Abstract

Chronic wound fluid (CWF) from chronic venous leg ulcers has been shown to inhibit dermal fibroblast growth by interfering with cell-cycle progression from G1 to S phase. CWF was found to reduce the levels of hyperphosphorylated retinoblastoma tumor-suppressor gene (Rb) and cyclin D1, both of which are required for the cell cycle to enter the S phase. To better understand the effects of CWF, researchers looked into a Rasmediated signalling pathway involving the mitogen-activated protein kinase kinase (MEK), which is known to modulate the expression of these cell-cycle-regulatory proteins [1-15]. The growth suppressive effects of CWF on hyperphosphorylated Rb (ppRb) and cyclin D1 were abolished by transient transfection of dermal fibroblasts with constitutively active Ras. in comparisonThe effects of CWF on these cell-cycle-regulatory proteins were mimicked by a MEK inhibitor, PD 98059. Concurrent administration of PD 98059 and CWF resulted in additive effects. These findings suggest that CWF inhibits dermal fibroblast growth, at least in part, by decreasing the level of active Ras, which results in lower levels of ppRb and cyclin D1. As a result, a Ras-dependent signalling pathway may mediate the growth inhibitory effect of CWF, and restoring Ras activity may overcome this effect. Wound fluid is thought to play an important role in wound healing Acute wound fluid has been shown to stimulate fibroblast and endothelial cell growth induce chemotaxis and increase extracellular matrix production.In contrast, chronic wound fluid (CWF) has been shown to inhibit cellular proliferation contributing to the poor healing of chronic ulcers .CWF inhibits the proliferation of newborn dermal fibroblasts (NbFb) and DNA synthesis in human neonatal fibroblasts .and it halts the cell cycle in the G1 phase (Phillips et al, 1998). Fibroblast proliferation is critical to wound healing,McClain et and any disruption can significantly alter proper wound healing.

The tightly regulated eukaryotic cell cycle can be broadly divided into two phases S (DNA synthesis) and M (mitosis), with a gap phase before S called G1 and a gap phase after M called G2. The product of the retinoblastoma tumor-suppressor gene (Rb), which modulates cell-cycle progression by sequestering transcription factors that regulate transcription of genes The transcription factors are released, allowing the cell to progress from G1 to S phase (Weinberg, 1995). The cyclin D1/CDK4 cyclin-dependent kinase complex is known to phosphorylate Rb and studies show that the cyclin D1/CDK4 complex is required for G1 progression in mammalian cells (Baldin et al, 1993; Sherr, 1993). The cyclin D1/CDK4 complex is made up of a catalytic subunit called CDK4 and a regulatory cyclin called cyclin D1 .Phosphorylation and association with a regulatory cyclin subunit are required for the complex to be activated. According to a recent report It has been proposed that CWF inhibits dermal fibroblast growth by decreasing the levels of cell-cycle-regulatory proteins such as ppRb and cycli.

Ras, a guanine nucleotide-binding protein with a molecular weight of 21kD, is a key regulator of cell growth in all eukaryotic cells (Lowy and Willumsen, 1993). Ras receives signals from a wide range of extracellular stimuli, and Ras mediates its effects by activating a cascade of protein kinases (acts as a molecular switch at the plasma membrane’s inner leaflet, and its activity is regulated by a guanosine mitogen-activated protein (MAP) kinase pathway Active Ras binds inactive Raf and translocates it to the plasma membrane where the Raf is activated (Many studies have established that cyclin D1 expression is induced by Ras through a Raf/MEK/MAP kinase-dependent pathway the ability of oncogenic Ras to shorten the G1 phase can be attributed to increased induction of cyclin D1. Furthermore, expression of dominant-negative Ras into cycling cells causes a decline in cyclin D1, accumulation of hypophosphorylated Rb and subsequent growth arrest in G1, which can be overcome with induction of Pathway of mitogen-activated protein (MAP) kinase Active Ras binds inactive Raf and transports it to the plasma membrane, where it activates Raf Many studies have shown that Ras induces cyclin D1 expression via a Raf/MEK/MAP kinaseand that the ability of oncogenic Ras to short.