WO2001057189A2 - Fas pathway genes - Google Patents

Abstract

A method of identifying compounds which stimulates or inhibits apoptosis by contacting the cell expressing a gene with the compound of interest and determining the ability of the compound to stimulate or inhibit apoptosis is provided. In addition a method and its pharmaceutical composition to treat degenerative disease, auto-immune disease and tumors in a subject by administering to the subject a compound which stimulates a gene in the Fas pathway is disclosed. The use of casein kinase inhibitor, dicumarol, sulfinpyrazone, Nrf-2 inhibitor or a glutathione precursor as a medicament is also disclosed. A method of identifying genes that encode for inhibitors of cell death by inactivating genes in cells by sensitizing cells to cell death, using gene inactivators, applying positive selection to the sensitized cells and using subtraction analysis to identify the genes that have been inactivated is also provided.

Description

FAS PATHWAY ΩFNFS

BACKGROUND OF THE INVENTION

1 FIELD OF THE INVENTION

The present invention relates to a method of identifying genes, specifically genes that maintain specific cell phenotypes

2 DESCRIPTION OF RELATED ART

There are methods available to isolate and identify specific genes However these methods are not efficient and rapid Applicant has previous disclosed the Technical Knock Out (TKO) selection method which has the advantage of rapid isolation of genes that inhibit proliferation in a specified restrictive environment [Deiss and Kimchi, 1991 , Deiss et al, 1995, Kissil et al, 1995, Deiss et al, 1996] However, this method has the limitation of requiring a phenotype that can be efficiently selected against, such as a cell growth arrest or cell killing phenotype

Recently Smith et al [1995] and United States Patent 5,612, 180 have described a method called genetic footpπnting to identify genes The method involves mutagenesis of potentially large numbers of genes followed by a genetic selection of the cells containing the mutated genes This is followed by retrospective analysis of the effect of individual gene inactivation on the behavior cells containing these inactivations From this information new genes are determined This method has significant disadvantages for large scale gene identification The genetic footpπnting method involves mutagenesis by gene insertion and because of this requires a haploid target which imposes a limitation on the method Second, the method of determining the effect of each gene inactivation on the fitness of the cells containing the mutation involves a PCR amplification of the target gene which requires prior knowledge of the nucleotide sequence of all the target genes that can be studied which limits the gene base which can be searched It would be useful to have a method which does not require a haploid target and does not require a known sequence

It would be useful to have a rapid method which can identify genes to be isolated that are essential for the maintenance of specific cell phenotypes where positive selection exists for the phenotypes These identified genes are excellent targets for the development of pharmacological inhibitors which would also act clinically to inhibit the specific phenotype In other words it would be useful to have a tool which can effectively identify pharmacological targets for inhibition of deleterious phenotypes

SUMMARY OF THE INVENTION

According to the present invention, a method for the identification of genes that are essential for the maintenance of specific cell phenotypes is disclosed The method includes the initial step of identifying a cell type with a phenotype of interest The method allows the phenotype of interest to be phenotypes relating to growth, phenotypes relating to release of factors and phenotypes relating to other basic cell functions

Gene inactivation is performed on an aliquot of cells of the cell type of interest Possible methods of gene inactivation include Genetic Suppressor Element (GSE) inactivation, Random Homozygous Knock-Out (RHKO) inactivation, or Technical Knock Out (TKO) inactivation Positive selection is then performed on an aliquot of the cell culture to which gene inactivation has been applied The positive selection includes manipulations that test the ability of cells to survive under specific culture conditions, ability to express a specific factor, changes in cell structure or differential gene expression

Cells which continue to maintain the phenotype following gene inactivation have not had the gene of interest inactivated whereas cells in which genes necessary for maintaining the phenotype have been inactivated have been lost Utilizing subtraction analysis between treated and untreated aliquots the gene in the cells which has been inactivated that affects the phenotype of interest is identified The subtraction analysis can include the methods of differential display, representational differential analysis (RDA), suppressive subtraction hybridization (SSH), serial analysis of gene expression (SAGE), gene expression microarray (GEM) nucleic acid chip technology, or direct sequencing

The invention further discloses the genes that are identified by the method of the present invention and for antibodies directed against the gene product of these identified genes The present invention also provides for a customized kit to practice the method of the present invention

DESCRIPTION OF THE DRAWINGS

Other advantages of the present invention can be readily appreciated as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings wherein Figure 1 is a schematic representation of a general outline of the method of the present invention,

Figure 2 is a schematic representation of the method of the present invention with a regulated anti-sense cDNA expression library,

Figure 3 is a schematic representation of the AHM method of the present invention,

Figures 4 A-C show the effect of the AHM method on cell survival, A shows that transfection of anti-sense bFGF sensitizes HeLa cells to Fas induced programmed cell death (PCD), B shows the levels of expression of bFGF, C shows the quantitation of the levels of the different bFGF forms,

Figures 5 A-H show the effect of the AHM method on cell survival, A shows that transfection of anti-sense Nrf2 sensitizes HeLa cells to Fas induced PCD, B shows the levels of expression of Nrf2, C shows that membrane permeable dominant negative Nrf2 polypeeeptide sensitizes HeLa cells to Fas induced PCD, D shows that transfection of Nrf2 protects HeLa cells from Fas induced PCD, E shows that Dicumarol sensitizes HeLa cells to Fas induced PCD as determined by the number of viable, trypan blue excluding cells, F shows that Dicumarol sensitizes HeLa cells to Fas induced PCD as measured by the apoptotic index (calculated as the ratio of the number of apoptotic cells to the total number of cells as determined by DAPI staining), G Shows that sulfinpyrazone sensitizes HeLa cells to Fas induced killing, and H shows that N-acetyl cysteine protects HeLa cells from Fas induced PCD,

Figures 6 shows the distribution of the differential abundance of cDNAs contained in the microarray, of the AHM method, and

Figure 7 shows that CKI-7 sensitizes HeLa cells to FAS induced PCD

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention, a method for the identification of which genes are essential for the maintenance of a specific cell phenotype is disclosed Phenotypes that can be studied are those for which changes can be monitored in either haploid or diploid cells The method requires two general steps The first is the inactivation of genes in the cell by any method known in the art and then in the second applying positive selection for the phenotype of interest followed by the identification via a subtraction analysis of the gene in the cells which has been inactivated that affects the phenotype of interest By this method, a collection of genes that are essential for the maintenance of a specific phenotype are identified at the conclusion of the procedure The invention further discloses the genes that are identified by the method of the present invention and for antibodies directed against the gene product of these identified genes

Briefly, the method includes initially the identification of a cell type for which genes controlling its phenotype are needed Once the cell type has been identified where required for the method an expression cDNA library is constructed of the cells as they are expressing the phenotype

In general three methods are available for the gene inactivation step Genetic Suppressor Element (GSE) [Holzmeyer et al, 1992, Roninson et al, 1995, Gudkov et al 1994], Random Homozygous Knock-Out (RHKO) [Li and Cohen, 1996], and Technical Knock Out (TKO) described herein above Of these methods the TKO method is preferred as it adaptable to the second step of the present method as described herein below in Example 1 Any method for gene inactivation can be used with existing or later derived methods which can be adapted to work with the second step of the present method is preferred

Following gene inactivation treatment, an aliquot of the treated cells are exposed to a positive selection That is, the cells are exposed to conditions requiring/activating the phenotype of interest A reserved aliquot of the treated cells is not exposed Following positive selection cells which continue to express the desired phenotype remain and those cells which cannot maintain the phenotype are lost The method then provides for determining the gene that was not expressed in the lost cells by a "subtraction" analysis by any method known in the art, generally utilizing a comparison between the reserved cell aliquot and the cells remaining after positive selection It should be noted that many aliquots can be tested and screened The gene(s) identified is at least one of the genes which controls the phenotype

The relative abundance of the differences between the "targeted" and "untargeted" aliquots are simultaneously compared using a "subtraction" analysis (differential analysis) technique such as differential display, representational difference analysis (RDA), GEM-Gene Expression Microarrays (Schena et al , 1995, Aiello et al , 1994, Shen et al , 1995, Bauer et al , 1993, Liang and Pardee, 1992, 1995, Liang et al , 1993, Braun et al , 1995, Hubank and Schatz, 1994, United States Patent Number 5,545,531), suppressive subtraction hybridization (SSH) and direct sequencing (WO96/17957)

In the preferred method the procedure involves the transfection of targets cells with an anti-sense expression library followed by the positive selection of cells which have maintained a specific phenotype in the face of a specific challenge to the phenotype It should be noted that one construct can be tested or many can be tested simultaneously in this method including over 100 000 constructs from an expression library Cells in which an anti-sense inactivation has targeted a "sense" gene essential for the selected phenotype can be lost during the selection Applicants have found that in general one cell has incorporated only one construct

In this embodiment the next steps are to identify and isolate anti-sense expression vectors that are lost from the cell population due to cell loss during positive selection, that is, that induce a disadvantage in transfected cells during specific, positive selection resulting in the loss of the cell carrying the vector These vectors are identified by subtracting the anti-sense expression vectors present after the selection from those present before the selection utilizing the reserved cell aliquot This difference represents the vectors that express anti- sense against gene(s) that are essential for the maintenance of the selected phenotype

These vectors are then recloned and sequenced The identified anti- sense expression vectors are re-tested individually for the ability to inactivate the specific phenotype With the sequence identified, the sense gene controlling the phenotype of interest can be identified using standard methods known in the art

More specifically, the first part of the method consists of transfecting a target cell culture aliquot with an anti-sense expression library The library is generated by cloning a cDNA library in the anti-sense orientation into an expression cassette that can express the anti-sense strand at a high efficiency The cassette also contains a resistance marker that allows for selection of cells that have been successfully transfected The cells that are transfected are ones that express a phenotype of interest

The transfection results in a pool of cells that can express anti-sense messages against a large number of the genes expressed in the cell These anti-sense messages can inactivate the functional expression of the corresponding sense message This results in a pool of cells "knocked out" for the expression of many different genes In many cases due to the vector system used, applicant have noted that the resulting cells can contain only a single anti-sense expressing vector

When a transfected cell looses a specific phenotype, the anti-sense identity of the sense gene that has been knocked out is identified by isolating and sequencing the anti-sense expression cassette in the reserved unselected (untreated) aliquot The anti-sense strand on the anti-sense expression cassette is the compliment of the sense gene If the anti-sense strand in not a full length anti-sense, or does not match a sequence of a known gene, then the gene fragment can be used as a hybridization probe in order to isolate the full length gene In essence, the anti-sense expression vector serves as a tag to identify the gene inactivation event of interest

The method involves the selection of the pool of anti-sense expressing cells for the specific phenotype The goal of the selection is to separate the majority of cells which continue to maintain a specific phenotype from the rare cells in which an anti-sense inactivation event has specifically knocked-out a gene that is essential for the maintenance of the specific phenotype

This can be based on virtually any kind of positive selection means These selection means can be varied as is known to those skilled in the art However, the following is a non-exhaustive list and is not to be construed as limiting the present invention to these listed means

The selection means is based on the ability of the cells to 1 Grow or survive under specific culture conditions that is the actual selection is for the growth or survival of the cells In an embodiment, this can be basic culture conditions, such that the selection is for growth or survival- essential genes The selection conditions could include sub-effective doses of specific factors which at effective doses can cause growth arrest or cell killing In this case the selection is for the identification of knock-outs which sensitize the cells to the specific added factor

In another embodiment, the selection can be in combination with a factor that normally does not cause an arrest or killing function In this case a knock- out could be selected which only in combination with the added factor are effective in arresting or killing cells

In a further embodiment, the selection can be for the inability to grow or survive when a parasite or infectious agent is added to the cell of interest In this case the selection is for knock-outs that are targeting genes that are specifically essential for some aspect of viral or parasitic function within a cell that are only essential when that cell is infected Since some viral infection result in the induction of survival factors (such as CrmA, p35) it is likely that at least some cell functions are different and potentially selectively needed during viral, parasite growth

2 The second type of selection means is for the expression of a specific factor that can be measured and this measurement can be adapted for a selection This factor can be anything that is accessible to measurement, including but not limited to, secreted molecules, cell surface molecules, soluble and insoluble molecules, binding activities, activities that induce activities on other cells or induce other organic or inorganic chemical reactions

3 The third type of selection means is for changes in cell structure that are detected by any means that could be adapted for a selection scheme This includes, but is not limited to, morphological changes that are measured by physical methods such as differential sedimentation, differential light scattering, differential buoyant density, differential cell volume selected by sieving

4 The fourth type of selection means is based on differences in gene expression that can be directly measured This includes changes in cell surface markers, changes in biochemical activities, any changes that can be re- selected in changes in binding of fluorescent labeled probes that could be used in conjunction with a Fluorescence Activated Cell Sorter (FACS) or any property that can be used as a basis for a selection

5 The fifth type of selection means is based on differences in gene expression that can be indirectly measured This includes changes in transcription factor activity that are measured by a synthetic gene construct encoding a selective marker (such as a drug resistance marker or a cell surface marker that could be used in a FACS selection) This category can also include changes in mRNA stability, mRNA localization, mRNA translation control All of these changes could be the basis of a selection because a synthetic construct which is controlled by one of these regulatory events could be constructed which can dπve the expression of an easily selected gene product

The third part of the method involves steps identifying the anti-sense knock-outs that specifically inhibit the phenotype of interest Since the selection of the anti-sense transfected cells is based on the maintenance of the phenotype of interest, the cells of interest (those loosing the phenotype) can not be present after the selection but is present before the selection Since the functional changes are caused by expression from anti-sense expression vectors and the inactivated genes can be identified by sequence analysis of the cloned anti-sense cDNA insert, the goal of this step is actually to identify the anti-sense expression vectors that are lost from the population of cells during the selection procedure

The anti-sense inserts are cloned into a defined position on the vector and the sequence elements surrounding the site are known, so all the cDNA inserts can be amplified with the use of a PCR amplification using primers from the sequences that surround the insert site Thus the goal becomes to identify DNA molecules present in one population and not in another This is accomplished by a variety of subtraction techniques Some of the methods that can be used are summarized below as is known to those skilled in the art However, the following is a non-exhaustive list and is not to be construed as limiting the present invention to these listed means Various differential hybridization methods as well as different subtractive hybridization techniques can be used They are summarized in some detail in the methods section

Once fragments are identified that are lost during the selection and are candidates for genes of interest their function must be confirmed and the gene identified in the fourth part of the method The fragments can be recloned into the anti-sense expression cassette and individually re-transfected into the target cell to determine whether the expression of the isolated fragment can really change phenotype If the phenotype is really lost as is predicted then the isolated fragment can be sequenced and used to isolate the full length sense gene It can also be determined whether the fragment is indeed anti-sense with the use of strand specific probes The sense gene fragment can be used to derive antibodies that can be used to monitor expression levels to determine if there has been a functional anti-sense knock-out [Deiss et al , 1995]

The present invention is a genetic method for identifying genes that are essential for the maintenance of specific cell phenotypes The method requires that the specific phenotype can be positively selected These identified genes are excellent targets for the development of pharmacological inhibitors which can also act clinically to inhibit the specific phenotype Thus the present invention provides a gene discovery tool which can effectively identify pharmacological targets for inhibition of deleterious phenotypes

The following are several examples but this list is not to be construed as limiting the present invention to these listed examples

Phenotypes related to growth or survival

Addressing the problem of unusual growth This includes the problem of cancer but is not limited to cancer but is applicable to all aberrant growth events The method of the present invention can be used to identify genes that are essential for the growth of cells transformed under general or specific conditions

To define genes that are essential specifically for transformed cells, an anti-sense cDNA library can be introduced into a transformed and the non- transformed cells that it was derived from The anti-sense constructs that interfere with transformed cell growth and not from the non-transformed cells are found by subtracting the anti-sense RNA molecules expressed in surviving cells from both transfections Knock-outs specifically absent in the transformed cells but present in the non-transformed cells are desired These are isolated by the methods described herein The selection can be a most specific selection such as one where sub-lethal doses of chemotherapeutics are added during the selection In this case the selection can include gene knock-outs that sensitize the cells to chemotherapeutic treatments

The factors added during the selection could be ones that are thought to be present at the site of tumors Thus the selection can include events that sensitize cells to a localized tumor effect and could increase the specificity of anti-cancer treatment Any growth or survival event could be used as a basis not just cancer related

The growth or survival phenotype can also be used as a way of eliminating populations of cells that are not necessarily growing improperly but which function in a manner that is deleterious Thus virally infected cells or parasite harboring cells could be used as a target and the un-infected or non- parasite containing cells used to subtract This can define all the genes that are specifically essential for the cell in the presence of these insults These can of course be excellent targets for inhibiting viral or parasite spread

Phenotypes related to the release of factors

This class of selections includes events that increase or decrease the production of secreted factors These include inflammatory mediators whose release could be modulated For example, if the production of a specific mediator is necessary for normal immune function but is produced at lethal levels in aberrant situations (such as septic shock), then one could use the production as a screen and look for events that knock-out or down-regulate productions In a further embodiment, the selection can be done in the presence of sub-optimal doses of other drugs in order to identify sensitization events

Phenotypes related to changes in cell functions

These selection events are designed to identify genes that are essential for many basic cell functions that depend on any changes that can be externally selected

Several permutations of the method of the present invention are possible and are presented as schematic diagrams in Figures 1-2 Figure 1 provides a general outline of the gene identification method of the present invention In this version a population of cells is first transfected with an anti- sense cDNA expression library The expression library in this scheme codes for a drug resistance marker that is used to select transfected cells This results in a population of cells (Population 1 ) that all contain anti-sense expression cassettes The population of transfected cells is then placed under a selection pressure Cells that survive this selection constitute population 2

Transfected cells that become sensitive to the selection procedure can be lost or at least reduced in abundance in population 2 In order to identify the constructs that induce this sensitization the following procedure is performed The expression cassettes contained in the two population are extracted from the cells The cDNA inserts are excised by PCR amplification using primers that flank the cDNA cloning sites This results in two pools of PCR fragments To identify the elements that are lost during the selection a subtraction is done between the two pools Elements are identified that are present in population 1 and absent or reduced in abundance in population 2 To confirm that the subtracted fragments do indeed induce a sensitization to the selection procedure, individual fragments are recloned into the identical vector and than individually retransfected into cells These cells are then individually assayed for sensitivity to the selection procedure A correctly cloned element can induce sensitization of the transfected clones to the selection procedure

