Explanation PolyADP-ribosylation is mediated by poly(ADP-ribose) (PAR) polymerases (PARPs) and may be involved in various cellular events, including chromosomal stability, DNA repair, transcription, cell death, and differentiation. The physiological level of PAR is difficult to determine in intact cells because of the rapid synthesis of PAR by PARPs and breakdown of PAR by PAR-degrading enzymes, including poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribosylhydrolase 3 (ARH3).In collaboration with Dr. Masanao Miwa, we developed analytical tools to study poly(ADP-ribosylation)and looked at conditions that modified poly(ADP-ribose) content. It was first necessary to better quantify PAR. Artifactual synthesis and/or degradation of PAR likely occurs during lysis of cells in culture. An enzyme-linked immunosorbent assaybased (ELISA) system was designed for determining the physiological level of PAR in cultured cells. We immediately inactivated enzymes that catalyze the synthesis and degradation of PAR. We validated that trichloroacetic acid is suitable for inactivating PARPs, PARG, and other enzymes involved in metabolizing PAR in cultured cells during cell lysis. The PAR level in cells harvested with the standard radio-immunoprecipitation assay buffer was increased by 450-fold compared to trichloroacetic acid for lysis, presumably because of activation of PARPs by DNA damage that occurred during cell lysis. This ELISA can be used to analyze the biological functions of polyADP-ribosylation under various physiological conditions in cultured cells. To better understand the utility of the assay, we determined the effect of mild temperature shift on poly(ADP-ribose) and gH2AX levels in cultured cells. PAR is rapidly synthesized by PARPs upon activation by DNA single- and double-strand breaks. In this study, we examined the quantitative amount of PAR in HeLa cells cultured within the physiological temperatures below 41C for verification of the effect of shifting-up or -down the temperature from 37C on the DNA breaks, whether the temperature-shift caused breaks that could be monitored by the level of PAR determined by the newly developed ELISA method. While PAR level did not change significantly when HeLa cells were cultured between 33.5C and 37.0C, it was significantly increased 2- and 3-fold when cells were cultured for 12 and 24 h at 40.5C as compared to 37.0C. Similar to the results with HeLa cells, PAR level was increased 2-fold in CHO-K1 cells cultured at 40.5C for 24 h as compared to 37.0C. As the cellular levels of PAR polymerase-1 (PARP-1) and PARG, a major degradation enzyme for PAR, did not seem to change significantly, this increase could be caused by activation of PARP-1 by DNA strand breaks. In fact, gH2AX, a marker of DNA double-strand breaks, was found in cell extracts of HeLa cells and CHO-K1 cells at elevated temperature vs. 37.0C, and these gH2AX signals were intensified in the presence of 3-aminobenzamide, a PARP inhibitor. The gH2AX immunohistochemistry results in HeLa cells were consistent with Western blot analyses. In HeLa cells, proliferation was significantly suppressed at 40.5C in 72 h-continuous cultures and decreased viabilities were also observed after 24-72 h at 40.5C. Flow cytometric analyses showed that the HeLa cells were arrested at G2/M after temperature shift-up to 40.5C. These physiological changes were potentiated in the presence of 3-aminobenzamide. Decrease in growth rates, increased cytotoxicity and G2/M arrest, were associated with the temperature-shift to 40.5C and are indirect evidence of DNA breaks. In addition to gH2AX, PAR could be a sensitive marker for DNA single- and double-strand breaks. These two molecular markers provide evidence of physiological changes occurring within cells. The ELISA appears to be a sensitive method for quantifying changes in PAR.

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