EP0850066A1 - Methods for selectively killing or inhibiting the growth of cells expressing the waf1 gene - Google Patents

Abstract

Methods of killing or inhibiting the growth of cells are disclosed. The invention provides the administration of a WAF1 inhibitor to cells which have induced a WAF1-dependent pathway. The amount of a WAF1 inhibitor which is administered to the cells is sufficient to inhibit the growth thereof or even kill the cells. The method can include subjecting the cells to a treatment which induces a WAF1-dependent pathway.

Description

METHODS FOR SELECTIVELY KILLING OR INHIBITING THE GROWTH OF CELLS EXPRESSING THE WAFl GENE

Field ofthe Invention

The present invention relates to the selective killing or to the selective inhibition ofthe growth of cells. More particularly, the present invention relates to methods and compositions useful in killing or inhibiting the growth of cells expressing the WAFl gene. The present invention has particular utility in the field of cancer therapy.

Background ofthe Invention

In recent years, much progress has been made toward understanding the cell cycle of eukaryotic cells. In brief, the cell cycle consists of four phases: (1) the mitotic phase, M, in which a cell with duplicated genetic material undergoes mitosis to produce daughter cells, (2) a first gap phase, G„ during which the cell grows and is, generally, metabolically active, (3) a synthesis phase, S, during which the cell duplicates its genetic material, and (4) a second gap phase, G₂, in which the cell prepares for mitosis and, perhaps, another cell cycle. The control or regulation ofthe cell cycle is a complex process involving dozens of intracellular and extracellular signals which appear to act at particular "checkpoints" at the transitions between different stages ofthe cell cycle. Amongst the known components of cell cycle regulation are the families of cyclins, cyclin-dependent kinases (CDKs), and cyclin-dependent kinase inhibitors (CDIs). Although the details of cell cycle control remain to be resolved, it is clear that the expression or inhibition of these molecules, as well as their stoichiometries, play a major role in controlling cell proliferation, differentiation and death (see, e.g., Hunter and Pines (1994) Cell 78:539-542).

At the same time, it has become clear that many of these same regulatory components of the normal cell cycle also play a role in oncogenesis. See, e.g.. Hunter and Pines (1994) Cell 79:573-582. The p53 "tumor suppressor" gene product, for example, not only has been linked to G, arrest but p53 mutants have been linked to a variety of cancers (see, e.g., Vogelstein, B. and Kinzler, K.W. (1992) CeU 70:523-526; Fisher, D.E. (1994) CeU 78:539-542). This gene appears to play a role in the normal cellular response to DNA damage in mammalian cells such that, when DNA damage has occurred, the p53 gene is expressed and the cell either arrests at the G, stage ofthe cell cycle or enters the apoptotic cell death pathway. Heritable defects in p53 are believed to be causally involved with oncogenesis either by allowing cells with damaged DNA to replicate and pass on the damaged genetic material or by preventing damaged cells from undergoing apoptosis. See, e.g., Canman, et ai. (1995) Genes & Devel. 9:600-611. The p53 protein is now known to be a transcriptional regulator and several genes have been identified with putative p53-binding sites (El-Deiry et al. (1993) CeU 75:817-825).

WAFl is a CDI which appears to be involved in the arrest ofthe cell cycle at a checkpoint in G,. Because ofthe varying means by which it has been identified, this CDI has been known by a variety of names in the recent literature: WAFl (Wild-type p53 activated fragment 1; El-Deiry, et al. (1993) CeU 75:817-825), Cipl (CDK-interacting protein 1; Harper, et al. (1993) £eli 75:805-816), SDH (Senescent cell-derived inhibitor 1; Noda, et al. (1994) Exp. Cell Res. 211 : 90-98) and p21 (21,000 Da cyclin-dependent kinase inhibitor protem; see, e.g., Jiang, et al. (1995) Oncogene 10:1855-1864). For consistency, this CDI will hereinafter be referred to as WAFl irrespective ofthe nomenclature used in any references cited.

Harper et al. (1993) disclosed the nucleic acid and amino acid sequences of a human WAFl gene and protein. These researchers found that WAFl is a potent inhibitor of a variety of cyclin-CDK complexes and can inhibit the phosphorylation ofthe retinoblastoma gene product (Rb). Inhibition ofthe CDKs and hypophosphorylation of Rb can lead to cell cycle arrest in Gj. Consonant with this, Harper et al. found that WAFl caused a dose-dependent in vitro decrease in human fibroblasts in the S phase ofthe cell cycle (implying WAFl -dependent G, arrest) and conclude that loss of WAFl function might contribute to cell proliferation even in the presence of negative growth signals.

El-Deiry et al. (1993) also disclosed the nucleic acid and amino acid sequences of a human WAFl gene. They noted that the gene included an upstream p53-binding site and, consonant with this, found that WAFl was strongly induced both by p53 and by UV irradiation (which also induces p53). In addition, these authors found that WAFl expression inhibited the growth of a variety of human tumor lines in vitro. These results, and others, suggested that WAFl might be a downstream mediator of Gj arrest controlled by the p53 tumor suppressor. Curiously, the introduction of WAFl -antisense had no effect on cell growth. Hence, WAFl is not required for normal cell proliferation. Furthermore, in the absence of wild-type p53, induction of G, arrest by serum starvation or treatment with mimosine did not induce WAF 1. This suggested that WAFl expression might be one of a series of mechanisms by which cells carry out G, arrest. Despite these complications, these authors conclude that the "identification of WAFl and its regulatory region potentially provides a novel drug discovery approach: compounds that activate expression of WAFl might bypass the p53 defect in tumors with endogenous p53 mutation" (emphasis added).

Sheikh et al. investigated the regulation of WAFl expression in human breast carcinomas (Sheikh et al. (1994) Oncogene 9:3407-3415). Cells expressing wild-type p53 were found to constitutively express WAFl at levels 26-33 fold higher than p53 mutants. In addition, exogenous mutant p53 (Val-143) counteracted the positive transcriptional effect of endogenous wild-type p53. Nonetheless, these authors found that WAFl could be induced both by p53- independent as well as p53-dependent signaling pathways. In particular, they found that the DNA-damaging agent etoposide and serum starvation could induce both WAFl expression and growth arrest even in p53 mutant cell lines.

Recently, Jiang et al. (1995) found that WAFl is differentially expressed during growth, differentiation and progression in human melanoma cells. Using subtractive cDNA hybridizations and anti-p53 and anti- WAFl antibodies, these authors found (1) lower levels of WAFl expression in proliferating and metastatic human melanomas as compared to normal or immortalized melanocytes and (2) an increase in WAFl protein levels in melanoma cells after induction of growth arrest and terminal differentiation (using recombinant human fibroblast β- interferon (IFN-β) and mezerein to induce terminal differentiation). In addition, levels of p53 and WAFl were found to be inversely correlated during growth arrest and differentiation of human melanoma cells, indicating that WAF 1 induction may occur independent of p53 expression. In light of these and other results, Jiang et al. suggest that agents that can increase WAFl expression may prove beneficial in metastatic melanoma therapy by directly inducing an irreversible loss of proliferative capacity and terminal cell differentiation.

Using a WAFl -antisense expression vector, Nakanishi et al. investigated the effect of WAFl inhibition on normal human fibroblasts grown to G₀ arrest in vitro (Nakanishi et al. (1995) Proc. Natl. Acad. Sci. (TJSA^{^}) 92:4352-4356). These authors found higher levels of WAFl protein in cells arrested in G₀ than in mitogen-stimulated cells in early S phase. In addition, Nakanishi et al. found that expression of WAFl -antisense RNA caused cells arrested in G₀ to resume the cell cycle and proliferate. The role of WAF 1 in the terminal differentiation and concomitant cell cycle arrest of normal tissue was investigated by Parker et al. (1995) Science 267:1024-1027. Using in situ hybridization with a WAFl probe, Parker et al. studied the tissue-specific pattern of WAFl expression in mouse embryos at varying stages of development. The expression of WAFl was found to correspond to the presence of post-mitotic, terminally differentiated cells (e.g., muscle, neurons). The pattern of WAFl expression did not, however, correspond to that of p53 expression and mouse embryos lacking the p53 gene showed normal WAFl expression and embryogenesis. Similarly, WAFl expression in adult mice was found to be localized to terminally differentiated tissues and to be unaltered in p53-knockouts. Thus, these authors concluded that WAFl has a role in normal terminal differentiation and embryogenesis but that WAFl induction is not dependent upon p53 induction.

Another example of p53 -independent induction of WAFl was demonstrated in embryonic fibroblasts from p53 knock-out mice (Michieli et al. (1994) Cancer Res. 54:3391- 3395). Michieli et al. found that serum, platelet-derived growth factor (PDGF), fibroblast growth factor (FGF) and epidermal growth factor (EGF) were able to induce WAFl in p53-deficient as well as normal cells. In contrast, γ-irradiation was able to induce WAFl in normal cells but not in cells lacking wild-type p53. These authors conclude that WAFl may be induced either by a p53-dependent pathway activated by DNA damage or by a p53 -independent pathway activated by mitogens. The fact that WAFl is induced by mitogens is curious in light of its apparent role in G] arrest and suggests that WAFl may have an unknown function independent of G, arrest.

Many additional reports in the last several years have supported the hypothesis that WAFl is an important element in regulating the cell cycle transition from G, to S phase and that WAFl can be induced by a p53 -dependent pathway activated by DNA damage as well as p53- independent pathways activated by cell differentiation signals. In addition, to those discussed above, p53 -independent inducers of WAFl have now been shown to include transforming growth factor β (TGFβ) in keratinocytes (Datto et al. (1995) Proc. Natl. Acad. Sci. fUSA^{^}l 92:5545-5549); 12-0-tetradecanoyl ρhorbol-13-acetate (TPA), l,25-dihydroxyvitamin D3 (Vit D3), retinoic acid (RA) and dimethyl-sulfoxide (DMSO) in promyelocytic leukemia cells (Jiang et al. (1994) Oncogene 9:3397-3406); and MyoD in skeletal muscle (Halevy et al. (1995) Science 267:1018-1021).

Summary of the Invention The present invention provides methods of killing or inhibiting the growth of cells in which the WAFl gene is being expressed. The methods comprise the administration a WAFl inhibitor to cells in which a WAFl -dependent pathway has been induced and wherein said inhibitor is administered in an amount sufficient to kill or inhibit the growth of said cells.

In one set of embodiments, the inhibitor comprises a WAFl -antisense oligonucleotide. In particular, the oligonucleotide may be a modified oligonucleotide in which the backbone linkages, termini or bases have been modified to increase resistance to degradation or to increase binding affinity. Particularly preferred modified oligonucleotides are those containing a plurality of phosphorothioate linkages. In all embodiments in which the WAFl inhibitor is an antisense oligonucleotide, it is preferred that the oligonucleotide is selected from the group consisting of (a) oligonucleotides comprising at least 10 consecutive bases from the WAFl sequence disclosed as SEQ ID NO.: 1 ; and (b) oligonucleotides capable of hybridizing to the complements ofthe oligonucleotides of (a) under physiological conditions. In another set of embodiments, the inhibitor is a vector which expresses a WAFl -antisense oligonucleotide.

In an alternative set of embodiments, the inhibitor is an inhibitor of WAFl gene transcription. In another set of embodiments, the inhibitor is an inhibitor of WAFl protein activity such as an intracellular anti- WAFl antibody or a fragment of either WAFl or a CDK which competitively inhibits the formation of complexes between the WAFl protein and endogenous CDKs. Other examples are described below.

In further embodiments, the method may include subjecting the cells to a treatment which induces a WAFl -dependent pathway. This pathway may be a p53-dependent pathway and the treatment may be one which induces p53 gene expression. Such treatments may include X- irradiation, γ-irradiation, UV-irradiation, administering to the cells an alkylating agent , administering to the cells cisplatin, administering to the cells bleomycin, doxorubicin, administering to the cells 5-fluorouracil, administering to the cells genistein, administering to the cells hydrogen peroxide, or administering to the cells methylmethane sulfonate. Alternatively, the pathway may be a p53 -independent pathway. For such pathways, the treatment may include administering to the cells differentiation-inducing agents or inhibitors of DNA synthesis. Such treatments may include administering to the cells a pharmaceutical composition selected from the group consisting of PDGF, FGF, EGF, NGF, β-interferon, TGFβ, TPA, Vit D3, RA, DMSO, MyoD, IL2, rapamycin, aphidicolin, etoposide, methotrexate, cytosine arabinoside, 6- thioguanine, 6-mercaptopurine.

For all ofthe above-described embodiments, the method is particularly intended for use with cells which abnormally proliferate, and in a particularly important embodiment with cancer cells in a human host. Preferred cancer cells include neuroblastoma, melanoma, epithelioma, fibroblastoma, carcinoma, leukemia and myeloma cells.