Figure 2 provides a diagram of the method with a regulated anti-sense cDNA expression library In this simple variation the object is to clone the anti- sense cDNA library into a vector in which expression of the anti-sense is regulatable The method is then modified so that during the original transfection, the expression of anti-sense is turned "OFF" After cells are selected for the presence of the vector an aliquot of cells is harvested and vectors are extracted and inserts excised by PCR This constitutes pool 1 The remaining transfected cells are treated to turn "ON" the expression of the anti- sense expression An aliquot of these cells are taken after several cell divisions (pool 2) Again the aliquot of cells are extracted and cDNA inserts excised by PCR Finally an aliquot of the cells with anti-sense turned "ON" is placed under a specific selection and cells after this selection are harvested Again following extraction and PCR amplification there is pool 3

In this case two kinds of subtractions can be performed The first subtraction can be pool 2 from pool 1 This identifies anti-sense inactivations that are lethal or growth arresting The second subtraction, can be subtracting pool 3 from pool 2 This identifies anti-sense knock-outs which sensitize cells to the specific selection

Additional permutations/variations on the method of the present invention can be made The method can be used to identify different gene expression backgrounds In this variation anti-sense induced sensitization in cells that express different genes is investigated This can be accomplished by transfecting into cells that contain an inducible gene expression cassette This cassette affords inducible expression of a specific gene construct called gene X for this example Following transfection and selection for the presence of an anti-sense cDNA library and aliquot of cells is harvested vector extracted and cDNA inserts excised by PCR This is pool 1 The remaining cells are induced to express gene X Allowing some time for expression, the cells are harvested, vectors extracted and cDNA inserts excised by PCR This generates pool 2 The subtraction of pool 2 from pool 1 yields inserts that specifically sensitize cells to the expression of gene X

In another variation the method of the present invention is used with different cell types This variation involves transfecting two different cell types This could be cells of different genetic background or of different tissue origins, or even from different organisms In the simple diagramed case two cell types are transfected with the same anti-sense cDNA expression library The different cell types are propagated in different containers Transfected cells are then selected for the presence of the library The cells containing the library are harvested, vectors extracted and cDNA inserts are excised by PCR For each cell type a different pool is generated The subtraction between these pools, both pool 1 from 2 and pool 2 from 1 identify anti sense knockouts that are specifically lethal or growth arresting to one cell type but not the other

In a further variation, the method of the present invention is used for determining the fitness of specific genes in a population In all the versions described above, populations of PCR fragments are generated which potentially differ by some number of elements due to the biological activity of those elements The subtraction of these pools is then used as a method to identify cDNA fragments which have biological effects when expressed It is also possible to use the same pools to determine whether an anti-sense construct directed against a specific gene could confer some biological effect during some sort of selection Specifically in this simple example when two pools 1 and 2 have been generated for the operation such as in the examples, there are a variety of tools available to individually measure the relative abundance of anti sense construct representing specific cDNAs in pool 1 and pool 2 A variety of methods are known for quantitating the abundance of DNA molecules in different samples Following is a non-exhaustive list Southern blot analysis either using a fragment of the gene of interest as a probe against the two pools, Quantitative PCR with specific primers identifying the gene of interest GEM analysis, using the pools 1 and 2 as the probes and hybridizing against chips of arrayed known genes If the abundance of the anti-sense construct significantly decreases after a selection then it is likely that anti-sense has sensitized the cells to that selection

As shown in Example 2 and Table 2 herein below, sequences of genes have been identified by the method of the present invention (SEQ ID Nos 15- 36) An antisense construct of these sequences delivered to a cell reduces a gene product (gene inactivation) and thereby provides sensitization of the cells to anti-Fas antibodies In a preferred embodiment the sequences are SEQ ID Nos 19,20,23,25,26,36 These antisense constructs can be used therapeutically to sensitize the cells for antibody therapy Antisense therapeutic construct can be delivered to the cells and can be rendered nudease resistant as is known in the art [Agrawal, 1996, Calabretta, et al, 1996, Crooke, 1995, Feigner, 1997, Gewirtz, 1993, Hanania, et al 1995, Lefebvre-d'Hellencourt et al, 1995, Lev-Lehman et al , 1997, Loke et al, 1989, Wagner et al , 1996, Wagner, 1994, Radhakπshnan et al , 1990 ] Also disclosed by the present invention is a method for the identification of genes that encode for inhibitors of cell death This method termed Achilles Heel Method (AHM) involves introducing an anti-sense library into a vector such as an episomal vector (Deiss and Kimchi 1991 ) and transfecting the library into target cells to generate a pool of cells with each cell expressing a different anti- sense fragment This pool of cells is known as Pool 1 (Figure 3) Then, the transfectants are treated with a sub-optimal dose of a killing inducer and the surviving cells are collected The surviving cells are known as Pool 2 The cells containing inactivation events that sensitize the cells to death are preferentially lost from Pool 2, and so are the anti-sense CDNA inserts that confer the sensitization These cDNA inserts are recovered by subtracting the cDNA inserts containing in Pool 2 from those in Pool 1 Following the subtraction of Pool 2 cDNAs from Pool 1 cDNAs, the potentially sensitizing cDNAs are cloned in a cDNA expression vector (an episomal expression vector) in the same orientation as in the original library The anti-sense cDNA containing episomes are individually transfected into target cells in order to confirm their ability to render the cells more sensitive to the killing inducer

Alternatively, Pool 1 and Pool 2 cDNAs are labeled and used as probes for hybridization to cDNA microarray filters Computer analysis identifies the cDNAs depleted from Pool 2 In both cases "function profiling" is being employed to identify signal pathway inhibitors

Recently, similar "function profiling" methods have been described for genetic analysis of S. cerevisae (Smith et al 1996) (Shoemaker et al 1996) These methods are well suited to yeast since they require prior knowledge of gene sequence and the ability to generate haploid cells By contrast, AHM does not require a priori knowledge of any gene sequence or haploid cells Thus, AHM is a powerful genetic tool for "functional profiling" in mammalian cells Moreover AHM can be easily scaled up to generate "function profiles" of all expressed human genes The AHM and the TKO methods are complementary and together can be used to identify the positive and negative regulators of any pathway that can be reconstructed in human cells in cell culture

AHM is used to identify of inhibitors of FAS induced apoptosis since the activation of the Fas pathway is relevant to several different pathologies Activation of the Fas pathway is physiologically associated with both detrimental and with protective processes For example, the activation of the Fas pathway is associated with liver damage in fulminate hepatitis and with immune mediated tissue destruction Conversely, activation of the Fas pathway is required for prevention of autoimmunity (elimination of auto- reactive T-cells) and suppression of tumoπgenesis (Askew et al 1991 , Evan et al 1992, Shi et al 1992, Wagner et al 1994) Thus, it is clinically beneficial to generate tools for enhancing or inhibiting the Fas pathway depending on the pathology of interest The identification of inhibitors of the Fas pathway can be used for both of these purposes The genes identified by the Fas AHM screen are putative survival agents As such, over-expression of these genes are predicted to prevent killing, and thus can be used for clinical benefit in situations where cell survival is desired such as organ failure, neurodegeneration etc Accelerating FAS induced PCD can ameliorate autoimmunity and enhance tumor suppression, thus, pharmacological inhibition of the FAS pathway inhibitors can be translated into significant clinical benefit, as they accelerate killing

One of the genes that was identified in the Fas AHM screen is a transcription factor Nrf2 Over-expression of Nrf2 and enhancing the Nrf2 pathway by a chemical compound (NAC) confers resistance to Fas induced killing (Figure 5D and 5H respectively) , Moreover, inhibition of Nrf2 sensitizes cells to killing induced by Fas which is demonstrated by four independent experimental approaches The approaches include expression of anti-sense Nrf2 (Figure 5A 5B) treatment with a membrane permeable fragment of Nrf2 that acts in a dominant-negative fashion (Figure 5C) and two drugs, namely Dicumarol (Figure 5E) and Sulfinpyrazone (Figure 5G) Thus, the present invention establishes that modulation of a target that is identified in the Fas AHM screen modulates the response of cells to Fas induced apoptosis Furthermore, there is established the benefit of employing available drugs for applications that has not been described before and the feasibility of designing new drugs, for example the dominant negative Nrf2 AHM opens new avenues for development and utilization of new drug targets and new applications to known drugs as demonstrated in the following data

Additionally, genes identified by AHM screens for any given pathway can then be used as targets to develop inhibitors that act as cofactors to activate the pathway For example, one can identify genes, which inhibit killing induced by chemotherapeutics Inhibition of such genes sensitizes tumors to chemotherapeutics Inhibitors of the inhibitor genes have utility in treating cancer patients

A list of genes identified as inhibitors of Fas induced killing were identified in the Fas AHM screen using the cDNA microarray analysis These genes are shown in TABLE 3

The present invention also discloses a novel gene sequence as set forth in SEQ ID No 37 The present invention also provides for a customized kit to practice the method of the present invention The kit can be assembled to include at least an expression cDNA library constructed for specified cells as they are expressing the phenotype Further a culture of cells of the requested phenotype could also be provided in the kit

The invention provides a method for producing a nucleic acid molecule involved in the regulation of the Fas pathway by nucleic acid sequence homology using a nucleic acid probe, the sequence of which is derived from the nucleic acid sequence encoding the nucleic acid molecule The method of making and using the probes are known in the art e g see references cited in EXAMPLES section under the heading GENERAL METHODS

The invention provides a method for producing a nucleic acid molecule involved in the regulation of the Fas pathway which includes the use of the polymerase chain reaction and oligonucleotide primers, the sequence of which is derived from the nucleic acid sequence encoding the nucleic acid molecule The method of making the primers and using them in the polymerase chain reaction are known in the art e g see references cited in EXAMPLES section under the heading GENERAL METHODS

In one embodiment of this invention, there is disclosed a screening method for identifying a compound which stimulates or inhibits a Fas-pathway gene including the steps of (a) contacting a cell expressing a gene with the compound, and (b) determining the ability of the compound to stimulate or inhibit apoptosis as compared to a control In the preferred embodiment, the gene is selected from the set of genes consisting of casein kinase alpha 1 , Nrf-2, basic fibroblast growth factor, TNF receptor associated factor 6, human COP9, antithrombin III, mucin 1 transmembrane, adenosine receptor A3, calcium/calmodulin-dependent protein kinase II human protein immunoreactive with anti-parathyroid hormone antibodies and retinoic acid receptor gamma 1

In preferred embodiments of the screening method of the present invention, the gene is transcribed to an mRNA, the corresponding cDNA of which comprises one of the nucleic acids selected from the set of nucleic acids having SEQ ID Nos 15, 16, 17, 18, 19 ,20, 21 , 22, 23, 24, 25, 26, 27, 28, 29,30, 31 , 32, 33 34, 35, 36 and 37

Alternatively, the screening method for compounds can be a cell-based method The cell-based method utilizes tetracyline-inducible gene expression or expression of a reporter gene Compounds identified by these methods are also contemplated as embodiments of the present invention

The Fas pathway genes disclosed in this application all oppose the Fas pathway, i e they all cause inhibition of Fas - induced killing A compound which stimulates such a Fas pathway gene is a compound which causes an increase in the amount and/or the activity of gene product, this compound renders the cells more resistant to Fas-induced apoptosis A compound which inhibits such a Fas pathway gene is a compound which causes an decrease in the amount and/or the activity of gene product, this compound renders the cells more sensitive to Fas-induced apoptosis PCD can also take place via pathways other than Fas, and the genes described here can also function in such pathways

In another embodiment of the present invention, a method is disclosed for treating a tumor in a subject This method includes administering to the subject a therapeutically effective amount of a compound which inhibits an gene (Fas pathway inhibitor) in the Fas pathway The gene (Fas pathway inhibitor) can be selected from but is not limited to, the set of genes consisting of casein kinase alpha 1 , Nrf-2, basic fibroblast growth factor, TNF receptor associated factor 6 human COP9, antithrombin III, mucin 1 transmembrane adenosine receptor A3 calcium/calmodulin-dependent protein kinase II, human protein immunoreactive with anti-parathyroid hormone antibodies and retinoic acid receptor gamma 1 Specifically, the gene can be casein kinase alpha 1 and the compound can be a casein kinase inhibitor, preferably CKI-7 The gene can also be Nrf-2 and the compound can be dicumarol, sulfinpyrazone or Nrf2 inhibitor Additionally, said contacting step utilises a gene (transcribed to an mRNA), the corresponding cDNA of which includes one of the nucleic acids selected from the set of nucleic acids having SEQ ID Nos 15, 16, 17, 18, 19 , 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29,30, 31 , 32, 33, 34, 35 , 36 and 37

The term "tumor" as used herein is intended to include, but is not limited to, cancers of various types including carcinoma, lymphoma, melanoma and leukemia, inter aha

In another embodiment of the present invention, there is provided a method of treating auto-immune diseases in a subject This method includes administering to the subject a therapeutically effective amount of a compound which inhibits a gene (Fas pathway inhibitor) in the Fas pathway The gene (Fas pathway inhibitor) can be selected from the set of genes including, but not limited to casein kinase alpha 1 , Nrf-2, basic fibroblast growth factor, TNF receptor associated factor 6, human COP9, antithrombin III, mucin 1 transmembrane, adenosine receptor A3, calcium/calmodulin-dependent protein kinase II, human protein immunoreactive with anti-parathyroid hormone antibodies and retinoic acid receptor gamma 1 When treating some auto-immune diseases, the preferred gene is casein kinase alpha 1 and the compound is a casein kinase inhibitor, preferably CKI-7 Alternatively, the preferred gene can be Nrf-2 and the compound is then dicumarol, sulfinpyrazone or Nrf2 inhibitor The gene utilized can be transcribed to an mRNA, the corresponding cDNA of which comprises one of the nucleic acids selected from the set of nucleic acids having SEQ ID Nos 15, 16, 17, 18, 19 ,20, 21 , 22, 23, 24, 25, 26, 27, 28, 29,30, 31 , 32, 33, 34, 35 36 and 37

In a further embodiment of the methods of the present invention, there is provided a method of treating degenerative disease in a subject This method includes administering to the subject a therapeutically effective amount of a compound which stimulates a gene (Fas pathway inhibitor) in the Fas pathway By "degenerative disease" as used herein, the term is intended to include, but is not limited to, degenerative disease of the liver, especially fulminate hepatitis, as well as Alzheimer's disease, Parkinson's disease and Amyotrophic lateral sclerosis (also termed ALS) Preferably, the gene which is stimulated is selected from the set of genes including, but not limited to, casein kinase alpha 1 , Nrf-2, basic fibroblast growth factor, TNF receptor associated factor 6, human COP9, antithrombin III, mucin 1 transmembrane, adenosine receptor A3, calcium/calmodulin-dependent protein kinase II, human protein immunoreactive with anti-parathyroid hormone antibodies and retinoic acid receptor gamma 1 Alternatively, the gene can be Nrf-2 and the compound can be a glutathione precursor, preferably N-acetyl Cysteine

The method of treating degenerative disease in a subject can include the use of a casein kinase inhibitor, preferably CKI-7, or dicumarol or sulfinpyrazone or a Nrf-2 inhibitor in the preparation of a medicament for anti-tumor therapy and/or for auto-immune disease therapy Alternatively, the method can use a glutathione precursor, preferably N-acetyl Cysteine, in the preparation of a medicament for treatment for degenerative disease

Also provided by the present invention is a method of preparing a pharmaceutical composition which includes the steps of determining whether a compound stimulates or inhibits a Fas-pathway gene by using the screening method described above, and admixing the compound with a pharmaceutically acceptable carrier

A pharmaceutically acceptable carrier encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline, water and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents Once the candidate drug has been shown to be adequately bio-available following a particular route of administration, for example, orally, nasally, rectally, or by injection ( for example intravenous, subcutanous ) and has been shown to be non-toxic and therapeutically effective in appropriate disease models, the drug can be administered to patients by that route of administration in an appropriate solid or liquid formulation, to gain the desired therapeutic benefit

The above discussion provides a factual basis for the method of identifying genes that are essential for the maintenance of specific cell phenotypes The methods used are shown below and can be shown by the following non-limiting examples and accompanying figures

EXAMPLES

GFNFRAI MFTHODS

General methods in molecular biology Standard molecular biology techniques known in the art and not specifically described were generally followed as in Sambrook et al , Molecular Cloning A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992) and in Ausubel et al , Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989)

Polymerase chain reaction (PCR) was carried out generally as in PCR Protocols A Guide To Methods And Applications, Academic Press, San Diego, CA (1990) Reactions and manipulations involving other nucleic acid techniques, unless stated otherwise, were performed as generally described in Sambrook et al , 1989, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory Press, and methodology as set forth in United States patents 4,666,828, 4,683,202, 4,801 ,531 , 5, 192,659 and 5,272,057 and incorporated herein by reference

Recombinant Protein Purification is undertaken as generally set forth in

Marshak et al, "Strategies for Protein Purification and Characterization A laboratory course manual " CSHL Press, 1996 unless otherwise specified

Vectors are constructed containing the cDNA of the present invention by those skilled in the art and should contain all expression elements necessary to achieve the desired transcription of the sequences (see below in specific methods for a more detailed description) Other beneficial characteristics can also be contained within the vectors such as mechanisms for recovery of the nucleic acids in a different form Phagemids are a specific example of such beneficial vectors because they can be used either as plasmids or as bacteπophage vectors Examples of other vectors include viruses such as bacteπophages baculoviruses and retroviruses, DNA viruses, cosmids, plasmids, liposomes and other recombination vectors The vectors can also contain elements for use in either procaryotic or eucaryotic host systems One of ordinary skill in the art knows which host systems are compatible with a particular vector

The vectors are introduced into cells or tissues by any one of a variety of known methods within the art (calcium phosphate transfection, electroporation, lipofection, protoplast fusion, polybrene transfection) The host cell can be any eucaryotic and procaryotic cells, which can be transformed with the vector and which supports the production of the enzyme Methods for transformation can be found generally described in Sambrook et al , Molecular Cloning A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1992), in

Ausubel et al , Current Protocols in Molecular Biology, John Wiley and Sons,

Baltimore, Maryland (1989), Chang et al , Somatic Gene Therapy, CRC Press,

Ann Arbor, Ml (1995), Vega et al , Gene Targeting, CRC Press, Ann Arbor, Ml

(1995) and Gilboa, et al (1986) and include, for example, stable or transient transfection lipofection, electroporation and infection with recombinant viral vectors In addition, see United States patent 4,866,042 for vectors involving the central nervous system and also United States patents 5,464,764 and

