P53 Ser15 Research Articles

Orabase-formulated gentian violet effectively improved oral potentially malignant disorder in vitro and in vivo

Oral cancer is a prevalent cancer in male worldwide. Oral potentially malignant disorders (OMPDs) are the oral mucosa lesions that have high malignant transformation rate to oral cancer. The mainstay for OMPDs treatment includes carbon dioxide (CO2) laser and surgery, which may lead to the side effects of scarring and impaired function of oral cavity in the patients and reduced their willingness to receive curative therapy. Therefore, developing a non-invasive and function-preserving therapy is clinically important. Since development of a novel chemotherapeutic drug requires a lot of time and cost, we applied the high-throughput screening (HTS) approach to identify new bioactivities for FDA-approved drugs, known as drug repurposing. Through this drug repurposing approach, we discovered that gentian violet (GV), which is well known for its antibacterial, antifungal, antihelminthic, antitrypanosomal and antiviral activities, was able to induce significant cell death in DOK oral precancerous cells through ROS production. Moreover, decreased phosphorylation of p53(Ser15) and NFκB(Ser536) was required for GV-induced cell death. In vivo, 3% GV orabase effectively suppressed the progression of DMBA-induced oral precancerous lesions. In conclusion, this new formulation of GV through drug repurposing has the potential to be further developed as a therapeutic drug for OPMD clinically.

Cell Fate Regulation upon DNA Damage: p53 Serine 46 Kinases Pave the Cell Death Road.

Mild and massive DNA damage are differentially integrated into the cellular signaling networks and, in consequence, provoke different cell fate decisions. After mild damage, the tumor suppressor p53 directs the cellular response to cell cycle arrest, DNA repair, and cell survival, whereas upon severe damage, p53 drives the cell death response. One posttranslational modification of p53, phosphorylation at Serine 46, selectively occurs after severe DNA damage and is envisioned as a marker of the cell death response. However, the molecular mechanism of action of the p53 Ser46 phospho-isomer, the molecular timing of this phosphorylation event, and its activating effects on apoptosis and ferroptosis still await exploration. In this essay, the current body of evidence on the molecular function of this deadly p53 mark, its evolutionary conservation, and the regulation of the key players of this response, the p53 Serine 46 kinases, are reviewed and dissected.

Anticancer Drug-Induced Epithelial-Mesenchymal Transition via p53/miR-34a axis in A549/ABCA3 Cells.

Several anticancer drugs including bleomycin (BLM) and methotrexate (MTX) cause serious lung diseases such as pulmonary fibrosis. Although evidences showing the association of epithelial-mesenchymal transition (EMT) with pulmonary fibrosis are increasing, the mechanism underlying anticancer drug-induced EMT has been poorly understood. On the other hand, miR-34a, a non-coding small RNA, has been highlighted as a key factor to regulate EMT in lung. In this study, we elucidated the role of miR-34a in anticancer drug-induced EMT using A549/ABCA3 cells as a novel type II alveolar epithelium model. Expression levels of α-smooth muscle actin (α-SMA) mRNA, miR-34a, and p53 were evaluated by real-time PCR and western blot analysis, respectively. BLM and MTX induced EMT-like morphological changes and increase in mRNA expression level of α-SMA, an EMT marker. Also, both drugs increased the expression level of miR-34a. Furthermore, mRNA expression level of α-SMA was enhanced by introduction of miR-34a mimic into A549/ABCA3 cells. To examine the mechanism underlying drug-induced enhancement of miR-34a expression, we focused on p53/miR-34a axis. Both drugs upregulated protein expression of p53, an inducer of miR-34a, as well as phosphorylation of Ser15 in p53. These findings indicated that p53/miR-34a axis may contribute to anticancer drug-induced EMT in type II alveolar epithelial cells.

