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ABSTRACT: INTRODUCTION: Prior work by our group has shown that HMGB1 release by urothelial carcinoma (UC) cells occurs as a consequence of BCG induced non-apoptotic cell death. Additional studies have demonstrated that HMGB1 release in response to BCG is required for the in vivo tumor response to BCG. This study evaluated the steps required for HMGB1 release by human UC cells in response to BCG exposure. MATERIALS AND METHODS: Two human UC cells lines T24 and 253 were employed. HMGB1 concentrations in cell culture supernatant, with and without BCG treatment, served as the principal end point to assess the role of potentially involved variables. Specific techniques were utilized to determine the role of α5β1 antigen receptor crosslinking, Toll Like Receptor signaling, BCG adherence, BCG internalization, BCG viability, iNOS expression/NO production, and p21 expression. RESULTS: Crosslinking of α5β1 integrin, or signaling through TLR2/4, did not contribute to HMGB1 release. Optimal HMGB1 release required both BCG adherence and internalization. BCG viability was correlated with the magnitude of the HMGB1 release. Inhibition of oxidative stress and p21 expression in response to BCG reduced the magnitude of HMGB1 release. CONCLUSIONS: BCG induced non-apoptotic cell death and HMGB1 release occurs as a consequence of a complex multi-step process. An understanding of the steps and mechanisms involved in BCG induced HMGB1 release affords an opportunity for targeted strategies to improve BCG treatment efficacy.
The Journal of urology 04/2013; · 4.02 Impact Factor
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ABSTRACT: BACKGROUND: Prior work has demonstrated that HMGB1 release by urothelial carcinoma (UC) cells in response to BCG is required for an in vivo antitumor effect. This study evaluated direct effects of HMGB1 on the in vitro response of UC cells to BCG. MATERIALS AND METHODS: Two human UC cell lines were used to study the effect of exogenous HMGB1, alone and in combination with BCG, on the tumor cell response to BCG. Antibody mediated blockade of receptors for HMGB1, or HMGB1 protein, was used to determine the contribution of paracrine HMGB1 release on BCG's biologic effects. The response endpoints evaluated included the activation of intracellular signaling pathways, gene transactivation, and cytotoxicity. RESULTS: UC cells express the receptor for HMGB1 signaling. Antibody blockade of the RAGE receptor confirmed the dependence of signaling in response to HMGB1 on RAGE function. Exogenous HMGB1 activated cell signaling pathways for NFκB, NRF2, and CEBP. Quantitative rtPCR on a panel of BCG responsive genes demonstrated peak expression resulting from the combination of BCG and HMGB1. Blockade of paracrine HMGB1 released in response to BCG, using HMGB1 and/or RAGE receptor blocking antibodies, demonstrated a significant reduction in gene expression relative to BCG alone. HMGB1 potentiated the cytotoxic effects of BCG. CONCLUSION: HMGB1 released by UC cells following BCG treatment functions as a paracrine factor to potentiate the UC cell response to BCG. This paracrine activity likely contributes to the dependence of an in vivo tumor response on HMGB1 release.
The Journal of urology 01/2013; · 4.02 Impact Factor
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ABSTRACT: BACKGROUND: Prior work has demonstrated that a portion of urothelial carcinoma (UC) cells exposed to BCG undergo non-apoptotic cell death and release of the chemokine HMGB1. This study evaluated the role of tumor cell derived HMGB1 in mediating thein vivo antitumor effect of BCG. MATERIALS AND METHODS: The murine UC cell line MB49 was engineered to express an shRNA construct targeting HMGB1. The shRNA expressing cell line underwent characterization to insure its comparability to the parental MB49 cell line. An orthotopic tumor model was employed to compare thein vivo antitumor efficacy of BCG in the parental cell line (N = 24 control and 24 BCG) vs. the HMGB1 knockdown line (N= 23 control and 21 BCG). RESULTS: Expression of the shRNA construct reduced HMGB1 expression and its release in response to BCG. The parental and shRNA cell lines demonstrated similarin vitro doubling times, and cytotoxicity in response to BCG. BCG treatment significantly reduced tumor volume relative to controls in parental MB49 tumor bearing animals (p=0.036). Tumor volumes inBCG treated animals inoculated with the shRNA cell line,were higher than sham treated shRNA controls (p=0.12). Among BCG treated animals tumor volumes were significantly lower in parental tumor bearing animals relative to the shRNA group (p<0.00001). Analysis of variance demonstrated a significant interaction between the cell line (shRNA vs. Parental) and BCG treatment effect (p=0.0076). CONCLUSION: The direct tumor response to BCG, culminating in HMGB1 release, may be an important contributor to the clinical efficacy of BCG.
