Curcumin analog, GO-Y078, overcomes resistance to tumor angiogenesis inhibitors.

Tumor angiogenesis inhibition is one of the most potent strategies in cancer chemotherapy. From past clinical studies, inhibition of the vascular endothelial growth factor pathway successfully treats malignant tumors. However, vascular endothelial growth factor inhibitors alone cannot cure tumors. Moreover, resistance to small molecule inhibitors has also been reported. Herein, we show the antiangiogenic potential of a newly synthesized curcumin analog, GO-Y078, that possibly functions through inhibition of actin stress fiber formation, resulting in mobility inhibition; this mechanism is different from that of vascular endothelial growth factor inhibition. In addition, we examined the detailed mechanism of action of the antiangiogenesis potential of GO-Y078 using human umbilical venous epithelial cells resistant to angiogenesis inhibitors (HUVEC-R). GO-Y078 inhibited the growth and mobility of HUVEC-R at 0.75 µmol/L concentration. Expression analyses by microarray and RT-PCR showed that expressions of genes including that of fibronectin 1 were significantly suppressed. Among these genes, fibronectin 1 is abundantly expressed and, therefore, seems to be a good target for GO-Y078. In a knockdown experiment using Si-oligo of fibronectin 1 (FN1), FN1 expression was decreased to half of that in mock experiments as well as GO-Y078. Knockdown of FN1 resulted in the suppression of HUVEC-R growth at 24 hours after treatment. Fibronectin is a key molecule contributing to angiogenesis that could be inhibited by GO-Y078. Thus, resistance to vascular endothelial growth factor inhibition can be overcome using GO-Y078.

Thymine DNA glycosylase modulates DNA damage response and gene expression by base excision repair-dependent and independent mechanisms.

Thymine DNA glycosylase (TDG) is a base excision repair (BER) enzyme, which is implicated in correction of deamination-induced DNA mismatches, the DNA demethylation process and regulation of gene expression. Because of these pivotal roles associated, it is crucial to elucidate how the TDG functions are appropriately regulated in vivo. Here, we present evidence that the TDG protein undergoes degradation upon various types of DNA damage, including ultraviolet light (UV). The UV-induced degradation of TDG was dependent on proficiency in nucleotide excision repair and on CRL4CDT2 -mediated ubiquitination that requires a physical interaction between TDG and DNA polymerase clamp PCNA. Using the Tdg-deficient mouse embryonic fibroblasts, we found that ectopic expression of TDG compromised cellular survival after UV irradiation and repair of UV-induced DNA lesions. These negative effects on cellular UV responses were alleviated by introducing mutations in TDG that impaired its BER function. The expression of TDG induced a large-scale alteration in the gene expression profile independently of its DNA glycosylase activity, whereas a subset of genes was affected by the catalytic activity of TDG. Our results indicate the presence of BER-dependent and BER-independent functions of TDG, which are involved in regulation of cellular DNA damage responses and gene expression patterns.

Differences in sustained alterations in protein expression between livers of mice exposed to high-dose-rate and low-dose-rate radiation.

Molecular mechanisms of radiation dose-rate effects are not well understood. Among many possibilities, long-lasting sustained alterations in protein levels would provide critical information. To evaluate sustained effects after acute and chronic radiation exposure, we analyzed alterations in protein expression in the livers of mice. Acute exposure consisted of a lethal dose of 8 Gy and a sublethal dose of 4 Gy, with analysis conducted 6 days and 3 months after irradiation, respectively. Chronic irradiation consisted of a total dose of 8 Gy delivered over 400 days (20 mGy/day). Analyses following chronic irradiation were done immediately and at 3 months after the end of the exposure. Based on antibody arrays of protein expression following both acute lethal and sublethal dose exposures, common alterations in the expression of two proteins were detected. In the sublethal dose exposure, the expression of additional proteins was altered 3 months after irradiation. Immunohistochemical analysis showed that the increase in one of the two commonly altered proteins, MyD88, was observed around blood vessels in the liver. The alterations in protein expression after chronic radiation exposure were different from those caused by acute radiation exposures. Alterations in the expression of proteins related to inflammation and apoptosis, such as caspase 12, were observed even at 3 months after the end of the chronic radiation exposure. The alterations in protein expression depended on the dose, the dose rate, and the passage of time after irradiation. These changes could be involved in long-term effects of radiation in the liver.

