U.S. patent application number 11/524618 was filed with the patent office on 2007-03-08 for methods of screening for risk of proliferative disease and methods for the treatment of proliferative disease.
This patent application is currently assigned to Duke University. Invention is credited to Andrea Amalfitano, Rose-Mary N. Boustany, Wei-Xing Guo.
Application Number | 20070054302 11/524618 |
Document ID | / |
Family ID | 37189176 |
Filed Date | 2007-03-08 |
United States Patent
Application |
20070054302 |
Kind Code |
A1 |
Boustany; Rose-Mary N. ; et
al. |
March 8, 2007 |
Methods of screening for risk of proliferative disease and methods
for the treatment of proliferative disease
Abstract
A method of screening a subject for a proliferative disease risk
factor comprises detecting the presence or absence of upregulation
of the CLN3 gene in the subject. The upregulation of the CLN3 gene
in the subject indicates the subject is at increased risk of
developing a proliferative disease. Methods of screening compounds
for the treatment of proliferative diseases based on the CLN3 gene
and its product are also disclosed, along with methods of treating
such diseases and vectors useful therefore.
Inventors: |
Boustany; Rose-Mary N.;
(Durham, NC) ; Guo; Wei-Xing; (Northridge, CA)
; Amalfitano; Andrea; (Durham, NC) |
Correspondence
Address: |
MYERS BIGEL SIBLEY & SAJOVEC
PO BOX 37428
RALEIGH
NC
27627
US
|
Assignee: |
Duke University
|
Family ID: |
37189176 |
Appl. No.: |
11/524618 |
Filed: |
September 21, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09830045 |
Jan 23, 2002 |
7129043 |
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PCT/US99/24695 |
Oct 21, 1999 |
|
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11524618 |
Sep 21, 2006 |
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60105262 |
Oct 22, 1998 |
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Current U.S.
Class: |
435/6.11 ;
435/6.14 |
Current CPC
Class: |
C12Q 1/6886 20130101;
C12Q 2600/136 20130101; C12Q 2600/118 20130101; G01N 2800/52
20130101 |
Class at
Publication: |
435/006 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Goverment Interests
[0002] The present invention was made with Government support under
grant RO1-NS30170 from the National Institutes of Health. The
Government has certain rights to this invention.
Claims
1-46. (canceled)
47. A method of identifying a human subject having an increased
risk of developing breast cancer, comprising detecting upregulation
of the CLN3 gene in said subject, wherein upregulation of the CLN3
gene in said subject identifies the subject as having an increased
risk of developing breast cancer.
48. The method of claim 47, wherein said subject has been
previously diagnosed with breast cancer.
49. The method of claim 47, wherein said subject has not been
previously diagnosed with breast cancer.
50. The method of claim 47, wherein said subject has been
previously identified to be at increased risk of developing breast
cancer.
51. The method of claim 47, wherein said subject has not been
previously identified to be at increased risk of developing breast
cancer.
52. The method of claim 47, wherein said detecting step is carried
out by detecting increased mRNA levels for said CLN3 gene.
53. The method of claim 47, wherein said subject has undergone
treatment for breast cancer.
Description
RELATED APPLICATIONS
[0001] This application is a continuation application of, and
claims priority to, U.S. application Ser. No. 09/830,045, filed
Apr. 20, 2001, which status is pending, which is a 35 U.S.C. .sctn.
371 national phase application of International Application No.
PCT/US99/24695 filed Oct. 21, 1999, which claims the benefit of
U.S. Provisional Application No. 60/105,262, filed Oct. 22, 1998.
The entire contents of each of these applications is incorporated
by reference herein.
FIELD OF THE INVENTION
[0003] The present invention involves methods of screening for risk
of proliferative diseases such as cancer. The screening may be a
diagnostic or prognostic screening. Methods of screening compounds
for the treatment of proliferative diseases and methods of treating
proliferative disease are also disclosed.
BACKGROUND OF THE INVENTION
[0004] The juvenile form of Batten disease is an autosomal
recessive neurodegenerative disease of childhood (Boustany R-M, et
al., Am. J. Med. Genet. Suppl. 5: 47-58 (1988)). It is clinically
characterized by onset at age 5-6 years with progressive blindness,
generalized and myoclonic seizures, cognitive and motor decline and
death in the mid to late twenties (Boustany R-M and Kolodny E H
Rev. Neurol. (Paris) 145: 105-110 (1989); Boustany R-M Am. J. Med.
Genet. 42: 533-535 (1992); Boustany R- M and Filipek P J Inher.
Metab. Dis. 16: 252-255 (1993)).
[0005] The CLN3 gene hypothesized to underly juvenile Batten
disease encodes a 438 amino acid protein containing six putative
hydrophobic transmembrane domains (The International Batten Disease
Consortium Cell 82: 949-957 (1995); Janes R W et al., FEBS Lett.
399: 75-77 (1996)). It is expressed in a variety of human tissues
including brain. The CLN3 protein is highly conserved across
species including dog, mouse, Caenorhabditis elegans and
Saccharomyces cerevisiae: (Altshul S F, et al., J. Mol. Biol. 215:
403-410 (1990); Mitchison H M, et al., Genomics 40: 346-350
(1997)).
[0006] It has been debated whether the function of the intact CLN3
gene is antiapoptotic, and that its integrity might be necessary
for neuronal and photoreceptor survival. (See, e.g., Howard M K, et
al., J. Neurochem. 60: 1783-1791 (1993); Kulkarni G V and McCulloch
C A J. Cell Sci. 107: 1169-1179 (1994); Kulkarni G V and McCulloch
C A J. Cell Sci. 107: 1169-1179 (1994) Kulkarni G V and McCulloch C
A J. Cell Sci. 107: 1169-1179 (1994); Walker P R, et al., Cancer
Res. 51: 1078-1085 (1991); Bertand R, et al., Exp. Cell Res. 211:
314-321 (1994); Stoll C, et al., Cancer Res. 36: 2710-2713 (1976)).
An antiapoptotic function for intact CLN3 is not generally
accepted.
SUMMARY OF THE INVENTION
[0007] As discussed in detail below, the present invention is based
in part on the demonstration of antiapoptotic function for CLN3, on
the surprising demonstration of upregulation of CLN3 in cancer
cells, and on the finding that downregulation of CLN3 cancer cells
inhibits the growth of cancer cells.
[0008] A first aspect of the present invention is a method of
screening a subject for a proliferative disease risk factor. The
method comprises detecting the presence or absence of upregulation
of the CLN3 gene in the subject. The upregulation of the CLN3 gene
in the subject indicates the subject is at increased risk of
developing a proliferative disease.
[0009] A second aspect of the present invention is a method of
screening a compound for efficacy in the treatment of a
proliferative disease. The method comprises providing a group of
subjects characterized by either (a) the presence of upregulation
of the CLN3 gene in the group or (b) the absence of upregulation of
the CLN3 gene in the group. The compound to be tested is then
administered to the subjects, and the efficacy of the compound in
the treatment of the proliferative disease is determined.
[0010] A third aspect of the present invention is an in vitro
method of screening compounds for efficacy in treating a
proliferative disease. The method comprises determining in vitro
whether the compound inhibits the expression of the CLN3 gene. The
inhibition of expression of the CLN3 gene indicates the compound is
useful in treating the proliferative disease.
[0011] A fourth aspect of the present invention is a method of
screening compounds for efficacy in treating a proliferative
disease. The method comprises determining in vitro whether said
compound specifically binds to the CLN3 gene product. The binding
of the compound to the CLN3 gene product indicates that the
compound is useful in treating the proliferative disease.
[0012] A fifth aspect of the present invention is a method of
inhibiting the growth of proliferative cells. The method comprises
administering to the cells a vector containing and expressing a
heterologous nucleic acid, wherein the heterologous encodes a
product such as an antisense oligonucleotide that inhibits the
expression of the CLN3 gene in the cells.
[0013] A sixth aspect of the present invention is a recombinant
vector useful for of inhibiting the growth of proliferative cells.
The vector contains and expresses in susceptible cells a
heterologous nucleic acid, wherein said heterologous nucleic acid
encodes a product that inhibits the expression of the CLN3 gene in
said cells.
[0014] The present invention is explained in greater detail in the
drawings herein and the specification set forth below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1. Overexpression of CLN3 enhances the rate of NT2 cell
growth.
[0016] FIG. 1A. Transient overexpression of CLN3: NT2 cells were
transiently transfected with CLN3 subcloned into the pCMV4 vector
by the calcium phosphate method. The left panel shows results of
semi-quantitative RT-PCR analysis expressed as CLN3 mRNA levels
relative to the internal control cyclophilin MRNA levels. NT2 cells
transfected with 10 .mu.g of CLN3 had a 4.5 times comparatively
higher level of CLN3 MRNA than control cells. The right panel shows
overexpression of CLN3 at the protein level as determined by
immunostaining of NT2 cells with polyclonal anti-CLN3 antibody.
Intense brown staining indicates higher levels of CLN3 protein in
CLN3 overexpressing compared to control cells (scale bar 5
.mu.m).
