High cyclin-dependent kinase inhibitors in Bcl-2 and Bcl-xL-expressing CD34+-proliferating haematopoietic progenitors.
We have previously described the isolation of primitive, slow-proliferating progenitors from normal, circulating CD34+ cells by using the fluorescent dye 5-6-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE). CFDA-SE(bright) (primitive) and CFDA-SE(dim) (differentiating) cells were isolated following cytokine stimulation on the basis of their different proliferation rates. In the present work we analysed the expression levels of a number of proteins involved with differentiation, proliferation and survival/apoptosis in CFDA-SE(bright)/CD34+/slow-proliferating cells that were previously defined as progenitors capable of differentiating into different lineages. The aim of this work was to gain a better understanding of our model system in order to define some of the important parameters that regulate differentiation in haematopoietic progenitors. GATA-1 and PU.1 RNA levels were similar in freshly isolated (d 0) CD34+ and in CFDA-SE(bright) (bright) cells, whereas they increased in CFDA-SE(dim) (dim) cells. Accordingly, Nm23 was expressed at higher levels in bright cells. Moreover, bright cells had higher p21WAF1/CIP1, p27KIP1 and p16Ink4 protein levels than dim cells. Consistently, Cdc2 and Cdk2 kinase activity was much higher in the dim than in the slower proliferating bright cells. C-myc and p53 levels were higher in bright cells than in d 0 CD34+ and dim cells, and so was Bcl-xL, which followed the trend we have previously described for Bcl-2. Thus, bright cells, despite having a higher proliferation rate than the starting d 0 CD34+ population, have strikingly elevated levels of cyclin-dependent kinase inhibitors, which are likely to also act as inhibitors of differentiation.
Marone
M
M
Department of Obstetrics and Gynecology and Department of Haematology, Catholic University, Rome, Italy. maria.marone@tiscalinet.it
Pierelli
L
L
Mozzetti
S
S
Masciullo
V
V
Bonanno
G
G
Morosetti
R
R
Rutella
S
S
Battaglia
A
A
Rumi
C
C
Mancuso
S
S
Leone
G
G
Giordano
A
A
Scambia
G
G
D018952:Antigens, CD34; D017209:Apoptosis; D015415:Biomarkers / Q000032:analysis; D015153:Blotting, Western; D018797:Cell Cycle Proteins; D002454:Cell Differentiation; D002455:Cell Division; D002470:Cell Survival; D002478:Cells, Cultured; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000032:analysis; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors*; D016213:Cyclins / Q000032:analysis; D004791:Enzyme Inhibitors / Q000032:analysis*; D005260:Female; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000032:analysis; D010051:Ovarian Neoplasms / Q000473:pathology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016159:Tumor Suppressor Protein p53 / Q000032:analysis; D025521:Tumor Suppressor Proteins; D051020:bcl-X Protein
D018952:Antigens, CD34; C494811:BCL2L1 protein, human; D015415:Biomarkers; C493956:CDKN1A protein, human; D018797:Cell Cycle Proteins; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D008869:Microtubule-Associated Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; D051020:bcl-X Protein; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases