JOURNAL OF CELLULAR PHYSIOLOGY 142:425-433 (1990)

Novel Monoclonal Antibodies Identify Antigenic Determinants Unique to CeIIular Senescence MARY BETH PORTER*, OLlVlA M. PEREIRA-SMITH,

AND

JAMES R. SMITH

Koy M. and Phyllis Cough Huffington Center on Aging IO.M.P.-5., /.R.5.), Division of Molecular Virology (M.B.P., O.M.P.-5., j.K.5.), and Departments of Cell Biology (l.R.S.1 and Medicine (0. M. P.-S., 1.R.S.), Baylor College of Medicine, Houston, Texas 77030 Normal human diploid fibroblasts exhibit a limited lifespan in vitro and are used a5 a model to study in vivo aging. Monoclonal antibodies were generated against

partially purified surface membranes from human diploid fibroblasts at the end of their lifespan (senescent). Three hybridomas were isolated that secreted antibodies reacting to cellular determinants expressed specifically on senescent human fibroblasts of different origin, including neonatal foreskin, embryonic lung, and adult skin punch biopsy, but not expressed on matched young cells. The antibodies did not bind to immortal human cells and normal young cells made reversibly nondividing, indicating the antigens are not expressed in cells that are not senescent. The antibodies identified senescent cells in a mixed cell population and expression of the senescent cell antigens correlated strongly with the cells inability to synthesize DNA at the onset of senescence. The antigens appeared to be cell surface or extracellular matrix associdted, and the epitopes were destroyed by mild trypsin treatment. Western analysis indicated all three antibodies reacted with fibronectin. Though the antigenic determinants on the fibronectin molecule were not accessible in (he intact young cell, the epitopes were present in fibronectin extracted from both senescent and young cells, a5 well as purified human plasma fibronectin. rhese antibodies and the senescent specific expression of the antigens provide powerful tools to investigate the mechanisms leading to in vitro ~ n e s c e n c e This . may enable us to investigate directly the relationship between cellular aging and aging of the individual.

Replicative senescence is well established for normal human diploid fibroblasts grown in tissue culture (Hayflick and Moorhead, 1961) and has been proposed as a n in vitro model for aging in vivo (Hayflick, 1965; Hayflick, 1977; Norwood and Smith, 1985). However, the mechanisms that result in senescence a t the cellular level are yet to be determined. There are no major differences between senescent and young cells that can readily explain the phenomenon of senescence. In fact, senescent cells that fail to respond to mitogenic stimuli and synthesize DNA, remain metabolically active, and can be maintained for at least a year in culture (Matsumura et al., 197913). Currently there are few well-defined morphological and biochemical differences between terminally nondividing senescent cells and proliferation competent cells. Morphological changes observed a t senescence include a n increase in overall size and loss of the spindle shape characteristic of fibroblasts (Cristofalo and Kritchevsky, 1969; Hayflick and Moorhead, 1961; Matsumura et al., 1979b). In addition, as the population becomes senescent, the cells become unable to synthesize DNA resulting in a decrease in the labeling index from >75% in young cell populations to 6% I in . senescent populations (Cristofalo and Sharf, 1973; Matsumura et al., 1979a). Studies of other metabolic and 5 1990 WILEY-LTSS, INC

biosynthetic parameters have revealed some differences between young and senescent cells (Smith and Pereira-Smith, 1985). One- and two-dimensional electrophoretic analysis has demonstrated the existence of a few proteins in senescent cells that are either not expressed or expressed a t lower levels in young cells (Bayreuther et al., 1988; Lincoln et al., 1984; Sottile et al., 1987; Wang, 1985). However, such differences are often observed in young cells made reversibly nondividing (quiescent) by removal of growth factors andlor contact inhibition, and are not unique to senescence. No direct evidence exists relating any of these changes to the mechanisms leading to senescence, a s distinct from changes that occur as a result of senescence. Though a few potential biologic markers for senescent cells have been identified (Brooks et al., 1987; Ching and Wang, 1988),these markers have not been demonstrated to be absent from quiescent cells. Therefore, there remains a significant need for additional biologic markers that specifically identify the senescent phenotype.

