Post-translational Stabilization Research Articles

Abstract 13051: A Lin28-let-7 Axis Modulates Adaptation of the Myocardium to Ischemia

Background: The RNA binding protein Lin28 and microRNA-let-7 each regulate glucose metabolism, tissue repair and pluripotency; however, their roles in cardiac adaptation to ischemia are unknown. Methods and Results: miR-let-7c was significantly downregulated in myocardium of mice following a 2 hour coronary occlusion, compared to sham-operated controls (0.69 fold, p<0.05). In parallel, Lin28 protein was induced in the ischemic myocardium compared to adjacent non-ischemic tissue (~2.0-fold, p<0.05). Consistent with this, mature but not precursor forms of miR-let-7c were downregulated (0.7 fold, p<0.05) in neonatal rat ventricular myocytes (NRVMs) during both hypoxia and hypoxia with glucose depletion, but not in similarly treated fibroblasts, together with a time-dependent 6-fold increase in Lin28 protein compared to normoxic controls. mRNA levels of Lin28 were also increased, but less strikingly (2.2 fold vs normoxia control, p<0.05). Ischemia induced acetylation of Lin28, both in vivo and in NRVMs that was potentiated by the non-selective histone deacetylase (HDAC) inhibitor, trichostatin A, suggesting that Lin28 is post-translationally stabilized. Mechanistically, transduction of NRVM with a let-7g lentiviral expression vector rescued let-7 levels, prevented ischemia-induced myocyte death and caspase activation, and promoted phosphorylation and activation of Stat3. Knockdown of the let-7 family in hypoxic NRVMs using a sponge vector containing 6 let-7 binding sites derepressed predicted target genes Bcl2l1, DUSP1, INSR, Ras at both mRNA and protein levels and depressed STAT3 phosphorylation. Using PAR-CLIP, in which the RISC complex is crosslinked to its target transcripts, we observed direct let-7 targeting of Lin28A in cardiac myocytes. Conclusion: During ischemic stress, let-7 is post-transcriptionally downregulated in a myocyte-specific manner through the ischemia-mediated induction of its repressor, Lin28. Induction of Lin28 occurs through transcriptional induction as well as post-translational acetylation and stabilization. This Lin28-let-7 axis constitutes a bidirectional feedback loop that directly and indirectly regulates signaling cascades of known importance to myocardial adaption to ischemic stress.

RM-01 * RADIATION INDUCED PRMT5 EXPRESSION IS REGULATED BY miRNA-96 IN GLIOBLASTOMA

BACKGROUND: Glioblastoma (GB) represent the most common and aggressive histological subtype among malignant gliomas and are associated with poor outcomes. It is composed of multiple types of tumor cells displaying multiple genetic and epigenetic defects affecting tumor suppressor gene expression, regulation of growth and apoptosis. Recurrence is the major concern with the current standard therapy. Protein arginine methyltransferase enzyme 5 (PRMT5), that regulates many cellular processes through its methylation-dependent and independent activity is overexpressed in this disease. We hypothesize that PRMT5 contributes to radioresistance in GB. Using patient derived GB neurosphere models we are probing the role of PRMT5 in inducing radioresistance in GB. METHODS: GB neurospheres dispersed into single cell, were treated with radiation or transfected with specified siRNAs. At appropriate time, cells were collected and probed for mRNA expression by qPCR and protein level by immunblotting. RESULTS: We found that PRMT5 associates with Hsp90 in GB neurospheres and that this binding is essential for PRMT5 stability. In addition to higher levels of baseline expression, radiation treatment increases PRMT5 mRNA level by 2 fold and protein expression by 60%. This increase is independent of its post-translational stability conferred by its association with Hsp90. However radiation treatment decreases miRNA-96 level significantly (p ≤ 0.01). Overxpression of miRNA-96 down regulates the expression of PRMT5 protein. Additionally we found that PRMT5 is important for regulation of HIF1alpha, a transcription factor induced post radiation and known to be involved in radiation resistance. We are currently investigating the implications of PRMT5 inhibition in conjunction with radiation in vivo. CONCLUSIONS: Based on our findings we conclude that radiation treatment induced suppression of miRNA-96 causes increased expression of PRMT5. This suggests that increased expression of PRMT5 may contribute for HIF-mediated radioresistance in GB.