Temporal and differential regulation of KAISO-controlled transcription by phosphorylated and acetylated p53 highlights a crucial regulatory role of apoptosis

Transcriptional regulator KAISO plays a critical role in cell cycle arrest and apoptosis through modulation of p53 acetylation by histone acetyltransferase p300. KAISO potently stimulates apoptosis in cells expressing WT p53, but not in p53-mutant or p53-null cells. Here, we investigated how KAISO transcription is regulated by p53, finding four potential p53-binding sites (p53-responsive DNA elements; p53REs) located in a distal 5'-upstream regulatory element, intron 1, exon 2 coding sequence, and a 3'-UTR region. Transient transcription assays of pG5-p53RE-Luc constructs with various p53REs revealed that p53 activates KAISO (ZBTB33) transcription by acting on p53RE1 (-4326 to -4227) of the 5'-upstream region and on p53RE3 (+2929 to +2959) of the exon 2 coding region during early DNA damage responses (DDRs). ChIP and oligonucleotide pulldown assays further disclosed that p53 binds to the p53RE1 and p53RE3 sites. Moreover, ataxia telangiectasia mutated (ATM) or ATM-Rad3-related (ATR) kinase-mediated p53 phosphorylation at Ser-15 or Ser-37 residues activated KAISO transcription by binding its p53RE1 or p53RE3 sites during early DDR. p53RE1 uniquely contained three p53-binding half-sites, a structural feature important for transcriptional activation by phosphorylated p53 Ser-15·Ser-37. During the later DDR phase, a KAISO-mediated acetylated p53 form (represented by a p53QRQ acetyl-mimic) robustly activated transcription by acting on p53RE1 in which this structural feature is not significant, but it provided sufficient KAISO levels to confer a p53 "apoptotic code." These results suggest that the critical apoptosis regulator KAISO is a p53 target gene that is differently regulated by phosphorylated p53 or acetylated p53, depending on DDR stage.

Sulfur Mustard Analog Mechlorethamine (Bis(2-chloroethyl)methylamine) Modulates Cell Cycle Progression via the DNA Damage Response in Human Lung Epithelial A549 Cells.

Nitrogen mustard, mechlorethamine (bis(2-chloroethyl)methylamine; HN2), and sulfur mustard are potent vesicants that modify and disrupt cellular macromolecules including DNA leading to cytotoxicity and tissue injury. In many cell types, HN2 upregulates DNA damage signaling pathways including ataxia telangiectasia mutated (ATM), ataxia telangiectasia mutated- and Rad3-related (ATR) as well as DNA-dependent protein kinase (DNA-PK). In the present studies, we investigated crosstalk between the HN2-induced DNA damage response and cell cycle progression using human A549 lung epithelial cells. HN2 (1-20 μM; 24 h) caused a concentration-dependent arrest of cells in the S and G2/M phases of the cell cycle. This was associated with inhibition of DNA synthesis, as measured by incorporation of 5-ethynyl-2'-deoxyuridine (EdU) into S phase cells. Cell cycle arrest was correlated with activation of DNA damage and cell cycle checkpoint signaling. Thus, HN2 treatment resulted in time- and concentration-dependent increases in expression of phosphorylated ATM (Ser1981), Chk2 (Thr68), H2AX (Ser139), and p53 (Ser15). Activation of DNA damage signaling was most pronounced in S-phase cells followed by G2/M-phase cells. HN2-induced cell cycle arrest was suppressed by the ATM and DNA-PK inhibitors, KU55933 and NU7441, respectively, and to a lesser extent by VE821, an ATR inhibitor. This was correlated with abrogation of DNA damage checkpoints signaling. These data indicate that activation of ATM, ATR, and DNA-PK signaling pathways by HN2 are important in the mechanism of vesicant-induced cell cycle arrest and cytotoxicity. Drugs that inhibit activation of DNA damage signaling may be effective countermeasures for vesicant-induced tissue injury.

