(Received for publication, June 26, 1995; and in revised form, July 31, 1995)
From the
Interferon-inducible membrane proteins of approximately 17 kDa have been suggested to play a role in the antiproliferative activity of interferons based on (1) their pattern of induction in interferon-sensitive and -resistant cell lines and (2) the ability of a membrane fraction enriched in 17-kDa proteins to inhibit cell growth. To gain insight into the nature of the proteins that mediate the antiproliferative activity of interferons, a monoclonal antibody, 13A5, was generated that reacted specifically with a 17-kDa interferon-inducible cell surface protein. The expression pattern of this 17-kDa protein by human cell lines correlated with sensitivity to the antiproliferative activity of interferons. To obtain information regarding the structure of this protein, the 13A5 antibody was used to screen COS cells transfected with a human cDNA expression library. Sequence analysis of a full-length cDNA clone revealed identity with the 9-27 cDNA, previously isolated on the basis of its interferon inducibility by differential screening. In addition, the 17-kDa protein encoded by the 9-27 gene was shown to be identical to the Leu-13 antigen. Leu-13 was previously identified as a 16-kDa interferon-inducible protein in leukocytes and endothelial cells and is a component of a multimeric complex involved in the transduction of antiproliferative and homotypic adhesion signals. These results suggest a novel level of cellular regulation by interferons involving a membrane protein, encoded by the interferon-inducible 9-27 gene, which associates with other proteins at the cell surface, forming a complex relaying growth inhibitory and aggregation signals.
Interferons (IFN) ()are multifunctional cytokines
that play a critical role in the defense against viral or parasitic
infections. These cytokines also exhibit antiproliferative and
differentiating activities, prompting an evaluation of their potential
as antitumor agents. While the results of animal testing have been
encouraging, clinical trials in humans have shown IFNs to be effective
for only a small subset of cancer types(1) . This dramatic host
difference in susceptibility to the antitumoral activity of IFNs
underscores the need to understand at the molecular level the mechanism
by which IFNs exert this activity. Exposure to IFNs leads to a
modulation in the levels of an estimated 50-100 cellular
proteins, which are thought to collectively mediate IFNs pleiotropic
effects(2) . Identifying the role and contribution of each
protein is a major challenge, but it will provide the basis for
designing better therapeutic strategies based on IFNs.
In addition to soluble cytokines and growth factors, cell-cell interactions involving specific membrane proteins are likely to play an important role in the control of cell growth and differentiation. Indeed, it has been shown that the antiproliferative activity of IFN could be transferred from cell to cell(3) . Moreover, it has been suggested that IFN-induced cell surface proteins of approximately 17 kDa could be involved in their antiproliferative activity, since their expression pattern in various cell lines correlates with the sensitivity to the inhibition of cell growth induced by IFNs(4, 5, 6) , and a membrane fraction enriched for 17-kDa proteins inhibits cell growth(7) .
The purpose of the present study was to investigate the possible contribution of IFN-inducible cell surface proteins to IFNs antiproliferative activity. Our approach was based on the generation of monoclonal antibodies (mAbs) directed against IFN-inducible cell surface proteins. The mAbs could then be used to analyze the expression and the function of their target and eventually to clone the corresponding cDNA.
Here we report the characterization of a mAb directed against a 17-kDa membrane antigen inducible by IFNs. The expression pattern of this 17-kDa protein by human cell lines correlated with sensitivity to the antiproliferative activity of IFNs. Expression cloning revealed that the 17-kDa protein is encoded by the 9-27 gene, which is a member of an IFN-inducible gene family isolated by differential screening and whose function was unknown(5, 6) . We also demonstrated that the product of the 9-27 gene is a protein previously identified as Leu-13, a leukocyte antigen that is part of a membrane complex of proteins involved in the transduction of antiproliferative and homotypic adhesion signals(4, 5, 8, 9, 10, 11, 12) .
Hybridoma
supernatants were screened for their differential reactivity to
IFN--treated or untreated Daudi cells by flow cytometry.
Positively selected hybridomas were repeatedly cloned by limiting
dilution in the presence of 100 units/ml of recombinant murine
interleukin-6 to maintain Ig secretion. Antibody isotype was determined
using the mouse mAb isotyping kit from Life Technologies. IgM mAbs were
purified by ammonium sulfate precipitation of ascites fluid followed by
exclusion chromatography on A-0,5 resin (Bio-Rad 200-400 mesh),
and their concentration was quantified by enzyme-linked immunosorbent
assay.
