HCRP1, a novel gene that is downregulated in hepatocellular carcinoma, encodes a growth-inhibitory protein

One of the most frequent allelic deletions in hepatocellular carcinoma (HCC) has been found at chromosome 8p21-23. We reported here the identification and characterization of a novel gene for a hepatocellular carcinoma related protein 1 (HCRP1) localized at 8p22, which was isolated by positional candidate cloning. The expression of the gene for HCRP1 was most abundant in normal human liver tissue and significantly reduced or undetected in HCC tissues. The analysis of subcellular distribution showed that HCRP1 diffused in the cytoplasm with a significant fraction accumulated in the nuclei. After introduction of the sense and antisense cDNA of HCRP1 into HCC cell line SMMC-7721, we observed that the overexpression of HCRP1 significantly inhibited both anchorage-dependent and anchorage-independent cell growth in vitro. Using the transgenic short hairpin RNA (shRNA) to knock down the expression of HCRP1 gene in the other HCC cell line BEL-7404 resulted in the cell growth greatly enhanced. Moreover, reduction of the HCRP1 gene expression could also elevate the invasive ability of BEL-7404 cells. Our results strongly suggest that HCRP1 might be a growth inhibitory protein and associated with decreasing the invasion of HCC cells.     

眼镜蛇毒组分C的抗瘤活性及其对肿瘤细胞存活率的影响

目的观察眼镜蛇毒组分Ｃ对ＢＡＬＢ／Ｃ型小鼠腹水型肝癌Ｈ２２的抑瘤作用及其体外对肝癌Ｈ２２细胞存活率的直接影响。方法通过半体内实验,观察眼镜蛇毒组分Ｃ对小鼠腹水型肝癌Ｈ２２的抑瘤率;通过细胞培养,以细胞存活率为指标观察中华眼镜蛇毒组分Ｃ对ＢＡＬＢ／Ｃ型小鼠腹水型肝癌Ｈ２２细胞在体外的直接杀伤作用。结果眼镜蛇毒组分Ｃ能明显抑制ＢＡＬＢ／Ｃ型小鼠腹水型肝癌Ｈ２２细胞的生长,其抑瘤作用随剂量增大而增强,ＩＣ５０为９５ｍｇ／Ｌ。在体外,眼镜蛇毒组分Ｃ的浓度对ＢＡＬＢ／Ｃ型小鼠腹水型肝癌Ｈ２２细胞存活率的影响随其作用剂量的增大而增强,ＩＣ５０为９ｍｇ／Ｌ;同时,这种影响还随其作用时间的延长而增强。结论眼镜蛇毒组分Ｃ对ＢＡＬＢ／Ｃ型小鼠腹水型肝癌Ｈ２２有显著的抑瘤作用。     

类卟啉稀土配合物对于小鼠腹水肝癌细胞光敏损伤的研究

研究了类卟啉稀土配合物（以下简称ＰＬＭ—Ｇｄ—Ａ）对小鼠腹水肝癌细胞（ＡＨ）的光敏作用及ＡＨ细胞对ＰＬＭ一Ｇｄ—Ａ的摄取表明：ＰＬＭ－Ｇｄ－Ａ被ＡＨ细胞摄取的速度快（大约１０ｍｉｎ可达到平衡）,用ＭＴＴ方法测定了细胞光敏存活曲线,杀伤细胞的能力与光照时间和光照强度以及ＰＬＭ—Ｇｄ的浓度密切相关,用ＦＡＤＵ方法和电镜观察结果证明：该光敏损伤细胞的靶主要是在细胞核;对于不同稀土离子配合物光敏能力比较表明：Ｇｄ＞Ｅｕ＞Ｓｍ;造成细胞死亡的原因包括１Ｏ２、在内的活性氧。     

人心肌酸性铁蛋白的分离纯化及临床应用

人心肌匀浆经热变性、酸化、硫酸铵盐析、超离心、ＳｅｐｈａｒｏｓｅＣＬ－４Ｂ柱层析和制备等电聚焦分离,得到酸性铁蛋白。经鉴定,所得酸性铁蛋白ｐＩ为５．０,Ｈ亚基分子量为２１ｋＤ,Ｌ亚基为１９ｋＤ,ＰＡＧＥ分析呈单一区带。制备了兔抗人酸性铁蛋白抗血清,用该抗血清建立的人酸性铁蛋白放射免疫分析可检测出８０％甲胎蛋白阴性肝癌病人。     

