Ribozymes designed to inhibit transformation of NIH3T3 cells by the activated c-Ha-ras gene.

We have designed hammerhead ribozymes that cleave c-Ha-ras mRNA mutated at codon 12 (GGU----GUU). Plasmids containing the ribozyme-encoding genes were expressed under the control of the long terminal repeats of Rous sarcoma virus in NIH3T3 cells transfected with the activated c-Ha-ras gene. These ribozymes were found to inhibit formation of foci (by about 50%) by cleaving the oncogene mRNA, rather than by hybridizing to it. Furthermore, when the activated c-Ha-ras gene was cotransfected with the ribozyme-encoding gene, three morphologically flat colonies were found and isolated. We also found that expression of c-Ha-ras was suppressed in cells containing ribozymes.     

Resistance to oncogenic transformation in revertant R1 of human ras-transformed NIH 3T3 cells.

A flat revertant, R1, was isolated from human activated c-Ha-ras-1 (hu-ac-Ha-ras) gene-transformed NIH 3T3 cells (EJ-NIH 3T3) treated with mutagens. R1 contained unchanged transfected hu-ac-Ha-ras DNA and expressed high levels of hu-ac-Ha-ras-specific mRNA and p21 protein. Transfection experiments revealed that NIH 3T3 cells could be transformed by DNA from R1 cells but R1 cells could not be retransformed by Kirsten sarcoma virus, DNA from EJ-NIH 3T3 cells, hu-ac-Ha-ras, v-src, v-mos, simian virus 40 large T antigen, or polyomavirus middle T antigen. Somatic cell hybridization studies showed that R1 was not retransformed by fusion with NIH 3T3 cells and suppressed anchorage independence of EJ-NIH 3T3 and hu-ac-Ha-ras gene-transformed rat W31 cells in soft agar. These results suggest that the reversion and resistance to several oncogenes in R1 is due not to cellular defects in the production of the transformed phenotype but rather to enhancement of cellular mechanisms that suppress oncogenic transformation.     

Transfer of active oncogenes and promoters into the mouse cell genome

Different cell DNA's (normal NIH 3T3 DNA; human osteosarcoma cell DNA; human malignant glioma cell DNA with amplified c-Ha-ras) were cotransfected onto NIH 3T3 cells with cloned long terminal repeat (LTR) sequences of Rous sarcoma virus. LTR RSV and normal NIH 3T3 DNA c-fos oncogen expression was detected in tumors induced in nude mice. In the same system human tumour cell DNA with amplified c-Ha-ras gene was used, that to the integration and amplification of LTP sequences with simultaneous maintenance of c-Ha-ras amplification. Nude mouse tumour DNA with integrated LTR sequences was active in successive rounds of transfection.     

The c-Ha-ras oncogene induces increased expression of beta-galactoside alpha-2, 6-sialyltransferase in rat fibroblast (FR3T3) cells.

Alteration in cell surface carbohydrates, and in particular cell surface sialylation, have been known to occur during oncogenic transformation. To examine the basis for such changes, we have transformed the rat fibroblast cell line FR3T3 with the oncogenes c-Ha-ras EJ, v-mycOK10, v-src, polyoma virus middle T or the transforming bovine papilloma virus 1 (BPV1), and measured the sialytransferase activities of cellular lysates. We found that, in contrast to all other oncogenes examined, c-Ha-ras induced a striking increase in beta-galactoside alpha-2,6-sialytransferase (Gal alpha-2,6-ST) activity in FR3T3 cells. This increase in Gal alpha-2,6-ST activity resulted in the increased expression of cell surface alpha-2,6-linked sialic acid on cell surface glycoconjugates, as determined by cell staining with fluorescein-labelled Sambucus nigra agglutinin. Immunoprecipitation and immunofluorescence experiments revealed that the increase in Gal alpha-2,6-ST activity was due to an elevation of expression of the enzyme. Moreover, Northern analysis suggested that the increased expression of this enzyme was the result of an increase in the steady-state mRNA level of the Gal alpha-2,6-ST gene. These results support the notion that alterations seen in cell surface glycoconjugates during oncogenic transformation can be the result of altered expression of glycosyltransferases.     