Received July 3, 1989; accepted October 25, 1989.

“To whom reprint requestsicorrespondence should be addressed.

426

PORTER ET AL.

TABLE 1. Normal human diploid fibroblast cell lines screened for production of Senescent specific cell determinants Cell line CSC303ClG CSC303 cSC303 CSC3Ol

HCA2 HCAl WI-38 IMR-90

HCA3

PDR (%I,SC)'' 3 (95) 40 (40) 3 (96) 70 1201 7 (92) 72 (20) 1(99) 59 (30) 0 (100) 48 (9) 2 197) 26 (59) 2 (97) 41 137) 4 (96)

SEN-2

SEN-3

++-

++-

Origin Nconatal foreskin

SEN-1

Neonatal foreskin

Neonatal foreskin

++ +

+

~

~

Adult skin punch biopsy

+

Embryonic lung

I

~

Embryonic lung

+ ++ ~

Embryonic lung Neonatal foreskin

76 I191

~

I -

+ + + ~

I -

+ ++~

+

-

t ~

+ ~

'Population doubhngs remaining.

'Percentage in vitro lifespan completed

We and others have shown that a protein capable of inhibiting DNA synthesis in proliferation competent normal cells exists (Burmer et al., 1982; Drescher-Lincoln and Smith, 1983, 1984) and is present in the plasma membrane of senescent fibroblasts (PereiraSmith et al., 1985; Stein and Atkins, 1986). This protein represents a significant difference between senescent and young cells. Therefore, we isolated surface membrane enriched preparations from senescent cells for use as antigen in order to generate monoclonal antibodies that identified antigens specific for the senescent phenotype, some of which might be against the DNA synthesis inhibitor protein. We report here the isolation and identification of three hybridomas secreting monoclonal antibodies that react to antigenic determinants expressed uniquely on the surface of senescent human diploid fibroblasts of different origin, but not on their young counterparts, immortal human cells, or quiescent young human cells.

28 mM Hepes, and 10% fetal bovine serum (FBS), or in 96 well microtiter plates containing MEM with Earle's balanced salts (EMEM), and 10% FBS in a CO, incubator. Both subconfluent and confluent monolayers were assayed. In all cases, cells were washed twice with calcium and magnesium free phosphate buffered saline (CMF-PBS), fixed with 1%glutaraldehyde in CMFPBS, and again washed twice with CMF-PBS. Cells were either assayed immediately or stored desiccated a t -20°C for future use. Immortal cells. The immortal cell lines (Table 2) were grown to confluence in 96 well microtiter plates containing EMEM and 10%FBS in a 37"C, C 0 2 incubator. Cells were washed with CMF-PBS, glutaraldehyde fixed as described above and assayed for antibody reactivity by the ABC-peroxidase method. Quiescent cells. Normal young CSC303 and HCA2 (established in our lab) human cells were made quiescent by various manipulations: growth factor deprivation (0.5%FBS versus 10% FBS in the culture medium) MATERIALS AND METHODS for 10 days to 2 weeks; density dependent inhibition (growth to confluence and maintenance in 10% FBS Cell culture techniques containing medium) for 8 days to 2 weeks; growth facNormal cells. The normal human diploid fibroblast tor deprivation of density dependent inhibited cells for cell line CSC303 isolated in our laboratory from neo- 2 weeks to 5 mont.hs. Cells were washed with CMFnatal foreskin and a clone derived from this line, PBS, glutaraldehyde fixed, and assayed for antibody CSC303ClG, are well characterized, and the population reactivity as described below. Cells at clonal density. Normal fibroblast cultures doublings required to reach senescence are known (Drescher-Lincoln and Smith, 1983). Senescent CSC303ClG established from skin punch biopsies taken from the cells were used to generate the hybridomas and the inner aspect of the upper arm of adult individuals from initial screenings utilized the senescent CSC303ClG ages 36 to 72 years were seeded a t clonal densities (20 and young CSC303 cells. Subsequently the antibodies cells/60 mm dish) in EMEM and 10% FBS (Smith et al., that were senescent cell specific were tested against 1978). The dishes were incubated for 2 weeks a t 37°C in young and senescent cultures of the cell lines listed in a C02 incubator. Cells were washed with CMF-PBS, Table 1. Young cells were generally used when less than fixed with 1% glutaraldehyde, and assayed by the 50% of their lifespan had been completed. Cell culture ABC-peroxidase method. DNA synthesis assay. Senescent CSC303ClG and conditions and determination of population doublings have been previously described (Drescher-Lincoln and young CSC303 cells were plated onto glass coverslips in 24 well plates at 2 x lo4 cells/well and cultured in Smith, 1983). Normal human diploid fibroblasts to be used in the EMEM and lo%, FBS in a 37"C, CO, incubator. Cells antibody staining assay (ABC-peroxidase, Vector Lab- were labelled with 1 pCi/ml of ["]-thymidine 24 hours oratories, described below) were grown a t 37°C in 25 prior to fixation. Cells were washed with CMF-PBS cm2 tissue culture flasks containing minimal essential and fixed with 1%glutaraldehyde a s described above. medium (MEM) with Hanks' balanced salts (HMEM), ABC assays were performed and the coverslips then