[0017] FIG. 1B. Stable overexpression of CLN3: NT2 cells were
stably transfected with CLN3 subcloned into the pOPRSV1CAT vector
by the calcium phosphate method. RT-PCR analysis shows the stably
transfected NT2 cells to have a comparatively 2.5 times higher
level of CLN3 mRNA (relative to cyclophilin) with respect to
control cells (left panel). The right panel shows overexpression of
CLN3 at the protein level as detected by immunostaining.
[0018] FIG. 1C. Western blot analysis of CLN3 protein
overexpression. Equal amounts of protein extracts from NT2 cells
overexpressing CLN3 and appropriate controls were analyzed by
immunoblotting with anti-CLN3 antibody. Cells were stably
transfected with CLN3 subcloned either in the pQPRSVI CAT vector
(lane 1) or the pCEP4 vector (lanes 3 and 4). The vector controls
are shown in lane 2 (pOPRSV1CAT) and lane 5 (pCEP4). High levels of
the CLN3 protein (.about.55 kDa), indicated by the arrowhead, were
observed in all three CLN3 overexpressing cell lines. Molecular
weight markers, in kDa, are shown on the left.
[0019] FIG. 1D. Effect of CLN3 overexpression on NT2 cell
proliferation: [.sup.3H] thymidine was added to NT2 cells stably
transfected with CLN3 either in the pOPRSV1CAT vector (left panel)
or the pCEP4 vector (right panel) at the indicated times and the
incubation continued for another 4 hours. The incorporated
[.sup.3H] thymidine is represented relative to that at 0 hour which
is taken as 100%. Each data point is an average of three samples.
The left panel shows results from one stable cell line
overexpressing CLN3. The right panel shows results from two
additional stable cell lines overexpressing CLN3 (CLN3 I-pCEP4 and
CLN3 II-pCEP4).
[0020] FIG. 2. CLN3 protects from serum starvation induced growth
inhibition.
[0021] FIG. 2A. Serum deprivation results in inhibition of NT2 cell
growth. The rate of growth of NT2 cells in the presence of serum
(open square) or absence of serum (open circle) was compared by
plating equal numbers of cells and counting at 6, 12, 18, 24 and 36
hours using the trypan blue method.
[0022] FIG. 2B. CLN3 overexpression enhances the survival of NT2
cells following serum withdrawal. NT2 cells stably transfected with
CLN3 or the vector (pOPRSV1CAT) alone were grown in serum free
media for 12, 24 and 36 hours and their viability assessed by the
trypan blue method. Results are expressed as cell survival rate,
which is the ratio of viable cells in serum free media to those in
media with serum. Each point is an average of two experiments each
of which was carried out in triplicate (p<0.05).
[0023] FIG. 3. CLN3 rescues NT2 cells from drug induced
apoptosis.
[0024] FIG. 3A. CLN3 rescues from etoposide induced apoptosis: (i)
For dose response curves, NT2 cells were treated with varying
concentrations of etoposide and harvested at 6, 12, 18 and 24 h
intervals. Viable cells were counted by the trypan blue method.
Each data point is an average of three samples; (ii) NT2 cells that
stably overexpress CLN3 were treated with etoposide (10 .mu.g/ml)
for 18 h and the viable cells were counted by the trypan blue
method. The bargrams show the survival rate (ratio of viable cells
in drug treated to untreated samples) of drug treated NT2 cells
overexpressing CLN3 compared to treated cells transfected with
vector alone. Results are shown from three separate experiments,
each carried out in triplicate.
[0025] FIG. 3B. CLN3 rescues from vincristine induced apoptosis:
(i) Dose response curve was determined by treating NT2 cells with
varying concentrations of vincristine and counting the viable cells
by the trypan blue method at various time intervals; (ii) NT2 cells
stably transfected with CLN3 or vector alone were treated with
vincristine (1 .mu.g/ml for 18 h) and the survival rate determined
by viability assay using trypan blue. The result shown is for three
separate experiments, each performed in triplicate. The bargram
compares the survival rate of CLN3-overexpressing and control cells
following treatment with vincristine.
[0026] FIG. 3C. CLN3 rescues from staurosporine induced apoptosis:
(i) Dose response curves for staurosporine in NT2 cells are shown.
Viability was assessed at various time intervals by the trypan blue
method; (ii) NT2 cells stably overexpressing CLN3 were treated with
500 nM staurosporine for 18 h. Survival rate was determined by the
trypan blue method and is shown for three separate experiments,
each carried out in triplicate. The bargram compares the survival
rate of CLN3 overexpressing and control cells following
staurosporine treatment, and the table shows protection and degree
of protection afforded by CLN3 overexpression.
[0027] FIG. 4. CLN3 overexpressing NT2 cells show less DNA
fragmentation than vector control NT2 cells in response to
treatment with etoposide, staurosporine and vincristine. NT2 cells
were transiently transfected with CLN3 (lanes 1, 3 and 5) or vector
alone (lanes 2, 4 and 6) followed by treatment with either 10
.mu.g/ml etoposide (lanes 1 and 2) or 500 nM staurosporine (lanes 3
and 4) or 1 .mu.g/ml vincristine (lanes 5 and 6). Low molecular
weight DNA was extracted and analyzed on a 2% agarose gel in
Tris-Borate-EDTA buffer (Rosenbaum et al, Ann. Neurol. 36, 864-870
(1994)).
[0028] FIG. 5. CLN3 modulates ceramide formation.
[0029] FIG. 5A. Overexpression of CLN3 lowers the level of
endogenous ceramide in NT2 cells. NT2 cells were either transiently
transfected with CLN3-CMV4 (left panel) or stably transfected with
CLN3-OPRSV1 CAT (right panel) and subjected to lipid extraction
followed by measurement of ceramide. Each experiment was performed
twice and in duplicate. The average percent drop in ceramide levels
of NT2 cells overexpressing CLN3 compared to control cells is
indicated within the bargraphs.
[0030] FIG. 5B. CLN3 overexpression prevents activation of ceramide
by vincristine. NT2 cells transiently (left panel) or stably (right
panel) overexpressing CLN3 and the appropriate controls were all
treated with vincristine (1 .mu.g/ml) prior to ceramide
quantitation. Absolute ceramide values were expressed as
pmoles/nmoles phospholipid and are shown below. The change in
ceramide level for each cell line following vincristine treatment
was calculated as the percent change in ceramide between
vincristine treated and untreated cells and plotted. Results are an
average of two experiments. TABLE-US-00001 Vincristine % Change
Untreated treated in ceramide pmoles/nmoles pmoles/nmoles
Treated-untreated/ phospholipid phospholipid untreated Transient
transfection CLN3-pCMV4 4.62 .+-. 0.34 5.45 .+-. 0.50 +18.0% pCMV4
4.68 .+-. 0.4 5.92 .+-. 0.46 +25.5% Stable transfection
CLN3-Poprsv1CAT 2.47 .+-. 0.13 2.03 .+-. 0.14 -17.8% pOPRSV1CAT
13.6 .+-. 0.04 20 .+-. 0.1 +47.0%
[0031] FIG. 5C. Ceramide causes apoptosis in NT2 cells. Equal
numbers of NT2 cells were plated and treated with increasing
concentrations of C.sub.2-ceramide. Cells were counted by the
trypan blue method at 6, 12, 18 and 24 hour intervals. Each data
point is an average of three samples.
[0032] FIG. 5D. CLN3 does not protect NT2 cells from ceramide
induced apoptosis. NT2 cells transiently transfected with CLN3 were
treated with 5 PM of either C.sub.2- or C.sub.6-ceramide for 18
hours. Cells were then harvested and viability assessed by the
trypan blue method. The data represents averages of three separate
experiments (p>0.2).
[0033] FIG. 6. CLN3 protects PARP from proteolysis in NT2 cells.
NT2 cells were transiently transfected with CLN3 or pCMV4 and
treated with etoposide (10 .mu.g/ml) for 18 hours. Total protein
was harvested from cells and analyzed for proteolysis of PARP from
116 kDa to the 85 kDa fragment by Western blotting using a
polyclonal anti-PARP antibody. Lanes 1 and 3 are transfected with
pCMV4, lanes 2 and 4 are transfected with CLN3-pCMV4, and lanes 3
and 4 are treated with etoposide.
[0034] FIG. 7. Proposed role for CLN3 in positive regulation of
cell growth. CLN3 acts upstream of ceramide and may regulate one of
a number of enzymes implicated in ceramide metabolism such as: acid
or neutral sphingomyelinase (1), ceramidase (2), ceramide synthase
(3), cerebroside synthase (4), and ceramide kinase (5).
[0035] FIG. 8. Leukemia cell line HL-60 shows an overexpression of
CLN3 compared to the transformed lymphoma cell line HS.
[0036] FIG. 8A. Column graph shows results of quantitative RT-PCR
analysis expressed as arbitrary units of CLN3 overexpression. HL-60
cells had 2.3 times higher level of CLN3 mRNA compared to HS
cells.
[0037] FIG. 8B. 10 .mu.l each of HS and HL-60 cDNA, synthesized
from the RT reaction, way hybridized with .alpha..sup.32P-dCTP in
the PCR reaction. The amplified DNAs were separated on 8%
polyacrylamide gels and visualized by autoradiography.
Autoradiogram shows the amplified 289 by fragment of CLN3 and 85 bp
fragment of cyclophilin (the internal control) for each cell line.
