Ubiquitin-dependent degradation of p73 is inhibited by PML.

p73 has been identified recently as a structural and functional homologue of the tumor suppressor p53. Here, we report that p73 stability is directly regulated by the ubiquitin-proteasome pathway. Furthermore, we show that the promyelocytic leukemia (PML) protein modulates p73 half-life by inhibiting its degradation in a PML-nuclear body (NB)-dependent manner. p38 mitogen-activated protein kinase-mediated phosphorylation of p73 is required for p73 recruitment into the PML-NB and subsequent PML-dependent p73 stabilization. We find that p300-mediated acetylation of p73 protects it against ubiquitinylation and that PML regulates p73 stability by positively modulating its acetylation levels. As a result, PML potentiates p73 transcriptional and proapoptotic activities that are markedly impaired in Pml-/- primary cells. Our findings demonstrate that PML plays a crucial role in modulating p73 function, thus providing further insights on the molecular network for tumor suppression.

Nucleofection is a highly effective gene transfer technique for human melanoma cell lines.

Despite the increasing use of gene transfer strategies in the study of cellular and molecular biology, melanoma cells have remained difficult to transfect in a safe, efficient, and reproducible manner. In the present study, we report the successful use of nucleofector technology to transfect human melanoma cell lines. This technology uses an empirically derived combination of cell line-specific solutions and nucleofector programmes to electroporate nucleic acid substrates directly into the cell nucleus. Using a colorimetric beta-galactosidase assay, we optimized nucleofection parameters for 13 melanoma cell lines, leading to maximum transfection efficiency and cell survival. The combinations of cell solutions NHEM or T and nucleofector programmes A-24 or U-20 produced the best results. We compared nucleofection with two commercially available lipid-based gene transfer systems, effectene and lipofectamine 2000 using a green fluorescent protein reporter vector. Nucleofection demonstrated a 3- to 40-fold improvement in transfection efficiency when compared with the lipid-based counterparts. Nucleofection was also superior in transfecting small-interfering RNA (siRNA) as determined by Western blot analysis. Lastly, we applied nucleofection to the simultaneous transfection of a p53-dependent luciferase plasmid and p53-siRNA. Experiments using dual transfection showed knockdown of p53 expression and silencing of the reporter plasmid. In conclusion, nucleofection is highly effective for the transfer of nucleic acid substrates, singly or in combination, into human melanoma cell lines.

Expression of p63 in diffuse large B-cell lymphoma.

The p63 gene, a homolog of the tumor suppressor gene TP53, maps to chromosome 3q27-28, a region frequently displaying genomic amplification in squamous cell carcinomas. p63 is expressed in a variety of epithelial tissues and has been reported to be critical for the normal development of stratified epithelia, including skin epidermis. In a previous study, the authors reported the expression of p63 in occasional cells in the germinal center of lymph nodes and also observed p63 expression in B-cell lymphomas, among other tumor types surveyed in that analysis. The present study was conducted to further analyze the potential clinical significance of identifying p63 expression, assessing a larger cohort of well-characterized patients with diffuse large B-cell lymphoma (DLBCL) (n = 172 cases) and a panel of established lymphoma cell lines. p63 expression at the microanatomic detail was examined by immunohistochemistry using a monoclonal antibody (clone 4A4), while distinction of p63 isoforms was analyzed by Western blotting and reverse transcription-polymerase chain reaction using isoform-specific primers. The authors found that a subset of DLBCL (32% of cases) expressed p63 in the nuclei of neoplastic lymphocytes. Examination of the different p63 isoforms revealed that the DeltaNp63 species was expressed by only one cell line, while the other p63 isoforms were found in most cell lines analyzed. The authors also observed that p63 expression correlated with high proliferative index, as assessed by Ki-67 immunostaining. Even though in univariate analysis p63 expression did not correlate with overall survival, the association of p63 with increased proliferative index suggests its involvement in DLBCL tumor progression.

p63 expression profiles in human normal and tumor tissues.

PURPOSE: The p63 gene, located on chromosome 3q27-28, is a member of the p53 gene family. The product encoded by the p63 gene has been reported to be essential for normal development. EXPERIMENTAL DESIGN: In this study, we examined the expression pattern of p63 in human normal and tumor tissues by immunohistochemistry using a monoclonal antibody (clone 4A4) that recognizes all p63 splice variants, and by reverse transcription-PCR using isoform-specific primers. RESULTS: We found that p63 expression was restricted to the nucleus, with a nucleoplasmic pattern. We also observed that the expression was restricted to epithelial cells of stratified epithelia, such as skin, esophagus, exocervix, tonsil, and bladder, and to certain subpopulations of basal cells in glandular structures of prostate and breast, as well as in bronchi. Consistent with the phenotype observed in normal tissues, we found that p63 is expressed predominantly in basal cell and squamous cell carcinomas, as well as transitional cell carcinomas, but not in adenocarcinomas, including those of breast and prostate. Interestingly, thymomas expressed high levels of p63. Moreover, a subset of non-Hodgkin's lymphoma was also found to express p63. Using isoform-specific reverse transcription-PCR, we found that thymomas express all isoforms of p63, whereas the non-Hodgkin's lymphoma tended to express the transactivation-competent isoforms. We did not detect p63 expression in a variety of endocrine tumors, germ cell neoplasms, or melanomas. Additionally, soft tissue sarcomas were also found to have undetectable p63 levels. CONCLUSIONS: Our data support a role for p63 in squamous and transitional cell carcinomas, as well as certain lymphomas and thymomas.

p73 Expression in human normal and tumor tissues: loss of p73alpha expression is associated with tumor progression in bladder cancer.

PURPOSE: To characterize the expression profile of p73 in human normal tissues by immunohistochemistry (IHC) and to analyze the correlation between p73 expression and bladder cancer progression. EXPERIMENTAL DESIGN: CJDp73 was characterized for p73alpha detection in Western blot and IHC through its application to isoform-transfected 293 cells. Normal tissues were analyzed by IHC with the CJDp73 antiserum. Transitional cell carcinoma (TCC)-derived cell lines were subjected to reverse transcription-PCR and Western blot. TCC tissue microarrays were analyzed for p73alpha expression by IHC, and the results were statistically analyzed. RESULTS: p73 immunostaining was nuclear and restricted to epithelial cells of certain organs such as squamous epithelium of the epidermis and transitional epithelium of the bladder. The expression was also observed in certain specialized glandular epithelia such as acinar cells of breast and parotid gland. Four of seven TCC-derived cell lines had low to undetectable p73alpha protein levels. We found undetectable or low p73alpha expression in 104 of 154 (68%) TCC cases, this phenotype being more frequently observed in invasive tumors when compared with superficial lesions. This association was statistically significant (P < 0.0001). We also observed a significant association between p53, p63, and p73alpha alterations with bladder cancer progression (P < 0.0001). CONCLUSIONS: p73alpha plays a tumor suppressor role in bladder cancer, and its inactivation occurs through an epigenetic mechanism, most probably involving protein degradation.

Comparative study of p63 and p53 expression in tissue microarrays of malignant melanomas.

p63 is a known homologue of p53. In contrast to p53, however, p63 mutations are rarely seen in tumours. There have been several reports that p63 plays a regulatory role in the normal differentiation of cells, whereas its role in tumour biology must still be elucidated. The main aim of this study was to compare p63 and p53 expression in tissue microarrays of malignant melanomas and to establish any prognostic significance. p63 expression was found in 2 out of 59 tumours, both pT4. The p63 index did not exceed 30%. p53 expression was found in 27 out of 59 melanomas, with maximal expression in up to 80% of tumour cells. There were no correlations observed between the two markers. Multivariate analysis confirmed the prognostically independent role of p53. This study also confirmed that tissue microarrays can be used effectively for evaluation of the expression of certain tumour markers.

The association of Tap42 phosphatase complexes with TORC1: another level of regulation in Tor signaling.

In the budding yeast Saccharomyces cerevisiae, rapamycin has been known to induce a rapid dephosphorylation of many downstream targets of Tor. The key components mediating this dephosphorylation process are the Tap42-associated phosphatases, which become active upon rapamycin treatment. However, the mechanism by which rapamycin rapidly activates phosphatases is unclear. A recent report has provided evidence demonstrating a physical association of the Tap42-phosphatase complexes with TORC1, which is sensitive to rapamycin treatment or nutrient starvation. This association adds another level of regulation in Tor signaling, and explains why rapamycin or nutrient availability is able to initiate a rapid and robust response in the cell.

Regulation of the cell integrity pathway by rapamycin-sensitive TOR function in budding yeast.

The TOR (target of rapamycin) pathway controls cell growth in response to nutrient availability in eukaryotic cells. Inactivation of TOR function by rapamycin or nutrient exhaustion is accompanied by triggering various cellular mechanisms aimed at overcoming the nutrient stress. Here we report that in Saccharomyces cerevisiae the protein kinase C (PKC)-mediated mitogen-activated protein kinase pathway is regulated by TOR function because upon specific Tor1 and Tor2 inhibition by rapamycin, Mpk1 is activated rapidly in a process mediated by Sit4 and Tap42. Osmotic stabilization of the plasma membrane prevents both Mpk1 activation by rapamycin and the growth defect that occurs upon the simultaneous absence of Tor1 and Mpk1 function, suggesting that, at least partially, TOR inhibition is sensed by the PKC pathway at the cell envelope. This process involves activation of cell surface sensors, Rom2, and downstream elements of the mitogen-activated protein kinase cascade. Rapamycin also induces depolarization of the actin cytoskeleton through the TOR proteins, Sit4 and Tap42, in an osmotically suppressible manner. Finally, we show that entry into stationary phase, a physiological situation of nutrient depletion, also leads to the activation of the PKC pathway, and we provide further evidence demonstrating that Mpk1 is essential for viability once cells enter G(0).

Loss of p63 expression is associated with tumor progression in bladder cancer.

p63, a member of the p53 gene family, encodes multiple proteins that may either transactivate p53 responsive genes (TAp63) or act as a dominant-negative factor toward p53 and p73 (Delta Np63). p63 is expressed in many epithelial compartments and p63(-/-) mice fail to develop skin, prostate, and mammary glands among other defects. It has been previously shown that p63 is expressed in normal urothelium. This study reports that p63 is regulated in bladder carcinogenesis and that p63 expression is lost in most invasive cancers whereas papillary superficial tumors maintain p63 expression. Examination of bladder carcinoma cell lines reveals that certain lines derived from invasive carcinomas maintain expression of Delta Np63, as demonstrated by both immunoblotting and confirmed by isoform-specific quantitative reverse transcriptase-polymerase chain reaction. Another novel finding reported in this study is the fact that p63(-/-) mice develop a bladder mucosa epithelial layer yet fail to complete uroepithelial differentiation, producing a nontransitional default cuboidal epithelium. These data indicate that in contrast to the skin and prostate, p63 is not required for formation of a bladder epithelium but is indispensable for the specific differentiation of a transitional urothelium.