Alkylator Resistance Research Articles

Transcriptional control of O6 -methylguanine DNA methyltransferase expression and temozolomide resistance in glioblastoma.

O6 -methylguanine DNA methyltransferase (MGMT) promoter methylation is a predictive biomarker for benefit from alkylating chemotherapy, specifically temozolomide (TMZ), in glioblastoma, the most common malignant intrinsic brain tumor. Glioma-initiating cells (GIC) with stem-like properties have been associated with resistance to therapy and progression. We assessed the levels of MGMT mRNA and MGMT protein by real-time PCR and immunoblot and evaluated the impact of MGMT on TMZ sensitivity in clonogenicity assays in GIC sphere cultures (S) or differentiated adherent monolayer cultures (M). Nuclear factor kappa B (NF-κB) signaling was assessed by reporter assay and immunoblot. Compared to M cells, S cells expressed higher levels of MGMT. Differentiation of GIC induced by S-to-M transition resulted in a gradual loss of MGMT expression and increased TMZ sensitivity. This transcriptional regulation of MGMT was restricted to cell lines without MGMT promoter methylation and was not coupled to any specific neurobasal (NB) stem cell medium supplement or loss of cell adhesion. Expression levels of p50/p65 subunits of NF-κB, a transcriptional regulator of MGMT, were increased in S cells. Inhibition of NF-κB by the small molecule inhibitor, BAY 11-7082, or siRNA-mediated gene silencing, reduced MGMT levels. In summary, alkylator resistance of S cells is mainly promoted by over-expression of MGMT which results from increased activity of the NF-κB pathway in this cell culture model of glioma stem-like cells. Read the Editorial Highlight for this article on page 688.

MicroRNA-138 promotes acquired alkylator resistance in glioblastoma by targeting the Bcl-2-interacting mediator BIM.

Glioblastoma is the most aggressive brain tumor in adults with a median survival below 12 months in population-based studies. The main reason for tumor recurrence and progression is constitutive or acquired resistance to the standard of care of surgical resection followed by radiotherapy with concomitant and adjuvant temozolomide (TMZ/RT→TMZ). Here, we investigated the role of microRNA (miRNA) alterations as mediators of alkylator resistance in glioblastoma cells. Using microarray-based miRNA expression profiling of parental and TMZ-resistant cultures of three human glioma cell lines, we identified a set of differentially expressed miRNA candidates. From these, we selected miR-138 for further functional analyses as this miRNA was not only upregulated in TMZ-resistant versus parental cells, but also showed increased expression in vivo in recurrent glioblastoma tissue samples after TMZ/RT→TMZ treatment. Transient transfection of miR-138 mimics in glioma cells with low basal miR-138 expression increased glioma cell proliferation. Moreover, miR-138 overexpression increased TMZ resistance in long-term glioblastoma cell lines and glioma initiating cell cultures. The apoptosis regulator BIM was identified as a direct target of miR-138, and its silencing mediated the induced TMZ resistance phenotype. Altered sensitivity to apoptosis played only a minor role in this resistance mechanism. Instead, we identified the induction of autophagy to be regulated downstream of the miR-138/BIM axis and to promote cell survival following TMZ exposure. Our data thus define miR-138 as a glioblastoma cell survival-promoting miRNA associated with resistance to TMZ therapy in vitro and with tumor progression in vivo.

446. Stable Correction of Alkylator Resistance in Fanconi Anemia Cells By a Mitotically Replicating Non-Integrating Lentiviral Vector

