RESUMEN
The early genes E6 and E7 from human papillomaviruses (HPVs) play a key role in the development of cervical cancer. Modulation of E6 and E7 gene expression may alter tumour progression; therefore, modifiers of viral transcription such as hormones or growth factors are potential risk factors in cancer development. We have analysed the effects of epidermal growth factor (EGF) on E6/E7 mRNA from human papillomavirus type 16 (HPV-16) by Northern blot in two cell lines, SiHa cervical carcinoma cells, and HPK IA, an HPV-16-immortalized keratinocyte cell line. E6/E7 mRNA is EGF-inducible in SiHa cells, with the earliest response after 2 h. In contrast, in HPK IA cells no increase in E6/E7 RNA is observed, suggesting a differential EGF response of viral transcription in tumour cells compared with keratinocytes. We demonstrate that the cell type-specific HPV-16 enhancer is a target of EGF-induced signals, as its activity is amplified by EGF in SiHa cell transfections. However, when transfected into HPK IA keratinocytes, the viral enhancer shows no EGF response. The enhancer contains two binding sites for the transcription factor AP-1, a potential mediator of the EGF signalling cascade. Enhancer subfragments with single AP-1 binding sites are also EGF-responsive in SiHa cells. Mutating either AP-1 site in the complete enhancer decreases the EGF response, whereas a double mutation causes a complete loss of EGF regulation, suggesting that the EGF induction of HPV-16 early transcription requires AP-1 activation. We conclude that alterations of EGF responsiveness that increase viral oncogene expression may contribute to cervical cancer progression.
Asunto(s)
Elementos de Facilitación Genéticos/genética , Factor de Crecimiento Epidérmico/farmacología , Proteínas Oncogénicas Virales/genética , ARN Mensajero/biosíntesis , Proteínas Represoras , Factor de Transcripción AP-1/metabolismo , Secuencia de Bases , Línea Celular Transformada , ADN de Neoplasias/metabolismo , Progresión de la Enfermedad , Elementos de Facilitación Genéticos/efectos de los fármacos , Femenino , Regulación Viral de la Expresión Génica , Humanos , Queratinocitos , Datos de Secuencia Molecular , Mutación , Papillomaviridae/genética , Proteínas E7 de Papillomavirus , ARN Viral/biosíntesis , Transducción de Señal/genética , Transcripción Genética/efectos de los fármacos , Transcripción Genética/genética , Transfección , Células Tumorales Cultivadas , Neoplasias del Cuello UterinoRESUMEN
A series of murine monoclonal antibodies were raised against purified human alpha v beta 3 integrin and against M21 human melanoma cells. Five notable hybridomas were identified by ELISA on purified integrins, and the isolated antibodies bound the alpha v-chain. These antibodies, 17E6, 20A9, 23G5, 14D9.F8 and 10G2, recognised the extracellular domains of the integrin, and were shown to be reactive in FACS, immunoprecipitation, ELISA, and ELISA on fixed cells with M21, M21-L4, and UCLA-P3, but not with the alpha v-deficient M21-L or M21-L-IIb (M21-L transfected with GpIIb integrin). One antibody, 17E6, strongly perturbed cell attachment mediated by alpha v integrins, reacting at least with alpha v beta 3, alpha v beta 5, and alpha v beta 1, and strongly inhibiting cell attachment to alpha v-ligands vitronectin and fibronectin with an IC50 of approximately 0.1 microgram ml-1. Furthermore, 17E6 at this concentration could induce cell retraction from the substrate, while LM609 (anti-alpha v beta 3) and control antibody 14E2 (anti-200 kDa melanoma surface protein) at 1,000-fold higher concentrations had minimal effects on cell morphology. The action of 17E6 was reversible and was not due to toxic effects: in vitro 17E6 at 0.1 mg ml-1 did not affect either cell proliferation or DNA synthesis. In two nude-mouse tumour models, subcutaneous tumour development and a lung colonisation ('experimental metastasis') assay, injection of 17E6 strongly inhibited tumour development, while isotype-matched controls had no effect. There was no obvious mechanism of cell or of complement-mediated tumour cytotoxicity; the antibody did not mediate ADCC or AECDC, or complement fixation. The data strongly support previous studies which have indicated the importance of alpha v-integrins, and especially alpha v beta 3, in the tumour progression of human melanoma.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Melanoma/patología , Receptores de Vitronectina/fisiología , Animales , Citotoxicidad Celular Dependiente de Anticuerpos , Complejo Antígeno-Anticuerpo , Línea Celular , Replicación del ADN/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Humanos , Inmunoglobulina G/farmacología , Cadenas kappa de Inmunoglobulina/farmacología , Melanoma/inmunología , Ratones , Ratones Endogámicos BALB C/inmunología , Ratones Desnudos , Pruebas de Precipitina , Receptores de Vitronectina/antagonistas & inhibidores , Receptores de Vitronectina/inmunología , Trasplante Heterólogo , Células Tumorales CultivadasAsunto(s)
Células de la Médula Ósea , Médula Ósea/inmunología , Células Dendríticas/citología , Células Dendríticas/inmunología , Animales , Anticuerpos Monoclonales , Antígenos CD/metabolismo , Antígeno B7-1/metabolismo , Antígeno B7-2 , Femenino , Citometría de Flujo , Inmunofenotipificación , Técnicas In Vitro , Activación de Linfocitos , Prueba de Cultivo Mixto de Linfocitos , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Fenotipo , Linfocitos T/inmunologíaRESUMEN
Low doses of cyclophosphamide (CPA) modulate immune responses and induce complete tumor regression and cures in mice. The mechanism of action is related to the development of a T-cell-dependent immune reaction. We started a trial with mafosfamide (MAF), the active metabolite of CPA and found that the response of Ehrlich ascites-tumor (EAT) cells in vivo to this compound is biphasic. The highest cure rate (70%) is obtained with a low i.p. dose of 7 mg/kg/d. Hematologic side effects were not observed. Arguments for an immune mechanism involved are that (1) mice treated with MAF showed a statistically significant increase in spleen weight compared with untreated controls, (2) after treatment large numbers of mononuclear cells appeared in the ascites, and (3) long-term surviving mice were resistant to further challenge with large inocula of EAT. We started a pilot trial in patients with metastasizing and advanced renal cell carcinoma - a disease which can be considered to be influenced by the immunologic response of the tumor host. The starting dose of MAF was 24 mg/m2/d administered i.v. Therapy was repeated at 14 days interval on an out-patient basis. Monitoring of mononuclear cells in the peripheral blood with monoclonal antibodies using a FACS IV were performed two times a week. There are eleven patients on study. Up to now, four out of them have been fully evaluated with respect to toxicity, immune modulation and tumor response. With respect to response to treatment, 2 patients had no change of disease, 1 patient had a mixed and 1 patient a partial response. No hematological or other toxicities could be observed. All patients showed an increase in monocytes/macrophages as well as NK-cells--clearly related to therapy.