Proc. Nati. Acad. Sci. USAVol. 86, pp. 182-186, January 1989Cell Biology

Cytolysis by tumor necrosis factor is preceded by a rapid andspecific dissolution of microfilaments

(actin/stress fibers/phalloidin/immunofluorescence/celi death)

MARY SCANLON*t, SCOTT M. LASTERt, JOHN G. WOOD*, AND LINDA R. GOODINGtDepartments of *Anatomy and Cell Biology, and *Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322

Communicated by Richard M. Krause, October 4, 1988

ABSTRACT Tumor necrosis factor (TNF) is cytotoxic tocertain transformed cells, whereas normal cells are resistant toits effects. The resistance of normal cells can often be overcomeby treatment with inhibitors of transcription or translationsuch as actinomycin D or cycloheximide (CHI), suggesting thatnormal cells produce a protein(s) that protects them fromTNF-induced cytolysis. In this report, we examine the mech-anism of cytolysis in a 3T3-like mouse cell line, C3HA, whichwas sensitized to TNF by treatment with CHI. We found thatan early change in TNF/CHI-treated cells was a significant lossof stress fibers in perinuclear areas of the cytoplasm. Thedisruption of microfflaments, which was observed within 15min of treatment, was not seen in untreated cells or in cellstreated with either TNF or CHI alone. The dissolution ofmicrofilaments spread peripherally over time and precededother TNF/CHI-induced effects such as cytoplasmic "boiling,"decrease in cell volume, and lysis of the plasma membrane. Thebreakdown of stress fibers occurred without a change in micro-tubules or intermediate filaments. Cytochalasin E, which dis-rupts microfilaments, induced cytolysis of TNF-treated cellseven in the absence of CHI; however, demecolcine, whichdepolymerizes microtubules, did not sensitize cells to TNF. Wepropose that the TNF-induced cytolysis of certain cell types ispreceded by a selective disruption of the microfllament lattice.

Tumor necrosis factor (TNF) is a protein, produced duringinflammation by macrophages and monocytes, that wasoriginally characterized for its ability to induce cytolysis ofcertain transformed cells (1). Normal cells are resistant to thecytolytic effects ofTNF (1). The resistance to TNF cannot beexplained by a lack of receptors for TNF or an inability tointernalize TNF (2). Since resistant cells can be madesensitive to TNF by treatment with inhibitors of transcriptionor translation, it has been suggested that resistant cells havean endogenous protective mechanism that is dependent uponprotein synthesis (3-5). In support of this hypothesis, Hahnet al. (5) showed that preincubation of SV-80 cells (a simianvirus 40-transformed cell line) with TNF rendered the cellsresistant to subsequent challenge with TNF and cyclohexi-mide (CHI), an inhibitor of protein synthesis. In vivo,pretreatment with TNF also induces protection to subse-quent challenge with TNF and inhibitors of protein synthesis(6). Kirstein and Baglioni (7) identified two proteins whoseconcentration in human fibroblasts increased severalfoldupon incubation with TNF. More recently, Beresini et al. (8)demonstrated the induction of nine proteins following treat-ment ofhuman fetal lung fibroblasts with TNF. Resistance toTNF can also be overcome by the expression ofthe oncogeneproducts ElA and pp60vs" (9, 10). Fletcher et al. (10)suggested that pp6Ov-src overcomes resistance by inhibitingthe formation of gap junctions.

In TNF-sensitized cells such as murine L929 cells (11),human breast carcinoma cells (12), and adenovirus-trans-formed fibroblasts (13), TNF-induced cell death is character-ized by breakdown of the nuclear membrane and DNAfragmentation. On the other hand, Laster et al. (13) identifiedtwo cell types in which low molecular weight DNA is notreleased following treatment with TNF, suggesting that thecytolytic effect of TNF on some cells may be directed atcytoplasmic rather than nuclear domains. In this report, weexamine the mechanism by which TNF induces cell death inC3HA cells, one ofthe cell types that undergoes TNF-inducedcytolysis without a change in nuclear integrity. C3HA cells area 3T3-like cell line derived from mouse embryo fibroblasts (14)and are resistant to the cytolytic effects ofTNF except in thepresence of inhibitors of transcription or translation (15). Thedeath sequence of C3HA cells incubated with TNF and CHIis characterized by violent cytoplasmic "boiling," loss of cellvolume, and lysis of the plasma membrane (13, 16). The lackof nuclear involvement and the dramatic morphologicalchanges suggest that the target of TNF action in sensitizedC3HA cells might be the cytoskeleton. In support of ourhypothesis, it has previously been shown that lymphotoxin,which has TNF-like effects on sensitive cells, induces arearrangement of actin within L cells (17). We have chosen toexamine TNF-induced effects on the cytoskeleton of C3HAcells because their large size (diameter, 100-200 ,um) andextensive cytoskeleton make C3HA cells the ideal modelsystem for studying TNF-induced effects on cytoarchitecture.We find that TNF induces a rapid and specific breakdown

