JOURNAL OF CELLULAR PHYSIOLOGY 144313-325 (1990)

Altered Regulation of Platelet-Derived Growth Factor A-Chain and c-fos Gene Expression in Senescent Progeria Fibroblasts JEFFREY A. WINKLES,* M A R Y L. O ' C O N N O R , AND ROBERT FRIESEL Laboratory of Molecular 6JO/Ogy, lerome H. Holland Ldbordtory tor the B~orned~cal Sciences, AnicrJran Red Cross, Rockville, Mdryland 20855 The study of human genetic disorders known as premature aging syndromes may provide insight into the mechanisms of cellular senescence. These diseases are clinically characterized by the premature onset and accelerated progression of numerous features normally associated with human aging. Previous studies have indicated that fibroblasts derived from premature aging syndrome patients have in vitro growth properties similar to senescent fibroblasts from normal individuals. As an initial approach to determine whether gene expression is altered in premature aging syndrome fibroblasts, RNA was prepared from various cell strains and used for gel blot hybridization experiments. Although normal fibroblasts only express platelet-derived growth factor (PDGF) A-chain mRNA for a brief period following mitogenic stimulation, one strain of Hutchinson-Cilford (progeria) syndrome fibroblasts, AG3.513, constitutively expresses PDGF A-chain mKNA and PDCF-AA homodimers. The PDGF A-chain gene does not appear to be amplified or rearranged in these fibroblasts. AG3513 progeria fibroblasts have propcrties characteristic of senescent cells, including an altered morphology and a diminished mitogenic response to growth promoters. The diminished response of AC3513 progeria fibroblasts to PDGF stimulation was examined in w m e detail. Studies using "51-PDGF-BB, which binds with high affinity to both A- and 6-type PDGF receptors, indicate that normal and AC3513 progeria fibroblasts have a similar number of PDGF receptors. Although receptor autophosphorylation occurs normally in PDGF-stimulated AG3513 progeria fibroblasts, c-fos mRNA induction does not. The senescent phenotype of AG3513 fibroblasts i s probably unrelated to their constitutive PDGF A-chain gene expression; further studie5 are necessary in order to directly address this issue. Also, additional analysis of this progeria fibroblast strain may provide information on the control of mitogeninducible gene expression in normal cells.

Normal human diploid cells have a finite in vitro replicative life span (Hayflick and Moorhead, 1961; Hayflick, 1965). As cells are repeatedly passaged, they eventually enter an irreversible nongrowing condition termed senescence. Although numerous cell types have been utilized in studies of cell aging, the properties of pre-senescent and senescent embryonic lung- or skinderived fibroblasts have been described in greatest detail. Various morphological, cytological, and biochemical changes have been reported to occur as fibroblasts age in vitro (reviewed in Stanulis-Praeger, 1987). As an alternative to comparing early and late passage fibroblasts derived from a single donor, some investigators have compared similar passage fibroblasts derived from either normal individuals or premature aging syndrome patients. Premature aging syndromes, such as Werner's syndrome and Hutchinson-Gilford Progeria syndrome (hereafter progeria), are rare human disorders characterized by the accelerated development of many features associated with normal aging (reviewed in Goldstein, 1978;Beauregard and Gilchrest, 1987).In comparison to fibroblasts from age-matched controls, Q 1990 WILEY-LISS, INC.

fibroblasts from premature aging syndrome patients frequently have low growth rates and a reduced replicative life span (Danes, 1971; Harley et al., 1981; Salk et al., 1981a; Brown et al., 1984). These findings support the notion that the biochemical lesion(s) responsible for the accelerated aging phenotype characteristic of these syndromes may be expressed by skin fibroblasts grown in culture. One diagnostic property of senescent fibroblasts is their diminished ability to synthesize DNA in response to growth factor stimulation. This property has been demonstrated in studies comparing early and late passage cells derived from a single donor (Phillips et al., 1984, 1987; Tsuji et al., 1984), similar passage cells

Received February 16, 1990; accepted May 2, 1990.

*To whom reprint requestsicorrespondence should be addressed.

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derived from young and old donors (Plisko and Gilchrest, 1983), and similar passage cells derived from age-matched normal individuals and premature aging syndrome patients (Harley et al., 1981; Bauer et al., 1986).The molecular basis for the diminished response to growth factors characteristic ofthe senescent state is unknown. Senescent cells may be deficient in some component(s) associated with cell proliferation; the reduced expression of c-fos and histone 3.2 mRNA (Seshadri and Campisi, 19901, a s well a s thymidine kinase mRNA (Chang and Chen, 1988), after mitogenic stimulation of senescent fibroblasts supports this explanation. Alternatively, or additionally, senescent cells may produce a factorb) which prevents a mitogenic response. Somatic cell hybridization (Norwood et al., 1974; Yanishevsky and Stein, 19801, cytoplast fusion (Burmer et al., 1983; Drescher-Lincoln and Smith, 1983; Pereira-Smith et al., 19851, and mRNA injection (Lumpkin et al., 1986) experiments indicate that senescent fibroblasts may in fact express genes encoding polypeptides with antiproliferative functions. The possibility that fibroblasts express senesccncespecific gene products with antiproliferative functions has renewed interest in the identification of polypeptides overexpressed in senescent fibroblasts. Although early studies using two-dimensional polyacrylamide gel electrophoresis identified a few proteins that appeared to be senescent cell-specific, the vast majority of the proteins detected were synthesized by both young and old fibroblasts (Sakagami et al., 1979; Engelhardt e t al., 1979; Lincoln et al., 1984). Senescent-specific polypeptides of 57 kDa and 55 kDa have been described by Ching and Wang (1988) and Sottile et al. (1987), respectively. West et al. (1989) have suggested that these proteins may be forms of procollagenase, a protein overexpressed in senescent normal fibroblasts (West et al., 1989) and fibroblasts derived from Werner’s syndrome patients (Bauer et al., 1986). Additional examples of mRNAs and/or polypeptides overexpressed in late passage fibroblasts include c-Ha-ras p21 (Srivastava e t al., 1985), statin (Wang, 1985), and varia n t forms of elongation factor ltr (Giordano and Foster, 1989) and histone 3 (Seshadri and Campisi, 1990). Platelet-derived growth factor (PDGP) is the major mitogen in serum for connective tissue-derived cells and one of the most extensively characterized polypeptide growth factors (reviewed in Ross e t al., 1986). There are two distinct PDGF polypeptides, denoted A and €3, that are approximately 60% similar in amino acid sequence (Hetsholtz et al., 1986). Biologically active PDGF exists as a disulfide-bonded dimer; all three possible PDGF dimer forms ( AA,BB,AB) have been identified and purified (Stroobant and Waterfield, 1984; Hcldin et al., 1986; Hammacher et al., 1988a).In this report, we describe the altered expression of PDGF A-chain mRNA and protein in one strain of progeria premature aging syndrome fibroblasts. Although these cells are senescent, as evaluated by their morphology and proliferative response to growth factor stimulation, i t is unlikely that this phenotype is related to their constitutive PDGF-AA secretion. However, PDCF-stimulated progeria fibroblasts express reduced levels of c-fos niRKA; this property could contribute to the diminished mitogenic response to growth factor stimulation characteristic of this cell strain.