5,487,992 for positive-negative selection methods

General methods in immunology Standard methods in immunology known in the art and not specifically described were generally followed as in Stites et al (eds), Basic and Clinical Immunology (8th Edition), Appleton & Lange, Norwalk, CT (1994) and Mishell and Shngi (eds), Selected Methods in Cellular Immunology, W H Freeman and Co , New York (1980)

Immunoassays In general, ELISAs are the preferred immunoassays employed to assess a specimen ELISA assays are well known to those skilled in the art Both polyclonal and monoclonal antibodies can be used in the assays Where appropriate other immunoassays, such as radioimmunoassays (RIA) can be used as are known to those in the art Available immunoassays are extensively described in the patent and scientific literature See, for example, United States patents 3,791 ,932, 3,839,153, 3,850,752, 3,850,578, 3,853,987, 3,867,517, 3,879,262, 3,901 ,654, 3,935,074, 3,984,533, 3,996,345, 4,034,074, 4,098,876, 4,879,219, 5,011 ,771 and 5,281 ,521 as well as Sambrook et al, Molecular Cloning A Laboratory Manual, Cold Springs Harbor, New York 1989

Polyclonal and Monoclonal Antibody Production

Antibody Production Antibodies can be either monoclonal or polyclonal Conveniently, the antibodies can be prepared against a synthetic peptide based on the sequence, or prepared recombinantly by cloning techniques or the natural gene product and/or portions thereof can be isolated and used as the immunogen Such proteins or peptides can be used to produce antibodies by standard antibody production technology well known to those skilled in the art as described generally in Harlow and Lane, Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1988 and Borrebaeck, Antibody Engineering - A Practical Guide, W H Freeman and Co , 1992

For producing polyclonal antibodies a host, such as a rabbit or goat, is immunized with the protein or peptide, generally with an adjuvant and, if necessary coupled to a carrier, antibodies to the protein are collected from the sera

For producing monoclonal antibodies the technique involves hypeπmmunization of an appropriate donor with the protein or peptide fragment, generally a mouse, and isolation of splenic antibody producing cells These cells are fused to a cell having immortality, such as a myeloma cell, to provide a fused cell hybrid which has immortality and secretes the required antibody The cells are then cultured, in bulk, and the monoclonal antibodies harvested from the culture media for use

The antibody can be bound to a solid support substrate or conjugated with a detectable moiety or be both bound and conjugated as is well known in the art (For a general discussion of conjugation of fluorescent or enzymatic moieties see Johnstone & Thorpe, Immunochemistry in Practice, Blackwell Scientific Publications, Oxford, 1982 ) The binding of antibodies to a solid support substrate is also well known in the art (see for a general discussion Harlow & Lane Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory Publications, New York, 1988 and Borrebaeck, Antibody Engineering - A Practical Guide, W H Freeman and Co , 1992) The detectable moieties contemplated with the present invention can include, but are not limited to, fluorescent, metallic, enzymatic and radioactive markers such as biotin, gold, ferπtin, alkaline phosphatase, a-galactosidase, peroxidase, urease, fluorescein, rhodamine, tritium, C and lodination

SPECIFIC METHODS

Construction of anti-sense expression vector

This method is not limited to any specific vector system The actual requirements are that the vector system express at high levels anti-sense molecules and that they can be identified In a preferred embodiment the Epstein Barr Virus (EBV) Episomal vector is used [Deiss et al 1991 ]

The EBV episomal vector consists of DNA segments that are necessary for the episomal maintenance of the episome both in bacteria (E coli) and in human cells (this include an origin of replication and a trans-acting factor (EBNA-1 ) The episome also includes genes encoding resistance markers for selection either in bacteria or in human cells Finally the vector contains a transcription cassette Initially it is based on the vector as described in Deiss et al [1991], but the present invention contemplates any transcription cassette that produces high levels of anti-sense expression The EBV episomal vector contains a RNA Polymerase II promoter and or enhancer driving the transcription of a synthetic transcript containing a set of cloning sites, a splice donor and acceptor site and a polyadenylafion signal, followed by a second set of enhancers This vector can be efficiently shuttled from animal cells to bacteria and vice versa One procedure that allows for rapid shuttling is using the method of Hirt [1967] to extract episomal vectors from animal cells and using this preparation to transform £ coli Applicants have observed that, on average, cells transfected with a library cloned into the vector contain only one expressing vector

The specific choice of promoters and enhancers are dependent on the exact selection condition and the cell line used This must be empirically determined for each selection condition as is known to those skilled in the art

In an embodiment, the EBV vector can also contain an inducible expressed promoter such that the expression of the anti-sense library can be inducibly expressed by a specific inducer This allows additional flexibility in designing selection protocols

Construction of Anti-sense cDNA library

There are several methods available to construct directional cDNA libraries Any of these methods can be sufficient since they result in the production of a directionally identified cDNA library and the practitioner can use the method they are most familiar with The directional cDNA is then cloned into the expression cassette in the anti-sense orientation A method that can be used is detailed in Deiss et al, 1991 Briefly, it consists of making cDNA by the method of Gubler and Hoffman [1983] and making the cDNA directional by the method of Meisner et al [1987]

The mRNA is extracted from cells that have been cultured under a variety of conditions that mimic the actual selection conditions This is designed to ensure that the library includes all the messages that are expressed in the target cell under selection conditions RNA is prepared at time points that can contain messages that are always present as well as messages that are induced by the selection procedure This is achieved by extracting RNA previous to the selection and at times during the selection The various pools of RNA are then mixed together so that all possible RNA molecules are present [Deiss and Kimchi, 1991]

An alternative method that can be used consists of deriving a library of genomic DNA fragments cloned into the expression cassette Since all the transcπbed messages are derived from genomic DNA (with the exception of RNA edited messages, this actually includes mitochondπal DNA as well) this method can generate all possible messages The directionality can be lost so the library can be only half anti-sense Since the sense fragments are unlikely to frequently encode full length proteins or have biological activity the anti- sense fragments can still likely produce the most frequent biological effects The genomic fragments are produced by restriction enzyme cleavage of genomic DNA Only one library per species is necessary to produce, since with ⁵ the exception of the B and T cell receptors, genomic DNA does not differ in different cell types at least in mammals (again erythrocytes or any cells that lack nuclei are an exception)

In the case of the genomic library it is necessary to determine whether ι ° any expressed fragments express a sense or an anti-sense message This is done by using the insert as a strand specific probe both from the expressed and non-expressed strand in a Northern analysis This indicates if the expressed fragment is sense or anti-sense in relation to the endogenously expressed gene

15

Some sequences match, some do not match, genes already deposited in the various databases In the case where the identified gene matches the sequence of a gene already in a database this information can then enable the determination if the insert is a sense or anti-sense insert

20

Transfection of the anti-sense library

There are a large variety of methods to transfect DNA into cell lines and cell cultures The most efficient method for each selection can be determined ²⁵ empirically based on experience and the known relative efficiency of each method

The method selected must both efficiently delivery DNA into cells and not effect the biological responses that can be selected following the transfection Viral vector system can also be used and this can entail producing infectious virus and infecting the target cells Applicant has found electroporation to be an efficient method, but other methods can be used as are known in the art

Identification of differentially expressed antisense messages

The methods of identifying the differentially expressed antisense messages in a preferred embodiment can include the methods described in Braun [1996] and as described in Diatchenko et al [1996] These methods include a PCR amplification of subtracted populations Appropriate restriction sites are included in the expression vector so that following PCR amplification of the cDNA inserts, the inserts are flanked by appropriate restriction sites Restriction digestion is then used to produce templates that are useful for these techniques

Another method that is used is the πGEMD gene expression microarray as described in Schena et al [1995] In this technique, PCR fragments corresponding to a set of specific plasmids (in this case antisense cDNA inserts contained in antisense expressing vectors or other DNAs as appropriate) are fixed to a glass template and this is hybridized with two fluorescently labeled probes In this specific case, the probes are reverse transcribed antisense transcripts derived from cells transfected with the antisense expression library either before or following a selection

Generation nf efficient antisense inhibitor

In addition to assaying expression cassettes where all the transcripts are directionally cloned in the antisense orientation, another strategy employed in the present invention is to generate randomly primed cDNA and cleave the cDNA with two restriction enzymes X and Y and clone the resulting mixture into two different expression cassette In the first cassette site, X, can transcnpfionally precede Y and the second cassette site, Y, can transcπptionally precede X In this arrangement, the cDNA is divided into sections that can have different abilities to serve as an efficient antisense inhibitor The strongest differential signal is likely to be produced by the fragment that is the most efficient antisense inhibitor Thus, the screening is more likely to produce a meaningful differential signal

EXAMPLE 1 DETERMINATION OF COMBINATION OF METHODS FOR PREFERRED EMBODIMENT

The method requires two distinct major steps as described herein above It is greatly advantageous if this method can be applied to a wide variety of cells It is therefore useful if both steps could be applied to a wide variety of situations

In the first step genes are inactivated in order to determine whether individual genes are essential for a specific phenotypic change It is advantageous if these inactivation can have a phenotype both in haploid cells and in diploid cells Since many cells of interest are diploid in nature Furthermore, it is also an advantage if the inactivation method allows for the rapid identification of the inactivated genes This can be achieved in a variety of manners The inactivation methods are generally based on one of three different principles

The first principle is that genes can be functionally inactivated by expressing mRNA that is derived from the anti-sense strand of the sense message This allows for inactivating the mRNA in the cell and does not require a specific gene dose This can work for single copy or multiple copy genes either from haploid or diploid organism It has been shown that anti-sense inactivation can be effective in a wide range of organisms including bacterial, plant and animal Applicants and others have extended the original observation by generating anti-sense expressing cDNA libraries Applicants termed this method Technical Knock Out (TKO) These libraries contain collections of many (usually 100,000 to 1 ,000,000) different anti-sense expression constructs that can individually express a single anti-sense RNA molecule when transfected into appropriate target cells Since these libraries contain large collections of these vectors they in effect can express anti-sense RNA to virtually all expressed mRNA molecules Several investigators have used these type of libraries to inactivate genes and change the phenotype of cells Once an altered cell is identified the expression cassette contained in the cells can be identified since the expression cassette DNA sequence is known Subsequently, the anti-sense expressed cDNA molecule that is contained in the expression cassette is identified This can be achieved by a variety of methods, applicants have used two methods The first method involves shuttling the vector from animal cells into bacterial cells Once the vector is in bacterial cells it is easy to produce large amounts of the vector for further analysis The second method employed involves PCR amplification of the cDNA inserts by designing PCR primers that flank the cDNA cloning site on the vector The flanking vector sequences are known so it is easy to chose appropriate primers PCR amplification with these primers amplifies any cDNA molecules that were present between the two primers The anti-sense approach also allows for tagging of the inactivation event That is the identity of the sense message generally can be determined by sequencing the anti sense construct This construct can then be identified and isolated from the phenotypically altered cells

The second gene inactivation method that fits these requirement is an inactivation method that relies on production of "dominant negative polypeptides" corresponding to fragments of genes from an expression cDNA library This method is called the Genetic Suppressor Element method (GSE) It is based on the observation that small fragments of a gene when expressed as polypeptides can interfere with the normal function of the full length gene product and in fact interfere with the normal function In this manner these gene fragments were called "dominant negatives" A GSE library thus consists of fragmented cDNA molecules which are cloned into an expression cassette When expressed from translation initiation signals in the cDNA molecule or from translation initiation signals present in the expression cassette these polypeptides corresponding to gene fragments can interfere with gene function In addition the libraries used in the GSE method also include some anti-sense fragments and therefore gene inactivation can occur either by anti-sense or by dominant negative inactivation of gene function

The third method of gene inactivation that could be used is called "Random Homozygous Knock-Out (RHKO)" In this method gene inactivation is achieved in two steps A retroviral vector is used to infect target cells The integration of the retroviral element itself can lead to inactivation of one copy of a gene if the integration event itself functionally disrupts the normal transcription or activity of the gene in which it integrates The retroviral vector used has an additional property that it encodes a transcription element that should transcribe into the chromosomal location in which it has integrated In the case that this generates and anti-sense RNA transcript, additional copies of the gene could be inactivated Thus this method also relies on anti-sense inactivation

The TKO method was chosen for generating mactivations in these examples as the preferred embodiment because the other methods described above are not as compatible with the second step in the method of the present invention as the TKO procedure However, as improvements become available in these methods they could be used

In the GSE method both sense and anti-sense gene fragments are generated which are expected to have different biological activities It is difficult to distinguish closely related gene fragments of this sort by the methods that can be used in the second part of the gene identification method of the present invention Thus the rare molecules that cause biological changes when expressed can be very difficult to distinguish from many similar molecules that do not have biological effects The molecules that do not have effects can in essence mask the active molecules

The RHKO method was also difficult to adapt to a high throughput subtractive procedure The potential anti- sense fragments generated in this procedure must be cloned out individually and this is a process that is hard to adapt to subtraction The TKO method was easily adapted to subtraction The cDNA inserts contained in expression vectors should be all or at least mostly anti-sense in nature The cloning procedure outlined in Deiss and Kimchi was used This generates anti-sense cDNA libraries and results in libraries that are biased to be anti-sense It is possible to obtain some sense cDNA inserts with this method Thus since most of the fragments are antisense the subtraction step is mainly between different cDNA fragments that were expressed as anti-sense constructs Again the principle of the present invention is that the abundance of an anti-sense construct(s) that induces a disadvantageous phenotype can be reduced after a biological selection It is important to understand the identity of these constructs Thus the TKO method was the method of choice for the gene inactivation step of the present invention It can be used in a variety of cell populations, both in haploid, diploid and aneuploid cells It can be easily scaled up to involve 100,000 events or more without undue expense And it can be easily adapted to the subtractive methods that are needed in the second part of the method of the present invention

The second step in the gene identification method requires identifying the loss of specific anti-sense gene constructs from a large population of anti- sense constructs that are not lost This can be accomplished in a variety of different ways Because it is a great advantage to be able to identify specific losses in the presence of large numbers of molecules that are not lost a method is needed that has a high throughput capacity One method that fits this requirement involves using high density arrayed chips such as the GEM chips These are arrayed dots containing specific DNA molecules corresponding to genes The dots are arrayed at high density on a glass covershp with the position of each dot and the identity of the DNA molecule fixed on each dot precisely determined Two probes derived from different population of DNA or RNA molecules are labeled with two different fluorescent dyes and hybridized to the arrays After appropriate washing the relative binding of the dyes at each dot is determined The amount of dye bound at each spot reflects the abundance of the gene fixed on that dot relative in the whole population Thus when two populations of DNA molecules are labeled with different dyes one can accurately determine whether there has been a change in relative abundance of individual molecules in the population If there is no change the ratio of the two dyes can be one If there has been a change in abundance then the ratio of the two different dyes can also change This method can rapidly measure the changes of large numbers of genes in a large population A copy of the DNA fix on each dot is stored and can be retrieved for further analysis Although in the following example the GEM method was not used to measure the loess of anti-sense constructs it is a method that can be used in the practice of the present invention

A second method uses to identify the loss of specific anti-sense gene constructs from a large population of anti-sense constructs that are not lost is called "Subtraction" This involves manipulating two populations of DNA or RNA molecules so that only molecules that are present in one population and not in another are recovered The version that was actually used is called PCR-Select which is a commercially available kit from CLONTECH Briefly

1 Two populations of double stranded DNA are generated It is assumed that some of the dsDNA molecules are present in only one of the populations (or at least much more abundant in one population)

2 These populations are separately processed The population that is assumed to have extra species of molecules is called the tester sample The second population that is assumed not to have these specific species is called the driver The tester population is separately hgated to two different linkers This generates tester population 1 and tester population 2 The driver is left without linkers

3 A series of manipulations including denaturation and renaturation of the driver and tester in various combinations is used This results in generating a series of DNA molecules that have different set of linkers at their ends The only product that can be effectively PCR amplified at the end of the manipulations are those that are present in the tester and absent (or reduced) in the driver These molecules are easily isolated after the last PCR step

The net result of this method is that a population of gene fragments that are present in one population and lost or absent in another population is rapidly isolated This is exactly what is needed in the gene identification method These PCR products can be isolated by standard techniques and be used for further analysis as shown in Example 2

EXAMPLE 2

IDENTIFICATION OF GENES IN HeLa CELLS THAT ARE INVOLVED IN fas ANTIBODY SENSITIVITY

The method of the present invention was applied to HeLa cells treated with anti-Fas antibody in order to identify genes that when knocked-out cause sensitization of HeLa cells to the action of anti-Fas antibodies

HeLa cells are derived from a human cervical carcinoma and were used in the original TKO [Deiss and Kimchi, 1991] HeLA cells were used as an exemplar of the method of the present system as they are easily grown in culture, are easily transfected and respond to anti-Fas antibody treatment Anti-Fas antibody (Kamiya Biomedical Company Seattle, Washington catalog number MC-060) is directed against Fas/CD95/Apo-1 a transmembrane receptor that is known to signal a death response in a variety of cell types This antibody is an activating antibody, that is, the binding of the antibody mimics the effects of binding of ligand Applying the appropriate dose to responding cells has been shown to lead to induction of cells death (Deiss et al , 1996) HeLa cells respond to this treatment

In this exemplar genes are identified that regulate the sensitivity of HeLa cells to killing by anti-Fas antibody Specifically, genes are identified whose loss sensitizes HeLa cells to anti-Fas treatment

The outline of the procedure is as follows

1 HeLa cells were transfected with an anti-sense cDNA library

2 Cells containing anti-sense expression vectors were isolated by selection with Hygromycin Since the vector contains the Hygromycin resistance marker the selection of the transfected cultures with Hygromycin generated a population of cells which contain the anti-sense expression cassettes

3 Aliquots of this pool of cells were treated with anti-Fas antibody under two different experimental conditions It should be noted that more conditions could be screened at the same time

a Treatment with a sub-lethal dose of anti-Fas antibody (10 ng/ml) Cells that are super-sensitive to treatment with anti-Fas antibody were killed whereas the majority of the population which is resistant to the treatment proliferated

b In the second condition, the cells were treated with a lethal dose of anti-Fas antibody (100 ng/ml) The cells were harvested at 24 hours, before the majority of cells had been killed In this case, applicants were looking for anti-sense events that accelerate the killing associated with anti-Fas treatment as another type of sensitization

4 Aliquots of the cells just before the treatment with anti-Fas antibody and just after the treatment with anti-Fas antibody were harvested The DNA contained in each cell population was extracted

5 The anti-sense cDNA inserts contained in these DNA samples were preferentially amplified through the use of PCR (see details below)

