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1.  The bromodomain and extra-terminal inhibitor CPI203 enhances the antiproliferative effects of rapamycin on human neuroendocrine tumors 
Cell Death & Disease  2014;5(10):e1450-.
Endogenous c-MYC (MYC) has been reported to be a potential pharmacological target to trigger ubiquitous tumor regression of pancreatic neuroendocrine tumors (PanNETs) and lung tumors. Recently inhibitors of bromodomain and extra-terminal (BET) family proteins have shown antitumor effects through the suppression of MYC in leukemia and lymphoma. In this paper, we investigated the antitumor activity of a BET protein bromodomain inhibitor (BETi) CPI203 as a single agent and in combination with rapamycin in human PanNETs. We found that exposure of human PanNET cell lines to CPI203 led to downregulation of MYC expression, G1 cell cycle arrest and nearly complete inhibition of cell proliferation. In addition, overexpression of MYC suppressed the growth inhibition caused by CPI203 and knockdown of MYC phenocopied the effects of CPI203 treatment. These findings indicate that suppression of MYC contributed to the antiproliferative effects of BETi inhibition in human PanNET cells. Importantly, CPI203 treatment enhanced the antitumor effects of rapamycin in PanNET cells grown in monolayer and in three-dimensional cell cultures, as well as in a human PanNET xenograft model in vivo. Furthermore, the combination treatment attenuated rapamycin-induced AKT activation, a major limitation of rapamycin therapy. Collectively, our data suggest that targeting MYC with a BETi may increase the therapeutic benefits of rapalogs in human PanNET patients. This provides a novel clinical strategy for PanNETs, and possibly for other tumors as well.
doi:10.1038/cddis.2014.396
PMCID: PMC4237236  PMID: 25299775
2.  Anti-inflammatory cytokines, pro-fibrogenic chemokines and persistence of acute HCV infection 
Journal of viral hepatitis  2013;20(6):10.1111/jvh.12052.
SUMMARY
Chemokines and cytokines play a vital role in directing and regulating immune responses to viral infections. Persistent hepatitis C virus (HCV) infection is characterized by the loss of anti-HCV cellular immune responses, while control of HCV infection is associated with maintenance of anti-HCV cellular immune responses. To determine whether plasma concentrations of 19 chemokines and cytokines controlling T-cell trafficking and function differed based on infection outcome, we compared them in at-risk subjects followed prospectively for HCV infection. Levels were compared over time in subjects who controlled HCV infection (Clearance) and subjects who developed persistent HCV infection (Persistence) at two time points during acute infection: (i) first viraemic sample (initial viraemia) and (ii) last viraemic sample in Clearance subjects and time-matched samples in Persistence subjects. At initial viraemia, increased pro-inflammatory tumour necrosis factor α (TNFα) plasma concentrations were observed in the Clearance group, while the plasma levels of anti-inflammatory interleukin (IL)-2, IL-10 and IL-13 were higher in the Persistence group. IL-13 was positively correlated with IL-2 and IL-10 at initial viraemia in the Persistence group. At the time of last viraemia, plasma levels of eotaxin, macrophage chemoattractant protein-4 (MCP-4), IL-5 and IL-10 were higher in the Persistence group and IL-10 and IL-5 levels were positively correlated. Collectively, these results suggest that the development of persistent infection is associated with an anti-inflammatory and pro-fibrogenic chemokine and cytokine profile that is evident at the onset of infection and maintained throughout acute infection.
doi:10.1111/jvh.12052
PMCID: PMC3793396  PMID: 23647957
acute infection; chemokine; cytokine; hepatitis c virus
3.  The diversity of zinc-finger genes on human chromosome 19 provides an evolutionary mechanism for defense against inherited endogenous retroviruses 
Cell Death and Differentiation  2013;21(3):381-387.
