(Received for publication, May 30, 1995; and in revised form, February 1, 1996)
From the
The human promyelocytic leukemia cell line HL-60 can be induced to differentiate toward neutrophils and subsequently die via apoptosis in vitro. In this paper, we investigated the roles of protein-tyrosine kinases (PTKs) in retinoic acid (RA)-induced granulocytic differentiation of HL-60 cells. Accompanying the RA-induced differentiation, activities of src family PTKs Lyn and Fgr became detected and reached a plateau 2 days after the stimulation. The immunoblotting using anti-phosphotyrosine antibody (PY-20) showed that the proteins of 56 and 53 kDa were predominantly tyrosine-phosphorylated at day 2. Adsorption and immunoprecipitation of the cell lysate by specific antibodies evidenced that these phosphotyrosine-containing proteins are Lyn and Fgr PTKs. The degree of both activities and tyrosine phosphorylation of these PTKs was reduced to be minimal at day 5 when the HL-60 cells start to die by apoptosis. The inhibitors of PTKs, herbimycin A and genistein, were demonstrated to cause premature cell death of HL-60 cells in the presence of RA. The death was the consequence of an apoptotic process. The RA-treated HL-60 cells, when incubated with specific c-lyn or c-fgr antisense oligodeoxynucleotide, also underwent premature death at day 2. These data implicate that Lyn and Fgr PTKs prevent programmed cell death to promote granulocytic differentiation of HL-60 cells.
Retinoic acid (RA) ()functions as an important
bioregulator in cell differentiation and
development(1, 2) . Due to its potent activity in
inducing differentiation and growth arrest of leukemic cells from acute
promyelocytic leukemia (APL M3) patients, RA has been utilized in
treatment of these leukemia patients (3, 4) .
The human promyelocytic leukemia cell line HL-60, upon stimulation with RA in vitro, undergoes differentiation toward neutrophils at day 3 to day 4 and dies via apoptosis at day 6 to day 8(5) . Neutrophil is the first cell to accumulate at the site of inflammation and plays a critical role in inducing various inflammatory events. As soon as neutrophils complete their roles at the inflammatory site, they die via apoptosis and are removed by macrophages to limit tissue injury since they release harmful molecules such as proteolytic enzymes(6, 7) . Therefore, molecular analysis for the events occurring after stimulation of HL-60 cells with RA will provide useful information on both the differentiation and the apoptosis of neutrophils.
Apoptosis requires active and coordinated regulation of specific molecules. Bcl-2 is a potent suppressor of death and the balances between Bcl-2 and a death-inducing signal determine the occurrence of apoptosis(8) . It was demonstrated that expression of Bcl-2 decreased markedly during RA-induced granulocytic differentiation of HL-60 cells (9, 10) .
RA binds
to specific nuclear receptors (RARs) and the RARAR complex is
thought to regulate the transcription of target
genes(11, 12) . However, little is known about the
intracellular events that lead to differentiation and apoptosis of the
responsive cells subsequent to RA
RAR interaction(13) .
The protein-tyrosine kinases (PTKs) play crucial roles in the intracellular signal transduction for growth and differentiation of the cells(14) . Recently, we reported that PTKs play essential roles in TPA-induced monocytic differentiation of HL-60 cells(15, 16, 17) , and that Ras and GTPase-activating protein complex function downstream of PTKs during the differentiation(18) . In addition, our previous paper described the induction of src family PTKs, lyn and fgr mRNA during RA-induced granulocytic differentiation(15) . We have therefore investigated the role of PTKs in the differentiation.
In the current study, during RA-induced granulocytic differentiation of HL-60 cells, both Fgr and Lyn PTKs were demonstrated to be superinduced and tyrosine-phosphorylated. In the presence of RA, treatment with specific c-lyn or c-fgr antisense oligodeoxynucleotides as well as that with herbimycin A or genistein, led to premature death of the HL-60 cells. Thus, Lyn and Fgr PTKs are assumed to exert as anti-apoptotic agents to promote granulocytic differentiation of HL-60 cells.
The P-labeled samples were
subjected to electrophoresis on 9% polyacrylamide-SDS gels. The gels
were treated with 1 M KOH at 55 °C for 2 h to detect
tyrosine-phosphorylated protein molecules, dried, and subjected to Fuji
RX film at -80 °C.
The antisense and sense PSNs were synthesized on a synthesizer (model 392; Applied Biosystems, Inc., Foster City, CA), precipitated with ethanol, and taken up in media containing 20 mM Hepes. Sense (S) or antisense (AS) c-lyn or S or AS c-fgr PSNs were added to the culture medium for HL-60 cells at the concentration of 20 µM. After 4 days of PSN treatment, the culture medium was replaced with fresh medium containing 20 µM S or AS PSNs, and 1 µM of RA was added to the cultures.
