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Proc. Nadl. Acad. Sci. USA Vol. 91, pp. 8641-8645, August 1994 Cell Expression of a specific marker of avian in both apoptosis and necrosis (development/immunofluorescence) PIERRE-ALAIN FERNANDEZ*t, Rocco J. ROTELLO*t, ZEHAVA RANGINI*t, ALLISON DOUPEt, HANNES C. A. DREXLER*t, AND JUNYING YUAN*t§ *Cardiovascular Research Center, Massachusetts General Hospital, 149 Thirteenth Street, Charlestown, MA 02129; tDepartment of Medicine, Harvard Medical School, Boston, MA 02115; and tDivision of Biology, California Institute of Technology, Pasadena, CA 91125 Communicated by H. Robert Horvitz, May 18, 1994 (receivedfor review December 3, 1993)

ABSTRACT Apoptosis and necrosis are two types of cell 8-week-old F1 BALB/cJ x C57/BL male mice (The Jackson death with different morphologic features. We report here the Laboratory) primed with incomplete Freund's adjuvant (Cap- isolation of a monoclonal antibody, BV2, that specifically rec- pel), and 2 weeks later, ascites fluid was collected and purified ognize cells undergoing developmental programmed cell death by E-Z-Sep (Middlesex Sciences, Foxborough, MA). in different tissues ofthe chicken and zebra-finch . The Immunofluorescence. Whole embryos and limbs were antigen recognized by BV2 monolonal antibody is detected in rinsed in cold phosphate-buffered saline (PBS) and fixed in vitro in primary chicken embryonic induced to die by 4% periodate/lysine/paraformaldehyde prepared in PBS for actinomycin D, as well as fibroblasts induced to die by chemical 12 hr at 4TC. Tissues were then washed in cold PBS and anoxia. The expression of this specific antigen during necrosis dehydrated in graded concentrations ofethanol (50%-95%) at appears to require active protein synthesis. These dinps 4TC. Infiltration was conducted with solution A ofJB-4 glycol provide evidence that cells from different embryonic tissues methacrylate embedding kit (Polyscience) in a light-protected undergoing prgrammed cell death during vertebrate develop- glass vial at 40C for several days (11). Tissues were embedded ment express simlr antigens and indicate that apoptosis and in glycol methacrylate and placed under vacuum (15 mmHg; necrosis may share similar biochemical features. 1 mmHg = 133 Pa) at 4°C and allowed to polymerize for 48 hr at 4°C (12). Sections were cut at 5 ,urm with glass knives on The morphological concept of apoptosis, defined as a phys- a Sorvall JB-4 microtome (DuPont), transferred via water to been as Superfrost/plus pretreated slides (Fisher Scientific), and iological type of cell death, has progressively used a air-dried at room temperature. For immunofluorescence synonym with the term programmed cell death, originally staining, the sections were treated with xylene for 30 min at described during normal development (1) and implying the room temperature, extensively washed with cold PBS, and existence of a genetic program of cell death (2). Apoptosis is digested with Pronase at 0.5 mg/ml (Sigma)/PBS for 1 hr at believed to account for most cell death during development 3rC. The enzyme reaction was stopped with cold 0.5 M Tris and in normal adult tissue turnover, and it can also be induced buffer on ice followed by washing in cold PBS. The slides experimentally by various biological, chemical, or physical were then treated with a blocking solution consisting of 1% agents (3). Necrosis, in contrast, has been defined as a bovine serum albumin (Sigma) and 1% heat-inactivated nor- passive degenerative phenomenon and is observed in a tissue mal goat serum (GIBCO) in PBS for 30 min at room temper- subjected to direct toxic or physical injury-such as hyper- ature. Incubation was conducted for 5 hr at room temperature thermia, hypoxia, and ischemia or to complement-mediated with the BV2 ascites fluid diluted in blocking buffer. After lysis (4-7). To investigate the mechanism of cell death, we several washes with PBS, affinity-purified goat anti-mouse have identified a monoclonal antibody (mAb), BV2, that antibody conjugated with fluorescein (1:100, Cappel) was specifically recognizes cells undergoing programmed cell diluted into blocking buffer and applied to the slides for 1 hr death in developing chicken embryos, and we used BV2 mAb at room temperature together with Hoechst 33258 (Ho 33258) as a specific marker of chicken programmed cell death to dye (0.5 jug/ml, Sigma), a DNA minor groove-binding ligand study the relationship between apoptosis and necrosis. that has strong adenine plus thymidine sequence specificity (13). After washing with PBS, the slides were mounted in 10o MATERIALS AND METHODS PBS/90% glycerol with p-phenylenediamine at 1 mg/ml (Sigma) to prevent photo bleaching. To verify the specificity Generation of mAbs. Chicken eggs were purchased from of the antibody labeling, control staining was done with Spafas, Norwich, CT. Zebra finches (Poephila guttata) were preimmune or nonimmune immunoglobulins as primary an- grown in the avian facilities of the California Institute of tibodies or with secondary antibody alone. No significant Technology. Chicken embryos were staged according to V. staining was obtained with these controls, even in compact Hamburger and H. L. Hamilton (8). The generation of the tissues. Positive controls included several antibodies specific cell-death-specific mAbs by neonatal tolerization (9) is de- for different chicken structures. Serial sections were coun- scribed in detail elsewhere (10). BV2 hybridoma was screened terstained by methylene blue-azure II and basic fuchsin (14). on sections of day 7 chicken hind limb foot plates, expanded, Sections were examined with a Zeiss epifluorescence Axio- and assayed several times for antibody reactivity and stabi- plan microscope with x20, x40, and xlO0 objectives and a lized by three cycles of cloning by limiting dilution. The filter set and photographed by using Kodak 1600 ASA film. immunoglobulin (IgM) type was determined by ELISA using Cell Cultures. Primary chicken cultures rat mAb anti-mouse immunoglobulin classes from Amersham. were freshly prepared for every experiment from 9-day-old BV2 hybridoma cells (2-5 x 106) were injected into 7- to embryos (15). Cells were plated out at different densities on culture slides with four chambers (Nunc) coated with type 1 The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviation: mAb, monoclonal antibody. in accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed. 8641 Downloaded by guest on September 28, 2021 8642 Cell Biology: Fernandez et al. Proc. Natl. Acad Sci. USA 91 (1994) collagen (Sigma, 500 Al of a 50 ,ug/ml solution for 8 hr) and adhering cells were harvested by trypsinization and com- were cultured in F-12 medium (GIBCO)/10% fetal bovine bined with floating cells present in the supernatant. After serum (HyClone)/2.5% chicken serum (Sigma), penicillin at centrifugation, the cell pellet was lysed in extraction buffer 100 units/ml (GIBCO)/streptomycin at 100 ,ug/ml (GIBCO) (100 mM NaCl/25 mM EDTA/10 mM Tris HCl, pH 8.0/0.5% at 370C in an atmosphere of 5% C02/95% air in a humidified SDS/proteinase K at 100 pg/ml for 3 hr at 55°C, extracted incubator. To mimic a state of cell anoxia, 1 mM KCN and twice with phenol/chloroform and once with chloroform; the 2 mM iodoacetate were added to the culture medium. Cell DNA was then ethanol-precipitated overnight at -80°C. The death was also induced with 10-6 M actinomycin D, and precipitated DNA was dissolved in 10 mM Tris, pH 8/1 mM inhibition of protein synthesis was done with cycloheximide EDTA containing RNase A at 10 pg/ml and analyzed on a at 50 Ag/mI (all chemicals from Sigma). Loss of cell viability 1.7% agarose gel. Electrophoresis was done for 3 hr at 140 V, was determined by failure to exclude Trypan blue and by and the DNA was visualized by staining with ethidium measuring the leakage of L-lactate dehydrogenase (EC bromide. DNA from untreated chicken embryonic fibroblasts 1.1.1.27) by Sigma procedure no. 500. For antibody staining, was extracted in parallel and served as a control. cell cultures were rinsed in cold PBS, fixed in freshly prepared 