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Experimental Therapeutics |
Laboratory of Molecular Tumor Biology, Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and IL-13R' chain (also known as
IL-13R1). However, no cell line expressed IL-2R common
-chain, which is known to be shared with IL-4R in immune cells.
IL-4R is functional because IL-4 strongly induced activation of signal
transducers and activators of transcription 6 (STAT-6) in these cell
lines. A fusion protein, IL4(38-37)-PE38KDEL, containing translocation
and enzymatic domains of Pseudomonas exotoxin and a
circularly permuted human IL-4 was found to be highly and specifically
cytotoxic to IL-4R-positive head and neck cancer cells, as determined
by protein synthesis inhibition assay and confirmed by clonogenic
assay. IL4(38-37)-PE38KDEL induced DNA fragmentation and condensation
of nuclei indicative of apoptotic cell death. These results establish
uniform expression of IL-4R on head and neck cancer cell lines and IL-4
toxin IL4(38-37)-PE38KDEL as a novel therapeutic agent for the possible
treatment of human head and neck cancers. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We have reported that a wide variety of murine and human carcinoma
cells expresses plasma membrane receptors for an immune regulatory
cytokine, IL-4, in vitro and in vivo
(5, 6, 7, 8)
and that IL-4 functions by signaling through its
receptors (9)
. We have also studied the subunit
composition of IL-4R on a variety of cell types (10, 11, 12, 13)
.
It was found that the IL-4R system could exist in three different
types. Type I receptors are shown to consist of a major
Mr 140,000 protein (IL-4Rß,
also known as IL-4R) and IL-2Rc. Type II
receptors are composed of IL-4Rß and IL-13R' (also known as
IL-13R1) chains. In a third type of IL-4R, all
three chains may form an IL-4R complex (11, 12, 13, 14, 15)
. Although
the importance of expression of IL-4Rs on solid tumor cells is not
known, we and others have observed that solid human tumors including
malignant melanoma, breast carcinoma, ovarian carcinoma, mesothelioma,
neuroblastoma, renal cell carcinoma, and AIDS-associated Kaposi’s
sarcoma respond to IL-4 (7
, 8
, 16, 17, 18, 19)
. IL-4, a unique
cytokine produced by activated T lymphocytes and mast cells (5
, 6
, 20)
, inhibits the in vitro growth of several tumor
cell lines. It has also been reported that IL-4 can induce apoptosis in
human breast cancer cell lines (21)
. In contrast, Myers
et al. (22)
reported that IL-4 could stimulate
the growth of 6 of 13 SCCHN cell lines.
Using a chimeric protein composed of circular permuted IL-4 and a truncated form of a powerful bacterial toxin called PE [IL-4 toxin called IL4 (38-37)-PE38KDEL], we have shown that this toxin is highly cytotoxic to IL-4R-positive tumor cells in vitro (23, 24, 25, 26, 27, 28) and in vivo (16 , 26 , 29) . In our previous study, we have reported that head and neck cancer cells also express IL-4R in situ (30) . However, IL-4R structure, function, and cytotoxic activity of IL-4 toxin in SCCHN have not been investigated. In the present study, we have examined the expression, structure, and function of IL-4R on head and neck cancer cell lines. In addition, we have investigated the cytotoxicity and mechanism of cytotoxicity of IL-4 toxin in SCCHN cell lines.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recombinant IL-4 Toxin.
The IL-4 toxin IL4(38-37)-PE38KDEL, containing the circularly permuted
IL-4 mutant in which amino acids 38–129 were linked to amino acids
1–37 via a GGNGG linker and then fused to truncated toxin PE38KDEL,
consisting of amino acids 253–364 and 381–608 of PE, followed by
KDEL, was expressed in Escherichia coli and purified as
described previously (24, 25, 26)
.
Cell Lines.