Abstract A46: HSP-90 inhibition in pediatric sarcomas

Abstract Hsp90 is a molecular chaperone that regulates post-translational folding, stability, and function of many client proteins, which play critical roles in key signal transduction pathways implicated in cell growth, differentiation, and survival. Several relevant oncogenic proteins are described as clients, such as insulin growth factor receptor 1, AKT/PKB, and src-family kinases. Signaling through these molecules is critical in the biology of rhabdomyosarcoma and Ewing's sarcoma, which are two of the most common pediatric sarcomas. Novel therapies for these tumors are clearly needed since patients with relapsed and metastatic disease continue to have poor outcomes and multimodality therapy, even when successful, is often accompanied by long-term morbidity. Ganetespib (Synta Pharmaceuticals) is a small molecule Hsp90 inhibitor that binds to the ATP pocket in the N-terminus of Hsp90, leading to down-regulation of Hsp90 client protein levels. We sought to evaluate the preclinical activity of ganetespib in a panel of Ewing's Sarcoma and rhabdomyosarcoma cell lines. We evaluated in vitro activity of ganetespib in Ewing Sarcoma cell lines (TC71, TC32, EW8, RDES) and in rhabdomyosarcoma (RMS) cell lines (RD, RH30) using kinetic proliferation assays and MTS. In all cases, marked inhibition of cell growth was achieved in a dose-dependent manner with drug IC50s in the low nanomolar range (3-6 nM). We found that ganetespib exposure is associated with both induction of apoptosis and alterations in cell cycle progression. In addition to upregulation of Hsp70, which is considered a biomarker of Hsp90 inhibition, ganetespib treatment was associated with loss of expression of IGF1R-beta as well as downregulation of key signaling pathways including mammalian target of rapamycin (mTOR), pAKT, and Src-family kinases, particularly YES1, a kinase we previously identified as critical in rhabdomyosarcoma biology. Moreover, we observed a cooperative effect between Hsp90 inhibition and IGF1R blockade with R1507, a monoclonal antibody targeting this receptor (Hoffman-La Roche). Furthermore, synergy was seen when ganetespib was combined with the SFK inhibitor dasatinib. While in-vivo validation of these results is ongoing, we conclude that Hsp90 inhibition with ganetespib is an attractive strategy to explore for the therapy of pediatric sarcomas, possibly in combination with other targeted therapies. Citation Format: Fernanda I. Arnaldez, Toby Terwilliger, Choh Yeung, Len Neckers, Lee Helman. HSP-90 inhibition in pediatric sarcomas. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A46.

Combined inhibition of Hsp90 and the proteasome affects NSCLC proteostasis and attenuates cell migration.

Lung cancer remains the most common cause of cancer-related death worldwide. This malignancy is a complex disease, and it is important to identify potential biological targets, the blockade of which would affect multiple downstream signaling cascades. A growing number of reports recognize novel therapeutic targets in the protein homeostasis network responsible for generating and protecting the protein fold. The heat shock protein 90 (Hsp90) is an essential molecular chaperon involved in the posttranslational folding and stability of proteins. It is required for conformational maturation of multiple oncogenic kinases that drive signal transduction and proliferation of cancer cells. However, in the case of unfolded protein accumulation endoplasmic reticulum (ER) stress is induced and several response pathways such as proteasome functions are activated. The ubiquitin-proteasome system orchestrates the turnover of innumerable cellular proteins. Here, we suggest that the therapeutic efficacy of Hsp90 inhibition may be augmented by coadministering proteasome inhibitor on human non-small-cell lung cancer (NSCLC) cell lines. Indeed, we showed that coadministration of the Hsp90 inhibitor 17-demethoxygeldanamycin (17-DMAG) and proteasome inhibitor (velcade) induced ER stress evidenced by increased unfolded protein response markers. The consequences were evident in multiple aspects of the NSCLC phenotype: reduced viability and cell count, increased apoptotic cell death, and most profoundly, synergistically decreased cell motility. Our findings provide proof-of-concept that targeting ER homeostasis is therapeutically beneficial in NSCLC cell lines.