4% periodate/lysine/paraformaldehyde in PBS for 15 min at room temperature, and then rinsed twice in cold RESULTS PBS for 5 min. Cells were permeabilized by treatment with Isolation of the mAb. The mAb BV2 was isolated, after 0.2% Triton X-100 in PBS at room temperature for 2 min (to neonatal tolerization, by immunization of mice with embry- preserve the cytoskeleton structure) to allow penetration of onic day 7 (stage 31) chicken foot plates. At this stage, antibodies into the cells and eliminate the danger of further developmental programmed cell death is easily identifiable in osmotic damage. Cell culture slides were then rinsed with the interdigital mesenchymal areas of foot plates (Fig. 1A). PBS, and immunofluorescence staining was done as de- Interdigital programmed cell death, a major process during scribed. Actin filaments were stained with tetramethyl- the morphogenesis of digits in all nonwebbed amniotes, rhodamine B isothiocyanate-phalloidin (10-7 M, Sigma). including the human, plays an important role in the separa- Rhodamine-conjugated wheat germ agglutinin (10 jug/ml, tion ofthe chondrifying digits (17, 18). To selectively enhance Sigma) was used as a marker for the Golgi apparatus because the production of antibodies specific for dying cells, the of its high affinity for the terminal glucosamine residues mouse immune response to living-cell antigens was sup- present in incompletely glycosylated proteins (16). pressed by injecting newborn mice with day 10 chicken foot DNA Fragmentation Assay. After treating primary chicken plate tissues (with virtually no cell death) before immuniza- embryonic fibroblasts for 24 hr (1 x 107 cells per 10-cm dish) tion. Potential cell-death-specific antibodies were screened with actinomycin D (2.5 x 10-7 M final concentration), by using indirect immunofluorescence staining on sections of

FIG. 1. Immunofluorescence staining of BV2 mAb on 5-,um glycol methacrylate sections of the chicken embryonic hind limb foot plates. (A) Structure of the hind limb foot plate at stage 31-32 (day 7) stained with methylene blue-azure II and basic fuchsin. Mesenchymal apoptotic dying cells are stained dark blue in the interdigital areas. (B-I) Double-staining immunofluorescence pictures: (B, D, F, H) Ho 33258 staining ofthe nuclear chromatin; (C, E, G, I) BV2 mAb staining ofcorresponding Ho 33258 dye staining ofB, D, F, and H, respectively. (B) Enlargement of the interdigital area of the hind limb foot plate shown in A. Nuclei of dying cells appear as shining condensed and fragmented dots and are not present in the digit (d). (C) BV2 mAb staining on the same section as in B. (D and E) Detection of BV2 mAb staining along the distal edge ofthe ofthe at stage 23-25 (day 4). (F and G) BV2 staining ofthe anterior necrotic zone ofthe chicken embryo hind limb at stage 23-25 (day 4). (H and I) Enlargement of the death area shown in F and G with apoptotic bodies with condensed shining chromatin (H) and the corresponding staining with BV2 mAb (I). Some degenerating cells lie among normal cells, but the majority are aggregated in the cytoplasm ofan engulfing cell (2, 17). Some ofthe dead cells in Ho 33258 dye staining and BV2 mAb staining are marked with small arrows. BV2 mAb staining is not detected in living cells with normal nucleus; nor is it detected in mitotic figures (m). [Bar = 445 pm (A), 220 Pm (B and C), 56 ,um (D-G), and 26 am (H and I).] Downloaded by guest on September 28, 2021 Cell Biology: Femandez et al. Proc. Natl. Acad. Sci. USA 91 (1994) 8643 day 7 chicken foot plates. Cells undergoing programmed cell mesoderm (17). Similar staining could be detected in many death show typical nuclear condensation and fragmentation, structures derived from both mesodermal and endodermal which can be revealed by costaining with Ho 33258 dye (Fig. origin-for example, in the tail bud (Fig. 2 A and B), the 1B and H). BV2 mAb was one ofseveral mAbs isolated in this and collecting ducts (Fig. 2 C and D), the gut way that showed specific staining of dying cells with apo- (Fig. 2 G-I), the ofthe