Tumor Necrosis Factor Increases Stability of Interleukin-1 mRNA by ...

Vol. 268, No. 9. Issue of March 25, pp. 62144220,1993 Printed in U.S A .

THEJOURNAL OF BIOLOGICAL CHEMISTRY

Q 1993 by

The American Society for Biochemistry and Molecular Biology, Inc.

Tumor Necrosis Factor IncreasesStability of Interleukin-1 mRNA by Activating Protein Kinase C* (Received for publication, August 31,1992)

Myriam Gorospe, Sanjay Kumar, and Corrado Baglioni From the Department of Biological Sciences, State University of New York, Albany, New York 12222

The mRNAs coding for interleukin-la (IL-la) and responses to foreign agents, several cytokines in a network IL-18 are constitutively transcribed but do not accu- are rapidly produced. These cytokines may be synthesized mulate in human diploid fibroblasts and in fibrosarquite rapidly when they are coded for by an unstable mRNA coma cells. Treatment ofthese cells with tumor necro- that is stabilized by post-transcriptional regulation. Such sis factor (TNF) induces accumulation ofIL-1 mRNA inducible expression was shown for the mRNA of granulocytebyanunknownmechanism.Thisinductionof IL-1 macrophage colony stimulating factor in T lymphocytes (6), mRNA was investigated in HT-1080 cells. The induc- for IL-3 mRNA in mast cells stimulated by phorbol esters (7), tion was quite fast, with maximum levels of IL- l a and and for interferon-@mRNA in macrophages stimulated by B mRNA reached 4 h after addition of TNF. Nuclear interferon-y (8). Conversely, the expression of many cytorun-off experiment showed that TNF did not increase the rate of transcription of IL-1 mRNA. This mRNA kines may be transient because of regulatory mechanisms that was apparently unstable in untreated cells, but it ac- result in mRNA destabilization, as reported for interferon-@ cumulated in cycloheximide-treatedcells. Phorbol es- mRNA in cells treated with glucocorticoids (9). ters induced IL-1 mRNA, suggesting that activation of Thestarting point for the present investigation is the protein kinaseC was responsible for the accumulationobservation that human diploid fibroblasts constitutively of this mRNA. This hypothesis was confirmed by ex- transcribe 1L-la and @ mRNA (10). However, these mRNAs periments with the PKC inhibitors staurosporine and do not accumulate in early passage fibroblasts but only in calphostin C, which prevented the induction of IL-1 aging cells. Treatment with TNF or IL-1 induces accumulamRNA by TNF and accelerated the decaythis of mRNA tion of IL-1 mRNA even in early passage fibroblasts by an in cells pretreated with TNF. Both IL-la and IL-18 unknown mechanism. The goal of the present investigation is the elucidation of the mechanism that promotes expression were detected in TNF-treated cells by Western blot analysis andenzyme-linkedimmunosorbent assay. of IL-1 mRNA in TNF-treated cells. To avoid working with These results indicate that the TNF-mediated induc- cells that express IL-1 mRNA with aging, we screened human tion of IL-1 can be entirely accounted for by stabili- tumor cell lines and observed constitutive transcription of zation of this mRNA. this mRNA in HT-1080 fibrosarcoma cells. In this study, we examine the mechanism responsible for TNF-induced production of IL-1 and conclude that it involves IL-1 mRNA stabilization and requires PKC activity. Tumor necrosis factor (TNF)’ and IL-1 are cytokines involved in inflammation and immune responses. TNF was MATERIALS ANDMETHODS originally defined by its tumoricidal activity in vivo and its Cell Culture and Treatment-Human fibrosarcoma HT-1080 cells cytotoxic activity i n vitro (1).Subsequently, TNF was found were cultured in Dulbecco’s modified Eagle’s medium supplemented to be a pleiotropic cytokine, capable of eliciting a wide variety with 10% heat-inactivated horse serum. Cells were treated with of biological responses (2). Several studies have shown that calphostin C (Kamiya Biochemical Co., Thousand Oaks, CA) under TNF does notoperate alone in the host defense against fluorescent light according to themanufacturer’s instructions. RNA Extraction and Analysis-Total cytoplasmic RNA was isomicrobial infections and other foreign agents, but it interacts with a complex network of mediators, including other cyto- lated from cells following the method previously described (11).The was extracted by lysing cells in a 25-cm2flask with 1ml of 2% kines (1,2).Among these cytokines, IL-1 is the initial media- RNA SDS, 0.2 M Tris buffer, pH 7.4,lm M EDTA. DNA and proteins were tor of immunological responses (3). Two structurally related precipitated at 0 “C by adding 0.3 ml of 5 M potassium acetate. After forms of IL-1 have been identified, IL-la and IL-l@,which 5-min centrifugation, thesupernatant was extracted twice in bind to thesame receptor (4) and manifest identical biological phenokch1oroform:isoamyl alcohol (25:24:1) and twice in chloroproperties in immunoregulation, tissue homeostasis, and in- fomxisoamyl alcohol (24:l). The RNA was precipitated with 1.3 ml flammation (3). Surprisingly, a wide range of biological ac- of isopropanol at -20 “C for 1h, recovered by centrifugation, washed 70% ethanol, dried, and resuspended in 100% formamide. The tivities of TNF and IL-1 overlap and are indistinguishable, with RNA was separated by electrophoresis in 1.2% agarose gels and even though these cytokines are neither structurally related, transferred to Genescreen Plus membranes (Du Pont-New England nor do they interact with related cellular receptors (5). Nuclear), that were vacuum-baked for 2 h. Probes were prepared by During certain biological processes such as inflammatory labeling PCR-amplified fragments of IL-la (816 bp), IL-10 (811bp),

