methylnitronitrosoguanidine and 1-(2-chloroethyl)-1-nitrosourea

The chloroethylnitrosoureas were developed in a synthetic program that began with the observation that N-methyl-N'-nitro-N-nitrosoguanidine was an effective agent against L1210 cells. The antitumor activity of the chloroethylnitrosoureas is based on their reactions with DNA, especially the formation of a cytosine-guanine crosslink in DNA. Resistance occurs when the enzyme, O6-alkylguanine-DNA alkyltransferase, repairs an intermediate in crosslink formation. Inhibition of O6-alkylguanine-DNA alkyltransferase often restores sensitivity to the chloroethlylnitrosoureas although evidence is accumulating that other repair mechanisms may also contribute to the resistance phenomenon. Continuing investigations in this field center on finding agents whose reactions with DNA are more specific, on elucidating other resistance mechanisms, and on overcoming resistance by developing new inhibitors of repair enzymes.

Topics: Alkyl and Aryl Transferases; Alkylation; Animals; Antineoplastic Agents; Cross-Linking Reagents; DNA; DNA Adducts; DNA Repair; Drug Resistance, Neoplasm; Ethylnitrosourea; Humans; Methylnitronitrosoguanidine; Nitrosourea Compounds

Topics: Alkylating Agents; Bacterial Proteins; Escherichia coli; Escherichia coli Proteins; Ethylnitrosourea; Methyl Methanesulfonate; Methylnitronitrosoguanidine; Mutagens; O(6)-Methylguanine-DNA Methyltransferase; Saccharomyces cerevisiae; Transcription Factors

A plasmid has been constructed in which the expression of human O6-methylguanine-DNA methyltransferase (MGMT) complementary DNA is driven by the Rous sarcoma virus promoter sequence. We had previously shown that transfection of this plasmid into Chinese hamster ovary (CHO) cells results in the expression of MGMT and in increased cellular resistance to N-methyl-N'-nitro-N-nitrosoguanidine and 1-(2-chloroethyl)-1-nitrosourea (CNU) but not N-nitroso-N-ethylurea (ENU). In the present study, the Rous sarcoma virus promoter-MGMT was transfected into DNA excision repair-deficient CHO UV41 cells to investigate the phenotype associated with MGMT expression in the absence of DNA excision repair. Both the UV41/MGMT and CHO/MGMT cells expressed similar levels of MGMT and exhibited a similar increased resistance to N-methyl-N'-nitro-N-nitrosoguanidine. The UV41 cells were 20-fold more sensitive to CNU than the wild-type CHO cells. Expression of MGMT increased the resistance to CNU about 6-fold in both cell lines, but the difference between the two cell lines attributable to the excision repair defect still persisted. The UV41 cells were 2- to 3-fold more sensitive than the wild-type CHO cells to the monofunctional alkylating agents 1-(2-hydroxyethyl)-1-nitrosourea and ENU, but the MGMT phenotype did not alter sensitivity. This suggests that alkylation at the O6 position of guanine has no role in cytotoxicity of ethylating agents and that monofunctional DNA damage has little role in the cytotoxicity of CNU. Since MGMT can prevent the formation of G-C interstrand cross-links formed by CNU, other excision repair-sensitive DNA adducts must play a major role in the sensitivity of UV41 cells to this bifunctional alkylating agent. These results suggest that DNA intrastrand cross-links may be major contributors to the cytotoxicity of CNU.

Topics: Animals; Cell Line; CHO Cells; Cricetinae; DNA; DNA Repair; Ethylnitrosourea; Humans; Methylnitronitrosoguanidine; Methyltransferases; O(6)-Methylguanine-DNA Methyltransferase; Phenotype; Transfection

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.

