Human tumor cell strains defective in the repair of alkylation damage

We have previously identified four human astrocytoma cell strainsas defective in the repair of N-methyl-N'-nitro-N-nitrosoguanidine(MNNG) damaged adenovirus 5. We now show that two of these strains(the only two tested), in comparison to other tumor strainsor normal human skin fibroblasts, are very sensitive to MNNG-producedkilling as measured by colony forming ability, but are normallysensitive to ultraviolet light. Further, such repair deficientcells may be cultured from tumors of the colon, lung, skin,and neck. The phenotype of deficient repair of MNNG-treatedadenovirus 5 has now been found in a subgroup of 9 of the 39human tumor strains tested. We propose to call this phenotypethe Mer^– phenotype. None of the 22 strains of normal humanskin fibroblasts tested showed deficient repair of MNNG damage.MNNG treatment (80 µM) causes a decrease in semi-conservativeDNA synthesis from which Mer^– tumor cells do not recover,but from which cells capable of normal repair of MNNG damage(Mer⁺) do. Somewhat paradoxically, Mer^–cells show moreMNNG-stimulated DNA synthesis (‘repair synthesis’)than do Mer⁺ cells. Besides being deficient in the repair ofMNNG-damaged adeno-viruses Mer– cells also have difficultyin repairing viruses damaged either by other N-alkyl-N'-nitro-N-nitrosoguanidines,or by N-methyl- or N-ethyl-N-nitrosoureas.     

Pretreatment of normal human fibroblasts and human colon carcinoma cells with MNNG allows chloroethylnitrosourea to produce DNA interstrand crosslinks not observed in cells treated with chloroethylnitrosourea alone

Chloroethylnitrosoureas (CNU) are antitumor agents which produceDNA interstrand crosslinks. We have proposed that crosslinksare produced in DNA via monoadduct formation at the guanine-O⁶position, followed by a delayed reaction with the opposite DNAstrand. Human cells are known to differ in their capacity torepair the O⁶-methylguanine lesion. One example of this repaircapacity is the ability of cells to reactivate adenovirus whichhas been damaged by in vitro treatment with N-methyl-N'-nitro-N-nitrosoguanidine(MNNG). Cells that repair the virus are designated Mer⁺ anddeficient cells Mer^–. In a recent report, we showed aclear correlation between CNU-induced DNA interstrand cross-linkingand the Mer phenotype. Mer^– cells produced consistentlyhigher levels of interstrand crosslinks than did Mer⁺ cells.In the present study we have measured the CNU-induced DNA interstrandcrosslinking in IMR-90 normal human fibroblasts (Mer⁺), HT-29human colon carcinoma cells (Mer⁺), and VA-13 SV-40 transformedhuman cells (Mer^–) following pretreatment with MNNG. Cellswere treated for 1 h with MNNG, then for an additional 1 h withCNU. Comparable levels of CNU-induced DNA interstrand crosslinkingwere observed in all cell lines. This crosslinking has beenpreviously undetected in the IMR-90 and HT-29 cells. Cytotoxicitystudies showed that MNNG pretreatment greatly enhanced the killingof IMR-90 and HT-29 cells by CNU, however, in VA-13 cells theincrease in cell kill was smaller. These data suggest that inMer⁺ cells a DNA repair system may remove chloroethyl monoadductsbefore the lethal DNA interstrand crosslinks can form. However,pretreatment of cells with MNNG may saturate this repair systemrendering it inoperable.     

