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M Bastin

Publications and source records attributed to M Bastin.

At least 37 records · Page 2Linked to original sources
PubMed1989

Effect of CpG-rich sequences in transformation and tumorigenesis by polyomavirus.

To address the question of the role of CpG-rich sequences in gene expression, we investigated the effect of an HTF island on the activity of the polyomavirus middle T (pmt) oncogene. pmt is less transformant and less tumorigenic when it is introduced into cultured cells or newborn rats in the presence of an HTF island. Transformed cells carrying pmt in the vicinity of the HTF island have a propensity to revert at a relatively high rate of about 2 X 10(-3) per cell per generation by a mechanism probably involving methylation of some CpG sites in the island. Our results suggest that HTF islands can function as transcriptional silencers.

Animals↗
PubMed1988

Site-directed mutagenesis of the polyomavirus genome: replication-defective large T mutants with increased immortalization potential.

We used the deletion loop mutagenesis procedure to direct point mutations into a small region of the polyomavirus genome, at 0.97 map units, affecting the structure of both the middle and large T antigens. We describe the construction of six middle T mutants which have retained the ability to transform rat cells in culture and four large T mutants, three of which are deficient in viral DNA replication and capable of immortalizing primary rat embryo fibroblasts very efficiently.

Antigens, Viral, Tumor↗
PubMed1988

Transfer of mlt mutations into polyomavirus intronless genomes by intramolecular recombination in bacteria.

We describe a modification of the procedure of Weber and Weissmann [Nucl. Acids Res. 11 (1983) 5661-5669] for the formation of hybrid genes by in vivo recombination to introduce two separate mutations into the same gene. The mutants of interest are inserted as head-to-tail tandems in a bacterial plasmid in such a way that the 5'-proximal mutation is located upstream from the mutant with the more distal mutation. Propagation of the plasmid in a rec+ strain of Escherichia coli allows recombination between homologous sequences in the insert. DNA with the size expected for the recombinant plasmid is isolated by agarose gel electrophoresis, cloned in a recA strain, and characterized by restriction endonuclease mapping. Using this procedure, we have transferred the deletion from polyomavirus mutant dl-8 into other mutant genomes lacking the intervening sequences for either middle T or large T.

Antigens, Polyomavirus Transforming↗
PubMed1988

Polyoma large T can activate middle T expression by a hit-and-run mechanism.

We studied the activation of polyoma middle T expression in revertant cells carrying transcriptionally inactive copies of the middle T (pmt) oncogene. Introduction of polyoma large T with neo into a rat cell line containing multiple copies of pmt stably integrated into the genome corrected the transformation defect in some of the transfected cells by activating the resident pmt gene. However, once the cells were transformed, continuous expression of the large T protein was not required for the maintenance of pmt expression and hence the maintenance of the transformed state. Transformants arising spontaneously as well as those induced by large T exhibited frequent rearrangements of the pmt inserts. Our results suggest that large T activated pmt expression by a hit-and-run mechanism involving recombination of sequences in the viral insert.

Animals↗
PubMed1987

Effect of ACTH on cholesterol and steroid synthesis in adrenocortical tissues.

Previous studies have established that under normal conditions, adrenal HMG-CoA reductase activity is higher in hamsters than in rats and humans. The hamster reductase activity follows a diurnal rhythm corresponding to that of plasma ACTH and glucocorticoids [Endocrinology 107 (1980) 215] but not to that of aldosterone. ACTH treatments to hamsters increased reductase activity after a latency of 60 min; this enhancement was prevented by cycloheximide [J. steroid Biochem. 24 (1986) 325]. Immunotitration and immunoblotting studies confirmed that ACTH caused an increase in reductase protein synthesis. In rats, long-term (1-9 days) and short-term (3 h) treatments with ACTH also induced increase in adrenal HMG-CoA reductase activity and reductase protein. In the presence of iodoacetamide and inhibitors of proteolytic enzyme, a main specific band of enzyme was evinced in the area of 102 +/- 6 kDaMr, by Western blotting, for both hamster homogenate and microsomal preparations (Endocrinology, 120 (1987]. Similarly Mr values were found with rat adrenal preparations. The concentration of mRNA, analyzed using the c-DNA pRed-10 coding for the Chinese hamster ovary reductase, was increased in adrenals of hamsters treated with ACTH. The reductase mRNA levels also fluctuated during the day in parallel with those of reductase activity and reductase protein. In conclusion, these results indicate that ACTH and other conditions inducing a change in hamster adrenal HMG-CoA reductase activity provoke parallel changes in reductase mRNA and reductase protein content. ACTH acts on the adrenal reductase of species synthesizing large as well as small quantities of cholesterol, thus indicating the general importance of this hormonal control.

