Basal P53 Research Articles

Expression of p53 target genes in the early phase of long-term potentiation in the rat hippocampal CA1 area.

Gene expression plays an important role in the mechanisms of long-term potentiation (LTP), which is a widely accepted experimental model of synaptic plasticity. We have studied the expression of at least 50 genes that are transcriptionally regulated by p53, as well as other genes that are related to p53-dependent processes, in the early phase of LTP. Within 30 min after Schaffer collaterals (SC) tetanization, increases in the mRNA and protein levels of Bax, which are upregulated by p53, and a decrease in the mRNA and protein levels of Bcl2, which are downregulated by p53, were observed. The inhibition of Mdm2 by nutlin-3 increased the basal p53 protein level and rescued its tetanization-induced depletion, which suggested the involvement of Mdm2 in the control over p53 during LTP. Furthermore, nutlin-3 caused an increase in the basal expression of Bax and a decrease in the basal expression of Bcl2, whereas tetanization-induced changes in their expression were occluded. These results support the hypothesis that p53 may be involved in transcriptional regulation during the early phase of LTP. We hope that the presented data may aid in the understanding of the contribution of p53 and related genes in the processes that are associated with synaptic plasticity.

NIAM-deficient mice are predisposed to the development of proliferative lesions including B-cell lymphomas.

Nuclear Interactor of ARF and Mdm2 (NIAM, gene designation Tbrg1) is a largely unstudied inhibitor of cell proliferation that helps maintain chromosomal stability. It is a novel activator of the ARF-Mdm2-Tip60-p53 tumor suppressor pathway as well as other undefined pathways important for genome maintenance. To examine its predicted role as a tumor suppressor, we generated NIAM mutant (NIAMm/m) mice homozygous for a β-galactosidase expressing gene-trap cassette in the endogenous gene. The mutant mice expressed significantly lower levels of NIAM protein in tissues compared to wild-type animals. Fifty percent of aged NIAM deficient mice (14 to 21 months) developed proliferative lesions, including a uterine hemangioma, pulmonary papillary adenoma, and a Harderian gland adenoma. No age-matched wild-type or NIAM+/m heterozygous animals developed lesions. In the spleen, NIAMm/m mice had prominent white pulp expansion which correlated with enhanced increased reactive lymphoid hyperplasia and evidence of systemic inflammation. Notably, 17% of NIAM mutant mice had splenic white pulp features indicating early B-cell lymphoma. This correlated with selective expansion of marginal zone B cells in the spleens of younger, tumor-free NIAM-deficient mice. Unexpectedly, basal p53 expression and activity was largely unaffected by NIAM loss in isolated splenic B cells. In sum, NIAM down-regulation in vivo results in a significant predisposition to developing benign tumors or early stage cancers. These mice represent an outstanding platform for dissecting NIAM's role in tumorigenesis and various anti-cancer pathways, including p53 signaling.

Basal p21 controls population heterogeneity in cycling and quiescent cell cycle states.

Phenotypic heterogeneity within a population of genetically identical cells is emerging as a common theme in multiple biological systems, including human cell biology and cancer. Using live-cell imaging, flow cytometry, and kinetic modeling, we showed that two states--quiescence and cell cycling--can coexist within an isogenic population of human cells and resulted from low basal expression levels of p21, a Cyclin-dependent kinase (CDK) inhibitor (CKI). We attribute the p21-dependent heterogeneity in cell cycle activity to double-negative feedback regulation involving CDK2, p21, and E3 ubiquitin ligases. In support of this mechanism, analysis of cells at a point before cell cycle entry (i.e., before the G1/S transition) revealed a p21-CDK2 axis that determines quiescent and cycling cell states. Our findings suggest a mechanistic role for p21 in generating heterogeneity in both normal tissues and tumors.

Aberrant autolysosomal regulation is linked to the induction of embryonic senescence: differential roles of Beclin 1 and p53 in vertebrate Spns1 deficiency.

