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.     

SHORT COMMUNICATION: O6-methyltransferase-deficient and -proficient CHO cells differ in their responses to ethyl-and methyl-nitrosourea-induced DNA alkylation

The mutagenic and cytotoxic effects of N-ethyl-N-nitrosourea(ENU) and N-methyl-N-nitrosourea (MNU) were compared in twoisogenic Chinese hamster ovary (CHO) cell lines differing forthe expression of the repair function for O⁶-methylguanine (O⁶-meGua),the O⁶-methyl-DNA-methyltransferase(MT). Survival and ouabainresistance (oua¹) mutation frequency were similar in the twocell lines after treatment with ENU while both effects werestrongly reduced in the MT-proficient (MT⁺) CHO cells afterexposure to MNU. The slow repair kinetics of O⁶-ethylguanine(O⁶-etGua) when compared to O⁶-meGua, i.e. 25% versus 88% removalat 20 h after treatment, could still account for the similarmutational curves reported in the two cell lines after ENU treatment.The number of ENU-induced sister chromatid exchanges (SCE) wasslightly reduced in the MT⁺as compared to MT-deficient CHO cellssuggesting a role for O⁶-etGua in SCE formation. Comparisionof survival after exposure to ENU and MNU showed that, at similarlevels of O⁶-alkylguanine on DNA, the ethyl-is more toleratedthan the methyl-adduct. These data focus the attention on theimportance of DNA damage processing in the cytotoxic responseto alkylating agents.     

Cytotoxicity, mutations and SCEs induced by methylating agents are reduced in CHO cells expressing an active mammalian O6-methylguanine-DNA methyltransferase gene

Alkylation at the O⁶ position of guanine leading to miscodingduring DNA replication has been shown to correlate with mutagenesisboth in bacteria and mammalian cells. The widely used Chinesehamster ovary cells (CHO) are unable to remove O⁶-meyhylguanine(O⁶-meG) due to the absence of O⁶ DNA methyltransferase (MT)activity. Recently Ding et al. [Mol. Cell. Biol. (1985) 5, 3293–3296]transfected CHO cells with human liver DNA obtaining a lineprovided with a function for the repair of O⁶-meG. We confirmedthe presence of MT activity in this particular clone (14 300molecules/cell). We used this MT-proficient cell line as comparedwith the original MT-deficient CHO cell line to analyse therelevance of repair of this lesion on cell killing, ouabain^{resistance (oua}r) mutations and sister chromatid exchanges (SCEs)induced by methylating agents. MT-proficient cells were moreresistant than MT-deficient ones to the cytotoxic and mutageniceffects of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and N-methyl-N-nitrosourea(MNU). Furthermore a lower number of MNNG-induced SCEs werefound in MT-proficient CHO than in MT-deficient cells. Similaroua^r mutation frequencies were recorded in the two cell linesafter 4-nitroquinoline-1-oxide (4NQO) treatment showing thatthe differences in cytotoxicity and mutagenesis are restrictedto treatment with alkylating agents.     

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 O6-methylguanine-DNA activity of rat hepatoma cells is increased after a single exposure to alkylating agents

The O⁶-methylguanine-DNA-methyltransferase activity was measuredin rat hepatoma cells (H4 cells) at different times after N-methyl-N'-nitro-N-nitrosoguanidine(MNNG), methyl- methane sulfonate (MMS) or ethylinethane sulfonate(EMS) treatment. Incubation with MNNG (10µM) first depletesthe methyltransferase activity, then the number of methyltransferasemolecules per cell increases and reaches-3-fold the constitutivelevel after 48 h. Incubations with MMS (0.5 or 1 mM) or withEMS (5 or 10 mM) do not modify or partially decrease the constitutivemethyltransferase level. However, an enhancement of the activityis also observed after 48 h: the activity is 5-and 4-fold higherthan the control value in MMS-and EMS-treated cells, respectively.The methyltransferase increase is due to de novo protein synthesis.It is not observed in cells constitutively lacking this protein.The data suggest that the O⁶-methylguanine (O⁶-meGua) repaircapacity of H4 cells can be increased after a single treatmentwith alkylating agents, by a process different to the adaptiveresponse.     