6 The pools of anti-sense cDNA fragments that were derived from cells after treatment were subtracted from those before treatment(see details below) This generated a set of cDNA fragments that were present in cells before treatment but were absent after treatment These fragments are good candidates for sensitizing cDNA fragments In other words, it is likely that expression of some of these fragments leads to the inactivation of genes which causes cells to become super-sensitive to anti-Fas antibody treatment These super-sensitive cells are quickly killed at a lower dose of anti-Fas antibody or more rapidly than the majority of cells These cells are therefore lost from the treated cultures but are present in the untreated population Likewise, the plasmids inducing this super-sensitivity are present in the cells before treatment but are absent from the cell sample taken after treatment Thus, these fragments are identified during the subtraction 7 The cDNA fragments generated by the subtraction were cloned into the original expression vector Appropriate restriction enzyme sites were generated or maintained during the subtraction procedure so that the recloned construct is exactly identical to the construct in the originally transfected cells The sequence of the isolated cDNA fragments was determined

8 The anti-sense expression plasmids containing the cDNA inserts that were identified in the method of the present invention were individually re- transfected into HeLa cells and the transfectant cells were assayed for sensitivity to anti-Fas antibody treatment

Specific Materials and Methods

HeLa cells were transfected with anti-sense cDNA library cloned in the episomal vector, anti-sense expression vector pTKO-1 This is the same library described in Deiss and Kimchi [1991] One million cells plated in a 100 mm dish were transfected with 15 ig of DNA containing the anti-sense cDNA library, by using the Superfect reagent (Qiagen, Santa Claπta, California) as suggested by the manufacturer Two days following transfection, cells were treated with Hygromycin B (200 ig/ml) (Calbiochem-Novabiochem Corporation, La Jolla, California) Following two weeks of selection, the population of cells was completely resistant to Hygromycin B

These cells were plated in triplicate at a density of 2 5D10⁶ cells per 150 mm dish in the absence of Hygromycin B One plate was treated with anti-Fas antibody at 10 ng/ml (clone CHI-11 Kamiya Biomedical Company, Seattle, Washington) for five days, the second plate was treated with 100 ng/ml of anti- as antibody for 24 hours and the third plate was UN-treated for 24 hours Following the treatments, the cells were harvested by washing twice with ice cold PBS (NaCI 8g/lιter KCI 0 2g/lιter, Na²HP0⁴ 1 44 g/liter, KH²P0⁴ 0 24 g/hter, final pH of solution adjusted to pH 7 4 with HCI) and concentrated by centπfugation (15,000 x g for 15 seconds) DNA was extracted by using solutions P1 , P2 and P3 from the Qiagen Plasmid Purification Kit (Qiagen, Santa Claπta, California) The cell pellet was resuspended in 200 II of solution P1 (50 mM Tπs-HCI, pH 8 0, 10 mM EDTA, 100 ig/ml RNase A) then mixed with 200 II of solution P2 (200 mM NaOH, 1 % SDS) and incubated five minutes at room temperature 200il of solution P3 (3 0M Potassium Acetate, pH 5 0) were added and incubated two minutes at room temperature, followed by a ten minute centπfugation at 15,000 x g The clear supernatant was mixed with an equal volume of isopropanol and centπfuged at 15,000 x g for ten minutes The precipitated DNA was resuspended in 100 il of water and stored frozen until use

For PCT amplification of the cDNA inserts contained in these DNA preparations, the following reaction was set in a total volume of 100 il 1 il of the DNA, 200 ιM of dATP, dGTP, dCTP, dTTP, 500 ng each of primers prLPD#64 (SE(SEQ ID No 2) and prLPD#65 (SEQ ID No 3), 10 mM Tπs-HCI pH 9 0, 0 1% Triton -100, 1 0 mM MgCI and 1 unit of Taq DNA polymerase (Gibco/BRL, Gaithersburg, Maryland) This reaction was incubated in a Thermocycler 2400 (Perkin-Elmer, Foster City, California) according to the following protocol First, the reaction was heated to 943 C for five minutes, then was cycled 25 times using the following three temperatures 58DC for one minute, 72DC for five minutes, 94 DC for one minute After 25 cycles, the reaction was incubated at 723C for seven minutes This resulted in amplification of the cDNA inserts The prLPD#64 and prLPD#65 primers were design such that the end of the cDNA insert that is proximal to the promoter in the pTKO-1 vector is exactly flanked by a Hindlll restriction site (this site is present in the vector) and the end of the cDNA that is distal to the promoter in pTKO-1 vector contains a BamHI restriction site The BamHI site was created by altering a single base in the sequence immediately adjacent to the distal cDNA insert site (prLPD#65), by PCR When the library was generated [Deiss and Kimchi 1991], this site distal to the promoter was generated by the fusion of a BamHI restriction site (derived from the cDNA fragments) and a Bglll site (derived from the vector) This fused site is resistant to cleavage by either enzymes, but a single base change restored the cleavage by BamHI Thus, the amplified cDNA fragments are flanked by a Hmdlll restriction site on the promoter proximal side of the cDNA and by a BamHI site on the promoter distal side This allows the exact re- cloning of the fragments into the pTKO-1 expression vector with exact conservation of sequence and orientation

Following the PCR reaction, the mixture was cleaved with BamHI and Hmdlll (Gibco/BRL, Gaithersburg, Maryland) as described by the manufacturer The digestion products were purified using the Wizard PCR Prep Kit (Promega, Madison, Wisconsin) This generated cDNA inserts with Hmdlll and BamHI ends

These nucleic acid fragments were subjected to subtraction using the PCR-Select Kit (Clontech, Palo Alto, California) according to the instructions of the manufacturer with the following modifications The driver was the PCR products derived from the untreated samples and two testers were used The first tester was derived from cells treated with 10 ng/ml anti-Fas antibody and the second tester was derived from cells treated with 100 ng/ml of anti-Fas antibody First modification the subtraction is done between dsDNA pools so no cDNA synthesis is required The fragments generated from the previous step were used directly in the subtraction Thus, applicants began at Step IV F3 in the instructions (preparation of the adapter ligated tester cDNA) The second modification was the replacement of the blunt end ligation of adapter 1 and adapter 2R with cohesive end adapters These cohesive end adapters were ligated to the BamHI and Hmdlll cleaved PCR fragments generated in the step above The cohesive ligafion is usually more efficient than blunt end hgation and since applicants use cDNA flanked by different restriction sites allowing the orientation of the fragments to be maintained when reclon g the subtracted products If the blunt end gation is used, it does not allow distinguishing one end from the other and applicants would not be able to determine the relative orientation of the cDNA in the original expression cassette Thus adapter 1 was replaced by an equal mixture of primers prLPD#80 (SEQ ID No 4), prLPD#81 (SEQ ID No 5), prLPD#83 (SEQ ID no 7) and prLPD#84 (SEQ ID No 8) Adapter 2R was replaced by an equal mixture of prLDP#82 (SEQ ID No 6), prLPD#88 (SEQ ID No 12), prLPD#89 (SEQ ID No 13) and prLPD#90 (SEQ ID No 14) The other primers were of identical sequence as described in the kit Thus, primer prLPD#85 (SEQ ID No 9) is the sequence of PCR primer 1 , primer prLPD#86 (SEQ ID No 10) is the sequence of nested PCR primer 1 and primer prLPD#87 (SEQ ID No 11 ) is the sequence of nested PCR primer 2R The manual supplied by the manufacturer with the kit was followed from the point of gation of the adapters to the tester (Section IV F3 in the Manual) 0 3 ig of the tester was taken for adapter hgation The initial hybridization included 0 9 ig of the driver and 0 03 ig of the adapted ligated tester At the conclusion of the subtraction, a final PCR reaction is done using nested PCR primer 1 (prLPD#86) and nested PCR primer 2R (prLPD#87) This material contains the cDNA fragments that were present in the untreated sample but absent from the treated samples The product of this PCR reaction were re-cloned into the anti-sense expression vector (Primers used in this example are set forth in Table 1 )

Re-cloning of the subtracted fragments was accomplished by cleaving the subtracted population with BamHI and Hmdlll and purifying the cleaved products with the Wizard PCR Prep Kit (Promega Madison Wisconsin) The cleaved products were then directly cloned into the pTK01 -DHFR vector between the Hmdlll and Bglll sites This replaced the DHFR sequences with the cDNA This is precisely the procedure that was used to generate the anti- sense cDNA expression library Thus, the fragments that were generated by the subtraction were exactly re-cloned into the original anti-sense expression vector that was used to transfect cells at the beginning of the procedure The re-cloned constructs exactly duplicate the constructs that were present in the library The re-cloned constructs were introduced into bacteria and DNA was extracted from the bacteria following conventional methods These DNA preparations were used as a template for sequencing in order to determine the nucleotide sequence of the isolated cDNA inserts Primer prLPD#51 (SEQ ID No 1 ) was used in Automated sequencing using Applied Biosystems 377XL DNA sequencer with Perkin-Elmer Dye Terminated Sequencing Kits (Perkin- Elmer, Applied Biosystems Division, Foster City, California) In addition, plasmids carrying the re-cloned inserts were transfected into HeLa cells to confirm their ability to induced super-sensitization to anti-Fas antibody treatment in HeLa cells

HeLa cells were transfected with 15 Ig of plasmids or control vectors as described for transfection of the original library The cells were selected for two weeks for resistance to Hygromycin B treatment (200 ig/ml) This selects for cells which contain expression cassettes One million cells were plated in a 100 mm dish and treated with anti-Fas antibody Effects of anti-Fas antibody on the transfected cultures were quantified by MTT assays as described by the manufacturer (Sigma, St Louis, Missouri)

ANAI YSIS OF THF ISOI ATFD DNA SFQUFNCFS

The clones were sequenced using primer prLPD#51 which anneals close to the edge of the cDNA which is distal to the promoter in the antisense expression cassette Thus, in the case that the sequence matches the sense strand of a known gene then the insert is in the antisense orientation Sequences were compared to the combined nonredundant database and the dbest compiled at the NCB1 using the Blastn program with default parameters (Internet address http //www ncbi nlm nih gov/cgi-bm/BLAST/nph- blast?Jform=0) The sequences determined by the method of the present invention are listed in Table 2

Clone LPD#599 (SEQ ID No 15) shows no match against known gene sequences in the nonredundant database as of November 9, 1997, but does match several EST sequences such as a 99% match against gene bank entry AA043612 Sequence analysis indicates that this fragment is oriented in the sense orientation in the antisense expression library Applicants have noticed that although the library is designed to preferentially express the antisense strand, there are some sense gene fragments included in the library [Levy- Strumpf et al , 1997]

Clone LPD#601 (SEQ ID No 16) shows no match against known genes in the nonredundant database as of November 7, 1997, but matches many genomic clone pieces and many EST entries as for example a 95% match to a portion of gene bank entry N20920

Clone LPD#602 (SEQ ID No 17) shows no match against known genes in the nonredundant database as of November 7, 1997 It does show some similarity to a large number of gene back entries such as gene bank entry Z68269 Many of these matches are in the 60-70% range and can indicate a repeated sequence

Clone LPD#606 (SEQ ID No 18) shows no match against known genes in the nonredundant database as of November 7, 1997 It does shown some matches against mouse EST in the 80% range (gene bank entry W71379, W29410 and AA409950) and a stretch of a good match against a human EST (gene bank W19764)

Clone LPD#607 (SEQ ID No 19) shows no match against known genes in the nonredundant database as of November 7, 1997 This clone does show a very good match against three EST (gene bank entries, T08248, H42827 and T30569) The sequence analysis indicates that this fragment was transcribed in the antisense oπentation in the original library Thus, reduction (inactivation) of the gene product that is encoded by the full length message representing this clone leads to supersensitization of cells to the treatment with anti-Fas antibodies

Clone I PD#608 and I PD#618 (SEQ ID No 20, SEQ ID No 26) show no match against known genes in the combined nonredundant database as of November 7, 1997 They do shown a match with a large number of EST entries (for example, gene bank entry, AA335297, H14907 and AA009451 ) The sequence analysis indicates that this fragment was transcribed in the antisense orientation in the original library Thus, reduction of the gene product that is encoded by the full length message representing this clone leads to supersensitization of cells to the treatment with anti-Fas antibodies

Clone I PD#609 (SEQ ID No 21) shows no good match against know genes or EST in the combined nonredundant database as of November 7, 1997

Clone I PD#610 (SEQ ID No 22) shows no good match against known genes in the combined nonredundant database as of November 7, 1997, but does show a good match against several EST entries including gene bank entries AA447349, H24439, R72995, H 17221 and R24985 Sequence analysis indicates that this fragment was in the sense orientation in the original library

Clone I PD#61 1 (SEQ ID No 23) shows no good match against known genes in the combined nonredundant database as of November 7, 1997, but does show good matches with a variety of EST (including gene bank entries R76164, R25241 N66591 and N66577) The sequence analysis indicates that this fragment was transcribed in the antisense orientation in the original library Thus, reduction of the gene product that is encoded by the full length message representing this clone leads to supersensitization of cells to the treatment with anti-Fas antibodies

Clone LPD#613 (SEQ ID No 24) shows no good match against known genes in the combined nonredundant database as of November 7, 1997, but a portion of the sequence shows homology to a large number of sequences and likely contains a repetitive element

Clone I PD#616 (SEQ ID No 25) shows an excellent match with human tryptophanyl-tRNA synthetase (see emb X67928 for example) The sequence analysis indicates that this fragment was transcribed in the antisense orientation in the original library Thus, reduction of tryptophany-tRNA synthetase leads to supersensitization of cells to the treatment with anti-Fas antibodies Clone I PD#619 (SEQ ID No 27) shows no good match against known genes in the combined nonredundant database as of November 7 1997, but does show good matches with the sequence of a human retroviral element called pHE 1 (for example emb Z95333, emb Z84475, gb M85205)

Clone I 7_10_1_l PP (SEQ ID No 28) shows no good match against known genes in the combined nonredundant database as of November 7, 1997, but does show very good matches against several EST (for example, see gb T7771 1 , gb T78724, gb AA324254) Sequence analysis indicates that the fragment inserted in the expression library was in the sense orientation

Clone I 7_10_?_l PP (SEQ ID No 29) shows no good match against known genes in the combined nonredundant database as of November 7, 1997, but does show good matches with several EST (for example, see gb

R12242 and gb H84498) Sequence analysis indicates that the fragment inserted in the expression library was in the sense orientation

Clone F7_100_1 1J PD (SEQ ID No 30) shows no good match against known genes or EST in the combined nonredundant database as of November 7, 1997

Clone L7_10_8_BS (SEQ ID No 31) shows no good match against known genes or EST in the combined nonredundant database as of November 7, 1997

Clone L7_10_3 (SEQ ID No 32) shows no good match against known genes or EST in the combined nonredundant database as of November 7, 1997 Clone F7_100_10 (SEQ ID No 33) shows a good match with mitochondria! DNA (see gb L00016)

Clone L7_100_4_BS and L7_100_5_BS (SEQ ID Nos 34-35) show no good match against known genes in the combined nonredundant database as of November 7, 1997 They do show good matches against several EST entries (for example see gb AA227149, gb N46661 , dbj C76104) Sequences analysis indicates that this gene fragment was oriented in the sense orientation in the original expression cassette

Clone E7_10_9 (SEQ ID No 36) shows no good match against known genes in the combined nonredundant database as of November 7, 1997 It does show good matches against two EST entries (see gb R54192, gb H39863) The sequence analysis indicates that this fragment was transcribed in the antisense orientation in the original library Thus, reduction of this protein leads to supersensitization of cells to the treatment with anti-Fas antibodies

As described above, the isolated fragments were recloned and then reassayed for sensitivity to treatment with anti-Fas antibody (50 ng/ml for 72 hrs) using the MTT assay These assays showed that expression of fragments LPD#599 (repeated in triplicate with two different transfectants) or LPD#606 (repeated in triplicate) resulted respectively in a 1 6 and 2 0 fold increase in sensitivity to anti-Fas antibody treatment whereas expression of CrmA (a protective protein) resulted in a 2 3 fold reduction in sensitivity This demonstrates that the method of the present invention can be successfully used to identify genes based on a positively selected phenotype

FXAMPI F 3 IDENTIFICATION OF GFNES INVOVFn IN THF FAS PATHWAY AND INHIBITORS OF THE GENES

The Achilles Heel Method utilizes functional profiling as diagrammed in Figure 3 The first step consists of introducing an anti-sense expression library (Deiss and Kimchi, 1991 ) into target cells to generate a pool of cells, each expressing a different anti-sense fragment (Pool 1 ) Then, the transfectants are treated with a sub-optimal dose of a PCD inducer and the surviving cells are collected (Pool 2) Cells containing inactivation events that sensitize the cells to killing are preferentially lost from Pool 2 Consequently, the anti-sense cDNAs contained in the sensitized cells are depleted from Pool 2 The "sensitizing" cDNA inserts that are present in Pool 1 but depleted from Pool 2 are identified by two methods, subtraction or hybridization to cDNA microarray Following the subtraction of Pool 2 cDNAs from Pool 1 cDNAs, the potentially sensitizing cDNAs are cloned in an anti-sense orientation in an episomal expression vector The anti-sense cDNA containing episomes are individually transfected into target cells in order to confirm their ability to render the cells more sensitive to the killing inducer Alternatively, Pool 1 and Pool 2 cDNAs are labeled and used as probes for hybridization to cDNA microarrays Computer analysis identifies the cDNAs depleted from Pool 2 In both cases "function profiling" is being employed to identify signal pathway inhibitors Recently, similar "function profiling" methods have been described for genetic analysis of S. cerevisae (Shoemaker et al 1996, Smith et al 1996) These methods are well suited to yeast since they require prior knowledge of gene sequence and the ability to generate haploid cells By contrast, AHM does not require a priori knowledge of any gene sequence or haploid cells Thus, AHM is a powerful genetic tool for "function profiling" in mammalian cells Moreover, AHM can be easily scaled up to generate "function profiles" of all expressed human genes AHM is used to identify inhibitors of the Fas induced programmed cell death (PCD) pathway Fas is a trans-membrane death receptor of the TNF super family The binding of Fas ligand to Fas results in the cascade of events that lead in most cell types to apoptosis Fas induced killing is utilized in different physiological processes as follows (for review see (Nagata 1997)) elimination of auto-reactive T-cells, tumor induced immune suppression and destruction of virally infected cells, transformed cells and b- cells in cases of Insulin Dependent Diabetes Melitus (IDDM) In addition, activation of the Fas pathway has been suggested to play a role in liver damage, brain damage, arteriosclerosis and tumor suppression Modulation of the Fas pathway has clinical implications in animal models inhibition of Fas induced PCD by caspase inhibitors limits liver damage in mice and acceleration of Fas induced killing ameliorates the auto-immune phenotype of gld mice Thus, identifying regulators of the Fas pathway that can be used as targets for drug development can have great clinical impact

