Mammalian MDM2 Binding Proteins and Uses Thereof

Abstract

Isolated nucleic acid sequences encoding mammalian MDM2 binding protein and polypeptide sequences for the mammalian MDM2 binding protein are provided. Also provided are vectors containing these nucleic acid sequences, host cells which express these proteins and antibodies targeted to these proteins. In addition, methods and compositions for modulating the G.sub.1 phase of the cell cycle via altering expression and/or activity of a mammalian MDM2 binding protein are provided.

Description

INTRODUCTION

[0001] This patent application is a divisional of U.S. patent application Ser. No. 11/876,901 filed Oct. 23, 2007, which is a divisional of U.S. patent application Ser. No. 11/650,159, filed Jan. 5, 2007, issued as U.S. Pat. No. 7,304,142, which is a divisional of U.S. patent application Ser. No. 10/312,954, filed Jun. 10, 2003, issued as U.S. Pat. No. 7,166,712, which is the U.S. National Phase of PCT application Ser. No. PCT/US01/22053, filed Jul. 12, 2001, which claims the benefit of priority from U.S. Provisional Application Ser. No. 60/230,894 filed Sep. 5, 2000, and U.S. Provisional Application Ser. No. 60/217,835 filed Jul. 12, 2000.

FIELD OF THE INVENTION

[0003] The present invention relates to the identification of mammalian genes, in particular a mouse and human gene, which encode a protein, referred to herein as MDM2 binding protein or MTBP, which is involved in the MDM2 growth regulatory pathway in cells. Specifically, it has been found that overexpression of MTBP can induce an arrest in the G.sub.1 phase of the cell cycle. Further, it has been found that overexpression of MDM2 can block the arresting effects of MTBP in the G.sub.1 phase of the cell cycle and that MDM2 can induce increased turnover of MTBP. Thus, it is believed that MTBP may have tumor suppressive activity. Accordingly, provided in the present invention are isolated nucleic acid sequences encoding MTBP, isolated polypeptide sequences for mammalian MTBP, vectors and host cells for expression of this cellular growth regulating protein and antibodies which target this cellular growth regulating protein.

[0004] Also provided in the present invention are methods and compositions for modulating the G.sub.1 phase of the cell cycle via altering expression levels and/or activity of MTBP.

BACKGROUND OF THE INVENTION

[0005] In tumors, loss of function of either p53 itself (Hollstein et al. Nucleic Acids Res. 1996 24:141-146; Nigro et al. Nature 1989 342:705-708), or of the p53 dependent pathway that activates G.sub.1 arrest, is one of the major and most frequent molecular events (reviewed in Sherr, C. J. Genes Dev. 1998 12:2984-2991). p53 function may be compromised directly, via genetic mutation and/or deletion of the p53 gene (Baker et al. Science 1989 244:217-221) and indirectly by changes in the regulation or level of the MDM2 protein (Oliner et al. Nature 1992 358:80-83). The MDM2 gene, itself a transcriptional target of p53 (Barak et al. EMBO J 1993 12:461-468; Juven et al. Oncogene 1993 8:3411-3416; and Wu et al. Genes Dev. 1993 7:1126-1132), encodes a protein, MDM2, that is a critical negative regulator of p53 function (Finlay, C. A. Mol. Cell. Biol. 1993 13:301-306; Momand et al. Cell 1992 69:1237-1245). MDM2 was originally discovered as an oncogene that was amplified on mouse double minute chromosomes (Cahilly-Snyder et al. Cell Mol. Genet. 1987 13:235-244). MDM2 was later found to be amplified and overexpressed in a variety of human cancers (Ladanyi et al. Cancer Res. 1993 1:16-18; Reifenberger et al. Cancer Res. 1993 53:2736-2739). MDM2 binds to the transcriptional activation domain of p53 and thus inhibits this function of p53 (Chen et al. Mol. Cell Biol. 1993 13:4107-4114; Oliner et al. Nature 1993 362:857-860). Moreover, MDM2 binding to p53 regulates the stability of the p53 protein such that p53 is ubiquitinated and is then degraded by the proteasome (Haupt et al. EMBO J. 1996 15:1596-1606; Kubbutat et al. Nature 1997 387:299-303). This, together with the observed effect upon p53 function, has led to a model in which an autoregulatory loop connects MDM2 and p53 (Barak et al. EMBO J 1993 12:461-468; Wu et al. Genes Dev. 1993 7:1126-1132).

[0006] MDM2 inhibits both p53 mediated G.sub.1 arrest and apoptosis (Chen et al. Mol. Cell. Biol. 1996 16:2445-2452; Haupt et al. EMBO J 1996 15:1596-1606). p53 induces G.sub.1 arrest by promoting transcriptional upregulation of the CDK inhibitor p21.sup.waf1/cip1 (Waldmann et al. Cancer Res. 1995 55:5187-5190). Therefore, it is likely that MDM2 prevents p53 from inducing G.sub.1 arrest by inhibiting p53 dependent transcriptional activation. MDM2 can prevent p53-mediated apoptosis, and this has been shown to be dependent upon the ability of MDM2 to inhibit transcriptional repression by p53 (Hsieh et al. Mol. Cell 1999 3:81-93). Moreover, a previously identified interaction with RB (Xiao et al. Nature 1995 375:694-698) was shown to be able to regulate this effect. By binding to MDM2, RB forms a stable ternary complex with p53 and this prevents the MDM2 promoted degradation of p53. The ternary complex can promote p53 dependent apoptosis but not p53 mediated transactivation.

[0007] The autoregulatory relationship between p53 and MDM2 suggests that MDM2 overexpression may be oncogenic because of the resulting inactivation of p53 (Wu et al. Genes Dev. 1993 7:1126-1132). This conclusion is supported by studies of human tumors which show that in the majority of cases either p53 is mutated/deleted or MDM2 is overexpressed (Leach et al. Cancer Res. 1993 53:2231-2234). Studies of allelic knockouts of these genes in mice further support the ability to negatively regulate p53 being a primary function of MDM2. Mice that possess a homozygous deletion of MDM2 die at around day 5 of embryogenesis whereas, mice that possess homozygous deletion of both MDM2 and p53 are viable and develop normally (Jones et al. Nature 1995 378:206-208; Montes de Oca Luna et al. Nature 1995 378:203-206). No differences have been detected between these p53 -/- and p53 -/-, MDM2 -/- mice in terms of the rate or spectrum of tumors developed (Jones et al. Proc. Natl Acad. Sci. USA 1996 93:14106-14111). Also, no differences could be detected between the embryonic fibroblasts derived from these animals in terms of their growth or cell cycle characteristics.

[0008] Collectively, these observations suggest that the primary function of MDM2 may be to regulate p53 activity and perhaps, during normal development, this is indeed the case. However, the situation appears to be different when MDM2 is expressed at abnormally high levels. Experiments in which MDM2 was overexpressed in NIH3T3 cells have shown that naturally occurring splice variants of MDM2 that lack the ability to bind to p53 are still able to transform these cells (Sigala et al. Nat. Med. 1996 2:912-917). Further support for the suggestion that MDM2 has p53 independent effects is derived from studies of transgenic mice. Mice transgenic for an MDM2 gene expressed from a .beta.-lactoglobulin promoter, exhibited abnormal mammary development, with cells becoming polyploid together with a multinucleate morphology, suggestive of DNA synthesis in the absence of mitosis (Lundgren et al. Genes Dev. 1997 11:714-725). The same results were obtained in both p53 wild type animals and in animals with homozygous deletion of p53. In addition, recent studies using a different transgenic system with multiple copies of the whole MDM2 gene being used to generate mice that overexpress MDM2 from the MDM2 promoter, have shown that these animals develop a different spectrum of tumors c.f. p53 null mice (Jones et al. Proc. Natl Acad. Sci. USA 1998 95:15608-15612). The same effect of MDM2 overexpression was observed regardless of the p53 status of these animals. Finally, in support of the existence of p53 independent effects of MDM2 upon overexpression, it has recently been shown that MDM2 has the ability to abrogate the growth inhibitory activities of Transforming Growth Factor-Beta1 (TGF.beta.1). This effect was p53 independent in cells in culture (Sun et al. Science 1998 282:2270-2272). Taken together, these results all suggest that overexpression of MDM2 acts not only upon p53 but also on additional pathways.

