(Received for publication, December 29, 1995; and in revised form, February 23, 1996)
From the
It has recently been shown that the alteration of the cell-redox
status affects the transcription factor expression and activity.
Dithiocarbamates (DTCs) are potent antioxidant agents that can switch
the expression of genes dependent on the activation of the
transcription factors AP-1 and NFB. In this study, we show that
these agents triggered the expression of genes involved in myeloid
differentiation of the promonocytic U-937 cell line. DTCs promoted
differentiation-associated changes that included the surface
up-regulation of
2-integrins (CD11a-c/CD18), cell growth arrest
concomitant with transferrin receptor (CD71) down-modulation, induction
of the nonspecific esterase enzyme, and a rapid drop in the mRNA levels
of c-myc. A further analysis, focused on the molecular
mechanisms leading to the activation of CD11c expression, revealed that
the pyrrolidine derivative of DTC (PDTC) increased CD11c mRNA
levels and augmented its gene promoter activity. Transfection
experiments with reporter constructs harboring different promoter
regions of CD11c gene, indicated the presence of a functional
DTC-responsive region located between positions -160 and +40
of the promoter. Gel retardation assays revealed that the PDTC-induced
DNA-protein complexes were restricted to members of the Fos and Jun
families that bound to an AP-1 site located at position -60 from
the transcription start site. A role for this site was confirmed by in vitro mutagenesis experiments that indicated the functional
importance of this site for the CD11c gene transcriptional
activation in response to PDTC. The effect of DTCs on myeloid cell
differentiation supports a possible role for these agents in the
therapy of some bone marrow-derived malignancies.
Differentiation of myeloid cells is a complex process that
involves the regulated expression of an array of genes responsible for
a series of cellular changes that drive precursor cells to different
functional and phenotypic cell stages. Several leukemic cell lines
arrested at different steps of the myeloid differentiation program,
such as the human U-937 and HL-60 cell lines
(Sundström and Nilsson, 1976; Collins, 1987), have
widely been used to dissect the molecular events accompanying this
process. A variety of differentiation inducers such as phorbol esters,
1,25-dihydroxyvitamin D, interferon-
, and tumor
necrosis factor-
have been employed to drive these leukemic cell
lines toward the macrophage differentiation pathways (Hass et
al., 1989; Dood et al., 1983; Ralph et al.,
1983; Schütze et al., 1988). During this
process, the expression of adhesion molecules implicated in
intercellular and cellular-extracellular matrix interactions is tightly
regulated (Hynes, 1992). Thus, the expression of different members of
adhesion receptors, such as the components of the
2 leukocyte
integrins subfamily LFA-1 (CD11a/CD18;
L/
2), Mac-1
(CD11b/CD18;
M/
2), and p150,95 (CD11c/CD18;
X/
2),
together with the member of the immunoglobulin superfamily ICAM-1
(CD54), are up-regulated, whereas VLA-4 (CD49/CD29,
4
1), a
member of the
1 integrin subfamily, is typically down-regulated
during differentiation (Miller et al., 1986; Dustin et
al., 1986; Ferreira et al., 1991). Other cell surface
molecules such as the transferrin receptor (CD71) are also
down-modulated (Hass et al., 1989). Additional changes
commonly linked with myeloid differentiation and often interrelated are
cell growth arrest and changes in the expression of the c-myc proto-oncogene. Thus, the HL-60 and U-937 cell lines exhibit high
basal levels of c-myc which are thought to be involved in the
high proliferation rate of these cells (Collins, 1987; Cotter et
al., 1994). A drop in the c-myc levels, accompanied by
cell growth arrest, takes place when cells are induced to differentiate
with a variety of agents, which appears to occur in normal bone marrow
myeloid progenitors undergoing terminal differentiation (Collins, 1987;
Rius et al., 1990).
