[Effect of TPT1 transduction on the biological behaviors of liver cell lines].

OBJECTIVE: The aim of this study was to transfect TPT1 into cell lines SMMC-7721 and L-02, seperately, and to observe the changes of biological behaviors of the cell lines. METHODS: Through lipofectamine, the eukaryotic report expression vector containing TPT1 ORF (open reading frame), pEGFP-N3TPT1, were transducted into hepatocarcinoma cell line SMMC-7721 cells and normal liver cell line L-02 cells, seperately. The transduction was repeated three times in 24 hrs. The differences of biological behaviors between the pEGFP-N3TPT1 and pEGFP-N3 groups were studied by RT-PCR, MTT assay, soft agar colony formation assay and cell cycle analysis. RESULTS: The pEGFP-N3TPT1 transfected cells had a high mRNA level in the two cell lines (P < 0.05) compared with the pEGFP-N3 controls. The ability of proliferation and the soft agar colony formation were enhanced in the SMMC-7721 transducted cells with pEGFP-N3TPT1 compared with that transducted with pEGFP-N3 (P < 0.05), and the cell cycle analysis showed that the cells in the phase G(2)+S/M increased after pEGFP-N3TPT1 transduction. In the L-02 cell line, we obtained similar results, pEGFP-N3TPT1 enhanced the colony formation in plate (P < 0.05), but not make it form colony in soft agar. CONCLUSIONS: TPT1 can enhance malignant phenotype of SMMC-7721 cells and promote the growth of L-02 cells, but not transform L-02 into malignant phenotype.

Messenger RNA expression of translationally controlled tumor protein (TCTP) in liver regeneration and cancer.

OBJECTIVE: Translationally controlled tumor protein (TCTP) was recently reported to be involved in tumor reversion. In order to understand TCTP mRNA transcript in proliferative tissue and malignant lesions, the mRNA expression of TCTP was determined in a rat model of liver regeneration and human liver cancer tissues. In addition, the potential role of TCTP on suppression of liver cancer was investigated. MATERIALS AND METHODS: Liver regeneration model was established by a two-thirds partial hepatectomy (PH) in adult, male Sprague-Dawley rat. TCTP mRNA expression was determined by semiquantitative RT-PCR analysis. Antisense mRNA of TCTP was transfected into the SMMC-7721 liver cancer cell line. Biological assay of proliferation and cell cycle were analysed by MTT and flow cytometry, respectively. RESULTS: After PH of rats, the level of TCTP mRNA transcript was upregulated slightly in liver tissue at 1 hour followed by a high expression from 3 to 12 hours, which then decreased dramatically at 24 hours before returning to original level during liver tissue proliferation. TCTP mRNA transcript increased significantly in liver cancer tissues when compared to non-cancerous adjacent tissues as control. The transfection with antisense oligodeoxynucleotides of TCTP led to decreased proliferation of SMMC-7721 cells resulting in cell cycle arrest and pro-apoptosis. CONCLUSION: The results suggested that TCTP mRNA expression might be stage-specific in the proliferation of liver tissue but alter abnormally in cancer lesions. TCTP could be used as a potential target for suppression of liver tumorigenicity.

Potential role of novel hepatocellular carcinoma-associated gene IDD01 in promoting tumorigenesis of HepG2 cell line.

BACKGROUND: We have used suppression subtractive hybridization to construct a subtracted cDNA library of hepatocellular carcinoma (HCC) and isolated a panel of differential expression sequence tag (ESTs). By using bioinformatics and rapid amplification of cDNA ends (RACE), we found a novel HCC-associated gene IDD01. To further investigate its function, a recombinant eukaryotic vector pEGFP/ORF was constructed and transfected into the HepG2 cell line. METHODS: The open reading frame (ORF) of IDD01 was amplified by RT-PCR, digested with Bamh I and Hind III, and subcloned into the pEGFP-C1 vector. The ligation reaction was conducted with T4 DNA ligase, and the recombinant vector was named pEGFP/ORF. Untransfer control (control group), pEGFP-C1 (HepG2/C1 group) and pEGFP/ORF (HepG2/ORF group) transfer groups were designed. Gene transfer was conducted with lipofectamine. To obtain stable transfection in HepG2 cells, selection was initiated with 500 microg/ml G418. Cellular IDD01 mRNA levels were assayed by semi-quantitative RT-PCR. The MTT colorimetric method and flow cytometry were used to determine the cell proliferation. The tumorigenic potential of transformed cells was determined from their ability to grow as anchorage-independent colonies on soft agar. Transient transfections were performed to observe subcellular location of GFP-IDD01 fusion protein. RESULTS: A 778 bp specific band of ORF was obtained by RT-PCR, and the positive clone of recombinant plasmid pEGFP/ORF (5.5 Kb) was identified by restriction endonuclease cleavage and sequence. The brightness ratio of IDD01 mRNA was not obvious between control and pEGFP/C1 groups, whereas the ratio of pEGFP/ORF was higher than that in the other two groups. After culture for 24 - 72 hours, the A(490) values in pEGFP/ORF were higher than those in the other two groups (P < 0.01). On histograms of flow cytometry, the S phase ratio of HepG2/ORF cells was significantly higher than that of the control and HepG2/C1 groups. The HepG2/ORF cells were able to form more colonies in soft agar compared with other HepG2 cell lines (P < 0.01). GFP-IDD01 fusion protein predominantly localized in the plasma, whereas EGFP protein diffused all over the cell. CONCLUSION: The IDD01 gene is a positive effector in cell proliferation and contributes to the carcinogenesis and progression of HCC. This gene may serve as a potential target for pharmaceutical intervention of HCC.

[Serum from partial hepatectomy rat and hepatocyte growth factor stimulate bone marrow cell expressing albumin and alpha fetoprotein].

OBJECTIVE: To explore the effect of serum from partial hepatectomy (PH) rat and hepatocyte growth factor (HGF) on expression of albumin and AFP of bone marrow cells. METHODS: The bone marrow mono-nucleated cells were separated from SD rats and cultured in three groups: (1) The medium only group as control was added normal fetal bovine serum; (2) Rat hepatic injury serum group (was added 15% rat serum from 2-AAF+PH model); (3) HGF group (HGF 20 ng/ml). The role of these factors was determined by RT-PCR, immunohistochemistry (IHC) and Western blot, using AFP and albumin as special hepatocytic markers. RESULTS: By immunohistochemical staining and Western blot, the fresh bone marrow cells were AFP-negative, same as the cells cultured with medium only group. While bone marrow cells, co-cultured with rat hepatic injury serum or HGF at day 10 and 20, expressed AFP protein. AFP mRNA expression could be found in bone marrow cells after 10 and 20 days cultured with rat hepatic injury serum or HGF, but not in fresh bone marrow cells and bone marrow cells cultured with medium only. Albumin mRNA expression was weak in fresh bone marrow cell and increased in groups 2 and 3. CONCLUSION: The rat hepatic injury serum or HGF could stimulate the expression of AFP protein and it's mRNA of bone marrow cells. Also they can stimulate albumin mRNA expression. It seems that, in bone marrow, there is a kind of cells so called bone marrow derived liver stem cell which can express albumin mRNA in a weak style.