Sun-Hee Yoon

STAT3 is a potential modulator of HIF-1-mediated VEGF expression in human renal carcinoma cells

Aberrantly enhanced vascular endothelial growth factor (VEGF) gene expression is associated with increased tumor growth and metastatic spread of solid malignancies, including human renal carcinomas. Persistent activation of STAT3 is linked to tumor‐associated angiogenesis, but underlying mechanisms remain unclear. Therefore, we examined whether STAT3 modulates the stability and activity of hypoxia‐inducible factor‐1α (HIF‐1α), and in turn enhances VEGF expression. We found that STAT3 was activated in ischemic rat kidneys and hypoxic human renal carcinoma cells. We also found that hypoxia‐induced activation of STAT3 transactivated the VEGF promoter and increased the expression of VEGF transcripts. Consistent with these findings, STAT3 inhibition attenuated the hypoxic induction of VEGF. Interestingly, activated STAT3 increased HIF‐1α protein levels due to the HIF‐1α stability by blocking HIF‐1α degradation and accelerated its de novo synthesis. The novel interaction of STAT3 with HIF‐1α was identified in hypoxic renal carcinoma cells. Furthermore, hypoxia recruited STAT3, HIF‐1α, and p300 to the VEGF promoter and induced histone H3 acetylation. Therefore, these findings provide compelling evidence that a causal relationship exists between STAT3 activation and HIF‐1‐dependent angiogenesis and suggest that therapeutic modalities designed to disrupt STAT3 signaling hold considerable promise for the blocking tumor growth and enhancing apoptosis of cancer cells and tissues.

Leukemic cell line, KG-1 has a functional loss of hOGG1 enzyme due to a point mutation and 8-hydroxydeoxyguanosine can kill KG-1

We tested the cytotoxic action of 8-hydroxyguanine (8ohG) by observing the viability of several leukemic cell lines (KG-1, U937, Jurkat and K 562) in the presence of 8-hydroxydeoxyguanosine (8ohdG), a nucleoside of 8ohG. It was found that 8ohdG showed cytotoxic action only to KG-1 and that only KG-1 showed a homozygous arginine 209 to glutamine mutation in the hOGG1 gene with an almost negligible hOGG1 enzyme activity. Possibly, the selective cytotoxicity in 8ohdG to KG-1 may be due to its low capacity to cope with an increase in the 8ohG level in DNA resulting from the incorporation of 8ohdG present in the culture media. The mutational impairment of hOGG1 in KG-1 is the first report in leukemic cell lines. Using KG-1 with impaired hOGG1, we demonstrated cytotoxicity of 8ohdG probably due to its incorporation into cellular DNA. This new property of KG-1 may allow it to serve as an useful tool for studies of OGG1, oxidative DNA damage and the cytotoxic action of 8ohG.

Gene-specific oxidative DNA damage in Helicobacter pylori-infected human gastric mucosa.

To study the status of oxidative DNA damage in Helicobacter pylori infection in more detail, we examined oxidative DNA damage to individual genes by determining the loss of PCR product of a targeted gene before and after gastric mucosal DNA was treated with 8‐hydroxyguanine glycosylase, which cleaves DNA at the 8‐hydroxyguanine residues. The results showed that, of the 5 genes tested, p53, insulin‐like growth factor II receptor and transforming growth factor‐β receptor type II showed significant oxidative DNA damage in H. pylori‐positive tissues and that the BAX and β‐ACTIN genes were relatively undamaged. These results suggest that in H. pylori infection, oxidative DNA damage does not occur homogeneously throughout the genomic DNA but, rather, in a gene‐specific manner. We conclude that the progressive accumulation of preferential oxidative DNA damage in certain genes, such as p53, likely contributes to gastric carcinogenesis.

8-oxo-7,8-dihydroguanosine triphosphate(8-oxoGTP) down-regulates respiratory burst of neutrophils by antagonizing GTP toward Rac, a small GTP binding protein.

