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S Bacchetti

Publications and source records attributed to S Bacchetti.

At least 55 records · Page 3Linked to original sources
PubMed1995

The transcriptionally competent U2 gene is necessary and sufficient for adenovirus type 12 induction of the fragile site at 17q21-22.

Adenovirus type 12 induces four fragile sites upon infection of human cells. The U2 locus, consisting of up to 20 tandem repeats of a 5.8-kbp monomer, maps at the most sensitive of these sites at 17q21-22. We have previously shown that an artificial U2 locus integrated into the human genome generates a new virus-induced fragile site. To determine which elements within the U2 monomer are responsible for fragility, we constructed loci consisting of tandem repeats of subfragments of the U2 monomer. With this approach, we demonstrate that a transcriptionally competent U2 gene is necessary and sufficient for virus-induced fragility and that no other element within the 5.8-kbp monomer contributes to this effect.

Adenovirus Infections, Human↗
PubMed1994

Establishment and characterization of four human epithelial ovarian carcinoma cell lines.

BACKGROUND: To develop in vitro models of human ovarian carcinoma, fresh tumour cells derived from malignant effusions were cultured in vitro in the presence or absence of serum to establish cell lines. METHODS: Cell lines established were characterized by morphology in culture, surface marker expression, cytogenetic analysis, and growth in anchorage-dependent and anchorage-independent conditions. RESULTS: Four cell lines (MAC-2, RIC-2, SCHM-1, and SIB-1) were established from tumor cells isolated from the malignant effusions of four patients with epithelial ovarian carcinoma. These lines were able to grow in the presence of low concentrations of serum (3%), and two lines grew in the absence of serum (MAC-2 and SIB-1). All cell lines have been grown continuously for longer than 6 months. One cell line (SCHM-1) grew only in liquid medium whereas the other lines grew in both liquid and semi-solid media. Chromosome analysis revealed aneuploidy in three of the four lines. All of the lines stained positively for CA-125 and HMFG-2, consistent with an epithelial origin. CONCLUSION: The ability of these cells to grow in low concentrations of serum or in serum-free conditions should prove useful for the in vitro study of factors affecting the growth of human ovarian carcinoma. The serum-free medium developed will be of use in the isolation of factors from the conditioned medium of these cell lines and previously established cell lines.

Adenocarcinoma↗
PubMed1994

Telomerase activity in human ovarian carcinoma.

Telomeres fulfill the dual function of protecting eukaryotic chromosomes from illegitimate recombination and degradation and may aid in chromosome attachment to the nuclear membrane. We have previously shown that telomerase, the enzyme which synthesizes telomeric DNA, is not detected in normal somatic cells and that telomeres shorten with replicative age. In cells immortalized in vitro, activation of telomerase apparently stabilizes telomere length, preventing a critical destabilization of chromosomes, and cell proliferation continues even when telomeres are short. In vivo, telomeres of most tumors are shorter than telomeres of control tissues, suggesting an analogous role for the enzyme. To assess the relevance of telomerase and telomere stability in the development and progression of tumors, we have measured enzyme activity and telomere length in metastatic cells of epithelial ovarian carcinoma. We report that extremely short telomeres are maintained in these cells and that tumor cells, but not isogenic nonmalignant cells, express telomerase. Our findings suggest that progression of malignancy is ultimately dependent upon activation of telomerase and that telomerase inhibitors may be effective antitumor drugs.

Ascites↗
PubMed1994

Stabilization of short telomeres and telomerase activity accompany immortalization of Epstein-Barr virus-transformed human B lymphocytes.

We have measured telomere length and telomerase activity throughout the life span of clones of human B lymphocytes transformed by Epstein-Barr virus. Shortening of telomeres occurred at similar rates in all populations and persisted until chromosomes had little telomeric DNA remaining. At this stage, some of the clones entered a proliferative crisis and died. Only clones in which telomeres were stabilized, apparently by activation of telomerase, continued to proliferate indefinitely, i.e., became immortal. Since loss of telomeres impairs chromosome function, and may thus affect cell survival, we propose that telomerase activity is required for immortality. We have now detected this enzyme in a variety of immortal human cells transformed by different viruses, indicating that telomerase activation may be a common step in immortalization.

