Illllllllno-
Immunogenetics 28: 215-220, 1988
genetics
© Springer-Verlag 1988
Cycloheximide, an inhibitor of protein synthesis, prevents y-interferon-induced expression of class II mRNA in a macrophage cell line Erik C. Biittger*,*, Michael A. Blanar*, and Richard A. Flavell* Biogen Research Corp., 14 Cambridge Center, Cambridge, MA 02142, USA
Abstract. To characterize the mechanisms by which interferon 3' (IFN-3`) upregulates major histocompatibility complex class II mRNA levels in mouse macrophages, we studied the effect of IFN-'y on the transcription rate of class II genes and investigated the requirement for ongoing protein synthesis for the induction of class II mRNA expression. Nuclear run-off assays demonstrate that IFN-3` induces class II mRNA at the transcriptional level. Treatment with cycloheximide, an inhibitor of protein synthesis, prevented the IFN-3`-mediated accumulation of E~ mRNA in the mouse macrophage cell line P388 D. 1, indicating that induction of E~ mRNA in P388 D. 1 cells requires de novo synthesis of a protein intermediate. Our studies suggest that this putative protein factor is labile and required throughout the induction period.
Introduction The mouse class II antigens are encoded by a family of genes - E,, E~, As, and A~ - that reside in the major histocompatibility complex. These genes, known collectively as the MHC class II locus, are expressed on cell surfaces as noncovalently associated heterodimers and regulate the immune response through their role as restriction elements in antigen presentation (reviewed in Unanue and Allen 1987, Flavell et al. 1986). A number of reports have suggested that the expression of class II antigens is controlled by transacting mechanisms (Walker et al. 1984, * denotes that this paper resulted from an equal contribution by the first two authors 1 Present address: Institut ffir medizinische Mikrobiologie, Medizinische Hochschule Hannover, Konstantyn-Gutschow-Str. 8, 3000 Hannover 61, FRG ~-Present address: Section for Immunobiology and Howard Hughes Medical Institute, Yale University School of Medicine, 310 Cedar St., New Haven, CT 06510, USA Abbreviations used in this paper: IFN-% interferon-),; CHX, cycloheximide; MHC, major histocompatibility complex; PK, pyruvate kinase Offprint requests to: E. C. B6ttger
Accolla et al. 1985). In antigen-presenting cells such as macrophages, the expression of class II antigens is regulated by IFN-3, (Steinman et al. 1980, King et al. 1985). Binding of IFN--y to its receptor leads to an increase in the level of particular mRNAs and the corresponding proteins (Revel and Chebath 1986, Friedmann et al. 1984). Accordingly, the changes in class II antigen which result from IFN-3` treatment are also reflected in the amount of class II mRNA (King et al. 1985). The level at which IFN3' regulates class II mRNA expression has, however, not yet been established. We are studying the IFN-3, mechanism of action by characterizing of the IFN-3"-induced expression of MHC class II gene mRNA in mouse macrophages. In this report we show that IFN-3" increases the transcription rate of class II genes. Treatment with cycloheximide, an inhibitor of protein synthesis, prevents IFN3"-mediated induction of E~ mRNA in the mouse macrophage-like cell line P388 D. 1, indicating that the IFN3`-mediated increase in E~ mRNA levels depends on the formation of an IFN-3`-induced protein intermediate.