Figure 1:
Induction of
p56 and p53/p56
PTKs
during RA-induced granulocytic differentiation of HL-60 cells. Lysates
of untreated HL-60 cells (None), the cells treated for 48 h
with retinoic acid (RA) or with TPA (TPA) were
immunoprecipitated with anti-Fgr, anti-Lyn, anti-Fyn, anti-Hck,
anti-Syk, or anti-Btk antibody. The immunoprecipitates were subjected
to immune complex kinase assay and analyzed by SDS-PAGE as described
under ``Materials and Methods.'' The autoradiograph was
exposed for 1 h at -80 °C with an intensifier screen. The
position of each PTK is indicated.
Both mRNAs and proteins of the Lyn and Fgr PTKs were also detected abundantly 2 days after RA treatment ((15) , Fig. 2). However, we could not determine whether the PTKs were activated or not since the degrees of their expressions were very low in untreated HL-60 cells.
Figure 2:
Upper, protein tyrosine phosphorylation in
HL-60 cells after stimulation with retinoic acid. The lysates from the
HL-60 cells (5 10
cells) stimulated with RA for 0,
1, 2, or 5 days were immunoblotted with
I-labeled PY-20.
The autoradiograph was exposed for 24 h at -80 °C with an
intensifier screen. The positions of major bands are indicated with an arrowhead. Molecular masses in kDa are indicated on the left. Lower, expressions of Lyn, Fgr, and actin in HL-60 cells
after stimulation with RA. The lysates from the HL-60 cells (5
10
cells) stimulated with RA for 0, 1, 2, or 5 days were
immunoblotted with anti-Lyn, anti-Fgr, and anti-actin. The bands were
detected by ECL assay system (Amersham).
Figure 3:
Lyn and Fgr were major
phosphotyrosine-containing proteins in RA-treated HL-60 cells. Left, p53/56 and p56
were tyrosine-phosphorylated in RA-treated HL-60 cells. The
lysates from the HL-60 cells (2.5
10
cells) treated
with TPA or RA for 48 h were immunoprecipitated with polyclonal
anti-Lyn or anti-Fgr antibodies and protein G-Sepharose 4B. The
immunoprecipitates were subjected to electrophoresis, transferred to a
polyvinylidene difluoride (PVDF) filter, and immunoblotted with
I-labeled PY-20. The autoradiograph was exposed for 12 h
at -80 °C with an intensifier screen. The positions of Lyn
and Fgr are indicated by arrowheads on the right.
Molecular masses in kDa are indicated on the left. Right, the
absorption of phosphorylated proteins with anti-Lyn plus anti-Fgr
antibodies. The lysates from RA-treated HL-60 cells (1
10
cells) were immunoprecipitated with polyclonal anti-Lyn plus
anti-Fgr (anti-Lyn + anti-Fgr) or rabbit IgG (RIg) and protein G-Sepharose 4B. The supernatant was
subjected to SDS-PAGE, transferred to a PVDF filter, and immunoblotted
with
I-labeled PY-20. The exposure time was 12 h at
-80 °C with an intensifier
screen.
Figure 4:
Tyrosine phosphorylation of
p95 in RA-treated HL-60 cells. Upper,
the RIPA lysates from RA- or TPA-treated HL-60 cells (2.5
10
cells) for 48 h were immunoprecipitated with monoclonal
anti-Vav or mouse IgG (mIg) as a control and protein
G-Sepharose. These immunoprecipitated proteins were subjected to
SDS-PAGE, transferred to a PVDF filter, and immunoblotted with
I-labeled PY-20. The autoradiograph was exposed for 12 h
at -80 °C with an intensifier screen. Lower, the
same whole lysates were subjected to SDS-PAGE and transferred to a PVDF
filter and then immunoblotted with anti-Vav antibody. The bands
detected by the ECL assay system (Amersham) showed that an almost equal
amount of Vav protein exists in each
lysate.
