Ser-15 Residue Research Articles

Enhancing the invasive traits of breast cancers by CYP1B1 via regulation of p53 to promote uPAR expression

Human cytochrome P450 1B1 (CYP1B1) catalyzes estrogen metabolism to produce metabolites that promote the progression of breast cancer. Since the invasive properties of cancer cells cause cancer relapse, which dramatically reduces patient survival, we investigated the new pro-invasive mechanism involving CYP1B1 in breast cancer. Exploring clinical data from invasive breast cancer patients revealed that CYP1B1 exhibits a potential correlation with urokinase-type plasminogen activator receptor (uPAR). Interestingly, uPAR mRNA expression was elevated in invasive breast cancer patients carrying TP53 genes with driver mutations, and our results showed that CYP1B1 activates the uPAR pathway following regulation of p53 according to its mutant status. CYP1B1 suppressed wild-type (WT) p53 whereas it induced the oncogenic gain-of-function mutant p53R280K, not only via transcriptional regulation but also the protein stabilization and activation following phosphorylation on Ser15 residue of p53R280K. Intriguingly, results from CYP1B1 polymorphic gene study and 4-hydroxyestradiol (4-OHE2) treatment showed that CYP1B1 regulates p53s and uPAR through its enzymatic activity. Furthermore, effects of DMBA and TMS on uPAR expression disappeared in HCT116p53−/− cells, indicating that p53 is critical for uPAR induction by CYP1B1. Collectively, our results demonstrate that CYP1B1 may reduce the relapse-free survival rate of breast cancer patients by inducing invasive traits in cancer cells via p53 regulation based on the mutation status of TP53 genes and further activation of the uPAR pathway. The elucidation of the previously unknown molecular mechanism of CYP1B1 may provide evidence for the development of effective anti-cancer therapeutic strategies that target the progression of cancer invasion.

The muscle clock: how does muscle physiology change in 24h in response to exercise?

The muscle clock: how does muscle physiology change in 24h in response to exercise?

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.

Protopine isolated from Nandina domestica induces apoptosis and autophagy in colon cancer cells by stabilizing p53.

The tumor suppressor p53 plays essential roles in cellular protection mechanisms against a variety of stress stimuli and its activation induces apoptosis or autophagy in certain cancer cells. Here, we identified protopine, an isoquinoline alkaloid isolated from Nandina domestica, as an activator of the p53 pathway from cell-based natural compound screening based on p53-responsive transcription. Protopine increased the p53-mediated transcriptional activity and promoted p53 phosphorylation at the Ser15 residue, resulting in stabilization of p53 protein. Moreover, protopine up-regulated the expression of p21WAF1/CIP1 and BAX, downstream genes of p53, and inhibited the proliferation of HCT116 colon cancer cells. Apoptosis was elicited by protopine as indicated by caspase-3/7 activation, poly ADP ribose polymerase cleavage, and increased population of Annexin V-FITC-positive cells. Furthermore, protopine induced the formation of microtubule-associated protein 1 light chain 3 (LC3) puncta and LC3-II turnover, typical biochemical markers of autophagy, in HCT116 cells. Our findings suggest that protopine exerts its antiproliferative activity by stimulating the p53 pathway and may have potential as a chemopreventive agent for human colon cancer.

Spasmolytic Mechanism of Aqueous Licorice Extract on Oxytocin-Induced Uterine Contraction through Inhibiting the Phosphorylation of Heat Shock Protein 27.