Spinster (Spin) in Drosophila or Spinster homolog 1 (Spns1) in vertebrates is a putative lysosomal H+-carbohydrate transporter, which functions at a late stage of autophagy. The Spin/Spns1 defect induces aberrant autolysosome formation that leads to embryonic senescence and accelerated aging symptoms, but little is known about the mechanisms leading to the pathogenesis in vivo. Beclin 1 and p53 are two pivotal tumor suppressors that are critically involved in the autophagic process and its regulation. Using zebrafish as a genetic model, we show that Beclin 1 suppression ameliorates Spns1 loss-mediated senescence as well as autophagic impairment, whereas unexpectedly p53 deficit exacerbates both of these characteristics. We demonstrate that ‘basal p53’ activity plays a certain protective role(s) against the Spns1 defect-induced senescence via suppressing autophagy, lysosomal biogenesis, and subsequent autolysosomal formation and maturation, and that p53 loss can counteract the effect of Beclin 1 suppression to rescue the Spns1 defect. By contrast, in response to DNA damage, ‘activated p53’ showed an apparent enhancement of the Spns1-deficient phenotype, by inducing both autophagy and apoptosis. Moreover, we found that a chemical and genetic blockage of lysosomal acidification and biogenesis mediated by the vacuolar-type H+-ATPase, as well as of subsequent autophagosome-lysosome fusion, prevents the appearance of the hallmarks caused by the Spns1 deficiency, irrespective of the basal p53 state. Thus, these results provide evidence that Spns1 operates during autophagy and senescence differentially with Beclin 1 and p53.

P63 and p73 coordinate p53 function to determine the balance between survival, cell death, and senescence in adult neural precursor cells.

The p53 family members p73 and p63 have been implicated in various aspects of stem cell regulation. Here, we have asked whether they work together to regulate stem cell biology, focusing upon neural precursor cells (NPCs) in the adult murine brain. By studying mice that are haploinsufficient for p63 and/or p73, we show that these two proteins cooperate to ensure appropriate NPC self-renewal and long-term maintenance in the hippocampus and forebrain, and that when both are haploinsufficient, the NPC deficits are significantly greater than haploinsufficiency for either alone. We show that, in the case of p63(+/-) mice, this decrease in adult NPCs is caused by enhanced apoptosis. However, when p73 is coincidently haploinsufficient, this rescues the enhanced apoptosis of p63(+/-) NPCs under both basal conditions and following genotoxic stress, instead causing increased cellular senescence. This increase in cellular senescence is likely due, at least in part, to increased levels of basal DNA damage and p53 activation, as genetic ablation of p53 completely rescues the senescence phenotype observed in p63(+/-); p73(+/-) mice. Thus, the presence of p73 determines whether p63(+/-) NPCs exhibit increased p53-dependent apoptosis or senescence. Together, these studies demonstrate that p63 and p73 cooperate to maintain adult NPC pools through regulation of p53 function; p63 antagonizes p53 to promote cellular survival, whereas p73 regulates self-renewal and p53-mediated apoptosis versus senescence.

Regulation of Mdm2 protein stability and the p53 response by NEDD4-1 E3 ligase

Mdm2 is a critical negative regulator of the tumor suppressor protein p53. Mdm2 is an E3 ligase whose overexpression leads to functional inactivation of p53. Mdm2 protein stability is regulated by several mechanisms including RING (Really Interesting New Gene) domain-mediated autoubiquitination. Here we report biochemical identification of NEDD4-1 as an E3 ligase for Mdm2 that contributes to the regulation of Mdm2 protein stability in cells. NEDD4-1 was identified from Jurkat cytosolic fractions using an enzyme-dead Mdm2 mutant protein as a substrate for in vitro E3 ligase assays. We show that lysates from Nedd4-1 knockout (KO) mouse embryonic fibroblasts (MEFs) have significantly diminished E3 ligase activity toward Mdm2 compared with lysates from wild-type (WT) MEFs. Recombinant NEDD4-1 promotes Mdm2 ubiquitination in vitro in a concentration- and time-dependent manner. In cells, NEDD4-1 physically interacts with Mdm2 via the RING domain of Mdm2. Overexpression of NEDD4-1, but not an enzyme-dead NEDD4-1CS mutant, increases ubiquitination of Mdm2. NEDD4-1 catalyzes the formation of K63-type polyubiquitin chains on Mdm2 that are distinct from K48-type polyubiquitination chains mediated by the Mdm2/MdmX complex. Importantly, K63-type polyubiquitination by NEDD4-1 competes with K48-type polyubiquitination on Mdm2 in cells. As a result, NEDD4-1-mediated ubiquitination stabilizes Mdm2. NEDD4-1 knockdown reduces the t1/2 (half-life) of endogenous Mdm2 from 20 to 12 min in U2OS cells. Nedd4-1 KO MEFs manifest increased p53 levels and activity, a more robust DNA damage response and increased G1 arrest compared with WT MEFs. Similarly, NEDD4-1 knockdown in WT-p53-bearing cells increases basal p53 levels and activity in an Mdm2-dependent manner, causes stronger p53 responses to DNA damage and results in p53-dependent growth inhibition compared with corresponding NEDD4-1-proficient control cells. This study identifies NEDD4-1 as a novel component of the p53/Mdm2 regulatory feedback loop that controls p53 activity during stress responses.

