biostudies-arrayexpressShamini SelvarajahHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-GEOD-7077Background: Osteosarcoma (OS) is a very aggressive bone tumor characterized by highly abnormal complex karyotypes. With the improved resolution offered by array comparative genomic hybridization (array CGH) platforms, it is possible to readily detect cryptic microaberrations in genomic DNA. The identification of these microaberrations in genetic syndromes is currently the focus of many array CGH studies, but there have been no analyses to date documenting the occurrence of microaberrations in tumors. Results: In this study we utilized high-resolution oligonucleotide array CGH to identify novel microaberrations under ~750 kb in four OS-derived cell lines: U-2 OS, HOS, MG-63 and SAOS-2. Comparative analysis of these alterations showed that SAOS-2 harbored the most microaberrations at 17, followed by MG-63 with 11, HOS with 9 and U-2 OS with 6. SAOS-2, which has a TP53 mutation, exhibited the highest level of chromosomal instability in previous studies by our group; whereas U-2 OS, which has wild-type p53 status, exhibited the least instability. A consensus region of gain at 5p15.33 was detected in three of the four OS-derived cell lines (HOS, MG-63 and SAOS-2) by aCGH. Of these consensus gains, one was a microaberration of 500 kb in SAOS-2, and confirmed by fluorescence in situ hybridization analysis. It should be noted that this microaberration is immediately telomeric to the TERT gene, which also showed gain. TERT is correlated with increased tumor aggression, as well as decreased progression free survival in OS patients. Experiment Overall Design: This genome-wide analysis is the first study to utilize oligonucleotide array CGH to identify microaberrations in OS, likely to contain genes involved in OS tumor oncogenesis. A better understanding of the underlying molecular genetic events leading to tumor initiation and progression could result in the identification of prognostic markers and therapeutic targets.biostudies-arrayexpressLabeling - Genomic DNA samples were digested with AluI (12.5 units) and RsaI (12.5 units) (Promega) for a minimum of 2 hours at 37C. Digestion quality was assessed by the DNA 1000 LabChip Kit (Agilent 2100 Bioanalyzer, Agilent Technologies). Individual reference and experimental samples were purified using the Qiaquick PCR Cleanup Kit (Qiagen Inc., Cologne, Germany). Labelling reactions were performed with purified digested DNA using the Bioprime labeling kit (Invitrogen, USA) according to the manufacturer's directions.Nucleic Acid Extraction - Cell line was grown in ATCC complete growth medium: Minimum essential medium (Eagle) with 2 mM L-glutamine and Earle's BSS adjusted to contain 1.5 g/L sodium bicarbonate, 0.1 mM non-essential amino acids, and 1.0 mM sodium pyruvate, 90%; fetal bovine serum, 10%; temperature: 37.0C; atmosphere: air, 95%; carbon dioxide (CO2), 5%. Samples not treated. Genomic DNA was prepared using the Puregene DNA Purification Tissue Kits (Gentra Systems, Inc., Minneapolis, USA). Samples were RNase A treated for 1 hour at 37C.Hybridization - Experimental and reference labelled DNA for each hybridization were pooled, mixed with 50 ug of human Cot-1 DNA (Invitrogen, USA), 52 ul of Agilent 10X Blocking Agent and 260 ul of Agilent 2X Hybridization Buffer. Prior to hybridization to the array, the 520 ul hybridization mixtures were denatured at 100C for 1 min 30 s and incubated at 37C for 30 min. The sample was applied to the array using an Agilent microarray hybridization chamber, and hybridized for 40h at 65C in a rotating oven (Robbins Scientific, Sunnyvale, USA) at 20 rpm.MIAME ScoreOrganizationAssays and DataMAGE-TAB FilesArray DesignsAssay Data Transformation - Data heading descriptions from GEO:<br>#ID_REF = Agilent platform feature number<br>#HOS Cy5 = log ratio from HOS Cy5 x normal male DNA Cy3<br>#HOS Cy3 = log ratio from HOS Cy3 x normal male DNA Cy5<br>#VALUE = normalized, log (test/ref) ratio representing the average of dye-flip experimentsAssay Data Transformation - Data heading descriptions from GEO:<br>#ID_REF = Agilent platform feature number<br>#U2OS Cy5 = log ratio from U-2 OS Cy5 x normal male DNA Cy3<br>#U2OS Cy3 = log ratio from U-2 OS Cy3 x normal male DNA Cy5<br>#VALUE = normalized, log (test/ref) ratio representing the average of dye-flip experimentsFeature Extraction - Arrays were washed according to the manufacturer's recommendations, air dried, and scanned using an Agilent 2565AA DNA microarray