Human head and neck cancer cell lines (KB, A253, RPMI 2650, and HEp-2)
were purchased from American Type Culture Collection (Manassas,
VA). The WSU-HN12 (termed HN12) cell line was a kind gift from
Dr. Andrew Yeudall (National Dental and Craniofacial Research
Institute, NIH, Bethesda, MD; Ref. 33
). Twelve head and
neck squamous cell cancer cell lines were established in the Department
of Otolaryngology, Yokohama City University School of Medicine or
Research Institute, Kanagawa Cancer Center (Yokohama, Japan;
Ref. 34
). The origin of the each cell lines is shown in
Table 1
. Cells were cultured in Eagle’s Modified Essential Medium (KB,
A253, RPMI 2650, HEp-2, and HN12) or RPMI 1640 (the other cell lines)
containing 10% fetal bovine serum (BioWhittaker Inc., Walkersville,
MD), 1 mM HEPES, 1 mM nonessential amino acids,
100 µg/ml penicillin, and 100 µg/ml streptomycin (BioWhittaker
Inc.).
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Northern Analysis.
Total RNA was isolated using TRIZOL reagent (Life Technologies,
Grand Island, NY). Equal amounts of total RNA were electrophoresed
through an 0.8% agarose, formaldehyde-denaturing gel, transferred to a
nylon membrane (S&S Nytran; Scheicher and Schuell, Keene, NH) by
capillary action, and immobilized by UV cross-linking (Stratagene, La
Jolla, CA). The cDNA for human IL-13R', common
c or glyceraldehyde-3-phosphate dehydrogenase,
was labeled with [-32P]dCTP (3000 Ci/mmol;
Amersham, Arlington Heights, IL). Membrane-bound RNA was prehybridized
for 30 min at 37°C and then hybridized with
32P-labeled cDNA probes for 1 h at 37°C in
ExpressHyb hybridization solution (Clontech Laboratories, Inc., Palo
Alto, CA). The membranes were washed and subsequently exposed to an
X-AR film for 12–72 h at -70°C to obtain an autoradiogram.
RT-PCR Analysis.
To detect the expression of IL-4Rß chain in head and neck cancer cell
lines, we performed RT-PCR analysis. Total RNA (2 µg ) was incubated
for 30 min at 42°C in 20-µl reaction buffer containing 10
mM Tris-HCl (pH 8.3), 5 mM
MgCl2, 50 mM KCl, 1 mM
each of dNTPs, 1 unit/µl RNase inhibitor, 2.5 µM random
hexamer, and 2.5 units/µl of MMLV RT (Perkin-Elmer Corp., Norwalk,
CT). A 10-µl aliquot of RT reaction was amplified in a final
100-µl volume of PCR mixture containing 10 mM
Tris-HCl (pH 8.3), 2 mM MgCl2, 50
mM KCl, 2.5 units of AmpliTaq Gold DNA polymerase
(Perkin-Elmer Corp.), and 0.1 µg of specific primer (5' primer
5'-GACCTGGAGCAACCCG TATC-3' and 3' primer 5'-CATAGCACAACAGGCAGACG-3';
Ref. 11
). PCR product (30 µl) was run on a 2% agarose
gel for UV analysis.
Electrophoretic mobility shift assay.
After incubation with IL-4 (50 ng/ml) for 10 min, cells were washed
with cold extraction buffer [1 mg/ml leupeptin, 5 mg/ml pepstatin A, 2
mg/ml aprotinin, 20 mM HEPES (pH 7.0), 10 mM
KCl, 300 mM NaCl, 0.5 mM DTT, 0.1% NP40, 1
mM phenylmethylsulfonyl fluoride, 1 mM
Na3VO4, and 20% glycerol).
DNA protein interactions were assessed by electrophoretic mobility
shift assay using a Bandshift kit (Pharmacia Fine Chemicals,
Piscataway, NJ). Briefly, 50 µg of sample proteins were incubated in
20 µl of binding buffer [10 mM Tris-HCl (pH 7.5), 50
mM NaCl, 0.5 mM DTT, 10% glycerol, 0.05%
NP40, and 0.05 mg/ml poly (dI-dC)2
; for 20 min at room
temperature with 1 ng of 32P-labeled
double-stranded oligonucleotide probe SBE1. SBE1 is a STAT-binding
element (5'-gatcGCTCTTCTTCCCAGGAACTCAATG-3';
3'-CGAGAAGAAGGGTCCTTGAGTTACagct-5'), which is from the region flanking
the transcription start site of the human sIL-1R
antagonist gene that is necessary for response to IL-4
(36)
. A 10x loading dye (2 µl) was added to samples
that were then applied to a 4% nonreducing polyacrylamide gel and run
at 150 V for 2.5 h. Gels were dried for 2 h and
autoradiographed overnight at room temperature.
