Genome-wide meta-analysis identifies variants associated with platinating agent susceptibility across populations.

Platinating agents are used in the treatment of many cancers, yet they can induce toxicities and resistance that limit their utility. Using previously published and additional world population panels of diverse ancestry totaling 608 lymphoblastoid cell lines (LCLs), we performed meta-analyses of over 3 million single-nucleotide polymorphisms (SNPs) for both carboplatin- and cisplatin-induced cytotoxicity. The most significant SNP in the carboplatin meta-analysis is located in an intron of NBAS (neuroblastoma amplified sequence; P=5.1 × 10(-7)). The most significant SNP in the cisplatin meta-analysis is upstream of KRT16P2 (P=5.8 × 10(-7)). We also show that cisplatin-susceptibility SNPs are enriched for carboplatin-susceptibility SNPs. Most of the variants that associate with platinum-induced cytotoxicity are polymorphic across multiple world populations; therefore, they could be tested in follow-up studies in diverse clinical populations. Seven genes previously implicated in platinating agent response, including BCL2 (B-cell CLL/lymphoma 2), GSTM1 (glutathione S-transferase mu 1), GSTT1, ERCC2 and ERCC6, were also implicated in our meta-analyses.

Heritable and non-genetic factors as variables of pharmacologic phenotypes in lymphoblastoid cell lines.

Publicly available genetic and expression data on lymphoblastoid cell lines (LCLs) make them a unique resource for understanding the genetic underpinnings of pharmacological outcomes and disease. LCLs have been used for pharmacogenomic discovery and validation of clinical findings associated with drug response. However, variation in cellular growth rate, baseline Epstein-Barr virus (EBV) copy number and ATP levels can all be confounders in such studies. Our objective is to better define confounding variables that affect pharmacological end points in LCLs. To this end, we evaluated the effect of these three variables on drug-induced cytotoxicity in LCLs. The drugs evaluated included daunorubicin, etoposide, carboplatin, cisplatin, cytarabine, pemetrexed, 5'-deoxyfluorouridine, vorinostat, methotrexate, 6-mercaptopurine, and 5-fluorouracil. Baseline ATP or EBV copy number were not significantly correlated with cellular growth rate or drug-induced cytotoxicity. In contrast, cellular growth rate and drug-induced cytotoxicity were significantly, directly related for all drugs except vorinostat. Importantly, cellular growth rate is under appreciable genetic influence (h²=0.30-0.39) with five suggestive linkage regions across the genome. Not surprisingly, a percentage of SNPs that significantly associate with drug-induced cytotoxicity also associate with cellular growth rate (P ≤ 0.0001). Studies using LCLs for pharmacologic outcomes should therefore consider that a portion of the genetic variation explaining drug-induced cytotoxicity is mediated via heritable effects on growth rate.

Genome scan implicates adhesion biological pathways in secondary leukemia.

The genetic risk factors for etoposide-induced leukemia with MLL translocations remain largely unknown. To identify genetic risk factors for and novel characteristics of secondary leukemia, we profiled 116,204 single nucleotide polymorphisms (SNPs) in germline and paired leukemic cell DNA from 13 secondary leukemia/myelodysplasia cases and germline DNA from 13 matched and 156 unmatched controls, all with acute lymphoblastic leukemia treated with etoposide. We analyzed global gene expression from a partially overlapping cohort. No single locus was altered in most cases. We discovered 81 regions of loss of heterozygosity (LOH) in leukemic blasts and 309 SNPs whose allele frequencies differed in cases vs controls. Candidate genes were prioritized on the basis of genes whose SNPs or expression differentiated cases from controls or showed LOH or copy number change in germline vs paired blast DNA from the 13 cases. Three biological pathways were altered: adhesion, Wnt signaling and regulation of actin. Validation experiments using a genome scan for etoposide-induced leukemogenic MLL chimeric fusions in 15 HapMap cell lines also implicated genes involved in adhesion, a process linked to de novo leukemogenesis. Independent clinical epidemiologic and in vitro genome-wide approaches converged to identify novel pathways that may contribute to therapy-induced leukemia.

The pharmacogenetics research network: from SNP discovery to clinical drug response.

