A mutant RAS gene acts through protein kinase C to augment interleukin-3 dependent proliferation in a fastidious immortal myeloid cell line.
The functional role of a mutant RAS gene in immortal myeloid cell proliferation was examined in a fastidious interleukin-3 (IL-3) dependent cell line (NFS/N1.H7) formed by forced proliferation in IL-3 of marrow cells of the NFS/N mouse. The NFS/N1.H7 cell line was strictly dependent upon IL-3 for growth, and the cell line could be activated by phorbol esters (PMA) to augment IL-3 dependent proliferation, but when pKC was downregulated, diminished IL-3 proliferative response resulted. Transfection (electroporation) of the T24 RAS-containing vector pAL8 to NFS/N1.H7 led to clones (H7 NeoRas.F3, H7 NeoRas.E2) that had incorporated the entire 6.6 Kb human mutant H-RAS genome. The mutant RAS-containing clones demonstrated greater proliferation than parent cells or cells containing a control (neo-resistance) vector over a range of suboptimal IL-3 does and in optimal IL-3 concentrations had a faster doubling rate than parent cells. The clone H7 NeoRas.F3 was studied biochemically, and found to constitutively form 3-fold more 3H-diacylglycerol than the parent cell line upon exposure to 3H-glycerol. PMA could partially repair the proliferative defect of NFS/N1.H7 compared to the RAS-expressor. These studies affirm a secondary, accelerating role for a mutant RAS gene product acting through pKC to promote clonal expansion of immortal myeloid cells stimulated by IL-3.
Boswell
H S
HS
Department of Medicine, Indiana University Medical Center, Indianapolis.
Harrington
M A
MA
Burgess
G S
GS
Nahreini
T L
TL
Derigs
H G
HG
Hodges
T D
TD
English
D
D
Crean
C D
CD
Gabig
T G
TG
D000818:Animals; D001854:Bone Marrow Cells; D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D002455:Cell Division / Q000187:drug effects*; D002460:Cell Line; D004075:Diglycerides / Q000502:physiology; D004789:Enzyme Activation; D011905:Genes, ras; D007377:Interleukin-3 / Q000494:pharmacology*; D051379:Mice; D009154:Mutation; D011493:Protein Kinase C / Q000502:physiology*; D011955:Receptors, Drug / Q000378:metabolism; D014162:Transfection
D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D004075:Diglycerides; D007377:Interleukin-3; D011955:Receptors, Drug; C083141:phorbol ester binding protein; C027776:phorbol ester receptor; D011493:Protein Kinase C