p53对乳腺癌耐药蛋白基因的转录调控

乳腺癌耐药蛋白（breast cancer resistance protein,BCRP）是ATP结合盒转运蛋白超家族成员之一,其通过主动外排化疗药物如米托蒽醌、托泊替康和甲氨蝶呤,进而介导肿瘤化疗耐受. 最近有研究发现,在野生型p53（wild type p53, wt-p53）低表达的乳腺癌细胞系MCF-7中,外源性wt-p53通过抑制核转录因子-κB （nuclear factor-κB, NF-κB）的活性进而抑制BCRP的表达,但其详细的分子机制有待进一步阐明. 本研究选用p53缺失的骨肉瘤细胞系Saos-2,通过瞬时转染技术发现,wt-p53可以激活BCRP的表达,而突变型p53的激活作用消失;报告基因试验显示,wt-p53可以上调BCRP启动子活性;通过生物信息学软件MatInspector对BCRP启动子区进行预测,未发现p53结合元件;同时,通过转染IκB抑制Saos-2细胞中NF-κB的活性后发现,Saos-2细胞中NF-κB活性越低,p53对BCRP启动子的激活作用越弱甚至完全消失. 上述结果提示,p53对Saos-2细胞中BCRP的激活作用是NF-κB依赖性的.     

Reduction of Orc6 Expression Sensitizes Human Colon Cancer Cells to 5-Fluorouracil and Cisplatin

Previous studies from our group have shown that the expression levels of Orc6 were highly elevated in colorectal cancer patient specimens and the induction of Orc6 was associated with 5-fluorouracil (5-FU) treatment. The goal of this study was to investigate the molecular and cellular impact of Orc6 in colon cancer. In this study, we use HCT116 (wt-p53) and HCT116 (null-p53) colon cancer cell lines as a model system to investigate the impact of Orc6 on cell proliferation, chemosensitivity and pathways involved with Orc6. We demonstrated that the down regulation of Orc6 sensitizes colon cancer cells to both 5-FU and cisplatin (cis-pt) treatment. Decreased Orc6 expression in HCT-116 (wt-p53) cells by RNA interference triggered cell cycle arrest at G1 phase. Prolonged inhibition of Orc6 expression resulted in multinucleated cells in HCT-116 (wt-p53) cell line. Western immunoblot analysis showed that down regulation of Orc6 induced p21 expression in HCT-116 (wt-p53) cells. The induction of p21 was mediated by increased level of phosphorylated p53 at ser-15. By contrast, there is no elevated expression of p21 in HCT-116 (null-p53) cells. Orc6 down regulation also increased the expression of DNA damaging repair protein GADD45β and reduced the expression level of JNK1. Orc6 may be a potential novel target for future anti cancer therapeutic development in colon cancer.     

Induction of apoptosis in p53-deficient human hepatoma cell line by wild-type p53 gene transduction: inhibition by antioxidant

We investigated the role of wild-type (wt)-p53 as an inducer of apoptotic cell death in human hepatoma cell lines. Following the retrovirus-mediated transduction of the wt-p53 gene, Hep3B cells lacking the endogenous p53 expression began to die through apoptosis in 4 h. They showed a maximal apoptotic death at 12 h, whereas HepG2 cells expressing endogenous p53 did not. However, the transduction of the wt-p53 gene elicited growth suppression of both Hep3B and HepG2 cells. P21(WAF1/CIP1), a p53-inducible cell cycle inhibitor, was induced, not only in Hep3B cells undergoing apoptosis, but also in HepG2 cells. The kinetics of the p21(WAF1/CIP1) induction, DNA fragmentation, and growth suppression of the Hep3B cells showed that DNA fragmentation and growth suppression progressed rapidly following p21(WAF1/CIP1) accumulation. N-acetyl-cysteine or glutathione, potent antioxidants, strongly inhibited the DNA fragmentation, but did not reduce the elevated level of p21(WAF1/CIP1). These findings suggested that p21(WAF1/CIP1) was not a critical mediator for the execution of p53-mediated apoptosis, although it contributed to the growth inhibition of cells undergoing apoptosis. Furthermore, p53-mediated apoptosis could be repressed by antioxidants.     

p53 plays a regulatory role in differentiation and apoptosis of central nervous system-associated cells.