The Role of p53 in Determining Mitochondrial Adaptations to Endurance Training in Skeletal Muscle

p53 plays an important role in regulating mitochondrial homeostasis. However, it is unknown whether p53 is required for the physiological and mitochondrial adaptations with exercise training. Furthermore, it is also unknown whether impairments in the absence of p53 are a result of its loss in skeletal muscle, or a secondary effect due to its deletion in alternative tissues. Thus, we investigated the role of p53 in regulating mitochondria both basally, and under the influence of exercise, by subjecting C57Bl/6J whole-body (WB) and muscle-specific p53 knockout (mKO) mice to a 6-week training program. Our results confirm that p53 is important for regulating mitochondrial content and function, as well as proteins within the autophagy and apoptosis pathways. Despite an increased proportion of phosphorylated p53 (Ser15) in the mitochondria, p53 is not required for training-induced adaptations in exercise capacity or mitochondrial content and function. In comparing mouse models, similar directional alterations were observed in basal and exercise-induced signaling modifications in WB and mKO mice, however the magnitude of change was less pronounced in the mKO mice. Our data suggest that p53 is required for basal mitochondrial maintenance in skeletal muscle, but is not required for the adaptive responses to exercise training.

Activated mitochondrial apoptosis in hESCs after dissociation involving the PKA/p-p53/Bax signaling pathway

Human embryonic stem cells (hESCs) are highly fragile with massive cell death after dissociation into single cells, which seriously hampers their applications. The mechanism underlying the massive cell death after dissociation still remains elusive. Here, the expression of apoptosis-related proteins, cell survival and mitochondrial membrane potential in dissociated hESCs before and after the treatments with a protein kinase A (PKA) inhibitor H89 and p53 inhibitor Pifithrin α were investigated, respectively. Protein interactions were identified by immunoprecipitation and immunofluorescence. The results show that the dissociation causes Caspase-dependent apoptosis in hESCs mediated by mitochondrial pathway with the up-regulation of pro-apoptotic proteins, decrease in mitochondrial membrane potential and elevation in pro-apoptotic Cyto c release, which are obviously suppresses by H89. The dissociation-induced increase of phosphorylated p53 Ser15 (p-p53) is suppressed by Pifithrin α which also rescues the elevated levels of pro-apoptotic proteins in mitochondrial pathway. During the dissociation-induced apoptosis, PKA/p-p53/Bax signaling pathway is identified by immunoprecipitation and immunofluorescence showing the most likely interaction between them. These results indicate that dissociation induces mitochondrial apoptosis in hESCs involving PKA/p-p53/Bax signaling pathway, which not only give new insights into the apoptosis mechanism of dissociated hESCs, but also provide clues for developing potential strategies to promote hESC survival after dissociation.

Differential proteomic profile of leukemic CD34+ progenitor cells from chronic myeloid leukemia patients.

Chronic Myeloid Leukemia (CML) is a stem cell disease sustained by a rare population of quiescent cells which are to some extent resistant to tyrosine kinase inhibitors (TKIs). BCR-ABL oncogene activates multiple cross-talking signal transduction pathways (STP), such as RAS/MEK/ERK, PI3K/Akt, Wnt and STAT5, contributing to abnormal proliferation of clonal cells. From this perspective, the aim of this study was to analyze the expression and activation profile of STP involved in the mechanisms of cell proliferation/quiescence and survival of the progenitor CD34+ cells from chronic phase (CP) CML.Our results showed that CP-CML CD34+ progenitors were characterized by significant lower phosphorylation of proteins involved in the regulation of growth and cell survival, such as tyrosine kinases of the Src family and members of STAT family, and by a significant higher phosphorylation of p53 (Ser15), compared to normal CD34+ cells from healthy donors. Consistent with these results, cell cycle analysis demonstrated that CP-CML CD34+ cells were characterized by higher percentage of cells in G0-phase compared to normal CD34+ cells. Analysis of expression profile on proteins involved in the apoptotic machinery revealed that, in addition, CD34+ cells from CP-CML were characterized by a significant lower expression of catalase and higher expression of HSP27 and FADD. In sum, we report that CD34+ cells from CP-CML are characterized by a proteomic and phospho-proteomic profile that promotes quiescence through the inhibition of proliferation and the promotion of survival. This differential signaling activation network may be addressed by novel targeted therapies aimed at eradicating CML stem cells.

The small molecule AU14022 promotes colorectal cancer cell death via p53-mediated G2/M-phase arrest and mitochondria-mediated apoptosis.

