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PubMed2004

Nuclear-localizing O6-benzylguanine-resistant GFP-MGMT fusion protein as a novel in vivo selection marker.

OBJECTIVE: We characterized a novel in vivo selectable fusion protein, green fluorescence protein-O6-benzylguanine (BG)-resistant O6-methylguanine-methyltransferase (GFP-MGMT* [*refers to mutant MGMT]) used to delineate optimum selection regimens for transduced hematopoietic stem cells (HSC) ex vivo and in vivo. MATERIALS AND METHODS: We transduced human or mouse HSC with retrovirus vector encoding GFP-MGMT* where BG-resistant forms of human P140K-hMGMT* and mouse P144K-mMGMT* were studied. We evaluated selection of transduced HSC ex vivo and in vivo using either BG/1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or BG/temozolomide (TMZ) combinations, evaluating transduction marking by flow cytometry and real-time TaqMan PCR. RESULTS: GFP-MGMT* transduction confers nuclear-localized GFP fluorescence and BG resistance. Optimum selection ex vivo of GFP-MGMT*-transduced HSC occurred with BG (2.5-10 microM)/BCNU (5-10 microM) or TMZ (100-200 microM), which increases marking while preserving maximum viable transduced cells. Starting at low levels (0.1%) or high levels (>30%) of in vivo bone marrow gene making in mice, in vivo selection with BG/BCNU (20/6 mg/kg) (weeks 4 and 5) or BG/TMZ (20/60 mg/kg) (daily x 5 at week 4) increased bone marrow marking to 8.58% +/- 3.52% or 82.0% +/- 3.4% GFP+ cells, respectively, in the low- or high-level initial marking mice. CONCLUSIONS: GFP-MGMT* is an informative tool to explore optimization of in vivo selection regimens using BG/BCNU or BG/TMZ to increase gene marking of HSC. Both timing and dosing of selection regimens and the starting level of marking may all be important to the level of selective increase of in vivo marking achieved.

Animals↗

PubMed2004

Primary cultures of microglial cells for testing toxicity of anticancer drugs.

Toxicity of anticancer agents on normal neural cells during chemotherapy of primary or secondary brain tumors is a clinical problem of increasing relevance and concern. In this perspective, here we used primary cultures of rat cortical microglia as an in vitro paradigm of normal glia to investigate the neurotoxicity of anticancer agents. The effects of two compounds frequently used for treatment of brain tumors, methotrexate (MTX) and temozolomide (TMZ), were compared to those of a known microglial activator, bacterial lipopolysaccharide (LPS); cell viability and metabolism was assessed by the MTS assay. We found that LPS, in the low-intermediate range of concentrations, strongly activates microglia cells, but a highly significant decrease in viability was observed from 100 ng/ml onward. TMZ has no effect at concentrations of clinical interest, whereas MTX significantly increases cell metabolism at 30 microM, a phenomenon possibly reflecting MTX neurotoxicity observed in patients.

Animals↗

PubMed1999

Measurement of troponin T to detect cardioprotective effect of trimetazidine during coronary artery bypass grafting.

