Mechanism Of P53 Inactivation Research Articles

P53 Isoforms and Their Implications in Cancer.

In this review we focus on the major isoforms of the tumor-suppressor protein p53, dysfunction of which often leads to cancer. Mutations of the TP53 gene, particularly in the DNA binding domain, have been regarded as the main cause for p53 inactivation. However, recent reports demonstrating abundance of p53 isoforms, especially the N-terminally truncated ones, in the cancerous tissues suggest their involvement in carcinogenesis. These isoforms are ∆40p53, ∆133p53, and ∆160p53 (the names indicate their respective N-terminal truncation). Due to the lack of structural and functional characterizations the modes of action of the p53 isoforms are still unclear. Owing to the deletions in the functional domains, these isoforms can either be defective in DNA binding or more susceptive to altered ‘responsive elements’ than p53. Furthermore, they may exert a ‘dominant negative effect’ or induce more aggressive cancer by the ‘gain of function’. One possible mechanism of p53 inactivation can be through tetramerization with the ∆133p53 and ∆160p53 isoforms—both lacking part of the DNA binding domain. A recent report and unpublished data from our laboratory also suggest that these isoforms may inactivate p53 by fast aggregation—possibly due to ectopic overexpression. We further discuss the evolutionary significance of the p53 isoforms.

Loss of LZAP inactivates p53 and regulates sensitivity of cells to DNA damage in a p53-dependent manner

Chemotherapy and radiation, the two most common cancer therapies, exert their anticancer effects by causing damage to cellular DNA. However, systemic treatment damages DNA not only in cancer, but also in healthy cells, resulting in the progression of serious side effects and limiting efficacy of the treatment. Interestingly, in response to DNA damage, p53 seems to play an opposite role in normal and in the majority of cancer cells—wild-type p53 mediates apoptosis in healthy tissues, attributing to the side effects, whereas mutant p53 often is responsible for acquired cancer resistance to the treatment. Here, we show that leucine zipper-containing ARF-binding protein (LZAP) binds and stabilizes p53. LZAP depletion eliminates p53 protein independently of its mutation status, subsequently protecting wild-type p53 cells from DNA damage-induced cell death, while rendering cells expressing mutant p53 more sensitive to the treatment. In human non-small-cell lung cancer, LZAP levels correlated with p53 levels, suggesting that loss of LZAP may represent a novel mechanism of p53 inactivation in human cancer. Our studies establish LZAP as a p53 regulator and p53-dependent determinative of cell fate in response to DNA damaging treatment.

Targeting Aggregation of Wilde-Type p53 and Mutant p53 with ReACp53 As a Novel Therapeutic Concept for AML

