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PURPOSE - Development of new treatments is critical to effective protection against radiation-induced injury. We investigate the potential of developing small-molecule inhibitors of glycogen synthase kinase 3beta (GSK-3beta)-SB216763 or SB415286-as radioprotective agents to attenuate intestinal injury.
METHODS AND MATERIALS - A survival study was done by use of C57BL/6J mice to evaluate the radioprotective effect of GSK-3beta inhibitors. Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay and immunohistochemical staining for Bax and Bcl-2 were used to assess apoptosis in the small intestines of the treated mice. A clonogenic survival study, apoptosis assays (staining with annexin V or 4',6-diamidino-2-phenylindole), and immunoblot analysis of beta-catenin, Bcl-2, Bax, and caspase 3 were done by use of Rat intestinal epithelial cell line IEC-6 cells.
RESULTS - Pretreatment with SB415286 significantly improved survival of mice irradiated with 8 and 12 Gy. Mice pretreated with SB216763 or SB415286 showed a significant reduction in TUNEL- and Bax-positive cells and an increase in Bcl-2-positive cells in intestinal crypts at 4 and/or 12 h after radiation with 4 and/or 8 Gy compared with radiation alone. Pretreatment of irradiated IEC-6 cells with GSK-3beta inhibitors significantly increased clonogenic survival compared with cells treated with radiation alone. This increase was due to the attenuation of radiation-induced apoptosis, as shown by annexin V and 4',6-diamidino-2-phenylindole assays, as well as immunoblot analysis of Bcl-2, Bax, and caspase 3.
CONCLUSIONS - Glycogen synthase kinase 3beta small-molecule inhibitors protect mouse intestine from radiation-induced damage in cell culture and in vivo and improve survival of mice. Molecular mechanisms of this protection involve attenuated radiation-induced apoptosis regulated by Bcl-2, Bax, and caspase 3. Therefore GSK-3beta inhibitors reduce deleterious consequences of intestinal irradiation and thereby improve quality of life during radiation therapy.
Copyright 2010 Elsevier Inc. All rights reserved.
The primary objectives of this study were to determine if erythropoietin (EPO) is neuroprotective to the photoreceptors in the retinal degeneration slow (rds) mouse in the absence of an increase in hematocrit and to determine if deglycosylated EPO (DEPO) is less neuroprotective. We performed subretinal injections of 10U EPO, DEPO or hyperglycosylated EPO (HEPO) in postnatal day 7 rds mice. Whole eye EPO levels were quantified by ELISA at specified time points post-injection. TUNEL analysis, hematocrit, and immunohistochemistry were performed at postnatal day 20. Half of the amount of EPO measured immediately after injection was detected less than 1 h later. Twenty four hours later, EPO levels were 1000 times lower than the amount originally detected. Uninjected rds mice contained 36 +/- 2 TUNEL-positive cells/mm retina and PBS injected mice contained 17 +/- 3 TUNEL-positive cells/mm retina. EPO, DEPO, and HEPO treated rds retinas contained 5 +/- 2, 9 +/- 2, and 3 +/- 1 TUNEL-positive cells/mm retina, respectively. The hematocrit was 43% in control and 41% in treated rds mice Previous studies have shown neuroprotection of the retina by treatment with as little as 24-39 mU EPO/mg total protein in the eye. In this study, we detected 40 mU/mg EPO in the eye 11 h after injection of 10 U EPO. Treatment with all forms of EPO tested was neuroprotective to the photoreceptors without a concomitant increase in hematocrit.