Thus, the present invention provides a method of treating a human patient having cancerous cells in which a WAFl -dependent pathway has been induced comprising administering a WAFl inhibitor to the patient in an amount sufficient to kill or inhibit the growth ofthe cells. The WAFl inhibitor may be any of those described above. In addition, the treatment may also include subjecting the patient to a treatment which induces a WAF1- dependent pathway. This additional treatment may include any of those described above. In particular, the additional treatment may include radiation or chemotherapy therapy which induces DNA damage in the cells or which induces growth arrest or differentiation ofthe cells. As in all embodiments described above, the preferred WAFl inhibitor a WAFl -antisense oligonucleotide and, preferably, a modified WAFl -antisense oligonucleotide with a plurality of phosphorothioate linkages.

The invention further involves use ofthe foregoing compositions in the preparation of medicaments and in particular the preparation of medicaments for treating abnormal cell proliferation such as cancer.

Detailed Description ofthe Invention

The present invention depends, in part, upon the surprising discovery that the selective inhibition ofthe expression of WAFl in cells in which a WAFl -dependent pathway has been induced does not lead to cell proliferation but, rather, to cell death. This result is particularly surprising in that WAFl is believed to play a role in the G, arrest and/or terminal differentiation of cells and, therefore, it has previously been proposed that inducing the WAFl gene or otherwise increasing the levels ofthe WAFl protein might be a useful means of controlling the growth of cell lines in vitro and, more important, tumor cells in vivo. The present invention, in contrast, provides methods of killing or inhibiting the growth of cells by inhibiting transcription ofthe WAFl gene, translation ofthe WAFl mRNA transcript, or activity ofthe WAFl protein. Without being bound to any particular theory ofthe invention, applicants believe that conditions or treatments which result in the induction of WAFl also induce other components of a G, arrest or differentiation pathway. Although WAFl is only one component in these "WAFl -dependent" pathways, it appears essential. Therefore, when WAFl is inhibited after induction of a WAFl- dependent pathway, it is believed that the cells cannot complete the Gj arrest or differentiation pathways and, instead, initiate programmed cell death or apoptosis.

Clearly, the present invention is useful only with cells in which the WAFl gene is being expressed. Thus, in some embodiments, the target cells ofthe invention may, without prior treatment, already be expressing WAF 1. In other embodiments, however, the methods of the invention include an additional treatment which induces the expression of a WAFl -dependent pathway. Such additional treatment will typically comprise radiation or chemotherapies.

Definitions: In order to more clearly and concisely describe the subject matter ofthe present invention, the following definitions are provided for specific terms used in the claims appended hereto:

WAFl. As used herein, the abbreviation "WAFl" means the human cyclin-dependent kinase inhibitor gene described in the various references cited herein and denoted as "WAFl," "Cipl," "CIPl," "SDIl," or "p21." A cDNA to one allele of WAFl was disclosed in Harper et al. (1993) and El-Deiry et al. (1993). In addition, one WAFl allele and the corresponding protein are disclosed herein as SEQ. ID NO.: 1 and SEQ ID NO.: 2, respectively. The translation initiation codon of this cDNA is found at base positions 76-78 and the stop codon is at positions 568-570, defining an open reading frame of 492 bases. As will be obvious to one of ordinary skill in the art, other functional alleles of WAFl are likely to exist in the human population and are embraced by the abbreviation "WAFl" as used herein.

WAFl -dependent pathway. As used herein, the term "WAFl -dependent pathway" means a biochemical pathway in human cells in which expression ofthe WAFl gene is induced. Such a pathway may require induction of WAFl expression by the p53 tumor suppressor protein, in which case the pathway is said to be "p53-dependent." Alternatively, the pathway may not require induction of WAFl expression by the p53 tumor suppressor protein, in which case the pathway is said to be "p53 -independent."

WAFl inhibitor. As used herein, the term "WAFl inhibitor" means a compound which, when present in a cell, inhibits the transcription ofthe WAFl gene, translation ofthe WAFl mRNA transcript, or activity ofthe WAFl protein product. Examples include WAFl -antisense, antibodies or fragments of antibodies which act intracellularly against the WAFl protein or WAFl-cyclin-CDK complexes, fragments of CDKs which would act as competitive inhibitors of WAF1 interaction with endogenous CDKs, small molecule inhibitors such as OK- 1035 [Biochemical and Biophysical Research Communications 221, 207-212 (1996)] and ribozymes which inhibit WAFl expression.

WAFl -antisense oligonucleotide. As used herein, the term "WAFl -antisense oligonucleotide" or "WAFl -antisense" describes an oligonucleotide that is an oligoribonucleotide, oligodeoxyribonucleotide or a modified oligonucleotide which hybridizes under physiological conditions to a WAFl mRNA transcript or to WAFl DNA and, thereby, acts as a WAFl inhibitor. The antisense molecule, of course, is constructed and arranged so as to interfere with transcription or translation of WAFl upon hybridization with the target. Those skilled in the art will recognize that the exact length ofthe antisense oligonucleotide and its degree of complementarity will depend upon the specific target selected, including the sequence ofthe target and the particular bases which comprise that sequence. It is preferred that the antisense oligonucleotide be selected so as to hybridize selectively with the target under physiological conditions, i.e., to hybridize substantially more with the target sequence than with any other sequence in the target cell under physiological conditions. I. Induction of WAFl -Dependent Pathways

The p53 protein is a potent inducer of WAFl expression. Therefore, p53 or inducers of p53 may be used to induce WAFl -dependent pathways. A variety of p53 inducers are known in the art and, because of p53's activity as a tumor suppressor, are already in use in the field of cancer therapy. Preferred p53 inducers are DNA-damaging radiation (e.g., X-rays, γ-rays, UV), DNA-damaging compounds (e.g., alkylating agents such as nitrogen mustard, chlorambucil, melphalan, cyclophosphamide, busulfan, and nitrosureas (BCNU, CCNU, methyl-CCNU); as well as etoposide, cisplatin, bleomycin, doxorubicin, 5 -fluorouracil, genistein, hydrogen peroxide, and methy lmethane sulfonate). In addition, as noted above, WAFl has been shown to be induced by p53 -independent pathways. Additional preferred inducers of WAFl therefore include WAFl -inducing growth factors and differentiation factors such as PDGF, FGF, EGF, nerve growth factor (NGF), β- interferon, TGFβ, TPA, Vit D3, RA, DMSO, MyoD and IL2, rapamycin and inhibitors of DNA synthesis such as aphidicolin, methotrexate, cytosine arabinoside, 6-thioguanine, 6- mercaptopurine. II. Inhibition of WAFl Expression

The present invention depends, in part, upon the discovery that the selective inhibition of the expression of WAFl in cells in which a WAFl -dependent pathway has been induced leads to the inhibition of cell growth and/or cell death. Thus, the present invention requires that the targeted cells be subject to conflicting conditions: conditions inducing WAFl -dependent G, arrest or differentiation and conditions under which WAFl expression is inhibited.

In most preferred embodiments, the WAFl -inhibiting conditions comprise treatment with WAFl -antisense oligonucleotides. Based upon SEQ. ID NO.:l, one of skill in the art can easily choose and synthesize any of a number of appropriate antisense molecules. In order to be sufficiently selective and potent for WAFl inhibition, such WAFl -antisense oligonucleotides should comprise at least 10 bases and, more preferably, at least 15 bases. Typically, antisense molecules are between 15 and 32 bases. Most preferably, the antisense oligonucleotides comprise 18-20 bases. Although oligonucleotides may be chosen which are antisense to any region ofthe WAFl gene or mRNA transcript, in preferred embodiments the antisense oligonucleotides correspond to the N-terminal or, more preferably, translation initiation region of the WAFl mRNA or to mRNA splicing sites. In addition, WAFl antisense may, preferably, be targeted to sites in which mRNA secondary structure is not expected (see, e.g., Sainio et al. (1994) Cell. Mol. Neurobiol. 14(5):439-457) and at which proteins are not expected to bind. As will be obvious to one of ordinary skill in the art, the WAFl -inhibiting antisense oligonucleotides ofthe present invention need not be perfectly complementary to the WAFl gene or mRNA transcript in order to be effective. Rather, some degree of mismatches will be acceptable if the antisense oligonucleotide is of sufficient length. In all cases, however, the oligonucleotides should have sufficient length and complementarity so as to selectively hybridize to a WAFl transcript under physiological conditions. Preferably, of course, mismatches are absent or minimal. In addition, although it is not recommended, the WAFl -antisense oligonucleotides may have one or more non-complementary sequences of bases inserted into an otherwise complementary WAFl -antisense oligonucleotide sequence. Such non-complementary sequences may "loop" out of a duplex formed by a WAFl transcript and the bases flanking the non-complementary region. Therefore, the entire oligonucleotide may retain an inhibitory effect despite an apparently low percentage of complementarity.

The WAFl -antisense oligonucleotides ofthe invention may be composed of deoxyribonucleotides, ribonucleotides, or any combination thereof. The 5' end of one nucleotide and the 3' end of another nucleotide may be covalently linked, as in natural systems, via a phosphodiester internucleotide linkage. These oligonucleotides may be prepared by art recognized methods such as phosphoramidate, H-phosphonate chemistry, or methylphosphoramidate chemistry (see, e.g., Uhlmann et al. (1990) Chem. Rev. 90:543-584; Agrawal (ed.) Meth. Mol. Biol.. Humana Press, Totowa, NJ (1993) Vol. 20; and U.S. Patent No. 5,149,798) which may be carried out manually or by an automated synthesizer (reviewed in Agrawal et al. (1992) Trends Biotechnol. 10:152-158).

The WAFl -antisense oligonucleotides ofthe invention also may include modified oligonucleotides. That is, the oligonucleotides may be modified in a number of ways which do not compromise their ability to hybridize to nucleotide sequences contained within the transcription initiation region or coding region ofthe WAFl gene. The term "modified oligonucleotide" as used herein describes an oligonucleotide in which at least two of its nucleotides are covalently linked via a synthetic linkage, i.e., a linkage other than a phosphodiester linkage between the 5' end of one nucleotide and the 3' end of another nucleotide. The most preferred synthetic linkages are phosphorothioate linkages. Additional preferred synthetic linkages include alkylphosphonates, phosphorodithioates, phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates, carbonates, phosphate triesters, acetamidate, and carboxymethyl esters. Oligonucleotides with these linkages or other modifications can be prepared according to known methods (see, e.g., Agrawal and Goodchild (1987) Tetrahedron Lett. 28:3539-3542; Agrawal et al. (1988) Proc. Natl. Acad. Sci. (USA) 85:7079-7083; Uhlmann et al. (1990) Chem. Rev. 90:534-583; Agrawal et al. (1992) Trends Biotechnol. 10:152-158; Agrawal (ed.) Meth. Mol. Biol.. Humana Press, Totowa, NJ (1993) Vol. 20).

The term "modified oligonucleotide" also encompasses oligonucleotides with a modified base and/or sugar. For example, modified oligonucleotides include oligonucleotides having the sugars at the most 3' and/or most 5' positions attached to chemical groups other than a hydroxyl group at the 3' position and other than a phosphate group at the 5' position. Other modified ribonucleotide-containing oligonucleotides may include a 2'-O-alkylated ribose group such as a 2'-O-methylated ribose, or oligonucleotides with arabinose instead of ribose. In addition, unoxidized or partially oxidized oligonucleotides having a substitution in one nonbridging oxygen per nucleotide in the molecule are also considered to be modified oligonucleotides. Such modifications may be at some or all ofthe internucleoside linkages, at either or both ends ofthe oligonucleotide, and/or in the interior ofthe molecule (reviewed in Agrawal et al. (1992) Trends Biotechnol. 10:152-158 and Agrawal (ed.) Meth. Mol. Biol.. Humana Press, Totowa, NJ (1993) Vol. 20). Also considered as modified oligonucleotides are oligonucleotides having nuclease resistance-conferring bulky substituents at their 3' and/or 5' end(s) and/or various other structural modifications not found in vivo without human intervention. Other modifications include additions to the internucleoside phosphate linkages, such as cholesteryl or diamine compounds with varying numbers of carbon residues between the amino groups and terminal ribose. The inhibition of WAF 1 expression need not be accomplished by means of a WAF 1 - antisense oligonucleotide. Rather, inhibitors of WAFl transcription or WAFl protein activity also may be employed to the same effect. For example, antibodies or fragments of antibodies which act intracellularly against the WAFl protein or WAFl-cyclin-CDK complexes, fragments of CDKs or recombinant genes encoding fragments of CDK which would act as competitive inhibitors of WAFl interaction with endogenous CDKs, and ribozymes which inhibit WAFl expression.