ARC Suppresses Cell Death By Antagonizing p53 Function and Suppressing TRAIL In AML Cells

The apoptosis repressor with caspase recruitment domain (ARC) protein is a unique antiapoptotic protein as it can suppress the activation of both the intrinsic and extrinsic apoptosis pathways. ARC levels are prognostic in AML (Carter BZ et al., Blood 2011) and inhibition of ARC sensitizes leukemic cells to chemotherapy-, to the Bcl-2 inhibitor ABT-737-, and to TRAIL-induced apoptosis in AML (Mak PY et al., ASH 2012 abstract #893). Here we investigated the connection between p53 and TRAIL in the antiapoptotic function of ARC in AML.Confocal microscopic analysis of wild type p53 OCI-AML3 and Molm13 AML cells showed co-localization of p53 and ARC, suggesting possible functional interactions between the two proteins. Contrary to a previous report (Li YZ, Mol Cell Biol, 2008), the activation of p53 by nutlin3a did not decrease ARC levels in AML cells. This was confirmed by Western blot and RT-PCR analysis.ARC knockdown (KD) in OCI-AML3 and Molm13 cells resulted in increased expression of basal and nultin3a-induced p53 and p53 target proteins, suggesting that ARC inhibits p53 expression and antagonizes its function. Consistent with this finding, ARC KD in AML cells sensitized these cells to nutlin3a-induced cell death (EC50 for ARC KD and control OCI-AML3 cells was 2.06 ± 0.50 µM and 4.86 ± 0.05 µM, respectively, and for ARC KD and control Molm13 cells 0.59 ± 0.03 µM and 1.09 ± 0.05 µM, respectively, at 48 hours) (p<0.05).CRM1 is known to transport p53 from the nucleus into the cytoplasm and decrease p53 transcriptional activity (Kojima K et al., Blood 2013). DR5, the TRAIL receptor is also a known transcriptional target of p53. ARC also regulated the expression of CRM1 and TRAIL. CRM1 protein levels are lower in ARC KD cells than in their controls. Conversely, TRAIL mRNA levels were lower in ARC overexpressing (OE) and higher in ARC KD cells. The combination of nutlin3a and TRAIL synergistically induced cell death in OCI-AML3 and Molm13 cells cultured alone or co-cultured with mesenchymal stromal cells (MSCs) (CI < 0.1), and this combination induced statistically significantly (P < 0.05) more cell death in ARC KD cells than in controls even when cells were co-cultured with MSCs. Similarly, combinations of nutlin3a and ABT-737 or TRAIL and ABT-737 synergistically induced cell death in AML cells cultured alone or co-cultured with MSCs (CI < 0.1). Cell death was more pronounced in ARC KD cells than in their respective controls (P < 0.05). Furthermore, the combination of TRAIL and ABT-737 synergistically induced cell death in KG-1 cells, and the cell death was significantly reduced in ARC OE KG-1 than the control cells. ConclusionsThis study identifies novel mechanisms by which ARC suppresses cell death in AML cells. Specifically, ARC antagonizes p53 function by suppressing its expression and that of its target proteins and by increasing CRM1 levels, and ARC inhibits TRAIL expression. These findings suggest that combined p53 activation and ARC inhibition can augment apoptosis induction and sensitize AML cells to chemotherapeutic agents. Disclosures:No relevant conflicts of interest to declare.

In human retinoblastoma Y79 cells okadaic acid–parthenolide co-treatment induces synergistic apoptotic effects, with PTEN as a key player

Retinoblastoma is the most common intraocular malignancy of childhood. In developing countries, treatment is limited, long-term survival rates are low and current chemotherapy causes significant morbidity to pediatric patients and significantly limits dosing. Therefore there is an urgent need to identify new therapeutic strategies to improve the clinical outcome of patients with retinoblastoma. Here, we investigated the effects of two natural compounds okadaic acid (OKA) and parthenolide (PN) on human retinoblastoma Y79 cells. For the first time we showed that OKA/PN combination at subtoxic doses induces potent synergistic apoptotic effects accompanied by lowering in p-Akt levels, increasing in the stabilized forms of p53 and potent decrease in pS166-Mdm2. We also showed the key involvement of PTEN which, after OKA/PN treatment, potently increased before p53, thus suggesting that p53 activation was under PTEN action. Moreover, after PTEN-knockdown p-Akt/ pS166Mdm2 increased over basal levels and p53 significantly lowered, while OKA/PN treatment failed both to lower p-Akt and pS166-Mdm2 and to increase p53 below/over their basal levels respectively. OKA/PN treatment potently increased ROS levels whereas decreased those of GSH. Reducing cellular GSH by l-butathionine-[S,R]-sulfoximine treatment significantly anticipated the cytotoxic effect exerted by OKA/PN. Furthermore, the effects of OKA/PN treatment on both GSH content and cell viability were less pronounced in PTEN silenced cells than in control cells. The results provide strong suggestion for combining a treatment approach that targets the PTEN/Akt/Mdm2/p53 pathway.