The NIH Pharmacogenetics Research Network (PGRN) is a collaborative group of investigators with a wide range of research interests, but all attempting to correlate drug response with genetic variation. Several research groups concentrate on drugs used to treat specific medical disorders (asthma, depression, cardiovascular disease, addiction of nicotine, and cancer), whereas others are focused on specific groups of proteins that interact with drugs (membrane transporters and phase II drug-metabolizing enzymes). The diverse scientific information is stored and annotated in a publicly accessible knowledge base, the Pharmacogenetics and Pharmacogenomics Knowledge base (PharmGKB). This report highlights selected achievements and scientific approaches as well as hypotheses about future directions of each of the groups within the PGRN. Seven major topics are included: informatics (PharmGKB), cardiovascular, pulmonary, addiction, cancer, transport, and metabolism.

Enhancement of nitrosourea activity in medulloblastoma and glioblastoma multiforme.

BACKGROUND: Although chemotherapy offers promise of increased survival for children with medulloblastoma and glioblastoma multiforme, drug resistance occurs frequently, resulting in tumor progression and death. Resistance to nitrosoureas and methylating agents, which damage DNA, can be mediated by a DNA repair protein, O6-alkylguanine-DNA alkyltransferase (AGAT). Depletion of this protein with alkylguanines or methylating agents, however, restores tumor cell sensitivity to the cytotoxicity of chloroethylnitrosoureas (e.g., carmustine [BCNU]). PURPOSE: This study was designed to determine whether resistance to the activity of nitrosourea (the drug BCNU) in BCNU-resistant human medulloblastoma (D341 Med) and human glioblastoma multiforme (D-456 MG) can be reversed by the methylating agent streptozocin and the O6-substituted guanines O6-methylguanine and O6-benzylguanine. METHODS: Xenografts were grown subcutaneously in athymic BALB/c mice. BCNU was administered as a single intraperitoneal injection at doses of 100 mg/m2, 75 mg/m2, or 38 mg/m2--i.e., 1.0, 0.75, or 0.38, respectively, of the dose lethal to 10% of treated animals (LD10). Mice were treated intraperitoneally with a single dose of O6-benzylguanine or O6-methylguanine (240 mg/m2) or with streptozocin (600 mg/m2) daily for 4 days. Response was assessed by tumor growth delay and tumor regression. AGAT activity in the xenografts was measured at 1 and 6 hours after pretreatment, at the time tumors were excised. RESULTS: Pretreatment with O6-benzylguanine, O6-methylguanine, or streptozocin reduced AGAT activity to 4%, 25%, and 95% of control values, respectively, in D341 Med and 0%, 0%, and 25% of control values, respectively, in D-456 MG 1 hour after injection. After 6 hours, levels changed to 7%, 61%, and 116% of control values in D341 Med and 0%, 79%, and 21% of control values in D-456 MG, respectively. Both D341 Med and D-456 MG xenografts were completely resistant to BCNU at its LD10. Pretreatment with O6-benzylguanine increased BCNU sensitivity in both types of xenograft. In contrast, treatment with BCNU plus O6-methylguanine or streptozocin did not produce growth delays substantially different from those produced by BCNU alone, reflecting the more efficient depletion of AGAT by O6-benzylguanine. Following therapy with BCNU plus O6-benzylguanine at 0.38 LD10, tumor regressions were seen in eight of 10 D341 Med and in all 10 D-456 MG xenografts. CONCLUSION: We recommend comprehensive clinical toxicologic evaluation of combination therapy with O6-benzylguanine plus BCNU, which would allow subsequent design of phase I clinical trials.

Thymidylate synthase expression and response to neoadjuvant chemotherapy in patients with advanced head and neck cancer.

