[CANCER RESEARCH 51. 4271-4278, August 15. 1991]
Expression of Three Major Protein Kinase C Isozymes in Various Types of Human
Leukemic Cells1
Fumihiko Komada, Masakatsu Nishikawa,2 Yasuhiro Uemura, Kouichi Monta. Hiroyoshi Hidaka,
and Shigeru Shirakawa
The 2nd Division, Department of Internal Medicine, Mie University School of Medicine, 2-174 Edobashi, Tsu, Mie 514 [F. K., M. N., Y. U., K. M., S. S.J, and
Department of Pharmacology, Nagoya University School of Medicine, Showa-ku, Nagoya 466 ¡H.H.J, Japan
ABSTRACT
We examined the levels of protein kinase C (PKC) activity and the
expressions of its three major isozymes, designated types I (7), II (/<).
and III (a), in the cytosol and particulate fractions of cells from patients
with acute myelogenous leukemia (AML), acute lymphocytic leukemia
(ALL), and chronic lymphocytic leukemia (CLL), in an attempt to
elucidate the cell type- or lineage-specific expression of these isozymes.
The levels of PKC activities in the cytosol and particulate fractions from
AML cells were higher than those from ALL or CLL cells. The average
PKC activities of AML cells, ALL cells, and CLL cells were 18.7, 12.2,
and 11.3 pmol/min/108 cells, respectively, in the cytosol fractions and
4.4, 3.1, and 2.6 pmol/min/108 cells, respectively, in their particulate
fractions. Ml cells (French-American-British classification) and AML
cells with T-lymphocyte-associated surface antigens, such as CD2 and
CD7, had significantly lower PKC activities among AML cells. Immunoblot analyses using monoclonal antibodies against each isozyme re
vealed that all three ¡sozymeswere broadly distributed on leukemic cells
with considerable variability in the level of expression. All lymphoid
leukemic cells expressed I'M -t in the cytosol fractions, albeit a minor
component; however, this type was observed in cells from only half the
number of AML patients. Those AML cells with cytosolic PKC-y usually
expressed lymphoid surface antigens, such as CD2, CD7, and CD19. On
the other hand, cytosolic PKC-/ÃŽ
and PKC-or were commonly observed in
all types of leukemic cells. AML cells expressed these two types at
almost equal levels, but in lymphoid cells, expressions of PKC-/? were
usually more abundant than those of PKC-a. These data suggest that
AML cells with lymphoid antigens might have a lower PKC activity but
more predominant expression of cytosolic PKC-7 than the usual AML
cells.
INTRODUCTION
PKC} is a ubiquitous serine/threonine-protein
kinase and is
thought to play a crucial role in cellular growth regulation,
differentiation, and malignant transformation (1-3). Studies on
leukemic cells from patients and on cultured leukemic cells
provided evidence that PKC expression is required for cellular
growth (4). Human myelogenous HL-60 leukemia cells differ
entiate into macrophage-like cells when exposed to phorbol
esters (5), and the activation, translocation, and subsequent
down-regulation of PKC appear to be essential for such HL-60
cell differentiation, as shown by pharmacological methods with
various PKC inhibitors (6, 7) and investigations on 12-0tetradecanoylphorbol-13-acetate-resistant
HL-60 cell variants
(8-10). On the other hand, the level of activity of this enzyme
was shown to increase during HL-60 cell differentiation by
Received 9/7/90; accepted 5/30/91.
The costs of publication of this article »eredefrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
' This work was supported in part by grants for research from the Ministry of
Education, Science, and Culture, Japan.
2To whom requests for reprints should be addressed.
3The abbreviations used are: PKC. protein kinase C; AML, acute myelogenous
leukemia: ALL, acute lymphocytic leukemia; CLL. chronic lymphocytic leukemia;
CD, cluster of differentiation antigen; CSF. colony stimulating factor: DTT.
dithiothreitol; EGTA, ethleneglycol bis((*-aminoethyl ether)-Ar.At,/V',A"-tetraacetic acid; FAB, French-American-British.
other inducers, such as dimethyl sulfoxide, retinole acid, and
1,25-dihydroxyvitamin D} (11, 12). Recent investigations sug
gested that PKC might play an essential role in the regulation
of granulocyte-macrophage-CSF-or
interleukin 3-induced sur
vival and proliferation of hematopoietic progenitor cells (13),
and the biological effects of these cytokines might be mediated
through the activation of this enzyme (14, 15).