MATERIALS AND METHODS Cell culture Human umbilical vein endothelial cells (kindly provided by Dr. T. Maciag, American Red Cross, Rockville, MD) were grown as previously described (Gay and Winkles, 1990). Human aortic smooth muscle cells (kindly provided by Dr. R. Weinstein, St. Elizabeth‘s Hospital, Boston, MA) were grown as described by Winkles et al. (1987). Skin fibroblasts derived from normal individuals (GM0498, 3-year-old male; GM0499, 8-year-old male; GM2037, 13-year-old male; AG6009, 59-year-old male) and patients with Hutchinson-Gilford syndrome (AG6917, 3-year-old male; AG6297, 8-year-old male; AG3513, 13-year-old male) or Werner’s syndrome (AG0780, 59-year-old male) were purchased from the Human Genetic Mutant Cell Repository, Camden, YJ. Standard growth medium was Dulbecco’s modified Eagle’s medium (DMEM; Mediatech) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Hyclone) and l x antibiotic/ antimycotic (Gibco). Fibroblasts were routinely cxpanded by trypsin treatment and subculturing at a 1:4 split ratio and were used at similar passage levels for all experiments. Most experiments employed a normal (GM2037) and progeria (AG3513) fibroblast strain derived from age- and sex-matched donors. These cells were used between passages 12 and 15 (approximately 24-30 cumulative population doublings).

RNA gel blot analysis RNA was prepared from endothelial cells, smooth muscle cells, and fibroblasts growing in their normal culture media and from PDGF-stimulated fibroblasts. For the PDGF-stimulation experiments, normal and progeria fibroblasts were grown to 80% confluence, serum-starved for 48 h r in DMEMi0.2% FBS, and either left unstimulated or stimulated with 20 ngi’ml PDGF (Bachem) for the indicated times. The PDGF purchased from Hachem was recombinant human PDGF-BB. Cells were harvested, total RNA was prepared, and 10 bg of each sample was subjected to electrophoresis in a 1.2% agarose gel containing 2.2 M formaldehyde a s described previously (Winkles et al., 1987). The gels were stained with ethidium bromide to verify that each lane contained similar amounts of undegraded ribosomal RNA. RKA was electroblotted onto Zetabind nylon filters (AMF Cuno) and cross-linked by UV irradiation. The restriction fragments used and the source of human DNA probes were as follows: 1)PDGF-A, 1.3-kb EcoRI fragment of pUC-13-D1, gift of B. Westermark, Ludwig Institute of Cancer Research, Uppsala, Sweden; 2 ) v-sis, 0.9-kb PstIiXbaI fragment of pv-sis, ATCC, Rockville, MD; 3) c-fos, 2.8-kb NcoLiXhoI fragment of pc-fos-1, ATCC; 4) glyceraldehyde 3-phosphate dehydrogenase (GAPDH), 0.8-kb PstIiXbaI fragment of pHcGAP, ATCC. The probes were labeled with [“PldCTP 13,000 Ciimmol, Amersham) using a multiprime labeling kit (Amersham). Hybridization and filter washes were a s described (Gay and Winkles, 1990). Blots were exposed to Kodak XAR5 films at - 70“ C. Autoradiographic signals were quantitated using a n LKB laser densitometer.

GENE EXPRESSION IN SENESCENT FIBROBLASTS

Immunoprecipitation analysis Normal and progeria fibroblasts were grown to confluence, rinsed twice with phosphate-buffered saline (PBS), and incubated for 3 h r in serum-freeicysteinefree medium containing 170 pCi/ml ["Slcysteine (1,200 Ciimmol; Amersham). Labeling medium was recovered, and the cells were incubated for a n additional 1 h r in the above medium without [''SJcysteine. Pulse and chase media were pooled, and phenylmethylsulfonyl fluoride (Sigma), aprotinin (Boehringer-Mannheim), and Triton X-100 (Sigma) were added to final concentrations of 0.5, 2.5, and 0.01%, respectively. The conditioned medium was then concentrated 30-fold using a Centricon-10 microconcentrator (Amicon). An equivalent number of trichloracetic acid-precipitable cpm from each cell type was then incubated at 4°C for 18 h r with normal rabbit serum, and protein ASepharose beads (Sigma) were added for 2 hr. Beads were removed by centrifugation and supernatants were incubated at 4°C for 18 h r with either nonimmune rabbit serum or PDGF A-chain antiserum (Genzyme). Samples were then incubated with protein ASepharose beads for 2 h r a t 4°C. The beads were washed four times with 0.5 M NaCl, 10 mM Tris-HC1, pH 7.6,5 mg!ml bovine serum albumin, 0.1% Tween 20 and once with 10 mM Tris-HC1, pH 7.6. Immunocomplexes were resuspended in 0.125 M Tris-HC1, pH 6.8, 4% sodium dodecyl sulfate (SDS), 20% glycerol, heated at 95°C for 5 min, and beads were removed by centrifugation. The supernatants were each divided into two aliquots; dithiothreitol (Sigma) was added to one aliquot (10 rnM final concentration) and the sample was again heated at 95°C for 3 min. The reduced and nonreduced samples were analyzed on a 15% polyacrylamide-SDS gel. The gel was stained, destained, and treated with Enlightening (Du Pont-New England Nuclear) prior to autoradiography.

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and hybridization (18 hr) were performed a t 65°C in 0.1 M NaH,PO,, 0.85 M NaCI, 1 0 Ilenhardt's ~ (0.2% Ficoll, 0.2% polyvinylpyrrolidone, 0.2% bovine serum albumin), 2 mM EDTA, 0.1% SDS, and 100 pgiml denatured salmon sperm DNA (Sigma). The PDGF Achain cDNA insert was radiolabeled as described for the RNA gel blots. The filter was extensively washed in 0.2 x SSPE at 65°C and exposed to Kodak XAR5 film a t -70°C.

