The expression of the ΔNp73β isoform of p73 leads to tetraploidy

The p73 locus gene has a complex structure encoding a plethora of isoforms. The different ΔN truncated isoforms of p73 may exert different activities depending on the cellular context. The β isoform of ΔNp73 seems to have a particular pattern of action even if its role in cell cycle and mitosis is still under investigation. To gain further knowledge of ΔNp73β’s function, we investigated the effects of its over-expression in tumour cellular models, using the tetracycline-inducible expression system. In the human lung carcinoma cell line H1299, ΔNp73β over-expression resulted in suppression of cell growth and in cell death. Surprisingly stable over-expression of ΔNp73β impaired the genomic stability of tumour cells, leading to the formation of tetraploid cells. The cells become enlarged and multinucleate, with incorrect mitotic figures, and died by apoptotic-independent pathways. Our data suggest that ΔNp73β-induced aberrant mitosis evades the control of the mitotic spindle assay checkpoint, leading to tetraploidy and cell death through mitotic catastrophe rather than apoptosis. The various C-terminal regions of ΔNp73 may influence the final cellular phenotype and we assume that the β one in particular could be important in both cell growth control and regulation of mitosis.     

ΔNp63 transcriptionally regulates ATM to control p53 Serine-15 phosphorylation

Background  

ΔNp63α is an epithelial progenitor cell marker that maintains epidermal stem cell self-renewal capacity. Previous studies revealed that UV-damage induced p53 phosphorylation is confined to ΔNp63α-positive cells in the basal layer of human epithelium.     

Hypoxia-induced β-catenin downregulation involves p53-dependent activation of Siah-1

Solid tumors contain extensive hypoxic areas and it is of considerable importance to decipher the potential role of hypoxia in signaling pathway regulation. In the present study, we examined the impact of hypoxia on β-catenin status and the mechanisms involved. Hypoxia significantly decreased β-catenin protein, but had no effect on glycogen synthase kinase (GSK)-3β or adenomatous polyposis coli (APC) levels. However, hypoxia-induced β-catenin downregulation seemed to require APC but not GSK-3β. Further investigation revealed that hypoxia significantly upregulated Siah-1, the human homolog of Drosophila seven in absentia. In addition, hypoxia augmented the interaction between β-catenin and SIP and Skp1. Silencing of Siah-1, as well as the use of a dominant negative Siah-1 mutant, attenuated these responses to hypoxia and rescued β-catenin transactivation. The Siah-1-mediated degradation of β-catenin during hypoxia may involve p53, but not hypoxia-inducible factor-1, activation. Together, the results suggest that hypoxia downregulates β-catenin by increasing Siah-1 expression in a p53-dependent manner.     

Are interactions with p63 and p73 involved in mutant p53 gain of oncogenic function?

Although still controversial, the presence of mutant p53 in cancer cells may result in more aggressive tumors and correspondingly worse outcomes. The means by which mutant p53 exerts such pro-oncogenic activity are currently under extensive investigation and different models have been proposed. We focus here on a proposed mechanism by which a subset of tumor-derived p53 mutants physically interact with p53 family members, p63 and p73, and negatively regulate their proapoptotic function. Both cell-based assays and knock-in mice expressing mutant forms of p53 support this model. As more than half of human tumors harbor mutant forms of p53 protein, approaches aimed at disrupting the pathological interactions among p53 family members might be of clinical value.     

Silibinin prevents ultraviolet B radiation-induced epidermal damages in JB6 cells and mouse skin in a p53-GADD45α-dependent manner

