Murine NIH3T3 (wildtype KRAS) and human CRC cells SW48 (wildtype KRAS) and SW620 (mutant KRAS) were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA). The expression of KRAS was measured by Western blot analysis of the corresponding protein in HME-1 WT or KI (knock-in) for KRAS G13D + Scramble shRNA (shSCRA), and in HME-1 WT cells infected with lentiviral control vector (LENTI Empty) or infected with lentiviral vector expressing the G13D KRAS cDNA (LENTI KRAS G13D). To further study the function of SIX4 in CRCs, SW48, and LoVo cells were transfected with siRNAs specific for SIX4 and control siRNA. (A) c-MET transcript is upregulated ∼12-fold in SW48 KRAS G12D cells compared with their isogenic parental counterparts. Antagonism by platinum drugs and cetuximab on proliferation of KRAS wild-type CRCs has been reported. We profiled the secretomes of SW48 KRAS-WT, KRAS-G13D, and KRAS-G12V cells in response to EGF and EGF/cetuximab (in both 2D and 3D). Among the cells with wild‐type KRAS, SW48 cells showed the highest EGFR and p‐EGFR expression levels. Isogenic SW48 KRAS wt, G12A, G12C, G12D, G12R, G12S, G12 V, and G13D cells were evaluated for ERK phosphorylation with and without EGF stimulation. IC 50 values were acquired for SW48 KRAS wild-type/mutant cell lines following 48 h of treatment with. KRAS proteins play a major role in human cancer, but have not yielded to therapeutic attack. Among a panel of human CRC cell lines, three with acquired resistance to cetuximab (SW48-CR, GEO-CR and Caco-2-CR) were highly sensitive to the Astragalus compound. KRAS is the most frequently mutated oncogene in cancer and KRAS mutation is commonly associated with poor prognosis and resistance to therapy. 2012-01-01. SW48 cells are wt for KRAS, whereas SW48 KRAS G13D cells were created by knock-in of an activating mutation into one KRAS allele (Di Nicolantonio et al. Patrick Casey (Duke Univer-. 04µM for Ox, 25 µg/mL for Cet, and C6-Cer concentrations ranged from 5 to 10 µM). 5% V600E and EGFR 20% T790M). Data represent the average of three independent experiments ± SD. This system uses endogenous promoters and enables panels of cell lines to be studied which differ only by the point mutation of interest, providing patient relevant in. Parental SW48 cells are wild type for KRAS and contain approximately 290,000 copies of Ras per cell (Fig 4A). Eight-day clonogenic assays demonstrate greater combination activity in SW48-KRAS G13D versus SW48-KRAS G12 mutant cell lines (Figure 5C). Active Motif's Ras GTPase Chemi ELISA Kit is the first ELISA-based kit designed to detect and quantify activated Ras GTPase. horizondiscovery. Densitometry measurements of EGFR and SFK relative to Colo320DM (1. Effect of KRAS mutation on TAK-931 antiproliferative activity, showing unique antiproliferative spectrum in cancer cells. PubMed Central. SW48 cells: not significant. ,LTD代理或销售的Biovector, Addgene, ATCC, Invi品牌的试剂,产品来源于Biovector Inc. How to cite this article: Park SH, Jo MJ, Kim BR, Jeong YA, Na YJ, Kim JL, Jeong S, Yun HK, Kim DY, Kim BG, Kang SH, Oh SC, Lee DH. G12C (H358). AML is a malignant disease in which hematopoietic precursors are arrested in an early stage of development. The IC50 was determined as the half inhibitory concentration of umbelliprenin that led to a 50% decrease in the OD of the test compound compared to the control. KRAS mutant and KRAS/PIK3CA double mutant (A) and KRAS wt (B) colorectal cancer cells were infected with two shRNA targeting KRAS (B, C) or control shRNA. Among the subtypes of KRAS mutations, substitution in codon 12, 35G>T was the most frequent, followed by substitution in codon 12, 35G>A and codon 12, 34G>A. Tan, Cong; Du, Xiang. Murine NIH3T3 (wildtype KRAS) and human CRC cells SW48 (wildtype KRAS) and SW620 (mutant KRAS) were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA). (A) c-MET transcript is upregulated ∼12-fold in SW48 KRAS G12D cells compared with their isogenic parental counterparts. Among the cells with wild‐type KRAS, SW48 cells showed the highest EGFR and p‐EGFR expression levels. For this purpose, we set up mice models of metastatic colorectal cancer by intrasplenic (to evaluate liver homing) and intrahepatic (to investigate liver colonization) implantation of human colorectal cancer cell lines (SW48 and LIM1215) in which oncogenic KRAS (G12D, G12V, G13D) variants have been somatically knocked-in. Cells were grown in growth medium alone or growth medium with cetuximab (20 μg/ml) for 48 hours. Cytotoxicity was measured using the MTT assay. Indeed, when we compared ERK phosphorylation in response to transfected KRAS G12V in SW48 and Caco2 CRC cells (that have no mutations in KRAS, NRAS or BRAF), we found that Caco2 cells were KRAS. In PP242-sensitive HCT 15 and SW48 cell lines, 1h of treatment with PP242 similarly reduced the phosphorylation of mTORC1 sub-strates S6K and 4E-BP1, as well as mTORC2 substrate AKT S473. However, one could easily extend the model to include wild-type forms of HRAS, NRAS, and KRAS using the same equations as above and utilizing the slightly different parameter values for each. A similar but greater effect is also observed in murine pancreatic organoids expressing mutant oncogenic Kras compared with controls ( Figure 7 E). Leibovitz, et al. Profiling Mutations in Known Signaling Pathways from Plasma Melissa de los Reyes, Sally Dow and Mark Parrish Covance Genomics Laboratory, Seattle, Washington Abstract Whole blood and plasma have long been a traditional noninvasive surrogate tissue for the identification of protein-based biomarkers. Of these, the mutation of valine 600 