O6-alkylguanine DNA Alkyltransferase Activity Research Articles

Phase I Clinical Trial of O6-Benzylguanine and Topical Carmustine in the Treatment of Cutaneous T-Cell Lymphoma, Mycosis Fungoides Type

To evaluate the toxic effects and maximum tolerated dose of topical carmustine [1,3-bis(2-chloroethyl)-1-nitrosourea] following intravenous O6-benzylguanine in the treatment of cutaneous T-cell lymphoma (CTCL), and to determine pharmacodynamics of O6-alkylguanine DNA alkyltransferase activity in treated CTCL lesions. Open-label, dose-escalation, phase I trial. Dermatology outpatient clinic and clinical research unit at a university teaching hospital. A total of 21 adult patients (11 male, 10 female)with early-stage (IA-IIA) refractory CTCL, mycosis fungoides type, treated with topical carmustine following intravenous O6-benzylguanine. Treatment once every 2 weeks with 120 mg/m(2) intravenous O6-benzylguanine followed 1 hour later by whole-body, low-dose topical carmustine starting at 10 mg, with 10-mg incremental dose-escalation in 3 patient cohorts. Cutaneous T-cell lymphoma lesional skin biopsy specimens were taken at baseline and 6 hours, 24 hours, and 1 week after the first O6-benzylguanine infusion for analysis of O6-alkylguanine-DNA alkyltransferase activity. Clinical response measured by physical examination and severity-weighted assessment tool measurements, safety data acquired by review of adverse events at study visits, and O6-alkylguanine-DNA alkyltransferase activity in treated lesion skin biopsy specimens. A minimal toxic effect was observed through the 40-mg carmustine dose level with 76% of adverse events being grade 1 based on the National Cancer Institute Common Terminology Criteria for Adverse Events. Mean baseline O6-alkylguanine-DNA alkyltransferase activity in CTCL lesions was 3 times greater than in normal controls and was diminished by a median of 100% at 6 and 24 hours following O6-benzylguanine with recovery at 1 week. Clinical disease reduction correlated positively with O6-alkylguanine-DNA alkyltransferase activity at 168 hours (P=.02) and inversely with area under the curve of O6-alkylguanine-DNA alkyltransferase over 1 week (P=.01). Twelve partial responses and 4 complete responses were observed (overall response, 76% [95% CI, 0.55-0.89]). Five patients discontinued therapy owing to adverse events with a possible, probable, or definite relationship to the study drug. O6-benzylguanine significantly depletes O6-alkylguanine-DNA alkyltransferase in CTCL lesions and in combination with topical carmustine is well tolerated and shows meaningful clinical responses in CTCL at markedly reduced total carmustine treatment doses.

Dietary variables associated with DNA N7-methylguanine levels and O6-alkylguanine DNA-alkyltransferase activity in human colorectal mucosa

Components of human diets may influence the incidence of colorectal adenomas, by modifying exposure or susceptibility to DNA-damaging alkylating agents. To examine this hypothesis, a food frequency questionnaire was used to assess the diet of patients recruited for a case-referent study where biopsies of normal colorectal mucosa were collected during colonoscopy and subsequently analysed for DNA N7-methylguanine (N7-MeG) levels, as an indicator of exposure, and activity of the DNA repair protein O6-alkylguanine DNA-alkyltransferase (MGMT), as an indicator of potential susceptibility. Cases with histologically proven colorectal adenomas (n = 38) were compared with referents (n = 35) free of gastrointestinal neoplasia. The case group consumed significantly more red meat (4.5 versus 3.4 servings/week, P < 0.05), processed meats, (4.7 versus 3.2 servings/week, P < 0.05) and % food energy as fat (34.9 versus 30.7%, P < 0.001). N7-MeG [mean: 95% confidence interval (CI)] levels were significantly lower in the group that consumed the highest proportion of dietary fibre/1000 kcal in comparison with the group with the lowest intake (0.61; 0.35-0.86 versus 1.88; 0.88-2.64 micromol/mol dG, P < 0.05). N7-MeG levels were also inversely associated with folate consumption (P < 0.05). MGMT activity (mean; 95% CI) was significantly higher in the group with the lowest consumption of vegetables than in the group with the greatest vegetable consumption (7.02; 5.70-8.33 versus 4.93; 3.95-5.91 fmol/microg DNA, P < 0.05). Our results are consistent with the hypothesis that dietary factors may modify exposure or susceptibility, respectively, to DNA damage by alkylating agents.