NSAIDs diclofenac, indomethacin, and meloxicam highly upregulate expression of ICAM-1 and COX-2 induced by X-irradiation in human endothelial cells.

BACKGROUND: It is well known that radiation exposure to the heart and the use of non-steroidal anti-inflammatory drugs (NSAIDs) increase the risk of myocardial infarction (MI). Some NSAIDs are also known to act synergistically with ionizing radiation and have radio-sensitizing effects in radiotherapy. These evidences suggest that NSAIDs may affect the risk of MI after radiation exposure to the heart. In the present study, we investigated effects of NSAIDs on radiation-induced expression of cell adhesion molecules and COX-2, which are associated with inflammation and an increased risk of MI, in human endothelial cells. METHODS: Effects of NSAIDs on radiation-induced expression of ICAM-1, VCAM-1, E-selectin, and COX-2 were investigated in human umbilical vein endothelial cells (HUVECs). As NSAIDs, diclofenac, etodolac, indomethacin, ketoprofen, meloxicam, and rofecoxib were used. RESULTS: Irradiation with 10 Gy increased expression of ICAM-1 and COX-2, but it did not affect expression of VCAM-1 or E-selectin. All the NSAIDs upregulated radiation-induced expression of ICAM-1 and COX-2. The extent of upregulation varied depending on the types of NSAIDs. Indomethacin, diclofenac, and meloxicam highly upregulated radiation-induced expression of ICAM-1 and COX-2. The extent of upregulation was not related to the degree of COX-2 selectivity. An NF-κB inhibitor BAY 11-7082 suppressed radiation-induced expression of ICAM-1, but it did not suppress upregulated expression of ICAM-1 or COX-2 by combination treatment with X-irradiation and meloxicam, suggesting the existence of NF-κB-independent pathways for ICAM-1 and COX-2 induction. CONCLUSION: Indomethacin, diclofenac, and meloxicam highly upregulated radiation-induced expression of ICAM-1 and COX-2 in HUVECs, which suggests that use of these NSAIDs may increase the effects of ionizing radiation and affect the risk of MI after radiation exposure to the heart.

A diarylpentanoid curcumin analog exhibits improved radioprotective potential in the intestinal mucosa.

PURPOSE: To best enhance the effects of radiotherapy, it is important to minimize adverse events, including free radical-induced intestinal cell damage. Given the threat of nuclear power plant accidents or nuclear terrorism, there is an urgent need for radioprotectants to counteract the radiation-induced toxicity and/or injuries. Curcumin exhibits protective effects against gamma irradiation; however, its in vivo efficacy is decreased due to the low bioavailability. We examined the radioprotective effect of a newly synthesized curcumin analog, GO-Y031, on 11-Gy X-ray-induced intestinal mucosal damage in mice. MATERIALS AND METHODS: The radioprotection experiments were conducted by using C57BL/6J or Jcl:ICR mice. Molecules related to radiation damage, including p53, Bax, Bcl-2, cleaved caspase-3, and reactive carbonyl species (RCS), were investigated immunohistochemically. RESULTS: GO-Y031 protected against crypt hypoplasia relative to a mock treatment at 0.5% (weight/weight); the number of crypts were 11.00 ± 2.00/circumference (mm) in treated versus 6.86 ± 0.99/mm in mock-treated C57BL/6 mice (p = 0.0079). GO-Y031 also reduced the levels of RCS, p53, and cleaved caspase-3 accumulation in the irradiated intestinal cells. CONCLUSIONS: GO-Y031 suppresses the accumulation of RCS and apoptosis-related molecules in irradiated cells. This compound may be a good primary radioprotective compound.

Activation of mTORC1 under nutrient starvation conditions increases cellular radiosensitivity in human liver cancer cell lines, HepG2 and HuH6.