Endogenous retroviruses (ERVs) are remnants of ancient retroviral infections of the germ line that can remain capable of replication within the host genome. In the soma, DNA methylation and repressive chromatin keep the majority of this parasitic DNA transcriptionally silent. However, it is unclear how the host organism adapts to recognize and silence novel invading retroviruses that enter the germ line. Krueppel-Associated Box (KRAB)-associated protein 1 (KAP1) is a transcriptional regulatory factor that drives the epigenetic repression of many different loci in mammalian genomes. Here, we use published experimental data to provide evidence that human KAP1 is recruited to endogenous retroviral DNA by KRAB-containing zinc-finger transcription factors (TFs). Many of these zinc-finger genes exist in clusters associated with human chromosome 19. We demonstrate that these clusters are located at hotspots for copy number variation (CNV), generating a large and continuing diversity of zinc-finger TFs with new generations. These zinc-finger genes possess a wide variety of DNA binding affinities, but their role as transcriptional repressors is conserved. We also perform a computational study of the different ERVs that invaded the human genome during primate evolution. We find candidate zinc-finger repressors that arise in the genome for each ERV family that enters the genomes of primates. In particular, we show that those repressors that gained their binding affinity to retrovirus sequences at the same time as their targets invaded the human lineage are preferentially located on chromosome 19 (P-value: 3 × 10−3).
doi:10.1038/cdd.2013.150
PMCID: PMC3921586  PMID: 24162661
Zinc-fingers; retrotransposons; germline
4.  Evaluation of the Accuracy of a Triaxial Accelerometer Embedded into a Cell Phone Platform for Measuring Physical Activity 
Background
Physical activity is important in health and weight management. Several cell phone platforms integrate an accelerometer onto the motherboard. Here we tested the validity of the cell phone accelerometer to assess physical activity in a controlled laboratory setting.
Methods
31 subjects wore the cell phone on their waist along with the validated Physical Activity Monitoring System (PAMS) with different body postures and during graded walking. Energy expenditure was measured using indirect calorimetry. 11 subjects also wore the iPhone at different locations such as arm, hand, pant pocket, etc.
Results
The cell phone accelerometer was accurate and precise compared to the PAMS, with an intra-class correlation coefficient (r2> 0.98). The cell phone accelerometer showed excellent sequential increases with increased in walking velocity and energy expenditure (r2>0.9).
Conclusion
An accelerometer embedded into a cell phone was accurate and reliable in measuring and quantifying physical activity in the laboratory setting. Data from free-living users shows promise for deployment of a comprehensive integrated physical activity promoting and weight loss platform using such mobile technologies.
PMCID: PMC3667221  PMID: 23730539
Energy expenditure; Obesity; Weight loss; Physical activity
5.  A role for primary cilia in glutamatergic synaptic integration of adult-born neurons 
Cilia  2012;1(Suppl 1):P31.
doi:10.1186/2046-2530-1-S1-P31
PMCID: PMC3555798
6.  Antiphospholipid antibodies are associated with enhanced oxidative stress, decreased plasma nitric oxide and paraoxonase activity in an experimental mouse model 
Rheumatology (Oxford, England)  2005;44(10):1238-1244.
Objective
Oxidative stress contributes to atherosclerosis, and evidence of enhanced oxidative stress exists in antiphospholipid syndrome (APS). In a non-lupus murine model, we evaluated whether anticardiolipin (aCL) antibodies could affect the oxidant/antioxidant balance as an early biochemical step of APS.
Methods
Hybridomas producing human and murine aCL and anti-β2-glycoprotein I (aβ2-GPI) monoclonal antibodies were injected into three groups of five female BALB/c severe combined immunodeficiency (SCID) mice. Corresponding hybridomas secreting non-antiphospholipid antibodies of the same isotype were employed as controls. Sera and organs were collected after 30 days. Paraoxonase (PON) activity, peroxynitrite, superoxide, nitric oxide (NO) and nitrotyrosine were measured in plasma. Expression of endothelial nitric oxide synthase and inducible nitric oxide synthase (iNOS) was assessed by western blot and immunohistochemistry.
Results
PON activity and NO (sum of nitrate and nitrite) levels were reduced in the human aCL IgG group (P<0.002 and P<0.04, respectively), whilst peroxynitrite and superoxide and expression of total antioxidant capacity of plasma were increased (P<0.01). PON and NO were decreased in the murine aβ2-GPI IgG and IgM aCL groups (P<0.03 and P<0.05, respectively). Nitrotyrosine was elevated in the human aCL IgG group (P<0.03). Western blotting showed reduced iNOS expression in the hearts of the IgG aCL group, confirmed by immunostaining. PON inversely correlated with IgG aCL titres (P<0.001), superoxide (P<0.008) and peroxynitrite levels (P<0.0009). Peroxynitrite and total IgG aCL were independent predictors of PON (P<0.0009 and P<0.02, respectively). Superoxide was the only independent predictor of NO (P<0.008) and of nitrotyrosine (P<0.002).
Conclusion
aCL antibodies are associated with the decreased PON activity and reduced NO that may occur in the preclinical phase of APS.
doi:10.1093/rheumatology/keh722
PMCID: PMC3465365  PMID: 15987712 CAMSID: cams2349
Antiphospholipid antibodies; Oxidative stress; Nitric oxide; Total antioxidant capacity; Paraoxonase
7.  Dispersion-relation phase spectroscopy of intracellular transport 
Optics Express  2011;19(21):20571-20579.
We used quantitative phase imaging to measure the dispersion relation, i.e. decay rate vs. spatial mode, associated with mass transport in live cells. This approach applies equally well to both discrete and continuous mass distributions without the need for particle tracking. From the quadratic experimental curve specific to diffusion, we extracted the diffusion coefficient as the only fitting parameter. The linear portion of the dispersion relation reveals the deterministic component of the intracellular transport. Our data show a universal behavior where the intracellular transport is diffusive at small scales and deterministic at large scales. Measurements by our method and particle tracking show that, on average, the mass transport in the nucleus is slower than in the cytoplasm.
doi:10.1364/OE.19.020571
PMCID: PMC3495870  PMID: 21997064
(180.0180) Microscopy; (170.0170) Medical optics and biotechnology; (000.2700) General science
8.  Proliferation and tissue remodeling in cancer: the hallmarks revisited 
Cell Death & Disease  2012;3(10):e397-.
Although cancers are highly heterogeneous at the genomic level, they can manifest common patterns of gene expression. Here, we use gene expression signatures to interrogate two major processes in cancer, proliferation and tissue remodeling. We demonstrate that proliferation and remodeling signatures are partially independent and result in four distinctive cancer subtypes. Cancers with the proliferation signature are characterized by signatures of p53 and PTEN inactivation and concomitant Myc activation. In contrast, remodeling correlates with RAS, HIF-1α and NFκB activation. From the metabolic point of view, proliferation is associated with upregulation of glycolysis and serine/glycine metabolism, whereas remodeling is characterized by a downregulation of oxidative phosphorylation. Notably, the proliferation signature correlates with poor outcome in lung, prostate, breast and brain cancer, whereas remodeling increases mortality rates in colorectal and ovarian cancer.
doi:10.1038/cddis.2012.140
PMCID: PMC3481128  PMID: 23034332
cancer pathways; cancer metabolism; cancer stem cell; meta-pathway; hallmarks
9.  The paths to death and differentiation 
Cell Death and Differentiation  2011;18(9):1391-1392.
doi:10.1038/cdd.2011.41
PMCID: PMC3178432  PMID: 21841805
10.  Autoantibodies to heat shock protein 60 promote thrombus formation in a murine model of arterial thrombosis 
Summary
Background and objectives
Anti-heat shock protein (HSP)60 autoantibodies are associated with atherosclerosis and are known to affect endothelial cells in vitro. However, their role in thrombus formation remains unclear. We hypothesized that anti-HSP60 autoantibodies could potentiate thrombosis, and evaluated the effect of anti-murine HSP60 antibodies in a ferric chloride (FeCl3)-induced murine model of carotid artery injury.
Methods
Anti-HSP60, or control, IgG was administered to BALB/c mice 48 h prior to inducing carotid artery injury, and blood flow was monitored using an ultrasound probe.
Results
Thrombus formation was more rapid and stable in anti-HSP60 IGG-treated mice than in controls (blood flow = 1.7% ± 0.6% vs. 34% ± 12.6%, P = 0.0157). Occlusion was complete in all anti-HSP60 IgG-treated mice (13/13), with no reperfusion being observed. In contrast, 64% (9/14) of control mice had complete occlusion, with reperfusion occurring in 6/9 mice. Thrombi were significantly larger in anti-HSP60 IgG-treated mice (P = 0.0001), and contained four-fold more inflammatory cells (P = 0.0281) than in controls. Non-injured contralateral arteries of anti-HSP60 IgG-treated mice were also affected, exhibiting abnormal endothelial cell morphology and significantly greater von Willebrand factor (VWF) and P-selectin expression than control mice (P = 0.0024 and P = 0.001, respectively).
