From the Instituto de Investigaciones Biomédicas, CSIC, Facultad Medicina Universidad Autónoma de Madrid, Arturo Duperier 4, 28029 Madrid, Spain
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ABSTRACT |
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Cot kinase is a protein serine/threonine kinase,
classified as a mitogen-activated protein kinase kinase kinase,
implicated in T lymphocyte activation. Here we show that an increase in
Cot kinase expression promotes tumor necrosis factor- (TNF-
)
production in Jurkat T cells stimulated by soluble anti-CD3 or by low
concentrations of phorbol 12,13-dibutyrate (PDBu) and calcium
ionophore. Overexpression of Cot kinase in Jurkat cells activates
TNF-
gene expression. Cot kinase promotes TNF-
promoter
activation to a similar extent as calcium ionophore and PDBu or soluble
anti-CD28 and PDBu. Neither phorbol esters nor calcium ionophore can
replace Cot kinase on TNF-
promoter-driven transcription. Expression
of a dominant negative form of Cot kinase inhibits TNF-
promoter
activation induced by stimulation with either calcium ionophore and
PDBu, soluble anti-CD28 and PDBu, or soluble anti-CD3 and PDBu. TNF-
promoter-driven transcription by Cot kinase is partially mediated by
MAPK/ERK kinase and is cyclosporin A-resistant. Cot kinase increases at
least the AP-1 and AP-2 response elements. These data indicate that Cot
kinase plays a critical role in TNF-
production.
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INTRODUCTION |
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Tpl-2/Cot kinase is a mitogen-activated protein kinase kinase kinase that activates both the ERK1 and JNK signal transduction pathways (1-4). The COT kinase gene was first cloned in a truncated form in transformed foci induced in SHOK cells by transfection of the genomic DNA of a human thyroid carcinoma cell line (5). The human COT gene is unique in the genomic sequence (5). The normal human cellular homologue has an open reading frame encoding 467 amino acids, being the first 397 identical to the truncated form, whereas the 69 amino acids from the C terminus are replaced by 18 amino acids in the truncated form (6-8). The rat homologue gene was identified as an oncogene associated with the progression of Moloney murine leukemia virus-induced T cell lymphomas in rats (Tpl-2) (9, 10). The provirus insertion occurs in the last intron of the Tpl-2 gene. Transgenic mice expressing the truncated oncogenic protein in thymocytes develop T cell lymphomas (11). As occurs with the human homologue, the disruption of the last coding exon of Tpl-2 appears to unmask the oncogenic potential of the protein (6, 10-12). However, overexpression of the human normal gene is also capable of conferring the transformed phenotype in established cell lines (6, 8).
At the amino acid level the identity between the human Cot kinase and the rat Tpl-2 homologue is >95% (1). Transient expression of Cot/Tpl-2 kinase activates ERK1 (1, 13, 14). Overexpression of the rat homologue in human Jurkat T cells also phosphorylates and activates JNKK, and consequently JNK is also phosphorylated and activated (1). This activation of JNK by Cot kinase leads to the phosphorylation of c-jun in its N-terminal region (1).
A variety of stimuli induce TNF- production in many cell types (15,
16), including T cells (17-22). Phorbol esters (23-25), calcium (26,
27), anti-CD3 antibody (20), or phorbol esters and anti-CD3 induce
TNF-
promoter transcription (21, 28). Enterotoxin (22), anti-AIM/CD
69 (29), or anti-CD2 pathway (30) also regulate TNF-
promoter
activity. The regulation of TNF-
gene transcription is cell
type-specific (15, 16, 19, 25), and different response elements such as
AP-1 (25, 31), AP-2 (25, 31), CRE (cAMP response element) (19, 20, 32), SP-1 (19, 20, 24, 25), Krox-24 (25, 33), NF-kappa B (34-37), and NFAT
(nuclear factor activated T cells) (19, 20, 26, 38) have been
identified in the TNF-
promoter.
