Differential role of repair proteins,BRCA1/NBS1 and Ku70/DNA‐PKcs,in radiation‐induced centrosome overduplication

Centrosomes are important cytoplasmic organelles involved in chromosome segregation, defects in which can result in aneuploidy, and contribute to tumorigenesis. It is known that DNA damage causes the supernumerary centrosomes by a mechanism in which centrosomes continue to duplicate during cell cycle arrest at checkpoints. We show here that ionizing radiation induces the overduplication of centrosomes in a dose‐dependent manner, and that the level of overduplication is pronounced in BRCA1‐ and NBS1‐deficient cells, even though their checkpoint control is abrogated. Conversely, marginal increases in overduplication were observed in Ku70‐ and DNA‐PKcs‐deficient cells, which are intact in checkpoint control. The frequency of radiation‐induced overduplication of centrosomes might be associated with DNA repair, as it was decreased with reduced cell killing after protracted exposures to radiation. As a result, when the frequency of radiation‐induced centrosome overduplication was plotted against radiation‐induced cell killing, similar curves were seen for both protracted and acute exposures in wild‐type cells, Ku70‐deficient, and DNA‐PKcs‐deficient cells, indicating a common mechanism for centrosome overduplication. However, the absence of either BRCA1 or NBS1 enhanced radiation‐induced overduplication frequencies by 2–4‐fold on the basis of the same cell killing. These results suggest that radiation‐induced centrosome overduplication is regulated by at least two mechanisms: a checkpoint‐dependent pathway involved in wild‐type cells, Ku70‐deficient and DNA‐PKcs‐deficient cells; and a checkpoint‐independent pathway as observed in BRCA1‐deficient and NBS1‐deficient cells. (Cancer Sci 2010; 101: 2531–2537)     

Differential expression and regulation of p53 in human prostatic cells

Although genetic analysis has convincingly shown the association possibly existing between alterations in p53 tumor suppressor gene and a broad spectrum of human tumors including prostate cancer, surprisingly little is known about ways in which p53 at the protein level is controlled. To determine factors that may play a role in its regulation and expression, changes in p53 protein was investigated by using the androgen-insensitive JCA-1, DU-145, PC-3 and the androgen-responsive LNCaP cells. With the exception of PC-3 cells in which p53 is missing, multiple distinct forms of p53 were found in the other 3 prostate cell lines. A single p53 band was detected in the JCA-1 cell extracts, whereas two and three p53 immunoreactive bands were correspondingly observed in the DU-145 and LNCaP cells. The relative abundance and distribution of the different forms of p53 in the latter two cell types varied with proliferation of cells in culture. In the presence of charcoal-stripped fetal bovine serum (cFBS), LNCaP took on the morphology of neuroendocrine cells, a phenotypic change which was accompanied by a greater than 80% reduction in p53 expression, concurrent with elimination of the two slow migrating forms of p53. Induction of apoptosis in JCA-1 cells by treatment with the retinoid 4-HPR caused the virtual disappearance of p53, which coincided with specific processing of p53 into lower molecular weight 28 kD fragments. We propose that rapid and dynamic posttranslational changes in p53 may actively participate in determining mutually exclusive functional cellular events such as proliferation, differentiation, and apoptosis.     

The role of XPD in cell apoptosis and viability and its relationship with p53 and cdk2 in hepatoma cells

We investigated the role of XPD in cell apoptosis of hepatoma and its relationship with p53 during the regulation of hepatoma bio-behavior. RT–PCR and Western blot were used to detect the expression levels of XPD, p53, c-myc, and cdk2. The cell apoptosis and cell cycle were analyzed with flow cytometry. Compared with the control cells, XPD-transfected cells displayed a lower viability and higher apoptosis rate. A decreased expression of p53 gene was detected in XPD-transfected cells. In contrast, both c-myc and cdk2 showed increased expressions of mRNAs and proteins in the transfected cells. Our results indicate that XPD may play an important role in cell apoptosis of hepatoma by inducing an over-expression of p53, but suppressing expressions of c-myc and cdk2.     

