Expression Of P53 Family Members Research Articles

Protective Effects of Cinnamaldehyde against Mesenteric Ischemia-Reperfusion-Induced Lung and Liver Injuries in Rats.

The aim of this study was to characterize and reveal the protective effects of cinnamaldehyde (CA) against mesenteric ischemia-reperfusion- (I/R-) induced lung and liver injuries and the related mechanisms. Sprague-Dawley (SPD) rats were pretreated for three days with 10 or 40 mg/kg/d, ig of CA, and then induced with mesenteric ischemia for 1 h and reperfusion for 2 h. The results indicated that pretreatment with 10 or 40 mg/kg of CA attenuated morphological damage in both lung and liver tissues of mesenteric I/R-injured rats. CA pretreatment significantly restored the levels of aspartate transaminase (AST) and alanine transaminase (ALT) in mesenteric I/R-injured liver tissues, indicating the improvement of hepatic function. CA also significantly attenuated the inflammation via reducing myeloperoxidase (MOP) activity and downregulating the expression of inflammation-related proteins, including interleukin-6 (IL-6), interleukin-1β (IL-1β), cyclooxygenase-2 (Cox-2), and tumor necrosis factor receptor type-2 (TNFR-2) in both lung and liver tissues of mesenteric I/R-injured rats. Pretreatment with CA significantly downregulated nuclear factor kappa B- (NF-κB-) related protein expressions (NF-κB p65, NF-κB p50, I kappa B alpha (IK-α), and inhibitor of nuclear factor kappa-B kinase subunit beta (IKKβ)) in both lung and liver tissues of mesenteric I/R-injured rats. CA also significantly downregulated the protein expression of p53 family members, including caspase-3, caspase-9, Bax, and p53, and restored Bcl-2 in both lung and liver tissues of mesenteric I/R-injured rats. CA pretreatment significantly reduced TUNEL-apoptotic cells and significantly inhibited p53 and NF-κB p65 nuclear translocation in both lung and liver tissues of mesenteric I/R-injured rats. CA neither induced pulmonary and hepatic histological alterations nor affected the parameters of inflammation and apoptosis in sham rats. We conclude that CA alleviated mesenteric I/R-induced pulmonary and hepatic injuries via attenuating apoptosis and inflammation through inhibition of NF-κB and p53 pathways in rats, suggesting the potential role of CA in remote organ ischemic injury protection.

P53-Family Proteins in Odontogenic Cysts: An Immunohistochemical Study.

The present study investigated the immunohistochemical expression of p53, p63, and p73 in different types of odontogenic cysts (OC), a group of common intraosseous jaw lesions, to provide a better understanding of p53-family functions in odontogenic lesions. We carried out immunohistochemical analysis to evaluate the expression of p53, p63, and p73 in 60 samples of OC, including dentigerous cysts, radicular cysts, orthokeratinized OC, and odontogenic keratocysts (OKC). The epithelial expression of p53-family members was evaluated both in the basal-parabasal and in the superficial layers, measuring the percentage of positive cells and the value of expression intensity. The expression of p53-family members showed a significant difference between the "OKC" and "non-OKC" groups. In particular, p53 positivity in the basal-parabasal layers, as well as p63 positivity in the superficial layers, were more common in OKC (P<0.0001; P=0.0237). p73 expression in the superficial layers was significantly more expressed in the "non-OKC" group (P<0.0001). No significant differences of staining intensity scores were reported between the groups. The Spearman test showed a positive correlation between p53 and p73 expression at the basal-parabasal level in all cysts (r=0.6626; P<0.0001). These results showed a significantly different expression of p53-family members in OC groups, in particular between the "OKC" and "non-OKC" groups, suggesting the existence of a p53-family pathway in the epithelial lining of OC.

