Key Specifications Table
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|A||FC, ICC, IHC, IH(P)||M||Purified||Monoclonal Antibody|
|Presentation||Purified Immunoglobulin. Liquid in PBS supplemented with 5% fetal bovine Serum (FBS), containing no preservatives.|
|Safety Information according to GHS|
|Storage and Shipping Information|
|Storage Conditions||Maintain frozen at -80°C in undiluted aliquots for up to 12 months after date of purchase.|
|Material Size||50 µg|
References | 20 Available | See All References
|Reference overview||Pub Med ID|
|Suppression of tumor growth in mice by rationally designed pseudopeptide inhibitors of fibroblast activation protein and prolyl oligopeptidase. |
Jackson, KW; Christiansen, VJ; Yadav, VR; Silasi-Mansat, R; Lupu, F; Awasthi, V; Zhang, RR; McKee, PA
Neoplasia (New York, N.Y.) 17 43-54 2015
Tumor microenvironments (TMEs) are composed of cancer cells, fibroblasts, extracellular matrix, microvessels, and endothelial cells. Two prolyl endopeptidases, fibroblast activation protein (FAP) and prolyl oligopeptidase (POP), are commonly overexpressed by epithelial-derived malignancies, with the specificity of FAP expression by cancer stromal fibroblasts suggesting FAP as a possible therapeutic target. Despite overexpression in most cancers and having a role in angiogenesis, inhibition of POP activity has received little attention as an approach to quench tumor growth. We developed two specific and highly effective pseudopeptide inhibitors, M83, which inhibits FAP and POP proteinase activities, and J94, which inhibits only POP. Both suppressed human colon cancer xenograft growth greater than 90% in mice. By immunohistochemical stains, M83- and J94-treated tumors had fewer microvessels, and apoptotic areas were apparent in both. In response to M83, but not J94, disordered collagen accumulations were observed. Neither M83- nor J94-treated mice manifested changes in behavior, weight, or gastrointestinal function. Tumor growth suppression was more extensive than noted with recently reported efforts by others to inhibit FAP proteinase function or reduce FAP expression. Diminished angiogenesis and the accompanying profound reduction in tumor growth suggest that inhibition of either FAP or POP may offer new therapeutic approaches that directly target TMEs.
|Chromatin collapse during caspase-dependent apoptotic cell death requires DNA fragmentation factor, 40-kDa subunit-/caspase-activated deoxyribonuclease-mediated 3'-OH single-strand DNA breaks. |
Iglesias-Guimarais, V; Gil-Guiñon, E; Sánchez-Osuna, M; Casanelles, E; García-Belinchón, M; Comella, JX; Yuste, VJ
The Journal of biological chemistry 288 9200-15 2013
Apoptotic nuclear morphology and oligonucleosomal double-strand DNA fragments (also known as DNA ladder) are considered the hallmarks of apoptotic cell death. From a classic point of view, these two processes occur concomitantly. Once activated, DNA fragmentation factor, 40-kDa subunit (DFF40)/caspase-activated DNase (CAD) endonuclease hydrolyzes the DNA into oligonucleosomal-size pieces, facilitating the chromatin package. However, the dogma that the apoptotic nuclear morphology depends on DNA fragmentation has been questioned. Here, we use different cellular models, including MEF CAD(-/-) cells, to unravel the mechanism by which DFF40/CAD influences chromatin condensation and nuclear collapse during apoptosis. Upon apoptotic insult, SK-N-AS cells display caspase-dependent apoptotic nuclear alterations in the absence of internucleosomal DNA degradation. The overexpression of a wild-type form of DFF40/CAD endonuclease, but not of different catalytic-null mutants, restores the cellular ability to degrade the chromatin into oligonucleosomal-length fragments. We show that apoptotic nuclear collapse requires a 3'-OH endonucleolytic activity even though the internucleosomal DNA degradation is impaired. Moreover, alkaline unwinding electrophoresis and In Situ End-Labeling (ISEL)/In Situ Nick Translation (ISNT) assays reveal that the apoptotic DNA damage observed in the DNA ladder-deficient SK-N-AS cells is characterized by the presence of single-strand nicks/breaks. Apoptotic single-strand breaks can be impaired by DFF40/CAD knockdown, abrogating nuclear collapse and disassembly. In conclusion, the highest order of chromatin compaction observed in the later steps of caspase-dependent apoptosis relies on DFF40/CAD-mediated DNA damage by generating 3'-OH ends in single-strand rather than double-strand DNA nicks/breaks.
