Downregulation Of P53 Expression Research Articles

Mentha piperita as a pivotal neuro-protective agent against gamma irradiation induced DNA fragmentation and apoptosis

Ionizing radiation is classified as a potent carcinogen, and its injury to living cells, in particular to DNA, is due to oxidative stress enhancing apoptotic cell death. Our present study aimed to characterize and semi-quantify the radiation-induced apoptosis in CNS and the activity of Mentha extracts as neuron-protective agent. Our results through flow cytometry exhibited the significant disturbance and arrest in cell cycle in % of M1: SubG1 phase, M2: G0/1 phase of diploid cycle, M3: S phase and M4: G2/M phase of cell cycle in brain tissue (p<0.05). Significant increase in % of apoptosis and P53 protein expression as apoptotic biomarkers were coincided with significant decrease in Bcl(2) as an anti-apoptotic marker. The biochemical analysis recorded a significant decrease in the levels of reduced glutathione, superoxide dismutase, deoxyribonucleic acid (DNA) and ribonucleic acid contents. Moreover, numerous histopathological alterations were detected in brain tissues of gamma irradiated mice such as signs of chromatolysis in pyramidal cells of cortex, nuclear vacuolation, numerous apoptotic cell, and neural degeneration. On the other hand, gamma irradiated mice pretreated with Mentha extract showed largely an improvement in all the above tested parameters through a homeostatic state for the content of brain apoptosis and stabilization of DNA cycle with a distinct improvement in cell cycle analysis and antioxidant defense system. Furthermore, the aforementioned effects of Mentha extracts through down-regulation of P53 expression and up-regulation of Bcl(2) domain protected brain structure from extensive damage. Therefore, Mentha extract seems to have a significant role to ameliorate the neuronal injury induced by gamma irradiation.

Endoplasmic reticulum stress stimulates p53 expression through NF-κB activation.

BackgroundInduction of apoptosis by endoplasmic reticulum (ER) stress is implicated as the major factor in the development of multiple diseases. ER stress also appears to be a potentially useful major response to many chemotherapeutic drugs and environmental chemical compounds. A previous study has indicated that one major apoptotic regulator, p53, is significantly increased in response to ER stress, and participates in ER stress-induced apoptosis. However, the regulators of p53 expression during ER stress are still not fully understood.Principal FindingsIn this report, we demonstrate that induction of p53 expression is mediated through NF-κB signaling pathways during ER stress in MCF-7 cells. Tunicamycin or brefeldin A, two ER stress inducers, increased p53 expression in MCF-7 and Hela cells. We found p53 nuclear localization, activity, and phosphorylation at serine 15 on p53 increased during ER stress. Nuclear translocation of NF-κB and activity of NF-κB were also observed during ER stress. ER stress-induced p53 expression was significantly inhibited by coincubation with the NF-κB inhibitor, Bay 11-7082 and downregulation of NF-κB p65 expression. The role of p53 in mediating Brefeldin A-induced apoptosis was also investigated. Induction of p53 expression by Brefeldin A was correlated to Brefeldin A-induced apoptosis. Furthermore, downregulation of p53 expression by p53 siRNA significantly reduced Brefeldin A-induced apoptosis in MCF-7 cells.SignificanceTaken together, NF-κB activation and induction of p53 expression is essential for ER stress-induced cell death which is important for therapeutic effects of clinical cancer drugs. Our results may provide insight into the mechanism of cancer chemotherapy efficacy that is associated with induction of ER stress.

In oral squamous cell carcinoma, high FAK expression is correlated with low P53 expression

Focal adhesion kinase (FAK) and p53 have been associated with metastatic activity and a poor prognosis in oral squamous cell carcinoma (OSCC). Recently, a feedback mechanism in which FAK regulates p53 has been proposed. The present study aims to determine the role of p53 in FAK regulation in these tumors. FAK and p53 expression was examined by immunohistochemistry in normal oral mucosa and in 67 oral squamous cell carcinomas. p16(INK4a) was also studied in view of its association with human papillomavirus infection. The association between FAK and p53 was subsequently analyzed. FAK expression in OSCCs was heterogeneous: 22 (33%) cases showed weak expression, 16 (24%) showed moderate expression, and 22 (33%) cases showed high expression. Regarding p53, 31 of 67 (46%) available tumor specimens showed negative staining, and 36 of 67 (54%) showed positive nuclear staining for p53. FAK expression was inversely correlated with p53 expression (Fisher's exact test, p = 0.005). There was no association between p16(INK4a) and p53 or FAK expression. In conclusion, our results support the hypothesis that FAK activity might be involved in the down-regulation of p53 expression in OSCC.

