MHY446 induces apoptosis via reactive oxygen species-mediated endoplasmic reticulum stress in HCT116 human colorectal cancer cells.

This study investigated the potential of a newly synthesized histone deacetylase (HDAC) inhibitor, MHY446, in inducing cell death in HCT116 colorectal cancer cells and compared its activity with that of suberoylanilide hydroxamic acid (SAHA), a well-known HDAC inhibitor. The results showed that MHY446 increased the acetylation of histones H3 and H4 and decreased the expression and activity of HDAC proteins in HCT116 cells. Additionally, MHY446 was confirmed to bind more strongly to HDAC1 than HDAC2 and inhibit its activity. In vivo experiments using nude mice revealed that MHY446 was as effective as SAHA in inhibiting HCT116 cell-grafted tumor growth. This study also evaluated the biological effects of MHY446 on cell survival and death pathways. The reactive oxygen species (ROS) scavenger N-acetyl-L-cysteine (NAC) confirmed that ROS play a role in MHY446-induced cell death by reducing poly(ADP-ribose) polymerase cleavage. MHY446 also induced cell death via endoplasmic reticulum (ER) stress by increasing the expression of ER stress-related proteins. NAC treatment decreased the expression of ER stress-related proteins, indicating that ROS mediate ER stress as an upstream signaling pathway and induce cell death. While MHY446 did not exhibit superior HDAC inhibition efficacy compared to SAHA, it is anticipated to provide innovative insights into the future development of therapeutic agents for human CRC by offering novel chemical structure-activity relationship-related information.

Microvirga terrestris sp. nov., and Microvirga arvi sp. nov., isolated from soil in South Korea.

Two novel Gram-stain-negative, aerobic, rod shaped bacterial strains BT290T and BT689T were isolated from soil collected in South Korea. Colony morphologies of both strains were circular and convex while the colors of BT290T and BT689T were light-pink and white, respectively. Phylogenetic analysis based on 16S rRNA gene sequences revealed that BT290T and BT689T belong to a distinct lineage within the genus Microvirga (family Methylobacteriaceae, order Rhizobiales, class Alphaproteobacteria, phylum Proteobacteria, kingdom Bacteria). The 16S rR NA gene sequence similarity between two strains was 97.9%. Both strains had the similar quinone system, with ubiquinone 10 (Q-10) as the major respiratory quinone. The major polar lipids of strains BT290T and BT689T were phosphatidylethanolamine (PE), diphosphatidylglycerol (DPG), phosphatidylcholine (PC), and phosphatidylglycerol (PG). The major cellular fatty acids of strain BT290T were C18:1 ω7c (58.2%) and C16:0 (17.7%), while those of strain BT689T were C18:1 ω7c (61.8%) and C16:0 (10.8%). On the bases of polyphasic analysis (phylogenetic, chemotaxonomic, and biochemical), strains BT290T and BT689T can be suggested as novel bacterial species within the genus Microvirga and the proposed names are Microvirga terrestris and Microvirga arvi, respectively. The type strain of Microvirga terrestris is BT290T (= KCTC 72367T = NBRC 114844T) and the type strain of Microvirga arvi is BT689T (= KACC 22016T = NBRC 114858T), respectively.

Hymenobacter telluris sp. nov., isolated from soil in South Korea.

Two novel bacterial strains, designated as BT186T and BT505, were isolated from a soil sample collected in South Korea and characterized. Both strains were Gram-stain-negative, rod-shaped, aerobic, circular, convex, and had red-colored colonies. The level of 16S rRNA gene sequence similarity between the strains BT186T and BT505 was 100%, indicating that they represent an identical species. 16S rRNA sequence analysis indicated that strains BT186T and BT505 belong to a distinct lineage within the genus Hymenobacter (family Hymenobacteraceae, order Cytophagales, class Cytophagia, phylum Bacteroidetes, Kingdom Bacteria). Both strains were closely related to Hymenobacter norwichensis DSM 15439T (98.3% 16S rRNA gene similarity), Hymenobacter aquaticus JCM 31653T (96.8%), and Hymenobacter perfusus LMG26000T (96.5%). Strain BT186T was found to have the MK-7 as the major respiratory quinone. The major polar lipid of strain BT186T was identified to be phosphatidylethanolamine (PE). The major cellular fatty acid profiles of strain BT186T were C16:1 ω5c (24.3%), iso-C15:0 (20.3%) and summed feature 3 (C16:1 ω6c/C16:1 ω7c) (19.9%). Characterization based on polyphasic analysis indicated that strains BT186T and BT505 represent novel species of the genus Hymenobacter and the name Hymenobacter telluris sp. nov. is proposed. The type strain of Hymenobacter telluris is BT186T (= KCTC 72338T = NBRC 114968T).

