Karyometric and cytophotometric studies on nucleic acids in the culture of Human lymphocytes with phytohemagglutinin

Abstract

Lymphocyte transformation with phytohemagglutinin (PHA) was analyzed by karyometry and cytophotometry of single cells. The former showed the distribution of various-sized transforming cells, and the latter the increase of DNA and RNA. Two different types of karyograms were obtained; one with three peaks corresponding to 2c, 4c, and 8c, and the other with only a 2c peak. Although small in number (8%), karyometry showed that DNA synthesis took place in the extremely early stage of transformation, 1 h after stimulation with PHA, which was proved by cytophotometry and ultrastructural findings. An increasing curve of DNA and RNA revealed that a lymphocyte continued to enlarge from 2c to 4c and divide again through the normal cell cycle in a suitable condition. The largest blast cell, a polyploid cell, may be induced by the lack of a sufficient amount of RNA and protein in need of cell division, and may be a degenerative cell in the strict sense. Among the large cells with blastlike appearance, there were a considerable number of so-called degenerative cells with a low amount of DNA. The ratio of RNA to DNA increased steeply in the actively DNA synthesizing S phase, which supports the morphologic findings of blast cell, the appearance of basophilic cytoplasm, and active-looking nucleoli.