Adrenal Cortex↗
PubMed1987

High-frequency changes in transcriptional activity in polyomavirus-transformed cell lines.

We applied the Luria and Delbruck fluctuation test to analyze high-frequency changes in the phenotype of rat cells transformed by a plasmid carrying the polyomavirus middle T (pmt) gene. All of the transformed cell lines analyzed were capable of switching to the normal state with rates ranging from 10(-3) to 10(-2) per cell per generation. Analysis of both middle T antigen and middle T transcripts indicated that the reversion occurred by a mechanism involving a transcriptional block of the pmt locus. Cell lines containing two separate loci reverted with a lower rate, suggesting that phenotypic switching in these cells involved two independent events affecting each locus. The flat revertants mutated to the transformed state with rates in the range of 10(-5) to 5 X 10(-5) per cell per generation. To determine whether changes in pmt expression would affect neighboring sequences, we transfected a hybrid plasmid carrying pmt linked to the neo marker and selected either for morphological transformants or for G418-resistant cells. Although their coordinate regulation was not absolute, both genes were usually subject to the same changes, reflected by loss and reacquisition of transcriptional activity.

Animals↗
PubMed1986

Expression of the malignant phenotype in rat fibroblasts transfected with the polyomavirus transforming genes.

As a step toward understanding the molecular mechanism of cooperation between viral and cellular genes in oncogenic transformation, we examined various properties of rat cells transfected with the polyomavirus transforming genes without selecting for a neoplastic phenotype. The cell lines displayed a phenotype ranging from nontumorigenic (flat) to fully transformed (tumorigenic). In the established FR3T3 cell line, acquisition of the fully transformed phenotype correlated with effective expression of the polyomavirus middle T (pmt) antigen. Flat cells carrying silent copies of pmt mutated spontaneously to the fully transformed state with a frequency of 2 to 6 X 10(-5) per cell per generation. In unestablished rat fibroblasts, simultaneous transfer of either pmt and small T or pmt and large T in the presence of the neo marker conferred only a partially transformed phenotype to most of the cell lines. The same results were obtained when wild-type genomic DNA was cotransfected with pSV2-neo. The flat transformants progressively acquired properties characteristic of fully transformed cells with passage in culture. However, in contrast to FR3T3 cells, the generation of fully transformed variants from the flat, unestablished fibroblasts was not caused by activation of pmt expression. This indicates that the functions conferred by the large and small T antigens, alone or in combination with each other, cannot substitute for all the functions expressed by the FR3T3 cell line as a result of in vitro establishment. Thus, polyomavirus-mediated transformation may require additional cellular alterations beyond the acquisition of the three viral oncogenes.

Animals↗
PubMed1986

Mutation in the polyomavirus genome that activates the properties of large T associated with neoplastic transformation.

We have constructed a polyomavirus mutant genome which exhibits an increased immortalization potential when transfected into primary rat embryo fibroblasts. The mutation is a 30-base-pair deletion (nucleotides 1367 through 1396) that inactivates the transforming potential of middle T but activates some of the properties of large T associated with neoplastic transformation. Unlike the wild-type large T, the mutant large T can fully complement polyoma middle T in the tumorigenic process in vivo as well as in the transformation of primary cells in vitro. The activity of the mutant can be explained by its inability to replicate in cells and, hence, its inability to exert a cytopathic effect after gene transfer at high multiplicity. A recombinant which encodes the middle and small T antigens, but not the large T antigen, can also elicit a fully transformed phenotype when introduced into primary rat fibroblasts. These results confirm previous observations from this laboratory indicating that two, and not three, viral gene functions are required for polyomavirus-mediated oncogenic transformation.

Animals↗
PubMed1986

Ability of a T-antigen transport-defective mutant of simian virus 40 to immortalize primary cells and to complement polyomavirus middle T in tumorigenesis.