* This work was supported by Grant CA-29895 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ‘‘aduertisernent” in accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. ‘The abbreviations used are: TNF, tumor necrosis factor; IL, interleukin; PMA, phorbol 12-myristate 13-acetate; PKC,protein kinase C; AUBF, AU-binding factors; bp, base pair(s).

and 0-actin (548 bp) by the random primer method; hybridization was carried out according to the manufacturer’s instructions. The blots were then washed twice with 0.1 X SSC, 1%SDS for 15 min at room temperature and twice with 0.1 X SSC, 0.1% SDS for 15 min a t 65 ‘C. The radioactivity was measured in aBetascope 603 (Betagen, Waltham, MA). To normalize for RNA loading, the membranes were boiled in 0.01 X SSC, 0.01% SDS and then hybridized to a 0-actin probe. Nuclear Run-off Analysis-Nuclei were prepared from 6 X lo’ HT-

6214

Stabilization of Interleukin-1 mRNA

6215

FIG. 1. Time course of induction of IL-1 mRNA by TNF in HT-1080 cells. The cells were treated with 10 ng/ ml TNF for the time indicated. RNA extraction and Northern blot analysis (inset) were carried out asdescribed under “Materials and Methods.” The blot was stripped and hybridized to a @-actin probe. The radioactivity in the bands was measured with a Betascope 603 and expressed as cpm in IL-1 mRNA/cpm in @-actinmRNA X 1000.0, IL-lamRNA; 0, IL-1@mRNA.

C

TNF

0

a

IL-I~

c

( I ,

IL” MnSOD

-

.-- 8-actin pUC19

FIG.2. Nuclear run-off assays. HT-1080 cells were either untreated (C)or treated for 3 h with 10 ng/ml TNF. IL-la, IL-la, manganese superoxide dismutase, and @-actin cDNA probes were prepared by polymerase chain reaction amplification. manganese superoxide dismutase and @-actincDNAs were included as positive controls; pUC19 DNA was included as a negative control. 1080 cells. The nascent RNA was labeled as previously reported (12). Denatured IL-la, IL-l@, manganese superoxide dismutase, and 8actin cDNAs (obtained by polymerase chain reaction amplification) were slot-blotted onto nitrocellulose membranes; pUC19 DNA was included as a negative control. The membranes were blockedwith 0.1

