RNA, Messenger ; biosynthesis ; genetics ; RNA, Viral ; chemistry ; genetics ; Ribonucleases ; biosynthesis ; genetics ; Virion ; chemistry ; genetics ; alpha-Fetoproteins ; biosynthesis ; genetics

Carcinoma, Hepatocellular ; blood ; Female ; Humans ; Liver Neoplasms ; blood ; Male ; alpha-Fetoproteins ; biosynthesis

To clone the murine alpha-fetoprotein (AFP) gene, construct the eukaryotic expression vector of AFP and express in CHO cells, total RNA were extracted from Hepa 1-6 cells, and then the murine alpha-fetoprotein gene was amplified by RT-PCR and cloned into the eukaryotic expression vector pcDNA3.1. The recombinant of vector was identified by restriction enzyme analysis and sequencing. After transient transfection of CHO cells with the vector, Western blotting was used to detect the expression of AFP. It is concluded that the 1.8 kb murine alpha-fetoprotein gene was successfully cloned and its eukaryotic expression vector was successfully constructed.
CHO Cells ; Cloning, Molecular ; Cricetinae ; DNA, Complementary ; Eukaryotic Cells/metabolism ; Genetic Vectors ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection ; alpha-Fetoproteins/*biosynthesis ; alpha-Fetoproteins/genetics

To comparatively investigate ultrastructural characteristics and expressions of AFP (alpha-fetoprotein) and Tn (Thomsen-Friedenreich-related antigen) protein in AFP negative (AFP-) and AFP positive (AFP+) primary hepatocellular carcinoma. Fourty-three cases of AFP- and AFP+ hepatocellular carcinoma (HCC) tissues and five cases of normal liver tissues were divided into three groups: control group (normal liver tissue, n=5); AFP+ HCC group (the serum AFP level was higher than 10 ng/ml, n = 22); AFP- HCC group (the serum AFP level was lower than 10 ng/ml, n=21). The ultrastructural morphology was studied by transmission electron microscopy, the expressions of AFP and Tn protein were detected by immunohistochemistry and cell image analysis. 1. The immunohistochemical study showed that (1) the expression intensity and positive rate of Tn protein in AFP- HCC group were markedly higher than that in AFP+ HCC group (P<0.01); (2) The expression intensity of AFP in AFP- HCC group was lower than that in AFP+ HCC group (P<0.01). 2. The transmission electron microscopy demonstrated that some AFP- HCC cells linked closely with each other, others dispersed loosely just as cultured cells, the remarkable morphologic features in AFP- HCC cells were simple organelles, but they were abundant in the free polyribosomes. In AFP+ HCC group, all the HCC cells linked closely together and were rich organelles in their cytoplasm, especially the rough endoplasmic reticula. In addition, mitochondria and Golgi complex were obviously observed. (1) The AFP and Tn protein had discrepancy distribution in AFP- and AFP+ HCC tissues, Tn protein may be one of the early diagnostic indicators in AFP- HCC; (2) The synthetic locations of the AFP and Tn protein were different in hepatocarcinoma cells by ultrastructural observation.
Antigens, Tumor-Associated, Carbohydrate ; biosynthesis ; genetics ; Biomarkers, Tumor ; Carcinoma, Hepatocellular ; metabolism ; ultrastructure ; Female ; Humans ; Immunohistochemistry ; Liver Neoplasms ; metabolism ; ultrastructure ; Male ; Tumor Cells, Cultured ; alpha-Fetoproteins ; biosynthesis ; genetics

OBJECTIVETo explore hepatitis C virus (HCV) non-structural protein 5A (NS5A)'s influence on inhibition of AFP expression executed by p53 protein and its possible molecular mechanism.

METHODSPlasmid transfection and MEIA were employed to observe p53's inhibitive effect on AFP expression of Huh7 cells and the HCV NS5A's influence on p53 function. Western blot was employed to find out if HCV NS5A affects p53 protein expression and GST pull down assay was applied to examine the interaction between HCV NS5A and p53.

RESULTSThe AFP concentration in the supernatant of the culture of the Huh7 cells transfected with pRc/CMV was (14322+/-2412) ng/ml, and that of the Huh7 cells transfected with pCNS5A was (13843+/-3218) ng/ml; no significant difference existed between these two groups (t = 1.42, P > 0.05). After transfection with pC53-NS3, the AFP level was decreased to (10 241+/-1326) ng/ml, and in comparison to the above two groups it had a statistically significant difference (t values were 2.41 and 2.38, P < 0.05). When co-transfected with pCNS5A and pC53-NS3, the AFP expression (14582+/-1238) ng/ml returned to the level of pRc/CMV transfected, and there was a remarkably significant difference between this and that of the pC53-NS3 transfected cells (t = 3.12, P < 0.01). HCV NS5A had no function on the p53 protein expression with Western blot experiment. In the GST pull down assay, an HCV NS5A protein band was found after GST-p53 was added, but not detected with GST only.