Conclusions
In summary, the presence of circulating anti-HSP60 autoantibodies resulted in increased P-selectin and VWF expression and altered cell morphology in endothelial cells lining uninjured carotid arteries, and promoted thrombosis and inflammatory cell recruitment in FeCl3-injured carotid arteries. These findings suggest that anti-HSP60 autoantibodies may constitute an important prothrombotic risk factor in cardiovascular disease in human vascular disease.
doi:10.1111/j.1538-7836.2009.03305.x
PMCID: PMC3429411  PMID: 19192108 CAMSID: cams2309
autoimmunity; endothelial cells; heat shock proteins; infection; thrombosis; von Willebrand factor
11.  International survey of seat belt use exemptions 
Injury Prevention  2006;12(4):258-261.
Background
Substantial evidence of seatbelt efficacy has been shown by several studies, and it is widely recommended that motor vehicle occupants use properly fitted seat belts. However, some (but a heretofore unknown number of) countries with national seat belt laws permit various exemptions which may lower use rates. The aim of this study was to survey the variety of exemptions to national seat belt laws.
Methods
This investigation relied on identifying respondents from national traffic safety agencies, other governmental and non‐governmental organizations, Internet searches, personal contacts, and other sources. Questionnaires were deployed through a web based survey supplemented by email and postal versions.
Results
Responses were received from 30 countries of which 28 (93.7%) had a national seat belt law. About two thirds (63.7%) of the 28 national laws applied to both front and back seat passengers. The leading exemption types included vehicles made before a certain year (n = 13), antique vehicles (n = 12), military vehicles (n = 11), buses (n = 9), and emergency vehicles (n = 8). Most responding countries reported one or more specific categories of individuals as exempt including those with medical exemptions (n = 20), taxi drivers (n = 11), police (n = 9), emergency medical personnel (n = 8), physically disabled people (n = 6), and pregnant women (n = 6). Out of 26 responses to the question regarding current level of enforcement, 42.3% felt enforcement was “very good or good” and 57.7% characterized it as “fair or poor”.
Conclusions
This study represents one of the largest international traffic law surveys reported. Most national seatbelt laws offer perilous exemptions to a broad array of vehicle types and road user groups. These findings, coupled with concern over the level of enforcement in the majority of countries surveyed, suggest that international road safety efforts have a long way to go to improve coverage and enforcement of national seat belt laws.
doi:10.1136/ip.2005.010686
PMCID: PMC2586772  PMID: 16887949
seat belts; motor vehicle crashes; pregnancy; road traffic policy
12.  Dendrites of Medial Olivocochlear (MOC) Neurons in Mouse 
Neuroscience  2008;154(1):147-159.
Stains for acetylcholinesterase (AChE) and retrograde labeling with Fluorogold (FG) were used to study olivocochlear neurons and their dendritic patterns in mice. The two methods gave similar results for location and number of somata. The total number of medial olivocochlear (MOC) neurons in the ventral nucleus of the trapezoid body (VNTB) is about 170 per side. An additional dozen large olivocochlear neurons are located in the dorsal periolivary nucleus (DPO). Dendrites of all of these neurons are long and extend in all directions from the cell bodies, a pattern that contrasts with the sharp frequency tuning of their responses. For VNTB neurons, there were greater numbers of dendrites directed medially than laterally and those directed medially were longer (on average, 25– 50% longer). Dendrite extensions were most pronounced for neurons located in the rostral portion of the VNTB. When each dendrite from a single neuron was represented as a vector, and all the vectors summed, the result was also skewed toward the medial direction. DPO neurons, however, had more symmetric dendrites that projected into more dorsal parts of the trapezoid body, suggesting that this small group of olivocochlear neurons has very different physiological properties. Dendrites of both types of neurons were somewhat elongated rostrally, about 20% longer than those directed caudally. These results can be interpreted as extensions of dendrites of olivocochlear neurons toward their synaptic inputs: medially to meet crossing fibers from the cochlear nucleus that are part of the MOC reflex pathway, and rostrally to meet descending inputs from higher centers.
doi:10.1016/j.neuroscience.2007.12.045
PMCID: PMC2587447  PMID: 18313859
superior olive; cochlear nucleus; efferent; descending pathway; auditory reflex
13.  Discrepancies Between Self-Report and Objective Measures for Stimulant Drug Use in HIV: Cognitive, Medication Adherence and Psychological Correlates 
Addictive behaviors  2007;32(12):2727-2736.