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.     

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.     

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.     

Inhibition of O6-alkylguanine-DNA-alkyltransferase activity potentiates cytotoxicity and induction of SCEs in human glioma cells resistant to 1,3-bis(2-chloroethyl)-1-nitrosourea

SF-188 is a human glioma-derived cell line resistant to thecytotoxic effects of and the induction of sister chromatid exchanges(SCEs) by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Pretreatmentof SF-188 cells with N-methyl-N-nitrosourea (MNU) for 1 h increasedthe cytotoxicity of a 1-h treatment with BCNU 2-to 10-fold anddoubled the number of SCEs; the magnitude of these effects wasdependent on the dose of both agents. Treatment of SF-188 cellswith MNU resulted in a dose-dependent inhibition of O⁶-alkylguanine-DNA-alkyltransferase(O⁶-AT) activity. Low doses of MNU, which did not significantlyinhibit O⁶-AT, did not potentiate SCE induction. Higher dosesof MNU inhibited O⁶-AT and potentiated cytotoxicity and theinduction of SCEs. These results are consistent with the hypothesisthat in resistant cells treated with BCNU, O⁶-AT repairs O⁶-chloroethylguaninebefore it can form a DNA interstrand cross-link. Inhibitionof this enzyme allows for the formation of BCNU-induced DNAinterstrand cross-links resulting in increases in cytotoxicityand induction of SCEs. The correlation between cytotoxicityand the induction of SCEs suggests that measurement of SCEsmay be useful for determining the cellular response of normaland tumor cells to in vivo treatment with combinations of chemotherapeuticagents.     

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.     

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.     

Transfectant CHO cells expressing O6-alkylguanine-DNA-alkyltransferase display increased resistance to DNA damage other than O6-guanine alkylation

BCNU [1,3-bis(2-chloroethyl)-1-nitrosourea, Carmustine] is anitrosourea that crosslinks DNA and is useful in cancer chemotherapy.Tumor cells resistant to BCNU produce high levels of O⁶-alkylguanine-DNA-alkltransferase(AT), a protein that removes the O⁶-guanine adduct formed byBCNU prior to crosslinking. By the transfection of a human cosmidlibrary into the Chinese hamster ovary cell line AA8, severaltransgenic cell lines which express the AT gene have been constructed.These ‘BR’ cells were isolated on the basis of theirresistance to G-418 and BCNU. Like human mer⁺ strains, BR cells(relative to the parental AA8 cells) are {small tilde}500 timesmore resistant to the cytotoxic effects of 80 µM BCNU.Treatment with exogenous O⁶-methylguanine (O⁶MG), which depletescellular AT, abolishes their BCNU resistance. Also consistentwith the mer⁺ phenotype, BR cells are resistant to the mutagenicand killing activity of N-methyl-N'-nitro-N-nitrosoguanidine(MNNG). Treatment with exogenous O⁶MG, while reversing the resistanceto MNNG mutation, does not reverse the resistance to MNNG killing.Unexpectedly, BR cells also exhibit resistance to killing bydimethylsulfate (DMS). The BR cells are not, however, detectablyresistant to UV light. These results suggest that AT activityin mammalian cells is dosely linked to the activity of otherDNA repair pathways.     

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.     

Genetic consequences of tolerance to methylation DNA damage in mammalian cells

We previously characterized a clone of CHO cells, clone B, thatdisplayed tolerance to the cytotoxic effects of N-methylnitrosourea(MNU) and 6-thioguanine (6-TG). To determine whether this phenotypeaffected the mutagenic response of the cells, MNU-induced mutationto 8-azaadenine resistance (8-AA^r) was measured in the parentaland clone B cells. Comparable mutation frequencies were foundin the two cell lines up to 0.5 mM MNU, while at higher MNUconcentrations mutations could be reproducibly measured onlyin clone B cells. Similar amounts of DNA methylated bases werefound in the two cell lines after a 30 min treatment with differentconcentrations of [³H]MNU and the same linear relationship wasobserved when mutation induction by MNU was plotted as a functionof the amount of O⁶-methylguanine (O⁶-MeGua) in DNA, indicatingthat mutation induction in both cell lines was related to thepresence of this methylated base. Fifteen MNU-induced 8-AA^rmutants were isolated from each cell line and the sequencesof the adenine phosphoribosyltransferase (aprt) mutations determined.The type (in 90% of the cases, GC to AT transitions), the sequencecontext and the strand localization of the mutations indicatedthat all mutations were targeted at O⁶-MeGua in DNA and no differencewas found between the two lines. These results are consistentwith a mechanism of tolerance of O⁶-MeGua that does not alterthe processing of this methylated base into a mutation. Growthin 6-TG induced point mutations in clone B but not in the parentalcells. A model is proposed in which the alkylation tolerantvariant is altered in a mismatch correction pathway responsiblefor the cytotoxicity of the methylated base.     