[0009] Using a yeast two hybrid screen a novel gene encoding a protein referred to herein as MTBP for MDM2 (Two) Binding Protein has now been identified. MTBP is demonstrated herein to be capable of negatively regulating growth by inducing G.sub.1 arrest in a p53 independent manner. Further, this negative regulation of growth can be suppressed by MDM2.

SUMMARY OF THE INVENTION

[0010] An object of the present invention is to provide isolated nucleic acid sequences encoding a mammalian MDM2 binding protein.

[0011] Another object of the present invention is to provide vectors and host cells expressing vectors which comprise nucleic acid sequences encoding a mammalian MDM2 binding protein.

[0012] Another object of the present invention is to provide isolated polypeptide sequences of a mammalian MDM2 binding protein.

[0013] Another object of the present invention is to provide antibodies which target a mammalian MDM2 binding protein or a fragment thereof.

[0014] Yet another object of the present invention is to provide methods and compositions for modulating the G.sub.1 phase of the cell cycle via altering expression of a mammalian MDM2 binding protein or levels and/or activity of a mammalian MDM2 binding protein. Examples of compositions capable of modulating expression of a mammalian MDM2 binding protein or levels or activity of this protein include, but are not limited to, antisense agents targeted to a gene encoding a mammalian MDM2 binding protein, ribozymes targeted to a gene encoding a mammalian MDM2 binding protein, peptide mimics of a mammalian MDM2 binding protein, antibodies targeted to a mammalian MDM2 binding protein and modulators of MDM2 expression.

DETAILED DESCRIPTION OF THE INVENTION

[0015] The MDM2 protein, through its interaction with p53 plays an important role in the regulation of the G.sub.1 checkpoint of the cell cycle. In addition to binding to and inhibiting the transcriptional activation function of the p53 protein, MDM2 binds, inter alia, to RB, and the E2F-1/DP-1 complex and in so doing is believed to promote progression of cells into S-phase. Mice transgenic for MDM2 possess cells that have cell cycle regulation defects and develop an altered tumor profile independent of their p53 status. MDM2 also blocks the growth inhibitory effects of TGF-PI in a p53 independent manner.

[0016] The present invention relates to a novel growth regulatory molecule which is also the target of MDM2 mediated inhibition. Using a yeast two-hybrid screen, a gene that encodes a cellular protein, referred to herein as MDM2 binding protein (MTBP), that binds to MDM2 has now been identified. As demonstrated herein, MTBP induces G.sub.1 arrest. As also demonstrated herein, MTBP induction can in turn be blocked by MDM2. These results indicate MTBP to be involved in a p53-independent growth control pathway regulated, at least in part, by MDM2. It is believed that the protein of the present invention, MTBP provides an additional link between MDM2 function and regulation of the G.sub.1 cell cycle checkpoint. Thus, like a number of tumor suppressor proteins such as p53, E2F-1, RB and p19.sup.Arf which interact with MDM2, MTBP when expressed at high levels can induce growth arrest in vitro. It is therefore believed that the MTBP protein of the present invention may also be a tumor suppressor protein.

[0017] The MTBP gene and protein of the present invention was first identified in experiments wherein a full length cDNA for murine MDM2 was subcloned into a GAL4 DNA binding domain (GAL4-DBD) yeast expression construct and used to screen a murine T cell lymphoma cDNA library. A carboxy terminal cDNA from a novel gene fused to the activation domain of GAL4 (GAL-4-AD-3'MTBP) was found to interact with GAL4-DBD-MDM2 but not with GAL4-DBD. This interaction was confirmed in a different system wherein the in vitro translated cDNA from the yeast two hybrid screen (pBBV-3'MTBP) was mixed with recombinant His.sub.6-tagged MDM2. pBBV-3'MTBP encodes a peptide that can bind in vitro to MDM2. Sequence analysis of this cDNA demonstrated that it is a novel sequence that encodes a predicted peptide of 380 amino acids. Northern analysis demonstrated that the carboxy terminal cDNA hybridized to a mRNA of approximately 3 kb. The rest of the cDNA for this gene was cloned using a RACE-based strategy. Analysis of 5' RACE products from mRNA obtained from a murine B cell line showed several clones possessing an authentic 5' end; the clones were identical and terminated upstream of a single long open reading frame that was in frame with the clone identified in the yeast two hybrid screen. The sequence of this murine clone has been deposited in the Genbank data base (AJ278508) and is depicted herein as SEQ ID NO:1. This cDNA encodes a protein (depicted in SEQ ID NO:4) with a predicted Mw of 104 kD, and this gene is referred to herein as MDM2 (Two) binding protein or MTBP. We have also referred to this gene as p104.

[0018] A cDNA for human MTBP has also been isolated and sequenced. The sequence of this cDNA and the polypeptide encoded thereby are depicted in SEQ ID NO:3 and 2, respectively.

[0019] Database analysis identified two yeast genes, BOI1 and BOI2, whose protein products possessed significant homology to murine MTBP,(Bender et al. J. Cell Biol. 1996 133:879-894; Matsui et al. J. Cell Biol. 133:865-878). The two proteins encoded by the genes, Boi1p and Boi2p, exhibit an overall amino acid identity of 38% which is concentrated into four regions (I-IV) that possess identities of 71%, 65%, 78% and 69%, respectively. Both Boi1p and Boi2p inhibit growth in yeast when expressed at high levels. More specifically, these yeast proteins are part of a pathway that is required for maintenance of cell polarity which is necessary for bud formation. This pathway is regulated by Cdc42p, a member of the rho family of GTPases together with an associated GTP-GDP exchange factor Cdc24p (reviewed in Cabib et al. Annu. Rev. Biochem. 1998 67:307-333).

[0020] The homology between Boi1p, Boi2p and MTBP is 21.2% and 21% amino acid identity in alignments of 401 and 400 amino acids, respectively, and is entirely contained within the carboxy terminal regions of all three proteins. Interestingly, the growth inhibitory function of Boi2p is entirely contained within the carboxy terminal moiety of the protein. Thus, it is believed that MTBP may also play a role in the regulation of a Cdc42p dependent pathway.

[0021] Domain three of Boi2p is a proline rich region that is essential for binding to the second src homology region 3 (SH3-2) of Bem1p. The corresponding region of MTBP is also proline rich. Given that many SH3 binding proteins use a region that is rich in proline residues for binding (Grossman et al. Mol. Cell 1998 2:405-415), it is believed that the homologous region of MTBP may also bind to SH3 domains.

[0022] Apart from ESTs, no other substantial homologies to MTBP were identified.

[0023] Numerous sequence motifs were identified within MTBP. In particular, six potential nuclear localization signals were detected (both mono- and bi-partite), which are compatible with a nuclear localization for this protein.

[0024] The full length cDNA for murine MTBP was used to examine the pattern of expression of this gene by northern blot. MTBP is expressed in a variety of normal tissues with the highest levels of expression being in the thymus, testis and ovary and low or almost undetectable expression in peripheral blood lymphocytes. Thymus, testis and ovary are sites of high levels of cell proliferation and differentiation and moreover are the same tissues that exhibit the highest levels of expression of MDM2(Fakharzadeh et al. EMBO J 1991 10:1565-1569). MTBP was also detected in pancreas, heart, liver, skeletal muscle, liver and relatively low expression was detected in brain.

[0025] The cDNA corresponding to amino acids 515 to 894 of MTBP was initially identified via its interaction with MDM2. To test whether the interaction detected between this carboxy terminal region of MTBP and MDM2 also occurred between the full length form of the protein, an in vitro binding assay was performed using recombinant His.sub.6-MDM2 and in vitro translated MTBP. Both this fragment and the full length protein bound to MDM2 in an in vitro assay. This indicates that the interaction of MTBP with MDM2 is likely to be direct. Further confirmation of the interaction between these two proteins came from analysis of mammalian cells transfected with MDM2 and a carboxy-terminal hemagglutinin (HA) tagged form of MTBP. Further, immunoprecipitation with either an anti HA MAb or an anti MDM2 MAb followed by western blot analysis, demonstrated that the two proteins could be co-precipitated. These results indicate that the newly identified protein of the present invention, MTBP, binds specifically to MDM2 under these conditions.

