P53 Ser15 Research Articles

Methylseleninic Acid Suppresses Pancreatic Cancer Growth Involving Multiple Pathways

As a potential novel agent for treating pancreatic cancer, methylseleninic acid (MSeA) was evaluated in cell culture and xenograft models. Results showed that MSeA induced G1 cell cycle arrest and apoptosis in a majority of human and mouse pancreatic cancer cell lines, but G2 arrest in human PANC-1 and PANC-28 cell lines. In contrast to our previous finding in human prostate cancer LNCaP cells having a lack of P53 activation by MSeA, induction of G2 arrest in PANC-1 cells was accompanied by increased mutant P53 Ser15 phosphorylation, upregulation of P53-targets P21Cip1 and GADD45 and G2 checkpoint kinase (Chk2) activation, suggestive of DNA damage responses. A rapid inhibition of AKT phosphorylation was followed by reduced mTOR signaling and increased autophagy in PANC-1 cells attenuating caspase-mediated apoptosis execution. Furthermore, daily oral treatment with MSeA (3 mg Se/kg body weight) significantly suppressed growth of subcutaneously inoculated PANC-1 xenograft in SCID mice. Immunohistochemical analyses detected increased p-Ser15 P53, P21Cip1, pS139-H2AX (DNA damage responses), and caspase-3 cleavage and decreased pSer473AKT and Ki67 proliferative index and reduced intratumor vascular density in MSeA-treated xenograft. These results provide impetus for further research of MSeA in the therapy and/or chemoprevention of pancreatic cancer.

P53 is a key regulator for osthole-triggered cancer pathogenesis.

Osthole has been reported to have antitumor activities via the induction of apoptosis and inhibition of cancer cell growth and metastasis. However, the detailed molecular mechanisms underlying the anticancer effects of osthole in human colon cancer remain unclear. In the present study, we have assessed osthole-induced cell death in two different human colon cancer cell lines, HCT116 and SW480. Our results also showed that osthole activated proapoptotic signaling pathways in human colon cancer cells. By using cell culture insert system, osthole reduced cell motility in both human colon cancer cell lines. This study also provides evidence supporting the potential of osthole in p53 activation. Expression of p53, an apoptotic protein, was remarkably upregulated in cells treated with osthole. Importantly, the levels of phosphorylation of p53 on Ser15 (p-p53) and acetylation of p53 on Lys379 (acetyl-p53) were increased under osthole treatment. Our results also demonstrated that p53 was activated followed by generation of reactive oxygen species (ROS) and activation of c-Jun N-terminal kinase (JNK). Our study provides novel insights of p53-mediated responses under osthole treatment. Taken together, we concluded that osthole induces cancer cell death and inhibits migratory activity in a controlled manner and is a promising candidate for antitumor drug development.

The Emerging Role of p53 in Exercise Metabolism

The major tumour suppressor protein, p53, is one of the most well-studied proteins in cell biology. Often referred to as the Guardian of the Genome, the list of known functions of p53 include regulatory roles in cell cycle arrest, apoptosis, angiogenesis, DNA repair and cell senescence. More recently, p53 has been implicated as a key molecular player regulating substrate metabolism and exercise-induced mitochondrial biogenesis in skeletal muscle. In this context, the study of p53 therefore has obvious implications for both human health and performance, given that impaired mitochondrial content and function is associated with the pathology of many metabolic disorders such as ageing, type 2 diabetes, obesity and cancer, as well as reduced exercise performance. Studies on p53 knockout (KO) mice collectively demonstrate that ablation of p53 content reduces intermyofibrillar (IMF) and subsarcolemmal (SS) mitochondrial yield, reduces cytochrome c oxidase (COX) activity and peroxisome proliferator-activated receptor gamma co-activator 1-α protein content whilst also reducing mitochondrial respiration and increasing reactive oxygen species production during state 3 respiration in IMF mitochondria. Additionally, p53 KO mice exhibit marked reductions in exercise capacity (in the magnitude of 50 %) during fatiguing swimming, treadmill running and electrical stimulation protocols. p53 may regulate contractile-induced increases in mitochondrial content via modulating mitochondrial transcription factor A (Tfam) content and/or activity, given that p53 KO mice display reduced skeletal muscle mitochondrial DNA, Tfam messenger RNA and protein levels. Furthermore, upon muscle contraction, p53 is phosphorylated on serine 15 and subsequently translocates to the mitochondria where it forms a complex with Tfam to modulate expression of mitochondrial-encoded subunits of the COX complex. In human skeletal muscle, the exercise-induced phosphorylation of p53(Ser15) is enhanced in conditions of reduced carbohydrate availability in association with enhanced upstream signalling through 5'adenosine monophosphate-activated protein kinase but not p38 mitogen-activated protein kinase. In this way, undertaking regular exercise in carbohydrate restricted states may therefore be a practical approach to achieve the physiological benefits of consistent p53 signalling. Although our knowledge of p53 in exercise metabolism has advanced considerably, much of our current understanding of p53 regulation and associated targets is derived from various non-muscle cells and tissues. As such, many fundamental questions remain unanswered in contracting skeletal muscle. Detailed studies concerning the time-course of p53 activation (including additional post-translational modifications and subsequent subcellular translocation), as well as the effects of exercise modality (endurance versus resistance), intensity, duration, fibre type, age, training status and nutrient availability, must now be performed so that we can optimise exercise prescription guidelines to strategically target p53 signalling. The emerging role of p53 in skeletal muscle metabolism therefore represents a novel and exciting research area for exercise and muscle physiologists.

