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Adrian C Begg

Publications and source records attributed to Adrian C Begg.

At least 19 recordsLinked to original sources
PubMed2006

Role for DNA polymerase beta in response to ionizing radiation.

Evidence for a role of DNA polymerase beta in determining radiosensitivity is conflicting. In vitro assays show an involvement of DNA polymerase beta in single strand break repair and base excision repair of oxidative damages, both products of ionizing radiation. Nevertheless the lack of DNA polymerase beta has been shown to have no effect on radiosensitivity. Here we show that mouse embryonic fibroblasts deficient in DNA polymerase beta are considerably more sensitive to ionizing radiation than wild-type cells, but only when confluent. The inhibitor methoxyamine renders abasic sites refractory to the dRP lyase activity of DNA polymerase beta. Methoxyamine did not significantly change radiosensitivity of wild-type fibroblasts in log phase. However, DNA polymerase beta deficient cells in log phase were radiosensitized by methoxyamine. Alkaline comet assays confirmed repair inhibition of ionizing radiation induced damage by methoxyamine in these cells, indicating both the existence of a polymerase beta-dependent long patch pathway and the involvement of another methoxyamine sensitive process, implying the participation of a second short patch polymerase(s) other than DNA polymerase beta. This is the first evidence of a role for DNA polymerase beta in radiosensitivity in vivo.

Animals↗
PubMed2006

Microsatellite alterations in head and neck squamous cell carcinoma and relation to expression of pimonidazole, CA IX and GLUT-1.

BACKGROUND AND PURPOSE: Because the locoregional control for HNSCC is still disappointing, research efforts focus on the exploration of new molecular markers located in both tumour and microenvironment, which could help stratify patients. The aim of the present work was therefore first to assess microsatellite alterations and hypoxia in HNSCC as possible molecular markers. Second, a relation between both was investigated, as hypoxia is known to select for genetic alterations. MATERIAL AND METHODS: Forty-eight patients with advanced HNSCC treated by surgery+/-radiotherapy were included. MSI and LOH were investigated with microsatellite markers using automatic fragment analysis. The presence of hypoxia was assessed by immunohistochemistry for pimonidazole, CA IX and GLUT-1. The mutual relationship between MSI/LOH and hypoxia was evaluated. RESULTS: No MSI was detected in this patient group. LOH occurred mostly on chromosomal arms 3p, 5q, 9p, 17p and 17q. Patients with LOH at D17S799, located in the near environment of p53, showed a higher CA IX expression (p=0.01). CONCLUSIONS: LOH is a possible molecular marker in HNSCC. The positive correlation between LOH at D17S799 and CA IX is in full concordance with previous publications linking hypoxia to selective pressure on the p53 gene.

Adult↗
PubMed2006

Prediction of treatment outcome by cisplatin-DNA adduct formation in patients with stage III/IV head and neck squamous cell carcinoma, treated by concurrent cisplatin-radiation (RADPLAT).

The purpose of our study was to test the predictive value of cisplatin-DNA adduct levels in head and neck squamous cell carcinoma (HNSCC) patients treated with cisplatin-radiation. Patients with advanced-stage HNSCC were treated within a randomized trial, investigating the optimal route of cisplatin administration, concurrently with radiation. Cisplatin was administered intra-arterially (IA, 150 mg/m2, with systemic rescue by sodium thiosulfate) or intravenously (IV, 100 mg/m2). In a subgroup, adducts were quantified in normal tissue and tumor. 32P-postlabeling was used to quantify intrastrand guanosine-guanosine adducts (GG-adducts) and adenosine-guanosine adducts (AG-adducts). Adduct levels were correlated with treatment outcome. Thirty-five patients were included (21 IV and 14 IA). At median follow-up of 27 months, locoregional (LR) control was 75% at 1 and 70% at 2 years. Adduct levels in tumor were 4-5-fold higher than in white blood cells (WBC) for both IA and IV treatment (p = 0.01). Adduct formation in WBC and buccal cells was higher in IV treated patients compared with IA infusion (p = 0.049 and 0.005 for GG-adducts in WBC and buccal cells, respectively). Adducts in tumors after IA infusion were not statistically different from those after IV. A strong correlation was observed between GG- and AG-adduct formation (r = 0.86, p < 0.001). Patients with higher GG adduct levels (>median) in primary tumor had significantly better disease free survival (DFS) than patients with lower (< or = median) adduct levels (p = 0.02). For overall survival (OS), a nonsignificant trend was observed, again in favor of patients with higher adduct levels (p = 0.06). In conclusion, cisplatin-DNA adduct formation in primary tumor appears to be predictive for DFS in HNSCC. No differences were observed in intratumoral adduct levels between IA and IV treatments, despite selective infusion of high-dose cisplatin with the IA procedure. However, systemic adduct levels (WBC and buccal cells) from IV patients were higher than in IA patients, consistent with less systemic exposure after IA administration.

