Cahya Yustisia Hasan, Prihartiningsih , dan Rahardjo

ABSTRAK Ameloblastoma merupakan tumor jinak odontogenik yang paling sering ditemukan secara klinis. Tumor ini secara histopatologis memperlihatkan tanda-tanda sebagai tumor jinak, tetapi secara klinis bersifat agresif, destruktif dan invasif lokal, mempunyai tingkat rekurensi yang tinggi, dan pernah dilaporkan adanya metastase. Gen p53 merupakan gen supresor tumor yang paling sering berubah pada tumor manusia, dan berperan pada mekanisme molekuler tumorigenesis. Adanya ekspresi berlebih p53 menggambarkan bahwa p53 berperan penting pada aktivitas proliferasi sel tumor. Tujuan penelitian ini adalah untuk mengetahui karakteristik ekspresi p53 pada ameloblastoma dan mencari korelasi antara imunoekspresi p53 dengan berbagai tipe histopatologis ameloblastoma. Penelitian ini dilakukan secara retrospektif pada 30 preparat ameloblastoma yang dipulas menggunakan pulasan imunohistokimia untuk melihat imunoekspresi p53 pada berbagai tipe histopatologis ameloblastoma. Analisa statistik yang digunakan Kruskal Wallis dan korelasi Spearman. Hasil yang diperoleh menunjukkan bahwa terdapat perbedaan yang bermakna (

Reactivation of wild-type and mutant p53 by tryptophanolderived oxazoloisoindolinone SLMP53-1:a novel anticancer small-molecule

Restoration of the p53 pathway, namely by reactivation of mutant (mut) p53, represents a valuable anticancer strategy. Herein, we report the identification of the enantiopure tryptophanol-derived oxazoloisoindolinone SLMP53-1 as a novel reactivator of wild-type (wt) and mut p53, using a yeast-based screening strategy. SLMP53-1 has a p53-dependent anti-proliferative activity in human wt and mut p53R280K-expressing tumor cells. Additionally, SLMP53-1 enhances p53 transcriptional activity and restores wt-like DNA binding ability to mut p53R280K. In wt/mut p53-expressing tumor cells, SLMP53-1 triggers p53 transcription-dependent and mitochondrial apoptotic pathways involving BAX, and wt/mut p53 mitochondrial translocation. SLMP53-1 inhibits the migration of wt/mut p53-expressing tumor cells, and it shows promising p53-dependent synergistic effects with conventional chemotherapeutics. In xenograft mice models, SLMP53-1 inhibits the growth of wt/mut p53-expressing tumors, but not of p53-null tumors, without apparent toxicity. Collectively, besides the potential use of SLMP53-1 as anticancer drug, the tryptophanol-derived oxazoloisoindolinone scaffold represents a promissing starting point for the development of effective p53-reactivating drugs

Cyclin B1/Cdk1 phosphorylation of mitochondrial p53 induces anti-apoptotic response.

The pro-apoptotic function of p53 has been well defined in preventing genomic instability and cell transformation. However, the intriguing fact that p53 contributes to a pro-survival advantage of tumor cells under DNA damage conditions raises a critical question in radiation therapy for the 50% human cancers with intact p53 function. Herein, we reveal an anti-apoptotic role of mitochondrial p53 regulated by the cell cycle complex cyclin B1/Cdk1 in irradiated human colon cancer HCT116 cells with p53(+/+) status. Steady-state levels of p53 and cyclin B1/Cdk1 were identified in the mitochondria of many human and mouse cells, and their mitochondrial influx was significantly enhanced by radiation. The mitochondrial kinase activity of cyclin B1/Cdk1 was found to specifically phosphorylate p53 at Ser-315 residue, leading to enhanced mitochondrial ATP production and reduced mitochondrial apoptosis. The improved mitochondrial function can be blocked by transfection of mutant p53 Ser-315-Ala, or by siRNA knockdown of cyclin B1 and Cdk1 genes. Enforced translocation of cyclin B1 and Cdk1 into mitochondria with a mitochondrial-targeting-peptide increased levels of Ser-315 phosphorylation on mitochondrial p53, improved ATP production and decreased apoptosis by sequestering p53 from binding to Bcl-2 and Bcl-xL. Furthermore, reconstitution of wild-type p53 in p53-deficient HCT116 p53(-/-) cells resulted in an increased mitochondrial ATP production and suppression of apoptosis. Such phenomena were absent in the p53-deficient HCT116 p53(-/-) cells reconstituted with the mutant p53. These results demonstrate a unique anti-apoptotic function of mitochondrial p53 regulated by cyclin B1/Cdk1-mediated Ser-315 phosphorylation in p53-wild-type tumor cells, which may provide insights for improving the efficacy of anti-cancer therapy, especially for tumors that retain p53

