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Prepublished online as a Blood First Edition Paper on October 3, 2002; DOI 10.1182/blood-2002-05-1374.
NEOPLASIA
From the Comprehensive Cancer Center and
Department of Laboratory Medicine, University of California, San
Francisco; Harvard Institutes of Medicine, Harvard Medical School,
Boston, MA; Division of Hematology/Oncology, Cedars-Sinai Medical
Center, University of California, Los Angeles School of Medicine;
Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas
Jefferson University, Philadelphia, PA; and Advanced Clinical Research
Center, Institute of Medical Science, University of Tokyo,
Japan.
CCAAT/enhancer binding proteins (C/EBPs) are a family of factors
that regulate cell growth and differentiation. These factors, particularly C/EBP CCAAT/enhancer binding proteins (C/EBPs) are a
family of proteins that play important roles in the development and
differentiation of many cell types, including granulocytes. C/EBP Given the part that C/EBP APL represents approximately 10% of human AMLs.16 This
leukemia is now defined by the presence of a t(15;17)(q22;q12)
chromosomal translocation, creating a PML-RARA fusion gene
(or rare variants that also result in fusions to
RARA).17 In 1987, all-trans retinoic acid (tRA) was discovered to be able to induce remissions in
APL18 by causing the leukemic cells to differentiate into
mature neutrophils.19 Subsequently, the combination of tRA
with chemotherapy has led to a marked increase in long-term survival
for patients with APL; tRA treatment represents a paradigm for
molecularly targeted therapy that restores normal behavior to malignant
cells.20
The promyelocytic leukemia-retinoic acid receptor Treatment of APL cells with pharmacologic doses of tRA results in the
restoration of PML nuclear bodies and transcriptional activation of
retinoic acid (RA)-responsive genes. Of these effects, transcriptional
activation appears central because differentiation takes place even
when PML nuclear bodies are not reassembled.23 C/EBPs
might be important targets of tRA. Consistent with this idea, C/EBPs
can arrest cell growth and induce differentiation of a variety of cell
types, including myeloid cell lines.8,24-28 Furthermore,
PML-RAR We assessed whether either C/EBP Plasmids
Cell culture
Retroviral transduction BOSC23 cells were transfected with retroviral constructs as previously described.37 Retroviral supernatants were filtered through 0.45-µm filters and stored at 80°C. FDC-P1 cells
were plated at 50 000 cells per well in 24-well tissue culture plates. On each of 2 consecutive days, the cells were transduced by incubation with 2 mL viral-containing supernatant with 4 µg/mL polybrene and
centrifugation at 1100 g for 1.5 hours at room temperature. Transduction of leukemic cells was similarly performed, except that
leukemic cells were plated at 2 × 106 cells per well.
Western blot analysis Whole-cell lysates were prepared by lysing 2 × 107 cells in 600 µL 2 × sample buffer, heating at 90°C to 95°C for 5 minutes, and shearing through a 20-gauge needle. Western blot analysis was performed as previously described38 with rabbit polyclonal antibodies to C/EBP
or C/EBP (Santa Cruz Biotechnology, Santa Cruz, CA).
Analysis of growth of FDC-P1 cells After 2 rounds of infection, transduced cells were pooled and washed once with buffered saline, and GFP+ cells were sorted using the FACSVantage cell sorter (Becton Dickinson, San Jose, CA). Culture of sorted cells started at 30 000 cells. At 48-hour intervals, the medium was changed, live cells were counted with trypan blue exclusion, and the percentage of GFP+ cells was assessed by flow cytometry.Analysis of differentiation For flow immunophenotyping, anti-Gr-1-phycoerythrin and anti-Mac-1-phycoerythrin (BD Pharmingen, San Diego, CA) were used. Cells were resuspended in 100 µL buffered saline and incubated with indicated antibodies for 20 to 30 minutes on ice in the dark, then washed and resuspended in buffered saline. Stained cells were analyzed on a FACScan, and at least 10 000 events were collected for each sample. Fluorescence-activated cell sorter (FACS) data were analyzed with CELLQUEST (Becton Dickinson). Differential cell counts of Wright Giemsa-stained bone marrow smears were performed according to published guidelines.39 The guidelines for the differential counts were modified for FDC-P1 cells as described in the legend to Table 1. The guidelines were also modified for leukemic cells cultured in vitro because an atypical cell type was present; myeloid cells with atypical nuclear segmentation and basophilic cytoplasm were enumerated separately from the intermediate forms and neutrophils of normal appearance.