BB0323 and novel virulence determinant BB0238: Borrelia burgdorferi proteins that interact with and stabilize each other and are critical for infectivity.

We have shown that Borrelia burgdorferi gene product BB0323 is essential for cell fission and pathogen persistence in vivo. Here we describe characterization of a conserved hypothetical protein annotated as BB0238, which specifically interacts with the N-terminal region of BB0323. We show that BB0238 is a subsurface protein, and similar to BB0323, exists in the periplasm and as a membrane-bound protein. Deletion of bb0238 in infectious B. burgdorferi did not affect microbial growth in vitro or survival in ticks, but the mutant was unable to persist in mice or transmit from ticks--defects that are restored on genetic complementation. Remarkably, BB0238 and BB0323 contribute to mutual posttranslational stability, because deletion of one causes dramatic reduction in the protein level of the other partner. Interference with the function of BB0238 or BB0323 and their interaction may provide novel strategies to combat B. burgdorferi infection.

A dual luciferase system for analysis of post-transcriptional regulation of gene expression in Leishmania

Gene expression in kinetoplastid parasites is regulated via post-transcriptional mechanisms that modulate mRNA turnover, translation rate, and/or post-translational protein stability. To facilitate the analysis of post-transcriptional regulation, a dual luciferase system was developed in which firefly and Renilla luciferase reporters genetically fused to compatible drug resistance genes are integrated in place of one allele of the gene of interest and of an internal control gene, respectively, in a manner that preserves the cognate pre-mRNA processing signals. The sensitivity and reproducibility of the assay coupled with the ability to rapidly assemble reporter integration constructs render the dual luciferase system suitable for analysis of multiple candidates derived from global expression analysis platforms. To demonstrate the utility of the system, regulation of three genes in response to purine starvation was examined in Leishmania donovani promastigotes. This dual luciferase system should be directly applicable to the analysis of post-transcriptional regulation in other kinetoplastids.

Heat-shock protein 90 inhibition in pediatric sarcomas.

10057 Background: Hsp90 is a molecular chaperone that regulates post-translational folding, stability, and function of many client proteins, which play critical roles in key signal transduction pat...

Differential role of TIMP2 and TIMP3 in cardiac hypertrophy, fibrosis, and diastolic dysfunction

Tissue inhibitor of metalloproteinases (TIMPs) can mediate myocardial remodelling, hypertrophy, and fibrosis in heart disease. We investigated the impact of TIMP2 vs. TIMP3 deficiency in angiotensin II (Ang II)-induced myocardial remodelling and cardiac dysfunction. TIMP2(-/-), TIMP3(-/-), and wild-type (WT) mice received Ang II/saline (Alzet pump) for 2 weeks. Ang II infusion resulted in enhanced myocardial hypertrophy and lack of fibrosis in TIMP2(-/-), and conversely, excess fibrosis without hypertrophy in TIMP3(-/-) mice. Echocardiographic imaging revealed preserved ejection fraction in all groups; however, exacerbated left ventricular (LV) diastolic dysfunction was detected in Ang II-infused TIMP2(-/-) and TIMP3(-/-) mice, despite the suppressed Ang II-induced hypertension in TIMP3(-/-) mice. Enhanced hypertrophy in TIMP2(-/-) mice impaired active relaxation, while excess fibrosis in TIMP3(-/-) mice increased LV passive stiffness. Adult WT cardiomyocytes, only when co-cultured with cardiac fibroblasts, exhibited Ang II-induced hypertrophy which was suppressed in TIMP3(-/-) cardiomyocytes. In vitro studies on adult cardiofibroblasts (quiescent and cyclically stretched), and in vivo analyses, revealed that the increased fibrosis in TIMP3(-/-)-Ang II hearts is due to post-translational stabilization and deposition of collagen by matricellular proteins [osteopontin and Secreted Protein Acidic and Rich in Cysteine (SPARC)], which correlated with increased inflammation, rather than increased de novo synthesis. Reduced cross-linking enzymes, LOX and PLOD1, could underlie suppressed collagen deposition in TIMP2(-/-)-Ang II hearts. TIMP2 and TIMP3 play fundamental and differential roles in mediating pathological remodelling, independent from their MMP-inhibitory function. TIMP2(-/-) and TIMP3(-/-) mice provide a unique opportunity to study myocardial hypertrophy and fibrosis independently, and their impact on cardiac dysfunction.