The small chromatin fragments released by micrococcal nuclease from hepatoma tissue cultured cell nuclei are strongly enriched in coding DNA sequences and are related to an actively transcribed single-stranded DNA fraction

It was shown with the use of specific probes that mild micrococcal nuclease digestion releases from chromatin actively-transcribed genes as small nucleosome oligomers. In the present work we demonstrate that most if not all of the active genes are accessible to the nuclease. It was found that the short released fragments are greatly enriched in transcribed DNA sequences, the most enriched being the dimers of nucleosomes since 35% of their DNA could be hybridized to cytoplasmic RNA. The results of cDNA-DNA hybridizations indicate that the monomers and dimers of nucleosomes contain most of the DNA sequences which encode poly(A⁺) RNAs, however larger released fragments include some transcribed sequences, while the nuclease-resistant chromatin is considerably impoverished in coding sites. These evidences and the finding that about 25% of the DNA from the dimers of nucleosomes are exclusively located in this class of fragments, tend to prove that the active chromatin regions are attacked in a non-random way by micrococcal nuclease. We have previously isolated, without using exogenous nuclease, an actively transcribed genomic fraction amounting to 1.5–2% of the total nuclear DNA, formed of single-stranded DNA. In the present study we show that all or nearly all the single-stranded DNA sequences could be reassociated with the DNA fragments present in the released monomers and dimers of nucleosomes. Our observations confirmed our previous finding that the greatest part of single-stranded DNA selectively originates from the coding strand of genomic DNA.     

Redifferentiation of human hepatoma cells induced by synthesized coumarin

The effects of synthesized 7-OH-4-CH(3)-coumarin on proliferation and differentiation of human hepatoma carcinoma cell line, SMMC-7721, were examined. Results showed that 7-OH-4-CH(3)-coumarin suppressed the proliferation of the SMMC-7721 cells in a dose-dependent manner, with an IC(50) value of 356 +/- 1.8 .M, while concentrations< or =200 mM could trigger differentiation. After treatment with this compound at 100 mM, the growth curve of human hepatoma cells decreased markedly. When treated with 50 and 100 mM, the cells' electrophoresis rate decreased from 2.2 mm/s/V/cm in the control group to 1.5 and 1.8 mm/s/V/cm, respectively, and the alpha-fetoprotein content decreased from 123 ng/mg in the control group to 68 and 45 ng/mg, respectively. The microvilli on the surface of treated cells were also reduced. All the above indexes related to cell malignancy were alleviated significantly. Results showed that 7-OH-4-CH(3)-coumarin could reverse human hepatoma cells' malignant phenotypic characteristics and induce redifferentiation.     

Integrative genomics based identification of potential human hepatocarcinogenesis-associated cell cycle regulators: RHAMM as an example

DNA microarray has been widely used to examine gene expression profile of different human tumors. The information generated from microarray analysis usually represents the overall range of cancer-associated abnormality associated with gene regulation. In order to identify key regulatory genes involved in carcinogenesis of human cancer, hypothesis driven data mining of the microarray data plus experimental validation becomes a critical approach in the post-genome era. Here, we present an integrative genomic analysis of published microarray data and homolog gene database. Over 20,000 genes were examined to reveal 16 genes specific to vertebrates, cell cycle G2/M regulated, and overexpressed in human HCC. Using Affymetrix microarray analysis, we found that all 16 genes were up-regulated in human HCC. Among these 16 genes, we experimentally validated the up-regulation of receptor for hyaluronan-mediated motility (RHAMM) in different cell model systems. We first confirmed elevation of RHAMM in the G2/M phase of synchronized HeLa cells. We also found that RHAMM had an elevated level of expression in all the HCC samples we examined and it was induced during the G2/M phase of regenerating mouse hepatocytes after partial hepatectomy. Thus, the expression of RHAMM appears to be tightly regulated during mammalian cell cycle G2/M progression. The ectopic overexpression of RHAMM in 293T cells resulted in the accumulation of cells at G2/M phase. RHAMM-induced mitotic arrest of cells was predominantly in the prophase. Taken together, using an integrated functional genomic approach, we have uncovered a set of genes that may play specific roles in cell cycle progression and in HCC development. To elucidate the function of these genes in cell cycle regulation may shed light on the control mechanism of human HCC in the future.     