Novel SIRT Inhibitor, MHY2256, Induces Cell Cycle Arrest, Apoptosis, and Autophagic Cell Death in HCT116 Human Colorectal Cancer Cells.

We examined the anticancer effects of a novel sirtuin inhibitor, MHY2256, on HCT116 human colorectal cancer cells to investigate its underlying molecular mechanisms. MHY2256 significantly suppressed the activity of sirtuin 1 and expression levels of sirtuin 1/2 and stimulated acetylation of forkhead box O1, which is a target protein of sirtuin 1. Treatment with MHY2256 inhibited the growth of the HCT116 (TP53 wild-type), HT-29 (TP53 mutant), and DLD-1 (TP53 mutant) human colorectal cancer cell lines. In addition, MHY2256 induced G0/G1 phase arrest of the cell cycle progression, which was accompanied by the reduction of cyclin D1 and cyclin E and the decrease of cyclin-dependent kinase 2, cyclin-dependent kinase 4, cyclin-dependent kinase 6, phosphorylated retinoblastoma protein, and E2F transcription factor 1. Apoptosis induction was shown by DNA fragmentation and increase in late apoptosis, which were detected using flow cytometric analysis. MHY2256 downregulated expression levels of procaspase-8, -9, and -3 and led to subsequent poly(ADP-ribose) polymerase cleavage. MHY2256-induced apoptosis was involved in the activation of caspase-8, -9, and -3 and was prevented by pretreatment with Z-VAD-FMK, a pan-caspase inhibitor. Furthermore, the autophagic effects of MHY2256 were observed as cytoplasmic vacuolation, green fluorescent protein-light-chain 3 punctate dots, accumulation of acidic vesicular organelles, and upregulated expression level of light-chain 3-II. Taken together, these results suggest that MHY2256 could be a potential novel sirtuin inhibitor for the chemoprevention or treatment of colorectal cancer or both.

ER stress contributes to autophagy induction by adiponectin in macrophages: Implication in cell survival and suppression of inflammatory response.

Adiponectin, the most abundant adipokine, exhibits various physiological functions. In addition to its critical role in lipid metabolism, recent studies have demonstrated its potent anti-inflammatory and cytoprotective properties. Accumulating evidence suggests that autophagy plays a critical role in various biological responses by adiponectin. However, the underlying mechanisms remain elusive. Herein, we investigated the role of ER stress in adiponectin-induced autophagy and its functional roles in biological responses by adiponectin in macrophages. In this study, globular adiponectin (gAcrp) significantly increased the expression of various ER stress markers in both RAW 264.7 and primary peritoneal macrophages. In addition, inhibition of ER stress by treatment with tauroursodeoxycholic acid (TUDCA) or gene silencing of CHOP prominently suppressed gAcrp-induced autophagy. Treatment with gAcrp also induced significant increase in sestrin2 expression. Interestingly, knockdown of sestrin2 prevented autophagy induction and inhibition of ER stress abrogated sestrin2 induction by gAcrp, collectively implying that ER stress critically contributes to gAcrp-induced autophagy activation via sestrin2 induction. Moreover, pretreatment with TUDCA restored suppression of TNF-α and IL-1ß expression and attenuated the enhanced viability of macrophages induced by gAcrp. Taken together, these findings indicate the potential role of ER stress in autophagy activation, modulation of inflammatory responses, and cell survival by gAcrp in macrophages.

MHY451 induces cell cycle arrest and apoptosis by ROS generation in HCT116 human colorectal cancer cells.

