Optimization of fluorescence in situ hybridization (fish) analysis to detect tert gene amplification in a cancer cell line model, K562
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...
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| Main Author: | |
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| Format: | Monograph |
| Language: | English |
| Published: |
Universiti Sains Malaysia
2009
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| Subjects: | |
| Online Access: | http://eprints.usm.my/51119/1/KALISELVI%20MANVEERAN%20-%2024%20pages.pdf http://eprints.usm.my/51119/ |
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| Summary: | 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. |
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