| Objectives 1. To establish a multidrug-resistant cell line(K562-n/VCR) derived from human leukemia cell line K562-n with high tumorigenicity in nude mice, characterize its biological feature, and explore the molecular mechanism of MDR by gene expression analysis. 2. To study the reverse role of MDR treated with cyclosporin A( CsA), tamoxifen (TAM) and interferon-α(IFN-() in K562-n/VCR cells. 3. To explore the possibility of drug resistance to STI571 and its reversal way in K562-n/VCR cells. To observe the inhibitory role of STI571 combined respectively with daunorubicin(DNR), vincristine(VCR), DNR+cytosine arabinoside (Ara-C) on leukemia cell line of both bcr-abl and mdr-1 positive. 4. A pilot study of pathogenesis,classification and prognosis of initial acute leukemia by cDNA microarray techniques. Methods K562-n cells were induced with a long-term ,intermittent, singular, uneffcient VCR addition into multidrug-resistant cell line K562-n/VCR. The biological features of K562-n/VCR were compared with K562-n, in the respect of cell growth curve ,tumorigenicity in nude mice, colony formation percentage in soft agar, ultrastructure by electron microscopy, cell cycle distribution and the intracellular accumulation of DNR by flow cytometer. The cytogenetics analysis of different strains was also done to observe the chromosome changes. MTT method was used to determine the cytotoxity activity of some anticancer drug, such as DNR, etoposide(VP16), mitozantrone(NVT) and homoharringtonin(HHT). Expression of p-gp was detected by the way of immunohistochemical method. cDNA microarray and RT-PCR were applied in gene expression analysis.The reversal effects of CsA,TAM and IFN-αon the drug resistance of K562-n/VCR were studied by MTT method.We detected wether K562-n/VCR cells were cross-resistant to STI571 and the reversal effect of CsA ,TAM and IFN -αwith MTT method. Synergism of STI571 and DNR, VCR and DNR+Ara-C on K562-n/VCR cells was examinined also by MTT method.The differential gene expression profiles on genomic scale were identified by the DNA microarry techniques to search the genes associated with prognosis of initial acute leukemia.Results K562-n cells were cultured with serially increasing concentrations up to 1.28 μg/ml of VCR .K562n/VCR cells were found to be cross-resistant to DNR, EPI, HHT, NVT, VP16 and VM26. There was no significant difference of cell growth curve and colony formation percentages in soft agar between the two cell lines. But the incubation period was shorter and the tumor was bigger in K562-n/VCR inoculated nude mice. Ultrastructurely, the nuclear was highly abnormal ,the euchromatic element of nuclear chromatin was increased and the heterochromatin was very rare in both cells, liberated ribosomes were increased ,intermediate filaments and microfilaments in cytoplasm were also increased and the arrangement was disturbed in K562n/VCR. The cell cycle analysis by flow cytometer showed higher S phase fractions of MDR cells. The cytogenetic analysis showed the chromosome numbers of K562-n/VCR cells varied from 54 to 65, the karyotype of stem line was hypotriploid, chromosomes model was 63±0.82. And the chromosome numbers of K562-n/VCR cells varied from 61 to 64, the karyotype of stem line was hypotriploid, chromosomes model was 63±0.99.No typical chomosome Ph was found in the two cell lines. Intracellular accumulation of DNR in K562n/VCR was less than that in the parent cells. DNR intracellular accumulation was partially reversed only by addition of CsA. The bcr-abl gene was positive in both cell line and P-glycoprotein (P-gp) was overexpressed only on K562-n/VCR cells. Multidrug- resistance was partially reversed by addition of CsA, TAM, and IFN-(. The reversal effectiveness of the modulator combinations was stronger than that of each modulator alone. The bcr-abl positive K562n/VCR was also resistant to STI571, an inhibitor of tyrosine kinase, and could be reversed by CsA, TAM, and IFN-(..Combination of STI571 with daunorubicin, VCR and DNR+ Ara-C had a greater Synergi...
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