Isolation of clones displaying enhanced resistance to methylating agents in O6-methylguanine-DNA methyltransferase-proficient CHO cells

O⁶-Methylguanine-DNA methyltransferase (MT)-proficient Chinesehamster ovary cells were grown in the presence of low, graduallyincreasing levels of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)with the aim of selecting MNNG resistant cell lines. Six resistantclones with two levels of resistance were isolated. A 3-foldincrease in survival was observed in clones 13, 14 and 15 anda > 10-fold increase in clones A, B and C. Cross resistanceto N-methyl-N-nitrosourea but not to mitomycin C was observed.By comparison with the parental MT-proficient cells, MT activitywas doubled in two resistant clones (13 and B) irrespectiveof then- resistance levels. DNA glycosylase activity responsiblefor the removal of 7-methylguanine and 3-methyladenine showedsimilar levels in resistant clones 13 and B, in the MT-proficientcells and in the original MT-deficient cells. Alkylation-inducedDNA damage, as measured by alkaline elution at the same MNNGdose, was higher in clones 13 and B than in the parental cells.The induction of sister chromatid exchanges by MNNG was inverselyrelated to the resistance levels, thus paralleling the inductionof cytotoxicity. These results suggest the existence in Chinesehamster ovary cells of at least two independent functions whichcontrol resistance to methylating agents, one possibly beingthe capacity to repair O⁶-methylguanine.     

The relationship between O6-alkylguanine alkyltransferase activity and sensitivity to alkylation-induced sister chromatid exchanges in human lymphoblastoid cell lines

We investigated the relationship between the ability to repairthe O⁶-alkylguanine lesions and sister chromatid exchange (SCE)induction. Six human lymphoblastoid cell lines, with O⁶-alkylguaninealkyltransferase (AGT) activities ranging from 0 to 13.2 fmol/µgDNA, were tested for their sensitivity to N-methyl-N'-nitro-N-nitrosoguanidine(MNNG)-, methyl methanesulfonate (MMS)- and 1,3-bis(2-chloroethyl)-1-nitrosourea(BCNU)-induced SCEs. L33, a long established lymphoblastoidcell line with no AGT activity, was sensitive to all three alkylatingagents. In the other more recently established Epstein-Barrvirus transformed cell lines, no correlation between AGT activity(ranging from 2.4 to 13.2 fmol/µg DNA) and sensitivityto MMS or MNNG was noted. In fact, of these five cell lines,the cell line with the highest AGT activity, line 852A, wasthe most sensitive to MNNG-induced SCEs. While cell lines differedin overall alkylation by MNNG, no relationship between overallakylation and sensitivity to MNNG-induced SCE formation wasnoted. In contrast to the results with the monofunctional alkylatingagents, there was a correlation between AGT activity and BCNU-sensitivityto SCE induction. Cell lines with low AGT activities were moresensitive to the bifunctional alkylating agent than cells withhigher activities. Therefore, while DNA interstrand cross-linksproduced by BCNU exposure probably underlie SCE induction bythis agent, the lesions and processes that lead to SCE inductionafter exposure to monofunctional alkylating agents remain unclear.     

