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Biomedical subjects

D Ayusawa

Publications and source records attributed to D Ayusawa.

At least 73 records · Page 4Linked to original sources
PubMed1986

Cell-cycle-directed regulation of thymidylate synthase messenger RNA in human diploid fibroblasts stimulated to proliferate.

Human diploid fibroblasts were synchronized in the resting phase by incubation in medium containing a low level of serum and then stimulated to proliferate by adding a high concentration of serum. DNA replication started 12 hours after addition of serum, and reached a maximum after 24 hours. Thymidylate synthase activity was very low in resting cells, but began to increase 12 hours after growth stimulation and thereafter continued to increase. Thymidylate synthase mRNA in the growing cells was compared with that in resting cells, using cloned human thymidylate synthase cDNA as a probe. Results showed that the mRNA content as a percentage of total RNA began to increase six hours after stimulation, reaching a level about 14 times that in unstimulated cells after 24 hours. However, the mRNA content relative to poly(A)+ RNA had increased two- to fourfold by 24 hours after growth stimulation. Transcription of the thymidylate synthase gene, determined by hybridizing labelled nascent transcripts obtained in isolated nuclei to immobilized human thymidylate synthase cDNA, was similar in the nuclei of resting and of growth-stimulated cells. These results show that the increase in thymidylate synthase mRNA in growth-stimulated cells is caused by an increase in post-transcriptional events.

Cell Division↗
PubMed1986

Aphidicolin-resistant mutants of mouse lymphoma L5178Y cells with a high incidence of spontaneous sister chromatid exchanges.

Two aphidicolin-resistant cell mutants (AC 12 and AC 41) with a fourfold increase in spontaneous frequency of sister chromatid exchanges (SCEs) were obtained out of over 400 aphidicolin-resistant mutants isolated from mouse lymphoma L5178Y cells. They also exhibited three- to fourfold increases in spontaneous frequency of chromosome aberrations (CAs). To determine whether the high level of SCE frequency in AC 12 is caused by 5-bromodeoxyuridine (BrdUrd) used for visualizing SCEs, the effect of BrdUrd incorporated into DNA on SCE induction was analyzed. The SCE frequencies in AC 12 remained constant at BrdUrd incorporation levels corresponding to 2-90% substitution for thymidine in DNA. In addition, the small amount of BrdUrd incorporated into both daughter and parenteral DNA strands in AC 12 had minimal effect on SCE induction. Furthermore, AC 12 and AC 41 were slightly resistant to BrdUrd with respect to the induction of CAs, the inhibition of cell-cycle progression and the decrease in mitotic activity. These findings suggest that the high incidence of SCEs in AC 12 and AC 41 is formed by their intrinsic defects, not by the effects of BrdUrd used. The analysis of SCE frequencies in hybrid cells between these mutants and the parental L5178Y revealed that the genetic defects in AC 12 and AC 41 appear to be recessive, and that these two mutants belong to the same complementation group. Furthermore, AC 12 belonged to a different complementation group from ES 4, which was isolated previously from L5178Y as an SCE mutant with a twofold higher frequency of spontaneous SCEs. This finding indicates that at least two different genetic defects participate in the formation of the high incidence of spontaneous SCEs in mouse cells. These SCE mutants would provide valuable cell materials for studying the molecular mechanism of SCE formation.

Animals↗
PubMed1986

Induction, by thymidylate stress, of genetic recombination as evidenced by deletion of a transferred genetic marker in mouse FM3A cells.

Studies were made on the genetic consequences of methotrexate-directed thymidylate stress, focusing attention on a human thymidylate synthase gene that was introduced as a heterologous genetic marker into mouse thymidylate synthase-negative mutant cells. Thymidylate stress induced thymidylate synthase-negative segregants with concomitant loss of human thymidylate synthase activity with frequencies 1 to 2 orders of magnitude higher than the uninduced spontaneous level in some but not all transformant lines. Induction of the segregants was suppressed almost completely by cycloheximide and partially by caffeine. Thymidylate stress did not, however, induce mutations, as determined by measuring resistance to ouabain or 6-thioguanine. Thymidylate synthase-negative segregants were also induced by other means such as bromodeoxyuridine treatment and X-ray irradiation. In each of the synthase-negative segregants induced by thymidylate stress, a DNA segment including almost the whole coding region of the transferred human thymidylate synthase gene was deleted in a very specific manner, as shown by Southern blot analysis with a human Alu sequence and a human thymidylate synthase cDNA as probes. In the segregants that emerged spontaneously at low frequency, the entire transferred genetic marker was lost. In the segregants induced by X-ray irradiation, structural alterations of the genetic marker were random. These results show that thymidylate stress is a physiological factor that provokes the instability of this exogenously incorporated DNA in some specific manner and produces nonrandom genetic recombination in mammalian cells.

