Báo cáo khoa học: Protein kinase Ch activity is involved in the 2,3,7,8tetrachlorodibenzo-p-dioxin-induced signal transduction pathway leading to apoptosis in L-MAT, a human lymphoblastic T-cell line potx

Protein kinase Ch activity is involved in the 2,3,7,8-
tetrachlorodibenzo-p-dioxin-induced signal transduction
pathway leading to apoptosis in L-MAT, a human
lymphoblastic T-cell line
Sohel Ahmed, Masahiko Shibazaki, Takashi Takeuchi and Hideaki Kikuchi
Department of Molecular Genetics, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan
The immune system is recognized as a consistent and
sensitive target for the toxic widespread environmental
pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)
and its congeners [1]. Triggering of apoptosis in both
thymocytes [2] and T cells [3,4] has clearly emerged as
a hallmark of TCDD immunotoxicity, as shown by
in vivo studies in animal models. Although an in vitro
study has also revealed that TCDD directly causes
apoptotic death in immature thymocytes [5], no such
direct effect of TCCD has been observed in vitro in T
cells from animal models [6]. However, we have clearly
shown that TCCD can directly induce apoptosis in
some cultured human T-cell lines [7]. In addition, we
have evaluated the immunotoxicity of TCDD and
Keywords
dioxin; apoptosis; PKCh; lymphoblastic T
cell; rottlerin
Correspondence
H. Kikuchi, Department of Biochemistry and
Biotechnology, Faculty of Agriculture and
Life Science, Hirosaki University, 3 Bunkyo-
cho, Hirosaki 036-8561, Japan
Fax: +81 172 39 3586
Tel: +81 172 39 3586
E-mail:

(Received 17 June 2004, revised 7 September
2004, accepted 8 December 2004)
doi:10.1111/j.1742-4658.2004.04519.x
The aromatic hydrocarbon receptor (AhR)-dependent pathway involved
in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced immunotoxicity
has been studied extensively, but the AhR-independent molecular mechan-
ism has not. In previous studies we found that the AhR is not expressed
in L-MAT, a human lymphoblastic T-cell line. In this report, we provide
the following evidence that the protein kinase C (PKC)h activity is func-
tionally involved in the AhR-independent signal transduction mechanism
that participates in the TCDD-induced L-MAT cell apoptosis. First, only
rottlerin, a novel PKC (nPKC)-selective inhibitor, blocked the apoptosis
completely, in a dose-dependent manner. Second, PKCh was the major
nPKC isoform (compared to PKCd) expressed in the L-MAT cell line.
Third, a cell-permeable myristoylated PKCh pseudosubstrate peptide
inhibitor also blocked the apoptosis completely, in a dose-dependent man-
ner. Fourth, both rottlerin and myristoylated PKCh pseudosubstrate pep-
tide inhibitor completely inhibited PKCh kinase activity in vitro at doses
that effectively blocked TCDD-induced L-MAT cell apoptosis. TCDD
treatment induced a time-dependent activation of nPKC kinase activity in
L-MAT cells, and moreover, TCDD induced a translocation of PKCh
from the cytosolic fraction to the particulate fraction in L-MAT cells.
Finally, transient over-expression of a dominant negative PKCh (a kinase-
dead mutant, K ⁄ R 409) in L-MAT cells conferred significant protection
against TCDD-induced apoptosis.
Abbreviations
AcDEVD-AMC, acetyl-Asp-Glu-Val-Asp ⁄ 7-amino-4-methylcoumarin; AhR, aromatic hydrocarbon receptor; DN PKC, dominant negative protein
kinase C; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; JNK, c-Jun N-terminal kinase; PKC, protein kinase C; NaCl ⁄ P
i
, phosphate-

buffered saline; nPKC, novel protein kinase C; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; myr-PKCh-PPI, myristoylated-PKCh-pseudosubstrate
peptide inhibitor.
FEBS Journal 272 (2005) 903–915 ª 2005 FEBS 903
some of its congeners by demonstrating the activation
of caspase-3, as a sensitive marker, using L-MAT as a
model T-cell line [8].
The role played by the aromatic hydrocarbon recep-
tor (AhR)-mediated pathway in TCDD immunotoxicity
has been well studied [1]. However, some of the immu-
notoxic effects induced by TCDD are known not to be
dependent on an AhR gene locus [9,10], and we have
already confirmed, by using the human lymphoblastic
T-cell line, L-MAT, as the model, that the AhR-medi-
ated pathway is in no way involved in TCDD-induced
apoptosis, [7,8]. Furthermore, neither TCDD-mediated
apoptosis in mouse thymoma cells (EL-4) [11] nor poly-
chlorinated biphenyl (Aroclor 1254)-mediated apoptosis
in mouse spleen cells [12], can be explained by the single
AhR pathway. The molecular mechanism involved in
the AhR-independent pathway(s) leading to TCDD-
induced immunotoxicity is not clearly understood, and
indeed the lack of a suitable system in which this immu-
notoxicity can be readily detected and demonstrated in
a regulated manner has hindered the research efforts.
The rapidity by which L-MAT cell apoptosis is
induced by TCDD, and the failure of actinomycin D
(an inhibitor of gene transcription) and cycloheximide
(an inhibitor of de novo protein synthesis) to block this
apoptosis, led us to focus on the possibility of a rapid
post-translational signal transduction mechanism [7,8].