Zoledronic Acid Produces Antitumor Effects on Mesothelioma Through Apoptosis and S-Phase Arrest in p53-Independent and Ras prenylation-Independent Manners

We examined whether zoledronic acid (ZOL), the third generation of bisphosphonates, produced cytotoxic effects on human mesothelioma cells in vitro and in vivo, and investigated a possible involvement of p53, Ras, and extracellular signal-regulated kinase1/2 (ERK1/2) pathways. Cytotoxicity and cell cycles were assessed with a colorimetric assay and flow cytometry, respectively. Expression levels of apoptosis-linked proteins and prenylation of small guanine-nucleotide-binding regulatory proteins were tested with p53-small interfering RNA, an ERK kinase1/2-inhibitor, and prenyl alcohols. The antitumor activity was examined in an orthotopic animal model. ZOL treatments suppressed growth of mesothelioma cells bearing the wild-type p53 gene through apoptosis induction accompanied by activation of caspases, or S-phase arrest by up-regulated cyclin A and B1. ZOL induced p53 phosphorylation and subsequent activation of the downstream pathways. Down-regulated p53 expression with the small interfering RNA, however, showed that both apoptosis and S-phase arrest were irrelevant to the p53 activation. Geranylgeranyl but not farnesyl pyrophosphate inhibited ZOL-induced apoptosis and S-phase arrest, and the geranylgeraniol supplement decreased ZOL-mediated Rap1A but not Ras unprenylation. Inhibition of ERK1/2 pathways suppressed ZOL-induced apoptosis but not S-phase arrest. We further demonstrated that ZOL, administrated intrapleurally, inhibited the tumor growth in the pleural cavity. These data indicate that ZOL induces apoptosis or S-phase arrest, both of which are independent of p53 activation and Ras unprenylation, and suggest that ZOL is a possible therapeutic agent to mesothelioma partly through non-Ras- and ERK1/2-mediated pathways.

WIP1 Enhances Tumor Formation in a Sonic Hedgehog–Dependent Model of Medulloblastoma

A significant number of medulloblastomas (MBs) originate from abnormal activation of the sonic hedgehog/patched (SHH/PTC) signaling pathway. Although p53 deficiency enhances tumor formation in mice, inactivation of the p53 gene is seen in a minority of MBs. Wild-type p53-induced phosphatase 1 (WIP1) downregulates p53 expression and has been shown to be overexpressed in MBs. We tested the hypothesis that overexpression of WIP1 enhances tumor formation in an SHH-dependent model of MB. We used the RCAS/Ntv-a system to study the effect of WIP1 in vitro and in vivo. We transfected A375-TVA cells with RCAS-WIP1 and then exposed these cells to cisplatin to determine the effect on p53 expression. We modeled ectopic WIP1 expression independently and in combination with SHH in the cerebella of newborn mice to assess the effect on tumor formation. Mice were observed for 12 weeks or until neurological symptoms developed. The brains were examined for tumor formation. A375-TVA cells infected with RCAS-WIP1 demonstrated reduced p53 expression after exposure to cisplatin compared with controls. We detected tumors in 12 of 35 mice (34%) injected with RCAS-WIP1 and RCAS-SHH. Tumors were detected in 3 of 40 mice (8%) injected with RCAS-SHH alone. The difference in tumor formation rates was significant (χ(2) test, P = < .01). Tumors did not form in mice injected with RCAS-WIP1 alone. We show that ectopic expression of WIP1 cooperates with SHH to enhance formation of MB, although it is insufficient to induce tumors independently. Our results verify the role of WIP1 in MB formation and provide a crucial link to the inactivation of p53 in MBs.

