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Adriamycin-induced Senescence in Breast Tumor Cells Involves Functional p53 and Telomere Dysfunction*

  • Lynne W. Elmore
    Affiliations
    Departments of Pathology,
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  • Catherine W. Rehder
    Footnotes
    Affiliations
    Human Genetics, and
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  • Xu Di
    Affiliations
    Pharmacology and Toxicology and the
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  • Patricia A. McChesney
    Affiliations
    Departments of Pathology,

    Massey Cancer Center, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298-0662
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  • Colleen K. Jackson-Cook
    Affiliations
    Departments of Pathology,

    Human Genetics, and

    Massey Cancer Center, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298-0662
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  • David A. Gewirtz
    Affiliations
    Pharmacology and Toxicology and the

    Massey Cancer Center, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298-0662
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  • Shawn E. Holt
    Correspondence
    V Foundation Scholar. To whom correspondence should be addressed: Depts. of Pathology and Human Genetics, Medical College of Virginia at Virginia Commonwealth University, 1101 E. Marshall St., Richmond, VA 23298-0662. Tel.: 804-827-0458; Fax: 804-828-5598;
    Affiliations
    Departments of Pathology,

    Human Genetics, and

    Pharmacology and Toxicology and the

    Massey Cancer Center, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298-0662
    Search for articles by this author
  • Author Footnotes
    * This work was supported in part by the Mary Kay Ash Charitable Foundation (to L. W. E. and S. E. H.), the Department of Defense Breast Cancer Research Program Grant DAMD 17-01-0441 (to D. A. G. and S. E. H.), Virginia's Commonwealth Health Research Board (to C. K. J.-C.), and NCI, National Institutes of Health Postdoctoral Training Grant CA 85159-01 (to P. A. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    ¶ A Glenn/American Federation for Aging Research Scholar.
      Direct experimental evidence implicates telomere erosion as a primary cause of cellular senescence. Using a well characterized model system for breast cancer, we define here the molecular and cellular consequences of adriamycin treatment in breast tumor cells. Cells acutely exposed to adriamycin exhibited an increase in p53 activity, a decline in telomerase activity, and a dramatic increase in β-galactosidase, a marker of senescence. Inactivation of wild-type p53 resulted in a transition of the cellular response to adriamycin treatment from replicative senescence to delayed apoptosis, demonstrating that p53 plays an integral role in the fate of breast tumor cells treated with DNA-damaging agents. Stable introduction of hTERT, the catalytic protein component of telomerase, into MCF-7 cells caused an increase in telomerase activity and telomere length. Treatment of MCF-7-hTERT cells with adriamycin produced an identical senescence response as controls without signs of telomere shortening, indicating that the senescence after treatment is telomere length-independent. However, we found that exposure to adriamycin resulted in an overrepresentation of cytogenetic changes involving telomeres, showing an altered telomere state induced by adriamycin is probably a causal factor leading to the senescence phenotype. To our knowledge, these data are the first to demonstrate that the mechanism of adriamycin-induced senescence is dependent on both functional p53 and telomere dysfunction rather than overall shortening.
      Most normal somatic cells continually shorten their telomeres after each cell division because of incomplete replication at the end of linear chromosomes (
      • Olovnikoff A.M.
      ,
      • Watson J.D.
      ). The original hypothesis stated that when telomeres have become sufficiently shortened, replicative senescence is induced (
      • Harley C.B.
      • Futcher A.B.
      • Greider C.W.
      ,
      • Hastie N.D.
      • Dempster M.
      • Dunlop M.G.
      • Thompson A.M.
      • Green D.K.
      • Allshire R.C.
      ). Tumor suppressor proteins such as p53 are required for this senescence arrest. Most cells with indefinite proliferative ability (e.g. human tumors and their derivative cell lines) express the enzyme telomerase to maintain telomeres, which allows for the continued cellular proliferation characteristic of human cancer (
      • Counter C.M.
      • Avilion A.A.
      • LeFeuvre C.E.
      • Stewart N.G.
      • Greider C.W.
      • Harley C.B.
      • Bacchetti S.
      ,
      • Kim N.W.
      • Piatyszek M.A.
      • Prowse K.R.
      • Harley C.B.
      • West M.D., Ho, P.L.
      • Coviello G.M.
      • Wright W.E.
      • Weinrich S.L.
      • Shay J.W.
      ). Telomerase is a cellular reverse transcriptase containing two strictly required elements: a protein component, hTERT, and an RNA element, hTR (
      • Feng J.
      • Funk W.D.
      • Wang S-S.
      • Weinrich S.L.
      • Avilion A.A.
      • Chiu C-P.
      • Adams R.R.
      • Chang E.
      • Allsopp R.C., Yu, J., Le, S.
      • West M.D.
      • Harley C.B.
      • Andrews W.H.
      • Greider C.W.
      • Villeponteau B.
      ,
      • Weinrich S.L.
      • Pruzan R., Ma, L.
      • Ouellette M.
      • Tesmer V.M.
      • Holt S.E.
      • Bodnar A.G.
      • Lichtsteiner S.
      • Kim N.W.
      • Trager J.B.
      • Taylor R.D.
      • Carlos R.
      • Andrews W.H.
      • Wright W.E.
      • Shay J.W.
      • Harley C.B.
      • Morin G.B.
      ,
      • Beattie T.L.
      • Zhou W.
      • Robinson M.
      • Harrington L.
      ). hTERT serves as the catalytic subunit, whereas hTR is utilized by hTERT as the template for catalyzing the addition of telomeric DNA to the end of the chromosome. The introduction of telomerase into normal human cells provides for telomere maintenance, prevention of senescence, and an extension of life span, indicating that gradual telomere shortening is one of the factors contributing to the onset of cellular senescence (
      • Bodnar A.G.
      • Ouellette M.
      • Frolkis M.
      • Holt S.E.
      • Chiu C-P.
      • Morin G.B.
      • Harley C.B.
      • Shay J.W.
      • Lichtsteiner S.
      • Wright W.E.
      ,
      • Vaziri H.
      • Benchimol S.
      ). Recent evidence suggests that although telomere length is an important trigger for the onset of senescence, increased telomere dysfunction results in a loss of chromosome end protection and induction of the senescence state (
      • Karlseder J.
      • Smogorzewska A.
      • de Lange T.
      ). These novel findings show that senescence can be induced without net telomere shortening, and that while length remains important, preservation of telomere integrity is critical regardless of telomere length.
      The mechanism(s) of action of the anthracycline antibiotic adriamycin, a drug that has long been a mainstay in the treatment of breast cancer (
      • DeVita V.T.
      • Hellman S.
      • Rosenberg S.A.
      ), have been studied extensively (
      • Gewirtz D.A.
      ). Adriamycin promotes apoptotic cell death in a variety of experimental tumor cell lines (
      • Ling Y-H.
      • Priebe W.
      • Perez-Solar R.
      ,
      • Zaleskis G.
      • Berleth E.
      • Verstovek S.
      • Ehrke M.J.
      • Mihich E.
      ,
      • Bose R.
      • Verheij M.
      • Haimovitz-Friedman A.
      • Scotto K.
      • Fuks Z.
      • Kolesnick R.
      ,
      • Jaffrezou J-P.
      • Levade T.
      • Bettaieb A.
      • Andrieu N.
      • Bezombes C.
      • Maestre N.
      • Vermeersch S.
      • Rousse A.
      • Laurent G.
      ). However, we have demonstrated that MCF-7 breast tumor cells fail to undergo a primary apoptotic response after either acute or chronic exposure to adriamycin (
      • Fornari F.A.
      • Jarvis W.D.
      • Grant S.
      • Orr M.S.
      • Randolph J.K.
      • White F.K.H.
      • Mumaw V.R.
      • Lovings E.T.
      • Freeman R.H.
      • Gewirtz D.A.
      ,
      • Fornari F.A.
      • Jarvis W.D.
      • Orr M.S.
      • Randolph J.K.
      • Grant S.
      • Gewirtz D.A.
      ). Here, we show that the growth-arrested state associated with acute adriamycin treatment of MCF-7 cells (
      • Fornari F.A.
      • Jarvis W.D.
      • Orr M.S.
      • Randolph J.K.
      • Grant S.
      • Gewirtz D.A.
      ) results in down-regulation of telomerase activity and induction of a senescence phenotype. MCF-7 cells expressing the human papillomavirus type 16 (HPV-16)
      The abbreviations used are: HPV, human papillomavirus; PBS, phosphate-buffered saline; TRAP, telomeric repeat amplification protocol; TRF, terminal restriction fragment; TUNEL, Tdt-mediated dNTP nick end labeling; kb, kilobase(s); MAPK, mitogen-activated protein kinase.
      1The abbreviations used are: HPV, human papillomavirus; PBS, phosphate-buffered saline; TRAP, telomeric repeat amplification protocol; TRF, terminal restriction fragment; TUNEL, Tdt-mediated dNTP nick end labeling; kb, kilobase(s); MAPK, mitogen-activated protein kinase.
      E6 protein show degradation of p53, a lack of overall p53 function, and conversion from a senescent phenotype to apoptosis after adriamycin treatment, demonstrating that p53 is critical for replicative senescence in drug-treated MCF-7 cells. Exogenous expression of hTERT provides for increased telomerase activity and elongated telomere lengths but does not protect MCF-7 cells from drug-induced cellular senescence. We find that adriamycin-induced DNA damage appears to preferentially target chromosome ends resulting in substantial telomere-related cytogenetic abnormalities, indicating that the observed senescence is because of telomere dysfunction rather than overall shortening. Our data clearly indicate that the senescence program observed in adriamycin-treated MCF-7 cells requires functional p53 and telomere dysfunction.