Licorice derived from the roots and rhizomes of Glycyrrhiza uralensis Fisch. (Fabaceae), is one of the most widely-used traditional herbal medicines in China. It has been reported to possess significant analgesic activity for treating spastic pain. The aim of this study is to investigate the spasmolytic molecular mechanism of licorice on oxytocin-induced uterine contractions and predict the relevant bioactive constituents in the aqueous extract. The aqueous extraction from licorice inhibited the amplitude and frequency of uterine contraction in a concentration-dependent manner. A morphological examination showed that myometrial smooth muscle cells of oxytocin-stimulated group were oval-shaped and arranged irregularly, while those with a single centrally located nucleus of control and licorice-treated groups were fusiform and arranged orderly. The percentage of phosphorylation of HSP27 at Ser-15 residue increased up to 50.33% at 60 min after oxytocin stimulation. Furthermore, this increase was significantly suppressed by licorice treatment at the concentration of 0.2 and 0.4 mg/mL. Colocalization between HSP27 and α-SMA was observed in the myometrial tissues, especially along the actin bundles in the oxytocin-stimulated group. On the contrary, the colocalization was no longer shown after treatment with licorice. Additionally, employing ChemGPS-NP provided support for a preliminary assignment of liquiritigenin and isoliquiritigenin as protein kinase C (PKC) inhibitors in addition to liquiritigenin, isoliquiritigenin, liquiritin and isoliquiritin as MAPK-activated protein kinase 2 (MK2) inhibitors. These assigned compounds were docked with corresponding crystal structures of respective proteins with negative and low binding energy, which indicated a high affinity and tight binding capacity for the active site of the kinases. These results suggest that licorice exerts its spasmolytic effect through inhibiting the phosphorylation of HSP27 to alter the interaction between HSP27 and actin. Furthermore, our results provide support for the prediction that potential bioactive constituents from aqueous licorice extract inhibit the relevant up-stream kinases that phosphorylate HSP27.

Hepatitis B virus X protein activates E3 ubiquitin ligase Siah-1 to control virus propagation via a negative feedback loop.

The seven in absentia homologue 1 (Siah-1) protein is an E3 ubiquitin ligase that induces ubiquitin-dependent proteasomal degradation of HBx, the principal regulatory protein of hepatitis B virus (HBV); however, its role in HBV propagation remains unknown. Here, we found that HBx upregulates Siah-1 levels in HepG2 but not in Hep3B cells, in which p53 is absent. For this effect, HBx sequentially activated ataxia telangiectasia mutated kinase and checkpoint kinase 2 via phosphorylation at the Ser-1981 and Thr-68 residues, respectively, which led to the activation of p53 via phosphorylation at the Ser-15 and Ser-20 residues. As a result, HBx was heavily ubiquitinated by Siah-1 and degraded by the ubiquitin-proteasome system in HepG2 cells, whereas this effect was marginal or undetectable in Hep3B cells. Knock-down of p53 in HepG2 cells downregulated Siah-1 levels and subsequently upregulated HBx levels, whereas ectopic p53 expression in Hep3B cells upregulated Siah-1 levels and subsequently downregulated HBx levels. In addition, Siah-1 knock-down impaired the ubiquitination and proteasomal degradation of HBx in HepG2 cells, whereas ectopic Siah-1 expression induced ubiquitin-dependent proteasomal degradation of HBx in Hep3B cells. The effects of HBx on p53 and Siah-1 were exactly reproduced in a 1.2-mer HBV replicon system, mimicking the natural course of HBV infection. In particular, Siah-1 knock-down upregulated the levels of HBx derived from the HBV replicon, resulting in an increase in HBV production. In conclusion, HBx modulates its own protein level via a negative feedback loop involving p53 and Siah-1 to control HBV propagation.

Investigation of the Molecular Interactions Regulating the Function of Human Cardiac Myosin