Materials and methods Peritoneal exudate cells were obtained from 8-12 week old Balb/c (H-2d) or C3H/HeJ (H-2k) mice (The Jackson Laboratory, Bar Harbor, Maine) that had been primed with 3 ml of a 3 % thioglycollate solution 4 days prior to collection. The thioglycollate-induced peritoneal exudate cells were cultured at a density of 2 x 105 cells/cm2 in RPMI 1640 supplemented with 10% fetal calf serum, 2 mM glutamine, 100 IU/ml penicillin, 100 ~tg/ml streptomycin, and 0.05 mM 2-mercaptoethanol. After 3--4 h of incubation at 37 °C to allow for adherence of the macrophages, nonadherent cells were washed off. P388 D. 1 cells were obtained from the American Tissue Culture Collection. This subline has been maintained in our laboratory in RPMI 1640 medium supplemented with 10% fetal calf serum and 2 ~ glutamine. The P388 D.1 tumor cell line has been characterized as a firmly adherent, monocyte/macrophage-like cell line showing macrophage properties such as phagocytosis and the presence of membrane Fc receptors (Koren et al. 1975). The cell line was routinely checked for mycoplasma (Genprobe, San Diego, California). Recombinant mouse IFN-3, was provided by the Shionogi Seiyaku Institute (Osaka, Japan) at a specific activity of 6 x 106 units/mg. Cy-
216 cloheximide (Sigma, St. Louis, Missouri) was used at a concentration of 5 gg/ml. This concentration resulted in a >_90% inhibition of protein synthesis as measured by 35S-cysteine incorporation. Actinomycin D (Sigma) was used at a concentration of 5 gg/ml to block transcription by RNA polymerase II (Dani et al. 1984). Cell viability was determined by trypan blue exclusion. RNA was isolated using the guanidium-isothiocyanate method (Maniatis et al. 1982). RNA concentrations were determined from the OD260 of each sample and were confirmed by ethidium bromide staining after electrophoresis through an agarose gel. For Northern analysis, RNA (20 gg/lane for P388; 10 ~tg/lane for peritoneal macrophages) was electrophoresed through a 1% agarose-formaldehyde gel, transferred to GeneScreen membrane (New England Nuclear, Boston, Massachusetts) according to the manufacturer's instructions, UV cross-linked, and prehybridized for 1 h at 65 °C. The solution used for prehybridization, hybridization, and first wash contained 0.2% polyvinylpyrrolidone, 0.2 % Ficoll-400, 0.2 % bovine serum albumin, 1% sodium dodecyl sulfate (SDS), 50 mM TRIS-HC1 pH 7.5, 1 M NaC1, 2.2 mM sodium pyrophosphate. For prehybridization, 100 gg/ml denatured salmon sperm DNA was added. For hybridization, 10% dextran sulfate, 100 gg/ml denatured salmon sperm DNA, and the denatured probe (at a concentration of i x 106 cpm]mi) were added. Washes were done as follows: 1 x 30 min at 65 °C with the above described solution, then 3 × 30 min at 65 °C with 0.5 x standard sodium citrate (SSC), 1% SDS. In order to rehybridize the blots, labeled probes were washed off according to the manufacturer's instructions. DNA fragments were prepared from plasmids by gel isolation and 32p-labeled by nick-translation (Bethesda Research Laboratories, Gaithersburg, Maryland) to a specific activity of 1x108-5x 108 cpm/gg DNA. The following probes were used: the E~ probe is a 1 kb Bgl II-Pst I genomic fragment containing exon 2 (Hyldig-Nielsen et al. 1983); the E¢ probe is a 2 kb Eco RI genomic fragment containing exon 2 (Widera and Flavell 1984); the A~ probe is a l kb Eco RI eDNA fragment which includes all coding exons (Benoist et al. 1983, provided by Dr. J. Woodward, University of Kentucky, Lexington); the A~ probe is a 1.6 kb Barn HI-Hind III genomic fragment containing exons 3 and 4 (Larhammar et al. 1983); the pyruvate kinase probe is a 2 kb Eco RI-Hind III cDNA fragment (provided by Dr. R. Cate, Biogen, Cambridge, Massachusetts). The nuclear run-off transcription assay was done according to Greenberg and Ziff (1984) with some modifications as suggested by Dr. B. Cochran (M.I.T. Cancer Research Institute, Cambridge, Massachusetts) and by Dr. J. Woodward (University of Kentucky, Lexington). Five micrograms of alkali-treated plasmid DNA was immobilized on nitrocellulose usir~g a minifold II slot-blotter (Schleicher and Schuell, Keene, New Hampshire). The plasmid probes were as follows: 1) B2-tubulin, a 2.2 kb genomic Sst I restriction fragment derived from pUC(L1.) [provided by Dr. N. Cowan (Lewis et al. 1985)], subcloned into Sst I-digested pSP64; 2) pyruvate kinase, a 2.3 kb bovine cDNA Xho I