The data shown here demonstrate that overexpression of Cot kinase
enhances TNF- secretion in Jurkat cells. Transfection of Cot kinase
in Jurkat T cells promotes TNF-
gene expression. We also demonstrate
that a dominant negative form of Cot kinase inhibits TNF-
promoter-driven transcription. Our data also further demonstrate that
Cot kinase activates TNF-
promoter-driven transcription in a
CSA-resistant way and that Cot kinase regulates at least the AP-1 and
AP-2 response elements.
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EXPERIMENTAL PROCEDURES |
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DNA Constructs-- The DraI fragment (222-1871 nt) of cot (8) was subcloned in the eukaryotic expression vector pEF-BOS, previously digested with BamHI, treated with CIP, and subsequently with Klenow enzyme. A pEF-BOS-cot construct, in the 5'-3' orientation relative to the EF promoter, was selected. A similar pEF-BOS-trunc-cot construct, in the 5'-3' orientation relative to the EF promoter, was obtained by digesting truncated cot with DraI (30-1302 nt) (5).
The inactive kinase form of full-length cot was generated by PCR. The AvaI-HindIII fragment of cot was cloned into the AvaI-HindIII sites of a normal pUC19 vector. A PCR with the mutagenic primers AGAATGGCGTGTGCACTGATCCCA and TAGTCTACCGAATTTAACTAGATG encompassing the substitution Lys-168 to Ala-168 was performed using this construct as template. Separately, cot cDNA (8) was ligated to a pUC19 vector devoid of AvaI and HindIII sites. The AvaI-HindIII fragment of cot was replaced by the mutated AvaI-HindIII fragment obtained by PCR, yielding inactive cot (inac-cot), which was subsequently cloned in the pEF-BOS vector as described for cot kinase and obtaining pEF-BOS-inac-cot(5'-3'). DNA sequencing of inac-cot was performed to verify the construct. Different constructs of the TNF-Generation of Antibodies-- The anti-CD3 monoclonal antibody was obtained by injecting 106 T3b hybridoma cells (41, 42), generously provided by Dr. F. Sanchez-Madrid in Balb/c mice, previously pristanized. Immunoglobulin from the ascitic fluid was purified by Sepharose-protein A chromatography.
Cells and Medium-- The human leukemia T cell line Jurkat was obtained from Dr. Abelardo López-Rivas and maintained in RPMI 1640 medium supplemented with 10% fetal calf serum (Life Technologies, Inc.), gentamycin (50 µg/ml), and L-glutamine (2 mM) (complete medium).
TNF- Production Assay--
DNA-mediated gene transfer into
Jurkat cells was accomplished by electroporation (43). Exponentially
growing cells were washed and resuspended in complete medium at a
concentration of 2 × 107/ml, and 1 ml of the cell
suspension was transferred into a 0.4-cm electroporation cuvette
(Bio-Rad), and unless otherwise indicated, 10 µg of the different
pEF-BOS constructs and 5 µg of the different pTNF
-Luc constructs
were added. Electroporation was accomplished with a Gene Pulser
apparatus (Bio-Rad) with a capacitance of 960 microfarads and an
electrical field of 300 V. The electroporated cells were transferred
into tissue culture flasks (Costar) in complete medium at a
concentration of 106 cells/ml. After 2 h in culture,
transfected cells were stimulated with calcium ionophore A23187
(Boehringer Mannheim) and different concentrations of PDBu (Sigma) or
with soluble anti-CD3 for 24 h. Culture supernatants were tested
for TNF-
production with the human TNF-
quantification
enzyme-linked immunosorbent assay kit (Genzyme), following the
manufacturer's instructions. The detection limit according to the
standard curve was over 73 pmol.
RT-PCR Assay-- Jurkat cells were electroporated with pEF-BOS (10 µg/ml), pEF-BOS-cot(5'-3') (10 µg/ml), or no plasmid. After 30 min incubation cells were stimulated for 6 h, with 0.25 µM calcium ionophore and 50 ng/ml PDBu or 0.1 µM calcium ionophore and 5 ng/ml PDBu, and total RNA was extracted using Ultraspec (Biotech), according to the manufacturer's instructions.