[0026] The MDM2 protein has a number of highly conserved regions and the function of these is not fully understood (reviewed in Freedman et al. Cell Mol. Life Sci. 1999 55:96-107). The region of MDM2 that binds to MTBP was determined using a series of carboxy terminal deletion mutants of GAL4-DBD-MDM2. The ability of the mutants to interact in yeast with GAL4-AD-MTBP was assessed. An interaction was detected with all mutants containing the amino terminal 304 amino acids of MDM2 but not with shorter mutants. A p53 containing construct, GAL4-AD-p53, was also demonstrated to interact with these mutants as well as mutants 1-199 and 1-166, thus indicating that the failure of MTBP to bind to these mutants of MDM2 does not merely reflect lower expression or other conformational problems. The ability of in vitro translated full length MTBP and carboxy terminal MTBP (pBBV-3'MTBP) to bind to both full length MDM2 and to a mutant that lacks the first 166 amino acids (.DELTA.166) was also examined. .DELTA.166 does not bind to p53. However, it does bind to both full length MTBP and to pBBV-3'MTBP. Thus, taken together, these results indicate that a region of MDM2 bounded by amino acids 167 to 304 is sufficient to bind to MTBP. This region of MDM2 contains a nuclear localization signal, a region identified as a nuclear export signal and an acidic region. Results in both yeast and in vitro suggest that the carboxy terminal region of MTBP (amino acids 515-894) is sufficient for binding to MDM2. Thus, it is believed that MDM2 binds to the carboxy terminal 380 amino acids of MTBP and that the region of MDM2 from amino acid 167 to 304 is sufficient for the binding interaction to occur. This region of MDM2 overlaps with the MDM2 binding sites for p300, TFIIE, RB and p19.sup.arf(Hsieh et al. Mol. Cell 1999 3:81-93; Xiao et al. Nature 1995 375:694-698; Grossman et al. Mol. Cell 1998 2:405-415; Pomerantz et al. Cell 1998 92:713-723; Zhang et al. Cell 1998 92:725-734; and Thut et al. Genes Dev. 1997 11:1974-1986). For example, the p300 binding region of MDM2 lies between amino acids 102 and 222. p300 binding to MDM2 has been shown to be necessary for MDM2 mediated degradation of p53. The region responsible for interaction of MDM2 with the 34 kD subunit of TFIIE lies between MDM2 amino acids 50-222. This interaction has been implicated in the ability of MDM2 to function as a transcriptional repressor. The region of MDM2 responsible for binding to RB, amino acids 272 to 320, also overlaps with the MTBP binding region. It has recently been shown that by binding to MDM2, RB (preferentially the hypophosphorylated form) can form a ternary complex with p53 that is distinct from the p19.sup.Arf/MDM2/p53 complex and appears to perform a distinct function. Experiments to determine whether MTBP can compete with RB or TFIIE for binding to MDM2 are in progress. p19.sup.Arf binds to a region of MDM2 that lies between amino acids 154-221 (plus a further interaction point contained with the carboxy terminal 271 amino acids) and in so doing prevents MDM2 from targeting p53 for degradation.

[0027] Several MDM2 binding proteins are regulators of cell growth. Further, both of the MTBP partial homologues, BOI1 and BOI2, have been shown to have growth inhibitory activity. Therefore the effect of MTBP expression upon cell growth in culture was examined. In these experiments, it was found that, in contrast to the empty vector controls, when an expression construct for MTBP was transfected into U20S cells no colonies were produced. Since U20S cells harbor wild type p53, it was believed that the observed effect of MTBP expression may be dependent upon p53. To examine this, H1299 cells that possess a homozygous deletion of the p53 gene were transfected with MTBP and for comparison, with p53, expressed from the same vector and a vector control. Expression of MTBP was found to reduce colony formation to approximately the same degree as p53. These results indicate that MTBP possesses similar growth inhibitory properties in these p53 null cells compared to U20S cells that contained wild type p53. Similar experiments were performed with Saos-2 cells. Only a slight reduction of approximately three- to four-fold in colony formation was observed in these cells. Since, amongst other alterations, these cells lack both p53 and RB it is believed that the growth inhibitory effect of MTBP may require the presence of RB.

[0028] A possible explanation for the growth inhibitory effect observed is that MTBP acts as a general suppressor of expression, for example by Asquelching.apprxeq. or competing for the availability of other transcription factors. To investigate this H1299 cells were transfected with a .beta.-galactosidase expression construct and the levels of .beta.-galactosidase were measured by western blot, in the presence of either the MTBP or p53 expression constructs and also with the pCEP vector. MTBP had no effect, whereas p53 reduces the level of .beta.-galactosidase expression. A similar analysis was performed in a more quantitative fashion using FACs and no reduction in the number of positive cells or signal strength of CD20 when co-transfected with MTBP was observed. In contrast, a 10% reduction was seen in both with p53.

[0029] An alternative explanation for the growth inhibitory effect observed in U20S cells is the activation of p53. In these cells p53 is wild type but is transcriptionally inactive because of the presence of high levels of MDM2 (Florenes et al. J. Natl Cancer Inst. 1994 86:1297-1302). Thus, transfection of MTBP could simply compete with p53 for binding of MDM2 and in so doing release the MDM2 mediated block. To examine the possibility, levels of p53 itself and of the p53 transcriptional target, p21.sup.waf1/cip1 in the presence of MTBP were measured. No alteration in the levels of either of these proteins was seen. Taken together, these observations indicate that high level expression of MTBP has a negative effect upon cell growth and that this is independent of p53.

[0030] Cell cycle analysis was performed to examine the possibility that MTBP might act at a specific point in the cell cycle. Cells were co-transfected with an expression construct for CD20 so that only the transfected cell population need be analyzed (van den Heuvel, S. And Harlow, E. Science 1993 262:2050-2054). At any given time in a rapidly cycling population of the cells which were used, typically 50% will have a 2N DNA complement. Initial experiments indicated that MTBP expression induces an increase in the percentage of cells with a 2N DNA complement. To examine this further and to facilitate detection of effects in this region of the cell cycle, cells were treated with the microtubule-disrupting drug nocodazole. The result of MTBP expression in U20S cells was compared to cells that were transfected with the vector alone and also with cells transfected with p53 expressed from the same vector. p53 expression induced an increase in the percentage of cells in G.sub.1 from 15.9 to 24.9%. MTBP expression induced a similar effect with an increase to 24.0%. As with the growth inhibition that was detected, the possibility existed that the effect observed for MTBP expression upon the cell cycle was in some manner dependent upon p53. To examine this, a similar experiment was performed with H1299 cells. It was found that both p53 and MTBP induce a comparable increase in the percentage of cells with a 2N DNA content from 22.4 to 35.8 and 38.3%, respectively. These experiments were performed on at least three occasions and similar results were obtained each time. These results indicate that p53 is not required for MTBP mediated cell cycle arrest.

[0031] The effect of MTBP expression upon Saos-2 cells was also examined since these cells appear to be resistant to the growth inhibitory effect of MTBP expression. Analysis of these cells clearly demonstrated that expression of MTBP had no effect upon their cell cycle. These results indicate that the ability of MTBP to inhibit colony formation is consistent with its ability to alter the cell cycle. Thus, from these experiments it is believed that MTBP induces G.sub.1 arrest in a p53 independent manner.

[0032] MDM2 blocks p53 mediated cell cycle arrest (Chen et al. Mol. Cell. Biol. 1996 16:2445-2452). Accordingly, its ability to inhibit the effect of MTBP was also examined. Little effect upon the level of MTBP protein was observed. However, it was found that MDM2 expression resulted in complete abrogation of the effect of MTBP in U20S cells. Thus, it is believed that MDM2 mediated inhibition of the MTBP induced cell cycle arrest does not require degradation of MTBP. Further, it is believed that MDM2 suppresses the G.sub.1 arrest mediated by MTBP and since this does not require degradation of MTBP, it seems likely that the effect is a consequence of the ability of MDM2 to bind directly to MTBP.