427

UNIQUE SENESCEN T CELL ANTIGENS

TABLE 2. Immortal cell lines reacted with monoclonal antibodies Cell line GM639 GM847

A' A

SVWF39

A

GM2096SV9

B

CMV-Mj-HEL.-1 C 293

D

WI-384x1 SUSM-1

D D

HT1080 T98G HeLa A549 A2182 A1698 TES5

A B B -2

D D C

Description SV40-transformed skin fibroblasts rIMR) SV40-transformed skin fibrohlasts (HPRT-, Lesch Nyhan) (IMR) Origin defective SV40 transformed bone fibroblasts (H Ozer) Origin detective SV40 transformed xeroderma pigmentosum skin fibroblasts (D Canaani) Cytomegalovirus-transformed lung fibroblast.; (F Rapp) Adenovirus-transformed embryonic kidney cells (ATCC) Co-irradiated lung fibroblasts ( M Namba) 4NQO-transformed liver fibroblasts (M Namba) Fibrosarcoma iN-ras+) ( R Baker) Glioblastoma (G Stein) Cervical carcinoma ( ATCC) Lung carcinoma (SAaronson) Lung carcinoma (Ki-ras + ) (S Aaronson) Bladder carcinoma (Ki-ras + ) rS Aaronson) Osteosarcoma tJ Foah)

'Complementation group assignment. 'Not assigned t o group A, B, or C. Group D assignment is being determined

subjected to standard autoradiographic analysis to determine the correlation between DNA synthetic activity and antibody reactivity.