Background: The tumor suppressor p53 is a master regulator of apoptosis, autophagy, cell cycle, and senescence. It is inactivated via mutation in approximately 50% of solid tumors, but only in 15% of hematopoietic malignancies including acute myeloid leukemia (AML). A recently proposed mechanism has linked loss of p53 function with its amyloid aggregation. Conceptually, certain p53 mutations can favor partial unfolding of the protein and expose a natively buried aggregation-prone segment. This can result in amyloidogenic aggregation and prevent p53 transcriptional activity and anti-tumor functions. The cell-permeable peptide ReACp53, has been recently developed to block p53 aggregation and restore its transcriptional function in the nucleus as well as its ubiquitination by MDM2. ReACp53 showed significant cytotoxicity in ovarian cancer but no toxicity to normal hematopoietic cells in animal experiment. We sought to determine the anti-tumor activity of ReACp53 in hematopoietic malignancies.Results: We examined the p53 status in 23 malignant hematopoietic cell lines by PCR, Sanger sequencing, and immunoblotting. Two cell lines were null for p53 expression, one harbored frame shift mutations, 11 cell lines expressed various missense p53 mutations, one cell line had an in frame deletion of p53, and eight cell lines expressed wild-type p53. Additionally, immunofluorescence staining (IF) with the conformation-specific PAb240 antibody revealed high levels of cytoplasmic, partially unfolded p53 in the cells expressing mutant p53. In p53 wild-type cells, p53 protein was mainly localized in the nucleus and was negative for PAb240. The p53 null and frame shift-mutant cells showed no p53 expression.All the cells were treated short-term with various concentrations of ReACp53, or a scrambled peptide, and assessed for apoptosis by flow cytometry. We found that ReACp53 was cytotoxic not only to the p53-mutant cells, but also to the wild-type p53 lines. In fact, all p53 wild type AML cell lines were highly sensitive. The p53 negative cell lines were seemingly resistant to short-term exposure to ReACp53.DeltaNp73, an isoform of p73 that antagonizes p53 and TAp73, is expressed in most AML cells and also has a similar aggregation-prone segment. We examined the levels of DeltaNp73 and total p73 in 12 AML cell lines by PCR, immunoblotting, and IF. Both proteins were overexpressed in all five wild-type p53 cell lines, and DeltaNp73 was predominately localized in the cytoplasm of these cells. After short-term treatment with ReACp53, DeltaNp73 expression and localization didn't change in wild type p53 AML cells. Over-expressing DeltaNp73 in HEK293T cells enhanced their level of Thioflavin T staining indicating amyloid aggregation of the protein. Compared to controls, the DeltaNp73 overexpressing HEK293T cells were more prone to apoptosis following ReACp53 treatment. Absent of transactivation domain, DeltaNp73 is not expected to be restored to function like TAp73. Mutant p53 is known to cross-aggregate p73 and p63 because of their highly similar aggregation-prone segments, therefore, we hypothesize that DeltaNp73 cross-aggregated p53 and p73 and ReACp53 inhibited the aggregation as to restore p53 and TAp73 function and exposure to MDM2.We chose two wild-type p53 AML cell lines, OCI-AML3 and MOLM-14, which express MDM2 and are sensitive to the MDM2 inhibitor DS3032b. After short-term treatment with ReACp53, p53 and p73 (also a MDM2 target) expression decreased significantly in both cells. We tested the anti-leukemia efficacy of the DS3032b and ReACp53 combinatorial treatment in these cells and found that DS3032b synergized with ReACp53 to efficiently kill the cells compared to the cytotoxic activity of DS3032b or ReACp53 treatment alone.Conclusions: We demonstrate a new mechanism of DeltaNp73 inhibition of wild-type p53 and TAp73 mediated by induction of amyloid aggregation. ReACp53 showed apoptogenic efficacy in malignant hematopoietic cells, both in cells expressing wild-type p53 as well as mutant p53. In the wild-type AML cells where p73 and DeltaNp73 were overexpressed, sensitivity to ReACp53 increased. ReACp53 also exhibited synergistic activity when combined with the MDM2 inhibitor DS3032b in wild-type p53 cells. Together, our data suggest a novel mechanism of p53 inactivation by amyloid formation, that can be corrected in acute myeloid leukemia carrying either wild-type or mutant p53. DisclosuresNo relevant conflicts of interest to declare.

Abstract 3693: Oligomerization status of p53 serves as an indicator of sensistivity of p53 wildtype tumors to the therapeutic combination of DNA damaging agent and checkpoint inhibitor

Abstract It is well established that cell cycle arrest in response to treatment with DNA damaging agents can be abrogated in p53-defective cells by treatment with the Chk1 inhibitors, but our more recent studies have shown that some p53 wild-type tumors are also sensitive to checkpoint abrogation. Two possible explanations are cytoplasmic sequestration of p53 or a defect in p53 oligomerization. In this study, we investigated the DNA damage response and UCN-01 sensitivity of two p53 wildtype neuroblastoma cell lines: SK-N-SH, and SH-SY5Y. In order to determine the responses of these cells to DNA damage, the cells were treated with concentrations of SN38 ranging from 0-30 ng/ml. Both lines arrested in G2, S, or G1 depending upon SN38 concentration and displayed an increase in p53 levels with increasing SN38. Additionally, p53 was phosphorylated on serine 15 and serine 20 following SN38 treatment in all three cell lines, suggesting that p53 is active. In order to determine whether these cells are susceptible to UCN-01-mediated abrogation of cell cycle arrest, cell were treated with 3 ng/ml SN38 for 24 hours, followed by 25 nM UCN-01 for 6 and 24 hours. The SK-N-SH showed now sensitivity to UCN-01 treatment whereas the SH-SY5Y abrogated S arrest within 6 hours and abrogated G2 arrest within 24 hours. We also analyzed the oligomerization status of p53 using glutaraldehyde crosslinking. The SK-N-SH cells possessed levels of p53 dimers and tetramers similar to what has previously been reported in p53 wildtype MCF10A cells. The SH-SY5Y, however, had extremely low levels of dimers and tetramers. Consistent with this, only SK-N-SH showed activation of p21waf1 and repression of cyclin B in response to SN38 treatment. Previous studies have reported cytoplasmic sequestration as a mechanism of p53 inactivation in p53 wildtype neuroblastomas. In order to determine the sub-cellular distribution of p53, we prepared nuclear and cytoplasmic extracts. Both the SK-N-SH and SH-SY5Y had primarily nuclear p53. The results of this study suggest that oligomerization status may serve as an indicator of sensitivity of p53 wildtype tumors to the therapeutic combination of DNA damage agent and checkpoint inhibitor. Citation Format: Pawan Puli, Robert Lipski, Aime A. Levesque. Oligomerization status of p53 serves as an indicator of sensistivity of p53 wildtype tumors to the therapeutic combination of DNA damaging agent and checkpoint inhibitor. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3693.

Deregulation of Internal Ribosome Entry Site-Mediated p53 Translation in Cancer Cells with Defective p53 Response to DNA Damage.

Synthesis of the p53 tumor suppressor and its subsequent activation following DNA damage are critical for its protection against tumorigenesis. We previously discovered an internal ribosome entry site (IRES) at the 5' untranslated region of the p53 mRNA. However, the connection between IRES-mediated p53 translation and p53's tumor suppressive function is unknown. In this study, we identified two p53 IRES trans-acting factors, translational control protein 80 (TCP80), and RNA helicase A (RHA), which positively regulate p53 IRES activity. Overexpression of TCP80 and RHA also leads to increased expression and synthesis of p53. Furthermore, we discovered two breast cancer cell lines that retain wild-type p53 but exhibit defective p53 induction and synthesis following DNA damage. The levels of TCP80 and RHA are extremely low in both cell lines, and expression of both proteins is required to significantly increase the p53 IRES activity in these cells. Moreover, we found cancer cells transfected with a shRNA against TCP80 not only exhibit decreased expression of TCP80 and RHA but also display defective p53 induction and diminished ability to induce senescence following DNA damage. Therefore, our findings reveal a novel mechanism of p53 inactivation that links deregulation of IRES-mediated p53 translation with tumorigenesis.

MP49-04 CRITICAL ROLES OF A NUCLEOLAR PROTEIN DDX31 IN BLADDER CARCINOGENESIS DEPENDING ON P53 MUTATION STATUS.

You have accessJournal of UrologyBladder Cancer: Basic Research III1 Apr 2015MP49-04 CRITICAL ROLES OF A NUCLEOLAR PROTEIN DDX31 IN BLADDER CARCINOGENESIS DEPENDING ON P53 MUTATION STATUS. Kei Daizumoto, Tomoya Fukawa, Hisanori Uehara, Toyomasa Katagiri, and Hiro-omi Kanayama Kei DaizumotoKei Daizumoto More articles by this author , Tomoya FukawaTomoya Fukawa More articles by this author , Hisanori UeharaHisanori Uehara More articles by this author , Toyomasa KatagiriToyomasa Katagiri More articles by this author , and Hiro-omi KanayamaHiro-omi Kanayama More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.508AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Bladder cancer (BCa) is the second common genitourinary tumor, yet the detailed mechanisms of its carcinogenesis is largely unknown. We previously identified that a novel nucleolar protein DDX31 (DEAD box polypeptide 31) contribute renal cell carcinoma (RCC) oncogenesis through its regulating the p53-MDM2 pathway, indicating a new mechanism of p53 inactivation in RCC cells. On the other hand, we also found frequent up-regulation of DDX31 in BCa although p53 mutations often occur. In this study, we report the distinct critical roles of DDX31 in bladder carcinogenesis, especially cancer progression. METHODS To investigate the significance of the DDX31 upregulation in BCa, we conducted immunohistochemical staining of 71 BCa specimens with the anti-DDX31 antibody, and estimated the association of DDX31 expression levels on the stained tissues based on a scoring method with cancer-disease specific survival by the Kaplan-Meier method and the log rank test. Next, we examined the siRNA mediated-knockdown effect of DDX31 expression on proliferation, migration and invasion abilities by MTT, wound healing and matrigel invasion assays. Moreover, to investigate biological function of DDX31, we examined the proteins changes in DDX31-depleted BCa cells with shot-gun proteome and bioinformatic analyses. RESULTS We found that DDX31 was significantly upregulated in 7 of 9 BCa cell lines, which possess p53 mutations. Immunohistochemical analysis revealed that DDX31 expression (score ≥4) was associated with a significantly worse cancer-disease specific survival (HR 3.565, p = 0.041) than those with low DDX31 protein expression (score ≤ 3 ; log-rank, p = 0.007). Deletion of DDX31 expression by siRNA led to p53 stabilization, resulting in significant growth suppression in p53 wild type BCa (WT) cells, but not in p53 mutant (MT) cells. Notably, in MT cells, deletion of DDX31 expression led to the significant suppression of both migration and invasion abilities (wound-healing, p = 0.0038; invasion, p = 0.0016, respectively). Through the proteome and bioinformatics analyses, DDX31 might be involved in molecular pathways regulating morphological changes and cytoskeletal organization. CONCLUSIONS Overexpression of a novel nucleolar protein DDX31 is critically involved in advanced events such as cell migration and invasion of BCa. Together, we suggest that DDX31 may play the distinct important roles in proliferation ability and cytoskeletal organization depending on p53 mutation status. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e604 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Kei Daizumoto More articles by this author Tomoya Fukawa More articles by this author Hisanori Uehara More articles by this author Toyomasa Katagiri More articles by this author Hiro-omi Kanayama More articles by this author Expand All Advertisement Advertisement PDF DownloadLoading ...