Response of the antioxidant defense system to tert-butyl hydroperoxide and hydrogen peroxide in a human hepatoma cell line (HepG2)

The aim of this work was to investigate the response of the antioxidant defense system to two oxidative stressors, hydrogen peroxide and tert-butyl hydroperoxide, in HepG2 cells in culture. The parameters evaluated included enzyme activity and gene expression of superoxide dismutase, catalase, glutathione peroxidase, and activity of glutathione reductase. Besides, markers of the cell damage and oxidative stress evoked by the stressors such as cell viability, intracellular reactive oxygen species generation, malondialdehyde levels, and reduced glutathione concentration were evaluated. Both stressors, hydrogen peroxide and tert-butyl hydroperoxide, enhanced cell damage and reactive oxygen species generation at doses above 50 microM. The concentration of reduced glutathione decreased, and levels of malondialdehyde and activity of the antioxidant enzymes consistently increased only when HepG2 cells were treated with tert-butyl hydroperoxide but not when hydrogen peroxide was used. A slight increase in the gene expression of Cu/Zn superoxide dismutase and catalase with 500 microM tert-butyl hydroperoxide and of catalase with 200 microM hydrogen peroxide was observed. The response of the components of the antioxidant defense system evaluated in this study indicates that tert-butyl hydroperoxide evokes a consistent cellular stress in HepG2.     

Reactive oxygen species stimulated human hepatoma cell proliferation via cross-talk between PI3-K/PKB and JNK signaling pathways

Reactive oxygen species (ROS) are important for intracellular signaling mechanisms regulating many cellular processes. Manganese superoxide dismutase (MnSOD) may regulate cell growth by changing the level of intracellular ROS. In our study, we investigated the effect of ROS on 7721 human hepatoma cell proliferation. Treatment with H2O2 (1-10 microM) or transfection with antisense MnSOD cDNA constructs significantly increased the cell proliferation. Recently, the mitogen-activated protein kinases (MAPK) and the protein kinase B (PKB) were proposed to be involved in cell growth. Accordingly, we assessed the ability of ROS to activate MAPK and PKB. PKB and extracellular signal-regulated kinase (ERK) were both rapidly and transiently activated by 10 microM H2O2, but the activities of p38 MAPK and JNK were not changed. ROS-induced PKB activation was abrogated by the phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002, suggesting that PI3-K is an upstream mediator of PKB activation in 7721 cells. Transfection with sense PKB cDNA promoted c-fos and c-jun expression in 7721 cells, suggesting that ROS may regulate c-fos and c-jun expression via the PKB pathway. Furthermore we found that exogenous H2O2 could stimulate the proliferation of PKB-AS7721 cells transfected with antisense PKB cDNA, which was partly dependent on JNK activation, suggesting that H2O2 stimulated hepatoma cell proliferation via cross-talk between the PI3-K/PKB and the JNK signaling pathways. However, insulin could stimulate 7721 cell proliferation, which is independent of cross-talk between PI3-K/PKB and JNK pathways. In addition, H2O2 did not induce the cross-talk between the PI3-K/PKB and the JNK pathways in normal liver cells. Taken together, we found that ROS regulate hepatoma cell growth via specific signaling pathways (cross-talk between PI3-K/PKB and JNK pathway) which may provide a novel clue to elucidate the mechanism of hepatoma carcinogenesis.     

三株肝癌单克隆抗体相应抗原在肝癌细胞中的表达与分布

应用免疫细胞化学方法、图像分析技术和免疫电镜技术,对3株肝癌mAs A10,E5和F11在肝癌细胞株中的表达情况及分布特点进行了研究。结果显示：3株单抗抗原在肝癌细胞株HHCC中均有较高表达;单抗A10,F11相应抗原主要分布在肝癌细胞的胞膜和胞浆中,而在胞核上无明显分布。单抗E5相应抗原除分布在胞膜和胞浆外,在细胞核上也有明显浓聚。本实验明确了3株肝癌单抗相应抗原在肝癌细胞中的分布特点,对于深入分析抗原的本质有重要参考价值。