Biomedical subjects

William F Benedict

Publications and source records attributed to William F Benedict.

14 recordsLinked to original sources

PubMed2006

Characterization of a panel of cell lines derived from urothelial neoplasms: genetic alterations, growth in vivo and the relationship of adenoviral mediated gene transfer to coxsackie adenovirus receptor expression.

PURPOSE: Cell lines have become an essential component for the investigation of cancer. We have developed a panel of cell lines derived from human urothelial cancers and we describe some of their important characteristics. MATERIALS AND METHODS: Ten human urothelial cancer cell lines were characterized by their growth in athymic nude mice, CAR expression and their susceptibility to adenoviral mediated transfer of the green fluorescence protein gene. TP53 mutation status and immunochemical analysis of p53, pRB and p16 were also examined. RESULTS: Five cell lines rapidly produced tumors in athymic nude mice. Two cell lines produced tumors in 1 month, 1 produced them in 3 months and 2 were nontumorigenic. The cell lines varied in CAR expression and in their susceptibility to adenoviral mediated gene transduction. There was no direct correlation between CAR expression and susceptibility to adenoviral mediated gene transduction. Seven cell lines had TP53 mutations, of which 2 had large deletions and did not express p53 protein by immunostaining. All cell lines expressed abnormal pRB by immunochemical analysis (3 had no staining and 7 had homogenously strong staining) and 8 did not express p16 (7 showed homogeneously strong pRB staining). CONCLUSIONS: Our panel of 10 human urothelial cell lines differed in genetic alterations, growth in nude mice, susceptibility to adenoviral mediated gene transduction, and expression of p53, p16 and pRB. The availability of various urothelial cancer cell lines with differing genotypic and phenotypic features will facilitate further research into bladder cancer.

Adenoviridae↗

PubMed2006

Evidence for alternative candidate genes near RB1 involved in clonal expansion of in situ urothelial neoplasia.

In this paper, we present whole-organ histologic and genetic mapping studies using hypervariable DNA markers on chromosome 13 and then integrate the recombination- and single-nucleotide polymorphic sites (SNPs)-based deletion maps with the annotated genome sequence. Using bladders resected from patients with invasive urothelial carcinoma, we studied allelic patterns of 40 microsatellite markers mapping to all regions of chromosome 13 and 79 SNPs located within the 13q14 region containing the RB1 gene. A whole-organ histologic and genetic mapping strategy was used to identify the evolution of allelic losses on chromosome 13 during the progression of bladder neoplasia. Markers mapping to chromosomal regions involved in clonal expansion of preneoplastic intraurothelial lesions were subsequently tested in 25 tumors and 21 voided urine samples of patients with bladder cancer. Four clusters of allelic losses mapping to distinct regions of chromosome 13 were identified. Markers mapping to the 13q14 region that is flanked by D13S263 and D13S276, which contains the RB1 gene, showed allelic losses associated with early clonal expansion of intraurothelial neoplasia. Such losses could be identified in approximately 32% bladder tumor tissue samples and 38% of voided urines from patients with bladder cancer. The integration of distribution patterns of clonal allelic losses revealed by the microsatellite markers with those obtained by genotyping of SNPs disclosed that the loss within an approximately 4-Mb segment centered around RB1 may represent an incipient event in bladder neoplasia. However, the inactivation of RB1 occurred later and was associated with the onset of severe dysplasia/carcinoma in situ. Our studies provide evidence for the presence of critical alternative candidate genes mapping to the 13q14 region that are involved in clonal expansion of neoplasia within the bladder antecedent to the inactivation of the RB1 gene.

Aged↗

PubMed2006

Combination therapy with IFN-alpha plus bortezomib induces apoptosis and inhibits angiogenesis in human bladder cancer cells.

