From the
Chimeric proteins provide a unique opportunity to target
therapeutic bacterial toxins to a subset of specific cells. We have
generated a new recombinant chimeric toxin composed of human
interleukin 13 (hIL13) and a Pseudomonas exotoxin A (PE)
mutant, PE38QQR. The hIL13-PE38QQR chimera is highly cytotoxic to cell
lines derived from several human epithelial carcinomas such as
adenocarcinoma of stomach, colon, and skin. The cytotoxic action of
hIL13-PE38QQR, which can only occur upon internalization of
ligand-receptor complex, is blocked by an excess of hIL13 but not of
hIL2. This action is not solely hIL13-specific because an excess of
hIL4 also blocks the cytotoxicity of hIL13-toxin. Conversely, hIL13
blocks the cytotoxicity of a hIL4-PE38QQR chimera. Binding studies
showed that hIL13 displaces competitively
Recent reports have described the isolation and cloning of
lymphocyte growth factor interleukin 13
(IL13)
We have recently reported
that a wide range of human cancer cell lines express receptors for
hIL4; these include hematopoietic and epithelial
malignancies(12, 13) . When the hIL4R was targeted with
PE-based chimeric toxins, we observed a specific killing of cancer
cells expressing hIL4R(12, 14) . We have also shown that
the PE-containing chimeric toxins have prominent antitumor activities
in a human solid tumor xenograft model(15) .
PE is a
single-chain bacterial protein made up of three major domains (for
review, see Ref. 16). A characteristic feature of PE is that the
N-terminally located domain Ia binds to the
Because hIL4R are richly represented among
malignancies and because of the similarities between hIL4 and hIL13, we
used human cancer cell lines to study the interaction of hIL13 and hIL4
with their receptors. We have used a novel recombinant chimeric toxin
in which hIL13 is fused to PE38QQR, since fusion proteins containing PE
are a powerful mean of plasma membrane receptors analysis(18) .
Protein concentration was determined by the
Bradford assay (Pierce ``Plus,'' Rockford, IL) using bovine
serum albumin as a standard.
To evaluate the effects of
cytokines on the protein synthesis, the assays were performed as
follows. 2
To construct the chimeric toxin, the coding region of the
hIL13 gene was fused to a gene encoding a mutated form of PE, PE38QQR
(Figs. 1 and 2A). The chimeric gene is in the bacterial vector
under the control of a bacteriophage T7 late promoter; the protein was
expressed in E. coli BL21 (
We have made a novel recombinant chimeric toxin,
hIL13-PE38QQR, composed of hIL13 and a mutated PE, PE38QQR. This
chimeric toxin exerts a potent cytotoxic activity on a spectrum of
human carcinomas. The cytotoxic activity of hIL13-PE38QQR, which can
only occur subsequent to the internalization of a ligand-receptor
complex(16) , is competed for by hIL13 and also by hIL4. Thus,
there is a common internalized binding site for the two cytokines on
many human carcinoma cells.
Our present results add new information
on the interrelatedness between receptors for IL13 and IL4, since we
demonstrate unequivocally that the wild-type hIL4 is capable of
inhibiting the action of hIL13 chimeric toxin on human carcinoma cells.
The inhibition is most likely conditioned by the binding of hIL4 to
hIL13R, and not by other conceivable mechanisms, because hIL2 does not
block the cytotoxic effect of hIL13 toxin. However, hIL4 has been shown
to be a very weak competitor for hIL13 binding sites on renal cell
carcinomas(8) . This contrasts the potent blocking activity of
hIL4 on the action of hIL13-toxin on the currently studied cancer cell
lines. As to this point, Puri and co-workers
On the other hand, two independent observations
demonstrate that hIL13 competes well for hIL4 binding sites: (i) hIL13
antagonizes the cytotoxic action of hIL4-PE38QQR and (ii) hIL13
displaces labeled hIL4 chimeric toxin on tumor cells. Thus, hIL13
blocks internalization of the hIL4-toxin by a direct binding to the
hIL4R; this finding supports original observation that hIL13 competes
for hIL4 binding sites(7) .
