Effects of oxidation agents and metal ions on binding of p53 to supercoiled DNA

Wild type human full length (f.l.) tumor suppressor p53 protein binds preferentially to supercoiled (sc) DNA in vitro both in the presence and absence of the p53 consensus sequence (p53CON). This binding produces a ladder of retarded bands on the agarose gel. Bands revealed by immunoblotting with antibody DO-1 corresponded to the ethidium stained retarded bands. The intensity and the number of bands of p53-scDNA complex were decreased by physiological concentrations of unchelated zinc ions. Nickel and cobalt ions inhibited binding of p53 to scDNA and to p53CON in linear DNA fragments less efficiently than zinc. Compared to the intrinsic zinc strongly bound to Cys 176, Cys 238, Cys 242 and His 179 in the p53 core domain, binding of additional Zn2+ to p53 was much weaker as shown by an easy removal of the latter ions by low concentrations of EDTA. Oxidation of the protein with diamide resulted in a decrease of the number of the retarded bands. Under the same conditions, no binding of oxidized p53 to p53CON in a linear DNA fragment was observed. In agreement with the literature oxidation of f.l. p53 with diamide was irreversible and was not reverted by an excess of DTT. We showed that in the presence of 0.1 mM zinc ions, oxidation of p53 became reversible. Other divalent cations tested (cadmium, cobalt, nickel) exhibited no such effect. We suggested that the irreversibility of p53 oxidation was due, at least in part, to the removal of intrinsic zinc from its position in the DNA binding domain (after oxidation of the three cysteines to which the zinc ion is coordinated in the reduced protein) accompanied by a change in the p53 conformation. Binding of C-terminal anti-p53 antibody also protected bacterially expressed protein against irreversible loss of activity due to diamide oxidation. Binding the human p53 core domain (segment 94-312) to scDNA greatly differed from that observed with the full-length p53. The core domain did not posses the ability to bind strongly to many sites in scDNA regardless of the presence or absence of p53CON suggesting involvement of some other domain (probably C-terminal) in binding of the full-length p53 to scDNA. Supershift experiments using antibodies against p53 N- or C-terminus suggested that in oxidized p53, scDNA binding through the C-terminus gained importance

∆Np63/p40 correlates with the location and phenotype of basal/mesenchymal cancer stem-like cells in human ER+ and HER2+ breast cancers

ΔNp63, also known as p40, regulates stemness of normal mammary gland epithelium and provides stem cell characteristics in basal and HER2‐driven murine breast cancer models. Whilst ΔNp63/p40 is a characteristic feature of normal basal cells and basal‐type triple‐negative breast cancer, some receptor‐positive breast cancers express ΔNp63/p40 and its overexpression imparts cancer stem cell‐like properties in ER+ cell lines. However, the incidence of ER+ and HER2+ tumours that express ΔNp63/p40 is unclear and the phenotype of ΔNp63/p40+ cells in these tumours remains uncertain. Using immunohistochemistry with p63 isoform‐specific antibodies, we identified a ΔNp63/p40+ tumour cell subpopulation in 100 of 173 (58%) non‐triple negative breast cancers and the presence of this population associated with improved survival in patients with ER−/HER2+ tumours (p = 0.006). Furthermore, 41% of ER+/PR+ and/or HER2+ locally metastatic breast cancers expressed ΔNp63/p40, and these cells commonly accounted for <1% of the metastatic tumour cell population that localised to the tumour/stroma interface, exhibited an undifferentiated phenotype and were CD44+/ALDH−. In vitro studies revealed that MCF7 and T47D (ER+) and BT‐474 (HER2+) breast cancer cell lines similarly contained a small subpopulation of ΔNp63/p40+ cells that increased in mammospheres. In vivo, MCF7 xenografts contained ΔNp63/p40+ cells with a similar phenotype to primary ER+ cancers. Consistent with tumour samples, these cells also showed a distinct location at the tumour/stroma interface, suggesting a role for paracrine factors in the induction or maintenance of ΔNp63/p40. Thus, ΔNp63/p40 is commonly present in a small population of tumour cells with a distinct phenotype and location in ER+ and/or HER2+ human breast cancers.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/153532/1/cjp2149_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/153532/2/cjp2149.pd

Serum antibodies against p53 in relation to cancer risk and prognosis in breast cancer: a population-based epidemiological study

To perform an epidemiological evaluation of the predictive value of p53 autoantibodies in breast cancer, we measured antibodies against p53 in serum samples from 165 breast cancer patients in comparison with serum samples from 330 healthy controls, selected from the same population as the cases and matched for age, sex and specimen storage time. Median age of patients was 51 years (range 25–64 years). Presence of serum p53 autoantibodies was analysed by enzyme-linked immunosorbent assay (ELISA) and confirmed by Western blotting. The lower ELISA reactivities were similar for cases and controls, but presence of high-level reactivity was more common among cases than among controls [odds ratio (OR) 9.03, 95% confidence interval (CI) 2.40–50.43]. Presence of Western blot-detected p53 autoantibodies had a very similar association (OR 10.8, CI 3.0–59.4). Among the cases, we also studied whether there was any correlation between level of anti-p53 antibodies and stage of the disease or survival. There was no significant correlation between presence of antibodies and stage of the disease. There was a significant negative correlation between presence of p53 antibodies and survival (P= 0.003). A stepwise multivariate Cox regression analysis showed that T-stage, age and presence of anti-p53 antibodies were significant independent prognostic variables, with a dose-dependent negative effect on survival for all three variables. We conclude that presence of anti-p53 antibodies are of significance both for the risk of having breast cancer and the risk of dying from breast cancer. 1999 Cancer Research Campaig