to glutamic acid (V600E) is the most prevalent. Riffo-Campos 1,2, Francisco Gimeno-Valiente 1, Fernanda M. While oncogenic RAS or BRAF cause sustained activation of the MEK1/2-ERK1/2 pathway, ERK5 is directly activated by MEK5. Stable knockdown of FOXO3, NCOA3, and TCF7L2 in SW48 colorectal cell lines devoid of KRAS mutations did not show changes in GLUT1 expression (Additional file 1: Figure S9A) and the phenotype in low glucose was similar to normal medium (Additional file 1: Figure S9B, C). Here we report the comparative and nucleotide-dependent interactomes of KRas4a and KRas4b. The antitumor activities on Tumor Volume of Erlotinib in the Treatment of SW48-parental, SW48 KRAS (G13D/+) and SW48 KRAS (G12V/+) Isogenic DualXeno models were summarized below in Table 4. Cell Culture Revival Rapidly thaw cells in a 37°C water bath. SW48 andSW48-KRAS-G13Dand SW48-KRAS-G12Vcellswereobtainedfrom HorizonDis-coveryLtd. Briefly, for a period of 8 months, SW48 and C99 cells were continuously exposed to cetuximab to increase the inhibition of 50% of cancer cell growth (IC50), and the final concentration was 12. CINECA IRIS Institutional Research Information SystemInstitutional Research Information System. Sonic hedgehog pathway activation is associated with cetuximab resistance and EPHB3 receptor induction in colorectal cancer. 0) for SFK are shown. All examined SW48 KRAS mutant cell Figure 1. (A) KRAS wild‐type SW48 and KRAS(G13D)‐mutated HCT116 cells were treated with increasing concentrations of 5‐FU for 72 hours. effectors of KRAS signaling for bypassing the response of KRAS-mutant mCRC cells to anti-EGFR therapy, almost nothing is known about the specific pathways employed by NRAS-mutant mCRC cells that render them unresponsive to EphA2 receptor activation with ephrin-A1 ligand restores cetuximab efficacy in NRAS-mutant colorectal cancer cells. Murine NIH3T3 (wildtype KRAS) and human CRC cells SW48 (wildtype KRAS) and SW620 (mutant KRAS) were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA). 2012-01-01. Among the subtypes of KRAS mutations, substitution in codon 12, 35G>T was the most frequent, followed by substitution in codon 12, 35G>A and codon 12, 34G>A. EC91072201-G0: 容量(包装) T-25フラスコ: 希望価格 (円/税別) 103,000円 旧注文Cat. Here, we found that LGR5, by imposing on cells an intestinal SC program, was required but not sufficient for efficient malignant transformation, which was optimized with KRAS mut activation of the. , Report on the clinical utility of reflex testing of resected stage I through III lung adenocarcinomas for EGFR and KRAS mutatations. Indeed, when we compared ERK phosphorylation in response to transfected KRAS G12V in SW48 and Caco2 CRC cells (that have no mutations in KRAS, NRAS or BRAF), we found that Caco2 cells were KRAS. Effect of KRAS mutation on TAK-931 antiproliferative activity, showing unique antiproliferative spectrum in cancer cells. TET1 in CRCs Ichimura N et al. A similar but greater effect is also observed in murine pancreatic organoids expressing mutant oncogenic Kras compared with controls ( Figure 7 E). HDC82, SW48, HCA7, and CAR1 are WT for KRAS/BRAF/PIK3CA, whereas the remainder have codons 12/13 KRAS mutations. ,LTD代理或销售的Biovector, Addgene, ATCC, Invi品牌的试剂,产品来源于Biovector Inc. SLC25A22 silencing inhibited colony formation in KRAS mutant CRC cells (DLD1, HCT116, LOVO, SW480, SW620, and SW1116), but had no effect on KRAS wild-type CRC cells (CACO-2 COLO205, HT29, and SW48) (middle and lower left). KRAS mutant (HCT116, MIA PaCa-2, and SW403) and KRAS WT (Caco-2 and SW48) cancer cell lines were transfected with various siRNA combinations targeting BRAF (B), CRAF (C), RAC1 (1), and ATG7 (7). Materials and methods. CCR-14-0361 pEGFR Is a Response Biomarker of Cetuximab Therapy in CRC Figure 1. Fiorentino at Department of Environmental Biological and Pharmaceutical Sciences and Technologies, Second University of Naples, (Caserta, Italy). 0151) as well as the number of CD19 and CD20 positive B cell follicles within the tumor environment (p = 0. Although the KRAS wild-type Caco-2 and SW48 cells were resistant to individual and combinations of RAF isoform knockdown , the PI3K node, on the other hand, in particular the PIK3CA gene, is critical for their viability. Centrifuge cells and seed into a 25cm2 flask containing pre-warmed media. Cell Culture. The average cell number was measured by determining ATP content in four replicate wells. Human CRC myofibroblasts cells (CRC-MF) were isolated from a primary human colorectal liver metastasis under an IRB approved protocol and expressed α-smooth muscle actin. Future studies will have to clarify whether KRAS can be used to guide sunitinib treatment or-in general-a treatment with a multityrosine kinase inhibitor in mCRC. exposure of SW48 and C99 cell lines to cetuximab according to the method of Troiani et al (30). Therefore, we investigated the effect of ZOL and/or CTX mainly in SW48 and LS174T cells. SW48 KRAS-G12D cells are approximately 7-fold more sensitive to Crizotinib in the presence of Poloppin compared with SW48 parental cells (Figures 7C and 7D). (D) MTT proliferation assays to assess dose responses of KRAS WT SW48 (WT) colon cancer cells and three derivative isogenic cell lines, each with one of the three most common KRAS mutants in colon cancer (G12D, G12V, and G13D), to the EGFR-blocking antibody cetuximab (CTX; at doses indicated for 48 hours). DKOB8 cells were acquired (09/19/03) from Dr. ,LTD代理或销售的Biovector, Addgene, ATCC, Invi品牌的试剂,产品来源于Biovector Inc. We have used SW48 and LIM1215 human colon cancer cell lines, quadruple wild-type for KRAS, NRAS, BRAF