JOURNAL OF CELLULAR PHYSIOLOGY 147516522 (1991)
Granular Presence of Terminin Is the Marker to Distinguish Between the Senescent and Quiescent States EUGENIA WANG* AND GEORGE TOMASZEWSKI The 6loomfield Center for Research in Aging, lady Davis institute for Medical Research, Sir Mortimer 6. Davis-Jewish General Hospital, and Departments of Medicine and Anatomy, McCill University, Montreal, Quebec H3 T 7 €2 We have previously identified statin, a nonproliferating-cell-specific nuclear protein of 57,000 dalton whose presence can be used to distinguish between growing and nongrowing cells. In this report we identify another protein, terminin, whose presence (by immunofluorescence microscopy) can be used to distinguish between temporarily and permanently growth-arrested cells. Thus terminin is a marker to separate the senescent from the quiescent state. By means of an unique monoclonal antibody (mAbl.2), the presence of terminin is recognized as granules in the cytoplasm of in vitro aged fibroblasts; these granules are not found in serum-starved, contact-inhibited, growing, or transformed fibroblasts, except for those cells experiencing the initiation of apoptosis due to long-term deprivation of nutrients. Preliminary histochemical studies show that terminin is also found in the superficial epithelial layer of the esophagus, where terminal differentiation is followed by apoptosis and sloughing off into the lumen. Biochemical characterization by Western blot shows the terminin antibody recognizing a protein of 84 kilodalton (kDa) in growing and quiescent cells, whereas in senescent cells a protein of 57 kDa is recognized; this result suggests that a senescence-dependent protease may cleave the 84 kDa proiein to 57 kDa. This proteolytic action seems to render the specific antigenic epitope exposed in its native state and accessible to the terminin antibody by immunofluorescence microscopy. It is this product o i posttranslational modification in the form of a cytoplasmic 57 kDa protein that is the marker distinguishing between senescence and quiescence.
Normal human fibroblasts grown in a laboratory dish exhibit a limited lifespan for proliferation (Swim and Parker, 1957; Hayflick and Moorhead, 1961). This principle restricting cells from further replication has been described as cellular or in vitro aging (Hayflick, 1965; Schneider and Mitsu, 1976).We and others have proposed that the aging process may not be simply the result of cumulative random deterioration; rather, a genetic program may be the underlying mechanism that makes cells become aged. Investigation along this line has led to the finding that senescence is a dominant phenoty e (Rabinovitch and Norwood, 1980; Matsumura et. a!, 1980; Pereira-Smith and Smith 1981; Stein and Yanishevsky, 1981), and that “senescencespecific gene expressions” may exist and be in direct control of the cessation of DNA synthesis and establishment of the associated in vitro aged phenotype of normal human fibroblasts (Lumpkin et. al., 1986). Our attempt to identify senescence-specificgene expression has resulted in the discovery of a novel protein, statin, associated with the nuclei of almost all nonproliferating cells (Wang, 1985a,b, 1987; Wang and Krueger, 1985; Wang and Lin, 1986). Unfortunately, statin cannot be deemed strictly senescence-specific;rather, it proves to be nonproliferation-specific, found in both 0 1991 WILEY-LISS, INC
senescent and temporarily growth-arrested quiescent cells (Wang, 1989). Obviously, whereas senescent and young quiescent fibroblasts may share many common features, including the most evident lack of DNA synthesis, the two cell ty es certainly demonstrate marked differences; speci ically, the old cells are in the state of irreversible growth-arrest. Except in the rare cases of transformation by oncogenic viruses, few workers have succeeded in extending the lifespan of senescent fibroblasts beyond one or two rounds of DNA synthesis (PerciraSmith et. al., 1985; Phillips et. al., 1987; Wang, unpublished results). Another major difference is the finding that young growth-arrested cells seem to be in the GO phase, whereas senescent cells are thought to be stalled in late G1 phase, shortly before entry to S phase (Rittling et al., 1986; Wang, 1989b). Results from the ongoing attempt at distinguishing senescent from quiescent fibroblasts are not yet adequate to provide a unifying clue to the underlying mechanisms contributing to the determination of these
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Received January 3, 1991; accepted February 25, 1991. *To whom reprint requestshhould be addressed.
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two distinct cell types; a major handicap is the lack of identified unique gene expression specific for senescence and absent from either young growing or temporarily growth-arrested cells. In this report we describe the identification of a cytoplasmic protein, terminin, whose unique presence in senescent fibroblasts can be used as a marker for in vitro aged cells and thus provide an initial lead in the investigation of “what makes cells irreversibly lose their growth capacity.” MATERIALS AND METHODS Cell systems Two specific cell strains, GMOOll (derived from a donor of 8 fetal weeks of age) and GM3529 (from a 66-year-old donor) were used in this study. The longitudinal assessment of in vitro lifespan for these two cell strains was performed by a rigid schedule of long-term serial passage of the monolayer cultures (Cristofalo and Charpentier, 1981); their in vitro life history has been published (Wang and Gundersen, 1984). A human glioma cell strain (U-251-4) was used for the transformed culture study. Both the fibroblasts and glioma cells are generally cultured in Eagle’s minimal essential medium supplemented with 10% fetal bovine serum, penicillin, and streptomycin; a humid atmosphere with standardized admixture of 5% CO, is maintained in an ambient environment of 37°C for culture conditions. Assay of proliferative activity Cultures of young and senescent fibroblasts in either sparse or confluent state, with and without serum, were analyzed for proliferative activity by measuring the index of DNA synthesis; the standard procedure (as reported previously) for assaying incorporation of 3Hthymidine was performed for most cultures used in the present study. Fibroblast cultures were determined to be senescent according to previously defined protocols: after serial passaging to reach the maximal population doubling level (PDL = 58 for the 0011cell strain, 28 for 3529), cultures were observed to have no mitotic activity for 14 days, and thymidine incorporation was at the background level. The nongrowing state of young fibroblasts (cumulative PDL less than 12 for the 0011 cell strain, less than 10 for 3529) was established by culturing either at confluency (with cell density of 35,550/cm2)or at low (0.05%) serum concentration for 72 hours; again, DNA synthesis as measured by thymidine incorporation was at background level. Production of monoclonal antibody (mAbl.2)to terminin Most cellular stage-specific proteins are of very small quantity; thus their detection can be masked by the presence of a lar e number of housekeeping proteins. Therefore, identi ication of specific novel proteins that are developmental or proliferation-dependent in their expression requires some amount of modification. Our aim in identifying senescence-specific but not quiescence-dependent antigens is the implementation in the standardized procedure of monoclonal antibody production of the principle of immunosuppression of antigenic reaction to the common “housekeeping” antigens. Once
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suppression to the common proteins is established, the immune system is then challenged again with another type of cell; manipulation following this regimen of immunization produces the simple subtraction of the highly abundant common antigens, thus allowing the increased probability of producing antibodies to unique stage-dependent gene roducts. The injection protoco consisted of an initial injection of detergent-insoluble extracts of confluent cultures of young fibroblasts (36,660 cells/cm2, maintained for at least 1week with basal level of 3H-thymidine incorporation); a pellet of cell specimen in 500 pl wet volume injected into two mice, accompanied by intraperitoneal injection of cyclophosphamide, 100 pgig of body wei ht in sterile phosphate-buffered saline. The cyclophospflamide kills the antibody-producing cells, whereas the elicited pattern of immunogenic reaction is retained in the memory of the animal’s immune system. A 40% weight loss indicates the effectiveness of this dose, but not the completeness of the induced immunotolerance; this is evaluated by characterizing the serum antibody reaction to the first antigen injection, on day 13 after the initial injection. After the mice recovered from the manipulation of the first immunization, and regained their physical health and weight, they were immunized with extracts of senescent cells prepared similarly by detergent extraction, in the same dosage; 2 weeks later, a similar booster injection was used. A simple criterion was designed to identify hybridoma clones of interest: negative antibody staining reaction in both young growing and young nongrowing cells, but positive reactivity in their senescent counterparts. Screening of antibody activity was performed by direct microscopic visualization of indirect immunofluorescence activity on appropriate cell specimens; monoclonal antibody mAbl.2 fit the above criteria. The mAbl.2 hybridoma has been cloned to single-cell stage through serial dilution and soft agar colony selection; the antibody is of the IgG type, and the ascites form of the antibody demonstrates a high titre (1:100,000 for immunofluorescence assays, 1:5,000 for immunoblotting procedures). For the sake of ease of terminology, we call the putative protein reacting with mAbl.2 terminin t o denote the nature of finality in the cellular aging process in which it was identified. Immunofluorescence microscopy For both purposes of antibody screening and cellular localization, fibroblasts of predetermined growth properties were cultured on glass substrate and processed for indirect staining as described previously (Wang, 1985a,b). In brief, the cells were fixed with methanol and acetone (1:l) at -20°C for 10 minutes; after air drying, the specimens were rehydrated with phosphatebuffered saline (PBS) at pH 7.2, and incubated with monoclonal antibody 1.2 overnight at room temperature. Afterward, the primary antibody was removed by repeated PBS rinses and fluorescein-conjugated goat antimouse immunoglobulin was applied and incubated for 30 minutes at room temperature, followed by PBS rinsing and mounting in glycerol-containing medium for epiillumination examination of positive reactions in a Nikon labophot microscope.