Without being bound to any particular theory ofthe invention, it is believed that treatments which result in the induction of WAFl also induce other components of a G, arrest or differentiation pathway. Although WAFl is only one component in these "WAFl -dependent" pathways, it appears essential. When WAFl is inhibited, it is believed that the cells cannot complete the G, arrest or differentiation pathways and, instead, initiate programmed cell death. As a general rule, therefore, the WAFl -inhibiting conditions must be more specific to WAFl than the WAFl -inducing conditions so that the WAFl -dependent pathway is induced while WAFl itself is inhibited. Thus, the WAFl -inducing conditions and WAFl -inhibiting conditions should not act at the same point or level in the WAFl -dependent pathway. For example, simultaneous administration ofthe p53 protein (as the WAFl -inducing condition) and administration of anti-p53 antibodies (as the WAFl -inhibiting condition) would be ineffective because the conditions would largely counteract each other at the same level. In contrast, induction ofthe p53 gene (as the WAFl -inducing condition) and administration of WAFl antisense oligonucleotides (as the WAFl -inhibiting condition) would be effective because p53 is a pleiotropic inducer and the WAFl -antisense oligonucleotides would not inhibit all components ofthe WAFl -dependent pathway. III. Methods of Treatment in Cancer Therapy

The methods ofthe present invention are particularly well suited for use in the field of cancer therapy. Because current radiation and chemotherapy methods typically involve treatments which cause DNA damage and/or induce terminal differentiation of tumor cells and/or inhibit proliferation of tumor cells, these treatments, in many cases, already induce expression of the WAFl gene. Therefore, by combining these treatments with administration of a WAF1- inhibitor, a more effective means of killing or inhibiting the growth of tumor cells is provided. In one set of preferred embodiments, a cancer patient is treated with ionizing radiation (e.g., X-rays or γ-rays) or an agent (e.g.,doxorubicin) which causes DNA damage, induces p53 expression and, thereby, induces a p53-dependent, WAFl -dependent pathway toward cell cycle arrest. At the same time or shortly thereafter, a WAFl inhibitor is administered to the patient. This administration may be oral, intravenous, parenteral, cutaneous or subcutaneous. The administration also may be localized to the region ofthe tumor by injection to or perfusion ofthe tumor site. Preferably, the WAFl inhibitor is WAFl -antisense administered in a pharmaceutically acceptable carrier or a recombinant vector with a WAF 1 -antisense gene which expresses a WAFl -antisense oligonucleotide.

In another series of embodiments, a cancer patient is treated with compounds which induce expression of a p53-independent, WAFl -dependent pathway. These embodiments may be of particular importance because p53 mutations are associated with many cancers and mutant p53 proteins may fail to induce WAFl expression. In preferred embodiments, the patient has been treated with an agent that induces p53 -independent G, arrest or differentiation ofthe tumor cells and at the same time, or shortly thereafter, is administered a WAFl inhibitor. Appropriate non-proliferation and differentiation agents are well known in the art and vary according to tumor type. For example, β-interferon and mezerein may be administered to melanoma patients, or TGFβ may be administered to any of a number of different types of cancer patients in order to induce a WAFl -dependent pathway. A WAFl inhibitor, as described above, may be simultaneously or subsequently administered to kill or further inhibit the growth ofthe tumor cells.

The cancer may be virtually any cancer, including, but not limited to, brain cancer including glioblastoma and medulloblastoma, breast cancer, cervical cancer, colon cancer, endometrial cancer, liver cancer, lung cancer, oral cancer, prostate cancer, sarcomas, skin cancer, and renal cancer. WAF1 -antisense oligonucleotides or other WAFl inhibitors may be administered as part of a pharmaceutical composition. Such a pharmaceutical composition may include the WAFl inhibitor in combination with any standard physiologically and/or pharmaceutically acceptable carriers which are known in the art. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness ofthe biological activity ofthe active ingredients. The term "physiologically acceptable" refers to a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or organism. The characteristics ofthe carrier will depend on the route of administration. Physiologically and pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials which are well known in the art. The pharmaceutical composition ofthe invention may also contain other active factors and/or agents which inhibit WAFl expression or otherwise inhibit cell growth or increase cell death. Such additional factors and/or agents may be included in the pharmaceutical composition to produce a synergistic effect or to minimize side-effects caused by the WAFl inhibitor ofthe invention. The pharmaceutical composition ofthe invention may be in the form of a liposome in which WAFl -antisense oligonucleotides are combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids wliich exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers which are in aqueous solution. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Patent No. 4,235,871; U.S. Patent No. 4,501,728; U.S. Patent No. 4,837,028; and U.S. Patent No. 4,737,323.

The pharmaceutical composition ofthe invention may further include compounds such as cyclodextrins and the like which enhance delivery of oligonucleotides into cells, as described by Zhao et al. (in press). When the composition is not administered systemically but, rather, is injected at the site ofthe target cells, cationic detergents (e.g. Lipofectin) may be added to enhance uptake.

When a therapeutically effective amount of a WAFl inhibitor is administered orally, the inhibitor will be in the form of a tablet, capsule, powder, solution or elixir. When administered in tablet form, the pharmaceutical composition ofthe invention may additionally contain a solid carrier such as a gelatin or an adjuvant. The tablet, capsule, and powder may contain from about 5 to 95% of a WAFl -antisense oligonucleotide and preferably from about 25 to 90% ofthe oligonucleotide. When administered in liquid form, a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, sesame oil, or synthetic oils may be added. The liquid form ofthe pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When administered in liquid form, the pharmaceutical composition may contain from about 0.5 to 90% by weight of a WAFl -antisense oligonucleotide and preferably from about 1 to 50% ofthe oligonucleotide.

When a therapeutically effective amount of a WAFl inhibitor is administered by intravenous, cutaneous or subcutaneous injection, the inhibitor will be in the form of a pyrogen- free, parenterally acceptable aqueous solution. The preparation of such parenterally acceptable solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art. A preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to the WAFl inhibitor, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or another vehicle as known in the art. The pharmaceutical composition ofthe present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art.

In preferred embodiments, when the target cells are readily accessible, administration of WAF 1 -antisense oligonucleotides is localized to the region of the targeted cells in order to maximize the delivery ofthe WAFl -antisense, minimize WAFl inhibition in non-target cells, and minimize the amount of WAFl -antisense needed per treatment. Thus, in one preferred embodiment, administration is by direct injection at or perfusion ofthe site ofthe targeted cells, such as a tumor. Alternatively, the WAFl -antisense oligonucleotides may be adhered to small particles (e.g.,microscopic gold beads) which are impelled through the membranes ofthe target cells (see, e.g., U.S. Pat. No. 5,149,655).

In another series of embodiments, a recombinant gene is constructed which encodes a WAFl -antisense oligonucleotide and this gene is introduced within the targeted cells on a vector. Such a WAFl -antisense gene may, for example, consist ofthe normal WAFl sequence, or a subset ofthe normal WAFl sequence, operably joined in reverse orientation to a promoter region. An operable WAFl -antisense gene may be introduced on an integration vector or may be introduced on an expression vector. In order to be most effective, it is preferred that the WAFl- antisense sequences be operably joined to a strong eukaryotic promoter which is inducible or constitutively expressed.

In all ofthe above-described methods of treatment, the WAFl inhibitors are administered in therapeutically effective amounts. In addition, in those methods in which the patient also is subjected to treatment that induces a WAFl -dependent pathway, these treatments are also administered in therapeutically effective amounts. As used herein, the term "therapeutically effective amount" means the total amount of each active component ofthe pharmaceutical composition or method that is sufficient to show a meaningful patient benefit, i.e., the killing or inhibition ofthe growth ofthe target cells. When applied to an individual active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts ofthe active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.