[0033] In addition to providing nucleic acid sequences and polypeptides encoded thereby of mammalian MTBP, the present invention also relates to vectors expressing this new protein as well as host cells comprising such vector which express these new proteins. Various vectors and host cells known in the art can be used and selection of appropriate vectors and host cells for expression of MTBP can be performed routinely by those of skill in the art.

[0034] Mammalian MTBP polypeptides prepared via these vectors and host cells or synthetically are useful in raising antibodies targeted to the mammalian MTBP polypeptides. Methods for raising both polyclonal and monoclonal antibodies are well known to those of skill in the art. Thus, raising antibodies specific for the mammalian MTBP polypeptides of the present invention can be performed routinely by those skilled in the art. Such antibodies are not only useful in further elucidation of the function of this protein, but also in methods for detecting these polypeptides and in methods for identifying modulators of the expression and/or activity of this proteins.

[0035] The vectors and host cells of the present invention are also useful in the development of methods and compositions for modulating the G.sub.1 phase of the cell cycle via altering expression of MTBP or levels and/or activity of MTBP. Examples of compositions capable of modulating expression of MTBP or levels or activity of MTBP include, but are not limited to antisense agents targeted to MTBP, ribozymes targeted to MTBP, peptide mimics of MTBP and modulators of MDM2 expression. Identification and development, as well as testing or screening, of such compositions can be performed routinely by those of skill in the art based upon the teachings provided herein relating to these new MTBP genes and proteins and their activity. Compositions which modulate MTBP levels or activity may be useful in suppressing tumors.

[0036] The following nonlimiting examples are provided to further illustrate the present invention.

EXAMPLES

Example 1

Cell culture, Plasmids and Antibodies

[0037] Cells were grown in RPMI supplemented with 10% fetal calf serum, and 1% penicillin, streptomycin, and neomycin (Gibco-BRL). H1299 (ATCC# CRL-5803), U20S (ATCC# HTB-96) and Saos-2 (ATCC# HTB-85) cells were obtained from the ATCC. pGAL4-DBD-MDM2 encodes full length mouse MDM2 cloned in-frame with the GAL4 DNA binding domain (DBD) of pGBT9 (Clontech). pGAL4-AD-MTBP-3' contains the carboxy terminal 380 amino acids of MTBP cloned into the XhoI site of pACT (Clontech). pBBV was generated by inserting an oligonucleotide containing the black beetle virus ribosome binding sequences from pBD7 (Dasmahapatra et al. Nucleic Acids Res. 1987 15:3933) into the HindIII and EcoRV sites of pcDNAI Neo (Invitrogen). pSK-BBV was generated by subcloning a HindIII/BglII DNA fragment containing the black beetle virus ribosome binding sequence from pBBV into the HindIII and BamHI sites of plasmid pBluescript SKII+ (Stratagene). Clones identified as encoding candidate MDM2 interacting molecules in the yeast two hybrid screen analysis were amplified from pACT with GAD5 (5' gag aga gat atc gcc aat ttt aat caa agt ggg aat att 3' (SEQ ID NO:11)) and GAD3 (5' gag aga gcg gcc get ttc agt atc tac gat tca tag atc tc 3' (SEQ ID NO:12)) primers and subcloned into the EcoRV and NotI sites of pBBV. pBBV-MTBP-3' was constructed by subcloning this PCR generated fragment from pGAL4-AD-MTBP-3' into pBBV. The pSK-MTBP construct used for in vitro translation of full length MTBP was made by sub-cloning the NotI fragment from pCEP-MTBP into the NotI site of pSK-BBV. Recombinant His.sub.6-tagged MDM2 (pQE32-MDM2) was generated by cloning an EcoRV/XhoI fragment from pBBV-MDM2 encoding the full length murine MDM2 cDNA into the SmaI site of pQE32 (Qiagen). Recombinant His.sub.6-tagged .DELTA.166 (pQE31-.DELTA.166 MDM2) contains a DNA fragment of murine MDM2 lacking the first 166 amino acid residues. The fragment was amplified from pCMVNeoBam-Mdm2 by PCR with primers MDM2 PstI (5' gag aga ctg cag gag aac aca gat gag cta cct gg 3' (SEQ ID NO:5)) and MDM2 HindIII (5' gag aga aag ct gtc agc tag ttg aag taa ctt agc a 3' (SEQ ID NO:6)) using rTth-XL polymerase (Perkin-Elmer) and cloned into the PstI and HindIII sites of pQE31 (Qiagen). MTBP (pCEP-MTBP-HA) contains full length murine cDNA for MTBP excised from the pCR-XL-TOPO vector and cloned into the NotI site of pCEP (Invitrogen). p53 contains full length human p53 cloned into the pCEP vector. The p53 antibody Ab-1 (PAb421), the MDM2 antibody used for western blotting Ab-1 (IF2) and the anti-.beta.-galactosidase antibody Ab-1 (200-193) were purchased from Oncogene Research Products. The MDM2 antibody used for immunoprecipitation, SMP14, and the antibody used to detect p21.sup.waf1/cip1 (F-5) were purchased from Santa Cruz Biotechnology, Inc. while the anti-Hemagglutinin A (HA) antibodies (12CA5 and 16BI2) used to detect HA-tagged MTBP were purchased from Roche Molecular Biologicals and BAbCO, respectively. The anti-CD20 antibody leu16 was purchased from Becton Dickinson and the anti-mouse-IgG-FITC conjugate was obtained from Pierce.

Example 2

Yeast Two Hybrid Screen

[0038] The MATCHMAKER system (Clontech) was used to screen a mouse T-cell lymphoma library (ML4001AE) and to assess interactions between the GAL4-DBD-MTBP and GAL4-AD-MDM2 deletion mutants.

Example 3

Cloning and Analysis of MTBP

[0039] The Marathon RACE system (Clontech) was used to amplify the 5' and 3' ends of MTBP from a murine B cell cDNA. Total cellular RNA was prepared from murine SP2 (ATCC# CRL-1646) cells using RNAZOL (MBI) and poly A+ RNA was isolated from this using OLIGOTEX beads (Qiagen). 5-prime RACE was performed using the gene specific oligonucleotides GSP-1 (5'tga aga ata agg ttc aac tgt acc 3' (SEQ ID NO:7)) and GSP-2 (5' cag ctt tca cgg tgt ctg ttt g 3' (SEQ ID NO:8)). PCR was performed with rTth-XL and products were cloned into pCR2.1 (Invitrogen). 3-prime RACE was also performed and confirmed the termination codon identified in the yeast two hybrid screen. Sequencing was performed using dye terminators and an ABI-373 sequencer.

[0040] Homology to BO11 and BO122 was identified using the FASTA program to examine the Saccharomyces cerevisiae database at Stanford University. The full length cDNA for MTBP was prepared by PCR amplification with the oligonucleotides MTBP-5'-NotI (5' gag aga gcg gcc gcg gcg cga aga gga tgg atc ggt act tgc tg 3' (SEQ ID NO:9)) and MTBP-3'-HA-NotI (5'gag aga gcg gcc gcc tac agg gag gcg taa tcg ggc aca tcg tag ggg tat ttc ttg ctc atc ttt tct acc acc 3' (SEQ ID NO:10)) using rTth-XL and the product was cloned into PCR-XL-TOPO (Invitrogen).

Example 4

In vitro Binding and Immunoassays

[0041] For in vitro binding assays, MDM2 or .DELTA.166-MDM2 were expressed in XL-1 bacteria (Stratagene) from the pQE32-MDM2 and pQE31-.DELTA.166-MDM2 constructs, respectively, captured on Ni.sup.++-agarose (Qiagen) and washed with buffers B, C and D as described by the manufacturer. Prior to all binding reactions, protein captured onto beads was run on a SDS-polyacrylamide gel and analyzed by both western blotting and staining with coomassie blue. Washed beads (100 .mu.l) were then mixed with 10 .mu.l of in vitro translated protein (TNT, Promega) for 3 hours at 30 C, followed by washing three times in Dignam's buffer D supplemented with 75 mM imidazole (Dignam et al. Nucleic Acids Res. 1983 11:1475-1489). Beads were then resuspended in loading buffer and analyzed by SDS-PAGE and fluorography using AMPLIFY (Amersham Pharmacia).

