Title

The expression and functional analysis of hyaluronic acid synthase in bladder cancer

Date of Award

2007

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

First Committee Member

Vinata Lokeshwar, Committee Chair

Abstract

Hyaluronic acid (HA) a glycosaminoglycan, with its degrading enzyme hyaluronidase (HYAL) are intricately associated with tumor growth and development. HA maintains a myriad of vital cell functions such as proliferation, migration, and angiogenesis. Bladder cancer (BCa), a common cancer of the urinary tract, has been shown to express high levels of HA and HYAL1 (a hyaluronidase isoform) in direct proportion to progression of tumor stage and grade. The three isoforms of hyaluronic acid synthase protein (HAS), synthesize HA polymers. An investigation into the expression and physiological functions of the three HAS protein isoforms was performed so as to bring forth a more elaborate picture of BCa progression and development possibly leading to their potential use as bladder tumor markers. Quantitative real time PCR analysis for HAS1, 2, & 3 expression revealed a 5, 12, and 5 respective fold level of increased expression in bladder tumor (TBL) tissue as compared to normal bladder (NBL) tissue. HA levels were also elevated in TBL tissue correlating with HAS1, 2 & 3 transcript expression. Quantitative HAS1, HAS2, and HAS3 real-time PCR on exfoliated bladder urothelial cells obtained from void patient urine and bladder wash revealed a 2-3 fold elevated level of HAS expression as compared to normal urine samples. No definitive data was found to firmly substantiate their use as bladder tumor markers. However, their ability in accurately detecting bladder cancer recurrence is still under investigation. The generation of HT1376 HAS1 stable sense and anti-sense transfectants allowed us the capability to understand the various roles and underlying function(s) that HAS1 offers the HT1376 bladder cancer cell line. HAS1-sense (HAS1-S) transfectants were observed to have an increased level of cell proliferation, greater amounts of HA production, increased invasive activity, as well as a thicker pericellular coat. On the contrary, HAS1-antisense (HAS1-AS) transfectants had greatly reduced levels of HA, slower rates of proliferation, a decreased level of invasive activity and an undetectable pericellular coat. In addition, FLOW cell cycle analysis revealed the affect of HAS1 on the cell cycle in either promoting (HAS1-S) or inhibiting (HAS1-AS) transgression through the G2-M phase. Western blot analysis of key G2-M cell cycle regulatory proteins helped to confirm observed cell cycle patterns.

Keywords

Biology, Cell

Link to Full Text

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