Colorectal cancer (CRC) is the third most frequently diagnosed cancer and cause of cancer-related deaths. Despite advancements in conventional therapeutic approaches to CRC, most patients with CRC die of their disease. There is a need to develop novel therapeutic agents for this malignancy. Therefore, the present study aimed to examine the anticancer effects and elucidate the underlying mechanism of MHY451 in HCT116 human colorectal cancer cells. Treatment with MHY451 inhibited cell growth in a time- and concentration-dependent manner. MHY451 increased the accumulation of cell cycle progression at the G2/M phase. This agent decreased the protein level of cyclin B1 and its activating partners, Cdc25c and Cdc2, whereas it increased the cell cycle inhibitor p21WAF/CIP. The induction of apoptosis was observed by decreased viability, cleavage of poly(ADP-ribose) polymerase (PARP), alteration in the ratio of Bax/Bcl-2 protein expression and reduction of procaspase-8 and -9. Pretreatment with Z-VAD-FMK, a pan-caspase inhibitor, inhibited MHY451-induced apoptosis, indicating that apoptotic cell death by MHY451 was mediated through caspases. Moreover, the apoptotic effect of MHY451 was reactive oxygen species (ROS)-dependent, evidenced by the inhibition of MHY451-induced PARP cleavage and ROS generation by N-acetylcysteine-induced ROS scavenging. Taken together, these results demonstrate that MHY451 exerts anticancer effects by regulating the cell cycle, inducing apoptosis through caspase activation and generating ROS. These results suggest that MHY451 has considerable potential for chemoprevention or treatment of CRC or both.

Serum 25-Hydroxyvitamin D3 and BAFF Levels Are Associated with Disease Activity in Primary Sjogren's Syndrome.

The study investigated the association between disease activity and serum 25-hydroxyvitamin D3 (25(OH)-D3), B cell activation of the tumor necrosis factor family (BAFF), or ß2 microglobulin in patients with primary Sjogren's syndrome (SS). Sixty-nine primary SS patients and 22 sicca control patients were included in the study. Disease activity was measured with EULAR Sjogren's syndrome disease activity index (ESSDAI). Serum levels of 25(OH)-D3 and ß2 microglobulin were measured by radioimmunoassay and BAFF was measured by an enzyme-linked immunosorbent assay. Serum levels of 25(OH)-D3 were significantly lower in SS patients compared to the sicca controls (p = 0.036). Serum levels of BAFF tended to be higher (p = 0.225) and those of ß2 microglobulin were significantly higher in patients with SS than in sicca controls (p = 0.023). In univariate regression analyses, ESSDAI was significantly associated with serum levels of 25(OH)-D3, BAFF, and ß2 microglobulin. After stepwise backward multivariate linear regression analyses including age and acute phase reactants, ESSDAI was associated with 25(OH)-D3 (ß = -0.042, p = 0.015) and BAFF (ß = 0.001, p = 0.015) in SS patients. In SS patients, ESSDAI is negatively associated with serum levels of 25(OH)-D3 and positively associated with BAFF.

Prevalence and clinical impact of fibromyalgia in patients with primary Sjögren's syndrome.

OBJECTIVES: Clinical features of primary Sjögren's syndrome (pSS) overlap with those of fibromyalgia (FM). This cross-sectional study was conducted to investigate the prevalence of FM in pSS patients and to compare the clinical features of pSS patients with FM to those without FM. METHODS: One hundred pSS patients were consecutively assessed to identify the presence of FM according to the American College of Rheumatology (ACR) 2010 criteria. Clinical and laboratory data were collected from all patients. Additional assessments included EULAR Sjögren's Syndrome Patient Reported Index (ESSPRI) and EULAR Sjögren's Syndrome Disease Activity Index (ESSDAI). The severity of depression was measured by Hamilton depression rating scale 17-items (HAM-D scale). RESULTS: The prevalence of FM was 31.0% (31/100) in pSS. Widespread pain index and symptom severity scale were significantly correlated with ESSPRI (r=0.6542 and r=0.7173, both p<0.0001) and HAM-D scale (r=0.6734 and r=0.6471, both p<0.0001) in pSS. In multivariate analysis, ESSPRI and HAM-D scale were independently associated with increase of tender point count and symptom severity scale. ESSPRI was significantly higher in pSS patients with FM compared to those without FM (p<0.0001). The prevalence of FM in pSS patients with moderate-to-severe depression was significantly higher than those with mild depression or without depression (odds ratio= 10.62, p=0.0009). Serum 25-hydroxy vitamin D3 levels in pSS patients with FM were significantly (p=0.0072) decreased compared to those without FM. CONCLUSIONS: Our study showed that FM was prevalent in pSS. FM was associated with higher ESSPRI and more severe depression.

Determination of EC95 of remifentanil for smooth emergence from propofol anesthesia in patients undergoing transsphenoidal surgery.

