[Construction and expression of fusion protein TRX-hJagged1 in E.coli BL21].

This study was purposed to construct prokaryotic expression vector and to investigate the expression of Notch ligand Jagged1 in E.coli. An expression vector pET-hJagged1 was constructed, which can be inserted in Jagged1 with different lengths, but the DSL domain of human Jagged1 should be contained. Then the recombinant plasmids were transformed into the competent cell of E.coli BL21, and the expression of the fusion protein was induced by IPTG. Fusion protein was purified from the supernatant of cell lysates via the Nickel affinity chromatography. The results showed that prokaryotic expression vectors pET-hJagged1 (Bgl II), pET-hJagged1 (Hind I) and pET-hJagged1 (Stu I) were successfully constructed, but only pET-hJagged1 (Stu I) could express the soluble TRX-hJagged1. The purified TRX-Jagged1 protein could be obtained via the Nickel affinity chromatography, and then confirmed by Western Blot. It is concluded that prokaryotic expression vector pET-hJagged1 is successfully constructed, but only pET-hJagged1 (Stu I) can express the soluble TRX-hJagged1 and the TRX-Jagged1 fusion protein is obtained through the prokaryotic expression system, which laid a solid foundation for further to explore the effects of Jagged1 in hematopoietic and lymphoid system.

[Effects of genistein on colon cancer cells in vitro and in vivo and its mechanism of action].

OBJECTIVE: To study the effects of genistein on the proliferation, apoptosis induction and expression of related gene proteins of human colon cancer cells in vitro and in vivo, and its mechanisms of action. METHODS: MTT colorimetric assay was used to detect the effects of genistein on the proliferation of human colon adenocarcinoma SW480 cells. Light and transmission electron microscopy were used to study the histological and ultrastructural changes. Flow cytometry was used to determine the effects of genistein on cell cycle and apoptosis. Flow cytometry and immunohistochemistry were used to determine the effects of genistein on apoptosis induction and expression of related gene proteins of colon cancer cells. RESULTS: The MTT colorimetric assay showed that genistein inhibited the proliferation of SW480 cells in a dose-dependent and time-dependent manner, and the highest inhibition rate was 60.2% after 80 microg/ml genistein treatment for 72 h. The light microscopy revealed that many genistein-treated cancer cells were shrunken, disrupted, or showing cytoplasmic vacuolization. The electron microscopic examination showed cell shrinkage, nuclear fragmentation and pronounced chromatin condensation, sometimes formed crescent chromatin condensation attached to the nuclear membrane. The results of flow cytometry showed that: after SW480 cells were treated with 0, 20, 40, 80 microg/ml genistein for 48 h, the FI values of PCNA were 1.49 +/- 0.02, 1.28 +/- 0.04, 1.14 +/- 0.03, and 0.93 +/- 0.08; the FI values of VEGF were 1.75 +/- 0.02, 1.34 +/- 0.06, 1.32 +/- 0.04, and 1.23 +/- 0.04; the fluorescence index (FI) values of p21 were 1.26 +/- 0.05, 1.36 +/- 0.06, 1.61 +/- 0.03, and 1.73 +/- 0.03, respectively. There were statistically significant differences between the control group and each treatment group (P < 0.05 or P < 0.01). The scores of immunohistochemical staining of PCNA and VEGF proteins were decreased, while p21 increased. There were statistically significant differences between the control group and each treatment group (P < 0.05 or P < 0.01). CONCLUSION: Genistein can inhibit the growth of colon cancer cells via apoptosis induction and cell cycle arrest at G(2)/M phase. The anti-tumor mechanisms of genistein may be related with the down-regulation of expression of VEGF and PCNA, and up-regulation of the expression of p21.

Notch signaling maintains proliferation and survival of the HL60 human promyelocytic leukemia cell line and promotes the phosphorylation of the Rb protein.

The Notch signaling pathway has been implicated in the development of several leukemia and lymphoma. In order to investigate the relationship between Notch signaling and acute myeloid leukemia (AML), in this study, we expressed a recombinant Notch ligand protein, the DSL domain of the human Jagged1 fused with GST (GST-Jag1). GST-Jag1 could activate Notch signaling in the human promyelocytic leukemia cell line HL60, as shown by a reporter assay and the induced expression of Notch effector gene Hes1 and Hes5. However, GST-Jag1 had no effect on the proliferation and survival of HL60 cells. HL60 cells expressed both Notch ligands and receptors, and had a potential of reciprocal stimulation of Notch signaling between cells. We, therefore, blocked Notch signaling in cultured HL60 cells using a gamma-secretase inhibitor (GSI). We found that GSI inhibited the proliferation of HL60 cells significantly by blocking the cell-cycle progression in the G1 phase. Furthermore, GSI induced remarkably apoptosis of HL60 cells. These changes in GSI-treated HL60 cells correlated with the down-regulation of c-Myc and Bcl2, and the low phosphorylation of the Rb protein. These results suggested that reciprocal Notch signaling might be necessary for the proliferation and survival of AML cells, possibly through the maintenance of the expression of c-Myc and Bcl2, as well as the phosphorylation of the Rb protein.