Recently, reactive oxygen intermediates
(ROIs) ()such as H
O
, superoxide
(O
), and hydroxyl radical
(OH
), have been described as common signal
transduction mediators in a number of activation pathways. The
involvement of these intermediates in such processes has been analyzed
on the basis of their effects on the activities of the transcription
factors NF
B and AP-1. Thus, NF
B behaves as an
oxidative-stress responsive factor that can be directly activated by
H
O
(Schreck et al., 1991; Meyer et
al., 1993) or through stimuli that increase intracellular ROIs
levels (Schreck et al., 1992b). These prooxidant stimuli can
also induce AP-1 activation as in the case of UV light and
H
O
(Devary et al., 1991),
proinflammatory cytokines (Brenner et al., 1989;
Muñoz et al., 1992) and
-radiation
(Datta et al., 1992). Strikingly, whereas the activation of
NF
B can be prevented by several antioxidant compounds with ROIs
scavenging properties, including DTCs (Meyer et al., 1993;
Schenk et al., 1994; Schreck et al., 1992a; Staal et al., 1990), these agents per se induce both AP-1
DNA binding activity and AP-1-dependent transcriptional activation by
mechanisms that involve de novo transcription of c-fos and c-jun (Meyer et al., 1993; Schenk et
al., 1994). An additional redox mechanism that modulates AP-1
activity is based on the presence of a critical redox-sensitive
cysteine residue that regulates the DNA binding of Fos and Jun proteins
(Abate et al., 1990). Strikingly, AP-1 is activated early on
when an antioxidative state is induced in cells under hypoxic
conditions (Yao et al., 1994). Hence, AP-1 can be activated by
signals generated under prooxidant and antioxidant conditions.
Cellular differentiation is mainly regulated by selective expression
of genes largely controlled at the transcriptional level. Since AP-1
can behave as an antioxidant-responsive transcription factor and
exogenous antioxidants can potentially switch on genes through the
activation of this transcription factor, we analyzed herein whether the
DTCs affect the expression of genes involved in myeloid
differentiation. Interestingly, we found that DTCs trigger the
expression of myeloid differentiation antigens, as well as other
changes associated to the differentiation of U-937 cells. To
characterize the molecular mechanisms involved in this process, we
analyzed the effect of these agents on the transcription of p150,95
-subunit (CD11c) gene. This analysis indicated that
DTCs stimulate CD11c gene promoter and identified the
transcription factor AP-1 as a central target implicated in this
activation.
Flow cytometry analysis was performed as
described previously (Postigo et al., 1991). Briefly, after
different treatments, cells were collected by centrifugation,
resuspended in phosphate-buffered saline, and incubated for 30 min with
100 µl (1 µg) of purified RR1/1 or 100 µl of the hybridoma
culture supernatants indicated above. Antibodies were incubated in the
presence of 50 µg/ml -globulin to prevent binding through the
Fc portion of the antibodies. Cells were washed, and bound antibodies
were detected using fluorescein isothiocyanate-conjugated rabbit
F(ab)`2 anti-mouse IgG (Dako A/S, Denmark), and finally resuspended in
300 µl of propidium iodide (2 ng/µl). The supernatant from the
myeloma P3X63 was included as a negative control. Samples were analyzed
by flow cytometry in a fluorescein-activated cell sorter
cytofluorometer (Becton Dickinson, Mountain View, CA). Analysis was
performed over viable cells (typically higher than 95%) as determined
by staining with the fluorochrome propidium iodide. Cell proliferation
was determined by counting in a hematocytometer the number of viable
cells by trypan blue exclusion.
For cytochemical staining, 10 cells were centrifuged in a Cytospin (Shandon, Shandon Southern
Products Ltd.) over microscope slides and stained for the detection of
the nonspecific esterase (NSE) following the instructions of a NSE kit
(Sigma). Cell preparations were observed in a Nikon Labophot-2
photomicroscope, and more than 300 cells were counted to score the
proportion of strongly positive stained cells.