of the microfilament lattice of C3HA cells treated with CHI.Initially, the disruption of stress fibers occurs withoutchanges in adherence or in the structural integrity of micro-tubules or intermediate filaments, suggesting that the mech-anism of action of TNF upon sensitized cells may bemediated, in part, by a direct effect upon microfilamentintegrity.

MATERIALS AND METHODSCells and Medium. The derivation of the C3HA cell line, a

contact-inhibited fibroblast cell line from C3H/HeJ mouseembryos, has been described (14). C3HA cells were grown inDulbecco's modified Eagle's medium (GIBCO) supple-mented with 10% fetal bovine serum (HyClone, Logan, UT)and maintained at 37°C in an 8% CO2 incubator.

Reagents. Human recombinant TNF was generously sup-plied by Cetus; TNF activity is 13 x 106 units/mg of protein.As defined by Cetus, 1 unit ofTNF induces 50% lysis of L929cells in the presence of actinomycin D at 1 ,ug/ml. CHI,cytochalasin E, demecolcine, L-a-lysophosphatidylcholine,and tetramethylrhodamine isothiocyanate (TRITC)-conju-

Abbreviations: CHI, cycloheximide; FITC, fluorescein isothiocya-nate; TNF, tumor necrosis factor; TRITC, tetramethylrhodamineisothiocyanate.tTo whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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gated phalloidin were purchased from Sigma. Rabbit anti-seaurchin tubulin was obtained from Polysciences (Warrington,PA) and used at a dilution of 1:20, as was mouse monoclonalanti-vimentin, which was obtained from ICN. Both fluores-cein isothiocyanate (FITC)-conjugated goat anti-rabbit IgGand FITC-conjugated goat anti-mouse IgG were obtainedfrom Organon and used at a dilution of 1:50.

Immunofluorescence. C3HA cells were incubated over-night in 8-well chamber slides (Miles Laboratories, Naper-ville, IL) at a density of 3-5 x 103 cells per well. The cellswere then treated at 370C with TNF (500 units/ml) and/orCHI (25 Ag/ml) for various times (15-120 min). Control cellswere incubated for identical lengths of time without TNF orCHI. The cells were fixed and stained for F-actin by additionof =100 Al of phosphate-buffered 10% formalin containingL-a-lysophosphatidylcholine (0.5 mg/ml) and TRITC-conjugated phalloidin (1 Ag/ml), for 10 min at room temper-ature. The cells were washed once with 50 mM Tris, pH7.3/0.15 M NaCl (Tris-buffered saline, TBS), the chamberscaffolding was removed, and the slide was mounted with10% (vol/vol) glycerol in TBS. In double-staining experi-ments, following fixation and staining with TRITC-phal-loidin, cells were incubated at room temperature for 60 minwith either anti-tubulin or anti-vimentin. The cells werethoroughly washed with TBS and incubated for 60 min with theappropriate FITC-conjugated secondary antibody. The cellswere then washed and mounted as above. Fluorescent andphase-contrast images ofthe stained preparations were viewedwith a Leitz Laborlux 12 microscope equipped for epifluores-cence and were photographed with a Wild MPS 51S automaticphotography system.

Cytotoxicity Assay. The cytotoxicity assay was performedas described (18). In brief, C3HA cells were labeled withNa51CrO4 (New England Nuclear) at 100,uCi/ml (1 jtCi = 37kBq) for 4 hr, harvested, and resuspended to 2 x 10- cells perml. Aliquots of the cell suspension were added to 96-wellflat-bottom assay plates (Flow Laboratories) with variousamounts of TNF (0.5-500 units/ml) and incubated for 16 hrat 37°C in combination with one or more of the followingdrugs: demecolcine (2,tM), cytochalasin E (4 ,uM), or CHI (25,ug/ml). The final volume of each well was 200 ,l. Theconcentration of CHI used in these experiments was deter-mined empirically to be the lowest necessary to induce thecytolysis of C3HA cells with TNF. The concentrations ofdemecolcine and cytochalasin E were determined empiricallyto be sufficient to depolymerize 90%o of microtubules andmicrofilaments, respectively, within 1 hr without inducing anincrease in spontaneous release of 51Cr.