Cell proliferation assays For thymidine incorporation experiments, normal and progeria fibroblasts were seeded at lo4 cells/well in 24-well plates, grown t o 80% confluence in standard growth medium, and then cultured in DMEM/0.2% FBS for 48 hr. They were then either left unstimulated or stimulated with DMEM containing either 10% FBS, 20 ngiml PDGF, 20 ng/ml heparin-binding growth factor (HBGF)-1, 20 ngiml HBGF-2, 10 ngiml epidermal growth factor (EGF), or 10 ngiml insulin-like growth factor-1 (IGF-I). PDGF (R&D Systems, Inc.) was from porcine platelets and thus consists predominantly of PDGF-BB (Stroobant and Waterfield, 1984; BowenPope et al., 1989). Bovine brain HBGF-1 (the a-form of endothelial cell growth factor (Burgess et al., 1985)) was provided by W. Burgess, American Red Cross, Rockville, MD. Recombinant bovine HBGF-2 (basic fibroblast growth factor) was from Amgen. EGF (murine) and IGF-I (human) were purchased from Boehringer-Mannheim and Imcera, respectively. After 8 hr of mitogenic stimulation, 1 KCiiml ['Hlthymidine (25 Ciimmol, Amersham) was added and the cultures were incubated with the growth promoters for a n additional 40 hr. Cells were washed once with PBS and then precipitated with 10% trichloracetic acid for 15 rnin at 4°C. After one rinse with PBS, precipitable material was solubilized with 0.5 ml 0.1 N NaOH for 30 min at 37°C. Samples were neutralized with 0.5 ml0.1 N HC1 and 10 ml Beckman Ready Safe scintillation fluid was added Genomic DNA gel blot analysis for counting in a Beckman LS3801. Human placental DNA was purchased from ClonFor cell growth assays, normal and progeria fibrotech. To isolate fibroblast genomic DNA, cells were in- blasts were seeded at l o 4 cellsiwell in 24-well plates cubated in 10 mM "ris-HC1 pH 7.6, 100 mM NaC1, 1 and cultured in DMEM containing 10% bovine platemM EDTA, 1% SDS, and 100 pgiml proteinase K (Worth- let-poor plasma-derived serum (Biomedical Technoloington) for 18 h r at 37°C. Samples were then extracted gies), 1x antibioticiantimycotic, in the presence or abonce with phenol, twice with 2:l (volivol) isobutanoli sence of 10 ngiml porcine PDGF. Cells were refed every isopropanol, and precipitated by the addition of 2 vol- other day with the appropriate media. Cell number was umes cold ethanol. Total nucleic acid was recovered by determined 4 h r after plating (day 0) and a t days 5 and centrifugation, dissolved in 10 mM Tris-HC1 pH 7.6, 1 10 using a hemocytometer. Neither cell type had mM EDTA, and incubated with 200 pgiml RNAse A reached confluency by day 10. (Worthington) for 3 h r at 37°C. Samples were then exPDGF receptor binding and tracted with 24:1 tvolivol) chloroformiisoamyl alcohol, autophosphorylation assays ethanol precipitated, and dissolved in 10 mM Tris-HC1 pH 7.6, 1 mM EDTA. DNA was quantitated by meaFor binding assays, normal and progeria fibroblasts suring absorbance at 260 nm. Placenta and fibroblast were seeded a t lo4 cellsiwell in 24-well dishes and DNA samples (15 p,g) were digested with BamHI, Xbal, grown to 80% confluency in standard fibroblast growth or SstI (BRL) and subjected t o electrophoresis on a 0.8% media. Cells were then incubated in DMEMi0.2% FBS agarose gel. The gel was sequentially treated with 0.2 for 48 hr. After two washes with ice-cold binding meN HCI (15 rnin), H 2 0 (15 min), 1.5 M NaCli0.5 N NaOH dium (DMEM, 0.1% bovine serum albumin, 25 mM (2 x 15 m i d , 2 M CH3CO2NH4(2 x 15 min), and then Hepes), each well received 0.2 ml binding medium, vartransferred by capillary blotting onto a nitrocellulose ious concentrations of l2'1-PDGF-BB (800 Ciimmol, filter (Schleicher and Schuell) in l o x SSCil M Amersham), and for those wells representing non-speCH3C0,NH4. After rinsing for 5 min in 0.9 M cific binding, 100-fold excess unlabeled PDGF-BB CH,C0,NH4, the filter was air dried and placed in a (Bachem).Cultures were incubated for 3 h r at O'C with vacuum oven for 2 h r at 80°C. Prehybridization (2 hr) gentle shaking. Binding was terminated by washing

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the cells four times with ice-cold binding medium and then 0.2 ml PBS containing 0.1% bovine serum albumin, 1%Triton X-100 was added. Cell-associated radioactivity was measured in a n LKB Model 1271 ycounter. The cell number of duplicate cultures was determined after trypsin treatment by counting in a hemocytometer chamber. Binding data was obtained from duplicate cultures representing total or non-specific binding and analyzed according to Scatchard (1949) using the LIGAND fitting program (Munson and Rodbard, 1980). Receptor autophosphorylation was examined by immunoblot analysis using anti-phosphotyrosine antibodies. The preparation and characterization of these antibodies is described in Friesel et al. (1989). Normal and progeria fibroblasts were grown to confluence in 10-cm plates and serum-starved a s described above. Cells were either left unstimulated or stimulated with 10 ngiml PDGF €or 10 min a t 37°C. Cell lysates were prepared as described (Friesel et al., 1989) and protein concentrations were determined using the BCA protein assay (Pierce Chemical Co.). Lysates (200 pg’lane) were subjected t o electrophoresis on a 7.5% polyacrylamide-SDS slab gel. Proteins were transferred to a nitrocellulose filter (Bio-Rad) by electroblotting and the filter was sequentially incubated with anti-phosphotyrosine antibody and then 1251-protein A as described previously (Friesel et al., 1989). Binding of lz5Iprotein A was visualized by autoradiography.

