Nuclear receptor PPARS function in stem cell differentiation and bone physiology

In adult, bone remodeling is a permanent process, reaching an annual turnover of about 10% of the skeleton. Bone remodeling requires the sequential and coordinated actions of the hematopoietic origin osteoclasts, to remove bone and the mesenchymal origin osteoblasts to replace it. An increased level of bone resorption is the primary cause of age-related bone loss often resulting in osteopenia, and is the major cause of osteoporosis.¦Peroxisome proliferator-activated receptors (PPARs), which are expressed in three isotypes, PPARa, PPARp and PPARy, are ligand-activated transcription factors that control many cellular and metabolic processes, more particularly linked to lipid metabolism. In bone, previous works has shown that PPARy inhibits osteogenesis by favoring adipogenesis from common mesenchymal progenitors. In addition, the pro-osteoclastogenesis activity of PPARy results in an increased bone resorption. Accordingly, treatment with PPARy agonist such as the anti-diabetic drug TZD causes bone loss and accumulation of marrow adiposity in mice as well as in postmenopausal women. The aim of the present thesis work was to elucidate the PPARs functions in bone physiology.¦The initial characterization of the PPARP" bone phenotype mainly revealed a decreased BMD. In vitro studies exploring the potency of mesenchymal stem cells to differentiate in osteoblast showed no differences depending on the genotype. However, we could demonstrate an effect of PPARp in partially inhibiting osteoclastogenesis. These results are further sustained by a study made in collaboration with the group of Dr Kronke, which showed an impressive protection against ovariectomy-generated bone loss when the females are treated with a PPARp agonist.¦Observations in PPARy null mice are more complex. The lab has recently been able to generate mice carrying a total deletion of PPARy. Intriguingly, the exploration of the bone phenotype of these mice revealed paradoxical findings. Whereas short bones such as vertebrae exhibit an elevated BMD as expected, long bones (tibia and femur) are clearly osteoporotic. According to their activity when set in culture, osteoblast differentiation normally occurs. Indeed the phenotype can be mainly attributed to a high density of osteoclasts in the cortical bone of PPARy null mice, associated to large bone resorption areas.¦Our explorations suggest a mechanism that involves regulatory processes linking osteoclastogenesis to adipogenesis, the latter being totally absent in PPARy null mice. Indeed, the lack of adipose tissue creates a favorable niche for osteoclastogenesis since conditioned medium made from differentiated adipocyte 3T3L1 inhibited osteoclastogenesis from both PPARy-/- and WT cells. Thus, adipokines deficiency in PPARy-/- mice contributes to de- repress osteoclastogenesis. Using specific blocking antibody, we further identified adiponectin as the major player among dozens of adipokines. Using flow cytometry assay, we explored the levels at which the osteoclastic commitment was perturbed in the bone marrow of PPARy-/- mice. Intriguingly, we observe a general decrease for hematopoietic stem cell and lineage progenitors but increased proportion of osteoclast progenitor in PPARy-/- bone marrow. The general decrease of HSC in the bone marrow is however largely compensated by an important extra-medullary hematopoeisis, taking place in the liver and in the spleen.¦These specific characteristics emphasize the key role of PPARy on a cross road of osteogenesis, adipogenesis and hematopoiesis/osteoclastogenesis. They underline the complexity of the bone marrow niche, and demonstrate the inter-dependance of different cell types in defining bone homeostasis, that may be overseen when experimental design single out pure cell populations.