Marked inactivation of O6-alkylguanine-DNA alkyltransferase activity with protracted temozolomide schedules.

Temozolomide, an oral DNA methylator that inactivates the DNA repair enzyme O6-alkylguanine-DNA alkyltransferase (AGAT), has demonstrated anticancer activity on protracted schedules. Protracted schedules may lead to an ‘autoenhancement’ of temozolomide's inherent cytotoxic potential by cumulative reduction of the cell's capacity for AGAT-mediated DNA repair and resistance. This study was undertaken to characterise AGAT inactivation and regeneration in the peripheral blood mononuclear cells (PBMCs) of patients treated on two protracted temozolomide schedules. O6-alkyl guanine-DNA alkyltransferase activity was measured in the PBMCs of patients treated on two phase I protracted temozolomide studies. Patients were treated daily for either 7 days every 2 weeks (Schedule A) or 21 days every 4 weeks (Schedule B). The effects of various temozolomide doses (75–175 mg m−2), treatment duration (7–21 days), and temozolomide plasma levels on AGAT inactivation and regeneration, as well as the relation between AGAT inactivation and toxicity, were studied. O6-alkyl guanine-DNA alkyltransferase activity in PBMCs was measured serially in 52 patients. Marked inactivation of AGAT occurred following 7 days of temozolomide treatment, with mean AGAT activity decreasing by 72% (P<0.0001). Similarly, mean AGAT activity decreased by 63 and 73% after 14 and 21 days of treatment, respectively (P<0.001 for both comparisons). O6-alkyl guanine-DNA alkyltransferase inactivation was greater after 7 days of treatment with higher doses of temozolomide than lower doses and remained markedly reduced 7 days post-treatment. However, AGAT inactivation following temozolomide treatment for 14 and 21 days was similar at all doses. On the continuous 21-day schedule, AGAT inactivation was significantly greater in patients who experienced severe thrombocytopenia than those who did not (90.3±5.5 vs 72.5±16.1%, P<0.045). In Conclusion, protracted administration of temozolomide, even at relatively low daily doses, leads to significant and prolonged depletion of AGAT activity, which may enhance the antitumour activity of the agent.

The effect of O6-alkylguanine-DNA alkyltransferase and mismatch repair activities on the sensitivity of human melanoma cells to temozolomide, 1,3-bis(2-chloroethyl)1-nitrosourea, and cisplatin.

The prognosis of advanced melanoma is generally poor, because this tumor commonly exhibits intrinsic or acquired resistance to chemotherapy. In an attempt to identify the underlying causes of this resistance, we studied the roles played by the DNA repair enzyme O(6)-alkylguanine-DNA alkyltransferase (OGAT) and the mismatch repair (MMR) system in the sensitivity of melanoma cells to temozolomide (TMZ), 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), or cis-diamminedichloroplatinum(II) (CDDP). To this end, OGAT levels and MMR efficiency of extracts of nine melanoma cell lines and selected clones derived from four of these lines were determined and correlated with the sensitivity of the respective cells to these drugs. The effectiveness of O(6)-benzylguanine (BG), a specific OGAT inhibitor, in potentiating TMZ- or BCNU-mediated cytotoxicity was also evaluated. Our results demonstrate that MMR efficiency and OGAT levels strongly affect melanoma cell sensitivity to TMZ. In MMR-proficient cells, a direct correlation between OGAT levels and TMZ IC(50) values was found. When OGAT activity was inhibited with BG, the sensitivity of these cells to TMZ increased and was then dictated largely by their MMR efficiency. MMR-deficient cells were highly resistant to the drug irrespective of their OGAT levels. Although OGAT activity and MMR status seemed to be the major determinants of melanoma sensitivity to TMZ, this was not the case for BCNU and CDDP; resistance to the latter drugs clearly involves processes other than the two DNA repair pathways analyzed in this study.