restriction fragment, subcloned into Sal I-digested pUC19 (provided by Dr. R. Cate); 3) K~, a 1.5 kb eDNA fragment snbcloned into Pst I-digested pBR327 [provided by Dr. P. Kourilsky (Lalanne et al. 1983)]; 4) E~, a 7.5 kb genomic Bgl I-Hind III fragment (Hyldig-Nielsen et al. 1983), containing 2 kb of 5' upstream sequences, was subcloned into Hinc H-Hind III-digested pUC18; 5) A~, a 967 bp cDNA Eco RI restriction fragment derived from pAAC6 (Benoist et al. 1983), subcloned into Eco RI-digested pGEM-blueTM (provided by Dr. J. Woodward); 6) pUC18. To prepare nuclei, cells were washed twice with cold phosphate-buffered saline. The pellet (1 × 108 cells) was resuspended in 10 ml NP40 lysis buffer [10 mM Tris-HCt pH 7.4, 10 mM NaC1, 3 mM MgC12, 0.5% (w/v) NP40], incubated for 5 rain on ice, and centrifuged at 500 g for 5 min. The nuclear pellet was washed once with 200 ml NP40 lysis buffer, resuspended in 250 gl 50 toM Tris-HC1 pH 8.3, 40% (v/v)
E.C. B6ttger et al.: CHX and class II mRNA expression glycerol, 5 mM MgClz, 0.1 mM ethylenediaminetetraacetate (EDTA), and immediately frozen in liquid nitrogen. For the run-off transcription assay, nuclei were thawed on ice, mixed with 250 gl reaction buffer [10 mM Tris-HC1 pH 8.0, 5 raM MgC12, 300 mM KC1, 1 mM each of ATP, GTP and CTP, 2.5 gM UTP and 500 p~Ciof [c~-32p]UTP (800 Ci mM -1, New England Nuclear], and incubated for 30 min at 30 °C. Fifteen units of RQI DNase (Promega Biotec, Madison, Wisconsin) were then added and the reaction incubated for 10 min at 30 °C. The reaction was stopped by adding 4.5 ml guanidinium-isothiocyanate and the [32pl-labeled RNA prepared (Maniatis et al. 1982). After precipitation with ethanol, the RNA was resuspended in 100 gl TE (10 mMTrisHC1 pH 7.4, 1 mM EDTA). NaOH was added to a final concentration of 0.2 M, kept for 3 min on ice, and neutralized by addition of HEPES (acid-free, final concentration 0.24 M). The RNA was precipitated with NH4Ac (final concentration 1.5 M) and 2 volumes of ethanol. The [32pl-labeled RNA pellet was resuspended in 500 gl hybridization ' buffer (10 mM Tris, 10 mM EDTA, 300 mM NaCI, 1 xDenhardt's, 100 units/mi RNasin, 0.25 mg/ml tRNA). Hybridization was performed in 1.5 ml hybridization buffer with an equal amount of trichloroacetic acid precipitable cpm run-off RNA (5 x 107) for 60 h at 65 °C. After hybridization, filters were washed with four changes of 2 x SSC for a total of 2 h at 65 °C. The filters were then incubated in 2 x SSC with RNaseA (10 gg/ml) for 30 min at 37 °C and were subsequently washed with three changes of 2 x SSC for 1 h at 65 °C. Filters were exposed to Kodak XAR-5 films at - 70 °C using intensifying screens (Cronex, DuPont, Boston, Massachusetts). Densitometric quantitation was determined from autoradiograms with a LKB ultrascan XL.
Results and Discussion
The mouse macrophage cell line P388 D. 1 was chosen for these studies because among other macrophage lines tested (WEHI-3 and J774 A. 1), this cell line was the only one able to withstand a cycloheximide treatment longer
Fig. 1. Effect of IFN-3" (250 units/ml) on accumulation of E~, E~, A~, and A~ mRNA in P388 D. 1 cells and peritoneal macrophages (Balb/c). P388 D. 1 cells were exposed to IFN- 7 for 20 h. In addition, a 56 h incubation with IFN-7 (to determine A n mRNA levels) is shown. C, untreated controls. Peritoneal macrophages were exposed to IFN-'y for 18 h. Blots were hybridized to E~-, E~-, A~-, and An-specific probes as described in Materials and methods
E.C. B6ttger et al.: CHX and class II mRNA expression
than 8 h. [Cell viability after 20 h of CHX treatment was 30-50%. The requirement for a prolonged CHX treatment is a result of the long incubation periods (612-20 h) required to induce class II mRNA expression by IFN-3,]. E~ and A s mRNAs were not found in untreated P388 D.1 cells, but were induced by IFN- 7. E~ and A;~ mRNAs, however, were constitutively expressed in this cell line; IFN-7 did not affect the level of these mRNAs for up to 20 h (Fig. 1). Only a prolonged (56 h) IFN-'y treatment revealed induction for A¢ (Fig. 1) as well as for E~ (data not shown). Together with our results concerning the effects of blocking protein synthesis with CHX on class II gene expression (see below), these data indicate that the expression of the various class II mRNAs may be independently regulated in the P388 D. 1 cell line. This is in contrast to our results with peritoneal macrophages (Fig. 1) as