Resistance to ionizing radiations (IR) is one major problem in the clinical management of osteosarcoma (OS) and marks the clonal evolution of tumor cells towards a more aggressive phenotype. Genome-wide screening techniques will be therefore helpful to uncover genomic unbalances eventually associated with specific characteristics of OS cells, including their radio- and/or drug-resistance. Here, we report on the results of a comparative genomic hybridization (CGH)-based technique to achieve informations on the genomic profile associated with radio-resistance in a human OS cel line, named SAOS, that lacks the p53-dependent radio-response. Radio-resistant SAOS subline was generated from the parental cell line following three rounds of low dose (10 Gy)/low dose rate (0.05 Gy/min) gamma irradiation performed at 24 hr intervals. Genomic unbalances associated with the development of radio-resistance were revealed by the co-hybridization of SpectrumGreen- labeled DNA from radioresistant SAOS and SpectrumRed-labeled DNA from parental cell line on AmpliOnc (GenoSensorTM Array 300) microarrays, representing the oncogenes and the tumor suppressor genes most frequently altered in human cancers (for the complete list of spotted targets see: Mao et al. , Genes Chromosomes Cancer 35,144-155,2002). The statistical analysis of obtained results was performed by normalized test-to-reference ratios of the included spots for each target calculated by the mass method according to a previously published work (Hattinger et al. , Europ J Celi BioI82,483-493,2003). The radio-resistance of SAOS cells was associated with the gain of the inherent DNA sequence copy number relative to 11 genes and no losses. In more detail, the amplification relative to tymidine kinase 1 (mapping at 17q25.2-q25.3), telomeric BAC and YAC regions (mapping at chromosomes 9 and 2, respectively) and EGR2 (a component of the mitochondrial apoptotic loop mapping at 10q21.3) was very significant, whereas the amplification relative to two telomeric sequences (mapping at 19p and 2p loci, respectively), to the chromosome segregation 1-like CA5 (mapping at 20q 13 locus), the cycloxigenase 2 (mapping at 1 q31.1), the ERBB2 sequence (Iocated at the 17q21.1 locus) and the Myc (mapping at 8q24.12-q24.13 locus) showed a borderline statistical significance. Genomic unbalances associated with radio-resistance exhibited discrete differences compared with methotrexate-resistance, suggesting that OS cells utilize different pathways to repair the damage induced by reactive oxygen species or metabolic defects. Additional studies currently in progress on gene expression of parental and radio-resistant SAOS cells, performed on U133A microarrays from Affymetrix, will help to elucidate whether in OS lacking p53-dependent response to IR the radioresistance has specific genomic markers.

GENOMIC UNBALANCES ASSOCIATED WITH THE RESISTANCE TO IONIZING RADIATIONS OF A HUMAN OSTEOSARCOMA CELL LINE (SAOS) DETECTED BY COMPARATIVE GENOMIC HYBRIDIZATION (CGH-BASED TECHNIQUE)

BRUSA, GIANLUCA;ASTOLFI, ANNALISA;CAMMELLI, SILVIA;SANTUCCI, MARIA ALESSANDRA;BARBIERI, ENZA
2004

Abstract

Resistance to ionizing radiations (IR) is one major problem in the clinical management of osteosarcoma (OS) and marks the clonal evolution of tumor cells towards a more aggressive phenotype. Genome-wide screening techniques will be therefore helpful to uncover genomic unbalances eventually associated with specific characteristics of OS cells, including their radio- and/or drug-resistance. Here, we report on the results of a comparative genomic hybridization (CGH)-based technique to achieve informations on the genomic profile associated with radio-resistance in a human OS cel line, named SAOS, that lacks the p53-dependent radio-response. Radio-resistant SAOS subline was generated from the parental cell line following three rounds of low dose (10 Gy)/low dose rate (0.05 Gy/min) gamma irradiation performed at 24 hr intervals. Genomic unbalances associated with the development of radio-resistance were revealed by the co-hybridization of SpectrumGreen- labeled DNA from radioresistant SAOS and SpectrumRed-labeled DNA from parental cell line on AmpliOnc (GenoSensorTM Array 300) microarrays, representing the oncogenes and the tumor suppressor genes most frequently altered in human cancers (for the complete list of spotted targets see: Mao et al. , Genes Chromosomes Cancer 35,144-155,2002). The statistical analysis of obtained results was performed by normalized test-to-reference ratios of the included spots for each target calculated by the mass method according to a previously published work (Hattinger et al. , Europ J Celi BioI82,483-493,2003). The radio-resistance of SAOS cells was associated with the gain of the inherent DNA sequence copy number relative to 11 genes and no losses. In more detail, the amplification relative to tymidine kinase 1 (mapping at 17q25.2-q25.3), telomeric BAC and YAC regions (mapping at chromosomes 9 and 2, respectively) and EGR2 (a component of the mitochondrial apoptotic loop mapping at 10q21.3) was very significant, whereas the amplification relative to two telomeric sequences (mapping at 19p and 2p loci, respectively), to the chromosome segregation 1-like CA5 (mapping at 20q 13 locus), the cycloxigenase 2 (mapping at 1 q31.1), the ERBB2 sequence (Iocated at the 17q21.1 locus) and the Myc (mapping at 8q24.12-q24.13 locus) showed a borderline statistical significance. Genomic unbalances associated with radio-resistance exhibited discrete differences compared with methotrexate-resistance, suggesting that OS cells utilize different pathways to repair the damage induced by reactive oxygen species or metabolic defects. Additional studies currently in progress on gene expression of parental and radio-resistant SAOS cells, performed on U133A microarrays from Affymetrix, will help to elucidate whether in OS lacking p53-dependent response to IR the radioresistance has specific genomic markers.
Haematologica
99
100
Brusa G.; Hattinger C.; Serra M.; Astolfi A.; Saponaro M.; Cammelli S.; Santucci M.A.; Barbieri E.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11585/18678
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