©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Fas- and Tumor Necrosis Factor-induced Apoptosis Is Inhibited by the Poxvirus crmA Gene Product (*)

(Received for publication, October 5, 1994; and in revised form, December 14, 1994)

Muneesh Tewari (§) Vishva M. Dixit (¶)

From the Department of Pathology and the Graduate Program in Cellular and Molecular Biology, University of Michigan Medical School, Ann Arbor, Michigan 48109

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

crmA is a cowpox virus gene that encodes a protease inhibitor of the serpin family. The only reported target for the CrmA protein is the cysteine protease interleukin-1beta converting enzyme (ICE). ICE, by virtue of its homology to the Caenorhabditis elegans cell death protein Ced-3, has been suggested to play a fundamentally important role in mammalian apoptosis. We hypothesized that a function of crmA may be to inhibit cell death, since a major mechanism of viral clearance is the immune system-mediated induction of apoptosis of infected cells. The tumor necrosis factor receptor and the Fas antigen are two cytokine receptors which, by engaging and activating the death pathway, can eliminate virus-infected cells. Remarkably, crmA was found to be an exceptionally potent inhibitor of apoptosis induced by both these receptors, capable of blocking the cell death program even at pharmacological doses of the death stimulus. Therefore, an important new function for crmA is the inhibition of cytokine-induced apoptosis. Further, the data suggest that a protease, either ICE or a related crmA-inhibitable protein, is a component of the Fas- and tumor necrosis factor-induced cell death pathways.


INTRODUCTION

Apoptosis, or programmed cell death (PCD), (^1)is critical to many biological processes, including embryogenesis, development of the immune system, elimination of virus-infected cells, and the maintenance of tissue homeostasis (reviewed in (1) and (2) ). Although the molecular components comprising the death pathway in mammalian cells await definition, recent work suggests that in the worm Caenorhabditis elegans, the ced-3 gene encodes a component of the death pathway(3) . ced-3 was found to be obligatory for apoptosis (4) and sequence analysis revealed it to be similar to the mammalian protease interleukin-1beta converting enzyme (ICE)(3) . ICE is a cysteine protease that catalyzes the proteolytic processing of the pro-inflammatory cytokine interleukin-1beta from an inactive precursor form to the active mature form(5, 6) . Sequence similarity between the predicted Ced-3 and ICE proteins was greatest in the active site region(3) , and the recent description of the crystal structure of ICE revealed that structurally important residues are conserved between the two proteins(7, 8) . Taken together, these data suggest that ced-3 encodes a cysteine protease and that a homologue of Ced-3, either ICE or a related protease, may be a component of the death pathway in mammalian cells.

Apoptosis is of particular relevance to viral pathogenesis, as a major mechanism for viral clearance by the mammalian immune system is the induction of apoptosis of infected cells(9) . It is becoming increasingly apparent that many viruses have evolved proteins that are capable of attenuating apoptosis(10) . The cowpox virus encodes a protein designated CrmA, which is an ICE inhibitor(11) . CrmA was originally described as a modulator of the host inflammatory response, presumably functioning by decreasing interleukin-1beta production through inactivation of ICE(11) . However, now given that ICE might be a component of the death pathway in mammalian cells, we hypothesized that crmA may additionally function to inhibit apoptosis. This line of thinking was substantiated by a recent report in which microinjection of crmA cDNA into chicken neuronal cells afforded partial protection from growth factor withdrawal-induced apoptosis(12) . There are multiple triggers for PCD, however, and it is not clear whether cells undergoing apoptosis triggered by different stimuli traverse biochemically identical or distinct pathways. Thus, it is not possible to extrapolate the finding in neuronal cells to cell death induced by other stimuli. In addition, the relevance of growth factor withdrawal-induced neuronal apoptosis to the physiologic function of crmA in the context of viral infection is not clear.

