Jae Hoon Park

Increased expression of cFLIPL in colonic adenocarcinoma

During tumour progression, cancer cells use diverse mechanisms to escape from apoptosis‐inducing stimuli, which may include receptor internalization, inhibition of signal pathways, and regulation of specific sets of genes. Substantial numbers of colon cancer cells have been observed to express Fas/Fas ligand, but are resistant to Fas‐mediated apoptosis, suggesting that colonic tumours might develop specific mechanisms to overcome Fas‐mediated apoptosis. Recently, cellular FLICE‐like inhibitory protein (cFLIP) has been identified as an endogenous inhibitor of Fas‐ or other receptor‐mediated apoptosis and its altered high expression has a suspected association with tumour development or progression. In an effort to investigate the prevalence of cFLIPL alterations in colon carcinomas and their possible implications for the progression of colon cancers, cFLIPL expression was analysed in adenocarcinomas and adenomatous polyps of colon, with matched normal tissues, at RNA and protein levels, by semi‐quantitative reverse transcription‐polymerase chain reaction (RT‐PCR) and immunohistochemistry. cFLIPL transcripts were constitutively expressed in colon cancers and expression levels were significantly higher in carcinomas than in normal tissues (p<0.05). Overexpression of cFLIPL protein was found exclusively in carcinoma cells in all matched sets analysed and approximately three‐fold induction was detected in cancer cells (p<0.05). The expression of cFLIPL protein was not significantly altered in adenomatous polyps compared with normal tissues. Taken together, these results strongly suggest that abnormal overexpression of cFLIPL is a frequent event in colon carcinomas and might contribute to in vivo tumour transformation. Copyright

Mutational abrogation of the PTEN/MMAC1 gene in gastrointestinal polyps in patients with Cowden disease

BACKGROUND & AIMS To understand the molecular etiology of Cowden disease-associated gastrointestinal polyps, we analyzed the mutational status of PTEN/MMAC1, a recently identified Cowden disease gene located at 10q23, in gastric hamartomas, colonic adenoma, and juvenile polyps of 3 patients with Cowden disease. METHODS Messenger RNA expression, gene deletion, and sequence alteration of PTEN/MMAC1 were evaluated by quantitative polymerease chain reaction (PCR), PCR-single-strand conformation polymorphism, and sequencing analysis. RESULTS Germline missense mutation at codon 289 (AAA to GAA, Lys to Glu) and deletion of the wild-type allele were detected in the polyps of 2 patients with Cowden disease in the same family. Germline allelic deletion and transcriptional silencing of the remaining allele, probably caused by abnormal methylation, were also observed in a gastric hamartoma of 1 patient. CONCLUSIONS The germline mutation and alteration of the remaining allele observed in this study strongly support that PTEN/MMAC1 functions as a tumor suppressor in Cowden disease. This study is the first to show that the mutational abrogation of PTEN/MMAC1 plays a causal role in the genesis of gastrointestinal polyps in Cowden disease, providing molecular genetic evidence that colonic adenoma, juvenile polyp, and gastric hamartoma could be included in the manifestations of Cowden disease.

Human carbonyl reductase 1 upregulated by hypoxia renders resistance to apoptosis in hepatocellular carcinoma cells

BACKGROUND & AIMS Human carbonyl reductase1 (CBR1) has been reported to protect cells against lipid peroxidation. Since human hepatocellular carcinoma (HCC) cells are under oxidative stress in hypoxic conditions, we tested if CBR1 is upregulated by hypoxia inducible factor (HIF)-1α, helps tumor growth under hypoxia, and renders chemoresistance to cisplatin and doxorubicin in HCC. METHODS Luciferase, EMSA, and chromatin immunoprecipitation (ChIP) assays were performed to analyze whether HIF-1α transactivates CBR1 promoter. CBR1 overexpression, siRNA, and inhibitors were used to study the role of CBR1 in tumor survival under hypoxia and chemoresistance to cisplatin and doxorubicin in HCC. FACS and Western blot analysis for oxidative stress markers were performed to measure ROS. Immunohistochemistry (IHC) was performed to analyze CBR1 expression in 78 cases of HCC and 123 cases of colon cancer tissues. RESULTS The CBR1 promoter was activated by HIF-1α. CBR1 overexpression enhanced cell survival by decreasing oxidative stress under hypoxia, cisplatin, and doxorubicin treatment. CBR1-siRNA increased apoptosis via increasing oxidative stress. Combinational therapy of CBR1 inhibitors with cisplatin or doxorubicin enhanced cell death in HCC cells. IHC showed CBR1 overexpression in 56 (72%) out of 78 HCC and 88 (72%) out of 123 colon cancer cases. CONCLUSIONS Overexpressed CBR1 by HIF-1α plays important roles in tumor growth under hypoxia and chemoresistance to anticancer drugs. The inhibition of CBR1 by specific inhibitors enhances anticancer drug efficacy in HCC. Therefore, we concluded that CBR1 is a good molecular target for the development of anticancer drugs for HCC patients.

TGF-β1 inhibition of apoptosis through the transcriptional up-regulation of Bcl-X L in human monocytic leukemia U937 cells

To characterize the TGF-β1 response of monocytic leukemia cells, we analyzed the effects of TGF-β1 on cell proliferation, differentiation, and apoptosis of human monoblastic U937 cells. Treatment of cells with TGF-β1 in the absence of growth factors significantly enhanced cell viability. Flow cytometric analysis of DNA content and CD14 expression revealed that TGF-β1 does not affect cell proliferation and differentiation. Consistent with these results was the finding that no transcriptional induction of Cdk inhibitors such as p21Waf1, p15Ink4b, and p27Kip1 was detected following TGF-β1 treatment. Interestingly, however, pretreatment of TGF-β1 significantly inhibited Fas-, DNA damage-, and growth factor deprivation-induced apoptosis. This antiapoptotic effect was totally abrogated by anti-TGF-β1 antibody. Quantitative RT-PCR analysis demonstrated a dose- and time-dependent transcriptional up-regulation of Bcl-X(L), suggesting its implication in the TGF-1-mediated antiapoptotic pathway. We also observed elevated expression of c-Fos and PTEN/MMAC1. But, no detectable change was recognized in expression of c-Jun, Fas, Fadd, Fap-1, Bcl-2, and Bax. Taken together, our study shows that TGF-β1 enhancement of cellular viability is associated with its antiapoptotic effect, which may result from the transcriptional up-regulation of Bcl-X(L).