[The methylation status of PEG10 in placentas of cloned transgenic calves].

The low efficiency of somatic cell nuclear transfer (SCNT) is a significant barrier to the production of highly valuable transgenic livestock. It is generally believed that the principal cause of the low SCNT efficiency is the aberrant nuclear epigenetic reprogramming of donor somatic cell. DNA methylation is a major epigenetic modification of the genome and plays a crucial role in nuclear reprogramming during SCNT. In order to assess whether the abnormal epigenetic modifications of the imprinted gene in placenta are correlated with the development abnormality and death of the cloned transgenic calves, the DNA methylation patterns of PEG10 were compared in the placentas from different kinds of cattle. This comparison included transgenic cloned calves died during perinatal stage and showed developmental defects (Death group), transgenic cloned calves survived and lived on healthily (Live group) and the normal reproduced calves (N group) used as the control group analyzed by Bisulfite Sequencing PCR (BSP) method and Combined Bisulfite Restriction Analy-sis (COBRA). Comparing to the control group, PEG10 gene in the Death group showed abnormal hypermethylation, but was not significant different in methylation level from the Live group. It can be postulated from the results that the incom-plete or abnormal DNA methylation epigenetic reprogramming of imprinting gene in placenta may be one of the main causes of the abnormal development and death of the transgenic cloned cattle.

[Relationship between tardive dyskinesia and the polymorphism of superoxide dismutase val9Ala and efficacy of Chaihu Taoren Capsules on it].

OBJECTIVE: To investigate the relationship between efficacy of Chaihu Taoren Decoction (CTD) and the polymorphism of valine-alanine missense mutation of 9th codan (Val9Ala, T1183C) of superoxide dismutase (SOD) in patients with tardive dyskinesia (TD). METHODS: Severity of TD was assessed by abnormal involuntary movement scale (AIMS), and the psychologic symptoms were rated by the positive and negative symptoms scale (PANSS). The sample size consisted of 119 patients with TD assigned to the TD group, 129 patients of chronic schizophrenia with the general condition matched strictly with that of the enrolled TD patients assigned to the non-TD group, and 148 healthy persons assigned to the normal group. The gene distribution rate of Val9Ala gene was analyzed using polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) analysis, and the therapeutic effect of CTD on 36 patients with TD was observed after 12 weeks treatment. RESULTS: There was no significant difference in genotype and allelic gene frequency of SOD Val9Ala among the TD, non-TD and normal groups (P > 0.05). Comparison of the AIMS score in TD patients with various Val9Ala genotypes showed that the difference of AIMS scores in patients with TT and CT genotype was not significant (P > 0.05), but CTD did show a better efficacy in TD patients with CT heterozygote than in those with TT homozygote (P < 0.05). CONCLUSION: The CTD could effectively relieve the symptoms of TD, its efficacy might be related with the genotype of SOD, and 9Ala is considered to be a protective factor for the susceptibility to TD.

Role of cell adhesion signal molecules in hepatocellular carcinoma cell apoptosis.

AIM: Cell adhesion molecules and their signal molecules play a very important role in carcinogenesis. The aim of this study is to elucidate the role of these molecules and the signal molecules of integrins and E-cadherins, such as (focal adhesion kinase) FAK, (integrin linked kinase) ILK, and beta-catenin in hepatocellular carcinoma cell apoptosis. METHODS: We first synthesized the small molecular compound, S-(1,2-dichlorovinyl)-L-cysteine (DCVC), and identified it, by element analysis and (1)H NMR. To establish the apoptosis model of the SMMC-7721 hepatocellular carcinoma cell, we treated cells with DCVC in EBSS for different concentrations or for various length times in the presence of 20 micromol/L N,N'-diphenyl-p-phenylenediamine, which blocks necrotic cell death and identified this model by flow cytometry and DNA ladder. Then we studied the changes of FAK, ILK, beta-catenin, and PKB in this apoptotic model by Western blot. RESULTS: We found that the loss or decrease of cell adhesion signal molecules is an important reason in apoptosis of SMMC-7721 hepatocellular carcinoma cell and the apoptosis of SMMC-7721 cell was preceded by the loss or decrease of FAK, ILK, PKB, and beta-catenin or the damage of cell-matrix and cell-cell adhesion. CONCLUSION: Our results suggested that the decrease of adhesion signal molecules, FAK, ILK, PKB, and beta-catenin, could induce hepatocellular carcinoma cell apoptosis.

Integrin beta1 subunit overexpressed in the SMMC-7721 cells regulates the promoter activity of p21(CIP1) and enhances its transcription.