well as to a recent report on the WEHI-3 line demonstrating coordinate class II mRNA induction by IFN-3, (King et al. 1985). Although the basis for this differential expression of class II genes in P388 D. 1 cells is unclear, it raises the interesting possibility that MHC class II genes are not only regulated by cell-type specific mechanisms but also by gene-specific mechanisms. The IFN-3,-induced accumulation of class II mRNA, as measured by Northern blot analysis, can be controlled at or after the level of transcription. Inhibition of transcription with actinomycin D completely blocked IFN3,-induced accumulation of E~, E¢, A~, and A~ mRNA in peritoneal macrophages, indicating that IFN--/-induction occurs at the transcriptional level (data not shown). To determine directly whether IFN-~/induces transcription of class II genes, nuclear run-off transcription assays were performed on nuclei isolated from uninduced and IFN-y-treated P388 D. 1 cells (Fig. 2). Transcription of the control genes, pyruvate kinase (PK) and/32-tubulin, was unaffected by IFN--y. In contrast, treatment with IFN-3, increased transcription of the E~ and A~ genes. As can be seen in Figure 2, transcription of the E~ gene was induced three-fold by a 48 h IFN-7 treatment. Similarly,
Fig. 2. Effect of IFN-3, (1000 units/ml) on E,, A s, and K d gene transcription. Nuclear run-off transcription assays and the gene-specific probes are described in Materials and methods. Run-off assays were done on nuclei isolated from P388 D. 1 cells with no treatment, with IFN-3, for 24 h or with IFN- 7 for 48 h. The results of two experiments are depicted
217
Fig. 3. Inhibition of IFN-7-induced E, mRNA expression by CHX in P388 D.1 cells. P388 D.1 cells were exposed to IFN-3, (250 units/ml) or CHX (5 gg/ml) alone, or to a combination of CHX/IFN- 7 for 12, 16, and 20 h. C, untreated controls. Blots were hybridized to E,- and As-specific probes and to a PK probe as a control
A,-gene transcription increased two- to threefold. Transcription of K d, an MHC class I gene, was also induced approximately twofold by IFN-3,. To ensure that this modest increase in transcription was reproducible, the experiment was performed several times and yielded identical results. The two- to threefold transcriptional induction of A~ and E~ contrasts with the increase in steady-state levels of the respective mRNA's. Presumably, the observed stability of class II mRNA (see Fig. 6) can account for the large elevation of class II mRNA levels despite low transcriptional induction. To characterize the initial steps in IFN-3/action, and to evaluate whether IFN-3,-induced class II mRNA expression represents a primary response not requiring protein synthesis or a secondary response dependent on protein synthesis (i. e., mediated by an IFN-3,-induced l~rotein intermediate; Ringold 1979), we examined the effect of CHX, an inhibitor of protein synthesis, on IFN"y-induced class II mRNA expression in the mouse macrophage cell line P388 D. 1. Treatment with CHX alone for 12, 16, or 20 h did not affect E~ mRNA levels (Fig. 3). Incubation with CHX and IFN-7 for 12, 16, or 20 h completely blocked the IFN--g-induced accumulation of E~ mRNA (Fig. 3). All RNA samples were analyzed for pyruvate kinase mRNA levels to ascertain that the lack of E~ mRNA was not due to an increased rate of non-
218 specific m R N A degradation in CHX-treated cells (Fig. 3). From these experiments we conclude that for P388 D. 1 cells, induction of E~ m R N A by IFN-7 is dependent on p r o t e i n synthesis. Surprisingly, CHX treatment by itself induced As m R N A accumulation in P388 D.1 cells (Fig. 3) as well as in the macrophage lines J774 A. 1 and WEHI-3 (unpublished results). Although further experiments are required to precisely define this effect of CHX, it has been shown that a number of other genes can be induced by inhibition of protein synthesis (Linial et al. 1985, Ringold et al. 1984, Sen and Baltimore 1986, Wall et al. 1986). In these studies CHX-mediated gene expression appears to involve transcriptional, post-transcriptional, as well as post-translational mechanisms. Due to the induction of A , m R N A expression by CHX alone, the effect of CHX on IFN--y-induced elevation of A~ m R N A levels could not be interpreted, but no superinduction was noted by simultaneous addition of CHX and IFN-3, (Fig. 3). To determine the time period during which E~ m R N A induction requires protein synthesis, we added CHX and IFN-3,, and at various points in time thereafter removed the CHX/IFN-3,-containing medium and replaced it with IFN-3,-containing medium to permit the recovery of protein synthesis. RNA was isolated after a total elapsed time of 20 h. Figure 4A shows that addition