For these reasons, we chose to address the hypothesis in the context of apoptosis driven by two cell-surface cytokine receptors: the receptor for tumor necrosis factor (TNF) and the Fas antigen. These have been shown to induce cell death upon activation by either their respective ligands or by cross-linking with agonist antibody(13, 14, 15, 16) . PCD triggered by these receptors is especially relevant to the viral life cycle since both receptors have been implicated in viral clearance. TNF itself is a potent antiviral agent (17) and cytotoxic T-cells, which comprise a major antiviral defense, utilize the Fas antigen to induce apoptosis of target cells(18, 19, 20, 21) . To investigate whether crmA could modulate cytokine-induced apoptosis, we expressed the gene for crmA in cell lines susceptible to cell death mediated through either the TNF receptor or Fas. crmA acted as a powerful inhibitor of apoptosis, capable of completely abrogating cell death induced by activation of either of these receptors. This finding has implications for understanding the physiologic function of crmA as well as for identifying the components that comprise the cytokine-driven death pathway.


MATERIALS AND METHODS

Analysis of Apoptosis

Apoptosis was assessed by the use of fluorescent DNA-staining dyes to reveal nuclear morphology and by transmission electron microscopy. For propidium iodide staining, MCF7 cells were grown on 22-mm^2 No. 1 glass coverslips (Corning) placed in 35-mm wells of a six-well culture dish (Costar). Following treatment with TNF, anti-Fas+cycloheximide (CHX), or no treatment, medium was removed and the wells were rinsed twice with phosphate-buffered saline (PBS), fixed in 100% methanol at -20 °C for 10 min., washed three times with PBS, and stained at room temperature for 10 min. in a 100 µg/ml solution of propidium iodide (Sigma) made in PBS. The coverslips were then washed three times with PBS, blotted dry, and mounted onto glass slides using Vectashield mounting medium for fluorescence (Vector Laboratories). BJAB cells were stained using acridine orange (Sigma) by preparing a wet mount of 30 µl of a cell suspension at a density of approximately 3 times 10^5 cells/ml mixed with 5 µl of a 100 µg/ml acridine orange solution made in PBS. Both propidium iodide-stained MCF7 and acridine orange-stained BJAB nuclei were visualized by fluorescence microscopy using a fluorescein isothiocyanate range barrier filter cube. Laser-scanning confocal microscopy was performed using the Bio-Rad MRC 600 confocal microscope, and digitized images obtained were artificially colorized.

For electron microscopy, cells were fixed and processed as per standard electron microscopy procedures.

Quantitative Apoptosis Assays

MCF7 cells or derived transfectants were plated at a concentration of 2.5 times 10^5 cells/well onto glass coverslips. Two days later, after the cells had adhered and spread, TNF or anti-Fas+CHX was added. TNF was added at a final concentration of 20 ng/ml, anti-Fas at 25 ng/ml, and CHX (Sigma) at 10 µg/ml. After 22 h for the TNF-treated samples or after 18 h for the anti-Fas+CHX-treated samples, cells were fixed, stained with propidium iodide and mounted as described above. Apoptotic and non-apoptotic cells were quantitated based on nuclear morphology using fluorescence microscopy and the percentage of non-apoptotic cells was calculated. A minimum of 100 cells was counted for each sample, and each experiment was done at least in duplicate. Since a small fraction of cells in any normally growing cell culture is undergoing apoptosis, spontaneous apoptosis in untreated or CHX alone-treated samples was also quantitated. The percentage of non-apoptotic cells in the TNF or anti-Fas+CHX-treated samples was then normalized by correcting for the frequency of spontaneous apoptosis in the untreated or CHX alone samples, respectively.

BJAB cells were grown at 3 times 10^5 cells/ml and treated with anti-Fas antibody at a concentration of 250 ng/ml (unless indicated otherwise) for 18 h after which an aliquot was stained with acridine orange as described above. Apoptotic cells and non-apoptotic cells were quantitated and normalized to untreated samples. Assays were done at least in duplicate.