Animals↗
PubMed1986

Establishment of mutant murine mammary carcinoma FM3A cell strains transformed with the herpes simplex virus type 2 thymidine kinase gene.

To establish cell systems appropriate for investigating the mode of action of antiherpetic nucleoside analogues, mutant cell strains were constructed from murine mammary carcinoma FM3A cells, which were deficient in TK, but were transformed with a recombinant plasmid DNA containing the HSV-2 TK gene. The transformed cells incorporated the viral DNA, expressed viral TK activity and showed unusually high sensitivity to the cytostatic action of the antiherpetic nucleoside analogues ACV and IVDU, both of which were only weakly inhibitory to the growth of the parent cells. Curiously, the FM3A cell strains transformed with HSV-2 TK gene showed a higher sensitivity to ACV and IVDU than the previously established cell line transformed with HSV-1 TK gene. This contrasts with the inhibitory effects of ACV and IVDU on acute HSV infection, since HSV-2 infection is slightly or considerably less susceptible than HSV-1 infection to inhibition by ACV or IVDU, respectively.

Acyclovir↗
PubMed1986

The cytotoxicity of 5-fluorouracil is due to its incorporation into RNA not its inhibition of thymidylate synthase as evidenced by the use of a mouse cell mutant deficient in thymidylate synthase.

The biochemical basis for the cytotoxicity of 5-fluorouracil is still controversial; it is not clear whether the mechanism involves interference with DNA metabolism or incorporation of the drug into RNA. To distinguish between these two possibilities, we took advantage of a mutant strain of the mouse mammary tumor cell line FM3A that is deficient in thymidylate synthase, which is widely believed to be the target of 5-fluorouracil. We demonstrated that the target of the cytotoxicity of 5-fluorouracil was not thymidylate synthase; instead, the cytotoxicity was positively correlated with drug incorporation into RNA under conditions where thymidine increased the incorporation of 5-fluorouracil into RNA concentration-dependently.

Animals↗
PubMed1986

[Biochemical modulation of 5-FU by thymidine and N-phosphoacetyl-L-aspartate: the relation between 5-FU incorporation into RNA and its cytocidal effect].

We attempted to study the relation between 5-FU incorporation into RNA or DNA and the cytocidal effect of 5-FU using thymidine (TdR) and the effect of N-phosphoacetyl-L-aspartate (PALA) on the cytotoxicity of 5-FU. A mouse mammary carcinoma cell line FM3A was used in all of the experiments. The results showed that: addition of TdR to cell cultures resulted in an increase of 5-FU incorporation into RNA and although 5-FU incorporation into nucleic acid decreased, addition of 10(-3) M TdR induced of complete incorporation of 5-FU into only RNA as revealed by alkaline hydrolysis and cesium gradient analysis; no addition of TdR caused 5-FU to be completely incorporated into DNA only; cytocidal effect of 5-FU, in a clonogenic assay, occurred only during the time when TdR was added to the cell culture; PALA possessed the capacity to enhance the cytocidal effect of 5-FU in a clonogenic assay. These results confirmed that 5-FU incorporation into RNA is necessary for the cytocidal effect of 5-FU and that both TdR and PALA are available as 5-FU-modulating agents for enhancement of the cytocidal effect.

Animals↗
PubMed1985

Murine mammary FM3A carcinoma cells transformed with the herpes simplex virus type 1 thymidine kinase gene are highly sensitive to the growth-inhibitory properties of (E)-5-(2-bromovinyl)-2'-deoxyuridine and related compounds.

Murine mammary carcinoma (FM3A TK-/HSV-1 TK+) cells, which are thymidine kinase (TK)-deficient but have been transformed with the herpes simplex virus type 1 (HSV-1) TK gene are inhibited in their growth by (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), (E)-5-(2-iodovinyl)-2'-deoxyuridine (IVDU) and (E)-5-(2-bromovinyl)-2'-deoxycytidine (BVDC) at 0.5, 0.5 and 0.8 ng/ml, respectively; i.e., a concentration 5000 to 20 000-fold lower than that required to inhibit the growth of the corresponding wild-type FM3A/0 cells. Hence, transformation of tumor cells with the HSV-1 TK gene makes them particularly sensitive to the cytostatic action of BVDU and related compounds.

