Georgina W. Sikorski

Aphidicolin inhibits eukaryotic DNA replication and repair — Implications for involvement of DNA polymerase α in both processes

Abstract Normal human lymphocytes were stimulated with phytohemagglutinin or treated with UV radiation or N-methyl-N′-nitro-N-nitroso guanidine then rendered permeable to exogenously supplied nucleotides and used to measure the replicative and repair modes of DNA synthesis. Aphidicolin, N-ethyl maleimide and several other compounds inhibited both DNA replication and repair. Since aphidicolin and N-ethyl maleimide are selective inhibitors of DNA polymerase α, these findings suggest that DNA polymerase α is involved in both the replicative and repair modes of DNA synthesis.

Nicotinamide stimulates repair of DNA damage in human lymphocytes

Abstract Nicotinamide stimulates the amount of DNA repair synthesis that occurs when freshly isolated, normal human lymphocytes are treated with UV irradiation, N-methyl-N′-nitro-N-nitroso guanidine, or dimethyl sulfate. Stimulation of DNA repair synthesis is concentration dependent and reaches a maximum between 2 to 5 mM nicotinamide. In contrast, DNA synthesis in cells that have not been subjected to DNA damage is not affected by nicotinamide at concentrations below 2 mM and is inhibited by concentrations between 2 to 5 mM. In the same concentration range, nicotinic acid has no effect on the rate of DNA synthesis in the presence or absence of DNA damage.

Synthesis of DNA and poly(adenosine diphosphate ribose) in normal and chronic lymphocytic leukemia lymphocytes.

Peripheral blood lymphocytes were isolated from 9 patients with chronic lymphocytic leukemia (CLL) and 12 normal control donors. The cells were assayed for synthesis of DNA and poly-(adenosine diphosphate ribose) (poly[ADPR]) immediately after isolation and on successive days following their treatment with phytohemagglutinin (PHA). Two different techniques were used to measure DNA synthesis. In the standard technique, DNA synthesis was measured by incubating intact cells with [(3)H]deoxythymidine. In the new technique, the lymphocytes were first rendered permeable to nucleotides, then DNA synthesis was measured by incubating them with [(3)H]deoxythymidine triphosphate in the presence of deoxyATP, deoxyGTP, deoxyCTP, ATP, and Mg(++). Both assays showed the anticipated rise in DNA synthesis after PHA stimulation of normal cells. PHA-stimulated lymphocytes from patients with CLL demonstrated low levels of DNA synthesis in both assay systems. The initial levels of poly(ADPR) synthesis were greater in CLL lymphocytes than in normal cells. Studies with a T-cell-depleted population of normal cells showed the same activity for poly(ADPR) synthesis that was demonstrated by the original population of normal cells. PHA stimulation produced an increase in poly(ADPR) synthesis in both the normal and CLL cells. The increase in poly(ADPR) synthesis in normal cells was coincident with the increase in DNA synthesis. The increase in poly(ADPR) synthesis in the CLL cells was dissociated from the delayed and diminished increase in DNA synthesis. Thus, CLL cells have higher than normal initial levels of poly(ADPR) synthesis. Poly(ADPR) synthesis is dissociated from DNA synthesis in CLL cells whereas it varies directly with DNA synthesis in normal lymphocytes.

Amplification of pyridine nucleotide pools in mitogen-stimulated human lymphocytes.

Abstract NAD+ levels in resting human lymphocytes obtained from 20 donors were found to be 69.9 ± 21.7 pmols/106 cells. After 3 days of phytohemagglutinin (PHA) stimulation the NAD+ levels rose to 452 ± 198 pmols/106 cells. NADH, NADP+ and NADPH also increased in mitogen-stimulated lymphocytes, but the major portion of the increase in total pyridine nucleotide pools was accounted for by the increase in NAD+. When PHA-stimulated lymphocytes were incubated in nicotinamide-deficient growth medium, there was no significant increase in their total pyridine nucleotide pools; however, the ratios of oxidized to reduced pyridine nucleotides changed in a similar fashion to cells grown in medium containing nicotinamide. When lymphocytes in nicotinamide-deficient medium were stimulated with PHA they increased their levels of DNA synthesis and cell replication in a similar fashion to cells growing in nicotinamide-supplemented media. Human lymphocytes were able to synthesize pyridine nucleotides from nicotinamide or nicotinic acid; however, in the absence of a preformed pyridine ring they did not efficiently use tryptophan for the synthesis of NAD. Uptake of [carbonyl-14C]nicotinamide and conversion to NAD was markedly increased in PHA-stimulated lymphocytes; these cells also showed a marked increase in activity of the enzyme adenosine-triphosphate-nicotinamide mononucleotide (ATP-NMN) adenylyl transferase.

Phosphonoacetate inhibition of deoxyribonucleic acid synthesis in intact and permeable eukaryotic cells.

Abstract Phosphonoacetate was evaluated as an inhibitor of DNA synthesis in L cells, L 1210 cells and phytohemagglutinin-stimulated human lymphocytes. L cells were most sensitive and lymphocytes least sensitive to inhibition by phosphonacetate. L cells showed rapid uptake of [ 14 C]phosphonoacetate compared to much slower uptake by L1210 cells and human lymphocytes. The rate of uptake in the three cell types correlated with the degree of inhibition of DNA synthesis. When the drug was studied in permeabilized cells, the DNA synthetic enzymes in all three cell types showed the same susceptibility to inhibition by phosphonoacetate. Thus, the differences in phosphonoacetate inhibition of DNA synthesis in intact L cells, L 1210 cells and human lymphocytes appear to be due to differences in drug uptake rather than to any differences in sensitivity to its inhibitory action.

ATP-independent DNA synthesis in vaccinia-infected L cells

Mouse L cells can be made permeable to exogenous nucleotides by a cold shock in 0.01 M Tris . HCl pH 7.8, 0.25 M sucrose, 1 mM EDTA, 30 mM 2-mercaptoethanol and 4 mM MgCl2. DNA synthesis in permeabilized L cells requires ATP whereas DNA synthesis in permeabilized L cells that are infected with Vaccinia virus is ATP-independent. Permeabilized L cells that are infected with ultraviolet-irradiated virus show a marked suppression of DNA synthesis which is not corrected by an excess of deoxynucleoside triphosphates and ATP. The ATP-dependent and ATP-independent processes of DNA synthesis are inhibited to the same extent by Mal-Net, pHMB, ara CTP and phosphonoacetate. Concentrations of daunorubicin and cytembena, which cause marked inhibition of the ATP-dependent enzymes, only cause partial inhibition of the ATP-independent enzymes.