Human p27Kip1 gene promoter

Details Human p27Kip1 gene promoter Human p27Kip1 gene promoter

1998-06-23

2001-05-01

Schwartzman, Robert A. (Department: 1636)

Chemistry: molecular biology and microbiology

Vector, per se

C435S325000, C536S023100, C536S024100

Reexamination Certificate

active

06225112

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a promoter of human p27
Kip1
gene and to a method of screening a compound capable of regulating activity of the promoter.
BACKGROUND OF THE INVENTION
In the eukaryotic cell cycle, several positive and negative factors control the cell cycle progression. Among the positive factors, the protein kinase family plays an important role. Each member of the family comprises a regulatory subunit, or cyclin, and a catalytic subunit named cyclin-dependent kinase (cdk). A number of reports have suggested that cyclin D-cdk4, cyclin D-cdk6, and cyclin E-cdk2 play important roles in promoting the transition from the G1 phase to the S phase by the phosphorylation of retinoblastoma protein (pRB). Recently, one further level of control has become apparent, namely the expression of cdk inhibitors (Sherr, C. J. and Roberts, J. M. (1995) Genes & Dev. 9:1149-1163). Two families of cdk inhibitor with different modes of action have already been identified in mammalian cells. One group, comprised of related proteins known as p21
Cip1
, p27
Kip1
, and p57
Kip2
, appears to function as specific inhibitors of the cyclin/cdk complexes (Harper, J. W., Adami, G. R., Wei, N., Keyomarsi, K. and Elledge, S. J. (1993) Cell 75: 805-816; Polyak, K., Lee, M.-H., Erdjument-Bromage, H., Koff, A., Roberts, J. M., Tempst, P., and Massague, J. (1994) Cell 78: 59-66; Toyoshima, H. and Hunter, T. (1994) Cell 78: 67-74; Matsuoka, S., Edwards, M. C., Bai, C., Parker, S., Zhang, P., Baldini, A., Harper, J. W., and Elledge, S. J. (1995) Genes & Dev. 9: 650-662). The second family of the cdk inhibitors is called INK4 family proteins. The four members of this family, called p15, p16, p18, and p19, bind directly to cdk4 and cdk6, and are therefore specific inhibitors of the cyclin D-dependent kinases (Hannon, G. J. and Beach, D. (1994) Nature 371: 257-261; Serrano, M., Hannon, G. J., and Beach, D. (1993) Nature 366: 704-707, Hirai, H., Roussel, M. F., Kato, J., Ashmun, R. A., and Sherr, C. J. (1995) Mol. Cell. Biol. 15: 2672-2681).
Although the precise roles of p27
Kip1
are far from clear, its level decreases when cells are stimulated to enter the cell cycle, and increases when cells are arrested by either the change in TGF-&bgr; concentration or contact inhibition (Polyak, K., Kato, J., Solomon, M. J., Sherr, C. J., Massague, J., Roberts, J. M., and Koff, A. (1994) Genes & Dev. 8: 9-22). p27
Kip1
was cloned as a binding protein with cyclin E-cdk2 (Polyak, K., Lee, M.-H., Erdjument-Bromage, H., Koff, A., Roberts, J. M., Tempst, P., and Massague, J. (1994) Cell 78: 59-66) or cyclin D-cdk4 (Toyoshima, H. and Hunter, T. (1994) Cell 78: 67-74). p27
Kip1
inhibits the activity of most cyclin-cdk complexes and can inhibit the phosphorylation of cyclin-cdk complexes by CAK (cdk-activation kinases) (Kato. J., Matsuoka, M., Polyak, K., Massague, J., and Sherr, C. J. (1994) Cell 79: 487-496). Therefore, p27
Kip1
functions as a negative regulator of the G1/S progression.
Tumor-specific mutations of the p27
Kip1
gene are rare, whereas several cell cycle regulators, such as p16, p53, and pRB, are frequently mutated in some cancers and have been shown to be tumor suppressor genes (Ponce-Castaneda, M. V., Lee, M.-H., Latres, E., Polyak, K., Lacombe, L., Montgomery, K., Mathew, S., Krauter, K., Sheinfeld, J., Massague, J., and Cordon-Cardo, C. (1995) Cancer Res. 55: 1211-1214). However, the p27
Kip1
-deficient mice were observed to have increased body size, multiple organ hyperplasia, retinal dysplasia, and formation of pituitary tumors (Fero, M. L., Rivkin, M., Tasch, M., Porter, P., Carow, C. E., Firpo, E., Polyak, K., Tsai, L.-H., Broudy, V., Perlmutter, R. M., Kaushansky, K., and Roberts, J. M. (1996) Cell 85: 733-744; Kiyokawa, H., Kineman, R. D., Manova-Todorova, K. O., Soares, V. C., Hoffman, E. S., Ono, M., Khanam, D., Hayday, A. C., Frohman, L. A., and Koff, A. (1996) Cell 85: 721-732; Nakayama, K., Ishida, N., Shirane, M., Inomata, A., Inoue, T., Shishido, N., Horii, I., Loh, D. Y., and Nakayama, K. (1996) Cell 85: 707-720). These data are in part similar to the case of the RB heterozygous knockout mice (Hu, N., Gutsmann, A., Herbert, D. C., Bradley, A., Lee, W.-H., and Lee, E. Y.-H. P. (1994) Oncogene 9:1021-1027). In addition, it has been shown that lower expression of the p27