While it has long been recognized that self-reported drug use may be at variance with objectively obtained evidence such as urine toxicology assays, few studies have explored the behavioral correlates of such discrepancies. Here we compared self-reported and objective measures of stimulant drug use for 162 HIV infected individuals and identified a sub-group with discrepancies between data obtained via the two methods. Results showed poorer neurocognitive performance (attention, learning/memory) and lower medication adherence rates for the discrepant group as compared to those who either acknowledged their drug use or accurately denied recent stimulant use. Using the Millon Clinical Multiaxial Inventory –III, it was also found that those in the discrepant group were more hesitant to reveal psychopathology. Comparisons of self-reported and objectively measured medication adherence data are also discussed.
doi:10.1016/j.addbeh.2007.04.006
PMCID: PMC2679746  PMID: 17499443
14.  Rat Optic Nerve Oligodendrocytes Develop in the Absence of Viable Retinal Ganglion Cell Axons 
The Journal of Cell Biology  1999;146(6):1365-1374.
Retinal ganglion cell axons and axonal electrical activity have been considered essential for migration, proliferation, and survival of oligodendrocyte lineage cells in the optic nerve. To define axonal requirements during oligodendrogenesis, the developmental appearance of oligodendrocyte progenitors and oligodendrocytes were compared between normal and transected optic nerves. In the absence of viable axons, oligodendrocyte precursors migrated along the length of the nerve and subsequently multiplied and differentiated into myelin basic protein–positive oligodendrocytes at similar densities and with similar temporal and spatial patterns as in control nerves. Since transected optic nerves failed to grow radially, the number of oligodendrocyte lineage cells was reduced compared with control nerves. However, the mitotic indices of progenitors and the percentage of oligodendrocytes undergoing programmed cell death were similar in control and transected optic nerves. Oligodendrocytes lacked their normal longitudinal orientation, developed fewer, shorter processes, and failed to form myelin in the transected nerves. These data indicate that normal densities of oligodendrocytes can develop in the absence of viable retinal ganglion axons, and support the possibility that axons assure their own myelination by regulating the number of myelin internodes formed by individual oligodendrocytes.
PMCID: PMC2156117  PMID: 10491397
oligodendrocyte progenitor cells; axon–glial interactions; myelination; optic nerve; program cell death
15.  Treatment of pneumothorax during pregnancy. 
Thorax  1996;51(3):338-341.
Pneumothorax during pregnancy is rare. A case report is presented and a novel way of managing the problem is discussed.
PMCID: PMC1090654  PMID: 8779146
16.  Preservation of macronutrient preferences in cancer anorexia. 
British Journal of Cancer  1998;78(5):579-581.
Indirect evidence suggests that cancer anorexia is associated with specific aversions to macronutrients. To investigate this, patients with cancer anorexia and hospitalized control subjects devised 3-day menus comprising foods that they wished to eat. These foods were then provided for 3 days and the intakes of each food carefully measured. As expected, patients with cancer anorexia consumed substantially less energy than hospitalized control subjects (6.0 +/- 0.9 MJ vs 9.5 +/- 0.5 MJ, P < 0.001). However, macronutrient composition was consistently maintained in the patients with cancer anorexia. These data argue against cancer anorexia representing a state of macronutrient aversion.
PMCID: PMC2063064  PMID: 9744494
17.  Lysophosphatidic acid is a major serum noncytokine survival factor for murine macrophages which acts via the phosphatidylinositol 3-kinase signaling pathway. 
Journal of Clinical Investigation  1998;102(4):716-727.
Lysophosphatidic acid (LPA) is the smallest and structurally simplest of all the glycerophospholipids. It occurs normally in serum and binds with high affinity to albumin, while retaining its biological activity. The effects of LPA are pleiotropic and range from mitogenesis to stress fiber formation. We show a novel role for LPA: as a macrophage survival factor with potency equivalent to serum. Administration of LPA protects macrophages from apoptosis induced by serum deprivation, and protection is equivalent to that with conventional survival factors such as macrophage colony stimulating factor. The ability of LPA to act as a survival factor is mediated by the lipid kinase phosphatidylinositol 3-kinase (PI3K), since LPA activated both the p85-p110 and p110gamma isoforms of PI3K and macrophage survival was blocked completely by wortmannin or LY294002, two mechanistically dissimilar inhibitors of PI3K. pp70(s6k), a downstream kinase activated by PI3K, also contributes to survival, because inhibitors of pp70(s6k), such as rapamycin, blocked macrophage survival in the presence of LPA. Modified forms of LPA and phospholipids, such as phosphatidylcholine and phosphatidylethanolamine, had no survival effect, thereby showing the specificity of LPA. These results show that LPA acts as a potent macrophage survival factor. Based on striking similarities between our LPA and serum data, we suggest that LPA is a major noncytokine survival factor in serum.