For the identification of inhibitors of Fas induced cell death, AHM was applied to HeLa cells that were treated with sub-lethal dose of Fas agonistic antibody The later mimics the binding of Fas ligand to Fas and induces apoptosis "Function profiling" (also termed functional profiling) was performed to identify "sensitizing" cDNA fragments by using subtraction and cDNA microarray analysis cDNA inserts from Pool 2 were subtracted from Pool 1 cDNAs and the recovered cDNAs were further analyzed Six out of seven randomly chosen cDNAs that were individually transfected into HeLa cells conferred increased sensitivity to Fas induced killing cells, ranging between 2 9 to 5 3 fold These fragments include three novel sequences and three fragments of previously described genes One of the cDNA inserts is an anti-sense fragment of human Basic Fibroblast Growth Factor (FGF-2, bFGF) and the other is an anti-sense fragment of the cap-n-collar b-zip transcription factor NF-E2 related factor 2 (NRF2)

bFGF is a potent survival factor that plays a role in development, angiogenesis, and in cell migration Previous reports have shown that down regulation of bFGF by anti-sense expression or by blocking antibodies result in loss of a transformed phenotype reduced tumor growth and reduced angiogenesis Five different polypeptides of 34kD, 24kD, 22 5kD, 22kD and 18 kD are translated from the human bFGF gene, initiating at different sites and terminating at the same position The anti-sense cDNA fragment isolated in the subtraction is 295 nucleotides long and corresponds to nucleotide 873 to 1167 of the bFGF gene (Genebank Accession Number NM_002006) It spans the last 60 nucleotides of the coding region (shared by all bFGF polypeptides) and a portion of the 3' un-translated region

In order to confirm that anti-sense bFGF confers sensitivity to Fas, pools of cells transfected with control vector (harboring no insert) or with anti- sense bFGF were generated and treated with a sub-optimal dose of anti-Fas antibody Analysis of two independent pools of transfectants demonstrates that under conditions that results in 59% and 29% killing of the vector transfected cells, anti-sense bFGF transfected cells are 3 7 and 4 4 fold more sensitive to killing (Figure 4A,) This significant increase sensitivity of anti- sense bFGF transfected cells was reproducible in six independent pools of transfectants It is not due to altered growth rate of the anti-sense bFGF transfected cells (compare the number of untreated cells in the control vector transfected pools to the number of untreated cells in the anti-sense bFGF transfected pools) or to a non-specific increase in sensitivity of anti-sense transfected cells to Fas induced killing since anti-sense cDNAs were previously isolated that render transfected cells resistant to Fas induced apoptosis Complementary to the bioassay experiments, quantitative Southern analysis of Pool 1 and Pool 2 indicates that the abundance of anti- sense bFGF cDNA is reduced by 1 9 fold in Pool 2 of cells surviving sub- lethal dose of anti-Fas antibody, compared to Pool 1

Western blot analysis of control vector transfected cells as well as anti- sense bFGF transfected cells revealed four polypeptides of 24kD, 22/22 5kD and 18kD while the 34kD form is not detected by the antibody used (Figure 4B) Quantitative analysis of the relative level of bFGF forms revealed that in the absence of anti-Fas antibody, expression of anti-sense bFGF results in reduction of approximately 25%-30% in the levels of each of the detected forms (Figure 4C) Interestingly, in cells treated with anti-Fas antibody a more significant reduction in the levels the 24kD form and 18kD is observed, 57% and 66% respectively, while the reduction of the levels of the 22/22 5kD is not altered Selective reduction in the level of some of the bFGF forms by an anti- sense fragment that overlaps the coding region of all the bFGF polypeptides can be due to a network of feedback regulation loops as previously reported for some bFGF forms

While previous studies has shown that over-expression of the 34kD form protects cells from serum deprivation induced killing and over- expression of the 24kD form protects cells from ionizing radiation, here it is demonstrated that bFGF is an inhibitor of Fas induced apoptosis, as identified by AHM

The second inhibitor of the Fas pathway that was identified by AHM is the cap-n-collar b-zip transcription factor NF-E2 related factor 2 (Nrf2) Nrf2 activates the transcription of phase II detoxifying enzymes such as NAD(P)H quinone oxireductase (NQ01 ) and Glutathione S-transferase (GST) by direct binding to the Antioxidant Response Element (ARE) in the promoter of these genes Studies of Nrf2 null mice indicate that Nrf2 is essential for the transcπptional activation of phase II enzymes in response to an anti-oxidant NQ01 and GST act in concert with phase I detoxifying enzymes (such as cytochrome p-450 monooxygenase) to mediate the cellular detoxification of xenobiotics In the absence of Nrf2, this coordinated detoxification is impaired and toxic products from phase I reactions can accumulate In the AHM screen an anti-sense fragment of Nrf2 corresponding to nucleotide 145 to 972 of the human Nrf2 (GeneBank Accession Number S74017, (Moi et al 1994)) was recovered

Bioassays of two pools of HeLa cells transfected with anti-sense Nrf2 clearly demonstrates that anti-sense Nrf2 render the cells 4 1 and 5 4 fold more sensitive to Fas induced apoptosis (Figure 5A) Again, this increased sensitivity is not a result of impaired growth, since there is only limited alteration in the growth rate of anti-sense Nrf2 transfected cells (Figure 5A) Sensitization by anti-sense Nrf2 was reproducible in seven independent pools of transfectants Western blot analysis indicated a significant 3 8 fold reduction in the level of Nrf2 protein in the anti-sense Nrf2 transfected cells (Figure 5B)

An alternative method of down regulation of Nrf2 was employed to confirm that inhibition of Nrf2 can sensitize cells to Fas induced killing Nrf2 is a transcription factor that contains an ammo terminal trans-activation domain and a carboxyl terminal DNA binding domain A dominant negative (DN) version of Nrf2 consisting of the DNA binding domain but lacking the trans- activation domain was generated Ohtsubo et al had shown that such a construct effectively inhibits the ability of wild-type Nrf2 to activate transcription (Ohtsubo et al , 1999) In order to efficiently introduce this dominant negative Nrf2 fragment into cells, a membrane solubilization domain of HIV TAT was added to it The membrane soluble DN Nrf2 was produced in bacteria and used to treat HeLa cells (Figure 5C) In the presence of low dose of vehicle (1x) or high dose vehicle (4x), an apoptotic index of approximately 10% is observed in the untreated cultures and 25% in cultures treated with anti-Fas antibody Similarly, the administration of low or high dose of the control membrane soluble Green Fluorescent Protein (GFP), did not alter the response of cells to treatment with anti-Fas antibody The addition of low dose of DN Nrf2 (1x) did not alter the response to anti-Fas antibody However higher dose of DN Nrf2 (4x), significantly sensitizes cells to killing induced by anti-Fas antibody while only modestly affected cell viability Thus using a different method, totally independent of anti-sense expression, it was confirmed that inhibition of Nrf2 leads to sensitization of cells to Fas mediated apoptosis Moreover, the membrane soluble version of DN Nrf2 can be used as a drug to sensitize human cells to apoptosis

Further testing to determine whether over-expression of Nrf2 can protect cells from Fas induced apoptosis was performed The coding region of Nrf2 was cloned into a retroviral vector Cells infected with the retrovirus were selected for resistance to puromycm, since the retrovirus carries a puromycin resistance marker Two pools of puromycm resistant cells, as well as the corresponding control vector infected cells were assayed for their response to Fas induced apoptosis The results shown in Figure 5D demonstrate that expression of Nrf2 protects cells from Fas induced apoptosis Untreated cells show a very low apoptotic index Anti-Fas antibody treatment of cells infected with vector alone (Control-1 , -2) results in apoptotic index of 89% and 87%, while cells infected with an Nrf2 encoding retrovirus (Nrf2-1 , -2) show apoptotic index of only 32% and 34% respectively The role of Nrf2 as an inhibitor of the Fas pathway was further validated by pharmacological agents It is predicted that treatment of cells with Dicumarol an inhibitor of GST and NQ01 can sensitize cells to Fas induced apoptosis, since Nrf2 up-regulates the levels of GST and NQ01 As shown in Figure 5E, HeLa cells treated with 100mM Dicumarol are 2 8 fold more sensitive to Fas induced killing compared to cells treated with vehicle as measured by the number of viable cells This result was further validated by staining the cells with DAPI, which is commonly used to detect apoptotic cells, as measured by chromosomal condensation and fragmentations, hallmarks of apoptosis (Figure 5F) Apoptotic index of approximately 25% is observed in cells treated with anti-Fas antibody in the presence of vehicle (OuM) and approximately 100% killing is observed in cells treated with anti-Fas antibody and 100uM Dicumarol Thus, Dicumarol significantly sensitizes HeLa cells to Fas induced programmed cell death

Since Nrf2 regulates genes involved in phase II detoxification, testing was conducted to determine whether other activities involved in detoxification can also influence the sensitivity of cells to Fas induced apoptosis It has been reported that the detoxification of some compounds involves the phase II gene GST and the action of a sulfinpyrazone sensitive export pump (Morrow et al 2000) Then it was directly tested whether sulfinpyrazone treatment of cells can sensitize to Fas induced apoptosis As is shown in Figure 5G, treatment of cells with 2mM sulfinpyrazone strongly sensitizes cells to the effect of Fas induced killing compared to treatment with vehicle alone, by approximately 4 fold Thus, treatment of cells with sulfinpyrazone can have clinical benefits in situations where enhanced cell killing can be beneficial

Since down regulation of Nrf2 sensitizes cell to Fas induced apoptosis, it was questioned whether increasing any of the activities induced by Nrf2 protects HeLa cells from apoptosis Nrf2 up-regulates GST that conjugates glutathione to the reactive products of phase I detoxification It was predicted that increased activity of GST protects cells from Fas induced apoptosis GST activity was elevated by treating HeLa cells with the glutathione precursor N- acetyl Cysteine (NAC) that increases the glutathione pool As shown in Figure 5H, NAC strongly protects HeLa cells from Fas induced apoptosis as previously reported for microgha, neutrophils and T-cells (Delneste et al 1996 Watson et al 1997, Spanaus et al 1998)

Thus, by using AHM, Nrf2 was identified as an inhibitor of Fas induced PCD in HeLa cells and this result was validated by genetic and pharmacological approaches Down regulation of Nrf2 sensitizes to killing while over-expression of Nrf2 protects from Fas induced apoptosis

A technically simpler alternative to "function profiling" by subtraction is analysis by microarray The relative abundance of cDNAs was measured in Pool 1 and in Pool 2 by radio-labeling each pool and hybridizing each of the probes to a cDNA microarray containing approximately 4,000 different known human genes Dark green spots indicate cDNAs absent in Pool 2, representing "sensitizing" anti-sense cDNAs The corresponding genes are predicted to be survival factors that inhibit Fas induced apoptosis Dark red spots indicate cDNAs that are enriched in Pool 2 These genes are positive mediators of killing and their inactivation by anti-sense results in resistance to PCD The abundance of such anti-sense cDNAs is therefore increased in Pool 2 that is comprised of cells that survived Fas induced apoptosis Most of the spots are of intermediate color indicating only modest changes in abundance in Pool 2 relative to Pool 1 A histogram representation of the results is shown in Figure 6 As seen, the abundance of the majority of cDNAs is not changed However, a small number of cDNAs are depleted from Pool 1 by 2 folds or more (left portion of the curve) A partial list of these genes is presented in TABLE 3. As predicated, these genes include survival factors For example, the most depleted cDNA (5 6-fold) corresponds to TNF receptor associated factor 6 that relays a strong survival signal via the activation of NFkB and AKT In addition casein kinase 1 alpha (CSNK1A1 ) and adenosine A3 receptor have been associated with survival in certain situations

Also validated was the role of CSNK1A1 as an mhibitoi of the Fas pathway Since CSNK1A1 is substantially reduced in Pool 2 by 5 2 fold, down regulation of CSNK1A1 by its pharmacological inhibitor CKI-7 (Chijiwa et al 1989) sensitizes cells to FAS induced apoptosis As shown in Figure 7, HeLa cells treated with CKI-7 are 3 9 fold more sensitive to FAS induced PCD than the untreated cells Again, this was validated, by a chemical inhibitor, that a gene identified by the AHM method involving the cDNA microarray analysis is an inhibitor of the Fas pathway In summary, AHM is a novel powerful tool for identifying signaling inhibitors in human cells Thus, a large gap in the genetic analyses of mammalian cells has now been filled AHM can be broadly used to identify inhibitors of any given selectable pathway for the purposes of basic research or clinical applications Moreover, since it does not require previous knowledge of any sequences, AHM can be employed as a high throughput method of gene discovery and "function profiling" as part of the ongoing effort of deciphering the human genome

Materials and Methods:

AHM: HeLa cells (10⁶ cells/100 mm plate) were transfected with 15 ug of anti-sense cDNA library in pTKO-1 (Deiss and Kimchi 1991 ) by Superfect reagent (Qiagen) Two days later cells were treated with 200 ug/ml Hygromycin B (Calbiochem-Novabiochem) for two weeks 2 5X10⁶ Hygromycιn^R cells were plated in a 150 mm plate 24 hours prior to treatment withl O ng/ml anti-Fas antibody (clone CH-1 1 , Kamiya Biomedical Company) (Pool 2) Five days post treatment approximately 30-40% of the cells were killed as estimated by microscopic examination A parallel culture was grown in the absence of anti-Fas antibody (Pool 1 ) After five days, cells were washed twice with PBS, scraped off the plate and stored as pellets at -80° 100 ul of frozen pellet were lysed by addition of 200 ul of solution P1 , followed by 200 ul of solution P2 After the lysate sat on ice for 5 minutes 200 ul of solution P3 was added (Qiagen plasmid purification kit) Following 5 minutes incubation on ice, the lysate was centπfuged for 10 minutes at 15,000xg, the supernatant was mixed with an equal volume of isopropanol and centπfuged at 15,000xg for 10 minutes The DNA pellet was rinsed with 70% ethanol and resuspended in 100 ul of water The cDNA inserts were amplified by PCR in a 100 ul reaction containing 1 ul DNA, 200 uM of d ATP, dGTP, dCTP, dTTP, 10 mM Tπs-HCI pH9 0, 0 1 % Triton X-100, 1 OmM MgCI, 1 unit Taq DNA polymerase (Gibco BRL) and 500 ng each of primers prLPD#64 (TGGAGGCCTAGGCTTTTGC) and prLPD#65

(GTAAGGTTCCTTCACAAGGATCC) These primers are derived from the sequences that flank the cDNA insertion site in the pTKO-1 anti-sense expression vector The primers are designed to restore a Hmdlll restriction site on the promoter proximal side of the cDNA and a BamHI site on the promoter distal side to conserve the orientation of the cDNA fragments upon their cloning in pTKO-1 The reaction was incubated 94°C for 5 minutes, subjected to 25 cycles of 94°C for one minute, 58°C for one minute and 72°C for five minutes, followed by 72°C for seven minutes The PCR products were cleaved by BamHI and Hmdlll, purified (Wizard PCR Prep Kit, Promega) and used in subtraction (PCR-Select kit, Clontech) The driver for the subtraction was the product of the PCR reaction derived from the untreated cells (Pool 1 ) and the tester was derived from treated cells (Pool 2) The following modifications to the manufacturer's instructions were made 1 The first step ⁵ was IV F 3, since no cDNA synthesis is required 2 The blunt ends adapters 1 and 2R were replaced with cohesive ends adapters as follows Adapter I was replaced by a mixture of primers prLPD#80 (CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAGGTA), prLPD#81 ⁱ c (CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAGGTG), prLPD#83 (AGCTTACCTGCCCGG) and prLPD#84

(GATCCACCTGCCCGG) Adapter 2R was replaced by a mixture of prLPD#82 (CTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAGGTG),

15 prLPD#88 (AGCTTACCTCGGCCG), prLPD#89 (GATCCACCTCGGCCG) and prLPD#90

(CTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAGGTA)

Cohesive end adapters hgate more efficiently to the cDNA and permit ²⁰ the directional cloning of the cDNA inserts 0 3 ug of the tester was used for adapter gation 3 The initial hybridization included 0 9 ug of the driver and 0 03 ug of the adapted ligated tester The products of the subtraction were cleaved with BamHI and Hmdlll, purified and cloned into the pTKO-1 between Bglll and Hmdlll sites Individual clones were sequenced and transfected into ²⁵ HeLa cells

Anti-sense transfection and Bioassays HeLa cells (2x10⁶ cells/100 mm plate) were plated 20 hours prior to transfection with either 17 ug of either anti-sense expressing vector or control vector harboring no cDNA insert, by calcium phosphate Forty eight hours post transfection cells were treated with 200 ug/ml Hygromycin B (Calbiochem-Novabiochem) for two weeks For bioassays, anti-sense transfected cells or control vector transfected cells (1 6 x10⁵ cells/ well in 6 wells plates) were plated 20-24 hours prior to the treatment with 200 ng/ml anti-Fas antibody (clone CH-1 1 , Kamiya Biomedical Company) The number of viable, trypan blue (Gibco/BRL) excluding cells that remained attached to the plate following rinsing with PBS was counted 24 hours post treatment

Nrf2 Infection and Bioassys To generate HeLa cells that over- express Nrf2 the human Nrf2 cDNA (Moi et al 1994) was cloned into the retrovirus expression vector pBABEpuro (Morgenstern and Land 1990) by standard methods The plasmid was transiently transfected into X and virus was collected at 36, 48, 60 and 72 hours post transfection At each time point, freshly collected virus stock was filtered through a 0 45fιlter, mixed with 4Dgr/ml polybrene (Sigma/Aldπch) and applied to HeLa cells 24 hours after the last addition of the virus, the cells were subjected to selection in 1gr/ml Puromycm (Sigma/Aldπch) The pool of Puromycm resistant colonies was expanded For bioassays (1 6x10⁵ cells/ well in 6 wells plates) were plated 20-24 hours prior to the treatment with 200 ng/ml anti-Fas antibody (clone CH-1 1 , Kamiya Biomedical Company) Three days later the cells were fixed by slowly adding 1/3 volume of formaldehyde (37%) to the medium Twenty four hours later the cells were gently washed twice with dH₂0 and stained with 2 5 g/ml DAPI (4',6-Dιamιdιno-2-phenylιndole, Sigma/Aldπch) in PBS for 5 minutes followed by two washes in dH₂0 Stained cells were viewed by Leica DMIRB fluorescent inverted microscope Apoptotic index was calculated as the ratio of number of dead cells (containing condensed and/or fragmented chromosomes) to the total number of cells Average apoptotic index calculated from ten fields from duplicate wells is presented Generation of membrane permeable dominant-negative Nrf2 and bioassays

