Oncogene-directed mutagenesis in vivo. Polyalkylating derivatives of short single-stranded polynucleotides, complementary E1-adeno-oncogene, in the normalization of adenovirus-transformed rodent cell lines]

Polyalkylating derivatives of single-stranded polynucleotides (30-200-mers) complementary to the long E1 oncogene sequences of simian adenovirus SA7 cause inherited normalization of SH2 and G11 cells transformed with adenovirus SA7; certain deletions in the integrated proviral E1A oncogene were observed in several cases during this process. The transformed cells are indifferent to reagents noncomplementary to the E1 region. Thus polyalkylating derivatives of single-stranded 30-200-mers act as addressed mutagenes which react in a specific way with the integrated complementary DNA sequences of E1 oncogene in transformed rodent cells and realize oncogene-directed mutagenesis in vivo. During this treatment temporary normalized cells reverting to the initial transformed phenotype are also produced.     

Integration of foreign genome fragments into cells transformed or cotransformed with fragmented adenoviral DNA

The integration of DNA of highly oncogenic simian adenovirus type 7 (SA7) and non-oncogenic human adenovirus type 6 (Ad6) into the genome of newborn rat kidney cells transformed by fragmented DNA preparations was studied using reassociation kinetics and spot hybridization. Transforming DNA was fragmented with the specific endonuclease SalI (SA7) and BglII (Ad6). In contrast to the cell transformation by intact viral DNA, transformation by fragmented DNA resulted in integration into the cellular genome of not only the lefthand fragment with the oncogene but also of other regions of the viral genome. Additionally integrated fragments were stable and preserved during numerous passages of cells lines, although they were no expressed, at least in the case of the Ad6-transformed cell line. The integration of the fragments of SA7 DNA was accompanied by loss of 25-50% of the mass of each fragment. Adding the linear form of the pBR322 plasmid to the preparation of transforming Ad6 DNA also contributed to its cointegration into the genome of the transformed cell. This technique of cell cotransformation with any foreign DNAs together with the viral oncogens may be used as an equivalent of an integration vector for eukaryotic cells.     

Complementary directed modifications of nucleic acids and oncogene-directed mutagenesis in vivo.

High reactivity of the polyalkylating ss oligomers that were sense or antisense 30-200-mers containing sequences complementary to E1 oncogenes of simian adenovirus SA7 and one alkylating residue -CH2CH2N(C2H5OH) (CH2)3N(Ph-p-CH2OH)CH2CH2Cl per each 25 bases of oligomers was demonstrated in vitro by alkylation of ss DNA of recombinant M 13 mp8E1 and mp9E1 phages with inserted E1 sequences of adenovirus oncogene and then by followed complete and selective elimination of E1 sequences from recombinant ss DNA. Treatment of rodent cell cultures transformed by oncogenic SA7 with polyalkylating oligomers which are complementary to the long region of the minus or plus chains of E1 DNA of SA7 revealed a rather high extent of mutant cell clones formation. The cells formed were normalized; they had lost some properties of the transformed cells. Dividing cell clones inherited the new phenotypic properties: morphology, slower and more limited proliferation, and higher dependence on bovine serum growth factors. Some of the mutant cell DNAs demonstrated different mutations in the E1A sequences of the integrated proviral oncogene. There were exchanges G to C (leu to val) in the 525 and C to A (asp to tyr) in the 555 positions of E1A oncogene. Besides a deletions in the 1057-1477 E1A region or/and a mutation in the 1457-1477 of E1A were observed. Thus the inherited cell normalization observed is performed due to oncogene-directed mutagenesis in vivo.     

Subcloning of DNA fragments of the simian adenovirus SA7 oncogene in bacteriophage M13

The XmaI/PstI and XmaI DNA fragments of adenovirus SA7 oncogene and the adjacent region (16.7% of the physical map of SA7 left end DNA) were recloned in M13 bacteriophages mp8 and mp9 in order to obtain the singlestranded fragments EIa and EIb from the DNA region of monkey adenovirus SA7 located on the recombinant plasmid pASP carrying the DNA APstI fragment including the adenovirus SA7 oncogene.     