Wild-type p53 induced sensitization of mutant p53 TNF-resistant cells: role of caspase-8 and mitochondria

In the present study, we have investigated the mechanisms by which the restoration of wild-type (wt) p53 functions in p53 mutant cells increases their susceptibility to the cytotoxic action of tumor necrosis factor (TNF). Our data indicate that the resistance of p53-mutated cl.1001 cells to TNF-induced cell death was not due to a defect in the expression of TRADD and FADD, yet correlated with a reduced caspase-8 activation as well as a deficient mitochondrial membrane permeabilization. Moreover, cl.1001 cells failed to translocate the mitochondrial AIF and cytochrome c to the nucleus and to the cytosol, respectively, in response to TNF. Sensitization of these cells, following infection with a recombinant adenovirus encoding wtp53, to TNF-induced cytotoxicity resulted in the restoration of caspase-8 cleavage and the reestablishment of mitochondrial signs of apoptosis. These findings suggest that the cross-talk between p53 and TNF-induced cell death depends on mitochondria and that the combination of TNF and Adwtp53 may be a potential strategy to sensitize mutant p53 TNF-resistant tumors to the cytotoxic action of this cytokine.     

Overexpression of Sp1 leads to p53‐dependent apoptosis in cancer cells

Numerous studies have documented that Sp1 expression level were elevated in various human cancers. However, the promoters of many pro‐apoptotic genes have been found to contain the Sp1 binding elements and are activated by Sp1 overexpression. To better understand the role and the mechanism of increased Sp1 levels on apoptosis, we used adenovirus to ectopically express GFP‐Sp1 protein in various cancer cell lines. First, in HeLa and A549 cells, we found that Sp1 overexpression suppressed the cell growth and increased the detection of sub‐G1 fraction, caspase‐3 cleavage, and annexin‐V signal revealed that apoptosis occurred. Furthermore, when cells entered the mitotic stage, the cell apoptosis was induced by Sp1 overexpression through affecting mitotic chromatin packaging. We also verified that p53 protein was accumulated and activated the p53‐dependent apoptotic pathways in the wild‐type p53 cells but not in the p53‐mutated or p53‐deleted cell lines when these cells were infected with adeno‐GFP‐Sp1 virus. In addition, A549 (p53^+/+) cells could be protected from apoptosis under Sp1 overexpression when p53 was knockdown by p53 shRNA. Finally, H1299 (p53^?/?) cell viability was significantly inhibited by adeno‐GFP‐Sp1 virus infection in the expression of p53. In conclusion, p53 was an essential factor for Sp1 overexpression‐induced apoptotic cell death in transforming cells. © 2009 UICC     

Expression of Hsp70 and Hdj1 chaperone proteins in human tumor cells

Heat shock proteins (Hsp), or "stress proteins", play an important role in maintenance of cellular homeostasis both under normal conditions and during cellular stress. We tested malignant female urogenital and breast tumors for Hsp70 and Hdj1 (Hsp40) chaperones. Immunoenzyme procedure (based on Hsp70 and ATP interaction) was used to assay Hsp70 levels. Hsp70 and Hdj1 expression was higher in malignant cells than in benign ones. We were the first to demonstrate the feasibility of using Hdj1 expression as a novel prognostic factor for neoplastic disease.     

Drug resistance in ovarian cancer — the role of p53

The aims were to determine the importance of p53 and bcl-2 expression on the response to chemotherapy with alkylating agents in patients with ovarian cancer. We have followed the response to chemotherapy in a series of 59 patients with ovarian adenocarcinoma designated as p53 and bcl-2 positive or negative by immunocytochemistry. Of these cases, 50 received either cisplatin + treosulfan or treosulfan alone. Immunocytochemistry for p53 was positive in 28/59 tumors. Patients were grouped according to their response to chemotherapy (stable or progressive disease) assessed at 6, 12, and 18 months. There was increasing divergence of p53+ and p53- tumors over time. Of those which were p53+, 25% showed progression at 6 months, 80% at 12 months and 89% progression at 18 months. In contrast, 23%, 50%, and 67% of p53-tumors showed progression at 6, 12 and 18 months respectively. For bcl-2, in 23/55 positive tumors there was progression in 35%, 78% and 94% compared with 25%, 57% and 59% in bcl-2 negative tumors at 6, 12 and 18 months respectively. Those tumors which were bcl-2 and p53 negative were most likely to progress, while those which were bcl-2 and p53 positive had the best prognosis. These differences did not translate into increased overall survival with minimum follow-up of 12 months. This data lends support to our suggestion that despite initially increased susceptibility to alkylating agents, enhanced genomic instability due to p53 inactivation may render tumors more likely to develop resistance to chemotherapy over time. This effect may be altered by bcl-2 function, lack of which will lead to a good response to chemotherapy as the tumor’s ability to undergo apoptosis will not be compromised.     

