DUCTAL CARCINOMA IN-SITU OF THE BREAST - HISTOLOGIC CATEGORIZATION AND ITS RELATIONSHIP TO PLOIDY AND IMMUNOHISTOCHEMICAL EXPRESSION OF HORMONE RECEPTORS, P53, AND C-ERBB-2 PROTEIN

Background. Ductal carcinoma in situ (DCIS) of the breast has been diagnosed increasingly since the advent of mammography. However, the natural history of these lesions remains uncertain. Ductal carcinoma in situ of the breast does not represent a single entity but a heterogeneous group with histologic and clinical differences. The histologic subtype of DCIS seems to have an influence on its biologic behavior, but there are few studies correlating subtype with biologic markers.Methods. The authors studied a consecutive series of 40 cases of DCIS and after its histologic categorization verified its relationship with ploidy using image analysis and analyzing estrogen receptor (ER), progesterone receptor (PR), p53 and c-erbB-2 expression using immunohistochemistry.Results. The three groups proposed according to the grade of malignancy were correlated significantly with some of the additional parameters studied, including aneuploidy and c-erB-2 expression. Aneuploidy was detected in 77.5% of cases of DCIS mainly in high and intermediate grade subtypes (100% and 80% vs. 35.7% in low grade) whereas immunoreactivity for c-erbB-2 was detected in 45% of cases of DCIS mainly in the high grade group. Expression of ER and PR were observed frequently in this study (63.9% and 65.7% respectively), but without correlation with the histologic subtype of DCIS, although we found a somewhat significant association between high grade DCIS and lack of ER. p53 protein expression was detected in 36.8% of these cases, but no relationship between this expression and histologic subtype or grading of DCIS was found.Conclusions. These results provide further evidence for the morphologic and biologic heterogeneity of DCIS. Besides histologic classification and nuclear grading, some biologic markers such as aneuploidy and c-erbB-2 expression constitute additional criteria of high grade of malignancy.

P53 PROTEIN EXPRESSION AND NUCLEAR-DNA CONTENT IN BREAST INTRADUCTAL PROLIFERATIONS

Immunohistochemical analysis of the p53 gene protein and cytometric assessment of nuclear DNA were performed in a series of 51 cases of intraductal breast proliferation. The series included 22 cases of intraductal hyperplasia without atypia, 6 cases of intraductal hyperplasia with atypia, and 23 cases of pure intraductal carcinoma. Expression of p53 protein was detected in one case of intraductal hyperplasia without atypia (4.5 per cent), one case of intraductal hyperplasia with atypia (16.6 per cent) and six cases of intraductal carcinoma (26.0 per cent). No significant correlation was observed between p53 expression and histological subtype of intraductal carcinoma. Aneuploidy was demonstrated in two cases of intraductal hyperplasia with atypia (33.3 per cent) and in 18 cases of intraductal carcinoma (78.2 per cent). All cases of intraductal hyperplasia without atypia were euploid. No significant association was observed between p53 protein expression and ploidy in intraductal hyperplasia. The only case of intraductal hyperplasia without atypia positive for p53 was euploid, whereas the only p53-positive case of intraductal hyperplasia with atypia was aneuploid. Among the intraductal carcinomas, only the aneuploid cases showed positivity for p53, regardless of histological subtype. The results suggest that some of the changes observed in invasive breast carcinoma, such as p53 expression and aneuploidy, are already present in breast intraductal proliferation, especially in areas with atypia and in intraductal carcinoma. The expression of p53 in breast intraductal proliferation may reflect the acquisition of p53 gene mutations in cells unable adequately to repair DNA damage, with genomic instability which would lead to clonal expansion and putative evolution to invasive disease.

Expression of P-glycoprotein, multidrug resistance-associated protein, glutathione-S-transferase pi and p53 in canine transmissible venereal tumor

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The structural and functional relationship of the p53 tumor suppressor protein

The tumor-suppressing function of p53 can be affected in a variety of manners. Here, we describe a novel mechanism of transformation by mutant p53. Previously, it had been believed that mutant p53 molecules transform cells by oligomerizing with wild-type p53 and inactivating it. However, we demonstrated that there exists an additional mechanism of inactivation of p53 available to p53 mutants. It involves sequestration of cofactors necessary to p53, and subsequent interruption of its transactivation and tumor suppression functions. The p53 amino or carboxyl termini, known to interact with a large number of cellular factors, can affect wild-type p53 in this manner. Although they are unable to oligomerize with wild-type p53, they transform cells containing p53, and inhibit its transactivation ability. In addition, they interrupt growth suppression by p53, but not RB, confirming that they specifically affect p53 function, rather than having a general growth-stimulatory phenomenon. Also, we have cloned a p53 tumor mutation which results in expression of the amino terminus of p53. This provides a means to study the factor-sequestration transforming mechanism in vivo. Additionally, we found that the published sequence of the mdm2 gene is in error. mdm2 is a gene intimately involved with p53, blocking its ability to transform cells. Finally, previous data had established the influence of cell-cycle status on p53 function. In growth-arrested cells, wild-type p53 expressed by a transgene cannot activate transcription, but if these cells are forced to cycle by addition of cyclin E, p53 once again becomes functional. In this study, we extend these findings by examining only those cells successfully transfected, using fluorescence-activated cell sorting. Our results support the previous data, that cyclin E pushes growth-arrested cells back into the cell cycle. In summary, we have demonstrated the potential importance of cofactor association and protein modification to the abilities of p53 to cause transcription activation and repression, inhibition of DNA replication and induction of DNA repair, and initiation of cell-cycle arrest and apoptosis. Further elucidation of these processes and their roles in tumor suppression will prove fascinating indeed. ^

Non-p53 p53RE binding protein, a human transcription factor functionally analogous to P53

The transactivation activity of the p53 tumor suppressor protein is critical for regulating cell growth and apoptosis. We describe the identification of a transcription factor that is functionally similar to p53 and contains the same DNA binding and transcription activities specific for the p53 responsive DNA element (p53RE). This protein was highly purified through chromatography from HeLa cell extracts. The purified protein was able to bind specifically to the p53RE derived from a p21waf1 promoter and to stimulate p53RE-dependent transcription but not basal transcription in vitro. Its DNA-binding activity was inhibited by the wild type but not mutant p53RE-containing DNA oligomers. Also, this p53RE-binding activity was found in human p53 null Saos-2 osteosarcoma and H1299 small cell lung carcinoma cells. Interestingly, this activity exhibited a p53RE sequence preference that was distinct from the p53 protein. The activity is neither p53 nor p73, because anti-p53 or anti-73 antibodies were unable to detect this purified protein nor were the antibodies able to alter the p53-like activity, the p53RE-protein complex. These results demonstrate that, besides p73, an additional p53-like protein exists in cells, which is named NBP for non-p53, p53RE binding protein.

Interaction between replication protein A and p53 is disrupted after UV damage in a DNA repair-dependent manner

Replication protein A (RPA) is required for both DNA replication and nucleotide excision repair. Previous studies have shown that RPA interacts with the tumor suppressor p53. Herein, we have mapped a 20-amino acid region in the N-terminal part of p53 that is essential for its binding to RPA. This region is distinct from the minimal activation domain of p53 previously identified. We also demonstrate that UV radiation of cells greatly reduces the ability of RPA to bind to p53. Interestingly, damage-induced hyperphosphorylated RPA does not associate with p53. Furthermore, down-regulation of the RPA/p53 interaction is dependent upon the capability of cells to perform global genome repair. On the basis of these data, we propose that RPA may participate in the coordination of DNA repair with the p53-dependent checkpoint control by sensing UV damage and releasing p53 to activate its downstream targets.

Hepatitis B virus X protein and p53 tumor suppressor interactions in the modulation of apoptosis

We have reported previously that the hepatitis B virus oncoprotein, HBx, can bind to the C terminus of p53 and inhibit several critical p53-mediated cellular processes, including DNA sequence-specific binding, transcriptional transactivation, and apoptosis. Recognizing the importance of p53-mediated apoptosis for maintaining homeostasis and preventing neoplastic transformation, here we further examine the physical interaction between HBx and p53 as well as the functional consequences of this association. In vitro binding studies indicate that the ayw and adr viral subtypes of HBx bind similar amounts of glutathione S-transferase-p53 with the distal C terminus of HBx (from residues 111 to 154) being critical for this interaction. Using a microinjection technique, we show that this same C-terminal region of HBx is necessary for sequestering p53 in the cytoplasm and abrogating p53-mediated apoptosis. The transcriptional transactivation domain of HBx also maps to its C terminus; however, a comparison of the ability of full-length and truncated HBx protein to abrogate p53-induced apoptosis versus transactivate simian virus 40- or human nitric oxide synthase-2 promoter-driven reporter constructs indicates that these two functional properties are distinct and thus may contribute to hepatocarcinogenesis differently. Collectively, our data indicate that the distal C-terminal domain of HBx, independent of its transactivation activity, complexes with p53 in the cytoplasm, partially preventing its nuclear entry and ability to induce apoptosis. These pathobiological effects of HBx may contribute to the early stages of hepatocellular carcinogenesis.

p53CP, a putative p53 competing protein that specifically binds to the consensus p53 DNA binding sites: A third member of the p53 family?

p53 tumor suppressor protein negatively regulates cell growth, mainly through the transactivation of its downstream target genes. As a sequence-specific DNA binding transcription factor, p53 specifically binds to a 20-bp consensus motif 5′-PuPuPuC(A/T) (T/A)GPyPyPyPuPuPuC(A/T)(T/A)GPyPyPy-3′. We have now identified, partially purified, and characterized an additional ≈40-kDa nuclear protein, p53CP (p53 competing protein), that specifically binds to the consensus p53 binding sites found in several p53 downstream target genes, including Waf-1, Gadd45, Mdm2, Bax, and RGC. The minimal sequence requirement for binding is a 14-bp motif, 5′-CTTGCTTGAACAGG-3′ [5′-C(A/T)(T/A)GPyPyPyPuPuPuC(A/T)(T/A)G-3′], which includes the central nucleotides of the typical p53 binding site with one mismatch. p53CP and p53 (complexed with antibody) showed a similar binding specificity to Waf-1 site but differences in Gadd45 and T3SF binding. Like p53, p53CP also binds both double- and single-stranded DNA oligonucleotides. Important to note, cell cycle blockers and DNA damaging reagents, which induce p53 binding activity, were found to inhibit p53CP binding in p53-positive, but not in p53-negative, cells. This finding suggested a p53-dependent coordinate regulation of p53 and p53CP in response to external stimuli. p53CP therefore could be a third member of the p53 family, in addition to p53 and p73, a newly identified p53 homolog. p53CP, if sequestering p53 from its DNA binding sites through competitive binding, may provide a novel mechanism of p53 inactivation. Alternatively, p53CP may have p53-like functions by binding and transactivating p53 downstream target genes. Cloning of the p53CP gene ultimately will resolve this issue.

Cytoplasmically sequestered wild-type p53 protein in neuroblastoma is relocated to the nucleus by a C-terminal peptide

Cytoplasmic sequestration of wild-type p53 protein occurs in a subset of primary human tumors including breast cancer, colon cancer, and neuroblastoma (NB). The sequestered p53 localizes to punctate cytoplasmic structures that represent large protein aggregates. One functional consequence of this blocked nuclear access is impairment of the p53-mediated G1 checkpoint after DNA damage. Here we show that cytoplasmic p53 from NB cells is incompetent for specific DNA binding, probably due to its sequestration. Importantly, the C-terminal domain of sequestered p53 is masked, as indicated by the failure of a C-terminally directed antibody to detect p53 in these structures. To determine (i) which domain of p53 is involved in the aggregation and (ii) whether this phenotype is potentially reversible, we generated stable NB sublines that coexpress the soluble C-terminal mouse p53 peptide DD1 (amino acids 302–390). A dramatic phenotypic reversion occurred in five of five lines. The presence of DD1 blocked the sequestration of wild-type p53 and relocated it to the nucleus, where it accumulated. The nuclear translocation is due to shuttling of wild-type p53 by heteroligomerization to DD1, as shown by coimmunoprecipitation. As expected, the nuclear heterocomplexes were functionally inactive, since DD1 is a dominant negative inhibitor of wild-type p53. In summary, we show that nuclear access of p53 can be restored in NB cells.

The catalytic subunit of DNA-dependent protein kinase selectively regulates p53-dependent apoptosis but not cell-cycle arrest

DNA damage induced by ionizing radiation (IR) activates p53, leading to the regulation of downstream pathways that control cell-cycle progression and apoptosis. However, the mechanisms for the IR-induced p53 activation and the differential activation of pathways downstream of p53 are unclear. Here we provide evidence that the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) serves as an upstream effector for p53 activation in response to IR, linking DNA damage to apoptosis. DNA-PKcs knockout (DNA-PKcs−/−) mice were exposed to whole-body IR, and the cell-cycle and apoptotic responses were examined in their thymuses. Our data show that IR induction of apoptosis and Bax expression, both mediated via p53, was significantly suppressed in the thymocytes of DNA-PKcs−/− mice. In contrast, IR-induced cell-cycle arrest and p21 expression were normal. Thus, DNA-PKcs deficiency selectively disrupts p53-dependent apoptosis but not cell-cycle arrest. We also confirmed previous findings that p21 induction was attenuated and cell-cycle arrest was defective in the thymoctyes of whole body-irradiated Atm−/− mice, but the apoptotic response was unperturbed. Taken together, our results support a model in which the upstream effectors DNA-PKcs and Atm selectively activate p53 to differentially regulate cell-cycle and apoptotic responses. Whereas Atm selects for cell-cycle arrest but not apoptosis, DNA-PKcs selects for apoptosis but not cell-cycle arrest.

Sustained activation of Ras/Raf/mitogen-activated protein kinase cascade by the tumor suppressor p53

The p53 tumor suppressor gene can inhibit proliferation transiently, induce permanent cell-cycle arrest/senescence, or cause apoptosis depending on the cellular context. The mitogen-activated protein kinase (MAPK) cascade is known to play a crucial role in cell proliferation and differentiation. Moreover, the duration and intensity of MAPK activation can profoundly influence the biological response observed. We demonstrated that a sustained activation of MAPK cascade could be induced by wild-type p53 expression but not by p21Waf1/Cip1. Furthermore, exposure of normal cells to DNA-damaging agents induced MAPK activation in a p53-dependent manner. Tumor-derived p53 mutants defective in DNA binding failed to activate MAPK, implying that p53 transcriptional activity is essential for this function. Finally, activation of MAPK by p53 was inhibited by expression of dominant-negative Ras (N17Ras) and Raf1 mutants, indicating that MAPK activation by p53 is mediated at a level upstream of Ras. All of these findings establish a biochemical link between p53 signaling and the Ras/Raf/MAPK cascade.

A meanfield approach to the thermodynamics of a protein-solvent system with application to the oligomerization of the tumor suppressor p53

The thermodynamic stability and oligomerization status of the tumor suppressor p53 tetramerization domain have been studied experimentally and theoretically. A series of hydrophilic mutations at Met-340 and Leu-344 of human p53 were designed to disrupt the hydrophobic dimer–dimer interface of the tetrameric oligomerization domain of p53 (residues 325–355). Meanfield calculations of the free energy of the solvated mutants as a function of interdimer distance were compared with experimental data on the thermal stability and oligomeric state (tetramer, dimer, or equilibrium mixture of both) of each mutant. The calculations predicted a decreasing stability and oligomeric state for the following amino acids at residue 340: Met (tetramer) > Ser Asp, His, Gln, > Glu, Lys (dimer), whereas the experimental results showed the following order: Met (tetramer) > Ser > Gln > His, Lys > Asp, Glu (dimers). For residue 344, the calculated trend was Leu (tetramer) > Ala > Arg, Gln, Lys (dimer), and the experimental trend was Leu (tetramer) > Ala, Arg, Gln, Lys (dimer). The discrepancy for the lysine side chain at residue 340 is attributed to the dual nature of lysine, both hydrophobic and charged. The incorrect prediction of stability of the mutant with Asp at residue 340 is attributed to the fact that within the meanfield approach, we use the wild-type backbone configuration for all mutants, but low melting temperatures suggest a softening of the α-helices at the dimer–dimer interface. Overall, this initial application of meanfield theory toward a protein-solvent system is encouraging for the application of the theoretical model to more complex systems.

Ultraviolet radiation, but not γ radiation or etoposide-induced DNA damage, results in the phosphorylation of the murine p53 protein at serine-389

Polyclonal antibodies were produced and purified that selectively react with a p53 epitope containing the murine phosphoserine-389 or the human phosphoserine-392 residue, but not the unphosphorylated epitope. These antibodies, termed alpha-392, were employed to demonstrate that the phosphorylation of this serine-389 residue in the p53 protein occurs in vivo in response to ultraviolet radiation of cells containing the p53 protein. After ultraviolet radiation of cells in culture, p53 levels increase and concomitantly serine-389 is phosphorylated in these cells. By contrast, the serine-389 phosphorylation of the p53 protein was not detected by these antibodies in the increased levels of p53 protein made in response to γ radiation or the treatment of cells with etoposide. These results demonstrate an ultraviolet responsive and specific phosphorylation site at serine-389 of the mouse or serine-392 of the human p53 protein. Previous studies have demonstrated that this phosphorylation of p53 activates the protein for specific DNA binding. This study demonstrates in vivo a unique phosphorylation site in the p53 protein that responds to a specific type of DNA damage.

Adenovirus E4orf6 oncoprotein modulates the function of the p53-related protein, p73

Recently, several proteins have been identified that are related in their sequence to the p53 tumor-suppressor protein. One of these proteins, which is termed p73, exhibits sequence homology to the p53 transcriptional activation, DNA binding, and oligomerization domains. The adenovirus E1B 55-kDa protein, the adenovirus E4orf6 protein, and SV40 T antigen each can bind to p53 and inhibit p53 function. Here we demonstrate that the adenovirus E4orf6 protein, but not the E1B 55-kDa protein or T antigen, interacts with p73. The E4orf6 protein inhibits p73-mediated transcriptional activation and cell killing in a manner similar to its effect on p53. Thus, only a subset of viral oncoproteins that antagonize p53 function also interacts with the related p73 protein.