Analysis of the intracellular localization of p73 N-terminal protein isoforms TAp73 and ∆Np73 in medulloblastoma cell lines

The protein homologous to the tumor suppressor p53, p73, has essential roles in development and tumorigenesis. This protein exists in a wide range of isoforms with different, even antagonistic, functions. However, there are virtually no detailed morphological studies analyzing the endogenous expression of p73 isoforms at the cellular level in cancer cells. In this study, we investigated the expression and subcellular distribution of two N-terminal isoforms, TAp73 and ΔNp73, in medulloblastoma cells using immunofluorescence microscopy. Both proteins were observed in all cell lines examined, but differences were noted in their intracellular localization between the reference Daoy cell line and four newly established medulloblastoma cell lines (MBL-03, MBL-06, MBL-07 and MBL-10). In the new cell lines, TAp73 and ΔNp73 were located predominantly in cell nuclei. However, there was heterogeneity in TAp73 distribution in the cells of all MBL cell lines, with the protein located in the nucleus and also in a limited non-random area in the cytoplasm. In a small percentage of cells, we detected cytoplasmic localization of TAp73 only, i.e., nuclear exclusion was observed. Our results provide a basis for future studies on the causes and function of distinct intracellular localization of p73 protein isoforms with respect to different protein–protein interactions in medulloblastoma cells.     

The p73 tumor suppressor is targeted by Pirh2 RING finger E3 ubiquitin ligase for the proteasome-dependent degradation

The p73 gene, a homologue of the p53 tumor suppressor, is expressed as TA and ΔN isoforms. TAp73 has similar activity as p53 and functions as a tumor suppressor whereas ΔNp73 has both pro- and anti-survival functions. While p73 is rarely mutated in spontaneous tumors, the expression status of p73 is linked to the sensitivity of tumor cells to chemotherapy and prognosis for many types of human cancer. Thus, uncovering its regulators in tumors is of great interest. Here, we found that Pirh2, a RING finger E3 ubiquitin ligase, promotes the proteasome-dependent degradation of p73. Specifically, we showed that knockdown of Pirh2 up-regulates, whereas ectopic expression of Pirh2 down-regulates, expression of endogenous and exogenous p73. In addition, Pirh2 physically associates with and promotes TAp73 polyubiquitination both in vivo and in vitro. Moreover, we found that p73 can be degraded by both 20 S and 26 S proteasomes. Finally, we showed that Pirh2 knockdown leads to growth suppression in a TAp73-dependent manner. Taken together, our findings indicate that Pirh2 promotes the proteasomal turnover of TAp73, and thus targeting Pirh2 to restore TAp73-mediated growth suppression in p53-deficient tumors may be developed as a novel anti-cancer strategy.     

p73 induces apoptosis by different mechanisms

p73, like its homologue, the tumor suppressor p53, is able to induce apoptosis in several cell types. This property is important for the involvement of p73 in cancer development and therapy. However, in contrast with p53, the TAp73 gene has two distinct promoters coding for two protein isoforms with opposite effects: while the transactivation proficient TAp73 shows pro-apoptotic effects, the amino-terminal-deleted DeltaNp73 has an anti-apoptotic function. Indeed, the relative expression of these two proteins is related to the prognosis of several cancers. Here we discuss recent developments in the control of p73-induced apoptosis. First, TAp73 induces ER stress via the direct transactivation of Scotin. Second, TAp73 induces the mitochondrial pathway by directly transactivating both Bax and the BH3 only protein PUMA promoters. While the first transactivation is weak, and not sufficient to trigger apoptosis (at least in the in vitro cellular models so far evaluated), the induction of PUMA is strong and lethal. Third, the promoter of the death receptor CD95 contains a p53 responsive element and preliminary experiments suggest that TAp73 also activates the death receptor pathway. In addition, TAp73 is able to transactivate its own second promoter, thus inducing the expression of the anti-apoptotic DeltaNp73 isoform. Therefore, the balance between TAp73 and DeltaNp73 finely regulates cellular sensitivity to death.     

Inhibition of apoptosis via CHIP-mediated proteasomal degradation of TAp73α

TAp73, a homologous of tumor suppressor p53, regulates apoptosis in a p53-independent manner and its suppressive as well as stimulatory role in promoting angiogenesis has been reported. It exists in multiple isoforms which varies structurally in their N-terminus and C-terminus region and crucial interplay among them guides the decision of cell survival and death. As molecular chaperones control both stability and degradation of TAp73, selective regulation of p73 isoforms has implication upon developing new therapeutic for hypoxic tumor. We have discovered that under DNA damage carboxy terminus Hsp70 interacting protein (CHIP's) antiapoptotic function is displayed via its E3 ligase activity that inhibits exclusively TAp73α-mediated apoptosis in cancer cell. The decrease in TAp73α level by CHIP as it is supported by increased ubiquitination pattern is reverted back by sh-CHIP. Further, the transactivation of p53-downstream apoptotic genes BAX, PUMA and PIG3 by TAp73α is also shown to be subsequently inhibited by CHIP. The tetratricopeptide TPR-domain of CHIP in its amino-terminus interacts with the carboxy-terminus of TAp73α and ΔNp73α and as a result, U-BOX domain of CHIP in the carboxy-terminus is able to ubiquitinate TAp73α for proteasomal degradation. Due to lack of C-terminus in TAp73β, CHIP fails to interact with and degrade it. In conclusion, we have thus uncovered for the first time a novel mechanism of chaperone-assisted regulation of p73 stability as well as its apoptotic functions by CHIP that might be utilized to develop new anticancer strategies.     

Evaluation of p63 and p73 antibodies for cross-reactivity

The tumor suppressor p53 is commonly mutated in human cancers. However, two homologs of p53, p63 and p73, are frequently over-expressed in tumors and are associated with tumor subtypes, clinical outcomes, and responses to therapy. There are many isoforms of p53, p63, and p73 (the p53 family). Proper detection of and discrimination between the members of this tumor suppressor family in human tissues is of critical importance to cancer research and clinical care. In this study, we assessed the specificity of several commercially available and newly generated p73 antibodies, focusing on antibodies that distinguish between the TAp73 and ?Np73 isoforms by Western analysis, immunohistochemistry, and immunofluorescence. In addition, we found that the pan-p63 and pan-p73 antibodies tested cross-react with p73 and p63 respectively. The results of this study have important implications for analysis of p63 and p73 expression and co-expression in human tumors, and for potential use of these reagents in molecular diagnostics and therapeutic decision-making.