Mice Mice were bred and maintained at the University of California at San Francisco, and their care was in accordance with University of California at San Francisco guidelines. Leukemias from MRP8 PML-RARA40 and MRP8 PML-RARAm441 transgenic mice were maintained by serial transplantation. Female FVB/N mice, 6 to 8 weeks old, received sublethal irradiation (4.5 Gy) and intravenous injection into the lateral tail vein with either leukemic cells (1 × 106 cells per animal for serial passaging) or sorted GFP+- transduced leukemic cells (25 000 or 50 000 cells per animal).Tamoxifen treatment As previously described,42 4-hydroxytamoxifen (4-HT; Sigma, St Louis, MO) was dissolved in ethanol at 100 mg/mL, then diluted in autoclaved sunflower seed oil (Sigma) at 10 mg/mL, sonicated for approximately 20 minutes, and stored at20°C. One milligram 4-HT per mouse was injected intraperitoneally on consecutive days.
Statistical analysis Statistical analyses were performed with Excel 2000 using the Student t test, one-tailed distribution, and unequal variance.
Expression of C/EBP and C/EBP,
have important roles in myeloid differentiation. We wished to assess
whether increasing the activity of either C/EBP or C/EBP could
suppress the leukemic phenotype of murine AML cells. We initially
examined whether the C/EBPs would suppress growth and/or induce
differentiation of the FDC-P1 cell line, a nonleukemic factor-dependent line of immature myeloid cells. FDC-P1 cells were
transduced with retroviruses designed to express only GFP (the MIG
control virus), hC/EBP, or hC/EBP (Figure
1A). Immunoblots of FDC-P1 whole-cell
lysates indicated that these retroviruses drove expression of the C/EBP
proteins (Figure 1B). Compared with control, overexpression of C/EBPs
in FDC-P1 cells repressed proliferation (Figure
2A). In addition, C/EBP and C/EBP
increased expression of both Ly-6G (Gr-1) and CD11b (Mac-1), which are
surface markers of myeloid differentiation (Figure 2B). Although
C/EBP and C/EBP both induced FDC-P1 cells to partially
differentiate, C/EBP-transduced cells showed greater morphologic
change, including the appearance of cells with nuclear segmentation
(Figure 2C; Table 1). The growth suppression and partial
differentiation observed in transduced FDC-P1 cells showed that
C/EBP and C/EBP proteins were expressed and functional.
Expression of C/EBP and C/EBP to induce
differentiation of leukemias derived from transgenic mice that expressed either PML-RAR (RA-sensitive leukemia no. 1111) or PML-RARm4 (RA-resistant leukemia no. 4048.2). Freshly harvested leukemic cells were transduced with control, hC/EBP-, or
hC/EBP-containing retroviruses. In vitro, the morphology of
transduced leukemia cells was assessed by differential counts of Wright
Giemsa-stained cytospins. Overexpression of C/EBP proteins induced
partial morphologic differentiation of both RA-sensitive and
RA-resistant leukemic cells (Figure 3).
Certain aspects of these results should be noted. First, in these
experiments, very few mature neutrophils were induced by the C/EBPs.