The c-Ha-ras oncogene and a tumor promoter activate the polyoma virus enhancer

A c-Ha-ras oncogene, to a lesser extent the c-Ha-ras proto-oncogene, and the tumor promoter 12-O-tetradecanoylphorbol-13-acetate activate the inactive polyoma virus (Py) enhancer in a myeloma cell line and the partially active Py enhancer in NIH 3T3 fibroblasts, but have no effect on the active Py enhancer in LMTK- fibroblasts. In addition, c-Ha-ras can stimulate the inactive Py enhancer in embryonal carcinoma F9 cells. c-Ha-ras activation in embryonal carcinoma cells does not appear to involve reversal of "E1A-like" inhibition of the enhancer. We suggest that modulation of cellular enhancer activity could play a key role in tumorigenesis by oncogenes.     

Biochemical correlates of phenotypic reversion in interferon-treated mouse cells transformed by a human oncogene

Mouse interferon (IFN) induced a phenotypic reversion in RS 485, a clonal line of NIH 3T3 oncogenically transformed by a human c-Ha-rasl gene activated by Ha-MuSV long terminal repeats (LTRs). Transfected c-Ha-ras DNA, unchanged in quantity and distribution, as compared to the parental RS 485 transformed cells, was still present in these revertants; however, there was a significant reduction in the amount of c-Ha-ras specific mRNA and of c-Ha-ras specified p21 protein.     

Differentiation of simian virus 40 transformed human mammary epithelial stem cell lines to myoepithelial-like cells is associated with increased expression of viral large T antigen

Cloned simian virus 40 (SV40)-transformed human breast epithelial cell lines can differentiate to myoepithelial-like cells, and these can be isolated as clonal cell lines. Immunofluorescent and immunocytochemical analysis of such cell lines growing on plastic surfaces, collagen gels, and as tumor-nodules in nude mice indicate that all the cell lines produce SV40 large T antigen, but that the production of this antigen is qualitatively increased in the myoepithelial-like cells and cell lines. The myoepithelial-like cell lines produce 4-6 times more immunoprecipitable large T antigen than the parental epithelial cells. The amount of mRNA for large T antigen is also increased by 3.5-5-fold in the myoepithelial-like cell lines when analysed by dot-blot or by Northern hybridisations. Thus, differentiation along the myoepithelial-like cell pathway is associated in these SV40-transformed cells with increased expression of the viral large T antigen. It is suggested that immortalization of primary breast epithelial cell cultures may be, in part, due to the expression of large T antigen preventing processes of terminal keratinization.     

Two separable functional domains of simian virus 40 large T antigen: carboxyl-terminal region of simian virus 40 large T antigen is required for efficient capsid protein synthesis.

The carboxyl-terminal portion of simian virus 40 large T antigen is essential for productive infection of CV-1 and CV-1p green monkey kidney cells. Mutant dlA2459, lacking 14 base pairs at 0.193 map units, was positive for viral DNA replication, but unable to form plaques in CV-1p cells (J. Tornow and C.N. Cole, J. Virol. 47:487-494, 1983). In this report, the defect of dlA2459 is further defined. Simian virus 40 late mRNAs were transcribed, polyadenylated, spliced, and transported in dlA2459-infected cells, but the level of capsid proteins produced in infected CV-1 green monkey kidney cells was extremely low. dlA2459 large T antigen lacks those residues known to be required for adenovirus helper function, and the block to productive infection by dlA2459 occurs at the same stage of infection as the block to productive adenovirus infection of CV-1 cells. These results suggest that the adenovirus helper function is required for productive infection by simian virus 40. Mutant dlA2459 was able to grow on the Vero and BSC-1 lines of African green monkey kidney cells. Additional mutants affecting the carboxyl-terminal portion of large T were prepared. Mutant inv2408 contains an inversion of the DNA between the BamHI and BclI sites (0.144 to 0.189 map units). This inversion causes transposition of the carboxyl-terminal 26 amino acids of large T antigen and the carboxyl-terminal 18 amino acids of VP1. This mutant was viable, even though the essential information absent from dlA2459 large T antigen has been transferred to the carboxyl terminus of VP1 of inv2408. The VP1 polypeptide carrying this carboxyl-terminal portion of large T could overcome the defect of dlA2459. This indicates that the carboxyl terminus of large T antigen is a separate and separable functional domain.     