      EXPERIMENTAL PROCEDURES

       Materials

      RPMI 1640 medium and trypsin-EDTA (0.5% trypsin, 5.3 mm EDTA) were obtained from Invitrogen.l-Glutamine, penicillin/streptomycin (10,000 units penicillin/ml and streptomycin 10 mg/ml), and fetal bovine serum were obtained from Whittaker BioProducts (Walkersville, MD). Adriamycin and retinoic acid were purchased from Sigma, reconstituted in molecular biology grade water, and stored as aliquots at −20 °C until dilution in culture medium immediately before cell treatments.

       Cell Culture and Adriamycin Treatment

      The MCF-7 and MDA-MB231 breast tumor cell lines were obtained from the NCI Frederick Cancer Research Facility. Cells were maintained as monolayers in RPMI 1640 medium supplemented with glutamine (0.292 mg/ml), penicillin/streptomycin (0.5 ml/100 ml medium), and 10% fetal bovine serum. All cells were cultured at 37 °C in 5% CO2 and 100% humidity. Twenty-four hours after plating, cells were exposed to 1 μm adriamycin for 2 h, rinsed once in PBS, and then maintained in supplemented RPMI 1640 medium. The retroviral packaging cells, PA317 HPV-16 E6 and PA317 hTERT, and their vector controls (pLXSN and pBABEpuro, respectively) were obtained from Dr. Jerry W. Shay (UT Southwestern, Dallas, TX) and were maintained in Dulbecco's modified Eagle's medium with 10% bovine calf serum as described above. Target MCF-7 cells were infected and selected using standard procedures as described previously (
      • Savre-Train I.
      • Gollahon L.S.
      • Holt S.E.
      ).