Adult↗
PubMed2006

Hypoxia in human colorectal adenocarcinoma: comparison between extrinsic and potential intrinsic hypoxia markers.

PURPOSE: To detect and quantify hypoxia in colorectal adenocarcinomas by use of pimonidazole and iododeoxyuridine (IdUrd) as extrinsic markers and carbonic anhydrase IX (CA IX), microvessel density (MVD), epidermal growth-factor receptor (EGFR), and vascular endothelial growth factor (VEGF) as intrinsic markers of hypoxia. METHODS AND MATERIAL: Twenty patients with an adenocarcinoma of the left colon and rectum treated by primary surgery were injected with pimonidazole and IdUrd. Serial sections of tumor biopsies were single stained for VEGF, EGFR, Ki67, and double stained for blood vessels in combination with either pimonidazole, IdUrd, or CA IX. Percentage of expression was scored as well as colocalization of pimonidazole with CA IX. RESULTS: The median percentage of hypoxia, as judged by pimonidazole staining, was 16.7% (range, 0-52.4%). The expression of pimonidazole correlated inversely with the total MVD and endothelial cord MVD (R = -0.55, p = 0.01; R = -0.47, p = 0.04). Good colocalization was found between pimonidazole and CA IX in only 30% of tumors, with no correlation overall between pimonidazole and CA IX, VEGF, or EGFR or between the different intrinsic markers. Cells around some vessels (0.08-11%) were negative for IdUrd but positive for Ki 67, which indicated their lack of perfusion at the time of injection. CONCLUSION: Chronic and acute hypoxic regions are present in colorectal tumors, as shown by pimonidazole and IdUrd staining. Only in a minority of tumors did an association exist between the areas stained by pimonidazole and those positive for CA IX. Pimonidazole also did not correlate with expression of other putative intrinsic hypoxia markers (VEGF, EGFR).

Adenocarcinoma↗
PubMed2006

Radiosensitization of squamous cell carcinoma by the alkylphospholipid perifosine in cell culture and xenografts.

PURPOSE: Combined modality treatment has improved outcome in various solid tumors. Besides classic anticancer drugs, a new generation of biological response modifiers has emerged that increases the efficacy of radiation. Here, we have investigated whether perifosine, an orally applicable, membrane-targeted alkylphospholipid, enhances the antitumor effect of radiation in vitro and in vivo. EXPERIMENTAL DESIGN: Several long-term and short-term in vitro assays (clonogenic survival, sulforhodamine B cytotoxicity, apoptosis, and cell cycle analysis) were used to assess the cytotoxic effect of perifosine in combination with radiation. In vivo, the response of human KB squamous cell carcinoma xenografts was measured after treatment with perifosine, irradiation, and the combination. Radiolabeled perifosine was used to determine drug disposition in tumor and normal tissues. At various intervals after treatment, tumor specimens were collected to document histopathologic changes. RESULTS: In vitro, perifosine reduced clonogenic survival, enhanced apoptosis, and increased cell cycle arrest after radiation. In vivo, radiation and perifosine alone induced a dose-dependent tumor growth delay. When combining multiple perifosine administrations with single or split doses of radiation, complete and sustained tumor regression was observed. Histopathologic analysis of tumor specimens revealed a prominent apoptotic response after combined treatment with radiation and perifosine. Radiation-enhanced tumor response was observed at clinically relevant plasma perifosine concentrations and accumulating drug disposition of >100 microg/g in tumor tissue. CONCLUSIONS: Perifosine enhances radiation-induced cytotoxicity, as evidenced by reduced clonogenic survival and increased apoptosis induction in vitro and by complete tumor regression in vivo. These data provide strong support for further development of this combination in clinical studies.