Secondary assays of cell death used were the MTT conversion assay and crystal violet staining and were done as described previously(22, 23) .

Plasmids, Transfections, and Selection of Stably Transfected Lines

The crmA gene (Dr. David Pickup, Duke University) was cloned into the pcDNA3 (Invitrogen) mammalian expression vector. The resulting expression construct or pcDNA3 itself was introduced into both MCF7 and BJAB cells by electroporation. MCF7 cells were electroporated at 330 V, 960 microfarads in 0.4-cm cuvettes (Bio-Rad), plated onto 100-mm dishes at varying dilutions and selected with G418 sulfate (Life Technologies, Inc.) at a concentration of 500 µg/ml. After selection for 3 weeks, pooled populations from each transfection were prepared by trypsinizing dishes containing several hundred colonies. Additionally, clonal cell lines were derived by picking individual colonies from selected dishes. BJAB cells were electroporated at 220 V, 960 microfarads in 0.4-cm cuvettes (Bio-Rad) and selected in 3 mg/ml G418 sulfate. One day following transfection, a portion of the cell population was diluted at a concentration of 2500 cells/well in 96-well dishes from which clonal cell lines were obtained after G418 selection. The remainder of the cells were retained as the pooled population.

Cell Lines, TNF, and Anti-Fas Antibody

The MCF7 cell line was a TNF-sensitive subclone obtained from Dr. David R. Spriggs (University of Wisconsin). The BJAB cell line was a gift of Dr. Fred Wang (Harvard). Recombinant TNF (specific activity 6.27 times 10^7 units/mg) was obtained from Genentech (South San Francisco, CA). Anti-Fas monoclonal antibody (clone CH-11, IgM) was obtained from PanVera (Madison, WI).

RNA Isolation and Northern Analysis

RNA isolation and Northern analysis were carried out as described previously(24) . Polymerase chain reaction was used to generate a probe spanning the coding region of the crmA gene. crmA hybridization signal was detected as a digitized image on a Molecular Dynamics PhosphorImager. beta-actin cDNA probe was purchased from Clontech (Palo Alto, CA), and the hybridization signal was visualized by autoradiography.


RESULTS

Induction of Apoptosis by TNF and Anti-Fas

A subclone of the MCF7 breast carcinoma cell line that expressed TNF receptor and was sensitive to TNF killing was chosen for these studies(25) . Further analysis revealed that Fas was also expressed on these cells and that cross-linking with an anti-Fas monoclonal antibody in the concomitant presence of the protein synthesis inhibitor cycloheximide induced cell death. (^2)Cycloheximide alone for the duration of the assay did not induce cell death beyond the negligible frequency of spontaneous apoptosis that is observed in any untreated cell culture. Anti-Fas alone was not cytotoxic, but this is not surprising, since induction of cell death in non-lymphoid cells by Fas activation has been reported to require the concomitant presence of either transcriptional or translational inhibitors(15) . Additionally, we examined a B-cell lymphoma cell line, BJAB, which expressed a high level of Fas and was killed by the addition of anti-Fas antibody in the absence of a protein synthesis inhibitor.

