A sensitive screening assay for secreted motility-stimulating factors.
Secreted motility-stimulating factors are often expressed and secreted at low concentrations that are difficult to detect by Northern or Western blot analysis. Autotaxin (ATX) is a tumor-secreted autocrine motility-stimulating factor that has been associated with tumor invasion and metastatic potential. Although ATX has a number of enzymatic activities, it is most sensitively detected by its induced chemotactic response. After transfecting ATX cDNA into NIH3T3 fibroblasts, we developed a motility-based method to screen the resulting cloned cells for secretion of active protein. We placed the cloned and transfected cells into the bottom wells of a modified Boyden chamber and placed responding cells (A2058 human melanoma cells) into the upper wells. After overnight incubation, the membrane that separated the two chambers was removed and stained. Simple densitometry measurements were sufficiently accurate to determine which clones secreted active protein. Utilizing this method, 4 positive cell lines were chosen out of 36 tested clones. Further tests on the expanded cell lines determined that all 4 were secreting ATX. Thus, this modified Boyden chamber assay appears to provide a rapid and highly adaptable means to identify cells that secrete motility-stimulating factors.
Nam
S W
SW
Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA. swnam@box-s.nih.gov
Clair
T
T
Schiffmann
E
E
Liotta
L A
LA
Stracke
M L
ML
D016475:3T3 Cells; D000818:Animals; D001681:Biological Assay / Q000379:methods*; D002465:Cell Movement; D002633:Chemotaxis; D017077:Culture Media, Conditioned; D005956:Glucose-6-Phosphate Isomerase / Q000235:genetics / Q000378:metabolism*; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008545:Melanoma / Q000201:enzymology / Q000378:metabolism; D051379:Mice; D009097:Multienzyme Complexes; D043264:Phosphodiesterase I; D010727:Phosphoric Diester Hydrolases; D011755:Pyrophosphatases; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014162:Transfection; D014407:Tumor Cells, Cultured; D000099189:Lysophospholipase D
D017077:Culture Media, Conditioned; D005956:Glucose-6-Phosphate Isomerase; D006023:Glycoproteins; D009097:Multienzyme Complexes; D043264:Phosphodiesterase I; D010727:Phosphoric Diester Hydrolases; D011755:Pyrophosphatases; D012333:RNA, Messenger; D000099189:Lysophospholipase D