Flow cytometric analysis of the kinetic effects of cisplatin on lung cancer cells

Abstract

The effects of cisplatin on the cell cycle and DNA synthesis of human lung adenocarcinoma cell line PC-9 were examined by flow cytometry. The cellular DNA content and the bromodeoxyuridine (BrdUrd) incorporation rate were measured simultaneously using a monoclonal anti-BrdUrd antibody. Following exposure to cisplatin (1.0 pg/ml) for 1 and 24 hr, the bivariate DNNBrdUrd distributions revealed a delayed S-phase transit and an accumulation of cells in the G2M phase. The BrdUrd-linked green fluorescence intensity continued to decrease with the lapse of time. However, earlyand mid-S-phase cells soon recovered DNA synthesis activity, and the former showed higher activity than the control cells. These findings suggested the vigorous DNA synthesis of cells in early S phase. However, for quantitative analysis of chemotherapeutic effects, some problems remained to be resolved regarding the condition for DNA denaturation and its alteration by the agents.Key terms: antibromodeoxyuridine, cell cycle, DNA synthesis rate, dual-parameter analysis Bromodeoxyuridine (BrdUrd) is a thymidine analogue that is incorporated specifically into DNA during the S phase of the cell cycle. In 1982, Gratzner (11) produced a monoclonal anti-BrdUrd antibody, and, in 1983, Dolbeare et al. (7) developed a procedure for simultaneous flow cytometric measurement of the cellular DNA content and the amount of incorporated BrdUrd using a n immunofluorescent antibody. They also showed that BrdUrd-linked green fluorescence was proportional to the amount of incorporated BrdUrd (7).Since then, many studies using this antibody have been reported (2,5,9,12,16,21,23). Dean et al. ( 5 ) reported that a mean BrdUrd-linked fluorescence could be taken as a n estimate of the mean DNA synthesis rate. Langer et al. (16) showed that the ratio of S-phase cells estimated by this method correlated well with the 3H-thymidine labeling index. However, there were few studies in which this technique was used to analyze the chemotherapeutic effects on cancer cell kinetics (12).We reported an analytical model of flow cytometric measurement of bivariate DNAiBrdUrd distribution for the study of the cell cycle and showed a good correlation (r = 0.85) between the "-thymidine labeling indices and the percent S-phase population obtained by this method (9). The purpose of this study was to analyze the effects of cisplatin, a n antineoplastic platinum derivative, on the cell cycle and DNA synthesis of a human lung cancer cell line. We applied the BrdUrd labeling method, simultaneous flow cytometric measurement of DNA content, and BrdUrd incorporation, for the quantitative analysis of the cancer cell kinetics. MATERIALS AND METHODS Cancer Cells and Cell Culture MethodsCultured human lung adenocarcinoma cell line PC-9 (kindly provided by Prof. Y. Hayata, Tokyo Medical College, Tokyo, Japan) was used. The cells were maintained in RPMI 1640 medium (GIBCO) supplemented with 10% heat-inactivated fetal bovine serum (FBS; GIBCO) and 40 Fgiml of gentamicin (Schering), a t 37°C in a humidified...

Treatment with the Chk1 inhibitor G�6976 enhances cisplatin cytotoxicity in SCLC cells

“…The differential cell cycle arrest between the two groups was interesting especially as previous studies in other human cancer cells suggest that cisplatin induces delayed S phase transit and ultimately G2 arrest (4,27). S phase arrest as seen in the chemoresistant cells in this study has only been documented in hamster cells and human cell lines modified to overexpress various repair proteins (13,28).…”

“…The six cell lines studied were characterised into two groups based on their sensitivity to the cancer chemotherapy agent cisplatin. The mean peak plasma concentration attained in clinical use of cisplatin is 2 µg/ml which is equivalent to 6.66 µM (27). The chemosensitive group exhibited decreased cell viability and increased levels of the apoptosis executioner protein, caspase 3 following 48 h of treatment with relatively low doses of cisplatin (IC 50 4-12 µM).…”

The role of apoptosis in cell killing by cisplatin: a flow cytometric study

“…One of the early effects of the platination of DNA is a reduction in the rate of DNA synthesis (Fraval & Roberts, 1978;Salles et al, 1983;Sorenson & Eastman, 1988a) and a consequent slow-down in the traverse of cells through the S-phase of their cycle; subsequently, there is a dosedependent arrest in G2 (Bergerat et al, 1979;Sorenson & Eastman, 1988a,b;Fujikane et al, 1989;Demarcq et al, 1992). It has been proposed that in G2 the cells reach a point of decision at which they either overcome the G2 block, divide and then cycle or remain in G2 and die (Sorenson & Eastman, 1988a,b;Sorenson et al, 1990).…”

Glutathione restores normal cell activation and cell cycle progression in cis-platinum treated human lymphocytes

“…In addition cell viability was monitored via flow cytometry. Many data are available on the proliferation of tumour cells or permanent cell lines treated with CDDP (Salles et al, 1983;Kanno et al, 1985;Sorenson & Eastman, 1988;Fujikane et al, 1989). However, none of the analytical techniques used were sufficient to reveal the cellular effects induced at the single cell level in heterogenous responding in vitro cell populations.…”