FIG. 3. Time course of accumulation of IL-1 mRNA in cycloheximide-treated HT-1080 cells. The cells were treated with 10 pg/ml cycloheximide for the time indicated. Northern blot analysis and measurement of radioactivity was carried out asdescribed in Fig. 1. 0, IL-la mRNA; 0, IL-l@ mRNA.

mg/ml tRNA before hybridization with 4 X 10’ cpm of nuclear RNA for 36 h and thoroughly washed in 1.5% SDS a t 65 “C before autoradiography Detectron of ZL-I-Cells were collected in phosphate-buffered saline, sonicated for 45 s, and centrifuged twice for 15 min at 10,000 X g. The supernatant was assayed for IL-la and IL-1@with enzymelinked immunosorbent assay kits purchased from R & D Systems (Minneapolis, MN). The culture medium wasconcentrated 10- to 60fold in a cell-flow concentrator (Amicon, PMlO membrane) and assayed similarly. The sensitivity of this assay is 0.3 pg for both ILla and IL-lp. For Western blot analysis, 100 pg of cell lysate proteins were separated in 15% polyacrylamide gels and transferred to Immobilon P membranes (Millipore). The membranes wereblocked with 5% non-fat dry milk for 2 h and incubated with a 1:500 dilution of either anti-human IL-la or IL-1@polyclonal antibodies (Endogen, Boston, MA) for 1 h, washed several times with 0.25% Tween-20 in Tris-buffered saline, pH 7.4, and incubated 1h with a 1:2000 dilution of horseradish peroxidase-conjugated secondary antibody (Amersham). Theblots were washed again and developed with an enhanced chemiluminescence kit (Amersham) according to the manufacturer’s specifications and autoradiographed for 15 s. RESULTS

Induction of ZL-1mRNA by TNF-In previous experiments, we observed that both TNF and IL-la induce expression of


6216

10 0 . C .-m

FIG. 4. Decay of IL-1mRNA after removal of cycloheximide. HT-1080 cells were treated for 6 h with 10 pg/ml cycloheximide,washed, and incubated in fresh medium for the times indicated in the abscissa. Northern blot analysis (inset) was performed as described in Fig. IL-la 1.0, mRNA; 0, IL-l/3 mRNA.

C -a

75.

E

9

1 =E

50.

1Jp

0.

'

2io

FIG. 5. Induction of IL-1 mRNA by PMA. HT-1080cells were treated for 3 h with 10 ng/ml TNF or with different concentrations of PMA. Northern blot analysis was carried out as in Fig. 1. Hatched burs, IL-la mRNA; solid bars, IL-16' mRNA.

control

TNF

1

10 PMA (nM)

IL-1 mRNA in human diploid fibroblasts (10). In the present investigation, we examined the mechanism responsible for this activity of TNF. Because of the changing pattern of IL1 expression with fibroblasts aging (lo), we used a human tumor cell line. In preliminary experiments,we detected TNFmediated IL-1 mRNA induction in HT-1080 fibrosarcoma, ME-180 epidermoid carcinoma, A-172 glioblastoma, and HeLa cells. This finding showed that many tumor cells express IL-1 mRNA in response to TNF.All subsequent experiments were carried out with fibrosarcoma HT-1080 cells. The induction of IL-la and@ mRNA was very fast, with maximum levels reached -4 h after addition of TNF (Fig. 1).The level of IL-la mRNA decreased after 8 h, even in the continuous presence of TNF, while that of IL-l@remained at a maximum even after 16 h. It should be pointed out that IL-la mRNA was undetectable in untreated HT-1080 cells, whereas a very low basal level of IL-lP mRNA was clearly detectable (Fig.

