U.S. patent application number 10/070676 was filed with the patent office on 2003-03-27 for genetic markers of toxicity, preparation and uses thereof.
Invention is credited to Bracco, Laurent, Schweighoffer, Fabien, Tocque, Bruno.
Application Number | 20030059788 10/070676 |
Document ID | / |
Family ID | 9549766 |
Filed Date | 2003-03-27 |
United States Patent
Application |
20030059788 |
Kind Code |
A1 |
Tocque, Bruno ; et
al. |
March 27, 2003 |
Genetic markers of toxicity, preparation and uses thereof
Abstract
The present invention describes new methods for the
determination of the potential toxicity of test compounds, as well
as the kits and tools for the implementation of these methods. The
invention also describes methods for generating nucleic acid
sequences that can be used as genetic markers of toxicity. The
invention is based in particular on the creation of differential
nucleic acid banks characteristic of situations in which cell
viability and/or proliferation are deregulated, and on the
demonstration that these banks can be used to evaluate the toxicity
profile of compounds with reliability and high sensitivity. The
invention is of special utility in the pharmaceutical industry for
analysis of the toxicity profile of compounds involved in drug
development and/or in pharmaceutical compositions.
Inventors: |
Tocque, Bruno; (Courbevoie,
FR) ; Bracco, Laurent; (Paris, FR) ;
Schweighoffer, Fabien; (Vincennes, FR) |
Correspondence
Address: |
CLARK & ELBING LLP
101 FEDERAL STREET
BOSTON
MA
02110
US
|
Family ID: |
9549766 |
Appl. No.: |
10/070676 |
Filed: |
August 2, 2002 |
PCT Filed: |
March 22, 2001 |
PCT NO: |
PCT/FR00/02503 |
Current U.S.
Class: |
435/6.11 ;
435/91.2; 702/20 |
Current CPC
Class: |
C12Q 1/6809 20130101;
C12Q 1/6837 20130101; C12N 15/1034 20130101 |
Class at
Publication: |
435/6 ; 435/91.2;
702/20 |
International
Class: |
C12Q 001/68; G06F
019/00; G01N 033/48; G01N 033/50; C12P 019/34 |
Claims
1. Method of analysis of the toxic potential of a test compound,
comprising at least one hybridization step between a) a nucleic
acid sample from cells treated with this compound and b) a nucleic
acid bank corresponding to genetic events characteristic of
deregulation(s) in cell signalling pathway(s), the hybridization
profile indicating the toxic potential of the test compound.
2. Method of analysis of the toxic potential of a test compound,
comprising at least a separate hybridization step between a)
labelled nucleic acid probes corresponding to RNA from untreated
cells and cells treated with said test compound and b) a nucleic
acid bank corresponding to genetic events (transcriptional and/or
splicing events) characteristic of a situation(s) of deregulation
in cell signalling pathway(s), the hybridization profile indicating
the toxic potential of the test compound.
3. Method according to claim 1 or 2, characterized in that the
nucleic probes a) correspond to messenger RNA from treated and
untreated cells.
4. Method according to one of claims 1 to 3, characterized in that
the nucleic probes a) are cDNA or cDNA fragments prepared from the
RNA of treated and untreated cells.
5. Method according to any one of the previous claims,
characterized in that the nucleic probes a) are amplification
products.
6. Method according to any one of the previous claims,
characterized in that the nucleic probes a) are labelled by
radioactive, fluorescent, enzymatic or calorimetric labels.
7. Method according to any one of the previous claims,
characterized in that the test compound is an individual compound
or is present in a mixture with other substances.
8. Method according to any one of the previous claims,
characterized in that the bank b) comprises nucleic acids
corresponding to genes whose level of expression is modified in
situations of deregulation(s) of cell signalling pathway(s).
9. Method according to one of claims 1 to 7, characterized in that
the bank b) comprises nucleic acids of which at least part of the
sequence corresponds to the sequence of genes that are
differentially spliced during deregulation(s) of cell signalling
pathway(s).
10. Method according to one of claims 1 or 2, characterized in that
the bank b) comprises nucleic acids according to claim 8 and
nucleic acids according to claim 9.
11. Method according to claim 9, characterized in that the bank b)
is prepared by hybridization between a nucleic acid population from
a cell in a situation of deregulation(s) of cell signalling
pathway(s), and a nucleic acid population from a cell in a control
situation, and separating, from the hybrids formed, nucleic acids
corresponding to differential splicings.
12. Method according to any one of the previous claims,
characterized in that the situation of deregulation is induced by
modification of the activation, preferably of the expression of a
gene that initiates or carries out apoptosis.
13. Method according to claim 12, characterized in that the
situation of deregulation is produced by induction or enhancement
of the activation, preferably of the expression of an
anti-oncogene.
14. Method according to claim 13, characterized in that the
anti-oncogene is chosen from among p53, Rb, p73, myc, TUPRO-2 and
NHTS.
15. Method according to claim 12, characterized in that the
situation of deregulation is induced by modification of the
activation, preferably of the expression of a gene involved in cell
growth or viability.
16. Method according to claim 12, characterized in that the
situation of deregulation is induced by constitutive or inducible
activation, preferably expression of all or part of a gene involved
in cell growth, cell viability or apoptosis.
17. Method according to any one of claims 1 to 7, wherein the bank
b) comprises at least 1 clone of sequence selected from SEQ ID Nos:
1 to 37, more preferably at least 5.
18. Method according to any one of claims 1 to 7, wherein the bank
b) comprises a set of probes, in particular 5 probes or more,
preferably 10 probes or more, each of said probes being
complementary to a part of a gene selected from the following
genes: Aldolase A; S4 subunit of proteasome 26S; Alpha-tubulin;
Glucosidase II; lamin B receptor homologue; EF1-alpha; Fra-1;
tyrosine kinase AX1 receptor; spliceosomale Protein SAP62; TRAF-3;
EF2; TEF-5; CDC25b; interleukine-1 receptor-associated kinase
(<<IRAK>>); WAF-1; c-fos (exon 4); ckshs1; PL16;
NFAR-2; phosphatidylinositol4-kinase, ERF, Eph type receptor
tyrosine kinase (hEphB1b); BAF60b protein of the SWI/SNF complex;
EB1; MSS1; retinoic acid alpha receptor (RARa); translation
initiation factor eiF4A; STE20 type kinase; protein HSP 90kda;
Lipocortin II; protein TPT1 (<<translationally controlled
tumor proteon >>); Hsc70; Cytokeratin 18; 2-oxoglutarate
dehydrogenase; mitochondrial gene NADH6; mitochondrial gene NADH
deshydrogenase 4; alpha subunit of mitochondrial ATP synthase.
19. Method according to any one of the previous claims,
characterized in that the treated or untreated cells a) and the
cells in a situation of deregulation b) are of a different
type.
20. Method according to any one of the previous claims,
characterized in that the treated or untreated cells are mammalian
cells, preferably of human origin.
21. Method according to any one of the previous claims,
characterized in that the treated or untreated cells are cell
lines.
22. Method according to any one of the previous claims,
characterized in that the treated or untreated cells are primary
cultures.
23. Method according to any one of the previous claims,
characterized in that the treated or untreated cells are cells
extracted from the organs or tissues of treated or untreated
animals.
24. Method of diagnosis of the toxic potential of a test compound,
comprising at least the hybridization between, on the one hand,
labelled nucleic probes corresponding to mRNA from untreated cells
and a nucleic acid bank corresponding to genetic events
characteristic of situation(s) of deregulation of cell signalling
pathway(s) and, on the other hand, labelled nucleic probes
corresponding to mRNA from cells treated with said test compound
and said nucleic acid bank corresponding to genetic events
characteristic of situation(s) of deregulation of cell signalling
pathway(s).
25. Method according to claim 24, characterized in that the
situation or situations of deregulation are situations of
deregulation of cell growth and/or cell viability.
26. Method according to claim 25, characterized in that the nucleic
acid bank characteristic of situation(s) of deregulation is a
nucleic acid bank characteristic of cells in situations of
deregulation of cell growth, notably transformed cells, in
particular tumor cells.
27. Method according to claim 24, characterized in that the nucleic
acid bank comprises at least 1 clone of sequence selected from SEQ
ID Nos: 1 to 37, more preferably at least 5.
28. Use of nucleic acid clones corresponding to genetic events
(transcriptional and/or splicing events) characteristic of
situation(s) of deregulation of cell signalling pathway(s), as
genetic markers of toxicity.
29. Use according to claim 28, of a clone of sequence selected from
SEQ ID Nos: 1 to 37.
30. Kit for the study of the toxic potential of a test compound,
comprising at least: a nucleic acid bank corresponding to genetic
events (transcriptional and/or splicing events) characteristic of
situation(s) of deregulation of cell signalling pathway(s)
31. Kit according to claim 30, characterized in that the bank is a
nucleic acid bank characteristic of cells in situations of
deregulation of cell growth or cell viability.
32. Kit according to claim 30, wherein the bank comprises at least
1 clone of sequence selected from SEQ ID Nos: 1 to 37, more
preferably at least 5.
33. Kit according to any one of claims 30 to 32, wherein the bank
is deposited on a support.
34. Nucleic acid bank comprising nucleic acid clones corresponding
to genetic events (transcriptional and/or splicing events) common
to cells in a situation(s) of deregulation of cell signalling
pathway(s) and a toxic situation.
35. Bank according to claim 34, wherein the bank comprises at least
1 clone of sequence selected from SEQ ID Nos: 1 to 37, more
preferably at least 5.
36. Process of production of genetic markers of toxicity,
comprising hybridization between a nucleic acid population derived
from cells in a situation(s) of deregulation of cell signalling
pathway(s), and a nucleic acid population derived from cells in a
control situation, the isolation from the hybridization product of
clones characteristic of the situation(s) of deregulation of cell
signalling pathway(s), and the hybridization of the clones obtained
with a nucleic acid sample derived from cells in a situation of
toxicity.
37. Process of preparation of a DNA chip that can be used to
diagnose the potential toxicity of a test compound, comprising the
application on a solid support of one or more nucleic acid
preparations characteristic of situation(s) of deregulation of cell
signalling pathway(s).
38. A method for the identification of SNPs or other mutations or
polymorphisms that allow the assessment of the response of a
subject to a given compound, the method comprising (i) the
identification in vitro of nucleic acids characteristic of splicing
events induced in a cell treated with said compound and (ii) the
identification of SNPs or other mutations or polymorphisms in the
gene or genes corresponding to nucleic acids identified in (i),
said SNPs or other mutations or polymorphisms allowing the
assessment of the response of a subject to said given compound.
39. A method for the evaluation of the sensitivity or of the
response of a subject to a test compound, comprising the analysis,
from a biological sample comprising DNA from said subject, of the
presence in the DNA of said subject of polymorphisms, SNPs, or
other genomic alterations present in genes whose splicing is
modified in response to said compound.
Description
[0001] The present invention is related to the technical areas of
biotechnology, medicine, biology and biochemistry. Its applications
concern the fields of human, animal and plant health. More
particularly, the invention sets forth new methods for determining
the potential toxicity of test compounds or for predicting the
sensitivity or response of patients to compounds, as well as the
kits and tools for the implementation of these methods. The
invention also describes methods for obtaining nucleic acid
sequences that can be used as genetic markers of toxicity. The
invention is of special utility in the pharmaceutical industry for
analysis of the toxicity profile of compounds involved in drug
development and/or in pharmaceutical compositions.
[0002] Toxicity is the major reason for abandoning candidate
therapeutic molecules during preclinical and clinical development.
To our knowledge, at the present time there are no tests by which
to rapidly determine the toxicity profile of a compound in man. Yet
the regulatory authorities require that new candidate drugs undergo
toxicity, mutagenicity, carcinogenicity and teratogenicity testing
in animals as well as clinical trials in man. These tests are long
and costly and are only partially satisfactory. For example, animal
toxicity is far from being a reflection of human toxicity.
Furthermore, the small number of patients enrolled in clinical
trials does not systematically allow identification of toxicities
associated with a small, specific population. The development,
perfection and use of such tests should make it possible to
identify and remove toxic compounds from the development process as
far upstream as possible. In this manner new drugs could be
marketed sooner and at a lesser cost to drug companies and, in
turn, to health care organizations and consumers. In addition, such
tests might also make it possible to detect some toxicities which
currently come to light only during the post-marketing period.
[0003] The tests which are currently available do not provide
sufficient characterization of toxicity markers or the potential
toxicity of compounds. Some tests in bacteria, such as the Ames
test, or in yeast, such as the test described in patent U.S. Pat.
No. 4,997,757, evaluate the mutagenic potential of compounds. These
tests can only detect damage at the level of the DNA. Yet many
drugs exert toxic effects without being mutagens and cannot
therefore be flagged by tests such as these. Other tests, such as
that described in application WO 94/17208, make use of certain
known eukaryotic gene promoters or response elements from these
promoters, induced under different conditions of stress, to
characterize drug toxicity. However, the small number of such
genetic markers and the process being measured (promoter activity)
do not allow prediction of the potential toxicity of the compounds.