Individuals with neurofibromatosis type 1 (NF1) are prone to develop optic pathway gliomas that can result in significant visual impairment. To explore the cellular basis for the reduced visual function resulting from optic glioma formation, we used a genetically engineered mouse model of Nf1 optic glioma (Nf1+/-(GFAP)CKO mice). We performed multimodal functional and structural analyses both before and after the appearance of macroscopic tumors. At 6 weeks of age, before obvious glioma formation, Nf1+/-(GFAP)CKO mice had decreased visual-evoked potential amplitudes and increased optic nerve axon calibers. By 3 months of age, Nf1+/-(GFAP)CKO mice exhibited pronounced optic nerve axonopathy and apoptosis of neurons in the retinal ganglion cell layer. Magnetic resonance diffusion tensor imaging showed a progressive increase in radial diffusivity between 6 weeks and 6 months of age in the optic nerve proximal to the tumor indicating ongoing deterioration of axons. These data suggest that optic glioma formation results in early axonal disorganization and damage, which culminates in retinal ganglion cell death. Collectively, this study shows that Nf1+/-(GFAP)CKO mice can provide a useful model for defining mechanisms of visual abnormalities in children with NF1 and lay the foundations for future interventional studies aimed at reducing visual loss.
Focal segmental glomerulosclerosis (FSGS) is a progressive renal disease, and the glomerular visceral cell hyperplasia typically observed in cellular/collapsing FSGS is an important pathological factor in disease progression. However, the cellular features that promote FSGS currently remain obscure. To determine both the origin and phenotypic alterations in hyperplastic cells in cellular/collapsing FSGS, the present study used a previously described FSGS model in p21-deficient mice with visceral cell hyperplasia and identified the podocyte lineage by genetic tagging. The p21-deficient mice with nephropathy showed significantly higher urinary protein levels, extracapillary hyperplastic indices on day 5, and glomerular sclerosis indices on day 14 than wild-type controls. X-gal staining and immunohistochemistry for podocyte and parietal epithelial cell (PEC) markers revealed progressive podocytopenia with capillary collapse accompanied by PEC hyperplasia leading to FSGS. In our investigation, non-tagged cells expressed neither WT1 nor nestin. Ki-67, a proliferation marker, was rarely associated with podocytes but was expressed at high levels in PECs. Both terminal deoxynucleotidyl transferase dUTP nick-end labeling staining and electron microscopy failed to show evidence of significant podocyte apoptosis on days 5 and 14. These findings suggest that extensive podocyte loss and simultaneous PEC hyperplasia is an actual pathology that may contribute to the progression of cellular/collapsing FSGS in this mouse model. Additionally, this is the first study to demonstrate the regulatory role of p21 in the PEC cell cycle.
Transforming growth factor beta (TGF-beta) ligands are known to regulate virgin mammary development and contribute to initiation of post-lactation involution. However, the role for TGF-beta during the second phase of mammary involution has not been addressed. Previously, we have used an MMTV-Cre transgene to delete exon 2 from the Tgfbr2 gene in mammary epithelium, however we observed a gradual loss of T beta RII deficient epithelial cells that precluded an accurate study of the role for TGF-beta signaling during involution timepoints. Therefore, in order to determine the role for TGF-beta during the second phase of mammary involution we have now targeted T beta RII ablation within mammary epithelium using the WAP-Cre transgene [T beta RII(WKO)Rosa26R]. Our results demonstrated that TGF-beta regulates commitment to cell death during the second phase of mammary involution. Importantly, at day 3 of mammary involution the Na-Pi type IIb co-transporter (Npt2b), a selective marker for active lactation in luminal lobular alveolar epithelium, was completely silenced in the WAP-Cre control and T beta RII(WKO)Rosa26R tissues. However, by day 7 of involution the T beta RII(WKO)Rosa26R tissues had distended lobular alveoli and regained a robust Npt2b signal that was detected at the apical luminal surface. The Npt2b abundance and localization positively correlated with elevated WAP mRNA expression, suggesting that the distended alveoli were the result of an active lactation program rather than residual milk protein and lipid accumulation. In summary, the results suggest that an epithelial cell response to TGF-beta signaling regulates commitment to cell death and suppression of lactation during the second phase of mammary involution.
(c) 2008 Wiley-Liss, Inc.
