Effects of diet-induced obesity on colitis-associated colon tumor formation in A/J mice.

OBJECTIVE: Studies have indicated that obesity is associated with a higher risk of colorectal cancer. This study was performed to determine the effect of diet-induced obesity on the formation of azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced colon tumors and to identify adiposity-related mechanisms. METHODS: Male A/J mice were placed on either a high-fat diet (HFD; 45% of total calories from fat) or a normal diet (ND; 15% of calories from fat) for 12 weeks. To induce colon tumors, AOM was administered at a dose of 10 mg/kg body weight, followed by two cycles of DSS supply. RESULTS: Study results indicated that the HFD group had twofold higher numbers of colonic tumors, as compared with the ND group. The HFD group also had significantly increased body weight and epididymal fat weight, which were associated with increases of serum insulin, insulin-like growth factor-1, leptin, epididymal fat pad leptin mRNA and colonic leptin receptor (Ob-R) mRNA. Animals on HFD showed higher expressions of Ob-R, insulin receptor, phosphorylated Akt, phosphorylated extracellular signal-regulated kinases, Bcl-xL and Cyclin D1 proteins in the colon. CONCLUSION: The results suggest that HFD-induced obesity facilitates colon tumor formation, possibly by regulating downstream targets of circulating adiposity-related factors via receptor-mediated signaling of the phosphatidylinositol 3-kinase/Akt pathway.

Base excision DNA repair defect in Gadd45a-deficient cells.

As one of a number of p53-regulated genes, Gadd45a (growth arrest and DNA damage inducible gene) has been shown to delay carcinogenesis and decrease mutation frequency. Gadd45a is known to regulate nucleotide excision DNA repair (NER) in response to UV radiation. Here, we report an emerging role for Gadd45a in base excision repair (BER). Gadd45a-null mouse embryo fibroblasts MEF and gadd45a-deficient human colon cancer cells exhibited slow BER after treatment with methyl methanesulfonate (MMS) a pure base-damaging agent. In addition, removal of AP sites by apurinic/apyrimidinic endonuclease 1/redox factor 1 (APE1/Ref1) was significantly delayed in gadd45a-null cells. Moreover, the localization of APE1/Ref1 within the nucleus was observed in gadd45a wild-type cells, whereas APE1 become mainly distributed in the cytoplasm, and there is a reduced interaction with proliferating cell nuclear antigen (PCNA) in Gadd45a-deficient cells. Inasmuch as p53 has been shown to regulate BER in addition to the NER pathway, our data suggest that p53-regulated gene Gadd45a contributes to the BER response by affecting the interaction of cellular APE1/Ref1 with PCNA. Gadd45a might be a key component gene of the p53 pathway involved in protection from carcinogenic base damage and maintenance of genomic stability, although the downstream mechanism including APE1/Ref1 will need further study.

The effect of methyl methanesulfonate (MMS)-induced excision repair on p53-dependent apoptosis in human lymphoid cells.

The tumor suppressor p53 product has been shown to play an important role in preventing carcinogenesis by at least two different mechanisms, by evoking cell cycle arrest and eliciting DNA repair on one hand, or by eliminating damaged cells by induction of apoptosis on the other hand. As a first step toward understanding the relationship between protective responses and apoptosis after genotoxic stress, we examined the effect of DNA strand breaks generated from repair processes in respect to acute cellular responses against DNA damage, and on p53-dependent apoptosis in human lymphoid cells. We used two isogenic cell lines, TK6 harboring wild-type p53, and WI-L2-NS, which carries a mutant p53. A significant difference in sensitivity was observed at 50 microg/ml methyl methane-sulfonate (MMS) between the two cell lines used. In addition, a clear p53-mediated contribution to apoptosis in MMS-induced cell death was observed. However, we did not observe any differences in repair of MMS-lesions, as determined by comet assay, between the two cell lines. These data suggest that the differences in apoptosis induction in the two lines are not a reflection of differences in strand-break frequency or repair capacity.

Sensitivity of cyclin E-overexpressing cells to cisplatin/taxol combinations.

Combinations of anticancer drugs, referred to as "combinatorial" chemotherapy, is often used in the treatment of cancer patients. Recent advances in our understanding of cell cycle and cellular stress responses may be viewed in the context of responses to anticancer drugs. A desired outcome is cell death preferentially occurring in cancer cells. We report that cyclin E, an oncogene that is deregulated in breast and other human cancers, can sensitize cancer cells to cisplatin/taxol combinations. The drug combination may selectively induce apoptosis in cyclin E-overexpressing cells.

p53-mediated DNA repair responses to UV radiation: studies of mouse cells lacking p53, p21, and/or gadd45 genes.