The amplified signal was quantitated on a Molecular Dynamics
phosphorimager using ImageQuant software.
[0038] FIG. 9. Both the breast cancer cell line BT-20 and the colon
cancer cell line HCT-116 shows an overexpression CLN3 compared to
the control 293, a kidney epithelial cell line. Column graph shows
results of quantitative RT-PCR analysis expressed as arbitrary
units of CLN3 overexpression. CLN3 mRNS levels were 2.8 and 4.1
times more in BT-20 and HCT-116, respectively, than in the
control.
[0039] FIG. 10. The breast cancer cell lines BT-20, BT-474, and
BT-549 all show an overexpression of CLN3 compared to the control
293, a kidney epithelial cell line. Column graph shows results of
quantitative RT-PCR analysis expressed as arbitrary units of CLN3
overexpression. BT-474, BT-20, and BT-549 had 1.9, 3.0, and 4.8
times more CLN3 expression than the control, respectively.
[0040] FIG. 11. The colon cancer cell lines HCT-116, SW480, and
SW1116 all show an overexpression of CLN3 compared to the control
293, a kidney epithelial cell line. Column graph shows results of
quantitative RT-PCR analysis expressed as arbitrary units of CLN3
overexpression. HCT-116 and SW480 had 3.0 and 7.7 times more CLN3
expression than the control, respectively. SW1116 had a dramatic
21.5 times the CLN3 mRNA level than did the control.
[0041] FIG. 12. Comparison of the CLN3 mRNA expression in two
melanoma cell lines, C32 and A-375, neuroblastoma cell line IMR-32,
glioma cell line Hs683, and glioblastoma cell line A-172 to the
control 293, a kidney epithelial cell line. Column graph shows
results of quantitative RT-PCR analysis expressed as arbitrary
units of CLN3 overexpression. C32 actually had less CLN3 expressed,
at 0.8 times, than did the control. Both A-375 and A-172 had a
slight overexpression of CLN3, with 1.2 times more than the
control. Hs683 had 1.7 times the CLN3 expression compared to the
control. IMR-32 had a notable 12.7 times the CLN3 expression
compared to the control.
[0042] FIG. 13 shows the inhibition of growth of SW1116 colon
cancer cells one day after infection with a CLN3 antisense
vector.
[0043] FIG. 14 shows the inhibition growth of SW1116 cells one day
after infection with a CLN3 antisense vector.
[0044] FIG. 15 shows the inhibition of growth of SW1116 at various
times after infection with a CLN3 antisense vector.
[0045] FIG. 16 shows the inhibition of growth of BT-20 breast
cancer cells one day after infection with a CLN3 antisense
vector.
[0046] FIG. 17 shows the cell growth curve of BT-20 cells at
various times after infection with a CLN3 antisense vector.
[0047] FIG. 18 shows the inhibition of growth of BT-20 cells at
various times after infection with a CLN3 antisense vector.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0048] Amino acid sequences disclosed herein are presented in the
amino to carboxy direction, from left to right. The amino and
carboxy groups are not presented in the sequence. Nucleotide
sequences are presented herein by single strand only, in the 5' to
3' direction.
[0049] The production of cloned genes, recombinant DNA, recombinant
vectors, proteins and protein fragments by genetic engineering is
well known, and can be carried out in accordance with known
techniques. See, e.g., U.S. Pat. No. 5,585,269 to Earp et al.; U.S.
Pat. No. 5,468,634 to Liu; and U.S. Pat. No. 5,629,407 to Xiong et
al. (the disclosures of all United States Patent references cited
herein are to be incorporated herein by reference in their
entirety.
[0050] The CLN3 gene referred to herein is known, and is also
referred to as the Batten Disease gene. See, e.g., The
International Batten Disease Consortium, Isolation of a Novel Gene
Underlying Batten Disease, CLN3, Cell, 82, 949-957 (1995). The gene
may be of any species depending upon the subject and/or the
particular use thereof, but is typically mammalian and preferably
human.
[0051] The term "risk factor" as used herein indicates subjects
that possess the indicated trait or factor face an increased risk
of developing the corresponding gene than subjects who do not
possess the risk factor.
[0052] The term "upregulation" as used herein with respect to the
CLN3 gene means greater levels of the gene product are produced as
compared to corresponding normal subjects.
[0053] The term "treat" as used herein refers to any type of
treatment that imparts a clinical improvement in the condition of
the patient, or delays the progression of the disease.
[0054] The term "proliferative disease" as used herein refers to
both cancer and non-cancer disease. Preferably the proliferative
disease is one characterized by increased expression of the CLN3
gene product in afflicted patients. Illustrative non-cancer
diseases include inflammatory and/or immunoproliferative disorders
such as arthritis, fibrosis, asthma and allergies. The invention
can be used to screen for risk of and/or treat a variety of
different types of cancer cells, particularly malignant (and
preferably solid) tumors of epithelial or mesenchymal cells.
Examples of cancers that can be screened for risk of and/or treated
by the present invention include breast cancer, melanoma, lung
cancer, colon cancer, leukemia (a liquid or non-solid tumor), soft
tissue and bone sarcomas, neuroendocrine tumors such as islet cell
carcinoma or medullary carcinoma of the thyroid, squamous
carcinomas (particularly of the head and neck), adenocarcinomas,
etc. The treatment of breast cancer is a particularly preferred
target for carrying out the present invention.
[0055] While the present invention is primarily concerned with the
screening and treatment of human subjects, the invention may also
be carried out on animal subjects such as dogs, cats, and horses
for veterinary purposes.
1. Screening Applications.
[0056] As noted above, the present invention provides a method of
screening a subject for a proliferative disease risk factor. The
method comprises detecting the presence or absence of upregulation
of the CLN3 gene in the subject. The upregulation of the CLN3 gene
in the subject indicates the subject is at increased risk of
developing a proliferative disease. Thus, the presence of the risk
fector is determined from the upregulation of the CLN3 gene in the
subject.
[0057] The method can be carried out whether or not the subject has
been previously diagnosed as being afflicted with a proliferative
disease, and whether or not the subject has been previously
prognosed to be at risk of developing the proliferative
disease.
[0058] The step of detecting whether the CLN3 gene is upregulated
(that is, the gene product thereof is found at increased levels as
compared to normal subjects), can be carried out by any suitable
means. For example, the step may be carried out by detecting
increased mRNA levels for the CLN3 gene in cells of the subject, or
by detecting increased levels of the protein product of the CLN3
gene in cells of the subject.
[0059] When the subject has previously been diagnosed as afflicted
with a proliferative disease, the method may be carried out to
monitor the progression of that disease, or monitor the efficacy of
drug treatments that the patient has undergone for the treatment of
that disease. Decreased levels of expression of the CLN3 gene would
be indicative of efficacy of the drug treatment.
[0060] As also noted above, the present invention also provides a
method of screening a compound for efficacy in the treatment of a
proliferative disease. The method comprises providing a group of
subjects characterized by either (a) the presence of upregulation
of the CLN3 gene in the group or (b) the absence of upregulation of
the CLN3 gene in the group. The compound to be tested is then
administered to the subjects, and the efficacy of the compound in
the treatment of the proliferative disease is determined. It will
be appreciated that not every member of the group need possess the
desired trait, as long as a sufficient number in the group possess
the desired trait so that the typical effect of the presence or
absence of the trait in the group can be discerned. By
incorporating this information into a drug trial, whether the
upregulation of the CLN3 is present or absent in the group (or the
group is divided into subgroups of those in whom upregulation is
present and those in whom upregulation is absent) more accurate
information can be obtained on the treatment of particular
patients. It will be appreciated that it can be equally valuable to
determine that a particular drug is efficacious for a particular
patient population as it is to determin that a particular drug is
not efficacious for a particular patient population, as the latter
information can at least be useful in directing therapy to more
promising treatments.
[0061] In vitro methods of screening compounds for efficacy in
treating a proliferative disease are also disclosed herein. In
general, in one embodiment, such methods comprise determining in
vitro whether the compound inhibits the expression of the CLN3 gene
(preferably the mammalian gene, and most preferably the human
gene). The inhibition of expression of the CLN3 gene indicates the
compound is useful in treating the proliferative disease. Numerous
such methods are available. The methods can be carried out in a
cell or cells, or can be carried out in essentially cell free
preparation. The method can be carried out by screening for
compounds that specifically disrupt either transcription or
translation of the CLN3 gene. The compound to be screened may be a
member of a library of compounds (the term "compound" as used in
this respect referring to both small organic compounds and other
therapeutic agents such as recombinant viral vectors). The method
may be carried out as a single assay, or may be implemented in the
form of a high throughput screen in accordance with a variety of
known techniques. In another embodiment, the method of screening
compounds for efficacy in treating a proliferative disease
comprises determining in vitro whether said compound specifically
binds to the CLN3 gene product (preferably the mammalian gene
product; most preferably the human gene product). The determining
step can be carried out by screening for binding of a test compound
or probe molecule to the entire full length CLN3 gene product, or
to a peptide fragment thereof (e.g., a fragment of from 5, 10 or 20
amino acids in length up to the full length of the CLN3 gene
product. The binding of the compound to the CLN3 gene product
indicates that the compound is useful in treating the proliferative
disease. Such techniques can be carried out by contacting a probe
compound to the CLN3 gene product or fragment thereof in any of the
variety of known combinatorial chemistry techniques (including but
not limited to split pool techniques, chip-based techniques and
pin-based techniques). Any suitable solid support can be used to
imobilize the CLN3 gene product or a fragment thereof to find
specific binding partners thereto (or immobilize the members of the
library against which the CLN3 gene product or fragment thereof is
contacted to find specific binding partners thereto), and numerous
different solid supports are well known to those skilled in the
art. Examples of suitable materials from which the solid support
may be formed include cellulose, pore-glass, silica gel,
polystyrene, particularly polystyrene cross-linked with
divinylbenzene, grafted copolymers such as polyethyleneglycol/
polystyrene, polyacrylamide, latex, dimethylacrylamide,
particularly cross-linked with N,N'bis-acrylolyl ethylene diamine
and comprising N-t-butoxycarbonyl-beta-alanyl-N'acrylolyl
hexamethylene diamine, composites such as glass coated with a
hydrophobic polymer such as cross-linked polystyrene or a
fluorinated ethylene polymer to which is grafted linear
polystyrene, and the like. Thus the term "solid support" includes
materials conventionally considered to be semi-solid supports.