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Histochemical studies To identify terminin presence in tissues, frozen sections of 8 to 10 km were obtained on a Reichert cryomicrotome. Tissues of interest were excised from 3-month-old Sprague-Dawley rats, and processed for immunocytochemistry by immediate immersion in liquid nitrogen. Thick sections of 20 km were used initially for staining with standard haematoxylin dye to evaluate tissue orientation and morpholo cal preservation; when these criteria were satisfie , serial sections of chosen tissue blocks were placed on gelatincoated microscope slides and processed for fixation and incubation with antibodies as described above for cultured fibroblasts. Preparation of cell samples for SDS gel electrophoresis a n d immunoblotting reaction Cultures of young cells (3529 cell strain, CPDL = 10) were separated into two groups to establish (1.)growin cultures by maintaining them in sparse state (3.5 x 105 cells/cm2)in 10%fetal bovine serum, and (2.) nongrowing cultures by maintaining them at the same density in 0.05% serum for 3 days. The senescent counterparts of these cultures were obtained by serial passaging to reach CPDL = 28. These three cultures were then processed for extraction, gel electrophoresis, and immunoblotting reaction as described previously (Wang and Lin, 1986).The terminin-specific monoclonal antibody was produced as described above. Ascites fluid of a hybridoma cell line secreting no specific antibody was used as a control antibody for the immune reaction described here. RESULTS Figure 1 demonstrates the t ical pattern of terminin staining in senescent fibro lasts. Comparison of the phase contrast and fluorescence images of the same cells reveals the dense granular structures stained by the terminin antibody mAbl.2; in some instances, it appears that these granular structures fill the interstices of the cytoskeletal framework. The senescencespecific nature of terminin is illustrated by Fig. 2, showing a confluent monolayer of young (CPDL less than 12) cell strain 0011 (Fig. 2a), and two separate intermediate states of the in vitro lifespan (CPDL less than 30, Fig. 2b, and CPDL = 48, Fig. 2c) and a senescent culture (CPDL = 58, Fig. 2d). To achieve the nonproliferating state, confluency was maintained for 1 week, and DNA synthesis activity as measured by 3H-thymidine incorporation was observed to be at the basal level. In these four cultures, cytoplasmic presence of terminin-containing granules were not present in the confluent cultures of young cells; however, terminin-positive granules were identified in a small number of cells in intermediate cultures, and the number of terminin-positive cells increased with in vitro age and reached 85% in senescent cultures. The unique presence of terminin-positive granules in senescent cells and not in quiescent young cells was also observed in growth-arrested conditions achieved by serum deprivation. As shown in Fig. 3a, terminin-positive granules were not detected in cultures of young cells maintained in the nonproliferating state by keeping the serum
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Fig. 1. Immunofluorescence microscopy of terminin distribution in the cytoplasm of senescent human fibroblasts. Phase contract (a) and fluorescence images (b) of the same cells show protein presence as dense granular structures occupying the interstices of the cytoskeletal network. Presence of the protein is clearly cytoplasmic and not associated with either the nucleus or the plasma membrane. Cultures of senescent human fibroblasts were obtained by serial passage according to the standard split ratio to reach the required cumulative population doubling levels (CPDL) for the in vitro aging process. The specific culture used here is at CPDL = 28; the original donor age of the cell strain (3529) was 66 years.
level at 0.05%. For the purpose of contrast, we have shown here the field of terminin-ne ative cells in the two intermediate states (Fig. 3b,c); owever, in these cultures a low level (15%) of cells show terminin granules in their cytoplasm. In similar culture conditions of low level serum, senescent cells show a high frequency (85%)of terminin-positive granules. The absence of terminin-positive granules was also observed in young growing cells of the same cultures used for Figures 2a and 3a, when the cells were kept in the sparse state or 10%serum allowed growth (Fig. 4a); a similar absence of terminin positivity was found in transformed cultures such as human glioma cells, as shown in Fi . 4b. The diffuse staining shown in Figures 3a a n 4a may suggest the presence of terminin in a nongranular form in these cells. The only unexpected instance of positive staining of terminin antibody in young cells was when cultures of an early passage (CPDL = 15) of the 0011 cell strain were left in the confluent state for 4 weeks without feeding (Fig. 51,
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Fig. 2. Immunofluorescence microscopy of the senescence-specific nature of terminin in cells of the 0011 strain a t cumulative population doubling levels (CPDL) of young (10, (a)), intermediate (24, (b)), presenescent (48, (c)),and senescent (58, (d)) stages; cultures with CPDL larger than 58 are considered aged. Cultures were allowed to grow to confluency and stay in this state for a week with no detectable activity of DNA synthesis. These cultures were then fixed and processed for staining with terminin antibody; for 3 days before fixation, no DNA synthesis was observed in any of the cultures used,
so that they are in the state of quiescence. Notice the absence of granular staining in young nongrowing cells (a); a small number of cells become positive for granular staining in the two cultures of intermediate life span (b and c). In contrast, a majority of cells show positive granular staining in the cytoplasm when cultures of senescent cells are reacted with terminin antibody (d). However, a small percentage of cells (10%) in senescent cultures remains negative for terminin, as repeatedly reported in the literature of a minority of nonaging phenotypes.