Animals↗
PubMed1985

Nucleotide sequence of a functional cDNA for human thymidylate synthase.

We have determined the nucleotide sequence of a cDNA clone, pcHTS-1, encoding human thymidylate synthase (5,10-methylenetetrahydrofolate: dUMP C-methyltransferase, EC 2.1.1.45) which was previously isolated from a human fibroblast expressible cDNA library and functional in mouse cells. The 1.6 kilobase cDNA insert of pcHTS-1 encodes a subunit protein of 313 amino acid (Mr = 35,706) and its predicted amino acid sequence is highly conserved in many regions including folylpolyglutamate and 5-fluoro-2'-deoxyuridylate binding sites, when compared with those of Lactobacillus casei, Escherichia coli, and bacteriophage T4. The cDNA contains in its 5'-untranslated region a triple tandemly repeated sequence consisting of 90 nucleotides, which starts immediately upstream of the ATG initiator codon, is very high in G+C content (80%), and can form three possible interconvertible stem-loop structures.

Amino Acid Sequence↗
PubMed1985

Thymineless death and genetic events in mammalian cells.

Thymidylate synthase-negative mutants of cultured mouse FM3A cells were immediately committed to cell death upon thymidine deprivation especially when the cells were synchronized in the S-phase. Thymidine deprivation induced single strand breaks in parental DNA strands, as measured by alkaline sucrose gradient sedimentation, giving rise to two peaks, one with large and the other with short fragments. Increase in the short DNA fragments paralleled that of thymineless death. Thymidine deprivation also accumulated double strand DNA fragments as determined by a method of neutral filter elution, and their extent paralleled that of cell death. Double-strand DNA eluted through the filter sedimented as a single peak both in a neutral and in an alkaline sucrose gradient that coincided with that of the above short DNA fragments. Therefore, the double strand breaks seemed to occur in some defined portions of the genome and in some specific manners in contrast to those induced by X-ray, which occurred rather randomly. Cycloheximide blocked thymineless death and accumulation of the double stranded DNA fragments in parallel. The double strand breaks induced by thymidine starvation were not repaired, but instead advanced on subsequent incubation of the cells in growth medium containing thymidine. Cytogenetically, thymidine deprivation induced chromosome aberrations such as chromatid breaks, chromatid interchanges, and chromosome fragmentation. Also, 5-bromodeoxyuridine deprivation induced sister chromatid exchange. Thymidylate stress also induced loss of a stably integrated human gene in mouse cells, possibly by DNA rearrangements, under the conditions where no point mutations were induced.

Animals↗
PubMed1985

Assignment of human gene encoding thymidylate synthase to chromosome 18 using interspecific cell hybrids between thymidylate synthase-negative mouse mutant cells and human diploid fibroblasts.

We have constructed interspecific somatic cell hybrids between a thymidine-auxotrophic mutant cell line of mouse FM3A cells that lacks thymidylate synthase and human diploid fibroblasts derived from a male patient with fragile X-linked mental retardation. Twenty primary hybrid clones were isolated independently, all of which exhibited the thymidine-prototrophic phenotype. Segregation of the hybrid cells in nonselective culture conditions gave rise to thymidine-auxotrophic hybrid clones. Both electrophoretic assay of thymidylate synthase activity and karyotype analysis of the segregants revealed a strong correlation between the expression of the human form of the enzyme and the presence of human chromosome 18. Thus, it is concluded that the functional gene for human thymidylate synthase, designated TS, is located on this chromosome.

Animals↗
PubMed1985

Purification and NH2-terminal amino acid sequence of human thymidylate synthase in an overproducing transformant of mouse FM3A cells.