The oncogenic potential of polyomavirus in newborn rats could not be expressed by a genome encoding only the middle T antigen but required the presence of one of the other two viral early genes, small T or large T. The tumorigenicity defect could also be complemented by other viral or cellular genes that are known to be implicated in immortalization and establishment functions. The simian virus 40(cT)-3 mutant (R. E. Lanford and J. S. Butel, Cell 37:801-813, 1984), which fails to localize to the nucleus, has the capacity to complement polyomavirus middle T in tumorigenesis and to immortalize primary rat embryo fibroblasts when it was cotransfected in the presence of pSV2-neo. Our data suggested that under the conditions of DNA-mediated tumor induction and cotransfection with a dominant selection marker, the cellular alterations achieved by nonnuclear oncogenes such as polyomavirus small T and simian virus 40(cT)-3 were sufficient to complement polyomavirus middle T in transformation and tumorigenesis.

Animals↗
PubMed1985

Malignant transformation of rat cells by the polyomavirus middle T gene.

To gain an insight into the molecular mechanism of cooperation between the polyomavirus middle T gene and cellular genes in the tumorigenic process, we have examined various properties of rat cell lines transformed by middle T alone. Middle T transformants display a phenotype ranging from nontumorigenic (flat) to fully transformed (tumorigenic) and the phenotype of a given cell line correlates very well with its cellular level of middle T antigen. Highly transformed, tumorigenic variants arise spontaneously in the flat cells during their growth with a mutation rate of 2.2 X 10(-5) per cell per generation. These variants contain elevated levels of both middle T antigen and middle T transcripts, suggesting that fully transformed cells arise as a consequence of an efficient mode of viral gene expression.

Animals↗
PubMed1985

Sequences from polyomavirus and simian virus 40 large T genes capable of immortalizing primary rat embryo fibroblasts.

We developed a procedure to evaluate quantitatively the capacity of subgenomic fragments from polyomavirus and simian virus 40 (SV40) to promote the establishment of primary cells in culture. The large T antigen from both of these viruses can immortalize primary rat embryo fibroblasts. Both antigens have amino-terminal domains that retain biological activity after deletion of other parts of the polypeptide chain. However, this activity varies considerably among various mutants, presumably because of alterations in the stability or conformation of the truncated polypeptides. The polyomavirus middle T gene alone immortalizes at a low efficiency, which indicates that this oncogene can have both immortalization and transformation potentials depending on the assay system chosen. We generated deletions in the polyomavirus and SV40 large T genes to localize more precisely the functional domains of the proteins involved in the immortalization process. Our results show that the region of the SV40 large T antigen involved in immortalization is localized within the first 137 amino acid residues. This region is encoded by the first large T exon and a small portion from the second exon which includes the SV40 large T nuclear location signal. The polyomavirus sequence involved in immortalization comprises a region from the second large T exon, mapping between nucleotides 1016 and 1213, which shares no homology with SV40 and is thought to be of cellular origin. We suggest that this region of the polyomavirus large T gene functions either as a nuclear location signal or as part of the large T protein sequence involved in DNA binding.

Amino Acid Sequence↗
PubMed1984

Tumorigenic activity of polyoma virus and SV40 DNAs in newborn rodents.

A procedure has been developed whereby the oncogenicity of the DNA from polyoma (Py) virus and Simian virus 40 (SV40) can be tested directly by injecting recombinant DNA into newborn rodents. Injection of 0.2-2.0 micrograms of linear DNA induced the development of subcutaneous liposarcomas and fibrosarcomas at the site of inoculation. Coinjection of high-molecular-weight rat DNA as carrier had little or no effect on tumor formation but plasmids pBR322, pAT153 , and pML2 behaved as strong inhibitors. Tumor induction by injecting DNA into newborn rodents provides an in vivo equivalent to a transformation assay but appears to be a more stringent and rigorous criterion of oncogenic transformation. The oncogenic potential of Py virus in newborn hamsters could be expressed by a recombinant encoding only the middle T protein, although with average tumor latencies 5-10 times longer than those observed with wild-type Py DNA. Py middle T required the cooperation from small T to induce tumors in newborn rats. SV40 DNA was tumorigenic only in newborn hamsters. delta 2005 DNA which is unable to produce the SV40 small T antigen was much less active and required a latent period about twice that of wild-type SV40 DNA. However, its tumorigenic potential was restored by addition of the Py small T antigen gene. This indicates that Py and SV40 small T antigens are interchangeable and that they probably play an identical role in malignant transformation. Finally, evidence was provided that intermolecular recombination or recombination between DNA fragments can occur in vivo.

