Induction of DNA polymerase in human lymphocytes by phytohemagglutinin.

The lymphocytes of human peripheral blood have been the subject of recent immunologic studies. Although these cells seldom grow or divide in vitro, the addition of phytohemagglutinin (PHA), an extract of the kidney bean (Phaseolus vulgaris), to the culture medium initiates a striking transformation: 90 per cent of the cells enlarge, synthesize DNA, and divide. ' Smaller percentages of cells show similar responses to many other mitogens, including appropriate immunologic stimuli.2 The effects of PHA have been extensively monitored by the morphologic transformation of the cells (blastogenesis) and by labeling the DNA of the cell upon the addition of radioactive thymidine to the culture. Changing patterns of RNA3-6 and protein', 6 metabolism as well as histone acetylation5 have also been well documented. The mitogenic effect of PHA has been extensively utilized for studies of human chromosomes. This communication is the first report that the DNA polymerase activity of human lymphocytes increases 30to 100-fold when these cells are cultured in the presence of phytohemagglutinin. The increase in DNA polymerase activity as measured in disrupted cell preparations is correlated both quantitatively and temporally with the ability of these cells to incorporate thymidine into DNA. Materials and Methods. -[a-P32]-labeled thymidine triphosphate was purchased from the International Chemical and Nuclear Corporation, City of Industry, California. H3and C14-labeled deoxynucleotides were purchased from Schwarz BioResearch, Inc., and unlabeled nucleotides from the California Foundation for Biochemical Research. Calf thymus DNA, E. coli DNA, crystalline pancreatic DNase, and micrococcal nuclease were acquired from Worthington Biochemical Corporation. Sea urchin nuclear DNA polymerase was purified about 300-fold from S. purpuratus embryos,7 and E. coli DNA polymerase was fraction IX in the procedure of Richardson et al.8 Poly dAT was made by the de novo reaction catalyzed by E. coli DNA polymerase.9 "Activated" calf thymus DNA was made by digesting the DNA with minute amounts of pancreatic DNase.Y0 The course of the reaction was monitored with sea urchin nuclear DNA polymerase. Maximum activation was achieved when about 2% of the DNA was rendered acid-soluble. The s2ot of the "activated" DNA was 2.4, as determined by boundary centrifugation on a Spinco analytical ultracentrifuge, and the increase in absorbancy at 260 m/A was 21% upon heating. "Activated" dAT was made in a similar manner, except that at a maximum activation 6% was acid-soluble. DNA was heat-denatured by keeping a solution of DNA (1 mg/ml in 0.01 M KCl) at 100'C for 10 min and then immediately cooling in an ice-water bath. Actinomycin D was a gift of Merck Sharp and Dohme, Rahway, New Jersey. Phytohemagglutinin M (1 mg protein/ml) was obtained from General Biochemicals, Chagrin Falls, Ohio (lot 685,291) and Difco Laboratories, Detroit, Michigan (lot 513, 461). Lymphocytes were isolated from human peripheral blood by the method of Bach and Hirschhorn. "' The lymphocytes were suspended at a concentration of 7.5 X 105 cells/ml in MEM Spinner medium supplemented with 20% fetal calf serum, penicillin, streptomycin, and glutamine. Aliquots of 2 ml were incubated with 0.05 ml of phytohemagglutinin in 15-ml Corex tubes at 370C for 3 days. These conditions resulted in maximal incorporation of thymidine into DNA, as well as maximum levels of DNA polymerase