General reviews of useful solid supports that include a
covalently-linked reactive functionality may be found in Atherton
et al., Prospectives in Peptide Chemistry, Karger, 101-117 (1981);
Amamath et al., Chem. Rev. 77: 183 (1977); and Fridkin, The
Peptides, Vol. 2, Chapter 3, Academic Press, Inc., pp 333-363
(1979). The solid support may take any suitable form, such as a
bead or microparticle, a tube, a plate, a microtiter plate well, a
glass microscope cover slip, etc.
[0062] The present invention can be used with probe molecules, or
libraries (where groups of different probe molecules are employed),
of any type. In general, such probe molecules are organic
compounds, including but not limited to that may be used to carry
out the present include oligomers, non-oligomers, or combinations
thereof. Non-oligomers include a wide variety of organic molecules,
such as heterocyclics, aromatics, alicyclics, aliphatics and
combinations thereof, comprising steroids, antibiotics, enzyme
inhibitors, ligands, hormones, drugs, alkaloids, opioids,
benzodiazepenes, terpenes, prophyrins, toxins, catalysts, as well
as combinations thereof. Oligomers include peptides (that is,
oligopeptides) and proteins, oligonucleotides (the term
oligonucleotide also referred to simply as "nucleotide, herein)
such as DNA and RNA, oligosaccharides, polylipids, polyesters,
polyamides, polyurethanes, polyureas, polyethers, poly (phosphorus
derivatives) such as phosphates, phosphonates, phosphoramides,
phosphonamides, phosphites, phosphinamides, etc., poly (sulfur
derivatives) such as sulfones, sulfonates, sulfites, sulfonamides,
sulfenamides, etc., where for the phosphorous and sulfur
derivatives the indicated heteroatom for the most part will be
bonded to C, H, N, O or S, and combinations thereof Numerous
methods of synthesizing or applying such probe molecules on solid
supports (where the probe molecule may be either covalently or
non-covalently bound to the solid support) are known, and such
probe molecules can be made in accordance with procedures known to
those skilled in the art. See, e.g., U.S. Pat. No. 5,565,324 to
Still et al., U.S. Pat. No. 5,284,514 to Ellman et al., U.S. Pat.
No. 5,445,934 to Fodor et al. (the disclosures of all United States
patents cited herein are to be incorporated herein by reference in
their entirety).
[0063] Test compounds used to carry out the present invention may
be of any type, including both oligomers or non-oligomers of the
types described above in connection with probe molecules above.
Again, such test compounds are known and can be prepared in
accordance with known techniques.
[0064] Where multiple different probe molecules are desired to be
tested, a screening substrate useful for the high throughput
screening of molecular interactions, such as in "chip-based" and
"pin-based" combinatorial chemistry techniques, can be prepared in
accordance with known techniques. All can be prepared in accordance
with known techniques. See, e.g., U.S. Pat. No. 5,445,934 to Fodor
et al., U.S. Pat. No. 5,288,514 to Ellman, and U.S. Pat. No.
5,624,711 to Sundberg et al.
[0065] In the alternative, screening of libraries of probe
molecules may be carried out with mixtures of solid supports as
used in "split-pool" combinatorial chemistry techniques. Such
mixtures can be prepared in accordance with procedures known in the
art, and tag components can be added to the discreet solid supports
in accordance with procedures known in the art. See, e.g., U.S.
Pat. No. 5,565,324 to Still et al.
2. Vectors and Administration.
[0066] Vectors used to carry out the present invention are, in
general, RNA virus or DNA virus vectors, such as lentivirus
vectors, papovavirus vectors (e.g., SV40 vectors and polyoma
vectors), adenovirus vectors and adeno-associated virus vectors.
See generally T. Friedmann, Science 244, 1275 16 (June 1989).
[0067] Examples of lentivirus vectors that may be used to carry out
the present invention include Moloney Murine Leukemia Virus
vectors, such as those described in U.S. Pat. No. 5,707,865 to
Kohn.
[0068] Any adenovirus vector can be used to carry out the present
invention. See, e.g., U.S. Pat. No. 5,518,913, U.S. Pat. No.
5,670,488, U.S. Pat. No. 5,589,377; U.S. Pat. No. 5,616,326; U.S.
Pat. No. 5,436,146; and U.S. Pat. No. 5,585,362. The adenovirus can
be modified to alter or broaden the natural tropism thereof, as
described in S. Woo, Adenovirus redirected, Nature Biotechnology
14, 1538 (Nov. 1996).
[0069] Any adeno-associated virus vector (or AAV vector) can also
be used to carry out the present invention. See, e.g., U.S. Pat.
No. 5,681,731; U.S. Pat. No. 5,677,158; U.S. Pat. No. 5,658,776;
U.S. Pat. No. 5,658,776; U.S. Pat. No. 5,622,856; U.S. Pat. No.
5,604,090; U.S. Pat. No. 5,589,377; U.S. Pat. No. 5,587,308; U.S.
Pat. No. 5,474,935; U.S. Pat. No. 5,436,146; U.S. Pat. No.
5,354,678; U.S. Pat. No. 5,252,479; U.S. Pat. No. 5,173,414; U.S.
Pat. No. 5,139,941; and U.S. Pat. No. 4,797,368.
[0070] The regulatory sequences, or the transcriptional and
translational control sequences, in the vectors can be of any
suitable source, so long as they effect expression of the
heterologous nucleic acid in the target cells. For example,
commonly used promoters are the LacZ promoter, and promoters
derived from polyoma, Adenovirus 2, and Simian virus 40 (SV40).
See, e.g., U.S. Pat. No. 4,599,308.
[0071] The heterologous nucleic acid may encode any product that
inhibits the expression of the CLN3 gene in cells infected by the
vector, such as an antisense oligonucleotide that specifically
binds to the CLN3 mRNA to disrupt or inhibit translation thereof, a
ribozyme that specifically binds to the CLN3 mRNA to disrupt or
inhibit translation thereof, or a triplex nucleic acid that
specifically binds to the CLN3 duplex DNA and disrupts or inhibits
transcription thereof. All of these may be carried out in
accordance with known techniques, as (for example) described in
U.S. Pat. Nos. 5,650,316; 5,176,996, or 5,650,316 for triplex
compounds, in U.S. Pat. Nos. 5,811,537; 5,801,154; and 5,734,039
for antisense compounds, and in U.S. Pat. Nos. 5,817,635;
5,811,300; 5,773,260; 5,766,942; 5,747,335; and 5,646,020 for
ribozymes (the disclosures of which are incorporated by reference
herein in their entirety). The length of the heterologous nucleic
acid is not critical so long as the intended function is achieved,
but the heterologous nucleic acid is typically from 5, 8, 10 or 20
nucleic acids in length up to 100, 200, 500 or 1000 nucleic acids
in length, up to a length equal the full length of the CLN3
gene.
[0072] Once prepared, the recombinant vector can be reproduced by
(a) propagating the vector in a cell culture, the cell culture
comprising cells that permit the growth and reproduction of the
vector therein; and then (b) collecting the recombinant vector from
the cell culture, all in accordance with known techniques. The
viral vectors collected from the culture may be separated from the
culture medium in accordance with known techniques, and combined
with a suitable pharmaceutical carrier for administration to a
subject. Such pharmaceutical carriers include, but are not limited
to, sterile pyrogen-free water or sterile pyrogen-free saline
solution. If desired, the vectors may be packaged in liposomes for
administration, in accordance with known techniques.
[0073] Any suitable route of administration can be used to carry
out the present invention, depending upon the particular condition
being treated. Suitable routes include, but are not limited to,
intraveneous, intrarterial, intrathecal, intraperitoneal,
intramuscular, and intralesional injection. Intralesional injection
is currently preferred.
[0074] The dosage of the recombinant vector administered will
depend upon factors such as the particular disorder, the particular
vector chosen, the formulation of the vector, the condition of the
patient, the route of administration, etc., and can be optimized
for specific situations. In general, the dosage is from about
10.sup.7, 10.sup.8, or 10.sup.9 to about 10.sup.11, 10.sup.12, or
10.sup.13 plaque forming units (pfu).