For
transfection experiments, a total of 35 10
U-937
cells were electroporated with 50 µg of the different constructs,
as described previously (López-Cabrera et
al., 1993), and cultured either in the absence or presence of
PDTC, 50 µM, or PMA, 20 ng/ml. After 13 h, cells were
collected by centrifugation, and luciferase activity was quantified
following instructions described in a luciferase assay kit (Promega,
Madison, WI). To determine transfection efficiency, 12 µg of
pCMV
-gal (Clontech Laboratories Inc., Palo Alto, CA), which
contains the cytomegalovirus promoter upstream to the
-galactosidase gene, were included in each transfection.
-Galactosidase activity was measured following the protocol
previously described (Sambrook et al., 1989).
Oligonucleotides (and their complementaries) from the CD11c gene promoter used as probes in EMSAs contained the following sequences: 5`-GCCCCCTCTGACTCATGCTGAC4-3` (nucleotides -68 to -46) (probe A); 5`-GACTCCGGTTGGGGGGTGGGGGCGTGTGGGAGCCGAGC-3` (nucleotides -136 to -99) (probe B); 5`-GCGTACTCTGCCCGCCCCCTCTGACTC-3` (nucleotides -81 to -55) (probe C); 5`-TCCTTCCCCTGGCCACCTCTCTGCCCACTTG-3` (nucleotides -39 to -8) (probe D); 5`-TGCTGACAATCTTCTTCCTTCCCCTGGCCAC-3` (nucleotides -54 to -23) (probe E); 5`-TCTGCCCACTTGCTTCCTCAGTACCTTGGT-3` (nucleotides -19 to +11) (probe F); and 5`-GGGAGCCGAGCCTGTCCTCGGATCAGTTG-3` (nucleotides -109 to -81) (probe G). Complementary double-stranded oligonucleotides were annealed and labeled using avian myeloblastosis virus reverse transcriptase.
Figure 1: Flow cytometry profiles of differentiation markers induced by dithiocarbamates. A, surface expression of the antigens indicated was analyzed by flow cytometry on either untreated U-937 cells or treated for 24 h with PMA, 20 ng/ml, or PDTC, 50 µM. B, effects of DDTC and disulfiram. Both DTCs were incubated for 24 h at doses of 50 and 20 µM, respectively. The monoclonal antibodies used were TS1/11 (anti-CD11a), Bear-1 (anti-CD11b), HC1/1 (anti-CD11c), RR1/1 (anti-CD54), HP2/4 (anti-CD49d), and FG2/12 (anti-CD71). Dotted and solid lines indicate the profiles of untreated and treated cells, respectively.
Since cell surface
changes induced by dithiocarbamates in U-937 and HL-60 cells were
characteristic of differentiation processes, we performed experiments
directed to determine whether DTCs exerted additional changes
associated to the myeloid differentiation program using PDTC. The
appearance of the acid -naphthyl acetate esterase (nonspecific
esterase) enzymatic activity is a typical marker of myeloid
differentiation toward monocyte-macrophages cells induced by PMA
(Rovera et al., 1979; Collins, 1987). Cytochemical experiments
performed in U-937 revealed that PDTC significantly induced the
expression of NSE at 24 h (Fig. 2A), although to lower
extent than that elicited by PMA (data not shown). At this time,
treatment with the antioxidant produced cell growth inhibition at doses
as low as 20 µM, being maximal with concentrations ranging
from 35 to 50 µM (Fig. 2A). Additionally,
dot blot experiments revealed that the levels of c-myc RNA
markedly declined after 6 h of treatment with 100 µM of
PDTC and remained at undetectable levels by 16 h, as occurred with PMA (Fig. 2B). At lower doses of DTC, c-myc levels
were also down-modulated after 6 h, and later reinduced (data not
shown). Altogether, these results demonstrate that PDTC not only
induces cell surface differentiation markers but also affects the
proliferation of U-937 cells as well as other
differentiation-associated events.