The amount of WAFl inhibitor in the pharmaceutical composition ofthe present invention will depend not only upon the potency ofthe inhibitor but also upon the nature and severity ofthe condition being treated, and on the nature of prior treatments which the patent has undergone. Ultimately, the attending physician will decide the amount of WAFl inhibitor with wliich to treat each individual patient. Initially, the attending physician will administer low doses ofthe inhibitor and observe the patient's response. Larger doses of a WAFl inhibitor may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further. In preferred embodiments, the WAFl inhibitor is a WAF1- antisense oligonucleotide and it is contemplated that the various pharmaceutical compositions used to practice the method ofthe present invention should contain about 1.0 μg to about 100 mg of oligonucleotide per kg body weight.

The duration of intravenous therapy using the pharmaceutical composition ofthe present invention will vary, depending on the severity ofthe disease being treated and the condition and potential idiosyncratic response of each individual patient. It is contemplated that the duration of each application of a WAFl -antisense oligonucleotide will be in the range of 12 to 24 hours of continuous intravenous administration. Ultimately the attending physician will decide on the appropriate duration of intravenous therapy using the pharmaceutical composition ofthe present invention. Experimental Examples

The efficacy ofthe present invention was demonstrated in vitro on a neuroblastoma cell line using conditions known to inhibit cell proliferation and to induce differentiation as the WAFl -inducing conditions and using treatment with WAFl -antisense oligonucleotides as WAFl -inhibiting conditions. As described above, this technique did not lead to cell proliferation but, on the contrary, increased cell death. SH-SY5Y (Biedler, J. et al. (1978) Cancer Res. 38:3751-3757), a neuroblastoma cell line, was used as a model for neuronal terminal differentiation (LoPresti P. et al., (1992) Cell Growth Diff. 3:627-635; Poluha, W. et al., (1995) Oncogene 10:185-189). These cells express low levels of both the low-affinity nerve growth factor receptor (LNGFR) and the TrkA NGF receptor (Baker, D. et al., (1989) 49:4142-4146; Poluha, W. et al., (1995) Oncogene 10:185-189). SH-SY5Y cells treated with nerve growth factor (NGF) and aphidicolin, a specific and reversible inhibitor of DNA polymerases (α and δ), cease cell proliferation and extend long neurites (Jensen, L. (1987) Dev. Biol. 120:56-64; LoPresti, P. et al., (1992) Cell Growth Diff. 3:627-635). Thus, "aphidicolin + NGF" is a WAF1- inducing treatment for these cells. The differentiated cells require NGF for survival and, in the presence of NGF, are stable for 4-6 weeks. These cells express neuronal markers and resemble sympathetic neurons. In contrast, NGF alone does not stop cell proliferation and induces only slight neurite extension (Chen, J. et al., (1990) Cell Growth Diff. 1:79-85; Sonnenfeld and Ishii, (1982) J. Neurosci. Res. 8:375-391). Treatment with aphidicolin does not induce neurite extension, and the cells resume proliferation, following removal of aphidicolin.

Cells were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM glutamine and 100 μg/ml of gentamicin. For differentiation studies, cells were plated on Primaria (Falcon Plastics) dishes or flasks and were treated with 100 ng/ml of NGF (2.5 S; Bioproducts for Science) and/or 0.3 μM of aphidicolin. Fresh (aphidicolin+NGF)-containing medium was added every 2-3 days.

To verify the role of WAFl in neuroblastic differentiation, expression ofthe mRNA for WAFl was assessed by Northern blotting. Expression ofthe WAFl mRNA transcript was up¬ regulated following treatment for 1 hr with aphidicolin+NGF and further increased as the aphidicolin+NGF treatment progressed. On day 6, the cells were changed from aphidicolin+NGF medium to NGF-containing medium. Despite the removal of aphidicolin from the medium, expression of WAFl transcripts was slightly greater on day 14 than on day 6. Treatment with aphidicolin alone also induced WAFl expression, but following removal on day 6 of aphidicolin from the medium, expression greatly declined. Treatment with NGF alone did not induce expression of WAFl. Ethidium bromide staining of RNA prior to transfer, as well as rehybridization of filters with a β-actin probe confirmed that the RNA was intact. These studies demonstrated that sustained expression of WAFl mRNA is specifically associated with terminal differentiation of SH-S Y5 Y cells.

Using Western blotting, we then determined whether the level ofthe WAFl protein was elevated. Treatments of SH-SY5Y cells with aphidicolin+NGF enhanced levels of WAFl (20- fold). Expression of WAFl protein persisted following removal of aphidicolin from the medium but at slightly lower levels. Treatment of cells with aphidicolin alone up-regulated the WAFl (14-fold) level. Following removal of aphidicolin from the medium, expression returned to starting levels. Treatment of cells with NGF alone did not induce expression of WAF 1.

As a WAFl -inhibitor, an antisense oligonucleotide with phosphorothioate linkages was employed. In addition, to enhance entry ofthe oligonucleotide into cells, the cationic detergent Lipofectin was used (Quattrone, A. et al., (1995) Biochemica 1:25-29; Wagner, R. (1994) Nature 372:333-335). The low concentration of detergent used in the experiments had no effect on cell viability or differentiation.

Phosphorothioate oligonucleotides (100 μM) and Lipofectin (1 mg/ml, a 1:1 (w/w) mixture of N-(l-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride and dioleoyl phosphatdylethanolamine (Gibco)) were incubated at 37 °C for 15 min. The oligonucleotide- detergent mix was diluted with serum-containing medium and added to the SH-S Y5 Y cells. In most cases, the dilution was 1:100 giving a final oligonucleotide concentration of 1 μM. Fresh oligonucleotide-containing medium was added to the cells each day. A WAFl -antisense oligonucleotide was employed which is complementary to the region around the translational start site (5'-TCC CCA GCC GGT TCT GAC AT-3' from Oligos, Etc.). For controls, an 18-mer Control- 1 (5'-TGG ATC CGA CAT GTC AGA-3') and an antisense oligonucleotide directed against M. tuberculosis (5'-CGC TTC ATC CTG CCG TGT CGG-3'), Control-2, were employed. Expression in the of WAFl -induced cells was reduced by the antisense oligonucleotide but not by two control oligonucleotides. As judged by Western blotting, the antisense oligonucleotide, but not the control, decreased WAFl expression by 2.0-2.5 fold. The antisense oligonucleotide had no apparent effect on the morphology or proliferation of control cells. However, cells treated with both WAFl -inducing and WAFl -inhibiting conditions differentiated into neuronal cells but the number of live cells per dish was much less than for cells treated with only WAFl -inducing conditions. Cells were stained with Hoechst 33342 to assay for apoptotic bodies which are characteristic of programmed cell death (Gregory, C. et al., (1991) Nature 349:612-614). In four independent experiments, the percentage of cells treated with both WAFl -inducing and WAFl -inhibiting conditions which exhibited apoptotic bodies was roughly twice that for cells treated with only inducing conditions or for cells treated with the inducing conditions and control oligonucleotides.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: WORCESTER FOUNDATION FOR BIOMEDICAL RESEARCH, INC.