After centrifugation of the plates at 200 x g for 10 min, 100,ul of the supernatant was taken for y counting (Gamma 4000,Beckman). Percent 51Cr released in each well was determinedas [(experimental release - spontaneous release)/(maximalrelease - spontaneous release)] x 100. Each point wasperformed in triplicate. Maximal release was determinedafter lysis of an aliquot of cells with 1 M HCl; spontaneousrelease was <40%.

RESULTSLaster et al. (13) found that treatment of C3HA cells withTNF and CHI induced a profound change in cellular mor-phology in the absence of nuclear disintegration. DyingC3HA cells underwent a violent "boiling" process that wasaccompanied by a significant loss of cell volume. During thisprocess, nuclear disintegration and DNA fragmentation didnot occur. In the experiments described here, we observedthat nuclear position was altered soon after addition ofTNF/CHI, although the nuclei ofthese cells remained visiblyintact. The nuclei of untreated cells were observed to lie inthe plane of focus of the entire cell (Fig. la); however, the

FIG. 1. Phase-contrast micrographs of formalin-fixed C3HAcells. (a) Control cell. The well-spread and flattened appearance wastypical of untreated cells or of cells treated with TNF or CHI. (b) Celltreated with TNF/CHI for 15 min. The nucleus and Golgi region ofthis cell were in sharp focus while the cytoplasm was not, suggestingthat the nucleus and adjacent regions extruded from the cell after ashort incubation with TNF/CHI. (x300.)

nuclei of treated cells bulged outward so that the nucleus andthe cytoplasm of the same cell could not be viewed simulta-neously (Fig. lb). We hypothesized that the nuclear extru-sion reflected an early change in the structural components ofthe cell (i.e., the cytoskeleton) that ordinarily act as a nuclearscaffold. To investigate this hypothesis, we employed immu-nofluorescence techniques to examine the cytoskeleton ofthe C3HA cell during TNF-induced cytolysis.

Microfilaments were stained with TRITC-conjugated phal-loidin, a fungal toxin that binds to F-actin (19, 20). The stressfibers of untreated C3HA cells (Fig. 2a) appeared as long,straight cables spanning the length of the cell in parallel, in amanner similar to that observed in normal mouse embryofibroblasts (21). However, the cable-like pattern of fluores-cence was disturbed in a significant number of cells treatedwith TNF and CHI (Fig. 2b). At a higher magnification, it was

FIG. 2. Micrographs of C3HA cells fixed and stained withTRITC-phalloidin. (a) Stress fibers appeared as long, fluorescentcables spanning the length of control cells. (b) In contrast, aftertreatment with TNF/CHI for 30 min, a loss of stress fibers wasvisible in central areas of nine cells (arrowheads) in this field. (X65.)

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clear that the distribution of microfilaments within cellstreated with TNF (Fig. 3b) or with CHI (Fig. 3c) was virtuallyidentical to that of untreated cells (Fig. 3a). In contrast, thereappeared to be a loss of staining of microfilaments in cellstreated with TNF and CHI (Fig. 3d). The loss of microfila-ments was not an artifact of focal plane; optical sectioning of

FIG. 3. Micrographs of C3HA cells fixed andstained with TRITC-phalloidin. Stress fibers ap-peared virtually identical in control cells (a) andcells treated with TNF (b) or CHI (c). Aftertreatment with TNF/CHI for 30 min (d), more than30% of C3HA cells exhibited a loss in TRITC-phalloidin staining in central areas of the cell. Therewas a marked decrease offluorescence in two of thecells in d (arrowheads). In a third cell (star), stressfibers were not visible. The punctate fluorescencemay reflect short or severed fragments of F-actin.(a-c, x 190; d, X95.)

these cells revealed a decrease in perinuclear microfilamentsthroughout the vertical axis of affected cells. Within 15 minof TNF/CHI treatment, 31.00 ± 1.53% (mean ± SEM, n =

2500) of cells exhibited a loss of stress fibers; in contrast, only7.33 ± 1.45% of untreated cells, 11.33 ± 1.20% of TNF-treated cells, and 9.33 ± 1.33% of CHI-treated cells exhibited

FIG. 4. Phase-contrast and fluorescent images of single cells: a control cell (a and b) and cells treated with TNF/CHI for 15 min (c andd), 90 min (e and f), or 120 min (g and h). As the incubation with TNF/CHI was lengthened, the number of stress fibers in the cytoplasmdecreased. In the end-stage cell (g and h), no stress fibers were visible. (x300.)

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FIG. 5. C3HA cells double-stained for F-actin(a) and tubulin (b) and for F-actin (c) and vimentin(d) after treatment with TNF/CHI for 15 (c and d)or 60 (a and b) min. Although both cells exhibitedthe typical loss of F-actin, the networks of bothmicrotubules and intermediate filaments appearednormal. The microtubule network of untreated cellstypically extended throughout the cytoplasm,whereas that of intermediate filaments was con-densed around the nucleus and Golgi apparatus.(x 300.)

such a change. Within the first 30 min of treatment, the lossof microfilaments in nuclear areas occurred without a changein elongation or adherence.

In Fig. 4, phase-contrast and fluorescent images of indi-vidual cells are illustrated. In contrast to the uniformlydistributed microfilaments typical of control cells (Fig. 4 a

and b), there was a marked decrease of stress fibers in theperinuclear area of a representative cell treated withTNF/CHI for 15 min (Fig. 4 c and d). With time, the loss ofmicrofilaments extended peripherally so that stress fiberswere observed only at the cell edge (Fig. 4 e and f). In cellstreated with TNF/CHI for an hour or more, the microfila-ments at the periphery were arranged unevenly and oftenacquired a circular distribution that followed the cellularperimeter. More than 50% of the nuclei of cells that exhibiteda loss of stress fibers were diffusely stained (Fig. 4 d and f).The nature of this fluorescence is unclear, and such fluores-cence may, in fact, exist in control cells where its appearancewould be obscured by overlying stress fibers. On the otherhand, since phalloidin does not bind to monomeric actin (19),the diffuse fluorescence may reflect the presence of short orsevered microfilaments in nuclear areas. Dying cells, char-acterized by "boiling" ofthe plasma membrane, massive lossof cell volume, and compression of the cytoplasm (Fig. 4g),were stained intensely with TRITC-phalloidin (Fig. 4h).

Initially, the striking changes observed in C3HA microfil-aments occurred without visible changes in microtubules orintermediate filaments. In all TNF/CHI-treated cells thatwere double-stained for F-actin and tubulin (Fig. 5 a and b)or for F-actin and vimentin (Fig. 5 c and d), the networks ofmicrotubules and intermediate filaments were indistinguish-able from those of control cells at the time that disruption ofthe microfilament lattice was observed.As mentioned previously, normal cells can be made sen-

sitive to the cytolytic effects of TNF by the addition ofinhibitors of transcription or translation. C3HA cells can also

be renoered sensitive to TNF by the addition of cytochalasinE, which induces microfilament dissolution, but not by theaddition of demecolcine, which induces microtubule depo-lymerization. Immunocytochemical studies revealed thataddition of 4 ,uM cytochalasin E or 2 ,uM demecolcine toC3HA cells effectively induced depolymerization of at least90% of the microfilaments or microtubules, respectively,within 60 min without causing nonspecific lysis of the cells(data not shown). Incubation of the cells with TNF alone didnot induce significant lysis; incubation with TNF and CHItogether induced cytolysis in a manner directly dependentupon the concentration ofTNF (Table. 1). A dose-dependentrelationship between cytolysis and the concentration ofTNFwas also observed when cells were incubated with TNF andcytochalasin E. Doses of cytochalasin E as low as 0.1 ,uMwere able to induce filament disruption and potentiate TNF-induced cytolysis. In contrast, the level of cytolysis ofTNF/demecolcine-treated cells was indistinguishable fromthat of cells treated with TNF alone.The results strongly suggest that C3HA cells can be

rendered sensitive to TNF by disruption of microfilamentsbut not microtubules. In conjunction with the immunofluo-rescence data, it appears that an early effect ofTNF on C3HAcells sensitized by CHI is dissolution of the normal micro-filament lattice. In the presence of TNF, disruption of themicrofilament lattice is sufficient to induce cytolysis.