¦Chez l'adulte, même après la fin de la croissance, le renouvellement des os se poursuit et porte sur environ 10% de l'ensemble du squelette adulte, par année. Ce renouvellement implique à la fois des mécanismes séquentiels et coordonnés des ostéoclastes d'origine hématopoïetique, qui dégradent l'os, et des ostéoblastes d'origine mésenchymale, qui permettent la régénération de l'os. La perte en densité osseuse due à l'âge entraîne un fort niveau de résorption, conduisant souvent à une ostéopénie, elle-même cause de l'ostéoporose.¦Les trois isotypes PPAR (Peroxisome proliferator-activated receptor, PPARa, PPARp, et PPARy) sont des récepteurs nucléaires qui contrôlent de nombreux mécanismes cellulaires et métaboliques, plus particulièrement liés au métabolisme lipidique. Au niveau osseux, des travaux précédents ont montré que PPARy inhibe l'ostéoblastogenèse en favorisant la formation d'adipocytes à partir de la cellule progénitrice commune. De plus, l'activité pro- ostéoclastogénique de PPARy induit une résorption osseuse accrue. Condormément à ces observations, les patients diabétiques traités par les thiazolidinediones qui agissent sur PPARy, ont un risque accrue d'ostéoporose liée à une perte osseuse accrue et un accroissement de l'adiposité au niveau de la moelle osseuse. Dans ce contexte, l'objectif de mon travail de thèse a été d'élucider le rôle des PPAR dans la physiologie osseuse, en s'appuyant sur le phénotype des souris porteuses de mutation pour PPAR.¦La caractérisation initiale des os des souris porteuses d'une délétion de ΡΡΑΕφ a principalement révélé une diminution de la densité minérale osseuse (DMO). Alors que l'ostéogenèse n'est pas significativement altérée chez ces souris, l'ostéoclastogenèse est elle augmentée, suggérant un rôle modérateur de ce processus par ΡΡΑΕΙβ. Ces résultats sont par ailleurs soutenus par une étude menée par le groupe du Dr Krônke en collaboration avec notre groupe, et qui monte une protection très importante des souris traitées par un activateur de PPARP contre l'ostéoporose provoquée par l'ovariectomie.¦Les observations concernant PPARy donnent des résultats plus complexes. Le laboratoire a en effet été capable récemment de générer des souris portant une délétion totale de PPARy. Alors que les os courts chez ces souris présentent une augmentation de la DMO, comme attendu, les os longs sont clairement ostéoporotiques. Ce phénotype corrèle avec une densité élevée d'ostéoclastes dans l'os cortical de ces os longs. Deux processus semblent contribuer à ce phénotype. En premier lieu, nous démontrons qu'un milieu conditionné provenant de cultures de cellules 3T3-L1 différenciées en adipocytes contiennent une forte activité inhibitrice d'osteoclastogenesis. L'utilisation d'anticorps neutralisant permet d'identifier l'adiponectine comme l'un des facteurs principaux de cette inhibition. Les souris PPARy étant totalement dépourvues d'adipocytes et donc de tissu adipeux, la sécrétion locale d'adiponectine dans la moelle osseuse est donc également absente, entraînant une désinhibition de l'ostéoclastogenèse. En second lieu, des analyses par FACS révèle une proportion accrue des cellules progénitrices d'ostéoclastes dans la moelle osseuse. Cela s'accompagne par une diminution globale des cellules souches hématopoïétiques, qui est cependant largement compensée par une importante hématopoëise extra-médullaire, dans le foie comme dans la rate.¦L'ensemble de notre travail montre toute l'importance de PPARy au carrefour de l'ostéogenèse, adipogenèse, et hématopoëise/osteoclastogenèse. Il souligne la complexité de la niche que représente la moelle osseuse et démontre l'inter-dépendance des différents types cellulaires définissant l'homéostasie osseuse, complexité qui peut facilement être masqué lorsque le travail expérimental se concentre sur le comportement d'un type cellulaire donné.

