X-ray irradiation and Rho-kinase inhibitor additively induce invasiveness of the cells of the pancreatic cancer line, MIAPaCa-2, which exhibits mesenchymal and amoeboid motility.

Tumor cells can migrate and invade tissue by two modes of motility: mesenchymal and amoeboid. X-ray or γ-ray irradiation increases the invasiveness of tumor cells with mesenchymal motility through the induction of matrix metalloproteinases (MMP), and this increase is suppressed by MMP inhibitors (MMPI). However, the effects of X-ray or γ-ray irradiation on the invasiveness of tumor cells with amoeboid motility remain unclear. We investigated the effect of irradiation on amoeboid motility by using cells of the human pancreatic cancer line, MIAPaCa-2, which exhibits both modes of motility. The X-ray-induced invasiveness of MIAPaCa-2 cells was associated with the upregulation of MMP2 at both the RNA and protein levels and was inhibited by MMPI treatment. Amoeboid-mesenchymal transition was slightly induced after irradiation. The MMPI treatment caused mesenchymal-amoeboid transition without significant increase in invasiveness, while the ROCK inhibitor (ROCKI) stimulated amoeboid-mesenchymal transition and enhanced invasiveness under both non-irradiated and irradiated conditions. This ROCKI-induced transition was accompanied by the upregulation of MMP2 mRNA and protein. Exposure to both irradiation and ROCKI further enhanced MMP2 expression and had an additive effect on the invasiveness of MIAPaCa-2 cells. Additionally, exposure to MMPI led to significant suppression of both radiation-induced and the basal invasiveness of MIAPaCa-2 cells. This suggests that ROCKI treatment, especially with concomitant X-ray irradiation, can induce invasion of cancer cells and should be used only for certain types of cancer cells. Simultaneous use of inhibitors, ROCKI and MMPI may be effective in suppressing invasiveness under both X-ray-irradiated and non-irradiated conditions.

Genome wide screen identifies microsatellite markers associated with acute adverse effects following radiotherapy in cancer patients.

BACKGROUND: The response of normal tissues in cancer patients undergoing radiotherapy varies, possibly due to genetic differences underlying variation in radiosensitivity. METHODS: Cancer patients (n = 360) were selected retrospectively from the RadGenomics project. Adverse effects within 3 months of radiotherapy completion were graded using the National Cancer Institute Common Toxicity Criteria; high grade group were grade 3 or more (n = 180), low grade group were grade 1 or less (n = 180). Pooled genomic DNA (gDNA) (n = 90 from each group) was screened using 23,244 microsatellites. Markers with different inter-group frequencies (Fisher exact test P < 0.05) were analyzed using the remaining pooled gDNA. Silencing RNA treatment was performed in cultured normal human skin fibroblasts. RESULTS: Forty-seven markers had positive association values; including one in the SEMA3A promoter region (P = 1.24 x 10(-5)). SEMA3A knockdown enhanced radiation resistance. CONCLUSIONS: This study identified 47 putative radiosensitivity markers, and suggested a role for SEMA3A in radiosensitivity.

The proangiogenic factor ephrin-A1 is up-regulated in radioresistant murine tumor by irradiation.

While the pre-treatment status of cancer is generally correlated with outcome, little is known about microenvironmental change caused by anti-cancer treatment and how it may affect outcome. For example, treatment may lead to induction of gene expression that promotes resistance to therapy. In the present study, we attempted to find a gene that was both induced by irradiation and associated with radioresistance in tumors. Using single-color oligo-microarrays, we analyzed the gene expression profiles of two murine squamous cell carcinomas, NR-S1, which is highly radioresistant, and SCCVII, which is radiosensitive, after irradiation with 137-Cs gamma rays or carbon ions. Candidate genes were those differentially regulated between NR-S1 and SCCVII after any kind of irradiation. Four genes, Efna1 (Ephrin-A1), Sprr1a (small proline-rich protein 1A), Srgap3 (SLIT-ROBO Rho GTPase activating protein 3) and Xrra1 [RIKEN 2 days neonate thymus thymic cells (NOD) cDNA clone E430023D08 3'], were selected as candidate genes associated with radiotherapy-induced radioresistance. We focused on Efna1, which encodes a ligand for the Eph receptor tyrosine kinase known to be involved in the vascular endothelial growth factor (VEGF) pathway. We used immunohistochemical methods to detect expression of Ephrin-A1, VEGF, and the microvascular marker CD31 in radioresistant NR-S1 tumor cells. Ephrin-A1 was detected in the cytoplasm of NR-S1 tumor cells after irradiation, but not in SCCVII tumor cells. Irradiation of NR-S1 tumor cells also led to significant increases in microvascular density, and up-regulation of VEGF expression. Our results suggest that radiotherapy-induced changes in gene expression related with angiogenesis might also modulate microenvironment and influence responsiveness of tumors.

p73 protein expression correlates with radiation-induced apoptosis in the lack of p53 response to radiation therapy for cervical cancer.

