Proteomic analysis of factors released from p21-overexpressing tumour cells

The p21Waf1/Cip1/Sdi1 cyclin-dependent kinase inhibitor is a key regulator of cell cycle progression and has also been observed to influence the expression of genes associated with several age-related disorders. Previous work has shown that expression of p21 in tumour cells mediates an antiapoptotic and mitogenic paracrine effect, which is in contrast to the arrested state of p21-expressing cells. Here, we have employed SELDI-MS technology to characterise, at a proteomic level, factors released from HT-1080 human fibrosarcoma cells displaying inducible p21 expression. Conditioned media from induced and noninduced cells were profiled on a range of diverse ProteinChip arrays and subjected to SELDI-MS analysis. Evaluation of proteins binding onto IMAC, Q10 or CM10 surfaces led to the discovery of a number of putative p21-regulated factors. We further validated three p21-regulated proteins observed at 10.2, 11.7 and 13.4 kDa. Using Q Ceramic HyperD fractionation columns, we were able to selectively enrich for each of these three proteins. Subsequent SDS-PAGE and MS analysis of tryptic digests identified the 13.4 kDa protein as cystatin C and the 10.2 kDa protein as pro-platelet basic protein (PPBP). Judging by the apparent MW and the pI of the 11.7 kDa protein, we reasoned that it may be beta-2-microglobulin, which was confirmed by subsequent identification. Increased levels of cystatin C and beta-2-microglobulin in conditioned media from p21-expressing cells was confirmed by antibody capture experiments using anticystatin C and anti-beta-2-microglobulin antibodies on preactivated PS-20 arrays. Western blot analysis demonstrated increased expression of intracellular and extracellular cystatin C and beta-2-microglobulin in p21-expressing cells, compared to noninduced controls. Increased levels of PPBP were validated in cell lysates from p21-expressing cells. The three secreted factors that we have identified in this study, have all been shown previously to have growth modulating effects and, as such, may contribute to the observed mitogenic and anti-apoptotic paracrine activity of p21-expressing [corrected] cells.