Jane C. Stewart

Isolation and Characterization of the Human Cytochrome P450 CYP1B1 Gene

Previously, we identified a novel human cytochrome P450 cDNA that is inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and represents the first member of a new subfamily designated cytochrome P4501B1 (CYP1B1; Sutter, T. R., Tang, Y. M., Hayes, C. L., Wo, Y. P., Jabs, E. W., Li, X., Yin, H., Cody, C. W., and Greenlee, W. F. (1994) J. Biol. Chem. 269, 13092-13099). Here, we report on the isolation and initial characterization of the CYP1B1 gene. The CYP1B1 gene maps to human chromosome 2 at 2p21-22 and contains three exons and two introns. The putative open reading frame starts in the second exon and is 1629 base pairs in length. Southern analysis using DNA probes directed to each of the three exons confirmed that CYP1B1 is a single copy gene. Human CYP1B1 differs from its two most closely related members of the cytochrome P450 superfamily, CYP1A1 and CYP1A2, in the number of exons (3 versus 7) and chromosome location (2 versus 15). A single transcription initiation site was identified by primer extension analysis and S1 nuclease mapping. Based on nucleotide sequence analysis, the CYP1B1 gene lacks a consensus TATA box in the promoter region and contains nine TCDD-responsive enhancer core binding motifs (5′-GCGTG-3′) located within a 2.5-kilobase pair genomic fragment 5′-ward of the transcription initiation start site. Deletion analysis of chloramphenicol acetyltransferase reporter gene constructs containing 5′ CYP1B1 genomic fragments indicates that a region from −1022 to −835 containing three of the nine core binding motifs contributes to the TCDD-inducible expression of CYP1B1.

E-Cadherin Binding Modulates EGF Receptor Activation

We found that E-cadherin and epidermal growth factor receptor (EGFR) are associated in mammary epithelial cells and that E-cadherin engagement in these cells induces transient activation of EGFR, as previously seen in keratinocytes (37). In contrast, EGFR does not associate with and is not activated by N-cadherin. Analysis of cells expressing chimeric cadherins revealed that the extracellular domain of E-cadherin is required for interaction with and activation of EGFR. This activation results in tyrosine phosphorylation of known EGFR substrates and reduction in focal adhesions. These interactions, however, are not necessary for suppression of cell motility by E-cadherin.

Cyclooxygenase inhibitors in urinary bladder cancer: in vitro and in vivo effects

More than 14,000 people die from invasive transitional cell carcinoma (TCC) of the urinary bladder yearly in the United States. Cyclooxygenase (COX)-inhibiting drugs are emerging as potential antitumor agents in TCC. The optimal in vitro or in vivo systems to investigate COX inhibitor antitumor effects have not been defined. The purpose of this study was to determine COX-1 and COX-2 expression and antitumor effects of COX inhibitors in human TCC cell lines (HT1376, RT4, and UMUC3 cells) and xenografts derived from those cell lines. COX-2 expression (Western blot, immunocytochemistry) was high in HT1376, modest in RT4, and absent in UMUC3 cells in vitro. Similarly, COX-2 expression was noted in RT4 but not UMUC3 xenografts. COX-2 expression in HT1376 xenografts was slightly lower than that observed in vitro. None of four COX inhibitors evaluated (celecoxib, piroxicam, valeryl salicylate, and NS398) reduced TCC growth in standard in vitro proliferation assays at concentrations that could be safely achieved in vivo (≤5 μmol/L). Higher celecoxib concentrations (≥50 μmol/L) inhibited proliferation and induced apoptosis in all three cell lines. Celecoxib or piroxicam treatment in athymic mice significantly delayed progression of HT1376 xenografts, which express COX-2, but not UMUC3 xenografts that lack COX-2 expression. In conclusion, standard in vitro assays were not useful in predicting COX inhibitor antitumor effects observed in vivo. Athymic mice bearing TCC xenografts provide a useful in vivo system for COX inhibitor studies. Results of this study provide justification for further evaluation of COX inhibitors as antitumor agents against TCC. [Mol Cancer Ther 2006;5(2):329–36]

Functional analysis of the promoter for the human CYP1B1 gene.

