Cytochemical observations on the nucleolus-ribosome system: Effects of actinomycin D and nitrogen mustard

Abstract

Cytochemical techniques have been developed for the quantitative determination of the DNA, RNA and proteins (dry mass) in individual cells in large cell populations. These methods have been employed for the study of the effects of nitrogen mustard and actinomycin D on the nucleolar system of Ehrlich ascites cells in vivo and a line of normal mouse fibroblasts maintained in cell culture. Parallel studies on the nucleoli of individual cells have been made with ultraviolet microscopy and spectrophotometry. Nitrogen mustard, in certain concentrations, blocked mitosis but did not block DNA synthesis, as adjudged by the accumulation of DNA to pre-mitotic levels in non-dividing cells. Concurrently, the total cellular RNA and the total cellular mass of such cells increased far beyond normal values, with an overdevelopment of the nucleolar apparatus. The experiments with actinomycin D support the conclusion that this antibiotic inhibits the total synthesis of DNA-dependent RNA, and consequently, the synthesis of cellular proteins. Exposure to effective concentrations of actinomycin D results in marked inhibition of the synthesis of nucleolar RNA and proteins, as well as cytoplasmic RNA and proteins. Cytochemical analyses of cell populations exposed to actinomycin D and nitrogen mustard, either alone or in combination, together with studies of the nucleolar apparatus in individual cells in such populations, support the concept that the nucleolus-associated chromatin is concerned primarily with the synthesis of ribosomal RNA—either directly, or by the mediation of a “messenger” fraction of RNA. Such a function is considered to be consistent with the polygenic character of the nucleolus-associated chromatin, suggesting the presence of many repeating DNA cistrons complementary to ribosomal RNA, as described recently in E. coli.