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• W1984107812 abstract "Skin provides an attractive organ system for exploring coordinated regulation of keratinocyte (KC) proliferation, differentiation, senescence, and apoptosis. Our main objective was to determine whether various types of cell cycle arrest confer resistance to apoptosis. We postulated that KC cell cycle and cell death programs are tightly regulated to ensure epidermal homeostasis. In this report, simultaneous expression of cyclin-dependent kinase inhibitors (p15, p16, p21, and p27), a marker of early differentiation (keratin 1), mediators of apoptosis (caspases 3 and 8), and NF-κB were analyzed in three types of KCs. By comparing the response of proliferating, senescent, and immortalized KCs (HaCaT cells) to antiproliferative agents followed by UV exposure, we observed: 1) Normal KCs follow different pathways to abrupt cell cycle arrest; 2) KCs undergoing spontaneous replicative senescence or confluency predominantly express p16; 3) Abruptly induced growth arrest, confluency, and senescence pathways are associated with resistance to apoptosis; 4) The death-defying phenotype of KCs does not require early differentiation; 5) NF-κB is one regulator of resistance to apoptosis; and 6) HaCaT cells have undetectable p16 protein (hypermethylation of the promoter), dysfunctional NF-κB, and diminished capacity to respond to antiproliferative treatments, and they remain highly sensitive to apoptosis with cleavage of caspases 3 and 8. These data indicate that KCs (but not HaCaT cells) undergoing abruptly induced cell cycle arrest or senescence become resistant to apoptosis requiring properly regulated activation of NF-κB but not early differentiation. Skin provides an attractive organ system for exploring coordinated regulation of keratinocyte (KC) proliferation, differentiation, senescence, and apoptosis. Our main objective was to determine whether various types of cell cycle arrest confer resistance to apoptosis. We postulated that KC cell cycle and cell death programs are tightly regulated to ensure epidermal homeostasis. In this report, simultaneous expression of cyclin-dependent kinase inhibitors (p15, p16, p21, and p27), a marker of early differentiation (keratin 1), mediators of apoptosis (caspases 3 and 8), and NF-κB were analyzed in three types of KCs. By comparing the response of proliferating, senescent, and immortalized KCs (HaCaT cells) to antiproliferative agents followed by UV exposure, we observed: 1) Normal KCs follow different pathways to abrupt cell cycle arrest; 2) KCs undergoing spontaneous replicative senescence or confluency predominantly express p16; 3) Abruptly induced growth arrest, confluency, and senescence pathways are associated with resistance to apoptosis; 4) The death-defying phenotype of KCs does not require early differentiation; 5) NF-κB is one regulator of resistance to apoptosis; and 6) HaCaT cells have undetectable p16 protein (hypermethylation of the promoter), dysfunctional NF-κB, and diminished capacity to respond to antiproliferative treatments, and they remain highly sensitive to apoptosis with cleavage of caspases 3 and 8. These data indicate that KCs (but not HaCaT cells) undergoing abruptly induced cell cycle arrest or senescence become resistant to apoptosis requiring properly regulated activation of NF-κB but not early differentiation. keratinocyte cylin-dependent kinase inhibitor transforming growth factor interferon 12-O -tetradecanoylphorbol-13-acetate terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid Normal human skin is covered by a multi-layered epidermis in which keratinocytes (KCs)1 undergo a continuous process of proliferation, differentiation and apoptosis (1Fuchs E. J. Cell Biol. 1990; 111: 2807-2814Crossref PubMed Scopus (588) Google Scholar, 2Dotto P. Front. Biosciences. 