Fanconi anemia (FA) is an inherited multisystem disorder leading to symptomatic hematopoietic defects by early school age in most patients. Observations of lymphoid mosaicism and spontaneous intragenic correction of stem cells leading to clonal hematopoiesis in a small number of patients indicate that phenotypically corrected cells enjoy a survival advantage over FA deficient cells. Accordingly, FA is a leading genetic disorder target for HSPC directed gene therapy. However, reports over the insertional complications of commonly used integrating vectors continue to raise concerns, while available non-integrating lentivectors (NILVs) are generally lost from dividing hematopoietic stem cells. We recently showed that the incorporation of human Scaffold/Matrix Attachment Region (S/MAR) sequences in NILVs results in the cellular retention of NILV episomes in actively dividing cell lines and HSPCs without selection. Southern analysis, Alu PCR and episome specific PCR all confirmed long-term episomal existence of this vector without genomic integration (NAR. 2014; PMID: 24474068). Here, we designed and tested an episome lentivector to phenotypically correct FA cells (aNILV GFP-FANCC). Results showed the vector expressing the human FANCC ORF fused with GFP could be readily packaged, with stable persistence of vector episomes and sustained GFP expression. Taking advantage of the selective expansion of phenotypically corrected cells, we transduced a FANCC -deficient human cell line pD331 by using aNILV-GFP-FANCC vector. Exaggerated sensitivity to DNA alkylating agents (e.g. Mitomycin C, MMC) is a hallmark of FA deficient cells and can be used as a selection agent. At week 2, we observed 15% retention of GFP expressing cells in MMC treated pD331-aNILV-FANCC population versus 3% retention in non-treated pD331-aNILV-FANCC cells. Long-term maintenance of cells up to 6 weeks under MMC selection resulted the enrichment of corrected cells up to 95% that is comparable to the enrichment observed by integrating LV expressingGFP-FANCC in pD331 cells under MMC selection. Molecular studies of aNILV episomes from long-term cultures by episome specific PCR revealed LTR episome persistence. Integration analysis by Alu-PCR from the bulk population with Southern analysis of clones is ongoing. We already reported that episomal S/MAR NILV transduced short-term murine hematopoietic stem and progenitor cells and are currently characterizing this vector in a murine model of Fanconi Anemia. In conclusion, epsiome maintenance under selective pressure and reversal of canonical alkylator sensitivity in FA cells make this novel aNILV-GFP-FANCC vector a consideration for expanded preclinical exploration.

O6-methylguanine-DNA methyltransferase activity is associated with response to alkylating agent therapy and with MGMT promoter methylation in glioblastoma and anaplastic glioma

BackgroundCpG methylation in the O6-methylguanine-DNA methyltransferase (MGMT) promoter is associated with better outcome following alkylating agent chemotherapy in glioblastoma (GBM) and anaplastic glioma (AG). To what extent improved response reflects low or absent MGMT activity in glioma tissue has not been unequivocally assessed. This information is central to developing anti-resistance therapies. MethodsWe examined the relationship of MGMT activity in 91 GBMs and 84 AGs with progression-free survival (PFS) following alkylator therapy and with promoter methylation status determined by methylation-specific PCR (MSP). ResultsCox regression analysis revealed that GBMs with high activity had a significantly greater risk for progression in dichotomous (P≤0.001) and continuous (P≤0.003) models, an association observed for different alkylator regimens, including concurrent chemo-radiation with temozolomide. Analysis of MGMT promoter methylation status in 47 of the GBMs revealed that methylated tumors had significantly lower activity (P≤0.005) and longer PFS (P≤0.036) compared to unmethylated tumors, despite overlapping activities. PFS was also significantly greater in methylated vs. unmethylated GBMs with comparable activity (P≤0.005), and among unmethylated tumors with less than median activity (P≤0.026), suggesting that mechanisms in addition to MGMT promote alkylator resistance. Similar associations of MGMT activity with PFS and promoter methylation status were observed for AGs. ConclusionsOur results provide strong support for the hypotheses that MGMT activity promotes alkylator resistance and reflects promoter methylation status in malignant gliomas. General significanceMGMT activity is an attractive target for anti-resistance therapy regardless of methylation status.

Understanding and targeting alkylator resistance in glioblastoma.

Alkylating chemotherapy is the mainstay in the treatment of pediatric and adult glioblastoma despite primary and acquired resistance and scientific efforts to precisely define therapies for individual patients. A focus on non-MGMT-mediated temozolomide resistance for pediatric glioblastoma suggests options for new drug combinations.

