Yonago Acta medica 1998;41:1–6

Tyrosine Phosphorylation Regulates the Expression of Major Histocompatibility Complex Antigens on a Human Lung Cancer Cell Line byInterferon−gamma
Yoshimasa Suzuki, Yoshiyuki Tanaka, Hiroshige Nakamura and Shigetsugu Ohgi
Second Department of Surgery, Faculty of Medicine, Tottori University, Yonago 683-0826, Japan
Expression of major histocompatibility complex (MHC) antigens on cancer cells isessential for cell-mediated immune function. However, these molecules are reduced oncancer cells enabling them to escape from host immune surveillance. It is well known
that interferon-gamma (IFN-γ) upregulates the expression of MHC molecules andrestores the immunogenicity of cancer cells. Nevertheless, the mechanism by which
IFN-γ modulates MHC expression on cancer cells is not clear. Therefore, in this report,
we examined the role of tyrosine protein kinases in IFN-γ-induced MHC expression ina human lung adenocarcinoma cell line, HLC-1. We found that a tyrosine protein
kinase inhibitor, herbimycin A, inhibited both IFN-γ-inducible MHC class I and class
II expression, as assessed by flow cytometry. Additionally, assessment of tyrosine
phosphorylation of cellular substrates by confocal laser microscopy using an antiphosphotyrosine monoclonal antibody (mAb) revealed that IFN-γ induced protein
tyrosine phosphorylation within 5 min of treatment. Herbimycin A inhibited this IFNγ-induced tyrosine phosphorylation. Thus, tyrosine phosphorylation plays an importantrole in IFN-γ-induced MHC class I and class II expression on HLC-1 cells.
Key words: human lung cancer; interferon-gamma; major histocompatibility complex; tyrosine
phosphorylation

host’s immunosurveillance system (Goodenowet al., 1985; Festenstein and Garrido, 1986).
Therefore, it is important to elucidate the mechanisms modulating the expression of MHC
molecules on cancer cells.
IFN-γ can restore the expression of MHC
molecules on many types of malignant cells.
Nevertheless the mechanisms of IFN-γ-induced
MHC expression on cancer cells have not been
elucidated, especially in the context of MHC
class I expression. Recent biochemical studies
indicate that tyrosine phosphorylation of Janus
kinases (JAK) is an important step in the signaltransduction system activated by IFN-γ (Shuaiet al., 1993a; Darnell et al., 1994). Further,
Hobart and colleagues (1997) suggested thatinterferon regulatory factor 1 might regulate theMajor histocompatibility complex (MHC)
antigens on cancer cells are involved in avariety of immune functions affecting tumor
immunity (Klein and Klein, 1985). MHC class
I and II molecules present antigens to CD8+ andCD4+ T cells in the form of peptide fragments
within their molecule-binding grooves, respectively (Matsumura et al., 1992; Madden et al.,
1993; Stern et al., 1994). MHC class I molecules have been shown to act as restriction
elements in the lysis of target cells by cytotoxic
T lymphocytes (Lurquin et al., 1989), while
MHC class II molecules present antigens tohelper T cells and regulate autologous T helper
cell activation (Eckels et al., 1983). Cancer cells,
however, have reduced expression of MHC
molecules, enabling them to escape from theAbbreviations: FCS, fetal calf serum; FITC, fluorescein isothiocyanate; IFN-γ, interferon-gamma; JAK,Janus kinase; mAb, monoclonal antibody; MHC, major histocompatibility complex; PBS, phosphatebuffered saline; PE, phycoerythrin; STAT, signal transducer and activator of transcription

1Y. Suzuki et al.

atmosphere of 5% CO2 in air. The number ofcells and their viability were evaluated by thetrypan blue exclusion method. Cells were pretreated for 2 h with or without various concentrations of herbimycin A before the addition ofIFN-γ. After 1 h of incubation with or without
IFN-γ (1000 U/mL), cells were washed 3 times
with FCS-free medium and cultured in thecomplete medium for an additional 48 h.

expression of MHC class I and class II genes ofmice. Therefore, in this report we examined therole of tyrosine phosphorylation in the signaltransduction of IFN-γ-induced MHC expression in a human lung adenocarcinoma cell line,HLC-1, using the tyrosine kinase inhibitor
herbimycin A.

Materials and Methods

Flow cytometric analysis
Cells

Following incubation, HLC-1 cells were harvested with 0.02% EDTA (Cosmo Bio, Tokyo)
and then washed 3 times with phosphatebuffered saline (PBS). The cells were incubated with an appropriate dilution of mAb W6/32
and I3-RD1 or MsIgG1-FITC and MsIgG1RD1 for 30 min at 4°C. After being washed 3times with PBS, the cells were analyzed by flow
cytometry with the FACSort system (BectonDickinson, Mountain View, CA). Data analyses by fluorescence intensity were based on
calculating 1 × 10 4 cells per sample by theLYSIS II software (Becton Dickinson). These
series of experiments were repeated 3 timeswith equivalent results. The results were presented as mean ± SE.

HLC-1, a human lung adenocarcinoma cell line,
was cultured in a complete medium consistingof RPMI 1640 (Kyokuto Pharmaceutical Industrial Co., Ltd., Tokyo, Japan) supplemented with
10% heat-inactivated fetal calf serum (FCS) andpenicillin (100 mg/mL).