|Apnea produces excitotoxic hippocampal synapses and neuronal apoptosis. |
Simon J Fung,Mingchu Xi,Jianhua Zhang,Sharon Sampogna,Michael H Chase
Experimental neurology 238 2012
Obstructive sleep apnea (OSA) results in the degeneration of neurons in the hippocampus that eventuates in neurocognitive deficits. We were therefore interested in determining the effects of apnea on monosynaptic excitatory processes in a hippocampal pathway (cornu ammonis 3-cornu ammonis 1, CA3-CA1) that has been shown to mediate the processing of cognitive information. In addition, to substantiate an anatomical basis for the cognitive dysfunction that occurs in OSA patients, we examined the effects of apnea with respect to neurodegenerative changes (apoptosis) in the same hippocampal pathway. In order to determine the effects of apnea, an automated system for the generation and analysis of single and recurrent periods of apnea was developed. Utilizing this system, the field excitatory postsynaptic potential (fEPSP) generated by pyramidal neurons in the CA1 region of the hippocampus was monitored in α-chloralose anesthetized rats following stimulation of glutamatergic afferents in the CA3 region. A stimulus-response (input-output) curve for CA3-CA1 synaptic activity was determined. In addition, a paired-pulse paradigm was employed to evaluate, electrophysiologically, the presynaptic release of glutamate. Changes in the synaptic efficacy were assessed following single episodes of apnea induced by ventilatory arrest (60 to 80s duration, mean=72s; mean oxygen desaturation was 53% of normoxia level). Apnea resulted in a significant potentiation of the amplitude (mean=126%) and slope (mean=117%) of the baseline CA1 fEPSP. This increase in the fEPSP was accompanied by a significant decrease in the amplitude (71%) and slope (81%) of normalized paired-pulse facilitation (PPF) ratios. Since the potentiation of the fEPSP is inversely proportional to changes in PPF ratio, the potentiated fEPSP accompanied by the reduced PPF reveals that apnea produces an abnormal increase in the preterminal release of glutamate that results in the over-activation (and calcium overloading) of hippocampal CA1 neurons. Thus, we conclude that individual episodes of apnea result in the development of excitotoxic processes in the hippocampal CA3-CA1 pathway that is critically involved in the processing of cognitive information. Morphologically, the deleterious effect of recurrent apnea was substantiated by the finding of apoptosis in CA1 neurons of apneic (but not normoxic) animals.
