Enhancement of platinum-induced cytotoxicity by O6-benzylguanine

O(6)-Benzylguanine (O(6)-BG), a potent inactivator of the DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (AGT), is presently in clinical trials combined with alkylating agents that modify the O(6) position of DNA guanine residues, i.e., 1,3-bis(2-chloroethyl)-1-nitrosourea and temozolomide. Previous work demonstrated that O(6)-BG also enhances the cytotoxicity of cyclophosphamide, ifosfamide, and nitrogen mustards in Chinese hamster ovary cells. We have extended this study to include other clinically relevant agents that form interstrand and intrastrand cross-links including cisplatin and carboplatin. Pretreatment of a series of head and neck tumor cell lines (i.e., SQ20b, JSQ3, SCC25, SCC35, and SCC61), Chinese hamster ovary cells, and HT29 human colon tumor cells with O(6)-BG (100 micro M for 2 h before treatment and 2 h during treatment) resulted in a 2-fold decrease in the ED(50) of cisplatin and a concomitant increase in the percentage of cells undergoing apoptosis. The enhancement was independent of AGT activity. Similar enhancement was observed with carboplatin, but no enhancement was seen in AGT-deficient cell lines with radiation or temozolomide, demonstrating the dependence of the effect on bifunctional, cross-linking agents. Furthermore, levels of platinum on DNA after treatment with cisplatin increased 1.4-fold in SQ20b cells and 4.5-fold in JSQ3 cells immediately after treatment with O(6)-BG plus cisplatin and remained elevated for 48 h. Consistent with greater cytotoxicity and apoptosis is the approximately 2-fold higher amount of DNA damage when cells are treated with O(6)-BG plus cisplatin compared with cisplatin alone. Modulation of cisplatin therapy with O(6)-BG might improve the prognosis of patients with head and neck, ovarian, testicular, or lung cancer who are treated with this drug.     

Characterization of the P140K, PVP(138-140)MLK, and G156A O6-methylguanine-DNA methyltransferase mutants: implications for drug resistance gene therapy

The G156A O6-alkylguanine-DNA alkyltransferase (AGT) mutant protein, encoded by the G156A O6-methylguanine-DNA methyltransferase gene (MGMT), is resistant to O6-benzylguanine (BG) inactivation and, after transduction into hematopoietic progenitors, transmits remarkable resistance to BG and BCNU. As a result, a clinical trial, in which the MGMT gene is transduced into CD34+ cells of patients with cancer, has been approved. A newly identified AGT mutation, P140K, generates dramatically increased BG resistance relative to G156A, and suggests that gene transfer of P140K may confer improved hematopoietic cell protection. To address this hypothesis, we measured BG + BCNU and BG + TMZ resistance in G156A, P140K, or P138M/V139L/P140K (MLK) MGMT-transduced K562 cells. In addition, we performed a detailed characterization of individual properties including BG resistance, activity, and protein stability of these mutants in human hematopoetic K562 cells and E86 retroviral producer cells. In K562 cell extracts, the MLK and P140K mutants retained full activity at doses up to 1 mM BG, while G156A had a BG ED50 of 15 microM, compared with 0.1 microM for wtAGT. In the absence of BG, the G156A protein possessed a 56% reduction in specific O6-methyltransferase activity compared with wtAGT. MLK, P140K, and wtAGT all possessed similar specific activities, although the O6-methyl repair rate of all mutants was reduced 4- to 13-fold relative to wtAGT. The wtAGT, MLK, and P140K proteins were stable, with half-lives of greater than 18 hr. In contrast, only 20% of the G156A protein was stable after 12 hr in cycloheximide and, interestingly, the remaining protein appeared to retain most of the activity present in non-cycloheximide-treated cells. Differences in BG resistance, activity, and stability between P140K, MLK, and G156A suggest that P140K may be the optimal mutant for drug resistance gene transfer. However, hematopoietic K562 cells transduced with MFG-G156A, P140K, or MLK had similar degrees of BG and BCNU as well as BG and TMZ resistance when treated with concentrations of BG (< or =25 microM) achieved in clinical trials, suggesting similar efficacy in many in vivo applications.     