A comprehensive analysis of human gene expression profiles identifies stromal immunoglobulin κ C as a compatible prognostic marker in human solid tumors.

PURPOSE: Although the central role of the immune system for tumor prognosis is generally accepted, a single robust marker is not yet available. EXPERIMENTAL DESIGN: On the basis of receiver operating characteristic analyses, robust markers were identified from a 60-gene B cell-derived metagene and analyzed in gene expression profiles of 1,810 breast cancer; 1,056 non-small cell lung carcinoma (NSCLC); 513 colorectal; and 426 ovarian cancer patients. Protein and RNA levels were examined in paraffin-embedded tissue of 330 breast cancer patients. The cell types were identified with immunohistochemical costaining and confocal fluorescence microscopy. RESULTS: We identified immunoglobulin κ C (IGKC) which as a single marker is similarly predictive and prognostic as the entire B-cell metagene. IGKC was consistently associated with metastasis-free survival across different molecular subtypes in node-negative breast cancer (n = 965) and predicted response to anthracycline-based neoadjuvant chemotherapy (n = 845; P < 0.001). In addition, IGKC gene expression was prognostic in NSCLC and colorectal cancer. No association was observed in ovarian cancer. IGKC protein expression was significantly associated with survival in paraffin-embedded tissues of 330 breast cancer patients. Tumor-infiltrating plasma cells were identified as the source of IGKC expression. CONCLUSION: Our findings provide IGKC as a novel diagnostic marker for risk stratification in human cancer and support concepts to exploit the humoral immune response for anticancer therapy. It could be validated in several independent cohorts and carried out similarly well in RNA from fresh frozen as well as from paraffin tissue and on protein level by immunostaining.

A phase I pharmacokinetic study of hypoxic abdominal stop-flow perfusion with gemcitabine in patients with advanced pancreatic cancer and refractory malignant ascites

PURPOSE: As no curative treatment for advanced pancreatic and biliary cancer with malignant ascites exists, new modalities possibly improving the response to available chemotherapies must be explored. This phase I study assesses the feasibility, tolerability and pharmacokinetics of a regional treatment of gemcitabine administered in escalating doses by the stop-flow approach to patients with advanced abdominal malignancies (adenocarcinoma of the pancreas, n = 8, and cholangiocarcinoma of the liver, n = 1). EXPERIMENTAL DESIGN: Gemcitabine at 500, 750 and 1,125 mg/m(2) was administered to three patients at each dose level by loco-regional chemotherapy, using hypoxic abdominal stop-flow perfusion. This was achieved by an aorto-caval occlusion by balloon catheters connected to an extracorporeal circuit. Gemcitabine and its main metabolite 2',2'-difluorodeoxyuridine (dFdU) concentrations were measured by high performance liquid chromatography with UV detection in the extracorporeal circuit during the 20 min of stop-flow perfusion, and in peripheral plasma for 420 min. Blood gases were monitored during the stop-flow perfusion and hypoxia was considered stringent if two of the following endpoints were met: pH </= 7.2, pO(2) nadir ratio </=0.70 or pCO(2) peak ratio >/=1.35. The tolerability of this procedure was also assessed. RESULTS: Stringent hypoxia was achieved in four patients. Very high levels of gemcitabine were rapidly reached in the extracorporeal circuit during the 20 min of stop-flow perfusion, with C (max) levels in the abdominal circuit of 246 (+/-37%), 2,039 (+/-77%) and 4,780 (+/-7.3%) mug/ml for the three dose levels 500, 750 and 1,125 mg/m(2), respectively. These C (max) were between 13 (+/-51%) and 290 (+/-12%) times higher than those measured in the peripheral plasma. Similarly, the abdominal exposure to gemcitabine, calculated as AUC(t0-20), was between 5.5 (+/-43%) and 200 (+/-66%)-fold higher than the systemic exposure. Loco-regional exposure to gemcitabine was statistically higher in presence of stringent hypoxia (P < 0.01 for C (max) and AUC(t0-20), both normalised to the gemcitabine dose). Toxicities were acceptable considering the complexity of the procedure and were mostly hepatic; it was not possible to differentiate the respective contributions of systemic and regional exposures. A significant correlation (P < 0.05) was found between systemic C (max) of gemcitabine and the nadir of both leucocytes and neutrophils. CONCLUSIONS: Regional exposure to gemcitabine-the current standard drug for advanced adenocarcinoma of the pancreas-can be markedly enhanced using an optimised hypoxic stop-flow perfusion technique, with acceptable toxicities up to a dose of 1,125 mg/m(2). However, the activity of gemcitabine under hypoxic conditions is not as firmly established as that of other drugs such as mitomycin C, melphalan or tirapazamine. Further studies of this investigational modality, but with bioreductive drugs, are therefore warranted first to evaluate the tolerance in a phase I study and later on to assess whether it does improve the response to chemotherapy.