[0042] Cells were transfected by either the calcium phosphate-DNA co-precipitation method (Sambrook et al. 1989 Molecular Cloning, A Laboratory Manual 2nd edition Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) or by using FUGENE-6 (Roche) according to the manufacturer's instructions. For immunoprecipitation experiments cells were typically transfected with 10 .mu.g of each plasmid and proteins were extracted 48-72 hours post transfection. Transfected cells were harvested and the cell pellet lysed in IP buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 10% glycerol, 0.1% TRITON-X100, 0.5 mg/ml BSA) in the presence of the protease inhibitors: 1-2 .mu.g/ml aprotinin, 1-2 .mu.g/ml leupeptin, 1 .mu.g/ml pepstatin A, 100 .mu.g/ml soybean trypsin inhibitor (Roche) and 1 mM phenylmethylsulfonylfluoride, for 10 minutes, on ice. The lysate was clarified by centrifugation for 10 minutes at 4 C and the concentration of total proteins determined by Bio-Rad Protein Assay (BioRad). Between 1 and 5 mg of protein was then pre-cleared by incubation with 50 .mu.l of protein G-sepharose (Amersham Pharmacia) for 1 hour at 4 C. Pre-cleared lysate was incubated with 1 .mu.g of primary antibody for 1 hour at 4 C, followed by incubation with 50 .mu.l of protein G-sepharose for 2 hours at 4 C. Immunoprecipitated complexes were washed three times with IP buffer, resuspended in 30 .mu.l of protein sample buffer (0.1 M

[0043] Tris-HCl, pH 6.8, 4% SDS, 0.2% bromophenol blue, 20% glycerol, 0.5 M DTT) and subjected to SDS-PAGE followed by transfer to Hybond-ECL membrane (Amersham Pharmacia). Following incubation with primary antibodies and subsequently with anti-mouse-IgG-HRP (Amersham Pharmacia), the signal was detected by enhanced chemiluminescence with RENAISSANCE (NEN).

Example 5

FACS, Cell Cycle Analysis and Colony Assays

[0044] Saos-2 and U20S cells were transfected using FUGENE-6 with the indicated plasmids. Cells were harvested and analyzed by FACS essentially as described by Chen et al. Mol. Cell Biol. 1996 16:2445-2452. Briefly, nocodazole was added to the indicated cells at 50 ng/ml for 12 hours prior to harvesting. Cells were harvested 48-72 hours after the addition of FUGENE-DNA complexes and washed in Dulbecco's phosphate buffered saline containing 1% bovine serum albumin (PB). CD20 positive cells were detected using anti-CD20 antibody and an anti-mouse-IgG-FITC conjugate. Cells were fixed in ethanol and then stained in propidium iodide. Cells were analyzed using a FACSCAN (Becton Dickinson) and LYSIS-II software. H1299 cells were transfected using either the calcium phosphate precipitation method or FUGENE-6. Typically for the calcium phosphate precipitate procedure, 24 hours after removal of precipitates, hygromycin B (Roche) was added to a final concentration of 200 .mu.g/ml. Cells were maintained under selective conditions for 72 hours, washed and refed with hygromycin-free complete media. Nocodazole (Sigma) was added as indicated at a concentration of 20 ng/ml, 16 hours before cells were harvested for analysis.

[0045] For colony formation assays, cells were transfected with the indicated plasmids and 24-48 hours after addition of DNA, hygromycin-B was added at a final concentration of 200 .mu.g/ml. Cells were refed every three days with media containing Hygromycin-B until colonies were visible. For some experiments cells were stained with Giemsa.