Abstract 1587: Loss of LZAP inactivates p53 in head and neck cancer and regulates sensitivity of cells to DNA damage in the p53-dependent manner.

Abstract We previously reported that tumor suppressor LZAP is lost in a portion of head and neck squamous cell carcinomas and that LZAP inhibits NF-kB, while activating p53 in ARF-dependent and -independent fashions. The ability of LZAP to regulate p53 in the absence of ARF has been largely unexplored. Here, we show that LZAP depletion diminished expression of mutant and wild-type p53 protein. LZAP activity toward p53 was independent of ARF and the known p53 regulators, Wip1 and HDM2. Loss of LZAP decreased p53 translation and destabilized p53 protein due, at least partially, to increased nucleolin expression. Knockdown of LZAP abrogated p53 stabilization induced by ionizing radiation, and LZAP depletion protected wild-type p53 cells from radiation or chemotherapeutic agents, while rendering cells expressing mutant or no p53 more sensitive. In human HNSCC, LZAP levels correlated with p53 levels, and remarkably, tumors with low LZ were less likely to have p53 mutations. These data suggest that loss of LZAP represents a novel mechanism of p53 inactivation in human cancer. Given these findings, inhibition of LZAP activity toward p53 for patients with tumors harboring p53 mutations may represent a therapeutic strategy to simultaneously sensitize tumors while protecting normal tissues from radiation or chemotherapy. Citation Format: Dan Liu, Natalia Issaeva, Wendell G. Yarbrough. Loss of LZAP inactivates p53 in head and neck cancer and regulates sensitivity of cells to DNA damage in the p53-dependent manner. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1587. doi:10.1158/1538-7445.AM2013-1587

Abstract LB-283: Aurora B binds p53 during both interphase and mitosis, phosphorylates it and promotes its degradation

Abstract Aurora B (AurB) is a mitotic checkpoint kinase that plays a pivotal role in the cell cycle, ensuring correct chromosome segregation and normal progression through mitosis. Tumor suppressor p53 is a genome guardian and an important negative regulator of the cell cycle. Although overexpression of AurB and loss or degradation of p53 have been described in many types of human cancers, it is not known whether AurB and p53 are coordinately regulated during the cell cycle. Recently has been shown that NIR, which is a transcriptional co-repressor of histone acetyltransferase activity, interact with and suppress p53 via interaction with AurB. Our results show that AurB directly interacts with p53 in a NIR-independent matter. To prove direct molecular interaction between AurB and p53 in whole cells, we employed the method of bimolecular fluorescent complementation (BiFC). AurB-p53 interaction occurs at the nucleus in interphase and at the centromeres in prometaphase of mitosis. We observed that AurB-p53 interaction co-localized with Survivin and CENP-A, suggesting that AurB and p53 interact at the centromeres. Our results also show that Aurora B phosphorylates p53 to accelerate p53's degradation through the polyubiquitination-proteasome pathway, thus functionally suppressing the expression of p53 target genes involved in cell cycle inhibition and apoptosis. Inhibition of Aurora B in cancer cells with wild-type p53 increased p53 protein level and expression of p53 target genes to inhibit tumor growth. Together, these results give a new insight of a novel mechanism of p53 inactivation during the cell cycle and imply that oncogenic hyperactivation or overexpression of AurB may compromise p53's tumor suppressor function. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-283. doi:1538-7445.AM2012-LB-283

Soluble Guanylyl Cyclase α1 and p53 Cytoplasmic Sequestration and Down-Regulation in Prostate Cancer