Cardiac myosin interacts cyclically with actin in the presence of ATP in the sarcomere to drive heart contraction. Previous fiber studies suggested that the structure and function of myosin in the thick filaments are regulated by its own regulatory light chain (RLC) phosphorylation and through its interaction with myosin binding protein-C (MyBP-C). Interestingly, ∼80% of mutations that cause hypertrophic cardiomyopathy (HCM), a relatively common genetic cardiac disorder characterized by hypercontractility, have been found in the genes encoding human β-cardiac myosin and human cardiac MyBP-C. It has been hypothesized that the heads of the two-headed motor can adopt a sequestered structure where the heads interact asymmetrically with their own tail as Ser-15 residue of RLC remains dephosphorylated, and the N-terminal domains of MyBP-C binds to myosin to stabilize the structure. We designed two different two-headed human β-cardiac myosin motor constructs differing in the length of their S2 tail domains and co-expressed them with both of the human light chains to test the hypothesis that the number of myosin heads available for interaction with actin is regulated by phosphorylation of the RLC and MyBP-C. The shorter construct (2-hep HMM) is devoid of the tail region needed for the interaction of S2 with its S1 heads whereas the longer one (25-hep HMM) has the required length of S2 tail region to allow such interactions. We found that the maximal actin-activated ATPase rate for 2-hep HMM and the single-headed myosin construct S1 are essentially the same, and are only slightly changed by RLC phosphorylation. On the other hand, de-phosphorylated 25-hep HMM has a significantly lower maximal actin-activated ATPase than the 2-hep HMM, consistent with the former having fewer actin-accessible S1 heads than the latter. Additionally, binding experiments of 2-hep and 25-hep HMM with the N-terminal C0-C2 domain of human cardiac MyBP-C showed that the affinity of complex formation is weakened by phosphorylation of either RLC or MyBP-C.

Hepatitis C virus core activates proteasomal activator 28γ expression via upregulation of p53 levels to control virus propagation.

The proteasomal activator 28γ (PA28γ), frequently overexpressed in hepatocellular carcinoma, is believed to play several important roles in hepatitis C virus (HCV) replication and viral pathogenesis. However, the underlying mechanism for PA28γ overexpression in hepatocellular carcinoma and its role during HCV replication are still unclear. In the present study, we found that HCV core derived from either ectopic expression or HCV infection upregulates PA28γ levels in p53-positive human hepatocytes. For this effect, HCV core sequentially activated ataxia telangiectasia mutated and checkpoint kinase 2 via phosphorylation at Ser-1981 and Thr-68 residues, respectively, resulting in stabilization of p53 via phosphorylation at Ser-15 and Ser-20 residues and subsequent transcriptional activation of PA28γ expression. The elevated PA28γ in turn downregulated HCV core levels by either inducing its ubiquitination-dependent proteasomal degradation via upregulation of E6AP levels in the presence of p53 or activating an ubiquitin-independent proteasomal degradation pathway in the absence of p53, which ultimately led to a decrease in HCV propagation. HCV core modulates its own protein level via a negative feedback loop involving p53 and PA28γ to control HCV replication in p53-positive hepatocytes, which may help HCV evade immune responses and establish chronic infection.

The Chromosomal parDE2 Toxin-Antitoxin System of Mycobacterium tuberculosis H37Rv: Genetic and Functional Characterization.

Mycobacterium tuberculosis H37Rv escapes host-generated stresses by entering a dormant persistent state. Activation of toxin-antitoxin modules is one of the mechanisms known to trigger such a state with low metabolic activity. M. tuberculosis harbors a large number of TA systems mostly located within discernible genomic islands. We have investigated the parDE2 operon of M. tuberculosis H37Rv encoding MParE2 toxin and MParD2 antitoxin proteins. The parDE2 locus was transcriptionally active from growth phase till late stationary phase in M. tuberculosis. A functional promoter located upstream of parD2 GTG start-site was identified by 5′-RACE and lacZ reporter assay. The MParD2 protein transcriptionally regulated the PparDE2 promoter by interacting through Arg16 and Ser15 residues located in the N-terminus. In Escherichia coli, ectopic expression of MParE2 inhibited growth in early stages, with a drastic reduction in colony forming units. Live-dead analysis revealed that the reduction was not due to cell death alone but due to formation of viable but non-culturable cells (VBNCs) also. The toxic activity of the protein, identified in the C-terminal residues Glu98 and Arg102, was neutralized by the antitoxin MParD2, both in vivo and in vitro. MParE2 inhibited mycobacterial DNA gyrase and interacted with the GyrB subunit without affecting its ATPase activity. Introduction of parE2 gene in the heterologous M. smegmatis host prevented growth and colony formation by the transformed cells. An M. smegmatis strain containing the parDE2 operon also switched to a non-culturable phenotype in response to oxidative stress. Loss in colony-forming ability of a major part of the MParE2 expressing cells suggests its potential role in dormancy, a cellular strategy for adaptation to environmental stresses. Our study has laid the foundation for future investigations to explore the physiological significance of parDE2 operon in mycobacterial pathogenesis.