Human Nrf-2 proteιn(amιno acid 367 to 589) was cloned between the

Kpnl and EcoRI sites of the bacterial expression vector pTAT-HA vector (obtained from Dr S Dowdy, U of Washington, St Louis) The GFP control protein expressed from the PTAT-HA-GFP vector (obtained from Dr S Dowdy, U of Washington, St Louis) as well as the Nrf2 dominant negative recombinant polypeptide were produced in bacteria as described (Schwarze et al 1999) Briefly, bacteria strain BL-21 (DE-3) pLysS was transformed by the plasmid of interest and an overnight culture was grown at 37°C Approximately 18 hours later, the culture was diluted into 500 ml of fresh LB media and grown to OD of 0 8-0 9 at 37^0C IPTG was added (0 5 mM final concentration) followed by incubation at 30^0C for additional three hours The bacteria pellet was washed in 20 ml of PBS The final pellet was resuspended in 10 ml of buffer Z (8M Urea, 100mM NaCI, 20 mM HEPES pH8 0) and sonicated on ice 4x30 sec pulses The sonicated lysate was centπfuged at 12,000 rpm for 30 minutes at 4°C Imidazole (20mM final concentration) was added to the supernatant The supernatant was then added to a Ni-NTA column (3ml) in Buffer Z containing 20mM imidazole The column had been pre-equihbrated with buffer Z containing 20mM imidazole Following binding of the lysate to the column, the column was washed with 50 ml of buffer Z containing 20 mM imidazole The fusion protein was eluted by washing the column with increasing concentrations of imidazole (100ιτιM, 250mM, 500 mM and 1 M imidazole) in buffer Z The proteins fraction eluted in 1 M imidazole was dialyzed against sterile PBS for 12-16 hours with two changes Glycerol (10% final concentration) was added to the dialyzed protein The concentration of the purified protein was estimated by SDS PAGE compared to standard BSA

For bioassays HeLa cells, 8 3x10⁴ cells /well were plated in 12 wells plates 20 hours later cells were treated with various concentrations of ⁵ recombinant protein or PBS 60 minutes prior to the addition of 200 ng/ml anti- Fas antibody (clone CH-1 1 , Kamiya Biomedical Company) (where indicated) 20 hours later the apoptotic index of treated cells was determined as described for the Nrf2 bioassays

° Western analysis: Anti-sense transfected cells or control vector transfected cells (2 5x10⁶ cells/150mm plate) were plated 24 hours prior to treatment with 200 ng/ml anti-Fas antibody (clone CH-11 , Kamiya Biomedical Company) 24 hours post treatment cells were washed with PBS and lysed in RIPA buffer (1% Nonidet P-40, 0 5% sodium deoxycholate, 0 1 % SDS, 1 mM PMSF, ⁵ 2mg/ml aprotonm and 2mg/ml pepstatm in PBS) Samples containing 50Dg protein were separated by SDS-PAGE and transferred to nitrocellulose membranes The immunoblots were probed with either antι-Nrf2 antibody (1 100, Santa Cruz, sc722) or antι-bFGF-2 antibody, (1 200, Santa Cruz, sc 079), incubated with goat anti rabbit conjugated to horseradish peroxidase ⁰ (Pierce) followed by incubation with SuperSignal substrate (Pierce) Following autoradiography, the probes were stripped (Amersham, ECL Western blotting protocols) and the membranes were hybridized with anti-actm antibody, (1 100, Sigma A4700 or A2066) The intensities of the bands were quantified by the National Institute of Health Image program 5

Treatment with N-acetyl cysteine HeLa cells (8 3x10⁴ cells /well in 6 wells plates) were plated 20-24 hours prior to treatment with various concentrations of NAC (Sigma/Aldπch) in the presence or absence of 50 ng/ml anti-Fas antibody (clone CH-11 , Kamiya Biomedical Company) The number of viable, trypan blue (Gibco/BRL) excluding cells that remained attached to the plate following rinsing with PBS was counted 5 days post treatment

Treatment with Dicumarol HeLa cells, 1 6x10⁵ cells /well were plated in 6 wells plates 20-24 hours later cells were treated with various concentrations of Dicumarol (Sigma/Aldπch) in 0 2 mM NaOH for 15 minutes prior to the addition of 200 ng/ml anti-Fas antibody (clone CH-1 1 , Kamiya Biomedical Company) The number of viable, trypan blue (Gibco/BRL) excluding cells that remained attached to the plate following rinsing with PBS was counted 17 hours post treatment Alternafively, the apoptotic index of treated cells was determined as described for the Nrf2 bioassays

Treatment with Sulfinpyrazone: HeLa cells, 1 6x10⁵ cells /well were plated in 6 wells plates 20-24 hours later cells were treated with various concentrations of sulfinpyrazone (Sigma/Aldπch) in 1% DMSO for 15 minutes prior to the addition of 200 ng/ml anti-Fas antibody (clone CH-11 , Kamiya Biomedical Company) The number of viable, trypan blue (Gibco/BRL) excluding cells that remained attached to the plate following rinsing with PBS was counted 17 hours post treatment

Treatment with CKI-7: HeLa cells, 1 6x10⁵ cells /well were plated in 6 wells plates in duplicates 20-24 hours later cells were treated with various concentrations of CKI-7 in 1 % DMSO (Seikagaku Corporation, Tokyo, Japan) an hour prior to the addition of 200 ng/ml anti-FAS antibody (clone CH-11 , catalog number MC-060, Kamiya Biomedical Company) The number of viable, trypan blue (Gibco/BRL) excluding cells that remained attached to the plate following rinsing with PBS was counted 17 hours post treatment

cDNA microarray analysis: Approximately 500ng of the PCR products of Pool 1 and Pool 2 (same preparations that were used for the subtraction, before their cleavage by BamHI and Hmdlll) were labeled with 100 mCi of [³³P] dCTP (3000 O/mmole ICN) by the random primers DNA labeling system (Gibco/BRL), purified (Amersham/Pharmacia, ProbeQuant G50 micro columns) and individually hybridized to Human GeneFilters (GF21 1 , Research Genetics) The filter was pre-hybπdized for 40-60 minutes at 68 °C in ExpressHyb Hybridization solution (Clontech), followed by hybridization for 3-5 hours at 68°C The filter was washed in 2xSSC, 0 05% SDS at room temperature 3-5 times for 10-15 minutes each time followed by 2 washes for 15 minutes each in 0 1x SSC, 0 1 % SDS at 55°C The image was generated by Molecular Dynamics phospho-imager In between hybridizations, the probe was stripped off by adding boiling solution of 0 5% SDS and incubating at room temperature for 1 hour Successful removal of probe was confirmed by phosphor-imager analysis Images processing and calculation of the ratio of the signals of Pool 2 probe to Pool 1 probe were performed by Pathways II software (Research Genetics) All the spots that showed significant differential abundance were visually inspected

EXAMPLE 4: NFW GENE SEQUENCF

Using methods described herein, the clone LPD #599 was obtained and sequenced The sequence is SEQ ID No 37, and contained within it is the complete sequence of SEQ ID No 15 A novel polypeptide is encoded by a DNA sequence comprising this DNA, which can be produced by cloning and expression methods known in the art EXAMPLE 5: SCREENING METHODS

Each of the genes identified by means of the present invention can be used as a candidate gene in a screening assay for identifying and isolating compounds which inhibit or stimulate PCD, in particular, Fas -induced apoptosis The compounds to be screened comprise inter aha small chemical molecules antibodies or fragments thereof including single chain antibodies, antisense o gonucleotides, antisense DNA or RNA molecules, proteins, polypeptides and peptides including peptido-mimetics and dominant negatives, and expression vectors (A synthetic antisense oligonucleotide drug can inhibit translation of mRNA encoding the gene product of a Fas pathway gene )

Many types of screening assays are known to those of ordinary skill in the art The specific assay which is chosen depends to a great extent on the activity of the candidate gene or the protein expressed thereby Thus, if it is known that the expression product of a candidate gene has enzymatic activity, then an assay which is based on inhibition (or stimulation) of the enzymatic activity can be used If the candidate protein is known to bind to a ligand or other mteractor, then the assay can be based on the inhibition of such binding or interaction When the candidate gene is a known gene, then many of its properties can also be known, and these can be used to determine the best screening assay If the candidate gene is novel, then some analysis and/or experimentation is appropriate in order to determine the best assay to be used to find inhibitors of the activity of that candidate gene The analysis can involve a sequence analysis to find domains in the sequence which shed light on its activity Other experimentation described herein to identify the candidate gene and its activity can also be engaged in so as to identify the type of screen that is appropriate to find inhibitors or stimulators (enhancers), as the case can be, for the candidate gene or the protein encoded thereby As is well known in the art, the screening assays can be cell-based or non-cell-based The cell-based assay is performed using eukaryotic cells and such cell-based systems are particularly relevant in order to directly measure the activity of candidate genes which are anti-apoptotic functional genes, i e , expression of the gene prevents apoptosis or otherwise prevent cell death in target cells One way of running such a cell-based assay uses tetracychne- mducible (Tet-mducible) gene expression Tet-mducible gene expression is well known in the art , see for example, Hofmann et al, 1996, Proc Natl Acad

≤£l 93(1 1 ) 5185-5190

Tet-mducible retroviruses have been designed incorporating the Self- inactivatmg (SIN) feature of a 3' Ltr enhancer/promoter retroviral deletion mutant Expression of this vector in cells is virtually undetectable in the presence of tetracychne or other active analogs However, in the absence of Tet, expression is turned on to maximum within 48 hours after induction, with uniform increased expression of the whole population of cells that harbor the inducible retrovirus, thus indicating that expression is regulated uniformly within the infected cell population

When dealing with candidate genes having anti-apoptotic function (as described in this application ), Tet-mducible expression prevents apoptosis in target cells One can screen for compounds able to rescue the cells from the gene-triggered inhibition of apoptosis

If the gene product of the candidate gene phosphorylates with a specific target protein, a specific reporter gene construct can be designed such that phosphorylation of this reporter gene product causes its activation, which can be followed by a color reaction The candidate gene can be specifically induced, using the Tet-mducible system discussed above, and a comparison of induced versus non-induced genes provides a measure of reporter gene activation

In a similar indirect assay, a reporter system can be designed that responds to changes in protein-protein interaction of the candidate protein If the reporter responds to actual interaction with the candidate protein, a color reaction occurs

One can also measure inhibition or stimulation of reporter gene activity by modulation of its expression levels via the specific candidate promoter or other regulatory elements A specific promoter or regulatory element controlling the activity of a candidate gene is defined by methods well known in the art A reporter gene is constructed which is controlled by the specific candidate gene promoter or regulatory elements The DNA containing the specific promoter or regulatory agent is actually linked to the gene encoding the reporter Reporter activity depends on specific activation of the promoter or regulatory element Thus, inhibition or stimulation of the reporter is a direct assay of stimulation/inhibition of the reporter gene, see, for example, Komarov et al (1999), Science vol 285,1733-7 and Storz et al (1999) Analytical Biochemistry, 216_^97-104

Various non-cell-based screening assays are also well within the skill of those of ordinary skill in the art For example, if enzymatic activity is to be measured, such as if the candidate protein has a kinase activity, the target protein can be defined and specific phosphorylation of the target can be followed The assay can involve either inhibition of target phosphorylation or stimulation of target phosphorylation, both types of assay being well known in the art, for example see Mohney et al (1998) J NeuroscienceJS, 5285 and Tang et al (1997) J Clin Invest 1Q0_,1180 for measurement of kinase activity

One can also measure in vitro interaction of a candidate protein with mteractors In this screen, the candidate protein is immobilized on beads An mteractor, such as a receptor ligand, is radioactively labeled and added When it binds to the candidate protein on the bead, the amount of radioactivity carried on the beads (due to interaction with the candidate protein) can be measured The assay indicates inhibition of the interaction by measuring the amount of radioactivity on the bead Any of the screening assays, according to the present invention, can include a step of identifying the compound (as described above) which tests positive in the assay and can also include the further step of producing as a medicament that which has been so identified It is considered that medicaments comprising such compounds are part of the present invention The use of any such compounds identified for inhibition or stimulation of PCD, in particular, Fas -induced apoptosis, is also considered to be part of the present invention

Throughout this application, various publications, including United States patents, are referenced by author and year and patents by number Full citations for the publications are listed below The disclosures of these publications and patents in their entireties are hereby incorporated by reference mto this application in order to more fully describe the state of the art to which this invention pertains

The invention has been described in an illustrative manner, and it is to be understood that the terminology which has been used is intended to be in the nature of words of description rather than of limitation

Obviously, many modifications and variations of the present invention are possible in light of the above teachings It is, therefore, to be understood that within the scope of the appended claims, the invention can be practiced otherwise than as specifically described

TABLE 1 SEQ ID No 1 prLPD#51 SEQUENCE TCTGTAGGTAGTTTGTC SEQ ID No 2 prLPD#64 SEQUENCE TGGAGGCCTAGGCTTTTGC SEQ ID No 3 prLPD#65 SEQUENCE GTAAGGTTCCTTCACAAGGATCC SEQ ID No 4 prLpd#80 SEQUENCE CTAATACGACTCACTATAGGGC TCGAGCGGCCGCCCGGGCAGGTA

SEQ ID No 5 prLPD#81 SEQUENCE CTAATACGACTCACTATAG GGCTCGAGCGGCCGCCCGGGCAGGTG

SEQ ID No 6 prLPD#82 SEQUENCE CTAATACGACTCACTATAGG GCAGCGTGGTCGCGGCCGAGGTG

SEQ ID No 7 prLpd#83 SEQUENCE AGCTTACCTGCCCGG SEQ ID No 8 prLPD#84 SEQUENCE GATCCACCTGCCCGG SEQ ID No 9 prLPD#85 SEQUENCE CTAATACGACTCACTATAGGGC SEQ ID No 10 prLpd#86 SEQUENCE TCGAGCGGCCGCCCGGGCAGGT SEQ ID No 1 1 prLPD#87 SEQUENCE AGCGTGGTCGCGGCCGAGGT SEQ ID No 12 prLPD#88 SEQUENCE AGCTTACCTCGGCCG SEQ ID No 13 prLPD#89 SEQUENCE GATCCACCTCGGCCG SEQ ID No 14 prLPD#90 SEQUENCE CTAATACGACTCACTAT

AGGGCAGCGTGGTCGCGGCCGAGGTA

TABLE 2

SEQUENCES SEQ ID No:15 seqLPD#599pr51 (N indicates uncertainty in sequence) GATCCAAGCGAAAAAGGGAATAAGAAGGGAGGAATGTAACTAGGAGCAG CTCCCAACAGTTTGCCTATGTATTTGCCAGCACCAAAATTTGTAGAGTAAG CCACTTACATTTCCACTGCTAGTATTAAGGAAAGACAGCAGTGGTGATTTT TATAAAGCGAGTATACATTTATTTTTATTCTGATATGTGAATTTTTCTTTCA CGAGTTAATTAACTGGTAATTTGTAAACAGTGGGAAGAAGATTAGAACAAT TATGGAGGTACTGAATTACACAAGGAGATTAAAATGAAATGAATCAATCTA CCTATCTTGTGGTTAGTTAATATTTACCATGATGCATACACTTGAGAAATG AGAAATGCCCAAATTGTATAATGCATTATCTTNNTTATTATTTATTNNNGTA AATAATTCTTGCTNNACATTANNCTNCAAGGNTAGCTNTATCTATACTTGN TAGCTANNTTCTTATACAAAGCANGNTNCTTTTGAANATGATTTACCNATT AANTANANNAGCTTAGGTGCCTNNTTTNACTCTGGNTNGTCATNTTGNTTN NCTTTTNNCNANATCATATATAATTTCCANNGAATGTTGATNTNTNTCCTCA NNTTNCATTNTANCCNGNGCNTATCCTNTNNNNTGNTNGNNTATGTC SEQ ID No:16 seqLPD#601 pr51

GATCCTGGTATAATGTGATTGAATTATGAAGGCAAATTTCTCATGAATGGT TCAGCACCATCCCCTTGTACCATCCTCACAATAATGAGTAACTTCTCATGA GATGTAGTCACTGAAATCTCTATATCACCTCCCCACTCTCCGTGTTTTCTC CTTGCCATGTGAGACAATTGATTCTTTCTTTGCCTTCCATGATTATTGAAA GATTTCTGAGGCCTAGAAGCAGAAGCACTGTGCTTAGAACCATGAGCCAA TTAAACCTCTTTTTCAAAATAAATCATACGGGAAATGGCAAATGAGGACTG GAGCGTTGCTATAAAGATACCTGGAAATGTGGAAGCAGCTTTGGAACCAG GTAATGGACGGAGGTTGGAAGAGTTTGGAGGGCTCAAAAGAAGACAGAT GAGAAAACTTTTGGACCATCTTAGAGTCTGGTTCAATGGTTGTGACAAAAA TCCTGACAGAAACATGGACAGTGAAGGCCAGGCTGAGGAGGTCTCAGAG AGAAATAAGAAGCTTTTTGCAA SEQ ID No:17 seqLPD#602pr51 AAACATTATGCACAGAGGAATAAAATTAAGAATGACCACAGTCTTCTCTTC