Many reports in the last decade have suggested that
protein phosphorylation by protein kinase C (PKC)
plays a key role in the regulation of the signal trans-
duction mechanism involved in TCDD-induced cellular
responses [13,14]. To date, a total of 11 isoforms of
PKC have been reported [15]. Although a few studies
have shown isoform-specific PKC activation as a cellu-
lar response to TCDD [16,17], a functional role for
isoform-specific PKC activity in the mediation of
TCDD immunotoxicity has not yet been shown at the
molecular level.
Recently, in an in vivo study, TCDD was implicated
in the enhancement of an activation-induced cell death
mechanism (AICD) involved in T-cell apoptosis [18].
Furthermore, activation of PKCh and its kinase acti-
vity have been implicated in the AICD mechanism in
human T-leukemic Jurkat cells [19,20]. PKCh, a novel
PKC (nPKC) isoform, is characterized both by its
unique tissue distribution (in skeletal muscle, lymphoid
organs, and hematopoietic cell lines, particularly
T cells [21]) and by its isoenzyme-specific activation
requirements and substrate preferences in vitro [21,22].
The unique expression profile and functional proper-
ties of PKCh led us to believe that it may play a spe-
cialized role in many signal transduction events in
T cells.
In the present study, we examined the functional
participation of the PKC pathway, and in particular
attempted to explore the possible role of PKCh in the
TCDD-induced AhR-independent signal transduction

mechanism involved in L-MAT cell apoptosis.
Results
Inhibition of TCDD-induced L-MAT cell apoptosis
by rottlerin, an nPKC inhibitor
12-O-Tetradecanoyl phorbol-13-acetate (TPA) was used
in combination with TCDD to clarify the involvement
of PKC in the signal transduction mechanism participa-
ting in TCDD-induced cellular responses [14]. We also
looked for evidence of the involvement of PKC in
the signal transduction mechanism of TCDD-induced
L-MAT cell apoptosis. We used several PKC-selective
inhibitors to determine whether PKC is functionally
involved in the L-MAT cell apoptosis induced by
TCDD and attempted to identify the specific PKC iso-
form(s) involved in the process. Pre-treatment with a
nonspecific PKC inhibitor, staurosporine [23], caused
only a partial inhibition of the apoptosis (Fig. 1A), and
no inhibitory effect was observed in the case of the clas-
sical PKC-selective inhibitor, Go
¨
6976 [24] (Fig. 1B).
Only pretreatment with rottlerin, an nPKC-selective
inhibitor [25], resulted in the complete inhibition of
TCDD-induced L-MAT cell apoptosis (Fig. 1C) at
doses suggestive of inhibition of PKCd and PKCh.
These results indicate that nPKC (PKCd and ⁄ or PKCh)
is involved in the TCDD-induced apoptosis of L-MAT
cells.
The incubation of L-MAT cells with 20 nm TCDD
resulted in morphological changes characteristic of

apoptosis upon staining with the DNA-specific fluoro-
chrome bis-benzinide (Fig. 2, TCDD). However, the
pretreatment of L-MAT cells with rottlerin (20 lm)
inhibited the alteration of morphology in nuclei treated
with TCDD (Fig. 2, TCDD + Rottlerin).
PKCh is a major isoform of nPKC in the L-MAT
cell line
The expression of PKCh was detected in L-MAT cells
at both mRNA (Fig. 3A) and protein (Fig. 3B) levels.
HepG2, a human hepatoma cell line, was used as a
negative control for PKCh. In HepG2 cells, neither
PKCh mRNA nor PKCh protein were detected, as
shown in Fig. 3. The Jurkat cell line was used as a
positive control for PKCh. Next, the expression level
of PKCh was compared to that of PKCd by using iso-
form-specific antibodies, with purified PKCh and
PKCh in TCDD-induced signaling for apoptosis S. Ahmed et al.
904 FEBS Journal 272 (2005) 903–915 ª 2005 FEBS
PKCd as standards. In L-MAT cells, PKCh was the
major isoform, the expression level of PKCd being less
than one-tenth that of PKCh (Fig. 3C).
TCDD-induced L-MAT cell apoptosis is completely
blocked by myristoylated-PKCh-pseudosubstrate
peptide inhibitor (myr-PKC h PPI)
Having made the above experimental observations, we
sought to confirm the functional involvement of nPKCs,
specifically PKCh, in the L-MAT T-cell apoptosis
induced by TCDD. Synthetic peptides corresponding to
the pseudosubstrate domains of PKC have been used as
specific inhibitors of PKC in in vitro assays. N-myristoy-

lation of such synthetic PKC-specific pseudosubstrate
peptides permits their use as selective, cell-permeable
inhibitors of PKC in intact cells [26]. Therefore, we
examined the effect of a PKCh pseudosubstrate peptide-
inhibitor myristoylated at its N-terminal site. The
L-MAT cell apoptosis induced by TCDD was com-
pletely inhibited in the presence of 20 lm myr-PKCh
PPI, as shown in Fig. 4, confirming the involvement of
nPKCs.
PKCh kinase activity is completely inhibited by
rottlerin and by myr-PKCh-PPI in vitro. To test
indirectly whether PKCh might be a target for both
rottlerin and myr-PKCh-PPI in the inhibition of
TCDD-induced L-MAT cell apoptosis, we performed
an in vitro kinase assay for PKCh. This confirmed that
both rottlerin and myr-PKCh PPI strongly inhibited
PKCh kinase activity (Fig. 5) at doses that completely
blocked TCDD-induced L-MAT cell apoptosis.
TCDD induces nPKC kinase activity in L-MAT
cells
To establish whether TCDD increases nPKC kinase
activity, we examined nPKC kinase activity by using
PKCh pseudo-substrate, which is preferentially phos-
phorylated by PKCh and PKCd, in whole L-MAT
cells exposed to TCDD for different time-periods. We
found that the kinase activity of nPKC increased in a
time-dependent manner, as shown in Fig. 6. In a previ-
ous report on the apoptotic cell-death mechanism in
immature CD4
+

CD8
+
mouse thymocytes, a specific
activation of nPKCs was shown to be responsible for
the induction of apoptosis by glucocorticoids and
diterpine ester ingenol 3, 20-dibenzoate (IDB) [27].
Therefore, both PKCh and PKCd may be activated in
L-MAT cells following treatment with TCDD.
PKCh is translocated in TCDD-treated L-MAT cells
We performed an nPKC translocation assay by fractiona-
ting L-MAT cells into cytosol and particulate fractions
and then examining the translocation of each of the
nPKC isoforms in L-MAT cells treated with TCDD (by
Fig. 1. Effects of protein kinase C (PKC) inhibitors on the
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced apoptosis of
L-MAT cells. L-MAT cells (10
5
cellsÆ100 lL
)1
per well in a 96-micro-
well plate in serum-free RPMI 1640) were preincubated with
PKC-selective pharmacological inhibitors (as indicated) for 30 min at
37 °C in 95% air and 5% CO
2
, followed by treatment with TCDD
for 3 h. Then, a caspase-3 activation assay was performed as
described in the Experimental procedures. (A) Staurosporine, (B)
Go
¨
6976, and (C) rottlerin. Data are shown as average values ± SD