Animals↗
PubMed2005

Resistance of hypoxic cells to ionizing radiation is influenced by homologous recombination status.

PURPOSE: To determine the role of DNA repair in hypoxic radioresistance. METHODS AND MATERIALS: Chinese hamster cell lines with mutations in homologous recombination (XRCC2, XRCC3, BRAC2, RAD51C) or nonhomologous end-joining (DNA-PKcs) genes were irradiated under normoxic (20% oxygen) and hypoxic (<0.1% oxygen) conditions, and the oxygen enhancement ratio (OER) was calculated. In addition, Fanconi anemia fibroblasts (complementation groups C and G) were compared with fibroblasts from nonsyndrome patients. RAD51 foci were studied using immunofluorescence. RESULTS: All hamster cell lines deficient in homologous recombination showed a decrease in OER (1.5-2.0 vs. 2.6-3.0 for wild-types). In contrast, the OER for the DNA-PKcs-deficient line was comparable to wild-type controls. The two Fanconi anemia cell strains also showed a significant reduction in OER. The OER for RAD51 foci formation at late times after irradiation was considerably lower than that for survival in wild-type cells. CONCLUSION: Homologous recombination plays an important role in determining hypoxic cell radiosensitivity. Lower OERs have also been reported in cells deficient in XPF and ERCC1, which, similar to homologous recombination genes, are known to play a role in cross-link repair. Because Fanconi anemia cells are also sensitive to cross-linking agents, this strengthens the notion that the capacity to repair cross-links determines hypoxic radiosensitivity.

Animals↗
PubMed2005

Molecular markers predicting radiotherapy response: report and recommendations from an International Atomic Energy Agency technical meeting.

PURPOSE: There is increasing interest in radiogenomics and the characterization of molecular profiles that predict normal tissue and tumor radioresponse. A meeting in Amsterdam was organized by the International Atomic Energy Agency to discuss this topic on an international basis. METHODS AND MATERIALS: This report is not completely exhaustive, but highlights some of the ongoing studies and new initiatives being carried out worldwide in the banking of tumor and normal tissue samples underpinning the development of molecular marker profiles for predicting patient response to radiotherapy. It is generally considered that these profiles will more accurately define individual or group radiosensitivities compared with the nondefinitive findings from the previous era of cellular-based techniques. However, so far there are only a few robust reports of molecular markers predicting normal tissue or tumor response. RESULTS: Many centers in different countries have initiated tissue and tumor banks to store samples from clinical trials for future molecular profiling analysis, to identify profiles that predict for radiotherapy response. The European Society for Therapeutic Radiology and Oncology GENEtic pathways for the Prediction of the effects of Irradiation (GENEPI) project, to store, document, and analyze sample characteristics vs. response, is the most comprehensive in this regard. CONCLUSIONS: The next 5-10 years are likely to see the results of these and other correlative studies, and promising associations of profiles with response should be validated in larger definitive trials.

Biomarkers, Tumor↗
PubMed2005

Hypoxic cell turnover in different solid tumor lines.

PURPOSE: Most solid tumors contain hypoxic cells, and the amount of tumor hypoxia has been shown to have a negative impact on the outcome of radiotherapy. The efficacy of combined modality treatments depends both on the sequence and timing of the treatments. Hypoxic cell turnover in tumors may be important for optimal scheduling of combined modality treatments, especially when hypoxic cell targeting is involved. METHODS AND MATERIALS: Previously we have shown that a double bioreductive hypoxic marker assay could be used to detect changes of tumor hypoxia in relation to the tumor vasculature after carbogen and hydralazine treatments. This assay was used in the current study to establish the turnover rate of hypoxic cells in three different tumor models. The first hypoxic marker, pimonidazole, was administered at variable times before tumor harvest, and the second hypoxic marker, CCI-103F, was injected at a fixed time before harvest. Hypoxic cell turnover was defined as loss of pimonidazole (first marker) relative to CCI-103F (second marker). RESULTS: The half-life of hypoxic cell turnover was 17 h in the murine C38 colon carcinoma line, 23 h and 49 h in the human xenograft lines MEC82 and SCCNij3, respectively. Within 24 h, loss of pimonidazole-stained areas in C38 and MEC82 occurred concurrent with the appearance of pimonidazole positive cell debris in necrotic regions. In C38 and MEC82, most of the hypoxic cells had disappeared after 48 h, whereas in SCCNij3, viable cells that had been labeled with pimonidazole were still observed after 5 days. CONCLUSIONS: The present study demonstrates that the double hypoxia marker assay can be used to study changes in both the proportion of hypoxic tumor cells and their lifespan at the same time. The present study shows that large differences in hypoxic cell turnover rates may exist among tumor lines, with half-lives ranging from 17-49 h.