AGAAATTATGCAAGCCAGAATAGAGTAGAGCAACATCTTTACAGTTCTTAA

AGAAAAAATATATCAACTTAGAATTCTATACCCAGCAAAAATATATTTCAAA

AAGAAGACACAATACTTTTTCAGACAGACAAAAGCCAAGAGAGTAATTCCA

GGGACGTGTAATATAAGAAATATTAGTGAAAGTAGTGTAAAGAATGAGAG

AGAGGAAATATAAGGATACTGTTATAATATCCCTACACTGTACCTTAAGTG

GATTGTAATATTATCTGAAGGTAAACTGTAATATGTTAAATAAAGATGTATA

TTTTTAATCCTAGAGAAACTGCTATGAAAACAAAAAGTAAAACAAAGTGGT

ATTACTAACAAGCCAATAATGGAGATAAAATGGGCAATAGAAAACAACAAC

AAAAATTCAATCCAAAAGAAGGAAG

SEQ ID No:18 seqLPD#606pr51

GATCCCTCTGGCACTGGGATGCTTCCACAGATGTACCTGAAAAGTCGTCA

CTCTCTCACCAAGGAGGAAGAAGCAGTACTTTTTTGTCTCATAGCATTAGT

AACCAAGCTAATGTTCACCAGTCTGTCATTAGCTCCTGGCTCAGCACTGA

TCCTGCAAAAGACCGAATTGAAATCACAAGCT

SEQ ID No:19 seqLPD#607pr51

GGGACATGGTTGGGCCATGCCACACCAGGGCTGGTGAGGCAACCAGTTT

TGATTTTGACAGAGTGGCTGGAGGAAAAGTGGCAATCAAGGTGCTGCTT

GGTTTGCTCTGAGTGCAAATGGAACCAACAGGTTTCTGCTGCAATCTGTG

TGTTCCCAGTGCCAGGTCACACCAGGAGGGGTGGGGCAGGGCTAACCA

AGTGGTCTCTGAACTCACCGAGCGTCTGCACTTGGTTGTGAAGTTAATGG

GAGTACAGAGAGCGTCTGGCCTTGGAGAGGGGTTGAGAGCCTCCTTTTT

GGTTCTTCATTCCTGAGCTCTTGCCTGCCCACAAATCTGACCTCTTTGAAT

GGGGACGCAGTCCTTCAACAGAGAAGTTTCTATGGCAAAGAAGTTTCTAT

TTAGCTCTAGATCCAGCAGAGTCATCCATTCTAACTGCCCTGAAGTCTAG

AGCAGGGGAGGGAACCCAGAGGCTGGGGATGAGACTAGGCAGACCCTG

GTTACCATATGGACAAGGACAGGGGAAAGCACCCCCTTCCTCAATTTCTG

AAAGTTCTATCTTTGGGTTCGCTGGACTTTGAGGATGATAAAGAACATNTA

GGTACTA SEQ ID No:20 seqLPD#608pr51

AAGTGGGACTCTGGGCCTCTGACCAGCTGTGCGGCATGGGCTAAGTCAC

TCTGCCCTTCGGAGCCTCTGGAAGCT

SEQ ID No:21 seqLPD#609pr51

GATCCTANACCTGCTCTTTCTCTAATCTTCTGTATCTTAGCAATCGGCCAA

AAAACTTGAAATCATACTTGATTTCTCCTGGCCCTCAAAGCCTACATTCAT

TCTGCCAAATCATGTAAGTCCATCTTTAAAATTTATACTGACTGTGCATTTC

TTCTTATCTCCATTACTATCACCCAAGTTCAAAATCCTTTCTTATTTTTCAC

CTGAGTATTGCAATATTTTCTTAGATGACCCTAACTGATCTTGCCGTTTCT

ACTCTTGCTTTCCTACAGTCAATTCTCTTCATATATGCCAACATTACCTTTT

AAAACTACAAACAAGTTCAGGTTACTTCTTCCTTGCTCAAAGTCTCAAATA

TCTTTTTATCACACACAAATCAAAGGTGGCTAAGAATGGTCTGGGTCCTG

CATACCTCTCCAATGTCATCTACAGCATCTCTGACTTTCTCGCACTGCTCT

GGTCACAGTGGTCTTCTTTTTCTCTAATACACTAAG

SEQ ID No:22 seqLPD#610pr51

TCCAGAGCAAACATTACATCATAGGCCATGGGGGTTGTCACTTAGCTGCA

TGCTAAGAATCCTCATATGCTGCTTTGAAAACGCCATAAAAAACTAAGATG

CCCTCATACACAGAAAAAAGGAGTTGGGCCAACAGCCTGCGAGAGAAGA

TCCTTCCCAACTCTGCCATTCTTACTGCAGGAGGGTTTGTAATGCTTCCC

CACTTCCTCAGGTCCAGCGACTGCACCTCCACCACTGGAGTGGGGGACA

CTTCCATTGCTGATGGAAGCTGCCTACTGCTTTTAAAAACACACACATGAG

CTGAGAAGTTCTGCTGAAGGTGGGGAGAGTGCTGCTCTTGGCTGCCAGC

CGGGTCTAGCGGCCCTAACAAGGGAAACTGGCTGATACAAAATGGATCA

CTGCCTGAGATATATGAACTGGACTGAGGATAGAAGTTGCATCCGCTGG

GCAAAATGTGCCAAAGAACAGAAAGTCTTTCAATTTATTCTTTTAGGCACC

AAGAATAAATAAATNCTAAGA

SEQ ID No:23 seqLPD#611 pr51

GGGTGAAGCATCTGNTTCATNACACAGGGCTATCATAATTGGGTTCTGGA

AATGGAATGGGTCTCTAAACATTTTAATGCCCCAGCTTGGTATTGGACNC AATGCAAGATGGCTAAAATCTTCATTTAAAATTCAAGGGTATTGCTTGCTA

TTTCACATTATATACTTTTTAAATACTATTGCTCTTTGCATATGAGGCATTT

CACTAACCTTGGATCATTGGATCTTAAACATGATAATATAGAATATCTGAA

CATTGGACTTGNTCTTTAGAGTATATGGCCTTTAGTTCTTGTGGAACTAAA

TACAGTGATTCTAAGATCAAAAGTCTTAGTGTTTGGGGTTTTTTTTTCCTCT

TTTTGGATATGGGGTGTT

SEQ ID o:24 seqLPD#613pr51

ATCCAGCGGNTCAGCACACACTGGGGCCTACCGGAGTGGGAGGGAGGG

ACAGGGATGAGGGAAAAATAACTAATGGGTACTAGGCTTAATACCTGGGT

GGTGAAATAATCTGTACAACAAACTCCCATGACACAAGTTTACCTTTACAA

ACCTGCACATGTACCCCTGAACTTAAAGAAAAGTTTTTTTAAAAAATCACA

CTTCCAGAGTTTGCTAAATAATTATGACCAGTATTTTACTACCATCTTCTTC

CTTGGTGAACTACTAAAATTAGTAAATTTATGTTAAAAATGAAGTTCTCCCT

AGAAATCAAAAGTGCAGTCTAAGCACTGAAAATGTTCTATCAACACTTGTT

AACTGAGAACCATTGAAACATCTAGGA

SEQ ID No:25 seqLPD#616pr51

TNCGGATGAGATAGTGAAAGAGTTCATGACTCCCCGGAAGCTGTCCTTCG

GCTTTCAGTAGCACTCGTTTTACATATGCTTATAAAAGAAGTGATGTATCA

GTAATGTATCAATAATCCCAGCCCAGTCAAAGCACCGCCACCTGTAGGCT

TCTGTCTCATGGTAATTACTGGGCCTGGCCCCTGTAAGCCTGTGTATGTT

ATCAATACTGTTTCTACCTGTGAGTTCCATTATTTCTATCTCTTATGGGCAA

AGCATTGTGGGTAATTGGTGCTGGCTAACATTGCATGGTCCGGATAGAGA

AGTCCAGCTGTGAGTCTCTCCCCAAAGCAGCCCCACAGTGGAGCCTTTG

GCTGGAAGTCCATGGGCCACCCTGTTCTTGTCCATGGAGGACTCCGAGG

GTTCCAAGTATACTCTTAAGACCCACTCTGTTTAAAAATATATATTCTATGT

ATGCGTATATGGAATTGGAAATGTCATTATTGGAACCTAGAAANGGCTTTG

GAATATTGATGTGGGGAGGNTTATTGAGCACCAGATGTATTTTANCCCAT

GCCCCCTCCAAAAAGAAATGGTTAGTNAAAAC

SEQ ID No:26 seqLPD#618pr51 GATCCTTNCAAGTGGGACTCTGGNCCTCTGACCAGCTGTGCGGCATGGG

CTAAGTCACTCTGCCCTTCGGAGCCTCTGGAAGCT

SEQ ID No:27 seqLPD#619pr51

CTNTGGANTACANCNATCCCTGGATTTAANGAGANGGCCAGGCCACTCTA

TACTCTAATCAAGGAGACCCAGAGGGCAAATACTCATCTAGGAGAATGGG

AACCAGAGGCAGAAACAGCCTTCAAAACCTTAAA

SEQ ID No:28 L7_10_1_LPD

AATAAGGTTCCTTCACAAANATCCAAGCCAGCACCTTAGTTTTCCTACAA

CATAAATGTAACAAAGTTATCTTCTACTGTATTGCACCTTAGTCCAAAAGT

AAAACAACTAAATGAAAATTTAAATAAATCAGACTGAAAAAGCCCAAAGA

GTAAGAGGAATACCTTATAAATGTGACTACCCACCTAAAAATTCTTGAAC

TACTTTGTTTTGCATAGGATTTTATGGGACTAACCAAATGTTCCATGAAC

CATGAGGTGAAAACTGCGATTTCATGATAGCACATTGTTTTACAATTCTG

ATTAGAAATCCTTCAGAAATATTTCTGTATC

SEQ ID No:29 L7_10_2_LPD

ANGTAAGGTTCCTTCACAAAAACCAAGCGGGACTCCNAAACAACCATGG

TGCAATGGTGGCAAAAACTGGGGACTGGGTCTTTCTCGCAACTTCTGTG

CCTCCTTTCATTCTCAAAAAAGGACTATTACCAAATGGGGGGGAAAAAAC

ATAAGCANAAAAAACCCCAAGGACAAACTGGAAATTTAATTCCTTNATGC

AAATCTTATCTTCATCTGGTGCCTCATTACCCTGGGCCCCAAGCCTTCTA

ATTAGGAAAAAATACTGATTTCTGTTAGGCAATTGCTTATNTTGGTGGCT

TCATCACATTTCA

SEQ ID No:30 E7_100_11_LPD

CAAAGTACCAAGCCCGAGAAAATACAGGTCTATTCAATTCCACCTGCTAG

CCTAAACTTCAAGTAGACTTCAAAGCAATACAAGTTTGTATGGTCTTTTTG

GCAATAGCAGGAAATGATACAAGCGAAATCAGTTCTTGATTAAGGGAGG

AGCAGAATTGCATTAGTTAGATATTCTGGTAGTGCTGCAAACTATAATGA

TAAATGAAGGCAGCCCAAGACTTAAGATGTAAAGTTATGTAGCTCATGCA

ATTCAATGTCAGTATTTGAAGGCTATATGATGAATATTTCCAGAATTATAA TGAAAAAAGTAAAAACAAATTTGCCTCTTACTGATGCTTCAAAAATCATTT

GTGTATATTTAACAAAAGAAGTGTCTAAATAGATCTGAATTTAAACCACAG

TACTGAACACACTACATGAGGTAACATTGAGTATTATCAAAGTACCAAGC

CCGAGAAAATACAGGTCTATTCAATTCCACCTGCTAGCCTAAACTTCAAG

TAGACTTCAAAGCAATACAAGTTTGTATGGTCTTTTTGGCAATAGCAGGA

AATGATACAAGCGAAATCAGTTCTTGATTAAGGGAGGAGCAGAATTGCA

TTAGTTAGATATTCTGGTAGTGCTGCAAACTATAATGATAAATGAAGGCA

GCCCAAGACTTAAGATGTAAAGTTATGTAGCTCATGCAATTCAATGTCAG

TATTTGAAGGCTATATGATGAATATTTCCAGAATTATAATGAAAAAAGTAA

AAACAAATTTGCCTCTTACTGATGCTTCAAAAATCATTTGTGTATATTTAA

CAAAAGAAGTGTCTAAATAGATCTGAATTTAAACCACAGTACTGAACACA

CTACATGAGGTAACATTGAGTATTAT

SEQ ID No:31 L7_10_8_BS

AAGCAAAAGGAATGCTTATACACTGTTGGTAGGAGTATTAGGATTACAG

GCGTGAGCCTCCACTTCCAGCCGTCCACAATCTATTGAGATGACCATGT

TGTTTTCTTCCTTCAGTCTGTTGCACTAATCATTTCCAGCTGATGAAATAA

CGTTGCATTCCAGGGATACACTGTACTGGGTACTGGTGTACATTCCTGT

TTATATGTTACGGTATTTGGTTTGCCAAAATTTACTAGGAAATTTCACAGA

TCCACTCATAAGCGATATTATCAGAGTTTTCCCTGTGTATGATGTTTCACT

TTGGTATCTAGATAACAATAGCATCATAGAACCAGTAAAAAATATTACATG

CTCTTCCAAGTTTTTAAGACATTTTTATCGAATTGGTAATTTTCATGTGTT

TGGTATAATATACAAAAGAATACATTTGAACTGGGAATTTACATGTGTGT

CAGGGGCAGGGGTGAGAGTTTTTAAATTACCAATTTATTGTCTTCACTTT

GTTCTTGAATGAGTTGTGGTAGTCAGTGTCTTTCTAGGAATTTGTCTACT

TCCACAAAGATATTTGTCAAAATACTAATCCCTTGTTATGGGAA

SEQ ID No:32 L7_10_3

AAGCGGCACTCATCACTTCTACCCTTATTTCTGCTGGCAGAAACCCAGTC

ACAAGCTCCACTGACATGCAAGGAGATTTGGGAAATGCAGTCTCTGTCA

GCTAGCCCAAACTCTAAGAAGCCAAAGGAAATATGTATTTTGGGTGGAG

! 0 ATCTAGCCATCTTACCACACTATGGTGGTCCAGTAGAGGTCTATC.AAAAT

ATTAATTCACAGAAGACTAAAGACACATTTACAATGAAGGTTTACAAAACT

TATCTGCAAAGAAAAAGCCAGAACATGTTTTATTGTGGAATAGTTCTAAA

ATTGCTTATAGATGAAAAGAACAAAACAAATATTTAAATCAGTCACCTCTA

GAATAGTGAAAGGCCAAAAACTGCATTTCAGAAATGAAATATCACTCTGG

GA

SEQ ID No:33 E7_100_10

AAGCGAAAACACCCTCATGTGAATGAGGGTTTTATGTTGTTAATGTGGTG

GGTGAGTGAGCCCCATTGTGTTGTGGTAAATATGTAGAGGGAGTATAGG

GCTGTGACTAGTATGTTGAGTCCTGTAAGTAGGAGAGTGATATTTGATCA

GGAGAACGTGGTTACTAGCACAGAGAGTTCTCCCAGTAGGTTAATAGTG

GGGGGTAAGGCGAGGTTAGCGAGGCTTGCTAGAAGTCATCAAAAAGCT

ATTAGTGGGAGTAGAGTTTGAAGCCCTTGAGAGAGGATTATGATGCGAC

TGTGAGTGCGTTCGTAGTTTGAGTTTGCTAGGCAGAATAGTAATGAGGA

TGTAAGTCCGTGGGCGATTATGAGAATGACTGCGCCGGTG

SEQ ID No:34 L7_100_4_BS

TACTGTGATGTTGTTGCTATCTTCATCATCTAACACCTGTGATTTTATATC

CATGGTCACATATGGAAAACCCCCAAGGACAGCCATAACCTCTTCATATT

TTTCATCTTCAAGGAAGTGCAGTAGAGTGTGACGATCTGATTCTTTTAAA

CTCACCAAATCCTGGATAGTTTTAATTTTATACTTCTTATGATTAGAAACC

CGTCTAAGATTGTCCTCTTCAATATGAGGGAGCTGCAGAAGGGGAGACT

TAAATTGCTGAAGTCCCTGAACGGCCATCTGAGA

SEQ ID No:35 L7_100_5_BS

TACTGTGATGTTGTTGCTATCTTCATCATCTAACACCTGTGATTTTATATC

CATGGTCACATATGGAAAACCCCCAAGGACAGCCATAACCTCTTCATATT

TTTCATCTTCAAGGAAGTGCAGTAGAGTGTGACGATCTGATTCTTTTAAA

CTCACCAAATCCTGGATAGTTTTAATTTTATACTTCTTATGATTAGAAACC

CGTCTAAGATTGTCCTCTTCAATATGAGGGAGCTGCAGAAGGGGAGACT

TAAATTGCTGAAGTCCCTGAACGGCCATCTGAGA SEQ ID No:36 E7_10_9

AAGCGGACTTTGGAGGGCAGTGTTATTTTCCCAAAGAAAGACGGCCAAG

GGCAGAGGCATGGATTCTTTGCAGAGCACTTCCTTTTGGTTTTTCAGTAC

TGTTTCATAGACAGTGGGCTCACATGTTCCTGATAGTGCTGCAGTTGCTT

AGAAAGCATCCCAGTTAATTGCAGTAATTAGAACTTCTGGAATATGCTAG

GGCAGAAGTATGTCAAGTATGTCACATGAAGAAAATGTGAAATTCAAGA

GTAATCCACACGTGAGAAACTAGACAATGTACATTCATGTGTTCTCTTGA

AAGGAAAGGGAGAGCTGTAAGCTATCGATACCGTC

SFQ ID NO: 37 I PD fiQQ - Spqiisncg of physical phagp clone

LPD_599 - Sequence of physical phage clone

TCAAATGCTATGGAAAAGTGCCTTTTTATCATTTATAATTTCATTTTTCACT

ATTTCCAAAAACACATAAACAAATAGTTTCAGTAGGTCCCAGCTTTTACTT

TTTCCATTTAAACCTTCTTTTCTCCATTTCTTCCCTTTGGCTTAAGAATAA

AAGAAAAGGTACATTGCTAGAATTGTTTCTTTGGGAGAGGGTAAAAGATT

ACAGAATTAGACTGTTCAGCCTTTATATAAACTAAATTTGTCTTCATCTCA

ACCAGCTAATGGTAGGTCTTATCTGAATACTCATGAGAATTTTAGCATCT

GTGAAACTCCATGCACCAGATGTGTGTAAATTTCAGGAAGAAAGTGTTG

AAAGCATTTTCTCTGATGTTAATTAGATGGAAATAAATCACTAAAACATAG

TTTAGGTAAAGCCTGATTATGCCACTTTTTTTTAACTAGACAGGGCAAAG

TTGTTTATGTTAGTGTACTTCTTGTCTATCCTCAGTTAATTTACCTAGACA

AAAAGTGTCAAAGGAAATGAGAAAAAGGTTATATCTGACTCCCTCCAGAC

CTAAGATAATTCCTTTTGATCAGATACAGTCAGATGGAGTGCCTTGGTTT

TTGTTAATTTTGCCTCTATTCCAGCTCCTTACCACAGCGGTGGTGCTTAA

AGAAAGGATCATCAGCAACAGGTCAGGATAGTTCTACCTTTGGGATAGG

GCTGCTTTCCCCGTGCTAGTACTTCTGTGACTGTTAGTGGCACTGAGGA

CTGCAAACTTTTATGCAATATTCTTAATACCCTATTGATATTATGCACTTT

AATCATTCCAAAGAAGCCAAGAATGCTGTATAGGGATGATTCCTTCCTAA

TGAATTCATCTTAACTATTTAGAATGTTATGTCCCTTTTCTTTTGGATAGC

CAACTTGGTATAAATGNTATATGGATGATTCTAAAAGGACTATATAGGAC TTAAGACTTTGAAATGTACATTTACTTATAAGGGGAAATAATTATGCTTTA

GCACATCATTTTAGAAACGTCACATTTTAGAAACATTCAGCTTGCTAACC

TACATGTTTGGGAATTCATTAAAACCAGTTGTCTATATATTTTGTGCCATG

TATATAAGAACATTACAATATATCTTTTTCTACATATGTAGTATGTGCAAC

CAGTGGTTCTCAGAGTATGGTTCTCAGCCCACCAGCTAGTATCAGTATC

ACCTGGGAACTAGTTAGAAATGTAAATTCTTTGGCCCCATCCCAGACATA

CTGAGTCAGAAACTCTGGAATAGGGCCCCGCAATCTGTTTTCACAAGCC

CTCCAGGTGATTCTGATGCACACTTTAAAGTTTAGGAACCACTGGGCTAA

GACTCTGTTGAGATATAGAGTTTTTCTTCCACTCAGACTGATATAGTTATA

CATTGTTCTTCATGGTAAATTCAGCTTAACCTGGTTATCTATAATCTTTTA

TTGGCAAAAGTTAATTCTCAGTACTGCCTATAGAGATACAGTGTATTTTAT

GTACATACACAATTAGTCTAATTCTTGATAATTCAGTTAATTTAGTTTGGC

ATTTTCCTACCACTTACTAAAAAGTTTACATTAAATGACTGATTTAAATATA

TAGGTGCAATGTTCTATGTTTATTTTAATTGTTATGACATTTAAGTAGCTA

ATATAATTGACCGGTGCTAAAGTCTCCTGTTTATCCATAAAATGGGTACA

TTATGGGCAGTGTAATACAAGCTTTCTTTTCATTGCCTAGTACTTTACCA

GCAGACCACAGTTTTGCCCTGGCTAGACCAACCCTCAGAACAAAATCAT

CATTCCTTGTATTTATATTTGTATCTGAGATAGTAAACAAGATGGCTGGC

CAGGTCAACATGGCACCTTAACTTATTTTTTTAATAGGTAAAACTTCTTCA

AAAGTAGCTTGCTTTGTATAAGAACTAAGCTATCAGTATAGATATAGCTA

TCCTTGGAGCTTATGTTTCAGACAAGAATTATTTACTAAAATAAATAATAA

ACAAGATAATGCATTATACAATTTGGGCATTTCTCGTTTCTCAAGTGTAT

GCATCATGGTAAATATAAACTAACCACAAGATAGGTAGATTGATTCATTT

CATTTTAATCTCCTTGTGTAATTCAGTACCTCCATAATTGTTCTAATCTTC

TTCCCACTGTTTACAAATTACCAGTTAATTAACTCGTGAAAGAAAAATTCA

CATATCAGAATAAAAATAAATGTATACTCACTTTATAAAAATCACCACTGC

TGTCTTTCCTTAATACTAGCAGTGGAAATGTAAGTGGCTTACTCTACAAA

TTTTGGTGCTGGCAAATACATAGGCAAACTGTTGGGAGCTGCTGTAGTT

ACATTCCTCCCTTCTTATTCCCTTTTTCTCTTCCTCACTTTATTGCATAAC ATATTCCTGTACCCAAAGCATTCTACCACAGTTCTATTTGACTCCCACTT

GTAATAACTCCTTTAAAAAATTCCATGTTTAACCATATGACCCTGCTTGCT

TACTCATATTCTCCCTCCCTCTCCCCTTCCTTTCTCTCTCTTCCAGAAGTC

ATTTGCCTGGTTTGAAATATTTTGTAGGGATTGCTTATTATATTATTTTAG

CTGATGAACCTCAGGACAACGTCTACACACACACACATACATACACGCA

CACAAAATCTCAGCTGTTGAAGAGTGGGCTTGGAATCAGACTTCTGTGT

CCAGTAAAAAACTCCTGCACTGAAGTCATTGTGACTTGAGTAGTTACAGA

CTGATTCCAGTGAACTTGATCTAATTTCTTTTGATCTAATGAATGTGTCTG

CTTACCTTGTTTCCTTTTAATTGATAAGCTCCAAGTAGTTGCTAATTTTTT

GACAACTTTAAATGAGTTTCATTCACTTCTTTTACTTAATGTTTTAAGTATA

GTACCAATAATTTCATTAACCTGTTCTCAAGTGGTTTAGCTACCATTCTGC

CATTTTTAATTTTTATTTAATTTTATTTGCTTGAGCACACTGATCAACCACT

GAACTGCCTTCTTCCATTGTCCTGCAATGATATAAGGGTTACATTTTTGT

GTATATGGCTTTCATAGTTGGGATTTCAGAGCACTGATACCAGATATTTT

CAGTTTGTTCTCTGGGGGAATTTCATTTGCATCTATGTTTTTAGCTATCTG

TGATAACTTGTTAAATATTAAAAAGATATTTTGCTTCTATTGGAACATTTG

TATACTCGCAACTATATTTCTGTAAACAGCTGCAGTCAAAAATAAAACACT

GAAAGTTTTCAAAAAAAAAAAAAAAAAA

Table 3:

Genes identified as inhibitors of Fas induced killing by the Fas AHM screen using the cDNA microarray analysis

REFFRFNCFS

Altschul et al (1990) Basic local alignment search tool .I Mnl Biol 215 403- 410

Braun et al (1 96) Molecular and Cellular Biology 15(8) 4623-4630

Cregg et al (1993) Recent Advances in the Expression of Foreign Genes in Pichia pastoπs Bio/Technology 11 905-910

Deiss et al (1996) Cathepsin D protease mediates programmed cell death induced by interferon-a, Fas/APO-1 and TNF-a EMBO 15(15) 3861-3870

Deiss et al (1995) Identification of a novel seπne/threonine kinase and a novel 15-kD protein as potential mediators of the a interferon-induced cell death Genes & Develop. 9 15-30

Deiss and Kimchi (1991 ) A Genetic Tool Used to Identify Thioredoxm as a Mediator of a Growth Inhibitory Signal Science.252 117-120

