Chemosensitivity of human small cell carcinoma of the lung detected by flow cytometric DNA analysis of drug‐induced cell cycle perturbations in vitro

Abstract

A method based on detection of drug-induced cell cycle perturbation by flow cytometric DNA analysis has previously been described in Ehrlich ascites tumors as a way to estimate chemosensitivity. The method is extended to test human small-cell carcinoma of the lung. Three tumors with different sensitivities to melphalan in nude mice were used. Tumors were disaggregated by a combined mechanical and enzymatic method and thereafter have incubated with different doses of melphalan. After incubation the cells were plated in vitro on agar, and drug induced cell cycle changes were monitored by flow cytometric DNA analysis. Melphalan produced a dose-related S phase accumulation in the two sensitive tumors, whereas no changes in the cell cycle distribution were found in the resis- In vitro tests, especially the human tumor cloning assay (12,13), have been increasingly used to optimize treatment of malignant diseases andor to test new and more potent non-cross-resistant drugs. However, the human tumor cloning assay is time-consuming and requires 2-3 wk before conclusive results are available. Furthermore, the method does not allow one to test how representative the growing cells are. In search of a faster technique to detect chemosensitivity, a method based on measurements of drug-induced cell cycle perturbations was examined in experimental tumors (5). This in vitro flow cytometry assay was found to be able to detect sensitivity to adriamycin in Ehrlich ascites tumors.The object of the present study was to determine whether the in vitro cell cycle perturbation assay could be extended to test the chemosensitivity of human solid tumors. This demands a valid procedure to disaggregate the solid tumors into single cell suspensions representative of the original tumors. Three heterotransplanted human small-cell carcinomas of the lung (SCCL) were selected for this study because of their different sensitivities to melphalan (9). The tumors were disaggregated by a combined mechanical and enzymatic method (7).Subsequently the cells were incubated in vitro with different doses of melphalan; flow cytometric DNA analysis was used to monitor drug-induced cell cycle pertubations.The results showed that melphalan induced dose-related S phase accumulations in two sensitive tumors, whereas no cell cycle changes were found in a resistant tumor. Furthermore, the size of the S phase accumulation in vitro reflected the previously found in vivo chemosensitivity of the tumors. Thus, the results suggest that the flow cytometry method applied on disaggregated tumor cells may be extended to sensitivity testing of human tumors in general, including screening for new agents. MATERIALS AND METHODS Tumors

Effectiveness of isolated liver perfusion with mitomycin C in the treatment of liver tumours of rat colorectal cancer

“…Increased intracellular concentration results in a higher number of cross-links (Dorr et al, 1985;Matsumoto et al, 1989). Evidently, the extent of mitomycin C induced S phase accumulation observed in the present study may result from the degree of cross-linking and the influx rate of tumour cells into the S phase (Engelholm et al, 1986). However, the sensitivity of tumour cells to mitomycin C induced DNA damage may also depend on their ability to remove DNA cross-links (Kaiser et al, 1982).…”

A review of techniques and results obtained in one laboratory by an integrated system of methods designed for routine clinical flow cytometric DNA analysis

“…Note that without a fixed location of F~, correct discrimination between Sand G2+,-phase would not have been possible. Other examples of perturbed S-phases can be found in references (13,15,(27)(28)(29)33,43).…”

“…Flow cytometric DNA analysis has been established as a useful research tool for a number years (36). The methods described in this paper have been used for monitoring the stability of cell lines, for sensitivity testing and for studying the action of anticancer drugs in vitro (13,(15)(16)(17)(27)(28)(29). Likewise the methods have been used to monitor the cell cycle perturbations produced by radiation (34), chemotherapy (13,271 and hormonal substances (6)(7)(8)(9) in murine and human tumors in experimental animals, and in malignant tumors in patients (43).…”

Flow cytometry: A useful technique in the study of multidrug resistance

“…Such information is most frequently obtained through analysis of the cell cycle. The majority of anticancer drugs, acting at different stages in the cycle, induce perturbations resulting in an accumulation of cells in one phase of the cycle [3,5,7]. For this, various techniques of DNA labeling are available, generally chosen as a function of the type of compounds studied, and the cell cycle perturbations supposed.…”