ABC-horseradishperoxidase assay Cells were grown and fixed as described above. For assay, the cells were rehydrated with PBS and blocked with a 0.5% (wt./vol.) solution of powdered milk made in PBS. The reagents of the ABC kit (Vector Laboratories) were added in the order specified by the manufacturer with washing (using PBS containing 0.05'3 TWEEN-20 [volivol])between each step. After the final wash, diaminobenzidine (DAB) was added and incubated for 30 minutes. The cells were rinsed a final time in deionized water and scored by light microscopy for antibody reactivity. Positive reactions were determined by a light-brown-colored DAB precipitate on the cells, whereas reactions in which no antibodies were bound to the cells had no color visible on the cells. Monoclonal antibody isolation Antigen preparation and immunization. Surface membrane enriched preparations of senescent CSC303ClG cells were used a s the immunogen to limit the number of potential antigens present, and because we and others have demonstrated that a protein inhibitor of DNA synthesis is present on surface membranes of senescent but not young cells (Pereira-Smith et al., 1985; Stein and Atkins, 1986). Expression of this protein represents a significant difference between senescent and young cells and may be an important regulatory step in the processes leading t o senescence. Membranes were isolated using the two-phase polymer system described by Brunette and Till (1971),replacing the ZnC1, with MgC1,. BALBic mice were immunized by intraperitoneal injections of senescent CSC303ClG membrane enriched preparations isolated from approximately 1x lo7 senescent cells per injection. In the first immunization, the membranes were resuspended in Freunds' complete adjuvant. This was followed by a

series of immunizations over the next six months (including hyperimmunizing injections at 6, 4, 3, and 2 days prior t o sacrificing the mice) using membranes resuspended in CMF-PBS. Hybridoma generation. Hybridomas were generated by modification of the procedure of Galfre and Milstein (1981). Mouse spleen cells were fused with a murine myeloma cell line (P3X63-Ag8.653, American Type Culture Collection) a t a 3:l ratio. The fusion products were plated into 96 well microtiter plates in selection medium containing hypoxanthine, aminopterin, thymidine, 20%)FBS and 2.55%Ewing sarcoma growth factor (Costar Europe) and cultured in a 37"C, CO, incubator for 9-12 days. Individual colonies were isolated and allowed to grow in fresh medium for 3-5 days prior to screening. Screening. Medium from each hybridoma colony was differentially screened against 1%glutaraldehyde fixed senescent CSC303ClG and young CSC303 cells by the ABC-peroxidase method. Only the hybridomas which had positive antibody reactivity against the senescent but not the young cells were retained and further subcultured. Hybridomas producing potentially senescent specific antibodies were twice cloned by limiting dilution. Ascites fluid was generated from cloned hybridoma lines by priming mice with 0.5 ml pristane 2 weeks prior t o injecting cells and then injecting intraperitoneally with 1 x lo6 cellshouse (Brodeur et al., 1984). Ascites fluid was collected 10-14 days after injection. The antibodies were purified using a protein-A affinity column (Pierce) according to the manufacturer's instructions.

Western analysis Senescent CSC303ClG and young CSC303 cell subconfluent monolayers were extracted into 1ml of sample buffer containing 4% SDS and 10% beta-mercaptoethanol by scraping the monolayers. Senescent and young extracts (10 pl per lane) were electrophoresed in 8% polyacrylamide gels (Laemmli, 1970). Purified human plasma fibronectin (Sigma) was run on the same gels (0.4 pg per lane) as a control. After electrophoresis, the proteins were transferred to nitrocellulose by modification of the Western transfer technique (Burnette, 1981; Towbin et al., 1979) and immunoblotted with either the senescent specific monoclonal antibodies, or a commercially available polyclonal anti-fibronectin antiserum (Collaborative Research). The immunoassay was completed using the reagents of the ABC kit (Vector) and DAB a s the substrate.

RESULTS Isolation of hybridomas producing novel antibodies Hybridoma clones were differentially screened for production of antibodies reactive to senescent cells but not young cells. Most of the clones produced antibodies that bound to both cell types in the initial screenings. However, monoclonal antibodies designated SEN-1, SEN-2, and SEN-3, secreted by three independently isolated hybridomas, reacted with senescent cells and did not react with young cells. They retained senescent cell specific antibody reactivity through all screenings

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PORTER ET AL.

Figs. 1--2.

