Differential Ganciclovir-Mediated Cytotoxicity and Bystander Killing in Human Colon Carcinoma Cell Lines Expressing Herpes Simplex Virus Thymidine Kinase

Overview summary The transfer of HSV-TK into tumor cells and the subsequent sensitization to GCV have resulted in successful antitumor effects both in vitro and in vivo for a variety of cancers. This study focuses on evaluating and comparing two colon carcinoma cell lines for their ability to metabolize GCV and transfer phosphorylated metabolites to neighboring non-HSV-TK-expressing cells (bystander effect). Here we demonstrate differences in HSV-TK expression, GCV triphosphate accumulation, and incorporation into DNA and their effect on cytotoxicity. We also provide evidence of the transfer of phosphorylated GCV to bystander cells in a cell line deficient in gap junctional intercellular communication.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63405/1/hum.1998.9.6-801.pd

The role of DNA synthesis inhibition in the cytotoxicity of 2′,2′-difluoro-2′-deoxycytidine

Cytotoxicity from the anticancer drug 2′,2′-difluoro-2′-deoxycytidine (dFdCyd) has been correlated with its incorporation into DNA. However, cytotoxicity may also result from inhibition of DNA synthesis, due to either (1) dFdCyd diphosphate-mediated inhibition of ribonucleotide reductase, or (2) direct inhibition of DNA polymerases by the 5′-triphosphate of dFdCyd (dFdCTP). To elucidate the role of DNA synthesis inhibition in the cytotoxicity of dFdCyd, we compared dFdCyd to hydroxyurea (HU), a ribonucleotide reductase inhibitor, and aphidicolin, an inhibitor of DNA polymerases, in the U251 and D54 human glioblastoma cell lines.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46929/1/280_2003_Article_661.pd

Affinity of the antiviral enantiomers of oxathiolane cytosine nucleosides for human 2'-deoxycytidine kinase

The two enantiomers of 2',3'-dideoxy-3'-thiacytidine (BCH-189) and their 5-fluoro analogs (FTC) were found to be good substrates for human 2'-deoxycytidine kinase with Km values in the 5.7 to 42.1 [mu]M range. The affinity of the (-)-enantiomers was greater than that of the (+)-compounds. These results may explain the greater in vitro antiviral potency against human immunodeficiency virus and hepatitis B virus of the (-)-enantiomers when compared to their (+)-counterparts. The (+)- and (-)-enantiomers of FTC and BCH-189 are the first nucleoside analogs for which we have observed lower apparent kinetic constants for this enzyme in the presence of ATP compared to UTP.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/30848/1/0000510.pd

Inhibition of fludarabine metabolism by arabinosylcytosine during therapy

The active 5′-triphosphate of arabinosyl-2-fluoroadenine (F-ara-ATP) increases the anabolism of arabinosylcytosine (ara-C), whereas ara-C 5′-triphosphate inhibits the phosphorylation of arabinosyl-2-fluoroadenine (F-ara-A) in human leukemia cells in vitro. These interactions have a potential impact on drug scheduling. Clinical trials of relapsed leukemia in which fludarabine (F-ara-A 5′-monophosphate) and ara-C were given in sequence provided the opportunity to evaluate the effects of ara-C infusion on two sequelae: the pharmacokinetics of F-ara-A in plasma and that of F-ara-ATP in leukemia cells. First, F-ara-A pharmacokinetics were altered by ara-C infusion. This was visualized as a transient increase in F-ara-A plasma levels during the ara-C infusion that was given 4 h after fludarabine. The perturbation in F-ara-A plasma levels was dependent on the dose of ara-C. Second, peak F-ara-ATP concentrations were lower in leukemia cells of patients who received ara-C in addition to fludarabine as compared with those who received fludarabine alone. The terminal half-life of F-ara-A in plasma and the half-life of intracellular F-ara-ATP were reduced after the ara-C infusion in a concentration-dependent manner. Studies using purified deoxycytidine kinase support the conclusion that the increase in plasma levels of F-ara-A is in part the result of an effective competition by ara-C for phosphorylation by this enzyme, leading to a perturbation of the pharmacokinetics of intracellular F-ara-ATP.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46924/1/280_2004_Article_BF00685547.pd

