|
|
 Previous Article | Table of Contents
Blood, Vol. 92 No. 12 (December 15), 1998:
pp. 4880-4881
CORRESPONDENCE
Expression of p53, Bcl-2, and Bax in CD34+ Cells
Recovering After Chemotherapy
 |
LETTER |
To the Editor:
We read with interest the report of Wlodarski et al1
published in a recent issue of Blood concerning the role of p53
in the recovery of hematopoieis after cytotoxic treatment. The
investigators used as an experimental model p53 wild-type and p53
knock-out mice treated with 5-fluorouracil (5-FU). They showed that
bone marrow (BM) mononuclear cells recovering in p53 knock-out mice contain a higher number of both primitive and multipotential
progenitors than BM cells recovering in p53 wild-type mice. Moreover,
p53 knock-out progenitors are responsible for the long-term engraftment of hematopoiesis when transplanted in lethally irradiated recipients. Thus, these data undoubtedly indicate that p53 is involved in the
induction of apoptosis of hematopoietic progenitors previously exposed
to DNA-damaging drugs.
To our knowledge, no data have yet been reported about the modulation
of the tumor-suppressor gene p53 or of its target genes in human
hematopoietic cells after in vivo chemotherapy administration. To this
purpose, we used flow cytometry to evaluate the expression of p53 and
of two apoptosis-related genes, bcl-2 and bax, in
CD34+ cells recovering after chemotherapy. We evaluated 5 patients affected by non-Hodgkin's lymphomas, candidates for
peripheral blood (PB) stem cell collection and transplantation. All
patients received cyclophosphamide (7 g/m2) and, from day
2 after chemotherapy, granulocyte colony-stimulating factor (G-CSF; 5 µg/kg). In all patients, BM and PB samples were collected on day 12 after chemotherapy. Data obtained in patients recovering from
chemotherapy were then compared with those obtained in
CD34+ cells isolated from normal BM and PB. All of the
samples were collected after informed consent was obtained.
CD34+ cells were isolated from mononuclear cell fractions
by the miniMACS starting kit and the CD34 multisort kit (Miltenyi Biotec Inc, Auburn, CA), according to the manufacturer. The purity of
immunoselected CD34+ cells was evaluated as previously
reported,2 and in all cases exceeded 90%. For the
measurement of intracellular antigens, immunoselected CD34+
cells were fixed and permeabilized by the Ortho Permeafix TM solution
(Ortho Diagnostic System, Raritan, NJ); cells were then incubated with
the following unconjugated monoclonal antibodies (MoAbs):
anti-wild-type p53 (Ab1 clone; Calbiochem, Cambridge, MA) and
anti-Bcl-2 (Ab2 clone; Calbiochem) or with the anti-Bax rabbit
polyclonal antibody (P-19 clone; Santa Cruz Biotechnology, Santa Cruz,
CA) for 1 hour at 4°C. Control samples were incubated in
the same experimental conditions with isotype-matched irrelevant unconjugated MoAb or with rabbit preimmune serum. After washing, cells
were further incubated with fluorescein isothiocyanate
(FITC)-conjugated goat antimouse MoAb (Ortho) or with FITC-conjugated
goat antirabbit Igs (Dako A/S, Glostrup, Denmark) for 1 hour at 4°C,
at 1:20 and 1:1,000 final dilution, respectively. All samples were
analyzed with a FACScan flow cytometer (Becton Dickinson, Mountain
View, CA) equipped with an argon laser emitting at 488 nm.
Results were expressed as the mean fluorescence intensity ratio
(MFI-R), which is calculated as the ratio between the MFI of test curve
and the MFI of control curve.
In addition, CD34+ cell samples were evaluated for the
content of apoptotic cells by in situ end-labeling of DNA strand breaks with the TdT-mediated biotin-nick-end-labeling (TUNEL), as previously described.3 The slides were counterstained with hematoxylin and 100 consecutive cells in 3 or more fields were counted. Three different experiments were performed. The statistical analysis of data
obtained was performed using the Mann Whitney test for unpaired data.
Results of flow cytometry analysis are shown in Fig
1. It emerged that p53, undetectable in
normal BM and PB CD34+ cells, was expressed in BM but not
in PB progenitors recovering after chemotherapy. Moreover, in BM
progenitors, the induction of p53 was accompained by a significant
reduction of the expression of Bcl-2 and by the appearance of Bax. On
the contrary, in PB CD34+ cells recovering after
chemotherapy, no differences in the protein levels of Bcl-2 and Bax
were detected when compared with normal PB CD34+ cells. In
addition, in normal PB CD34+ progenitors, Bax was expressed
at a higher level than in normal BM CD34+
progenitors, but its expression did not further increase after chemotherapy. The upregulation of Bax has been reported in progenitor B
cells detached from the stromal layer.4 Similarly, we
can hypothesize that Bax is expressed in
CD34+ progenitors released from the BM microenvironment to
the PB. Data obtained by flow cytometry were consistant with the
content of apoptotic cells evaluated by the TUNEL method (Table
1). Actually, BM CD34+ cells
recovering after chemotherapy showed a percentage of apoptotic cells
significantly higher than that observed in the other samples evaluated.
