Adenovirus type 9 E4 open reading frame 1 encodes a transforming protein required for the production of mammary tumors in rats.

The E4 region of human adenovirus type 9 (Ad9) transforms established rat embryo fibroblasts and encodes an essential determinant for the production of estrogen-dependent mammary tumors in rats. Testing of the seven Ad9 E4 open reading frames (ORFs) individually for transformation of the established rat embryo fibroblast cell line CREF indicated that only Ad9 E4 ORF1 possessed a significant ability to generate transformed foci on these cells. In contrast, the E4 ORF1 sequences from human Ad5 and Ad12 lacked the transforming potential exhibited by Ad9 E4 ORF1. Cell lines derived from Ad9 E4 ORF1-transformed foci expressed the 14-kDa Ad9 E4 ORF1 protein and formed colonies in soft agar. In addition, the Ad9 E4 ORF1 protein was required for initiation of mammary oncogenesis in vivo, as E4 ORF1 mutant viruses failed while E4 ORF2 and ORF3 mutant viruses succeeded in eliciting mammary tumors in animals. A role for Ad9 E4 ORF1 in tumor maintenance was suggested by the fact that 100% of virus-induced mammary tumors expressed the E4 ORF1 protein. Taken together, the facts that the Ad9 E4 ORF1 protein exhibits transforming potential in culture and is required by Ad9 to produce mammary tumors in animals suggest that Ad9 E4 ORF1 is a new viral oncoprotein.     

Transformation of rat cells by fusion-infection with Rous sarcoma virus

The transformation of a rat cell line, 3Y1, by nonmammalian tropic strains of avian sarcoma virus was tested using cell-virus fusion mediated by Sendai virus or polyethylene glycol. Furthermore, the establishment of several transformed 3Y1 cell clones induced by the Schmidt-Ruppin strain of Rous sarcoma virus (RSV), its derivative mutants, and the Bryan high-titer strain of RSV is reported. The presence and expression of the viral genomes in these cells were examined, and all transformed cell clones tested were found to contain rescuable RSV genomes when they had been fused with normal chicken embryo fibroblast cells or those preinfected with Rous-associated virus type 1. However, the gag gene product, pr76, was barely detectable in wild-type RSV-transformed cells, whereas it was produced in considerable amounts in cells transformed by env-deleted mutants, the Bryan high-titer strain of RSV and NY8 derived from the Schmidt-Ruppin strain of RSV.     

Adenovirus type 5 E1A immortalizes primary rat cells expressing wild-type p53

Adenovirus (Ad) E1A induces apoptosis in cells expressing wild-type p53, and stable transformation by Ad E1A requires the co-introduction of an anti-apoptotic gene such as Ad E1B 19K. Thus, cells immortalized by Ad E1A alone might have lost functional p53. In order to analyze the p53 in rat cells expressing Ad E1A, we established rat cell lines by transfecting primary rat embryo fibroblast (REF) and baby rat kidney (BRK) cells with cloned Ad5 E1A. By using a yeast functional assay, we analyzed p53 in six primary REF and three BRK cell lines immortalized by Ad5 E1A as well as five spontaneously immortalized rat cell lines (REF52, NRK, WFB, Rat-1 and 3Y1). The yeast functional assay revealed that all of the spontaneously and Ad5 ElA-immortalized rat cell lines except for 3Y1 expressed wild-type p53. All of the Ad5 E1A-immortalized rat cell lines contained p53 detectable by immunoprecipitation. Recombinant adenovirus expressing rat p53 cloned from a REF cell line immortalized by Ad5 E1A, as well as that expressing murine wild-type p53, induced apoptosis in p53-null cells in collaboration with E1A. Thus, it is suggested that the mutation of p53 appears to be not frequent in the spontaneous immortalization of primary rat cells, and that the functional loss of wild-type p53 is not a prerequisite of E1A-mediated immortalization.     

Immunological characterization of proteins detected by phosphotyrosine antibodies in cells transformed by Rous sarcoma virus.