What's more, these tests are difficult to implement because they
involve the culturing of transformed cell lines comprising one or
more genetic constructions.
[0004] The present invention now describes rapid, effective methods
by which to determine the potential toxicity of test compounds, as
well as the tools and kits for the implementation of such methods.
In the context of the invention, the term "toxicity" refers to any
adverse and/or side effect of a compound on the metabolism of a
cell or a tissue such as, in particular, its mutagenic,
carcinogenic or teratogenic potential, and more generally any
alteration in metabolism that can result in a harmful effect of the
compound on the cell or the tissue. The present invention is based
more specifically on genomics and on the development of genetic
markers of toxicity that can be used to predict the toxic potential
of any type of compound on any type of cell. The present invention
also sets forth new methods for obtaining nucleic acid sequences
that allow determination of the toxicity of compounds (eg., genetic
markers of toxicity), particularly compounds entering into drug
development and/or pharmaceutical compositions.
[0005] The present invention is based in part on the demonstration
that genetic markers can be created and used to evaluate the
toxicity of test compounds.
[0006] In particular, and in an advantageous manner, these markers
can be used regardless of the toxic compound being tested, and
regardless of the type of cell in which the test compound is being
studied. Such markers, as well as the supports, kits and methods of
the invention advantageously lead to the rapid generation of
toxicity profiles that are particularly thorough and reliable.
Furthermore, these markers, supports, kits and methods of the
invention also make it possible to determine and assign a toxicity
index to the test compounds.
[0007] In particular, the present invention demonstrates that there
exist genetic events that are common to situations of toxicity and
to cellular metabolic pathways induced in the absence of toxic
compounds. Such genetic events can therefore be induced and then
used as markers of toxicity. As will be described in detail herein,
such markers can further be selected or modified for the
constitution of improved banks allowing a more highly predictive
diagnosis of the toxicity of a compound. More specifically, the
present invention now shows that genetic markers induced in a cell
in a situation where cell signalling pathways are deregulated,
particularly a cell in which cell viability and/or proliferation
are deregulated (for example, in a situation of apoptosis), can be
efficiently used to characterize the toxicity profile of test
compounds. In an advantageous manner, the invention also shows that
these markers can be used in toxicity tests irrespectively of the
type of compound and the type of cell used. The invention also
provides for the constitution of differential banks of nucleic
acids characteristic of deregulated cell signalling pathway(s),
particularly situations in which cell viability and/or
proliferation are deregulated, and demonstrates that these banks
can be used for a reliable, highly sensitive evaluation of the
toxicity profile of compounds.
[0008] The invention also allows the identification and/or the
characterization of mutations or polymorphisms (in particular of
SNPs "Single Nucleotide Polymorphisms") which are characteristic of
the profile of subjects, particularly of the response profile to a
compound or treatment, of a sensitivity profile to a toxic effect,
etc. In this regard, the libraries produced represent an extraction
of sequences which are mobilised during deregulations of cell
signalling and, particularly, during stress that can lead to
apoptosis. The invention shows that the expression of these
sequences is modified in a specific manner in response to given
compounds. Mutations or polymorphisms, also termed SNPs (Single
Nucleotide Polymorphisms), can affect genes from which these
sequences derive. Individuals presenting such polymorphisms are
thus susceptible to exhibit altered responses to compounds that
mobilise or recruit the expression of these genes. One aspect of
this invention is thus also to enable a pertinent selection of the
genes involved in toxic phenomenon. More precisely, the invention
allows to further select, within this selection, genes that are
involved in the response to a given compound. The invention
provides a basis for the identification of polymorphisms in a
limited (10 to 50) number of genes whose expression modifications
are linked to the toxic impact of a given compound, said compound
being of any pharmacological class and of any chemical family.
[0009] One object of the invention is therefore based more
specifically on the use of genetic markers characteristic of
situation(s) of deregulation of cell signalling pathway(s)
(situation(s) of "deregulation") to characterize the toxicity
profile of test compounds. The invention more preferably concerns
the use of genetic markers induced in a situation of deregulation
to characterize the toxicity profile of test compounds. The
invention more preferably concerns the use of nucleic acid clones
corresponding to genetic events, such as transcriptional and/or
splicing events, that are characteristic of situations of
deregulation, as genetic markers of toxicity.
[0010] The invention also concerns methods for analysis of the
potential toxicity of a test compound, comprising at least one
hybridization step between a) a sample of nucleic acids from cells
treated with this compound and b) a preparation (for example, a
bank) of nucleic acids corresponding to genetic events
characteristic of situation(s) of deregulation, the hybridization
profile indicating the toxic potential of the test compound.
[0011] The invention further concerns the kits for the
implementation of these methods, as well as the compositions and
nucleic acid banks comprising genetic markers of deregulation(s) of
cell signalling pathways, and also the methods by which to generate
such markers.
[0012] The invention also relates to the use of clones or nucleic
acids as disclosed, for the identification of mutations or
polymorphisms, particularly SNPS, that are correlated to the
response of subjects to pharmaceutical compounds. The invention
indeed allows the identification of a set of genes whose expression
is altered in response to a given compound. These genes may present
mutations or polymorphisms (including SNPs) in human populations.
The invention allows the selection of genes and their polymorphisms
to be studied in order to segregate patients with respect to their
genotype for their capacity to respond in a more or less optimal
manner to a compound and, more particularly, with limited risks of
toxicity.
[0013] In this respect, the invention relates, more generally, to
the use of nucleic acids representative of splicing events
characteristic of a given physio-pathological situation, for the
identification or the characterisation of SNPs correlated to said
situation.
[0014] The invention also concerns a method for the identification
of SNPs or other mutations or polymorphisms that allow the
assessment of the response of a subject to a given compound, the
method comprising (i) the identification in vitro of nucleic acids
characteristic of splicing events induced in a cell treated with
said compound and (ii) the identification of SNPs or other
mutations or polymorphisms in the gene or genes corresponding to
nucleic acids identified in (i). The invention thus provides
methods and means to select genes for which polymorphisms are
associated to a toxicity for a given compound.
[0015] The invention also relates to a population of nucleic acids
specific for polymorphisms or SNPs thus identified, in particular
of nucleotide primers for the selective amplification of said
polymorphisms, or of probes for the selective hybridisation to said
polymorphisms.
[0016] The invention also concerns methods for the analysis (e.g.,
the prediction, the evaluation or the determination) of the
response of a subject to a test compound, comprising the analysis
of polymorphisms, in particular SNPs, as defined above.
[0017] The present invention is therefore based in particular on
the identification and development of genetic markers of toxicity
that can be used for predictive toxicity testing of test compounds,
or as targets for the study of the mechanisms of cell death or the
research and development of therapeutic agents.
[0018] 1. Identification and Development of Genetic Markers of
Toxicity
[0019] Cell viability and differentiation are regulated by the
balance that exists between mitosis and apoptosis. In the body,
tissue homeostasis is also regulated by this equilibrium between
cell proliferation and cell death. Alterations in this equilibrium
form the basis of disease, whether they be related to an excess
(neurodegenerative disorders) or to a defect in apoptosis
(rheumatoid arthritis, cancer). The present invention is based in
particular on the hypothesis that disturbances in this equilibrium
can also be involved in toxic phenomena and that genetic markers
characteristic of these events of deregulation of cell signalling
pathways (especially cell viability and/or proliferation) might
represent efficient markers of toxicity.
[0020] The present invention now shows that there are genetic
events common to these situations of deregulation of cell
signalling pathways, and to situations of toxicity induced by
compounds. The present invention also provides for methods by which
to identify, characterize, select and isolate nucleic acid clones
corresponding to these genetic events, and shows that they can be
efficiently used as genetic markers of toxicity. The invention also
describes the establishment of banks of such clones, particularly
on solid supports, and their use in methods for evaluation of the
toxicity of test compounds.
[0021] 2. Definitions
[0022] In the context of the present invention, the term situation
of deregulation, or "deregulation", refers to any situation in
which one or more cell signalling pathways are deregulated or
altered, and in particular any situation in which cell growth
and/or viability are deregulated. This therefore concerns any cell
in which one or more cell signalling pathways have been altered, in
particular induced or repressed, for example a situation of
apoptosis, as will be described hereafter in further detail.
[0023] In the context of the present invention, the term "genetic
event characteristic of a situation of deregulation" refers more
specifically to any modification of gene activity, particularly of
gene expression (enhancement or repression, inhibition or
induction, etc.), post-transcriptional regulation (particularly
splicing), replication, appearance of deletions, etc.,
characteristic of deregulation, i.e. induced in a cell in a
situation of deregulation. It is understood that each individual
genetic event characteristic of deregulation is not necessarily
specific to this situation, in so far as some cell signalling
pathways participate in a number of cellular processes. However,
the nucleic acid banks described by the invention contain different
clones and therefore effectively represent the genetic signatures
characteristic of such situations. The clones described by the
invention more preferably correspond to transcriptional and/or
splicing genetic events characteristic of deregulation(s). In the
context of the invention, the term "transcriptional event"
encompasses more specifically any activity of gene expression, i.e.
the production of primary transcripts, premessengers and messengers
from coding regions. The term "splicing event" refers more
particularly to any splicing event related to a situation of
deregulation, i.e. the appearance of specific splicing forms, the
disappearance of specific splicing forms, or possibly the
modulation of the quantity of splicing forms, etc.
[0024] The invention therefore describes the production of nucleic
acid clones corresponding to such genetic events characteristic of
situations where one or more cell signalling pathways are altered,
and their use as genetic markers of toxicity. In the context of the
invention, a nucleic acid clone corresponding to such a genetic
event refers to any nucleic acid or nucleic acid fragment
comprising at least one region whose sequence is specific to this
event. For example, it may be any nucleic acid comprising a
sequence specific for a splicing form or a deletant characterizing
a situation of deregulation, or even a gene which is specifically
regulated or expressed in a situation of deregulation. Such clones
are therefore nucleic acids that can hybridize, in the nucleic acid
test sample, with splicing forms characteristic of the situation of
deregulation or with genes preferentially or specifically expressed
in a situation of deregulation. Through hybridization, these clones
thereby reveal the presence of targeted genetic events in a test
nucleic acid population.
[0025] 3. Preparation of Genetic Markers of Toxicity
[0026] As noted above, the present invention shows that genetic
markers characteristic of situations of deregulation can be used as
markers of toxicity, and in this regard it describes the
development of methods by which to generate and isolate such
markers.
[0027] One subject of the invention is therefore based on the
methods by which to generate genetic markers of toxicity. The
methods of the invention more specifically comprise the
establishment of clones and nucleic acid banks characteristic of
deregulation(s) in cell signalling pathway(s). These clones and
banks can be generated in different ways, as described hereafter.
Furthermore, the banks of the invention can contain variable
quantities of nucleic acid clones. In addition, these banks are
advantageously deposited on supports to facilitate hybridization
and analysis of the hybridization profile.
[0028] The starting material used for the production of the clones
and banks primarily comprises populations of nucleic acids derived
from cells in a situation of deregulation, and populations of
nucleic acids derived from cells in a control situation. In a more
specific embodiment, the process of production of genetic markers
of toxicity according to the invention advantageously comprises a
hybridization step between a nucleic acid sample derived from a
cell in a situation of deregulation, and a nucleic acid sample
derived from a cell in a control situation. This hybridization step
enables the generation of (banks of) nucleic acid clones
characteristic of the cell in the situation of deregulation
relative to the control situation.
[0029] The nucleic acid populations used are, for example, total
RNA or messenger RNA from cells in a situation of deregulation and
a control situation, or nucleic acids derived from this total or
messenger RNA by reverse transcription, amplification, cloning into
vectors, etc. These populations can be prepared by conventional
methods known to those skilled in the art, such as reverse
transcription, amplification, etc.
[0030] The choice of the situation or situations of deregulation,
the control situation or situations, and the methods for production
and isolation of the markers, is important because it determines
the relevance of the markers generated, and therefore of the banks,
and also their ease of use.
[0031] 3.1. Starting Material
[0032] As indicated above, the genetic markers of toxicity
according to the invention are generally prepared initially from
nucleic acid preparations derived from cells in a situation of
deregulation. It is understood that these markers, once isolated
and characterized, can be reproduced or amplified by artificial
means such as PCR, artificial synthesis (when the sequences are
available), amplification in bacteria, replication of banks, etc.,
as illustrated hereafter.
[0033] As noted above, in the context of the present invention the
term "situation of deregulation" refers to any situation in which
cell growth and/or viability are deregulated, for example any cell
in which at least one cell signalling pathway has been altered.
[0034] According to a specific embodiment of the invention, the
cell in the situation of deregulation is a cell in which the
expression of all or part of a gene involved in cell growth or
viability has been modified. According to another specific
embodiment of the invention, the cell in the situation of
deregulation is a cell in which the expression of all or part of a
relevant oncogene or anti-oncogene has been modified.