mandible, the wing bud, ptotic morphology (Fig. 1 C and I). around vessels, and in the heart (data not shown). Cell death Speciflcity of the Antibody in Vivo. To determine the was intense in the mesenchymal regions undergoing active specificity of BV2 mAb, we stained glycol methacrylate proliferation and migration, such as the sclerotomes ofday 4 sections ofday 3 and 4 chicken embryo and ofday 15 chicken embryos (Fig. 2 A and B, as well as J-L). In all these areas, retina with BV2 mAb and Ho 33258 dye by using immuno- the cellular fragments of dying cells stained by the antibody, fluorescence. The staining of BV2 mAb in the developing were generally aggregated (Fig. 1 H and I), most likely in the embryo was evident in many different areas of cell death cytoplasm of an engulfing cell (2, 17). Cell death is also a previously mapped by embryologists by vital staining in ovo major morphogenetic feature in neurogenesis (19). BV2 an- (17,18). In the hind limb bud, BV2 mAb staining was detected tigen could be detected in the , in cells with not only in the mesenchymal interdigital areas at stages 31-32 pyknotic nuclear morphology revealed by Ho 33258 staining. (day 7) (Fig. 1 B and C) but also at stages 23-25 (day 4) in the Fig. 2 (E and F) shows the staining in the bipolar layer ofthe anterior necrotic zone (Fig. 1 F-I) in the centrally located embryonic retina at day 4. BV2 mAb was then tested on opaque patch (Fig. 2 A and B) and in the posterior necrotic frozen sections ofdifferent avian species and showed intense zone (data not shown). These mesodermal areas ofcell death cross-reactivity in zebra-finch embryos and similar patterns are probably involved in the regulation of the limb size by ofexpression in different embryonic tissues (data not shown). controlling the amount of distal mesenchyme available for All areas ofpyknosis detected in the embryos with Ho 33258 digit formation and in skeletal patterning, such as the initial dye on serial sections of 5 ,m were stained with the mAb, separation of tibia and fibula (18). Another area of cell death without exception. No staining was detected in living cells or in the limb that was stained with the antibody was along the in mitotic figures (Fig. 1 H and I). Taken together, these edge of the apical ectodermal ridge (Fig. 1 D and E), a results indicate that cells undergoing programmed cell death thickening of the of the distal end of the limb bud in avian embryonic tissue, derived from the three embryonic that is known to induce the outgrowth of the underlying germ layers, actively express at least one similar antigen.

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FIG. 2. Detection of BV2 mAb staining in different tissues of the chicken embryo derived from all three embryonic germ layers. Pictures are from 5-,um glycol methacrylate parasaggital sections ofstage 23 to 25 (day 4) embryos, stained with methylene blue-azure II and basic fuchsin (G, J) or by double immunofluorescence staining with chromatin dye Ho 33258 (blue) and BV2 mAb (green). (A and B) General view of the posterior part of a chicken embryo showing the extension of cell death during . All areas of pyknosis detected by the dye are stained with the mAb. Cell death is intense in the somitic derived mesoderm (s) and in the tail bud (t). An important area of cell death at the base of the primitive limb bud (I) is probably related to the centrally located opaque patch (k, mesonephros). (C and D) Double staining of intensive cell death with BV2 mAb and Ho 33258 in mesonephric ducts (d) (t, ). (E and F) Double staining of BV2 mAb and Ho 33258 of a section of neuroectoderm: BV2 staining in embryonic retina (r), at the level of the optic nerve (on). In the nervous tissue, the pyknotic bodies stained with the dye or the mAb are more dispersed than in the mesodermal-derived tissues (see for example, Fig. 1 F and G). (G-I) Histological staining (G), Ho 33258 (H), and immunofluorescence of BV2 mAb (I) of a section of : dying cells in the of the hind gut endoderm (g) are evident. (n, ; nt, ; v, blood