This study demonstrated the involvement of the tumor suppressor protein p53 in differentiation and programmed cell death of neurons and oligodendrocytes, two cell types that leave the mitotic cycle early in development and undergo massive-scale cell death as the nervous system matures. We found that primary cultures of rat oligodendrocytes and neurons, as well as of the neuronal PC12 pheochromocytoma cell line, constitutively express the p53 protein. At critical points in the maturation of these cells in vitro, the subcellular localization of p53 changes: during differentiation it appears mainly in the nucleus, whereas in mature differentiated cells it is present mainly in the cytoplasm. These subcellular changes were correlated with changes in levels of immunoprecipitated p53. Infection of cells with a recombinant retrovirus encoding a C-terminal p53 miniprotein (p53 DD), previously shown to act as a dominant negative inhibitor of endogenous wild-type p53 activity, inhibited the differentiation of oligodendrocytes and of PC12 cells and protected neurons from spontaneous apoptotic death. These findings suggest that p53, upon receiving appropriate signals, is recruited into the nucleus, where it plays a regulatory role in directing primary neurons', oligodendrocytes, and PC12 cells toward either differentiation or apoptosis in vitro.     

Radiation response and cell cycle regulation of p53 rescued malignant keratinocytes

Mutations in the tumor suppressor gene p53 were found in more than 90% of all human squamous cell carcinomas (SCC). To study the function of p53 in a keratinocyte background, a tetracycline-controlled p53 transgene was introduced into a human SCC cell line (SCC15), lacking endogenous p53. Conditional expression of wild-type p53 protein upon withdrawal of tetracycline was accompanied with increased expression of p21(WAF1/Cip1) resulting in reduced cell proliferation. Flow-cytometric analysis revealed that these cells were transiently arrested in the G1/S phase of the cell cycle. However, when SCC15 cells expressing p53 were exposed to ionizing radiation (IR), a clear shift from a G1/S to a G2/M cell cycle arrest was observed. This effect was greatly depending on the presence of wild-type p53, as it was not observed to the same extent in SCC15 cells lacking p53. Unexpectedly, the p53- and IR-dependent G2/M cell cycle arrest in the keratinocyte background was not depending on increased expression or stabilization of 14-3-3sigma, a p53-regulated effector of G2/M progression in colorectal cancer cells. In keratinocytes, 14-3-3sigma (stratifin) is involved in terminal differentiation and its cell cycle function in this cell type might diverge from the one it fulfills in other cellular backgrounds.     

Regulation of p53-mediated apoptosis and cell cycle arrest by Steel factor.

Activation of the p53 protein can lead to apoptosis and cell cycle arrest. In contrast, activation of the signalling pathway controlled by the Kit receptor tyrosine kinase prevents apoptosis and promotes cell division of a number of different cell types in vivo. We have investigated the consequences of activating the Kit signalling pathway by its ligand Steel factor on these opposing functions of the p53 protein in Friend erythroleukemia cells. A temperature-sensitive p53 allele (Val-135) was introduced into the Friend erythroleukemia cell line (DP-16) which lacks endogenous p53 expression. At 38.5 degrees C, the Val-135 protein maintains a mutant conformation and has no effect on cell growth. At 32 degrees C, the mutant protein assumes wild-type properties and induces these cells to arrest in G1, terminally differentiate, and die by apoptosis. We demonstrate that Steel factor inhibits p53-mediated apoptosis and differentiation but has no effect on p53-mediated G1/S cell cycle arrest. These results demonstrate that Steel factor functions as a cell survival factor in part through the suppression of differentiation and apoptosis induced by p53 and suggest that cell cycle arrest and apoptosis may be separable functions of p53.