Department of Pharmacology and Therapeutics
Roswell Park Cancer Institute
Buffalo, NY 14263
Tel: 716-845-8542
Fax: 716-845-8857
E-mail: gokul.das@roswellpark.org

Education
PhD, Baylor College of Medicine, Houston, Texas
Post-Doctoral, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

Program

Tumor suppressor protein p53/Transcriptional regulation/ Genomic damage, DNA repair, and cell cycle control/Interaction between p53 and estrogen receptor signaling pathways

The p53 tumor suppressor protein is at the center of a major anti-cancer defecnce mechanism, as is evident by the fact that functional inactivation of p53 is the most frequently observed phenomenon in human cancer.  Extensive research over the past two decades has placed p53 at the hub of a very complex network of signaling pathways that integrate variety of intracellular and extracellular inputs.  It is becoming increasingly clear that the balance of opposing effects of various components in the signaling net work dictates the cellular function of p53. 

Our laboratory focuses on analyzing how interaction between two major cellular signaling pathways – one mediated by p53 and the other mediated by estrogen receptor – affect cellular proliferation and oncogenesis. Most of the biological effects of p53 are due to its ability to function as a transcriptional regulator of various genes that control cell division, cell death (apoptosis), cell differentiation and senescence. Effect of p53 on the target genes can range from transcriptional activation to repression depending on the gene promoter context and interaction with other regulatory molecules and signaling pathways.  Estrogen receptor is another important player in regulating cell proliferation, differentiation, and apoptosis. Estrogen receptor is known to stimulate transcription by binding directly to response elements on target gene promoters or by interacting with other transcriptional regulators (genomic effect).  Besides, estrogen receptor can mediate non-genomic effect of estrogen via signaling pathways such as Src/Ras/Erk pathway.  There are two estrogen receptors encoded by independent genes: Estrogen receptor-a was the first receptor to be isolated and characterized, whereas estrogen receptor-b was more recently discovered and its functional importance is being studied intensively. 

Our studies in cell culture model systems have revealed that p53 and estrogen receptor can physically interact and can repress each other’s function at the transcriptional level.  Using various assays, we have shown that p53 can interact in vitro and in vivo on target gene promoters. To complement our cell culture model systems, we intend to develop a novel mouse model where p53 signaling to specific target gene transcription is disrupted (unlike the conventional p53-knock-out mouse model where all p53-dependent pathways are obliterated).  Since we will be disrupting p53 signaling to specific gene transcription, this model will be suitable for analyzing gene-specific effect of p53 signaling.  We will also be able to combine the disruption in p53 pathway with disruptions in other signaling pathways.  The model will enable us to analyze physiological consequences of gene-specific disruption of p53 function in terms of developmental abnormalities, susceptibility to carcinogenesis and aging, DNA repair/replication defects, and response to radiation and cancer chemotherapeutic agents. It will also be possible to analyze, at the molecular level, the role of, if any, of specific p53 regulated gene expression in determining effects of carcinogens such as xenobiotic estrogenic compounds.

The specific aims of research in my laboratory are: (1) To elucidate cellular and molecular mechanisms underlying the interaction and mutual functional antagonism between p53 and estrogen receptor, (2) To investigate how cellular stress and genomic damage (caused by, for example, radiation and chemotherapeutic agents) affect cross-talk between p53 and estrogen receptor signaling pathways, (3) to examine the role of cell type cell cycle context in determining functional antagonism between p53 and estrogen receptor, (4) To identify downstream effectors of p53-estrogen receptor interaction and analyze their profile in cancer patients, (5) To develop a mouse model where p53-signaling to specific target gene (e.g., p21) is disrupted in a tissue-specific (e.g., breast) manner and analyze cellular and physiological consequences of such disruption.

Progress

Studies are ongoing to elucidate the molecular mechanism of interaction and resultant functional antagonism between p53 and estrogen receptor. Our recent experiments have revealed that the interaction between p53 and estrogen receptor is exquisitely responsive to genomic damage. Chromatin immunoprecipitation (ChIp) assay has enabled us to test in vivo binding of p53 and estrogen receptor on target gene promoters.   We are actively pursuing analysis of such chromatin-bound complexes as part of our effort to understand the molecular mechanisms underlying antagonism between p53 and estrogen receptor  as well as downstream consequences.  In collaboration with Dr. Margot Ip, we have started to analyze p53-estrogen receptor interaction in rat mammary epithelial cells in culture.

