Biomedical subjects

M E Dolan

Publications and source records attributed to M E Dolan.

101 records · Page 6Linked to original sources

PubMed1985

Depletion of O6-methylguanine-DNA-methyltransferase in human fibroblasts increases the mutagenic response to N-methyl-N'-nitro-N-nitrosoguanidine.

We showed previously that resistance of a series of human fibroblast cell lines to the cytotoxic and mutagenic effects of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) is highly correlated with their level of O6-methylguanine-DNA-methyltransferase (MT) activity. In the present study, MT activity in normal fibroblasts was decreased to between 40 and 20% of the constitutive level by 15 or 24 h exposure of the cells to exogenous O6-methylguanine (O6-MeG). MT-depleted and non-depleted populations were then challenged with various doses of MNNG and assayed for cytotoxicity and mutagenicity. At every dose the frequency of 6-thioguanine resistant cells induced by MNNG was higher in the MT-depleted populations than in the controls. Since the MT activity in these cells does not remove methyl from the O4 position of thymine, these results strongly support the hypothesis that O6-methylguanine is the principal mutagenic lesion induced by MNNG. Cells with decreased levels of MT were not significantly more sensitive to the cytotoxic effect of MNNG. If O6-methylguanine is a potentially cytotoxic lesion, this lack of increased sensitivity may reflect the fact that regeneration of MT protein occurred rapidly enough to remove these lesions before they resulted in cell death (i.e., inability to form a clone). Consistent with this explanation is the fact that 7 h after removal of the exogenous O6-MeG, the level of MT activity had regenerated to 51% of normal; by 18 h, it was 65% of normal.

Cell Line↗

PubMed1985

Repair of O6-propylguanine and O6-butylguanine in DNA by O6-alkylguanine-DNA alkyltransferases from rat liver and E. coli.

DNA substrates containing O6-n-butylguanine, O6-iso-butylguanine, O6-n-propylguanine and O6-iso-propylguanine were prepared by reaction of calf thymus DNA with the appropriate N-alkyl-N-nitrosourea. These substrates were used to test the ability of O6-alkylguanine-DNA alkyltransferases from Escherichia coli and rat liver to remove such alkyl groups from the O6-position of guanine. It was found that all of these adducts were removed by the alkyltransferases, but the branched alkyl chain iso-butyl- and iso-propyl adducts were removed very slowly. Also, when tested with a DNA substrate containing both O6-n-propylguanine and O6-iso-propylguanine, the alkyltransferases removed almost all of the n-propyl-adduct before the iso-propyl-adduct was attacked. Both alkyltransferases showed a decreasing rate of reaction as the size of the alkyl group increased, but there was a significant difference between the rat liver and E. coli alkyltransferase in the relative rates. The rat liver alkyltransferase repaired O6-methylguanine more slowly than the E. coli protein, but was considerably more rapid than the bacterial equivalent when acting on n-propyl- and n-butyl-adducts. The relative rates of repair were methyl much greater than ethyl greater than n-propyl greater than n-butyl greater than iso-propyl, iso-butyl for the E. coli alkyl-transferase and methyl greater than ethyl, n-propyl greater than n-butyl greater than iso-propyl, iso-butyl greater than 2-hydroxyethyl for the rat liver protein. These results indicate that differential rates of repair may contribute to the relative risks of carcinogenesis and mutagenesis by exposure to alkylating agents of different size and that rates of repair may be species specific and must be determined from specific measurements rather than extrapolated from data on other organisms.

Animals↗

PubMed1985

Studies of the repair of O6-alkylguanine and O4-alkylthymine in DNA by alkyltransferases from mammalian cells and bacteria.

O6-Methylguanine in DNA is repaired by the action of a protein termed O6-alkylguanine-DNA alkyltransferase (AT) which transfers the methyl group to a cysteine residue in its own sequence. Since the cysteine which is methylated is not regenerated rapidly, if at all, the capacity for repair of O6-methylguanine is limited by the number of molecules of the AT available within the cell. The level and inducibility of the AT differed greatly in different mammalian cell types and species with the highest levels in human tissues and in liver and the lowest levels in brain. Only a small induction occurred in rat liver in response to exposure to alkylating agents. In E. coli such exposure increased the activity more than 100-fold. The At was not specific for methyl groups but also removed ethyl, 2-hydroxyethyl, n-propyl, isopropyl and n-butyl groups from the O6-position in DNA. The protein isolated from E. coli removed methyl groups much more rapidly than the larger alkyl groups but the mammalian AT isolated from rat liver showed much less difference in rate with adducts of different size. Ethyl and n-propyl groups were removed by the rat liver AT only three to four times more slowly than methyl groups. Another important difference between the bacterial and mammalian ATs is that the bacterial protein was also able to remove methyl groups from the O4-position of thymine in methylated DNA or poly(dT) but the AT from rat liver or human fibroblasts did not repair O4-methylthymidine.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1985

Reduction of O6-alkylguanine-DNA alkyltransferase activity in HeLa cells treated with O6-alkylguanines.