Defective Nucleotide Metabolism Contributes To p53 Activation In Diamond-Blackfan Anemia

Diamond-Blackfan Anemia (DBA) is a rare childhood bone marrow failure disorder, characterized by the presence of red cell aplasia, congenital abnormalities, and increased levels of adenosine deaminase (ADA). Haploinsufficiency of ribosomal proteins (RPs) due to mutations in RPS19 and RPL11 occurs approximately 25% and 5% of DBA patients, respectively. The pathogenesis of DBA has been associated with activation of p53, but the mechanism of how this leads to the erythroid defect in DBA patients is not well understood.To understand the molecular pathways leading to DBA, we used previously published zebrafish models of RPS19 and RPL11 deficiency, in addition to primary human fetal liver CD34+ cells transduced with RPS19 shRNA to evaluate signaling pathways upstream of p53. One of the earliest responses to RP deficiency was upregulation of rrm1, a subunit of ribonucleotide reductase (RNR), responsible for de novosynthesis of dNTPs. Since ADA expression is increased in DBA and ADA is involved in nucleotide catabolism, we hypothesized that RP deficient zebrafish and primary human fetal liver CD34+ cells have defects in nucleotide metabolism, which result in replication stress and activation of p53 through the ATR/ATM/Chk1/2 pathways.Several genes involved in de novo nucleotide synthesis, nucleotide catabolism, and purine salvage are upregulated in RPL11 and RPS19 deficient zebrafish, including rrm1 (2-7 fold), ada (2.8 fold), xdh (2.3 fold), and hprt1 (3-fold). In contrast, genes involved in pyrimidine salvage, such as tk1 and dck were downregulated in RP deficient zebrafish (0.4 and 0.8 fold, respectively). RP deficient zebrafish also had imbalanced dNTP pools, with increased dTTP and decreased dCTP levels compared to controls. To test whether these alterations in nucleotide metabolism lead to replication stress, we evaluated DNA damage and stress-induced proteins in primary human fetal liver CD34+ cells transduced with RPS19 shRNA and found increased phosphorylation of p53 (Ser15 and Ser37), ATM (Ser1981), 53BP1 (Ser1778), Chk1 (Ser345), and Chk2 (Thr68), indicating activation of the ATR/ATM/Chk1/2/p53 pathway in RP deficient cells. To rescue the RP-deficient zebrafish, we treated them with a mixture of exogenous nucleosides. Nucleoside treatment decreased p53 expression, restored rrm1, ada, tk1, and dck to normal levels, decreased number of apoptotic cells, and increased the number of Gata1-expressing cells in RP deficient zebrafish. Our data suggest that defective nucleotide metabolism contributes to p53 upregulation in DBA, and that nucleoside supplements alleviate replication stress and may prove beneficial for patients with DBA. Disclosures:No relevant conflicts of interest to declare.

17α-Estradiol에 의한 인체 대장암 세포주 HCT116의 에폽토시스에 수반되는 Bak/Bax의 활성화에 미치는 종양억제단백질 p53의 강화효과

<TEX>$17{\alpha}$</TEX>-estradiol (<TEX>$17{\alpha}-E_2$</TEX>)의 에폽토시스 유도활성에 미치는 종양억제단백질 p53의 조절효과를 조사하고자, <TEX>$17{\alpha}-E_2$</TEX>에 의해 유도되는 에폽토시스 현상들을 인체 대장암 세포주 유래 클론인 HCT116 (<TEX>$p53^{+/+}$</TEX>) 및 HCT116 (<TEX>$p53^{-/-}$</TEX>) 세포에서 비교하였다. HCT116 (<TEX>$p53^{+/+}$</TEX>) 및 HCT116 (<TEX>$p53^{-/-}$</TEX>) 세포를 <TEX>$17{\alpha}-E_2$</TEX> (<TEX>$2.5{\sim}10{\mu}M$</TEX>)로 처리하거나 혹은 HCT116 (<TEX>$p53^{+/+}$</TEX>) 및 HCT116 (<TEX>$p53^{-/-}$</TEX>) 세포를 <TEX>$10{\mu}M$</TEX> <TEX>$17{\alpha}-E_2$</TEX>로 시간 별로 처리한 결과, HCT116 (<TEX>$p53^{+/+}$</TEX>)에 있어서는 세포독성과 에폽토시스-관련 sub-G1 peak의 비율은 처리농도와 시간에 의존적으로 나타났다. 그러나 HCT116 (<TEX>$p53^{-/-}$</TEX>) 세포의 경우는 이러한 현상이 미약하게 나타났다. <TEX>$17{\alpha}-E_2$</TEX>에 의해 유도되는 비정상적 유사분열방추사 형성, 중기판 염색체 배열의 미완성, 이에 따른 유사분열정지(<TEX>$G_2/M$</TEX> arrest) 등의 현상은 HCT116 (<TEX>$p53^{+/+}$</TEX>) 및 HCT116 (<TEX>$p53^{-/-}$</TEX>) 세포에서 유사한 수준으로 나타났다. 이에 반해, <TEX>$17{\alpha}-E_2$</TEX>에 의해 유도되는 Bak과 Bax의 활성화, 미토콘드리아의 막전위 상실(<TEX>${\Delta}{\Psi}m$</TEX> loss), 그리고 PARP 분해 등의 현상은 HCT116 (<TEX>$p53^{-/-}$</TEX>) 세포에 비해 HCT116 (<TEX>$p53^{+/+}$</TEX>) 세포에서 훨씬 높은 수준으로 확인되었다. 아울러 <TEX>$17{\alpha}-E_2$</TEX>로 처리된 HCT116 (<TEX>$p53^{+/+}$</TEX>) 세포에서 확인되는 p53 (Ser-15)의 인산화 및 p53 수준의 증가와 일치하여, 세포 내의 p21및 Bax 수준도 현저히 증가하였다. 이때 <TEX>$17{\alpha}-E_2$</TEX>로 처리된 HCT116 (<TEX>$p53^{-/-}$</TEX>) 세포에서는 p21 및 Bax의 발현수준이 매우 낮았다. 한편, 에폽토시스 억제단백질인 Bcl-2 단백질 수준은 HCT116 (<TEX>$p53^{-/-}$</TEX>) 세포에 비해 HCT116 (<TEX>$p53^{+/+}$</TEX>) 세포에서 다소 낮았으나, 이러한 Bcl-2 단백질 수준은 <TEX>$17{\alpha}-E_2$</TEX> 처리 후에도 크게 변화하지 않는 것으로 나타났다. 이러한 결과들은 <TEX>$17{\alpha}-E_2$</TEX> 처리에 의해 유도되는 에폽토시스 유도 경로의 구성원들의 변화, 즉 비정상적 유사분열방추사 형성 및 이에 따른 유사분열정지(<TEX>$G_2/M$</TEX> arrest), 뒤이은 Bak 및 Bax의 활성화, 미토콘드리아의 막전위 상실, 그리고 이에 수반되는 caspase cascade 활성화 및 PARP 분해로 진행되는 에폽토시스 현상들 중에서, Bak 및 Bax의 활성화 단계가 종양억제단백질 p53의 에폽토시스 증진 활성에 의해 양성적으로 조절되는 작용 타켓임을 보여준다. The regulatory effect of the tumor-suppressor protein p53 on the apoptogenic activity of <TEX>$17{\alpha}$</TEX>-estradiol (<TEX>$17{\alpha}-E_2$</TEX>) was compared between HCT116 (<TEX>$p53^{+/+}$</TEX>) and HCT116 (<TEX>$p53^{-/-}$</TEX>) cells. When the HCT116 (<TEX>$p53^{+/+}$</TEX>) and HCT116 (<TEX>$p53^{-/-}$</TEX>) cells were treated with <TEX>$2.5{\sim}10{\mu}M$</TEX> <TEX>$17{\alpha}-E_2$</TEX> for 48 h or with <TEX>$10{\mu}M$</TEX>for various time periods, cytotoxicity and an apoptotic sub-<TEX>$G_1$</TEX> peak were induced in the HCT116 (<TEX>$p53^{+/+}$</TEX>) cells in a dose- and time-dependent manner. However, the HCT116 (<TEX>$p53^{-/-}$</TEX>) cells were much less sensitive to the apoptotic effect of <TEX>$17{\alpha}-E_2$</TEX>. Although <TEX>$17{\alpha}-E_2$</TEX> induced aberrant mitotic spindle organization and incomplete chromosome congregation at the equatorial plate, <TEX>$G_2/M$</TEX> arrest was induced to a similar extent in both cell types. In addition, <TEX>$17{\alpha}-E_2$</TEX>-induced activation of Bak and Bax, <TEX>${\Delta}{\Psi}m$</TEX> loss, and PARP degradation were more dominant in the HCT116 (<TEX>$p53^{+/+}$</TEX>) than in the HCT116 (<TEX>$p53^{-/-}$</TEX>) cells. In accordance with enhancement of p53 phosphorylation (Ser-15) and p53 levels, p21 and Bax levels were elevated in the HCT116 (<TEX>$p53^{+/+}$</TEX>) cells treated with <TEX>$17{\alpha}-E_2$</TEX>. The HCT116 (<TEX>$p53^{-/-}$</TEX>) cells exhibited barely or undetectable levels of p21 and Bax, regardless of <TEX>$17{\alpha}-E_2$</TEX> treatment. On the other hand, although the level of Bcl-2 was slightly lower in the HCT116 (<TEX>$p53^{+/+}$</TEX>) than in the HCT116 (<TEX>$p53^{-/-}$</TEX>) cells, it remained relatively constant after the <TEX>$17{\alpha}-E_2$</TEX> treatment. Together, these results show that among the components of the <TEX>$17{\alpha}-E_2$</TEX>-induced apoptotic-signaling pathway, which proceeds through mitotic spindle defects causing mitotic arrest, subsequent activation of Bak and Bax and the mitochondria-dependent caspase cascade, leading to PARP degradation, <TEX>$17{\alpha}-E_2$</TEX>-induced activation of Bak and Bax is the upstream target of proapoptotic action of p53.

Src kinase modulates the apoptotic p53 pathway by altering HIPK2 localization

Non-receptor tyrosine kinase Src is a master regulator of cell proliferation. Hyperactive Src is a potent oncogene and a driver of cellular transformation and carcinogenesis. Homeodomain-interacting protein kinase 2 (HIPK2) is a tumor suppressor mediating growth suppression and apoptosis upon genotoxic stress through phosphorylation of p53 at Ser46. Here we show that Src phosphorylates HIPK2 and changes its subcellular localization. Using mass spectrometry we identified 9 Src-mediated Tyr-phosphorylation sites within HIPK2, 5 of them positioned in the kinase domain. By means of a phosphorylation-specific antibody we confirm that Src mediates phosphorylation of HIPK2 at Tyr354. We demonstrate that ectopic expression of Src increases the half-life of HIPK2 by interfering with Siah-1-mediated HIPK2 degradation. Moreover, we find that hyperactive Src binds HIPK2 and redistributes HIPK2 from the cell nucleus to the cytoplasm, where both kinases partially colocalize. Accordingly, we find that hyperactive Src decreases chemotherapeutic drug-induced p53 Ser46 phosphorylation and apoptosis activation. Together, our results suggest that Src kinase suppresses the apoptotic p53 pathway by phosphorylating HIPK2 and relocalizing the kinase to the cytoplasm.

OSU-03012, a non-cox inhibiting celecoxib derivative, induces apoptosis of human esophageal carcinoma cells through a p53/Bax/cytochrome c/caspase-9-dependent pathway

OSU-03012 is a celecoxib derivative devoid of cyclooxygenase-2 inhibitory activity. It was previously reported to inhibit the growth of some tumor cells through the AKT-signaling pathway. In the current study, we assessed the ability of OSU-03012 to induce apoptosis in human esophageal carcinoma cells and the mechanism by which this occurs. A cell proliferation assay indicated that OSU-03012 inhibited the growth of human esophageal carcinoma cell lines with an IC50 below 2 μmol/l and had the most effective cytotoxicity against Eca-109 cells. Terminal deoxynucleotidyl transferase-mediated nick-end labeling assay and flow cytometry analysis showed that OSU-03012 could induce the apoptosis in Eca-109 cells. After treatment of Eca-109 cells with 2 μmol/l OSU-03012 for 24 h, the apoptosis index increased from 14.07 to 53.72%. OSU-03012 treatment resulted in a 30-40% decrease in the mitochondrial membrane potential and caused cytochrome c release into the cytosol. Further studies with caspase-9-specific and caspase-8-specific inhibitors (z-LEHDfmk and z-IETDfmk, respectively) pointed toward the involvement of the caspase-9 pathway, but not the caspase-8 pathway, in the execution of OSU-03012-induced apoptosis. Immunoblot analysis demonstrated that OSU-03012-induced cellular apoptosis was associated with upregulation of Bax, cleaved caspase-3, and cleaved caspase-9. Ser-15 of p53 was phosphorylated after 24 h of treatment of the cancer cells with OSU-03012. This increase in p53 was associated with the decrease in Bcl-2 and increase in Bax. An inhibitor of p53, pifithrin-α, attenuated the anticancer effects of OSU-03012 and downregulated the expression of Bax and cleaved caspase-9. Altogether, our results show that OSU-03012 could induce apoptosis in human esophageal carcinoma cells through a p53/Bax/cytochrome c/caspase-9-dependent pathway.

Distinct single cell signal transduction signatures in leukocyte subsets stimulated with khat extract, amphetamine-like cathinone, cathine or norephedrine

BackgroundAmphetamine and amphetamine derivatives are suggested to induce an immunosuppressive effect. However, knowledge of how amphetamines modulate intracellular signaling pathways in cells of the immune system is limited. We have studied phosphorylation of signal transduction proteins (Akt, CREB, ERK1/2, NF-κB, c-Cbl, STAT1/3/5/6) and stress sensors (p38 MAPK, p53) in human leukocyte subsets following in vitro treatment with the natural amphetamine cathinone, the cathinone derivatives cathine and norephedrine, in comparison with a defined extract of the psychostimulating herb khat (Catha edulis Forsk.). Intracellular protein modifications in single cells were studied using immunostaining and flow cytometry, cell viability was determined by Annexin V-FITC/Propidium Iodide staining, and T-lymphocyte proliferation was measured by 3H-thymidine incorporation.ResultsCathinone, cathine and norephedrine generally reduced post-translational modifications of intracellular signal transducers in T-lymphocytes, B-lymphocytes, natural killer cells and monocytes, most prominently affecting c-Cbl (pTyr700), ERK1/2 (p-Thr202/p-Tyr204), p38 MAPK (p-Thr180/p-Tyr182) and p53 (both total p53 protein and p-Ser15). In contrast, the botanical khat-extract induced protein phosphorylation of STAT1 (p-Tyr701), STAT6 (p-Tyr641), c-Cbl (pTyr700), ERK1/2 (p-Thr202/p-Tyr204), NF-κB (p-Ser529), Akt (p-Ser473), p38 MAPK (p-Thr180/p-Tyr182), p53 (Ser15) as well as total p53 protein. Cathinone, cathine and norephedrine resulted in unique signaling profiles, with B-lymphocytes and natural killer cells more responsive compared to T-lymphocytes and monocytes. Treatment with norephedrine resulted in significantly increased T-lymphocyte proliferation, whereas khat-extract reduced proliferation and induced cell death.ConclusionsSingle-cell signal transduction analyses of leukocytes distinctively discriminated between stimulation with cathinone and the structurally similar derivatives cathine and norephedrine. Cathinone, cathine and norephedrine reduced phosphorylation of c-Cbl, ERK1/2, p38 MAPK and p53(Ser15), and norephedrine induced T-lymphocyte proliferation. Khat-extract induced protein phosphorylation of signal transducers, p38 MAPK and p53, followed by reduced cell proliferation and cell death. This study suggests that protein modification-specific single-cell analysis of immune cells could unravel pharmacologic effects of amphetamines and amphetamine-like agents, and further could represent a valuable tool in elucidation of mechanism(s) of action of complex botanical extracts.

P53 Ser15 phosphorylation and histone modifications contribute to IR-induced miR-34a transcription in mammary epithelial cells

Previous studies have demonstrated that miR-34a is a direct transcriptional target of tumor suppressor p53 and plays a crucial role in p53-mediated biological processes, such as cell cycle arrest, apoptosis and senescence. However, the role of p53 phosphorylation at Ser15 and histone modifications in ionizing radiation (IR)-induced miR-34a transcription in human mammary epithelial cells remains unknown. The present study showed that IR triggers miR-34a induction in rat mammary gland tissue and human mammary epithelial cells in a dose- and time-dependent fashion. Gene copy number and CpG methylation exhibit no effect on IR-inducible miR-34a expression, while the levels of phosphorylated p53 at Ser15 are markedly elevated in human mammary epithelial cells 96 h post-IR, which correlates with IR-inducible miR-34a transcription and the p38 MAPK pathway. Conversely, suppression of p38 MAPK with SB239063 inhibits IR-induced p53 phosphorylation at Ser15 and miR-34a expression in a dose-dependent manner. Our study found that wild-type p53 is enriched at miR-34a promoter, and luciferase activity of miR-34a promoter reporter is attenuated by either mutant p53 (Ser15Ala) or mutant miR-34a promoter. Furthermore, IR also triggers phosphorylation, tri-methylation and acetylation of histone H3 and acetylation of histone H4, which correlates with IR-inducible miR-34a transcription, while SAHA potentiates IR-inducible miR-34a expression. Moreover, acetyl-histone H3 is significantly enriched at miR-34a promoter in IR-exposed HMEC cells. Yet, we show that there is no correlation between IR-inducible miR-34a expression and IR-induced rapid and transient G2/M arrest. In sum, our novel data for the first time demonstrate that IR-induced p53 Ser15 phosphorylation via p38 MAPK is essential for its functional regulation of IR-inducible miR-34a transcription in human mammary epithelial cells, and that histone modifications may also play a key role in IR-inducible miR-34a expression.

Mechanisms of ceramide‐induced COX‐2‐dependent apoptosis in human ovarian cancer OVCAR‐3 cells partially overlapped with resveratrol

Ceramide is a member of the sphingolipid family of bioactive molecules demonstrated to have profound, diverse biological activities. Ceramide is a potential chemotherapeutic agent via the induction of apoptosis. Exposure to ceramide activates extracellular-signal-regulated kinases (ERK)1/2- and p38 kinase-dependent apoptosis in human ovarian cancer OVCAR-3 cells, concomitant with an increase in the expression of COX-2 and p53 phosphorylation. Blockade of cyclooxygenase-2 (COX-2) activity by siRNA or NS398 correspondingly inhibited ceramide-induced p53 Ser-15 phosphorylation and apoptosis; thus COX-2 appears at the apex of the p38 kinase-mediated signaling cascade induced by ceramide. Induction of apoptosis by ceramide or resveratrol was inhibited by the endocytosis inhibitor, cytochalasin D (CytD); however, cells exposed to resveratrol showed greater sensitivity than ceramide-treated cells. Ceramide-treated cells underwent a dose-dependent reduction in trans-membrane potential. Although both ceramide and resveratrol induced the expressions of caspase-3 and -7, the effect of inducible COX-2 was different in caspase-7 expression induced by ceramide compared to resveratrol. In summary, resveratrol and ceramide converge on an endocytosis-requiring, ERK1/2-dependent signal transduction pathway and induction of COX-expression as an essential molecular antecedent for subsequent p53-dependent apoptosis. In addition, expressions of caspase-3 and -7 are observed. However, a p38 kinase-dependent signal transduction pathway and change in mitochondrial potential are also involved in ceramide-induced apoptosis.

Down-regulation of Wild-type p53-induced Phosphatase 1 (Wip1) Plays a Critical Role in Regulating Several p53-dependent Functions in Premature Senescent Tumor Cells

Premature or drug-induced senescence is a major cellular response to chemotherapy in solid tumors. The senescent phenotype develops slowly and is associated with chronic DNA damage response. We found that expression of wild-type p53-induced phosphatase 1 (Wip1) is markedly down-regulated during persistent DNA damage and after drug release during the acquisition of the senescent phenotype in carcinoma cells. We demonstrate that down-regulation of Wip1 is required for maintenance of permanent G2 arrest. In fact, we show that forced expression of Wip1 in premature senescent tumor cells induces inappropriate re-initiation of mitosis, uncontrolled polyploid progression, and cell death by mitotic failure. Most of the effects of Wip1 may be attributed to its ability to dephosphorylate p53 at Ser(15) and to inhibit DNA damage response. However, we also uncover a regulatory pathway whereby suppression of p53 Ser(15) phosphorylation is associated with enhanced phosphorylation at Ser(46), increased p53 protein levels, and induction of Noxa expression. On the whole, our data indicate that down-regulation of Wip1 expression during premature senescence plays a pivotal role in regulating several p53-dependent aspects of the senescent phenotype.

