U.S. patent application number 08/463404 was filed with the patent office on 2002-09-12 for therapy and diagnosis of conditions related to telomere length and/or telomerase activity.
Invention is credited to BLACKBURN, ELIZABETH H., SHAY, JERRY, WEST, MICHAEL D., WRIGHT, WOODRING.
Application Number | 20020127634 08/463404 |
Document ID | / |
Family ID | 26715521 |
Filed Date | 2002-09-12 |
United States Patent
Application |
20020127634 |
Kind Code |
A1 |
WEST, MICHAEL D. ; et
al. |
September 12, 2002 |
THERAPY AND DIAGNOSIS OF CONDITIONS RELATED TO TELOMERE LENGTH
AND/OR TELOMERASE ACTIVITY
Abstract
Method and compositions are provided for the determination of
telomere length and telomerase activity, as well as the ability to
inhibit telomerase activity in the treatment of proliferative
diseases. Particularly, primers are elongated under conditions
which minimize interference from other genomic sequences, so as to
obtain accurate determinations of telomeric length or telomerase
activity. In addition, compositions are provided for intracellular
inhibition of telomerase activity and means are shown for slowing
the loss of telomeric repeats in aging cells.
Inventors: |
WEST, MICHAEL D.; (BELMONT,
CA) ; SHAY, JERRY; (DALLS, TX) ; WRIGHT,
WOODRING; (ARLINGTON, TX) ; BLACKBURN, ELIZABETH
H.; (SAN FRANCISCO, CA) |
Correspondence
Address: |
LYON & LYON LLP
633 WEST FIFTH STREET
SUITE 4700
LOS ANGELES
CA
90071
US
|
Family ID: |
26715521 |
Appl. No.: |
08/463404 |
Filed: |
June 5, 1995 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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08463404 |
Jun 5, 1995 |
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08060952 |
May 13, 1993 |
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5695932 |
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08060952 |
May 13, 1993 |
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08038766 |
Mar 24, 1993 |
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5489508 |
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08038766 |
Mar 24, 1993 |
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07882438 |
May 13, 1992 |
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Current U.S.
Class: |
435/46 ; 356/320;
436/15; 436/43; 436/50; 436/8 |
Current CPC
Class: |
Y02A 50/409 20180101;
G01N 2333/91245 20130101; Y10S 977/927 20130101; Y10T 436/105831
20150115; C12N 5/0018 20130101; A61P 35/00 20180101; C12Q 1/6886
20130101; Y02A 50/411 20180101; C12Q 1/703 20130101; C12Y 207/07049
20130101; C12Q 1/6827 20130101; Y10T 436/115831 20150115; A61K
31/70 20130101; A61K 38/00 20130101; Y10S 977/801 20130101; A61P
43/00 20180101; C12N 2510/04 20130101; C12N 9/1241 20130101; C12Q
1/6876 20130101; Y10T 436/11 20150115; C12Q 2600/112 20130101; C12N
15/113 20130101; C12Q 1/686 20130101; Y10S 977/918 20130101; Y10S
977/773 20130101; Y10T 436/10 20150115; C12N 15/10 20130101; C12N
5/163 20130101; C12N 2501/70 20130101; Y02A 50/30 20180101; C12Q
1/48 20130101; A61K 31/522 20130101; A61K 31/711 20130101; A61K
31/7076 20130101; C12Q 1/68 20130101; C12N 15/1137 20130101; C12Q
2600/136 20130101; C12Q 1/6827 20130101; C12Q 2521/113 20130101;
C12Q 1/68 20130101; C12Q 2521/113 20130101 |
Class at
Publication: |
435/46 ; 356/320;
436/43; 436/50; 436/8; 436/15 |
International
Class: |
G01N 031/00; G01N
035/00; G01N 035/02; G01J 003/42 |
Claims
1. Method for treatment of a condition associated with an elevated
level of telomerase activity within a cell comprising the step of:
administering to said cell a therapeutically effective amount of an
inhibitor of said telomerase activity.
2. Method for treatment of a condition associated with an increased
rate of proliferation of a cell, comprising the step of:
administering to said cell a therapeutically effective amount of an
agent active to reduce loss of telomere length within said cell
during said proliferation.
3. Method for extending the ability of a cell to replicate,
comprising the step of: administering to said cell a replication
extending amount of an agent active to reduce loss of telomere
length within said cell during cellular replication.
4. A pharmaceutical composition comprising a therapeutically
effective amount of an inhibitor of telomerase activity in a
pharmaceutically acceptable buffer.
5. A pharmaceutical composition comprising a therapeutically
effective amount of an agent active to reduce loss of telomere
length within a cell during proliferation of said cell, in a
pharmaceutically acceptable buffer.
6. Method for diagnosis of a condition in a patient associated with
an elevated level of telomerase activity within a cell, comprising
the step of: determining the presence or amount of telomerase
within said cells in said patient.
7. Method for diagnosis of a condition associated with an increased
rate of proliferation in a cell in an individual, comprising the
steps of determining the length of telomeres within said cell.
8. Method for determining telomere length of an animal chromosome
or group of chromosomes, said method comprising: bringing together
in a reaction mixture said chromosome(s) or telomere comprising
fragment(s) thereof, a primer having at least two telomeric repeat
units, and nucleoside triphosphates having the same nucleotides as
the non-protruding strand of said telomere, wherein at least one of
said nucleoside triphosphates or primer is labeled with a
detectable label; and a DNA polymerase; incubating said reaction
mixture for sufficient time for said primer to be extended to
provide a primer extended sequence; separating said primer extended
by size; and determining the size of said primer extended sequence
by means of said label.
9. Method according to claim 8, wherein one of nucleoside
triphosphates is labeled with a radioisotope and said size is
determined by the level of radioactivity in relation to the amount
of DNA present.
10. Method according to claim 8, wherein said nucleosides are
combinations of A, T and C, or A, T and G.
11. Method of determining telomere length of an animal chromosome
or group of chromosomes, said method comprising: fragmenting said
chromosome(s) by a restriction endonuclease having a four base
recognition site absent in the telomere sequence; bringing together
said fragments and a primer for said telomeric sequence, wherein
said primer is labeled to allow for binding of said primer to a
surface; cross-linking said primer to said telomeric sequence;
isolating said telomeric sequence by means of said label; and
determining the size of said telomeric sequence bound to said
surface.
12. Method according to claim 11, wherein said primer is conjugated
with (1) an agent capable of cross-linking nucleic acids upon
irradiation; and (2) a specific binding pair member; and said
surface is conjugated with the complementary specific binding pair
member.
13. Method according to claim 11, wherein said primer is conjugated
with (1) an agent capable of cross-linking nucleic acids upon
irradiation; and (2) a particle.
14. Method of reducing the rate of telomere shortening in a
proliferating cellular composition, said method comprising:
introducing into cells of said cellular composition primers having
from 2 to 3 repeats of the repeating unit of the cellular
telomere.
15. Method of measuring the telomerase activity of a composition,
said method comprising: combining 1 or more repeats of the telomere
unit sequence and nucleoside triphosphates lacking cytidine
nucleotide, wherein at least one of said primer or nucleoside
triphosphates is labeled with a detectable label, with the proviso
that when said composition lacks a telomere sequence complementary
to said probe, said telomere sequence is added to said composition;
incubating said composition for a predetermined time for said
primer to be extended to provide an extended sequence; and
determining the rate of formation of said extended sequence.
16. Method according to claim 15, wherein one of said nucleoside
triphosphates is labeled with a radioisotope, and said determining
is by measuring radioactivity per unit weight of DNA.
17. Method of inhibiting the proliferation of telomerase-comprising
immortalized cells, said method comprising: contacting said
immortalized cells with a telomerase inhibitor under conditions
wherein said inhibitor enters said cells; whereby said
proliferation of said cells is inhibited.
18. Method according to claim 17, wherein said inhibitor inhibits
expression of telomerase.
19. Method of according to claim 17, wherein said inhibition is an
oligonucleotide sequence comprising the complementary sequence of
the telomerase RNA.
20. Method according to claim 17, wherein said oligonucleotide
sequence is a ribozyme.
21. Method for extending the proliferative capability of a
mammalian cell population, said method comprising: adding to said
cells oligonucleotides comprising at least two repeats
complementary to the sequence of the protruding strand of the
telomere of the chromosomes of said cells, whereby the shortening
of said telomere is slowed.
22. Method for treatment of a disease or condition associated with
cell senescense, comprising the steps of: administering a
therapeutically effective amount of an agent active to derepress
telomerase in the senescing cells.
23. Method for screening for a telomerase derepression agent,
comprising the steps of: contacting a potential agent with a cell
lacking telomerase activity, and determining whether said agent
increases the level of said activity.
24. The method of claim 23, wherein said cell is a cell expressing
an inducible T antigen.
25. The method of claim 1, wherein said cell if a fungal cell, and
said administering reduces viability of said cell.
26. The method of claim 25, wherein said cell is a C. albicans
cell.
27. Method for screening for agents useful in treatment of a human
disease associated with an elevated level of telomerase activity in
a human cell, comprising the step of testing potential said agents
for activity in inhibiting telomerase activity.
Description
[0001] This application is a continuation-in-part of Michael D.
West et al., entitled "Therapy and diagnosis of conditions related
to telomere length and/or telomerase activity, filed Mar. 24, 1993,
and assigned U.S. Ser. No. 08/038,766, which is a
continuation-in-part of Michael D. West et al., entitled
"Telomerase Activity Modulation and Telomere Diagnosis", filed May
13, 1992, and assigned U.S. Ser. No. 07/882,438, both (including
drawings) hereby incorporated by reference herein.
[0002] This invention relates to methods for therapy and diagnosis
of cellular senescence and immortalization.
BACKGROUND OF THE INVENTION
[0003] The following is a general description of art relevant to
the present invention. None is admitted to be prior art to the
invention. Generally, this art relates to observations relating to
cellular senescence, and theories or hypothesis which explain such
aging and the mechanisms by which cells escape senescence and
immortalize.
[0004] Normal human somatic cells (e.g., fibroblasts, endothelial,
and epithelial cells) display a finite replicative capacity of
50-100 population doubling characterized by a cessation of
proliferation in spite of the presence of abundant growth factors.
This cessation of replication in vitro is variously referred to as
cellular senescence or cellular aging, See, Goldstein, 249 Science
1129, 1990; Hayflick and Moorehead, 25 Exp. Cell Res. 585, 1961;
Hayflick, ibid., 37:614, 1985; Ohno, 11 Mech. Aging Dev. 179, 1979;
Ham and McKeehan, (1979) "Media and Growth Requirements", W. B.
Jacoby and I. M. Pastan (eds), in: Methods in Enzymology, Academic
Press, N.Y., 58:44-93. The replicative life span of cells is
inversely proportional to the in vivo age of the donor (Martin et
al., 23 Lab. Invest. 86, 1979; Goldstein et al., 64 Proc. Natl.
Acad. Sci. USA 155, 1969; and Schneider and Mitsui, ibid., 73:3584,
1976), therefore cellular senescence is suggested to play an
important role in aging in vivo.
[0005] Cellular immortalization (the acquisition of unlimited
replicative capacity) may be thought of as an abnormal escape from
cellular senescence, Shay et al., 196 Exp. Cell Res. 33, 1991.
Normal human somatic cells appear to be mortal, i.e., have finite
replicative potential. In contrast, the germ line and malignant
tumor cells are immortal (have indefinite proliferative potential).
Human cells cultured in vitro appear to require the aid of
transforming viral oncoproteins to become immortal and even then
the frequency of immortalization is 10.sup.-6 to 10.sup.-7. Shay
and Wright, 184 Exp. Cell Res. 109, 1989. A variety of hypotheses
have been advanced over the years to explain the causes of cellular
senescence. While examples of such hypotheses are provided below,
there appears to be no consensus or universally accepted
hypothesis.
[0006] For example, the free radical theory of aging suggests that
free radical-mediated damage to DNA and other macromolecules is
causative in critical loss of cell function (Harmon, 11 J.
Gerontol. 298, 1956; Harmon, 16 J. Gerontol. 247, 1961). Harman
says (Harman, 78 Proc. Natl. Acad. Sci. 7124, 1981) "aging is
largely due to free radical reaction damage . . . "
[0007] Waste-product accumulation theories propose that the
progressive accumulation of pigmented inclusion bodies (frequently
referred to as lipofuscin) in aging cells gradually interferes with
normal cell function (Strehler, 1 Adv. Geront. Res. 343, 1964;
Bourne, 40 Prog. Brain Res. 187, 1973; Hayflick, 20 Exp. Gerontol.
145, 1985).
[0008] The gradual somatic mutation theories propose that the
progressive accumulation of genetic damage to somatic cells by
radiation and other means impairs cell function and that without
the genetic recombination that occurs, for instance, during meiosis
in the germ line cells, somatic cells lack the ability to
proliferate indefinitely (Burnet, "Intrinsic Mutagenesis--A Genetic
Approach to Aging", Wile, N.Y., 1976; Hayflick, 27 Exp. Gerontol.
363, 1992). Theories concerning genetically programmed senescence
suggest that the expression of senescent-specific genes actively
inhibit cell proliferation (Martin et al., 74 Am. J. Pathol. 137,
1974; Goldstein, 249 Science 1129, 1990).
[0009] Smith and Whitney, 207 Science 82, 1980, discuss a mechanism
for cellular aging and state that their data is:
[0010] "compatible with the process of genetically controlled
terminal differentiation. . . . The gradual decrease in
proliferation potential would also be compatible with a continuous
build up of damage or errors, a process that has been theorized.
However, the wide variability in doubling potentials, especially in
mitotic pairs, suggests an unequalled partitioning of damage or
errors at division."
[0011] Shay et al., 27 Experimental Gerontology 477, 1992, and 196
Exp. Cell Res. 33, 1991 describe a two-stage model for human cell
mortality to explain the ability of Simian Virus 40 T-antigen to
immortalize human cells. The mortality stage 1 mechanism (M1) is
the target of certain tumor virus proteins, and an independent
mortality stage 2 mechanism (M2) produces crisis and prevents these
tumor viruses from directly immortalizing human cells. The authors
utilized T-antigen driven by a mouse mammary tumor virus promoter
to cause reversible immortalization of cells. The Simian Virus 40
T-antigen is said to extend the replicative life span of human
fibroblast by an additional 40-60%. The authors postulate that the
M1 mechanism is overcome by T-antigen binding to various cellular
proteins, or inducing new activities to repress the M1 mortality
mechanism. The M2 mechanism then causes cessation of proliferation,
even though the M1 mechanism is blocked. Immortality is achieved
only when the M2 mortality mechanism is also disrupted.
[0012] It has also been proposed that the finite replicative
capacity of cells may reflect the work of a "clock" liked to DNA
synthesis in the telomere (end part) of the chromosomes. Olovnikov,
41 J. Theoretical Biology 181, 1973, describes the theory of
marginotomy to explain the limitations of cell doubling potential
in somatic cells. He states that an:
[0013] "informative oligonucleotide, built into DNA after a
telogene and controlling synthesis of a repressor of
differentiation, might serve as a means of counting mitosis
performed in the course of morphogenesis. Marginotomic elimination
of such an oligonucleotide would present an appropriate signal for
the beginning of further differentiation. Lengthening of the
telogene would increase the number of possible mitoses in
differentiation."
[0014] Harley et al., 345 Nature 458, 1990, state that the amount
and length of telomeric DNA in human fibroblasts decreases as a
function of serial passage during aging in vitro, and possibly in
vivo, but do not know whether this loss of DNA has a causal role in
senescence. They also state:
[0015] "Tumour cells are also characterized by shortened telomeres
and increased frequency of aneuploidy, including telomeric
associations. If loss of telomeric DNA ultimately causes cell-cycle
arrest in normal cells, the final steps in this process may be
blocked in immortalized cells. Whereas normal cells with relatively
long telomeres and a senescent phenotype may contain little or no
telomerase activity, tumour cells with short telomeres may have
significant telomerase activity. Telomerase may therefore be an
effective target for anti-tumour drugs.
[0016] . . .
[0017] There are a number of possible mechanisms for loss of
telomeric DNA during ageing, including incomplete replication,
degradation of termini (specific or nonspecific), and unequal
recombination coupled to selection of cells with shorter telomeres.
Two features of our data are relevant to this question. First, the
decrease in mean telomere length is about 50 bp per mean population
doubling and, second, the distribution does not change
substantially with growth state or cell arrest. These data are most
easily explained by incomplete copying of the template strands at
their 3' termini. But the absence of detailed information about the
mode of replication or degree of recombination at telomeres means
that none of these mechanisms can be ruled out. Further research is
required to determine the mechanism of telomere shortening in human
fibroblasts and its significance to cellular senescence."
[Citations]omitted.)
[0018] Hastie et al., 346 Nature 866, 1990, while discussing colon
tumor cells, state that:
[0019] "[T]here is a reduction in the length of telomere repeat
arrays relative to the normal colonic mucosa from the same
patient.
[0020] . . .
[0021] Firm figures are not available, but it is likely that the
tissues of a developed fetus result from 20-50 cell divisions,
whereas several hundred or thousands of divisions have produced the
colonic mucosa and blood cells of 60-year old individuals. Thus the
degree of telomere reduction is more or less proportional to the
number of cell divisions. It has been shown that the ends of
Drosophila chromosomes without normal telomeres reduce in size by
.sub.--4 base pairs (bp) per cell division and that the ends of
yeast chromosomes reduce by a similar degree in a mutant presumed
to lack telomerase function. If we assume the same rate of
reduction is occurring during somatic division in human tissues,
then a reduction in TRA by 14 kb would mean that 3,500 ancestral
cell divisions lead to the production of cells in the blood of a
60-year old individual; using estimates of sperm telomere length
found elsewhere we obtain a value of 1,000-2,000. These values
compare favourably with those postulated for mouse blood cells.
Thus, we propose that telomerase is indeed lacking in somatic
tissues. In this regard it is of interest to note that in maize,
broken chromosomes are only healed in sporophytic (zygotic) tissues
and not in endosperm (terminally differentiated), suggesting that
telomerase activity is lacking in the differentiated tissues."
[Citations omitted.]
[0022] The authors propose that in some tumors telomerase is
reactivated, as proposed for HeLa cells in culture, which are known
to contain telomerase activity. But, they state:
[0023] "One alternative explanation for our observations is that in
tumours the cells with shorter telomeres have a growth advantage
over those with larger telomeres, a situation described for
vegetative cells of tetrahymena." [Citations omitted.]
[0024] Harley, 256 Mutation Research 271, 1991, discusses
observations allegedly showing that telomeres of human somatic
cells act as a mitotic clock shortening with age both in vitro and
in vivo in a replication dependent manner. He states:
[0025] "Telomerase activation may be a late, obligate event in
immortalization since many transformed cells and tumour tissues
have critically short telomeres. This, telomere length and
telomerase activity appear to be markers of the replicative history
and proliferative potential of cells; the intriguing possibility
remains that telomere loss is a genetic time bomb and hence
causally involved in cell senescence and immortalization.
[0026] . . .
[0027] Despite apparently stable telomere length in various tumour
tissues or transformed cell lines, this length was usually found to
be shorter than those of the tissue of origin. These data suggest
that telomerase becomes activated as a late event in cell
transformation, and that cells could be viable (albeit genetically
unstable) with short telomeres stably maintained by telomerase. If
telomerase was constitutively present in a small fraction of normal
cells, and these were the ones which survived crisis or became
transformed, we would expect to find a greater frequency of
transformed cells with long telomeres."[Citations omitted.]
[0028] He proposes a hypothesis for human cell aging and
transformation as "[a] semi-quantitative model in which telomeres
and telomerase play a causal role in cell senescence and cancer"
and proposes a model for this hypothesis.
[0029] De Langa et al., 10 Molecular and Cellular Biology 518,
1990, generally discuss the structure of human chromosome ends or
telomeres. They state:
[0030] "we do not know whether telomere reduction is strictly
coupled to cellular proliferation. If the diminution results from
incomplete replication of the telomere, such a coupling would be
expected; however, other mechanisms, such as exonucleolytic
degradation, may operate independent of cell division. In any
event, it is clear that the maintenance of telomeres is impaired in
somatic cells. An obvious candidate activity that may be reduced or
lacking is telomerase. A human telomerase activity that can add
TTAGGG repeats to G-rich primers has recently been identified (G.
Morin, personal communication). Interestingly, the activity was
demonstrated in extracts of HeLa cells, which we found to have
exceptionally long telomeres. Other cell types have not been tested
yet, but such experiments could now establish whether telomerase
activity is (in part) responsible for the dynamics of human
chromosome ends."
[0031] Starling et al., 18 Nucleic Acids Research 6881, 1990,
indicate that mice have large telomeres and discusses this length
in relationship to human telomeres. They state:
[0032] "Recently it has been shown that there is reduction in TRA
length with passage number of human fibroblasts in vitro and that
cells in a senescent population may lack telomeres at some ends
altogether. Thus in vitro, telomere loss may play a role in
senescence, a scenario for which there is evidence in S. cerevisae
and Tetrahymena.
[0033] Some of the mice we have been studying are old in mouse
terms, one and a half years, yet they still have TRA's greater than
30 kb in all tissues studied. In humans, telomeres shorten with age
at a rate of 100 bp per year, hence, it is conceivable that the
same is happening in the mouse, but the removal of a few 100 bps of
terminal DNA during its lifetime would not be detectable."
[Citations omitted.]
[0034] D'Mello and Jazwinski, 173 J. Bacteriology 6709, 1991,
state:
[0035] "We propose that during the life span of an organism,
telomere shortening does not play a role in the normal aging
process. However, mutations or epigenetic changes that affect the
activity of the telomerase, like any other genetic change, might
affect the life span of the individual in which they occur.
[0036] . . .
[0037] In summary, the telomere shortening with age observed in
human diploid fibroblasts may not be a universal phenomenon.
Further studies are required to examine telomere length and
telomerase activity not only in different cell types as they age
but also in the same cell type in different organisms with
differing life spans. This would indicate whether telomere
shortening plays a causal role in the senescence of a particular
cell type or organism."
[0038] Hiyama et al., 83 Jpn. J. Cancer Res. 159, 1992, provide
findings that "suggest that the reduction of telomeric repeats is
related to the proliferative activity of neuroblastoma cells and
seems to be a useful indicator of the aggressiveness of
neuroblastoma. . . . Although we do not know the mechanism of the
reduction and the elongation of telomeric repeats in neuroblastoma,
we can at least say that the length of telomeric repeats may be
related to the progression and/or regression of neuroblastoma."
[0039] Counter et al., 11 EMBO J. 1921, 1992, state "loss of
telomeric DNA during cell proliferation may play a role in ageing
and cancer." They propose that the expression of telomerase is one
of the events required for a cell to acquire immortality and note
that:
[0040] This model may have direct relevance to tumourigenesis in
vivo. For example, the finite lifespan of partially transformed
(pre-immortal) cells which lack telomerase might explain the
frequent regression of tumours after limited growth in vivo. In
bypassing the checkpoint representing normal replicative
senescence, transformation may confer an additional 20-40
population doubling during which an additional .apprxeq.2 kbp of
telomeric DNA is lost. Since 20-40 doubling (10.sup.6-10.sup.12
cells in a clonal population) potentially represents a wide range
of tumour sizes, it is possible that many benign tumours may lack
telomerase and naturally regress when telomeres become critically
shortened. We predict that more aggressive, perhaps metastatic
tumours would contain immortal cells which express telomerase. To
test this hypothesis, we are currently attempting to detect
telomerase in a variety of tumour tissues and to correlate activity
with proliferative potential. Anti-telomerase drugs or mechanisms
to repress telomerase expression could be effective agents against
tumours which depend upon the enzyme for maintenance of telomeres
and continued cell growth.
[0041] Levy et al., 225 J. Mol. Biol. 951, 1992, state that:
[0042] "Although it has not been proven that telomere loss
contributes to senescence of multicellular organisms, several lines
of evidence suggest a causal relationship may exist.
[0043] . . .
[0044] It is also possible that telomere loss with age is
significant in humans, but not in mice." [Citations omitted.]
[0045] Windle and McGuire, 33 Proceedings of the American
Association for Cancer Research 594, 1992, discuss the role of
telomeres and state that:
[0046] "These and other telomere studies point in a new direction
regarding therapeutic targets and strategies to combat cancer. If
the cell can heal broken chromosomes preventing genomic disaster,
then there may be a way to facilitate or artificially create this
process. This could even provide a preventive means of stopping
cancer which could be particularly applicable in high risk
patients. The difference in telomere length in normal versus tumor
cells also suggests a strategy where the loss of telomeres is
accelerated. Those cells with the shortest telomeres, such as those
of tumor metastasis would be the most susceptible."
[0047] Goldstein, 249 Science 1129, 1990, discusses various
theories of cellular senescence including that of attrition of
telomeres. He states:
[0048] "However, such a mechanism is not easily reconciled with the
dominance of senescent HDF over young HDF in fusion hybrids,
particularly in short-term heterokaryons. One could again invoke
the concept of dependence and the RAD9 gene example, such that
complete loss of one or a few telomeres leads to the elaboration of
a negative signal that prevents initiation of DNA synthesis,
thereby mimicking the differentiated state. This idea, although
speculative, would not only explain senescent replicative arrest
but also the chromosomal aberrations observed in senescent HDS that
would specifically ensue after loss of telomeres." [Citations
omitted.]
[0049] The role of telomere loss in cancer is further discussed by
Jankovic et al. and Hastie et al., both at 350 Nature 1991, in
which Jankovic indicates that telomere shortening is unlikely to
significantly influence carcinogenesis in men and mice. Hastie et
al. agree that if telomere reduction does indeed reflect cell
turnover, this phenomenon is unlikely to play a role in pediatric
tumors, and those of the central nervous system. Hastie et al.,
however, feel "our most original and interesting conclusion was
that telomere loss may reflect the number of cell division in a
tissue history, constituting a type of clock."
[0050] Kipling and Cooke, 1 Human Molecular Genetics 3, 1992,
state:
[0051] "It has been known for some years that telomeres in human
germline cells (e.g. sperm) are longer than those in somatic tissue
such as blood. One proposed explanation for this is the absence of
telomere repeat addition (i.e. absence of telomerase activity) in
somatic cells. If so, incomplete end replication would be expected
to result in the progressive loss of terminal repeats as somatic
cells undergo successive rounds of division. This is indeed what
appears to happen in vivo for humans, with both blood and skin
cells showing shorter telomeres with increasing donor age, and
telomere loss may contribute to the chromosome aberrations
typically seen in senescent cells. Senescence and the measurement
of cellular time is an intriguingly complex subject and it will be
interesting to see to what extent telomere shortening has a causal
role. The large telomeres possessed by both young and old mice
would seem to preclude a simple relationship between telomere loss
and ageing, but more elaborate schemes cannot be ruled
out."[Citations omitted.]
[0052] Greider, 12 BioEssays 363, 1990, provides a review of the
relationship between telomeres, telomerase, and senescence. She
indicates that telomerase contains an RNA component which provides
a template for telomere repeat synthesis. She notes that an
oligonucleotide "which is complementary to the RNA up to and
including the CAACCCCAA sequence, competes with d(TTGGGG)n primers
and inhibits telomerase in vitro" (citing Greider and Blackburn,
337 Nature 331, 1989). She also describes experiments which she
believes "provide direct evidence that telomerase is involved in
telomere synthesis in vivo." She goes on to state:
[0053] "Telomeric restriction fragments in many transformed cell
lines are much shorter than those in somatic cells. In addition,
telomere length in tumor tissues is significantly shorter than in
the adjacent non-tumor tissue. When transformed cell lines are
passaged in vitro there is no change in telomere length. Thus if
untransformed cells lack the ability to maintain a telomere length
equilibrium, most transformed cells appear to regain it and to
reset the equilibrium telomere length to a size shorter than seen
in most tissues in vivo. The simplest interpretation of these data
is that enzymes, such as telomerase, involved in maintaining
telomere length may be required for growth of transformed cells and
not required for normal somatic cell viability. This suggests that
telomerase may be a good target for anti-tumor drugs." [Citations
omitted.]
[0054] Blackburn, 350 Nature 569, 1991, discusses the potential for
drug action at telomeres stating:
[0055] "The G-rich strand of the telomere is the only essential
chromosomal DNA sequence known to be synthesized by the copying of
a separate RNA sequence. This unique mode of synthesis, and the
special structure and behavior of telomeric DNA, suggest that
telomere synthesis could be a target for selective drug action.
Because telomerase activity seems to be essential for protozoans or
yeast, but not apparently for mammalian somatic cells, I propose
that telomerase should be explored as a target for drugs against
eukaryotic pathogenic or parasitic microorganisms, such as
parasitic protozoans or pathogenic yeasts. A drug that binds
telomerase selectively, either through its reverse-transcriptase or
DNA substrate-binding properties, should selectively act against
prolonged maintenance of the dividing lower eukaryote, but not
impair the mammalian host over the short term, because telomerase
activity in its somatic cells may normally be low or absent.
Obvious classes of drugs to investigate are those directed
specifically against reverse transcriptases as opposed to other DNA
or RNA polymerases, and drugs that would bind telomeric DNA itself.
These could include drugs that selectively bind the G.degree.G
base-paired forms of the G-rich strand protrusions at the
chromosome termini, or agents which stabilize an inappropriate
G.degree.G base-paired form, preventing it from adopting a
structure necessary for proper function in vivo. Telomeres have
been described as the Achilles heel of chromosomes: perhaps it is
there that drug strategies should now be aimed." [Citations
omitted.]
[0056] Lundblad and Blackburn, 73 Cell 347, 1993, discuss
alternative pathways for maintainance of yeast telomers, and state
that:
[0057] ". . . the work presented in this paper demonstrates that a
defect in telomere replication need not result in the death of all
cells in a population, suggesting that telomere loss and its
relationship to mammalian cellular senescence may have to be
examined further."
[0058] Other review articles concerning telomeres include Blackburn
and Szostak, 53 Ann. Rev. Biochem. 163, 1984; Blackburn, 350 Nature
569, 1991; Greider, 67 Cell 645, 1991, and Moyzis 265 Scientific
American 48, 1991. Relevant articles on various aspects of
telomeres include Cooke and Smith, Cold Spring Harbor Symposia on
Quantitative Biology Vol. LI, pp. 213-219; Morin, 59 Cell 521,
1989; Blackburn et al., 31 Genome 553, 1989; Szostak, 337 Nature
303, 1989; Gall, 344 Nature 108, 1990; Henderson et al., 29
Biochemistry 732, 1990; Gottschling et al., 63 Cell 751, 1990;
Harrington and Grieder, 353 Nature 451, 1991; Muller et al., 67
Cell 815, 1991; Yu and Blackburn, 67 Cell 823, 1991; and Gray et
al., 67 Cell 807, 1991. Other articles or discussions of some
relevance include Lundblad and Szostak, 57 Cell 633, 1989; and Yu
et al., 344 Nature 126, 1990.
