Quantitative analysis of Fas and bcl-2 expression in hematopoietic precursors.
OBJECTIVE
We investigated the expression of bcl-2 and CD95 (Apo1-/Fas) on CD34+ cells obtained from bone marrow (BM), mobilized peripheral blood (MPB), and umbilical cord blood (UCB) samples. The expression of bcl-2 and Fas was then compared with that of other markers usually associated with immaturity; functional tests using the agonistic antibody anti- Fas CH11 were also carried out.


METHODS
The analysis was performed by flow cytometry on purified CD34+ cells in a three (CD95 PE, CD34 APC and CD71 FITC) and in a four (CD38 PE, HLA-DR PerCP, CD34 APC and bcl-2 FITC) fluorescence assay.


RESULTS
The results were expressed as mean fluorescence index (MFI); bcl-2 expression was significantly higher (p < 0.001) in BM (3.73 +/- 0.63) than in MPB (2.47 +/- 0.39) and UCB (2.38 +/- 0.58); Fas was significantly less expressed (p < 0.001) in UCB (1.27 +/- 0.78) than in MBP (3.63 +/- 2.19) and BM (4.56 +/- 1.69). CD34 expression was significantly (p < 0.001) brighter in UCB compared to in MBP and BM, while CD38 and CD71 were significantly (p = 0.005 and p < 0.001, respectively) more expressed in BM than in MPB and UCB. Fas values were directly correlated to CD38; both Fas and bcl-2 were directly related to CD71 and inversely to CD34. Culture assays showed that hematopoietic precursor cells from BM, MPB and UCB had a low susceptibility to undergo Fas-mediated apoptosis.


CONCLUSIONS
In conclusion, bcl-2 and Fas are less expressed in UCB than in MPB and BM; early hematopoietic precursor cells are relatively resistant to CD95-triggered apoptosis; the observed correlation between Fas/bcl-2 and markers of immaturity suggests that they may be determinants of commitment in early hematopoietic precursors.
Cattedra di Ematologia, Università Tor Vergata, Divisione di Ematologia, Ospedale S. Eugenio, piazzale Umanesimo 10, 00144 Rome, Italy. lucamaurillo@tiscalinet.it
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Maurillo
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Venditti
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Buccisano
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Battaglia
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Santinelli
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Caravita
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Epiceno
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Del Moro
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Tamburini
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Picardi
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Catalano
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Bruno
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Amadori
D000328:Adult; D018952:Antigens, CD34 / Q000032:analysis; D001773:Blood Cells / Q000276:immunology / Q000378:metabolism; D001854:Bone Marrow Cells / Q000276:immunology / Q000378:metabolism; D005312:Fetal Blood / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D006412:Hematopoietic Stem Cells / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D007945:Leukemia, Lymphoid / Q000473:pathology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis* / Q000378:metabolism; D019014:fas Receptor / Q000096:biosynthesis* / Q000378:metabolism
D018952:Antigens, CD34; D019253:Proto-Oncogene Proteins c-bcl-2; D019014:fas Receptor