Abstract 2246: Steam distilled ginger extract inhibits endometrial cancer cell proliferation by activating P53 and causing apoptosis.

Abstract Phenolic compounds present in dry ginger powder or solvent extracts of ginger roots induce cell cycle arrest and apoptosis in skin, breast, prostate, colon, and ovarian cancer cells. Another class of bioactive compounds, the terpenes, can be isolated by steam distillation of the ginger rhizomes. In this study, we examine the anti-cancer properties of Steam Distilled Ginger Extracts (SDGE) on endometrial cancer cells. SDGE at an IC-50 of 1.5 micrograms/ml inhibited the proliferation of two endometrial cancer cell lines, ECC-1 and Ishikawa. Decreased proliferation of Ishikawa and ECC-1 cells was a direct result of SDGE-induced-apoptosis as demonstrated by Annexin V FITC staining and increased expression of cleaved caspase 3. GC-MS analysis allowed us to identify 22 distinct terpenes in SDGE. Gingerols and other non-terpene phenolic compounds were not present in SDGE. Neral and Geranial constituted 25-35% of the total SDGE terpenes and were found to inhibit endometrial cancer cell proliferation. On the other hand, camphene and α-pinene, which together constitute 10% of SDGE, showed no effect on the proliferation of ECC-1 and Ishikawa cells. SDGE at concentrations as low as 25 nanograms/ml caused a rapid increase in intracellular calcium levels and an approximate decrease of 20% in the mitochondrial membrane potential. Ser-15 of p53 was phosphorylated after a 15 minute treatment of the cancer cells with SDGE (250 nanograms/ml). This activation of p53 was associated with a 90% decrease in Bcl2, whereas no effect was observed on Bax. Pifithrin-α, an inhibitor of p53, attenuated the anti-cancer effects of SDGE that result from apoptosis. In addition, SDGE (1.5 microgram/ml) was unable to induce apoptosis in the p53-negative ovarian cancer cell line, SKOV3. Our data therefore indicates that specific terpenes present in SDGE are highly efficient in controlling the growth of p53-expressing cancer cells. We therefore propose that SDGE and its constituent bioactive terpenes should be further investigated as anti-cancer agents. Citation Format: Yang Liu, Rebecca J. Whelan, Bikash R. Pattnaik, Kai David Ludwig, Rosalina V. Landeros, Enkateswar Subudhi, Helen Rowland, Nicholas Claussen, Noah Zucker, Shitanshu Uppal, David M. Kushner, Mildred Felder, Manish S. Patankar, Arvinder K. Kapur. Steam distilled ginger extract inhibits endometrial cancer cell proliferation by activating P53 and causing apoptosis. [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 2246. doi:10.1158/1538-7445.AM2013-2246

P53 activation by Ni(II) is a HIF-1α independent response causing caspases 9/3-mediated apoptosis in human lung cells

Hypoxia mimic nickel(II) is a human respiratory carcinogen with a suspected epigenetic mode of action. We examined whether Ni(II) elicits a toxicologically significant activation of the tumor suppressor p53, which is typically associated with genotoxic responses. We found that treatments of H460 human lung epithelial cells with NiCl2 caused activating phosphorylation at p53-Ser15, accumulation of p53 protein and depletion of its inhibitor MDM4 (HDMX). Confirming the activation of p53, its knockdown suppressed the ability of Ni(II) to upregulate MDM2 and p21 (CDKN1A). Unlike DNA damage, induction of GADD45A by Ni(II) was p53-independent. Ni(II) also increased p53-Ser15 phosphorylation and p21 expression in normal human lung fibroblasts. Although Ni(II)-induced stabilization of HIF-1α occurred earlier, it had no effect on p53 accumulation and Ser15 phosphorylation. Ni(II)-treated H460 cells showed no evidence of necrosis and their apoptosis and clonogenic death were suppressed by p53 knockdown. The apoptotic role of p53 involved a transcription-dependent program triggering the initiator caspase 9 and its downstream executioner caspase 3. Two most prominently upregulated proapoptotic genes by Ni(II) were PUMA and NOXA but only PUMA induction required p53. Knockdown of p53 also led to derepression of antiapoptotic MCL1 in Ni(II)-treated cells. Overall, our results indicate that p53 plays a major role in apoptotic death of human lung cells by Ni(II). Chronic exposure to Ni(II) may promote selection of resistant cells with inactivated p53, providing an explanation for the origin of p53 mutations by this epigenetic carcinogen.

The effect of ATM kinase inhibition on the initial response of human dental pulp and periodontal ligament mesenchymal stem cells to ionizing radiation

Purpose: This study evaluates early changes in human mesenchymal stem cells (MSC) isolated from dental pulp and periodontal ligament after γ-irradiation and the effect of ataxia-telangiectasia mutated (ATM) inhibition.Methods: MSC were irradiated with 2 and 20 Gy by 60Co. For ATM inhibition, specific inhibitor KU55933 was used. DNA damage was measured by Comet assay and γH2AX detection. Cell cycle distribution and proteins responding to DNA damage were analyzed 2–72 h after the irradiation.Results: The irradiation of MSC causes an increase in γH2AX; the phosphorylation was ATM-dependent. Irradiation activates ATM kinase, and the level of p53 protein is increased due to its phosphorylation on serine15. While this phosphorylation of p53 is ATM-dependent in MSC, the increase in p53 was not prevented by ATM inhibition. A similar trend was observed for Chk1 and Chk2. The increase in p21 is greater without ATM inhibition. ATM inhibition also does not fully abrogate the accumulation of irradiated MSC in the G2-phase of the cell-cycle.Conclusions: In irradiated MSC, double-strand breaks are tagged quickly by γH2AX in an ATM-dependent manner. Although phosphorylations of p53(ser15), Chk1(ser345) and Chk2(thr68) are ATM-dependent, the overall amount of these proteins increases when ATM is inhibited. In both types of MSC, ATM-independent mechanisms for cell-cycle arrest in the G2-phase are triggered.

