The specificity and patterns of staining in human cells and tissues of p16INK4a antibodies demonstrate variant antigen binding

The validity of the identification and classification of human cancer using antibodies to detect biomarker proteins depends upon antibody specificity. Antibodies that bind to the tumour-suppressor protein p16INK4a are widely used for cancer diagnosis and research. In this study we examined the specificity of four commercially available anti-p16INK4a antibodies in four immunological applications. The antibodies H-156 and JC8 detected the same 16 kDa protein in western blot and immunoprecipitation tests, whereas the antibody F-12 did not detect any protein in western blot analysis or capture a protein that could be recognised by the H-156 antibody. In immunocytochemistry tests, the antibodies JC8 and H-156 detected a predominately cytoplasmic localised antigen, whose signal was depleted in p16INK4a siRNA experiments. F-12, in contrast, detected a predominately nuclear located antigen and there was no noticeable reduction in this signal after siRNA knockdown. Furthermore in immunohistochemistry tests, F-12 generated a different pattern of staining compared to the JC8 and E6H4 antibodies. These results demonstrate that three out of four commercially available p16INK4a antibodies are specific to, and indicate a mainly cytoplasmic localisation for, the p16INK4a protein. The F-12 antibody, which has been widely used in previous studies, gave different results to the other antibodies and did not demonstrate specificity to human p16INK4a. This work emphasizes the importance of the validation of commercial antibodies, aside to the previously reported use, for the full verification of immunoreaction specificity

Mapping H4K20me3 onto the chromatin landscape of senescent cells indicates a function in control of cell senescence and tumor suppression through preservation of genetic and epigenetic stability

Background: Histone modification H4K20me3 and its methyltransferase SUV420H2 have been implicated in suppression of tumorigenesis. The underlying mechanism is unclear, although H4K20me3 abundance increases during cellular senescence, a stable proliferation arrest and tumor suppressor process, triggered by diverse molecular cues, including activated oncogenes. Here, we investigate the function of H4K20me3 in senescence and tumor suppression. Results: Using immunofluorescence and ChIP-seq we determine the distribution of H4K20me3 in proliferating and senescent human cells. Altered H4K20me3 in senescence is coupled to H4K16ac and DNA methylation changes in senescence. In senescent cells, H4K20me3 is especially enriched at DNA sequences contained within specialized domains of senescence-associated heterochromatin foci (SAHF), as well as specific families of non-genic and genic repeats. Altered H4K20me3 does not correlate strongly with changes in gene expression between proliferating and senescent cells; however, in senescent cells, but not proliferating cells, H4K20me3 enrichment at gene bodies correlates inversely with gene expression, reflecting de novo accumulation of H4K20me3 at repressed genes in senescent cells, including at genes also repressed in proliferating cells. Although elevated SUV420H2 upregulates H4K20me3, this does not accelerate senescence of primary human cells. However, elevated SUV420H2/H4K20me3 reinforces oncogene-induced senescence-associated proliferation arrest and slows tumorigenesis in vivo. Conclusions: These results corroborate a role for chromatin in underpinning the senescence phenotype but do not support a major role for H4K20me3 in initiation of senescence. Rather, we speculate that H4K20me3 plays a role in heterochromatinization and stabilization of the epigenome and genome of pre-malignant, oncogene-expressing senescent cells, thereby suppressing epigenetic and genetic instability and contributing to long-term senescence-mediated tumor suppression