Identification of an extracelular o-n-acetyl-glycosylase responsible for an increased level of O-linked n-acetylglucosamine modified proteins in the cartilage of patients with osteoarthritis

Identification of an extracelular o-n-acetyl-glycosylase responsible for an increased level of O-linked n-acetylglucosamine modified proteins in the cartilage of patients with osteoarthritis

2-Hydroxyestradiol slows progression of experimental polycystic kidney disease

Male gender is a risk factor for progression of polycystic kidney disease (PKD). 17β-Estradiol (E2) protects experimentally, but clinical use is limited by adverse effects. Novel E2 metabolites provide many benefits of E2 without stimulating the estrogen receptor, and thus may be safer. We hypothesized that E2 metabolites are protective in a model of PKD. Studies were performed in male control Han:SPRD rats, and in cystic males treated with orchiectomy, 2-methoxyestradiol, 2-hydroxyestradiol (2-OHE), or vehicle, from age 3 to 12 wk. Cystic rats exhibited renal functional impairment (∼50% decrease in glomerular filtration and renal plasma flow rates, P < 0.05) and substantial cyst development (20.5 ± 2.0% of cortex area). 2-OHE was the most effective in limiting cysts (6.0 ± 0.7% of cortex area, P < 0.05 vs. vehicle-treated cystic rats) and preserving function, in association with suppression of proliferation, apoptosis, and angiogenesis markers. Downregulation of p21 expression and increased expression of Akt, the mammalian target of rapamycin (mTOR), and some of its downstream effectors were significantly reversed by 2-OHE. Thus, 2-OHE limits disease progression in a cystic rodent model. Mechanisms include reduced renal cell proliferation, apoptosis, and angiogenesis. These effects may be mediated, at least in part, by preservation of p21 and suppression of Akt and mTOR. Estradiol metabolites may represent a novel, safe intervention to slow progression of PKD.

CYR61 controls p53 and NF-κB expression through PI3K/Akt/mTOR pathways in carboplatin-induced ovarian cancer cells

CYR61 over-expression promotes cell proliferation by inhibiting carboplatin-induced apoptosis, decreasing Bax expression, and increasing Bcl-xL, Mcl-1, and Bcl-2. At the same time, down-regulating p53 expression, while up-regulated NF-κB expression. Additionally, p21 and p53 promoter activities were reduced, while NF-κB and Bcl-2 activities increased. In parallel, CYR61-expressing cells, during carboplatin-induced apoptosis, resulted in an increase of Akt phosphorylation, while rapamycin-treated cells were not affected. Carboplatin effectively inhibited the activation of mTOR signaling cascade, which includes mTOR, 4E-BP1, p70S6K, HIF-1α, and VEGF. These results provide evidence that CYR61 promotes cell proliferation and inhibits apoptosis.

Cardiomyocyte Specific Ablation of p53 Is Not Sufficient to Block Doxorubicin Induced Cardiac Fibrosis and Associated Cytoskeletal Changes

Doxorubicin (Dox) is an anthracycline used to effectively treat several forms of cancer. Unfortunately, the use of Dox is limited due to its association with cardiovascular complications which are manifested as acute and chronic cardiotoxicity. The pathophysiological mechanism of Dox induced cardiotoxicity appears to involve increased expression of the tumor suppressor protein p53 in cardiomyocytes, followed by cellular apoptosis. It is not known whether downregulation of p53 expression in cardiomyocytes would result in decreased rates of myocardial fibrosis which occurs in response to cardiomyocyte loss. Further, it is not known whether Dox can induce perivascular necrosis and associated fibrosis in the heart. In this study we measured the effects of acute Dox treatment on myocardial and perivascular apoptosis and fibrosis in a conditional knockout (CKO) mouse model system which harbours inactive p53 alleles specifically in cardiomyocytes. CKO mice treated with a single dose of Dox (20 mg/kg), did not display lower levels of myocardial apoptosis or reactive oxygen and nitrogen species (ROS/RNS) compared to control mice with intact p53 alleles. Interestingly, CKO mice also displayed higher levels of interstitial and perivascular fibrosis compared to controls 3 or 7 days after Dox treatment. Additionally, the decrease in levels of the microtubule protein α-tubulin, which occurs in response to Dox treatment, was not prevented in CKO mice. Overall, these results indicate that selective loss of p53 in cardiomyocytes is not sufficient to prevent Dox induced myocardial ROS/RNS generation, apoptosis, interstitial fibrosis and perivascular fibrosis. Further, these results support a role for p53 independent apoptotic pathways leading to Dox induced myocardial damage and highlight the importance of vascular lesions in Dox induced cardiotoxicity.

