12-O-Tetradecanoylphorbol-13-acetate and UV Radiation-induced Nucleoside Diphosphate Protein Kinase B Mediates Neoplastic Transformation of Epidermal Cells
2004; Elsevier BV; Volume: 279; Issue: 7 Linguagem: Inglês
10.1074/jbc.m310820200
ISSN1083-351X
AutoresSung‐Jen Wei, Carol S. Trempus, Robin C. Ali, Laura A. Hansen, Raymond W. Tennant,
Tópico(s)Metastasis and carcinoma case studies
ResumoThe molecular changes associated with early skin carcinogenesis are largely unknown. We have previously identified 11 genes whose expression was up- or down-regulated by 12-O-tetradecanoylphorbol-13-acetate (TPA) in mouse skin keratinocyte progenitor cells (Wei, S.-J., Trempus, C. S., Cannon, R. E., Bortner, C. D., and Tennant, R. W. (2003) J. Biol. Chem. 278, 1758-1768). Here, we show an induction of a nucleoside diphosphate protein kinase B (NDPK-B) gene in response to TPA or UV radiation (UVR). TPA or UVR significantly induced the expression of NDPK-B both in vivo hyperplastic mouse skin and in vitro mouse JB6 Cl 41-5a epidermal cells. Indeed, this gene was also up-regulated in TPA or UVR-mediated skin tumors including papillomas, spindle cell tumors, and squamous cell carcinomas, relative to adjacent normal skins. Functional studies by constitutive expression of nm23-M2/NDPK-B in TPA susceptible JB6 Cl 41-5a and TPA-resistant JB6 Cl 30-7b preneoplastic epidermal cell lines showed a remarkable gene dosage-dependent increase in foci-forming activity, as well as an enhancement in the efficiency of neoplastic transformation of these cells in soft agar but no effect on proliferation in monolayer cultures. Interestingly, stable transfection of the nm23-M2/NDPK-B del-RGD or G106A mutant gene in JB6 Cl 41-5a cells selectively abrogated NDPK-B-induced cellular transformation, implicating a possible Arg105-Gly106-Asp107 regulatory role in early skin carcinogenesis. The molecular changes associated with early skin carcinogenesis are largely unknown. We have previously identified 11 genes whose expression was up- or down-regulated by 12-O-tetradecanoylphorbol-13-acetate (TPA) in mouse skin keratinocyte progenitor cells (Wei, S.-J., Trempus, C. S., Cannon, R. E., Bortner, C. D., and Tennant, R. W. (2003) J. Biol. Chem. 278, 1758-1768). Here, we show an induction of a nucleoside diphosphate protein kinase B (NDPK-B) gene in response to TPA or UV radiation (UVR). TPA or UVR significantly induced the expression of NDPK-B both in vivo hyperplastic mouse skin and in vitro mouse JB6 Cl 41-5a epidermal cells. Indeed, this gene was also up-regulated in TPA or UVR-mediated skin tumors including papillomas, spindle cell tumors, and squamous cell carcinomas, relative to adjacent normal skins. Functional studies by constitutive expression of nm23-M2/NDPK-B in TPA susceptible JB6 Cl 41-5a and TPA-resistant JB6 Cl 30-7b preneoplastic epidermal cell lines showed a remarkable gene dosage-dependent increase in foci-forming activity, as well as an enhancement in the efficiency of neoplastic transformation of these cells in soft agar but no effect on proliferation in monolayer cultures. Interestingly, stable transfection of the nm23-M2/NDPK-B del-RGD or G106A mutant gene in JB6 Cl 41-5a cells selectively abrogated NDPK-B-induced cellular transformation, implicating a possible Arg105-Gly106-Asp107 regulatory role in early skin carcinogenesis. Mouse skin carcinogenesis is a complex multistage process that progresses through distinct stages of initiation, promotion, and progression to malignancy (1DiGiovanni J. Pharmacol. Ther. 1992; 54: 63-128Crossref PubMed Scopus (582) Google Scholar, 2Yuspa S.H. J. Dermatol. Sci. 1998; 17: 1-7Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). The