The p53 tumor suppressor plays critical roles in cell cycle regulation and apoptotic cell death, with its activation capable of sensitizing cancer cells to radiotherapy or chemotherapy. To identify small molecules that induce apoptosis via increased p53 transcriptional activity, we used a novel in-house library containing 96 small-molecule compounds. Using a cell-based screening method with a p53-responsive luciferase-reporter assay system involving benzoxazole derivatives, we found that AU14022 administration significantly increased p53 transcriptional activity in a concentration-dependent manner. Treatment with AU14022 increased p53 protein expression, p53 Ser15 phosphorylation, p53-mediated expression of downstream target genes, and apoptosis in p53-wild-type HCT116 human colon cancer cells, but not in p53-knockout HCT116 cells. Additionally, p53-wild-type HCT116 cells treated with AU14022 exhibited mitochondrial dysfunction, including modulated expression of B-cell lymphoma-2 family proteins and cytochrome c release. Combination treatment with AU14022 and ionizing radiation (IR) synergistically induced apoptosis as compared with IR or AU14022 treatment alone, with further investigation demonstrating that cell cycle progression was significantly arrested at the G2/M phase following AU14022 treatment. Furthermore, in a mouse p53-wild-type HCT116 colon cancer xenograft model, combined treatment with AU14022 and IR inhibited tumor growth more effectively than radiation alone. Therefore, AU14022 treatment induced apoptosis through p53-mediated cell cycle arrest involving mitochondrial dysfunction, leading to enhanced radiosensitivity in colon cancer cells. These results provide a basis for further assessments of AU14022 as a promising anticancer agent.

Disruption of nNOS-NOS1AP protein-protein interactions suppresses neuropathic pain in mice.

Elevated N-methyl-D-aspartate receptor (NMDAR) activity is linked to central sensitization and chronic pain. However, NMDAR antagonists display limited therapeutic potential because of their adverse side effects. Novel approaches targeting the NR2B-PSD95-nNOS complex to disrupt signaling pathways downstream of NMDARs show efficacy in preclinical pain models. Here, we evaluated the involvement of interactions between neuronal nitric oxide synthase (nNOS) and the nitric oxide synthase 1 adaptor protein (NOS1AP) in pronociceptive signaling and neuropathic pain. TAT-GESV, a peptide inhibitor of the nNOS-NOS1AP complex, disrupted the in vitro binding between nNOS and its downstream protein partner NOS1AP but not its upstream protein partner postsynaptic density 95 kDa (PSD95). Putative inactive peptides (TAT-cp4GESV and TAT-GESVΔ1) failed to do so. Only the active peptide protected primary cortical neurons from glutamate/glycine-induced excitotoxicity. TAT-GESV, administered intrathecally (i.t.), suppressed mechanical and cold allodynia induced by either the chemotherapeutic agent paclitaxel or a traumatic nerve injury induced by partial sciatic nerve ligation. TAT-GESV also blocked the paclitaxel-induced phosphorylation at Ser15 of p53, a substrate of p38 MAPK. Finally, TAT-GESV (i.t.) did not induce NMDAR-mediated motor ataxia in the rotarod test and did not alter basal nociceptive thresholds in the radiant heat tail-flick test. These observations support the hypothesis that antiallodynic efficacy of an nNOS-NOS1AP disruptor may result, at least in part, from blockade of p38 MAPK-mediated downstream effects. Our studies demonstrate, for the first time, that disrupting nNOS-NOS1AP protein-protein interactions attenuates mechanistically distinct forms of neuropathic pain without unwanted motor ataxic effects of NMDAR antagonists.

The association between p53 protein phosphorylation at serine 15, serine 20 and sensitivity of cells isolated from patients with ovarian cancer and cell lines to chemotherapy in in vitro study.