Tumor suppressor p53 binds with high affinity to CTG-CAG trinucleotide repeats and induces topological alterations in mismatched duplexes

DNA binding is central to the ability of p53 to function as a tumor suppressor. In line with the remarkable functional versatility of p53, which can act on DNA as a transcription, repair, recombination, replication, and chromatin accessibility factor, the modes of p53 interaction with DNA are also versatile. One feature common to all modes of p53-DNA interaction is the extraordinary sensitivity of p53 to the topology of its target DNA. Whereas the strong impact of DNA topology has been demonstrated for p53 binding to sequence-specific sites or to DNA lesions, the possibility that DNA structure-dependent recognition may underlie p53 interaction with other types of DNA has not been addressed until now. We demonstrate for the first time that conformationally flexible CTG·CAG trinucleotide repeats comprise a novel class of p53-binding sites targeted by p53 in a DNA structure-dependent mode in vitro and in vivo. Our major finding is that p53 binds to CTG·CAG tracts by different modes depending on the conformation of DNA. Although p53 binds preferentially to hairpins formed by either CTG or CAG strands, it can also bind to linear forms of CTG·CAG tracts such as canonic B DNA or mismatched duplex. Intriguingly, by binding to a mismatched duplex p53 can induce further topological alterations in DNA, indicating that p53 may act as a DNA topology-modulating factor

Structural analysis of MDM2 RING separates degradation from regulation of p53 transcription activity

MDM2–MDMX complexes bind the p53 tumor-suppressor protein, inhibiting p53's transcriptional activity and targeting p53 for proteasomal degradation. Inhibitors that disrupt binding between p53 and MDM2 efficiently activate a p53 response, but their use in the treatment of cancers that retain wild-type p53 may be limited by on-target toxicities due to p53 activation in normal tissue. Guided by a novel crystal structure of the MDM2–MDMX–E2(UbcH5B)–ubiquitin complex, we designed MDM2 mutants that prevent E2–ubiquitin binding without altering the RING-domain structure. These mutants lack MDM2's E3 activity but retain the ability to limit p53′s transcriptional activity and allow cell proliferation. Cells expressing these mutants respond more quickly to cellular stress than cells expressing wild-type MDM2, but basal p53 control is maintained. Targeting the MDM2 E3-ligase activity could therefore widen the therapeutic window of p53 activation in tumors

Defeating Cytoplasmic Sequestration of p53 in Human Breast Cancer Cells; Is Mortalin Involved?

Cytoplasmic sequestration of p53, possibly caused by p53 interacting with mortalin, can prevent p53 from functioning in DNA repair and apoptosis, causing aberrant growth. This project treated SKBR3 breast cancer cells with MKT-077, a dye that is a competitive binder to mortalin to see if it would result in the release of p53 from the cytoplasm and restoration of p53 function. Treatment resulted in partial translocation of a protein suspected to be p53 to the nucleus and apoptosis initiated at the mitochondria

Mdm2 Is Required for Survival and Growth of p53-Deficient Cancer Cells.

