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K Keyomarsi

Publications and source records attributed to K Keyomarsi.

45 records · Page 3Linked to original sources
PubMed1990

Quinazoline folate analogs inhibit the catalytic activity of thymidylate synthase but allow binding of 5-fluorodeoxyuridylate.

We have investigated some unusual aspects of the inhibition of mammalian thymidylate synthase (TS) by the folate antimetabolite, 10-propargyl-5,8-dideaza-folic acid (CB 3717). From our results, we conclude that binding of CB 3717 metabolites to one subunit of L1210 TS modified the conformation of the second active site of this enzyme so that it retained the ability to bind 5-fluro-2'-deoxyuridine-5'-monophosphate (FdUMP) but not its catalytic activity. Exposure of intact mouse L1210 cells to CB 3717 resulted in inactivation of cellular TS activity, yet desalted cytosol preparations from these cells retained the ability to bind FdUMP. The same effect was found with several analogs of CB 3717. Complexes of FdUMP formed in vitro with TS from cells exposed to CB 3717 were covalent and co-migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with complexes of FdUMP, folate cofactor, and TS from cells not exposed to CB 3717. In the presence of dUMP, a tightly bound complex rapidly formed between isolated pure TS and the pentaglutamate of CB 3717 but not the monoglutamate form of this compound. Binding experiments using CB 3717 pentaglutamate-inhibited TS suggested a stoichiometry of 1 mol of FdUMP bound per mol of dimeric TS.

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PubMed1990

An efficient deletion mutant packaging system for defective herpes simplex virus vectors: potential applications to human gene therapy and neuronal physiology.

We have previously described a defective herpes simplex virus (HSV-1) vector system that permits the introduction of virtually any gene into nonmitotic cells. pHSVlac, the prototype vector, stably expresses Escherichia coli beta-galactosidase from a constitutive promoter in many human cell lines, in cultured rat neurons from throughout the nervous system, and in cells in the adult rat brain. HSV-1 vectors expressing other genes may prove useful for studying neuronal physiology or performing human gene therapy for neurological diseases, such as Parkinson disease or brain tumors. A HSV-1 temperature-sensitive (ts) mutant, ts K, has been used as helper virus; ts mutants revert to wild type. In contrast, HSV-1 deletion mutants essentially cannot revert to wild type; therefore, use of a deletion mutant as helper virus might permit human gene therapy with HSV-1 vectors. We now report an efficient packaging system for HSV-1 vectors using a deletion mutant, D30EBA, as helper virus; virus is grown on the complementing cell line M64A. pHSVlac virus prepared using the deletion mutant packaging system stably expresses beta-galactosidase in cultured rat sympathetic neurons and glia. Both D30EBA and ts K contain a mutation in the IE3 gene of HSV-1 strain 17 and have the same phenotype; therefore, changing the helper virus from ts K to D30EBA does not alter the host range or other properties of the HSV-1 vector system.

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PubMed1990

Progression through the cell cycle: an overview.

Tissues in adults can be maintained at constant mass or they can increase or decrease in size because of imbalances of synthetic and degradative processes acting at the cellular and molecular levels. Some size changes are caused by physiologic conditions to which the tissue must adjust. Alternatively, the balance may be distorted in favor of net tissue increase in pathologic situations such as cancer. Strict regulatory mechanisms are required to keep proliferation responsive to the organism's needs; these mechanisms may be defective in disease. Net tissue proliferation requires repeated rounds of cell duplication in excess of that necessary to counterbalance cell death. Duplication of a cell requires a net doubling of its every molecule and structure. The myriad of molecular events required for cell proliferation such as DNA duplication and its partitioning at mitosis are tightly regulated in normal cells. One may conceive of two classes of molecules: those required for "housekeeping," which constitute the cell's structural and functional machinery, and those such as growth factors, their receptors, and second messengers involved in signal transduction responsible for regulating the activities of the housekeeping molecules. These molecular events and the cascade of processes that control them can be organized within the sequence of the cell cycle. In this brief overview, we illustrate these issues with a few examples taken from very recent discoveries of novel proteins that appear to have major regulatory roles. Most of these results have been obtained with mammalian fibroblasts, but some have originated with discoveries made using two very different yeasts.(ABSTRACT TRUNCATED AT 250 WORDS)

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PubMed1988

Mechanism of the cytotoxic synergism of fluoropyrimidines and folinic acid in mouse leukemic cells.

We have investigated the mechanism by which reduced folates, such as folinic acid, enhance the cytotoxicity of fluoropyrimidines in L1210 mouse leukemic cells. Exposure of L1210 cells to folinic acid resulted in expansion of intracellular pools of 5,10-CH2-H4PteGlun, delayed the reappearance of catalytically active thymidylate synthase (TS) following 5-fluoro-2'-deoxyuridine exposure, and stabilized inhibited TS complexes over the same concentration range that augmented the cytotoxic effects of fluorodeoxyuridine and 5-fluorouracil. The data showed that, in intact L1210 cells, fluorodeoxyridylate behaves as an inhibitor whose complexes with TS dissociated with a biologically significant rate. However, these complexes become functionally irreversible in cells incubated with high levels of folinic acid. It was also found that bound and total TS levels increased in cells treated with fluorodeoxyuridine to an extent that substantially exceeded the increase in protein content per cell under the same conditions. These results are in accord with the concept that folinic acid augments the effects of the fluoropyrimidines by expansion of cellular 5,10-CH2-H4PteGlun pools with subsequent stabilization of ternary complexes among 5-fluoro-2'-deoxyuridine 5'-monophosphate, TS, and 5,10-CH2-H4PteGlun. In light of the accumulation of TS that occurs following exposure to fluoropyrimidines, this stabilization may be needed for efficient tumor cell killing by these agents.

