Analysis of the mammalian G1 checkpoint

Abstract

In order to address the functional role of components of the p53 and Rb pathways, this thesis study utilized two strategies that rely upon the differential oncogenic activities in the Rat Embryo Fibroblast (REF) Assay as well as the unique experimental attributes of the ocular lens as a developmental genetic system. In the former assay, the impact of cell cycle regulators was analyzed with regard to their impact on the transforming activity of Ras in cooperation with an oncogene (Myc) or with Rb and/or p53 inactivation (through expression of E1A, SV40 LTag, or dominant-negative forms of p53).;The regional compartmentalization of growth, differentiation, and apoptosis in the developing murine lens provided an ideal system in which to examine more closely the genetic control of these processes. The Rb-deficient state is associated with unchecked proliferation, impaired expression of differentiation markers, and p53-dependent apoptosis in lens fiber cells. A candidate CKI inhibiting Rb phosphorylation (and thus inactivation) is p57, as it is the most highly expressed CKI in the lens. p57 loss-of-function in the lens resulted in a phenotype similar to that observed with the Rb-deficiency, demonstrating that p57 is important for controlling lens fiber cell growth control and functionally crucial to proper control of the Rb-regulated G{dollar}\sb1{dollar}-S transition.;In an effort to fully exploit the power of the lens system and overcome early embryonic lethality inherent to the established gene targeting approach, a genetic approach called Lens Complementation System (LCS) was developed which makes use of a fertile mouse strain (Ak) that harbors a mutation that results in a failure of lens formation. Injection of wild type Embryonic Stem (ES) cells into Ak blastocysts resulted in lenses that are wild type, derived from the injected ES cell. The injection of Rb-null ES cells into Ak blastocysts resulted in lenses of the Rb-deficient phenotype, demonstrating that this system faithfully recapitulates the phenotypic effect obtained in classical knockout studies. Aside from faithfully recapitulating the phenotype of a known knockout in the lens, LCS offers several advantages over the conventional approach including substantial reductions in the time necessary to generate and evaluate potential phenotypes resulting from homozygous inactivation of new genes, lack of a requirement for germ-line transmission of a mutant allele, and elimination of the need to genotype each sample prior to analysis and to document ES-cell contribution to the organ under study. Most significantly, LCS allows for increased longevity of a homozygous null condition by virtue of host blastocyst contribution to other organ systems of the chimera. Survival to much later stages of development permits the detailed study of cancer-relevant genes whose loss of function results in early embryonic lethality. (Abstract shortened by UMI.).