MNNG-induced partial phenotypic reversion of Mer- cells

The effect of pretreatment with N-methyl-N'-nitro-N-nitrosoguanidine(MNNG) on MINNG sensitivity of the surviving population wascompared in two HeLa lines, one of the Mer⁺ phenotype (HeLaS3) and one of the Mer^– phenotype (HeLa MR). Whereas MNNGpretreatment of HeLa Mer⁺ cells had no effect on the MNNG sensitivityof surviving cells, Mer^– cells surviving a first exposureto MNNG became much more resistant to MNNG Comparison of thesensitivity of individual HeLa MR clones with their MNNG-pretreatedpopulation and analysis of the composition of the pretreatedpopulation showed that the majority of cells surviving the MNNG-pretreatmentnow displayed the Mer⁺ phenotype in respect to sensitivity toMINNG. One MNNG-resistant clone derived from a pretreated HeLaMR population (Cl 4) was characterized further. It had a similarsensitivity to the Mer⁺ line to all monofunctional alkylatingagents, but was as sensitive as the Mer^– line to the crosslinkingagent chloroethylntrosourea. Cl 4 cells, like the Mer^–cells, did not repair O⁶-methylguanine (O6MeG). The resultssuggest that the two characteristics which are usually coupledwith the Mer^– phenotype - lack of O⁶MeG repair and hypersensitivityto MNNG - can be separated.     

Human tumor strains defective in the repair of alkylated DNA fail to regenerate rapidly-sedimenting nucleoids after N-methyl-N' -nitro-N-nitrosoguanidine treatment

Upon treatment with N-methyl-N'-nitro-N-nitroso-guanidine (MNNG),human cell strains characterized as either proficient or defectiveboth in repair of alkyla-tion-damaged DNA and in supportingthe growth of MNNG-treated adenovirus (Mer⁺ and Mer^– pheno-types(1,2), all underwent a rapid relaxation of nucleokl DNA, asjudged by sedimentation in 15–30% neutral sucrose gradients.DNA in the repair-proficient Mer⁺ strains (normal fibroblastand tumor) was restored to the rapidly-sedimenting (control)form within 2–4 h after the removal of MNNG. In contrast,nucleoid DNA of the repair-deficient Mer^– tumor strainsremained slowly-sedimenting even after 48 h of incubation. Thedelayed recovery of Mer^– nucleoid DNA was specific forMNNG damage, since after u.v. irradiation, to which Mer⁺ andMer^– strains are equally resistant (2), all cell linestested underwent DNA relaxation within the first hour afterirradiation (3 J/m²) and regenerated rapidly-sedimenting nucleoidswithin 4–6 h of repair incubation.     

Hypersensitivity of human tumor xenografts lacking O6-alkylguanine-DNA alkyltransferase to the anti-tumor agent 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea

Human tumor cell strains having different activities of O⁶-alkylguanine-DNAalkyltransferase (ATR) were transplanted into nude mice andchemotherapeutic responses of tumor xenografts were comparedafter intraperitoneal injection of the anti-tumor drug 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea(ACNU). The tumor strains used were four Mer⁺ strains possessinghigh ATR activity and three Mer^– strains lacking thisactivity. Included in these Mer⁺ strains was a clone 5'dD whichexpresses the Escherichia coli ATR in Mer^– HeLa cellsand thus shows the Mer⁺phenotype. All the Mer^– tumor xenograftswere much more sensitive than tumors of Mer⁺ strains, includingthe clone 5’dD; after the highest ACNU dose (three injectionsof 50 mg/kg), some Mer^– tumors disappeared completelyand the growth of other tumors was severely retarded, whereasall Mer⁺ tumors continued to grow. These results demonstratethat ATR activity in tumor cells is a major determinant of tumorresponse to ACNU, and further suggest that measurement of ATRactivity in biopsy specimens may provide a useful guide to predictthe response to chemotherapy.     

Repair of O6-methylguanine in DNA by demethylation is lacking in Mer- human tumor cell strains

The ability of extracts of human tumor cells to demethylateO6-methylguanine (O⁶-MeG) in DNA was assayed using the syntheticDNA polymer poly(dC,dG,m⁶dG). Cell strains proficient in repairof O⁶-MeG in vivo (Mer+ phenotype) contained a methyltransferaseactivity while repair deficient cells (Mer^– phenotype)had little or no activity. Mixing extracts of different Mer^–strains did not result in the appearance of the activity. Extractsof Mer^– cells did not inhibit the activity in extractsof Mer⁺ cells. Both Mer⁺ and Mer^– strains contained methylnitrosourea-damage-specificendonudease activity. The data suggest that the Mer⁺ strainsare deficient in methyltransferase and that this is the fundamentalreason for their hypersensitivity to the cytotoxic effects ofDNA alkyla-tion. The activity was partially purified from aMer⁺ colon carcinoma cell strain. Its kinetics parallel therepair of O⁶-MeG in DNA in vivo and suggest that the activityis inactivated during repair of DNA.     