Longitudinal variation in O(6)-alkylguanine DNA-alkyltransferase activity in the human colon and rectum.

In a systematic study of O6-alkylguanine DNA-alkyltransferase activity in the human colon and rectum, tumours were found to occur in regions of low activity. These results are consistent with the hypothesis that O6-alkylguanine DNA-alkyltransferase levels and alkylating agent exposure may be important determinants of large bowel tumorigenesis.British Journal of Cancer (2002) 87, 168–170. doi:10.1038/sj.bjc.6600455 www.bjcancer.com© 2002 Cancer Research UK

Heterogeneity of O6-alkylguanine DNA-alkyltransferase expression in human breast tumours.

An important determinant of cellular resistance to chemotherapeutic O6-alkylating agents, which comprise methylating and chloroethylating agents, is the ability of cells to repair alkylation damage at the O6-position of guanine in DNA. This is achieved by a specific DNA repair enzyme O6-alkylguanine DNA-alkyltransferase. In this study O6-alkylguanine DNA-alkyltransferase expression was measured in human breast tumours using both biochemical and immunohistochemical techniques. O6-alkylguanine DNA-alkyltransferase activity was then compared with known clinical prognostic indices to assess the potential role of O6-alkylguanine DNA-alkyltransferase in predicting the behaviour of this common malignancy. The application of both biochemical and immunohistochemical techniques was feasible and practical. Most breast tumours expressed high levels of O6-alkylguanine DNA-alkyltransferase. Immunohistochemical analysis showed marked variation in expression not only between individuals but also within individual tumours, and in the same patient, between metastases and between primary tumour and metastatic site. O6-alkylguanine DNA-alkyltransferase activity in tissue extracts significantly correlated not only with immunohistochemical staining intensity determined by subjective quantitation, but also with measures of protein levels using a computerised image analysis system including mean grey (P<0.001), percentage of cells positive for O6-alkylguanine DNA-alkyltransferase (P<0.001), and integrated optical density (P<0.001). O6-alkylguanine DNA-alkyltransferase expression did not correlate with any of the established clinical prognostic indicators for current treatment regimens. However, immunohistochemical offers a rapid and convenient method for assessing potential utility of O6-alkylating agents or O6-alkylguanine DNA-alkyltransferase inactivating agents in future studies of breast cancer treatment.British Journal of Cancer (2002) 86, 1797–1802. doi:10.1038/sj.bjc.6600324 www.bjcancer.com© 2002 Cancer Research UK

Mutation of the mismatch repair gene hMSH2 and hMSH6 in a human T‐cell leukemia line tolerant to methylating agents

Cell killing by monofunctional methylating agents is due mainly to the formation of adducts at the O6 position of guanine. These methyl adducts are removed from DNA by the O6-alkylguanine DNA alkyltransferase (OGAT). The mechanism by which O6-methylguanine (O6meG) induces cell death in OGAT-deficient cells requires a functional mismatch repair system (MRS). We have previously reported that depletion of OGAT activity in the human T-cell leukemic Jurkat line does not sensitize these cells to the cytotoxic and apoptotic effects of the methylating triazene temozolomide (Tentori et al., 1995). We therefore decided to establish whether the tolerance of Jurkat cells to O6meG could be associated with a defect in MRS. The results of mismatch repair complementation studies indicated that Jurkat cells are defective in hMutSα, a heterodimer of the hMSH2 and hMSH6 proteins. Cytogenetic analysis of two Jurkat clones revealed a deletion in the short arm of chromosome region 2p15–21, indicating an allelic loss of both hMSH2 and hMSH6 genes. DNA sequencing revealed that exon 13 of the second hMSH2 allele contains a base substitution at codon 711, which changes an arginine to a termination codon (CGA→TGA). In addition, a (C)8→(C)7 frameshift mutation in codon 1085–1087 of the hMSH6 gene was also found. Although both hMSH2 and hMSH6 transcripts could be detected in Jurkat clones, the respective polypeptides were absent. Taken together, these data indicate that tolerance of Jurkat cells to methylation damage is linked to a loss of functional hMutSα. Genes Chromosomes Cancer 23:159–166, 1998. © 1998 Wiley-Liss, Inc.

