RESUMO
Imatinib mesylate (imatinib), previously known as STI571 (Gleevec), is currently utilized in the treatment of chronic myeloid leukemia (CML). However, its effect on telomerase activity and the correlation of this to its observed antitumor effect has yet to be defined. We investigated the effects of this agent on human telomerase reverse transcriptase (hTERT) expression and telomerase activity and found that it significantly down-regulated telomerase activity in both K562 cells and primary leukemic cells. The telomerase activity of primary leukemic cells from CML patients in blastic crisis showed less suppression than that of cells from patients in chronic phase. Additionally, data also demonstrate that inhibition of telomerase was due to the direct action of imatinib on hTERT transcription, rather than an increase in cell death. These results suggest a novel mechanism in the antitumor activity of imatinib and may provide a basis for future development of anti-telomerase therapies, as well as leading to better understanding of the regulation of telomerase in leukemic cells.
Assuntos
Antineoplásicos/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Piperazinas/uso terapêutico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Pirimidinas/uso terapêutico , Telomerase/metabolismo , Apoptose/efeitos dos fármacos , Benzamidas , Crise Blástica , Feminino , Regulação Leucêmica da Expressão Gênica , Humanos , Mesilato de Imatinib , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Masculino , Pessoa de Meia-Idade , Leucemia-Linfoma Linfoblástico de Células Precursoras B/enzimologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Regiões Promotoras Genéticas , Proteínas Tirosina Quinases/antagonistas & inibidores , Telomerase/antagonistas & inibidores , Telomerase/genética , Telômero/genética , Transcrição Gênica/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
Leukemic stem cells that expressed endogenous telomerase activity were induced to show overexpression of exogenous hTERT and were analyzed for biological changes in order to assess the possible influence of telomerase gene therapy on the transplantation of normal hematopoietic stem cells. Introduction of hTERT into K562, a telomerase-positive immortal cell line, resulted in a 2.5-fold elevation of telomerase activity and the lengthening of telomeres by 6 kb to 23 kb. Real-time fluorescent PCR, which could perform quantitative analysis of transcripts, revealed a 175-fold increase in hTERT expression, suggesting the posttranscriptional regulation of telomerase. Ectopic expression of hTERT in K562 cells showed a survival advantage during culture in the absence of serum. Expression of mRNA for the telomeric-repeat binding factor 1 (TRF1) and caspase-3 activity were both decreased in hTERT-transfected K562 cells. Transduced cells retained their usual phenotypic characteristics, differentiation ability, and signal transduction response to TPA. These data suggest that ectopic expression of hTERT by normal hematopoietic stem cells may confer a survival advantage without changing their innate biological characteristics.
Assuntos
Diferenciação Celular/genética , Regulação Neoplásica da Expressão Gênica , Telomerase/genética , Telômero/genética , Proteína 1 de Ligação a Repetições Teloméricas/genética , Caspase 3 , Caspases/metabolismo , Sobrevivência Celular/genética , Senescência Celular/genética , Proteínas de Ligação a DNA , Humanos , Células K562 , Piridinas/farmacologia , Interferência de RNA/fisiologia , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Supressão Genética/genética , Telomerase/uso terapêutico , Transdução Genética/métodos , Regulação para Cima/genéticaRESUMO
A high-pressure freezing method was used to observe the ultrastructure of pathogenic yeasts, Cryptococcus neoformans and Exophiala dermatitidis, after freeze-substitution and ultrathin sectioning. The method well preserved the cell structure in its natural state, since the capsule, cell wall, plasma membrane, nucleus, outer and inner nuclear membranes, nuclear pores, nucleolus, mitochondria, mitochondrial membrane and cristae, vacuoles, endoplasmic reticulum, Golgi apparatus, spindle pole body, ribosomes, lipid droplets, microtubules, actin filaments, and glycogen granules were clearly visible. The method was shown to freeze cells as deep as 0.1 mm by sectioning the sample perpendicular to specimen surface. The quality of the cell image was similar to that obtained by a rapid freezing method when compared using the same materials. Thus, high-pressure freezing would be useful for making serial ultrathin sections for three-dimensional analysis of cells, which should give basic information of structure and function of pathogenic yeast cells necessary for finding an effective therapy for mycoses.
Assuntos
Criopreservação/métodos , Cryptococcus neoformans/ultraestrutura , Exophiala/ultraestrutura , Microscopia Eletrônica/métodos , Cryptococcus neoformans/patogenicidade , Exophiala/patogenicidadeRESUMO
Mato's FGP cells surrounding cerebral arterioles play a significant role in the maintenance of a homeostatic microenvironment in the brain. In this study, the perivascular cells were isolated from rat cerebral microvessels and cultured in vitro to characterize their phenotype. Autofluorescence of the intracellular granules in cultured cells and the uptake of HRP and DiI-Ac-LDL by these cells were observed. The cells reacted positively to an anti-scavenger receptor A antibody. Positive immunoreactions of cultured cells to ED1 and ED2 antibodies were observed, whereas they were weak or negative to ED3 and OX42 antibodies. Acid phosphatase activity was detected in the granules of cultured cells. In conclusion, the cells cultivated under the present conditions revealed very similar characteristics to Mato's FGP cells in situ and therefore are useful for studies on FGP cells.
