RESUMEN
The earliest genetic alteration in human astrocytoma progression is mutation of the p53 tumour suppressor gene, while one of the earliest phenotypic changes is the stimulation of neovascularization. Here, we tested the role of p53 in the angiogenic process by introducing a tetracycline-regulated wild type p53 gene into null glioblastoma cells. The parental cells expressed strong angiogenic activity while upon induction of wild type, but not mutant, p53 expression, the cells secreted a factor able to neutralize the angiogenicity of the factors produced by the parental cells as well as of basic fibroblast growth factor.
Asunto(s)
Neoplasias Encefálicas/patología , Regulación Neoplásica de la Expresión Génica , Glioblastoma/patología , Proteínas de Neoplasias/biosíntesis , Neovascularización Patológica , Biosíntesis de Proteínas , Proteína p53 Supresora de Tumor/fisiología , Inhibidores de la Angiogénesis , Animales , Movimiento Celular , Córnea/irrigación sanguínea , Progresión de la Enfermedad , Endotelio Vascular/patología , Femenino , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiología , Neovascularización Patológica/fisiopatología , Proteínas/farmacología , Ratas , Ratas Endogámicas F344 , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/biosíntesis , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Approximately 30% of human breast and ovarian cancers have amplification and/or overexpression of HER-2/neu gene which encodes a cell surface growth-factor receptor. Overexpression of this receptor, p185HER-2/neu, is associated with poor outcome and may predict clinical response to chemotherapy. Antibodies to HER-2/neu receptor have a cytostatic effect in suppressing growth of cells with overexpression of p185HER-2/neu. To elicit a cytocidal effect, therapy with antireceptor antibody was used in combination with the DNA-damaging drug, cisplatin, and this combined treatment produced a synergistic decrease in cell growth. In addition, antibody mediated an increased sensitivity to cisplatin in drug-resistant ovarian carcinoma cells containing multiple copies of HER-2/neu gene. To evaluate the mechanism for this synergy, unscheduled DNA synthesis was measured in cancer cells using incorporation of [3H]thymidine and autoradiography, and formation and repair of cisplatin-induced DNA adducts was also measured. Treatment with cisplatin led to a marked, dose-dependent increase in unscheduled DNA synthesis which was significantly reduced by combined treatment with antireceptor antibody in HER-2/neu-overexpressing cells. Therapy with antibody to HER-2/neu receptor also led to a 35-40% reduction in repair of cisplatin-DNA adducts after cisplatin exposure and, as a result, promoted drug-induced killing in target cells. This phenomenon which we term receptor-enhanced chemosensitivity may provide a rationale for more selective targeting and exploitation of overexpressed growth factor receptors in cancer cells, thus leading to new strategies for clinical intervention.
Asunto(s)
Anticuerpos Antineoplásicos/inmunología , Neoplasias de la Mama/patología , Cisplatino/farmacología , Aductos de ADN , Reparación del ADN , Receptores ErbB/inmunología , Neoplasias Ováricas/patología , Proteínas Proto-Oncogénicas/inmunología , Animales , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/inmunología , Neoplasias de la Mama/genética , Neoplasias de la Mama/inmunología , ADN , Reparación del ADN/efectos de los fármacos , ADN de Neoplasias/biosíntesis , ADN de Neoplasias/efectos de los fármacos , Resistencia a Medicamentos , Receptores ErbB/genética , Femenino , Expresión Génica , Humanos , Ratones , Ratones Desnudos , Neoplasias Ováricas/genética , Neoplasias Ováricas/inmunología , Proteínas Proto-Oncogénicas/genética , Receptor ErbB-2 , Células Tumorales CultivadasRESUMEN
The HER-2/neu proto-oncogene (also known as c-erb B-2) is homologous with, but distinct from, the epidermal growth factor receptor. Amplification of this gene in node-positive breast cancers has been shown to correlate with both earlier relapse and shorter overall survival. In node-negative breast cancer patients, the subgroup for which accurate prognostic data could make a significant contribution to treatment decisions, the prognostic utility of HER-2/neu amplification and/or overexpression has been controversial. The purpose of this report is to address the issues surrounding this controversy and to evaluate the prognostic utility of overexpression in a carefully followed group of patients using appropriately characterized reagents and methods. In this report we present data from a study of HER-2/neu expression designed specifically to test whether or not overexpression is associated with an increased risk of recurrence in node-negative breast cancers. From a cohort of 704 women with node-negative breast cancer who experienced recurrent disease (relapsed cases) 105 were matched with 105 women with no recurrence (disease-free controls) after the equivalent follow-up period. Immunohistochemistry was used to assess HER-2/neu expression in archival tissue blocks from both relapsed cases and their matched disease-free controls. Importantly, a series of molecularly characterized breast cancer specimens were used to confirm that the antibody used was of sufficient sensitivity and specificity to identify those cancers overexpressing the HER-2/neu protein in this formalin-fixed, paraffin-embedded tissue cohort. In addition, a quantitative approach was developed to more accurately assess the amount of HER-2/neu protein identified by immunostaining tumor tissue. This was done using a purified HER-2/neu protein synthesized in a bacterial expression vector and protein lysates derived from a series of cell lines, engineered to express a defined range of HER-2/neu oncoprotein levels. By using cells with defined expression levels as calibration material, computerized image analysis of immunohistochemical staining could be used to determine the amount of oncoprotein product in these cell lines as well as in human breast cancer specimens. Quantitation of the amount of HER-2/neu protein product determined by computerized image analysis of immunohistochemical assays correlated very closely with quantitative analysis of a series of molecularly characterized breast cancer cell lines and breast cancer tissue specimens.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Biomarcadores de Tumor/análisis , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/patología , Carcinoma Lobular/patología , Receptores ErbB/biosíntesis , Expresión Génica , Proteínas Proto-Oncogénicas/biosíntesis , Proto-Oncogenes , Western Blotting , Neoplasias de la Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Carcinoma Lobular/metabolismo , Línea Celular , Receptores ErbB/análisis , Femenino , Amplificación de Genes , Humanos , Inmunohistoquímica , Invasividad Neoplásica , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas/análisis , Receptor ErbB-2 , Recurrencia , Células Tumorales CultivadasRESUMEN
It has long been debated whether the accumulation of allelic losses in tumors involves the selection of cells which have stochastically lost chromosomal regions or whether there is, inherent to the neoplastic state, a process which predisposes to genetic instability. Changes in DNA methylation are commonly seen in human tumors and can alter chromosome structure. We now have examined specific types of primary neural tumors which allow us to determine relationships between abnormal DNA hypermethylation and allelic loss. In primary brain tumors which frequently lose chromosome 17p (30-50%) even in the earliest stages, we now show that 84% (21 of 25) exhibit hypermethylation at locus D17S5, on 17p. However, in primary neuroblastomas, a tumor type which does not lose chromosome 17p, no regional hypermethylation is observed. These data suggest that on chromosome 17p, regional D17S5 hypermethylation constitutes a molecular change which is associated with genetic instability.
Asunto(s)
Alelos , Astrocitoma/genética , Neoplasias Encefálicas/genética , Deleción Cromosómica , Cromosomas Humanos Par 17 , ADN de Neoplasias/metabolismo , Glioblastoma/genética , Neuroblastoma/genética , Astrocitoma/metabolismo , Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Humanos , Metilación , Neuroblastoma/metabolismoRESUMEN
Amplification of the HER-2 (c-erbB-2) gene and overexpression of the p185HER-2 gene product is found in approximately one-third of primary human breast and ovarian cancers and is associated with a poor clinical outcome of early relapse and death. The HER-2 gene encodes a cell-surface growth factor receptor with intrinsic tyrosine kinase activity. Wild-type human HER-2 has been shown to act as a potent oncogene when over-expressed in mouse fibroblasts. Recent data suggest that the mechanism by which HER-2 mediates transformation requires the interaction of the epidermal growth factor (EGF) receptor. To test whether overexpression of normal human HER-2 can transform cells independently of the EGF receptor, we have introduced multiple copies of HER-2 into the EGF receptor-negative cell line, NR6, and have performed assays for both transformation and tumorigenicity. Engineered NR6 cells that overexpress the HER-2 gene product display a highly transformed and tumorigenic phenotype as compared with control cells. Additionally, a monoclonal antibody to the extracellular domain of the HER-2 receptor is able to inhibit the proliferation of the overexpressing cells in vitro as well as tumor growth in vivo. This study provides clear evidence that HER-2-mediated transformation can be achieved independently of the EGF receptor.
Asunto(s)
Transformación Celular Neoplásica , Receptores ErbB/fisiología , Proteínas Proto-Oncogénicas/genética , Proto-Oncogenes , Expresión Génica , Humanos , Receptor ErbB-2 , Células Tumorales CultivadasRESUMEN
The mechanism by which HER2/neu overexpressing tumor cells resist NK, LAK, and LDCC cytotoxic lymphocytes was investigated. Resistance was not explained by a delay in kinetics of lysis, concurrent resistance to TNF, or a diminished expression of the transferrin receptor. HLA-class I expression, however, was markedly elevated compared to HER2 nonexpressing targets suggesting a reason for resistance. To test the role of class I, we selectively decreased expression by incubation of targets with beta-2 microglobulin anti-sense oligonucleotides. Anti-sense-treated HER2+ targets, displaying levels of class I comparable to HER2- targets, were still markedly resistant to cytotoxic effectors. Down-regulation of class I expression in HER2- carcinoma cells also had no effect on sensitivity to cytotoxicity by anti-sense treatment of Raji and U937 targets resulted in enhanced sensitivity to NK and LAK effectors but not to T cells mediating LDCC. These data indicate resistance to cytotoxicity in HER2-expressing targets cannot be solely explained by heightened expression of class I. The data also support the concept that class I expression regulates sensitivity to NK and LAK cells (but not LDCC effectors) in selected targets.
Asunto(s)
ADN sin Sentido/inmunología , Antígenos HLA/inmunología , Proteínas Oncogénicas Virales/análisis , Linfocitos T Citotóxicos/inmunología , Microglobulina beta-2/inmunología , Animales , Secuencia de Bases , Citotoxicidad Inmunológica , ADN sin Sentido/farmacología , Regulación de la Expresión Génica/inmunología , Antígenos HLA/efectos de los fármacos , Antígenos HLA/genética , Humanos , Datos de Secuencia Molecular , Oligonucleótidos/inmunología , Receptor ErbB-2 , Linfocitos T Citotóxicos/efectos de los fármacos , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/inmunología , Microglobulina beta-2/genéticaRESUMEN
Osmotic pumps containing Escherichia coli-derived recombinant human granulocyte colony-stimulating factor (rhG-CSF) were attached to indwelling jugular vein catheters and implanted subcutaneously into Golden Syrian hamsters. Within 3 days, peripheral granulocyte counts had increased greater than 10-fold with a concomitant 4-fold increase in total leukocytes. Microscopic examination of Wright-Giemsa-stained blood smears from rhG-CSF hamsters showed that only the neutrophil subpopulation of granulocytes had increased. No significant changes in lymphocyte or monocyte counts were observed during the course of continuous rhG-CSF treatment. After subcutaneous injection at rhG-CSF doses of up to 10 micrograms X kg-1 X day-1 only granulocyte counts were affected. However, at higher dose levels, a transient thrombocytopenia was noted. Erythrocyte had lymphocyte/monocyte counts remained unaffected by rhG-CSF over the entire dose range (0.3-300 micrograms X kg-1 X day-1) studied. Total leukocyte counts increased 3-fold within 12 hr after a single s.c. injection of rhG-CSF. This early effect was associated with an increase in the total number of colony-forming cells and the percent of active cycling cells in the marrow. A sustained elevation of peripheral leukocyte and marrow progenitor counts was observed following seven daily s.c. injections of rhG-CSF. The ability of rhG-CSF to increase the production and release of granulocytes from the marrow may underlie the beneficial effect it produced on the restoration of peripheral leukocyte counts in hamsters made leukopenic by treatment with 5-fluorouracil.
Asunto(s)
Granulocitos/citología , Hematopoyesis/efectos de los fármacos , Interleucina-3/farmacología , Proteínas Recombinantes/farmacología , Animales , Células de la Médula Ósea , Ensayo de Unidades Formadoras de Colonias , Cricetinae , Escherichia coli/genética , Granulocitos/efectos de los fármacos , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Cinética , Recuento de Leucocitos , Masculino , MesocricetusRESUMEN
Human or rodent bone marrow treated with recombinant human granulocyte colony-stimulating factor (rhG-CSF) in a CFU-GM assay yield predominantly granulocytic colonies. The specificity for granulocyte progenitors in vitro is also demonstrated in vivo by a five- to six-fold elevation in hamster peripheral blood neutrophils. Other cell types (monocytes, lymphocytes and eosinophils) remain stable. Analysis of mRNA from the bladder carcinoma cell line 5637 (1A6) shows the predominant species of mRNA codes for a mature protein of 174 amino acids. A small fraction of the mRNA can code for an alternative form of hG-CSF containing additional three amino acids between positions 35 and 36.
Asunto(s)
Factores Estimulantes de Colonias/genética , Granulocitos/fisiología , Animales , Línea Celular , Ensayo de Unidades Formadoras de Colonias , Cricetinae , Hematopoyesis/efectos de los fármacos , Humanos , Recuento de Leucocitos , Ratones , ARN Mensajero/genética , Proteínas Recombinantes/genética , Especificidad de la Especie , Neoplasias de la Vejiga Urinaria/genéticaRESUMEN
Experiments were conducted to isolate and characterize the gene and gene product of a human hematopoietic colony-stimulating factor with pluripotent biological activities. This factor has the ability to induce differentiation of a murine myelomonocytic leukemia cell line WEHI-3B(D+) and cells from patients with newly diagnosed acute nonlymphocytic leukemia (ANLL). A complementary DNA copy of the gene encoding a pluripotent human granulocyte colony-stimulating factor (hG-CSF) was cloned and expressed in Escherichia coli. The recombinant form of hG-CSF is capable of supporting neutrophil proliferation in a CFU-GM assay. In addition, recombinant hG-CSF can support early erythroid colonies and mixed colony formation. Competitive binding studies done with 125I-labeled hG-CSF and cell samples from two patients with newly diagnosed human leukemias as well as WEHI-3B(D+) cells showed that one of the human leukemias (ANLL, classified as M4) and the WEHI-3B(D+) cells have receptors for hG-CSF. Furthermore, the murine WEHI-3B(D+) cells and human leukemic cells classified as M2, M3, and M4 were induced by recombinant hG-CSF to undergo terminal differentiation to macrophages and granulocytes. The secreted form of the protein produced by the bladder carcinoma cell line 5637 was found to be O-glycosylated and to have a molecular weight of 19,600.