RESUMO
To investigate how overexpression of p27KIP1, a downstream effector of TGF-beta and a universal cyclin-dependent kinase (CDK) inhibitor could influence the malignant phenotype of malignant human brain tumor cells, an adenovirus vector system was used to transfer the human p27KIP1 gene (Adp27KIP1) into the human astrocytoma cell line, U-373MG. Inhibition of CDK activity in Adp27KIP1-infected cells was indicated by inhibition of [3H]thymidine incorporation, an increase in cell doubling time and by cell cycle arrest in G1. Notably, ectopic overexpression of p27KIP1 was associated with a marked decrease in the accumulation of aneuploid cells. Diminished malignant potential of Adp27KIP1-infected cells was manifested by the loss of anchorage-independent growth in soft agar and by the inability to induce tumorgenesis in a xenograft model. These studies suggest that p27KIP1 is a tumor suppressor gene and supports the use of Adp27KIP1 for gene therapy of human brain tumors.
Assuntos
Aneuploidia , Astrocitoma/patologia , Neoplasias Encefálicas/patologia , Proteínas de Ciclo Celular , Inibidores Enzimáticos , Proteínas Associadas aos Microtúbulos/biossíntese , Proteínas Supressoras de Tumor , Animais , Astrocitoma/genética , Western Blotting , Neoplasias Encefálicas/genética , Ciclo Celular , Divisão Celular , Linhagem Celular , Sobrevivência Celular , Inibidor de Quinase Dependente de Ciclina p27 , Quinases Ciclina-Dependentes/antagonistas & inibidores , DNA de Neoplasias/biossíntese , Expressão Gênica , Humanos , Camundongos , Camundongos Nus , Proteínas Associadas aos Microtúbulos/análise , Protamina Quinase/metabolismo , Proteínas Recombinantes/biossíntese , Timidina/metabolismo , Transfecção , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
p21WAF1/CIP1 is a downstream effector of the p53 tumor suppressor gene and a universal cyclin-dependent kinase (CDK) inhibitor. To determine the ability of p21WAF1/CIP1 to function as a tumor suppressor, we constructed a replication-defective adenovirus vector containing p21WAF1/CIP1 (Adp21WAF1/CIP1) to effect ectopic overexpression in a p53-defective human astrocytoma cell line, U-373MG. We observed a marked decrease in CDC2 and CDK2 kinase activity associated with a corresponding decrease in the amount of CDC2 but not CDK2 protein; a decreased growth potential of Adp21WAF1/CIP1-infected cells demonstrated by diminished [3H]thymidine incorporation, increased cell doubling time and G1-arrested cell cycle; an association between Adp21WAF1/CIP1-infected cells and inhibition of aneuploid cell accumulation; and an alteration of the malignant phenotype of cells was evidenced by the loss of anchorage-independent growth in soft agar and the failure to induce tumorigenesis in both peripheral and intracerebral xenograft models, including the prevention of tumor formation Adp21WAF1/CIP1 infection 2 days post tumor cell implantation. Adp21WAF1/CIP1. Adp21WAF1/CIP1 appears to be a strong candidate for gene therapy studies based on these studies indicating that Adp21WAF1/CIP1 inhibits proliferation, tumorigenicity and aneuploidy in human brain tumor cells.
Assuntos
Aneuploidia , Astrocitoma/metabolismo , Astrocitoma/terapia , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/terapia , Ciclinas/metabolismo , Animais , Apoptose , Astrocitoma/genética , Neoplasias Encefálicas/genética , Proteínas de Ciclo Celular/metabolismo , Divisão Celular/genética , Inibidor de Quinase Dependente de Ciclina p21 , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/fisiologia , Vírus Defeituosos , Fase G1/fisiologia , Genes p53/fisiologia , Terapia Genética , Vetores Genéticos , Humanos , Camundongos , Camundongos Nus , Fenótipo , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
BACKGROUND: In human brain tumors, sensitivity to procarbazine as measured by sensitivity in a xenograft tumor model correlated inversely with amounts of the DNA repair enzyme O6-alkylguanine DNA alkyltransferase (AT). METHODS: To test the hypothesis that mutations of the p53 tumor suppressor gene in human tumors also can correlate with the response to chemotherapy, p53 mutations2 were identified in primary human malignant brain tumors and cell lines in which AT activity and procarbazine sensitivity in a xenograft model was ascertained. RESULTS: Mutations were identified in 7 of 21 (33%) specimens tested. Specimens containing p53 mutations tended to exhibit an increased growth delay in procarbazine-treated xenografts and lower amounts of AT. CONCLUSIONS: p53 mutations in brain tumors may contribute to procarbazine sensitivity by failing to induce arrest at the G1/S cell-cycle checkpoint, thereby preventing the repair of procarbazine-induced genetic alterations.