Introduction We develop a multiplex quantitative realtime PCR for synchronized analysis of mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) to investigate relative mtDNA abundance in paired normal and cancerous breast tissues.
Materials and methodsThe amounts of nDNA and mtDNA in 102 tissue samples were quantiWed for both glyceraldehype-3-phosphodehydrogenase (GAPDH) gene and mtDNA encoded ATPase (MTATP) 8 gene. The average threshold cycle (Ct) number values of the nDNA and mtDNA were used to calculate relative mtDNA content in breast tissues. Results The median delta Ct ( Ct) and the median mtDNA content for normal and cancerous breast tissues were 6.73 and 2.54, as well as 106.50 and 5.80 (P = 0.000, respectively). The mtDNA content was decreased in 82% of cancerous breast tissues compared with the normal ones. The changes were associated with hormone receptor status. Conclusion Our Wnding suggests that decreased mtDNA content in breast cancer may have diagnostic and prognostic value for the disease.
Over the last decade, the rapidly expanding interest in the involvement of DNA methylation in developmental mechanisms, human diseases, and malignancies has highlighted the need for an accurate, quantitative, and high-throughput assay. Existing methods are limited and are often too laborious for high-throughput analysis or inadequate for quantitative analysis of methylation. Recently, a MassCLEAVE assay has been developed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to analyze base-specific methylation patterns after bisulfite conversion. To find an efficient and more cost-effective high-throughput method for analyzing the methylation profile in breast cancer, we developed a method that allows for the simultaneous detection of multiple target CpG residues by using thymidine-specific cleavage mass array on matrix-assisted laser desorption/ionization time-of-flight silicon chips. We used this novel quantitative approach for the analysis of DNA methylation patterns of four tumor suppressor genes in 96 breast tissue samples from 48 patients with breast cancer. Each individual contributed a breast cancer specimen and corresponding adjacent normal tissue. We evaluated the accuracy of the approach and implemented critical improvements in experimental design. (Mol Cancer Res 2008;6(11):1702 -9)
Unregulated cell growth, a major hallmark of cancer, is coupled with telomere shortening. Measurement of telomere length could provide important information on cell replication and proliferation state in cancer tissues. Telomere shortening and its potential correlation with downregulation of cell-cycle regulatory elements were studied by the examination of relative telomere length and methylation status of the TP53, P21 and P16 promoters in tissues from breast cancer patients. Telomere length was measured in 104 samples (52 tumors and paired adjacent normal breast tissues) by quantitative PCR. Methylation profile of selected genes was analyzed in all samples using a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Our results demonstrated a significant shortening of tumor telomere regions compared with paired adjacent normal tissues (Po0.001). Similarly, telomere lengths were significantly shorter in advanced stage cases and in those with higher histological grades (Po0.05). Telomere shortening in cancer tissues was correlated with a different level of hypermethylation in the TP53, P21 and P16 promoters (r ¼ À0.33, P ¼ 0.001; r ¼ À0.70, Po0.0001 and r ¼ À0.71, Po0.0001, respectively). The results suggested that inactivation of p16/Rb and/or p53/p21 pathways by hypermethylation may be linked to critical telomere shortening, leading to genome instability and ultimately to malignant transformation. Thus, telomere shortening and promoter hypermethylation of related genes both might serve as breast cancer biomarkers.
The enzyme 5,10-methylene-tetrahydrofolate reductase (MTHFR) is linked to DNA methylation, synthesis, and repair. C677T is one of the most important polymorphisms in the MTHFR gene. The single nucleotide polymorphism C677T has been found to be associated with decreased enzyme activity and plasma folate, and thus may play a crucial role in the etiology of colorectal cancer. This decrease was observable in people with either high or low folate status. We aimed to test the hypothesis that the C677T genotype is involved in colorectal cancer. Using pyrosequencing, we analyzed the MTHFR genotypes in 234 colorectal cancer patients and 257 matched controls. We examined the polymorphisms in MTHFR and folate intake in relation to risk of colon cancer in an Iranian population-based case-control study. Our finding revealed that the CC, CT, and TT genotypes of MTHFR among the colorectal cancer patients were 50%, 29.1%, and 20.9%, respectively. On the other hand, we could find 29.5% of 677CC, 46% of 677CT and 24.5% of 677TT in the controls. A decreased risk of colon cancer for participants with wild-type genotype was observed. Interestingly, this association was stronger at higher levels of folate intake. Our study corroborates previous findings of an inverse association of the MTHFR 677TT genotype with colorectal cancer, in particular at high levels of folate.
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