Tumor proteinases are considered to be important in the process of cancer invasion and metastasis. We have proposed that the surface membrane localization of these proteinases places them in an optimal site to facilitate the invasion of surrounding extracellular matrix. In this study, we have used the organic solvent, n-butanol, and the detergent, n-octyl-glucoside, to sequentially extract metalloproteinases from crude plasma membranes of human RWP-I pancreatic cancer cells. Anion exchange chromatography and gel permeation chromatography were employed to further purify enzymes with the capacity to degrade gelatin, type-IV collagen, and carboxymethylated transferrin. Gelatin zymography was used to demonstrate proteinase bands of 92, 70 and 62-kDa. Immunoblotting of solubilized, partially purified pancreatic cancer plasma membrane proteins using polyclonal rabbit antibodies, which have specificity for type-IV collagenase/gelatinase, resulted in the recognition of a 70-kDa protein, but not the 92-kDa gelatinase. A type-IV collagenase/gelatinase of 68-kDa was similarly identified in A2058 human melanoma cancer cell plasma membranes.
Inhibition of HbS polymerization is a major target for therapeutic approaches in sickle cell anemia. Toward this goal, initial efforts at pharmacological elevation of fetal hemoglobin (HbF) has shown therapeutic efficacy. In order to identify well-tolerated, novel agents that induce HbF in patients, we developed a high-throughput screening approach based on induction of gamma-globin gene expression in erythroid cells. We measured gamma-globin transcription in K562 cells transfected with either gamma promoter elements fused with the locus control region hypersensitivity site 2 and luciferase reporter gene (HS2 gamma) or a beta-yeast artificial chromosome in which the luciferase reporter gene was recombined into the gamma-globin coding sequences (gamma YAC). Corresponding pharmacological increases in HbF protein were confirmed in both K562 cells and in human primary erythroid progenitor cells. Approximately 186,000 defined chemicals and fungal extracts were evaluated for their ability to increase gamma gene transcription in either HS2 gamma or gamma YAC models. Eleven distinct classes of compounds were identified, the majority of which were active within 24-48 hr. The short chain hydroxamate-containing class generally exhibited delayed maximal activity, which continued to increase transcription up to 120 hr. The cyclic tetrapeptide OSI-2040 and the hydroxamates were shown to have histone deacetylase inhibitory activity. In primary hematopoietic progenitor cell cultures, OSI-2040 increased HbF by 4.5-fold at a concentration of only 40 nM, comparable to the effects of hydroxyurea at 100 microM. This screening methodology successfully identifies active compounds for further mechanistic and preclinical evaluation as potential therapeutic agents for sickle cell anemia.
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