PMCID: PMC508934  PMID: 9710440
18.  Adipocyte macrophage colony-stimulating factor is a mediator of adipose tissue growth. 
Journal of Clinical Investigation  1998;101(8):1557-1564.
Adipose tissue growth results from de novo adipocyte recruitment (hyperplasia) and increased size of preexisting adipocytes. Adipocyte hyperplasia accounts for the severalfold increase in adipose tissue mass that occurs throughout life, yet the mechanism of adipocyte hyperplasia is unknown. We studied the potential of macrophage colony-stimulating factor (MCSF) to mediate adipocyte hyperplasia because of the profound effects MCSF exerts on pluripotent cell recruitment and differentiation in other tissues. We found that MCSF mRNA and protein were expressed by human adipocytes and that adipocyte MCSF expression was upregulated in rapidly growing adipose tissue that encircled acutely inflamed bowel and in adipose tissue from humans gaining weight (4-7 kg) with overfeeding. Localized overexpression of adipocyte MCSF was then induced in rabbit subcutaneous adipose tissue in vivo using adenoviral-mediated gene transfer. Successful overexpression of MCSF was associated with 16-fold increases in adipose tissue growth compared with a control adenovirus expressing beta-galactosidase. This occurred in the absence of increased cell size and in the presence of increased nuclear staining for MIB-1, a marker of proliferation. We conclude that MCSF participates in adipocyte hyperplasia and the physiological regulation of adipose tissue growth.
PMCID: PMC508735  PMID: 9541484
19.  The major surface glycoprotein of Pneumocystis carinii induces release and gene expression of interleukin-8 and tumor necrosis factor alpha in monocytes. 
Infection and Immunity  1997;65(11):4790-4794.
Recent studies suggest that interleukin-8 (IL-8) and tumor necrosis factor alpha (TNF-alpha) may play a central role in host defense and pathogenesis during Pneumocystis carinii pneumonia. In order to investigate whether the major surface antigen (MSG) of human P. carinii is capable of eliciting the release of IL-8 and TNF-alpha, human monocytes were cultured in the presence of purified MSG. MSG-stimulated cells released significant amounts of IL-8 within 4 h, and at 20 h, cells stimulated with MSG released 45.5 +/- 9.3 ng of IL-8/ml versus 3.7 +/- 1.1 ng/ml for control cultures (P = 0.01). In a similar fashion, MSG elicited release of TNF-alpha. Initial increases were also seen at 4 h, and at 20 h, TNF-alpha levels reached 6.4 +/- 1.1 ng/ml, compared to 0.08 +/- 0.01 ng/ml for control cultures (P < 0.01). A concentration-dependent increase in IL-8 and TNF-alpha secretion was observed at 20 h with 0.2 to 5 microg of MSG/ml (P < 0.01). Secretion of IL-8 and TNF-alpha from MSG-stimulated monocytes at 20 h was inhibited by 60 and 86%, respectively, after coincubation with soluble yeast mannan (P = 0.01). With an RNase protection assay, increases in steady-state mRNA levels for IL-8 and TNF-alpha were detectable at 4 h. These data show that recognition of MSG by monocytes involves a mannose-mediated mechanism and results in the release of the proinflammatory cytokines IL-8 and TNF-alpha.
PMCID: PMC175687  PMID: 9353066
20.  The CDK7-cycH-p36 complex of transcription factor IIH phosphorylates p53, enhancing its sequence-specific DNA binding activity in vitro. 
Molecular and Cellular Biology  1997;17(10):5923-5934.