Fig. 4 A and B. Kinetics of inhibition of IFN-T-mediatedaccumulation of E~ mRNA by CHX in P388 D. 1 cells. A) Cells were incubated first with IFN-3, (250 units/ml) and CHX (5 Ixg/ml), after which they were washed at the indicated times and incubated solely in the presence of IFN-3, for a total time (IFN~/CHX+IFN-7) of 20 h. B) Cells were incubated first with IFN-3, (250 units/ml). At the indicated times CHX (5 ~tg/ml) was added for a total elapsed time (IFN-3,+IFN--y/CHX)of 20 h. C, untreated controls; IFN, IFN--ytreatment for 20 h
E.C. B6ttger et al.: CHX and class II mRNA expression
Fig. 5. Effect of CHX and actinomycinD on continuous accumulation of E~ mRNA. Cells were incubated first with IFN-3, (250 units/ml) for 24 h, at which time either actinomycin D (5 gg/ml) or CHX (5 ~tg/m/) was added for a total elapsed time (IFN-q~ +IFN-7/actinomycin D or IFN-3,+IFN-3~ /CHX) of 33 h. IFN24, IFN-'ytreatment for 24 h; IFN33, IFN-7 treatment for 33 h; IFN33/CHX24-33, cells were incubated with IFN-'), for 33 h, 24 h after the initial IFN-7 addition CHX was added; IFN33/actinomycin D 24-33, cells were incubated with IFN-7 for 33 h, 24 h after the initial IFN-3, addition actinomycin D was added of CHX for only 8 h results in a significant inhibition of E~ m R N A production. Extending the exposure to CHX results in further inhibition of the expression of E~ mRNA. In the next set of experiments we investigated whether E~ induction requires only an initial period of protein synthesis or whether continued synthesis in response to IFN--f is required. We first added IFN-T to P388 D. 1 cells and, at various points in time thereafter, CHX was added to prevent any further protein synthesis. RNA was isolated at 20 h following the initial addition of IFN-7 and analyzed for E~ mRNA. As can be seen in Figure 4B, addition of CHX as late as 12 h after the initial addition of IFN-3, prevented E~ m R N A accumulation. (In some cases, a low level of E~ m R N A was observed in CHX/IFN-7 treated cells. We believe that since CHX itself does not increase E , m R N A levels, this low level of residual E, m R N A is most likely due to the residual protein synthesis not blocked by CHX.) These results suggest that maximal induction of E~ m R N A by IFN-7 requires protein synthesis throughout the entire induction period. To confirm that ongoing protein synthesis is required throughout the period of exposure to IFN-3,, P388 D. 1 cells were treated with IFN-7 for time periods longer than 24 h, thereby leading to increased accumulation of E~ mRNA. As shown in Figure 5, IFN--y treatment for 33 h results in E~ m R N A levels that exceed those of IFN-7 treatment for 24 h. Northern analysis of RNA isolated after a total elapsed time of 33 h showed that addition of CHX at 24 h after the initial addition of IFN- 7 abolished the further increase of E , m R N A between 24 and 33 h (Fig. 5), indicating that even once E , m R N A is induced by IFN-% further accumulation of E , m R N A still requires protein synthesis. As expected, the further increase in the E~ m R N A level between 24 and 33 h was blocked
E.C. B6ttger et al.: CHX and class II mRNA expression by adding actinomycin D at 24 h after the initial addition of IFN-% Thus, the induction and continued transcription of E s m R N A by IFN- T is apparently dependent on continuous protein synthesis. To investigate whether the CHX effect on IFNy-mediated accumulation of E s m R N A is due to destabilization of IFN-T-induced E s mRNA, E s m R N A was induced in P388 D.1 cells with IFN-7 for 24 h, then actinomycin D alone or actinomycin D with CHX was added for various periods. Total cellular R N A was prepared and subjected to Northern blot analysis. Since actinomycin D inhibits R N A polymerase I1 and the subsequent synthesis of new transcripts, the decrease in m R N A levels in the presence of this inhibitor should be a measure of RNA stability (Dani et al. 1984). Figure 6 shows that within 14 h of treatment with actinomycin D alone or actinomycin D and CHX, CHX did not appreciably affect the stability of E~ mRNA. It is unlikely, therefore, that the inhibition of IFN-3,-induced accumulation