On the contrary, PB CD34+ progenitors recovering after
chemotherapy showed few apoptotic cells. Finally, according to their
increased Bax expression, normal PB CD34+ cells
showed a higher content of apoptotic cells than did normal BM
progenitors and PB progenitors isolated after chemotherapy.
View larger version (27K):
[in this window]
[in a new window]
| Fig 1.
Expression of p53, Bcl-2, and Bax in CD34+
cells isolated from BM and PB of normal individuals and of patients
treated with cyclophosphamide. Results are expressed as the MFI-R (mean
value ± SD) of five experiments.
|
|
View this table:
[in this window]
[in a new window]
|
Table 1.
Mean Percentage of Apoptotic Cells in BM and PB
CD34+ Cells Isolated From Normal Individuals and From
Patients Recovering After Chemotherapy
|
|
Our observations agree with those of Wlodarski et al1 and
confirm that p53 is induced in vivo in hematopoietic CD34+
cells after chemotherapy. Moreover, the differences that we found between BM and PB CD34+ cells suggest that hematopoietic
progenitors that recover from the cytostatic damage (p53 and Bax
negative) leave BM, whereas those still suffering from the toxic insult
(p53 and Bax positive) are retained in the BM microenvironment.
Interestingly, Wlodarski et al1 reported in BM cells from
p53 knock-out mice an increased expression of cyclin-dependent kinases
inhibitor (CDK-I) p16INK4A and suggested that other
regulatory mechanisms involving this CDK-I may compensate for the loss
of p53. In a previous study, we demonstrated that the cell cycle arrest
of CD34+ progenitors mobilized by chemotherapy is
associated with the expression of the CDK-I
p15INK4B.5 Thus, these observations suggest
that p53 and CDK-Is (p15INK4B or p16INK4A)
operate differently depending on whether cells are
induced to or recover from apoptosis, respectively.
In conclusion, our data further support the possibility that
hematopoietic recovery could be improved by interfering with the
expression of p53 gene.
Luciana Teofili
Sergio Rutella
Rossana Putzulu
Carlo Rumi
Giuseppe Leone
Department of Hematology
Luigi Maria Larocca
Nicola Maggiano
Department of Pathology
Catholic University
Rome,
Italy
| |
ACKNOWLEDGMENT |
This work has been supported by a grant from A.I.R.C. (Associazione
Italiana per la Ricerca sul Cancro).
| |
REFERENCES |
1.
Wlodarski P, Wasik M, Ratajczak MZ, Sevignani C, Hoser G, Kawiak J, Gerwitz AM, Calabretta B, Skorski T:
Role of p53 in hematopoietic recovery after cytotoxic treatment.
Blood
91:2998, 1998[Abstract/Free Full Text]
2.
Rumi C, Rutella S, Teofili L, Etuk B, Ortu La Barbera E, Voso MT, Leone G:
RhG-CSF mobilized CD34+ peripheral blood progenitors are myeloperoxidase-negative and non-cycling irrespectively of CD33 or CD13 coexpression.
Exp Hematol
25:246, 1997[Medline]
[Order article via Infotrieve]
3.
Larocca LM, Ranelletti FO, Maggiano N, Rutella S, Ortu La Barbera E, Rumi C, Serra F, Voso MT, Piantelli M, Teofili L, Leone G:
Differential sensitivity of leukemic and normal hematopoietic progenitors to the killing effect of hyperthermia and quercetin used in combination: Role of heat-shock protein-70.
Int J Cancer
73:75, 1997[Medline]
[Order article via Infotrieve]
4.
Gibson LF, Piktel D, Narayanan R, Nunez G, Landreth KS:
Stromal cells regulate bcl-2 and bax expression in pro-B cells.
Exp Hematol
24:628, 1996[Medline]
[Order article via Infotrieve]
5. Teofili L, Rutella S, Ranelletti FO, Rumi C, Ortu La Barbera E,
Leone G, Larocca LM: Expression of P15INK4B in normal
hematopoiesis. Exp Hematol (in press)
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
S. Yada, N. Takamura, K. Inagaki-Ohara, M. K. O'Leary, C. Wasem, T. Brunner, D. R. Green, T. Lin, and M. J. Pinkoski
The Role of p53 and Fas in a Model of Acute Murine Graft-versus-Host Disease
J. Immunol.,
February 1, 2005;
174(3):
1291 - 1297.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. A. Sohn, R. Bansal, G. H. Su, K. M. Murphy, and S. E. Kern
High-throughput measurement of the Tp53 response to anticancer drugs and random compounds using a stably integrated Tp53-responsive luciferase reporter
Carcinogenesis,
June 1, 2002;
23(6):
949 - 958.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Stahnke, S. Fulda, C. Friesen, G. Strau{beta}, and K.-M. Debatin
Activation of apoptosis pathways in peripheral blood lymphocytes by in vivo chemotherapy
Blood,
November 15, 2001;
98(10):
3066 - 3073.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. I. Pestina, J. L. Cleveland, C. Yang, G. P. Zambetti, and C. W. Jackson
Mpl ligand prevents lethal myelosuppression by inhibiting p53-dependent apoptosis
Blood,
October 1, 2001;
98(7):
2084 - 2090.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
