Anti-H2AFX monoclonal antibody, clone 091DU2.6.6

Purpose: DNA damage responses are relevant to prostate cancer initiation, progression and treatment. Few models of the normal and malignant human prostate that maintain stromal-epithelial interactions in vivo exist in which to study DNA damage responses. We evaluated the feasibility of maintaining tissue slice grafts at subcutaneous vs subrenal capsular sites in RAG2(-/-)gamma C-/- mice to study the DNA damage responses of normal and malignant glands. Materials and Methods: We compared the take rate and histology of tissue slice grafts from fresh, precision cut surgical specimens that were maintained for 1 to 4 weeks in subcutaneous vs subrenal capsular sites. Induction of gamma H2AX, p53, ATM and apoptosis was evaluated as a measure of the DNA damage response after irradiation. Results: The take rate of subcutaneous tissue slice grafts was higher than typically reported but lower than at the subrenal capsular site. Subcutaneous tissue slice grafts frequently showed basal cell hyperplasia, squamous metaplasia and cystic atrophy, and cancer did not survive. In contrast, normal and malignant histology was well maintained in subrenal capsular tissue slice grafts. Regardless of implantation site the induction of gamma H2AX and ATM occurred in tissue slice graft epithelium 1 hour after irradiation and decreased to basal level by 24 hours, indicating DNA damage recognition and repair. As observed previously in prostatic ex vivo models, p53 was not activated. Notably, tumor but not normal cells responded to irradiation by undergoing apoptosis. Conclusions: To our knowledge this is the first study of DNA damage responses in a patient derived prostate tissue graft model. The subrenal capsular site of RAG2(-/-)gamma C-/- mice optimally maintains normal and malignant histology and function, permitting novel studies of DNA damage responses in a physiological context.

Spermiogenesis includes transcriptional silencing, chromatin condensation and extensive morphological changes as spermatids transform into sperm. Chromatin condensation involves histone hyperacetylation, transitory DNA breaks, histone H2AX (also known as H2AFX) phosphorylation at Ser139 (gamma H2AX), and replacement of histones by protamines. Previously, we have reported that the spermatid protein kinase TSSK6 is essential for fertility in mice, but its specific role in spermiogenesis is unknown. Here, we show that TSSK6 expression is spatiotemporally coincident with gamma H2AX formation in the nuclei of developing mouse spermatids. RNA-sequencing analysis demonstrates that genetic ablation of Tssk6 does not impact gene expression or silencing in spermatids. However, loss of TSSK6 blocks gamma H2AX formation, even though the timing and level of the transient DNA breaks is unaltered. Further, Tssk6-knockout sperm contained increased levels of histones H3 and H4, and protamine 2 precursor and intermediate(s) indicative of a defective histone-to-protamine transition. These results demonstrate that TSSK6 is required for gamma H2AX formation during spermiogenesis, and also link gamma H2AX to the histone-to-protamine transition and male fertility.