Y. Suzuki et al.

Herbimycin A (–)

Herbimycin A (+)

01515(min)TimeFig. 3. Effects of herbimycin A on the interferongamma (IFN-γ)-induced phosphorylation of tyrosine. As shown in the upper panel, IFN-γ induced
phosphorylation of tyrosine on HLC-1 cells. In thelower panel, pretreatment with herbimycin A appeared to inhibit the phosphorylation of tyrosine.

These findings indicate that IFN-γ induced
tyrosine phosphorylation of cellular substrates
which was inhibited by herbimycin A.

Discussion

Mean fluorescence intensity

65Three intracellular signal transduction pathways have been considered in the induction ofMHC antigens by IFN-γ in several types of cells(Koide et al., 1988; Klein et al., 1990; Nezu et al.,
1990; Towata et al., 1991; Lahat et al., 1993); thatis, the protein kinase A, Ca2+-calmodulin andprotein kinase C pathways. However, thesepathways have not been shown to be decisiveon the IFN-γ-induced expression of MHC class
I and II antigens. There is only one reportwhich states that tyrosine kinase pathways may
be involved in the IFN-γ-induced expression ofMHC class II (Ryu et al., 1993). Nevertheless,the detailed signal transduction mechanisms
responsible for the IFN-γ-inducible expressionof MHC antigens on cancer cells have not been
elucidated, especially for MHC class I.
Recent biochemical studies have shown that
IFN-γ activated the JAK1 and JAK2 tyrosine
kinases which phosphorylate a down stream
signal transducer and activator of transcription

Herbimycin A (–)
Herbimycin A (+)

432

10010Time20(min)Fig. 4. Effects of herbimycin A on interferongamma (IFN-γ)-induced phosphorylation of tyrosine. Analysis of tyrosine phosphorylation by mean
fluorescence intensity shows that IFN-γ increased
the tyrosine phosphorylation five fold within 5 min.
Herbimycin A inhibited the production of IFN-γinducible phosphotyrosine.

4

Tyrosine phosphorylation in MHC induction(STAT) in the IFN-γ response signal pathway.
Phosphorylated STAT1α dimerizes, translocates to the nucleus, and binds specific DNA
elements, i.e., IFN-γ activation sites, thereby
activating transcription factors, e.g., interferon
regulatory factor 1 (Shuai et al., 1993a, 1993b;
Silvennoinen et al., 1993; Darnell et al., 1994;
Taniguchi et al., 1995).
We used an inhibitor of tyrosine protein
kinase, herbimycin A (Uehara et al., 1989;
Obinata et al., 1991), to determine whether
tyrosine phosphorylation may be involved inthe signal transduction for IFN-γ-induced expression of MHC class I and II molecules on
HLC-1 cells. However, while herbimycin A is
useful in determining whether tyrosine phosphorylation is involved in the mechanisms for
activation by receptor-mediated signal transduction, it is not specific for JAK tyrosine kinases.
We observed that IFN-γ induced the expression of MHC class I and II on HLC-1 cells andthe phosphorylation of protein tyrosine in theircellular substrates within 5 min. Herbimycin A
inhibited both these expressions and the enhancement of tyrosine phosphorylation. Thefindings that IFN-γ activated the protein
tyrosine kinases that phosphorylate the cellularsubstrates and induce MHC class I and II
molecules suggest that tyrosine phosphorylation is involved in the induction of the expression of MHC molecules on HLC-1 cells byIFN-γ. Therefore, a JAK-STAT signal transduction pathway must be involved in the IFN-γinduced expression of MHC class I and IImolecules. However, we cannot exclude thepossibility of involvement by other pathways
because herbimycin A did not completely inhibit IFN-γ-inducible expression of MHC molecules. While the expression of MHC molecules
is known to be regulated by other cytokines
such as tumor necrosis factor alpha and interleukin 1 alpha (Seong et al., 1991; Sedlak et al.,
1992; Wolchok and Vilcek, 1992), little is
known about the signal transduction mechanisms which induce MHC antigens by thesecytokines. Further, there are some reports
which state that c-myc oncogene regulates theexpression of MHC molecules on cancer cells(Versteeg et al., 1988; Gaforio et al., 1991). We

think it should also be necessary in the future toclarify the signal transduction pathways bythese cytokines and oncogenes for understanding the more detailed mechanisms which induce
MHC molecules.
The expression of MHC class I and II antigens on cancer cells are involved in a variety of
immune functions affecting tumor immunity.
Cancer cells have reduced expression of MHC
molecules enabling them to escape from thehost’s immunosurveillance system (Goodenowet al., 1985; Festenstein et al., 1986). Therefore, we think it is important to elucidate boththe mechanisms modulating the expression of
MHC molecules and the methods restoring theexpression of these molecules on cancer cells
for improvement of clinical problems about
tumor immunity. In the present study, we elucidated the fact that tyrosine phosphorylation
played an important role as the mechanism
which restores the expression of MHC molecules on cancer cells.
In conclusion, tyrosine phosphorylation isconsidered to be an essential factor in the IFNγ-induced expression of MHC class I and IImolecules on HLC-1 cells.
Acknowledgments: The authors wish to express theirsincere gratitude to Emeritus Prof. Tohru Mori, Second Dept. of Surgery, Faculty of Medicine, Tottori
Univ., for his kind advice and valuable suggestions
during the course of this study. We would also liketo thank Prof. Hisao Ito, First Dept. of Pathology,Faculty of Medicine, Tottori Univ. and Prof. Kenzo
Sato, Dept. of Molecular Biology, Faculty of Medicine, Tottori Univ., for reading this manuscript andhelpful suggestions.
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