ANTICANCER RESEARCH 28 : 957-964 (2008)

Characterization of Akt Overexpression in MiaPaCa-2 Cells: Prohibitin Is an Akt Substrate both In Vitro and in Cells EDWARD KYU-HO HAN, THOMAS MCGONIGAL, CHRIS BUTLER, VINCENT L. GIRANDA and YAN LUO

Abbott Laboratories, Department of R47S, Cancer Research, Global Pharmaceutical Research Division, Abbott Park, IL 60064, U.S.A.

Abstract. Akt (PKB) is a serine/threonine protein that is activated by growth factors, cytokines and . that plays an important role in the transduction of signals Activated PI-3K produces PI-3,4,5-P 3 and PI-3,4,-P 2 that affecting , cell proliferation and survival. The Akt interact with Akt and recruit Akt from the cytosol to the is frequently hyperactivated in tumors and has been plasma membrane. Following membrane localization, Akt is shown to be amplified in a number of types of human phosphorylated at Thr-308 in the T-loop (kinase activation cancers. Furthermore, Akt activity is elevated in cell lines loop) and Ser-473 in the carboxy-terminal tail (hydrophobic with the mutated PTEN tumor suppressor gene. These studies motif). Thr-308 phosphorylation is required for Akt establish Akt as an attractive target for cancer therapy. To activation, while Ser-473 phosphorylation is only needed for determine the roles of Akt1, Akt2 and Akt3 in signal maximal activity. Thr-308 is phosphorylated by 3- transduction, constitutively active Akt1, Akt2 and Akt3 was phophoinositide dependent kinase (PDK1), which is ectopically overexpressed in human pancreatic MiaPaCa-2 activated by PI-3K lipid products (2). cells. The three Akt stable clones were characterized to AKT is emerging as a key player in human cancer. Akt1 and determine their effects on transformation and proliferation. Akt2 are amplified in a human gastric and ovarian cancer Compared to a vector control, the three Akt clones were able (7). Akt3 activity and expression were up-regulated in estrogen to drive cellular proliferation even in reduced serum receptor-negative breast tumors and breast carcinomas and conditions. Furthermore, in soft-agar assays, the Akt clones androgen-insensitive prostate cell lines (14). Furthermore, Akt showed an 25-38% increase in colony formation in 2% transforms cells. For example, overexpression of constitutively serum. Our results indicate that all three forms of Akt may active Akt1 or Akt2 transforms NIH3T3 fibroblasts (1, 8, 11). have protective effects within the cell depending on the type Akt-3 overexpression in MCF-7 cells results in estrogen- of apoptotic stimuli. Using 2D-PAGE comparisons between independence for tumor formation (4). parental and Akt overexpressing cells, we attempted to Using two-dimensional-polyacry la mide gel electrophoresis determine novel targets of Akt phosphorylation. In this study, (2D-PAGE) and an Akt substrate phosphor specific antibody, we identified prohibitin as a substrate for Akt both in vitro we identified a number of potential new targets of Akt and in vivo. These studies suggest that Akt may regulate the phosphorylation. One of these targets was identified as cellular function of prohibitin via its phosphorylation. prohibitin-1 and additional studies were undertaken to confirm this relationship. The prohibitins, prohibitin-1 and The serine/threonine (Akt/PKB) plays a prohibitin-2 are highly conserved proteins in eukaryotic cells major role in cellular processes such as cell growth, cell present in multiple compartments. The prohibitins act as proliferation and apotosis (3). In humans, there are three chaperones in the assembly of subunits of mitochondrial closely related, highly conserved homologues designated as respiratory chain complexes (15). Furthermore, prohibitin-1 Akt1, Akt2, and Akt3. The Akt kinase is a major downstream is required for ras-induced raf-MEK-ERK activation and target of the phosphatidylinositol 3-kinase (PI-3K) pathway epithelial cell migration (16, 17). Recent studies indicated that prohibitins are localized in the nucleus and can modulate transcriptional activity by interacting with various transcriptional factors (12, 13). Prohibitin-2 inhibits muscle Correspondence to: Edward K. Han, Ph.D., Abbott Laboratories, differentiation by repressing the transcriptional activity of Dep artment of R47S, Bldg. AP9A, 100 Abbott Park Road, Abbott Park, IL 60064, U.S.A. Tel: +1 847 935 4733, Fax: +1 847 938 both MyoD and MEF2. In other work, Sun et al. (18) have 2365, e-mail: [email protected] demonstrated that Akt associaties with prohibitin-2 but in this case, prohibitin-2 does not seem to be a target for Akt Key Words: Akt, prohibitin, transformation. phosphorylation.