BACKGROUND: The aim of the present study was to evaluate potential myocardial protection by trimetazidine by measurement of the cardiac marker protein troponin T (TnT) during coronary bypass operations. METHODS: We conducted a double-blind, placebo-controlled study on 30 randomized patients who had aorta-coronary artery bypass operations. The TMZ group was composed of 15 patients and the placebo group of 15 patients in New York Heart Association class III or IV. Pretreatment was started 3 weeks preoperatively with trimetazidine (60 mg orally per day) or the placebo. In the trimetazidine TMZ group, there were 2 women and 13 men with a mean age of 57.1+/-2.2 years and mean cross-clamp time of 44+/-1.8 minutes. In the placebo group, there were 5 women and 10 men with a mean age of 58.4+/-1.2 years and a mean cross-clamp time of 42+/-2.4 minutes. Serial blood samples were collected before and after the operation, and serum concentrations of cardiac TnT were measured. RESULTS: The preoperative serum concentration of TnT was 0 to 0.39 ng/mL in all patients. The mean TnT levels were measured 5 minutes after completion of cardiopulmonary bypass (1.5+/-0.3 ng/mL) and 12 (1.4+/-0.1 ng/mL), 24 (0.9+/-0.1 ng/mL), and 48 hours postoperatively (0.1+/-0.1 ng/mL) in the trimetazidine group. Troponin T levels in the placebo group measured at the same time periods were 4.4+/-0.4, 4.8+/-0.7, 2.8+/-0.4, and 0.7+/-0.1 ng/mL. In the trimetazidine group, TnT levels were significantly less than those of the placebo group (p < 0.001). The levels of TnT were tested by creatine kinase-MB levels of both groups. Mean cardiac index was evaluated in all patients preoperatively and postoperatively. There was no significant difference in perioperative hemodynamics (blood pressure and cardiac index) between groups. CONCLUSIONS: These results obtained by measurement of cardiac TnT suggested that pretreatment with trimetazidine reduces ischemic-reperfusion damage during coronary bypass operations but did not affect postoperative hemodynamics.

Cardiopulmonary Bypass↗

PubMedDate not supplied

Assessing nighttime vigilance through a three-letter cancellation task (3-LCT): effects of daytime sleep with temazepam or placebo.

Nocturnal sleepiness is a common complaint suffered by night-shift workers, especially in conditions of an abrupt shift of the wake-sleep cycle. Alertness management strategies can minimize the adverse effects of sleep loss and circadian rhythm desynchronization and promote optimal vigilance in operational settings. Within these strategies. one possibility is to use short periods of "prophylactic sleep" (before long periods of work), which can be facilitated by hypnotics. Vigilance can be evaluated by means of several tests which, sometimes, imply procedures and devices not easily employable in operational settings. In such conditions pencil and paper tests of vigilance can be very useful in the assessment of attentional performance degradation due to sleep loss and/or inversion of the sleep-wake cycle. In this study we evaluated the sensitivity of a three-Letter Cancellation Task (3-LCT) in revealing nighttime variations of vigilance in a laboratory simulation of acute night shift, after a diurnal sleep with placebo (PLC) or temazepam (TMZ). Nocturnal levels of vigilance were also assessed using the Maintenance of Wakefulness Test (MWT) and the Multiple Sleep Latency Test (MSLT). All tests were administered four times at 2-h intervals during nighttime after a daytime sleep. Results show that the 3-LCT is sensitive to variations of vigilance occurring during a laboratory simulation of acute night shift. We also found some effects of TMZ, which in the first nocturnal session caused a slowing down of visuoattentive performance. Nocturnal variations of vigilance detected through the 3-LCT were similar to those revealed by means of MSLT, while the ability to maintain wakefulness was substantially spared during the night.

Adult↗

PubMed2003

Effects of trimetazidine on submaximal exercise test in patients with acute myocardial infarction.

BACKGROUND: It was demonstrated that the novel metabolic agent, trimetazidine, could lessen the incidence and severity of angina, whether used in monotherapy or combination. Although the animal studies demonstrated that trimetazidine reduces myocardial infarct size anf improves recovery of mechanic function after ischemia, little is known on the potential benefits of trimetazidine in patients with myocardial infarction (AMI). The aim of this study was to evaluate the efficacy of trimetazidine on AMI by sub-maximal exercise test. METHODS: A double-blind crossover trimetazidine versus placebo trial was carried out in 44 patients with AMI. Patients with randomly allotted into trimetazidine (23 patients) or placebo (21 patients) for 5 days and underwent an initial sub-maximal exercise test. Exercise tests according to the modified Bruce protocol were performed. Exercise end points included completion of stage II or 75% of maximum predicted heart rate whichever came first. An average 12-lead ECG was obtained at rest, every minute during exercise, at the onset of anginal symptoms, at the onset of 1-mm ST segment depression, at peak exercise and every 2-minute during recovery. After the initial exercise tests, study groups resumed the drugs in the opposite order for 4 to 5 days and underwent a second sub-maximal exercise test. RESULTS: Exercise induced ST segment depression was noted in 17 patients (38.6%) receiving placebo. However, exercise induced ST-segment depression was observed in 8 patients (18.1%) taking TMZ. Positive exercise test results were significantly higher on placebo group than TMZ group (p=0.018). Additionally, trimetazidine prolonged the time to 1-mm ST-segment depression (6.1 +/-0.5 vs 4.9 +/-0.4, P< 0.031) and exercise duration (7.2+/-0.9 vs 5.8 +/-0.9, p<0.025). CONCLUSION: Trimetazidine therapy improves the exercise capacity and reduces evidence ischemia derived from sub-maximal post-infarction exercise testing.