|Involvement of autophagy in the pharmacological effects of the mTOR inhibitor everolimus in acute kidney injury. |
Shunsaku Nakagawa,Kumiko Nishihara,Ken-Ichi Inui,Satohiro Masuda
European journal of pharmacology 696 2012
Inhibitors of mammalian target of rapamycin (mTOR) have immunosuppressive and anti-cancer effects, but their effects on the progression of kidney disease are not fully understood. Using cells from normal kidney epithelial cell lines, we found that the antiproliferative effects of mTOR inhibitor everolimus accompanied the accumulation of a marker for cellular autophagic activity, the phosphatidylethanolamine-conjugated form of microtubule-associated protein 1 light chain 3 (LC3-II) in cells. We also showed that the primary autophagy factor UNC-51-like kinase 1 was involved in the antiproliferative effects of everolimus. Levels of LC3-II decreased in the kidneys of rats treated with ischemia-reperfusion or cisplatin; however, renal LC3-II levels increased after administration of everolimus to rats subjected to ischemia-reperfusion or cisplatin treatment. Simultaneously, increased signals for kidney injury molecule-1 and single-stranded DNA and decreased signals for Ki-67 in the proximal tubules were observed after treatment with everolimus, indicating that everolimus diminished renal function after acute tubular injury. We also found leakage of LC3 protein into rat urine after treatment with everolimus, and urinary LC3 protein was successfully measured between 0.1 and 500ng/mL by using an enzyme-linked immunosorbent assay. Urinary LC3 levels were increased after administration of everolimus to rats subjected to ischemia-reperfusion or cisplatin treatment, suggesting that renal LC3-II and urinary LC3 protein are new biomarkers for autophagy in acute kidney injury. Taken together, our results demonstrated that the induction of autophagy by everolimus aggravates tubular dysfunction during recovery from kidney injury.
|Heat shock protein 27 confers resistance to androgen ablation and chemotherapy in prostate cancer cells through eIF4E. |
C Andrieu,D Taieb,V Baylot,S Ettinger,P Soubeyran,A De-Thonel,C Nelson,C Garrido,A So,L Fazli,F Bladou,M Gleave,J L Iovanna,P Rocchi
Oncogene 29 2010
One strategy to improve therapies in advanced prostate cancer (PC) involves targeting genes that are activated by androgen withdrawal to delay the emergence of the androgen-independent (AI) phenotype. Heat shock protein 27 (Hsp27) expression becomes highly upregulated in PC cells after androgen withdrawal or chemotherapy, in which it functions as a cytoprotective chaperone to confer broad-spectrum treatment resistance. The purpose of this study is to elucidate anti-apoptotic pathways regulated by Hsp27 that are activated during PC progression. Using two-hybrid experiment, we found that Hsp27 was having a major role in the protein translational initiation process. Furthermore, using complementary DNA (cDNA) microarray analysis, 4E binding protein 1 was identified as being proportionately and highly regulated by Hsp27. These data led us to analyze the protein synthesis initiation pathway, which is a prerequisite for cell growth and proliferation. Using northern and western blot analysis, we found that Hsp27 downregulation decreased eukaryotic translation initiation factor 4E (eIF4E) expression at the protein, but not mRNA, level. The cytoprotection afforded by Hsp27 overexpression was attenuated by eIF4E knockdown using specific eIF4E short interfering RNA (siRNA). Co-immunoprecipitation and co-immunofluorescence confirmed that Hsp27 colocalizes and interacts directly with eIF4E. Hsp27-eIF4E interaction decreases eIF4E ubiquitination and proteasomal degradation. By chaperoning eIF4E, Hsp27 seems to protect the protein synthesis initiation process to enhance cell survival during cell stress induced by castration or chemotherapy. Forced overexpression of eIF4E induces resistance to androgen-withdrawal and paclitaxel treatment in the prostate LNCaP cells in vitro. These findings identify Hsp27 as a modulator of eIF4E and establish a potential mechanism for the eIF4E-regulated apoptosis after androgen ablation and chemotherapy. Targeting Hsp27-eIF4E interaction may serve as a therapeutic target in advanced PC.