To perform the RT reaction (45), 2 µg of total RNA from nonstimulated or stimulated cells were heated at 37 °C for 30 min in the presence of 100 mM dithiothreitol, 0.5 mM dNTPs, 50 units of ribonuclease inhibitor (Life Technologies, Inc.), 2.5 units of DNase (Life Technologies, Inc.) and RT buffer (Life Technologies, Inc.), and then treated at 95 °C for 5 min. Ten µM random primers (Life Technologies, Inc.) were subsequently added, and the mixture was heated at 70 °C for 10 min. After chilling on ice, 200 units of reverse transcriptase (Superscript RNase HTNF- Promoter-driven Transcription Assay--
DNA-mediated
gene transfer into Jurkat cells was performed as explained above.
Jurkat cells were cotransfected with different TNF-
promoter-Luc
reporter constructs (5 µg/ml) together with control construct
(pEF-BOS) (10 µg/ml) or different pEF-BOS-cot constructs (10 µg/ml). After 30 min in culture, cells were stimulated for 12 h
with different stimuli as follows: soluble anti-CD3, soluble anti-CD28
9.3 antibody (generously donated by Dr. C. June), calcium ionophore
A23187, or PHA (Sigma) in the presence or absence of different
concentrations of PDBu (Sigma). 8-Br-cAMP (Boehringer Mannheim), Dex
(Sigma), CSA, or PD 98059 (MEK inhibitor) (Calbiochem) were added to
the cells 30 min after electroporation and then the cells were
stimulated 2 h later. Twelve hours after stimulation cells were
collected by centrifugation, and Luc activity was determined by the
luciferase assay kit (Promega), according to the manufacturer's instructions. Cell extracts were normalized by protein measurements with the Dc protein assay (Bio-Rad).
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RESULTS |
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Cot Kinase Promotes TNF- Production in Submaximally Stimulated
Jurkat T Cells--
Table I illustrates
the effect of Cot expression in Jurkat T cells on TNF-
production.
Jurkat cells stimulated with 0.25 µM calcium ionophore
and 50 ng/ml PDBu (maximum stimulation) produced about 240 pmol/5 × 105 cells of TNF-
, independently of whether cells
were electroporated with pEF-BOS-cot(5'-3'), pEF-BOS, or no plasmid.
Stimulation with suboptimal concentrations of calcium ionophore and
PDBu only increased TNF-
production in cells overexpressing Cot
kinase (Table I). Soluble anti-CD3 (10 µg/ml) alone did not stimulate
TNF-
production in pEF-BOS or non-plasmid electroporated cells.
However, with this stimulus TNF-
production was detected in
pEF-BOS-cot(5'-3')-electroporated cells. In the absence of any stimuli
pEF-BOS-cot(5'-3')-transfected cells were not able to produce TNF-
,
indicating that overexpression of Cot kinase by itself was not able to
induce TNF-
production.
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Cot Kinase Induces TNF- Gene Expression in Jurkat T
Cells--
We then decided to investigate whether Cot kinase regulated
TNF-
gene expression in Jurkat cells. RNA from pEF-BOS,
pEF-BOS-cot(5'-3'), or non-plasmid electroporated cells stimulated or
not with different concentrations of calcium ionophore and PDBu was
isolated to perform RT-PCR assays. In the different electroporated
cells stimulated with 0.25 µM calcium ionophore and 50 ng/ml PDBu (maximum stimulation), TNF-
mRNA was detected (Fig.
1). A similar amount of TNF-
PCR product was obtained in Cot-transfected cells stimulated with 0.1 µM calcium ionophore and 5 ng/ml PDBu (submaximal
stimulation). However, in pEF-BOS or no plasmid-transfected cells
incubated with these concentrations of stimuli, a significant decrease
in the intensity of the band corresponding to the TNF-
PCR product was detected. Without stimuli, the TNF-
PCR product was only detected in cells overexpressing Cot kinase (Fig. 1).