Additional murine mAbs used in this study include the
following: anti-Leu-13, an IgG originally characterized by
Chen et al.(10) ; anti-TAPA-1 antibody
5A6(17) , an IgG
(kind gift of Dr. S. Levy,
Stanford University School of Medicine, Stanford CA); anti-CD3 mAb
OKT3, an IgG
(obtained from the American Type Culture
Collection); and two isotype-specific control antibodies that were
nonreactive against human cell lines, A4 (an IgG
that is an
anti-idiotypic antibody) and M104E (an IgM directed against B1355S
dextran, kindly provided by Dr. R. Ward (Roswell Park Cancer Institute,
Buffalo, NY)).
One mAb termed 13A5, an IgM, reacted with a
protein that was highly inducible by IFN-
on Daudi cells. The
antigen recognized by this mAb was characterized by immunoprecipitation
of
I-labeled surface proteins from IFN-
-treated or
untreated Daudi cells. SDS-polyacrylamide gel electrophoresis analysis
of the immunoprecipitation products revealed that the molecular mass of
the protein recognized by 13A5 was approximately 17 kDa and that its
expression on Daudi cells was strongly induced upon IFN-
treatment (Fig. 1, A and B). The antigen recognized by
13A5 had the same mobility as a predominant IFN-inducible protein that
can be detected in whole
I-labeled IFN-
-treated
Daudi cell lysates (Fig. 1, A and C). An
IFN-inducible 17-kDa protein on the surface of Daudi cells has been
previously described and implicated in the antiproliferative activity
of IFN(5, 7) .
Figure 1:
Immunoprecipitation of Daudi
cells' proteins by 13A5. Untreated(-) or IFN--treated
(+) Daudi cells were
I-labeled and lysed in Nonidet
P-40 buffer. Immunoprecipitates or crude lysates were resolved on 20%
polyacrylamide gel. A, immunoprecipitation of cell lysates
with 13A5 or a control mAb. B, longer exposure of the gel
shown in A. C, crude lysates. Molecular mass markers
are indicated in kDa on the left.
The results of the 17-kDa protein expression
analysis are presented in Table 1. Fig. 2shows the
fluorescence histograms of four representative cell lines on a
logarithmic scale. We observed that IFN- treatment increased the
expression of the 13A5 antigen on most of the cell lines tested. As
much as a 20-fold increase was observed on Daudi, Jurkat, Reh, and
FS-7F cells. Interestingly, the level of induction was very weak on
several resistant cell lines (e.g. on DaudiR or U937) or
undetectable on DIF8. The pattern of expression appeared to correlate
closely with the ability of IFN-
to inhibit cell growth. IFN-
also significantly affected the expression of the 17-kDa protein
although to a lesser extent than did IFN-
. Induction of the 17-kDa
protein by IFN-
was observed in most cell lines tested, except in
those derived from B or T cells. This result is consistent with our
previous observation that genes that are predominantly induced by type
I IFN are not or very poorly inducible by IFN-
in cell lines of
lymphoblastoid origin(26) . We concluded that the 17-kDa
protein is inducible by both types of IFNs on sensitive cells.
Figure 2:
IFN- or IFN-
induction of the
17-kDa protein. Each cell line was reacted with 13A5 antibody (heavylines) or with a control IgM antibody (thinlines), followed by fluorescein-conjugated
anti-mouse antibody and immunofluorescence was assessed by flow
cytometry.
Some
of the cell lines tested expressed a basal level of the 17-kDa protein,
which was particularly prominent on K562 cells. Confirming the
fluorocytometric analysis, immunoprecipitation of I-labeled surface proteins with 13A5 showed the high
basal level of the 17-kDa protein on K562 cells (Fig. 3).
Overexposure of the autoradiogram allowed the detection of a 28-kDa
component, similar to the one observed in the proteins
immunoprecipitated by 13A5 from IFN-
-treated Daudi cells (compare Fig. 1B and Fig. 3). Immunoprecipitated proteins
of 17 and 28 kDa were also detected under nonreducing conditions (Fig. 3B). These data suggest that the 17-kDa protein
is noncovalently associated in the cell membrane with a protein of 28
kDa.
Figure 3:
Immunoprecipitation of K562 cell proteins
by 13A5. Untreated(-) or IFN--treated (+) K562 cells
were
I-labeled and lysed in Nonidet P-40 buffer. 13A5
immunoprecipitates from K562 extracts were resolved on 20%
polyacrylamide gel under reducing conditions (A) or under
nonreducing conditions (B). Molecular mass markers are
indicated in kDa on the right of each gel.
Figure 4: 13A5 and anti-Leu-13 reacted with 9-27- but not 1-8u-transfected cells. A, COS cells were transfected with the pse1.9-27 plasmid (heavylines) or with the control pse1 plasmid (thinlines) and reacted with 13A5 or anti-Leu-13 as indicated. Cells were counterstained with a GAM-Ig-FITC antibody and analyzed by flow cytometry. B, same experiment as in A except that cells were transfected with the pse1.1-8 plasmid (heavylines). The control plasmid (thinlines) was pse1.