Exposure of HeLa cells to 0.2 mM O6-methylguanine for 4 h or longer led to a 70-80% loss in the activity of the DNA-repair protein, O6-alkylguanine-DNA alkyltransferase. The decline in alkyltransferase activity brought about by O6-methylguanine was reversible on removing the base but at least 48 h were required for complete restoration. This loss of activity could also be brought about by other O6-alkylguanines including ethyl, n-propyl, and n-butyl, but the isopropyl and 2-hydroxyethyl derivatives were considerably less active. The rate of decline of alkyltransferase activity produced by O6-methylguanine was much more rapid than the rate of loss when protein synthesis was inhibited indicating that it was not brought about by blocking the synthesis of the protein. The loss of alkyltransferase activity was not prevented by the addition of inhibitors of nucleic acid or protein synthesis suggesting that it did not require protein synthesis or the incorporation of the O6-alkylguanine into nucleic acids. When cell free O6-alkylguanine-DNA alkyltransferase preparations were incubated in vitro with O6-methylguanine they became inactivated and when O6-[3H]methylguanine was used, [3H]guanine was produced. The inactivation was concentration dependent requiring 0.4 mM for a maximal rate and was quite slow requiring 3-4 hours for completion. These results suggest that the loss of O6-alkylguanine-DNA alkyltransferase activity is brought about by the free base acting as a very weak substrate for the protein. Exposure of mammalian cells to O6-methylguanine or O6-n-butylguanine provides a means by which the level of O6-alkylguanine-DNA-alkyltransferase can be regulated experimentally. This should enable the design of experiments to examine the role of O6-alkylguanine adducts in mutagenesis, carcinogenesis, and cell toxicity after administration of alkylating agents.

Female↗

PubMed1984

Comparison of repair of methylated pyrimidines in poly(dT) by extracts from rat liver and Escherichia coli.

Partially purified preparations of O6-alkylguanine-DNA alkyltransferase from rat liver and E. coli were tested for their ability to repair O4-methylthymine in a methylated poly(dT) X poly(dA) substrate. The bacterial preparation readily carried out this reaction, but no loss of O4-methylthymine was obtained with the rat liver protein. These results indicate a significant difference in specificity between the mammalian and bacterial proteins which could have important consequences for carcinogenesis and mutagenesis by alkylating agents in mammalian cells.

Animals↗

PubMed1984

Correlation between O6-methylguanine-DNA-methyltransferase activity and resistance of human cells to the cytotoxic and mutagenic effect of N-methyl-N'-nitro-N-nitrosoguanidine.

Cells from Gardner's syndrome (GS) and familial polyposis coli (FP) patients, persons with a hereditary predisposition to colon cancer, were compared to those of normal persons for sensitivity to the cytotoxic and mutagenic action of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a model compound chosen because methylating agents have been implicated in colon carcinogenesis. FP cell line GM2355 and GS cell lines 2938 and GM3948 exhibited normal sensitivity to the cytotoxic and mutagenic effects of MNNG. In contrast, GS cell line GM3314 and cells from an apparently normal fetus GM0011 showed extreme sensitivity to the killing and mutagenic effect of this alkylating agent. To determine if the resistance of the various cell lines to MNNG correlated with their ability to remove methyl groups from the O6-position of guanine, we measured their O6-methylguanine-DNA methyltransferase (MT) activity. The resistant cell lines exhibited normal levels of MT; the sensitive strains showed virtually non-detectable levels of this activity. We also compared fibroblasts from a xeroderma pigmentosum (XP) patient (XP12BE, complementation group A), an SV40 virus-transformed XP cell line (XP12ROSV) and a normal cell line transformed by this virus (GM637) for their response to the cytotoxic and mutagenic effect of MNNG and for MT activity. XP12BE cells showed normal sensitivity and a normal level of MT; GM637 cells showed an intermediate level of sensitivity and a reduced level of MT activity; XP12ROSV cells were extremely sensitive to the cytotoxic and mutagenic effect of MNNG and showed virtually non-detectable levels of MT activity. The MT did not remove methyl group from O4-methyl-thymine. These results suggest that O6-methylguanine and/or any other adduct repaired by the methyltransferase, is a potentially cytotoxic and mutagenic lesion. They also indicate that the predisposition to colon cancer of FP and GS patients is not necessarily correlated with an increased sensitivity of their fibroblasts to mutations induced by methylating carcinogens.

Cell Line↗

PubMed1984

Comparison of the rates of repair of O6-alkylguanines in DNA by rat liver and bacterial O6-alkylguanine-DNA alkyltransferase.

