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S Bacchetti

Publications and source records attributed to S Bacchetti.

At least 19 recordsLinked to original sources
PubMed2003

Herpes simplex virus type-1 infection upregulates cellular promoters and telomerase activity in both tumor and nontumor human cells.

Targeted gene expression through viral vectors has been a promising approach for gene therapy. However, the effects of viral gene products expressed from virus vectors on the expression of the host gene are not well known. In the present study, we examined the activities of cellular promoters, including the promoter for genes of human telomerase reverse transcriptase (hTERT), tyrosinase and probasin, in both tumor and normal cells after infection with herpes simplex virus type 1 (HSV-1) vectors. Our results showed that infection with replication-defective HSV-1 vectors significantly upregulated the activity of all three cellular promoters in a nonsequence specific fashion in all cell types tested. Furthermore, viral infection upregulated activities of the hTERT promoter and endogenous telomerase in nontumoral cells. Additional experiments suggested that the viral immediate-early gene product, infected cell protein 0, might be responsible for the deregulation of cellular promoter activity and activation of telomerase. Our study alerts to the potential risk of oncogenesis through deregulation of host gene expression, such as the telomerase by viral vectors in normal cells.

Androgen-Binding Protein↗
PubMed2003

Natural history of the neoplastic locoregional disease: clinical and pathological patterns.

A great number of locoregional treatments are currently carried out to treat a variety of locoregional neoplastic diseases. Indications are the treatment of primary and metastatic liver tumors, peritoneal mesotheliomas, peritoneal spread of ovarian carcinomas, peritoneal recurrences of gastrointestinal cancers, peritoneal spread of retroperitoneal sarcomas, melanomas and sarcomas of the limbs, some primary tumors of the brain, breast, kidney, lung, bladder. But to deal with locoregional therapy demands to clarify some features of these malignancies. At this regard, the knowing of their natural history can be crucial to guide the choice of the correct locoregional treatment. For instance peritoneal carcinomatosis is considered as a main step of disease progression for ovarian cancer and often for gastrointestinal tumors as well. However when the tumors are confined on the surface of the peritoneum, basing on their own natural history, they can be considered as localized diseases. Selected patients with peritoneal neoplastic seeding, previously considered in a preterminal condition, can be considered as candidates for curative treatment, using cytoreductive surgical tecniques (16) and hyperthermic intraperitoneal chemotherapy (19). The same can be thought about others primary or metastatatic tumors when the neoplastic deposits are confined within a definite site or region of the body. In this paper the main aspects of liver metastases and peritoneal carcinomatosis natural history, two of the most frequently recognized indications for locoregional therapy, are presented.

Gastrointestinal Neoplasms↗
PubMed2003

Epirubicin and its metabolites levels in experimental liver metastases after different administration routes.

Locoregional chemotherapy in the 80's was considered an effective palliative treatment for unresectable hepatic metastases. With the advent of new drugs supporting effective systemic chemotherapy it was disregarded for many years. Recently, following the advent of new drugs and the developing of new association scheme, it has regained interests also for its adjuvant and neoadjuvant role to hepatic resections. Current schemes of locoregional and systemic chemotherapy for liver metastases are based on continuous infusions using implantable pumps but confirmation, in term of tissue drug concentration, that continuous infusions do better than bolus infusions is still lacking. To address this specific aspect we have experimentally compared these two different administration modalities using an anthracyclin, Epiadryamicin (EPI), with high plasmatic clearance and main biliary escretion (8,16) and infused through arterial, portal and systemic routes. The most high EPI concentration within the tumour was obtained after bolus-arterial infusion but also for continuous infusions the artery resulted better than other routes. Differently the most high EPI liver concentration resulted after portal infusion both if infused with a bolus or in 5 minutes time. This experiment may therefore legitimate the clinical use of this drug with bolus repeated infusions through the hepatic artery.

Animals↗
PubMed2003

Epiadryamicin concentration in experimental hepatic metastases after bolus or continous infusion through systemic or locoregional routes.

Locoregional chemotherapy in the 80's was considered an effective palliative treatment for unresectable hepatic metastases: it significantly improved the response rates if compared with systemic chemotherapy but didn't modify the survival (7,19). With the advent of new drugs supporting effective systemic chemotherapy it was disregarded for many years. Recently, following the advent of new drugs and the developing of new association scheme, it has regained interests also for its adjuvant and neoadjuvant role to hepatic resections (1,2,3,9,13,14,15,18). Loco-regional drug administration is feasible through two different administration routes, portal system and hepatic artery; the hepatic arterial infusion, in terms of tumor tissue antiblastic concentration, seems to be the most effective (6) Current schemes of chemotherapy for liver metastases are based on continuous infusions using implantable pumps (11, 12) but confirmation, in term of tissue drug concentration, that continuous infusions do better than bolus infusions is still lacking. To address this specific aspect we have experimentally compared these two different administration modalities using an anthracyclin, Epiadryamicin (EPI), with high plasmatic clearance and main biliary escretion (8,16).