Inhibition of the Kit Ligand/c-Kit Axis Attenuates Metastasis in a Mouse Model Mimicking Local Breast Cancer Relapse after Radiotherapy.

PURPOSE: Local breast cancer relapse after breast-saving surgery and radiotherapy is associated with increased risk of distant metastasis formation. The mechanisms involved remain largely elusive. We used the well-characterized 4T1 syngeneic, orthotopic breast cancer model to identify novel mechanisms of postradiation metastasis. EXPERIMENTAL DESIGN: 4T1 cells were injected in 20 Gy preirradiated mammary tissue to mimic postradiation relapses, or in nonirradiated mammary tissue, as control, of immunocompetent BALB/c mice. Molecular, biochemical, cellular, histologic analyses, adoptive cell transfer, genetic, and pharmacologic interventions were carried out. RESULTS: Tumors growing in preirradiated mammary tissue had reduced angiogenesis and were more hypoxic, invasive, and metastatic to lung and lymph nodes compared with control tumors. Increased metastasis involved the mobilization of CD11b(+)c-Kit(+)Ly6G(high)Ly6C(low)(Gr1(+)) myeloid cells through the HIF1-dependent expression of Kit ligand (KitL) by hypoxic tumor cells. KitL-mobilized myeloid cells homed to primary tumors and premetastatic lungs, to give rise to CD11b(+)c-Kit(-) cells. Pharmacologic inhibition of HIF1, silencing of KitL expression in tumor cells, and inhibition of c-Kit with an anti-c-Kit-blocking antibody or with a tyrosine kinase inhibitor prevented the mobilization of CD11b(+)c-Kit(+) cells and attenuated metastasis. C-Kit inhibition was also effective in reducing mobilization of CD11b(+)c-Kit(+) cells and inhibiting lung metastasis after irradiation of established tumors. CONCLUSIONS: Our work defines KitL/c-Kit as a previously unidentified axis critically involved in promoting metastasis of 4T1 tumors growing in preirradiated mammary tissue. Pharmacologic inhibition of this axis represents a potential therapeutic strategy to prevent metastasis in breast cancer patients with local relapses after radiotherapy. Clin Cancer Res; 18(16); 4365-74. ©2012 AACR.

Phenotypic stability and genetic gains in six-year girth growth of Hevea clones

Rubber tree [Hevea brasiliensis (Willd. ex Adr. de Juss.) Müell. Arg.] budgrafts of seven clones were evaluated on five contrasting sites in the plateau region of the São Paulo State, Brazil. The objective of this work was to study the phenotypic stability for girth growth. The experimental design was a randomized block design with three replications and seven treatments. Analysis of variance of girth at six-year plant growth indicated a highly significant clone x site interaction. Only linear sites and clone x site components of clone x year interaction were significant, indicating that the performance of clones over sites for this trait could be predicted. The clones GT 1 and PB 235 showed the greatest stability in relation to girth growth, with foreseen responses to change, introduced in the sites. The clones PB 235 and IAN 873 showed significative difference in relation to regression coefficient, representing clones with specific adaptability on favorable and unfavorable sites respectively. The clone GT 1 became the most promissory one in the study of stability and adaptability even showing low girth growth. Expected genetic gains from planting sites, along with estimates of clonal variance and repeatability of clonal means are generally greatest or close to the greatest when selection is done at the same site.

