Published in Cancer Detection and Prevention 2000; 24(Supplement 1).

Chromosomal instability attributed to the Ha-ras oncogene is associated to the abnormal maintenance of telomeres in Balb/3T3 transfected cells

ET Sakamoto-Hojo PhD 1,2, SS Mello BC 1,2, P Peitl Jr MS 1, ML Camparoto BC 1, GAS Passos Jr PhD 1,3, MP Hande PhD 4, RS Cardoso MS 1

1 FMRP,, 2 FFCLRP,, 3 FORP, University de São Paulo, Ribeirão Preto, SP, Brasil;, 4 Columbia University, Center for Radiological Research, New York NY, USA,

AIMS: Balb/3T3 fibroblasts cells transfected with the Ha-ras oncogene (B61 clone) overexpressing the mRNA ras were used as a model to study the genomic instability attributed to the ras gene associated to the maintenance of telomere integrity, compared to the parental cells (A31 clone). METHODS: Both cell lines were provided by Prof. M. A. Sogayar (IQ-USP). The Ha-ras insert was originally cloned in pBR322, and the plasmid was replaced by PUC18 to perform gene sequencing and use it as a probe in the FISH (fluorescent in situ hybridization) method to detect the ras transfection. Chromosomal rearrangements involving telomeric DNA sequences were also studied by FISH. RESULTS: PUC18 ras insert demonstrated 87% of identity with the human p21 Ha-ras-1 gene. The transfected cells presented well defined fluorescent signals corresponding to extra copies of the ras gene, confirming their integration in tandem array; 82.2 and 15.3% of cells showed one and two signals, respectively, and the remaining 2.5% presented 3-12 signals. The frequencies of rearrangements involving telomeric sequences were 17.5 and 5.9 deletions/cell, 1.95 and 0.25 RLC (Robertsonian fusion-like configurations)/cell, and 5.7 and 4.1 ITS (interstitial telomeric signals) for B61 and A31 cells, respectively. CONCLUSIONS: The high frequencies of chromosomal rearrangements involving telomeric sequences demonstrated the role of the ras oncogene associated to the abnormal maintenance of telomeres. ITS located randomly in the genome indicate the possibility of the occurrence of break-fusion-bridge mechanism, followed by (TTAGGG)n addition at the breakpoints (chromosome healing).(Research supported by FAPESP and CNPq).

KEY WORDS: Genomic instability, Ha-ras-1 oncogene, telomere.

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Paper presented at the International Symposium on Impact of Biotechnology on Cancer Diagnostic & Prognostic Indicators; Geneva, Switzerland; October 28 - 31, 2000; in the section on genetic instability.