BACKGROUND: In patients undergoing pituitary surgery using a transsphenoidal approach, anesthesia emergence should be smooth with minimal coughing. Recent studies demonstrated that a target-controlled infusion of remifentanil effectively suppresses coughing induced by the endotracheal tube. We investigated the EC95 of remifentanil for smooth emergence without coughing from propofol anesthesia in patients undergoing transsphenoidal hypophysectomy. MATERIALS AND METHODS: A total of 41 patients undergoing transsphenoidal hypophysectomy, aged 20 to 65 years, with an ASA physical status of I or II, were enrolled. For all participants, anesthesia was induced and maintained with a target-controlled infusion of remifentanil and propofol using predicted effect-site concentration (Ce). A biased coin design up-and-down sequential allocation and isotonic regression method were used to determine the remifentanil EC95 to prevent emergence coughing. In addition, we observed recovery profiles after anesthesia. RESULTS: According to the study design, 19 patients received remifentanil 2.6 ng/mL Ce and 22 patients received a lower Ce, ranging from 1.0 to 2.2 ng/mL. The EC95 of remifentanil to prevent coughing was estimated as 2.51 ng/mL (95% confidence interval, 2.28-2.57 ng/mL). Despite the exclusion of 1 case because of delayed emergence, 17 of 18 patients receiving 2.6 ng/mL of remifentanil had bradypnea (<10 breaths/min) until 3 minutes after extubation. However, end-tidal carbon dioxide was maintained below 55 mm Hg during anesthetic emergence and respiratory rate recovered within 20 minutes of admission to the postanesthetic care unit. CONCLUSIONS: The EC95 of remifentanil for smooth emergence from anesthesia was 2.51 ng/mL after transsphenoidal hypophysectomy.

Complete conversion of major protopanaxadiol ginsenosides to compound K by the combined use of α-L-arabinofuranosidase and ß-galactosidase from Caldicellulosiruptor saccharolyticus and ß-glucosidase from Sulfolobus acidocaldarius.

The ginsenoside compound K has pharmaceutical activities, including anti-tumor, anti-inflammatory, anti-allergic, and hepatoprotective effects. To increase the production of compound K, the α-L-arabinofuranoside-hydrolyzing α-L-arabinofuranosidase (CS-abf) and/or the α-L-arabinopyranoside-hydrolyzing ß-galactosidase from Caldicellulosiruptor saccharolyticus (CS-bgal) were mixed with the ß-D-glucopyranoside-hydrolyzing ß-glucosidase from Sulfolobus acidocaldarius (SA-bglu). The optimum conditions for the production of ginsenoside compound K from ginsenoside Rc or Rb2, or from major protopanaxadiol ginsenosides in ginseng root extract were determined to be pH 6.0 and 75°C with 8 mg ml⁻¹ ginsenoside Rc, 8 mg ml⁻¹ Rb2, or 10% (w/v) ginseng root extract; and 10.5 U ml⁻¹ CS-abf or CS-bgal supplemented with 4.5 U ml⁻¹ SA-bglu, or 10.5 U ml⁻¹ CS-abf and 10.5 U ml⁻¹ CS-bgal supplemented with 4.5 U ml⁻¹ SA-bglu, respectively. Under optimum conditions, ginsenosides Rc and Rb2, and major protopanaxadiol ginsenosides in ginseng root extract were completely converted to compound K after 12, 14, and 20 h, respectively, with the respective productivities of 388, 328, and 144 mg l⁻¹ h⁻¹. This is the first report of the complete conversion of major protopanaxadiol ginsenosides to compound K.

Pharmacological activation of Sirt1 ameliorates polyglutamine-induced toxicity through the regulation of autophagy.

Intracellular accumulation of polyglutamine (polyQ)-expanded Huntingtin (Htt) protein is a hallmark of Huntington's disease (HD). This study evaluated whether activation of Sirt1 by the anti-cancer agent, ß-lapachone (ß-lap), induces autophagy in human neuroblastoma SH-SY5Y cells, thereby reducing intracellular levels of polyQ aggregates and their concomitant cytotoxicity. Treatment of cells with ß-lap markedly diminished the cytotoxicity induced by forced expression of Htt exon 1 containing a pathogenic polyQ stretch fused to green fluorescent protein (HttEx1(97Q)-GFP). ß-lap increased autophagy in SH-SY5Y cells, as evidenced by the increased formation of LC3-II and autolysosomes. Furthermore, ß-lap reduced HttEx1(97Q)-GFP aggregation, which was significantly prevented by co-incubation with 3-methyladenine, an inhibitor of autophagy. ß-lap increased Sirt1 activity, as shown by the increased deacetylation of the Sirt1 substrates, PARP-1 and Atg5, and the nuclear translocation of FOXO1. Both the induction of autophagy and attenuation of HttEx1(97Q)-GFP aggregation by ß-lap were significantly prevented by co-incubation with sirtinol, a general sirtuin inhibitor or by co-transfection with shRNA against Sirt1. The pro-autophagic actions of ß-lap were further investigated in a transgenic Caenorhabditis elegans (C. elegans) line that expressed Q67 fused to cyanine fluorescent protein (Q67). Notably, ß-lap reduced the number of Q67 puncta and restored Q67-induced defects in motility, which were largely prevented by pre-treatment with RNAi against sir-2.1, the C. elegans orthologue of Sirt1. Collectively, these data suggest that ß-lap induces autophagy through activation of Sirt1, which in turn leads to a reduction in polyQ aggregation and cellular toxicity. Thus, ß-lap provides a novel therapeutic opportunity for the treatment of HD.