vessels). (J-L) Histological staining (J), Ho 33258 staining (K), and immunofluorescence of BV2 mAb (L) of cell-death areas in the sclerotome (s). (dm, Dermomyotome; 1, limb; dorsal is up.) [Bar = 220 Am (A, B, G, and J), 130 ,um (E and F), and 110 ,um (C, D, H, I, K, and L).] Downloaded by guest on September 28, 2021 8644 Cell Biology: Fernandez et al. Proc. Natl. Acad. Sci. USA 91 (1994) Detection of BV2 mAb Durng Celi Death in Vitro. To study Detection of BV2 mAb During Necrosis in Vitro. The other possible conditions that specifically induce the expression of cell death condition we tested was chemical anoxia induced the antigen recognized by BV2 mAb, we analyzed chicken by potassium cyanide and iodoacetate, which serve as typical embryonic fibroblasts undergoing cell death in vitro. Nuclear examples of necrotic stimuli (21-23). Upon addition of these and cytoplasmic morphological changes were followed by metabolic inhibitors of respiration and glycolysis, chicken staining the chromatin with Ho 33258 dye and staining the fibroblasts grown on collagen swell and detach in <3 hr actin filaments with phalloidin. In several situations in vivo or without nuclear condensation (Fig. 3 D and E), as described in vitro, apoptosis can be suppressed or postponed by inhib- for necrosis (2, 21). The antigen recognized by the BV2 mAb itors of RNA or protein synthesis, suggesting that cell death was dramatically induced in dying fibroblasts =20 min after is an active process requiring new gene expression. In other the induction of necrosis (Fig. 3 F and K). Removal of examples, however, cells undergo apoptosis in the presence cyanide and iodoacetate after 20 min did not rescue the dying of protein or RNA synthesis inhibitors (20). In the latter cells, nor did it suppress expression ofthe antigen recognized instances, presumably, proteins that suppress cell death need by BV2 mAb. BV2 staining of necrotic cells was in certain to be constantly synthesized while cell-death machinery is localized regions of cytoplasm, in contrast to the staining in cell death induced by actinomycin D. Rhodamine-conjugated already in place. We induced cell death ofchicken embryonic wheat germ aggiutinin was used as a marker for the Golgi fibroblasts in vitro by addition ofactinomycin D, an inhibitor apparatus because of its high affinity for the terminal glucos- ofRNA polymerase and a cancer chemotherapeutic agent (3). amine residues present in incompletely glycosylated proteins Nuclear and cytoplasmic condensation of the cells in vitro (16). Analysis of the BV2 mAb staining in necrotic cells, by can be observed 2 to 3 hr after treatment (Fig. 3 G and H). costaining with the lectin, suggested that the antigen recog- Genomic DNA isolated from such cells showed typical DNA nized by BV2 mAb may be localized in the Golgi apparatus fragmentation patterns (data not shown). BV2 mAb staining (Fig. 3 J-L). Pretreatment ofthe cultures with cycloheximide was detected in dying cells 6-8 hr after drug addition (Fig. (50 ptg/ml for 2 hr), an inhibitor of protein synthesis, com- 31). No staining was detected in control living fibroblasts pletely suppressed BV2 staining, even though cells still (Fig. 3C). Thus, the antigen recognized by BV2 mAb is detach and die as untreated necrotic fibroblasts (data not expressed in dying cells in vivo and in vitro. shown). The brief treatment of cells with cycloheximide did

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FIG. 3. Correlation between cell death in vitro induced by actinomycin D and necrosis using BV2 mAb staining. All views are triple immunofluorescence staining pictures of chicken embryonic fibroblasts grown on collagen and induced to die by drug treatment. Ho 33258 staining of the chromatin is shown in blue; BV2 mAb staining is shown in green. Phalloidin staining of actin filaments (B, E, and H) or wheat germ agglutinin staining of the Golgi apparatus (L) is shown in red. (A-C) Control cells. BV2 staining (C) was not detectable on untreated fibroblasts. (D-F) Necrosis. Fibroblasts were induced to die