(B) STREET: 222 MAPLE AVENUE

(C) CITY: SHREWSBURY (D) STATE: MASSACHUSETTS

(E) COUNTRY: UNITED STATES OF AMERICA

(F) ZIP: 01545

(ii) TITLE OF INVENTION: METHODS AND PRODUCTS FOR SELECTIVELY KILLING OR INHIBITING THE GROWTH OF CELLS EXPRESSING THE

WAFl GENE

(iii) NUMBER OF SEQUENCES: 2

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: WOLF, GREENFIELD & SACKS, P.C.

(B) STREET: 600 ATLANTIC AVENUE

(C) CITY: BOSTON

(D) STATE: MA (E) COUNTRY: USA

(F) ZIP: 02210

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 60/001,248

(B) FILING DATE: 20-JULY-1995

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: GATES, EDWARD R.

(B) REGISTRATION NUMBER: 31,616

(C) REFERENCE/DOCKET NUMBER: W0461/7028WO

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 617-720-3500

(B) TELEFAX: 617-720-2441

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2121 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:

(A) ORGANISM: HOMO SAPIENS

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 76..570

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

GCCGAAGTCA GTTCCTTGTG GAGCCGGAGC TGGGCGCGGA TTCGCCGAGG CACCGAGGCA 60

CTCAGAGGAG GCGCC ATG TCA GAA CCG GCT GGG GAT GTC CGT CAG AAC CCA 111

Met Ser Glu Pro Ala Gly Asp Val Arg Gin Asn Pro 1 5 10

TGC GGC AGC AAG GCC TGC CGC CGC CTC TTC GGC CCA GTG GAC AGC GAG 159 Cys Gly Ser Lys Ala Cys Arg Arg Leu Phe Gly Pro Val Asp Ser Glu 15 20 25

CAG CTG AGC CGC GAC TGT GAT GCG CTA ATG GCG GGC TGC ATC CAG GAG 207 Gin Leu Ser Arg Asp Cys Asp Ala Leu Met Ala Gly Cys Ile Gin Glu 30 35 40

GCC CGT GAG CGA TGG AAC TTC GAC TTT GTC ACC GAG ACA CCA CTG GAG 255 Ala Arg Glu Arg Trp Asn Phe Asp Phe Val Thr Glu Thr Pro Leu Glu 45 50 55 60

GGT GAC TTC GCC TGG GAG CGT GTG CGG GGC CTT GGC CTG CCC AAG CTC 303 Gly Asp Phe Ala Trp Glu Arg Val Arg Gly Leu Gly Leu Pro Lys Leu

65 70 75 -22-

TAC CTT CCC ACG GGG CCC CGG CGA GGC CGG GAT GAG TTG GGA GGA GGC 351 Tyr Leu Pro Thr Gly Pro Arg Arg Gly Arg Asp Glu Leu Gly Gly Gly 80 85 90

AGG CGG CCT GGC ACC TCA CCT GCT CTG CTG CAG GGG ACA GCA GAG GAA 399 Arg Arg Pro Gly Thr Ser Pro Ala Leu Leu Gin Gly Thr Ala Glu Glu 95 100 105

GAC CAT GTG GAC CTG TCA CTG TCT TGT ACC CTT GTG CCT CGC TCA GGG 447 Asp His Val Asp Leu Ser Leu Ser Cys Thr Leu Val Pro Arg Ser Gly 110 115 120

GAG CAG GCT GAA GGG TCC CCA GGT GGA CCT GGA GAC TCT CAG GGT CGA 495 Glu Gin Ala Glu Gly Ser Pro Gly Gly Pro Gly Asp Ser Gin Gly Arg 125 130 135 140

AAA CGG CGG CAG ACC AGC ATG ACA GAT TTC TAC CAC TCC AAA CGC CGG 543 Lys Arg Arg Gin Thr Ser Met Thr Asp Phe Tyr His Ser Lys Arg Arg 145 150 155

CTG ATC TTC TCC AAG AGG AAG CCC TAATCCGCCC ACAGGAAGCC TGCAGTCCTG 597 Leu Ile Phe Ser Lys Arg Lys Pro

160 165

GAAGCGCGAG GGCCTCAAAG GCCCGCTCTA CATCTTCTGC CTTAGTCTCA GTTTGTGTGT 657

CTTAATTATT ATTTGTGTTT TAATTTAAAC ACCTCCTCAT GTACATACCC TGGCCGCCCC 717

CTGCCCCCCA GCCTCTGGCA TTAGAATTAT TTAAACAAAA ACTAGGCGGT TGAATGAGAG 777

GTTCCTAAGA GTGCTGGGCA TTTTTATTTT ATGAAATACT ATTTAAAGCC TCCTCATCCC 837 GTGTTCTCCT TTTCCTCTCT CCCGGAGGTT GGGTGGGCCG GCTTCATGCC AGCTACTTCC 897