Our laboratory has cloned the cDNA (Sutter, T. R., Tang, Y. M., Hayes, C. L., Wo, Y.-Y. P., Jabs, E. W., Li, X., Yin, H., Cody, C. W., and Greenlee, W. F. (1994) J. Biol. Chem. 269, 13092–13099) and gene (Tang, Y. M., Wo, Y.-Y. P., Jabs, E. W., Stewart, J. C., Sutter, T. R., and Greenlee, W. F. (1996) J. Biol. Chem. 271, 28324–28330) for humanCYP1B1, a new member of the cytochrome P450 superfamily. Here, we report on the mapping and function of the CYP1B1promoter. The CYP1B1 promoter is fully functional, when it is uncoupled from upstream enhancer elements. Deletion analysis and site-directed mutagenesis identified four regulatory elements required for maximum promoter activity: two antisense Sp1 sites (−84 to −89 and −68 to −73), a TATA-like box (−34 to −29), and an initiator motif (−5 to +3). The initiator and the TATA-like elements are both required for basal promoter activity, with enhanced activity mediated by the two antisense Sp1 elements. The CYP1B1 initiator was demonstrated by in vitro transcription analysis to be a positioning element that maintained fidelity of transcription from a single site. Specific binding to a CYP1B1 initiator probe by human nuclear extract proteins was competed either by the highly homologous murine terminal deoxynucleotidyl transferase initiator or, to a lesser extent, by the adenovirus major late initiator. Taken together, these results indicate that the structure and function of theCYP1B1 promoter confers constitutive expression of the gene and assures fidelity of transcription initiation from a single site. The CYP1B1 promoter is distinct from the promoters of the closely related cytochrome P450s CYP1A1 andCYP1A2 and is structurally and functionally similar to the promoters of constitutively expressed genes and at least two viruses.

Clinical Trial of Vinblastine in Dogs with Transitional Cell Carcinoma of the Urinary Bladder

BACKGROUND Transitional cell carcinoma (TCC) of the urinary bladder of dogs can be a difficult cancer to treat, and effective therapies are limited. Vinblastine has been used in humans with TCC and has potent anti-proliferative effects against canine TCC cells in vitro. OBJECTIVES To determine the antitumor activity and toxicoses of vinblastine in dogs with urinary bladder TCC. ANIMALS Animals selected were 28 privately owned dogs that presented to the Purdue University Veterinary Teaching Hospital (PUVTH) with measurable, histologically confirmed TCC. METHODS Prospective clinical trial: The starting vinblastine dosage was 3.0 mg/m(2) i.v. every 2 weeks. Treatment continued until cancer progression or unacceptable toxicoses occurred. Complete evaluations (physical exam, complete blood count [CBC], serum biochemical profile, urinalysis, thoracic radiography, abdominal ultrasound [US]) were performed at 8-week intervals. Urinary tract US with bladder tumor mapping was performed monthly. Toxicoses were graded according to Veterinary Co-Operative Oncology Group (VCOG) criteria. RESULTS Tumor responses included 10 (36%) partial remission, 14 (50%) stable disease, and 4 (14%) progressive disease. The median progression free interval was 122 days (range, 28-399 days). The median survival time was 147 days (range, 28-476 days) from 1st vinblastine treatment to death and 299 days (range, 43-921 days) from diagnosis to death. The majority of dogs (27 of 28) did not have clinically relevant adverse effects. Seventeen of 28 (61%) dogs required dosage reductions because of neutropenia. CONCLUSION AND CLINICAL IMPORTANCE Vinblastine has antitumor activity against TCC in dogs and can be considered another treatment option for this cancer.

