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1.
Clin Cancer Res ; 7(6): 1600-9, 2001 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-11410496

RESUMO

PURPOSE: Preclinical and clinical investigation of the combination of the antiangiogenesis/anti-invasion agent carboxyamido-triazole (CAI) administered with the cytotoxic agent paclitaxel (PAX). EXPERIMENTAL DESIGN: Colony-forming assays were used to test the activity of CAI plus PAX on A2780 human ovarian cancer. The sequence of CAI followed by PAX (CAI>Pax) was modeled in nude mice to test for potential additive toxicity. The Phase I clinical dose escalation schema tested p.o. administered CAI in PEG-400 (50-100 mg/m(2)) or micronized CAI (250 mg/m(2)) for 8 days followed by a 3-h infusion of PAX (110-250 mg/m(2)) every 21 days. Patients were assessed for toxicity, pharmacokinetics of CAI and PAX, and disease outcome. RESULTS: In preclinical studies, CAI>Pax was additive in A2780 human ovarian cancer cell lines when CAI (1 or 5 microM) preceded subtherapeutic doses of PAX. CAI did not reverse PAX resistance and collateral resistance to CAI was documented in PAX-resistant cells. CAI>PAX administration had no overt additive toxicity in nude mice. Thirty-nine patients were treated on a dose-escalation Phase I trial using daily oral CAI for 8 days followed by the PAX infusion. Pharmacokinetic analysis revealed that PAX caused an acute increase in circulating CAI concentrations in a dose-dependent fashion. No additive or cumulative toxicity was observed, and grade 3 nonhematological toxicity was rare. Three partial responses and two minor responses were observed. CONCLUSIONS: The sequential combination of CAI and PAX is well tolerated, and the activity observed suggests that further study of the combination is warranted.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Interações Medicamentosas , Neoplasias/tratamento farmacológico , Paclitaxel/administração & dosagem , Paclitaxel/uso terapêutico , Recidiva , Triazóis/administração & dosagem , Triazóis/uso terapêutico , Adulto , Idoso , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/uso terapêutico , Antineoplásicos Fitogênicos/administração & dosagem , Antineoplásicos Fitogênicos/uso terapêutico , Relação Dose-Resposta a Droga , Feminino , Humanos , Concentração Inibidora 50 , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Neovascularização Patológica , Neoplasias Ovarianas/tratamento farmacológico , Fatores de Tempo , Resultado do Tratamento , Células Tumorais Cultivadas
2.
Biochem Pharmacol ; 60(11): 1611-9, 2000 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-11077043

RESUMO

Tumor tissue specimens from human ovarian cancer patients were assessed for relative mRNA abundance levels of several genes thought to be involved in the development of in vitro drug resistance in this disease. Higher mRNA levels of Xeroderma pigmentosum group B (XPB), which links DNA repair with DNA transcription, and of Cockayne's syndrome group B (CSB), which is essential for gene-specific repair, were observed in tumor tissues that were clinically resistant to platinum-based chemotherapy, as compared with tissues from patients responding to therapy. In a cohort of 27 patients, mRNA levels of XPB averaged 5-fold higher in platinum-resistant tumors (P = 0.001); and for CSB, mRNA levels averaged 6-fold higher but with greater variability (P = 0.033). Concurrently, these platinum-resistant tumor tissues did not exhibit significantly higher mRNA levels for the MDR1 (multidrug-resistance) gene (P = 0.134) or of the metallothionein-II (MT-II) gene (P = 0.598). Since these platinum-resistant tumors also show higher mRNA levels of ERCC1 and XPA, platinum resistance appears to be associated with concurrent up-regulation of four genes (XPA, ERCC1, XPB, and CSB). These four genes participate in DNA damage excision activity, gene-specific repair, and linkage of DNA repair with DNA transcription. These data suggest that concurrent up-regulation of genes involved in nucleotide excision repair may be important in clinical resistance to platinum-based chemotherapy in this disease.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Resistencia a Medicamentos Antineoplásicos/fisiologia , Metalotioneína/genética , Neoplasias Ovarianas/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , DNA Helicases/metabolismo , Reparo do DNA , Enzimas Reparadoras do DNA , Proteínas de Ligação a DNA/metabolismo , Feminino , Expressão Gênica , Humanos , Metalotioneína/metabolismo , Neoplasias Ovarianas/metabolismo , Platina/farmacologia , Proteínas de Ligação a Poli-ADP-Ribose , RNA Mensageiro/metabolismo , Transcrição Gênica
3.
Oncol Rep ; 7(5): 1123-8, 2000.
Artigo em Inglês | MEDLINE | ID: mdl-10948350