Sequence CWU 1

1

1215768DNAMus sp. 1aatttcccac aataattggc gggaaaaccg acgtaggaca ttattttgca gccaacctga 60gcggcaggaa accggaagtg cacgttgcgc gcggctcttt tgagacctaa cactgagtcc 120gggcgcgaag aggatggatc ggtacttgct gctggtcacc tggagggaag ggaagtttcg 180atccgtggcg ggtggggaga tcgagcctgg cactgaggcg acatccctgg agagcaccga 240caaacagccc gatttgaccg caaccaatat ttatcacctc ttgaagagaa gcatcagcga 300ttcaatccat ccagatgaca gtacattccc tgcttgttca gtgggtggca cacctcattc 360caggaagtgg ttctttgcgg tgcaagcaat atgcggattt taccagtttt gtagttctga 420ttggcaagag atacattttg atgctgaaaa agataaaatc gaagatgttc ttcaagcaaa 480tatagaagaa cgtcagagtg ctgttgagtg ttttgaagaa gatgacagta atagcaggga 540atccttaccc ttggctgacg tatatgaaga atcagcagaa aatttgcatc agttatcaga 600caagcttccc gctcctggta gagcaatgat agacataata ctgttgcctt ctgacaaaga 660ccctcgtaag ctaaaagagt gcttgcccat tgtaggggcc ttgaaacatc tgaaggaatg 720gcattcagca aaagttatca tagcaggaag ttactgtgag ataaattgtc agaaaattgc 780tgaatacctt tcagctagtg ttgtgccttt agaagaattc agaaatgcca ttgatccgag 840ggaagtgtgg cggggagaga ttcagatgcg ggaacgaaag tttggatttg aaattagttt 900acctgaattt tgtttaaaag gagttacgcc tacgaatgtt agtgcgtata atttaaatac 960ctgcttcctt gccaagaaga tagcatcttc taaggttttc cattattatg gtcctgcttt 1020ggaatttgtg cagatgataa aactatcaga tcttccctcc tgttacatgt cggatatcga 1080gtttgagtta gaggtgactg ggcactgcac gaggcagaat tccatgctgc tgttggaaca 1140gatctcttcc ctgtgtggca aggttggtgc tctctttgtg ctgccgtgta ctgttagcaa 1200tgtactcatc ccacctccca gccaactggc ctcaagaaag tggaaggaat acatggctaa 1260gaagcccaag accatcagtg ttcccgatgt tgccgtgaag ggagagtttt ctggctatca 1320tctcctgctg caaggtatgg gcaagagaaa atgcagagcc accctgctgc actcggccag 1380ccagatcaat ggctcgtttg cactcagtgt cattcatggg aagatgaaaa caaaggcagg 1440agaagccaga ccgagtttcc cctttgactt ctcgtcactc ccaaggtttt cggaggagca 1500ggttttacag agagagaaac aattagccag ctttcaagtt ttggctttga aagaatgcct 1560gaaaagaaga aaggctgcaa accagcccga agccttttct gccgatgaac tcaaaagtct 1620gttggcactc acgagggagc gcttcctagg tcactttgat gttctcccca ctgaagcagc 1680tttagcacaa acagacaccg tgaaagctgc cggcgtggta aatgatgacg gtacagttga 1740accttattct tcaagcctaa tggaaaccaa tcctctggaa tggccagaaa gacatgttct 1800tcagaattta gaaacttctg aaaaagctaa acaaaaaatg agaactggct cattaccgcg 1860ttcgtctgaa cagttgctgg gccataaaga gggtccccgg gactcactca cattactgga 1920tgctaaggag ctgctgaagt atttcacctc ggatgggtta ccagtcggag atcttcagcc 1980gttacacatt caacgggggg aaaagccttt tgttttgaca ccagagctta gtcctggaaa 2040acttcaggtc ttaccttttg aaaaagcctc ggaatgccat taccacggga ttgaatattg 2100cctggatgac caaaaagctt tagaaagaga tgggggattt tctgaacttc agtcgcgcct 2160tattcgttac gagacgcaga ccacctgcac cagagacagt tttccagtcc ccaccgtgct 2220gagccctctt ccatcccctg cagttctgtc agagcctcaa agtgtccccg aaggagaagc 2280actgcaaggc gaactgagga ctgaagtttc tggattgaag cggagatcta aagaccccag 2340ctgcctgtac ccccagaaaa gacttacgag atcagaaagt tctgattgtt tgccttccca 2400agcgagttgc aatagtaatc atcaccatca cacagggaaa cccaggaagc ctcaggcaga 2460gcgctgtgtg tcagggcttc ctctgcctgg ccgggaagct tccaaagata cctcaaagac 2520cagttcagga caaaaacgag cacacgaatc aaaatcatca aagcaaatga aggaatcacg 2580gtcccagaaa cacacacgga tgctgaagga ggtggtaaaa gacaccttga agaggcacca 2640catcacggag gcccatgaga gcttcacggc ctgcagccag agactcttcg acatctccaa 2700aatttcccac aataattggc gggaaaaccg acgtaggaca ttattttgca gccaacctga 2760gcggcaggaa accggaagtg cacgttgcgc gcggctcttt tgagacctaa cactgagtcc 2820gggcgcgaag aggatggatc ggtacttgct gctggtcacc tggagggaag ggaagtttcg 2880atccgtggcg ggtggggaga tcgagcctgg cactgaggcg acatccctgg agagcaccga 2940caaacagccc gatttgaccg caaccaatat ttatcacctc ttgaagagaa gcatcagcga 3000ttcaatccat ccagatgaca gtacattccc tgcttgttca gtgggtggca cacctcattc 3060caggaagtgg ttctttgcgg tgcaagcaat atgcggattt taccagtttt gtagttctga 3120ttggcaagag atacattttg atgctgaaaa agataaaatc gaagatgttc ttcaagcaaa 3180tatagaagaa cgtcagagtg ctgttgagtg ttttgaagaa gatgacagta atagcaggga 3240atccttaccc ttggctgacg tatatgaaga atcagcagaa aatttgcatc agttatcaga 3300caagcttccc gctcctggta gagcaatgat agacataata ctgttgcctt ctgacaaaga 3360ccctcgtaag ctaaaagagt gcttgcccat tgtaggggcc ttgaaacatc tgaaggaatg 3420gcattcagca aaagttatca tagcaggaag ttactgtgag ataaattgtc agaaaattgc 3480tgaatacctt tcagctagtg ttgtgccttt agaagaattc agaaatgcca ttgatccgag 3540ggaagtgtgg cggggagaga ttcagatgcg ggaacgaaag tttggatttg aaattagttt 3600rcctgaattt tgtttaaaag gagttacgcc tacgaatgtt agtgcgtata atttaaatac 3660ctgcttcctt gccaagaaga tagcatcttc taaggttttc cattattatg gtcctgcttt 3720ggaatttgtg cagatgataa aactatcaga tcttccctcc tgttacatgt cggatatcga 3780gtttgagtta gaggtgactg ggcactgcac gaggcagaat tccatgctgc tgttggaaca 3840gatctcttcc ctgtgtggca aggttggtgc tctctttgtg ctgccgtgta ctgttagcaa 3900tgtactcatc ccacctccca gccaactggc ctcaagaaag tggaaggaat acatggctaa 3960gaagcccaag accatcagtg ttcccgatgt tgccgtgaag ggagagtttt ctggctatca 4020tctcctgctg caaggtatgg gcaagagaaa atgcagagcc accctgctgc actcggccag 4080ccagatcaat ggctcgtttg cactcagtgt cattcatggg aagatgaaaa caaaggcagg 4140agaagccaga ccgagtttcc cctttgactt ctcgtcactc ccaaggtttt cggaggagca 4200ggttttacag agagagaaac aattagccag ctttcaagtt ttggctttga aagaatgcct 4260gaaaagaaga aaggctgcaa accagcccga agccttttct gccgatgaac tcaaaagtct 4320gttggcactc acgagggagc gcttcctagg tcactttgat gttctcccca ctgaagcagc 4380tttagcacaa acagacaccg tgaaagctgc cggcgtggta aatgatgacg gtacagttga 4440accttattct tcaagcctaa tggaaaccaa tcctctggaa tggccagaaa gacatgttct 4500tcagaattta gaaacttctg aaaaagctaa acaaaaaatg agaactggct cattaccgcg 4560ttcgtctgaa cagttgctgg gccataaaga gggtccccgg gactcactca cattactgga 4620tgctaaggag ctgctgaagt atttcacctc ggatgggtta ccagtcggag atcttcagcc 4680gttacacatt caacgggggg aaaagccttt tgttttgaca ccagagctta gtcctggaaa 4740acttcaggtc ttaccttttg aaaaagcctc ggaatgccat taccacggga ttgaatattg 4800cctggatgac caaaaagctt tagaaagaga tgggggattt tctgaacttc agtcgcgcct 4860tattcgttac gagacgcaga ccacctgcac cagagacagt tttccagtcc ccaccgtgct 4920gagccctctt ccatcccctg cagttctgtc agagcctcaa agtgtccccg aaggagaagc 4980actgcaaggc gaactgagga ctgaagtttc tggattgaag cggagatcta aagaccccag 5040ctgcctgtac ccccagaaaa gacttacgag atcagaaagt tctgattgtt tgccttccca 5100agcgagttgc aatagtaatc atcaccatca cacagggaaa cccaggaagc ctcaggcaga 5160gcgctgtgtg tcagggcttc ctctgcctgg ccgggaagct tccaaagata cctcaaagac 5220cagttcagga caaaaacgag cacacgaatc aaaatcatca aagcaaatga aggaatcacg 5280gtcccagaaa cacacacgga tgctgaagga ggtggtaaaa gacaccttga agaggcacca 5340catcacggag gcccatgaga gcttcacggc ctgcagccag agactcttcg acatctccaa 5400gttctacctg aaggatctga aaacatcaag gggtctcttt gaagaaatga agaaaacagc 5460caacaacaat gtggtgcagg tgatcgaatg ggtggtagaa aagatgagca agaaataagc 5520tactgcaccc cctttcctta gagaactgta cacagcctgt aaactttccc caaagagcag 5580atgcctcatt tgtaaggagg gtccatggtg tcctgtatct atatagtttt gtgacactca 5640gttattttca actaatgcac ttatttttac agttttacaa tattttaatc tatgaaattt 5700ttatatattt taaatgtttt aactcttggt tatttaattg ttaaataaac agtaaggtaa 5760atctcgag 57682914PRTHomo sapiens 2Met Asp Arg Tyr Leu Leu Leu Val Ile Glu Trp Gly Glu Gly Lys Phe 1 5 10 15Pro Ser Ala Ala Ser Arg Glu Ala Glu His Gly Pro Glu Val Ser Ser 20 25 30Gly Glu Gly Thr Glu Asn Gln Pro Asp Phe Thr Ala Ala Asn Val Tyr 35 40 45His Leu Leu Lys Arg Ser Ile Ser Ala Ser Ile Asn Pro Glu Asp Ser 50 55 60Thr Phe Pro Ala Cys Ser Val Gly Gly Ile Pro Gly Ser Lys Lys Trp 65 70 75 80Phe Phe Ala Val Gln Ala Ile Tyr Gly Phe Tyr Gln Phe Cys Ser Ser 85 90 95Asp Trp Gln Glu Ile His Phe Asp Thr Glu Lys Asp Lys Ile Glu Asp 100 105 110Val Leu Gln Thr Asn Ile Glu Lys Cys Leu Gly Ala Val Glu Cys Phe 115 120 125Glu Glu Glu Asp Ser Asn Ser Arg Glu Ser Leu Ser Leu Ala Asp Leu 130 135 140Tyr Glu Glu Ala Ala Glu Asn Leu His Gln Leu Ser Asp Lys Leu Pro145 150 155 160Ala