Yearbook 05/06


Mario Kramer
Home Institution Eberhard-Karls-Universität, Tübingen Department of Urology Host Institution(s) University of Miami, Miller School of Medicine, Department of Urology, Miami Mentor: Vinata B. Lokeshwar, Ph.D. E-Mail: mariokramer@gmail.com
Research Topic see Abstract
Personal Reactions to the U.S. Experience Miami is not a typical U.S. city. With only 20% of its population US-born and with Spanish being by far the most spoken language, Miami has developed into a place where you find a mixture of American laws and attitudes, stewed with the typical South American behaviour and lifestyle. What enhances living in Miami Beach is its tropical weather and its beautiful beaches, which attract American tourists during the winter and European ones in the Summer, with the result that one never really feels that he is living in an actually "city", but rather in a never-resting vacation Mecca. It has been a very special experience living among tourists, hundreds of bars, restaurants and clubs which create this summer vacation lifestyle. I was lucky to live in such unique part of the city, because Miami's strong immigration from South America and Cuba has brought with it wide-spread poverty, and vast areas of the city are far poorer, and more run-down and dangerous than what we are used to in Europe. My life in Miami was divided into two parts, one in South Beach and one at my lab. I experienced that lab-work is undertaken with much more efficiency, seriousness and thoughtfulness, compared to the ease of my SoBe life, where practically nothing is scheduled or programmed. In my lab, I was happy to witness the attention and the kindness that I received from everyone around me: my mentor, my colleagues and staff from different departments all helped me and facilitated my duties. Everyone was pleased to help me. Above all, both my professor and my mentor showed me how much support you can truly receive when you show honest interest in the subject and you are willing to work hard.
Greatest Difficulties Encountered I never had to deal with too many complications. In the very beginning, organizing life can be annoying, especially with all the paperwork revolving around owning a car. But after some weeks life became absolutely normal. In fact, my greatest problem was combining life in the lab with my social life in South Beach. Don't tell me that it's not problematic dealing with the desire to enjoy the weather and the beach, to go out and meet people while many projects in the lab are waiting to be done.
Most humorous incident(s) I have had many funny moments during my stay here, but I can only present a handful of them: 1) Many of my friends in Miami are Italians and one of them is a 40-year-old, still single, masseur who used to be a model. These traits could already provide enough ingredients for many funny stories, but what makes Fabrizio truly special is his super funny dyslexia, which gave birth to some memorable sentences, such as, when one night he had to go to Segafredo (a popular bar here in SoBe) to meet some French girls and he boasted: "I'm late, I have to go to France to meet Segafredo!" 2) After one week my name was changed from Mario to Marione, literally "Big Mario", and, honestly, nobody calls me Mario anymore. The reason for my nickname is deeper and more of a compliment than what one may first think. I am called Marione not because I am physically a big guy, but because everyone recognized that I am a good, worthy boy... hence I earned the "MarioNE"! 3) I lived several months in a camping atmosphere, since my roommate was renovating his whole apartment. During this time (>2 months) I was not only the cook for a starving roommate, but I also had the temporary kitchen in my room!!! 4) I tried to speak to a handyman one time but he only looked at me and said: "You should learn Spanish!" Am I in the USA??!!
Helpful Hints for Future Students If you find a former BMEP student who lived in the same city, ask her/him for advice Organize as much as you can from Germany before you come http://www.roommates.com Work hard and many doors will open up Try to find ways of getting some money because you cannot only rely on the money that you get from the BMEP. You should not hesitate to ask your mentor for a supplemental stipend Miami is great!!

