THU0104 <i>In vivo</i>and<i>in vitro</i>investigations of the tumour suppressor p16 in rheumatoid arthritis
Authors: P Künzler, CA Seemayer, S Kuchen, M Neidhart, M Pruschy, BA Michel, RE Gay, S Gay
Journal: Annals of the Rheumatic Diseases (2001)

<h3>Background</h3> The progression of the cell cycle is tightly controlled by cyclins and cyclin-dependent kinases. The p16Ink4a protein was identified as a specific inhibitor of cdk4. P16 inhibits the cdk4-dependent phosphorylation of the tumour suppressor retinoblastoma protein (Rb) and thereby leads to a G1 arrest. It is suggested that p16 acts as a tumour suppressor. Despite several approaches have been undertaken to inhibit cell growth in rheumatoid arthritis synovial fibroblasts (RA-SF) by gene transfer of p16, its role in RA is poorly understood. Very few data are available concerning the expression of p16 in RA tissue. <h3>Objectives</h3> The aim of the study was to investigate whether the tumour suppressor p16 plays a role in RA and to get some basic information about its expression level and pattern. We determined the <i>in vivo</i> expression of p16 in tissue sections and its <i>in vitro</i> expression in cultured RA-SF. The inducibility of p16 was evaluated by X-ray irradiation. <h3>Methods</h3> Anti-human p16Ink4a antibodies (PharMingen, clone G175–1239) were used for immunohistochemistry (IHC) as well as for immunofluorescence (IF). Sections from 13 RA, 1 osteoarthritis (OA) and 2 normal synovia were investigated by IHC. Tissue specimens from normal uterus and pancreas were used as positive controls. RA-SF (n = 6), normal synovial fibroblasts (n = 1) and foreskin fibroblasts (FSFB, n = 1) from passages 3–7 were grown on chamberslides and further analysed by IF. HeLa cervix carcinoma cells (ATCC CCL 2) and the osteosarcoma cell line Saos-2 (ATCC HTB 85) served as positive controls. Six RA-SF and 1 control (FSFB) were irradiated with 10 Gy (X-rays) and analysed after 25 h for expression of p16. Moreover, 2 RA-SF and 1 control were irradiated with the same dose, but kept in culture for 13 days before they were investigated for p16. In both experiments, non-irradiated cultures served as negative controls. The percentage of p16 positive cells on tissue sections and fibroblast cultures was evaluated by a scoring system. <h3>Results</h3> P16 was expressed in 10/13 RA specimens in 0–6% of the cells <i>in vivo</i>. Less than 1% stained positively for p16 in OA tissue and in normal synovial tissue. However, in three samples derived from patients with RA, p16 was present in 14–23% of the cells. The positive cells were predominantly located in the sublining and in one case also in the lining. <i>In vitro</i>, cultured RA-SF and controls did not spontaneously express p16, not even after serum starvation or in senescent cultures (data not shown). When irradiated with 10 Gy (X-rays), no p16 could be detected after 25 h, whereas after 13 days p16 was present in about 30–50% of the cells. No major differences were found between RA-SF and FSFB in terms of response to irradiation. <h3>Conclusion</h3> This is, according to our knowledge, the first report investigating the expression of p16 in RA tissue sections by IHC. The <i>in vitro</i> experiments demonstrated no constitutive expression of this protein in RA-SF. However, p16 could be induced and detected in irradiated cultures of RA-SF and normal fibroblasts. Based on our <i>in vivo</i> data, we suggest that in a subset of patients the expression of p16 might be responsible for the low rate of proliferation of synovial cells in RA.