Kip1
protein correlated with poorer survival in breast cancer and colorectal cancer (Porter, P. L., Malone, K. E., Heagerty, P. J., Alexander, G. M., Gatti, L. A., Firpo, E. J., Daling, J. R., and Roberts, J. M. (1997) Nature Medicine 3: 222-225; Catzavelos, C., Bhattacharya, N., Ung, Y. C., Wilson, J. A., Roncari, L., Sandhu, C., Shaw, P., Yeger, H., Morava-Protzner, I., Kapsuta, L., Franssen, E., Pritchard, K. I., and Slingerland, J. M. (1997) Nature Medicine3: 227-230; Loda, M., Cukor, B., Tam, S. W., Lavin, P., Fiorentino, M., Draetta, G. F., Jessup, J. M., and Pagano, M. (1997) Nature Medicine 3: 231-234). These results clearly indicate that p27
Kip1
plays an important role in inhibiting tumor formation and tumor progression. There have also been reports on the importance of the p27
Kip1
gene in enhancing the susceptibility of tumor cells to anticancer drugs and in influencing the prognosis factors of cancers (Croix, B. S., Florenes, V. A., Rak, J. W., Flanagan, M., Bhattacharya, N., Slingerland, J. M., and Kerbel, R. S. (1996) Nature Medicine 2: 1204-1210; Loda, M., Cukor, B., Tam, S. W., Lavin, P., Fiorentino, M., Draetta, G. F., Jessup, J. M., and Pagano, M. (1997) Nature Medicine 3: 231-234; Hengst, L. and Reed, S. I. (1996) Science 271: 1861-1864; Pagano, M., Tam, S. W., Theodoras, A. M., Beer-Romero, P., Sal, G. D., Chau, V., Yew, P. R., Draetta, G. F., and Rolfe, M. (1995) Science 269: 682-685). Consequently, it has been desired to develop drugs that regulate the transcription of the p27
Kip1
gene in order to prevent or treat malignant tumors.
Recent reports showed that p27
Kip1
mRNA is induced by vitamin D3 in U937 cells (Liu, M., Lee, M.-H., Cohen, M., Bommakanti, M., and Freedman, L. P. (1996) Genes & Dev. 10: 142-153) and by neuronal differentiation (Poluha, W., Poluha, D. K., Chang, B., Crosbie, N. E., Schonhoff, C. M., Kilpatrick, D. L., and Ross, A. H. (1996) Mol. Cell. Biol. 16: 1335-1341). These facts suggest that the transcriptional regulation of the p27
Kip1
gene might also be important in cellular differentiation.
SUMMARY OF THE INVENTION
An object of the present invention is to provide the promoter of the human p27
Kip1
gene and a method of screening a compound capable of regulating the activity of the promoter.
The present inventors earnestly studied to achieve the above object. As a result, the inventors succeeded in isolating an upstream region of the human p27
Kip1
gene by preparing a partial fragment of the p27
Kip1
cDNA and screening a human leukocyte genomic library using this fragment as a probe. Furthermore, the inventors succeeded in identifying the basal promoter activity region within the upstream region by preparing deletion mutants of the upstream region and detecting their promoter activities. In addition, the inventors found that it is possible to screen compounds capable of regulating the promoter activity by using the isolated promoter region.
Namely, the present invention relates to a promoter region of the human p27
Kip1
gene and a method of screening a compound using the promoter region. More specifically, the invention relates to:
(1) a DNA comprising at least part of the nucleotide sequence of SEQ ID NO:1;
(2) a DNA comprising at least part of the nucleotide sequence of SEQ ID NO:1 and having the promoter activity;
(3) a vector comprising the DNA of (2) above;
(4) a cell carrying the vector of (3) above;
(5) a method of screening a protein capable of regulating the promoter activity of the DNA of (2) above, which comprises the steps selected from:
(a) steps of bringing a test sample into contact with the DNA of
(2) above and selecting a protein that binds to the DNA of (2) above;
(b) steps of introducing a test DNA into cells carrying a repo

Affiliated with

Also associated with

Rating

Human p27Kip1 gene promoter does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Human p27Kip1 gene promoter, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Human p27Kip1 gene promoter will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2529184