Ligand-dependent stabilization of androgen receptor in a novel mouse ST38c Sertoli cell line

Mature Sertoli cells (SC) are critical mediators of androgen regulation of spermatogenesis, via the androgen receptor (AR) signaling. Available immortalized SC lines loose AR expression or androgen responsiveness, hampering the study of endogenous AR regulation in SC. We have established and characterized a novel clonal mouse immortalized SC line, ST38c. These cells express some SC specific genes (sox9, wt1, tjp1, clu, abp, inhbb), but not fshr, yet more importantly, maintain substantial expression of endogenous AR as determined by PCR, immunocytochemistry, testosterone binding assays and Western blots. Microarrays allowed identification of some (146) but not all (rhox5, spinlw1), androgen-dependent, SC expressed target genes. Quantitative Real-Time PCR validated regulation of five up-regulated and two down-regulated genes. We show that AR undergoes androgen-dependent transcriptional activation as well as agonist-dependent posttranslational stabilization in ST38c cells. This cell line constitutes a useful experimental tool for future investigations on the molecular and cellular mechanisms of androgen receptor signaling in SC function.

Lactam‐Based HDAC Inhibitors for Anticancer Chemotherapy: Restoration of RUNX3 by Posttranslational Modification and Epigenetic Control

Expression and stability of the tumor suppressor runt-related transcription factor 3 (RUNX3) are regulated by histone deacetylase (HDAC). HDAC inhibition alters epigenetic and posttranslational stability of RUNX3, leading to tumor suppression. However, HDAC inhibitors can nonselectively alter global gene expression through chromatin remodeling. Thus, lactam-based HDAC inhibitors were screened to identify potent protein stabilizers that maintain RUNX3 stability by acetylation. RUNX activity and HDAC inhibition were determined for 111 lactam-based analogues through a cell-based RUNX activation and HDAC inhibition assay. 3-[1-(4-Bromobenzyl)-2-oxo-2,5-dihydro-1H-pyrrol-3-yl]-N-hydroxypropanamide (11-8) significantly increased RUNX3 acetylation and stability with relatively low RUNX3 mRNA expression and HDAC inhibitory activity. This compound showed significant antitumor effects, which were stronger than SAHA, in an MKN28 xenograft model. Thus, we propose a novel strategy, in which HDAC inhibitors serve as antitumor chemotherapeutic agents that selectively target epigenetic regulation and protein stability of RUNX3.

Direct Contact With Bone Marrow Stromal Cells Protects CML Progenitors From Imatinib Through Cytoplasmic Stabilization Of β-Catenin