Phosphorylation is believed to be one of the mechanisms by which p53 becomes activated or stabilized in response to cellular stress. Previously, p53 was shown to interact with three components of transcription factor IIH (TFIIH): excision repair cross-complementing types 2 and 3 (ERCC2 and ERCC3) and p62. This communication demonstrates that p53 is phosphorylated by the TFIIH-associated kinase in vitro. The phosphorylation was found to be catalyzed by the highly purified kinase components of TFIIH, the CDK7-cycH-p36 trimeric complex. The phosphorylation sites were mapped to the C-terminal amino acids located between residues 311 and 393. Serines 371, 376, 378, and 392 may be the potential sites for this kinase. Phosphorylation of p53 by this kinase complex enhanced the ability of p53 to bind to the sequence-specific p53-responsive DNA element as shown by gel mobility shift assays. These results suggest that the CDK7-cycH-p36 trimeric complex of TFIIH may play a role in regulating p53 functions in cells.
PMCID: PMC232440  PMID: 9315650
21.  Expression of wild-type alpha-catenin protein in cells with a mutant alpha-catenin gene restores both growth regulation and tumor suppressor activities. 
Molecular and Cellular Biology  1997;17(8):4501-4508.
Recent studies indicate that disruption of the E-cadherin-mediated cell-cell adhesion system is frequently associated with human cancers of epithelial origin. Reduced levels of both E-cadherin and the associated protein, alpha-catenin, have been reported in human tumors. This report describes the characterization of a human ovarian carcinoma-derived cell line (Ov2008) which expresses a novel mutant form of the alpha-catenin protein lacking the extreme N terminus of the wild-type protein. The altered form of alpha-catenin expressed in Ov2008 cells fails to bind efficiently to beta-catenin and is localized in the cytoplasm. Deletion mapping has localized the beta-catenin binding site on alpha-catenin between amino acids 46 and 149, which encompasses the same region of the protein that is deleted in the Ov2008 variant. Restoration of inducible expression of the wild-type alpha-catenin protein in these cells caused them to assume the morphology typical of an epithelial sheet and retarded their growth in vitro. Additionally, the induction of alpha-catenin expression in Ov2008 cells injected into nude mice attenuated the ability of these cells to form tumors. These observations support the classification of alpha-catenin as a growth-regulatory and candidate tumor suppressor gene.
PMCID: PMC232303  PMID: 9234707
22.  Differential regulation of the p21/WAF-1 and mdm2 genes after high-dose UV irradiation: p53-dependent and p53-independent regulation of the mdm2 gene. 
Molecular Medicine  1997;3(7):441-451.
BACKGROUND: DNA damage in mammalian cells stabilizes the p53 protein which then functions as a cell cycle checkpoint by leading to growth arrest or apoptosis. p53 is a transcription factor and positively regulates the expression of the p21/WAF-1 gene and the mdm2 gene. After high-dose UV irradiation, p53 increases the expression of the p21/WAF-1 gene immediately (2 to 5 hours after irradiation) while the induction of the mdm2 gene is delayed (8 to 12 hours after irradiation). Experiments presented here explore this differential expression of two different p53-regulated genes. MATERIALS AND METHODS: IP-Western (protein) and Northern (mRNA) blot experiments are used to follow mdm2 and p21/WAF-1 expression in primary rat or mouse cells after a low-dose (4 J/m2) or a high-dose (20 J/M2) of UV irradiation. Northern blot and nuclear run-on experiments are employed to study mRNA stability as well as transcription rates of selected genes. RESULTS: After high-dose UV irradiation, p53 is rapidly stabilized and the expression of p21/WAF1 is immediately increased. By contrast, both protein and mRNA levels of mdm2 first decrease in a p53-independent manner, and later increase in a p53-dependent manner. The initial decline of mdm2 expression following high-dose UV irradiation is UV-dosage dependent and regulated at the level of transcription. CONCLUSION: p53 regulates two genes, p21/WAF1 (blocks cell cycle progression) and mdm2 (reverses p53 activity), that mediate opposite actions. This process is regulated in a temporal fashion after high-dose UV irradiation, so that cell cycle progression can be halted while DNA repair continues prior to reversal of p53-mediated arrest by mdm2.
Images
PMCID: PMC2230214  PMID: 9260156
23.  A genetic approach to mapping the p53 binding site in the MDM2 protein. 
Molecular Medicine  1997;3(4):248-259.
