S100 calcium-binding proteins such as S100B are elevated in primary malignant melanoma and are used as markers for this and numerous other cancers. Wild-type p53 protein levels are relatively low in these cancer cells (i.e. when compared with cells without S100B) but are elevated when RNA antisense to S100B is introduced. This result implicates S100B in the down-regulation of p53 and is consistent with the large decreases in p53 protein levels observed previously in transient cotransfections of p53 and S100B (Lin, J., Blake, M., Tang Down-regulation of p53 in primary malignant melanoma cells is likely the result of a direct interaction with S100B, which was observed by co-immunoprecipitation experiments. Furthermore, p53 binds regions of the S100B promoter, one of which matches the 20-nucleotide p53-binding consensus DNA sequence perfectly. Therefore, when p53 levels increase, it contributes to its own demise by up-regulating the transcription of S100B as part of a negative feedback loop. This is analogous to what is found for another protein that down-regulates p53, namely hdm2 (human double mutant 2).

The levels of S100 Ca 2؉ -binding proteins correlate with the progression of certain tumors, but their role, if any, in carcinogenesis is still poorly understood. S100B protein associates with both the p53 oligomerization domain (residues 325-355) and the extreme C terminus of the tumor suppressor p53 (residues 367-392). Consequently, S100B inhibits p53 tetramer formation and p53 phosphorylation mediated by protein kinase C, on p53 C-terminal end. In this report, we show that the S100B protein decreases p53 DNA binding and transcriptional activity. The effect of S100B is reflected in vivo by a reduced accumulation of p53, p21, and MDM2 protein levels in co-transfection assays and in response to bleomycin. The S100B can still interact with p53 in the absence of p53 extreme C-terminal end and reduce the expression of p53 downstream effector genes. These data indicate that S100B does not require p53 extreme C-terminal end to inhibit p53 activity. Collectively, these findings imply that elevated levels of S100B in tumors such as astrocytomas and gliomas could inhibit p53 functions and contribute to cancer progression.The S100 proteins are dimeric Ca 2ϩ -binding proteins (ϳ10 kDa/subunit) initially characterized by their solubility in 100% ammonium sulfate (S100) (1). Deregulation of Ca 2ϩ homeostasis has been associated with different pathologies including neurodegenerative disorders, hypertension, and cancer (2). The S100 proteins are overexpressed in many tumor cells and have been used as a marker for the classification of tumors (3). A possible role for the S100 proteins in carcinogenesis has often been suspected, but their specific involvement is still ill defined. Evidence has indicated that S100B interacts with the tumor suppressor p53 (4). p53 plays a pivotal role in the maintenance and regulation of normal cellular functions, and its inactivation can affect cell cycle checkpoints, apoptosis, gene amplification, centrosome duplication, and ploidy (5). p53 interacts with a number of proteins to mediate its pleiotropic effects. The interactions of p53 with S100 calcium-binding proteins are of particular interest because like p53, the S100 proteins affect cell cycle progression, are overexpressed in numerous tumor cells, and are associated with tumor progression (2). The S100B protein interacts with the p53 C-terminal end and inhibits both,p53 tetramerization and phosphorylation by PKC 1 (4). Because these two events are known to be important for p53 activation (6), we wanted to determine the effect of S100B on p53 transcriptional activity in vivo. Our data indicate that overexpression of S100B can reduce p53 transcriptional activity by more than 50%. This effect is correlated with a decrease in p53 DNA binding activity and a reduction in the accumulation of MDM2 and p21 protein levels. Interaction of the S100B protein with p53 may thus impede p53 cellular functions. Such an interaction could especially be detrimental in astrocytomas and gliomas, where S100B levels are significantly elevated (7). EXPERIMENTAL PR...

S100B is an EF-hand containing calcium-binding protein of the S100 protein family that exerts its biological effect by binding and affecting various target proteins. A consensus sequence for S100B target proteins was published as (K/R)(L/I)xWxxIL and matches a region in the actin capping protein CapZ (V.V. Ivanenkov, G.A. Jamieson, Jr., E. Gruenstein, R.V. Dimlich, Characterization of S-100b binding epitopes. Identification of a novel target, the actin capping protein, CapZ, J. Biol. Chem. 270 (1995) 14651-14658). Several additional S100B targets are known including p53, a nuclear Dbf2 related (NDR) kinase, the RAGE receptor, neuromodulin, protein kinase C, and others. Examining the binding sites of such targets and new protein sequence searches provided additional potential target proteins for S100B including Hdm2 and Hdm4, which were both found to bind S100B in a calcium-dependent manner. The interaction between S100B and the Hdm2 and/or the Hdm4 proteins may be important physiologically in light of evidence that like Hdm2, S100B also contributes to lowering protein levels of the tumor suppressor protein, p53. For the S100B-p53 interaction, it was found that phosphorylation of specific serine and/or threonine residues reduces the affinity of the S100B-p53 interaction by as much as an order of magnitude, and is important for protecting p53 from S100B-dependent down-regulation, a scenario that is similar to what is found for the Hdm2-p53 complex.

