Jean Y.J. Wang
Regulation and Pathology of Steroid ReceptorsWhile the proliferation of all eukaryotic cells involves three fundamental processes: cytoplasmic growth, DNA replication and mitosis, which are coordinated by the orderly activation of cyclin-dependent protein kinases, unique strategies have evolved to regulate the cell cycle machinery in complex organisms such as the mammals. Aberration in the cell cycle regulatory programs is linked to many defects including the development of cancer. The formation of malignant tumors is initiated and driven by mutations in cellular genes which play critical roles in growth control. By elucidating the regulatory function of cancer genes, it is possible to gain insight into some of the unique strategies by which mammalian cell proliferation is controlled. Our research has been focused on two human cancer genes, the proto-oncogene c-abl and the tumor suppressor gene, Rb. Both genes are widely expressed in a variety of cell types. Homozygous mutations of either gene causes embryonal or neonatal lethality in mice. These two essential genes play critical roles in complex regulatory programs: the c-abl gene is involved in coordinatingthe cellular responses to adhesion, the cell cycle status and DNA damage, whereas the Rb gene is involved in establishing the long-term survival of terminally growth arrested cells.
The c-abl gene encodes a tyrosine kinase which contains binding sites for a number of cellular components. In the cytoplasm, this tyrosine kinase interacts with the globular and filamentous actin. The c-Abl protein contains three nuclear localization sequences and a DNA binding domain. In the nucleus, c-Abl binds to DNA and RB, the product or the retinoblastoma susceptibility gene. The cytoplasmic c-Abl is regulated by the adherent status of a cell. When cells are attached to the extracellular matrix (ECM), c-Abl is active. When deprived of matrix contact, c-Abl is quickly inactivated. Interestingly, the nuclear pool of c-Abl tyrosine kinase is regulated by at least three signals, i.e., adhesion, the cell cycle, and DNA damage. The highest nuclear c-Abl kinase activity is achieved when adherent cells are exposed to DNA damaging agents such as ionizing radiation after entry into S phase. Irradiation does not activate c-Abl in non-adherent cells, nor does it activate c-Abl in resting or G1 cells. When activated, the nuclear c-Abl phosphorylates the catalytic subunit of RNA polymerase II and the TAFI100 of RNA polymerase I to activate transcription. Thus, the nuclear c-Abl functions as an "integrator" of adhesion, cell cycle and DNA damage signals to regulate the synthesis of mRNA and rRNA. Cells lacking c-Abl are more sensitive to irradiation in that they fragment their DNA at a radiation dosage that is very well tolerated by the wild type cells. Adhesion to the ECM has been shown to protect cells against radiation-induced death. We propose that the c-Abl tyrosine kinase plays an essential role in the adhesion-dependent protection of cells against radiation damage. Experiments to test this hypothesis will be the major focus of our research in the next few years.
The retinoblastoma susceptibility gene product, RB, has no catalytic activity but plays an important role in the regulation of gene expression. RB regulates transcription by promoting the assembly of specific transcription factor complexes. In the presence of RB, the assembled complexes function as repressors of transcription. When RB becomes phosphorylated by cyclin-dependent kinases and dissociates from the complex, the assembled factors can activate transcription. We have shown that disruption of the assembly function of RB inactivates its biological effects, which include the suppression of cell growth and the protection against cell death. RB has been proposed to regulate G1/S transition during cell cycle progression. However, our evidence suggests that the critical role of RB is to hold cells in the resting state. Re-entry into the cell cycle requires the inactivation of RB, but the transition from G1 to S during continuous proliferative cycles can occur normally with or without a functional RB. Expression of RB is maintained in terminally differentiated cells, especially those which survive for a long time in a resting state, e.g., neurons and cadiomyocytes. Wehave found that RB is a target of the death effector protease during programmed cell death, suggesting that RB may play an active role in promoting the long-term survival of specific mammalian cell types and the degradation of RB contributes to cell death. Experiments in the next few years will be focused on identifying RB-regulated genes and their roles in the establishment of permanent growth arrest and long-term survival.