A β-catenin expression signature is associated with primary resistance to tyrosine kinase inhibitors (TKIs) (McWeeney et al. 2010), but its role in TKI resistance is not completely understood. To assess the role of β-catenin in TKI resistance, we used shRNA targeting β-catenin (shβcat) in in vitro models of BCR-ABL1 kinase-independent resistance. To model resistance in the absence of bone marrow (BM)-derived factors, we used TKI-resistant K562R cells that are adapted for growth in the presence of imatinib, as well as primary CD34+ progenitors from CML patients who had failed treatment with two or more TKIs. These cells are cultured in regular medium (RM), proliferate in 1.0-2.5 μM imatinib, and exhibit β-catenin expression that is independent of BCR-ABL1 kinase activity. To model resistance mediated by the BM microenvironment, we cultured TKI-sensitive parental K562 cells and CD34+ progenitors from newly diagnosed CML patients in direct contact (DC) with HS-5 BM stromal cells. HS-5 co-culture increases β-catenin protein levels and clonogenic potential by >3-fold despite continued suppression of BCR-ABL1 kinase activity. All cells lack BCR-ABL1 kinase domain mutations and undergo TKI-mediated kinase inhibition as detected by immunoblot analyses.CML cell lines and primary cells with BCR-ABL1-independent resistance were lentivirally transduced with shβcat or a scrambled control (shSCR), and knockdown was confirmed by immunoblot and/or qRT-PCR. In RM, shβcat reduced the clonogenicity of TKI-sensitive K562 cells and CD34+ cells from newly diagnosed CML patients by 49% and 39%, respectively, compared to shSCR controls. In TKI-resistant K562R cells and CD34+ cells from TKI-resistant CML patients, shβcat reduced clonogenicity by 60% and 50%, respectively, in the presence or absence of imatinib (0-10 μM), suggesting a role for β-catenin in the development or maintenance of TKI resistance. In contrast to cells grown in RM, clonogenicity of cell lines and patient samples cultured in HS-5 DC was unaffected by shβcat compared to imatinib alone. Immunoblot analyses revealed that β-catenin protein levels were fully restored in HS-5 DC, despite the continued presence of shβcat. qRT-PCR revealed that while cells in HS-5 DC have high amounts of β-catenin protein, the mRNA levels remained similar to shβcat-expressing cells cultured in RM, consistent with post-translational stabilization of β-catenin. Importantly, increased β-catenin was not observed when cells were cultured in HS-5 conditioned medium, indicating that stabilization requires DC with the bone marrow stroma. These data are consistent with a role for β-catenin in TKI resistance mediated by DC with the BM microenvironment, similar to a recent report (Zhang et al., 2013).To understand nuclear versus cytoplasmic distribution following HS-5 DC, CD34+ cells from newly diagnosed CML patients were cultured in RM or HS-5 DC for 36 hours and analyzed for β-catenin localization by immunfluorescence. As expected, cells cultured in RM had low levels of β-catenin in the nucleus and cytoplasm that decreased upon treatment with imatinib (2.5 μM). In contrast, cells cultured in HS-5 DC had a marked increase of β-catenin that was unaffected by treatment with imatinib. While detectable in the nucleus, the majority of β-catenin protein was localized in the cytoplasm and at the cell membrane, consistent with its role in cell-cell junctions. Accordingly, in CD34+ cells from newly diagnosed CML patients, an N-cadherin blocking antibody impaired the clonogenic potential of cells cultured in HS-5 DC, with no significant effect on cells grown in RM. Affymetrix Human Gene 1.0 ST arrays revealed high levels of genes encoding the CDH2 and CDH13 cadherins, which may be involved in N-cadherin-mediated β-catenin stabilization, even in the presence of shβcat. Preliminary data also suggests that HS-5 DC reduces luciferase reporter activity from a construct harboring sequential β-catenin binding elements (pGF1-Lef/Tcf-eFGP-luc), further supporting a role for cytoplasmic β-catenin in TKI resistance. These data demonstrate a critical role for β-catenin in BCR-ABL1 kinase-independent TKI resistance, and suggest new strategies for targeting TKI resistance in the absence of BCR-ABL1 kinase domain mutations. Disclosures:Deininger:Bristol-Myers Squibb: Advisory Boards Other, Consultancy, Research Funding; Ariad Pharmaceuticals: Advisory Boards, Advisory Boards Other, Consultancy; Novartis: Advisory Boards, Advisory Boards Other, Consultancy, Research Funding; Celgene: Research Funding; Gilead Sciences: Research Funding.

