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

Abstract

Rat 3Y1 cells were transfected with recombinant gARC (pSV2gpt carrying the adenovirus 12 early region 1 [El] 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 ElY cell lines were then established from these foci. The ElY 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 ElY 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 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 molecular weight 11,000 (E4-11K) by immunoprecipitation with monoclonal antibodies. The above results also suggest that expression of the E4 gene gives cells the advantage of forming colonies in soft-agar culture. A tendency was noticed for ElB 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. Recently, evidence has accumulated showing that the transforming genes of human adenovirus are located in only a small region of the left end of the viral genome (5, 10, 29, 32, 34, 37, 39, 40). The transforming genes of the highly oncogenic adenovirus 12 (Adl2) are located in the left end of Adl2 DNA (early region 1A [ElA] and E1B). Rat cells transformed by the EcoRI-C (0 through 16.4 map units) (CY cells), HindIII-G (0 through 6.8 map units) (GY cells), and AccI-H fragments (0 through 4.7 map units) (HY cells) were established (34, 37, 40). CY and GY cells showed the phenotype of completely transformed cells, showing foci in culture, forming large colonies in ...

Two Distinct Activities Contribute to the Oncogenic Potential of the Adenovirus Type 5 E4orf6 Protein

“…The transforming properties of Ad5 E1B-55kDa involve direct binding to p53 (22,50), repression of p53-activated transcription (66,67) and maybe sequestration of the tumor suppressor protein in cytoplasmic bodies (1,69,70). Although expression of the E1A and E1B oncoproteins is sufficient to oncogenically transform cells, earlier work by Shiroki et al already demonstrated that the in vitro growth properties and tumorigenicity of rat 3Y1 cells transformed by E1 gene products of highly oncogenic Ad12 are substantially enhanced by coexpression of Ad12 E4 (52). Further, it was shown that the E4 genes of nononcogenic Ad2 potentiate Ad2 E1-induced focus formation in CREF cells (42) and that the presence of E4 is not only a prerequisite but even the major determinant for the tumorigenic capacity of Ad9 (19,58).…”

An insertion mutation in the adenovirus type 12 early region 1A 13S mRNA unique region

“…pACE1 refers to pACYC177 into which was inserted the Adl2 DNA BamHI-PstI fragment (12to 3,578base-pair [bp] position). gARC refers to pSV2gpt into which was inserted the EcoRI-BamnHI fragment (12to 5,600-bp position) (39). pSVElB refers to pSV2gpt into which was inserted the DdeI-Hinfl fragment (1,536to 3,837-bp position) between the HindlIl and EcoRI sites of the vector.…”

“…1). gE2, gE3, and gE4-2 refer to pSV2 gpt into which were inserted BamHI-C (59.6 to 73.0 map units) and -B (73.0 to 88.2 map units) and HintdIII-E (90.2 to 100 map units) fragments, respectively (39).…”

“…Preparation and blot analysis of mRNA. Cytoplasmic RNAs were extracted as described previously (35,39). A sample of cytoplasmic RNA (10 ,ug) was dissolved in 25 [lI of 10 mM sodium phosphate (pH 7.4)-50% formamide-2.2 M formaldehyde-0.5 mM EDTA, denatured at 65°C for 10 min, and applied to a 1.0 to 1.4% agarose gel containing 1.1 M formaldehyde-10 mM sodium phosphate (pH 7.4)-0.5 mM EDTA.…”

Altered expression of adenovirus 12 DNA-binding protein but not DNA polymerase during abortive infection of hamster cells

“…Total DNA was isolated from whole cells in a manner that reduced shearing (Ausubel et al, 1987). To avoid detecting integrated Ad5 E1 DNA when samples were from 983.2 cells, Ad12 DNA was detected by Southern hybridization of either undigested or restriction enzymedigested DNA (Maniatis et al, 1982) using plasmid gE4-2, which contains the Ad12 E4 region (Shiroki et al, 1984).…”