whereas all cells were observed positive for statin, approximately 50% of the cultures were observed to contain terminin-positive granules. For histochemical studies of terminin presence in tissues, we chose to focus on two systems: esophageal e ithelium and liver. The former is a well-defined type o stratified epithelium composed of three distinct stages of differentiation: the basal layer of stem cells, the suprabasal layer of transient amplifying cells, and the superficial layer of terminally differentiated cells; active proliferation is mostly associated with the basal cells. Although both suprabasal and superficial cells show a positive reaction to statin antibody and lack DNA synthesis, these two types differ in many other ways; clearly the cornified superficial cells represent the final stage of esophageal epithelium before detaching and being sloughed off into the lumen of the esophagus. Figure 6 shows the nonnuclear cytoplasmic presence of terminin in the superficial layer of esophageal epithelium, whereas the protein is absent from cells in the suprabasal and basal layers of the esophagus; although the staining looks like a punctate mem-
branous pattern, it nevertheless is confirmed to be cytoplasmic granular staining. The absence of terminin found in the liver of 3-month-old rats may be due to the fact that the liver is composed of a mostly homogeneous population of hepatocytes possessing high regenerative potential (Fig. 7). Biochemical characterization of the protein identity of terminin was performed by immunoblotting experiments with extracts of young growing, young nongrowing and senescent cells, such as those specimens used for Figures 1, 3, and 4.Senescent cultures and young cells grown in 10% and 0.05% serum were harvested and extracted as described in Materials and Methods. Equal amounts of protein from specimens of the three cultures were loaded onto 10%SDS polyacrylamide gel, and the electrophoretically separated proteins were transferred onto nitrocellulose (NC) papers, which were then reacted with terminin monoclonal antibody. The results show the reactivity of mAb 1.2 with a band at 84 kDa in cell extracts from young growing and young nongrowing cells (Fig. 8A,B,C); however, the same antibody reacts with a protein at 57 kDa in extracts of
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Fig. 3. Immunofluorescence microscopy of the senescence-specific nature of terminin. The cells used here in (a-d) are in the same cumulative population doubling levels (CPDL) as those seen in Figure 2, i.e., (a) young at (CPDL = 10);(b) intermediate (CPDL = 24); (c) presenescent (CPDL) = 48; and (d) senescent (CPDL = 58). However, the quiescence in cultures used here was reached by serum
senescent cells (Fig. 8D,E). No detectable bands were identified when the NC filter bearing the same types of extracts was reacted with a control monoclonal antibody produced from hybridoma cells bearing no specific antibody-producing capability (data not shown).
starvation. Cultures were maintained in medium containing 0.05% serum for 4 days, and DNA synthesis was not observed for at least 48 hours before fixation and processing for immunofluorescence microscopy. Again, similar to that shown in Figure 2, the granular pattern of terminin staining was not observed in the majority of cells until senescence was reached.
gest that the cytoplasmic staining seen by immunofluorescence microscopy is due to a possible posttranslational modification to 57 kDa from a preexisting 84 kDa protein. It is this modifying process, possibly proteolytic cleavage, which allows the specific epitope to become accessible to react with the antibody. To be DISCUSSION more precise, it is rather the putative posttranslational The search for a unique marker for senescent fibro- modification process that is senescence-specific; the blasts has been a long-term pursuit for many investi- identification of the existence of terminin merely progators in the hope of obtaining a handle to study the vides us the means to characterize this unique cellular complex mechanism involved in the aging process of process. individual cells. Here, we report the result of our The production of monoclonal antibody 1.2 to termiendeavor by identifying the senescence-dependent nin involves the lengthy processes of antigen preparapresence of a cytoplasmic protein, terminin. Morpho- tion and immunosuppression. The 2 years of endeavour logically, the presence of terminin was found in cyto- involved in this study prove the possibility of identifyplasmic granules of senescent fibroblasts, and its ab- ing stage-specific, differentiation- or developmentsence was found in a wide variety of cells including dependent gene expression by the immunological apyoung growing and nongrowing and transformed cells. proach. The incorporation of the immunosuppression In tissues of esophageal epithelium, terminin is present scheme in the regimen of hybridoma production proin the cytoplasm of only those cells at the last stage of vides a logical methodology to produce antibodies tardifferentiation. The biochemical characterization of getting stage-specific proteins in minute quantity. The terminin identity, however, indicates antibody recog- subtraction of a large number of abundant housekeepnition of a protein at 84 kDa in young growing and ing proteins by the “self” recognition of an animal’s nongrowing cells, but a protein of 57 kDa in their immune system avoids the conventional procedure senescent counterparts. These results lead us to sug- described as a “hit-or-miss”approach. Recently, by this
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Fig. 4. Immunofluorescence microscopy of the absence of terninin positive granules in young growing cells (a) as well as transformed cultures (b). (a) shows the lack of terminin positive granule3 in a growing culture of human fibroblasts (3529 cell line a t CpDL = 10) kept in 10%serum-containing medium, (b) shows absence of terminin positive granules in transformed human glioma cells (U-251-4) also kept in 10% serum-containing medium.
approach of immunosuppression, we have produced another monoclonal antibody (S6) that recognizes a nuclear protein of 100 kDa present in all except senescent cells (data not shown). The major difference between the techniques of immunoblotting vs. immunofluorescence reaction is that the former procedure recognizes the denatured protein, including both soluble and insoluble forms, whereas the latter recognizes native protein in the insoluble cellular scaffold. Our current working model for termi-
Fig. 5 . Immunofluorescence microscopy of statin presence in quiescent cells and additional terminin presence in the same type of culture maintained long term without feeding. Cells used here were of young cells of 3529 cell strain at CPDL of 10. (a) and (b) are phase contrast and fluorescence images of'the same culture of young fibroblasts kept in confluent quiescent state for a week. At this time, as shown in Figures 2 and 3, terminin was not detected. However, if the cultures are kept at this state for 4 weeks without feeding new medium, the cells then become terminin positive with approximately 508 of cells showing granular structure after staining with the antibody (c).
nin presence is as follows: in young cells, the extra protein mass of 24 kDa may mask the epitope in the in situ state, thus preventing recognition by the antibody during the fluorescence staining reaction. Processing of the protein to 57 kDa may cause the rearran ement of protein conformation or association with ce lular organelles in a way allowing the antigenic sites to become
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Fig. 6. Immunofluorescence microscopy of terminin staining reaction seen in the superficial layer (in area indicated by the arrows) of 3-month-old rat esophageal epithelium. Phase contrast (A) and fluorescence (B) show the same area indicating the histological morphology as well as the location of the terminin positive layer. The strong fluorescence in the area below the basal cell layer is due to non-specific reaction of the antibody, as supported by the same staining intensity of a control antibody.
accessible to react with the antibody. Alternatively, the 84 kDa protein may be in the soluble portion of young cells, which is extracted with detergent during the preparation for immunofluorescence microscopy, and therefore lost from the cell specimen. In senescent cells, the change from 84 to 57 kDa may make the protein detergent-insoluble, thus remaining associated with the cell specimens and readily detected by the antibody. Future investigation of the biochemical relationship between the two forms (84 and 57 kDa) of the protein should provide us some answers to these hypotheses. The 57 kDa protein described here is not the same polypeptide reacting with the statin S44 antibody; 57 kDa terminin is exclusively located in cytoplasmic
granules, whereas statin of the same size is found mostly in the nucleus. In addition, statin is found in both nongrowing young and senescent cells, while terminin is found only in senescent cells. Furthermore, terminin antibody 1.2 does not react with the immunoprecipitation complex that results from the reaction between statin antibody and senescent cell extracts (data not shown). Therefore, it is quite unlikely that the two 57 kDa polypeptides reacting with either the S44 or the 1.2 antibodies are the same protein. The distinct granular distribution of terminin in the cytoplasm of senescent cells poses a series of interesting questions: do the granules exist in young cells as well, but not associated with the protein? Are the granules of
TERMININ AS MARKER FOR SENESCENT STATE
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Fig. 7. Immunofluorescence microscopy of terminin absence in the liver of 3-month-old rat. The frozen sections of the liver were processed the same way as the esophageal tissue, and the identical staining conditions and antibody strength were used for reaction. However, little specific staining pattern was observed, indicating absence of terminin in liver tissue from rats of this age. a and b are phasecontrast and fluorescence images of the same field. The nuclei appear as black holes; the cytoplasm is faintly stained with the antibody, as also with the control antibody.