|Method of specific detection of apoptosis using formamide-induced DNA denaturation assay. |
Ito, Y; Shibata, MA; Kusakabe, K; Otsuki, Y
The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society 54 683-92 2006
We compared the reliability between apoptosis detection methods, namely, the terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) method and formamide-induced DNA denaturation assay using a monoclonal antibody (MAb) to single-stranded DNA (ssDNA) (formamide-MAb assay). Reaction targets in these methods are different: the TUNEL method recognizes free 3'-OH DNA ends, whereas the formamide-MAb assay detects ssDNA itself (25-30 bp). We found that the formamide-MAb assay immunohistochemically detected apoptotic cells, whereas the TUNEL method detected apoptotic cells as well as mitotic and necrotic cells. The TUNEL method recognized not only 3'-OH DNA ends cleaved by DNase during apoptosis but also constitutive physiological nicking that occurs in DNA duplication and histone posttranslational modifications during mitosis and random DNA breaks during necrotic execution. By electron microscopy, the mean labeling density (the number of 3'-OH DNA ends/nuclear area) obtained by the TUNEL method was determined to be consistently higher than that (the number of ssDNAs/nuclear area) obtained by the formamide-MAb assay. On the basis of these findings, we conclude that the formamide-MAb assay was more specific than the TUNEL method for the detection of apoptotic cells using electron microscopy; however, the labeling intensity of the formamide-MAb assay was slightly weaker than that of the TUNEL method.
|Small interference RNA targeting heat-shock protein 27 inhibits the growth of prostatic cell lines and induces apoptosis via caspase-3 activation in vitro. |
Palma Rocchi, Paul Jugpal, Alan So, Shannon Sinneman, Susan Ettinger, Ladan Fazli, Colleen Nelson, Martin Gleave
BJU international 98 1082-9 2006
OBJECTIVES: To evaluate synthetic small interference RNA (siRNA) compounds targeting heat-shock protein 27 (Hsp27) as an alternative approach to Hsp27 'knockdown' in prostate cancer cells, as Hsp27 expression is highly up-regulated in prostate cancer cells after androgen withdrawal or chemotherapy, to become uniformly highly expressed in androgen-independent (AI) prostate cancer. MATERIALS AND METHODS: We recently showed that targeting Hsp27 by a 2'-methoxyethyl modified phosphorothioate antisense oligonucleotide, OGX-427, inhibits Hsp27 expression and enhances hormone- and chemotherapy in prostate cancer xenograft models. In the present study, a 'gene walk' screening different siRNAs was initially used in PC-3 and LNCaP cells to determine the most potent sequence to down-regulate Hsp27 mRNA and protein levels. The effects of Hsp27 silencing on in vitro growth rates were studied by tetrazolium-blue and crystal violet assays. Apoptosis was determined by single-stranded DNA nuclear and cleaved caspase-3 immunostaining, as well as flow cytometry. Spotted microarrays with 14,000 human oligonucleotides were used to examine changes in gene expression. RESULTS: Low concentrations of 1 nm siRNA decreased Hsp27 mRNA levels by 19-fold and suppressed protein expression to undetectable levels. Silencing of Hsp27 in prostate cancer cells by siRNA # 2 increased apoptotic rates 2.4-4 fold and caused 40-76% inhibition of cell growth in LNCaP and PC-3 cells. Characteristic cleavage of caspase-3 occurred after treatment with Hsp27 siRNA (1 nm). cDNA microarray analysis from LNCaP and PC-3 cell lines revealed differential gene expression profiles after Hsp27 down-regulation that could be used to identify various survival pathways involved in androgen-dependent and AI growth. CONCLUSIONS: These findings illustrate the potential utility of Hsp27-silencing therapy and highlight Hsp27 siRNA strategies as a novel and highly effective tool, with the potential for future targeted therapy in enhancing the efficacy of chemotherapy in advanced prostate cancer.