Abstract B73: The monoterpene, citral, increases intracellular oxygen radicals and inhibits cancer cell proliferation by inducing apoptosis and endoplasmic reticulum stress .

Abstract The terpene-enriched steam distilled extract of ginger rhizomes has potent anti-tumor activity. Screening of the components of ginger extract demonstrated citral, a commonly used food flavoring agent, as the major monoterpene that inhibits proliferation of human and murine cancer cell lines A2780, ECC-1, OVCAR-3, SKOV-3, ID8 and MOVCAR. Citral initiates a rapid decrease in intracellular levels of glutathione and a corresponding increase in oxygen radicals. In wild-type p53-expressing ECC-1 cells, citral treatment increases phosphorylation of the Ser-15 residue of p53 and causes apoptosis. Inhibition of intracellular oxygen radicals by N-acetylcysteine and p53 activity by pifithrin-α inhibits citral-induced apoptosis in ECC-1 cells. Apoptosis in mutant p53 (Arg248Glu) expressing OVCAR-3 cells was also inhibited by N-acetylcysteine and was associated with a decrease in phosphorylation of Ser-15 of p53. Finally, in cells not expressing p53, citral inhibited proliferation, but not through apoptosis. Instead in the p53-negative SKOV-3 cells, citral treatment caused endoplasmic reticulum stress indicated by increase in phosphorylation of eIF2α and the expression of CHOP, GADD45, EDEM, ATF4 and Hsp90 and increased ATG5 and LC3-II suggesting the cells were undergoing autophagy. The study identifies the mechanism that allows citral to produce its cytotoxic effects. Combined with our recent results that nanoparticle-encapsulated citral decreases in vivo growth of 4T1 murine breast tumors, the results from the current study suggest that citral and its analogs be considered as chemotherapeutic agents. Since citral mediates its cytotoxic effects primarily by inducing oxidative stress, it is likely that this monoterpenes and its derivatives can be used for the treatment of a variety of solid tumors including ovarian cancer. Citation Format: Arvinder Kapur, Mildred Felder, Lucas Fass, Justanjyot kaur, Austin Czarnecki, Kavya Rathi, Manish Patankar. The monoterpene, citral, increases intracellular oxygen radicals and inhibits cancer cell proliferation by inducing apoptosis and endoplasmic reticulum stress . [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B73.

Abstract 3211: Citral is the major component of ginger-derived terpenes to mediate p53-dependent apoptosis in cancer cells