Animals↗
PubMed1984

mlt Mutation in the polyomavirus genome impairing a function of the middle T protein.

The DNA from polyomavirus mlt mutant P155 transforms cells in culture as efficiently as wild-type DNA but has a much lower tumorigenic potential when injected into newborn rodents. The mutant has a 12-base-pair deletion between nucleotides 1347 and 1360, i.e., in a region which encodes parts of the middle and large T antigens (G elinas et al., J. Virol. 43:1072-1081, 1982). To determine which of the two viral gene functions was affected by the mutation, we transferred the latter into a modified polyomavirus genome encoding exclusively the middle T protein. Our results show that the P155 mutation alters a function of the polyomavirus middle T protein required for the induction of the tumorigenic process in vivo. Beside the 12-base-pair deletion at 96.3 map units, there is no other alteration in the coding sequence of P155 middle T with respect to that of P16, the wild-type parental strain. We conclude, therefore, that the deletion is the lesion affecting the tumorigenic potential of mutant P155 .

Amino Acid Sequence↗
PubMed1984

Polyoma middle T antigen requires cooperation from another gene to express the malignant phenotype in vivo.

The oncogenic potential of polyomavirus in newborn hamsters can be expressed by a recombinant encoding only the middle T protein. However, polyoma middle T requires the cooperation from small T to induce tumors in newborn rats. Similar complementary functions such as cocarcinogens or tumor promotors can be exerted by the simian virus 40 T antigens as well as by one or several products of the early region 1A of adenovirus 2.

Adenoviruses, Human↗
PubMed1983

Role of the three polyoma virus early proteins in tumorigenesis.

A modified polyoma virus genome which can encode the middle T protein but not the large or small T proteins transforms rat cells in culture with an efficiency about 20% that of the wild-type genome. Although middle T-transformed cells grow as tumors when transplanted into nude mice or syngeneic rats, the middle T gene alone is totally inactive when used in a more stringent and rigorous assay for tumorigenicity such as the injection of DNA into newborn rats. Thus, functions other than those expressed by middle T antigen are required for the elaboration of all the properties associated with tumorigenesis. To assess whether a complementary function could be exerted by the large or the small T antigen, we constructed plasmids containing two modified early regions which independently encoded middle T and one of the two other proteins. Both recombinants were tumorigenic in newborn rats. Cell lines derived by transfer of these plasmids under no special selective conditions did not acquire the property of growth in low-serum medium but exhibited the same tumorigenic properties as wild-type polyoma DNA-transformed cells. Furthermore, a recombinant which encoded the middle and small T antigens, but not the large T antigen, was tumorigenic in newborn rats. Although the small T antigen provides a complementary function for tumorigenicity, it cannot complement the middle T antigen for an efficient induction of transformation of cultured cells. This suggests that the complementary function exerted by the small T antigen is different from that of the N-terminal fragment of the large T protein.

Animals↗
PubMed1982

Polyoma virus mutant with normal transforming ability but impaired tumorigenic potential.

Cloned DNA from the P155 mutant of polyoma virus transforms cells in culture as efficiently as wild-type DNA, but has a much lower tumorigenic potential when injected into newborn rats. Like cells transformed by wild-type DNA, cells transformed by the mutant DNA grow in low serum concentrations, form colonies in agar suspension, and grow to high saturation densities compared with untransformed cells. They are, however, much less tumorigenic since they transplant 100- to 2,000-fold less efficiently than cells transformed by wild-type DNA. Substitution of the region between 89.7 and 1.8 map units by the corresponding region of P155 DNA decreased the tumorigenicity of wild-type DNA. When this region was isolated from wild-type DNA and substituted in P155 DNA, the tumorigenicity of the latter increased to values comparable to those of wild-type DNA. This showed that the lesion affecting tumorigenicity occurred between 89.7 and 1.8 map units on the polyoma virus genome. Sequence analysis in this region revealed a 12-base-pair deletion between nucleotides 1,347 and 1,360. This identified P155 as an mlt mutant, i.e., a mutant with a deletion from a region which encodes parts of the large and middle T antigens.

Animals↗