1998; 3: 502-508Crossref PubMed Scopus (13) Google Scholar). In this life-long self-renewing process, quiescent stem cells are triggered to produce transiently amplifying cells, which then give rise to early stages of differentiation followed by terminal differentiation and death. This process is responsible for maintaining the proper cutaneous thickness and barrier function of stratified epithelium (2Dotto P. Front. Biosciences. 1998; 3: 502-508Crossref PubMed Scopus (13) Google Scholar). Such dynamic tissue homeostasis involving cells in the basal layer and throughout suprabasilar levels requires a delicate balance of epidermal cells entering the proliferating poolversus cells in various stages of differentiation, senescence, and death (3Haake A.R. Polakowska R.R. J. Invest. Dermatol. 1993; 101: 107-112Abstract Full Text PDF PubMed Google Scholar). These cellular transitions take place within a few cell layers each month over the span of several decades. Although the relationship between proliferation, replicative senescence and apoptosis has been extensively explored in dermal fibroblasts, less is known regarding these complex processes in epidermis (4Wang E. Lee M.J. Pandey S. J. Cell. Biochem. 1994; 54: 432-439Crossref PubMed Scopus (74) Google Scholar, 5Linskens M.H. Harley C.B. West M.D. Campisi J. Hayflick G. Science. 1995; 267: 17Crossref PubMed Scopus (78) Google Scholar, 6Alcorta D.A. Xiong Y. Phelps D. Hannon G. Beach D. Barrett J.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13742-13748Crossref PubMed Scopus (804) Google Scholar, 7Smith J.R. Pereira-Smith D.M. Science. 1996; 273: 63-67Crossref PubMed Scopus (471) Google Scholar, 8Brown J.P. Wei W. Sedivy J.M. Science. 1997; 277: 831-834Crossref PubMed Scopus (685) Google Scholar, 9Zhu J. Woods D. McMahon M. Bishop J.M. Genes Dev. 1998; 12: 2997-3007Crossref PubMed Scopus (661) Google Scholar). Furthermore, because dermal fibroblasts do not undergo terminal differentiation like KCs, it is unclear whether differentiation-inducing treatments influence apoptotic-related events. To fill this experimental void, various types of skin-derived KCs were used to explore molecular mechanisms that regulate cell proliferation, differentiation, senescence, and apoptosis in skin (10Campisi J. J. Invest. Dermatol. 1998; 3: 1-5Scopus (176) Google Scholar). Our working hypothesis extends observations by previous investigators who established there are multiple pathways leading to senescence and protection from apoptosis in other organ systems (4Wang E. Lee M.J. Pandey S. J. Cell. Biochem. 1994; 54: 432-439Crossref PubMed Scopus (74) Google Scholar, 5Linskens M.H. Harley C.B. West M.D. Campisi J. Hayflick G. Science. 1995; 267: 17Crossref PubMed Scopus (78) Google Scholar, 6Alcorta D.A. Xiong Y. Phelps D. Hannon G. Beach D. Barrett J.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13742-13748Crossref PubMed Scopus (804) Google Scholar, 7Smith J.R. Pereira-Smith D.M. Science. 1996; 273: 63-67Crossref PubMed Scopus (471) Google Scholar, 8Brown J.P. Wei W. Sedivy J.M. Science. 1997; 277: 831-834Crossref PubMed Scopus (685) Google Scholar, 9Zhu J. Woods D. McMahon M. Bishop J.M. Genes Dev. 1998; 12: 2997-3007Crossref PubMed Scopus (661) Google Scholar), and that KCs can also follow different biochemical pathways leading to cell cycle arrest. Furthermore, we postulated that these pathways will have distinctive characteristics as regards subsequent cell fate decisions such as differentiation, senescence, and susceptibility/resistance to apoptosis. The goal of this project was to begin to elucidate the phenotype and biochemical pathways regulating KC growth arrest, differentiation, and apoptosis. We postulated that there would be biochemical links between KC replication, senescence, and apoptosis and designed experiments to address how various types of KCs would respond to rapidly induced growth arrest or spontaneous replicative senescence, followed by exposure to high levels of ultraviolet radiation (UV light) that acutely triggers apoptosis. Primary KC cultures proliferate for several passages in a low calcium, serum-free medium (11Nickoloff B.J. Mitra R.S. Riser B.L. Dixit V.M. Varani J. Am. J. Pathol. 