Instead, the C/EBPs led to the appearance in culture of partially
differentiated cells, including many with nuclear segmentation. Second,
a greater number of differentiated cells was seen with the PML-RARm4
leukemia than with the PML-RAR leukemia, including the control
transduced cells. This finding is consistent with our previous
observation that leukemias induced by an MRP8 PML-RARAm4
transgene showed somewhat more differentiation in vivo than leukemias
induced by MRP8 PML-RARA.41
C/EBPs suppress the leukemic phenotype of PML-RAR and C/EBP to
limit leukemic cell growth in vivo. In addition to the RA-sensitive and
RA-resistant leukemias described in the previous paragraph, a
second RA-responsive PML-RAR leukemia was studied, no. 935. Leukemic
cells were harvested and then transduced on 2 consecutive days with
MIG, hC/EBP, or h/CEBP retroviruses. GFP+ cells were
sorted, and 25 000 or 50 000 cells were injected intravenously into
sublethally irradiated histocompatible recipient animals. Recipients of
PML-RAR leukemias transduced with C/EBP survived longer than
recipients of leukemias transduced with the MIG control (Figure
4A). C/EBP modestly prolonged survival
(mean increase in survival of 3 days, median 2.5 days, maximum 10 days;
P = .01), whereas C/EBP had a more substantial impact
on survival (mean increase in survival of 13 days, median 6 days,
maximum 59 days; P = .004). Results in Figure 4A are
pooled data from 3 independent experiments. The results of the
individual experiments are shown in a supplemental figure on the
Blood website; see the Supplemental Figure link at the top
of the online article. Of note, RA treatment of
RA-sensitive leukemias prolonged survival more than did transduction by
C/EBPs (mean increase in survival of 40 days [median 42 days] with a
5-mg 21-day tRA pellet; mean increase in survival of 74 days [median
41 days] with a 10-mg 21-day tRA pellet).41,44 The impact
of C/EBP transduction was, however, likely to be attenuated by the
presence of untransduced cells, loss of retroviral expression, and/or
down-regulation of protein expression (see below). Despite these limitations, 2 recipients of cells transduced with C/EBP had
their survival prolonged to a degree comparable to that of RA
treatment.
Consistent with previous observations, recipients of a
PML-RAR When possible, leukemic cells were harvested from moribund recipients,
and the cells were assessed for the expression of GFP (Table
2A). There appeared to be selection
against cells that expressed the MSCV-hC/EBP
PML-RAR recipients and in some C/EBP recipients, it was
important to assess whether C/EBP proteins were in fact present in the
leukemic cells. Neither C/EBP nor C/EBP proteins were detected by
Western blot analysis (data not shown). Because the C/EBPs and GFP are
encoded by a bi-cistronic transcript, the lack of C/EBP protein
expression might be due to decreased translation. In fact, initiation
of C/EBP translation can be inhibited by a short open reading frame
and spacer (uORF) upstream of the initiation codon for C/EBP. We
hypothesized that deletion of the uORF might create additional
selective pressure against the C/EBP retrovirus. Constructs
expressing rat C/EBP either with or without the uORF (rC/EBP-WT
and rC/EBP- uORF; Figure 1A) were used to test this idea. Both
constructs were able to produce C/EBP protein in FDC-P1 cells
(Figure 1B). PML-RAR leukemic cells (no. 935) were transduced with
control, rC/EBP-WT, and rC/EBP-uORF retroviruses, and 25 000
GFP+ cells were injected into sublethally irradiated
recipients. Although deletion of the uORF did not significantly
influence survival (control, n = 4, mean survival 37 days;
rC/EBP-WT, n = 6, mean survival 40 days; rC/EBP-uORF,
n = 6, mean survival 38 days), deletion of the uORF led to markedly
diminished GFP expression in the leukemias that arose (Table
2).
A tamoxifen-inducible version of C/EBP has been proposed to be a central mechanism
by which tRA causes the differentiation of APL cells. To address whether C/EBP can induce neutrophilic maturation of leukemic cells in
vivo, we used a tamoxifen-inducible form of C/EBP,
hC/EBP-ERTM. PML-RAR leukemic cells (no. 1111) were
transduced with control and C/EBP-ERTM retroviruses,
then transplanted into sublethally irradiated histocompatible mice.