VP22-mediated intercellular transport of p53 in hepatoma cells in vitro and in vivo

The capacity of VP22 chimeric proteins to spread from the primary transduced cell to surrounding cells could improve gene therapy approaches, especially in cancer therapy. However, there are conflicting data about VP22-mediated intercellular trafficking in different studies. To assess the role of VP22 in gene therapy of hepatocellular carcinomas (HCCs) we constructed expression vectors for N- and C-terminal versions of VP22-p53 fusion proteins and investigated the VP22-mediated shuttle effect in hepatoma cells by cotransfection experiments. VP22-mediated trafficking was not detectable in hepatoma cells in vitro by fluorescence microscopy, but reporter gene transactivation assays demonstrated intercellular trafficking of functional VP22-p53 in vitro. For in vivo experiments, the recombinant adenoviruses Ad5CMVp53 and Ad5CMVp53-VP22 were constructed. In contrast to the in vitro experiments intercellular trafficking of VP22-p53 could be observed in subcutaneous tumors of hepatoma cells by fluorescence microscopy, indicating a stronger shuttle effect in solid tumors compared to cell culture experiments. Because spread of p53-VP22 in liver tumors was correlated with enhanced apoptosis of hepatoma cells VP22-mediated trafficking of potential therapeutic proteins may improve the results of gene therapy of HCCs.     

Synergic CSE1L/CAS, TNFR-1, and p53 apoptotic pathways in combined interferon-gamma/adriamycin-induced apoptosis of Hep G2 hepatoma cells

Many cancers are chemotherapy-resistant. Chemotherapy combined with immunotherapy offers a potential avenue for the treatment of chemotherapy-resistant cancers. In this study, we investigated the apoptotic pathways induced by combined interferon-gamma/adriamycin treatment in Hep G2 cells. Our data showed that Hep G2 cells treated with combined interferon-gamma/adriamycin enhanced cell apoptosis in comparison with that of cells treated with adriamycin. Interferon-y increased TNFR-1, CSE1L/CAS (cellular apoptosis susceptibility protein), Bax, and Bad levels. Adriamycin increased p53 and Bax, but not TNFR- 1 and CAS levels. Interferon-y did not increase p53 accumulation; nevertheless it enhanced adriamycin-induced p53 accumulation. Overexpression of IRF-1 augmented the combined interferon-gamma/adriamycin-induced p53 accumulation. Interferon-gamma co-treatment increased the stability of p53 protein induced by adriamycin. Our data suggest that TNF-gamma may greatly enhance the combined interferon-gamma/chemotherapeutic drug-induced apoptosis of cancers. Our findings also indicate that CAS, TN-FR-1, p53, Bax, and Bad may be the targets for the interferon-y-based chemo-immunotherapy of the chemotherapy-resistant cancers.     

Molecular targets of apigenin in colorectal cancer cells: Involvement of p21, NAG-1 and p53

Persuasive epidemiological and experimental evidence suggests that dietary flavonoids have anti-cancer activity. Since conventional therapeutic and surgical approaches have not been able to fully control the incidence and outcome of most cancer types, including colorectal neoplasia, there is an urgent need to develop alternative approaches for the management of cancer. We sought to develop the best flavonoids for the inhibition of cell growth, and apigenin (flavone) proved to be the most promising compound in colorectal cancer cell growth arrest. Subsequently, we found that pro-apoptotic proteins (NAG-1 and p53) and cell cycle inhibitor (p21) were induced in the presence of apigenin, and kinase pathways, including PKCδ and ataxia telangiectasia mutated (ATM), play an important role in activating these proteins. The data generated by in vitro experiments were confirmed in an animal study using APC^MIN+ mice. Apigenin is able to reduce polyp numbers, accompanied by increasing p53 activation through phosphorylation in animal models. Our data suggest apparent beneficial effects of apigenin on colon cancer.     