Figure 5:
A,
morphological characteristics of cell death of the HL-60 cells after
treatment with RA plus PTK inhibitors. Morphological characteristics of
granulocytes were observed when HL-60 cells were incubated with RA for
0 days (RA 0d.), 2 days (RA 2d.), 3 days (RA
3d.), and 7 days (RA 7d.); proliferating HL-60 cells 2
days after treatment with 0.2 µg/ml herbimycin A alone (Herbimycin A 2d.); progressive cell death of HL-60 cells
treated with RA plus 0.2 µg/ml herbimycin A for 2 days (RA
+ Herbimycin A 2d.), cell death of HL-60 cells treated with
RA plus 10 µg/ml genistein for 2 days (RA + Genistein
2d.). B, DNA fragmentation in the HL-60 cells treated
with RA plus herbimycin A. The high molecular mass DNA extracted from
untreated HL-60 cells (None), from HL-60 cells treated with
0.2 µg/ml herbimycin A (H0.2), from HL-60 cells induced to
differentiate with 1 µM RA for 2 days (RA), from
HL-60 cells induced to differentiate with 10 ng/ml TPA (TPA)
for 2 days, from HL-60 cells treated with TPA plus herbimycin A (TPA+H0.2), or fragmented DNA from HL-60 cells treated
with RA plus herbimycin A (RA+H0.2) for 2 days were
subjected to agarose gel electrophoresis. The phage DNA digested
with restriction endonucleases EcoRI and HindIII is
indicated as a DNA molecular mass marker (Marker).
Apoptosis is in many cases associated with a characteristic oligonucleosomal DNA fragmentation induced by intrinsic endonuclease(s). Apoptotic cell death of HL-60 cells treated with herbimycin A plus RA was further confirmed by detecting the ladder pattern of DNA cleavage in these cells (Fig. 5B), whereas DNA remained unfragmented in preparations obtained from HL-60 cells treated with RA, herbimycin A, or TPA plus herbimycin A for 48 h (Fig. 5B).
Figure 6:
A, expressions of the
p53/p56and p56
in HL-60
cells treated with the c-lyn S or AS or c-fgr S or
AS. Sense (S) or antisense (AS) c-lyn or S or ASc-fgr PSNs were added to the culture medium of HL-60 cells at the
concentration of 20 µM. After 4 days of PSN treatment, the
culture medium was replaced with fresh medium containing 20 µM S or AS PSNs, and 1 µM RA was added to the cultures.
Lysates of the untreated (Untreated) or PSN-treated HL-60
cells (lyn S or AS, or fgr S or AS)
were obtained at 24 h after RA stimulation and subjected to SDS-PAGE,
transferred to a PVDF filter, and immunoblotted with anti-Lyn,
anti-Fgr, or anti-Btk, respectively. The position of Lyn, Fgr, or Btk
was indicated by an arrowhead. B, viability of HL-60 cells
cultured for 2 days in the absence (None) or presence (RA) of RA after treatment with c-lyn or c-fgr S or AS oligomer. Cell viability was assessed by the ability of
the cells to exclude trypan blue. The result is a typical one, and the
experiment was repeated three times with similar results. The average
and S.E. of triplicate determinations are
shown.
The regulation system of apoptosis is highly organized, and the balances between inducers and repressors determine the occurrence of apoptosis. In the current study, two cytoplasmic tyrosine kinases, Lyn and Fgr, were demonstrated to be members of the repressors for apoptosis of neutrophils. Both Lyn and Fgr PTKs were shown to be induced and tyrosine-phosphorylated during differentiation of HL-60 cells toward neutrophils. After completion of the differentiation, the expressions of these PTKs were reduced to be minimal and the cells die via apoptosis. Using antisense oligonucleotides specific for Lyn or Fgr PTK, it was demonstrated that inhibition of expression of either PTK upon RA stimulation leads to premature cell death via apoptosis. Consistent with the results, herbimycin A in combination with RA was exhibited to cause premature cell death of the HL-60 cells. These data imply that Lyn and Fgr PTKs exert an antiapoptotic effect to promote differentiation of HL-60 cells toward neutrophils. Antiapoptotic function of PTKs in neutrophils was also suggested by Yousefi et al.(25) who demonstrated that GM-CSF-induced tyrosine phosphorylation prevents apoptosis of human peripheral blood neutrophils. In our study, at least two possibilities exist to explain antiapoptotic roles of Lyn and Fgr PTKs in the granulocytic differentiation of HL-60 cells; one is their functioning in independent signal transduction pathways which converge to join each other at the terminal, and the other is their direct interaction or ordered functions on the same pathway which is required for antiapoptotic function. As yet, we have no evidence for supporting the one.
In
neutrophils, Lyn has been demonstrated to be activated and associated
with phosphatidylinositol 3-kinase accompanying the stimulation with
granulocyte macrophage-colony stimulating factor (GM-CSF)(26) .