1998; 132: 543-551Google Scholar). Freshly isolated, proliferating, and relatively undifferentiated KCs can become growth-arrested and subsequently follow at least five different pathways. First, if growth supplements are removed, KCs maintained in basal medium will become quiescent and remain viable for at least several days, but retain the capacity to re-enter the cell cycle (12Ruisser F. Erp P.E.J. Jongh G.J. Boezeman J.B.M. Kerkhof P.C.M. Schalkwijk J. J. Cell Sci. 1994; 107: 2219-2228PubMed Google Scholar). Another distinct method for inducing reversible growth inhibition of KCs is exposure to anti-proliferative agents such as transforming growth factor β (TGF-β) (13Missero C. Calautti E. Eckner R. Chin J. Tsai L.H. Livingston D.M. Dotto G.P. Proc. Natl. Acad. Sci. 1995; 92: 5451-5455Crossref PubMed Scopus (329) Google Scholar). These initial two growth-arresting pathways can be reversed if quiescent cells are subsequently stimulated to re-enter the cell cycle and proliferate by addition of competence and progression factors following withdrawal of TGF-β (14Nickoloff B.J. Misra P. Morhenn V.B. Hintz R. Rosenfeld R. Dermatologica. 1988; 177: 265-273Crossref PubMed Scopus (30) Google Scholar). A third pathway involves growth arrest such that no further proliferation is possible, and growth arrest does not induce early markers of diffentiation (i.e. keratin 1), such as after exposure to phorbol ester and/or interferon γ (IFN-γ) (15Sark M.W.J. Fisher D.F. Demeijer E. Vandepatte P. Backendorf C. J. Biol. Chem. 1998; 273: 24687-24692Abstract Full Text Full Text PDF Scopus (68) Google Scholar,16Saunders N. Dahler A. Jones S. Smith R. Jetten A. J. Dermatol. Sci. 1996; 13: 98-106Abstract Full Text PDF PubMed Scopus (30) Google Scholar). A fourth pathway involves irreversible growth arrest and early differentiation, which occurs when extracellular calcium ion concentration is increased (17Rosenthal D.S. Steinert P.M. Ching S. Huff C.A. Johnson J. Yuspa S.H. Roop D.R. Cell Growth. Differ. 1991; 2: 107-113PubMed Google Scholar). A fifth pathway for KCs is to undergo replicative senescence, in which case they remain viable and metabolically active but not capable of any further replicative expansion (18Loughran O. Malliri A. Owens D. Gallimoe P.H. Stanley M.A. Ozanne B. Frame M.C. Parkinson E.K. Oncogene. 1996; 13: 561-568PubMed Google Scholar). The purpose of this investigation was to delineate the response of KCs to various stimuli that can influence all five potential pathways and to examine the interactive behavior of both cell cycle regulatory proteins that predominantly localize to the nucleus, with members of the caspase family that are present in the cytoplasm and regulate apoptosis (19Mitra R.S. Wrone-Smith T. Foreman K.E. Nunez G. Nickoloff B.J. Lab. Invest. 1997; 76: 99-107PubMed Google Scholar). Because NF-κB is a key transcription regulator in KCs and plays a critically important role in both regulation of the cell cycle as well as influencing cell death pathways, particular focus was directed at this transcription factor (20Beg A.A. Baltimore D.M. Science. 1996; 274: 782-784Crossref PubMed Scopus (2940) Google Scholar, 21Seitz C.S. Lin Q. Deng M. Khavari P.