The Journal of urology 10/2012; · 4.02 Impact Factor
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ABSTRACT: The mouse urothelial carcinoma cell line MB49 is widely used as an in vitro and in vivo model of urothelial carcinoma. Little comparative data exist on the molecular and phenotypic responses of this cell line relative to human cell lines. We compared the effect of bacillus Calmette-Guerin on the MB49 cell line relative to responses previously observed in the human urothelial carcinoma lines T24 (ATCC) and 253J.
Molecular end points in MB49 cells after bacillus Calmette-Guerin exposure were signaling pathway activation (NF-kappaB, AP1 and C/EBP), gene expression (IL-6 and p21), HMGB1 release/responsiveness and gene expression profiling at 6 hours. Phenotypic response end points were direct cytotoxicity using dye exclusion, viability on MTT assay, apoptotic sensitivity and cell cycle compartmentalization.
NF-kappaB, AP1, C/EBP, IL-6 and p21 reporter constructs were activated in MB49 cells in response to bacillus Calmette-Guerin. Gene expression profiles showed an inflammatory/immune clustering response. Bacillus Calmette-Guerin decreased cell viability and induced G1 cell cycle arrest. Treatment of MB49 cells with bacillus Calmette-Guerin induced caspase independent cell death while simultaneously decreasing sensitivity to pro-apoptotic agents. Cell death was associated with release of the necrotic cell death marker HMGB1. MB49 cells expressed HMGB1 receptors and activated intracellular NF-kappaB signaling pathways in response to bacillus Calmette-Guerin.
MB49 cells show molecular and phenotypic responses to bacillus Calmette-Guerin that replicate those observed in human urothelial carcinoma lines. MB49 cells appear to be an excellent model in which to study bacillus Calmette-Guerin as an antitumor agent for urothelial carcinoma.
The Journal of urology 12/2009; 182(6):2932-7. · 4.02 Impact Factor
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ABSTRACT: The direct phenotypic effects of BCG on human urothelial carcinoma (UC) cells include cell cycle arrest, apoptotic resistance, and caspase-independent cell death. These effects are associated with increased expression of the cyclin dependant kinase inhibitor (CDKI) p21. This study assessed the role of p21 expression in mediating the phenotypic effects observed in response to BCG.
Inducible systems for the autocrine expression of p21, or the blockade of p21 expression in response to BCG, were established in the human UC line T24. The effect of increasing or inhibiting p21 expression on tumor phenotype was assessed using assays for cell cycle compartmentalization (flow cytometry), apoptotic sensitivity (caspase 3 activation), and cytotoxicity (vital dye exclusion).
p21 Overexpression resulted in cell cycle arrest with an increase in the percentage of the cell in G0/G1 phase when compared with the untreated group. p21 Expression decreased basal caspase-3 expression compared with the untreated group, and reversed camptothecin-induced caspase-3 activity compared with camptothecin alone group. p21 Overexpression increased BCG's direct cytotoxicity. shRNA-mediated inhibition of p21 expression in response to BCG failed to reverse BCG-induced changes in cell cycle compartmentalization. p21 Inhibition partially reversed the antiapoptotic effect of BCG. The expression of p21 was required for the direct cytotoxic effect of BCG.
p21 Expression is sufficient but not necessary for BCG-induced cell cycle arrest. It is both sufficient and necessary for the full antiapoptotic effect of BCG. p21 Expression alone is not sufficient for caspase-independent cytotoxicity but is necessary for BCG's direct cytotoxic effect.
Urologic Oncology 06/2009; 28(5):526-33. · 3.22 Impact Factor
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ABSTRACT: To evaluate the ability of bacille-Calmette Guèrin (BCG) to induce caspase-independent cell death and release the necrosis-associated chemokine high molecular group box protein 1 (HMGB1) from urothelial carcinoma (UC) cells; a correlative clinical trial determined if BCG treatment resulted in increased urinary levels of HMGB1.