SUMMARY OF THE INVENTION
[0059] This invention concerns methods for therapy and diagnosis of
cellular senescence and immortalization utilizing techniques
associated with control of telomere length and telomerase activity.
Therapeutic strategies of this invention include reducing the rate
or absolute amount of telomere repeat length loss or increasing the
telomere repeat length during cell proliferation, thereby providing
for the postponement of cellular senescence and reducing the level
of chromosomal fusions and other chromosomal aberrations. In
addition, inhibition of telomerase activity in vivo or in vitro may
be used to control diseases associated with cell immortality, such
as neoplasia, and pathogenic parasites.
[0060] Applicant has determined that the inhibition of telomere
shortening in a cell in vitro is causally related to increasing the
length of the replicative lifespan of that cell. Applicant has also
determined that inhibition of telomerase activity in a cell in
vitro is causally related to reducing the ability of that cell to
proliferate in an immortal manner. Thus, applicant is the first to
provide data which clearly indicates that inhibition of telomere
shortening in vivo or in vitro, and that inhibition of telomerase
activity in vivo or in vitro, is therapeutically beneficial. Prior
to applicant's experiments, as indicated above, there was no
consensus by those in the art that one could predict that such
experiments would provide the data observed by applicant, or that
such manipulations would have therapeutic utility.
[0061] The invention also concerns the determination of cellular
status by diagnostic techniques that analyze telomere length and
telomerase activity, as a diagnostic of cellular capacity for
proliferation. Assays for telomere length are performed to provide
useful information on the relative age and remaining proliferative
capability of a wide variety of cell types in numerous tissues.
Sequences are also described from the telomeres of budding yeasts
which are highly variable from strain to strain and provide
sequences for oligonucleotide probes that would enable the rapid
identification of yeast strains, and in the case of human and
veterinary pathogens, the diagnosis of the strain of the
pathogen.
[0062] Telomerase activity and the presence of the enzyme is used
as a marker for diagnosing and staging neoplasia and detecting
pathogenic parasites. Applicant's experiments have, for the first
time, determined a correlation between telomerase activity and the
tumor cell phenotype, as well as a correlation between telomere
length and the in vivo aged status of cells. As noted above, there
was no consensus in the art that one could predict that such a
relationship existed. In contrast, applicant has defined this
relationship, and thus has now defined useful diagnostic tools by
which to determine useful clinical data. Such data can be used to
define a therapeutic protocol, or the futility of such a
protocol.
[0063] Thus, in a first aspect, the invention features methods for
the treatment of a condition associated with cellular senescence or
increased rate of proliferation of a cell (e.g., telomere repeat
loss associated with cell proliferation in the absence of
telomerase). A first method involves administering to the cell a
therapeutically effective amount of an agent active to reduce loss
of telomeric repeats during its proliferation. Such therapeutics
may be especially applicable to conditions of increased rate of
cell proliferation.
[0064] By "increased rate of proliferation" of a cell is meant that
the cell has a higher rate of cell division compared to normal
cells of that cell type, or compared to normal cells within other
individuals of that cell type. Examples of such cells include the
CD4.sup.+ cells of HIV-infected individuals (see example below),
connective tissue fibroblasts associated with degenerative joint
diseases, age-related macular degeneration, astrocytes associated
with Alzheimer's Disease and endothelial cells associated with
atherosclerosis (see example below). In each case, one particular
type of cell or a group of cells is found to be replicating at an
increased level compared to surrounding cells in those tissues, or
compared to normal individuals, e.g., individuals not infected with
the HIV virus. Thus, the invention features administering to those
cells an agent which reduces loss of telomere length in those cells
while they proliferate. The agent itself need not slow the
proliferation process, but rather allow that proliferation process
to continue for more cell divisions than would be observed in the
absence of the agent. The agent may also be useful to slow telomere
repeat loss occurring during normal aging (wherein the cells are
proliferating at a normal rate and undergoing senescence late in
life), and for reducing telomere repeat loss while expanding cell
number ex vivo for cell-based therapies, e.g., bone marrow
transplantation following gene therapy.
[0065] As described herein, useful agents can be readily identified
by those of ordinary skill in the art using routine screening
procedures. For example, a particular cell having a known telomere
length is chosen and allowed to proliferate, and the length of
telomere is measured during proliferation. Agents which are shown
to reduce the loss of telomere length during such proliferation are
useful in this invention. Particular examples of such agents are
provided below. For example, oligonucleotides which are able to
promote synthesis of DNA at the telomere ends are useful in this
invention. In addition, telomerase may be added to a cell either by
gene therapy techniques, or by of the enzyme or its equivalent into
a cell administration, e.g., by injection or lipojection.
[0066] A second method for the treatment of cellular senescence
involves the use of an agent to derepress telomerase in cells where
the enzyme is normally repressed. Telomerase activity is not
detectable in any normal human somatic cells, but is detectable in
cells that have abnormally reactivated the enzyme during the
transformation of a normal cell into an immortal tumor cell.
Telomerase activity may therefore be appropriate only in germ line
cells and some stem cell populations (though there is currently no
evidence of the latter in human tissues). Since the loss of
telomeric repeats leading to senescence in somatic cells is
occuring due to the absence of adequate telomerase activity, agents
that have the effect of activating telomerase would have the effect
of adding arrays of telomeric repeats to telomeres, thereby
imparting to mortal somatic cells increased replicative capacity,
and imparting to senescent cells the ability to proliferate and
appropriately exit the cell cycle (in the absence of growth factor
stimulation with associated appropriate regulation of cell
cycle-linked genes typically inappropriately expressed in
senescence e.g., collagenase, urokinase, and other secreted
proteases and protease inhibitors). Such factors to derepress
telomerase may be administered transiently or chronically to
increase telomere length, and then removed, thereby allowing the
somatic cells to again repress the expression of the enzyme
utilizing the natural mechanisms of repression.
[0067] Such activators of telomerase may be found by screening
techniques utilizing human cells that have the M1 mechanism of
senescence abrogated by means of the expression of SV40 T-antigen.
Such cells when grown to crisis, wherein the M2 mechanism is
preventing their growth, will proliferate in response to agents
that derepress telomerase. Such activity can be scored as the
incorporation of radiolabeled nucleotides or proliferating clones
can be selected for in a colony forming assay.
[0068] Such activators of telomerase would be useful as therapeutic
agents to forestall and reverse cellular senescence, including but
not limited to conditions associated with cellular senescence,
e.g., (a) cells with replicative capacity in the central nervous
system, including astrocytes, endothelial cells, and fibroblasts
which play a role in such age-related diseases as Alzheimer's
disease, Parkinson's disease, Huntington's disease, and stroke, (b)
cells with finite replicative capacity in the integument, including
fibroblasts, sebaceous gland cells, melanocytes, keratinocytes,
Langerhan's cells, and hair follicle cells which may play a role in
age-related diseases of the integument such as dermal atrophy,
elastolysis and skin wrinkling, sebaceous gland hyperplasia, senile
lentigo, graying of hair and hair loss, chronic skin ulcers, and
age-related impairment of wound healing, (c) cells with finite
replicative capacity in the articular cartilage, such as
chondrocytes and lacunal and synovial fibroblasts which play a role
in degenerative joint disease, (d) cells with finite replicative
capacity in the bone, such as osteoblasts and osteoprogenitor cells
which play a role in osteoporosis, (e) cells with finite
replicative capacity in the immune system such as B and T
lymphocytes, monocytes, neutrophils, eosinophils, basophils, NK
cells and their respective progenitors, which may play a role in
age-related immune system impairment, (f) cells with a finite
replicative capacity in the vascular system including endothelial
cells, smooth muscle cells, and adventitial fibroblasts which may
play a role in age-related diseases of the vascular system
including atherosclerosis, calcification, thrombosis, and
aneurysms, and (g) cells with a finite replicative capacity in the
eye such as pigmented epithelium and vascular endothelial cells
which may play an important role in age-related macular
degeneration.
[0069] In a second aspect, the invention features a method for
treatment of a condition associated with an elevated level of
telomerase activity within a cell. The method involves
administering to that cell a therapeutically effective amount of an
inhibitor of telomerase activity.
[0070] The level of telomerase activity can be measured as
described below, or by any other existing methods or equivalent
methods. By "elevated level" of such activity is meant that the
absolute level of telomerase activity in the particular cell is
elevated compared to normal cells in that individual, or compared
to normal cells in other individuals not suffering from the
condition. Examples of such conditions include cancerous
conditions, or conditions associated with the presence of cells
which are not normally present in that individual, such as
protozoan parasites or opportunistic pathogens, which require
telomerase activity for their continued replication. Administration
of an inhibitor can be achieved by any desired means well known to
those of ordinary skill in the art.
[0071] In addition, the term "therapeutically effective amount" of
an inhibitor is a well recognized phrase. The amount actually
applied will be dependent upon the individual or animal to which
treatment is to be applied, and will preferably be an optimized
amount such that an inhibitory effect is achieved without
significant side-effects (to the extent that those can be avoided
by use of the inhibitor). That is, if effective inhibition can be
achieved with no side-effects with the inhibitor at a certain
concentration, that concentration should be used as opposed to a
higher concentration at which side-effects may become evident. If
side-effects are unavoidable, however, the minimum amount of
inhibitor that is necessary to achieve the inhibition desired may
have to be used.
[0072] By "inhibitor" is simply meant any reagent, drug or chemical
which is able to inhibit a telomerase activity in vitro or in vivo.
Such inhibitors can be readily identified using standard screening
protocols in which a cellular extract or other preparation having
telomerase activity is placed in contact with a potential
inhibitor, and the level of telomerase activity measured in the
presence or absence of the inhibitor, or in the presence of varying
amounts of inhibitor. In this way, not only can useful inhibitors
be identified, but the optimum level of such an inhibitor can be
determined in vitro for further testing in vivo.
[0073] One example of a suitable telomerase inhibitor assay is
carried out in 96-well microtiter plates. One microtiter plate is
used to make dilutions of the test compounds, while another plate
is used for the actual assay. Duplicate reactions of each sample
are performed. A mixture is made containing the appropriate amount
of buffer, template oligonucleotide, and Tetrahymena or human
telomerase extract for the number of the samples to be tested, and
aliquots are placed in the assay plate. The test compounds are
added individually and the plates are pre-incubated at 30.degree.
C. .sup.32P-dGTP is then added and the reaction allowed to proceed
for 10 minutes at 30.degree. C. The total volume of each reaction
is 10 .mu.l. The reaction is then terminated by addition of Tris
and EDTA, and half the volume (5 .mu.l) spotted onto DE81 filter
paper. The samples are allowed to air dry, and the filter paper is
rinsed in 0.5 M NaPhosphate several times to wash away the
unincorporated labeled nucleotide. After drying, the filter paper
is exposed to a phosphor imaging plate and the amount of signal
quantitated. By comparing the amount of signal for each of the test
samples to control samples, the percent of inhibition can be
determined.
[0074] In addition, a large number of potentially useful inhibitors
can be screened in a single test, since it is inhibition of
telomerase activity that is desired. Thus, if a panel of 1,000
inhibitors is to be screened, all 1,000 inhibitors can potentially
be placed into microtiter wells. If such an inhibitor is
discovered, then the pool of 1,000 can be subdivided into 10 pools
of 100 and the process repeated until an individual inhibitor is
identified. As discussed herein, one particularly useful set of
inhibitors includes oligonucleotides which are able to either bind
with the RNA present in telomerase or able to prevent binding of
that RNA to its DNA target or one of the telomerase protein
components. Even more preferred are those oligonucleotides which
cause inactivation or cleavage of the RNA present in a telomerase.
That is, the oligonucleotide is chemically modified or has enzyme
activity which causes such cleavage. The above screening may
include screening of a pool of many different such oligonucleotide
sequences.
[0075] In addition, a large number of potentially useful compounds
can be screened in extracts from natural products. Sources of such
extracts can be from a large number of species of fungi,
actinomyces, algae, protozoa, plants, and bacteria. Those extracts
showing inhibitory activity can then be analyzed to isolate the
active molecule.
[0076] In related aspects, the invention features pharmaceutical
compositions which include therapeutically effective amounts of the
inhibitors or agents described above, in pharmaceutically
acceptable buffers much as described below. These pharmaceutical
compositions may include one or more of these inhibitors or agents,
and be co-administered with other drugs. For example, AZT is
commonly used for treatment of HIV, and may be co-administered with
an inhibitor or agent of the present invention.
[0077] In a related aspect, the invention features a method for
extending the ability of a cell to replicate. In this method, a
replication extending amount of an agent which is active to reduce
loss of telomere length within the cell is provided during cell
replication. As will be evident to those of ordinary skill in the
art, this agent is similar to that useful for treatment of a
condition associated with an increased rate of proliferation of a
cell. However, this method is useful for the treatment of
individuals not suffering from any particular condition, but in
which one or more cell types are limiting in that patient, and
whose life can be extended by extending the ability of those cells
to continue replication. That is, the agent is added to delay the
onset of cell senescence characterized by the inability of that
cell to replicate further in an individual. One example of such a
group of cells includes lymphocytes present in patients suffering
from Downs Syndrome (although treatment of such cells may also be
useful in individuals not identified as suffering from any
particular condition or disease, but simply recognizing that one or
more cells, or collections of cells are becoming limiting in the
life span of that individual).
[0078] It is notable that administration of such inhibitors or
agents is not expected to be detrimental to any particular
individual. However, should gene therapy be used to introduce a
telomerase into any particular cell population, or other means be
used to reversibly de-repress telomerase activity in somatic cells,
care should be taken to ensure that the activity of that telomerase
is carefully regulated, for example, by use of a promoter which can
be regulated by the nutrition of the patient. Thus, for example,
the promoter may only be activated when the patient eats a
particular nutrient, and is otherwise inactive. In this way, should
the cell population become malignant, that individual may readily
inactivate telomerase of the cell and cause it to become mortal
simply by no longer eating that nutrient.
[0079] In a further aspect, the invention features a method for
diagnosis of a condition in a patient associated with an elevated
level of telomerase activity within a cell. The method involves
determining the presence or amount of telomerase within the cells
in that patient.
[0080] In yet another aspect, the invention features a method for
diagnosis of a condition associated with an increased rate of
proliferation in that cell in an individual. Specifically, the
method involves determining the length of telomeres within the
cell. The various conditions for which diagnosis is possible are
described above. As will be exemplified below, many methods exist
for measuring the presence or amount of telomerase within a cell in
a patient, and for determining the length of telomeres within the
cell. It will be evident that the presence or amount of telomerase
may be determined within an individual cell, and for any particular
telomerase activity (whether it be caused by one particular enzyme
or a plurality of enzymes). Those in the art can readily formulate
antibodies or their equivalent to distinguish between each type of
telomerase present within a cell, or within an individual. In
addition, the length of telomeres can be determined as an average
length, or as a range of lengths much as described below. Each of
these measurements will give precise information regarding the
status of any particular individual.
[0081] Thus, applicant's invention has two prongs--a diagnostic and
a therapeutic prong. These will now be discussed in detail.
[0082] The therapeutic prong of the invention is related to the now
clear observation that the ability of a cell to remain immortal
lies in the ability of that cell to maintain or increase the
telomere length of chromosomes within that cell. Such a telomere
length can be maintained by the presence of sufficient activity of
telomerase, or an equivalent enzyme, within the cell. Thus,
therapeutic approaches to reducing the potential of a cell to
remain immortal focus on the inhibition of telomerase activity
within those cells in which it is desirable to cause cell death.
Examples of such cells include cancerous cells, which are one
example of somatic cells which have regained the ability to express
telomerase, and have become immortal. Applicant has now shown that
such cells can be made mortal once more by inhibition of telomerase
activity. As such, inhibition can be achieved in a multitude of
ways including, as illustrated below, the use of oligonucleotides
which, in some manner, block the ability of telomerase to extend
telomeres in vivo.
[0083] Thus, oligonucleotides can be designed either to bind to a
telomere (to block the ability of telomerase to bind to that
telomere, and thereby extend that telomere), or to bind to the
resident oligonucleotide (RNA) present in telomerase to thereby
block telomerase activity on any nucleic acid (telomere). Such
oligonucleotides may be formed from naturally occurring
nucleotides, or may include modified nucleotides to either increase
the stability of the therapeutic agent, or cause permanent
inactivation of the telomerase, e.g., the positioning of a chain
terminating nucleotide at the 3' end of the molecule of a
nucleotide with a reactive group capable of forming a covalent bond
with telomerase. Such molecules may also include ribozyme
sequences. In addition, non-oligonucleotide based therapies can be
readily devised by screening for those molecules which have an
ability to inhibit telomerase activity in vitro, and then using
those molecules in vivo. Such a screen is readily performed and
will provide a large number of useful therapeutic molecules. These
molecules may be used for treatment of cancers, of any type,
including solid tumors and leukemias (including those in which
cells are immortalized, including: apudoma, choristoma, branchioma,
malignant carcinoid syndrome, carcinoid heart disease, carcinoma
(e.g., Walker, basal cell, basosquamous, Brown-Pearce, ductal,
Ehrlich tumor, in situ, Krebs 2, merkel cell, mucinous, non-small
cell lung, oat cell, papillary, scirrhous, bronchiolar,
bronchogenic, squamous cell, and transitional cell), histiocytic
disorders, leukemia (e.g., b-cell, mixed-cell, null-cell, T-cell,
T-cell chronic, HTLV-II-associated, lyphocytic acute, lymphocytic
chronic, mast-cell, and myeloid), histiocytosis malignant,
Hodgkin's disease, immunoproliferative small, non-Hodgkin's
lymphoma, plasmacytoma, reticuloendotheliosis, melanoma,
chondroblastoma, chondroma, chondrosarcoma, fibroma, fibrosarcoma,
giant cell tumors, histiocytoma, lipoma, liposarcoma, mesothelioma,
myxoma, myxosarcoma, osteoma, osteosarcoma, Ewing's sarcoma,
synovioma, adenofibroma, adenolymphoma, carcinosarcoma, chordoma,
craniopharyngioma, dysgerminoma, hamartoma, mesenchymoma,
mesonephroma, myosarcoma, ameloblastoma, cementoma, odontoma,
teratoma, thymoma, trophoblastic tumor, adenocarcinoma, adenoma,
cholangioma, cholesteatoma, cylindroma, cystadenocarcinoma,
cystadenoma, granulosa cell tumor, gynandroblastoma, hepatoma,
hidradenoma, islet cell tumor, leydig cell tumor, papilloma,
sertoli cell tumor, theca cell tumor, leiomyoma, leiomvosarcoma,
myoblastoma, myoma, myosarcoma, rhabdomyoma, rhabdomyosarcoma,
ependymoma, ganglioneuroma, glioma, medulloblastoma, meningioma,
neurilemmoma, neuroblastoma, neuroepithelioma, neurofibroma,
neuroma, paraganglioma, paraganglioma nonchromaffin, angiokeratoma,
angiolymphoid hyperplasia with eosinophilia, angioma sclerosing,
angiomatosis, glomangioma, hemangioendothelioma, hemangioma,
hemangiopericytoma, hemangiosarcoma, lymphangioma, lymphangiomyoma,
lymphangiosarcoma, pinealoma, carcinosarcoma, chondrosarcoma,
cystosarcoma phyllodes, fibrosarcoma, hemangiosarcoma,
leiomyosarcoma, leukosarcoos arliposarcoma, lymphangiosarcoma,
myosarcoma, myxosarcoma, ovarian carcinoma, rhabdomyosarcoma,
sarcoma (e.g., Ewing's, experimental, Kaposi's, and mast-cell),
neoplasms (e.g., bone, breast, digestive system, colorectal, liver,
pancreatic, pituitary, testicular, orbital, head and neck, central
nervous system, acoustic, pelvic, respiratory tract, and
urogenital), neurofibromatosis, and cervical dysplasia), and for
treatment of other conditions in which cells have become
immortalized.
[0084] Applicant has also determined that it is important to slow
the loss of telomere sequences, in particular, cells in association
with certain diseases (although such treatment is not limited to
this, and can be used in normal aging and ex vivo treatments). For
example, some diseases are manifest by the abnormally fast rate of
proliferation of one or more particular groups of cells. Applicant
has determined that it is the senescence of those groups of cells
at an abnormally early age (compared to the age of the patient),
that eventually leads to death of that patient. One example of such
a disease is AIDS, in which death is caused by the early senescence
of CD4.sup.+ cells. It is important to note that such cells age,
not because of abnormal loss of telomere sequences (although this
may be a factor), but rather because the replicative rate of the
CD4.sup.+ cells is increased such that telomere attrition is caused
at a greater rate than normal for that group of cells. Thus,
applicant provides therapeutic agents which can be used for
treatment of such diseases, and also provides a related diagnostic
procedure by which similar diseases can be detected so that
appropriate therapeutic protocols can be devised and followed.
[0085] Specifically, the loss of telomeres within any particular
cell population can be reduced by provision of an oligonucleotide
which reduces the extent of telomere attrition during cell
division, and thus increases the number of cell divisions that may
occur before a cell becomes senescent. Other reagents, for example,
telomerase, may be provided within a cell in order to reduce
telomere loss, or to make that cell immortal. Those of ordinary
skill in the art will recognize that other enzymatic activities may
be used to enhance the lengthening of telomeres within such cells,
for example, by providing certain viral sequences which activate
telomerase or can otherwise function to synthesize telomere
sequences within a cell. In addition, equivalent such molecules, or
other molecules may be readily screened to determine those that
will reduce loss of telomeres. Such screens may occur in vitro, and
the therapeutic agents discovered by such screening utilized in the
above method in vivo.
[0086] Other therapeutic treatments relate to the finding of
unusual telomeric DNA sequences in a group of fungi, specifically a
group of budding yeasts that includes some pathogens--Candida
albicans, Candida tropicalis and Candida paratropicalis--as well as
nonpathogenic fungi. These results are described in more detail
below. Drugs or chemical agents can be used to specifically exploit
the unusual nature of the telomeric DNA of fungi. This includes the
introduction of antisense polynucleotides specific to the telomeric
repeat DNA sequences, in order to block telomere synthesis in these
and any related pathogens. Such a block will lead to fungal
death.
[0087] This approach is advantageous because of the unusual nature
of the telomeric DNA in these fungi. The unusually high DNA
sequence complexity of the telomeric repeats of these fungi
provides specificity, and potential for minimal side effects, of
the antifungal agent or the antisense DNA or RNA.
[0088] Agents that are potentially useful antifungal agents
include: AZT, d4T, ddI, ddC, and ddA. The telomere synthesis of
these fungi is expected to show differential inhibition to these
drugs, and in some cases to be more sensitive than the telomere
synthesis in the human or other animal or plant host cells.
[0089] We performed a preliminary test of the use of antisense
techniques in living fungal cells. A stretch of 40 bp of telomeric
DNA sequence, imbedded in a conserved sequence flanking a region of
Candida albicans chromosomal DNA, was introduced on a circular
molecule into Candida albicans cells. The transformed cells had
high copy numbers of the introduced telomeric DNA sequence. 10% of
the transformants exhibited greatly (.about.3-fold) increased
length of telomeric DNA. This result indicates that telomeric DNA
can be modulated in vivo by introduction of telomeric sequence
polynucleotides into cells. This demonstrates the need to test a
particular oligonucleotide to ensure that it has the desired
activity.
[0090] With regard to diagnostic procedures, examples of such
procedures become evident from the discussion above with regard to
therapy. Applicant has determined that the length of the telomere
is indicative of the life expectancy of a cell containing that
telomere, and of an individual containing that cell. Thus, the
length of a telomere is directly correlated to the life span of an
individual cell. As discussed above, certain populations of cells
may lose telomeres at a greater rate than the other cells within an
individual, and those cells may thus become age-limiting within an
individual organism. However, diagnostic procedures can now be
developed (as described herein) which can be used to indicate the
potential life span of any individual cell type, and to follow
telomere loss so that a revised estimate to that life span can be
made with time.
[0091] In certain diseases, for example, the AIDS disease discussed
above, it would, of course, be important to follow the telomere
length in CD4.sup.+ cells. In addition, the recognition that
CD4.sup.+ cells are limiting in such individuals allows a
therapeutic protocol to be devised in which CD4.sup.+ cells can be
removed from the individual at an early age when AIDS is first
detected, stored in a bank, and then reintroduced into the
individual at a later age when that individual no longer has the
required CD4.sup.+ cells available. These cells can be expanded in
number in the presence of agents which slow telomere repeat loss,
e.g., C-rich telomeric oligonucleotides or agents to transiently
de-repress telomerase to ensure that cells re-administered to the
individual have maximum replicative capacity. Thus, an individual's
life can be extended by a protocol involving continued
administration of that individual's limiting cells at appropriate
time points. These appropriate points can be determined by
following CD4.sup.+ cell senescence, or by determining the length
of telomeres within such CD4.sup.+ cells (as an indication of when
those cells will become senescent). In the case of AIDS, there may
be waves of senescent telomere length in peripheral blood
lymphocytes with bone marrow tem cells still having replicative
capacity. In this way, rather than wait until a cell becomes
senescent (and thereby putting an individual at risk of death)
telomere length may be followed until the length is reduced below
that determined to be pre-senescent, and thereby the timing of
administration of new CD4.sup.+ cells or colony stimulating factors
can be optimized.
[0092] Thus, the diagnostic procedures of this invention include
procedures in which telomere length in different cell populations
is measured to determine whether any particular cell population is
limiting in the life span of an individual, and then determining a
therapeutic protocol to insure that such cells are no longer
limiting to that individual. In addition, such cell populations may
be specifically targeted by specific drug administration to insure
that telomere length loss is reduced, as discussed above.
[0093] Other diagnostic procedures include measurement of
telomerase activity as an indication of the presence of immortal
cells within an individual. A more precise measurement of such
immortality is the presence of the telomerase enzyme itself. Such
an enzyme can be readily detected using standard procedures,
including assay of telomerase activities, but also by use of
antibodies to telomerase, or by use of oligonucleotides that
hybridize to the nucleic acid (template RNA) present in telomerase,
or DNA or RNA probes for the mRNAs of telomerase proteins.
Immunohistochemical and insitu hybridization techniques allow the
precise identification of telomerase positive cells in histological
specimens for diagnostic and prognostic tests. The presence of
telomerase is indicative of cells which are immortal and frequently
metastatic, and such a diagnostic allows pinpointing of such
metastatic cells, much as CD44 is alleged to do so. See, Leff,
3(217) BioWorld Today 1, 3, 1992.
[0094] It is evident that the diagnostic procedures of the present
invention provide the first real method for determining how far
certain individuals have progressed in a certain disease. For
example, in the AIDS disease, this is the first methodology which
allows prior determination of the time at which an HIV positive
individual will become immunocompromised. This information is
useful for determining the timing of drug administration, such as
AZT administration, and will aid in development of new drug
regimens or therapies. In addition, the determination of the
optimum timing of administration of certain drugs will reduce the
cost of treating an individual, reduce the opportunity for the drug
becoming toxic to the individual, and reduce the potential for the
individual developing resistance to such a drug.
[0095] In other related aspects, the invention features a method
for treatment of a disease or condition associated with cell
senescence, by administering a therapeutically effective amount of
an agent active to derepress telomerase in senescing cells. A
related aspect involves screening for a telomerase derepression
agent by contacting a potential agent with a cell lacking
telomerase activity, and determining whether the agent increases
the level of telomerase activity, e.g., by using a cell expressing
an inducible T antigen. Such an assay allows rapid screening of
agents which are present in combinatorial libraries, or known to be
carcinogens.
[0096] Applicant recognizes that known agents may be useful in
treatment of cancers since they are active at telomerase itself, or
at the gene expressing the telomerase. Thus, such agents can be
identified in this invention as useful in the treatment of diseases
or conditions for which they were not previously known to be
efficacious. Indeed, agents which were previously thought to lack
utility because they have little if any effect on cell viability
after only 24-48 hours of treatment, can be shown to have utility
if they are active on telomerase in vivo, and thus affect cell
viability only after several cell divisions.
[0097] Other features and advantages of the invention will be
apparent from the following description of the preferred
embodiments thereof, and from the claims.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0098] The drawings will first briefly be described.
DRAWINGS
[0099] FIGS. 1-3 are graphs where the cell type and/or the culture
conditions are varied, plotting days in culture (horizontal axis)
length versus cell number (vertical axis).
[0100] FIG. 4 is a linear plot of mean terminal restriction
fragment (TRF) length versus PDL for human umbilical vein
endothelial cell cultures. The plot had a slope (m) of -190.+-.10
bp/PD, r=-0.98, P=0.01.
[0101] FIG. 5 is a plot of mean TRF of endothelial cell cultures
from human iliac arteries and iliac veins as a function of donor
age. Parameters for iliac arteries are: m=-102 bp/yr, r=-0.98,
P=0.01 and for iliac veins are: m=-42 bp/yr, r=-0.71, P=0.14.
[0102] FIG. 6 is a plot of decrease in mean TRF of medial tissue
from the aortic arch, abdominal aorta, iliac artery and iliac vein
as a function of donor age. Parameters for linear plot are: m=-47
bp/yr, r=-0.85, P=0.05.
[0103] FIG. 7 is a plot of mean TRF length from PBLs plotted as a
function of donor age. The slope of the linear regression line
(-41.+-.2.6 bp/y) is significantly different from 0
(p<0.00005).
[0104] FIG. 8 is a plot showing accelerated telomere loss in Down's
Syndrome (DS) patients. Genomic DNA isolated from PBLs of DS
patients was analyzed as described in FIG. 7. Mean TRF length is
shown as a function of donor age, for DS patients (open squares),
and age-matched controls (solid squares). The slope of the linear
regression lines (-133.+-.15 bp/y, trisomy, vs -43.+-.7.7, normals)
are significantly different (p<0.0005)
[0105] FIG. 9 is a plot showing decrease in mean TRF length in
cultured T-lymphocytes as a function of population doubling (shown
for DNA from two normal individuals). Donor ages for these cells
were not available. The slopes of these lines (-80.+-.19 (.degree.)
and -102.+-.5.4 (O) bp/doubling) are significantly different from
zero (p<0.0001). Mean TRF length at terminal passage from a
third donor for which multiple passages were not available is also
shown (upsidedown V-symbol).
[0106] FIG. 10 is a copy of an autoradiogram showing TRF lengths of
ovarian carcinoma and control normal cells. DNA from cells in
ascitic fluid from 2 patients (cas and wad) was digested with HinfI
and RsaI separated by electrophoresis, hybridized to the telomeric
probe .sup.32P(CCCTAA).sub.3, stringently washed and
autoradiographed. The cells of ascitic fluid from 7 other patients
were separated into adhering normal cells (N) and tumour clumps in
the media (T). The DNA was extracted and run as above. DNA from
patient was obtained from both the first and forth paracentesis.
Tumour cells from patients were cultured and DNA was obtained at
the respected population doubling (pd).