Co-expression for intracellular processing in microbial protein production

The biological activity of a recombinant protein is highly dependent on its biophysical properties including post-translational modifications, solubility, and stability. Production of active recombinant proteins requires careful design of the expression strategy and purification schemes. This is often achieved by proper modification of the target protein during and/or after protein synthesis in the host cells. Such co-translational or post-translational processing of recombinant proteins is typically enabled by co-expressing the required enzymes, folding chaperones, co-factors and/or processing enzymes in the host. Various applications of the co-expression technology in protein production are discussed in this review with representative examples described.

Treatment of Niemann--pick type C disease by histone deacetylase inhibitors.

Niemann-Pick type C disease (NPC) is a devastating, recessive, inherited disorder that causes accumulation of cholesterol and other lipids in late endosomes and lysosomes. Mutations in 2 genes, NPC1 and NPC2, are responsible for the disease, which affects about 1 in 120,000 live births. About 95% of patients have mutations in NPC1, a large polytopic membrane protein that is normally found in late endosomes. More than 200 missense mutations in NPC1 have been found in NPC patients. The disease is progressive, typically leading to death before the age of 20 years, although some affected individuals live well into adulthood. The disease affects peripheral organs, including the liver, spleen, and lungs, but the most severe symptoms are associated with neurological disease. There are some palliative treatments that slow progression of NPC disease. Recently, it was found that histone deacetylase (HDAC) inhibitors that are effective against HDACs 1, 2, and 3 can reduce the cholesterol accumulation in fibroblasts derived from NPC patients with mutations in NPC1. One example is vorinostat. As vorinostat is a Food and Drug Administration-approved drug for treatment of cutaneous T-cell lymphoma, this opens up the possibility that HDAC inhibitors could be repurposed for treatment of this rare disease. The mechanism of action of the HDAC inhibitors requires further study, but these drugs increase the level of the NPC1 protein. This may be due to post-translational stabilization of the NPC1 protein, allowing it to be transported out of the endoplasmic reticulum.

Insulin Regulates Hypoxia-Inducible Factor-1α Transcription by Reactive Oxygen Species Sensitive Activation of Sp1 in 3T3-L1 Preadipocyte

Oxygen sensing transcription factor HIF-1 is activated due to accumulation of regulatory subunit HIF-1α by posttranslational stability mechanism during hypoxia or by several other stimuli even in normoxia. HIF-1α is also regulated by NF-kB mediated transcription mechanism. Reactive oxygen species (ROS) act as an important regulator of HIF-1 either by affecting prolyl hydroxylase activity, the critical determinant of HIF-1α stabilization or by activating NF-kB to promote HIF-1α transcription. Insulin is known to activate HIF-1 by a ROS dependent mechanism but the molecular mechanism of HIF-1α regulation is not known so far. Here we show that insulin regulates HIF-1α by a novel transcriptional mechanism by a ROS-sensitive activation of Sp1 in 3T3-L1 preadipocyte. Insulin shows little effect on HIF-1α protein stability, but increases HIF-1α promoter activity. Mutation analyses, electrophoretic mobility shift assay and chromatin immunoprecipitation assay confirm the role of Sp1 in HIF-1α transcription. We further demonstrate that insulin-induced ROS generation initiates signaling pathway involving phosphatidylinositol 3-kinase and protein kinase C for Sp1 mediated HIF-1α transcription. In summary, we reveal that insulin regulates HIF-1α by a novel transcriptional mechanism involving Sp1.

Abstract 748: Studying the significance of human p53-Sin3B interaction under the condition of DNA damage.