SKIN FIBROBLASTS

B

RESULTS Expression of PDGF A-chain mRNA in AG3513 progeria fibroblasts As a n approach to identify specific genes that are differentially expressed in pre-senescent and senescent fibroblasts, total RNA was prepared from normal and premature aging syndrome fibroblasts and equivalent amounts were used in gel blot hybridization experiments. The results obtained using DNA probes for the PDGF A-chain and B-chain mRNAs are shown in Figure 1. Since a previous study demonstrated that normal human dermal fibroblasts in culture do not express PDGF mRNA (Betsholtz et al., 1986), human endothelial and smooth muscle cell RNA samples were included as positive hybridization controls. PDGF Achain transcripts of identical size (,approximately 2.8-, 2.4-, and 1.7-kb) were detected in both vascular cell types and in one strain (AG3513) of progeria fibroblasts (Fig. 1A). PDGF A-chain mRNA expression was undetectable in two other progeria fibroblast strains, a strain of Werner’s syndrome fibroblasts, and in ageand sex-matched normal fibroblasts corresponding to each of the four premature aging syndrome strains. The expression level in one of these normal strains (GM20371,the control for AG3513 fibroblasts, is shown in this figure. PDGF B-chain mRNA was expressed by endothelial cells, but not by smooth muscle cells or any of the normal or premature aging syndrome fibroblast strains examined (Fig. 1B). Previous studies have shown t h a t PDGF (Paulsson et al., 1987a), EGF (Paulsson et al., 1987a), transforming growth factor-p (Paulssonet al., 1988; Soma and Grotendorst, 1989), interleukin-1 (Raines et al., 1989), and tumor necrosis factor (Paulsson et al., 1989) rapidly

Fig. 1. Expression of PDGF A- and B-chain mRNAs in normal and premature aging syndrome fibroblasts. Total RNA was prepared from endothelial cells (EC), smooth muscle cells (SMC), normal fibroblasts (GM20371, Hutchinson-Gilford (progeria) syndrome fibroblasts (AG6917, 6297, 3513), and Werner’s syndrome fibroblasts (AG0780) and equivalent amounts of each sample were used for RNA gel blot analysis. Blots were hybridized to either radiolabeled PDGF A-chain cl)NA (A) or PDGb’ B-chain (v-sis) DNA (B). In this and subsequent RNA gel blot figures, the upper and lower tick marks on the right side of each panel represent the position of 28 and 18s rRNA, respectively.

and transiently increase PDGF A-chain mRNA levels in human fibroblasts. To assay PDGF A-chain mRNA levels in quiescent and growth factor-induced AG3513 fibroblasts, normal and progeria fibroblasts were serum-starved for 48 h r and then stimulated with PDGF. In this and subsequent experiments using PDGF we have used either porcine PDGF (which is predominantly PDGF-BB (Stroobant and Waterfield, 1984; Bowen-Pope et al., 1989) or recombinant human PDGFBB. Previous studies have demonstrated that human skin fibroblasts show a strong mitogenic response to this PDGF isoform (Heldin et al., 1988; Hosang e t al., 1989; Seifert et al., 1989; Raines et al., 1989). Cells were collected at various times post-stimulation, RNA was prepared, and RNA gel blots were hybridized to the PDGF A-chain cDNA probe. In agreement with the study of Paulsson et al. (1987a), PDGF A-chain mRNA was detected a t 2 and 4 h r after PDGF stimulation of normal fibroblasts (Fig. 2A). However, PDGF A-chain mRNA was expressed at a similar level in serum-

GENE EXPRESSION IN SENESCENT FIBROBLASTS

PDGF I

I

0' 3 0 ' 2 h 4 h 8h 16h

A

Fig. 2. Expression of PDGF A-chain mRNA in PDGF-stimulated normal and progeria fibroblasts. Serum-starved normal (GM2037) and progeria (AG3513) fibroblasts were either left unstimulated or stimulated with 20 ng/ml PDGF for the indicated times. RNA was prepared and equivalent amounts of each sample were used for RNA gel blot analysis using radiolabeled PDGF A-chain cDNA probe. The result obtained with normal fibroblasts is shown in A, and with progeria fibroblasts in B.

starved and PDGF-stimulated progeria fibroblasts (Fig. 2B).

Secretion of PDGF-AA homodimers by AG3513 fibroblasts Direct evidence that the constitutive expression of PDGF A-chain mRNA results in constitutive PDGFAA production was obtained by immunoprecipitation experiments using A-chain-specific antiserum. Normal and progeria fibroblasts were cultured for 3 h r in the presence of [35Slcysteine; the medium was then collected, concentrated, and incubated with either nonimmune rabbit serum or PDGF A-chain-specific antiserum. This PDGF antiserum can recognize both PDGFAA and PDGF-AB. Immunoprecipitates were adsorbed to protein A-Sepharose and analyzed on 15% polyacrylamide-SDS gels. When samples were not treated with reducing agent prior to electrophoresis, a major species of 31 kDa, and minor species of 35 and 33 kDa, were specifically precipitated from the culture medium of AG3513 fibroblasts, but not GM2037 fibroblasts (Fig. 3A). When immunoprecipitates were reduced prior to gel loading, a diffuse band at 17 kDa was detected, again only in the AG3513 fibroblast sample (Fig, 3B). Additional experiments revealed that this diffuse band contains two closely migrating polypeptides. Identical immunoprecipitation experiments using PDGF B-

