Senescent mesenchymal cells accumulate in tumour susceptible oral fibrosis

Objectives: Oral submucous fibrosis (OSMF) is a cancer-susceptible fibrotic condition caused by chewing of the Areca Nut. Areca nut extract can cause keratinocyte senescence in vitro and there is evidence that senescent fibroblasts can stimulate in growth of pre-malignant keratinocytes, even when present at a low frequency. However, a quantitative study of senescence in tumour or fibrotic mesenchyme has never been carried out. Methods: Immunofluorescence studies were conducted using antibodies against histone repressor A (HIRA), 53BP1, GammaH2A.X, P16, 8-oxo-guanine and Ki67, on cultured cells from normal, non diseased chewers and OSMF and on frozen sections of OSMF and control samples. Cells were stained for senescence associated beta-Galactosidase. Results: Fibroblasts possessing senescence-associated heterochromatic foci (SAHFs), as visualized by HIRA staining, accumulated during the progression of OSMF and prior to the presence of dysplasia in the epithelium or the presence of an inflammatory cell infiltrate. Markers of DNA damage foci (Gamma H2AX and 53BP1), oxidative damage (8-oxo-guanine) and p16INK4A accumulated with disease progression. Ki67 staining did not reveal excessive proliferation in the mesenchymal compartment of OSMF at any stage, arguing against replicative senescence or mitotic stress but an antioxidant reduced the frequency of senescent cells All the senescence-associated phenotypes were retained in vitro and cultures from non-diseased Areca Nut users were near normal and the cultures were uncontaminated by keratinocytes, endothelial or inflammatory cells. Therefore, the cause of senescence was intrinsic to the fibroblast population and independent of the disease stimulus. In addition, the replicative lifespan of OSMF fibrblasts was truncated compared to normal, suggesting that they possessed cryptic damage. Conclusion: Our results suggest that the intrinsic generation of reactive oxygen species causes senescence in OSMF. The results suggest that proteins secreted by senescent fibroblasts could be early indicators of neoplasia and that anti-oxidants may be useful in the treatment of fibrosis.