Protein Synthesis Inhibition Assay.
The cytotoxic activity of IL-4 toxin was tested as described previously
(37)
. Typically, 104 cells were
cultured in leucine-free medium with or without various concentrations
of IL4(38-37)-PE38KDEL for 20–22 h at 37°C. For blocking
experiments, cells were preincubated with IL-4 or IL-13 (2 µg/ml) for
1 h before the addition of IL-4 toxin. Then 1 µCi of
[3
H]leucine (NEN Research Products, Boston, MA)
was added to each well and incubated for an additional 4 h. Cells
were harvested and radioactivity incorporated into cells was measured
by a ß plate counter (Wallac).
Detection of Apoptotic Cells.
Apoptotic cells were assessed morphologically by fluorescent
microscopy. KB cells (2 x 106)
were plated in 100-mm Petri dishes with 7 ml of medium and were allowed
to attach for 24 h. The cells were exposed to IL-4 toxin (1000
ng/ml) or IL-4 (100 ng/ml) for 48 h, then washed, fixed with 1%
glutaraldehyde, and stained with 1 mM bis-Benzimide
(Hoechst no. 33342; Sigma Chemical Co., St. Louis, MO). The samples
were examined by fluorescent microscopy under an UV filter.
DNA Fragmentation Assay.
After a 48-h incubation of KB cells (2 x 106) with IL-4 toxin, the cell extracts
containing fragmented DNA were incubated with 0.5 mg/ml RNase A at
37°C for 60 min, then with 0.5 mg/ml proteinase K at 37°C for 60
min. After incubations, fragmented DNA was precipitated by isopropanol
and dissolved in 10 mM Tris-HCl (pH 8.0), 1 mM
EDTA, 5% glycerol, and 0.05% bromphenol blue. DNA fragments (2 µg),
separated by 1% agarose gel electrophoresis, were stained with
ethidium bromide and photographed on a UV transilluminator.
Clonogenic Assay.
The in vitro cytotoxic activity of IL4(38-37)-PE38KDEL on KB
cells was also determined by colony-forming assay. The cells were
plated in triplicates in 100-cm2
Petri dishes
with 7 ml of medium containing 20% fetal bovine serum and allowed to
attach for 20–22 h. The number of cells/plate was chosen such that
>100 colonies were obtained in the control group. The cells were
exposed to different concentrations of IL-4 toxin (0–100 ng/ml) or
IL-4 (0–100 ng/ml) for 9 days at 37°C in a humidified incubator. The
cells were washed, fixed, and stained with crystal violet (0.25% in
25% alcohol). Colonies consisting of >50 cells were scored. The
percentage of colony survival was determined from the number of
colonies formed in the control and treated groups.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 125I-IL-4 bound to SCCHN cells at
varying degrees and excess of unlabeled IL-4 displaced the binding of
125I-IL-4. Because IL-4R and IL-13R share two
chains with each other, we also examined whether IL-13 can
displace the IL-4 binding in SCCHN cells (14
, 15)
. As
shown in Fig. 1A
, IL-13 also displaced for
125I-IL-4 binding, however, IL-4 was slightly
superior to IL-13 in displacing 125I-IL-4
binding.
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). YCUM862 cells bound IL-4 in a
concentration-dependent manner. Scatchard analysis of the binding data
showed a single type of receptor with a kd value
of 37.9 ± 0.4 nM. The number of
IL-4Rs was calculated as 476,000 ± 5,000 IL-4 molecules
bound/cell (mean ± SD, n = 2).
Subunit Structure of IL-4R on Head and Neck Cancer Cells.
Seventeen head and neck cancer cell lines were examined for the
expression of various putative IL-4R subunits. By Northern analysis, we
found that mRNA for IL-13R' chain was uniformly present in all of
the cell lines examined. However, no SCCHN cell lines showed presence
of c mRNA (Fig. 2A
). H9 T lymphoma cells that express
c mRNA served as a positive control. We also
examined the mRNA expression of IL-4Rß chain by RT-PCR analysis and
found that all of the cell lines examined expressed IL-4Rß chain
(Fig. 2B
).