Cell death can occur by two biochemically and morphologically distinct processes: apoptosis and necrosis(26) . We first confirmed that death seen in response to TNF or anti-Fas occurred by apoptosis. Although various markers of apoptosis have been reported, the phenomenon is ultimately defined at the morphological level and is characterized by chromatin condensation and margination along the inner nuclear membrane, cytoplasmic condensation, and membrane blebbing without disintegration of the cellular membrane(26) . Necrosis, conversely, is defined by cytoplasmic swelling and lysis of the cell membrane and, importantly, does not exhibit the chromatin margination characteristic of apoptosis(26) . DNA laddering, representative of cleavage at internucleosomal intervals, is seen in some but not all forms of apoptosis, further emphasizing the importance of morphological criteria in defining apoptosis(27) . Nuclear morphology of cells dying in response to TNF or anti-Fas antibody was examined following staining with the DNA-binding dyes propidium iodide (MCF7 cells) and acridine orange (BJAB cells). Fluorescence microscopy and laser-scanning confocal microscopy demonstrated marked changes in nuclear morphology in the MCF7 cells in response to either TNF or anti-Fas+CHX and in the BJAB cells in response to anti-Fas. Chromatin condensation was clearly visible by immunofluorescence microscopy in both cell lines and formed the basis for our later assays of apoptosis in transfected cell lines (Fig. 1B for BJAB and data not shown for MCF7). Confocal microscopy confirmed margination along the inner nuclear membrane (Fig. 1, A and B (inset)). These morphological criteria of apoptotic cell death were further confirmed by transmission electron microscopy. The MCF7 cells clearly demonstrated chromatin condensation and margination along the inner nuclear membrane, cytoplasmic condensation, and increased membrane blebbing in response to either TNF or anti-Fas+CHX (Fig. 1A). BJAB cells treated with anti-Fas antibody demonstrated chromatin margination and cellular shrinkage typical of apoptosis in lymphoid cells (data not shown). Thus, both TNF and Fas triggered genuine apoptotic death in these cell lines.


Figure 1: A, TNF and anti-Fas+CHX induce apoptosis in MCF7 cells. Treatment with TNF or anti-Fas+CHX was carried out as described under ``Materials and Methods.'' Upperrow, nuclei of untreated, TNF-treated, or anti-Fas+CHX-treated MCF7 cells stained with propidium iodide and visualized by laser-scanning confocal microscopy. Arrows indicate examples of apoptotic nuclei. Lowerrow, transmission electron microscopy of untreated, TNF-treated or anti-Fas+CHX-treated MCF7 cells. B, anti-Fas antibody induces apoptosis in BJAB cells. Anti-Fas treatment was carried out as described under ``Materials and Methods.'' Fluorescence microscopy of untreated or anti-Fas-treated BJAB cells stained with acridine orange. Arrows indicate examples of apoptotic nuclei. Inset, laser scanning confocal microscopy of untreated or anti-Fas-treated BJAB cells stained with acridine orange.



crmA Blocks TNF- and Anti-Fas-induced Apoptosis

To determine whether crmA can function to inhibit cytokine-induced apoptosis, MCF7 and BJAB cell lines were transfected with either the expression vector pcDNA3 by itself or as a crmA expression construct. Stable transfectants were generated by neomycin (G418) selection, and pooled populations of neomycin-resistant cells from each transfection were assayed for crmA expression by Northern analysis (data not shown). These pooled populations were analyzed for their sensitivity to TNF- and anti-Fas-induced apoptosis by direct quantitation of apoptotic cells based on nuclear morphology following staining with DNA-binding dyes and visualization by fluorescence microscopy. Initial experiments demonstrated dramatic resistance of the crmA-transfected pools to apoptosis mediated by either TNF or Fas in both cell lines (Fig. 2). This was remarkable, given that in each pooled population of neomycin-resistant cells transfected with crmA, a significant fraction of cells were likely not expressing crmA due to, among other reasons, nonproductive integration of the expression construct into genomic DNA.


Figure 2: Inhibition of apoptosis by crmA in pooled populations of transfectants. Pooled populations of the vector- or crmA-transfected cells indicated were analyzed for sensitivity to TNF- or Fas-mediated apoptosis.