Molecular cloning of several complementary DNAs for PKC
and isolation of its multiple isozymes revealed that this enzyme
is a family of multiple isozymes (16-18), and type I, II, and III
PKCs have been identified as products of 7, ß,and «genes,
respectively (19). These multiple isozymes exhibit considerable
tissue specificities (16, 20-22), different subcellular distribu
tions (18, 22), different developmental expressions (22), and
individual enzymological characteristics (23, 24), thereby sug
gesting that these isozymes do not share common functional
roles. Although PKC-7 was thought to be expressed solely in
the brain and spinal cord (16), several laboratories including
ours reported that leukemic cells, such as HL-60 (10, 12), U937 (25), or an HL-60 variant cell line (10, 26), contain this
type of PKC, mostly in the particulate fraction, albeit as a
relatively minor component.
To clarify the possible role of PKC in leukemogenesis, we
measured PKC activity and the expressions of PKC isozymes
in the cytosol and particulate fractions of leukemic cells from
patients with various types of leukemia. Leukemic cells are
believed to arise from a clonal expansion of cells after transfor
mation and arrest at a certain stage of differentiation (27).
However, in some cases, they express inappropriate differentia
tion antigens, which are usually confined to another lineage
(27, 28). We also investigated the possibility that such morpho
logical and phenotypical variants of human leukemic cells may
be affected by altered levels of activities or distinct expressions
of PKC isozymes.
MATERIALS AND METHODS
Cells. Leukemic cells from 39 adult Japanese patients (24. 6, and 9
cases of AML, ALL, and B-CLL, respectively) referred to our labora
tory between October 1987 and September 1989 were evaluated in this
study. Informed consent was obtained for these studies from all patients.
AML and ALL diagnoses were made by morphological and cytochemical analyses according to conventions of the FAB Cooperative Group
(29, 30). CLL diagnosis was based on established hematological criteria
(31) and was confirmed by an immunophenotypic analysis. Heparinized
venous blood or bone marrow was obtained from these patients prior
to chemotherapy, and mononuclear cells were separated using FicollHypaque density gradient centrifugation. The samples containing more
than 95% leukemic cells were frozen in liquid nitrogen and stored at
—70°C
until PKC activity was measured. Selection of patient samples
was based on the availability of large cell numbers (>2 x IO8),necessary
to measure PKC activity, and the isozyme expression. Immunopheno
typic analyses were performed using an immunofluorescence-activated
cell sorter (Cytron; Ortho Diagnostic System, Tokyo, Japan). CD1 Ib
(recognized by OKM1), CD13 (MCS2), CD14 (My4), CD15 (LeuMl),
4271
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PROTEIN KINASE C ISOZYMES IN HUMAN LEUKEMIAS
CD33 (My9), and CD34 (MylO) were tested as myeloid cell markers.
CD2 (Til), CD3 (Leu4), CD4 (Leu3), CDS (Leul), CD7 (WT1 or
TP40), and CDS (Leu2) were tested as T-cell markers. Immunoglobulins CD19 (by B4 or Leul2), CD20 (by Leulo), and CD22 (Leul4)
were tested as B-cell markers. CD10 and HLA-DR were examined by
J5 and OKIal. respectively.
Subccllular Fractionation of Protein Kinase C. All procedures were
carried out at 0-4°C.Cells (2 x 10s each) were washed three times with
ice-cold Dulbecco's phosphate-buffered saline and then homogenized
in a buffer containing 20 niM Tris-HCl (pH 7.5). 2 mM DTT, 2 mM
EDTA, 2 mM EGTA, 0.25 M sucrose, and several protease inhibitors
(75 mg/liter phenylmethylsulfonyl fluoride, 10 mg/liter leupeptin, 10
mg/liter /V-tosyllysine chloromethyl ketone, and 5 mg/liter /V-tosylphenylalanine chloromethyl ketone) in a glass to glass Potter-Elvehjem
homogenizer. The homogenate was centrifuged at 2,000 x g for IO min
to remove nuclei and cell debris; then the supernatant was collected
and centrifuged at 100.000 x g for l h to separate the cytosol and
paniculate fractions. The paniculate fraction was subsequently resuspended in homogenization buffer containing 20 mM n-octyl-ff-D-glucoside (Dojindo Laboratories, Ruttiamolo, Japan) and kept on ice for
1 h. These preparations were applied to a DEAE-cellulose (DE-52;
Whatman, Maidstone. England) minicolumn (1.0 x 2.0 cm), and the
column was washed with 10 ml of 20 mM Tris-HCl (pH 7.5), 2 mM
DTT, 2 mM EGTA, 2 mM EDTA, and protease inhibitors (buffer A).