BACKGROUND: Thymidylate synthase (TS), an essential enzyme in DNA synthesis, is a target for the fluoropyrimidines, an important group of antineoplastic agents used widely in the treatment of head and neck cancer. PURPOSE: We evaluated relationships between the level and/or pattern of tumor TS expression and response to fluorouracil (5-FU)-based neoadjuvant chemotherapy in patients with advanced head and neck cancer. METHODS: Tumor specimens from 86 patients were available for this retrospective analysis. The patients were enrolled in four consecutive phase II studies that tested combinations of 5-FU, leucovorin, and cisplatin with or without added methotrexate plus piritrexim or interferon alfa-2b (IFN alpha-2b). TS protein expression in the tumors was assessed by use of the TS 106 monoclonal antibody and standard immunohistochemical staining techniques. TS immunostaining was classified according to its level of intensity (TS 0-1 = low, TS 2 = intermediate, and TS 3 = high) and according to its extent (focal pattern = less than 25% of tumor cells positive; diffuse pattern = greater than or equal to 25% of tumor cells positive). Data from 79 patients were available for an analysis of tumor TS expression and patient/tumor characteristics; 70 patients were assessable for their response to neoadjuvant chemotherapy. RESULTS: There was a statistically significant association between the level of tumor TS expression and the degree of tumor differentiation; a higher proportion of patients whose tumors exhibited TS 0-1 immunostaining had undifferentiated or poorly differentiated tumors than patients whose tumors exhibited TS 2 or TS 3 immunostaining (P = .04, Jonckheere-Terpstra trend test). Among the 70 patients who were assessable for response to neoadjuvant chemotherapy, TS 3 tumor immunostaining was associated with a lower rate of complete response (i.e., complete disappearance of clinically detectable disease for a minimum of 4 weeks from time of initial determination) than was TS 2 or TS 0-1 immunostaining, but this association was not statistically significant (P = .09, exact trend test); among the 39 patients who were treated with regimens that included 5-FU, leucovorin, cisplatin, and IFN alpha-2b, this inverse association between TS immunostaining intensity and response was statistically significant (P = .02, exact trend test). Tumor TS immunostaining intensity and overall survival were not found to be associated. Patients with tumors exhibiting a focal pattern of TS immunostaining have experienced significantly longer survival than patients with tumors exhibiting a diffuse pattern; for the 53 patients with diffuse tumor TS immunostaining, the median survival was 24.7 months, whereas the median survival has not yet been reached for the 22 patients with focal tumor TS immunostaining (P = .04, two-tailed logrank test). However, the survival advantage for the focal versus diffuse TS immunostaining pattern was limited to patients whose tumors also exhibited a TS 3 level of immunostaining intensity. CONCLUSIONS AND IMPLICATIONS: Characterization of tumor TS expression may be of value in identifying patients with advanced head and neck cancer who would benefit from fluoropyrimidine-based neoadjuvant chemotherapy.

Use of oligodeoxynucleotides containing O6-alkylguanine for the assay of O6-alkylguanine-DNA-alkyltransferase activity.

A sensitive assay procedure was developed for the measurement of the activity of mammalian O6-alkylguanine-DNA-alkyltransferase. The procedure utilized oligodeoxynucleotides containing O6-methylguanine as substrates for the reaction. The oligodeoxynucleotides were end labeled with 32P by the reaction with polynucleotide kinase and [gamma-32P]ATP and allowed to react with organ or cell extracts containing the alkyltransferase. The unmethylated product which was formed was separated from the substrate by reverse-phase high-pressure liquid chromatography. Since the repair by the alkyltransferase is bimolecular, the second order rate constants for the reaction between the labeled oligomer and repair protein from several different sources were determined. The amount of alkyltransferase present was then calculated from the amount of product formed and the appropriate second order rate constant for the reaction. Excellent agreement was obtained between the alkyltransferase levels determined in this procedure and those measured by conventional assay procedures in a variety of cell lines having both high and low activity. The method also gave results in good agreement with other assay procedures for a number of rat tissues, although a few tissues gave anomalous results owing to a high level of nuclease activity which degraded the substrate. This method should prove useful for the measurement of alkyltransferase activity in samples in which the activity is very low or the amount of material available is limited.

Effect of O6-alkylguanine pretreatment on the sensitivity of human colon tumor cells to the cytotoxic effects of chloroethylating agents.

