RESUMO
Dysfunction of the endoplasmic reticulum (ER) has been reported in a variety of human pathologies, including cancer. However, the contribution of the ER to the early stages of normal cell transformation is largely unknown. Using primary human melanocytes and biopsies of human naevi (moles), we show that the extent of ER stress induced by cellular oncogenes may define the mechanism of activation of premature senescence. Specifically, we found that oncogenic forms of HRAS (HRAS(G12V)) but not its downstream target BRAF (BRAF(V600E)), engaged a rapid cell-cycle arrest that was associated with massive vacuolization and expansion of the ER. However, neither p53, p16(INK4a) nor classical senescence markers--such as foci of heterochromatin or DNA damage--were able to account for the specific response of melanocytes to HRAS(G12V). Instead, HRAS(G12V)-driven senescence was mediated by the ER-associated unfolded protein response (UPR). The impact of HRAS on the UPR was selective, as it was poorly induced by activated NRAS (more frequently mutated in melanoma than HRAS). These results argue against premature senescence as a converging mechanism of response to activating oncogenes and support a direct role of the ER as a gatekeeper of tumour control.
Assuntos
Retículo Endoplasmático/metabolismo , Genes ras/genética , Melanoma/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Ciclo Celular , Proliferação de Células , Senescência Celular , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Dano ao DNA , Fibroblastos/metabolismo , Heterocromatina/genética , Heterocromatina/metabolismo , Humanos , Lactente , Melanócitos/patologia , Melanoma/metabolismo , Melanoma/patologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Transdução de SinaisRESUMO
Phosphorylation of the cell adhesion protein CEACAM1 increases insulin sensitivity and decreases insulin-dependent mitogenesis in vivo. Here we show that CEACAM1 is a substrate of the EGFR and that upon being phosphorylated, CEACAM1 reduces EGFR-mediated growth of transfected Cos-7 and MCF-7 cells in response to EGF. Using transgenic mice overexpressing a phosphorylation-defective CEACAM1 mutant in liver (L-SACC1), we show that the effect of CEACAM1 on EGF-dependent cell proliferation is mediated by its ability to bind to and sequester Shc, thus uncoupling EGFR signaling from the ras/MAPK pathway. In L-SACC1 mice, we also show that impaired CEACAM1 phosphorylation leads to ligand-independent increase of EGFR-mediated cell proliferation. This appears to be secondary to visceral obesity and the metabolic syndrome, with increased levels of output of free fatty acids and heparin-binding EGF-like growth factor from the adipose tissue of the mice. Thus, L-SACC1 mice provide a model for the mechanistic link between increased cell proliferation in states of impaired metabolism and visceral obesity.
Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação/metabolismo , Moléculas de Adesão Celular/metabolismo , Divisão Celular/fisiologia , Receptores ErbB/metabolismo , Insulina/metabolismo , Animais , Antígenos CD/genética , Antígenos de Diferenciação/genética , Células COS , Antígeno Carcinoembrionário , Moléculas de Adesão Celular/genética , Linhagem Celular Tumoral , Receptores ErbB/genética , Humanos , Fígado/citologia , Fígado/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Camundongos , Camundongos Transgênicos , Obesidade/metabolismo , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Gap junctional intercellular communication and expression of gap junction proteins (connexins) are decreased frequently in neoplastic cells including human ovarian carcinoma cells. In order to test the hypothesis that these changes contribute to the neoplastic phenotype of ovarian carcinoma cells, we transfected human ovarian carcinoma SKOV-3 cells with connexin43. Stable, connexin43-expressing transfectants were characterized for cell proliferation in vitro in normal, low-serum, and serum-free culture medium, for tumorigenicity in nude mice, and for sensitivity to adriamycin in vitro. Transfected clones expressed higher levels of connexin43 and gap junctional intercellular communication, reduced proliferation and greater dependence upon serum for growth in vitro, decreased tumor formation, increased sensitivity to adriamycin, and reduced expression of p-glycoprotein. These data suggest that gap junctional intercellular communication and/or connexin43 expression suppresses the neoplastic phenotype of ovarian carcinoma cells and their downregulation is involved in neoplastic transformation of ovarian epithelial cells. The increased sensitivity to adriamycin and elevated expression of p-glycoprotein by the transfected cells also suggest that gap junctional intercellular communication and connexin43 expression are involved in drug sensitivity and might be manipulated to enhance the clinical response.
Assuntos
Carcinoma/metabolismo , Conexina 43/biossíntese , Neoplasias Ovarianas/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Antineoplásicos/farmacologia , Carcinoma/tratamento farmacológico , Carcinoma/patologia , Comunicação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Clonais/efeitos dos fármacos , Células Clonais/metabolismo , Células Clonais/patologia , Conexina 43/genética , Conexina 43/farmacologia , Meios de Cultura Livres de Soro/farmacologia , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Corantes Fluorescentes , Junções Comunicantes/efeitos dos fármacos , Humanos , Camundongos , Camundongos Nus , Microinjeções , Transplante de Neoplasias , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Transfecção , Células Tumorais CultivadasRESUMO
The New Zealand obese (NZO) mouse strain shares with the related New Zealand black (NZB) strain a number of immunophenotypic traits. Among these is a high proportion of B-1 B lymphocytes, a subset associated with autoantibody production. Approximately 50% of NZO/HlLt males develop a chronic insulin-resistant type 2 diabetes syndrome associated with 2 unusual features: the presence of B lymphocyte-enriched peri-insular infiltrates and the development of anti-insulin receptor autoantibodies (AIRAs). To establish the potential pathogenic contributions of B lymphocytes and AIRAs in this model, a disrupted immunoglobulin heavy chain gene (Igh-6) congenic on the NZB/BlJ background was backcrossed 4 generations into the NZO/HlLt background and was then intercrossed to produce mice that initially segregated for wild-type versus the mutant Igh-6 allele and thus permitted comparison of syndrome development. A new flow cytometric assay (AIRA binding to transfected Chinese hamster ovary cells stably expressing mouse insulin receptor) showed IgM and IgG subclass AIRAs in serum from Igh-6 intact males, but not in Igh-6null male serum. However, the absence of B lymphocytes and antibodies distinguishing mutant from wild-type males failed to significantly affect diabetes-free survival. The Igh-6null males gained weight less rapidly than wild-type males, probably accounting for a retardation, but not prevention, of hyperglycemia. Thus, AIRA and the B-lymphocyte component of the peri-insulitis in chronic diabetics were not essential either to development of insulin resistance or to eventual pancreatic beta cell failure and loss. A new substrain, designated NZL, was generated by inbreeding Igh-6 wild-type segregants. Currently at the F10 generation, NZL mice exhibit the same juvenile-onset obesity as NZO/HlLt males, but develop type 2 diabetes at a higher frequency (> 80%). Also, unlike NZO/HlLt mice that are difficult to breed, the NZL/Lt strain breeds well and thus offers clear advantages to obesity/diabetes researchers.
Assuntos
Autoanticorpos/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/imunologia , Receptor de Insulina/imunologia , Animais , Linfócitos B/patologia , Células CHO , Cricetinae , Cricetulus , Diabetes Mellitus Tipo 2/patologia , Genes de Imunoglobulinas/genética , Masculino , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Obesidade/genética , Receptor de Insulina/metabolismo , Fatores de Tempo , TransfecçãoRESUMO
Inactivation of CEACAM1 in L-SACC1 mice by a dominant-negative transgene in liver impairs insulin clearance and increases serum free fatty acid (FFA) levels, resulting in insulin resistance. The contribution of elevated FFAs in the pathogenesis of insulin resistance is herein investigated. Treatment of L-SACC1 female mice with carnitine restored plasma FFA content. Concomitantly, it normalized insulin levels without directly regulating receptor-mediated insulin internalization and prevented glucose tolerance in these mice. Similarly, treatment with nicotinic acid, a lipolysis inhibitor, restored insulin-stimulated receptor uptake in L-SACC1 mice. Taken together, these data suggest that chronic elevation in plasma FFAs levels contributes to the regulation of insulin metabolism and action in L-SACC1 mice.