Evidence has been emerging to suggest that integrin could induce growth inhibition in some cell types. Some of the molecular mechanisms underlying growth arrest have been elucidated. We reported here that overexpression of integrin beta1 imposed a growth inhibitory effect on the hepatocellular carcinoma cell line SMMC-7721, and this phenomenon was mainly attributed to the cyclin-dependent kinase inhibitor p21(CIP1). Furthermore, our findings suggested that transcription activity of the p21(CIP1) gene could be upregulated in the integrin beta1-overexpressing cells, and possibly controlled by the cis-elements in the core region of the p21(CIP1) promoter.

Expression of focal adhesion kinase and alpha5 and beta1 integrins in carcinomas and its clinical significance.

AIM: To detect the expression pattern of FAK (focal adhesion kinase) and integrin alpha5 and beta1 subunits in different kinds of cancerous tissues and to study their correlation with clinicopathological data including tumor type, grade and lymph node status. METHODS: Using an immunohistochemical technique, we examined the expression of FAK and integrin and subunits in cancerous and noncancerous tissues obtained from 75 patients with gastric carcinomas, 21 colorectal carcinomas, 16 hepatocellular carcinomas, 20 uterocervical carcinomas, and 20 breast carcinomas. RESULTS: The staining of FAK was stronger in cancerous than in noncancerous areas. Enhanced expression of FAKwas detected in poor-differentiated carcinoma of the stomach and colorectum. Tumors with lymph node metastases had more FAK protein than those without metastases. In addition, the deeper the extent of tumor infiltration, the higher the FAK expression. The expression of integrin alpha5 and beta1 subunits was lower in cancerous areas than in noncancerous areas, but it was higher in well-differentiated cancerous tissues than in poor differentiated tissues. The relationship between the expression of integrin alpha5 and beta1 subunits and infiltration or metastasis was not significant. Cancerous tissues with stronger FAK expression (++ or +++) also had a higher expression of integrin alpha5 and beta1 subunits in the tumor and its unaffected margins. CONCLUSION: FAK is a better marker for carcinogenesis and the progression of cancer than integrin alpha5 and beta1 subunit, and it may be not only a transformation-linked enzyme but also a progression-linked enzyme.

S-phase delay in human hepatocellular carcinoma cells induced by overexpression of integrin beta1.

AIM: To clarify the mechanisms of integrin overexpression in negatively regulating the cell cycle control of hepatocellular carcinoma cells SMMC-7721. METHODS: The cell cycle pattern was determined by flow cytometry. The mRNA and protein expression levels were assayed by RT-PCR and Western blot, respectively. Stable transfection was performed by Lipofectamine 2000 reagent, and cells were screened by G418. RESULTS: Overexpression of alpha5beta1 or beta1 integrin induced S-phase delay in SMMC-7721 cells, and this delay was possibly due to the accumulation of cyclin-dependent kinase inhibitors (CKIs) p21(cip1) and p27(kip1). The decrease of protein kinase B (PKB) phosphorylation was present in this signaling pathway, but focal adhesion kinase (FAK) was not involved. When phosphorylation of PKB was solely blocked by wortmannin, p27(kip1) protein level was increased. Moreover, S-phase delay was recurred when attachment of the parental SMMC-7721 cells was inhibited by the preparation of poly-HEME, and this cell cycle pattern was similar to that of beta1-7721 or alpha5beta1-7721 cells. CONCLUSION: S-phase delay induced by overexpression of integrin beta1 subunit is attributed to the decrease of PKB phosphorylation and subsequent increases of p21(cip1) and p27(kip1) proteins, and may be involved in the unoccupied alpha5beta1 because of lack of its ligands.

Effects of the removal of cytoplasm on the development of early cloned bovine embryos.

Oocyte cytoplasm plays a prominent role in cloned embryonic development. To investigate the influence of oocyte cytoplasmic amount on cloned embryo development, we generated bovine somatic cell nuclear transfer (SCNT) embryos containing high (30-40% of the cytoplasm was removed), medium (15-25% of the cytoplasm was removed) and low (<10% of the cytoplasm was removed) nucleocytoplasmic volume ratios (N/C) using enucleated metaphase II oocyte as recipient, and fibroblast as donor nucleus, and analyzed the expression levels of ND1, Cytb and ATPase6, as well as the embryonic quality. The results indicated: (1) the process of embryonic development was not influenced by <40% of cytoplasm removal; (2) the rate of blastocyst formation, the total number of blastomere and the ratio of ICM to TE were inversely proportional to the N/C; (3) SCNT embryos with reduced volume equal to 75-85% or >90% of an intact oocyte volume showed similar karyotype structure of the donor cells; (4) the number of mtDNA copy was larger in low N/C embryos than that in medium or high N/C embryos, and the expression levels of each gene hardly varied from the 2-cell to 8-cell stage, while the expression levels increased dramatically at the blastocyst stage; (5) from 16-cell to the blastocyst stage, the change of the expression level of each gene was not significant between low N/C embryos and IVF embryos, but it was more significant than those of high or medium N/C embryos. The results suggest that the decrease of mtDNA copy number and mitochondrial gene expression may be related to the impairment in early embryonic development, and removal of <10% adjacent cytoplasm volume may be optimal for bovine SCNT embryo development.