Effect of cellular determination on oncogenic transformation by chemicals and oncogenes.

Three developmentally determined myogenic cell lines derived from C3H 10T1/2 C18 (10T1/2) mouse embryo cells treated with 5-azacytidine were compared with the parental 10T1/2 line for their susceptibility to oncogenic transformation by 3-methylcholanthrene or the activated human c-Ha-ras oncogene. Neither the 10T1/2 cells nor the myogenic derivatives grew in soft agar or formed tumors in nude mice. In contrast to 10T1/2 cells, the three myogenic derivatives were not susceptible to transformation by 3-methylcholanthrene, so that cellular determination altered the response of 10T1/2 cells to chemical carcinogen. On the other hand, all cell types were transformed to a tumorigenic phenotype following transfection with the activated c-Ha-ras gene. The transfected myogenic cells expressed both the c-Ha-ras gene and the muscle determination gene MyoD1. In contrast to other reports, the presence of as many as six copies of the c-Ha-ras gene per genome did not prevent the formation of striated muscle cells which expressed immunologically detectable muscle-specific myosin. The expression of the c-Ha-ras gene does not therefore necessarily preclude the expression of the determination gene for myogenesis or prevent end-stage myogenic differentiation.     

多胺生物合成的抑制对转化细胞c－Ha－ras癌基因表达的调控

抗多胺代谢剂──二氟甲基鸟氨酸（ＤＦＭＯ）作用于经含点突变的Ｈａ－ｒａｓ基因片段转染的转化细胞（ＨＲ－１细胞）引起细胞生长的抑制,其抑制率随ＤＦＭＯ浓度的增加而增大,此时细胞多停滞于Ｇ_１期;多胺合成的关键酶鸟氨酸脱羧酶（ＯＤＣ）活性显著下降;Ｈａ－ｒａｓ癌基因ｍＲＮＡ及ｒａｓＰ~（２１）蛋白的表达受到抑制;而外源性腐胺与ＤＦＭＯ的同时加入可防止上述一系列改变的发生,说明ＤＦＭＯ使ＨＲ－１细胞某些表型向亲本细胞逆转的作用是与细胞多胺生物合成的抑制直接相关。     

Ability of nonpermissive mouse cells to express a simian virus 40 late function(s)

Mouse cells are fully nonpermissive for simian virus 40 (SV40). Infection does not lead to detectable virus replication. In this report, it was shown, first, that spliced 16S and 19S SV40 late mRNA were present in cytoplasmic and polysomal polyadenylated acid+ RNA preparations from SV40-infected baby mouse kidney cells. The 16S and 19S SV40 late mRNA's produced in infected baby mouse kidney cells were identical to or similar to the 16S and 19S SV40 late mRNA's produced in permissive monkey cells as judged by their S1 mapping patterns performed with the late strand of HpaII-BamHI fragment B and by their sedimentation patterns in a sucrose gradient. It was also shown that the 16S late mRNA from infected baby mouse kidney cells could be translated into a polypeptide which was identical to or similar to virion protein VP1 in every aspect examined, including the patter of peptide mapping by limited proteolysis. Second, we reported that mouse kidney cells produced detectable, although low, levels of SV40 virion protein VP1, as shown by the sodium dodecyl sulfate-polyacrylamide gel autoradiogram of [35S]methionine-labeled proteins immunoprecipitated by a rabbit antiserum directed against SV40 virion proteins. Third, it was reported that infected baby mouse kidney cells produced late mRNA's either (i) when the infection was done at a restrictive temperature with the nonleaky tsA58 mutant or (ii) in cells treated with 100 microgram of cycloheximide per ml, in which large T antigen synthesis was inhibited by more than 99.9%. This suggested that large T antigen was not required for the synthesis of late mRNA in mouse cells.     

