Genetic analysis of resistance to total bromodeoxyuridine substitution in mammalian cell hybrids.

Somatic cell hybrids derived from the fusion of Chinese hamster ovary cells (CHO) and mutant Syrian hamster melanoma cells (2E) were tested for their ability to grow with all of the thymidine (dThd) in their DNA replaced with 5-bromo-2'-deoxyuridine (BrdU), a phenotypic capability of the 2E cells but not of the CHO cells. Under these conditions, the 2E cells survived and grew, all of the hybrid clones survived and grew to varying degrees, and the CHO cells did not survive at all. When 2E cells were tested, they were also found to be resistant to the toxic effects of BrdU substitution and white light irradiation, relative to CHO cells. Thus, when the DNAs of 2E and CHO cells were equally (50%) substituted with BrdU, and the two cell lines irradiated with identical doses of white light, the survival of CHO cells was reduced to less than 1% of that of unirradiated cells, while 40% of the 2E cells survived. The 2E x CHO hybrid clones were found to survive at values from 10% to 40% under these identical conditions. Thus, the phenotypic characteristics of 2E cells involving total substitution and resistance to the toxic effects of BrdU substitution and white light irradiation appear to be expressed in a codominant fashion in somatic cell hybrids.

Induction of sister chromatid exchanges by the thymidine analog 5-hydroxymethyl-2'-deoxyuridine.

The thymidine analog, 5-hydroxymethyl-2'-deoxyuridine (hmdUrd), was tested for its ability to induce sister chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells. When tested, hmdUrd was found to be a potent inducer of SCEs in CHO cells under nontoxic conditions. Under these same conditions, hmdUrd was found to be nonmutagenic, as no increase above the background frequency of 6-thioguanine-resistant mutants was observed. The induction of SCEs by hmdUrd was suppressed by thymidine. Simultaneous exposure of the cells to low concentrations of hmdUrd and to high concentrations of the free pyrimidine 5-hydroxymethyluracil (hmUrd), which had no effect alone, had a strong synergistic effect on the induction of SCEs. Simultaneous exposure of cells to hmdUrd and to 3-aminobenzamide, a potent inhibitor of poly(ADP-ribose) polymerase, was found to increase the level of SCEs induced by the hmdUrd. These findings support the hypothesis that the formation of hmUra residues in DNA may be an important factor in the genotoxicity of radiation and oxidative damage in mammalian cells.

The role of deoxyribonucleotide metabolism in 5-bromo-2'-deoxyuridine mutagenesis in mammalian cells.

The effects of deoxyribonucleoside triphosphate (dNTP) pool imbalance on the induction of mutations and sister-chromatid exchanges (SCEs) by 5-bromo-2'-deoxyuridine (BrdUrd) in mammalian cells is reviewed. The INC BrdUrd mutagenesis protocol involves the incorporation of BrdUrd into DNA under conditions of specific dNTP pool imbalance, while the REP BrdUrd mutagenesis protocol involves the replication of 5-bromouracil (BrUra)-substituted DNA in the presence of specific (but different) dNTP pool imbalance. Biochemical and genetic analyses of both the INC and REP mutagenesis protocols provided evidence that (1) INC mutagenesis resulted from errors of incorporation due to the mispairing of BrdUTP with a guanine residue in replicating DNA leading to GC to AT transitions and (2) REP mutagenesis resulted from errors of replication due to the mispairing of dGTP with a BrUra residue in replicating DNA leading to AT to GC transitions. Further analyses involving different cell lines has led to an hypothesis describing the role of mismatch repair in the induction of mutations and SCEs.

Analysis of mutagenesis and sister-chromatid exchanges induced by 5-bromo-2'-deoxyuridine in somatic hybrids derived from Syrian hamster melanoma cells and Chinese hamster ovary cells.