BACKGROUND: The MDM2 oncoprotein binds to the tumor suppressor p53 and inhibits its anti-oncogenic functions. MATERIALS AND METHODS: To determine the amino acids of MDM2 that are critical for binding to p53, a modified two-hybrid screen was performed in yeast. Site-directed mutagenesis was then performed to identify MDM2 residues important for p53 interaction. Mutant MDM2 proteins were subsequently tested for their ability to bind to p53 in vitro and for their ability to regulate p53-mediated transcription in vivo. RESULTS: The yeast genetic screen yielded two Mdm2 mutations (G58D and C77Y) which disrupted binding to p53 in vitro without altering the conformation of MDM2 as determined with conformation-sensitive monoclonal antibodies. Site-directed mutagenesis yielded mutations of two additional amino acids of MDM2 (D68 and V75) that prevented binding to p53 in vitro. The mutant MDM2 proteins were unable to inhibit p53-dependent transcription in vivo, which is consistent with prior indications that a physical interaction between the two proteins is required for MDM2's inhibition of p53. Finally, the crystal structure of the MDM2-p53 complex shows that two of the four critical residues identified here contact p53 directly, while the remaining two residues play important structural roles in the MDM2 domain. CONCLUSIONS: MDM2 residues G58, D68, V75, and C77 are critical for MDM2's interaction with the p53 protein. Mutation of these residues to alanine prevents MDM2's interaction with p53 in vitro, and MDM2's regulation of p53's transcriptional activity in vivo.
Images
PMCID: PMC2230064  PMID: 9131587
24.  The MDM2 oncoprotein binds specifically to RNA through its RING finger domain. 
Molecular Medicine  1996;2(4):439-451.
BACKGROUND: The cellular mdm2 gene has transforming activity when overexpressed and is amplified in a variety of human tumors. At least part of the transforming ability of the MDM2 protein is due to binding and inactivating the p53 tumor suppressor protein. Additionally, this protein forms a complex in vivo with the L5 ribosomal protein and its associated 5S ribosomal RNA and may be part of a ribosomal complex. MATERIALS AND METHODS: A RNA homopolymer binding assay and a SELEX procedure have been used to characterize the RNA-binding activity of MDM2. RESULTS: The MDM2 protein binds efficiently to the homopolyribonucleotide poly(G) but not to other homopolyribonucleotides. This binding is independent of the interaction of MDM2 with the L5 protein, which occurs through the central acidic domain of MDM2. An RNA SELEX procedure was performed to identify specific RNA ligands that bind with high affinity to the human MDM2 (HDM2) protein. After 10 rounds of selection and amplification, a subset of RNA molecules that bound efficiently to HDM2 was isolated from a randomized pool. Sequencing of these selected ligands revealed that a small number of sequence motifs were selected. The specific RNA binding occurs through the RING finger domain of the protein. Furthermore, a single amino acid substitution in the RING finger domain, G446S, completely abolishes the specific RNA binding. CONCLUSIONS: These observations, showing that MDM2 binds the L5/5S ribosomal ribonucleoprotein particle and can also bind to specific RNA sequences or structures, suggest a role for MDM2 in translational regulation in a cell.
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PMCID: PMC2230168  PMID: 8827714
25.  mdm-2 inhibits the G1 arrest and apoptosis functions of the p53 tumor suppressor protein. 
Molecular and Cellular Biology  1996;16(5):2445-2452.
The mdm-2 gene encodes a 90-kDa polypeptide that binds specifically to the p53 tumor suppressor protein. This physical interaction results in the inhibition of the transcriptional functions of p53 (J. Chen, J. Lin, and A. J. Levine, Mol. Med. 1:142-152, 1995, and J. Momand, G. P. Zambetti, D. C. Olson, D. George, and A. J. Levine, Cell 69:1237-1245, 1992). Experiments are described that demonstrate the ability of mdm-2 to abrogate both the p53-mediated cell cycle arrest and the apoptosis functions. In addition, the results presented here suggest that mdm-2 binding to p53 and the resultant inhibition of p53 transcription functions are critical for reversing p53-mediated cell cycle arrest. The N-terminal half or domain of the mdm-2 protein is sufficient to regulate these biological activities of p53, consistent with the possibility that the highly conserved central acidic region and the C-terminal putative zinc fingers of mdm-2 may encode other functions.
PMCID: PMC231233  PMID: 8628312

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