0250-7005/2008 $2.00+.40 957 ANTICANCER RESEARCH 28 : 957-964 (2008)

In the present study, we examined the role of each Akt Proliferation assay. Cell proliferation assays were performed in family member by ectopic overexpression in MiaPaCa-2 96-well microtiter plates using the colorimetric method. To 3 pancreatic cancer cells. Akt overexpressing clones were measure cell proliferation, 5x10 cells were plated in the presence of different concentrations of serum. The cells were examined in cell proliferation and soft-agar assays. cultured for 72 h, after which time, cell culture medium was removed and 100 μl of 10% Alamar Blue reagent (Biosource Materials and Methods International, Camarillo, CA, USA) was added. After 2 h, plates were read at excitation of 544 nm/emission at 590 nm on F-Max Cell culture, isolation of AKT clones and in vitro kinase assay. fluorescent plate reader (Molecular Devices, Sunnyvale, CA, MiaPaCa-2 pancreatic cells were obtained from the ATCC USA). All values are representative of 2 to 3 independent (Rockville, MD, USA) and maintained in Dulbecco’s modified experiments. Eagle’s medium with 10% fetal bovine serum and 5% C O2 at 37˚C. pCIneo-LCK-HA-AKT1, AKT2 and AKT3 constructs were Soft-agar assay. provided by IDUN Pharmaceuticals (San Diego, CA, USA). These The soft agar assay was performed as described constructs were also used in 3T3 cells as described elsewhere (8). elsewhere (5). For the bottom layer of agar, 1 ml of 0.5% agar Recombinant Akt protein was purified as described (9). In vitro containing medium and different concentrations of serum was kinase assays were performed as described (9). MiaPaCa-2 cells added to each 35 mm-diameter well of six-well plates. The bottom agar was solidified (~30 min) in the hood. For the top were transfected with the above constructs using the 4 Lipofectamine Reagent according to the manufacturer’s agar, 2 ml of 0.35% top agar containing 1x10 cells and medium instructions (Invitrogen, Carlsbad, CA, USA). Following with serum (final 2%) was layered on top of the solidified bottom transfection, cells were selected with 1 mg/ml G418 and the AKT agar and after setting, the plates were incubated at 37 ˚C for 10 expressing clones were isolated and expanded. 293T Cells (ATCC) days. Colonies were stained with P-iodonitrotetrazolium violet were used for expression of exogenous prohibitin during and the number of stained colonies (>200 μm) were scored using cotransfection experiments with pAKT. an image analysis program (Image-Pro Plus; Media Cybernetics, Silver Springs, MD, USA). Construction of prohibitin plasmids. Full-length cDNA for Two -dimensional gel analysis. prohibitin was obtained from Research Genetics (Huntsville, AL, Two-dimensional gel electro- USA) as Image clone 42313. The following primers were used to phoresis was performed according to the method of O’Farrell by generate a BamHI, EcoRI pcr fragment to subclone the gene into Kendrick Labs, Inc. (Madison, WI, USA) as follows: Isoelectric plasmid pcDNA 3.1, adding an N-terminal HA tag and C-terminal focusing (IEF) was carried out in glass tubes of inner diameter 6-His tag to the construct: 5’gtgtggatccatgtatccttacgacgtgc 2.0 mm using 2% pH 4-8 ampholines (BDH from Hoefer ctgactacgccgctgc caaagtgtttgagtcc-3’ and 5’-gtgtgaattcagtgatgatg