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Biomedical subjects

R S Day

Publications and source records attributed to R S Day.

At least 55 records · Page 3Linked to original sources
PubMed1991

Effect of passaging on Mer phenotype of human fetal cell cultures.

With increasing passage in culture, the human fibroblast cell strain GM11 lost the Mer+ phenotype (the ability to support the growth of adenovirus 5 damaged prior to infection by MNNG). All of 46 embryonic strains prepared either from various organs of 20 fetuses from 6-7 to 13-14 weeks of gestational age or from two hydatidiform moles showed normal repair of MNNG-treated virus. We conclude that human fetal strains are not usually deficient in such repair, and that the behavior of GM11 is atypical.

Adenoviruses, Human↗
PubMed1991

The sensitivities of SV40-transformed human fibroblasts to monofunctional and DNA-crosslinking alkylating agents.

4 repair-deficient (Mer-) and 2 repair-proficient (Mer+) lines of SV40-transformed human fibroblasts were assayed for colony-forming ability after treatment with MNNG, methyl methanesulfonate (MMS), 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU), and 1-(2-chloroethyl)-3-(2-hydroxyethyl)-1-nitrosourea (HECNU). The sensitivities to MMS, BCNU and HECNU of these SV40-transformed lines were similar to those of comparably treated human tumor cells observed previously. However, unlike human tumor lines, whose post-MNNG survival is strongly dependent upon Mer phenotype, SV40-transformed lines showed a lack of dependence of post-MNNG colony-forming ability on Mer phenotype. No differences in glutathione levels that might explain these differences were detected. The amounts of SV40-specific DNA and RNA among the lines were found to vary widely, but no correlation with Mer phenotype was found.

Carmustine↗
PubMed1989

Synergistic killing of virus-transformed human cells with interferon and N-methyl-N'-nitro-N-nitrosoguanidine.

Interferons potentiate the cytotoxic effects of certain antineoplastic drugs on human tumor cells both in vitro and in vivo, although the mechanism of interferon's synergistic action is unknown. Interferon may act by modulating the expression of DNA repair activity in cells. To test this hypothesis, we maintained parallel cultures of normal O6-methylguanine repair-proficient human fibroblasts and tumor cells, or RSV-and SV40-transformed repair-deficient Mer- human fibroblasts in medium containing 0, 100, 500 or 680 U/ml human interferon alpha or beta; after 1-10 weeks, cultures were challenged with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG, CAS: 70-25-7) and assayed for colony-forming ability. Based on the dose at 99% lethality, MNNG cytotoxicity was potentiated from 1.3- to 9-fold in interferon-treated cultures, compared with control cultures (no interferon). A significant potentiation was observed both with Mer+ normal fibroblasts (KD strain) and tumor cells (HOS) and with Mer- SV40-transformed fibroblasts (IMR90-830 and GM638) as well as with RSV-transformed cells (RHOS). However, the degree of potentiation was greater in Mer- virus-transformed cells than in Mer+ cells. The greatest effects were observed with Mer- IMR90-830 cells (5- to 9-fold reduction of dose at 99% lethality). Therefore, because the Mer+ phenotype is not required in order for HuIFNs to sensitize cells to killing by MNNG, interferon does not act by modulating O6-methylguanine repair. However, the effect of interferon on O6-methylguanine-DNA methyltransferase levels and on DNA excision repair should be examined in future experiments.

Cell Survival↗
PubMed1988

Potentiation of cytotoxicity by 3-aminobenzamide in DNA repair-deficient human tumor cell lines following exposure to methylating agents or anti-neoplastic drugs.