8-Oxo-7,8-dihydroguanosine triphosphate (8-oxoGTP) has been regarded simply as a oxidative mutagenic byproduct. The results obtained in this study imply that it may act as a down-regulator of respiratory burst of neutrophils. Human neutrophils treated with PMA produced superoxides and at the same time, the cytosol of these cells was intensely immunostained by 8-oxo-7,8-dihydroguanosine(8-oxoG) antibody, indicating that 8-oxoG-containing chemical species including 8-oxoGTP are produced. Human neutrophil lysates treated with PMA also produced superoxides, which was stimulated by GTPγS but inhibited by 8-oxoGTPγS. Moreover, 8-oxoGTPγS suppressed the stimulatory action of GTPγS. Likewise, GTPγS stimulated Rac activity in neutrophil lysates but 8-oxoGTPγS and GDP inhibited it. The inhibitory effect of GDP was one tenth that of 8-oxoGTPγS. Here again, 8-oxoGTPγS also suppressed the stimulatory action of GTPγS on Rac activity. These results imply the possibility that 8-oxoGTP is formed during respiratory burst of neutrophils and limits neutrophil production of superoxides by antagonizing GTP toward Rac.

Measurement of oxidative damage at individual gene levels by quantitative PCR using 8-hydroxyguanine glycosylase (OGG1).

In this study, an attempt was made to develop a method to estimate oxidative damage of individual genes for assessing chemopreventive potential of dietary or medicinal plants. Oxidative damage was investigated on the two genes in gastric mucosal tissue infected with Helicobacter pylori, which were genes of glyceraldehydes-3-phosphate dehydrogenase (GAPDH), a house-keeping gene, and gene of insulin-like growth factor II receptor (IGFIIR), a gene known to be mutated frequently in gastric carcinoma. The oxidative damage in genomic DNA in the above tissue was confirmed by immunohistochemical study using monoclonal antibody to 8-hydroxyguanine (oh(8)G), which showed much higher degree of staining in their nuclei. Using the method we developed, it was demonstrated that the number of oh(8)G (indicated by 8-hydroxyguanine glycosylase (OGG1) sensitive sites) in GAPDH was almost not changed in H. pylori-infected tissue but in IGFIIR, it increased significantly. These results indicate that this method is valid for the estimate of oxidative damage of individual genes and also showed that the susceptibility of genomic DNA to attack of reactive oxygen species is not homogeneous but different depending upon the region of DNA. We expect to use this method in studies of carcinogenic mechanism and chemoprevention since it can provide more specific information pertaining to individual genes we are interested in.

MutY is down-regulated by oxidative stress in E. coli

In Escherichia coli, MutM (8-oxoG DNA glycosylase/lyase or Fpg protein), MutY (adenine DNA glycosylase) and MutT (8-oxodGTPase) function cooperatively to prevent mutation due to 7, 8-dihydro-8-oxoguanine (8-oxoG), a highly mutagenic oxidative DNA adduct. MutM activity has been demonstrated to be induced by oxidative stress. Its regulation is under the negative control of the global regulatory genes, fur, fnr and arcA. However, interestingly the presence of MutY increases the mutation frequency in mutT- background because of MutY removes adenine (A) from 8-oxoG:A which arises from the misincorporation of 8-oxoG against A during DNA replication. Accordingly we hypothesized that the response of MutY to oxidative stress is opposite to that of MutM and compared the regulation of MutY activity with MutM under various oxidative stimuli. Unlike MutM, MutY activity was reduced by oxidative stress. Its activity was reduced to 30% of that of the control when E. coli was treated with paraquat (0.5 mM) or H2O2 (0.1 mM) and induced under anaerobic conditions to more than twice that observed under aerobic conditions. The reduced mRNA level of MutY coincided with its reduced activity by paraquat treatment. Also, the increased activity of MutY in anaerobic conditions was reduced further in E. coli strains with mutations in fur, fnr and arcA and the maximum reduction in activity was when all mutations were present in combination, indicating that MutY is under the positive control of these regulatory genes. Therefore, the down-regulation of MutY suggests that there has been complementary mechanism for its mutagenic activity under special conditions. Moreover, the efficacy of anti-mutagenic action should be enhanced by the reciprocal co-regulation of MutM.