AU689840B2 - Association of kinesin with sensitivity to chemotherapeutic drugs - Google Patents

Description

I -1 I WO 95/18857 PCT/US95/00092 -1- ASSOCIATION OF KINESIN WITH SENSITIVITY TO CHEMOTHERAPEUTIC DRUGS BACKGROUND OF THE INVENTION 1. Field Of The Invention The invention relates to genetic factors associated with sensitivity to chemotherapeutic drugs. More particularly, the invention relates to methods for identifying such factors as well as to uses for such factors. The invention specifically provides genetic suppressor elements derived from mammalian kinesin genes, and therapeutic and diagnostic uses related thereto.

2. Summary Of The Related Art A broad variety of chemotherapeutic agents are used in the treatment of human cancer. For example the textbook CANCER: Principles Practice of Oncology, 2d Edition, (De Vita et al., eds.), J.B. Lippincott Company, Philadelphia, PA (1985) discloses as major antineoplastic agents the plant alkaloids vincristine, vinblastine, and vindesine; the antibiotics actinomycin-D, doxorubicin, daunorubicin, mithramycin, mitomycin C and bleomycin; the antimetabolites methotrexate, fluorouracil, 5-fluorodeoxyuridine, 6-mercaptopurine, 6-thioguanine, cytosine arabinoside, 5-aza-cytidine and hydroxyurea; the alkylating agents cyclophosphamide, melphalan, busulfan, CCNU, MeCCNU, BCNU, streptozotocin, chlorambucil, bisdiaminedichloro-platinum, azetidinylbenzoquinone; and the miscellaneous agents dacarbazine, mAMSA and mitoxantrone.

These and other chemotherapeutic agents such as etoposide and amsacrine have proven to be very useful in the treatment of cancer. Unfortunately, some ttmor cells become resistant to specific chemotherapeutic agents, in some instances even to multiple chemotherapeutic agents. Such drug resistance or multiple drug resistance can theoretically arise from either the presence of genetic factors that confer resistance to the drugs, or from the absence of genetic factors that confer sensitivity to the drugs. The former type of factors have been identified, and include the multiple drug resistance gene mdr-1 (see Chen et al., 1986, Cell 47: 381-389).

However, the latter type of factor remains largely unknown, perhaps in part because the absence of such factors would tend to be a recessive trait.

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WO 95/18857 PCTIUS95/00092 -2- Identification of genes associated with sensitivity to chemotherapeutic agents is desirable, because the discovery of such genes can lead to both diagnostic and therapeutic approaches for cancer cells and for drug resistant cancer cells, as well as to improvements in gene therapy and rational drug design. Recently, some developments have been made in the difficult area of isolating recessive genetic elements, including those involved in cytotoxic drug sensitivity. Roninson et al., U.S. Patent No. 5,217,889 (issued June 8, 1993) teach a generalized method for obtaining genetic suppressor e'"-ents (GSEs), which are dominant negative factors that confer the recessive-type "..notype for the gene to which the particular GSE corresponds. (See also Holzmayer et al,, 1992, Nucleic Acids Res. 20: 711-717).

Gudkov et al., 1993, Proc. Natl. Acad. Sci. USA 90: 3231-3235 teach isolation of GSEs from topoisomerase II cDNA that induce resistance to topoisomerase IIinteractive drugs. Co-pending U.S. Patent Application Serial No. 08/033,986, filed March 3, 1993, discloses the discovery by the present inventors of a novel and unexpected result of experiments performed to identify GSEs isolated from RNA of cells resistant to the anticancer DNA damaging agent, etoposide. This reference discloses that a GSE encoding an antisense RNA homologous to a portion of a mouse kinesin heavy chain gene has the capacity to confer etoposide resistance to cells expressing the GSE. The experiments described in this reference also demonstrate that under-expression of the particular kinesin heavy chain gene disclosed therein was associated with naturally-occurring etoposide resistance in cultures of drug-selected human adenocarcinoma cells. These results were particularly unexpected because the role of kinesin genes in etoposide resistance was unknown in the art prior to the instant inventors' discoveries.

The kinesins comprise a family of motor proteins involved in intracellular movement of vesicles or macromolecules along microtubules in eukaryotic cells (see Vale, 1987, Ann. Rev. Cell Biol. 3: 347-378; and Endow, 1991, Trends Bibchem.

Sci. 16: 221-225 for reviews). Among the family of kinesin genes are encoded kinesin light chains and kinesin heavy chains that assemble to form mature kinesin.

A number of kinesin genes have been isolated in the prior art.

Gauger and Goldstein, 1993, J. Biol. Chem. 268: 13657-13666 disclose cloning and sequencing of a Drosophila kinesin light chain gene.

lulr I WO 95/18857 PCT/US95/00092 -3- Navone et al., 1992, J. Cell. Biol. 117: 1263-1275 disclose cloning and sequencing of a human kinesin heavy chain gene, Kato, 1991, J. Neurosci. 2: 704-711 disclose cloning and sequencing of a mouse kinesin heavy chain gene.

Cyr et al., 1991, Proc. Natl. Acad. Sci. USA 88: 10114-10118 disclose cloning and sequencing of a rat kinesin light chain gene.

McDonald Goldstein, 1990, Cell 61: 991-1000 disclose isolation of a Drosophila kinesin heavy chain gene.

Kosik et al., 1990, J. Biol. Chem. 265: 3278-3283 disclose isolation of a squid kinesin heavy chain gene.

The present inventors have demonstrated that a heretofore unexpected gene, a kinesin heavy chain gene, is involved in cellular sensitivity to the anticancer drug etoposide, and that down-regulation of functional expression of this kinesin heavy chain gene is associated with resistance to this drug. Further experiments, disclosed herein, have suggested that the role of kinesin genes in chemotherapeutic drug resistance may not be limited to this single member of the kinesin gene family.

These results further underscore the power of the GSE technology developed by these inventors to elucidate unexpected mechanisms of drug resistance in cancer cells, thereby providing the opportunity and the means for overcoming drug resistance in cancer patients. Reagents and methods directed towards such goals are provided in this disclosure.

BRIEF SUMMARY OF THE INVENTION The invention provides genetic suppressor elements (GSEs) that are random fragments derived from genes associated with sensitivity to chemotherapeutic drugs, and that confer resistance to chemotherapeutic drugs and DNA damaging agents upon cells expressing such GSEs. The invention specifically provides GSEs derived from cDNA and genomic DNA encoding kinesin genes. Diagnostic assays useful in determining appropriate candidate cancer patients bearing tumors likely to be successfully reduced or eliminated by administration of particular anticancer treatment modalities, including chemotherapeutic drugs and other DNA damaging agents, are provided by the invention, on the basis of levels of kinesin gene -L II, I C WO 95118857 PCT/US95/00092 -4expression in the tumor cells borne by such cancer patients. In vitro drug screening and rational drug design methods are also within the scope of the instant disclosure.

The invention is based in part on the discoveries disclosed in co-pending U.S.

Patent Application Serial No. 08/033,086, filed March 3, 1993 and incorporated by reference, providing a method for identifying and isolating GSEs that confer resistance to any chemotherapeutic drug for which resistance is possible. Particularly provided herein are methods for identifying GSEs derived from any kinesin gene, said GSEs being capable of conferring resistance to DNA damaging agents on cells expressing the GSEs. This method utilizes chemotherapeutic drug selection of cells that harbor clones from a random fragment expression library derived from kinesinspecific cDNA, and subsequent rescue of library inserts from drug-resistant cells.

In a second aspect, the invention provides GSEs comprising oligonucleotides and/or peptides derived from kinesin genes that function as GSEs in vivo and confer on cells expressing said GSEs resistance to DNA damaging agents, including certain chemotherapeutic drugs. In a third aspect, the invention provides a method for obtaining GSEs having optimized suppressor activity for a kinesin gene associated with sensitivity to a chemotherapeutic drug. This method utilizes chemotherapeutic drug selection of cells that harbor clones from a random fragment expression library derived from DNA of a kinesin gene associated with sensitivity to that chemotherapeutic drug, and subsequent rescue of the library inserts from drug resistant cells. Particularly and preferably provided are such optimized GSEs derived from a mouse or human kinesin gene. In a fourth aspect, the invention provides synthetic peptides and oligonucleotides that confer upon cells resistance to DNA damaging agents, including certain chemotherapeutic drugs. These synthetic peptides and oligonucleotides are designed based upon the sequence of a drugresistance conferring GSE derived from a mouse or human kinesin gene according to the invention.

In a fifth aspect, the invention provides a diagnostic assay for tumor cells that are resistant to one or more therapeutic DNA damaging agents and, at the same time, sensitive to therapeutic anti-microtubular agents, due to the absence of expression or under-expression of a kinesin gene. This diagnostic assay comprises quantitating the level of expression of any particular kinesin gene product in a particular tumor cell

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WO 95118857 PCT/US95/00092 sample to be tested, and comparing the expression levels so obtained with a standardized set of cell lines expressing varying amounts of kinesin gene mRNA and/or protein and having different degrees of resistance to chemotherapeutic drugs and DNA damaging agents associated with their levels of kinesin gene expression.

In preferred embodiments, such a standardized set of cell lines is matched by tissue type with the tissue type of the tumor cells to be evaluated.

In a sixth aspect, the invention provides methods for determining the appropriateness of candidates for particular cancer chemotherapeutic treatment modalities. In one preferred embodiment, the invention provides a means for determining whether a cancer patient is an appropriate candidate for treatment with DNA damaging chemotherapeutic drugs or other DNA damaging agents such as radiation, the method determining whether a kinesin gene, such as the kinesin heavy chain gene disclosed herein and in co-pending U.S. Patent Application Serial No.

