Long interspersed element-1 is differentially regulated by food-borne carcinogens via the aryl hydrocarbon receptor.

A single human cell contains more than 5.0 × 10(5) copies of long interspersed element-1 (L1), 80-100 of which are competent for retrotransposition (L1-RTP). Recent observations have revealed the presence of de novo L1 insertions in various tumors, but little is known about its mechanism. Here, we found that 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,8-dimethyl-imidazo[4,5-f]quinoxaline (MeIQx), food-borne carcinogens that are present in broiled meats, induced L1-RTP. This induction was dependent on a cellular cascade comprising the aryl hydrocarbon receptor (AhR), a mitogen-activated protein kinase, and CCAAT/enhancer-binding protein ß. Notably, these compounds exhibited differential induction of L1-RTP. MeIQx-induced L1-RTP was dependent on AhR nuclear translocator 1 (ARNT1), a counterpart of AhR required for gene expression in response to environmental pollutants. By contrast, PhIP-induced L1-RTP did not require ARNT1 but was dependent on estrogen receptor α (ERα) and AhR repressor. In vivo studies using transgenic mice harboring the human L1 gene indicated that PhIP-induced L1-RTP was reproducibly detected in the mammary gland, which is a target organ of PhIP-induced carcinoma. Moreover, picomolar levels of each compound induced L1-RTP, which is comparable to the PhIP concentration detected in human breast milk. Data suggest that somatic cells possess machineries that induce L1-RTP in response to the carcinogenic compounds. Together with data showing that micromolar levels of heterocyclic amines (HCAs) were non-genotoxic, our observations indicate that L1-RTP by environmental compounds is a novel type of genomic instability, further suggesting that analysis of L1-RTP by HCAs is a novel approach to clarification of modes of carcinogenesis.

Involvement of JNK pathway in the promotion of the early stage of colorectal carcinogenesis under high-fat dietary conditions.

BACKGROUND AND AIMS: The molecular mechanisms underlying the promotion of colorectal carcinogenesis by a high-fat diet (HFD) remain unclear. We investigated the role of the insulin-signal pathway and the c-Jun N-terminal kinase (JNK) pathway, which reportedly play crucial roles in insulin resistance, during colorectal carcinogenesis in the presence of hyperinsulinaemia induced by a HFD. METHODS: Azoxymethane-induced aberrant crypt foci formation and cell proliferation in the colonic epithelium were compared between mice fed a normal diet (ND) and mice fed a HFD. A western blot analysis was performed to elucidate the mechanism affecting colorectal carcinogenesis by a HFD. RESULTS: The number of aberrant crypt foci and the colonic epithelial cell proliferative activity were significantly higher in the HFD group than in the ND group. While the plasma insulin level was significantly higher in the HFD group than in the ND group, a western blot analysis revealed the inactivation of Akt, which is located downstream of the insulin receptor, in the colonic epithelia of the HFD group. On the other hand, JNK activity was significantly higher in the HFD group than in the ND group. A JNK specific inhibitor significantly suppressed the increase in epithelial cell proliferation only under a HFD, but not under a ND. CONCLUSIONS: Colonic cell proliferation was promoted via the JNK pathway in the presence of a HFD but not in the presence of a ND. This novel mechanism may explain the involvement of the JNK pathway in the effect of dietary fat intake on colon carcinogenesis.

Adiponectin suppresses colorectal carcinogenesis under the high-fat diet condition.