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chain-specific antiserum failed to detect PDGF B-chain secretion by either GM2037 or AG3513 fibroblasts (data not shown). P D G F A-chain gene structure in AG3513 fibroblasts The molecular mechanism responsible for altered PDGF A-chain mRNA levels in AG3513 fibroblasts is presently unknown, but could reflect constitutive gene transcription and/or stabilization of PDGF A-chain transcripts. Since fibroblasts cultured from Werner's syndrome patients frequently contain chromosomal translocations, inversions, and deletions (Salk et al., 1981b; Fukuchi et al., 1989) we determined whether PDGF A-chain gene structure was grossly altered in this progeria fibroblast strain. DNA prepared from GM2037 and AG3513 fibroblasts was digested with three restriction endonucleases t h a t recognize nonpolymorphic sites within the PDGF A-chain locus (Bonthron et al., 1988; Rorsman et al., 1988; Ferns and ROSS,1990). Digested DNA was fractionated by electrophoresis, blotted, and hybridized to radiolabeled PDGF A-chain cDNA. As shown in Figure 4, a similar pattern of hybridizing fragments was detected using placenta DNA and both fibroblast DNA samples; fragment sizes corresponded to those predicted from the physical map in Rorsman et al. (1988). Morphology and mitogenic responsiveness of GM2037 and AG3513 fibroblasts During the early stage of this work, it became obvious that progeria fibroblasts had a n altered morphology and a longer generation time than normal fibroblasts. In comparison to normal fibroblasts, progeria fibroblasts were larger, flatter, more vacuolated, and had more cytoplasmic processes (Fig. 5). These morphological properties are characteristic of senescent normal fibroblasts (Crusberg et al., 1979; Wang and Gundersen, 1984; Sherwood et al., 1978; Angello et al., 1989). It should be emphasized that the morphology of progeria fibroblast strain AG3513 is not noticeably different from that of progeria fibroblast strains that do not continually secrete PDGF-AA. As human fibroblasts age in vitro there is a progressive decline in their ability to replicate DNA in response to mitogenic stimulation (Phillips e t al., 1984,1987; Tsuji et al., 1984). The response of serumstarved GM2037 and AG3513 fibroblasts to various growth promoters was examined by measuring the incorporation of ['Hlthymidine into DNA. As shown in Figure 6A, AG3513 progeria fibroblasts had a diminished mitogenic response to FBS and all purified mitogens tested; the response varied from 73% (IGF-I) to 35% (PDGF) of that observed in GM2037 normal fibroblasts. Since the difference observed with PDGF was particularly striking, the response of AG3513 fibroblasts t o this mitogen was examined in more detail. A PDGF dose-response experiment was performed to test whether AG3513 fibroblasts show a normal proliferative response at higher PDGF concentrations. The incorporation of [3Hlthymidine into DNA of normal f i broblasts stimulated with 10 ngiml PDGF was 420% that of unstimulated cells, while t h a t of progeria fibroblasts stimulated with 50 ng/ml PDGF was 210% that of unstimulated cells (Fig. 6B). Therefore, a 5-fold in-

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GM 2037

GM 2037

AG 35 13

A

AG 3513

B

43-

29-

18.4-

14.3-

Fig. 3. PDGF-AA secretion by normal and progeria fibroblasts. Norma1 (GM2037) and progeria (AG3513) fibroblasts were labeled with [35S]cystcine and the conditioned media were collected. Immunoprecipitation using either nonimmune rabbit serum or PDGF A-chain antiserum was performed as described under “Materials and Methods.” Immunoprecipitated proteins were either left rionreduced (A) or

reduced with dithiothreitol (B) prior to electrophoresis on a 15% polyacrylamide-SDS gel. Radiolabeled immunoprecipitates were visualized by autoradiography. Molecular masses (in kilodaltons) of 14C-labeled molecular weight markers (BRL) are shown on the left side of A.

crease in the concentration of PDGF still fails to elicit a mitogenic response in AG3513 progeria fibroblasts similar to that observed in GM2037 normal fibroblasts. Although the [3H]thymidine incorporation assays indicate a diminished mitogenic response by progeria fibroblasts, i t is possible that these cells are defective in the uptake or metabolism of exogenously supplied [3H]thymidine. To address this issue, we measured the PDGF-induced proliferative response of normal and progeria fibroblasts using a cell growth assay. GM2037 and AG3513 fibroblasts were grown in DMEM/lO% platelel-poor plasma in the absence or presence of 10 ngiml PDGF and cell number was determined at 5 and 10 days (Fig. 7). After 10 days of growth in the absence of PDGF, progeria fibroblasts grew to 75% of the control fibroblast cell number. PDGF stimulated normal fibroblast growth another 1.8-fold, but stimulated progeria fibroblast growth only 1.2-fold. The above results indicate that, as assayed by either L3Hlthymidine incorporation or cell growth, AG3513 fibroblasts have a diminished mitogenic response to PDGF stimulation.

tors. In general, pre-senescent and senescent fibroblasts contain a similar number of growth factor receptors (Phillips et al., 1983, 1987; Paulsson et al., 1986); however, in the case of AG3513 fibroblasts, a decreased number of PDGF binding sites could result from secreted PDGF-AA homodimers causing receptor downregulation (Heldin et al., 1982; Hart et al., 1988). PDGF binding studies using both normal cells (Hart et al., 1988; Heldin et al., 1988; Hosang e t al., 1989; Seifert et al., 1989, Hammacher e t al., 1989) and cells transfected with each of two distinct PDGF receptor cDNA clones (Escobedo et al., 1988; Claesson-Welsh et al., 1988, 1989a; Gronwald et al., 1988; Matsui et al., 1989a,b; Severinsson et al., 1989) demonstrate the existence of multiple PDGF receptor classes. Seifert e t al. (1989) have proposed that the two PDGF receptor polypeptides, which they call the a- and p-subunits, can dimerize to form three receptor classes. The aa receptor, corresponding to the A-type receptor described initially by Heldin et al. (1988), can bind all three PDGF isoforms with high affinity, the aP receptor binds both PDGF-AB and PDGF-BB, and the PP receptor binds only PDGF-BB. The P-subunit-containing receptor classes would correspond to the B-type receptor described by Heldin et al. (1988). Human dermal fibroblasts contain predominantly p-subunits (B-type receptors); PDGF-AA can bind to only 5% of the sites

Characterization of PDGF-BB binding sites on GM2037 and AG3513 fibroblasts The diminished proliferative response of AG3513 fibroblasts after mitogenic stimulation could reflect a significantly reduced number of growth factor recep-

GENE EXPRESSION IN SENESCENT FIBROBLASTS

BamHI 23.1

-

9.4

-

6.6

-

4.4

-

2.3 2.0

-

s st I

XbaI

Fig. 4 Genomic structure of the PDGF A-chain gene in normal and progeria fibroblasts DNA from human placenta (PI, the normal fibroblast strain GM2037 (N), and the progeria strain AG3513 (Pr) was digested with the indicated restriction enzymes and used for genomic DNA gel blot analysis using radiolabeled PDGF A-chain cDNA probe A HindIII-digested h size standard is shown (kbj

available to PDGF-BB (Seifert et al., 1989) and consequently is a relatively poor mitogen for these cells (Heldin et al., 1988; Raines et al., 1989; Hosang et al., 1989; Seifert et al., 1989). On the basis of these studies, one would predict that AG3513 fibroblast-derived PDGF-AA would downregulate only a minor fraction of the total PDGF binding sites on these cells. To test this prediction, PDGF-BB binding studies were done using both GM2037 and AG3513 fibroblasts. Data obtained using this PDGF isoform should represent the combined number of Aand B-type receptors and their average dissociation constant (K,&. The K, for the binding of PDGF-BB to GM2037 normal fibroblasts was 4.8 0.3 x lop1' M with 3.40 t 0.02 x lo5 sites per cell (mean SD). The AG3513 progeria fibroblasts had approximately 20% fewer binding sites per cell (2.72 0.16 x lo5) of similar ligand affinity (Kd of 3.5 ? 0.3 x 10-l0M) (Fig. 8A). The attenuated mitogenic response to PDGF in progeria cells is therefore not attributable to a large reduction in PDGF receptor number or ligand affinity. Although AG3513 fibroblasts contain receptors capable of binding PDGF-BB, they may still be defective. PDGF binding induces rapid receptor autophosphorylation on tyrosine residues (Ek and Heldin, 1982,1984; Frackelton et al., 1984; Daniel et al., 1985; Bishayee et al., 1986; Kazlauskas and Cooper, 1989). As a n assay for receptor function, we determined whether the PDGF receptors on AG3513 fibroblasts would autophosphorylate in response to ligand occupancy. GM2037 and AG3513 fibroblasts were serum-starved and then either left unstimulated or stimulated with PDGF for 10 min. Cell lysates were prepared and receptor tyrosine kinase activity assayed by immunoblot-

*

*

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ting using anti-phosphotyrosine antibody. I n both GM2037 and AG3513 fibroblasts a polypeptide of approximately 180 kDa was tyrosine-phosphorylated after PDGF addition (Fig. 8B). The apparent molecular weight of this polypeptide is similar to that reported for both the A- and B-type PDGF receptors on human fibroblasts (Claesson-Welsh et al., 1989b). Therefore, the PDGF receptors on AG3513 fibroblasts are not defective in their ability to autophosphorylate after PDGFBB binding.

Expression of c-fos mRNA in PDGF-stimulated GM2037 and AG3513 fibroblasts AG3513 fibroblasts have a diminished mitogenic response to multiple growth factors. This may reflect a deficiency in some post-receptor signal transduction event common to many growth promoting agents; one such event is the enhanced expression of specific genes, including proto-oncogenes (reviewed in Rollins and Stiles, 1989). One of the best characterized growth factor-inducible proto-oncogenes is c-fos. Increased levels of c-fos mRNA occur when many diverse cell types are exposed to different growth stimuli (reviewed in Rollins and Stiles, 1988). Recently, Seshadri and Carnpisi (1990) reported that c-fos expression was diminished in serum-induced senescent fibroblasts. Therefore, we assayed c-fos mRNA levels after PDGF stimulation of serum-starved GM2037 and AG3513 fibroblasts by RNA gel blot analysis. The intensity of autoradiographic signals was quantitated by densitometry. To correct for slight differences in the amount of RNA per lane, c-fos mRNA levels were normalized to GAPDH mRNA levels. As shown in Figure 9, the c-fos expression pattern we observed in skin fibroblasts derived from the normal donor were similar to results obtained by Paulsson et al. (1987b) using foreskin fibroblasts and Seshadri and Campisi (1990) using fetal lung fibroblasts. Specifically, c-fos mRNA was expressed transiently, with the maximal level evident at 0.5 h r post-stimulation. When AG3513 progeria fibroblasts were stimulated with PDGF, the temporal pattern of c-fos expression was similar but the induction level was reduced, representing 1 0 8 of that observed in normal fibroblasts.

DISCUSSION PDGF A-chain mRNA is expressed by various tumor cell lines (Betsholtz et al., 1986; Bronzert e t al., 1987; Matoskova e t al., 19891, endothelial cells (Sitariset al., 1987; Collins et al., 1987; Starksen et al., 1987; Gay and Winkles, 1990), and smooth muscle cells (Sejersen et al., 1986; Sjolund et al., 1988; Valente et al., 1988; Majesky et al., 1988) proliferating under normal cell culture conditions. In contrast, PDGF A-chain transcripts are only detectable for a brief period following mitogenic stimulation of serum-starved normal fibroblasts (Paulsson et al., 1987a, 1988,1989; Raines et al., 1989). The functional significance of this transient PDGF-AA production is unknown, although i t could act in a n autocrine manner to amplify the initial mitogenic signal. However, PDGF-AA secretion also increases in mitogen-stimulated endothelial cells, which do not contain PDGF receptors (Gay and Winkles, 1990). We have identified a strain of human skin fibroblasts (AG3513) that expresses PDGF A-chain mRNA

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Fig. 5. Morphology of normal and progeria fibroblasts. Cells were plated at low density in identical growth media and photographed. A Normal fibroblast strain GM2037. B: Progeria fibroblast strain AG3513. C: Progeria fibroblast strain AG6917. D: Progeria fibroblast strain AG6297. Magnification = x 200.

in a constitutive manner. Immunoprecipitation analysis of conditioned medium from metabolically labeled AG3513 fibroblasts demonstrated that these cells also secrete PDGF A-chain homodimers. The molecular masses of the major immunoprecipitable species (nonreduced conditions, 31 kDa; reduced conditions, 17 kDa) agree well with those reported previously (Westermark et al., 1986; Paulsson et al., 1987a; Hammacher et al., 1988b; Bywater et al., 1988). The AG3513 fibroblasts were derived from a patient with a premature aging syndrome known as Hutchinson-Gilford syndrome (progeria). Progeria is a rare disease with an estimated incidence of 1per 8 million live births; individuals manifest certain features of advanced age during their 1st year of life and typically survive until the age of 12 years (Debusk, 1972). Genetic considerations indicate that progeria results from a sporadic autosoma1 dominant mutation (Brown et al., 1984). Altered PDGF A-chain gene expression was detected in one of three different progeria strains analyzed and

therefore is not a general property of the progeria phenotype. Fibroblasts derived from both parents of the AG3513 donor do not express PDGF A-chain mRNA in a constitutive manner (data not shown). We cannot determine whether altered PDGF A-chain gene expression occurred in skin fibroblasts in situ since tissue samples are not available from this donor (T. Brown, personal communication). It is possible that the altered expression evident in vitro is due to the differential outgrowth of unique myofibroblast- andlor fibroblastlike cells. Alternatively, this property may be related to a fibroblast derived from a sub-type of progeria, which is thought to be a genetically heterogeneous disease. The altered expression of PDGF A-chain mRNA in AG3513 fibroblasts could reflect transcript stabilization andior constitutive gene transcription. Experiments are in progress to address these two possibilities. Regardless of the mechanism, there could be a genetic alteration in the PDGF A-chain gene itself or in any of the genes encoding factors acting at the transcriptional

GENE EXPRESSION IN SENESCENT FIBROBLASTS

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Fig. 7. PDGF-stimulated growth response of normal and progeria fibroblasts. Normal (GM2037) and progeria (AG3513) fibroblasts were cultured in DMEM supplemented with 10%platelet-poor serum. After 4 hr, one set of cultures were counted in a hemocytometer chamber (day 0). The remaining cells were then incubated in media alone (open bars) or in media containing 10 ngiml PDGF (stippled bars). Cells were refed every other day and counted on days 5 and 10. The data points represent the mean -t SD of duplicate cultures.