08/033,086, is over-expressed or under-expressed in tumor cells borne by a cancer patient, relative to a standardized set of cell lines as disclosed herein. Using this method, appropriate candidates for treatment with DNA damaging agents, including certain chemotherapeutic drugs, will be those patients whose tumor cells over-express the kinesin gene. In another embodiment, the invention provides a means for determining whether a cancer patient is an appropriate candidate for treatment with anti-microtubular chemotherapeutic drugs. Using this aspect of the method, appropriate candidates for anti-microtubular agent treatment will be those patients whose tumor cells under-express the kinesin gene compared with expression levels in a standardized set of cell lines. In a particularly useful embodiment of this aspect of the invention, potential candidate cancer patients for treatment with antimicrotubular anticancer agents will have failed or proven resistant to a course of cancer chemotherapy using DNA damaging agents.

In a seventh aspect, the invention provides a starting point for the rational design of pharmaceutical products that are useful against tumor cells that are resistant to chemotherapeutic drugs. By examining the structure, function, localization and pattern of expression of kinesin genes associated with resistance to DNA damaging agents and sensitivity to anti-microtubular chemotherapeutic drugs, strategies can be developed for creating pharmaceutical products that will overcome drug resistance II I II 6 in tumor cells in which such kinesin genes are either over-expressed or under-expressed.

Specific preferred embodiments of the present invention will become evident from the following more detailed description of certain preferred embodiments and the claims.

According to a first embodiment of the invention there is provided a method of identifying genetic suppressor elements derived from a kinesin gene that confer upon a cell resistance to one or more DNA damaging agents, the method comprising the steps of: generating a set of random fragments of cDNA encoding the kinesin gene; transferring the DNA fragments to an expression vector to yield a library, wherein the expression vector is capable of expressing the DNA fragments in a living cell; genetically modifying living cells by introducing the random fragment library of step into the living cells; isolating or enriching for genetically modified living cells containing kinesinderived genetic suppressor elements by selecting cells in the presence of a DNA damaging 15 agent; and g obtaining the genetic suppressor element from the genetically modified cells.

According to a second embodiment of the invention there is provided a genetic suppressor element identified by the method according to the first embodiment of the invention.

According to a third embodiment of the invention there is provided a diagnostic assay comprising the eps of: isolating cellular RNA comprising messenger RNA from a tumor or malignant cells from an animal; o measuring a level of expression of kinesin mRNA in the tumor or malignant cells in the animal; determining whether the level of expression of kinesin mRNA in the tumor or malignant cells indicates that the kinesin gene is over-expressed or under-expressed in the tumor or malignant cells in the animal.

According to a fourth embodiment of the invention there is provided a diagnostic assay comprising the steps of: isolating cellular protein from a tumor or malignant cells from an animal; measuring an amount of a kinesin protein in the tumor or malignant cells in the animal; determining whether the amount of the kinesin protein in the tumor or malignant cells indicates that the kinesin gene is over-expressed or under-expressed in the tumor or malignant cells in the animal.

According to a fifth embodiment of the invention there is provided a method of identifying a multiplicity of genetic suppressor elements derived from a kinesin gene, that confer upon a cell resistance to one or more DNA damaging agents, the method HAr4R comprising the steps of: [N:\LIBFF)OO858:MCN 1 6a generating a set of random fragments of cDNA encoding the kinesin gene; transferring the DNA fragments to a retroviral expression vector to yield a retroviral random fragment expression library, wherein the retroviral expression vector is capable of expressing the DNA fragments in a living cell; genetically modifying a culture of living cells by introducing the retroviral random fragment expression library of step into the living cells of the culture, wherein the living cells of the culture are capable of propagating each of the retroviral expression vectors comprising the library; isolating or enriching for genetically modified living cells containing kinesinderived genetic suppressor elements by selecting cells in the presence of a DNA damaging agent; and allowing the drug-resistant, genetically modified living cells of step to propagate the kinesin-derived genetic suppressor elements of the retroviral random fragment expression library to form an amplified random fragment expression library 15 comprising a multiplicity of kinesin-derived genetic suppressor elements.

Brief Description of the Drawings Figures 1A and 1B show a scheme for construction of a random fragment expression library (RFEL) from NIH 3T3 cDNA. Figure 1A shows the overall construction scheme. Figure 1B shows normalization of the cDNA fragments. In Figure 1B, t represents total unfractionated cDNA, s and d represent the single-stranded and double-stranded fractions separated by hydroxyapatite, time points indicate the period of reannealing, and tubulin, c-myc, and c-fos indicate the probes used in Southern S hybridization with the total, single-stranded and double-stranded fractions.

Figure 2 shows the structure of the LNCX vector and the adaptor used in cDNA cloning. The nucleotide sequences are shown for the ATG-sense (SEQ ID NO:1) and ATG-antisense (SEQ ID NO: 2) strands of the adaptor.

Figure 3 shows the overall scheme for selecting cell lines containing chemotherapeutic drug resistance-conferring GSEs and rescuing the GSEs from these cells.

Figures 4A and 4B show etoposide resistance conferred by preselected virus (Figure 4A) and PCR analysis of the selected and unselected populations (Figure 4B). Figure 4C illustrates a schema dor recloning individual PCR-amplified fragments into the LNCX vector in the same position and orientation as in the original plasmid.

Figures 5A and 5B show resistance to various concentrations of etoposide, conferred upon the cells by the GSE anti-khcs under an IPTG-inducible promoter (Figure and the scheme for this experiment (Figure Figure 6 shows the nucleotide sequence of the GSE anti-khcs (SEQ ID NO. 3).

[N:\LIBFF!00858:MCN

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PC~II I WO 95/18857 PCT/US95/00092 -7- Figure 7 shows the nucleotide sequence of most of the coding region of the mouse khcs cDNA (SEQ.ID.No.:4).

Figures 8A through 8D show the dot matrix alignments of khcs protein sequence deduced from the nucleotide sequence in Figure 7 with kinesin heavy chain sequences from human (Figure 8A), mouse (Figure 8B), fruit fly (Figure 8C), and GSE-C (Figure 8D).

Figures 9A and 9B illustrates the experimental protocol for drug-selected production of kinesin-derived GSEs (Figure 9A) and the structure of the adaptors used for the preparation of a random fragment KHCS cDNA library (Figure 9B).

Figure 10 shows etoposide resistance in HT1080 cells carrying insert-free vector virus or a random fragment library of human KHCS cDNA.

Figure 11 shows the effects of different drugs on 4-day growth of NIH 3T3 cells infected with insert-free vector virus or with a virus encoding anti-khcs. Cell growth in the absence of the drug differed less than 5% for the compared populations. Drug concentrations are given in ng/mL. A representative series of parallel assays, carried out in triplicate, is shown.

Figure 12 shows growth of cells carrying anti-khcs (solid lines) and control cells (broken lines) after treatment with colchicine or vinblastine. Cells were incubated with the drugs for 4 days, followed by 2 or 4 days in drug-free media, as indicated. Cell growth, presented in arbitrary units, was evaluated by methylene blue staining.

Figure 13 shows a kinetic analysis of cell growth of anti-khcs GSE-carrying cells (black lines) and control cells (grey lines) incubated with different drugs. Cell growth was measured as described for Figure 13. Cells were plated and one day later (indicated by the first arrow) the indicated drugs were added at concentrations as described. Four days later (indicated by the second arrow), the drugs were removed from some of the plates. Solid lines indicate cell growth in the continuous presence of the drug, and broken lines indicate cell growth after removal of the drug.

Figure 14 demonstrates increased immortalization of primary mouse embryo fibroblasts by infection with the LNCX vector containing the anti-khcs GSE, relative to cells infected with the LNCX vector alone or uninfected (control) cells.

I I IP III WO 95/18857 PCT/US95/00092 8- Figure 15 demonstrates increased immortalization of primary human skin fibroblasts by infection with the LNCX vector containing the anti-khcs GSE (antikhcs) at the 4th passage after infection, relative to human skin fibroblasts in growth crisis (control).

Figure 16 shows cDNA-PCR quantitative analysis of expression of the human khcs gene in various unselected and etoposide-selected human HeLa cells. Lanes a shows results for clone CS(O), lands a' for clone CX(200), lanes b for clone E/11(0), lanes b' for clone E11 (1000), lanes c for clone lanes c' for clone 6(1000), lanes d for clone E20(0) and lanes d' for clone E20 (1000). The numbers in parentheses for each clone name indicate the concentration of etoposide (ng/ml) present in the growth media. Bands indicative of khcs expression are shown along with bands for 0-2 macroglobulin expression as an internal control.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The invention relates to means for suppressing specific gene functions that are associated with sensitivity and resistance to chemotherapeutic drugs. The invention provides genetic suppressor elements (GSEs) derived from kinesin genes that have such suppressive effect and thus confer resistance to DNA damaging agents including chemotherapeutic drugs. The invention further provides methods for identifying such GSEs, as well as methods for their use.

For the purposes of this invention, the term "kinesin gene" will be understood to encompass any kinesin gene, particularly mammalian, and preferably mouse or human, kinesin genes. Kinesins comprise a family of related genes encoding a number of related motor proteins involved in intracellular movement of vesicles or macromolecules along microtubules in eukaryotic cells (see Background of the Invention). The mature, functional kinesin molecule is comprised of products of a kinesin heavy chain gene and a kinesin light chain gene. The instant invention encompasses GSEs derived from both kinesin light chain and kinesin heavy chain genes. The invention specifically is intended to contain within its scope all kinesin genes and GSEs derived therefrom that are capable of causing resistance or sensitivity to DNA damaging agents.

I I II L I -Y I WO 95/18857 PCT/US95/00092 -9- The DNA damaging agents that fall within the scope of this invention are all DNA damaging agents, including but not limited to ionizing and ultraviolet radiation, and certain chemotherapeutic drugs, including amsacrine, etoposide, doxorubicin (Adriamycin), cisplatin, and camptothecin.

