Detecting DNA methylation of the BCL2, CDKN2A and NID2 genes in urine using a nested methylation specific polymerase chain reaction assay to predict bladder cancer.

PURPOSE: Detection of methylated DNA has been shown to be a good biomarker for bladder cancer. Bladder cancer has the highest recurrence rate of any cancer and, as such, patients are regularly monitored using invasive diagnostic techniques. As urine is easily attainable, bladder cancer is an optimal cancer to detect using DNA methylation. DNA methylation is highly specific in cancer detection. However, it is difficult to detect because of the limited amount of DNA present in the urine of patients with bladder cancer. Therefore, an improved, sensitive and noninvasive diagnostic test is needed. MATERIALS AND METHODS: We developed a highly specific and sensitive nested methylation specific polymerase chain reaction assay to detect the presence of bladder cancer in small volumes of patient urine. The genes assayed for DNA methylation are BCL2, CDKN2A and NID2. The regions surrounding the DNA methylation sites were amplified in a methylation independent first round polymerase chain reaction and the amplification product from the first polymerase chain reaction was used in a real-time methylation specific polymerase chain reaction. Urine samples were collected from patients receiving treatment at Wolfson Medical Center in Holon, Israel. RESULTS: In a pilot clinical study using patient urine samples we were able to differentiate bladder cancer from other urogenital malignancies and nonmalignant conditions with a sensitivity of 80.9% and a specificity of 86.4%. CONCLUSIONS: We developed a novel methylation specific polymerase chain reaction assay for the detection and monitoring of bladder cancer using DNA extracted from patient urine. The assay may also be combined with other diagnostic tests to improve accuracy.

SirT3 is a nuclear NAD+-dependent histone deacetylase that translocates to the mitochondria upon cellular stress.

In humans, there are at least seven Sir2-like proteins (SirT1-7) with diverse functions, including the regulation of chromatin structure, and metabolism. SirT3 levels have been shown to correlate with extended life span, to localize to the mitochondria, and to be highly expressed in brown adipose tissue. In humans, SirT3 exists in two forms, a full-length protein of approximately 44 kDa and a processed polypeptide lacking 142 amino acids at its N terminus. We found that SirT3 not only localizes to the mitochondria, but also to the nucleus under normal cell growth conditions. Both the full-length and processed forms of SirT3 target H4-K16 for deacetylation in vitro and can deacetylate H4-K16 in vivo when recruited to a gene. Using a highly specific antibody against the N terminus of SirT3, we found that SirT3 is transported from the nucleus to the mitochondria upon cellular stress. This includes DNA damage induced by Etoposide and UV-irradiation, as well as overexpression of SirT3 itself.

SirT2 is a histone deacetylase with preference for histone H4 Lys 16 during mitosis.

The mammalian cytoplasmic protein SirT2 is a member of the Sir2 family of NAD+-dependent protein deacetylases involved in caloric restriction-dependent life span extension. We found that SirT2 and its yeast counterpart Hst2 have a strong preference for histone H4K16Ac in their deacetylation activity in vitro and in vivo. We have pinpointed the decrease in global levels of H4K16Ac during the mammalian cell cycle to the G2/M transition that coincides with SirT2 localization on chromatin. Mouse embryonic fibroblasts (MEFs) deficient for SirT2 show higher levels of H4K16Ac in mitosis, in contrast to the normal levels exhibited by SirT1-deficient MEFs. The enzymatic conversion of H4K16Ac to its deacetylated form may be pivotal to the formation of condensed chromatin. Thus, SirT2 is a major contributor to this enzymatic conversion at the time in the cell's life cycle when condensed chromatin must be generated anew.