In addition to the pools, clonal lines were derived from both MCF7 and BJAB transfectants and challenged by activation of the TNF and Fas death pathways. In the MCF-7 cell line, vector clones were uniformly sensitive to apoptosis induced by either TNF or anti-Fas+CHX, whereas among the transfected clones, those that expressed detectable crmA transcript were markedly resistant (Fig. 3A). Indeed, lines expressing the highest levels of crmA were totally resistant to apoptosis (Fig. 3A) and showed no morphologic cytopathic effects (Fig. 3B), demonstrating that crmA can completely block the TNF- and Fas-mediated death pathways. Similarly, among BJAB-transfected clones, the crmA-expressing lines were markedly resistant to anti-Fas-induced apoptosis, whereas the vector clones were universally sensitive (Fig. 4A). Importantly, in both MCF7 and BJAB transfectants, those clones expressing the highest levels of crmA were the most resistant, while those clones expressing little or undetectable levels were the most sensitive (Fig. 3A and Fig. 4A). Although direct visual quantitation of apoptotic nuclei is the most accurate measure of apoptosis, comparable results were obtained when either an MTT conversion-based death assay (Fig. 3A, inset, and 4A, inset) or crystal violet staining (Fig. 3C) was employed to assess cell survival.


Figure 3: A, inhibition of TNF- and anti-Fas+CHX-induced apoptosis of MCF7 cells by crmA. Top, sensitivity of MCF7 vector-transfected clones (V1-V5) or crmA-transfected clones (crmA1-crmA5) to TNF- and anti-Fas+CHX-induced cell death assessed by propidium iodide apoptosis assay. Inset, sensitivity of selected clones to TNF- and anti-Fas+CHX-induced cell death assessed by MTT conversion assay. Middle, Northern analysis of corresponding cell lines to detect crmA transcript. Bottom, Northern analysis to detect beta-actin transcript to assess loading of RNA. B, crmA inhibits TNF- and anti-Fas+CHX-induced cytopathic effects on MCF7 cells. Phase contrast photomicrographs of cultures of a vector-transfected clone (MCF7V4) or a crmA-expressing clone (MCF7crmA3) treated with TNF, anti-Fas+CHX, or no treatment (UnRx) for 36 h. C, crmA inhibits TNF and anti-Fas+CHX-induced death of MCF7 cells when assessed by crystal violet staining. A vector-transfected clone (MCF7 V4) and a crmA-expressing clone (MCF7crmA3) were grown in a six-well culture dish and treated with TNF, anti-Fas+CHX, or no treatment (UnRx) for 48 h. Surviving cells were stained with crystal violet as described under ``Materials and Methods.''




Figure 4: A, inhibition of anti-Fas-induced apoptosis in BJAB cells by crmA. Top, analysis of sensitivity of BJAB vector-transfected clones (V1-V6) or crmA-transfected clones (crmA1-crmA10) to anti-Fas-induced cell death assessed by acridine orange apoptosis assay. Inset, sensitivity of selected clones to anti-Fas-induced cell death assessed by MTT conversion assay. Middle, Northern analysis of corresponding cell lines for detection of expression of crmA transcript. Bottom, Northern analysis to detect beta-actin transcript to assess loading of RNA. B, crmA inhibits apoptosis induced by increasing doses of anti-Fas antibody. The acridine orange apoptosis assay was used to analyze the sensitivity of vector-transfected (BJAB V1) or crmA-transfected (BJABcrmA2, BJABcrmA3) clones to apoptosis induced by the indicated doses of anti-Fas antibody.



We proceeded to determine if protection conferred by crmA from cytokine-induced apoptosis could be attenuated by increasing the dose of the death stimulus. Remarkably, crmA afforded comparably high levels of protection from anti-Fas-induced apoptosis in response to doses of antibody 250 times greater than those needed to kill greater than 95% of the vector-transfected cells (Fig. 4B). Similar results were obtained when the dose of TNF was varied for the MCF7 transfectants (data not shown), implying that crmA is functioning as an exceptionally potent inhibitor of cell death at a presumably critical step in the death pathway.


DISCUSSION

Our findings describe an important new function for crmA: the blockade of TNF- and Fas-mediated apoptosis. Given the importance of both TNF and Fas in the host anti-viral response, it is likely that this function of crmA is important for productive viral infection in vivo. crmA represents yet another example of viral economy in which two important functions, namely the inhibition of interleukin-1beta production and the prevention of apoptosis, are embedded in one protein.