Animals↗
PubMed2002

Cytokine, infiltrating macrophage and T cell-mediated response to development of primary and secondary human liver cancer.

BACKGROUND: Kupffer cells, monocytes and infiltrating T cells have been considered the major source of interleukin-1beta and tumour necrosis factor-alpha in the liver. AIMS; To explore the expression of interleukin-1beta and tumour necrosis factor-alpha and to evaluate the density and the distribution of T lymphocytes and monocytes/macrophages in the liver of patients with primary and secondary tumours. METHODS: Tumoural and peritumoural liver samples were examined from 21 patients with hepatocellular carcinoma, 10 with hepatic metastases, 5 with benign focal liver lesions and 4 healthy adult livers. Interleukin-1beta and tumour necrosis factor-alpha mRNAs were detected by a semiquantitative comparative reverse transcriptase polymerase chain reaction. T lymphocytes and monocytes/macrophages were detected by immunohistochemistry. RESULTS: Higher levels of interleukin-1beta, tumour necrosis factor-alpha, CD3+ and CD68+ cells were found in the tissue surrounding hepatocellular carcinoma and metastases than in the tumour itself. A strong expression of CD68+ and CD3+ cells was found mainly along the tumour-host interface but the highest expression of CD3+ cells was found at the metastasis interfaces. Interleukin-1beta expression, CD3+ and CD68+ cell densities were higher in peritumoural samples than in so-called "normal" liver tissue. CONCLUSIONS: An increased production of interleukin-1beta and, to a lesser extent, of tumour necrosis factor-alpha mRNA coincides with the presence of cancer be it primary or secondary, both in healthy and cirrhotic livers. The presence of cancer, irrespective of the presence of underlying liver damage, appears to play the most important role.

Adult↗
PubMed2001

Telomere maintenance by telomerase and by recombination can coexist in human cells.

Immortal human cells maintain their telomeres by two independent mechanisms, a prevalent one dependent on de novo synthesis of telomeric DNA by telomerase, and a rarer one based on telomere recombination [alternative lengthening of telomeres (ALT)]. Studies with yeast have indicated that expression of telomerase inhibits telomere recombination. In the present study, we have investigated whether expression of telomerase in cells that use ALT would similarly reveal dominance of telomere elongation by telomerase over telomere recombination. Telomerase-negative WI38 VA13/2RA ALT cells were reconstituted for telomerase activity through ectopic expression of the enzyme subunits, hTERT and hTR, and the presence and function of telomerase and ALT were monitored during long term cell growth by enzymatic assays, detection of the ALT-associated PML bodies (APBs) and analysis of telomere dynamics. Our results indicate that telomerase activity and APBs persisted in the cells over at least 90 population doublings. The activity of both pathways on telomeres was determined by analysis of telomere length versus time by gel electrophoresis and in situ hybridization. ALT cells are characterized by very heterogeneous telomeres with a much longer average size than the telomeres of telomerase-positive cells. Telomere dynamics in our cells were compatible with both ALT and telomerase being biologically active since the long telomeres typical of ALT were maintained, while short telomeres, thought to be the preferential substrate of telomerase, were elongated. These findings, indicating that human cells may be capable of concomitantly utilizing both mechanisms of telomere maintenance without effects on their growth and viability, have implications for cancer therapy.

Cell Division↗
PubMed2001

Transient expression of wild-type or biologically inactive telomerase allows the formation of artificial telomeres in mortal human cells.

Telomere seeding, the formation of artificial telomeres, has been routinely successful in immortalized but not normal human cells. We compared seeding efficiencies in preimmortal and immortal SV40-transformed cells using plasmid telomeres with T(2)AG(3) tracts of 1600 and 3200 bp. Seeding occurred only in immortal cells, indicating that transformed preimmortal cells behave like normal cells vis à vis formation of new telomeres and that T-antigen inhibition of cellular checkpoints is insufficient to allow seeding. Telomerase is active in immortal but not preimmortal cells, which do not express the reverse transcriptase hTERT. Upon transient expression of hTERT, seeds with 1600 bp of T(2)AG(3) formed telomeres in preimmortal cells. Comparable seeding efficiencies were obtained with wild-type hTERT or the HA-tagged protein that is catalytically active but unable to maintain endogenous telomeres. No seeding occurred with catalytically inactive hTERT. Given that telomerase expression was transient and that longer seeds did not form telomeres in the absence of the enzyme, seeding may not be elicited merely by elongation of telomeric sequences. We propose that modification of the telomeric terminus by telomerase may contribute to telomere seeding by leading to formation of a structure that impedes rejoining of this terminus with chromosomal sequences.