DISCUSSION

The apparent dissolution of stress fibers within C3HA cellstreated with TNF and CHI preceded such cytolytic indicatorsas cytoplasmic boiling by 2-4 hr and 51Cr release by 6-8 hr(13, 16). Initially, the loss of microfilaments occurred withoutperturbations in microtubules or intermediate filaments andwell before changes in adherence. The dissolution of stressfibers was vectorial, spreading from perinuclear to peripheral

Table 1. Sensitivity of C3HA cells to cytolysis by TNF

% spontaneous % TNF-induced releaseInhibitor release 0.5 u/ml 5 u/ml 50 u/ml 500 u/ml

None 28 3.3 ± 0.9 2.2 ± 1.8 3.1 ± 2.0 5.3 ± 5.7CHI (90 ,iM) 39 12.9 ± 2.3 23.2 ± 1.6 31.0 ± 3.2 53.7 ± 4.5Cytochalasin E (4 AM) 32 3.8 ± 0.6 5.7 ± 2.5 14.9 ± 4.6 26.5 ± 5.5Demecolcine (2 ,uM) 28 0 0.1 ± 1.2 1.4 ± 0.8 0.7 ± 1.0

Cells were loaded with 51Cr. After incubation with TNF at 0.5, 5, 50, or 500 units (u)/mI in the presence of inhibitor asindicated, the percentage of cells lysed was determined from the release of 51Cr into the supernatant. % spontaneous releaserepresents the level of lysis in the absence of TNF. % TNF-induced release is derived from the expression described inMaterials and Methods. Values are expressed as the average of three cultures ± SEM.

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areas. Within 2-4 hr of TNF/CHI treatment, cytoplasmicboiling occurred and cell volume decreased markedly. At thisstage, stress fibers were not observed; however, these cellswere intensely stained with TRITC-phalloidin, suggestingthat dying cells contain short or severed microfilaments.

In a preliminary report, Rossomando et al. (22) reportedthat treatment of periodontal fibroblasts with TNF for 24 hrinduced degeneration of microfilaments in a manner virtuallyidentical to that observed in C3HA cells. Leopardi et al. (17)showed that lymphotoxin, a cytokine closely related to TNF,induced a rearrangement of F-actin without a change in itsconcentration in L cells, a cell line that is spontaneouslysensitive to TNF and that undergoes nuclear disintegrationduring cytolysis. This evidence suggests that dissolution ofmicrofilaments may be a common mechanism of TNF-mediated cytolysis, even in cells undergoing nuclear disin-tegration. If so, then this hypothesis would predict thatdissolution of stress fibers should induce cytolysis in TNF-treated cells. In support of this hypothesis, we found thatcytochalasin E, like CHI, sensitized C3HA cells to thecytolytic effects of TNF but demecolcine did not. Neithercytochalasin E nor demecolcine was toxic to the cells in theabsence of TNF, since levels of lysis induced by either drugwere virtually identical to levels of spontaneous lysis (Table1). The resistance of demecolcine-treated cells to TNFcannot be attributed to a nonspecific effect ofdemecolcine onreceptor binding or internalization since at the doses ofTNFused in these experiments, levels of cytolysis ranged from78% to 100% in cells treated with TNF/CHI and demecolcine(unpublished data).

In contrast to our results, Kull and Cuatrecasas (4) showedthat L-M cells, which are spontaneously sensitive to TNF,were less sensitive to TNF in the presence of colchicine,Colcemid, and cytochalasin B. The resolution of the discrep-ancy between the two sets of results may lie in the cell typesused in each study and in the manner of TNF-induced celldeath. L-M cells treated with TNF undergo necrosis, aprocess in which the cell swells to a balloon-like shapeindicative of homeostatic injury (13). This type of death isdifferent from that of C3HA cells (13, 16) and thus, thedifferential sensitivity of L-M cells and C3HA cells tocytoskeletal inhibitors may reflect differences in the type ofcell death induced by TNF.The mechanism ofTNF/CHI-induced dissolution of stress

