Sw48 Kras

HT-29 cells are WT for KRAS. KRAS WT SW-48 (SW48 [SW48] (ATCC ® CCL231TM)) and KRAS-Mut CRC cells SW480 ([SW480] (ATCC ® CCL228TM)) colorectal cancer cell lines (CRC cell lines) were obtained from American Type Culture Collection (ATCC). 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. It was reported that 5-aza-dC-induced reexpression of Mlh1, a MMR protein, restores sensitivity of SW48 CRC cells to 5-fluorouracil. For this purpose, SW48 cells were stably transduced with the same retroviral particles used to generate HCT116 stable miRNAs overexpressing cells, resulting in cells overexpressing miR-143 (SW48-miR-143) and miR-145 (SW48-miR-145), and the. Methods: Cells were incubated with IC50 (0. Deals with the establishment, characterization, deposition, storage, and distribution of cell lines, management of a database, and holds related workshops, and conferences. A, SW48-KRas G12D cells were serum starved (0% FBS) over the final 18 hours in the presence of fatty acid and stimulated with EGF (25 ng/mL) for 5 minutes, then immediately fixed in ice-cold 100% methanol. Giacomini a,c , Stephanie Huang a ,. RESEARCH ARTICLE KRAS Genotype Correlates with Proteasom Inhibitor Ixazomib. In contrast, in KRAS-mutant CRC cells (DLD-1G13D and SW480G12V), KRAS knocked-down by KRAS-siRNA led to ERCC1 upregulation and increased oxaliplatin resistance. 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. SW480 was established from a primary adenocarcinoma of the colon. Parise2, Edward Chu1,2 and John C. 7 Additional mutations ALK P1543S 50. f miR-193a-3p expression was decreased in the KRAS/BRAF-wild-type SW48 and DiFi cells, but not in the KRAS-mutant HCT8 cells and the BRAF-mutant LIM2405 cells 72 h after overexpression of mutant BRAF protein. Technologies) at 37°C in a humidified incubator with. G13D) but the ΔCq between the tested and the cross-reactive probe was superior to 10 and had no consequence on ?nal result. KRAS/BRAF WT cell lines exhibited limited to no inhibition of colony formation, using either U0126 (SW48 and SNU-C1) or CI-1040 (COLO-320-HSR and SNU-C1) (Figs. One notable example of specific up-regulation in KRAS codon 12 mutant SW48 cells is provided by the short form of the colon cancer stem cell marker doublecortin-like Kinase 1 (DCLK1) that can be reversed by suppression of KRAS. 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. SW48-WT RAS, SW48-KRAS G12V, and SW48 KRAS G13D cells were cultured in T-75 adherent culture flasks. 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) that are cetuximab-sensitive, as well as in their derived cells with acquired resistance to cetuximab (GEO-CR and SW48-CR). Tan, Cong; Du, Xiang. We evaluated the antitumor activity of selumetinib (AZD6244, ARRY-142886), a potent and selective MEK1/2 inhibitor, on a panel of colorectal carcinoma (CRC) cells and found no inhibition of KRAS mutant CRC cell anchorage-independent. As for KRAS, five supplementary mutations were detected (one p. 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. The human CACO2 (KRAS, NRAS, BRAF and PIK3CA WT), colon cancer cell line was obtained from Dr. cetuximab, whereas two cells (GEO and SW48) were highly sensitive. Sorafenib Overcomes Irinotecan Resistance in Colorectal Cancer by Inhibiting the ABCG2 Drug-Efflux Pump Thibault Mazard1, Annick Causse2, Joelle Simony5, Wilhem Leconet2, Nadia Vezzio-Vie2, Adeline Torro2, Marta Jarlier3, Alexandre Evrard6, Maguy Del Rio2, Eric Assenat1, Pierre Martineau2, Marc Ychou4, Bruno Robert 2, and Celine Gongora Abstract. Transfer contents into a tube containing pre-warmed media. 18 Generated the multiplex standards using the same background (RKO or SW48) Base mutations present at >10% allelic frequency Can be titrated up or down to analyse LOD of platform/panel Gene Variant Freq % Core mutations KRAS G12D 16. Our digital PCR assays are hydrolysis probe–based assays that were designed by experts in the digital PCR field. SW48 and HCT116 cells were maintained by implantation into the right axilla of nude mice at 3-week intervals. Because cMet was upregulated in a colon cancer cell line (SW48) expressing KRAS G12D, Narvaez, et al investigated the combination of MET knockdown and cMet inhibition with Poloppin. 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). 過去の報告(Cancer Res 2008;68(6):1953-61)に基づき、Kras wildな感受性群(LIM1215、CAC02、SW948)、非感受性群(RKO、SW48、HT29)に分類し、スタンフォード大学が運営するGenePatternというマイクロアレイ解析ソフトを使用し、cetuximab感受性を反映したheat mapおよびgene signatureを. