RESUMO
Protein kinase C (PKC) modulates growth, differentiation and apoptosis in a cell-specific fashion. Overexpression of PKC-alpha in MCF-7 breast cancer cells (MCF-7-PKC-alpha cell) leads to expression of a more transformed phenotype. The response of MCF-7 and MCF-7-PKC-alpha cells to phorbol esters (TPA) was examined. TPA-treated MCF-7 cells demonstrated a modest cytostatic response associated with a G1 arrest that was accompanied by Cip1 expression and retinoblastoma hypophosphorylation. While p53 was detected in MCF-7 cells, evidence for TPA-induced stimulation of p53 transcriptional activity was not evident. In contrast, TPA treatment induced death of MCF-7-PKC-alpha cells. Bryostatin 1, another PKC activator, exerted modest cytostatic effects on MCF-7 cells while producing a cytotoxic response at low doses in MCF-7-PKC-alpha cells that waned at higher concentrations. TPA-treated MCF-7-PKC-alpha cells accumulated in G2/M, did not express p53, displayed decreased Cip1 expression, and demonstrated a reduction in retinoblastoma hypophosphorylation. TPA-treated MCF-7-PKC-alpha cells expressed gadd-45 which occurred before the onset of apoptosis. Thus, alterations in the PKC pathway can modulate the decision of a breast cancer cell to undergo death or differentiation. In addition, these data show that PKC activation can induce expression of gadd45 in a p53-independent fashion.
Assuntos
Apoptose , Genes p53/fisiologia , Isoenzimas/análise , Proteína Quinase C/análise , Proteínas/fisiologia , Acetato de Tetradecanoilforbol/farmacologia , Neoplasias da Mama/patologia , Briostatinas , Ciclo Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/biossíntese , Relação Dose-Resposta a Droga , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Lactonas/farmacologia , Macrolídeos , Fosforilação , Proteína do Retinoblastoma/metabolismo , Células Tumorais Cultivadas , Proteínas GADD45RESUMO
Increased protein kinase C (PKC) activity in malignant breast tissue and positive correlations between PKC activity and expression of a more aggressive phenotype in breast cancer cell lines suggest a role for this signal transduction pathway in the pathogenesis and/or progression of breast cancer. To examine the role of PKC in the progression of breast cancer, human MCF-7 breast cancer cells were transfected with PKC-alpha, and a group of heterogenous cells stably overexpressing PKC-alpha were isolated (MCF-7-PKC-alpha). MCF-7-PKC-alpha cells expressed fivefold higher levels of PKC-alpha as compared to parental or vector-transfected MCF-7 cells. MCF-7-PKC-alpha cells also displayed a substantial increase in endogenous expression of PKC-beta and decreases in expression of the novel delta- and eta-PKC isoforms. MCF-7-PKC-alpha cells displayed an enhanced proliferative rate, anchorage-independent growth, dramatic morphologic alterations including loss of an epithelioid appearance, and increased tumorigenicity in nude mice. MCF-7-PKC-alpha cells exhibited a significant reduction in estrogen receptor expression and decreases in estrogen-dependent gene expression. These findings suggest that the PKC pathway may modulate progression of breast cancer to a more aggressive neoplastic process.
Assuntos
Neoplasias da Mama/enzimologia , Neoplasias da Mama/etiologia , Isoenzimas/biossíntese , Isoenzimas/genética , Proteína Quinase C/biossíntese , Proteína Quinase C/genética , Animais , Northern Blotting , Western Blotting , Neoplasias da Mama/patologia , Neoplasias da Mama/ultraestrutura , Adesão Celular , Ciclo Celular , Feminino , Humanos , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , Metástase Neoplásica , Fenótipo , Proteína Quinase C-alfa , Proteínas Recombinantes/biossíntese , TransfecçãoRESUMO
PU.1, a member of the ets transcription factor family, has been previously shown to be necessary for tetradecanoylphorbol-13 acetate (TPA)-induced U937 leukemic cell maturation. We examined the effects of TPA on PU.1 content and PU.1 DNA binding activity in U937 cells. Unstimulated cells expressed PU.1 mRNA transcripts and TPA did not increase these levels. However, TPA treatment induced phosphorylation of PU.1. Gel-shift analysis using a labeled PU.1 oligomer showed that TPA induced a unique PU.1 binding activity. This binding activity was phosphorylation-dependent, as indicated by the ability of phosphatase treatment to abolish its detection. The PU.1 binding activity was generated at TPA-13 concentrations stimulating growth arrest and was blocked by the PKC inhibitor GF109203X, which antagonized TPA-induced growth inhibition. Bryostatin 1, another protein kinase C activator, induced only a modest degree of U937 growth inhibition and antagonized TPA-stimulated growth arrest. Bryostatin 1 was unable to induce this TPA-generated PU.1 binding activity. High bryostatin 1 concentrations inhibited generation of this TPA-induced band shift. These data suggest that TPA-induced growth inhibition is associated with phosphorylation of PU.1 and generation of a unique PU.1 binding activity.