Optimization of fluorescence in situ hybridization (fish) analysis to detect tert gene amplification in a cancer cell line model, K562

• Main Author: Kalaiselvi, Manveeran
• Format: Monograph
• Language: English
• Published: Universiti Sains Malaysia 2009
• Subjects: R Medicine (General)
• Online Access: http://eprints.usm.my/51119/1/KALISELVI%20MANVEERAN%20-%2024%20pages.pdf; http://eprints.usm.my/51119/

TERT gene located on chromosome 5p15.33 is one of the core components oftelomerase enzyme which encodes human telomerase reverse transcriptase (hTERT) and commonly amplified in human cancers. With this regards, the detection ofTERT gene amplification in cancer cell may have useful application in cancer diagnosis and prognosis. Hence, in this study, K562 cell line, a chronic leukemic cell line was used to demonstrate the TERT gene amplification by using Fluorescence in situ hybridization (FISH) method in interphase stage. Maintenance and cultivation of the K562 cell line was performed where the cells were grown in tissue culture flask as suspension culture in RF 10 medium. Normal peripheral blood also was used in this study as the normal control. In this study, TERT gene amplification was examined in K562 cell line by using a dual-color probe (Poseidon ™) that covered the genomic region of TERT gene at region 5p15 together with the control DNA probe, EGRI (5q31) gene to facilitate identification of chromosome 5. FISH analysis was successfully performed based on the recommendation protocol provided by the manufacturer with a minor modification. The FISH signals were visualized using fluorescence microscope with Leica system and Cyto Vision system. Amplification involving the TERT gene region showed several red signals, while the control at the chromosome 5q31 region (EGRl) will provide 2 green signals. In normal cells, the FISH signal pattern indicated the presence of2 red (R) 2 green (G) signals. Our fmdings showed that the ratio ofTERT/5q31 signal varied between 1 and 3 per cell. The cells which have 3- 4 red (TERT) signals/cell and two green (5q31) signals/cell were considered to be a low grade of amplification. There was also an occurrence of chromosome 5 aneusomy, where there were 3 green signals indicating the presence of 3 copies of chromosome 5 or trisomy 5. Some cells also showed 3 green signals with more than 2 red signals. Our study suggests that TERT gene amplification was detected in K562 cells but further investigation by observing the frequency of signal pattern in 200 cells or using metaphase FISH or cytospin samples should be done to reveal the pattern ofTERT gene amplification in K562 cells or other types of cancer cells. Additional optimization of FISH technique should be done to ensure cost-effectiveness before utilizing it for routine diagnosis of TERT gene amplification in cancer patients.