Topics: Antineoplastic Agents; Benzamides; Busulfan; Cell Line; Cell Survival; DNA Repair; Drug Synergism; Ethylnitrosourea; Humans; Kinetics; Methyl Methanesulfonate; Methylation; Methylnitronitrosoguanidine; Tumor Cells, Cultured; Tumor Stem Cell Assay

A subclone of a human glioma tumor cell strain which was deficient at O-6-alkylguanine repair was transfected with DNA from an O-6-alkylguanine DNA alkyltransferase-positive human colon tumor cell line (HT29). The transfected subclone, which was selected by its resistance to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), acquired increased resistance to chloroethylnitrosourea (CNU), contained O-6-methylguanine-DNA methyltransferase activity, and failed to accumulate interstrand crosslinks in its DNA upon treatment with CNU. The subclone morphologically resembled its CNU-sensitive parental glioma cell strain. The close correlation between the increased cellular resistance, increased O-6-alkylguanine repair activity and the absence of crosslinking suggests that the formation of DNA crosslinks is one important mechanism of cytotoxicity produced by CNU, and that repair of DNA lesions by O-6-alkylguanine DNA alkyltransferase may partially prevent CNU-induced cytotoxicity.

Topics: Cell Line; Cell Survival; DNA Repair; DNA, Neoplasm; Drug Resistance; Ethylnitrosourea; Humans; Methylnitronitrosoguanidine; Methyltransferases; O(6)-Methylguanine-DNA Methyltransferase; Transfection

The sensitivities of Escherichia coli K-12 strain AB1157, its uvrA-deficient mutant AB1886, and its recA mutant AB2463 to N,N'-bis(2-chloroethyl)-N-nitrosourea, N-(2-chloroethyl)-N-nitrosourea, and N-ethyl-N-nitrosourea have been determined. These data indicate that loss of either uvr excision repair or recA-dependent DNA repair greatly increases sensitivity to the haloethylnitrosoureas. At the same time, loss of recA-dependent DNA repair increases sensitivity to N-ethyl-N-nitrosourea significantly while loss of uvr excision repair increases sensitivity to this agent only marginally. Adapting the uvrA-deficient and recA-deficient mutants by growth in N-methyl-N'-nitro-N-nitrosoguanidine increases survival after exposure to either N-methyl-N'-nitro-N-nitrosoguanidine or N-ethyl-N-nitrosourea, but neither adapted strain loses its sensitivity to N,N'-bis(2-chloroethyl)-N-nitrosourea. Taken together, these data indicate that the haloethylnitrosoureas cause other important cytotoxic lesions in DNA in addition to those involving alkylation of the O6 position of guanine and that the uvrA and recA gene products are involved in the repair of these lesions.

Topics: Alkylation; Carmustine; DNA Repair; Dose-Response Relationship, Drug; Escherichia coli; Ethylnitrosourea; Methylnitronitrosoguanidine; Mutation; Rec A Recombinases; Recombination, Genetic

In two recent reports we have shown that pretreatment of MER+ cells [cells proficient at: reactivating N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-treated adenovirus; removing O-6 methylguanine from their DNA; and preventing DNA interstrand crosslinks produced by the chloroethylnitrosoureas (CNUs)] with MNNG apparently inhibits the repair process that these cells utilize to prevent CNU-induced DNA interstrand crosslinking. The MNNG pretreatment, accompanied by a subsequent CNU treatment, resulted in a synergistic increase in cell kill of 2-3 logs. In the present study we have examined whether or not conditions which inhibit the ability of a cell to prevent CNU-induced DNA interstrand crosslinking can also prevent DNA interstrand crosslinking induced by four clinically used alkylating anti-tumor agents. The agents used in the present study include cis-diamminedichloroplatinum(II) (cis-Pt), L-phenylalanine mustard (L-PAM), nitrogen mustard (HN-2) and 4-S-(propionic acid)-sulfidocyclophosphamide (C-2), a derivative of cyclophosphamide. Alkaline elution analysis was used to measure DNA interstrand crosslinking, and colony formation assays to estimate cell survival. Unlike the CNUs, all four agents produced DNA interstrand crosslinks in a Mer+ cell line in the absence of MNNG pretreatment. MNNG pretreatment did not alter the levels of DNA interstrand crosslinks formed. Similar results were seen with a Mer- cell line. In cytotoxicity studies, in contrast to the CNUs, MNNG pretreatment did not appreciably increase the cell kill produced by the four agents. Since all four agents studied are thought to react primarily at the N-7 position in guanine, these data suggest that: the DNA repair system which prevents CNU-induced crosslinking is specific for methyl, ethyl, and chloroethyl monoadducts; this DNA repair system is specific for adducts only at the O-6 position of guanine and does not recognize and remove adducts at other positions in DNA; or a combination of the two explanations.