Cross-Over Studies↗

PubMed1999

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.

Antineoplastic Agents↗

PubMed1999

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.

Antigens, CD↗

PubMed2006

Irradiation and hypoxia promote homing of haematopoietic progenitor cells towards gliomas by TGF-beta-dependent HIF-1alpha-mediated induction of CXCL12.

Previously we defined a pathway of transforming growth factor beta (TGF-beta) and stromal cell-derived factor-1/CXC chemokine ligand 12 (SDF-1alpha/CXCL12) dependent migration of adult haematopoietic stem and progenitor cells (HPC) towards glioma cells in vitro and their homing to experimental gliomas in vivo. Hypoxia is a critical aspect of the microenvironment of gliomas and irradiation is an essential part of the standard therapy. To evaluate the therapeutic potential of HPC as vectors for a cell-based therapy of gliomas, we investigated the impact of hypoxia and irradiation on the attraction of HPC by glioma cells. Temozolomide (TMZ) treatment and hyperthermia served as controls. Supernatants of irradiated or hypoxic LNT-229 glioma cells promote HPC migration in vitro. Reporter assays reveal that the CXCL12 promoter activity is enhanced in LNT-229 cells at 24 h after irradiation at 8 Gy or after exposure to 1% oxygen for 12 h. The irradiation- and hypoxia-induced release of CXCL12 depends on hypoxia inducible factor-1 alpha (HIF-1alpha), but not on p53. Induction of transcriptional activity of HIF-1alpha by hypoxia or irradiation requires an intact TGF-beta signalling cascade. This delineates a novel stress signalling cascade in glioma cells involving TGF-beta, HIF-1alpha and CXCL12. Stress stimuli can be irradiation, hypoxia or TMZ, but not hyperthermia. Cerebral irradiation of nude mice at 21 days after intracerebral implantation of LNT-229 glioma induces tumour satellite formation and enhances the glioma tropism of HPC to the tumour bulk and even to these satellites in vivo. These data suggest that the use of HPC as cellular vectors in the treatment of glioblastoma may well be combined with irradiation or other anti-angiogenic therapies that induce tumour hypoxia.

Animals↗

PubMed2026

Mutational signature stratification of recurrent gliomas reveals distinct patterns of genomic traits.

BACKGROUND: Although temozolomide (TMZ) is widely used for glioma treatment, its therapeutic benefit is limited by acquired resistance and recurrence, facilitated by intratumor heterogeneity. Mutational signatures (MSs) inform tumor evolution and reveal alterations associated with treatment response. METHODS: We performed molecular analyses of 96 glioma recurrences with sufficient private single-nucleotide variants relative to their matched primary tumors, stratified by their dominant MS. RESULTS: Four groups were identified: MS11/TMZ-related (n = 38), MS1/5/aging-related (n = 32), MS6/15/21/26/microsatellite instability (MSI)-related (n = 13), and other MS-related recurrences (n = 13). MS11/TMZ-related recurrences showed higher acquired mutational counts than the other groups (1338 vs 59 (MS1/5/aging) vs 57 (MS6/15/21/26/MSI) vs 57 (other MSs); P < .01). Mutations in SYNE2, SZT2, and FBN3 were restricted to recurrences with dominant or second-dominant MS11/TMZ-related signature (n = 41), and 85% (35/41) harbored mutations in these genes. In MS11/TMZ-related recurrences with RNA sequencing data (n = 17), mRNA co-expression analyses identified SYNE2-ATAD5 and SZT2-MAPKBP1 associations. Among MS11/TMZ-related recurrences, MS23 was frequent (44%, 18/41) and associated with higher acquired mutational counts (2089 vs 1188; P = .018) and more IDH-wildtype tumors (67% vs 30%; P = .037). MAPKBP1 mutations were enriched in MS23-positive recurrences (56% (10/18) vs 0% (0/23); P < .001). MS1/5/aging-related recurrences showed more frequent acquired chromosome 16q losses (22% vs 8% (TMZ) vs 0% (MSI) vs 0% (other); P < .05), which were associated with an increased fraction of genome altered relative to 16q-diploid cases (15% vs 7%; P = .01). CONCLUSIONS: These findings show that MS-based stratification of recurrences refines molecular characterization after therapy and nominates candidate biomarkers and pathways for functional studies of treatment-associated glioma evolution.