In a first aspect, the invention provides a GSE derived from the cDNA of a mouse kinesin heavy chain gene isolated from a normalized, random fragment expression library made from total cellular mRNA from NIH 3T3 cells and isolated on the basis of its ability to confer resistance to the topoisomerase II drug, etoposide (described in Examples 1-4 herein and co-pending U.S. Patent Application Serial No.

08/033,086, filed March 3, 1993 and incorporated by reference). Prior to the discovery by the present inventors, there was no suspicion that kinesin was in any way implicated in etoposide sensitivity. These results demonstrated the ability of the general method for identifying GSEs to provide much new and surprising information about the genetic basis for resistance to chemotherapeutic drugs.

In addition, the kinesin-derived GSE conferring resistance to etoposide caused cellular effects suggesting that kinesin may be involved in programmed cell death.

The method according to this aspect of the invention therefore also provides valuable information about the genetic basis for senescence and cell death. This may have important implications for studying genes involved in development, since GSEs used to identify genes associated with chemotherapeutic drug resistance or senescence can also be expressed as transgenes in embryos to determine the role of such genes in development. The elucidation of the structure of the mouse kinesin heavy chain gene corresponding to this drug resistance-related GSE is described in Example 5 and functional analyses of the drug resistance capacity of this GSE is disclosed in Example 7.

In a second aspect, the invention provides a method for identifying kinesin gene-derived GSEs that confer resistance to a DNA damaging agent. The GSEs identified by this method will be homologous to a kinesin gene. For purposes of the invention, the term "homologous to" a kinesin gene has two different meanings, depending on whether the GSE acts through an antisense mechanism or antigene mechanism through a mechanism of interference at the protein level). In the former case, a GSE that is an antisense or antigene oligonucleotide or polynucleotide 0 ~r~C I a WO 95/18857 PCT/US95/00092 is homologous to a gene if it has a nucleotide sequence that hybridizes under physiological conditions to the gene or its mRNA transcript by Hoogsteen or Watson- Crick base-pairing. In the latter case, a GSE that interferes with a protein molecule is homologous to the gene encoding that protein molecule if it has an amino acid sequence that is the same as that encoded by a portion of the gene encoding the protein, or that would be the'same, but for conservative amino acid substitutions.

In either case, as a practical matter, whether the GSE is homologous to a gene is determined by assessing whether the GSE is capable of inhibiting or reducing the function of gene; in particular, any kinesin gene, preferably any mouse or human kinesin gene, as disclosed herein.

The method according to this aspect of the invention comprises the step of screening a kinesin-specific cDNA or kinesin-specific genomic DNA random fragment expression library phenotypically to identify clones that confer resistance to a DNA damaging agent such as certain chemotherapeutic drugs. Preferably, the library of random fragments of kinesin-specific cDNA or kinesin-specific genomic DNA is cloned into a retroviral expression vector. In this preferred embodiment, retrovirus particles containing the library are used to infect cells and the infected cells are tested for their ability to survive in a concentration of a DNA damaging agent that kills uninfected cells. Preferably, the inserts in the library will range from about 100 b.p. to about 700 b.p. and more preferably, from about 200 b.p. to about 500 b.p. Once a clonal population of cells that are resistant to the DNA damaging agent has been isolated, the library clone encoding the GSE is rescued from the cells.

At this stage, the nucleotide sequence of the insert of the expression library may be determined; in clones derived from a kinesin gene-specific cDNA random fragment expression library, the nucleotide sequence is expected to be homologous to a portion of the kinesin gene cDNA nucleotide sequence. Alternatively, the rescued library clone may be further tested for its ability to confer resistance to DNA damaging agents and chemotherapeutic drugs in additional transfection or infection and selection assays, prior to nucleotide sequence determination. Determination of the nucleotide sequence, of course, results in the identification of the GSE. This method is further illustrated in Example 6.

-I I Lli~s WO 95/18857 )'CTIUS95/00092 11 Thus, the invention provides a method for obtaining kinesin gene-derived GSEs having optimized suppressor activity. By screening a random fragment expression library made exclusively from kinesin gene-specific fragments, a much greater variety of GSEs derived specifically from the kinesin gene can be obtained, compared with a random fragment library prepared from total cDNA as in Example 1. Consequently, the likelihood of obtaining optimized GSEs, those kinesinderived GSEs conferring an optimal level of resistance to a chemotherapeutic drug, is maximized using the single gene random fragment library approach, as is shown in greater detail in Example 6.

An additional feature of this aspect of the invention is the production of a multiplicity of kinesin-specific GSEs by drug selection of cells producing infectious retroviral embodiments of the kinesin-derived GSEs of the invention. In this aspect, ecotropic cells infected with a kinesin cDNA or kinesin genomic DNA-specific random fragment expresoion library are subjected to selection with a DNA damaging agent, preferably and most practically a chemotherapeutic drug such as etoposide.

A population of resistant clones are thereby obtained, each containing a drug resistance-conferring, kinesin-derived GSE. Since these cells are capable of producing infection retroviral embodiments of the GSEs of the invention, a multiplicity of kinesin-derived GSEs, pre-selected for the ability to confer drug resistance, can be easily and efficiently produced.

In a fourth aspect, the invention provides synthetic peptides and oligonucleotides that are capable of inhibiting the function of kinesin genes associated with sensitivity to chemotherapeutic drugs. Synthetic peptides according to the invention have amino acid sequences that correspond to amino acid sequences encoded by GSEs according to the invention. Synthetic oligonucleotides according to the invention have nucleotide sequences corresponding to the nucleotide sequences of GSEs according to the invention. Once a GSE has been discovered and sequenced, and its orientation is determined, it is straightforward to prepare an oligonucleotide corresponding to the nucleotide sequence of the GSE (for antisenseoriented GSEs) or amino acid sequence encoded by the GSE (for sense-oriented GSEs). In certain embodiments, such synthetic peptides or oligonucleotides may have the complete sequence encoded by the GSE or may have only part of the -T I l WO 95/18857 P'CTUS95/10092 12sequence present in the GSE, respectively. In certain other embodiments, the peptide or oligonucleotide may have only a portion of the GSE-encoded or GSE sequence. In such latter embodiments, undue experimentation is avoided by the observation that many independent GSE clones corresponding to a particular gene will have the same 5' or 3' terminus, but generally not both. This suggests that many GSE's have one critical endpoint, from which a simple walking experiment will determine the minimum size of peptide or oligonucleotide necessary to inhibit gene function. For peptides, functional domains as small as 6-8 amino acids have been identified for immunoglobulin binding regions. Thus, peptides or peptide mimetics having these or larger dimensions can be prepared as GSEs. For antisense oligonucleotides, inhibition of gene function can be mediated by oligonucleotides having sufficient length to hybridize to their corresponding mRNA under physiological conditions. Generally, oligonucleotides having about 12 or more bases will fit this description. Preferably, such oligonucleotides will have from about 12 to about 100 nucleotides. As used herein, the term oligonucleotide includes modified oligonucleotides having nuclease-resistant internucleotide linkages, such as phosphorothioate, methylphosphonate, phosphorodithioate, phosphoramidate, phosphotriester, sulfone, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate and bridged phosphorothioate internucleotide linkages. The synthesis of oligonucleotides containing these modified linkages is well known in the art. (See, Uhlmann and Peyman, 1990, Chemical Reviews 90: 543-584; Schneider and Banner, 1990, Tetrahedron Letters 31: 335). The term oligonucleotides also includes oligonucleotides having modified bases or modified ribose or deoxyribose sugars.

In a fifth aspect, the invention provides dominant selectable markers that are useful in gene co-transfer studies. Since GSEs according to the invention confer resistance to chemotherapeutic drugs, the presence of a vector that expresses the GSE can readily be selected by growth of a vector-transfected cell in a concentration of the appropriate cyrotoxic drug that would be cytotoxic in the absence of the GSE.

GSEs according to the invention are particularly well suited as dominant selectable markers because their small size allows them to be easily incorporated along with a gene to be co-transferred even into viral vectors having limited packaging capacity.

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-L II C- -I- WO 95118857 PCT/US95/00092 13 In a sixth aspect, the invention provides a diagnostic assay for tumor cells that are resistant to one or more DNA damaging agents, including certain chemotherapeutic drugs, due to the absence of expression or the under-expression of a kinesin gene. In particular, the class of DNA damaging agents resistance to which involves under-expression of a kinesin gene includes but is not limited to cisplatin, etoposide and camptothecin. To determine whether absence of expression or underexpression of a kinesin gene is a naturally occurring, and thus medically significant basis for chemotherapeiitic drug resistance, human tumor cells can be treated with cytotoxic quantities of an appropriate chemotherapeutic drug to select for spontaneous drug resistant mutants. These mutants can then be assessed for their level of expressing of the particular gene of interest. Absence of expression or significantly reduced expression indicates a natural mechanism of chemotherapeutic drug resistance. The description of such an experiment, disclosing that under-expression of the human kinesin heavy chain gene disclosed herein is associated with naturallyoccurring resistance to the chemotherapeutic drug etoposide in cultures of etoposideresistant human adenocarcinoma (HeLa) cells, is disclosed in Example 11 herein and in co-pending U.S. Patent Application Serial No. 08/033,086,filed March 3, 1993 and incorporated by reference. In a preferred embodiment of this assay, a standardized set of tissue-specific cell lines, wherein the levels of drug resistance and kinesin gene expression have been quantitated and correlated with each other, are provided for tumors from each tissue type to be assayed.

Alternatively, and preferably, collections of naturally occurring treatmentresponding and non-responding tumor tissue samples can be examined for expression levels of kinesin genes, and correlations established between treatment outcome, and presumably the drug-resistant mechanisms thereof, and kinesin gene expression.