The cDNA sequence was
obtained by the dideoxy termination method. Comparison with the EMBL
data bank showed that the cDNA encoding the 17-kDa protein was nearly
identical to the 9-27 cDNA. The two sequences diverge at
positions 280 (AT), 391 (C
T), 499 (G
A), 624
(C
T), and 643 (G
A) of the previously cloned 9-27
cDNA. Only the replacement at position 624 results in a modification in
the peptide sequence (Ser
Leu). These divergences
are probably due to allelic variations. The 9-27 cDNA was first
isolated on the basis of its IFN inducibility (28) and is part
of a family containing at least two other members, 1-8u and
1-8d, both IFN-inducible as well. The isolated cDNA was
full-length, containing the entire open reading frame of 9-27
preceded by a 5`-untranslated region corresponding to the second start
site of transcription reported for the 9-27 gene(29) .
The 9-27, 1-8u, and 1-8d coding sequences diverge at the amino and carboxyl termini. Thus, it was theoretically possible that 13A5 and anti-Leu-13 could recognize an epitope common to both 9-27 and 1-8 proteins. An reverse transcription polymerase chain reaction approach using primers designed to amplify the 1-8u and 1-8d mRNAs resulted in the obtention of the 1-8u but not of the 1-8d cDNA, which is less abundant and less homologous. As shown in Fig. 4B, neither anti-Leu-13 nor 13A5 reacted with COS cells transfected with the 1-8u cDNA. These results were confirmed by immunoprecipitation experiments performed on in vitro synthesized 9-27 and 1-8u proteins. Both mAbs were able to precipitate 9-27 but not 1-8u products (data not shown). Therefore, the epitope recognized by 13A5 and anti-Leu-13 on the 9-27 protein is not present in the 1-8u protein. Thus, we conclude that the leukocyte antigen, Leu-13, is composed of only one polypeptide encoded by the IFN-inducible 9-27 gene.
Figure 5:
Analysis of proteins coprecipitated by
mAbs 13A5 and 5A6. A, K562 cells were I-labeled
and lysed in Nonidet P-40 buffer (lanes1 and 2) or in CHAPS buffer (lanes3 and 4). These cell extracts were immunoprecipitated by 5A6, the
anti-TAPA-1 antibody (lanes1 and 3), or by 13A5 (lanes2 and 4). Arrows, from higher to lower molecular mass, correspond to
p28, TAPA-1, 9-27. Molecular masses are given in kDa. B,
the same immunoprecipitates shown in A were separated by
SDS-polyacrylamide gel electrophoresis under nonreducing conditions,
transferred to a nitrocellulose sheet, and the presence of TAPA-1 was
revealed using the 5A6 antibody, a horseradish peroxidase-conjugated
anti-mouse antibody, and ECL chemiluminescence. Upperbands correspond to the antibodies used in the
immunoprecipitation.
Figure 6:
Effect of 13A5 and anti-Leu-13 mAbs on
homotypic adhesion and proliferation. A, induction of
homotypic adhesion by mAbs. K562, Jurkat, and U937 cells were cultured
for 24 h with or without 500 units/ml IFN- (+ifn or
-ifn) in the presence of the indicated mAbs at a final
concentration of 5 µg/ml. Control IgG1 and control IgM had no
effect on cell-to-cell adhesion. B, anti-Leu-13 inhibits
anti-CD3-driven proliferation of human PBMC. Human PBMC were cultured
in the absence or presence of 0.5 µg/ml of anti-CD3 mAb.
Anti-Leu-13, 13A5, or isotype-matched control antibodies were included
in cultures at a concentration of 10 µg/ml.
[
H]thymidine incorporation was measured on day 3
of culture. Results represent the mean ±S.D. of replicate
cultures. Data are representative of three independent
experiments.
Modulations of gene expression in cells exposed to IFNs play
an essential role in the ability of these cytokines to affect vital
biological processes. Several proteins whose synthesis is induced by
IFNs have been implicated in the antiviral activity shared by all IFNs
(for review, see (2) and (31) -33). The
inhibition of cell growth by IFNs is also likely to result from an
action on multiple pathways affecting different steps at which cell
growth can be regulated. Interestingly, two double-stranded
RNA-dependent antiviral pathways induced by IFNs have been recently
implicated in their antiproliferative activity as
well(34, 35, 36) . IFN treatment also
directly affects the function of two genes known to be involved in the
control of the cell-cycle, the protooncogene c-myc and the
tumor-suppressor retinoblastoma gene, resulting in arrest at the
G/G
phase of the cell
cycle(37, 38, 39, 40) .