We studied the potentiation by 3-aminobenzamide (3AB) of killing of nine human cell lines exposed to alkylating agents. Cell lines included normal, transformed and DNA repair-proficient and -deficient phenotypes. 3AB potentiated cell killing by the methylating agents methylmethanesulfonate (MMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in all lines tested. The degree of potentiation ranged from 1.7- to 3.8-fold, based on the LD99. The average potentiation observed with MMS (2.7-fold) was greater than with MNNG (2.2-fold). On average the potentiation of MMS and MNNG killing of repair-deficient Mer- lines (2.4-fold) was similar to that of repair-proficient Mer+ lines. The degree of 3AB potentiation of MNNG killing (2.0-fold) was similar in Mer+ Rem- lines and in Mer+ Rem+ lines. Mer+ Rem+, Mer+ Rem-, Mer- Rem+, and Mer- Rem- strains all appeared proficient in a 3AB-sensitive DNA repair pathway. Within experimental error, 20 mM 3AB did not inhibit the removal of the MNNG-induced methylpurines 7-methylguanine, O6-methylguanine and 3-methyladenine from the DNA of repair-proficient Mer+ Rem+ HT29 cells, consistent with evidence that 3AB inhibits the ligation step of excision repair. 3AB potentiated cell killing by the bifunctional alkylating agents 1-(2-chlorethyl)-1-nitrosourea or busulfan, two anti-neoplastic drugs, by only 0.9- to 1.5-fold. These drugs therefore produce DNA damage which is not efficiently repaired by the pathways that repair methylated bases.

Antineoplastic Agents↗
PubMed1987

Differential sensitivities of transformed and untransformed murine cell lines to DNA cross-linking agents relative to repair of O6-methylguanine.

Sensitivities of several murine cell strains to killing by the DNA cross-linking agents 1-(2-chloroethyl)-1-nitrosourea (CNU), cis-diamminedichloroplatinum (II) (Cis-Pt) and mitomycin C (MMC) were measured by post-treatment colony-formation. Virally-transformed murine cells were usually more sensitive to cell killing by these agents than were the parental 3T3 cell strains. The hypersensitivity to CNU of some virally-transformed murine cell strains correlated well with the reduced ability to repair O6-methylguanine (O6mGua), a phenomenon similar to that in human cells. The loss of ability to repair O6mGua, as well as the increased sensitivity of transformed strains to cell killing, may not be due to a mutation but rather due to a change of gene expression associated with transformation by viruses or activation of oncogenes.

Animals↗
PubMed1987

Exogenous O6-methylguanine inhibits adduct removal and sensitizes human cells to killing by the chemical carcinogen N-methyl-N'-nitro-N-nitrosoguanidine.

We partially depleted the O6-methylguanine-DNA methyltransferase activity in four O6-methylguanine (O6-mGua) repair-proficient (Mer+) human cell strains with exogenous O6-mGua (2 mM for 3 h, a non-toxic regimen) and then challenged them with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). MT-partially depleted HT29 cells removed O6-mGua from DNA at about half the rate of control cells, while removal of 3-methyladenine was unaffected. In spite of partial depletion of MT, however, cell killing by MNNG in a colony-forming assay with HT29, A549, A498 or KD cells was not greatly affected. (This is in contrast to the dramatic potentiation of CNU cytotoxicity observed previously.) In an attempt to sensitize Mer+ strains to killing by MNNG, we treated cells with O6-mGua following MNNG exposure (0.4 mM for 4 days), in addition to the pre-MNNG treatment of 2 mM O6-mGua for 3 h. This sensitized KD and HT29 cells 2-fold to killing by MNNG, based on the dose at 10% survival, but did not sensitive Mer- A1336. However, post-treatment alone was as effective as combined pre- and post-treatment in sensitizing KD cells to killing. Thus, when the O6-mGua post-treatment was begun, greater than 50% of O6-mGua was already removed from cell DNA. Our findings may be accounted for by at least two schemes, one in which nonlethal O6-mGua are removed from DNA rapidly, while potentially lethal O6-mGua are repaired later. The other scheme proposes that exogenous O6-mGua increases the lethality of a non-O6-mGua lesion by reducing its repair both in Mer+ and Mer- cells. Both schemes are consistent with the hypothesis that O6-mGua may be a lethal DNA lesion in human cells.

Cell Survival↗
PubMed1987

The role of O6-methylguanine in human cell killing, sister chromatid exchange induction and mutagenesis: a review.