UNIQUE SENESCENT CELL ANTIGENS

and twice cloning by limiting dilution. These three clones were used for expansion and ascites generation. The staining patterns observed after incubating cells with control antibodies are shown in Figure 1.Mouse serum, obtained after the last injection of the surface membrane enriched preparation, was used as a positive control and stained senescent (Fig. l a ) and young cells (Fig. l b ) equally well. This was expected, a s most of the antigens to which this polyclonal antiserum is directed should be common to both senescent and young cells. The brown-colored DAB precipitate stained the entire cell surface of both cell types. Reaction with a mouse monoclonal antibody generated against SV40 T-Ag (negative control antibody) resulted in no staining of either senescent (Fig. lc) o r young (Fig. Id) cells. These preparations were counter stained with brilliant green which gave the cells a blue-green appearance and enhanced their visualization. All three of the monoclonal antibodies showed similar staining patterns (Fig. 2). The DAB staining on senescent CSC303ClG cells appeared uniform over both nucleus and cytoplasm (Fig. 2a,c,e), suggesting the antigens were cell surface associated. Analysis of the distribution of antigens in senescent cells by immunofluorescence (not shown) showed fibril-like patterns indicative of cell surface or extracellular matrix association. Antibody binding was lost after a mild trypsin treatment (0.125%twice crystallized trypsin, 1 minute, 4°C) designed t o cleave only cell surface proteins and not allow entry of the enzyme into the cell (Fig. 2g). This further implicated antibody reaction to cell surface or extracellular matrix associated antigens. The young CSC303 cells (Fig. 2b,d,f), after reaction with each of the three monoclonal antibodies, showed no significant staining with DAB in the ABC assay. The density of seeding of the young cells had no effect on staining, a s both subconfluent and confluent monolayers (Fig. 2b,d,f) yielded negative results.

429

tion the antibodies were tested for binding with human diploid fibroblast cell lines derived from different tissues including neonatal foreskin, embryonic lung, and adult skin inner upper arm (Table 1). The antibodies reacted with the senescent cells of each line and showed little t o no reactivity with their young counterparts. Although there were a few (200 KDa Mr) protein and young cells derived from different origins (described in Table 1) with the monoclonal antibody SEN-3. Antibodies SEN-1 and SEN-2 doublet present in both the senescent and young cell extracts (Fig. 6b,c) that was thought to be fibronectin. had similar patterns to SEN-3 [shown). The DAB staining patterns observed on the senescent cells of each line, CSC303 mass culture (a), Observations in our lab West et al., in preparation) HCAl (b), and HCAZ ( c ) are similar t o those observed with the orig- and by others (Chandrasekhar et al., 1983; Edick and inal line CSC303ClG (Fig. 2). The young cells of each line, CSC303 (d),HCAl (e), and HCAZ (0again show no reactivity with the mono- Millis, 1984; Sorrentino and Millis, 1984) had shown there were some functional and structural differences clonal antibodies. between the fibronectin molecules secreted by senesFig. 4. Cell lines which do not express the senescent specific anti- cent and young cells in culture. To confirm that the gens and therefore do not react with SEN-1, SEN-2 (shown), and SEN-3. Young CSC303 (a) and HCA:! (b) cells made reversibly non- high molecular weight bands observed were fibronecdividing by culture in medium containing 0.5% FBS for 4 weeks and tin, two different analyses were performed. First, the 70 days, respectively; and immortal cell lines representative of Table senescent specific antibodies were reacted with puri2, WI-38-CT1 (c),HT1080 (d), HeLa (e),and TE85 (f). fied human plasma fibronectin and senescent cell and Fig. 5. Ability of monoclonal antibodies to recognize senescent cells young cell extracts that had been electrophoresed in polyacrylamide gels and transferred to nitrocellulose. within a mixed cell population. Cells were seeded a t clonal density, cultured for two weeks, fixed and reacted with SEN-1 (shown),SEN-2, SEN-1 reacted with the plasma fibronectin and the and SEN-3. The small clone consisting of mostly morphologically se- slightly higher molecular weight proteins from senesnescent cells showed positive reaction with the monoclonal antibodcent and young cells (Fig. 6,b-d). SEN-2 and SEN-3 ies: x 4 magnification (a), and x 20 magnification (b). Large clones gave the same results (data not shown). In addition, the containing young proliferation competent cells did not react with the monoclonal antibodies: x 4 magnification (c). plasma fibronectin and cell extracts were electro-