increased rate of transcription. Both IL-la and j3 mRNA were actively transcribed in untreated control cells, but the treatment with TNF did not appear to increase this rate of transcription (Fig. 2). As a positive control, we examined the rate of transcription of the manganese superoxide dismutase mRNA that is induced by TNF (13). As expected, treatment with TNF increased the transcription rate of this mRNA (Fig. 2). In furthercontrols, nonspecific hybridization to thevector pUC19 was negligible and the rateof transcription of j3-actin mRNA was not affected by TNF treatment. It should be pointed out that the IL-1 mRNAs were apparently transcribed a t a faster rate than @-actinmRNA (Fig. 2). These nuclear run-off experiments indicated thatTNF did not induce expression of IL-1 mRNA by increasing its rate of transcription. As an alternative explanation, TNF could increase the stability of IL-1 mRNA. Instability of IL-la and@ mRNA-A commonly used assay to detect unstable mRNAs is based on cell treatment with an 1). Nuclear run-off experiments were performed to establish inhibitor of protein synthesis, such as cycloheximide, that whether the induction of IL-1 mRNA by TNF was due to an increases the half-life of short-lived mRNAs. IL-la and j3

6217

Stabilization of Interleukin-1 mRNA 25

20

SP TNF

"

-

10 5 0

+ - -

IO

50

+ +

-1L-Id

-1L-1p

FIG. 6. Inhibition by staurosporine of TNF-mediated induction of IL-1 mRNA. HT-1080 cells were treated for 3 h with 10 ng/ml TNF, and 10 or 50 nM staurosporine (SP). Northern blot analysis (inset) was carried out as described in Fig. 1. Hatched bars, ILla mRNA; solid bars, IL-10 mRNA.

.E 15 > .c

2

.-0

3

10

5

0

150

-

+ +

- -

+

200 400

TNF ColpbmtinC

125

100

FIG. 7. Inhibition by calphostin C of TNF-mediated induction of IL-1 mRNA. HT-1080cells were treated for 3 h with 10 ng/ml TNF and 200 or 400 nM calphostin C. Northern blot analysis (inset) was carried out as described in Fig. 1. Hatched bars, IL-lamRNA; solid bars, IL-10 mRNA.

.E

.-c> 00

75

5 rd U

50

25

0

Calphostin C (nM)

200

expression of IL-1 mRNA was mRNA accumulated in HT-1080 cells, reaching a plateau PKC,staurosporine.The after 8 h of cycloheximide treatment (Fig. 3). The level of greatly inhibited by staurosporine in TNF-treated cells (Fig. these mRNAs remained unchanged for up to 16 h, but de6). Furthermore, a treatment with staurosporine reduced the creased rapidlywhen cycloheximide was removed bywashing basal level of IL-1/3 mRNA in control cells, suggesting that the cells (Fig. 4). The apparenthalf-life of IL-la and6 mRNA PKC activitywas required forthe expression of both inducible was -60 min in these cells, indicating that they were short- and constitutively expressed IL-1 mRNA. However, the concentration of staurosporine used in these experiments was lived mRNAs. Previous investigators have shown that activators of protein relatively high,and itcould not be excludedthat staurosporine kinase C (PKC), such as phorbol esters, increase the expres-inhibited in addition to PKC (IC5o = 2.7 nM), also CAMPsion of some cytokines. Shaw and Kamen (6) first reported dependent protein kinase (IC50 = 8.2 nM) or other kinases that the stability of granulocyte-macrophage colony stimulat- (14). This limited selectivity of staurosporine for different with the homologous ing factor mRNA was increased in PMA-treated cells and protein kinases is due to its interaction proposed that phorbol esters could modulate a degradation catalytic domain of these enzymes (14). T o show that PKC pathway specific for a class of unstable mRNAs. This expla- activity was specifically required for the expression of IL-1, nation could account for the increased level of both IL-la and we treated HT-1080 cells with the inhibitor calphostin C. 6 mRNA in PMA-treated HT-1080 cells (Fig. 5 ) . The increase This compound is highly specific for PKC (IC50 = 50 nM) regulatorydomain and does not in IL-1 mRNAlevel was correlated with the PMA concentra- since it interacts with its tion added to the cultures. inhibit CAMP-dependent protein kinase or otherkinases even The role of PKC in mediating the expression of IL-1 mRNA at 50 p~ (14). Additionof 200 or 400 nM calphostin C blocked was further documented by experiments with an inhibitorof the increase in IL-1 mRNA induced by T N F (Fig. 7).