Effects of Deicing Salt Trace Compounds on Deterioration of Portland Cement Concrete, HR-271, 1987

A study was made of the detrimental effects of trace amounts of calcium sulfate (occurring naturally in halite deposits used for deicing) on portland cement concrete pavements. It was found that sulfate introduced as gypsum with sodium chloride in deicing brines can have detrimental effects on portland cement mortar. Concentrations of sulfate as low as 0.5% of the solute rendered the brine destructive. Conditions of brine application were critical to specimen durability. The mechanisms of deterioration were found to be due to pore filling resulting from compound formation and deposition. A field evaluation of deteriorating joints suggests that the sulfate phenomena demonstrated in the laboratory also operates in the field. A preliminary evaluation was made of remedies: limits on sulfates, fly ash admixtures, treatment of existing pavement, and salt treatments. This report gives details of the research objectives, experimental design, field testing, and possible solutions. Recommendations for further study are presented.

Methylation of the hTERT Promoter: A Novel Cancer Biomarker for Leptomeningeal Metastasis Detection in Cerebrospinal Fluids.

PURPOSE: The diagnosis of leptomeningeal metastases is usually confirmed by the finding of malignant cells by cytologic examination in the cerebrospinal fluid (CSF). More sensitive and specific cancer biomarkers may improve the detection of tumor cells in the CSF. Promoter methylation of the human telomerase reverse transcriptase (hTERT) gene characterizes most cancer cells. The aim of this study was to develop a sensitive method to detect hTERT methylation and to explore its use as a cancer biomarker in CSF. EXPERIMENTAL DESIGN: In 77 CSF specimens from 67 patients, hTERT promoter methylation was evaluated using real-time methylation-sensitive high-resolution melting (MS-HRM) and real-time TaqMan PCR and MS-HRM in a single-tube assay. RESULTS: Real-time MS-HRM assay was able to detect down to 1% hTERT-methylated DNA in a background of unmethylated DNA. PCR products were obtained from 90% (69/77) of CSF samples. No false positive hTERT was detected in the 21 non-neoplastic control cases, given to the method a specificity of 100%. The sensitivity of the real-time MS-HRM compared with the cytologic gold standard analysis was of 92% (11/12). Twenty-six CSFs from 22 patients with an hTERT-methylated primary tumor showed cytologic results suspicious for malignancy; in 17 (65%) of them, a diagnosis of leptomeningeal metastases could be confirmed by the hTERT methylation test. CONCLUSION: The hTERT real-time MS-HRM approach is fast, specific, sensitive, and could therefore become a valuable tool for diagnosis of leptomeningeal metastases as an adjunct to the traditional examination of CSF. Clin Cancer Res; 19(8); 2216-23. ©2013 AACR.

Analysis of experiments in square lattice with emphasis on variance components. i. Individual analysis

This paper focused on four alternatives of analysis of experiments in square lattice as far as the estimation of variance components and some genetic parameters are concerned: 1) intra-block analysis with adjusted treatment and blocks within unadjusted repetitions; 2) lattice analysis as complete randomized blocks; 3) intrablock analysis with unadjusted treatment and blocks within adjusted repetitions; 4) lattice analysis as complete randomized blocks, by utilizing the adjusted means of treatments, obtained from the analysis with recovery of interblock information, having as mean square of the error the mean effective variance of this same analysis with recovery of inter-block information. For the four alternatives of analysis, the estimators and estimates were obtained for the variance components and heritability coefficients. The classification of material was also studied. The present study suggests that for each experiment and depending of the objectives of the analysis, one should observe which alternative of analysis is preferable, mainly in cases where a negative estimate is obtained for the variance component due to effects of blocks within adjusted repetitions.

Clinical trial substantiates the predictive value of O-6-methylguanine-DNA methyltransferase promoter methylation in glioblastoma patients treated with temozolomide.