PDGF (nglml)

Fig. 6. Mitogenic response of normal and progeria fibroblasts. A Serum-starved normal (GM2073) and progeria (AG3513) fibroblasts were stimulated with DMEM containing either 10% FBS, 20 ngiml PDGF, 20 n g h l HBGF-1, 20 ngiml HBGF-5, 10 ngiml EGF, or 10 ngiml IGF-1 and the amount of r3H1thymidine incorporated into trichloracetic acid-precipitable material was measured. The amount of [3Hlthymidine incorporated by normal (open bars) and progeria (stippled bars) fibroblasts is shown as a percentage of the incorporation in unstimulated cells. The data points represent the mean SD of three independent experiments, each of which was performed using triplicate cultures. B Serum-starved normal (solid circles) and progeria (open circles) fibroblasts were stimulated with increasing concentrations of PDGF and the amount of [3Hlthymidine incorporation was measured. The data points represent the mean 2 SD of triplicate cultures.

does not normally express PDGF A-chain mRNA (AG1972) has similar properties (Figs. 1, 5 and data not shown). In a previous study Bauer e t al. (1986) reported that Werner's syndrome fibroblasts have a full mitogenic response to FBS but a n attenuated response to whole human serum, PDGF, EGF, and FGF. * The diminished mitogenic response observed in AG3513 fibroblasts occurs with various distinct growth factors, and thus is unlikely to reflect deficiencies at the level of cell surface receptor. Nevertheless, since these cells were secreting PDGF-AA into their culture media, we examined their PDGF receptor number, binding affinity, and tyrosine kinase activity. Previous studies using early and late passage normal fibroblasts or post-transcriptional level to regulate PDGF A-chain indicate that the number of EGF, IGF-I, and PDGF mRNA abundance. Genomic DNA gel blot analysis in- receptor sites per cell is slightly increased in senescent dicates t h a t the PDGF A-chain gene is not amplified or cells, presumably due to their larger size (Phillips et rearranged in AG3513 fibroblasts, but this method al., 1983, 1987; Paulsson et al., 1986; Cristofalo et al., cannot detect point mutations or small restriction frag- 1989). The AG3513 progeria fibroblasts examined here actually contained 20% fewer PDGF-BB binding sites ment size differences. In comparison to similar passage fibroblasts derived than GM2037 normal fibroblasts. This difference is from a n age- and sex-matched healthy donor, the probably only partially due to A-type (a&)receptor AG3513 strain of progeria fibroblasts had a n altered downregulation by PDGF-AA, since there are few Amorphology, grew slower, and had a diminished mito- type receptors on human fibroblasts (Seifert et al., genic response to growth promoters. These properties 1989) and suramin treatment of AG3513 fibroblasts are diagnostic of senescent fibroblasts derived from does not significantly increase PDGF-BB binding (data normal individuals (Crusberg et al., 1979; Wang and not shown). Since the mitogenic effect of PDGF-BB Gundersen, 1984; Phillips et al., 1984, 1987; Tsuji et only requires partial receptor occupancy (as estimated al., 1984; Sherwood et al., 1988; Angello et al., 1989). from the data in Severinsson et al., 1989), this reducAlthough it is possible that the senescent phenotype of tion in receptor number is probably insufficient to acAG3513 cells is due to the autocrine effects of PDGF- count for the diminished PDGF responsiveness. The K, AA, a t least one other progeria fibroblast strain that values we obtained for PDGF-BB binding were similar

WINKLES ET AL.

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Fig. 8. Characterization of the PDGF-BB binding sites on normal and progeria fibroblasts. A Serum-starved normal (GM2037; solid circles) and progeria (AG3513; open circles) fibroblasts were incubated with increasing amounts of Iz5I-PDGF-BBand specific cell surface binding was measured. Data obtained from duplicate cultures was analyzed as described in “Materials and Methods.” B: Serumstarved normal (GM2037) and progeria (AG3513) fibroblasts were incubated for 10 min in the absence ( ~) or presence ( + of 10 ngiml PDGF. Cell lysates were prepared and equivalent amounts of protein were subjected to electrophoresis on a 7.5%polyacrylamide-SDS gel. Immunoblotting using anti-phosphotyrosine antibody and detection with l2”1-protein A was performed as described in “Materials and Methods.” Positions of the molecular weight standards (BioRad; in kilodaltons) are indicated at the left.

for both fibroblast strains and in agreement with previous reports (Gronwald et al., 1988; Severinsson et al., 1989). The addition of PDGF to quiescent fibroblasts induces numerous biochemical responses, including the stimulation of receptor tyrosine kinase activity (reviewed in Williams, 1989). The PDGF receptor can phosphorylate itself (Ek and Heldin, 1982, 1984; Frackelton et al., 1984; Daniel et al., 1985; Bishayee et al., 1986; Kazlauskas and Cooper, 1989) and additional substrates such a s phospholipase C-7 (Meisenhelder et al., 19891, raf-1 (Morrison et al., 1989) and GTPase activating protein (Kaplan e t al., 1990). The PDGF receptors on AG3513 fibroblasts appear to be indistinguishable from those on GM2037 fibroblasts in their ability to autophosphorylate after PDGF-BB binding. However, we have not determined if the receptors on both cell strains are phosphorylated on identical tyrosine residues, nor have we assessed the ability of the AG3513 fibroblast receptors to phosphorylate additional cellular substrates. Previous studies have demonstrated that the EGF (Brooks et al., 1987) and PDGF (Paulsson e t al., 1986) receptors on senescent normal fibroblasts can also autophosphorylate after ligand binding. We examined one additional PDGF-induced cellular response, the enhanced expression of the c-fos protooncogene, in GM2037 and AG351.1 fibroblast,s. Four studies have compared proto-oncogene expression in serum- or growth factor-stimulated early and late pas-