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Cot Kinase Activates 1185 TNF-
Promoter-driven Transcription
in Jurkat T Cells--
Next, we decided to determine whether Cot
kinase enhanced TNF-
promoter-driven transcription. Jurkat cells
electroporated with
1185 pTNF
-Luc alone or together with
pEF-BOS-cot(5'-3') or pEF-BOS were subjected to different stimuli, and
Luc activity was measured.
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Regulation of 1185 TNF
Promoter-driven Transcription by
8-Br-cAMP, Dex, CSA, and MEK Inhibitor in Cot Kinase and Truncated Cot
Kinase-transfected Jurkat Cells--
Several compounds were used to
study if overexpression of Cot kinase stimulated the TNF-
promoter-driven transcription through the same mechanism as PDBu and
calcium ionophore. Cells electroporated with
1185 pTNF
-Luc and
either pEF-BOS-cot(5'-3'), pEF-BOS-trunc-cot(5'-3'), pEF-BOS, or no
additional plasmid were incubated with 8-Br-cAMP, Dex, CSA, or MEK
inhibitor. Cells transfected with
1185 pTNF
-Luc alone or together
with pEF-BOS were also incubated with 50 ng/ml PDBu and 0.25 µM calcium ionophore.
Soluble Anti-CD28 or Soluble Anti-CD3 Does Not Cooperate with Cot
Kinase for Transactivation of the TNF- Promoter--
To determine
whether stimulation with soluble anti-CD28 (1 µg/ml) or with soluble
anti-CD3 (10 µg/ml) further enhanced Cot kinase activation of the
TNF-
promoter, cells were electroporated with either
pEF-BOS-cot(5'-3') or pEF-BOS, and
1185 pTNF
Luc or only with the
1185 pTNF
Luc, and different stimuli were added.
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Expression of Kinase-inactive Cot Inhibits TNF- Promoter
Activation in Stimulated Jurkat Cells--
Jurkat cells electroporated
with
1185 pTNF
-Luc alone or together with pEF-BOS-inac-cot(5'-3')
or pEF-BOS were subjected to stimulation with different additives, and
TNF-
promoter-driven transcription of the Luc gene was measured.
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Cot Kinase Regulation of Different TNF- Promoter Constructs in
Jurkat Cells--
To determine the sites in the TNF-
promoter that
Cot kinase could regulate, cells were electroporated with different
constructs of the TNF-
promoter linked to the Luc gene (
1185,
615,
362,
105, or
36 bp pTNF
-Luc) and with
pEF-BOS-cot(5'-3') or with pEF-BOS. Cells electroporated with pEF-BOS
and the different TNF-
promoter constructs were stimulated with
calcium ionophore (0.25 µM) and PDBu (50 ng/ml), or with
soluble anti-CD28 (1 µg/ml) and PDBu (50 ng/ml). No stimulus was
added when cells were transfected with pEF-BOS-cot(5'-3') and the
different pTNF
-Luc constructs. The
1185 pTNF
-Luc construct
exhibited the highest Luc activity (100%) when compared with the other
TNF-
promoter constructs.
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Cot Kinase Activation of the AP-1 Response Element in Jurkat
Cells--
In Cot kinase-transfected cells the 105 pTNF
-Luc
construct exhibited a much higher Luc activity than the
36
pTNF
-Luc construct. Several response elements have been identified
in the
105- to
36-bp region of the human TNF-
promoter (19, 20,
25, 37), including the
60 bp AP-1 response element. Rhoades et
al. (25) have shown, in different cell systems, that this AP-1
response element plays an important role in the transcriptional
regulation of the human TNF-
promoter. On the other hand, Cot/Tpl-2
kinase is a mitogen-activated protein kinase kinase kinase that
activates the ERK1 and the JNK pathways and consequently induces JNK to phosphorylate c-jun (1). All these data indicated that Cot/Tpl-2 kinase
could regulate the AP-1 response element. To test this hypothesis we
decided to study the regulation of the AP-1 element by Cot kinase in
Jurkat cells. To perform this study two different constructions were
used. One was the
73 col promoter linked to the Luc gene that
contains only an AP-1 site as response element (39, 50). The other
construct was the NFAT-AP-1 composite element from the IL-2 promoter
linked to the Luc gene (40, 51, 52).