O6-methylguanine (O6mG) produced in DNA by such SN1 methylating agents as N-methyl-N-nitrosourea and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) has been suggested by some to be the lesion that leads to certain biological endpoints in mammalian cells: cell killing, sister chromatid exchange (SCE) production, mutagenesis and cellular transformation. Other evidence is interpreted as inconsistent with this point of view. The finding of Karran & Williams (1985) that O6mG delivered to cells in culture resulted in the depletion of the activity of the protein responsible for repair of O6mG in DNA (O6mG-DNA methyltransferase, O6MT) provided a tool for the assessment of the role of O6mG in producing biological endpoints. In this paper we review much of the literature on human cells pertinent to this question. In addition we present our survival data obtained using the depletion technique of Karran & Williams as well as data supporting a model invoking a mismatch and excision response to O6mG proposed by Sklar & Strauss (1980). Although data linking O6mG to causation are inconclusive, it is premature to conclude that O6mG is not a lesion lethal to certain cultured cells.

Adenoviridae↗
PubMed1986

Protoporphyrinogen oxidase and porphobilinogen deaminase in variegate porphyria.

Two enzymes of the haem biosynthetic pathway were investigated in patients with variegate porphyria. Protoporphyrinogen oxidase in cultures of Epstein-Barr virus transformed lymphoblasts from twenty-seven patients showed a mean maximal velocity (Vmax) of 0.39 +/- 0.08+ nmol of protoporphyrin mg protein-1 h-1, a 52% reduction (P less than 0.001) from a non-porphyric control group (0.82 +/- 0.10). Km values (1.00 +/- 0.27 microM) did not differ significantly (P greater than 0.05) from control values in any of the patients. The mean Vmax of porphobilinogen deaminase in the cultures was 1.50 +/- 0.18 nmol of uroporphyrin mg protein-1 min-1, a 24% reduction (P less than 0.001) from controls (1.94 +/- 0.14). Mean porphobilinogen deaminase activity in the erythrocytes of twenty-one patients with variegate porphyria was 8.37 +/- 1.99 nmol of uroporphyrin 1 erythrocytes-1 s-1, a 28% reduction (P less than 0.001) from normal (11.98 +/- 2.11). The reduced activities of these two enzymes comply with the expression of variegate porphyria during its quiescent and acute phases.

Ammonia-Lyases↗
PubMed1986

Treatment sequencing, asymmetry, and uncertainty: protocol strategies for combination chemotherapy.

This paper summarizes a large number of simulations which relax the "symmetry" assumptions in the Goldie-Coldman treatment model, which was symmetrical with respect to two drugs considered for use together. The results are that, under relevant violations of the assumptions, nonalternating treatment schedules frequently outperform alternation and combination substantially. The magnitudes of these effects are at least as large as the improvements made by shortening the time periods between treatment changes. Three protocol design strategies derived from these results are described: (a) when there is no knowledge of parameters, empirical trials in search of the best schedule can be contemplated, if patients in such trials have fundamentally "similar" tumors, in a special sense given the name "pattern homogeneity"; (b) when minimal knowledge of cell kill parameters is available, the "worst drug rule" could perform remarkably well. This strategy is contrary to much of current practice, but a clear rationale is proposed; and (c) when detailed knowledge of tumor parameters is available for each individual, detailed modeling to predict optimal schedule promises great improvements in treatment outcome. Additional considerations addressed include factors determining the merit of these strategies, suggestions for new laboratory research, and implications for future clinical chemotherapy research.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1986

Inactivation of O6-methylguanine-DNA methyltransferase and sensitization of human tumor cells to killing by chloroethylnitrosourea by O6-methylguanine as a free base.

Human fibroblasts and tumor cells with constitutive levels of the DNA repair protein O6-methylguanine-DNA methyltransferase were incubated with mM concentrations of the free base O6-methylguanine for up to 24 h. This treatment depleted the cells of their transferase activity, and sensitized the cells to killing by the antineoplastic drug 1-[2-chloroethyl]-1-nitrosourea. Cells constitutively lacking the methyltransferase were not sensitized to cell killing. Cell free extracts incubated with O6-methylguanine also lost methyltransferase activity. Other alkylpurines, such as O6-methylguanosine, S6-methylthioguanine, O6-ethylguanine, and 3-methyladenine, did not have this effect on extracts of human tumor cells, while O6-methylguanosine and O6-methylguanine inactivated purified methyltransferase from Escherichia coli. The data suggest that the free base O6-methylguanine is probably a substrate for the methyltransferase. Calculation of the second order rate constants for free base versus O6-methylguanine in DNA, and experiments in which the free base was mixed with DNA containing O6-methylguanine before reaction with methyltransferase, indicated that the base in DNA is about 4 X 10(7) better as a substrate than is the free base. These results demonstrate that DNA repair capacity of tumor cells can be diminished without DNA damage, and suggest a method for increasing the efficiency of chemotherapy.