PKC was then eluted batchwise with 10 ml of buffer A containing 0.15
M NaCI, and fractions of 1 ml each were collected. This elution yields
approximately 95% of PKC observed in NaCI gradient elution. Since
the content of PKC in leukemic cells appears to be lower than that in
the brain, this Chromatographie step serves to concentrate the kinase
activity and to eliminate substances that possibly interfere with PKC
activity. Protein concentrations were determined by the dye-binding
method of Bradford (32), using bovine serum albumin as a standard.
Assay of Protein Kinase C. Activity of PKC was examined using
histone HI (Boehringer Mannheim) as the substrate. The reaction
mixture contained 20 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 200 MM
CaClz, 10 Mg/ml phosphatidylserine. 1 Mg/ml 1.2-diolein, 400 Mg/ml
histone HI, 50 JIM [v"P]ATP (New England Nuclear, Boston. MA),
and appropriate amounts of an enzyme fraction from the DEAE minicolumn. The reaction was initiated by the addition of ATP following 2
min of preincubation at 30°C,and incubation was carried out for 3 min
at 30°C.Time course studies indicated that reactions were linear during
this time period. The reaction was halted by the addition of 20%
trichloroacetic acid. The acid-precipitable materials were collected on
Millipore Titters and counted in a liquid scintillation spectrometer. All
assays were done in duplicate. Protein kinase activity is expressed as
nmol/min/mg protein, determined in the cytosolic or paniculate eluate,
respectively. The values given in the tables have been corrected for the
blank values by subtraction of the radioactivity obtained in the absence
of diolein and phosphatidylserine.
Immunoblot Analysis. Monoclonal antibodies MC-la, MC-2a, and
MC-3a, which reacted with types I (7), II (ß),
and III (a) rabbit brain
PKC isozymes, respectively, were used. All antibodies were used at a
1:100 dilution. These antibodies were reactive only with their respective
isozymes. tested for cross-reactivity with different isozymes by immunoblotting (21). Immunoblot analysis was performed according to the
method of Towbin et al. (33). The pooled active fractions were centri
fuged at 2.000 x g after the addition of 100% trichloroacetic acid (final
concentration, 10%). The resulting pellet was washed three times with
1 ml of acetone and suspended in appropriate amounts of 20 m\i TrisHCl (pH 7.5). 2 mM DTT, 0.5 mM EGTA, and 10% glycerol after
vacuum evaporation using a centrifugal concentrator (Taiyo Kagaku
Co., Tokyo, Japan). The protein concentration of this sample was
determined by the dye-binding method of Bradford (32), and appropri
ate amounts of protein (usually 50 jig) were subjected to 0.1% sodium
dodecyl sulfate and 12.5% polyacrylamide gel followed by electrophoretic transfer to a nitrocellulose membrane. The membranes were
reacted with the antibodies, and immunoreactive proteins were stained
using the avidin-biotin peroxidase complex method (Vectastain; Vector
Laboratories, Burlingame, CA). Prestained sodium dodecyl sulfate-
polyacrylamide gel electrophoresis standards (Bio-Rad, Richmond, CA)
were used as the molecular weight standards. Quantitative estimation
of the level of each PKC isozyme was carried out densitometrically
with a Bio-Rad videodensitometer by scanning the immunoreactive
band after immediately photographing the visualized band. The area of
an individual peak was measured above background in densitometric
tracings and was expressed as the absorbance x mm. The signal was
then compared with that of known amounts of purified rat brain PKC
containing PKC-7, -ß,
and -a [30, 39, and 31%, respectively, as deter
mined by enzyme immunoassay (34)]. Routinely, at least three different
amounts of the purified rat brain PKC were included as standards to
quantify the level of each isozyme. The immunoreactive signals deter
mined by this method were proportional to increasing amounts of each
isozyme (Fig. 1). If the signal of the sample was off the limit of linearity
shown in Fig. 1, the loaded quantity of sample was adjusted until a
signal in the linear range was obtained.