BACKGROUND AND AIMS: The effect of adiponectin on colorectal carcinogenesis has been proposed but not fully investigated. We investigated the effect of adiponectin deficiency on the development of colorectal cancer. METHODS: We generated three types of gene-deficient mice (adiponectin-deficient, adiponectin receptor 1-deficient, and adiponectin receptor 2-deficient) and investigated chemical-induced colon polyp formation and cell proliferation in colon epithelium. Western blot analysis was performed to elucidate the mechanism which affected colorectal carcinogenesis by adiponectin deficiency. RESULTS: The numbers of colon polyps were significantly increased in adiponectin-deficient mice compared with wild-type mice fed a high-fat diet. However, no difference was observed between wild-type and adiponectin-deficient mice fed a basal diet. A significant increase in cell proliferative activity was also observed in the colonic epithelium of the adiponectin-deficient mice when compared with wild-type mice fed a high-fat diet; however, no difference was observed between wild-type and adiponectin-deficient mice fed a basal diet. Similarly, an increase in epithelial cell proliferation was observed in adiponectin receptor 1-deficient mice, but not in adiponectin receptor 2-deficient mice. Western blot analysis revealed activation of mammalian target of rapamycin, p70 S6 kinase, S6 protein and inactivation of AMP-activated protein kinase in the colon epithelium of adiponectin-deficient mice fed with high-fat diet. CONCLUSIONS: Adiponectin suppresses colonic epithelial proliferation via inhibition of the mammalian target of the rapamycin pathway under a high-fat diet, but not under a basal diet. These studies indicate a novel mechanism of suppression of colorectal carcinogenesis induced by a Western-style high-fat diet.

Bcl-2 overexpression in PhIP-induced colon tumors: cloning of the rat Bcl-2 promoter and characterization of a pathway involving beta-catenin, c-Myc and E2F1.

Beta-catenin/T-cell factor (Tcf) signaling is constitutively active in the majority of human colorectal cancers, and there are accompanying changes in Bcl-2 expression. Similarly, 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP)-induced colon tumors in the rat have increased beta-catenin and elevated Bcl-2. To examine the possible direct transcriptional regulation of rat Bcl-2 by beta-catenin/Tcf, we cloned and characterized the corresponding promoter region and found 70.1% similarity with its human counterpart, BCL2. Bcl-2 promoter activity was increased in response to LiCl and exogenous beta-catenin, including oncogenic mutants of beta-catenin found in PhIP-induced colon tumors. Protein/DNA arrays identified E2F1, but not beta-catenin/Tcf, as interacting most strongly with the rat Bcl-2 promoter. Exogenous E2F1 increased the promoter activity of rat Bcl-2, except in mutants lacking the E2F1 sites. As expected, beta-catenin induced its downstream target c-Myc, as well as E2F1 and Bcl-2, and this was blocked by siRNA to c-Myc or E2F1. These findings suggest an indirect pathway for Bcl-2 over-expression in PhIP-induced colon tumors involving beta-catenin, c-Myc and E2F1.

Poly(ADP-ribosyl)ation in relation to cancer and autoimmune disease.

Carcinogenesis involves multiple steps and pathways with functional alterations in a variety of genes. There is accumulating evidence that a deficiency of poly(ADP-ribose) polymerase (PARP)-1 leads to DNA repair defects, genomic instability, failure of induction of cell death and modulation of gene transcription. PARP-1 also supports the growth of tumor cells in certain situations. Genetic analyses of the PARP-1 gene have demonstrated alterations in neoplasms, and a mutation affecting the conserved amino acid E251 in germ cell tumors, as well as an association of a single-nucleotide polymorphism V762A with risk of prostate cancer. Recent development of a selective inhibitor of poly(ADP-ribose) glycohydrolase (PARG), the enzyme primarily responsible for degradation of poly(ADP-ribose), and PARG-deficient animals should facilitate studies of the relationship of poly(ADP-ribose) with carcinogenesis. Inhibitors of PARP have also suggested roles in the pathogenesis of autoimmune disease, and a promoter haplotype of PARP-1 confers a higher risk of rheumatoid arthritis. Further analysis of PARP-1, PARG and other PARP family genes should extend our understanding of the pathogenesis of cancer and autoimmune diseases. Furthermore, there is potential for sensitization to chemo- and radiation therapy of cancers as well as the treatment of autoimmune disease with development of stronger PARP inhibitors.

Detection and isolation of minisatellite Pc-1 binding proteins.