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2307-2312Crossref PubMed Scopus (382) Google Scholar, 22Tomic-Canic M. Komine M. Freedberg J.M. Blumenberg M. J. Dermatol. Sci. 1998; 17: 167-181Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). In addition to the aforementioned cell cycle arresting agents, UV light was also used because it can induce apoptosis in human skin and is regarded as an important etiological factor in development of skin cancer (23Rehemtulla A. Hamilton C.A. Chinnaiyan A.M. Dixit V.M. J. Biol. Chem. 1997; 272: 25783-25786Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar, 24Aragane Y. Kulms D. Metze D. Wilkes G. Poppelmann B. Luger T.A. Schwarz T. J. Cell Biol. 1998; 140: 171-182Crossref PubMed Scopus (432) Google Scholar, 25Denning M.F. Wang Y. Nickoloff B.J. Wrone-Smith T. J. Biol. Chem. 1998; 273: 29995-30002Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). The death defying behavior of normal skin-derived KCs was compared with senescent KCs and immortalized HaCaT cells (26Boukamp P. Petrussevska R.T. Breitkreutz D. Hornung J. Markham A. Fusenig N.E. J. Cell Biol. 1988; 106: 761-771Crossref PubMed Scopus (3493) Google Scholar, 27Fusenig N.E. Boukamp P. Mol. Carcinogen. 1998; 23: 144-158Crossref PubMed Scopus (231) Google Scholar). Because the orderly process of KC proliferation, differentiation, and apoptosis occurs with a high degree of fidelity in skin, we postulated that the regulatory mechanisms involved in maintaining homeostasis would reveal a remarkable degree of coordination among the pathways that regulate the cell cycle (i.e. proliferation), and several key molecular participants involved in caspase cascades (i.e. cell death). Interestingly, just as KC differentiation is not required for cells to undergo apoptosis (19Mitra R.S. Wrone-Smith T. Foreman K.E. Nunez G. Nickoloff B.J. Lab. Invest. 1997; 76: 99-107PubMed Google Scholar), we observed that early stages of differentiation were not required for growth-arrested or senescent KCs to acquire an apoptotic-resistant phenotype. Because the c-myc proto-oncogene is a central regulator of cell proliferation, differentiation, and apoptosis, expression of c-Myc protein levels were also examined (28Henriksson M. Luscher B. Adv. Cancer Res. 1996; 68: 109-182Crossref PubMed Google Scholar, 29Vlach J. Hennecke S. Alevizopoulos K. Conti D. Amati B. EMBO J. 1996; 15: 6595-6604Crossref PubMed Scopus (298) Google Scholar). By comparing the behavior and response of normal KCs, senescent KCs, and immortalized KCs to antiproliferative agents followed by UV light, new insights were gained into the complex interactions of molecular mediators that regulate KC proliferation, growth arrest, differentiation, senescence, and apoptosis. Primary KCs were isolated from freshly excised neonatal foreskins as described previously (11Nickoloff B.J. Mitra R.S. Riser B.L. Dixit V.M. Varani J. Am. J. Pathol. 1998; 132: 543-551Google Scholar) HaCaT cells, an immortalized KC cell line, were obtained from Professor N. Fusenig (Heidelberg, Germany) (26Boukamp P. Petrussevska R.T. Breitkreutz D. Hornung J. Markham A. Fusenig N.E. J. Cell Biol. 1988; 106: 761-771Crossref PubMed Scopus (3493) Google Scholar). Both normal KCs and HaCaT cells were maintained in a low calcium (0.15 mm) KC growth medium (KGM, Clonetics, San Diego, CA). Cells were treated with either KGM alone or KGM containing various treatments for 8 and 48 h. In some experiments, KGM was replaced with a basal medium (KBM) lacking growth supplements. Treatments to reduce cell proliferation included addition of recombinant IFN-γ (103 units/ml, Genentech Inc., San Francisco, CA), 12-O -tetradecanoylphorbol-13-acetate (TPA, 5 nmol/liter; Sigma) or transforming growth factor-β (10 ng/ml; Upstate Biotechnology, Inc., Lake Placid, NY) or increasing the extracellular calcium ion concentration to 2.0 mm. Counting