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 killingof nine human cell lines exposed to alkylating agents. Celllines included normal, transformed and DNA repairproficientand -deficient pbenotypes. 3AB potentiated cell killing by themethylating agents methyhnethanesulfonate (MMS) and N-methyl-N'-nitro-N-nitrosoguanidine(MNNG) in all lines tested. The degree of potentiation rangedfrom 1.7- to 3.8-fold, based on the LD₉₉. The average potentiationobserved with MMS (2.7-fold) was greater than with MNNG (2.2-fold).On average the potentiation of MMS and MNNG killing of repair-deficientMer^– lines (2.4-fold) was similar to that of repair-proficientMer⁺ 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-sensitiveDNA repair pathway. Within experimental error, 20 mM 3AB didnot inhibit the removal of the MNNG-induced methylpurines 7-methylguanine,O⁶-methylguanine and 3-methyladenine from the DNA of repair-proficientMer₊ Rem⁺ HT29 cells, consistent with evidence that 3AB inhibitsthe ligation step of excision repair. 3AB potentiated cell killingby the bifunctional alkylating agents 1-(2-chlorethyl)-1-nitrosoureaor busulfan, two antineoplastic drugs, by only 0.9- to 1.5-fold.These drugs therefore produce DNA damage which is not efficientlyrepaired by the pathways that repair methylated bases.     

Increased O6-methylguanine-DNA methyltransferase activity and reduced mutability in 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea-resistant HeLa S3 cells

To clarify the involvement of O⁶-methylguanine (O⁶-MeG) in mutagenesis,we have been trying to isolate Mer⁺ cells from a HeLa S3 Mer^–cell line, and to compare the mutation frequencies between thecell lines. We previously isolated the N-methyl-^N'-nitro-^N-nitrosoguanidine(MNNG)-resistant cells, MR10–1, from HeLa S3 Mer^–cells. However, the MR10–1 cells still had only a littleO⁶-MeG-DNA methyltransferase (MT) activity. In the present study,we have isolated two 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-mitrosourea(ACNU)-reslstant cells, AC^r41 and AC^r42, from the MR10–1cells. The two AC^r cells had increased MT activities. The AC^rcells were also significantly more resistant to 1-(2-chloroethyl)-1-nitrosoureaand slightly more resistant to MNNG than the MR10–1 cells.When the mutation frequencies were tested at the hypoxanthine–guanine phosphoribosyl transferase and ouabain loci in thesecell lines, the two AC^r cells were more resistant to the mutageniceffect of MNNG than the MIR10–1 cells. These results showthe linkage between the resistance to the cytotoxic effect ofnitrosourea compounds and MT activity, and strongly supportthe hypothesis that O⁶-MeG is the main pre-mutagenic lesioninduced by MNNG.     

The methylation status of the gene for O6-methylguanine-DNA methyltransferase in human Mer+ and Mer- cells

O⁶-Methylguanine-DNA methyltransferase (MGMT) plays an importantrole in protecting cells from the mutagenic potency of alkylatingagents. This study addresses the role of DNA methylation inthe expression of the human MGMT gene. Southern blot analysisof DNA from human Mer⁺ (MGMT proficient) and Mer^– (MGMTdeficient) cell lines demonstrated that the methylation stateof a unique SmaI site in the MGMT gene promoter, previouslyshown by others to be invariably unmethylated in Mer⁺ cellsand methylated in Mer^– cells, did not correlate with theMer phenotype. Neither was there any significant differencein the density of CpG methylation in the MGMT gene 5'-flankingsequences between Mer⁺ and Mer^– cells. On the other hand,the body of the MGMT gene was less methylated in mod Mer cellsrelative to Mer⁺ cells, and in three of six Mer^– cellLines the gene was essentially methylationfree. Interestingly,the Mer^– cells that were hypomethylated in the MGMT genealso tended to be less methylated at other loci. Widespreadhypomethylation is a frequent trait in carcinogenesis, and maybe involved in development of the frequently found Mer^–phenotype.     

