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Biomedical subjects

G M Carbone

Publications and source records attributed to G M Carbone.

9 recordsLinked to original sources
PubMed2001

Imbalanced DNA synthesis induced by cytosine arabinoside and fludarabine in human leukemia cells.

Previous studies have demonstrated that cytosine arabinoside (araC) induces an accumulation of Okazaki fragments, while fludarabine (FaraA) inhibits Okazaki fragment synthesis. We extended these observations in the present study to provide insights into various mechanisms by which these anticancer drugs affect DNA replication and induce genomic instability in human CEM leukemia cells. Neither araC nor FaraA induced a detectable amount of re-replicated DNA in S-phase cells, which indicated that drug-induced alterations in Okazaki fragment synthesis were not accompanied by DNA re-replication. Synthesis on both leading and lagging DNA strands within the c-myc locus was measured in cells incubated with equitoxic concentrations of araC or FaraA. In araC-treated cells, nascent DNA from the lagging strand was enriched about 5-fold compared with the leading strand. In contrast, FaraA did not induce any replication imbalance. AraC- and FaraA induced changes in the frequency of N-(phosphonacetyl)-l-aspartate (PALA) resistance and the extent of CAD gene amplification were monitored as markers of drug-induced genomic instability. At concentrations that reduced cloning efficiency by 50% (IC(50)), araC increased the frequency of PALA resistance about 4-fold, while FaraA did not have a significant effect on the frequency of PALA resistance. Pretreatment with araC also increased the extent of CAD gene amplification. We propose that the imbalanced DNA synthesis induced by araC leads to the accumulation of Okazaki fragments on the lagging arms and single-stranded DNA regions on the leading arms of replication forks. The formation of these abnormal replication structures was associated with the generation of genomic instability.

Antineoplastic Agents↗
PubMed2000

Antigene and antiproliferative effects of a c-myc-targeting phosphorothioate triple helix-forming oligonucleotide in human leukemia cells.

The c-myc gene is frequently deregulated and overexpressed in human cancers, and strategies designed to inhibit c-myc expression in cancer cells may have considerable therapeutic value. The purpose of the present work was to characterize the antigene and antiproliferative activity of a triple helix-forming oligonucleotide (TFO) targeted to a homopurine-homopyrimidine sequence in the P2 promoter of the c-myc gene. The TFO was synthesized with phosphorothioate (PS) internucleotide linkages to confer resistance to intra- and extracellular nucleases. This property is required of oligonucleotides designed for in vivo testing and therapeutic applications. The PS-TFO was found to form triplex DNA with affinity and specificity comparable with that of the corresponding phosphodiester TFO, as shown by gel mobility shift and footprinting assays. Fluorescence microscopy and polyacrylamide gel analysis showed that the fluorescein-labeled PS-TFO accumulated in nuclei of CEM leukemia cells and remained intact for at least 72 h. Incubation of CEM cells with PS-TFO reduced c-myc RNA and protein levels. A single exposure of leukemia cells to the PS-TFO was sufficient to induce dose-dependent growth inhibitory effects. Growth inhibition correlated with accumulation of cells in S phase and with induction of cell death by apoptosis. The PS-TFO was also effective in other leukemia and lymphoma cell lines. Control oligonucleotides had minimal effects in all assays. These data indicate that the c-myc-targeted PS-TFO is an effective antigene and antiproliferative agent, with potential for testing in vivo as a novel approach to cancer therapy.

Antigens↗
PubMed2000

Inhibition of gene expression and cell proliferation by triple helix-forming oligonucleotides directed to the c-myc gene.

