Synthesis of DNA and Poly(Adenosine Diphosphate Ribose) in Normal and Chronic Lymphocytic Leukemia Lymphocytes

Abstract

A B S T R A C T Peripheral blood lymphocytes were isolated from 9 patients with chronic lymphocytic leukemia (CLL)

Association of Poly(Adenosine Diphosphoribose) Synthesis with DNA Damage and Repair in Normal Human Lymphocytes

“…To measure DNase-responsive poly(ADPR) synthesis, the reaction system was adjusted to contain 0.05% Triton X-100 (Sigma Chemical Co.) and 30 jig of DNase I for a final concentration of 300 gg/ml. This concentration of DNase has been previously demonstrated to produce maximal stimulation of poly(ADPR) synthesis (4,12).…”

“…Hypaque (Winthrop Laboratories, New York) gradients (12,14). Purified lymphocytes were suspended at a final concentration of 2 x 106 cells/ml in a-modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum, 50 U/ml penicillin, 50 ,ug/ml streptomycin, and 25 mM Hepes buffered to a final pH of 7.2, and then incubated at 37°C.…”

“…Synthesis of DNA and poly(ADPR) was measured after the cells were rendered permeable to exogenously supplied nucleotides. Details of this technique, the proof that the cells become permeable to exogenously supplied phosphorylated compounds, the proof that the reaction products are distinctly DNA or poly(ADPR), and the kinetics of each reaction have already been presented in detail (4,12,13,16 (4,12). To measure DNase-responsive poly(ADPR) synthesis, the reaction system was adjusted to contain 0.05% Triton X-100 (Sigma Chemical Co.) and 30 jig of DNase I for a final concentration of 300 gg/ml.…”

“…Samples were sonicated, collected on Whatman GF/C filter discs, and washed five times with 10% TCA with 2% Na4P207 in the case of DNA, or five times with 20% TCA in the case of poly(ADPR). All discs were washed twice with ethanol; then the radioactivity was determined in a toluenebased scintillation fluid as previously described (4 90 ,ug/ml Proteinase K. The solutions were adjusted to a pH above 12 by addition of 0.5 ml 2 N NaOH. Solid CsCl was added to a refractive index of 1.4080, then 6.5 ml of the DNA solutions in alkaline CsCl were transferred to polyallomer tubes and centrifuged in a Beckman type 50 rotor (Beckman Instruments, Inc., Spinco Div., Palo Alto, Calif.) at 44,000 rpm at 250C for 40 h (16).…”

Effect of nicotinamide on RNA and DNA synthesis and on poly(ADP‐ribose) polymerase activity in normal and phytohemagglutinin stimulated human lymphocytes

“…A higher poly(ADP-ribose) polymerase activity in leukemic leukocytes than in lymphocytes from normal donors was observed [6,7]. Similarly, in human and pig lymphocytes, the activity of poly(ADP-ribose) polymerase increased together with DNA synthesis, induced by phytohemagglutinin PHA [7,8]. Finally, an increase in poly(ADP-ribose) polymerase activity in parallel to cellular NAD decrease after treatment of cells with DNA-damaging agents was reported [9,10].…”