The human UC cell lines 253 J and T24 were pretreated with apoptosis inhibitors, exposed to BCG, and cell viability and ultrastructural changes measured. HMGB1 levels were assessed in cell culture supernatant after BCG treatment. The expression/function of HMGB1 receptors on the UC cell lines was determined by reverse transcription-polymer chain reaction and the ability of exogenous HMGB1 to activate nuclear factor (NF)-kappaB signalling assessed. An HMGB1 enzyme-linked immunosorbent assay was used to measure HMGB1 levels in urine obtained from BCG-treated patients.
Inhibition of apoptotic pathways failed to inhibit BCG-induced cell death in UC cells. Electron microscopy showed BCG-dependent ultrastructural changes consistent with cellular necrosis. BCG exposure resulted in a binary increase in cell culture supernatant levels of HMGB1. UCs expressed multiple HMGB1 receptors. Treatment of UCs with HMGB1 activated NF-kappaB. In the clinical setting, six of seven patients had increased urinary levels of HMGB1 at 24 h after BCG treatment.
BCG causes direct cytotoxicity in a subpopulation of UC cells. This cytotoxicity is caspase-independent and associated with ultrastructural changes and cellular protein release (HMGB1), characteristic of necrosis. Urinary levels of HMGB1 can be elevated in patients after BCG treatment. The expression and function of HMGB1 receptors in UC cells, coupled with the known role of HMGB1 on the host immune response, suggest a role for necrosis and HMGB1 release in the antitumour effect of BCG.
BJU International 01/2009; 103(12):1714-20. · 2.84 Impact Factor
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ABSTRACT: By inducing cell cycle arrest at the G1/S transition bacillus Calmette-Guerin has been shown to have a direct antiproliferative effect on urothelial carcinoma cell lines. In other systems cell cycle arrest has been shown to confer a relative state of apoptotic resistance. We assessed the effect of bacillus Calmette-Guerin on the susceptibility of urothelial carcinoma cells to apoptotic stimuli.
The human UC cell lines T24 and 253J (American Type Cell Culture, Rockville, Maryland) were used to evaluate the effect of bacillus Calmette-Guerin or antibody mediated alpha5beta1cross-linking on apoptosis and apoptotic sensitivity. Following treatment baseline apoptosis and the response to the apoptotic inducing agent camptothecin was evaluated using assays for caspase 3 activation and DNA fragmentation. Pharmacological blockade of signaling pathways known to be activated in response to bacillus Calmette-Guerin/alpha5beta1 cross-linking was used to assess the role of these pathways in the bacillus Calmette-Guerin apoptotic response. A final series of experiments used the MTT assay to study the impact of bacillus Calmette-Guerin pretreatment on the cytotoxicity of the antineoplastic agent mitomycin C.
Treatment with bacillus Calmette-Guerin failed to induce apoptosis, as measured by caspase 3 activation or DNA laddering. Bacillus Calmette-Guerin pretreatment significantly inhibited the induction of apoptosis in response to camptothecin. These effects were reproduced by antibody mediated cross-linking of alpha5beta1 integrin. Pharmacological inhibition of nuclear factor kappaB and/or AP1 signaling pathways reversed the anti-apoptotic effect of bacillus Calmette-Guerin. Mitomycin C cytotoxicity was significantly decreased by bacillus Calmette-Guerin pretreatment.
Bacillus Calmette-Guerin exerts a direct anti-apoptotic effect on human urothelial carcinoma cell lines. The ability of antibody mediated cross-linking to reproduce the effect and the ability of signal transduction inhibitors to block it are consistent with a mechanism involving integrin mediated signaling. Apoptotic resistance represents a therapeutic target for modulating the response to bacillus Calmette-Guerin and it may have clinical implications in the sequencing of intravesical therapies.
The Journal of Urology 12/2007; 178(5):2166-70. · 3.75 Impact Factor
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ABSTRACT: Bacillus Calmette-Guerin (BCG) binds to the tumor cell as a result of mycobacterial receptors for fibronectin (FN). Cell surface bound FN serves as a bridge through which BCG attaches to the tumor cell. Despite the importance of FN studies have demonstrated an idiosyncratic decrease in BCG adherence in response to exogenous FN. We evaluated the effect of exogenous and autocrine FN on the ability of BCG to adhere to the tumor cell surface and initiate cellular signaling.