Diatchenko et al (1996) Proc Natl Acad Sci. USA , 93 6025-6030

Gilboa et al (1986) Transfer and expression of cloned genes using retroviral vectors BioTechniques 4(6) 504-512

Gubler and Hoffman (1983) Gene (Amst.) 25 263

Gudkov et al (1994) Cloning mammalian genes by expression selection of genetic suppressor elements association of kinesin with drug resistance and cell immortalization Proc Natl. Acad. Sci U A , 91 3744-3748

Hirt (1967) Selective extraction of polyoma DNA from infected mouse cell cultures J. Mol Biol., pp 365-9

Holzmayer et al (1992) Isolation of dominant negative mutants and inhibitory antisense RNA sequences by expression selection of random DNA fragments Nucleic Acids Res., 20 71 1-717

Izant and Wemtraub (1984) Inhibition of thymidine kinase gene expression by anti-sense RNA a molecular approach to genetic analysis Ceil, 36 1007- 1015

Kissil et al (1995) "Isolation of DAP3, a Novel Mediator of Interferon-a-induced Cell Death" J Biol Chem 270(46) 27932-27936

Levy-Strumpf et al (1997) DAP-5, a novel homolog of eukaryotic translation initiation factor 4G isolated as a putative modulator of gamma terferon- mduced programmed cell death Mol. Cell. Biol , 17 1615-1625

Li and Cohen (1996) Tsg101 A novel tumor susceptibility gene isolated by controlled homozygous functional knockout of allehc loci in mammalian cells CeJ, 85 319-329

Meissner et al (1987) PNAS (USA) 84 4171

Ronmson et al (1995) Genetic suppressor elements new tools for molecular oncology - thirteenth Cornelius P Rhoads Memorial Award Lecture Cancer Res., 55 4023-4028 Schena et al 1995 Aiello et al 1994, Shen et al 1995, Bauer et al , 1993 Liang and Pardee, 1992 Liang and Pardee, 1995 Liang et al , 1993, Braun et al 1995 Hubank and Schatz, 1994

Smith et al (1995) genetic footpπnting a genomic strategy for determining a gene's function given its sequence PNAS 92 6479-6489

Su et al (1993) LYAR, a novel nucleolar protein with zinc finger DNA-bindmg motifs, is involved in cell growth regulation Genes Dev., 7 735-748 Schena et al (1995) Science, 270 467-470

Agrawal, 1996 Antisense ohgonucleotides towards clinical trials, TIBTECH, 14 376

Calabretta, et al, 1996 Antisense strategies in the treatment of leukemias Semm Oncol 23 78

Crooke, 1995 Progress in antisense therapeutics, Hematol Pathol 2 59

Feigner, 1997 Nonviral Strategeies for Gene Therapy Scientific American June, 1997, pgs 102-106

Gewirtz, 1993 Oligodeoxynucleotide-based therapeutics for human leukemias, Stem Cells Dayt 11 96

Hanania, et al 1995 Recent advances in the application of gene therapy to human disease Am J Med 99 537 Lefebvre-d'Hellencourt et al, 1995 Immunomodulation by cytok e antisense ohgonucleotides Eur Cytokme Netw 6 7

Lev-Lehman et al 1997 Antisense Oligomers in vitro and in vivo In Antisense Therapeutics A Cohen and S Smicek eds (Plenum Press, New York)

Loke et al, 1989 Characterization of oligonucleotide transport into living cells PNAS USA 86 3474

Wagner et al , 1996 Potent and selective inhibition of gene expression by an antisense heptanucleotide Nature Biotechnology 14 840-844

Wagner, 1994 Gene inhibition using antisense ohgodeoxynucleotides Nature 372 333

Radhakπshnan et al , 1990 The automated synthesis of sulfur-containing ohgodeoxyribonucleotides using 3/ -1 ,2-Benzodιthιol-3-0ne 1 ,1 Dioxide as a sulfur-transfer reagent J Org Chem 55 4693-4699

Askew, D S , R A Ashmun, B C Simmons, and J L Cleveland 1991 Constitutive c-myc expression in an IL-3-dependent myeloid cell line suppresses cell cycle arrest and accelerates apoptosis Oncogene 6 1915- 1922

Chijiwa, T , M Hagiwara, and H Hidaka 1989 A newly synthesized selective casein kinase I inhibitor, N-(2-amιnoethyl)-5-chloroιsoquιnolιne-8- sulfonamide, and affinity purification of casein kinase I from bovine testis J Biol Chem 264 4924-4927 Deiss, L P and A Kimchi 1991 A genetic tool used to identify thioredox as a mediator of a growth inhibitory signal Science 252 1 17-120

Delneste, Y , P Jeannm, E Sebille, J P Aubry, and J Y Bonnefoy 1996 Thiols prevent Fas (CD95)-medιated T cell apoptosis by down-regulating membrane Fas expression Eur J Immunol 26 2981 -2988

Evan, G I , A H Wyllie, C S Gilbert, T D ϋttlewood, H Land, M Brooks, C M Waters, L Z Penn, and D C Hancock 1992 Induction of apoptosis in fibroblasts by c-myc protein Cell 69 119-128

Moi, P , K Chan, I Asunis, A Cao, and Y W Kan 1994 Isolation of NF-E2- related factor 2 (Nrf2), a NF-E2-hke basic leucme zipper transcπptional activator that binds to the tandem NF-E2/AP1 repeat of the beta-globm locus control region Proc Natl Acad Sci U S A 91 9926-9930

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Wagner, A J , J M Kokontis, and N Hay 1994 Myc-mediated apoptosis requires wild-type p53 in a manner independent of cell cycle arrest and the ability of p53 to induce p21waf1/cιp1 Genes Dev 8 2817-2830 Watson, R W , O D Rotstem, M Jimenez, J Parodo, and J C Marshall 1997 Augmented intracellular glutathione inhibits Fas-triggered apoptosis of activated human neutrophils S/oocf 89 4175-4181

Claims

What is claimed is

1 A method for identifying a compound which stimulates or inhibits cell apoptosis by

(a) contacting a cell expressing a gene with the compound, and

(b) determining the ability of the compound to stimulate or inhibit apoptosis of the cell as compared to a control

2 The method according to claim 1 wherein said contacting step includes contacting a cell expressing a gene selected from the group consisting essentially of casein kinase alpha 1 , NF-E2 related factor 2 (Nrf-2), basic fibroblast growth factor, TNF receptor associated factor 6, human COP9, antithrombin III, mucin 1 transmembrane, adenosine receptor A3, calcium/calmodulin-dependent protein kinase II, human protein immunoreactive with anti-parathyroid hormone antibodies and retinoic acid receptor gamma 1

3 The method according to claim 2, wherein said method includes utilizing methods selected from the group consisting essentially of tetracyhne- ducible gene expression and expression of a reporter gene

4 The method according to claim 1 , wherein said contacting step utilizes a gene the corresponding cDNA of which comprises a nucleic acid selected from the group of nucleic acids consisting essentially of SEQ ID Nos 15, 16, 17, 18, 19 ,20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36 and 37 5 A compound identified by the method of claim 1

6 A method of treating a tumor in a subject by administering to the subject a therapeutically effective amount of an compound which inhibits a gene in the Fas pathway

7 The method according to claim 6, said administering step includes administering an effective amount of a compound which inhibits a gene selected from the group consisting essentially of casein kinase alpha 1 , NF- E2 related factor 2 (Nrf-2), basic fibroblast growth factor, TNF receptor associated factor 6, human COP9, antithrombin III, mucin 1 transmembrane, adenosine receptor A3, calcium/calmodulin-dependent protein kinase II, human protein immunoreactive with anti-parathyroid hormone antibodies and retinoic acid receptor gamma 1

8 The method according claim 6, wherein said administering step includes administering an effective amount of a compound which inhibits a gene, the corresponding cDNA of which comprises a nucleic acid selected from the group of nucleic acids consisting essentially of SEQ ID Nos 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32 , 33, 34, 35, 36 and 37

9 The method according to claim 7, said administering step includes administering an effective amount of a compound selected from the group consisting essentially of dicumarol, sulfinpyrazone, NF-E2 related factor 2 (Nrf2) inhibitor, CKI-7 and casein kinase inhibitor

10 A method of treating a tumor in a subject which comprises administering to the subject a therapeutically effective amount of a compound selected from the group consisting essentially of dicumarol, sulfinpyrazone, an Nrf2 inhibitor and a casein kinase inhibitor

1 1 A method of treating auto-immune disease in a subject by administering to the subject a therapeutically effective amount of a compound which inhibits a gene in the Fas pathway

12 The method according to claim 11 , wherein said administering step includes administering an effective amount of a compound which inhibits a gene selected from the group consisting essentially of casein kinase alpha 1 , Nrf-2 , basic fibroblast growth factor, TNF receptor associated factor 6 , human COP9, antithrombin III, mucin 1 transmembrane, adenosine receptor A3, calcium/calmodulin-dependent protein kinase II, human protein immunoreactive with anti-parathyroid hormone antibodies and retinoic acid receptor gamma 1

13 The method according to claim 1 1 , said administering step includes administering an effective amount of a compound selected from the group consisting essentially of dicumarol, sulfinpyrazone, Nrf2 inhibitor, CKI-7 and casein kinase inhibitor

14 The method according to claim 11 , wherein the corresponding cDNA of the gene inhibited comprises a nucleic acid selected from the group of nucleic acids consisting essentially of SEQ ID Nos 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36 and 37

15 A method of treating degenerative disease in a subject by administering to the subject a therapeutically effective amount of a compound which stimulates a gene in the Fas pathway 16 The method according to claim 15 wherein said administering step includes administering an effective amount of a compound which stimulates a gene selected from the group consisting essentially of casein kinase alpha 1 , Nrf-2 basic fibroblast growth factor, TNF receptor associated factor 6, human COP9, antithrombin III mucin 1 transmembrane, adenosine receptor A3 calcium/calmodulin-dependent protein kinase II human protein immunoreactive with anti-parathyroid hormone antibodies and retinoic acid receptor gamma 1

17 The method according to claim 16, said administering step includes administering an effective amount of a compound which is a glutathione precursor

18 The method according to claim 15 wherein said administering step includes administering an effective amount of compound for treating degenerative diseases selected from the group consisting essentially of degenerative disease of the liver, fulminate hepatitis, Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis

19 Use of a casein kinase inhibitor in the preparation of a pharmaceutical

20 The use according to claim 19, wherein said pharmaceutical is used for therapies selected from the group consisting essentially of anti-tumor therapy and auto-immune disease therapy

21 Use of dicumarol in the preparation of a pharmaceutical for therapies selected from the group consisting essentially of anti-tumor therapy and autoimmune disease therapy 22 Use of sulfinpyrazone in the preparation of a pharmaceutical

23 The use according to claim 22, wherein the pharmaceutical is used for therapies selected from the group consisting essentially of anti-tumor therapy and auto-immune disease therapy

24 Use of Nrf-2 inhibitor in the preparation of a pharmaceutical

25 The use according to claim 24, wherein the pharmaceutical is used for therapies selected from the group consisting essentially of anti-tumor therapy and auto-immune disease therapy

26 Use of a glutathione precursor in the preparation of a pharmaceutical

27 The use according to claim 26, wherein said pharmaceutical is used for treatment of degenerative disease selected from the group consisting essentially of degenerative disease of the liver, fulminate hepatitis, Alzheimer's disease, Parkinson's disease, and Amyotrophic lateral sclerosis

28 The use according to claim 26, wherein said glutathione precursor is N- acetyl Cysteine

29 A method of preparing a pharmaceutical composition which comprises determining whether a compound stimulates or inhibits a Fas-pathway gene using the method of claims 1 , and admixing said compound with a pharmaceutically acceptable carrier

30 A method for the identification of genes that encode for inhibitors of cell death comprising the steps of inactivating genes in cells by sensitizing cells to cell death, using gene inactivation means, applying positive selection means to the sensitized cells, and utilizing subtraction analysis means to identify the genes that have been inactivated

31 The method according to claim 30, wherein said method is further used for identifying drug interactions

32 The method according to claim 30, wherein said method is further used for identifying survival factors

33 The method according to claim 30, wherein said method is further used for identifying targets that inhibit signaling

34 A gene identified by the method of claim 30