Abstract Steam distillation of ginger yields a mixture of twenty two terpenes. This mixture has anti-proliferative effect on over fifteen different human and mouse cancer cell lines tested in our previous studies. Since the mixture is heterogeneous, it cannot be developed into a therapeutic drug. Hence, we tested individual terpenes from the ginger extract to identify its bioactive component(s). Camphene and alpha-pinene together constitute 10% of the ginger terpene extract and have no effect on cancer cell proliferation. On the other hand, citral a mixture of the two isomers, neral and geranial which constitute 30-50% of the ginger terpene extract, inhibits proliferation of ECC-1, OVCAR-3, OVCAR5, OVCAR 433 with IC50 between 20-50 μM. The decrease in proliferation of the cells is due to induction of apoptosis by citral as measured by monitoring Annexin V and propidium iodide staining and expression of cleaved caspase3. Treatment of cancer cells with citral results in a rapid increase in intracellular Reactive Oxygen Species (ROS). The cellular stress resulting from ROS generation leads to phosphorylation of the Ser-15 residue of p53. Activation of p53 via this phosphorylation event leads to apoptosis in cancer cells. When cells are pre-treated with ROS inhibitor, N-acetyl cysteine (NAC), citral induced apoptosis is inhibited. Similarly inhibition of p53 by pifithrin-α or knockdown of the tumor suppressor by siRNA attenuates the pro-apoptotic response of citral. There is a significant increase in p53 protein expression in cancer cells when treated with Citral This increase in p53 is likely due to an increase in p53 gene expression as determined in our qPCR assays, even though increase due to inhibition of proteasomal degradation of the protein cannot be completely ruled out at this point. An increase in p53 expression in response to citral is associated with an increase in expression of the p53 responsive genes BAX, PUMA and NOXA. All of these experiments demonstrate a novel p53-dependent mechanism for citral to induce apoptosis in cancer cells. Anti-proliferative effects of citral were observed in cancer cells expressing wild-type p53 (ECC-1) as well as in cell lines with R248Q (OVCAR3), ins224 (OVCAR5) p53 mutations. Based on these experimental results we propose that citral should be considered as a novel p53 activating agent that can be used as an adjunct to conventional chemo- and biologic therapies against cancer. Our on-going efforts will identify sensitivity of commonly found p53 mutations to activation by citral and also the specific p53 kinases triggered subsequent to the ROS surge mediated by this terpene. Citation Format: Lucas Fass, Mildred Felder, Manish S. Patankar, Arvinder K. Kapur. Citral is the major component of ginger-derived terpenes to mediate p53-dependent apoptosis in cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3211. doi:10.1158/1538-7445.AM2014-3211

Akt phosphorylation in human chondrocytes is down-regulated via p53r2 in response to mechanical stress

Purpose: p53 tumor suppressor protein is activated in response to DNA damage. When the DNA is damaged, Ser15 residue is phosphorylated and p53R2 is induced. The role of p53R2 in chondcotytes is unknown. In this study, we evaluated p53R2 expression in chondrocytes in response to mechanical stress. Furthermore, we investigated the function of p53R2 in response to mechanical stress. Methods: OA cartilage samples were obtained from total knee replacement surgery, and normal cartilage samples were from femoral neck fracture. The expression of p53R2 was analyzed by immunohistochemistry. Chondrocytes were isolated from OA and normal cartilage and grown. The expression of p53R2 in chondrocytes was detected by western blotting and real-time PCR. Normal and OA chondrocytes were introduced 5% tensile strain using FX-2000. After the strain, expressions of p53R2, ERK1/2, JNK, Akt, phospho-ERK1/2, phospho-JNK and phospho-Akt were detected by western blotting respectively. OA chondrocytes after transfection of p53R2 siRNA or control siRNA were introduced the same strain. Expressions of p53R2 and the same protein kinases were detected by western blotting. Furthermore, glycosaminoglycan(GAG) quantification was performed after the strain. Results: p53R2 was highly expressed in OA cartilage in comparison with normal cartilage by immunohistochemistry. Western blotting and real-time PCR showed p53R2 expression in chondrocytes was higher in OA chondrocytes than normal chondrocytes, too. (Fig.1) p53R2 expression in OA and normal chondrocytes was increased after 5% tensile strain. On the other hand, Akt phosphorylation was down-regulated after the strain. However other protein kinases were not regulated significantly. (Fig.2) Akt-phosphorylation was up-regulated after transfection of p53R2 siRNA. (Fig.3) GAG was down-regulated by the 5% strain, however up-regulated after p53R2 siRNA transfection. Conclusions: p53R2 could regulate Akt signaling in chondrocyte mechanotransduction. Down-regulation of p53R2 expression might increase production of glycosaminoglycan. We consider that the regulation of p53R2 might be a strategy for OA treatment. View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT)

P53 and DNA-dependent protein kinase catalytic subunit independently function in regulating actin damage-induced tetraploid G1 arrest