Abstract 786: Genomic and functional characterization identify different manifestations of p53 pathway in 17p13 deletion cases of multiple myeloma.

Abstract Multiple myeloma (MM) is an incurable hematologic malignancy. p53, being one of the most commonly mutated tumor suppressor gene in human cancer, is found to be wild type (WT) in almost 90% of MM. Nevertheless, the frequency of p53 abnormalities is more prevalent in the advanced stage of MM, suggesting its crucial role in the disease progression. Of paramount importance, hemizygous deletion of 17p13 (17p13(del)), which harbours the TP53 gene, has been identified in more than 30% of MM patients and is significantly associated with poor prognosis. To date, there is no conclusive evidence that p53 is the critical gene. Essentially, the integrity of p53 pathway in 17p13(del) cases remains obscure. This study utilized three human myeloma cell lines (HMCL) which harbour 17p13(del) encompassing TP53, as study models. Concurrently, HMCLs with various different p53 status (WT, mutation and null) were recruited as reference model. We have identified a spectrum of unique protein expression profile amongst cells with different p53 status upon induction of the pathway. Genotoxic and non-genotoxic stress confer an intact p53 pathway in WT/WT p53 cells; whereas, p53 mutant cells and p53 null cells demonstrated a defective p53 activity, indicating that functional p53 is exclusively required in triggering the downstream response. Using the WT/WT and the mutant/null profile as reference, the three 17p13(del) cell lines exhibited distinctive phenotypes. XG6, which have basal p53 expression half of that of the WT/WT cell line, displayed a partially compromised p53 pathway. Differentially, KMS18, which has a very low level of basal p53, and JJN3 which has a complete absence of basal p53, displayed a virtual abrogation of p53 response, as evidenced by defective transcriptional activities of p53 as well as resistance to growth arrest and apoptosis. Physiological investigations revealed that the remaining single copy of p53 was likely to be negatively regulated by MDM2 overexpression and epigenetic factors. In conclusion, this study has identified different behaviors in the p53 pathway in 17p13(del) cases. p53 appears to possess haploinssufficiency properties whereby hemizygous p53 loss is sufficient to render a partially defective p53 response. p53 basal level was shown to be the critical determinant of the integrity of the pathway. This has significant clinical implications as this finding can be manipulated to identify patients with different outcome and treatment response. Citation Format: Phaik Ju Teoh, Junli Yan, Wee Joo Chng. Genomic and functional characterization identify different manifestations of p53 pathway in 17p13 deletion cases of multiple myeloma. [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 786. doi:10.1158/1538-7445.AM2013-786

Transcription Factor Sp3 Represses Expression of p21CIP1 via Inhibition of Productive Elongation by RNA Polymerase II

Like that of many protein-coding genes, expression of the p21(CIP1) cell cycle inhibitor is regulated at the level of transcription elongation. While many transcriptional activators have been shown to stimulate elongation, the mechanisms by which promoter-specific repressors regulate pausing and elongation by RNA polymerase II (RNA PolII) are not well described. Here we report that the transcription factor Sp3 inhibits basal p21(CIP1) gene expression by promoter-bound RNA PolII. Knockdown of Sp3 led to increased p21(CIP1) mRNA levels and reduced occupancy of the negative elongation factor (NELF) at the p21(CIP1) promoter, although the level of binding of the positive transcription elongation factor b (P-TEFb) kinase was not increased. Sp3 depletion correlated with increased H3K36me3 and H2Bub1, two histone modifications associated with transcription elongation. Further, Sp3 was shown to promote the binding of protein phosphatase 1 (PP1) to the p21(CIP1) promoter, leading to reduced H3S10 phosphorylation, a finding consistent with Sp3-dependent regulation of the local balance between kinase and phosphatase activities. Analysis of other targets of Sp3-mediated repression suggests that, in addition to previously described SUMO modification-dependent chromatin-silencing mechanisms, inhibition of the transition of paused RNA PolII to productive elongation, described here for p21(CIP1), is a general mechanism by which transcription factor Sp3 fine-tunes gene expression.