In a recent study, we showed that the proteasome inhibitor bortezomib sensitizes human bladder cancer cells to IFN-induced cell death. Here, we characterized the molecular mechanisms underlying the antitumoral effects of the combination in more detail. Bortezomib synergized with IFN-alpha to promote apoptosis via a tumor necrosis factor-related apoptosis-inducing ligand-associated mechanism but did not inhibit production of proangiogenic factors (vascular endothelial growth factor, basic fibroblast growth factor, and interleukin-8) in human UM-UC-5 cells. In contrast, exposure to the combination did not increase the levels of apoptosis in human UM-UC-3 cells but did inhibit the production of basic fibroblast growth factor and vascular endothelial growth factor. Studies with tumor xenografts confirmed that combination therapy with bortezomib plus IFN-alpha was effective in both models but that the effects were associated with differential effects on tumor necrosis factor-related apoptosis-inducing ligand-associated apoptosis (predominant in UM-UC-5) versus inhibition of angiogenesis (predominant in UM-UC-3). Together, our results show that combination therapy with IFN-alpha plus bortezomib is effective but can work via different mechanisms (apoptosis versus angiogenesis inhibition) in preclinical models of human bladder cancer.

Animals↗

PubMed2004

p53, p21, pRB, and p16 expression predict clinical outcome in cystectomy with bladder cancer.

PURPOSE: To determine whether p53, p21, pRB, and/or p16 expression is associated with bladder cancer stage, progression, and prognosis. PATIENTS AND METHODS: Immunohistochemical staining for p53, p21, pRB, and p16 was carried out on serial sections from archival specimens of 80 patients who underwent bilateral pelvic lymphadenectomy and radical cystectomy for bladder cancer (median follow-up, 101 months). RESULTS: p53, p21, and pRB or p16 expression was altered in 45 (56%), 39 (49%), and 43 (54%) tumors, respectively. Sixty-six patients (83%) had at least one marker altered, and 21 patients (26%) had all three altered. Abnormal expressions of p53, p21, and pRB/p16 expression were associated with muscle-invasive disease (P=.007, P=.003, and P=.003, respectively). The alteration of each marker was independently associated with disease progression (P< or =.038) and disease-specific survival (P< or =.039). In multivariable models that included standard pathologic features and p53 with p21 or p53 with pRB/p16, only p53 and lymph node metastases were associated with bladder cancer progression (P< or =.026) and death (P< or =.028). In models that included p21 and pRB/p16, only p21 and lymph node metastases were associated with bladder cancer progression (P< or =.022) and death (P< or =.028). In a model that included the combined variables p53/p21 and pRB/p16, only p53/p21 and lymph node status were associated with bladder cancer progression (P< or =.047) and death (P< or =.036). The incremental number of altered markers was independently associated with an increased risk of bladder cancer progression (P=.005) and mortality (P=.007). CONCLUSION: Although altered expression of each of the four cell cycle regulators is associated with bladder cancer outcome in patients undergoing radical cystectomy, p53 is the strongest predictor, followed by p21, suggesting a more pivotal role of the p53/p21 pathway in bladder cancer progression.

Adult↗

PubMed2004

Focus on bladder cancer.

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Animals↗

PubMed2004

Intravesical Ad-IFNalpha causes marked regression of human bladder cancer growing orthotopically in nude mice and overcomes resistance to IFN-alpha protein.

We have produced prolonged, high local concentrations of interferon in vivo by intravesical instillation of adenoviruses encoding interferon-alpha (Ad-IFNalpha) together with the gene transfer-enhancing agent Syn3. We found sustained interferon protein levels for days, both in normal mouse urothelium and in human bladder cancer cells growing as superficial bladder tumors in nude mice using an orthotopic bladder model developed by us. Tumor burden in the bladder was determined utilizing cancer cells containing the green fluorescent protein. Marked tumor regression was observed following two 1-h exposures of Ad-IFNalpha/Syn3 and little or no cytotoxicity was detected in normal cells. Similar intravesical instillation of clinically relevant concentrations of IFN protein alone or Ad-IFNalpha without Syn3 was ineffective. Surprisingly, in vitro, Ad-IFNalpha also caused caspase-dependent death of bladder cancer cell lines that were resistant to high concentrations of IFN-alpha protein, including the cell line used in vivo. These findings demonstrate that Ad-IFNalpha can overcome resistance to IFN-alpha protein both in vitro and in vivo and support evaluation of intravesical Ad-IFNalpha/Syn3 for the treatment of superficial bladder cancer.

Adenoviridae↗

PubMed2004

Correlation of metastasis related gene expression and relapse-free survival in patients with locally advanced bladder cancer treated with cystectomy and chemotherapy.

