Role of p21 RAS in p210 bcr-abl transformation of murine myeloid cells.
The p21 RAS product has been implicated as part of the downstream signaling of certain nonreceptor tyrosine kinase oncogenes and several growth factor receptor-ligand interactions. We have reported that the chronic myelogenous leukemia oncogene p210 bcr-abl transforms a growth-factor-dependent myeloid cell line NFS/N1.H7 to interleukin-3 (IL-3) independence. In these p210 bcr-abl-transformed cells (H7 bcr-abl.A54) and in two other murine myeloid cell lines transformed to IL-3 independence by p210 bcr-abl, endogenous p21 RAS is activated as determined by an elevated ratio of associated guanosine triphosphate (GTP)/guanosine diphosphate (GDP), assayed by thin-layer chromatography of the nucleotides eluted from p21 RAS after immunoprecipitation with the Y13-259 antibody. Treatment of p210 bcr-abl-transformed cells with a specific tyrosine kinase inhibitor herbimycin A resulted in diminished tyrosine phosphorylation of p210 bcr-abl and associated proteins, without major reduction in expression of the p210 bcr-abl protein itself. Inhibition of p210 bcr-abl-dependent tyrosine phosphorylation resulted in a reduction of active p21RAS-GTP complexes in the transformed cells, in diminished expression of the nuclear early response genes c-jun and c-fos, and in lower cellular proliferation rate. To further implicate p21 RAS in these functional events downstream of p210 bcr-abl tyrosine phosphorylation, we targeted G-protein function directly by limiting the availability of GTP with the inosine monophosphate dehydrogenase inhibitor, tiazofurin (TR). In p210 bcr-abl-transformed cells treated for 4 hours with TR, in which the levels of GTP were reduced by 50%, but GDP, guanosine monophosphate, and adenosine triphosphate (ATP) were unaffected, p210 bcr-abl tyrosine phosphorylation was at control levels. However, expression of c-fos and c-jun nuclear proto-oncogenes were strongly inhibited and p21 RAS activity was downregulated. These findings show that p210 bcr-abl transduces proliferative signals, in part, through downstream activation of p21 RAS. Furthermore, p21 RAS activity is linked to pathways that regulate c-jun and c-fos expression.
Mandanas
R A
RA
Division of Hematology-Oncology, Walther Oncology Center, Indianapolis, IN.
Leibowitz
D S
DS
Gharehbaghi
K
K
Tauchi
T
T
Burgess
G S
GS
Miyazawa
K
K
Jayaram
H N
HN
Boswell
H S
HS
D000818:Animals; D016227:Benzoquinones; D015152:Blotting, Northern; D001853:Bone Marrow; D002460:Cell Line; D002471:Cell Transformation, Neoplastic; D015342:DNA Probes; D016044:Fusion Proteins, bcr-abl / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D011905:Genes, ras; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D047029:Lactams, Macrocyclic; D051379:Mice; D009857:Oncogenes; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000302:isolation & purification / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011809:Quinones / Q000494:pharmacology; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D017828:Rifabutin / Q000031:analogs & derivatives; D015398:Signal Transduction; D014158:Transcription, Genetic
D016227:Benzoquinones; D015342:DNA Probes; D047029:Lactams, Macrocyclic; D011809:Quinones; D006153:Guanosine Diphosphate; D017828:Rifabutin; D012313:RNA; C020754:herbimycin; D006160:Guanosine Triphosphate; D011505:Protein-Tyrosine Kinases; D016044:Fusion Proteins, bcr-abl; D016283:Proto-Oncogene Proteins p21(ras)