Expression of the E4 gene is required for establishment of soft-agar colony-forming rat cell lines transformed by the adenovirus 12 E1 gene

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Abstract
Rat 3Y1 cells were transfected with recombinant gARC (pSV2gpt carrying the adenovirus 12 early region 1 [E1] gene), and focus formation was observed in monolayer cultures after culture of cells in gpt-selective medium (Eagle medium containing 10% fetal calf serum, xanthine, thymidine, aminopterin and mycophenolic acid) for 10 days, followed by focus formation. Transformed E1Y cell lines were then established from these foci. The E1Y cells were transformed morphologically similarly to cells transformed with intact adenovirus 12 DNA, but formed no colonies in soft-agar culture and induced tumors in transplanted rats only after a long incubation period. For the establishment of completely transformed cells, 3Y1 cells were transformed with combinations of gARC, pE3 (pBR322 carrying the adenovirus 12 E3 gene) and gE4 (pSV2gpt carrying the adenovirus 12 E4 gene) DNA. E1-3Y cells (3Y1 cells transformed with gARC and pE3 DNA), E1-4Y cells (3Y1 cells transformed with gARC and gE4 DNA) and E1-3-4Y cells (3Y1 cells transformed with gARC, pE3 and gE4 DNA) were established. These transformed cell lines were compared for growth in Eagle medium with 2 or 10% fetal calf serum, colony formation in soft-agar culture and tumor growth in rats transplanted with the transformed cells. Several transformed cell lines of E1-4Y and E1-3-4Y cells showed colony formation in soft-agar culture and abundant expression of the E1B gene. T antigen f was seen by immunofluorescence as flecks in these cells, in which the E4 gene was transcribed, but was not seen in E1Y cells, suggesting that T antigen f was encoded by the E4 gene. The suggestion was confirmed by the observation that T antigen f was detected in SV40-transformed monkey kidney COS-1 cells transfected singly with gE4 DNA by immunofluorescence with polyclonal and monoclonal antibodies. Transcription of the E4 gene was confirmed in gE4-transfected COS-1 cells. T antigen f, one of the E4 gene products, was identified as a polypeptide of MW 11,000 (E4-11K) by immunoprecipitation with monoclonal antibodies. Evidently, expression of the E4 gene gives cells the advantage of forming colonies in soft-agar culture. A tendency was noticed for E1B gene expression to be enhanced by E4 gene expression. The relationship between enhancement of colony formation in soft-agar culture and enhancement of E1B gene expression is discussed.