The association between p53 protein phosphorylated at serine 15 (Ser15), serine 20 (Ser20) and ovarian tumor cell sensitivity after chemotherapy was analyzed in order to define the influence of p53 activation on tumor cell sensitivity to chemotherapy. The study was performed on ovarian cancer cell line (OvBH-1), colon adenocarcinoma metastasis to ovary (SW626) and on cells isolated from ascitic fluids from patients with ovarian cancer: with (p53+) or without (p53-) p53 nuclear protein accumulation. p53 protein, Ser15, Ser20, Bax, Noxa and PgP protein expression was evaluated by means of immunocytochemical staining before and after chemotherapy. Cell viability after treatment was estimated using MTT assay. Cell lines and tumor cells p53+, p53- revealed a significant decrease in cell survival after camptothecin, paclitaxel, cisplatin treatment, compared to the control group (p < 0.01). In p53+ group, the expression of Ser20 significantly increased after camptothecin and paclitaxel (p < 0.05). Ser15, Ser20, Bax, Noxa expression correlated with MTT and depended on p53+, p53- tumor cell and the drug used (p < 0.05). Expression of Bax and Noxa were dependent on the type of tumor cells and drug used. The correlation between Ser15, Ser20 and Bax, Noxa expression was found in cell lines and tumor cells (p < 0.05). Our study suggests that the relation between Ser15 or Ser20 and tumor cell viability might reflect their role in tumor sensitivity on chemotherapy in dependent p53 protein status. Revealed association between p53 protein phosphorylated at Ser15, Ser20 and Bax, Noxa protein expression determined the apoptotic activity of tumor cells.

Resveratrol induces sumoylated COX-2-dependent anti-proliferation in human prostate cancer LNCaP cells

Cyclooxygenase (COX)-2 has been implicated in cancer development. However, resveratrol-induced nuclear accumulation of COX-2 enhances p53-dependent anti-proliferation in different types of cancers. Treatment with resveratrol leads to phosphorylation and nuclear translocation of mitogen-activated protein kinase (ERK1/2), and accumulation of nuclear COX-2 to complex with pERK1/2 and p53. The consequence is Ser-15 phosphorylation of p53 (pSer15-p53), and induction of anti-proliferation in cancer cells. We investigated the mechanisms by which resveratrol-inducible COX-2 facilitates p53-dependent anti-proliferation in prostate cancer LNCaP cells. Resveratrol treatment caused nuclear accumulation and complexing of ERK1/2, pSer15-p53 and COX-2 which was activated ERK1/2-dependent. Knockdown of SUMO-1 by shRNA also reduced nuclear accumulation of COX-2. Inhibition of nuclear accumulation by the COX-2 specific inhibitor, NS-398, inhibited co-localization of nuclear COX-2 and SUMO-1. Similar results were observed in the PD98059-treated cells. Finally, inhibition of SUMO-1 expression also reduced resveratrol-induced expression of pro-apoptotic genes but increased the expression of proliferative genes. In summary, these results demonstrate that inducible COX-2 associates with phosphorylated ERK1/2 to induce the phosphorylation of Ser-15 in p53 and then complexes with p53 and SUMO-1 which binds to p53-responsive pro-apoptotic genes to enhance their expression. The inhibition of COX-2 expression and activity significantly blocks the pro-apoptotic effect of resveratrol.

Abstract 2580: Ceramide-rubusoside nano-micelles restores p53-mediated tumor suppression in p53R248W mutant ovarian cancer cells

Abstract Ceramide (Cer), a sphingolipid metabolite, has crucial functions in anti-proliferation, cell differentiation and apoptosis. Cellular Cer levels highly correlates to therapeutic efficacy of anticancer drugs, however, the hydrophobicity Cer has limits to develop it as a therapeutic agent. Here, we report that Cer-rubusoside (Cer-RUB) nanomicelles enhanced Cer bioavailability and restored p53-mediated tumor suppression. These nanomicelles significantly increased the levels of NBD-C6-Cer by more than 50-fold in serum, 40-fold in tumor and 70-fold in lung of tumor-bearing mice (1 mg/kg, i.p., 24 hr), respectively. Cer-RUB (3 µM) significantly increased OVCAR-3 (p53 R248W) cell sensitivity to cisplatin, and the IC50 values decreased by 3-fold, even though these values were no significant changes in A2780 (wt p53) cells. Furthermore, Cer-RUB treatments (1 mg/kg, i.p. every 3 days for 24 days) significantly increased cisplatin efficacy in tumor growth-arrest in OVCAR-3 tumor-mice, and the tumor volumes reduced by 52% (1653 vs. 805 mm3) in combination with cisplatin, compared to cisplatin alone. We found that ovarian cancer stem cells (CD44+/CD133+) decreased by 10-fold (0.85 vs. 0.08% of total cells) in OVCAR-3 tumors treated with Cer-RUB combined with cisplatin, as compared to cisplatin alone, respectively. Western blotting showed that Cer-RUB treatments selectively increased the expression levels of phosphorylated p53 (Ser15), BAX and p21 in OVCAR-3 cancer cells and tumors, respectively. These studies indicates that the gain-of-function (GOF) of p53 R248W leads OVCAR-3 cell resistance to cisplatin and tumor growth. Cer-RUB can restore p53 and eliminates the GOF of p53 R248W in cancers. Citation Format: sachin K. khiste, Zhijun Liu, Yong-Yu Liu. Ceramide-rubusoside nano-micelles restores p53-mediated tumor suppression in p53R248W mutant ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2580. doi:10.1158/1538-7445.AM2017-2580