(n ¼ 3). *P < 0.05; **P > 0.01 vs. TCDD alone (Student’s t-test).
S. Ahmed et al. PKCh in TCDD-induced signaling for apoptosis
FEBS Journal 272 (2005) 903–915 ª 2005 FEBS 905
comparison with untreated L-MAT cells). Our data
clearly revealed that PKCh, not PKCd, was the nPKC
isoform activated in L-MAT cells treated with TCDD,
as shown in Fig. 7. Although the level of PKCd was low
because of the low expression of this isoform (Fig. 7),
the loading amounts (40 lg of protein) were sufficient to
allow the detection of change of PKCd in the particulate
fraction. Finally, we sought to verify the functional
involvement of PKC h kinase activity in TCDD-induced
L-MAT cell apoptosis at the molecular level.
Transfected L-MAT cells express H-2K
K
on their
surface
To test whether PKCh kinase activity is required in the
pathway of TCDD-induced L-MAT cell apoptosis, we
examined the effect of a dominant negative PKCh (DN
PKCh; a kinase-dead mutant, K ⁄ R 409) [22]. To separ-
ate (on the miniMACS column) transfected cells from
the mixture of electroporated cells containing pMACS
plasmid DNA, expression of H-2K
K
on the cell surface
is essential. Therefore, prior to their magnetic separation
we checked the L-MAT cells transfected with empty
pMACSK
K

.II or DN PKCh-FLAG ⁄ pMACSK
K
.II
DNA to determine whether they expressed the H-2K
K
molecule on their surface. Direct immunofluorescence
microscopy of these transfected cells did indeed reveal
the expression of H-2K
K
on their surface (Fig. 8A).
Over-expression of DN PKCh in L-MAT cells
confers protec tion a gainst TCDD-induced apoptosis
Over-expression of DN-PKCh would interfere with
endogenously expressed PKCh and specifically sup-
press its kinase activity. To confirm that PKCh kinase
activity really does participate in the signal transduc-
tion mechanism involved in TCDD-induced L-MAT
cell apoptosis, transfected L-MAT cells expressing
H-2K
K
were separated from the nontransfected cells in
the miniMACS column and then tested for caspase-3
activation by treatment with TCDD. In this experi-
ment, TCDD-induced caspase-3 activation (that is,
apoptosis) was significantly reduced in L-MAT cells
transfected with DN PKCh-FLAG ⁄ pMACSK
K
.II
DNA (as compared to that in cells transfected with
empty pMACSK

K
.II) (Fig. 8B). As the next step, the
expression of DN PKCh-3·FLAG of the same con-
struct in L-MAT cells was confirmed by immunopre-
cipitation and Western blotting (Fig. 8C).
Discussion
Participation of PKCh in TCDD-induced apoptosis
The L-MAT cell apoptosis induced by TCDD was
completely blocked by rottlerin, now well established
as an nPKC inhibitor [25]. A number of studies have
demonstrated that rottlerin acts solely as a specific
inhibitor of many PKCh functions in T cells [28].
Rottlerin has also been shown to block the activation-
induced cell death process in T cells, indicating a func-
tional role for PKCh in the cell-death mechanism
[19,20]. In our study, PKCh expression was detected at
both the mRNA and protein levels in L-MAT T cells.
This observation strengthens the possibility of a func-
tional involvement of PKCh in TCDD-induced
L-MAT cell apoptosis. However, rottlerin was origin-
ally reported as a novel ATP-competitive protein
Fig. 2. Morphological alterations in chroma-
tin. L-MAT cells were treated with solvent
only (Cont), with 20 n
M 2,3,7,8-tetrachlo-
rodibenzo-p-dioxin (TCDD) or 20 l
M rottlerin,
or with the combination of TCDD + rottlerin.
Cells were collected after 4 h and fixed in
paraformaldehyde, then stained with the

DNA-specific fluorochrome, bis-benzimide
(Hoechst 33258).
PKCh in TCDD-induced signaling for apoptosis S. Ahmed et al.
906 FEBS Journal 272 (2005) 903–915 ª 2005 FEBS
kinase inhibitor with a very high selectivity for PKCd
[25], and it was later used widely as a selective inhib-
itor for nPKCd, both in vitro and in studies of intact
cells [29]. Human primary T cells have been reported
to express PKCd [30], and we found here that L-MAT
cells did indeed express PKCd, as we were able to
detect PKCd mRNA and protein by RT ⁄ PCR and
Western blotting methods, respectively (Fig. 3A,B).
However, the involvement of PKCd seemed to be less
important, as judged from our analysis of the dose–
response effect of rottlerin on L-MAT cell apoptosis.
If PKCd really is involved, a much lower concentra-
B
A
C
Fig. 3. Detection of the expression of endogenous protein kinase
Ch (PKCh). The expression of endogenous PKCh and PKCd was
detected in the L-MAT cell line at the mRNA and protein levels. (A)
RT-PCR analysis of L-MAT cell mRNA for PKCh. HepG2 cell mRNA
was used as a negative control. Glyceraldehyde 3-phosphate dehy-
drogenase (GAPDH) detection was performed as a control. Total
RNA was extracted from 2 · 10
7
L-MAT or HepG2 cells. Then,
10 lg of the total RNA was reverse transcribed to cDNA and ana-
lyzed for PKCh mRNA along with GAPDH. (B) Western blotting ana-