MGMT testing--the challenges for biomarker-based glioma treatment.

Many patients with malignant gliomas do not respond to alkylating agent chemotherapy. Alkylator resistance of glioma cells is mainly mediated by the DNA repair enzyme O(6)-methylguanine-DNA methyltransferase (MGMT). Epigenetic silencing of the MGMT gene by promoter methylation in glioma cells compromises this DNA repair mechanism and increases chemosensitivity. MGMT promoter methylation is, therefore, a strong prognostic biomarker in paediatric and adult patients with glioblastoma treated with temozolomide. Notably, elderly patients (>65-70 years) with glioblastoma whose tumours lack MGMT promoter methylation derive minimal benefit from such chemotherapy. Thus, MGMT promoter methylation status has become a frequently requested laboratory test in neuro-oncology. This Review presents current data on the prognostic and predictive relevance of MGMT testing, discusses clinical trials that have used MGMT status to select participants, evaluates known issues concerning the molecular testing procedure, and addresses the necessity for molecular-context-dependent interpretation of MGMT test results. Whether MGMT promoter methylation testing should be offered to all individuals with glioblastoma, or only to elderly patients and those in clinical trials, is also discussed. Justifications for withholding alkylating agent chemotherapy in patients with MGMT-unmethylated glioblastomas outside clinical trials, and the potential role for MGMT testing in other gliomas, are also discussed.

Double akylating agents treatment with ifosfamide (IFOS) and cyclophosphamide (CYCLO) for relapsed pediatric solid tumors.

10057 Background: CYCLO and IFOS are two of the most active agents in childhood cancer. There is non-cross resistance between CYCLO and IFOS and they have dissimilar toxic side effects. In vitro, tumor model systems document that alkylator resistance may be overcome by several fold increases in drug concentration. We developed a strategy in attempt to limit side effects and increase anticancer activity of high doses oxazaphosphorines therapy: an association of IFOS plus CYCLO, giving an equivalent to 20g/m2 of IFOS or 5g/m2 of CYCLO. The schedule is the association of these two drugs: CYCLO 2,5g/m2 (corresponding to 10g/m2 of IFOS) plus IFOS 10g/m2. Methods: Eligibility included recurrent/refractory measurable disease, life expectancy > 6 weeks, adequate renal, hepatic and bone marrow function. CYCLO (2,5g/m2) and IFOS (10g/m2) with Mesna, with interval of 21 days. Responses were evaluated after 2 cycles.So far, 13 patients were enrolled: median age 17 years (5-26), 9M:4F,6 osteosarcoma, 1 hemangioperycitoma, 2 medulloblastoma, 1 nasopharyngeal carcinoma, 1 Wilms tumor,1 synoviosarcoma, 1 retinoblastoma. Six patients received IFOS previously (Total= 38 - 63 g/m²) and 5 CYCLO. Results: Thirty four cycles were evaluated. Toxicity was tolerable with no death. Main adverse event was neutropenia grade (GR) 4 in all cycles, median duration of seven days (3-15), GCS-F was used in all cycles; anemia GR 3 and 4 and thrombocytopenia GR 4 in 14 cycles; infection GR 3 and 4 in 15 cycles; hemorrhage cystitis GR 1 in 2 cycles and neurologic toxicity GR 2 in 6 cycles. No acute renal toxicity was observed. Responses were 2 complete response (CR), 5 partial responses (PR), 3 stable disease, and 3 progress disease. Conclusions: This schedule is feasible with high response rate (CR+PR=54%). Due to lack of new agents, innovative approaches for high risk patients can have a potential benefit. More patients are warranted.