Autophagy and Cellular Senescence Mediated by Sox2 Suppress Malignancy of Cancer Cells

Autophagy is a critical cellular process required for maintaining cellular homeostasis in health and disease states, but the molecular mechanisms and impact of autophagy on cancer is not fully understood. Here, we found that Sox2, a key transcription factor in the regulation of the “stemness” of embryonic stem cells and induced-pluripotent stem cells, strongly induced autophagic phenomena, including intracellular vacuole formation and lysosomal activation in colon cancer cells. The activation occurred through Sox2-mediated ATG10 gene expression and resulted in the inhibition of cell proliferation and anchorage-independent colony growth ex vivo and tumor growth in vivo. Further, we found that Sox2-induced-autophagy enhanced cellular senescence by up-regulating tumor suppressors or senescence factors, including p16INK4a, p21 and phosphorylated p53 (Ser15). Notably, knockdown of ATG10 in Sox2-expressing colon cancer cells restored cancer cell properties. Taken together, our results demonstrated that regulation of autophagy mediated by Sox2 is a mechanism-driven novel strategy to treat human colon cancers.

Indicators of replicative damage in equine tendon fibroblast monolayers

BackgroundSuperficial digital flexor tendon (SDFT) injuries of horses usually follow cumulative matrix microdamage; it is not known why the reparative abilities of tendon fibroblasts are overwhelmed or subverted. Relevant in vitro studies of this process require fibroblasts not already responding to stresses caused by the cell culture protocols. We investigated indicators of replicative damage in SDFT fibroblast monolayers, effects of this on their reparative ability, and measures that can be taken to reduce it.ResultsWe found significant evidence of replicative stress, initially observing consistently large numbers of binucleate (BN) cells. A more variable but prominent feature was the presence of numerous gammaH2AX (γH2AX) puncta in nuclei, this being a histone protein that is phosphorylated in response to DNA double-stranded breaks (DSBs). Enrichment for injury detection and cell cycle arrest factors (p53 (ser15) and p21) occurred most frequently in BN cells; however, their numbers did not correlate with DNA damage levels and it is likely that the two processes have different causative mechanisms. Such remarkable levels of injury and binucleation are usually associated with irradiation, or treatment with cytoskeletal-disrupting agents.Both DSBs and BN cells were greatest in subconfluent (replicating) monolayers. The DNA-damaged cells co-expressed the replication markers TPX2/repp86 and centromere protein F. Once damaged in the early stages of culture establishment, fibroblasts continued to express DNA breaks with each replicative cycle. However, significant levels of cell death were not measured, suggesting that DNA repair was occurring. Comet assays showed that DNA repair was delayed in proportion to levels of genotoxic stress.ConclusionsResearchers using tendon fibroblast monolayers should assess their “health” using γH2AX labelling. Continued use of early passage cultures expressing initially high levels of γH2AX puncta should be avoided for mechanistic studies and ex-vivo therapeutic applications, as this will not be resolved with further replicative cycling. Low density cell culture should be avoided as it enriches for both DNA damage and mitotic defects (polyploidy). As monolayers differing only slightly in baseline DNA damage levels showed markedly variable responses to a further injury, studies of effects of various stressors on tendon cells must be very carefully controlled.

Terpenoids from Zingiber officinale (Ginger) induce apoptosis in endometrial cancer cells through the activation of p53.

Novel strategies are necessary to improve chemotherapy response in advanced and recurrent endometrial cancer. Here, we demonstrate that terpenoids present in the Steam Distilled Extract of Ginger (SDGE) are potent inhibitors of proliferation of endometrial cancer cells. SDGE, isolated from six different batches of ginger rhizomes, consistently inhibited proliferation of the endometrial cancer cell lines Ishikawa and ECC-1 at IC50 of 1.25 µg/ml. SDGE also enhanced the anti-proliferative effect of radiation and cisplatin. Decreased proliferation of Ishikawa and ECC-1 cells was a direct result of SDGE-induced apoptosis as demonstrated by FITC-Annexin V staining and expression of cleaved caspase 3. GC/MS analysis identified a total of 22 different terpenoid compounds in SDGE, with the isomers neral and geranial constituting 30–40%. Citral, a mixture of neral and geranial inhibited the proliferation of Ishikawa and ECC-1 cells at an IC50 10 µM (2.3 µg/ml). Phenolic compounds such as gingerol and shogaol were not detected in SDGE and 6-gingerol was a weaker inhibitor of the proliferation of the endometrial cancer cells. SDGE was more effective in inducing cancer cell death than citral, suggesting that other terpenes present in SDGE were also contributing to endometrial cancer cell death. SDGE treatment resulted in a rapid and strong increase in intracellular calcium and a 20–40% decrease in the mitochondrial membrane potential. Ser-15 of p53 was phosphorylated after 15 min treatment of the cancer cells with SDGE. This increase in p53 was associated with 90% decrease in Bcl2 whereas no effect was observed on Bax. Inhibitor of p53, pifithrin-α, attenuated the anti-cancer effects of SDGE and apoptosis was also not observed in the p53neg SKOV-3 cells. Our studies demonstrate that terpenoids from SDGE mediate apoptosis by activating p53 and should be therefore be investigated as agents for the treatment of endometrial cancer.