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PORTER ET AL

phoresed, transferred to nitrocellulose and probed with a rabbit fibronectin antiserum. The bands identified with the antiserum (Fig. 6,e-g) were identical to those observed with the senescent specific monoclonal antibodies (Fig. 6,b-d). Therefore, the specificity of SEN-1, SEN-2, and SEN-3 was demonstrated by positive reaction with a doublet in a total protein extract (Fig 6,a), and the identity of this band was confirmed to be fibronectin by both antibody and plasma fibronectin analyses. The monoclonal antibodies interact with epitopes that are common among the three types of fibronectin, even though the antibodies do not react with young cell fibronectin in situ. Possible explanations are discussed below.

DISCUSSION The results reported here demonstrate that senescent human fibroblasts cultured in vitro express cell surface antigenic determinants, as identified by monoclonal antibodies SEN-1, SEN-2, and SEN-3, that are not expressed on young cells in culture. The antigens are not cell type specific, since monoclonal antibodies react with the determinants expressed on senescent human fibroblasts from a variety of cell sources. Expression of the antigen is not a n artifact of time in culture or culture conditions a s the antigens are not expressed in any of the immortal human cell lines tested. Moreover, the antigens are not expressed in young cells made reversibly nondividing or quiescent by experimental manipulation. The expression of the antigens is not related to the cell cycle and, therefore, is not simply a result of non-division per se. Rather the antigens are expressed only by senescent cells as part of the senescent phenotype. These represent the first antibodies generated that react only to cells exhibiting the senescent phenotype. Whether or not the expression of these antigenic determinants is part of the mechanisms leading to senescence or a result of cellular senescence is yet to be determined. Nevertheless, the senescent specific expression of the antigenic determinants will provide reliable biomarkers for the in vitro senescent phenotype. This represents a n important new development in the field of aging, a s very few markers for senescence exist. There is a strong correlation between expression of the antigens and the inability of senescent cells to synthesize DNA, and expression of the antigens allows for identification of senescent cells in a mixed population. This may allow for the first time a feasible approach to relate in vitro with in vivo aging. For example, if these antibodies recognize senescent cells in vivo, they may be used to directly examine tissues for the presence of aging cells. We have identified fibronectin as the antigen to which all three monoclonal antibodies react. Since the antibodies react with fibronectin extracted from both young and old cells, as well as plasma fibronectin, it is apparent that the antibodies do not recognize a new epitope expressed exclusively in senescent cells. Rather, it appears that fibronectin on the surface of senescent fibroblasts presents a n epitope that is sequestered in young cells. This could be due t o changes in conformation resulting from 1) change in primary amino acid structure in a region of the molecule distinct from the epitope, 2) post-translational modifica-

tions, or 3) altered interaction with the cell surface or extracellular matrix due to changes in molecules other than fibronectin. Studies are in progress to determine what these differences are and to map the epitopes to which the antibodies bind. Fibronectin may be important in growth regulation (Rouslahti, 1988), and because the molecule is common to both senescent and young cells but has a n altered expression in senescent cells, identification of the specific epitopes involved may help in understanding the mechanisms involved in cellular senescence.