The rates of loss of O6-methylguanine and O6-ethylguanine from rat liver DNA were determined over a time period of 15 min to 4 hr after various doses (5 micrograms/kg to 2 mg/kg) of dimethylnitrosamine and diethylnitrosamine which produced total amounts of these adducts in the range of 300 to 16,000 molecules/cell. This amount is considerably less than the content of O6-alkylguanine-DNA alkyltransferase protein (approximately 60,000 molecules/hepatocyte), and during the time period studied, the adducts were found to be lost with pseudo-first order kinetics. The half-life for O6-methylguanine was 47 min. O6-Ethylguanine was removed 3.6 times more slowly with a half-life of 172 min. The ability of partially purified rat liver O6-alkylguanine-DNA alkyltransferase to remove O6-methylguanine and O6-ethylguanine from [3H]alkyl-labeled DNA substrates in vitro was measured, and it was found that O6-methylguanine was removed 3.4 times more rapidly than was O6-ethylguanine. These results are consistent with the hypothesis that most, if not all, of the repair of these adducts which occurs within the first 4 hr after treatment is due to the alkyltransferase protein. Diethylnitrosamine, which is slightly more potent as a carcinogen to rat liver, produced a total amount of O6-ethylguanine of 3.7 mumol/mol guanine/mg compared to O6-methylguanine (28 mumol/mol guanine/mg) given by dimethylnitrosamine. The slower rate of loss of the ethyl adduct is not sufficient to account for this difference, and the results, therefore, support the concept that other DNA adducts (possibly O-alkylpyrimidines) contribute to the initiation of tumors by diethylnitrosamine. Preliminary evidence that the rat liver alkyltransferase can also remove hydroxyethyl groups from DNA at a rate slower than removal of ethyl groups was also obtained. Bacterial O6-alkylguanine-DNA alkyltransferase was shown to remove methyl, ethyl, and hydroxyethyl groups from the O6 position of guanine in DNA using fluorescence detection to quantitate these adducts. The bacterial protein removed methyl groups very rapidly but was much slower than the rat liver protein on the larger adducts. These results suggest that the relative rates of repair of different alkyl groups may be species specific and must be determined experimentally in the cell of interest before conclusions concerning biological effects can be drawn.

Alkylation↗

PubMed1983

An analysis of 1-(2-chloroethyl)-1-nitrosourea activity at the cellular level.

The effect of five different 1-(2-chloroethyl)-1-nitrosoureas on the growth of cultured P388 cells has been analyzed in terms of physical, chemical, and kinetic parameters that are related to the mechanism of action of this class of cancer chemotherapeutic agent. This study correlates structure with activity at the cellular level by using a dose function that is related to the amount of active species, the (2-chloroethyl)diazonium ion, that is formed during the period of exposure of cells to drug rather than to the initial drug dose. 1-(2-Chloroethyl)-1-nitrosourea analogues that rapidly enter the P388 cells are shown to have the same activity relative to the amount of active species formed. When analyzed in this way, activity is not influenced by the structure of the N-3 substituent, lipophilicity, or carbamoylating activity. The agents 1-(2-chloroethyl)-1-nitrosourea (CNU), 1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl)-1-nitrosourea (PCNU), 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU), and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) all produce a 50% cell growth inhibition at 6 to 7 microM active species formed per cell volume. Chlorozotocin required a twofold higher effective dose to produce the same toxic effect. This decreased activity is attributed to the slow uptake of the water-soluble chlorozotocin into P388 and L1210 cells relative to the rate of chlorozotocin conversion to active species in medium. The yields to 2-chloroethanol from CNU, BCNU, and chlorozotocin were shown to be the same, indicating that these agents generate the same yield of alkylating intermediate at 37 degrees C and pH 7.4.

Animals↗

PubMedDate not supplied

Effect of O6-alkylguanine-DNA alkyltransferase on the frequency and spectrum of mutations induced by N-methyl-N'-nitro-N-nitrosoguanidine in the HPRT gene of diploid human fibroblasts.