It has been shown that the antiproliferative activity of IFN could be transferred from cell to cell, not by diffusion of a soluble mediator but through direct contact between cells(3) . To explore the role of membrane proteins in the antiproliferative activity of IFNs, we generated mAbs directed against IFN-inducible membrane antigens. We obtained an antibody that reacted specifically with a 17-kDa protein. This 17-kDa protein was induced by type I and II IFNs on a wide range of cell lines sensitive to the antiproliferative effect of IFNs, but not significantly on resistant cell lines. Screening of an expression library yielded a full-length cDNA clone encoding the 17-kDa protein, and sequence analysis revealed identity with the 9-27 cDNA.
The 9-27 gene is induced by both type I and II IFNs, and the mRNA level can increase as much as a 100-fold upon induction by IFNs. Induction by IFNs is transcriptionally controlled by a single IFN-stimulated response element present in the promoter of the gene (41) . The protein encoded by the 9-27 gene has a calculated molecular mass of 13.9 kDa, in close agreement with the observed 16-17 kDa(7, 10) . In addition, we demonstrated that 9-27 was coding for the leukocyte antigen Leu-13. In most cell types studied thus far, Leu-13 was shown to be part of a membrane complex containing several distinct proteins. One of these proteins is the 26-kDa TAPA-1 protein, the target of an antiproliferative antibody(12) . TAPA-1 is strongly related at the sequence level to two other surface proteins involved in the regulation of cell growth, the ME491 melanoma-associated antigen, and CD37, another leukocyte antigen(17) . Leu-13 and TAPA-1 are involved in a mechanism controlling cellular adhesion since anti-Leu-13 and anti-TAPA-1 mAbs each triggers a general homotypic adhesion phenomenon. Interestingly, homotypic adhesion induced by anti-Leu-13 or anti-TAPA-1 is not dependent on adhesion pathways involving known adhesion molecule including the leukocyte function-associated antigen-1, the intercellular adhesion molecule-1, CD44, or VLA-4(4, 5, 12) .
Along with their
involvement in the regulation of cell aggregation, Leu-13 and TAPA-1
are likely to also play a role in the control of cell growth. Indeed,
anti-Leu-13 and anti-TAPA-1 mAbs can directly inhibit cell growth,
although this effect is observed in a more restricted subset of cell
lines(4, 12, 17) . Furthermore, anti-Leu-13
mAbs were shown to potentiate the antigrowth effect of IFN- on
leukemic B cells(4) . Insensitivity to the growth inhibitory
effect of anti-Leu-13 and anti-TAPA-1 mAbs in some cell lines can
probably be accounted for by the alterations in the control of cell
growth that occur when a cell line is established for in vitro growth. We also attempted to establish cell lines stably
expressing 9-27/Leu13 under the control of a constitutive
promoter. Although some clones did express heterogeneous levels of the
protein early in the selection procedure as revealed by staining,
expression rapidly declined to undetectable levels (data not shown).
This result suggested that stable expression of 9-27/Leu13 might
be hindering cell growth.
Evidence that antibodies against 9-27/Leu-13 and TAPA-1 can induce a biological response suggests that the multimeric cell surface complex containing 9-27/Leu-13, TAPA-1, and other molecules is a receptor for an as yet unidentified ligand. Indeed, activation of a signal transduction pathway by extracellular signals often requires ligand-induced dimerization or oligomerization of the corresponding receptor. Kinase(s) associated with the receptor are brought together, resulting in their reciprocal phosphorylation and the subsequent activation of further downstream components of the signal transduction pathway(42) . Therefore, antibodies against cell surface receptors that are activated by dimerization can sometimes function as an agonist because they artificially induce dimerization of the receptor, whereas Fab fragments of such antibodies have no activity(42) .
Taken together, these results show that the 9-27 gene is coding for a cell surface protein that associates with other membrane proteins, forming a multimeric complex that relays growth inhibitory and cell adhesion signals.
Interferons seem to exert their inhibition on cell growth by acting at many different levels, (1) directly affecting the function of protein such as c-myc and Rb that are intimately involved in cell-cycle control, (2) increasing the level of enzymes such as the double-stranded RNA-dependent protein kinase and the 2-5A synthetases that inhibit cell anabolism, and (3) inducing cell surface proteins such as 9-27/Leu-13 that relay other growth inhibitory signals. While the activation of a single pathway might be sufficient to inhibit the growth of a given cell, activation of multiple pathways by IFNs allows for both more efficiency and flexibility in the control of cell growth. Indeed, in a physiological setting, IFNs are acting on a population of cells at distinct stages and in different programs of differentiation, cells that are continuously exposed to various other stimuli and have to maintain the ability to perform other essential functions, hence the requirement for efficiency and flexibility, i.e. multiple pathways.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X84958[GenBank].