DNA alkylation repair and the induction of cell death and sister chromatid exchange in human cells

Mer^– human cells, which lack O⁶-methylguanine DNA methyltransferaseactivity, are extremely sensitive to alkylation induced killing,mutation and sister chromalid exchange. We have analyzed a Mer⁺, a Mer^–, and a Mer^– revertant HeLa cell line andfound that the methyltransferase deficiency correlates withincreased levels of mutation and sister chromatid exchange,but does not correlate with increased killing of Mer^–HeLa cells by alkylating agents. Furthermore, we show that HeLaMer^– cells repair N-3-methylguanine and N-3-methyladeninejust as efficiently as HeLa Mer⁺ cells.     

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)-producedO⁶-methylguanine (O⁶-MeG) in DNA and its correlation with MNNG-producedcell-killing and sister chromatid exchange (SCE) induction werecompared in mouse and reference human tumor cell strains. Asa result, mouse cell strains were divided into three groups:(i) cells proficient in O⁶-MeG-repair and insensitive to MNNGsimilar to human Mer⁺ Rem⁺ strains; (ii) cells deficient inO⁶-MeG-removal and sensitive to MNNG similar to human Mer^–Rem^– strains; (iii) cells deficient in O⁶-MeG-removalbut insensitive to MNNG similar to some SV40-transformed humanstrains. Attempts at correlating lack of capacity for O⁶-MeG-removal,MNNG-sensitivity and high SCE induction showed that O⁶-MeG inDNA may be a lesion common to cell-killing and SCE inductiononly in mouse cells of groups i and ii. Levels of O⁶-MeG-DNAmethyttransferase activity in mouse cells were measured andthe enzyme had the same molecular weight as that in human cells.     

A human cell line proficient in O6-methylguanine-DNA-methyltransferase and hypersensitive to alkylating agents

The involvement of O⁶-methylguanine (O⁶-meGua) in mutagenesisis well established, while the toxic effect of these residuesis still controversial. In this study, we compare the cytotoxicityof N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and N-methyl-N-nitrosourea(MNU) on three cell lines of different origin, which have differentabilities to repair O⁶-meGua residues (Mer phenotype): a humanhepatoma cell line (LICH cells, Mer⁺), a rat hepatoma cell line(H4 cells, Mer⁺) and a Chinese hamster cell line (CHO cells,Mer^– phenotype). LICH and CHO cells show the same sensitivityto the killing effect of MNNG and MNU and are 5-fold more sensitivethan H4 cells. However, LICH and H4 cells share similar sensitivitiesto the toxic effect of 1, 3-bis(2-chloroethyl)-l-nitrosourea.O⁶-meGua residues are removed at the same rate from the DNAof [³H]MNU-treated LICH and H4 cells, which also do not differin the rate of removal of N3-methyladenine residues nor in overallDNA repair synthesis. The results suggest that MNNG and MNUproduce a lethal lesion that is repaired by a process that doesnot involve the alkyltransferase.     

Comparison of mutation spectra induced by N-ethyl-N-nitrosourea in the hprt gene of Mer+ and Mer- diploid human fibroblasts