Experimental study on the suppression of sodium nitroprussiate-induced chondrocyte apoptosis by Tougu Xiaotong Capsule (透骨消痛胶囊)-containing serum

To study the mechanism of action of Tougu Xiaotong Capsule (透骨消痛胶囊, TGXTC) ex vivo in suppressing chondrocyte (CD) apoptosis induced by sodium nitroprussiate (SNP). Thirty New Zealand rabbits, 2 months old, were randomized by lottery into five groups, six in each: the blank group treated with saline, the positive control group treated with Zhuanggu Guanjie Pill (壮骨关节丸, 70 mg/kg), and the three experimental groups, EGA, EGB, and EGC, treated with low dose (35 mg/kg), moderate dose (70 mg/kg), and high dose (140 mg/kg) of TGXTC, respectively. All treatments were administered via gastrogavage twice a day for 3 days. Arterial blood was collected from the abdominal aorta and drug or drug metabolites-containing serum was prepared. CDs obtained from knee joints of 16 four-week-old New Zealand rabbits were cultured to the third passage and confirmed by toluidine blue staining. SNP of various final concentrations (0, 0.5, 1.0, and 2.0 mmol/L) was used to induce CD apoptosis, and the dosage-effect relationship of SNP in inducing CD apoptosis was determined. Serum samples from the blank, control, and three dosages of TGXTC-treated rabbits were tested in the CD culture in the presence of SNP. Cell apoptosis was determined by Hoechst 33342 staining, viability of CDs was quantified by MTT, CD apoptosis rate was determined by annexin V-FITC/PI staining, levels of p53 and Bcl-2 mRNA expression in CDs were determined with RT-PCR, and contents of caspase-3 and caspase-9 proteins were determined by colorimetry. CD apoptosis was induced by SNP at all concentrations tested and in a dose-dependent manner. The SNP concentration of 1 mmol/L and treatment duration of 24 h appeared to be optimal and were selected for the study. Serum samples from the positive control rabbits and from the two higher doses of TGXTC-treated rabbits showed reduction of SNP-induced CD apoptosis, decrease in p53 mRNA expression, inhibition of catalytic activities of caspase-3 and caspase-9, and increase in Bcl-2 mRNA expression when compared with the serum from the blank group (P<0.05). TGXTC-containing sera antagonized SNP-induced CD apoptosis and the molecular basis for the action was associated with up-regulation of Bcl-2, down-regulation of p53 expression, and inhibition of caspase-3 and caspase-9 catalytic activities.

Abstract 2638: Association between Adriamycin-induced autophagy and senescence in breast cancer cells