Endogenous butyrylcholinesterase in SV40 transformed cell lines: COS-1, COS-7, MRC-5 SV40, and WI-38 VA13

Summary Comparison of proteins expressed by SV40 transformed cell lines and untransformed cell lines is of interest because SV40 transformed cells are immortal, whereas untransformed cells senesce after about 50 doublings. In MRC-5 SV40 cells, only seven proteins have previously been reported to shift from undetectable to detectable after transformation by SV40 virus. We report that butyrylcholinesterase is an 8th protein in this category. Butyrylcholinesterase activity in transformed MRC-5 SV40 cells increased at least 150-fold over its undetectable level in MRC-5 parental cells. Other SV40 transformed cell lines, including COS-1, COS-7, and WI-38 VA13, also expressed endogenous butyrylcholinesterase, whereas the parental, untransformed cell lines, CV-1 and WI-38, had no detectable butyrylcholinesterase activity or mRNA. Infection of CV-1 cells by SV40 virus did not result in expression of butyrylcholinesterase, showing that the butyrylcholinesterase promoter was not activated by the large T antigen of SV40. We conclude that butyrylcholinesterase expression resulted from events related to cell immortalization and did not result from activation by the large T antigen.     

Monoclonal antibody analysis of simian virus 40 small t-antigen expression in infected and transformed cells.

The monoclonal antibody PAb280 binds to small t antigen but not to large T antigen. Its binding site within the unique region of small t antigen was localized by studying its reaction with simian virus 40 mutants, other papovaviruses, and bacterial expression vectors coding for fragments of small t antigen. The antibody was used to define the cellular location of small t antigen by immunocytochemistry and by immunoprecipitation of subcellular extracts of infected cells. PAb280 reacts strongly with a cytoplasmic form of small t antigen that appears to be associated with the cytoskeleton and is not detected by antibodies directed to the common N terminus of small t and large T antigens. Immunoperoxidase staining of cells infected by the simian virus 40 defective strain SV402 with PAb280 and other anti-T antibodies demonstrated that this virus produced an N-terminal fragment of large T antigen as well as small t antigen. In cells infected by the virus, this fragment was located in the cell nucleus but was very unstable. These results suggest that the activity of the SV402 virus in transformation assays may not be entirely due to the action of small t antigen alone.     

Simian virus 40 large T antigen associates with cyclin A and p33cdk2.

In this paper we provide evidence that a fraction of large T antigen of simian virus 40 (SV40) interacts with cyclin A and p33cdk2 in both virus-infected and stably transformed cells. Immunoprecipitates of SV40 large T antigen from SV40-infected or SV40 large-T-antigen-transformed cells contain cyclin A, p33cdk2, and histone H1 kinase activity. Conversely, immunoprecipitates of cyclin A from these cells contain SV40 large T antigen. In this respect, SV40 large T antigen has properties similar to those of the E1A oncogene of adenoviruses and the E7 oncogene of human papillomaviruses.     

Mutation causing premature termination of the polyoma virus medium T antigen blocks cell transformation

We used site-specific mutagenesis to introduce a termination codon, TGA, into the reading frame for the polyoma virus medium T antigen. We induced this mutation in a region of the polyoma genome in which the overlapping coding regions for the large and medium TE antigens are translated in different reading frames. Therefore, the mutation terminated translation of the medium T antigen, but it caused only a single amino acid substitution in the large T antigen and did not affect the small T antigen. Cells infected by the mutant virus produced normal-size small and large T antigens. The infected cells produced a 28,000-dalton fragment of the 48,000-dalton medium T antigen, whose size and tryptic peptide map were consistent with its being a truncated N-terminal fragment terminating at the new termination codon of the mutant. Immunoprecipitates of mutant-infected cell extracts did not show medium-T-antigen-associated protein kinase activity. The mutant virus replicated normally in mouse 3T6 cells and induced cellular DNA synthesis in resting mouse 3T3 cells, but it failed to transform rat or hamster cells, as judged by focus formation and growth in agar. The mutant complemented a tsA mutant which affects the large T antigen for transformation, implying that the mutant defect for transformation was in the medium T antigen. These results imply that the small T antigen and the large T antigen together are insufficient to cause transformation and support the conclusion that the medium T antigen is essential for cell transformation by polyoma virus.