Selective formation of tumor necrosis factor-alpha (TNF) degradation products contributes to TNF mediated cytotoxicity.
We compared tumor necrosis factor (TNF) metabolism by wild-type MCF-7 (WT) cells, by 40-fold doxorubicin resistant (40F) breast cancer cells and by PC3 and LNCaP prostate cancer cell lines. MCF-7 WT and LNCaP cell lines were sensitive to TNF cytotoxicity and both lines produced two major intracellular TNF degradation products of 15 kDa and 5.5 kDa. The MCF-7 40F and the PC3 cell lines were resistant to TNF and produced multiple TNF degradation products with molecular weights lower than 15 kDa. Both the breast and prostate lines showed TNF receptor crosslinking patterns consistent with a molecular weight of 55 kDa. The breast and LNCaP lines expressed TNF receptors with an apparent dissociation constant (Kd) of 0.4 to 0.6 nM, while the TNF resistant line had a Kd of 2 nM. Similar receptor numbers per cell were found for all cell types (4,000 to 8,000/cell), and comparable levels of TNF internalization were noted. TNF-conditioned medium from the TNF-sensitive cell types was cytotoxic toward both the TNF-sensitive and TNF-resistant lines, and the toxicity was significantly blocked by an anti-TNF monoclonal antibody. Hydrophobic interaction column HPLC fractionation of the TNF-degradation products produced by MCF-7 WT and LNCaP cells revealed that the trimeric, monomeric, and 5.5 kDa fractions possessed the greatest in vitro antitumor activity. These findings suggest that a TNF degradation product, produced selectively by TNF-sensitive cells, may contribute to the antitumor action of TNF.
Fruehauf
J P
JP
Section of Biochemical and Molecular Pharmacology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland.
Sinha
B K
BK
D000911:Antibodies, Monoclonal; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D002455:Cell Division / Q000187:drug effects; D003602:Cytotoxicity, Immunologic / Q000187:drug effects; D004317:Doxorubicin / Q000494:pharmacology; D004351:Drug Resistance; D004586:Electrophoresis; D066246:ErbB Receptors / Q000235:genetics / Q000502:physiology; D005260:Female; D015870:Gene Expression / Q000235:genetics; D006801:Humans; D007457:Iodine Radioisotopes; D008297:Male; D009857:Oncogenes / Q000235:genetics; D011471:Prostatic Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D011956:Receptors, Cell Surface / Q000378:metabolism; D018124:Receptors, Tumor Necrosis Factor; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism / Q000633:toxicity; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism* / Q000633:toxicity
D000911:Antibodies, Monoclonal; D007457:Iodine Radioisotopes; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D018124:Receptors, Tumor Necrosis Factor; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D004317:Doxorubicin; D066246:ErbB Receptors