CONCLUSIONWe found that p53 has an inhibitive function on the AFP expression in Huh7 cells and HCV NS5A minimized this p53 function. HCV NS5A did not affect p53 protein expression, but was able to form a complex with p53, by which HCV NS5A inactivated this p53 function.

Carcinoma, Hepatocellular ; metabolism ; virology ; Hepacivirus ; genetics ; Humans ; Liver Neoplasms ; metabolism ; virology ; Tumor Cells, Cultured ; Tumor Suppressor Protein p53 ; genetics ; pharmacology ; Viral Nonstructural Proteins ; genetics ; alpha-Fetoproteins ; biosynthesis ; genetics

To clone the murine alpha-fetoprotein (AFP) gene, construct the eukaryotic expression vector of AFP and express in CHO cells, total RNA were extracted from Hepa 1-6 cells, and then the murine alpha-fetoprotein gene was amplified by RT-PCR and cloned into the eukaryotic expression vector pcDNA3.1. The recombinant of vector was identified by restriction enzyme analysis and sequencing. After transient transfection of CHO cells with the vector, Western blotting was used to detect the expression of AFP. It is concluded that the 1.8 kb murine alpha-fetoprotein gene was successfully cloned and its eukaryotic expression vector was successfully constructed.
Animals ; CHO Cells ; Cloning, Molecular ; Cricetinae ; DNA, Complementary ; Eukaryotic Cells ; metabolism ; Genetic Vectors ; Humans ; Mice ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection ; alpha-Fetoproteins ; biosynthesis ; genetics

OBJECTIVESTo study the biological behavior of hepatocarcinoma stem cells in rats.

METHODSPrimary liver carcinomas were induced in rats using diethylnitrosamine. Tumor cells from 8 rats were separated according to rats oval cell (OVC) markers CD34, c-Kit, Thy-1, AFP, CK7, CK8, CK14, CK18, CK19 and GGT and then they were separately injected into the livers of nude mice. The tumors grown from the different subpopulation of OVC markers in the nude mice livers (10 OVC markers negative or positive cells) were weighted 1 month after the inoculations. The hepatocarcinoma cell subpopulations with higher ability in causing tumor growths were further studied in vitro. The cell cycles and DNA content of those subpopulation cells were investigated using flow cytometry.

RESULTS(1) Subpopulation cells with CK7(-), Thy-1(+) and AFP(+) markers had a higher ability in causing tumors in nude mice; (2) Subpopulation cells, exhibiting characters of TSC, had a low growth rate in vitro.

CONCLUSIONS(1) Different subpopulations of hepatocarcinoma cells had different abilities in causing tumors in rats. Some subpopulation cells, such as CK7(-), Thy-1(+) and AFP(+) cells, have characteristics of tumor stem cells. (2) The hepatocarcinoma stem cells may have a low growth rate in vitro.

Animals ; Cell Cycle ; Cyclin-Dependent Kinases ; biosynthesis ; Liver Neoplasms, Experimental ; pathology ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplastic Stem Cells ; pathology ; physiology ; Rats ; Rats, Sprague-Dawley ; Thy-1 Antigens ; biosynthesis ; Tumor Cells, Cultured ; alpha-Fetoproteins ; biosynthesis

Adult ; Bone Marrow Cells ; cytology ; metabolism ; Cells, Cultured ; Culture Media ; Female ; Hepatitis, Viral, Human ; blood ; Humans ; Male ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Serum ; Serum Albumin ; biosynthesis ; genetics ; alpha-Fetoproteins ; biosynthesis ; genetics

OBJECTIVETo investigate the expressions of glypican-3 (GPC3) mRNA in hepatocellular carcinoma (HCC) tissues and peripheral blood cells (PBCs), and to determine the values of GPC3 mRNA in the diagnosis of HCC and HCC micrometastasis.

METHODSUsing semi-quantitative and nested reverse transcription polymerase chain reactions (RT-PCR), we detected the expressions of AFP and GPC3 genes in the tissues of 41 HCC, 41 paracancer and 52 non-HCC liver samples (41 far from HCC tissues and 11 normal liver tissues), and in the PBCs of 67 specimens from subjects.

