Analisis Gen Penindas Tumor P16 Dan Asai Aktiviti Telomerase Di Kalangan Pesakit Tumor Sistem Saraf Pusat Di Malaysia
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Date
2003-04
Authors
Sarina Sulong
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Abstract
The p16 gene is a tumour suppressor gene that encodes a negative regulator involved in
regulating the cell cycle at the G}-S checkpoint. Both of p 16 gene
(MTSlICDKN2NINK4a) inactivation and presence oftelomerase enzyme are proposed
to be involved in tumourigenesis. Telomerase is an enzyme that stabilizes telomere
length and may be useful as a tumour marker for malignancy. Its activity has been
detected in germ line cells and most cancer cells, but not in most of the normal somatic
cells. Our objectives are to investigate the possible p16 gene alterations via homozygous
deletion and mutation analysis; and to detect telomerase activity in central nervous
system tumours.ยท A total of 50 tumour tissues collected between 1997 to 2002 from the
Brain Tumour Tissue Bank of Neuroscience Unit in USM were used in p16 gene
analysis and 23 of these were also utilized in telomerase analysis. Homozygous deletion
of p 16 gene was determined by the absence of PCR product of the gene while the
GAPDH gene amplification was used as an internal control by multiplex- PCR analysis.
PCR-SSCP (Single-Strand Conformation Polymorphism) analysis was performed to
screen for p 16 gene mutation at exon I and 2, which was confirmed by DNA
sequencing analysis. Telomerase activity was detected based on a PCR-Telomeric
Repeat Amplification Protocol (TRAP) using a TRAPEZE Telomerase Detection Kit
(Intergen, Co). The PCR products were electrophoresed on a 12% polyacrylamide gel
and stained with silver staining. Positive amplification of p 16 gene and GAPDH gene
were observed in all samples, suggested no homozygous deletion occurred. Mutation
screening analysis of p 16 gene indicated that there was no mobility shift and suggested
no mutation at exon 1 and 2 had occurred in all samples. DNA sequencing results on all
high-grade tumours showed that all base sequences of exon I and 2 were normal.
Telomerase activity was detected in 26.1 % of the tumour samples and mostly present in
high-grade tumours (66.7%). There was a significant association (Fisher's exact test)
between telomerase activity status with tumour grade (p=O.008). This activity was
detected in the analysed tumours, supporting the fact that activation of telomerase is an
important feature for tumourigenesis. However, alteration of the p16 gene was not
identified in all the samples and suggested that the gene may not playa major role in
tumourigenesis of these tumours in Malaysia. It is possible that there is another
mechanism of p 16 gene inactivation involved in the development of these tumours such
as hypermethylation of this gene in promoter region. This study indicates that there is
no inactivation of p 16 gene, while there is a presence of te10merase activity among the
central nervous system tumour's patients in Malaysia.
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Keywords
The p16 gene is a tumour suppressor gene that encodes a negative regulator , involved in regulating the cell cycle at the G}-S checkpoint.