PURPOSE: p73 belongs to the p53 tumor suppressor family of genes and can inhibit cell growth in a p53-like manner by inducing apoptosis or cell cycle arrest. Here, we investigated whether p73 could compensate for impaired p53 function in apoptosis induced by radiation therapy (RT) for cervical cancer. METHODS AND MATERIALS: Sixty-eight patients with squamous cell carcinoma of the cervix who received definitive RT combined with (n=37) or without (n=31) cisplatin were investigated. Biopsy specimens were excised from the cervical tumor before RT and after 9 Gy. RESULTS: Mean apoptosis index (AI) was 0.93% before RT and 1.97% after 9 Gy with a significant increase (p<0.001). For all patients, there was a significant correlation between p73 expression positivity after 9 Gy and AI ratio (AI after 9 Gy/AI before RT) (p=0.021). Forty-one patients were regarded as the p53-responding group according to the expression of p53 after 9 Gy, whereas the remaining 27 patients were regarded as the p53-nonresponding group. A significant correlation between p73 expression after 9 Gy and AI ratio was observed in the p53-non-responding group (p<0.001) but not in the p53-responding group (p=0.940). CONCLUSION: Our results suggest that p73 plays an important role in compensating for the lack of p53 function in radiation-induced apoptosis of cervical cancer.

Prediction of lymphatic metastasis based on gene expression profile analysis after brachytherapy for early-stage oral tongue carcinoma.

BACKGROUND AND PURPOSE: The management of lymphatic metastasis of early-stage oral tongue carcinoma patients is crucial for its prognosis. The purpose of this study was to evaluate the predictive ability of lymphatic metastasis after brachytherapy (BRT) for early-stage tongue carcinoma based on gene expression profiling. PATIENTS AND METHODS: Pre-therapeutic biopsies from 39 patients with T1 or T2 tongue cancer were analyzed for gene expression signatures using Codelink Uniset Human 20K Bioarray. All patients were treated with low dose-rate BRT for their primary lesions and underwent strict follow-up under a wait-and-see policy for cervical lymphatic metastasis. Candidate genes were selected for predicting lymph-node status in the reference group by the permutation test. Predictive accuracy was further evaluated by the prediction strength (PS) scoring system using an independent validation group. RESULTS: We selected a set of 19 genes whose expression differed significantly between classes with or without lymphatic metastasis in the reference group. The lymph-node status in the validation group was predicted by the PS scoring system with an accuracy of 76%. CONCLUSIONS: Gene expression profiling using 19 genes in primary tumor tissues may allow prediction of lymphatic metastasis after BRT for early-stage oral tongue carcinoma.

In-gel multiple displacement amplification of long DNA fragments diluted to the single molecule level.

The isolation and multiple genotyping of long individual DNA fragments are needed to obtain haplotype information for diploid organisms. Limiting dilution of sample DNA followed by multiple displacement amplification is a useful technique but is restricted to short (<5 kb) DNA fragments. In the current study, a novel modification was applied to overcome these problems. A limited amount of cellular DNA was carefully released from intact cells into a mildly heated alkaline agarose solution and mixed thoroughly. The solution was then gently aliquoted and allowed to solidify while maintaining the integrity of the diluted DNA. Exogenously provided Phi29 DNA polymerase was used to perform consistent genomic amplification with random hexameric oligonucleotides within the agarose gels. Simple heat melting of the gel allowed recovery of the amplified materials in a solution of the polymerase chain reaction (PCR)-ready form. The haplotypes of seven SNPs spanning 240 kb of the DNA surrounding the human ATM gene region on chromosome 11 were determined for 10 individuals, demonstrating the feasibility of this new method.

Gene expression analysis in human malignant melanoma cell lines exposed to carbon beams.