Modulation of chemotherapy resistance in regional therapy: a novel therapeutic approach to advanced extremity melanoma using intra-arterial temozolomide in combination with systemic O6-benzylguanine

This study investigated whether the therapeutic index of regional melanoma therapy using parenteral temozolomide could be improved by chemomodulation with O6-benzylguanine (O6BG), an inhibitor of the DNA repair enzyme O6-alkylguanine-DNA alkyltransferase (AGT). Using a nude rat s.c. human melanoma xenograft model of the extremity, tumors were analyzed for AGT level 2 to 3 hours after the i.p. injection of 3.5 to 70.0 mg/kg O6BG to inhibit AGT activity. Survival studies were conducted using animals that were treated with a 15-minute isolated limb infusion with 10% DMSO in PBS (control), temozolomide alone, or temozolomide in conjunction with single or multiple doses of i.p. O6BG. Tumor volume and toxicity level were monitored every other day. Administration of 3.5 mg/kg O6BG depleted tumor AGT activity by 93.5% (P < 0.01). Groups treated with regional temozolomide alone (350 mg/kg), systemic temozolomide with O6BG, or vehicle combined with O6BG showed no significant tumor responses compared with controls. Whereas use of regional temozolomide alone at a higher dose (750 mg/kg) showed some degree of tumor response, regional temozolomide given in conjunction with multiple dosages of O6BG showed a marked (P < 0.01) reduction in tumor growth with minimal toxicity. Our findings suggest that AGT modulation by the administration of O6BG in combination with temozolomide regional chemotherapy leads to a significant improvement in melanoma antitumor responses. Clinical trials using chemotherapy modulation may improve response rates in future regional infusion and perfusion drug trials.     

Potentiation of antitumor drug action by centrophenoxine: specificity.

The cytotoxic action of certain antitumor agents is potentiated by centrophenoxine although centrophenoxine itself is not an antitumor agent. Previous investigations have suggested that centrophenoxine might potentiate the cytotoxicity produced by antitumor drugs that alkylate, and other modalities that damage, DNA, but that it would not potentiate the cytotoxicity produced by antitumor drugs that inflict cellular damage in other ways. To test this hypothesis, the antitumor effects of X-irradiation UV-irradiation, alkylating agents and antitumor drugs that are not ordinarily considered to be alkylating agents were determined in the presence and absence of centrophenoxine. Mouse P388 lymphoma cells growing in static suspension culture were used as the experimental tumor. The cytotoxic action of most alkylating agents was found to be potentiated by centrophenoxine; Included in this group were several difunctional nitrogen mustards, two ethylenimines, a nitrosourea and mitomycin C. Greatest enhancement, 7-fold, was of chlorambucil antitumor activity. Centrophenoxine did not potentiate the lethality of X- or UV-irradiation or the cytotoxicity of several antineoplastic drugs that are not alkylating agents.     

Inactivation of human O(6)-alkylguanine-DNA alkyltransferase by modified oligodeoxyribonucleotides containing O(6)-benzylguanine

Inactivation of the DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (AGT) enhances tumor cell killing by therapeutic alkylating agents. O(6)-Benzylguanine (b(6)G) can inactivate AGT and is currently in clinical trials to enhance therapy. Short oligodeoxyribonucleotides containing b(6)G are much more effective inactivators, but their use for therapeutic purposes is likely to be compromised by metabolic instability. We have therefore examined the ability to inactivate AGT of an 11-mer oligodeoxyribonucleotide containing b(6)G (11-mpBG) when modified with terminal methylphosphonate linkages to protect it from nucleases. This modification did not reduce the ability to serve as a substrate/inactivator for AGT, and 11-mpBG had an ED(50) value of 1.3 nM, more than 300-fold lower than that for b(6)G. A similar oligodeoxyribonucleotide containing O(6)-methylguanine (m(6)G) was also found to be a good substrate (ED(50) value of 10 nM), but the benzylated form was repaired more rapidly and preferentially. When added to HT29 cell cultures, 5 microM 11-mpBG was able to cause a prolonged inactivation of cellular AGT for at least 72 h and to greatly sensitize the cells to killing by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). The 11-mpMG was ineffective at up to 20 microM, suggesting that the benzyl group allows better uptake into the cell. However, even with 11-mpBG, the 1000-fold decrease in potency toward AGT in HT29 cells compared to that toward the protein in vitro suggests that uptake may be a limiting factor. These results suggest that oligodeoxyribonucleotides such as 11-mpBG may prove to be useful drugs for potentiation of alkylating agent chemotherapy if uptake can be improved.     