Phosphotyrosine antibodies were used to identify tyrosine-phosphorylated proteins in Rous sarcoma virus (RSV)-transformed chicken embryo fibroblasts. A large number of tyrosine phosphoproteins were detected. A similar set of proteins was observed in RSV-transformed murine cells. An 85,000-dalton protein, however, was present in transformed avian cells but missing in transformed murine cells. Neither the 85,000-dalton protein nor any of the other tyrosine phosphoproteins appeared to be viral structural proteins. Use of RSV mutants encoding partially deleted src gene products enabled us to identify a 60,000-dalton cellular tyrosine phosphoprotein that comigrated with wild-type pp60v-src. With the exception of calpactin I, the major tyrosine phosphoproteins detected in immunoblots appeared to be different from several previously characterized substrates of pp60v-src with similar molecular masses (ezrin, vinculin, and the fibronectin receptor).     

78-kilodalton glucose-regulated protein is induced in Rous sarcoma virus-transformed cells independently of glucose deprivation.

To identify mRNAs with altered expression in Rous sarcoma virus (RSV)-transformed cells, we screened a chicken embryo fibroblast (CEF) cDNA library by differential hybridization. One clone, designated R1H, showed markedly elevated mRNA expression in RSV-transformed cells. Nucleotide sequence analysis indicated that R1H mRNA encodes 78-kilodalton glucose-regulated protein (GRP78). Chicken GRP78 was found to be very highly conserved in comparison with rat GRP78 (96% identity between chicken and rat amino acid sequences). In contrast to previous observations, we found that GRP78 was induced in RSV-transformed cells in the absence of glucose deprivation. When cells were grown in glucose-supplemented medium, the level of GRP78 mRNA was approximately fivefold higher in RSV-transformed CEF than in transformation-defective virus-infected or uninfected CEF. Similar changes in GRP78 protein content were also found. Using a temperature-sensitive mutant of RSV and supplemental glucose, we found a gradual increase in the level of GRP78 mRNA beginning at 4 h after shiftdown to permissive temperature. Uridine supplementation did not block the induction seen in CEF infected with a temperature-sensitive mutant. These results indicate that GRP78 is induced by p60v-src in the absence of glucose deprivation.     

Association between the cellular p53 and the adenovirus 5 E1B-55kd proteins reduces the oncogenicity of Ad-transformed cells.

The association of the cellular p53 protein with the E1B-55kd protein of adenovirus 5 (Ad5) is thought to result in inactivation of the p53 recessive oncogene product. Here we show that Ad5 E1-transformed 3Y1 rat cells which express low levels of the 55 kd E1B protein do not contain the p53-E1B 55kd complex. These cells have nuclearly located p53 and are highly oncogenic in nude mice. In 3Y1 cells expressing the E1B protein at a sufficiently high level, association between p53 and E1B-55kd occurs, resulting in an almost complete trapping of p53 into a discrete cytoplasmic body. These cells only form tumors after a very long latency period and in the tumors that eventually appear selection has occurred in favor of cells lacking the complex and containing free nuclear p53. Comparable results were found when highly oncogenic Ad12-transformed cells were supertransfected with the Ad5 E1B region. In none of the Ad-transformed mouse, rat and human cell lines examined, could we detect p53 of abnormal molecular weight or association with hsc70, neither could we immunoprecipitate p53 by the mutant specific antibody PAb240. These data suggest that a high level of nuclear p53 with a wild-type conformation contributes to the oncogenicity of Ad transformed cells.     

Alterations in cellular phenotype induced by the type 5 adenovirus E1A and the beta 1 protein kinase C genes in cloned rat embryo fibroblast cells.

The E1A gene of adenovirus type 5 (Ad5) induces morphological transformation and anchorage-independent growth in cloned rat embryo fibroblast (CREF) cells. In contrast, CREF cells transfected with a beta 1 protein kinase C (PKC) gene and expressing low-levels of beta 1 PKC display a CREF-like morphology and do not form colonies when grown in agar. The combination of Ad5 E1A and low-level beta 1 PKC expression in the same CREF cell line results in an enhanced ability to grow when suspended in agar. In Ad5 E1A and Ad5 E1A + low-level beta 1 PKC expressing CREF clones, the tumor promoting agent 12-0-tetradecanoyl-phorbol-13-acetate (TPA) further enhances anchorage-independence. In contrast, TPA does not induce CREF cells or transfected CREF cells expressing low-levels of beta 1 PKC to grow in agar. Low-level beta 1 PKC expression in transfected CREF cells is associated with a modest 1.2 to 1.6-fold increase in binding of [3H]-phorbol-12,13-dibutyrate (PDBu) and only a 2.3-fold increase in PKC enzymatic activity. In contrast, specific beta 1 PKC-retroviral vector transformed CREF clones (CREF-RV-PKC) display higher levels of PKC mRNA, PDBu binding and PKC enzymatic activity. A majority of CREF-RV-PKC clones exhibit a transformed morphology and grow more rapidly in monolayer culture, form macroscopic colonies in agar in the absence of TPA and in many independent clones TPA further enhances anchorage-independent growth. This effect is not directly related to the level of enhanced [3H]-PDBu binding. The present study indicates that the effect of beta 1 PKC on cellular phenotype in immortal rat embryo cells is complex and is affected by its mode of insertion into CREF cells, i.e. transfection versus retroviral insertion. In addition, the combination of a transfected Ad5 E1A and a beta 1 PKC gene in the same CREF clone results in an enhanced expression of the transformed phenotype in both the absence and presence of TPA.     