Human thymidylate synthase [EC 2.1.1.45] was purified to homogeneity and its NH2-terminal amino acid sequence was determined taking advantage of the following facts: i) The source of the enzyme was a transformant of mouse FM3A mutant cells which lacks mouse thymidylate synthase but overproduces human thymidylate synthase. ii) The enzyme could be purified on two kinds of affinity column, Cibacron blue dye-bound agarose and methotrexate-bound Sepharose. iii) The enzyme could finally be separated from a trace of impurities by electrophoresis on polyacrylamide gel containing sodium dodecyl sulfate. The purified human thymidylate synthase had a subunit with a molecular weight of 33,000, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was subjected to Edman degradation and the NH2-terminal 24 amino acids were sequenced by successive use of a high-sensitivity gas-phase protein sequencer and high performance liquid chromatography to be as follows: Pro-Val-Ala-Gly-Ser-Glu-Leu-Pro-Arg-Arg-Pro-Leu-Pro-Pro-Ala-Ala-Gln-Glu- Arg-Asp -Ala-Glu-Pro-Arg-.

Amino Acid Sequence↗
PubMed1985

Establishment of mutant FM3A murine mammary carcinoma cell strains transformed with the herpes simplex virus type 1 thymidine kinase gene.

To establish cell systems appropriate for investigating the mode of action of anti-herpetic nucleoside analogs, mutants were constructed from murine FM3A mammary carcinoma cells, which were deficient in both thymidine kinase (EC 2.7.1.21) and thymidylate synthase (EC 2.1.1.45), but were transformed with a recombinant plasmid DNA containing the herpes simplex virus type 1 thymidine kinase gene. The transformed cells expressed viral thymidine kinase activity and showed unusually high sensitivity to the cytostatic action of the antiherpetic agent (E)-5-(2-iodovinyl)-2'-deoxyuridine, which was only weakly inhibitory to the growth of the parent cells.

Animals↗
PubMed1985

Highly selective cytostatic activity of (E)-5-(2-bromovinyl)-2'-deoxyuridine derivatives for murine mammary carcinoma (FM3A) cells transformed with the herpes simplex virus type 1 thymidine kinase gene.

(E)-5-(2-Bromovinyl)-2'-deoxyuridine (BVDU) and various structurally related analogues thereof, i.e., (E)-5-(2-iodovinyl)-2'-deoxyuridine (IVDU) and (E)-5-(2-bromovinyl)-2'-deoxycytidine (BVDC), and the carbocyclic analogues of BVDU, IVDU, and BVDC, were evaluated for their inhibitory effects on the growth of murine mammary carcinoma FM3A cells, deficient in thymidine kinase (TK) activity but transformed with the herpes simplex virus type 1 (HSV-1) TK gene (designated FM3A/TK-/HSV-1 TK+). BVDU and its congeners were much more inhibitory to the growth of FM3A/TK-/HSV-1 TK+ than to the growth of the wild type (FM3A/0) cells. For BVDU, for example, the 50% inhibitory dose for the FM3A/TK-/HSV-1 TK+ cells was 0.5 ng/ml, as compared to 11 micrograms/ml for the FM3A/0 cells. Evidently, BVDU and its congeners required phosphorylation by the HSV-1 TK to exert their cytostatic action. In attempts to evaluate further the mechanism of this cytostatic action, BVDU, IVDU, and their carbocyclic analogues were evaluated for their inhibitory effects on thymidylate synthetase (TS) and their incorporation into DNA. TS was identified as one, but not the sole, target in the cytostatic activity of BVDU and its derivatives. With [125I]IVDU and its carbocyclic analogue C-[125I]IVDU, clear evidence was obtained for the incorporation of these radiolabeled analogues into DNA of the FM3A/TK-/HSV-1 TK+ cell line and a TS-deficient mutant thereof, FM3A/TK-/HSV-1 TK+/TS-. No incorporation was detected with [125I]IVDU or C-[125I]IVDU into DNA of FM3A/0 and FM3A/TS- cells. To what extent the incorporation of [125I]IVDU and C-[125I]IVDU contributed to their cytostatic action against FM3A/TK-/HSV-1 TK+ cells remains the subject of further study.

Animals↗
PubMed1985

Selective inhibition of the proliferation of herpes simplex virus type 1 thymidine kinase gene-transformed murine mammary FM3A carcinoma cells by (E)-5-(2-bromovinyl)-2'-deoxyuridine and related compounds.

Mouse mammary carcinoma FM3A cells deficient in thymidine kinase were transformed by a cloned gene for herpes simplex virus type 1 thymidine kinase. Among several anti-herpetic nucleoside analogues, (E)-5-(2-bromovinyl)-2'-deoxyuridine, (E)-5-(2-iodovinyl)-2'-deoxyuridine and (E)-5-(2-bromovinyl)-2'-deoxycytidine inhibited the growth of the transformed cells at concentrations 5000- to 20000-fold lower than those required to inhibit the growth of the corresponding wild-type cells. The selective inhibitory action of these compounds was due to a specific phosphorylation by the viral thymidine kinase. From the transformed cells, thymidine-auxotrophic mutants that are deficient in thymidylate synthase were isolated. These mutant cell lines should prove useful in elucidating the mechanism of action of the antiherpetic nucleoside analogues.