Pro Gly Arg Ala Met Val Asp Ile Ile Leu Leu Leu Ser Asp Lys 165 170 175Asp Pro Pro Lys Leu Lys Asp Tyr Leu Pro Thr Val Gly Ala Leu Lys 180 185 190His Leu Arg Glu Trp Tyr Ser Ala Lys Ile Thr Ile Ala Gly Asn His 195 200 205Cys Glu Ile Asn Cys Gln Lys Ile Ala Glu Tyr Leu Ser Ala Asn Val 210 215 220Val Ser Leu Glu Asp Leu Arg Asn Val Ile Asp Ser Lys Glu Leu Trp225 230 235 240Arg Gly Lys Ile Gln Ile Trp Glu Arg Lys Phe Gly Phe Glu Ile Ser 245 250 255Phe Pro Glu Phe Cys Leu Lys Gly Val Thr Leu Lys Asn Phe Ser Thr 260 265 270Ser Asn Leu Asn Thr Asp Phe Leu Ala Lys Lys Ile Ile Pro Ser Lys 275 280 285Asp Lys Asn Ile Leu Pro Lys Val Phe His Tyr Tyr Gly Pro Ala Leu 290 295 300Glu Phe Val Gln Met Ile Lys Leu Ser Asp Leu Pro Ser Cys Tyr Met305 310 315 320Ser Asp Ile Glu Phe Glu Leu Gly Leu Thr Asn Ser Thr Lys Gln Asn 325 330 335Ser Val Leu Leu Leu Glu Gln Ile Ser Ser Leu Cys Ser Lys Val Gly 340 345 350Ala Leu Phe Val Leu Pro Cys Thr Ile Ser Asn Ile Leu Ile Pro Pro 355 360 365Pro Asn Gln Leu Ser Ser Arg Lys Trp Lys Glu Tyr Ile Ala Lys Lys 370 375 380Pro Lys Thr Ile Ser Val Pro Asp Val Glu Val Lys Gly Glu Cys Ser385 390 395 400Ser Tyr Tyr Leu Leu Leu Gln Gly Asn Gly Asn Arg Arg Cys Lys Ala 405 410 415Thr Leu Ile His Ser Ala Asn Gln Ile Asn Gly Ser Phe Ala Leu Asn 420 425 430Leu Ile His Gly Lys Met Lys Thr Lys Thr Glu Glu Ala Lys Leu Ser 435 440 445Phe Pro Phe Asp Leu Leu Ser Leu Pro His Phe Ser Gly Glu Gln Ile 450 455 460Val Gln Arg Glu Lys Gln Leu Ala Asn Val Gln Val Leu Ala Leu Glu465 470 475 480Glu Cys Leu Lys Arg Arg Lys Leu Ala Lys Gln Pro Glu Thr Val Ser 485 490 495Val Ala Glu Leu Lys Ser Leu Leu Val Leu Thr Arg Lys His Phe Leu 500 505 510Asp Tyr Phe Asp Ala Val Ile Pro Lys Met Ile Leu Arg Lys Met Asp 515 520 525Lys Ile Lys Thr Phe Asn Ile Leu Asn Asp Phe Ser Pro Val Glu Pro 530 535 540Asn Ser Ser Ser Leu Met Glu Thr Asn Pro Leu Glu Trp Pro Glu Arg545 550 555 560His Val Leu Gln Asn Leu Glu Thr Phe Glu Lys Thr Lys Gln Lys Met 565 570 575Arg Thr Gly Ser Leu Pro His Ser Ser Glu Gln Leu Leu Gly His Lys 580 585 590Glu Gly Pro Arg Asp Ser Ile Thr Leu Leu Asp Ala Lys Glu Leu Leu 595 600 605Lys Tyr Phe Thr Ser Asp Gly Leu Pro Ile Gly Asp Leu Gln Pro Leu 610 615 620Pro Ile Gln Lys Gly Glu Lys Thr Phe Val Leu Thr Pro Glu Leu Ser625 630 635 640Pro Gly Lys Leu Gln Val Leu Pro Phe Glu Lys Ala Ser Val Cys His 645 650 655Tyr His Gly Ile Glu Tyr Cys Leu Asp Asp Arg Lys Ala Leu Glu Arg 660 665 670Asp Gly Gly Phe Ser Glu Leu Gln Ser Arg Leu Ile Arg Tyr Glu Thr 675 680 685Gln Thr Thr Cys Thr Arg Glu Ser Phe Pro Val Pro Thr Val Leu Ser 690 695 700Pro Leu Pro Ser Pro Val Val Ser Ser Asp Pro Gly Ser Val Pro Asp705 710 715 720Gly Glu Val Leu Gln Asn Glu Leu Arg Thr Glu Val Ser Arg Leu Lys 725 730 735Arg Arg Ser Lys Asp Leu Asn Cys Leu Tyr Pro Arg Lys Arg Leu Val 740 745 750Lys Ser Glu Ser Ser Glu Ser Leu Leu Ser Gln Thr Thr Gly Asn Ser 755 760 765Asn His Tyr His His His Val Thr Ser Arg Lys Pro Gln Thr Glu Arg 770 775 780Ser Leu Pro Val Thr Cys Pro Leu Val Pro Ile Pro Ser Cys Glu Thr785 790 795 800Pro Lys Leu Ala Thr Lys Thr Ser Ser Gly Gln Lys Ser Met His Glu 805 810 815Ser Lys Thr Ser Arg Gln Ile Lys Glu Ser Arg Ser Gln Lys His Thr 820 825 830Arg Ile Leu Lys Glu Val Val Thr Glu Thr Leu Lys Lys His Ser Ile 835 840 845Thr Glu Thr His Glu Cys Phe Thr Ala Cys Ser Gln Arg Leu Phe Glu 850 855 860Ile Ser Lys Phe Tyr Leu Lys Asp Leu Lys Thr Ser Arg Gly Leu Phe865 870 875 880Glu Glu Met Lys Lys Thr Ala Asn Asn Asn Ala Val Gln Val Lys Lys 885 890 895Leu Phe Pro Arg Asn Tyr Ile Gln Leu Asn Trp Tyr Phe Ile Ser Gly 900 905 910Leu Leu32975DNAHomo sapiens 3ggatgtggaa gccgagacct aaagttgggg ggtgatctct gaggagatgg atcggtacct 60gctgctggtg atctgggggg aaggaaaatt cccgtcggcg gccagtaggg aggcagaaca 120tgggccagag gtgtcgtcgg gtgagggtac tgagaatcag ccggacttca cagcagcaaa 180tgtttatcac ctcttgaaaa gaagcattag tgcttcaatt aatccagaag atagtacttt 240ccctgcctgt tcagtgggag gtatacctgg ttccaagaag tggttctttg cagtgcaggc 300aatatatgga ttttatcagt tttgtagttc tgattggcaa gagatacatt ttgatacaga 360aaaagataaa attgaagatg ttcttcaaac gaatatcgaa aaatgtttgg gtgctgttga 420gtgttttgaa gaagaagaca gtaatagcag ggaatcatta tccttggctg atctctatga 480agaagctgca gaaaatttgc atcagctgtc agacaagctt cctgctcctg gtagagcaat 540ggtagatata atactgttgc tttctgacaa agatcctcct aaattgaaag actatttacc 600tactgtagga gcattaaaac atttgagaga atggtattca gcaaagatca ctatagcagg 660aaatcattgt gaaataaact gtcagaaaat tgcagaatac ctttctgcta atgttgtatc 720tttagaagat ctcagaaatg ttattgactc aaaggaatta tggaggggga aaatacagat 780atgggaaaga aagtttggat ttgaaattag ctttcctgaa ttttgtttaa agggagtcac 840acttaagaat tttagtactt ctaatttaaa tactgacttc cttgccaaaa agatcatacc 900atcaaaggat aagaatattt tgccaaaggt tttccattat tatggccctg ctttagaatt 960tgtgcagatg ataaaattat cagatctacc ctcctgctat atgtcggata ttgaatttga 1020gttaggattg acaaacagta ccaaacagaa ttctgtgttg ctgttggagc agatttcttc 1080tctgtgtagc aaggttggtg ctctttttgt attgccatgt accattagta acatactgat 1140tccacctccc aaccaactca gttcaagaaa atggaaggaa tatatagcta aaaagcctaa 1200aacaatcagt gttccagatg ttgaagtgaa aggagagtgt tctagctatt atctcttgtt 1260acaaggtaat ggcaatagaa gatgtaaagc cacattgatt cactcagcca accagatcaa 1320tggctcattt gcactcaatt taattcatgg aaagatgaaa acaaagacag aagaagccaa 1380attgagtttt ccttttgact tattatcact tccacatttt tctggggagc agattgtaca 1440gagagagaaa cagttagcta atgttcaagt tttagctttg gaagaatgcc taaaaagacg 1500aaagttggca aagcagcctg aaacagtttc tgttgctgaa ctcaaaagtc tgttagtact 1560cacaaggaaa cactttttag attattttga tgctgtgatt cctaaaatga ttctaagaaa 1620gatggacaaa attaaaacct tcaatatatt aaatgatttt agtccagtgg aacctaattc 1680ctcaagtcta atggaaacca atcctctgga atggccagaa aggcatgttc ttcaaaattt 1740ggaaactttt gaaaaaacta aacaaaaaat gagaactggt tcattacctc attcatctga 1800acagttgctg ggccacaaag agggtcctcg ggactcaatc acattgttgg atgctaaaga 1860attgctgaag tactttacct cagatggatt acccattgga gatcttcaac ctttaccgat 1920tcaaaagggg gaaaagactt ttgttttgac accagaactt agtcctggga aacttcaggt 1980cttacctttt gagaaagcct cagtatgtca ttatcatgga attgaatatt gcttggatga 2040ccgaaaagct ttggaaagag atggaggatt ttctgaactt cagtctcgtc ttattcgtta 2100tgaaactcaa actacctgca ccagagaaag ttttccagta cctactgtgt tgagccctct 2160tccatctcct gtagtttcgt cagatcctgg aagtgtccct gacggagaag ttttacaaaa 2220tgaacttcga actgaagtat cccgattgaa acggagatct aaagatctga attgccttta 2280tcccagaaaa agacttgtga aatctgaaag ttcagagtct cttctttctc agacaactgg 2340taatagtaat cactatcatc atcatgtgac atccagaaag ccacaaacag agcggtcctt 2400accagtgact tgtccattgg ttccaattcc tagctgtgaa actccaaaac ttgctacaaa 2460gaccagttca ggtcaaaaaa gtatgcatga atcaaaaaca tcaaggcaaa ttaaggaatc 2520aagatcacag aaacacacac ggatactgaa agaagtagtt actgaaaccc tgaagaaaca 2580cagtattacc gagactcatg aatgtttcac tgcatgcagc cagcgtctct ttgaaatctc 2640taagttctat ctaaaggatc ttaaaacttc aaggggtcta tttgaagaaa tgaagaaaac 2700agcaaacaac aatgctgtac aggtaaagaa attattccca agaaactata ttcaattaaa 2760ttggtatttt atttcagggt tgctctgatg ccattatata tgcagcagta atcagttact 2820ggtctttttc aactctgtcc ctatgaaaac tctttaagaa acttcatagg cttatttagg 2880ctgagtattt catagtattt agtagattgc tctgtcagca aaagagtttc taaagtgaca 2940ctaagaaagc aaaaaaaaaa aaaaaaaaaa aaaaa 29754418PRTMus sp.unsure(418)X=any amino acid 4Arg Arg Lys Ala Ala Asn Gln Pro Glu Ala Phe Ser Ala Asp Glu Leu 1 5 10 15Lys Ser Leu Leu Ala Leu Thr Arg Glu Arg Phe Leu Gly His Phe Asp 20 25 30Val Leu Pro Thr Glu Ala Ala Leu Ala Gln Thr Asp Thr Val Lys Ala 35 40 45Ala Gly Val Val Asn Asp Asp Gly Thr Val Glu Pro Tyr Ser Ser Ser 50 55 60Leu Met Glu Thr Asn Pro Leu Glu Trp Pro Glu Arg His Val Leu Gln 65 70 75 80Asn Leu Glu Thr Ser Glu Lys Ala Lys Gln Lys Met Arg Thr Gly Ser 85 90 95Leu Pro