mutational signatures↗

PubMed2004

Protective roles of polyethylene glycol and trimetazidine against cold ischemia and reperfusion injuries of pig kidney graft.

Ischemia-reperfusion injury (IRI) represents an allo-independent risk factor which favors chronic allograft nephropathy (CAN). Here we analyzed the influence of preservation solutions on the function of autotransplanted pig kidneys over 1-16 weeks after surgery. Kidneys were cold-flushed and cold-stored for 24 or 48 h either in University of Wisconsin (UW), modified-UW Hôpital Edouard Herriot, polyethylene glycol 20 kDa (PEG)-supplemented preservation solutions with low K+ (ECPEG) or high K+ (ICPEG) content. Animals autotransplanted with kidneys cold-stored for 24 h in ECPEG exhibited the greatest levels of creatinine clearance (Ccr: 161 +/- 12 mL/min, n=10) and the lowest levels of proteinuria (0.5 +/- 0.03 mg/mL) 16 weeks after surgery as compared with pigs autotransplanted with kidneys cold-stored in the other solutions tested (Ccr ranging from 80 and 140 mL/min). Similar differences, but with lower Ccr levels, were achieved after a prolonged period of cold-storage(48 h). ECPEG better preserved the kidneys from monocytes/macrophages and CD4+ T cells infiltrations, VCAM-1 and MHC class II overexpressions and occurrence of renal interstitial fibrosis (2%) as compared with the other preservation solutions (5%-20%). Adding the anti-ischemic drug trimetazidine (TMZ) to the preservation solutions, particularly ECPEG, further improved the quality of the week-16 post-transplanted kidneys (Ccr: 182 +/- 12 mL/min, n=10). These findings demonstrated that adding PEG to extracellular-like (with low K+ content) preservation solutions in combination with TMZ significantly improved the long-term outcome of kidney grafts in this model of autotransplanted pig kidney.

Animals↗

PubMed2003

The p38 mitogen-activated protein kinase pathway links the DNA mismatch repair system to the G2 checkpoint and to resistance to chemotherapeutic DNA-methylating agents.

Although human cells exposed to DNA-methylating agents undergo mismatch repair (MMR)-dependent G(2) arrest, the basis for the linkage between MMR and the G(2) checkpoint is unclear. We noted that mitogen-activated protein kinase p38alpha was activated in MMR-proficient human glioma cells exposed to the chemotherapeutic methylating agent temozolomide (TMZ) but not in paired cells made MMR deficient by expression of a short inhibitory RNA (siRNA) targeted to the MMR protein Mlh1. Furthermore, activation of p38alpha in MMR-proficient cells was associated with nuclear inactivation of the cell cycle regulator Cdc25C phosphatase and its downstream target Cdc2 and with activation of the G(2) checkpoint, actions which were suppressed by the p38alpha/beta inhibitors SB203580 and SB202590 or by expression of a p38alpha siRNA. Finally, pharmacologic or genetic inhibition of p38alpha increased the sensitivity of MMR-proficient cells to the cytotoxic actions of TMZ by increasing the percentage of cells that underwent mitotic catastrophe as a consequence of G(2) checkpoint bypass. These results suggest that p38alpha links DNA MMR to the G(2) checkpoint and to resistance to chemotherapeutic DNA-methylating agents. The p38 pathway may therefore represent a new target for the development of agents to sensitize tumor cells to chemotherapeutic methylating agents.