atg Scientific Instruments, San Francisco, CA, USA) for 9600 Vh. atggtgctggggcagctggaggagc-3’. Threonine 238 was mutagenized to One microgram of an IEF internal standard, tropomyosin, was alanine using the Quick Change Mutagenesis Kit (Stratagene, La added to each sample. This protein migrates as a doublet with Jolla, CA, USA) with the following primers: 5’-gctctcgga lower polypeptide spot of MW 33,000 and s pI of 5.2. The acatcgcctacctgccagcg-3’ and 5’-cgctggcaggtaggcgatgt tccg ag agc3’. enclosed tube gel pH gradient plot for this set of ampholines was determined with a surface pH electrode. After equilibration Protein extraction and Western blot analysis. Cells were scraped for 10 min in Buffer ‘O’ (10% glycerol, 50 mM dithiothreitol, into cold phosphate-buffered saline, pelleted, and then resuspended 2.3% sodium dodecyl sulfate and 0.0625 M Tris, pH 6.8) the in 100-200 μl ice-cold insect cell lysis buffer supplememented with tube gels were sealed to the top of stacking gels on top of 10% protease inhibitors (BD Biosciences Pharmingen, San Diego, CA, acrylamide slab gels (0.75 mm thick) and SDS slab gel USA). Cells were lysed by sonication and the debris cleared in a electrophoresis carried out for about 4 h at 12.5 mA/gel. The microcentrifuge. A total of 40 μg of lysate were loaded into each following proteins (Sigma Chemicals Co., St. Louis, MO, USA) well of 6% or 10% Tris-glycine polyacrylamide minigels were added as MW standards to a well in the agarose, which (Invitrogen) for SDS-PAGE analysis. Proteins were transferred to sealed the tube gel to the slab gel: myosin (220,000), PVDF membranes (Invitrogen), blocked for 1 h in TBS-T plus 5% phosphorylase A (94,000), catalase (60,000), actin (43,000) (w/v) powdered blotting grade milk (Bio-Rad Laboratories, carbonic anhydrase (29,000) and lysozyme (14,000). These Hercules, CA, USA), and then probed overnight at 40˚C with standards appear as horizontal lines on the Coomassie Brilliant primary antibodies at a 1:2000 dilution in blocking solution. Blots Blue R-250 stained 10% acrylamide slab gels. The silver- were developed using enhanced chemiluminescence (ECL Plus) stained gels were dried between sheets of cellophane with the reagents from Amersham Biosciences (GE Healthcare, acid edge to the left. After slab gel electrophoresis, duplicate Buckinghamshire, UK). gels were transferred to transfer buffer (12.5 mM Tris, pH 8.8, 86 mM glycine, 10% methanol) transblotted onto PVDF Antibodies and reagents. Antibodies for Akt, Influenza membranes overnight at 200 mA and approximately 100 V/two hemagglutinin epitope tag (HA), phospho-Akt Ser-473, phospho- gels. Anti-phospho Akt substrate antibody was used in the GSK3α/β were obtained from Cell Signaling (Beverly, MA, Western blot analysis. Spot #1 was removed and mass USA). β-Actin antibody was obtained from Sigma-Aldrich (St. spectroscopic analysis was performed. Louis, MO, USA). Synthetic peptides (prohibitin sequence from 247 to 269) were generated by Genemed Synthesis (San Statistical analysis. Statistical analyses were done with Student’s t- test Francisco, CA, USA). Phosphoprohibitin antibody was generated using GraphPad Software program (San Diego, CA, USA ). Two-tailed by AnsPec (San Jose, CA, USA). p< 0.05 was considered statistically significant.