BACKGROUND: The presence of unperfused regions containing cells under hypoxic and nutrient starvation conditions contributes to radioresistance in solid human tumors. It is well known that the hypoxia causes cellular radioresistance. However, the effects of nutrient starvation conditions on cellular radiosensitivity remain unclear. METHODS: Human liver cancer cell lines, HepG2 and HuH6, and a SV40-transformed human fibroblast cell line, LM217 were used to examine the effects of nutrient starvation conditions on cellular radiosensitivity and on activity of mammalian target of rapamycin complex 1 (mTORC1) that senses cellular nutrient conditions and affects radiosensitivity. RESULTS: In contrast to suppressed mTORC1 activity under nutrient starvation conditions in LM217, HepG2 and HuH6 cells showed increased mTORC1 activity under nutrient starvation conditions. Both AMP-activated protein kinase (AMPK) and Akt were activated under nutrient starvation conditions in all the three cell lines. Under starvation conditions, increased radiosensitivity was observed in HepG2 and HuH6 cells, in contrast to decreased radiosensitivity in LM217 cells. Knockdown of mTOR using siRNA for mTOR or treatment with a mTOR inhibitor, rapamycin, suppressed the increased radiosensitivity under starvation conditions in HepG2 cells. CONCLUSION: Our data show for the first time that nutrient starvation conditions activate mTORC1 and increase radiosensitivity through mTORC1 activation in liver cancer cell lines, HepG2 and HuH6.

Inhibition of ß-catenin and STAT3 with a curcumin analog suppresses gastric carcinogenesis in vivo.

BACKGROUND: Potent chemotherapy for advanced gastric cancer has not been completely established. Many molecularly targeted therapies are under investigation, but their therapeutic outcomes are not promising because they do not target specific and/or critical targets of gastric carcinogenesis. Although the molecular basis of gastric carcinogenesis remains poorly understood, nuclear localization of ß-catenin was observed in approximately 50 % of gastric cancer specimens. Recent studies have suggested that activation of signal transducer and activator of transcription 3 (STAT3) contributes to gastric carcinogenesis in a mouse model. A newly synthesized curcumin analog has inhibitory potential against ß-catenin and STAT3. METHODS: Using a transgenic mouse model of gastric cancer in which ß-catenin, cyclooxygenase 2, and microsomal prostaglandin E synthase 1 activation is induced, we examined a curcumin analog with the most enhanced potential for treating gastric cancer through oral administration. Inhibition of these targets was demonstrated using microarray and immunohistochemical analyses. RESULTS: The curcumin analog GO-Y031 decreased the incidence of gastric carcinogenesis to 54.5 % of that of the control (50.0 % vs 91.7 %, p = 0.043), and tumor size was reduced to 51.6 % of that of the control (1.6 mm vs 3.1 mm, p = 0.03). ß-Catenin and STAT3 levels were suppressed to 26.2 % (p = 0.00023) and 44.8 % (p = 0.025), respectively, of those of the control. Moreover, macrophage infiltration was suppressed with GO-Y031. CONCLUSION: ß-Catenin and STAT3 can be pharmacologically inhibited in vivo with a curcumin analog, which effectively inhibits ß-catenin and STAT3.

Low-dose total-body carbon-ion irradiations induce early transcriptional alteration without late Alzheimer's disease-like pathogenesis and memory impairment in mice.

The cause and risk factors of Alzheimer's disease (AD) are largely unknown. Studies on possible radiation-induced AD-like pathogenesis and behavioral consequences are important because humans are exposed to ionizing radiation (IR) from various sources. It was reported that total-body irradiations (TBI) at 10 cGy of low linear energy transfer (LET) X-rays to mice triggered acute transcriptional alterations in genes associated with cognitive dysfunctions. However, it was unknown whether low doses of IR could induce AD-like changes late after exposure. We reported previously that 10 cGy X-rays induced early transcriptional response of several AD-related genes in hippocampi without late AD-like pathogenesis and memory impairment in mice. Here, further studies on two low doses (5 or 10 cGy) of high LET carbonion irradiations are reported. On expression of 84 AD-related genes in hippocampi, at 4 hr after TBI, 5 cGy induced a significant upregulation of three genes (Abca1, Casp3, and Chat) and 10 cGy led to a marked upregulation of one gene (Chat) and a downregulation of three genes (Apoe, Ctsd, and Il1α), and, at 1 year after TBI, one gene (Il1α) was significantly downregulated in 10 cGy-irradiated animals. Changes in spatial learning ability and memory and induction of AD-like pathogenesis were not detected by in vivo brain imaging for amyloid-ß peptide accumulation and by immunohistochemical staining of amyloid precursor protein, amyloid-ß protein, tau, and phosphorylated tau protein. These findings indicate that low doses of carbon-ion irradiations did not cause behavioral impairment or AD-like pathological change in mice.