Figure 2:
Effects of PDTC on cell growth,
nonspecific esterase staining, and c-myc RNA levels in U-937
cells. A, cells were treated with different doses of PDTC for
24 h and processed for -naphthyl acetate esterase (NSE)
staining. Percents of strongly stained cells and cell growth are
represented. Cell number was determined by seeding 3
10
cells in the presence and the absence of PDTC at the
concentrations indicated. After 24 h, cells were counted in a
hematocytometer. Cell viability, determined by trypan blue exclusion,
was higher than 95% for the different treatments. B, dot blot
analysis was performed with total RNA isolated from cells untreated or
treated with PMA, 20 ng/mL, and PDTC, 100 µM, for 6 or 16
h. RNA was hybridized with cDNA probes of the human c-myc and
-actin genes.
Since PDTC exerted similar differentiation-associated changes than those triggered by PMA, we performed experiments to determine whether protein kinase C (PKC) was mediating the effect of PDTC. As expected, treatment of the cells with the PKC-specific inhibitor bisindolylmaleimide resulted in a dramatic inhibition of CD11c and CD54 surface expression induced by PMA. By contrast, the induction of these markers by PDTC remained unaffected in the presence of the same concentrations of the inhibitor (Fig. 3). Furthermore, microscopic examination of cultures of U-937 cells revealed additional differences in the signals displayed by both inducers. Thus, PMA-mediated differentiation was accompanied with strong cell-aggregation and cell adherence to plastic surfaces, whereas PDTC, failed to induce such changes (not shown). These data indicate that the signaling pathway triggered by the dithiocarbamate is not dependent on PKC activity and differs from that elicited by PMA.
Figure 3: Effects of the PKC inhibitor bisindolylmaleimide on CD11c and CD54 expression induced by PDTC and PMA. U-937 cells were pretreated or not for 2 h with the inhibitor (2 µM) before addition of PDTC (50 µM) or PMA (20 ng/ml). After 24 h cells were analyzed for surface expression markers by fluorescein-activated cell sorter analysis. Dotted lines indicate profiles of untreated cells and solid lines represent the profiles of treated cells as indicated.
Figure 4:
Transcriptional response of CD11c gene to PDTC. A, Dot blot analysis was performed with
total RNA isolated from U-937 cells untreated or treated with PDTC for
different times. A 0.8-kb cDNA fragment of the human CD11c gene was used as a probe. Hybridization with the -actin is included as a control. B, luciferase-based recombinant
plasmids harboring different regions of CD11c gene promoter
were transiently transfected into U-937 cells and seeded in the absence
or presence of PDTC and PMA. After 13 h of treatment, luciferase
activity was measured in extracts from lysed cells. Fold inductions of
luciferase activity in PDTC or PMA treated versus untreated
cells are shown. Results are representative of four independent
experiments that yielded substantially the same results. Location of
consensus binding sites for AP-1, Sp1, Oct-1, and AP-2, as well as the
major transcription start site (+1), are
indicated.
To further investigate the effect of PDTC on CD11c gene transcription, we transfected U-937 cells with reporter plasmids containing the regions of the CD11c promoter spanning from -361 to +40, -253 to +40, and -160 to +40 (López-Cabrera et al., 1993). All of these constructs displayed basal promoter activity that was clearly increased in PDTC-treated transfected cells (Fig. 4B). This inducible activity was qualitatively similar although less potent than that exerted by PMA, which was used as a positive control. The transfection of longer fragments of the CD11c gene promoter spanning from -640 to +40 and -960 to +40 yielded similar levels of induction (data not shown) suggesting that the cis-acting elements responsible for the transcriptional activation mediated by PDTC were located within the -160 to +40 proximal region of the CD11c promoter.