RESULTS
Activity of Protein Kinase C from Leukemic Cells. Tables 1
and 2 summarize the PKC activities and the expressions of
three major isozymes recovered in the cytosolic and paniculate
fractions of various human leukemic cells. The activity of PKC
was recovered mainly from the cytosol fraction (average, 81%)
of all the leukemic cells. The averages of total amount of PKC
activities (expressed in pmol/min/108 cells) of all cases was
17.1 (range, 3.7-62.9) in the cytosol and 4.0 (range, 0.3-20.2)
in the particulate fraction, and those of specific activities (ex
pressed in nmol/min/mg protein) were 4.0 (range, 1.1-8.2) in
the cytosol and 1.7 (range, 0.3-4.1) in the particulate fraction.
Although there were considerable variations in the level of PKC
activity, even from patients with a similar clinical diagnosis,
the levels of the total amounts of PKC activity in both cytosol
and particulate fractions from AML cells were significantly
higher than those from ALL or B-CLL cells (P < 0.05). The
FAB classification and immunophenotypes of AML cells are
summarized in Table 3. AML cells, which simultaneously ex
pressed lymphoid surface antigens, such as CD2, CD7, and
CD 19, had a lower total amount of activity than did the usual
AML cells without lymphoid antigens, as shown in Table 4. In
the unusual AML cells, those with T-lymphoid surface antigens
(such as CD2 and CD7), which are thought to represent the
2.0
_*
20
40
60
80
Protein Kinase C Content
100
( ng )
Fig. 1. Quantification of protein kinase C isozymes by immunoblotting. PKC
purified from rat brain, which contained PKC--y. -rf, and -«(30. 39, and 31%,
respectively) was separated by sodium-dodecyl sulfate-polyacry lamide gel electro
phoresis and electrophorelically transferred to nitrocellulose sheets. Each sheet
was incubated with PKC->-, -rf-, and -«-specificantibodies (MC-la, MC-2a, and
MC-3a). and the immunoreactive bands were scanned with a Bio-Rad videoden
sitometer after photographing these bands. •.PKC--y; A, PKC-/}; •PKC-a.
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PROTEIN KINASE C ISOZYMES IN HUMAN LEUKEM1AS
Table I Activities and isozyme expressions of protein kinase C in cytosolic and paniculate fractions from AML cells
Isozyme expressions in these fractions were evaluated by immunoblot analysis with monoclonal antibodies specific for each isozyme and the levels were determined
using densitometric tracing of immunoreactive bands with purified rat brain protein kinase C as the standard.
cells)PKC-v32400021716236317135100000000406839533160330003202470°
expression (ng/108
cells14.48.49.86.98.28.716.59.326.920.128.98.519.228.121.425.412.310.131.162.95.99.742.113.35.91.10.71.71.92.44.22.29.10.32.73.
proteinCytosolic
Patient123456789101112131415161718192021222324123456789101112131415161718192021222324DiagnosisMlMlMlMlM2M
fraction4.42.23.83.63.15.75.51.16.35.55.73.34.46.72.74.01.71.83.87.53.52.57.93.1Paniculate
2M
2M
2M
2M2M2M2M
2M2M3M3M3M4M4M4M5aM5aM5bMSbMIMIMIMIM2M2M2M
fraction3.10.40.30.50.70.62.51.22.52.70.82.13.21.34.11.41.81.21.60.91.30.91.91.6Isozyme
2M2M2M
2M2M2M2M3M3M3M4M4M4M5aM5aM5bM5bpmol/min/10*
* No immunoreactive bands were detected in Patients 3 and 10.
transformed counterpart of an early stem cell before lineage
commitment and which exhibit distinct clinical and biological
features (35, 36), had significantly lower cytosolic and particulate total activities than did AML cells without such surface
antigens (P < 0.01). In contrast, in other unusual AML cells
associated with B-lymphoid surface antigens (such as CD 19),
the total amount of activity was similar to findings in the AML
cells. When we examined differences with respect to the specific
activity (nmol/min/mg protein), similar results were obtained,
but these differences were not as significant as the total amount
of PKC activity expressed on a per cell basis.
Fig. 2 shows differences in the level of total amount of PKC
activity in different FAB subgroups of AML. In the cytosol
fraction, FAB Ml cells exhibited lower and FAB M3, M4, or
M5b cells often exhibited higher PKC activity than did other
FAB subgroups. On the other hand, in the paniculate fraction,
Ml cells often showed lower and M3 cells higher activity than
other subgroups. In FAB subgroups, except for M3 and M5b,
blasts with T-lymphoid surface antigens were often observed
(40%), and such cells usually exhibited a lower level of activity.