[0107] FIG. 11 shows telomerase activity in ovarian carcinoma
cells. S100 extracts from the previously studied transformant cell
line 293 CSH, the tumor cell line HEY, purified tumour cell
population and cells directly from the ascitic fluid from patients
were incubated with the telomere primer (TTAGGG).sub.3 in the
presence of DATP and TTP, .sup.32PdGTP and buffer. The reaction
products were separated on a sequencing gel and exposed to a
PhosphoImager screen. Either single (1) or double reactions (2)
were tested.
[0108] FIG. 12 is a copy of an autoradiogram showing TRF lengths in
HME-31 cells and HME31-E6 cells to extended lifespan (PD68) and
subsequent immortalization and stabilization of telomere length
(PD81, 107).
[0109] FIG. 13 is a copy of an autoradiogram showing the effect of
CTO on telomere length during the senescence of HME31:E6 cells. An
intermediate time point is chosen to show the dose-dependent
protective effect of CTO oligonucleotide.
[0110] FIG. 14 is a graph showing extension of the life span of
IMR90 lung fibroblast cells in response to the CTO
oligonucleotide.
[0111] FIGS. 15 and 16 are copies of autoradiograms showing the
effect of GTO on telomere length in IDH4 cells.
[0112] FIG. 17 is a graph showing extension of the life span of
HME31:E6 human breast epithelial cells in response to the CTO
oligonucleotide.
[0113] FIG. 18A. shows the templating portion of the Tetrahymena
telomerase RNA with residues numbered 1 (5') through 9 (3') below
it. The oligonucleotide primer with the sequence
T.sub.2G.sub.4T.sub.2G.sub.4 binds to the template by the
base-pairing shown. Elongation followed by template translocation
are thought to occur as indicated.
[0114] FIG. 18B shows positions of major chain termination on the
telomerase RNA template by different nucleoside triphosphate
analogs. The telomerase RNA template sequence is shown as in FIG.
18A. Arrows indicate the position of maximal chain termination for
each nucleoside triphosphate (derived from the nucleoside) analog
shown.
[0115] FIG. 19A-F are graphs showing that nucleoside analog
triphosphates inhibit incorporation of a .sup.32p label in a
Tetrahymena telomerase assay. The effect of adding increasing
concentrations of the analog, unlabeled dGTP or unlabeled TTP on
the incorporation of labeled nucleotides was measured using a
quantitative telomerase reaction assay. Radioactivity incorporated
(cpm) was plotted against the concentration of competitors
indicated in each panel. (A. labeled with [.alpha.-.sup.32P]TTP.
B-F. labeled with [.alpha.-.sup.32P]dGTP. F. Effect of streptomycin
sulfate on the telomerase reaction. The incorporation in the
presence of 40 mM sodium sulfate is shown as the control for
streptomycin sulfate).
[0116] FIG. 20A and B show the effect of nucleoside triphosphate
analogs on pausing patterns and processivity of telomerase in
vitro. Specifically, FIG. 20A shows telomerase reactions in the
presence or absence of the indicated nucleoside triphosphate
analogs. Unlabeled TTP competitor was also analyzed as a control,
with and without primer in the reaction mix. Products were then
analyzed on a denaturing polyacrylamide gel. FIG. 20B shows
standard telomerase reactions were performed in the presence of
ddGTP (lanes 4-6), ddITP (lanes 7-9), or DMSO (lane 1). DMSO was
the solvent for ddGTP and at the highest concentration tested (1%)
showed no effect on the reactions compared with control reactions
run without analog or DMSO (control lanes 2-3). Products were
analyzed on a denaturing polyacrylamide gel.
[0117] FIG. 21A-D shows Southern blot analysis to demonstrate the
effect of nucleoside analogs on telomere length in vivo, using a
nick-translated [.alpha.-.sup.32P]-labeled plasmid containing a 3'
rDNA fragment as probe. Genomic DNA was digested with PstI and
BamHI and the rDNA telomeres analyzed. The telomeric PstI fragment
from the rDNA is between the 1.6 and 1.0 kb markers, indicated as
lines on both sides of each panel. The constant 2.8 kb band is the
adjacent internal PstI rDNA fragment. Specifically, FIG. 21A shows
results with a clone of Tetrahymena thermophila grown in 2% PPYS in
the absence (-) and three clones in the presence (+) of 5 mM AZT.
Each set of three lanes shows the results for a single cell clone
grown vegetatively and transferred after 3 days (lanes 1, 4, 7,
10), 10 days (lanes 2, 5, 8, 11) and 16 days (lanes 3, 6, 9, 12).
FIG. 21B shows that growth in different concentrations of AZT
consistently resulted in concentration-dependent shortening of
telomeres in log phase cells grown in thymine-deficient broth
(Isobroth) plus AZT. DNA made from cells sampled at 6, 10, and 16
days show that shortened telomere lengths remain constant between 6
and 16 days in culture. Lanes 1, 5, 9, 0 mM AZT control; lanes 2,
6, 10, 0.01 mM AZT; lanes 3, 7, 11, 0.1 mM AZT; lanes 4, 8, 12, 1
mM AZT. FIG. 21C shows cells grown vegetatively in 2% PPYS with no
addition (lane 1), with 1% DMSO, the solvent for Ara-G, ("C", lanes
2 and 5), and with Ara-G (lane 3, 1 mM; lanes 4 and 6, 2 mM ) at 14
and 27 days in culture. FIG. 21D shows analysis of DNA from
single-cell cultures grown in Isobroth plus 1 mm AZT (lanes 2 and
3) segregated into two classes based on growth rate: "slow" ("S",
0-1 doubling per day, lane 2) or "fast" ("F", 2-4 doubling per day,
lane 3). DNA from control cultures grown in the absence of AZT are
indicated ("C", 2-4 doubling per day, lane 1). Several cultures
were pooled in order to obtain sufficient DNA for analysis.
[0118] FIG. 22 shows PCR analysis of DNA from Tetrahymena cells
conjugated in the presence of analog and starved for the duration
of mating. A Telomeric a primer and a 5' rDNA primer were used in
PCR reactions with DNA from cells conjugated in the presence or
absence of analog to detect the addition of telomeres to the 11 Kb
rDNA formed during macronuclear development. A reaction was run
without DNA as a control. Tests included use of 5 mM AZT; 1 mM
Ara-G, and 1 mM Acyclo-G. SB210 cells were also mock-conjugated as
a control. The expected product is approximately 1400 bp. In
addition, 3' micronuclear rDNA primers were used on the same DNA to
demonstrate the presence and competence of the DNA samples for PCR.
The expected band is 810 bp. In the figure southern blot analysis
of the 5' rDNA telomeric PCR reactions using a random-primed
.sup.32P-labeled 5'rDNA probe confirmed the 1400 bp PCR product as
part of the 5'rDNA with telomeres, from the 11 Kb rDNA species
formed transiently during macronuclear development. No
hybridization is seen in the no DNA control (lane 1) or the SB210
mock-conjugated control (lane 6). Lane 2, no added analog; lane 3,
5 mM AZT, lane 4, 1 mM Ara-G; lane 5, 1 mM Acyclo-G; lane 6,
mock-conjugated SB210 cell DNA. These results were reproduced in
three separate experiments.
[0119] FIG. 23 shows growth of cultured JY lymphoma cells with RPMI
medium and no added agents (control) and with a relatively low dose
of ddG, AZT, ara-G, and ddI. The DMSO is a control for ddG.
[0120] FIG. 24 shows the growth of cultured JY lymphoma cells
cultured in an analogous manner to those in FIG. 23, but treated
with relatively higher doses of potential telomerase
inhibitors.
[0121] FIG. 25 shows Southern blot of DNA isolated from JY lymphoma
cells at weeks one and three probed with the telomeric repeat
sequence (TTAGGG).sub.3. The first lane is DNA from the cells at
the start of the experiment, the second is the RPMI control, and
the third is cells treated with AZT for the times indicated.
[0122] FIG. 26 shows fibroblast DNA hybridized by Southern blot to
the telomeric (TTAGGG).sub.3 probe. Lane labeled "HinfI" is DNA
digested with the restriction enzyme HinfI, the lane labeled "O"
had no treatment, the lane labeled "P only" was treated with
piperidine, and the lane labelled "P+DMS" was piperidine and
dimethyl sulfate treated.
[0123] FIG. 27 shows the inhibition of human telomerase achieved by
the agent ddG at various dosages in three separate experiments. The
telomerase was derived from the tumor cell line 293.
[0124] FIG. 28 shows hybridization of C. albicans telomeric repeats
to genomic DNAs of a variety of other Candida species. Genomic DNAs
of eight species of yeasts were digested with EcoRI,
electrophoresed on 0.8% agarose, blotted, and then probed with a
.sup.32P-labeled telomeric fragment from C. albicans WO-1.
Hybridization was carried out at 55.degree. C. and washes were at
the same temperature in Na.sub.2HPO.sub.4 at 200 mM Na+ and 2% SDS.
DNA size markers, measured in kilobase pairs (kb), are shown at the
right. The species used here are C. quillermondii, S. cerevisiae,
C. pseudotropicalis, Kluyveromyces lactis, C. lusitaniae, C.
maltosa, C. tropicalis, and C. albicans. Asterisks indicate
particular strains from which telomeres were cloned. Strains
beginning with "B" are N.I.H. strains obtained from B. Wickes.
[0125] FIG. 29 shows Bal31 sensitivity of genomic copies of the
tandem repeats in K. lactis ATCC 32143 (left panel) and C.
guillermondii B-3163 (right panel). Uncut yeast genomic DNAs were
incubated with Bal3l nuclease for increasing periods of time (given
in minutes above each lane), then digested with EcoRI and
electrophoresed on a 0.8% agarose gel, and blotted onto a nylon
membrane. For K. lactis, probing was done with a .sup.32P-kinased
25 base oligonucleotide identical in sequence to the K. lactis
telomeric repeat shown in FIG. 30. Hybridization and washes were
carried out at 49.degree. C. For C. guillermondii, probing was done
with .sup.32P-labeled pCgui3, a pBluescript vector (Stratagene,
LaJolla, Calif.) carrying a .alpha.-2 kb telomeric clone from C.
guillermondii. Hybridization and washing (in 200 mM Na.sup.+) were
carried out at 54.degree. C. Most bands are gone by the 1 min. time
point. Approximately three other bands are shortening but are not
gone at 3 min. These latter bands presumably are homologous to the
particular subtelomeric sequences present in pCgui3. DNA size
markers (in kb) are indicated at the right of each panel.
[0126] FIG. 30 shows sequences of telomeric repeats from several
budding yeast species. Specifically, telomere-enriched libraries
were constructed from genomic DNA by standard methods. Uncut yeast
genomic DNA was ligated to a blunt-ended linearized plasmid vector
and then this ligated mix was digested with a restriction enzyme
that cleaves both within the vector's polylinker and within a few
kilobases of at least some of the putative telomeric ends of the
species in question. No enzymatic pre-treatment was done to produce
blunt-ends of the telomeres in the genomic DNA prior to the initial
ligations. Plasmids were then recircularized with T4DNA ligase, and
transformed into E. coli cells prior to screening for putative
telomere clones by colony hybridization. The libraries from C.
maltosa, C. pseudotropicalis, two strains of C. tropicalis, and K.
lactis ATCC 32143, species which showed multiple bands that cross
hybridized to the C. albicans telomeric repeat probe, were screened
with this probe. A cloned S. cerevisiae telomere probe (repeat unit
TG.sub.2-3(GT).sub.1-3) was used to screen the telomere-enriched
library from C. glabrata, whose genomic DNA cross-hybridized with
this, but not with the C. albicans telomeric repeat probe. C.
guillermondii DNA did not appreciably cross-hybridize with either
the C. albicans or the S. cerevisiae telomeric probes at the
stringencies tested. The telomere-enriched library from this
species was screened using total genomic C. guillermondii DNA as a
probe. This procedure can be used to identify all clones containing
repetitive sequences and we reasoned that telomeres should be a
reasonable percentage of the repetitive sequences found in telomere
enriched libraries. Typically, a few hundred E. coli transformants
were obtained for each small library and up to nine putative
telomere clones were obtained from each. Nine repetitive DNA clones
were obtained from C. guillermondii, three of which proved to be
telomeric.
[0127] FIG. 31 shows two types of telomeric repeats present in
certain C. tropicalis strains. Genomic DNAs from ten (only five
here are shown) C. tropicalis strains and C. albicans WO-1 were
digested with ClaI, eletrophoresed on a 0.8% agarose gel, blotted,
and probed with oligonucleotides specific to either the "AC form of
C. tropicalis telomeric repeat (left panel) or to the "AA" form of
repeat (right panel). Sequences of these two oligonucleotides are:
5'ACGGATGTCACG ("AC") and 5'GTGTAAGGATG ("AA") with the position of
the dimorphic base shown underlined. Hybridization with the kinased
"AC" probe was at 47.degree. C., and hybridization with the "AA"
probe at 24.degree. C. Washes for both were in 2% SDS with 500 mM
Na.sup.+. The specificity of the "AA" probe is indicated by its
failure to hybridize with the C. albicans telomeres, despite only
one base mismatch and the fact that the C. albicans cells used here
have much longer telomeres (and therefore many more telomeric
repeats) than do C. tropicalis strains. The shortness of the C.
tropicalis telomeres may explain why they appear to be particularly
homogeneous in size, as is suggested by the relative sharpness of
individual telomeric bands.
[0128] FIG. 32 shows a Southern blot of DNA isolated from JY cells
hybridized to the (TTAGGG).sub.3 probe. Cells were treated over a
10 week period with either 10 .mu.M ddG in 0.01% DMSO or medium
with 0.01% DMSO only. Cells treated with ddG showed a marked
decrease in mean telomere length consistant with the inhibition of
telomerase activity.
[0129] FIG. 33 shows telomerase activity in cells from ascitic
fluid. Specifically, S100 extracts were prepared, protein
concentrations determined and telomerase activity assayed by
incubating S100 extracts with an equal volume of reaction mixture
containing buffer, telomere primer (TTAGGG).sub.3,
.alpha..sup.32PdGTP, TTP and DATP, at 30.degree. C. for 1 hour. The
reactions were terminated with RNase followed by deproteination
with proteinase K. Unincorporated .alpha..sup.32PdGTP was removed
using NICK SPIN columns (Pharmacia) according to the supplier's
direction. Products were resolved on a sequencing gel and exposed
to either a PhosphorImager screen (Molecular Dynamics). A ladder
(L) and kinased 5'.sup.32P(TTAGGG).sub.3 (O) were run as markers.
FIG. 33A shows telemerase assayed in S100 extracts with equal
protein concentration (.apprxeq.11 mg/ml) prepared from the control
human cell line 293 CSH, a subline of 293 cell line, and from
unfractionated ascitic fluid cells from patient Dem-1 and Rud-1. In
lanes 1, 3 and 5 RNase was added to the extracts prior to addition
of .alpha..sup.32PdGTP. FIG. 33B shows S100 extracts isolated and
assayed for telomerase activity from the early passage cultures of
cells from patients Pres-3 and Nag-1 compared to 293 cells. All
extracts were assayed at a protein concentration of .apprxeq.2-3
mg/ml.
TELOMERES AND TELOMERASE
[0130] All normal diploid vertebrate cells have a limited capacity
to proliferate, a phenomenon that has come to be known as the
Hayflick limit or replicative senescence. In human fibroblasts,
this limit occurs after 50-100 population doublings, after which
the cells remain in a viable but non-dividing senescent state for
many months. This contrasts to the behavior of most cancer cells,
which have escaped from the controls limiting their proliferative
capacity and are effectively immortal.
[0131] One hypothesis to explain the cause of cellular senescence
concerns the role of the distal ends of chromosomes called
telomeres. The hypothesis is that somatic cells lack the ability to
replicate the very ends of DNA molecules. This results in a
progressive shortening of the ends of the chromosomes until some
function changes, at which time the cell loses the capacity to
proliferate.
[0132] DNA polymerase synthesizes DNA in a 5' to 3' direction and
requires a primer to initiate synthesis. Because of this, the
"lagging strand" does not replicate to the very ends of linear
chromosomes. The chromosome is thus shortened with every cell
division. The ends of chromosomes are called telomeres, and are
composed of long TTAGGG repeats. The enzyme telomerase can add
TTAGGG repeats to the 3' end of the telomeric DNA, thus extending
the DNA and preventing shortening.
[0133] Germline cells have long telomeres and active telomerase.
Somatic cells lack telomerase activity, and their telomeres have
been found to shorten with cell division both in vivo and in
culture. Cancer cells are immortal, and have regained telomerase
activity and thus can maintain their chromosome ends. Examples are
provided below of definitive experiments which indicate that
telomere shortening and telomerase activity are key factors in
controlling cellular senescence and immortalization.
[0134] Methods
[0135] As noted above, the present invention concerns diagnosis and
therapy associated with measuring telomeric length and manipulating
telomerase-dependent extension or telomerase-independent
shortening. While the invention is directed to humans, it may be
applied to other animals, particularly mammals, such as other
primates, and domestic animals, such as equine, bovine, avian,
ovine, porcine, feline, and canine. The invention may be used in
both therapy and diagnosis. In this case of therapy, for example,
telomere shortening may be slowed or inhibited by providing DNA
oligonucleotides by reactivating or introducing telomerase
activity, or their functional equivalent, or indefinite
proliferation can be reduced by inhibiting telomerase. In the case
of diagnostics, one may detect the length of telomeres as to a
particular chromosome or group of chromosomes, or the average
length of telomeres. Diagnosis may also be associated with
determining the activity of telomerase, or the presense of the
components of the enzyme either on a protein or RNA level, in
cells, tissue, and the like.
[0136] Information on the relative age, remaining proliferative
capacity, as well as other cellular characteristics associated with
telomere and telomerase status may be obtained with a wide variety
of cell types and tissues, such as embryonic cells, other stem
cells, somatic cells (such as hepatocytes in the context of
cirrhosis), connective tissue cells (such as fibroblasts,
chondrocytes, and osteoblasts), vascular cells (such as endothelial
and smooth muscle cells), cells located in the central nervous
system (such as brain astrocytes), and different neoplastic
tissues, and parasitic pathogens where it is desirable to determine
both the remaining replicative capacity of the hyperplastic cells
and their capacity for immortal growth to predict growth
potential.
[0137] Maintaining Telomere Length
[0138] Telomere length in cells in vitro or in vivo may be usefully
maintained by a variety of procedures. These include those methods
exemplified below. These examples, however, are not limiting in
this invention since those in the art will recognize equivalent
methods. It is expected that all the methods will be useful in
manipulating telomere length now that applicant has demonstrated
this experimentally. Such methods may be based upon provision of
oligonucleotides or other agents which interact with telomeres to
prevent shortening during cell division. In addition, the methods
include treatment with agents which will include telomerase, or its
equivalent activity, within a cell to prevent shortening. Finally,
the methods also include modulation of gene expression associated
with cell senescence.
[0139] Useful agents can be determined by routine screening
procedures. For example, by screening agents which interact in an
in vitro system with telomeres, and block loss of telomere ends, or
aid increase in telomere length. Non-limiting examples of such
methods are provided below. All that is necessary is an assay to
determine whether telomere end shortening is reduced during cell
division. The mechanism by which such agents act need not be known,
so long as the desired outcome is achieved. However, by identifying
useful target genes (e.g., the M2 mortality modulation gene(s)),
antisense and equivalent procedures can be designed to more
appropriately cause desired gene expression or non-expression
(e.g., the de-repression of telomerase).
[0140] In a particular example (non-limiting in this invention) one
can reduce the rate of telomere shortening, by providing a nucleic
acid, e.g., DNA or RNA (including modified forms), as a primer to
the cells. Such nucleic acid will usually include 2 to 3 repeats,
more usually 2 repeats, where the repeats are complementary to the
G-rich DNA telomere strand. Such oligonucleotides may be used to
extend the proliferative capability of cells.
[0141] The oligonucleotides can be transferred into the cytoplasm,
either spontaneously (i.e., without specific modification) or by
the use of liposomes which fuse with the cellular membrane, or are
endocytosed by employing ligands which bind to surface membrane
protein receptors of the cell resulting in endocytosis.
Alternatively, the cells may be permeabilized to enhance transport
of the oligonucleotides into the cell, without injuring the host
cells. Another way is to use a DNA binding protein, e.g., HBGF-1,
which is known to transport an oligonucleotide into a cell. In this
manner, one may substantially reduce the rate of telomere
shortening from an average of about 50 bp per division, to an
average of about 6-12 bp per division (see examples below), thus
significantly extending the number of divisions occurring before
induced cellular senescence.
[0142] By "senescence" is meant the loss of ability of a cell to
replicate in the presence of normally appropriate replicative
signals, and may be associated with the expression of degradative
enzymes, such as collagenase. The term does not include quiescent
cells which might be induced to replicate under appropriate
conditions. This term is exemplified below in the examples, where
the number of cell doubling prior to senescence is increased.
[0143] The above processes are useful in vivo. As already
indicated, by using liposomes, particularly where the liposome
surface carries ligands specific for target cells, or the liposomes
will be preferentially directed to a specific organ, one may
provide for the introduction of the oligonucleotides into the
target cells in vivo. For instance, utilizing lipocortin affinity
for phosphatidyl serine, which is released from injured vascular
endothelial cells, the oligonucleotides may be directed to such
site. Alternatively, catheters, syringes, depots or the like may be
used to provide high localized concentrations. The introduction of
such oligonucleotides into cells resulting in decreased senescence
in response to cell division can have therapeutic effect.
[0144] The maintenance of telomere length has application in tissue
culture techniques to delay the onset of cellular senescence. For
instance, cell-based therapies which require the clonal expansion
of cells for reintroduction into an autologous patient are limited
to about 20-30 doublings. This invention allows, the expansion of
cells in the case of gene therapy, both prior to genetic
manipulation and then expansion of the manipulated cells, the
maintenance of telomere length. This in turn allows normal cells to
be cultivated for extended doublings in vitro. Experiments
described below demonstrate the utility of this method in vitro,
and demonstrate its applicability in vivo.
[0145] Critical shortening of telomeres leads to a phenomenon
termed "crisis" or M2 senescence. See, Shay et al., 1992, supra.
Among the cells in crisis, rare mutants may become immortalized in
which M2 genes have altered regulation, and where expression of
telomerase is reactivated and stabilizes the telomere length. An M2
regulatory gene may be modulated to provide a useful means of
modulating telomere length and telomerase activity. The M2 genes
may be identified by means of insertional mutagenesis into cells in
M2 crisis utilizing a retrovirus. Cells wherein the M2 gene has
been knocked out will then grow in response to the reactivation of
telomerase, and such cells can supply a source or DNA from which to
clone the M2 genes. This technique has yielded numerous cell clones
in which the retrovirus has inserted into a common restriction
fragment. The repression of the M2 regulatory gene(s) by antisense
or other means can provide a means of activating telomerase
reversibly, such that telomeres may be extended and then telomerase
again repressed. In this manner, proliferative capacity may be
extended with or without the addition of oligonucleotides to slow
the telomere shortening. Such cells may then be used in cell-based
therapies, such as bone marrow transplantation, reconstitution of
connective tissue, and transplantation of early passage adrenal
cortical cells, fibroblasts, epithelial cells, and myoblasts.
[0146] Telomerase Modulation
[0147] As discussed above, cancer cells contain telomerase activity
and are thereby immortal. In addition, numerous types of parasitic
pathogens are immortal and have active telomerase. Thus, it is
useful to modulate (e.g., decrease) telomerase activity in such
cells to impart a finite replicative life span. In contrast to the
long telomeric tracts in normal human cells, tracts of telomeric
DNA in protozoan cells, fungal cells, and some parasitic worms, as
well as many cancer cells, are typically shorter. This makes these
cells more vulnerable to telomerase inhibitors than normal human
cells (e.g. germ line cells).
[0148] Thus, inhibition or induction of telomerase has applications
in various situations. By inhibiting telomerase intracellularly,
one may reduce the ability of cancer cells to proliferate.
Telomerase may be competitively inhibited by adding synthetic
agents, e.g., oligonucleotides comprising 2 or more, usually not
more than about 50 repeats, of the telomeric motif of the 5'-3'
G-rich strand (the strand which acts as the template). The
oligonucleotides may be synthesized from natural or unnatural
units, e.g., the derivatives or carbon derivatives, where a
phosphate-oxygen is substituted with sulfur or methylene, modified
sugars, e.g., arabinose, or the like. As discussed above, other
equivalent agents may also be used to inhibit or cause expression
of telomerase activity.
[0149] The oligonucleotides may be introduced as described above so
as to induce senescence in the immortalized cells, in culture and
in vivo. Where growing cells in culture, where one wishes to
prevent immortalized cells from overgrowing the culture, one may
use the subject oligonucleotides to reduce the probability of such
overgrowth. Thus, by maintaining the oligonucleotides in the
medium, they will be taken up by the cells and inhibit telomerase
activity. One may provide for linkage to the telomeric sequence
with a metal chelate, which results in cleavage of nucleic acid
sequences. Thus, by providing iron chelate bound to the telomeric
motif, the telomerase RNA will be cleaved, so as to be
non-functional. Alternatively, a reactive group may be coupled to
the oligonucleotide that will covalently bind to telomerase, or the
3' residue may be made to be dideoxy so as to force chain
termination.
[0150] Alternatively, one may introduce a ribozyme, having 5' and
3'-terminal sequences complementary to the telomerase RNA, so as to
provide for cleavage of the RNA. In this way, the telomerase
activity may be substantially inhibited, so as to result in a
significant limitation of the ability of the cancer cells to
proliferate. Telomerase may also be inhibited by the administration
of an M2 regulator gene product. By modulating the expression of
any of the proteins directly regulating telomerase expression, one
may also modulate cellular telomerase activity.
[0151] Alternatively, one may use a screening assay utilizing human
or tetrahymena telomerase to screen small molecules e.g.,
nucleoside analogs like ava-G, ddG, AZT, and the like and RNA and
DNA processing enzyme inhibitors, alkylating agents, and various
potential anti-tumor drugs. These may then be further modified.
[0152] The nucleic acid sequences may be introduced into the cells
as described previously. Various techniques exist to allow for
depots associated with tumors. Thus, the inhibiting agents or
nucleic acids may be administered as drugs, since they will only be
effective only in cells which include telomerase. Since for the
most part, human somatic cells lack telomerase activity they will
be unaffected. Some care may be required to prevent entry of such
drugs into germ cells, which may express telomerase activity.
[0153] The subject compositions can therefore be used in the
treatment of neoplasia wherein the tumor cells have acquired an
immortal phenotype through the inappropriate activation of
telomerase, as well as various human and veterinary parasitic
diseases; including human protozoal pathogens such as; amebiasis
from Entamoeba histolytica, amebic meningoencephalitis from the
genus Naegleria or Acanthamoeba, malaria from Plasmodium vivax,
Plasmodium ovale, Plasmodium malariae, and Plasmodium falciparum,
Leishmaniasis from such protozoa as Leishmania donovani, Leishmania
infantum, Leishmania chagasi, Leishmania tropica, Leishmania major,
Leishmania aethiopica, Leishmania mexicana, and Leishmania
braziliensis, Chagas' disease from the protozoan Trypanosoma cruzi,
sleeping sickness from Trypanosoma brucei, Trypanosoma gambiense,
and Trypanosoma rhodesiense, Toxoplasmosis from Toxoplasma gondii,
giardiasis from Giardia lamblia, cryptosporidiosis from
Cryptosporidium parvum, trichomoniasis from Trichomonas vaginalis,
Trichomonas tenax, Trichomonas hominis, pneumocystis pneumonia from
Pneumocystis carinii, bambesosis from Bambesia microti, Bambesia
divergens, and Bambesia boris, and other protozoans causing
intestinal disorders such as Balantidium coli and Isospora belli.
Telomerase inhibitors would also be useful in treating certain
helminthic infections including the species: Taenia solium, Taenia
saginata, Diphyllobothrium lata, Echinococcus granulosus,
Echinococcus multilocularis, Hymenolepis nana, Schistosoma mansomi,
Schistosoma japonicum, Schistosoma hematobium, Clonorchis sinensis,
Paragonimus westermani, Fasciola hepatica, Fasciolopsis buski,
Heterophyes heterophyes, Enterobius vermicularis, Trichuris
trichiura, Ascaris lumbricoides, Ancylostoma duodenale, Necator
americanus, Strongyloides stercoralis, Trichinella spiralis,
Wuchereria bancrofti, Onchocerca volvulus, Loa loa, Dracunculus
medinensis, and fungal pathogens such as: Sporothrix schenckii,
Coccidioides immitis, Histoplasma capsulatum, Blastomyces
dermatitidis, Paracoccidioides brasiliensis, Candida albicans,
Cryptococcus neoformans, Aspergillus fumigatus, Aspergillus flavus,
fungi of the genera Mucor and Rhizopus, and species causing
chromomycosis such as those of the genera Phialophora and
Cladosporium, and important veterinary protozoal pathogens such as:
Babesia caballi, Babesia canis, Babesia egui, Babesia felis,
Balantidium coli, Besnoitia darlingi, Eimeria acervulina, Eimeria
adenoeides, Eimeria ahsata, Eimeria alabamensis, Eimeria
auburnensis, Eimeria bovis, Eimeria brasiliensis, Eimeria brunetti,
Eimeria canadensis, Eimeria cerdonis, Eimeria crandallis, Eimeria
cylindrica, Eimeria debliecki, Eimeria despersa, Eimeria
ellipsoidalis, Eimeria fauvei, Eimeria gallopavonis, Eimeria
gilruthi, Eimeria granulosa, Eimeria hagani, Eimeria illinoisensis,
Eimeria innocua, Eimeria intricata, Elmeria leuskarti, Eimeria
maxima, Eimera meleagridis, Eimeria meleagrimitis, Eimeria mitis,
Eimeria mivati, Eimeria necatrix, Eimeria neodebliecki, Eimeria
ninakohlyakimorae, Eimeria ovina, Eimeria pallida, Eimeria parva,
Eimeria perminuta, Eimeria porci, Eimeria praecox, Eimeria
punctata, Eimeria scabra, Eimeria spinoza, Eimeria subrotunda,
Eimeria subsherica, Eimeria suis, Eimeria tenella, Eimeria
wyomingensis, Eimeria zuernii, Endolimax gregariniformis, Endolimax
nana, Entamoeba bovis, Entamoeba gallinarum, Entamoeba histolytica,
Entamoeba suis, Ciardia bovis, Giardia canis, Giardia cati, Giardia
lamblia, Haemoproteus meleagridis, Hexamita meleagridis, Histomonas
meleagridis, Iodamoeba buetschili, Isospora bahiensis, Isospora
burrowsi, Isospora canis, Isospora felis, Isospora ohioensis,
Isospora rivolta, Isospora suis, Klossiella equi, Leucocytozoon
caallergi, Leucocytozoon smithi, Parahistomonas wenrichi,
Pentatrichomonas hominis, Sarcocystis betrami, Sarcocystis
bigemina, Sarcocystis cruzi, Sarcocystis fayevi, hemionilatrantis,
Sarcocystis hirsuta, Sarcocystis miescheviana, Sarcocystis muris,
Sarcocystis ovicanis, Sarcocystis tenella, Tetratrichomonas
buttreyi, Tetratrichomonas gallinarum, Theileria mutans, Toxoplasma
gondii, Toxoplasma hammondi, Trichomonas canistomae, Trichomonas
gallinae, Trichomonas felistomae, Trichomonas eberthi, Trichomonas
equi, Trichomonas foetus, Trichomonas ovis, Trichomonas rotunda,
Trichomonas suis, and Trypanosoma melophagium. In addition, they
can be used for studying cell senescence, the role of telomeres in
the differentiation and maturation of cells from a totipotent stem
cell, e.g., embryonic stem cells, or the like, and the role of
telomerase in spermatogenesis.