The relationship between hIL13R
and hIL4R is a matter of extensive
investigations(7, 8, 11, 24) . Our
current data and previous
studies(7, 8, 12)
We have
demonstrated the lack of hIL13 activity while hIL4, at high
concentrations, inhibited the protein synthesis in two carcinoma cell
lines. Differential effects of hIL13 and hIL4 have recently been
reported(24) . The reason for this phenomenon is not clear at
present. It is possible that hIL13 and hIL4 and their binding sites(s)
may resemble other growth factors in that the formation of binding
sites is determined by the expression of different protein variants and
modulated by ligands(25) . In consequence, various cellular
responses can be seen with the same ligands. Alternatively, the number
of receptors for the cytokines responsible for intracellular signaling
is significantly different on the studied cancer cell lines. This may
be the case for A431 epidermoid carcinoma cells, which has 1000 binding
sites/cell for hIL4 (15) and only 200 binding sites/cell for
hIL13(8) . Further studies are needed to identify the reason for
dissimilar effects of hIL13 and hIL4 on cancer cells.
In conclusion,
we have demonstrated that a variety of human carcinoma cell lines
express hIL13R because hIL13-toxin is highly cytotoxic to these cells.
This cytotoxicity is hIL13- and hIL4-specific, indicating that the two
cytokines bind to a common form of the receptor that is present on the
studied cell lines. Finally, our data suggest that hIL13-PE38QQR merits
evaluation as a therapeutic agent for the treatment of human
carcinomas, since binding sites for hIL13 on normal cells appear to be
limited even if the cells are responsive to hIL13(8) .
We thank Sonia Girouard for excellent technical
assistance and Pamela Leland for performing some of the binding and
cytotoxicity experiments. The readiness of the DNAX Research Institute
to share recombinant material is greatly appreciated.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
I-labeled
hIL4-PE38QQR on carcinoma cells. These results indicate that IL4 and
IL13 compete for a common binding site on the studied human cell lines.
Despite this competition, hIL4 but not hIL13 decreased protein
synthesis in malignant cells susceptible to the cytotoxicity of both
hIL13- and hIL4-PE38QQR. Our results suggest that a spectrum of human
carcinomas express binding sites for IL13. Furthermore, hIL13 and hIL4
compete reciprocally for a form of the receptor that is internalized
upon binding a ligand. Thus, cancer cells represent an interesting
model for studying receptors for these two growth factors. Finally,
hIL13-PE38QQR may be a useful agent in the treatment of several
malignancies.
(
)(1, 2) . IL13 is a
potentially glycosylated peptide of M
12,000, and
it bears a limited but significant homology to IL4 at the N and C
termini of the protein chain. Both ends of IL4 are important for
binding to receptor (3, 4). Functional responses of normal blood cells
to IL13 are pleiotropic and often, but not always, overlapping with
those observed using IL4(5, 6) . Contrary to IL4, IL13
is not species-specific and does not regulate T
lymphocytes(1, 2) . It has been suggested that receptors
for hIL4 and hIL13 share a subunit that is important for ligand-induced
intracellular signaling(7) . This is based on observation that
an antagonist of IL4, a mutated form of hIL4, blocked the mitogenic
effect of both hIL4 and hIL13 on pre-leukemic TF 1 cells. As hIL13
weakly displaced labeled hIL4 from binding sites, nonmutated hIL4
competition for hIL13 sites was not reported(7) . In fact, our
experiments showed lack of displacement of labeled hIL13 by a wild-type
hIL4 on TF 1 cells and a very weak or nil displacement of hIL13 by hIL4
on two renal cell carcinoma cell lines(8) . Also, a possibility
has been raised that hIL13R may use the
subunit of
the hIL2R, as has been observed with hIL4R and
hIL7R(9, 10, 11) .