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Fig. 9. Expression of c-fos mRNA in PDGF-stimulated normal and progeria fibroblasts. Serum-starved normal (GM2037) and progeria (AG3513) fibroblasts were left unstimulated or stimulated with 20 ng/ml PDGF for 0.5, 2, and 4 hr. RNA was prepared and equivalent amounts were used for RNA gel blot analysis. Hybridization to the radiolabeled c-fos DNA probe is shown in A, and hybridization to GAPDH DNA in B.

sage fibroblasts. They all report that c-myc mRNA induction occurs normally in senescent cells (Paulsson et al., 1986; Rittling et al., 1986; Chang and Chen, 1988; Seshadri and Campisi, 1990). Two of these studies also examined c-fos expression; Paulsson et al. (1986) reported that PDGF stimulation of early and late passage foreskin fibroblasts induces similar levels of c-fos mRNA, while Seshadri and Campisi (1990) reported that c-fos gene activation is repressed in senescent fetal lung fibroblasts. We have also found that senescent fibroblasts, in this case cultured from a progeria patient, have reduced c-fos mRNA levels after mitogenic stimulation. The c-fos polypeptide is a “leucine-zipper”containing transcriptional factor that can associate with the AP-1 protein c-jun (reviewed in Mitchell and Tjian, 1989). Although various approaches have implicated c-fos in the control of cell proliferation (Miller et al., 1984; Holt et al., 1986; Nishikura and Murray, 1987; Riabowol et al., 1988), c-fos induction can occur without subsequent DNA synthesis (Curran and Morgan, 1985; Severinsson et al., 1990). We do not yet know whether reduced c-fos mRNA levels contribute to the diminished mitogenic response to growth factors characteristic of AG3513 fibroblasts. In summary, we have identified a strain of fibroblasts derived from a premature aging syndrome patient that expresses two PDGF-inducible genes in a manner distinct from normal fibroblasts. PDGF Achain transcripts, normally only expressed after growth factor stimulation of quiescent fibroblasts, are expressed at a similar level in quiescent, PDGF-stim-

GENE EXPRESSION IN SElNESCENT FIBROBLASTS

ulated, and normally proliferating AG3513 fibroblasts. Constitutive PDGF-AA production has no effect on cellular morphology and does not confer a growth advantage; consistent with the properties described for human fibroblasts that express high levels of PDGF-AA as a result of infection with a retrovirusiPDGF A-chain cDNA construct (Bywater et al., 1988). In fact, as judged by three criteria (morphology, growth rate, proliferative response to growth factor stimulation) these cells appear senescent. In agreement with a recent study comparing serum-stimulated early and late passage fibroblasts (Seshadri and Campisi, 19901, c-fos mRNA induction levels are 10-fold lower in PDGFstimulated AG3513 fibroblasts compared to GM2037 normal fibroblasts. The altered regulation of two PDGF-inducible genes suggests that one or more of the intracellular pathways involved in PDGF signal transduction may be deficient in AG3513 progeria fibroblasts.

ACKNOWLEDGMENTS We are grateful to T. Maciag and R. Weinstein for providing endothelial cells and smooth muscle cells, respectively. We thank W. Burgess for HBGF-1 and B. Westermark for the PDGF A-chain plasmid. We also thank B. Hampton, C. Gay, and P. Donohue for technical assistance and S. Young for excellent secretarial assistance. LITERATURE CITED Angello, J.C., Pendergrass, W R , Norwood, T.H , and Prothero, J. (1989) Cell enlargement One possible mechanism underlying cellular senescence J Cell Physiol , 140 288-294 Bauer, E A , Silverman, W , Busiek, D F , Kronberger, A , and Deuel, T F (1986) Diminished response of Werner’s syndrome fibroblasts to growth factors PDGF and FGF Science, 234 1240-1243 Beauregard, S , and Gilchrest, B A (1987) Syndromes of premature aging. Derm. Clinics, 5:109-121. Betsholtz, C., Johnsson, A,, Heldin, C-H., Westermark, B., Lind, P., Urdea, M.S., Eddv. R.. Shows. T.B.. Phihott. K., Mellor. A.L.. Knott, T.J., and Scott, J. (1986)cDNA sequence and chromosomal localization o f human platelet-derived growth factor A-chain and its expression in tumor cell lines. Nature, 320:695-699. Bishayee, S., Ross, A.H., Womer, R., and Scher, C.D. (1986) Purified platelet-derived growth factor receptor has ligand-stimulated tyrosine kinase activity. Proc. Natl. Acad. Sci. U.S.A., 83t6756-6760. Bonthron, D.T., Morton, C.C., Orkin, S.H., and Collins, T. (1988) Platelet-derived growth factor A chain: Gene structure, chromosomal location, and basis for alternative mRNA splicing. Proc. Natl. Acad. Sci. U.S.A., 85:1492-1496. Bowen-Pope, D.F., Hart, C.E., and Seifert, R.A. (1989) Sera and conditioned media contain different isoforms of platelet-derived growth factor (PDGF) which bind to different classes of PDGF receptor. J. Biol. Chem.! 264.2502-2508. Bronzert, D.A., Pantazis, P., Antoniades, H.N., Kasid, A., Davidson, N., Dickson, R.B., and Lippman, M.E. (1987) Synthesis and secretion of platelet-derived growth factor by human breast cancer cell lines. Proc. Natl. Acad. Sci. U.S.A.,84:5763-5767. Brooks, K.B., Phillips, P.D., Carlin, C.C., Knowles, B.B., and Cristofalo, V.J. (1987) EGF-dependent phosphorylation of the EGF receptors in plasma membranes isolated from young and senescent WI38 cells. J . Cell. Physiol., 133:523-531. Brown, W.T., Zebrower, M., and Kieras, F.J. (1984) Progeria, a model disease for the study of accelerated aging. In: Molecular Biology of Aging. A.D. Woodhead, A.D. Blackett, and A. Hollaender, eds. Plenum Press, New York, pp. 375-396. Burgess, W.H., Mehlman, T., Friesel. R., Johnson, W.V., and Maciag, T. (1985) Multiple forms of endothelial cell growth factor. J. Biol. Chem., 260:11389-11392. Burmer, G.C., Motulsky, H., Zeigler, C.J., and Norwood, T.H. (1983) Inhibition of DNA synthesis in young cycling human diploid fibroblast-like cells upon fusion to enucleate cytoplasts from senescent cells. Exp. Cell Res., 145.79-84,

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~

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Altered regulation of platelet-derived growth factor A-chain and c-fos gene expression in senescent progeria fibroblasts.

The study of human genetic disorders known as premature aging syndromes may provide insight into the mechanisms of cellular senescence. These diseases...
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