lysis of endogenous PKCh and PKCd expression. Whole cell lysates
were prepared from 2 · 10
7
HepG2, L-MAT and Jurkat cells, and
100 lg of total protein was probed for PKCh expression by using
the Western blotting method. HepG2 and Jurkat cell lysates were
used as negative and positive controls, respectively. (C) Evaluation
of the protein levels of PKCh and PKCd in L-MAT cells by using a
quantitative Western blotting method. The assay system was opti-
mized to resolve 20 and 40 lg of total L-MAT WCL (whole cell
lysate) protein for the evaluation of comparable expressions of
PKCh and PKCd, respectively. Suitable dilutions of the purified
enzymes were used as standards for PKCh and PKCd. The upper
panel shows the amount of PKCh (in 20 lg of total protein)
expressed in L-MAT cells, which seemed to be around 5 ngÆlg
)1
of
total L-MAT cell protein. The lower panel shows the amount of pro-
tein PKCd (in 40 lg of total protein) expressed in L-MAT cells,
which seemed to be 500 pgÆlg
)1
of total L-MAT cell protein.
Fig. 4. The effect of myristoylated-PKCh pseudosubstrate peptide
inhibitor (myr-PKCh-PPI) on the apoptosis of 2,3,7,8-tetrachloro-
dibenzo-p-dioxin (TCDD)-induced L-MAT cells. L-MAT cells (10
5
cellsÆ
100 lL
)1
per well in serum-free RPMI 1640 in a 96-microwell plate)

were pretreated with myr-PKCh-PPI (as indicated) for 30 min at
37 °C in 95% air and 5% CO
2
, followed by treatment with TCDD
for 3 h. Then, a caspase-3 activation assay was performed for the
evaluation of apoptosis. Data are presented as average values ± SD
(n ¼ 3). (*P < 0.05; **P > 0.01 vs. TCDD alone; Student’s t-test.)
S. Ahmed et al. PKCh in TCDD-induced signaling for apoptosis
FEBS Journal 272 (2005) 903–915 ª 2005 FEBS 907
tion of rottlerin should have blocked the apoptosis
completely, as demonstrated by others [29].
Moreover, we found that PKCh, but not PKCd, was
activated in TCDD-treated L-MAT cells. We suggest
that TCDD treatment of L-MAT cells induces signal
transduction, leading to very rapid activation of PKCh,
as a significant translocation of PKCh to the particu-
late fraction from the cytosolic fraction (Fig. 7) was
observed within 1 min of TCDD treatment in L-MAT
cells (with the peak being reached at 20 min and the
level sustained until 120 min, after which it decreased).
However, there was no significant change in the case of
PKCd. We also compared the expression level of
PKCd, in the form of protein, to that of PKCh in
L-MAT cells (Fig. 3C), and found the PKCd expres-
sion level ( 500 pgÆlg
)1
) to be at least 10 times lower
than that of PKCh (5 ngÆlg
)1
). Altogether, it was

suggested that the time-dependent increase of nPKC
kinase activity in TCDD-treated L-MAT cells (Fig. 6)
was mainly a result of the activation of PKCh.
Apoptosis is a multistage process. The increase of
nPKC kinase activity (100 min, Fig. 6) preceded the
apoptotic responses, as described below. Early change
was observed in the induction of JNK activity within
30 min upon TCDD treatment [7]. The caspase-3 acti-
vation by proteolytic cleavage [the maximal decrease
of procaspase-3 at 240 min of treatment with TCDD
was detected by Western blotting, as was the caspase-3
activity (peak activity at 240 min of TCDD-treatment]
was detected by using the kinetic assay (S. Ahmed,
PhD Thesis, 2004, Department of Molecular Genetics,
Institute of Development, Aging and Cancer, Tohoku
University, Sendai, Japan). The appearance of apop-
totic morphology (peak at 180–240 min of treatment
of TCDD) was observed by fluorescence microscopy
(shown in Fig. 2, TCDD only at 240 min). Although
we do not have any evidence of a direct interaction, it
is possible that these events produce a cascade reaction
to the TCDD-mediated apoptosis of L-MAT cells.
Considering the results of all the experiments des-
cribed above, we can conclude that a major part of the
signal transduction to this apoptosis was mediated by
PKCh, although we cannot completely rule out the
participation of PKCd in the apoptosis. Therefore,
even if PKCd is involved in TCDD-induced L-MAT
T-cell apoptosis, it would seem to be far less important
than PKCh.

Fig. 5. Effects of rottlerin and myristoylated-PKCh pseudosubstrate
peptide inhibitor (myr-PKCh-PPI) on PKCh kinase activity in vitro.
The kinase assay was performed by using 10 ng of a purified
human recombinant PKCh enzyme in a reaction mixture that con-
tained 50 l
M ATP, 40 lM of a biotinylated PKCh pseudosubstrate
peptide, and 4 lg of phosphatidylserine with or without rottlerin
(20 l
M) or myr-PKCh-PPI (20 lM). Data are presented as average
values ± SD (n ¼ 3). (**P > 0.01 vs. PKCh alone; Student’s t-test.)
Fig. 6. Time-dependent effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin
(TCDD) on L-MAT cell novel protein kinase C (nPKC) activity. Whole
cell lysate was prepared from 1 · 10
7
L-MAT cells [treated (j)or
not treated (s) with TCDD (20 n
M) on ice] by sonication for 5 s
with a Branson Sonifier Cell Disruptor (Branson Ultrasonic Corpora-
tion, Danbury, CT, USA) equipped with a microtip (output set at 4)
in 100 lL of buffer containing 50 m
M Tris ⁄ HCl, pH 7.5, 150 mM
NaCl, 0.1 mM Na
3
VO
4
, 0.1 mM Na
4
P
2
O