Better preventive strategies are required to reduce ultraviolet (UV)-caused photodamage, the primary etiological factor for non-melanoma skin cancer (NMSC). Accordingly, here we examined the preventive efficacy of silibinin against UVB-induced photodamage using mouse epidermal JB6 cells and SKH1 hairless mouse epidermis. In JB6 cells, silibinin pretreatment protected against apoptosis and accelerated the repair of cyclobutane pyrimidine dimers (CPD) induced by moderate dose of UVB (50 mJ/cm(2)), which we are at risk of daily exposure. Silibinin also reversed UVB-induced S phase arrest, reducing both active DNA synthesizing and inactive S phase populations. In mechanistic studies, UVB-irradiated cells showed a transient upregulation of both phosphorylated (Ser-15 and Ser-392) and total p53, whereas silibinin pretreatment led to a more sustained upregulation and stronger nuclear localization of p53. Silibinin also caused a marked upregulation of GADD45α, a downstream target of p53, implicated in DNA repair and cell cycle regulation. Importantly, under p53 and GADD45α knockdown conditions, cells were more susceptible to UVB-induced apoptosis without any significant S phase arrest, and protective effects of silibinin were compromised. Similar to the in vitro results, topical application of silibinin prior to or immediately after UVB irradiation resulted in sustained increase in p53 and GADD45α levels and accelerated CPD removal in the epidermis of SKH1 hairless mice. Together, our results show for the first time that p53-mediated GADD45α upregulation is the key mechanism by which silibinin protects against UVB-induced photodamage and provides a strong rationale to investigate silibinin in reducing the risk and/or preventing early onset of NMSC.     

HIPK2 phosphorylates ΔNp63α and promotes its degradation in response to DNA damage

Homeodomain-interacting protein kinase 2 (HIPK2) is an emerging player in cell response to genotoxic agents that senses damage intensity and contributes to the cell's choice between cell cycle arrest and apoptosis. Phosphorylation of p53 at S46, an apoptosis-specific p53 posttranslational modification, is the most characterized HIPK2 function in response to lethal doses of ultraviolet (UV), ionizing radiation or different anticancer drugs, such as cisplatin, roscovitine and doxorubicin (DOX). Indeed, like p53, HIPK2 has been shown to contribute to the effectiveness of these treatments. Interestingly, p53-independent mechanisms of HIPK2-induced apoptosis were described for UV and tumor growth factor-β treatments; however, it is unknown whether these mechanisms are relevant for the responses to anticancer drugs. Because of the importance of the so-called 'p53-independent apoptosis and drug response' in human cancer chemotherapy, we asked whether p53-independent factor(s) might be involved in HIPK2-mediated chemosensitivity. Here, we show that HIPK2 depletion by RNA interference induces resistance to different anticancer drugs even in p53-null cells, suggesting the involvement of HIPK2 targets other than p53 in response to chemotherapy. In particular, we found that HIPK2 phosphorylates and promotes proteasomal degradation of ΔNp63α, a prosurvival ΔN isoform of the p53 family member, p63. Indeed, effective cell response to different genotoxic agents was shown to require phosphorylation-induced proteasomal degradation of ΔNp63α. In DOX-treated cells, we show that HIPK2 depletion interferes with ΔNp63α degradation, and expression of a HIPK2-resistant ΔNp63α-Δ390 mutant induces chemoresistance. We identify T397 as the ΔNp63α residue phosphorylated by HIPK2, and show that the non-phosphorylatable ΔNp63α-T397A mutant is not degraded in the face of either HIPK2 overexpression or DOX treatment. These results indicate ΔNp63α as a novel target of HIPK2 in response to genotoxic drugs.     

Increased ΔNp63 expression is predictive of malignant transformation in oral epithelial dysplasia and poor prognosis in oral squamous cell carcinoma

This study examined immunohistochemical expression of ΔNp63, a keratinocyte stem cell marker, in oral leukoplakia (OL) and oral squamous cell carcinoma (OSCC) and then to elucidate usefulness of ΔNp63 as a marker for diagnosis and prognosis. One-hundred and twelve cases of OL and 81?cases of OSCC were analyzed by immunohistochemical staining for ΔNp63, Ki-67, and cytokeratin 14. These labeling indices (LIs) were calculated, and the association of these LIs with clinicopathologic characteristics in the OL and OSCC was evaluated. In the OL, these LIs increased significantly according to the severity of epithelial dysplasia (p<0.0001). ΔNp63-LI in the OL with malignant transformation was significantly higher than that in the OL without (49.3 vs. 34.2%; p<0.01). In the OSCC, the LIs increased significantly in association with the histologic grade (p<0.0001). A significant difference between the high and low ΔNp63-LI groups was found in the incidence of cervical lymph node and distant metastasis (p<0.05). The prognosis of the high ΔNp63-LI (mean value >73.8%) group is poorer than that of the low ΔNp63-LI (mean value ≤73.8%) group (p<0.05). These results suggested that increased ΔNp63 expression is involved in malignant transformation in epithelial dysplasia and poor prognosis in OSCC.     