The B6 -vitamer Pyridoxal is a Sensitizer of UVA-induced Genotoxic Stress in Human Primary Keratinocytes and Reconstructed Epidermis.

UVA-driven photooxidative stress in human skin may originate from excitation of specific endogenous chromophores acting as photosensitizers. Previously, we have demonstrated that 3-hydroxypyridine-derived chromophores including B6 -vitamers (pyridoxine, pyridoxamine and pyridoxal) are endogenous photosensitizers that enhance UVA-induced photooxidative stress in human skin cells. Here, we report that the B6 -vitamer pyridoxal is a sensitizer of genotoxic stress in human adult primary keratinocytes (HEKa) and reconstructed epidermis. Comparative array analysis indicated that exposure to the combined action of pyridoxal and UVA caused upregulation of heat shock (HSPA6, HSPA1A, HSPA1L, HSPA2), redox (GSTM3, EGR1, MT2A, HMOX1, SOD1) and genotoxic (GADD45A, DDIT3, CDKN1A) stress response gene expression. Together with potentiation of UVA-induced photooxidative stress and glutathione depletion, induction of HEKa cell death occurred only in response to the combined action of pyridoxal and UVA. In addition to activational phosphorylation indicative of genotoxic stress [p53 (Ser15) and γ-H2AX (Ser139)], comet analysis indicated the formation of Fpg-sensitive oxidative DNA lesions, observable only after combined exposure to pyridoxal and UVA. In human reconstructed epidermis, pyridoxal preincubation followed by UVA exposure caused genomic oxidative base damage, procaspase 3 cleavage and TUNEL positivity, consistent with UVA-driven photooxidative damage that may be relevant to human skin exposed to high concentrations of B6 -vitamers.

P53 induces miR199a-3p to suppress SOCS7 for STAT3 activation and renal fibrosis in UUO

The role of p53 in renal fibrosis has recently been suggested, however, its function remains controversial and the underlying mechanism is unclear. Here, we show that pharmacological and genetic blockade of p53 attenuated renal interstitial fibrosis, apoptosis, and inflammation in mice with unilateral urethral obstruction (UUO). Interestingly, p53 blockade was associated with the suppression of miR-215-5p, miR-199a-5p&3p, and STAT3. In cultured human kidney tubular epithelial cells (HK-2), TGF-β1 treatment induced fibrotic changes, including collagen I and vimentin expression, being associated with p53 accumulation, p53 Ser15 phosphorylation, and miR-199a-3p expression. Inhibition of p53 by pifithrin-α blocked STAT3 activation and the expression of miR-199a-3p, collagen I, and vimentin during TGF-β1 treatment. Over-expression of miR-199a-3p increased TGFβ1-induced collagen I and vimentin expression and restored SOCS7 expression. Furthermore, SOCS7 was identified as a target gene of miR-199a-3p, and silencing of SOCS7 promoted STAT3 activation. ChIp analyses indicated the binding of p53 to the promoter region of miR-199a-3p. Consistently, kidney biopsies from patients with IgA nephropathy and diabetic nephropathy exhibited substantial activation of p53 and STAT3, decreased expression of SOCS7, and increase in profibrotic proteins and miR-199a-3p. Together, these results demonstrate the novel p53/miR-199a-3p/SOCS7/STAT3 pathway in renal interstitial fibrosis.