N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) reacts with 12 nucleophilic sites in DNA to induce a variety of lesions, but O6-methylguanine (O6-MeG) and O4-methylthymine are the most effective premutagenic lesions produced, mispairing with thymine and guanine, respectively. O6-MeG is repaired by O6-alkylguanine-DNA alkyltransferase (AGT), which removes the methyl group from the O6 position and transfers it to itself, rendering the transferase inactive. When diploid human fibroblasts were exposed to 25 microM, O6-benzylguanine (O6-BzG) in the medium for 3 h, their level of AGT activity was dramatically reduced, to a level of at most 1.6% of the control. Populations of cells pretreated with this level of O6-BzG for 2 h or not pretreated, were exposed to MNNG at a concentration of 2, 4 or 6 microM in the presence or absence of O6-BzG and assayed for survival of colony-forming ability and the frequency of 6-thioguanine-resistant cells (mutations induced in the HPRT gene). O6-BzG (25 microM) was also present in the appropriate half of the cells during the 24 h immediately following exposure to MNNG. This 27-h exposure to O6-BzG alone had no cytotoxic or mutagenic effect on the cells but significantly increased the cytotoxicity and mutagenicity of MNNG, increasing the mutant frequency to that found previously in human cells constitutively devoid of AGT activity. At doses of 2 microM and 4 microM MNNG, the mutant frequency observed with the AGT-depleted cells was 120 x 10(-6) and 240 x 10(-6), respectively; in the cells with abundant AGT activity, these values were 10 x 10(-6) and 20 x 10(-6), respectively. DNA-sequence analysis of the coding region of the HPRT gene in 36 independent mutants obtained from MNNG-treated AGT-depleted populations and 36 from the control populations showed that even though AGT repair lowered the frequency of mutants by more than 90%, it did not affect the kinds of mutations induced by MNNG nor the strand distribution of the premutagenic guanine lesions. In mutants from the AGT-depleted cells, there were 26 base substitutions and 13 putative splice site mutations; in the control, there were 25 base substitutions and 11 splice site mutations. All but two substitutions involved G.C with 92% being G.C----A.T. In both sets, approximately 73% of the premutagenic lesions were located in the nontranscribed strand.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗

PubMedDate not supplied

Intracellular activation of cytotoxic agents: kinetic models for methylnitrosoureas and N-methyl-N'-nitro-N-nitrosoguanidine in cell culture.

The cytotoxic activity of N-methyl-N-nitrosourea (MNU), streptozotocin, and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was determined in cell culture by using a P388 cell growth rate inhibition assay. These agents appear to have very different activities when inhibition is related to the agent concentration in the culture medium: ED50(C0) = 40 microM for MNNG to 875 microM for streptozotocin. The mechanism of action of these three agents involves conversion to the active methanediazonium ion and subsequent methylation of cellular macromolecules. As a consequence, the rates of conversion of the parent agent to the methylating species in the medium and within the cell are important parameters that also need to be considered to reach a more detailed understanding of the mechanism of action. In order to do this, a kinetic model has been developed to calculate the concentration of drug that is converted to active methylating species within the cell during the assay incubation period. The use of cell culture kinetic models was extended from simple compounds activated through solvolytic reactions (nitrosoureas) to an agent that undergoes selective intracellular activation (MNNG). By use of measured values for initial drug concentration, incubation time, and cell volume, as well as extracellular and intracellular chemical activation rate constants, the intracellular concentration, [P4], which represents the cumulative intracellular reaction products formed during the incubation period, was calculated and related to cytotoxicity. All three agents showed an ED50[P4] between 140 and 180 microM, and for MNNG, this ED50 was independent of extracellular sulfhydryl concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMedDate not supplied

Metabolism and disposition of O6-benzyl-2'-deoxyguanosine in Sprague-Dawley rats.

O6-Benzyl-2'-deoxyguanosine is a potential antitumor drug modulator that is intended to reduce or eliminate O6-alkylguanine-DNA alkyltransferase activity in tumors prior to treatment with genotoxic chemotherapeutic alkylating agents. The rationale for using this compound instead of the more active O6-benzylguanine and its substituted benzyl derivatives at the benzyl ring is its greater solubility in aqueous media and potential pharmacologic advantage. Metabolism and disposition of O6-benzyl-2'-deoxyguanosine was determined in adult male Sprague-Dawley rats following an ip injection of 100 mg/kg. Under these conditions, the compound was partially metabolized to yield a glucuronic acid conjugate, which was secreted exclusively in the bile. Removal of the 2'-deoxyribose or the benzyl group to yield O6-benzylguanine and 2'-deoxyguanosine, respectively, occurred to a lesser extent. Metabolism accounted for the clearance of at least 58% of the total dose and took place primarily in the liver. Direct excretion of unchanged drug, mainly in urine, accounted for the remainder of the dose. Analysis of venous blood showed the presence of O6-benzyl-2'-deoxyguanosine and O6-benzylguanine at concentrations which are considered to be effective in depleting alkyltransferase activity. Levels of the nucleoside reached a maximum of 45 microM at 2 h, while those of O6-benzylguanine peaked to 20 microM at 4 h and remained at that level for at least 4 more hours. Transport of O6-benzyl-2'-deoxyguanosine in C6 glioma cells increased linearly with the extracellular concentration of the drug up to 600 microM. Intracellular levels of the drug reached 1.2 pmol per microM of extracellular compound per 10(6) cells as soon as 30 s after exposure and remained as high for at least 1 h. Such levels indicate that entrapment of the nucleoside inside cells by either phosphorylation or other means is probably not an important feature for this drug.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