We investigated the role of Aurora kinase A (AURKA) in regulating p73-dependent apoptosis using the p53-deficient cancer cell lines H1299, TE7, and HCT116p53(-/-). Overexpression of AURKA led to down-regulation of the TAp73-induced activation of the p53/p73-dependent luciferase reporter plasmid (pG13-luc). The reduction in the TAp73 transcription activity was confirmed by measuring the activity of luciferase reporters for p21/WAF1, and PUMA. The siRNA knockdown of endogenous AURKA reversed these effects and Western blot analysis showed a significant increase in the protein level of TAp73 and its downstream transcription targets, PUMA, NOXA, and p21/WAF1. The coexpression of AURKA together with TAp73 inhibited the activation of the pG13-luc, PUMA-luc, and p21/WAF1-luc reporter plasmids with reduction in the protein levels of TAp73 transcription targets. Treatment with AURKA-selective small molecule inhibitor MLN8054 led to a significant increase in the activities of pG13-luc, PUMA-luc, and p21/WAF1-luc reporter plasmids. This effect was accompanied by a significant increase in the mRNA and protein levels of several TAp73 transcription targets: p21/WAF1, PUMA, and NOXA. Flow cytometry cell cycle analysis, after MLN8054 treatment, showed more than a 2-fold increase in cell death. The apoptotic outcome was corroborated by showing an increase in cleaved caspase-3 protein levels by Western blot. Using terminal deoxynucleotidyl-transferase-mediated dUTP nick-end labeling assay, we showed that the expression of dominant-negative mutant TAp73 expression plasmid (p73DD) counteracted the MLN8054-induced cell death. Taken together, our results indicate that AURKA regulates TAp73-dependent apoptosis and highlight the potential of the AURKA inhibitor MLN8054 in treating cancers that are defective in p53 signaling.
PURPOSE - Frizzled-5 (Fzd5) is expressed in the developing retina of multiple species and appears to play species-specific roles during eye development. The present study analyzed the effects of tissue-specific deletion of Fzd5 on mammalian eye development.
METHODS - To generate Fzd5 conditional knockout (CKO) mice, Fzd5(+/-) mice carrying the Six3-Cre transgene were crossed with Fzd5(LoxP/LoxP) mice. To determine which cell lineages in the eye had Cre recombinase activity, Six3-Cre transgenic mice were crossed with ROSA-26 reporter mice, and lacZ activity was assayed. Histologic analysis, immunofluorescence, and TUNEL labeling were performed from embryonic day (E)12.5 to postnatal stages to analyze vascularization, cell proliferation, retinal organization, and apoptosis.
RESULTS - On conditional disruption of Fzd5 specifically in the retina, but not in vitreous hyaloid vasculature (VHV), an abnormal accumulation consisting of pericytes and endothelial cells was observed in the vitreous as early as E12.5. The abundant retrolental cells persisted into postnatal stages and appeared as a pigmented intravitreal mass. In Fzd5 CKO mice there was failure of normal apoptosis of the VHV, and cells in the persistent VHV were maintained in the cell cycle up to postnatal day 23. Moreover, morphogenesis of the retina adjacent to the vasculature was disrupted, leading to retinal folds, detachment, and abnormal lamination. This phenotype is similar to that of human eye disease persistent hyperplastic primary vitreous (PHPV).
CONCLUSIONS - Selective loss of Fzd5 in the retina results in PHPV and retinal defects through an apparently cell-nonautonomous effect, revealing a potential requirement for retina-derived signals in regulating the development of the VHV.
BACKGROUND & AIMS - Raf-1 kinase is a key regulator of a number of cellular processes, which promote the maintenance of a healthy colon epithelium. This study addresses the role of Raf in epithelial cell survival in response to dextran sulfate sodium (DSS)-induced injury and inflammation.