Animals↗
PubMed2005

Thymidine analogues to assess microperfusion in human tumors.

PURPOSE: To validate the use of the thymidine analogues as local perfusion markers in human tumors (no labeling indicates no perfusion) by comparison with the well-characterized perfusion marker Hoechst 33342. METHODS AND MATERIALS: Human tumor xenografts from gliomas and head-and-neck cancers were injected with iododeoxyuridine (IdUrd) or bromodeoxyuridine (BrdUrd) and the fluorescent dye Hoechst 33342. In frozen sections, each blood vessel was scored for the presence of IdUrd/BrdUrd labeling and Hoechst in surrounding cells. The percentage of analogue-negative vessels was compared with the fraction of Hoechst-negative vessels. Collocalization of the two markers was also scored. RESULTS: We found considerable intertumor variation in the fraction of perfused vessels, measured by analogue labeling, both in the human tumor xenografts and in a series of tumor biopsies from head-and-neck cancer patients. There was a significant correlation between the Hoechst-negative and IdUrd/BrdUrd-negative vessels in the xenografts (r = 85, p = 0.0004), despite some mismatches on a per-vessel basis. CONCLUSIONS: Thymidine analogues can be successfully used to rank tumors according to their fraction of perfused vessels. Whether this fraction correlates with the extent of acute hypoxia needs further confirmation.

Animals↗
PubMed2005

Radiosensitization by a dominant negative to DNA polymerase beta is DNA polymerase beta-independent and XRCC1-dependent.

BACKGROUND AND PURPOSE: DNA base damages and single strand breaks after ionizing radiation are repaired by base excision repair (BER) and single strand break repair (SSBR), with both DNA polymerase beta (polbeta) and XRCC1 playing key roles. We previously showed that a dominant negative to polbeta (polbetaDN) sensitized human tumor cells to ionizing radiation. However, polbeta-deficient cells, in contrast to XRCC1-deficient cells, are not more radiosensitive. The purpose of the present study was to further elucidate the mechanism of action of the polbetaDN to better understand the roles of BER and SSBR in determining radiosensitivity. MATERIALS AND METHODS: Mouse embryonic fibroblasts, both polbeta wildtype and knockout, and hamster XRCC1-deficient EM9 cells together with its parental line, were transfected with the polbetaDN. Clones with equal polbetaDN expression levels were selected and used in clonogenic assays to determine radiosensitivity. RESULTS: Radiosensitization of polbeta deficient cells by the polbetaDN is shown here, demonstrating inhibition of a polbeta-independent pathway. In addition, we observed radiosensitization of wildtype hamster cells but no radiosensitization of the XRCC1-deficient EM9 cells. CONCLUSIONS: The polbetaDN acts independently of polbeta status and inhibits a pathway, which is dependent on XRCC1, consistent with inhibition of BER and/or SSBR. The data further indicate involvement of other polymerases, which are inhibited by polbetaDN. Finally, they demonstrate that inhibition of BER and SSBR can increase radiosensitivity, with potential clinical relevance.

Animals↗
PubMed2004

Differentiation-associated staining with anti-pimonidazole antibodies in head and neck tumors.