B-Lymphocytes↗
PubMed1994

Induction of genomic instability in SV40 transformed human cells: sufficiency of the N-terminal 147 amino acids of large T antigen and role of pRB and p53.

Genomic instability is an early event in the transformation of human cells by SV40 and may contribute, as a mutagenic process, to the generation of the rare cells which survive crisis and yield immortal populations. We have previously reported that expression of large T antigen is responsible for induction of chromosome aberrations and aneuploidy. In the present study we have demonstrated that the amino terminal 147 amino acids of the protein are as proficient as full length T antigen for this destabilization of the cell genome. Analysis of mutants within this region indicated that T antigens defective for binding to pRB or lacking the first 127 amino acids are significantly reduced in their ability to induce aneuploidy and/or aberrations, whereas a cytoplasmic T antigen is less severely impaired. In addition, we have shown that binding of T antigen to p53 is dispensable for genome destabilization but may be required for continued proliferation of genetically aberrant cells.

Amino Acids↗
PubMed1993

Generation of a new adenovirus type 12-inducible fragile site by insertion of an artificial U2 locus in the human genome.

Infection with adenovirus type 12 (Ad12) induces four fragile sites in the human genome (H.F. Stich, G.L. van Hoosier, and J.J. Trentin, Exp. Cell Res. 34:400-403, 1964; H. zur Hausen, J. Virol. 1:1174-1185, 1967). The major site, at 17q21-22, contains the U2 gene cluster, which is specifically disrupted by infection in at least a percentage of the cells (D.M. Durnam, J.C. Menninger, S.H. Chandler, P.P. Smith, and J.K. McDougall, Mol. Cell. Biol. 8:1863-1867, 1988). For direct assessment of whether the U2 locus is the target of the Ad12 effect, an artificial locus, constructed in vitro and consisting of tandem arrays of the U2 6-kbp monomer, was transfected into human cells. We report that integration of this artificial locus on the p arm of chromosome 13 creates a new Ad12-inducible fragile site.

Adenoviruses, Human↗
PubMed1992

Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity.

Loss of telomeric DNA during cell proliferation may play a role in ageing and cancer. Since telomeres permit complete replication of eukaryotic chromosomes and protect their ends from recombination, we have measured telomere length, telomerase activity and chromosome rearrangements in human cells before and after transformation with SV40 or Ad5. In all mortal populations, telomeres shortened by approximately 65 bp/generation during the lifespan of the cultures. When transformed cells reached crisis, the length of the telomeric TTAGGG repeats was only approximately 1.5 kbp and many dicentric chromosomes were observed. In immortal cells, telomere length and frequency of dicentric chromosomes stabilized after crisis. Telomerase activity was not detectable in control or extended lifespan populations but was present in immortal populations. These results suggest that chromosomes with short (TTAGGG)n tracts are recombinogenic, critically shortened telomeres may be incompatible with cell proliferation and stabilization of telomere length by telomerase may be required for immortalization.

Cell Transformation, Neoplastic↗
PubMed1991

Synergism between aphidicolin and adenoviruses in the induction of breaks at fragile sites on human chromosomes.

Infection of human embryonic kidney cells with adenoviruses results in the induction of gaps and breaks in cell chromosomes. With adenovirus type 12, cytogenetic damage is known to occur primarily at fragile sites on chromosomes 1 and 17. We have mapped adenovirus type 5-induced breaks and have observed that, although they occur on all chromosomes, they are localized primarily on bands where fragile sites have been mapped. The susceptibility of fragile sites to adenovirus led us to investigate their expression upon combined treatments with virus and aphidicolin, a frequently used inducer of fragile sites. Under these experimental conditions, the frequency of damage at all sites was found to increase significantly, and the magnitude of such increases indicated a synergistic effect between drug and virus.

Adenoviridae Infections↗
PubMed1991

Expression of SV40 large T antigen, but not small t antigen, is required for the induction of chromosomal aberrations in transformed human cells.