Minisatellites (MNs) are arrays of 5-100 nucleotide repeats that are dispersed throughout the genome of vertebrates. They demonstrate alteration in tumors and in cells exposed to various carcinogens, but the molecular mechanisms underlying the induction of mutations at MNs are largely unknown. Hypervariable MN Pc-1 isolated from the mouse genome consists of tandem repeats of d(GGCAG) flanked with locus-specific sequences at both ends. We have found that MN mutations are induced in NIH3T3 cells by treatment with okadaic acid using a Pc-1 MN fragment as a probe. In order to shed light on the molecular mechanisms, we isolated six MN Pc-1 binding proteins, pA, pB, pD, pE, pF and pG, from nuclear extracts of NIH3T3 cells treated with okadaic acid. While pA and pB bound to the G-rich strand of Pc-1, pD, pE, pF and pG bound to the complementary C-rich strand. Sequence specificities for DNA binding were revealed and one base substitution and insertion into the Pc-1 repeat unit dramatically changed the affinity of each protein, suggesting that they bind to Pc-1 and Pc-1-like MNs in vivo.

High susceptibility of Scid mice to colon carcinogenesis induced by azoxymethane indicates a possible caretaker role for DNA-dependent protein kinase.

Severe combined immunodeficiency (Scid) mice have defects in V(D)J recombination and DNA double-strand breaks repair caused by an inherited genetic defect in the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). Scid mice are highly susceptible to development of T-cell lymphomas, and because of the nature of its association with DNA repair and recombination, DNA-PKcs is considered to belong to the caretaker class of tumor suppressor genes. In the present study, the susceptibility of Scid mice to colon carcinogenesis due to administration of azoxymethane (AOM) was investigated. Significantly higher susceptibility in terms of induction of both aberrant crypt foci (ACFs), putative pre-cancerous lesions of the colon and colon cancers was observed as compared with the isogenic strain, C.B-17 mice. The incidences of colon tumors, either adenomas or adenocarcinomas, in Scid and C.B-17 mice after administration of AOM (10 mg/kg body weight/week) for 6 weeks were 87% (26 of 30) and 50% (15 of 30), respectively, by experimental week 22 (P < 0.01). The multiplicity of colon tumors in Scid mice was also significantly higher than in C.B-17 mice, being 2.2 +/- 1.5 and 0.9 +/- 1.2, respectively (P < 0.001). The present study clearly demonstrated high susceptibility of Scid mice to colon carcinogenesis, which might be attributable to disruption of the caretaker role of DNA-PK in colonic epithelial cells.

Involvement of poly(ADP-ribose) polymerase in trophoblastic cell differentiation during tumorigenesis.

Poly(ADP-ribose) polymerase (Parp) monitors DNA strand breaks and poly(ADP-ribosyl)ates nuclear proteins using NAD as a substrate. The participation of Parp in DNA damage responses has been demonstrated by recent studies using Parp knockout mice. On the other hand, accumulated evidence has shown that Parp is involved in the regulation of gene expression and cell differentiation. In this study, the role of Parp in tumorigenesis and differentiation was studied with Parp-/- embryonic stem (ES) cells. When Parp+/+, Parp+/-, and Parp-/- ES cells were injected subcutaneously into nude mice, teratocarcinoma-like tumors developed from ES cells. However, only tumors derived from Parp-/- ES cells showed trophoblast giant cells (TGCs) containing single or multiple megalo-nuclei. These TGCs are located in a large blood-lake like hemorrhage. This example suggests that Parp is not essential for tumor formation, however, it is involved in trophoblastic cell differentiation and could consequently affect tumor phenotype.

Early detection of 2-amino-1-methyl-6-phenylimidazo (4,5-b)pyridine(PhIP)-induced mutations within the Apc gene of rat colon.