       Telomerase Activity Assay

      Telomerase activity was determined by the telomeric repeat amplification protocol (TRAP) using the TRAPeze kit (Intergen, Purchase, NY) as described previously (
      • Kim N.W.
      • Piatyszek M.A.
      • Prowse K.R.
      • Harley C.B.
      • West M.D., Ho, P.L.
      • Coviello G.M.
      • Wright W.E.
      • Weinrich S.L.
      • Shay J.W.
      ,
      • Holt S.E.
      • Norton J.C.
      • Wright W.E.
      • Shay J.W.
      ). The reactions were extended for 30 min at 30 °C, and extension products were PCR-amplified for 27 cycles and resolved on a 10% polyacrylamide gel. The gel was then exposed to a PhosphorImaging cassette (Amersham Biosciences) and directly scanned and analyzed using ImageQuant Software (Amersham Biosciences). A positive result indicating telomerase activity was shown by the presence of a 6-bp progressive ladder. A 36-bp internal standard verified successful amplification and served as a useful standard for relative quantitation. Telomerase activity was semi-quantitatively calculated using the ratio of the intensity of the telomerase ladder to the intensity of the 36-bp internal standard.

       Reverse Transcription-PCR Analysis of Endogenous Versus Exogenous hTERT Expression

      Total RNA was isolated from cells in logarithmic growth phase using TRIzol (Invitrogen) as recommended by the manufacturer. According to the manufacturer's established protocol, 3 μg of total RNA from each cell culture were reverse-transcribed in a 20-μl reaction volume using decamers and the regular reaction buffer provided in the first strand synthesis kit, RETROscript (Ambion Inc., Austin, TX). A 2.5-μl aliquot of cDNA was used for PCR amplifications. hTERT was amplified using the oligonucleotide primer hTERT (5′-GACTCGACACCGTGTTCACCTAC-3′) paired with either Endo 1 (5′-ACGTAGAGCCCGGCGTGACAG-3′) or pBABE (5′-GACACACATTCCACAGGTCG-3′) (
      • Hahn W.C.
      • Counter C.M.
      • Lundberg A.S.
      • Beijersbergen R.L.
      • Brooks M.W.
      • Weinberg R.A.
      ), which selectively amplify endogenous or exogenous hTERT, respectively. For both primer pairs, the thermocycling conditions were: 94 °C for 5 min followed by 34 cycles of 94 °C for 45 s, 62 °C for 30 s, and 72 °C for 45 s. Amplification of 18 S RNA was performed as a control with 18 S PCR primer pairs (Ambion, Inc.) using the same thermocycling conditions described above with only 22 cycles completed. Amplified products (exogenous hTERT: 175-bp; endogenous hTERT: 219-bp; 18 S: 488-bp) were resolved on a 1.5% agarose gel and visualized by staining with ethidium bromide.

       Telomere Length Analysis

      Telomere length was determined using terminal restriction fragment (TRF) analysis as noted before (
      • Bodnar A.G.
      • Ouellette M.
      • Frolkis M.
      • Holt S.E.
      • Chiu C-P.
      • Morin G.B.
      • Harley C.B.
      • Shay J.W.
      • Lichtsteiner S.
      • Wright W.E.
      ,
      • Savre-Train I.
      • Gollahon L.S.
      • Holt S.E.
      ). DNA was isolated from cells using genomic tips (Qiagen, Santa Clarita, CA) digested with a mixture of HinfI,AluI, and RsaI restriction enzymes (Invitrogen) and resolved on a 0.7% agarose gel. The DNA ladder and G-rich telomere probe (TTAGGG)4 were labeled with [γ-32P]ATP (6000 Ci/mmol) with unincorporated label removed from the reaction using the QIAquick nucleotide removal kit (Qiagen). The dried agarose gel was subjected to in-gel hybridization with the labeled probe, washed repeatedly with varying concentrations of SSC buffer, and exposed to a PhosphorImaging cassette for 2–24 h. An estimation of median telomere length for comparison purposes only was made by measuring the radioactive smear, taking the midpoint of its length for characterization of isogenic cell lines. A more rigorous determination of average telomere length was accomplished using an established protocol as described previously (
      • Ouellette M.
      • Liao M.
      • Shea-Herbert B.
      • Johnson M.
      • Holt S.E.
      • Liss H.S.
      • Shay J.W.
      • Wright W.E.
      ).

       β-Galactosidase Histochemical Staining

      MCF-7 cells were washed twice with PBS and fixed with 2% formaldehyde, 0.2% glutaraldehyde for 5 min. The cells were then washed again with PBS and stained with a solution of 1 mg/ml 5-bromo-4-chloro-3-inolyl-β-galactosidase in dimethylformamide (20 mg/ml stock), 5 mm potassium ferrocyanide, 150 mm NaCl, 40 mm citric acid/sodium phosphate, pH 6.0, and 2 mm MgCl2 as described previously (
      • Bodnar A.G.
      • Ouellette M.
      • Frolkis M.
      • Holt S.E.
      • Chiu C-P.
      • Morin G.B.
      • Harley C.B.
      • Shay J.W.
      • Lichtsteiner S.
      • Wright W.E.
      ,
      • Dimri G.P.
      • Xinhau L.
      • Basile G.
      • Acosta M.
      • Scott G.
      • Roskelley C.
      • Medrano E.E.
      • Linskens M.
      • Rubej J.
      • Pereira-Smith O.
      • Peacocke M.
      • Campisi J.
      ). Following overnight incubation at 37 °C, the cells were washed twice with PBS, and the percentage of positively stained cells was determined after counting three random fields of 100 cells each. As a positive control for β-galactosidase expression, MCF-7 cells were exposed to 200 nm retinoic acid for 4 days and then stained. Representative microscopic fields were photographed under a ×20 objective.