Expression of the Simian virus 40 (SV40) early region in human cells results in the induction of chromosomal aberrations and polyploidy, and in transformation. To understand how genetic damage occurs and what role it plays in transformation, human diploid fibroblasts and embryonic kidney cells were transfected with plasmids encoding wild type or mutant forms of the viral early region, and the neo gene. Clones selected for G418 resistance and expressing viral genes were initially analyzed within 20 cell divisions. Our results demonstrate that expression of the SV40 large T antigen is sufficient for the induction of chromosomal damage and ploidy changes, and that small t does not contribute to these processes. Mutant plasmids lacking the SV40 origin of DNA replication were as proficient as wild type plasmids, indicating that viral DNA replication is not required for cytogenetic damage. We have also shown that chromosome aberrations, but not necessarily polyploidy, increase in frequency and complexity upon subculturing of the clones regardless of whether such populations arrest at crisis or yield immortal lines. Our results are compatible with the hypothesis that large T antigen destabilizes the cellular genome, and that specific mutations arising from this process may contribute to cell immortalization.

Antibodies, Monoclonal↗
PubMed1990

Definition of adenovirus type 5 functions involved in the induction of chromosomal aberrations in human cells.

Infection of human embryonic kidney cells with adenovirus type 5 (Ad5) induces aberrations (gaps and breaks) in the cell chromosomes. We have conducted a study utilizing a large number of Ad5 mutants to identify the viral functions that are responsible for the occurrence of cytogenetic damage. The results of our investigation have indicated that expression of the gene products of the Ad5 early region 1A (E1A) is necessary for the induction of chromosomal aberrations and that other early viral gene products do not appear to contribute to this phenotype. We have also shown that expression of both the major E1A gene products, the 243 amino acid and the 289 amino acid proteins, is required for induction of damage at wild-type levels, although the 289 amino acid protein appears to retain detectable activity on its own. Lastly, we have observed that deletions in the amino-terminal region of the E1A proteins and in the transactivating domain of the 289 amino acid protein prevent the occurrence of cytogenetic damage, whereas mutations elsewhere in the proteins do not affect this process.

Adenovirus Early Proteins↗
PubMed1990

Chromosomal damage induced by human adenovirus type 12 requires expression of the E1B 55-kilodalton viral protein.

Infection of human embryonic kidney cells with adenovirus type 12 results in the induction of damage at specific (17q21-22, 1p36, 1q21, and 1q42-43) and random sites in the cellular chromosomes. A previous study by Durnam et al. (D. M. Durnam, P. P. Smith, J. C. Menninger, and J. K. McDougall, Cancer Cells 4:349-354, 1986) indicated that the expression of viral early region 1 (E1) is sufficient for the induction of damage at band 17q21-22. In the present report we used an adenovirus type 12-adenovirus type 5 recombinant with E1A hybrid sequences as well as viruses with mutations in the adenovirus type 12 E1B genes to map adenovirus type 12 E1 functions involved in the induction of genetic damage. Our results show that the expression of the E1A proteins is not sufficient for this effect. On the other hand, mutations within the E1B 55-kilodalton protein but not the E1B 19-kilodalton protein affect the ability of the virus to induce both specific and random chromosomal damage.

Adenoviruses, Human↗
PubMed1989

Cloning of the herpes simplex virus ICP4 gene in an adenovirus vector: effects on adenovirus gene expression and replication.

To assess the ability of the herpes simplex virus ICP4 protein to complement adenovirus E1a mutants we have constructed an adenovirus type 5 vector containing a temperature-sensitive ICP4 gene, under control of its own promoter, within the E1 region of the genome. The recombinant virus expresses ICP4 in cells which are permissive (293) or nonpermissive (KB and R970-5) for viral replication, and at levels which approximate those obtained in herpes simplex infection. The adenovirus-encoded protein is functional in that it complements an ICP4 deletion mutant of herpes simplex virus; however, it is incapable of complementing adenovirus E1a mutants for viral growth or DNA replication. At the level of activation of gene expression, ICP4 stimulates the expression of the adenovirus E2a gene but not that of other early genes. Our results indicate that ICP4 does not possess all of the functions of the E1a proteins and, furthermore, that adenovirus early genes differ in their susceptibility to heterologous trans-activators.

Adenovirus Early Proteins↗
PubMed1988

Expression of the HSV-2 ribonucleotide reductase subunits in adenovirus vectors or stably transformed cells: restoration of enzymatic activity by reassociation of enzyme subunits in the absence of other HSV proteins.

We have cloned the large subunit (RR1) of the HSV-2 ribonucleotide reductase into a helper-independent adenovirus 5 vector under control of the viral major late promoter. Infection of 293 cells with the AdRed-1 recombinant virus resulted in the expression of the HSV-2 RR1 protein. We have also produced cells which constitutively express the small (RR2) subunit of the HSV-2 enzyme by transfecting 293 cells with a plasmid encoding this protein and the neo resistance marker (pSV2neo-RR2). Infection of the A439-14 producer cells with AdRed-1 resulted in the expression of enzymatically active HSV-2 ribonucleotide reductase. HSV-2 reductase activity could also be detected upon mixing of extracts from cells expressing either subunit. Our results indicate that the HSV-2 holoenzyme can be reconstituted in vivo and in vitro and that no HSV-2 proteins, beyond the enzyme subunits, are required for the formation and activity of the viral reductase.

Adenoviridae↗
PubMed1988

Oligopeptides inhibit the ribonucleotide reductase of herpes simplex virus by causing subunit separation.

It has recently been reported that a nonapeptide (H-Tyr-Ala-Gly-Ala-Val-Val-Asn-Asp-Leu-OH) identical in sequence to the last nine amino acid residues of RR2, the small subunit of herpes simplex virus (HSV) ribonucleotide reductase (E.C. 1.17.4.1; RR), can inhibit HSV RR activity in vitro. It has been postulated that this peptide acts by interfering with the interaction of the large subunit, RR1, and RR2, but direct demonstration of its mechanism of action has not been reported. We present data which show that the inhibition of HSV RR by this oligopeptide is due to induced subunit dissociation.

Macromolecular Substances↗
PubMed1988

The responsiveness of human papillomavirus upstream regulatory regions to herpes simplex virus immediate early proteins.

We have tested the responsiveness of the upstream regulatory regions (URR) of human papillomaviruses (HPV) to transactivation by the herpes simplex virus type 1 (HSV-1) immediate early proteins ICP4 (Vmw175) and ICP0 (Vmw110), using recombinant plasmids which contain URRs of HPV 1, 11 or 16 upstream of an SV40-promoted CAT gene. Transfection of the HPV-CAT plasmids into mouse cells expressing a temperature-sensitive ICP4 protein enabled us to demonstrate that ICP4 enhances transcription of the HPV 1 and 16 URRs, but not of the HPV 11 URR. Cotransfection of the HPV CAT plasmids with a plasmid encoding ICP0 indicated that only the URR of HPV 16 responded to this protein, and that the response was host cell dependent.

Animals↗
PubMed1987

In cell lines constitutively synthesizing a temperature-sensitive ICP4 protein of herpes simplex virus type 1, amount and function of ICP4 are both regulated by temperature.

We have established two cell lines that constitutively synthesize a temperature-sensitive form of ICP4, the herpes simplex virus immediate-early protein that activates early and late transcription. ICP4 in both cell lines was confirmed to be functionally temperature sensitive when tested by complementation of an ICP4 deletion mutant virus for expression of viral early and late genes. When grown at the permissive temperature the two cell lines contained approximately 5 and 25%, respectively, of the ICP4 present in control HSV-infected cells. If the cells were grown at the nonpermissive temperature, ICP4 levels were reduced by approximately fourfold; a twofold reduction was observed in control cells synthesizing the wild-type protein. The lower levels of ICP4 at the nonpermissive temperature were the result of two effects: a decrease in mRNA which was similar in cells producing the mutant or wild-type form of ICP4 and a more rapid turnover of the protein which was greater for the mutant than for the wild-type form. Our observations of lower levels of ICP4 in producer cells differ from published reports of overproduction of immediate-early proteins at the nonpermissive temperature in human or hamster cells infected with ICP4 temperature-sensitive mutant viruses. This discrepancy may be related to cell species differences since we observed only a modest twofold overproduction of immediate-early proteins at the nonpermissive temperature in infections of mouse cell lines with an ICP4 temperature-sensitive mutant virus.

Animals↗
PubMed1986

Identification and separation of the two subunits of the herpes simplex virus ribonucleotide reductase.

The herpes simplex virus ribonucleotide reductase is associated with two viral proteins which are both immunoprecipitated by monoclonal antibodies specific for the enzyme. We separated the two proteins and showed that individual antibodies react solely with one or the other. In addition, antibodies to either protein can neutralize enzymatic activity. Our data demonstrate that the proteins are associated in a complex and constitute the subunits of the enzyme.

Animals↗