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PubMed1988

Tumor cell responses to inhibition of thymidylate synthase.

Whether inhibition of thymidylate synthase is lethal to a population of tumor cells depends upon three factors: 1) the dependence of the cells upon de novo synthesis of thymidine nucleotides; 2) the length of time enzyme is inhibited and the requirement for thymidine nucleotides during this period; and 3) the biochemical responses of the cells to the initial inhibition of enzyme, many of which interfere with maintenance of thymidylate synthase in an inhibited state. Following inhibition of thymidylate synthase, deoxyuridylate accumulates, as does the cellular content of thymidylate synthase. In addition, the initially formed enzyme-inhibitor complexes dissociate. These biochemical sequelae alter the effectiveness of the blockade of thymidylate synthase in a time-dependent, continuously-changing manner. Whether cell kill occurs depends on whether the dynamic balance of these factors allows a sufficiently low enzymatic activity to be maintained for a long enough period of time. An analysis of this interaction of factors leads us to the conclusions that efficient tumor cell kill with fluoropyrimidines is best attained by combination with reduced folate cofactors and inhibitors of deoxypyrimidine biosynthesis. Each of these agents modifies the response of tumor cells with the result that the fluorodeoxyuridylate-induced inhibition of thymidylate synthase is maintained. This analysis also suggests that folate analogs inhibitory to thymidylate synthase are more compatible than pyrimidine analogs with inhibition of thymidylate synthase as an approach to cancer chemotherapy.

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PubMed1987

Biochemical rationale for the synergism of 5-fluorouracil and folinic acid.

The fluoropyrimidines, FUra and 5-fluoro-2'-deoxyuridine (FUdR), have been found to be more growth inhibitory and cytotoxic to both mouse and human tumor cells when grown in cell culture medium containing folinic acid. The increment in the activity of these drugs observed in folinate-containing medium was similar for a mouse leukemia cell line and for 4 human leukemia cell lines. This suggests that the mechanism of action of the fluoropyrimidines against these mouse and human cell lines is similar. The most probable mechanism of the interaction between folinic acid and the fluoropyrimidines is stabilization of thymidylate synthase (TS) in inactive complexes with 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP) and folate cofactor. Such trapping of enzyme in inactive form would negate the effects of the accumulation of the reaction substrate 2'-deoxyuridine-5'-monophosphate. It is suggested that the combination of FUra with folinic acid and, in addition, an inhibitor of ribonucleotide reductase such as hydroxyurea may be more effective than FUra and folinic acid alone.

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PubMed1986

Folinic acid augmentation of the effects of fluoropyrimidines on murine and human leukemic cells.

The effects of the fluoropyrimidines on leukemic cells of mouse and human origin have been studied in the presence of folinic acid. This reduced folate enhanced the cytotoxicity and the growth inhibitory potency of 5-fluorouracil (5-FUra) and of 5-fluoro-2'-deoxyuridine (FUdR) against all cell lines examined. The human leukemic cell lines used (two T- and two B-cells) were affected by these fluoropyrimidines only at substantially higher concentrations than were found to be inhibitory to mouse L1210 cells; however, the enhancement of the activity of the fluoropyrimidines occurred over the same range of folinic acid concentrations in mouse and human cells. Whereas the total intracellular folate pool increased continuously with every increment of folinic acid added to the medium, the enhancement of the potency of the fluoropyrimidines was limited. Augmentation of the effects of FUdR exceeded that of 5-fluorouracil in the human leukemic cells studied. The cytotoxicity of the fluoropyrimidines (as defined by cloning efficiency) was enhanced to a greater extent than was growth inhibition so that an impressive lethal synergism was noted; for instance, exposure of L1210 cells to nontoxic concentrations of 5-fluorouracil or FUdR in the presence of folinic acid resulted in a 98 or 99.9% cell kill, respectively. In contrast to previous predictions, the fluoropyrimidines were more inhibitory to mouse leukemic cells containing folate pools that were suboptimal for growth than for folate-replete cells. Growth rate experiments showed that cells exposed to moderate concentrations of FUdR were initially inhibited but recovered with time, whereas in cells exposed to both FUdR and folinic acid, the initial growth inhibitory effects were sustained. We conclude that folinic acid stabilizes the effects of the fluoropyrimidines on thymidylate synthase of both mouse and human leukemic cell populations and that this enhancement is reflected in both inhibition of the growth of and the lethality to these cells. We suggest that only doses of the fluoropyrimidines that are capable of initially inhibiting thymidylate synthase to a high degree will be synergistic with excess reduced folates.

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