Reagents
Herbimycin A, an inhibitor of protein tyrosine
kinase, was purchased from Wako Pure Chemical (Osaka, Japan). Recombinant human IFN-γ
was kindly supplied by Shionogi PharmaceuticalCo., Ltd. (Osaka).

Antibodies
Laser microscopic analysis

The following monoclonal antibodies (mAb)
were used: fluorescein isothiocyanate (FITC)labeled W6/32 (Serotec, Oxford, United Kingdom)
against MHC class I; phycoerythrin (PE)-labeled
I3-RD1 (Coulter Immunology, Hialeah, FL)against MHC class II; FITC-labeled 6D12(MBL, Nagoya, Japan) against phosphotyrosine; MsIgG1-FITC (Coulter Immunology),
FITC-labeled anti-mouse immunoglobulin G1,and MsIgG1-RD1 (Coulter Immunology), PElabeled anti-mouse immunoglobulin G1, as
controls for the FITC- and PE-labeled mAbs,respectively.

HLC-1 cells were cultured in a glass-bottomed
microwell dish (MatTek Corp., Ashland, MA),1 × 104 cells/dish at 37°C for 24 h in a humidified atmosphere of 5% CO2 in air. Cells werepretreated for 2 h with or without herbimycin A(0.1 mg/mL) before the addition of IFN-γ (1000U/mL). After being stimulated with IFN-γ,cells were fixed with 70% ethanol. After being
washed 3 times with PBS, the cells were incubated with an appropriate dilution of mAb
6D12 for 30 min at 4°C. After incubation, cells
were washed 3 times with PBS and analyzed byconfocal laser microscopy, ACAS Ultima(Meridian Instruments, Okemos, MI). These
series of experiments were also repeated 3times with equivalent results, and the data presented were representative of one such experiment.

Induction of MHC class I and II antigens
HLC-1 cells were cultured in the tissue culture
dish (Sumitomo Bakelite, Tokyo, Japan), 1 ×
105 cells/dish at 37°C for 24 h in a humidified2

Tyrosine phosphorylation in MHC induction

MHC class II
Mean fluorescence intensity

Mean fluorescence intensity

MHC class I
50

4030

20

100IFN-γ(1000 U/mL)

Herbimycin A–+++++––

0.0125

0.025

0.05

0.1

10864

20IFN-γ(1000 U/mL)

Herbimycin A(μg/mL)–+++++––

0.01250.0250.05

0.1(μg/mL)

Fig. 1. Interferon-gamma (IFN-γ) induced the expression of major histocompatibility complex (MHC)class I molecules on HLC-1 cells. The MHC expression was analyzed by FACsort system after 48 h exposition to IFN-γ. Herbimycin A (0.0125–0.1 mg/mL)
prevented such expression in a dose-dependent manner.

Fig. 2. Interferon-gamma (IFN-γ) induced the expression of major histocompatibility complex(MHC) class II molecules on HLC-1 cells. TheMHC expression was analyzed by FACsort systemafter 48 h exposition to IFN-γ. Herbimycin A(0.0125–0.1 mg/mL) prevented such expression.

that IFN-γ increased the expression of both
MHC class I and II molecules by 3.6 and 1.9
times respectively. Herbimycin A inhibited
IFN-γ-inducible expression of MHC class I
molecules in a dose-dependent manner andprevented IFN-γ-inducible expression of MHC
class II molecules (Figs. 1 and 2).

Results

Flow cytometric analysis
Cells were induced to express MHC antigens ina dose-dependent manner following incubationfor 48 h in the presence of IFN-γ, 50, 100, 500,
1000 and 2000 U/mL. The maximum expression was observed at IFN-γ concentrations of1000 and 2000 U/mL (data not shown). The
induction of the MHC expression was initiallyobserved after 12 h of exposure to IFN-γ; theexpression of MHC molecules graduallyintensified during 48 h of incubation (data notshown). IFN-γ at 2000 U/mL affected cell
growth and viability, so the cells were stimulated with IFN-γ at 1000 U/mL, followed by culture for 48 h.
HLC-1 cells had fundamentally reduced expression of MHC class I and II molecules.
However, when HLC-1 cells were stimulatedwith IFN-γ, the expression of these molecules
increased immensely. IFN-γ-inducible expression of MHC class I and class II molecules wasinhibited by herbimycin A. Analysis of thesedata by mean fluorescence intensity showed

Laser microscopic analysis
After stimulation with IFN-γ (1000 U/mL),tyrosine phosphorylation of cellular substrates
in HLC-1 cells was assessed by confocal laser
microscopy. IFN-γ induced protein tyrosine
phosphorylation within 5 min (Fig. 3). However, pretreatment of HLC-1 cells with herbimycin A (0.1 μg/mL) appeared to prevent tyrosine phosphorylation. Analyzing these data bymean fluorescence intensity based on calculating 1 × 102 cells per sample (Fig. 4), protein
tyrosine phosphorylation by IFN-γ was observed within 1 min and peaked by 5 min. After 15
min, protein tyrosine phosphorylation returned
to baseline levels as assessed by mean fluorescence intensity. Herbimycin A almost completely inhibited the IFN-γ-inducible tyrosine
protein phosphorylation.
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