NMR-based metabolomics study of canine bladder cancer

Bladder cancer is one of the leading lethal cancers worldwide. With the high risk of recurrence for bladder cancer following the initial diagnoses, lifelong monitoring of patients is necessary. The lack of adequate sensitivity and specificity of current noninvasive monitoring approaches including urine cytology, other urine tests, and imaging, underlines the importance of studies that focus on the detection of more reliable biomarkers for this cancer. The emerging area of metabolomics, which deals with the analysis of a large number of small molecules in a single step, promises immense potential for discovering metabolite markers for screening and monitoring treatment response and recurrence in patients with bladder cancer. Since naturally-occurring canine transitional cell carcinoma of the urinary bladder is very similar to human invasive bladder cancer, spontaneous canine transitional cell carcinoma has been applied as a relevant animal model of human invasive transitional cell carcinoma. In this study, we have focused on profiling the metabolites in urine from dogs with transitional cell carcinoma and healthy control dogs combining nuclear magnetic resonance spectroscopy and statistical analysis methods. (1)H NMR-based metabolite profiling analysis was shown to be an effective approach for differentiating samples from dogs with transitional cell carcinoma and healthy controls based on a partial least square-discriminant analysis of the NMR spectra. In addition, there were significant differences in the levels of six individual metabolites between samples from dogs with transitional cell carcinoma and the control group based on the Students t-test. These metabolites were selected to build a separate partial least square-discriminant analysis model that was then used to test the classification accuracy. The result showed good classification between transitional cell carcinoma and control groups with the area under the receiver operating characteristic curve of 0.85. The sensitivity and specificity of the model were 86% and 78%, respectively. These results suggest that urine metabolic profiling may have potential for early detection of bladder cancer and of bladder cancer recurrence following treatment, and may enhance our understanding of the mechanisms involved.

Cyclooxygenase-2 dependent and independent antitumor effects induced by celecoxib in urinary bladder cancer cells

Transitional cell carcinoma of the urinary bladder is the second most common genitourinary malignancy in people in the United States. Cyclooxygenase-2 (COX-2) is overexpressed in bladder cancer. COX-2 inhibitors have had antitumor activity against bladder cancer, but the mechanisms of action are unclear. Clinically relevant concentrations of COX-2 inhibitors fail to inhibit proliferation in standard in vitro assays. In pilot experiments, different culture conditions [standard monolayer, modified monolayer, soft agar, collagen, and poly(2-hydroxyethyl methacrylate)–coated plates] were assessed to determine conditions suitable for the study of COX inhibitor growth-inhibitory effects. This was followed by studies of the effects of clinically relevant concentrations of a selective COX-2 inhibitor (celecoxib) on urinary bladder cancer cell lines (HT1376, TCCSUP, and UMUC3). Celecoxib (≤5 μmol/L) inhibited proliferation of COX-2–expressing HT1376 cells in soft agar and modified monolayer cell culture conditions in a COX-2–dependent manner. COX-2 expression, however, did not always correlate with response to celecoxib. TCCSUP cells that express COX-2 were minimally affected by celecoxib, and UMUC3 cells that lack COX-2 expression were modestly inhibited by the drug. When UMUC3Cox-2/Tet cells overexpressing COX-2 under the control of tetracycline-inducible promoter were treated with celecoxib in modified monolayer cell culture, growth inhibition was found to be associated with changes in the expression of pRb. Not surprisingly, the proliferation of all cell lines was inhibited by excessively high concentrations of celecoxib. In conclusion, the modified culture conditions allowed detection of COX-2–dependent and COX-2–independent growth-inhibitory activity of celecoxib in urinary bladder cancer cells. [Mol Cancer Ther 2008;7(4):897–904]

Metronomic administration of chlorambucil for treatment of dogs with urinary bladder transitional cell carcinoma.