Mechanisms of resistance to 1,3-bis(2-chloroethyl)-1-nitrosourea in human medulloblastoma and rhabdomyosarcoma

Medulloblastoma (D-341 MED) and rhabdomyosarcoma (TE-671) cell lines, which are resistant to either 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or the combination of BCNU and O6-benzylguanine (O6-BG), were generated by serial escalation of BCNU. The activities of O6-alkylguanine-DNA alkyltransferase (AGT), glutathione-S-transferase (GST), and total glutathione (GSH) of the parental, BCNU-resistant (BR), and BCNU + O6-BG-resistant (OBR) cells were measured. No significant differences in GST activity or total GSH were seen between the parental, BR, and OBR cells of both TE-671 and D-341 MED. The AGT activities of D-341 MED (BR) and TE-671 (BR) were twice those of D-341 MED and TE-671, respectively, confirming the importance of this enzyme for BCNU resistance. The D-341 MED (OBR) cells did not exhibit any AGT activity, suggesting that another mechanism must play a role in the drug resistance. Fewer DNA interstrand cross-links (ICLs) were observed in D-341 MED (OBR) than in D-341 MED after 8 h BCNU (100-400 microM) treatment. However, the amounts of DNA ICLs observed in D-341 MED and D-341 MED (OBR) were stable after 24 h. Microarray analysis showed the increased expressions of several metallothionein genes and down-regulation of several proapoptotic genes. The AGT activity of TE-671 (OBR) was 223 fmol/mg when the cells were grown in 10 microM O6-BG and decreased to about half this value when the O6-BG concentration was increased 60 microM. The AGT cDNA of TE-671 (OBR) cells was cloned and found to contain a G-to-T transversion at codon 156, resulting in conversion of glycine to cysteine (G156C). In vitro mutagenesis has shown that the G156C AGT mutant is resistant to inactivation by O6-BG. Thus, the selection of a mutant AGT with decreased sensitivity to O6-BG is a significant contributing factor to BCNU + O6-BG resistance.     

In vivo selection of MGMT(P140K) lentivirus-transduced human NOD/SCID repopulating cells without pretransplant irradiation conditioning

Infusion of transduced hematopoietic stem cells into nonmyeloablated hosts results in ineffective in vivo levels of transduced cells. To increase the proportion of transduced cells in vivo, selection based on P140K O6-methylguanine-DNA-methyltransferase (MGMT[P140K]) gene transduction and O6-benzylguanine/1,3-bis(2-chloroethyl)-1-nitrosourea (BG/BCNU) treatment has been devised. In this study, we transduced human NOD/SCID repopulating cells (SRCs) with MGMT(P140K) using a lentiviral vector and infused them into BG/BCNU-conditioned NOD/SCID mice before rounds of BG/BCNU treatment as a model for in vivo selection. Engraftment was not observed until the second round of BG/BCNU treatment, at which time human cells emerged to compose up to 20% of the bone marrow. Furthermore, 99% of human CFCs derived from NOD/SCID mice were positive for provirus as measured by PCR, compared with 35% before transplant and 11% in untreated irradiation-preconditioned mice, demonstrating selection. Bone marrow showed BG-resistant O6-alkylguanine-DNA-alkyltransferase (AGT) activity, and CFUs were stained intensely for AGT protein, indicating high transgene expression. Real-time PCR estimates of the number of proviral insertions in individual CFUs ranged from 3 to 22. Selection resulted in expansion of one or more SRC clones containing similar numbers of proviral copies per mouse. To our knowledge, these results provide the first evidence of potent in vivo selection of MGMT(P140K) lentivirus-transduced human SRCs following BG/BCNU treatment.     

Defining regional infusion treatment strategies for extremity melanoma: comparative analysis of melphalan and temozolomide as regional chemotherapeutic agents

Five different human melanoma xenografts were used in a xenograft model of extremity melanoma to evaluate the variability of tumor response to regionally administered melphalan or temozolomide and to determine if various components of pertinent drug resistance pathways for melphalan [glutathione S-transferase (GST)/glutathione] and temozolomide [O(6)-alkylguanine DNA alkyltranferase (AGT)/mismatch repair (MMR)] could be predictive of tumor response. Xenograft-bearing rats underwent regional isolated limb infusion with either melphalan (90 mg/kg) or temozolomide (2,000 mg/kg). The levels of AGT activity, GST activity, glutathione level, and GST/AGT expression were examined in this group of xenografts and found to be quite heterogeneous. No correlation was identified between melphalan sensitivity and the GST/glutathione cellular detoxification pathway. In contrast, a strong correlation between the levels of AGT activity and percentage increase in tumor volume on day 30 (r = 0.88) was noted for tumors treated with temozolomide. Regional therapy with temozolomide was more effective when compared with melphalan for the xenograft with the lowest AGT activity, whereas melphalan was more effective than temozolomide in another xenograft that had the highest AGT activity. In three other xenografts, there was no significant difference in response between the two chemotherapy agents. This study shows that AGT activity may be useful in predicting the utility of temozolomide-based regional therapy for advanced extremity melanoma tumors. Our observations also point out the limited ability of analysis of the GST/glutathione pathway to predict response to chemotherapies like melphalan whose resistance is primarily mediated through a complex mechanism of detoxification.     