An important implication of our data is that a protease is a component of both the TNF- and Fas-induced cell death pathways. Earlier experiments with small molecule protease inhibitors and with overexpression of plasminogen activator inhibitor-2 (28, 29, 30) provided evidence for protease activation in TNF-induced cytotoxicity, although protection was partial and the physiologic relevance of the studies unclear. crmA is a powerful inhibitor capable of conferring complete protection and, by virtue of its known inhibition of ICE, has particular relevance to apoptosis. Although the only reported target for CrmA is ICE, it is becoming increasingly clear that ced-3 and ICE are members of a gene family, of which at least one other member, the murine nedd2 gene, has been reported(31) . Hence, it is possible that the protease involved in TNF- and Fas-mediated apoptosis may not be ICE itself but instead one of its relatives. Furthermore, a recent report suggests that CrmA functions as a ``cross-class inhibitor'' and may be capable of inhibiting serine proteases as well as ICE(32) . Thus, the possibility that the relevant CrmA target is a serine protease cannot be excluded.

Additionally, the findings have implications for the unification of death pathways in general. First, the fact that crmA blocks both TNF- and Fas-mediated apoptosis, especially in the MCF7 cells which possess both receptors, suggests that the two receptors signal death through a biochemically common pathway. This hypothesis is supported by the finding that the cytoplasmic regions of both these receptors encompass a region of homology which has been defined by mutational analysis as a ``death domain'' and which presumably interacts with a common set of signal transduction molecules(23, 33) . Furthermore, it is now apparent that crmA blocks cell death triggered by two very different stimuli: growth factor withdrawal in neuronal cultures (12) and, from our studies, activation of cytokine receptors. It is of note that apoptosis in these two systems has been suggested to occur through biochemically distinct pathways, in that apoptosis in the former system is dependent on new protein synthesis and death is blocked by cycloheximide(34) , whereas in contrast TNF- and Fas-mediated cytotoxicity is independent of new protein synthesis and is, in fact, enhanced by cycloheximide(13) . Our data would suggest that at some point, the death pathway in both systems converges upon a crmA-inhibitable step, likely the activation of a protease. Indeed, given that in C. elegans the ced-3 gene is required for all apoptosis in the organism during development(4) , it is possible that the functional homologue of ced-3 in mammalian cells might well play a similar universal role. Therefore, the identification of the apoptosis-relevant CrmA target, be it ICE or another protein, may define the first component of a general mammalian cell death pathway.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant CA61348. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Fellow of the Medical Scientist Training Program. Supported by a Young Scientist M.D./Ph.D. Fellowship from the Life and Health Insurance Medical Research Fund.

Established Investigator of the American Heart Association. To whom correspondence should be addressed: Dept. of Pathology, University of Michigan Medical School, 1301 Catherine St., Box 0602, Ann Arbor, MI 48109. Tel.: 313-747-2921; Fax: 313-764-4308; vishva.dixit{at}med.umich.edu.

(^1)
The abbreviations used are: PCD, programmed cell death; ICE, interleukin-1beta converting enzyme; TNF, tumor necrosis factor; CHX, cycloheximide; PBS, phosphate-buffered saline; MTT, (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide).

(^2)
M. Tewari and V. M. Dixit, unpublished observations.


ACKNOWLEDGEMENTS

We thank David J. Pickup for the crmA gene, David R. Spriggs for the TNF-sensitive MCF7 cells, Fred Wang for BJAB cells and advice on electroporation, Bruce Donohoe and Lisa Riggs for electron microscopy, and Tom Komorowski and Walter Meixner for assistance with confocal microscopy. We are grateful to Neil Perkins for assistance with the use of the phosphorimager, to Ian Jones for help in preparing the figures, and to S. Suchard for advice on photography. We thank K. Farmanfarmaian for critical review of the manuscript and Karen O'Rourke, Vidya Sarma, Akhilesh Pandey, Claudius Vincenz, Arul Chinnaiyan, and Zhi Zheng for helpful discussions and encouragement.


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