These findings suggest that the level of PKC activity expressed
on a per cell basis may be relatively low in leukemic cells which
4273
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PROTEIN KINASE C ISOZVMES IN HUMAN LEUKEMIAS
Table 2 Activities and isozyme expressions of protein kinase C in cytosolic and
paniculate fractions from ALL and CLL cells
commonly observed in M4 and M5a cells. PKC-0 was a major
subtype in the cytosol of Ml, M5a, and M5b cells, and PKC-a
expression
was more abundant than PKC-ßin M3 cells. On the other
ActivityPatientpmol/min/10* cells)PKC-7PKC-t)PKC-nCytosolic
(ng/108
hand, in the particulate fraction, PKC-7 was usually a major
subtype in all FAB subgroups.
Diagnosis
cellsnmol/min/mg
proteinIsozyme
In the cytosol fractions from ALL cells, PKC-7, -ß,and -a
fractions252627282930313233343536373839CALL"CALLCALLcALLCALLT-ALLB-CLLBCLLB-CLLB-CLLB-CLLB-CLLB-CLLB-CLLB-CLL11.76.26.832.811.93.78.46.95.37.16.45.622.323.116
were present in 100, 100, and 83% of 6 patients, and, in the
particulate fractions, they were present in 83, 83, and 67% of 6
patients, respectively. On the other hand, in the cytosol frac
tions from B-CLL cells, PKC-7, -ft and -«were present in 100,
100, and 89% of 9 patients, and in the particulate fractions,
they were present in 89, 100, and 89% of 9 patients, respec
tively. In the cytosol fraction of either ALL or B-CLL cells,
PKC-7 was observed in all cases, but the level was relatively
low (less than 30%) as compared to findings in PKC-/3 and
PKC-«.PKC-ßwas usually the most predominant subtype of
cytosolic PKC from these lymphoid leukemic cells, while that
of membrane-associated PKC was 7-subtype in ALL or 0subtype in B-CLL cells.
fractions252627282930313233343536373839CALLCALLcALLCALLcALLT-ALLB-CLLB-CLLB-CLLB-CLLB-CLLBCLLB-CLLB-CLLB-CLL2.92.50.75.74.91.71.40.70.91.32.21.15.83.95.91.41.1
We found that 54% of the cells from 24 AML patients
meeting conventional FAB criteria expressed some T- or Blymphoid surface antigens (Table 3). It was reported that such
unusual AML were often associated with high peripheral WBC
(35). A large number of cells (2 x IO8cells) from each patient
" cALL. common ALL.
are considered to be morphologically and immunophenotypically immature and that PKC activity increases during the
course of myeloid cell differentiation.
Expression of Protein Kinase C Isozymes. The expression of
PKC isozymes in leukemic cells was examined by an immunoblot method using monoclonal antibodies MC-la, MC-2a, and
MC-3a which react specifically with PKC-7, -ft and -«from
rabbit brain, respectively (21). Quantification of these isozymes
by immunoblotting appears to be adequate because the immunoreactive signal is proportional to the amount of antigen
present (Fig. 1). Fig. 3 shows the results of immunoblotting in
representative cases. In cytosol fractions from the AML cells,
PKC-7, -ft and -a were present in 48, 96, and 96% of 24
patients, respectively, and, in the particulate fractions from
these cells, PKC-7, -ft and -«were present in 88, 84, and 72%
of 24 patients, respectively. These three isozymes exhibited
different modes of subcellular distribution. PKC-7 was re
covered mainly from the particulate fraction, as noted with
other tissues ( 18, 22), and usually a major subtype of membraneassociated PKC. On the other hand, PKC-ßand -«were dis
tributed mainly in the cytosol fraction, and either was usually
a major subtype of cytosolic PKC. The amount of PKC (7 + ß
+ a) in each leukemic cell type, determined by immunoblot
analysis, appeared to be consistent with the total amount of
PKC activity (Fig. 4).
We then analyzed the correlation between isozyme expres
sions and FAB subgroups of AML. In the cytosol fraction, FAB
Ml and M2 cells sometimes expressed PKC-7 (43% patients).