Involvement of p53 in oroxylin A‐induced apoptosis in cancer cells

Oroxylin A, a naturally occurring monoflavonoid extracted from Scutellariae radix, exhibits anticancer activity and induces apoptosis in human hepatocellular carcinoma HepG2 cells according to our previous data. In this study, we investigate whether p53 is involved in oroxylin A‐triggered viability inhibition and apoptosis induction in cancer cells. In a panel of different cancer cell lines, more potent inhibitory effects of oroxylin A were observed in wtp53 cells than those in mtp53 or p53‐null cells. Moreover, p53‐siRNA‐transfected HepG2 cells showed lower levels of apoptosis induced by oroxylin A than control‐siRNA‐transfected cells. Likewise, after oroxylin A treatment, p53‐null K‐562 cells displayed promoted apoptosis rate when transfected with wtp53 plasmid. Western blot and real‐time RT‐PCR assay revealed that oroxylin A markedly upregulated p53 protein expression in HepG2 and p53‐overexpressing K‐562 cells, but had no influence on p53 mRNA synthesis. Furthermore, after co‐treatment with cycloheximide, oroxylin A still exerted a little effect on p53 expression. The negative regulator of p53, MDM2 protein was detected, and downregulated expression was observed. In the presence of MG132, an inhibitor of proteasome‐mediated proteolysis, no change in p53 expression was obtained. Additionally, the antioxidant N‐acetyl‐L‐cysteine could obviously abrogate p53 stabilization triggered by oroxylin A. Therefore, it is summarized that oroxylin A stabilized p53 expression and induced apoptosis at the posttranslational level via downregulating MDM2 expression and interfering MDM2‐modulated proteasome‐related p53 degradation. This indicated that oroxylin A could be served as a potential, novel agent candidate for cancer therapy. © 2009 Wiley‐Liss, Inc.     

Evidence of an interaction between Mos and Hsp70: a role of the Mos residue serine 3 in mediating Hsp70 association.

c-Mos is a germ cell-specific MAP kinase kinase kinase (MAPKKK) that plays an essential role during meiotic divisions of oocytes. c-Mos is a key component of an activity, cytostatic factor, required for metaphase II arrest of unfertilized eggs in vertebrates. To understand the regulation of c-Mos, we are investigating c-Mos-interacting proteins. We provide evidence that mouse c-Mos binds to Hsp70, a molecular chaperone. Hsp70 was found to associate with Mos ectopically expressed in COS-1 cells. Mos-Hsp70 complexes could be immunoprecipitated with both Mos and Hsp70 antibodies. Despite a low-abundance of Mos, the Hsp70 antibody immunoprecipitated Mos as the major protein. Of importance, the Mos protein present in anti-Hsp70 immunoprecipitates functioned as an active MAPKKK indicating that it is not grossly misfolded. It is known that c-Mos protein kinase activity in cell extracts of transfected COS-1 or NIH3T3 cells is labile. We found that the inclusion of adenosine triphosphate (ATP) in cell extracts protected against the loss of Mos kinase activity. In the absence of ATP from cell extracts, protein kinase activity of Mos was lost within 6 h on ice even though the Mos protein was not degraded and remained bound to Hsp70. Based on our identification of c-Mos-Hsp70 interaction, one of the roles of ATP may be to assist the regulation of c-Mos via ATP involvement in the protein-folding function of Hsp70 and possibly other molecular chaperones. We also detected by coimmunoprecipitation a physical association between endogenous c-Mos and Hsp70 in Xenopus eggs. To provide further evidence for the functional significance of Hsp70 interaction to Mos function, we show that the residue serine 3 in Mos, which is important for the regulation of protein kinase activity of Mos is also important for Hsp70 association.     

Ginsenoside-Rs4, a new type of ginseng saponin concurrently induces apoptosis and selectively elevates protein levels of p53 and p21WAF1 in human hepatoma SK-HEP-1 cells

In this paper, we present evidence that ginsenoside-Rs₄ (G-Rs4; an acetylated analogue of ginsenoside-Rg₅), a new ginseng saponin isolated from Panax ginseng C. A. Meyer, elevates protein levels of p53 and p21^WAF1, which are associated with the induction of apoptosis in SK-HEP-1 cells. Flow cytometric analyses showed that G-Rs4 initially arrested the cell cycle at the G1/S boundary, but consequently induced apoptosis as evidenced by generating an apoptotic peak. The induction of apoptosis was confirmed by the results of DNA fragmentation assays and alterations in cell morphology after treatment of the cells with G-Rs4. Immunoblot assays showed that G-Rs4 significantly elevated protein levels of p53 and p21^WAF1, concurrently with the downregulation of both cyclins E- and A-dependent kinase activities and induction of apoptosis. We suggest that G-Rs4 induces apoptosis, the effect of which is closely related to the downregulation of both cyclins E- and A-dependent kinase activity as a consequence of selectively elevating protein levels of p53 and p21^WAF1 in SK-HEP-1 cells.