Animals↗
PubMed1985

Expression of fragile site on the human X chromosome in somatic cell hybrids between human fragile X cells and thymidylate synthase-negative mouse mutant cells.

Fragile sites appear to be associated with a higher rate of breakage and specific chromosome rearrangement in cancer. A fragile site located on the human X chromosome at band Xq27 is known to be expressed under conditions of thymidylate stress. In order to obtain a model cell system suitable for studying the mechanism of expression of the fragile X site, interspecific somatic cell hybrids were constructed by cell fusion between human skin fibroblasts derived from a male patient with fragile X-linked mental retardation and thymidylate synthase-negative mouse mutant cells. The primary isolated hybrid clones were thymidine-prototrophic and expressed the fragile X site under conditions of thymidylate stress caused by 5-fluoro-2'-deoxyuridine treatment. In a thymidine-auxotrophic hybrid clone segregated from a thymidine-prototrophic hybrid clone, the expression of the fragile X site was induced in thymidylate stress conditions achieved by thymidine deprivation alone. This result provides direct evidence that expression of the fragile X site is dependent upon a lowered supply of thymidylate.

Animals↗
PubMed1985

Incorporation of 5-substituted pyrimidine nucleoside analogues into DNA of a thymidylate synthetase-deficient murine FM3A carcinoma cell line.

Various 5-substituted 2'-deoxyuridine (dUrd), 2'-deoxycytidine (dCyd), 1-beta-D-arabinofuranosyluracil (araU) and 1-beta-D-arabinofuranosylcytosine (araC) analogues have been investigated for their stimulatory effect on the growth of a thymidylate (dTMP) synthetase-deficient murine mammary carcinoma cell line (FM3A/TS-) that is auxotrophic for thymidine (dThd). Such stimulatory effect may be considered as indicative for the incorporation of the nucleoside analogue into host cell DNA. Based on this premise, several dUrd analogues were found to be incorporated into FM3A/TS- cell DNA (in decreasing order of incorporation): 5-bromo-dUrd greater than 5-chloro-dUrd greater than 5-(3-hydroxy-1-propynyl)-dUrd greater greater than 5-(1-pentynyl)-dUrd approximately 5-(1-propynyl)-dUrd approximately 5-iodo-dUrd greater than 5-(5-carboxy-1-hexenyl)-dUrd greater than 5-(3,3-dimethyl-1-butynyl)-dUrd greater than 5-ethyl-dUrd greater than 5-(5-chloro-1-pentynyl)-dUrd greater than 5-ethynyl-dUrd approximately 5 vinyl-dUrd greater than 5-phenylethynyl-dUrd greater than 5-(5-cyano-1-pentenyl)-dUrd greater than 5-(1-propenyl)-dUrd greater than 5-(1-hexynyl)-dUrd greater than 5-(5-hexyn-1-enyl)-dUrd. Among the 5-substituted dCyd analogues, 5-methyl-dCyd, 5-chloro-dCyd, 5-bromo-dCyd and 5-iodo-dCyd were also found to stimulate cell growth, and are therefore assumed to be incorporated into FM3A/TS- cell DNA. Since the stimulatory effects of these compounds on FM3A/TS- cell proliferation were suppressed in the presence of a Cyd deaminase inhibitor (tetrahydrouridine) or dCMP deaminase inhibitor (2'-deoxytetrahydrouridine), it is surmised that the dCyd analogues are first deaminated to the corresponding dUrd analogues before they are incorporated into DNA. None of the 5-substituted araU or araC analogues tested were able to sustain the growth of FM3A/TS- cells. It is postulated, therefore, that these araU or araC analogues are not incorporated to any appreciable extent into the DNA of FM3A/TS- cells, or, if they are incorporated, prevent cell growth. Thus, the dTMP synthetase-deficient FM3A/TS- cell line represents a unique system to distinguish those pyrimidine nucleoside analogues that are able to sustain cell growth and, therefore, assumed to be incorporated into the host cell DNA from those pyrimidine nucleoside analogues that are not.

Animals↗