Human mesenchymal stem cells provide stromal support for efficient CD34+ transduction

Human mesenchymal stem cells (hMSC)-nonhematopoietic cells within the bone marrow microenvironment that can be culture expanded to a uniform population of fibroblastic cells-have been shown to support long-term hematopoiesis of CD34+ cells. Because direct contact between stromal elements and CD34+ cells enhances long-term engraftment, we postulated that hMSC would be a good alternative to the more heterogeneous stroma currently used in gene transfer studies. We used hMSC to support retroviral gene transfer of the G156A MGMT (deltaMGMT) gene encoding an alkyltransferase (AGT), which confers drug resistance to a combination of O6-benzylguanine (BG) plus the alkylating agents BCNU and temozolomide (TMZ) in human hematopoietic progenitors. In the presence of IL-3, IL-6, SCF, or leukemia inhibitory factor (LIF) and Flt-3 ligand, hMSC facilitated expansion and retroviral transduction of human peripheral blood-mobilized CD34+ cells. Furthermore, the transduced cells expressed AGT in 29% of hematopoietic cells and were 5-fold more resistant to BCNU and TMZ than were untransduced cells. Unirradiated hMSC present as support cells were simultaneously transduced and expressed AGT in 26% of the cells. Thus, the homogeneous nature of hMSC, and their ability to support gene transfer and be transduced themselves suggest they may be useful in clinical gene transfer protocols and have broad therapeutic applications.     

Cytotoxicity,DNA damage,and apoptosis induced by new fotemustine analogs on human melanoma cells in relation to O6-methylguanine DNA-methyltransferase expression

Fotemustine is a third generation chloroethylnitrosourea that has demonstrated significant antitumoral effects in malignant melanoma. However, its use is somewhat limited by its toxic side effects and chemoresistance caused by direct repair of O6-alkyl groups by the enzyme O6-methylguanine DNA-methyltransferase (MGMT). The aim of this work was to determine to what extent the expression of MGMT influences cytotoxicity, DNA damage, and apoptosis induced by new nitrososulfamide analogs of fotemustine (compounds 4 and 8), which have previously demonstrated interesting antiproliferative properties. We carried out complementary strategies that consisted of MGMT cDNA transfection in CAL77 Mer- melanoma cells and of MGMT inhibition with O6-benzylguanine (BG) in A375 Mer+ melanoma cells. MGMT-transfected cells were 7 to 9 times less sensitive to fotemustine than parent cells, whereas no difference between the transfected and parent cells was observed for nitrososulfamide analogs. The cytotoxicity of these analogs vis à vis a MGMT-proficient A375 melanoma cell line was approximately 3 times greater than that of fotemustine. Coincubation of these cells with O6-benzylguanine significantly increased the cytotoxicity of fotemustine and compound 8, whereas BG had little effect on the cytotoxicity of compound 4. Furthermore, DNA fragmentation determined by a comet assay was greater with nitrososulfamide analogs than with fotemustine. O6-benzylguanine increased DNA fragmentation for fotemustine and compound 8, but not for compound 4, which induced comets with a typical apoptotic appearance. The ability of this compound to induce apoptosis in the absence of BG was confirmed by a specific enzyme-linked immunosorbent assay apoptotic assay using a single-stranded DNA monoclonal antibody.     

Prolonged inhibition of O(6)-methylguanine DNA methyltransferase in human tumor cells by O(6)-benzylguanine in vitro and in vivo

We previously demonstrated that sustained depletion of methylguanine DNA methyltransferase (MGMT) activity is required for optimal reversal of chloroethylnitrosourea resistance in tumor cells. The purpose of this study was to design O(6)-benzylguanine (BG) treatments that deplete MGMT activity in tumor cells and xenograft tumors in a prolonged manner. When SF767 cells were treated with a bolus dose of BG (25 microM for 1 h), >95% of MGMT activity was depleted but 33% of the activity recovered within 24 h. In contrast, MGMT activity was completely depleted for 24 h when cells were pretreated with a low dose of BG (2.5 microM) for 24 h, followed by the bolus dose and same low-dose treatment for 24 h. This combination regimen of pre- and post-treatments with a bolus dose sensitized cells N,N'-bis(2-chloroethyl)-N-nitrosourea in vitro by approximately 2-fold more than the bolus dose alone. Similar BG treatment with Alzet micro-osmotic pumps produced sustained inhibition of MGMT activity in vivo. In xenograft SF767 tumors, low-dose pre- and post-treatments (8 mg/kg over 24 h) combined with an i.p. bolus dose (80 mg/kg) of BG inhibited >95% of MGMT activity for 24 h after the bolus. The bolus dose alone did not deplete MGMT for 24 h. These results demonstrate that combination low-dose and bolus BG treatment is superior to the bolus dose alone in depleting MGMT activity in a sustained manner in vitro and in vivo. When combined with N,N'-bis(2-chloroethyl)-N-nitrosourea treatment, this BG regimen also should also produce greater antitumor activity than the single bolus dose evaluated clinically.     

Brain tumor cell lines resistant to O6-benzylguanine/1,3-bis(2-chloroethyl)-1-nitrosourea chemotherapy have O6-alkylguanine-DNA alkyltransferase mutations

The chemotherapeutic activity of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU or carmustine) may be improved by the addition of O6-benzylguanine (O6-BG). The reaction of O6-BG with O6-alkylguanine-DNA alkyltransferase (AGT) prevents the repair of O6-chloroethyl lesions caused by BCNU. In clinics, the combination of O6-BG and BCNU is now being tested for the treatment of brain tumors. However, the effectiveness of this drug regimen may be limited by drug resistance acquired during treatment. To understand the possible mechanisms of resistance of brain tumor cells to the O6-BG/BCNU combination, we generated medulloblastoma cell lines (D283 MED, D341 MED, and Daoy) resistant to the combination of O6-BG and BCNU [O6-BG/BCNU resistant (OBR)]. DNA sequencing showed that all of the parent cell lines express wild-type AGTs, whereas every OBR cell line exhibited mutations that potentially affected the binding of O6-BG to the protein as evidenced previously by in vitro mutagenesis and structural studies of AGT. The D283 MED (OBR), Daoy (OBR), and D341 MED (OBR) cell lines expressed G156C, Y114F, and K165T AGT mutations, respectively. We reported previously that rhabdomyosarcoma TE-671 (OBR) also expresses a G156C mutation. These data suggest that the clonal selection of AGT mutants during treatment with O6-BG plus an alkylator may produce resistance to this intervention in clinical settings.     

DeltaMGMT-transduced bone marrow infusion increases tolerance to O6-benzylguanine and 1,3-bis(2-chloroethyl)-1-nitrosourea and allows intensive therapy of 1,3-bis(2-chloroethyl)-1-nitrosourea-resistant human colon cancer xenografts

O6-Benzylguanine (BG) is a potent inhibitor of the DNA repair protein 06-alkylguanine DNA alkyltransferase (AGT), and sensitizes tumors to BCNU in vitro and in xenografts. The combination of BG and BCNU is now undergoing phase I clinical testing. The maximally tolerated dose of BCNU given after BG is expected to be lower then the doses tolerated as a single agent owing to BG sensitization of hematopoietic progenitors. We have previously shown that retroviral expression of G156A mutant MGMT (deltaMGMT) in mouse and human marrow cells results in significant BG and BCNU resistance. In this study we evaluated the effect of deltaMGMT-transduced marrow infusion on the therapeutic index of multiple BG and BCNU treatments in tumor-bearing nude (nu/nu athymic) mice. Prior to subcutaneous implantation of BCNU-resistant SW480 human colon cancer cells, cohorts of mice were given intraperitoneal injections of nonablative doses of BG (30 mg/kg) and BCNU (10 mg/kg, one-half of the LD10) and then infused with 1-2 x 10(6) isogeneic deltaMGMT (n = 29 mice) or lacZ-transduced (n = 20 mice) marrow cells. The xenograft-bearing mice were treated with multiple cycles of BG (30 mg/kg) and BCNU (10-25 mg/kg). After three cycles, deltaMGMT mouse bone marrow was repopulated with CFU containing the provirus, and demonstrated a 2.7-fold increase in AGT activity and a 5.5-fold increase in BCNU IC90 compared with LacZ mice. After five cycles, the BCNU IC90 of CFU cells increased nine-fold over control cells, indicating selective enrichment of CFU precursor cells expressing high levels of deltaMGMT. Starting with the third cycle of therapy, tolerance to BG and BCNU was significantly improved in deltaMGMT mice compared with LacZ mice, as evidenced by preserved peripheral blood counts, bone marrow cellularity, and CFU content 1 and 2 weeks posttreatment and a significantly higher survival rate. Xenograft growth was significantly delayed in mice tolerating multiple cycles and higher dose intensity of BG and BCNU as compared with mice receiving less intensive therapy. We conclude that deltaMGMT-transduced marrow cells can improve the therapeutic index of BG and BCNU by selectively repopulating the marrow and providing significant marrow tolerance to this combination, allowing intensive therapy of a BCNU-resistant tumor.     