An engineered viral protease exhibiting substrate specificity for a polyglutamine stretch prevents polyglutamine-induced neuronal cell death.

BACKGROUND: Polyglutamine (polyQ)-induced protein aggregation is the hallmark of a group of neurodegenerative diseases, including Huntington's disease. We hypothesized that a protease that could cleave polyQ stretches would intervene in the initial events leading to pathogenesis in these diseases. To prove this concept, we aimed to generate a protease possessing substrate specificity for polyQ stretches. METHODOLOGY/PRINCIPAL FINDINGS: Hepatitis A virus (HAV) 3C protease (3CP) was subjected to engineering using a yeast-based method known as the Genetic Assay for Site-specific Proteolysis (GASP). Analysis of the substrate specificity revealed that 3CP can cleave substrates containing glutamine at positions P5, P4, P3, P1, P2', or P3', but not substrates containing glutamine at the P2 or P1' positions. To accommodate glutamine at P2 and P1', key residues comprising the active sites of the S2 or S1' pockets were separately randomized and screened. The resulting sets of variants were combined by shuffling and further subjected to two rounds of randomization and screening using a substrate containing glutamines from positions P5 through P3'. One of the selected variants (Var26) reduced the expression level and aggregation of a huntingtin exon1-GFP fusion protein containing a pathogenic polyQ stretch (HttEx1(97Q)-GFP) in the neuroblastoma cell line SH-SY5Y. Var26 also prevented cell death and caspase 3 activation induced by HttEx1(97Q)-GFP. These protective effects of Var26 were proteolytic activity-dependent. CONCLUSIONS/SIGNIFICANCE: These data provide a proof-of-concept that proteolytic cleavage of polyQ stretches could be an effective modality for the treatment of polyQ diseases.

Phosphorylation of Ser 21 in Fyn regulates its kinase activity, focal adhesion targeting, and is required for cell migration.

The tyrosine kinase Fyn is a member of the Src family kinases which are important in many integrin-mediated cellular processes including cell adhesion and migration. Fyn has multiple phosphorylation sites which can affect its kinase activity. Among these phosphorylation sites, the serine 21 (S21) residue of Fyn is a protein kinase A (PKA) recognition site within an RxxS motif of the amino terminal SH4 domain of Fyn. In addition, S21 is critical for Fyn kinase-linked cellular signaling. Mutation of S21A blocks PKA phosphorylation of Fyn and alters its tyrosine kinase activity. Expression of Fyn S21A in cells lacking Src family kinases (SYF cell) led to decreased tyrosine phosphorylation of focal adhesion kinase resulting in reduced focal adhesion targeting, which slowed lamellipodia dynamics and thus cell migration. These changes in cell motility were reflected by the fact that cells expressing Fyn S21A were severely deficient in their ability to assemble and disassemble focal adhesions. Taken together, our findings indicate that phosphorylation of S21 within the pPKA recognition site (RxxS motif) of Fyn regulates its tyrosine kinase activity and controls focal adhesion targeting, and that this residue of Fyn is critical for transduction of signals arising from cell-extracellular matrix interactions.

In situ thermal gelling polypeptide for chondrocytes 3D culture.