RESUMO

Nucleotide excision repair (NER) is the DNA repair pathway through which cisplatin-DNA intrastrand adduct is repaired. Clinical studies have shown that increased mRNA expression of selected genes involved in the rate-limiting step of NER, appear to be closely associated with clinical resistance to platinum agents. These specific studies have led to the possibility of an assessment of the order in which, the appearance of mRNAs of selected NER genes may occur. Included in this assessment are ERCC1, XPB, CSB, and XPA, studied in 28 ovarian cancer tumor tissue specimens. The study of these four genes, in pairs, from 28 ovarian cancer specimens, results in 168 separate observations. Based on the mRNA expression patterns of these genes in these tissues, it is suggested that ERCC1 mRNA may appear before the mRNA of any of the other genes, in an obligate fashion. This is followed by XPB mRNA; which appears before the mRNA of XPA; which in turn, may appear before CSB. This pattern is consistent with what we have reported previously, in non-malignant human bone marrow specimens from a cohort of 52 patients.


Assuntos
Adenocarcinoma/genética , Reparo do DNA/genética , Endonucleases , Neoplasias Ovarianas/genética , RNA Mensageiro/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Autorradiografia , DNA Helicases/biossíntese , DNA Helicases/genética , Enzimas Reparadoras do DNA , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Feminino , Expressão Gênica , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Estadiamento de Neoplasias , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Proteínas de Ligação a Poli-ADP-Ribose , Biossíntese de Proteínas , Proteínas/genética , RNA Mensageiro/biossíntese , Proteínas de Ligação a RNA/biossíntese , Proteínas de Ligação a RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína de Xeroderma Pigmentoso Grupo A
4.
Anticancer Res ; 20(2A): 645-52, 2000.
Artigo em Inglês | MEDLINE | ID: mdl-10810335

RESUMO

Nucleotide excision repair (NER) is responsible for the repair of platinum-DNA lesions. ERCC-1 is a critical gene within the NER pathway, and cells without a functional ERCC-1 do not repair cisplatin-caused DNA damage. The present study was therefore designed to evaluate the relationship between the expression of ERCC-1 and the repair of cisplatin-induced DNA adducts in human ovarian cancer cells in vitro. One hour exposure of MCAS cells to cisplatin yielded an approximately two-fold increment in the levels of ERCC-1 mRNA and ERCC-1 protein, as determined, respectively, by Northern and Western blottings. In addition, nuclear run-on assay showed that ERCC-1 gene transcription rate was increased to about the same extent as steady-state ERCC-1 mRNA and protein, in response to cisplatin treatment. However, the levels of ERCC-1 mRNA, ERCC-1 protein, and ERCC-1 transcript in MCAS cells are two-fold lower than those in A2780/CP70 cells, as previously reported. Furthermore, the repair of cisplatin-DNA adducts in MCAS cells, as measured by atomic absorption spectrometry, is also nearly two-fold less than that in A2780/CP70 cells, indicating a strong association between the level of ERCC-1 expression and the activity of excision repair in these two human ovarian tumor cell lines. These results suggest that ERCC-1 may be a useful marker to monitor the repair of platinum-DNA damage in tumor cells, and further highlight that potential pharmacological approaches which specifically inhibit ERCC-1 expression may increase cellular sensitivity to cisplatin.


Assuntos
Cisplatino/análise , Cisplatino/toxicidade , Adutos de DNA/análise , Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA , Endonucleases , Regulação Neoplásica da Expressão Gênica , Proteínas/genética , Núcleo Celular/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Cinética , Neoplasias Ovarianas , RNA Mensageiro/genética , Transcrição Gênica , Células Tumorais Cultivadas
5.
Cancer Lett ; 151(2): 127-32, 2000 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-10738106

RESUMO

We have previously reported on mRNA expression of ERCC1, XPA and XPD in human ovarian cancer cells and tissues. Several factors can influence mRNA expression for any given gene. Alterations in gene copy number for ERCC1 and/or XPD have been reported to occur in malignant glioma specimens. Human ovarian cancer cell lines and tissues were therefore examined for evidence of altered gene copy number in selected genes within the nucleotide excision repair (NER) pathway. Six ovarian cancer cell lines were studied: A2780, A2780/CP70, SKOV3, MCAS, QvCar3 and Caov4. Cellular sensitivity to cisplatin varies by more than 1 log between some of these cells. In each of these cell lines, the genes examined included ERCC1, XPA, XPB, XPD, XPG, CSB and p53. Genomic DNA was also extracted from ovarian cancer specimens taken from 22 patients and assessed for evidence of allelic loss and/or allelic gain for ERCC1 and XPD. Twelve of the clinical specimens were from patients with platinum-sensitive tumors and ten were from patients with platinum-resistant tumors. In no case could we demonstrate a reproducible variation in gene copy number in any cell line. Among the human tissues studied, there was one case of allelic gain out of 22 specimens. We therefore conclude that alterations in gene copy number is not a common event in human ovarian cancer. Other mechanisms must be invoked to explain differences in mRNA expression for these genes.


Assuntos
DNA Helicases , Proteínas de Ligação a DNA , Endonucleases , Perda de Heterozigosidade/genética , Neoplasias Ovarianas/genética , Proteínas/genética , Fatores de Transcrição , Southern Blotting , Cisplatino/farmacologia , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Dosagem de Genes , Humanos , Neoplasias Ovarianas/patologia , Platina/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células Tumorais Cultivadas , Proteína Grupo D do Xeroderma Pigmentoso
6.
Int J Oncol ; 16(3): 555-60, 2000 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-10675489

RESUMO

ERCC1 is an essential gene within the nucleotide excision repair process. We studied two human ovarian carcinoma cell lines for cisplatin resistance, which differed with respect to ERCC1. The A2780/CP70 cell line has been extensively studied previously, and has the wild-type ERCC1 sequence. The MCAS cell line has a recently described ERCC1 polymorphism at codon 118, which is associated with an approximate 50% reduction in codon usage. These cells did not differ with respect to p53 sequence nor p53 mRNA induction following cisplatin exposure. The induction of ERCC1 mRNA was markedly reduced in MCAS cells as compared to A2780/CP70 cells. At the IC50 cisplatin dose for each cell line, MCAS cells were less proficient at cisplatin-DNA adduct repair than A2780/CP70 cells. In absolute terms, A2780/CP70 cells repaired 3-fold as much adduct (2.7 pg/microgram DNA over 6 h vs 0.86 pg/microgram DNA); and when expressed in terms of the maximal DNA adduct load, A2780/CP70 cells repaired 50% more adduct than MCAS cells. MCAS cells had increased cytosolic inactivation of drug at the IC50 dose level, which has been previously suggested to be a compensatory cellular response for reduced DNA repair capacity. These data suggest the possibility that this specific ERCC1 polymorphism, may be associated with reduced DNA repair capacity in human ovarian cancer cells. This association may be effected through a reduction in peak production of ERCC1 mRNA, and a consequent reduction in the translation of ERCC1 mRNA into protein.


Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Códon , Proteínas de Ligação a DNA , Endonucleases , Neoplasias Ovarianas/genética , Proteínas/genética , Cisplatino/metabolismo , Adutos de DNA/análise , Reparo do DNA , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , RNA Mensageiro/análise , Células Tumorais Cultivadas
7.
Cell Mol Life Sci ; 55(3): 456-66, 1999 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-10228559

RESUMO

ERCC-1 is an essential gene in the nucleotide excision repair pathway, and may be essential for life. However, the mechanism of transcriptional activation and regulation of ERCC-1 gene expression is unclear. We therefore investigated the effect of the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on the expression of the ERCC-1 gene in A2780/CP70 human ovarian carcinoma cells. TPA induced a four- to sixfold increase in steady-state ERCC-1 messenger RNA (mRNA) levels that was time- and concentration-dependent. Nuclear run-on experiments demonstrated that the rate of transcription of ERCC-1 was approximately 2.8-fold higher in TPA-treated cells than in the controls. TPA stimulation of A2780/CP70 cells also resulted in a rapid but transient induction of c-jun and c-fos as determined by Northern and Western blot analyses, which peaked about 2 h before the peak in ERCC-1 expression. Electrophoretic mobility shift assays of nuclear extracts from TPA-treated cells revealed an increase in DNA-binding activity specific for the AP-1-like binding site in the 5'-flanking region of ERCC-1. c-Jun and c-Fos proteins were confirmed to be the components of the activated AP-1 complex by supershift analysis. The increase in AP-1 activity occurs immediately before the increase in ERCC-1 transcription. The increase in AP-1 DNA-binding activity and the increase in ERCC-1 mRNA expression were prevented by pretreatment with cycloheximide. These data suggest that AP-1 may contribute to the upregulation of ERCC-1 in response to TPA in human ovarian cancer cells.


Assuntos
Carcinógenos/farmacologia , Reparo do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA , Endonucleases , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Neoplasias/genética , Neoplasias Ovarianas/patologia , Proteínas/genética , RNA Mensageiro/biossíntese , RNA Neoplásico/biossíntese , Acetato de Tetradecanoilforbol/farmacologia , Fator de Transcrição AP-1/fisiologia , Transcrição Gênica/efeitos dos fármacos , Amanitinas/farmacologia , Sítios de Ligação , Cicloeximida/farmacologia , Feminino , Humanos , Inibidores da Síntese de Ácido Nucleico/farmacologia , Neoplasias Ovarianas/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/genética , RNA Neoplásico/genética , Células Tumorais Cultivadas
8.
Biochem Pharmacol ; 57(4): 347-53, 1999 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-9933022

RESUMO

Excision repair cross complementation group 1 (ERCC-1) is a DNA repair gene that is essential for life, and it appears to be a marker gene for nucleotide excision repair activity. Overexpression of ERCC-1 during cisplatin-based chemotherapy is associated with clinical and cellular drug resistance. We therefore began to assess the influence of various pharmacological agents on the induction of ERCC-1 mRNA in A2780/CP70 human ovarian carcinoma cells. Cisplatin exposure in culture resulted in a 4- to 6-fold induction for the steady-state level of ERCC-1 mRNA in A2780/CP70 cells. ERCC-1 mRNA induction was concentration and time dependent. Cyclosporin A and herbimycin A, which suppress c-fos and c-jun gene expressions, respectively, blocked the cisplatin-induced increase in ERCC-1 mRNA. This effect of cyclosporin A or herbimycin A on the down-regulation of ERCC-1 correlates with enhanced cytotoxicity of cisplatin in this system. The products of c-fos and c-jun are components of the transcription factor AP-1 (activator protein 1). 12-O-Tetradecanoylphorbol 13-acetate (TPA), a known AP-1 agonist, induced ERCC-1 mRNA to the same extent as cisplatin, but did not synergize with cisplatin in this regard. The TPA effect was biphasic, with an initial increase during the first 1-6 hr, followed by decreasing mRNA levels at 24-72 hr. These data suggest that the effects of these pharmacological agents on ERCC-1 gene expression may be mediated through the modulation of AP-1 activities.


Assuntos
Cisplatino/farmacologia , Reparo do DNA , Proteínas de Ligação a DNA , Endonucleases , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas/genética , Benzoquinonas , Ciclosporina/farmacologia , Humanos , Lactamas Macrocíclicas , Biossíntese de Proteínas , Quinonas/farmacologia , RNA Mensageiro/biossíntese , Rifabutina/análogos & derivados , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais Cultivadas
9.
Int J Mol Med ; 1(3): 617-20, 1998 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-9852275

RESUMO

Alternative splicing is a common natural tool for the inhibition of function of full length gene products. We explored whether there was evidence that alternative splicing of ERCC1 may serve such a function for nucleotide excision repair. The ratio of alternatively spliced species to full length species was assessed for the protein and/or for the mRNA, for a series of human cell lines and tissues. This ratio was plotted against the amount of cisplatin-DNA adduct repair in each cell line (n=9), as measured by atomic absorbance spectrometry. As the percentage of alternatively spliced protein and/or mRNA increased, the amount of cisplatin-DNA adduct that was repaired was reduced. This inverse relationship was associated with a substantial amount of scatter (r=0.635), particularly at low levels of repair. These data demonstrate an association between alternative splicing of ERCC1, and reduction in cellular capability to repair cisplatin-DNA adduct.


Assuntos
Processamento Alternativo , Cisplatino/metabolismo , Adutos de DNA/metabolismo , Reparo do DNA/genética , Proteínas de Ligação a DNA , Endonucleases , Proteínas/genética , Western Blotting , DNA de Neoplasias/análise , DNA de Neoplasias/genética , Humanos , Modelos Lineares , Proteínas/análise , RNA Mensageiro/análise , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas
10.
Int J Oncol ; 13(5): 987-92, 1998 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-9772291

RESUMO

Nucleotide excision repair (NER) is the DNA repair pathway by which cisplatin-induced damage is removed from DNA in human cells. ERCC-1 is one of the essential proteins in NER, and is essential for life. Enhanced ERCC-1 expression has been associated with clinical and cellular resistance to cisplatin. We therefore carried out this study to investigate the effect of cisplatin on ERCC-1 protein expression in A2780/CP70 human ovarian cancer cells. Western blot analysis showed that ERCC-1 protein levels were increased to more than 3 times control after a 1 h cisplatin exposure to A2780/CP70 cells in culture. This increase was time- and concentration-dependent. The effect of cisplatin was maximal at 40 mM and peaked 24-48 h after exposure to the drug. These results extend our previous observations that ERCC-1 mRNA expression is induced by cisplatin in this system. TPA, a known AP-1 activator and tumor-promoting phorbol ester, also induced ERCC-1 protein to the same extent as cisplatin, but did not synergize with cisplatin in this regard. These findings suggest that ERCC-1 gene up-regulation in these cells can result through a DNA damage-response pathway, or through the induction of AP-1 activity, independent of the occurrence of DNA damage.


Assuntos
Cisplatino/farmacologia , Proteínas de Ligação a DNA , Endonucleases , Neoplasias Ovarianas/metabolismo , Biossíntese de Proteínas , Acetato de Tetradecanoilforbol/farmacologia , Antineoplásicos/farmacologia , Western Blotting , Carcinógenos/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas de Neoplasias/biossíntese , Neoplasias Ovarianas/genética , Proteínas/metabolismo , Células Tumorais Cultivadas
11.
Anticancer Res ; 18(4A): 2283-7, 1998.
Artigo em Inglês | MEDLINE | ID: mdl-9703867

RESUMO

Enhanced expression of the human excision repair enzyme ERCC-1 is associated with cellular and clinical resistance to cisplatin in human ovarian cancer. High levels of expression of ERCC-1 appear to be associated with increased activity of the nucleotide excision repair pathway. We therefore began to examine the effect of some cisplatin resistance modulators on cisplatin-induced ERCC-1 mRNA expression in the human ovarian carcinoma cell line, A2780/CP70. Cisplatin exposure to A2780/CP70 cells in culture resulted in a four- to five-fold induction for steady-state ERCC-1 mRNA, that was dose- and time-dependent. The biological agents interleukin (IL)-1 alpha and tumour necrosis factor (TNF)-alpha have been shown to enhance cisplatin cytotoxicity in vitro. IL-1 alpha inhibited cisplatin induction of ERCC-1 mRNA levels in our system. The effect of IL-1 alpha was sequence dependent, in that the maximum inhibitory effect was observed with 24-hour pretreatment with IL-1 alpha. By contrast, TNF-alpha had little effect on ERCC-1 mRNA induction by cisplatin. Low-temperature hyperthermia (42 degrees C) almost completely suppressed ERCC-1 mRNA induction in these cells. These findings suggest that the enhancement effect of some agents on cisplatin sensitivity in ovarian tumour cells may be through downregulating ERCC-1 expression.


Assuntos
Cisplatino/farmacologia , Proteínas de Ligação a DNA , Endonucleases , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Interleucina-6/farmacologia , Biossíntese de Proteínas , Transcrição Gênica/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Reparo do DNA , Feminino , Humanos , Cinética , Neoplasias Ovarianas , RNA Mensageiro/biossíntese , Células Tumorais Cultivadas
12.
J Biol Chem ; 273(36): 23419-25, 1998 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-9722577

RESUMO

ERCC-1 is a critical gene within the nucleotide excision repair pathway, and cells without a functional ERCC-1 do not perform cisplatin-DNA adduct repair. We therefore investigated the cisplatin effect on ERCC-1 mRNA expression in vitro. In response to a 1-h cisplatin exposure, A2780/CP70 human ovarian cancer cells showed a 6-fold increase in steady-state level of ERCC-1 mRNA. This rise was attributable to increased transcription as measured by nuclear run-on assays and a 60% increase in ERCC-1 mRNA half-life. The increase in ERCC-1 mRNA was preceded by a 4-5-fold rise in mRNA expressions of c-fos and c-jun, a 14-fold increase in c-Jun protein phosphorylation, and an increase in in vitro nuclear extract binding activity to the AP-1-like site of ERCC-1. These data suggest that the induction of ERCC-1 expression in A2780/CP70 cells exposed to cisplatin results from two major factors: (a) an increase in the expression of transactivating factors that bind the AP-1-like site in the 5'-flanking region of ERCC-1 and (b) an increase in the level of c-Jun phosphorylation that enhances its transactivation property.


Assuntos
Cisplatino/farmacologia , Proteínas de Ligação a DNA , Endonucleases , Regulação Neoplásica da Expressão Gênica , Neoplasias Ovarianas/genética , Biossíntese de Proteínas , Amanitinas/farmacologia , Cisplatino/metabolismo , Cicloeximida/farmacologia , Adutos de DNA/metabolismo , Feminino , Meia-Vida , Humanos , Ligação Proteica , Proteínas Proto-Oncogênicas c-fos/biossíntese , Proteínas Proto-Oncogênicas c-jun/biossíntese , RNA Mensageiro/farmacocinética , Fator de Transcrição AP-1/metabolismo , Regulação para Cima/efeitos dos fármacos
13.
Mutat Res ; 382(1-2): 13-20, 1997 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-9360634

RESUMO

We studied the DNA sequence of the entire coding region of ERCC1 gene, in five cell lines established from human ovarian cancer (A2780, A2780/CP70, MCAS, OVCAR-3, SK-OV-3), 29 human ovarian cancer tumor tissue specimens, one human T-lymphocyte cell line (H9), and non-malignant human ovary tissue (NHO). Samples were assayed by PCR-SSCP and DNA sequence analyses. A silent mutation at codon 118 (site for restriction endonuclease MaeII) in exon 4 of the gene was detected in MCAS, OVCAR-3 and SK-OV-3 cells, and NHO. This mutation was a C-->T transition, that codes for the same amino acid: asparagine. This transition converts a common codon usage (AAC) to an infrequent codon usage (AAT), whereas frequency of use is reduced two-fold. This base change was associated with a detectable band shift on SSCP analysis. For the 29 ovarian cancer specimens, the same base change was observed in 15 tumor samples and was associated with the same band shift in exon 4. Cells and tumor tissue specimens that did not contain the C-->T transition, did not show the band shift in exon 4. Our data suggest that this alteration at codon 118 within the ERCC1 gene, may exist in platinum-sensitive and platinum-resistant ovarian cancer tissues.


Assuntos
Proteínas de Ligação a DNA , Endonucleases , Neoplasias Ovarianas/genética , Polimorfismo Conformacional de Fita Simples , Proteínas/genética , Antineoplásicos/uso terapêutico , Asparagina , Carboplatina/uso terapêutico , Linhagem Celular , Cisplatino/uso terapêutico , DNA/análise , DNA/genética , DNA de Neoplasias/análise , DNA de Neoplasias/genética , Éxons/genética , Feminino , Genes/genética , Humanos , Dados de Sequência Molecular , Neoplasias Ovarianas/tratamento farmacológico , Ovário/química , Mutação Puntual/genética , Polimorfismo de Fragmento de Restrição , Análise de Sequência de DNA , Linfócitos T/química , Células Tumorais Cultivadas
14.
Mol Carcinog ; 17(1): 1-7, 1996 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-8876669

RESUMO

Nucleotide excision repair (NER) is an ordered process in nonmalignant cells, in both human and nonhuman systems. We previously reported that in human brain there is discordant mRNA expression of excision repair cross-complementing (ERCC) 1 and ERCC2 in malignant tissues, concurrent with excellent concordance of these genes in nonmalignant tissues from the same patients. Here we have extended these studies to compare low-grade tumors to high-grade tumors and to include ERCC3 (which links DNA repair with DNA transcription) and ERCC6 (which is essential for gene-specific repair). Glial tumor and adjacent normal brain specimens from 19 individuals were studied. Paired malignant and nonmalignant tissues were obtained from 12 of these patients. For ERCC3, there was excellent concordance of mRNA expression between malignant and nonmalignant tissues from the same individuals (P = 0.003). For ERCC6, no concordance was observed (P = 0.314). Tumor tissue from patients with high-grade gliomas exhibited marked discordance of mRNA expression patterns in situations in which good concordance was observed in tumor tissue from low-grade gliomas. We previously established that malignant brain tumors show increased disorder of genes in the NER process, as compared with nonmalignant tissues. These data suggest that increasing disorder in the NER process may occur as cells move from low-grade to high-grade malignancy.


Assuntos
Neoplasias Encefálicas/genética , Encéfalo/fisiologia , DNA Helicases/genética , Reparo do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila , Regulação Neoplásica da Expressão Gênica , Glioma/genética , Astrocitoma/genética , Astrocitoma/terapia , Neoplasias Encefálicas/terapia , Enzimas Reparadoras do DNA , Glioblastoma/genética , Glioblastoma/terapia , Glioma/terapia , Humanos , Oligodendroglioma/genética , Oligodendroglioma/terapia , Proteínas de Ligação a Poli-ADP-Ribose , RNA Mensageiro/genética , RNA Neoplásico/genética , Análise de Regressão
15.
J Clin Invest ; 94(2): 703-8, 1994 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-8040325

RESUMO

Nucleotide excision repair is a DNA repair pathway that is highly conserved in nature, with analogous repair systems described in Escherichia coli, yeast, and mammalian cells. The rate-limiting step, DNA damage recognition and excision, is effected by the protein products of the genes ERCC1 and XPAC. We therefore assessed mRNA levels of ERCC1 and XPAC in malignant ovarian cancer tissues from 28 patients that were harvested before the administration of platinum-based chemotherapy. Cancer tissues from patients whose tumors were clinically resistant to therapy (n = 13) showed greater levels of total ERCC1 mRNA (P = 0.059), full length transcript of ERCC1 mRNA (P = 0.026), and XPAC mRNA (P = 0.011), as compared with tumor tissues from those individuals clinically sensitive to therapy (n = 15). In 19 of these tissues, the percentage of alternative splicing of ERCC1 mRNA was assessed. ERCC1 splicing was highly variable, with no difference observed between responders and nonresponders. The alternatively spliced species constituted 2-58% of the total ERCC1 mRNA in responders (median = 18%) and 4-71% in nonresponders (median = 13%). These data suggest greater activity of the DNA excision repair genes ERCC1 and XPAC in ovarian cancer tissues of patients clinically resistant to platinum compounds. These data also indicate highly variable splicing of ERCC1 mRNA in ovarian cancer tissues in vivo, whether or not such tissues are sensitive to platinum-based therapy.


Assuntos
Carboplatina/uso terapêutico , Cisplatino/uso terapêutico , Reparo do DNA , Proteínas de Ligação a DNA/genética , Endonucleases , Neoplasias Ovarianas/tratamento farmacológico , Proteínas/genética , RNA Mensageiro/análise , Feminino , Humanos , Neoplasias Ovarianas/genética , Proteína de Xeroderma Pigmentoso Grupo A
16.
Cancer Res ; 53(16): 3694-9, 1993 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-8339278

RESUMO

Using atomic absorbance spectrometry with Zeeman background correction, we measured platinum-DNA adduct levels in leukocyte DNA of 49 patients receiving therapy consisting of only carboplatin and cisplatin. Twenty-four histological types of malignancy were included in the cohort. Peripheral blood leukocytes were collected at defined times during the first two cycles of treatment. The relationship between adduct level and disease response was highly statistically significant during cycle 1 of therapy (two-sided P = 0.007 at day 2), but statistical significance was lost during cycle 2. On all days studied, median and mean adduct levels were consistently higher in responders as compared to nonresponders (summary two-sided P = 0.0004). These data suggest that the processes which protect cellular DNA may be common to malignant and nonmalignant rapidly dividing tissues of the same individual, regardless of the type of tumor that individual may harbor.


Assuntos
Carboplatina/metabolismo , Cisplatino/sangue , Adutos de DNA , DNA/sangue , Leucócitos/metabolismo , Neoplasias/sangue , Carboplatina/uso terapêutico , Cisplatino/análise , DNA/análise , Humanos , Leucócitos/química , Neoplasias/tratamento farmacológico , Estudos Prospectivos , Espectrofotometria Atômica
17.
Cancer Res ; 53(2): 242-7, 1993 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-8417816

RESUMO

The human ovarian cancer cell lines A2780 and A2780/CP70 were studied to investigate the cellular basis for their relative sensitivities to tetrachloro(DL-trans)-1,2-diamminecyclohexaneplatinum(IV) (ormaplatin). Cells were exposed to ormaplatin for 1 h in all experiments. As assessed by colony formation assays, the A2780/CP70 cell line [50% inhibitory dose (IC50) = 3.6 microM] was 9.5-fold more resistant to ormaplatin than the A2780 cell line [IC50 = 0.38 microM]. For cisplatin, the IC50 doses were 40 and 3 microM, respectively. Both cell lines were treated with ormaplatin at doses ranging from 0.10 to 40 microM, for the purpose of studying drug accumulation and efflux, and DNA adduct formation and repair. When these cell lines were treated at their respective IC50 doses, drug accumulation was greater in the resistant cells. When treated at equal microM doses, the sensitive cells formed 8-fold more DNA adduct than the resistant cells. When cells were treated with ormaplatin so as to achieve equivalent levels of platinum-DNA modification, sensitive cells removed 53% of the platinum-DNA damage in the first 6 h after drug exposure, compared to 68% in the resistant cells. We conclude that in human ovarian cancer cells made resistant to cisplatin, there is moderate cross-resistance to ormaplatin. This cross-resistance is not explained by differences in drug accumulation but is associated with reduced platinum-DNA adduct formation, which may be attributable in part to cytosolic inactivation of drug.


Assuntos
Compostos Organoplatínicos/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Cisplatino/metabolismo , Cisplatino/farmacologia , DNA/metabolismo , Resistência a Medicamentos , Feminino , Humanos , Técnicas In Vitro , Compostos Organoplatínicos/metabolismo , Neoplasias Ovarianas/metabolismo , Células Tumorais Cultivadas/metabolismo
18.
Mutat Res ; 293(2): 151-60, 1993 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-7678143

RESUMO

The patterns of expression of 3 human DNA-repair genes (ERCC1, ERCC2, ERCC6) were assessed in 52 bone-marrow specimens obtained from cancer patients prepared for autologous bone-marrow transplantation. Marrow was collected prior to the initiation of treatment in patients with sarcoma or testicular cancer; marrow was collected after initial cytoreductive therapy for patients with non-Hodgkin's lymphoma, Hodgkin's disease, and other tumors. Slot-blot analysis of marrow RNA showed a bimodal pattern of ERCC1, ERCC2 and ERCC6 gene expression with relative expression values ranging more than 200-fold. This pattern was seen in all patient groups and appeared to be independent of whether or not patients had received prior chemotherapy. In all patient groups, when expression was low for ERCC1, expression was also low for ERCC2 and ERCC6, suggesting that expression of these genes may be coordinated within an individual although they are located on two different chromosomes. Southern blot analyses of Pst I digests of DNA from 6 bone-marrow samples indicate no differences in ERCC1 gene copy number between high expressors and low expressors. There is absence of restriction fragment length polymorphism for ERCC1 suggesting that the different levels of expression in high and low expressors were not due to major deletions or rearrangements of the ERCC1 gene. We conclude that expression of these ERCC genes may vary widely between individuals, and that within an individual, their expression may be linked and coordinated by a common regulatory mechanism.


Assuntos
Medula Óssea/fisiologia , Reparo do DNA , Neoplasias/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Neoplásico/genética , Análise de Regressão
19.
J Natl Cancer Inst Monogr ; (15): 83-8, 1993.
Artigo em Inglês | MEDLINE | ID: mdl-7912534

RESUMO

Taxol and cisplatin are the two most effective agents discovered to date for treating advanced-stage cancer of the ovary. Learning how best to combine these agents is the focus of preclinical and clinical studies conducted at a number of institutions. Taxol's effect on cellular sensitivity to cisplatin was studied in paired cisplatin-sensitive A2780 and cisplatin-resistant A2780/CP70 human ovarian cancer cell lines. Cisplatin growth curves were generated under conditions of specific sequencing with Taxol, and IC50s (concentrations at which growth is inhibited to 50% of control) for cisplatin were obtained and compared. Taxol was used at an IC10 dose in all experiments. Taxol treatments were for 24 hours and cisplatin treatments were for 1 hour in all experiments. Dimethyl sulfoxide (DMSO) was the diluent for all Taxol stock solutions. Separately, the effects of Taxol and DMSO on cisplatin cellular accumulation were measured. End points reported include measures of cytotoxicity and Taxol effects on cisplatin cellular accumulation. Using a microculture tetrazolium assay, cisplatin growth curves were obtained under the influence of Taxol, at a Taxol dose of 3 nM for both cell lines. DMSO alone had no effect on tumor cell growth. In A2780 cells, the influence of Taxol on cisplatin cytotoxicity was modest, whereas cisplatin-induced cell kill was augmented 1.5-fold when cisplatin was given immediately after Taxol. In A2780/CP70 cells, Taxol augmented cisplatin-induced cell kill by 30-fold when cisplatin was given immediately after Taxol; 75-fold when cisplatin was given 24 hours after completion of Taxol; and 19-fold when cisplatin was given 48 hours after completion of Taxol.(ABSTRACT TRUNCATED AT 250 WORDS)


Assuntos
Cisplatino/farmacologia , Neoplasias Ovarianas/patologia , Paclitaxel/farmacologia , Cisplatino/farmacocinética , Dano ao DNA , Interações Medicamentosas , Feminino , Humanos , Neoplasias Ovarianas/metabolismo , Células Tumorais Cultivadas
20.
Environ Health Perspect ; 98: 53-9, 1992 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-1486863

RESUMO

We have extended our studies on the relationship between cisplatin/carboplatin-induced DNA damage in readily accessible tissue(s) and clinical response to therapy. Such an approach may assist in the study of cancer drug resistance and in establishing parameters for assessing human populations for sensitivity to DNA damaging agents in the environment. Platinum-DNA adduct levels were measured by atomic absorbance spectrometry. DNA repair capacity was assessed in human T-lymphocytes by the ability to repair cisplatin lesions in cellular DNA or in transfected plasmid DNA. In a "blinded" study of 21 patients receiving combination cisplatin/carboplatin drug therapy, there was a direct relationship between DNA damage in leukocytes and disease response (summary two-sided p = 0.00011). The cohort of patients had 15 different tumor types, suggesting that blood tissue and tumor tissue of an individual may process platinum-DNA damage similarly regardless of the tissue of origin of the tumor. In leukocytes in vivo, persistence and accumulation were prominent features of the cisplatin-DNA adduct profile. Functional DNA repair capacity has been studied in eight human leukocyte cell lines in vitro (three, T-cells; three, B-cells; one, monocytic; one, promyelocytic), using a host cell reactivation assay with cisplatin-damaged pRSVcat. In the three T cell lines studied, host cell reactivation efficiency was directly related to the cells' abilities to repair cisplatin-damaged cellular DNA (correlation coefficient = 0.993).(ABSTRACT TRUNCATED AT 250 WORDS)


Assuntos
Cisplatino/análise , Adutos de DNA , Dano ao DNA , Reparo do DNA , DNA de Neoplasias/efeitos dos fármacos , DNA/análise , Neoplasias/tratamento farmacológico , Linfócitos T/química , Carboplatina/administração & dosagem , Linhagem Celular , Cisplatino/administração & dosagem , Estudos de Coortes , Humanos , Neoplasias/sangue
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