Abstract Previous studies from this laboratory demonstrated that the topoisomerase II poison, Doxorubicin, at a clinically relevant concentration of 1µM, induced a pronounced senescence response in breast cancer cells. Doxorubicin was also observed to generate a time-dependent increase of autophagy within 72 hrs by multiple approaches, including acridine orange staining (AOS), downregulation of p62 (indicative of autophagic flux), LC3 puncta formation and electron microscopy. The current studies were designed to investigate the potential relationship between senescence and autophagy in response to Doxorubicin in breast cancer cells. Silencing of ATM by the pharmacological inhibitors, caffeine or KU55933, or by small interfering RNA abrogated doxorubicin-induced senescence, as demonstrated by decreased β -galactosidase staining as well as down-regulation of p21 expression. At the same time, silencing/attenuation of ATM suppressed autophagy, based on decreased conversion of LC3-I to LC3-II by Western blotting, and reduced acridine orange staining. Free radicals are well-established to induce senescence, and Doxorubicin-induced senescence has been shown to be associated with free radical generation. We demonstrate that the antioxidants, glutathione (GSH) or N-acetyl cysteine (NAC), were able to collaterally suppress Doxorubicin-induced senescence and autophagy, based on reduced β-Gal staining, LC3-I to II conversion and activation of ATG5, respectively. Similarly, both senescence and autophagy induced by the topoisomerase I inhibitor, camptothecin, were suppressed by GSH or NAC. Silencing of p53 or p21 expression inhibited both Doxorubicin-induced senescence and autophagy in both breast and colon cancer cells. In contrast, a blockade of autophagy either by the pharmacological inhibitors, 3-methyl adenine or chloroquine, or genetic silencing of ATG5 or ATG7 expression, only served to delay the onset of Doxorubicin-induced senescence. Similarly, a delayed senescence response was observed when autophagy induced by camptothecin or H2O2 was blocked by chloroquine or bafilomycin in MCF-7 cells. Our studies suggest that: 1. Free radicals, ATM, p53 and p21 are involved in the signaling pathways that promote the autophagic and senescence responses to chemotherapeutic agents; 2. autophagy acts to accelerate senescence; and 3) while chemotherapy induced autophagy and senescence are closely associated, senescence can likely occur in the absence of autophagy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2638. doi:10.1158/1538-7445.AM2011-2638

Zeranol Down-Regulates p53 Expression in Primary Cultured Human Breast Cancer Epithelial Cells through Epigenetic Modification

Epidemiological studies have suggested that there are many risk factors associated with breast cancer. Silencing tumor suppressor genes through epigenetic alterations play critical roles in breast cancer initiation, promotion and progression. As a growth promoter, Zeranol (Z) has been approved by the FDA and is widely used to enhance the growth of beef cattle in the United States. However, the safety of Z use as a growth promoter is still under debate. In order to provide more evidence to clarify this critical health issue, the current study investigated the effect of Z on the proliferation of primary cultured human normal and cancerous breast epithelial cells (PCHNBECs and PCHBCECs, respectively) isolated from the same patient using MTS assay, RT-PCR and Western blot analysis. We also conducted an investigation regarding the mechanisms that might be involved. Our results show that Z is more potent to stimulate PCHBCEC growth than PCHNBEC growth. The stimulatory effects of Z on PCHBCECs and PCHBCECs may be mediated by its down-regulating expression of the tumor suppressor gene p53 at the mRNA and protein levels. Further investigation showed that the expression of DNA methylatransferase 1 mRNA and protein levels is up-regulated by treatment with Z in PCHBCECs as compared to PCHNBECs, which suggests a role of Z in epigenetic modification involved in the regulation of p53 gene expression in PCHBCECs. Our experimental results imply the potentially adverse health effect of Z in breast cancer development. Further study is continuing in our laboratory.

MEF/ELF4 transactivation by E2F1 is inhibited by p53

Myeloid elf-1-like factor (MEF) or Elf4 is an E-twenty-six (ETS)-related transcription factor with strong transcriptional activity that influences cellular senescence by affecting tumor suppressor p53. MEF downregulates p53 expression and inhibits p53-mediated cellular senescence by transcriptionally activating MDM2. However, whether p53 reciprocally opposes MEF remains unex-plored. Here, we show that MEF is modulated by p53 in human cells and mice tissues. MEF expression and promoter activity were suppressed by p53. While we found that MEF promoter does not contain p53 response elements, intriguingly, it contains E2F consensus sites. Subsequently, we determined that E2F1 specifically binds to MEF promoter and transactivates MEF. Nevertheless, E2F1 DNA binding and transactivation of MEF promoter was inhibited by p53 through the association between p53 and E2F1. Furthermore, we showed that activation of p53 in doxorubicin-induced senescent cells increased E2F1 and p53 interaction, diminished E2F1 recruitment to MEF promoter and reduced MEF expression. These observations suggest that p53 downregulates MEF by associating with and inhibiting the binding activity of E2F1, a novel transcriptional activator of MEF. Together with previous findings, our present results indicate that a negative regulatory mechanism exists between p53 and MEF.