|Rapid in vivo Taxotere quantitative chemosensitivity response by 4.23 Tesla sodium MRI and histo-immunostaining features in N-Methyl-N-Nitrosourea induced breast tumors in rats. |
Sharma, R; Kline, RP; Wu, EX; Katz, JK
Cancer cell international 5 26 2005
Sodium weighted images can indicate sodium signal intensities from different features in the tumor before and 24 hours following administration of Taxotere.To evaluate the association of in vivo intracellular sodium magnetic resonance image intensities with immuno-biomarkers and histopathological features to monitor the early tumor response to Taxotere chemotherapy in Methyl-Nitroso-Urea induced rat xenograft breast tumors.Methyl-Nitroso-Urea (MNU) induced rat xenograft breast tumors were imaged for sodium MRI and compared with tumor histology, immunostaining after 24 hours chemotherapy.Sodium MRI signal intensities represented sodium concentrations. Excised tumor histological sections showed different in vitro histological end points i.e. single strand DNA content of cell nuclei during cell cycle (G1/S-G2/M), distinct S or M histograms (Feulgen labeling to nuclear DNA content by CAS 200), mitotic figures and apoptosis at different locations of breast tumors. Necrosis and cystic fluid appeared gray on intracellular (IC) sodium images while apoptosis rich regions appeared brighter on IC sodium images. After 24 hours Taxotere-treated tumors showed lower 'IC/EC ratio' of viable cells (65-76%) with higher mitotic index; apoptotic tumor cells at high risk due to cytotoxicity (greater than 70% with high apoptotic index); reduced proliferation index (270 vs 120 per high power field) associated with enhanced IC sodium in vivo MR image intensities and decreased tumor size (3%; p less than 0.001; n = 16) than that of pre-treated tumors. IC-Na MR signal intensities possibly indicated Taxotere chemosensitivity response in vivo associated with apoptosis and different pre-malignant features within 24 hours of exposure of cancer cells to anti-neoplastic Taxotere drug.Sodium MRI imaging may be used as in vivo rapid drug monitoring method to evaluate Taxotere chemosensitivity response associated with neoplasia, apoptosis and tumor histology features.
|Increased Hsp27 after androgen ablation facilitates androgen-independent progression in prostate cancer via signal transducers and activators of transcription 3-mediated suppression of apoptosis. |
Rocchi, P; Beraldi, E; Ettinger, S; Fazli, L; Vessella, RL; Nelson, C; Gleave, M
Cancer research 65 11083-93 2005
One strategy to improve therapies in prostate cancer involves targeting cytoprotective genes activated by androgen withdrawal to delay the emergence of the androgen-independent (AI) phenotype. The objectives of this study were to define changes in Hsp27 levels after androgen ablation and to evaluate the functional relevance of these changes in AI progression. Using a tissue microarray of 232 specimens of hormone-naïve and post-hormone ablation-treated prostate cancer, we found that Hsp27 levels increase after androgen ablation to become highly expressed (greater than 4-fold, P less than or = 0.01) in AI tumors. Hsp27 overexpression rendered LNCaP cells highly resistant to androgen withdrawal both in vitro and in vivo. Tumor volume and serum prostate-specific antigen levels increased 4.3- and 10-fold faster after castration when Hsp27 was overexpressed. Treatment of LNCaP tumor cells in vitro with Hsp27 antisense oligonucleotides (ASO) or short-interfering RNA suppressed Hsp27 levels in a dose-dependent and sequence-specific manner increased the apoptotic sub-G0-G1 fraction and caspase-3 cleavage greater than 2-fold, as well as decreased signal transducers and activators of transcription 3 (Stat3) levels and its downstream genes, c-fos and sPLA-2. The cytoprotection afforded by Hsp27 overexpression was attenuated by Stat3 knockdown using specific Stat3 ASO. Coimmunoprecipitation and immunofluorescence confirmed that Hsp27 interacts with Stat3 and that Stat3 levels correlated directly with Hsp27 levels. Hsp27 ASO treatment in athymic mice bearing LNCaP tumors significantly delayed LNCaP tumor growth after castration, decreasing mean tumor volume and serum prostate-specific antigen levels by 57% and 69%, respectively. These findings identify Hsp27 as a modulator of Stat3-regulated apoptosis after androgen ablation and as a potential therapeutic target in advanced prostate cancer.