       Western Analysis for p53 and p21waf-1Proteins

      MCF-7 cells (parental, HPV-16 E6, and pLXSN) were treated with 1 μm adriamycin for 2 h, washed with PBS, and then cultured for an additional 2 h prior to lysing in a standard radioimmune precipitation assay buffer (50 mmTris, pH 7.4, 150 mm NaCl, 1% Triton X-100, 0.1% SDS, 100 mm dithiothreitol, and protease inhibitors). The respective isogenic untreated MCF-7 cultures were included for assessing the constitutive levels of p53 and p21waf1. Protein concentrations were determined using a Lowry-based spectrophormetric assay (Bio-Rad) according to the manufacturer's protocol. A 15-μg aliquot of each sample was separated by SDS-PAGE and electrotransferred onto nitrocellulose membrane. A standard blotting protocol was then performed using pantropic p53 (1:500 of Ab-6, Oncogene Research Products) and a p21waf-1 monoclonal (1:500, Signal Transduction Laboratories) antibodies followed by peroxidase-conjugated anti-mouse IgG (1:10,000, Amersham Biosciences). A chemiluminescent reaction (ECL Reagent, Amersham) was used for detection. To control for protein loading, the membrane was subsequently probed with an actin antibody (1:5000, Sigma) and processed as described above.

       TUNEL Assay

      MDA-MB231, MCF-7, and MCF-7-E6 cells were directly seeded onto 4-well chamber slides, treated with 1 μm adriamycin for 2 h, and then after 5 days, stained by TUNEL (Roche Molecular Biochemicals) according to the manufacturer's instructions. Cells were counterstained with DAPI (Sigma) to allow easy determination of the total number of cells per field. Three random fields of at least 300 cells in monolayer culture were scored to determine the percentage of cells undergoing apoptosis. Values represent the mean ± S.D.

       Cytogenetic Analysis

      Metaphase chromosomes were harvested from the MCF-7 cell cultures using standard methods (
      • Rooney D.E.
      • Czepulkowski B.H.
      ). Actively dividing cells were blocked in mitosis with 0.1 μg/ml colcemid for 2 h, incubated in 0.075 m KCl hypotonic solution for 20 min, and fixed in methanol:glacial acetic acid (3:1). Slides were made using standard procedures, and metaphase chromosomes were visualized using conventional Geimsa staining (
      • Barch M.J.
      ). Metaphase spreads were scored for chromosomal findings from both the MCF-7-hTERT cell cultures before and after adriamycin treatment. A total of 200 metaphase spreads (100 each) were evaluated (
      • Tawn E.J.
      • Holdsworth D.
      ). The frequency of the types of chromosomal abnormalities seen in the cells with and without adriamycin treatment were compared using a contingency chi-square test with an α < 0.05 significance level.

      RESULTS

       Acute Adriamycin Treatment Induces Senescence and Represses Telomerase Activity

      In this study, our initial objective was to determine whether MCF-7 cells undergo replicative senescence following acute exposure to 1 μm adriamycin. β-Galactosidase expression, a marker of cellular senescence (
      • Bodnar A.G.
      • Ouellette M.
      • Frolkis M.
      • Holt S.E.
      • Chiu C-P.
      • Morin G.B.
      • Harley C.B.
      • Shay J.W.
      • Lichtsteiner S.
      • Wright W.E.
      ,
      • Dimri G.P.
      • Xinhau L.
      • Basile G.
      • Acosta M.
      • Scott G.
      • Roskelley C.
      • Medrano E.E.
      • Linskens M.
      • Rubej J.
      • Pereira-Smith O.
      • Peacocke M.
      • Campisi J.
      ), was present in the MCF-7 cells as early as 2 days after acute exposure to adriamycin (data not shown). After 1 week, histochemical staining was intense and expressed in virtually every cell of the culture (Fig.1). Cells expressing this senescence marker were typically much larger in size and multinucleated, both of which are morphological features indicative of a senescent state. Continuous exposure to retinoic acid used as a positive control (
      • Chang B.D.
      • Xuan Y.
      • Broude E.V.
      • Zhu H.
      • Schott B.
      • Roninson I.B.
      ) induced β-galactosidase activity in MCF-7 breast tumor cells as expected (data not shown). Because one could argue that β-galactosidase staining in this system may only reflect a lack of cell division rather than true senescence, MCF-7 cells were held in a non-dividing state by serum removal for 7 days and stained for β-galactosidase. In contrast to the adriamycin-treated cells, β-galactosidase expression was detected only very rarely in non-dividing MCF-7 cells, consistent with that observed for untreated MCF-7 cells (0.3% + 0.2).
      Figure thumbnail gr1
      Figure 1Expression of senescence-associated β-galactosidase in MCF-7 cells following acute exposure to adriamycin. Cells were exposed to 1 μm adriamycin for 2 h, and β-galactosidase expression was assessed 4 days after adriamycin treatment. Shown are representative microscopic fields from untreated (left panel) and adriamycin-treated (right panel) cells. Original magnification for both is ×20. Note the substantial increase in cellular volume and the blue staining of the treated (senescent) cells.
      Because induction of senescence has been closely associated with the suppression of telomerase activity (
      • Holt S.E.
      • Wright W.E.
      • Shay J.W.
      ,
      • Holt S.E.
      • Aisner D.L.
      • Shay J.W.
      • Wright W.E.
      ), we also assessed the influence of acute adriamycin treatment on telomerase activity in the MCF-7 cells. Adriamycin produced a time-dependent decline of telomerase activity in MCF-7 cells (Fig.2) that differs from the recently described early daunorubicin-mediated telomerase inhibition in lung cancer cells (85% reduction at 24 h) (
      • Ogretmen B.
      • Kraveka J.M.
      • Schady D.
      • Usta J.
      • Hannun Y.A.
      • Obeid L.M.
      ). Although the effects of adriamycin on telomerase activity were not significant within the first 1–3 days of drug exposure, exhibiting a half-life consistent with previous results (
      • Holt S.E.
      • Wright W.E.
      • Shay J.W.
      ,
      • Holt S.E.
      • Aisner D.L.
      • Shay J.W.
      • Wright W.E.
      ), telomerase activity was reduced 90% after 7 days with a greater than 95% reduction after 10 days (Fig.2 B).
      Figure thumbnail gr2
      Figure 2Influence of adriamycin on telomerase activity in the MCF-7 breast tumor cell line. Left panel, cells were exposed to 1 μm adriamycin for 2 h and then assayed for telomerase activity using the TRAP assay (
      • Kim N.W.
      • Piatyszek M.A.
      • Prowse K.R.
      • Harley C.B.
      • West M.D., Ho, P.L.
      • Coviello G.M.
      • Wright W.E.
      • Weinrich S.L.
      • Shay J.W.
      ,
      • Holt S.E.
      • Norton J.C.
      • Wright W.E.
      • Shay J.W.
      ). One thousand cell equivalents were used for each reaction with a representative experiment shown. IC denotes the 36-bp internal control band that serves to normalize sample to sample variation. Nonspecific banding between the 36-bp IC and the initial 50-bp telomerase band (seen in the 10-day sample) is unrelated to telomerase activity and is not included in the quantitation.Right panel, relative quantitation of telomerase activity levels after adriamycin treatment of MCF-7 cells using the 0 time point as 100%. LB, lysis buffer; AdR, adriamycin.