[0075] In addition to their pharmaceutical or veterinary use, the
recombinant vectors of the present invention (sometimes also
referred to as "active agents" herein) are useful in vitro to
distinguish cells in culture based on their response to the active
agents, to induce apoptosis, etc. Such techniques are useful for
both carrying out cell culture procedures and for drug screening
purposes.
[0076] The present invention is described in further detail in the
following non-limiting Examples. Abbreviations used herein are as
follows: JNCL, juvenile neuronal ceroid lipofuscinosis; NT2,
Ntera2/D1; NGF, nerve growth factor; ICE, interleukin-1 converting
enzyme; PARP, poly (ADP-ribose) polymerase; RT-PCR, reverse
transcription-polymerase chain reaction; PBS, phosphate buffered
saline; DAB, 3,3'-diaminobenzidine; TUNEL, Tdt mediated dUTP nick
end labeling. Drs. Terry Lerner and Jim Gusella graciously provided
the CLN3 cDNA.
EXAMPLE 1
CLN3 Defines a Novel Antiapoptotic Pathway
Operative in Neurodegeneration and Mediated by Ceramide
[0077] This example establishes a direct link between apoptosis and
neurodegeneration in Batten disease at a molecular level.
I. Experimental Procedures
[0078] Transfections: NT2 cells (ATCC# CRL1973) were plated on 60
mm dishes at a density of 0.5.times.10.sup.6 and grown at
37.degree. C. in a 5% CO.sub.2 atmosphere in DMEM supplemented with
10% fetal bovine serum and 100 units each of penicillin and
streptomycin. The CLN3 cDNA was subcloned in the pCMV4 vector for
transient transfections and in the pOPRSV1CAT vector or the pCEP4
vector (Invitrogen, Carlsbad, Calif.) for stable transfections
(Andersson S, et al., J. Biol. Chem. 264: 8222-8229 (1989); Fieck
A, et al., Nucleic Acid Res. 20: 1785-1791 (1992)). Transfections
with the CLN3-pCMV4 construct or the CLN3-pOPRSV1CAT construct were
carried out by the calcium phosphate method according to the
manufacturer's protocol using the MBS transfection kit (Stratagene,
LaJolla, Calif.). Transfections with the CLN3-pCEP4 construct were
carried out using Superfect reagent (Qiagen, Valencia, Calif.)
according to the manufacturer's method. Transiently transfected
cells were harvested for all analyses within 48 hours of
transfection. Stable clones were selected either with 500 .mu.g/ml
geneticin (CLN3-pOPRSV1CAT) or with 100 .mu.g/ml hygromycin
(CLN3-pCEP4) and subsequently grown under selective pressure.
[0079] Drug treatments: Dose response curves were established for
etoposide, vincristine and staurosporine in NT2 cells (FIGS. 3Ai,
3Bi and 3Ci). Stably transfected NT2 cells and vector control cells
were seeded at a density of 5.times.10.sup.4 cells/well in 12-well
plates, allowed to attach and then were treated with etoposide (10
.mu.g/ml) or vincristine (1 .mu.g/ml) or staurosporine (500 nM) for
18 h and the number of viable cells counted by the trypan blue
assay. Survival represents the number of drug treated viable cells
divided by the number of untreated viable cells and expressed as a
percentage. Protection is calculated as the difference in survival
between CLN3-overexpressing cells and the vector-transfected
control cells. The degree of protection is calculated as the
difference in survival between treated CLN3-overexpressing and
vector control cells divided by the survival of treated vector
control cells. The values are from three separate experiments, each
of which was carried out in triplicate (FIGS. 3Aii, 3Bii, and
3Cii).
[0080] RT-PCR: Messenger RNA was isolated from NT2 cells by the
oligo-dT-cellulose method using the QuickPrep Micro MRNA
Purification kit (Pharmacia Fine Chemicals, Piscataway, N.J.). The
mRNA was converted to cDNA by the reverse transcription (RT)
reaction (Estus et al, 94). PCR reactions were set up with the
first strand cDNA, 1U of Taq polymerase and 5 .mu.Ci of
.alpha..sup.32P-dCTP in each reaction. The reactions were performed
in Taq buffer containing 1.5 mM MgCl.sub.2, 2.5 mM dCTP and 5 mM
each of dATP, dGTP and dTTP. The primers used for amplification of
CLN3 were 5' primer: 5'-GGTGGACAGTATTCAAGGG-3' (958-976) and 3'
primer: 5'-CTTGGCAGAAAGACGAAC-3' (1229-1246). Cyclophilin was used
as the internal control and primers used for cyclophilin
amplification were the 5' primer: 5'-AAATGCTGGACCCAACAC-3'
(317-334) and 3' primer: 5'-AAACACCACATGCTTGCC-3' (384-401). The
reaction conditions used were 1 minute at 94.degree. C., 1 minute
at 50.degree. C. and 2 minutes at 72.degree. C. for 20 cycles. The
PCR amplified products were analyzed on an 8% non-denaturing
polyacrylamide gel that was dried and visualized by
autoradiography. The amplified signal was quantitated using a
Phosphorlmager (Molecular Dynamics Inc., Sunnyvale, Calif.). The
results are expressed as the ratio of CLN3 signal to that of the
internal control cyclophilin. This provides a reproducible and
comparative, semi-quantitative measure of CLN3 expression.
[0081] Western Blottingfor CLN3 and PARP detection: The CLN3
antibody used in this study is a polyclonal antibody raised against
the peptide sequence AAHDILSHKRTSGNQSHVDP corresponding to amino
acids 58-77 of the CLN3 protein (Research Genetics, Huntsville,
Ala.). Total cellular extracts for CLN3 detection were prepared
from NT2 cells transfected with CLN3 or the appropriate vector
control. Cells were washed with cold PBS and lysed in buffer (250
mM NaCl, 0.1% NP40, 50 mM Hepes, pH 7.0, 5 mM EDTA, 1 mM DTT, 1 mM
PMSF) on ice for 10 min. The lysate was collected and clarified by
centrifugation at 12,000 .times.g for 10 min at 4.degree. C. The
supernatant was quantitated for total protein by the BioRad protein
assay method. Equal amounts of total protein from each sample were
electrophoresed on a 9% SDS-polyacrylamide gel in buffer containing
0.092 M glycine, 0.125 M Tris-OH and 2% SDS. The gel was
transferred onto nitrocellulose membrane by semidry electroblotting
and blocked by incubation in solution containing 3% BSA in TBST (10
mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween-20) buffer for one
hour at 25.degree. C. followed by incubation with the CLN3 antibody
(IgG fraction) diluted in TBST, for 15 hours at 25.degree. C. After
extensive washes with TBST buffer, the membrane was incubated in a
1:5000 dilution of goat-anti-rabbit IgG conjugated with horseradish
peroxidase for 30 minutes at 25.degree. C. The blot was washed and
developed using the chemiluminescence detection system (Amersham,
Arlington Heights, Ill.).
[0082] For the PARP analysis, transfected NT2 cells were harvested
by scraping in PBS followed by lysis in gel loading buffer
containing 62.5 mM Tris-HCl pH 6.8, 6 M urea, 10% glycerol, 2% SDS,
0.003% bromophenol blue and 5% 2-mercaptoethanol. The samples were
incubated at 65.degree. C. for 15 min prior to being
electrophoresed on a 9% SDS-polyacrylamide gel as above. The
primary antibody for detection of PARP (obtained from Enzyme
System, Dublin, Calif.) was used at a dilution of 1:5000. Western
blotting was carried out as described above.
[0083] Immunocytochemistry and TUNEL Staining: For immunostaining,
NT2 cells transfected with CLN3 or the vector alone were grown on
glass coverslips, and fixed in 2% formaldehyde and 0.2%
glutaraldehyde for 15 minutes at 4.degree. C. NT2 cells stably
overexpressing CLN3 were also grown on coverslips and then fixed.
The cells were permeabilized by treatment with 0.1% Triton X-100
for 30 minutes at 25.degree. C. followed by incubation in 3% bovine
serum albumin (BSA) in phosphate buffered saline (PBS) for 1 hour
at 25.degree. C. The cells were then incubated for 16 hours with
the CLN3 antibody (IgG fraction) made up in PBS containing 3% BSA.
After removal of the primary antibody and washes, the cells were
incubated in a 1:500 dilution of biotin conjugated goat-anti-rabbit
IgG for 1 hour at 25.degree. C. This was followed by incubation
with avidin conjugated horseradish peroxidase for 30 minutes at
25.degree. C. The cells were then washed and incubated with
3,3'-diaminobenzidine (DAB). The cells were counterstained with
Hematoxylin Blue, rinsed in xylene and the coverslips mounted on
slides. The slides were viewed with a light microscope. TUNEL
staining was carried out on transient- or stable-transfected NT2
cells grown on glass coverslips using the fluorescent Apoptag
Direct kit (Oncor, Gaithersburg, Md.) following the manufacturer's
protocol (Schmitz G Anal. Biochem. 192: 222-231 (1991)). After
counterstaining with propidium iodide, the coverslips were mounted
on slides and the cells visualized under a fluorescent
microscope.