A new O6-alkylguanine-DNA alkyltransferase inhibitor associated with a nitrosourea (cystemustine) validates a strategy of melanoma-targeted therapy in murine B16 and human-resistant M4Beu melanoma xenograft models

Chemoresistance to O(6)-alkylating agents is a major barrier to successful treatment of melanoma. It is mainly due to a DNA repair suicide protein, O(6)-alkylguanine-DNA alkyltransferase (AGT). Although AGT inactivation is a powerful clinical strategy for restoring tumor chemosensitivity, it was limited by increased toxicity to nontumoral cells resulting from a lack of tumor selectivity. Achieving enhanced chemosensitization via AGT inhibition preferably in the tumor should protect normal tissue. To this end, we have developed a strategy to target AGT inhibitors. In this study, we tested a new potential melanoma-directed AGT inhibitor [2-amino-6-(4-iodobenzyloxy)-9-[4-(diethylamino) ethylcarbamoylbenzyl] purine; IBgBZ] designed as a conjugate of O(6)-(4-iododbenzyl)guanine (IBg) as the AGT inactivator and a N,N-diethylaminoethylenebenzamido (BZ) moiety as the carrier to the malignant melanocytes. IBgBZ demonstrated AGT inactivation ability and potentiation of O(6)-alkylating agents (cystemustine, a chloroethylnitrosourea) in M4Beu highly chemoresistant human melanoma cells both in vitro and in tumor models. The biodisposition study on mice bearing B16 melanoma, the standard model for the evaluation of melanoma-directed agents, and the secondary ion mass spectrometry imaging confirmed the concentration of IBgBZ in the tumor and in particular in the intracytoplasmic melanosomes. These results validate the potential of IBgBZ as a new, more tumor-selective, AGT inhibitor in a strategy of melanoma-targeted therapy.     

Enhancement of cisplatin [cis-diammine dichloroplatinum (II)] cytotoxicity by O6-benzylguanine involves endoplasmic reticulum stress

O6-Benzylguanine (BG) enhances cisplatin [cis-diammine dichloroplatinum (II)]-induced cytotoxicity and apoptosis in head and neck cancer cell lines by an unknown mechanism. We investigated the effect of cisplatin with and without BG on two targets of damage: DNA and the endoplasmic reticulum (ER). We chose three cancer cell lines to ascertain the mechanism of BG-enhanced cytotoxicity: SQ20b head and neck and SKOV-3x ovarian cancer cell lines, where BG enhanced cisplatin cytotoxicity, and A549 nonsmall cell lung cancer line, where BG did not enhance cisplatin cytotoxicity. All three lines had an increase in DNA damage when BG was added to cisplatin treatment, as evidenced by increased platination and phosphorylated histone H2AX formation. The increase in cisplatin-induced DNA damage after treatment with BG plus cisplatin is not sufficient to increase cytotoxicity or apoptosis in A549 cells. We evaluated the effect of cisplatin on the ER and observed increased caspase 12 cleavage in SQ20b and SKOV-3x cells, but not in A549 cells, after treatment with BG plus cisplatin versus cisplatin alone. Growth arrest and DNA damage inducible (GADD) 153, an ER stress-response gene, is up-regulated after treatment with BG plus cisplatin compared with cisplatin alone in SQ20b and SKOV-3x cells, but not in A549 cells. ER stress-induced apoptosis is an integral part of the mechanism by which BG enhances cisplatin. Inhibition of ER stress in the SQ20b cell line by salubrinal, an inhibitor of eIF2alpha dephosphorylation, or GADD153 small interfering RNA, abrogated BG-enhancement of cisplatin cytotoxicity and apoptosis through caspase 3 and 12 cleavage. These data indicate GADD153 up-regulation plays an important role in BG-enhanced cisplatin cytotoxicity and apoptosis.     