Accordingly, such reduced or absent expression can be the basis for a diagnostic assay for tumor cell resistance to a DNA damaging agent or chemotherapeutic drug or drugs of interest. A first embodiment of a diagnostic assay according to this aspect of the invention utilizes an oligonucleotide or oligonucleotides that is/are homologous to the sequence of a kinesin gene. In this embodiment, RNA is extracted from a tumor sample, and RNA specific for a particular kinesin gene is quantitated by standard filter hybridization procedures, an mIC WO 95/18857 PCT/US95/00092 14- RNase protection assay, or by quantitative cDNA-PCR (see Noonan et al., 1990, Proc. Natl. Acad. Sci. USA 87: 7160-7164). In a second embodiment of a diagnostic assay according to this aspect of the invention, antibodies are raised against a synthetic peptide having an amino acid sequence that is identical to a portion of the kinesin heavy chain or kinesin light chain protein. Antibodies specific for the human kinesin heavy chain have in fact been disclosed (see Navone et al., supra). These antibodies are then used in a conventional quantitative immunoassay RIA or immunohistochemical assays) to determine the amount of the gene product of interest present in a sample of proteins extracted from the tumor cells to be tested, or on the surface or at locations within the tumor cells to he tested.

A particular utility for such diagnostic assays of this invention are their clinical use in making treatment decisions for the alleviation of malignant disease in humans. For example, a determination that the kinesin heavy chain gene of this invention is under-expressed in a tumor compared with the levels of expression found in normal cells comprising that tissue would suggest that a patient nearing such a tumor might be a poor candidate for therapeutic intervention using DNA damaging agents, since it would be expected that such kinesin under-expressing cells of the tumor would be resistant to such agents. Similarly, tumor cells which fortuitously over-express the kinesin heavy chain gene of the invention would be expected to be sensitive to such agents and thus to be susceptible to tumor cell killing by these agents. On the other hand, the instant disclosure provides experimental evidence that kinesin heavy chain gene under-expressors are sensitive to the cytocidal action of anti-microtubular agents, including for example colchicine, colcemide, vinblastine, vincristine and vindesine. These results suggest that patients bearing tumors whose cells under-express the kinesin heavy chain gene of the present invention may be responsive to treatment with DNA damaging agents. The present invention thus enables intelligent and informed therapeutic intervention based on properties of an individual cancer patient's tumor resistance or sensitivity to DNA damaging agents and other chemotherapeutic treatment modalities, where treatment choices can be made prior to initiation of treatment based on mechanisms of resistance specific for DNA damaging agents and mediated by kinesin heavy chain gene over- or underexpression. Particularly useful in this aspect of the invention are kinesin-specific I I-- Ib I F WO 95/18857 PCTIUS95/00092 15 antibodies, such as the anti-kinesin heavy chain antibodies described in Navone, et al., supra, for detection of kinesin expression levels in tumor samples.

In a seventh aspect, the invention provides a starting point for the rational design of pharmaceutical products that can counteract resistance by tumor cells to chemotherapeutic drugs.

Understanding the biochemical function of the kinesin genes that are involved in drug sensitivity is likely to suggest pharmaceutical means to stimulate or mimic the function of such genes and thus augment the cytotoxic response to anticancer drugs. One may also be able to up-modulate gene expression at the level of transcription. This can be done by cloning the promoter region of each of the corresponding kinesin genes and analyzing the promoter sequences for the presence of cis elements known to provide the response to specific biological stimulators. Due to the structure of the kinesins in eukaryotic cells, comprised of both kinesin heavy chain and kinesin light chain proteins, coordinate up-regulation of the expression of both of the appropriate kinesin light chain and heavy chain proteins would be required for efficacious therapeutic intervention based on modulating expression of kinesin genes.

Alternatively, kinesin expression in a cancer cell can be increased by cointroduction of recombinant expression constructs encoding functional, full-length copies of a kinesin heavy chain and a kinesin light chain, whereby coordinate coexpression of such exogenous kinesins would result in increased expression of functional kinesin molecules in the cancer cells.

The protein structure deduced from the cDNA sequence can also be used for computer-assisted drug design, to develop new drugs that affect this protein in the same manner as the known anticancer drugs. The purified protein, produced in a convenient expression system, can also be used as the critical component of in vitro biochemical screen systems for new compounds with anticancer activity.

Accordingly, mammalian cells that express chemotherapeutic drug resistanceconferring GSEs according to the invention are useful for screening compounds for the ability to overcome drug resistance. As with pharmaceutical intervention methods, both kinesin light chains and heavy chains should be present in such in I '-I r WO 95/18857 PCT/US95/00092 16vitro screening systems in amounts capable of reconstituting mature kinesin molecules in vitro.

Specific preferred embodiments of the present invention will become evident from the following more detailed description of certain preferred embodimerns and the claims.

EXAMPLE 1 Generation Of a Normalized Random Fragment cDNA Library In A Retroviral Vector A normalized cDNA population was prepared using a modification of the protocol of Patanjali et al. (1991, Proc. Natl. Acad. Sci. USA 88: 1943-1947), illustrated in Figure 1A and lB. Poly(A) RNA was extracted from NIH 3T3 cells.

To obtain mRNAs for different genes expressed at various stages of the cell growth, one half of the RNA was isolated from a rapidly growing culture and the other half from quiescent cells that had reached complete monolayer confluence. To avoid overrepresentation of the 5'-end sequences in a randomly primed cDNA population, RNA was fragmented by boiling to an average size range of 600-1,000 nucleotides.

These RNA fragments were .then used for preparing randomly primed doublestranded cDNA. This randomly primed cDNA was then ligated to a synthetic adaptor providing ATG codons in all three possible reading frames and in a proper context for translation initiation. The structure of the adaptor (see Figure 2) determined its ligation to the blunt-ended fragments of the cDNA in such a way that each fragment started from initiation codons independently from its orientation. The adaptor was not supplied with termination codons in the opposite strand since the cloning vector pLNCX, contained such codons immediately downstream of the cloning site. (This vector has been described by Miller and Rosman, 1989, Biotechniques 7: 980-986.) The ligated mixture was amplified by PCR, using the "sense" strand of the adaptor as a PCR primer, (in contrast to the method of Patanjali et al., which utilized cloning the initial cDNA preparation into a phage vector and then using vector-derived sequences as PCR primers to amplify the cDNA population.) The PCRs were carried out in 12 separate reactions that were subsequently combined, to minimize random over- or under-amplification of specific sequences and to increase the yield of the product. The PCR-amplified mixtures was I I I- c -I WO 95/18857 PCT/US95/00092 17size-fractionated by gel electrophoresis, and 200-500 bp fragments were selected for subsequent manipulations, (in contrast to Patanjali's fragment size range of from 400 to 1,600 bp.) For normalization, the cDNA preparation was denatured and reannealed, using different time points for reannealing, as described by Patanjali et al., supra, and shown in Figure 1A. The single-stranded and double-stranded DNAs from each reannealed mixture were separated by hydroxyapatite chromatography. The singlestranded DNA fractions from each time point of reannealing were PCR-amplified using the adaptor-derived primer and analyzed by Southern hybridization for the relative abundance of different mRNA sequences. The fraction that contained similar proportions of tubulin, c-myc and c-fos cDNA sequences (see Figures 1A and 1B), corresponding to high-, medium- and low-expressed genes, respectively, was used for the library preparation.

The normalized cDNA preparation was cloned into a ClaI site of the MoMLV-based retroviral vector pLNCX, which carries the neo (G418 resistance) gene, transcribed from the promoter contained in the retroviral long terminal repeat (LTR), and which expresses the inserted sequence from a strong promoter of the cytomegalovirus (CMV) (see Figure The ligation mixture, divided into five portions, was used for five subsequent large-scale transformations of E. coli. The transformed bacteria were plated on the total of 500 agar plates (150 mm in diameter) and the plasmid population (18 mg total) was isolated from the colonies washed off the agar. A total of approximately 5 x 107 clones were obtained, more than 60% of which carried the inserts of normalized cDNA, as estimated by PCR amplification of inserts from 50 randomly picked colonies. These results demonstrate the feasibility of generating a normalized cDNA library of as many as 3 x 107 recombinant clones in a retroviral plasmid expression vector.

EXAMPLE 2 Transduction Of A Retroviral Random Fragment Library Into Virus-Packaging Cell Lines And NIH 3T3 Cells The plasmid library prepared according to Example 1 was converted into a mixture of retroviral particles by transfection into virus-packaging cells (derivatives of NIH 3T3) that express retroviral virion proteins. (Examples of such cell lines have i--I 1 I WO 95/18857 PCTIUS95/00092 18been described by Markowitz et al., 1988, Virology 167: 400-406.) Ecotropic and amphotropic virus-packaging cell lines, GP+ E86 and GP+envAml2, respectively, were mixed at a 1:1 ratio and 107 cells of this mixture were transfected with the plasmid library under standard calcium phosphate coprecipitation conditions. This transfection resulted in the packaging and secretion of ecotropic and amphotropic virus particles, which rapidly spread through the packaging cell population, since ecotropic viruses are capable of infecting amphotropic packaging cells and vice versa. The yield of the virus, as measured by the number of G418-resistant colonies obtained after the infection of NIH 3T3 cells, reached 105 infectious units per 1 mL of media during the stage of transient transfection (1-3 days), then decreased (4-8 days) and then rapidly increased due to the expression of proviral genomes that became stably integrated in most of the packaging cells. The yield of the virus 9-12 days after transfection reached 10 6 per 1 mL of media supernatant. At this stage, the library showed fairly even representation of different fragments, but at later stages individual virus-producing clones began to predominate in the population, leading to uneven representation of cDNA-derived inserts. The uniformity of sequence representation in the retroviral population was monitored by rapid extraction of DNA from cells infected with the virus-containing supernatant, followed by PCR amplification of inserts. The inserts were analyzed first by the production of a continuous smear in ethidium bromide-stained agarose gel and then by Southern hybridization with different probes, including topoisomerase II, c-myc and tubulin.

As long as each gene was represented by a smear of multiple fragments, the representativity of the library was considered to be satisfactory.