Our laboratory has previously identified soluble guanylyl cyclase α1 (sGCα1) as a novel androgen-regulated gene essential for prostate cancer cell proliferation. sGCα1 expression is highly elevated in prostate tumors, contrasting with the low expression of sGCβ1, with which sGCα1 dimerizes to mediate nitric oxide (NO) signaling. In studying its mechanism of action, we have discovered that sGCα1 can inhibit the transcriptional activity of p53 in prostate cancer cells independent of either classical mediators of NO signaling or the guanylyl cyclase activity of sGCα1. Interestingly, sGCα1 inhibition of p53-regulated gene expression was gene specific, targeting genes involved in apoptosis/cell survival. Consistent with this, overexpression of sGCα1 makes prostate cancer cells more resistant to etoposide, a chemotherapeutic and apoptosis-inducing drug. Immunoprecipitation and immunocytochemistry assays show a physical and direct interaction between sGCα1 and p53 in prostate cancer cells. Interestingly, sGCα1 induces p53 cytoplasmic sequestration, representing a new mechanism of p53 inactivation in prostate cancer. Analysis of prostate tumors has shown a direct expression correlation between sGCα1 and p53. Collectively, these data suggest that sGCα1 regulation of p53 activity is important in prostate cancer biology and may represent an important mechanism of p53 down-regulation in those prostate cancers that express significant levels of p53.

Cytoplasmic sequestration of wild-type p53 in a patient with therapy-related resistant AML: first report

p53 inactivation is a key factor in human tumorigenesis and chemotherapy resistance. The traditionally described mechanisms of p53 inactivation in acute myeloid leukemia (AML) include TP53 mutations and abrogation of p53 pathway. Malfunction of wild-type (wt) p53, due to its cytoplasmic mislocalization, has been described, thus far, only in solid tumors. Herein, we present a patient with therapy-related resistant AML, monosomal karyotype, wt TP53, and cytoplasmic sequestration of p53 protein. Proposed mechanisms of p53 mislocalization and their probable clinical and therapeutic implications are discussed. In view of the relative rareness of TP53 mutations in AML, the cytoplasmic sequestration of p53 protein offers an additional inactivating mechanism, which might be more frequent than currently diagnosed. This notion warrants confirmation by prospective studies in large cohorts of patients. We recommend that evaluation of p53 subcellular localization and function should be included in the diagnostic work-up of AML with wt p53.

Mutational Analysis of ASPP1 and ASPP2 Genes, a p53-related Gene, in Gastric and Cololorectal Cancers with Microsatellite Instability

To the editor, In most human cancers, functions of p53 tumor suppressor are inactivated by many mechanisms, most commonly by somatic mutation.1 Alternatively, p53 is inactivated by genes whose protein products interact with p53.1 Apoptosis-stimulating protein of p53-1 (ASPP1) and -2 (ASPP2) promote p53-mediated apoptosis by enhancing its transactivation function.2 ASPP2+/- mice suffer from an increased incidence of tumorsm,3 suggesting it as a haploinsufficient tumor suppressor gene. However, neither of ASPP1 or ASPP2 mutation has been reported in cancers. Microsatellite instability (MSI) is characterized by length alterations in repeated DNA sequences, and 10-30% of colorectal cancer (CRC) and gastric cancer (GC) are categorized as MSI-positive cancers.4 In a public database, we found an A7 repeat in exon 8 of ASPP1 and an A7 repeat in exon 6 of ASPP2. To see whether these A7 are mutated in GC and CRC with MSI, we analyzed them by polymerase chain reaction (PCR)-based single-strand conformation polymorphism (SSCP) as described previously.5 We analyzed 32 high-MSI (MSI-H) and 13 low-MSI (MSI-L) GC, and 38 MSI-H and 15 MSI-L CRC. We used two primer pairs that amplified each A7 of ASPP1 and the ASPP2. Tumor and normal DNA from microdisscted cells were amplified by the PCR. Radioisotope ([32P]dCTP) was incorporated into the PCR for detection by SSCP. Direct DNA sequencing was performed in the cancers with mobility shifts in the SSCP. The PCR-SSCP analysis of ASPP2 identified aberrant bands in one GC with MSI-H (3.1%) and two CRC with MSI-H (5.3%), but none in those with MSI-L (Fig. 1). Normal DNA of the patients showed no shifts, indicating the mutations had risen somatically. Direct DNA sequencing identified an identical ASPP2 deletion mutation in the A7 repeat (c.576delA), which would result in a premature stop (p.Val193fsX1) and resembled a typical loss-of-function mutation. For ASPP1, there was no aberrantly migrating SSCP band in the cancer tissues. Fig. 1 Mutation of ASPP2 in a gastric carcinoma with high-microsatellite instability. (A) PCR product of ASPP2 from a colon carcinoma shows aberrant bands (arrows in lane T) as compared to single-strand conformation polymorphism from normal tissue (lane N) of ... As a possible mechanism for p53 inactivation, we analyzed ASPP1 and ASPP2 mutations in their repeat sequences and found some of the cancers with MSI-H harbored ASPP2 frameshift mutation. In colon, tumors with MSI have frameshift mutations in specific target genes, and fewer mutations are found in K-RAS and p53.4 Although the incidence of the ASPP2 mutation is not high, our data indicate that the ASPP2 mutation could partially explain a p53-inactivating mechanism in GC and CRC with MSI. Four ankylin repeats (amino acids 930-1047) and an SH3 domain (amino acids 1064-1114) of ASPP2 are essential for the interaction of ASPP2 with p53.2,3,6 The frameshift mutation identified in this study (p.Val193fsX1) would remove ankylin repeats and SH3 domain, and pro-apoptotic function of ASPP2 may be decreased in the affected cancers. Our data suggest that decreased p53 functions by the ASPP2 mutation might possibly contribute to pathogenesis in MSI-H cancers. Whether inactivation of ASPP genes further contributes to GC and CRC development in addition to the frameshift mutation of ASPP2, mutational analysis of entire coding region of ASPP1 and ASPP2 genes is required in future studies.