Two human colon carcinoma cell lines which differ greatly in their content of O6-alkylguanine-DNA alkyltransferase were analyzed for their response to 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) and 2-chloroethyl(methylsulfonyl)methanesulfonate (ClEtSoSo) before and after treatment with O6-alkylguanines. HT29 cells contained about 17 times more alkyltransferase activity than BE cells. The alkyltransferase activity of HT29 cells was reduced by 60-80% by treatment for 24 h with 0.05-0.4 mM O6-methylguanine or O6-n-butylguanine. Such pretreatment prior to exposure to CCNU or ClEtSoSo increased the sensitivity of HT29 cells to these drugs. The exposure to O6-alkylguanines gave a greater enhancement of the toxic effects of ClEtSoSo than of CCNU. There was no significant increase in the toxicity of these agents towards the BE cells which contained much lower levels of the alkyltransferase. When added alone neither O6-methylguanine nor O6-n-butylguanine showed any toxicity towards HT29 or BE cells at the doses used. These results provide strong evidence that the formation of adducts at the O6-position of guanine by these agents contributes significantly to their lethality and that this reaction is more critical for ClEtSoSo than CCNU. The enhancement of the activity of chloroethylating agents by pretreatment with nontoxic doses of O6-alkylguanines may be clinically useful in terms of increasing their therapeutic efficacy towards cells containing high levels of alkyltransferase.

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

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

Extended depletion of O6-methylguanine-DNA methyltransferase activity following O6-benzyl-2'-deoxyguanosine or O6-benzylguanine combined with streptozotocin treatment enhances 1,3-bis(2-chloroethyl)-1-nitrosourea cytotoxicity.

We have recently suggested that optimal reversal of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) resistance might require complete inactivation of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) for at least 24 h following BCNU administration (22). This study was undertaken to further evaluate the functional importance of the regeneration rate of MGMT activity following O6-benzylguanine (BG), O6-benzyl-2'-deoxyguanosine (dBG), and streptozotocin (STZ) in determining the potentiation of BCNU cytotoxicity in the highly resistant colon carcinoma cell line HT-29. To this end, we measured the enhancement of BCNU cytotoxicity utilizing regimens which provided complete inhibition, with partial or complete recovery of MGMT activity by 24 h. We were able to modulate the recovery rate of MGMT activity following BG or dBG administration by repeated washing of cells with complete medium. Subsequent to equally inhibitory doses of BG (100 microM) or dBG (1.0 mM) treatment without washing, MGMT activity was completely inactivated for 24 h. However, MGMT activity recovered to control levels by 24 h when cells were treated with BG or dBG and washed 4 times with complete medium. This recovery was completely inhibited for 24 h by combining BG or dBG with 2.5 mM STZ. These differential repletion profiles produced disparate potentiation of BCNU cytotoxicity. The regimens which produced complete inactivation of MGMT for 24 h produced the greatest enhancement of BCNU cytotoxicity. BG or dBG (without a wash) potentiated BCNU cytotoxicity by approximately 3 logs of synergistic cell kill. When the recovery rate of MGMT activity was markedly enhanced via washing of cells, BG-BCNU or dBG-BCNU produced less than 1 log of synergistic cell kill. The addition of STZ to BG or dBG inhibited this temporal recovery for 24 h and potentiated BCNU cytotoxicity by approximately 4 logs. These data further demonstrate that extended depletion of MGMT is required for optimal reversal of BCNU resistance. Because a three-drug combination of BG-STZ-BCNU or dBG-STZ-BCNU consistently produced greater cytotoxicity than any two-drug regimen, clinical testing of these combinations is warranted. Additionally, our data suggest that the design of clinical regimens targeting the inactivation of MGMT and the reversal of BCNU resistance should consider the functional importance of extended depletion of MGMT in order to increase the possibility of antitumor responses.

Effect of O6-benzylguanine on the sensitivity of human tumor xenografts to 1,3-bis(2-chloroethyl)-1-nitrosourea and on DNA interstrand cross-link formation.