BCG adherence to parental 253J and FN over expressing 253JTGFbeta1-8 cells as well as to the intrinsic FN expressing cell line 647V was quantified using green fluorescent protein-BCG. Experiments were performed to assess the effect of FN on BCG initiated signal transduction through nuclear factor kappaB and AP1. Finally, the integrity of the BCG activated signaling pathway in transforming growth factor-beta1/FN over expressors was assessed using antibody mediated cross-linking of the FN receptor.
BCG adherence was decreased in cell lines with high autocrine expression of FN. Exogenous FN prevented BCG induced transactivation of nuclear factor kappaB and AP1 reporter constructs. No BCG stimulated signaling to these reporters could be detected in FN over expressing 253J cells. NonFN dependent alpha5beta1 cross-linking initiated signal transduction in FN over expressing cells.
We propose that by saturating cellular and BCG receptors excess FN expression decreases the ability of cellular or mycobacterial bound FN to bind vacant receptors on BCG or on the cell. Excess FN inhibits BCG adherence and BCG initiated signal transduction.
The Journal of Urology 11/2004; 172(4 Pt 1):1496-500. · 3.75 Impact Factor
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ABSTRACT: Prior studies have shown that bladder trauma occurring during transurethral bladder tumor resection increases urinary levels of the cytokine transforming growth factor (TGF)-beta1. This study used complementary deoxyribonucleic acid micro-array technology to identify additional genes in human transitional cell carcinoma (TCC), whose expression is altered as a consequence of increased urinary levels of TGF-beta1.
The human TCC line 253J was cultured in standard media, or media spiked with either 10% post-transurethral bladder tumor resection urine (PTU), or PTU and anti-TGF-beta1 neutralizing antibody. Messenger ribonucleic acid from these conditions, together with messenger ribonucleic acid from stably transfected 253J cells over-expressing TGF-beta1, was hybridized with ATLAS micro-array membranes (Clontech, Palo Alto, CA) containing 588 human genes. Hybridization signal intensity was quantified using phospho-imaging. An analytic strategy based on the variance in the signal intensity ratio of specific housekeeping genes in control and experimental comparisons was used to identify significant changes in gene expression. Reverse transcriptase polymerase chain reaction of target genes was used to confirm gene over-expression and TGF-beta1 responsiveness.
Seven genes were identified on micro-array: v-RAF-1, colony stimulating factor-1 receptor, v-FGR, insulin growth factor-1 receptor, epidermal growth factor receptor, alpha5 integrin, and interferon receptor-1. Reverse transcriptase polymerase chain reaction confirmed over-expression in the autocrine TGF-beta1 producing cell line and increased expression in response to exogenous TGF-beta1.
TGF-beta1 in PTU alters the expression of multiple genes in human TCC in vitro. The impact of these changes on the biologic phenotype of the malignant cell and the efficacy of adjuvant therapies requires further evaluation.
Urologic Oncology 23(6):413-8. · 3.22 Impact Factor
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ABSTRACT: Work by our group has shown that bacillus Calmette-Guérin (BCG) induces cell cycle arrest at the G1/S interface in human transitional carcinoma cell lines. This study evaluated the effect of BCG on cell cycle regulatory proteins relevant to this effect.
The effect of BCG on selected cell cycle regulatory proteins, including cyclin D1, p27, and p21, was assessed in 2 human cell lines. After the identification of p21 as a candidate protein, p21 regulation was evaluated using a combination of Western, reverse transcriptase polymerase chain reaction, and promoter-reporter analysis. The immediate early versus delayed nature of p21 induction was determined. Finally, given the known potential for p21 to be regulated by both p53 dependent and independent pathways, the role of p53 in BCG induced expression of p21 was evaluated.
BCG increased p21 expression 2-fold relative to controls as measured by Western, and promoter-reporter analysis. Inhibition of protein synthesis had no effect on p21 messenger ribonucleic acid induction in response to BCG. T24 cells contained a previously reported mutation in p53. In the p53 wild-type 253J cells, deletion of one or both p53 response elements in the p21 reporter had no effect on BCG induced reporter transactivation.
BCG up-regulates expression of p21 in human transitional cell carcinoma lines. The transactivation of p21 in response to BCG occurs through an immediate early, p53 independent pathway. The finding of increased p21, together with the observation that BCG induces cell cycle arrest at the G1/S interface, supports a role for this protein in the biologic response to BCG.
Urologic Oncology 25(3):221-7. · 3.22 Impact Factor
