Expression and physiologic significance of Kit ligand and stem cell tyrosine kinase-1 receptor ligand in normal human CD34+, c-Kit+ marrow cells.
To determine the potential role of autocrine growth factor production in regulating primitive human hematopoietic cell development, we examined highly purified CD34+, c-Kit+ marrow mononuclear cells for expression of c-Kit ligand (KL) and stem cell tyrosine kinase 1 (stk1) ligand (STK1-L). Normal marrow mononuclear cells coexpressing CD34 and c-Kit were isolated by a combination of immunomagnetic bead isolation and fluorescence-activated cell sorting. Purified cells were then screened for expression of KL and stk1-L mRNA using a sensitive reverse transcription-polymerase chain reaction method. Using this approach, expression of both cytokine genes at the mRNA level was found in this highly enriched cell population. We then examined the functional significance of these mRNAs by inhibiting their expression with antisense (AS) oligodeoxynucleotides (ODN). In comparison to untreated or control ODN treated cells, inhibition of KL led to a 70% and 89% inhibition in burst-forming unit-erythroid (BFU-E) and colony-forming unit-Mix (CFU-Mix) colonies but had no significant effect on CFU-granulocyte-macrophage (CFU-GM) cloning efficiency. In contrast, inhibition of STK1-L alone had no effect on colony formation. However, when STK1-L AS ODN was combined with KL AS ODN, additive inhibition of CFU-GM and CFU-MIX but not of BFU-E colonies was observed. These findings, along with those of our previous studies showing inhibition of primitive hematopoietic cell growth with antisense ODN directed towards the stk1 receptor, suggest the possibility that both receptor/ligand axes regulate primitive hematopoietic cell growth via an autocrine growth loop.
Ratajczak
M Z
MZ
Department of Pathology, University of Pennsylvania School of Medicine, Philadelphia 19104, USA.
Kuczynski
W I
WI
Sokol
D L
DL
Moore
J S
JS
Pletcher
C H
CH
Gewirtz
A M
AM
D015703:Antigens, CD / Q000032:analysis; D018952:Antigens, CD34; D001483:Base Sequence; D001854:Bone Marrow Cells; D002455:Cell Division; D002469:Cell Separation; D005434:Flow Cytometry; D016298:Hematopoietic Cell Growth Factors / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D018189:Immunomagnetic Separation; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D011518:Proto-Oncogene Proteins / Q000502:physiology; D019009:Proto-Oncogene Proteins c-kit; D012333:RNA, Messenger / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D020794:Receptor Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D016184:Receptors, Colony-Stimulating Factor / Q000502:physiology; D019089:Stem Cell Factor; D051941:fms-Like Tyrosine Kinase 3
D015703:Antigens, CD; D018952:Antigens, CD34; D016298:Hematopoietic Cell Growth Factors; D008565:Membrane Proteins; D016376:Oligonucleotides, Antisense; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D016184:Receptors, Colony-Stimulating Factor; D019089:Stem Cell Factor; C084881:flt3 ligand protein; C497970:FLT3 protein, human; D011505:Protein-Tyrosine Kinases; D019009:Proto-Oncogene Proteins c-kit; D020794:Receptor Protein-Tyrosine Kinases; D051941:fms-Like Tyrosine Kinase 3