Alkylation Sensitivity Screens Reveal a Conserved Cross-species Functionome

To identify genes that contribute to chemotherapy resistance in glioblastoma, we conducted a synthetic lethal screen in a chemotherapy-resistant glioblastoma-derived cell line with the clinical alkylator temozolomide (TMZ) and an siRNA library tailored toward "druggable" targets. Select DNA repair genes in the screen were validated independently, confirming the DNA glycosylases uracil-DNA glycosylase (UNG) and A/G-specific adenine DNA glycosylase (MYH) as well as methylpurine-DNA glycosylase (MPG) to be involved in the response to high dose TMZ. The involvement of UNG and MYH is likely the result of a TMZ-induced burst of reactive oxygen species. We then compared the human TMZ sensitizing genes identified in our screen with those previously identified from alkylator screens conducted in Escherichia coli and Saccharomyces cerevisiae. The conserved biologic processes across all three species compose an alkylation functionome that includes many novel proteins not previously thought to impact alkylator resistance. This high-throughput screen, validation and cross-species analysis was then followed by a mechanistic analysis of two essential nodes: base excision repair (BER) DNA glycosylases (UNG, human and mag1, S. cerevisiae) and protein modification systems, including UBE3B and ICMT in human cells or pby1, lip22, stp22 and aim22 in S. cerevisiae. The conserved processes of BER and protein modification were dual targeted and yielded additive sensitization to alkylators in S. cerevisiae. In contrast, dual targeting of BER and protein modification genes in human cells did not increase sensitivity, suggesting an epistatic relationship. Importantly, these studies provide potential new targets to overcome alkylating agent resistance.

Inhibition of STAT3 reverses alkylator resistance through modulation of the AKT and β-catenin signaling pathways

Activation of signal transducer and activator of transcription 3 (STAT3) is associated with poor clinical outcome of glioblastoma (GBM). However, the role of STAT3 in resistance to alkylator-based chemotherapy remains unknown. Here, we retrospectively analyzed the phosphorylated STAT3 (p-STAT3) profile of 68 GBM patients receiving alkylator therapy, identifying p-STAT3 as an independent unfavorable prognostic factor for progression-free and overall survival. Additionally, elevated p-STAT3 expression correlated with resistance to alkylator therapy. In vitro analysis revealed that U251 and U87 human glioma cells were refractory to treatment with the common alkylating agent temozolomide (TMZ), with only a modest impact on AKT and β-catenin activation in the context of high p-STAT3. Inhibition of STAT3 in these cells significantly enhanced the effect of TMZ. Inhibition of STAT3 dramatically decreased the IC50 of TMZ, increasing TMZ-induced apoptosis while up-regulating expression of Bcl-2 and down-regulating expression of Bax. Furthermore, inhibition of STAT3 increased TMZ-induced G₀-G₁ arrest and decreased Cyclin D1 expression compared to TMZ alone. Together, these results indicate that inhibition of STAT3 sensitizes glioma cells to TMZ, at least in part, by blocking the p-AKT and β-catenin pathways. These findings strongly support the hypothesis that STAT3 inhibition significantly improves the clinical efficacy of alkylating agents.

Mismatch repair deficiency is an uncommon mechanism of alkylator resistance in pediatric malignant gliomas: A report from the children's oncology group

Alkylating agents are commonly used in the treatment of childhood malignant gliomas. Overexpression of O(6)-methylguanine-DNA methyltransferase (MGMT) constitutes an important mechanism for resistance to such agents, and MGMT status has been associated with outcome in several recent trials. Deficiency in mismatch repair (MMR) function has been implicated in preclinical studies as an additional potential mechanism of resistance to methylating agents, such as temozolomide, independent of tumor MGMT status. However, the frequency of this abnormality as a clinical resistance mechanism in childhood malignant gliomas has not been well characterized. To address this issue, we examined the frequency of microsatellite instability (MSI), a marker of defective MMR, in a series of 68 tumors, derived from newly diagnosed patients treated on the Children's Cancer Group 945 study, and the Children's Oncology Group ACNS0126 and 0423 studies. MSI was assessed using a panel of six microsatellite markers, including BAT-25, BAT-26, CAT-25, D2S123, D5S346, and D17S250. MGMT immunoreactivity was assessed in parallel to allow comparison of the relative incidence of MGMT overexpression and MSI. Only three tumors had high-level MSI involving three or more markers; the remainder had no MSI at any of the loci examined. These children did not have unusual features in terms of their outcome. In contrast to the infrequency of MSI, 25 tumors (37%) exhibited MGMT overexpression as assessed by immunohistochemistry. None of the tumors with MSI exhibited overexpression of MGMT. MMR deficiency is an infrequent contributor to initial alkylator resistance in children with malignant gliomas.

Loss of the mismatch repair protein MSH6 in human glioblastomas is associated with tumor progression during temozolomide treatment.

Glioblastomas are treated by surgical resection followed by radiotherapy [X-ray therapy (XRT)] and the alkylating chemotherapeutic agent temozolomide. Recently, inactivating mutations in the mismatch repair gene MSH6 were identified in two glioblastomas recurrent post-temozolomide. Because mismatch repair pathway inactivation is a known mediator of alkylator resistance in vitro, these findings suggested that MSH6 inactivation was causally linked to these two recurrences. However, the extent of involvement of MSH6 in glioblastoma is unknown. We sought to determine the overall frequency and clinical relevance of MSH6 alterations in glioblastomas. The MSH6 gene was sequenced in 54 glioblastomas. MSH6 and O(6)-methylguanine methyltransferase (MGMT) immunohistochemistry was systematically scored in a panel of 46 clinically well-characterized glioblastomas, and the corresponding patient response to treatment evaluated. MSH6 mutation was not observed in any pretreatment glioblastoma (0 of 40), whereas 3 of 14 recurrent cases had somatic mutations (P = 0.015). MSH6 protein expression was detected in all pretreatment (17 of 17) cases examined but, notably, expression was lost in 7 of 17 (41%) recurrences from matched post-XRT + temozolomide cases (P = 0.016). Loss of MSH6 was not associated with O(6)-methylguanine methyltransferase status. Measurements of in vivo tumor growth using three-dimensional reconstructed magnetic resonance imaging showed that MSH6-negative glioblastomas had a markedly increased rate of growth while under temozolomide treatment (3.17 versus 0.04 cc/mo for MSH6-positive tumors; P = 0.020). Loss of MSH6 occurs in a subset of post-XRT + temozolomide glioblastoma recurrences and is associated with tumor progression during temozolomide treatment, mirroring the alkylator resistance conferred by MSH6 inactivation in vitro. MSH6 deficiency may therefore contribute to the emergence of recurrent glioblastomas during temozolomide treatment.

The glutathione system in alkylator resistance.

One of the greatest obstacles in the effective chemotherapy of neoplastic disease is the presence of tumor chemoresistance. In some instances, a tumor can be intrinsically resistant to chemotherapy or, in other cases, develop resistance during the course of antineoplastic treatment. This acquired resistance is thought to occur through the selection of a subpopulation of resistant tumor cells as the tumor is exposed to chemotherapy. To complicate chemotherapeutic drug selection, it has been found that once a tumor demonstrates resistance to one class of drugs, it will often be resistant to other classes that share structural or functional homology

Buthionine sulfoximine and myeloablative concentrations of melphalan overcome resistance in a melphalan-resistant neuroblastoma cell line.