Abstract Sin3, a global regulator of transcription plays an important role in mediating gene regulation, by binding to various transcription factors through its highly conserved Paired Amphipathic Helices (PAH) domains. Physical association of Sin3 corepressor molecule with human p53, a tumour suppressor protein is now well established. In response to stimuli that activate p53, cells can undergo repair processes, cell cycle arrest or apoptosis. In order to combat the stress, p53 gets activated by posttranslational stabilization and modulate the expression levels of its target genes. There are number of genes which are negatively regulated by p53, but the mechanism of repression is not well characterized. We hereby show that p53 interacts with Sin3/HDAC corepressor complex in-vivo and expression of Sin3B is up regulated in response to DNA damaging stimuli in p53 dependent manner. Increase in the levels of Sin3B upon different kind of DNA damage suggests that Sin3B might be an important protein involved in DNA damage pathway. Sin3/HDAC corepressor complex is recruited by p53 onto the promoters of its target genes, resulting in the down regulation of p53 target genes. In our study we propose that the mechanism of repression of p53 target genes is through recruitment of Sin3/HDAC corepressor complex which leads to context specific chromatin modification. Such modifications results in enrichment of epigenetic marks like H3K9me3, H3K27me3, which are marker of repression under condition of DNA damage induced by Bleomycin. Knockdown of Sin3B using shRNA causes derepression of p53 target genes, suggesting that Sin3B is a key player in p53 mediated transrepression. Based on our studies we propose that Sin3B is an important corepressor molecule recruited by p53 in response to DNA damage and Increased Sin3B expression is an early event in DNA damage induced by Bleomycin. We further provide evidence that p53 regulate its target genes, namely HSPA8, MAD1, CRYZ, ANLN and CDC25c through its interaction with Sin3/HDAC corepressor complex which leads to chromatin modification by recruiting proteins involved in epigenetic modifications. Citation Format: Rama Kadamb, Shilpi Mittal, Nidhi Bansal, Bilikere Srinivasa Rao Dwarakanath, Daman Saluja. Studying the significance of human p53-Sin3B interaction under the condition of DNA damage. [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 748. doi:10.1158/1538-7445.AM2013-748

P53 protein subcellular localization and apoptosis in rodent corneal epithelium cell culture following ultraviolet irradiation

The tumor-suppressor gene p53 encodes a phosphoprotein involved in the control of cell growth. p53 expression and function have been documented in malignancy, apoptosis and the aging processes. Recently, p53 has been mapped and characterized in the normal cornea across different species. In the present study, high levels of cytoplasmic p53 protein were noted in normal primary corneal epithelium cultures by immunohistochemistry and western blot analysis. Following ultraviolet(UV) irradiation, the level of cytoplasmic p53 protein expression was increased beginning from 30min and lasting until 6h post-irradiation and then returned close to control levels by 24h. Cytoplasmic p53 phosphorylation was detected from 30min following UV treatment until 6h post-irradiation. p53 protein became apparent in the nucleus in a fraction of these cultured cells beginning 30min following UV irradiation and was still present 24h later. We also found that p53 colocalized with mitochondria 2h following UV irradiation in some of the cells and remained there up to 24h. As the expression levels of p53 transcription following UV irradiation were not significantly altered, the increase in cytoplasmic p53 protein expression may be conditional only upon post-translational stabilization. We also observed that the apoptotic index increased following UV irradiation in the same time frame as the p53 nuclear transfer and was partially suppressed by pifithrin-α, which is a reversible inhibitor of p53-mediated apoptosis and p53-dependent gene transcription. The present study offers new evidence suggesting that cytoplasmic p53 in rodent corneal epithelium is functionally active.

Preferential Retention of Circadian Clock Genes during Diploidization following Whole Genome Triplication in Brassica rapa