Model systems of carcinogenesis in vitro: the interaction of the ras oncogene with immortalized cells

Gene transfer experiments have shown that ras effector functions are sufficient to transform cells from a variety of established lines (e. g., mouse NIH3T3 cells). In contrast, primary cells and early passage rodent cells can be transformed by ras oncogenes only at low frequencies, unless cotransfected with collaborating genes such as adenovirus early region IA (EIA) or myc retroviral oncogene homologue. Primary rat embryo fibroblasts (REF) were chosen as a model for the analysis of multistep cellular transformation. Transfection of REF, immortalized by early region of simian adenovirus SA7 with c-Ha-ras oncogene cannot induce their morphological transformation. This phenomenon is observed only after second transfection with the same oncogene. These different cell lines can be used for further analysis of the mechanisms of carcinogenesis.     

Modification of steroidogenesis in a mouse adrenal cell line (Y-1) transformed by simian adenovirus SA-7

Transformation of a steroidogenic mouse adrenal cell line (Y-1) by simian adenovirus SA7 produced a cell line with low apparent steroidogenic activity. The effect of ACTH and cholera toxin on cyclic AMP production was similar in both not transformed and virus-transformed cells and activity of cyclic AMP-dependent protein kinase was also similar in both cells. In transformed cells, cholesterol was metabolized to delta 5-3 beta-hydroxysteroids, mainly 20 alpha-dihydropregnenolone while in not transformed cells, the major metabolites were delta 4-3 ketosteroids (20 alpha-dihydro- and 11 beta-hydroxy-20 alpha-dihydroprogesterone). In both cell lines ACTH increased the metabolism of cholesterol. Further studies with labelled pregnenolone and progesterone revealed a loss of delta 5-3 beta-hydroxysteroid dehydrogenase/isomerase and 11 beta-hydroxylase activity in the transformed cells.     

Influence of the deprivation of a single amino acid on cellular proliferation and survival in rat 3Y1 fibroblasts and their derivatives transformed by a wide variety of agents

We compared proliferation and survival of various syngeneic transformed cell lines under conditions of depletion of 15 amino acids in Dulbecco-Eagle's medium. We used a normal fibroblast line 3Y1 and 22 transformed sublines of 3Y1 which had been induced by one of seven transforming agents--simian virus 40, mouse polyomavirus, adenovirus type 12, E1A gene of adenovirus type 12, cDNA of Harvey murine sarcoma virus, Rous sarcoma virus, or N-methyl-N'-nitro-N-nitrosoguanidine. Unlike other untransformed cells examined (mouse BALB/c-3T3 line, mouse NIH-3T3 line, and primary Fischer rat embryo fibroblasts), 3Y1 ceased to proliferate and accumulated in a viable state with a G1-phase DNA content under 14 singular deprivations of amino acid. None of the transformed 3Y1 lines completely arrested in the G1 phase of the cell cycle and each showed different levels of survival, depending on each transforming agent. As for transformed 3Y1 cells induced by a given virus or a given transforming gene, any one of the three sublines shared the same trend with respect to proliferation and survival. Transformed derivatives induced by N-methyl-N'-nitro-N-nitrosoguanidine showed almost the same trend in proliferation, but the patterns of survival were not uniform. Our observations suggest that the unique responses of 3Y1 to amino acid depletion are differently modified by different transforming agents.     

Complementary-addressed elimination of E1a sequence of simian adenovirus oncogene SA7 from circular single-stranded DNA of recombinant phage M13

The G fragment of the simian adenovirus SA7 oncogene corresponding to E1a region was cloned into M13mp8 and M13mp9 phages. Single-stranded DNAs of the recombinant phages thus obtained (mp8G and mp9G) partially digested with DNAse II were used to synthesize polyalkylating derivatives capable of specific hybridisation and subsequent alkylation of complementary G sequences of corresponding phage DNAs. After incubation of complementary alkylated DNA in the presence of lysine, the preselected region (G fragment) was specifically eliminated without damaging vector sequences. The method of complementary-addressed cleavage proved to be useful for precise analysis of reactions of polyalkylating derivatives within complementary complexes.     