Antineoplastic Agents, Alkylating↗

PubMed2025

Infectious bursal disease virus (IBDV) as a novel oncolytic virotherapy in glioblastoma.

BACKGROUND: Glioblastoma (GBM) is the most aggressive form of cancer of the central nervous system. Despite advances in immunotherapies and standard-of-care treatments for GBMs, clinical outcomes remain limited-owing to the immunosuppressive tumor microenvironment and the intrinsic resistance of GBM to conventional approaches. As a result, there is growing interest in rational combination strategies, particularly those pairing oncolytic viruses with immune-based therapies or established treatment modalities. Oncolytic viruses, by displaying conditionally enabled tumor cell-restricted replication, while stimulating antitumor immune responses and leaving healthy tissue unharmed, have the potential to reshape the therapeutic landscape in GBM and aid in achieving more durable benefits for patients. This study investigates the use of infectious bursal disease virus (IBDV) as a potential virotherapy for GBM. METHODS AND RESULTS: In vitro, IBDV infects and replicates within murine GBM cells and patient-derived GBM stem cells, inducing direct oncolysis and activating proinflammatory gene expression programs. IBDV also enhances the cytolytic activity of temozolomide (TMZ) in treated GBM cells, complementing TMZ chemotherapeutic activity. In vivo, treatment with IBDV in CT-2A GBM-bearing syngeneic mice significantly reduced tumor growth and improved survival compared with control mice. Intratumoral administration of IBDV induces a deep remodeling of the tumor immune microenvironment, reducing immunosuppressive M2-like macrophages and increasing the ratio of CD8+T cells to regulatory T cells. This reversion of immunosuppression linked to monocyte-derived macrophages has been confirmed on experimental ex vivo infections of explants derived from human GBM donors. CONCLUSION: These findings support further consideration of IBDV as a novel virotherapeutic agent for GBM.

Oncolytic Virotherapy↗

PubMed2005

Mitochondrial targeting of human O6-methylguanine DNA methyltransferase protects against cell killing by chemotherapeutic alkylating agents.

DNA repair capacity of eukaryotic cells has been studied extensively in recent years. Mammalian cells have been engineered to overexpress recombinant nuclear DNA repair proteins from ectopic genes to assess the impact of increased DNA repair capacity on genome stability. This approach has been used in this study to specifically target O(6)-methylguanine DNA methyltransferase (MGMT) to the mitochondria and examine its impact on cell survival after exposure to DNA alkylating agents. Survival of human hematopoietic cell lines and primary hematopoietic CD34(+) committed progenitor cells was monitored because the baseline repair capacity for alkylation-induced DNA damage is typically low due to insufficient expression of MGMT. Increased DNA repair capacity was observed when K562 cells were transfected with nuclear-targeted MGMT (nucl-MGMT) or mitochondrial-targeted MGMT (mito-MGMT). Furthermore, overexpression of mito-MGMT provided greater resistance to cell killing by 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) than overexpression of nucl-MGMT. Simultaneous overexpression of mito-MGMT and nucl-MGMT did not enhance the resistance provided by mito-MGMT alone. Overexpression of either mito-MGMT or nucl-MGMT also conferred a similar level of resistance to methyl methanesulfonate (MMS) and temozolomide (TMZ) but simultaneous overexpression in both cellular compartments was neither additive nor synergistic. When human CD34(+) cells were infected with oncoretroviral vectors that targeted O(6)-benzylguanine (6BG)-resistant MGMT (MGMT(P140K)) to the nucleus or the mitochondria, committed progenitors derived from infected cells were resistant to 6BG/BCNU or 6BG/TMZ. These studies indicate that mitochondrial or nuclear targeting of MGMT protects hematopoietic cells against cell killing by BCNU, TMZ, and MMS, which is consistent with the possibility that mitochondrial DNA damage and nuclear DNA damage contribute equally to alkylating agent-induced cell killing during chemotherapy.