RESULTSThe semi-quantitative RT-PCR displayed GPC3 mRNA was expressed in all samples of tissues and PBCs, and the relative intensities of its expressions in HCC, paracancer, non-HCC liver tissues were 78.9 +/- 35.5, 30.6 +/- 21.6, 23.8 +/- 15.5 respectively. The AFP mRNA expression values were 61.2 +/- 32.6, 31.5 +/- 23.6, and 21.2 +/- 15.9 respectively. The expression of each gene in HCC differed significantly from those in other two kinds of tissue samples (P < 0.01). The expressions of GPC3 mRNA and AFP mRNA, accounting for 80.5% and 63.4% in all the HCC tissues, were higher than their respective peak values in the tissues of non-HCC liver (+1.96s), but the expressions of at least one of the two genes was elevated in 92.7% of all the HCC tissues. There was a significant difference between combined detection of two genes and single AFP mRNA detection in HCC tissues (P < 0.01). Clinicopathologically, AFP mRNA was related with the grade of HCC and serum AFP, while GPC3 mRNA was related with not only the grade of HCC but also the invasion of HCC. The relative intensities of GPC3 mRNA expressions in PBCs of 67 specimens was 15.9 +/- 9.0, and GPC3 mRNA expressed in three kinds of tissue samples were all stronger than its counterparts in PBCs (P < 0.01). The GPC3 mRNA expression values in PBCs of the HCC group and the non-HCC group were respectively 16.1 +/- 8.3, 15.6 +/- 10.2, there was no significant difference between the two groups. Of the HCC metastasis group and the HCC non-metastasis group, the respective GPC3 mRNA expression values in PBCs were 16.0 +/- 9.0 and 16.3 +/- 7.7, there was also no significant difference between the two groups. The nested RT-PCR showed that the positive rates of AFP mRNA expressions in PBCs from the HCC group and the non-HCC group were 56.1% and 23.1%, and the difference between the two groups was significant (P = 0.011). The positive rates of AFP mRNA expressions in PBCs from the HCC metastasis group and the HCC non-metastasis group were 80.9% and 30.0%, and there was also a significant difference between the two groups (P = 0.002).

CONCLUSIONSAlthough GPC3 mRNA is expressed broadly, it still may serve as a potential tissue biomarker in the diagnosis of HCC. Detecting the expression of the two genes in the tissues will improve the screening and diagnosis of HCC. GPC3 is prevalently transcribed in the PBCs, but we have not found any relationship between the GPC3 expression in PBCs and the metastasis or recurrence of hepatocellular carcinoma, thus we can not identify HCC micrometastasis with GPC3 mRNA.

Adult ; Aged ; Carcinoma, Hepatocellular ; diagnosis ; metabolism ; pathology ; Female ; Glypicans ; biosynthesis ; genetics ; Humans ; Liver Neoplasms ; diagnosis ; metabolism ; pathology ; Male ; Middle Aged ; Neoplasm Metastasis ; RNA, Messenger ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; alpha-Fetoproteins ; biosynthesis ; genetics

Twenty-one cases of seminoma (including testicular seminoma, ovarian dysgerminoma and extragonadal germinoma) were reviewed for the cell types responsible for the production of alpha-fetoprotein (AFP) and human chorionic gonadotropin (HCG). Histologically the cases included seventeen classical seminomas and 4 anaplastic seminomas. The latter had some mononuclear and multinuclear giant cells. All 4 patients with anaplastic seminoma had elevated levels of serum AFP, and each of these cases contained AFP producing tumor cells identified by immunoperoxidase staining. All seminomas of patients with elevated serum levels of HCG were of the classical type but HCG producing tumor cells could not be identified by immunoperoxidase staining. Immunoreactivity to anti-AFP was found in some large mononuclear cells and anaplastic cells. To explain these results, we propose that the large mononuclear cell is a multipotential cell capable of differentiating into a germ cell, yolk sac and embryo, and that the anaplastic seminoma cells might represent a stage on the continuum of cellular differentiation from the large mononuclear cells to germ cells. The multinuclear giant cell does not appear to be essential for the production of either AFP or HCG in seminoma.
Adolescent ; Adult ; Child ; Chorionic Gonadotropin/metabolism ; Dysgerminoma/metabolism/*pathology ; Female ; Human ; Immunohistochemistry ; Male ; Middle Age ; Ovarian Neoplasms/metabolism/pathology ; Support, Non-U.S. Gov't ; Testicular Neoplasms/metabolism/pathology ; alpha-Fetoproteins/biosynthesis