The Checkpoint Kinase Inhibitor AZD7762 Potentiates Chemotherapy-Induced Apoptosis of p53-Mutated Multiple Myeloma Cells

DNA cross-linking agents are frequently used in the treatment of multiple myeloma-generating lesions, which activate checkpoint kinase 1 (Chk1), a critical transducer of the DNA damage response. Chk1 activation promotes cell survival by regulating cell-cycle arrest and DNA repair following genotoxic stress. The ability of AZD7762, an ATP-competitive Chk1/2 inhibitor to increase the efficacy of the DNA-damaging agents bendamustine, melphalan, and doxorubicin was examined using four human myeloma cell lines, KMS-12-BM, KMS-12-PE, RPMI-8226, and U266B1. The in vitro activity of AZD7762 as monotherapy and combined with alkylating agents and the "novel" drug bortezomib was evaluated by studying its effects on cytotoxicity, signaling, and apoptotic pathways. The Chk1/2 inhibitor AZD7762 potentiated the antiproliferative effects of bendamustine, melphalan, and doxorubicin but not bortezomib in multiple myeloma cell lines that were p53-deficient. Increased γH2AX staining in cells treated with bendamustine or melphalan plus AZD7762 indicates a greater degree of DNA damage with combined therapy. Abrogation of the G(2)-M checkpoint by AZD7762 resulted in mitotic catastrophe with ensuing apoptosis evidenced by PARP and caspase-3 cleavage. In summary, the cytotoxic effects of bendamustine, melphalan and doxorubicin on p53-deficient multiple myeloma cell lines were enhanced by the coadministration of AZD7762. These data provide a rationale for testing these combinations in patients with relapsed and/or refractory multiple myeloma. Mol Cancer Ther; 11(8); 1781-8. ?2012 AACR.     

Retroviral-mediated expression of the P140A, but not P140A/G156A, mutant form of O6-methylguanine DNA methyltransferase protects hematopoietic cells against O6-benzylguanine sensitization to chloroethylnitrosourea treatment.

O(6)-Benzylguanine (6-BG) inactivates mammalian O(6)-methylguanine DNA methyltransferase (MGMT), an important DNA repair protein that protects cells against chloroethylnitrosourea (CENU) cytotoxicity. 6-BG is being tested as an approach to treat CENU-resistant tumors that overexpress endogenous MGMT. However, in addition to restoring CENU tumor cell sensitivity, 6-BG also increases the cytotoxic effects of CENUs on hematopoietic cells. Several 6-BG-resistant human MGMT mutants have been characterized in Escherichia coli and are predicted to protect mammalian cells against the combination of 6-BG and CENU treatment in vivo. Two mutants, P140A and P140A/G156A, demonstrated 20- and 1200-fold more resistance to 6-BG depletion of MGMT activity compared with wild-type MGMT (WTMGMT). Here, we analyzed retroviral vectors that express either WTMGMT, the P140A or P140A/G156A mutant forms of MGMT. Retroviral-infected L1210 hematopoietic cells demonstrated similar levels of RNA in all transduced clones. However, the amount of MGMT protein and DNA repair activity was reduced in clones expressing the P140A/G156A mutant compared with those expressing WTMGMT or P140A. Expression of P140A was associated with a 4- to 8-fold increase in resistance to 6-BG depletion of MGMT in transduced L1210 clones and a 1, 3-bis(2-chloroethyl)-1-nitrosourea IC(50) of 50 microM (compared with 27.5 microM for WTMGMT) in primary murine hematopoietic cells. These results demonstrate the utility of screening 6-BG-resistant MGMT proteins in hematopoietic cells and provide evidence that the P140A mutant form of MGMT generates 6-BG- and CENU-resistant hematopoietic cells. Retrovirus vectors expressing this mutant may be useful in future human gene therapy trials.     

Hematopoietic expression of O(6)-methylguanine DNA methyltransferase-P140K allows intensive treatment of human glioma xenografts with combination O(6)-benzylguanine and 1,3-bis-(2-chloroethyl)-1-nitrosourea