ZIKA virus elicits P53 activation and genotoxic stress in human neural progenitors similar to mutations involved in severe forms of genetic microcephaly and p53.

Epidemiological evidence from the current outbreak of Zika virus (ZIKV) and recent studies in animal models indicate a strong causal link between ZIKV and microcephaly. ZIKV infection induces cell-cycle arrest and apoptosis in proliferating neural progenitors. However, the mechanisms leading to these phenotypes are still largely obscure. In this report, we explored the possible similarities between transcriptional responses induced by ZIKV in human neural progenitors and those elicited by three different genetic mutations leading to severe forms of microcephaly in mice. We found that the strongest similarity between all these conditions is the activation of common P53 downstream genes. In agreement with these observations, we report that ZIKV infection increases total P53 levels and nuclear accumulation, as well as P53 Ser15 phosphorylation, correlated with genotoxic stress and apoptosis induction. Interestingly, increased P53 activation and apoptosis are induced not only in cells expressing high levels of viral antigens but also in cells showing low or undetectable levels of the same proteins. These results indicate that P53 activation is an early and specific event in ZIKV-infected cells, which could result from cell-autonomous and/or non-cell-autonomous mechanisms. Moreover, we highlight a small group of P53 effector proteins that could act as critical mediators, not only in ZIKV-induced microcephaly but also in many genetic microcephaly syndromes.

Abstract A20: Excess centrosomes induce p53-dependent senescence in endothelial cells

Abstract Excess centrosomes (&amp;gt;2 centrosomes/cell), which are prevalent in both tumor cells and tumor endothelial cells, lead to aneuploidy and chromosome instability. Therefore, understanding how excess centrosomes affect cell behaviors is critical for studying tumor progression and tumor angiogenesis. As demonstrated by previous literature, cells with excess centrosomes routinely undergo bipolar cell division to produce viable progeny. However, we found that endothelial cells were not able to maintain the percentage of cells with excess centrosomes, suggesting that excess centrosomes had a negative impact on endothelial cell cycle. We used a tetracycline-inducible Polo-like kinase 4 (Plk4)-expressing system to induce excess centrosomes in human umbilical vein endothelial cells (HUVEC). Doxycycline treatment induced ~30% centrosome over-duplication, which decreased to &amp;lt;5% in 6 days. We found that HUVEC with excess centrosomes activated p53 by phosphorylating p53 at Ser33. Complete down-regulation of p53 helped maintain the frequency of cells with excess centrosomes. Excess centrosomes-induced p53 activation was independent of DNA damage, as demonstrated by lack of p53 Ser15 phosphorylation or γH2AX foci. Excess centrosomes induced senescence but not apoptosis, as shown by senescence-associated beta-galactosidase (SA-β-gal) activity and Galactosidase Beta 1 (GLB1) antibody staining, and the induced senescence can be blocked by p53 down-regulation. Since endothelial cells maintain different cell shapes and behavior in a 3-dimensional (3D) environment compared to 2D, we asked whether excess centrosomes induce senescence in 3D. We used a sprouting angiogenesis assay and found that centrosome over-duplicated ECs in 3D sprouts showed SA-β-gal activity, suggesting that they underwent senescence. In summary, we show that excess centrosomes induced p53-dependent senescence in endothelial cells in both 2D and 3D. Our work provides a novel association between excess centrosomes and senescence, which will help to better understand regulation of the tumor microenvironment. Citation Format: Zhixian Yu, Erich Kushner, Victoria Bautch. Excess centrosomes induce p53-dependent senescence in endothelial cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr A20.