Figure 5:
Effects of PDTC on binding activity to the
-68/-46 and -136/-99 regions of CD11c promoter and on the RNA levels of c-fos and
c-jun. A, nuclear extracts prepared from U-937 cells
untreated and treated for 6 h with PDTC (50 and 100 µM)
were incubated with probes including the -68 to -46 and
-136 to -99 regions (probes A and B) of
the CD11c gene promoter. DNA-protein complexes were resolved
by EMSA. A 30-fold molar excess of unlabeled homologous oligonucleotide
was used as competitor in binding reactions. B, probe A,
containing an AP-1 consensus site was incubated with nuclear extracts
from PDTC (100 µM) treated cells in the presence of
preimmune rabbit antiserum or rabbit antisera against Fos family (RR
26/8) or Jun family (6 36/6) members. C, Dot blot analysis was
performed with total RNA isolated from U-937 untreated (C) or
treated with PDTC 50 µM and PMA 20 ng/ml for the time
points indicated. RNA was hybridized with cDNA probes of
c-fos, c-jun, and -actin.
In order to better characterize the mechanism involved in the induction of AP-1 DNA binding activity mediated by PDTC, we examined the effect of the antioxidant on the steady state mRNA levels of c-jun and c-fos. We performed dot-blot analysis using RNA from cells treated with PDTC or PMA. PDTC strongly increased c-jun mRNA levels as early as 30 min of treatment, reaching its highest levels between 4 and 7 h. Similarly, the dithiocarbamate also induced an early expression of c-fos mRNA that declined after 2 h of treatment (Fig. 5C).
Figure 6: Role of AP-1 in CD11c gene promoter activity in response to PDTC. A, sequences of the wild type (white box) and mutated regions (black box) (-68 to -46) of the CD11c promoter. The substitutions introduced within the AP-1 site (-61 to -54) are indicated. B, recombinant wild type plasmids, containing -361/+40 and -160/+40 regions of CD11c gene promoter and the respective mutated plasmids, were transiently transfected into U-937 cells, and after 13 h of treatment with PDTC, luciferase activity was measured. Relative luciferase activity was expressed as fold induction over the activity of the promoterless plasmid pXP2 in untreated and treated cells. Results are representative of three independent experiments.
Alteration of the redox status of the cells by antioxidant
agents such as DTCs, has previously been shown to interfere with the
activity and expression of the transcription factors NFB and AP-1,
which appear to be involved in cell differentiation and activation
processes (Baeuerle and Henkel, 1994). The results presented in this
report show that DTCs simultaneously induce the expression of myeloid
differentiation markers in U-937 cells and inhibit cell proliferation.
To characterize the mechanisms responsible for DTC-mediated cell
differentiation, we have analyzed as a model the activation of CD11c gene transcription. These studies suggest that PDTC
induces the transcriptional activation of the CD11c gene
through the activation of the AP-1 transcription factor.
The
comparative analysis of differentiation markers induced by DTCs and PMA
indicated that both elicit a similar pattern of cell surface antigen
expression. In addition, PDTC and PMA induced the NSE enzymatic
activity, inhibited cell growth, down-modulated c-myc RNA
levels, and increased those of c-fos and c-jun.
However, several lines of evidence indicate that both inducers act
through different signaling mechanism. (i) Whereas the changes in the
expression of myeloid surface antigens by PMA have been shown to be
dependent on the activity of PKC (Bellón et
al., 1994) (Fig. 3), those mediated by PDTC were not
affected by the specific PKC inhibitor bisindolylmaleimide. In
accordance, it has been previously shown that the treatment of Jurkat T
cells with PDTC does not affect the activity of PKC (Meyer et
al., 1993). (ii) PMA triggers strong homotypic aggregation, cell
adhesion, and spreading to plastic surface (Collins, 1987; Miller et al., 1986; Nueda et al., 1995). In contrast, PDTC
induced only a weak homotypic aggregation. These results also support
that PKC-independent signals are mediating the effect of DTCs, since
the aggregation and adhesion processes induced by PMA have been
proposed to be dependent on a PKC-induced conformational change of the
2 integrin molecules (Keizer et al., 1988). (iii) Flow
cytometry analysis using dichlorofluorescein dye revealed that PMA
induces an increase of the intracellular H
O
levels in U-937 cells whereas PDTC has the opposite effect. (