PURPOSE: Patients with locally advanced (ie clinically extravesical) transitional cell carcinoma are at high risk for recurrence after cystectomy. Although randomized trials have established an incremental benefit from the addition of chemotherapy in this setting, many patients still have disease relapse, and therefore it is necessary to determine patient and tumor characteristics that correlate with outcome in this setting. We investigated the tumor expression of several metastasis related genes and the association of gene expression with disease specific survival of patients with locally advanced transitional cell carcinoma treated randomized to either neoadjuvant or adjuvant chemotherapy and radical cystectomy. MATERIALS AND METHODS: Archival paraffin embedded specimens were available for 64 patients enrolled in a clinical trial of the methotrexate, vinblastine, doxorubicin and cisplatin regimen and cystectomy. Only samples obtained before exposure to chemotherapy were studied. The expression of several metastasis related genes, including basic fibroblast growth factor, vascular endothelial growth factor (VEGF), interleukin-8, matrix metalloproteinase (MMP)-9, and E-cadherin were assayed on paraffin sections using a colorimetric in situ hybridization assay. RESULTS: Expression of basic fibroblast growth factor, interleukin-8 and MMP-9 did not correlate with outcome. Expression of VEGF and E-cadherin were strongly related to disease specific survival. In addition, the ratio of MMP-9-to-E-cadherin was strongly prognostic for disease specific survival. CONCLUSIONS: These data advance the hypotheses that VEGF expression and an "invasive phenotype" characterized by the ratio of MMP-9-to-E-cadherin expression are mechanistically relevant to clinically aggressive locally advanced bladder cancers that are not cured by currently available combined modality treatment. Thus, in our view there is a compelling rationale to target these aspects of the malignant phenotype in this patient population.

Adult↗

PubMed2003

Efficient therapeutic gene delivery after systemic administration of a novel polyethylenimine/DNA vector in an orthotopic bladder cancer model.

Successful systemic gene therapy has been hindered by vector-related limitations, including toxicity and inefficient gene delivery to tumor cells after i.v. administration. To circumvent these problems, we developed a novel formulation between the polycation polyethyleneimine and DNA that mediates high-level tumor cell transduction in vitro and efficient i.v. gene delivery in that greater reporter gene expression occurred in tumor than in lung. Strikingly, administration of just 6 micro g of the polyethyleneimine/DNA-p53 vector every 3 days for 3 weeks indicated restoration of normal cell cycle regulation and apoptotic mechanisms as demonstrated by efficient p53 expression, increased apoptosis, and a 70% reduction in tumor size in an orthotopic bladder cancer model. This novel vector formulation represents a new method to increase i.v. delivery of genes to tumors.

Animals↗

PubMed2003

Adenoviral-mediated retinoblastoma 94 produces rapid telomere erosion, chromosomal crisis, and caspase-dependent apoptosis in bladder cancer and immortalized human urothelial cells but not in normal urothelial cells.

Retinoblastoma (RB)94, which lacks the NH(2)-terminal 112 amino acid residues of the full-length RB protein (RB110), is a more potent tumor and growth suppressor than RB110. In this study, Ad-RB94, but not Ad-RB110, produced marked growth inhibition, cytotoxicity, caspase-dependent apoptosis, and G(2)-M block in the human RB-negative, telomerase-positive bladder cancer cell line UM-UC14. This effect was completely inhibited by pretreatment with caspase inhibitors (P < 0.0001). Similar results were seen in RB-positive and other RB-negative bladder cancer cell lines. Ad-RB94 produced rapid telomere length shortening and loss of telomere signal, which was associated with polyploidy and chromosomal aberrations (P < 0.001). Ad-RB94, however, showed no cytotoxicity to telomerase-negative human normal urothelium cells but was highly cytotoxic to telomerase-positive human E6 and E7 immortalized urothelial cells (P < 0.0001). In addition, telomerase-negative cells, which maintain their telomere length through an alternative lengthening of telomeres DNA recombination pathway, showed no cytotoxicity to RB94. These results suggest that the induction of rapid telomere erosion and chromosomal crisis by RB94 in telomerase-positive cancer and in telomerase-expressing immortalized human cells is a major factor in its selective and potent tumor suppression and cytotoxic activity. The lack of cytotoxicity to normal cells should also provide a high therapeutic index when used in gene therapy protocols for the treatment of bladder and other cancers.