and PI3KCA genes, and their HER2–amplified (LIM1215-HER2 and SW48-HER2) derived cells to perform in vitro and in vivo studies in order to identify novel therapeutic strategies in HER2 gene amplified human colorectal cancer. distinct signature, with the most variability seen between G13D and the codon 12 KRAS mutants. V600E mutations were identi?ed without ambiguity up to 0. It is a secreted pentameric globular protein that forms part of the extracellular matrix, and functions in calcium binding, cell attachment, cell migration, cellular proliferation, cytoskeletal organisation, neurite growth, binding of other extracellular matrix components and cell to cell interactions [1. Densitometry measurements of EGFR and SFK relative to Colo320DM (1. Antitumor activity was accompanied by inhibition of the MAPK and MEK pathways. com or tel: +44 (0)1223 655 580Genotype Cell Line Description Catalogue IDB-Raf (BRAF)BRAF (V600A/+) HCT116 Heterozygous knock-in of BRAF mutation (V600A) HD 104-075BRAF (V600G/+) HCT116 Heterozygous knock-in of BRAF. View Essay - McCormick from DEPARTMENT BIOL3704 at Mid America Nazarene University. 52606881166759,CII,Leukemia,Leukemia. To investigate the differential effects upon cell status associated with KRAS mutations we performed a quantitative analysis of the proteome and phosphoproteome of isogenic SW48 colon cancer cell lines in which one allele of the endogenous gene has been edited to harbor specific KRAS mutations (G12V, G12D, or G13D). Endometrial cancer and somatic G>T KRAS transversion in patients with constitutional MUTYH biallelic mutations. Material and methods SW48 (KRAS WT) and the isogenic SW48 KI G12V (KRAS mutant) cell lines were used (Horizon Discovery Ltd). To test whether Ubc9 is require for KRAS tumor growth in vivo, we established KRAS mutant DLD-1 cells and KRAS WT SW48 cells that stably express shUbc9#4 under the control of a tet-inducible promoter. Design of “one armed scFab” bispecific antibodies XGFR and XGFR* targeting EGFR and IGF-1R simultaneously. SW48 KRAS WT tumors, but neither SW48-KRAS-G13D tumors nor SW48-KRAS-G12V tumors, were sensitive to ixazomib in vivo. がん創薬に有用な解析結果。細胞株の増殖を指標にした化合物のプロファイリング。化合物の効果と変異遺伝子との関連性. For invitro use,ixazomibwasformulated inDMSOand diluted inmedia todesired concentration. The number of invaded cells significantly decreased in SW48 and LoVo cells expressing SIX4 siRNA than in the control cells (Figs. Our digital PCR assays are hydrolysis probe–based assays that were designed by experts in the digital PCR field. G13D was selected, since it is known that p. The human CRC cell lines HCT116 and DLD‐1, which harbor a KRAS mutation at codon 13, and their isogenic derivatives lacking mutated KRAS (Hkh2, DKO‐4, and DKs‐8) have been previously described. Similar reduction in MUC2 expression was seen with knockdown of mutant KRAS alone ( , red arrows) (note that the KRAS western blot shown in was probed with an anti-KRASG12D-specific antibody that binds only to mutant KRAS protein). Results: SW48, LIM 1215, and GEO colorectal cancer cell lines were engrafted into nude mice and treated for 3 weeks with irinotecan and/or cetuximab. Citation Guide If the product is used for publication, it should be cited in the publication as: "AcceGen Biotech Cat. Activity of regorafenib was evaluated in isogenic SW48 KRAS wild-type (WT) and mutant cells. スピンオフ・バイオベンチャー企業。大手製薬企業の研究部門が母体となって2003年4月に設立されたバイオベンチャー企業. Experimental Design: We have tested, in vitro and in vivo, the effects of regorafenib in a panel of human colorectal cancer cell lines with a KRAS mutation (SW480, SW620, HCT116, LOVO, and HCT15) or with a BRAF mutation (HT29), as models of intrinsic resistance to cetuximab treatment, and in two human colorectal cancer cell lines (GEO and SW48. The antitumor activities on Tumor Volume of Erlotinib in the Treatment of SW48-parental, SW48 KRAS (G13D/+) and SW48 KRAS (G12V/+) Isogenic DualXeno models were summarized below in Table 4. 7 Additional mutations ALK P1543S 50. Peter Camaj, Stefano Primo, Yan Wang, Volker Heinemann, Yue Zhao, Ruediger Paul Laubender, Sebastian Stintzing, Clemens Giessen-Jung, Andreas Jung, Sebastian Gamba, Christiane Josephine Bruns & Dominik Paul Modest; Pages: 1919–1929. One way of dealing with that heterogeneity is to test large numbers of patient-derived cell lines for common vulnerabilities. Human CRC cell lines with KRAS mutations (LS153, LS174T, DLD1, LoVo, SW403, SW480, SNU175, and LS1034) or with v-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutations (DiFi, SW48, HT29, and RKO) were used to test the effect of cetuximab, simvastatin, and cetuximab plus simvastatin on cell proliferation and apoptosis in vitro. KRAS G13D mutant SW48 cells represented the KRAS subspecies with the lowest grade of resistance. Cell Culture Revival Rapidly thaw cells in a 37°C water bath. AML is a malignant disease in which hematopoietic precursors are arrested in an early stage of development. To provide independent validation of the accuracy of the quantitation, we compared the summed values from isoform-specific peptides with those peptides present in more than one isoform and observed no significant difference between the pairs of. DepMap ID,TPM (log2),Cell Line Name,Primary Disease,Lineage ACH-001097,1. KRAS Multiplex FFPE 5% - HD301 KRAS Multiplex FFPE - Exome Data EGFR Multiplex FFPE 5% - HD300 EGFR Multiplex FFPE 5% - Exome Data. Try again. The AZ-Merck Collaboration Institute of Medicine Washington, D. When KRAS is targeted with shRNA in these mutant SW48 cells, DCLK1 expression decreases in a dose-dependent fashion. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country. [Journal Article] High level expression of AMAP1 protein correlates with poor prognosis and survival after surgery of head and neck squamous cell carcinoma patients. VP Oncology, Merck and Co. The parental, KRAS wt cell line SW48 has low to medium EGFR expression levels, but is very sensitive to EGFR inhibition by cetuximab already in. Densitometry measurements of EGFR and SFK relative to Colo320DM (1. Every digital PCR assay has been experimentally validated to ensure optimal performance for its target application:. To investigate the impact of different KRAS mutations on treatment with the tyrosine kinase inhibitor sunitinib in SW48 colorectal cancer cell line variants. Mol Cancer. 描述 Doxorubicin, an antibiotic anthracycline, is commonly considered to exert its anti-tumor activity at two fundamental levels, altering DNA and producing free radicals to trigger apoptosis of cancer cells through DNA damage. com or by email: info@horizondiscovery. Bevacizumab-erlotinib as maintenance therapy in metastatic colorectal cancer. All examined SW48 KRAS mutant cell Figure 1. Biomarkers of resistance to anti-EGFR in wild type KRAS/BRAF colorectal cancer cell lines Thesis submitted for the degree of Doctor of Philosophy Shalini Sree Kumar. For this purpose, we set up mice models of metastatic colorectal cancer by intrasplenic (to evaluate liver homing) and intrahepatic (to investigate liver colonization) implantation of human colorectal cancer cell lines (SW48 and LIM1215) in which oncogenic KRAS (G12D, G12V, G13D) variants have been somatically knocked-in. Soft agar assays, growth rates and colony formation on plastic for stable 100 knockdown lines for KRAS, NCOA3, FOXO3 and TCF7L2 in DLD-1 (KRAS G13D) 101 cells. KRAS mutation testing in metastatic colorectal cancer. La Biblioteca Virtual en Salud es una colección de fuentes de información científica y técnica en salud organizada y almacenada en formato electrónico en la Región de América Latina y el Caribe, accesible de forma universal en Internet de modo compatible con las bases internacionales. Schmitz1,2*. Using site-specific homologous recombination, we inserted the rs61764370:T>G KRAS gene variant in the colorectal cancer cell line SW48 (SW48 +SNP) and assessed the cellular and biochemical phenotype. Thereafter, protein samples were isolated, and phosphorylation of YB‐1 was analyzed. The percentage of animals developing lymph node metastasis was higher in KRas G12V than in KRas G13D mice. 28 µM, respectively. Consistent with the in vitro effects, panitumumab/TAS-102 combination caused tumor regression in LIM1215 and COL-01-JCK colon cancer patient-derived xenograft models. Transfer contents into a tube containing pre-warmed media. Thrombospondin-4 (THBS4) is a member of the extracellular calcium-binding protein family. We found a modest cross reactivity between probes for 3 assays (p. The human CRC cell lines HCT116 and DLD‐1, which harbor a KRAS mutation at codon 13, and their isogenic derivatives lacking mutated KRAS (Hkh2, DKO‐4, and DKs‐8) have been previously described. A proliferation assay was used to examine the sensitivity of KRAS wild-type SW48 cells, KRAS(G12V)-mutated SW480 cells and KRAS(G13D)-mutated HCT116 cells to cetuximab. Moreover, in the cetuximab plus MEKi-treated SW48 xenograft group, KRAS mutations as a mechanism of acquired resistance were detected in 25% of cases compared with 75% KRAS mutations in the MEKi-treated group. 5% V600E and EGFR 20% T790M). was effective as a single agent or in combination with zoledronic acid (ZOL) in human CRC cell lines with different KRAS sta-tus. The human CACO2 (KRAS, NRAS, BRAF and PIK3CA WT), colon cancer cell line was obtained from Dr. 1 nM for human and canine, respectively. Panitumumab/FTD cotreatment showed additive antiproliferative effects in LIM1215 and synergistic antiproliferative effects in SW48 colon cancer cells. RESEARCH Open Access Herbal formula Huang Qin Ge Gen Tang enhances 5-fluorouracil antitumor activity through modulation of the E2F1/TS pathway Haizhou Liu1,2, Hui Liu1,2,3, Zhiyi Zhou1,2,4, Robert A. The lack of testing in these two studies might cause a small negative bias on outcome of patients with unmutated tumors. Centrifuge cells and seed into a 25cm2 flask containing pre-warmed media. Human CRC myofibroblasts cells (CRC-MF) were isolated from a pri-mary human colorectal liver metastasis under an IRB approved protocol and expressed α-smooth muscle actin. Many mutation sites, such as KRAS and EGFR, have been discovered in circulating DNA of cancer patients. We did not use SW48 in our studies of CI-1040 due to its poor growth in soft agar. IRIS è il sistema di gestione integrata dei dati della ricerca (persone, progetti, pubblicazioni, attività) adottato dall'Università degli Studi di Torino. Biomarkers of resistance to anti-EGFR in wild type KRAS/BRAF colorectal cancer cell lines Thesis submitted for the degree of Doctor of Philosophy Shalini Sree Kumar. Conversely, HCT116 p53+/+ and HCT116 p53−/−, cell lines with activating KRAS G13D mutations, exhibited higher levels of KLF5, cyclin D1 and b-catenin ( Fig. distinct signature, with the most variability seen between G13D and the codon 12 KRAS mutants. , index of species), while by clicking on a specific term (e. Consequently, dual targeting of RSK and Akt efficiently inhibited cell proliferation in KRAS(G13D)-mutated HCT116 and KRAS wild-type SW48 cells. (D) MTT proliferation assays to assess dose responses of KRAS WT SW48 (WT) colon cancer cells and three derivative isogenic cell lines, each