[0084] Cell proliferation assay by [.sup.3H] thymidine
incorporation: NT2 cells stably tranfected with CLN3 or the
appropriate vector control were plated in 12 well plates at a
density of 1.times.10.sup.5 cells/well and incubated with 0.5
.mu.Ci/ml [.sup.3H] thymidine at the indicated times for 4 h. The
cells were then washed twice with ice-cold PBS and the DNA
precipitated with 5% trichloroacetic acid. The TCA precipitate was
dissolved in 0.4 ml of 0.25 M NaOH and the incorporated [.sup.3H]
thymidine measured by liquid scintillation counting. For each time
point three separate samples were measured.
[0085] Ceramide determination: NT2 cells transiently or stably
transfected with CLN3 or the vector alone were harvested and the
total lipid extracted according to the Bligh & Dyer method
(Bligh E G and Dyer W J Can. J. Biochem. Physiol. 37: 911-917
(1959)). The ceramide assay was performed as previously described
(Zhang J, et al., Proc. Natl. Acad. Sci. U.S.A. 93: 5325-5328
(1996)). The labeled ceramide was viewed by autoradiography and
quantitated using a liquid scintillation counter. Ceramide was
expressed as pmole per nmole of total phospholipid. For measurement
of activated ceramide, both the appropriate vector and
CLN3-overexpressing cells (transient and stable) were treated with
vincristine (1 .mu.g/ml) for 18 h and subjected to lipid extraction
followed by ceramide measurements. The data from these experiments
is represented as change in ceramide level and reflects the
percentage difference in ceramide values before and after
vincristine treatment.
II. Results and Discussion
A. Overexpression of CLN3 Increases NT2 Cell Survival
[0086] Transient overexpression of CLN3 subcloned into the pCMV4
vector in NT2 cells was assessed by RT-PCR as shown in the left
panel of FIG. 1A. NT2 cells were stably transfected with CLN3
subcloned into pOPRSV1CAT, which resulted in a CLN3 signal 2.5
times stronger in CLN3 transfected NT2 cells compared to vector
transfected cells as determined by RT-PCR (FIG. 1B, left panel).
CLN3 protein overexpression was demonstrated by Western blot and
immunocytochemistry. The Western blot shows that CLN3 protein is
expressed at a higher level in three stable NT2 cell clones
(CLN3-pOPRSV1CAT seen in FIG. 1D left panel, and CLN3 I-pCEP4 and
CLN3 II-pCEP4 seen in FIG. 1D left panel) overexpressing CLN3 in
comparison to those transfected with vector alone (FIG. 1C).
Immunocytochemistry confirms CLN3 overexpression in both a
transient overexpression system (FIG. 1A, right panel) and a stable
overexpressing system (FIG. 1B, right panel). Three different
stable NT2 cell clones overexpressing CLN3 had a higher rate of
thymidine incorporation compared to corresponding vector controls:
for the CLN3-pOPRSV1CAT it was double, and for both pCEP4 clones it
was five-fold at 36 h compared to the vector controls. This
exceeded 100% at 36 hours for all three clones. This indicates that
CLN3 positively modulates the rate of growth of NT2 cells.
B. CLN3 Rescues from Serum Withdrawal Induced Growth Inhibition
[0087] Serum deprivation is known to induce apoptosis in lymphoid
(Obeid L M, et al., Science 259: 1769-1771 (1993)), neuronal and
other cell lines (Howard M K, et al., J. Neurochem. 60: 1783-1791
(1993); Kulkarni G V and McCulloch C A J. Cell Sci. 107: 1169-1179
(1994)). At the very least serum starvation inhibits growth. We
demonstrate that serum starvation induces growth arrest in NT2
cells. NT2 cells grown in DMEM with 10% FBS exhibit logarithmic
growth for 38 hours with a doubling time of 14 hours; serum
withdrawal blunts logarithmic growth of NT2 cells (FIG. 2A). The
effect of stable CLN3 overexpression on growth inhibition induced
by serum withdrawal was assessed using the trypan blue dye
exclusion method. The survival rate was calculated as the number of
viable cells grown in serum free medium divided by the number of
viable cells grown in serum supplemented medium. The amount of
protection consisted of the difference between survival rate of
CLN3- and vector-overexpressing NT2 cells. The degree of protection
consists of the amount of protection divided by the survival rate
of the vector control cells. CLN3 stably transfected NT2 cells
grown in medium lacking serum had a much higher survival rate as
compared to NT2 cells stably transfected with vector alone. The
degree of protection from serum starvation induced growth
inhibition exceeded 100% in cells stably overexpressing CLN3 at 12
hours and was even higher at 36 hours (FIG. 2B).
C. CLN3 Protects from Drug Mediated Apoptosis
[0088] The drugs etoposide and vincristine are chemotherapeutic
agents known to induce apoptosis. The effect of etoposide is
mediated by blocking topoisomerase II (Walker P R, et al., Cancer
Res. 51: 1078-1085 (1991)), whereas vincristine induces cell death
by interfering with spindle formation and the progression of the
cell cycle (Harmon B V et al., Cell Prolif. 25: 523-536 (1992)).
NT2 cells were stably transfected with CLN3 or vector alone
followed by treatment with 10 .mu.g/ml of etoposide or 1 .mu.g/ml
of vincristine, which are the doses resulting in 50% killing at 18
hours (FIGS. 3Ai and 3Bi). The protection conferred by stable CLN3
overexpression on NT2 cell viability was measured by trypan blue
exclusion: CLN3-overexpressing NT2 cells had a degree of protection
from cell death exceeding up to 78% following treatment with
etoposide (FIG. 3Aii). After vincristine treatment, stable
CLN3-overexpressing NT2 cells had up to a 52% degree of protection
as compared to vector transfected cells (FIG. 3Bii). Transiently
transfected NT2 cells were harvested after 18 hours of drug
treatment with etoposide, and stained by the TUNEL technique (data
not shown). A much larger number of green apoptotic nuclei is seen
in the treated vector cells. Conversely, transient overexpression
of antisense CLN3 cDNA decreased the survival rate of NT2 cells
treated with etoposide by 10% and vincristine by 16%.
Staurosporine, a potent protein kinase inhibitor, has been shown to
trigger both the morphological changes and DNA fragmentation
associated with apoptosis in many different cell lines (Bertand R,
et al., Exp. Cell Res. 211: 314-321 (1994)). The optimal
staurosporine concentration for causing apoptosis in NT2 cells was
established as being 500 nM (FIG. 3Ci). Viability was determined in
NT2 cells stably transfected with CLN3 or vector following
treatment with 500 nM staurosporine for 18 hours (FIG. 3Cii). The
degree of protection of stable CLN3-overexpressing NT2 cell clones
ranged from 42% to 129% compared to the corresponding
vector-transfected NT2 cells.
[0089] A hallmark of apoptosis is the fragmentation of nuclear DNA
into nucleosome-sized fragments creating a DNA ladder of 180 bp and
its concatamers (Arends M J, et al., Am. J. Pathol. 136: 593-608
(1990)). The drugs etoposide, staurosporine and vincristine cause
DNA fragmentation in NT2 cells. NT2 cells were transiently
transfected with CLN3 or vector alone, then treated with the drugs
prior to the extraction of low molecular weight DNA (Rosenbaum D M,
et al., Ann. Neurol. 36: 864-870 (1994)). Transient overexpression
of CLN3 resulted in a reduction in the extent of DNA fragmentation
caused by these drugs (FIG. 4). CLN3 was also found to be
protective against vincristine and/or etoposide induced apoptosis
in PC-12 neuronal cells, 293 kidney epithelial cells and U937
lymphoid cells. This was seen by either the TUNEL method or DNA
ladder formation (results not shown).
D. The Biologic Function of CLN3
[0090] These findings assign a major biologic function to the novel
gene CLN3 found to be defective in the juvenile form of Batten
disease. When overexpressed, CLN3 protects cells from growth arrest
induced by serum starvation and from death induced by treatment
with the proapoptotic agents vincristine, etoposide and
staurosporine. The protective function of CLN3 appears to be vital
for maintenance of cell survival in the central nervous system. The
evidence for massive and progressive loss of neurons and
photoreceptors in patients homozygous for the 1.02 kb CLN3 deletion
provides a naturally occurring model where both alleles are knocked
out. The neuropathologic lesion in the juvenile form of Batten
disease underscores the importance of the CLN3 protein for survival
of both neurons and photoreceptor cells. However, we show that the
antiapoptotic effect of CLN3 is not just restricted to neurons but
is operative in other mammalian cells. We have confirmed it in
pheochromocytoma derived PC-12 cells, epithelial 293 cells and
lymphoid U937 cells. CLN3 seems to be crucial for the survival of
postmitotic, fully differentiated nondividing cells, particularly
cortical neurons in the brain and photoreceptor cells in the eye.
One possible explanation for this is that the eye and the brain are
both immune-privileged sites that cannot tolerate destructive
inflammatory responses: Fas-Fas ligand interactions in the eye and
brain promote apoptosis and normally protect these organs from
tissue damage induced by inflammation (Griffith T S, et al.,
Science 270: 1189-1192 (1995); Griffith T S, et al., Immunity 5:
7-16 (1996)). Both neurons and photoreceptors are known to express
Fas ligand and lymphoid cells express Fas receptor. This could be
one of multiple mechanisms of immune privilege and the reason why
loss of function of the antiapoptotic gene, CLN3, is phenotypically
expressed only in the eye and brain in Batten disease. The absence
or defects in key locations of the CLN3 gene in juvenile Batten may
be sufficient for the apoptotic demise of both neurons and
photoreceptors.