-macroglobulin receptor, and the ligand-receptor
complex undergoes receptor-mediated internalization (endocytosis) to
allow intracellular routing and processing of the toxin(16) .
Domain II is a site of proteolytic cleavage that activates PE and is
necessary to catalyze the translocation of the toxin into the cytosol.
The C-terminal domain, domain III, contains the REDLK sequence that
directs the processed fragment of the toxin to the endoplasmic
recticulum and possesses an ADP ribosylation activity that inactivates
elongation factor 2 and leads to cell death(16) . Since domain
Ia of PE binds to many types of eukaryotic cells, several mutated forms
of the exotoxin have been constructed that do not bind to its
ubiquitous receptor and can be attached at a gene level to various
internalized ligands. One of the derivatives of PE used in the present
study is PE38QQR in which domain Ia (amino acids 1-252) and amino
acids 365-380 of PE are deleted, and lysine residues at positions
590 and 606 are replaced by glutamine and at 613 replaced by
arginine(17) .
Materials
Restriction endonucleases and DNA
ligase were obtained from New England Biolabs (Beverly, MA), Life
Technologies, Inc., and Boehringer Mannheim.
[H]Leucine and
I were purchased
from Amersham Corp. Fast protein liquid chromatography columns and
media were purchased from Pharmacia Biotech Inc. Oligonucleotide
primers were synthesized at Pharmacia's Gene Assembler at the
Research Center, Hôtel-Dieu Hospital, Montreal. PCR kit was from
Perkin-Elmer.
Plasmids, Bacterial Strains, and Cell
Lines
Plasmids carry a T7 bacteriophage late promoter, a T7
transcription terminator at the end of the open reading frame of the
protein, a f1 origin of replication and gene for ampicillin
resistance(19) . The gene encoding hIL13 (a generous gift of Dr.
René de Waal Malefyt of the DNAX Research Institute of Molecular
and Cellular Biology, Palo Alto, CA to W.D.; plasmid JFE14-SR) was
PCR-cloned into plasmid pWDMH-QQR to produce hIL13-PE38QQR or into
plasmid pSGC242FdN1(20) , to make hIL13 alone. hIL4 was cloned
into an expression vector in a similar way to hIL13 using plasmid
pWDMH4 (12) as a template for PCR amplification. Recombinant
proteins were expressed in Escherichia coli BL21 (
DE3)
under control of the T7 late promoter(12) . Plasmids were
amplified in E. coli (HB101 or DH5
high efficiency
transformation) (Life Technologies, Inc.) and DNA was extracted using
Qiagen kits (Chatsworth, CA).
Plamids Construction
The chimeric gene encoding
hIL13-PE38QQR (phuIL13-Tx) was constructed as outlined (see Fig. 1). hIL13 was PCR-amplified from plasmid JFE14-SR in
its mature form. New sites were introduced for the restriction
endonucleases NdeI and HindIII at the 5` and 3` ends
of the hIL13 gene, respectively. The 336-base pair DNA fragment was
obtained during 25 cycles of PCR and digested with appropriate
restriction enzymes. The digested fragment was subcloned into the
vector obtained by digestion of plasmid pWDMH4-QQR (15) with NdeI and HindIII to produce plasmid
phuIL13-Tx. The 5` end of the gene fusion was sequenced and showed the
correct DNA of hIL13.
Figure 1:
Scheme for cloning plasmid
phuIL13-Tx, which encodes hIL13-PE38QQR. prohIL13,
propeptide of hIL13; mhIL13, mature hIL13; N, NdeI; H, HindIII; p1,
primer 1; p2, primer 2.