[0035] According to another specific embodiment of the invention,
the cell in the situation of deregulation is a cell in which the
expression of all or part of a gene involved in apoptosis has been
modified.
[0036] Apoptosis is a term familiar to those skilled in the art; it
refers to the process of programmed cell death. Apoptosis
encompasses all the mechanisms, genes and metabolic pathways that
lead to cell death. Examples of factors that initiate or carry out
apoptosis include the anti-oncogenes, Fas and TNF receptors,
adaptors (particularly "death domain"), members of the bcl2 family,
or caspases, which are involved in ontogenic morphogenesis,
pathological deregulations and cell death.
[0037] Within the scope of the invention, a situation of apoptosis
can be any situation involving initiation, execution or termination
of apoptosis, i.e. a situation of apoptosis at any stage in the
process of cell death, including very early stages in which only
some metabolic pathways (or cell signalling pathways) have been
altered, for example by activation, repression, stimulation, etc.
The clones of the invention can therefore be produced from any cell
in which at least one cell signalling pathway has been altered,
particularly in which at least one apoptotic pathway has been
initiated. These can be cells in which a metabolic pathway involved
in apoptosis has been induced or stimulated, even if this induction
or stimulation is carried out in sublethal conditions (i.e. not
resulting in cell death).
[0038] In this regard, the cells can be in a natural situation of
apoptosis, as for example a population of tumor cells, certain
nerve cells in pathological situations, etc., or cells in which
apoptosis has been artificially induced. In the latter case, the
situation of apoptosis can be induced by different types of
treatments, particularly by modulation of the activity, preferably
of the expression of genes that initiate or carry out
apoptosis.
[0039] In a particular embodiment, the genetic markers of the
invention are produced initially from a population of cells in
which a situation of apoptosis has been artificially induced, by
the induction or repression of the activity, preferably of the
expression of all or part of a gene that initiates or carries out
apoptosis.
[0040] According to a preferred embodiment of the invention, the
cell in a situation of deregulation is a cell in which the
activation, preferably the expression of all or part of an
anti-oncogene (or tumor suppressor gene) has been induced or
enhanced, for instance by over-expression, post-translational
modification(s) (phosphorylation, glycosylation, etc.), deregulated
intra-cellular localisation, association with different functional
partners, etc.
[0041] In an especially preferred embodiment of the present
invention, the genetic events characteristic of a situation of
deregulation are genetic events induced by modification of the
expression of one or more genes that initiate or carry out
apoptosis, particularly one or more tumor suppressor genes (or a
functional variant of such genes).
[0042] This can particularly be any cell in which overexpression of
an anti-oncogene has been induced. In particular, this can be a
cell in which a gene construct has been introduced to induce or
enhance the quantity of an anti-oncogene in this cell. Specific
examples of anti-oncogenes (also called tumor suppressor genes)
include p53 (Eliyahu et al., Proc Natl Acad Sci USA (1989) 86:
8763-7; Finlay et al., Cell (1989) 57: 1083-93); Rb (Lee et al.,
Science (1987) 235: 1394-9); p73 (Kaghad et al., Cell (1997) 90:
809-19); TUPRO-2 (U.S. Pat. No. 5,849,899); NHTS (U.S. Pat. No.
5,892,016); p15 (Hannon et al., Nature 371 (1994) 257); p16 (Ivison
et al., Nature Genet. 371 (1994) 180); etc., but this list is not
exhaustive.
[0043] A typical example of a situation of deregulation according
to the invention is generated by the induction or enhancement of
p53 activity, preferably of p53 expression. The p53 tumor
suppressor gene, or anti-oncogene, is situated at the crossroads
between cell life and death, negatively regulating cell growth by
inducing cell cycle arrest at the end of the G1 phase. This cell
cycle arrest is characterized by repression of the expression of
genes involved in G1 to S progression and by induction of specific
genes. Abolition of these p53 functions is one of the mechanisms
involved in the development of a large number of tumors in which
p53 deletion or mutations are observed. Overexpression of p53 can
induce apoptosis in different cell types. This overexpression
induced by infection with a virus permitting expression of wild
type p53 forms is the basis for the gene therapy of cancer.
Induction of p53 expression can also occur physiologically,
particularly during restructuring of the mammary glands after
lactation has ceased. Apoptosis induction by removal of serum, a
situation which affects a large number of metabolic pathways, has
also been reported to involve p53 stabilization.
[0044] p53 induction triggers programmed cell death in cells when
DNA damage saturates cell repair systems. This apoptosis induction
involves the Fas system and the caspases in particular, whose
messengers must be spliced to isoforms that are competent for cell
death.
[0045] Furthermore, p53 is known to regulate the expression of
genes involved in other various cell processes, such as cell cycle
regulation, angiogenesis, response to oxidative stress, DNA
alteration or cell growth. P53 also plays a direct role on the cell
response to DNA alterations, hypoxia, cellular redox potential, or
cell adhesion. In addition, it has been shown that p53 can migrate
from the cell nucleus towards mitochondria, where it can interact
with hsp70 protein, leading to cell death. The signatures obtained
with the present invention upon induction of p53 thus enable the
study of all of these signalling pathways and to provide a complete
and predictive response on the toxic potential of compounds.
[0046] The invention more specifically describes the use of genetic
markers induced by p53 activation, preferably by p53 expression (in
particular wild type p53) to characterize the toxicity profile of
test compounds. The invention more preferably concerns the use of
nucleic acid clones corresponding to genetic events, such as
transcriptional and/or splicing events, induced by p53 activation,
preferably p53 expression (in particular wild type p53), as genetic
markers of toxicity.
[0047] The invention more specifically describes the obtaining and
use of cDNA clones corresponding to qualitative and/or quantitative
genetic events that differ between control cells (for example
growing cells) and cells engaged in apoptosis through induction of
wild type p53.
[0048] It is understood that there are other possibilities by which
to subject cells to a stress that can engage them in programmed
cell death (apoptosis) or alter cell signalling pathways. The
invention therefore also concerns the use of other cell models such
as those based on inducible (or constitutive) expression of all or
part of anti-oncogenes, promotors, initiators and mediators of
apoptosis. Among these genes can be advantageously chosen RB, the
retinoblastoma gene product; p73, a p53 homolog; myc; or any other
protein or protein fragment capable of interfering with cell growth
and viability. As noted above, this can also be a tumor cell or a
degenerated nerve cell, for example.
[0049] Other types of starting material consist, for example, of
samples containing cells in which proliferation has been
deregulated by activation of oncogene cascades (such as the ras
signalling pathway), inactivation of tumor suppressor genes,
inhibition of apoptotic cascades, inhibition or activation or
kinase-mediated pathways (such as p13 kinase which stimulates Akt
protein), inhibition of gene expression by antisense RNA or
oligonucleotides, etc. A specific example of cells consists of
tumor cells obtained from tumor biopsies, in which case the genetic
markers according to the invention are those characteristic of the
tumor cell relative to a specimen of control tissue, particularly
healthy tissue, obtained by biopsy.
[0050] Furthermore, the situation of deregulation can also be
induced by one or more selected toxic compounds that act on cell
viability via processes that alter genome integrity (genotoxicity),
cell redox potential, cell surface receptor response, protein
conformation or energy status. In particular, such compounds can be
used in combination with the aforementioned induction methods based
on modification of gene expression.
[0051] The cell population used to induce a situation of
deregulation can be of diverse origin and nature, such as
epithelial, hepatic, pulmonary, nerve, muscle, fibroblastic cells,
etc. Preferably these are human cells. They can be primary cultures
or established lines, in which deregulation is induced under the
aforementioned conditions. They can also be tumor specimens which
will contain cells in a situation of deregulation, or
serum-deprived cells. As indicated below, the genetic markers of
deregulation for use as markers of toxicity according to the
invention can be produced from different cell types.
[0052] The control situation used generally corresponds to a
population of cells of the same type as the cells used for the
situation of deregulation, although different cell types can be
used. The control situation can be a normal, quiescent or
proliferative situation or even a situation of deregulation, but at
a degree or stage less advanced than the reference situation of
deregulation.
[0053] The situation of deregulation is typically a situation of
induction of the activation, preferably of the expression of a
tumor suppressor gene such as p53 or Rb, and the control situation
is a situation in which induction of this activation, preferably of
this expression is absent, or at a lower level. In another example,
the situation of deregulation is a population of tumor cells and
the control situation is a population of the same but healthy
cells. Specific examples of cells are given in the experimental
section.
[0054] 3.2. Production of Markers and Banks
[0055] As noted above, the process of production of genetic markers
of toxicity according to the invention advantageously comprises a
hybridization step between a nucleic acid sample derived from a
cell in a situation of deregulation, and a nucleic acid sample
derived from a cell in a control situation. This hybridization step
enables the generation of nucleic acid clones characteristic of the
cell in the situation of deregulation relative to the control
situation.
[0056] The nucleic acid populations are, for example, total RNA or
messenger RNA from cells in a situation of deregulation and a
control situation, or nucleic acids derived from this total or
messenger RNA by reverse transcription, amplification, cloning into
vectors, etc. These nucleic acids can be prepared according to
methods familiar to those skilled in the art. Briefly, these
methods generally comprise lysis of the cells, tissue or sample and
isolation of the RNA by extraction. In particular, this can consist
of a treatment with chaotropic agents such as guanidium
thiocyanate, which destroys the cells and protects the RNA,
followed by RNA extraction with solvents such as phenol or
chloroform. Such methods are well known to those skilled in the art
(see Maniatis et al., Chomczynski et al., Anal. Biochem. 162 (1987)
156), and can be easily put into practice by using commercially
available kits such as the kit US73750 (Amersham) for total RNA.
The RNA used does not have to be perfectly pure, and in particular
the presence in the preparation of traces of genomic DNA or other
cell components such as protein, etc. is not a problem so long as
they do not significantly affect RNA stability. In addition, in an
optional manner it is also possible to use not total RNA
preparations but rather messenger RNA preparations. These can be
isolated either directly from the biological sample or from total
RNA by means of polyT sequences, according to conventional methods.
In this regard, messenger RNA can be obtained through the use of
commercially available kits such as the kit US72700 (Amersham). The
RNA can also be obtained directly from banks or from other samples
prepared in advance and/or available in collections, and stored
under suitable conditions.
[0057] The hybridization can be carried out under different
conditions which can be adjusted by those skilled in the art. The
hybridization preferably uses an excess of the nucleic acid
population derived from the situation of deregulation relative to
the nucleic acid population derived from the control situation.
[0058] Using the product of the hybridization reaction, two main
types of approaches can be used to isolate the clones
characteristic of deregulation according to the invention. The
first, which is strictly quantitative, enables the generation of a
nucleic acid preparation comprising all (or a significant part) of
the clones resulting from a difference in the level of expression
between the two situations. Such clones (and banks) are obtained by
known subtractive hybridization methods consisting primarily of
eliminating the hybrids formed during the hybridization step and
keeping only non-hybridized clones characteristic of the
deregulated situation relative to the chosen control situation.
[0059] In a preferred embodiment, however, a qualitative process is
used for the generation of a nucleic acid preparation comprising
all (or a large part) of the clones resulting from functional gene
mutations characteristic of the deregulated situation relative to
the chosen control situation. More particularly, such a qualitative
bank comprises not the entire group of clones whose expression is
modified, but for example clones corresponding to splicing or
deletion events that differ between the two situations. Considering
the role of alternative splicing in cell regulatory and
transformation pathways, such preparations (and banks)
advantageously comprise clones having an important functional value
and therefore likely to reflect the genetic modifications involved
in the situation of deregulation. Such clones therefore enable the
constitution of banks with greater predictive power and the
generation of more representative genetic markers.
[0060] Such qualitative banks can be constituted by the isolation
from the hybrids formed during the hybridization step of nucleic
acid regions corresponding to differential splicing or to
deletions. Depending on the methods used, these regions correspond
either to unpaired regions or to paired regions.
[0061] These two approaches are described below in more detail.
[0062] 3.2.1. Production and Use of Differential Quantitative
Banks
[0063] In a first embodiment, the present invention therefore makes
use of a differential quantitative bank, i.e. a bank comprising
nucleic acid clones corresponding to genes whose level of
expression is modified in cells in the situation of deregulation
relative to a control situation. Such banks can also be derived
from differential quantitative analyses, pooling sequences whose
expression is increased or decreased in cellular deregulation
phenomena. The methods to establish this type of bank are familiar
to those skilled in the art and can be broken down into the
following categories:
[0064] High Flow Sequencing Electronic Subtraction
[0065] This process is based on the random sequencing of a certain
number of cDNAs. A computer search engine can then be used to
perform a subtraction between the two situations under analysis.
Serial Analysis of Gene Expression (SAGE) This process is based on
the recognition of a signature associated with each cDNA by using
restriction enzymes and oligonucleotide adaptors. This label
corresponds to a part of the cDNA sequence (10 nucleotides long so
as to unambiguously identify the corresponding cDNA). The labels
are then assembled for sequencing and analysis (Velculescu et al.,
Science, 1995, 270: 484-487). This approach therefore represents a
short-cut to systematic sequencing.
[0066] Nucleic Acid Arrays
[0067] This method is based on the application at more or less high
density of nucleic acids such as oligonucleotides, PCR fragments or
cDNAs on solid supports such as membranes, glass plates or
bio-chips. Messenger RNA probes from the healthy or pathological
samples are then used in hybridization to identify messengers that
are overexpressed or underexpressed.
[0068] Differential Display
[0069] This method makes use of an oligo-dT primer and random
primers to perform PCR on cDNA populations. The PCR products are
then compared on very high resolution gels. Differentially
expressed fragments are then isolated and their presence confirmed
by northern blot analysis prior to sequencing. Several variants of
this method have been described (Prashar and Weissman, PNAS, 1996,
93: 659-663). These variants differ in terms of the primer and
restriction enzymes and adaptor used. Like the SAGE method, they
make use of the 3'-ends of cDNAs. This approach is made accessible
by the existence of several commercially available kits.
[0070] Subtractive Cloning
[0071] This method is based on the elimination of cDNAs that are
common to the two samples under comparison. Thus, different kits in
which the "tester" cDNA is hybridized with an excess of "driver"
cDNA are available (Clontech). The final product consists of a pool
of PCR-amplified fragments derived from differentially expressed
cDNAs, which can be cloned in a suitable vector for subsequent
analysis. RDA (Representational Difference Analysis) is another
method based on this principle of subtraction (Lisitsyn et al.,
Science, 1993, 259: 946-951).
[0072] The implementation of these differential analytical methods
therefore enables the generation of quantitative banks and clones,
i.e. comprising all the sequences whose expression is increased or
decreased in cellular deregulation phenomenon or phenomena.
[0073] 3.2.2. Production and Use of Differential Qualitative
Banks
[0074] In another embodiment, the present invention advantageously
uses a differential qualitative bank, i.e. a bank comprising
nucleic acid clones of which at least part of the sequence
corresponds to the sequence of the genes differentially spliced in
the cells in a situation of deregulation. This type of bank
therefore comprises sequences that are differentially spliced in
deregulatory processes.
[0075] The use of this type of bank is particularly advantageous.
In fact, the different cellular stresses leading to cell death or
to other deregulatory phenomena involve initiators and mediators of
signalling pathways and regulate key cell cycle components,
including p53. Regulation of the level of expression of this
protein occurs mainly through stabilization and transcriptional
enhancement. While Fas receptors, members of the bcl2 family and
caspases are regulated at the transcriptional level, they are also
regulated at the level of premessenger RNA maturation and splicing.
This post-transcriptional regulation is critical because, from a
same transcription event, it determines the creation of a pro- or
anti-apoptotic form of each member of the concerned protein
families. These transcriptional and splicing modifications are
regulated by cell signalling and metabolism. The transcription and
splicing regulatory factors which provide "trans" regulation of
these key steps of gene expression are proteins whose own activity
is regulated, notably by phosphorylation, according to the state of
proliferation, differentiation and viability of the cell. The
splicing program which is engaged during cell deregulation and
which mobilizes different initiators and mediators of cell death
therefore reflects modifications in cell signalling due to
different damage. By affecting the transcriptional and splicing
machinery, these modifications affect the expression of many genes
which are not limited to the genes known to play a role as
initiators or mediators of deregulation, but participate at
different levels of the signalling cascades. Likewise, the cascades
altered during oncogenesis, particularly by expression of splicing
variants, mobilize markers whose expression is not restricted to
tumors.
[0076] To take into account these phenomena and this complexity,
and to thereby provide a more predictive evaluation of the toxic
potential of a test compound, the process of the invention
advantageously uses splicing events characteristic of deregulated
situations, as genetic markers of toxicity.
[0077] To do so, the present invention uses, for example,
differential qualitative nucleic acid banks produced according to
"DATAS" methodology described in international patent application
no WO99/46403, incorporated in the present by reference. In
particular, such banks can be prepared by hybridization between the
nucleic acid population derived from cells in a situation of
deregulation, and the nucleic acid population derived from cells in
a control situation, and isolation, from the hybrids formed, of the
nucleic acids corresponding to differential splicing.
[0078] In this approach, hybridization is preferably carried out in
liquid phase. Furthermore, it can be carried out in any suitable
device such as tubes (Eppendorf tubes, for example), plates or any
other suitable support commonly used in molecular biology. The
hybridization is advantageously carried out in volumes of between
10 and 1000 .mu.l, for example between 10 and 500 .mu.l. It is
understood that the device and volumes used can be easily adapted
by those skilled in the art. The quantities of nucleic acids used
for the hybridization are also known to those skilled in the art.
In general, microgram quantities of nucleic acids suffice, for
example between approximately 0.1 and 100 .mu.g. Furthermore, it is
possible to use the nucleic acids in a driver/tester ratio ranging
from approximately 50 to 0.02, preferably from 40 to 0.1. Even more
advantageously, this ratio is preferably close to or greater than
1, advantageously between approximately 1 and approximately 10. It
is understood that this ratio can be adapted by those skilled in
the art according to the conditions of the process (available
quantities of nucleic acids, physiological situations, purpose,
etc.). The other hybridization parameters, including time,
temperature and ionic strength, can also be adapted by those
skilled in the art. As a general rule, following denaturation of
the tester and driver, for example by heating, the hybridization is
carried out for approximately 2 to 24 hours at a temperature of
approximately 37.degree. C. (possibly submitted to temperature
spikes), and in standard conditions of ionic strength, which can
range from 0.1 to 5 M NaCl, for example. Ionic strength is known to
be one of the factors that determines the stringency of a
hybridization, especially in the case of hybridization on a solid
support.
[0079] According to a specific embodiment of the invention, the
hybridization is carried out in a phenol emulsion, for example
according to the PERT method ("Phenol Emulsion DNA Reassociation
Technique) described by Kohne D. E. et al. (Biochemistry, Vol. 16,
No. 24, pp 5329-5341, 1977). The hybridization is avantageously
carried out in a phenol emulsion maintained by thermocycling
(temperature increase from approximately 37.degree. C. to
approximately 60-65.degree. C.) and not by agitation, according to
the method described by Miller and Riblet (NAR 23 (1995) 2339).
[0080] Any other hybridization method in liquid phase, preferably
in emulsion, can be used within the scope of the present invention.
Furthermore, the hybridization can also be done with one of the
strands immobilized on a support. Advantageously, it is the driver
that is immobilized. This can be done notably thanks to
biotinylated primers or by any other immobilization technique known
to those skilled in the art.
[0081] Using the nucleic acid populations generated by
hybridization, the genetic markers of the invention (the clones
characteristic of qualitative genomic alterations) can be
identified by any method familiar to those skilled in the art. In
the case of RNA/DNA heteroduplex, these regions are mainly regions
of unpaired RNA (RNA loops) and can be identified and cloned by
separation of the heteroduplex and the single-stranded nucleic
acids (excess nucleic acids which did not react), selective
digestion of the double-stranded RNA (domains participating in the
heteroduplex), followed by separation of the resulting
single-stranded RNA and the single-stranded DNA. In the case of
heterotriplex, these differential splicing regions consist mainly
of regions of double-stranded DNA and can be identified and cloned
by treatment with suitable enzymes such as an enzyme that digests
RNA, followed by an enzyme that digests single-stranded DNA. The
nucleic acids so obtained are directly in the form of
double-stranded DNA and can be cloned in any suitable vector.
[0082] It is understood that other specific variants and conditions
for the isolation of nucleic acids, hybridization and obtaining of
qualitative clones, are indicated in WO99/46403, incorporated in
the present by reference.
[0083] These methods enable the generation of nucleic acid banks
and clones corresponding to qualitative genetic markers of a
situation(s) of deregulation. As indicated in the experimental
section, these nucleic acid preparations are especially useful
markers to characterize the toxicity profile of compounds.
[0084] 4. Diversity of the Banks
[0085] The aforementioned methods therefore enable the generation
of groups of nucleic acid clones characteristic of situations of
deregulation. Each method of preparation generates many clones
which constitute banks. These banks can be used as is, deposited on
supports, modified by the addition or removal of clones, or
different banks can be pooled or control clones added, etc.
[0086] The banks provided for by the invention can comprise 10 to
50,000 clones, more generally 10 to 10,000 clones, and even more
preferably 50 to 5,000 clones. The clones are generally deposited
in a well-ordered fashion on one or more supports so as to
facilitate analysis of the hybridization results. The support can
be composed of glass, nylon, plastic, fiber, etc. or generally be
any solid support suitable for the deposit of nucleic acids. The
banks can be deposited on the supports by conventional methods
known to those skilled in the art, as described for example in the
international application No. WO99/46403.
[0087] The banks used can comprise both the nucleic acid clones
corresponding to genes whose level of expression is altered in
cells in a situation of deregulation (quantitative genetic markers)
and the nucleic acid clones of which at least part of the sequence
corresponds to genes that are differentially spliced in cells in a
situation of deregulation (qualitative genetic markers). Thus, the
genetic markers can be generated by different approaches, then
pooled to obtain a response that is as predictive as possible.
[0088] In this regard, it is also possible to use banks comprising
clones derived from different situations of deregulation. For
example, as noted above, the situations of deregulation can be
induced in different manners (increased p53 gene expression,
increased Rb gene expression, use of different cell populations,
use of tumor cells, etc.). These different situations of
deregulation do not always induce exactly the same genetic events,
and therefore do not always produce exactly the same nucleic acid
banks according to the invention. Although each of these banks can
be used separately within the scope of the present invention, it is
also possible to pool them on the same support (or on separate
supports) so as to further increase the number of genetic markers
of toxicity and thereby improve the predictability of the methods
of the invention.
[0089] A subject of the present invention is therefore also based
on a nucleic acid preparation comprising qualitative and
quantitative genetic markers characteristic of cellular
deregulation(s). A specific subject concerns a bank comprising
genetic markers characteristic of different situations of
deregulation. Another subject of the invention is any solid support
on which at least two banks of nucleic acids characteristic of two
situations of deregulation have been deposited. In this regard, the
invention further concerns a process of preparation of a DNA chip
used to determine the toxic potential of a test compound,
comprising the application, on a solid support, of one or more
nucleic acid preparations characteristic of situation(s) of
deregulation.
[0090] Furthermore, in a preferred embodiment, use is made of
nucleic acid banks refined through use by the selection of clones
based on their actual involvement in toxicity phenomena. The
initial banks can in fact comprise all the clones characteristic of
genetic events of a situation of deregulation. Implementation of
the diagnostic process set forth by the invention then makes it
possible to observe that some of the clones hybridize very rarely,
if ever, with the nucleic acid test samples. Such clones can
eventually be removed from the bank in order to generate more
specific, and therefore more universal and predictive banks.
[0091] Another advantageous subject of the invention is therefore
based on a nucleic acid bank comprising nucleic acid clones
corresponding to genetic events common to a situation of
deregulation and a situation of toxicity.
[0092] Another subject concerns a method of preparation of banks of
genetic markers of toxicity, comprising hybridization between a
nucleic acid population derived from cells in a situation of
deregulation, and a nucleic acid population derived from cells in a
control situation, isolation from the hybridization product of
clones characteristic of the situation of deregulation, and
hybridization of the clones obtained with a nucleic acid sample
derived from cells in a situation of toxicity. The above process
advantageously further comprises a selection step, comprising
hybridizing the bank with different nucleic acid samples derived
from cells in a situation of toxicity induced by different
compounds, and identification of the clones in the bank that
hybridize most frequently with said samples.
[0093] More specifically, the present invention now describes the
identification and characterization of such clones, which can be
used as genetic markers of toxicity.
[0094] In this regard, the invention discloses the identification,
preparation, cloning and characterization of particular clones,
whose sequences are represented by SEQ ID NOs: 1 to 37. These
clones can be used as genetic markers of toxicity, according to the
present invention. These clones have been isolated by qualitative
methods and comprise sequences corresponding to qualitative genetic
alterations characteristic of deregulation events that affect
different cellular genes, such as genes coding for proteins,
enzymes, mitochondrial genes, transcription factors, pathological
genes, stress-response genes, genes involved in signal(s)
transduction, etc. Such clones, the preparation and libraries
containing the same, as well as their uses, represent particular
objects of this invention.
[0095] In this regard, a particular object of this invention
resides in a nucleic acid preparation or library, comprising a
plurality of nucleic acid clones that are genetic markers of
toxicity, said preparation or library comprising at least one clone
of sequence selected from SEQ ID Nos: 1 to 37.
[0096] A particular object of the invention resides, more
generally, in any library of nucleic acids comprising at least one
clone of sequence selected from SEQ ID n.degree.:1 to 37.
[0097] A clone of sequence SEQ ID n.degree.: X designates, within
the context of this invention, (i) any clone comprising all of said
sequence SEQ ID n.degree.: X (or of a sequence complementary
thereto), optionally flanked by additional sequences, preferably a
clone consisting in all of said sequence SEQ ID n.degree.: X (or a
sequence complementary thereto); as well as (ii) any clone
comprising a part of said SED ID n.degree.: X (or of a sequence
complementary thereto), optionally flanked at one end by additional
sequence(s) (in particular originating from the sequence of the
corresponding gene or messenger), said part of the sequence being
sufficient to allow a specific hybridization (for instance at least
10 bases); or (iii) any other nucleic acid sequence specific for
the gene or for a genetic event of deregulation of the gene
identified by SEQ ID n.degree.: X, without necessarily overlapping
with SEQ ID n.degree.: X. Sequences (iii) are, for instance,
sequences from a distinct region of said gene, allowing its
detection in similar conditions. Clones (i) to (iii) of this
invention typically comprise between 10 and 1000 bases, more
generally between 20 and 500 bases, a portion of which at least
being specific for the genetic event under consideration.
[0098] The preparations or libraries of the invention comprise more
preferably at least 5, more preferably at least 10, even more
preferably at least 15, even further preferably at least 20 clones
of sequence selected from SEQ ID n.degree.: 1 to 37. As indicated
before, the libraries comprise, typically, between 10 and 50 000
clones, more generally between 10 and 5 000 clones, in particular
between 50 and 1 000 clones, a part thereof at least being
represented by one or several clones of sequence selected from SEQ
ID n.degree.: 1 to 37.
[0099] The libraries and preparations of this invention can be
deposited on supports and used as disclosed in this
application.
[0100] One of the major applications of the identification and
cloning of these genetic markers concerns the evaluation of the
toxic potential of a test compound. This evaluation can be carried
out by hybridizing a probe corresponding to the messenger RNA of a
cell culture treated with this product, with one or more banks of
signatures characteristic of situation(s) of deregulation, such as
those described above. An other application resides in the
identification of genomic alterations (in particular SNPs) that are
correlated to a response of a subject to a given compound, for
instance the response to a treatment, the sensitivity to a
compound, etc. These applications are described in more detail
below.
[0101] 5. Methods of Analysis and Diagnosis of Toxicity
[0102] The invention therefore also concerns a method for analysis
of the toxic potential of test compounds, using the aforementioned
genetic markers. In particular, the invention allows to determine
the toxic potential of any compound by hybridizing a sample of
nucleic acids from cells treated with this compound, with the
aforementioned genetic markers, the observed hybridization profile
indicating the toxic potential of the compound. To this end, the
genetic markers used are preferably combined into banks so that the
response can be as predictive as possible. One of the advantages of
the present invention also concerns the large number of markers
used, which makes the information obtained even more predictive.
The predictive nature of the information is further strengthened by
the type of markers used and prepared.
[0103] A specific subject of the invention is based on a method of
analysis of the toxic potential of a test compound, comprising at
least one hybridization step between a) a nucleic acid sample from
cells treated with this compound and b) a nucleic acid bank
corresponding to genetic events characteristic of situation(s) of
deregulation of cell signalling pathway(s), the hybridization
profile indicating the toxic potential of the test compound. In
particular, the greater the number of hybridization-positive
clones, the greater the potential toxicity of the test
compound.
[0104] The analysis of the toxicity profile of the compound is more
generally done by comparing the hybridization profile on the bank
of a nucleic acid sample from cells treated with this compound,
with that of a nucleic acid sample from cells not treated with the
compound. This comparison makes it possible to demonstrate a
difference between the number of hybridizing clones in the toxic
situation and in the control situation, by which a toxicity index
for the test compound can be established.
[0105] More specifically, the invention therefore concerns a method
of analysis of the toxic potential of a test compound, comprising
at least
[0106] a hybridization step between a) a nucleic acid sample from
cells treated with this compound and b) a nucleic acid bank
corresponding to genetic events characteristic of situation(s) of
deregulation of cell signalling pathway(s),
[0107] a hybridization step between c) a nucleic acid sample from
cells not treated with this compound and b) a nucleic acid bank
corresponding to genetic events characteristic of situation(s) of
deregulation of cell signalling pathway(s),
[0108] comparison of the hybridization profiles obtained indicates
the toxic potential of the test compound.
[0109] The hybridization profile can be detected by any method
familiar to those skilled in the art, and particularly by labelling
of the nucleic acid test samples and detection of the label in the
bank. Thus, the invention more specifically concerns a method of
analysis of the toxic potential of a test compound comprising at
least the separate hybridization between a) labelled nucleic acid
probes corresponding to RNA from cells not treated and cells
treated with said test compound and b) a nucleic acid bank
corresponding to genetic events, particularly transcriptional
and/or splicing events, characteristic of deregulation(s), the
hybridization profile indicating the toxic potential of the test
compound.
[0110] The nucleic acids can be labelled by any method known to
those skilled in the art, particularly through the use of
radioactive, fluorescent, enzymatic or calorimetric labels. In
general, radioactive labelling is used and the hybridization is
visualized by detection of radioactivity on the support.
Fluorescent labelling can also be used, in which case the
hybridization is visualized by detection of the fluorescence
emitted on the support.
[0111] As indicated above, in the methods set forth by the
invention, the nucleic acid banks corresponding to genetic events
characteristic of deregulation(s) can be, in particular, nucleic
acid banks characteristic of situations of deregulation of cell
growth and/or viability. More specifically, these are nucleic acid
banks characteristic of cells in a situation of deregulation of
cell growth, notably transformed cells, tumor cells in
particular.
[0112] In the methods of the invention described above, the nucleic
acid banks are more preferably nucleic acid banks corresponding to
genetic events characteristic of alterations in signalling pathways
induced by the activation, preferably the expression of all or part
of a tumor suppressor gene, preferably p53. In particular these are
banks characteristic of apoptosis induced by a tumor suppressor
gene, preferably p53. The results presented in the examples
demonstrate in particular that, through the use of a specific
differential qualitative screening strategy, a situation of
apoptosis resulting from induction of p53 protein expression leads
to many modifications in splicing, in addition to quantitative
modifications related to transcriptional regulation of different
genes. The invention shows that probes from cells treated with
different toxic compounds hybridize with clones from banks derived
from quantitative and qualitative screening prepared from control
cells, on the one hand, and from cells in which wild type p53
expression is induced, on the other hand. This indicates that the
transcriptional and splicing events are common to toxic situations
and to situations where wild type p53 protein expression is
induced.
[0113] Moreover, the results obtained also show that when the
treated cells are of the same type as those in which p53 expression
has been induced, the probes prepared from the messenger RNA from
cells treated with increasing concentrations of the toxic compounds
hybridize a larger number of clones in the quantitative and
qualitative banks when the concentrations are high, and therefore
the toxicity important. This demonstrates that there is a
correlation between the level of toxicity and the hybridization
profile on the banks of the invention.
[0114] It is clear however that the application of the methods,
kits and banks of the invention is not restricted to a specific
cell type. In particular, it is not restricted to tests of products
on the same cells as those from which the quantitative or
qualitative banks and clones are derived. In fact, the results
presented show that the clones of the invention advantageously
enable a diagnosis of toxicity independent of the toxic compound
and the type of test cell.
[0115] In a specific embodiment, the nucleic acid library is a
library comprising at least one clone of sequence selected from SEQ
ID Nos: 1 to 37, as defined above.
[0116] In a specific embodiment of the methods of the invention,
the cells treated or not treated with the test compound and the
cells used to generate the banks are of a different type.
[0117] More specifically, the cells treated or not treated with the
test compound can be of different origin and type. They are
preferably mammalian cells, even more preferably human cells. They
can be primary cultures or cell lines. The cell type used, its
concentration, density and physiological state (resting,
stimulated, etc.) can be chosen by the user of the methods of the
invention according to the test compounds and desired applications.
The cells can also be cells extracted from organs or tissues of
animals treated or not treated with the compound.
[0118] The test compound can also be highly varied in nature. For
example, it can be an individual compound or a mixture of
compounds. The compound can be chemical or biological. In
particular, it can be a peptide, polypeptide, nucleic acid, lipid,
carbohydrate, a chemical molecule, plant extract, combinatorial
libraries, etc. The test compound can also be a treatment applied
to the cells, such as irradiation or UV. To implement the process
of the invention, the test compound can be applied at different
concentrations chosen by the user. Furthermore, the nucleic acid
samples derived from the treated cells can be prepared at various
time points after treatment with the compound. In this way, kinetic
studies can be carried out as needed.
[0119] The nucleic acids and corresponding probes are prepared by
conventional methods. Hybridization can then be carried out, also
under conditions that can be adapted by those skilled in the art
and by the user. In this regard, the hybridization can be carried
out under high, intermediate or low stringency, depending on the
desired level of sensitivity, quantity of available material, etc.
Furthermore, according to the present invention the hybridizations
between the bank and the probes can be homologous (when the bank
and probes are composed of nucleic acids from the same species, for
example human) or heterologous (when the bank and probes are
composed of nucleic acids from different species, for example a
human bank and probes prepared from material derived from another
mammalian species). Although the banks and probes are preferably
constructed from material of human origin, it is in fact possible
to use other sources, particularly for the probes (for example,
murine origin). For such heterologous hybridizations, the
hybridization conditions can be adjusted by those skilled in the
art according to conventional techniques, particularly by
decreasing the hybridization temperature and/or by increasing the
salt concentration of the hybridization buffer.
[0120] 6. Determination of a Toxicity Index
[0121] An advantage of the methods of the invention is based on the
possibility of determining a toxicity index for a given compound on
any cell type. For this application, the clones corresponding to
elements identified from cells used to establish banks specific for
cell viability, as well as those specific for the induced
deregulation, are hybridized with a probe derived from the
untreated cells under study. Their hybridization serves as the
reference RNA expression common to both the model of deregulation
and the cell model under study. Comparison of RNA expression in the
untreated cell situation with that of situations treated with
different products at different concentrations and treatment times
can be easily done by comparing the hybridization profiles of each
probe from each situation under study. The toxicty index of a
situation can be evaluated by assigning a value of 1 to clones that
hybridize with the untreated probe and do not hybridize with the
treated situation, on the one hand, and by also assigning a value
of 1 to clones not hybridizing with the untreated probe but
hybridizing with the treated probe, on the other hand. The index is
then the sum of the individual 1 values expressed as a percentage
of the number of clones subjected to hybridization. This index can
therefore range from 0 to 100. This example of calculation of the
toxicity index is not limiting. Other mathematical methods,
algorithms or calculation processes such as neuronal networks can
also be used.
[0122] The value of determining such an index by hybridizing clones
derived from differential qualitative screening between two states
of a same cell type (proliferation and apoptosis-induced) with
probes from other cell types, is confirmed by the ability to
determine an index that is all the greater for a given compound
when the concentration applied to the test cells is high.
Naturally, the value of the index depends on the threshold at which
a clone is assigned a value of 1 or 0. The index is all the greater
when the factor of repression or induction that the clone requires
for consideration is low. The robustness of the method is
nonetheless illustrated by the fact that there is a linear relation
between the chosen factor and the calculated toxicity index. This
linearity reflects the number of independent events which
constitute the qualitative bank, which makes it possible to avoid
giving too much weight to a particular clone with an overly high
differential expression between the treated and untreated
situation.
[0123] 7. Identification of Polymorphisms Linked to the Toxicity
for a Given Compound
[0124] The determination of the toxic potential of a compound
according to the procedure disclosed in 5, and in the examples
allows the identification of the sequences which derive from genes
involved in mechanisms of stress and cell death, whose expression
is altered by this compound.
[0125] These genes represent candidates of choice for the
identification of polymorphisms that may affect the response to
said compound. These polymorphisms allow to segregate candidate
patients for the administration of this compound in different
groups that are more or less susceptible of developing a pronounced
toxicity, in particular hepatic.
[0126] For example, it can be envisioned that a polymorphism which
affects a gene whose expression is decreased by a compound renders
the patient more susceptible to the toxic activity of this
compound. Without being limited to such particular example, the
importance of polymorphisms in the genes selected according to the
invention is assessed by determining the correlation existing
between said polymorphisms and the toxicities observed during
clinical trials conducted with the compound under
consideration.
[0127] An object of the invention is thus the use of clones or
nucleic acids disclosed, for the identification of mutations or
polymorphisms, in particular SNPs, correlated to the response of
subjects to pharmaceutical compounds.
[0128] The invention concerns, more generally, the use of nucleic
acids representative of splicing events characteristic of a given
physiopathological situation, for the identification or the
characterization of SNPs correlated to said situation.
[0129] The identification may be performed using different methods
or approaches known to the skilled person. In particular, it can be
performed by sequencing, from biological samples derived from
subjects that respond and from subjects that do not respond to a
compound, the genes corresponding to the selected clones or nucleic
acids, followed by the identification of polymorphisms in said
genes. The identification can also be performed from SNP libraries,
by searching for SNPs present in said genes.
[0130] The invention also concerns a method for the identification
of SNPs or other mutations or polymorphisms that allow the
assessment of the response of a subject to a given compound, the
method comprising (i) the identification in vitro of nucleic acids
characteristic of splicing events induced in a cell treated with
said compound and (ii) the identification of SNPs or other
mutations or polymorphisms in the gene or genes corresponding to
nucleic acids identified in (i), said SNPs or other mutations or
polymorphisms allowing the assessment of the response of a subject
to said given compound. The invention thus provides methods and
means to select genes for which polymorphisms are linked to a
toxicity for a given compound.
[0131] The invention also relates to a population of nucleic acids
specific for polymorphisms or SNPs thus identified, in particular a
population of nucleotide primers for the selective amplification of
said polymorphisms, or of probes for the selective hybridization to
the considered polymorphisms.
[0132] The invention also relates to methods for the analysis
(e.g., the prediction, evaluation or determination) of the response
of a subject to a test compound, comprising the study of
polymorphisms, in particular the analysis of SNPs, as defined
above. A particular method relates to the analysis of the
sensitivity or of the response of a subject to a compound, in
particular to the toxicity of a compound, comprising the analysis,
from a biological sample comprising DNA from said subject, of the
presence in the subject's DNA of polymorphisms, SNPs or other
genomic alternations present in genes whose splicing is modified in
response to said compound. As indicated above, the presence of
polymorphisms, SNPs or other genomic alterations in a subject's DNA
may be determined by different techniques known to the skilled
person, such as sequencing, amplification, hybridization,
separation or chromatographic methods, etc. A preferred approach
consists in the amplification of the subject's DNA using primers
specific for the alteration (in particular the SNP) under
consideration. An other approach consists in the use of a
nucleotide probe whose sequence is specific for the alteration (in
particular the SNP) under consideration. It should be understood
that the invention is not to be limited to particular methods.
[0133] 8. Kits
[0134] The present invention also concerns kits for the
implementation of the aforementioned methods. The invention more
specifically concerns kits for the study of the toxic potential of
a test compound, comprising at least:
[0135] a nucleic acid bank corresponding to genetic events
(transcriptional and/or splicing) characteristic of alteration(s)
in one or more cell signalling pathways, notably situation(s) of
deregulation of cell growth and/or viability.
[0136] The kits of the invention more generally comprise a bank of
genetic markers characteristic of deregulation(s), for example
apoptosis, such as defined in the invention, deposited on a solid
support.
[0137] The kits of the invention more preferably comprise a nucleic
acid bank characteristic of several situations of deregulation
induced in different conditions.
[0138] According to another specific embodiment, the kits of the
invention comprise at least one nucleic acid bank comprising clones
corresponding to genetic events common to cells in a situation of
deregulation and in a situation of toxicity.
[0139] According to a particular embodiment, the invention relates
to any kit comprising a library of nucleic acids, the library
comprising at least one clone of sequence selected from SEQ ID Nos:
1 to 37, as defined above. Advantageously, the library is deposited
on a support, preferably a solid support, in particular, glass,
silica, filter, membrane, nylon, plastic, etc.
[0140] In the kits according to the invention, the banks further
advantageously comprise control clones for calibration of the
detected signals. These controls can more particularly comprise
three types of material:
[0141] genomic DNA clones,
[0142] cDNA used for construction of probes that will hybridize the
banks, and/or
[0143] cDNA corresponding to mRNA encoding control proteins, for
example housekeeping proteins (especially enzymes) such as
.beta.-actin or GAPDH (Glyceraldehyde Phosphate DeHydrogenase).
[0144] The controls provided by the housekeeping enzymes are
classically used by those skilled in the art in cDNA display
experiments However, during the deregulation phenomena according to
the invention, the quantity of mRNA of these enzymes can vary.
Therefore, to normalize the hybridizing power (and therefore the
signal) of each probe, the present invention proposes the use of
one or more other control systems. In this regard, the use of
genomic DNA and/or cDNA (unlabelled) corresponding to the probe
(i.e. to the DNA population from the treated or untreated
biological sample) makes it possible, according to the invention,
to obtain hybridization controls independent of any qualitative or
quantitative variations in gene expression. Equivalent quantities
of cDNA with the same specific activity after radiolabelling should
in principle generate equivalent signals by hybridizing with both
genomic DNA and with themselves.
[0145] The kits of the invention can also comprise:
[0146] oligonucleotides for use in the preparation of the probes,
and/or
[0147] a hybridization protocol, and/or
[0148] items and information for establishment of a toxicity index
for each test product, notably a signal processing software
program.
[0149] The oligonucleotides are preferably semi-random, such as
oligonucleotides with the formula X-N.sub.n-AGGT, where X is a
stabilizing sequence such as GAGAAGCGTTATG or CCACGCTACAAG(C), N is
a base, and n is a whole number between 3 and 8 inclusive.
[0150] The feasibility, realization and other advantages of the
invention are depicted in further detail in the examples that
follow, which should be considered illustrative and
non-limiting.
LEGENDS TO FIGURES
[0151] FIG. 1. Differential hybridization of probes from HepG2
cells untreated (A), treated with ethanol (B) or treated with
cyclosporine on filters containing PCR fragments corresponding to
clones from the qualitative screening of a p53-induced apoptosis
system.
[0152] FIG. 2. Toxicity index calculated for different toxic
compounds on the p53 apoptosis filter.
[0153] FIG. 3. Differential hybridization of probes from HepG2
cells untreated (A), treated with clenbuterol (B), R-propranolol
(C) or D,L-propranolol (D) on filters containing PCR fragments
corresponding to clones from the qualitative screening of tumor
biopsies.
9. EXAMPLES
[0154] 9.1. Identification of Genetic Markers of Toxicity from
Qualitative Screening on a Cellular Apoptosis System
[0155] An example of the contribution of differential qualitative
analysis to the identification of genetic markers associated with
drug toxicity is given by the use of DATAS in a model of apoptosis
induction via induction of wild type p53 expression. This cell
model was established by transfection of a system of inducible
expression of the p53 tumor suppressor gene. So as to identify the
qualitative differences specifically related to p53-induced
apoptosis, qualitative screening was implemented on messenger RNA
extracted from induced (ml) and non-induced (mNI) cells. This
messenger RNA is converted to the complementary DNA (cl) and (cNI)
using reverse transcriptase (RT) and biotinylated oligonucleotide
primers. ml/cNI and cl/mNI hybrids are then obtained in liquid
phase.
[0156] The required quantities of RNA and cDNA (in this case, 200
ng of each) are combined and ethanol-precipitated. The samples are
taken up in 30 .mu.l of hybridization buffer (40 mM Hepes pH 7.2,
400 mM NaCl, 1 mM EDTA) supplemented with deionized formamide (80%
VN). After denaturation for 5 min at 70.degree. C., samples are
incubated overnight at 40.degree. C.
[0157] Streptavidin beads (Dynal) are washed and then reconditioned
in binding buffer (2.times.=10 mM Tris-HCl pH 7.5, 2 M NaCl, 1 mM
EDTA). Hybridization samples are adjusted to a volume of 200 pi
with water and added to 200 .mu.l of beads, then incubated at
30.degree. C. for 60 min. The beads are captured on a magnet and
washed, then suspended in 150 .mu.l of RnaseH buffer and incubated
at 37.degree. C. for 20 min. After capture on the magnet,
non-hybridized regions are released in the supernatant which is
treated with Dnase, then extracted with acidic phenol/chloroform
and ethanol-precipitated. Ethanol precipitations of small
quantities of nucleic acids are done with the commercial polymer
SeeDNA (Amersham Pharmacia Biotech) for quantitative recovery of
nucleic acids from very dilute solutions (ng/ml
concentrations).
[0158] cDNA synthesis from the RNA samples derived via the action
of RnaseH is carried out by using random hexanucleotides and
Superscript II Reverse Transcriptase. The RNA is then degraded in a
mixture of RnaseH and Rnase T1. The primer, unincorporated
nucleotides and enzymes are separated from the cDNA on a GlassMAX
Spin cartridge. The cDNA corresponding to splicing loops is then
used for PCR with semi-random oligonucleotides with the following
structure: (FS3)N7AGGT, where (FS3)=CCACGCTACMG.
[0159] PCR is performed with taq polymerase for 30 cycles:
[0160] Initial denaturation: 94.degree. C. for 1 min.
[0161] 94.degree. C. for 30 s
[0162] 55.degree. C. for 30 s
[0163] 72.degree. C. for 30 s
[0164] Final elongation: 72.degree. C. for 5 min.
[0165] The PCR products are cloned in a pGEM-T vector (Promega)
with a floating T at the 3' ends to facilitate cloning of fragments
derived from the action of taq polymerase. Following transformation
in competent JM109 bacteria (Promega), colonies are stored on 96
well plates. The PCR products generated with the T7 and Sp6
oligonucleotides located on the cloning plasmid and surrounding the
insertion cassettes are deposited on nitrocellulose filters.
[0166] HepG2 cells are then individually treated with different
compounds with different levels of toxicity. In this example, the
concentration used was calculated by a cell viability test (MTT)
and corresponds to an IC80. Table 1 gives the concentrations of the
different compounds and the exposure times.
1 TABLE 1 Compound Concentration Time Aspirin 2 mM 18 h Paracetamol
1 mM 18 h Cyclosporine 2.5 .mu.M 18 h Diclofenac 50 .mu.g/ml 18 h
Beclomethazone 50 nM 18 h Methotrexate 500 nM 18 h Erythromycin 2.5
.mu.g/ml 18 h Vinblastine Sulfate 30 .mu.M 18 h Clonidine 150 .mu.M
18 h Valinomycin 15 nM 18 h Etoposide 50 .mu.g/ml 18 h Camptothecin
1 .mu.g/ml 4 h PMA 1 .mu.g/ml 18 h Ethanol 0.1 M/0.5 M 4 h 5 FU 10
.mu.M 24 h Cycloheximide 5 .mu.g/ml 18 h FCCP 5 .mu.M 18 h
Staurosporine 50 nM 18 h Terbutaline Sulfate 200 ng/ml 18 h
Epinephrine Hydrate 50 .mu.g/ml 18 h AZT 50 .mu.g/ml 18 h H2O2 0.1
mM 18 h Actinomycin D 0.02 .mu.g/ml 18 h
[0167] Labelled RNA probes are prepared from these situations. To
do so, the first cDNA strand obtained from total RNA by methods
known to those skilled in the art serves as matrix for the PCR
reaction using the oligonucleotides (FS4)GN3AGGT and FS3CN3AGGT, in
which (FS4)=GAGAAGCGTTAT, according to the following program:
[0168] Initial denaturation: 95.degree. C. for 5 min
[0169] 5 cycles with: denaturation: 94.degree. C. for 30 sec
[0170] annealing: 30.degree. C. for 30 sec
[0171] temperature increase from 30 to 72.degree. C. in 30 sec
[0172] elongation: 72.degree. C. for 30 sec
[0173] 35 cycles with: denaturation: 94.degree. C. for 30 sec
[0174] annealing: 57.degree. C. for 30 sec
[0175] elongation: 72.degree. C. for 30 sec
[0176] Final elongation: 72.degree. C. for 7 min
[0177] The PCR products are labelled with the Redyprime kit
(Amersham).
[0178] Filters are prehybridized in hybridization buffer (Rapid
Hybrid Buffer, Amersham) containing 100 .mu.g/ml of salmon sperm
DNA at 65.degree. C. for 30 min. The PCR probe is then applied to
the membrane (0.5.times.10.sup.6 to 1.times.10.sup.6 cpm/ml) and
incubated at 65.degree. C. for 2 to 18 hours. Filters are washed in
5.times. SSC buffer, 0.1% SDS at 65.degree. C. for 30 min then in
0.2.times. SSC buffer, 0.1% SDS. The hybridization profiles are
analyzed by measuring the radioactivity with an InstantImager
(Packard Instruments) (FIG. 1). Quantification of the individual
hybridization intensities allows calculation of an index according
to the procedure described above (FIG. 2). This result shows that
it is possible to classify different products according to their
ability to induce the expression of markers that are differentially
spliced during p53-induced toxicity. For example, in HepG2 cells,
diclofenac generates more splicing events identical to those
induced by p53-related toxicity than aspirin. The use of banks
containing cDNA sequences differentially spliced in a reference
(healthy) situation and a situation involving a major route of
toxicity, therefore enables the classification of products whose
actions are not distinguishable using standard toxicity tests such
as MTT.
[0179] 9.2. Identification of Genetic Markers of Toxicity from
Qualitative Screening on Tumor Biopsies
[0180] A second example of the use of the invention for the
identification of genetic markers of toxicity consists in the
generation of differential nucleic acid banks for healthy tissue
and tumor tissue. The value of identifying sequences specifically
expressed in control (healthy) tissue on the one hand, and in tumor
tissue on the other hand, is related to the fact that within a
tumor, information characteristic of deregulated cell growth and
cell death is present in different cells but also within the same
cells. As compared to a model of apoptosis induced by deregulated
expression of p53 or any other protein with a negative effect on
cell growth and viability, the use of tumor specimens therefore
additionally provides filters for use in predictive toxicology
bearing sequences whose expression is characteristic of cellular
hyperproliferation or carcinogenesis.
[0181] The working material comprised tumor biopsy specimens from 7
patients with head and neck cancers together with biopsy specimens
from healthy uvula of these same patients. The procedure was
identical to that described in example 9.1 although this is not a
quantitative considering the scarcity of material available from
human biopsy specimens.
[0182] The nucleic acid fragments generated by application of the
invention to tumor and normal tissues were deposited on
nitrocellulose filters. These filters were hybridized with the same
radioactive probes as those used in example 9.1, i.e. generated
from the genetic material of HepG2 cells treated with different
compounds displaying different toxic potential. Differential
hybridization profiles were observed for the different compounds,
thereby demonstrating the utility of these nucleic acid banks for
evaluation of the toxicity of a given compound.
[0183] FIG. 3 depicts the hybridization signals obtained with
probes derived from normal HepG2 cells, or cells treated with
clenbuterol, the inactive propranolol enantiomer, or a racemic
mixture of the active and inactive propranolol enantiomers. The
compounds were used at the concentrations indicated in Table 1 and
the hybridization probes and protocols were identical to those in
the paragraph describing the use of filters containing specific
signatures of p53-induced apoptosis (example 9.1).
[0184] The hybridization signals shown in FIG. 3 can be quantified
and serve as a basis for determining a toxicity index for each
compound using the same procedure as that used to obtain the
indices shown in FIG. 2. Thus, the hybridizations shown in FIG. 3
reveal that the effects of the different compounds on HepG2 cells,
which cannot be distinguished by standard toxicological methods
such as MTT staining, can be classified according to their ability
to mobilize splicing events specific for the differences that exist
between cells from healthy tissue and tumor cells.
[0185] 9.3. Description of Genetic Markers of Toxicity
[0186] The implementation of the techniques disclosed in examples
9.1 and 9.2 allowed the constitution and use of libraries of
clones, genetic markers of toxicity. The clones of these libraries
have been sequenced and analyzed, and the sequence of a portion
thereof is presented in the sequence listing included in the
present application (SEQ ID n.degree.: 1 to 37).
[0187] The analysis of these clones shows that they correspond to
qualitative genetic events that affect varied cellular genes, thus
confirming (i) the efficacy of the methods of this invention, (ii)
the existence of "universal" genetic markers within the meaning of
this invention and (iii) the importance of the diversity of the
clones to obtain a predictive response.
[0188] In particular, the sequences identified enable the
detection, through hybridization, of expression variations or of
alterations in the following human cellular genes: Aldolase A (exon
9) (SEQ ID NO: 1); S4 subunit of proteasome 26S (SEQ ID NO: 2);
Alpha-tubulin (SEQ ID NO: 3); Glucosidase II (SEQ ID NO: 4); lamin
B receptor homologue (SEQ ID NO: 5); EF1-alpha (SEQ ID NO: 6);
Fra-1 (SEQ ID NO: 7); tyrosine kinase AX1 receptor (SEQ ID NO: 8);
spliceosomale Protein SAP62 (SEQ ID NO: 9); TRAF-3 (SEQ ID NO: 10);
EF2 (SEQ ID NO: 11); TEF-5 (SEQ ID NO: 12); CDC25b (SEQ ID NO: 13);
interleukine-1 receptor-associated kinase (<<IRAK>>)
(SEQ ID NO: 14); WAF-1 (SEQ ID NO: 15); c-fos (exon 4) (SEQ ID NO:
16); ckshs1 (SEQ ID NO: 17); PL16 (SEQ ID NO: 18); NFAR-2 (SEQ ID
NO: 19); phosphatidylinositol4-kinase (SEQ ID NO: 20), ERF (SEQ ID
NO: 21), Eph type receptor tyrosine kinase (hEphBlb) (SEQ ID NO:
22); BAF60b protein of the SWI/SNF complex (SEQ ID NO: 23); EB1
(SEQ ID NO: 24); MSS1 (SEQ ID NO: 25); retinoic acid alpha receptor
(RARa) (SEQ ID NO: 26); translation initiation factor eiF4A (SEQ ID
NO: 27); STE20 type kinase (SEQ ID NO:28); protein HSP 90kda (SEQ
ID NO:29); Lipocortin II (SEQ ID NO:30); protein TPT1
(<<translationally controlled tumor proteon>>) (SEQ ID
NO:31); Hsc70 (SEQ ID NO:32); Cytokeratin 18 (SEQ ID NO:33);
2-oxoglutarate dehydrogenase (SEQ ID NO:34); mitochondrial gene
NADH6 (SEQ ID NO:35); mitochondrial gene NADH deshydrogenase 4 (SEQ
ID NO:36); alpha subunit of mitochondrial ATP synthase (SEQ ID
NO:37).
[0189] A particular object of the invention resides in the use of a
nucleic probe specific for all or part of a gene coding for a
protein or a factor as mentioned above, for the detection or the
evaluation of the toxic potential of a compound, and for the
detection of a toxicity situation in a biological sample.
[0190] Preferably, the probe comprises less than 1 000 bases, more
particularly less than 500 bases, and comprises a sequence
complementary to a portion of an above-mentioned gene. More
preferably, the probe is labeled.
[0191] Advantageously, the invention relates to a set of probes, in
particular of 5 probes or more, preferably of 10 probes or more,
each being complementary to a portion of a gene selected from the
above-mentioned genes. Even more preferably, the probes are
immobilized on a support, as disclosed in the present application.
Sequence CWU 1
1
37 1 225 DNA Homo sapiens 1 tgggcggagg ggacaggaga caggagcaga
gcagcagctg agcagcgtcc ctccccggcc 60 agctctccac agcccacctc
cggccaacag ccttgcctgt caaggaaagt acactccgag 120 cggtcaggct
ggggctgctg ccagcgagtc cctcttcgtc tctaaccacg cctattaagc 180
ggaggtgttc ccaggctgcc cccaacactc caggccctca ctcgc 225 2 186 DNA
Homo sapiens 2 tggggggagg gagtggaaat tcaattttac ggtccagccg
tcctggccgt agcagggccg 60 gatccagggt gtctgctctg tttgtggcca
tgattacctt gacattgaca ttctgatcaa 120 atccatccat ctgattcagc
agctccagca ggatcctctg aacctccctg tcggccccta 180 ccacgc 186 3 206
DNA Homo sapiens misc_feature 67 n = A,T,C or G 3 ggatgatgag
gagagcgttt cggtcggagg ggatggagga aggcgagttt tagaggcccg 60
tgaaganatg gctgcccttg agaaggatta tgaggaggtt ggtgtggatt ctgttgaagg
120 agagggtgag gaagaaggag aggaatctaa ttatccattc cttttggccc
tgcccctcat 180 aacgcttctc tcatcatcca atcact 206 4 237 DNA Homo
sapiens 4 cgggtggagg tacagggtct ggggaccatg atgcttctgg tagctttgaa
tgtcatacca 60 cacctcccct tggccaggca gatagacctg gacaccatgg
gctccagagt ctgatacagg 120 gtgaaccagc aacgcatccc caagcaagta
ctgatcatct atattgaagg tagtcacatc 180 ctgagggtac tgcacccaga
ggggcctcat gacaggaata cctcccacga aacgctt 237 5 152 DNA Homo sapiens
5 agccccagtt tcggccctgg cacctggggg gaactcaggc aatccgattt acgacttttt
60 tctgggacga gagctcaacc ctcgtatctg tttcttcgac ttcaaatatt
tctgtcaact 120 gcgacccggc ctcatcggct gggtcctcat ca 152 6 241 DNA
Homo sapiens misc_feature 20, 49 n = A,T,C or G 6 tcaaaggtgg
atagtctgan agctctcaac acacatgggc ttgccaggna accatatcaa 60
caatggcagc atcaccagac ttcaagaatt tagggccatc ttccagcttt ttaccagaac
120 ggcgatcaat cttttccttc agctcagcaa acttgcatgc aatgtgagcc
gtgtggcaat 180 ccaatacagg ggcatagccg gcgcttattt ggcctggatg
gttcaggata atcacctccc 240 c 241 7 301 DNA Homo sapiens misc_feature
171, 261 n = A,T,C or G 7 tgcgttgagg gggctggtgg gctgctggtg
ccctggtact gcctgtgtcc ccctccttgg 60 ttccttccgg gattttgcag
atgggtcggt gggcttccgc accagctcta ggcgctcctt 120 ctgcttctgc
agctcctcaa tctctcgctg cagcccagat ttctatcttc ngtttgtcag 180
tctccgcctg caggaaagtc ggtcagttcc ttcctccggt tcctgcactt ggccgcagcc
240 agcttgttcc gctcgcgcct nctcggcggc gctcctcttc ctccgggctg
atctgttaca 300 c 301 8 149 DNA Homo sapiens misc_feature 88 n =
A,T,C or G 8 tgggggcagg gcttgcctta cttcctggag gaagcccgaa gacaggactg
tggccgccaa 60 cacccccttc aacctgagct gccaagcntc agggaccccc
agagcccgtg gacctactct 120 ggctccagga tgctgtcccc ctccgtgca 149 9 271
DNA Homo sapiens misc_feature 66 n = A,T,C or G 9 atgaggaggg
ggcaaaccgt tctcagcggg ggtggaggcc gcttcacccc agggggccag 60
cagggnaggc tgggtggacc gggggcttct ccatcttaaa gtggaactgg aggaagaact
120 gcttggtctc ccggttccag tgtgtccaga acttgccctc cgccttgtcg
atctctctgc 180 tcggcacctt gaaggcaatg gtctcgtagg gctcagcggc
catgagcagg tactgccagc 240 gccggtccgg aggctcgatc ccctgccgca c 271 10
224 DNA Homo sapiens 10 gcgggctagg ggactaagtt gtcaaataca gtctcaccta
attacaggag cggtacagcg 60 atcctggact tctagcctct tttcacggtg
agagttcaca agacagacta gacacagtgc 120 agcaggagaa atgaaacgca
ggctctgctt ggccccgggg cctcctcacc cgcacacctg 180 ccagccccga
gacggccgag gcttacacgt ctgccctccc acta 224 11 288 DNA Homo sapiens
11 tggactgagg ggctgaagcg gctggccaag tccgacccca tggtgcagtg
catcatcgag 60 gagtcgggag agcatatcat cgcgggcgcc ggcgagctgc
acctggagat ctgcctgaag 120 gacctggagg aggaccacgc ctgcatcccc
atcaagaaat ctgacccggt cgtctcgtac 180 cgcgagacgg tcagtgaaga
gtcgaacgtg ctctgcctct ccaagtcccc caacaagcac 240 aaccggctgt