molecular changes associated with the early stages of skin tumor formation have yet to be determined. Tg·AC mice, which carry the coding region of the v-Ha-ras oncogene fused to a fetal ζ-globin gene promoter (3Leder A. Kuo A. Cardiff R.D. Sinn E. Leder P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9178-9182Crossref PubMed Scopus (279) Google Scholar), are considered to be genetically initiated and have a higher sensitivity to promotional stimuli including TPA 1The abbreviations used are: TPA12-O-tetradecanoylphorbol-13-acetateFBSfetal bovine serummAbmonoclonal antibodyMEMminimal essential mediumNDPKnucleoside diphosphate kinasepAbpolyclonal antibodyRGDArg-Gly-AspRIPAradioimmunoprecipitation assayRTreverse transcriptionUVRUV radiationWTwild typeNSnormal skin. (3Leder A. Kuo A. Cardiff R.D. Sinn E. Leder P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9178-9182Crossref PubMed Scopus (279) Google Scholar) and full thickness wounding (4Cannon R.E. Spalding J.W. Trempus C.S. Szczesniak C.J. Virgil K.M. Humble M.C. Tennant R.W. Mol. Carcinog. 1997; 20: 108-114Crossref PubMed Scopus (47) Google Scholar), or carcinogens such as UV radiation (UVR) (5Trempus C.S. Mahler J.F. Ananthaswamy H.N. Loughlin S.M. French J.E. Tennant R.W. J. Invest. Dermatol. 1998; 111: 445-451Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar) and 7,12-dimethylbenz[a]anthracene (6Spalding J.W. Momma J. Elwell M.R. Tennant R.W. Carcinogenesis. 1993; 14: 1335-1341Crossref PubMed Scopus (121) Google Scholar). These features establish the in vivo Tg·AC mouse model as a valuable tool to study the early stages of skin carcinogenesis. 12-O-tetradecanoylphorbol-13-acetate fetal bovine serum monoclonal antibody minimal essential medium nucleoside diphosphate kinase polyclonal antibody Arg-Gly-Asp radioimmunoprecipitation assay reverse transcription UV radiation wild type normal skin. In an earlier study with a combination of fluorescence-activated cell sorting, switching mechanism at the 5′-end of RNA templates cDNA amplification, and mouse cDNA array technology, we identified 11 genes whose expression changed significantly in α6+ CD34+ keratinocytes harvested from TPA-treated mice relative to cells from untreated mice. Nine genes, including galectin-7, nm23-M2/NDPK-B, cytoskeletal epidermal keratin 14, deleted in split hand/split foot gene 1 (Dss1), DNA double strand break repair RAD21 homolog, transcription termination factor 1, thymosin β4, calpactin I light chain, and 40 S ribosomal protein SA, were up-regulated, and two genes, apolipoprotein E precursor and acidic keratin complex 1 gene 15, were down-regulated by TPA (7Wei S.-J. Trempus C.S. Cannon R.E. Bortner C.D. Tennant R.W. J. Biol. Chem. 2003; 278: 1758-1768Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Dss1, a gene associated with a heterogeneous limb developmental disorder called split hand/split foot malformation (8Crackower M.A. Scherer S.W. Rommens J.M. Hui C.-C. Poorkaj P. Soder S. Cobben J.M. Hudgins L. Evans J.P. Tsui L.-C. Hum. Mol. Genet. 1996; 5: 571-579Crossref PubMed Scopus (173) Google Scholar), has recently been identified as a novel TPA-inducible gene expressed in keratinocyte progenitor cells, with possible involvement in early skin tumorigenesis (7Wei S.-J. Trempus C.S. Cannon R.E. Bortner C.D. Tennant R.W. J. Biol. Chem. 2003; 278: 1758-1768Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). This novel approach was highly effective in the in vivo identification of TPA-inducible effector genes that might lead to neoplastic transformation. The protein kinase nm23-M2/NDPK-B was another one of nine TPA-up-regulated genes and was selected for further characterization. Nm23 is a large family of structurally and functionally conserved proteins consisting of 4-6 identical subunits of 17-20 kDa each, known also as nucleoside diphosphate kinases (NDPKs; EC 2.7.4.6) (9Parks J.R.E. Agarwal R.P. Boyer P.D. The Enzymes. 