Amplification of pyridine nucleotide pools in mitogen-stimulated human lymphocytes

Berger, Nathan A.; Berger, Sosamma J.; Sikorski, Georgina W.; Catino, Donna M.

doi:10.1016/0014-4827(82)90010-6

Cited by 51 publications

(18 citation statements)

References 29 publications

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“…1) is consistent with the proposed action of these chemicals as inhibiting ADPRT [18,19] by competing with the enzyme's substrate, NAD (average concentration inside lymphocytes 0.35 mM, calculated from the data in [29]). The inhibitors do not affect lymphoid cell cycle events at these concentrations as shown by their lack of inhibition of proliferation of already activated lymphocytes (later additions in Fig.…”

Section: Discussionsupporting

confidence: 80%

“…If they are constantly being rejoined and broken again, the continual ADPRT activity required for efficient ligation might explain the markedly low level of NAD in lymphocytes, which increases to normal following PHA stimulation [29]. The DNA breaks may also be relevant to the unusual sensitivity of lymphocytes to radiation, which diminishes after mitogeninduced activation [31].…”

Section: Discussionmentioning

confidence: 99%

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Rejoining of DNA strand breaks is an early nuclear event during the stimulation of quiescent lymphocytes

Johnstone

1984

Eur J Biochem

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Activation of quiescent human peripheral blood lymphocytes or purified T cells by the mitogen, phytohemagglutinin (PHA), involves a rapid rejoining of DNA breaks present in the resting cells as detected by both nucleoid sedimentation analysis and rate of strand unwinding in alkali. Inhibitors of the enzyme ADP-ribosyltransferase (ADPRT) prevent activation of peripheral lymphocytes or T cells by PHA or concanavalin A in a dose-dependent manner, but only if present during the early stages. They do not affect subsequent proliferation if added later, nor do they inhibit the growth of lymphoblastoid cell lines. The inhibitors slow the rejoining of DNA breaks but do not affect the binding of mitogen to the cell surface or the early PHA-stimulated turnover of plasma membrane inositol phospholipids. DNA breaking and rejoining, regulated by ADPRT, may be involved in controlling gene expression during differentiation.Cell differentiation is the process by which genetic information is selectively expressed to produce cells with various morphologies and functions. This process is fundamentally important not only in the development of multicellular organisms but also in the life cycles of many single-celled eukaryotes. It has been known for some time that different types of differentiated cells contain different populations of messenger RNA, which are translated to produce the proteins peculiar to each type. However, many of the processes involved in the integrated changes, necessary for selectively expressing genetic information during differentiation, are still obscure.From work on chick embryo muscle cells the transient appearance of single-strand breaks in DNA, regulated by nuclear ADP-ribosyltransferase (ADPRT), has been proposed as part of a general mechanism for eukaryotic differentiation [l, 21. ADPRT is a DNA-dependent enzyme, which covalently attaches ADP-ribose moieties, derived from NAD, to many proteins [3,4]. Breaks in DNA strongly stimulate ADPRT [5,6] and it has been proposed that the enzyme regulates DNA repair [7,8] by increasing the activity of DNA ligase I1 [9,10].The stimulation of quiescent lymphocytes in vitro to change their morphology, proliferate and acquire effector functions has been widely studied at the molecular level [l I -141 because of its relevance to the immune system and also as a model for cell differentiation in general. This paper supports the hypothesis of Farzaneh et al. [1,2] by demonstrating the rejoining of DNA breaks soon after mitogen stimulation of human peripheral blood lymphocytes using two independent assays. Furthermore, the process of lymphocyte activation is blocked by competitive inhibitors of ADPRT. The relationship of DNA break rejoining and ADPRT activity to each other and to other molecular events in lymphocyte activation is investigated and the study also extended to purified T lymphocytes and lymphoblastoid cell lines to simplify interpretation of the data. A preliminary report of one aspect of this work has been published [15]. MATERIALS AND METHODS CellsPeripher...

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Section: Discussionsupporting

confidence: 80%

Section: Discussionmentioning

confidence: 99%

Rejoining of DNA strand breaks is an early nuclear event during the stimulation of quiescent lymphocytes

Johnstone

1984

Eur J Biochem

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“…After informed consent, 150 ml of blood was drawn from healthy donors into plastic syringes. Blood was anticoagulated with heparin or was defibrinated in the presence of50 ml 5% dextran in order to remove platelets as previously described (37). Preparation ofcells by either method gave similar results.…”

Section: Methodsmentioning

confidence: 94%

“…The defibrinated buffy coat or heparinized whole blood was layered over Ficoll-Hypaque (specific gravity = 1.077, Sigma Chemical Co.) and separated by density gradient centrifugation at 1,000 g X 30 min at 22°C as previously described (38). Granulocytes in the pellet were isolated after osmotic shock lysis of the erythrocytes by incubation at 4°C for 3 min in 4 vol of lysing buffer consisting of 0.155 M NH4Cl, 0.015 M NH4HCO3, and 0.1 mM EDTA followed by dilution into phosphate-buffered saline (PBS) (37) and centrifugation at 500 g X 10 min. Low density mononuclear cells were recovered at the Ficoll-Hypaque interface.…”

Section: Methodsmentioning

confidence: 99%

O6 alkylguanine-DNA alkyltransferase activity in human myeloid cells.