In other experiments, for transducing the random-fragment normalized cDNA library into NIH 3T3 cells, without loss of representativity, NIH 3T3 cells were infected either with a virus produced at the transient stage of transfection (days 1-3), or with the high-titer virus collected 10-12 days after transfection. In the latter case, 100 ml of viral suspension contained more than 108 infectious units. In the case of the "transient" virus, NIH 3T3 cells were infected with at least 107 recombinant retroviruses by using 500 ml of media from virus-producing cells (five rounds of infection, 100 ml of media in each). These results demonstrate the feasibility of IWIP~I r WO 95/18857 PCT/US95/00092 19converting a large and complex random fragment library into retroviral form and delivering it to a non-packaging cell line without loss of complexity.

EXAMPLE 3 Isolation of GSEs Conferring Resistance To The Chemotherapeutic Drug Etoposide The overall scheme for the selection of GSEs conferring etoposide resistance is illustrated in Figure 3. This selection was carried out directly on virus-producing packaging cells, in the expectation that cells whose resistant phenotype is caused by the GSE expression will produce virus particles carrying such a GSE. The mixture of amphotropic and ecotropic packaging cells was transfected with the cDNA library in the LNCX vector, prepared according to Example 1, and the virus was allowed to spread through the population for 9 days. Analysis of a small part of the population for G418 resistance showed that practically 100% of the cells carried the neo-containing provirus. The cells were then exposed to 350ng/mL etoposide for days and then allowed to grow without drug for two more weeks. No difference was observed between the numbers of colonies obtained in the experiment and in the control (uninfected cells or cells infected with the insert-free LNCX virus) after etoposide selection. The virus present in the media supernatant of the surviving cells was then used to infect NIH 3T3 cells followed by etoposide selection using essentially the same protocol. NIH 3T3 cells infected with the library-derived virus produced by packaging cells that were selected with etoposide showed a major increase in the number of etoposide-resistant cells relative to the control cells infected with the insert-free LNCX virus, indicating the presence of biologically active GSEs in the preselected virus population (see Figure 4A).

The proviral inserts contained in the etoposide-selected NIH 3T3 cells were analyzed by PCR. This analysis (see Figure 4B) showed an enrichment for specific fragments, relative to the unselected population of the infected cells. Individual PCR-amplified fragments wk re recloned into the LNCX vector in the same position and orientation as in the original plasmid, as illustrated in Figure 4C. A total of 42 proviral inserts, enriched after etoposide selection, were thus recloned, and tested either in batches or individually for the ability to confer increased etoposide resistance after retroviral transduction into NIH 3T3 cells. Three non-identical 1 WO 95/18857 PCT/US95/00092 clones were found to induce etoposide resistance, indicating that they contained biologically active GSEs. These GSEs were named anti-khcs, VPA and VP9-11.

Etoposide resistance induced by the clone named anti-khcs is illustrated in Figure The ability of one of the anti-khcs GSE to induce etoposide resistance was further documented by using the isopropyl g-D-thiogalactopyranoside (IPTG)inducible promoter/activator system, as described by Bairn et al. (1991, Proc. Natl.

Acad. Sci. USA 88: 5072-5076). The components of this system include an enhancer-dependent promoter, combined in cis with multiple repeats of the bacterial lac operator, and a gene expressing LAP265, an artificial regulatory protein derived from the lac repressor and a mammalian transcriptional activator. The anti-khcs GSE was cloned into the plasmid pX6.CLN, which contains the inducible promotor used by Baim et al., supra (a gift of Dr. T. Shenk) which expresses the inserts from an enhancerless SV40 early gene promoter supplemented with 21 repeats of the lac operator sequence. The resulting plasmid, which contains no selectable markers, was co-transfected into NIH 3T3 cells together with the LNCX plasmid carrying the neo gene. The mass population of G418-selected transfectants, along with control cells transfected with the insert-free vector, was exposed to increasing concentrations of etoposide, in the presence or in the absence of 5 mM IPTG. Even though the cotransfection protocol usually leads to the integration of the GSE in only a fraction of the G418-resistant cells, transfection with anti-khcs resulted in a clearly increased etoposide resistance, which was dependent on IPTG (see Figure EXAMPLE 4 Sequence Analysis of GSEs Conferring Resistance To The Chemotherapeutic Drug Etoposide The GSE anti-khcs, cloned as described in Example 3, was sequenced by the standard dideoxy sequencing procedure, and the deduced sequence is shown in Figure 6. The nucleotide sequence of the "sense" and "antisense" strands, as well as amino acid sequence of the predicted peptides encoded by each of these strands, were analyzed for homology to the nucleic acid and protein sequences present in the National Center for Biotechnology Information data base, using the BLAST network program for homology search. The sequence corresponding to the "antisense" strand I Ir Bb~ II S W 95118857 PCT/US95/00092 -21 of the anti-khcs GSE, showed strong homology with several genes encoding the heavy chain of kinesins, a family of microtubule motor proteins involved in intracellular movement of organelles or macromolecules along the microtubules of eukaryotic cells. The highest homology was found with the human kinesin heavy chain (KHC) gene, as described by Navone et al. (1992, J. Cell Biol. 117: 1263- 1275). Anti-khcs therefore encodes antisense RNA for a mouse khc gene, which we have termed khcs for khc associated with sensitivity (to drugs) or senescence. We refer to the kinesin molecule, formed by the associate of the KHCS protein with kinesin light chains, as kinesin-S, to distinguish it from the other kinesins present in mammalian cells. These results demonstrate that chemotherapeutic drug selection for GSEs can lead to the discovery of novel genetic elements, and can also reveal roles of genes in drug sensitivity that had never before been suspected.

EXAMPLE Cloning And Analysis Of The Gene From Which Anti-khcs GSE Gene Was Derived The anti-khcs GSE isolated in Example 3 was used as a probe to screen 400,000 clones from each of two cDNA libraries in the lambda gtl0 vector. These libraries were prepared by conventional procedures from the RNA of mouse BALB/c 3T3 cells, either unsynchronized or at Go G, transition, as described by Lau and Nathans (1985, EMBO J. 4: 3145-3151 and 1987, Proc. Natl. Acad. Sci. USA 84: 1182-1186, a gift of Dr. L. Lau). Screening of the first library yielded no hybridizing clones, but two different clones from the second library were found to contain anti-khcs sequences. These clones were purified and sequenced. Sequence analysis showed that we have isolated the bulk of the mouse khcs cDNA, corresponding to 796 codons (the full-length human KHC cDNA encodes 963 amino acids). This sequence is shown in Figure 7; an additional 252 nucleotides encoding 84 amino acids from the amino terminus have been determined from cDNA isolated using the "anchored PCR" technique, as described by Ohara et al.

(1989, Proc. Natl. Acad. Sci. USA 86: 5763-5677.) Additional missing 3' terminal se.~ences are currently being isolated using this technique.

I la~ 111' -I WO 95/18857 PCT/US95/00092 -22- The dot-matrix alignment of the sequenced portion of the khcs protein with previously cloned KHC proteins from the human (see Navone et al., 1992, J. Cell.

Biol. 117: 1263-1275), mouse (see Kato, 1991, J. Neurosci. 2: 704-711), and Drosophila (McDonald Goldstein, 1990, Cell 61: 991-1000) is shown in Figures 8A through 8C; homology to GSE-C is shown as a control (Figure 8D). The portion corresponding to the anti-khcs GSE, is shown in brackets. The khcs gene is most highly homologous to the human gene (97% amino acid identity), suggesting that the human KHC (KHCS) gene is functionally equivalent to the mouse khcs. The alignment also shows that the anti-khcs GSE corresponds to the region which is the most highly diverged between different kinesins (shown in the Figure by brackets around these sequences.) EXAMPLE 6 Generation of a Random Fragment KHCS cDNA Retroviral Library and Isolation of KHCS-derived GSEs As described in Example 5 above, the murine khcs gene is highly homologous to the human KHC (or KHCS) gene described by Navone et al. (1992, J. Cell Biol.

117: 1263-1275). The functional equivalence of these genes was also suggested by the observation that the levels of KHCS mRNA are decreased in human cells selected for etoposide resistance (see Example To determine conclusively that the human KHCS gene represents the functional equivalent of the mouse khcs, it was determined whether any random fragment of human KHCS cDNA could function as an etoposide-resistance GSE.

A library of random DNAaseI-generated fragments of a full-length human KHCS cDNA (2.9 kb in length; provided by Dr. R.Vale, University of California at San Francisco) was generated essentially as described above for topoisomerase II cDNA (see Example 1 in co-pending U.S. Patent Application Serial No. 08/033,086, incorporated by reference), using the protocol illustrated in Figure 9A, with the following modifications. Specifically, two synthetic adaptors, instead of one, were used for ligation with DNAase I-generated cDNA fragments. One adaptor, containing three ATG codons, carried a HindIII cloning site (Figure 9B). The other adaptor had translation stop codons in all three reading frames and carried a ClaI i C WO 95/18857 PCTIUS95/90092 -23 cloning site (Figure 9B). After ligation with the equimolar mixture of both adaptors, cDNA fragments were amplified by PCR using sense and antisense strands of the first and second adaptor, respectively. PCR products were digested with ClaI and HindIII and cloned into the corresponding sites of the pLNCX plasmid. This modification of the cloning strategy resulted in avoiding the formation of inverted repeats at the ends of the cDNA inserts after cloning into the retroviral vector.

A plasmid library of 20,000 independent insert-carrying clones was obtained and transfected into ecotropic packaging cells using the calcium phosphate precipitation technique. Virus released by transiently transfected cells was used to infect HT1080/pJET-2TGH cells, clone 2, a derivative of human HT1080 fibrosarcoma cell line transfected with a plasmid expressing the murine ecotropic receptor (Albritton et al., 1989, Cell 57: 659-666) and susceptible to infection with ecotropic retroviruses (provided by Dr. G.R. Stark, Cleveland Clinic Foundation).