The major mechanism of tumor cell resistance to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) is the DNA repair protein O(6)-methylguanine DNA methyltransferase (MGMT). This repair system can be temporarily inhibited by the free base O(6)-benzylguanine (BG), which depletes cellular MGMT activity and sensitizes tumor cells and xenografts to BCNU. In clinical studies, the combination of BG and BCNU enhanced the myeloid toxicity of BCNU, thereby reducing the maximum tolerated dose. We have shown previously that retroviral expression of the P140K mutant of MGMT (MGMT-P140K) in murine and human hematopoietic cells produces significant resistance of bone marrow cells to low-dose, combination BG and BCNU treatment in vivo. In the current study, we investigated the ability of bone marrow transplantation with MGMT-P140K-transduced hematopoietic cells to protect against an intensive antitumor treatment regimen of combination BG and BCNU in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. The donor marrow cells underwent in vivo BG and BCNU selection before transplantation, allowing infusion of a highly selected population of transduced cells. Tolerance to the intensive BG and BCNU treatment was markedly improved in secondary MGMT-P140K-transplanted mice (n = 19) compared to untransplanted mice (n = 15), as indicated by blood counts and survival rate. The dose-intensified BG and BCNU therapy produced significant growth delays of glioma xenografts in MGMT-P140K-transplanted mice, extending the tumor doubling time by >40 days. These results demonstrate that MGMT-P140K-transduced bone marrow protects against BG and BCNU combination therapy in vivo and allows dose-intensified treatment of tumor xenografts.     

Blockage of abasic site repair enhances antitumor efficacy of 1,3-bis-(2-chloroethyl)-1-nitrosourea in colon tumor xenografts

Methoxyamine (MX) has been shown to potentiate the antitumor effect of temozolomide (TMZ) in human tumor xenograft models. This potentiation is due to the reactivity of MX with apurinic/pyrimidinic (AP) sites in DNA, which are formed following DNA glycosylase removal of TMZ-induced methyl-purine adducts. MX-bound AP sites cannot be further processed by base excision repair (BER), resulting in cell death. On the basis of this finding, we investigated in vivo whether MX enhanced therapeutic efficacy of other agents, such as 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) that generates AP sites during DNA repair. Nude mice carrying human colon tumor xenografts, HCT116 and HCT116-Ch3, were treated by a single injection (i.p) of BCNU alone (30 mg/kg) or MX (2 mg/kg) combined with BCNU. The effect on tumor growth of BCNU alone was very moderate. Combined administration of MX and BCNU produced significant inhibition of tumor growth. Tumor growth delays were 14 +/- 3 days in HCT116 and 16 +/- 2 days in HCT116-Ch3 tumors, respectively (P < 0.05 versus control or BCNU alone groups). Similar results were also observed in SW480 and DLD1 tumors. Importantly, no systemic toxicity was noted with BCNU and MX. In contrast, BCNU (at dose of 25 mg/kg) combined with O(6)-benzylguanine (BG), an inhibitor of O(6)-alkylguanine-DNA alkyltransferase (AGT) being tested in clinical trials, caused toxic death in all treated mice. However, a lower dose BCNU (10 mg/kg) combined with BG and MX had significant antitumor effect without toxic death. Thus, targeting BER with MX is a promising strategy to improve the antitumor activity of BCNU and perhaps other DNA-damaging agents.     

Rescue from enhanced alkylator-induced cell death with low molecular weight sulfur-containing chemoprotectants

Modulation of glutathione has been proposed as a mechanism to alter the efficacy and toxicity of chemotherapeutic agents. We investigated in vitro cytoenhancement of chemotherapy toxicity by reducing cellular glutathione levels with L-buthionine-[S,R]-sulfoximine (BSO), and chemoprotection with small molecular weight sulfur-containing agents that mimic or replace glutathione. Cytotoxicity, caspase-2 enzymatic activity, and in situ DNA staining for apoptosis were assessed in cultured human small cell lung carcinoma cells and fibroblasts. BSO treatment reduced the half-maximal cytotoxic dose of the alkylating chemotherapeutics melphalan, carboplatin, and cisplatin, and increased the total magnitude of cell death. Melphalan was more sensitive than carboplatin or cisplatin to BSO. The chemoprotective agents sodium thiosulfate, N-acetylcysteine, and glutathione ethyl ester reduced the cytotoxicity of all three alkylating chemotherapeutics regardless of BSO treatment, but D-methionine was effective only against the platinum agents. N-Acetylcysteine was the most effective protectant tested. Chemoprotection against melphalan toxicity was maximally effective only if administered concurrent with chemotherapy, whereas chemoprotection for the platinum agents remained effective if delayed 4 h after chemotherapy. BSO enhancement and N-acetylcysteine chemoprotection for melphalan toxicity occurred at least partially through an apoptotic mechanism. Modulation of glutathione levels will be valuable in the clinical setting if chemotherapy and chemoprotectant can be physically and/or temporally separated. Cytoenhancement and chemoprotection may be particularly useful in the central nervous system where the blood-brain barrier of the cerebral vasculature creates two compartments, for cytoenhancement in brain tumors and systemic chemoprotection.