fibers is unknown; however, the dependence upon CHIsuggests that a protective protein(s), which normally rendersC3HA cells resistant to TNF, may do so by preventing thedisruption of microfilaments. Yin and coworkers (23, 24)demonstrated that microfilaments are sensitive to severing bygelsolin in the presence of micromolar Ca2". Gelsolin nucle-ates filament growth and thus induces the redistribution ofF-actin to shorter filament lengths. Although we have notmeasured the content of F-actin within TNF/CHI-treatedC3HA cells, the intense staining of end-stage cells (Figs. 3dand 4h) suggests that, as in L cells treated with lymphotoxin(17), the microfilaments of TNF/CHI-treated cells mayundergo a redistribution of F-actin to shorter filament lengthsrather than a complete depolymerization to actin monomers.An increase in Ca2+ concentration upon treatment with TNFhas been observed in Erlich ascites cells (25) and in a sublineof L929 cells (26). It is possible that the combined effect ofTNF and CHI upon C3HA cells increases intracellular Ca2+and/or activates gelsolin. This hypothesis suggests that therole of the protective protein(s) may be to maintain intracel-lular Ca2' at submicromolar levels and/or to prevent theactivation of gelsolin by another mechanism.The loss of microfilaments in TNF/CHI-treated C3HA

cells can explain, in part, the ensuing morphological changes

that accompany cell death. Cytoplasmic boiling may resultfrom the loss of cellular scaffolding as the microfilamentlattice collapses. However, microfilament dissolution bycytochalasin E is not accompanied by a loss of adherence(unpublished data) and thus suggests that TNF must have adirect effect on cellular adhesion, possibly through a changein the integrity of adhesion plaques.

In summary, our results suggest that TNF induces cytolysisin CHI-treated C3HA cells by disrupting the microfilamentlattice. Our findings suggest that dissolution of the microfil-ament lattice is an early effect ofTNF upon sensitized C3HAcells and may be a necessary event in a general type of celldeath exhibited by TNF/CHI-treated C3HA cells.

This work was supported by National Institutes of Health GrantsA126035 (L.R.G.), CA40266 (L.R.G.), AG06383 (J.G.W.), andNS17731 (J.G.W.). S.M.L. was supported as a trainee by NationalInstitutes of Health Grant A107265.

1. Carswell, E. A., Old, L. J., Kassel, R. L., Green, S., Fiore, N.& Williamson, B. (1975) Proc. Nail. Acad. Sci. USA 72, 3666-3670.

2. Tsujimoto, M., Yip, Y. K. & Vildek, J. (1985) Proc. Nail.Acad. Sci. USA 82, 7626-7630.

3. Ostrove, J. M. & Gifford, G. E. (1979) Proc. Soc. Exp. Biol.Med. 160, 354-358.

4. Kull, F. C., Jr., & Cuatrecasas, P. (1981) CancerRes. 41, 4885-4890.

5. Hahn, T., Toker, L., Budilovsky, S., Aderka, D., Eshhar, Z.& Wallach, D. (1985) Proc. Nail. Acad. Sci. USA 82, 3814-3818.

6. Wallach, D., Holtmann, H., Engelmann, H. & Nophar, Y.(1988) J. Immunol. 140, 2994-2999.

7. Kirstein, M. & Baglioni, C. (1986) J. Biol. Chem. 261, 9565-9567.

8. Beresini, M. H., Lempert, M. J. & Epstein, L. B. (1988) J.Immunol. 140, 485-493.

9. Chen, M. J., Holskin, B., Strickler, J., Gorniak, J., Clark,M. A., Johnson, P. J., Mitcho, M. & Shalloway, D. (1987)Nature (London) 330, 581-583.

10. Fletcher, W. H., Shiu, W. W., Ishida, T. A., Haviland, D. L.& Ware, C. F. (1987) J. Immunol. 139, 956-962.

11. Schmid, D. S., Hornung, R., McGrath, K. M., Paul, N. &Ruddle, N. H. (1987) Lymphokine Res. 6, 195-202.

12. Dealtry, G. B., Naylor, M. S., Fiers, W. & Balkwill, F. R.(1987) Eur. J. Immunol. 17, 689-693.

13. Laster, S. M., Wood, J. G. & Gooding, L. R. (1988) J. Immu-nol. 141, 2629-2634.

14. Gooding, L. R. (1979) J. Immunol. 122, 1002-1008.15. Gooding, L. R., Elmore, L. W., Tollefson, A. E., Brady,

H. A. & Wold, W. S. M. (1988) Cell 53, 341-346.16. Laster, S. M., Scanlon, M., Wood, J. G. & Gooding, L. R.

(1988) in Monokines and Other Non-Lymphocytic Cytokines,eds. Powanda, M. C., Oppenheim, J. J., Kluger, M. J. &Dinarello, C. A. (Liss, New York), pp. 285-290.