A combination of IFN-β and temozolomide in human glioma xenograft models: implication of p53-mediated MGMT downregulation

Purpose Methylation of the O⁶-methyguanine-DNA methyltransferase (MGMT) gene promoter in gliomas has been reported to be a useful predictor of the responsiveness to temozolomide (TMZ). In our previous experiments, we observed that IFN-β sensitized TMZ-resistant glioma cells with the unmethylated MGMT promoter and that the mechanism of action was possibly due to attenuation of MGMT expression via induction of TP53. In this study, (1) we explored the synergistic effect of IFN-β and TMZ in the animal model, and (2) clarified the role of IFN-β induced TP53 in the human MGMT promoter. Methods (1) Nude mice with either subcutaneous T98 (TMZ-resistant) or U251SP (TMZ-sensitive) tumor were treated with IFN-β/TMZ for 5 consecutive days. (2) The MGMT promoter activity was assayed by a luciferase reporter system in Saos2 (p53-null) cells transduced with a p53-adenoviral vector, and T98 glioma cells treated with IFN-β. Results (1) A combination of IFN-β/TMZ had significant synergistic antitumor activity on the growth of both T98 and U251SP tumors. (2) MGMT promoter activity was suppressed by either adenovirally transduced p53 or IFN-β. Conclusions It would be appealing to consider a prospective clinical trial in which genetic markers are used for personalized drug selection, eliciting other forms of treatment or inhibition of MGMT for those with MGMT expression. In this context, IFN-β inactivates MGMT via p53 gene induction and enhances the therapeutic efficacy to TMZ.     

The interaction between C35 and ΔNp73 promotes chemo-resistance in ovarian cancer cells

Background:

The purpose of this study was to characterise the oncogenic roles of C35, a novel protein binding partner of ΔNp73, in ovarian cancer and to investigate the functional significance of C35–ΔNp73 interaction in the regulation of chemo-resistance.

Methods:

C35 expression was evaluated by quantitative real-time PCR in human ovarian cancer tissues and cell lines. The aggressiveness of ovarian cancer cells overexpressing C35 was examined by cell proliferation, migration, soft agar and nude mouse xenograft. The significance of C35–ΔNp73 interaction in chemo-resistance was evaluated by apoptosis assays and cell viability after cisplatin treatment.

Results:

The expression of C35 was significantly enhanced in human ovarian cancer tissues. Overexpression of C35 augmented proliferation, migration and tumourigenicity in ovarian cancer cell lines. C35 knockdown inhibited cell motility and cell growth. The co-expression of C35 and ΔNp73 by transient or stable transfection in ovarian cancer cells induced greater resistance to cisplatin treatment than did transfection with C35 or ΔNp73 alone. The cisplatin resistance was demonstrated to be caused by increased AKT and NFκB activity induced by C35–ΔNp73.

Conclusion:

Our results suggest that ΔNp73 might cooperate with C35 to promote tumour progression and contribute to cisplatin resistance in ovarian cancer cells. Future studies of the functional roles of ΔNp73 and C35 will provide insight that will aid in the establishment of new strategies and more effective therapies.     

Wogonin induced apoptosis in human nasopharyngeal carcinoma cells by targeting GSK-3β and ΔNp63

Purpose  

Wogonin, a plant flavonoid, has antitumor activity in various cancers. Dysregulation of GSK-3β has been implicated in tumorigenesis and cancer progression. In this study, we investigated the antitumor activity and the mechanistic action of wogonin in human nasopharyngeal carcinoma (NPC) cells.     