Antigens, CD34↗

PubMed2006

Phase II study of temozolomide in relapsed or refractory high-risk neuroblastoma: a joint Société Française des Cancers de l'Enfant and United Kingdom Children Cancer Study Group-New Agents Group Study.

PURPOSE: To determine the response rate (RR) of neuroblastoma (NB) in children to temozolomide (TMZ), and evaluate the duration of response and tolerance of the drug in this patient population. PATIENTS AND METHODS: A multicenter, phase II evaluation of an oral, daily schedule of TMZ (200 mg/m2/d x 5 days every 28 days) was undertaken in children with refractory or relapsed high-risk NB (metastatic or localized with Myc-N amplification). Response assessment was based on imaging with two-dimentional measurement of disease and meta-iodobenzylguanidine (MIBG) score. Activity was defined by a reduction in lesion size or isotope uptake at anytime. Methodology included a two-step design using Fleming's method with a first step of 15 patients and a second of 10 additional patients if two to four responses had been observed in the first cohort. All data was centrally reviewed by a panel. RESULTS: Twenty-five assessable patients were recruited over a 14-month period in 14 centers and received 94 cycles of chemotherapy. Twenty-three patients had metastatic NB either refractory (n = 9) or in relapse (n = 14). Grade 3 or 4 thrombocytopenia was the most frequent toxicity (16% of cycles). Myelosuppression resulted in treatment delays and dose reductions (24% and 21% of cycles, respectively). Response (complete response, very good partial response, or partial response) was observed in five patients (RR = 20% +/- 8%) with a median duration of 6 months and an objective or mixed response in five additional patients. CONCLUSION: Temozolomide shows activity in heavily pretreated patients with NB, and deserves further evaluation in combination with another drug.

Adolescent↗

PubMed2002

Chemotherapy-induced O(6)-benzylguanine-resistant alkyltransferase mutations in mismatch-deficient colon cancer.

The ability of O(6)-benzylguanine (BG) to inactivate alkyltransferase (AGT) to potentiate the antitumor efficacy of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) is being tested in clinical trials. As of now, there are no examples of acquired resistance to BG+BCNU in the clinical setting. However, we hypothesized that genetically unstable tumors might develop resistance to the combination after repeated drug-exposures to achieve therapeutic efficacy. To evaluate this possibility, we treated three colon cancer cell lines that are either proficient in mismatch repair (MMR) [SW480 (MMR wild type)] or deficient in MMR [HCT116 (hMLH1 mutant) and HCT15 (hMSH6 mutant)] with three cycles of BG+BCNU. After drug-treatments, HCT116 and HCT15 were completely resistant to BG-potentiated cytotoxicity of BCNU. In these two cell lines, the acquired BG resistance resulted from two de novo and different mutations at amino acid 165 in AGT: 165-lysine (K) to glutamic acid (E) (K165E in HCT116), and 165-lysine to asparagine (N) (K165N in HCT15). Both K165-mutated AGTs had markedly decreased enzymatic activity because of unstable AGT protein but were remarkably resistant to BG inactivation. FISH analysis showed that only one copy of MGMT gene exists in HCT116 cells, and the status of promoter methylation of MGMT in HCT15 showed that one allele of the MGMT promoter has an aberrant methylation. Thus, the MGMT gene expressing AGT either from one copy (HCT116) or from unmethylated allele (HCT15) was mutated because of the exposure to BG+BCNU in these two MMR-deficient cell lines. Conversely, MMR-proficient SW480 cells, treated with three cycles of BG+BCNU, maintained wt AGT and the sensitivity to BG-potentiated BCNU-cytotoxicity. To confirm that K165-mutated AGT proteins were responsible for resistance to BG+BCNU, we transfected K165E and K165N MGMT cDNAs into Chinese hampster ovary (CHO) cells. Transfected CHO cells had low AGT activity but increased IC(50) for either BCNU or temozolomide (TMZ), compared with parental CHO cells. BG did not potentiate the cytotoxicity of these two alkylating agents at concentrations up to 200 microM; in contrast, BG, at 25 microM, sensitized CHO-AGT (transfected with wt MGMT cDNA) cells to BCNU or TMZ-cytotoxicity by 3-4 fold. These results suggest that K165 AGT mutants arising in MMR-deficient tumor cells after treatment with chemotherapeutic agents are both resistant to BG-inactivation and are active in the repair of alkylated DNA adducts.

