Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Patent
1996-02-26
1999-02-16
Eisenschenk, Frank C.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
435 15, 435 21, 435 29, 435194, 435375, 530358, 53038824, 5303892, C12S 912
Patent
active
058719016
DESCRIPTION:
BRIEF SUMMARY
This invention is based on the transcription factor DP-1 and its role in controlling the cell cycle. It also relates to assays for DP-1, DP-2 and DP-3 inhibitors that interfere with cell growth, as well as for modulators of the phosphorylation state of the DP proteins.
The cellular transcription factor DRTF1/E2F integrates cell cycle events with the transcription apparatus through its cyclical interactions with important regulators of cellular proliferation. Two sequence specific DNA binding proteins, DP-1 and E2F-1, are components of DRTR1/E2F which synergistically interact in a DP-1/E2F-1 heterodimer. In this application it is shown that DP-1 is a very frequent, possibly universal, component of DRTF1/E2F in 3T3 cells since it is present in all forms of the DNA binding activity which occur during cell cycle progression. Furthermore, the DP-1 polypeptide, which is phosphorylated, undergoes a phosphorylation-dependent mobility shift during the cell cycle suggesting that its level of phosphorylation is regulated during cell cycle progression. This finding can be employed in assays to detect changes in the phosphorylation state of DP-1. A C-terminal region in DP-1 can interact with pRb which, in the context of the DP-1/E2F-1 heterodimer, contributes to the efficiency of pRb binding. The DP-1/E2F-1 heterodimer specifically interacts with the adenovirus type 5 E4 orf 6/7 protein, to produce a DNA binding activity which binds co-operatively to and transcriptionally activates through two appropriately positioned E2F sites in a manner which resembles the regulation of DRTF1/E2F by E4 orf 6/7 during adenovirus infection. One can also conclude that DP-1 is a frequent and cell cycle-regulated component of DRTF1/E2F, and that in the DP-1/E2F-1 heterodimer it is functionally important for recognition by pRb and the E4 orf 6/7 protein.
The molecular events that occur during the cell cycle need to be integrated with the transcription apparatus so that gene expression can be synchronised with cell cycle progression.
Control of the cell cycle is fundamental to the growth and maintenance of eukaryotic organisms, including mammalians and amphibians. Before a typical cell can divide (undergo mitosis), it must double its mass and duplicate all its contents. Most of the work involved in preparing for division goes on invisibly during the growth phase of the cell cycle, denoted as interphase. Interphase is divided into 3 periods. G.sub.1 is used to denote the gap between the completion of the previous division and the start of DNA synthesis. The length of G.sub.1 can be variable and cells can be held in a quiescent state of hours, days or longer. Cells in such a quiescent state are sometimes referred to as being in the G.sub.o phase.
Following G.sub.1, cells enter the S phase during which DNA synthesis occurs. This is followed by a second gap, G.sub.2, which is then followed by the M phase, i.e. mitosis. Generally, once cells enter the S phase they are committed to mitosis.
An important aspect of understanding and controlling the uncontrolled growth of cells is thus understanding the mechanisms by which cells undergo transition from G.sub.1 to S phase.
Recently, a transcription factor called DRTF1 or E2F has been identified and shown to bind to pRb, the protein product of the retinoblastoma susceptibility gene, an anti-oncogene or tumour suppressor gene (see for example Wagner and Green, Nature 352, 189-190, 1991). It is widely believed that the cellular transcription factor DRTF1/E2F functions as a key component in cell cycle control because it associates with important cell cycle regulating proteins, such as the retinoblastoma gene product (pRb), p107, cyclins and cyclin-dependent kinases and furthermore its transcriptional activity is modulated by certain viral oncoproteins, such as adenovirus Ela, SV40 large T antigen, and the human papilloma virus E7 protein.
It is widely believed that the cellular transcription factor DRTF1/E2F plays a pivotal role in co-ordinating cell cycle progression through its interactions with impor
REFERENCES:
The Embo Journal vol. 13, No. 13 pp 3104-3114, 1994 Bandara et al "DP-1: a cell cycle-regulated and phosphorylated..".
Oncogens (1995) 10, 1529-1536 Jooss et al "Proto-oncogenic properties of the DP family of proteins".
A Protein Phosphorylation: A Practical Approach--D.G. Hardie, IRL Press (at Oxford University Press), 1993 (pp 126-9, 140-3, 154-5, 202-9, 214-9, 242-5, 278-283).
Krek et al (Cell, 1994, 78 (Jul. 15, 1994) 161-172).
Dynlacht, et al (Genes in Development, 1994 8; 1772-1786.
Eisenschenk Frank C.
Medical Research Council
Nolan Patrick J.
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