Alkylator resistance contributes to treatment failure in high-risk neuroblastoma. Buthionine sulfoximine (BSO) can deplete glutathione and synergistically enhance in vitro sensitivity to the alkylating agent melphalan (L-PAM) for many neuroblastoma cell lines, but optimal use of this combination needs to be defined because clinical responses have been less frequent and not durable. The authors established and characterized a neuroblastoma cell line (CHLA-171) from a patient who died of progressive disease after treatment with BSO and low-dose L-PAM. CHLA-171 lacks MYCN amplification, expresses PGP (P-glycoprotein) 9.5 RNA, and shows cell surface antigen expression (human leukocyte antigen class I weakly positive, but HSAN 1.2 (hybridoma, SAN 1.2) and anti-GD2 (anti-ganglioside GD2 antibody) strongly positive) characteristic of neuroblastoma cell lines. Twenty-four hours of BSO treatment (0-1,000 micromol/L) maximally depleted CHLA-171 glutathione to 36% of baseline. The cytotoxic response of CHLA-171 to BSO and L-PAM, alone and in combination, was measured by digital image microscopy (DIMSCAN) over a range of drug concentrations and compared with drug levels obtained in the patient during BSO/L-PAM therapy. As single agents, CHLA-171 was highly resistant to L-PAM (LD90 = 42 micromol/L; peak plasma concentration in the patient equals 3.9 micromol/L) and moderately resistant to BSO (LD90 = 509 micromol/L; steady-state concentration in the patient equals 397 micromol/L). Treatment with a 10:1 (BSO:L-PAM) fixed ratio combination synergistically overcame resistance (3-4 logs of cell kill, combination index <1) at clinically achievable levels of BSO (100-400 micromol/L) and levels of L-PAM (10-40 micromol/L) clinically achievable only with hematopoietic stem cell support. The in vitro results obtained for CHLA-171 suggest that BSO/L-PAM therapy may be optimally effective for drug-resistant neuroblastoma using myeloablative doses of L-PAM.

Synergism of buthionine sulfoximine and melphalan against neuroblastoma cell lines derived after disease progression.

Despite intensive-alkylator based regimens, >50% of patients with high-risk neuroblastoma (NB) die from recurrent disease that is probably due, in part, to acquired alkylator resistance. Using buthionine sulfoximine (BSO)-mediated, glutathione (GSH) depletion to modulate melphalan (L-PAM) resistance, we examined six NB cell lines established after progressive disease following either standard chemotherapy, BSO/L-PAM therapy, or myeloablative therapy and autologous hematopoietic stem cell transplant (AHSCT). Four of the six cell lines (three p53-nonfunctional and one p53-functional) showed high-level L-PAM resistance. Fixed ratio analysis demonstrated BSO/L-PAM synergy (combination index >1) for all cell lines tested. In L-PAM-resistant cell lines, the minimal cytotoxicity observed for BSO combined with nonmyeloablative concentrations of L-PAM was markedly enhanced (>4 logs total cell kill) when BSO was combined with myeloablative concentrations of L-PAM. In alkylator-resistant NB, the optimal use of BSO may require dose escalation of L-PAM to levels requiring AHSCT.

Induction of Alkylator (Melphalan) Resistance in HL60 Cells Is Accompanied by Increased Levels of Topoisomerase II Expression and Function

Human leukemic HL60 cells were selected for resistance to alkylating agents by stepwise exposure to increasing concentrations of L-phenylalanine mustard (melphalan). The resulting resistant cell line (R-HL60) was 4-fold resistant (melphalan IC50 value, 27.84 +/- 4.2 microM) to melphalan compared with parental HL60 cells (melphalan IC50 value, 6.9 +/- 1.78 microM). Nuclear extracts from R-HL60 cells possess a approximately 4-fold increase in DNA topoisomerase II activity compared with parental HL60 cells. As determined using Western blot analysis, the level of topoisomerase IIalpha protein expressed in R-HL60 cells was approximately 3-fold that of parental HL60 cells. However, there were no differences observed in the level of topoisomerase IIbeta protein, in the topoisomerase I activity, or in the level of topoisomerase I protein expression comparing the two cell lines. R-HL60 cells were 5-fold more sensitive than parental HL60 cells to the cytotoxic effect of the topoisomerase II inhibitor doxorubicin. The sensitivity to the cytotoxic effects of the topoisomerase I inhibitor camptothecin did not differ in R-HL60 and parental HL60 cell lines. Preincubation with doxorubicin significantly increased melphalan-induced interstrand DNA cross-link formation and cytotoxicity in R-HL60 cells compared with the parental HL60 cells. The affinity of topoisomerase II for UV-irradiated cross-linked HL60 DNA was increased by approximately 2.5-fold compared with that of HL60 native DNA. The affinity of topoisomerase II for both UV-irradiated (cross-linked) and native DNA was significantly decreased after doxorubicin pretreatment. Elevated topoisomerase II activity and the increased affinity of topoisomerase II for cross-linked DNA in melphalan-resistant cells seems to contribute to alkylator resistance by changing DNA topology, thereby facilitating DNA repair.