of E~ m R N A by CHX is caused by a destabilization of E s mRNA. We next determined the effects of protein synthesis inhibition in a natural, although heterogeneous, macrophage population (Lee 1982) and isolated peritoneal macrophages from Balb/c mice. This mouse strain has the same haplotype as the P388 D. 1 cells (H-2d). As shown in Figure 7, IFN-3, induced the expression of E s and A s mRNAs in this cell population (the low level of constitutive class II m R N A in peritoneal macrophages may be due to a minor subpopulation of activated macrophages). In contrast to the P388 D. 1 cell line, however, CHX did not affect the IFN-y-induced accumulation of E~ and A s mRNAs to a significant extent (Fig. 7), indicating that class H m R N A expression by IFN-'y seems to be a primary response in thioglycollate-elicited peritoneal macrophages. In addition, CHX alone caused a small increase in class II m R N A levels (two- to threefold by densitomet-
Fig. 6. Estimation of E~ mRNA stability. P388 D. l cells were treated with IFN-7 (250 units/ml) for 24 h to induce E mRNA, after which either actinomycinD (5 gg/ml) or actinomycin D and CHX (5gg/ml) were added for the indicated periods. C, untreated controls; IFN 24, IFN-T for 24 h. (The half-life of A~ mRNA, determined by the same approach, was estimated to be approximately 6-8 h; data not shown)
219
Fig. 7. Effect of CHX on IFN-T-induced class II mRNA expression in Balb/c (//-2d) peritoneal macrophages. Peritoneal macrophages were exposed to IFN-7 (250 units/ml) or CHX (5 gg/ml) alone, or to a combination of CHX/IFN-3,for 18 h. C, untreated controls ric analysis). This observation contradicts a recent report in which A~ m R N A induction by IFN-3, in peritoneal macrophages was sensitive to CHX (Fertsch et al. 1987). We considered that this may be related to the use of different strains of mice as the source of thioglycollate-elicited peritoneal macrophages (Balb/c versus C3H/HeJ mice). Indeed, as shown in Figure 8, the effects of CHX treatment are different on peritoneal macrophages derived from C3H/HeJ mice. CHX by itself not only failed to increase class II m R N A levels, but its addition also resulted in an approximately 45% reduction (as measured by densitometry) for IFN-3,-induced accumulation of A~ mRNA. The reasons of these puzzling strain differences are unknown, although such a strain dependency of peritoneal macrophages has been demonstrated for other experimental systems (Varesio 1986). The requirement for ongoing protein synthesis for E~ m R N A induction by IFN- T in P388 D. 1 cells suggests that IFN-'y acts on E s gene expression in P388 D. 1 cells by causing the de novo synthesis of a protein intermediate. The exact nature of this protein remains to be clarified. If our notion of synthesis of a novel protein intermediate
Fig. 8. Effect of CHX on IFN-3, class II mRNA expression in C3H/HeJ (H-2k) peritoneal macrophages. Peritoneal macrophages were exposed to IFN-3, (250 units/ml) or CHX (5gg/ml) alone, or to a combinationof CHX/IFN-T for 18 h. C, untreated controis
220
is correct, our data, which show that the further accumulation of E~ mRNA still requires protein synthesis even once E~ mRNA is induced by IFN-'),, also suggest that this putative protein is turned over rapidly, perhaps as a consequence of a short half-life or rapid conversion to an inactive form. Of course, we cannot rule out the possibility that the CHX sensitivity of IFN-'g-induced E~ expression in P388 D.I cells might be due to an aberration in this tumor cell line as the result of transformation or expression of endogenous viruses. Whether or not this is the case, identification and characterization of the protein factor that controls the response of E£ to IFN-3, is likely to be important to our understanding of the regulation of MHC gene expression. Acknowledgments. We thank Drs. Brent Cochran and Jerry Woodward for help with the nuclear transcription assays, Drs. Richard Cate, Nicholas Cowan, Philippe Kourilsky, and Jerry Woodward for providing plasmids, Dr. John LeBowitz for help with the densitometric analysis, and Drs. Linda Burkly and Gerry Waneck for stimulating discussions and for comments on the manuscript. ECB was supported by a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft, MAB is a recipient of an Arthritis Foundation Postdoctoral Fellowship. This work was funded by Biogen N.V.
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