Faculty Bibliography

A number of proteins are found attached to the plasma membrane of mammalian cells by a glycosylphosphatidylinositol (GPI) anchor that can be cleaved by GPI specific phospholipase D (GPI-PLD). There are no known specific inhibitors of GPI-PLD. We examined some inhibitors of phosphatidylinositol specific phospholipase C (PI-PLC) for their ability to inhibit human serum and human bone marrow cell GPI-PLD. Azo analogues of suramin were found to be potent inhibitors of GPI-PLD. One compound had an IC50 of 3.7 microM that was 10-fold lower than the IC50 required to inhibit PI-PLC. The azo suramin analogues inhibited cancer cell growth at concentrations similar to those required to inhibit GPI-PLD, and below concentrations required to inhibit growth factor binding. It is possible that inhibition of cell growth might be related to the ability of the compounds to inhibit GPI-PLD

The high molecular weight (HMW) forms (24, 22.5, and 22 kDa) of basic fibroblast growth factor-2 (FGF-2) contain an N-terminal extension responsible for their predominantly nuclear localization. These forms of FGF-2 are post-translationally modified, resulting in a 1- to 2-kDa increase in apparent molecular mass. Here we show that this post-translational modification is inhibited by methionine starvation and by the methyltransferase inhibitors 5'-deoxy-5'-methylthioadenosine (MTA) and 3-deaza-adenosine. Inhibition of the methylation-dependent modification results in a significant decrease in HMW FGF-2 nuclear accumulation, suggesting that methylation is relevant to the intracellular distribution of these forms of FGF-2. Treatment with MTA does not affect either the synthesis or the intracellular fate of another nuclear protein, the SV40 large T antigen, demonstrating that this drug does not have a generalized effect on nuclear protein accumulation. These results link HMW FGF-2 post-translational modification to its intracellular distribution

Most cell types express transforming growth factor-beta (TGF-beta) as a large latent TGF-beta complex that must be converted to an active form before TGF-beta can interact with cell surface TGF-beta receptors. This conversion involves the release of mature TGF-beta from the complex by disrupting noncovalent interactions between mature TGF-beta and its propeptide, latency associated peptide. A critical step in regulating TGF-beta effects may be the activation of the large latent TGF-beta complex. Activation of the complex can be achieved by chemical and enzymatic treatments, or by various cell systems. We have identified that coculturing bovine endothelial and smooth muscle cells generates active TGF-beta. Coculture activation of the large latent TGF-beta complex occurs through a plasmin-dependent mechanism that requires concentration of reactants on the cell surface and/or extracellular matrix. The mechanism of latent TGF-beta activation self-regulates through effectors of plasmin generation

Retinoic acid (RA)-treated HL-60 cells were used as a model to study differentiation of granulocytic leukemias. RA induces these cells to mature into granulocytes and to decrease growth. Mediators of these RA effects have not been identified definitively, but transforming growth factor-beta (TGF-beta) has been implicated in regulating proliferation and differentiation of myelogenous leukemic cells. The role of TGF-beta in RA-dependent differentiation and cessation of growth was examined by adding neutralizing anti-TGF-beta IgG to RA-treated HL-60 cells, followed by assessing cell growth and markers of granulocytic differentiation over 5 days. After addition of neutralizing anti-TGF-beta IgG, growth of RA-treated HL-60 cells was maintained at control levels, but granulocytic differentiation continued. These experiments demonstrated that the antiproliferative activity of RA was TGF-beta dependent but that differentiation was not. Because most cell types secrete TGF-beta in a biologically inactive complex, a TGF-beta-dependent effect requires cells to activate the latent form of TGF-beta. Active and total TGF-beta levels were quantitated in media harvested from control and RA-treated cells using a luciferase-based bioassay for TGF-beta activity. Similar levels of total TGF-beta were observed between control and RA-treated cells. RA-treated cells produced active TGF-beta (18-24 pg/ml) after 1, 2, and 3 days of treatment, whereas negligible levels were produced by control cultures. Activation of endogenous latent TGF-beta by RA-treated cells occurred through a plasmin-independent mechanism

We have examined the binding of FGF-2 to ribosomes and have found that in NIH 3T3 cells that synthesize high amounts of all FGF-2 forms, both 18 kDa and HMW forms of FGF-2 bind to ribosomes. Ribosomes purified from these cells were treated with RNase or puromycin to identify the binding site of FGF-2 on the ribosome. Neither RNase nor puromycin treatment affected the in vivo binding of FGF-2 to ribosomes suggesting that FGF-2 binds ribosomal protein or rRNA, but not mRNA. The stoichiometry of binding in these cells was approximately 1 FGF-2 molecule bound per 1 ribosome. Binding was unaffected by high salt treatment indicating that FGF-2 tightly associates with polysomes. An in vitro binding experiment performed with purified ribosomes and recombinant FGF-2 suggested that the binding site is saturable. HBNF, a protein with similar charge and size to FGF-2, bound 15-fold less than FGF-2 to purified ribosomes. These results indicate that the binding of FGF-2 to ribosomes is specific

Angiogenesis is defined as the formation of capillaries from preexisting blood vessels. This involves a balanced spatiotemporal modulation of endothelial cell adhesion, migration, and proliferation. An array of proteolytic enzymes expressed from endothelial cells including those of the plasminogen activator/plasmin system are required for angiogenesis to occur. In this review we focus on the growth factors that are involved in the angiogenic process and that modulate the expression and/or the activity of the plasminogen activator/plasmin system. The elucidation of the interactions between angiogenic growth factors, endothelial cell proteolytic enzymes, and the extracellular environment could ultimately lead to the therapeutic approaches to block angiogenesis and the pathophysiological conditions associated with it

In the initial stages of capillary formation (angiogenesis) microvascular endothelial cells of preexisting blood vessels locally degrade the underlying basal lamina and invade into the stroma of the tissue to be vascularized. A consistent body of experimental evidence has shown that this process requires a wide array of dedradative enzymes. Components of the plasminogen activator (PA)-plasmin system and of the matrix metalloproteinase (MMP) family play important roles. PAs trigger a proteinase cascade that results is the generation of high local concentrations of plasmin and active MMPs. This increase in proteolytic activity has three major consequences: it permits endothelial cell degradation and invasion of the vessel basal lamina, generates extracellular matrix (ECM) degradation products that are chemotactic for endothelial cells, and activates and mobilizes growth factors localized in the ECM. In addition, urokinase-type PA modulates some endothelial cell functions, including proliferation and migration, with a mechanism independent of proteolytic activity. PA and MMP activities are modulated in endothelial cells by complex mechanisms, including transcriptional regulation by a variety of growth factors and cytokines with angiogenic activity, extracellular control of the proteolytic activities by tissue inhibitors, and interaction with binding sites on the cell membrane and ECM