of cells was performed manually using a calibrated slide chamber hemacytometer. The caspase inhibitor Z-VAD-FMK and IEDT were purchased from Enzyme System Products (Livermore, CA), and DEVD was purchased from Calbiochem (La Jolla, CA). In some experiments KCs were pre-exposed for 2 h to the proteasome inhibitor MG132 (0.1–1 μm, BIOMOL Research Laboratories, Inc., Plymouth Meeting, PA) to inhibit NF-κB activation as described previously (30Kothny-Wilkes G. Kulms D. Poppelmann B. Luger T.A. Kubin M. Schwarz T. J. Biol. Chem. 1998; 273: 2925-29247Abstract Full Text Full Text PDF Scopus (98) Google Scholar). SW13 cells were obtained from American Type Culture Collection. The anti-caspase-3/CPP32 antibody was used at 1:2,000 (c31720, Transduction Laboratories, Lexington, KY), and the anti-caspase 8 and anti-poly(ADP)ribose polymerase antibodies were purchased from CLONTECH (Palo Alto, CA). Antibody against Bcl-x was obtained from Craig Thompson (University of Chicago) and used as described previously (19Mitra R.S. Wrone-Smith T. Foreman K.E. Nunez G. Nickoloff B.J. Lab. Invest. 1997; 76: 99-107PubMed Google Scholar). Antibodies against p15 (sc-612R), p16 (sc-468R), p21 (sc-397R), and p27 (sc-528R) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA), as were antibodies against the p50 (sc-7178) and p65 (sc-109) subunits of NF-κB, the antibody against c-Myc (clone 9E10), and the antibody against heat shock protein 27 (sc-1048). Flow cytometry was performed on single cell suspensions obtained using trypsin/EDTA as described previously (19Mitra R.S. Wrone-Smith T. Foreman K.E. Nunez G. Nickoloff B.J. Lab. Invest. 1997; 76: 99-107PubMed Google Scholar). Briefly, for cell cycle analysis propidium iodide staining (50 μg/ml, Sigma) was performed following the manufacturer's instructions. Also, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay positive cells were detected following fixation and staining as described previously (19Mitra R.S. Wrone-Smith T. Foreman K.E. Nunez G. Nickoloff B.J. Lab. Invest. 1997; 76: 99-107PubMed Google Scholar). Apoptosis was induced by irradiating KCs with a Panelite Unit (Ultralite Enterprises, Inc., Lawrenceville, GA) equipped with four UVB bulbs (FS36T12/UVB-VH0) that have the majority of their output in the UVB range (65%), with minor output in the UVA and UVC range (34 and 1%, respectively). KCs were irradiated with dish lids removed using a dose of 25 mJ/cm2. The UV dose was monitored with an International Light Inc. (Newburyport, MA) radiometer fitted with a UVB detector. In selected experiments, cells were pretreated with caspase inhibitors for 30 min prior to irradiation as described previously (25Denning M.F. Wang Y. Nickoloff B.J. Wrone-Smith T. J. Biol. Chem. 1998; 273: 29995-30002Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). Another method for inducing apoptosis was to place a single cell suspension of KCs in KGM medium containing 1.68% methylcellulose (4000 centipoises, Sigma) for 48 h as described previously (19Mitra R.S. Wrone-Smith T. Foreman K.E. Nunez G. Nickoloff B.J. Lab. Invest. 1997; 76: 99-107PubMed Google Scholar). Nuclear cell lysate and whole cell lysate were prepared to detect different proteins. In brief, for nuclear lysates cells were washed with phosphate-buffered saline, pelleted in buffer A (20 mm Hepes, pH 7.9, 1.5 mm MgCl2, 0.2 mm EDTA, 0.5 mm phenylmethylsulfonyl fluoride, 0.5 mmdithiothreitol with 0.1% Nonidet P-40), incubated in ice for 15 min, and microcentrifuged, and the supernatant was discarded. The pellet was resuspended in buffer C (20 mm Hepes, 0.4 mNaCl, 1 mm EDTA, 1 mm EGTA, 1 mmdithiothreitol, 1 mm phenylmethylsulfonyl fluoride) for 15 min on ice. Cells were vortexed and microcentrifuged, and the supernatant was saved and frozen at −80 °C. For the whole cell lysate, KCs were washed with phosphate-buffered saline and were incubated in ice for 15 min in CHAPS buffer (31Liles W.C. Ledbetter J.A. Waltersdoeph A.W. Klebanoff S.J. J. Immunol. 