CONCLUSIONS Antigenic determinants expressed specifically in senescent cells provide biomarkers for the senescent phenotype. These unique antigenic determinants, together with the monoclonal antibodies reacting to them, may also represent powerful new tools to obtain direct evidence correlating the in vitro cellular aging model with in vivo cellular aging in the individual. ACKNOWLEDGMENTS We would like to thank John W. Burns for his assistance in generating the monoclonal antibodies. This work was supported by the Nobel Foundation, and grants from the National Institutes of Health, AG04749, AG-05333, and P01-AG-07123. LITERATURE CITED Absher, P.M., Absher, R.G., and Barnes, W.D. (1974) Genealogies of clones of diploid fibroblasts. Cinemicrophotographic observations of cell division patterns in relation to population age. Exp. Cell Rcs., 88t95-104. Bayreuther, K., Rodemann, H.P., Hommel, R., Dittman, K., Albiez, M., and Francz, P.I. (1988) Human skin fibroblasts in vitro differentiate along a terminal cell lineage. Proc. Natl. Acad. Sci. U.S.A., 85t5112-6116. Rrodeur, B.H., Tsang. P., and Larose, Y. (1984) Parameters affecting ascites tumor formation in mice and monoclonal antibody production. J. Immunol. Methods, 71t265-272. Brooks, K.M., Phillips, P.D., Carlin, C.R., Knowles, B.B., and Cristofalo, V.J. (1987)EGF-dependent phosphorylation of the EGF receptor in plasma membranes isolated from young and senescent WI-38 cells. J. Cell. Physiol., 133t523-531. Brunette, D.M., and Till, J.E. (1971) A rapid method for the isolation of L-cell surface membranes using an aqueous two-phase polymer system. J. Membr. Biol., 5:215--224. Burmer, G.C., Zeigler, C.J., and Norwood, T.H. 11982) Evidence for endogenous polypeptide-mediated inhibition of cell-cycle transit in human diploid cells. J. Cell Biol., 94t187-192. Burnette, W.N. (1981)“Western Blotting:” Electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal. Biochem., 112t195-203. Chandrasekhar, S., Sorrentino, J.A., and Millis, A.J.T. (1983) Interaction o f fibronectin with collagen: Age-specific defect in the biological activity of human fibroblast fibronectin. Proc. Natl. Acad. Sci. U.S.A., 8Ot4747-4751. Ching, G . ,and Wang, E. (1988)Absence of three secreted proteins and presence o f a 57-kDa protein related to irreversible arrest of cell growth. Proc. Natl. Acad. Sci. U.S.A., 85:151-155. Cristofalo, V.J., and Kritchevsky, D. (1969) Cell size and nucleic acid content in the diploid human cell line WI-38 during aging. Med. Exp. 19,313-320. Cristofalo, V.J., and Sharf, B.B. (1973) Cellular senescence and DNA synthesis. Thymidine incorporation a s a measure of population age in human diploid cells. Exp. Cell Res., 76,419-427, Drescher-Lincoln, C.K., and Smith, J.R. (1983) Inhibition of DNA synthesis in proliferating human diploid fibroblasts by fusion with senescent cytoplasts. Exp. Cell Res., 144t455-462. Drescher-Lincoln, C.K., and Smith, J.R. (1984) Inhibition of DNA synthesis in senescent-proliferating human cybrids is mediated by endogenous proteins. Exp. Cell Res., 153:208 -217.