17. Leopardi, E., Friend, D. S. & Rosenau, W. (1984) J. Immunol.133, 3429-3436.

18. Chapes, S. K. & Gooding, L. R. (1985) J. Immunol. 135, 2192-2198.

19. Wieland, Th. & Faulstich, H. (1978) CRC Crit. Rev. Biochem.5, 185-260.

20. Wulf, E., Deboben, A., Bautz, F. A., Faulstich, H. & Wieland,Th. (1979) Proc. Nail. Acad. Sci. USA 76, 4498-4502.

21. Verderame, M., Alcorta, D., Egnor, M., Smith, K. & Pollack,R. (1980) Proc. Natl. Acad. Sci. USA 77, 6624-6628.

22. Rossomando, E. F., Gronowicz, G., Hadjimichael, J. & Ruth-erford, R. B. (1987) J. Leukocyte Biol. 42, 558-559 (abstr.).

23. Yin, H. L., Zaner, K. S. & Stossel, T. P. (1980) J. Biol. Chem.255, 9494-9500.

24. Janmey, P. A., Chaponnier, C., Lind, S. E., Zaner, K. S.,Stossel, T. P. & Yin, H. L. (1985) Biochemistry 24, 3714-3723.

25. Anghileri, L. J. & Robert, J. (1987) Tumori 73, 269-271.26. Kobayashi, Y., Utsunomiya, N., Nakanishi, M. & Osawa, T.

(1987) Immunol. Lett. 15, 53-57.

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Correction. In the article "Cytolysis by tumor necrosisfactor is preceded by a rapid and specific dissolution ofmicrofilaments" by Mary Scanlon, Scott M. Laster, John G.Wood, and Linda R. Gooding, which appeared in number1, January 1989, of Proc. Natl. Acad. Sci. USA (86, 182-186), Fig. 5 was poorly reproduced due to a printer's error.Fig. 5 is shown below with the accompanying figure leg-end. In the figure, representative C3HA cells double-labeledfor F-actin (a) and tubulin (b) and for F-actin (c) and vi-mentin (d) are illustrated following treatment with tumornecrosis factor plus cycloheximide (TNF/CHI) for 15 (c andd) or 60 (a and b) min. The striking changes observed inC3HA microfilaments occurred without visible changes in

microtubules or intermediate filaments. In fact, the networksof microtubules and intermediate filaments in the cells treatedwith TNF/CHI were indistinguishable from those of controlcells. The microtubules of control cells and of cells treatedwith TNF/CHI for 60 min extended uniformly throughoutthe cytoplasm to the periphery of the cell. The intermediatefilaments of control cells and of cells treated with TNF/CHI for 15 min were condensed around the nucleus and Golgiapparatus. It is clear that after treatment with TNF/CHI(at least for the times indicated), there is no change in thenetworks of microtubules and intermediate filaments, whilea profound dissolution of the microfilament lattice is occur-ring.

FIG. 5. C3HA cells double-stained for F-actin (a) and tubulin (b) and for F-actin (c) and vimentin (d) after treatment with TNF/CHI for15 (c and d) or 60 (a and b) min. Although both cells exhibited the typical loss of F-actin, the networks of both microtubules and intermediatefilaments appeared normal. The microtubule network of untreated cells typically extended throughout the cytoplasm, whereas that ofintermediate filaments was condensed around the nucleus and Golgi apparatus. (x300.)

Correction. In the article "Detection of two growth hormonereceptor mRNAs and primary translation products in themouse," by William C. Smith, Daniel I. H. Linzer, andFrank Talamantes, which appeared in number 24, December1988, of Proc. Natl. Acad. Sci. USA (85, 9576-9579), a lineof text was deleted in a printing error subsequent to theauthor-galley stage. The corrected sentences, which begin online 14 of the left column on p. 9579, read as follows: The invitro translation data suggest that the 3.9-kb mRNA encodesthe Mr 95,000 polypeptide, whereas the 1.2-kb mRNA en-codes the Mr 31,000 form of the mouse GHR. The increase inamount of these mRNAs in the liver during pregnancy issufficient to account for the increase in GHR-binding activity(1) and in the concentration of GHR polypeptides duringgestation (3).

3664 Cell Biology: Corrections