Total body 100-mGy X-irradiation does not induce Alzheimer's disease-like pathogenesis or memory impairment in mice.

The cause and progression of Alzheimer's disease (AD) are poorly understood. Possible cognitive and behavioral consequences induced by low-dose radiation are important because humans are exposed to ionizing radiation from various sources. Early transcriptional response in murine brain to low-dose X-rays (100 mGy) has been reported, suggesting alterations of molecular networks and pathways associated with cognitive functions, advanced aging and AD. To investigate acute and late transcriptional, pathological and cognitive consequences of low-dose radiation, we applied an acute dose of 100-mGy total body irradiation (TBI) with X-rays to C57BL/6J Jms mice. We collected hippocampi and analyzed expression of 84 AD-related genes. Mouse learning ability and memory were assessed with the Morris water maze test. We performed in vivo PET scans with (11)C-PIB, a radiolabeled ligand for amyloid imaging, to detect fibrillary amyloid beta peptide (Aß) accumulation, and examined characteristic AD pathologies with immunohistochemical staining of amyloid precursor protein (APP), Aß, tau and phosphorylated tau (p-tau). mRNA studies showed significant downregulation of only two of 84 AD-related genes, Apbb1 and Lrp1, at 4 h after irradiation, and of only one gene, Il1α, at 1 year after irradiation. Spatial learning ability and memory were not significantly affected at 1 or 2 years after irradiation. No induction of amyloid fibrillogenesis or changes in APP, Aß, tau, or p-tau expression was detected at 4 months or 2 years after irradiation. TBI induced early or late transcriptional alteration in only a few AD-related genes but did not significantly affect spatial learning, memory or AD-like pathological change in mice.

Role of the Msh2 gene in genome maintenance and development in mouse fetuses.

In an attempt to evaluate the roles of the mismatch repair gene Msh2 in genome maintenance and in development during the fetal stage, spontaneous mutations and several developmental indices were studied in Msh2-deficient lacZ-transgenic mouse fetuses. Mutation levels in fetuses were elevated at 9.5 dpc (days post coitum) when compared to wild-type mice, and the level of mutations continued to increase until the fetuses reached the newborn stage. The mutation levels in 4 different tissues of newborns showed similar magnitudes to those in the whole body. The levels remained similar after birth until 6 months of age. The molecular nature of the mutations examined in 12.5 dpc fetuses of Msh2(+/+) and Msh2(-/-) revealed unique spectra which reflect errors produced during the DNA replication process, and those corrected by a mismatch repair system. Most base substitutions and simple deletions were reduced by the presence of the Msh2 gene, whereas G:C to A:T changes at CpG sequences were not affected, suggesting that the latter change was not influenced by mismatch repair. On the other hand, analysis of developmental indices revealed that there was very little effect, including the presence of malformations, resulting from Msh2-deficiencies. These results indicate that elevated mutation levels have little effect on the development of the fetus, even if a mutator phenotype appears at the organogenesis stage.

Embryonic lethality in mice lacking mismatch-specific thymine DNA glycosylase is partially prevented by DOPS, a precursor of noradrenaline.

Thymine DNA glycosylase (TDG) is involved in the repair of G:T and G:U mismatches caused by hydrolytic deamination of 5-methylcytosine and cytosine, respectively. Recent studies have shown that TDG not only has G-T/U glycosylase activities but also acts in the maintaining proper epigenetic status. In order to investigate the function of TDG in vivo, mice lacking Tdg, Tdg (-/-), were generated. Tdg mutant mice died in utero by 11.5 days post coitum (dpc), although there were no significant differences in the spontaneous mutant frequencies between wild type and Tdg (-/-) embryos. On the other hand, the levels of noradrenaline in 10.5 dpc whole embryos, which is necessary for normal embryogenesis, were dramatically reduced in Tdg (-/-) embryos. Consequently, we tested the effect of D, L-threo-3, 4-dihydroxyphenylserine (DOPS), a synthetic precursor of noradrenaline, on the survival of the Tdg (-/-) embryos. DOPS was given to pregnant Tdg (+/-) mice from 6.5 dpc through drinking water. Most of the Tdg (-/-) embryos were alive at 11.5 dpc, and they were partially rescued up to 14.5 dpc by the administration of DOPS. In contrast, the administration of L-3, 4-dihydroxyphenylalanine (L-DOPA) had marginal effects on Tdg (-/-) embryonic lethality. No embryo was alive without DOPS beyond 11.5 dpc, suggesting that the lethality in (-/-) embryos is partially due to the reduction of noradrenaline. These results suggest that embryonic lethality in Tdg (-/-) embryos is due, in part, to the reduction of noradrenaline levels.