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shows that IL-4 induced STAT6-DNA interaction in KB, A253, and YCUM862
cell lines (Lanes 2, 4, and
6), whereas STAT6 activation was not detectable in HEp-2
cells that expressed a low number of IL-4Rs (Lane
8).
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shows the
IC50s (the protein concentration required for the
inhibition of protein synthesis by 50%) in the 17 cell lines studied
that ranged from 0.1–600 ng/ml. Fifteen of 17 cell lines were
extremely sensitive to IL-4 toxin in which the
IC50s were 
10 ng/ml. The cytotoxic activity of
IL4(38-37)-PE38KDEL was neutralized by excess IL-4 and IL-13 in the
cell lines examined (Fig. 4
). Extremely high numbers of IL-4R on YCUM862 cells might account for
the high sensitivity to IL-4 toxin (IC50 = 0.75 ng/ml).
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, IL-4 toxin induced pronounced programmed cell death as
evidenced by fragmented and condensed nuclei, whereas no apoptotic
cells could be detected after incubation with IL-4. Fig. 5B
shows DNA fragmentation in the KB cell line after treatment with
various concentrations of IL4(38-37)-PE38KDEL. Inter-nucleosomal DNA
fragmentation, a biochemical feature of the apoptotic process, was
observed when cells were treated with 100 or 1000 ng/ml of IL-4 toxin
(Lanes 5 and 6). Lower concentrations
of IL-4 toxin also showed slight DNA smears (Lanes 2–4).
However, IL-4 at concentrations up to 100 ng/ml did not cause DNA
fragmentation (Lane 7 and data not shown for
lower concentrations).
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, IL4(38-37)-PE38KDEL inhibited colony formation in KB cells
in a concentration-dependent manner. These results were comparable with
the dose-kinetics seen in the cytotoxicity assay (Fig. 6A
).
In both assays IL-4 had no effect on proliferation of KB cells.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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' chains, our studies imply that IL-4R complex on SCCHN is
of type II form where the IL-4Rß chain forms a complex with the
IL-13R' chain. The common c chain was not
identified in these cells. This receptor structure is similar to that
observed on a variety of human solid tumor cell types, e.g.,
colon cancer, renal cell carcinoma, AIDS-associated Kaposi’s sarcoma,
glioblastoma multiforme, and breast carcinoma (8
, 10
, 12
, 16
, 27)
. Thus, it is reasonable to assume that all solid cancers
that express IL-4R may express type II receptors. The IL-4R on SCCHN
seemed to be related to the IL-13R because IL-13 was able to displace
125I-IL-4 binding and competed for IL-4
toxin-mediated cytotoxicity.
Typically, a ligand must bind to the extracellular domain of cytokine
receptors to generate a biological response. In the case of IL-4, such
interaction in immune cells (e.g., B cells and T cells)
induces growth stimulation through the binding to type I IL-4Rs in
which the IL-4Rß chain forms a functional complex with the common
c and through STAT 6 activation. However,
unlike other solid cancer cells, including some head and neck cancer
cell lines in which IL-4 has been shown to modulate growth, IL-4 did
not modulate growth of head and neck cancer cell lines studied in our
laboratory (30)
. Nevertheless, IL-4 was able to signal
through activation of STAT6 protein in these cells. Thus, our studies
indicate that IL-4 uses a similar distal pathway of signaling in cancer
cells and immune cells and that IL-4Rs are functional on head and neck
cancer cells.
The IL-4R expressed on head and neck cancer cells served as an efficient target for a cytotoxic agent. As seen in several solid cancers, IL4(38-37)-PE38KDEL was also highly and specifically cytotoxic to head and neck cancer cells (23, 24, 25, 26, 27) . Fourteen of 17 cell lines exhibited remarkable sensitivity to the cytotoxic activity of IL-4 toxin. The cytotoxic activity was further confirmed in a clonogenic assay. Furthermore, the IC50 was similar in protein synthesis inhibition and clonogenic assays. The cytotoxic activity of IL4(38-37)-PE38KDEL was mediated through apoptotic cell death, whereas IL-4 had no apoptotic activity. Others have reported that B3(Fv)-PE38 immunotoxin (in which Lewisy is recognized by the Fv fragment of an antibody connected to a mutated form of PE) induces apoptosis in breast cancer cells through the caspase pathway (39) . Our study confirms these observations. However, in contrast to previous studies, IL-4 by itself did not cause apoptosis in SCCHN cell lines (21) . Similarly, in contrast to previous studies, our study has not identified growth stimulatory effects of IL-4 in four SCCHN cell lines (e.g., KB, A253, RPMI 2650, and HEp-2; Ref. 22 , 30 ). The reason for this differential effect is not known; however, it is possible that the IL-4 effect is tumor specific representing the heterogeneous nature of this cancer.