E. Modulation of Endogenous Ceramide by CLN3
[0091] Elevation of proapoptotic ceramide levels in the brain of
patients with JNCL suggested a possible link between CLN3 and
ceramide (Puranam K, et al., Neuropediatrics 28: 37-41 (1997)). We
have shown that ceramide levels when measured as pmoles/nmoles of
phospholipid drop by 18% in NT2 cells transiently transfected with
CLN3 compared to NT2 cells just transfected with vector (FIG. 5A,
left panel). Endogenous ceramide levels in NT2 cells stably
transfected with CLN3 dropped by 69% (FIG. 5A, right panel). Also,
both transient and stable overexpression of CLN3 prevented
vincristine-induced activation of ceramide in NT2 cells (FIG. 5B).
The attenuation of vincristine-induced activation of ceramide by
CLN3 parallels the protective effect of CLN3 overexpression on NT2
cell survival seen following treatment with vincristine (FIGS.
3Bii). We show that C.sub.2-ceramide is an effective apoptotic
agent in NT2 cells (FIG. 5C). Overexpression of CLN3 did not rescue
NT2 cells from exogenous ceramide-induced killing (FIG. 5D). This
suggests that CLN3 inhibits apoptosis proximally at the level of
ceramide signaling in NT2 cells. The proteolytic cleavage of PARP
or poly (ADP-ribose) polymerase by the protease caspase-3 has been
identified as one of the key downstream events in the execution of
apoptotic cell death (White E Genes Develop. 10: 1-15 (1996)). The
chemotherapeutic drug etoposide has been shown to act via this
pathway and cause proteolysis of the 116 kDa PARP protein to
fragments that are 85 kDa and 25 kDa in size (Kaufmann S, et al.,
Cancer Res. 53: 3976-3985 (1993)). In order to investigate whether
CLN3 protection is conferred along this pathway, we studied the
effect of overexpression of CLN3 on the cleavage of PARP in NT2
cells. NT2 cells were transiently transfected with CLN3 or vector
alone and subsequently treated with 10 .mu.g/ml etoposide for 18
hours. Total cellular extracts were analyzed using a polyclonal
anti-PARP antibody by Western blotting (FIG. 6). In the absence of
etoposide, the 116 kDa PARP protein is intact in both CLN3 and
vector transfected cells. Following etoposide treatment, NT2 cells
transiently overexpressing CLN3 show a decrease in the amount of
cleaved 85 kDa PARP fragment as compared to control cells. This
indicates that CLN3 modulates ceramide levels which in turn lead to
a blunting of the activation of the executionary phase of apoptosis
by indirectly inhibiting activation of caspase-3, the enzyme that
cleaves PARP into the apoptosis specific 85 kDa fragment.
F. Mechanism of Action of CLN3 on Cell Survival
[0092] Without wishing to be bound to any particular theory for the
instant invention, the following observations are offered.
Antiapoptotic agents may protect the cell via one or more of the
following: antioxidant effects; regulation of calcium transport;
inhibition of the protease cascade; interference with protein
translocation, ubiquination or degradation; modulation of signal
transduction pathways and /or regulation of release of
mitochondrial cytochrome c oxidase (White E Genes Develop. 10: 1-15
(1996); Hannun Y A Science 274: 1855-1859 (1996); Liu X, et al.,
Cell 86: 147-157 (1996)). The coexistence of elevation of
proapoptotic ceramide and apoptosis in brain from patients with
JNCL (Lane S C, et al., J. Neurochem. 67: 677-683 (1996); Puranam
K, et al., Neuropediatrics 28: 37-41 (1997)) led us to suggest the
following: the signaling lipid second messenger ceramide and the
neuroprotective protein CLN3 partake in the same pathway of cell
growth and regulation. Significant drops in endogenous and
vincristine-activated ceramide levels in response to overexpression
of the CLN3 protein in NT2 cells strongly supports this hypothesis.
Ceramide formation or breakdown in the cell has at least five, if
not more, known origins: 1) sphingomyelin hydrolysis via neutral or
acid sphingomyelinase (Rena L A, et al., Biochem. Pharm. 53:
615-621 (1997)); 2) breakdown to sphingosine by ceramidase; 3)
generation of cerebrosides via cerebroside synthase, 4) de novo
synthesis of ceramide by ceramide synthase, and 5) formation of
ceramide phosphate by ceramide kinase. CLN3, predicted to reside in
the membrane, probably fine-tunes regulation of the apoptotic
pathway by blunting or attenuating ceramide generation. CLN3 could
be acting as a dimmer-switch for one of the reactions responsible
for ceramide formation. One of these enzymes could be a target for
the action of the CLN3 protein, or CLN3 could actually be one of
those enzymes (FIG. 7). Ceramide leads to activation of caspase-3
(Smyth M J, et al., Biochem. J. 316: 25-28 (1996)). We have also
shown that the overexpression of CLN3 inhibits the activation of
caspase-3 following etoposide treatment as demonstrated in FIG. 7.
This occurs probably because CLN3 modulates endogenous ceramide
levels further upstream and is most likely not a direct effect of
CLN3 on caspase-3. The fact that CLN3 overexpression does not
rescue NT2 cells from killing in response to exogenous C.sub.2- or
C.sub.6-ceramide confirms that CLN3 is acting upstream of ceramide
in the apoptotic pathway. Once killing of cells has been set in
motion by exogenously supplied ceramide, CLN3 is impotent in
blocking cell death (FIG. 8). This is in contrast to Bcl-2, which
acts downstream of ceramide, but upstream of caspase-3 (Perry D K,
et al., Cell Death Differen. 4: 29-33 (1997); Zhang J, et al.,
Proc. Natl. Acad. Sci. U.S.A. 93: 5325-5328 (1996)). The
upregulation of Bcl-2 in surviving neurons from brains of Batten
patients actually implies the following: Bcl-2 and CLN3 protect
neurons from apoptosis via two separate mechanisms that probably
operate independently of one another (Puranam et al, supra). In our
hands, PKC .alpha. is the predominant form of protein kinase C in
NT2 cells. We found no effect of stable CLN3 overexpression on
translocation of PKC .alpha. in NT2 cells upon stimulation with
phorbol esters (0.1 .mu.M PMA) even after 2 hours of stimulation
(Lee J Y, et al., J. Biol. Chem. 271: 13169-13174 (1996)). Also,
stable CLN3 overexpression had no effect on basal or calcium and
lipid stimulated PKC kinase activity (unpublished data). This
suggests that the effect of CLN3 on ceramide generation and
ceramide-induced apoptosis in NT2 cells is independent of PKC
signal transduction pathways identifying an alternate route of
sphingomyelin signal transduction.
EXAMPLE 2
CLN3 is Overexpressed in Cancer Cells
I. Materials and Methods
[0093] Cell Culture. All cells used are human cell lines. The
EB-virus transformed lymphoblast cell line, HS, was grown in
suspension in RPMI 1640 media supplemented with 10% fetal bovine
serum (FBS). 293 (ATCC# CRL-1573), a kidney epithelial cell line,
was grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented
with 10% FBS. The following cell lines were obtained from the
American Type Culture Collection (Rockville, Md.). HL-60 cells
(ATCC# CCL-240) were grown in RPMI 1640 supplemented with 15% FBS.
BT-20 cells (ATCC# HTB-19) were grown in RPMI 1640 supplemented
with 5% FBS. BT-474 cells (ATCC# HTB-121) were grown in RPMI 1640
with Insulin and L-Glutamine supplemented with 10% FBS. BT-549
cells (ATCC# HTB-122) were grown in ROMI 1640 supplemented with 10%
FBS. HCT-116 cells (ATCC# CCL-247) were grown in McCoy's 5A medium
supplemented with 10% FBS. SW1116 (ATCC# CCL-233) and SW480 (ATCC#
CCL-288) were both grown in Leibovitz's L-15 medium supplemented
with 10% FBS. Hs 683 cells (ATCC# HTB-138), A-172 cells (ATCC#
CRL-1620), and A-375 cells (ATCC# CRL-1619) were grown in DMEM
supplemented with 10% FBS. IMR-32 cells (ATCC# CCL-127) and C32
cells (ATCC# 1585) were both grown in Eagle's Minimum Essential
Medium with non-essential amino acids supplemented with 10%
FBS.
[0094] All cell lines were supplemented with 100 units each of
penicillin and streptomycin and maintained at 37% C in a 5%
CO.sub.2 atmosphere with the exception of SW1116, which required a
free gas exchange environment of CO.sub.2 and atmospheric air.
Cells were fed 2-3 times/week.