7
12H
2
O, 1 mM NaF, 0.1 mM
phenylmethanesulfonyl fluoride freshly supplemented with
1 · Complete EDTA-free Protease Inhibitor Cocktail (Roche Diag-
nostics GmbH, Mannheim, Germany). Following centrifugation
(16 000 g,20min,4°C), the supernatant was transferred to
fresh microcentrifuge tubes. Then, 8 lg of total cell protein was
examined in an in vitro nPKC kinase assay, using as the substrate
biotin-PKCh pseudosubstrate peptide, which is a rather selective
substrate for PKCh and PKCd.
PKCh in TCDD-induced signaling for apoptosis S. Ahmed et al.
908 FEBS Journal 272 (2005) 903–915 ª 2005 FEBS
Possible events downstream of PKCh
in TCDD-induced apoptosis
In a previous report, we showed that c-Jun N-terminal
kinase 1 (JNK1) is rapidly activated in L-MAT cells,
and that a dominant negative mutant of JNK prevented
TCDD-induced cell death [7]. Ghaffari-Tabrizi et al.
and others have demonstrated that the transfection of
constitutively active PKCh A408E activates both JNK1
and its upstream activating kinase, SEK1 ⁄ MKK4, in
a T-cell-specific manner [31], although the immediate
target for PKCh-mediated phosphorylation in the
SEK1 ⁄ JNK pathway is unknown [28]. Therefore, in
TCDD-induced L-MAT cell apoptosis it is possible that
PKCh activation somehow conveys its signal to JNK1,
leading finally to caspase 3 activation. However, we still
do not know the details of the signal pathway upstream

of PKCh, or the identity of the first molecule that inter-
acts with TCDD and conveys the signal on the down-
stream side.
Significance of TCDD-induced L-MAT cell
apoptosis
Although it is generally considered that the AhR-
dependent pathway mediates the major part of TCDD
immunotoxicity [32], not all examples of this immuno-
toxicity can be explained by using the single AhR
model, as AhR-independent mechanisms exist by
which TCDD can exert immunotoxic effects [9,10]. In
previous studies, we attested that AhR cannot be
involved in TCDD-induced apoptosis in the human
T-cell line, L-MAT, because AhR does not exist in
L-MAT [7,8]. In view of our previous findings and the
very high susceptibility to TCDD among T-cell lines
(A. Hossain, unpublished data), we would like to
emphasize the value of L-MAT as a model for study-
ing the AhR-independent molecular mechanism
involved in the immunotoxicity of TCDD.
In summary, we suggest that PKCh kinase activity is
functionally involved in the TCDD-induced signal
transduction mechanism leading to L-MAT cell apopto-
sis. This study clearly demonstrates the importance of
the PKC pathway in TCDD-induced immunotoxicity.
Experimental procedures
Cell culture
L-MAT T cells were cultured, as described previously [7],
with some modifications. Briefly, they were grown in
25 mm Hepes-supplemented RPMI 1640 (ICN Biomedicals

Inc., Irvine, CA, USA), pH 7.4, containing 5% fetal bovine
serum, 100 IU ÆmL
)1
penicillin, and 0.1% (v ⁄ v) streptomy-
cin at 37 °C in 95% air and 5% CO
2
. Jurkat T cells were
maintained under the same conditions in RPMI 1640 of
similar composition, pH 7.4, containing 10% (v ⁄ v) fetal
bovine serum. HepG2 cells were maintained in DMEM
(Dulbecco’s modified Eagle’s medium) (Gibco, Invitrogen
Corporation, Grand Island, NY, USA), pH 7.4, supplemen-
ted with 10% (v ⁄ v) fetal bovine serum, 100 IUÆmL
)1
peni-
cillin, and 0.1% (v ⁄ v) streptomycin at 37 °C in 95% air
and 5% CO
2
.
Apoptosis assay by determination of acetyl-
Asp-Glu-Val-Asp ⁄ 7-amino-4-methylcoumarin
(AcDEVD-AMC) cleavage
Throughout the study we used the detection of caspase-3
activation to evaluate apoptosis in L-MAT cells, as des-
cribed previously [8]. This entailed some modifications of
the method of Nicholson et al. [33]. We observed that the
apoptosis of L-MAT cells by TCDD could be induced in
the presence of 5% (v ⁄ v) fetal bovine serum; however,
serum starvation resulted in sensitization of the cells to
TCDD. Therefore, serum starvation conditions were used

in these experiments. Cells growing exponentially in RPMI
1640 containing 5% (v ⁄ v) fetal bovine serum were collec-
ted, and fresh medium without serum was added. Under
these conditions, cells were grown for another 4–6 h at
Fig. 7. The effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on
L-MAT cells. Novel protein kinase C (nPKC) activation by transloca-
tion. L-MAT cells (1 · 10
7
), treated or not treated with TCDD (as
indicated in the text), were fractionated. Then, 40 lg of the particu-
late (membrane + cytoskeleton) fraction protein was examined by
Western blotting to determine whether TCDD treatment caused
translocation of PKCh and PKCd from the cytosolic to the particu-
late fraction in L-MAT cells. The upper panel represents PKCh, the
lower panel PKCd.
S. Ahmed et al. PKCh in TCDD-induced signaling for apoptosis
FEBS Journal 272 (2005) 903–915 ª 2005 FEBS 909
37 °C in 95% air and 5% CO
2
. Then, after collection and
washing once with phosphate-buffered saline (NaCl ⁄ P
i
),
cells were incubated at a density of 10
5
Æ100 lL
)1
per well in
96-microwell plates (Falcon 3072; Becton Dickinson,
Franklin Lakes, NJ, USA) in serum-free RPMI 1640, either

in the presence of TCDD or in the presence of an equal
1
2
34
A
B
C
Fig. 8. Effect of the over-expression of dominant negative protein kinase C h (DN PKCh) on 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-
induced L-MAT cell apoptosis. (A) Detection of H-2KK expression in L-MAT cells by direct immunofluorescence microscopy. L-MAT cells
were transfected with empty pMACSKK.II or with DN PKCh-FLAG ⁄ pMACSKK.II DNA (10 lg) by using electroporation. After 44 h of transfec-
tion, L-MAT cells were directly immunostained with anti-mouse H-2KK immunoglobulin conjugated to fluorescein isothiocyanate (FITC), then
examined under a fluorescence microscope. The two pools of L-MAT cells were photographed using a digital camera at the same exposure.
(A1,3) Bright field, (A2,4) green fluorescence. (A1,2) L-MAT cells transfected with empty pMACSKK.II (A3,4) L-MAT cells transfected with
DN PKCh-FLAG ⁄ pMACSKK.II DNA. (B) Over-expression of DN-PKCh suppressed TCDD-induced L-MAT cell apoptosis. L-MAT cells trans-
fected with empty pMACSKK.II or with DN PKCh-FLAG ⁄ pMACSKK.II DNA were collected at 44 h, starved for 4 h in serum-free RPMI 1640,
labeled with MACSelect KK MicroBeads, then separated magnetically by means of the miniMACS Separation System. L-MAT cells retained
on the miniMACS column were immediately eluted with serum-free RPMI 1640 and counted for viability by using the Trypan blue exclusion
assay. These L-MAT cells were then distributed as 10
5
cellsÆ100 lL
)1
of serum-free RPMI 1640 per well in a 96-microwell plate, and subse-
quently treated with TCDD (as indicated) for 3 h. Finally, the effect of over-expression of DN-PKCh in L-MAT cells on TCDD-induced apopto-
sis was evaluated by assessing caspase-3 activation. Data are shown as average values ± SD (n ¼ 3). (**P > 0.01 vs. TCDD alone;
Student’s t-test.) The filled columns represent L-MAT cells transfected with empty pMACSKK.II; the open columns represent those trans-
fected with DN PKCh-FLAG ⁄ pMACSKK.II DNA. (C) Detection of the over-expression of DN PKCh in L-MAT cells by immunoprecipitation and
Western blotting methods. Whole cell lysates were prepared from L-MAT cells at 48 h of transfection, and 1.0 mg of total cell protein was
then probed by immunoprecipitation and Western blotting to detect the 3·FLAG peptide fused at the C terminus of DN PKCh.
PKCh in TCDD-induced signaling for apoptosis S. Ahmed et al.
910 FEBS Journal 272 (2005) 903–915 ª 2005 FEBS