[0154] Telomere Length
[0155] Procedures for measuring telomere length are known in the
art and can be used in this invention. Typically, restriction
endonuclease digestion is used (with enzymes which do not cleave
telomeric DNA), and the length of the fragment having detectable
telomere DNA is separated according to molecular weight by agarose
gel electrophoresis. Given that the DNA sequence of a telomere is
known, detection of such DNA is relatively easy by use of specific
oligonucleotides. Examples of these methods are provided below.
[0156] For diagnosis, in detection of the telomeric length, one may
study just a particular cell type, all cells in a tissue (where
various cells may be present), or subsets of cell types, and the
like. The preparation of the DNA having such telomeres may be
varied, depending upon how the telomeric length is to be
determined.
[0157] Conveniently, the DNA may be isolated in accordance with any
conventional manner, freeing the DNA of proteins by extraction,
followed by precipitation. Whole genomic DNA may then be melted by
heating to at least about 80.degree. C., usually at least about
94.degree. C., or using high salt content with chaotropic ions,
such as 6.times.SSC, quanidinium thiocyanate, urea, and the like.
Depending upon the nature of the melting process, the medium may
then be changed to a medium which allows for DNA synthesis.
[0158] (a) DNA Synthesis
[0159] In one method, a primer is used having at least about 2
repeats, preferably at least about 3 repeats of the telomeric
sequence, generally not more than about 8 repeats, conveniently not
more than about 6 repeats. The primer is added to the genomic DNA
in the presence of only 3 of the 4 nucleoside triphosphates (having
the complementary nucleosides to the protruding or G-rich strand of
a telomere, e.g., A, T and C for human chromosomes), DATP, dTTP and
dCTP. Usually at least the primer or at least one of the
triphosphates is labeled with a detectable label, e.g., a
radioisotope, which label is retained upon incorporation in the
chain. If no label is used, other methods can be used to detect DNA
synthesis. The primer is extended by means of a DNA polymerase,
e.g., the Klenow fragment of DNA polymerase I, T7 DNA polymerase or
Taq DNA polymerase
[0160] The length of the extended DNA can then be determined by
various techniques, e.g., those which separate synthesized DNA on
the basis of its molecular weight, e.g., gel electrophoresis. The
DNA synthesized may then be detected based on the label, e.g.,
counts incorporated per .mu.g of DNA, where the counts will be
directly proportional to telomere length. Thus, the measure of
radioactivity in relation to the amount of DNA will suffice to
quantitate telomere length.
[0161] If desired, telomeres of known length may be used as
standards, whereby a determination of radioactivity may be read off
a standard curve as related to telomere length. Instead, one may
prepare tissues where individual cells may be assayed for relative
telomere length by in situ hybridization. In this approach, for
example, the primer is labeled with a detectable label, usually
biotin or digoxygenin. Following annealing to prepared tissue
sections or cells, the label is revealed histochemically, usually
using autoradiography (if the label were radioactive), using
avidin/streptavidin (if the label were biotin) or using
antidigoxygenin antibodies (if the label were digoxygenin). The
amount of signal per cell is proportional to the number of
telomeric repeats, and thus to the telomere length. This can be
quantitated by microfluorometry or analogous means, and compared to
the signal from standard cells of known telomere length to
determine the telomere length in the test sample.
[0162] (b) Restriction Endonuclease Digestion
[0163] Alternatively, one may use primers which cause covalent
cross-linking of the primer to telomere DNA. In this situation, one
may totally digest the DNA with restriction endonucleases which
have 4 base recognition sites, which results in the production of
relatively short fragments of DNA, except for telomeric DNA which
lacks the recognition site. Restriction endonucleases which may
find use include AluI, HinfI, MspI, RsaI, and Sau3A, where the
restriction endonucleases may be used individually or in
combination. After digestion of the genomic DNA, the primer may be
added under hybridizing conditions, so as to bind to the protruding
chain of the telomeric sequence. By providing for two moieties
bound to the primer, one for covalent bonding to the telomeric
sequence and the other for complex formation with a specific
binding pair member, one can then provide for linking of a
telomeric sequence to a surface. For example, for covalent bonding
to the telomeric sequence, psoralen, or isopsoralen, may be linked
to one of the nucleotides by a bond or chain and upon UV-radiation,
will form a bridge between the primer and the telomere.
[0164] The specific binding pair member will normally be a hapten,
which binds to an appropriate complementary member, e.g., biotin
and strept/avidin, trinitrobenzoic acid and anti-trinitrobenzamide
antibody, or methotrexate and dihydrofolate reductase. Rather than
having the moiety for covalent bonding covalently bonded to the
primer, one may add a compound into the medium which is
intercalatable into the nucleic acid, so as to intercalate between
double-stranded nucleic acid sequences. In this manner, one may
achieve the same purpose. Use of a substantial excess of the
intercalatable compound will cause it to also intercalate into
other portions of DNA which are present. Various modifications of
this process may be achieved, such as size separation, to reduce
the amount of label containing DNA.
[0165] The specific binding pair member may be used for separation
of telomeric DNA free of contaminating DNA by binding to the
complementary pair member, which may be present on beads, on
particles in a column, or the like. In accordance with the nature
of the separation, the covalently bonded telomere strand may now be
purified and measured for size or molecular weight. Again, if
desired, standards may be employed for comparison of distribution
values.
[0166] The specific binding pair member hapten can be present at
the 5'-terminus of the primer or at intermediate nucleotides.
Specifically, biotin-conjugated nucleotides are generally available
and may be readily introduced into synthetic primer sequences in
accordance with known ways.
[0167] The above-described techniques can also be used for
isolating and identifying DNA contiguous to the telomere.
[0168] (c) Average Telomere Length
[0169] In methods of this invention it may be useful to determine
average telomere length by binding a primer to a telomere prior to
separation of the telomeric portion of the chromosomes from other
parts of the chromosomes. This provides a double-stranded telomeric
DNA comprising the telomeric overhang and the primer. A reaction
may then be carried out which allows for specific identification of
the telomeric DNA, as compared to the other DNA present. The
reaction may involve extension of the primer with only 3 of the
nucleotides (dNTPs), using a labeled nucleotide, covalent bonding
of the primer to the telomeric sequence, or other methods which
allow for separation of the telomeric sequence from other
sequences. The length of the synthesized DNA detected then
represents the average telomere length.
[0170] Telomere length can also be measured directly by the
"anchored terminal primer" method. In this method, the 3' ends of
genomic DNA are first "tailed" with dG nucleotides using terminal
transferase. Telomeres, which are known to have 3' overhangs, then
would have one of the three following conformations:
[0171] . . . 5'TTAGGGTTAGGGTTAGGGGGGGGGGG . . . 3'
[0172] . . . 5'TTAGGGTTAGGGTTGGGGGGGGGGGG . . . 3'
[0173] . . . 5'TTAGGGTTAGGGTGGGGGGGGGGGGG . . . 3'
[0174] Other ends of the genomic DNA which were generated by
shearing would be tailed with G's but would not have the adjacent
TTAGGG repeats. Thus, a mix of the following 3 biotinylated
oligonucleotides would anneal under stringent conditions
specifically to all possible telomere ends:
[0175] 5'B-CCCCCCCCTAACCCTA
[0176] 5'B-CCCCCCCCAACCCTAA oligo Mix [M]
[0177] 5'B-CCCCCCCCACCCTAAC
[0178] Oligo mix [M] consists of 16-base oligonucleotides with 5'
biotin (B), but other combinations of 5'-C-tracts adjacent to the
C-rich telomeric repeats could provide specific hybridization to
the 3' end of the native telomeres.
[0179] Extension of the primer with a DNA polymerase such as
Klenow, DNA Polymerase I, or Taq polymerase, in the presence of
dCTP, dATP, dTTP (no dGTP, and with or without ddGTP) would
stabilize the primer-template configuration and allow selection,
using streptavadin beads, of the terminal fragments of DNA
containing the telomeric DNA. The length of primer extension using
Klenow (monitored with labeled nucleotides) would indicate the
length of the telomeric (GTR) 3' overhang, since Klenow lacks 5'-3'
exonuclease activity and would stall ac the CTR. This length
distribution could be indicative of the level of telomerase
activity in telomerase-positive cells (i.e., longer extensions
correspond to greater telomerase activity). In contrast, extension
of the primer with DNA polymerase I, an enzyme with 5'-3'
exonuclease activity as well as polymerase activity, would allow
extension through the CTR until C's are encountered in the template
strand (subtelomeric to the GTR). The length distribution of this
reaction, monitored by labeled nucleotides, would be indicative of
the length distribution of the GTR. In both cases, labeled products
arising from biotinylated primers are selected with the
streptavadin beads to reduce the signal from non-specific priming.
Alternatively, re-priming and extension of the tailed chromosome
end can take place after selection of the partially extended
products with the streptavadin beads, and after denaturation of the
C-rich strand from the duplex.
[0180] Experiments have confirmed that the G-tailing of chromosome
ends can be carried out efficiently such that about 50 G residues
are added per end, that the priming with the junction
oligonucleotide mix is highly specific for the tailed telomeric
ends, and that streptavadin beads select specifically for the
extension products that originate from the biotinylated primers and
not from other fortuitous priming events. The length of the
extension products under the conditions outlined above thus provide
a direct estimate of the length of the terminal TTAGGG repeat
tract. This information is especially important in cases where
stretches of TTAGGG repeats occur close to but not at the termini
of chromosomes. No other method described to date is capable of
distinguishing between the truly terminal TTAGGG repeats and such
internal repeats.
[0181] The determination of telomere length as described above can
be associated with a variety of conditions of diagnostic interest.
Following telomere length in tumor cells provides information
regarding the proliferative capacity of such cells before and
following administration of inhibitors of telomerase (or other
treatments which destabilizes the telomere length as discussed
above). It also provides a means of following the efficacy of any
treatment and providing a prognosis of the course of the
disease.
[0182] Where diseased tissue is involved, the native tissue can be
evaluated as to proliferative capability. By "proliferative
capability" is meant the inherent ability of a cell or cells in a
tissue to divide for a fixed number of divisions under normal
proliferation conditions. That is, the "Hayflick" number of
divisions, exemplified below in the examples. Thus, despite the
fact that the tissue may have a spectrum of cells of different
proliferative capability, the average value will be informative of
the state of the tissue generally. One may take a biopsy of the
tissue and determine the average telomeric length. Using the value,
one may then compare the value to average normal healthy tissue as
to proliferative capability, particularly where the tissue is
compared to other tissue of similar age.
[0183] In cases of cellular diseases, such as liver disease, e.g.,
cirrhosis, or muscle disease, e.g., muscular dystrophy, knowledge
of the proliferative capability can be useful in diagnosing the
likely recuperative capability of the patient. Other situations
involve injury to tissue, such as in surgery, wounds, burns, and
the like, where the ability of fibroblasts to regenerate the tissue
will be of interest. Similarly, in the case of loss of bone,
osteoarthritis, or other diseases requiring reformation of bone,
renewal capability of osteoblasts and chondrocytes will be of
interest.
[0184] While methods are described herein to evaluate the
proliferative capacity of a tissue by taking an average measure of
telomere length it is noted that the tissue may have a spectrum of
cells of different proliferative capability. Indeed, many tissues,
including liver, regenerate from only a small number of stem cells
(less than a few percent of total cells). Therefore, it is useful
in this invention to use in situ hybridization (such as with
fluorescently labeled telomeric probes), to identify and quantitate
such stem cells, and/or the telomeric status of such cells on an
individual, rather than collective basis. This is performed by
measuring the fluorescent intensity for each individual cell
nucleus using, e.g., automated microscopy imaging apparatus. In
addition to in situ hybridization, gel electrophoresis is useful in
conjunction with autoradiography to determine not only the average
telomere length in cells in a tissue sample, but also the longest
telomere lengths (possibly indicating the presence of stem cells)
and the size distribution of telomere lengths (which may reflect
different histological cell types within a tissue, see FIGS.
10-11). Thus, the autoradiogram, or its equivalent provides useful
information as to the total telomere status of a cell, or group of
cells. Each segment of such information is useful in diagnostic
procedures of this invention.
[0185] d) Modified Maxam-Gilbert Reaction
[0186] The most common technique currently used to measure telomere
length is to digest the genomic DNA with a restriction enzyme with
a four-base recognition sequence like HinfI, electrophorese the DNA
and perform a Southern blot hybridizing the DNA to a radiolabeled
(TTAGGG).sub.3 probe. A difficulty with this technique is that the
resulting terminal restriction fragments (TRFs) contain a 3-5 kbp
stretch of subtelomeric DNA that lacks restriction sites and
thereby adds significantly to the size of the measured telomere
length. Another approach to eliminate this DNA and improve accuracy
of telomere length assays utilizes the fact that this subtelomeric
DNA contains G and C residues in both strands, and thus should be
cleaved under conditions that cause breaks at G residues In
contrast, DNA composed exclusively of telomeric repeats will have
one strand lacking G residues, and this strand should remain intact
under G-cleavage conditions. The Maxam-Gilbert G-reaction uses
piperidine to cleave guanine residues that have been methylated by
dimethylsulfate (DMS) treatment. Although the original conditions
of the Maxam-Gilbert G-reaction (treatment in 1M piperidine for 30
min. at 90.degree. C.) breaks unmethylated DNA into fragments of
1-2 kbp and is thus non-specific, milder conditions (0.1M
piperidine for 30 min. at 37.degree. C.) leave untreated DNA
intact. The DNA is therefore treated with DMS and piperidine as
described above, precipitated with ethanol, electrophoresed, and
hybridized on a Southern blot to the a (TTAGGG).sub.3 probe. The
results of such a test are shown in FIG. 26.
[0187] Telomerase Activity
[0188] Telomerase activity is useful as a marker of growth
potential, particularly as to neoplastic cells, or progenitor
cells, e.g., embryonic stem cells. Human telomerase activity may be
determined by measuring the rate of elongation of an appropriate
repetitive sequence (primer), having 2 or more, usually 3 or more,
repeats of the telomere unit sequence, TTAGGG. The sequence is
labeled with a specific binding pair member at a convenient site,
e.g., the 5'-terminus, and the specific binding pair member allows
for separation of extended sequences. By using one or more
radioactive nucleoside triphosphates or other labeled nucleoside
triphosphate, as described previously, one can measure the
incorporated radioactivity as cpm per unit weight of DNA as a
function of unit of time, as a measure of telomerase activity. Any
other detectable signal and label may also be used, e.g.,
fluorescein.
[0189] The activity may be measured with cytoplasmic extracts,
nuclear extracts, lysed cells, whole cells, and the like. The
particular sample which is employed and the manner of pretreatment
will be primarily one of convenience. The pretreatment will be
carried out under conditions which avoids denaturation of the
telomerase, so as to maintain the telomerase activity. The primer
sequence will be selected or labeled so as to allow it to be
separated from any other DNA present in the sample. Thus, a
haptenic label may be used to allow ready separation of the
elongated sequence, which represents the telomerase activity of the
sample. The nucleoside triphosphates which may be employed may
include at least one nucleoside triphosphate which is labeled. The
label will usually be radiolabel, but other labels may also be
present. The labels may include specific binding pair members,
where the reciprocal member may be labeled with fluorescers,
enzymes, or other detectable label. Alternatively, the nucleoside
triphosphates may be directly labeled with other labels, such as
fluorescent labels.
[0190] The sequence elongation usually will be carried out at a
convenient temperature, generally from about 20.degree. C. to
40.degree. C., and for a time sufficient to allow for at least
about 100 bp to be added on the average to the initial sequence,
generally about 30-90 minutes. After the incubation time to allow
for the telomerase catalyzed elongation, the reaction may be
terminated by any convenient means, such as denaturation, e.g.,
heating, addition of an inhibitor, rapid removal of the sequence by
means of the label, and washing, or the like. The separated DNA may
then be washed to remove any non-specific binding DNA, followed by
a measurement of the label by any conventional means.
[0191] The determination of telomerase activity may be used in a
wide variety of ways. It can be used to determine whether a cell is
immortalized, e.g., when dealing with tissue associated with
neoplasia. Thus, one can determine at the margins of a tumor,
whether the cells have telomerase activity and may be immortalized.
The presence and activity of the telomerase may also be associated
with staging of cancer or other diseases. Other diagnostic
interests associated with telomerase include measurement of
activity as an assay for efficacy in treatment regimens designated
to inhibit the enzyme.
[0192] Other techniques for measuring telomerase activity can use
antibodies specific for the telomerase protein, where one may
determine the amount of telomerase protein in a variety of ways.
For example, one may use polyclonal antisera bound to a surface of
monoclonal antibody for a first epitope bound to a surface and
labeled polyclonal antisera or labeled monoclonal antibody to a
second epitope dispersed in a medium, where one can detect the
amount of label bound to the surface as a result of the telomerase
or subunit thereof bridging between the two antibodies.
Alternatively, one may provide for primers to the telomerase RNA
and using reverse transcriptase and the polymerase chain reaction,
determine the presence and amount of the telomerase RNA as
indicative of the amount of telomerase present in the cells.
[0193] The following examples are offered by way of illustration
and not by way of limitation.
EXAMPLES
[0194] The following are examples of specific aspects of the
invention to merely illustrate this invention to those in the art.
These examples are not limiting in the invention, but provide an
indication of specific methodology useful in practice of the
invention. They also provide clear indication of the utility of the
invention and of the correlation between telomere length,
telomerase activity and cellular senescence. Such correlation
indicates to those in the art the breadth of the invention beyond
these examples.
Example 1
Telomere Length and Cell Proliferation
[0195] The effects of telomere length modulation on cellular
proliferation were studied. An average of 50 bp are lost per cell
division in somatic cells. The telomere end is thought to have a
single-stranded region as follows (although the amount of overhang
is unknown):
[0196] 5'TTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTAGGGTTAGGGTTAG GGTTA
GGG 3'AATCCCAATCCC (Seq. ID No. 1)
[0197] Applicant postulated that loss of this single-stranded
overhang should be significantly slowed if cells were provided with
a synthetic oligonucleotide of the sequence CCCTAACCCTAA (Seq. ID
No. 2). This oligonucleotide should hybridize to the exposed
single-stranded region, and serve as a primer for DNA synthesis by
the normal DNA polymerase present in somatic cells. In this way,
rather than shortening by an average of 50 bp per division, the
telomeres may only shorten by a lesser amount per division, thus
significantly extending the number of divisions required before
telomere shortening induced cellular senescence. This hypothesis
was tested by measuring both the change in proliferative lifespan
and rate of telomere shortening in cultured cells treated with this
indicated oligonucleotide, versus control oligonucleotides.
[0198] The efficacy of the CTO-12 oligonucleotide
(5'-CCCTAACCCTAA-3' Seq. ID No. 2) to reduce telomere shortening
associated with cellular senescence (FIG. 1) was studied using
target cells cultured under standard cell culture conditions in
minimal essential medium supplemented with 10% fetal calf serum.
The cells were subcultivated every four days by trypsinization upon
reaching confluency and were fed new medium at subcultivation or
every two days, whichever came first. Cells at various population
doubling levels were seeded at 10,000 cells per well and fed medium
containing oligonucleotides at various concentrations.
oligonucleotides studied were the cytidine-rich terminal
oligonucleotide (CTO-12), guanidine-rich terminal
oligonucleotide-12 bp (GTO-12, having the sequence
5'-TTAGGGTTAGGG-3' (Seq. ID No. 3)), and a 12 base pair randomer
with a random nucleotide in every position. As an additional
control, cells were fed identical medium without oligonucleotide.
Cells were fed oligonucleotide every 48 hours from 10X stocks.
(Such oligonucleotides may be modified to enhance stability, e.g.,
with phosphorothioates, dithioate and 2-O-methyl RNA.) In the case
of phosphorothioates it would be desirable to use longer CTO
primers such as 5'-CCCTAACCCTAACCCT-3', 5'-CCCTAACCCTAACCCTAA-3',
or 5'-CCCTAACCCTAACCCTAACC-3'.
[0199] Specifically, IMR-90 human lung fibroblasts with a
proliferative capacity of approximately 55 population doubling (PD)
were seeded at PD45 at 10,000 cells per well in a 48 well tissue
culture dish, and fed medium only or medium supplemented with
CTO-12 (at 1.0 .mu.M and 0.1 .mu.M) and 12 base pair randomer at
1.0 .mu.M. As shown in FIG. 1, cells grown in medium without
oligonucleotide, or with CTO-12 at less than 1.0 .mu.M or with
oligonucleotide of random sequence reached replicative senescence
in a similar fashion at about 52 population doubling. Cells fed the
CTO-12 oligonucleotide at 1.0 .mu.M, however, continued to
proliferate for approximately 10 doubling more than control
cells.
Example 2
Inhibition of Telomerase in Cancer Cells
[0200] One way by which cancer cells are able to escape cellular
senescence is by regaining telomerase activity, which permits them
to maintain the length of their telomeres in the face of multiple
rounds of cell division. The enzyme telomerase contains an RNA
complementary to TTAGGG, which allows it to recognize the telomeres
and extend them by the addition of additional TTAGGG repeats. In
fact, one assay for telomerase uses a TTAGGGTTAGGG primer and
measures the ability of cell extracts to synthesis a ladder of 6 bp
additions to this substrate. Telomerase activity in cancer cells is
likely to be present in limiting amounts since telomere length is
relatively stable (thus only about 50 bp per telomere are added, so
that lengthening and shortening are balanced).
[0201] Applicant hypothesized that feeding cells a synthetic
TTAGGGTTAGGG oligonucleotide (Seq. ID No. 3) should competitively
inhibit the ability of telomerase to elongate chromosome ends, and
thus should lead to telomere shortening and senescence in cancer
cells. Since somatic cells lack telomerase activity, the effects of
this treatment should be strictly limited to cancer cells and the
germ line.
[0202] Specifically, MDA 157 human breast cancer cells with an
immortal phenotype were seeded at 10,000 cells per well in 12 well
tissue culture dishes and fed medium only or medium supplemented
with GTO-12 (at 1.0 .mu.M, 0.1 .mu.M, and 0.01 .mu.M). As shown in
FIG. 2, cells grown in medium without oligonucleotide, or with
doses of less than 1.0 .mu.M continued replicating in an immortal
phenotype. Cells fed the CTO-12 oligonucleotide, at 1.0 .mu.M,
however, ceased to proliferate after less than 10 doubling. Cells
grown in the presence of 1.0 .mu.M CTO-12 or 1.0 .mu.M CTO-12 and
1.0 .mu.M GTO-12 (G+C) continued to express the immortal phenotype
suggesting that the GTO-12 oligonucleotide was not intrinsically
toxic (FIG. 3). The lack of effect of the G+C mixture may reflect
the CTO-12 oligonucleotide, competing with or base pairing with the
GTO-12 oligonucleotide, this preventing its inhibitory effect on
the cancer cell telomerase.
Example 3
Telomere Length as a Biomarker
[0203] In the U.S. and Western Europe, atherosclerosis is the
principal contributor to mortality from cardiovascular diseases
(Ross, 314 N. Engl. J. Med. 488, 1986). Atherosclerosis is
characterized by the mural and focal formation of lipid and
cell-rich lesions or "plaques" on the intimal surfaces of arterial
tissues. This is followed by an age-dependent expansion of the
lesion into the lumen, potentially leading to occlusion and to
myocardial and/or cerebral infarction (Haust, (1981) in Vascular
Injury and Atherosclerosis, ed. Moore, S. (Marcel Dekker Inc., New
York), pp. 1-22; Ross and Glomset, 295(7) N. Engl. J. Med. 369,
1976; and Ross, 295(8) N. Engl. J. Med. 420, 1976). Prominent among
the mechanisms proposed to explain the pathogenesis of
atherosclerosis is the "response-to-injury" hypothesis (Ross, 314
N. Engl. J. Med. 488, 1986; Moore, (1981) in Vascular Injury and
Atherosclerosis, ed. Moore, S. (Marcel Dekker Inc., New York), pp.
131-148; and Moore, 29(5) Lab. Invest. 478, 1971) in which repeated
mechanical, hemodynamic and/or immunological injury to the
endothelium is the initiating event.
[0204] A prediction of this hypothesis is that the intimal and
medial tissue in the area comprising the atherosclerotic plaque
will have a higher rate of cell turnover than the surrounding
normal tissue. Several lines of evidence support this prediction.
Ross et al., (Ross and Glomset, 295(7) N. Engl. J. Med. 369, 1976;
Ross, 295(8) N. Engl. J. Med. 420, 1976) showed that cultured
smooth muscle cells from fibrous plaques displayed lower
responsiveness to growth serum when compared to cells from the
underlying medial layer. Moss and Benditt 78(2) (1973) Am. J.
Pathol. 175, 1973, showed that the replicative life-span of cell
cultures from arterial plaques were equal to or less than the
replicative life-spans from cells of nonplaque areas. Dartsch et
al., 10 Arteriosclerosis 62, 1992, showed that human smooth muscle
cells obtained from primary stenosing lesions became senescent in
culture far later than smooth muscle cells from restenosing
lesions. These results suggest that cells derived from regions of
atherosclerotic plaques undergo more cellular divisions than cells
from non-plaque areas hence rendering them older and nearer to
their maximum replicative capacity.
[0205] Thus, to understand the pathogenesis of atherosclerosis, one
must examine the alterations in the behavior of cell turnover on
and adjacent to the arterial lesions. One requires a biomarker for
the cell turnover of intimal and medial tissue. Several workers
have examined hiomarkers for the progression of atherosclerosis or
for the propensity of an individual to develop atherosclerosis. The
former objective entailed the measurement of a number of
biochemical compounds which are detected in the plasma but
originate from the endothelium. Examples are serum Type III
collagen (Bonnet et al., 18 Eur. J. Clin. Invest. 18, 1988), von
Willebrand's Factor (Factor VIII) (Baron et al., 10
Arteriosclerosis 1074, 1990), cholesterol, triglycerides,
apolipoprotein B (Stringer and Kakkar, 4 (1990) Eur. J. Vasc. Surg.
513, 1990), lipoprotein (a) (Breckenridge, 143 Can. Med. Assoc. J.
115, 1990; Mezdour et al., 48 Ann. Biol. Clin. (Paris) 139, 1990;
and Scanu, 14 Clin. Cardiol. 135, 1991), endothelin (Lerman et al.,
325 N. Engl. J. Med. 997, 1991) and heparin-releasable Platelet
Factor 4 (Sadayasu et al., 14 (1991) Clin. Cardiol. 725, 1991). A
number of markers originate from the cell surface (Hanson et al.,
11 (1991) Arterioscler. Thromb. 745, 1991; and Cybulsky and
Girnbrone, 251 Science 788, 1991). Other markers monitor
physiological aberrations as a result of atherogenesis (Vita et
al., 81 (1990) Circulation 491, 1990). Candidate genes used to
delineate the RFLP profile of those susceptible to atherogenesis
(Sepehrnia et al., 38 (1988) Hum. Hered. 136, 1988; and Chamberlain
and Galton, 46 Br. Med. Bull. 917, 1990) have also been
established. However, there have been relatively few markers
developed to monitor directly cell turnover.
[0206] Applicant now shows that telomere length may serve as a
biomarker of cell turnover in tissues involved in atherogenesis.
The results show that endothelial cells lose telomeres in vitro as
a function of replicative age and that in vivo telomere loss is
generally greater for tissues of the atherosclerotic plaques
compared to control tissue from non-plaque regions.
[0207] In general, telomere lengths were assessed by Southern
analysis of terminal restriction fragments (TRF, generated through
HinfI/RsaI digestion of human genomic DNA. TRFs were resolved by
gel electrophoresis and hybridized with a telomeric oligonucleotide
(.sup.32P-(CCCTAA).sub.3) (Seq. ID No. 4). Mean TRF length
decreased as a function of population doubling in human endothelial
cell cultures from umbilical veins (m=-190 bp/PD, P=0.01), and as a
function of donor age in iliac arteries (m=-120 bp/PD, P=0.05) and
iliac veins (m=-160 bp/PD, P=0.05). Thus, mean TRF length decreased
with the in vitro age of all cell cultures. When early passage cell
cultures were assessed for mean TRF length as a function of donor
age, there was a significant decrease for iliac arteries (m=-102
bp/y, P=0.01) but not for iliac vein (m=47 bp/y, P=0.14). Mean TRF
length of medial tissue decreased significantly (P=0.05) as a
function of donor age. Intimal tissues from one individual who
displayed extensive development of atherosclerotic plaques
possessed mean TRF lengths close to those observed for senescent
cells in vitro (-6 kbp). These observations indicate that telomere
size indeed serves as a biomarker for the replicative history of
intima and media and that replicative senescence of endothelial
cells is involved in atherogenesis.
[0208] Specifically, the following materials and methods were used
to achieve the results noted below.
[0209] Endothelial Cell Cultures Human umbilical vein endothelial
cells (HUVEC) were obtained from Dr. Thomas Maciag of the Jerome H.
Holland Laboratory of the American Red Cross. Human endothelial
cells from the iliac arteries and iliac veins were obtained from
the Cell Repository of the National Institute of Aging (Camden,
N.J.). Cells were grown at 37.degree. C. in 5% CO.sub.2 on 100 mm
tissue plates whose interiors were treated with an overnight
coating of 0.4% gelatin (37.degree. C.). The supplemented media
consisted of M199, 15% fetal bovine serum, 5 U/ml heparin and 20
.mu.g/ml crude Endothelial Cell Growth Supplement (Collaborative
Research) or crude Endothelial Cell Growth Factor
(Boehringer-Mannheim). Cultures were trypsinized (0.05%, 3 minutes)
at confluence, reseeded at 25% of the final cell density and refed
every 2-3 days.
[0210] Tissue Samples Tissue samples from the aortic arch,
abdominal aorta, iliac artery and iliac vein were obtained from
autopsies at the Department of Pathology, Health Sciences Center,
McMaster University. Post-mortem times ranged from 5 to 8 hours.