We have previously shown that O6-benzylguanine can be used to deplete cells of the DNA repair protein O6-alkylguanine-DNA alkyltransferase and to enhance the sensitivity of human glioma (SF767) and colon tumor (HT29) cells to the cytotoxic effects of alkylnitrosoureas. In the present study, the combination of O6-benzylguanine and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) was evaluated in vitro to determine the number of DNA interstrand cross-links formed and in vivo to compare the therapeutic index with that of BCNU alone. The number of DNA interstrand cross-links, as measured by alkaline elution, was increased in HT29 cells treated with 10 microM O6-benzylguanine for 2 h prior to BCNU exposure compared to cells treated with BCNU only. The number of single strand breaks was not increased by prior exposure to O6-benzylguanine. To evaluate the therapeutic index, HT29 and SF767 cells were grown as xenografts in nude mice and the tumor growth rate after treatment with BCNU alone was compared with the rate after treatment with O6-benzylguanine and BCNU. Treatment was administered i.p. when tumors reached 100-200 mm3. For animals bearing HT29 xenografts that were treated with 60 mg/kg O6-benzylguanine 1 h prior to 20 mg/kg BCNU, the average time for tumor volume to increase by 200% was 25 days, compared to 10 days for animals treated with 20 mg/kg BCNU alone. For animals bearing SF767 xenografts, the tumor growth of controls was not significantly different from that of animals treated with O6-benzylguanine alone or BCNU alone up to the maximally tolerated dose (50 mg/kg). For these 3 groups, the average time for tumors to reach 300 mm3 was 9-12 days. However, when animals were treated with 80 mg/kg O6-benzylguanine 1 h prior to receiving 20 mg/kg BCNU tumor size did not increase for at least 21 days. Our studies demonstrate that the therapeutic index of BCNU can be increased when given in combination with O6-benzylguanine.

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

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

Prolonged depletion of O6-methylguanine DNA methyltransferase activity following exposure to O6-benzylguanine with or without streptozotocin enhances 1,3-bis(2-chloroethyl)-1-nitrosourea sensitivity in vitro.

This study was undertaken to ascertain the importance of prolonged depletion of O6-methylguanine DNA methyltransferase (MGMT) activity, following O6-benzylguanine (BG) and streptozotocin (STZ) exposure, in reversing 1,3 bis(2-chloroethyl)-1-nitrosourea (BCNU) resistance in vitro. We evaluated BCNU-induced cytotoxicity and measured the temporal recovery of MGMT activity in human colon carcinoma HT-29 cells following treatment with BG, STZ, or the combination of BG and STZ. The pretreatment regimens which provided the greatest potentiation of BCNU cytotoxicity were those exhibiting the greatest temporal inhibition of MGMT activity. The combination of BG (10 microM) and STZ (1.0 mM) produced sustained inhibition of MGMT activity through 24 h and potentiated BCNU cytotoxicity by at least one log greater than either agent alone. Similarly, BG (10-100 microM) produced marked reductions in MGMT activity and increased BCNU cytotoxicity in a dose-dependent fashion. A 100-microM dose of BG inhibited MGMT activity for 48 h and potentiated BCNU induced cell kill by 3 logs greater than BCNU alone. In addition, we observed that during the period of sustained inhibition of MGMT activity, no changes in the steady-state MGMT mRNA levels occurred. We conclude that prolonged inhibition of MGMT activity is an important determinant in reversing BCNU resistance and that chemotherapeutic regimens targeting the inactivation of MGMT activity should be optimized such that MGMT activity is depleted for at least 24 h following BCNU administration.

Effect of O6-methylguanine on DNA interstrand cross-link formation by chloroethylnitrosoureas and 2-chloroethyl(methylsulfonyl)methanesulfonate.

Exposure of HT29 cells in culture to O6-methylguanine is known to result in a reduction in O6-alkylguanine-DNA alkyltransferase (AGT) activity and an enhancement of sensitivity to the cytotoxic effects of chloroethylating agents. Since cytotoxicity of these agents may be mediated by the formation of interstrand cross-links, alkaline elution analysis was performed on HT29 cells treated with 1-(2-chloroethyl)-1-nitrosourea, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, and Clomesone [2-chloroethyl(methylsulfonyl)methanesulfonate] in the presence or absence of O6-methylguanine pretreatment to determine if the enhanced toxicity was due to an increase in the number of cross-links formed. Interstrand cross-linking by 1-(2-chloroethyl)-1-nitrosourea or 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea was increased by pretreatment with 0.4 mM O6-methylguanine for 24 h. Cross-linking by Clomesone was observed only in cells exposed to 0.4 mM O6-methylguanine for 24 h prior to administration of the drug and for 12 h after administration, suggesting that the resynthesis of the AGT may prevent the cross-linking by Clomesone. Complete recovery of AGT activity after reduction to 20 to 30% of the basal level upon treatment with 0.4 mM O6-methylguanine required between 8 h and 15 h in both HT29 cells and in Raji cells which were also sensitized to 1-(2-chloro-ethyl)-3-cyclohexyl-1-nitrosourea by exposure to O6-methylguanine. These data suggest that the enhancement of chloroethylnitrosourea toxicity after treatment with O6-methylguanine may be related to an increase in the number of DNA cross-links and that the relatively rapid rate of AGT recovery plays a role in prevention of cross-links resulting from Clomesone.