Selective inhibition of cell death in malignant vs normal B-cell precursors: implications for cAMP in development and treatment of BCP-ALL

AbstractB-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most commonly occurring pediatric cancer. Despite its relatively good prognosis, there is a steady search for strategies to improve treatment effects and prevent the undesired side effects on normal cells. In the present paper, we demonstrate a differential effect of cyclic adenosine monophosphate (cAMP) signaling between normal BCPs and BCP-ALL blasts, pointing to a potential therapeutic window allowing for manipulation of cAMP signaling in the treatment of BCP-ALL. By studying primary cells collected from pediatric BCP-ALL patients and healthy controls, we found that cAMP profoundly decreased basal and DNA damage-induced p53 levels and cell death in malignant cells, whereas normal BCP counterparts displayed slightly augmented cell death when exposed to cAMP-increasing agents. We did not find evidence for a selection process involving generation of increased basal cAMP levels in BCP-ALL cells, but we demonstrate that paracrine signaling involving prostaglandin E2-induced cAMP generation has the potential to suppress p53 activation and cell death induction. The selective inhibitory effect of cAMP signaling on DNA damage-induced cell death in BCP-ALL cells appears to be an acquired trait associated with malignant transformation, potentially allowing the use of inhibitors of this pathway for directed killing of the malignant blasts.

60 Binding and transactivation by the tumor suppression protein p53 are encoded in the differential structural properties of its response elements

The tumor suppressor p53 is one of the most central hubs in human cells, connected to a complex network in the living cell. Mutations in the p53 tumor suppressor gene are the most frequent genetic alterations in human cancer. In response to cellular stress, p53 acts as a transcription factor by binding to defined DNA targets, thereby activating the expression of genes leading to diverse cellular outcomes. How p53 decides between its various cellular functions and what determines the strength of binding of p53 to its response elements (REs) at various levels of protein is currently unknown. We will discuss our recent studies and will show that differential structural properties of p53 REs affect both p53 binding mechanism as well as the transactivation (TA) level (Beno et al. 2011; Jordan et al. 2012; Menendez et al. 2012). Transactivation by p53 is considered to be dependent on its cellular level, but until recently how the binding strength of p53 to its REs is related to transactivation level was unknown. We recently discovered that this relationship is protein-level dependent. At high protein level, we observed the expected positive relationship between TA level and binding affinity of p53 to its REs, but at low protein level this relationship breaks down and instead we observed a positive relationship between TA level and the torsional flexibility of p53 REs. In addition, we discovered two sequences that supported high TA levels even at basal p53 concentration (“super-transactivating” REs, or STA-REs), in both a highly controlled functional assay in yeast and when transfected into human cells. The transcriptional capacity of many p53 mutants associated with breast cancers could be discriminated by transactivation from these REs. Our findings establish that p53 transactivation is kinetically determined at low levels and thermodynamically driven at high levels, and that the DNA sequence itself can strongly affect p53 transactivation even under conditions where the number of p53 molecules is small, suggesting that DNA structural properties can be critical factors in p53-dependent gene regulation. The mechanistic implications of these findings for p53 binding on its target sequences will be discussed.

Androgen receptor primes prostate cancer cells to apoptosis through down-regulation of basal p21 expression

The androgen receptor (AR) for the male hormone androgen plays an important role in regulation of cell survival or death depending on the nature of cellular context and extracellular stimuli. The pro-survival function of AR is mediated mainly by transcriptional regulation of its target genes. By contrast, the pro-death function of AR can be transcription-dependent or -independent, although the underlying mechanism of the latter is incompletely understood. Here we report that, in androgen-independent prostate cancer cells, AR promotes UV-induced apoptosis through down-regulation of basal expression of p21 independently of its transcriptional activity. Down-regulation of basal p21 expression depends on AR N-terminal interacting protein PIRH2, an E3 ligase for proteasomal degradation of p53. Silencing of PIRH2 up-regulates p53, which in turn activates p21 transcription. Consistent with this, knockdown of PIRH2 suppresses UV-induced AR-dependent apoptosis. Our data suggest that AR primes androgen-independent prostate cancer cells to DNA damage-induced apoptosis through the PIRH2-p53-p21 axis.