N-Ethyl-N-nitrosourea (ENU) forms several major adducts uponreaction with DNA, of which ethylation at the O⁶ position ofguanine and the O⁴, O² and N³ positions of thymine have beenimplicated to be mutagenic lesions. To investigate what specifickinds of ENU-induced mutations were affected by the repair abilityof O⁶-alkylguanine-DNA alkyltransferase (AGT), we examined themutations in the hypoxanthlne (guanine) phosphoribosyltransferasegene (hprt) in 87 independent mutants derived from ENU-treatedAGT proficient (Mer⁺) or deficient (Men^–) diploid humanfibroblasts. Of the characterized mutations, 97% were singlebase substitutions. The major difference in the mutation spectrawas that the frequency of G·C to A·T transitionswas significantly higher in Mer^– mutants (16/38) thanin Mer mutants (4/33). The results indicate that AGT removesO⁶-ethylguanine, thus protecting human cells from parts of thecytotoxic and mutagenic effects of ENU. A high frequency ofT· to A·T transversions induced by ENU was observedin both Mer⁺ (52%) and Mer^– (34%) mutants. This type ofmutation was less frequently observed (10%) in N-methyl N'-nitro-N-nitrosoguanidine(MNNG)-induced mutants derived from the same Mer⁺ cells in ourprevious report (J. Mol. Biol., 221, 421, 1991). Comparisonof alkylating lesions formed by MNNG and ENU indicates thatO² and N³-ethylthymine are potent mutational adducts for T toA transversions. The occurrence of ENU induced T·A basepair transverslons showed a strong strand bias; 35/37 were locatedon the non-transcribed strand, assuming thymine is the mutageniclesion. The result suggests a difference in repair capacityof ethyithymine on the two strands. In addition, this type ofmutation preferentially occurred at 5'-Pu-T sequences.     

Specific DNA repair mechanisms may protect some human tumor cells from DNA interstrand crosslinking by chloroethylnitrosoureas but not from crosslinking by other anti-tumor alkylating agents

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 theirDNA; and preventing DNA interstrand crosslinks produced by thechloroethyl-nitrosoureas (CNUs)] with MNNG apparently inhibitsthe repair process that these cells utilize to prevent CNU-inducedDNA interstrand crosslinking. The MNNG pretreatment, accompaniedby a subsequent CNU treatment, resulted in a synergistic increasein cell kill of 2–3 logs. In the present study we haveexamined whether or not conditions which inhibit the abilityof a cell to prevent CNU-induced DNA interstrand crosslinkingcan also prevent DNA interstrand crosslinking induced by fourclinically used alkylating anti-tumor agents. The agents usedin 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 analysiswas used to measure DNA interstrand crosslinking, and colonyformation 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 pretreatmentdid not alter the levels of DNA interstrand crosslinks formed.Similar results were seen with a Mer^– cell line. In cytotoxicitystudies, in contrast to the CNUs, MNNG pretreatment did notappreciably increase the cell kill produced by the four agents.Since all four agents studied are thought to react primarilyat the N-7 position in guanine, these data suggest that: theDNA repair system which prevents CNU-induced crosslinking isspecific for methyl, ethyl, and chloroethyl monoadducts; thisDNA repair system is specific for adducts only at the O-6 positionof guanine and does not recognize and remove adducts at otherpositions in DNA; or a combination of the two explanations.     

Differential cytotoxicity and DNA-damaging effects produced in human cells of the Mer+ and Mer- phenotypes by a series of alkyltriazenylimidazoles

A series of alkyltriazenylimidazoles have been investigatedfor their differential cytotoxicity towards the HT-29 (Mer⁺)and BE (Mer^–) cell lines and for their ability to causeDNA strand breaks and cross-links. A monomethyltriazene, andsome hydroxymethyltriazene derivatives capable of generatingthe monomethyltriazene in situ, were preferentially cytotoxictowards the BE cell line compared with the HT-29 cell line,with very close similarity in the differential toxicity to theanalogous monochloroethyltriazene. In contrast, the dimethyl-andmonoethyltriazenes in the series display reduced toxicity towardsthe BE cell line with little or no differential toxicity betweenBE and HT-29 cell lines. With another pair of human cell lines,the IMR-90 (Mer⁺⁾ and VA-13 (Mer^–) cells, the monomethyl-and monochloroethyltriazenes were again more cytotoxic to theMer– cells. Neither the formation of DNA single-strandbreaks or DNA-protein cross-links could account for the differentialcytotoxicity oberved in the Mer⁺ and Mer^– cells. Moreimportantly, the inability of the monofunctional monomethyltriazeneto cross-link DNA tends to question the role of DNA inter-strandcross-linking as a mechanism for cell killing by chloroethylatingagents.     