OBJECTIVE To determine the antitumor effects and toxicoses of metronomic oral administration of a low dose of chlorambucil in dogs with transitional cell carcinoma (TCC). DESIGN Prospective clinical trial. ANIMALS 31 client-owned dogs with TCC for which prior treatments had failed or owners had declined other treatments. Procedures-Chlorambucil (4 mg/m2, PO, q 24 h) was administered to dogs. Before and at scheduled times during treatment, evaluations of dogs included physical examination, CBC, serum biochemical analyses, urinalysis, thoracic and abdominal imaging including cystosonography for measurement of TCCs, and grading of toxicoses. RESULTS 29 of 31 dogs had failed prior TCC treatment. Of the 30 dogs with available data, 1 (3%) had partial remission (≥ 50% reduction in tumor volume), 20 (67%) had stable disease (< 50% change in tumor volume), and 9 (30%) had progressive disease (≥ 50% increase in tumor volume or development of additional tumors); 1 dog was lost to follow-up. The median progression-free interval (time from the start of chlorambucil treatment to the day progressive disease was detected) for the dogs was 119 days (range, 7 to 728 days). The median survival time of dogs from the time of the start of chlorambucil treatment was 221 days (range, 7 to 747 days). Few toxicoses were detected; chlorambucil administration was discontinued because of toxicoses in only 1 dog. CONCLUSIONS AND CLINICAL RELEVANCE Metronomic administration of chlorambucil was well tolerated, and 70% of dogs had partial remission or stable disease. Metronomic administration of chlorambucil may be a treatment option for dogs with TCC.

Targeting Folate Receptors to Treat Invasive Urinary Bladder Cancer

Folate receptors (FR) may be of use for targeted delivery of cytotoxic drugs in invasive urothelial carcinoma (iUC), for which improved therapy is needed. FR expression and function in iUC were explored and the antitumor activity and toxicity of a folate-targeted vinblastine conjugate were evaluated in dogs with naturally occurring iUC, an excellent model for human iUC. FR immunohistochemistry was carried out on iUC and normal human and dog bladder tissues together with nuclear scintigraphy in dogs to monitor iUC folate uptake. Dose escalation of a folate-targeted vinblastine compound, EC0905, was conducted in dogs with biopsy-confirmed, FR-positive iUC. FRs were detected by immunohistochemistry (PU17) in most primary iUC and many nodal and lung metastases from dogs, and scintigraphy confirmed folate uptake in both primary and metastatic lesions. The maximum tolerated dose of EC0905 in dogs was 0.25 mg/kg IV weekly, with neutropenia at higher doses. Tumor responses included partial remission (≥ 50% reduction in tumor volume) in five dogs and stable disease (<50% change in tumor volume) in four dogs. Immunoreactivity to PU17 was similar in humans (78% of primary iUC, 80% of nodal metastases). Less immunoreactivity to mab343 (22% of cases) occurred. FR-β was noted in 21% of human iUC cases. Our findings suggest folate-targeted therapy holds considerable promise for treating iUC, where FR-β may be important in addition to FR-α.

Canine invasive transitional cell carcinoma cell lines: In vitro tools to complement a relevant animal model of invasive urinary bladder cancer

OBJECTIVES Urinary bladder cancer is the fifth most common form of cancer in humans in the United States. Urinary bladder cancer also occurs in pet dogs, and naturally-occurring bladder cancer in pet dogs very closely resembles invasive bladder cancer (intermediate to high grade invasive transitional cell carcinoma, InvTCC) in humans. Pet dogs with InvTCC offer a highly relevant resource for preclinical studies in bladder cancer. For translational research in which findings are moved from in vitro experiments through in vivo studies in dogs to human trials, access to human and canine bladder cancer cell lines is important. Cell lines derived from canine InvTCC have been lacking. Here we describe eight such cell lines. MATERIALS AND METHODS Eight cell lines were established from canine InvTCC. Cells were characterized using immunocytochemistry, evaluated for anchorage independent growth in soft agar, and assessed for tumorigenicity in athymic mice. Western blotting was used to identify expression of proteins of interest in human InvTCC. RESULTS The cell lines were confirmed to be of epithelial origin by their expression of cytokeratin and E-cadherin. Seven cell lines were found to be tumorigenic in athymic mice, and 4 of these cell lines grew in an anchorage independent manner. The cell lines expressed several proteins of interest associated with bladder cancer prognosis and progression in humans, including p53, cox-2, and pRb protein. CONCLUSIONS These established cell lines can be used for comparative bladder cancer research and to evaluate new therapy approaches in vitro prior to in vivo testing.