Somatic cell hybrids were derived from the fusion of Chinese hamster ovary (CHO) cells and Syrian hamster melanoma cells (2E). These two cell lines had previously been shown to differ in their response to the induction of mutations and sister-chromatid exchanges (SCEs) by 5-bromo-2'-deoxyuridine (BrdUrd) (Kaufman, 1987). The parental cells and a number of representative, independent hybrid clones were tested for their response to both the INC and REP mutagenesis protocols. INC mutagenesis involves the incorporation of BrdUrd into DNA under conditions of deoxyribonucleoside triphosphate (dNTP) pool imbalance, while REP mutagenesis involves the replication of 5-bromouracil-substituted DNA in the presence of dNTP pool imbalance. When tested for the toxic effects of high concentrations of BrdUrd and for the induction of mutations by the INC protocol, the hybrid clones all expressed the 2E phenotype, i.e., sensitivity to relatively low concentrations of BrdUrd and thymidine for the induction of mutations, dNTP pool perturbation, and the toxic effects of BrdUrd. When the hybrid clones were tested for the induction of mutations and SCEs by the REP protocol, it was found that they expressed the 2E phenotype for the induction of mutations and the CHO phenotype for the induction of SCEs. Thus, various aspects of the 2E phenotype, such as high mutation frequencies associated with large dNTP pool perturbations, appeared to be dominantly expressed in the cell hybrids, while the lack of induction of SCEs by these mutagenic conditions in 2E cells was found to be a recessive characteristic.

Report of two cases of acute myelogenous leukemia immunized with autologous leukemia-derived hybrid cells.

Reports that immunizations with leukemia-derived hybrid cells prolonged the survival of leukemic mice led us to attempt an analogous approach in two adult patients with acute myeloid leukemia (AML). Hybrid cells were prepared from the pretreatment marrows of the newly-diagnosed patients with D98OR cells, in the first case, and with KR12 cells, in the second case. (D98OR and KR12 cells are human cell-lines.) Hybrids formed with KR12 cells expressed HLA antigens of both parental sources and some of the clonal isolates expressed myeloid-associated determinants. The immunizations were performed during the first complete clinical remission; the patients were demonstrably immunocompetent. Positive delayed type hypersensitivity responses to both (X-irradiated) hybrid cells and to (X-irradiated) autologous pretreatment marrow were observed following the immunizations. Mixed lymphocyte reactions toward autologous marrow were positive in one of the patients. In both, relapse occurred approximately two months after the first immunization and eight months after first diagnosis. The first patient remained in complete remission for two and one-half years following reinduction chemotherapy; reinduction chemotherapy was unsuccessful in the second patient.

Resistance to toxic effects of 5-hydroxymethyl-2'-deoxyuridine in mammalian cells.

A spontaneously arising clone, stably resistant to the toxic effects of the thymidine analog, 5-hydroxymethyl-2'-deoxyuridine (hmdU), was isolated from unmutagenized V79.5 Chinese hamster fibroblast cells by a single-step selection procedure. The hmdUr cells were selected in the continuous presence of 30 microM hmdU, a concentration which reduces the plating efficiency of wild-type cells to less than 1% after a 24-h exposure. A line of human HeLa cells were found to be intrinsically resistant to concentrations of hmdU as high as 100 microM. All of the hmdUr cells were found to grow normally in HAT medium, which requires the expression of thymidine kinase activity; be sensitive to the toxic effects of high concentrations of 5-bromo-2'-deoxyuridine, another thymidine analog; have unaltered hmdU nucleotide metabolism, as measured by HPLC analysis of acid-soluble cell extracts; and have decreased levels of hmdU incorporation into DNA. Although high concentrations of 5-hydroxymethyluracil (hmUra) were found to be nontoxic for both wild-type and hmdUr cells, the resistance phenotype could be suppressed by exposing the cells to hmdU and high concentrations of hmUra simultaneously.

Uncoupling of the induction of mutations and sister-chromatid exchanges by the replication of 5-bromouracil-substituted DNA.

The REP mutagenesis protocol, which involves the replication of 5-bromouracil (BrUra)-substituted DNA in the presence of deoxyribonucleoside triphosphate (dNTP) pool imbalance, has been shown to induce both mutations and sister-chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells. However, when a Syrian hamster melanoma-derived cell line, called 2E, which was selected for its ability to replace all of the thymine residues in DNA with BrUra, was subjected to the REP mutagenesis protocol, the correlation between the induction of mutations and SCEs was no longer observed. The 2E cells were found to be much more sensitive to the induction of mutations by REP mutagenesis than were the CHO cells. This increased sensitivity to REP mutagenesis was found to correlate with increased perturbations of the dNTP pools that have been shown to be involved in the mutagenic mechanism of this protocol. In contrast, when the induction of SCEs by the REP protocol was measured, it was found that although a baseline level of SCEs was detected in 2E cells, no significant induction of SCEs due to dNTP pool perturbation was observed. It was shown that high levels of SCEs were readily induced in 2E cells by other agents, e.g. mitomycin C. A model, which discusses the fate of mismatched bases thought to be generated by the REP mutagenesis protocol as the determining factor for the induction of mutations of SCEs, is proposed to explain the uncoupling of mutagenesis and SCE induction in 2E cells.

