Objective Alkaline comet assay was used to evaluate DNA repair (nucleotide excision repair, NER) capacity of human fresh lymphocytes from 12 young healthy non-smokers (6 males and 6 females). Methods Lymphocytes were ...Objective Alkaline comet assay was used to evaluate DNA repair (nucleotide excision repair, NER) capacity of human fresh lymphocytes from 12 young healthy non-smokers (6 males and 6 females). Methods Lymphocytes were exposed to UV-C (254 nm) at the dose rate of 1.5 J/m2/sec. Novobiocin (NOV) and aphidicolin (APC), DNA repair inhibitors, were utilized to imitate the deficiency of DNA repair capacity at the incision and ligation steps of NER. Lymphocytes from each donor were divided into three grougs: UVC group, UVC plus NOV group, and UVC plus APC group. DNA single strand breaks were detected in UVC irradiated cells incubated for 0, 30, 60, 90, 120, 180, and 240 min after UVC irradiation. DNA repair rate (DRR) served as an indicator of DNA repair capacity. Results The results indicated that the maximum DNA damage (i.e. maximum tail length) in the UVC group mainly appeared at 90 min. The ranges of DRRs in the UVC group were 62.84%-98.71%. Average DRR value was 81.84%. The DRR difference between males and females was not significant (P<0.05). However, the average DRR value in the UVC plus NOV group and the UVC plus APC group was 52.98% and 39.57% respectively, which were significantly lower than that in the UVC group (P<0.01). Conclusion The comet assay is a rapid, simple and sensitive screening test to assess individual DNA repair (NER) capacity. It is suggested that the time to detect DNA single strand breaks in comet assay should include 0 (before UV irradiation), 90 and 240 min after exposure to 1.5 J·m-2 UVC at least. The DRR, as an indicator, can represent the individual DNA repair capacity in comet assay.展开更多
To study the resistant mechanisms of cisplatin in human lung adenocarcinoma cell line A 549 DDP. A 549 DDP cells was established by stepwise increasing concentration of cisplatin (CDDP) in medium. Interstran...To study the resistant mechanisms of cisplatin in human lung adenocarcinoma cell line A 549 DDP. A 549 DDP cells was established by stepwise increasing concentration of cisplatin (CDDP) in medium. Interstrand cross linked DNA (ICL) was measured by ethidium bromide fluorescence assay. The intracellular and intranuclear accumulation of cisplatin was measured by atomic absorption spectrometry. The removal of GS X was determined by FCM and fluorescence microscopy. Results: The A 549 DDP cell line was 8.9 fold resistance relative to the parental A 549 cell line. The formation of ICL in A 549 was 6.28 times higher than that in A 549 DDP cells. The intracellular and intranuclear accumulation of cisplatin in A 549 cells was 5.9 times and 4.1 times higher than that in A 549 DDP cells, respectively. The ability of GS X pump pumped GS X complex (GS Pt) in A 549 DDP cells was higher than that in A 549 . The repair rate in A 549 DDP cells was 2 times higher than that in A 549 . Conclusions: Decreased accumulation and increased export of cisplatin might be the main mechanism of cisplatin resistant A 549 DDP cells while the enhanced repair capacity of DNA may play a role in CDDP resistance.展开更多
文摘Objective Alkaline comet assay was used to evaluate DNA repair (nucleotide excision repair, NER) capacity of human fresh lymphocytes from 12 young healthy non-smokers (6 males and 6 females). Methods Lymphocytes were exposed to UV-C (254 nm) at the dose rate of 1.5 J/m2/sec. Novobiocin (NOV) and aphidicolin (APC), DNA repair inhibitors, were utilized to imitate the deficiency of DNA repair capacity at the incision and ligation steps of NER. Lymphocytes from each donor were divided into three grougs: UVC group, UVC plus NOV group, and UVC plus APC group. DNA single strand breaks were detected in UVC irradiated cells incubated for 0, 30, 60, 90, 120, 180, and 240 min after UVC irradiation. DNA repair rate (DRR) served as an indicator of DNA repair capacity. Results The results indicated that the maximum DNA damage (i.e. maximum tail length) in the UVC group mainly appeared at 90 min. The ranges of DRRs in the UVC group were 62.84%-98.71%. Average DRR value was 81.84%. The DRR difference between males and females was not significant (P<0.05). However, the average DRR value in the UVC plus NOV group and the UVC plus APC group was 52.98% and 39.57% respectively, which were significantly lower than that in the UVC group (P<0.01). Conclusion The comet assay is a rapid, simple and sensitive screening test to assess individual DNA repair (NER) capacity. It is suggested that the time to detect DNA single strand breaks in comet assay should include 0 (before UV irradiation), 90 and 240 min after exposure to 1.5 J·m-2 UVC at least. The DRR, as an indicator, can represent the individual DNA repair capacity in comet assay.
文摘To study the resistant mechanisms of cisplatin in human lung adenocarcinoma cell line A 549 DDP. A 549 DDP cells was established by stepwise increasing concentration of cisplatin (CDDP) in medium. Interstrand cross linked DNA (ICL) was measured by ethidium bromide fluorescence assay. The intracellular and intranuclear accumulation of cisplatin was measured by atomic absorption spectrometry. The removal of GS X was determined by FCM and fluorescence microscopy. Results: The A 549 DDP cell line was 8.9 fold resistance relative to the parental A 549 cell line. The formation of ICL in A 549 was 6.28 times higher than that in A 549 DDP cells. The intracellular and intranuclear accumulation of cisplatin in A 549 cells was 5.9 times and 4.1 times higher than that in A 549 DDP cells, respectively. The ability of GS X pump pumped GS X complex (GS Pt) in A 549 DDP cells was higher than that in A 549 . The repair rate in A 549 DDP cells was 2 times higher than that in A 549 . Conclusions: Decreased accumulation and increased export of cisplatin might be the main mechanism of cisplatin resistant A 549 DDP cells while the enhanced repair capacity of DNA may play a role in CDDP resistance.