PURPOSE: In the setting of a prospective clinical trial, we determined the predictive value of the methylation status of the O-6-methylguanine-DNA methyltransferase (MGMT) promoter for outcome in glioblastoma patients treated with the alkylating agent temozolomide. Expression of this excision repair enzyme has been associated with resistance to alkylating chemotherapy. EXPERIMENTAL DESIGN: The methylation status of MGMT in the tumor biopsies was evaluated in 38 patients undergoing resection for newly diagnosed glioblastoma and enrolled in a Phase II trial testing concomitant and adjuvant temozolomide and radiation. The epigenetic silencing of the MGMT gene was determined using methylation-specific PCR. RESULTS: Inactivation of the MGMT gene by promoter methylation was associated with longer survival (P = 0.0051; Log-rank test). At 18 months, survival was 62% (16 of 26) for patients testing positive for a methylated MGMT promoter but reached only 8% (1 of 12) in absence of methylation (P = 0.002; Fisher's exact test). In the presence of other clinically relevant factors, methylation of the MGMT promoter remains the only significant predictor (P = 0.017; Cox regression). CONCLUSIONS: This prospective clinical trial identifies MGMT-methylation status as an independent predictor for glioblastoma patients treated with a methylating agent. The association of the epigenetic inactivation of the DNA repair gene MGMT with better outcome in this homogenous cohort may have important implications for the design of future trials and supports efforts to deplete MGMT by O-6-benzylguanine, a noncytotoxic substrate of this enzyme.

Effect of soil-water tension on herbaceous cotton yield

A field experiment was conducted during two years, 1990/91, in an alluvial soil, in the State of Paraíba, Brazil, to study the effect of the levels of soil-water tension, 50, 100, 200, 300, 400 and 600 kPa, at 20 cm depth, on upland cotton (Gossypium hirsutum L.r. latifolium Hutch, cv. CNPA-6H) yield. The experimental design was a complete randomized block with six treatments and four repetitions. There was an effect of the treatments on plant height, leaf area index and cotton yield, but the precocity index was not modified. Water should be applied when the soil-water tension, measured at 20 cm depth, reaches values around 200 kPa. There was a quadratic (R² = 0.893**) response of cotton yields to soil water tension, with the maximum when water was applied at 52% of soil water depletion.

R functions for quantifying nonindependence in standard dyadic and SRM designs

Interdependence is the main feature of dyadic relationships and, in recent years, various statistical procedures have been proposed for quantifying and testing this social attribute in different dyadic designs. The purpose of this paper is to develop several functions for this kind of statistical tests in an R package, known as nonindependence, for use by applied social researchers. A Graphical User Interface (GUI) is also developed to facilitate the use of the functions included in this package. Examples drawn from psychological research and simulated data are used to illustrate how the software works.

Diseños multivariables en evaluación de programas

En este escrito se describe la problematica relativa al Diseño experimental multivariable como alternativa a la investigación clasica dentro del ambito experimental. Se discuten 10s diferentes modelos de Diseño multivariable, planteándose la fórmula general que recoge tanto la estructura de cruzamiento como la estructura de anidación. A continuación se desarrolla el modelo de la Regresión Multivariable como estructura basica para el contraste de hipótesis de los Diseños experimentales multivariables. Al mismo tiempo se plantean los diferentes estadisticos aplicables a 10s datos de estos esquemas de investigación. Por último, se analiza un ejemplo practico como guia para la utilización del procedimiento descrito a lo largo del escrito

Biochemical traits useful for the determination of genetic variation in a natural population of Myracrodruon urundeuva