lysosomal nature? If so, are they the manifestation of a unique senescence-specific lysosomal process? Could the protein be of a secretory nature? Immunogold labelling in combination with ultrastructural studies may reveal the nature of these cellular organelles. Identification of the presence or absence of terminin in the culture medium of senescent cells may also help us to unravel the question of whether terminin can be secreted from cells. The fact that terminin positive staining is found in senescent fibroblasts, the superficial layer of squamous epithelial cells, and in cultures of prolonged period quiescence shows the usefulness of antibody in identifying a particular state of physiology following natural course of development or injury.
Fig. 8. Biochemical characterization of the electrophoretic mobility of terminin in extracts from young growing, young nongrowing, and old cells of the 3529 cell line. Same amount of proteins were loaded in ELD. A and E are the prestained molecular weight standards ranging from 116 to 49 kilodaltons. B is the extract from young growing 3529 cells CPDL = 10. C is the extract from the young confluent cultures of nongrowing 3529 a t CPDL = 10. D is the extract from aged cultures (CPDL = 28) of 3529 cells. After the cell extracts were run on the 10% polyacrylamide gel, they were transferred electrophoretically onto nitrocellulose paper and processed for reaction with terminin antibody. As shown here, the antibody reacts with a band at 84 kilodalton (arrow) in B and C, but with a band at 57 kilodalton(arrow) in D.
The presence of terminin antibody staining in senescent cells and terminally differentiated esophageal epithelium points to the fact that these cells are at the last cellular state before death takes place. From this point of view, terminin presence may be used as a marker for those cells predisposed to irreversible growth arrest leading to death. It is therefore not surprising to find the absence of terminin antibody staining in liver cells, which in general maintain a high degree of flexibility to shift back into the growth cycle. Similarly, in long-term unfed cultures of young cells, the appearance of terminin antibody staining may indicate the putative events of conversion from reversible to irreversible growth arrest, and many cells may experience accelerated senescence and the subsequent death processes. Nature has provided readily available systems such as the neuronal and thymus systems where developmentally regulated cell death is a necessary process. Investigation of terminin antibody staining in these systems will allow us to define whether indeed senescence or terminal differentiation is en route to apoptosis on programmed cell death.
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ACKNOWLEDGMENTS The authors express thanks to Ms. Lucia Badolato for typing this manuscript, to Mr. Michael Morcos for the preparation of photographic reproduction, and to Mr.
okaryon study of cellular senescence and the serum-deprived state. Exp. Cell Res. 13O:lOl-109. Rittling, S.R., Brooks, K.M., Cristofalo, V.J., and Baserga, R. (1986) Expression of cell cycle dependent genes in young and senescent WI-38 fibroblasts. Proc. Natl. Acad. Sci. U.S.A. 83:33163320. E.L., and Mitsu, J. (1976)The relationship between in vitro Alan Bloch for proofreading the manuscript. This work Schneider, cellular aging and in vivo human age. Proc. Natl. Acad. Sci. U.S.A. was supported by a grant (AGO92781from the National 73:3584-3588. Institute on Aging of the National Institutes of Health. Stein, G.H., and Yanishevsky, R.M. (1981) Quiescent human diploid cells an inhibit entry into S phase in replicative nuclei in heterodikaryons. Proc. Natl. Acad. Sci. U.S.A. 78:3025-3029. Swim, H.E., and Parker, R.F. (1957)Culture characteristics of human LITERATURE CITED fibroblasts propagated serially. Amer. J . Hyg. 66:235-243. Cristofalo, V.J., and Charpentier, R. (1981) A standard procedure for Wang, E. (1985a) A 57,000-mol-wt. protein uniquely present in cultivating human diploid fibroblastlike cells to study cellular nonproliferating cells and senescent human fibroblasts. J. Cell Biol. aging. J. Tissue Culture Methods. fi:117-121. 100:545-551. Hayflick, L. (1965) The limited in vitro lifetime of human diploid cell Wang, E. (1985b3 Rapid disappearance of statin, a nonproliferating strains. Exp. Cell Res. 37:61&636. and senescent cell-specific protein, upon reentering the process of Hayflick, L., and Moorhead, P.S. (1961) The serial cultivation of cell cycling. J. Cell Biol. 101:1695-1701. human diploid cell strains. Exp. Cell Res. 25:585-621. Wang, E. (1987)Contact-inhibition-induced quiescent state is marked by intense nuclear expression of statin. J . Cell Physiol. 133:151Lumpkin, C.K. Jr., McGlung, J.K., Pereira-Smith, O.M., and Smith, J.R. (1986)Existence of high abundance antiproliferative mRNA’s 157. in senescent human diploid fibroblasts. Science. 232:393-397. Wang, E. (1989a) Statin, a nonproliferation-spccific protein, is associated with the nuclear envelope and is heterogeneously distributed Matsumura, T., Pfendt, A., Zerrudo, Z., and Hayflick, L. (1980). in cells leaving quiescent state. J. Cell Physiol. 140:418-126. Senescent human diploid cells (WI38) attempted induction of proliferation bv infection with SV40 and bv fusion with irradiated Wang, E. (1989) Programmed gene expressions suggest multiple blocks to replication during cell aging. In Growth Control During continuous ’cell lines. Exp. Cell Res. 125:453437. Cell Aging, CRC Press, Boca Raton, Section 111, Chapter 11, 163Pereira-Smith, O.M., and Smith, J.R. (1981) Expression of SV40 T 178. antigen is finite lifespan hybrids of normal and SV 40-transformed Wang, E. and Gundersen, D. (1984) Increased organization of cyfibrGblasts. Somatic Cell Genet. 7:411-421. toskeletal architecture accompanying in vitro aging in human Pereira-Smith, O.M., Fisher, S.F., and Smith, J.R. (1985). Senescent fibroblasts. Exp. Cell Res. 154:191-202. and quiescent cell inhibitors of DNA synthesis, Exp. Cell Res. 160:297-305. Wang, E., and Krueger, J.G. (1985) Application of an unique monoclonal antibody as a marker for nonproliferating subpopulations of Phillips, P.D. Pignolo, R.J., and Cristofalo, J.J. (1987). Insulin-like cells of some tissues. J. Histochem. Cytochem. 33.587694. growth factor-1: specific binding to high and low affinity sites and mitogenic action throughout the like span of WI-38 cells. J. Cell Wang, E., and Lin, S.L. (1986) Disappearance of statin, a protein Psysiol. 133:135-143. marker for nonproliferating and senescent cells, following serumRabinocitch, P.S., and Norwood, T.H. (1980) Comparative heterstimulated cell cycle entry. Exp. Cell Res. 167:135-143.