Expression of p53-family members in odontogenic cysts

Expression of p53-family members in odontogenic cysts

PO-159 Expression profiles of P53/P73 isoforms in human melanoma cell lines

IntroductionTP53 is the most frequently mutated gene in human cancer. However, in metastatic melanoma mutations of TP53 occur infrequently and p53 fails to function as a tumour suppressor. The altered expression of p53 family members, including p53/p73 isoforms, as well as of the interactions among them could affect normal function of p53. Furthermore, somatic BRAF mutations have been found in 37%–50% of all melanomas, of which almost 90% harbour the activating V600E mutation. Although initial response to BRAF inhibitors is highly effective, the resistant clones frequently develop, and, in treated patients disease progression is observed within 6 to 8 months. To address this, a better understanding of the genetic basis of melanoma initiation and progression is needed.Material and methodsThe expression profile of p53 and its potential interaction partners - p53 and p73 isoforms was determined in a panel of melanoma cell lines by western blot analysis and quantitative RT-PCR. We have determined the protein levels of p53/p73 isoforms in response to DNA damage treatment (γ-irradiation and etoposide) in cell lines with different TP53 mutational status using western blot analysis. Furthermore, vemurafenib resistant cells are generated and resistance was confirmed by MTT assay. Expression of p53/p73 isoforms was determined in these cells.Results and discussionsRelative expression analysis of metastatic melanoma cell lines revealed that the most expressed p53 isoforms are p53α and Δ133p53α, while Δ40p53ß, Δ40p53γ and Δ133p53γ are least expressed. Also, interestingly, relative expression of full length TAp73 was higher than ΔNp73. Furthermore, the most expressed proteins were p53α, Δ40p53α, Δ133p53γ and Δ160p53γ. Contrary to gene expression, the most expressed p73 isoform is oncogenic ΔNp73β. γ-irradiation induced accumulation of all p53α isoforms in p53 mutant melanoma cell lines, but not in p53 wild type cells. Levels of p53 beta isoforms remained the same, while gamma isoforms were undetectable. Upon γ-irradiation, accumulation of ΔNp73 isoforms was observed in p53 mutant cells. Treatment with etoposide induced expression of p53α isoform, and both TAp73 and ΔNp73 isoforms in p53 wild type cells. Furthermore, in vemurafenib resistant clones the changes in p53/p73 protein expression were observed.ConclusionTaken together, these analyses enabled us to detect p53/p73 isoforms in melanoma cell lines and gave us insight into their abundance in melanoma cell lines for further analyses of p53 interacting partners.

Diverse p63 and p73 isoforms regulate Δ133p53 expression through modulation of the internal TP53 promoter activity

In response to stress, p53 binds and transactivates the internal TP53 promoter, thus regulating the expression of its own isoform, Δ133p53α. Here, we report that, in addition to p53, at least four p63/p73 isoforms regulate Δ133p53 expression at transcriptional level: p63β, ΔNp63α, ΔNp63β and ΔNp73γ. This regulation occurs through direct DNA-binding to the internal TP53 promoter as demonstrated by chromatin immunoprecipitation and the use of DNA-binding mutant p63. The promoter regions involved in the p63/p73-mediated transactivation were identified using deleted, mutant and polymorphic luciferase reporter constructs. In addition, we observed that transient expression of p53 family members modulates endogenous Δ133p53α expression at both mRNA and protein levels. We also report concomitant variation of p63 and Δ133p53 expression during keratinocyte differentiation of HaCat cells and induced pluripotent stem cells derived from mutated p63 ectodermal dysplasia patients. Finally, proliferation assays indicated that Δ133p53α isoform regulates the anti-proliferative activities of p63β, ΔNp63α, ΔNp63β and ΔNp73γ. Overall, this study shows a strong interplay between p53, p63 and p73 isoforms to orchestrate cell fate outcome.

Expression of p53 family members and CD44 in oral squamous cell carcinoma (OSCC) in relation to tumorigenesis.

Oral squamous cell carcinomas (OSCCs) are described as the result of a multistep tumorigenesis process. In order to develop useful diagnosis of pre-malignant lesions, expression of p53 family members and the cancer stem cell (CSCs) marker, CD44v6, were studied in histologically normal oral epithelium, precancerous lesions and succeeding invasive OSCCs. p53 was expressed focally in normal epithelium adjacent to tumors, while expression was high in intra-epithelial neoplasia and moderate in OSCC. p63 nuclear staining was important in basal and suprabasal layers of histologically normal oral mucosa and in immature compartments of premalignant lesions and cancer. In epithelium without neoplasia, intense p73 staining was observed in the basal layer, while focal expression was present in suprabasal layers. Most immature dysplastic areas showed either high or moderate staining, whereas those in OSCCs expressed low and moderate p73 level expression. CD44v6 was only expressed in poorly differentiated areas of epithelium, altered or not. p53, p63 and p73 positive stainings were statistically related in intra-epithelial neoplasia to tumours. Analysis of TP53 mutations in 17% of tumours principally revealed G>A and A>G transitions. No relation was observed between this mutational profile and different immunostainings. In conclusion, our results support that immunostaining of p53 family members might be helpful in diagnosis and monitoring of high-risk pre-malignant lesions of oral epithelium. The combination of staining patterns of p63, p73alpha and CD44v6 enabled us to isolate phenotypic undifferentiated or transient amplifying areas, reflecting the immaturity of the tumour cell lineage. While CD44v6 expression is an interesting marker of such epithelial cells, it is not specific enough to be useful alone and other phenotypic markers are needed.