Triple helix-forming oligonucleotides (TFOs) bind with high affinity and specificity to homopurine-homopyrimidine sequences in DNA and have been shown to inhibit transcription of target genes in various experimental systems. In the present study, we evaluated the ability of 3'-amino-modified phosphodiester TFOs directed to four sites in the c-myc gene to inhibit gene expression and proliferation of human leukemia (CEM, KG-1, and HL-60) and lymphoma (Raji and ST486) cells. GT-rich TFOs were designed to target sequences located either upstream (myc1 and -2) or downstream (myc3 and -4) of the P2 promoter, which is the major c-myc promoter. Myc2, which was directed to a site immediately upstream of this promoter, inhibited c-myc expression and proliferation of CEM cells. The effects of this TFO were sequence- and target-specific, since control oligonucleotides and TFOs directed to other sites were less or not active. Myc2 was also effective in KG-1, HL-60, and Raji cells. In contrast, ST486 cells were more sensitive to myc3, which targets a sequence in intron 1 upstream of the P3 promoter, than myc2. As result of a chromosomal translocation, P3 is the active promoter in ST486 cells. This study demonstrates the activity and specificity of TFOs designed to act as repressors of c-myc gene expression in human leukemia and lymphoma cells. Our results suggest that this is a valid approach to selectively inhibit gene expression and cancer cell growth, and encourage further investigation of its potential applications in cancer therapy.

Cell Division↗
PubMed1998

Staging of primary colorectal carcinomas with fluorine-18 fluorodeoxyglucose whole-body PET: correlation with histopathologic and CT findings.

PURPOSE: To evaluate the diagnostic usefulness of positron emission tomography (PET) with fluorine-18 fluorodeoxyglucose (FDG) in patients with primary colorectal carcinomas. MATERIALS AND METHODS: Forty-eight patients with biopsy-proved (n = 44) or high clinical suspicion for (n = 4) colorectal cancer underwent whole-body PET after intravenous administration of 10 mCi (370 MBq) of FDG. FDG PET results were correlated with computed tomographic (CT), surgical, and histopathologic findings. RESULTS: PET depicted all known intraluminal carcinomas in 37 patients (including two in situ carcinomas) (sensitivity, 100%), but findings were false-positive in four of seven patients without cancer (three with inflammatory bowel conditions, one who had undergone polypectomy). Specificity was 43% (three of seven patients); positive predictive value, 90% (37 of 41 patients); and negative predictive value, 100% (three of three patients). No FDG accumulation was noted in 35 hyperplastic polyps. FDG PET depicted lymph node metastases in four of 14 patients (sensitivity, 29%). Results were similar to those obtained with CT (true-positive, two of seven patients [sensitivity, 29%]; true-negative, 22 of 26 patients [specificity, 85%]). FDG PET depicted liver metastases in seven of eight patients and was superior to CT, which depicted liver metastases in three patients (sensitivity of 88% and 38%, respectively). FDG PET and CT, respectively, correctly depicted the absence of liver metastases in 35 and 32 patients (specificity, 100% and 97%; negative predictive value, 97% and 86%). CONCLUSION: FDG PET has a high sensitivity and specificity for detection of colorectal carcinomas (primary and liver metastases) and appears to be superior to CT in the staging of primary colorectal carcinoma.

Adenocarcinoma↗
PubMed1997

Arrest of replication fork progression at sites of topoisomerase II-mediated DNA cleavage in human leukemia CEM cells incubated with VM-26.

Recent studies have shown that the anticancer drugs VM-26 and mitoxantrone stabilize preferentially the binding of topoisomerase IIalpha to replicating compared to nonreplicating DNA. To further understand the mechanisms by which cleavable complex-forming topoisomerase II inhibitors interfere with DNA replication, we examined the effects of VM-26 on this process in human leukemia CEM cells. Both the inhibition of DNA synthesis and cell survival were directly related to the total amount of drug-stabilized cleavable complexes formed in VM-26-treated cells. DNA chain elongation was also inhibited in a concentration-dependent fashion in these cells, which suggested that VM-26-stabilized cleavable complexes interfered with the movement of DNA replication forks. To test this hypothesis directly, we monitored replication fork progression at a specific site of VM-26-induced DNA cleavage. A topoisomerase II-mediated cleavage site was detected in the first exon of the c-myc gene in VM-26-treated cells. This cleavage site was downstream of a putative replication origin located in the 5' flanking region of the gene. Replication forks, which moved through this region of the c-myc gene in the 5' to 3' direction, were specifically arrested at this site in VM-26-treated cells, but not in untreated or aphidicolin-treated cells. These studies provide the first direct evidence that a VM-26-stabilized topoisomerase II-DNA cleavable complex acts as a replication fork barrier at a specific genomic site in mammalian cells. Furthermore, the data support the hypothesis that the replication fork arrest induced by cleavable complex-forming topoisomerase II inhibitors leads to the generation of irreversible DNA damage and cytotoxicity in proliferating cells.