A Monoclonal Antibody against p53 Cross-Reacts with Processing Bodies

The p53 tumor suppressor protein is an important regulator of cell proliferation and apoptosis. p53 can be found in the nucleus and in the cytosol, and the subcellular location is key to control p53 function. In this work, we found that a widely used monoclonal antibody against p53, termed Pab 1801 (Pan antibody 1801) yields a remarkable punctate signal in the cytoplasm of several cell lines of human origin. Surprisingly, these puncta were also observed in two independent p53-null cell lines. Moreover, the foci stained with the Pab 1801 were present in rat cells, although Pab 1801 recognizes an epitope that is not conserved in rodent p53. In contrast, the Pab 1801 nuclear staining corresponded to genuine p53, as it was upregulated by p53-stimulating drugs and absent in p53-null cells. We identified the Pab 1801 cytoplasmic puncta as P Bodies (PBs), which are involved in mRNA regulation. We found that, in several cell lines, including U2OS, WI38, SK-N-SH and HCT116, the Pab 1801 puncta strictly colocalize with PBs identified with specific antibodies against the PB components Hedls, Dcp1a, Xrn1 or Rck/p54. PBs are highly dynamic and accordingly, the Pab 1801 puncta vanished when PBs dissolved upon treatment with cycloheximide, a drug that causes polysome stabilization and PB disruption. In addition, the knockdown of specific PB components that affect PB integrity simultaneously caused PB dissolution and the disappearance of the Pab 1801 puncta. Our results reveal a strong cross-reactivity of the Pab 1801 with unknown PB component(s). This was observed upon distinct immunostaining protocols, thus meaning a major limitation on the use of this antibody for p53 imaging in the cytoplasm of most cell types of human or rodent origin

p21WAF1/CIP1expression in stage I cutaneous malignant melanoma: its relationship with p53, cell proliferation and survival

The expression of p21, p53 and proliferating cell nuclear antigen (PCNA) was analysed by immunohistochemistry in a consecutive series of 369 clinical stage I cutaneous malignant melanoma patients. Correlation of the detected expression levels with each other, with clinicopathological data and with melanoma survival were statistically evaluated. p21 expression was significantly associated with p53 and PCNA expression levels. In addition, high levels of p53 and PCNA were significantly interrelated. Tumour thickness, recurrent disease, high TNM category and older (≥ 55 years) age at diagnosis were inversely associated with p21 expression. Gender, bleeding, tumour thickness, Clark's level of invasion, TNM category and p53 index were all important predictors of both recurrence-free and overall survival of melanoma. In Cox's multivariate analysis including 164 patients with a complete set of data, only high tumour thickness and bleeding predicted poor recurrence-free survival (P= 0.0042 and 0.0087 respectively) or overall survival (P= 0.0147 and 0.0033 respectively). Even though elevated p21 expression may be associated with more favourable prognosis in clinical stage I cutaneous melanoma, our results suggest that cell cycle regulatory effects of p21 can be overcome by some other and stronger, partly yet unknown, mechanisms. 1999 Cancer Research Campaig

An animal model to evaluate the function and regulation of the adaptively evolving stress protein SEP53 in oesophageal bile damage responses

Squamous epithelium in mammals has evolved an atypical stress response involving down-regulation of the classic HSP70 protein and induction of sets of proteins including one named SEP53. This atypical stress response might be due to the unusual environmental pressures placed on squamous tissue. In fact, SEP53 plays a role as an anti-apoptotic factor in response to DNA damage induced by deoxycholic acid stresses implicated in oesophageal reflux disease. SEP53 also has a genetic signature characteristic of an adaptively and rapidly evolving gene, and this observation has been used to imply a role for SEP53 in immunity. Physiological models of squamous tissue are required to further define the regulation and function of SEP53. We examined whether porcine squamous epithelium would be a good model to study SEP53, since this animal suffers from a bile-reflux disease in squamous oesophageal tissue. We have (1) cloned and sequenced the porcine SEP53 locus from porcine bacterial artificial chromosome genomic DNA, (2) confirmed the strikingly divergent nature of the C-terminal portion of the SEP53 gene amongst mammals, (3) discovered that a function of the conserved N-terminal domain of the gene is to maintain cytoplasmic localisation, and (4) examined SEP53 expression in normal and diseased porcine pars oesophagea. SEP53 expression in porcine tissue was relatively confined to gastric squamous epithelium, consistent with its expression in normal human squamous epithelium. Immunohistochemical staining for SEP53 protein in normal and damaged pars oesophagea demonstrated significant stabilisation of SEP53 protein in the injured tissue. These results suggest that porcine squamous epithelium would be a robust physiological model to examine the evolution and function of the SEP53 stress pathway in modulating stress-induced responses in squamous tissue