PURPOSE: To elucidate the molecular changes in response to carbon beams (C-ions) in melanoma. MATERIALS AND METHODS: We examined expression profiles of 6 melanoma cell lines exposed to C-ions or X-rays with 2 Gy using single-color microarrays. RESULTS: Twenty-two genes, including nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (NFKBIA), responded to C-ions in all six cell lines, based on analysis of variance (ANOVA) filtering (p < 0.001). We found 173 genes that responded in common to C-ions in four cell lines. We identified many down-regulated genes including the cell cycle - related genes that were more responsive to C-ions than X-rays. In contrast, most of the up-regulated genes including the tumor protein p53 (p53) target genes responded to both C-ions and X-rays. C-ions induced G2/M arrest significantly more than X-rays at 30 h (p < 0.05). CONCLUSION: Our findings suggest that down-regulation of gene expression plays a key role in the response to C-ions. Regulation of cell cycle - related genes and induction of prolonged G2/M arrest may be responsible for the extra sensitivity to C-ions, whereas p53-related genes may have similar roles in the sensitivities to both C-ions and X-rays.

Expression profiles are different in carbon ion-irradiated normal human fibroblasts and their bystander cells.

Evidence has accumulated that ionizing radiation induces biological effects in non-irradiated bystander cells having received signals from directly irradiated cells; however, energetic heavy ion-induced bystander response is incompletely characterized. Here we performed microarray analysis of irradiated and bystander fibroblasts in confluent cultures. To see the effects in bystander cells, each of 1, 5 and 25 sites was targeted with 10 particles of carbon ions (18.3 MeV/u, 103 keV/microm) using microbeams, where particles traversed 0.00026, 0.0013 and 0.0066% of cells, respectively. diated cells, cultures were exposed to 10% survival dose (D), 0.1D and 0.01D of corresponding broadbeams (108 keV/microm). Irrespective of the target numbers (1, 5 or 25 sites) and the time (2 or 6h postirradiation), similar expression changes were observed in bystander cells. Among 874 probes that showed more than 1.5-fold changes in bystander cells, 25% were upregulated and the remainder downregulated. These included genes related to cell communication (PIK3C2A, GNA13, FN1, ANXA1 and IL1RAP), stress response (RAD23B, ATF4 and EIF2AK4) and cell cycle (MYCN, RBBP4 and NEUROG1). Pathway analysis revealed serial bystander activation of G protein/PI-3 kinase pathways. Instead, genes related to cell cycle or death (CDKN1A, GADD45A, NOTCH1 and BCL2L1), and cell communication (IL1B, TCF7 and ID1) were upregulated in irradiated cells, but not in bystander cells. Our results indicate different expression profiles in irradiated and bystander cells, and imply that intercellular signaling between irradiated and bystander cells activate intracellular signaling, leading to the transcriptional stress response in bystander cells.

CD44 and Bak expression in IL-6 or TNF-alpha gene knockout mice after whole lung irradiation.

To understand the molecular mechanisms that underlie radiation pneumonitis, we examined whether knockout of the TNF or the IL-6 gene could give mice an inherent resistance to radiation in the acute phase of alveolar damage after thoracic irradiation. The temporal expression of inflammation (CD44) and apoptosis (Bak) markers in lung after thoracic irradiation was measured to determine the degree of alveolar damage. At 4 weeks post-irradiation (10 Gy), small inflammatory foci were observed in all mice, but there were no obvious histological differences between control (C57BL/6JSlc), TNF-alpha knockout (TNF KO), and IL-6 knockout (IL-6 KO) mice. However, immunohistochemical analysis of CD44 and Bak expression over a time course of 2 weeks highlighted significant differences between the three groups. C57BL/6JSlc and TNF KO mice had increased numbers of both CD44-positive and Bak-positive cells after irradiation, while the IL-6 KO mice showed stable levels of CD44 and Bak. In conclusion, the radioresistant status of IL-6 KO mice in the acute phase of alveolar damage after irradiation suggested an important role for IL-6 in radiation pneumonitis.

Visible Genotype Sensor Array.

A visible sensor array system for simultaneous multiple SNP genotyping has been developed using a new plastic base with specific surface chemistry. Discrimination of SNP alleles is carried out by an allele-specific extension reaction using immobilized oligonucleotide primers. The 3'-ends of oligonucleotide primers are modified with a locked nucleic acid to enhance their efficiency in allelic discrimination. Biotin-dUTPs included in the reaction mixture are selectively incorporated into extending primer sequences and are utilized as tags for alkaline phosphatase-mediated precipitation of colored chemical substrates onto the surface of the plastic base. The visible precipitates allow immediate inspection of typing results by the naked eye and easy recording by a digital camera equipped on a commercial mobile phone. Up to four individuals can be analyzed on a single sensor array and multiple sensor arrays can be handled in a single operation. All of the reactions can be performed within one hour using conventional laboratory instruments. This visible genotype sensor array is suitable for "focused genomics" that follows "comprehensive genomics".