Depletion of Deoxyribonucleotide Pools is an Endogenous Source of DNA Damage in Cells Undergoing Oncogene-Induced Senescence

In normal human cells, oncogene-induced senescence (OIS) depends on induction of DNA damage response (DDR). Oxidative stress and hyper-replication of genomic DNA have been proposed as major causes of DNA damage in OIS cells. Here we report that down-regulation of deoxyribonucleoside pools is another endogenous source of DNA damage in normal human fibroblasts (NHF) undergoing HRAS[superscript G12V]-induced senescence. NHF-HRAS[superscript G12V] cells under-expressed thymidylate synthase (TS) and ribonucleotide reductase (RR), two enzymes required for the entire de novo deoxyribonucleotide biosynthesis, and possessed low dNTP levels. Chromatin at the promoters of the genes encoding TS and RR was enriched with RB tumor suppressor protein and histone H3 tri-methylated at lysine 9. Importantly, ectopic co-expression of TS and RR or addition of deoxyribonucleosides substantially suppressed DNA damage, senescence-associated phenotypes and proliferation arrest in two types of NHF expressing HRAS[superscript G12V]. Reciprocally, shRNA-mediated suppression of TS and RR caused DNA damage and senescence in NHF although less efficiently than HRAS[superscript G12V]. However, overexpression of TS and RR in quiescent NHF did not overcome proliferation arrest, suggesting that unlike quiescence, OIS requires depletion of dNTP pools and activated DNA replication. Our data identify a previously unknown role of deoxyribonucleotides in regulation of oncogene-induced senescence.This is the author's peer-reviewed final manuscript, as accepted by the publisher. The published article is copyrighted by Elsevier and can be found at: http://www.journals.elsevier.com/the-american-journal-of-pathology/

Ribonucleotide reductase and thymidylate synthase or exogenous deoxyribonucleosides reduce DNA damage and senescence caused by C‐MYC depletion

The down‐regulation of dominant oncogenes, including C‐MYC, in tumor cells often leads to the induction of senescence via mechanisms that are not completely identified. In the current study, we demonstrate that MYC‐depleted melanoma cells undergo extensive DNA damage that is caused by the underexpression of thymidylate synthase (TS) and ribonucleotide reductase (RR) and subsequent depletion of deoxyribonucleoside triphosphate pools. Simultaneous genetic inhibition of TS and RR in melanoma cells induced DNA damage and senescence phenotypes very similar to the ones caused by MYC‐depletion. Reciprocally, overexpression of TS and RR in melanoma cells or addition of deoxyribonucleosides to culture media substantially inhibited DNA damage and senescence‐associated phenotypes caused by C‐MYC depletion. Our data demonstrate the essential role of TS and RR in C‐MYC‐dependent suppression of senescence in melanoma cells.Keywords: ribonucleotide reductase, oncogene‐induced senescence, dNTP, myc, melanoma, thymidylate synthas

Rapid communication : Inhibition of adenosine deaminase by azapurine ribonucleosides

We have synthesized several 8-azapurine nucleosides as inhibitors of adenosine deaminase. The presence of a nitrogen on the imidazole ring decreased the Ki value for nebularine by 100-fold but did not lower the Ki value for coformycin. Evaluation of these compounds in a MOLT-4 growth assay revealed that 2-azacoformycin was as effective as 2'-deoxycoformycin in potentiating growth inhibition by 2'-deoxyadenosine. The azapurine nucleosides merit further study as antitumor agents.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29732/1/0000068.pd

cDNA and Amino Acid Sequence of Human Adenosine Deaminase a

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/74342/1/j.1749-6632.1985.tb27114.x.pd