After infection and G418 selection, these cells (further referred to as HT1080/ER) were plated at 10' cells per 100 mm plate and cultivated for 12 days in different concentrations of etoposide (200-500 ng/mL). After removal of the drug, cells were allowed to grow in media without drug for 7 more days. At this point, some of the plates were fixed and stained with crystal violet, to determine the number of surviving colonies (Figure 10). As illustrated in Figure 10, a T :lrug concentrations of 250 ng/mL etoposide, there were only several colonies in control plates, compared with about a hundred times more colonies in the plates containing GSE-containing cells.

In a parallel experiment, virus-producing mixtures of packaging cells were subjected to similar etoposide selection. At all drug concentrations tested, there were many more colonies surviving etoposide treatment in the GSE-carrying cells than. in the control cell population.

These results indicated that the retroviral library of random fragments of KHCS cDNA contained numerous GSEs inducing drug resistance in human cells, confirming that human KHCS is associated with drug resistance. Some of these GSEs are likely to be more potent as selectable markers of drug resistance that the original single GSE from the murine khcs gene. The virus isolated from such etoposide-resistant cells represents a collection of a multiplicity of kinesin-derived,

I

PIBI~A WO 95/18857 PCT/US95/00092 -24drug resistance-conferring GSEs, which multiplicity is itself an aspect of the present invention and is useful in conferring risistance to DNA damaging agents, including chemotherapeutic drugs, as disclosed herein.

EXAMPLE 7 Generation of Cell Populations Carrying Multiple Copies of anti-khcs GSEs A 1:1 mixture of ecotropic and amphotropic packaging cells was transfected with retroviral vector pLNCX carrying the anti-khcs GSE using a standard calcium phosphate procedure. Two weeks later, the virus titer, as measured by the formation of G418-resistant NIH 3T3 colonies, reached 106 infectious units per mL as a result of "ping-pong" infection (see Bodine et al., 1990, Proc. Natl. Acad. Sci. USA 87: 3738-3742). This virus-containing supernatant was used to infect NTH 3T3 cells, times with 12 hour intervals. Control cells were infected in parallel with the insert-free vector virus obtained by the same procedure. G418 selection showed that 100% of NIH 3T3 cells became infected with the virus. DNA from the infected cells was analyzed by Southern blot hybridization with a virus-specific probe. This analysis showed that the infected cells contained multiple copies of the integrated provirus.

Freshly-obtained multiply-infected NIH 3T3 cells were characterized by a decreased growth rate and plating efficiency. After several passages, however, their growt. parameters became indistinguishable from the control cells, suggesting the elimination of slowly growing cells from the population. At this stage, the cells were frozen and used for the experiments described below.

EXAMPLE 8 Drug Resistance Pattern of NIH 3T3 Cells Carrying Multiple Copies of Anti-khcs GSEs The infected cell populations described in Example 7 were analyzed for resistance to several anticancer drugs by a growth inhibition assay. For this assay, 104 cells per well were plated in 12-well plates and exposed to increasing concentrations of different drugs for 4 days. Relative cell numbers were measured by the methylene blue staining assay (Perry et al., 1992, Mutation Res. 276: 189- 197). Despite the relative insensitivity of this assay when carried out with unselected

II

ICI ~auL- WO 95/18857 'CTIUS95/00092 heterogeneous cell populations, infection with the virus carrying an anti-khcs GSE induced a pronounced increase in the resistance to the cytostatic effects of etoposide and amsacrine and, to a lesser extent, of Adriamycin, camptotbhcin and cisplatin (Figure 11). All of these drugs are known to induce DNA damage, albeit by different mechanisms. Under the same; assay conditions, no increase in resistance was observed with colchicine or actinomycin D (Figure 11).

To further characterize the nature of drug resistance conferred by anti-khcs, the above-described short-term growth inhibition assays were followed by long-term growth inhibition assays which measured both the cytostatic and the cytotoxic effects of different drugs. These assays were carried out by incubating the cells for four days in the presence of the drugs, followed by either two or six days in the absence of the drug, to allow for programmed cell death, which is frequently associated with recovery from drug-induced inhibition (Kung et al., 1990, Cancer Res. 50: 7307- 7317). These assays showed that the GSE-carrying cells were resistant to etoposide and adriamycin, but not to cisplatin, camptothecin or actinomycin D. Furthermore, the GSE-carrying cells were found to be hypersensitive to colchicine and vinblastine, said hypersensitivity being increasingly more evident with increasing length of the assay.

Thus, in the experiment shown in Figure 12, NIH 3T3 cells carrying the antikhcs GSE and control cells without the GSE were plated at a density of 2 x 10' per culture dish and grown for five days in concentrations of either colchicine or vinblastine. The cells were then fixed and stained, and the number of surviving cells determined and shown as a percentage of cell growth in the absence of drug. These results clearly show that expression of the anti-khcs GSE in these cells was accompanied by hypersensitivity to both colchicine and vinblastine.

To further investigate the discrepancy between the results obtained in the short-term and long-term drug assays, analyses of the dynamics of cell growth during and after treatment with 250 ng/mL etoposide, 20 ng/mL camptothecin and ng/mL colchicine were performed. In these experiments (shown in Figure 13), NIH 3T3 cells were treated with the corresponding drugs for 5 days, and then incubated for additional 6 days either in the presence or in the absence of the drug. The selected drug concentrations resulted in growth inhibition but little detectable cell I ~I I WO 95/18857 PCT/US95/00092 26 death in continuous presence of the drug. After 11 days of drug exposure the total cell number in the etoposide- or camptothecin-treated populations of control cells was a little lower than after 5 days of drug exposure, but in the colchicine-treated control cell population a slow cell growth was still detectable. In the experiments where the drug was removed after 5 days, cell growth was initially activated for all three drugs. After about two days of growth in the absence of the drug, the number of cells treated with etoposide or camptothecin decreased, however, due to extensive cell death, so that the final cell number in cell populations incubated in the absence of the drugs was practically the same as in the cells continuously maintained in the presence of colchicine or camptothecin. In contrast, cells pre-treated with colchicine undergo only limited cell death after removal from the drug, resulting in a relatively minor slowdown in cell growth two days after removal from the drug, and a major increase in the total cell number in the populations removed from the drug relative to those that were constantly maintained in colchicine (Figure 13).

The expression of the anti-khcs GSE had different effects on the growth inhibition and recovery-associated cell death induced by these drugs. In cells treated with etoposide, anti-khcs decreased the growth-inhibitory effect of the drug to the same, relatively small extent after 5 or 11 days of continuous exposure. In addition, the anti-khcs GSE decreased the amount of cell death in the populations released from etoposide inhibition. As a result, the increase in etoposide resistance conferred by this GSE was much more pronounced after release from the drug than under the conditions of continuous exposure (Figure 13). In cells treated with camptothecin, the GSE resulted in a small decrease of the growth-inhibitory effect of the drug, which was more pronounced after 5 days of exposure but was reduced to negligible levels after 11 days of continuous exposure. The decrease in growth inhibition after days of exposure was accompanied by a slight increase in cell death after the removal from the drug, so that the GSE produced no significant long-term difference in camptothecin resistance. In the populations treated with colchicine, the anti-khcs GSE made cells more susceptible to the growth-inhibitory effect of the drug; this effect was increased with prolonged exposure and was equally apparent in the populations released from the drug after 5 days or continuously maintained in colchicine (Figure 13).

I r- __CIC~ WO 95/18857 PCT/US95/00092 -27- The results of the above experiments indicated that the anti-khcs GSE acts by inhibiting the cytostatic effects of different DNA-damaging drugs. In addition, this GSE appears to decrease the extent of programmed cell death occurring after the release from the drug in cells treated with some (etoposide) but not other (camptothecin) DNA-damaging agents.

The finding that cells carrying the anti-khcs GSE become hypersensitive to the cytostatic effects of colchicine has potentially significant therapeutic implications.

The observed hypersensitivity to colchicine, an anti-microtubular agent, in cells with GSE-mediated inhibition of kinesin is likely to be mechanistically related to the essential functions of kinesin, which moves various structures along the microtubules and may be involved in the sliding of microtubules relative to each other, as well as the assembly and disassembly of microtubules. The pronounced effect of the antikhcs GSE on the sensitivity of cells to anti-microtubule agents also indicates that this GSE affects the general kinesin function in the cells, and is not limited to a particular drug-response-specific isoform of kinesin. Since we have demonstrated that downregulation of the KHCS gene represents a natural mechanism of drug resistance in human tumor cells (see Example 11), the hypersensitivity to colchicine provides an approach to overcoming this type of resistance in human cancer.

EXAMPLE 9 Assessment of Cellular Effects Of Anti-kinesin GSEs The virus carrying the anti-khcs GSE was tested for the ability to increase the life span of primary mouse embryo fibroblasts (MEF). MEF were prepared form day old mouse embryos by a standard trypsinization procedure and senescent cells were frozen at different passages prior to crisis. Senescent MEF, two weeks before crisis, were infected with recombinant retroviruses carrying LNCX vector either without an insert or with anti-khcs. Figure 14 shows MEF cell colonies two weeks after crisis. Relative to uninfected MEF cells, or cells infected with a control LN A, virus, cells infected with the anti-khcs showed a great increase in the proportion of cells surviving the crisis. Post-crisis cells infected with the anti-khcs virus showed no microscopically visible features of neoplastic transformation. These results indicate that anti-khcs promotes the immortalization of normal senescent fibroblasts.

LII--L- _1I._II IIIIC~- WO 95/18857 PCT/US95/00092 -28- These results suggest that the normal function of kinesin-S may be associated with the induction of programmed cell death occurring after exposure to certain cytotoxic drugs or in the course of cellular senescence. These results also indicate that isolation of GSEs that confer resistance to chemotherapeutic drugs can provide insight into the cellular genes and processes involved in cell growth regulation.