by chemical anoxia after exposure to 1 mM potassium cyanide and 2 mM iodoacetate. BV2 mAb staining is detected 20 min after induction of necrosis (F). (G-I) Fibroblasts were induced to die by exposure to 10-6 M actinomycin D. Nuclear (G) and cytoplasmic (H) condensation are seen 2-3 hr after drug addition. (I) BV2 mAb staining, detected after 6-8 hr. (J-L) Localization of BV2 antigen in the Golgi apparatus (K) during necrosis by costaining with wheat germ lectin (L). [Bar = 70 pm (A-C and G-I), 56 ,m (D-F), and 28 pLm (J-L).] Downloaded by guest on September 28, 2021 Cell Biology: Femandez et al. Proc. Natl. Acad. Sci. USA 91 (1994) 8645 not affect the viability of the control chicken fibroblasts. 1. Ellis, R. E., Yuan, J. & Horvitz, H. R. (1991) Annu. Rev. Cell Thus, the expression of the antigen recognized by BV2 mAb Biol. 7, 663-698. in cells undergoing necrosis appears to require protein syn- 2. Wyllie, A. H., Kerr, J. F. R. & Currie, A. R. (1980) Int. Rev. thesis, rather than unmasking of preexisting determinant, Cytol. 68, 251-306. although the determinant itself could be a carbohydrate or 3. Searle, J., Kerr, J. F. R. & Bishop, C. J. (1982) Pathol. Annu. any other protein 17, 229-259. modification. 4. Buckley, I. K. (1972) Lab. Invest. 26, 201-209. 5. Kloner, R. A., Ganote, C. E., Whalen, D. A., Jr., & Jennings, DISCUSSION R. B. (1974) Am. J. Pathol. 74, 399-422. 6. Prieto, A., Kornblith, P. L. & Pollen, D. A. (1967) Science 157, The molecular mechanisms of cell death in vertebrates are 1185-1187. still largely not understood. In the nematode Caenorhabditis 7. Batistatou, A. & Greene, L. A. (1993) J. Cell Biol. 122, elegans, a genetic pathway of programmed cell death has 523-532. been described. Eleven genes identified in this pathway affect 8. Hamburger, V. & Hamilton, H. L. (1951) J. Morphol. 88, all cells that undergo programmed cell death during C. 49-92. elegans development (1), suggesting that all cells undergoing 9. Hockfield, S. (1987) Science 237, 67-70. programmed 10. Rotello, R. J., Fernandez, P.-A. & Yuan, J. (1994) Develop- cell death share a common mechanism. Many ment (Cambridge, U.K.) 120, 1421-1431. cells die at predicted times during vertebrate development, 11. Van der Velde, S. (1980) Am. J. Clin. Pathol. 73, 119-121. but it is not clear how many different mechanisms have 12. Carnegie, J. A., McCully, M. E. & Robertson, H. A. (1980) J. evolved to execute cell death in vertebrates. Expression of Histochem. Cytochem. 28, 308-310. the antigen recognized by BV2 mAb is specifically detected 13. Neidle, S., Pearl, L. H. & Skelly, J. V. (1987) Biochem. J. 243, on apoptotic cells in all the areas of cell death in developing 1-13. chicken embryos and zebra-finch embryos so far examined 14. Humphrey, C. D. & Pittman, F. E. (1974) Stain Technol. 42, by us, suggesting that during vertebrate development, pro- 9-14. grammed cell death in tissues derived from all embryonic 15. Hunter, T. (1970) Methods Enzymol. 58, 379-393. 16. Virtanen, I., Ekblom, P. & Laurila, P. (1978) J. Cell Biol. 85, germ layers may share a common mechanism as well. Ex- 429-434. pression of several genes has been associated with experi- 17. Saunders, J. W., Jr., & Fallon, J. F. (1967) in Major Problems mentally induced apoptosis or developmental cell death, but in , ed. Locke, M. (Academic, New the expression of all those genes have been detected in living York), pp. 289-314. cells as well (24-31). In contrast, based on our developmental 18. Hinchliffe, J. R. (1981) in CellDeath in Biology andPathology, studies, using immunofluorescence techniques, BV2 mAb eds. Bowen, I. D. & Lockshin, R. A. (Chapman & Hall, appears as a different specific marker of apoptotic cells London), pp. 35-78. during avian development. Although it is unlikely that the 19. Oppenheim, R. W. (1991) Annu. Rev. Neurosci. 