Genotoxic stress is a common aspect of life that mammalian cells have to contend with, Paradoxically DNA-damage inducing agents, such as gamma-irradiation and alkylating agents, are also used in cancer therapy. It became evident that both the molecular basis for the initial increase in the susceptibility of malignant cells to anti-cancer agents, and the development of treatment resistance originate from genetic lesions that alter cell cycle arrest and apoptotic set points. Understanding the molecular-genetic pathways which mediate the response of mammalian cells to genotoxic and other types of environmental stress is, thus of high priority. In response to genotoxic stress mammalian cells have evolved an intricate defense mechanism, including activation of 01/S and G2/M cell cycle checkpoints &/or activation of a cell death program. How stress response pathways interact to signal cells to undergo either cell cycle arrest or programmed cell death is still not understood. Recently, the MyD1 18/CR6/GADD45 family of nuclear proteins [also termed GADD45f3, GADD45y, Gadd45cx] has been implicated in mediating the response of mammalian cells to genotoxic stress, either dependent or independent of p53. Evidence has accumulated that MyD1 18/CR6/GADD45 display a complex array of physical interactions with other proteins such as PCNA, p21, Cdc2, & MEKK4. To what extent the stress response function of each of the MyD1 18/CR6/GADD45 proteins is unique or overlaps with the functions of the other proteins, is unclear. Also, not understood is how the nature of the stress stimulus encountered, the cell type, its physiological state, and its genetic makeup, notably p53 status, modulate MyD1 18/CR6/GADD45 function to determine if the outcome will be cell cycle arrest, DNA repair and survival or apoptotic cell death. Thus, the specific aims are:
Aim I : Elucidate the physiological functions of MyD118 and CR6 (and Gadd4S when in combination with MyDI18) in normal development, growth control and the response to prototype stress agents. This will be done by analyzing the phenotype of mouse model systems deficient for either one or more of MyD1 18/CR6/GADD4S genes, and analyzing the phenotype of cells (primarily MEFs) obtained from such mice, untreated or following treatment with stress agents.
AIM II : Dissect the role of MyD II 8/CR6/GADD45 interactions with cdc2/cyclinB 1, p21, PCNA and MEKK4 in cellular stress responses, including cell cycle arrest, DNA repair, cell survival & apoptosis. MEFs null for MyD1 l8/CR6/Gadd45 will be infected at high efficiency with retroviral vectors encoding for MyD1 18/CR6/GADD4S, either wt or interaction/function deficient, and the stress response will be analyzed. In vitro cell free systems for apoptosis and DNA repair, using null cell extracts spiked with recombinant wt or interaction/function deficient proteins, also will be used.
AIM III : Understand how pS3 & p53 target genes; implicated in cell cycle control (p21, 14-3-3delta) or apoptosis (Bax), modulate MyDI18/CR6/GADD4S stress functions. MEFs, null for p53, p21, or Bax, and the HCT1 16 cells, null for 14-3-3s, will be infected with retroviral vectors encoding for MyD1 18/CR6/GADD45, and the response to stress will be analyzed. Following through on this research plan, should result in an increased understanding of negative growth control in response to genotoxic stress, how perturbing these controls may contribute to oncogenicity, and how treatment resistance in cancer therapy can arise.
| Gupta, Mamta; Gupta, Shiv K; Balliet, Arthur G et al. (2005) Hematopoietic cells from Gadd45a- and Gadd45b-deficient mice are sensitized to genotoxic-stress-induced apoptosis. Oncogene 24:7170-9 |
| Liebermann, Dan A; Hoffman, Barbara (2003) Myeloid differentiation (MyD) primary response genes in hematopoiesis. Blood Cells Mol Dis 31:213-28 |
