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Objective To investigate the role of methylated DNA mismatch repair gene MLH1 and MSH2 in the acquired multidrug-resistance of human small cell lung cancer cells H446.Methods The reverse transcription polymerase chain reaction(RT-PCR)and Western blot were applied to measure MLH1 and MSH2 mRNA and protein expressions of the multidrug-resistant cells H446/DDP and its parental cells H446.The promoter methylation status of the genes was assessed by methylation-specific PCR(MSP).Results The expressions of MLH1 and MSH2 significantly decreased both in mRNA level and protein level.Promoter methylation of MLH1 was observed in H446/DDP cells but not in H446 cells.Promoter semi-methylation of MSH2 in H446 cells was transformed to methylation in H446/DDP cells.Conclusion The downregulation of DNA mismatch repair gene MLH1 and MSH2 induced by its promoter methylation may play an important role in the acquired multidrug resistance of human small cell lung cancer.
ABSTRACT
Objective To establish a multidrug-resistant cell subline NCI-H446/CDDP of human small cell lung cancer and characterize its biological properties. Methods The cell subline NCI-H446/CDDP was derived from human small cell lung cancer cell line NCI-H446 by exposing it to high concentration first followed by being cultured with gradually increasing dose of cisplatin, which used to be the first-line chemotherapeutic drug for lung cancer. The cell growth curve and the doubling time were determined by cell counting. The chemosensitivities of NCI-H446/CDDP and NCI-H446 to cisplatin, hydroxycamptothecine, vincristin, 5-fluorouracil and topotecan were tested and IC 50 measured by MTT. Changes in cellular morphology and ultrastructure were observed under inverted-microscope, scanning electron microscope and transmission electron microscope, respectively. Results Multidrug-resistant cell subline (NCI-H446/CDDP) of human small cell lung cancer was established successfully after culturing NCI-H446 in a high concentration of cisplatin first, followed by subjecting it to increasing concentrations of CDDP until they could stably grow in the culture medium containing 0.5?g/mL CDDP. The rate of cell proliferation of NCI-H446/CDDP was similar to that of NCI-H446, but the number of the former cells exhibiting S phase increased (20.24% vs 18.42% P
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Background and purpose:Regulation of MMR activity under hypoxia may play an important role in genetic instability of cancer,but the mechanism is still unclear.We investigated the expression of DNA mismatch repair genes MLH1 and MSH2 in human SCLC cell line H446 under hypoxic condition and explore the role of promoter methylation of genes in hypoxia.Methods:RT-PCR and Western blot were applied to detect MLH1 and MSH2 expression in human SCLC cell line H446 at the mRNA and the protein level,respectively,under either hypoxic condition or after 5-Aza-CdR treatment.Meanwhile,methylation-specific PCR(MSP)was used to determine promoter methylation of MLH1 and MSH2.Results:The expression of MLH1 and MSH2 in H446 cells significantly decreased both at the mRNA and the protein level under hypoxic condition.5-Aza-CdR treatment led to the restoration of MLH1 and MSH2 expression,while,both MLH1 and MSH2 were down-regulated again after removing 5-Aza-CdR.Conclusions:The promoter methylation of MLH1 and MSH2 may play an important role in its defective expression in H446 cells under hypoxic condition.And 5-Aza-CdR could restore MLH1 and MSH2 expression.