Establishment Of Homoharringtonine-resistant Human SKM-1 Cell Line And Its Biological Characteristics Research

Objective:To estabilish a homoharringtonine(HHT)-resistant SKM-1 cell line and explore the biologic characteristics and mechanisms for drug resistance.Methods:The HHT-resistant SKM-1 cell line was established by repeatedly exposing the cells to comparatively large doses of HHT with a short-time duration, and gradually elevating the drug concentration to an endurable level.The morphology of the resistant and parental cell lines were observed through optical microscope.The MTT assay was used to determine the doubling time,the resistance index,and to draw growth curve.The immunophenotype, cell cycle distribution and DNR accumulation between SKM-1 and SKM-1/HHT were analyzed by flow cytometry and the karyotypes by R-banding.Both cell lines were compared in the nude mice nodulation.To evaluate the expression level of mdr-1, MRP and topo-IIa, semi-quantitative real-time PCR was performed.Results:Through 7-month drug induction, the HHT-resistant cell line,SKM-1/HHT,was eventually established. In morphology, the resistant cel line was the same to the parental cell line, both growing in suspension with big volume, size heterogeneity and large nuclear-plasma ratio. Vacuoles were seen in both the cytoplasma and nucleus cavity. The immunophenotpyes of two cell types were similar, SKM-1:CD2-,CD3-,CD4-,CD5-,CD7-,CD8-,CD11b+(42.28%),CD13+(65.35%),CD14-,CD33+(99.45%), SKM-1/HHT:CD2-,CD3-,CD4-,CD5-,CD7-,CD8-,CD11b+(46.81%), CD13+(83.37%). CD14-,CD33+(99.61%).The karyotypes were roughly the same, but with some differences.The resistant cell line had more G1 phase cells, less S phase and G2 phase cells compared to the parental cell line.Both cell lines cannot get nodulation in nude mice within 30 days.The drug resistance of SKM-1/HHT cell to HHT,VCR, DNR, and etoposide all increased. The resistance indices of HHT, VCR, DNR, and etoposide were 17.94,8.78,5.99, and 13.76,respectively.DNR accumulation was impaired in SKM-1/HHT. Both mdr-1 and MRP increased at gene level, though mdr-1 was prominent (20.1-fold). And there was a slightly decrease of topo-IIa (1:0.619) Conclusion:A HHT-resistant MDS-AML cell line,SKM-1/HHT,was established. The prominent overexpression of mdr-1 may be the main cause for multidrug resistance.