Gerson¹,

Miller²,

Berger³

1985

J. Clin. Invest.

103

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The association between alkylating agent exposure and acute nonlymphocytic leukemia in humans indicates that myeloid cells may be particularly susceptible to mutagenic damage. Alkylating agent mutagenesis is frequently mediated through formation and persistence of a particular DNA base adduct, O6alkylguanine, which preferentially mispairs with thymine rather than cytosine, leading to point mutations. O6alkylguanine is repaired by O6alkylguanine-DNA alkyltransferase (alkyltransferase), a protein that removes the adduct, leaving an intact guanine base in DNA. We measured alkyltransferase activity in myeloid precursors and compared it with levels in other cells and tissues.In peripheral blood granulocytes, monocytes, T lymphocytes, and B lymphocytes, there was an eightfold range of activity between individuals but only a twofold range in the mean activity between cell types. Normal donors maintained stable levels of alkyltransferase activity over time. In bone marrow T lymphocytes and myeloid precursors, there was an eightfold range of alkyltransferase activity between donors. Alkyltransferase activity in the two cell types was closely correlated in individual donors, r = 0.69, P < 0.005, but was significantly higher in the T lymphocytes than the myeloid precursors, P < 0.05. Liver contained the highest levels of alkyltransferase of all tissues tested. By comparison, small intestine contained 34%, colon 14%, T lymphocytes 11%, brain 11%, and myeloid precursors 6.6% of the activity found in liver. Thus, human myeloid precursors have low levels of Oalkylguanine-DNA alkyltransferase compared with other tissues. Low levels of this DNA repair protein may increase the susceptibility of myeloid precursors to malignant transformation after exposure to certain alkylating agents.

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“…Because of the important requirement for NAD and poly(ADP-ribose) metabolism in the DNA repair process (16- (20,21).…”

Section: Introductionmentioning

confidence: 99%

Modulation of nicotinamide adenine dinucleotide and poly(adenosine diphosphoribose) metabolism by the synthetic "C" nucleoside analogs, tiazofurin and selenazofurin. A new strategy for cancer chemotherapy.

Berger¹,

Berger²,

Catino³

et al. 1985

J. Clin. Invest.

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Tiazofurin (2-8-D-ribofuranosylthiazole4-carboxamide) and selenazofurin (2-%-D-ribofuranosylselenazole4-crboxmiude) are synthetic 'C" nucleosides whose antineoplastic activity depends on their conversion to tiazofurin-adenine dinucleotide and selenazofurin-adenine dinucleotide which are analogs of NAD. The present study was conducted to determine whether these nucleoside analogs and their dinucleotide derivatives interfere with NAD metabolism and in particular with the NADdependent enzyme, poly(ADP-ribose) polymerase. Incubation of L1210 cells with 10 gM tiazofurin or selenazofurin resulted in inhibition of cell growth, reduction of cellular NAD content, and interference with NAD synthesis. Using 114Clnicotinamide to study the uptake of nicotinamide and its conversion to NAD, we showed that the analogs interfere with NAD synthesis, apparently by blocking formation of nicotinamide mononucleotide. The analogs also serve as weak inhibitors of poly(ADPribose) polymernse, which is an NAD-utilizing, chromatinbound enzyme, whose function is required for normal DNA repair processes. Continuous incubation of L1210 cells in tiazofurin or selenazofurin resulted in progressive and synergistic potentiation of the cytotoxic effects of DNA-damaging agents, such as 1,3-bis(2-chloroethyl)-1-nitrosourea or N-methyl-N'-nitro-N-nitrosoguanidine. These studies provide a basis for designing chemotherapy combinations in which tiazofurin or selenazofurin are used to modulate NAD and poly(ADP-ribose) metabolism to synergistically potentiate the effects of DNA strand-disrupting agents.

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Amplification of pyridine nucleotide pools in mitogen-stimulated human lymphocytes

Cited by 51 publications

References 29 publications

Rejoining of DNA strand breaks is an early nuclear event during the stimulation of quiescent lymphocytes

Rejoining of DNA strand breaks is an early nuclear event during the stimulation of quiescent lymphocytes

O6 alkylguanine-DNA alkyltransferase activity in human myeloid cells.

Modulation of nicotinamide adenine dinucleotide and poly(adenosine diphosphoribose) metabolism by the synthetic "C" nucleoside analogs, tiazofurin and selenazofurin. A new strategy for cancer chemotherapy.

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