Preparation of single-stranded E1A-oncogene DNA fragments from simian adenovirus SA7 by endonuclease hydrolysis recombinant phage M13 SS-DNA complexed with oligonucleotides, containing restriction sites]

The ss-DNA of the (+) and (-) chains of Ela DNA fragment was obtained by hydrolysis of the recombinant bacteriophages M13 mp8G and mp9G (where G is 1-1750 bp:, E1a region of oncogene SA7) in complexes with the 16 bp oligonucleotides containing AluI and BspRI sites of restriction and sequences complementary to E1a SA7. The obtained fragments overlap the E1a zones associated with the immortalizing potential of SA7.     

Specific interactions between rotavirus outer capsid proteins VP4 and VP7 determine expression of a cross-reactive, neutralizing VP4-specific epitope.

We previously reported that the expression of rotavirus phenotypes by reassortants was affected by recipient genetic background and proposed specific interactions between the outer capsid proteins VP4 and VP7 as the basis for the phenotypic effects (D. Chen, J. W. Burns, M. K. Estes, and R. F. Ramig, Proc. Natl. Acad. Sci. USA 86:3743-3747, 1989). A neutralizing, cross-reactive VP4-specific monoclonal antibody (MAb), 2G4, was used to probe the protein-protein interactions. The VP4 specificity of 2G4 was confirmed by immunoblot analysis. MAb 2G4 reacted with both standard (SA11-C13) and variant rotavirus SA11 (SA11-4F) but did not react with bovine rotavirus B223 as determined by plaque reduction neutralization (PRN) and enzyme-linked immunosorbent assay (ELISA). When a panel of SA11-4F/B223 and SA11-Cl3/B223 reassortants in purified or crude lysate form that had been grown in the presence or absence of trypsin was analyzed with MAb 2G4 by PRN and ELISA, the results with some reassortants were unexpected. That is, MAb 2G4 reacted with VP4 of SA11 parental origin (4F or C13) when it was assembled into capsids with the homologous SA11 VP7 but failed to react with VP4 of SA11 assembled into capsids with heterologous B223 VP7. Conversely, MAb 2G4 failed to react with VP4 of B223 parental origin when it was assembled into capsids with homologous B223 VP7 but did react with B223 VP4 assembled into capsids with the heterologous SA11 VP7. Similar reactivity was observed when 2G4 was used to immunoprecipitate purified double-shelled virions. When soluble unassembled viral proteins were analyzed by ELISA, the 2G4 reactive pattern was as predicted from the parental origin of VP4. That is, 2G4 reacted with the soluble VP4 of reassortants having VP4 from SA11-Cl3 or SA11-4F and failed to react with VP4 of B223 origin, regardless of the origin of VP7. PRN and ELISA results obtained with nonglycosylated viruses revealed that the unexpected reactivity of 2G4 with virus particles was not the result of differential glycosylation of VP7 and epitope masking. These results indicate that the 2G4 epitope existed in the soluble form of VP4 encoded by SA11-Cl3 or SA11-4F but not in soluble B223 VP4. On the other hand, in assembled virions, the presentation of the 2G4 epitope on VP4 was unexpected in some reassortants and was affected by the specific interactions between VP4 and VP7 of heterologous parental origin.     

Transforming Potential of the Adenovirus Type 5 E4orf3 Protein

Previous observations that the adenovirus type 5 (Ad5) E4orf6 and E4orf3 gene products have redundant effects in viral lytic infection together with the recent findings that E4orf6 possesses transforming potential prompted us to investigate the effect of E4orf3 expression on the transformation of primary rat cells in combination with adenovirus E1 oncogene products. Our results demonstrate for the first time that E4orf3 can cooperate with adenovirus E1A and E1A plus E1B proteins to transform primary baby rat kidney cells, acting synergistically with E4orf6 in the presence of E1B gene products. Transformed rat cells expressing E4orf3 exhibit morphological alterations, higher growth rates and saturation densities, and increased tumorigenicity compared with transformants expressing E1 proteins only. Consistent with previous results for adenovirus-infected cells, the E4orf3 protein is immunologically restricted to discrete nuclear structures known as PML oncogenic domains (PODs) in transformed rat cells. As opposed to E4orf6, the ability of E4orf3 to promote oncogenic cell growth is probably not linked to a modulation of p53 functions and stability. Instead, our results indicate that the transforming activities of E4orf3 are due to combinatorial effects that involve the binding to the adenovirus 55-kDa E1B protein and the colocalization with PODs independent from interactions with the PML gene product. These data fit well with a model in which the reorganization of PODs may trigger a cascade of processes that cause uncontrolled cell proliferation and neoplastic growth. In sum, our results provide strong evidence for the idea that interactions with PODs by viral proteins are linked to oncogenic transformation.     

