[Frontiers in Bioscience E2, 1042-1053, June 1, 2010]

Regulation of p53 isoform expression in renal cell carcinoma

Linda van den Berg1, Adebowale Daniel Segun1, Sabrina Mersch1, Nina Blasberg1, Edgar Grinstein2, Daniel Wai3, Martin Anlauf1, Helmut Erich Gabbert1, Csaba Mahotka1, Sebastian Heikaus1

1Institute of Pathology, Heinrich-Heine University Hospital, 40225 Duesseldorf, Germany, 2Department of Pediatric Oncology, Hematology and Clinical Immunology, Heinrich-Heine University Hospital, 40225 Duesseldorf, Germany, 3 Childrens Hospital Los Angeles, 4650 Sunset Blvd, Los Angeles, CA 90027, USA

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Material and methods
3.1. Patients and specimens
3.2. Cells and culture
3.3. RNA extraction
3.4. Reverse transcription of the p53 isoforms
3.5. Reverse transcription of the p53 inducible genes
3.6. Quantitative PCR
3.7. Western Blot analysis
3.8. Caspase Assays
3.9. Quantification of p53 isoform and p53 inducible gene expression
4. Results
4.1. p53, p53beta and p53gamma expression is upregulated in RCCs
4.2. del133p53beta and del133p53gamma expression is downregulated in early tumor stages
4.3. del40p53 and del40p53gamma expression is increased in late tumor stages
4.4. Induction of p53 target genes in RCC cell lines exhibiting a similar sensitivity towards Topotecan-induced cell death
4.5. Apoptosis induction by Topotecan in clearCa-6 and clearCa-3
4.6. Different basal expression levels of the p53 isoforms in clearCa-6 and clearCa-3
4.7. Regulation of mRNA expression of the p53 isoforms in clearCa-6 and clearCa-3
5. Discussion
6. References

1. ABSTRACT

Differential expression of p53 isoforms might participate in the marked resistance towards conventional chemotherapy of renal cell carcinomas (RCCs). Therefore, we analysed their differential expression and regulation in RCCs. RCCs expressed a more p53 activating isoform pattern during tumor initiation and progression, in vivo. In vitro, two cell lines exhibiting a similar sensitivity towards Topotecan-induced cell death revealed a similar induction of p53 target genes but strongly differed in their extent of apoptosis. Furthermore, they strongly differed in their basal expression patterns and differential regulation of the isoforms. In conclusion, our study examined for the first time the differential expression and regulation of all p53 isoforms in a tumor in vivo. Furthermore, novel results in our in vitro studies show that p53 isoforms are strongly differentially regulated by chemotherapy in RCCs and that expression and regulation of so-called "p53-target genes" are obviously at least in part regulated by other transcription factors. In addition, our original findings show that p53 isoform expression in RCC cell lines is of minor importance for sensitivity towards chemotherapy.