Differential function of Myc-family oncoproteins in proliferation and differentiation in a well-defined cell type, in vivo

Abstract

Myc-family genes appear to control cellular growth, differentiation and apoptosis. This thesis has addressed whether Myc proteins regulate these processes in vivo, whether their functions are distinct or redundant, and it sought to determine the significance of the coordinated expression of two myc genes. These questions were studied in the mouse lens because proliferation and differentiation are compartmentalized--there are post-mitotic, differentiated lens fiber cells (LFCs) and proliferating, immature epithelial cells. Epithelial cells express c- and L-myc, but when they cease proliferating and differentiate into LFCs, they downregulate these genes. In order to study whether c- and L-myc downregulation is required for proliferative arrest and/or differentiation, I used the lens-specific {dollar}\alpha{dollar}A-crystallin promoter to direct their expression to murine LFCs.;{dollar}\alpha{dollar}A/L-myc mice developed centrally-located cataracts, suggesting a perturbation in early lens development. Morphological analysis revealed that while embryonic lenses appeared normal, adult LFCs were disorganized. While there was a significant reduction in level of the late-stage marker MIP26, the expression of {dollar}\alpha{dollar}-crystallins (early-stage markers) and {dollar}\beta{dollar}- and {dollar}\gamma{dollar}-crystallins (late-stage markers) were unaffected. Notably, BrdU-incorporation and TUNEL assays demonstrated that embryonic LFCs did not undergo ectopic proliferation and apotosis, respectively. In addition, LFC-specific expression of a transactivation-deficient L-Myc mutant ({dollar}\alpha{dollar}A/L-zip) did not result in any lens anomalies, indicating that L-Myc's action relied upon the transactivation domain.;{dollar}\alpha{dollar}A/c-myc mice developed cataracts that were similar in location and latency to those of {dollar}\alpha{dollar}A/L-myc mice. In contrast, the {dollar}\alpha{dollar}A/c-myc adult lenses were reduced in size. While histological analysis of embryonic lenses showed that LFC elongation and denucleation were unaffected, their nuclei displayed an abnormal morphology. Moreover, while the BrdU assay demonstrated that the LFCs were inappropriately proliferating, TUNEL failed to identify apoptotic cells. Notably, crystallin and MIP26 expression were unaltered during embryogenesis.;These experiments demonstrated that the expression of L- and c-myc in LFCs resulted in different phenotypes, indicating that L- and c-Myc perform distinct functions in the same cell type. The {dollar}\alpha{dollar}A/L-myc experiment suggests that L-myc downregulation in the lens is not required for withdrawal from the cell cycle, but may be necessary for specific differentiation processes. In contrast, the {dollar}\alpha{dollar}A/c-myc studies indicate that c-myc downregulation is required for proliferative arrest, but not for differentiation.