Identification of phosphotyrosine-containing proteins in untransformed and Rous sarcoma virus-transformed chicken embryo fibroblasts

Phosphorylation on tyrosine residues mediated by pp60src appears to be a primary biochemical event leading to the establishment of the transformed phenotype in Rous sarcoma virus (RSV)-infected cells. To identify the cellular proteins that undergo tyrosine phosphorylation during transformation, a 32P-labeled RSV-transformed chicken embryo cell extract was analyzed by electrophoresis on a polyacrylamide gel. After slicing the gel into approximately 60 slices, phosphoamino acid analyses were carried out on the protein recovered from each gel slice. Phosphotyrosine was found in every gel slice, with two major peaks of this phosphoamino acid around M(r)'s of 59 and 36 kilodaltons. When the same analysis was performed with cells infected with a transformation-defective src deletion mutant of RSV (tdNY101), significant and reproducible peaks of phosphotyrosine were found in only 2 of 60 gel slices. These gel slices corresponded to M(r)'s of 42 and 40 kilodaltons. Identical results were obtained with normal uninfected chicken embryo fibroblasts. We conclude from these observations that pp60src or the combined action of pp60src and pp60src-activated cellular protein kinases cause the tyrosine-specific phosphorylation of a very large number of cellular polypeptides in RSV-transformed cells. In addition, untransformed cells appear to possess one or more active tyrosine-specific protein kinases which are responsible for the phosphorylation of a limited number of proteins. These proteins are different from the major phosphotyrosine-containing proteins of the transformed cells.     

Isolation of a nondefective recombinant between adenovirus type 5 and early region 1A of adenovirus type 12.

A nondefective recombinant between adenovirus type 5 (Ad5) and type 12 (Ad12), rc-1 (Ad5 dl312, carrying the Ad12 E1A gene), was isolated from hamster cell foci transformed by a defective recombinant, rcB-1 (dl312, carrying the Ad12 E1 gene). The recombinant rc-1 grew in human embryo kidney and KB cells in the absence of helper and synthesized Ad12 T antigen g, the product of the E1A gene. The genome of rc-1 has a deletion between 79.9 and 82.5 map units of Ad5 dl312 DNA with an insertion of 0.1 to 5.5 map units of Ad12 DNA at the deletion site. The mRNAs of Ad12 E1A were transcribed from the Ad12 E1A promoter, and unusual RNAs were abundantly transcribed from the Ad5 E3 promoter on the opposite strand. The frequency of cell transformation with rc-1 was lower than those with Ad5 and Ad12 wild types.     

Tumorigenicity of adenovirus-transformed cells: collagen interaction and cell surface laminin are controlled by the serotype origin of the E1A and E1B genes.

A library of cells transformed with recombinant adenoviruses was used to study tumorigenicity and interaction with extracellular matrix. Cells expressing the complete E1 region of highly oncogenic adenovirus type 12 (Ad12) are tumorigenic, adhere preferentially to type IV collagen, and express cell surface laminin. Weakly tumorigenic cells, which express the E1A oncogene of Ad12 and the E1B genes of Ad5, also attach preferentially to type IV collagen but do not contain laminin on their surface. Cells which express the E1A oncogene of Ad5 and the E1B genes of Ad12 are nontumorigenic and do not preferentially attach to type IV versus type I collagen but have laminin on their surface. There is no significant difference in the amounts of laminin secreted into the culture medium among cells expressing the E1B genes of Ad5 or Ad12. In vitro assays show that cells which express the E1B genes of Ad12, irrespective of the origin of the E1A genes, can bind three times more exogenously added laminin than cells expressing the E1B genes of nononcogenic Ad5. The interaction of adenovirus-transformed cells with collagen is controlled by the serotype origin of the E1A oncogene, whereas cell surface laminin is controlled by the serotype origin of the E1B genes.     