with one of the three most common KRAS mutants in colon cancer (G12D, G12V, and G13D), to the EGFR-blocking antibody cetuximab (CTX; at doses indicated for 48 hours). Active RAS was pulled. tion of KRAS mutations in the blood of colorectal cancer patients, predicting lack of response to targeted treat-ment [8], screening for metastatic breast cancer by small increases in HER2 copy number in plasma samples [9], gene expression analyses to detect hepatocellular carcin-oma from circulating tumor cells [10], and detection of. β-catenin is then shuttled into the nucleus where it activates the transcription of its target genes, including the proto. KRAS Multiplex FFPE 5% - HD301 KRAS Multiplex FFPE - Exome Data EGFR Multiplex FFPE 5% - HD300 EGFR Multiplex FFPE 5% - Exome Data. PCR products were purified by. 52606881166759,CII,Leukemia,Leukemia. Endogenous mutation data (lignées) Endogenous Mutation Data (HCT116) Endogenous Mutation Data (SW48) Endogenous Mutation Data (RKO) Euroclone: Milieux de culture P et M (sangs et MO) Synchroset FICHE DE STRESS. clinically actionable genes: KRAS, NRAS and BRAF. Box plot of the percentage of nonamplificable (NA) NSCLC paraffin-embedded samples recovered by alternative techniques of the 74 tested. HDC82, SW48, HCA7, and CAR1 are WT for KRAS/BRAF/PIK3CA, whereas the remainder have codons 12/13 KRAS mutations. Following subcutaneous preconditioning, recombinant clones of the SW48 CRC cell line [Kras wild-type (Kras WT)] expressing the KRas G12V or KRas G13D allele were microinjected in the mouse cecum. Legend for data file S1; References (43, 44) [Download PDF] Other Supplementary Material for this manuscript includes the following: Data file S1 (Microsoft Excel format). SW48 KRAS WT (D) and p. We compared the DNA methylation profiles of. Technologies) at 37°C in a humidified incubator with. Centrifuge cells and seed into a 25cm2 flask containing pre-warmed media. (A, B) Viability of SW48 (A) and LIM1215 (B) cells after cotreatment with panitumumab and FTD for 72 h. E−, E+, C−, C+ refer to the absence or presence of EGF and cetuximab respectively. We developed an intact protein assay for the detection and quantitation of KRAS protein forms (proteoforms), enabling the measurement of how genetically encoded mutations affect posttranslational modifications on the same protein molecule. KRAS wild-type SW48 and KRAS(G13D)-mutated HCT116 cells were treated with 5-FU (1 µM), a combination of the RSK and Akt inhibitors (5 µM LJI308 for SW48 and 10 µM LJI308 for HCT116 cells; 5 µM MK2206) or a combination of the inhibitors with 5-FU for 72 h. APC Adenomatous polyposis coli (APC) is a tumor suppressor gene that plays a critical role in both cell division and adhesion, through the regulation of the Wnt-. Targeted knockdown of PLEKHA7 using siRNA resulted in growth inhibition for mut-KRAS cells (HCT116, DLD-1, SK-CO-1, SW480, HCT15, and LOVO), but not for wt-KRAS cells (SW48, CACO-2, SW1417, and HT29). The studies presented in this thesis aimed to determine other biomarkers of resistance to anti-EGFR therapy in wild type KRAS and BRAF CRC cell lines. (D) MTT proliferation assays to assess dose responses of KRAS WT SW48 (WT) colon cancer cells and three derivative isogenic cell lines, each with one of the three most common KRAS mutants in colon cancer (G12D, G12V, and G13D), to the EGFR-blocking antibody cetuximab (CTX; at doses indicated for 48 hours). For one such study, RAS Initiative researchers constructed GFP-labeled derivatives of dozens of cell lines that express mutant KRAS. We found a modest cross reactivity between probes for 3 assays (p. Each DNA cell line was serially diluted in wild-type DNA (derived from the SW48 cell line) to produce mixtures with 50%, 20% and 5% mutant alleles, the last per-. kras Since Akt aberrant activation is responsible for the resistant of many classic chemotherapy drugs like 5-FU, inhibiting Akt activity by combinational treatment come to our consideration. SW48-WT RAS, SW48-KRAS G12V, and SW48 KRAS G13D cells were cultured in T-75 adherent culture flasks. The KRAS G13D Reference Standard is a highly-characterized, biologically-relevant quality control material used to assess the performance of assays that detect somatic mutations, such as Sanger and qPCR sequencing assays. Results: SW48, LIM 1215, and GEO colorectal cancer cell lines were engrafted into nude mice and treated for 3 weeks with irinotecan and/or cetuximab. PubMed Central. PDF | ABSTRACT Aim: To investigate the impact of KRAS mutation variants on the activity of regorafenib in SW48 colorectal cancer cells. 描述 Doxorubicin, an antibiotic anthracycline, is commonly considered to exert its anti-tumor activity at two fundamental levels, altering DNA and producing free radicals to trigger apoptosis of cancer cells through DNA damage. Murine NIH3T3 (wildtype KRAS) and human CRC cells SW48 (wildtype KRAS) and SW620 (mutant KRAS) were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA). Since activated KRAS is known to be associated with metabolic reprogramming, we compared metabolite profiling of SW48-WT and SW48-KRAS-G13D tumors treated with or without ixazomib. McCormick, Frank. Antibodies. KRAS WT and KRAS mutant (KRAS Mut) CRC cell lines (SW48 and SW480, respectively). 