TCCTCCCCAC TTGTCCGCTG GGTGGTACCC TCTGGAGGGG TGTGGCTCCT TCCCATCGCT 957

GTCACAGGCG GTTATGAAAT TCACCCCCTT TCCTGGACAC TCAGACCTGA ATTCTTTTTC 1017

ATTTGAGAAG TAAACAGATG GCACTTTGAA GGGGCCTCAC CGAGTGGGGG CATCATCAAA 1077

AACTTTGGAG TCCCCTCACC TCCTCTAAGG TTGGGCAGGG TGACCCTGAA GTGAGCACAG 1137

CCTAGGGCTG AGCTGGGGAC CTGGTACCCT CCTGGCTCTT GATACCCCCC TCTGTCTTGT 1197

GAAGGCAGGG GGAAGGTGGG GTACTGGAGC AGACCACCCC GCCTGCCCTC ATGGCCCCTC 1257

TGACCTGCAC TGGGGAGCCC GTCTCAGTGT TGAGCCTTTT CCCTCTTTGG CTCCCCTGTA 1317

CCΠTTTGAGG AGOCCCAGCT TACCCTTCTT CTCCAGCTGG GCTCTGCAAT TCCCCTCTGC 1377

TGCTGTCCCT CCCCCTTGTC TTTCCCTTCA GTACCCTCTC ATGCTCCAGG TGGCTCTGAG 1437

GTGCCTGTCC CACCCCCACC CCCAGCTCAA TGGACTGGAA GGGGAAGGGA CACACAAGAA 1497

GAAGGGCACC CTAGTTCTAC CTCAGGCAGC TCAAGCAGCG ACCGCCCCCT CCTCTAGCTG 1557

TGGGGGTGAG GGTCCCATGT GGTGGCACAG GCCCCCTTGA GTGGGGTTAT CTCTGTGTTA 1617

GGGGTATATG ATGGGGGAGT AGATCTTTCT AGGAGGGAGA CACTGGCCCC TCAAATCGTC 1677

CAGCGACCTT CCTCATCCAC CCCATCCCTC CCCAGTTCAT TGCACTTTGA TTAGCAGCGG 1737

AACAAGGAGT CAGACATTTT AAGATGGTGG CAGTAGAGGC TATGGACAGG GCATGCCACG 1797 TGGGCTCATA TGGGGCTGGG AGTAGTTGTC TTTCCTGGCA CTAACGTTGA GCCCCTGGAG 1857

GCACTGAAGT GCTTAGTGTA CTTGGAGTAT TGGGGTCTGA CCCCAAACAC CTTCCAGCTC 1917

CTGTAACATA CTGGCCTGGA CTGTTTTCTC TCGGCTCCCC ATGTGTCCTG GTTCCCGTTT 1977

CTCCACCTAG ACTGTAAACC TCTCGAGGGC AGGGACCACA CCCTGTACTG TTCTGTGTCT 2037

TTCACAGCTC CTCCCACAAT GCTGAATATA CAGCAGGTGC TCAATAAATG ATTCTTAGTG 2097

ACTTTAAAAA AAAAAAAAAA AAAA 2121

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 164 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Ser Glu Pro Ala Gly Asp Val Arg Gin Asn Pro Cys Gly Ser Lys 1 5 10 15

Ala Cys Arg Arg Leu Phe Gly Pro Val Asp Ser Glu Gin Leu Ser Arg 20 25 30

Asp Cys Asp Ala Leu Met Ala Gly Cys Ile Gin Glu Ala Arg Glu Arg 35 40 45

Trp Asn Phe Asp Phe Val Thr Glu Thr Pro Leu Glu Gly Asp Phe Ala 50 55 60

Trp Glu Arg Val Arg Gly Leu Gly Leu Pro Lys Leu Tyr Leu Pro Thr 65 70 75 80

Gly Pro Arg Arg Gly Arg Asp Glu Leu Gly Gly Gly Arg Arg Pro Gly 85 90 95

Thr Ser Pro Ala Leu Leu Gin Gly Thr Ala Glu Glu Asp His Val Asp 100 105 110

Leu Ser Leu Ser Cys Thr Leu Val Pro Arg Ser Gly Glu Gin Ala Glu 115 120 125

Gly Ser Pro Gly Gly Pro Gly Asp Ser Gin Gly Arg Lys Arg Arg Gin 130 135 140

Thr Ser Met Thr Asp Phe Tyr His Ser Lys Arg Arg Leu Ile Phe Ser 145 150 155 160

Lys Arg Lys Pro

Claims

1. A method of killing or inhibiting the growth of cells comprising administering a WAFl inhibitor to cells in which a WAFl -dependent pathway has been induced and wherein said inhibitor is administered in an amount sufficient to kill or inhibit the growth of said cells.

2. A method as in claim 1 wherein said inhibitor comprises a WAFl -antisense oligonucleotide.

3. A method as in claim 2 wherein said inhibitor is a modified oligonucleotide containing a plurality of phosphorothioate linkages.

4. A method as in either claim 2 or claim 3 wherein said oligonucleotide is selected from the group consisting of

(a) oligonucleotides comprising at least 10 consecutive bases from SEQ ID NO.: 1; and

(b) oligonucleotides capable of hybridizing to the complements ofthe oligonucleotides of (a) under physiological conditions.

5. A method as in claim 1 wherein said inhibitor is a vector which expresses a WAF 1 - antisense oligonucleotide.

6. A method as in claim 1 wherein said inhibitor is an inhibitor of WAFl gene transcription.

7. A method as in claim 1 wherein said inhibitor is an inhibitor of WAFl protein activity.

8. A method as in claim 1 wherein said method further comprises subjecting said cells to a treatment which induces a WAFl -dependent pathway.

9. A method as in claim 8 wherein said WAFl -dependent pathway is a p53-dependent pathway and said treatment induces p53 gene expression.

10. A method as in claim 9 wherein said treatment is selected from the group consisting of X-irradiation, γ-irradiation, UV-irradiation, administering to said cells an alkylating agent , administering to said cells cisplatin, administering to said cells bleomycin, doxorubicin, administering to said cells 5-fluorouracil, administering to said cells genistein, administering to said cells hydrogen peroxide, and administering to said cells methylmethane sulfonate.

11. A method as in claim 8 wherein said WAF 1 -dependent pathway is a p53 -independent pathway.

12. A method as in claim 11 wherein said treatment comprises administering to said cells differentiation-inducing agents.

13. A method as in claim 11 wherein said treatment comprises administering to said cells inhibitors of DNA synthesis.

14. A method as in claim 11 wherein said treatment comprises administering to said cells a pharmaceutical composition selected from the group consisting of PDGF, FGF, EGF, NGF, β- interferon, TGFβ, TPA, Vit D3, RA, DMSO, MyoD, IL2, rapamycin, aphidicolin, etoposide, methotrexate, cytosine arabinoside, 6-thioguanine, 6-mercaptopurine.

15. A method as in claim 1 wherein said cells are cancer cells in a human host.

16. A method as in claim 15 wherein said cancer cells are selected from the group consisting of neuroblastoma, melanoma, epithelioma, fibroblastoma, carcinoma, leukenia and myeloma cells.

17. A method of treating a human patient having cancerous cells in which a WAF1- dependent pathway has been induced comprising administering a WAFl inhibitor to said patient in an amount sufficient to kill or inhibit the growth of said cells.

18. A method as in claim 17 further comprising subjecting said patient to a treatment which induces a WAF 1 -dependent pathway.

19. A method as in claim 18 wherein said treatment comprises radiation therapy which induces DNA damage in said cells.

20. A method as in claim 19 wherein said treatment comprises chemotherapy which induces growth arrest or differentiation of said cells.

21. A method as in any one of claims 17 to 20 wherein said inhibitor is a WAF1- antisense oligonucleotide.