Multiple DNA repair mechanisms and alkylator resistance in the human medulloblastoma cell line D-283 Med (4-HCR).

We have previously reported preferential repair of DNA interstrand crosslinks in the 4-hydroperoxycyclophosphamide-resistant human medulloblastoma cell line D-283 Med (4-HCR). We now report further studies that explored the potential mechanisms underlying this repair. Limiting dilution assays and Western, Southern, and Northern blots were used to compare specific differences between D-283 Med (4-HCR) and its parental line D-283 Med. D-283 Med (4-HCR) was cross-resistant to melphalan and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), with O6-alkylguanine-DNA alkyltransferase (AGT) levels of 466+/-164 fmol/mg protein; AGT levels in the parental line, D-283 Med, were 76+/-96 fmol/mg. The increase in AGT activity was not a result of gene amplification. Depleting AGT with O6-benzylguanine partially restored sensitivity to BCNU. Both cell lines were deficient in the human mismatch protein MutLalpha. ERCC4 mRNA and poly(ADP-ribose) polymerase levels were similar in both cell lines, and ERCC1 mRNA levels were 2- to 2.5-fold lower in D-283 Med (4-HCR). Topoisomerase I levels were 2- to 2.5-fold higher in D-283 Med compared with D-283 Med (4-HCR). These results, while illustrating the multiple differences between D-283 Med and D-283 Med (4-HCR), do not explain the enhanced DNA interstrand crosslink repair seen in D-283 Med (4-HCR).

Identification of the Yc1 glutathione S‐Transferase mRNA as the overexpressed species in a nitrogen mustard–resistant rat mammary carcinoma cell line

Glutathione transferases (GSTs) have been shown to be overexpressed in a number of tumor cell lines selected for resistance to chemotherapeutic drugs and have been implicated in some studies of clinical specimens. In tumor cell lines selected for resistance to chemicals that alkylate DNA, the isoform most frequently overexpressed is GST-Yc, a member of the α class GSTs. To date, two variations of the cDNA designated Yc1 with subtle differences have been described, and Yc2 is shown to be clearly distinct. Transfection of a Yc1 cDNA constitutively expressed in rat liver into rat mammary cancer cells confers resistance to alkylators, however, to a lesser extent than is observed in the cells selected for resistance. It has therefore been widely suggested that the GST that is overexpressed in selected resistant cells represents a distinct and novel isoform. We have previously described a rat mammary carcinoma cell line (MLNr) that is resistant to alkylating agents, and overexpresses a GST with characteristics similar to GST-Yc1 and not Yc2. It has many features common to the several other GST-Yc overexpressing alkylator resistant cell lines. We have cloned the specific Yc cDNA overexpressed in MLNr and analyzed it in detail and found that it is identical to one of the previously reported Yc1 cDNAs, suggesting that there is no additional Yc gene specifically induced by nitrogen mustards. Another hypothesis to explain the difference in the level of resistance in selected versus GST-Yc transfected cells is the lack of concurrent increased glutathione (GSH) in the transfectants, which is a common feature in the selected resistant cells. Experiments in which we modulated GSH levels suggest that this is not likely. These studies add to our speculation that other mechanisms may be involved in alkylator resistance. © 1997 John Wiley & Sons, Inc. J Biochem Toxicol 12: 11–17, 1998

Identification of the Yc1 glutathione S-transferase mRNA as the overexpressed species in a nitrogen mustard-resistant rat mammary carcinoma cell line.