This type was not observed in M3 and M5b cells but was
was needed for the present investigations; therefore, these un
usual AML patients were more frequent in number than stated
in previous reports. Most of the usual AML cells without
lymphoid surface antigens did not express cytosolic PKC-7,
whereas 73% of the AML cells with such aberrant antigens
expressed PKC-7, 'ike lymphoid leukemic cells. However, the
expression of cytosolic PKC-7 did not seem to be obligatory
for the expression of such lymphoid surface antigens, since even
AML cells without PKC-7 sometimes expressed such lymphoid
surface antigens. In addition, in the cytosol of lymphoid anti
gen-associated AML cells, PKC-/S was usually a major subtype
as in ALL or CLL cells. These data indicate that the mode of
PKC isozyme expression in lymphoid antigen-associated AML
cells appears to resemble that of lymphoid leukemic cells.
Therefore, from the aspect of intracellular signal transduction
systems, such leukemic cells with mixed lymphoid and myeloid
phenotypes may be regarded as a distinct subgroup.
DISCUSSION
A multilineage hematopoietic growth factor, interleukin 3
has been shown to translocate and activate PKC in factordependent cell lines (14), and this translocation is associated
with increased survival and proliferation of the cells in the
absence of interleukin 3 (37). Using protein kinase inhibitors,
we found that the sequence of activation of protein kinase C
may be involved in the in vitro proliferation of myeloid progen
itor cells stimulated by four different CSFs, interleukin 3,
granulocyte-macrophage
CSF, granulocyte CSF, and macro
phage CSF (13). The majority of human myeloid leukemias
retain an absolute dependence on CSFs for growth, at least in
vitro, although the patterns of responsiveness of AML cells
from different patients to these three CSFs, whether alone or
together, can show a striking variability (38). PKC activation
was also required for T-cell proliferation in response to T-cell
receptor activation (39). Moreover, the overexpression of pro
tein kinase C is involved in the disordered proliferation of rat
fibroblasts (3). Investigations focusing on PKC activity and its
isozymic pattern as a signal transduction after stimulation with
4274
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PROTEIN KINASE C ISOZYMES IN HUMAN LEUKEMIAS
Results of immunophenotyping
Table 3 ¡mmunophenolypesofleukemic cells from AML patients
are given as a percentage of positive cells.
antigensCD34
CD13
Patient123456789101112131415161718192021222324FAB
typeMlMlMlMlM CD1457
622
ND"13
91
78
110
ND8
24
95
CD24286
CD19
5573
2ND371ND08471663503
ND
112
131
10
40
00
479
019
2233
50
2M2M2M
02
93
14
19
00
83
2M2M
00
59
08
92
037
64
2M
14337
89
2M2M2M2M3M3M3M4M4M4M5aM5aM5bM5bCDU
028
523
66
63
65
0ND
6ND
ND
03145265443295524
ND
33
21
065
94
96
58
40
00
06
4298
9460
547
99
99
75
360
530
51
140
94
40
687
74
229
4762
19
0152
ND
12CD7698239078526611840602201760928908la999552444556635799758163591223357905298989024"
78
66CD331186ND89950987787169587576999998787988694719320Surface
ND, not determined.Table
cellsResults
4Subcellular
SE.DiagnosisAML
are expressed as the averages ±
distribution and isozyme expression of protein kinase C in various human leukemic
cells)pmol/min/108
protein22.7
expression (ng/10s
cells
nmol/min/mg
Cytosol24
withoutlymphoid
Ag.°AML
withlymphoid
Ag.CD7(+)CD7
(-)ALLB-CLLAML
J9.5±2.4
>'269±24 i
±3.9
>r15.2±5.1 1
l207 ±47
±•
75404±
63>
±
80189
±
»
48697
346 ±
116661
±
45751
±
121270
±
±4.6
3.4
±3.3
39278
±
9189
±
|11.3 ±4.3
±4.1
i106 ±13
83549
±
93110
±
J5.9±2.4
±1.7
±20
25234
±
25131
+
121.9±1.2
i12.2 ±3.4
i>
75523
±
JB.
Paniculate375
withoutlymphoid
Ag.AML
withlymphoid
Ag.CD7(+)CD7
'L3.2±1.6 )
|J2.2±0.7
±1.5iU0.3
±+
r"J0.3
52255
±
41117±
±
1966
±
29188
±
21105
3432
±
o1.1
±2.0
17220
±
j4.3 ±0.4
10.4
*0.60.3PKC-7A.
f
'3.1 ±1.3 J
(-)ALLB-CLLNo.1113766911137669ActivityIsozyme
±2.0
±71113±
2577
12.6±0.8
±1.5
±0.50.50.60.61.00.80.3 ±14PKC-/Ì884
±0.7 J4.4
°Lymphoid Ag.. lymphoid cell surface-associated antigens, such as CD2, CD7. and CD 19.