1997; 155: 2175-2184Google Scholar). Cells were microcentrifuged, and supernatants were saved and frozen at −80 °C. Protein concentration of each sample was determined by Lowry assay. 30 μg of protein were loaded on 8–12.5% SDS-polyacrylamide gel, transferred to Immobilon-p (polyvinylidene difluoride) membrane and blocked in 5% nonfat powdered milk in TBST (50 mm Tris, pH 7.5, 150 mm NaC1, 0.01% Tween 20). The membrane was incubated with the primary antibody in 2.5% powdered milk in TBST and was washed extensively with TBST and then incubated with 1:1500 diluted anti-rabbit or mouse horseradish peroxidase (Amersham Pharmacia Biotech). Proteins were visualized with ECL reagents (Amersham Pharmacia Biotech) according to manufacturer's instruction. Loading of proteins to verify equivalent distribution of proteins in each well was confirmed by Ponceau S staining. Total cellular RNA was extracted using Trizol Reagent (Life Technologies, Inc.). The RNase protection assay was performed according to the supplier's instructions (PharMingen, San Diego, CA). Briefly, human apoptosis template set hAP0–5 was labeled with [α-32P]uridine triphosphate. RNA (10 μg) and 8 × 105 cpm of labeled probes were used for hybridization, and after RNase treatment, the protected probes were resolved on a 5% sequencing. Electrophoretic mobility shift assays were performed as described previously (24Aragane Y. Kulms D. Metze D. Wilkes G. Poppelmann B. Luger T.A. Schwarz T. J. Cell Biol. 1998; 140: 171-182Crossref PubMed Scopus (432) Google Scholar). In brief, 1 μg of poly(dI-dC) (Amersham Pharmacia Biotech) and 104 cpm of 32P-labeled double-stranded oligonucleotide were incubated with 5 μg of nuclear protein on ice for 30 min. The reaction mixture was separated on 4% native polyacrylamide gel, dried, and autoradiographed. The NF-κB oligonucleotide had the following sequence: 5′-AGT TGA GGG GAC TTT CCC AGG C-3′. Competition analysis was performed by adding excess unlabeled oligonucleotides. For supershift experiments, 4 μg of rabbit polyclonal antibodies against p50, p65, RelB, and c-Rel (Santa Cruz) subunits of NF-κB were incubated with 5 μg of nuclear proteins for 30 min on ice, prior to the addition of 32P-labeled probe. Examination of the p16 gene in HaCaT cells was performed initially by sequence analysis of exons 1 and 2 using an automated DNA sequencer as described previously (32Timmermann S. Hinds P.W. Munger K. Oncogene. 1998; 17: 3445-3453Crossref PubMed Scopus (69) Google Scholar). In addition, detection of p16 promoter methylation was performed by Southern blot analysis as described previously (33Otterson G.A. Khlief S.N. Chen W. Coxon A.B. Karge F.J. Oncogene. 1995; 11: 1211-1216PubMed Google Scholar). Briefly, 10 μg of total genomic DNA was isolated from HaCaT cells, normal KCs, and a cell line known to have p16 DNA hypermethylation (SW13). DNA was subjected to restriction endonuclease digestion with Eco RI alone or Eco RI and Sac II or Eco RI andEag I (Life Technologies, Inc.). After running on 1% agarose gel, DNA was transferred to nitrocellulose membrane. This membrane was analyzed with a genomic DNA fragment (1.1-kilobase probe) containing the promoter and exon 1 of human p16 gene as described previously (33Otterson G.A. Khlief S.N. Chen W. Coxon A.B. Karge F.J. Oncogene. 