958 Han et al : Characterization of Akt Overexpression in MiaPaCa-2 Cells

Figure 1. A. Cell extracts from Akt1, Akt2 and Akt3 transfected MiaPaCa-2 clones were prepared and electrophoresed on SDS-PAGE followed by Western blot analyses as described in Materials and Methods. Exp=exponentially growing cells; V8, V9=vector clones 8 & 9; 1-28=Akt1 clone 28; 2-5=Akt2 clone 5; 2-26=Akt2 clone 26, 3-16=Akt 3 clone16. B. Cell proliferation was determined by MTT assay in the presence of different concentrations of serum. After 3 days, Alama r Blue was added and read at 545-590 nM using a plate reader.

Results GSK-3 β at serine 9. As shown in Figure 1A, GSK-3 β phosphorylation was also increased in Akt clones. The effect Overexpression and characterization of Akt1, Akt 2 and Akt3 of serum on cell growth of the Akt clones was also examined. in MiaPaCa-2 cells. To understand the role of Akt in Cell proliferation was determined in the presence of 0.1%, transformation and cell proliferation, stable MiaPaCa-2 cells 0.5%, 2.0% and 10% serum. All three Akt isoform clones were generated that overexpress constitutively active Akt1 survived better than the control cells in the presence of (clones 1-28 and 1-31), Akt2 (clones 2-5 and 2-26) or Akt3 different serum concentrations (Figure 1B). This effect was (clone 3-16). As shown in Figure 1A, compared to vector most pronounced in cells grown in 0.1% serum where there control clones (V8 and V9) all Akt clones display the was a 3- to 6.5-fold increase in survival when compared to expression of HA tagged AKT protein and an increase in Akt the V8 vector control clone. Soft-agar colony formation activity as determined by Akt phosphorylation at the serine assays were performed to examine if Akt could enhance 473 site. Glycogen synthase kinase-3 (GSK-3) is a colony formation in soft-agar. Anchorage independent growth downstream substrate of Akt and its activity can be inhibited is one of the hallmarks of the cancer phenotype. As shown in by Akt-mediated phosphorylation of GSK-3 α at serine 21 and Figure 2A and B, colony formation in soft-agar was increased

959 ANTICANCER RESEARCH 28 : 957-964 (2008)

Figure 2. MiaPaCa-2 control and Akt clone cells were plated at 1x10 4 cells/well, in each well of 6-well plates containing 0.5% bottom agar and 0.35% top agar. After 10 days, photos of colonies were taken (x4), (Panel A). The means ±s tandard deviations from triplicate determination for colony formation are shown (B). Two independent experiments were carried out with the same results, and a representative experiment is shown. Student’s t-test (two-tailed) was used to determine the statistical significance ( p<0.05) of control versus Akt clones.

for all three Akt clones compared to vector controls by 25 to In vitro phosphorylation of prohibitin by Akt. The protein 38 %. These data indicated that Akt overexpression can sequence of human prohibitin-1 revealed that there is a potential promote anchorage-independent growth of these cells. Akt substrate sequence RSRNIT (amino acids 252-258) that follows the proposed consensus sequence derived for Akt Two-dimensional analysis of Akt1 overexpressing MiaPaCa- phosphorylation (R-X-R-X-X-S/T). Wild-type (WT) and 2 cells. To further understand what effect Akt overexpression mutant (MT) peptide sequences that encompass the putative Akt might have on cellular signaling pathways, 2-dimensional gel phosphorylation site were synthesized (Figure 4A). The MT electrophoresis of cell lysates followed by Western blotting peptide sequence has alanine (A) instead of threonine (T) at were performed. Blots were then probed using a position 258. These two peptides were tested in a 32 P-ATP in phosphospecific antibody that recognizes a peptide vitro kinase assay using the recombinant Akt protein followed consensus motif phosphorylated by Akt. This Akt phospho- by protein gel electrophoresis and X-ray autoradiography. As substrate antibody has been very useful in identifying novel shown in Figure 4B, the WT peptide was phosphorylated substrates for Akt (10). As shown in Figure 3, the antibody whereas the MT peptide was not phosphorylated by Akt. The demonstrated an increase in phosphorylation of spot 1 recombinant Akt protein also undergoes autophosphorylation as (circled in Figure 3) by the Akt overexpressing clone relative shown in Figure 4B. Our data indicated that Akt phosphorylated to the control cell line. Subsequent analysis of the spot by prohibitin-1 at threonine 258 in vitro . MALDI-mass spectroscopy identified it as prohibitin-1 (data not shown). Prohibitin1 has a molecular weight (Mr) of 30.4 In vivo phosphorylation of prohibitin-1 by Akt. To kDa and an isoelectric point (pI) of 6.02. determine whether Akt can phosphorylate prohibitin-1 in

960 Han et al : Characterization of Akt Overexpression in MiaPaCa-2 Cells

Figure 3. Cell extracts from vector control and Akt1 clone 28 were prepared and run on two-dimensional gel electrophoresis followed by Western blot analysis as described in Materials and Methods. Spot 1 was identified as prohibitin by mass spectrometric analysis.

Figure 4. Prohibitin peptides that contained either WT or MT sequences were generated. Threonine (T) at p osition at 258 is a putative phosphorylation site. This site was changed to alanine (A) in the MT peptide (A). Peptides were incubated with or without Akt in the presence of 32 P-ATP. Samples were run on SDS-PAGE and the autoradiogram is shown in (B).