p53 deletion prevents the embryonic lethality of normal tissues lacking Mdm2, suggesting that cells can survive without Mdm2 if p53 is also absent. Here we report evidence challenging this view, with implications for therapeutically targeting Mdm2. Deletion of Mdm2 in T-cell lymphomas or sarcomas lacking p53 induced apoptosis and G2 cell-cycle arrest, prolonging survival of mice with these tumors. p53-/- fibroblasts showed similar results, indicating that the effects of Mdm2 loss extend to pre-malignant cells. Mdm2 deletion in p53-/- cells upregulated p53 transcriptional target genes that induce apoptosis and cell-cycle arrest. Mdm2 deletion also increased levels of p73, a p53 family member. RNAi-mediated attenuation of p73 rescued the transcriptional and biological effects of Mdm2 loss, indicating that p73 mediates the consequences of Mdm2 deletion. In addition, Mdm2 deletion differed from blocking Mdm2 interaction with p53 family members, as Nutlin-3 induced G1 arrest but did not activate apoptosis in p53-/- sarcoma cells. Our results indicate that, in contrast to current dogma, Mdm2 expression is required for cell survival even in the absence of p53. Moreover, our results suggest that p73 compensates for loss of p53 and that targeting Mdm2 in p53-deficient cancers has therapeutic potential. ©2017 AACR

The degradation of p53 and its major E3 ligase Mdm2 is differentially dependent on the proteasomal ubiquitin receptor S5a.

p53 and its major E3 ligase Mdm2 are both ubiquitinated and targeted to the proteasome for degradation. Despite the importance of this in regulating the p53 pathway, little is known about the mechanisms of proteasomal recognition of ubiquitinated p53 and Mdm2. In this study, we show that knockdown of the proteasomal ubiquitin receptor S5a/PSMD4/Rpn10 inhibits p53 protein degradation and results in the accumulation of ubiquitinated p53. Overexpression of a dominant-negative deletion of S5a lacking its ubiquitin-interacting motifs (UIM)s, but which can be incorporated into the proteasome, also causes the stabilization of p53. Furthermore, small-interferring RNA (siRNA) rescue experiments confirm that the UIMs of S5a are required for the maintenance of low p53 levels. These observations indicate that S5a participates in the recognition of ubiquitinated p53 by the proteasome. In contrast, targeting S5a has no effect on the rate of degradation of Mdm2, indicating that proteasomal recognition of Mdm2 can be mediated by an S5a-independent pathway. S5a knockdown results in an increase in the transcriptional activity of p53. The selective stabilization of p53 and not Mdm2 provides a mechanism for p53 activation. Depletion of S5a causes a p53-dependent decrease in cell proliferation, demonstrating that p53 can have a dominant role in the response to targeting S5a. This study provides evidence for alternative pathways of proteasomal recognition of p53 and Mdm2. Differences in recognition by the proteasome could provide a means to modulate the relative stability of p53 and Mdm2 in response to cellular signals. In addition, they could be exploited for p53-activating therapies. This work shows that the degradation of proteins by the proteasome can be selectively dependent on S5a in human cells, and that this selectivity can extend to an E3 ubiquitin ligase and its substrate

Mutant p53 establishes targetable tumor dependency by promoting unscheduled replication

Gain-of-function (GOF) p53 mutations are observed frequently in most intractable human cancers and establish dependency for tumor maintenance and progression. While some of the genes induced by GOF p53 have been implicated in more rapid cell proliferation compared with p53-null cancer cells, the mechanism for dependency of tumor growth on mutant p53 is unknown. This report reveals a therapeutically targetable mechanism for GOF p53 dependency. We have shown that GOF p53 increases DNA replication origin firing, stabilizes replication forks, and promotes micronuclei formation, thus facilitating the proliferation of cells with genomic abnormalities. In contrast, absence or depletion of GOF p53 leads to decreased origin firing and a higher frequency of fork collapse in isogenic cells, explaining their poorer proliferation rate. Following genome-wide analyses utilizing ChIP-Seq and RNA-Seq, GOF p53–induced origin firing, micronuclei formation, and fork protection were traced to the ability of GOF p53 to transactivate cyclin A and CHK1. Highlighting the therapeutic potential of CHK1’s role in GOF p53 dependency, experiments in cell culture and mouse xenografts demonstrated that inhibition of CHK1 selectively blocked proliferation of cells and tumors expressing GOF p53. Our data suggest the possibility that checkpoint inhibitors could efficiently and selectively target cancers expressing GOF p53 alleles