6218

FIG. 8. Effect of staurosporine on the decay of IL-1 mRNA. HT-1080 cells were treated for 3 h with 10 ng/ml TNF, washed and incubated in fresh medium (solid lines) or medium containing 10 nM staurosporine (broken lines) for the time indicated. Northern blot analysis and measurement of radioactivity was performed as described in Fig. 1 . 0 , ILla mRNA; 0, IL-lP mRNA.

9U

E

ae

60-

\

40-

20-

"

I

15

0

3b

45

gb

75

!30

minutes

T m r c

CHX TNF

IL-I

TNF [L-la

IL-1

0

I control

p

El CHX

B

T

TNF

CHX+TNF

FIG. 9. Induction of IL-1 mRNA in cells treated with cycloheximide, TNF, or IL-1. HT-1080 cells were 10 pg/mlcycloheximide treatedwith ( C H X ) , 10 ng/ml TNF, or IL-lafor 4 h. Northern blot analysis(inset) and measurement of radioactivity was carried out as described in Fig. 1. Hatched bars, ILla mRNA; solid bars, IL-16 mRNA.

_I 11-1 oc

Evidence for a destabilizing activity of 10 nM staurosporine on IL-1 mRNA was obtained by following the decay of this mRNA after aninitial inductionby TNF. HT-1080 cells were treated for 3 h with 10 ng/ml of TNF, washed, and incubated in fresh medium minus or plus staurosporine (Fig. 8). After removing TNF, both IL-la and p mRNA decayed with an apparent half-life of -75 min. In the presence of staurosporine, however, these mRNAs decayed with an apparent halflife of -30 min. Therefore, inhibitionof PKC activity resulted in anaccelerated decay of IL-1 mRNA. TNF andcycloheximide appeared to increase the stability of IL-1 mRNA by different mechanisms. This was suggested by the synergistic effect of these agents in combination (Fig. 9). Concentrations of cycloheximide and TNF that had a relatively modest effect on the expression of IL-la mRNA, had a much greater than additive effect when administered together. This synergistic effect was less evident for IL-lp mRNA since T N F alonegreatly increased its expression. Addition of staurosporine together with cycloheximide had

no effect on the increased expression of IL-1 mRNA (data not shown). Therefore, the mechanism by which cycloheximide increased mRNA stability was apparently independent of PKC activity. It should be pointed out that IL-la had no effect on the expression of either IL-laor /3 mRNA, confirming a previous report that HT-1080 cells do not respond to IL-1 (15). The increased stability of IL-1 mRNA induced by TNF or PMA led to the synthesis of IL-la and /3 that were detected by specific enzyme-linked immunosorbent assay in cell extracts (Fig. 9). IL-la remains cell-associated (16), while ILl p is secreted by different cells (15) but not by human lung fibroblasts (17). In HT-1080 fibrosarcoma cells neither IL-la nor IL-1p could be detected inthe culture medium, even when concentrated up to 60-fold. Addition of staurosporine inhibited the TNF-mediated induction of IL-1 and decreased the basal level of expression of IL-1 in untreated cells (Fig. 10). This finding suggests that PKC activity is required for both constitutive and induced synthesis of IL-1. Similar results


.- I

1

I

C TNF TNF P Y A

+ SP

12

control

TNF

SP+W

PMA

SP

FIG.10. Presence of IL-1in cells treated with TNF and/or staurosporine. The HT-1080 cells were treated for 6 h with 10 ng/ ml TNF, 10 nM PMA, and 10 nM staurosporine. IL-la and IL-10 were measured incell extracts with an enzyme-linked immunosorbent assay. Hatchedbars, IL-la; solid bars, IL-10. The inset shows a Western blot analysis of cell extract probed with anti-IL-la or antiIL-10 antibodies. The position of a protein marker of M,= 27,500 is indicated by the arrowhead.

were obtained by Western blot analysis (see inset in Fig. 10). However, only the precursor forms of both IL-la andP were detected, suggesting that thesecytokines are not processed to the secretory forms by HT-1080 cells. DISCUSSION