Glutathione transferase (GSTs) have been shown to be overexpressed in a number of tumor cell lines selected for resistance to chemotherapeutic drugs and have been implicated in some studies of clinical specimens. In tumor cell lines selected for resistance to chemicals that alkylate DNA, the isoform most frequently overexpressed is GST-Yc, a member of the alpha class GSTs. To date, two variations of the cDNA designated Yc1 with subtle differences have been described, and Yc2 is shown to be clearly distinct. Transfection of a Yc1 cDNA constitutively expressed in rat liver into rat mammary cancer cells confers resistance to alkylators, however, to a lesser extent than is observed in the cells selected for resistance. It has therefore been widely suggested that the GST that is overexpressed in selected resistant cells represents a distinct and novel isoform. We have previously described a rat mammary carcinoma cell line (MLNr) that is resistant to alkylating agents, and overexpresses a GST with characteristics similar to GST-Yc1 and not Yc2. It has many features common to the several other GST-Yc overexpressing alkylator resistant cell lines. We have cloned the specific Yc cDNA overexpressed in MLNr and analyzed it in detail and found that it is identical to one of the previously reported Yc1 cDNAs, suggesting that there is no additional Yc gene specifically induced by nitrogen mustards. Another hypothesis to explain the difference in the level of resistance in selected versus GST-Yc transfected cells is the lack of concurrent increased glutathione (GSH) in the transfectants, which is a common feature in the selected resistant cells. Experiments in which we modulated GSH levels suggest that this is not likely. These studies add to our speculation that other mechanisms may be involved in alkylator resistance.

In vitro assessment of melphalan induced cytotoxicity and interstrand-DNA-cross-links in lymphocytes from patients with lymphoproliferative disorders

The relationship between in vitro cytotoxicity and melphalan induced interstrand-DNA-cross-linking was studied in lymphocytes from healthy persons and from patients with a variety of lymphoproliferative disorders. Interstrand-DNA-cross-link formation, as measured by ethidium bromide fluorescence assay showed a highly significant correlation with in vitro cytotoxicity in normal lymphocytes an in those from patients with lymphoproliferative disorders. However, the melphalan concentration at which such cross-linking occurred, differed significantly when normal lymphocytes and those in patients with lymphoproliferative disorders were compared. The kinetics of interstrand-DNA-cross-link formation and removal following treatment with melphalan also differed, with lymphocytes from patients with lymphoproliferative disorders, particularly those having had prior alkylating agent exposure, showing a more rapid rate of disappearance of cross-links as compared to normal lymphocytes. These findings suggest an increased rate of DNA repair occurring in lymphocytes from patients with lymphoproliferative disorders. In a clinical correlation study in vitro melphalan resistance correlated with in vivo resistance to treatment with alkylating agent based chemotherapy. These results suggest a rapid and simple method for determining alkylator resistance in patients with lymphoproliferative disorders.

Modulation of resistance to alkylating agents in cancer cell by gossypol enantiomers

Several cell lines resistant to alkylating agents possess increased activity of glutathione-S-transferase (GST) drug detoxifying enzymes. Inhibition of certain enzymes of the glutathione redox system may affect cellular sensitivity to alkylators. We report that the (-.)enantiomer of gossypol is a potent and selective inhibitor of GST alpha and GST pi isozymes, and that in combination with buthionine sulfoximine (BSO), causes the enhanced modulation of alkylator resistance in two drug resistant cell lines with increased GST activity. The use of (-)gossypol alone had no effect on the 2-5-fold resistance of MCF-7 Adr and Walker resistant cells to chlorambucil, melphalan and BCNU. Cellular depletion of glutathione with BSO resulted in a 2-4-fold modulation of cell sensitivity to these alkylators. However, the combination of (-)gossypol with BSO resulted in a markedly greater modulation of alkylator sensitivity than with either inhibitor alone. Therefore, the complementary inhibition of glutathione and GST by BSO and (-)gossypol, respectively, produced a synergistic modulation of alkylator cytotoxicity in these drug resistant cell lines. The favorable clinical pharmacokinetics of (-)gossypol suggest its further evaluation for use in combination with BSO and alkylating agents in clinical trials.