RESUMEN

Identifying sources of variability in the response to cancer chemotherapy requires knowledge of all variables, including concomitant medications, that can alter the metabolism and pharmacokinetics of chemotherapy. This study investigated the accuracy of the lists of concomitant medications in the charts of cancer patients. Information collated from a questionnaire, patient interview, and the patient's medical chart was used to obtain validated medication lists. Patients took an average of 4.8 prescription drugs, 1.6 nonprescription drugs, and 1.6 other remedies within the 3 days prior to chemotherapy. Of the concomitant drugs actually taken, the medical records did not report 24% of prescription drugs, 84% of nonprescription drugs, and 83% of other remedies. Electronic medical records (EMRs) were more complete than paper charts, but even these omitted >75% of nonprescription drugs and other remedies. Potential drug interactions were noted in this study. This study documents the extent and complexity of the concomitant drugs taken by patients undergoing chemotherapy and the deficiencies in recording this information in medical charts.

Asunto(s)

Antineoplásicos/farmacocinética , Antineoplásicos/uso terapéutico , Suplementos Dietéticos , Anamnesis/métodos , Neoplasias/tratamiento farmacológico , Medicamentos sin Prescripción , Medicamentos bajo Prescripción , Adulto , Anciano , Anciano de 80 o más Años , Interacciones Farmacológicas , Monitoreo de Drogas , Registros Electrónicos de Salud , Femenino , Humanos , Masculino , Registros Médicos , Persona de Mediana Edad , Neoplasias/complicaciones , Farmacología Clínica/métodos , Autoinforme , Caracteres Sexuales , Encuestas y Cuestionarios

RESUMEN

We have proposed that the nephrotoxicity of cisplatin, a widely used chemotherapy drug, is the result of the binding of cisplatin to glutathione and the subsequent metabolism of the cisplatin-glutathione complex via a gamma-glutamyl transpeptidase (GGT)-dependent pathway in the proximal tubules. To test the hypothesis that GGT activity is essential for the nephrotoxicity of cisplatin, the effects of cisplatin were examined in wild-type and GGT-deficient mice. Mice were treated with 15 mg cisplatin/kg. Five days after treatment, renal histopathology, blood urea nitrogen levels, serum creatinine, platinum excretion, and platinum accumulation in the kidney were examined. Half the mice were supplemented with N-acetylcysteine, which has been shown to correct low levels of tissue glutathione in GGT-deficient mice. The data show that cisplatin was nephrotoxic in wild-type mice but not in GGT-deficient mice. The wild-type mice, with and without N-acetylcysteine supplementation, had significantly elevated levels of blood urea nitrogen, serum creatinine, and renal tubular necrosis. There was no evidence of nephrotoxicity in the GGT-deficient mice regardless of N-acetyl cysteine supplementation. No differences in platinum excretion were seen comparing wild-type and GGT-deficient mice, nor was there any significant difference in renal platinum accumulation. These data suggest that renal cisplatin toxicity is dependent on GGT activity, and is not correlated with uptake. The results support our hypothesis that the nephrotoxicity of cisplatin is the result of the metabolism of the drug through a GGT-dependent pathway.

Asunto(s)

Antineoplásicos/envenenamiento , Cisplatino/envenenamiento , Riñón/efectos de los fármacos , gamma-Glutamiltransferasa/deficiencia , Acetilcisteína/farmacología , Animales , Nitrógeno de la Urea Sanguínea , Peso Corporal , Creatinina/sangre , Resistencia a Medicamentos , Femenino , Riñón/metabolismo , Riñón/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados/genética , Platino (Metal)/metabolismo , Platino (Metal)/orina , Valores de Referencia , gamma-Glutamiltransferasa/genética

RESUMEN

Previous studies have shown that the enzyme-glutamyl transpeptidase (GGT) is essential for the nephrotoxicity of cisplatin. This study was designed to determine whether GGT activity is necessary for the therapeutic effect of the drug. The relationship between GGT expression and clinical response to platinum-based chemotherapy was examined in 41 human germ cell tumours. Sections of formalin-fixed, paraffin-embedded tumours were immunohistochemically stained with an antibody directed against human GGT. There was no expression of GGT in any of the 17 seminomas or four dysgerminomas; whereas, 12/12 ovarian yolk sac tumours and 4/4 embryonal carcinomas of the testis were GGT-positive. In stage I tumours fewer tumour cells expressed GGT than in later stage tumours. In four germ cell tumours of mixed histology, the seminomatous and dysgerminoma areas were GGT-negative while the areas of the tumour with yolk sac or embryonal histology contained GGT-positive tumour cells. The patients with seminomas or dysgerminomas who were treated with cisplatin-based chemotherapy, all had a complete response despite the absence of GGT expression in these tumours. Fifteen of the 16 patients with yolk sac or embryonal carcinomas received cisplatin-based chemotherapy following surgery. Twelve had a complete response, while three failed to respond to platinum-based therapy. There was no correlation between the level of GGT-expression and response to therapy in this group. Three of the four patients with tumours of mixed histology were treated with cisplatin-based therapy, and had a complete response. Therefore, expression of GGT is not necessary for the therapeutic effect of cisplatin in germ cell tumours. The results from this study suggest that systemic inhibition of GGT would inhibit the nephrotoxic side-effect of cisplatin without interfering with its activity towards germ cell tumours.

Asunto(s)

Cisplatino/uso terapéutico , Germinoma/tratamiento farmacológico , Germinoma/enzimología , gamma-Glutamiltransferasa/biosíntesis , Adolescente , Adulto , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Niño , Cisplatino/administración & dosificación , Cisplatino/efectos adversos , Resistencia a Antineoplásicos , Tumor del Seno Endodérmico/tratamiento farmacológico , Tumor del Seno Endodérmico/enzimología , Tumor del Seno Endodérmico/patología , Tumor del Seno Endodérmico/cirugía , Femenino , Germinoma/patología , Germinoma/cirugía , Humanos , Inmunohistoquímica , Masculino , Estadificación de Neoplasias , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/enzimología , Neoplasias Ováricas/patología , Neoplasias Ováricas/cirugía , Adhesión en Parafina , Neoplasias Testiculares/tratamiento farmacológico , Neoplasias Testiculares/enzimología , Neoplasias Testiculares/patología , Neoplasias Testiculares/cirugía , gamma-Glutamiltransferasa/antagonistas & inhibidores

RESUMEN

We have shown previously that gamma-glutamyl transpeptidase (GGT) activity is essential for the nephrotoxicity of cisplatin. In this study we asked whether GGT activity was necessary for the antitumor activity of cisplatin. GGT was transfected into PC3 cells, a human prostate tumor cell line. Two independent GGT-positive cell lines were isolated and characterized. GGT cleaves extracellular glutathione providing the cells with access to additional cysteine. Expression of GGT had no effect on the growth rate of the cells in vitro where the culture medium contains high levels of cysteine. However, when the cells were injected into nude mice the GGT-positive tumors grew at more than twice the rate of the GGT-negative tumors. Weekly treatment with cisplatin was toxic to both GGT-positive and -negative tumors. The GGT-positive tumors were significantly more resistant to the toxicity of cisplatin than the GGT-negative tumors. Therefore, expression of GGT is required for the nephrotoxicity of cisplatin, but diminishes the tumor toxicity of the drug. These results indicate that the nephrotoxicity and the tumor toxicity of cisplatin are via two distinct pathways.

Asunto(s)

Antineoplásicos/farmacología , Cisplatino/farmacología , Cisteína/metabolismo , Resistencia a Antineoplásicos , Glutatión/metabolismo , gamma-Glutamiltransferasa/fisiología , Animales , Antineoplásicos/efectos adversos , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapéutico , Cisplatino/efectos adversos , Cisplatino/farmacocinética , Cisplatino/uso terapéutico , Medios de Cultivo , Humanos , Necrosis Tubular Aguda/inducido químicamente , Necrosis Tubular Aguda/enzimología , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/enzimología , Masculino , Ratones , Ratones Desnudos , Neoplasias de la Próstata/patología , Proteínas Recombinantes de Fusión/fisiología , Transfección , Células Tumorales Cultivadas/efectos de los fármacos , gamma-Glutamiltransferasa/deficiencia , gamma-Glutamiltransferasa/genética

RESUMEN

Elevated levels of gamma-glutamyl transpeptidase (GGT) activity and intracellular glutathione in tumor cells have been correlated with resistance to several classes of chemotherapeutic drugs. In this study, the first comprehensive analysis of GGT expression in human malignant neoplasms, 451 tumors were immunostained with an antibody directed against a c-terminus peptide of the human GGT protein. Analysis of the immunostaining revealed that GGT was expressed in 22 of 44 lung carcinomas and 16 of 22 ovarian surface epithelial carcinomas, although normal pulmonary and ovarian epithelium are GGT-negative. The tumor samples were obtained from patients before the start of therapy; therefore, GGT was not induced by radiation or chemotherapy. There was no GGT expression in mesotheliomas, Hodgkin's disease, non-Hodgkin's lymphomas, melanomas, basal cell carcinomas, and most soft tissue sarcomas, all of which are derived from GGT-negative cells. Carcinomas arising from some GGT-positive epithelium retained their GGT-positive phenotype. These included renal cell carcinomas, hepatocellular and cholangiocarcinomas, and carcinomas of the prostate and thyroid whereas both pancreatic adenocarcinomas and infiltrating carcinomas of the breast showed a wide range of GGT expression. Further studies are underway to determine whether expression of GGT plays a role in the inherent resistance of some tumors to alkylating agents and other classes of chemotherapeutic drugs.

Asunto(s)

Neoplasias/enzimología , gamma-Glutamiltransferasa/metabolismo , Femenino , Humanos , Inmunohistoquímica , Masculino

RESUMEN

OBJECTIVE: Gamma-glutamyl transpeptidase activity has been shown to be essential for the nephrotoxicity of cisplatin. The purpose of this study was to determine whether expression of gamma-glutamyl transpeptidase in ovarian carcinomas is necessary for the antitumor effect of cisplatin. STUDY DESIGN: Tumor tissue from 18 patients with stage III or IV ovarian serous papillary carcinoma or poorly differentiated adenocarcinoma was analyzed for expression of gamma-glutamyl transpeptidase by histochemical or immunohistochemical staining. Response to cisplatin-based combination chemotherapy was evaluated on the basis of clinical response, progression-free interval, and survival. RESULTS: Gamma-glutamyl transpeptidase expression in the tumors ranged from 0% to 100% of the tumor cells gamma-glutamyl transpeptidase positive. Patient survival ranged from 15 months to 9 years. Twelve of the 18 patients had a complete response to the initial course of cisplatin-based combination chemotherapy. There was no statistically significant correlation between either response or time to relapse and gamma-glutamyl transpeptidase expression. However, there was a correlation between high levels of gamma-glutamyl transpeptidase in the tumor and acute ototoxicity in patients treated with cisplatin. Expression of high levels of gamma-glutamyl transpeptidase in the tumor was also found to be associated with shorter patient survival, suggesting that gamma-glutamyl transpeptidase might have a role in resistance to drugs used in second- and third-line therapy. CONCLUSIONS: Expression of gamma-glutamyl transpeptidase in ovarian serous papillary or poorly differentiated adenocarcinomas is not necessary for the antitumor activity of cisplatin. A correlation was found between high levels of gamma-glutamyl transpeptidase in the tumor and both increased ototoxicity from cisplatin and decreased patient survival. These data suggest that administering an inhibitor of gamma-glutamyl transpeptidase activity to block the nephrotoxicity of cisplatin would not interfere with its therapeutic effect.

Asunto(s)

Adenocarcinoma/tratamiento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Cisplatino/efectos adversos , Cistadenocarcinoma Papilar/tratamiento farmacológico , Neoplasias Ováricas/tratamiento farmacológico , gamma-Glutamiltransferasa/fisiología , Adenocarcinoma/enzimología , Adulto , Anciano , Cistadenocarcinoma Papilar/enzimología , Femenino , Audición/efectos de los fármacos , Humanos , Riñón/efectos de los fármacos , Persona de Mediana Edad , Estadificación de Neoplasias , Neoplasias Ováricas/enzimología , gamma-Glutamiltransferasa/antagonistas & inhibidores

RESUMEN

In carcinogen-treated rats, gamma-glutamyl transpeptidase (GGT) is induced in preneoplastic liver lesions and liver tumors. However, in mice, GGT is rarely detected during hepatocarcinogenesis. Data in this study reveal that GGT is not induced in mouse hepatocytes when they are maintained in vitro under the same conditions that induce GGT activity in primary cultures of rat hepatocytes. GGT activity in rat hepatocytes increased 20-fold during the first 7 days in culture, but there was no induction of GGT in primary cultures of mouse hepatocytes. Comparison of intracellular glutathione levels in rat and mouse liver cells showed that the glutathione level was higher in the mouse liver cells than the rat. Blocking glutathione synthesis with buthionine sulfoximine reduced the intracellular glutathione concentration in mouse liver cells but did not trigger an induction of GGT. Analysis of the GGT mRNA in primary cultures of rat hepatocytes showed that only GGT mRNA(III) is induced. This is the same GGT mRNA species present in preneoplastic hepatic lesions and liver tumors in the rat (1-3). Therefore activation of promoter III in the GGT gene is responsible for induction of GGT in both hepatocytes in vitro and liver tumors in vivo. These data show that primary cultures of rat and mouse hepatocytes provide a model system with which to study interspecies differences in the regulation of this enzyme and to better understand the role of GGT in normal and neoplastic processes.

Asunto(s)

Neoplasias Hepáticas Experimentales/enzimología , Hígado/enzimología , gamma-Glutamiltransferasa/biosíntesis , Animales , Células Cultivadas , Inducción Enzimática , Femenino , Glutatión/metabolismo , Neoplasias Hepáticas Experimentales/etiología , Ratones , Ratones Endogámicos C57BL , Regiones Promotoras Genéticas/fisiología , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Ribonucleasas/metabolismo , Especificidad de la Especie , Factores de Tiempo , gamma-Glutamiltransferasa/metabolismo

RESUMEN

We examined the correlation between response to platinum-based chemotherapy and expression of glutathione S-transferase (GST), gamma-GGT (both by immunohistochemistry) and gamma-GCS (by in situ hybridization) in 51 patients with head and neck cancer, who received a total of 56 courses of chemotherapy. The overall response rate for the 56 chemotherapy treatment courses was 48%. The overall response rate (CR, PR) for patients with low GST scores was 88% (21 of 24), while among the patients with high GST scores, the overall response rate was 19% (6 of 32, P = 0.001). Patients with a low GST score were 4.7 times more likely to respond to chemotherapy than patients with high GST scores. GST scores corresponded to response in 84% of cases. Among 33 patients treated with chemotherapy for relapsed disease, the overall response rate for patients with low GST score was 70% (7 of 10), while among the patients with high GST scores, the overall response rate was 8.7% (2 of 23), P < 0.001). In contrast, both gamma-GCS and gamma-GGT showed a range of expression in these samples, but there was no significant correlation with treatment response. We conclude that GST expression correlates well with response to platinum based chemotherapy in head and neck cancer.

Asunto(s)

Antineoplásicos/uso terapéutico , Glutatión/metabolismo , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/metabolismo , Compuestos de Platino/uso terapéutico , Anciano , Femenino , Expresión Génica , Glutamato-Cisteína Ligasa/genética , Glutatión Transferasa/metabolismo , Neoplasias de Cabeza y Cuello/enzimología , Humanos , Inmunohistoquímica , Hibridación in Situ , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , gamma-Glutamiltransferasa/metabolismo

RESUMEN

gamma-Glutamyl transpeptidase (GGT) is found throughout the plant and animal kingdoms. It is a cell surface glycoprotein that cleaves gamma-glutamyl amide bonds. The most abundant physiologic substrates for the enzyme are glutathione and glutathione-conjugated compounds. GGT initiates the cleavage of extracellular glutathione into its constituent amino acids which can then be transported into the cell. It also catalyzes the initial step in the conversion of glutathione-conjugated compounds to mercapturic acids. GGT is expressed at high levels in many human tumors and in many carcinogen-induced tumors in animals. These observations have lead an increased focus on the role of the enzyme in the development and treatment of tumors. This chapter begins with an overview of the structure and function of GGT in normal tissues. A summary of its expression in neoplastic tissues and the ways in which GGT effects the response of tumors to chemotherapy follows. The chapter concludes with a discussion of strategies for using GGT to activate and target chemotherapy drugs to tumors as a means of improving treatment for common human malignancies.

Asunto(s)

Neoplasias/enzimología , Neoplasias/etiología , gamma-Glutamiltransferasa/genética , gamma-Glutamiltransferasa/fisiología , Animales , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapéutico , Biotransformación , Resistencia a Medicamentos , Expresión Génica , Humanos , Neoplasias/tratamiento farmacológico , Profármacos/farmacocinética , Profármacos/uso terapéutico , gamma-Glutamiltransferasa/química

RESUMEN

GGT 129, a polyclonal antibody directed against gamma-glutamyl transpeptidase (GGT), was used to study GGT expression in formalin-fixed paraffin-embedded tissues from normal breast, 24 benign lesions, 27 in situ carcinomas or atypical hyperplasias, and 79 infiltrating mammary carcinomas. Epithelium of the ducts and ductules in normal breast tissue showed immunoreactivity along the apical surface. There was a strong correlation (P < 0.01) between the histologic classification of the tissue and GGT expression. All of the benign breast lesions stained positive for GGT. Among in situ carcinomas and atypical hyperplasias, 5/27 (19%) were negative for GGT while 22/27 were immunopositive. Infiltrating carcinomas showed the greatest deviation from normal tissue with 23/79 (29%) negative for GGT. GGT expression in benign and malignant breast tissue was not correlated with the age of the patient, suggesting that menopausal status does not influence expression of GGT. Correlation of GGT immunoreactivity with tubule formation, nuclear pleomorphism, mitoses, grade, size of tumor, lymph node status, and ER/PR status was performed for 69 cases of infiltrating ductal adenocarcinoma. There were no statistically significant relationships between the level of GGT immunoreactivity and any of the parameters. The loss of GGT in some of the cases is evidence that this enzyme is not required for mammary tumor development or maintenance. However, as GGT is a component of the pathways that metabolize glutathione and glutathione-conjugates, the difference in levels of the enzyme in invasive breast cancers may be one explanation for the variation in chemotherapy response that has been observed in patients treated for advanced mammary cancer.

Asunto(s)

Enfermedades de la Mama/enzimología , Neoplasias de la Mama/enzimología , Mama/enzimología , Carcinoma Ductal de Mama/enzimología , gamma-Glutamiltransferasa/análisis , Neoplasias de la Mama/tratamiento farmacológico , Carcinoma Ductal de Mama/tratamiento farmacológico , Femenino , Humanos , Inmunohistoquímica , Persona de Mediana Edad

RESUMEN

gamma-Glutamyl transpeptidase (GGT) is a cytoplasmic membrane-bound enzyme important in the metabolism of glutathione and other gamma-glutamyl compounds. It is present in highest levels in the kidney and is also expressed prominently in reproductive organs such as the prostate gland. Because GGT has never been examined in prostatic carcinoma, we used a new polyclonal antibody (GGT 129) for immunohistochemical localization of GGT in normal prostate gland and 72 prostatic carcinomas. The normal lining secretory cells of ducts and acini showed apical immunoreactivity for GGT, but the basal epithelial cells were negative. Most of the prostatic adenocarcinomas had GGT staining patterns and intensity similar to those of normal prostatic secretory cells, because the majority of neoplastic cells showed immunopositivity in more than 80% of the adenocarcinomas. In comparing the percentage of cells staining for GGT with clinicopathologic parameters, there was no correlation between the number of positive cells and the Gleason score, the percentage of intraglandular carcinoma, capsule penetration, or seminal vesicle invasion. Immunostaining for GGT lacks value as a prognostic factor and does not correlate with standard clinicopathologic parameters. GGT may be important, however, for growth and maintenance of both normal and neoplastic prostatic cells. Modulating GGT levels or administering drugs that can be activated by GGT may have therapeutic value.

Asunto(s)

Adenocarcinoma/enzimología , Próstata/enzimología , Neoplasias de la Próstata/enzimología , gamma-Glutamiltransferasa/análisis , Adenocarcinoma/patología , Glutatión Transferasa/análisis , Humanos , Sueros Inmunes/inmunología , Inmunohistoquímica , Masculino , Próstata/patología , Neoplasias de la Próstata/patología

RESUMEN

We developed a new, highly specific antibody that localizes gamma-glutamyl transpeptidase (GGT) in formalin-fixed sections of human tissue. The specificity of the antibody, GGT129, is demonstrated by immunostained Western blots of whole cell homogenate from five different tissues. The utility of the antibody is shown by a comprehensive study of GGT expression in normal human tissue. This study reveals GGT expression for the first time in a number of tissues, including glands in the endocervix, endometrium, and adrenals. Strong immunoreactivity was observed on the surface of renal proximal tubule cells, hepatic bile canaliculi, and capillary endothelial cells within the nervous system. Secretory or absorptive cells in sweat glands, prostate, salivary gland ducts, bile ducts, pancreatic acini, intestinal crypts, and testicular tubules were also GGT-positive. Small bands of positively stained stromal cells and GGT-positive histiocytes were seen in some tissues. Analysis of human fetal tissue shows that the developmental expression of GGT differs in humans and rodents. These findings form the basis for further work on GGT induction in tumors and the effect of GGT expression on the response of tumors to chemotherapy.

Asunto(s)

gamma-Glutamiltransferasa/análisis , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos , Western Blotting , Epitelio/química , Femenino , Proteínas Fetales/análisis , Formaldehído , Humanos , Técnicas para Inmunoenzimas , Masculino , Datos de Secuencia Molecular , Proteínas de Neoplasias/análisis , Especificidad de Órganos , Adhesión en Parafina , Fragmentos de Péptidos/inmunología , Conejos , Fijación del Tejido , Vísceras/química , gamma-Glutamiltransferasa/inmunología

RESUMEN

Determination of breast cancer estrogen receptor (ER) status as a predictor of tumor response to adjuvant endocrine therapy remains a mainstay of breast cancer management. Recent second generation anti-ER antibodies and new epitope retrieval methods have produced paraffin-based immunohistochemical results that correlate closely with the dextran-coated charcoal (DCC) assay and appear to represent a superior method of ER assay. The authors determined the ER status of 103 invasive breast cancers by paraffin-based, automated immunohistochemistry on the Ventana ES using a new monoclonal antibody, CC4-5, and compared the results to those of parallel DCC biochemical analysis and manual immunohistochemical analysis using anti-ER monoclonal antibody ER1D5. The specificity of the CC4-5 antibody for ER protein was confirmed by Western blot analysis. Sixty of 103 cases were positive for ER by CC4-5 automated immunohistochemistry. With a ligand binding assay threshold value of 20 fmol/mg protein, there were 50 positive cases by biochemical assay. The biochemical results corresponded to an 88% rate of agreement with automated CC4-5 staining. Analysis of discordant cases revealed that the majority of CC4-5 immunopositive only cases (8 of 11) were strongly positive, stroma rich tumors, suggesting that corresponding biochemical measurements were diluted by non representative stromal tissue. There was only one immunonegative, biochemically positive case (27 fmol/mg protein). Semiquantitation of CC4-5 staining using percent positive tumor cells or weighted average staining intensity (HSCORE) showed moderate to good correlation with quantitative DCC results (r = 0.64 and 0.62, P < .0001). ER1D5 was not suitable for use on the Ventana ES, most likely due to temperature constraints of the instrument. By manual ER1D5 staining, 40 of 79 examined cases were positive corresponding to a 99% rate of agreement with automated CC4-5 staining. Semiquantitation of ER1D5 staining by percent positive tumor cells and weighted average staining intensity (HSCORE) showed excellent correlation with semiquantitation of automated CC4-5 results (r = 0.90 and 0.88, P < .0001). Automated immunohistochemistry using the Ventana ES and monoclonal antibody CC4-5 is a reliable method for determining breast cancer ER status. As with other immunohistochemical methods, direct correlation with morphology precludes errors due to tissue sampling, allowing for accurate analysis of stroma-rich or partially necrotic tumors and small neoplasms that otherwise would yield insufficient tumor tissue for biochemical analysis.

Asunto(s)

Anticuerpos Monoclonales , Neoplasias de la Mama/química , Inmunohistoquímica/métodos , Receptores de Estrógenos/análisis , Neoplasias de la Mama/patología , Femenino , Humanos , Reproducibilidad de los Resultados , Coloración y Etiquetado

RESUMEN

OBJECTIVE: Inhibition of gamma-glutamyl transpeptidase activity by acivicin or a large bolus of intravenous glutathione blocks the nephrotoxicity of cisplatin. The purpose of this study was to determine whether these compounds inhibit nephrotoxicity by reducing the amount of platinum retained by the kidney. STUDY DESIGN: The platinum concentration in urine and kidney of cisplatin-treated rats was determined by graphite furnace atomic absorption spectroscopy. Tissues from three experimental groups of rats were analyzed. The first group was treated with a nephrotoxic dose of cisplatin. The second group was treated with acivicin before cisplatin. The third group received a bolus of glutathione before cisplatin. Urine collected for 3 hours after the injection of cisplatin and kidney tissue from animals 5 days after treatment were analyzed for platinum content. RESULTS: Urine from animals pretreated with acivicin had the same concentration of platinum as that of control animals treated with cisplatin alone. Analysis of kidney tissue, blood urea nitrogen and serum creatinine 5 days after treatment showed that pretreatment with acivicin or glutathione blocked the nephrotoxicity of cisplatin. However, these agents did not alter the concentration of platinum in the kidney. CONCLUSIONS: The data in this study reveal that pretreatment with acivicin or glutathione does not block the uptake of platinum into the kidney nor do these agents reduce the concentration of platinum retained by the kidney. The mechanism by which these agents may inhibit the nephrotoxicity of cisplatin is discussed.

Asunto(s)

Cisplatino/toxicidad , Riñón/efectos de los fármacos , Riñón/metabolismo , Platino (Metal)/metabolismo , gamma-Glutamiltransferasa/antagonistas & inhibidores , Animales , Cisplatino/antagonistas & inhibidores , Glutatión/farmacología , Isoxazoles/farmacología , Masculino , Platino (Metal)/orina , Ratas , Ratas Sprague-Dawley , gamma-Glutamiltransferasa/metabolismo

RESUMEN

Cells from the GGT-negative mouse hepatoma cell line, Hepa 1-6, were transfected with a human GGT cDNA and stably transformed clones were isolated. In standard tissue culture medium the GGT-positive cells and GGT-negative controls grew equally well. However, when the cysteine concentration of the medium was reduced to physiologic levels the GGT-positive cells had a growth advantage. Further investigation revealed that the medium of the GGT-negative Hepa 1-6 cells contained glutathione that had been excreted by the cells, but no glutathione was present in the medium of the GGT-positive cells. We have previously shown that expression of GGT enables cells to use extracellular glutathione as a source of cysteine (Hanigan and Ricketts, Biochem., 32:6302, 1993). These new data reveal that physiologic levels of cysteine can be limiting for cell growth and expression of GGT can provide the cells with a selective growth advantage. These data explain the observation that cells transfected with GGT grow at the same rate as the GGT-negative controls in tissue culture medium which contains a high level of cysteine, but the GGT-positive cells grow more rapidly than the GGT-negative cells when transplanted into animals (Warren et al., Proc. Soc. Exp. Biol. Med., 202:9, 1993). GGT-positive tumor cells have a selective growth advantage in vivo in comparison to GGT-negative tumor cells because they are able to use serum glutathione as a secondary source of cysteine thereby overcoming the growth restriction imposed by serum levels of cysteine.

Asunto(s)

Cisteína/biosíntesis , Cisteína/farmacología , Neoplasias Hepáticas Experimentales/enzimología , gamma-Glutamiltransferasa/metabolismo , Animales , División Celular/fisiología , Medios de Cultivo , ADN Complementario/genética , Espacio Extracelular/metabolismo , Glutatión/metabolismo , Cinética , Neoplasias Hepáticas Experimentales/patología , Metionina/metabolismo , Ratones , Transfección , Células Tumorales Cultivadas/efectos de los fármacos , gamma-Glutamiltransferasa/genética

RESUMEN

Cisplatin [cis-dichlorodiammineplatinum(II)] is a widely used chemotherapeutic drug that is toxic to the proximal tubule cells of the kidney. gamma-Glutamyl transpeptidase (GGT) is localized to the luminal surface of the renal proximal tubules. GGT catalyzes the initial step in the metabolism of glutathione-conjugated drugs to mercapturic acids, some of which are severely nephrotoxic. We proposed that the nephrotoxicity of cisplatin was dependent on the cleavage of a cisplatin-glutathione conjugate by GGT. To test this hypothesis, renal GGT activity was blocked in male Sprague-Dawley rats by acivicin, a non-competitive inhibitor of GGT. Treatment with cisplatin alone caused extensive acute necrosis of the proximal tubules, but the proximal tubule cells appeared normal in rats treated with acivicin prior to cisplatin. Blood urea nitrogen and serum creatinine levels confirmed the protective effect of acivicin. Glutathione is a physiological substrate for GGT. Administration of an 83-fold excess of glutathione 30 min prior to cisplatin also inhibited cisplatin-induced nephrotoxicity. These data provide important new evidence that a large bolus of glutathione blocks the nephrotoxicity of cisplatin by competitively inhibiting GGT. These results indicate that cisplatin is conjugated to glutathione in vivo. The platinum-glutathione conjugate is nontoxic until metabolized by the proximal tubule cells. Formation of the nephrotoxic derivative of cisplatin requires GGT activity.

Asunto(s)

Cisplatino/antagonistas & inhibidores , Isoxazoles/farmacología , Riñón/efectos de los fármacos , gamma-Glutamiltransferasa/antagonistas & inhibidores , Animales , Nitrógeno de la Urea Sanguínea , Peso Corporal/efectos de los fármacos , Cisplatino/efectos adversos , Cisplatino/metabolismo , Creatinina/sangre , Ingestión de Alimentos , Glutatión/metabolismo , Riñón/enzimología , Riñón/patología , Túbulos Renales Proximales/efectos de los fármacos , Masculino , Ratas , Ratas Sprague-Dawley

RESUMEN

Gamma-glutamyl transpeptidase (GGT) is a cell surface enzyme that initiates the cleavage of extracellular glutathione, thereby providing the cell with the amino acids necessary for increased synthesis of glutathione. GGT is induced in ovarian tumor cell lines selected in vitro for resistance to cisplatin. No study has examined GGT expression in primary human ovarian tumors. We analyzed frozen sections of 80 normal human ovaries and 56 ovarian tumors for expression of GGT. Histochemical staining showed that GGT was not expressed in the cells of the follicle or surface germinal epithelium of the normal ovary. GGT was expressed in some epithelial inclusion glands and occasionally in a small subset of stromal cells. Granulosa-stromal cell tumors were largely GGT-negative. In contrast, GGT-positive neoplastic cells were observed in 33 of 45 common epithelial ovarian tumors. None of the patients had been treated with chemotherapy. Some of the tumors had only rare GGT-positive cells, while others consisted almost entirely of GGT-positive cells. Among the low malignant potential and invasive tumors, at least one-half of the cells were GGT-positive in 6 of 9 serous borderline tumors (2 with mucinous foci), 0 of 1 borderline mucinous tumor, 3 of 12 serous papillary carcinomas, 2 of 3 mucinous carcinomas, 1 of 2 endometrioid carcinomas, 2 of 2 clear cell carcinomas, 0 of 2 transitional cell carcinomas, and 4 of 5 undifferentiated carcinomas. There was no correlation between the stage of the tumor and GGT expression, indicating that a GGT-negative tumor does not become GGT-positive as it progresses to a more widely disseminated lesion. In addition, there was no correlation between serum levels of CA 125 and GGT expression. These data show that GGT is expressed in many common ovarian epithelial neoplasms. We are currently following the response of these patients to chemotherapy to determine if expression of GGT serves as a marker for identifying neoplasms with enhanced resistance to platinum-based therapy.

Asunto(s)

Neoplasias Ováricas/enzimología , Ovario/enzimología , gamma-Glutamiltransferasa/análisis , Adenocarcinoma Mucinoso/enzimología , Adenocarcinoma Mucinoso/patología , Adolescente , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Carcinoma Endometrioide/enzimología , Carcinoma Endometrioide/patología , Cistadenocarcinoma Seroso/enzimología , Cistadenocarcinoma Seroso/patología , Femenino , Humanos , Persona de Mediana Edad , Neoplasias Ováricas/patología

Asunto(s)

Sangre , gamma-Glutamiltransferasa/sangre , Animales , Bovinos , Medios de Cultivo , gamma-Glutamiltransferasa/metabolismo

RESUMEN

We show that gamma-glutamyl transpeptidase (GGT) is a glutathionase that enables cells to use extracellular glutathione as a source of cysteine. We transfected NIH/3T3 mouse fibroblasts with a plasmid containing cDNA for human GGT, and obtained stably transformed cell lines that expressed GGT in its proper orientation on the outer surface of the cell. NIH/3T3 fibroblasts require cysteine for growth and are unable to use extracellular glutathione as a source of cysteine. We demonstrate GGT-positive fibroblasts are able to grow in cysteine-free medium supplemented with glutathione. Cysteine derived from the cleavage of extracellular glutathione can be used to maintain intracellular levels of glutathione. GGT-positive NIH/3T3 cells were able to replenish intracellular glutathione when incubated in cysteine-free medium containing glutathione. GGT-negative cells could not. Therefore, GGT is a glutathionase that provides the cell with access to a secondary source of cysteine.

Asunto(s)

Cisteína/metabolismo , Glutatión/metabolismo , gamma-Glutamiltransferasa/biosíntesis , Células 3T3 , Animales , Medios de Cultivo , ADN , Humanos , Riñón/enzimología , Ratones , Ratas , gamma-Glutamiltransferasa/genética

RESUMEN

Although expression of the enzyme gamma-glutamyl transpeptidase (GGT) is the most common phenotypic marker of preneoplastic foci in the livers of carcinogen-treated rats, it is not generally expressed in mouse liver tumors or hepatic foci. However, several carcinogens, including safrole and ortho-azoaminotoluene (OAT), have been reported to induce GGT-positive foci in mice. We asked whether safrole and OAT induce GGT expression in preneoplastic foci or if these compounds select for a distinct set of lesions that can be identified by their GGT-positive phenotype. We treated 12-day-old male and female C57BL/6J mice with N,N-diethylnitrosamine (DEN) (0.20 mumol/g body wt) to initiate hepatocarcinogenesis. From 6 to 24 weeks of age, during the promotion phase of hepatocarcinogenesis, groups of mice were treated with 3,4,5,3',4',5'-hexabromobiphenyl (HBB), safrole or OAT. Additional groups of female mice were ovariectomized at 6 weeks of age with or without subsequent chronic treatment with testosterone. All the animals were killed at 24 weeks of age and serial liver sections were stained for glucose-6-phosphatase (G6Pase) or GGT. Both testosterone and HBB were strong promoters of the development of G6Pase-deficient foci. No GGT-positive foci were observed in animals treated with these agents or with DEN alone. In mice fed safrole or OAT during the promotion period, female mice developed more G6Pase-deficient foci than male mice, and GGT-positive foci were observed. Analysis of serial sections revealed that the G6Pase-deficient foci and the GGT-positive foci were independent populations. The relative number of these two classes of foci varied according to the treatment regimen. In females fed safrole, 7% of the foci in the liver were GGT-positive while in female mice fed OAT, 45% were GGT-positive. In all groups of mice in which we observed GGT-positive foci and in ovariectomized female mice, we noted a third independent population of foci which demonstrated significantly increased expression of G6Pase relative to surrounding normal liver. These data indicate that different treatments during the promotion stage of hepatocarcinogenesis in the mouse may give rise to distinct populations of preneoplastic lesions. Further studies of the molecular events giving rise to these distinct lesions will provide insights into the multiple pathways that result in hepatocarcinogenesis.

Asunto(s)

Carcinógenos/toxicidad , Neoplasias Hepáticas/inducido químicamente , Hígado/patología , Lesiones Precancerosas/inducido químicamente , Animales , Dietilnitrosamina/toxicidad , Femenino , Hígado/efectos de los fármacos , Hígado/enzimología , Neoplasias Hepáticas/enzimología , Neoplasias Hepáticas/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ovariectomía , Bifenilos Polibrominados/toxicidad , Lesiones Precancerosas/enzimología , Lesiones Precancerosas/patología , Safrol/toxicidad , Factores Sexuales , Testosterona/farmacología , gamma-Glutamiltransferasa/análisis , gamma-Glutamiltransferasa/metabolismo , o-Aminoazotolueno/toxicidad