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Effect of 8-Br-cAMP, Dex, CSA, and MEK Inhibitor on the AP-1 Response Element Activated by Cot Kinase in Jurkat T Cells-- To study whether overexpression of Cot kinase and calcium ionophore stimulated the AP-1 response element through the same mechanism as PDBu and calcium ionophore, several inhibitors were added to electroporated cells prior to stimulation.
Cells transfected with pEF-BOS-cot(5'-3') and
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DISCUSSION |
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Cot/Tpl-2 kinase is a protein serine/threonine kinase that belongs
to the mitogen-activated protein kinase kinase kinase family (2-4).
Cot/Tpl-2 kinase has been involved in IL-2 production (43) and
lymphocyte activation (1, 9, 10). This paper demonstrates that Cot
promotes TNF- production in Jurkat cells stimulated by soluble
anti-CD3 or by low concentrations of PDBu and calcium ionophore. In
addition, the data also show that Cot kinase, by itself, promotes
TNF-
mRNA expression. The data also show that Cot kinase induces
TNF-
promoter activation as it enhanced transcription of a reporter
gene linked to the TNF-
promoter in Jurkat T cells. Here, we also
demonstrate that expression of a kinase-inactive form of Cot inhibits
TNF-
promoter activation. Together these data indicate that Cot
kinase plays a critical role in T cell TNF-
production.
Overexpression of Cot kinase in Jurkat cells is sufficient to induce
TNF- gene expression (Fig. 1) and TNF-
promoter activation (Fig.
2) but not for TNF-
secretion (Table I), confirming that TNF-
production is not only regulated at transcriptional levels but that
some additional events in TNF-
production are regulated. Supporting
this view, calcium appears to control pre-TNF-
processing and
TNF-
secretion (53-55), and the data shown here demonstrate that
Cot kinase does not replace calcium-generated signals (see below).
We have recently shown that Cot kinase enhances IL-2 promoter
activation, although, in contrast with the TNF- promoter, Cot kinase
alone could not regulate IL-2 promoter activation in Jurkat T cells
(43). All these data also demonstrate that in the activation of TNF-
and IL-2 promoters some common intracellular pathways are shared, but
other(s) are cytokine-specific.
Different signals regulate TNF- gene expression in different cell
types (15, 16, 19, 27). Calcium influx alone, by regulating at least
the NFAT response elements in a CSA-dependent manner, is
sufficient for the rapid gene induction in A20 B cells and Ar-5 T cells
(19, 26, 27). On the other hand, stimulation of MLA 144 T cells, U937
macrophages, or 729-6 B cells by phorbol esters is sufficient to induce
TNF-
promoter activation; the AP-1 site plays an important role in
this transcriptional regulation (25). Activation of the AP-1 of the
73 col promoter site in Jurkat T cells requires phorbol esters and
calcium ionophore (Fig. 6) (50). The data shown here demonstrate that
in Jurkat T cells stimulation with calcium ionophore or PDBu alone is
not sufficient for induction of TNF-
transcription, which requires
the addition of both stimuli together. Soluble anti-CD28 antibody or
soluble anti-CD3 antibody are not sufficient either to stimulate
expression of this gene, and in Jurkat T cells addition of PDBu is also
needed.
