QSG-7701 human hepatocytes form polarized acini in three-dimensional culture.

Hepatocytes are polarized and fulfill a variety of liver-specific functions in vivo; but the polarized tissue structure and many of these functions are lost when the cells are cultured on plastic. To recapitulate the polarized structure and tissue-specific function of liver cells in culture, we established a three-dimensional (3D) culture assay with the human hepatocyte line QSG-7701. In 3D Matrigel culture, QSG-7701 cells formed polarized spheroids with a center lumen, which is reminiscent of bile canaliculi in the liver.

[Preparation of a novel telomerase inhibitory protein LPTS-L].

The gene for LPTS is originally cloned as a human liver-related putative tumor suppressor (LPTS) gene that encodes a full length protein of 328 amino acids (LPTS-L). LPTS-L is also identified as a telomerase inhibitor to regulate telomere length in the cells. To facilitate the functional and structural studies of LPTS-L protein, the cDNA for LPTS-L was cloned into the expression vector pET-24 in frame to generate a recombinant plasmid pET-24-LPTS.

Genes encoding Pir51, Beclin 1, RbAp48 and aldolase b are up or down-regulated in human primary hepatocellular carcinoma.

Aim: To reveal new tumor markers and target genes from differentially expressed genes of primary tumor samples using cDNA microarray.

Methods: The (33 )P labeled cDNAs were synthesized by reverse transcription of message RNA from the liver cancerous tissue and adjacent non-cancerous liver tissue from the same patient and used to hybridize to LifeGrid 1.0 cDNA microarray blot containing 8400 known and unique human cDNA gene targets, and an expression profile of genes was produced in one paired human liver tumor tissue.

[The cloning and expression of a novel mouse gene mLPTS and its subcellular localization].

A novel mouse gene mLPTS was cloned by EST assembling, RT-PCR and DNA sequencing. The gene fragment for mLPTS is 1244 bp in length, encoding a protein of 332 amino acids. The amino acid sequence of mLPTS has 78% homologue with that of LPTS gene, which is a novel liver cancer-related gene identified through positional candidate cloning stratage by our laboratory.

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

Aim: To find the point mutations meaningful for inactivation of liver-related putative tumor suppressor gene (LPTS) gene, a human novel liver-related putative tumor suppressor gene and telomerase inhibitor in hepatocellular carcinoma.

Methods: The entire coding sequence of LPTS gene was examined for mutations by single strand conformation polymorphism (SSCP) assay and PCR products direct sequencing in 56 liver cancer cell lines, 7 ovarian cancer and 7 head neck tumor cell lines and 70 pairs of HCC tissues samples. The cDNA fragment coding for the most frequent mutant protein was subcloned into GST fusion expression vector.

p73beta inhibits transcriptional activities of enhancer I and X promoter in hepatitis B virus more efficiently than p73alpha.

Aim: p73, as a novel member of a family of p53-related transcription factors, shares redundant functions with p53, such as the abilities of inducing apoptosis and suppressing growth. It is well known that p53 can repress HBV expression and transcription efficiently. The aim of this paper is to investigate the transcriptional effect of p73alpha and p73beta on hepatitis B virus (HBV) and to understand the correlation between HBV and p73.

Over-expression of LPTS-L in hepatocellular carcinoma cell line SMMC-7721 induces crisis.

Aim: To evaluate the function of the longer transcripts LPTS-L in hepatocellular carcinoma cell line SMMC-7721.

Methods: SMMC-7721 cells were transfected with LPTS-L expression construct and stably transfected cells were selected by G418. Multiple single clones formed and were checked for their phenotype.

The Autonomous Replication Function (ARS) of the Scaffold-associated Region (SAR) of Silkworm Attacus ricini rDNA in Yeast.

The one kilo-base scaffold-associated region (SAR) of silkworm Attacus ricini rRNA gene (rDNA) has been identified previously([1]). To investigate the critical sequence and the relative activity of ARS (autonomously replicating sequence), a set of restriction from covered the whole rRNA gene unit were subcloned into the nonreplicative pSKY vector. Among the seven plasmids constructed, the plasmid pSEY, having SAR, gave obvious positive replication activity in yeast as determined by the transformation efficiency.

Detection of Lis1 Gene Frame Shift Mutation in Human Hepatocarcinoma.

GST fusion protein expression system combined with protein truncation test(PTT) protocol was used to detect gene frame shift mutation. The RT-PCR products of Lis1 genes from hepatocarcinoma samples were respectively cloned into a GST fusion protein expression vector pGEX-1, then expressed in E.coli.

Regulation of Gene Expression by SAR of Silkworm Attacus ricini rRNA Gene.

The 1 kb scaffold-attachment region (SAR) at 5' non-transcription region of rRNA gene of silkworm Attacus ricini was cloned into eukaryotic expression vector pLu, which contained luciferase report gene and neo(R) selecting marker. After transfection of constructs into NIH3T3 cell line by using cation liposome, the luciferase activity was monitored to check the SAR's function. The results demonstrated that the SAR could enhance gene expression up to 15-fold in stable transformed cells, but no obvious gene expression was observed in transient transfection.

Identification of Up-regulated Genes in Rat Regenerating Liver Tissue by Suppression Subtractive Hybridization.

Suppression subtractive hybridization (SSH) technique was used to screen up-regulated genes in regenerating liver. The cDNA from rat regenerating liver tissue was used as the tester, and that from normal liver was used as the driver. After cloning, a subtraction cDNA library of 900 clones was obtained.

Cloning and Characterization of fup1, A Gene Highly Expressed in Hepatocellular Carcinoma.

Primary hepatocellular carcinoma(HCC) is one of the common malignant tumors in China. In our previous work, a gene named fup1(function-unknown protein 1) was isolated that was expressed differently in HCC and in normal liver. We assumed that it might be a candidate oncogene for the HCC.

The DNA Sequence and Structural Characteristic of The 5'-Nontranscribed Spacer of Silkworm Attacus ricini rDNA.

We have found that the SacII-EcoRI fragment in the nontranscribed spacer (NTS) of silkworm Attacus ricini rDNA is a nuclear scaffold-associated region (SRA) and showed the function as the ARS element in yeast. This paper reports the sequence of this NTS region and the various characteristic potential functional motifs as analyzed by computer. It is 1 025 bp long and AT-Rich.