Mutation of the mismatch repair genehMSH2 andhMSH6 in a human T-cell leukemia line tolerant to methylating agents

Cell killing by monofunctional methylating agents is due mainly to the formation of adducts at the O6 position of guanine. These methyl adducts are removed from DNA by the O6-alkylguanine DNA alkyltransferase (OGAT). The mechanism by which O6-methylguanine (O6meG) induces cell death in OGAT-deficient cells requires a functional mismatch repair system (MRS). We have previously reported that depletion of OGAT activity in the human T-cell leukemic urkat line does not sensitize these cells to the cytotoxic and apoptotic effects of the methylating triazene temozolomide (Tentori et al., 1995). We therefore decided to establish whether the tolerance of Jurkat cells to O6meG could be associated with a defect in MRS. The results of mismatch repair complementation studies indicated that Jurkat cells are defective in hMutSalpha, a heterodimer of the hMSH2 and hMSH6 proteins. Cytogenetic analysis of two Jurkat clones revealed a deletion in the short arm of chromosome region 2p15-21, indicating an allelic loss of both hMSH2 and hMSH6 genes. DNA sequencing revealed that exon 13 of the second hMSH2 allele contains a base substitution at codon 711, which changes an arginine to a termination codon (CGA-->TGA). In addition, a (C)8-->(C)7 frameshift mutation in codon 1085-1087 of the hMSH6 gene was also found. Although both hMSH2 and hMSH6 transcripts could be detected in Jurkat clones, the respective polypeptides were absent. Taken together, these data indicate that tolerance of Jurkat cells to methylation damage is linked to a loss of functional hMutSalpha.

Inhibition of O6-Alkylguanine DNA-Alkyltransferase or Poly(ADP-ribose) Polymerase Increases Susceptibility of Leukemic Cells to Apoptosis Induced by Temozolomide

High levels of expression of the DNA repair enzyme O6-alkylguanine DNA-alkyltransferase (OGAT) (EC 2.1.1.63) account for tumor cell resistance to methylating agents. Previous studies suggested that methylating triazenes might have a potential role for the treatment of acute leukemias with low levels of OGAT. In the current study, we transduced the human OGAT cDNA in OGAT-deficient leukemia cell clones. OGAT-transduced cells were more resistant than their OGAT-deficient counterparts to apoptosis triggered by the methylating triazene temozolomide (TZM), as indicated by the results of flow cytometry, terminal deoxynucleotidyl transferase assay, and analysis of DNA fragmentation. Depletion of OGAT activity by O6-benzylguanine increased leukemia cell sensitivity to TZM-mediated apoptosis. Moreover, combined treatment of cells with TZM and benzamide, an inhibitor of the poly(ADP-ribose) polymerase (EC 2.4.2.30), increased the apoptosis induced by the methylating agent. These results demonstrate for the first time that methyl adducts at the O6 position of guanine, which are specifically removed by OGAT, are the principal DNA lesions responsible for the induction of apoptosis on treatment of leukemic cells with the methylating triazene TZM. This study also supports the possible use of TZM for the treatment of acute leukemias and suggests new strategies to increase the susceptibility of tumor cells to methylating triazenes in the clinic.

Low O6-alkylguanine DNA-alkyltransferase activity in normal colorectal tissue is associated with colorectal tumours containing a GC-->AT transition in the K-ras oncogene.