Effects of calorie restriction on the age-dependent accumulation of mutations in the small intestine of lacZ-transgenic mice.

To understand the effect of calorie restriction on genome maintenance systems, the age-dependent accumulation of mutations in animals maintained on high and low calorie diets was examined using lacZ-transgenic mice. Mice were fed a diet of 95 kcal/w or 65 kcal/w from 2 to 17 months of age. The mutation frequencies in the lacZ gene in epithelial tissues from the small intestine were examined at 12 and 17 months. Mutation frequencies were found to be lower in mice fed with a low calorie diet than in mice fed with a high calorie diet at the two age points. The molecular nature of the mutations was examined with DNA sequencing. It showed a predominance of transversions from G:C to T:A, and this is a typical type of mutation induced by reactive oxygen species. The fraction of this type of mutation among the different types of mutations detected was not affected by calorie restriction. The percentage of the other types of mutation was not influenced either. These results suggest that calorie restriction reduces the age-dependent accumulation of mutations by stimulating or inducing various types of DNA protection and repair systems rather than protecting cells against any specific type of DNA alteration.

Transcription factor-recognition sequences potentially involved in modulation of gene expression after exposure to low-dose-rate γ-rays in the mouse liver.

In vivo modulation of gene expression profiles after low-dose and low-dose-rate irradiation has been observed in a variety of experimental systems. However, few studies actually investigated the underlying mechanisms for these genetic responses. In this study, we used pre-existing microarray data and searched for gene modulations in response to long-term, low-dose-rate irradiation. Nucleotide sequences in the neighboring region of the up-regulated, down-regulated, and unaffected genes were retrieved from the Entrez Gene database, and recognition sequences for transcription factors (TFs) were searched using the TFSEARCH database. As a result, we suggested 21 potential TF-binding sites with significantly different incidence between the three gene groups (up-regulated, down-regulated and unaffected gene groups). The binding sites for sterol regulatory element-binding protein 1 (SREBP-1), aryl hydrocarbon receptor (AhR/Ar) and olfactory 1 (Olf-1) were suggested to be involved in up-regulation, while the binding sites for glucocorticoid receptor (GR(GGTACAANNT GTYCTK) ) and hepatocyte nuclear factor 1 (HNF-1) were suggested to be involved in down-regulation of the genes. In addition, the binding sites for activating enhancer-binding protein 4 (AP-4), nuclear factor-κB (NFκB), GR (NNNNNNCNNTNTGTNCTNN) and early growth response 3 (Egr-3) were correlated with modulation of gene expression regardless of the direction of modulation. Our results suggest that these TF-binding sites are involved in gene modulations after long-term continuous irradiation with low-dose-rate γ rays. GR and/or SREBP-1 might be associated with the altered metabolic process observed in liver after exposure to low-dose-rate irradiation.

Curcumin analog GO-Y030 is a novel inhibitor of IKKß that suppresses NF-κB signaling and induces apoptosis.

Curcumin is a dietary constituent with tumor-suppressing potential, inhibiting various pathways involved in carcinogenesis. However, because of its low bioavailability, the use of curcumin in in vivo trials has been limited. To overcome this problem, we synthesized more than 50 analogs and identified a monoketone analog, GO-Y030, which has a 30-fold higher potential to suppress tumor cell growth compared with curcumin. We investigated the inhibitory effect of GO-Y030 on NF-κB activation. In thyroid, pancreatic cancers and cholangiocarcinoma cells, in which NF-κB is activated, NF-κB activation was suppressed to 8-62% of the control value following treatment with 1 µM GO-Y030, an effect comparable to that of 10 µM curcumin. Direct inhibition of IKKß kinase activity and suppression of nuclear translocation of the NF-κB p65 subunit were observed. The 50% growth inhibition concentrations of GO-Y030 ranged from one-11th to one-14th of those of curcumin. GO-Y030 also induced cell death comparable to that induced by curcumin but at a 10-fold lower concentration. In pancreatic and thyroid cancer cells, the growth-inhibitory effect of GO-Y030 was 4- and 15-fold greater, respectively, than that of curcumin. GO-Y030 was a much stronger inducer of apoptosis compared with curcumin. The enhanced potency of GO-Y030 may make it more useful than curcumin, which suffers from low bioavailability. GO-Y030 is a good lead compound for the development of useful compounds for practical cancer chemotherapy.