Addition of PDBu to Cot-transfected cells does not further induce
transactivation of the TNF- promoter, the
73 col promoter, or the
NFAT-AP-1 composite element, indicating that the effects of PDBu on
the activation of the TNF-
promoter and the two AP-1 containing
sequences tested can be replaced in Jurkat cells by overexpression of
Cot kinase. However, Cot kinase does not mimic the effects of PDBu
exactly, because Cot kinase overexpression alone activates the TNF-
promoter constructs, the collagenase promoter construct, and to a
certain extent the NFAT-AP-1 composite element and PDBu does not.
Calcium influx activates TNF- promoter through the CSA-sensitive
NFAT response element (26, 27, 38, 56). Stimulation of TNF-
promoter
by calcium and PDBu is CSA-sensitive, but Cot kinase TNF-
promoter
activation is CSA-insensitive suggesting that Cot kinase activation of
this promoter is independent of the calcium pathway. This hypothesis is
reinforced by the fact that addition of calcium ionophore in
Cot-transfected cells further stimulates the TNF-
promoter, the
73
col promoter, and also the NFAT-AP-1 composite element.
The results obtained with a kinase-inactive form of Cot (Fig. 4) and
with MEK inhibitor and CSA (Fig. 2B) indicate that Cot kinase and stimulation by PDBu and calcium share some signal pathways (the ERK and probably the JNK transduction pathways), but not other(s),
leading to TNF- promoter activation.
To our knowledge this is the first time that TNF- promoter has been
shown to be activated by stimulation with anti-CD28 and PDBu. Cot
kinase mimics better the activation of this promoter by anti-CD28 and
PDBu than by calcium ionophore and PDBu, because activation of TNF-
promoter by Cot kinase or by anti-CD28 and PDBu is not regulated by
CSA. On the other hand, we have also demonstrated that expression of a
kinase-inactive form of Cot inhibits the TNF-
promoter-driven
transcription stimulated by anti-CD28 and PDBu. Furthermore,
unstimulated Cot-transfected cells or anti-CD28 and PDBu-stimulated
pEF-BOS electroporated cells activated, through its AP-2 site, the
36
TNF-
promoter construct to a similar extent. Cot kinase-transfected
cells exhibit an induction of about 20-fold in the NFAT-AP-1 composite
element (Fig. 6B), a similar level of activation as that
achieved by anti-CD28 and phorbol esters in Jurkat T cells (57). We
have previously demonstrated that Jurkat cells express the cot gene
prior to stimulation (43), but the mechanism by which Cot kinase is
activated is still unknown. All these data indicate that anti-CD28
together with another stimulus could regulate Cot kinase activation in T cells.
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ACKNOWLEDGEMENTS |
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We thank Dr. Miyoshi for providing the truncated cot cDNA probe; Dr. Chan for the cot (est) cDNA probe; Dr. Arnero (Sandoz, España) for the cyclosporin A; and Dr. C. June for the 9.3 anti-CD28 antibody. We thank V. Calvo and M. Lopez Cabrera for the critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported in part by Plan Nacional, Comunidad de Madrid, and Europharma.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Both authors contributed equally to this work.
§ Recipient of a fellowship from CSIC and Plan Nacional.
¶ To whom correspondence should be addressed: Instituto Investigaciones, Biomédicas, CSIC, Arturo Duperier 4, 28029 Madrid, Spain. Tel.: 34-1-3975445; Fax: 34-1-5854587; E-mail: Salemany{at}biomed.iib.uam.es.
1
The abbreviations used are: ERK, extracellular
signal-regulated kinase; TNF-, tumor necrosis factor-
; MEK,
MAPK/ERK kinase; JNK, c-Jun N-terminal kinase; PDBu, phorbol
12,13-dibutyrate; Luc, luciferase; CSA, cyclosporin A; IL-2,
interleukin 2; bp, base pair(s); RT, reverse transcriptase; PCR,
polymerase chain reaction; Dex, dexamethasone; nt, nucleotide(s);
PHA, phytohemagglutinin; inac-cot, inactive cot.
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REFERENCES |
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