Heightened DNA damage response impairs hematopoiesis in Fanconi anemia

Since its first description by Guido Fanconi in 1927,1 Fanconi anemia (FA) has become one of the best characterized inherited bone marrow failure syndromes.2–3 Numerous studies over the years have documented its clinical and biological features. It is recognized as being clinically heterogeneous and patients can have a wide range of developmental abnormalities. Many patients develop progressive bone marrow failure and there is a high predisposition to cancer. FA cells characteristically display a high frequency of spontaneous chromosomal breakage and hypersensitivity to DNA cross-linking agents4 such as mitomycin C and diepoxybutane. This FA cell hallmark has facilitated diagnosis in the clinic and scientific advances in the laboratory. FA is now known to be caused by biallelic mutations in one of 15 genes, the products of which interact in the FA/BRCA pathway in response to cellular stress and DNA damage to maintain genomic stability.5 FA can, therefore, be regarded as an inherited DNA repair disorder. Despite these advances, the precise pathophysiology of the bone marrow failure in FA has remained elusive. A recent international study by Ceccaldi and colleagues6 now provides new insights into the pathogenesis of bone marrow failure in FA. They analyzed bone marrow samples from a large number of FA patients and controls. By undertaking quantitative and qualitative assessment of hematopoietic function they established that FA patients have a significant impairment of hematopoiesis that begins early and is progressive throughout life. In view of the DNA repair defect that has been observed in FA cells, together with the observation that p53 plays an important role in the response to DNA damage, Ceccaldi and colleagues6 decided to analyze p53. They found strong basal and mitomycin C-induced p53 expression in primary cells from FA patients and in bone marrow cells of FA mouse (Fancg−/− and Fancd2−/−) models. To confirm that the p53 induction was directly related to the FA/BRCA pathway defect they knocked down FANCD2 (a key target of the FA pathway) in a human cell line (293T cells) and observed that p53, as well as p21, was induced in these cells. To mimic the response of FA cells to endogenous DNA damage, and to determine the link to the progressive cell impairment observed in patients, they exposed EBV-immortalized FA cells to interstrand cross-linking agents and studied the cellular response at several time points. These EBV-immortalized FA cells displayed the classic excess G2 arrest at 24 h; however, at later time points the G2 arrest resolved and the cells shifted to G0/G1 cell cycle arrest. Interestingly, in p53-silenced FA cells, p21 induction and G0/G1 accumulation were less marked. Complementary studies in FA mice showed that p53 impairs the hematopoietic and mitotic potential of Fanc-deficient murine hematopoietic stem and progenitor cells. Additional studies (including on bone marrow from young FA patients and fetal liver of FA patients) demonstrated constitutive activation of p53 and p21 as well as G0/G1 cell cycle arrest in hematopoietic progenitor cells in vivo. In conclusion, this study identifies an exacerbated p53/p21 DNA damage response as an important factor in the progressive impairment of hematopoiesis in FA; this begins prenatally and typically results in overt bone marrow failure in childhood. These new observations have important implications for the management of FA patients.

Docetaxel and 5-fluorouracil induce human p53 tumor suppressor gene transcription via a short sequence at core promoter element

The p53 tumor suppressor protein is involved in cellular defense against agents that can cause genetic damage. Induction of p53 gene expression at transcriptional and post-transcriptional levels by such agents results in p53-regulated gene activation or suppression. Docetaxel (DOC), a member of the taxanes family that is widely used in cancer chemotherapy, activates p53 at the transcriptional level. We demonstrated that p53 is induced by low dose DOC treatment, resulting in MDR-1 gene suppression in human lung cancer cells. To identify the cis-element of p53 promoter that responds to DOC, p53 promoter region was cloned and promoter activity was analyzed on luciferase gene reporter assay. Promoter region (−78 to +129) contained the highest basal p53 promoter activity and deletion of +86 to +129 severely reduced basal promoter activity. Basal promoter region included the 21-bp element (PE21) that determines UV-inducible expression of p53 and mediates DOC-inducible p53 expression. On site-specific mutagenesis of PE21 (−78 to −58), with mutation of ATTG (−62 to −59) to CGGT, completely diminished the response to DOC. The same mutations also inhibited 5-fluorouracil (5-FU)-inducible p53 expression. Our data revealed that a sequence located at PE21 of p53 core promoter regulates p53 induction by chemotherapeutic agents.