Haplotype-based analysis of genes associated with risk of adverse skin reactions after radiotherapy in breast cancer patients.

PURPOSE: To identify haplotypes of single nucleotide polymorphism markers associated with the risk of early adverse skin reactions (EASRs) after radiotherapy in breast cancer patients. METHODS AND MATERIALS: DNA was sampled from 399 Japanese breast cancer patients who qualified for breast-conserving radiotherapy. Using the National Cancer Institute-Common Toxicity Criteria scoring system, version 2, the patients were grouped according to EASRs, defined as those occurring within 3 months of starting radiotherapy (Grade 1 or less, n = 290; Grade 2 or greater, n = 109). A total of 999 single nucleotide polymorphisms from 137 candidate genes for radiation susceptibility were genotyped, and the haplotype associations between groups were assessed. RESULTS: The global haplotype association analysis (p < 0.05 and false discovery rate < 0.05) indicated that estimated haplotypes in six loci were associated with EASR risk. A comparison of the risk haplotype with the most frequent haplotype in each locus showed haplotype GGTT in CD44 (odds ratio [OR] = 2.17; 95% confidence interval [CI], 1.07-4.43) resulted in a significantly greater EASR risk. Five haplotypes, CG in MAD2L2 (OR = 0.55; 95% CI, 0.35-0.87), GTTG in PTTG1 (OR = 0.48; 95% CI, 0.24-0.96), TCC (OR = 0.48; 95% CI, 0.26-0.89) and CCG (OR = 0.50; 95% CI, 0.27-0.92) in RAD9A, and GCT in LIG3 (OR = 0.46; 95% CI, 0.22-0.93) were associated with a reduced EASR risk. No significant risk haplotype was observed in REV3L. CONCLUSION: Individual radiosensitivity can be partly determined by these haplotypes in multiple loci. Our findings may lead to a better understanding of the mechanisms underlying the genetic variation in radiation sensitivity and resistance among breast cancer patients.

Correlation between single nucleotide polymorphisms and jejunal crypt cell apoptosis after whole body irradiation.

PURPOSE: To identify loci concerned with radiosensitivity in a mouse model using single nucleotide polymorphism (SNP) markers. MATERIALS AND METHODS: We subjected 276 second filial generation (F2) mice descended from two inbred mouse strains, radiation-induced apoptosis sensitive C57BL/6JNrs (B6) and radiation-induced apoptosis resistant C3H/HeNrs (C3H), to 2.5 Gy whole-body irradiation. We quantified jejunal crypt apoptosis, performed a genome-wide survey, and identified quantitative trait loci (QTL) associated with radiation sensitivity. We expressed apoptosis levels as an apoptotic score (AS), which was equal to the number of apoptotic bodies divided by the number of crypts. We genotyped the mice for 109 SNP markers. RESULTS: AS values were 97.7+/-32.9 in B6 mice and 49.0+/-24.9 in C3H mice (p < 0.01). Genome-wide analysis revealed 8 markers (2 on chromosome 9, 4 on 15, 1 on 17, and 1 on 18) affecting radiation-induced jejunal apoptosis with log odds (LOD) scores ranging from 2.11+/-3.91. We found a significant locus on chromosome 15, which was previously reported by Weil and colleagues. CONCLUSION: These findings support the view that the radiosensitivity of clinically normal tissue depends on variations in several genes.

Gene expression profile changes correlating with radioresistance in human cell lines.

PURPOSE: To identify gene expression profiles specific to radioresistance of human cells. METHODS AND MATERIALS: Global gene expression profiles of a total of 15 tumor and normal fibroblast cell lines were analyzed using DNA microarrays and statistical clustering methods. Initially, six of the cell lines were categorized into radioresistant (RG) or nonradioresistant (NRG) groups according to the radiation dose required to reduce their survival to 10% (D10). Genes for which expression was specific to each group at 1 or 3 h after irradiation were identified using statistical procedures including analysis of variance and a two-dimensional hierarchical clustering method. The remaining nine cell lines were subjected to the k-nearest neighbor pattern classification. RESULTS: The nine test cell lines were successfully classified by their D10 value using 46 and 44 genes for which transcription levels had significantly changed at 1 and 3 h after irradiation, respectively. Of these genes, 25 showed altered expression at both time points in the NRG or RG, but independently were unable to classify the test cell lines. CONCLUSIONS: Radioresistant cell lines analyzed in this study showed certain radiation-induced changes in gene expression profiles that are different from the profile changes of the more-sensitive cell lines.