Granular Presence of Terminin Is the Marker to Distinguish Between the Senescent and Quiescent States EUGENIA WANG* AND GEORGE TOMASZEWSKI The 6loomfield Center for Research in Aging, lady Davis institute for Medical Research, Sir Mortimer 6. Davis-Jewish General Hospital, and Departments of Medicine and Anatomy, McCill University, Montreal, Quebec H3 T 7 €2 We have previously identified statin, a nonproliferating-cell-specific nuclear protein of 57,000 dalton whose presence can be used to distinguish between growing and nongrowing cells. In this report we identify another protein, terminin, whose presence (by immunofluorescence microscopy) can be used to distinguish between temporarily and permanently growth-arrested cells. Thus terminin is a marker to separate the senescent from the quiescent state. By means of an unique monoclonal antibody (mAbl.2), the presence of terminin is recognized as granules in the cytoplasm of in vitro aged fibroblasts; these granules are not found in serum-starved, contact-inhibited, growing, or transformed fibroblasts, except for those cells experiencing the initiation of apoptosis due to long-term deprivation of nutrients. Preliminary histochemical studies show that terminin is also found in the superficial epithelial layer of the esophagus, where terminal differentiation is followed by apoptosis and sloughing off into the lumen. Biochemical characterization by Western blot shows the terminin antibody recognizing a protein of 84 kilodalton (kDa) in growing and quiescent cells, whereas in senescent cells a protein of 57 kDa is recognized; this result suggests that a senescence-dependent protease may cleave the 84 kDa proiein to 57 kDa. This proteolytic action seems to render the specific antigenic epitope exposed in its native state and accessible to the terminin antibody by immunofluorescence microscopy. It is this product o i posttranslational modification in the form of a cytoplasmic 57 kDa protein that is the marker distinguishing between senescence and quiescence.
Normal human fibroblasts grown in a laboratory dish exhibit a limited lifespan for proliferation (Swim and Parker, 1957; Hayflick and Moorhead, 1961). This principle restricting cells from further replication has been described as cellular or in vitro aging (Hayflick, 1965; Schneider and Mitsu, 1976).We and others have proposed that the aging process may not be simply the result of cumulative random deterioration; rather, a genetic program may be the underlying mechanism that makes cells become aged. Investigation along this line has led to the finding that senescence is a dominant phenoty e (Rabinovitch and Norwood, 1980; Matsumura et. a!, 1980; Pereira-Smith and Smith 1981; Stein and Yanishevsky, 1981), and that “senescencespecific gene expressions” may exist and be in direct control of the cessation of DNA synthesis and establishment of the associated in vitro aged phenotype of normal human fibroblasts (Lumpkin et. al., 1986). Our attempt to identify senescence-specificgene expression has resulted in the discovery of a novel protein, statin, associated with the nuclei of almost all nonproliferating cells (Wang, 1985a,b, 1987; Wang and Krueger, 1985; Wang and Lin, 1986). Unfortunately, statin cannot be deemed strictly senescence-specific;rather, it proves to be nonproliferation-specific, found in both 0 1991 WILEY-LISS, INC
senescent and temporarily growth-arrested quiescent cells (Wang, 1989). Obviously, whereas senescent and young quiescent fibroblasts may share many common features, including the most evident lack of DNA synthesis, the two cell ty es certainly demonstrate marked differences; speci ically, the old cells are in the state of irreversible growth-arrest. Except in the rare cases of transformation by oncogenic viruses, few workers have succeeded in extending the lifespan of senescent fibroblasts beyond one or two rounds of DNA synthesis (PerciraSmith et. al., 1985; Phillips et. al., 1987; Wang, unpublished results). Another major difference is the finding that young growth-arrested cells seem to be in the GO phase, whereas senescent cells are thought to be stalled in late G1 phase, shortly before entry to S phase (Rittling et al., 1986; Wang, 1989b). Results from the ongoing attempt at distinguishing senescent from quiescent fibroblasts are not yet adequate to provide a unifying clue to the underlying mechanisms contributing to the determination of these
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Received January 3, 1991; accepted February 25, 1991. *To whom reprint requestshhould be addressed.
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two distinct cell types; a major handicap is the lack of identified unique gene expression specific for senescence and absent from either young growing or temporarily growth-arrested cells. In this report we describe the identification of a cytoplasmic protein, terminin, whose unique presence in senescent fibroblasts can be used as a marker for in vitro aged cells and thus provide an initial lead in the investigation of “what makes cells irreversibly lose their growth capacity.” MATERIALS AND METHODS Cell systems Two specific cell strains, GMOOll (derived from a donor of 8 fetal weeks of age) and GM3529 (from a 66-year-old donor) were used in this study. The longitudinal assessment of in vitro lifespan for these two cell strains was performed by a rigid schedule of long-term serial passage of the monolayer cultures (Cristofalo and Charpentier, 1981); their in vitro life history has been published (Wang and Gundersen, 1984). A human glioma cell strain (U-251-4) was used for the transformed culture study. Both the fibroblasts and glioma cells are generally cultured in Eagle’s minimal essential medium supplemented with 10% fetal bovine serum, penicillin, and streptomycin; a humid atmosphere with standardized admixture of 5% CO, is maintained in an ambient environment of 37°C for culture conditions. Assay of proliferative activity Cultures of young and senescent fibroblasts in either sparse or confluent state, with and without serum, were analyzed for proliferative activity by measuring the index of DNA synthesis; the standard procedure (as reported previously) for assaying incorporation of 3Hthymidine was performed for most cultures used in the present study. Fibroblast cultures were determined to be senescent according to previously defined protocols: after serial passaging to reach the maximal population doubling level (PDL = 58 for the 0011cell strain, 28 for 3529), cultures were observed to have no mitotic activity for 14 days, and thymidine incorporation was at the background level. The nongrowing state of young fibroblasts (cumulative PDL less than 12 for the 0011 cell strain, less than 10 for 3529) was established by culturing either at confluency (with cell density of 35,550/cm2)or at low (0.05%) serum concentration for 72 hours; again, DNA synthesis as measured by thymidine incorporation was at background level. Production of monoclonal antibody (mAbl.2)to terminin Most cellular stage-specific proteins are of very small quantity; thus their detection can be masked by the presence of a lar e number of housekeeping proteins. Therefore, identi ication of specific novel proteins that are developmental or proliferation-dependent in their expression requires some amount of modification. Our aim in identifying senescence-specific but not quiescence-dependent antigens is the implementation in the standardized procedure of monoclonal antibody production of the principle of immunosuppression of antigenic reaction to the common “housekeeping” antigens. Once
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suppression to the common proteins is established, the immune system is then challenged again with another type of cell; manipulation following this regimen of immunization produces the simple subtraction of the highly abundant common antigens, thus allowing the increased probability of producing antibodies to unique stage-dependent gene roducts. The injection protoco consisted of an initial injection of detergent-insoluble extracts of confluent cultures of young fibroblasts (36,660 cells/cm2, maintained for at least 1week with basal level of 3H-thymidine incorporation); a pellet of cell specimen in 500 pl wet volume injected into two mice, accompanied by intraperitoneal injection of cyclophosphamide, 100 pgig of body wei ht in sterile phosphate-buffered saline. The cyclophospflamide kills the antibody-producing cells, whereas the elicited pattern of immunogenic reaction is retained in the memory of the animal’s immune system. A 40% weight loss indicates the effectiveness of this dose, but not the completeness of the induced immunotolerance; this is evaluated by characterizing the serum antibody reaction to the first antigen injection, on day 13 after the initial injection. After the mice recovered from the manipulation of the first immunization, and regained their physical health and weight, they were immunized with extracts of senescent cells prepared similarly by detergent extraction, in the same dosage; 2 weeks later, a similar booster injection was used. A simple criterion was designed to identify hybridoma clones of interest: negative antibody staining reaction in both young growing and young nongrowing cells, but positive reactivity in their senescent counterparts. Screening of antibody activity was performed by direct microscopic visualization of indirect immunofluorescence activity on appropriate cell specimens; monoclonal antibody mAbl.2 fit the above criteria. The mAbl.2 hybridoma has been cloned to single-cell stage through serial dilution and soft agar colony selection; the antibody is of the IgG type, and the ascites form of the antibody demonstrates a high titre (1:100,000 for immunofluorescence assays, 1:5,000 for immunoblotting procedures). For the sake of ease of terminology, we call the putative protein reacting with mAbl.2 terminin t o denote the nature of finality in the cellular aging process in which it was identified. Immunofluorescence microscopy For both purposes of antibody screening and cellular localization, fibroblasts of predetermined growth properties were cultured on glass substrate and processed for indirect staining as described previously (Wang, 1985a,b). In brief, the cells were fixed with methanol and acetone (1:l) at -20°C for 10 minutes; after air drying, the specimens were rehydrated with phosphatebuffered saline (PBS) at pH 7.2, and incubated with monoclonal antibody 1.2 overnight at room temperature. Afterward, the primary antibody was removed by repeated PBS rinses and fluorescein-conjugated goat antimouse immunoglobulin was applied and incubated for 30 minutes at room temperature, followed by PBS rinsing and mounting in glycerol-containing medium for epiillumination examination of positive reactions in a Nikon labophot microscope.