DNA Damage↗
PubMed1995

Effect of chronic infusion of cortisol on renin gene expression and renin response to hemorrhage in fetal lambs.

In the ovine fetus, plasma renin levels increase close to term, and renin responses to various stimuli are enhanced when compared with responses earlier in gestation. These changes are accompanied by increases in renal renin gene expression and renin content, and they occur in conjunction with elevations in fetal plasma cortisol. Thus, the purpose of this study was to test the hypothesis that a chronic, physiologic elevation in fetal plasma cortisol in early gestation would increase activity in the renin-angiotensin system prematurely. We studied fetuses (control, n = 8; cortisol infused, n = 11) at 94 +/- 2 d of gestation. Fetal vessels were catheterized, and cortisol or saline solution was infused for 6 d. At the end of infusion, fetuses were hemorrhaged approximately 30% of estimated blood volume. Blood samples were collected to measure plasma renin concentration. Then the animals were killed, and kidneys were removed to measure renin mRNA and renin content. Plasma cortisol concentrations in the control and cortisol-treated animals were 7.2 +/- 0.8 and 57.7 +/- 8.6 nmol/L (p < 0.01), respectively. Basal plasma renin concentrations were similar in the two groups 3.2 +/- 0.4 versus 4.4 +/- 1.8 ng of angiotensin I/mL/h, and there was a significant increase after hemorrhage in the cortisol-treated group only. Renal renin content and mRNA levels were similar in the two groups. These data indicate that chronic increases in cortisol in fetal lambs at 0.65 gestation significantly enhance the renin response to hemorrhage but do not alter renal renin gene expression.

Animals↗
PubMed1994

Expression of manganese superoxide dismutase in ovine kidney cortex during development.

Manganese superoxide dismutase (MnSOD) is one of the main antioxidant enzymes in mammalian tissue. Previous studies have shown that the activity of MnSOD increases in the rat kidney during development. To define further the developmental change in MnSOD activity and better understand some of the steps involved in the control of MnSOD expression during kidney development, we measured MnSOD messenger RNA and enzyme activity in the ovine kidney cortex during fetal life, in the newborn period, and in adults. MnSOD mRNA and enzyme activity were detected at 0.65 gestation. Hybridization of the Northern blot with a human MnSOD cDNA probe showed evidence of two transcripts of 4.0 and 1.5 kb, respectively. There was a significant increase with age of MnSOD activity and MnSOD mRNA (p < 0.0001). The abundance of each MnSOD transcript significantly increased with age (p < 0.001). In the fetuses, both transcripts increase in parallel; in newborns and adults the 1.5-kb increase was significantly greater than the 4.0-kb increase. Enzyme activity and mRNA were strongly correlated (r = 0.89, p < 0.0001). These data indicate that the expression of MnSOD is developmentally regulated in the ovine kidney cortex. This increase seems to be dependent largely on pretranslational events.

Animals↗
PubMed1993

Developmental changes in renin gene expression in ovine kidney cortex.

The ontogeny of renin mRNA and renin content from renal cortical slices was studied in two groups of ovine fetuses at 92-94 days (0.64 gestation) and at 138-142 days (0.96 gestation), newborn lambs (0.4-2 days old), and adult sheep. Renal renin mRNA was identified by hybridization with a 32P-labeled full length rat renin cDNA. Renal renin content was measured as nanograms of angiotensin I generated per hour (active renin). There was a significant age effect on renin mRNA levels (F = 10.0, P < 0.001); values increase significantly between 0.64 and 0.95 g (P < 0.005), remain elevated in the newborns (P < 0.05), and subsequently decline in adulthood (P < 0.005). Likewise, renal renin content was significantly higher in late gestation fetuses and newborn lambs than in early gestation and adults (F = 8.3, P < 0.003). The renal renin content was strongly correlated with renin mRNA levels (R = 0.88, P < 0.0001). These results suggest that 1) the renin gene is developmentally regulated in the ovine kidney and 2) the renal content of active renin in basal conditions is regulated, at least in part, by events at the transcriptional level.

Animals↗