Blotting, Western↗
PubMed2001

Expression of mutant telomerase in immortal telomerase-negative human cells results in cell cycle deregulation, nuclear and chromosomal abnormalities and rapid loss of viability.

We have reconstituted wild type or mutant telomerase activity in two human cell lines that lack constitutive expression of both core subunits of the enzyme and maintain telomeres by a telomerase-independent mechanism (ALT cells). Wild type telomerase RNA and four telomerase RNAs with single point mutations in their template domain were used to express enzymes specifying different telomeric DNA sequences. Expression of wild type telomerase for up to 32 days had no detectable effect on cell growth or viability. In contrast, cells expressing mutant telomerases had slower growth rate, abnormal cell cycle and reduced viability. Dramatically aberrant nuclei, typical of cells undergoing mitotic catastrophe, and large numbers of fused chromosomes were also characteristic of these populations. Notably, all phenotypes were apparent within the first few cell divisions after expression of the enzymes. Unlike wild type, mutant telomerase activity was progressively selected against with cell culturing, and this correlated with the disappearance of cells with aberrant phenotypes. Our results suggest that even very limited synthesis of mutated sequences can affect telomere structure in human cells, and that the toxicity of mutant telomerases is due to telomere malfunction.

Cell Cycle↗
PubMed2001

Patterns of recurrence after resection of colorectal liver metastases: prediction by models of outcome analysis.

Various series have reported similar survival and recurrence rates after resection of colorectal liver metastases (CRLM). If outcomes were predictable, indications for surgery could be improved. This hypothesis was tested in 135 consecutive patients with CRLM who underwent "curative" resection from 1977 to 1997. Among the 132 patients available for follow-up, three groups were identified on the basis of outcome: (1) survival of more than 5 years disease-free (n = 32; 24%); (2) diffuse recurrences within the first 6 months (n = 24; 18%); and (3) discrete recurrences for which reresection was performed (n = 16; 12%). As our results are similar to those reported in the literature, we assumed that about 50% of patients with resectable lesions have recognizable patterns of recurrence. At multivariate analysis, factors significant for disease-free survival (DFS) were the percentage of liver invasion, metastases to lymph nodes at the primary site, number of metastases, preoperative glutamic pyruvic transaminase (GPT) level, and type of liver resection. On the basis of the relative risk (RR) expressed by significant prognostic factors, a score model was developed, and three prognostic groups were defined: Group A, with the best prognostic score, included 23 of 32 (72%) patients who survived more than 5 years, and that with the worst prognostic score (group C) included 22 of 24 (92%) patients with early diffuse recurrences. Extreme (especially unfavorable) outcomes can therefore be predicted. By using improved models of outcome analysis, many patients could be spared surgery as first-line treatment, and stratification criteria could be worked out for future trials.

Adenocarcinoma↗
PubMed2000

Histone deacetylation is involved in the transcriptional repression of hTERT in normal human cells.

Trancriptional regulation of the human telomerase reverse transcriptase (hTERT) gene, encoding the catalytic protein of human telomerase, plays a critical role in the activation of the enzyme during cell immortalization and tumorigenesis. However, the molecular mechanisms involved in the regulation of hTERT expression are still not fully understood. We have previously cloned and characterized the genomic sequences and promoter of the hTERT gene. Here, we provide evidence that histone deacetylation is involved in the repression of hTERT in human cells. Inhibition of histone deacetylases by trichostatin A in telomerase-negative cells resulted in activation of telomerase activity and up-regulation of hTERT mRNA. Transient transfection experiments with a reporter under control of the hTERT promoter indicated that this promoter can be activated by trichostatin A. Finally, our results show that repression of the hTERT promoter by the Mad protein requires histone deacetylase activity, whereas de-repression by trichostatin A is independent of the E-boxes located in its core region.

Acetylation↗
PubMed2000

Induction of hTERT expression and telomerase activity by estrogens in human ovary epithelium cells.

