AROS Is a Significant Biomarker for Tumor Aggressiveness in Non-cirrhotic Hepatocellular Carcinoma

Despite a low risk of liver failure and preserved liver function, non-cirrhotic hepatocellular carcinoma (HCC) has a poor prognosis. In the current study, we evaluated an active regulator of SIRT1 (AROS) as a prognostic biomarker in non-cirrhotic HCC. mRNA levels of AROS were measured in tumor and non-tumor tissues obtained from 283 non-cirrhotic HCC patients. AROS expression was exclusively up-regulated in recurrent tissues from the non-cirrhotic HCC patients (P=0.015) and also in tumor tissues irrespective of tumor stage (P<0.001) or BCLC stage (P<0.001). High mRNA levels of AROS were statistically significantly associated with tumor stage (P<0.001), BCLC stage (P=0.007), alpha fetoprotein (AFP) level (P=0.013), microvascular invasion (P=0.001), tumor size (P=0.036), and portal vein invasion (P=0.005). Kaplan-Meir curve analysis demonstrated that HCC patients with higher AROS levels had shorter disease-free survival (DFS) in both the short-term (P<0.001) and long-term (P=0.005) compared to those with low AROS. Cox regression analysis demonstrated that AROS is a significant predictor for DFS along with large tumor size, tumor multiplicity, vascular invasion, and poor tumor differentiation, which are the known prognostic factors. In conclusion, AROS is a significant biomarker for tumor aggressiveness in non-cirrhotic hepatocellular carcinoma.

A Comparative Study of Intra-arterial and Intramuscular Administration of phVEGF165 in a Chronic Ischemic Model of Rabbit Hindlimb

PURPOSE: To obtain phVEGF165 for angiogenesis and to compare the effects of its intra-arterial and intramuscular administration in a chronic ischemic rabbit hindimb model. MATERIALS AND METHODS: Chronic ischemic models were constructed in the left hindlimb of rabbits and divided into control (n=6), intra-arterial (n=7) and intramuscular groups (n=5). Plasmid DNA (phVEGF165) expressing vascular endothelial growth factor (VEGF) was obtained from HL60 cells, and transfection into CHO cells and western blot analysis of the medium, as well as proliferation assay of CPAE cells were performed. Two weeks after construction of the models, 500 mug phVEGF165 was injected into both the left common iliac artery and thigh muscles. Angiography was performed and the number of vessels counted, and ELISA was used to determine the quantity of VEGF in blood samples. Wilcoxon signed rank test was employed for statistical analysis. RESULTS: VEGF165 was expressed on western blot of the culture medium. Proliferation assay showed that optical densities were 0.73+/-0.043 in the control study and 1.09+/-0.015 in phVEGF165. The angiographic scores were 1.32+/-0.13 (pre-gene therapy) and 1.30+/-0.07 (post-gene therapy) in the control group, 1.42+/-0.15 and 1.59+/-0.09 in the intra-arterial group, 1.59+/-0.27 and 1.14+/-0.12 in the intramuscular group. The differences were not statistically significant. In the intra-arterial group, serum VEGF levels were 39.96+/-1.08 pg/ml (pregene therapy), 44.99+/-2.13 pg/ml (4th day), 48.18+/-1.49 pg/ml (1st week), 45.70+/-3.77 pg/ml (2nd week), and 46.54+/-5.47 pg/ml (3rd week), but in the control and intramuscular groups there were no increases. CONCLUSION: phVEGF165 affected the proliferation of CPAE cells. There was no difference in angiographic scores and serum VEGF levels between intra-arterial and intramuscular administrations.

An experimental model of ischemia in rabbit hindlimb

This study was performed to establish an experimental model of ischemia for the investigation of new treatment modality of limb-threatening ischemia. We produced ischemia in the hindlimbs of 8 New Zealand white rabbits. Under general anesthesia, the left femoral artery was exposed, freed, and excised from distal external iliac artery to proximal popliteal and saphenous arteries. And then both hindlimbs were serially examined to assess the ischemia according to the time table until postoperative 6 weeks. We assessed clinical observation, blood pressure, radioisotopic perfusion scan, and angiography. Clinical ischemic changes of the operated feet were observed in 63%. The blood pressure of left calves was measurable on postoperative day 3 (p<0.05, vs preoperative day 2) and then gradually increased to reach a plateau in postoperative week 6. Radioisotopic arterial perfusion showed similar profiles as in blood pressure. Angiography of ischemic hindlimbs demonstrated a few collateral vessels arising from the internal iliac artery with the reconstitution of the posterior tibial artery in postoperative week 2. In postoperative week 6, collaterals remained the same in number. However, these became dilated and tortuous and showed reconstitution in distal hindleg. In conclusion, this is a reproducible, measurable, and economical animal model of hind limb ischemia.

p53 Mutation and Functional Analyses by Using Yeast Functional Assay / 대한암학회지

PURPOSE: Mutation of the p53 tumor suppressor gene is the most common genetic defect in all human tumors. Because of the widespread mutations and polymorphism in the p53 gene, the conventional screening methods cannot distinguish between polymorphisms or functionally silent mutations and inactivating mutations. It is well known that plasmids can be generated by homologous recombination in vivo in the yeast by cotransforming the PCR product with a linearized yeast expression vector encoding part of a gene and a selectable marker gene. The aim of this study is to develop more easy and reliable method for functional assay of p53 mutation. MATERIALS AND METHODS: We constructed a gap vector which can reliably and conveniently be used to screen p53 mutations in a simple yeast growth assay. The gap vector was constructed as follows: About 100 bp DNA fragments containing parts of N- and C- terminal portion of p53 were cloned into XbaI/SmaI and HindIII/XhoI sites of yeast expressing vector, respectively. The gap vector was obtained by double cutting with SmaI and HindIII followed by gel elution. Yeast was transformed with the reporter vector containing three tandem copies of the consensus p53 binding site by lithium acetate-mediated method. RT-PCR amplification of p53 transcripts from cell lines or tumor tissues was carried out. To investigate whether p53 gene is mutated or not, yeast containing reporter gene was cotransformed with PCR product and linearized gap vector, plated on SD medium minus histidine, and incubated for 3 days. The colonies on selective media were isolated and characterized. RESULTS: The tumor tissues examined were one hepatocellular carcinoma, three breast cancers, two stomach cancers and two colon cancers. One hepatocellular carcinoma tissue had mutation in both alleles of the p53 gene, and 7 cancer tissues had heterozygous mutations in the p53 gene. The result of functional assay was well correlated with mutational analysis by sequencing. CONCLUSION: p53 functional assay system might be easy and reliable method for functional screening of p53 on tumor tissues and this might be used for screening of other mutated gene. This technique, FASAY, requires only a few steps, can be automated readily and should permit screening for germline or somatic heterozygous mutations in any gene whose function can be monitored in yeast.

Early Diagnosis of Rifampin-Resistant Mycobacterium tuberculosis by Gene Analysis of RNA Polymerase B Subunit

PURPOSE: The control of tuberculosis is seriously threatened worldwide by the recently emerging multidrug-resistant Mycobacterium tuberculosis. As a result, early detection of drug resistant M.tuberculosis strain has become very important but conventional laboratory methods are time consuming and delayed results often affect patients adversely in controlling tuberculosis. The authors studied the usefulness of the line probe assay to determine the mutaion in rpoB gene of rifampin resistant M.tuberculosis and to find out if this method can substitute conventional methods in the detection of resistant strain. METHODS: This study employed 40 clinical samples of M.tuberculosis which had been determined by culture and drug sensitivity test. After amplification of rpoB-the gene for the B subunit of the RNA polymerase-by PCR, the amplified products were hybridized with specific oligonucleotide probes immobilized on nitrocellulose strip and direct DNA sequencing was also performed. The results were compared with those of the classical susceptibility test. RESULTS: Among the 40 samples, 10 were identified as drug resistant strain by classical drug susceptibility test. Three of the ten resistant samples were rifampin resistant strains, which were identified by either method. All mutations were clustered within the region of 69bp of rpoB and all were single nucleotide mutations. Two isolates had a TCG->TTG(serine->leucine) mutation in codon 522. One isolate had a CAC->CTC(histidine->leucine) mutation in codon 526. CONCLUSION: In contrast to culture and sensitivity tests, line probe assay is an easy and speedy method for detecting rifampin resistant M.tuberculosis in clinical samples as well as a helpful tool for choosing antituberculosis drug in children.

Deletion of the Importin-alpha Gene in the Breast Cancer Cell

BACKGROUND: BRCA1 (breast-cancer gene 1) is a tumor suppressor gene that accounts for nearly all families of both early onset breast and ovarian cancer and about 45% of families with breast cancer only. Sporadic nonhereditary breast cancer is recognized as the most common form of this malignancy. However, presence of germ-line mutations in the BRCA1 gene of these tumors is an infrequent event. The BRCA1 protein includes a ring domain and an acidic domain, both of which are characteristics of certain transcription factors, as well as two putative nuclear localization signals (NLS) that interact with importin-alpha. The normal BRCA1 protein is located in the nucleus of most breast-cell types whereas the BRCA1 protein of breast cancer cells is aberrantly localized in the cytoplasm. This mislocation of the BRCA1 protein in breast cancer cells may be due to defects in the NLS receptor-mediated pathway for the nuclear import of the BRCA1 gene product. Identification of importin-alpha mutations as a cellular protein responsible for the nuclear import of BRCA1 in breast-cancer cell lines and primary breast cancers is the focus of this investigation. METHODS: A series of 15 surgical samples of breast cancer and 3 samples of breast-cancer cell lines (Hs578T, ZR75-1, MCF-7) was assayed for the presence of the deletion mutant in importin-alpha by using both RT-PCR amplification of importin-alpha transcripts and sequencing analysis. RESULTS: Three of the 15 primary breast cancers and 1 of the 3 breast-cancer cell lines showing deletions in importin-alpha transcripts produced two different truncated transcripts. 1208 bp deletions were observed in transcripts from breast cancer (T-1, T-3) and ZR75-1, which is specified by the nucleotide 251-1458 of the transcript. Another transcript encoded by primary breast cancer (T-2) included a 1312 bp deletion in the nucleotide 61-1372 of the transcript. CONCLUSIONS: The deletions eliminated part of the importin-alpha transcript segment encoding the putative NLS-binding domain but not the importin-beta binding domain, suggesting that these deletion mutants could not bind to NLS of the BRCA1 protein. These results suggest that the composite effects of mislocationof the BRCA1 protein by deletion of the NLS-binding domain in importin-alpha may contribute to tumorigenesis in sporadic breast cancer.