EXAMPLE Biological Effects of Mouse Anti-khcs GSE on Human Senescent Fibroblasts The ability of the anti-khcs GSE described in Example 4 to promote immortalization of primary mouse embryo fibroblasts (demonstrated in Example 9) suggested that kinesin-S may act as a tumor suppressor by preventing immortalization of normal mouse fibroblasts. To determine if this gene may play the same role in human cells, the ability of the anti-khcs GSE to affect the life span of primary human fibroblasts was investigated.

Primary human fibroblasts, derived from human skin, were obtained from the Aging Cell Repository of the National Institutes of Aging. The cells were grown in DMEM with 20% fetal calf serum supplemented with twice the concentration of amino acids and vitamins normally used to supplement normal human skin fibroblast growth in culture. Cells at the fifth passage were infected with either the control pLNCX virus or the virus carrying mouse anti-khcs GSE (produced as described above in Example Four passages later, the control cells went into crisis, but GSE-carrying cells continued to grow (Figure 15) and have so far survived at least five additional passages.. These results demonstrated that the anti-khcs GSE of this invention is also capable of prolonging life span of primary human fibroblasts, indicating that KHCS is a potential tumor suppressor in human cells.

EXAMPLE 11 Assessment Of The Role Of Decreased khcs Gene Expression In Naturally Occurring Mechanisms Of Drug Resistance To test whether decreased khcs gene expression is associated with any naturally occurring mechanisms of drug resistance, an assay was developed for measuring khcs mRNA levels by cDNA-PCR. This assay is a modification of the IllaBaPcb~---~

~II

WO 95/18857 PCTIUS95/00092 -29quantitative assay described by Noonan et al. (1990, Proc. Natl. Acad. Sci. USA 87: 7160-7164) for determining mdr-1 gene expression. The oligonucleotide primers had the sequences: AGTGGCTGGAAAACGAGCTA (SEQ.ID.No.:5) and CTTGATCCCTTCTGGTTGAT (SEQ.ID.No.:6).

These primers were used to amplify a 327 bp segment of mouse khcs cDNA, corresponding to the anti-khcs GSE. These primers efficiently amplified the mouse cDNA template but not the genomic DNA, indicating that they spanned at least one intron in the genomic DNA. Usir~ these primers, we determined that khcs mRNA is expressed at a higher level in the mouse muscle tissue than in the kidney, liver or spleen.

In another experiment a pair of primers amplifying a homologous segment of the human KHCS cDNA was selected, based on the reported human KHC sequence published by Navone et al. (1992, J. Cell. Biol. 117: 1263-1275). The sequences of these primers are: AGTGGCTTGAAAATGAGCTC (SEQ.ID.No.:7) and CTTGATCCCTTCTGGTAGATG (SEQ.ID.No.:8), and they amplify a 327 bp cDNA fragment. These primers were used to test for changes in the KHCS gene expression in several independently isolated populations of human HeLa cells, each selected for spontaneously acquired etoposide resistance;

P

2 -microglobulin cDNA sequences were amplified as an internal control. Figure 16 shows the results of the cDNA-PCR assay on the following populations: CX(0), HeLa population infected with the LNCX vector virus and selected with G418; CX (200), the same cells selected for resistance to 200 ng/ml etoposide; E11(O), and E21(0), populations obtained after infection of HeLa cells with recombinant retroviruses carrying different GSEs derived from topoisomerase a cDNA, as described in Example 1 of co-pending U.S. Patent Application Serial No.

08/033,086, incorporated by reference, and selected with G418:E11 (1000), 6(1000) and E21(1000), the same populations selected for resistance to 1 pg/ml etoposide.

As shown in Figure 16, the yield of the PCR product specific for the khcs gene was significantly lower in each of the etoposide-selected populations than in the control

I

FY9C I U~I' WO 95/18857 PCT/US95/00092 cells. This result indicates that a decrease in the khcs gene expression is a common natural mechanism for drug resistance.

EXAMPLE 12 Diagnostic Assay The results presented in the above Examples suggest the utility of diagnostic assays for determining the expression levels of kinesin genes in tumor cells of a cancer patient, relative to a standardized set of cell lines in vitro having wellcharacterized levels of kinesin heavy chain gene expression correlated with their level of resistance to certain chemotherapeutic drugs such as etoposide. One such standardized set of cell lines comprise the HeLa cell lines described in Example 11.

Alternatively, different, tissue-specific standardized sets of cell lines are developed by drug selection for each cell type to be evaluated, for example, using human K562 cells for evaluating patients having chronic myelogenous leukemia, or human cells for patients having acute promyelocytic leukemia.

The assay for kinesin would assess the appropriateness of treatment of human cancer patients with certain anticancer therapeutic regimens. Patients whose tumor cells under-express kinesin may be refractory to treatment with DNA damaging agents, including radiation and the chemotherapeutic drugs etoposide, camptothecin, cisplatin and adriamycin. Such patient, however, may be particularly responsive to treatment with anti-microtubular agents such as colchicine, colcemide, vinblastine, vincristine or vindesine. On the other hand, patients whose tumor cells over-express kinesin, for example, may be responsive to treatment with DNA damaging agents and refractory to treatment with anti-microtubular agents. These assays provide, for the first time, a basis for making such therapeutic judgments before the fact, rather than after a therapeutic regimen has been tried and failed. The assay also provide a basis for determining which patients, previously refractory to treatment with DNA damaging agents, particularly certain anticancer drugs, would benefit from further chemotherapy using anti-microtubular agents, by distinguishing kinesin genemediated drug resistance from other mechanisms of drug resistance expected to result in cross-resistance to both DNA damaging agents and anti-microtubular drugs.

I WO 95/18857 PCT/US95/00092 -31 It should be understood that the foregoing disclosure emphasizes certain specific embodiments of the invention and that all modifications or alternatives equivalent thereto are within the spirit and scope of the invention as set forth in the appended claims.

I WO 95/18857 PCT/US95/00092 32 SEQUENCE LISTING GENERAL INFORMATION:

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(ii) TITLE OF INVENTION: Association of Kinesin with Sensitivity To Chemotherapeutic Drugs (iii) NUMBER OF SEQUENCES: 8 (iv) COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (EPO) CURRENT APPLICATION DATA: APPLICATION NUMBER: INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: AATCATCGAT GGATGGATGG INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: I a C~ILP91P- II-- C WO 95/18857 PTIUS9/00092 33 CCATCCATCC ATCGATGATT AAA INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 327 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

CTTGATCCCT

TCTCTACCAA

ATTTAGCAAG

CGACTGCAGC

CCAAATTAGC

AACGGTTTAG

TCTGGTTGAT

TTGGCTTTGT

TTCTTCTTCA

AGCTGGTTTA

TTTCTCTTTG

CTCGTTTTCC

GCCAGAAGCT CTTCCTGATC TGGTTAATCT CTTCATCCTT CACTTTCTTC TTTCAGCATC TCACTGGTAA TAGCAATATC TCAAACTGCT CATCAATAGG

AGCCACT

CAGCATTTGT

GTCATCAAGT

GGTAAAACTA

TTTATCCGCT

CACTGTCTCC

ATCTTCAATT

TGTTTATACA

CCAGCCATTC

GTGAAGGCTT

CCGTTACGCC

120 180 240 300 327 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 2389 base pairs TYPE: nucleic acid STRANDEDNESS: sing.e TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

CGACAAACAT

ATGGGAATTA

AATTTGGAAT

TTGTTAGATG

GTAAAGGGGT

GAAGGGAAAT

CACAGCATAT

GGAAAGCTTT

GGTGCTGTGC

CATCTGGGAA GACCCACACG ATGGAGGGTA TTCCAAGAAT AGTGCAAGAT ATTTTTAATT TTCATATTAA GGTTTCATAT TTTGAAATAT TTTCAAAGAC TAACCTTTCA GTCCATGAAG GCACAGAACG TTTCGTGTGT AGTCCAGATG CCAACAGAGA TGTCGCAGTT ACAAATATGA TTCTTATTAA TGTAAAACAA GAGAATACAC ATCTGGTTGA TTTAGCTGGC AGTGAGAAGG TGGATGAAGC TAAGAACATC AAGAAGTCAC