The intima was obtained by cutting open the arteries or veins and
carefully scraping off the lumenal surface with a No. 10 scalpel
(Lance Blades, Sheffield) (Ryan, 56 Envir. Health Per. 103, 1984).
The resulting material was either treated directly for extraction
of DNA or processed for cell culture.
[0211] The adventitial layer was removed by cutting or scraping the
non-lumenal side of the vessel. The remaining medial layer was
prepared for DNA extraction by freezing it in liquid-N.sub.2 and
grinding it in a liquid-N.sub.2 chilled mortar and pestle (Kennedy
et al., 158 Exp. Cell Res. 445, 1985). After the tissue was ground
to a powder, 5 ml of frozen digestion Buffer (10 mM Tris; 100 mM
NaCl; 25 mM EDTA; 0.5% SDS; pH 8.0) was added and ground into the
powderized tissue. The powder was then transferred to a 50 ml
Falcon tube and incubated at 48.degree. C. until thawed. Proteinase
K (10 mg/ml) was added to a final concentration of 0.2 mg/ml. After
a 12-16 hour incubation, the solution was removed from the water
bath and either prepared for DNA extraction or stored at 20.degree.
C.
[0212] Extraction and Restriction Enzyme Digestion of Genomic
DNA
[0213] DNA was extracted as described previously (Harley et al.,
345 Nature 458, 1990; Allsopp et al., 89 Proc. Natl. Acad. Sci. USA
10114, 1992). In brief, proteinase K-digested lysates were
extracted twice with one volume of phenol:chloroform:isoamyl
alcohol (25:24:1) and once with chloroform. Nucleic acid was
precipitated by adding 2 volumes of 100% EtOH to the aqueous layer,
washed once with 70% EtOH and finally resuspended in 100-200 .mu.l
of 10 mM Tris-HCl, 1 mM EDTA, pH 7.5. DNA was quantified by
fluorometry and 1 .mu.g was digested with 1 unit each of HinfI/RsaI
for 3-24 hours at 37.degree. C. Complete digestion was monitored by
gel electrophoresis. The integrity of the DNA before and after
digestion was monitored in control experiments by gel
electrophoresis.
[0214] Southern Blot Hybridization
[0215] Electrophoresis of digested genomic DNA was performed in
0.5% agarose gels in a standard Tris, sodium borate, EDTA buffer
for a total of 650-700 V/hr as described previously (Harley et al.,
345 Nature 458, 1990; Allsopp et al., 89 Proc. Natl. Acad. Sci. USA
10114, 1992). After electrophoresis, the gel was placed onto 3 mm
Whatman filter paper and dried under vacuum for 25 minutes at
60.degree. C. Gels were denatured by soaking in 0.5 M NaOH, 1.5 M
NaCl for 10 minutes at room temperature and then neutralized
through immersion in 0.5 M Tris, 1.5 M NaCl. Genomic DNA was
immersed in standard hybridization solution (Harley et al., 345
Nature 458, 1990) (6.times.SSC) with the telomeric
.sup.32P-(CCCTAA).sub.3 probe (Seq. ID No. 4) for 12-16 hours at
37.degree. C. The telomeric smears were visualized through
autoradiography on pre-flashed (OD.sub.545=0.15) Kodak XAR-5 film.
The mean lengths of the terminal restriction fragments (TRFs) were
calculated from densitometric scans of the developed films as
described previously (Harley et al., 345 Nature 458, 1990).
[0216] In vitro Results
[0217] To determine the feasibility of employing telomere length as
a biomarker for cell turnover in atherosclerosis, we first examined
the change in telomere length in cultured endothelial cells where
cell division can be directly monitored in vitro. The DNA was
digested with HinfI and RsaI, and the resulting terminal
restriction fragments (TRF) were subjected to Southern analysis. As
in human skin fibroblasts (Allsopp et al., 89 Proc. Natl. Acad.
Sci. USA 10114, 1992), mean TRF length decreased as a function of
population doubling (PD). Thus, telomere length decreases with in
vitro age of human umbilical vein endothelial cells. Mean TRF
length decreased linearly (P=0.01) at a rate of 190.+-.10 bp/PD
(see FIG. 4). The Y-intercept, which signifies the mean TRF at 0
PDL is 14.0 kbp while mean TRF at senescence was 5.7.+-.0.4
kbp.
[0218] To prove that telomere length decrease occurred in
endothelial cells from other arterial and venous sources, mean TRF
length versus population doubling level (PDL) was determined for
several strains of endothelial cells from human iliac artery and
human iliac vein. In both iliac arteries and iliac veins there was
a significant (P=0.05) linear decrease in mean TRF length with age
of culture: 120.+-.60 bp per population doubling for the iliac
artery and 160.+-.30 bp per population doubling for the iliac veins
from endothelial cells.
[0219] In vivo Results
[0220] Formation of atherosclerotic plaques occurs more often in
the iliac artery than in the iliac vein (Crawford, (1982) Pathology
of Atherosclerosis (Butterworth and Co. Ltd., U.K.), p. 187-199),
thus it is expected that turnover of intimal tissue in vivo from
the iliac artery should be greater than that from the iliac veins.
To test this, nine different strains of endothelial cell cultures
from iliac arteries and veins of donors ranging in age from 14-58
years of age were cultivated and TRF lengths from the earliest
possible PDL were determined (FIG. 5).
[0221] Consistent with the hypothesis of greater cell turnover in
vivo in arteries than in veins, the rate of decrease in mean TRF
length, was significant over the age range 20-60 years for iliac
arteries (-100 bp/yr, P=0.01) and greater than for the iliac veins
(-47 bp/yr, P=0.14). Among the nine strains of endothelial cells,
there were cultures from the iliac artery and iliac vein from the
same individuals for 3 of the donors, aged 21, 47 and 49 years.
There was a significantly shorter mean TRF length in the cultures
of iliac artery cells as compared to the venous cells for the two
older donors. The younger donor showed no significant difference in
mean TRF length between the two cultures, possibly reflecting
relatively little difference in cell turnover between the vessels
of the 21-year old donor.
[0222] Differences in mean TRF length of the cell cultures from
iliac arteries and iliac veins in donors of different ages will
reflect not only differences in original mean TRF length of the
primary tissues but also differences in the rate of telomere loss
between the different cultures in vitro during the time required to
collect sufficient cells for analysis (approximately 5-10 PDL). To
determine if there is a relationship between cell turnover and the
extent of atherosclerotic plaque formation, we examined mean TRF
length in primary tissue. Autopsies from 3, 11, 12, 14, 18, 26,
75-year old females and a 77-year old male were performed. Sections
of the aortic arch, abdominal aorta, iliac artery and iliac vein
were taken and the intimal and medial tissues separated and
assessed for TRF length.
[0223] Sufficient intimal tissue could be obtained from the aortic
arch, abdominal aorta, iliac arteries and iliac veins of 3 donors
(aged 27, 75 and 77 years) for TRF analysis. There was a striking
difference between the mean TRF lengths averaged over these sites
in the 27-year old female (10.4.+-.0.7 kbp) versus the 75-year old
(8.8.+-.0.6 kbp) and the 77-year old male (6.3+0.4kbp). It is
noteworthy that the 77-year old male had extensive atherosclerotic
lesions in his vasculature and that the mean TRF length of his
intimal tissue is close to that of endothelial cells, at senescence
in vitro (approximately 6 kbp, FIG. 4).
[0224] FIG. 6 shows that mean TRF of medial tissue (from the aortic
arch) decreases with donor age at a small but significant rate (47
bp/yr, P=0.05). Thus, medial cells turnover in vivo occurs at a
rate less than that of the venous or arterial endothelial
cells.
[0225] In general, telomere loss in medial tissue underlying an
atherosclerotic plaque was greater than those in non-plaque regions
(Table 1). With the 75-year old female, mean TRF was significantly
reduced in medial DNA from the plaque regions versus the non-plaque
regions of both the aortic arch (P=0.04) and the abdominal aorta
(P=0.01). For the 77-year old male, this was observed in the
abdominal aorta (P=0.01).
1TABLE 1 Mean TRF values for primary medial tissues of plaque and
non-plaque areas Plaque Region Non-Plaque Region P 75-year old
Donor Aortic Arch 10.2 .+-. 0.5 11.1 .+-. 0.1 0.04 Abdominal Aorta
9.5 .+-. 0.6 11.0 .+-. 0.1 0.01 77-year old Donor Aortic Arch 8.2
.+-. 0.4 8.4 .+-. 0.2 NS Abdominal Aorta 7.1 .+-. 0.1 8.2 .+-. 0.4
0.01
[0226] These results show that mean TRF length decreases as a
function of donor age for primary medial and intimal tissue,
suggesting that cell turnover does occur in cardiovascular tissue.
The decrease in mean TRF length for plaque regions versus clear
regions of medial tissue from the same blood vessel is consistent
with augmented cell turnover of tissue associated with
atherosclerotic plaques. Thus, the results indicate that
measurement of telomere length provides a biomarker for alterations
of cellular turnover in tissues associated with cardiovascular
diseases, i.e., cells of the intima and media.
[0227] Measurement of telomere length is a direct register of
proliferative history but to obtain telomeric DNA one must obtain a
biopsy of endothelial tissue. Since removal of the endothelium in
itself can induce plaque formation, the biopsy strategy obviously
entails ethical and practical problems. Based upon experience with
autopsy samples one requires a minimal area of 1 cm.sup.2 in order
to perform a Southern analysis as described in this paper. For a
practical biopsy, this is untenable. A detection technique to
circumvent this problem may be confocal fluorescent microscopy.
Example 4
Simplified Test for Telomere Length
[0228] Telomere length has been found to be the best predictor of
the remaining lifespan of cells cultured from donors of different
ages. The ability to measure telomere length thus has significant
clinical use. Because of their simple repetitive nature, telomeres
lack DNA sequences recognized by many restriction enzymes. One way
to measure telomere length is to digest DNA with restriction
enzymes with 4-base recognition sites, which cuts most of the DNA
into very small pieces and leaves the telomeres in relative large
TRFs (Terminal Restriction Fragments). A Southern blot of the DNA
is then probed with a radioactive TTAGGGTTAGGGTTAGGG (Seq. ID No.
5) oligonucleotide, and the size of the TRF determined.
[0229] A much simpler method to measure telomere length exploits
the fact that the telomere sequence lacks guanidine residues in the
C-rich strand. Genomic DNA can be melted and mixed with the DNA
synthesis primer CCCTAACCCTAACCCTAACCCTAA (Seq. ID No. 6) in the
presence of DNA polymerase and only three deoxynucleotides (DATP,
dTTP and radioactive dCTP). Rare complementary sequences scattered
throughout the genome would fail to extend due to the lack of dGTP.
The length of the extended DNA can then be determined from a simple
gel electrophoresis. The amount of DNA synthesized (counts
incorporated per .mu.g of DNA) will be directly proportional to
telomere length, and for diagnostic purposes a simple measure of
radioactivity would then suffice to quantitate telomere length.
Example 5
Identification of DNA Sequences Near Telomeres
[0230] There are good reasons to believe that the regulatory
factors that control cellular and organismal senescence are located
near telomeres, and are themselves regulated by the length of the
adjacent telomere. It is thus important to identify and clone them
in order to be able to understand and manipulate the aging process.
In addition, there is great interest in identifying unique
telomeric DNA within the human genome project, since telomeric
markers for mapping purposes are lacking for the ends of the
chromosomes.
[0231] In one method, large telomeric DNA is purified as follows. A
biotinylated CCCTAACCCTAA (Seq. ID No. 7) oligonucleotide is used
to prime DNA synthesis in double-stranded genomic DNA. The only
sequences with which this oligonucleotide can anneal will be the
single-stranded base overhangs at telomere ends. The extended DNA
will then be digested with a restriction enzyme such as NotI to
produce large restriction fragments. Biotinylated fragments are
retrieved using streptavidin coated magnetic beads, and analyzed by
pulsed field electrophoresis. 46 fragments (one for each end of the
23 human chromosomes) are produced.
[0232] Multiple strategies can be used to pursue the successful
isolation of large telomeric DNA. The DNA can be labeled and used
to screen cDNA libraries in order to identify genes located near
telomeres. The expression of these cDNAs can then be examined in
young versus old cells in order to identify those which are
differentially expressed as a function of cellular senescence, and
which are thus candidates to be regulatory factors that control
aging.
[0233] The purified telomeric DNA can also be digested with
additional restriction enzymes, mixed with 100-fold excess of
genomic DNA, melted and reannealed. Under these circumstances, the
repetitive sequences in the telomeric DNA will anneal with genomic
DNA while unique sequences in the purified DNA will self-anneal.
Only the self-annealed unique sequences will contain restriction
overhangs at each end, and thus a simple cloning of the annealed
DNA will result in the successful cloning of only unique
fragments.
Example 6
Telomere Loss in Down's Syndrome Patients
[0234] Loss of telomeric DNA from human chromosomes may ultimately
cause cell cycle exit during replicative senescence. Since
lymphocytes have a limited replicative capacity and blood cells
were previously shown to lose telomeric DNA during aging in vivo,
we wished to determine whether accelerated telomere loss is
associated with the premature immunosenescence of lymphocytes in
individuals with Down's Syndrome (DS), and whether telomeric DNA is
also lost during aging of lymphocytes in vitro.
[0235] To investigate the effects of aging and trisomy 21 on
telomere loss in vivo, genomic DNA was isolated from peripheral
blood lymphocytes of 140 individuals (0-107 y) and 21 DS patients
(0-45 y). Digestion with restriction enzymes HinfI and RsaI
generated terminal restriction fragments (TRFs) which can be
detected by Southern analysis using a telomere-specific probe,
(.sup.32P-(CCCTAA).sub.3). The rate of telomere loss was calculated
from the decrease in mean TRF length as a function of donor age. DS
patients showed a significantly higher rate of telomere loss with
donor age (133.+-.15 bp/y) compared to age-matched controls
(41.+-.7.7 bp/y) (P<0.0005), indicating that accelerated
telomere loss is a biomarker of premature immunosenescence of DS
patients, and may play a role in this process.
[0236] Telomere loss during aging in vitro was calculated for
lymphocytes from two normal individuals grown in culture for 20-30
population doubling. The rate of telomere loss was 90 bp/cell
doubling, that is, it was comparable to that seen in other somatic
cells. Telomere lengths of lymphocytes from centenarians and from
older DS patients were similar to those of senescent lymphocytes in
culture, which suggests that replicative senescence could partially
account for aging of the immune system in DS patients and elderly
individuals.
[0237] The following materials and methods were used to obtain the
results provided below.
[0238] Culture of Human Peripheral Blood T Lymphocytes
[0239] Adult peripheral blood samples were collected, and
mononuclear cells were isolated by Ficoll-Hypaque gradient
centrifugation then cryopreserved in liquid nitrogen. Cultures were
initiated by mixing 10.sup.-6 mononuclear cells with 10.sup.6
irradiated (8000 Rad) lymphoblastoid cells (Epstein-Barr virus
transformed B cells), or 10.sup.6 mononuclear cells with 10
.mu.g/ml phytohemagglutinin (PHA-P, Difco) in each well of a
48-well cluster plate (Costar). After 8 to 11 days, cells were
washed and plated in 2 ml wells of 24-well cluster plates at a
concentration of 2-4.times.10.sup.5/ml. Cultures were passaged
every three to four days, or whenever viable cell concentration
(determined by trypan blue exclusion) reached
.gtoreq.8.times.10.sup.5/ml- . Cultures were terminated when they
showed no proliferative response to irradiated lymphoblastoid cells
and/or when there were no viable cells present in the entire visual
field of the haemocytometer. Once transferred to the 2 ml wells,
cells were continuously exposed to 25 U/ml of recombinant
interleukin-2 (Amgen). The media used were (a) RPM1 (Irvine
Scientific) supplemented with 10 to 20% fetal calf serum, 2 mM
glutamine, and 1 mM Hepes; (b) AIM V.TM., a DMEM/nutrient mixture
F-12 basal medium, containing purified human albumin, transferrin,
and recombinant insulin (Gibco), supplemented with 25% Ex-cyte (an
aqueous mixture of lipoprotein, cholesterol, phospholipids, and
fatty acids, (Miles Diagnostics).
[0240] At each cell passage, the number of population doubling (PD)
was calculated according to the formula: PD=ln (final viable cell
no. initial cell no.)/ln2.
[0241] Isolation of DNA
[0242] PBLs (including=15% monocytes) were isolated using
Ficoll-Hypaque gradient centrifugation (Boyum et al., 21(97) Scan.
J. Clin. Lab. Invest. 77, 1968) and washed 3 times in PBS. Cell
pellets were resuspended in 500 .mu.l of proteinase K digestion
buffer (100 mM NaCl, 10 mM Tris pH 8, 5 mM EDTA, 0.5% SDS)
containing 0.1 mg/ml proteinase K and incubated at 48.degree. C.
overnight. Lysates were extracted twice with
phenol/chloroformisoamyl alcohol (25:24:1 v/v/v) and once with
chloroform. DNA was precipitated with 95% ethanol and dissolved in
TE (10 mM Tris, 1 mM EDTA, pH=8).
[0243] Analysis of Telomeric DNA
[0244] Genomic DNA (10 .mu.g) was digested with HinfI and RsaI
(BRL) (20 U each), re-extracted as above, precipitated with 95%
ethanol, washed with 70% ethanol, dissolved in 50 .mu.l TE, and
quantified by fluorometry. One .mu.g of digested DNA was resolved
by electrophoresis in 0.5% (w/v) agarose gels poured on Gel Bound
(FMC Bioproducts) for 700 V-h. Gels were dried at 60.degree. C. for
30 minutes, denatured, neutralized, and probed with 5'end-labeled
.sup.32P-(CCCTAA) as described above. Autoradiograms exposed within
the linear range of signal response were scanned with a Hoefer
densitometer. The signal was digitized and subdivided into 1 kbp
intervals from 2 kbp to 21 kbp for calculation of the mean TRF
length (L) using the formula L=.SIGMA.,
(OD.sub.i-L.sub.i)/.SIGMA.OD.sub.i, where OD.sub.i=integrated
signal in interval i, and L=TRF length at the mid-point of interval
i.
[0245] TRF Length vs. Age
[0246] When measured as a function of donor age, mean TRF length in
PBS of 140 unrelated normal individuals (aged 0-107 y) declined at
a rate of 41.+-.2.6 bp/y (p<0.00005, r=0.83). This rate of TRF
loss for PBLs is close to that previously found for peripheral
blood cells by Hastie et al., 346 Nature 866, 1990. When our data
were separated according to gender it was noticed that males lost
telomeric DNA at a rate slightly faster than that of females
(50.+-.4.2 vs 40.+-.3.6 bp/y), but this difference did not reach
statistical significance (p=0.1). The 18 centenarians (aged 99-107
y) among our population of normal individuals had a mean TRF length
of 5.28.+-.0.4 kbp (FIG. 7). Interestingly, the standard deviation
of mean TRF values for the centenarians (0.4 kbp) was much smaller
than that of other age groups. Although it is possible that this
represents selection of a more homogeneous population of cells with
age, it is also possible that the group of centenarians were less
genetically diverse than the younger populations in our study.
[0247] Mean TRF length was also analyzed in PBLs of 21 Down's
Syndrome individuals (aged 2-45 y) and the rate of loss was
compared to 68 age-matched controls (aged 0-43 y). We found that
cells from DS patients showed a significantly greater rate of
telomere loss (133.+-.15 bp/y vs 41.+-.7.7 bp/y; one tailed t-test,
t=5.71, p<0.0005) (FIG. 8).
[0248] To determine the rate of telomere loss as a function of cell
doubling, we cultured normal lymphocytes from 2 individuals in
vitro until replicative senescence and measured mean TRF length at
several population doubling levels (FIG. 9). Mean TRF length
decreased 90 bp/population doubling in these strains, within the
range observed for other human somatic cell types. The mean TRF
length at senescence for the lymphocyte cell strains shown here and
one other analyzed at terminal passage (FIG. 9), was 5.1.+-.0.35
kbp. The observed TRF values in vivo for PBLs of centenarians
(5.3.+-.0.4 kbp) and old DS patients (4.89.+-.0.59 kbp), were close
to this value, suggesting that a fraction of the cells from these
individuals were close to the limit of their replicative
capacity.
[0249] The results showing that telomeres in PBLs from normal
individuals shorten during aging in vivo and in vitro extend
similar observations on human fibroblasts (Harley et al., 345
Nature 458, 1990) and support the hypothesis that telomere loss is
involved in replicative senescence. We also found that in Down's
Syndrome, the rate of telomere loss in PBS in vivo was
significantly higher than that in age-matched normal donors. Thus,
accelerated telomere loss in PBS of trisomy 21, a syndrome
characterized by premature immunosenescence and other features of
accelerated aging (Martin, "Genetic Syndromes in Man with Potential
Relevance to the Pathobiology of Aging", in: Genetic Effects on
Aging, Bergsma, D. and Harrison D. E. (eds.), pp. 5-39, Birth
Defects: Original article series, no. 14, New York: Alan R. Liss
(1978)), could reflect early senescence of lymphocytes.
[0250] The increased rate of telomere loss in PBS from DS patients
could reflect a higher turnover rate of cells in vivo due to
reduced viability of the trisomy 21 cells. However, it is also
possible that the rate of telomere loss in PBS from DS patients is
greater per cell doubling than that in normal individuals.
[0251] The pathology of DS is similar in many ways to normal aging.
Premature senescence of the immune system possibly plays a role in
this similarity since DS patients have a high incidence of cancer
and suffer from autoimmunity. In support of this idea, lymphocytes
of older DS patients and old individuals share several
characteristics, including diminished response of T-cells to
activate and proliferate in response to antigen, low replicative
capacity, and reduced B- and T-cell counts (Franceschi et al., 621
Ann. NY Acad. Sci. 428, 1991). Our finding that telomere length
decreased faster in DS patients than normal individuals, and that
the mean TRF length in centenarians and old DS patients in vivo
were similar to that of senescent lymphocytes in vitro (=5 kbp)1
extends these observations. Moreover, these data suggest that
replicative senescence within the lymphoid lineage in vivo
contributes to the compromised immune system of both elderly
individuals and Down's Syndrome patients.
Example 7
Ovarian Cancer and Telomerase Activity
[0252] The following is an example of a method by which telomerase
activity is shown to correlate with the presence of cancer cells.
In addition, the length of TRF was determined as an indication of
the presence of tumor cells. Generally, it was found that tumor
cells had significantly lower TRF values than surrounding normal
cells, and had telomerase activity. Thus, these two features are
markers for the presence of tumor cells.
[0253] The following methods were used to obtain these results:
[0254] Separation of Tumor and Non-tumor Cells
[0255] In one method, ascitic fluid was obtained by either
diagnostic laparotomy or therapeutic paracentesis (from patients
diagnosed as having ovarian carcinoma), and centrifuged at
600.times.g for 10 minutes at 4.degree. C. The cell pellet was
washed twice in 10 to 30 ml of phosphate buffered saline (PBS: 2.7
MM KCl, 1.5 mM KH.sub.2PO.sub.4, 137 mM NaCl and 8 mM
Na.sub.2HPO.sub.4) and centrifuged at 570.times.g for 4 minutes at
4.degree. C. After the final wash the cell pellet was resuspended
in 20 ml of PBS and filtered through a 30 or 10 .mu.m nylon mesh
filter (Spectrum) which retains the tumor clumps but not single
cells. The filters were backwashed to liberate highly purified
tumor clumps. The flow-through was a combination of fibroblasts,
lymphocytes and tumor cells.
[0256] In another method ascitic fluid cells were collected and
washed as described above. The cellular pellet was resuspended in
a-MEM with 10% fetal calf serum and cultured in 150 mm dishes.
After 12 hours the media was removed and new plates were used to
separate the adhering fibroblasts from the non-adhering cells in
the medium. After 12 hours the media containing mostly tumor clumps
was removed from the second plates and allowed to adhere in DMA F12
medium supplemented with 3% fetal calf serum, 5 ng/ml EGF, 5
.mu.g/ml insulin, 10 .mu.g/ml human transferrin, 5.times.10.sup.-5
M phosphoethanolamine and 5.times.10.sup.-5 M ethanolamine. These
tumor cells were cultured for DNA analysis and S100 extracts.
[0257] DNA Extraction
[0258] Cells were lysed and proteins were digested in 10 mM
Tris-Hcl (pH 8.0), 100 mM NaCl, 25 mM EDTA, 0.5% SDS, 0.1 mg/ml
proteinase K at 48.degree. C. overnight. Following 2 extractions
with phenol and 1 with chloroform, DNA was precipitated with
ethanol and dissolved in 10 mM Tris-HCl (pH 8.0), 1 mM EDTA
(TE).
[0259] Determination of TRF Length and Amount of Telomeric DNA
[0260] Genomic DNA was digested with HinfI and RsaI, extracted and
precipitated as above, and redissolved in TE. DNA concentration was
measured by fluorometry (Morgan et al., 7 Nucleic Acids Res. 547,
1979). DNA samples (1 .mu.g each) were loaded onto a 0.5% agarose
gel and electrophoresed for 13 hours at 90 V. The gel was dried at
60.degree. C. for 30 minutes, denatured in 1.5 M NaCl and 0.5 M
NaOH for 15 minutes, neutralized in 1.5 M NaCl, 0.5 M Tris-HCl (pH
8.0) for 10 minutes and hybridized to a 5' .sup.32P(CCCTAA).sub.3
telomeric probe in 5.times.SSC (750 mM NaCl and 75 mM sodium
citrate), 5.times.Denhart's solution (Maniatis et al., Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold
Spring Harbor (1982)) and 0.1.times.P wash (0.5 mM pyrophosphate,
10 mM Na.sub.2HPO.sub.4) at 37.degree. C. for 12 hours. Following
three high stringency washes in 0.24.times.SSC at 20-22.degree. C.
(7 minutes each), the gel was autoradiographed on pre-flashed
(OD=0.15) Kodak XAR-5 X-ray films for 3 days with enhancing
screens. Each lane was scanned with a densitometer and the data
used to determine the amount of telomeric DNA and the mean TRF
length as previously described (Harley et al., 345 Nature 458,
1990).
[0261] Preparations of S-100 Cell Extracts
[0262] A minimum of 6.times.10.sup.8 cells were used for each
extract. Ascitic fluid or purified ascitic fluid tumor cells (by
the first method described above) were centrifuged at 570.times.g
for 4 minutes at 4.degree. C. Ascitic fluid tumor cells separated
by the second method described above (grown in monolayer) were
harvested by scraping with a rubber policeman, and centrifuged as
above. The pellets were rinsed twice in cold PBS followed by
centrifugation as above. The final pellet was rinsed in cold
2.3.times.Hypo buffer (1.times.Hypo buffer: 10 mM Hepes (pH 8.0)),
3 mM KCl, 1 mM MgCl.sub.2, 1 mM DTT, 0.1 mM PMSF and 10 U/ml of
RNAsin, 1 .mu.M leupeptin and 10 .mu.M pepstatin A, centrifuged for
5 minutes and resuspended in 0.75 volumes of 2.3.times.Hypo buffer.
After incubation on ice for 10 minutes the sample was transferred
to an ice cold 7 or 1 ml Dounce homogenizer and homogenized on ice
using a B pestle (25-55 .mu.m clearance). After a further 30
minutes on ice the samples having a volume larger than 1 ml were
centrifuged for 10 minutes at 10,000 rpm (16,000.times.g) at
4.degree. C. in a Beckman J3-13.1 swinging bucket rotor.
One-fiftieth volume of 5 M NaCl was added, and the samples
supernatant were centrifuged for 1 hour at 38,000 rpm
(100,000.times.g) at 4.degree. C. in a Beckman Ti50 rotor. Glycerol
was added to a final concentration of 20% and the extract aliquoted
and stored at -70.degree. C. Samples less than 1 ml were
centrifuged at 55,000 rpm for 1 hour at 4.degree. C. in a TLA 100.2
rotor (Beckman) and NaCl and glycerol were added to the supernatant
as above. Protein concentration in a typical extract was
approximately 4 mg/ml.
[0263] Telomerase Assay
[0264] Telomerase activity was assayed by a modification of the
method of Morin, 59 Cell 521, 1989. Aliquots (20 .mu.l) of S-100
cell extract were diluted to a final volume of 40 .mu.l containing
2 mM dATP, 2 mM dTTP, 1 mM MgCl.sub.2, 1 .mu.M (TTAGGG).sub.3
primer, 3.13 .mu.M (50 .mu.Ci) a-.sup.32P-dGTP (400 Ci/mmole), 1 mM
spermidine, 5 mM .beta.-mercaptoethanol, 50 mM potassium acetate,
and 50 mM Tris-acetate (pH 8.5). In some experiments reaction
volumes were doubled. The reactions were incubated for 60 minutes
at 30.degree. C. and stopped by addition of 50 .mu.l of 20 mM EDTA
and 10 mM Tris-HCl (pH 7.5) containing 0.1 mg/ml RNAseA, followed
by incubation for 15 minutes at 37.degree. C. To eliminate
proteins, 50 .mu.l of 0.3 mg/ml Proteinase K in 10 mM Tris-HCl (pH
7.5), 0.5% SDS was added for 10 minutes at 37.degree. C. Following
extraction with phenol and chloroform, unincorporated
a-.sup.32P-dGTP was separated by centrifuging the samples for 4
minutes at 500 g in a swinging bucket rotor through NICK SPIN
columns (Pharmacia). DNA was precipitated by the addition of 5.3
.mu.l of 4 M NaCl, 4 .mu.g of carrier tRNA and 500 .mu.l of ethanol
at -20.degree. C. DNA pellets were resuspended in 3 .mu.l of
formamide loading dye, boiled for 1 minute, chilled on ice and
loaded onto an 8% polyacrylamide, 7 M urea sequencing gel and run
at 1700 V for 2 hours using 0.6.times.TBE buffer. Dried gels were
exposed to Kodak XAR-5 pre-flashed film at -70.degree. C. with
enhancing screen or to phosphoimager screens (Molecular Dynamics)
for 7 days.
[0265] The results of the above experiments are shown in tables 2
and 3 below:
2TABLE 2 Characteristics of ATCC Ovarian Carcinoma Cell Lines Cell
line Mean TRF Length (kbp) Telomerase Activity HEY stable at 3.7 +
CAOV-3 stable at 3.7 N.D. SKOV-3 Increases at 60 bp/pd N.D.
[0266]
3TABLE 3 Characteristics of Ovarian Carcinoma Tumor Cells from
Ascitic Fluid Mean TPF Telomerase Patient Description Length (kbp)
Activity Pres-3 Purified tumor cells 3.7 + Mac-2 Purified tumor
cells 3.7 N.D. Sib-1 Purified tumor cells 4.2 N.D. Ric 207 Purified
tumor cells 3.3 N.D. Cra-1 Purified tumor cells 5.2 N.D. Ing-1
Purified tumor cells 5.8 N.D. Lep-1 Purified tumor cells 5.8 N.D.