14, 453-501. antigen recognized by BV2 mAb is directly involved in 20. Martin, S. J. (1993) Trends Cell Biol. 3, 141-144. controlling cell death, because the inhibition ofits expression 21. Trump, B. F., Berezesky, I. K. & Osornio-Vargas, A. R. (1981) in Cell Death in Biology and Pathology, eds. Bowen, during necrosis by cycloheximide does not block cell death, I. D. & Lockshin, R. A. (Chapman & Hall, London), pp. the identification of a specific marker of cell death will allow 209-242. us to characterize cell death under different conditions. 22. Nicotera, P., Thor, H. & Orrenius, S. (1989) FASEB J. 3, The unexpected result that the expression of the antigen 59-64. recognized by BV2 mAb is also induced during cell death 23. Laiho, K. U. & Trump, B. F. (1975) Lab. Invest. 32, 163-182. caused by chemical anoxia and its dependency upon protein 24. Sawczuk, I. S., Hoke, G., Olsson, C. A., Connor, J. & synthesis suggests that these types of necrosis and apoptosis Buttyan, R. (1989) Kidney Int. 35, 1315-1319. may share certain common biochemical elements. The endog- 25. Schwartz, L. M., Myer, A., Kosz, L., Engelstein, M. & Maler, enous machinery of apoptosis may also be activated and C. (1990) Neuron 5, 411-419. 26. Rennie, P. S., Bruchovsky, N., Buttyan, R., Benson, M. & contribute to cell death under certain necrotic conditions. Cheng, H. (1988) Cancer Res. 48, 6309-6312. Overexpression of the bcl-2 gene has been found to not only 27. Bandyk, M. G., Sawczuk, I. S., Olsson, C. A., Katz, A. E. & inhibit cell death induced by serum or growth factor depriva- Buttyan, R. (1990) J. Urol. 143, 407-413. tion but also protect certain vertebrate cells from sodium 28. Briehl, M. M. & Miesfeld, R. L. (1991) Mol. Endocrinol. 5, azide, glucocorticoids, phorbol ester, methotrexate, heat 1381-1388. shock, ethanol, radiation, antimetabolite 1-#-D-arabinofura- 29. Harrigan, M. T., Baughman, G., Campbell, N. F. & Bour- nosylcytosine, calcium ionophore, vincristine, glucose with- geois, S. (1989) Mol. Cell. Biol. 9, 3438-3446. drawal, membrane peroxidation, free radical-induced damage, 30. Owens, G. P., Hahn, W. E. & Cohen, J. J. (1991) Mol. Cell. and tumor necrosis factor-mediated cytotoxicity (32-39). Biol. 11, 4177-4188. 31. Fesus, I., Thomazy, V. & Falus, A. (1987) FEBS Lett. 224, Thus, some ofthe apparent differences between apoptosis and 104-108. necrosis on the basis of morphological criteria may not be 32. Strasser, A., Harris, A. W. & Cory, S. (1991) Cell 67, 889-899. reflected entirely on a mechanistic level, and both pathways 33. Zhong, L.-T., Sarafian, T., Kane, D. J., Charles, A. C., Mah, may share certain common molecular players. If so, our S. P., Edwards, R. H. & Bredesen, D. E. (1993) Proc. Nati. findings suggest that one should look at traditional necrotic cell Acad. Sci. USA 90, 4533-4537. death, such as cell death during ischemic diseases ofthe heart, 34. Hennet, T., Bertoni, G., Richter, C. & Peterhans, E. (1993) kidney, and brain, the primary causes of mortality and mor- Cancer Res. 53, 1456-1460. bidity in industrialized nations (40), in a different way. 35. Henderson, S., Rowe, M., Gregory, C., Croom-Carter, D., Wang, F., Longnecker, R., Kieff, E. & Rickinson, A. (1991) P.-A.F. and R.R. contributed equally to this paper. We are grateful Cell 65, 1107-1115. to S. Hockflield for many valuable insights on neonatal tolerization and 36. Miyashita, T. & Reed, J. C. (1992) CancerRes. 52, 5407-5411. generation of mAbs, to E. Harlow for suggestion on generation of 37. Sentman, C. L., Shutter, J. R., Hockenbery, D., Kanagawa, mAbs, and to H. R. Horvitz for helpful comments during manuscript 0. & Korsmeyer, S. J. (1991) Cell 67, 879-888. preparation. We thank V. Gagliardini, C. Tuffereau, C. Fankhauser, 38. Alnemri, E. S., Fernandes, T. F., Haldar, S., Croce, C. M. & M. Igarashi, M. Miura, M. Pack, and C. Simpson for stimulating Litwack, G. (1992) Cancer Res. 52, 491-495. discussions. J.Y. was supported by a grant from the National Institute 39. Tsujimoto, Y. (1989) Oncogene 4, 1331-1336. of Aging (AG11017) and a grant from Bristol-Myers/Squibb. P.-A.F. 40. Cheung, J. Y., Bonventre, J. V., Malis, C. D. & Leaf, A. L. was supported by a fellowship from Rh6ne-Poulenc. (1986) N. Engl. J. Med. 314, 1670-1676. Downloaded by guest on September 28, 2021