Characterization of CPT-11 hydrolysis by human liver carboxylesterase isoforms hCE-1 and hCE-2.

7-Ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxy-camptothecin (irinotecan; CPT-11) is a prodrug activated by carboxylesterase enzymes. We characterized the hydrolysis of CPT-11 by two recently identified human carboxylesterase (hCE) enzymes, hCE-1 and hCE-2. Km and Vmax for hCE-1 and hCE-2 are 43 microM and 0.53 nmol/min/mg protein and 3.4 microM and 2.5 nmol/min/mg protein, respectively. hCE-2 has a 12.5-fold higher affinity for CPT-11 and a 5-fold higher maximal rate of CPT-11 hydrolysis when compared with hCE-1. In cytotoxicity assays, incubation of 1 microM CPT-11 with hCE-2 (3.6 microg/ml) resulted in a 60% reduction in survival of SQ20b cells. No significant reduction in cell survival was observed after incubation of CPT-11 with hCE-1. These data indicate that hCE-2 is a high-affinity, high-velocity enzyme with respect to CPT-11. hCE-2 likely plays a substantial role in CPT-11 activation in human liver at relevant pharmacological concentrations.

Effect of O6-benzylguanine analogues on sensitivity of human tumor cells to the cytotoxic effects of alkylating agents.

The effect of O6-benzylguanine, O6-(p-chlorobenzyl)guanine, and O6-(p-methylbenzyl)guanine on the sensitivity of various human tumor cell lines to alkylating agents is evaluated. The sensitivity of human colon tumor cells, HT29, to the chloroethylating agents, 1,3-bis(2-chloroethyl)-1-nitrosourea, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, 2-chloroethyl(methylsulfonyl) methanesulfonate (clomesone), and chlorozotocin was increased by pretreatment for 2 h with 25 microM of each analogue. O6-Benzylguanine was slightly more effective as a sensitizer in HT29 cells than the p-chlorobenzyl and p-methylbenzyl analogues. However, all analogues sensitized SF767 glioma cells to the cytotoxic effects of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, 1,3-bis(2-chloroethyl)-1-nitrosourea, and clomesone to the same degree. Both cell lines were sensitized to the methylating agents streptozotocin and 5-(3-methyl-1-triazeno)imidazole-4-carboxamide, the active intermediate of 5-(3,3-dimethyl-1-triazenyl)imidazole-4-carboxamide, by pretreatment with 10 microM O6-benzylguanine for 2 h. The number of Raji cells surviving 50 microM clomesone decreased 3-fold upon pretreatment for 2 h with 1 microM O6-benzylguanine. The degree of enhancement was dependent on the amount of alkyltransferase protein present in cell lines. For example, HT29 cells (alkyltransferase activity, 381 fmol/mg protein) exhibited a greater degree of enhancement when treated with O6-benzylguanine than SF767 (77 fmol/mg protein) and M19-MEL melanoma (36 fmol/mg protein) cells. There was no enhancement observed in mer- cell lines, U251 (less than 2 fmol/mg protein), and BE (3 fmol/mg protein), or with alkylating agents which did not produce a cytotoxic lesion at the O6 position of guanine in DNA such as cisplatin or 4-hydroperoxycyclophosphamide. Our studies suggest that O6-benzylguanine analogues may have utility in mer+ tumors as an adjuvant to a variety of alkylating agents which produce a toxic lesion at the O6 position of guanine.

Effect of O6-benzylguanine on alkylating agent-induced toxicity and mutagenicity. In Chinese hamster ovary cells expressing wild-type and mutant O6-alkylguanine-DNA alkyltransferases.

The DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT) has been shown to protect cells from the toxic and mutagenic effect of alkylating agents by removing lesions from the O6 position of guanine. O6-Benzylguanine (BG) is a potent inactivator of AGT, resulting in an increase in the sensitivity of cells to the toxic effects of chemotherapeutic alkylating agents. Chinese hamster ovary (CHO) cells and CHO cells transfected with wild-type AGT (CHOWTAGT) and a mutant AGT [P138 M/V139I/P140K (CHOMIK)] known to be resistant to BG were treated with BG and various alkylating agents. BG treatment alone dramatically decreased AGT activity in CHOWTAGT cells but resulted in no depletion in AGT activity in CHOMIK cells. In the absence of AGT, these cells are highly sensitive to the toxic and mutagenic effects of temozolomide and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), and no further sensitization occurs in the presence of BG. In contrast, CHOWTAGT cells are resistant to temozolomide and BCNU, and treatment with BG resulted in a significantly higher cell killing and mutation frequency. CHOMIK cells were completely resistant to temozolomide or BCNU in the presence and absence of BG. Both cell killing and mutation frequency of 4-hydroperoxycyclophosphamide (4-HC) in CHO, CHOWTAGT, and CHOMIK cells were increased in the presence of BG. 4-HC generates two active metabolites, phosphoramide mustard (PM) and acrolein. BG had no effect on 4hydroperoxydidechlorocyclophosphamide (which generates acrolein and a nonalkylating form of PM) in CHO cells and CHOMIK cells, but enhancement of toxicity was observed with PM in both these cell lines. Therefore, we attribute the enhancement to the PM metabolite of 4-HC. Our results demonstrate that wild-type AGT plays an important role in protecting against the toxic and mutagenic effect of O6 alkylating agents and that a mutant AGT resistant to inactivation by BG effectively prevents BG-enhanced toxicity and mutagenicity induced by these agents. Expression of the AGT protein contributes to resistance of 4-HC. BG also enhances the toxicity of 4-HC and PM by a mechanism that may not involve the AGT repair protein.

O6-benzylguanine potentiates the antitumor effect of locally delivered carmustine against an intracranial rat glioma.

Local delivery of carmustine (BCNU) via biodegradable polymers prolongs survival against experimental brain tumors and in human clinical trials. O6-benzylguanine (O6-BG), a potent inhibitor of the DNA repair protein, O6-alkylguanine-DNA alkyltransferase (AGT), has been shown to reduce nitrosourea resistance and, thus, enhance the efficacy of systemic BCNU therapy in a variety of tumor models. In this report, we demonstrate that O6-BG can potentiate the activity of BCNU delivered intracranially via polymers in rats challenged with a lethal brain tumor. Fischer 344 rats received a lethal intracranial challenge of 100,000 F98 glioma cells (F98 cells have significant AGT activity, 328 fmol/mg protein). Five days later, animals receiving an i.p. injection of O6-BG (50 mg/kg) 2 h prior to BCNU polymer (3.8% BCNU by weight) implantation had significantly improved survival (n = 7; median survival, 34 days) over animals receiving either O6-BG alone (n = 7; median survival, 22 days; P = 0.0002) or BCNU polymer alone (n = 8; median survival, 25 days; P = 0.0001). Median survival for the control group (n = 8) was 23.5 days. Moreover, there was no physical, behavioral, or pathological evidence of treatment-related toxicity. These findings suggest that O6-BG can potentiate the effects of interstitially delivered BCNU and, for tumors expressing significant AGT, may be necessary for the BCNU to provide a meaningful therapeutic benefit. Given the clinical use of BCNU polymers against malignant gliomas, concurrent treatment with O6-BG may provide an important addition to our therapeutic armamentarium.

Role of O6-alkylguanine-DNA alkyltransferase in protecting against cyclophosphamide-induced toxicity and mutagenicity.