Expression and Purification of Recombinant
Proteins
E. coli BL21 (DE3) cells were transformed
with plasmids of interest and cultured in 1.0 liter of Terrific Broth
(Life Technologies, Inc.). hIL13 and hIL13-PE38QQR were localized to
the inclusion bodies. The procedure for the recombinant proteins
isolation from the inclusion bodies was described
previously(12) . After dialysis, the renatured protein of
hIL13-PE38QQR was purified on Q-Sepharose Fast Flow and by size
exclusion chromatography on Sephacryl S-200 HR (Pharmacia). The initial
step of hIL13 or hIL4 purification was conducted on SP-Sepharose Fast
Flow (Pharmacia).
Protein Synthesis Inhibition Assay
The cytotoxic
activity of chimeric toxins, such as hIL13-PE38QQR, were tested as
follows. Usually 1 10
cells/well were plated in a
24-well tissue culture plate in 1 ml of medium, and various
concentrations of the chimeric toxins were added 20-28 h
following cell plating. After 20 h of incubation with chimeric toxins,
[
H]leucine was added to cells for 4 h, and the
cell-associated radioactivity was measured. For blocking studies,
rhIL2, -4, or -13 was added to cells for 30 min before the chimeric
toxin addition. Data were obtained from the average of duplicates, and
the assays were repeated several times.
10
cells/well were plated in a 24-well
tissue culture plate in 1 ml of medium, and the cytokines were added 20
h following cell plating. After 72 h of incubation with the cytokines,
[
H]leucine was added to the cells for 4 h, and
the cell-associated radioactivity was measured.
Competitive Binding Assay
We determined the
binding ability of hIL13 as compared with hIL4-PE38QQR. The recombinant
hIL4-PE38QQR was labeled with I using the lactoperoxidase
method exactly as described previously(19) . The specific
activity of
I-hIL4-PE38QQR was 6.2 µCi/µg of
protein. Binding assays were performed by a standard saturation and
displacement curves analysis. A431 epidermoid carcinoma cells were
seeded at 10
cells/well in a 24-well tissue culture plates
at 24 h before the experiment. The plates were placed on ice, and cells
were washed with ice-cold PBS without Ca
or
Mg
in 0.2% bovine serum albumin, as described
previously(19) . Then, increasing concentrations of hIL13 or
hIL4-PE38QQR were added to cells and incubated 30 min prior to the
addition of fixed amount of
I-hIL4-PE38QQR for 2-3
h. After incubation, the cells were washed twice and lysed with 0.1 NAOH, and the radioactivity was counted in a
counter.
DE3) as described previously (12) (Fig. 1). hIL13 alone was subcloned into the same
expression plasmid. hIL13 and hIL13-PE38QQR were expressed at high
levels in bacteria as seen in SDS-polyacrylamide gel electrophoresis
analysis of the total cell extract (Fig. 2B). After
initial purification on SP-Sepharose (hIL13) or Q-Sepharose
(hIL13-PE38QQR), the renatured recombinant proteins were applied onto a
Sephacryl S-200 HR Pharmacia column (Fig. 2B). hIL13 and
hIL13-PE38QQR appeared as single entities demonstrating the very high
purity of the final products. The chimeric toxin migrated within
somewhat lower than expected for 50-kDa protein M
range, which may be related to the hydrophobicity of the
molecule. The biologic activity of the rhIL13 was exactly the same as
one obtained from Sanofi Elf Bio Recherche(8) .
Figure 2:
A, Schematic drawing of
multi-domain proteins: (i) PE, (ii) its derivative, PE38QQR, and (iii)
hIL13-PE38QQR. Circles, structural domains of PE; domain Ia (amino acids 1-252), binding domain; domain II (amino acids 253-364), place of the proteolytic cleavage
shown by an interrupted line; the dispensable domain Ib (amino acids 365-404); and domain III (amino acids
405-613), the ADP-ribosylating domain. PE38QQR, domain
Ia and amino acids 365-380 in Ib are deleted, plus the three
lysine residues in domain III at positions 590, 606, and 613 are
changed to two glutamines and arginine (QQR) (17). The square symbolizes hIL13. B, expression in E. coli and
purification of hIL13 and hIL13-PE38QQR. 15% nonreduced
SDS-polyacrylamide gel electrophoresis stained with Coomassie Blue.