O6-alkylguanine DNA-alkyltransferase (ATase) provides protection against the toxic, mutagenic and carcinogenic effects of alkylating agents, principally by removing the promutagenic lesion O6-alkylguanine from DNA. Differences in ATase activity in human tissue may thus determine mutational susceptibility. As GC-->AT transitions, which can be induced by O6-alkylguanine in DNA, are commonly observed in the K-ras oncogene of alkylating agent induced animal tumours and in human colorectal tumours, we have examined whether differences in ATase activity may affect the risk of K-ras mutations in humans with colorectal tumours. NTase activity in normal tissue from individuals with a K-ras mutation in colorectal tissue and more specifically a GC-->AT transition (but not a transversion mutation) was significantly lower than that in individuals without a mutation (P < 0.01). Thus, individuals with low ATase activity in normal tissue (i.e. below the median) were at increased risk of having a transition (OR 10.1; 95% CI 1.9-99.0), but not a transversion mutation (OR 1.7; 95% CI 0.3-12.2). There were no significant differences in tumour ATase activity in individuals with or without a mutation. These results suggest that ATase can protect colorectal tissue against the mutagenic effects of alkylating agents and furthermore, that alkylating agent exposure plays a role in the aetiology of colorectal tumours containing a GC-->AT transition in the K-ras oncogene.

O6-alkylguanine DNA alkyltransferase activity in student embalmers.

O6-Alkylguanine-DNA alkyltransferase (AGT) activity was assessed in peripheral blood lymphocytes among 23 mortuary science students before and after 9 weeks in a laboratory course in techniques of embalming. Formaldehyde exposure was established by environmental monitoring. The average air concentration of formaldehyde during embalming was about 1.5 ppm. At the pre-exposure sampling, baseline DNA repair capacity tended to be reduced in subjects who reported a prior history of embalming (p = 0.08). From pre- to post-exposure, 17 subjects decreased in DNA repair capacity, while only 6 increased (p < 0.05). Analysis of variance, including adjustment for age, sex, and smoking status, confirmed these findings. Among the eight subjects who had no embalming experience during the 90 days before study, seven had decreased and one had increased AGT activity during the period of study (p < 0.05). For those with prior embalming experience, 10 subjects decreased in AGT activity, while 5 increased (p < 0.05). Although the major chemical exposure in embalming practice was to formaldehyde, no clear link was established between amount of formaldehyde exposure and AGT activity.

Sensitization of human colon tumour cell lines to carmustine by depletion of O6-alkylguanine-DNA alkyltransferase.

In nine human colon tumour cell lines the relationship between O6-alkylguanine-DNA alkyltransferase (O6-AGT) activity and carmustine resistance was investigated. Three lines with O6-AGT activity below 25 fmol/mg protein had carmustine ED50 values ranging from 5.0 microM to 11.9 microM; six lines displaying an O6-AGT activity above 240 fmol/mg protein had ED50 values ranging from 28.9 microM to 69.5 microM. In lines with low O6-AGT activity, depletion of the repair protein by O6-benzylguanine resulted in a marginal increase of carmustine cytotoxicity (sensitization factors less than 2). In the majority of cell lines with high O6-AGT activity, pretreatment with O6-benzylguanine resulted in a pronounced sensitization to carmustine. In one line, an optimal time schedule for sensitization of colon tumour cells by O6-benzylguanine was assessed; continuous exposure to O6-benzylguanine > or = 16 h after carmustine exposure) was superior to short-term exposure limited to a period before carmustine treatment.

Chemosensitivity to triazene compounds and O6-alkylguanine-DNA alkyltransferase levels: Studies with blasts of leukaemic patients

A clinical pilot study performed by our group showed that dacarbazine can induce a marked reduction of blast cells in patients with acute myelogenous leukaemia (AML). Leukaemic blasts (LB) from responsive patients showed low levels of O6-alkylguanine-DNA alkyltransferase (OGAT). An in vitro study was performed to evaluate OGAT levels and sensitivity to temozolomide (a triazene compound that spontaneously decomposes into the active metabolite of dacarbazine) in a relatively large number of LB samples. OGAT levels varied widely among the LB of different patients, with a mean value higher in acute lymphoblastic leukaemias than in AML. About 25% of LB obtained from patients with AML showed low OGAT activity, in the range corresponding to that observed in leukaemic patients responsive to dacarbazine in vivo. A reasonable inverse correlation was found between OGAT levels and LB sensitivity to temozolomide. Triazenes could have a therapeutic potential in human leukaemias. Moreover, OGAT determination could provide rapid and reliable information about a patient's susceptibility to these antitumor agents.