Lep-4 Purified tumor cells 5.6 N.D. Sal-1 Purified tumor cells 5.6
N.D. Rud-1 Ascitic fluid cells 3.4 + Murr-1 Ascitic fluid cells 3.8
+ Dem-1 Ascitic fluid cells N.D. + Cas-1 Ascitic fluid cells 5.3 +
Wad-1,2 Ascitic fluid cells 4.9 N.D.* N.D. = not determined *High
background precluded detection
[0267] Table 4 shows the TRF length of cells from ascitic fluid. A
minimum of 2 autoradiographs were scanned with a densitometer over
the size range 2-21 kbp, and the densitometric values used to
determine mean TRF length in kbp. Average standard deviation of the
data was 0.5 kbp with the largest deviation being 2 kbp. The value
following the three character patient code refers to the
paracentesis number (i.e., OCl-1 is the first sample from patient
OCI). Samples defined as E (early) were obtained near the time of
presentation while samples L (late) were obtained near death.
Paracenteses were performed 4 to 15 times over the course of 4 to
22 months.
4TABLE 4 Unfractionated Fractionated Cultured cells TRF Normal
Tumour Tumour* Patient (kbp) Patient TRF (kbp) TRF (kbp) Patient
TRF (kbp) OC1-1 3.8 OC5-1 7.0 5.0 OC18-2 3.4 OC2-1 5.5 OC6-1 9.2
5.4 OC19-3 3.4 OC3-1 5.4 OC7-1 8.0 5.4 OC20-1 4.2 -2 4.4 OC8-1 7.7
4.3 OC21-1 3.3 OC4-1 4.5 OC9-1 5.2 OC5-1 4.3 OC10-1 3.9 OC22-13 6.9
OC11-2 3.7 OC12-1 3.8 OC13-1 5.1 Serial Samples OC14-1 (E) 9.4 5.0
-4 (L) 9.3 5.2 OC15-1 (E) 7.3 4.1 -5 (L) 4.7 OC16-1 (E) 3.9 -2 (E)
3.4 -7 (L) 3.9 OC17-1 (E) 7.7 4.3 -15 (L) 4.7 Means.sup.554: 4.7
.+-. 0.7 8.2 .+-. 0.9 4.5 .+-. 0.8 3.7 .+-. 0.5 (4.2 .+-.
1.4).sup..dagger-dbl. *mean TRF length was determined for each of
the samples over the course of at least 30 PD. Values were averaged
since TRFs were stable in all populations .sup..dagger.average and
standard deviation of the mean TRF lengths of all samples
.sup..dagger-dbl.mean value including OC22-13
[0268] Table 5 shows the telomerase activity in normal and tumor
cells. Leukocytes and acsites cells were isolated and ascitic fluid
cells fractionated into normal and tumor fractions and assayed for
telomerase activity. Protein concentration in all extracts was
<2 mg/ml, i.e., 20 fold higher than the lowest concentration at
which activity was detected in control 293 CSH extract.
5 TABLE 5 Unfractionated Fractionated Telomerase Telomerase
activity Patient activity Patient Normal Tumour OC4-1 + OC19-3 N.D.
+ -5 + OC17-1 -- N.D. OC2-1 .+-. OC8-1 -- N.D. OC1-1 + LEK -- N.D.
OC23-1 +
[0269] In the TRF assay, each tumor clump had significantly lower
TRF lengths than associated normal cells. (See FIG. 10).
[0270] In the telomerase assay, significantly greater telomerase
activity was evident in the ascitic fluid of certain patients than
in the control tumor lines HEY and PRES, or the control cell line
293 CSH (FIGS. 11, 33).
Example 8
Effect of HIV Infection on TRF Length
[0271] HIV infection leads to an acute viral infection manifesting
itself as a virus-like syndrome, followed by a prolonged period of
latency characterized by an absence of signs and symptoms. During
this prolonged asymptomatic period (lasting usually 7-10 years),
there is no diagnostic available for staging the course of the
infection other than the presence or absence of antibodies to viral
coat proteins. This does little to stage the disease or to help the
physician measure the effectiveness of prophylactic agents.
[0272] While Meyaard et al., 257 Science 217, 1992, propose a
programmed cell death for CD4.sup.+ and CD8.sup.+ cells of an
HIV-infected individual, we propose that during those 7 to 10 years
the immune system is able to keep the infection relatively
repressed, but there is markedly increased turnover of the infected
CD4.sup.+ T-destruction. We propose that this essentially
accelerates the replicative senescence of this particular
subpopulation of T-cells, and with time results in a population of
precursor pluripotent cells with markedly reduced proliferative
capacity. Finally, this results in CD4.sup.+ T-cells that are
relatively unresponsive to stimuli to proliferate, as is typical of
the replicative senescence of the cells observed in vitro.
[0273] We also propose that the replicative capacity of total
peripheral lymphocytes or CD4.sup.+ cells in particular, can be
effectively determined by assaying telomere repeat length utilizing
the method described above, e.g., with the oligonucleotide probe 5'
TTAGGGTTAGGGTTAGGGTTAGGG (or one of similar or complementary
sequence) hybridized to CD4.sup.+ lymphocyte DNA isolated from the
patient along with molecular size markers. These assays allow the
physician to chart the course of the disease during the long
intervening asymptomatic period, and to score the effectiveness of
prophylactic therapeutics.
[0274] In order to determine whether TRF length is a useful marker
in diagnosis of HIV infection, CD4.sup.+ cell counting was
performed on asymptomatic HIV-infected individuals, and compared to
TRF length, measured as discussed above. As shown above, peripheral
lymphocytes start with around 10 kb TRF length at birth, and reach
a TRF length of 5.0 at approximately age 120. The results were as
follows:
[0275] A 30 year old HIV+ with a CD4 count of 476 had a TRF of
7.6.
[0276] A 46 year old HIV- control, had a TRF of 7.0.
[0277] A 34 year old HIV+ with a CD4 count of 336, had a TRF of
7.7.
[0278] A 46 year old HIV- control, had a TRF of 7.1.
[0279] A 32 year old HIV+ with a CD4 count of 448, had a TRF of
6.9.
[0280] A 33 year old HIV+ with a CD4 count of 358, had a TRF of 5.0
(i.e., at a length observed for senescent cells)
[0281] The results indicate that the 33 year old HIV+ patient has a
senescent telomere length in his CD4.sup.+ cells, which means that
they are at the end of their replicative capacity. In contrast, the
CD4.sup.+ count provided no indication of the status of this
patient. Indeed, one patient actually had a lower CD4.sup.+
count.
[0282] Two weeks after the assay was performed, this patient
experienced a precipitous drop in CD4.sup.+ count, going from 358
to 159, and was therefore diagnosed AIDS, and rapidly acquired
leukoplakia on the tongue. The other patients remain asymptomatic.
Thus, this diagnostic procedure is able to distinguish patients
near the end of the course of HIV infection, whereas the previously
used marker (CD4.sup.+ count) could not.
[0283] The accelerated replicative senescence of CD4.sup.+
lymphocytes during the course of HIV infection provides an
appropriate indication for therapies designed to forestall telomere
shortening, e.g., utilizing the CTO oligonucleotide described
above. In addition, as described above, CD4.sup.+ cells of an
individual at an early stage of infection can be banked for later
administration to the individual. The efficacy of drugs, such as
AZT, may also be determined to study whether the drug slows the
rate of proliferation of CD4.sup.+ cells, and is thus useful at all
stages of the disease. If not, it can be administered only when
necessary during the course of the disease.
Example 9
Telomere Shortening in Human Mammary Epithelial (HME) Cells
[0284] Referring to FIG. 12, when digested with a restriction
enzyme having a 4-base recognition site (like Hinfl), most genomic
DNA is digested into small fragments. However, because the
repetitive telomeric sequences lack restriction sites, telomeres
retain relatively large terminal restriction fragments (TRFs)
composed of 2-5 Kb of subtelomeric DNA and age-dependent amounts of
telomeric repeats. As previously described for human fibroblasts,
lymphocytes and endothelial cells, telomere length shortens in
normal human mammary epithelial cells during in vitro cellular
senescence (compare TRF length in lanes 1 (PDL 21) and 2 (PDL 40)).
In human mammary epithelial cells expressing E6 of human papilloma
virus 16, the TRF length continues to shorten during the extended
lifespan period until crisis and subsequently immortalization
occurs (lane 3 (PDL 68)). The TRFs generally stabilize in
immortalized cells (lane 4 (PDL 81) and lane 5 (PDL 107))
consistent with the re-expression of telomerase activity.
Example 10
Slowing Telomere Loss in Mammary Epithelial Cells Results in
Increased Replicative Lifespan
[0285] Normal human mammary epithelial cells can be established
from organoids (obtained from reduction mammoplasty) and can be
cultured in defined condition in a standard medium (MCDB170) devoid
of serum. Epithelial cells with typical cobblestone morphology
spread around organoids plated in this medium. After the first
subcultivation these cultures enter a period of growth arrest for
2-3 weeks until a population of small, highly birefringent and
rapidly dividing cells expand among larger cells. The medium (MCDB
104) apparently selects for a less differentiated cell type with
increased growth potential. These cells can be subcultured for
40-45 additional doubling before undergoing cellular
senescence.
[0286] As in Example 1, the change in proliferative lifespan and
rate of telomere shortening in cultured mammary epithelial cells
treated with the indicated amounts of CTO (occasionally referred to
as C-Rich Terminal Repeat (CTR)) versus control random
oligonucleotides. Normal human mammary epithelial cells from a
donor (31) were infected with the E6 gene of human papilloma virus
16. This gene product binds p53 protein and permits HME31 cells to
have extended life span by proliferating from PDL 42 to PDL 62 when
crisis occurs. During this extended lifespan period the TRFs
shorten from an average of approximately 5 kb to 2.5 kb (compare in
FIG. 12 HME31 PD 40 to HME31E6 PD 68).
[0287] As is demonstrated in FIG. 13, experiments initiated using
HME31E6 cells at PDL 36 were cultured in the presence of 3, 10, 30
and 100 .mu.M CTO. As controls the cells were cultured without
oligonucleotides (nil) or with 30 .mu.M random oligonucleotide.
FIG. 13 demonstrates that compared to the nil control and the 30
.mu.M random oligonucleotide, there was a dose related retardation
of TRF shortening between PDL 36 and 50. This is most easily seen
by examining the subpopulation of telomere TRFs that migrate more
slowly than the rest, giving a discrete trailing band. Cells were
maintained in logarithmic growth with medium changed and fresh
oligonucleotide added three times per week.
[0288] Human mammary epithelial cells expressing HPV16 E6 bypass M1
and have extended replicative lifespan. HME31 cells normally
senesce at PDL 42-45. When expressing E6 they will bypass Ml and
divide until they reach crisis (M2) at PDL 53-62. The TRFs in HME31
(E6) cells at PDL 40 are approximately 5-6 Kb while at PDL 62 they
are 3-4 Kb (see FIG. 12). As is demonstrated in FIG. 17,
experiments initiated using HME31E6 cells at PDL 36 were cultured
in the presence of 30 .mu.M and 100 .mu.M CTR in defined medium
without serum. As controls, the cells were cultured without
oligonucleotide (control), or with a 30 .mu.M random
oligonucleotide with the base content matched to the CTR
oligonucleotide. FIG. 17 demonstrates that compared to the control
and the 30 .mu.M random oligonucleotide, there was a dose-related
extension of the replicative lifespan in cells treated with CTR
oligonucleotides. The control cells divided approximately 20 times
during the experiment, whereas the CTR-treated cells divided at
least 40-50 times. These results correlate well with the
retardation of telomere shortening observed in FIG. 13.
Example 11
Extension of Life Span of IMR90 Fibroblasts
[0289] Referring to FIG. 14, IMR-90 lung fibroblasts TRFat PDL 30
were treated with 10 .mu.M, 30 .mu.M or 100 .mu.M phosphodiester
CT0 or with only media addition (control). The cells were cultured
in medium containing regular defined supplemented calf serum. The
cells were passaged in 24 well dishes and subcultivated by
trypsinization upon reaching confluency at 25,000 cells per well.
The cells were fed medium containing oligonucleotides at various
concentrations daily. As a control, cells were fed identical medium
without oligonucleotides. As is illustrated in FIG. 14, there was
approximately a 12-15% extension of total life span with CTO. In
these experiments the control cells divided approximately 15-18
times during the experiment, whereas the treated cells divided
23-26 times. IMR-90 telomeres shorten approximately 50 b.p. per
division and the TRF length of the control IMR-90 fibroblasts at
senescence was approximately 9 kb. Since the 100 .mu.M CTO-treated
IMR-90 cells senesced at PDL 55, the predicted difference in the
rate of TRF loss between the control and the 100 .mu.M CTO (9 kb vs
9.4 kb) is too small to be resolved using current techniques.
Example 12
GTO Experiments
[0290] As in Example 2, an immortalized human fibroblast cell line,
IDH4, which has very short TRFs, was incubated with GTO
oligonucleotide. Referring to FIGS. 15 and 16, cells were incubated
in regular culture medium containing serum in the presence of 10
.mu.M, 30 .mu.M and 100 .mu.M GTO. The cells were fed fresh
phosphodiester GTO oligonucleotide every other day and subcultured
when confluent for a total of 90 days. The cells were still growing
in GTO after 90 days at all concentrations used even though they
grew more slowly at the higher GTO concentrations and went through
fewer population doubling (control, 45 PDL; 10 .mu.M GTO 40 PDL; 30
.mu.M 35 PDL; 100 .mu.M 25 PDL). When TRF analysis was performed
after 90 days the IDH4 cells regained TRF length in a dose
dependent manner with 30 AM and 100 AM being approximately the same
(FIG. 15). This suggests that the presence of excess
single-stranded TTAGGG DNA in the cell was probably influencing the
feedback regulation of telomerase and actually increasing
telomerase activity and extending telomere length. The control and
30 .mu.M GTO were passaged without oligonucleotide addition for an
additional 90 days (approximately 35-40 PDL). As is illustrated in
FIG. 16, the TRFs slowly shorten.
[0291] These data and those in Example 2, indicate that cell lines
differ in their response to GTO oligonucleotide. Thus, prior to use
of such an oligonucleotide in therapeutic compositions it is
important to ensure that the target cells respond as desired.
Should the effect seen above occur, then the oligonucleotide should
be chosen to change the response to that shown in Example 2. This
can be done by choosing an oligonucleotide which binds to
telomerase at a different site from that bound by GTO. Applicant
believes that the effect observed above is caused by binding of GTO
to required proteins, allowing telomerase to be active to expand
the telomeres. Thus, by choosing an oligonucleotide which does not
bind such proteins the desired effect of reducing telomerase
activity can be achieved.
Example 13
Small Molecule Inhibition of Telomerase
[0292] The following is an example of a method for screening for
activity of small molecules as inhibitors of telomerase. Similar
examples will be evident to those in the art. Compounds that can be
screened include those which are not thought to be cytotoxic
because they do not cause immediate cell death. Rather, such
compounds act only after several generations of inhibition of
telomerase activity. Thus, previous drugs tested by standard means
should now be retested to determine their utility as claimed
herein. Drugs which inhibit telomerase activity, or in some cases
activate it in vivo (e.g.. at the level of transcription) are
useful in treatment of disease are discussed herein.
[0293] We analyzed the effects of various nucleoside analogs, which
are chain-terminating inhibitors of retroviral reverse
transcriptases, on Tetrahymena thermophila telomerase activity in
vitro, and on telomere length and maintenance, cell division and
conjugation of Tetrahymena cells in vivo. In vitro assays of
telomerase activity showed that arabinofuranyl-guanosine
triphosphate (Ara-GTP) and ddGTP were both very efficient
inhibitors of incorporation of labeled nucleotides into telomeric
DNA repeats, even at low inhibitor concentrations, while
azidothymidine triphosphate (AZT-TP), dideoxyinosine triphosphate
(ddITP) or ddTTP were less efficient inhibitors of incorporation.
All of these nucleoside triphosphate analogs, however, produced
analog-specific alterations of the normal banding patterns seen
upon gel electrophoresis of the synthesis products of telomerase,
suggesting that the competitive and/or chain terminating action
differed at different positions along the RNA template.
[0294] The effects of these analogs in nucleoside form on
Tetrahymena cell growth, conjugation, and telomere length were
tested. Although cell division rates and viability were unaffected
after several weeks in culture with Ara-G, telomeres were
consistently and rapidly shortened in cultures containing AZT or
Ara-G, and growth rates and viability of a fraction of cells were
decreased in AZT. In short-term experiments with cultures
containing ddG, ddI, or 3' deoxy-2',3'-didehydrothymidine (d4T) ,
d4T also showed shortened telomeres. ddG or ddI had no effect on
telomere length. AZT, Ara-G, Acycloguanosine (Acyclo-G), ddG and
ddI were added to conjugating cells, but none showed any
irreversible disruption of conjugation or macronuclear development,
as shown by quantitation of the efficiency of formation of progeny
cells. PCR analysis of DNA from cells mated in AZT did show a
decrease in the formation of 11 Kb rDNA, a marker for telomere
addition during Macronuclear developement.
[0295] The following materials and methods were used to obtain
these results:
[0296] Tetrahymena thermophila strains SB210(VI) and PB9R(II),
where numbers in parentheses indicate mating type, were maintained
as stocks at room temperature in 1% PPYS (1% proteose peptone
(Difco), 0.1% yeast extract (Difco) and 0.0015% Sequestrine
(Ciba-Geigy)). Stocks were passaged every three to four weeks.
[0297] For analysis of macronuclear DNA from cultures containing
the nucleoside analog AZT (Sigma), or controls lacking analog, at
various timepoints during vegetative divisions, cells from
stationary stock cultures were inoculated into 25 ml
thymine-deficient Iso-sensitest broth (`Isobroth`, Oxoid USA) in
250 ml flasks. Cultures were incubated at 30.degree. C. with
shaking (100 rpm) for 48 hours. Cells were counted and plated at
1000 cells/1.5 ml in 24-well plates (Falcon) and grown at
30.degree. C., without shaking, for 48 hours. 5 .mu.l of these log
phase cells were used to inoculate 1 ml cultures (Isobroth)
containing varied concentrations of nucleoside analog. Thereafter,
every 2-4 days cells were transferred, either 5 .mu.l per well, or
1-3 .mu.l using a multi-pronged replicator into fresh 1 ml broth
containing AZT. Remaining cells were pelleted and stored at
-80.degree. C. until processed for DNA analysis.
[0298] For analysis of macronuclear DNA from vegetative cultures
containing the nucleoside analogs Ara-G (Calbiochem), ddG
(Calbiochem), or ddI (Calbiochem), or controls lacking analog,
stock cultures were grown overnight in 2% PPYS as described. Cells
were counted and plated at 100 cells/2 ml in 2% PPYS containing
varied amounts of analog, 1% DMSO (Fisher) (as a control for ddG
and Ara-G), or 2% PPYS alone. Cells were replica plated into fresh
medium every 2-6 days, and remaining cells were pelleted and stored
at -80.degree. C. until processed for DNA analysis.
[0299] For analysis of macronuclear DNA from vegetative cultures
containing d4T (Sigma) or control lacking the analog, stock
cultures (SB210 VI) were grown overnight in Isobroth as described.
Cells were then counted and duplicate cultures inoculated at 500
cells/5 ml Isobroth in 50 ml conical tubes, and grown at 30.degree.
C., shaking 80 rpm. 500-2000 cells were transfered to fresh broth
every 2-4 days, and the remainder pelleted and stored at
-80.degree. C. until processed for DNA analysis.
[0300] For analysis of rDNA from cells conjugated in the presence
of nucleoside analogs, 50 ml overnight cultures (2% PPYS) were
starved by pelleting cells and resuspending in an equal volume of
Dryl's solution before returning to 30.degree. C. shaking (100 rpm)
incubator for 18 hours. (1.times.Dryl's solution=0.5 g Na citrate,
0.16 g NaH.sub.2PO.sub.4H.sub.2O, 0.14 g Na.sub.2HPO.sub.4 per
liter, plus 15 ml of 9.98 g CaCl.sub.2.2H.sub.2O/500 ml). Cells
were then counted and equal numbers mixed before pelleting (6
minutes in an IEC tabletop centrifuge, 3/4 speed), and resuspended
in Dryl's to 1.5-2.times.10 .sup.6/ml. Cells were plated at an
average density of 1.5 cells/well into 6-well plates (Falcon) and
allowed to conjugate 6 hours, 30.degree. C. without shaking.
Mock-conjugated SB210 cells were treated identically but not mixed
with PB9R cells. At 6 hours the cultures were checked for pairing
(>90%, except SB210 controls) and either 1 ml Dryl's solution or
2% PPYS containing the nucleoside analog (Acyclo-G purchased from
Sigma) or no added drug as control were added slowly with gentle
swirling. Cultures were returned to 30.degree. C. for an additional
18 hours before being harvested for DNA analysis.
[0301] For analysis of vegetative growth and macronuclear DNA from
single-cell cultures containing the nucleoside analogs AZT or
Ara-G, SB210 (VI) cells were grown from stationary stock cultures
overnight at 30.degree. C. with shaking (100 rpm) in 50 ml 2% PPYS
or Isobroth. Cells were counted and added to the appropriate medium
plus analog (Ara-G to 1 mM or DMSO to 1% as control in 2% PPYS; AZT
to 10 .mu.M or 1 mM, or no addition as control in Isobroth) and
plated in 96-well plates (Falcon), 100 .mu.l per well at a density
of 1 cell per well. 5 plates were prepared for each analog or
control. Wells were scored for cell growth and plates were replica
plated every 1-2 days (Ara-G and DMSO plates) or every 2-4 days
(AZT and Isobroth control plates) to maintain approximate
inoculation densities of 1-10 cells per well for each passage.
Occasionally individual wells were passaged by hand (1 .mu.l
inoculated per well using a pipettor) into several blank wells, to
expand the number of live wells per plate as single-cell cultures
were lost over time due to low probability of being transferred at
each passage. After passaging, cells were pooled, pelleted and
stored at -80.degree. C. until processed for DNA analysis.
[0302] Total cellular DNA was prepared essentially as described by
Larson 50 Cell, 477, 1987, except that the Hoechst 33258-CsCl
gradient purification step was omitted.
[0303] Restriction digests, agarose gel electrophoresis, transfer
of DNA to Nytran filters (Schleicher and Schuell), and
hybridization with .sup.32P-nick-translated or random-primed probes
were carried out using standard procedures (Maniatis et.al. 1989).
Telomere length was analyzed as described previously for
Tetrahymena [Larson 50 Cell, 477, 1987].
[0304] For analysis of cycloheximide (CHX) sensitivity of cells
conjugated in the presence of analog, 50 ml cultures of each cell
type were grown overnight in 2% PPYS, starved in Dryl's for 18
hours, mated (5.times.10.sup.5cells/ml) for 6 hours, then analog
was added. Cells were allowed to complete mating in the presence of
the analog. Twenty-four hours after mixing, cells were diluted in
Dryl's solution, counted and plated at 1 cell per well of 96-well
plates in 1% PPYS without analog. Cells were grown for 4 days in a
humid chamber at 30.degree. C., without shaking. Cells were then
replica plated into 1% PPYS plus 15 .mu.g/ml cycloheximide, allowed
to grow for four days before scoring, and percent of CHX-resistant
wells was calculated. Because generation of progeny expressing the
cycloheximide marker requires successful production of a new
macronucleus, cells whose macronuclear development was disrupted by
the analog are killed in CHX.
[0305] For PCR analysis of the llkb form of the rDNA from cultures
conjugated in the presence of analog, 1.25 .mu.M each of the
telomeric primer (C.sub.4A.sub.2).sub.4 and a 25-mer rDNA primer
(5' GTGGCTTCACACAAAATCTAAGCGC 3') located 1371 nucleotides from the
5' end of the rDNA were used in a "hot start" reaction containing 1
mM MgCl.sub.2, 0.2 mM each dNTP, 1.times.PCR reaction buffer
(Perkin Elmer Cetus), and 0.5 .mu.l Amplitaq polymerase (Perkin
Elmer Cetus). Sample DNA and polymerase were kept separate by the
use of Ampliwax PCR Gem 100 wax beads (Perkin Elmer Cetus),
following manufacturer's instructions. The samples were heated to
95.degree. C. for 1 minute, and then cycled 40 rounds in a
Perkin-Elmer thermocycler as follows: 1 minute at 94.degree. C., 30
seconds at 58.degree. C., 3 minutes at 68.degree. C. Identical
reactions were done using 3' micronuclear rDNA primers, 9610
nucleotides from the 5'end, and (5'
CAATAATGTATTAAAAATATGCTACTTATGCATTATC 3'), 10300 nucleotides from
the 5' end.
[0306] Synthetic oligomers were prepared as described Greider 43
Cell, 405, 1985. Extracts were prepared as described by Blackburn
et.al., 31 Genome 553, 1989.
[0307] A standard assay contained 50% by volume of heparin-agarose
purified telomerase, 25 .mu.M TTP, 1.25 .mu.M .sup.32P-labeled dGTP
(400 Ci/mMol, Amersham), 1 .mu.M oligo (either
(T.sub.2G.sub.4).sub.4 or (T.sub.2G.sub.4).sub.2 mixed with water
and heated at 90.degree. C. for two minutes and cooled at
30.degree. C. for 10 minutes), and 0.1 .mu.l RNasin (40 U/ml,
Promega) in a no-salt buffer. AZT-triphosphate was obtained from
Burroughs Wellcome, N.C. Ara-G-triphosphate was purchased from
Calbiochem and ddNTPs from Sigma. Reaction mixes were kept on ice
until ready for use, and then mixed into tubes containing analog
for incubation at 30.degree. C. Reaction times were thirty minutes.
Reaction rates under these conditions were determined previously to
be linear over time for thirty minutes. Identical reactions were
run without primers as controls. The reactions were then processed
essentially as described by Greider and Blackburn 337 Nature, 331,
1989. For quantitative assays, aliquots of the reaction mixture
were spotted in triplicate onto DE81 paper and washed as described
Greider 43 Cell, 405, 1985. Incorporation of .sup.32P label from
either .sup.32P-TTP or .sup.32P-dGTP was measured to monitor the
reaction rate. For visualization of the elongation reaction
products, samples were heated to 95.degree. C. for 2 minutes and
cooled on ice before loading onto a 12% polyacrylamide/8 M urea
gel.
[0308] The model for the mechanism of the telomerase
ribonucleoprotein enzyme from Tetrahymena is shown in FIG. 18A. The
enzyme synthesizes TTGGGG repeats onto the 3' end of a suitable DNA
primer by copying a template sequence in the RNA moiety of the
enzyme. For ease of reference in discussing the results, the
residues in the template region are numbered 1 to 9 (5' to 3' along
the RNA). The standard telomerase assay used in this example
consists of incorporation of dGTP and TTP substrates, one
triphosphate .sup.32P-labeled, into synthesized DNA in the reaction
shown in FIG. 18A. For the experiments discussed in this example we
used as the DNA primer either 1 .mu.M (T.sub.2G.sub.4).sub.4 or
(T.sub.2G.sub.4).sub.2, under conditions in which the overall rate
of incorporation of label was determined previously to be linear
over time. Incorporation of .sup.32P label from either .sup.32P-TTP
or .sup.32P-dGTP was measured to monitor the reaction rate, and the
distributions of elongation products were analyzed by denaturing
polyacrylamide gel electrophoresis.
[0309] The effect of adding increasing amounts of AZT-triphosphate
(AZT-TP) to the standard assay for telomerase activity is shown in
FIG. 19A. A series of control reactions using unlabeled TTP added
at the same concentrations as the AZT-TP was run in parallel (FIG.
19A). The unlabeled TTP inhibits incorporation of the
.sup.32P-labeled TTP by simple competition. Quantitation of label
incorporated into product in this experiment enabled us to
determine the K.sub.m for TTP to be -5 .mu.M. Compared with
addition of unlabeled TTP competitor, AZT-TP had only a modest
quantitative effect on the incorporation of .sup.32P-labeled TTP
(FIG. 19A). Since AZT incorporation leads to chain termination,
this result indicates that AZT-triphosphate competes less
efficiently for telomerase than TTP. Similar results were obtained
when incorporation of .sup.32P-dGTP was monitored (FIG. 19B), with
50% inhibition occurring at -80 .mu.M AZT-TP.
[0310] In similar experiments in which increasing concentrations of
arabinofuranyl-guanosine triphosphate (Ara-GTP) were added to the
reaction, significant reduction of overall incorporation occurred
even at low concentrations of the analog (FIG. 19C). From parallel
experiments in which unlabeled dGTP was added as competitor (FIG.
19C), the K.sub.m for dGTP under these reaction conditions was
found to be 1-2 .mu.M. 50% inhibition occurred with 0.7 .mu.M
Ara-GTP; thus Ara-GTP potentially competes as well as unlabeled
dGTP for .sup.32P-dGTP. However, as incorporation of Ara-G causes
chain termination, each Ara-G incorporated is expected to have a
greater impact on total incorporation than competition with
unlabeled dGTP.
[0311] We also tested the effects of dideoxynucleoside
triphosphates (ddNTPs) on the telomerase reaction. As shown
previously for telomerase [Greider 43 Cell, 405, 1985], and as is
the case for many other reverse trancriptases, ddNTPs are
recognized by the enzyme and incorporated, causing chain
termination with a subsequent shift in banding patterns and
reduction of the average product length. Consistent with previous
qualitative analyses of Tetrahymena and human telomerases [Greider
43 Cell, 405, 1985; Morin 59 Cell, 521, 1989], ddGTP and ddTTP each
inhibited the incorporation of labeled .sup.32P-NTP into elongation
products (FIG. 19D and E). ddGTP was a much more efficient
inhibitor than ddTTP: under these reaction conditions 50%
inhibition occurred at <0.1 and 5 .mu.M ddGTP and ddTTP
respectively. As observed previously for Tetrahymena telomerase
[Greider 43 Cell, 405, 1985], no significant effects were seen with
either ddCTP or ddATP. In addition, ddITP inhibited telomerase
(FIG. 19E), although less efficiently than ddGTP, with 50%
inhibition occurring at 3 .mu.M ddITP.
[0312] The size distribution of labeled products was then analyzed
by denaturing polyacrylamide gel electrophoresis. Consistent with
the expectation for a chain-terminator, the proportion of longer
telomerase products was decreased in the presence of AZT compared
with cold TTP competitor controls (FIG. 20A; compare lanes 1 and 2
with lanes 3 to 5), and in the presence of Ara-G (FIG. 20A; lanes 7
and 8). Average product length also decreased in the presence of
Ara-GTP, ddGTP and ddITP (FIG. 20A and B). In addition, each
nucleoside triphosphate analog produced distinctive and
characteristic patterns of chain termination, as shown by analysis
of the shifts in the banding patterns of the elongation products.