Effect of 5-azacytidine on expression of the human DNA repair enzyme O6-methylguanine-DNA methyltransferase

The role of methylatlon of CpG dinucleotides in the regulationof O⁶-methylguanine-DNA methyltransferase (MGMT) gene expressionhas been investigated. A previous observation, that cell linesdeficient in MGMT (Men^– are methylated in a SmaI sitein the MGMT gene promoter whereas MGMT expressing cells (Mer⁺)are unmethylated in the same site, has been extended to a totalof 30 cell lines, tumors and normal tissues. To examine furtherthe association between methylation in the MGMT promoter andthe Mer^– phenotype we have treated Mer⁺ and Mer^–cell lines with 5-azacytidine to inhibit DNA methylation. Reducedmethylation in the SmaI site coincided with induction of MGMTexpression for one of three Mer^– cell lines. MGMT increasedseveral-fold further upon continued culture of the induced cellsin the absence of 5-azacytidlne, coincident with an abrupt increasein methylation in the body of the MGMT gene even though theSmaI site remained demethylated. These results, and those ofother previous studies, suggest that methylation of sequenceswithin the MGMT gene promoter and methylation within the bodyof the gene have opposite effects.     

Pretreatment of human colon tumor cells with DNA methylating agents inhibits their ability to repair chloroethyl monoadducts

We have recently shown that a variety of human tumor cell linesare capable of preventing coloroethylnitrosourea (CNU)-inducedDNA crosslinks, presumably by removing guanine O⁵ choloroethylDNA monoadducts before crosslinks can form. Those cells capableof preventing crosslinking were of the Mer⁺ (Methylation repair)phenotype, and have been shown to be proficient at repair ofO⁶-methyl-guanine adducts by the repair enzyme guanine-O⁶-methyl-transferase.Mer^- tumor cell lines are: deficient at 06-methyl-guanine repair,incapable of preventing CNU Interstrand crosslinking, and haverecently been shown to lack the repair enzyme O⁶methyl-transferase.We wish to report that pretreatment of Mer⁺ cells (HT-29 humancolon carcinoma cells and IMR-90 normal human fibroblasts) withthe DNA methylating agent MNNG, under conditions which shouldinactivate methyltransferase, apparently saturates the monoadductrepair system, and allows CNU to form interstrand crosslinksin these cells. This effect was also seen when MNU pretreatmentwas used, but not with MMS or streptozotocin. The formationof CNU-induced interstrand crossllnks following MNNG or MNUpretreatment was coincident with a dramatic increased in cytotoxityas measured by colony formation assays. In contrast, cytotoxicitywas only slightly increased when MMS or streptozotocin pretreatmentwas used.     

N-Methyl-N'-nitro-N-nitrosoguanidine-resistant HeLa S3 cells still have little O6-methylguanine-DNA methyltransferase activity and are hypermutable by alkylating agents

To clarify the involvement of O⁶-methylguanine (O⁶-MeG) in mutagenesis,we isolated N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-resistantcells, MR10-1 from HeLa S3 mer^– cells. MR10-1 cells were40 times more resistant to MNNG than the parental cells. MR10-1cells were also significantly more resistant to N-methyl-N-nitrosoureaand slightly more resistant to methyl methanesulfonate and dimethylsulfate than parental cells. However, we found that MR10-1 cellshad still little O⁶-MeG-DNA methyltransferase activity and weresensitive to 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosoureahydrochloride, like HeLa mer^– cells, thereby showing thatMR10-1 cells are still mer^–. When induced 6-thioguanine(6TG)-resistant colonies were plotted as a function of the correspondingpercentage survival, the resistant colonies of MR10-1 cellswere induced much more frequently than in the case of HeLa mer^–cells. However, induction of 6TG-resistant cells in the bothcell lines did not differ significantly in terms of mutant cellsper 0.1 µM MNNG. On the contrary, MR10-1 cells (mer^–)and two HeLa S3 mer⁺ cells lines differed in the induction ofmutation as a function of MNNG concentration. The HeLa mer⁺cell lines were not mutable, while MR10-1 cells were highlymutable. These above results clearly show that the HeLa mer^–cell has at least two defects in the repair of the alkylatedadducts which are related to cell killing and mutation, andalso suggest that O⁶-MeG is involved in the induction of mutation.