Induction of sister-chromatid exchanges by the replication of 5-bromouracil-substituted DNA under conditions of nucleotide-pool imbalance.

The induction of sister-chromatid exchanges (SCEs) by the replication of 5-bromouracil(BrUra)-containing DNA under conditions of nucleotide-pool imbalance was investigated. A modification of a protocol developed for the induction of mutations under these conditions (E.R. Kaufman, Mol. Cell. Biol., 4, 2449-2454, 1984) was used. To induce SCEs, Chinese hamster ovary cells were grown under non-mutagenic conditions which allowed the uniform incorporation of BrUra into their DNA at specific levels of substitution for thymine residues (25, 50 and 75% BrUra substitution). After 4 and 5 days of growth, the cells, which had incorporated BrUra into their DNA, were washed free of 5-bromodeoxyuridine (BrdUrd) and provided with fresh culture medium supplemented with various concentrations of thymidine (10 microM to 3 mM) and no BrdUrd. The cells were allowed to replicate their BrUra-containing DNA under these conditions, in the absence of BrdUrd, for two rounds of DNA synthesis to achieve sister-chromatid differentiation, and second-division metaphases were scored for SCEs. The results of these studies indicated that the SCEs observed were proportional to the level of BrUra substituted for thymine in the cellular DNA, were induced by increasing concentrations of thymidine in the culture medium during replication of the BrUra-containing DNA, correlated well with the induction of mutations to thioguanine resistance and to ouabain resistance, correlated with increases in the intracellular levels of dTTP and dGTP generated by the high concentrations of thymidine. These findings provide direct evidence for the induction of SCEs by the replication of BrUra-containing DNA and for the importance of the pools of nucleoside triphosphate precursors for DNA replication in these processes. When the effects of 3-aminobenzamide, a potent inhibitor of poly(ADP-ribose) synthesis, were tested, it was found that 3-aminobenzamide significantly increased SCEs, but it had no effect on mutations induced.

Biochemical analysis of toxic effects of 5-hydroxymethyl-2'-deoxyuridine in mammalian cells.

Cellular and biochemical analyses of the toxic effects of the thymidine analog, 5-hydroxymethyl-2'-deoxyuridine (hmdUrd), were carried out using V79.5 Chinese hamster cells. It was found that the toxic effects of hmdUrd could be totally suppressed by the addition of thymidine at 1/10th the concentration of hmdUrd. When other pyrimidines were tested, deoxyuridine was found to also suppress toxicity, although not as well as thymidine, while orotate, uridine, cytidine, and deoxycytidine did not have significant effect. Biochemical analyses of the metabolic fate of hmdUrd demonstrated low but significant levels of hmdUrd triphosphate and the incorporation of 5-hydroxymethyluracil (hmUra) residues into DNA. Surprisingly, in addition to these metabolites, relatively high levels of free hmUra were also detected in the acid-soluble cell extracts. Further analysis demonstrated that when V79.5 cells were exposed to hmdUrd significant amounts of hmUra were released into the culture medium. In vitro assays provided evidence that hmdUrd was first phosphorylated to its monophosphate and then degraded to hmUra, possibly via the action of a new enzyme, hydroxymethyldeoxyuridylate phosphorylase. Exposure of cells to hmUra alone, at concentrations as high as 3 mM, had no effect on viability. However, when V79.5 cells were simultaneously exposed to low, nontoxic concentrations of hmdUrd and high, nontoxic concentrations of hmUra, a synergistic reduction in viability was observed. This synergistic effect was found to correlate with increased incorporation of hmUra into DNA, possibly via end-product inhibition of an hmUra-DNA glycosylase.

Altered CTP synthetase activity confers resistance to 5-bromodeoxyuridine toxicity and mutagenesis.