P53 mutations, O6-alkylguanine DNA alkyltransferase activity, and sensitivity to procarbazine in human brain tumors

In human brain tumors, sensitivity to procarbazine as measured by sensitivity in a xenograft tumor model correlated inversely with amounts of the DNA repair enzyme O6-alkylguanine DNA alkyltransferase (AT). To test the hypothesis that mutations of the p53 tumor suppressor gene in human tumors also can correlate with the response to chemotherapy, p53 mutations2 were identified in primary human malignant brain tumors and cell lines in which AT activity and procarbazine sensitivity in a xenograft model was ascertained. Mutations were identified in 7 of 21 (33%) specimens tested. Specimens containing p53 mutations tended to exhibit an increased growth delay in procarbazine-treated xenografts and lower amounts of AT. p53 mutations in brain tumors may contribute to procarbazine sensitivity by failing to induce arrest at the G1/S cell-cycle checkpoint, thereby preventing the repair of procarbazine-induced genetic alterations.

Inhibition of O6-alkylguanine-DNA alkyltransferase in animal and human ovarian tumor cell lines by O6-benzylguanine and sensitization to BCNU.

O6-Alkylguanine-DNA alkyltransferase (O6-AGT) activity in rat ovarian tumor lines O-342 and O 342/DDP was 103.4 +/- 18.4 and 240.9 +/- 40.2 fmol/mg protein, respectively; thus, cisplatin (DDP) resistance was paralleled by an increase in O6-AGT activity by a factor of approximately 2.3. The DDP-resistant line expressed a collateral resistance to BCNU. Both lines could be sensitized to BCNU by O6-BG, with sensitization factors of 6.0 and 2.1, respectively. In neither line did depletion of O6-AGT have any sensitizing effect towards DDP. In the human ovarian cancer lines SK-OV-3 and OAW 42, O6-AGT activity was 337.6 +/- 18.2 and 180.0 +/- 39.9 fmol/mg protein, respectively; in these lines depletion of O6-AGT activity by O6-BG treatment resulted in sensitization factors of 3.0 and 4.1, respectively. The increase in sensitivity of ovarian tumor cell lines against a chloroethylating agent by O6-AGT depletion and possible pharmacological advantages of regional (i.p.) administration of this combination might be beneficial in advanced ovarian cancer.

Increased resistance to the toxic effects of alkylating agents in tobacco expressing the E. coli DNA repair gene ada

The protein coding region of the E. coli gene ada has been transferred to tobacco plants by a leaf disc transformation procedure involving an Agrobacterium tumefaciens Ti plasmid. Transformed plants were shown to be transgenic for the ada message and had increased levels of O6-alkylguanine DNA alkyltransferase activity. The N-methyl-N-nitrosourea- or taurinechlorethylnitrosourea-induced inhibition of growth of calluses or of cells in suspension was considerably lower in ada-transformed than in non-transformed plants. This indicates that O6-alkylguanine, O4-alkylthymine or phosphotriesters are growth-inhibitory lesions in tobacco.

Monoclonal antibody-mediated solid-phase assay for mammalian O6-alkylguanine DNA alkyltransferase activity

We describe a sensitive, rapid, and simple assay for mammalian O 6-alkylguanine DNA alkyltransferase ( O 6-AGT) utilizing solid-phase DNA as the substrate and a monoclonal antibody (Mab)-based immuno-slotblot (ISB) for quantitation of O 6-ethylguanine ( O 6-EtG). γ-phage DNA was treated with N-ethyl- N-nitrosourea and immobilized on newly developed hydrophilic latex beads. After incubation with cell extracts to be assayed for O 6-AGT activity, the substrate DNA could be isolated easily by a brief centrifugation through 50% glycerol. The amount of O 6-EtG retained in the substrate DNA was determined by ISB using the anti-( O 6-ethyl-2′-deoxyguanosine) Mab ER-6. As little as 2 fmol of O 6-AGT per reaction tube can be reproducibly measured by this procedure, which is suitable for handling large numbers of samples within a short time (e.g., 80 samples within 2 days). In normal and malignant cells, respectively, O 6-AGT activity protects against O 6-alkylguanine-mediated mutagenesis and oncogenesis following exposure to N-nitroso carcinogens or confers resistance against cytocidal anti-cancer drugs such as chloroethylnitrosoureas and related compounds. The analysis of cellular O 6-AGT activity by a highly sensitive, routinely applicable method is, therefore, of particular interest in studies related to carcinogenesis, molecular epidemiology, and clinical oncology.

