Abstract
The cytotoxic action of 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) is considered to be mainly the result of alkylation of deoxyribonucleic acid (DNA). If ACNU acts chiefly on DNA, sensitivity to ACNU might have a close relation to nuclear DNA content. In this study, the relative DNA content in three established human glioma cell lines, NP-1, NP-2, and NP-3, was investigated by flow cytometry and correlated to their sensitivity to ACNU. For flow cytometric analysis, the cells were fixed in 75% ethanol, treated with ribonuclease and pepsin, and stained with ethidium bromide. Human peripheral mononuclear cells were used as an internal standard for determining the DNA index. Sensitivity to ACNU was evaluated by the cell growth curve and perturbed DNA histograms.
DNA indices were 1.8 for NP-1 and 1.0 for NP-3. A DNA histogram of NP-2 showed three peaks. Therefore two clonal cell lines, clone A and clone B, were segregated from NP-2; their DNA indices were 1.4 and 2.4, respectively. The growth-inhibitory effect of ACNU was dose-dependent but varied greatly with each cell line. Analysis of the alteration of the DNA histograms after exposure to various doses of ACNU showed common features in all cell lines; the more the cell growth was inhibited by treatment with a higher dosage of ACNU, the more the cells accumulated in S and G2M phases and the less the pattern of the DNA histogram returned to that of the control with the lapse of time. Based on this principle, the histograms of each cell line were compared and the sensitivities were determined together with the result of the growth curve. The sequence was NP-2 clone A, NP-2 clone B, NP-1, NP-3 in the order of high to low sensitivity to ACNU.
Flow cytometric analysis revealed some interesting characteristics of these cell lines. It was suggested that sensitivity to ACNU of human glioma cells might increase with nuclear DNA content deviating from the normal amount to a higher one. The difference in sensitivity to ACNU between NP-2 clone A and clone B, which were representative of different populations in terms of DNA content, showed the heterogeneity and complexity of human malignant gliomas.
