Reversion of autocrine transformation by a dominant negative platelet-derived growth factor mutant.
A non-receptor-binding mutant of the platelet-derived growth factor (PDGF) A chain, PDGF-0, was generated by exchanging 7 amino acids in the sequence. The mutant chains formed dimers that were similar to wild-type PDGF-AA with regard to stability and rate of processing to the mature 30-kDa secreted forms. Moreover, the mutant chains formed disulfide-bonded heterodimers with the PDGF B chain in NIH 3T3 cells heterodimer underwent the same processing and secretion as PDGF-AB. Transfection of c-sis-expressing 3T3 cells with PDGF-0 significantly inhibited the transformed phenotype of these cells, as determined by the following criteria. (i) Compared with PDGF-0-negative clones, PDGF-0-producing clones showed a reverted morphology. (ii) Clones producing PDGF-0 grew more slowly than PDGF-0-negative clones, with a fivefold difference in cell number after 14 days in culture. (iii) The expression of PDGF-0 completely inhibited the ability of the c-sis-expressing 3T3 cells to form colonies in soft agar; this inhibition was overcome by the addition of recombinant PDGF-BB to the culture medium, showing that the lack of colony formation of these cells was not due to a general unresponsiveness to PDGF. The specific expression of a PDGF-0/PDGF wild-type heterodimer in COS cells revealed that the affinity of the mutant heterodimer for the PDGF alpha receptor was decreased by approximately 50-fold compared with that of PDGF-AA. Thus, we show that a non-receptor-binding PDGF A-chain mutant neutralizes in a trans-dominant manner the autocrine transforming potential of the c-sis/PDGF B chain by forming low-affinity heterodimers with wild-type PDGF chains. This method of specifically antagonizing the effect of PDGF may be useful in investigations of the role of PDGF in normal and pathological conditions.
Vassbotn
F S
FS
Ludwig Institute for Cancer Research, Biomedical Center, Uppsala, Sweden.
Andersson
M
M
Westermark
B
B
Heldin
C H
CH
Ostman
A
A
D016475:3T3 Cells; D000818:Animals; D002460:Cell Line; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D006801:Humans; D007700:Kinetics; D051379:Mice; D009154:Mutation; D010641:Phenotype; D010982:Platelet-Derived Growth Factor / Q000235:genetics* / Q000378:metabolism; D011233:Precipitin Tests; D017479:Receptors, Platelet-Derived Growth Factor / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured; D015854:Up-Regulation
D010982:Platelet-Derived Growth Factor; D017479:Receptors, Platelet-Derived Growth Factor