RESUMO
Earlier reports have suggested that the BCR/ABL oncogene, associated with chronic myeloid leukemia, induces a mutator phenotype; however, it is unclear whether this leads to long-term changes in chromosomes and whether the phenotype is found in primary chronic myelogeneous leukemia (CML) cells. We have addressed both these issues. BCR/ABL-expressing cell lines show an increase in DNA breaks after treatment with etoposide as compared to control cells. However, although BCR/ABL-expressing cell lines have an equivalent cell survival, they have an increase in chromosomal translocations after DNA repair as compared to control cells. This demonstrates that BCR/ABL expression decreases the fidelity of DNA repair. To see whether this is true in primary CML samples, normal CD34+ progenitor cells and CML progenitor cells were treated with etoposide. CML progenitor cells have equivalent survival but have an increase in DNA double-strand breaks (DSBs). Spectral karyotyping demonstrates new chromosomal translocations in CML cells, but not normal progenitor cells, consistent with error-prone DNA repair. Taken together, these data demonstrate that BCR/ABL enhances the accumulation of DSBs and alters the apoptotic threshold in CML leading to error-prone DNA repair.
Assuntos
Instabilidade Cromossômica/genética , Dano ao DNA/genética , Proteínas de Fusão bcr-abl , Morte Celular/genética , Sobrevivência Celular , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Etoposídeo/farmacologia , Células-Tronco Hematopoéticas/patologia , Humanos , Translocação Genética , Células Tumorais CultivadasRESUMO
Retinoids, the analogues of vitamin A, have a broad range of effects on different cell types. One biologically active form of vitamin A is all-trans-retinoic acid (ATRA), which binds to retinoic acid receptors, as does its intracellular metabolite, 9-cis-RA. Earlier studies have documented G1 cell cycle arrest and the induction of apoptosis in human adult T-cell leukemia cells after ATRA treatment. Previous work exploring the growth-inhibitory activity of ATRA in human malignancies has implicated several mechanisms that can arrest cells in the G1 phase of the cell cycle, including activation of p21Waf1 and inhibition of cyclin D1 expression. Therefore, we decided to examine the effects of ATRA exposure on G1 cell cycle components in human adult T-cell leukemia cells. Our data demonstrate a correlation between cyclin/cyclin-dependent kinase activity and subunit complex formation with duration of drug exposure. We also observed an increase in p53 protein levels that were not associated with an increase in p21Waf1 levels. Furthermore, we observed a differential effect on cell cycle progression that was temporally related to length of ATRA exposure. These observations, consistent with a bimodal effect of ATRA on cell cycle progression, may have important implications for the clinical application of ATRA.
Assuntos
Antineoplásicos/farmacologia , Leucemia-Linfoma de Células T do Adulto/tratamento farmacológico , Leucemia-Linfoma de Células T do Adulto/patologia , Proteínas Proto-Oncogênicas , Tretinoína/farmacologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Transformada , Ciclina D1/metabolismo , Quinase 4 Dependente de Ciclina , Quinase 6 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p21 , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Fase G1/efeitos dos fármacos , Vírus Linfotrópico T Tipo 1 Humano , Humanos , Leucemia-Linfoma de Células T do Adulto/enzimologia , Leucemia-Linfoma de Células T do Adulto/metabolismo , Testes de Precipitina , Proteínas Serina-Treonina Quinases/metabolismo , Linfócitos T/enzimologia , Linfócitos T/metabolismo , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/metabolismoRESUMO
We have recently identified a novel candidate oncogene, MCT-1, in the HUT 78 T-cell line. When overexpressed in NIH3T3 fibroblasts, the MCT-1 gene shortens the G1 phase of the cell cycle and promotes anchorage-independent growth. Progression of cells through a late G1 phase restriction point is regulated by G1 cyclins whose phosphorylation of the retinoblastoma gene product facilitates entry into S phase. Deregulated expression of G1 cyclins and their cognate cdk partners is often found in human tumor cells. In order to address the potential relationship of MCT-1 to cell cycle regulatory molecules, we analyzed the ability of MCT-1 overexpression to modulate cdk4 and cdk6 kinase activity in NIH3T3 fibroblasts constitutively overexpressing MCT-1. We observed an increase in the kinase activity of both cdk4 and cdk6 in asynchronously growing transformed cells compared with the parent cells. This increased kinase activity was accompanied by an elevated level of cyclin D1 protein and increased G1 cyclin/cdk complex formation. We also observed a correlation between increased protein levels of MCT-1 with cyclin D1 expression in a panel of lymphoid cell lines derived from T-cell malignancies. These results demonstrate that constitutive expression of MCT-1 is associated with deregulation of protein kinase-mediated G1 phase checkpoints.
Assuntos
Proteínas de Ciclo Celular/genética , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Proteínas Oncogênicas/genética , Oncogenes , Proteínas Proto-Oncogênicas , Células 3T3 , Animais , Ciclina D1/metabolismo , Ciclina G , Ciclina G1 , Quinase 4 Dependente de Ciclina , Quinase 6 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p21 , Fase G1/fisiologia , Expressão Gênica , Humanos , Camundongos , Proteínas Serina-Treonina Quinases/metabolismo , TransfecçãoRESUMO
Using the arbitrarily primed-PCR (AP-PCR) assay to detect genetic abnormalities that occur in a panel of lymphoid cell lines, we identified an amplified stretch of genomic DNA that contained a putative open reading frame. Northern blot analysis with this genomic clone revealed widespread low level expression in normal human tissue. The full cDNA sequence was obtained with no significant homology to any known genes in the genome database. We termed this novel gene with multiple copies in a T-cell malignancy as MCT-1. MCT-1 was localized to the long arm of chromosome Xq22-24 by flourescence in situ hybridization analysis. Although there was no significant homology at the primary sequence level, there was a limited degree of amino acid homology with a domain of cyclin H that appears to specify protein-protein complexes. This relationship between MCT-1 and cyclin H implied a potential role for MCT-1 in cell cycle regulation. Overexpression of MCT-1 increased the proliferative rate of cells by decreasing the length of the G1 phase without a reciprocal increase in the S and G2-M phases. Recent work has established the role of cell cycle regulatory molecules in the development of certain human malignancies. Therefore, we investigated the transforming ability of MCT-1 overexpression using soft agar growth assays and demonstrated that only MCT-1-overexpressing cells were able to establish colonies. Taken together, MCT-1 is a novel candidate oncogene with homology to a protein-protein binding domain of cyclin H.