Leukemias were allowed to develop in recipients of either control or
C/EBP-ERTM-transduced leukemic cells. Leukemic mice
were then treated with either vehicle or 4-HT. In contrast to findings
in the other mice, bone marrow examination revealed that 4-HT caused
neutrophilic differentiation of the leukemic cells that had been
transduced with C/EBP-ER. Morphologically, only the 4-HT-treated
C/EBP-ERTM samples showed numerous mature neutrophils
(Figure 5A). In addition, the cells
exhibited increased CD11b (Mac-1) expression (Figure 5B). Differential
counts of bone marrow smears are presented in Figure 5C. 4-HT induced
substantial neutrophilic differentiation and allowed nonmyeloid
hematopoietic cells (erythroid cells and lymphocytes) to repopulate the
bone marrow. These findings parallel the effects of RA on this mouse
model of human APL.40
Differentiation therapy of APL with tRA has transformed this leukemia into a highly curable illness. This remarkable impact of differentiation therapy on one subtype of AML has created hope that molecularly-targeted therapies can be developed for other types of AML, including those with complex karyotypic abnormalities and poor prognosis. C/EBPs are central mediators of neutrophil maturation. Our results show that C/EBPs induce differentiation of retinoic acid responsive and resistant leukemias. Moreover, in an animal model of AML, C/EBPs suppressed the leukemic phenotype. Thus, developing therapeutics that stimulate C/EBP expression and activity should extend the range of leukemias that can be treated with differentiation therapy. In U937 and 32Dcl3 cell lines, overexpression of either C/EBP Perrotti et al10 have shown that BCR-ABL inhibits C/EBP The relationships among C/EBPs, RARs, mutations that contribute to AML,
and the therapeutic effects of retinoids are complex. In APL,
PML-RAR The ability of C/EBPs to suppress the leukemic phenotype indicates that C/EBP induction may be critical to the tRA response. However, overexpression of these proteins may provide a more powerful stimulus to growth suppression and differentiation than the effect of pharmacologic tRA on C/EBPs. In other words, induction of C/EBP activity may be central to the response of APL to tRA, but may not represent the only important effect of tRA: it may be that the combination of C/EBP induction along with additional effects causes differentiation. Overexpression of C/EBPs could obviate the requirement for these additional effects of tRA by causing supraphysiologic effects on C/EBP targets. In some settings, C/EBP activity appears sufficient to induce complete
neutrophil differentiation whereas in other contexts C/EBPs induce only
partial differentiation. Expression of C/EBP C/EBPs likely suppress the leukemic phenotype through a combination of
induction of transcriptional targets and direct inhibition of cell
cycle progression. Target genes of C/EBP
We thank H. Jeffrey Lawrence and Nancy Berliner for helpful discussions and Adam Olshen for advice on statistical analyses. We also acknowledge the continuing support of J. Michael Bishop, Frank McCormick, Kevin Shannon, and Daphne Haas-Kogan.
Submitted May 10, 2002; accepted September 14, 2002.
Prepublished online as Blood First Edition Paper, October 3, 2002; DOI 10.1182/blood-2002-05-1374.
Supported by the 32nd Edward Mallinckrodt Junior Scholar award (S.C.K.) and by a Burroughs Wellcome Fund Career Award (S.C.K.). Additional support was provided by grants K08-CA75986, U01-CA84221, and R01-CA88046 from the National Institutes of Health and by the Parker Hughes Trust Fund.
The online version of the article contains a data supplement.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Scott C. Kogan, University of California San Francisco Comprehensive Cancer Center, 2340 Sutter St, Room N-361, Box 0128, San Francisco, CA 94143-0128; e-mail: skogan{at}cc.ucsf.edu.
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Top
Abstract
Introduction
.
retroviral constructs
, or