With AZT-triphosphate, we saw increased relative intensities of the
bands corresponding to the incorporation of T residues (copying the
A residues at positions 2 and 3 on the template RNA (see FIG.
18A)). This change in banding pattern is consistent with simple
chain termination, which is predicted to increase the intensity of
bands corresponding to the position of both incorporated T
residues. Similar effects were seen with ddTTP. We interpret this
to mean that AZT-triphosphate was recognized by the enzyme and
incorporated into the correct positions in the growing telomeric
sequence, causing chain termination. However it cannot be excluded
that the increase in relative intensity of the band corresponding
to position 3 on the template, which precedes addition of the
second T, is also attributable to pausing caused by competition
with TTP and a slower reaction rate with AZT-triphosphate at
position 2.
[0313] The results with Ara-GTP were also consistent with
incorporation of Ara-G and consequent chain termination (FIG. 20A,
lanes 7 and 8). Although there are four positions at which a G
residue can be incorporated and therefore at which chain
termination could occur, the strongest increase was in the band
corresponding to the G residue specified by position 4, in the
middle of the telomerase RNA template (see FIG. 18A). With ddGTP,
chain termination appeared to occur most efficiently at positions 6
and 5 (FIG. 20B, compare lane 1 with lanes 4 to 6), and with ddITP,
at position 5 (lanes 7 to 9).
[0314] FIG. 18B summarizes schematically the effects of the various
triphosphate analogs on polymerization at each of the six positions
along the template. There was no correlation between the efficiency
of a nucleoside analog as an inhibitor and the position of its
maximal chain termination on the template. For example, the potent
inhibitors ddg-and Ara-G-triphosphates cause maximal chain
termination at different positions on the telomerase RNA template
(5 and 6 for ddG, and 4 for Ara-G).
[0315] In addition to nucleoside triphosphate analogs expected to
act as chain terminators, we also tested rifampin, an inhibitor of
bacterial RNA polymerase, and streptomycin sulfate. Streptomycin
sulfate is known to inhibit the activity of group I self-splicing
introns at high concentrations [von Ahsen 19 Nucl. Acids Res.,
2261, 1991], and has a guanidino group that might be recognized by
telomerase as part of the enzyme's specificity for G-rich DNA
primers (Greider 51 Cell, 887, 1987). Adding rifampin at
concentrations up to 100 .mu.g/ml did not affect the quantitative
incorporation of label or change the banding pattern of the
elongation products. Streptomycin sulfate at 40 mM dramatically
reduced the amount (FIG. 19F) and average length of elongation
products, with little decrease in activity being seen in a 40 mM
sodium sulfate control. However, unlike the nucleoside triphosphate
analogs, inhibition by streptomycin did not appear to affect
incorporation at specific positions in the repeat. The inhibition
by streptomycin may be useful experimentally as a criterion for
telomerase activity in vitro. However, the significance of the
inhibition by streptomycin is unclear, as it is difficult to rule
out that its effect is the result of nonspecific binding to either
the RNA moiety of telomerase or the DNA primer.
[0316] Because the triphosphate forms of the analogs AZT, Ara-G,
ddT, ddG and ddI each inhibited (with varying efficiencies)
telomerase in vitro, we tested whether supplying each of these
nucleoside analogs in the cell growth medium caused in vivo changes
in telomere length or senescence. Additionally, Acyclo-G and d4T
were tested on conjugating and vegetative cells, respectively.
[0317] Previous work with Tetrahymena showed that at least one
alteration of the telomerase RNA causes telomere shortening and
cellular senescence [Yu 344 Nature 126, 1990]. To test whether such
a phenotype could be produced by inhibitors of telomerase in
Tetrahymena, duplicate log-phase phase cultures were grown for
prolonged periods in the presence of varying concentrations of
analogs. The growth and cell morphology of these cultures were
monitored, and DNA was isolated at different times for telomere
length analysis. AZT at 5 or 10 mM added to Isobroth medium
strongly inhibited cell growth and killed cells within a day, and
thus at these concentrations acted in a manner suggestive of
immediate toxicity to cells, rather than of senescence. AZT added
to Isobroth medium at lower concentrations (up to 1 mM) did not
result in senescence of cultures maintained by subculture of
-10.sup.3 cells per transfer, over a 50-day period of continuous
growth and subculturing of these cell cultures. From growth rate
measurements it was calculated that the cells went through 150 to
250 cell generations in the course of this 50 day period. In
similar mass transfer experiments no effects on cell doubling rate,
morphology or long term viability were obtained with cells grown in
2% PPYS plus up to 2 mM Ara-G, the highest concentration tested
that did not cause immediate toxicity.
[0318] Telomere lengths in cells grown in the presence of the
different analogs were monitored by Southern blot analysis of DNA
samples extracted at a series of time points during the
subculturings. The telomeres of cells grown vegetatively in 1 and 5
mM AZT in 2% PPYS medium were reproducibly shortened by up to an
average of 170 base pairs compared with the control cultures grown
in 2% PPYS in the absence of the drug (FIG. 21A and B). This
shortening of telomeres occured in a concentration-dependent manner
(FIG. 21B), with at least 50% of the maximal shortening effect
occurring by 10 .mu.M AZT, the lowest concentration tested. For
each AZT concentration tested, the full decrease was seen within 3
days of culturing in the presence of the drug (15 to 30 cell
divisions), but after this initial length adjustment, at each drug
concentration telomeres thereafter showed no statistically
significant shortening over time, and mean telomere length
consistently remained static for at least 28 days of mass transfer
subculturing.
[0319] Similar degrees and timing of telomere shortening were
produced with 1 or 2 mM Ara-G added to 2% PPYS culture medium (FIG.
21C). d4T added to Isobroth culture medium in concentrations
ranging from 10 .mu.M to 1 mM produced shortened telomeres at 100
.mu.M and 1 mM, again in a concentration dependent manner, after 5
days (16 generations) in culture. In contrast, up to 1 mM ddG or
ddI produced no changes in telomere length compared with control
cultures, over a period of 5 days of subculturing (15-20 cell
generations) in 2% PPYS medium.
[0320] Because we had found previously that telomerase is strongly
inhibited in vitro by at least some of the analogs tested, and
telomere length is affected in vivo within an estimated 15 to 30
cell generations by these analogs, it was possible that telomere
addition was in fact being disrupted in vivo, but that our failure
to find any evidence of progressive telomere shortening or
senescence was attributable to a subset of the cell population that
escapes an inhibitory effect of the analog on telomerase. We have
shown previously that impairing telomerase in vivo by mutating the
telomerase RNA produced senescence in most cells, but only
-10.sup.2 single cell subclones were analyzed in these experiments,
[Yu 344 Nature 126, 1990]. Under our mass transfer subculturing
regime, in which about 10.sup.3 cells were transferred per passage,
if a fraction as small as -1% of the cells escaped senescence, and
if their growth advantage was sufficiently high compared with cells
losing telomeres, they could become the predominant population in
any cell passage and we would not have detected any phenotype.
[0321] To test whether we had missed such a subpopulation of cells,
we carried out the same experiments on vegetatively dividing
Tetrahymena cells in the presence and absence of drug, but in these
experiments the subculturing was carried out by plating cells at an
average of 1 to 10 cells per well in microtiter plate wells in the
presence of 10 .mu.M and 1 mM AZT, and 10 .mu.M and 1 mM Ara-G. For
each drug, cells were plated out in this manner for 30 consecutive
days (90 to 150 cell generations) and 16 consecutive days (50 to 80
cell generations) respectively for the 10 .mu.M and 1 mM drug
concentrations. DNA was isolated at intervals from combined
samplings of the wells for analysis of telomere length.
[0322] Compared with control medium lacking the nucleoside analog,
no changes in the plating efficiency were observed over the course
of the experiment for cells grown in 10 .mu.M AZT and 10 .mu.M or 1
mM Ara-G. However, in the presence of 1 mM AZT, monitoring growth
rates of cells maintained in this way by single cell transfers
allowed us to identify two general growth classes, which we
designated as slow (0 to 1 cell doubling per day) and fast (2 to 4
cell doubling per day). The growth rate of fast cells was similar
to that of the controls grown in Isobroth containing no AZT. Over
time, the proportion of wells with slow cells decreased, as would
be expected if they simply had a lower probability of being
transferred, since they were present in lower cell densities than
fast cells, which grew to higher cell densities and for which the
timing of the plating protocols had been worked out. However,
monitoring the cells remaining in wells after transfers had been
made from them showed that the slow cells lost viability over time.
In addition, throughout the course of the transfers, slow cells
appeared from formerly fast cell wells. We pooled cells from the
slow growing wells (pooling of several microtiter wells was
necessary to obtain sufficient DNA for Southern analysis) and
compared their telomere length distribution with that of pooled
fast cells. The mean length and size distribution of telomeric DNA
from pooled fast cells were indistinguishable from those of control
cells grown without AZT. In contrast, the pooled slow cell DNA
showed a slight decrease in mean telomere length and heterogeneity
(FIG. 21D). Control cells grown in Isobroth medium had telomeres
that were an average of 165 bp shorter than cells grown in 1% PPYS
medium. We believe that because the telomeric G.sub.4T.sub.2repeat
tracts in cells grown in Isobroth medium are already markedly
shorter than those of cells grown in the richer PPYS medium, the
additional amount of telomere shortening caused by growth in 1 mM
AZT is sufficient to reduce continually and stochastically a
fraction of the telomeres below a critical lower threshold required
for function, thus causing the decreased viability of a
subpopulation of the cells.
[0323] We examined the effects of AZT, Ara-G, Acyclo-G, ddI and ddG
on progeny formation by cells that have undergone conjugation. This
process involves de novo formation of new macronuclear telomeres in
the progeny cells. Mracronuclear development in ciliated protozoans
such as Tetrahymena involves developmentally programmed,
site-specific fragmentation of germline chromosomes into linear
subchromosomes, whose ends are healed by de novo addition of
telomeres. We showed previously that telomerase not only elongates
pre-existing telomeres in vivo during vegetative cell divisions [Yu
344 Nature, 126, 1990], but also functions to directly add
telomeric DNA onto non-telomeric sequences during this
developmentally-controlled chromosome healing. Because of the
immediate requirement for telomere addition to fragmented DNA, it
is possible that the latter process might be more sensitive to
telomerase inhibition than telomere maintenance during vegetative
growth. To test whether nucleoside analogs cause inhibition of
macronuclear development due to a disruption of telomere formation,
we mated two strains of Tetrahymena which are sensitive to
cycloheximide, but whose progeny after mating are resistant to
cycloheximide. Synchronized mated cells were treated with AZT at
concentrations ranging from 10 .mu.M to 5 mM for a period beginning
just prior to when macronuclear development begins and continuing
during macronuclear development (the period 6-24 hours after mating
was initiated). At this point cells were diluted out in microtiter
plate wells in fresh medium lacking the analog, at an average cell
density of one cell per well, and allowed to grow for the minimum
period before selection for cells that had successfully produced
progeny. In attempts to maximize the effect of AZT, cells were
either refed at 6 hrs with 2% PPYS or Isobroth, or starved until 24
hrs (the duration of the AZT treatment). Such starvation arrests
macronuclear development at an intermediate stage. When refed,
macronuclear development would then be forced to proceed in the
presence of the AZT. Control, unmated parental cells were also
plated and exposed to drug. Similar experiments were performed with
Ara-G, Acyclo-G, ddI and ddG. The results are shown in Table 4.
[0324] The control plates showed 99%-100% cell death in CHX, while
the majority of cells that were mated with or without analog
survived. None of the nucleoside analogs had any statistically
significant effect on progeny formation. The design of this
experiment would prevent takeover of the culture by a minority
population that evaded the effects of the drug, as described above.
Therefore little or no irreversible disruption of macronuclear
development due to impaired telomerase activity and telomere
formation occurred in the presence of AZT, Ara-G, Acyclo-G, ddG, or
ddI.
[0325] Although macronuclear development was not significantly
disrupted, analysis of the formation of a marker for telomere
addition during macronuclear development suggests that AZT reduces
the efficiency of telomere addition.
[0326] DNA from cells mated in the presence or absence of analog,
and either refed at 6 hours or starved fully for the duration of
conjugation were used in PCR with a telomeric primer and a 5' rDNA
primer. This selected for a fragment of the llkb rDNA to which
telomeres had been added. The 11 kb rDNA is either a by-product of
the 21 kb rDNA formed during macronuclear development or an
intermediate of this process. It is present only transiently during
new macronuclear development and as such is a good marker for
telomere addition in vivo. Knock-down of relative amounts of the
1400 nucleotide PCR-generated fragment from 11 kb-rDNA was seen in
DNA from cells conjugated in the presence of AZT, but not in those
containing Ara-G, Acyclo-G, H.sub.2O or DMSO controls or in
mock-conjugated SB210 cells. To show that the DNA used in the PCR
reactions was present and competent for PCR, identical reactions
were run using primers from the 3'-micronuclear copy of the rDNA.
In all samples the expected 810 nucleotide fragment was produced in
substantial quantities (FIG. 22), indicating that the decrease in
the 1400 nucleotide telomere-containing PCR product in samples from
cells mated in AZT is due to the presence of analog rather than
contaminants in the DNA or reagents. Southern blotting with a
5'-rDNA probe confirmed that the telomere-containing PCR product
was from the expected rDNA sequence, (FIG. 22B) and no
cross-hybridization occurred to the 3' PCR product. An overall
decrease in telomere-containing PCR products was seen in all
samples that were re-fed at 6 hours post-mixing, but the decrease
was more pronounced in samples that had been mated in the presence
of AZT.
6TABLE 6 Effects of nucleoside analogs on progeny formation. CELL
TREATMENT # CHX-R # TOTAL % CHX-R SB210 (NOT MATED) 1 215 0.5 PB9R
(NOT MATED) 3 307 1 AZT (mM) 0 139 212 66 0.01 121 169 72 0.1 100
148 68 1.0 91 166 55 5.0 75 120 63 SB210 (NOT MATED) 0 57 0 AZT
(mM) 0 165 214 77 0.01 67 92 73 0.1 128 190 67 1.0 60 125 48 5.0 89
168 53 1% DMSO 84 109 77 ARA-G (mM) 0.01 114 141 81 0.1 134 167 80
1.0 89 161 55 1% DMSO 51 75 68 ARA-G (mM) 1.0 51 86 59 2.0 40 92 43
SB210 (NOT MATED) 0 9 0 PB9R (NOT MATED) 0 37 0 ddI (mM) 0 63 75 84
0.001 59 71 83 0.01 85 96 89 0.1 83 106 78 1.0 100 110 91 1% DMSO
21 44 48 ddG (mM) 0.001 86 102 84 0.1 73 86 85 1.0 51 66 77
ACYCLO-G (mM) 0 36 45 80 0.017 80 107 75 0 78 116 67 0.017 101 146
69
Example 14
G-Reaction for Reducing the Size of the Terminal Restriction
Fragment
[0327] Human fibroblast DNA digested with restriction enzymes,
electrophoresed, and hybridized by Southern blot makes possible the
resolution of terminal restriction fragments (TRFs) which in turn
reflect the relative length of telomeric repeat sequences (See FIG.
26, HinfI digested DNA, labeled "HinfI"; DNA not digested, labeled
"O"). This Southern analysis is complicated by the fact that human
and many other species have long stretches of subtelomeric
repetitive sequences that add to the TRF size. As a means of
eliminating the artifactual inclusion of this subtelomeric repeats
in a measurement of telomeric repeat length, a modified
Maxam-Gilbert reaction is employed to hydrolyze the DNA at G
residues. In the lane labeled "P only" (underloaded) the DNA is
treated with piperidine in mild conditions which does not in itself
decrease the size of the DNA. In the lane labeled "P+DMS" the
samples are pretreated with DMS. Not the substantial reduction in
TRF size compared to the HinfI digest relecting the deletion of
subtelomeric sequences in the C-rich strand containing G residues.
All lanes were probed with (TTAGGG).sub.3. This assay is thus
useful for analysis of telomere lengths in diagnostic
procedures.
Example 15
Fungal Telomeres
[0328] The following example illustrates various specific telomeric
sequences which can be used to identify specific fungi. Those in
the art will recognize that such sequences can be probed with
oligonucleotides to specifically diagnose the presence of a
selected fungus. In addition, specific treatment of fungi can be
effected by use of agents which bind to such sequences and reduce
the long term viability of the fungal cell.
[0329] As described herein telomeric DNA is an attractive target
for specific drug therapy. Telomeres are short single-stranded
protrusions which are accessible to specific drugs. Binding by such
drugs will interfere with normal telomere function and thus fungal
cell viability. In similar experiments (routine to those in the art
when conducted as described herein) inhibitors or facilitators of
such telomere replication (or telomerase activity) can be
discovered and used as anticancer, antiparasite and antifungal
agents.
[0330] The significantly increased length of fungal telomeres makes
them ideal targets for antisense therapy or diagnosis. In addition,
this different telomere structure indicates a different mechanism
of action of the telomerase, and thus its availability as a target
for antifungal agents which are inactive on human or other animal
cells.
[0331] Telomeric DNA sequences have generally been found to be
remarkably conserved in evolution, typically consisting of
repeated, very short sequence units containing clusters of G
residues. Recently however the telomeric DNA of the budding yeast
Candida albicans was shown to consist of much longer repeat units.
Here we report the identification of seven additional new telomeric
sequences from budding yeasts. Although within the budding yeasts
the telomeric sequences show more phylogenetic diversity in length
(8-25 bp), sequence and composition than has been seen previously
throughout the whole phylogenetic range of other eukaryotes, we
show that all the known budding yeast telomeric repeats contain a
strikingly conserved 6 bp motif of T and G residues resembling more
typical telomeric sequences. We propose that G clusters in
telomeres are conserved because of constraints imposed by their
mode of synthesis, rather than by a fundamental requirement for a
specific common structural property of telomeric DNA.
[0332] The DNA sequences of telomeres, the ends of eukaryotic
chromosomes, have been found previously to be conserved even
between very diverse eukaryotes, typically consisting of tandem
arrays of 5-8 bp repeating units characterized by clusters of G
residues, producing a marked strand composition bias. However, the
telomeric repeats of the opportunistic pathogen Candida albicans
were shown to consist of homogeneous repeats of a 23 bp sequence
that lacks any noticeable strand composition bias.
[0333] To determine the relationship of the apparently exceptional,
complex telomeric repeat sequence of Candida albicans to the more
usual, simple telomeric sequences, genomic DNA from budding yeast
species related to both C. albicans and S. cerevisiae were analyzed
by Southern blotting, using cloned C. albicans telomeric repeats as
the hybridization probe. Under low-stringency hybridization
conditions we detected multiple cross-hybridizing bands in several
species FIG. 28. In some cases, the cross-hybridizing bands clearly
were broad, a characteristic feature of telomeric restriction
fragments caused by different numbers of telomeric repeats in
individual telomeres among a population of cells.
[0334] Telomere-enriched libraries were constructed from genomic
DNA from seven budding yeast species and strains. Telomeric clones
were identified by their ability to hybridize to known yeast
telomeric repeats (either the 23 bp C. albicans repeat or the
TG.sub.1-3 repeat of S. cerevisiae), or by screening for end-linked
repetitive DNA sequences without the use of a specific probe.
Sequencing putative telomere fragment inserts from seven species
identified clones that contained tandem repeats with unit lengths
of 8-25 bp. With a single exception, the repeats showed no sequence
variations within a species. In every case the repeat array was
present at the very end of the insert, directly abutting vector
sequences, as would be expected for cloned telomeres. The
repeat-containing clone from each species hybridized back to the
same pattern of restriction fragments observed originally with the
C. albicans or the S. cerevisiae probe used for library screening.
Most of the bands were preferentially sensitive to Bal31 nuclease
(FIG. 29) indicating that the bulk of the repeat sequences are
present at the ends of chromosomes. The lengths of the tracts of
repeats cloned from the different yeast species were typically
between 250-600 bp, although those from the two C. tropicalis
strains were only 130-175 bp. That this species has particularly
short telomeres is also supported by their very rapid loss during
Bal31 digestion and by the relatively weak hybridization, even with
species-specific telomere probes.
[0335] FIG. 30 shows an alignment of these newly discovered
telomeric repeat unit sequences together with those of C. albicans
and S. cerevisiae. Two striking features are apparent: the much
greater variety of the budding yeast telomeres, with respect to
repeat unit lengths and sequence complexities, compared to other
eukaryotes, and a conserved six-base cluster of T and G residues
that most resembles typical telomeric sequences.
[0336] The sequence relationships among the telomeric repeats are
generally consistent with the phylogenetic relationships of these
budding yeasts. The telomeric repeats of the two C. tropicalis
strains differ by only a single base polymorphism. The 25 bp
telomeric repeats of the closely related K. lactis and C.
pseudotropicalis differ at only one position. The telomeric repeat
sequences from C. albicans, C. maltosa, C. pseudotropicalis, C.
tropicalis and K. lactis are 23-25 bp in length, with differences
largely or entirely confined to the central part of the repeat. The
16 bp repeat unit from C. glabrata, the species in this study that
may be most closely related to S. cerevisiae, is very G-rich, which
probably contributes to its cross-hybridization to the
heterogeneous and smaller S. cerevisiae telomeric repeats. All the
budding yeast sequences, including the irregular S. cerevisiae
repeats, have a perfect or 5/6 match to a 6 bp T/G sequence
(boxed).
[0337] In the cloned telomere from C. tropicalis strain B-4414, we
found two telomeric repeat sequences that differed at the second
base position of the repeat, as shown in FIG. 30 repeat units in
the B-4414 telomere were homogeneous (and will be termed the "AC
repeat"), but the remaining repeat (henceforth termed the "AA
repeat") was identical to the homogeneous telomeric repeats cloned
from strain C. tropicalis B-4443.
[0338] To determine the distribution of these variant repeats among
the telomeres and strains of C. tropicalis, genomic DNA from
several C. tropicalis strains including B-4414 and B-4443, and a
control C. albicans strain were probed with oligonucleotide probes
specific for either the AA or the AC repeat (FIG. 31 left panel).
Only strains B-4414 and 1739-82, and to some extent the C. albicans
telomeres, hybridized with the AC repeat-specific oligonucleotide
probe (FIG. 31 left panel). However, genomic DNA from all of the C.
tropicalis strains tested, including B-4443, but not from C.
albicans, hybridized well with the oligonucleotide specific for
"AA" repeats (FIG. 31 right panel). These results clearly indicate
that both B-4414 and 1739-82 contain at least two forms of
telomeric repeats, which are most likely variably interspersed in
different telomeres, as signal rations with the two probes differed
between individual telomeric fragments (FIG. 31A and B, lanes 1 and
2).
Example 16
Effects of Telomerase Inhibitors on Human Tumor Cell Growth
[0339] Agents that were shown to inhibit telomerase from
Tetrahymena e.g., AZT, ddG, and ara-G were tested to determine
their effect on human telomerase activity, telomere repeat length,
and cell growth immortality. Of the compounds tested ddG and ara-G
were effective inhibitors of human telomerase obtained from the
tumor cell line 296. The data for ddG is shown in FIG. 27. The
effect of the agents on telomerase activity in intact cells was
then studied utilizing the lymphoma cell line JY 616 which were
maintained in RPMI 1640 with 0.25M Hepes, 10% FCS, and
penicillin/streptomycin (Gibco). The cells were cultured in 6-well
plates (Falcon) with 5.0 ML of medium per well in duplicate. Cells
were passaged every 7-10 days which corresponded to 5-7 mean
population doublings (MPD), and seeded at 3.times.10.sup.4 cells
per well into fresh medium containing analog or control. Cell
viability was monitored prior to harvest utilizing trypan blue
stain (Gibco) during counting with a hemocytometer. The average
ratio of stained: unstained cells (dead:alive) was >90%. The
intactness of the DNA was measured on a parallel gel by observing
its mobility in a gel prior to digestion by a restriction
enzyme.
[0340] As seen in FIG. 23, all JY cells grew in an immortal fashion
in the presence of a low concentration of the potential telomerase
inhibitors. At high concentrations (FIG. 24), the cells ceased
proliferating in the presence of 50 .mu.M AZT and displayed a
slowed growth in the presence of 20 .mu.M ara-G. In support of the
belief that this inhibition of cell growth in the presence of 50
.mu.M AZT, is due to telomerase inhibition, is the observation that
the cells grew at a normal rate until week 3 and then ceased
dividing. This is the effect one would expect if the inhibition of
cell growth was via telomerase inhibition (i.e., the cells require
multiple rounds of cell division to lose their telomeric repeats).
Also in support of the belief that AZT inhibited the growth of the
cells via the inhibition of telomerase is the finding shown in FIG.
25 where compared to week 1, and week 3 where the cells stopped
dividing, the AZT treated cells had a marked decrease in mean
telomere length compared to the control medium "R" at the same
time.
[0341] In addition, 10 .mu.M ddG was shown to cause a decrease in
telomere length compared to the control (in this case a DMSO
control). In FIG. 32 it can be seen that JY cells studied in a
manner similar to that described above, and treated with ddG,
showed a markedly shorter telomere repeat length after 9 and 10
weeks compared to the DMSO control. It should be noted that while
JY cells are immortal, when cultured under the conditions
described, they lose some telomeric repeats over 10 weeks. The
addition of ddG markedly accelerated this loss.
Example 17
Assay for Telomerase Inhibitors Utilizing Human Telomerase
[0342] The following is a micro assay for the rapid screening of
potential inhibitors of human telomerase. The reaction consists of
the following components (given as a final concentration in 10
.mu.l reaction volume): 1.times.Human Telomerase Buffer (HTB; 50 mM
Tris.Cl pH 7.5, 1 mM Spermidine, 5 mM .beta.-mercaptoethanol (BME),
1 mM MgCl.sub.2, 50 mM potassium acetate (K-OAc), 2 mM DATP, dTTP,
0.625 .mu.M [.sup.32P]-dGTP (800 Ci/mmol, 10 mCi/ml stock), 1 .mu.M
(TTAGGG).sub.3, 1 .mu.l test compound, 5 .mu.l of 1:5 diluted 293
S-100 extract, and diethylpyrocarbonate(depc)-treated H.sub.2O to
10 .mu.l.
[0343] The HTB, dATP, dTTP, 293 S-100 diluted extract, oligo and
depc H.sub.2O are combined, and aliquots are distributed to
individual wells of a 96-well microtiter plate. Different
concentrations of the test compound are added to the wells and the
plate is preincubated at 30.degree. C. for 20 minutes to allow for
inhibition to occur. The [.sup.32P]-dGTP is then added to the
wells, and the reactions are incubated at 30.degree. C. for a
further 30-60 minutes. At the end of this time, duplicate aliquots
are spotted onto DE81 filter paper and allowed to dry. The filter
paper is then washed extensively in 0.5M Na.sub.2HPO.sub.4 to wash
away the unincorporated label, and briefly rinsed in water before
drying. The filter is then exposed to a phosphorimaging screen for
30-60 minutes, and the samples are quantitated.
[0344] By comparing the sample signal to the RNase pretreated
controls and the no extract controls, a percent decrease in
telomerase-generated signal can be calculated. Any sample causing a
change in the amount of radioactivity detected is electrophoresed
on a DNA sequencing gel to confirm inhibition and to observe the
effect of the agent on telomerase processivity. This will include
determining if the inhibitory effect is a direct effect on the
telomerase enzyme, or on one of the other components in the
reaction, by performing titrations of these components and
observing the percent inhibition. When a direct in vitro effect on
telomerase is detected, the inhibitor is then be tested on cultured
cells and in animal models such as tumor-bearing nude mice. As seen
in FIG. 27, ddG was shown to inhibit human telomerase in a
dose-dependent manner. Similar results were obtained with
ara-G.
[0345] Compositions
[0346] Compositions or products according to the invention may
conveniently be provided in the form of solutions suitable for
parenteral or nasal or oral administration. In many cases, it will
be convenient to provide an agent in a single solution for
administration.
[0347] If the agents are amphoteric they may be utilized as free
bases, as acid addition salts or as metal salts. The salts must, of
course, be pharmaceutically acceptable, and these will include
metal salts, particularly alkali and alkaline earth metal salts,
e.g., potassium or sodium salts. A wide variety of pharmaceutically
acceptable acid addition salts are available. These include those
prepared from both organic and inorganic acids, preferably mineral
acids. Typical acids which may be mentioned by way of example
include citric, succinic, lactic, hydrochloric and hydrobromic
acids. Such products are readily prepared by procedures well known
to those skilled in the art.
[0348] The agents (and inhibitors) of the invention will normally
be provided as parenteral compositions for injection or infusion.
They can, for example, be suspended in an inert oil, suitably a
vegetable oil such as sesame, peanut, or olive oil. Alternatively,
they can be suspended in an aqueous isotonic buffer solution at a
pH of about 5.6 to 7.4. Useful buffers include sodium
citrate-citric acid and sodium phosphate-phosphoric acid.
[0349] The desired isotonicity may be accomplished using sodium
chloride or other pharmaceutically acceptable agents such as
dextrose, boric acid, sodium tartrate, propylene glycol or other
inorganic or organic solutes. Sodium chloride is preferred
particularly for buffers containing sodium ions.
[0350] If desired, solutions of the above compositions may be
thickened with a thickening agent such as methyl cellulose. They
may be prepared in emulsified form, either water in oil or oil in
water. Any of a wide variety of pharmaceutically acceptable
emulsifying agents may be employed including, for example acacia
powder, or an alkali polyether alcohol sulfate or sulfonate such as
a Triton.
[0351] The therapeutically useful compositions of the invention are
prepared by mixing the ingredients following generally accepted
procedures. For example, the selected components may be simply
mixed in a blender or other standard device to produce a
concentrated mixture which may then be adjusted to the final
concentration and viscosity by the addition of water or thickening
agent and possibly a buffer to control pH or an additional solute
to control tonicity.
[0352] For use by the physician, the compositions will be provided
in dosage unit form containing an amount of agent which will be
effective in one or multiple doses to perform a desired function.
As will be recognized by those in the field, an effective amount of
therapeutic agent will vary with many factors including the age and
weight of the patient, the patient's physical condition, the blood
sugar level to be obtained, and other factors.
[0353] Administration
[0354] Selected agents, e.g., oligonucleotide or ribozymes can be
administered prophylactically, or to patients suffering from a
target disease, e.g., by exogenous delivery of the agent to an
infected tissue by means of an appropriate delivery vehicle, e.g.,
a liposome, a controlled release vehicle, by use of iontophoresis,
electroporation or ion paired molecules, or covalently attached
adducts, and other pharmacologically approved methods of delivery.
Routes of administration include intramuscular, aerosol, oral
(tablet or pill form), topical, systemic, ocular, intraperitoneal
and/or intrathecal. Expression vectors for immunization with
ribozymes and/or delivery of oligonucleotides are also
suitable.
