Rabbit Anti-Human H2AFX Polyclonal Antibody

Renal cell carcinoma (RCC) is the most common primary cancer arising from the kidney in adults, with clear cell renal cell carcinoma (ccRCC) representing approximately 75% of all RCCs. Increased expression of the hypoxia-induced factors-1 alpha (HIF1 alpha) and HIF2 alpha has been suggested as a pivotal step in ccRCC carcinogenesis, but this has not been thoroughly tested. Here, we report that expression of a constitutively activated form of HIF2 alpha (P405A, P530A, and N851A, named as HIF2 alpha M3) in the proximal tubules of mice is not sufficient to promote ccRCC by itself, nor does it enhance HIF1 alpha M3 oncogenesis when coexpressed with constitutively active HIF1 alpha M3. Neoplastic transformation in kidneys was not detected at up to 33 months of age, nor was increased expression of Ki67 (MKI67), gH2AX (H2AFX), or CD70 observed. Furthermore, the genome-wide transcriptome of the transgenic kidneys does not resemble human ccRCC. We conclude that a constitutively active HIF2 alpha is not sufficient to cause neoplastic transformation of proximal tubules, arguing against the idea that HIF2 alpha activation is critical for ccRCC tumorigenesis. (C) 2013 AACR.

Spermatocytes normally sustain many meiotically induced double-strand DNA breaks (DSBs) early in meiotic prophase; in autosomal chromatin, these are repaired by initiation of meiotic homologous-recombination processes. Little is known about how spermatocytes respond to environmentally induced DNA damage after recombination-related DSBs have been repaired. The experiments described here tested the hypothesis that, even though actively completing meiotic recombination, pachytene spermatocytes cultured in the absence of testicular somatic cells initiate appropriate chromatin remodeling and cell-cycle responses to environmentally induced DNA damage. Two DNA-damaging agents were employed for in vitro treatment of pachytene spermatocytes: gamma-irradiation and etoposide, a topoisomerase II (TOP2) inhibitor that results in persistent unligated DSBs. Chromatin modifications associated with DSBs were monitored after exposure by labeling surface-spread chromatin with antibodies against RAD51 (which recognizes DSBs) and the phosphorylated variant of histone H2AFX (herein designated by its commonly used symbol, H2AX), gamma H2AX (which modifies chromatin associated with DSBs). Both gamma H2AX and RAD51 were rapidly recruited to irradiation- or etoposide-damaged chromatin. These chromatin modifications imply that spermatocytes recruit active DNA damage responses, even after recombination is substantially completed. Furthermore, irradiation-induced DNA damage inhibited okadaic acid-induced progression of spermatocytes from meiotic prophase to metaphase I (MI), implying efficacy of DNA damage checkpoint mechanisms. Apoptotic responses of spermatocytes with DNA damage differed, with an increase in frequency of early apoptotic spermatocytes after etopo-side treatment, but not following irradiation. Taken together, these results demonstrate modification of pachytene spermatocyte chromatin and inhibition of meiotic progress after DNA damage by mechanisms that may ensure gametic genetic integrity.