A V79 Chinese hamster fibroblast cell line selected for resistance to the toxic effects of 5-fluorouracil (Kaufman, 1984b) was found to be cross-resistant to the toxic effects of the thymidine analog 5-bromodeoxyuridine (BrdUrd). When tested for sensitivity to BrdUrd mutagenesis, the fluorouracil-resistant cells were found to be resistant to mutagenesis induced by high concentrations of BrdUrd in the medium (INC mutagenesis) but not to mutagenesis induced by the replication of DNA containing 5-bromouracil (REP mutagenesis). Analyses of deoxyribonucleoside triphosphate pools indicated that high endogenous dCTP levels in the mutant prevented the high BrdUTP/dCTP ratio associated with INC mutagenesis. However, the mutant phenotype had no effect on the nucleotide pool imbalance associated with REP mutagenesis. This mutant provides further genetic evidence for the existence of two independent mechanisms for BrdUrd mutagenesis in mammalian cells.

Right to healthy birth: medico-legal issues in phencyclidine ingestion.

Child abuse is a multifaceted issue which requires a multidisciplinary approach. To date, child abuse has dealt only with the born child. With the panepidemic abuse of phencyclidine (PCP), and the knowledge that it will remain in the body greater than one year, the concept of child abuse in utero and preconception must be considered. This paper addresses this topic. Legal intervention concerning "wrongful birth and wrongful life" is raised. Legislative solutions permitting further research as well as assisting the healthy birth of viable fetuses on phencyclidine are also recommended.

Reversion analysis of mutations induced by 5-bromodeoxyuridine mutagenesis in mammalian cells.

Two protocols have been developed, both of which utilize the thymidine analog 5-bromodeoxyuridine (BrdUrd) to induce mutations in mammalian cells in culture (E. R. Kaufman and R. L. Davidson, Proc. Natl. Acad. Sci. USA 75:4982-4986, 1978; E. R. Kaufman, Mol. Cell. Biol. 4:2449-2454, 1984). The first protocol, termed incorporational (INC) mutagenesis, utilizes high concentrations of BrdUrd in the culture medium to generate a high intracellular ratio of BrdUTP/dCTP. The second protocol, termed replicational (REP) mutagenesis, entails the incorporation of BrdUrd into DNA under nonmutagenic conditions, the removal of all BrdUrd from the culture medium, and the subsequent replication of the bromouracil-containing DNA in the presence of high intracellular levels of dTTP and dGTP. Genetic studies using reversion analysis at the hypoxanthine-guanine phosphoribosyltransferase locus were used to determine whether the mechanisms of these two BrdUrd mutagenesis protocols had enough specificity to be distinguishable by their ability to revert various mutants. The results of these studies indicated that (i) mutants induced by INC mutagenesis were induced to revert only by REP mutagenesis and not by INC mutagenesis, (ii) mutants induced by REP mutagenesis were more efficiently reverted by INC mutagenesis than by REP mutagenesis, and (iii) both spontaneous mutants and mutants induced by the chemical mutagen ethyl methanesulfonate showed a high degree of specificity when tested for reversion by the BrdUrd mutagenesis protocols.

Isolation and characterization of a mutant Chinese hamster cell line resistant to the glutamine analog 6-diazo-5-oxo-L-norleucine.

A mutant cell line, called don801, was isolated from a wild-type population of V79.5 Chinese hamster cells by its ability to grow in the presence of the glutamine analog 6-diazo-5-oxo-L-norleucine (DON), which is toxic for V79.5 cells. The don801 cells were found not to be cross-resistant to another glutamine analog, O-diazoacetyl-L-serine (azaserine, AS). It was shown that guanine but neither hypoxanthine nor adenine protected V79.5 cells from the toxic effects of DON, while hypoxanthine and adenine, but not guanine protected them against AS toxicity. Exposure of wild-type cells to DON was shown to result in a specific reduction of intracellular GTP pools, while in the mutant cells there was no effect on GTP levels. These results strongly suggested that DON was specifically inhibiting guanylate synthetase (GMP synthetase;xanthosine-5'-phosphate: L-glutamine amidoligase, EC 6.3.5.2) in V79.5 cells and that the enzyme in don801 cells was resistant to inhibition. In vitro assays of GMP synthetase activities from V79.5 and don801 cells confirmed this hypothesis. The mutant phenotype was also found to be dominant in intraspecific cell hybrids.

Replication of DNA containing 5-bromouracil can be mutagenic in Syrian hamster cells.