METHODS - Inducible intestinal epithelium-specific Raf knockout mice were generated and subjected to acute colitis followed by a short recovery period. Colon sections were analyzed by in situ oligo ligation or immunostaining for Ki67, phospho-extracellular signal regulated kinase, and nuclear factor-kappaB p65. Western blot analysis and terminal deoxynucleotidyl transferase nick-end labeling assays were performed on Raf small interfering RNA-transfected young adult mouse colon cells following DSS treatment.
RESULTS - We report that Raf protects against epithelial injury and inflammation and promotes recovery from acute DSS-induced colitis by both MAPK/ERK kinase (MEK)-dependent and -independent pathways. Furthermore, we demonstrate that Raf induces novel cell survival responses through activating nuclear factor-kappaB in a MEK-independent manner.
CONCLUSIONS - These novel findings indicate a protective role for Raf in colon epithelium following ulcerative damage through inhibiting cell apoptosis and promoting proliferation with important implications for responses such as inflammation-associated carcinogenesis.
Human herpesvirus-8 (HHV-8) is the causative agent of Kaposi's sarcoma and is associated with the angioproliferative disorders primary effusion lymphoma and multicentric Castleman's disease. Evidence of HHV-8 infection within the pulmonary vasculature of patients with idiopathic pulmonary arterial hypertension (IPAH) has been described. We hypothesize that HHV-8 infection of pulmonary microvascular endothelial cells results in an apoptotic-resistant phenotype characteristic of severe pulmonary arterial hypertension. Our objective was to investigate the ability of HHV-8 to infect human pulmonary microvascular endothelial cells in vitro and characterize the phenotypic effect of this infection. Human pulmonary microvascular endothelial cells were exposed to HHV-8 using two methods (direct virus and co-culture technique). The presence of lytic and latent infection was confirmed. Changes in endothelial cell gene and protein expression and effects on cellular apoptosis were measured. HHV-8 can both lytically and latently infect primary human pulmonary microvascular endothelial cells in vitro. HHV-8 infection results in significant changes in gene expression, including alterations of pathways important to cellular apoptosis. HHV-8 infection also alters expression of genes integral to the bone morphogenic protein pathway, including down-regulation of bone morphogenic protein-4. Other genes previously implicated in the development of PAH are affected by HHV-8 infection, and cells infected with HHV-8 are resistant to apoptosis.
PURPOSE - We have investigated the role of t-DARPP in trastuzumab resistance in ERBB2-amplified and overexpressed breast cancer cell lines.
EXPERIMENTAL DESIGN - We have used the HR-5 and HR-6 trastuzumab-resistant cells that were established from tumors that recurred in the presence of trastuzumab therapy following xenografts of BT-474 cells in nude mice. In addition, SKBR-3 cells, engineered for stable expression of t-DARPP, and HCC-1569 cells, which have constitutive expression of t-DARPP and are de novo resistant to trastuzumab, were used.
RESULTS - We reported > or =15-fold up-regulation of mRNA and protein levels of t-DARPP in HR-5 and HR-6 cells compared with their progenitor BT-474 trastuzumab-sensitive cells. The t-DARPP expression was not regulated by changes in its promoter DNA methylation levels. The SKBR-3 cells stably expressing t-DARPP developed resistance to trastuzumab compared with their parental cells and empty vector controls (P < 0.01). The trastuzumab-resistant cell lines showed a significant increase in pAKT (Ser(473)) and BCL2 protein levels. The small interfering RNA knockdown of t-DARPP in all trastuzumab-resistant cells led to a significant reduction in ERBB2, pAKT (Ser(473)), and BCL2 protein levels with a significant decrease in cell viability (P < or = 0.001) and an increase in cleaved caspase-3 levels, indicating the progression of these cells toward apoptosis. The t-DARPP protein was associated with both heat shock protein 90 and ERBB2 forming a potential protein complex. This association may play a role in regulating ERBB2 protein in trastuzumab-resistant cells.
CONCLUSION - We conclude that t-DARPP is a novel molecular target that can mediate the therapeutic resistance to trastuzumab in breast cancer cells.