Chinese hamster cells harbouring the Escherichia coli O6-alkylguanine alkyltransferase gene are less susceptible to sister chromatid exchange induction and chromosome damage by methylating agents

Clones of Chinese hamster V79 cells harbouring the Escherichiacoli O⁶-alkylguanine (O⁶-AG) alkylphosphotriester (AP) alkyltransferase(ATase) gene (clone 8) or a subclone of it that codes only forO⁶-AG ATase activity (clone SB) have been exposed to increasingdoses of N-methyl-N-nitrosourea (MNU) or methybnethanesulphonate(MMS) and the frequencies of induced sister chromatid exchanges(SCEs) measured. In control (clone 2) cells, SCE induction wasalmost linearly proportional to dose of MNU or MMS and at thehighest doses used (15 or 80 µg/ml) SCE frequencies were6 or 8 times background levels, respectively. Slightly lowerlevels of MMS-induced SCEs were seen in clone 8 and clone SBcells whilst, in contrast, MNU-induced SCE levels in these twoclones were drastically reduced being less than twice backgroundlevels at 15 µg/ml. After treatment with N-butyl-N-nitrosourea,SCE frequency was similar in all three clones. At higher doses,MNU treatment produced less chromatid aberrations and micronucleiin clone SB than in clone 2 cells. These results suggest thatATase-repairable damage is involved in the induction of SCE,chromosome aberrations and micronuclei in V79 cells.     

Analysis of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced DNA damage in tumor cell strains from Japanese patients and demonstration of MNNG hypersensitivity of Mer-xenografts in athymic nude mice

Among 15 human tumor cell strains from Japanese patients, onestrain derived from a patient with thyroid cancer showed inabilityto support the growth of adenovirus 5 treated with N-methyl-N'-nitro-N-nitrosoguanidine(MNNG). When plated on this Mer^– strain, adenovirus 5showed 3–4 times higher sensitivity to MNNG-induced killingthan when plated on any of the other 14 Mer⁺ tumor cell strains.Biochemical analysis showed that the Mer^– strain was defectivein demethylation repair of O⁶-methylguanine produced by MNNGtreatment. The sensitivities of 12 of the 15 human tumor strains,including the Mer^– strain, to MNNG were compared by measuringtheir colony-forming abilities. All the strains tested showedthe Rem^– phenotype (having higher sensitivity to MNNG-producedcell killing than normal fibroblasts). The differential killingeffects of MNNG on Mer^– and Mer⁺ tumor cells under invivo conditions were tested using the Mer⁺ HeLa S3 strain andits Mer^– variant. Mer⁺ cells and Mer^– cells wereimplanted sub-cutaneously into the left and right flanks, respectively,of 10 nude mice and the next day, MNNG solution (0.25 ml at1 mg/ml) was injected into the implantation sites of eight mice.Mer^– tumor cells in six of eight treated mice showed nogrowth and those in the other two mice did grow, but regressedafter 3 weeks. In contrast, Mer⁺ tumor cells continued to growin all the eight mice treated, indicating that Mer^– tumorcells may be selectively inactivated by suitable therapeuticregimens with appropriate methylating drugs.     

DNA adduct formation and assessment of aberrant crypt foci in vivo in the rat colon mucosa after treatment with N-methyl-N-nitrosourea

N-Nitroso-compound DNA adduct formation in vivo and occurrenceof aberrant crypt foci (ACF) were studied in the rat colon mucosaafter a single, local treatment with a carcinogen, N-methyl-N-nitrosourea(MNU), using a simple surgical approach. A segment of F344 ratcolon was ligated to make a pouch and injected with MNU solution.For the study of DNA adduct formation, the solution contained50 µCi of [³H]MNU. The results demonstrated that similarranges of carcinogen dose, i.e. 0.15x10^–2 –1.5x10^–2MMNU, could induce both DNA adduct formation and appearanceof ACF in the rat colon with both parameters showing a nearbylinear dose dependence. HPLC analysis revealed the DNA adductsto include both 7-methylguanine (7-mGua) and O⁶-methylguanine(O⁶-mGua) with the 7-mGua/O⁶-mGua ratio being 8.2–11.3:1in the system used. Assessment of ACF development from 4 to16 weeks after MNU treatment at a dose of 7.5x10^–2 M showedthe numbers to increase up to the 8th week, followed by a decreaseat weeks 12 and 16, when 40% of the ACF counted at the peaktime point were still present. The percentage of large ACF (