       Inactivation of p53 Results in Induction of Apoptosis Rather Than Senescence After Adriamycin Treatment

      Because MCF-7 cells express wild-type p53 but lack functional caspase 3 protein (
      • Kagawa S., Gu, J.
      • Honda T.
      • McDonnell T.J.
      • Swisher S.G.
      • Roth J.A.
      • Fang B.
      ), the induction of replicative senescence during adriamycin treatment may be attributed to a combination of p53-mediated senescence (
      • Shay J.W.
      • Pereira-Smith O.M.
      • Wright W.E.
      ) and the inability to progress down the apoptotic pathway. We tested this hypothesis using another p53-positive breast tumor cell line with functional caspase 3 (ZR-75) and found that adriamycin treatment results in an identical senescence pattern without detectable apoptosis (data not shown), consistent with that observed for the MCF-7 cells (quantitation in Fig.3 A). In addition, adriamycin-treated breast tumor cells with mutant p53 (MDA-MB231) exhibit a delayed apoptosis rather than senescence (Fig.3 A). These observations taken together suggested a pivotal role for p53 in the induction of senescence in breast tumor cells and that inactivation of p53 in MCF-7 cells may allow conversion to an apoptotic pathway following DNA damage (
      • Bunz F.
      • Dutriaux A.
      • Lengauer C.
      • Waldman T.
      • Zhou S.
      • Brown J.P.
      • Sedivy J.M.
      • Kinzler K.W.
      • Vogelstein B.
      ). To test this hypothesis, MCF-7 cells were infected with HPV-16 E6, which resulted in the degradation and inactivation of p53 as denoted by the lack of p53-mediated transcriptional activation of its downstream target p21waf-1 (Fig. 3 B) after drug treatment. Even though we observe a slight increase (2–3-fold) in p21waf-1independent of p53, acute treatment of MCF-7-E6 cells with 1 μm adriamycin resulted in a delayed apoptotic event rather than senescence 5 days post-treatment using the TUNEL assay (quantification shown in Fig. 3 C). Collectively, these data suggest that wild-type p53 activity is necessary for the induction of the replicative senescence phenotype observed in the adriamycin-treated MCF-7 cells.
      Figure thumbnail gr3
      Figure 3Functional p53 is required for induction of senescence after adriamycin treatment. A, comparison of breast tumor cells with differing p53 status, MCF-7 (wild-type p53) and MDA-MB231 (mutant p53), for response to adriamycin treatment. Five days post-treatment, cells were assessed for senescence as indicated by β-galactosidase induction and for apoptosis using the TUNEL assay. Three independent fields of stained cells were counted, and the average percent positivity compared with untreated controls (% of control) was calculated. B and C, MCF-7-derived cells were treated with 1 μm adriamycin for 2 h and tested for p53 and p21waf-1 induction (B) and induction of apoptosis (C). B, parental MCF-7 (parental), MCF-7 with vector pLXSN only, and MCF-7 infected with HPV-16 E6 (E6) were treated and harvested 2 h after treatment with adriamycin (+) and compared with untreated controls (−). Cells were extracted, and 15 μg of total protein were electrophoresed (12% SDS-PAGE). Transferred blots were probed using antibodies specific to p53, p21waf-1, and β-actin followed by detection using chemiluminescence. The modest increase in p21waf-1 levels observed in the MCF-7-E6 cells after adriamycin treatment is within the range of experimental error, possibly representing a p53-independent increase in p21waf-1. C, quantitation of TUNEL positivity in the MCF-7 and MCF-7-E6 cells at 5 days post-treatment. Values represent the mean ± S.D. based on three random fields with 300 cell counts/field.