Gene expression profiles in mouse liver after long-term low-dose-rate irradiation with gamma rays.

Changes in gene expression profiles in mouse liver induced by long-term low-dose-rate γ irradiation were examined by microarray analysis. Three groups of male C57BL/6J mice were exposed to whole-body radiation at dose rates of 17-20 mGy/day, 0.86-1.0 mGy/day or 0.042-0.050 mGy/day for 401-485 days with cumulative doses of approximately 8 Gy, 0.4 Gy or 0.02 Gy, respectively. The gene expression levels in the livers of six animals from each exposure group were compared individually with that of pooled sham-irradiated animals. Some genes revealed a large variation in expression levels among individuals within each group, and the number of genes showing common changes in individuals from each group was limited: 20 and 11 genes showed more than 1.5-fold modulation with 17-20 mGy/day and 0.86-1.0 mGy/day, respectively. Three genes showed more than 1.5-fold modulation even at the lowest dose-rate of 0.04-0.05 mGy/day. Most of these genes were down-regulated. RT-PCR analysis confirmed the expression profiles of the majority of these genes. The results indicate that a few genes are modulated in response to very low-dose-rate irradiation. The functional analysis suggests that these genes may influence many processes, including obesity and tumorigenesis.

Radiation dose-rate effect on mutation induction in spleen and liver of gpt delta mice.

The effect of dose rate on radiation-induced mutations in two somatic tissues, the spleen and liver, was examined in transgenic gpt delta mice. These mice can be used for the detection of deletion-type mutations, and these are the major type of mutation induced by radiation. The dose rates examined were 920 mGy/min, 1 mGy/min and 12.5 microGy/min. In both tissues, the number of mutations increased with increasing dose at each of the three dose rates examined. The mutation induction rate was dependent on the dose rate. The mutation induction rate was higher in the spleen than in the liver at the medium dose rate but was similar in the two tissues at the high and low dose rates. The mutation induction rate in the liver did not show much change between the medium and low dose rates. Analysis of the molecular nature of the mutations indicated that 2- to 1,000-bp deletion mutations were specifically induced by radiation in both tissues after high- and low-dose-rate irradiation. The occurrence of deletion mutation without any sequence homology at the break point was elevated in spleen after high-dose-rate irradiation. The results indicate that the mutagenic effects of radiation in somatic tissues are dependent on dose rate and that there is some variability between tissues.

XPC is involved in genome maintenance through multiple pathways in different tissues.

In an attempt to evaluate the role of the Xpc gene in maintaining genomic stability in vivo under normal conditions, the age-dependent accumulation of spontaneous mutations in different tissues was analyzed in Xpc-deficient lacZ-transgenic mice. Brain, testis, and small intestine revealed no effects from the Xpc-deficiency, whereas liver, spleen, heart, and lung showed an enhanced age-related accumulation of mutations in Xpc-deficient mice. In the spleen, the effect was not obvious at 2 and 12 months of age, but became apparent at 23 months. The magnitude of the observed effect at an advanced age was similar in the liver, spleen and heart, but was comparatively smaller in the lung. Haploinsufficiency was observed in liver and spleen but not in heart and lung. Analysis of DNA sequences in the mutants revealed that the frequency of G:C to T:A changes were elevated in the liver and heart of Xpc-deficient aged mice, supporting the possible involvement of XPC in base excision repair of oxidized guanine. The occurrence of two or more mutations within a single lacZ gene was termed a multiple mutation and was also elevated in old Xpc-deficient mice. Among the clones examined, two mutant clones showed as many as four mutations within a short stretch of DNA. This is the first demonstration to support suggestions for the existence of a role for XPC in the suppression of multiple mutations. These multiple mutations could conceivably be generated by error-prone trans-lesional DNA synthesis. Overall, these results indicate that there may be diverse roles or mechanisms through which XPC participates in genome maintenance in different tissues.