The adenovirus type 12 early-region 1B 58,000-Mr gene product is required for viral DNA synthesis and for initiation of cell transformation.

An E1B 58K mutant of adenovirus type 12 (Ad12), dl207, was constructed by the deletion of 852 base pairs in the E1B 58K coding region. The mutant could grow efficiently in 293E1 cells but not in HeLa, KB, or human embryo kidney (HEK) cells. Viral DNA replication of dl207 was not detected in HeLa and KB cells and was seldom detected in HEK cells. Analysis of viral DNA synthesis in vitro showed that the Ad12-DNA-protein complex replicated by using the nuclear extract from Ad12 wild-type (WT)-infected HeLa cells but not by using the nuclear extract from dl207-infected cells. In dl207-infected HeLa and KB cells, early mRNAs were detected, but late mRNAs were not detected. The mutant induced fewer transformed foci than the WT in rat 3Y1 cells. Cells transformed by dl207 could grow efficiently in fluid medium, form colonies in soft agar culture, and induce tumors in rats transplanted with the transformed cells at the same efficiency as WT-transformed cells. Tumors were induced in hamsters injected with WT virions but were not induced in hamsters injected with dl207 virions. The results indicate that the E1B 58K protein is required both for viral DNA replication in productive infection and for initiation of cell transformation, but not for maintenance of the transformed phenotype.     

Different activities of the adenovirus types 5 and 12 E1A regions in transformation with the EJ Ha-ras oncogene.

We have compared the capacities of the E1A regions of nononcogenic adenovirus type 5 (Ad5) and highly oncogenic Ad12 to cooperate with the EJ bladder carcinoma Ha-ras-1 oncogene in the transformation of primary baby rat kidney cells. Both E1A regions, when cotransfected with the Ha-ras oncogene, transformed the primary cells with a low frequency. Ad5 E1A plus Ha-ras-transformed cells differed in phenotype from cells transformed by Ad12 E1A plus Ha-ras. The cells expressing Ad5 E1A appeared highly transformed and practically failed to adhere to plastic. This phenotype may be due to the virtually complete absence of fibronectin gene expression in these cells. In contrast, the cells expressing Ad12 E1A were flatter and adhered to plastic, whereas fibronectin gene expression was reduced but not absent. The oncogenic potential of the two types of E1A plus ras-transformed cells was tested by their injection into both athymic nude mice and weanling syngeneic rats. The Ad5 E1A plus ras-transformed cells were found to be highly oncogenic in both animal species, whereas the Ad12 E1A plus ras-transformed cells were only weakly oncogenic in both syngeneic rats and nude mice. The difference in oncogenic potential of the Ad5 E1A plus ras- and the Ad12 E1A plus ras-transformed cells is discussed in terms of the different capacities of the Ad5 and Ad12 E1A-encoded proteins to modulate cellular gene expression.     

Human adenovirus early region 4 open reading frame 1 genes encode growth-transforming proteins that may be distantly related to dUTP pyrophosphatase enzymes.

