RESUMEN
AIM: To investigate the expression of CD44 isoforms containing variant exon 6 (v6) in a well characterised cohort of patients with non-Hodgkin's lymphoma (NHL) and chronic lymphocytic leukaemia (CLL), and to correlate this with phenotype and disease course. METHODS: Cryostat sections of OCT embedded diagnostic nodal material from NHL patients and cryopreserved mononuclear preparations from CLL patients were used as sources of RNA. After reverse transcription, PCR was carried out with amplimers positioned at either side of the variant exon insertion site to amplify all possible CD44 isoforms. Those isoforms containing v6 were identified after Southern blotting and hybridisation with a radiolabelled oligonucleotide. RESULTS: Of 32 NHL samples analysed, 16 did not express CD44 isoforms containing v6, six expressed an isoform containing exon v6 alone, and 10 expressed v6 long isoforms which contained exon v6 in addition to other variant exons. These data did not correlate with lymphoma classification, disease staging, or the presence or absence of extranodal disease. However, those patients expressing v6 long CD44 isoforms had a worse overall survival than those that did not. The plateau of the survival curves was 50% compared with 82%. No v6 long isoforms were detected in the 21 CLL samples investigated. CONCLUSIONS: The expression of v6 long CD44 isoforms is associated with aggressive disease in NHL, independent of grade, stage, or presence of extranodal disease.
Asunto(s)
Empalme Alternativo , Antígenos de Neoplasias/genética , Receptores de Hialuranos/genética , Leucemia Linfocítica Crónica de Células B/genética , Linfoma no Hodgkin/genética , Antígenos de Neoplasias/metabolismo , Southern Blotting , Humanos , Receptores de Hialuranos/metabolismo , Leucemia Linfocítica Crónica de Células B/metabolismo , Linfoma no Hodgkin/metabolismo , Pronóstico , Isoformas de Proteínas/metabolismo , Estudios Retrospectivos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tasa de SupervivenciaRESUMEN
AIMS: To produce a method to measure and quantify enzymatically active topoisomerase II in normal and neoplastic human cells. METHODS: A crude cell lysate from density separated mononuclear cells from either peripherial blood or bone marrow was prepared as a source of topoisomerases. Using the lysate, minicircles from the Crithedia kinetoplast DNA complex were decatenated before being separated by agarose gel electrophoresis and visualised using ethidium bromide/ultraviolet fluorescence. RESULTS: Cell number, sample volume and drug inhibition concentration required to produce reliable and reproducible assay conditions were established. Intra- and interassay standards were included which permitted the quantification of active topoisomerase II in matched peripheral blood, bone marrow, presentation, and relapse samples from patients with acute lymphoblastic leukaemia. Active topoisomerase II has been converted to a unit scale which has been used to compare topoisomerase II activities in cells from patients with normal blood and bone marrow samples. CONCLUSIONS: There was no change in topoisomerase II activities between samples taken at presentation and those taken during a recurrence. However, topoisomerase II activity in leukaemic blast populations was increased compared with topoisomerase II activity in normal cells.
Asunto(s)
Bioquímica/métodos , ADN-Topoisomerasas de Tipo II/análisis , Leucemia-Linfoma Linfoblástico de Células Precursoras/enzimología , Adolescente , Adulto , Anciano , Animales , Blastómeros/enzimología , Crithidia fasciculata , ADN-Topoisomerasas de Tipo II/metabolismo , ADN de Cinetoplasto/metabolismo , Doxorrubicina/farmacología , Elipticinas/farmacología , Etilmaleimida/farmacología , Etopósido/farmacología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Novobiocina/farmacología , Recurrencia , Sensibilidad y Especificidad , Inhibidores de Topoisomerasa IIRESUMEN
In contrast to most other types of cancer, metastatic testicular germ cell tumours (TGCT) are cured in most patients using cisplatin-based combination chemotherapy. The biochemical mechanisms underlying this sensitivity have not been defined. Drug detoxification can modulate response to chemotherapy in vivo and in vitro, and therefore we measured levels of glutathione (GSH), glutathione-S-transferase (GST) and both constitutive and cisplatin- and dexamethasone-induced levels of metallothionein (MT) in five human testis tumour cell lines. The levels were compared with those in five human bladder cancer cell lines and two cell lines with cisplatin resistance acquired in vitro. GSH levels were relatively low in the testis tumour cell lines, as might be expected in drug-sensitive cells, and there was a 77-fold increase in GSH levels in the cisplatin-resistant testis tumour cell line. GST levels were similar in the two cell types, while metallothionein levels were relatively high in the testis tumour cell lines. These data indicate that GSH may contribute to the sensitivity of TGCT to chemotherapy, and that GSH expression may be involved in the acquisition of cisplatin resistance in these tumours.