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Abstract on Research Topic
Analysis of the diagnostic and prognostic potential of HA, HYAL1, HYAL1-v1 and HAS-1 for urologic malignancies

Authors:
Mario Kramer, Roozbeh Golshani, Vinata B. Lokeshwar

Introduction:
According to the American Cancer Society, more than 63,000 new cases of bladder cancer are diagnosed in the USA each year. However, no method perfectly diagnosis the disease. Cystoscopy and biopsy are still the “gold standards” for detecting bladder cancer, however, new methods such as urine analysis assays have been developed in the past but never introduced into daily clinical work. Approximately 80% of the tumors are diagnosed in an early stage but 20-30% of recurrent tumors will subsequently become muscle invasive. Therefore, there is need to find markers that can predict recurrence and progression. Hyaluronic acid (HA) and HYAL1 hyaluronidase have proved to be useful markers to detect bladder cancer. HA is a glycosaminoglycan that promotes tumor metastasis. It is synthesized by HA Synthases (HAS) 1, 2 or 3. Hyaluronidase is an enzyme that degrades HA into angiogenic fragments. HYAL1 type HAase is expressed by tumor cells. Our lab has previously shown that the measurement of urinary HA & HAase levels by two very similar ELISA-like assays (HA-HAase test), detects bladder cancer with ~ 90% accuracy. Furthermore, HYAL1 is a molecular determinant of bladder cancer growth, infiltration and angiogenesis.

In this research fellowship we are working on three projects, as described below.

Project 1: Role of HYAL1 splice variant HYAL1-v1 in BCa.

We have previously described 5 alternatively spliced variants of HYAL1 which encode enzymatically inactive proteins. In this study, we examined whether HYAL1-v1 expression affects bladder cancer growth and invasion by stably transfecting HT1376 bladder cancer cells with a HYAL1-v1 cDNA construct. Although HYAL1-v1 transfectants expressed equivalent levels of enzymatically active HYAL1 protein, when compared to vector transfectants, there was 4-fold less HAase activity present in their condition media, due to a non-covalent complex formation between HYAL1 and HYAL1-v1 proteins. HYAL1-v1 transfectants grew 3-4-fold slower due to a cell cycle block in the G2-M phase and showed higher apoptosis. In xenografts, HYAL1-v1 tumors grew 3-4-fold slower and tumor weights at day 35 were 3-6-fold less than vector tumors (P < 0.001). While vector tumors were infiltrating and showed high mitoses and microvessel density, HYAL1-v1 tumors were necrotic, infiltrated with leukocytes and showed low mitoses and microvessel density. These results show that the expression of enzymatically inactive HYAL1 variants may negatively regulate bladder tumor growth and its progression.

Project 2: HA, HYAL1, HYAL1-v1 & HAS-1 as prognostic markers for BCa.

We are examining the expression of 4 bladder tumor markers i.e., HA, HYAL1, HYAL1-v1 and HAS-1 in bladder tissues in comparison to evaluate their prognostic potential for predicting tumor recurrence and progression. Tissue microarrays (TMA) were generated using 266 bladder tissues. 30 different specimens of bladder cancer and normal bladder were also collected and sectioned as standard pathology slides for comparison with the TMAs. The expression levels of each marker are being identified by immunohistochemistry. Initial results seem to indicate that HA, HYAL1 and HAS-1 correlate with tumor aggressiveness while HYAL-1-v1 might show a negative correlation.

Project 3: Development of a urine test for BCa involving HYAL1 and HAS-1, 2 and 3.

In this study, we are investigating whether the expression of HYAL1, HAS-1, 2 and 3 mRNAs in either exfoliated urothelial cells in urine specimens and/or bladder tissue correlates with clinical information. Over 100 urine specimens are being collected involving bladder cancer patients, normals and patients with other genitourinary conditions. HYAL1, HAS-1, 2, and 3 mRNA levels in urine and tissue specimens are being measured by a quantitative real-time PCR assay. These results will be compared with the inferences of the HA-HAase urine test. Comparing both tests will give us an idea how these markers are expressed at the transcriptional (real-time PCR) and translational (HA-HAase test) level and what role they might play in predicting tumor aggressiveness. Our preliminary results show that HYAL-1, HAS-1 and 2 RNA transcript levels are elevated in bladder tumor specimens. Therefore, we suggest an increased expression of these markers when compared to exfoliated urine cells from bladder cancer patients.