|Fluorosis: a new model and new insights. |
J D Bartlett, S E Dwyer, E Beniash, Z Skobe, T L Payne-Ferreira, J D Bartlett, S E Dwyer, E Beniash, Z Skobe, T L Payne-Ferreira
Journal of dental research 84 832-6 2005
Fluoride is an effective agent for the prevention of dental caries. However, the mechanism of how excessive fluoride exposure causes fluorosis remains uncertain. Zebrafish (Danio rerio) exhibit periodic tooth replacement throughout their lives, thereby providing continuous access to teeth at developmental stages susceptible to fluoride exposure. Zebrafish teeth do not contain true enamel, but consist of a hard enameloid surface. Therefore, we asked whether zebrafish could be used as a model organism for the study of dental fluorosis. Scanning electron microscopy of fluoride-treated teeth demonstrated that the enameloid was pitted and rough, and FTIR analysis demonstrated that the teeth also contained a significantly higher organic content when compared with untreated controls. Furthermore, we demonstrate for the first time that decreased expression of an important signaling molecule (Alk8) in tooth development may contribute to the observed fluorotic phenotype, and that increased cell apoptosis may also play a role in the mechanism of fluorosis.
|Increased airway epithelial and T-cell apoptosis in COPD remains despite smoking cessation. |
Hodge, S; Hodge, G; Holmes, M; Reynolds, PN
The European respiratory journal 25 447-54 2005
There is heterogeneity in the propensity of smokers to develop chronic obstructive pulmonary disease (COPD), and improved treatment strategies are hindered by limited understanding of COPD pathogenesis, especially as distinct from the effects of smoking per se. Although apoptosis is essential for tissue homeostasis, increased apoptosis may cause tissue damage and inflammation. This study addressed whether airway T-lymphocytes and airway epithelial cells (AEC) show an increased likelihood of undergoing apoptosis in COPD and if this was related to smoking. Apoptosis (7-amino-actinomycin D, Annexin, single-stranded DNA and caspase), Bcl-2, Bax and p53 were assessed in cells obtained from bronchial bushing and bronchoalveolar lavage from ex- and continuing smokers with COPD, and nonsmoking controls, using flow cytometry. A mean 87% increase in apoptosis of AEC and a 103% increase in T-lymphocyte apoptosis were found in COPD. There were no significant differences in apoptosis of AEC between current and ex-smokers with COPD. Apoptosis may contribute to chronic obstructive pulmonary disease pathogenesis, and continued excess apoptosis after smoking cessation may offer a new target for therapeutic interventions. Whether the persistence of increased apoptosis after smoking cessation results from changes in the pulmonary milleau after years of noxious insult, or whether some individuals have a natural predisposition toward increased apoptosis and possible development of chronic obstructive pulmonary disease remains to be determined.
|Increased peripheral blood T-cell apoptosis and decreased Bcl-2 in chronic obstructive pulmonary disease. |
Hodge, S; Hodge, G; Holmes, M; Reynolds, PN
Immunology and cell biology 83 160-6 2005
Chronic obstructive pulmonary disease (COPD) is an inflammatory airway disease, usually associated with cigarette smoking. Stimulated peripheral blood T cells from patients with COPD have an increased propensity to undergo apoptosis. The mitochondrial apoptotic pathway is regulated by pro-apoptotic proteins (including p53 and Bax) as well as anti-apoptotic proteins (e.g. Bcl-2) and cytokines (IL-2, IL-4 and IL-7). We hypothesized that alterations in expression of these apoptosis-related proteins, cytokines and cytokine receptors may be important in determining the susceptibility of T cells to undergoing apoptosis in COPD. We further hypothesized that inhaled corticosteroids (GCS) contribute to the increased rates of T-cell apoptosis observed in COPD. The process of apoptosis (assessed by Annexin V and ssDNA staining), as well as Bcl-2, Bax, p53, IL-2, IL-4 and receptors IL-7R, IL-4R and IL-2Rgamma were investigated in PHA-stimulated peripheral blood-derived T cells, using flow cytometry. Fifteen patients with COPD receiving inhaled GCS (four of who received additional prednisolone), eight patients with COPD receiving symptom control medication, and 16 control subjects were studied. T cells (CD4(+) and CD8(+)) from GCS-treated COPD patients showed an increased propensity to undergo apoptosis, associated with significantly decreased Bcl-2 and IL-7 receptor expression. No significant differences were observed for the COPD patients who were receiving symptom control medication. These findings may suggest a negative peripheral effect of inhaled GCS on the immune system in COPD, although the clinical significance of these effects remains uncertain.