       Ectopic Expression of hTERT in MCF-7 Cells Results in Increased Telomerase Activity and Telomere Length

      To assess the involvement of telomerase and telomere length in the adriamycin-induced senescence response, we retrovirally infected the hTERT gene into MCF-7 cells and selected for stable integration using puromycin. The introduction of hTERT into MCF-7 cells resulted in elevated telomerase activity (nearly 5-fold) (Fig. 4 A), continuous expression of exogenous hTERT (Fig. 4 B), and a substantial increase in telomere length from a median length of 3.5 to 7 kb (Fig.4 C). Because MCF-7 cells already have a substantial amount of telomerase activity, it was possible that hTERT expression would not result in an increase in telomerase activity or telomere elongation. However, our data clearly show that hTERT is the limiting factor for telomerase elevation and telomere elongation in MCF-7 cells.
      Figure thumbnail gr4
      Figure 4Expression of hTERT in MCF-7 cells elevates telomerase activity and elongates telomeres. MCF-7 cells were retrovirally infected with hTERT, and a stable population of cells was selected with puromycin. A, a representative TRAP assay with MCF-7 parental (uninfected), vector only (pBABEpuro), and hTERT-selected populations at 1000 cells/reaction. A lysis buffer (LB) only sample served as a negative control. IC denotes the 36-bp internal control band. Two independent experiments yielded elevated activity of ∼5–7-fold compared with parental or vector controls. B, Reverse Transcription-PCR for hTERT was accomplished using primers specific for endogenous (endo, 219-bp, present in both pBABEpuro and hTERT cells) and exogenous (exo, 175-bp, present only in the hTERT cells) hTERT as described under “Experimental Procedures.” Levels of 18-s amplification were indistinguishable between the four samples (data not shown). C, TRF analysis to assess telomere sizes in vector controls (pBABEpuro) and hTERT MCF-7 cells. H1299 is a lung adenocarcinoma cell line that serves as a positive control.White bars indicate the median size of telomere length. Thenumbers on the left show the positions of a DNA-sizing ladder (in kb).

       Senescence in Adriamycin-treated MCF-7 Cells Is Induced by Telomere Dysfunction but Not Telomere Shortening

      We hypothesized that because telomere length has been shown to be a primary cause of cellular senescence in normal cells (
      • Bodnar A.G.
      • Ouellette M.
      • Frolkis M.
      • Holt S.E.
      • Chiu C-P.
      • Morin G.B.
      • Harley C.B.
      • Shay J.W.
      • Lichtsteiner S.
      • Wright W.E.
      ,
      • Vaziri H.
      • Benchimol S.
      ), elongation of telomeres using hTERT would prevent the induction of the senescence pathway in adriamycin-treated breast cancer cells or, at a minimum, postpone it. Both MCF-7- pBABEpuro cells and MCF-7-hTERT cells with elongated telomeres senesced with the same frequency and timing (Fig.5, A and B) nearly identical to uninfected MCF-7 controls. As expected, adriamycin treatment of MCF-7-hTERT cells did not result in a decline in telomerase activity levels (Fig. 5 C), whereas vector-only (pBABE) controls exhibited a decline in activity similar to uninfected MCF-7 cells (compare with Fig. 1).
      Figure thumbnail gr5
      Figure 5Adriamycin-induced senescence in MCF-7 and MCF-7-hTERT cells is independent of telomere length. A, MCF-7-pBABEpuro and MCF-7-hTERT cells were histochemically stained for β-galactosidase expression at day 7 after treatment. Original magnification, ×20. B, quantitation of β-galactosidase expression in the MCF-7-pBABEpuro and MCF-7-hTERT cells over time. Values represent the mean ± S.D. based on three random field with 100 cell counts/field. C, MCF-7-pBABEpuro and MCF-7-hTERT cells were acutely treated with adriamycin (1 μm), harvested at the indicated days, and analyzed by the TRAP assay using 500 cell equivalents/sample. IC denotes the 36-bp internal control band. D, TRF analysis of MCF-7-pBABEpuro and MCF-7-hTERT cells before and 72 h after acute adriamycin treatment. The numbers on the leftshow the positions of a radiolabeled DNA marker in kb, and thewhite bars indicate the position of the measured mean telomere length (
      • Ouellette M.
      • Liao M.
      • Shea-Herbert B.
      • Johnson M.
      • Holt S.E.
      • Liss H.S.
      • Shay J.W.
      • Wright W.E.
      ). AdR, adriamycin.
      Because there was no lag in the timing of the onset of senescence in the MCF-7-hTERT cells, overall telomere shortening does not appear to be involved in the senescence process. To show this conclusively, at 24 (data not shown) and 72 h when the majority of cells are β-galactosidase-positive after acute treatment with adriamycin, changes in telomere length were determined for treated and untreated cells (representative TRF shown in Fig. 5 D). Mean telomere length calculations (
      • Ouellette M.
      • Liao M.
      • Shea-Herbert B.
      • Johnson M.
      • Holt S.E.
      • Liss H.S.
      • Shay J.W.
      • Wright W.E.
      ) provide a quantitative estimate of changes in telomere length after treatment: MCF-7-pBABE (untreated 3.4 kb and treated 3.4 kb) and MCF-7-hTERT (untreated 8.3 kb and treated 8.1 kb). The 200-bp difference in calculated average telomere length in the MCF-7-hTERT cells is not significantly different and is within the range of experimental error. Thus, we found no substantial change in telomere length in treated and untreated MCF-7 and MCF-7-hTERT cells, demonstrating that the senescence phenotype observed in adriamycin-treated cells is not directly related to overall telomere shortening.
      Because recent evidence implicates telomere dysfunction as a cause of replicative senescence (
      • Karlseder J.
      • Smogorzewska A.
      • de Lange T.
      ), we determined the types of chromosomal changes induced by adriamycin treatment. To eliminate the possible contributions of shortened telomeres, we used the MCF-7-hTERT cells and evaluated the frequency and location of structural chromosomal changes in MCF-7-hTERT cells. As expected, adriamycin induced many structural anomalies related to chromosomal ends such as end:end fusions, end breaks, and radial chromosomes (TableI). Interestingly, the number of telomere-associated breaks and rearrangements (77 total observations excluding dicentrics) were overrepresented compared with interstitial breaks (24 observations) (p ≤ 0.0001). These data suggest that adriamycin, a potent topoisomerase II inhibitor, preferentially produced breaks in distal chromosomal sequences and that these telomeric changes may ultimately contribute to the onset of replicative senescence in p53 wild-type breast tumor cells.
      Table ICharacterization of cytogenetic abnormalities in adriamycin-treated MCF-7 cells
      Cell lineMetaphases scoredMetaphases with abnormalities
      Several spreads had more than one structural change, so the total number of anomalies seen is greater than the number of abnormal spreads.
      Structural anomalies involving chromosome endsDicentrics
      The end versusinterstitial nature of the breaks giving rise to dicentric chromosomes could not always be clearly delineated. Thus, these aberrations are not categorized as a structural change clearly arising from an end compared to an interstitial region of the chromosome.
      Breaks involving interstitial sites
      End fusionsRingsRadialsEnd breaksChromatidChromosome
      MCF-7 hTERT10093100320
      MCF-7 hTERT + AdR
      AdR, adriamycin.
      100725536135222
      a Several spreads had more than one structural change, so the total number of anomalies seen is greater than the number of abnormal spreads.
      b The end versusinterstitial nature of the breaks giving rise to dicentric chromosomes could not always be clearly delineated. Thus, these aberrations are not categorized as a structural change clearly arising from an end compared to an interstitial region of the chromosome.
      c AdR, adriamycin.