P53 is Nuclear and Functional in Both Undifferentiated and Differentiated Neuroblastoma

Aberrant cytoplasmic sequestration has been reported as an alternative mechanism of p53 inactivation to mutation in neuroblastoma. We hypothesized that p53 localization and function in neuroblastoma is related to differentiation status. Eighty-two untreated and 24 paired pre and post-chemotherapy neuroblastomas were studied by immunocytochemistry for p53, p21WAF1, BAX, Bcl2 and Ki67. Predominantly nuclear p53 was detected in undifferentiated neuroblastoma, and both nuclear and cytoplasmic p53 in differentiating neuroblastoma. The nuclear p53 labeling index (LI) correlated with the Ki67 LI (r = 0.51, p

Repression of Dmp1 and Arf transcription by anthracyclins: critical roles of the NF-κB subunit p65

Both genotoxic and oncogenic stress activates the nuclear factor-kappa B (NF-kappaB) and p53 proteins; however, the p53 activity is antagonized by NF-kappaB signaling. Dmp1 is a Myb-like transcription factor that activates the Arf-p53 pathway. The Dmp1 promoter was activated by a classical NF-kappaB activator tumor necrosis factor alpha, but repressed by treatment of cells with non-classical NF-kappaB activators, anthracyclins and UV-C. p65 and other subsets of NF-kappaB proteins were bound to the Dmp1 promoter following anthracyclin/UV-C treatment of rodent fibroblasts. This resulted in the downregulation of Dmp1 mRNA and protein. Repression of the Dmp1 transcription by anthracyclins depended on the unique NF-kappaB site on the promoter. Downregulation of p65 significantly attenuated the repression of the Dmp1 promoter by anthracyclins/UV-C. The amount of Dmp1 bound to the Arf promoter decreased significantly upon anthracyclin treatment; this, in turn, downregulated the Arf levels. Repression of the Arf promoter by p65 or anthracyclins depended on Dmp1, which was significantly attenuated in Dmp1(-/-) cells. Both Dmp1(-/-)and Arf(-/-)cells showed resistance to anthracyclin-induced cell death compared to wild-type cells; non-immortalized p65-knockdown cells were much more sensitive. Thus, the Dmp1-Arf pathway is repressed by p65 in response to genotoxic stress, which implicates a novel mechanism of p53 inactivation by NF-kappaB.

PCTAIRE3: a putative mediator of growth arrest and death induced by CTS-1, a dominant-positive p53-derived synthetic tumor suppressor, in human malignant glioma cells

Chimeric tumor suppressor-1 (CTS-1) is based on the sequence of p53 and was designed as a therapeutic tool resisting various mechanisms of p53 inactivation. We previously reported that an adenovirus expressing CTS-1 (Ad-CTS-1) has superior cell death-inducing activity in glioma cells compared with wild-type p53. Here, we used cDNA microarrays to detect changes in gene expression preferentially induced by Ad-CTS-1. The putative serine threonine kinase, PCTAIRE3, and the quinone oxireductase, PIG3, were strongly induced by Ad-CTS-1 compared with wild-type p53. An adenoviral vector encoding PCTAIRE3 (Ad-PCTAIRE3) induced growth arrest and killed a minor proportion of the glioma cells. Ad-PIG3 alone affected neither growth nor viability. However, coinfection with Ad-PCTAIRE3 and Ad-PIG3 resulted in enhanced growth inhibition compared with Ad-PCTAIRE3 infection alone. Ad-CTS1, Ad-PCTAIRE3 or Ad-PIG3 induced the formation of free reactive oxygen species (ROS). However, the prevention of ROS formation induced by Ad-PCTAIRE3 and Ad-CTS-1 did not block growth arrest and cell death, suggesting that ROS formation is not essential for these effects. Altogether, these data identify PCTAIRE3 as one novel growth-inhibitory and death-inducing p53 response gene and suggest that changes in the expression of specific target genes contribute to the superior anti-glioma activity of CTS-1.