We previously reported that the p53 tumor suppressor protein plays an essential role in the induction of tetraploid G1 arrest in response to perturbation of the actin cytoskeleton, termed actin damage. In this study, we investigated the role of p53, ataxia telangiectasia mutated protein (ATM), and catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) in tetraploid G1 arrest induced by actin damage. Treatment with actin-damaging agents including pectenotoxin-2 (PTX-2) increases phosphorylation of Ser-15 and Ser-37 residues of p53, but not Ser-20 residue. Knockdown of ATM and DNA-PKcs do not affect p53 phosphorylation induced by actin damage. However, while ATM knockdown does not affect tetraploid G1 arrest, knockdown of DNA-PKcs not only perturbs tetraploid G1 arrest, but also results in formation of polyploidy and induction of apoptosis. These results indicate that DNA-PKcs is essential for the maintenance of actin damage induced-tetraploid G1 arrest in a p53-independent manner. Furthermore, actin damage-induced p53 expression is not observed in cells synchronized at G1/S of the cell cycle, implying that p53 induction is due to actin damage-induced tetraploidy rather than perturbation of actin cytoskeleton. Therefore, these results suggest that p53 and DNA-PKcs independently function for tetraploid G1 arrest and preventing polyploidy formation.

Melatonin triggers p53Ser phosphorylation and prevents DNA damage accumulation

Several epidemiological studies have shown that high levels of melatonin, an indolic hormone secreted mainly by the pineal gland, reduce the risks of developing cancer, thus suggesting that melatonin triggers the activation of tumor-suppressor pathways that lead to the prevention of malignant transformation. This paper illustrates that melatonin induces phosphorylation of p53 at Ser-15 inhibiting cell proliferation and preventing DNA damage accumulation of both normal and transformed cells. This activity requires p53 and promyelocytic leukemia (PML) expression and efficient phosphorylation of p53 at Ser-15 residue. Melatonin-induced p53 phosphorylation at Ser-15 residue does not require ataxia telangiectasia-mutated activity, whereas it is severely impaired upon chemical inhibition of p38 mitogen-activated protein kinase activity. By and large, these findings imply that the activation of the p53 tumor-suppressor pathway is a critical mediator of melatonin and its anticancer effects. Therefore, it provides molecular insights into increasing observational evidence for the role that melatonin has in cancer prevention.

EDD Inhibits ATM-mediated Phosphorylation of p53

The EDD (E3 identified by differential display) gene, first identified as a progestin-induced gene in T-47D breast cancer cells, encodes an E3 ubiquitin ligase with a HECT domain. It was reported that EDD is involved in the G(2)/M progression through ubiquitination of phospho-katanin p60. Previous study has also shown that EDD can act as a transcription cofactor independently of its E3 ligase activity. In this study, we uncover a new role for EDD during cell cycle progression in an E3 ligase-independent manner. We demonstrate that EDD can physically interact with p53 and that this interaction blocks the phosphorylation of p53 by ataxia telangiectasia mutated (ATM). Silencing of EDD induces phosphorylation of p53 at Ser(15) and activates p53 target genes in fibroblasts and some transformed cells without activation of DNA damage response. The G(1)/S arrest induced by EDD depletion depends on p53. On the other hand, overexpression of EDD inhibits p53-Ser(15) phosphorylation and suppresses the induction of p53 target genes during DNA damage, and this effect does not require its E3 ligase activity. Thus, through binding to p53, EDD actively inhibits p53 phosphorylation by ATM and plays a role in ensuring smooth G(1)/S progression.