A large proportion of human cancers result from exposure of individuals to environmental or occupational carcinogens. The early detection of carcinogen-induced mutations is a prerequisite for the identification of individuals at risk for developing cancer. Short G-rich repetitive sequences have been previously identified as hot-spots for frameshift mutagenesis induced by a large variety of carcinogens belonging to several families of widespread environmental pollutants. In order to test if these sequences, when mutated, might serve as biomarkers for carcinogen exposure, we designed a sensitive PCR-based strategy that allows the detection of rare mutational events within a whole genome. 2-Amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP), the most abundant carcinogenic heterocyclic amine generated in cooked meat, induces mammary and colon carcinoma in F344 rats. About 25% of male rats exposed to 400 p.p.m. PhIP in the diet for >43 weeks present colon tumors with specific -1G mutations within 5'-GGGA-3' sequences of the APC: gene. Using our PCR assay we have assessed the occurrence of such specific events in rats exposed to PhIP for only 1, 2, 4 and 6 weeks. A specific amplification signal was already observed in the 1 week-treated population and increases in a treatment time-dependent manner. These data validate this approach for the early detection of mutations and demonstrate its usefulness for molecular epidemiology and early diagnosis.

Increased susceptibility of poly(ADP-ribose) polymerase-1 knockout mice to nitrosamine carcinogenicity.

The involvement of poly(ADP-ribose) polymerase-1 (Parp-1), one of the poly(ADP-ribose) polymerase family proteins, in genomic stability, DNA repair and cell death triggered by DNA damage has been well documented. However, the potential role of Parp-1 in carcinogenesis has not been well evaluated. In this study the carcinogenic activity of N:-nitrosobis(2-hydroxypropyl)amine (BHP) was studied in Parp-1(-/-) mice, generated by disrupting P:arp-1 gene exon 1. Parp-1(-/-) and Parp-1(+/+) male mice received 0, 250 and 500 p.p.m. BHP in their drinking water for 20 weeks and were then killed. The percentage of animals bearing hemangiomas and hemangiosarcomas in the liver and numbers of tumors per mouse were markedly higher in the Parp-1(-/-) groups given 250 or 500 p.p.m. BHP than in their Parp-1(+/+) counterparts. Hemangiosarcomas developed only in Parp-1(-/-) mice. In the lung the numbers of adenomas per mouse were increased in Parp-1(-/-) mice given BHP at 250 and 500 p.p.m. (P < 0.01) compared with the Parp-1(+/+) case. The results show that susceptibility to BHP is significantly elevated in Parp-1(-/-) mice, thus providing direct evidence that Parp-1 is relevant to carcinogenesis.

Characterization of adriamycin-induced G2 arrest and its abrogation by caffeine in FL-amnion cells with or without p53.

We investigated the effect of Adriamycin on FL-amnion (FL) cells. After treatment with the drug, the cells arrested at G2, but we did not detect an increase in the p21 levels. We established a p53-deficient derivative of these cells, in which G2 arrest also occurred after treatment with Adriamycin, suggesting that the arrest we observed in these cells is independent of the p53 pathway. Low doses of Adriamycin (100-200 ng/ml) induced G2 arrest, while late S-phase arrest was observed at high doses (500-1000 ng/ml) in both FL and p53-deficient FL cells. Accumulation of cyclin B1 was detected only in cells arrested at G2, and not in those arrested at S phase, suggesting that the S-phase checkpoint functioned efficiently even in p53-deficient FL cells. In both cell lines, caffeine-induced activation of CDC2 kinase was detected only in cells arrested at G2 and CDC2 kinase-activated cells died exhibiting features of apoptosis. CDC2 kinase activation was inhibited by cycloheximide. Furthermore, cycloheximide inhibited activation of CDK2:cyclin A, which normally precedes CDC2 kinase activation in caffeine-treated cells. These results suggest that p53 and p21 do not have special roles in the S- and G2-phase checkpoints and that CDK2:cyclin A could be the target of the G2-phase DNA damage checkpoint.

Expression in hepatomas and chromosomal localization of rat protein phosphatase 5 gene.