S-phase sensing of DNA-protein crosslinks triggers TopBP1-independent ATR activation and p53-mediated cell death by formaldehyde

We examined genotoxic signaling and cell fate decisions in response to a potent DNA-protein crosslinker formaldehyde (FA). DNA-protein crosslinks (DPC) are poorly understood lesions produced by bifunctional carcinogens and several cancer drugs. FA-treated human cells showed a rapid activation of ATR kinase that preferentially targeted the p53 transcription factor at low doses and CHK1 kinase at more severe damage, producing bell-shaped and sublinear responses, respectively. CHK1 phosphorylation was transient, and its loss was accompanied by increased p53 accumulation and Ser15 phosphorylation. Activation of p53 was insensitive to inhibition of mismatch repair and nucleotide and base excision repair, excluding the role of small DNA adducts in this response. The p53-targeted signaling was transcription-independent, absent in quiescent cells and specific to S-phase in cycling populations. Unlike other S-phase stressors, FA-activated p53 was functional transcriptionally, promoted apoptosis in lung epithelial cells and caused senescence in normal lung fibroblasts. FA did not induce ATR, RAD1 or RPA foci, and p53 phosphorylation was TopBP1-independent, indicating a noncanonical mode of ATR activation. Replication arrest by FA caused a dissociation of ATR from a chromatin-loaded MCM helicase but no PCNA monoubiquitination associated with stalled polymerases. These results suggest that unlike typical DNA adducts that stall DNA polymerases, replication inhibition by bulkier DPC largely results from blocking upstream MCM helicase, which prevents accumulation of ssDNA. Overall, our findings indicate that S-phase-specific, TopBP1-independent activation of the ATR-p53 axis is a critical stress response to FA-DPC, which has implications for understanding of FA carcinogenesis.

P53 Ser15 phosphorylation disrupts the p53–RPA70 complex and induces RPA70-mediated DNA repair in hypoxia

Cellular stressors are known to inhibit the p53-RPA70 (replication protein A, 70 kDa subunit) complex, and RPA70 increases cellular DNA repair in cancer cells. We hypothesized that regulation of RPA70-mediated DNA repair might be responsible for the inhibition of apoptosis in hypoxic tumours. We have shown that, in cancer cells, hypoxia disrupts the p53-RPA70 complex, thereby enhancing RPA70-mediated NER (nucleotide excision repair)/NHEJ (non-homologous end-joining) repair. In normal cells, RPA70 binds to the p53-NTD (N-terminal domain), whereas this binding is disrupted in hypoxia. Phosphorylation of p53-NTD is a crucial event in dissociating both NTD-RPA70 and p53-RPA70 complexes. Serial mutations at serine and threonine residues in the NTD confirm that p53(Ser15) phosphorylation induces dissociation of the p53-RPA70 complex in hypoxia. DNA-PK (DNA-dependent protein kinase) is shown to induce p53(Ser15) phosphorylation, thus enhancing RPA70-mediated NER/NHEJ repair. Furthermore, RPA70 gene silencing induces significant increases in cellular apoptosis in the resistant hypoxic cancer cells. We have thus elucidated a novel pathway showing how DNA-PK-mediated p53(Ser15) phosphorylation dissociates the p53-RPA70 complex, thus enhancing NER/NHEJ repair, which causes resistance to apoptosis in hypoxic cancer cells. This novel finding may open new strategies in developing cancer therapeutics on the basis of the regulation of RPA70-mediated NER/NHEJ repair.

Combination of p53(ser15) and p21/p21(thr145) in peripheral blood lymphocytes as potential Alzheimer's disease biomarkers

Alzheimer's disease (AD) is still difficult to be precisely diagnosed in its early stage to date. Establishing of reliable and manageable disease-specific biological markers is required to improve diagnostic accuracy. Based on the hypothesis of cell cycle regulatory failure at the early stage of AD, we tested whether cell cycle regulating proteins p53, p21 and their phosphorylated forms p53(ser15), p21(thr145) were changed in AD patients and whether these proteins could be used as diagnostic biomarkers. Western bolt, Enzyme-linked immunosorbent assay (ELISA), immunofluorescent staining and flow cytometry (FCM) analysis were employed to analyze levels of these proteins in peripheral blood lymphocytes (PBLs) from 95 controls, 94 AD, 12 Parkinson's disease (PD) and 15 vascular dementia (VaD) patients. Compared with controls, p53(ser15) and p21(thr145) levels were significantly increased and p21 level was significantly decreased in PBLs of AD patients but not in PD or VaD, while p53 was increased in both AD and VaD patients. The receiver operating characteristic (ROC) curve analysis showed that the specificity and sensitivity were 76% and 84% for p53, 88% and 82% for p53(ser15), 80% and 75% for p21 and 84% and 68% for p21(thr145) in identifying AD patients. The relatively high diagnostic accuracy support these proteins, especially p53(ser15) and p21 in PBLs may become potential biomarkers for diagnosis of AD.