Abstract 5364: Zeranol Down-regulates p53 Expression in Primary Cultured Human Breast Cancer Epithelial Cells through Epigenetic Modification

Abstract Despite great improvements in diagnosis, treatment advances, and healthier life-styles, breast cancer remains the most common cancer among women in the United States. It is estimated that 192,370 new cases arise and 40,170 women will die from breast cancer in 2009. Epidemiological studies have suggested that there are many risk factors associated with breast cancer such as dietary fat and environmental estrogenic endocrine disruptors. Zeranol (Z), as one of the six growth promoters approved by FDA, has been widely used to enhance the growth of beef cattle. Researchers are still in disagreement whether the growth promoters pose any adverse health risk from consuming beef products produced from Z-implanted beef cattle. Our previous results showed that pre-adipocytes isolated from the cattle 60 days post Z-implantation (72 mg/pellet/animal) grow about 12 fold faster than the pre-adipocytes isolated from the control cattle. Treatment with 0.2, 1 and 5% sera (Z-Sera) harvested from the cattle 30 days post implantation in culture medium significantly stimulated MCF-10A, a human normal breast epithelial cells, and MCF-7 cell growth. Proliferation might be caused by Z-Sera down-regulating the expression of a tumor suppressor gene, protein tyrosine phosphatase γ (PTP γ). The current study investigated the effects of Z on primary cultured human normal and cancerous breast epithelial cell growth (PCHNBECs, PCHBCECs), and the regulation of p53 expression, as well as the potential mechanism. Using non-radioactive cell proliferation assay, real time PCR and western blot analysis, we demonstrated that PCHBCECs are more sensitive in response to treatment with Z than PCHNBECs. Treatment with 7.5, 15 and 30 nM Z increased the growth of PCHNBECs at 27, 45, 50%, respectively, as compared to the control group. The same concentrations enhanced the proliferation of PCHBCECs at 25, 84 and 91%. The expression of p53 protein in PCHNBECs did not decrease by the treatment of Z. However, in PCHBCECs, treatment with 7.5, 15 and 30 nM Z significantly down-regulated the expression of p53 protein by 10, 20 and 40%, respectively. Further investigation suggests that the effect of Z on decreasing the expression of p53 in PCHBCECs might be mediated through its epigenetic modification. Our results showed that Z treatment up-regulates the expression of DNMT1 mRNA by 36, 58 and 160%, and protein by 14, 25 and 42% in PCHBCECs after 24 hour exposure. Our data suggest that the stimulatory effect of Z on PCHBCECs growth might be via down-regulation of p53 expression as a result of epigenetic modification mediated through Z enhancing the expression of DNMT1 mRNA and protein levels. These results imply the potential adverse health risks of the consumption of beef products with Z or its bio-active metabolites. Further investigation on this critical issue is in-progress in our laboratory (Supported by NIH grant R01 ES015212-01). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5364.

Downregulation of p53 by phosphatase of regenerating liver 3 is mediated by MDM2 and PIRH2

Aims The phosphatase of regenerating liver (PRL) family is related to tumorigenesis and metastasis in various cancer types. Its overexpression increases cell motility and proliferation via the downregulation of p21 expression. In a previous study, we reported that PRL-1 downregulates p53 and is a target gene of p53. In this study, we investigated whether a member of the PRL family, PRL-3, could regulate p53 like PRL-1 in cancer cells. Main methods To elucidate the role of PRL-3 in regulating p53 in cancer cells, we used a cell culture system to measure protein level, transcriptional level, apoptosis or localization. Key findings We determined that PRL-3 overexpression reduced the activity of the p21 and p53 reporters. Additionally, the levels of endogenous and exogenous p53 protein were reduced in cells transiently expressing PRL-3, whereas the ablation of PRL-3 by siRNA increased levels of the p53 protein. The downregulation of p53 by PRL-3 inhibited p53-mediated apoptosis. However, the phosphatase-dead mutant C104S, prenylated-site mutant C170S, and C104S/C170S PRL-3 evidenced minimal effects on the downregulation of p53 protein as compared with wild-type PRL-3. Further examinations revealed that PRL-3 expression reduced the stability of p53 by inducing the transcription of p53 induced protein with a RING-H2 domain (PIRH2) through early growth response (EGR) and by increasing the phosphorylation of mouse double minute 2 (MDM2), and then both negatively regulated p53. Significance These findings demonstrated that PRL-3, like PRL-1, can negatively regulate p53 via the activation of PIRH2 and MDM2 in cancer cells.