In mammals, molecular mechanisms and factors involved in the tight regulation of telomerase expression and activity are still largely undefined. In this study, we provide evidence for a role of estrogens and their receptors in the transcriptional regulation of hTERT, the catalytic subunit of human telomerase and, consequently, in the activation of the enzyme. Through a computer analysis of the hTERT 5'-flanking sequences, we identified a putative estrogen response element (ERE) which was capable of binding in vitro human estrogen receptor alpha (ERalpha). In vivo DNA footprinting revealed specific modifications of the ERE region in ERalpha-positive but not ERalpha-negative cells upon treatment with 17beta-estradiol (E2), indicative of estrogen-dependent chromatin remodelling. In the presence of E2, transient expression of ERalpha but not ERbeta remarkably increased hTERT promoter activity, and mutation of the ERE significantly reduced this effect. No telomerase activity was detected in human ovary epithelial cells grown in the absence of E2, but the addition of the hormone induced the enzyme within 3 h of treatment. The expression of hTERT mRNA and protein was induced in parallel with enzymatic activity. This prompt estrogen modulation of telomerase activity substantiates estrogen-dependent transcriptional regulation of the hTERT gene. The identification of hTERT as a target of estrogens represents a novel finding which advances the understanding of telomerase regulation in hormone-dependent cells and has implications for a potential role of hormones in their senescence and malignant conversion.

3T3 Cells↗
PubMed1999

Construction of a recombinant adenovirus for efficient delivery of the I-SceI yeast endonuclease to human cells and its application in the in vivo cleavage of chromosomes to expose new potential telomeres.

We have constructed a replication-defective adenovirus vector encoding the yeast I- Sce I endonuclease under the control of the murine cytomegalovirus immediate-early gene promoter (AdM Sce I) for efficient delivery of this enzyme to mammalian cells. We present evidence of AdM Sce I-mediated I- Sce I protein expression and cleavage activity in replication-permissive 293 cells, and of cleavage of chromosomes in vivo in both 293 cells and in non-permissive human cells. We have exploited this system for the generation of chromosomes capped by artificial telomeric sequences in cells with integrated plasmids containing telomeric DNA arrays adjacent to an I- Sce I recognition site. The properties of the AdM Sce I virus described here make it a useful tool for studying biological processes involving induction of DNA breaks, recombination and gene targeting in cells grown in culture and in vivo.

Adenoviridae↗
PubMed1999

Genetic interactions between error-prone and error-free postreplication repair pathways in Saccharomyces cerevisiae.

Evidence obtained from recent studies supports the existence of an error-free postreplication repair (PRR) and a mutagenesis pathway within the Saccharomyces cerevisiae RAD6 DNA repair group. The MMS2 gene is the only known yeast gene involved in error-free PRR that, when mutated, significantly increases the spontaneous mutation rate. In this study, the mutational spectrum of the mms2 mutator was determined and compared to the wild type strain. In addition, mutagenenic effects and genetic interactions of the mms2 mutator and rev3 anti-mutator were examined with respect to forward mutations, frameshift reversions as well as amber and ochre suppressions. It was concluded from these results that the mms2 mutator phenotype is largely dependent on the functional REV3 gene. The synergistic effects of mms2 and rev3 mutations towards killing by a variety of DNA-damaging agents ruled out the possibility that MMS2 simply acts to suppress REV3 activity and favored the hypothesis that MMS2 and REV3 form two alternative subpathways within the RAD6 DNA repair pathway. Taken together, we propose that two pathways represented by MMS2 and REV3 deal with a similar range of endogenous and environmental DNA damage but with different biological consequences, namely, error-free repair and mutagenesis, respectively.

Base Sequence↗
PubMed1999

The human telomerase catalytic subunit hTERT: organization of the gene and characterization of the promoter.

Telomerase, the enzyme that synthesizes telomeric DNA, is not expressed in most human somatic cells but is activated with in vitro immortalization and during tumorigenesis, and repressed by cell differentiation. Of the two components of the core enzyme, the catalytic protein hTERT is limiting for activity. To investigate mechanisms of hTERT gene regulation, we have cloned genomic sequences encompassing the complete hTERT transcription unit. The hTERT gene consists of 16 exons and 15 introns spanning approximately 35 kb. Transient transfections of immortal human cells with potential regulatory 5' sequences linked to a reporter, combined with deletion analysis of these sequences, indicated that elements responsible for promoter activity are contained within a region extending from 330 bp upstream of the ATG to the second exon of the gene. Assays in different cell types have shown that the hTERT promoter is inactive in normal and in transformed pre-immortal cells, but, like telomerase, it is activated with cell immortalization. Sequence analysis revealed that the hTERT promoter is GC-rich, lacks TATA and CAAT boxes but contains binding sites for several transcription factors that may be involved in its regulation. The abundance of these sites suggests the possibility that hTERT expression may be subject to multiple levels of control and be regulated by different factors in different cellular contexts.

Amino Acid Sequence↗
PubMed1998

Up-regulation of CIR1/CROC1 expression upon cell immortalization and in tumor-derived human cell lines.

Acquisition of the immortal phenotype by tumor cells represents an essential and potentially rate-limiting step in tumorigenesis. To identify changes in gene expression that are associated with the early stages of cell immortalization, we compared genetically matched pairs of pre-immortal and immortal human cell clones by mRNA differential display. Two transcripts, denoted CIR1 and CIR2, were identified which were up-regulated in immortal cells. Sequence analysis revealed CIR1 to be identical to the recently cloned CROC1/UEV-1 gene, whereas CIR2 corresponds to an as yet uncharacterized 1.2 kb mRNA. A 5-6-fold elevation in CIR1/CROC1 expression and a 2-3-fold elevation in CIR2 expression were observed in SV40-transformed human embryonic kidney cells immediately following proliferative crisis, suggesting a potential role for these genes in immortalization. Expression of CIR1/CROC1 was found to be elevated also in a variety of immortal human tumor-derived cell lines, as compared to their normal tissue counterparts. These results are compatible with induction of CIR1/CROC1 being an early event in the acquisition of immortality and with a role for this gene in the immortal phenotype of tumor cells.

Base Sequence↗
PubMed1998

Dissociation of telomere dynamics from telomerase activity in human thyroid cancer cells.

Prevention of telomere erosion through acquisition of telomerase activity is thought to be an essential mechanism in most human cancer cells for avoidance of cellular senescence and crisis. It has been generally assumed that once telomerase has been activated, no further telomere shortening should ensue. We show here, however, that a much more complex pattern of telomere dynamics can exist in telomerase-positive immortal cancer cells. Using a panel of subclones derived from a human thyroid cancer cell line, K1E7, we found that some clones show persistent decline in mean telomere restriction fragment (TRF) length by up to 2 kb over 450 population doublings (pd), despite sustained high telomerase activity (as assessed by the in vitro "TRAP" assay). TRF length subsequently stabilized at around 5 kb, but with no corresponding increase in telomerase activity. One clone showed an even more unexpected biphasic time course, with the mean TRF length initially increasing by 1.5 kb over 90 pd, before "plateauing" and then returning over a similar period to its original value, again without any correlation to TRAP activity. Such dissociations between telomere dynamics and telomerase activity support the existence of additional controls on telomere length in the intact cell. Our observations are consistent with current negative-feedback models of telomere length regulation by telomere binding proteins and these cell lines should prove useful experimental tools for their further evaluation.

Humans↗
PubMed1998

Prognostic factors affecting long term outcome after liver resection for hepatocellular carcinoma: results in a series of 100 Italian patients.

BACKGROUND: Long term results after liver resection for hepatocellular carcinoma (HCC) are disappointing because the disease tends to recur. In this study, the authors assessed prognostic factors affecting long term outcome, in the hope that these factors might be used in selecting HCC patients for surgery. METHODS: During the period 1977-1995, 100 consecutive patients underwent curative liver resection; 78 of 100 had HCC arising on preexisting cirrhosis (53 Child's Class A and 25 Child's Class B). Thirty-five prognostic factors were evaluated for their association with overall survival (OS) and disease free survival (DFS) in univariate and multivariate analysis (Cox proportional hazards model). RESULTS: There were four postoperative deaths. Seven patients died in hospital of hepatorenal failure: six had Child's Class B cirrhosis and had undergone preoperative chemoembolization. Of the remaining 89 patients, 50 developed recurrence. All surviving Child's Class B patients had recurrence. Five-year OS, postoperative deaths included, was 38% (median, 36 months). Five-year DFS, postoperative deaths excluded, was 26% (median, 21 months). Independent prognostic factors for DFS were Child's class, glutamic-oxaloacetic transaminase, gamma-glutamyltransferase, alpha-fetoprotein, number of tumor nodules, width of resection margins, preoperative chemoembolization, and experience of the team that performed the surgery. Factors with an independent effect on OS were Child's class and width of resection margins. CONCLUSIONS: Liver resection can provide long term DFS in HCC patients with normal liver function. In patients with liver function impairment or an inadequate resection margin, recurrences are almost certain to occur. Preoperative chemoembolization significantly prolongs DFS but may increase the risk of postoperative liver failure in patients with liver function impairment.

Adult↗