Our previous studies have demonstrated that in vitro sensitivity to IL-4 toxin correlates with in vivo antitumor activity in brain tumor, AIDS-associated Kaposi’s sarcoma, epidermoid carcinoma, and breast tumor models in nude mice (16 , 26 , 29 , 40) . On the basis of these observations, it is reasonable to predict that IL4(38-37)-PE38KDEL will have significant antitumor activity in SCCHN in vivo in athymic nude mice with s.c.-growing tumors. We are currently examining the antitumor activity of IL4(38-37)-PE38KDEL by systemic administration in a SCCHN xenograft model.
Various innovative approaches, including gene transfer, are being tested for SCCHN. Although these techniques seem to be promising, currently no approach seems to be more effective. In addition, these approaches are limited due to vector-related toxicities and suboptimal gene transfer. Because IL4(38-37)-PE38KDEL is a small molecule and does not involve an indirect mechanism of tumor cell kill, we believe that it may have superior antitumor activity without producing unknown virus or plasmid-related toxicities.
In conclusion, we demonstrate that 100% of SCCHN tumor cell lines examined express surface IL-4Rs that seem to be biologically functional. Because IL-4 toxin IL4(38-37)-PE38KDEL has profound cytotoxic activity against all of the tested cell lines, we conclude that IL-4R can serve as a unique target for the delivery of a cytotoxic agent to SCCHN. Additional studies must be performed to reveal the antitumor activity of IL-4 toxin in animal models, and perhaps a Phase I clinical trial should be undertaken to study its antitumor activity.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 To whom requests for reprints should be
addressed, at Laboratory of Molecular Tumor Biology, Division of
Cellular and Gene Therapies, Center for Biologics Evaluation and
Research, Food and Drug Administration, Building 29B, Room 2NN10, 29
Lincoln Drive MSC 4555, Bethesda, MD 20892. Phone: (301) 827-0471; Fax:
(301) 827-0449; E-mail: puri{at}cber.fda.gov 
2 The abbreviations used are: SCCHN, squamous cell
carcinoma of the head and neck; IL-4, interleukin-4; IL-4R, IL-4
receptor; IL-13R, IL-13 receptor; PE, Pseudomonas
exotoxin A; c, common -chain; RT, reverse
transcription; STAT, signal transducers and activators of
transcription.
3 B. H. Joshi, Y. Oshima, and R. K. Puri,
unpublished results.
Received 11/ 8/99. Accepted 4/ 3/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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chain shared by receptors for interleukins 2, 4, 7, 9, and 15. J. Biol. Chem., 270: 8797-8804, 1995. chain, and IL-4 induces the phosphorylation of Jak2 tyrosine kinase in human colon carcinoma cells. J. Biol. Chem., 270: 30829-30836, 1995.' but not chain: a functional component of interleukin-4 receptors. Blood, 91: 3884-3891, 1998.-interferon or tumor necrosis factor inhibits cell growth and modulates cell surface antigens on human renal cell carcinomas. Cancer Res., 51: 5687-5693, 1991.c chain in RCC cells does not improve sensitivity. Cell. Immunol., 171: 80-86, 1996.[Medline]
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, IL4(38-37)-PE38KDEL+IL-4; 
,
IL4(38-37)-PE38KDEL+IL-13.
, IL-4. Data are means; bars, SD. B,
Five hundred KB cells were allowed to adhere in Petri dishes, and
medium was replaced with medium containing various concentrations of
IL4(38-37)-PE38KDEL or IL-4. Cells were cultured for 9 days, and
colonies consisting of at least 50 cells were scored after staining
with crystal violet. 
, IL-4. Data are
means of triplicate determinations; bars, SD.