Adenoviridae↗

PubMed2003

Noninvasive detection of bladder cancer in an orthotopic murine model with green fluorescence protein cytology.

PURPOSE: Orthotopic models of bladder cancer mimic the normal microenvironment and provide an opportunity to study new therapies for superficial bladder cancer. The use of green fluorescent protein (GFP) transduced cells provides a sensitive way of monitoring this disease. We investigated whether examining voided urine for GFP expressing cells would indicate the presence of GFP producing tumors in an orthotopic bladder tumor model in nude mice. MATERIALS AND METHODS: The human bladder cancer cell lines KU-7, UM-UC-3 and UM-UC-14 were used. GFP transductants were generated after transfection with pEGFP-N3, followed by G418 selection. After the cells were inoculated in an orthotopic model of superficial bladder cancer voided urine was collected on slides weekly for 3 weeks and observed for GFP expressing cells by fluorescence microscopy. Bladder tumor imaging for GFP was performed in surgically exposed bladders to determine the tumor incidence. RESULTS: KU-7 GFP cells produced tumors in all 16 mice on whole bladder GFP imaging. UM-UC-3 and UM-UC-14 GFP cells produced tumors in 8 of 12 (67%) and 18 of 25 (72%) mice, respectively. The rate of GFP positive cells in spontaneously voided urine varied by cell line and increased with time but it was generally less than the rate of detection by whole bladder GFP imaging. All mice with GFP expressing cells in the urine had GFP expressing bladder tumors. CONCLUSIONS: Examining urine for GFP expressing cells is less sensitive than imaging surgically exposed bladders but it is 100% specific.

Animals↗

PubMed2003

Correlation of cyclooxygenase-2 expression with molecular markers, pathological features and clinical outcome of transitional cell carcinoma of the bladder.

PURPOSE: We investigated the relationship between cyclooxygenase-2 (COX-2) expression and molecular alterations commonly found in transitional cell carcinoma (TCC) of the bladder and determined whether COX-2 immunoreactivity is associated with cancer stage, progression and survival in patients undergoing radical cystectomy. MATERIALS AND METHODS: Immunohistochemical staining for COX-2 was done in archival tumor specimens from 80 patients who underwent radical cystectomy. Immunoreactivity was categorized as positive (reactivity in greater than 10% tumor cells) or negative. Microvessel density, E-cadherin, pRB, p16, p21, p53 and transforming growth factor (TGF)-beta1 and its receptors (types I and II) were also studied because evidence suggests a biological association between COX-2 and alteration of these molecules. RESULTS: COX-2 was over expressed in 62 patients (78%). COX-2 over expression was associated with muscle invasive pathological stage (p = 0.022), TGF-beta1 over expression (p = 0.004), decreased E-cadherin expression (p < 0.001), and altered expression of pRB (p = 0.003) and p16 (p = 0.006). At a median followup of 101 months COX-2 over expression was associated with disease progression (p = 0.038) and bladder cancer specific survival (p = 0.042). However, when adjusted for the effects of standard pathological features, only lymph node metastasis was associated with bladder cancer progression (p = 0.027) and mortality (p = 0.042). CONCLUSIONS: COX-2 is commonly expressed in patients with bladder TCC. Using the cutoff of 10% abnormal COX-2 expression is associated with the degree of invasiveness, alterations in TGF-beta1 and pRB/p16 pathways, and loss of cell adhesion. While COX-2 expression has limited prognostic value in patients with bladder TCC, it may serve as a target for therapy with selective COX-2 inhibitors.

Blotting, Western↗

PubMed2002

Syn3 provides high levels of intravesical adenoviral-mediated gene transfer for gene therapy of genetically altered urothelium and superficial bladder cancer.

Using our model to grow superficial human bladder cancer in the mouse bladder, we have found that the polyamide compound, Syn3, when injected intravesically for 1 hour at 1 mg/mL on two consecutive days, markedly increases rAd-beta-gal intravesical gene transfer and expression. This enhanced transgene expression was much greater than obtain by the use of 22% ethanol, which had previously been shown to increase intravesical adenoviral gene transfer, whereas little or no gene expression was seen with exposure to only rAd-beta-gal. beta-Galactosidase staining was seen in virtually every normal urothelial and superficial tumor cell present, including tumors that express little or no coxsackie-adenovirus receptors when Syn3 was present. High adenoviral-mediated gene transfer was also documented in the pig bladder using Syn3 in a similar protocol. Therefore, Syn3 may overcome the limitations of adequate intravesical adenoviral-mediated gene transfer and, when combined with an appropriate adenoviral-mediated gene, could offer an effective approach to the treatment of superficial bladder cancer and perhaps even genetically altered precursor lesions.