Cyclooxygenase-2 impairs treatment effects of radiotherapy for cervical cancer by inhibition of radiation-induced apoptosis.

PURPOSE: Cyclooxygenase-2 (COX-2) plays a pivotal role in regulation of radiation-induced apoptosis. The aim of this study was to analyze the relationship between COX-2 expression and postradiotherapy outcomes of patients with cervical cancer. METHODS AND MATERIALS: Biopsy specimens from 47 consecutive patients who had undergone definitive radiotherapy alone or radiotherapy combined with chemotherapy between October 2002 and November 2004 were investigated. RESULTS: The COX-2 expression rate of the pretreatment samples was 46.1% +/- 21.0%, and the apoptotic index (AI) 1 week after start of radiotherapy was 2.1% +/- 0.9%. There was a significant negative correlation between the pretreatment COX-2 expression and the AI during radiotherapy (r = -0.52, p = 0.0002). Complete response rates were 59% for COX-2-positive patients compared with 80% for COX-2-negative patients (p = 0.12). The 2-year local control rate for COX-2-positive patients was 71.3%, whereas the corresponding rate for COX-2-negative patients was 96.0% (p = 0.06). CONCLUSIONS: To the best of our knowledge, this is the first report to prove clinically that COX-2 can make cervical squamous cell carcinomas more refractory to radiotherapy by inhibition of radiation-induced apoptosis. Furthermore, expression of COX-2 may be a good indicator to predict local tumor control after radiotherapy. Although long-term results are ultimately needed, the combination therapy of radiotherapy with use of a COX-2 inhibitor could yield improved outcomes for patients with COX-2 expressing cervical cancer.

Analysis of non-genetic risk factors for adverse skin reactions to radiotherapy among 284 breast cancer patients.

OBJECTIVES: We analyzed non-genetic risk factors for adverse skin reactions to irradiation at 4 collaborating Japanese institutions, to design future investigation into genetic risk factors for adverse skin reactions to irradiation in a multicenter setting. METHODS: From April 2001, 284 breast cancer patients, who underwent radiotherapy with breast-conserving surgery, were enrolled from 4 collaborating institutions in Japan. We graded skin reactions according to international scoring systems. Clinical factors were tested against adverse effects. RESULTS: Grade 1+ skin reactions were observed in 261 (92%) of the patients in less than 3 months, 118 (42%) at 3 months, and 29 (10%) at 6 months in the late phase. Univariate analysis of treatment risk factors (such as the use of a multi-leaf colimeter, wedge-filter, or immobilization device) for skin reactions revealed a significant association (p< 0.0001). After a variable selection procedure with logistic regression, the institution, operative procedure, and magnitude of photon energy remained significantly associated with acute skin reactions. Only the institution was an explanatory variable for skin reactions at 3 and 6 months in the final logistic model. CONCLUSION: After stratification, substantial remaining variations in the occurrence of skin reactions of a given level suggested that individual genetic factors contribute markedly to individual radiosensitivity. Analysis of genetic factors associated with adverse effects would be possible by stratifying patients according to institution. Selection of eligible institutions, where appropriate treatment modalities could be performed, would also be possible when planning such a study.

Reliable and fast allele-specific extension of 3'-LNA modified oligonucleotides covalently immobilized on a plastic base, combined with biotin-dUTP mediated optical detection.

In the present work, a convenient microarray SNP typing system has been developed using a plastic base that covalently immobilizes amino-modified oligonucleotides. Reliable SNP allele discrimination was achieved by using allelic specificity-enhanced enzymatic extension of immobilized oligonucleotide primer, with a locked nucleic acid (LNA) modification at the SNP-discriminating 3'-end nucleotide. Incorporation of multiple biotin-dUTP molecules during primer extension, followed by binding of alkaline phosphatase-conjugated streptavidin, allowed optical detection of the genotyping results through precipitation of colored alkaline phosphatase substrates onto the surface of the plastic base. Notably, rapid primer extension was demonstrated without a preliminary annealing step of double-stranded template DNA, allowing overall processes to be performed within a couple of hours. Simultaneous evaluation of three SNPs in the genes TGFB1, SOD2 and APEX1, previously investigated for association with radiation sensitivity, in 25 individuals has shown perfect assignment with data obtained by another established technique (MassARRAY system).