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Histochemical studies To identify terminin presence in tissues, frozen sections of 8 to 10 km were obtained on a Reichert cryomicrotome. Tissues of interest were excised from 3-month-old Sprague-Dawley rats, and processed for immunocytochemistry by immediate immersion in liquid nitrogen. Thick sections of 20 km were used initially for staining with standard haematoxylin dye to evaluate tissue orientation and morpholo cal preservation; when these criteria were satisfie , serial sections of chosen tissue blocks were placed on gelatincoated microscope slides and processed for fixation and incubation with antibodies as described above for cultured fibroblasts. Preparation of cell samples for SDS gel electrophoresis a n d immunoblotting reaction Cultures of young cells (3529 cell strain, CPDL = 10) were separated into two groups to establish (1.)growin cultures by maintaining them in sparse state (3.5 x 105 cells/cm2)in 10%fetal bovine serum, and (2.) nongrowing cultures by maintaining them at the same density in 0.05% serum for 3 days. The senescent counterparts of these cultures were obtained by serial passaging to reach CPDL = 28. These three cultures were then processed for extraction, gel electrophoresis, and immunoblotting reaction as described previously (Wang and Lin, 1986).The terminin-specific monoclonal antibody was produced as described above. Ascites fluid of a hybridoma cell line secreting no specific antibody was used as a control antibody for the immune reaction described here. RESULTS Figure 1 demonstrates the t ical pattern of terminin staining in senescent fibro lasts. Comparison of the phase contrast and fluorescence images of the same cells reveals the dense granular structures stained by the terminin antibody mAbl.2; in some instances, it appears that these granular structures fill the interstices of the cytoskeletal framework. The senescencespecific nature of terminin is illustrated by Fig. 2, showing a confluent monolayer of young (CPDL less than 12) cell strain 0011 (Fig. 2a), and two separate intermediate states of the in vitro lifespan (CPDL less than 30, Fig. 2b, and CPDL = 48, Fig. 2c) and a senescent culture (CPDL = 58, Fig. 2d). To achieve the nonproliferating state, confluency was maintained for 1 week, and DNA synthesis activity as measured by 3H-thymidine incorporation was observed to be at the basal level. In these four cultures, cytoplasmic presence of terminin-containing granules were not present in the confluent cultures of young cells; however, terminin-positive granules were identified in a small number of cells in intermediate cultures, and the number of terminin-positive cells increased with in vitro age and reached 85% in senescent cultures. The unique presence of terminin-positive granules in senescent cells and not in quiescent young cells was also observed in growth-arrested conditions achieved by serum deprivation. As shown in Fig. 3a, terminin-positive granules were not detected in cultures of young cells maintained in the nonproliferating state by keeping the serum
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Fig. 1. Immunofluorescence microscopy of terminin distribution in the cytoplasm of senescent human fibroblasts. Phase contract (a) and fluorescence images (b) of the same cells show protein presence as dense granular structures occupying the interstices of the cytoskeletal network. Presence of the protein is clearly cytoplasmic and not associated with either the nucleus or the plasma membrane. Cultures of senescent human fibroblasts were obtained by serial passage according to the standard split ratio to reach the required cumulative population doubling levels (CPDL) for the in vitro aging process. The specific culture used here is at CPDL = 28; the original donor age of the cell strain (3529) was 66 years.
level at 0.05%. For the purpose of contrast, we have shown here the field of terminin-ne ative cells in the two intermediate states (Fig. 3b,c); owever, in these cultures a low level (15%) of cells show terminin granules in their cytoplasm. In similar culture conditions of low level serum, senescent cells show a high frequency (85%)of terminin-positive granules. The absence of terminin-positive granules was also observed in young growing cells of the same cultures used for Figures 2a and 3a, when the cells were kept in the sparse state or 10%serum allowed growth (Fig. 4a); a similar absence of terminin positivity was found in transformed cultures such as human glioma cells, as shown in Fi . 4b. The diffuse staining shown in Figures 3a a n 4a may suggest the presence of terminin in a nongranular form in these cells. The only unexpected instance of positive staining of terminin antibody in young cells was when cultures of an early passage (CPDL = 15) of the 0011 cell strain were left in the confluent state for 4 weeks without feeding (Fig. 51,
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Fig. 2. Immunofluorescence microscopy of the senescence-specific nature of terminin in cells of the 0011 strain a t cumulative population doubling levels (CPDL) of young (10, (a)), intermediate (24, (b)), presenescent (48, (c)),and senescent (58, (d)) stages; cultures with CPDL larger than 58 are considered aged. Cultures were allowed to grow to confluency and stay in this state for a week with no detectable activity of DNA synthesis. These cultures were then fixed and processed for staining with terminin antibody; for 3 days before fixation, no DNA synthesis was observed in any of the cultures used,
so that they are in the state of quiescence. Notice the absence of granular staining in young nongrowing cells (a); a small number of cells become positive for granular staining in the two cultures of intermediate life span (b and c). In contrast, a majority of cells show positive granular staining in the cytoplasm when cultures of senescent cells are reacted with terminin antibody (d). However, a small percentage of cells (10%) in senescent cultures remains negative for terminin, as repeatedly reported in the literature of a minority of nonaging phenotypes.