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Figure 5. A total of 3x10 6 293 cells were transfected with either Akt or prohibitin cDNA. Two days following transfection, cells were harvested and analyzed by Western blot using prohibitin, phospho-prohibitin and phospho-Akt substrate antibody.

vivo , HEK-293T cells were transfected with a combination Akt has been shown to play a role in numerous and of myristylated Akt and WT prohibitin-1 or Akt and MT diverse cellular functions such as cell survival, growth, prohibitin-1 plasmids. As shown in Figure 5, only the WT proliferation, migration, metabolism and angiogenesis (10). prohibitin-1 co-transfected with Akt was phosphorylated Clearly, Akt has the capacity to interact with substrates as determined by both phospho-prohibitin-1 antibody and affecting multiple cellular signaling pathways and it is of phospho-Akt substrate antibody. These results suggest that interest to us to identify as many of these interactions as Akt can phosphorylate prohibitin-1 in cells. We also possible to elucidate the full spectrum of Akt activity. In our observed the binding of prohibitin-1 to Akt in an study, we compared lysates from parental and Akt immuoprecipitation experiment (data not shown). It will overexpressing cell lines using Western blots of 2- be interesting to examine the role of prohibitin-1 dimensional gels probed with a phospho-Akt substrate phosphorylation by Akt. antibody to identify prohibitin-1 as a potential Akt substrate. Furthermore, a prohibitin-1 peptide that contained a putative Discussion Akt phosphorylation site was phosphorylated by Akt in vitro . In cells , co-transfection of Akt and prohibitin-1 cDNA To examine the different roles of Akt isoforms in cell plasmids induced prohibitin-1 phosphorylation in HEK-293T proliferation, we generated pancreatic MiaPaCa-2 cells that cells. It has been shown that Akt binds prohibitin-2 and stably overexpressed either Akt1, Akt2 or Akt3. In this paper, relieves its repression of MyoD and muscle differentiaton we demonstrated that Akt1, Akt2 or Akt3 overexpressing (18). In the same paper, the authors mentioned that Akt did MiaPaCa-2 clones displayed several characteristics. Firstly, not seem to phosphorylate prohibitin-2 in spite of the all three isoforms of Akt clones survived better under low presence of the Akt consensus site. This was determined in serum conditions as compared to the vector control clone. vitro by an immune-complex kinase assay or in vivo using Secondly, all three Akt clones increased colony formation in an antibody raised against the phosphorylated peptides soft-agar assays. Our results indicated that all three isoforms containing the Akt recognition site phospho-Akt antibody of Akt clones enhanced the transforming capability of (18). It would be interesting to see if a peptide containing MiaPaCa-2 cells. It was shown previously that Akt consensus sites from prohibitin-2 could be overexpression of constitutively active Akt1, Akt2 or Akt3 in phosphorylated by the recombinant Akt protein in vitro . normal chicken fibroblasts resulted in a similar degree of We are interested in determing the functional significance transformation (11). of prohibitin-1 phosphorylation by Akt. Prohibitin has been