Enhancement of radiation sensitivity in lung cancer cells by celastrol is mediated by inhibition of Hsp90

The radiosensitizing activity of celastrol, a quinone methide triterpene was examined. We found that celastrol treatment of the NCI-H460 lung cancer cell line increased radiation-induced cell killing. The increased radiosensitivity was correlated with decreased levels of Hsp90 clients, such as EGFR, ErbB2 and survivin as well as with increased p53 expression. Celastrol inhibited the ATP-binding activity of Hsp90. Furthermore, celastrol treatment dissociated an Hsp90 client protein, EGFR, and this in turn resulted in degradation of the client protein. These results were not observed with another structurally similar triterpenoid, 6β-acetonyl-22β-hydroxytingenol (TG), suggesting that a specific structural feature of the triterpenoid is required for radiosensitization. Moreover celastrol treatment increased p53 levels by phosphorylating Ser15 and Ser20 residues as well as by inhibiting its proteasomal degradation. Celastrol may be considered an effective radiosensitizer acting as an inhibitor of Hsp90 and a p53 activator. The two activities could be applicable to a broad range of cancer cells with either wild-type or mutant p53 because either activity could be effective for the enhancement of radiation cell killing. Further analysis with other triterpenoids should identify the functional moiety of the structure and additional candidates for effective radiosensitizers, which can be used in combined radiotherapy.

Ubiquitin proteasomal pathway mediated degradation of p53 in melanoma

Ubiquitin proteasomal pathway (UPP) is the principle mechanism for protein catabolism and affects cellular processes critical for survival and proliferation. Levels of tumor suppressor protein p53 are very low in cells due to its rapid turnover by UPP-mediated degradation. While p53 is mutated in human cancers, most human melanomas maintain wild-type conformation. In this study, to investigate the effects of UPP inhibitor in vitro and in vivo, we used a genetically-engineered mouse model (GEMM) that has the same genetic alterations as those of human melanomas . Melanoma cells were established from mouse tumors and named 8B20 cells. Treatment of 8B20 cells with the UPP inhibitors, MG132 and clasto-lactacystin-β-lactone, led to an increase in levels of p53 while treatment with non-proteasomal inhibitors did not alter p53 levels. UPP inhibitors induced formation of heavy molecular weight ubiquitinated proteins, a hallmark of UPP inhibition, and p53-specific poly-ubiquitinated products in 8B20 cells. To further decipher the mechanism of p53 stabilization, we investigated half-life of p53 in cells treated with cycloheximide to block de novo protein synthesis. Treatment of 8B20 cells with MG132 led to an increase in the half-life of p53. Further analysis revealed that p53 stabilization was not mediated by phosphorylation of Ser-15 and Ser-20 residues. In vivo studies showed that MG132 induced p53 overexpression and reduced tumor growth, suggesting an important role of p53 stabilization in controlling melanoma. Taken together, our studies provide a proof of principle for using a GEMM to address the mechanisms of action and efficacy of melanoma treatment.

Dose-dependent mutual regulation between Wip1 and p53 following UVC irradiation

DNA damage stabilizes and activates p53, which selectively induces downstream targets to modulate the cellular response. As a homeostatic regulator of cell cycle checkpoint, the p53 target Wip1 plays essential roles in releasing cells from DNA damage-induced checkpoints after appropriate repair of the damaged-DNA. It is unknown how Wip1 performs when the DNA damage is beyond repair. Here we address that Wip1 displays dose-dependent responses to UVC irradiation. A low dose of UVC, which stimulates intra-S phase cell cycle arrest, transiently induces the Wip1 protein levels in a p53-dependent manner. In contrast, a high dose of UVC, which induces apoptosis, suppresses the Wip1 protein levels in a p53-independent manner. The UVC dose-dependent response of Wip1 correlates not only with the cellular response but also with the activity of p53. Wip1 dephosphorylates p53 on its Ser15 residue. However, the mutual regulation between Wip1 and p53 is only triggered by a low dose of UVC. In response to a high dose of UVC, the sustained activation of p53 fails to induce the downstream targets, including Wip1, Mdm2, p21 and GADD45α. Nonetheless, the reduced Wip1 level contributes to the sustained accumulation of phospho-p53 (Ser15) in response to a high dose of UVC. Our results suggest that Wip1 is regulated by UVC in a dose-dependent manner. Moreover, the mutual regulation between Wip1 and p53 is highly dose-dependent upon UVC irradiation, and this contributes to the different outcomes of the cellular response to UVC.