The induction of IL-1 by TNF appearsto be entirely mediated by mRNA stabilization. This induction is quite rapid, since IL-la mRNA is almost maximally induced 2 h after TNF addition and IL-lP mRNA after 4 h (Fig. 1).The nuclear run-off experiments performed (Fig. 2) clearly show that both IL-la and fi mRNA are constitutively transcribed by HT-1080 cells and that TNF does not increase their transcription rate.IL-1 mRNA is also expressed in cells treated with cycloheximide, because of its increased stability when protein synthesisis inhibited. However, the IL-1mRNA expressed either in cycloheximide or TNF-treated cells is rather unstable when theseagents are removed, since its apparent half-life is reduced to 60-75 min (Figs. 4 and 8). This half-life is presumably longer than that of IL-1 mRNA in untreated HT-1080 cells, since otherwise we would expect to observe some accumulation of IL-1 mRNA in these cells. Presumably, newly transcribed IL-1 mRNA is rapidly degraded, since IL-la mRNA is undetectable and only small amounts of IL-lP mRNA are found in untreated cells. Once IL-1 mRNA is accumulated a t relatively high levels, however, it may decay at a slower rate than newly transcribed mRNA. The increased stability of IL-1 mRNA induced by TNF requires PKC activity, since the PKCinhibitor staurosporine blocks the activity of TNF. It islikely that PKCactivation is sufficient to induce IL-1 mRNA stabilization, since phorbol esters have an inducing activity equal to or even greater than that of TNF. Previous studies have shown that TNFactivates PKC (18) and may therefore stabilize IL-1 mRNA in this way. Furthermore, some basal level of PKC activity is required in untreated cells to allow constitutive expression of IL-1P mRNA, as shown by the effect of a treatment with staurosporine. This PKC inhibitor increases the turnover of IL-1 mRNA in cells initially treated with TNF, indicating that the decay of this unstable mRNA is tightly regulated by PKC activity (Fig. 8). The levels of both IL-la and IL-lP mRNA

6219

correlate with the level of IL-1 assayed by enzyme-linked immunosorbent assayor Western blot (Fig. 10) in these cells. This protein is presumably biologically inactive since both IL-la andIL-1P remain cell-associated and unprocessed (Fig. 10). The mechanisms that regulate the stability of IL-1 mRNA are not yetfully understood. These mechanisms may operate on aclass of mRNAs that containAUUUA motifs within AUrich sequences in the3'- untranslated region. Specificproteins (AUBF) may bind to the AUUUA motifs, as shown by the formation of complexes with synthetic oligonucleotides containing multiple AUUUA stretches or with the AU-rich sequences of cytokine and oncogene mRNAs (19,20). Stephens et al. (21) recently reported that TNFinduces stabilization of the glucose transporter mRNA and increases the activity of AUBF. This observation suggests that the AUBF maybe involved in protecting mRNAs containing AUUUA motifs from degradation. Malter and Hong (22) had previously reported that AUBF are up-regulated post-translationally by phorbol ester-mediated phosphorylation. Therefore, aplausible hypothesis that takes intoaccount these observations and our results is that PKC activated by TNF or phorbol esters mediates an increase in AUBF binding to IL-1 mRNA. Such an increase in AUBF activity and in the half-life of the glucose transporter mRNA from 50 min to 5 h has been reported in phorbol ester-treated preadipocytes (21). Treatment of these cells with a serine-threonine phosphatase inhibitor, okadaic acid, also results inAUBF activation and mRNA stabilization; this suggests that a kinase-phosphatase systemis involved in AUBF regulation (21). Other investigators have reported multiple AUBF differing in their binding specificity and affinity (23). The activity of a specific AUBF apparently decreases in phorbol ester-treated T cells despite increased lymphokine mRNA stability (24). Therefore, it is possible that various AUBF may function by different mechanisms and may either increase or decrease the stability of mRNAs that interact with them. It is likely that TNF and IL-1 increase IL-1 mRNA stability by an identical mechanism, since these cytokines activate common multiple protein kinases in human fibroblasts(25). Activation of PKC by IL-1 may thus explain the induction of IL-1 in IL-1-treated rabbits in uiuo and human mononuclear cells in vitro (26), and the synergistic stimulation of IL-lP mRNA expression by IL-1 and TNF in fibroblasts (17). Furthermore, IL-1 increases the stability of nerve growth factor mRNA in fibroblasts isolated from rat sciatic nerve (27). Therefore, it is likely that TNF and IL-1may stabilize several mRNAs that are constitutively transcribed,but arecharacterized by a rapid turnover and by the presence of AU-rich sequences in the 3'untranslated region. REFERENCES 1. Old, L.J. (1985)Science 230,630-632 2. Beutler, B., and Cerami, A. (1988)Annu. Rev. Biochem. 57,505-518 3. Dinarello, C. A. (1988)FASEB J. 2, 108-115 4. Sims, J. E., March, C. J., Cosman, D., Widmer, M. B., MacDonald, H. R., Mc Mahan, C. J., Grubin, C. E., Wiall, J. M., Jackson, J. L.,Call, Friend, D., Alpert, A. R., Gillis, S., Ur%, D. L., and Dower, S. K. (1988) Science 241,585-58 5. Vilcek, J., and Lee, T. H.(1991)J. Biol. Chem. 266,7313-7316 6. Shaw, C., and Kamen, R. (1986)Cell 46,659-667 7. Wodnar-Filipowiccz, A., andMoroni, C. (1990) Proc. Natl. Acad. Sci. U.S. A. 87,777-786 8. Gessani, S., Di Marzio, P., Rizza, P., Belardelli, F., and Baglioni, C. (1991) J. ViroL 65,989-991 9. Gessani. S.. McCandless. S.. and Baelioni. C. (1988)J. Biol. Chem. 263.