The objectives of this work were to analyze seeds from 20 trees of aroeira (Myracrodruon urundeuva Fr. All.) of a natural population located in the region of Selvíria, State of Mato Grosso do Sul, Brazil, in order to evaluate their protein, lipid and carbohydrate contents, and to estimate their genetic variation. A completely randomized experimental design consisting of 20 treatments (families) was set up, with two replications. Four types of proteins were detected: albumin (35.0 to 107.3 mg/g seed), globulin (3.4 to 9.3 mg/g), prolamin (60.0 to 135.2 mg/g) and glutelin (118.0 to 286.0 mg/g). The lipid content varied between 200 and 334 mg/g seed. The total sugars also varied (26.5 to 46.3 mg/g seed), with a predominance of polyols (arabinitol, mannitol, glucitol and xylitol). The main monosaccharides detected were glucose and arabinose. Total hydrolysis of the sugars indicated the presence of neutral arabinan and xylan oligosaccharides. The starch content varied from 0.35 to 1.58 mg/g seed. These biochemical traits showed considerable genetic variability, indicating that only the collection of seeds from many different trees can provide a representative sample of the population for conservation and genetic improvement.

Analysis of variance of primary data on plant growth analysis

Plant growth analysis presents difficulties related to statistical comparison of growth rates, and the analysis of variance of primary data could guide the interpretation of results. The objective of this work was to evaluate the analysis of variance of data from distinct harvests of an experiment, focusing especially on the homogeneity of variances and the choice of an adequate ANOVA model. Data from five experiments covering different crops and growth conditions were used. From the total number of variables, 19% were originally homoscedastic, 60% became homoscedastic after logarithmic transformation, and 21% remained heteroscedastic after transformation. Data transformation did not affect the F test in one experiment, whereas in the other experiments transformation modified the F test usually reducing the number of significant effects. Even when transformation has not altered the F test, mean comparisons led to divergent interpretations. The mixed ANOVA model, considering harvest as a random effect, reduced the number of significant effects of every factor which had the F test modified by this model. Examples illustrated that analysis of variance of primary variables provides a tool for identifying significant differences in growth rates. The analysis of variance imposes restrictions to experimental design thereby eliminating some advantages of the functional growth analysis.

Temozolomide-mediated radiation enhancement in glioblastoma: a report on underlying mechanisms.

PURPOSE: In this study, we investigated the mechanisms by which temozolomide enhances radiation response in glioblastoma cells. EXPERIMENTAL DESIGN: Using a panel of four primary human glioblastoma cell lines with heterogeneous O(6)-methylguanine-DNA methyltransferase (MGMT) protein expression, normal human astrocytes, and U87 xenografts, we investigated (a) the relationship of MGMT status with efficacy of temozolomide-based chemoradiation using a panel of in vitro and in vivo assays; (b) underlying mechanisms by which temozolomide enhances radiation effect in glioblastoma cells; and (c) strategies to overcome resistance to radiation + temozolomide. RESULTS: Temozolomide enhances radiation response most effectively in glioblastomas without detectable MGMT expression. On concurrent radiation + temozolomide administration in MGMT-negative glioblastomas, there seems to be decreased double-strand DNA (dsDNA) repair capacity and enhanced dsDNA damage compared either with radiation alone or with sequentially administered temozolomide. Our data suggest that O(6)-benzylguanine can enhance the antitumor effects of concurrent radiation + temozolomide in MGMT-positive cells by enhancing apoptosis and the degree of dsDNA damage. O(6)-Benzylguanine was most effective when administered concurrently with radiation + temozolomide and had less of an effect when administered with temozolomide in the absence of radiation or when administered sequentially with radiation. Our in vivo data using U87 xenografts confirmed our in vitro findings. CONCLUSIONS: The present study shows that temozolomide enhances radiation response most effectively in MGMT-negative glioblastomas by increasing the degree of radiation-induced double-strand DNA damage. In MGMT-positive glioblastomas, depletion of MGMT by the addition of O(6)-benzylguanine significantly enhances the antitumor effect of concurrent radiation + temozolomide. These are among the first data showing mechanisms of synergy between radiation and temozolomide and the effect of MGMT.