Arun Fotedar, M.D., Ph.D., Professor
Director of Cancer Cell Biology Program

Dr. Fotedar's Active Grants
Dr. Fotedar's Publications
afotedar@skcc.org

Molecular Biology Program

Laboratory Staff: Howard Brickner, Mickaël Howell, Farid Menaa-Touati

This laboratory is clarifying how stress signals modulate the cell's ability to progress through the cell cycle, undergo apoptosis and DNA repair.

Apoptosis, Cell cycle and transformation

Radiation stabilizes the expression of p53 which leads to cell cycle arrest and apoptosis. The cyclin kinase inhibitor p21 is upregulated by the p53 tumor suppressor protein. Cyclin dependent kinases regulate the progression of eukaryotic cells through the cell cycle. p21 is an inhibitor of cdk-cyclin kinase activity, and has been shown to form complexes with cdk-cyclins and with PCNA, an accessory protein of DNA polymerase d. A functional characterization of p21 mutants in the N-terminal domain reveals that p21 can associate with cyclin-cdk kinases in two functionally distinct forms, one in which the kinase activity is inhibited and the other in which the kinase is still active. The cdk2 and cyclin binding sites on p21 are both required to inhibit kinase activity. The second type of interaction, in which an active cyclin-cdk complex only interacts with p21 either via the cyclin or the cdk2 binding site but not through both, does not lead to inhibition of cyclin kinase activity. These results thus provide a basis for understanding the mechanism by which p21, and perhaps other cdk-cyclin kinase inhibitory proteins, suppress kinase activity.

To investigate the role of p21 in apoptosis, we have generated transgenic mice in which the p21 transgene is expressed in the T cell lineage. Thymic cellularity and cell cycle progression of T cells was reduced in p21 transgenic mice. Thymocytes from p21 transgenic mice were hypersensitive to cell death induced by DNA damaging agents. p53 dependent death of thymocytes by ionizing radiation is suppressed by inhibitors of transcription/translation, consistent with a role for transcriptional targets of p53. The Bcl2 transgene rescues the radiation hypersensitivity of p21 transgenic thymocytes. p21 modulates apoptosis through a p53-dependent pathway. These results further suggest that additional signals are required for p53-dependent thymocyte death. We used Lck transgenic mice to investigate the influence of the p21 transgene on the generation of tumours in the T cell lineage. Lck transgenic mice develop thymomas and die around three months after birth. In contrast, Lck/p21 double transgenic mice have dramatically prolonged life spans compared to Lck transgenic mice.

Stress Kinases

POU domain proteins have been implicated as key regulators during development and lymphocyte activation. The POU domain protein TCFß1, which we have described earlier to bind octamer and octamer-related sequences, is a potent transactivator. We have recently found that TCFß1 is phosphorylated by stress-induced signals. Phosphorylation of TCFß1 occurred predominantly at serine/threonine residues. Signals which upregulate JNK (c-Jun N-terminal kinase) activity also lead to association of JNK with TCFß1. JNK associates with the activation domain of TCFß1 and phosphorylates its DNA binding domain. The phosphorylation of recombinant TCFß1 by recombinant JNK enhances the ability of TCFß1 to bind to a consensus octamer motif. These results suggest that stress-induced signals and T cell activation induce JNK activity which then acts on multiple cis sequences by modulating distinct transactivators like c-Jun and TCFß1. This demonstrates a coupling between the JNK activation pathway and POU domain proteins and implicates TCFß1 as a physiological target in the JNK signal transduction pathway leading to coordinated biological responses.

DNA Replication.

Cell cycle progression is regulated by cyclin dependent kinases. Using in vitro replication of SV40 origin containing DNA as a model system, we have performed a detailed analysis of the dependency on cyclin associated kinases of mammalian DNA replication. Depletion of cyclin A from human S phase cell extracts decreases replication, which can be restored by the addition of purified cyclin A. Cyclin dependent kinase does not influence the assembly of initiation complexes but acts at a stage prior to elongation. We have also been investigating the mechanisms controlling DNA elongation. Replication factor C [RF-C], a complex of five polypeptides is essential for DNA elongation. We have cloned the large subunit of human RF-C [RF-Cp145 and mapped the PCNA binding domain. The PCNA binding domain of RF-Cp145 acts like a dominant negative mutant of RFC activity in vitro and in vivo. This provides us a powerful tool to study replication in mammalian cells.