Adenovirus Transformation of Hamster Embryo Cells

Inoculation of hamster embryo cell cultures with human adenovirus type 12 (Ad12) or simian adenovirus (SA7) resulted in the formation of foci of morphologically transformed cells within 12 days. The rapid appearance of well-defined foci was dependent upon the transfer of cells into new plates, with sufficient dilution after virus adsorption, and was independent of virus dose. Dose-response studies showed linearity of focus formation with dilution of Ad12 or SA7. Results averaged from several experiments show plaque-forming unit to focus-forming unit ratios of approximately 1.8 x 10(6) for Ad12 and 2.6 x 10(5) for SA7. Other experiments showed that most of the adenovirus involved in transformation was adsorbed by 3 hr. Cell lines derived from SA7 transformed cells produced tumors within 19 days when inoculated intradermally into young adult hamsters. Such cell-induced tumors histologically resembled SA7 virus-induced hamster tumors. Formation of tumors with SA7 transformed cells was inhibited by prior immunization of test animals with SA7 or Ad12 virus.     

Transformation of BALB/3T3 cells with EJ/T24/H-ras oncogene inhibits adenylate cyclase response to beta-adrenergic agonist while increases muscarinic receptor dependent hydrolysis of inositol lipids

Balb/3T3 murine fibroblasts transformed by transfection with the EJ/T24 human bladder carcinoma oncogene were assayed in terms of adenylate cyclase response and hydrolysis of polyphosphoinositides dependent on specific agents. Transformed cells were much less responsive to beta-adrenergic agonists in rising cAMP than normal cells. They are instead much more sensitive to muscarinic receptor agonists, inducing a rapid intracellular accumulation of inositol phosphates. These results suggest that the functional alteration of the cell membrane caused by the product of the point mutated H-ras oncogene concerns in 3T3 fibroblasts both inhibitory and stimulatory effects, respectively on adenylate cyclase and phosphoinositide-phosphodiesterase.     

Transformation of Hamster Embryo Cells and Tumor Induction in Newborn Hamsters by Simian Adenovirus SV11

Simian adenovirus, SV11, readily transformed hamster embryo cell cultures in vitro and produced tumors in vivo when inoculated into newborn hamsters. Foci consisting of small, loosely attached, rounded cells could be seen as early as 7 days postinoculation. Many of these cells contained several nuclei or the nucleus was multilobed. The cells grew without extensive cell to cell contact or formed small chains or clusters when passaged in vitro. This pattern of cell morphology and growth has not been reported with other simian or human adenovirus-transformed cells. Linearity of foci formation with virus dilution was observed when the virus multiplicity was less than 3 plaque-forming units (PFU)/cell. The PFU to focus-forming units ratio for SV11 was found to be 2 x 10(4) to 4 x 10(4), which is approximately 5- to 10-fold and 50- to 100-fold lower than those reported for simian adenovirus, SA7, and human adenovirus type 12, respectively. Cells transformed by SV11: (i) produced tumors when inoculated into young hamsters, (ii) contained tumor antigen which reacts with serum obtained from hamsters bearing SV11 passaged tumors, and (iii) could be propagated in vitro through an indefinite number of generations.     