Topics: Alkylating Agents; Antineoplastic Agents; Cell Line; Cell Survival; Cross-Linking Reagents; DNA; DNA Repair; Ethylnitrosourea; Humans; Methylnitronitrosoguanidine; Neoplasms; Nitrosourea Compounds

We have recently shown that a variety of human tumor cell lines are capable of preventing chloroethylnitrosourea (CNU)-induced DNA crosslinks, presumably by removing guanine O6-chloroethyl DNA monoadducts before crosslinks can form. Those cells capable of preventing crosslinking were of the Mer+ (Methylation repair) phenotype, and have been shown to be proficient at repair of O6-methyl-guanine adducts by the repair enzyme guanine-O6-methyl-transferase. Mer- tumor cell lines are: deficient at O6-methyl-guanine repair, incapable of preventing CNU interstrand crosslinking, and have recently been shown to lack the repair enzyme O6-methyl-transferase. We wish to report that pretreatment of Mer+ cells (HT-29 human colon carcinoma cells and IMR-90 normal human fibroblasts) with the DNA methylating agent MNNG, under conditions which should inactivate O6-methyl-transferase, apparently saturates the monoadduct repair system, and allows CNU to form interstrand crosslinks in these cells. This effect was also seen when MNU pretreatment was used, but not with MMS or streptozotocin. The formation of CNU-induced interstrand crosslinks following MNNG or MNU pretreatment was coincident with a dramatic increase in cytotoxicity as measured by colony formation assays. In contrast, cytotoxicity was only slightly increased when MMS or streptozotocin pretreatment was used.

Topics: Alkylating Agents; Cell Survival; Cells, Cultured; Cross-Linking Reagents; DNA; DNA Repair; Ethylnitrosourea; Humans; Methylation; Methylnitronitrosoguanidine; Nitrosourea Compounds

Chloroethylnitrosoureas (CNU) are antitumor agents which produce DNA interstrand crosslinks. We have proposed that crosslinks are produced in DNA via monoadduct formation at the guanine-O6 position, followed by a delayed reaction with the opposite DNA strand. Human cells are known to differ in their capacity to repair the O6-methylguanine lesion. One example of this repair capacity is the ability of cells to reactivate adenovirus which has been damaged by in vitro treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Cells that repair the virus are designated Mer+ and deficient cells Mer-. In a recent report, we showed a clear correlation between CNU-induced DNA interstrand crosslinking and the Mer phenotype. Mer- cells produced consistently higher levels of interstrand crosslinks than did Mer+ cells. In the present study we have measured the CNU-induced DNA interstrand crosslinking in IMR-90 normal human fibroblasts (Mer+), HT-29 human colon carcinoma cells (Mer+), and VA-13 SV-40 transformed human cells (Mer-) following pretreatment with MNNG. Cells were treated for 1 h with MNNG, then for an additional 1 h with CNU. Comparable levels of CNU-induced DNA interstrand crosslinking were observed in all cell lines. This crosslinking has been previously undetected in the IMR-90 and HT-29 cells. Cytotoxicity studies showed that MNNG pretreatment greatly enhanced the killing of IMR-90 and HT-29 cells by CNU, however, in VA-13 cells the increase in cell kill was smaller. These data suggest that in Mer+ cells a DNA repair system may remove chloroethyl monoadducts before the lethal DNA interstrand crosslinks can form. However, pretreatment of cells with MNNG may saturate this repair system rendering it inoperable.

Topics: Cell Line; Cell Survival; Colonic Neoplasms; DNA; Ethylnitrosourea; Female; Fibroblasts; Humans; Methylnitronitrosoguanidine; Nitrosourea Compounds; Pregnancy; Time Factors