1 Volume 58 Journal of Number 1 Investigative Medicine January 2010 Contents Comings and Goings 1 Brigham and Women_s Hospital and Faulkner Hospital Appoint New President 1 UNC Executive Associate Dean for Faculty Affairs and Faculty Development Stepping Down 2 University of Arkansas for Medical Sciences Names Chancellor 2 LSU School of Medicine Appoints New Dean 3 UCR Names. Box plot of the percentage of nonamplificable (NA) NSCLC paraffin-embedded samples recovered by alternative techniques of the 74 tested. Following injection into NOD/SCID mice and. Treatment with BAY 86-9766 determined dose-dependent growth inhibition in all cancer cells, including two human colorectal cancer cells with acquired resistance to cetuximab (GEO-CR and SW48-CR), with the exception of HCT15 cells. It is a secreted pentameric globular protein that forms part of the extracellular matrix, and functions in calcium binding, cell attachment, cell migration, cellular proliferation, cytoskeletal organisation, neurite growth, binding of other extracellular matrix components and cell to cell interactions [1. ; Whitling, Nicholas A. KRAS mutant (HCT116, MIA PaCa-2, and SW403) and KRAS WT (Caco-2 and SW48) cancer cell lines were transfected with various siRNA combinations targeting BRAF (B), CRAF (C), RAC1 (1), and ATG7 (7). Professor Mariano Barbacid's lab constructed a mouse embryonic fibroblast (MEF) line in which the HRAS and NRAS genes have been deleted, and the KRAS gene has been flanked by loxP sites (Drosten, et al. β-catenin is then shuttled into the nucleus where it activates the transcription of its target genes, including the proto. We're committed to accelerating cancer research by pioneering improvements that may impact tumor detection and. cetuximab, whereas two cells (GEO and SW48) were highly sensitive. Table 5 summarized antitumor activities on Tumor Volume of Cetuximab in the Treatment of SW48-parental, SW48 KRAS (G13D/+) and SW48 KRAS (G12V/+) Isogenic. 厦门大学 硕士学位论文 熔解曲线分析技术用于基因型的研究 姓名:张换敬 申请学位级别:硕士 专业:化学生物学 指导教师:郑立谋;曾骥孟 201106 摘要 摘 熔解曲线分析法(Melting 要 curve analysis)是一种PCR后产物分析技术,具 有操作简单、结果判断直观等特点,已经被广泛用于突变扫描、甲基化. Here, Caco-2 and SW48 cells harboring wild-type KRAS and BRAF, exhibited growth suppression following double blockage of GPNMB and EGFR; however, no growth suppression was observed in LS174T cells harboring mutant KRAS or in SW1417 cells harboring mutant BRAF (Fig. Using site-specific homologous recombination, we inserted the rs61764370:T>G KRAS gene variant in the colorectal cancer cell line SW48 (SW48 +SNP) and assessed the cellular and biochemical phenotype. In a previous study, BRAF and KRAS mutation status, as well as CIMP status, have been determined for 12 of the 24 cell lines reported here. KRAS-WT HT29 and SW48 cells (Figure S5) confirmed a marked decrease in KRAS, p-ERK, p-AKT, HIF1α, and YAP1 expression by DDX3 manipulation in KRAS-mutated HCT116 cells and an increase in DLD1 cells. Paradoxically, targeted knock-down of KRAS caused an increase in PLEKHA7 mRNA levels in mut-KRAS cells. The IC50 was determined as the half inhibitory concentration of umbelliprenin that led to a 50% decrease in the OD of the test compound compared to the control. Analytical sensitivity defined by serial dilution of cancer cell line DNAs Figure 6. 89917563048051,KARPAS384,Lymphoma,Lymphoma ACH-001485,2. , from a lymph node in the same manner as was the primary adenocarcinoma from which SW480 was derived the previous year. Dasatinib sensitizes KRAS mutant colorectal tumors to cetuximab in vitro. We have used SW48 and LIM1215 human colon cancer cell lines, quadruple wild-type for KRAS, NRAS, BRAF and PI3KCA genes, and their HER2–amplified (LIM1215-HER2 and SW48-HER2) derived cells to perform in vitro and in vivo studies in order to identify novel therapeutic strategies in HER2 gene amplified human colorectal cancer. Generally, there are multiple treatment procedures for lung cancer, such as surgery, immunotherapy, radiotherapy and chemotherapy. The human CACO2 (KRAS, NRAS, BRAF and PIK3CA WT), colon cancer cell line was obtained from Dr. We find that in a panel of isogenic SW48 colorectal cancer cells, endogenous Ras proteins are highly abundant with ≥260,000 total Ras protein copies per cell and the rank order of isoform abundance is KRAS>NRAS≥HRAS. 14, 19 The switches between these two states are controlled by two classes of proteins: guanosine nucleotide exchange factors (known as GEFs) and GTPase-activating proteins (known as GAPs). The lack of mutant KRAS-induced effector activation observed in SW48 cells appears to be representative of a broad panel of colon cancer cell lines harboring mutant KRAS. Charles River Tumor Model Compendium. HOME; 이용약관; 개인정보취급방침; 이메일집단수집거부; 사이트맵; Korean Cell Line Bank, Korean Cell Line Research Foundation, 101, Daehak-ro. Leibovitz, et al. しかし、kras 13d の発現上昇によって、複数のkrasエフェクター経路が活性化されることから、kras変異がもたらす治療上の課題が強調される。これらの結果は、併用療法の使用に影響を及ぼす可能性がある。. Table 5 summarized antitumor activities on Tumor Volume of Cetuximab in the Treatment of SW48-parental, SW48 KRAS (G13D/+) and SW48 KRAS (G12V/+) Isogenic. , The presence of KRAS mutations and loss of PTEN expression were not associated with impaired response to cetuximab-based. 2%) harbored a somatic mutation of the PI3K gene. It attempts to describe all cell lines used in biomedical research, including immortalized cell lines, naturally immortal cell lines (ie stem cell lines), finite life cell lines when those are distributed and widely used, vertebrate cell lines with an emphasis on human, mouse and rat cell lines, and invertebrate (insect and tick) cell lines. To investigate this, we depleted RAD51 alone or in combination with KRAS by siRNA in SW480 (KRASmt) and SW48 (KRASwt) cell lines and found that combined depletion decreased cell viability in mutant, but not WT cells (Fig. Biomarkers such as mutant KRAS or BRAF predict resistance to anti-EGFR therapy in only a subset of patients and we hypothesise that other biomarkers for resistance to EGFR targeted therapies exist. Active RAS was pulled. 