acatgaaggc gcggcccttc cccgacggcc tcccacag 288 12 219 DNA Homo
sapiens 12 ggggctgagg ggacatggac gccatgctct gaagggcttt gtccttggag
acctggtccg 60 gttcatggcc ttgatgccaa cctggtactc ccgcaccttc
ttccgagcta gaacctgtat 120 gtggctggac acctgtcttc tcgtccgagt
cttccccgtc ctcagtttaa tatagcgtgc 180 aatcaactca tttcggccgt
acatcttgcc ctcctcact 219 13 111 DNA Homo sapiens 13 ctgtggcagg
ctgtctgctc aacaaaacgc tcccacctgg tttgggtatg caaggcactg 60
cgcatccacg gccatccacg gccatccacc catccatcca acctccccca t 111 14 297
DNA Homo sapiens misc_feature 6, 10 n = A,T,C or G 14 gggaangaan
gttggctttg ggtgcgtgta ccgggcggtg atgaggaaac acggtgtatg 60
ctgtgaagag gctgaaggag aacgctgacc tggagtggac tgcagtgaag cagagcttcc
120 tgaccgaggt ggagcaggct gtccaggttt cgtcacccaa acattgtgga
ctttgctggc 180 tctgtgctca gaacggcttc tactgcctgg tgtacggctt
cctgcccaac ggctccctgg 240 aggaccgtct ccactgccag acccaggcct
gcccacctct ctcctggcct cccggca 297 15 331 DNA Homo sapiens
misc_feature 6, 19, 24, 61, 331 n = A,T,C or G 15 ggggancagg
tcagcatgna cagnatttct accactccaa acgccggttg atcttctcca 60
ngaggaagcc ctaatccgcc cacaggaagc ctgcagtcct ggaagcgcga ggggcctcaa
120 aggcccgctc tacatcttct gccttagtct agtttgtgtg tcttaattat
tatttgtgtt 180 ttaatttaaa cacctcctca tgtacatacc ctggccgccc
cctgccccac tcatttacac 240 caaccaccca actatctata aacctagcca
tggccatccc cttatgaagc gggcacagtg 300 attataggct ttcgctctaa
gaattaaaga n 331 16 273 DNA Homo sapiens 16 cggtaggagg gtgaaggcct
cctcagactc cggggtggca acctctggca ggcccccagt 60 cagatcaagg
gaagcccaga catctcttct gggaagccca ggtcatcagg gatcttgcag 120
gcaggtcggt gagctgccag gatgaactct agtttttcct tctccttcag caggttggca
180 atctcggtct gcaaagcaga cttctcatct tctagttggt ctgtctccgc
ttggagtgaa 240 taagtcagct ccctctccgg ttgcggaatt tgg 273 17 145 DNA
Homo sapiens misc_feature 8, 9, 18, 24, 32, 33, 85 n = A,T,C or G
17 tcgcgaanng ggctgaangc tagncaaacc gnncgatcat gtcgcacaaa
caaatttact 60 attcggacaa atacgacgac gaggnagttt gagtatcgac
atgtcatgct gcccaaggac 120 atagccaagc tggtccctac ctccc 145 18 334
DNA Homo sapiens misc_feature 64 n = A,T,C or G 18 tttgtgcagg
ggggctgtcc ccttggcccc agactcctct tcatcatcat cctgcctggg 60
ccgnatggac tggtcttccc tctcttcagc cgctcattga gtgccttcag ggccagttgc
120 cttctccgct cggcgtcttg agggtctgtg cctggcaggc tgatggtgat
ggaggatggg 180 gcacccacat cgtagcgctt caccgtcttc tggcatatct
ttaccttcac caggaggctg 240 tgcaccaagt tcgccagcaa acccaccaca
ggctgcagga tctcagggaa gaaagtggcg 300 aaagcaaagt ggtcagccat
gtcccctctc gcac 334 19 245 DNA Homo sapiens misc_feature 43 n =
A,T,C or G 19 agcgtttgga gttgaggggc cgatcctgac aaagccggca
agnaacccag tcatggagcc 60 tgaacgagaa agaggcgtgg gcctcaagta
cgagctcatc tccgagaccg ggggcagcca 120 cgacaagcgc ttcgtcacgg
aggtcgaagt ggatggacag aagttccaag gtgctggttc 180 caacaaaaag
gtggcgaagg cctacgctgc tcttgctgcc ctagaaaagc ttttccctcc 240 catga
245 20 178 DNA Homo sapiens misc_feature 15 n = A,T,C or G 20
gggggtaagg cgganatgag atgggggccg ctgtggcctc aggcacagcc aaaggagcaa
60 gaagacggcg gcagaacaac tcagctaaac agtcttggct gctgaggctg
tttgagtcaa 120 aactgtttga catctccatg gccatttcat acctgtataa
ctccaaggag cctgattt 178 21 163 DNA Homo sapiens misc_feature 21, 22
n = A,T,C or G 21 ggttgcaggc ccacacccaa nncgtctaca actaccacct
cagtccccgc gccttcctgc 60 actaccctgg gctggtggtg ccccagcccc
agcgccctga caagtgcccg ctgccgccca 120 tggcacccga gaccccaccg
gtcccctcct cggcctgcca ggc 163 22 296 DNA Homo sapiens misc_feature
8, 11, 16, 25, 283 n = A,T,C or G 22 ggattcgngg ncgaantgcc
gtggnacatt actggcactg gcacctgtgc tgggactgcc 60 aattccccgc
agctcacggc actcagctta cttgagagtt tgaccataga ctcccgggtg 120
gcatcaggtg actcaagcag tggtggggac ttcactgctt gctggctgtc tgagcgtctc
180 agagtacccc ccacccgccg gcgcagcatc ttcctgatac tgccgccaga
tttcttacca 240 tcagttcatc aaccatggac tgcaagcaga tgctaataat
ganagcctcc ccacaa 296 23 310 DNA Homo sapiens misc_feature 3, 33,
34 n = A,T,C or G 23 aangcgatct cctgctgatg gtggtagagg ccnnaaaatt
gctcatttgg gccttcagtg 60 ggtcgtccac ctccacatcg atgtcgtaac
aggctgtctt cttctggtcg ttagggtcga 120 cactaatgac atggttgatg
acaatggggt ctggatgctg cagcaaccct gccagcttca 180 tgggaatctc
ggagaaacgg agtcggccca actgaagatc tggcggaagt aacggttgca 240
gttgatgtac tcccgctcgt gcccatcctg cagctggttg tgcttgatgt aaagccacag
300 ggcctgccac 310 24 232 DNA Homo sapiens misc_feature 230 n =
A,T,C or G 24 gtgaggtagg cagctgagtt gatgcagcag gtcaacgtat
tgaaacttac tgttgaagac 60 ttggagaaag agagggattt ctcttcggaa
agctcggaac attgaattga tttgccagga 120 gacgaggggg aaaacgaccc
tgtattgcag aggattgtag acattctgta tgcccagatg 180 aaggctttgt
gatcctgatg aagggggccc acaggaggag caagaagagn at 232 25 231 DNA Homo
sapiens misc_feature 18, 203, 220 n = A,T,C or G 25 tccggcgagg
cttttggngc tgctaaaatg ccggattcct cggtgccgat cagcggaaga 60
ccaaagagga tgagaaggac gacaagccca tccgagctct ggatgagggg gatattgcct
120 tgttgaaaac ttatggtcag agcacttatt ctaggcagat caagcaagtt
gacgatgaca 180 ttcagcaact tctcaagaaa atnatgagct cactggtatn
aagaatctga c 231 26 301 DNA Homo sapiens misc_feature 26 n = A,T,C
or G 26 aatgagggtg gtgaagccgg gcagcntgct tggcgaactc cacagtctta
atgatgcact 60 tggtggagag ttcactgaac ttgtcccaga ggtcaatgtc
cagagagaca cgttgttctg 120 agctgttgtt cgtagtgtat ttgcccagct
ggcagagggc agggaaggtt tcctggtgcg 180 ctttgcgcac cttctcaatg
agctccccca cctccggcgt cagcgtgtag ctctcagagc 240 actcgggctt
gggcacctcc ttcttcttct tgtttcggtc gtttctacag actccctggc 300 c 301 27
279 DNA Homo sapiens misc_feature 4, 6, 18, 198 n = A,T,C or G 27
gggnangggt acccacgnat gatgtgggga gcttcctctg cagtttctgc acctcacacg
60 cacgttggtg cccccgatac aggcgtgaca ggaggcgccc atgtagtctc
ctagtgccat 120 gaccaccttc tgtatctgct gagccaattc tcgagtgggt
gctaggacca aggcctgggt 180 ggcttttaga tctaattnat ctgctgcaga
atcgatatgg caaatgtggc cgttttccca 240 gtcccagatt gggcttgagc
aatcacatca taacccttc 279 28 295 DNA Homo sapiens misc_feature 6, 7,
11, 17, 18, 31, 32, 41, 74 n = A,T,C or G 28 atgggnnggg naccagnntg
tctgccttcg nntcataagg nccgactgtt tgatgacctc 60 gggtgccatc
cagnaatggg gtgcccacga aggtgttcct tttgatctgg gtgtctgtca 120
gctggccagc cacgccaaag tccgccagct tcacctcgcc atgctcagac agcaggacgt
180 tggccgcttt aatgtctctg tggattttct tctccgaatg gagataatcg
agtcctttca 240 gtatttctct taatataagt aagcgatctg ggtttcatct
aatgggccag gttct 295 29 348 DNA Homo sapiens 29 gcttgtaagg
kgggacaggg gcmcccgagg ctgcagkggg agcccatggg gacactatac 60
argggcacaa gttttccaac tatraactcc taacctaatc gacttyttcc atgcraracs
120 catcctcatc gccctcgaga ggggggatct matcaggaac tgcagcattg
ggttcctctg 180 ctgccacttc atyttcatca atacctaaac ctagcttgat
catgcaataa atgcggttgg 240 agtgggtctg gggatcctca ggggaaaagc
cagaagatag cagggcggtt tcaaacagca 300 gcaccaccag gtccttaact
gccttatcat tcttgtcggc ctaacctt 348 30 450 DNA Homo sapiens
misc_feature 385, 408, 411, 422, 434 n = A,T,C or G 30 gcgggggagg
tgtcttcaat aggcccaaaa tcaccgtctc caggtggcca gataaggctg 60
acttcagtgc tgatgcaagt tcctttttgg tccttctctg gtaggcgaag gcaatatcct
120 gtctctgtgc attgctgcgg ttggtcaaaa tgttgacaat ggtgacctca
tccacacctt 180 tggtcttgat ggctgtttca atgttcaaag catcccgctc
agcatcaaag ttagtatagg 240 ctttgacaga cccatatgca cttgggggtg
tagagtgatc accctccaag ctgagcttgc 300 acaggatttc gtgaacagta
gacattttga aggaagcttc ctgaggccaa tgtgttcaac 360 caagcggaaa
ctctccgggt agagngaaac ccaagttgct atctcaanaa ncctgcaaaa 420
anacgctttt aatnctagtg cgccgcctga 450 31 492 DNA Homo sapiens
misc_feature 188, 436, 468, 477, 478 n = A,T,C or G 31 ccagaggtgg
aggggaatat ggtcagtagg acagaaggta acattgatga ctcgctcatt 60
ggtggaaatg cctccgctga aggccccgag ggcgaaggta ccgaaagcac agtaatcact
120 ggtgtcgata ttgtcatgaa ccatcacctg caggaaacaa gtttcacaaa
agaagcctac 180 aagaagtnca tcaaagatta catgaaatca atcaaaggga
aacttgaaga acagagacca 240 gaaagagtaa aaccttttat gacaggggct
gcagaacaaa tcaagcacat ccttgctaat 300 ttcaaaaact accagttctt
tattggtgaa aacatgaatc cagatggcat ggttgctcta 360 ttggactacc
gtgaggatgg tgtgccccat aatgatttct ttaaggtggt taaaaatgga 420
aaatgtacca atgtgnaata ttttgactat cccttgcccg ataccttnta atctagnngg
480 ccctgagtca ct 492 32 251 DNA Homo sapiens misc_feature 187,
211, 233 n = A,T,C or G 32 cagcattcag ttcttcaaat cgggcacggg
taatggaggt atagaagtcg attccttcat 60 agagagaatc gatctcaata
ctggcctggg tgctggaaga gagggtacgc ttagcacgtt 120 cacaagcagt
acggaggcgt cttacagctc tcttgttctc actgatgtcc ttcttatgct 180
tgcgctnaac tcagcaataa aatggttgac nattcggttg taaaatcttc tcncccaagt
240 gggtgtctcc a 251 33 212 DNA Homo sapiens 33 gaaagcgtta
ttgtggccgg tcgatctcca agactggact gtacgtctca gctctgtgag 60
cgtcgtctca gcagctccaa cctcagcaga ctgtgtggtg accactgtgg tgctctcctc
120 aatctgctga gaccagtact tgtctagctc ctctcggttc ttccgagcca
gctcgtcata 180 ttgggcccgg atgtctgcca tgatcttggc ga 212 34 186 DNA
Homo sapiens 34 actgatccct gccctcaaga ctcattgaca agtctagtga
gaaatggcgt ggactacgtg 60 atcatgggca tgccacacag agggcggctg
aacgtgcttg caaatgtcat caggaaggag 120 ctggaacaga tcttctgtca
attcgattca aagctggagg cagctgatga gggctccgga 180 gatgtg 186 35 120
DNA Homo sapiens 35 ggatgatgag gagaacgtta tggggaggag gggttgaggt
cttggtgagt gttttagtgg 60 ggttagcgat ggaggtagga ttggtgctgt
gggtgaaaga gtatgatggg gtggtggttg 120 36 314 DNA Homo sapiens 36
ggggtgtagg gggacttcaa actctactcc cactaatagc tttttgatga cttctagcaa
60 gcctcgctaa cctcgcccta ccccccacta ttaacctact gggagaactc
tctgtgctag 120 taaccacgtt ctcctgatca aatatcactc tcctacttac
aggactcaac atactagtca 180 cagccctata ctccctctac atatttacca
caacacaatg gggctcactc acccaccaca 240 ttaacaacat aaaaccctca
ttcacacgag aaaacaccct catgttcata cacctatccc 300 ccattctcct ccta 314
37 258 DNA Homo sapiens 37 ggatgatgag gagagcgtat ggggcggagg
tttaggtatt gtagcgcgtg gctcgtaggc 60 ccaccgagga acagggcgga
gtagcggccg agcttggatg agcggagaga cctgcaccgg 120 tggcaccatc
ttgtcctgac ctccccggat acgctttcct catcatcaat cactagtgcg 180
gcgctgcagg tcgaccatat gggagagctc ccaacgcgtt ggatgcatag cttgagtatc
240 tatagtgtca cctaaata 258
* * * * *