Modulation of cell cycle-regulatory proteins by chronic wound fluid

Wound fluid collected from chronic venous leg ulcers (CWF) has been shown t o inhibit the growth of newborn dermal fibroblasts (NbFb) by arresting cell s in the G1/G0 phase and preventing entry into the S phase of the cell cycl e, To characterize the molecular mechanisms through which CWF suppresses th e growth of fibroblasts, we examined the effects of CWF on levels of the ce ll cycle-regulatory proteins, retinoblastoma protein (Rb), and cyclin D1, k nown to be critical for entering the S phase of the cell cycle. CWF from fi ve separate patients was added to NbFb (250 mug/mL) for various time period s, using bovine serum albumin (BSA) as control. Immunoblot analyses reveale d that CWF treatment reduced the levels of phosphorylated Rb and cyclin D1, a protein that modulates the level of phosphorylated Rb. In contrast, CWF elevated the level of the cell cycle inhibitory protein p21(Cip1/Waf1). Tak en together, these results suggest that CWF suppresses the growth of dermal fibroblasts at least in part by reducing the levels of phosphorylated Rb a nd cyclin D1 and increasing the level of p21(Cip1/Waf1).