A Deacetylase-Deficient SIRT1 Variant Opposes Full-Length SIRT1 in Regulating Tumor Suppressor p53 and Governs Expression of Cancer-Related Genes

SIRT1 is an NAD-dependent deacetylase and epigenetic regulator essential for normal mammalian development and homeostasis. Here we describe a human SIRT1 splice variant, designated SIRT1-Δ2/9, in which the deacetylase coding sequence is lost due to splicing between exons 2 and 9. This work aimed to determine if SIRT1-Δ2/9 is a novel functional product of the SIRT1 gene. Endogenous SIRT1-Δ2/9 protein was identified in human cell lysate by immunoblotting and splice variant-specific RNA interference (RNAi). SIRT1-Δ2/9 mRNA is bound by CUGBP2, which downregulates its translation. Using pulldown assays, we demonstrate that SIRT1-Δ2/9 binds p53 protein. SIRT1-Δ2/9 maintains basal p53 protein levels and supports p53 function in response to DNA damage, as evidenced by RNAi-mediated depletion of SIRT1-Δ2/9 prior to damage. In turn, basal p53 downregulates SIRT1-Δ2/9 RNA levels, while stress-activated p53 eliminates SIRT1-Δ2/9. Loss of wild-type (wt) p53 has been correlated with overexpression of SIRT1-Δ2/9 in a range of human cancers. Exogenous SIRT1-Δ2/9 protein associates with specific promoters in chromatin and can regulate cancer-related gene expression, as evidenced by chromatin immunoprecipitation analysis and RNAi/genomic array data. SIRT1 is of major therapeutic importance, and potential therapeutic drugs are screened against SIRT1 deacetylase activity. Our discovery of SIRT1-Δ2/9 identifies a new, deacetylase-independent therapeutic target for SIRT1-related diseases, including cancer.

2A Peptide-based, Lentivirus-mediated Anti-death Receptor 5 Chimeric Antibody Expression Prevents Tumor Growth in Nude Mice

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily, induces tumor cell death via death receptors on target cells, without adverse effects on most normal cells. Its receptors are therefore an attractive target for antibody-mediated tumor therapy. Here, we report the creation of a lentivirus vector constructed by linking the heavy chain and the light chain of the antibody with a 2A/furin self-processing peptide in a single open reading frame that expresses a novel chimeric antibody (named as zaptuximab) with tumoricidal activity, which is consisted of the variable region of a mouse anti-human DR5 monoclonal antibody, AD5-10, and the constant region of human immunoglobulin G1. Lentivirus-expressed zaptuximab bound specifically to its antigen, DR5, and exhibited significant apoptosis-inducing activity in various tumor cell lines. The packaged recombinant virus lenti-HF2AL showed strong apoptosis-inducing activity in vitro. Meanwhile, inoculated subcutaneous human colon HCT116 tumor formation in nude mice were inhibited significantly. Moreover, there was a synergistic effect of mitomycin C (MMC) on the observed tumoricidal efficacy, prolonging the life span of nude mice with orthotopic human lung tumor cancers. These data suggest that lentivirus-mediated, 2A peptide-based anti-DR5 chimeric antibody expression may have clinical utility as an anticancer treatment and may represent a rational adjuvant therapy in combination with chemotherapy.

Oncogenic functions of hMDMX in in vitro transformation of primary human fibroblasts and embryonic retinoblasts

BackgroundIn around 50% of all human cancers the tumor suppressor p53 is mutated. It is generally assumed that in the remaining tumors the wild-type p53 protein is functionally impaired. The two main inhibitors of p53, hMDM2 (MDM2) and hMDMX (MDMX/MDM4) are frequently overexpressed in wild-type p53 tumors. Whereas the main activity of hMDM2 is to degrade p53 protein, its close homolog hMDMX does not degrade p53, but it represses its transcriptional activity. Here we study the role of hMDMX in the neoplastic transformation of human fibroblasts and embryonic retinoblasts, since a high number of retinoblastomas contain elevated hMDMX levels.MethodsWe made use of an in vitro transformation model using a retroviral system of RNA interference and gene overexpression in primary human fibroblasts and embryonic retinoblasts. Consecutive knockdown of RB and p53, overexpression of SV40-small t, oncogenic HRasV12 and HA-hMDMX resulted in a number of stable cell lines representing different stages of the transformation process, enabling a comparison between loss of p53 and hMDMX overexpression. The cell lines were tested in various assays to assess their oncogenic potential.ResultsBoth p53-knockdown and hMDMX overexpression accelerated proliferation and prevented growth suppression induced by introduction of oncogenic Ras, which was required for anchorage-independent growth and the ability to form tumors in vivo. Furthermore, we found that hMDMX overexpression represses basal p53 activity to some extent. Transformed fibroblasts with very high levels of hMDMX became largely resistant to the p53 reactivating drug Nutlin-3. The Nutlin-3 response of hMDMX transformed retinoblasts was intact and resembled that of retinoblastoma cell lines.ConclusionsOur studies show that hMDMX has the essential properties of an oncogene. Its constitutive expression contributes to the oncogenic phenotype of transformed human cells. Its main function appears to be p53 inactivation. Therefore, developing new drugs targeting hMDMX is a valid approach to obtain new treatments for a subset of human tumors expressing wild-type p53.