Functional characterization of human oncoprotein gankyrin in Zebrafish

Gankyrin is an oncoprotein containing seven ankyrin repeats that is overexpressed in hepatocellular carcinoma (HCC). Gankyrin binds to Mdm2, which results in accelerated ubiquitylation via degradation of p53, and it also plays an important role in cell proliferation. However, little is known about the relationships between p53 levels, cell proliferation, and gankyrin over-expression. In order to investigate the influence of gankyrin protein on p53 and Mdm2 in a zebrafish model, we injected human gankyrin (hgankyrin) containing expression vectors (pCS2-hgankyrin, pCS2- hgankyrin-EGFP) into zebrafish embryos. To measure p53 and Mdm2 expression in hgankyrin-injected embryos, RT-PCR, Northern blot and in-situ hybridization and BrdU immunostaining were used. In addition, to know the effect of hgankyrin on cell proliferation in vitro, cell viability assays such as MTT, trypan blue staining and RT-PCR following transfection of hgankyrin-containing vector into HEK 293 cell line were performed. In vivo results indicated that p53 mRNA levels decreased but those of Mdm2 were not decreased in the presence of hgankyrin. These results suggest that gankyrin downregulates p53 expression and not Mdm2 expression. In the study of cell proliferation, BrdU-positive cells were predominantly increased in the head and tail regions in hgankyrin-injected zebrafish. Additional in vitro studies using trypan blue staining and MTT assay showed that gankyrin-expressing HEK 293 cells proliferated at a faster rate, indicating that gankyrin promotes cell proliferation. Our results demonstrate that hgankyrin overexpression downregulates p53 expression and promotes cell proliferation in zebrafish. Gankyrin may play an important role in tumorigenesis via its effects on p53 and cell proliferation.

Nuclear expression of E2F4 induces cell death via multiple pathways in normal human intestinal epithelial crypt cells but not in colon cancer cells

E2F transcription factors control cell cycle progression. The localization of E2F4 in intestinal epithelial cells is cell cycle dependent, being cytoplasmic in quiescent differentiated cells but nuclear in proliferative cells. However, whether nuclear translocation of E2F4 alone is sufficient to trigger intestinal epithelial cell proliferation remains to be established. Adenoviruses expressing fusion proteins between green fluorescent protein (GFP) and wild-type (wt)E2F4 or GFP and nuclear localization signal (NLS)-tagged E2F4 were used to infect normal human intestinal epithelial crypt cells (HIEC). In contrast to expression of wtE2F4, persistent expression of E2F4 into the nucleus of HIEC triggered phosphatidylserine exposure, cytoplasmic shrinkage, zeiosis, formation of apoptotic bodies, and activation of caspase 9 and caspase 3. Inhibition of caspase activities by zVAD-fmk partially inhibited cell death induced by E2F4-NLS. An induction of p53, phosphorylated Ser15-p53, PUMA, FAS, BAX, RIP, and phosphorylated JNK1 was also observed in HIEC expressing E2F4-NLS compared with wtE2F4-expressing cells. E2F1 and p14ARF expression remained unaltered. Downregulation of p53 expression by RNA interference attenuated cell death induced by E2F4-NLS. By contrast, the level of cell death was negligible in colon cancer cells despite the strong expression of E2F4 into the nucleus. In conclusion, deregulated nuclear E2F4 expression induces apoptosis via multiple pathways in normal intestinal epithelial cells but not in colon cancer cells. Hence, mutations that deregulate E2F4 localization may provide an initial proliferative advantage but at the same time accelerate cell death. However, intestinal cells acquiring mutations (e.g., p53, Bax loci, etc.) may escape apoptosis, thereby revealing the full mitogenic potential of the E2F4 transcription factor.

The over-expression of p53 H179Y residue mutation causes the increase of cyclin A1 and Cdk4 expression in HELF cells

Down-regulation of p53 expression has been found in a broad range of human cancers and cell proliferation disorders, indicating that p53 plays a key role in cell cycle regulation and tumor suppression. In our current study, we transfected human embryonic lung fibroblast (HELF) cells with pcDNA3-wild-type p53 (pcDNA3-wtp53) plasmid, or pcDNA3-H179Y-mutated p53 (pcDNA3-mtp53) plasmid that mimics the mutation found in some human lung tumors, and further studied the role of p53 in the regulation of cell proliferation. Over expression of wild-type p53 caused cell cycle arrest at G1 phase with reduced cell size, decreased expression of cyclin D3, cyclin E, Cdk2 and Cdk4, and increased expression of p21. In contrast, over expression of H179Y-mutant p53 promoted G1 to S phase transition with enlarged cell size and increased cyclin A1 and Cdk4 expression in HELF cells. These results indicate that mutation at the p53 H179Y residue up-regulates cyclin A1 and Cdk4 expression, and promotes HELF cell proliferation.

Growth-inhibitory effects of curcumin on Raji cells and its mechanisms

Objective: To study the growth-inhibitory effects of curcumin on B-NHL cell line Raji cells in vitro and its molecular mechanisms. Methods: The growth inhibition rates of Raji cells, after being treated with 6.25 µmol/L ∼ 50 µmol/L curcumin for 12 h ∼ 48 h, were examined by MTT assay. The apoptosis rate was detected by flow cytometry (FCM), the protein expression levels of bcl-2 and p53 in Raji cells were examined by SP immunohistochemistry. The expression of p53 in Raji cell were checked by RT-PCR. Results: After being treated by various concentrations of curcumin, the growth of Raji cells was inhibited significantly. The rates of apoptosis were 11.8%∼79.7% (P<0.01), the down regulation of p53 expression was observed within 24 h after the treatment of curcumin by RT-PCR. The expression of bcl-2 and p53 was decreased, which depended on the action time. Conclusion: Curcumin could significantly inhibit the growth of Raji cells. The induction of apoptosis by down-regulating the expression of bcl-2 and p53 was probably one of its molecular mechanisms.

Conditional transformation of mouse pancreatic epithelial cells: an in vitro model for analysis of genetic events in pancreatocarcinogenesis

Pancreatic ductal adenocarcinomas arise through the accumulation of certain genetic alterations including ras, p16, p53, and DPC4. We found that activation of ras and inactivation of p53 could cooperatively induce in vitro tumorigenicity in conditionally immortalized pancreatic epithelial (IMPE) cells. IMPE cells were established from transgenic mice bearing a temperature-sensitive mutant SV40 Large T (LT) antigen. IMPE cells grew continuously under permissive conditions (33°C with interferon-γ), but rapidly suffered growth arrest under non-permissive conditions (39°C without interferon-γ). The cells showed strong expression of E-cadherin and β-catenin as epithelial markers, and cytokeratin 19, a specific ductal cell marker. Cell proliferation under permissive conditions was associated with down-regulation of p21 expression through inactivation of p53 after overexpression of LT antigen. Intriguingly, the shift from the permissive to non-permissive culture conditions caused G2/M arrest of IMPE cells. Although the cells did not form colonies when cultured in soft agar without activation of ras, cells with ras activation via an adenovirus vector formed colonies under permissive conditions. These findings suggest that activation of ras and inactivation of p53 can cooperatively induce anchorage-independent growth of IMPE cells. This cell line might be useful for studying the processes involved in pancreatocarcinogenesis.