UNIQUE SENESCENT CEI,L ANTIGENS Edick, G.F.. and Millis, A.J.T. (1984) Fibronectin distribution on the surfaces of young and old human fibroblasts. Mech. Ageing Dev., 27.249-256. Galfre, G., and Milstein, C. (1981) Preparation of monoclonal antibodies: Strategies and procedures. Methods Enzyrnol., 73:3-46. Grove. G.. and Cristofalo. V.J. (1977) Characterization ofthe cell cvcle of cultured human diploid cells: Effects of aging and hydrocortisone. J. Cell. Physiol., 90t415-422. Hayflick, L. (1965) The limited in vitro lifetime of human diploid cell strains. Exp. Cell Res., 37t6ll-636. Hayflick, L. (1977) The cellular basis for biological aging. In: Handbook of the Biology of Aging. C.E. Finch and L. Hayflick, eds. Van Nostrand and Rheinhold, New York, pp. 159-186. Hayflick, L., and Moorhead, P.S. (1961) The serial cultivation of human diploid cell strains. Exp. Cell Res., 25585-621. Laemmli, U.K. (1970) Cleavage o f structural proteins during the assembly of the head of the bacteriophage T4. Nature, 227:680-685. Lincoln, D.W., 11, Braunschweiger, K.I., Braunschweiger, W.R., and Smith, J.R. ( 19843 The two-dimensional polypeptide profile of terminally nondividing human diploid cells. Exp. Cell Res., 154:136146. Macieira-Coelho, A,, Ponten, J.,and Philipson, L. (1966)The division cycle and RNA-synthesis in diploid human cells a t different passage levels in vitro. Exp. Cell Res., 421673-684. Matsumura, T., Pfendt, E.A., and Hayflick, L. (1979a3 DNA synthesis in the human diploid cell strain WI-38 during in vitro aging: An autoradiography study. J. Gerontol., 34:323-327. Matsumura, T., Zerrudo. Z., and Hayflick, L. (1979b) Senescent human diploid cells in culture: Survival, DNA synthesis and morphology. J. Gerontol., 34:328-334. Norwood, T.H., and Smith, J.R. (1985) The cultured fibroblast-like cell as a model for the study of aging. In: Handbook of the Biology of Aging (2nd Ed.). C.E. Finch and E.L. Schneider, eds. Van Nostrand, New York, pp. 291-311.

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Pereira-Smith, O.M.. Fisher, S.F., and Smith, J.R. (19851 Senescent and quiescent cell inhibitors of DNA synthesis: Membrane-associated proteins. Exp. Cell Res., lfiOi0t297-306. Pereira-Smith, O.M., and Smith, J.R. (1988, Genetic analysis of indefinite divisinn in human cells: Identification of four complernentation groups. Proc. Natl. Acad. Sci. U.S.A., 85t6042-6046. Rouslahti E. 11988) Fibronectin and its receptore. Annu. Rev. Biocheni., 57t375-413. Smith, J.R., and Hayflick, I,. (1974) Variation in the life-span of clones derived from human diploid cell strains. J. Cell Biol., 62: 48-53. Smith, J.R., and O.M. Pereira-Smith (1985) Lung-derived fibroblastlike cells in culture. In: CRC Handbook of Cell Biology. V.J. Cristofalo, ed. CRC Press, Boca Raton, Florida, pp. 375-423. Smith. J.R., Pereira-Smith, O.M., and Schneider, E.L. (1978) Colony size distributions as a measure of in vivo and in vitro aging. Proc. Natl. Acad. Sci. U.S.A., 75t1353-1356. Sorrentino, J.A., and Millis, A.J.T. (1984) Structural comparisons of fibronectins isolated from early and late passage cells. Mech. Ageing Dev. 28t83-97. Sottile, J.,Hoyle, M., and Millis, A.J.T. (1987) Enhanced synthesis of a Mr 55,000 Dalton peptide by senescent human fibroblasts. J . Cell. Physiol., 131:210-217. Stein, G.H., Atkins, L. (1986)Membrane-associated inhibitor of DNA synthesis in senescent human diploid fibroblasts: Characterization and comparisnn to quiescent cell inhibitor. Proc. Natl. Acad. Sci. U.S.A., 83:9030-9034. Towbin, H., Staehelin, T., and Gordon, J. (1979)Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. U.S.A.. 76: 4350-4354. Wang. E. (1985) A 57,000-mol-wt-proteinuniquely present in nonproliferating cells and senescent human fibroblasts. J. Cell Biol., 100: 545-551.

Novel monoclonal antibodies identify antigenic determinants unique to cellular senescence.

Normal human diploid fibroblasts exhibit a limited lifespan in vitro and are used as a model to study in vivo aging. Monoclonal antibodies were genera...
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