* P < 0.05.
<P<Q.O\.
CSFs could be pertinent to clarify the regulatory abnormalities
of growth of leukemic cells.
The present data indicate that lymphoid leukemic cells from
ALL and CLL patients had significantly lower total amounts
of PKC activity than did the leukemic cells from AML patients,
although considerable variations were found in PKC activities
of leukemic cells from different patients. These findings may
be attributed to variations in PKC activity itself and differences
in the expression of PKC isozymes, but other factors such as
substrate specificities and cellular inhibitors may also affect the
altered level of activity. Differences in the specific activities of
2081
±
171 ±
±2998
8363
74 ±
261 ±
1743
±
18 ±20PKC-«825
±12
PKC between myeloid and lymphoid leukemic cells were not as
remarkable as those of the total amounts of the PKC activity.
The small differences in specific activity may mean that PKC
plays important roles in fundamental cellular responses.
Immunoblot analyses showed that the patterns of expressions
of PKC-7, -ß,
and -a isozymes also varied considerably among
leukemic cells from different patients and that the expression
of PKC isozymes was not differently affected in leukemic cells
in which the level of PKC activity was altered. Blast cells from
patients with the FAB subtype of M l had a lower cytosolic and
membrane-associated PKC activity than did those from patients
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PROTEIN KINASE C ISOZYMES IN HUMAN LEUKEMIAS
i/iu
A.
Cytosol^^
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The Amount of PKC Activity
cells)20
(pmol/min/108
cells)
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The Amount
(pg/108
0
0
0.5
ir
of PKC
1
i
without
Ag.AML
Lymphoid
4.00£•
with
Lymphoid
Ag.ALL
3Ou
r^^llli^^^11li^§IJ1
20Ci1
1
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iAML
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o1
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M 1
M 2
M 3
M 4
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M5
The Amount of PKC Activity
cells)10
(pmol/min/108
cells)
Fig. 1. Total amount of PKC activity in subcellular fractions of AML cells
grouped according to FAB classification.
0.5'
5
'LZ
The Amount of PKC
(pg/10»
0
0
without
Lymphoid
Ag.AML
with
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MC-2a
MC-3a
123456789
Fig. 4. Comparison between the total amount of PKC activity and the amount
of PKC in various leukemic cells. The amount of PKC in the cytosol (A) and the
particulate (B) fractions from various types of leukemic cells was determined by
kinase assay and by immunoblot analysis. Each column shows the average of the
amount of PKC activity per cell, as determined by kinase assay, and average of
the amount of PKC (the total of 7, ß,and a isozymes), as determined by
immunoblot analysis. Lymphoid Ag., lymphoid cell-surface associated antigen.
with the FAB subtype of M2 through M5, although the limited
number of cases does not allow for a definite conclusion re
garding the relationship of PKC activity (and isozyme pattern)
and FAB subtype of AML. There was no direct correlation
between the potential of the cells to respond to growth factors
and the level of PKC activity (or pattern of the isozymic
expression) in AML cells.4 AML cells with T-lymphoid features
10 11
B
MC-1a
(such as CD7) exhibited a reduced activity of PKC in both the
cytosol and particulate fractions. Such leukemic cells are
thought to arise from malignant transformation of early pro
genitor cells capable of differentiating into more than one
lineage ("lineage promiscuity") (28). Shoji et al. (40) found that
MC-2a
MC-3a
123456789
10 11
Fig. 3. Immunoblot analysis of protein kinase C fractions from various leukemic cells. The cytosol (A) and paniculate (B) fractions prepared from various
Icukemic cells (2 X 10*each) were fractionated batchwise with 0.15 M NaCI from
DEAE-cellulose column, and protein kinase C fractions (SO >igof protein) were
subjected to immunoblot analysis with monoclonal antibodies MC-la, MC-2a,
and MC-3a (specific for PKC--y, -fi, and -«,respectively), as indicated in the
corresponding lanes. Arrows, positions of protein kinase C, as determined by the
molecular weight standards. Lane I, AML M l (Patient 4); Lane 2, CD7(+) AML
Ml (Patient 2); Lane 3. AML M2 (Patient 13); Lane 4, CD7(+) AML M2
(Patient 6); Lane 5, CD19(+) AML M2 (Patient 9); Lane 6, AML M3 (Patient
16); Lane 7, AML M4 (Patient 19); Lane 8, CD7(+) AML M5a (Patient 21);
Lane 9. AML M5b (Patient 23); Lane 10, common ALL (Patient 28); Lane II.