Cytophotometric study of changes in the structure of nuclear chromatin induced by the oncogenic adenovirus SA7 (C8)

Infection of primary murine embryonic cell cultures by adenovirus SA7 (C8) results in an increase in chromatin condensation Average optical density of Feulgen stained nuclei 24 h following virus absorption increased for G0/1, S, and G2 cells by 16.1, 11.3 and 13.1%, respectively. This phenomenon is associated with the stimulation of proliferation, with an increase of the S cell amount by 50% of the control values and a decrease of average cell nuclei areas in all phases of cell cycle.     

Role of tumor necrosis factor-alpha in E1A oncogene-induced susceptibility of neoplastic cells to lysis by natural killer cells and activated macrophages

NIH-3T3 cells transfected with adenovirus E1A oncogene cDNA were found to exhibit cytolytic susceptibility to murine NK cells and activated macrophages associated with a threshold level of oncogene product expression exceeding that required for morphological transformation. A similar correlation was observed between threshold levels of E1A gene product expression and target cell susceptibility to direct cytotoxicity by rTNF. Inhibition of splenic NK cell and peritoneal macrophage cytolysis by antisera specific for murine rTNF confirmed the importance of E1A-induced TNF susceptibility as one determinant of target cell cytolytic susceptibility. Anti-TNF antibody was, however, unable to block killing of E1A-expressing targets by the NK cell line, NKB61A2. These results suggest a direct link between the functions of E1A oncogene products and cellular mechanisms of action of TNF elaborated by host effector cells and indicate that E1A expression also affects target cell susceptibility to TNF-independent cytolytic mechanisms.     

Biological effects accompanying the microinjection of adenovirions, adenoviral and plasmid DNA into mammalian cells

Microinjection of either type 1 human adenovirus, type SA7 monkey adenovirus virions or circular adenovirus DNA, obtained by the treatment of DNA-terminal protein complexes with glutaraldehyde, into nuclei of permissive cells results in the complete cycle of virus reproduction. Microinjection of neither linear native, condensed adenovirus DNA nor the DNA-terminal protein complexes under the same conditions initiates the adenovirus reproduction thought the synthesis of early and some late viral antigens is observed in the injected cells. Integration of injected adenovirus DNA into the cellular DNA occurs as far as 30 min after injection. Microinjection of either adenovirus DNA or its oncogene containing fragments into nuclei of semipermissive cells induces the transformation of these cells. In this case the time of the first appearance of transformation foci is decreased.     

Isolation of a cDNA encoding the adenovirus E1A enhancer binding protein: a new human member of the ets oncogene family.

The cDNA encoding adenovirus E1A enhancer-binding protein E1A-F was isolated by screening a HeLa cell lambda gt11 expression library for E1A-F site-specific DNA binding. One cDNA clone produced recombinant E1A-F protein with the same DNA binding specificity as that endogenous to HeLa cells. Sequence analysis of the cDNA showed homology with the ETS-domain, a region required for sequence-specific DNA binding and common to all ets oncogene members. Analysis of the longest cDNA revealed about a 94% identity in amino acids between human E1A-F and mouse PEA3 (polyomavirus enhancer activator 3), a recently characterized ets oncogene member. E1A-F was encoded by a 2.5kb mRNA in HeLa cells, which was found to increase during the early period of adenovirus infection. In contrast, ets-2 mRNA was significantly reduced in infected HeLa cells. The results indicate that E1A enhancer binding protein E1A-F is a member of the ets oncogene family and is probably a human homologue of mouse PEA3.     

A small nuclear RNA, U5, can transform cells in vitro.

Low-molecular-weight RNA exhibiting transforming potential was identified in chemically induced lymphoma cells by the transformation of mink lung cells after transfection. The RNA was sequenced by the direct chemical method and was shown to be a small nuclear RNA, U5. The transforming potential of the RNA was further studied in quantitative transformation assays using 3Y1, a rat fibroblastic cell line. Transformed foci appeared with a latency of 3 to 4 weeks after transfection. U5-transformed 3Y1 cells frequently carried an amplified c-myc oncogene. In addition, U5 induced chromosome aberrations in transfected cells, indicating that the RNA acts as a clastogen. Transforming and clastogenic potentials were specifically inactivated when U5 was incubated with RNase H in the presence of a complementary oligonucleotide. We discuss a possible mechanism of U5-induced cell transformation.