|A novel Mtd splice isoform is responsible for trophoblast cell death in pre-eclampsia. |
Soleymanlou, N; Wu, Y; Wang, JX; Todros, T; Ietta, F; Jurisicova, A; Post, M; Caniggia, I
Cell death and differentiation 12 441-52 2005
Pre-eclampsia is a serious disorder of human pregnancy, characterized by decreased utero-placental perfusion and increased trophoblast cell death. Presently, the mechanisms regulating trophoblast cell death in pre-eclampsia are not fully elucidated. Herein, we have identified a novel Mtd/Bok splice isoform (Mtd-P) resulting from exon-II skipping. Mtd-P expression was unique to early-onset severe pre-eclamptic placentae as assessed by quantitative real-time-PCR and immunoblotting. Mtd-P overexpression in cell lines (BeWo: cytotrophoblast-derived; and CHO: ovary-derived) resulted in increased apoptotic cell death as assessed by caspase-3 cleavage, internucleosomal DNA laddering and mitochondrial depolarization. Moreover, Mtd-P expression increased under conditions of low oxygenation/oxidative stress in human villous explants. Antisense knockdown of Mtd under conditions of oxidative stress resulted in decreased caspase-3 cleavage. We conclude that under conditions of reduced oxygenation/oxidative stress, Mtd-P causes trophoblast cell death in pre-eclampsia and hence may contribute to the molecular events leading to the clinical manifestations of this disease.
|Comparison of chondrocyte apoptosis in vivo and in vitro following acute osteochondral injury. |
Costouros, John G, et al.
J. Orthop. Res., 22: 678-83 (2004) 2004
The objective of the present study was to directly compare levels of chondrocyte apoptosis produced by osteochondral injury in vivo and in vitro. Adult New Zealand White rabbits underwent 2 mm osteochondral drilling of the medial and lateral femoral condyles of a single hind limb. Animals were euthanized, and specimens were harvested at 0, 2, 4, 7, 10, and 14 days following injury. At the time of euthanasia, identical injuries were created in the femoral condyles of the contralateral hind limb. These condyles were maintained in vitro under standard tissue culture conditions until harvesting at time points corresponding to the in vivo specimens (i.e. after 0, 2, 4, 7, 10 and 14 days in culture). The extent of apoptosis in the in vivo and in vitro specimens was quantified by TUNEL analysis. The amount and distribution of TUNEL positive cells followed similar patterns in both in vivo and in vitro specimens with a maximal percentage of apoptotic chondrocytes observed on post-injury day 4. On post-injury day 4, in vivo specimens displayed a statistically significant increased overall level of apoptosis compared to in vitro specimens [in vivo=32.5+/-8.6%; in vitro=22.2+/-4.8%; (p=0.03)]. These experiments suggest that the majority of programmed cell death observed after osteochondral injury can be attributed to processes intrinsic to the cartilage itself; however, additional factors present within the acutely traumatized joint also appear to potentiate chondrocyte apoptosis following injury.