It was shown that granulocyte-colony stimulating factor (G-CSF)
activated Lyn and Syk (p72), both of which were then
recruited into G-CSF receptor signaling complex in human peripheral
neutrophils(27) . While the evidence has been reported that Fgr
also function in human neutrophils, a chemotactic agonist induces
translocation of Fgr from an intracellular compartment to the plasma
membrane and that Fgr is associated to Fc
IIR on
neutrophils(28) . Recently, Berton et al.(29) demonstrated that agonists of
2 integrin
activation such as tumor necrosis factor, TPA, and fMet-Leu-Phe enhance
the kinase activity of Fgr in human neutrophils. These data suggest
that Fgr PTK plays a crucial role in the expression of function by
peripheral neutrophils at the inflammatory site. Our data on the
antiapoptotic role of Lyn and Fgr PTK in the granulocytic
differentiation of HL-60 cells will add a new insight into the function
of these PTKs in the neutrophils. Further studies on the analyses of
HL-60 transfectants expressing active Lyn or Fgr or both PTKs will
provide more information on the roles of these PTKs in the apoptosis
and differentiation of human neutrophils. Identification of the
molecules interacting with these PTKs in RA-stimulated HL-60 cells will
also give information on the function of these PTKs in neutrophils.
These studies are now in progress in our laboratory.
While our study
is in progress, Manfredini et al.(30) demonstrated
that the HL-60 cells which had been induced to differentiate into
granulocytes underwent premature death when incubated with antisense
oligonucleotide specific for c-fes gene which encodes a Fes
PTK (p92). In our system, accompanying the
differentiation of HL-60 cells toward neutrophils, expression of Fes
PTK is increased without detectable levels of the tyrosine
phosphorylation of Fes PTK (data not shown). To get a clue to
understanding the relation of Lyn, Fgr, and Fes PTK in the
antiapoptotic signaling pathway, we tried to investigate the possible
co-immunoprecipitation with each other during RA-induced
differentiation. However, no association with each other was observed.
Several genes have been identified that participate as either
inducers or repressors of programmed cell death. Among these, Bcl-2 and
Bax are homologous proteins that have opposing effects on cell life and
death. Bcl-2 serves to prolong cell survival while Bax acts as an
accelerator of apoptosis(31, 32) . Previous papers
reported that Bcl-2 decreased during RA-induced differentiation of
HL-60 cells into granulocytes which subsequently undergo apoptosis and
that HL-60 cells which hyperexpressed Bcl-2 showed little evidence for
apoptosis(9, 10) . In our study, we also observed the
prompt decrease in expression of Bcl-2 protein by HL-60 cells after RA
stimulation (data not shown). Expression of Bax protein was shown to be
reduced far more slowly accompanying the differentiation (data not
shown). Thus, the ratio of Bcl-2 and Bax was markedly reduced at an
early phase of the differentiation. Therefore, some molecules, for
which we propose Lyn and Fgr PTK, should protect cells from apoptosis
instead of Bcl-2 to promote differentiation toward neutrophils.
Recently, various proteins such as Bcl-x,
Bcl-x
, Bad, and Bag-1 were cloned as molecules associated
with Bcl-2 or Bax and demonstrated to be involved in programmed cell
death(33, 34, 35) . Thus, the mechanisms of
the antiapoptotic function of Lyn and Fgr PTK should be considered by
taking the balances of the above Bcl-2-related molecules into account.
In addition to Lyn and Fgr PTKs, we identified preferentially
tyrosine-phosphorylated proteins as p95 (Vav). Vav is
expressed specifically in hematopoietic cells and has the guanine
nucleotide releasing factor activity for Ras which is regulated by
tyrosine kinase(36) . In our previous work, we could not find
activation of Ras during RA-induced differentiation of HL-60 cells
while the activation of Ras was observed accompanying TPA-induced
differentiation(18) . As the degree of tyrosine phosphorylation
of Vav upon RA stimulation is higher than that upon TPA stimulation,
Vav might have functions other than working as a guanine nucleotide
releasing factor for Ras. In this term, study on the differentiation of
HL-60 cells will provide useful information on physiological function
of Vav.
As RA induces growth arrest and terminal differentiation in some promyelocytic leukemia cell lines, RA has been utilized as a differentiation therapy for treatment of patients with acute promyelocytic leukemia(3, 4) . This apparently resulted in remission of the patients without causing marrow aplasia(3, 4) . In the current study, RA in combination with a small amount of herbimycin A was found to be a potent agent to induce apoptosis of promyelocytic leukemia cell line HL-60. Although its validity and toxicity should be scrutinized using experimental animals, careful treatment with the combination of the agents will improve the remedial value exhibited by RA alone. For more specifically refined treatment, further studies on the molecular mechanisms of antiapoptotic functions of tyrosine kinases should be required.