The Histone Deacetylase Inhibitor FR901228 (Depsipeptide) Restores Expression and Function of Pseudo-Null p53

We have previously described a novel mechanism of p53 dysfunction, characterized by repression of mRNA and protein expression effectively leading to functional inactivation of wt p53 in SW-1736 human anaplastic thyroid cancer cells (pseudo-null p53). Here we demonstrated that treatment of SW-1736 cells with sub-cytotoxic concentrations of FR901228, a histone deacetylase (HDAC) inhibitor, results in marked induction of p53 mRNA and protein. The p53 induced by FR901228 was functional as evidenced by mdm-2 and p21 transactivation, and its further accumulation following DNA damage by doxorubicin. Furthermore, pretreatment with FR901228 sensitized SW-1736 cells to doxorubicin. This study validates the concept of pseudo-null p53, as a mechanism of p53 inactivation, and demonstrates that pseudo-null p53 can be rescued pharmacologically.

Alteration of cell cycle in cervical tumor associated with human papillomavirus: cyclin-dependent kinase inhibitors.

The ability of viral oncoproteins to subvert cell cycle checkpoints may constitute a mechanism by which viral oncoproteins induce genetic instability. HPV 16 E6 and E7 disrupt cell cycle checkpoints, particularly affecting nearly all cyclin-dependent kinase inhibitors linked to the G1- and G2- checkpoints, in each case by means of a different mechanism. HPV 16 E7 shows homology with the pRb binding sites of cyclin D1, which consequently releases E2F. In addition, E7 directly binds to p21, and releases PCNA and other S-phase promoting genes. In turn, released E2F activates cyclin E, and cyclin E accelerates p27 proteolysis as a function of the antagonistic reaction of its own inhibitor. The induction of p16 expression is assumed to be indirectly associated with E7, which is upregulated only after prolonged inactivation of Rb. HPV 16 E6 decreased the fidelity of multiple checkpoints controlling both entry into and exit from mitosis, with the mechanism of p53 inactivation. In addition, HPV 16 E6 increased the sensitivity to chemically induced S-phase premature mitosis and decreased mitotic spindle assembly checkpoint function. Alongside the impressive advances made in the understanding of the molecular mechanisms, which HPV disrupts, the validity of these conclusions should be evaluated in the diagnostic and prognostic fields.

Loss of heterozygosity frequency at the Trp53 locus in p53-deficient (+/-) mouse tumors is carcinogen-and tissue-dependent.

Mutagenic carcinogens rapidly induced tumors in the p53 haploinsufficient mouse. Heterozygous p53-deficient (+/-) mice were exposed to different mutagenic carcinogens to determine whether p53 loss of heterozygosity (LOH) was carcinogen-and tissue-dependent. For 26 weeks, C57BL/6 (N4) [corrected] p53-deficient (+/-) male or female mice were exposed to p-cresidine, benzene or phenolphthalein. Tumors were examined first for loss of the wild-type p53 allele. p-cresidine induced p53 LOH in three of 13 bladder tumors, whereas hepatocellular tumors showed p53 LOH in carcinomas (2/2), but not in adenomas (0/3). Benzene induced p53 LOH in 13 of 16 tumors examined. Finally, phenolphthalein induced p53 LOH in all tumors analyzed (21/21). Analysis of the p-cresidine-induced bladder tumors by cold single-strand conformation polymorphism (SSCP) analysis of exon 4-9 amplicons failed to demonstrate polymorphisms associated with mutations in tumors that retained the p53 wild-type allele. p-cresidine induced a dose-related increase in lacI mutations in bladder DNA. In summary, these data demonstrate that loss of the wild-type allele occurred frequently in thymic lymphomas and sarcomas, but less frequently in carcinomas of the urinary bladder. In the bladder carcinomas other mechanisms may be operational. These might include (i) other mechanisms of p53 inactivation, (ii) inactivating mutations occurring outside exons 4-9 or (iii) p53 haploinsufficiency creating a condition that favors other critical genetic events which drive bladder carcinogenesis, as evidenced by the significant decrease in tumor latency. Understanding the mechanisms of p53 LOH and chemical carcinogenesis in this genetically altered model could lead to better models for prospective identification and understanding of potential human carcinogens and the role of the p53 tumor suppressor gene in different pathways of chemical carcinogenesis.

