Constitutive and stimulated production of VEGF by human megakaryoblastic cell lines: effect on proliferation and signaling pathway.
Release of vascular endothelial growth factor (VEGF) and other candidate angiogenic factors such as basic fibroblast growth factor and transforming growth factor beta, may play a role in sustaining neoplastic cell proliferation and tumor growth. We evaluated VEGF expression and synthesis in the two erythromegakaryocytic cell lines B1647, HEL and one megakaryocytic cell line MO7 expressing erythroid markers. In this study RT-PCR was performed to evaluate VEGF expression and that of its receptor KDR; VEGF production was assayed by Elisa test and western blot analysis; sensitivity to VEGF was tested by thymidine incorporation. VEGF and its receptor KDR were expressed in B1647 and HEL, both as mRNAs and as proteins, while only KDR transcript was found in MO7 cells. Only B1647 and HEL cells showed a strong spontaneous proliferating activity. In fact, measurable amounts of VEGF were present in the unstimulated cell medium, thus suggesting an autocrine production of VEGF by B1647 and HEL cells, but not by MO7, which was inhibited in mRNA-silencing conditions. This production could not be further boosted by other growth factors, whereas it was inhibited by TGF-beta1. Finally, analysis of Shc signal transduction proteins following stimulation with VEGF indicated that only p46 was tyrosine phosphorylated. These data indicate that leukemic cells may be capable of autocrine production of VEGF which, in turn, maintains cell proliferation, possibly mediated by Shc p46 phosphorylation.
Bonsi
L
L
Department of Histology, Embryology and Applied Biology, University of Bologna, Italy. bonsi@alma.unibo.it
Pierdomenico
L
L
Biscardi
M
M
Marchionni
C
C
Gavazzi
S
S
Fossati
V
V
Ghinassi
B
B
Alviano
F
F
Rondelli
D
D
Franchina
M
M
Bagnara
G P
GP
Grossi
A
A
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000378:metabolism; D015415:Biomarkers / Q000097:blood / Q000378:metabolism; D015153:Blotting, Western; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008533:Megakaryocytes / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055765:Shc Signaling Adaptor Proteins; D015398:Signal Transduction / Q000187:drug effects*; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D053773:Transforming Growth Factor beta1; D014443:Tyrosine / Q000737:chemistry; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis* / Q000378:metabolism / Q000494:pharmacology*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D015415:Biomarkers; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C528024:SHC1 protein, human; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; C508960:TGFB1 protein, human; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D042461:Vascular Endothelial Growth Factor A; D014443:Tyrosine; D040301:Vascular Endothelial Growth Factor Receptor-2