A new protocol for inducing mutations in mammalian cells in culture by exposure to the thymidine analog 5-bromodeoxyuridine (BrdUrd) was established. This protocol, called "DNA-dependent" mutagenesis, involved the incorporation of BrdUrd into DNA under nonmutagenic conditions and the subsequent replication of the 5-bromouracil (BrUra)-containing DNA under mutagenic conditions but with no BrdUrd present in the culture medium. The mutagenic conditions were induced by allowing BrUra-containing DNA to replicate in the presence of high concentrations of thymidine. This generated high intracellular levels of dTTP and dGTP, causing nucleotide pool imbalance. The mutagenesis induced by this protocol was found to correlate with the level of BrUra substituted for thymine in DNA.

Resistance to 5-fluorouracil associated with increased cytidine triphosphate levels in V79 Chinese hamster cells.

Clones stably resistant to the toxic effects of 5-fluorouracil have been isolated from V79.5 Chinese hamster fibroblast cells by a single-step selection procedure. The 5-fluorouracil-resistant lines were found to (a) have an auxotrophic requirement when grown in dialyzed fetal calf serum that was satisfied by the addition of either thymidine, deoxyuridine, or deoxycytidine to the medium, (b) be cross-resistant to the toxic effects of 1-beta-D-arabinofuranosylcytosine and to high concentrations of thymidine, (c) have increased intracellular levels of cytidine 5'-triphosphate (CTP) and deoxycytidine 5'-triphosphate and decreased levels of uridine 5'-triphosphate, (d) also be resistant to 5-fluorouridine but not to 5-fluorodeoxyuridine, and (e) incorporate less 5-fluorouracil into RNA than do the wild-type cells. The primary lesion in these mutant appears to be an altered CTP synthetase activity which is no longer sensitive to negative regulation by CTP. The resulting increased CTP levels appear to be responsible for the various phenotypic characteristics of these mutants, including the resistance to 5-fluorouracil.

Phencyclidine in umbilical cord blood: preliminary data.

The epidemic abuse of phencyclidine (PCP) has become a major psychiatric issue within the past decade. With the assistance of highly sensitive capillary gas chromatographic-nitrogen detector measurements, PCP's true pervasiveness is only now being appreciated. To further quantitate the severity of the problem, the authors analyzed samples of umbilical cord blood from 200 patients on the obstetrics service of a major university medical center. Preliminary results revealed that 24 (12%) of the samples were positive for PCP (.10-5.80 ng/ml). The authors discuss the significance of this finding with regard to psychiatry, obstetrics, pediatrics, and juvenile law.

Physical mapping of drug resistance mutations defines an active center of the herpes simplex virus DNA polymerase enzyme.

The genome structures of herpes simplex virus type 1 (HSV-1)/HSV-2 intertypic recombinants have been previously determined by restriction endonuclease analysis, and these recombinants and their parental strains have been employed to demonstrate that mutations within the HSV DNA polymerase locus induce an altered HSV DNA polymerase activity, exhibiting resistance to three inhibitors of DNA polymerase. The viral DNA polymerases induced by two recombinants and their parental strains were purified and shown to possess similar molecular weights (142,000 to 144,000) and similar sensitivity to compounds which distinguish viral and cellular DNA polymerases. The HSV DNA polymerases induced by the resistant recombinant and the resistant parental strain were resistant to inhibition by phosphonoacetic acid, acycloguanosine triphosphate, and the 2',3'-dideoxynucleoside triphosphates. The resistant recombinant (R6-34) induced as much acycloguanosine triphosphate as did the sensitive recombinant (R6-26), but viral DNA synthesis in infected cells and the viral DNA polymerase activity were not inhibited. The 2',3'-dideoxynucleoside-triphosphates were effective competitive inhibitors for the HSV DNA polymerase, and the Ki values for the four 2',3'-dideoxynucleoside triphosphates were determined for the four viral DNA polymerases. The polymerases of the resistant recombinant and the resistant parent possessed a much higher Ki for the 2',3'-dideoxynucleoside triphosphates and for phosphonoacetic acid than did the sensitive strains. A 1.3-kilobase-pair region of HSV-1 DNA within the HSV DNA polymerase locus contained mutations which conferred resistance to three DNA polymerase inhibitors. This region of DNA sequences encoded for an amino acid sequence of 42,000 molecular weight and defined an active center of the HSV DNA polymerase enzyme.