Much has been learned about the architecture and function of the circadian clock of Arabidopsis thaliana, a model for plant circadian rhythms. Circadian rhythms contribute to evolutionary fitness, suggesting that circadian rhythmicity may also contribute to agricultural productivity. Therefore, we extend our study of the plant circadian clock to Brassica rapa, an agricultural crop. Since its separation from Arabidopsis, B. rapa has undergone whole genome triplication and subsequent diploidization that has involved considerable gene loss. We find that circadian clock genes are preferentially retained relative to comparison groups of their neighboring genes, a set of randomly chosen genes, and a set of housekeeping genes broadly conserved in eukaryotes. The preferential retention of clock genes is consistent with the gene dosage hypothesis, which predicts preferential retention of highly networked or dose-sensitive genes. Two gene families encoding transcription factors that play important roles in the plant core oscillator--the PSEUDO-RESPONSE REGULATORS, including TIMING OF CAB EXPRESSION1, and the REVEILLE family, including CIRCADIAN CLOCK ASSOCIATED1 and LATE ELONGATED HYPOCOTYL--exhibit preferential retention consistent with the gene dosage hypothesis, but a third gene family, including ZEITLUPE, that encodes F-Box proteins that regulate posttranslational protein stability offers an exception.

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Abstract 4208: Post-translational modification of E2F1 in malignant melanoma

Abstract Malignant melanoma has the highest fatality among all skin cancers. No current treatment significantly enhances survival once metastasis is established. Previous studies have shown that E2F1, a transcription factor, is over-expressed in melanoma cells. However, the mechanism through which E2F1 is elevated remains unknown. E2F1 is known to be involved in many important biological processes, including cell cycle progression, DNA damage, DNA repair, differentiation, development, autophagy and apoptosis. This underscores the importance of E2F1 in carcinogenesis. Normally in late S phase, E2F1 is recognized by the E3 ligase p45 SKP2 and undergoes ubiquitination and finally degradation in 26S proteasome. Our overall hypothesis is that E2F1 escapes proteasomal degradation in melanoma cells due to changes in post-translational modification. In this study we have used melanoma cells of increasing disease progression. Our results show that although the primary melanoma cells have high level of E2F1 message and protein, it is unstable. On the other hand the more metastatic melanoma cells have low levels of E2F1 which is however very stable. Consistent with this result, post-translational modification of E2F1 including the phosphorylation at Serine 337 and the acetylation at Lysine 117 increase with melanoma progression, which suggests that these post-translational modification sites might play an important role in stabilization of E2F1 and keep it functionally active in the more metastatic cell lines. E2F1 knockdown data also shows that E2F1 KD can inhibit cell proliferation in early stage melanoma cells but not in the more metastatic cells, which also suggest that inhibition of post-translational modifications and stability of E2F1 may be a more effective way to inhibit melanoma progression to more advanced stages. Abrogation of acetylation is expected to cause E2F1 protein instability. On the other hand, if acetylation is potentiated, the stabilized E2F1 will continue to promote progression through the cell cycle. We are currently testing whether the ectopic expression of the continuously acetylated E2F1 protein can rescue proliferation inhibition caused by E2F1 down-regulation, and drive metastasis and a mutant E2F1 with abrogated acetylation can block rescue of the proliferation inhibition in the E2F1 down-regulated melanoma cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4208. doi:1538-7445.AM2012-4208

Abstract 211: Ubiquitin-proteolytic regulation of hematopoietic stem cell expansion

Abstract Direct transduction of the homeobox protein HOXB4 promotes the proliferation of hematopoietic stem cells (HSCs) without induction of leukemogenesis, but requires frequent administration to overcome its short protein half-life. We demonstrate here that the CUL4A ubiquitin ligase regulates the post-translational stability of multiple HOX proteins, including HOXB4, and we defined the degron of HOXB4 required for CUL4A-mediated degradation. Ectopic expression of degradation-resistant HOXB4 in myeloid progenitor cells confers a growth advantage over wild-type HOXB4-expressing cells. Finally, direct transduction of recombinant degradation-resistant HOXB4 protein to human adult HSCs significantly enhanced their maintenance in a more primitive state both in vitro and in vivo compared to transduction with wild-type HOXB4 protein. Our studies demonstrate the feasibility of engineering a stable HOXB4 variant to overcome a major technical hurdle in the ex vivo expansion of adult HSCs and early progenitors for human therapeutic use. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 211. doi:1538-7445.AM2012-211