      DISCUSSION

      Replicative senescence in normal human cells involves the action of the tumor suppressors p53 and pRB, presumably in response to the DNA damage signal elicited by shortened telomeres and/or telomere dysfunction. We have previously demonstrated prolonged growth arrest and the absence of an initial apoptotic response after exposure of MCF-7 breast cancer cells to either chronic treatment of a sublethal (50 nm) concentration of adriamycin (
      • Fornari F.A.
      • Jarvis W.D.
      • Grant S.
      • Orr M.S.
      • Randolph J.K.
      • White F.K.H.
      • Mumaw V.R.
      • Lovings E.T.
      • Freeman R.H.
      • Gewirtz D.A.
      ) or a single acute dose (1 μm) for 2–4 h (
      • Fornari F.A.
      • Jarvis W.D.
      • Orr M.S.
      • Randolph J.K.
      • Grant S.
      • Gewirtz D.A.
      ). Others have recently reported (
      • Chang B.D.
      • Xuan Y.
      • Broude E.V.
      • Zhu H.
      • Schott B.
      • Roninson I.B.
      ,
      • Chang B-D.
      • Broude E.V.
      • Dokmanovic M.
      • Zhu H
      • Ruth A.
      • Xuan Y.
      • Kandel E.S.
      • Lausch E.
      • Christov K.
      • Roninson I.B.
      ) that chronic exposure to sublethal concentrations of adriamycin promotes senescence in a variety of solid tumor cell lines based on β-galactosidase expression and cell morphology. Here, we clearly demonstrate that acute treatment of breast tumor cells with a clinically relevant dose of adriamycin (1 μm) results in the induction of replicative senescence and down-regulation of telomerase activity.
      To determine whether telomere shortening was the ultimate cause of the observed senescence following acute adriamycin exposure, we generated an isogenic strain of MCF-7 with elongated telomeres. The catalytic subunit of telomerase, hTERT, was over-expressed in MCF-7 cells, which provided for increased telomerase activity and elongated telomere lengths. Upon treatment with adriamycin, the MCF-7-hTERT cells underwent senescence with identical frequency and periodicity as the parental or vector-only controls. These data clearly indicate that constitutive expression of telomerase does not delay or prevent the senescence program from occurring, suggesting a telomere length-independent cause for the senescence growth arrest in treated breast tumor cells. In addition, because adriamycin-treated MCF-7-hTERT cells continue to express telomerase activity even after treatment and growth arrest, the suppression of telomerase activity in parental MCF-7 cells is probably transcriptional rather than through proteolysis. In fact, our preliminary data suggest that endogenous hTERT mRNA levels are reduced within the first 24 h after treatment.
      L. W. Elmore, D. A. Gewirtz, and S. E. Holt, unpublished data.
      We also found no detectable shortening in telomere lengths after drug treatment in either the control cells with short telomeres or the MCF-7-hTERT cells with elongated telomeres. These results provide direct experimental evidence that the senescence observed in adriamycin-treated MCF-7 cells is not telomere length-based, a result consistent with the inability of exogenous telomerase to prevent the premature senescence that occurs in normal cells strains after overexpression of oncogenic Ha-Ras (
      • Wei S.
      • Wei W.
      • Sedivy J.M.
      ). It is possible that an individual chromosome within the cell has shortened telomeres after treatment because of chromosome breaks at the telomere (
      • Hemann M.T.
      • Strong M.A.
      • Hao L.Y.
      • Greider C.W.
      ), which is beyond the limits of detection for our assay. However, given that virtually all of the MCF-7-pBABEpuro and MCF-7-hTERT cells senesced within the identical time frame, it is unlikely that this drug-induced senescence is the result of telomere shortening. The elongated telomeres would require significantly more population doublings to shorten telomeres enough to induce a telomere length-based senescence.
      Instead, we propose that the senescence induced in these solid tumor-derived cell lines by adriamycin is in fact attributed to a reduction in the protective function of the chromosome ends (i.e. telomere dysfunction). As a result of adriamycin treatment, chromosomal ends were preferentially targeted for DNA damage, presumably induced by deregulation of topoisomerase II, where single strand and double strand breaks accumulate and force deprotection at the telomere. Consistent with previous findings for normal cell cultures with genetic manipulation (
      • Karlseder J.
      • Smogorzewska A.
      • de Lange T.
      ), we find karyotypic instability as a result of telomere dysfunction and a lack of chromosome end protection after chemotherapeutic treatment. Our data clearly show that adriamycin-induced cytogenetic abnormalities at chromosome ends were significantly elevated compared with interstitial changes. Furthermore, one can speculate that the mechanism of the adriamycin-induced telomere-related abnormalities is probably the result of deregulation of telomere binding proteins and disruption of the telomere loop structure (
      • Griffith J.D.
      • Comeau L.
      • Rosenfield S.
      • Stansel R.M.
      • Bianchi A.
      • Moss H.
      • de Lange T.