whereas all cells were observed positive for statin, approximately 50% of the cultures were observed to contain terminin-positive granules. For histochemical studies of terminin presence in tissues, we chose to focus on two systems: esophageal e ithelium and liver. The former is a well-defined type o stratified epithelium composed of three distinct stages of differentiation: the basal layer of stem cells, the suprabasal layer of transient amplifying cells, and the superficial layer of terminally differentiated cells; active proliferation is mostly associated with the basal cells. Although both suprabasal and superficial cells show a positive reaction to statin antibody and lack DNA synthesis, these two types differ in many other ways; clearly the cornified superficial cells represent the final stage of esophageal epithelium before detaching and being sloughed off into the lumen of the esophagus. Figure 6 shows the nonnuclear cytoplasmic presence of terminin in the superficial layer of esophageal epithelium, whereas the protein is absent from cells in the suprabasal and basal layers of the esophagus; although the staining looks like a punctate mem-
branous pattern, it nevertheless is confirmed to be cytoplasmic granular staining. The absence of terminin found in the liver of 3-month-old rats may be due to the fact that the liver is composed of a mostly homogeneous population of hepatocytes possessing high regenerative potential (Fig. 7). Biochemical characterization of the protein identity of terminin was performed by immunoblotting experiments with extracts of young growing, young nongrowing and senescent cells, such as those specimens used for Figures 1, 3, and 4.Senescent cultures and young cells grown in 10% and 0.05% serum were harvested and extracted as described in Materials and Methods. Equal amounts of protein from specimens of the three cultures were loaded onto 10%SDS polyacrylamide gel, and the electrophoretically separated proteins were transferred onto nitrocellulose (NC) papers, which were then reacted with terminin monoclonal antibody. The results show the reactivity of mAb 1.2 with a band at 84 kDa in cell extracts from young growing and young nongrowing cells (Fig. 8A,B,C); however, the same antibody reacts with a protein at 57 kDa in extracts of
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Fig. 3. Immunofluorescence microscopy of the senescence-specific nature of terminin. The cells used here in (a-d) are in the same cumulative population doubling levels (CPDL) as those seen in Figure 2, i.e., (a) young at (CPDL = 10);(b) intermediate (CPDL = 24); (c) presenescent (CPDL) = 48; and (d) senescent (CPDL = 58). However, the quiescence in cultures used here was reached by serum
senescent cells (Fig. 8D,E). No detectable bands were identified when the NC filter bearing the same types of extracts was reacted with a control monoclonal antibody produced from hybridoma cells bearing no specific antibody-producing capability (data not shown).
starvation. Cultures were maintained in medium containing 0.05% serum for 4 days, and DNA synthesis was not observed for at least 48 hours before fixation and processing for immunofluorescence microscopy. Again, similar to that shown in Figure 2, the granular pattern of terminin staining was not observed in the majority of cells until senescence was reached.
gest that the cytoplasmic staining seen by immunofluorescence microscopy is due to a possible posttranslational modification to 57 kDa from a preexisting 84 kDa protein. It is this modifying process, possibly proteolytic cleavage, which allows the specific epitope to become accessible to react with the antibody. To be DISCUSSION more precise, it is rather the putative posttranslational The search for a unique marker for senescent fibro- modification process that is senescence-specific; the blasts has been a long-term pursuit for many investi- identification of the existence of terminin merely progators in the hope of obtaining a handle to study the vides us the means to characterize this unique cellular complex mechanism involved in the aging process of process. individual cells. Here, we report the result of our The production of monoclonal antibody 1.2 to termiendeavor by identifying the senescence-dependent nin involves the lengthy processes of antigen preparapresence of a cytoplasmic protein, terminin. Morpho- tion and immunosuppression. The 2 years of endeavour logically, the presence of terminin was found in cyto- involved in this study prove the possibility of identifyplasmic granules of senescent fibroblasts, and its ab- ing stage-specific, differentiation- or developmentsence was found in a wide variety of cells including dependent gene expression by the immunological apyoung growing and nongrowing and transformed cells. proach. The incorporation of the immunosuppression In tissues of esophageal epithelium, terminin is present scheme in the regimen of hybridoma production proin the cytoplasm of only those cells at the last stage of vides a logical methodology to produce antibodies tardifferentiation. The biochemical characterization of getting stage-specific proteins in minute quantity. The terminin identity, however, indicates antibody recog- subtraction of a large number of abundant housekeepnition of a protein at 84 kDa in young growing and ing proteins by the “self” recognition of an animal’s nongrowing cells, but a protein of 57 kDa in their immune system avoids the conventional procedure senescent counterparts. These results lead us to sug- described as a “hit-or-miss”approach. Recently, by this
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Fig. 4. Immunofluorescence microscopy of the absence of terninin positive granules in young growing cells (a) as well as transformed cultures (b). (a) shows the lack of terminin positive granule3 in a growing culture of human fibroblasts (3529 cell line a t CpDL = 10) kept in 10%serum-containing medium, (b) shows absence of terminin positive granules in transformed human glioma cells (U-251-4) also kept in 10% serum-containing medium.
approach of immunosuppression, we have produced another monoclonal antibody (S6) that recognizes a nuclear protein of 100 kDa present in all except senescent cells (data not shown). The major difference between the techniques of immunoblotting vs. immunofluorescence reaction is that the former procedure recognizes the denatured protein, including both soluble and insoluble forms, whereas the latter recognizes native protein in the insoluble cellular scaffold. Our current working model for termi-
Fig. 5 . Immunofluorescence microscopy of statin presence in quiescent cells and additional terminin presence in the same type of culture maintained long term without feeding. Cells used here were of young cells of 3529 cell strain at CPDL of 10. (a) and (b) are phase contrast and fluorescence images of'the same culture of young fibroblasts kept in confluent quiescent state for a week. At this time, as shown in Figures 2 and 3, terminin was not detected. However, if the cultures are kept at this state for 4 weeks without feeding new medium, the cells then become terminin positive with approximately 508 of cells showing granular structure after staining with the antibody (c).
nin presence is as follows: in young cells, the extra protein mass of 24 kDa may mask the epitope in the in situ state, thus preventing recognition by the antibody during the fluorescence staining reaction. Processing of the protein to 57 kDa may cause the rearran ement of protein conformation or association with ce lular organelles in a way allowing the antigenic sites to become
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Fig. 6. Immunofluorescence microscopy of terminin staining reaction seen in the superficial layer (in area indicated by the arrows) of 3-month-old rat esophageal epithelium. Phase contrast (A) and fluorescence (B) show the same area indicating the histological morphology as well as the location of the terminin positive layer. The strong fluorescence in the area below the basal cell layer is due to non-specific reaction of the antibody, as supported by the same staining intensity of a control antibody.
accessible to react with the antibody. Alternatively, the 84 kDa protein may be in the soluble portion of young cells, which is extracted with detergent during the preparation for immunofluorescence microscopy, and therefore lost from the cell specimen. In senescent cells, the change from 84 to 57 kDa may make the protein detergent-insoluble, thus remaining associated with the cell specimens and readily detected by the antibody. Future investigation of the biochemical relationship between the two forms (84 and 57 kDa) of the protein should provide us some answers to these hypotheses. The 57 kDa protein described here is not the same polypeptide reacting with the statin S44 antibody; 57 kDa terminin is exclusively located in cytoplasmic
granules, whereas statin of the same size is found mostly in the nucleus. In addition, statin is found in both nongrowing young and senescent cells, while terminin is found only in senescent cells. Furthermore, terminin antibody 1.2 does not react with the immunoprecipitation complex that results from the reaction between statin antibody and senescent cell extracts (data not shown). Therefore, it is quite unlikely that the two 57 kDa polypeptides reacting with either the S44 or the 1.2 antibodies are the same protein. The distinct granular distribution of terminin in the cytoplasm of senescent cells poses a series of interesting questions: do the granules exist in young cells as well, but not associated with the protein? Are the granules of
TERMININ AS MARKER FOR SENESCENT STATE
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Fig. 7. Immunofluorescence microscopy of terminin absence in the liver of 3-month-old rat. The frozen sections of the liver were processed the same way as the esophageal tissue, and the identical staining conditions and antibody strength were used for reaction. However, little specific staining pattern was observed, indicating absence of terminin in liver tissue from rats of this age. a and b are phasecontrast and fluorescence images of the same field. The nuclei appear as black holes; the cytoplasm is faintly stained with the antibody, as also with the control antibody.