Cell Survival↗
PubMed1985

Quantitative assessment of the role of O6-methylguanine in the initiation of carcinogenesis by methylating agents.

Induction of transformation, cell lethality, and DNA lesions were quantitatively compared in Syrian hamster embryo cells (HEC) treated with three different methylating agents: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-methyl-N-nitrosourea (MNU), or methyl methanesulfonate (MMS). Each induced transformation in a dose-dependent manner. On a molar basis, MNNG was approximately equal to 100- and 500-fold more effective than MNU and MMS, respectively. For each carcinogen the induction and repair of O6- and N7-methylguanine (O6- and N7-MeGua) relative to total guanine content was compared. At concentrations that induced equivalent transformation frequencies, the induction of O6-MeGua was the same for all three carcinogens, but N7-MeGua induction was 30-fold higher with MMS than with MNNG or MNU. The capacity to repair methylation lesions in HEC is limited because only between 50% and 70% of both O6- and N7-MeGua lesions were removed from the DNA within 24 hr after treatment, independent of methylating carcinogen. No consistent effect on either the rate of DNA replication or the size distribution of nascent strands correlated with O6-MeGua induction. These data support the hypothesis that O6-MeGua is the critical lesion for initiation of carcinogenesis by methylating agents. The frequency of transformation relative to O6-MeGua induction is 40- to 750-fold more than that of mutation. Based on the quantitative data for induction of O6-MeGua and transformation, the target size for initiation of carcinogenesis was calculated as a minimum of 10(4) nucleotides. This suggests that one of many genes can initiate carcinogenesis or that initiation is not the result of a single base mutation.

Animals↗
PubMed1985

Human tumor cell strains both unable to repair O6-methylguanine and hypersensitive to killing by human alpha and beta interferons.

Human brain tumor cell strains were previously found by others to be sensitive to growth inhibition by human interferon-beta (HuIFN-beta). We noticed that the sensitive strains were some that we had found deficient in the repair of O6-methylguanine (O6MeG), a characteristic of 20% of the human tumor cell strains we have studied. We confirmed this sensitivity to HuIFN-beta, and have further shown that human brain tumor cells which repair O6MeG are resistant to the growth inhibitory effects of HuIFN-beta. In addition, treatment with HuIFN-alpha or HuIFN-beta resulted in more killing (reproductive inactivation) of six human tumor cell strains deficient in repairing O6-methylguanine in DNA than did such treatment of six strains of cells proficient in such repair. Further, we found two human lines, altered to become O6MeG repair deficient after establishment of the primary tumor cell culture, that were resistant to interferon. IFN treatment produced no DNA damage detectable by either chemical or biological assays. It is suggested that the genes responsible for resistance to IFN treatment and to agents that produce O6MeG are often coordinately shut down.

Brain Neoplasms↗
PubMed1985

Human O6-alkylguanine-DNA alkyltransferase fails to repair O4-methylthymine and methyl phosphotriesters in DNA as efficiently as does the alkyltransferase from Escherichia coli.

The repair of alkylated poly(dT) annealed to poly(dA) and alkylated DNA was studied using human cells in culture, extracts of human liver and Escherichia coli, and partially purified O6-alkylguanine-DNA alkyltransferase from human liver. The human alkyltransferase behaved as if the transferring and accepting functions of the repair process resided on the same molecule. Extracts of E. coli efficiently repaired O6-methylguanine, O4-methylthymine and methyl phosphotriesters in DNA. In contrast, cultured human cells and extracts from human liver repaired O6-methylguanine but did not repair O4-methylthymine or methyl phosphotriesters in DNA nearly as efficiently as did the E. coli extracts.