Asunto(s)
Antineoplásicos/farmacología , Germinoma/metabolismo , Neoplasias Testiculares/metabolismo , Antineoplásicos/farmacocinética , Western Blotting , Cisplatino/farmacología , Dexametasona/farmacología , Resistencia a Antineoplásicos/fisiología , Germinoma/patología , Glutatión/metabolismo , Glutatión Transferasa/metabolismo , Humanos , Inactivación Metabólica , Masculino , Metalotioneína/metabolismo , Neoplasias Testiculares/patología , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
OCT embedded cryostat sections of stored pathological specimens of non-Hodgkin's lymphoma were used to provide RNA. After reverse transcription to produce cDNA, the polymerase chain reaction was performed with primers for standard and variant forms of the CD44 molecule. Using Southern transfer and hybridisation with a probe specific for exon 4 of the CD44 gene, both standard and variant forms were visualised by autoradiography. This method was shown to be applicable to other gene products by using primers specific for the abl and bcr genes. This technique permits retrospective analysis of RNA from small amounts of stored pathological samples.
Asunto(s)
Biomarcadores de Tumor/análisis , Criopreservación , Receptores de Hialuranos/análisis , Ganglios Linfáticos/química , Reacción en Cadena de la Polimerasa , ARN Neoplásico/análisis , Southern Blotting , Electroforesis , Humanos , Receptores de Hialuranos/genética , Linfoma no Hodgkin/inmunología , Estudios RetrospectivosRESUMEN
Haematological tumours in the CB17 scid mouse produce a disseminated blood-borne disease analogous to that seen in humans. The CB17 scid mouse model has been applied to study the efficacy of chemotherapeutic agents on tumours. Using three human tumour-cell lines of haemopoietic origin (CCRF-CEM, Raji, HS-Sultan), we established disseminated tumours in scid mice and studied the in vivo response of these tumours to four chemotherapeutic agents (daunorubicin, idarubicin, ifosfamide, etoposide). The in vitro drug-resistance profiles of the same cell lines to these drugs were also determined by the tetrazolium-dye (MTT) assay. Differences were found in the patterns of resistance and sensitivity of the cell lines in the in vivo and in vitro systems tested. Since the scid mouse model determines the in vivo response of both host and tumour to cytotoxic agents, it may be more valid than the other models in determining drug resistance of haematological malignancies.
Asunto(s)
Antineoplásicos/farmacología , Linfoma de Burkitt/tratamiento farmacológico , Modelos Animales de Enfermedad , Ensayos de Selección de Medicamentos Antitumorales/métodos , Leucemia Experimental/tratamiento farmacológico , Mieloma Múltiple/tratamiento farmacológico , Animales , Antineoplásicos/uso terapéutico , Colorantes , Daunorrubicina/uso terapéutico , Resistencia a Antineoplásicos , Etopósido/uso terapéutico , Femenino , Humanos , Idarrubicina/uso terapéutico , Masculino , Ratones , Ratones SCID , Sales de Tetrazolio , Pruebas de Toxicidad , Células Tumorales CultivadasRESUMEN
BACKGROUND: Glutathione S-transferases (GST) are known to play a role in the detoxification of carcinogens. Individual isoenzymes of the alpha-, mu-, and pi-class vary in substrate specificities, tissue distribution, and activities among individuals. GST-pi expression has been shown to be increased in preneoplastic and neoplastic lesions. GST-mu is known to play a role in detoxification of epoxides released from cigarette smoke, and individuals with low GST-mu activity have a relatively high risk to develop smoking-related lung and laryngeal cancer. The occurrence of a second primary tumor (SPT) in the whole respiratory and upper aerodigestive tract is an important factor for mortality in head and neck squamous cell carcinoma (HNSCC), and, at present, there are no markers that are available to predict which patient has increased chances of developing an SPT. Risk-assessment by use of biomarkers, particularly the ones that can be obtained with noninvasive techniques, are of great value in predicting prognosis and hence possibly more aggressive treatment and follow-up in selected patient groups. EXPERIMENTAL DESIGN: In a nested case control study, 20 patients who had previous history of oral cancer were used; 10 of the 20 had developed an SPT, and the other 10 patients were minimally 7 years free of disease. The expression of GST-pi, GST-mu, and GST-alpha was immunohistochemically analyzed using apparently normal oral mucosa, free of tumor or dysplasia, obtained from the resection edges around the primary tumor. In another experiment, the three GST isoenzymes were immunohistochemically analyzed using exfoliated cells, obtained noninvasively from several sites of the upper aerodigestive tract of the apparently normal-looking mucosa of HNSCC patients (n = 25) and of control individuals (n = 10). RESULTS: The expression of all GST was significantly higher (p < 0.001) in the suprabasal and superficial layers of the mucosa at risk. Also, in cell scrapes of clinically healthy mucosa of HNSCC patients, we observed a significantly higher expression (p < 0.001) of GST-pi and GST-mu compared with their matched controls. For GST-alpha, we observed a more heterogenous expression pattern in these exfoliated cells. CONCLUSIONS: Expression of GST-pi, -mu, and -alpha in normal tissue in the direct vicinity of the first tumor seems to have predictive value for the development of an SPT.
Asunto(s)
Carcinoma de Células Escamosas/diagnóstico , Glutatión Transferasa/análisis , Neoplasias de Cabeza y Cuello/diagnóstico , Mucosa Bucal/enzimología , Neoplasias Primarias Secundarias/diagnóstico , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/análisis , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/prevención & control , Estudios de Casos y Controles , Femenino , Neoplasias de Cabeza y Cuello/patología , Neoplasias de Cabeza y Cuello/prevención & control , Humanos , Inmunohistoquímica , Masculino , Tamizaje Masivo , Persona de Mediana Edad , Mucosa Bucal/citología , Neoplasias de la Boca/patología , Neoplasias Primarias Secundarias/patología , Neoplasias Primarias Secundarias/prevención & control , Valor Predictivo de las Pruebas , Factores de Riesgo , Fumar/efectos adversosRESUMEN
Expression of the main classes (pi, mu, and alpha) of glutathione S-transferase (GST) was assessed in the blasts of children presenting with acute lymphoblastic leukemia using an immunohistochemical technique. Bone marrow trephine biopsies obtained at presentation from 71 cases were studied (42 boys, 29 girls; age range, 6 months-14 years; median age, 4 years) and expression was correlated with event-free survival. The period of follow-up was 12-108 months, during which time 21 patients (30%) relapsed. All the samples examined were negative for alpha class GST. Samples from 8 patients, all of whom remained in remission at the time of analysis, were found to be negative for pi class GST at presentation. Samples from 44 (patients were negative for mu class GST (62%); of these, 36 patients (82%) remained in remission. In comparison, of the 27 patients who were positive for mu class GST, only 14 (52%) remained in remission. Analysis of event-free survival demonstrated that expression of mu class GST predicts a 3-fold increased risk of relapse (95% confidence interval, 1.25-7.26). This risk factor appears to be independent of other recognized prognostic factors.
Asunto(s)
Glutatión Transferasa/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidad , Adolescente , Niño , Preescolar , Supervivencia sin Enfermedad , Femenino , Humanos , Lactante , Masculino , Ploidias , Leucemia-Linfoma Linfoblástico de Células Precursoras/enzimología , Leucemia-Linfoma Linfoblástico de Células Precursoras/genéticaRESUMEN
Using the C.B.17 scid mouse strain, we have developed a model of disseminated leukaemia and myeloma using five human cell lines, CCRF-Cem, Molt-4, Raji, IM9 and HS-Sultan. Introduction of any of these cell lines by either an intravenous or an intraperitoneal route eventually kills the mouse due to leukaemia or myeloma cell load. Neoplastic cells can be found in the blood, liver and bone marrow. Intraperitoneal transfer produces a local solid tumour whereas intravenous transfer produces foci of neoplastic cells in the spine and brain. A single dose of melphalan is able to increase survival time from infection of a lethal dose of the T-cell leukaemia cell line, CCRF-Cem.
Asunto(s)
Leucemia Experimental/patología , Mieloma Múltiple/patología , Animales , Modelos Animales de Enfermedad , Femenino , Humanos , Inmunofenotipificación , Leucemia Experimental/tratamiento farmacológico , Masculino , Melfalán/uso terapéutico , Ratones , Ratones SCID , Mieloma Múltiple/tratamiento farmacológico , Trasplante de NeoplasiasRESUMEN
Antisera to each of the three main cytosolic forms of glutathione S-transferase (GST; alpha, mu, and pi) has been used to characterise GST expression by metastatic neuroblastoma in bone marrow trephine biopsies taken from 15 patients at presentation and from five of this group at relapse. There was no correlation between expression of extra-nuclear alpha or mu GST and outcome, and no consistent pattern at relapse. Seven of eight expressing nuclear pi GST at presentation died of resistant disease. Three of five cases with no detectable nuclear pi class GST remain alive and disease free. The results provide no encouragement for further investigation of alpha or mu GST in this disease but larger studies of uniformly treated patients may show whether nuclear pi GST expression at presentation indicates likely relapse.
Asunto(s)
Biomarcadores de Tumor/análisis , Enfermedades de la Médula Ósea/enzimología , Glutatión Transferasa/análisis , Neuroblastoma/enzimología , Neuroblastoma/secundario , Adolescente , Enfermedades de la Médula Ósea/mortalidad , Núcleo Celular/enzimología , Niño , Preescolar , Citosol/enzimología , Humanos , Lactante , Neuroblastoma/mortalidadRESUMEN
The glutathione S-transferases (GSTs) are a family of detoxification and metabolising enzymes, which have been linked with the susceptibility of tissues to environmental carcinogens and resistance of tumours to chemotherapy. Environmental carcinogens have been implicated in the pathogenesis of pancreatic carcinoma, which is also a tumour characterised by marked chemotherapeutic drug resistance. In this study 26 pancreatic adenocarcinoma and 12 normal pancreatic samples were examined immunohistochemically for expression of pi (acidic), alpha (basic), and mu (neutral) GST. Fourteen (54%) of the tumours expressed pi GST alone, two (8%) expressed both pi and alpha GST, and two (8%) showed immunoreactivity with alpha GST alone. In the normal pancreas the intralobular ducts and centroacinar cells expressed pi GST alone whereas the large ducts expressed both pi and alpha GST. The acinar cells showed immunoreactivity only with anti-alpha GST. Mu GST was not expressed by normal or malignant pancreas. Expression of pi GST by pancreatic carcinoma may be a marker of the malignant phenotype and be induced during neoplastic transformation. Alternatively it could possibly reflect cell of origin, suggesting that the tumour arises from the centroacinar cells or intralobular ducts, or both rather than the large ducts.
Asunto(s)
Adenocarcinoma/enzimología , Glutatión Transferasa/análisis , Páncreas/enzimología , Neoplasias Pancreáticas/enzimología , Adulto , Anciano , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana EdadRESUMEN
AIMS: To investigate the distribution of alpha and pi class glutathione S-transferases (GST) in normal fetal, neonatal, and adult liver; and to examine changes in GST expression in neonatal liver disease. METHODS: alpha and pi class GST were immunolocalised in sections of formalin fixed liver tissue obtained from human fetuses (n = 21), neonates (n = 8), young children (n = 9) and adults (n = 10), and from neonates with extrahepatic biliary atresia (n = 15) and neonatal hepatitis (n = 12). Monospecific rabbit polyclonal antibodies were used with a peroxidase-antiperoxidase method. RESULTS: Expression of pi GST was localised predominantly within biliary epithelial cells of developing and mature bile ducts of all sizes from 16 weeks' gestation until term and in neonatal and adult liver. Coexpression of pi and alpha GST was seen in hepatocytes of developing fetal liver between 16 and 34 weeks' gestation. Although pi GST was seen in occasional hepatocytes up to six months of life, this isoenzyme was not expressed by hepatocytes in adult liver. By contrast, alpha GST continued to be expressed by hepatocytes in adult liver; this isoenzyme was also seen in some epithelial cells of large bile ducts in adult liver. No change was observed in the distribution of alpha GST in either neonatal hepatitis or extrahepatic biliary atresia. However, aberrant expression of pi GST was identified in hepatocytes of all but one case of extrahepatic biliary atresia but in only two cases of neonatal hepatitis. CONCLUSIONS: The phenotypic alterations noted in extrahepatic biliary atresia may result from the effect of cholate stasis. Evaluation of the pattern of pi and alpha GST distribution by immunohistochemical staining may provide valuable information in distinguishing between these two forms of neonatal liver disease.
Asunto(s)
Atresia Biliar/enzimología , Glutatión Transferasa/análisis , Hepatitis/enzimología , Hígado/enzimología , Adulto , Factores de Edad , Conductos Biliares Intrahepáticos/enzimología , Preescolar , Feto , Edad Gestacional , Humanos , Lactante , Recién Nacido , Isoenzimas/análisisRESUMEN
We have isolated a multidrug-resistant derivative of Chinese hamster ovary CHO-K1 cells by exposure to progressively increasing concentrations of Adriamycin. This cell line, designated CHO-Adrr, was 27-fold more resistant than the parental line to Adriamycin and showed similar degrees of cross-resistance to several other topoisomerase II (topo II) inhibitors, including mitoxantrone, daunomycin and etoposide. CHO-Adrr cells showed a lower (4-fold) level of cross-resistance to vincristine and colchicine, drugs associated with the multidrug-resistant phenotype. While CHO-Adrr cells showed no enhanced resistance to several mono- and bi-functional alkylating agents or to UV and ionizing radiation, they were greater than 80-fold resistant to mitomycin C (MMC). There was a 5-fold decreased level of daunomycin accumulation in CHO-Adrr cells compared to CHO-K1 cells and this was associated with increased drug efflux. The resistant cells had amplified multidrug resistance gene (mdr) sequences and overexpressed (mdr) mRNA. Verapamil was able to completely reverse Adriamycin resistance but reversal of MMC resistance was only partial, with residual 23-fold resistance. CHO-Adrr cells expressed a 4-fold reduced level of topo II protein but overexpressed an alpha class (basic) glutathione S-transferase (GST). Analysis of cell hybrids showed that while the level of resistance to Adriamycin dropped by a factor of 3 in CHO-K1/CHO-Adrr hybrids compared to CHO-Adrr/CHO-Adrr hybrids, resistance to MMC dropped 10-fold. Thus, CHO-Adrr cells appear to exhibit simultaneously several different drug resistance mechanisms including MDR and GST overexpression, and topo II reduction.
Asunto(s)
ADN-Topoisomerasas de Tipo II/metabolismo , Glutatión Transferasa/metabolismo , Mitomicina/farmacología , Amsacrina/farmacología , Animales , Células CHO/efectos de los fármacos , Células CHO/enzimología , Ensayo de Unidades Formadoras de Colonias , Cricetinae , Daño del ADN , Daunorrubicina/farmacología , Doxorrubicina/farmacología , Resistencia a Medicamentos/genética , Expresión Génica/efectos de los fármacos , Humanos , Células Híbridas/efectos de los fármacos , Recién Nacido , Verapamilo/farmacologíaRESUMEN
Sixty-eight patients with advanced breast cancer were treated with mitoxantrone and clinical responses assessed. Expression of c-erbB-2 protein and cytosolic glutathione S-transferase (GST) isoenzymes pi, alpha and mu by the primary tumours of these patients was determined immunohistochemically, and correlated with treatment response. Tumours overexpressing c-erbB-2 (n = 16, 23%) showed a lower response rate (50% vs 58%) and shorter duration of response to treatment, compared with c-erbB-2 negative tumours. These associations were not statistically significant but survival following start of treatment was significantly shorter in the c-erbB-2 positive group. For each GST isoenzyme, the response rate and duration of response of the group showing enzyme expression did not differ significantly from those with negatively staining tumours. These data do not support a role for expression of GSTs alone in resistance to mitoxantrone monotherapy in advanced breast cancer. The poorer post treatment survival of patients with c-erbB-2 positive tumours suggests they could be selected for more intensive treatment regimens.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Glutatión Transferasa/metabolismo , Isoenzimas/metabolismo , Mitoxantrona/uso terapéutico , Proteínas Proto-Oncogénicas/metabolismo , Adulto , Anciano , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Femenino , Humanos , Persona de Mediana Edad , Estadificación de Neoplasias , Receptor ErbB-2 , Análisis de SupervivenciaRESUMEN
The expression of cytosolic glutathione S-transferase (GST) isoenzymes has been assessed in a series of 74 primary human breast carcinomas using an immunohistochemical method. GST pi was detected in sections from all 74 tumours; it was expressed by non-epithelial (stromal and inflammatory) cells in 62 tumours (84 per cent), but by tumour epithelium in only 35 (47 per cent). Non-neoplastic mammary epithelium was uniformly positive for GST pi. Expression of GST alpha and mu was observed in 19 and 42 per cent of the tumours, respectively, and was largely confined to the neoplastic component. Lack of staining of tumour epithelium for GST pi was significantly associated with poorer tumour differentiation (higher grade). There was no association between expression of any of the three isoenzymes and either menopausal status or expression of c-erbB-2 oncogene protein product. Immunohistochemistry is a useful method for the investigation of expression and cellular localization of GSTs within tumours; such data are needed to improve our understanding of the role of these enzymes in neoplasia and in resistance to cytotoxic drug therapy.
Asunto(s)
Neoplasias de la Mama/enzimología , Mama/enzimología , Glutatión Transferasa/análisis , Isoenzimas/análisis , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Epitelio/enzimología , Femenino , Humanos , Técnicas para Inmunoenzimas , Menopausia/fisiología , Persona de Mediana Edad , Proteínas Proto-Oncogénicas/análisis , Receptor ErbB-2RESUMEN
This study evaluates the relationship between bone marrow growth in a long-term bone marrow culture (LTBMC) system and speed of engraftment of the same marrow following autologous bone marrow transplantation (ABMT). Bone marrow from 21 patients transplanted with unmanipulated, non-cryopreserved autologous marrow was cultured. Samples from 21 normal donors were cultured to establish the normal supernatant cell count range. Supernatant counts from LTBMCs established from marrow taken from patients at the time of bone marrow harvest were compared with the time to neutrophil and platelet engraftment. Supernatant counts, particularly after 1 week in culture, showed close correlation with time to neutrophil and platelet engraftment following ABMT (r = 0.733, p less than 0.01; r = 0.735, p less than 0.01 respectively). Where supernatant cell counts were within the normal range rapid engraftment was predicted (neutrophils greater than 0.5 x 10(9)/l within 21 days, platelets greater than 50 x 10(9)/l within 28 days) and if supernatant counts were below this range, engraftment was predicted to be delayed. After 1 week in culture, the speed of neutrophil and platelet engraftment were correctly predicted in 19 and 18 cases respectively. Preliminary data suggest that LTBMC of marrow obtained 2-6 weeks before harvesting provides similar data, thus allowing the opportunity to intervene, for example with growth factors, in selected patients.
Asunto(s)
Células de la Médula Ósea , Trasplante de Médula Ósea/fisiología , Adolescente , Adulto , Plaquetas/citología , Plaquetas/fisiología , Médula Ósea/fisiología , Recuento de Células , División Celular/fisiología , Células Cultivadas , Femenino , Humanos , Masculino , Persona de Mediana Edad , Neutrófilos/citología , Neutrófilos/fisiología , Factores de Tiempo , Trasplante AutólogoRESUMEN
Although considerable advances have been made over the past 20 years in the treatment of leukaemia, many patients still die either of their disease, or of the attempts made to cure it. A major contribution to this unacceptable level of mortality is the presence of drug resistance in the residual leukaemic cells. Although many laboratory studies have been performed which have indicated possible cellular mechanisms for the development of resistance, comparatively little is known of the relevance of these processes to resistance as it occurs in patients. Information from such studies should provide a basis for the rational design of agents capable of reversing resistance, and thereby improving the chances of achieving sustainable remission in the majority of patients presenting with leukaemia.
Asunto(s)
Antineoplásicos/uso terapéutico , Resistencia a Medicamentos , Leucemia/tratamiento farmacológico , Antineoplásicos/farmacocinética , Ensayos de Selección de Medicamentos Antitumorales , HumanosRESUMEN
Ten paired samples of primary human colorectal carcinoma and adjacent non-neoplastic mucosa were analysed for total glutathione S-transferase (GST) activities as determined by 1-chloro-2,4-dinitrobenzene assays. These tissues were also investigated for the expression of acidic (pi), basic (alpha) and neutral (mu) GSTs using Western blotting procedures and immunohistochemical staining. For each of the paired samples examined the total GST activity was higher in tumour than in adjacent non-neoplastic mucosa. Western blotting, using an antibody against acidic GST also showed strong immunoreactivity in all the samples with more intense reactions in tumour compared to mucosa in nine out of the ten paired samples. Low levels of basic GST were also expressed in all samples of tumour and mucosa. Neutral GST was not detectable in two samples of tumour and corresponding mucosa, but low levels of expression were demonstrated in the remaining eight. Immunohistochemical staining for acidic GST showed a dark brown reaction in all tumour cells; in non-neoplastic mucosa there was positive immunoreactivity for epithelial cells situated deep within the crypts and a negative reaction for surface epithelial cells. Immunohistochemical staining for basic GST was negative except for one sample of tumour and two of mucosa. Neutral GST was expressed only in two samples of tumour and two samples of mucosa. We therefore conclude that there is enhanced expression of GSTs, acidic GST being the predominant form, in tumour compared to normal mucosa, in keeping with a role for GSTs in colonic carcinogenesis and acquired or innate drug resistance.
Asunto(s)
Neoplasias Colorrectales/enzimología , Glutatión Transferasa/análisis , Mucosa Intestinal/enzimología , Western Blotting , Humanos , InmunohistoquímicaRESUMEN
The glutathione S-transferases are a group of enzymes involved in the detoxification of a wide range of xenobiotics. Elevation of the level of activity of glutathione S-transferases within the cytosol has been associated with the development of resistance to a number of cytotoxic drugs, including some commonly used in the treatment of leukaemia. In this paper we describe the purification and characterization of an anionic (p class) form of the enzyme from the peripheral blood of patients with acute myeloid leukemia, chronic myeloid leukaemia, and acute lymphocytic leukaemia and the spleen of a patient with chronic lymphocytic leukaemia. We present evidence that the form of enzyme purified closely resembles pi class glutathione S-transferase purified from human placenta. Immunoblotting performed on cytosol from the leukaemic cells from a range of cases of leukaemia at presentation, or on treatment, demonstrated that this form of glutathione S-transferase was the predominant isoenzyme expressed in all cases studied. However, in the limited number of cases studied there was no correlation between the level of expression and response to chemotherapy, suggesting that increased expression of pi class GST is not the sole cause of resistance to bifunctional alkylating agent in human leukaemias.
Asunto(s)
Glutatión Transferasa/aislamiento & purificación , Leucemia/enzimología , Anciano , Resistencia a Medicamentos/fisiología , Electroforesis en Gel de Poliacrilamida , Femenino , Glutatión Transferasa/metabolismo , Humanos , Immunoblotting , Focalización Isoeléctrica , Leucemia/tratamiento farmacológico , Masculino , Persona de Mediana EdadRESUMEN
Using an indirect immunohistochemical technique on paraffin sections, employing a polyclonal antibody to the acidic (placental) form of glutathione-S-transferase (GST), we have evaluated cytoplasmic and nuclear staining in a series of 67 cervical biopsies including normal non neoplastic tissue, immature squamous metaplasia, all grades of cervical intraepithelial neoplasia (CIN) and invasive carcinomas of keratinising and non-keratinising types. No differences in cytoplasmic staining between the varied lesions studied were seen. However, there were marked differences in nuclear staining. While normal non-neoplastic stratified squamous epithelium showed weak staining of the lower one-third of the epithelium only, in immature squamous metaplasia and in all grades of CIN there was intense nuclear staining in all layers of the epithelium. Invasive carcinomas showed generally less intense nuclear staining than CIN lesions. Endocervical cell nuclei also showed intense nuclear staining. These findings indicate that GST is of limited use as a marker of transformation in the human cervix uteri.
Asunto(s)
Biomarcadores de Tumor/análisis , Transformación Celular Neoplásica , Cuello del Útero/enzimología , Glutatión Transferasa/análisis , Neoplasias del Cuello Uterino/enzimología , Carcinoma/enzimología , Femenino , Humanos , InmunohistoquímicaRESUMEN
A program has been developed to allow the control, the storage of method files, file text, peak and graphical data generated by a Pharmacia FPLC. The program allows for the remote control of a LCC 500 and the storage of up to 9,999,999 different method files. The program can either be used to start the FPLC and store text and peaks or also store monitor data from two monitors including percentage pump B, graphically. System requirements are an IBM PC or fully compatible microcomputer, mouse, CGA or EGA card and GEM Desktop software.