BACKGROUND AND PURPOSE: Hypoxia is a strong negative prognostic factor for all three major treatment modalities for cancer. The bioreductive drug pimonidazole is currently under clinical investigation as a hypoxia marker. In human head and neck tumors, in addition to staining patterns typical of chronic hypoxia, staining was seen specifically around areas of keratinization, raising the question of whether this is hypoxia-related. This could influence quantitative hypoxia estimates using this marker. We investigated here whether the differentiation-related staining was caused by locally high reductive enzyme levels. PATIENTS AND METHODS: The nitrotetrazolium compound NBT was used, which is reduced by nitroreductases to yield a blue color. The assay was validated on three genetically related MDA231 human mammary carcinoma cell lines: wildtype, overexpressing DT-diaphorase (DT1), and overexpressing cytochrome p450 reductase (R4). Increased NBT staining under normoxia was indeed seen for both R4 and DT1 lines. Pimonidazole staining under normoxia was only seen in the R4 line. RESULTS: Frozen tumor sections from 20 patients with head and neck cancer injected with pimonidazole were incubated with NBT. Parallel sections were stained for pimonidazole. Staining patterns were then compared on matched images, and areas of keratinization scored for the presence or absence of pimonidazole and NBT. Pimonidazole staining was seen in 56% of keratinized areas, and of these, 78% showed increased NBT staining, indicating that high reductase levels are not a necessary requirement for differentiation-associated pimonidazole staining. In a second series, frozen sections of tumors from 15 patients not receiving pimonidazole were incubated with NBT and compared with staining after incubation with pimonidazole under both oxic and hypoxic conditions. Pimonidazole staining of some keratinizing areas under oxic conditions was seen. Of these areas, only a proportion (70%) showed increased NBT staining, confirming the lack of correspondence between keratin-associated pimonidazole staining and reductase levels. CONCLUSION: Hypoxia-independent pimonidazole staining can occur in more differentiated head and neck tumors, necessitating caution in hypoxia quantification. These data argue against a causative role for locally high reductase levels in differentiation-associated staining. DT-diaphorase appears to play no role in pimonidazole reduction.

Antibodies, Anti-Idiotypic↗
PubMed2002

HIF-1A, pimonidazole, and iododeoxyuridine to estimate hypoxia and perfusion in human head-and-neck tumors.

PURPOSE: Tumor hypoxia measured by microelectrodes has been shown to indicate poor patient outcome. Here we investigated four potentially more widely applicable immunohistochemical parameters of tumor oxygenation and perfusion in human head-and-neck tumors. METHODS: Twenty patients with squamous cell carcinomas of the head and neck treated with primary surgery were injected with pimonidazole and IdUrd the evening before operation. Consecutive paraffin-embedded sections were stained for blood vessels, pimonidazole, IdUrd, and HIF-1alpha. IdUrd labeling and Ki-67 labeling around individual blood vessels were scored. The spatial relationship between HIF-1alpha and pimonidazole was studied, as well as the distribution of both markers as a function of distance from the nearest blood vessel. RESULTS: Measurement of all four parameters (diffusion-limited fraction, pimonidazole fraction, HIF-1alpha fraction, IdUrd-negative vessels) was feasible, and a significant difference between tumors was found for all parameters. IdUrd-labeled cells were absent around some vessels, indicating lack of perfusion, because these regions were positive for Ki-67. There was a positive correlation between diffusion-limited fraction and pimonidazole area for all images from all tumors, although no correlation for mean values per tumor. Colocalization of pimonidazole and HIF-1alpha was low (0.02%-25%). Most expression profiles showed a more homogenous distribution for HIF-1alpha than pimonidazole. There was no significant correlation between the pimonidazole and HIF-1alpha fractions in the 10 tumors studied. CONCLUSIONS: Simultaneous immunohistochemical measurements related to hypoxia and perfusion are feasible (and easily applicable) in resected human tumors. The different geographic distributions of HIF-1alpha and pimonidazole indicate that HIF-1alpha might not be suitable as a marker for chronic hypoxia. Each parameter will be correlated with outcome in a larger ongoing study on head-and-neck tumors treated with surgery with or without postoperative radiotherapy.

Carcinoma, Squamous Cell↗
PubMed2002

Phase 1 study to identify tumour hypoxia in patients with head and neck cancer using technetium-99m BRU 59-21.