scanner (Agilent Technologies, Inc).Assay Data Transformation - Data heading descriptions from GEO:<br>#ID_REF = Agilent platform feature number<br>#SaOS2 Cy3 = log ratio from SAOS-2 Cy3 x normal male DNA Cy5<br>#SaOS2 Cy5 = log ratio from SAOS-2 Cy5 x normal male DNA Cy3<br>#VALUE = normalized, log (test/ref) ratio representing the average of dye-flip experimentsAssay Data Transformation - Data heading descriptions from GEO:<br>#ID_REF = Agilent platform feature number<br>#MG63 Cy5 = log ratio from MG63 Cy5 x normal male DNA Cy3<br>#MG63 Cy3 = log ratio from MG63 Cy3 x normal male DNA Cy5<br>#VALUE = normalized, log (test/ref) ratio representing the average of dye-flip experimentsMetabolomicsUnknownTranscriptomicsGenomicsProteomicsOsteosarcoma (OS) is an aggressive bone tumor characterized by complex abnormal karyotypes and a high level of genomic instability. Using high-resolution array comparative genomic hybridization (aCGH), a novel class of localized copy number variations called microaberrations has been detected. These genomic anomalies typically involve DNA imbalances affecting 700 kb to 1 Mb DNA, and are often associated with some type of genetic syndromes. Because the origin of instability in OS is poorly understood, we used aCGH to determine whether microaberrations were a characteristic of four OS cell lines: U-2 OS, HOS, MG-63, and SAOS-2. TP53 is mutated in SAOS-2, a line in which 17 microaberrations were found. In contrast, U-2 OS, which has a wild-type TP53, had only six such anomalies, the lowest incidence. A 500-kb microaberration within a region of gain at 5p15.33 in SAOS-2 was confirmed by fluorescence in situ hybridization. Significantly, this genomic location is close to the TERT gene, a region of gain in all four cell lines. To our knowledge, this is the first systematic analysis of the incidence of microaberrations in OS. The high levels of these anomalies detected suggest that the instability processes in OS that lead to a highly abnormal karyotypes may also be associated with acquisition of genomic microaberrations.comparative genomic hybridization by arrayHomo sapiensIdentification of cryptic microaberrations in osteosarcoma by high-definition oligonucleotide array comparative genomic hybridization.Shamini SelvarajahShamini Selvarajah, Maisa Yoshimoto, Georges Maire, Jana Paderova, Jane Bayani, Jeremy A Squire, Maria ZielenskafalseComparative genomic hybridization of human osteosarcoma cell lines to identify microaberrationsBackground: Osteosarcoma (OS) is a very aggressive bone tumor characterized by highly abnormal complex karyotypes. With the improved resolution offered by array comparative genomic hybridization (array CGH) platforms, it is possible to readily detect cryptic microaberrations in genomic DNA. The identification of these microaberrations in genetic syndromes is currently the focus of many array CGH studies, but there have been no analyses to date documenting the occurrence of microaberrations in tumors. Results: In this study we utilized high-resolution oligonucleotide array CGH to identify novel microaberrations under ~750 kb in four OS-derived cell lines: U-2 OS, HOS, MG-63 and SAOS-2. Comparative analysis of these alterations showed that SAOS-2 harbored the most microaberrations at 17, followed by MG-63 with 11, HOS with 9 and U-2 OS with 6. SAOS-2, which has a TP53 mutation, exhibited the highest level of chromosomal instability in previous studies by our group; whereas U-2 OS, which has wild-type p53 status, exhibited the least instability. A consensus region of gain at 5p15.33 was detected in three of the four OS-derived cell lines (HOS, MG-63 and SAOS-2) by aCGH. Of these consensus gains, one was a microaberration of 500 kb in SAOS-2, and confirmed by fluorescence in situ hybridization analysis. It should be noted that this microaberration is immediately telomeric to the TERT gene, which also showed gain. TERT is correlated with increased tumor aggression, as well as decreased progression free survival in OS patients. Experiment Overall Design: This genome-wide analysis is the first study to utilize oligonucleotide array CGH to identify microaberrations in OS, likely to contain genes involved in OS tumor oncogenesis. A better understanding of the underlying molecular genetic events leading to tumor initiation and progression could result in the identification of prognostic markers and therapeutic targets.2008-06-18T00:00:00Z2023-09-13T15:45:05.262Z2021-09-28T09:15:50ZE-GEOD-7077GSE707717981215EFO_000074917981215