|Heat shock protein 27 increases after androgen ablation and plays a cytoprotective role in hormone-refractory prostate cancer. |
Rocchi, P; So, A; Kojima, S; Signaevsky, M; Beraldi, E; Fazli, L; Hurtado-Coll, A; Yamanaka, K; Gleave, M
Cancer research 64 6595-602 2004
Heat shock protein 27 (Hsp27) is a chaperone implicated as an independent predictor of clinical outcome in prostate cancer. Our aim was to characterize changes in Hsp27 after androgen withdrawal and during androgen-independent progression in prostate xenografts and human prostate cancer to assess the functional significance of these changes using antisense inhibition of Hsp27. A tissue microarray was used to measure changes in Hsp27 protein expression in 232 specimens from hormone naive and posthormone-treated cancers. Hsp27 expression was low or absent in untreated human prostate cancers but increased beginning 4 weeks after androgen-ablation to become uniformly highly expressed in androgen-independent tumors. Androgen-independent human prostate cancer PC-3 cells express higher levels of Hsp27 mRNA in vitro and in vivo, compared with androgen-sensitive LNCaP cells. Phosphorothioate Hsp27 antisense oligonucleotides (ASOs) and small interference RNA potently inhibit Hsp27 expression, with increased caspase-3 cleavage and PC3 cell apoptosis and 87% decreased PC3 cell growth. Hsp27 ASO and small interference RNA also enhanced paclitaxel chemosensitivity in vitro, whereas in vivo, systemic administration of Hsp27 ASO in athymic mice decreased PC-3 tumor progression and also significantly enhanced paclitaxel chemosensitivity. These findings suggest that increased levels of Hsp27 after androgen withdrawal provide a cytoprotective role during development of androgen independence and that ASO-induced silencing can enhance apoptosis and delay tumor progression.
|Cyclic AMP differentiation of the oligodendroglial cell line N20.1 switches staurosporine-induced cell death from necrosis to apoptosis |
Studzinski DM and Benjamins JA
J. Neuroscience Research, 66:691-697 (2001) 2001
|Numerical density of cardiomyocytes in chronic nitric oxide synthesis inhibition. |
Mandarim-De-Lacerda, C A and Meirelles Pereira, L M
Pathobiology, 68: 36-42 (2000) 2000
OBJECTIVE: The myocardial effect on the normalization of arterial blood pressure in animals with hypertension previously induced by nitric oxide synthesis (NOS) inhibition is still unknown. The purpose of this study was to estimate the numerical density of cardiomyocytes that is able to show cardiomyocyte loss as a consequence of NOS inhibition. METHODS: Sixty rats were divided into the following groups: control for 25 days (C25), control for 40 days (C40) control for 80 days (C80) and three other groups in which the rats received the NOS inhibitor N(G)-nitro-L-arginine-methyl-ester hydrochloride L-NAME; 50 mg/kg/day for 25 days (L25), 40 days (L40) and 40 days, respectively, the latter group having another 40 days of only water and food ad libitum (without L-NAME; L80 group). The detection of apoptotic cells was performed using a monoclonal antibody. RESULTS: In the L25, L40 and L80 groups, blood pressure was 74.5, 90.2 and 56.3% higher than the respective age-matched control rats, the myocardium had hypertrophy of the cardiomyocytes and scattered areas of fibrosis, and apoptosis occurred in isolated cells. Compared to the controls, the heart mass/body mass ratio was significantly greater in the L-NAME groups L25, L40 and L80, i.e. 31, 26 and 21%, respectively, the numerical density of cardiomyocytes in L-NAME rats was 32.7, 48.8 and 41.7% lower and the mean volume of cardiomyocytes was 33, 53 and 48% greater. CONCLUSION: The cardiomyocyte loss in NOS inhibition seems to be mainly due to necrosis, although apoptosis is also present.
|Apoptosis in breast carcinomas detected with monoclonal antibody to single-stranded DNA: relation to bcl-2 expression, hormone receptors, and lymph node metastases |
Frankfurt, O.S. et al.
Clin Cancer Res., 3:465-471 (1997) 1997
|Decreased stability of DNA in cells treated with alkylating agents |
Exp. Cell. Res., 191:181-185 (1990) 1990
|Detection of DNA damage in individual cells by flow cytometric analysis using anti-DNA monoclonal antibody |
Exp. Cell. Res., 170:369-380 (1987) 1987