Total cell extract of transformed E. coli BL21 (DE3) was
loaded at 2 µl/lane (sucrose suspension of the pellet obtained from
1.0 liter of culture; total volume was 75 ml). Sephacryl S-200
HR-purified proteins were loaded at 5.5 µg/lane of hIL13 and 11
µg/lane hIL13-PE38QQR, respectively.
hIL13-PE38QQR Is Cytotoxic to Many Cancer Cell
Lines
There have been no reports about the presence of IL13R on
human solid cancers besides on renal cell carcinomas(8) .
Therefore, we tested several established cancer cell lines to determine
if hIL13-PE38QQR is cytotoxic to them. We examined cancers derived from
colon, skin, and stomach; the cancer cells were sensitive to
hIL13-PE38QQR with IC values ranging from less than 1
ng/ml to 300 ng/ml (20 pM to 6.0 nM). A colon
adenocarcinoma cell line, Colo201, was very responsive with an
IC
of 1 ng/ml (Fig. 3). A431 epidermoid carcinoma
cells were also very sensitive to the action of hIL13-toxin; the
IC
for hIL13-PE38QQR ranged from 6 to 10 ng/ml. A gastric
carcinoma CRL1739 cell line responded moderately to the hIL13-toxin
with an IC
of 50 ng/ml. Another colon carcinoma cell line,
Colo205, had a poorer response with an IC
of 300 ng/ml.
The cytotoxic action of hIL13-PE38QQR was specific as it was blocked by
a 10-fold excess of hIL13 on all cells (Fig. 3). These data
suggest that a specturm of human cancer cells possess hIL13 binding
sites and such cells are sensitive to hIL13-PE38QQR chimeric toxin.
Figure 3:
Cytotoxic activity of hIL13-PE38QQR on
several cancer cell lines and an inhibition of this cytotoxicity by
hIL13. hIL13 was added at a concentration of 1.0 µg/ml. The dashed line shows 50% of [H]leucine
incorporation.
hIL13-PE38QQR;
, with
hIL13.
Because the hIL13R has been suggested to share subunit of the IL2R(11) , we explored further the
specificity of hIL13-PE38QQR action on A431 and CRL1739 cells, the two
cell lines with different sensitivities to the chimeric toxin (Fig. 4). The cells were treated with hIL13-PE38QQR with or
without rhIL2 (a gift from Cetus) at a concentration of 1.0 or 10
µg/ml. The rhIL2 did not have any blocking action on hIL13-PE38QQR
on the two cell lines, even at 10,000-fold molar excess over the
chimeric toxin (Fig. 4). These results indicate that the cell
killing by the hIL13-toxin is independent of the presence of hIL2.
Figure 4:
Failure
to block the cytotoxicity of hIL13-PE38QQR on cancer cells by 1.0
µg/ml or 10 µg/ml of hIL2. The dashed line shows 50%
of [H]leucine incorporation.
hIL13-PE38QQR;
, with hIL2 (1 µg/ml);
, with hIL2 (10
µg/ml).
hIL4, Unlike hIL2, Blocks the Action of
hIL13-PE38QQR
We also added native hIL4 to cells and then
treated with hIL13-PE38QQR, as another measure of specificity (Fig. 5). Unexpectedly, we found that hIL4 inhibited very
potently the cytotoxic activity of the hIL13-toxin. This phenomenon was
seen on all of the tested cell lines, which includes Colo201, A431 (Fig. 5), and CRL1739.(
)To investigate the
possibility that hIL13 and hIL4 may compete for the same binding site,
we also treated the cells with hIL4-based recombinant toxin,
hIL4-PE38QQR (15) (Fig. 5). The cytotoxic action of
hIL4-PE38QQR had already been shown to be blocked by an excess of hIL4
but not of hIL2(15) . In the present experiment, we examined
hIL13 and found that it blocked potently the cytotoxic activity of
hIL4-PE38QQR (Fig. 5). Also, the action of another hIL4-based
chimeric toxin, hIL4-PE4E(12) , was blocked by an excess of
hIL13 on Colo201 and A431 cells.
Thus, the cytotoxicity of
hIL13-PE38QQR is blocked by an excess of hIL13 or hIL4, and the
cytotoxic action of hIL4-PE38QQR is also blocked by the same two growth
factors. However, IL2 does not block the action of either chimeric
toxin. These results strongly suggest that hIL4 and hIL13 have
affinities for the same binding site.
Figure 5:
Blocking the cytotoxicity of hIL13-PE38QQR
by hIL4 and blocking the cytotoxicity of hIL4-PE38QQR by hIL13 on
cancer cell lines. hIL4 was added at a concentration of 0.25 µg/ml
and hIL13 at a concentration of 1.0 µg/ml. The dashed line shows 50% of [H]leucine incorporation.
hIL13-PE38QQR;
, with hIL4;
, hIL4-PE38QQR;
,
with hIL13.
This conclusion was supported
by the observation of one cytokine blocking the effect of a mixture of
the two chimeric toxins. Fig. 6illustrates experiments in which
we used both hIL13- and hIL4-PE38QQR at equimolar concentrations on
A431 cells. When the cells were incubated with both chimeric toxins
concomitantly, the cytotoxic action was preserved, and additive effect
was observed as expected. An excess of hIL13 efficiently
blocked the action of a mixture of the two chimeric toxins (Fig. 6). Moreover, neither hIL13 (Fig. 6) nor hIL4
blocked the cell killing by another mixture composed of
hIL13-PE38QQR and TGF
-PE40, a chimeric toxin that targets the
epidermal growth factor receptor (TGF
-based chimeric toxin,
TGF
-PE40)(21) . The same was observed on Colo201
cells.
Figure 6:
hIL13 blocks the cytotoxic action of a
mixture of hIL13-PE38QQR and hIL4-PE38QQR, but it does not block the
cytotoxicity of a mixture of hIL13-PE38QQR and TGF-PE40. The dashed line shows 50% of [
H]-leucine
incorporation.
, with hIL13.
Blocking the Action of Chimeric Toxins by the
Cytokines Is Due to the Competition for Binding Sites
It was
important to determine whether the binding of hIL4 chimeric toxin is
affected by hIL13, since receptor binding is the first step in the
cytotoxic action of chimeric protein. To investigate this, we performed
competitive binding assays. The iodination of hIL4-PE38QQR and the
binding experiments were carried out as described under
``Experimental Procedures.'' hIL4-PE38QQR competed for the
binding of I-hIL4-PE38QQR to A431 cells with an apparent
IC
of 4
10
M (Fig. 7). In addition, hIL13 also competed for the
I-hIL4-PE38QQR binding site with a comparable potency to
that exhibited by the chimeric protein. More extensive binding studies
have shown that hIL13 competes for hIL4 binding sites on human renal
carcinoma cell lines also(8) .
Figure 7:
Competitive binding assay on A431 cells.
Data are expressed as a percentage of total I-hIL4-PE38QQR binding to cells. The points are the
average of experiments performed in
duplicate.
We excluded the possibility of
an influence of hIL13 or hIL4 on the process of receptor-mediated
endocytosis and post-binding PE cellular toxicity steps by adding the
following to cells: (i) native PE (PE binds to the
-macroglobulin receptor), (ii) TGF
-PE40, and
(iii) a recombinant immunotoxin C242rF(ab`)-PE38QQR(20) .
C242rF(ab`)-PE38QQR binds a tumor-associated antigen that is a
sialylated glycoprotein(19) . We observed the expected potent
cytotoxic actions of these recombinant toxins and neither hIL13 nor
hIL4 blocked these actions on A431 and Colo205 cells.
hIL4 and hIL13 Compete for a Common Binding Site on
Carcinoma Cells but May Evoke Different Biological
Effects
Despite being competitors for the same binding site,
some differences were observed in hIL13- and hIL4-induced cellular
effects. Protein synthesis was inhibited in A431 epidermoid carcinoma
cells in a dose-dependent manner by hIL4 alone (Fig. 8) or by a
ADP ribosylation-deficient chimeric toxin containing hIL4(15) .