Cyclophosphamide is used to treat a wide range of human malignancies. However, it is also a known carcinogen associated with induction of therapy-related leukemia and bladder cancer. The DNA repair protein, O6-alkylguanine-DNA alkyltransferase (AGT), protects cells from the toxic and mutagenic effects of O6-alkylating agents. We report here the contribution of AGT in protecting against the toxic and mutagenic effects of cyclophosphamide. CHO cells transduced with wild-type human AGT (CHO(AGT)) and pcDNA3 (CHOpcDNA3) were treated with activated cyclophosphamide derivatives, 4-hydroperoxycyclophosphamide (4-HC), 4-hydroperoxydidechlorocyclophosphamide (4-HDC), a progenitor of acrolein, and phosphoramide mustard (PM). The results show that CHO(AGT) is 7- or 20-fold less sensitive to the toxic effects of 30 microM 4-HC or 300 microM 4-HDC, respectively, than CHOpcDNA3 cells as measured by cell survival using a colony-forming assay. CHO(AGT) cells treated with 20 microM 4-HC or 200 microM 4-HDC produced 4- or 7-fold lower mutation frequency as measured at the HPRT locus than CHOpcDNA3 cells treated with the same dose of drugs. At 30 microM acrolein, the cell survival for CHO(AGT) was 30% compared with 18.7% for CHOpcDNA3. The mutation frequency of acrolein at the same dose was 57 mutants/10(6) cells in CHOpcDNA3 compared with no mutants in CHO(AGT). In contrast, CHO(AGT) and CHOpcDNA3 cells treated with PM had similar survival curves and exhibited no difference in mutation frequency. The present study demonstrates that AGT plays an important role in protecting against the toxic and mutagenic effect of cyclophosphamide and suggests that acrolein, not PM, is responsible for generating the toxic and mutagenic lesion(s) protected by the AGT protein.

Elevated DNA polymerase alpha, DNA polymerase beta, and DNA topoisomerase II in a melphalan-resistant rhabdomyosarcoma xenograft that is cross-resistant to nitrosoureas and topotecan.

Previous investigations have revealed that the human TE-671 MR human rhabdomyosarcoma xenograft selected in vivo for melphalan resistance (M. C. Rosenberg, et al., Cancer Res., 49: 6917-6922, 1989) is cross-resistant to a wide variety of alkylating agents and to bleomycin, but is collaterally sensitive to etoposide. Although glutathione levels were noted to be elevated in TE-671 MR compared to the melphalan-sensitive parental TE-671 xenograft, treatment with buthionine sulfoximine to deplete glutathione levels did not fully restore melphalan sensitivity in the TE-671 MR xenograft. The present studies were undertaken to search for additional mechanisms of resistance in the TE-671 MR xenograft. Drug sensitivity testing performed at the dose of agents that was lethal to 10% of the animals revealed that the TE-671 MR xenograft maintained resistance to the bifunctional cross-linking agent 1,3-bis(2-chloroethyl)-1-nitrosourea and was cross-resistant to the topoisomerase I poison topotecan. Treatment with buthionine sulfoximine did not sensitize the TE-671 MR xenograft to 1,3-bis(2-chloroethyl)-1-nitrosourea. Further, even though O6-alkylguanine-DNA alkyltransferase levels were high in both the TE-671 and TE-671 MR xenografts, depletion of O6-alkylguanine-DNA alkyltransferase activity by treatment with O6-benzylguanine substantially sensitized the TE-671 xenografts but not the TE-671 MR xenografts, suggesting an additional mechanism of resistance. Measurement of additional enzyme activities that might be involved in DNA repair revealed significant elevations in DNA polymerase alpha (46 +/- 8 (SD) units/mg protein in TE-671, 69 +/- 6 units/mg protein in TE-671 MR, P < 0.05) and DNA polymerase beta (0.43 +/- 0.01 units/mg protein in TE-671, 0.78 +/- 0.12 units/mg protein in TE-671 MR, P < 0.05) but not DNA polymerase delta or total DNA ligase. Examination of topoisomerases by activity assays and Western blotting revealed a 2-fold increase in topoisomerase II and a 2-fold decrease in topoisomerase I in the TE-671 MR xenograft compared to the parental xenograft, apparently explaining the collateral sensitivity to etoposide and cross-resistance to topotecan. These results suggest that TE-671 MR xenografts contain multiple changes in activities of DNA repair-related proteins and other nuclear proteins that could contribute to alkylating agent resistance.