Purification and analysis of growth regulating proteins secreted by a human melanoma cell line.
Supernatants of a human malignant cell line established from a CNS metastasis, contained several proteins with putative growth regulating functions. BioGel P-10 gel filtration chromatography, reverse phase HPLC purification, and amino-terminal sequencing of purified peptides resulted in characterization of beta 2-microglobulin (beta 2M, 10 kD), ubiquitin (6 kD), and tissue inhibitor of metalloproteinases 2 (TIMP-2, 21 kD). In addition, CNBr cleavage and purification of resulting peptides revealed diazepam binding inhibitor (DBI, 8 kD) and melanoma inhibiting activity (MIA, 11 kD). The secretion of beta 2M as part of the HLA-class I complex may be related to impaired autologous anti-tumour immune function; ubiquitin may play a role in activation or deactivation of extracellular proteins or cell-cell interactions. As HTZ-19 cells respond in a dose-dependent manner to midozolam, DBI may interfere with growth regulation mediated by diazepam receptor sites. In a collagenolytic assay, TIMP-2 interfered with metalloproteinase functions, which are required for degradation of collagen type IV and organotopic metastasis. MIA is clearly associated with a proliferation inhibiting effect on HTZ-19 cells. In conclusion, although this tumour shows a degree of progression, several proteins with putative functions at different cellular levels were identified, related to proliferation as well as to the type of metastasis.
Apfel
R
R
Department of Neurology Tumorbiology-Laboratory, Würzburg, Germany.
Lottspeich
F
F
Hoppe
J
J
Behl
C
C
Dürr
G
G
Bogdahn
U
U
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002455:Cell Division / Q000187:drug effects; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006131:Growth Inhibitors / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D006133:Growth Substances / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D008545:Melanoma / Q000378:metabolism*; D008874:Midazolam / Q000494:pharmacology; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D010446:Peptide Fragments / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D013936:Thymidine / Q000378:metabolism; D014357:Trypsin; D014407:Tumor Cells, Cultured
D006131:Growth Inhibitors; D006133:Growth Substances; D009363:Neoplasm Proteins; D010446:Peptide Fragments; D014357:Trypsin; D008874:Midazolam; D013936:Thymidine