7 KRAS A146T 16. Activity of regorafenib was evaluated in isogenic SW48 KRAS. Among cancers, lung cancer is the most morbidity and mortality disease that is remaining the fatalist. Human CRC myofibroblasts cells (CRC-MF) were isolated from a primary human colorectal liver metastasis under an IRB approved protocol and expressed α-smooth muscle actin. in KRAS wild-type SW48 colon cancer cells, which are sensitive to cetuximab. Cell cultures The study is a test system measuring the cell growth of KRAS WT and KRAS Mut cells. We compared the DNA methylation profiles of. しかし、kras 13d の発現上昇によって、複数のkrasエフェクター経路が活性化されることから、kras変異がもたらす治療上の課題が強調される。これらの結果は、併用療法の使用に影響を及ぼす可能性がある。. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country. kras Since Akt aberrant activation is responsible for the resistant of many classic chemotherapy drugs like 5-FU, inhibiting Akt activity by combinational treatment come to our consideration. (A) Active RAS was significantly increased in both DLD1-based and SW48-based isogenic cell lines of KRAS mutations. Treatment with 5-fluorouracil (5-FU) significantly enhanced YB-1 phosphorylation in KRAS(G13D)-mutated HCT116 cells but not in KRAS wild-type SW48 cells. La Biblioteca Virtual en Salud es una colección de fuentes de información científica y técnica en salud organizada y almacenada en formato electrónico en la Región de América Latina y el Caribe, accesible de forma universal en Internet de modo compatible con las bases internacionales. For one such study, RAS Initiative researchers constructed GFP-labeled derivatives of dozens of cell lines that express mutant KRAS. Oncogenic mutations of Ras at codons 12, 13, or 6. , from a lymph node in the same manner as was the primary adenocarcinoma from which SW480 was derived the previous year. For this purpose, we set up mice models of metastatic colorectal cancer by intrasplenic (to evaluate liver homing) and intrahepatic (to investigate liver colonization) implantation of human colorectal cancer cell lines (SW48 and LIM1215) in which oncogenic KRAS (G12D, G12V, G13D) variants have been somatically knocked-in. AbstractERK5, encoded by MAPK7, has been proposed to play a role in cell proliferation, thus attracting interest as a cancer therapeutic target. Further, we observed an unexpected correlation of driver alterations (KRAS, BRAF, ALK and EGFR) with the number of infiltrating CD19 positive B cells (p = 0. Final results of the GERCOR DREAM study SW48 (KRAS wt,. In accordance with findings from the COIN, NORDIC, and NEW EPOC studies, we observed negative effects combining cetuximab with oxaliplatin in SW48 and DLD-1 KRAS wild-type cells. Panitumumab/FTD cotreatment showed additive antiproliferative effects in LIM1215 and synergistic antiproliferative effects in SW48 colon cancer cells. Human CRC myofibroblasts cells (CRC-MF) were isolated from a primary human colorectal liver metastasis under an IRB approved protocol and expressed α-smooth muscle actin. Indeed, when we compared ERK phosphorylation in response to transfected KRAS G12V in SW48 and Caco2 CRC cells (that have no mutations in KRAS, NRAS or BRAF), we found that Caco2 cells were KRAS. 描述 Doxorubicin, an antibiotic anthracycline, is commonly considered to exert its anti-tumor activity at two fundamental levels, altering DNA and producing free radicals to trigger apoptosis of cancer cells through DNA damage. SW48 was the control cell line used in the in silico analysis. Shackelford, Rodney E. 2012-01-01. Overexpression of TGF-α, a specific EGFR ligand, was involved in the acquisition of cetuximab resistance in GEO-CR and SW48-CR cells. All examined SW48 KRAS mutant cell Figure 1. ,LTD代理或销售的Biovector, Addgene, ATCC, Invi品牌的试剂,产品来源于Biovector Inc. PCR products were purified by. to KRAS status and to investigate the prognostic and predic-tive value of Cdk5 in representative patients cohorts. Differences in the signaling functions of the two KRas proteins are poorly understood. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country. Data represent the average of three independent experiments ± SD. The KRAS mutation status of SW48. Vangl2 5'UTR was then subjected to bisulfite sequencing in these and other cell lines (SW48, HCT116, LOVO) and methylation specific PCR (MSP) was performed on 6 more CRC (DLD1, LS174T, SW620, Caco2, HT29, SW837) and one normal colon cell line (NCM460). ; Whitling, Nicholas A. How to cite this article: Park SH, Jo MJ, Kim BR, Jeong YA, Na YJ, Kim JL, Jeong S, Yun HK, Kim DY, Kim BG, Kang SH, Oh SC, Lee DH. To investigate the differential effects upon cell status associated with KRAS mutations we performed a quantitative analysis of the proteome and phosphoproteome of isogenic SW48 colon cancer cell lines in which one allele of the endogenous gene has been edited to harbor specific KRAS mutations (G12V, G12D, or G13D). PI3K mutational profiling. The in silico results were experimentally validated by quantitative real?time PCR. Human CRC cell lines with KRAS mutations (LS153, LS174T, DLD1, LoVo, SW403, SW480, SNU175, and LS1034) or with v-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutations (DiFi, SW48, HT29, and RKO) were used to test the effect of cetuximab, simvastatin, and cetuximab plus simvastatin on cell proliferation and apoptosis in vitro. Abstract 3073: Potential predictive biomarkers of clinical responses for a novel CDC7-selective inhibitor TAK-931 Kenichi Iwai , Tadahiro Nambu , Osamu Kurasawa , Noriko Uchiyama , Ryo Dairiki , Yukiko Yamamoto , Satoru