The serine/threonine protein phosphatase type 5 (PP5) gene expression was ubiquitously observed among all the tissues examined, but being at the lowest level in the liver. The levels of PP5 mRNA were markedly elevated in rat highly malignant ascites hepatomas, while those in several rat primary hepatomas were slightly increased and those in the regenerating livers were not elevated at all compared to the control liver. The PP5 gene was mapped to rat chromosome 1q22.1. This region has been identified to have linkage homology among human, mouse and rat, and is known to be associated with several tumor types. Taken together, the present results strongly suggest important roles of PP5 in tumorigenesis.

Effect of imbalance in activities between ON- and OFF-center LGN cells on orientation map formation.

It has been reported that the OFF responses of cells in the visual pathway are stronger, on average, than the ON responses early in the life of cats and ferrets. In this study, we theoretically investigate the effects of this imbalance in activity on the orientation map formation. We carry out computer simulations based on our previously proposed self-organization model, in which the correlated activities between ON- and OFF-center cells in the lateral geniculate nucleus regulate the formation of orientation maps in the visual cortex. When imbalance between the activities of these ON- and OFF-center cells is assumed, we obtain orientation maps with spatial periodicity, as observed in the experiments. On the other hand, when balanced activities are assumed, orientation maps do not show periodicity. This suggests that the imbalance in activities between ON- and OFF-center cells contributes to the elaboration of orientation maps during the critical period.

The response of Parp knockout mice against DNA damaging agents.

Gene-disruption studies involving poly(ADP-ribose) polymerase (Parp) have identified the various roles of Parp in cellular responses to DNA damage. The partial rescue of V[D]J recombination process in SCID/Parp(-/-) double mutant mice indicates the participation of Parp in the repair of DNA strand break. Parp(-/-) mice are more sensitive to the lethal effects of alkylating agents. Parp is also thought to be involved in base-excision repair after DNA damage caused by alkylating agents. On the other hand, resistance of Parp(-/-) mice to DNA damage induced by reactive oxygen species implicates the contribution of Parp to cell death through NAD depletion. Parp(-/-) mice with two different genetic backgrounds also show enhanced sensitivity to the lethal effects of gamma-irradiation. Parp(-/-) mice show more severe villous atrophy of the small intestine compared to the wild-type counterpart in a genetic background of 129Sv/C57BL6. Other forms of enhanced tissue damage have been identified in Parp(-/-) mice with a genetic background of 129Sv/ICR. For example, Parp(-/-) mice exhibit extensive hemorrhage in the glandular stomach and other tissues, such as the testes, after gamma-irradiation. Severe myelosuppression is also observed in both Parp(+/+) and Parp(-/-) mice, but Parp(+/+) mice show extensive extramedullary hematopoiesis in the spleen during the recovery phase of post-irradiation, whereas the spleen of Parp(-/-) mice exhibits severe atrophy with no extramedullary hematopoiesis. The absence of extramedullary hematopoiesis in the spleen is probably the underlying mechanism of hemorrhagic tendency in various tissues of Parp(-/-) mice. These findings suggest that loss of Parp activity could contribute to post-irradiation tissue hemorrhage.

Molecular approach in cancer epidemiology: early detection of carcinogen-induced mutations in a whole genome (Review).

Chronic exposure of organisms to endo- or exogenous genotoxic products results in the accumulation of mutations in the genome and eventually to the development of cancers. Early detection of these mutations would allow the identification of at risk individuals who present a high load of mutations either because of an occupational or environmental exposure, or because of less efficient DNA repair processes. However, highly specific and sensitive assays are required to allow the detection of point mutations in a whole genome. We review a long-term study on the mutagenesis induced in E.coli by an aromatic amide, the N-2-acetylaminofluorene. A major contribution of this work was to reveal the presence of specific mutation hot spot sequences. Taking advantage of this observation, we designed a specific, sensitive and semi-quantitative in vitro assay allowing the detection of carcinogen induced mutations. This assay has been validated in vivo and demonstrate the sensitivity of the technique in early detection of mutations and its usefullness in molecular epidemiology, early diagnostic and prognosis.

Syncytiotrophoblastic giant cells in teratocarcinoma-like tumors derived from Parp-disrupted mouse embryonic stem cells.