3rd Ed. 8. Academic Press, Inc., New York1973: 307-334Google Scholar). 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Recently, nm23-M2/NDPK-B was also identified as a novel potential disease locus that was involved in mouse leukemic transformation (42Joosten M. Vankan-Berkhoudt Y. Tas M. Lunghi M. Jenniskens Y. Parganas E. Valk P.J.M. Löwenberg B. van den Akker E. Delwel R. Oncogene. 2002; 21: 7247-7255Crossref PubMed Scopus (38) Google Scholar). The biological functions of Nm23-M2/NDPK-B in cellular transformation still remain unknown. To gain insights into the contribution of this protein to early skin carcinogenesis, we characterized and studied the biological properties of Nm23-M2/NDPK-B. In this study, our data showed that Nm23-M2/NDPK-B was significantly induced either in vivo animal models or in vitro cell cultures by TPA or UVR and appeared to have a critical role in mediating neoplastic transformation of epidermal cells in the early stages of skin carcinogenesis. Using site-directed mutagenesis analysis, we further identified an RGD consensus sequence domain site in Nm23-M2/NDPK-B that was involved in the potentiation of cellular transformation activity. Nm23-M2/NDPK-B represents an attractive candidate mediator of TPA- or UVR-induced tumor promotion. Nm23-M2/NDPK-B rat monoclonal antibodies (mAb) were obtained from United States Biological (Swampscott, MA). V5 tag mouse mAb was from Invitrogen; α-tubulin mouse mAb was from Zymed Laboratories Inc. (San Francisco, CA); actin rabbit polyclonal antibody (pAb) was from Sigma. Horseradish peroxidase-conjugated secondary antibodies were from Amersham Biosciences. Nm23-H2/NDPK-B mouse mAb (233.1) was a kind gift from Dr. Michel Véron (Institute Pasteur, Paris, France). Uridine 5′-[α-35S]thiotriphosphate triethylammonium salt (SP6/T7 grade) (∼800 Ci/mmol) was purchased from Amersham Biosciences. Restriction enzymes, including BamHI, XbaI, EcoRI, and HindIII, were obtained from New England Biolabs (Beverly, MA). The primers used in this study were purchased from Proligos Corp. (La Jolla, CA). Noble agar was purchased from Difco. The tumor promoter TPA was from Sigma. TPA-susceptible JB6 Cl 41-5a and TPA-resistant JB6 Cl 30-7b mouse epidermal cell clonal variants were from the American Type Culture Collection (Manassas, VA) and grown at 37 °C in a 95% air plus 5% CO2 atmosphere in Eagle's minimal essential medium (MEM) supplemented with 5% heat-inactivated fetal bovine serum (FBS) containing 2 mm glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin sulfate (Invitrogen). Mouse fibroblast Rat-1, human keratinocyte HaCaT (generously provided by Dr. Norbert Fusenig, German Cancer Research Center, Heidelberg, Germany), and human epidermoid carcinoma cell A431 were cultured in Dulbecco's modified Eagle's medium containing 10% FBS. Tg·AC43 (a TPA-induced Tg·AC squamous cell carcinoma cell line) and FVB/N217 (an FVB/N carcinoma cell line) (43French J.E. Libbus B.L. Hansen L. Spalding J.W. Tice R.R. Mahler J. Tennant R.W. Mol. Carcinog. 1994; 11: 215-226Crossref PubMed Scopus (14) Google Scholar) were cultured in RPMI1640/Dulbecco's modified Eagle's medium (1:1) with 20% FBS. NIH/3T3 cells were maintained as described previously (44Paules R.S. Buccione R. Moschel R.C. Vande Woude G.F. Eppig J.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5395-5399Crossref PubMed Scopus (141) Google Scholar). Cell lines used in this study were free of mycoplasma infection. 8-10-week-old female homozygous Tg·AC mice were obtained from the Taconic Laboratory of Animals and Services (Germantown, NY). Animal studies were carried out in compliance with the National Institutes of Health Guidelines for Humane Care and Use of Laboratory Animals. The dorsal skin surface of groups of four homozygous female Tg·AC mice were dosed twice weekly for 2 weeks with 5 μg of TPA in 200 μl of acetone. Untreated control mice were sacrificed on day 1 (designated as NS). Four dosing protocols were used as follows. Mice were dosed on day 1 and sacrificed on day 5 (designated as 1TPA); mice were dosed on days 1 and 5 and sacrificed on day 8 (designated as 2TPA); mice were dosed on days 1, 5, and 8 and sacrificed on day 12 (designated as 3TPA); and mice were dosed on days 1, 5, 8, and 12 and sacrificed respectively at 48 h (designated as 4TPA), 7 days (designated as 4TPA + 7 days), 14 days (designated as 4TPA + 14 days), and 21 days (designated as 4TPA + 21 days) after the last dose. TPA-induced papillomas and malignant tumors (spindle cell tumors and squamous cell carcinomas) were identified, removed, and characterized as described previously (6Spalding J.W. Momma J. Elwell M.R. Tennant R.W. Carcinogenesis. 1993; 14: 1335-1341Crossref PubMed Scopus (121) Google Scholar). Tg·AC mice were irradiated with combination of 30-40% UVA and 60-70% UVB, as described previously (5Trempus C.S. Mahler J.F. Ananthaswamy H.N. Loughlin S.M. French J.E. Tennant R.W. J. Invest. Dermatol. 1998; 111: 445-451Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). After three exposures to UVA/UVB with 8.67 kJ/m2 per exposure, total cumulative dose of 26 kJ/m2, skin tissues (designated as UV1-UV4) were collected 24 h after the last exposure. Some animals were held for papilloma development (designated as UVP1-UVP6), and tumors were collected and stored at -80 °C. The full-length mouse nm23-M2/NDPK-B cDNA was generated by reverse transcription (RT) and PCR amplification using total RNA isolated from untreated (normal) skins harvested from Tg·AC mice. Because the nucleotide similarity (83%) and amino acid identity (88%) between nm23-M1/NDPK-A and nm23-M2/NDPK-B are relatively high (27Urano T. Takamiya K. Furukawa K. Shiku H. FEBS Lett. 1992; 309: 358-362Crossref PubMed Scopus (80) Google Scholar), mouse nm23-M2/NDPK-B-specific primers were designed to exclude the possibility of cross-reactivity with mouse nm23-M1/NDPK-A. The specific primers used for PCR were as follows: forward (5′-CAC CAT GGC CAA CCT CGA GCG TA-3′) and reverse (5′-CTC GTA CAC CCA GTC ATG GGC ACA A-3′) for full-length mouse nm23-M2/NDPK-B cDNA cloning; forward (105→123) (5′-CAC CG105G TGG CCA TGA AGT TCC TT123-3′) and reverse (321→297) (5′-A321TC CCC ACG GAT GGT GCC TGG TTT T297-3′) for in situ hybridization assay. The amplified cDNA was cloned directly into pcDNA3.1D/V5-His-TOPOc mammalian expression vector (Invitrogen). For the in situ hybridization assay, a construct expressing 217 bp of mouse nm23-M2/NDPK-B sense or antisense RNA probe was generated as follows. pcDNA3.1D/nm23-M2/NDPK-B (105→321)-V5-His was digested with BamHI and XbaI and the mouse nm23-M2/NDPK-B cDNA fragment (105→321) was cloned into T3/T7-U19 plasmid (Ambion, Austin, TX). The deletion or point substitution mutations were introduced into mouse nm23-M2/NDPK-B cDNA by the ExSite™ PCR-based site-directed mutagenesis kit, as described in the manufacturer's instructions (Stratagene, La Jolla, CA). The mutagenic primers were 24-29 bases long and had a greater than 50% GC content. The oligonucleotides used to generate the different mouse Nm23-M2/NDPK-B mutants were as follows (base changes underlined): delRGD (RGD consensus sequence domain), 5′-TTC TGC ATT CAA GTT GGC AGG AAC-3′ (forward) and 5′-pGAT GGT GCC TGG TTT TGA ATC AGC-3′ (reverse); G106A (RGD consensus sequence domain), 5′-CAC CAT CCG TGC GGA TTT CTG CAT-3′ (forward) and 5′-pCCT GGT TTT GAA TCA GCT GGA TTG GTC TC-3′ (reverse); S122P (phosphoryl transfer activity site), 5′-AGT GAT CCG GTG GAG AGT GCT GAG AAA-3′ (forward) and 5′-pGCC ATG AAT GAT GTT CCT GCC AAC TTG-3′ (reverse); H118F (kinase activity site), 5′-CAT CAT TTT TGG CAG TGA TTC AGT GGA GAG TGC-3′ (forward) and 5′-pTTC CTG CCA ACT TGA ATG CAG AAA TCC CC-3′ (reverse). DNA sequences were verified using an automated Applied Biosystems sequencer and the BigDye™ Terminator Kit (PerkinElmer Life Sciences). Plasmid DNAs were purified using purification kits from Qiagen (Stanford Valencia, CA) and were endotoxin-free when used for transfection into mammalian cells. Cells were transfected with empty vector pcDNA3.1, wild type nm23-M2/NDPK-B (WT) or mutant types nm23-M2/NDPK-B including delRGD, G106A, S122P, and H118F plasmid DNAs using LipofectAMINE PLUS™ reagents (Invitrogen). Transfected cells were cultured for at least 2 weeks in medium containing 400 μg/ml of geneticin (G418) (Invitrogen). Cells were analyzed by immunoblot to confirm the expression of mouse NDPK-B. Single-stranded cDNA was prepared from total RNA using the Moloney murine leukemia virus reverse transcriptase SuperScript II (Invitrogen) with oligo(dT) primer and used as a template for PCR. PCR primers for mouse nm23-M2/NDPK-B were as described above. The forward and reverse primers of β2-microglobulin gene (217 bp in size), used as an internal control, are 5′-GAC TGG TCT TTC TAT ATC CTG G-3′ and 5′-CTT TCT GCG TGC ATA AAT TG-3′, respectively. PCR cycling was as follows: denaturation (94 °C, 45 s), annealing (58 °C, 45 s), and extension (72 °C, 2 min) for 30 cycles. The reaction was carried out in a PerkinElmer-9600 thermal cycler, and PCR products were analyzed using 2% agarose gels. DNA was quantified using Quantity One software version 4.0 (Bio-Rad). An in situ hybridization assay was performed as previously described (45Hansen L.A. Tennant R.W. Mol. Carcinog. 1994; 9: 143-154Crossref PubMed Scopus (56) Google Scholar). Briefly, skin tissues were removed from Tg·AC mice treated or untreated with multiple doses of TPA and fixed overnight in 10% neutral buffered formalin. Tissues were paraffin-embedded, and sections (6 μm) were cut onto SuperFrost Plus microscope slides (Daigger, Vernon Hills, IL). The sections were deparaffinized and rehydrated by successive washes in xylene and graded alcohols to 2× SSC, and then 2 × 106 cpm of [α-35S]UTP-labeled mouse nm23-M2/NDPK-B sense or antisense riboprobes was applied to slides. Riboprobes were prepared from T7/T3-U19/nm23-M2/NDPK-B (105→321) plasmid linearized with EcoRI (antisense) or HindIII (sense) using an in vitro T7 or T3 Riboprobe kit (Promega, Madison, WI). Following 40 °C overnight hybridization, tissues were washed in 2× SSC plus 50% formamide at 40 °C and then in 2× SSC, 1× SSC, 0.5× SSC, and 0.5× SSC for 30 min each wash at room temperature. To remove unbound probe, tissues were incubated with 20 μl of RNase (10 mg/ml). After several washes, the slides were dehydrated in graded alcohols and completely air-dried. The slides were then dipped into NTB-3 autoradiographic emulsion (Eastman Kodak Co.), exposed for 10 days at room temperature in the dark, dried in a light-tight container, and developed in Kodak D19 developer and fixer. The sections were counterstained with hematoxylin, covered with coverslips, and photographed under dark-field illumination (model BX51, Olympus Optical