The S100B-p53 protein complex was discovered in C8146A malignant melanoma, but the consequences of this interaction required further study. When S100B expression was inhibited in C8146As by siRNA (siRNA S100B ), wt p53 mRNA levels were unchanged, but p53 protein, phosphorylated p53, and p53 gene products (i.e. p21 and PIDD) were increased. siRNA S100B transfections also restored p53-dependent apoptosis in C8146As as judged by poly(ADP-ribose) polymerase cleavage, DNA ladder formation, caspase 3 and 8 activation, and aggregation of the Fas death receptor (؉UV); whereas, siRNA S100B had no effect in SK-MEL-28 cells containing elevated S100B and inactive p53 (p53R145L mutant). siRNA S100B -mediated apoptosis was independent of the mitochondria, because no changes were observed in mitochondrial membrane potential, cytochrome c release, caspase 9 activation, or ratios of pro-and anti-apoptotic proteins (BAX, Bcl-2, and Bcl-X L ). As expected, cells lacking S100B (LOX-IM VI) were not affected by siRNA S100B , and introduction of S100B reduced their UV-induced apoptosis activity by 7-fold, further demonstrating that S100B inhibits apoptosis activities in p53-containing cells. In other wild-type p53 cells (i.e. C8146A, UACC-2571, and UACC-62), S100B was found to contribute to cell survival after UV treatment, and for C8146As, the decrease in survival after siRNA S100B transfection (؉UV) could be reversed by the p53 inhibitor, pifithrin-␣. In summary, reducing S100B expression with siRNA was sufficient to activate p53, its transcriptional activation activities, and p53-dependent apoptosis pathway(s) in melanoma involving the Fas death receptor and perhaps PIDD. Thus, a well known marker for malignant melanoma, S100B, likely contributes to cancer progression by down-regulating the tumor suppressor protein, p53.In addition to regulating numerous genes and pathways involved in cell cycle control (1), the tumor suppressor protein, p53, is an important component for inducing apoptosis (2-4). The p53 protein activates the transcription of pro-apoptotic factors (BAX, Bak, Fas/APO-1, PIDD, etc.) as well as suppresses the transcription of anti-apoptotic genes (Bcl-2, Bcl-X L , etc.) (5, 6). In addition, p53 itself up-regulates apoptosis, without transcription activation, by directly localizing to mitochondria following DNA damage and interacting with anti-apoptotic proteins such as Bcl-X L to free pro-apoptotic proteins like BAX (2, 3, 5, 7). Under stress, the p53 protein can also contribute to apoptosis by facilitating the transport of death receptors such as Fas/APO-1 and/or Killer/DR5 from cytoplasmic stores to the cell surface as required for programmed cell death (5, 8). Although, it is now clear that p53-dependent pathways of apoptosis are numerous and are regulated in a cell-type and signalspecific manner (5).Apoptosis is initiated by a variety of stimuli, including withdrawal of growth factors, activation of specific receptors, such as Fas antigen and TNF receptor, and/or by exposure to UV radiation, ␥ irradiation, DNA-da...

Objective: Nasopharyngeal carcinoma (NPC) is one of the dominant cancers in South China and Taiwan. Although NPC is highly chemosensitive, the use of chemotherapy for treating patients with recurrent or metastatic NPC has not been very successful. The emergence of drug resistance may be one of the major reasons. However, the mechanisms of drug resistance of NPC have never been addressed before. In this study, we sought to clarify the role of classical drug resistance markers in predicting the chemosensitivity and the prognosis of patients with advanced NPC. Methods: In a cohort of 202 consecutive patients diagnosed at the Department of Pathology of the National Taiwan University Hospital, 44 patients with adequately preserved pretreatment tumor tissues and complete clinical information regarding the details of chemotherapy and tumor response were identified. The expression of multidrug resistance (MDR1), glutathione-S-transferase-π (GSTπ), and p53 were determined by immunohistochemistry. Tumor response to chemotherapy and survival of the patients were the endpoints of this analysis. Results: Thirty-four patients received cisplatin-based regimens, and 28 of them were enrolled in a prospective trial using a doxorubicin-containing regimen. The overall response rate was 70%. Expression of MDR1 was seen in only 5 cases (11%) and was associated with a significantly worse overall survival, yet did not appear to predict chemoresistance to the doxorubicin-containing regimen. Overexpression of p53 was seen in 22 patients, and surprisingly, was correlated with chemoresponse and a trend towards better survival. GSTπ expression was demonstrated in 13 cases (30%) and was not correlated with chemoresistance to cisplatin-containing regimens and overall survival. Conclusion: In this relatively small cohort, positive MDR1 immunostaining predicted a poor overall survival for recurrent or metastatic NPC patients receiving chemotherapy. Overexpression of p53 by immunohistochemical staining, however, was associated with a better response rate to systemic chemotherapy and a trend towards better survival.