      ). Direct proof of this hypothesis is currently under investigation.
      The absence of an initial apoptotic response to adriamycin in the MCF-7 breast tumor cells may reflect the generalized refractoriness of breast tumor cells to apoptosis induced by DNA damage. One of the general criticisms with using the MCF-7 breast tumor cell line as a model is its lack of functional caspase 3 expression (
      • Kagawa S., Gu, J.
      • Honda T.
      • McDonnell T.J.
      • Swisher S.G.
      • Roth J.A.
      • Fang B.
      ), reasoning that in response to adriamycin, these cells are unable to undergo apoptosis and that because they express wild-type p53, senescence is induced. Our work and that of others demonstrate an essentially identical pattern of response to adriamycin (initial non-apoptotic cell death followed by prolonged growth arrest) in a variety of p53-positive breast tumor cells including MCF-7 and ZR-75–1 cells (data not shown) (
      • Kagawa S., Gu, J.
      • Honda T.
      • McDonnell T.J.
      • Swisher S.G.
      • Roth J.A.
      • Fang B.
      ). We also find that MCF-7 cells ectopically expressing caspase 3 undergo drug-induced senescence comparable to parental controls.
      L. W. Elmore and S. E. Holt, unpublished observations.
      Preliminary studies have demonstrated that adriamycin induces MAPK and promotes the phosphorylation of the BAD protein, both of which may confer protection against apoptosis in breast tumor cells. It is possible that cells responding to adriamycin through a senescence arrest rather than apoptosis may ultimately die through another process such as reproductive cell death (
      • Chang B.D.
      • Xuan Y.
      • Broude E.V.
      • Zhu H.
      • Schott B.
      • Roninson I.B.
      ).
      Because p53 is intimately involved in the senescence process in primary normal cell strains (
      • Shay J.W.
      • Pereira-Smith O.M.
      • Wright W.E.
      ), we sought to define its role in the adriamycin-induced senescence phenotype in MCF-7 cells. We found that non-isogenic cell lines with mutant p53 (MDA-MB231) exhibited very little senescence after treatment with adriamycin but instead displayed a delayed apoptosis effect. To isogenically determine whether p53 was critical in the difference between senescence and apoptosis induction, MCF-7 cells were generated, which exogenously expressed the HPV-16 E6 oncogene to eliminate p53. Introduction of HPV-16 E6 resulted in the degradation and inactivation of the p53 protein, abolishing a sustained p53-mediated DNA damage response when treated with adriamycin. Yet, instead of undergoing senescence as before, treated MCF-7-E6 cells underwent a delayed programmed cell death, similar to other breast tumor cell lines without functional p53 (MDA-MB231). Seemingly inconsistent with our findings, a previous report concludes that MCF-7 cells with disrupted p53 (MCF-7-E6) are not sensitized to adriamycin (
      • Fan S.
      • Smith M.L.
      • Rivet D.J.
      • Duba D.
      • Zhan Q.
      • Kohn K.W.
      • Fornace A.J.
      • O'Connor P.M.
      ). However, unlike our study that employed a comparative quantitative assessment of the frequency of apoptosis and senescence in isogenic cell lines, these investigators utilized clonogenic survival as their end point assay, which is a more limited approach that would not distinguish between a senescent or apoptotic cell.
      Although it is clear that E6 has a number of functions unrelated to p53 inactivation, the currently defined role for HPV-16 E6 is mainly associated with the prevention of apoptosis during transformation (reviewed in Ref.
      • Hengstermann A.
      • Linares L.K.
      • Ciechanover A.
      • Whitaker N.J.
      • Scheffner M.
      ). Thus, although it is formally possible that E6 is partially responsible for the shift of MCF-7 cells from senescence to apoptosis after adriamycin treatment, it is more probable that the elimination of p53 function is the mechanism for the conversion. This conclusion is supported by the recent data on p53 and p16INK4a in murine tumors that indicates p53 status is critical in determining drug-induced cellular fate (
      • Schmitt C.A.
      • Fridman J.S.
      • Yang M.
      • Lee S.
      • Baranov E.
      • Hoffman R.M.
      • Lowe S.W.
      ). Although they find that p53 and p16INK4a play an equally important role in murine tumors, MCF-7 cells treated with adriamycin require wild-type p53 to undergo senescence in the absence of p16INK4a, suggesting that adriamycin-induced senescence in breast tumor cells does not call for p16INK4a. Taken together, our results indicate the following: 1) Programmed cell death after adriamycin treatment is possible in the absence of functional caspase 3; 2) Functional p53 is critical for the senescence phenotype observed in MCF-7 cells after adriamycin treatment. Our experimental data suggest that breast tumors with a loss of wild-type p53 function would be significantly more sensitive to adriamycin-induced apoptosis. Thus, assessing the p53 status in clinical specimens may have value for tailoring chemotherapeutic treatments for breast cancer patients, especially those involving adriamycin.

      ACKNOWLEDGEMENT

      We thank Melissa Landon for critical experimental assistance.

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