volume of the solvent dimethylsulfoxide [the concentra-
tion of which never exceeded the 1% (v ⁄ v) level)] or
NaCl ⁄ P
i
. Three hours later, the cells were centrifuged at
1190 g for 10 min at room temperature. Then, 75 lLof
the medium was removed and frozen at )80 °C for
30 min, followed by thawing on ice for 30 min. Next,
50 lL of 100 mm Hepes (pH 7.25), 20% (w ⁄ v) sucrose,
5mm dithiothreitol, 0.1% (v ⁄ v) CHAPS, 10
–6
%(v⁄ v)
Nonidet P-40 (NP-40) containing 100 lm AcDEVD–AMC
(Calbiochem, San Diego, CA, USA) was added to each
well. Substrate cleavage to release free AMC was monit-
ored against time at 37 °C, by using a Fluoroscan Ascent
(Labsystems, Helsinki, Finland). The amount of AMC
released was calculated from the emission at 460 nm
(excitation at 355 nm), using a standard curve for AMC.
Fluorescence units were converted to pmoles of AMC
with the aid of a standard curve generated using free
AMC.
RNA isolation and RT-PCR
Total RNA was extracted from exponentially growing
L-MAT and HepG2 cells (2 · 10
7
) by using the acid guani-
dium thiocyanate ⁄ phenol ⁄ chloroform method, as described
by Chomczynski & Sacchi [34]. The prepared RNA (50 lg)
was first treated with RNase-free DNaseI (Boehringer

Mannheim GmbH, Mannheim, Germany) to remove the
genomic DNA contaminant. Then, 10 lg of total RNA
was reverse-transcribed to synthesize cDNA by means of
AMV (avian myeloblastosis virus)-reverse transcriptase
from Pharmacia using random hexamer, oligo (dN)6-pri-
ming in a final reaction volume of 50 lL supplemented
with RNase inhibitor (Boehringer Mannheim GmbH). For
human PKCh and PKCd, primer sequences were from a
published source [35]. For human glyceraldehyde 3-phos-
phate dehydrogenase (GAPDH), primers were designed as
follows: forward primer, 5¢-CATCACCATCTTCCAGG
AGC-3¢; reverse primer, 5¢-GGATGATGTTCTGGAGC-3¢.
PCR reactions were prepared as a final volume of 20 lL
containing 1.0 lL of the reverse-transcribed sample, 2.0 lL
of 10· Taq buffer, MgCl
2
(1.5 mm for PKCh and 2.0 mm
for GAPDH), 200 lm of each dNTP mixture in the pres-
ence of 0.5 lm of each primer, and 2.5 units of Taq DNA
polymerase (TaKaRa Bio Inc., Tokyo, Japan). The PCR
reaction was performed under the following conditions: ini-
tial denaturation for 5 min at 94 °C (1 cycle), followed by
35 cycles of amplification, each comprising denaturation
for 30 s at 94 °C, annealing for 1 min at 55 °C for GAPDH,
and elongation for 1 min plus a 5 s extension at 72 °C,
then finally one more cycle of a 5 min elongation at 72 °C,
followed by cooling to 4 ° C. The PCR products (10 lL)
were then subjected to electrophoresis in a 1.5% (w ⁄ v)
agarose gel supplemented with ethidium bromide
(0.5 lgÆmL

)1
). GAPDH expression was checked as the
internal control.
Cell lysis, immunoprecipitation, and Western
blotting analysis
Cells were lysed as previously described [36], with some
modifications, for detection of the endogenous expression
of PKCh and PKCd by using direct Western blotting.
About 2 · 10
7
cells were collected in a 15 mL conical tube
(Nalge Nunc International, Rochester, NY, USA) at
154 g for 5 min and, after the removal of culture medium,
were washed once with NaCl ⁄ P
i
and collected again. All the
subsequent steps for protein preparation were conducted in
a cold room. The cell pellet was resuspended by gentle
pipetting in 1.0 mL of ice-cold cell lysis buffer (20 mm
Tris ⁄ HCl, pH 7.5, 150 mm NaCl, 5 mm EDTA, 5 mm
Na
4
P
2
O
7
12H
2
O, 1 mm Na
3