[0095] Quantitative RT-PCR. RT-PCR was used to assess differences
in CLN3 messenger RNA levels in cell lines. Approximately
2.5.times.10.sup.5 cells were used per sample for the mRNA
harvesting. The mRNA was isolated from each sample by the
oligo-dT-cellulose method using the Quick Prep Micro MRNA
Purification kit (Pharmacia Fine Chemicals, Piscataway, N.J.). The
mRNA was then converted to cDNA by the reverse transcription (RT)
reaction in accordance with known techniques. For each sample,
one-fourth of the RNA obtained, or 5 .mu.l, was used for the RT
reaction. The RT reaction was carried out in buffer containing 50
mM Tris-HCL, pH 8.3, 40 mM KCL, 6 mM MgCl.sub.2, 1 mM DTT, 0.5 mM
each of dATP, dCTP, dGTP and dTTP, 16 .mu.M random primers
(haxamers) and 20 U Rnasin (Promega Corp., Madison, Wis.) in a
final volume of 30 .mu.l. After addition of 1 U of Superscript
reverse transcriptase (GIBCO BRL), the sample was incubated at
20.degree. C. for 10 minutes followed by 50 minutes at 42.degree.
C. The reaction was terminated by adding 70 .mu.l DEPC treated
water and incubating at 94.degree. C. for 5 minutes. The PCR
reactions were set up with 10 .mu.l of the cDNA, synthesized in the
RT reaction above, 1 U of Taq polymerase (Perkin Elmer), and 5
.mu.Ci of .alpha..sup.32P-dCTP in each reaction. The reactions were
performed in Taq buffer (Perkin Elmer), containing 15. mM
MgCl.sub.2, 2.5 mM dCTP and 5 mM each of dATP, dGTP, and dTTP. The
primers use for amplification of CLN3 and cyclophilin, an internal
control for MRNA, are listed below. The reaction conditions used
for amplification were 1 minute at 94.degree. C., 1 minute at
50.degree. C., and 2 minutes at 72.degree. C. for 20 cycles in a
9600 Perkin Elmer Thermocycler (Foster City, Calif.). The PCR
amplified products were analyzed on an 8% nondenaturing acrylamide
gel, which as dried and visualized by autoradiography. The
amplified signal was quantitated on a Molecular Dynamics
phosphorimager using ImageQuant software (Sunnyvale, Calif.). The
results are expressed as the ratio of CLN3 signal to that of the
internal control, cyclophilin; the quantitated value of CLN3
present in the sample was divided by the quantitated value of
cyclophilin.
[0096] Primers used for PCR amplification of human CLN3 were:
[0097] 5' primer: 5'-GGTGGACAGTATTCAGGG-3' (958-976)
[0098] 3' primer: 5'-CTTGGCAGAAAGACGAAC-3' (1229-1246)
Primers used for amplification of human cyclophilin
amplification:
[0099] 5' primer: 5'-AAATGCTGGACCCAACAC-3' (317-334)
[0100] 3' primer: 5'-AAACACCACATGCTTGCC-3' (384-401)
II. Results.
[0101] An initial look at EB-virus transformed lymphoblasts (HS)
and a leukemia cell line (HL-60) show that HL-60 cells had 2.3
times more CLN3 expression than HS after normalizing to the
internal control, cyclophilin (FIG. 8A). Next, 293, a kidney
epithelial cell line, was chosen as a control because a wide
variety of cancer cell morphology is epithelial. To test its
suitability and viability as a control, the level of CLN3 expressed
in 293 was compared to the levels expressed in HCT-116, a colon
cancer cell line, and BT-20, a breast cancer cell line. CLN3 levels
were 4.1 and 2.8 times more in HCT-116 and BT-20, respectively,
than in 293 (FIG. 9A).
[0102] Based on the elevated CLN3 expression of BT-20 compared to
293, two more breast cancer cell lines, BT-474 and BT-549, were
selected for comparison with 293 along with BT-20. Results show
that all three breast cancer cells have upregulated CLN3 MRNA
levels compared to 293 (FIG. 10A). BT-474, BT-20, and BT-549 had
1.9, 3.0, and 4.8 times more CLN3 expression than 293,
respectively. To better substantiate the elevated CLN3 expression
in HCT-116, a well differentiated colon cancer cell line, SW480,
and a poorly differentiated colon cancer cell line, SW1116, were
chosen, along with HCT-116, for comparison with 293 (FIG. 11A).
HCT-116 again showed over-expression of CLN3 compared to 293, with
3.0 times more CLN3 expressed. SW480 showed a more dramatic
over-expression, with 7.7 times the level of 293. SW1116 had the
largest difference in CLN3 expression compared to 293, with 21.5
times more CLN3 expressed.
[0103] A further screening of other cancer types was conducted. Two
melanoma cell lines, C32 and A-375, a neuroblastoma cell line,
IMR-32, a glioma cell line, Hs683, and a glioblastoma cell line,
A-172, were compared against 293 (FIG. 12A). Interestingly, C32
(melanoma) actually showed 0.8 times the CLN3 expression compared
to 293. It was the only cell line examined in this experiment that
had less CLN3 expression than 293. The other melanoma cell line,
A-375, and the glioblastoma cell line A-172, both had a very slight
upregulation of CLN3, with 1.2 times more than 293. Hs683, the
glioma cell line, had 1.7 times the CLN3 expression compared to
293. The neuroblastoma cell line, IMR-32, had a notable 12.7 times
the CLN3 expression compared to 293.
EXAMPLE 3
Preparation of Adenoviral CLN3 Antisense Vectors
[0104] A CMV enhancer/promoter element, juxtaposed to the CLN3 DNA
in an anti-sense configuration, followed by the SV-40
polyadenylation signal was subcloned as a minigene cassette into
the AscI site of the shuttling plasmid pAdAscLwt. PAdAscLwt
contains as a circular plasmid: nucleotides 1-358 of the AdS
genome, followed by the AscI restriction enzyme subcloning site,
followed by nucleotides 3329-15671 of the Ad5 genome, followed by
the bacterial plasmid backbone containing the ampicillin resistance
gene and bacterial origin of replication derived from the commonly
used bacterial plasmid pAT153. The resultant plasmid was designated
pAdAscLwtCMV-antisenseCLN3pA. The shuttle plasmid was linearized by
restriction enzyme digestion, and contransfected into human 293
cells (supplying adenoviral E1 functions in trans) along with ClaI
restriction enzyme digested full length adenoviral DNA. Successful
recombination between the homologous portions of the shuttle
plasmid and the Ad viral DNA sequences resulted in the generation
of a recombinant, E1 deleted Ad vector capable of transducing the
CMV-driven antisense CLN3 cDNA to all cells amenable to Ad vector
infection.
EXAMPLE 4
CLN3 Antisense Vectors Inhibit
Growth of Cancer Cells
[0105] Cell proliferation was assayed by counting cell numbers and
measuring [.sup.3H]-thymidine incorporation. Data were expressed as
the mean values of three wells.
[0106] To observe a dose-response relationship, BT-20 breast cancer
cells and SW1116 colon cancer cells were plated overnight in
24-well plates at a density of 1.times.10.sup.5 cells/well. Then,
cells were infected with Ad-Antisense-CLN3 or adenovirus (vector)
at 8, 40, and 80 MOI. After 24 hours, 0.5 .mu.Ci/ml
[.sup.3H]thymidine (DuPont) was added to each well for 4 hours. To
measure [.sup.3H]thymidine incorporation, each well was washed with
two changes of ice-cold PBS. Thereafter, DNA was precipitated with
5% trichloroacetic acid and dissolved in 0.4 ml of 0.25 M NaOh.
Incorporated radioactivity was detected by liquid scintillation
counting. Data for SW1116 is given in FIG. 13.
[0107] To study a time-response relationship, BT-20 and SW1116
cells were plated overnight in 12 well plates at a density of
2.times.10.sup.5 cells/well. Then, cells were infected with
Ad-Antisense-CLN3 or adenovirus (vector) at 10 MOI. At the
indicated dates, cell prolilferation was assayed by
[.sup.3H]thymidine incorporation and/or by counting cells using a
hemocytometer. Cell growth of SW116 after one-day infection is
given as thymidine uptake in FIG. 14 and as cell numbers at various
days after infection in FIG. 15. Cell growth of BT-20 cells after
one-day infection is given in FIG. 16, and as cell numbers at
various days after infection in FIG. 17. Finally, thymidine uptake
of BT-20 cells at various days after infection is given in FIG.
18.
[0108] These data indicate that the proliferation of cancer cells,
specifically colon cancer cells and breast cancer cells, is
inhibited by infection with a vector that expresses an antisense
construct to the CLN3 gene transcript.
[0109] The foregoing is illustrative of the present invention, and
is not to be construed as limiting thereof. The invention is
defined by the following claims, with equivalents of the claims to
be included therein.
Sequence CWU 1
1
5 1 19 DNA Artificial sequence Synthetic oligonucleotide 1
ggtggacagt attcaaggg 19 2 18 DNA Artificial sequence Synthetic
oligonucleotide 2 cttggcagaa agacgaac 18 3 18 DNA Artificial
sequence Synthetic oligonucleotide 3 aaatgctgga cccaacac 18 4 18
DNA Artificial sequence Synthetic oligonucleotide 4 aaacaccaca
tgcttgcc 18 5 20 PRT Artificial sequence Synthetic polypeptide 5
Ala Ala His Asp Ile Leu Ser His Lys Arg Thr Ser Gly Asn Gln Ser 1 5
10 15 His Val Asp Pro 20
* * * * *