Microarray analysis of differentially expressed genes in the kidneys and testes of mice after long-term irradiation with low-dose-rate gamma-rays.

Measuring global gene expression using cDNA or oligonucleotide microarrays is an effective approach to understanding the complex mechanisms of the effects of radiation. However, few studies have been carried out that investigate gene expression in vivo after prolonged exposure to low-dose-rate radiation. In this study, C57BL/6J mice were continuously irradiated with gamma-rays for 485 days at dose-rates of 0.032-13 microGy/min. Gene expression profiles in the kidney and testis from irradiated and unirradiated mice were analyzed, and differentially expressed genes were identified. A combination of pathway analysis and hierarchical clustering of differentially expressed genes revealed that expression of genes involved in mitochondrial oxidative phosphorylation was elevated in the kidney after irradiation at the dose-rates of 0.65 microGy/min and 13 microGy/min. Expression of cell cycle-associated genes was not profoundly modulated in the kidney, in contrast to the response to acute irradiation, suggesting a threshold in the dose-rate for modulation of the expression of cell cycle-related genes in vivo following exposure to radiation. We demonstrated that changes to the gene expression profile in the testis were largely different from those in the kidney. The Gene Ontology categories "DNA metabolism", "response to DNA damage" and "DNA replication" overlapped significantly with the clusters of genes whose expression decreased with an increase in the dose-rate to the testis. These observations provide a fundamental insight into the organ-specific responses to low-dose-rate radiation.

Absence of Ku70 gene obliterates X-ray-induced lacZ mutagenesis of small deletions in mouse tissues.

With the goal of understanding the role of non-homologous end-joining repair in the maintenance of genetic information at the tissue level, we studied mutations induced by radiation and subsequent repair of DNA double-strand breaks in Ku70 gene-deficient lacZ transgenic mice. The local mutation frequencies and types of mutations were analyzed on a lacZ gene that had been chromosomally integrated, which allowed us to monitor DNA sequence alterations within this 3.1-kbp region. The mutagenic process leading to the development of the most frequently observed small deletions in wild-type mice after exposure to 20 Gy of X rays was suppressed in Ku70(-/-) mice in the three tissues examined: spleen, liver and brain. Examination of DNA break rejoining and the phosphorylation of histone H2AX in Ku70-deficient and -proficient mice revealed that Ku70 deficiency decreased the frequency of DNA rejoining, suggesting that DNA rejoining is one of the causes of radiation-induced deletion mutations. Limited but statistically significant DNA rejoining was found in the liver and brain of Ku70-deficient mice 3.5 days after irradiation, showing the presence of a DNA double-strand break repair system other than non-homologous end joining. These data indicate a predominant role of non-homologous end joining in the production of radiation-induced mutations in vivo.

Spontaneous mutations in digestive tract of old mice show tissue-specific patterns of genomic instability.

In an attempt to evaluate the possible role of mutations in the age-dependent increase of tumor incidence, we studied the mutational burden that accumulates in the aging process in different parts of the digestive tract in mice. The mutations were monitored in lacZ genes integrated in the mouse genome. The digestive tract was divided into the esophagus, stomach, proximal, medial, and distal part of the small intestine, and the colon. Epithelial tissues were separated from these tissues with the exception of the esophagus, in which case the whole tissue was examined. At a young age, the mutant frequencies as well as the molecular nature of the mutations were similar among the tissues examined. In old age, on the other hand, mutant frequencies were elevated to different degrees among the tissues; they were high in the small intestine and colon, intermediate in the stomach, and low in the esophagus. The molecular characteristics of the mutations also revealed distinct tissue-specificity; there were elevated rates of a small deletion mutation in the esophagus, G:C to T:A transversion in the proximal small intestine, and multiple mutations in the distal small intestine and colon. The results indicate that different parts of the digestive tract suffer from different kinds of mutational stress in the aging process. The nature of the multiple mutations suggests the presence of a mutator phenotype based on an imbalance in deoxyribonucleotide pools.