RESUMO
Recent studies suggest that physiological and tumorigenic proliferation of mammalian cells is controlled by multiple cyclin-dependent kinases (CDKs) largely in tissue-specific manners. We and others previously demonstrated that adult mice deficient for the Cyclin D partner CDK4 (Cdk4(-/-) mice) exhibit hypoplasia in the pituitary and pancreatic islet due to primary postnatal defects in proliferation. Intriguingly, those neuroendocrine tissues affected in Cdk4(-/-) mice are the primary targets of tumorigenesis in the syndrome of multiple endocrine neoplasia type-1 (MEN1). Mice with heterozygous disruption of the tumor suppressor Men1 gene (Men1(+/-)) develop tumors in the pituitary, pancreatic islets and other neuroendocrine tissues, which is analogous to humans with MEN1 mutations. To explore the genetic interactions between loss of Men1 and activation of CDKs, we examined the impact of Cdk4 or Cdk2 disruption on tumorigenesis in Men1(+/-) mice. A majority of Men1(+/-) mice with wild-type CDKs developed pituitary and islet tumors by 15 months of age. Strikingly, Men1(+/-); Cdk4(-/-) mice did not develop any tumors, and their islets and pituitaries remained hypoplastic with decreased proliferation. In contrast, Men1(+/-); Cdk2(-/-) mice showed pituitary and islet tumorigenesis comparable to those in Men1(+/-) mice. Pituitaries of Men1(+/-); Cdk4(-/-) mice showed no signs of loss of heterozygosity (LOH) in the Men1 locus, whereas tumors in Men1(+/-) mice and Men1(+/-); Cdk2(-/-) mice exhibited LOH. Consistently, CDK4 knockdown in INS-1 insulinoma cells inhibited glucose-stimulated cell cycle progression with a significant decrease in phosphorylation of retinoblastoma protein (RB) at specific sites including Ser780. CDK2 knockdown had minimum effects on RB phosphorylation and cell cycle progression. These data suggest that CDK4 is a critical downstream target of MEN1-dependent tumor suppression and is required for tumorigenic proliferation in the pituitary and pancreatic islet, whereas CDK2 is dispensable for tumorigenesis in these neuroendocrine cell types.
Assuntos
Transformação Celular Neoplásica/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Quinase 4 Dependente de Ciclina/metabolismo , Insulinoma/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Hipofisárias/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/metabolismo , Animais , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Quinase 2 Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/genética , Feminino , Humanos , Insulinoma/genética , Insulinoma/patologia , Masculino , Camundongos , Camundongos Knockout , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Fosforilação/genética , Neoplasias Hipofisárias/genética , Neoplasias Hipofisárias/patologia , Proteínas Proto-Oncogênicas/genética , Ratos , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Fatores de Transcrição/genéticaRESUMO
A diacetyldapsone-2-hydroxypropyl-beta-cyclodextrin complex (DADDS-CD) was evaluated with regard to the ability of cultured lung cells to activate DADDS to the active metabolite dapsone. The same system was used to assess the effect of the complex on cell growth. The complex was incubated with cells for periods of 24 to 72 h, followed by extraction of metabolites from the incubation medium and analysis by HPLC. In addition, the Trypan Blue exclusion technique was used to assess cell viability during this time period. Results indicated that lung cells could activate DADDS to dapsone and that, while the complex appeared to delay cell growth in the first 24 h period, no significant difference was seen between cells incubated in the presence and absence of the complex at 72 h. These results indicate that DADDS-CD has significant potential as a drug-delivery system for DADDS in the lung based upon the ability of the cells to activate DADDS. The mixed effects of the complex on cell growth may have important implications when considering the frequency of administration of the complex to the lung.