1995; 11: 1211-1216PubMed Google Scholar). When neonatal foreskin-derived KCs are maintained at subconfluent density in a low calcium serum-free medium, they proliferate and are highly sensitive to induction of apoptosis by UV irradiation. A representative cell cycle profile for proliferating normal KCs is presented in Fig.1 A in which less than 1% of the cells have a sub-G0 DNA content and 5% are TUNEL-positive. Typically, proliferating KCs have 45–55% of cells in G1, 30–40% of cells in S, and 5–15% of cells in G2M. 18 h after UV irradiation (25 mJ/cm2), subconfluent KCs undergo apoptosis with over 55% of the KCs having sub-G0 DNA, and 39% becoming TUNEL-positive (Fig. 1 B ). However, if KCs become confluent and then exposed to UV (Fig. 1 C ), substantially less apoptosis (17% sub G0 DNA, 22% TUNEL-positive) is induced. If confluent KCs cultures have their growth supplements removed (i.e. washed and maintained in KBM for 24 h), the resistance to apoptosis observed for the confluent cultures is reduced, and over 70% of KCs have sub-G0 DNA and greater than 40% of the cells are TUNEL-positive after UV exposure (Fig.1 D ). To further assess the consequences of growth-arresting treatments, normal KCs at 50–60% confluence were treated for 24 h with 2 mm Ca2+, TPA, IFN-γ, or TGF-β and then washed and maintained in KGM for an additional 24 h. In this scenario, there is irreversible growth arrest for all of the treatments except TGF-β. KC cultures exposed to elevated Ca2+, TPA, IFN-γ, or TGF-β had a reduction in proliferation assessed by manual cell counting of viable cells revealing substantial reductions compared with untreated cells in KGM of 79 ± 8, 92 ± 8, 93 ± 6, and 83 ± 6%, respectively. After the pulse/wash treatments, the dishes were then exposed to UV light (25 mJ/cm2), and the KCs were examined as before. KCs pulsed/washed with Ca2+ (Fig. 1 E ) were highly resistant to UV-induced apoptosis (10% sub G0; 14% TUNEL-positive), as were KCs pulsed/washed after exposure to TPA (Fig. 1 F ) or IFN-γ (Fig. 1 G ). However, TGF-β-treated KCs were not as consistently protected as revealed by 45% sub-G0 DNA content and over 32% TUNEL-positive cells (Fig. 1 H ). Resistance to apoptosis for these treatments was not unique to UV light treatment, because similar results were observed when different KC cultures treated as above with identical pulse/wash protocols were trypsinized followed by 48 h of suspension in methylcellulose (data not shown). When the results using 24 h pulse/wash were compared with 48 h of continuous exposure similar levels of protection from UV-induced apoptosis were observed, with the exception that continuous growth arrest produced by 48 h of treatment with TGF-β enhanced the resistance to apoptosis (29% sub G0; 23% TUNEL-positive) compared with the pulse/wash protocol. Interestingly, in cultures of normal KCs that underwent spontaneous replicative senescence (i.e. passages 3–5), the exposure to UV did not induce apoptosis in these subconfluent cultured cells (Fig.1 I ). Pretreatment of senescent KCs with the aforementioned growth-arresting agents did not alter this resistance to UV-induced apoptosis (data not shown). To determine whether a similar phenotypic response would occur in immortalized KCs, HaCaT cells were treated following the same protocol as described above for early passage normal human KCs. HaCaT cells were also growth-arrested by exposure to elevated Ca2+, TPA, or IFN-γ as determined by manual cell counting, although with an overall diminished antiproliferative response. Compared with untreated HaCaT cells, cultures (n = 3) exposed for 72 h to elevated Ca2+, TPA, IFN-γ, or TGF-β had reductions in cell proliferation of 38 ± 5, 31 ± 85, 68 ± 8, and 54 ± 11%, respectively. In marked contrast to growth-arrested normal KCs, none of the treatments (i.e. either pulse/wash or continuous) reduced the extent of apoptosis present in HaCaT cells after UV exposure (Fig. 2). To better view the HaCaT cultures, only cell cycle DNA profiles are presented (although similar trends in TUNEL assays were also identified; data