Analysis of 9p21 homozygous deletions in urinary tract carcinoma cells: the role of LINE-1 retrotransposons

AR Florl, MSc, WA Schulz, PhD

Department of Urology, Heinrich Heine University, Duesseldorf, Germany

AIM: Alterations of the CDKN2A locus on chromosome 9p21 encoding the p16INK4A cell cycle regulator and the p14ARF1 p53 activator are frequently found in transitional cell carcinoma (TCC) and in renal carcinoma (RCC) cell lines. Homozygous deletion (HD) is the most frequent mechanism of inactivation. The mechanisms causing HD are unknown, although homologous recombination between repetitive sequences such as LINE-1 retrotransposons has been suggested. METHODS: 17 distinct TCC and 7 RCC cell lines were investigated for HD by PCR of CDKN2A exons 1-3, CDKN2B exons 1-2, and adjacent microsatellite markers on 9p21. Following mapping and Southern blot analysis, deletion end-points were cloned by vectorette PCR. RESULTS: 14 cell lines showed HD involving CDKN2A. Deletion sizes ranged from 95 kb to more than 3.5 Mb. Centromeric deletion ends were located from c5.3 to beyond D9S171. Telomeric deletion ends extended up to D9S782, 7 clustering in a 80 kb LINE-1-rich region between CDKN2A and MTAP. Three of 4 cloned telomeric deletion ends were located in LINE-1 sequences; all sequences at the centromeric ends were not homologous, with only 2 bp identical at each junction. All cloned deletions were interstitial. CONCLUSIONS: As predicted, LINE-1 elements seem to be preferentially involved in the genesis of HD around CDKN2A. However, the structure of homozygous deletions ends strongly suggests that they originate from non-homologous end joining (NHEJ) of double-strand breaks rather than from homologous recombination. Clustered LINE-1 sequences may therefore constitute preferential sites either for DNA double strand breaks or for inserting open DNA ends during NHEJ repair in carcinoma cells.

KEY WORDS: homologous recombination, non-homologous end joining, bladder carcinoma, renal cell carcinoma.

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Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Paris, France; February 9 - 12, 2002; in the section on Chromosomal Aberrations.