Adaptor Proteins, Signal Transducing↗

PubMed2003

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.

Animals↗

PubMed2004

Combination of caspase transfer using the human telomerase reverse transcriptase promoter and conventional therapies for malignant glioma cells.

Recently, we have reported the therapeutic efficacy of delivering initiator caspase (caspase-8) or executioner active caspase (rev-caspase-6) to telomerase-positive malignant glioma cells using the human telomerase reverse transcriptase (hTERT) gene promoter system (hTERT/caspase-8 or hTERT/rev-caspase-6). In the present study, we investigated if conventional treatments for malignant gliomas augment the efficacy of the hTERT/caspase therapy. First, we demonstrated that hTERT/rev-caspase-6 exhibited a greater ability to induce apoptosis in malignant glioma U87-MG and U373-MG cells than hTERT/caspase-8. Next, as conventional treatments to combine with hTERT/rev-caspase-6, apoptosis-inducing agents [cisplatin (CDDP), paclitaxel (PTX), and BCNU] and non-apoptosis-inducing therapies [temozolomide (TMZ) and gamma-irradiation (IR)] were used. Combination of hTERT/rev-caspase-6 gene therapy with PTX yielded a dose-dependent additive effect, while CDDP and BCNU had additive effect only when tumor cells were treated at IC75 of each agent. A decline in the combination effect of CDDP and BCNU at IC50 was due to decreased activity of telomerase in treated tumor cells prior to the hTERT/rev-caspase-6 transfer. On the other hand, TMZ or IR had no significant additive effect on induction of apoptosis. These results suggest that agents, which induce apoptosis without inhibiting telomerase activity are a promising counterpart to combine with hTERT/rev-caspase-6 therapy for the management of malignant gliomas.

Antineoplastic Agents↗

PubMed2005

Generation of an immortalized human endothelial cell line as a model of neovascular proliferating endothelial cells to assess chemosensitivity to anticancer drugs.

Assessment of chemosensitivity of neovessel endo-thelium associated to tumor mass is hindered by the limited availability of experimental models of actively proliferating endothelial cells. In fact, primary endothelial cells possess a limited lifespan and replicative senescence represents a major limit to their long-term culture. Moreover, non-dividing senescent cells undergo a gradual loss of phenotypic markers and become unable to respond to mitogenic stimuli. We report the generation of an immortalized human endothelial cell line by transfection of human umbilical vein endothelial cells (HUVEC) with both SV40 large/small T antigens and the catalytic subunit of human telomerase. This cell line (HUV-ST) possesses stabilized telomere length and increased proliferation rate with respect to parental cells or to cells transfected with SV40 T antigens only (HUV-S). Nevertheless, even at PD > 100 it is not tumorigenic and displays all major endothelial phenotypic markers, such as von Willebrand factor, CD31, vascular endothelial growth factor (VEGF) receptors (VEGFR1/Flt-1, VEGR2/KDR) and CD105/endoglin. HUV-ST cells are capable of organizing into tubule-like networks with branching morphology in response to appropriate stimuli and migrate upon exposure to VEGF. Interestingly, HUV-ST cells over-express the tumor endothelial marker-1/endosialin which is regarded as the most differentially expressed molecule in tumor-derived endothelium versus normal-derived endothelium. Analysis of chemosensitivity to the wide spectrum methylating agent temozolomide (TMZ), an anticancer drug more effective against actively dividing cells than against resting or slowing proliferating cells, indicated that HUV-ST cells are more susceptible to the drug with respect to HUVEC or HUV-S cells. Abrogation of poly(ADP-ribose) polymerase activity significantly enhances growth inhibition induced by TMZ. In conclusion, the immortalized human endothelial line HUV-ST represents a suitable model for studying the efficacy of anti-neovascular therapy, mimicking proliferating neovascular endothelial cells associated to the tumor mass.

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