Autocrine tumor cell growth-inhibiting activities from human malignant melanoma.
Autocrine-secreted tumor cell growth-inhibiting activities were isolated from supernatants of a malignant melanoma cell line, HTZ 19-dM, established from a central nervous system melanoma metastasis. HTZ 19-dM was characterized by cyto- and immunocytochemistry and karyotyping; cells were propagated in defined serum-free tissue culture medium for up to 8 months. Supernatants were ultrafiltrated, dialyzed, lyophilized, and purified by Bio-Gel P-10 gel permeation chromatography, leading to three active fraction pools, MIAI [melanoma-inhibiting activity (MIA), 2 kDa), MIAII (Mr 11,500-17,000) and MIAIII (proteins at the cutoff of Bio-Gel P-10) inhibiting growth of 19-dM cells with 50% inhibitory concentrations of 0.79 microgram/ml (MIAI), 0.13 microgram/ml (MIAII), and 16.7 micrograms/ml (MIAII). MIAII could be further purified by reverse phase high pressure liquid chromatography; the main activity displayed a 50% inhibitory concentration of 0.33 microgram/ml. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis one major band (molecular weight about 14,000) and two minor bands (up to Mr 17,000) were identified. Macromolecular synthesis was inhibited in 19-dM cells up to greater than 99.5%; tumor stem cell colony formation was reduced by 99.89%; the inhibitory effect of MIAII was irreversible, nonsaturable, and partially antagonized by a serum factor (depending on purification stage). MIAII was heat stable (3 min at 100 degrees C) and trypsin labile. The effect of MIAII on allogeneic neuroectodermal tumors was also investigated; proliferation of two of three malignant melanomas and two of four glioblastomas was inhibited up to 85.2%; proliferation of a neuroblastoma cell line could be inhibited to 33.8%, whereas normal fibroblasts and low grade gliomas were not influenced in their proliferation.
Bogdahn
U
U
Department of Neurology, Julius-Maximilians-University, Würzburg, Federal Republic of Germany.
Apfel
R
R
Hahn
M
M
Gerlach
M
M
Behl
C
C
Hoppe
J
J
Martin
R
R
D001685:Biological Factors / Q000032:analysis* / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D006131:Growth Inhibitors / Q000032:analysis* / Q000494:pharmacology; D006801:Humans; D008545:Melanoma / Q000032:analysis* / Q000473:pathology; D008565:Membrane Proteins / Q000032:analysis* / Q000494:pharmacology; D009363:Neoplasm Proteins / Q000096:biosynthesis; D014411:Neoplastic Stem Cells / Q000187:drug effects; D013936:Thymidine / Q000378:metabolism
D001685:Biological Factors; D006131:Growth Inhibitors; D008565:Membrane Proteins; D009363:Neoplasm Proteins; C031717:growth inhibitory proteins; D013936:Thymidine