The roles of two nuclear oncogenes, E1A andmyc, in multistep cellular transformation
Abstract
To determine whether the E1A and myc immortalizing oncoproteins act through similar mechanisms in oncogene-mediated multistep transformation, I have compared the abilities of myc 2nd E1A to cooperate with ras to transform C3H10T1/2 cells in several different assays. Coexpression of ras and E1A generated a transformed phenotype that could be scored by colony assays and by soft agarose assays but not by standard focus assays. The ras/E1A transformed phenotype in C3H10T1/2 cells relies on sequences present in conserved regions 1 and 2 of the E1A proteins. The contrast between the ras/myc- and ras/E1A-transformed phenotypes suggests that myc and E1A cooperate with ras to transform C3H10T1/2 cells by mechanisms that can be distinguished using this established cell line as a model system. Unlike myc expression, E1A expression, in the absence of activated ras expression, inhibits C3H10T1/2 cell growth. However, two C3H10T1/2-E1A clonal cell lines have been isolated that express high levels of E1A and display characteristics of transformed cells, including high saturation densities and high levels of anchorage-independent growth. Two C3H10T1/2-myc clonal cell lines analyzed displayed less pronounced alterations in phenotype and growth properties than C3H10T1/2-E1A cell lines. Notably, C3H10T1/2-myc clonal cell lines displayed a predisposition to transformation by ras which suggests that these cell lines express functional myc protein that cooperates with ras in transformation assays. The max protein, an oligomerization partner for the myc protein, appears to be a ubiquitous protein, and therefore may be required for basic mechanisms of cell growth. C3H10T1/2-myc clonal cell lines expressed levels of max mRNA similar to C3H10T1/2 cells yet displayed high levels of transformation following transfection of C3H10T1/2-myc cell lines with ras. In addition, cotransfection of C3H10T1/2 cells with ras, myc, and max did not result in an increase in the frequency of transformation compared to cotransfection of cells with ras and myc. Based on these observations, the levels of max protein are sufficient for myc cooperation with ras in C3H10T1/2 cells.
Degree
Ph.D.
Advisors
Taparowsky, Purdue University.
Subject Area
Molecular biology|Cellular biology
Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server.