.

-1.

. ."

10. Kumar, S.,Millis, A. J. T., and Baglioni, C. (1992)Proc. Natl Acad. Sci. U.S. A. 89,4683-4687 11. Peppel, K., and Baglioni, C. (1990)Biotechniques 9,711-713 12. Groudine, M., Beretz, M., and Weintraub, H.(1981)Mol. Cell. Bio. 1,2819QQ

13. W, G. H.W., and Goeddel, D. V. (1988)Science 242,941-944 14. Tamaoki, T. (1991)Methods Enzymol. 201,340-347

6220


15. Kumar, S., and Baglioni, C. (1991)J. Bid. Chem. 266,20960-20964 22. 16. Lepe-Zuniga, B., and Geri, I. (1984)Clin. Immunol. Immunopathol. 31, 222-230 17. Elias, J. A., Reynolds, M.M., Kotloff, R. M., and Kern, J. A. (1989)Proc. Natl. Acad. Sci. U. S. A. 86,6171-6175 18. Schiitze, S., Nottrott, S., Ptizenmaier, K., and Kronke, M. (1990)J. Immunol. 144,2604-2608 19. Malter, J. S . (1989)Science 246,664-666 20. Gillis, P., and Malter, J. S. (1991)J. Biol. Chem. 266,3172-3177 21. Stephens, J. M., Carter, B. Z., Pekala, P. H., and Malter, J. S. (1992)J. Bwl. Chem. 267,8336-8341

Malter, J. S., and Hong, Y. (1991)J. Biol. Chem. 266,3167-3171 23. Bohjanen, P. R., Petryniak, B., June, C. H., Thompson, C. B., andLindsten, T. (1992)J. Biol. Chem. 267,6302-6309 24. Bohjanen, P. R., Petryniak, B., June, C. H., Thompson, C. B., and Lindsten, T. (1991)Mol. Cell Bwl. 11,3288-3295 25. Guy, G. R.,Chua, S. P., Wong,N. S., Ng, S. B., and Tan, Y.H. (1991)J. Biol. Chem. 266,14343-14352 26. Dinarello, C. A,, Ikejima, T., Warner, S. J. C., Orencole, S. F., Lonnemann, G., Cannon, J. G., and Libby, P. (1987)J. Immunol. 139,1902-1910 27. Lindholm, D.,Heumann, R., Hengerer, B., and Thoenen, H.(1988)J. Biol. Chem. 263,16348-16351