VO
4
, and 1% NP-40) freshly
supplemented with 1· Complete EDTA-free Protease Inhib-
itor Cocktail (Roche Diagnostics GmbH, Mannheim, Ger-
many). Then, 10 lLof10mgÆmL
)1
phenylmethanesulfonyl
fluoride was added, and the cells were further disrupted and
homogenized by passing them 15 times through a 1.0 mL
syringe fitted with a 21-gauge needle. They were then main-
tained on ice for 30 min, transferred to microcentrifuge
tubes, and centrifuged (4 °C, 20 min, 16 000 g). The super-
natant was collected by filtration through a 0.45 lm Acro-
disk Syringe Filter (Pall Corporation, East Hills, NY,
USA), and total protein was estimated by using the Brad-
ford protein assay method. Then, 100 lg of protein was
mixed with an equal volume of 2· SDS ⁄ PAGE buffer
[100 mm Tris ⁄ HCl, pH 6.8, 4% (w⁄ v) SDS, 1.728 mm
b-mercaptoethanol, 20% (v ⁄ v) glycerol, and 0.2% (w ⁄ v)
Bromophenol blue], boiled for 3 min, resolved by electro-
phoresis on a 7.5% (w ⁄ v) SDS gel, and transferred onto a
poly(vinylidene difluoride) membrane. The membrane was
blocked with 5% (w ⁄ v) skimmed milk in 1 · TTBS [50 mm
Tris ⁄ HCl, pH 7.5, 0.15 m NaCl, 0.1% (v ⁄ v) Tween 20] for
30 min at room temperature on a shaker, then subjected to
immunoblot analysis by incubation overnight with anti-
human PKCh immunoglobulin (goat polyclonal, sc-1875;
Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) at
4 °C. The membrane was washed four times (15 min each

wash) with 1 · TTBS at room temperature, followed by
another incubation with an anti-goat immunoglobulin G
(IgG) horseradish peroxidase (HRP)-conjugated antibody
(donkey polyclonal; Santa Cruz Biotechnology Inc.) for
60 min at room temperature. Finally, the signal was detec-
ted by using an enhanced chemiluminescence kit (ECL Plus;
Amersham Biosciences, London, UK).
For detection of the exogenous expression of
PKCh)3·FLAG fusion protein, protein was prepared from
transfected L-MAT cells as described above. About 1.0 mg
of protein was immunoprecipitated overnight at 4 °C with
50 lL of anti-FLAG M2 affinity gel (Product No. A2220;
Sigma, St Louis, MO, USA). Before immunoprecipitation,
anti-FLAG M2 affinity gel resin was prepared as a 2 : 1
ratio of suspension to packed gel volume, as described in
S. Ahmed et al. PKCh in TCDD-induced signaling for apoptosis
FEBS Journal 272 (2005) 903–915 ª 2005 FEBS 911
the attached protocol. The resin was washed once with
0.5 mL of 0.1 m glycine HCl, pH 3.5, for 1 min, while thor-
oughly tapping and inverting to remove traces of unbound
anti-FLAG immunoglobulin from the resin. The superna-
tant was aspirated out using a Hamilton syringe after
centrifugation (4 °C, 5 s, 7300 g). The resin was immedi-
ately washed twice with 0.5 mL of NaCl ⁄ Tris (TBS) buffer
and once with 0.5 mL of cell lysis buffer, as described in
the attached protocol, without inhibitors. After immuno-
precipitation overnight, the affinity gel was washed three
times with the same cell lysis buffer without the inhibitors,
then mixed with 50 lLof2· SDS ⁄ PAGE buffer, boiled for
3 min, resolved in a similar way to that described above,

and subjected to immunoblot analysis by incubation with
mouse monoclonal anti-FLAG M2 immunoglobulin
(Sigma) at room temperature for 90 min. The membrane
was washed in a similar way to that described above,
followed by a further 90 min incubation with an anti-mouse
IgG ⁄ HRP-conjugated antibody (goat polyclonal; Santa
Cruz Biotechnology Inc.) at room temperature. Finally, the
signal was detected as described above.
In vitro kinase assay for nPKC
A biotinylated PKCh pseudosubstrate peptide (Biotin-
LHQRRGSIKQAKVHHVKC) was used as substrate for an
in vitro PKC kinase assay to evaluate the inhibitory effects
exerted by rottlerin (Calbiochem) and myr-PKCh-PPI
(Calbiochem) on the kinase activity of PKCh. The biotinyl-
ated PKCh pseudosubstrate can be phosphorylated, because
alanine (A) of real pseudosubstrate is replaced with serine (S)
at position 7 from the biotinylated N terminus. The reaction
mixture consisted of 10 ng of purified human recombinant
PKCh (PanVera, Madison, WI, USA) in a reaction volume
of 25 lL containing 50 mm Tris ⁄ HCl, pH 7.5, 5 mm MgCl
2
,
0.1 mm Na
3
VO
4
, 0.1 mm Na
4
P
2

O
7
12H
2
O, 1 mm NaF,
0.1 mm phenylmethanesulfonyl fluoride, 50 lm ATP, 5 lCi
[c-
32
P]dATP[cP], 4 lg of phosphatidyl-l-serine (Sigma), and
40 lm biotinylated-PKCh pseudosubstrate peptide, with or
without rottlerin (20 lm) or myr-PKC h -PPI (20 lm). The
reaction, which took place at 30 °C in a water bath for
10 min, was terminated by using Protocol A, as previously
described [37]. Reactions were centrifuged (in a microcentri-
fuge) at 14 300 g for 10 s. Then, 25 lL of the reaction mix-
ture was spotted onto streptavidin-coated square-cut
membrane (Promega, Madison, WI, USA), washed with
95% (v ⁄ v) ethanol, dried using a heat-lamp, and counted in
an LSC6000C Scintillation Counter (Beckman Coulter Inc.,
Fullerton, CA, USA), as previously described [37]
.
Subcellular fractionation for nPKC translocation
assay
L-MAT cells were fractionated, as previously described
[30], with minor modifications, to examine the translocation
of PKCh and PKCd during the TCDD-induced apoptosis
mechanism. In brief, L-MAT cells growing exponentially in
RPMI 1640 containing 5% (v ⁄ v) fetal bovine serum were
collected, resuspended in the same serum-free medium, and
cultured for another 4 h at 37 °C in 95% air and 5% CO

2
.
Then, the cells were collected and washed once with
NaCl ⁄ P
i
. First, 1 · 10
7
cells without any TCDD treatment
were collected as a control. Then, at each time-point,
1 · 10
7
cells were incubated in 5 mL of serum-free RPMI
in a 60 mm cell-culture dish (Falcon 3002; Becton Dickin-
son) in the presence of 20 nm TCDD. At the end of the
incubation, each dish was placed on chilled-ice and taken
to a cold room. Cells were collected in a 15 mL precooled
centrifuge tube (Nalge Nunc International), spun at
270 g for 10 min at 4 °C, and washed once with ice-cold
NaCl ⁄ P
i
. Finally, the cell pellet was resuspended in 0.5 mL
of buffer B [5 mm Na
3
VO
4
,5mm Na
2
P
2
O