Inactivation of Tumor Suppressor p53 by Mot-2, a hsp70 Family Member

The mortalin genes, mot-1 and mot-2, are hsp70 family members that were originally cloned from normal and immortal murine cells, respectively. Their proteins differ by only two amino acid residues but exhibit different subcellular localizations, arise from two distinct genes, and have contrasting biological activities. We report here that the two proteins also differ in their interactions with the tumor suppressor protein p53. The pancytosolic mot-1 protein in normal cells did not show colocalization with p53; in contrast, nonpancytosolic mot-2 and p53 overlapped significantly in immortal cells. Transfection of mot-2 but not mot-1 resulted in the repression of p53-mediated transactivation in p53-responsive reporter assays. Inactivation of p53 by mot-2 was supported by the down-regulation of p53-responsive genes p21(WAF-1) and mdm-2 in mot-2-transfected cells only. Furthermore, NIH 3T3 cells transfected with expression plasmid encoding green fluorescent protein-tagged mot-2 but not mot-1 showed an abrogation of nuclear translocation of wild-type p53. These results demonstrate a novel mechanism of p53 inactivation by mot-2 protein.

No evidence for functional inactivation of wild-type p53 protein by MDM2 overexpression in gastric carcinogenesis.

Inactivation of wild-type p53 during gastric carcinogenesis is usually caused by mutations within exons 5-8 of the p53 gene leading to mutated, usually immunohistochemically detectable p53 proteins. However, functional inactivation of wild-type p53, mimicking mutational inactivation, may also result from binding to overexpressed MDM2 protein. While these two mechanisms of p53 inactivation are considered to be mutually exclusive, no data exist as to whether MDM2 overexpression occurs during gastric carcinogenesis. MDM2 protein overexpression was therefore studied in relation to p53 protein accumulation in gastric carcinogenesis. Forty-five paraffin-embedded gastrectomy specimens from early gastric carcinomas were examined for the presence of chronic active gastritis, chronic atrophic gastritis, subtypes of intestinal metaplasia, and dysplasia. The Lauren type was reassessed for all early carcinomas. p53 protein accumulation was examined using the monoclonal antibody DO-7. MDM2 protein overexpression was assessed with the monoclonal antibody SMP-14. Complete absence of nuclear p53 protein accumulation was observed in chronic active gastritis, chronic atrophic gastritis, and intestinal metaplasia, irrespective of the subtype. In gastric dysplasia (one mild, two moderate, one severe), only severe dysplasia was p53-positive. Intestinal-type (n = 20) and diffuse-type early gastric carcinoma (n = 25) were p53-positive in 70 and 52 per cent of the cases, respectively. MDM2 protein overexpression was not observed during gastric carcinogenesis, either in the p53-positive or in the p53-negative cases. In conclusion, it appears that functional inactivation of wild-type p53 by MDM2 protein overexpression plays no role in (early) gastric carcinogenesis.

Review : p53 in the pathogenesis, diagnosis, and treatment of cancer

Objective. The cellular functions of p53, the conse quences of the loss of p53 function, and the potential impact of p53 in oncology are reviewed within the framework of an overview of the molecular basis of cancer and cell cycle control. Data Sources. A MEDLINE search of articles from 1976 to the present was conducted using the terms p53 protein and p53 gene. The search was restricted to the English language. Oncology and molecular biology textbooks were used as additional references. Data Extraction. We reviewed the literature to discuss the cellular function of p53, the mechanisms of p53 inactivation, the cellular consequences of the loss of p53 function, the role of p53 loss in tumori genesis, and the potential applications of this knowl edge. Data Synthesis. p53 mutations are found in ~ 50% of human cancers. Knowledge of p53 functions and defects provides the basis for potential applica tions in the areas of cancer epidemiology, cancer diagnosis, and determination of prognosis. An under standing of the functions and defects of p53 also presents a host of opportunities for the design of novel cancer therapies. Therapeutic approaches be ing studied include the restoration of p53 by gene therapy, the alteration of mutant p53 expression by antisense therapy, and the use of p53 mutations as a target for directing therapy to cancer cells; some of these approaches are already under phase I investiga tion. As knowledge of p53 unfolds, additional thera peutic approaches will certainly be developed. The story of p53 illustrates that the manipulation of mo lecular interactions is a new frontier in therapeutics and offers an additional role for oncology pharmacy specialists.