Molecular characterization of primary breast cancer in older women using partitional clustering and correlation with long-term clinical outcome.

10549 Background: Recent advances in molecular classification of breast cancer have given more insights into management strategies and outcome. However, most of the reported studies are focused on younger patients. We now report its application in a series of older women managed in a single centre with long-term follow-up. Methods: Over 36 years (1973-2009), 2,000+ older (≥70 years) women with early operable primary breast cancer were managed in a dedicated clinic. Of these 831 underwent surgery as primary therapy (and received adjuvant therapies as per unit protocol at the time of diagnosis) and 575 had good quality tumour samples available for tissue microarray (TMA) analysis using indirect immunohistochemistry. H-scores of 19 markers (ER, PR, HER2, Ki67, p53, CK5,5/6,7/8,14,17,18,19, EGFR, bcl2, E-Cadherin, MUC1, CD44, HER3, HER4) were clustered using R (2.11.1, USA). Results: Unsupervised clustering showed 6 clusters. Following identification of their main drivers, 12 markers were selected and the same 6 clusters were reproduced, using K-means and Partitioning Around Medoids (PAM) methods, in 367 patients. Using Predictive Analytics Software (PASW 18, Chicago, Illinois), the following correlation with their clinical outcome was obtained, at a median follow-up of 60 months (longest=106) (Table). Conclusions: When compared with known molecular classification, the cluster with low hormone receptors and high luminal cytokeratins (“low ER luminal”) appears unique to older women, associated with different clinical outcome. Cluster (N/%) Pattern of key markers Breast cancer-specific survival Hormone receptors Luminal markers Basal markers p53 and HER2 5-year (%) 10-year (%) Luminal A (139/37.9) High High Low Low 95 76 Luminal B (92/25.1) ER=High PR=Low High Low Low 90 68 “Low ER luminal” (56/15.3) Low High Low Low 83 63 Basal (30/8.2) Low CK18=Low CK19=High CK7/8=Moderate High p53=High HER2=Low 73 73 Normal like (22/6.0) Low Low Low Low 84 47 HER2 overexpressive (28/7.6) Low High Low High 73 65

Tumor-infiltrating lymphocytes are correlated with response to neoadjuvant chemotherapy in triple-negative breast cancer

The purpose of the present study was to identify histological surrogate predictive markers of pathological complete response (pCR) to neoadjuvant chemotherapy (NAC) in triple-negative breast cancer (TNBC). Among 474 patients who received NAC and subsequent surgical therapy for stage II-III invasive breast carcinoma between 1999 and 2007, 102 (22%) had TNBC, and 92 core needle biopsy (CNB) specimens obtained before NAC were available. As controls, CNB specimens from 42 tumors of the hormone receptor-negative and HER2-positive (HR-/HER2+) subtype and 46 tumors of the hormone receptor-positive and HER2-negative (HR+/HER2-) subtype were also included. Histopathological examination including tumor-infiltrating lymphocytes (TIL) and tumor cell apoptosis, and immunohistochemical studies for basal markers were performed, and the correlation of these data with pathological therapeutic effect was analyzed. The rates of pCR at the primary site were higher for TNBC (32%) and the HR-/HER2+ subtype (21%) than for the HR+/HER2- subtype (7%) (P = 0.006). Expression of basal markers and p53, histological grade 3, high TIL scores, and apoptosis were more frequent in TNBC and the HR-/HER2+ subtype than in the HR+/HER2- subtype (P = 0.002 for TIL and P < 0.001 for others). In TNBC, the pCR rates of tumors showing a high TIL score and of those showing a high apoptosis score were 37 and 47%, respectively, and significantly higher or tended to be higher than those of the tumors showing a low TIL score and of the tumors showing a low apoptosis score (16 and 27%, respectively, P = 0.05 and 0.10). In a total of 180 breast cancers, the pCR rates of the tumors showing a high TIL score (34%) and of those showing a high apoptosis score (35%) were significantly higher than those of the tumors showing a low TIL score (10%) and those of the tumors showing a low apoptosis score (19%) (P = 0.0001 and 0.04, respectively). Histological grade and basal marker expression were not correlated with pCR. Although the whole analysis was exploratory, the degree of TIL correlated with immune response appear to play a substantial role in the response to NAC in TNBC.