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Publication Date



UM campus only

Embargo Period


Degree Type


Degree Name

Master of Science (MS)


Cancer Biology (Medicine)

Date of Defense


First Committee Member

Vinata B. Lokeshwar

Second Committee Member

Ralf Landgraf

Third Committee Member

Robert B. Levy


While cigarette smoking remains the most important risk factor for the development of urothelial neoplasms, the precise mechanisms of urothelial carcinogenesis induced by tobacco smoke toxicants remain obscure. Cigarette smoke is a heterogeneous mix of thousands of compounds, over 60 of which are known human carcinogens. Many of these compounds are electrophilic and form adducts with DNA, leading to mutation in oncogenes and tumor suppressors alike. While this theory remains the primary model of smoke-induced carcinogenesis, recent evidence suggests that cigarette smoke, and the many toxicants contained within, may induce epigenetic and transcriptional changes in smoke-exposed epithelial cells. This thesis aims to elucidate the gene expression modulation of the family of molecules responsible for the synthesis, degradation, and signal transduction of the extracellular glycosaminoglycan, hyaluronic acid. The expression of members of this family of biomolecules is elevated in cases of bladder cancer (BCa) and has been correlated to disease recurrence after resection, progression, and disease-specific mortality. Moreover, several members of this family have been shown in vitro and in vivo to be functionally responsible for BCa growth, angiogenesis, metastasis, and invasion. Despite this knowledge, studies of smoke-induced modulation of this family of biomolecules in the urothelium are yet to be conducted, until now. In this thesis, I have used qPCR and immunoblotting to investigate the transcriptional and translational changes, respectively, in a model of a normal urothelium exposed to whole cigarette smoke, as well as to individual constituents of tobacco smoke. To model the normal urothelium, I have used an SV40 Large T-antigen immortalized normal human urothelial cell line, which maintains similar morphology and stress gene expression to in situ urothelium, but is incapable of soft agar colony formation and does not form tumors upon xenotransplantation into mice. Culturing this cell line, termed UROtsa, in serum-free media results in a multi-layer epithelial sheet, which can be used as a model of a normal transitional urothelium. Using this model, we investigated the transcriptional and translational changes in HA family molecules in confluent UROtsa cultures chronically exposed to a cigarette smoke extract (CSE), generated via bubbling whole smoke through serum-free culture media. After only two weeks of continuous exposure to 0.1% CSE, we found statistically significant elevations in both transcript and protein levels of HA receptor splice variants, CD44v. These elevated levels of CD44v were observed throughout the duration of chronic exposure (up to 6 weeks). This finding is the first evidence of smoke-mediated elevation of these HA receptor variants in urothelial cells. Additionally, we investigated the modulation of the HA family of molecules in response to exposure to two individual constituents of tobacco smoke: BBN and acrolein. After only 1 hour of exposure to these carcinogenic compounds, confluent UROtsa cultures exhibited significant elevations in HA family molecules. Exposure to 2.5 uM BBN, a metabolic derivative of members of the tobacco specific nitrosamine family highly present in tobacco smoke, resulted in an elevated UROtsa cell expression of both CD44 standard and variant, as well as the HA synthase, HAS2, transcript levels. Moreover, exposure to the volatile organic compound, acrolein, resulted in the elevated expression of HAS1 and HYAL1 transcript levels. After 24 hours of exposure to acrolein, confluent UROtsa cultures exhibited elevated levels of both HAS1 and HYAL1, at both the transcript and protein levels. These results indicate that HA family molecules are, indeed, modulated by exposure to tobacco smoke. Because the induction of HA family transcript levels in response to CSE (within 2 weeks), BBN, and acrolein (within 1 hour) are rapid, it is probable that these elevations in transcript levels precede phenotypic alteration in smoke-exposed urothelial cells. Given the known roles of the HA family molecules in BCa growth and progression, the elevated transcript levels of these HA family molecules in exfoliated urothelial cells collected in urine may provide an opportunity to detect BCa prior to the detection of lesions using current clinical diagnostic procedures. Therefore, our study may provide evidence for use of these biomarkers in screening high-risk individuals, like smokers, for BCa. Moreover, our observation of individual tobacco smoke toxicants revealed these compounds are independently capable of inducing some HA family members. This discovery may support the reduction of these toxicants as a harm reduction strategy in tobacco control.


bladder cancer; biomarkers; smoking; toxicants