Human cells lacking functional p53 exhibit a partial deficiency in nucleotide excision repair (NER), the pathway for repair of UV-induced DNA damage. The global genomic repair (GGR) subpathway of NER, but not transcription-coupled repair (TCR), is mainly affected by p53 loss or inactivation. We have utilized mouse embryo fibroblasts (MEFs) lacking p53 genes or downstream effector genes of the p53 pathway, gadd45 (Gadd45a) or p21 (Cdkn1a), as well as MEFs lacking both gadd45 and p21 genes to address the potential contribution of these downstream effectors to p53-associated DNA repair. Loss of p53 or gadd45 had a pronounced effect on GGR, while p21 loss had only a marginal effect, determined by measurements of repair synthesis (unscheduled DNA synthesis), by immunoassays to detect removal of UV photoproducts from genomic DNA, and by assays determining strand-specific removal of CPDs from the mouse dhfr gene. Taken together, the evidence suggests a role for Gadd45, but relatively little role for p21, in DNA repair responses to UV radiation. Recent evidence suggests that Gadd45 binds to UV-damaged chromatin and may affect lesion accessibility. MEFs lacking p53 or gadd45 genes exhibited decreased colony-forming ability after UV radiation and cisplatin compared to wild-type MEFs, indicating their sensitivity to DNA damage. We provide evidence that Gadd45 affects chromatin remodelling of templates concurrent with DNA repair, thus indicating that Gadd45 may participate in the coupling between chromatin assembly and DNA repair.

Mild hyperthermia-induced apoptosis is dependent on p53 in human lymphoid cells.

Mild hyperthermia is known to enhance apoptosis. The p53 tumor-suppressor gene product has been shown to function in apoptosis in response to genotoxic stress. However, there is little information regarding the mechanism of p53-dependent apoptosis induced by heat stress. In present study, a p53 contribution in mild hyperthermia-induced apoptosis was investigated in human lymphoid system. After 30-minute exposure at 44 degrees C, the accumulation of p53 protein was clearly observed in TK6 and ML-1 cells. Using comet assay, the more significant sensitivity to hyperthermic apoptosis was found in TK6 (wild-type p53) than in WI-L2-NS (mutated in p53). Furthermore, the significantly rapid shifting from early apoptotic phase to late apoptotic was observed in heat-induced p53 TK6 cells. These findings suggest that p53-dependent apoptosis is efficaciously induced by mild hyperthermia as non-genotoxic stress in human lymphoid system.

Effect of p53 tumor suppressor on nucleotide excision repair in human colon carcinoma cells treated with 4-nitroquinoline 1-oxide.

In probing the mechanism of nucleotide excision repair (NER) in response to 4-nitroquinoline 1-oxide (4NQO)-induced DNA damage, the effect of p53 tumor suppressor was investigated. The effect of p53 protein on the repair of damaged DNA was examined by comet assay. Expression of p53 and p21(Waf1/Cip1) proteins was measured by the Enzyme-linked immunosorbent assay (ELISA) and immunocytochemistry, respectively. Compared to RKO cells having the wild-type p53 gene, increased cytotoxicity by 4NQO was observed in RKOmp53 cells with a mutation in p53 protein. DNA single strand breaks (SSB), indicative of the DNA repair, were considerably increased in 4NQO-treated RKO cells. Also, the expression of p53 and p21 proteins was significantly increased in 4NQO-treated RKO cells. In RKOmp53 cells, no effect of 4NQO on p21 expression was observed. Our findings suggest that 4NQO-induced NER is p53-dependent and involves up-regulation of its downstream regulator, p21(Waf1/Cip1) proteins.

Nickel (II)-induced apoptosis and G2/M enrichment.

Treatment with certain DNA-damaging agents induce a complex cellular response comprising pertubation of cell cycle progression and/or apoptosis on proliferating mammalian cells. Our studies were focused on the cellular effects of nickel (II) acetate, DNA-damaging agent, on Chinese hamster ovary (CHO) cells. Fragmented DNAs were examined by agarose gel electrophoresis and cell cycle was determined by DNA flow cytometry using propidium iodide fluorescence. Apparent DNA laddering was observed in cells treated with 240 microM nickel (II) and increased with a concentration-dependent manner. Treatment of nickel (II) acetate resulted in apoptosis which was accompanied by G2/M cell accumulation. Proportion of CHO cells in G2/M phase was also significantly increased in cells exposed to at least 480 microM nickel (II) from 57.7% of cells in the G0/G1 phase, 34.7% in the S phase, and 7.6% in the G2/M1 phase for 0 microM nickel (II), to 58.6%, 14.5%, and 26.9% for 640 microM nickel (II). These findings suggest that nickel (II) can modulate cellular response through some common effectors involving in both apoptotic and cell cycle regulatory pathways.