This effect of hIL4 or enzymatically deficient chimeric toxin can be
best seen with a prolonged time of incubation (24 h) and requires
concentrations of hIL4 many fold higher than that of the active
chimeric toxin in order to cause a substantial decrease in tritium
incorporation. When, however, A431 cells were treated with various
concentrations of hIL13, no inhibition (or stimulation) of protein
synthesis was observed, even at concentrations as high as 10 µg/ml
of hIL13 for a 72-h incubation (Fig. 8). The same lack of
response to hIL13 was found on renal cell carcinoma cells PM-RCC.
(
)Thus, hIL13 and hIL4 possess a common site but may
transduce differently in carcinoma cells expressing this common site,
such as A431 and PM-RCC cells.
Figure 8:
hIL4, but not hIL13, inhibits protein
synthesis in A431 epidermoid carcinoma cells. Data represent the
average of quadruplicates. Standard deviations are marked as vertical bars.
have recently
found that the action of hIL13-PE38QQR is poorly blocked by the
wild-type hIL4 on the very same renal cell carcinomas on which hIL4
also poorly or not at all displaced labeled hIL13(8) . Thus, the
binding data may actually predict the competition for the activity of
chimeric toxin. These data together suggest the presence of different
cytokine receptors variants among various human malignant cells. The
nature of the receptor forms on cancer cells is under further
investigation.
suggest a
possible form of the common for hIL13 and hIL4 internalized receptor on
cancer cells. There are several lines of evidence suggesting the
presence of (i) more than one binding protein and (ii) a common
receptor by virtue of having a common receptor component(s). First, our
results showed a potent killing of phytohaemagglutinin-activated
peripheral blood lymphocytes by a hIL4-based chimeric toxin (12) but not by hIL13-toxin.
This finding has been
substantiated by the demonstration of a negligible amount of hIL13
binding sites on phytohaemagglutinin-activated peripheral blood
lymphocytes, which in turn bind hIL4 avidly(8) . Second, there
is no parallelism between the cytotoxic potencies of hIL13- and
hIL4-PE38QQR on most of the studied cancer cell lines of various
origins.
Third, a truncated chain of the major hIL4R
140-kDa protein(22) , which contains the extracellular and
transmembrane portions of the receptor(23) , mediates the
binding and internalization of hIL4 that is not blocked by hIL13 (7).
Fourth, the cross-linking experiments revealed interesting patterns of
binding proteins for hIL13 and hIL4(8) . For example, the
is not a component of hIL3R and, unexpectedly, the
chain is not associated with hIL4R on renal cell
carcinomas(8) . Furthermore, hIL4 cross-linked to two proteins:
the already identified 140-kDa protein and a novel 70-kDa plasma
membrane protein(8) . hIL13 cross-linked to only one protein of
70 kDa, which is similar in size to a lower molecular weight
hIL4-cross-linked protein (8). It is thus plausible that a common
internalized species of the hIL13R and hIL4R is the hIL4R 140-kDa
protein complexed with a
70 kDa hIL13-responsive subunit different
from
. Since there are so many carcinomas expressing
receptors for hIL13 and hIL4 (12-15), it will be important to
establish the exact nature of interrelatedness between the two types of
receptors and their possible molecular arrangements. This should help
in designing strategies targeting these internalized receptors for
anticancer therapy using, for example, chimeric toxins.
,
-subunit of the interleukin 2 receptor; PE, Pseudomonas exotoxin A; r, recombinant; PCR, polymerase chain
reaction; IC
, 50% inhibitory concentration; TGF,
transforming growth factor.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.