Adenoviridae↗

PubMed2002

Visualizing superficial human bladder cancer cell growth in vivo by green fluorescent protein expression.

There has been no reliable orthotopic model available to visualize the growth of human superficial bladder cancer over time and to evaluate the efficacy of intravesical therapies. We have developed a novel approach to accomplish this task by generating human superficial bladder tumor cells to stably express high levels of green fluorescent protein (GFP) in vivo. Superficial bladder tumors were produced in athymic mice by intravesical instillation. In our initial studies tumors were quantitated by image analysis at a single time point, and the results compared to the estimation of the percentage of GFP cells present using flow cytometry after obtaining single cell suspensions of normal and tumor cells in the same bladder. A high correlation between the two methods was seen. Therefore, in subsequent studies, approximately 1 week after the intravesical instillation of the GFP expressing cancer cells a small incision was made to expose the bladder. The anterior, posterior, and lateral images of each bladder were captured to visualize GFP-expressing tumors. The ratio of green fluorescence pixel area, which represented the tumor burden, to the total area of the bladder was then calculated. A similar procedure was performed at 2, 3, and 4 weeks after instillation of the tumor cells. Using this procedure tumor progression over time could be measured in each mouse. By using this approach, it will now be possible to monitor the initial tumor sizes in the bladder of each mouse and then to evaluate the efficacy of various intravesical therapy protocols including intravesical gene therapy alone or in combination with other treatment modalities.

Cell Division↗

PubMed2002

Inhibition of tumorigenicity and metastasis of human bladder cancer growing in athymic mice by interferon-beta gene therapy results partially from various antiangiogenic effects including endothelial cell apoptosis.

We determined whether the IFN-beta gene could suppress angiogenesis, tumor growth, and metastasis of human bladder transitional cell carcinoma. The highly tumorigenic and metastatic 253J B-V(R) human bladder transitional cell carcinoma (TCC) cell line (resistant to the antiproliferative effects of IFN-beta) was infected in vitro with adenoviral beta-galactosidase (Ad-LacZ), murine adenoviral IFN-beta (Ad-mIFN-beta), or human adenoviral IFN-beta (Ad-hIFN-beta) and implanted into the bladders of athymic nude mice. Ad-mIFN-beta and Ad-hIFN-beta were used because of the species specificity of IFN-beta. The transient production of mIFN-beta and hIFN-beta from the infected 253JB-V(R) tumor cells significantly inhibited tumorigenicity and spontaneous lymph node metastasis. Subsequently, the 253J B-V(R) cells were implanted into the subcutis of athymic nude mice, and established tumors were treated by direct intratumoral injection with Ad-mIFN-beta, Ad-hIFN-beta, Ad-LacZ, or PBS. By in situ hybridization (ISH) and immunohistochemical analysis (IHC), expression of hIFN-beta and mIFN-beta mRNA and protein within the tumors was demonstrated after Ad-hIFN-beta and Ad-mIFN-beta gene therapy, respectively. The therapy also induced necrosis in both the Ad-mIFN-beta- and Ad-hIFN-beta-treated tumors. IHC revealed decreased tumor cell proliferation and the sequestration of activated macrophages within the tumors after Ad-mIFN-beta therapy. In addition, the expression of the proangiogenic factors bFGF, and MMP-9 protein (by IHC) was significantly down-regulated by Ad-hIFN-beta gene therapy. Double-immunofluorescent IHC for terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) and CD-31 demonstrated tumor and endothelial cell apoptosis in those tumors treated with Ad-hIFN-beta gene therapy. Tumor-induced angiogenesis, as determined by the microvessel density, was decreased in tumors treated with both Ad-mIFN-beta and Ad-hIFN-beta. These data suggest that the inhibition of tumorigenicity and the metastasis of the 253J B-V(R) cells after infection with Ad-IFN-beta is caused by the inhibition of angiogenesis and the activation of host effector cells.

Animals↗