Expression and methylation status of imprinted genes in placentas of deceased and live cloned transgenic calves.

Placental deficiencies are linked with developmental abnormalities in cattle produced by somatic cell nuclear transfer (SCNT). To investigate whether the aberrant expression of imprinted genes in placenta was responsible for fetal overgrowth and placental hypertrophy, quantitative expression analysis of six imprinted genes (H19, XIST, IGF2R, SNRPN, PEG3, and IGF2) was conducted in placentas of: 1) deceased (died during perinatal period) transgenic calves (D group, n = 4); 2) live transgenic calves (L group, n = 15); and 3) conventionally produced (control) female calves (N group, n = 4). In this study, XIST, PEG3 and IGF2 were significantly over-expressed in the D group, whereas expression of H19 and IGF2R was significantly reduced in the D group compared to controls. The DNA methylation patterns in the differentially methylated region (DMR) from H19, XIST, and IGF2R were compared using Bisulfite Sequencing PCR (BSP) and Combined Bisulfite Restriction Analysis (COBRA). In the D group, H19 DMR was significantly hypermethylated, but XIST DMR and IGF2R ICR were significantly hypomethylated compared to controls. In contrast, there were no noticeable differences in the expression and DNA methylation status of imprinted genes (except DNA methylation level of XIST DMR) in the L group compared to controls. In conclusion, altered DNA methylation levels in the DMRs of imprinted genes in placentas of deceased transgenic calves, presumably due to aberrant epigenetic nuclear reprogramming during SCNT, may have been associated with abnormal expression of these genes; perhaps this caused developmental insufficiencies and ultimately death in cloned transgenic calves.

Scriptaid improves in vitro development and nuclear reprogramming of somatic cell nuclear transfer bovine embryos.

The present study evaluated the effect of Scriptaid, a novel histone deacetylase inhibitor (HDACi), on the in vitro development of somatic cell nuclear transfer (SCNT) bovine embryos. Average fluorescence intensity of two epigenetic markers (H3K9ac and H3K9m2) at two-cell, eight-cell, and blastocyst stages, and the expression levels of two developmental important genes (Oct4 and IFN-t) at the blastocyst stage were also examined to assess the influence of Scriptaid on the nuclear reprogramming of bovine SCNT embryos. The results showed that treatment with 500 nM Scriptaid for 14 h after activation significantly increased the cleavage rate, blastocyst formation rate, and blastocyst hatching rate of SCNT embryos compared with those of nontreated counterparts, but the total number of blastomeres per blastocyst did not differ. Scriptaid treatment also significantly increased the immunofluorescent signal for H3K9ac in SCNT embryos at two-cell, eight-cell, and blastocyst stages, and the fluorescent signal for H3K9m2 was decreased at two-cell, eight-cell, and blastocyst stages. The expression levels of Oct4 and IFN-t were significantly higher in Scriptaid-treated SCNT blastocysts than in Scriptaid nontreated SCNT blastocysts. The results indicated that Scriptaid treatment improved the in vitro developmental capacity and the nuclear reprogramming of bovine SCNT embryos.

Transforming growth factor-beta1 stimulated protein kinase B serine-473 and focal adhesion kinase tyrosine phosphorylation dependent on cell adhesion in human hepatocellular carcinoma SMMC-7721 cells.

Transforming growth factor-beta1 (TGF-beta1) is a potent growth inhibitor and apoptosis inducer for most normal cells. However, tumor cells are commonly nevertheless sensitive to the tumor-suppressing effects of TGF-beta1. In this paper, we focus on the effects of TGF-beta1 on two important anti-apoptotic protein kinases, protein kinase B (PKB), and focal adhesion kinase (FAK), in SMMC-7721 cells. We found that PKB-Ser-473 phosphorylation was apparently up-regulated by TGF-beta1. In the meantime, PKB-Thr-308 phosphorylation was slightly up-regulated by TGF-beta1. TGF-beta1 could also enhance FAK-Tyr phosphorylation. We observed that integrin-linked kinase (ILK) was also up-regulated by TGF-beta1 in good accordance with PKB-Ser-473 phosphorylation. We first found that TGF-beta1 could stimulate PKB-Ser-473 phosphorylation possibly via up-regulating ILK expression. Furthermore, we also failed to detect PKB-Ser-473 and FAK-Tyr phosphorylation with various concentrations of TGF-beta1 treatment when cells were kept in suspension. The above results indicate that PKB-Ser-473 and FAK-Tyr phosphorylation stimulated by TGF-beta1 are both dependent on cell adhesion.