Mutation analysis of novel human liver-related putative tumor suppressor gene in hepatocellular carcinoma

AIM: To find the point mutations meaningful for inactivationof liver-related putative tumor suppressor gene (LPTS) gene,a human novel liver-related putative tumor suppressor geneand telomerase inhibitor in hepatocellular carcinoma.METHODS: The entire coding sequence of LPTS genewas examined for mutations by single strand conformationpolymorphism (SSCP) assay and PCR products directsequencing in 56 liver cancer cell lines, 7 ovarian cancerand 7 head & neck tumor cell lines and 70 pairs of HCCtissues samples. The cDNA fragment coding for the mostfrequent mutant protein was subcloned into GST fusionexpression vector. The product was expressed in E. coliand purified by glutathione-agarose column. Telomericrepeat amplification protocol (TRAP) assays wereperformed to study the effect of point mutation totelomerase inhibitory activity.RESULTS: SSCP gels showed the abnormal shifting bandsand DNA sequencing found that there were 5 differentmutations and/or polymorphisms in 12 tumor cell lineslocated at exon2, exon5 and exon7. The main alterationswere A(778)A/G and A(880)T in exon7. The change in siteof 778 could not be found in HCC tissue samples, while themutation in position 880 was seen in 7 (10 %) cases. Themutation in the site of 880 had no effect on telomeraseinhibitory activity.CONCLUSION: Alterations identified in this study arepolymorphisms of LPTS gene. LPTS mutations occur in HCCbut are infrequent and of little effect on the telomeraseinhibitory function of the protein. Epigenetics, such asmethylation, acetylation, may play the key role in inactivationof LPTS.

Overexpression of p28/gankyrin in human hepatocellular carcinoma and its clinical significance

AIM: To investigate the expression of p28/gankyringene and its role in the carcinogenetic process of humanhepatocellular carcinoma (HCC).METHODS: 64 specimens of HCC and para-carcinomatissues, 22 specimens of non-tumor liver tissues (7normal, 15 cirrhosis), 10 specimens of normal humantissues and 5 hepatoma cell lines were studied for theexpression of p28/gankyrin by Northern blot. Theexpression of p28/gankyrin protein was detectedimmunohistochemically by using the specificpolyclonal antibody. RESULTS: Northern blot analysis indicated that theexpression of p28/gankyrin mRNA was intensivelydistributed in brain and heart, weakly in lung, spleenand muscle, undetectable in digestive system includingliver, pancreas, stomach, small and large intestines.p28/gankyrin mRNA was absent in normal liver, weaklydetected in liver cirrhosis and in 18 of 64 para-carcinoma liver tissues. In contrast, the expression ofp28/gankyrin mRNA was intensively detected in ali 5hepatoma cell lines tested, markedly increased in 57of 64 and moderately increased in 5 of 64 HCC samples.In comparison with liver cirrhosis and para-carcinomaliver tissues, the average expression of p28/gankyrin mRNA in HCC was increased 3.6- (2.901+0.507 vs 0.805 + 0.252, P＜0.05) and 5.2-fold (2.901 +0.507 vs 0.557+0.203, p＜0.01), respectively, Inaddition, p28/gankyrin mRNA expression level washigher in HCC with portal vein tumor thrombus andmicroscopic hepatic vein involvement (P--0.021 andP=-0.047, respectively). The overexpression of p28/gankyrin protein in HCC was targeted in hepatic tumorcells, not in bile duct cells and other interstitial cells.CONCLUSION: Overexpression of p28/gankyrin in HCCplays an important role and contributes to themetastasis potential in the process of carcinogenesis.p28/gankyrin may become a specific biological tissuemarker for the pathological diagnosis of HCC.

Different alterations of cytochrome P450 3A4 isoform and its gene expression in livers of patients with chronic liver diseases

AIM: To determine whether parenchymal cells or hepaticcytochrome P450 protein was changed in chronic liverdiseases, and to compare the difference of CYP3A4 enzymeand its gene expression between patients with hepaticcirrhosis and obstructive jaundice, and to investigate thepharmacologic significance behind this difference.METHODS: Liver samples were obtained from patientsundergoing hepatic surgery with hepatic cirrhosis (n=6) andobstructive jaundice (n=6) and hepatic angeioma (controls,n=6). CYP3A4 activity and protein were determined by Nashand western bloting using specific polychonal antibody,respectively. Total hepatic RNA was extracted andCYP3A4cDNA probe was prepared according the methodof random primer marking, and difference of cyp3a4expression was compared among those patients byNorthern blotting.RESULTS: Compared to control group, the CYP3A4 activityand protein in liver tissue among patients with cirrhosis wereevidently reduced. (P＜0.01) Northern blot showed the samechange in its mRNA levels. In contrast, the isoenzyme andits gene expression were not changed among patients withobstructive jaundice.CONCLUSION: Hepatic levels of P450s and its CYP3A4isoform activity were selectively changed in different chronicliver diseases. CYP3A4 isoenzyme and its activity declinedamong patients with hepatic cirrhosis as expression of cyp3a4gene was significantly reduced. Liver's ability to eliminatemany clinical therateutic drug substrates would declineconsequently, These findings may have practical implicationsfor the use of drugs in patients with cirrhosis and emphasizethe need to understand the metabolic fate of therapeuticcompounds. Elucidation of the reasons for these differentchanges in hepatic CYP3A4 may provide insight into morefundamental aspects and mechanisms of imparied liverfunction.

Expression of cytochrome P4502E1 gene in hepatocellular carcinoma

AIM: To investigate cytpchrome P4502E1 (CYP2E1) gene expression in occurrence and progression of hepatocellular carcinoma (HCC). METHODS: The human liver arrayed library was spotted onto the nylon membranes to make cDNA array. Hybridization of cDNA array was performed with labeled probes synthesized from RNA isolated from HCC and adjacent liver tissues. Sprague-Dawley rats were administrated diethyInitrosamine (DENA) to induce HCC. CYP2E1 expression was detected by the method of RT-PCR and Northern blot analysis. RESULTS: CYP2E1 was found by cDNA array hybridization to express differently between HCC and liver tissues. CYP2E1 only expressed in liver, but did not express in HCC tissues and expressed lowly in cirrhotic tissues. In the progression of cirrhosis and HCC, the expression level of CYP2E1 was gradually decreased and hardly detected until the late stage of HCC. CONCLUSION: Using arrayed library to make cDNA arrays is an effective method to find differential expression genes. CYP2E1 is a unique gene expressing in liver but did not express in HCC. CYP2E1 expression descended along with the initiation and progression of HCC, which is noteworthy further investigations in its significance in the development of HCC.

c-src activating mutation analysis in Chinese patients with colorectal cancer

AIM: To investigate the occurrence of cellular src (c-src)activating mutation at codon 531 in colorectal cancer patients from Chinese mainland.METHODS: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay followed by sequencing and single-strand conformation polymorphism analysis were carried out to screen 110 samples of primary colorectal cancer and 20 colorectal liver metastases.RESULTS: Only one sample showed PCR-RFLP-positive results and carried somatic codon 531 mutations. No additional mutation of c-src exon 12 was found.CONCLUSION: c-src codon 531 mutation in colorectal cancer is not the cause of c-src activation.