Nishizawa , Mengkun Zhang , Yuko Ishii , Huifeng Niu and Akihiro Ohashi. CRC cell lines SW48 (wild-type KRAS) and LS174T (mutant KRAS) were treated with ZOL, CTX, and a combination of both drugs. For MAP kinase pathway activation in KRAS-mutant cells, the requirement for coincident growth factor stimulation occurs at an early point in the Raf activation cycle. E−, E+, C−, C+ refer to the absence or presence of EGF and cetuximab respectively. B ) KRAS and BRAF mutational status was determined via pyrosequencing. therapies exist. The aim of this study was to investigate the impact of different KRAS mutations on the inhibitory potential of afatinib and gefitinib in SW48 colorectal cancer cells. mutations in the APC and K-ras genes and hMLH1 expression, were all successfully and completely analysed, 103 colorectal tumours did not harbour a truncating or missense APC mutation, an activating K-ras mutation or showed lack of hMLH1 expression, as depicted in figure 1. Riffo-Campos 1,2, Francisco Gimeno-Valiente 1, Fernanda M. , index of species), while by clicking on a specific term (e. growth assays. 1158/1078-0432. Among a panel of human CRC cell lines, three with acquired resistance to cetuximab (SW48-CR, GEO-CR and Caco-2-CR) were highly sensitive to the Astragalus compound. In order to further examine the efficacy of cetuximab, we expanded our studies by generating xenograft mouse models with either SW48 or HCT8 colon cancer cells, which harbor either EGFR G719S or KRAS G13D mutation, respectively. The8othercelllinesused asxenograftswereobtained from ATCC,Manassas, VA. We profiled the secretomes of SW48 KRAS-WT, KRAS-G13D, and KRAS-G12V cells in response to EGF and EGF/cetuximab (in both 2D and 3D). DepMap ID,TPM (log2),Cell Line Name,Primary Disease,Lineage ACH-001097,1. Citation Guide If the product is used for publication, it should be cited in the publication as: "AcceGen Biotech Cat. Herman3, Enqiang Linghu1, Yunsheng Yang1, François Fuks4, Fuyou Zhou5,. SW48: SW480: SW579: disease: squamous cell carcinoma SW620: SW780: disease: transitional cell carcinoma SW837: SW900: SW948: T173: T3M10: A immortal human cell line cell that has the characteristics: Lung large cell carcinoma producing CSF. V600E mutations were identi?ed without ambiguity up to 0. HDAC2 functions in SW48 cell phenotypes were evaluated after co-transfection with pEGFP-RasQ61L/T35S and si-HDAC2 vectors. (C) SW48 cells were injected subcutaneously into both flanks of BALB/c nu−/nu− mice (5 × 10 6 /injection) and treated with PBS or cetuximab (0. Antibodies. Technologies) at 37°C in a humidified incubator with. Tumors with a CpG island methylator phenotype (CIMP), a distinct subgroup with extensive DNA methylation, show characteristic features in the case of colorectal cancer. Genomic DNAs were extracted from the cell-line SW48 bearing wild-type KRAS alleles (ATCC CCL-231) and the heterozygotous cell-line LoVo (LoVo (ATCC CCL-229) bearing a mutant allele of the KRAS oncogene (G12C) and a wild-type allele. GSK-923295 (GSK923295) is a first-in-class, specific, allosteric inhibitor of CENP-E kinesin motor function. Using site-specific homologous recombination, we inserted the rs61764370:T>G KRAS gene variant in the colorectal cancer cell line SW48 (SW48 +SNP) and assessed the cellular and biochemical phenotype. profound synthetic lethaleffects, namelyamedianZscoreacross thep. , retionoblastoma cell lines). lovo ccl-229 apc, fbxw7, kras, msh2 結腸直腸 結腸直腸腺がん ls 174t cl-188 ctnnb1, kdm6a, kras, pik3ca 結腸直腸 結腸直腸腺がん sw48 ccl-231 ctnnb1, egfr, fbxw7 結腸直腸 結腸直腸腺がん sw480 ccl-228 結腸直腸 結腸直腸腺がん sw620 ccl-227 apc, kras, map2k4, smad4, tp53, myc 結腸直腸 結腸直腸腺がん. ,LTD代理或销售的Biovector, Addgene, ATCC, Invi品牌的试剂,产品来源于Biovector Inc. The combined treatment induced a significant reduction of tumor size. Therefore, we investigated whether CTX treatment was effective as a single agent or in combination with zoledronic acid (ZOL) in human CRC cell lines with different KRAS status. Epigenetic and genetic features of colon cancer cell lines. PubMed Central. LY364947 in a panel of human HCC and other gastrointestinal cancer cells Materials and Methods: The human colorectal cancer cell lines SW48 by MTT assay, baseline and phosphorylated (p-) protein levels by western (KRAS/BRAF non-mutated), Caco-2 (BRAF V600E) and HCT-116 (KRAS blot analysis, mRNA expressions by qRT-PCR, motility by wound-healing. Dual targeting of Akt and RSK sensitized HCT116 cells to 5-FU by stimulating 5-FU-induced apoptosis and inhibiting repair of 5-FU-induced DNA damage. Herman3, Enqiang Linghu1, Yunsheng Yang1, François Fuks4, Fuyou Zhou5,. This is the complete description of the cell line that you selected. Error: Network error: undefined. Gly12Asp (c. Briefly, for a period of 8 months, SW48 and C99 cells were continuously exposed to cetuximab to increase the inhibition of 50% of cancer cell growth (IC50), and the final concentration was 12. 00 ic 50 (µ m) kras nras braf pi3k. 5B,C left). isogenic human cell lines comprising mutated cancer alleles and process using the cell lines. cell line gene mutant allele a549 kras g12s h1666 braf g466v h1781 erbb2 g776vc h1975 egfr t790m h1975 egfr l858r h2228 alk eml4 h2347 nras q61r h3122 alk eml4 hcc78 ros1 slc34a2 hct116 kras g13d hct116 pik3ca h1047r lc2 ret ccdc6 pc9 egfr kelrea745k skmel28 braf v600e skmel28 egfr p753s sw48 egfr. The Ras Project: Overview The clinical need High incidence of RAS mutations in cancer No drugs that target RAS. DKOB8 cells were acquired (09/19/03) from Dr. For invitro use,ixazomibwasformulated inDMSOand diluted inmedia todesired concentration.