An essential oncogenic determinant of subgroup D human adenovirus type 9 (Ad9), which uniquely elicits estrogen-dependent mammary tumors in rats, is encoded by early region 4 open reading frame 1 (E4 ORF1). Whereas Ad9 E4 ORF1 efficiently induces transformed foci on the established rat embryo fibroblast cell line CREF, the related subgroup A Ad12 and subgroup C Ad5 E4 ORF1s do not (R. T. Javier, J. Virol. 68:3917-3924, 1994). In this study, we found that the lack of transforming activity associated with non-subgroup D adenovirus E4 ORF1s in CREF cells correlated with significantly reduced protein levels compared to Ad9 E4 ORF1 in these cells. In the human cell line TE85, however, the non-subgroup D adenovirus E4 ORF1s produced protein levels higher than those seen in CREF cells as well as transforming activities similar to that of Ad9 E4 ORF1, suggesting that all adenovirus E4 ORF1 polypeptides possess comparable cellular growth-transforming activities. In addition, searches for known proteins related to these novel viral transforming proteins revealed that the E4 ORF1 proteins had weak sequence similarity, over the entire length of the E4 ORF1 polypeptides, with a variety of organismal and viral dUTP pyrophosphatase (dUTPase) enzymes. Even though adenovirus E4 ORF1 proteins lacked conserved protein motifs of dUTPase enzymes or detectable enzymatic activity, E4 ORF1 and dUTPase proteins were predicted to possess strikingly similar secondary structure arrangements. It was also established that an avian adenovirus protein, encoded within a genomic location analogous to that of the human adenovirus E4 ORF1s, was a genuine dUTPase enzyme. Although no functional similarity was found for the E4 ORF1 and dUTPase proteins, we propose that human adenovirus E4 ORF1 genes have evolved from an ancestral adenovirus dUTPase and, from this structural framework, developed novel transforming properties.     

Dependence of tumor-forming capacities of cells transformed by recombinants between adenovirus types 5 and 12 on expression of early region 1.

Recombinants between an adenovirus type 5 (Ad5) deletion mutant and the Ad12 DNA fragment containing early region 1 (E1) were isolated from cells cotransfected with the EcoRI-C fragment of Ad12 DNA and Ad5 dl312 (deletion in E1A) DNA (rcA) and from cells cotransfected with the SalI-C fragment of Ad12 DNA and Ad5 dl312 DNA (rcB). No recombinant was isolated from cells cotransfected with Ad5 dl313 (deletion in E1B) DNA and restriction fragments of Ad12 DNA. Both rcA and rcB are defective and able to replicate in human embryo kidney (HEK) and KB cells with complementation by dl312. Both rcA and rcB formed Ad12 T antigen g, but not T antigen f, in infected HEK and KB cells. In rcA- and rcB-infected cells, Ad5 E1B and Ad12 E1A genes are transcribed. Heteroduplex and size analyses of rcA-1 or rcB-1 DNA fragments hybridized with Ad12 DNA revealed that rcA-1 DNA has a deletion between 5 and 15 map units with an insertion of a portion of Ad12 DNA (10%) and that rcB-1 DNA has a deletion between 70 and 80 map units with an insertion of a portion of Ad12 DNA (10%). The transformed cell lines, RCAY and RCBY, were established after infection of rat 3Y1 cells with rcA and rcB, respectively. Both Ad5 and Ad12 DNA sequences are contained in these cells. In RCAY cells, Ad12 T antigen g is detected, but Ad12 T antigen f is not. In RCBY cells, both Ad12 T antigen g and f are detected. Only the Ad12 E1A gene is transcribed in RCAY cells, whereas Ad5 E1B, Ad12 E1A, and Ad12 E1B genes are transcribed in RCBY cells. In soft-agar cultures, RCBY cells form large colonies, whereas RCAY cells form only tiny colonies. RCBY cells form tumors as efficiently as 12WY cells in transplanted rats. RCAY cells formed tumors inefficiently. Ad5-transformed 5WY cells do not form tumors. These observations indicate that the efficient tumor formation by RCBY cells is dependent on the expression of the Ad12 E1A and E1B genes, whereas the inefficient tumor formation by RCAY cells is due to the expression of only the Ad12 E1A gene.     

Transformation by Rous sarcoma virus: a cellular substrate for transformation-specific protein phosphorylation contains phosphotyrosine

Transformation of chicken embryo fibroblasts by Rous sarcoma virus (RSV) is caused by a single viral gene, src, which encodes a phosphoprotein, pp60src, with the enzymatic activity of a protein kinase. The relative abundance of a 36,000 molecular weight (36K) phosphorylated polypeptide which can be detected by two-dimensional electrophoresis of 32P-labeled phosphoproteins is greatly increased in RSV-transformed fibroblasts. We have reported previously that phosphorylation of the 36K polypeptide is an early event in the process of transformation and that protein synthesis is not required for its appearance. Here we identify a nonphosphorylated 36K polypeptide, present in both uninfected and transformed cells, which is homologous to the 36K phosphoprotein as judged by limited proteolysis and by tryptic peptide mapping. We conclude that the 36K phosphoprotein is generated by phosphorylation of this 36K polypeptide. It has recently been shown that pp60src phosphorylates tyrosine residues in vitro: phosphotyrosine and also phosphoserine are present in the 36K phosphoprotein isolated from RSV-transformed cells. On the basis of these results we propose that the 36K polypeptide present in chicken fibroblasts is a substrate for the protein kinase activity of pp60src. Phosphorylation of this polypeptide may be important in cellular transformation by Rous sarcoma virus.     