B-CLL (Patient 32).
the levels of PKC activity in the cytosol and particulate fractions
from human myelogenous leukemia KG-1 cells were about 3
times higher than those from its immature subline, KG-la cells.
HL-60 variant subline, which is resistant to the induction of
differentiation by phorbol ester, has a 3 times lower PKC
activity than the parental HL-60, due to a decreased expression
of PKC-/? (10). PKC activity may be relatively lower in the
leukemic cells arresting at the stage of early pluripotent stem
cells and may increase during the course of maturation toward
myelomonocytic lineage. Blast cells separated from an individ
ual patient are thought to consist of various subgroups, with
subtle differences in immunophenotypes, molecular genetics,
or responses to hematopoietic growth factors. More detailed
4 Unpublished observation.
4276
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PROTEIN KINASE C ISOZYMES IN HUMAN LEUKEMIAS
fractionations of leukemic cells based on these characteristics
may reveal more apparent differences in the levels of PKC
activity of various leukemic cells.
A growing body of literature on the characteristics of individ
ual PKC isozymes suggests that each isozyme may regulate
individually a unique cellular physiological response. Use of
monoclonal antibodies specific for the three protein kinase C
isozymes has shown that the three isoforms do not share a
common distribution pattern in leukemic cells. PKC-« was
expressed to variable extents and ubiquitously in the cytosol of
leukemic cells. This type, commonly observed in various tissues
(16), phosphorylates an epidermal growth factor receptor most
rapidly among the three major isozymes (41). These findings
suggest that this isozyme may play roles in fundamental cellular
responses, such as cell proliferation. PKC-/3 was also expressed
ubiquitously in the cytosol and paniculate of leukemic cells and
was usually a major subtype of cytosolic PKC, especially in
leukemic cells with lymphoid phenotypes. This isozyme was
activated preferentially, translocated from the cytosol to the
paniculate, and down-regulated most rapidly in the course of
phorbol ester-induced HL-60 cell differentiation (IO).5 Other
investigators reported that PKC-/3 might be a critical signal
component in the secretory response of rat leukemic cell line
(42). It had been reported that PKC-7 was expressed solely in
the brain and spinal cord (16), but, recently, several investiga
tors, including our group, reported that some myeloid leukemic
cells expressed this isozyme, albeit a relatively minor compo
nent (10, 12, 25, 26). This type PKC expressed in COS cells
transfected with 7-complementary DNA displayed a higher
affinity for the paniculate fraction than did other types (18).
PKC-7 appears to be more lipophilic than PKC-/3 and -«.We
also found that PKC-7 was commonly observed in the panicu
late fractions from all types of leukemic cells. The expression
of this type in the cytosol appeared to be related to the lymphoid
phenotype of the leukemic cells. It is tempting to speculate that
these differences in PKC isozyme expression between AML
cells with and without lymphoid phenotypes, especially CD7,
may account for one of the distinct biological and clinical
features of mixed lineage leukemia.
Worthy of consideration is the possibility that the expression
of protein kinase C isozymes may be regulated differently
during the maturation of a hematopoietic cell. It remains to be
determined whether variability in the levels of PKC isozyme
expression is regulated at the level of transcription, mRNA
stabilization, or protein turnover. Moreover, at least three other
isozymes (6, t, and f) have been isolated from a rat brain library
in addition to a, ß,and 7 isozymes (16). We are currently
investigating the subcellular expression of these additional iso
zymes in leukemic cells from patients, in parallel with analyses
of other protein kinases, such as cyclic AMP-dependent protein
kinase and tyrosine protein kinase in these leukemic cells.
Further investigations on these intracellular protein kinases in
various leukemias will reveal the roles of these enzymes in the
processing of various biological responses not only in trans
formed leukemic cells but also in normal hematopoietic cells.
ACKNOWLEDGMENTS
We thank Dr. K. Kita for kind cooperation and M. Ohara for critical
comments.
' Unpublished data.
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Expression of Three Major Protein Kinase C Isozymes in
Various Types of Human Leukemic Cells
Fumihiko Komada, Masakatsu Nishikawa, Yasuhiro Uemura, et al.
Cancer Res 1991;51:4271-4278.
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