Co., Tokyo, Japan). Cells were washed with ice-cold phosphate-buffered saline and lysed in ice-cold modified radioimmunoprecipitation (RIPA) buffer consisting of 50 mm Tris-HCl (pH 7.4), 1% Nonidet P-40, 150 mm NaCl, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml each aprotinin, leupeptin, and pepstatin, 1 mm Na3VO4, and 1 mm NaF. Cell suspensions were gently rocked on an orbital shaker in a cold room for 15 min to lyse cells. Lysates were centrifuged at 14,000 × g for 15 min at 4 °C. The skin tissues or tumors were homogenized and sonicated in ice-cold RIPA buffer and ultracentrifuged at 100,000 × g for1hat4 °C. Protein concentration was determined by Bradford assay (Bio-Rad). Proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene difluoride membranes (Amersham Biosciences). Membranes were stained with primary antibodies, detected using horseradish peroxidase-conjugated secondary antibodies (1:3000) (Amersham Biosciences) and enhanced chemiluminescence (Amersham Biosciences). Membranes were stripped and rehybridized with anti-α-tubulin mouse mAb (1:1000) or anti-actin rabbit pAb (1:1000) as a control to confirm equal loading. Protein quantitation was determined by ImageQuant software version 5.1 (Amersham Biosciences), and relative quantity is shown below the panels of Figs. 3B, 4, 5, 6, and 8.Fig. 4In vitro dose response and kinetics of TPA-induced expression of nm23-M2/NDPK-B in epidermal cells. JB6 Cl 41-5a (A and B) or JB6 Cl 30-7b epidermal cells (C) were treated with the indicated TPA concentrations or exposed to 10 ng/ml TPA at indicated time points. Cells were harvested, and whole cell lysates were extracted using modified RIPA buffer. Sixty micrograms of whole-cell lysates were analyzed by immunoblotting for the detection of mouse Nm23-M2/NDPK-B (top panel). α-Tubulin (50 kDa) (lower panel) served as an internal control.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 5Elevated NDPK-B expression in skin tumor cell lines and Tg·AC neoplasms.A, skin tumor cell lines. Two mouse skin tumor cell lines, FVB/N217 and Tg·AC43, and one human epidermoid carcinoma cell line A431 were examined for NDPK-B gene expression by immunoblotting. HaCaT keratinocytes and TPA-untreated normal keratinocytes [TPA(-)KCs] (isolated from Tg·AC mice) were used as negative controls for humans and mice, respectively. TPA-treated Tg·AC mouse keratinocytes (TPA(+)KCs) served as a positive control to indicate the position of NDPK-B. KCs, keratinocytes. B, TPA-mediated skin tumors in Tg·AC mice. Total protein lysates were prepared from seven normal skin tissues of Tg·AC mice (NS1-NS7) and tumors, including 15 papillomas (P1-P15) and three malignancies (M1-M3) with one spindle cell tumor (M1) and two squamous cell carcinomas (M2 and M3). All total protein lysate (60 μg) immunoblots were probed with anti-mouse Nm23-M2/NDPK-B rat mAb (1:200) or anti-human Nm23-H2/NDPK-B mouse mAb (1:1000), anti-actin rabbit pAb (1:1000), or anti-α-tubulin mouse mAb (1:1000).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 6Induction of nm23-M2/NDPK-B by UVR.A, Nm23-M2/NDPK-B increase in UVR-mediated hyperplastic skins. Tg·AC mouse skins were irradiated with a combination of UVA and UVB (30-40% UVA and 60-70% UVB). After three exposures to UVA/UVB with 8.67 kJ/m2 per exposure (total 26 kJ/m2), the skin tissues were collected 24 h after the last exposure and snap frozen in liquid nitrogen. UV1-UV4, UV-exposed skin tissues from mouse 1-4. B, nm23-M2/NDPK-B overexpression in UVR-induced papilloma in Tg·AC mice. Tg·AC mice were irradiated with a combination of 30-40% UVA and 60-70% UVB. After three exposures to UVA/UVB w
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