Arg Ser Ser Glu Gln Leu Leu Gly His Lys Glu Gly Pro Arg 100 105 110Asp Ser Leu Thr Leu Leu Asp Ala Lys Glu Leu Leu Lys Tyr Phe Thr 115 120 125Ser Asp Gly Leu Pro Val Gly Asp Leu Gln Pro Leu His Ile Gln Arg 130 135 140Gly Glu Lys Pro Phe Val Leu Thr Pro Glu Leu Ser Pro Gly Lys Leu145 150 155 160Gln Val Leu Pro Phe Glu Lys Ala Ser Glu Cys His Tyr His Gly Ile 165 170 175Glu Tyr Cys Leu Asp Asp Gln Lys Ala Leu Glu Arg Asp Gly Gly Phe 180 185 190Ser Glu Leu Gln Ser Arg Leu Ile Arg Tyr Glu Thr Gln Thr Thr Cys 195 200 205Thr Arg Asp Ser Phe Pro Val Pro Thr Val Leu Ser Pro Leu Pro Ser 210 215 220Pro Ala Val Leu Ser Glu Pro Gln Ser Val Pro Glu Gly Glu Ala Leu225 230 235 240Gln Gly Glu Leu Arg Thr Glu Val Ser Gly Leu Lys Arg Arg Ser Lys 245 250 255Asp Pro Ser Cys Leu Tyr Pro Gln Lys Arg Leu Thr Arg Ser Glu Ser 260 265 270Ser Asp Cys Leu Pro Ser Gln Ala Ser Cys Asn Ser Asn His His His 275 280 285His Thr Gly Lys Pro Arg Lys Pro Gln Ala Glu Arg Cys Val Ser Gly 290 295 300Leu Pro Leu Pro Gly Arg Glu Ala Ser Lys Asp Thr Ser Lys Thr Ser305 310 315 320Ser Gly Gln Lys Arg Ala His Glu Ser Lys Ser Ser Lys Gln Met Lys 325 330 335Glu Ser Arg Ser Gln Lys His Thr Arg Met Leu Lys Glu Val Val Lys 340 345 350Asp Thr Leu Lys Arg His His Ile Thr Glu Ala His Glu Ser Phe Thr 355 360 365Ala Cys Ser Gln Arg Leu Phe Asp Ile Ser Lys Phe Tyr Leu Lys Asp 370 375 380Leu Lys Thr Ser Arg Gly Leu Phe Glu Glu Met Lys Lys Thr Ala Asn385 390 395 400Asn Asn Val Val Gln Val Ile Glu Trp Val Val Glu Lys Met Ser Lys 405 410 415Lys Xaa535DNAArtificial SequenceDescription of Artificial Sequence Synthetic 5gagagactgc aggagaacac agatgagcta cctgg 35636DNAArtificial SequenceDescription of Artificial Sequence Synthetic 6gagagaaagc tgtcagctag ttgaagtaac ttagca 36724DNAArtificial SequenceDescription of Artificial Sequence Synthetic 7tgaagaataa ggttcaactg tacc 24822DNAArtificial SequenceDescription of Artificial Sequence Synthetic 8cagctttcac ggtgtctgtt tg 22944DNAArtificial SequenceDescription of Artificial Sequence Synthetic 9gagagagcgg ccgcggcgcg aagaggatgg atcggtactt gctg 441075DNAArtificial SequenceDescription of Artificial Sequence Synthetic 10gagagagcgg ccgcctacag ggaggcgtaa tcgggcacat cgtaggggta tttcttgctc 60atcttttcta ccacc 751139DNAArtificial SequenceDescription of Artificial Sequence Synthetic 11gagagagata tcgccaattt taatcaaagt gggaatatt 391241DNAArtificial SequenceDescription of Artificial Sequence Synthetic 12gagagagcgg ccgctttcag tatctacgat tcatagatct c 41

Claims

1. A composition for modulating the G.sub.1 phase of cell cycling comprising a compound which alters levels or activity of a mammalian MDM2 binding protein.

2. The composition of claim 1 wherein the compound is an antisense agent targeted to a gene encoding a mammalian MDM2 binding protein, a ribozyme targeted to a gene encoding a mammalian MDM2 binding protein, a peptide mimic of a mammalian MDM2 binding protein or a modulator of MDM2 expression.