AACTTCATGA

ATATTTACTC

ATTTGGATAA

ACAAAAACCG

AAGTCATGGA

ATGAACATAG

AAACGGAACA

TTAGTAAGAC

TTTCTGCACT

TCCAGAAGGC

CATGGATGAA

GATAAGGGAC

TGTTCCCTAT

TACCATAGAT

CTCTAGGAGC

GAAACTCAGT

TGGGGCTGAA

TGGAAATGTC

120 180 240 300 360 420 480 540 i, Il I PCTIUS95/00092 WO 95/18857 34

ATTTCTGCTT

ATTCTTCAAG

TCATCATACA

ATTAAGAACA

GAAAAAGAAA

CTAAACCGTT

GCTAATTTGG

GCTGCAGTCG

CTTGCTAAAT

TTGGTAGAGA

AGGGATCAAG

AAAGAAG: .G

AAGTCTCAGG

CAAAAATCTG

AACCACCAGA

ATAGGAATTG

GAAGAGTTTA

GTGAAACGCT

AATGAGAAAG

TCACTGACTG

GATTCCCTTG

AAAGAGCACT

ATCCAGAGTC

GCAAAGGAAA

GAACGGCTAA

CTGCATGAGC

TTGGAGGAGA

CAGGACTTGG

GTGCTGCACA

TGCACAAGCA

AACGGCTTAG

TGGCAGAGGG

ATTCATTAGG

ATGAGTCTGA

CAGTCTGTGT

AGGAAAJAAA

GGCGTAACGG

AAGCCTTCAC

GAATGGCTGG

TGTATAAACA

AATTGAAGAC

ATAATATGCA

TCAAAGAAGT

AAGTTGAAGA

CAACTTTAGC

AGAAACGAGC

CTGTGGGGAA

CTGTTGCAAG

GCAAACAGCT

AGTTAGCAGC

AGTACCTTCA

GTGAGGAGCT

TGAACAAGGT

ACAGAGAAAC

AGCTAATCAC

GGGTGGAGCA

TCACGGTTAT

CCGTGGCAAA

CTACCAGGGT

GAAGCAGAAA

GTTGGTACGT

AGCTACTGCA

CAGTACCTAT

TGGCAACTGT

GACAAAGTCA

CAATGTAGAG

TAAGACTCTA

GGAGACAGTG

AGCGGATAAA

TAGTTTTACC

GCTTGATGAC

ACAAATGCTG

AGCTGAACTG

TTTACAGGCC

CAAAACAAAG

AAGTATTGAT

AGCTGAAATG

TAACGATGTG

ACTCTACATT

AGAAAGCACG

ATGCCAGCTT

GAATGTAGAA

AGTCCAACTC

TCAGACTGCA

CCACCAAAAA

TGACCTCCAA

TGAGAGGCTG

GCAAGACAGA

AGAACTTCAG

GATAAAAGAGG

ATCTCCTTCC

GATAATGCAG

GAAAGAGTGA

GTTCCTTATC

AGGACCACTA

ACACTCCTCT

TTAACTGCAG

CGGAACACTA

CCTATTGATG

GATACTGCTA

GATGCTGAAA

AAGGATGAAG

GATCAGGAAG

AATCGCCTCC

TTAGAGGAAC

GAATATGAAT

GCTGAGCTTC

ATGGCATCAT

AAGCAACCAG

AGCAAAATGA

CAGACTGAGA

CGGATCTCC.C

CAAAAGAAGA

CGAGCACAAG

AATGAAGTCA

CAAATCAGTA

GACCAAAACC

AAGGCTACAG

CGAGAACAAG

ACTTTACACA

CCGAGGTCGA

TTGAAAACAA

ATCTTCGCTG

AAGCTTTGGA

GAGATAGTAA

TTGTCATATG

TTGGTC',>-,-

AGCAG'.AGAA

TTCAGTGGCT

AGCAGTTTGA

TTACCAGTGA

GAAGAAAGTG

AGATTAACCA

AGCTTCTGGC

AAGCAGAAAA

TGGCTGTTAA

TGCTTAGTGA

AGAAGCTGAA

TATTAAAAGA

AAGGAACTGG

AATCAGAAGT

GCAACAAAAA

AACATGAAGC

GGCAGCTGGA

AGAPIAGTCCA

AGCAAGCTGT

GCTTGCGAGA

AGAAGATGGT

ACCAAGAGAA

CAAGACAAGA

ACCTGCGTAA

CTCTGACGAC

CCTTGAACAG

TGAGCTTCCT

GTCAGCCCG

AATGACCAGA

CTGCTCTCCA

GGCCAAAACA

AAAGAAGTAT

GGAAAACGAG

CAAAGAGAAA

TAAACCAGCT

TGAAGAAGAA

ACAAAGCCAA

ATCAACCAGA

TGATGCTTCT

TTATGATCAG

TGAATTGAAT

GGAAATGACC

CCTTGCAGAA

TATGATAGAT

AAAGACCATG

AATGGAAGAA

CAAAATCAAG

GGAATCTGTT

TGAAATGGAA

TGAGCAGCAG

TGAAGTTGAG

GTTGGAGCAG

GA(GCAGGAAG

CTTGJAAGGGT

GCTCTTTGTT

ACTGGCGGCA

CTCACCAAAG

AAGTTAGAGA

600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2389 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid ~L -L C 1 WO 95/18857 PCT/US95/00092 35 STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID CTCCCAAGCT TATGGATGGA TG 22 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 25 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE CESCRIPTION: SEQ ID NO:6: CATCCATCCA TAAGCTTGGG AGAAA INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: TGAGTGAGTG AATCGATGAT TAAA 24 INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO WO 95/18857 PCT/US95/00092 36 (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: AATCATCGAT TCACTCACTC A 21

Claims (20)

1. A method of identifying genetic suppressor elements derived from a kinesin gene that confer upon a cell resistance to one or more DNA damaging agents, the method comprising the steps of: generating a set of random fragments of cDNA encoding the kinesin gene; transferring the DNA fragments to an expression vector to yield a library, wherein the expression vector is capable of expressing the DNA fragments in a living cell; genetically modifying living cells by introducing the random fragment library of step into the living cells; isolating or enriching for genetically modified living cells containing kinesin-derived genetic suppressor elements by selecting cells in the presence of a DNA damaging agent; and obtaining the genetic suppressor element from the geneticatly modified cells,

2. A genetic suppressor element identified by the method of ciatm 1.

3. A genetic suppressor element according to claim 2 having a nucleotide sequence that is homologous to a portion of the human or mouse khcs gene, 4, A mammalian cell that expresses a OSEl according to claim 2. A mammalian cell that expressea a GSE according to claim 3.

6. The method of Claim 1, wherein the genetic suppressor element is sense- oriented,

7. The method of Claim 1, wherein the genetic suppressor element is antisense-oriented, i I lr WO095/18857 PCTIUS9S/00092 38

8. A syndietic peptide having an amino acid sequence corresponding to from about 6 amino acids to all of the amino acid sequence encoced by the GSE produced according to the method of Claim 6.

9. A synthetic oligont~cleotide having a nucleotide sequence from about 12 nucleotides to all of thc nucleotide sequence of the antisense RNA encockod by the OSE produced by Claim 7. A diagnostic assay comprising thle steps of (it) Isolating cellular RNA comprlising inessnger RNA from) a tumnor or inaliguant cells froml aU animial;, measuring a level of expression of kinesin niRNA Ii thle tunm or malignait, cells In tile a,11blal; detormining whether the level of expression of kinesin m.RNA In flie tumior or inalignant cells indicates tha the kineshi Aene Is over- expressed or undiur'expressed inl the tumoor or malign81ant cells Inl the anlial It. A diagnostic assay comprising thue stops of (at) Isolating cellular protein fromn a tumor or mnalignant, cells from all allnial; mecasuring an amiount of a kiiuesln protein in thie tumlor or mnalignant cells In thle "Animal; dletermining whether th iount of thie kinesin Protoin Ini tile tumor or malignant tlsIndicates that tlie kinesin gene Is over-expressed or under-expressed In the tumor or malignant cells In tlid animial. 12, A mnethod of treating an animial having a malignant tumior or malignant cells in the animal's body, thie method comprising admiinistering ,,in anticancer drug that, is a DNA damlaging agent to thie animal if the tumor or malignant cells over- express a kinesi gene as detcrminted using (lie assay of Clailms 10 Or It. cr~ WO 95/18857 PCTUS95/00092 39

13. A method of treating an animal having a malignant tumor or malignant cells in the animal's body, the method comprising administering an anticancer drug that is an anti-microtubule agent to the animal if the tumor or malignant cells under- express a kinesin gene as determined using the assay of Claims 10 or 11.

14. A me2.n of treating an animal having a malignant tumor or malignant cells in the animal's body, the method comprising administering an anticancer drug that is an anti-microtubule agent to the animal if the tumor or malignant cells under- express a kinesin gene as determined using the assay of Claims 10 or 11 and the animal was previously unsuccessfully treated with an anticancer drug that is a DNA damaging agent. A method of identifying a multiplicity of genetic suppressor elements derived from a kinesin gene, that confer upon a cell resistance to one or more DNA damaging agents, the method comprising the steps of: generating a set of random fragments of cDNA encoding the kinesin gene; transferring the DNA fragments to a retroviral expression vector to yield a retroviral random fragment expression library, wherein the retroviral expression vector is capable of expressing the DNA fragments in a living cell; genetically modifying a culture of living cells by introducing the retroviral random fragment expression library of step into the living cells of the culture, wherein the living cells of the culture are capable of propagating each of the retroviral expression vectors comprising the library; isolating or enriching for genetically modified living cells containing kinesin-derived genetic suppressor elements by selecting cells in the presence of a DNA damaging agent; and allowing the drug-resistant, genetically modified living cells of step (d) to propagate the kinesin-derived genetic suppressor elements of the retroviral random fragment expression library to form an amplified rl ~BF Il ~(II II r-i-i; WO 95/18857 Vc'IU895/00092 40 random fragment expression library comprising a multiplicity of kinesin-derived genetic suppressor elements.

16. A genetic suppressor element first identified by the method of claim

17. A genetic suppressor element according to Claim 16 having a nucleotide sequence that is homologous to a portion of the human or mouse khcs gene.

18. A mammalian cell that expresses a GSE according to claim 16.

19. A mammalian cell that expresses a GSE according to claim 17. The method of Claim 15, wherein the genetic suppressor element is sense-oriented.

21. The method of Claim 15, wherein the genetic suppressor element is antisense-oriented.

22. A synthetic peptide having an amino acid sequence corresponding to from about 6 amino acids to all of the amino acid sequence encoded by the GSE produced according to the method of Claim 21.

23. A synthetic oligonucleotide having a nucleotide sequence from about 12 nucleotides to all of the nucleotide sequence of the antisense RNA encoded by the GSE produced by Claim 22. 41

24. A method of identifying genetic suppressor elements derived from a kinesin gene that confer upon a cell resistance to one or more DNA damaging agents, substantially as hereinbefore described with reference to any one of the Examples. A genetic suppressor element, substantially as hereinbefore described with reference to any one of the Examples.

26. A mammalian cell that expresses a GSE, substantially as hereinbefore described with reference to any one of the Examples.

27. A diagnostic assay, ,substantially as hereinbefore described with reference to any one of the Examples.

28. A method of identifying a multiplicity of genetic suppressor elements derived from a kinesin gene, that confer upon a cell resistance to one or more DNA damaging agents, substantially as hereinbefore described with reference to any one of the Examples. Dated 30 July, 1996 Board of Trustees of the University of Illinois Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON 0 S S **o [N:\LIBC]01390:MER M