The enzyme poly(ADP-ribose) polymerase (Parp) catalyzes poly(ADP-ribosyl)ation reaction and is involved in DNA repair and cell death induction upon DNA damages. Meanwhile, poly(ADP-ribosyl)ation of chromosome-associated proteins is suggested to be implicated in the regulation of gene expression and cellular differentiation, both of which are important in tumorigenesis. To investigate directly the role of Parp deficiency in tumorigenicity and differentiation of embryonic stem (ES) cells during tumor formation, studies were conducted by using wild-type J1 (Parp(+/+)) ES cells and Parp(+/-) and Parp(-/-) ES clones generated by disrupting Parp exon 1. These ES cells, irrespective of the Parp genotype, produced tumors phenotypically similar to teratocarcinoma when injected s.c. into nude mice. Remarkably, all tumors derived from Parp(-/-) clones contained syncytiotrophoblastic giant cells (STGCs), which possess single or multiple megalo-nuclei. The STGCs were present within large areas of intratumoral hemorrhage. In contrast, neither STGC nor hemorrhage was observed in tumors of both wild-type J1 cells and Parp(+/-) clones. Electron microscopic examination showed that the STGCs possess microvilli on the cell surface and contained secretory granules in the cytoplasm. Furthermore, the cytoplasms of STGCs were strongly stained with antibody against mouse prolactin, which could similarly stain trophoblasts in placenta. These morphological and histochemical features indicate that the STGCs in teratocarcinoma-like tumors derived from Parp(-/-) clones belong to the trophoblast cell lineage. Our findings thus suggest that differentiation of ES cells into STGCs was possibly induced by the lack of Parp during the development of teratocarcinoma.

Intramolecular quadruplex formation of the G-rich strand of the mouse hypervariable minisatellite Pc-1.

The minisatellite Pc-1, isolated from the mouse genome consisting of a tandem repeat of d(GGCAG), is hypervariable with a mutation rate of 0.15/generation. Here we describe a structural characterization of the G-rich strand of Pc-1 by biochemical and physicochemical methods. It was found to be comparatively resistant to both single-stranded DNA-binding protein binding and digestion by single-stranded DNA-specific nuclease and to cause arrest of DNA synthesis. The guanine imino proton NMR signals observed on the Pc-1 G-rich strand and their slow (1)H/(2)H exchange profiles pointed to a quadruplex structure with guanine quartets. The melting temperature of the quadruplex determined by CD was not dependent on DNA concentration. These results indicate that the G-rich strand of Pc-1 forms an intramolecular folded-back quadruplex structure under physiological conditions. Possible mechanisms of the Pc-1 mutations implicated with the formation of the quadruplex structure are discussed.

Isolation and cloning of rat poly(ADP-ribose) glycohydrolase: presence of a potential nuclear export signal conserved in mammalian orthologs.

Poly(ADP-ribose) glycohydrolase (Parg) is the main enzyme of poly(ADP-ribose) degradation. To understand its structure-and-function relationship, we purified Parg from rat testis 9,740-fold using an improved affinity column; the purified product was a 60 kDa protein. Based on the determined sequences of three peptide fragments, degenerated primers were synthesized and a Parg cDNA comprising 3,974 nucleotides, encoding a 109 kDa protein, was isolated. The 60 kDa Parg purified from rat testes corresponded to the C-terminal half of the 109 kDa deduced peptide. When recombinant rat Parg was expressed as a glutathione S-transferase fusion protein in Escherichia coli, Parg activity was observed for the full-length and C-terminal half proteins but not in for the N-terminal half protein. Taken together, these data indicate that the catalytic domain of Parg is located in the C-terminal half. Further, we newly identified the presence of a potential nuclear export signal in the N-terminal half in addition to the previously reported nuclear localization signals in rat and other mammalian Pargs. Northern blot analysis showed the ubiquitous expression of a single 4.0 kb Parg mRNA in various rat tissues. The findings suggest that the 60 kDa Parg is produced by post-transcriptional processing.