not shown). Briefly, subconfluent HaCat cells had only approximately 3% of the cells undergoing spontaneous apoptosis (sub-G0 DNA: Fig. 2 A ), whereas after UV exposure subconfluent (Fig.2 B ) or confluent cultures (Fig. 2 C ) or cells placed in KBM (Fig. 2 D ) had markedly increased numbers of cells with sub-G0 DNA (48–58%). Pulse/wash treatments using Ca2+, TPA, IFN-γ, or TGF-β prior to UV exposure provided no protective effects (Fig. 2, E–H , respectively). Also, 48 h of continuous exposure to these antiproliferative treatments did not change the ability of UV light to induce apoptosis in the HaCaT cells (data not shown). To determine the molecular mediators involved in the UV-induced apoptosis, Western blot analysis was performed on whole cell extracts before and after UV exposure with a focus on the caspase 8, caspase 3, and poly(ADP)ribose polymerase. UV-induced apoptosis in KCs is caspase-dependent, because it can be blocked by caspase inhibitors (25Denning M.F. Wang Y. Nickoloff B.J. Wrone-Smith T. J. Biol. Chem. 1998; 273: 29995-30002Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). Because caspase 3 is a primary executioner intermediate in this apoptotic pathway, the change in caspase 3 will be highlighted (Fig. 3), although similar changes were also observed for caspase 8 and poly(ADP)ribose polymerase (data not shown). Prior to UV irradiation, proliferating normal KCs and HaCaT cells had intact caspase 3, but after UV exposure both types of cells undergoing apoptosis had proteolysis (i.e. activation) of caspase 3. Normal KCs in either a confluent state or after pretreatment/wash with Ca2+, TPA, IFN-γ, or TGF-β had less evidence of caspase 3 cleavage upon subsequent UV exposure, which correlated with diminished induction of apoptosis. HaCaT cells treated in a similar fashion had no or barely detectable intact caspase 3 levels, consistent with the induction of apoptosis under these conditions induced by UV. To determine whether HaCaT cells were capable of acquiring an apoptotic resistant phenotype, several different caspase inhibitors such as z-VAD, DEVD, and IETD, were utilized (25Denning M.F. Wang Y. Nickoloff B.J. Wrone-Smith T. J. Biol. Chem. 1998; 273: 29995-30002Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). Pretreatment of HaCaT cells with these compounds prior to UV exposure decreased the apoptotic response (data not shown), with concomitant prevention of the cleavage of caspase 3 (Fig. 3). Overall, the degree of protection from UV-B-induced apoptosis and extent of caspase 3 degradation using these inhibitors was similar between KCs and HaCaT cells (data not shown). Two differentiation-related proteins were measured before and after the pulse/wash or continuous treatments including keratin-1 (present in suprabasilar KCs undergoing early differentiation) and keratin-14 (detectable in basal layer KCs in vivo and relatively undifferentiated cells in culture). Over 90% of cultured normal KCs were keratin 14-positive (expect for small foci of stratified clusters of KCs), and HaCaT cells were diffusely and uniformly expressed keratin-14 (Fig. 4,upper panel ). In contrast, only rare focal clusters of keratin-1-positive normal KCs maintained in KGM were" @default.
• W1984107812 created "2016-06-24" @default.
• W1984107812 creator A5032356581 @default.
• W1984107812 creator A5034146754 @default.
• W1984107812 creator A5048981189 @default.
• W1984107812 creator A5065426791 @default.
• W1984107812 creator A5069688610 @default.
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• W1984107812 date "1999-08-01" @default.
• W1984107812 modified "2023-10-14" @default.
• W1984107812 title "Apoptosis in Proliferating, Senescent, and Immortalized Keratinocytes" @default.
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