Abstract 1248: 14-3-3 interacts with TC1(C8orf4) to protect the protein from rapid degradation and regulates cancer cell invasiveness

Abstract 14-3-3 interacts with TC1(C8orf4) to protect the protein from rapid degradation and regulates cancer cell invasiveness. Jungtae Kim, Yusun Jung, Dong Wook Kim, Hyunju Lee, Kyuyoung Song, Inchul Lee. Asan Institute for Life Sciences, Asan Medical Center, University of Ulsan, College of Medicine, Seoul, Republic of Korea TC1(C8orf4) is a multifunctional regulator associated with aggressive biological behavior of cancer and poor survival of patients (Clin Cancer Res 2006;12:3541-8). It regulates the Wnt/β-catenin pathway (Cancer Res 2006;66:723-8).and induces endothelial inflammation (J Immunol 2009;183:3996-4002). TC1 is up-regulated by NF-kB (FEBS letters 2006;580:3519-24), and heat shock and other cellular stress (Biochem Biophys Res Commun 2007;360:447-52). TC1 is a small protein present in vertebrates, which is degraded rapidly. TC1 interacted with 14-3-3β upon yeast two hybrid screening. The interaction of TC1 with 14-3-3β and 14-3-3ε was confirmed in HEK293T cells upon cotransfection. The deletion mutant analysis showed that the N-terminus of TC1 and C-terminus of 14-3-3 interacted with each other. The binding site of TC1 appeared to involve the Ser15 residue. The interaction was inhibited by serum starvation, staurosporine, and GSK3β inhibitor, indicating the requirement of TC1 phosphorylation, probably of Ser15, for the interaction. The half-life of transfected HA-TC1 was 2.49 hours in HEK293T cells, while it was elongated to 5.07 hours upon co-transfection with 14-3-3β. 14-3-3 knock down reduced TC1 expression. Our data together suggested that 14-3-3 protected TC1 in vivo. 14-3-3 proteins have been implicated in the regulation of various biological functions. However, the role in cancer is not clear. TC1 was shown to enhance the Matrigel invasiveness of cancer cells (Clin Cancer Res 2006;12:3541-8). 14-3-3 knock down using si14-3-3β reduced the Matrigel invasiveness of HeLa cells significantly (p<0.0001). Cancer cell proliferation was not affected by 14-3-3 transfection or knock down. Our data suggest the requirement of 14-3-3 in cancer cell invasiveness potentially by regulating TC1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1248.

Loss of Cyclin G1 Expression in Human Uterine Leiomyoma Cells Induces Apoptosis

Observations from the authors' laboratory suggest a physiological role for increased cyclin G1 protein levels in human uterine leiomyoma. The hypothesis of the present study is that the strategic modulation of cyclin G1 by antisense technology will inhibit the survival of in vitro-grown uterine leiomyoma cells. Cultured uterine leiomyoma cells were transfected with cyclin G1 ribbon-type antisense oligonucleotide (cyclin G1 RiAS) to effectively reduce cyclin G1 expression. Cell viability, in situ terminal deoxyuridine nick end-labeling (TUNEL) assay, flow cytometry, DNA fragmentation, and expression of cell cycle regulatory-related proteins were evaluated by Western blot. Antisense oligonucleotides compromised uterine leiomyoma cell viability and inducted apoptosis in a caspase-independent mechanism. In situ TUNEL and DNA fragmentation revealed apoptosis induction, and fluorescent-activated cell sorting analysis showed increased sub-G1-phase cells. Furthermore, abrogation of cyclin G1 enhanced p53 accumulation, phosphorylation of p53 at Ser-15 residue, and increased expression of cyclin-dependent kinase inhibitors p21 and p27. These data imply that cyclin G1 expression is associated with growth promotion and the potential utility and novelty of using ribbon-type antisense oligonucleotides as a gene therapy strategy to treat human uterine leiomyoma.