The aim of this study was to assess the safety and biodistribution of technetium-99m BRU 59-21, a novel radioactively labelled 2-nitro-imidazole hypoxic marker, in head and neck cancer patients and to correlate uptake with pimonidazole staining. (99m)Tc-BRU 59-21 was administered intravenously (mean dose 824 MBq, range 780-857 MBq) to ten head and neck cancer patients scheduled for primary surgery, and whole-body images and SPET scans were then obtained. Uptake of radioactivity in the regions of interest was determined and tumour to normal tissue ratios were calculated after correlative evaluation with MRI/CT. Twelve to 16 h before surgery (up to 2 weeks after the scan), patients received pimonidazole intravenously. Tumour sections were stained immunohistochemically for pimonidazole binding. No serious adverse events were reported. In five patients there were ten adverse events, which were mild in intensity and resolved completely without intervention. Uptake of (99m)Tc-BRU 59-21 was observed in eight of the ten primary tumours. Tumour to normal tissue ratios on the SPET scans for primary tumour and lymph nodes increased from 1.8 (range 0.9-2.7) to 2.1 (range 0.8-3.7) between 30 min and 3 h post injection. Tumour to normal tissue ratios in the primary tumour were significantly correlated with pimonidazole staining for SPET scans performed 30 min and 3 h post injection ( P=0.016 and P=0.037, respectively). When primary tumour and involved lymph nodes were considered in conjunction, correlation between the tumour to normal tissue ratio and pimonidazole staining was observed for early ( P<0.001) but not for late SPET scans ( P=0.076). However, late scans showed better tumour delineation than early scans. Administration of (99m)Tc-BRU 59-21 in head and neck cancer patients appears to be safe and feasible. Uptake and retention in tumour tissue was observed, suggestive of tumour hypoxia, and this was supported by correlations with staining for the hypoxic marker pimonidazole.

Cell Hypoxia↗
PubMed2002

The role of DNA polymerase beta in determining sensitivity to ionizing radiation in human tumor cells.

Lethal lesions after ionizing radiation are thought to be mainly unrepaired or misrepaired DNA double-strand breaks, ultimately leading to lethal chromosome aberrations. However, studies with radioprotectors and repair inhibitors indicate that single-strand breaks, damaged nucleotides or abasic sites can also influence cell survival. This paper reports on studies to further define the role of base damage and base excision repair on the radiosensitivity of human cells. We retrovirally transduced human tumor cells with a dominant negative form of DNA polymerase beta, comprising the 14 kDa DNA-binding domain of DNA polymerase beta but lacking polymerase function. Radiosensitization of two human carcinoma cell lines, A549 and SQD9, was observed, achieving dose enhancement factors of 1.5-1.7. Sensitization was dependent on expression level of the dominant negative and was seen in both single cell clones and in unselected virally transduced populations. Sensitization was not due to changes in cell cycle distribution. Little or no sensitization was seen in G(1)-enriched populations, indicating cell cycle specificity for the observed sensitization. These results contrast with the lack of effect seen in DNA polymerase beta knockout cells, suggesting that polDN also inhibits the long patch, DNA polymerase beta-independent repair pathway. These data demonstrate an important role for BER in determining sensitivity to ionizing radiation and might help identify targets for radiosensitizing tumor cells.

Cell Cycle↗
PubMed2002

Nbs1 promotes ATM dependent phosphorylation events including those required for G1/S arrest.

Cell lines from Nijmegen Breakage Syndrome (NBS) and ataxia telangiectasia (A-T) patients show defective S phase checkpoint arrest. In contrast, only A-T but not NBS cells are significantly defective in radiation-induced G1/S arrest. Phosphorylation of some ATM substrates has been shown to occur in NBS cells. It has, therefore, been concluded that Nbs1 checkpoint function is S phase specific. Here, we have compared NBS with A-T cell lines (AT-5762ins137) that express a low level of normal ATM protein to evaluate the impact of residual Nbs1 function in NBS cells. The radiation-induced cell cycle response of these NBS and 'leaky' A-T cells is almost identical; normal G2/M arrest after 2 Gy, intermediate G1/S arrest depending on the dose and an A-T-like S phase checkpoint defect. Thus, the checkpoint assays differ in their sensitivity to low ATM activity. Radiation-induced phosphorylation of the ATM-dependent substrates Chk2, RPAp34 and p53-Ser15 are similarly impaired in AT-5762ins137 and NBS cells in a dose dependent manner. In contrast, NBS cells show normal ability to activate ATM kinase following irradiation in vitro and in vivo. We propose that Nbs1 facilitates ATM-dependent phosphorylation of multiple downstream substrates, including those required for G1/S arrest.

Ataxia Telangiectasia↗