O6-Alkylguanine-DNA Alkyltransferase Activity in Human Brain Tumors.

It is well known that resistance in tumor cells to alkylating agents and, in particular, chloroethylnitrosoureas (CENUs), which are widely used in the chemotherapy of brain tumors, correlate well with activity of the DNA repair enzyme O6-alkylguanine-DNA alkyltransferase (O6-AT). We measured O6-AT activity in human brain tumors in order to obtain basic knowledge of whether or not CENU chemotherapy can be applied selectively on brain tumors. The subjects included 17 gliomas (seven malignant astrocytomas, two glioblastomas, two medulloblastomas, two oligodendrogliomas, two ependymomas, one fibrillary astrocytoma, one primitive neuroectodermal tumor) and five non-glial tumors (three meningiomas, two neurinomas). The value of O6-AT activity for the gliomas varied widely and indicated 111 +/- 65 fmol of 3H-methyl adducts transferred/mg protein extract/hr (mean +/- S.D., range 0-258, 18 tumors), while the non-glial tumors showed a relatively high value of 270 +/- 43 fmol/mg/hr (range 225-330, 5 tumors). A significant difference in the O6-AT activity was noted between the gliomas and the nonglial tumors at the p-value of 0.001. Six (38%) out of 17 glioma cases showed a value below 100 fmol/mg/hr and four cases (24%) a value below 60 fmol/mg/hr. These results provide a biological basis for applying CENU chemotherapy on glioma patients with a lower value of O6-AT enzyme.

Predominance of Mex+ cells in newly-established human lymphoblastoid cell lines.

Previous studies have demonstrated that approximately one-third of human lymphoblastoid cell lines (LCLs) are deficient in removing O6-methylguanine residues because of the lack of O6-alkylguanine-DNA alkyltransferase (O6-AGT) activity. Such LCLs have been designated Mex-, while the proficient LCLs are Mex+. Our determinations of O6-AGT activity as a function of cellular protein concentration on 37 previously-established LCLs disclosed that the expression of the enzyme was high in 14 (Mex+) and barely detectable in 16 (Mex-). The other seven LCLs showed intermediate activity of the enzyme. By contrast, all of the 28 LCLs that we newly established contained high enzyme activity, implying that they consisted of mainly Mex+ cells. Since the conventional O6-AGT assay on Mex+ cell populations was not capable of detecting the co-existence of Mex- cells as a minor component, we attempted to determine the proportion of Mex- phenotype in newly-immortalized lymphoblastoid cell clones which had been established directly on semisolid agar. All of the 15 independent clones derived from a single blood sample also showed high O6-AGT activity, rendering it unlikely that Epstein-Barr virus transformation per se was responsible for the generation of Mex- LCLs. These results collectively indicate that Mex+ cells predominate in LCLs shortly after establishment and also suggest that the possible growth advantage for Mex- cells should play an important role in the subsequent development of Mex- LCLs during the long-term culture in vitro.

Inactivation of O6-alkylguanine-DNA alkyltransferase in HeLa cells by cisplatin.

Inactivation of O6-alkylguanine-DNA alkyltransferase (O6-AGT) in HeLa CCL2 cells by cisplatin was studied. HeLa CCL2 cells treated with cisplatin showed a dose-dependent decline in O6-AGT activity. After cisplatin was removed and replaced with fresh medium, the transferase level began to rise slowly. By 72 h slightly more than 80% of the activity was recovered. It seems that the activity of the alkyltransferase can be inactivated by platinated DNA adducts. The data suggest that the O6-platinum-guanine formation and the O6-alkyltransferase depletion are not responsible for cytotoxicity but may result in a base substitution mutation in mammalian cells.