Alkylation↗
PubMed1984

Relationship of methyl purines produced by MNNG in adenovirus 5 DNA to viral inactivation in repair-deficient (Mer-) human tumor cell strains.

Adenovirus 5 treated with MNNG (N-methyl-N'-nitro-N-nitrosoguanidine) has greater plaque-forming ability in cell strains having the Mer+ phenotype than in strains having the Mer- phenotype. MNNG-treated Mer- strains repair the N3-methyladenine (N3MeA) but not the O6-methylguanine (O6MeG) produced in their DNA, while MNNG-treated Mer+ strains repair both of these adducts. The fate of N7-methylguanine (another DNA adduct produced by MNNG) is similar in Mer+ and Mer- strains. We show in this paper that 2.3 +/- 0.4 O6MeG and 1.4 N3MeA per adenovirus genome correlate with one lethal hit when the survival assay is done using Mer- strains as viral hosts. We suggest that O6MeG is the lesion lethal to the virus.

Adenoviruses, Human↗
PubMed1984

O6-Methylguanine-DNA methyltransferase in human cells.

O6-Methylguanine-DNA methyltransferase activity was measured in extracts of human tumor cells and was partially purified from human placenta. Repair of O6-methylguanine in DNA inactivated the methyltransferase, and treatment of cells with MNNG, which produces this alkylated base in DNA, depleted the cells of active methyltransferase. RNA and protein synthesis were required for restoration of methyltransferase activity, which transiently exceeded the original levels by 50% 48 h after treatment. One species of methyltransferase of Mr = 22 kd was present in human tumor cells and human placenta.

Astrocytoma↗
PubMed1984

Comparison of repair of O6-methylguanine produced by N-methyl-N'-nitro-N-nitrosoguanidine in mouse and human cells.

The repair of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-produced O6-methylguanine (O6-MeG) in DNA and its correlation with MNNG-produced cell-killing and sister chromatid exchange (SCE) induction were compared in mouse and reference human tumor cell strains. As a result, mouse cell strains were divided into three groups: (i) cells proficient in O6-MeG-repair and insensitive to MNNG similar to human Mer+ Rem+ strains; (ii) cells deficient in O6-MeG-removal and sensitive to MNNG similar to human Mer-Rem- strains; (iii) cells deficient in O6-MeG-removal but insensitive to MNNG similar to some SV40-transformed human strains. Attempts at correlating lack of capacity for O6-MeG-removal, MNNG-sensitivity and high SCE induction showed that O6-MeG in DNA may be a lesion common to cell-killing and SCE induction only in mouse cells of groups i and ii. Levels of O6-MeG-DNA methyltransferase activity in mouse cells were measured and the enzyme had the same molecular weight as that in human cells.

Animals↗
PubMed1984

Hybrids between human tumor cell strains differing in repair of MNNG-produced DNA damage.

Human tumor cell strains with differing responses to MNNG damage in their DNA were treated with precipitates of the plasmids pSV2gpt or pSV2neo . Transfected clones were selected on the basis of the drug resistance which each plasmid confers. Cells with different drug resistances were fused and hybrids were selected in medium requiring the expression of both markers. Hybrids produced by fusion of two different strains hypersensitive to MNNG-produced cytotoxicity and which lack the DNA repair enzyme O6-methylguanine-DNA methyltransferase ( O6MT ) failed to show complementation, suggesting that these strains share a common genetic defect. Hybrids from fusions of each of three strains containing O6MT activity with the same strain lacking O6MT activity were of surprising character. In one case the hybrid had resistance to MNNG-produced cell killing and O6MT activity similar to the parent strain possessing O6MT activity. In a second case, the hybrid had greater resistance to MNNG produced cytotoxicity than either parent strain although the level of O6MT activity was not higher. In a third case, the hybrid had little or no O6MT and as great hypersensitivity to MNNG-produced cytotoxicity as the parent strain lacking O6MT activity. We conclude that the survival of human tumor cell strains after MNNG-produced DNA damage is controlled by several genes. Even individual repair enzymes, like O6MT , are likely to be regulated by the interaction of these genes.

Animals↗