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.

DNA repair capacity measured by high throughput alkaline comet assays in EBV-transformed cell lines and peripheral blood cells from cancer patients and healthy volunteers.

We collected peripheral blood (PB) from 556 patients with various types of cancer who had undergone radiotherapy and from 81 healthy volunteers. We exposed whole PB and Epstein-Barr virus-transformed lymphoblastoid cell lines (EBLs) derived from the PB mononucleocytes to X-irradiation (5 Gy). Using the alkaline comet assay, we measured the immediate DNA damage and, at 15 min, the % residual damage. In PB, the immediate damage was similar in patients and healthy volunteers while the % residual damage (mean+/-S.D.) was significantly higher in patients with breast (54.3+/-A23.9), cervical (54.7+/-A23.9), head/neck (56.8+/-A24.4), lung (60.1+/-23.5), or esophageal cancers (59.5+/-A33.7) than in healthy donors (42.9+/-19.6) (P<0.05). We did not observe such differences in the EBV-transformed cell lines. Thus, radiation sensitivity of fresh PB cells measured by the alkaline comet assay was related to cancer status.

Modified radiosensitivity of pancreatic cancer xenografts by farnesyl protein transferase inhibitor and MEK inhibitor.

We investigated the effects of the farnesyl transferase inhibitor (FTI) manumycin and the MEK inhibitor PD98059 on growth of human pancreatic cancer, with mutant (SUIT2) or wild-type (BxPC-3) K-ras, xenografted into nude mice. Tumor growth was not reduced by either of the agents at a dose of 3 mg/kg without irradiation. Growth of SUIT2 irradiated at 15 Gy or 30 Gy was reduced by manumycin and PD98059: at 15 Gy, tumor volume doubling time (TVDT) increased from 18.6+/-3.8 to 36.3+/-14.2 days with PD98059 (p<0.05); at 30 Gy, TVDT increased from 32.8+/-6.8 to 70.5+/-10.5 days and 70.7+/-1.5 days, respectively. Manumycin tended to reduce growth of BxPC-3, but the difference in TVDT was not statistically significant. PD98059 significantly increased the TVDT of BxPC-3 at 30 Gy from 34.4+/-18 to 62.6+/-9.8 at 30 Gy. The present results suggest that Ras signaling pathways are potential targets for manipulation of radiosensitivity, and that induction of an alternative pathway may enhance radiosensitivity of pancreatic cancer.

Strain dependent differences in a histological study of CD44 and collagen fibers with an expression analysis of inflammatory response-related genes in irradiated murine lung.

Using a mouse model, we investigated the mechanisms of heterogeneity in response to ionizing radiation in this research. C57BL/6J and C3H/HeMs mice were irradiated with gamma rays at 10 and 20 Gy. The animals were sacrificed at times corresponding to the latent period, the pneumonic phase, and the start of the fibrotic phase for histological investigation. Small areas of fibrosis initially appeared in C57BL/6J mice at 4 weeks postirradiation with 20 Gy, whereas small inflammatory lesions appeared at 4 and 8 weeks after 20 and 10 Gy, respectively. The alveoli septa were thickened by an infiltration of inflammatory cells, and alveoli were obliterated in lungs from C57BL/6J mice after 20 Gy irradiation. At 24 hours and from 2 to 4 weeks postirradiation, fourfold more CD44 positive cells had accumulated in the lungs of C3H/HeMs than in C57BL/6J mice. Hyaluronan accumulated 12 hours after irradiation, and the rapid resolution was achieved within 2 weeks in the lungs in both strains of mice. C57BL/6J mice lungs accumulated dense collagen at 8 weeks. Quantitative RT-PCR assay was performed for several genes selected by cDNA microarray analysis. The expression of several genes, such as Cap1, Il18, Mmp12, Per3, Ltf, Ifi202a, and Rad51ap1 showed strain-dependent variances. In conclusion, a histological investigation suggested that C3H/HeMs mice were able to induce a more rapid clearance of matrix after irradiation than C57BL/6J mice. The expression analysis showed that the several genes are potentially involved in interstrain differences in inflammatory response causing radiation-induced lung fibrosis.