962 Han et al : Characterization of Akt Overexpression in MiaPaCa-2 Cells implicated in diverse cellular functions including cell 8 Liu X, Powlas J, Shi Y, Oleksijew AX, Shoemaker AR, DeJong proliferation, molecular chaperone function in the R, Oltersdorf T, Giranda VL and Luo Y: Rapamycin inhibits Akt- mitochondria, and regulation of transcriptional activity (12, mediated oncogenic transformation and tumor growth. Anticancer Res 24 : 2697-2704, 2004. 13). Akt phosphorylation of prohibitin-1 could potentially 9 Luo Y, Shoemaker AR, Liu X, Woods KW, Thomas SA, de Jong have an impact on stability of the prohibitin-1 protein, R, Han EK, Li T, Stoll VS, Powlas JA, Oleksijew A, Mitten MJ, transcriptional regulation of E2F and localization. It could Shi Y, Guan R, McGonigal TP, Klinghofer V, Johnson EF, also have an additional role for prohibitin in activation of the Leverson JD, Bouska JJ, Mamo M, Smith RA, Gramling-Evans Ras-Raf pathway (16, 17). Likewise, prohibitin is thought to EE, Zinker BA, Mika AK, Nguyen PT, Oltersdorf T, Rosenberg have a role as a molecular chaperone and perhaps prohibitin SH, Li Q and Giranda VL: Potent and selective inhibitors of Akt has a regulatory role on Akt. Recently discovered small slow the progress of tumors in vivo . Mol Cancer Ther 4: 977-986, 2005. molecule Akt inhibitors will be useful in elucidating the 10 Manning BD and Cantley LC: Akt/PKB signaling: navigating function of prohibitin-1 phosphorylation by Akt (6, 9). downstream. Cell 129 : 1261-1274, 2007. 11 Mende I, Malstrom S, Tsichlis PN, Vogt PK and Aoki M: Acknowledgements Oncogenic transformation induced by membrane-targeted Akt2 and Akt3. Oncogene 20 : 4419-4423, 2001. We thank IDUN Pharmaceuticals for providing the AKT constructs. 12 Mishra S, Murphy LC and Murphy LJ: The prohibitins: emerging roles in diverse functions. J Cell Mol Med 10 : 353- References 363, 2006. 13 Mishra S, Murphy LC, Nyomba BLG and Murphy LJ: Prohibitin: a potential target for new therapeutics. Trends Mol 1 Cheng JQ, Altomare DA, Klein MA, Lee WC, Kruh GD, Lissy Med 11 : 192-197, 2005. NA and Testa JR: Transforming activity and mitosis-related 14 Nakatani K, Thompson DA, Barthel A, Sakaue H, Liu W, Weigel expression of the AKT2 oncogene: evidenc e suggesting a link RJ and Roth RA: Up-regulation of Akt3 in estrogen receptor- between cell cycle regulation and oncogenesis. Oncogene 14 : deficient breast cancers and androgen-independent prostate 2793-1801, 1997. cancer lines. J Biol Chem 274 : 21528-21532, 1999. 2 Cheng JQ, Lindsley CW, Cheng GZ, Yang H and Nicosia SV: 15 Nijtmans LGJ, Sanz MA, Grivell LA and Coates PJ: The The Akt/PKB pathway: molecular target for cancer drug mitocondiral PHB complex: roles in mitochondrial respiratory discovery. Oncogene 14 : 7482-7492, 2005. complex assembly, ageing and degenerative disease. Cell Mol 3 Crowell JA, Steele VE and Fay JR: Targeting the Akt protein Life Sci 59 : 143-155, 2002. kinase for cancer chemoprevention. Mol Cancer Ther 6: 2139- 16 Rajalingam K, Wunder C, Brinkmann V, Churin Y, Hekman M, 2148, 2007. Sievers C, Rapp UR and Rudel T: Prohibitin is required for Ras- 4 Faridi J, Wang L, Endemann G and Roth RA: Expression of induced Raf-MEK-ERK activation and epithelial cell migration. constitutively active Akt-3 in MCF-7 breast cancer cells reverses Nat Cell Biol 7: 837-843, 2005. the estrogen and tamoxifen responsivity of these cells in vivo . 17 Rajalingam K and Rudel T: Ras-Raf signaling needs prohibitin. Clin Canc Res 9: 2933-2939, 2003. Cell Cycle 4: 1503-1505, 2005. 5 Han EK-H, McGonigal T, Wang J, Giranda VL and Luo Y: 18 Sun D, Liu L, Yang X-J and Wu Z: Akt binds prohibitin 2 and Functional analysis of focal adhesion kinase (FAK) reduction by relieves its repression of MyoD and muscle differentiation. J Cell small inhibitory RNAs. Anticancer Res 24 : 3899-3906, 2004. Sci 117 : 3021-3029, 2004. 6. Han EK, Leverson JD, Shah OJ, Woods KW, Hunter T, Giranda VL and Luo Y: Akt inhibitor A-443654 induces rapid Akt Ser- 473 phosphorylation independent of mTORC1 inhibition. Oncogene 16 : 5655-5661, 2007. 7 Hennessy BT, Smith DL, Ram PT, Lu Y and Mills GB: Received November 21, 2007 Exploiting the PI3K/AKT pathway for cancer drug discovery. Revised January 18, 2008 Nat Rev Drug Discov 4: 988-1004, 2005. Accepted February 4, 2008

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