Modulation of the level of expression of cellular genes in adenovirus 12-infected and transformed human cells.

The level of expression of thymidine kinase (TK), heat shock protein 70 (HSP70), beta-tubulin and p53 was assessed in human embryo kidney cells (HEKs) infected with adenovirus type 12 (Ad 12) and Ad 12 early region 1 (E1) mutants. HSP70, beta-tubulin and p53 levels were unchanged but TK activity was dramatically increased following wild-type infection. The initial activation of TK required the expression of the product of the E1A 13S mRNA but sustained expression only occurred with those viruses expressing the E1B proteins as well. A number of human cell lines transformed with either Ad 12 or Ad 5 E1 DNA were also assessed for the level of expression of HSP70, beta-tubulin and p53. Both HSP70 and beta-tubulin levels were greatly increased compared with primary human cells although there was considerable variation between lines. p53 was only expressed at high levels in Ad 12-transformed lines expressing E1A and E1B proteins.     

Transformation of chicken myelomonocytic cells by a retrovirus expressing the v-myb oncogene from the long terminal repeats of avian myeloblastosis virus but not Rous sarcoma virus.

To test the effect of long terminal repeat (LTR) regulatory sequences on the transforming capability of the v-myb oncogene from avian myeloblastosis virus (AMV), we have constructed replication-competent avian retroviral vectors with nearly identical structural genes that express v-myb from either AMV or Rous sarcoma virus (RSV) LTRs. After transfection into chicken embryo fibroblasts, virus-containing cell supernatants were used to infect chicken myelomonocytic target cells from preparations of 16-day-old embryonic spleen cells. Both wild-type AMV and the virus expressing v-myb from AMV LTRs (RCAMV-v-myb) were able to transform the splenocyte cultures into a population of immature myelomonocytic cells. The transformed cells expressed the p48v-Myb oncoprotein and formed compact foci when grown in soft agar. In contrast, the virus expressing v-myb from RSV LTRs (RCAS-v-myb) was repeatedly unable to transform the same splenocyte cells, despite being able to infect fibroblasts with high efficiency. This difference in the transforming activities of v-myb-expressing viruses with different LTRs most likely results from the presence of a factor (or factors) within the appropriate myelomonocytic target cell that promotes specific expression from the AMV but not from the RSV LTR.     

Regulation of cellular morphology by the Rous sarcoma virus src gene: analysis of fusiform mutants.

We have been interested in how Rous sarcoma virus (RSV) influences transformed cell morphology and compared the molecular properties of chicken embryo cells (CEC) infected with mutants of RSV that induce the fusiform transformed cell morphology with those of CEC infected by wild-type RSV, which induces the more normal round transformed cell morphology. We looked for properties shared by all fusiform mutant-infected cells, because these may be responsible for maintaining the fusiform morphology. Five different fusiform mutants, two wild-type RSVs, and one wild-type back revertant of a fusiform mutant were studied. In the fusiform mutant-infected cells, the localization and myristylation of pp60src were determined and the extent of expression of the extracellular matrix protein fibronectin was examined at both the mRNA and protein levels. The phosphorylation of vinculin on tyrosine also was examined in the same CEC. Within all fusiform mutant-transformed CEC, pp60src was dramatically absent from the adhesion plaque sites normally seen in cells transformed with wild-type RSV, and these transformed CEC all expressed more fibronectin mRNA and protein in the extracellular matrix than did the wild-type RSV-transformed CEC. The absence of pp60src from the adhesion plaques was not due to lack of myristylation of the src protein, and tyrosine phosphorylation of vinculin was not related to fibronectin expression. These results suggest that the inverse relationship between pp60src in the adhesion plaques and fibronectin expression in the extracellular matrix may be interconnected phenomena and could be related to the maintenance of the fusiform transformed morphology.