In the search for a cell-instructive or cell-interactive artificial extracellular matrix, synthetic hydrogels have been extensively investigated to apply three-dimensional (3D) cell culture and tissue engineering. Here, we are reporting a reverse thermal gelling l/dl-polyalanine block copolymer aqueous solution for chondrocyte 3D culture. The polymer aqueous solution undergoes sol-to-gel transition as the temperature increases, thus forming a 3D cell encapsulating scaffold in situ at 37 °C. In particular, the fraction of the ß-sheet structure of the polyalanine dictated the population and thickness of fibrous nanostructure of the hydrogel, which in turn affected the proliferation and protein expression of the encapsulated chondrocytes. As an injectable tissue engineering system of chondrocytes, very promising results were confirmed for nude mice, using the current polypeptide aqueous solution. This paper not only provides important clues in designing an artificial extracellular matrix but also proves the significance of thermal gelling polypeptide as a minimally-invasive tissue engineering scaffold.

v-Crk induces Rac-dependent membrane ruffling and cell migration in CAS-deficient embryonic fibroblasts.

Crk-associated substrate (CAS) is a focal adhesion protein that is involved in integrin signaling and cell migration. CAS deficiency reduces the migration and spreading of cells, both of which are processes mediated by Rac activation. We examined the functions of v-Crk, the oncogene product of the CT10 virus p47gag-crk, which affects cell migration and spreading, membrane ruffling, and Rac activation in CAS-deficient mouse embryonic fibroblasts (CAS-/- MEFs). CAS-/- MEFs showed less spreading than did CAS+/+ MEFs, but spreading was recovered in mutant cells that expressed v-Crk (CAS-/-v-Crk MEF). We observed that the reduction in spreading was linked to the formation of membrane ruffles, which were accompanied by Rac activation. In CAS-/- MEFs, Rac activity was significantly reduced, and Rac was not localized to the membrane. In contrast, Rac was active and localized to the membrane in CAS-/-v-Crk MEFs. Lamellipodia protrusion and ruffle retraction velocities were both reduced in CAS-/- MEFs, but not in CAS-/-v-Crk MEFs. We also found that microinjection of anti-gag antibodies inhibited the migration of CAS-/-v-Crk MEFs. These findings indicate that v-Crk controls cell migration and membrane dynamics by activating Rac in CAS-deficient MEFs.

Focal adhesion targeting of v-Crk is essential for FAK phosphorylation and cell migration in mouse embryo fibroblasts deficient src family kinases or p130CAS.

We examined the consequences of v-Crk expression in mouse embryo fibroblasts deficient Src family kinases or p130CAS. We found that Src kinases are essential for p130CAS/v-Crk signaling leading to FAK phosphorylation and cell migration in which Src is likely to mediate the focal adhesion targeting of v-Crk. SYF cells showed only low levels of FAK phosphorylation and cell migration, even in the presence of v-Crk. Expression of v-Crk restored migration of p130CAS-deficient cells to the level of wild-type cells, most likely through the targeting of v-Crk to focal adhesions by cSrc. In addition, we identified a new v-Crk-interacting protein that mediates v-Crk signaling in p130CAS-deficient cells. Using RT-PCR and caspase cleavage assays, we confirmed that this protein is not p130CAS and is responsible for maintaining v-Crk/Src signaling and migration in these. These findings suggest that focal adhesion targeting of v-Crk is essential in v-Crk-mediated cellular signaling and that v-Crk must form a complex with p130CAS or a p130CAS substitute to transduce signaling from the extracellular matrix.

Modulation of beta-catenin by cyclin-dependent kinase 6 in Wnt-stimulated cells.

Beta-catenin is implicated in quite different cellular processes, which require a fine-tuned regulation of its function. Here we demonstrate that cyclin-dependent kinase 6 (CDK6), in association with cyclin D1 (CCND1), directly binds to beta-catenin. We showed that CCND1-CDK6 phosphorylates beta-catenin on serine 45 (S45). This phosphorylation creates a priming site for glycogen synthase kinase 3beta (GSK3beta) and is both necessary and sufficient to initiate the beta-catenin phosphorylation-degradation cascade. Moreover, co-immunoprecipitation assays using Wnt3a-conditioned medium reveals that while Wnt stimulation leads to the dissociation of beta-catenin from axin and casein kinase Ialpha (CKIalpha), Wnt treatment promotes an increase in CCND1 level and the association of beta-catenin with CCND1-CDK6. Furthermore, Wnt3a-stimulated cytosolic beta-catenin levels were higher in CDK6 knockout mouse embryonic fibroblasts (CDK6-/- MEFs) compared to wild-type MEFs. Thus, the CCND1-CDK6 complex is like to negatively regulate Wnt signaling by mediating beta-catenin phosphorylation and its subsequent degradation in Wnt-stimulated cells.