PS2 - 201 Bone Marrow-Derived Mesenchymal Stem Cells-Mediated Radioresistance in Glioma

Ionizing radiation (IR) is one of the conventional post-surgical treatments for Glioblastoma Multiforme (GBM). Mesenchymal stem cells (MSCs) constitute a subpopulation of bone marrow derived cells which are actively recruited to the site of radiation and/or tumour microenvironment (TME), both of which have important implications for neovascularization and tumor progression. The goal of this project is to investigate the functional contribution of MSCs in the TME. We postulate that Bone Marrow-MSCs promote radio-resistance in GBM via cell cycle arrest. We tested the effect of MSC on U87 glioblastoma cell line in response to IR. We found that MSC co-culture, MSC-conditioned media (MSCCM) and irradiated MSC-conditioned media (MSCIRCM) did not reduce IR-induced p53 (ser15) phosphorylation, signifying intact p53-dependent DNA damage pathway in all conditions. However, both MSCCM and MSCIRCM temporally increased phospho-Chk2, a kinase involved in ATM-dependent cascade and cell cycle arrest. This increase occurred at 24 hours and reverted to baseline levels by 48 hours. Interestingly, IR (15Gy) caused transiently heightened metabolic rate under MSC and MSC IRCM as opposed to IR-null treatment at 48 hours elevated cell proliferation. MSCCM, but not MSCIRCM, marginally reduced caspase 3/7-dependent apoptotic levels. The combination of IR and MSCCM as well as MSCIRCM first increased protein level of phospho-Chk2 at 24 hours; followed by increased metabolic rate at 48 hours; and lastly, boosted proliferation at 72 hours. This data combined proposes plausible machinery for BM-MSC mediated radio-resistance by initiating cell cycle arrest in tumour cells for DNA damage repair.

ZIKA virus elicits P53 activation and genotoxic stress in human neural progenitors similar to mutations involved in severe forms of genetic microcephaly.

Epidemiological evidence from the current outbreak of Zika virus (ZIKV) and recent studies in animal models indicate a strong causal link between ZIKV and microcephaly. ZIKV infection induces cell-cycle arrest and apoptosis in proliferating neural progenitors. However, the mechanisms leading to these phenotypes are still largely obscure. In this report, we explored the possible similarities between transcriptional responses induced by ZIKV in human neural progenitors and those elicited by three different genetic mutations leading to severe forms of microcephaly in mice. We found that the strongest similarity between all these conditions is the activation of common P53 downstream genes. In agreement with these observations, we report that ZIKV infection increases total P53 levels and nuclear accumulation, as well as P53 Ser15 phosphorylation, correlated with genotoxic stress and apoptosis induction. Interestingly, increased P53 activation and apoptosis are induced not only in cells expressing high levels of viral antigens but also in cells showing low or undetectable levels of the same proteins. These results indicate that P53 activation is an early and specific event in ZIKV-infected cells, which could result from cell-autonomous and/or non-cell-autonomous mechanisms. Moreover, we highlight a small group of P53 effector proteins that could act as critical mediators, not only in ZIKV-induced microcephaly but also in many genetic microcephaly syndromes.

Investigation of inducing apoptosis in human lung cancer A549 cells and related mechanism of a ruthenium(II) polypyridyl complex

Two Ru(II) polypyridyl complexes [Ru(bpy)2(pztp)]2+ (Ru1) and [Ru(bpy)2(pytp)]2+ (Ru2) were synthesized and characterized. Our data demonstrated that Ru2 displayed relatively higher cytotoxic activity against lung cancer A549 cells and had higher selectivity between tumor and normal cells in comparison with cisplatin. Studies on the molecular mechanism revealed that Ru2 caused cell cycle arrest at G2/M in A549 cells and induced apoptosis through a ROS-mediated mitochondrial dysfunction pathway. The further studies by comet assay at single cell level indicated that DNA damage in A549 cells was triggered by Ru2, following with the up-regulation of phosphorylated ATM (Ser1984), Histone H2A.X (Ser139) and p53 (Ser15). Western blot analysis suggested that MAPKs signaling pathways, especially ERK, were involved in Ru2-induced apoptosis. Moreover, both DNA damage and MAPKs signaling pathways were regulated by the level of ROS. To our knowledge, this is the first report of the ruthenium (II) polypyridyl complex which induces apoptosis partly through the activation of ERK.