Factors which interact with oncoproteins

Details Factors which interact with oncoproteins Factors which interact with oncoproteins

1999-03-26

2001-03-20

Schwartzman, Robert A. (Department: 1635)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Carbohydrate doai

C435S069100, C435S091100, C435S325000, C435S375000, C435S006120, C514S04400A, C536S023100

Reexamination Certificate

active

06204253

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to the oncoprotein mdm2 (murine double minutes), and more specifically, agents which interact therewith and which can thus influence cell growth regulation and which have use particularly, but not exclusively, as therapeutics, diagnostics, prognostics and in assays and as models designed to elucidate cell growth regulation.
BACKGROUND OF THE INVENTION
The oncoprotein mdm2 (murine double minutes) was originally isolated through its ability to transform mouse BALB/c fibroblast cells (Fakharzadeh, et al., 1991). In the transformed cells, the mdm2 gene is often amplified and exists as a double minute chromosome. The mdm2 gene encodes a protein of 491 amino acids and contains all the domains necessary for being a transcription factor. It has a consensus nuclear translocation signal, two zinc binding domains and acidic and basic domains (Brown, et al., 1993). The mdm2 gene is located on human chromosome 12q13-14 and is often seen to be co-amplified with the CDK4 gene (a cyclin dependent kinase gene which is located within the same region) in human malignant gliomas (He, et al., 1994, Reifenberger, et al., 1995). Amplification of the mdm2 gene has also been found in a variety of human sarcomas (Ollner, et al., 1992; Ladanyl, et al., 1993; Khatib, et al., 1993). Amplification of the mdm2 gene is, however, not universal; many tumours, including some types of leukaemia, were found to have no amplification of the mdm2 gene (Ridge, et al., 1994). Nevertheless, abnormal expression of the mdm2 gene has been found in many types of human tumour. Abnormal expression of mdm2 has been reported in chronic lymphocytic leukaemia (Watanbe, et al., 1994; Huang, et al., 1994). Elevated mdm2 expression was also found in Hodgkin's and non-Hodgkin's lymphomas at both mRNA and protein levels (Chllosl, et al., 1994, Finnegan, et al., 1994). High levels of expression of the mdm2 gene have been linked to a poor response to chemotherapy and short survival in haematological malignancies (Quesnel, et al., 1994).
SUMMARY OF THE INVENTION
The best known function of mdm2 is its ability to bind to the tumour suppressor protein p53 via its transcriptional activation domain (Lin, et al., 1994), thus inhibiting the p53 transcription activity (Momand et al., 1992). In addition, mdm2 has been shown to be able to block p53 induced apoptosis in some cell lines. A recent study also demonstrated that inactivation of the mdm2 gene can result in the embryonic lethality, a phenotype that could be rescued by a p53 null background. All these observations suggest that one of the important functions of mdm2 is its ability to block the activity of p53. However, recent reports showed that mdm2 can also bind to another tumour suppressor gene product Rb (Xiao, et al., 1995) as well as a cell cycle transcription factor E2F1 (Martin, et al., 1995). Interestingly, all three tumour suppressor proteins, p53, Rb and E2F1 (Field et al, 1996), are key players in controlling cell cycle progression and apoptosis, suggesting that mdm2 may play a key role in regulating cell growth. Although the mdm2 protein has many features characteristic of transcription factors and the phenomena listed above are well established, there is little understanding of the true biological function of mdm2. It is, however, clear that mdm2 occupies a key role in cell growth regulation.
A biological role for mdm2 is suggested by the following data. Using FACS analysis as described in Hseih et al 1997, cells selected containing a sub-G1 DNA content (typical of cells undergoing apoptosis) it has been shown that in the presence of Rb the anti-apoptotic function of mdm2 is abolished. It is known that mdm2 targets p53 for degradation, possibly via the ubiquitination pathway. In the presence of Rb, p53 is stabilised. Therefore interaction between Rb and mdm2 can promote the tumour suppressor activity of p53.
Mdm2 interacts with and is phosphorylated by cyclinA-cdk2 (a S-phase cyclin-cdk complex Lu unpublished data). It has been shown previously that cyclins are targets for ubiquitination. As p53 is also a target for ubiquitination it is possible that mdm2 interaction is causally related to degradation of these important cell-cycle proteins or indeed to the ubiquitination of mdm2. Saos-2 and HI299 cells (null for p53) treated with the proteosome inhibitor ALLN (a calpain proteinase inhibitor) results in an increase in the cellular levels of mdm2. This suggests that mdm2 is a target for ubiquitination in the absence of p53. It may therefore be possible to regulate the negative effects of mdm2 on p53 by alternative strategies.
It is therefore an object of the invention to identify agents which interact with mdm2 and thus have therapeutic, diagnostic or prognostic application.
It is yet a further object of the invention to identify agents which can influence cell growth and regulation via binding to mdm2.
It is yet a further object of the invention to elucidate the role of mdm2 in cell growth regulation by identifying agents that interact therewith with a view to then determining the nature of the pathway involved and thus agents which may be of potential use in regulating said pathway.
It is yet a further object of the invention to provide a methodology for identifying the aforementioned agents.
DETAILED DESCRIPTION OF THE INVENTION
It will be apparent that agents which interact with mdm2 i.e. those sequences identified in Tables 1 and 2 have potential as therapeutic agents in various processes relating to cell growth and division. A relatively recent alternative strategy to traditional therapies is the use of antisense molecules to regulate the production and/or availability of translatable mRNA to targeted nucleic acid sequences. Clearly some of the identified sequences show elevated expression in certain transformed cells and their down regulation has been linked to a reversion of the transformed phenotype.
As mentioned above, the invention embraces antisense oligonucleotides that selectively bind to a nucleic acid molecule presented in Tables 1 and 2, to decrease transcription and/or translation of these genes. This is desirable in virtually any medical condition wherein a reduction in gene product expression is desirable, including to reduce any aspect of a tumour cell phenotype attributable to the expression of that sequence. Antisense molecules, in this manner, can be used to slow -down or arrest such aspects of a tumour cell phenotype.
As used herein, the term “antisense oligonucleotide” or “antisense” describes an oligonucleotide that is an oligoribonucleotide, oligodeoxyribonucleotide, modified oligoribonucleotide, or modified oligodeoxyribonucleotide which hybridizes under physiological conditions to DNA comprising a particular gene or to an mRNA transcript of that gene and thereby, inhibits the transcription of that gene and/or the translation of that mRNA. The antisense molecules are designed so as to interfere with transcription or translation of a target gene upon hybridization with the target gene. Those skilled in the art will recognise that the exact length of the antisense oligonucleotide and its degree of complementarity with its target will depend upon the specific target selected, including the sequence of the target and the particular bases which comprise that sequence. It is preferred that the antisense oligonucleotide be constructed and arranged so as to bind selectively with the target under physiological conditions, i.e., to hybridize substantially more to the target sequence than to any other sequence in the target cell under physiological conditions. Based upon the DNA sequence presented in Tables 1 and 2 or upon allelic or homologous genomic and/or DNA sequences, one of skill in the art can easily choose and synthesize any of a number of appropriate antisense molecules for use in accordance with the present invention. In order to be sufficiently selective and potent for inhibition, such antisense oligonucleotides should comprise at least 7 (Wagner et al.,
Nature Biotechnology
14:840-844, 1996

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