Unique domain functions of p63 isotypes that differentially regulate distinct aspects of epidermal homeostasis

p63 is critical for squamous development and exists as multiple isotypes of two subclasses, TA and DeltaN. DeltaNp63 isotypes can antagonize transcription by TAp63 and p53, and are highly expressed in squamous cell cancers. Using mouse keratinocytes as a biological model of squamous epithelium, we show that multiple p63 isotypes, DeltaN- and TA-containing, are expressed and differentially modulated during in vitro murine keratinocyte differentiation. DeltaNp63alpha declines with Ca2+-induced differentiation, while a smaller DeltaN-form, DeltaNp63s, persists, suggesting unique functions of the two DeltaN-forms. To investigate the impact of dysregulated p63 expression that is observed in cancers and to define the biological contribution of the different domains of the p63 isotypes, DeltaNp63alpha, DeltaNp63p40, TAp63alpha, TAp63gamma or beta-galactosidase were overexpressed in primary murine keratinocytes. Microarray, RT-PCR and western blot analyses revealed that overexpression of DeltaNp63p40, which lacks the entire alpha-tail present in DeltaNp63alpha, permits expression of a full panel of differentiation markers. This is in contrast to overexpression of the full-length DeltaNp63alpha, which blocks induction of keratin 10, loricrin and filaggrin. These findings support a role for the alpha-tail of DeltaNp63alpha in blocking differentiation-specific gene expression. Overexpression of either TAp63 isotype permits keratin 10 and loricrin expression, thus the alpha-terminus requires the cooperation of the DeltaN domain in blocking early differentiation. However, both TA isotypes block filaggrin induction. The DeltaN-terminus is sufficient to maintain keratinocytes in a proliferative state, as both DeltaN forms block Ca2+-mediated p21WAF1 induction and S-phase arrest, while sustaining elevated PCNA levels. No alteration in cell cycle regulation was observed in keratinocytes overexpressing TAp63alpha or TAp63gamma. Clarifying the functional distinctions between p63 isotypes and domains will help to elucidate how their dysregulation impacts tumor biology and may suggest novel therapeutic strategies for modulating behavior of tumor cells with altered expression of p53 family members.

Expression of p53-family members and associated target molecules in breast cancer cell lines in response to vincristine treatment

As the antimitotic agent vincristine (VCR) has been reported to induce a weak p53 response in some studies, we hypothesised that p73 and p63, the recently described p53 homologues, may replace p53 in triggering apoptosis or cell cycle arrest effectors in VCR-treated cell lines. To address this issue, we measured p53, p73 and p63 mRNA and protein levels in two VCR-treated breast cancer cell lines, one p53-proficient (MCF7) and the other p53-deficient (MDA-MB157). We found an increase of p53 mRNA and protein levels in VCR-treated MCF7 cells, while, as expected, no p53 protein was detected in VCR-treated MDA-MB157 cells. Surprisingly, the p73 mRNA and protein expression levels decreased in both cell lines during VCR treatment, whereas p63 protein levels remained unchanged. In both cell lines, up-regulations of the canonical p53-target genes, such as p21 and GADD45, were consistently observed. We conclude that, in response to VCR treatment: (1) p53 is markedly induced in MCF7 cells, with the same extent than after DNA damaging drugs treatments; and (2) p63 is not involved, while p73 expression is down-regulated regardless of the p53 status of the cell lines. Our results therefore suggest the involvement of a fourth member of the p53 gene family, or the use of another pathway able to trigger canonical p53-target genes in response to VCR in p53-deficient cells.