Immunohistochemical analysis of the mutant epidermal growth factor, ΔEGFR,in glioblastoma

The naturally occurring mutated form of the epidermal growth factor receptor, ΔEGFR (also named EGFRvIII and de2-7EGFR), greatly enhances glioblastoma (GBM) cell growth in vivo through several activities, such as down-regulating p27 and up-regulating BclX(L) while increasing signaling through the RAS-MAPK and PI3-K cascades. More than half of GBMs, especially of the de novo type, overexpress EGFR, and 50%–70% of these express ΔEGFR. However, little is known about the distribution of ΔEGFR-expressing tumor cells within surgical specimens. In order to address this clinically important issue, we performed immunohistochemical analyses of 53 GBMs obtained during surgery using the anti-ΔEGFR monoclonal antibody, DH8.3. We also simultaneously analyzed wild-type EGFR expression in these tissues using the anti-EGFR monoclonal antibody, EGFR. 113. ΔEGFR and wild-type EGFR expression were observed in 20/53 (38%) and 29/53 (55%), respectively. Nineteen (95%) of the ΔEGFR-positive tumors also expressed wild-type EGFR; one case was ΔEGFR-positive but wild-type EGFR-negative. In 13/20 (65%) of the ΔEGFR-positive tumors, tumor cells were scattered diffusely within the tumors, 6/20 showed geographical distribution of ΔEGFR-positive tumor cells, and one case showed homogeneous staining. In the wild-type EGFR-positive cases, almost all tumor cells expressed EGFR. The differential distribution of cells expressing the two receptors observed here may suggest either that ΔEGFR arises at a low frequency from wild-type EGFR-expressing cells, perhaps during the process of gene amplification, or that there is a paracrine-type of interaction between them.     

14-3-3σ and p63 play opposing roles in epidermal tumorigenesis

14-3-3σ plays a regulatory role in epidermal epithelial differentiation and loss of 14-3-3σ leads to increased proliferation and impaired differentiation. A tumor suppressor function for 14-3-3σ has been proposed based on the fact that some epithelial-derived tumors lose 14-3-3σ expression. p63, a p53 family member, is a master regulator of epidermal epithelial proliferation and differentiation and is necessary for the epidermal development. The function of p63 in tumorigenesis is still controversial and poorly defined as multiple isoforms have been found to play either collaborative or opposing roles. By using 'repeated epilation' heterozygous (Er/+) mice containing a dominant-negative 14-3-3σ mutation, the functional relationship of p63 with 14-3-3σ in epidermal proliferation, differentiation and tumorigenesis was investigated. It was found that p63, particularly the ΔNp63α isoform, was strongly expressed in 14-3-3σ-deficient keratinocytes and knockdown of p63 remarkably inhibited proliferation in these cells. To study the functional roles of 14-3-3σ and p63 in epidermal tumorigenesis, we adopted a 7,12-dimethylbenzanthracene/12-O-tetradecanoyl-phorbol-13-acetate (DMBA/TPA) two-stage tumorigenesis procedure to induce formation of skin papillomas and squamous cell carcinomas in Er/+ mice and identified strong p63 expression in resultant tumors. The loss of one allele of p63 caused by the generation of Er/+/p63(+/-) double compound mice decreased the sensitivity to DMBA-/TPA-induced tumorigenesis as compared with Er/+ mice. This study shows that p63 and 14-3-3σ play opposing roles in the development of skin tumors and that the accumulation of p63 is essential for Ras/14-3-3σ mutation-induced papilloma formation and squamous cell carcinoma carcinogenesis.     

The limitless role of p53 in cell cycle machinery: good news or bad news?

The p53 tumor suppressor gene acts as a great protagonist in deciding how cells undergo either cell cycle arrest or apoptosis after experiencing various stress signals, including DNA damage, hypoxia, oncogene activation, and hyperproliferation. Research on p53 is in steady expansion, as evidenced by the continual flood of papers claiming novel mutations, gain or loss of p53 functions, and gene interactions. The latest study carried out by Spurgers of Texas University and his Colleagues (J Biol Chem 2006; 281:25134-42) emphasizes the strong impact of p53 in the complicated machinery that regulates cell cycle progression. In this paper, microarray data and well-evaluated statistical procedures on PC3 and LNCaP prostate carcinoma cells, open new perspectives in p53 mechanisms and bring the simultaneous identification of novel p53-repressed cell cycle genes, hopefully providing significant improvements in the study of DNA damage response, multistep carcinogenesis, and treatment rationales and outcomes.