Immortalization of primary epithelial cells by E1A 12S requires late, second exon-encoded functions in addition to complex formation with pRB and p300

Immortalization of primary cells is an early and important event in multistep tumorigenesis and is itself a multistep process. Adenovirus E1A 12S encodes an oncoprotein that can rescue cells from senescence and overcome apoptosis, leading to their immortalization. Five regions of 12S, located in both exons, are required for immortalization. Two regions in the first exon are necessary to activate the cell cycle, increase the number of population doublings, and overcome the M1 stage of mortality. However, extension of life span requires overcoming crisis or M2, which can be accomplished by the expression of the second exon. Several cellular proteins associate with the peptide encoded by the first exon of 12S including pRB, p107, p130, and p300. The importance of pRB-E1A and p300-E1A complexes in transformation is well established; however, their roles in 12S-mediated immortalization remain undefined. Results obtained from the present study using a panel of second exon immortalization-defective mutants demonstrate that formation of pRB-E1A and p300-E1A complexes is insufficient for immortalization of primary cells. We further demonstrate that the expression levels of another tumor suppressor protein, p53, also do not correlate with the inability of the mutants to immortalize. Thus, mutations in the second exon of 12S do not affect the early steps in the immortalization pathway. The second exon mutants are defective in performing a late function in immortalization, involving the reactivation of the cell cycle, indicating that it is a crucial event in immortalization.