Tissue or cell lysates (1 mg/ml protein) in IP incubation buffer (Active Motif, Carlsbad CA), were incubated overnight at 4°C with 20 ?g of antibodies against POLG, PARP1, or PAR molecules. Samples were then loaded on to Protein G Agarose Prepacked Columns (Active Motif), and columns were washed five times with IP wash buffer (Active Motif), to remove non-binding proteins. Immune complexes were eluted from the columns in 0.2 M glycine (pH 2.5) solution. The eluents were pooled (total 100 ?l), neutralized with 20 ?l of 1 M Tris-HCl (pH 9.0), and used for Western blotting.
For ChIP assay, Imprint Chromatin Immunoprecipitation Kit was employed (Sigma). Briefly, Hela cells (5 X 107) or tissue sections (10 mg) were incubated with 9 ml of 1% buffered formaldehyde for 30 min at room temperature on a rocking platform to cross-link DNA/proteins, neutralized by adding 1 ml of 1.25 M glycine buffer, homogenized, and then sonicated on a Misonix XL2020 sonicator (30 pulses, 30 sec on/off, 100% power) to fragment the chromatin DNA. Cross-linked samples were subjected to immune-precipitation with mouse hPARP1 N-terminus-specific monoclonal antibody (sc-74470, Santa Cruz). Mouse IgG (Sigma) was used as negative control. The formaldehyde cross-link was reversed by incubating for 15 min at 65°C. Samples were then treated with RNase H and protease K, to digest RNA and proteins, respectively, and DNA was extracted with phenol/chloroform and purified by using QIAquick PCR Purification Kit (28104, Qiagen). ChIP DNA was used as substrate for PCR amplification of PARP1-bound sequences by using the oligonucleotides listed in S1 Table.
But after taking into account other factors -- such as site of the cancer and prostate-specific antigen (PSA) levels -- the researchers found that black men had a 19 percent lower risk of dying during the study period than white men.
"By pooling data across clinical trials, this study provided a unique opportunity to evaluate how race might affect prostate cancer response to treatment," said lead study author Susan Halabi, a professor of biostatistics and bioinformatics at Duke University.
Earlier studies found that black men with advanced prostate cancer who received treatment died sooner than white men, but the evidence has been inconsistent, the researchers noted.
"This study underscores the importance of increasing the participation of racial minorities in clinical trials. Every patient who participates in a clinical trial contributes to improving care, and all patients should have the opportunity to receive needed therapies," she said in an American Society of Clinical Oncology, or ASCO, news release.
Black men have a 60 percent higher rate of getting prostate cancer. In addition, they are more likely to be diagnosed at a younger age and with advanced, aggressive cancer, the researchers said.
Because black men had more problems that could limit their survival, such as higher PSA levels and worse measures of general well-being, the researchers compared outcomes in black men to white men using the same prognostic factors.
According to ASCO expert Dr. Robert Dreicer, "This study adds to the growing body of evidence showing that black men with advanced prostate cancer who participate in clinical trials have the same, if not better, chances of survival as white men."
In addition, he said, "This research shows that by providing equal access to treatment, we can reduce racial disparities in outcomes for men with advanced prostate cancer."
The findings were to be presented Friday at the American Society of Clinical Oncology meeting, in Chicago. Research presented at meetings is considered preliminary until published in a peer-reviewed journal.More information
Visit the American Cancer Society for more on prostate cancer.
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Demi said: “I have done ballet since I was a little girl and a huge part of my eating disorder came from that.
“At about 15, I started to realise that a lot of dancers are very, very thin. I felt they got a lot further and got a lot more attention.
“I was so passionate about ballet, I’d have done anything to get where I wanted to be.”