Based on our observation that estrogen receptor can antagonize p53-mediated transcriptional activation, we analyzed the effect of estrogen receptor on p53-mediated transcriptional repression.  Interestingly, p53-mediated repression of human multi drug resistance (hmdr1) gene is not affected by estrogen receptor.  Survivin is another p53-repressed gene.  Survivin is a member of the inhibitor of apoptosis family and is well conserved as a mitotic spindle checkpoint protein.  In collaboration with Dr. Fengzhi Li, we have started analyzing the interaction of p53 and estrogen receptor pathways on transcriptional regulation of this important p53 target gene.
 Although role of p53 in G1 and G2/M checkpoints to conserve genomic integrity is well characterized, its role in S-phase checkpoint is less well understood.  Our experiments in five different cell types (NHOST, normal human osteoblasts; MCF-7, human breast cancer cells, HMEC; human mammary epithelial cells; RKO, human colon cancer cells; HCT116, human colon cancer cells) have shown that p53 accumulates and transactivates its target genes such as p21, gadd45 and bax in response to replication blockade in normal and cancer cells (Nayak and Das, Oncogene, 21, 7226-7229). Lack of transcriptional activation under similar conditions in cells lacking p53 showed that p53-target gene activation during replication blockade was indeed p53-dependent. Further, transactivation of p21 in response to replication blockade by hydroxyurea and aphidicolin is similar to that in response to ionizing radiation except that the latter is more immediate compared to the response to replication blockade. These findings suggest that p53 is stabilized and remain transcriptionally active in response to replication blockade in S phase.

In another study, we analyzed expression of some of the important p53 target genes in response to DNA damage by ionizing radiation in normal as well as cancer cells.  Our studies revealed that cell cycle phase at the time of exposure to genomic damage determined G1 or G2/M arrest. Importantly, transcriptional upregulation of proliferating cell nuclear antigen (PCNA), an essential component of DNA replication and repair machineries, in response to genomic damage was p53-dependent both in normal human mammary epithelial cells and MCF7 breast cancer cells in contrast to p53-independent increase in PCNA transcription during mitogen-induced S-phase.  Based on our results thus far, it is clear that transcription of PCNA gene in cell cycle-specific manner is determined by the type of signal to which the cells are exposed.  For example, during mitogenic stimulation, PCNA transcription is induced prior to entry into S phase, and that induction is p53-independent in contrast to the p53-dependent transcriptional activation during G1 and G2/M phases in response to genomic damage. Transcription of cyclins A and B was repressed while cyclin G transcription was increased in normal human osteoblast cells and MCF-7 cells in response to genomic damage.  Intriguingly, transcription of none of these cyclins responded to genomic damage in normal mammary epithelial cells.  Together, these observations suggest that cell-type and cell cycle status during exposure to DNA damaging agent are major determinants of the type of cellular response to genomic damage.

Another area of research in our laboratory is to analyze the importance of p53 and estrogen receptor cross talk in proliferation of breast cancer cells.  Our initial studies in human breast cancer cells suggest that cell proliferation status has an important bearing on p53 and estrogen receptor pathways.   Surprisingly, contrary to the widespread notion that low levels of p53 is associated with high rate of proliferation, our experiments showed that in fact p53 levels are at its peak in breast cancer cells when proliferation rate is very high.  We are in the process of analyzing the mechanism underlying elevation of p53 levels concomitant with cell proliferation. 
 The major goal of this research program is to understand the mechanism and consequences of interaction between p53 and estrogen receptor signaling pathways.  Besides gaining insight into the mechanisms and consequences of cross talk between two major cellular signaling pathways, these studies have the potential to unravel novel targets for cancer preventive and therapeutic strategies.

Select Publications

• Das G, Hinkley CS, Herr W. Basal promoter elements as a selective determinant of transcriptional activator function.  Nature 314: 657-659, 1995.
• Karuppayil S, Moran E, Das GM.  Differential Regulation of p53-dependent and -independent proliferating cell nuclear antigen gene transcription by 12S E1A oncoprotein requires CBP.  J. Biol. Chem. 273:17303-17306, 1998.
• Giabernardi TA, Sakaguchi AY, Gluhak J, Pavlin D, Troyer D, Das G, Rodeck U, Klebe RJ.  MMP-8 (Collagenase-2), the first fibrillar collagenase expressed during development, is found in cells of neural crest origin and melanoma cells.  Matrix Biology 20: 577-587, 2001.
• Thavathiru E, Das GM.  Activation of pRL-TK by 12S E1A oncoprotein: drawbacks of using an internal reference reporter in transcription assays.  BioTechniques 31:528-532, 2001. 
• Nayak BK, Das GM.  Stabilization of p53 and transactivation of its target genes in response to replication blockade.  Oncogene 21: 7226-7229, 2002.