7
,5mm NaF,
5mm EGTA, 2 mm EDTA, 1 mm dithiothreitol, 20 mm
Tris ⁄ HCl, pH 7.5, freshly supplemented with 10 mm benz-
amidine; Sigma), and 1· Complete EDTA-free Protease
Inhibitor Cocktail (Roche Diagnostics GmbH)] by gentle
pipetting, and further sheared by passing 30 times through
a 1.0 mL syringe fitted with a 25-gauge needle. Then, 5 lL
of phenylmethanesulfonyl fluoride (10 lgÆlL
)1
) was added
to the sheared cell suspension on ice. After 15 min of incu-
bation on ice, the cell suspension was transferred to a
1.5 mL microcentrifuge tube. Cell nuclei were removed
by centrifugation (10 min, 1190 g,4°C). Fractionation
into cytosolic (cyt) and particulate (pt) fractions was
achieved by centrifugation for 30 min at 35 000 r.p.m.
( 100 000 g), as previously described [21]. After two wash-
ing steps with buffer B, 0.25 mL of buffer B containing 1%
NP-40 was added to the particulate fraction (membrane +
cytoskeleton), and this was solubilized by pipetting and vig-
orous vortexing for 1 min. Then, the protein content of the
particulate fraction collected at each time-point was estima-
ted by using the Bio-Rad Protein Assay Standard Procedure
(Bio-Rad Laboratories, Hercules, CA, USA), and 40 lgof
the particulate fraction was examined by using the Western
blotting method, as described above.
Plasmids and subcloning
We used pMACSKK.II (Miltenyi Biotec GmbH, Bergisch
Gladbach, Germany) as the vector for gene expression and

functional analysis. The kinase-dead mutant of PKCh
(K ⁄ R 409 mutant), established as a DN mutant [22], was
subcloned into pMACSKK.II from a pEF-neo ⁄ DN PKCh
construct (kindly provided by G Baier, University of Inns-
bruck, Austria). First, a SalI-FLAG-Stop oligo with XhoI
at the N terminus and HindIII at the C terminus was sub-
cloned into pMACSKK.II. Then, PCR was performed to
create XhoI at the N terminus, upstream of the start codon,
and SalI at the C terminus, just before the stop codon of
PKCh, with pEFneo-DN PKCh being used as the template.
PKCh in TCDD-induced signaling for apoptosis S. Ahmed et al.
912 FEBS Journal 272 (2005) 903–915 ª 2005 FEBS
Next, the XhoI-DN PKCh-SalI was subcloned into
pMACSKK.II at the XhoI ⁄ SalI site in-frame with the SalI-
FLAG-Stop, and finally a DN PKCh-FLAG construct was
made in the pMACSKK.II vector. In a similar manner, a
DN PKCh)3·FLAG construct was also made. The sequence
was confirmed by restriction enzyme digestion and sequen-
cing analyses at all the steps involved in subcloning by using
standard procedures.
Transient transfection assay
Plasmid DNAs were transfected into L-MAT cells by using
the electroporation method. Exponentially growing L-MAT
cells were collected and resuspended at 5 · 10
6
cells per
10 mL of RPMI [containing 5% (v ⁄ v) fetal bovine serum]
in each 100 mm dish, then cultured overnight at 37 °Cin
95% air and 5% CO
2

for transfection the next day.
L-MAT cells were collected, resuspended at 5 · 10
6
cells in
200 lL of intracellular buffer CYTOMIX, pH 7.6, prepared
as previously described [38], then transfected with 10 lgof
empty pMACSKK.II or DN PKCh-FLAG ⁄ pMACSKK.II
DNA at 280 V, 72 ohm, 1050 lF in each 2 mm gap electro-
poration cuvette (Molecular BioProducts Inc., San Diego,
CA, USA) using the BTX Electroporation System (BTX
Inc., San Diego, CA, USA). Transfected L-MAT cells were
collected at optimal time-points for further analysis.
Immunofluorescence microscopy for transient
gene expression analysis
Transfected L-MAT cells were collected at 44 h and imme-
diately prepared for immunofluorescence staining with anti-
mouse H-2KK-fluorescein isothiocyanate (FITC) antibody
(clone H100-27.R55; Miltenyi Biotec), as described in the
attached protocol. Immunostained cells were mounted and
examined by using a Leica DMLB (Leica, Mikroskopie und
Systeme GmbH, Wetzlar, Germany) fluorescent microscope.
Magnetic separation of transfected L-MAT cells
for functional assay
Transfected L-MAT cells were collected at 44 h, resuspended
in RPMI 1640 without fetal bovine serum, and incubated for
another 4 h at 37 °C in 95% air and 5% CO
2
. Cells were
collected again, washed once with NaCl ⁄ P
i

, and labeled with
MACSelect KK magnetic MicroBeads for separation by the
MiniMACS Separation System, as described in the attached
protocol (Miltenyi Biotec). The only modification to this
process was that we used serum-free RPMI 1640 instead of
PBE (phosphate-buffered saline supplemented with 2 mm
EDTA). Then, the transfected L-MAT cells retained within
the MiniMACS Column were eluted with serum-free
RPMI 1640 and counted for viability by using a Trypan Blue
exclusion assay. Finally, the effect of TCDD on caspase-3
activation was examined, as described above in the apoptosis
assay section.
Acknowledgements
We thank Dr Gottfried Baier (University of Innsbruck,
Austria) for generously providing the dominant negat-
ive PKCh construct of plasmid DNA. This work was
supported, in part, by Grants-in-Aid for Scientific
Research (B) [Nos 11558068 and 12480153 from the
Japanese Ministry of Education, Culture, Sports,
Science and Technology (Monbu Kagakusho)] and
supported by a Scholarship for foreign students from
Monbu Kagakusho (S.A.).
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