lysosomal nature? If so, are they the manifestation of a unique senescence-specific lysosomal process? Could the protein be of a secretory nature? Immunogold labelling in combination with ultrastructural studies may reveal the nature of these cellular organelles. Identification of the presence or absence of terminin in the culture medium of senescent cells may also help us to unravel the question of whether terminin can be secreted from cells. The fact that terminin positive staining is found in senescent fibroblasts, the superficial layer of squamous epithelial cells, and in cultures of prolonged period quiescence shows the usefulness of antibody in identifying a particular state of physiology following natural course of development or injury.
Fig. 8. Biochemical characterization of the electrophoretic mobility of terminin in extracts from young growing, young nongrowing, and old cells of the 3529 cell line. Same amount of proteins were loaded in ELD. A and E are the prestained molecular weight standards ranging from 116 to 49 kilodaltons. B is the extract from young growing 3529 cells CPDL = 10. C is the extract from the young confluent cultures of nongrowing 3529 a t CPDL = 10. D is the extract from aged cultures (CPDL = 28) of 3529 cells. After the cell extracts were run on the 10% polyacrylamide gel, they were transferred electrophoretically onto nitrocellulose paper and processed for reaction with terminin antibody. As shown here, the antibody reacts with a band at 84 kilodalton (arrow) in B and C, but with a band at 57 kilodalton(arrow) in D.
The presence of terminin antibody staining in senescent cells and terminally differentiated esophageal epithelium points to the fact that these cells are at the last cellular state before death takes place. From this point of view, terminin presence may be used as a marker for those cells predisposed to irreversible growth arrest leading to death. It is therefore not surprising to find the absence of terminin antibody staining in liver cells, which in general maintain a high degree of flexibility to shift back into the growth cycle. Similarly, in long-term unfed cultures of young cells, the appearance of terminin antibody staining may indicate the putative events of conversion from reversible to irreversible growth arrest, and many cells may experience accelerated senescence and the subsequent death processes. Nature has provided readily available systems such as the neuronal and thymus systems where developmentally regulated cell death is a necessary process. Investigation of terminin antibody staining in these systems will allow us to define whether indeed senescence or terminal differentiation is en route to apoptosis on programmed cell death.
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ACKNOWLEDGMENTS The authors express thanks to Ms. Lucia Badolato for typing this manuscript, to Mr. Michael Morcos for the preparation of photographic reproduction, and to Mr.
okaryon study of cellular senescence and the serum-deprived state. Exp. Cell Res. 13O:lOl-109. Rittling, S.R., Brooks, K.M., Cristofalo, V.J., and Baserga, R. (1986) Expression of cell cycle dependent genes in young and senescent WI-38 fibroblasts. Proc. Natl. Acad. Sci. U.S.A. 83:33163320. E.L., and Mitsu, J. (1976)The relationship between in vitro Alan Bloch for proofreading the manuscript. This work Schneider, cellular aging and in vivo human age. Proc. Natl. Acad. Sci. U.S.A. was supported by a grant (AGO92781from the National 73:3584-3588. Institute on Aging of the National Institutes of Health. Stein, G.H., and Yanishevsky, R.M. (1981) Quiescent human diploid cells an inhibit entry into S phase in replicative nuclei in heterodikaryons. Proc. Natl. Acad. Sci. U.S.A. 78:3025-3029. Swim, H.E., and Parker, R.F. (1957)Culture characteristics of human LITERATURE CITED fibroblasts propagated serially. Amer. J . Hyg. 66:235-243. Cristofalo, V.J., and Charpentier, R. (1981) A standard procedure for Wang, E. (1985a) A 57,000-mol-wt. protein uniquely present in cultivating human diploid fibroblastlike cells to study cellular nonproliferating cells and senescent human fibroblasts. J. Cell Biol. aging. J. Tissue Culture Methods. fi:117-121. 100:545-551. Hayflick, L. (1965) The limited in vitro lifetime of human diploid cell Wang, E. (1985b3 Rapid disappearance of statin, a nonproliferating strains. Exp. Cell Res. 37:61&636. and senescent cell-specific protein, upon reentering the process of Hayflick, L., and Moorhead, P.S. (1961) The serial cultivation of cell cycling. J. Cell Biol. 101:1695-1701. human diploid cell strains. Exp. Cell Res. 25:585-621. Wang, E. (1987)Contact-inhibition-induced quiescent state is marked by intense nuclear expression of statin. J . Cell Physiol. 133:151Lumpkin, C.K. Jr., McGlung, J.K., Pereira-Smith, O.M., and Smith, J.R. (1986)Existence of high abundance antiproliferative mRNA’s 157. in senescent human diploid fibroblasts. Science. 232:393-397. Wang, E. (1989a) Statin, a nonproliferation-spccific protein, is associated with the nuclear envelope and is heterogeneously distributed Matsumura, T., Pfendt, A., Zerrudo, Z., and Hayflick, L. (1980). in cells leaving quiescent state. J. Cell Physiol. 140:418-126. Senescent human diploid cells (WI38) attempted induction of proliferation bv infection with SV40 and bv fusion with irradiated Wang, E. (1989) Programmed gene expressions suggest multiple blocks to replication during cell aging. In Growth Control During continuous ’cell lines. Exp. Cell Res. 125:453437. Cell Aging, CRC Press, Boca Raton, Section 111, Chapter 11, 163Pereira-Smith, O.M., and Smith, J.R. (1981) Expression of SV40 T 178. antigen is finite lifespan hybrids of normal and SV 40-transformed Wang, E. and Gundersen, D. (1984) Increased organization of cyfibrGblasts. Somatic Cell Genet. 7:411-421. toskeletal architecture accompanying in vitro aging in human Pereira-Smith, O.M., Fisher, S.F., and Smith, J.R. (1985). Senescent fibroblasts. Exp. Cell Res. 154:191-202. and quiescent cell inhibitors of DNA synthesis, Exp. Cell Res. 160:297-305. Wang, E., and Krueger, J.G. (1985) Application of an unique monoclonal antibody as a marker for nonproliferating subpopulations of Phillips, P.D. Pignolo, R.J., and Cristofalo, J.J. (1987). Insulin-like cells of some tissues. J. Histochem. Cytochem. 33.587694. growth factor-1: specific binding to high and low affinity sites and mitogenic action throughout the like span of WI-38 cells. J. Cell Wang, E., and Lin, S.L. (1986) Disappearance of statin, a protein Psysiol. 133:135-143. marker for nonproliferating and senescent cells, following serumRabinocitch, P.S., and Norwood, T.H. (1980) Comparative heterstimulated cell cycle entry. Exp. Cell Res. 167:135-143.