)
Several antioxidants have been shown to influence the
activity of the inducible transcription factors NFB and AP-1 (Beg et al., 1993; Meyer et al., 1993; Schreck et
al., 1992a, 1992b; Staal et al., 1990). In HeLa cells
PDTC, N-Acetyl-L-Cysteine (NAC) and the antioxidative
enzyme ADF/thioredoxin potently activates the transcription factor AP-1
involving induction of c-fos and c-jun (Meyer et
al., 1993). Our results indicate that PDTC stimulates the surface
expression of CD11c/CD18 (p150,95), increasing its
-subunit (CD11c) mRNA steady state levels by transcriptional mechanisms
acting on the CD11c gene promoter. The induction of c-fos and c-jun mRNA levels by PDTC, as well as the presence of
an AP-1 site at -60, the elimination of which greatly decreases
the PDTC responsiveness of the CD11c promoter, supports an
important role of AP-1 transcription factor in the activation and
differentiation process elicited by DTCs. In this context, this
transcription factor has been suggested to play a role in the myeloid
differentiation triggered by different inducers such as PMA or
etoposide (Bellón et al., 1994;
Pérez et al., 1994). Furthermore, PMA
has been shown to induce AP-1 DNA binding activity to the functional
AP-1 site at -60 on the CD11c promoter, concomitant with
the appearance of Fos protein detected by EMSA, while only Jun
components, that account for the basal levels of AP-1, are detected in
undifferentiated U-937 cells.
Thus, AP-1 may represent a
transcription factor that can integrate different signals elicited by
several differentiation agents that converge at this point. However, in
order to explain the distinct patterns of differentiation observed,
additional signaling different to AP-1 activation must be produced by
the different inducers.
A thorough study of the nuclear factors that bind to the responsive region of the CD11c promoter spanning from -160 to +40, revealed that PDTC restrictively induced DNA binding activity to the AP-1 site. Hence, AP-1 is stimulated in a specific manner and can function as an antioxidant-responsive transcription factor in U-937 cells. Similarly, the genes of the antioxidative enzymes NAD(P)H:quinone oxidoreductase, EC 1.6.99.2, previously known as DT-diaphorase and the glutathione S-transferase Ya subunit, are controlled at the transcriptional level through antioxidant responsive elements in their promoter regions. These elements are required for the transcriptional activation mediated by several antioxidants (Rushmore et al., 1991). Since antioxidants can induce a hypoxic status in the cells, it is noteworthy that the DT-diaphorase gene is transcriptionally activated under hypoxic conditions in human colon cancer cells. Moreover, this effect involves early induction of jun family genes followed by a latter elevation of c-fos m-RNA levels (Yao et al., 1994). Interestingly, the gene promoter of the DT-diaphorase contains an AP-1 motif that has been shown to be functionally involved in the transcriptional induction of the gene by polycyclic aromatic hidrocarbons and phenolic antioxidants (Li and Jaiswal, 1992). Mutation of this site results in the loss of basal and induced transcriptional activity of the promoter. As shown in this paper, the AP-1 site of the CD11c promoter behaves in a similar fashion to that of the DT-diaphorase promoter and could, therefore, be considered an antioxidant responsive element. In this regard, it would be interesting to study whether the activation of the AP-1 transcription factor under hypoxic conditions could trigger the expression of CD11c.
Finally, DTCs are drugs that have already been pharmacologically used in heavy metal poisoning and AIDS treatment (Lang et al., 1985; Sunderman et al., 1967). It will be important to determine whether the effects of the DTCs that we have described in U-937 and HL-60 cells are also operative in leukemic cells derived from patients. In this regard, retinoic acid has been used in the treatment of patients with acute promyelocytic leukemia yielding a high rate of transient clinical remissions (Chen et al., 1991; Warrel et al., 1991). However, after prolonged periods of treatment, resistance to RA appears and the disease is reactivated (Delva et al., 1993). The effects of DTCs on myeloid cell differentiation supports a therapeutical potential of these agents in bone marrow-derived malignancies. Future experiments on fresh leukemic cells will aid in the elucidation of this interesting matter.