[0355] The specific delivery route of any selected agent will
depend on the use of the agent. Generally, a specific delivery
program for each agent will focus on naked agent uptake with regard
to intracellular localization, followed by demonstration of
efficacy. Alternatively, delivery to these same cells in an organ
or tissue of an animal can be pursued. Uptake studies will include
uptake assays to evaluate, e.g., cellular oligonucleotide uptake,
regardless of the delivery vehicle or strategy. Such assays will
also determine the intracellular localization of the agent
following uptake, ultimately establishing the requirements for
maintenance of steady-state concentrations within the cellular
compartment containing the target sequence (nucleus and/or
cytoplasm). Efficacy and cytotoxicity can then be tested. Toxicity
will not only include cell viability but also cell function.
[0356] Some methods of delivery, e.g., for oligonucleotides, that
may be used include:
[0357] a. encapsulation in liposomes,
[0358] b. transduction by retroviral vectors,
[0359] c. conjugation with cholesterol,
[0360] d. localization to nuclear compartment utilizing antigen
binding site found on most snRNAs,
[0361] e. neutralization of charge of oligonucleotides by using
nucleotide derivatives, and
[0362] f. use of blood stem cells to distribute oligonucleotides
throughout the body.
[0363] At least three types of delivery strategies are useful in
the present invention, including: agent modifications, particle
carrier drug delivery vehicles, and retroviral expression vectors.
Unmodified agents may be taken up by cells, albeit slowly. To
enhance cellular uptake, the agent may be modified essentially at
random, in ways which reduces its charge but maintains specific
functional groups. This results in a molecule which is able to
diffuse across the cell membrane, thus removing the permeability
barrier.
[0364] Modification of agents to reduce charge is just one approach
to enhance the cellular uptake of these larger molecules. The
structural requirements necessary to maintain agent activity are
well understood by those in the art. These requirements are taken
into consideration when designing modifications to enhance cellular
delivery. The modifications are also designed to reduce
susceptibility to enzymatic degradation. Both of these
characteristics should greatly improve the efficacy of the
agent.
[0365] Chemical modifications of the phosphate backbone of
oligonucleotides will reduce the negative charge allowing free
diffusion across the membrane. This principle has been successfully
demonstrated for antisense DNA technology. In the body, maintenance
of an external concentration will be necessary to drive the
diffusion of the modified oligonucleotides into the cells of the
tissue. Administration routes which allow the diseased tissue to be
exposed to a transient high concentration of the oligonucleotide,
which is slowly dissipated by systemic adsorption are preferred.
Intravenous administration with a drug carrier designed to increase
the circulation half-life of the oligonucleotides can be used. The
size and composition of the drug carrier restricts rapid clearance
from the blood stream. The carrier, made to accumulate at the site
of infection, can protect the oligonucleotides from degradative
processes.
[0366] Drug delivery vehicles are effective for both systemic and
topical administration. They can be designed to serve as a slow
release reservoir, or to deliver their contents directly to the
target cell. An advantage of using direct delivery drug vehicles is
that multiple molecules are delivered per uptake. Such vehicles
have been shown to increase the circulation half-life of drugs
which would otherwise be rapidly cleared from the blood stream.
Some examples of such specialized drug delivery vehicles which fall
into this category are liposomes, hydrogels, cyclodextrins,
biodegradable nanocapsules, and bioadhesive microspheres.
[0367] From this category of delivery systems, liposomes are
preferred. Liposomes increase intracellular stability, increase
uptake efficiency and improve biological activity.
[0368] Liposomes are hollow spherical vesicles composed of lipids
arranged in a similar fashion as those lipids which make up the
cell membrane. They have an internal aqueous space for entrapping
water soluble compounds and range in size from 0.05 to several
microns in diameter. Several studies have shown that liposomes can
deliver agents to cells and that the agent remains biologically
active.
[0369] For example, a liposome delivery vehicle originally designed
as a research tool, Lipofectin, has been shown to deliver intact
mRNA molecules to cells yielding production of the corresponding
protein.
[0370] Liposomes offer several advantages: They are non-toxic and
biodegradable in composition; they display long circulation
half-lives; and recognition molecules can be readily attached to
their surface for targeting to tissues. Finally, cost effective
manufacture of liposome-based pharmaceuticals, either in a liquid
suspension or lyophilized product, has demonstrated the viability
of this technology as an acceptable drug delivery system.
[0371] Other controlled release drug delivery systems, such as
nanoparticles and hydrogels may be potential delivery vehicles for
an agent. These carriers have been developed for chemotherapeutic
agents and protein-based pharmaceuticals.
[0372] Topical administration of agents is advantageous since it
allows localized concentration at the site of administration with
minimal systemic adsorption. This simplifies the delivery strategy
of the agent to the disease site and reduces the extent of
toxicological characterization. Furthermore, the amount of material
to be applied is far less than that required for other
administration routes. Effective delivery requires the agent to
diffuse into the infected cells. Chemical modification of the agent
to neutralize negative or positive charges may be all that is
required for penetration. However, in the event that charge
neutralization is insufficient, the modified agent can be
co-formulated with permeability enhancers, such as Azone or oleic
acid, in a liposome. The liposomes can either represent a slow
release presentation vehicle in which the modified agent and
permeability enhancer transfer from the liposome into the targeted
cell, or the liposome phospholipids can participate directly with
the modified agent and permeability enhancer in facilitating
cellular delivery. In some cases, both the agent and permeability
enhancer can be formulated into a suppository formulation for slow
release.
[0373] Agents may also be systemically administered. Systemic
absorption refers to the accumulation of drugs in the blood stream
followed by distribution throughout the entire body. Administration
routes which lead to systemic absorption include: intravenous,
subcutaneous, intraperitoneal, intranasal, intrathecal and
ophthalmic. Each of these administration routes expose the agent to
an accessible diseased or other tissue. Subcutaneous administration
drains into a localized lymph node which proceeds through the
lymphatic network into the circulation. The rate of entry into the
circulation has been shown to be a function of molecular weight or
size. The use of a liposome or other drug carrier localizes the
agent at the lymph node. The agent can be modified to diffuse into
the cell, or the liposome can directly participate in the delivery
of either the unmodified or modified agent to the cell.
[0374] Most preferred delivery methods include liposomes (10-400
nm), hydrogels, controlled-release polymers, microinjection or
electroporation (for ex vivo treatments) and other pharmaceutically
applicable vehicles. The dosage will depend upon the disease
indication and the route of administration but should be between
10-2000 mg/kg of body weight/day. The duration of treatment will
extend through the course of the disease symptoms, usually at least
14-16 days and possibly continuously. Multiple daily doses are
anticipated for topical applications, ocular applications and
vaginal applications. The number of doses will depend upon disease
delivery vehicle and efficacy data from clinical trials.
[0375] Establishment of therapeutic levels of agent within the
target cell is dependent upon the rate of uptake and degradation.
Decreasing the degree of degradation will prolong the intracellular
half-life of the agent. Thus, chemically modified agents, e.g.,
oligonucleotides with modification of the phosphate backbone, or
capping of the 5' and 3' ends of the oligonucleotides with
nucleotide analogues may require different dosaging.
[0376] It is evident from the above results, that by modulating
telomerase activity and monitoring telomere length and telomerase
activity, one may provide therapies for proliferative diseases and
monitor the presence of neoplastic cells and/or proliferative
capacity of cells, where one is interested in regeneration of
particular cell types. Assays are provided which allow for the
determination of both telomere length, particularly as an average
of a cellular population, or telomerase activity of a cellular
population. This information may then be used in diagnosing
diseases, predicting outcomes, and providing for particular
therapies.
[0377] All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference.
[0378] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be readily apparent to those of ordinary
skill in the art in light of the teachings of this invention that
certain changes and modifications may be made thereto without
departing from the spirit or scope of the appended claims.
Example 18
An Alternative Method of Measuring Telomere Repeat Length
[0379] An alternative method to measure telomere length exploits
the fact that the telomere sequence lacks guanine residues in the
C-rich strand. Unmelted genomic DNA can be mixed with a
biotinylated oligonucleotide containing the sequence
Biotinyl-X-CCCTAACCCTAA which will anneal to the single stranded
G-rich overhang, followed by extension with the Klenow fragment of
DNA polymerase in the presence of dTTP, DATP and radioactive dCTP.
The DNA is then mixed with streptavidin-coated magnetic beads, and
the DNA-biotin-streptavidin complexes recovered with a magnet. This
procedure purifies the telomeres and the radioactivity recovered at
this step is proportional to the number of telomeres. The DNA is
then melted, and DNA synthesis primed with fresh CCCTAACCCTAA
oligonucleotide, dTTP, dATP and radioactive dCTP. The radioactivity
incorporated during this step is proportional to the number of
telomeric repeats (telomere length) after correction for the number
of telomeres present as determined during the first step. This
value can then be converted into an actual telomere length by
comparison to a standard curve generated from telomeres of
previously determined lengths.
Example 19
An Alternative Method to Isolate Telomeric Sequences
[0380] Large telomeric DNA is purified as follows. A biotinylated
oligonucleotide with the sequence biotinyl-X-CCCTAACCCTAA is used
to prime DNA synthesis in double-stranded DNA. The only sequences
with which this oligonucleotide can anneal will be the
single-stranded base overhangs as telomere ends. The extended DNA,
which now has a more stable structure than that provided by the
initial 12 bp overlap, is then recovered using streptavidin. For
large DNA, the DNA could be digested with a rare-cutting
restriction endonuclease such as Not1, then subjected to
pulse-field electrophoresis, Streptavidin, covalently attached to a
block of agarose near the origin, would bind to the biotinylated
DNA and restrict the migration of the telomeres while permitting
the bulk of genomic DNA to migrate into the gel. Telomeric DNA
could then be recovered, cloned and characterized.
[0381] Alternately, smaller telomeric DNA fragments are recovered
from sheared DNA using streptavidin coated magnetic beads. The
following method was used to obtain these results:
[0382] Pilot experiments had indicated that the shearing forces
generated during the mixing and separation procedure yielded DNA
fragments approximately 20 kbp long. In order to maximize the
amount of subtelomeric DNA obtained, DNA from a T-antigen
immortalized cell line (IDH4, derived from IMR90 human lung
fibroblasts) that had very few telomeric repeats (short TRFs) were
used as the source of the DNA. 50 .mu.g of IDH4 DNA was mixed with
1.25 pmol of biotinylated CCCTAACCCTAA primer, 33 .mu.M each of
dATP, dTTP and dCTP, and 2 U of the Klenow fragment of DNA
polymerase, in a final volume of 100 .mu.l of Boehringer Mannheim
restriction endonuclease Buffer A and extended for three hours at
37.degree. C. A similar amount of a biotinylated TTAGGGTTAGGG
primer (which should not anneal to the G-rich telomeric overhang)
was added to a second reaction as a negative control. Five .mu.l of
M-280 Streptavidin-coated magnetic beads (Dynal, Inc.) were then
added and gently mixed for 2 hours at room temperature, then
biotinylated DNA-streptavidin-bead complexes were recovered by
holding a magnet against the side of the tube, and washed first
with isotonic saline containing 0.1% Triton X-100 and 0.1% bovine
serum albumin, and then with Sau3a restriction enzyme digestion
buffer. The DNA was then suspended in 20 .mu.l Sau3a digestion
buffer (New England Biolabs) and digested with 3 U of Sau3a in
order to release the subtelomeric DNA, leaving the terminal
restriction fragments attached to the beads. The bead-TRF complexes
were removed with a magnet, and the supernatant containing the
subtelomeric DNA was heated at 70.degree. C. for one hour to
inactivate the Sau3a. PCR linkers were added to the subtelomeric
DNA fragments by adjusting the buffer to 5 mM DTT and 0.5 mM ATP,
adding 25 pmol annealed PCR linkers plus 1.5 U of T4 DNA ligase,
and incubating overnight at 16.degree. C. The sequence of the PCR
linkers used is:
[0383] OLM2: 5' TGGTACCGTCGAAAGCTTGACTG 3'
[0384] DMO1: 3' ATGAACTGACCTAG 5'
[0385] These linkers are designed such that the annealed linkers
have a Sau3a compatible end (5' GATC 3'), the 3' end of OLM2 will
become ligated to the subtelomeric DNA fragment, while the 5' end
of DMO1 (which is not phosphorylated) will remain unligated. The
overlap between OLM2 and DMO1 has an approximate melting point of
24.degree. C., so that heating the ligated mixture to 70.degree. C.
for 20 minutes both inactivates the ligase and dissociates DMO1.
Half of the ligation mix was then diluted in PCR buffer with 100
pmol OLM2/100 .mu.l as the only primer. After three thermal cycles
of 72.degree. C..times.1 min then 85.degree. C..times.1 min (in
order to fill in the complementary sequence to OLM2 before melting
the DNA) the DNA was PCR amplified for 20 cycles (94.degree.
C..times.1 min, 55.degree. C..times.1 min, 72.degree. C..times.3
min).
[0386] The purity of the PCR amplified subtelomeric library was
assessed by in situ hybridization to metaphase chromosomes. Three
probes were prepared by amplifying the libraries in the presence of
digoxigenin labelled UTP: a positive control in which PCR linkers
had been ligated to a concatenated TTAGGG oligonucleotide to
produce an amplified mixture containing an average size of about 1
kbp of telomeric repeats ("Concatenated GTR"); a negative control
of the DNA selected with the biotinylated TTAGGGTTAGGG primer
("GTR-selected"); and the experimental library selected with the
biotinylated CCCTAACCCTAA primer ("CTR-selected"). The slides were
hybridized to the different probes, stained with an
anti-digoxigenin monoclonal antibody followed by an alkaline
phosphatase conjugated anti-mouse antibody, then coded and scored
for the presence of signal at internal sites versus telomeric ends.
Only after being analyzed was the code broken. The results are
shown in Table 7:
7TABLE 7 In Situ Hybridization Analysis of Subtelomeric DNA (two
experiments) Internal Probe End Signal Signal % Telomeric
Concatenated 104, 46 20, 19 81%, 71% GTR GTR-selected 20, 32 90, 95
18%, 25% CTR-selected 76, 79 57, 29 57%, 73%
[0387] The CTR-selected PCR amplification products were then
cloned, and 37 individual clones were picked and analyzed by in
situ hybridization. 10/37 (27%) of these clones gave telomeric
signals. The reason why a much smaller fraction of the individual
clones were telomeric than the fraction of signals in Table 7 is
due to the complexity of the PCR amplified material: Actual
telomeric DNA would be relatively abundant and thus be able to give
a signal, while contaminating internal sequences would be highly
diverse and thus each individual sequence in the mixture would tend
to be too rare to give a signal. The 20 kbp of DNA at the end of
each of 46 chromosome ends represents approximately 1/3000 of the
genome. The telomeric location of approximately 1/3 of the cloned
CTR-enriched DNA thus indicates that using the biotinylated CTR
resulted in a 1000-fold enrichment for telomeric DNA.
[0388] Seven of the telomeric clones were present on individual
telomeres, while three hybridized to multiple telomeres. The
characteristics of the ten telomeric clones are listed in Table 8,
and partial DNA sequences from all but clone CSITU6 are shown in
Table 9.
8TABLE 8 Characteristics of Subtelomeric Clones Number of Clone
Approx. Size Telomeric Signals CSITU5 1.5 Kbp single CSITU6 0.5 Kbp
multiple CSITU9 0.9 Kbp single CSITU13 0.9 Kbp single CSITU22 0.9
Kbp multiple CSITU24 0.9 Kbp multiple CSITU33 0.8 Kbp single
CSITU37 0.9 Kbp single CSITU38 0.9 Kbp single CSITU51 1.5 Kbp
single
[0389]
9TABLE 9 Sequences of Subtelomeric 5a Clones CSITU5 1
GATCTAGGCACAGCTGCTTCTCATTAGGCAGGTCTCAGC- TAGAAGACCAC 51
TTCCCTCCCTGAGGAAGTCAACCCTTCTGCCACCCCATGGCC- TTGCTTAA 101
TTTTCAGACTGTCGAATTGGAATCCTACCTCCATTAGCTACTAGC- TTGGG 151
CAAGATACAGAGCCCTCCC Total number of bases is: 169 DNA sequence
composition: 39 A; 54 C; 33 G; 43T; 0 OTHER CSITU9 1
ATATATGCGCTACATAAATGTATCTAGATGCAATTATCTA- GATACATATA 51
AGAAAGTATTTGAAGGCCTTCTACAAGGCTTAGTTATTATATT- GGTTCAT 101
ACAAGTTCTTCTTCAG Total number of bases is: 116 DNA sequence
composition: 39 A; 17 C, 18 G; 42 T; 0 Other CSITU13 1
ATCCTTCTCCGCAAACTAACAGGAACAGAAAACCAAACACTGCATGT- TCT 51
CACATCATTGTGGGAGTTGAACAATGAGAACACATGGACACAGGGAGGGG 101
AACATCACACACTCGGGGTGTCAGCCGGGTGGGAGGGTAGAGGAGGAGAA 151
ATACCTAAGTTCCAGATGACAGGTTG Total number of bases is: 176 DNA
sequence composition: 58 A; 37 C; 50 G; 31 T; 0 Other CSITU22 1
GATCTATGCTACCTCTAGGGATGGCACCATTCACAAGCACAAAGGAG- ATG 51
TCAGTGATTAAAAACACATGCTCTGGAGTCTGAGAGACTTTGACACTTGC 101
TAGCTTGTGGACTCTAGAGTTTAAGGTATCTGGACCCCTTTTTTCCCTCA 151
TGTGCATAATGAAGAGATT Total number of bases is: 169 DNA sequence
composition: 47 A; 35 C; 39 G; 48 T; 0 Other CSITU24 1
GATCAACACTGTTAGTTGAGTACCCACATCACAAACGTGATTCTCAAAT 51
GCCTTCCTTCCTGTCTAGTTTCTATAGGTATATATTTCCTTTTTCGCAT 101
AGGCCTGAAAAGCCGCCTCCAAATGCCGCCTTCCAGACACTATAAAAAG 151
AGGGTTCAAACCTACTCTATGAAAGGGAATGTTCAACACAGA Total number of bases
is: 192 DNA sequence composition: 58 A; 49 C; 33G; 52 T; 0 Other
CSITU33 1 GATCTGTTTATTATTCTTCCAATATCTCCCCATCTCTTAAA- AATTGGTTA 51
TTTCTTCGTTCATACATTTTTATCTCCCAAATTANNNNTGAGAC- TGGTTT 101
GAAGAGAGGAAAGCAATGTACACACTTTTATATTCCACCATGTATAT- CCG 151 GATATCC
Total number of bases is: 157 DNA sequence composition: 43 A; 32 C;
19 G; 59 T; 4 Other CSITU37 1
AATCCTCCTACCCCTCCCTTTGTTAGCCTGCCATTACAGGTGTGAG 51
CCACCATTGCTCATTCGTCCGTTTTCATTCAACAATCATCCATCTA 101
TTACATGTGAGGGATCTTCAGGTCATGGAAATTC Total number of bases is: 135
DNA sequence composition: 32 A; 37 C; 22 G; 44 T; 0 Other CSITU38 1
GATCACTTGAGCCCAGGAGTTTGAGACCAGCCTGGGTGACATGGCAAAAC 51
CCCATCTCTACCAAAAGAAAAAAANNNNACAAATTGGTGGTGTTGATGGT 101
CGGCGACCATTGATCCC Total number of bases is: 117 DNA sequence
composition: 35 A; 27 C; 28 G; 23 T; 4 Other CSITU51 1
GATCAGGGAGGGGCCGAAAACTGGAGATGCAGGTGTGCTGTAAGACACTG 51
CAGAGAGGGCATTTACCTGCCCCATCATCCAGCACAGGAACAGCGACTGA 101
CAGCGCTCACCCACCCACCATCGCCAGTCACACTGGG Total number of bases is: 137
DNA sequence composition: 37 A; 42 C; 39 G; 19 T; 0 Other
[0390] The CTR-enriched subtelomeric PCR amplified library has also
been used to screen a cDNA library. 32 clones have been isolated,
and partial sequence has been obtained form five clones. Their
sequences are shown in Table 10.
[0391] Two of these clones, PhC4 and PHCS, have been characterized
on Northern blots. Both hybridize to the same two mRNAs of
approximately 6.2 and 7.7 Kb. Since the 3' sequences of PHC4 and
PHC5 are different, this suggests they may represent alternative
splicing products of the same gene. Both messages are abundant in
PDL 38 IMR90 cells, which have relatively long telomeres, and
neither is expressed in the immortal IDH4 cells (which have very
short telomeres) that were derived from IMR90. This supports the
hypothesis that the expression of genes located in the subtelomeric
DNA are regulated by telomeric length. This data is evidence that
the above mentioned procedure provides a means of obtaining
sequences located in the proximity of telomeres, some of which
encode mRNA. Those sequences which are unique to individual
chromosomes will be useful in genomic mapping. Those which are
active genes and differentially expressed in cells with differing
telomere length, may play an important role in communicating
information relating to telomere length to the cell. Genes that
regulate the onset of M1 senescence can be isolated by these means,
as will as genes which modulate telomerase activity. The function
of the telomeric genes can be identified by overexpression and
knock-out in young senescent and immortal cells. Such cDNAs,
antisense molecules, and the encoded proteins may have important
therapeutic and diagnostic value in regard to their modulation of
cell proliferation in age-related disease and hyperplasias such as
cancer.
10TABLE 10 Partial Sequence of subtelorneric cDNA clones. PHC4-5'
end 1 GGCTCGAGAACGGGAGGAGGGGGCTCTTGTATCAGGGCCCGTTGTCACAT 51
CTGCTCTCAGCTTGTTGAAAACTCATAATC Total number of bases is: 80 DNA
sequence composition: 17 A; 19 0; 24 G; 20 T; 0 Other PHC5-3'END 1
AGGTCCCTTGGTCGTGATCCGGGAAGGGGCCTGACGTTGCGGGAGATCGA 51
GTTTTCTGTGGGCTTGGGGAACCTCTCACGTTGCTGTGTCCTGGTGAGCA 101
GCCCGGACCAATAAACCTGCTTTTCTTAAAGGAAAAAAAAAAAAAAAAAA 151 AAAAAAA
Total number of bases is: 157 DNA sequence composition: 47 A; 31 C;
44 G; 35 T; 0 Other PHC 7 1
ATCTAGGTTTTTTAAAAAAGCTTTGAGAGGTAATTACTTGCATATGAGAG 51
AATAAAACATTTGGCACATTGTTAAAAAAAAAAAAAAAAAAAAAAAAAAA 101
AAAAAAAAAAAAAAAAAAAA Total number of bases is: 120 DNA sequence
composition: 73 A; 7 C; 14 G; 26 T; 0 Other PHC8 1
CTCATTTACTTTTCTCTTATAGCGTGGCTTTAAACATATATACATTTGTA 51
TATATGTATATATGAATATAATGTATAAAATGTATGTAGATGTATATACA 101
AAAAATAAACGAGATGGGTTAAAGATATGTAAAAAAAAAAAAAAAAAAAA Total number of
bases is: 149 DNA sequence composition: 69 A; 11 C; 19 G; 50 T; 0
Other PHC9 1 AGTCCCAGCTACTCGGGAGGGCTGAGGCAGGAGAATGGCGTGAA- CCCAGG
51 AGGCGAAGCTTGCAGTGAGCTGAGATCGCGCCACTGCACTCCAGCCT- GGA 101
CGACAGAGCGAGACTCTGTCTCAAAAAAAAAAAAAAAAAAAA Total number of bases
is: 169 DNA sequence composition: 47 A; 35 C; 39 G; 48T; 0
Other
Example 20
Isolation of Factors that Derepress Telomerase
[0392] The M2 mechanism of cellular senescence occurs when
insufficient numbers of telomeric repeats remain to support
continued cellular proliferation. Escape from the M2 mechanism and
immortalization occur concomitantly with the induction of
telomerase activity and stabilization of telomere length, and thus
the inactivation of the M2 mechanism directly or indirectly
derepresses telomerase.
[0393] The gene(s) regulating the M2 mechanism have been tagged
with retroviral sequences. The methods by which this was
accomplished consisted of first determining the frequency at which
a clone of SV40 T-antigen transfected human lung fibroblasts was
able to escape M2 and become immortal (T-antigen blocks the Ml
mechanism, thus the M2 mechanism is the sole remaining block to
immortality in these cells). The pre-crisis cells were then
infected with a defective retrovirus in order to insertionally
mutagenize potential M2 genes, and it was shown that the frequency
of immortalization was increased by almost three-fold. Finally,
pulse-field electrophoresis of different immortalized insertionally
mutagenized lines was used to identify which of the lines became
immortal due to an insertion into the same M2 gene. Since an M2
mechanism gene has now been tagged with retroviral sequences, those
with ordinary skills in the art can now clone and identify the
specific gene. The methods used were as follows:
[0394] The frequency of escape from crisis (e.g., the
immortalization frequency of T-antigen expressing cells) was
estimated using an approach based on what is essentially a
fluctuation analysis as previously described (Shay, J. W., and
Wright, W. E. (1989) Exp. Cell Res. 184, 109-118). SW26 cells (a
clone isolated from IMR 90 normal human lung fibroblasts
transfected with a vector expressing SV40 large T antigen) were
expanded approximately 15 PDL's before crisis into multiple series
at a constant cell density of 6667 cells/cm.sup.2. Each series was
subsequently maintained as a separate culture, so that at the end
of the experiment the fraction of each series that gave rise to
immortal cell lines could be determined. Cultures were split at or
just prior to confluence at 6667 cells/cm.sup.2. Once cells reached
crisis they were split at least once every three weeks until
virtually no surviving cells remained or the culture had
immortalized. When too few cells were obtained, all of the cells
were put back into culture in a single dish. Fibroblasts were
considered immortal if vigorous growth occurred after crisis during
two subcultivations in which 1000 cells were seeded into 50
cm.sup.2 dishes and allowed to proliferate for three weeks for each
cycle.
[0395] SW 26 cells enter crisis at approximately PDL 82-85.
Numerous vials of SW26 cells (8.times.10.sup.6 cells/vial) were
frozen at PDL 71, and testing verified that spontaneous
immortalization events had not yet occurred. Five vials were
thawed, scaled up for 4 days to approximately 10.sup.8 cells (thus
to approximately PDL 74), than trypsinized and combined into a
single pool of cells in 40 ml of medium and distributed into 200 10
cm.sup.2 dishes. Thirty dishes were treated with 25 .mu.g/ml
bleomycin sulfate (a chemical mutagen) for two hours in serum free
medium one day later. Since this concentration of bleomycin sulfate
resulted in approximately 50% of the IMR-90 SW26 cells dying, these
dishes had been plated at twice the cell density as the rest.
[0396] The remainder of the dishes were used as controls (70
dishes) or infected with LNL6 defective retrovirus (100 dishes).
LML6 was generated in the amphotrophic packaging line PA317
according to previously described procedures (Miller and Rosman,
1989, Biotechniques 7, 980-990). Culture supernatant from LNL6
infected PA317 cells were used to infect one hundred dishes
containing approximately 5.times.10.sup.5 cells in the presence of
2 .mu.g/ml of polybrene. Control medium supernatant from uninfected
PA317 cells containing polybrene were used to treat 70 dishes and
served as controls. Within a few days after infection, all control
and experimental dishes were counted and each dish contained
1-2.times.10.sup.6 cells. The PDL of each dish was calculated and
cells were then replated at 0.33.times.10.sup.6 in 50 cm.sup.2
dishes and maintained separately to conduct the fluctuation
analysis.
[0397] Bleomycin treated SW26 cells escaped crisis with an
approximately two-fold higher frequency (7.7.times.10.sup.-7) than
the spontaneous rate (4.7.times.10.sup.-7). Pre-crisis SW26 cells
infected with the defective retrovirus LNL6 in order to produce
insertional mutations yielded a frequency of escape from crisis
(10.9.times.10.sup.-7) that was 2-3 fold greater than the rate from
simultaneous control series mock-infected with culture supernatant
from the non-infected packaging line.
11TABLE 10 Bleomycin Sulfate Exrosure and Retrovirus Infection
Increase Immortalization Frequency Addition Immortalization
Frequency Nil 10/68 4.4 .times. 10.sup.-7 Bleomycin 7/27 7.7
.times. 10.sup.-7 sulfate LNL6 retrovirus 36/99 10.9 .times.
10.sup.-7 Immortalization is expressed as the number of immortal
lines per number of culture series, each series being derived from
a single dish at the initiation of the experiment. Frequency is
expressed as the probability of obtaining an immortal cell line
based on the number of cells plated at each passage (not per cell
division).
[0398] DNA has been isolated from 23 of the 36 independent cell
lines obtained following insertional mutagenesis with LNL6, and 7
of these (30%) did not contain retroviral sequences when analyzed
on Southern Blots, while most of the remainder contained single
insertions. Given that those without retroviral insertions had to
represent spontaneous immortalization events, most of the remaining
clones with retroviral insertions should be due to insertional
mutagenesis if the frequency of immortalization was actually
increased 2-3 fold. DNA from 12 lines has been digested with the
rare-cutting enzyme Sfi1, followed by pulse-field electrophoresis,
transfer to nylon membranes and probing with the retrovirus LTR.
Six of the 12 lines contained a common band of approximately 350
Kbp that hybridized to the retroviral LTR. Four of these six have
also been analyzed following BamHl digestion, and three of these
four also contained a common band of approximately 20 Kbp. Given
that the retrovirus is 6 Kbp long, this strongly suggests that the
retrovirus has inserted multiple times within 14 Kbp region of
DMNA, which is strongly suggestive of a single gene. Digestion with
EcoR1, which cuts within the retrovirus, yields different size
fragments for each line, establishing that they represent different
insertional events and are truly independent isolates. The use of
retroviral sequences to clone the genomic DNA flanking the
insertion sites should now permit positive identification of a gene
involved in the M2 mechanism. Interference with the function of
that gene (for example, using antisense techniques) should result
in the derepression of telomerase and the ability to extend the
lifespan of normal human cells. This gene should also prove to be
mutated in a variety of cancer cells, and is thus likely to be of
diagnostic and therapeutic value in cancer as well.
Example 21
Tissue Distribution of Telomerase Activity in Primates
[0399] S100 extracts were prepared from a 12 year old healthy male
Rhesus Macaque to determine the tissue distribution of telomerase
activity. Abundant telomerase activity was detected only from the
testis. Samples of tissue from the brain, kidney, and liver
displayed no detectable activity. This suggests that telomerase
inhibition as a therapeutic modality for cancer has the unique
advantage of not being abundant in normal tissues with the
exception of the germ line. Therefore telomerase inhibitors should
be targeted away from the germ cells in reproductive aged
individuals to decrease the chance of birth defects. Such targeting
may be accomplished by localized injection or release of the active
agent near the site of the tumor. The effect of the telomerase
inhibitors in the male may be easily determined by measuring
telomere repeat length in the sperm.
Sequence CWU 1
1
* * * * *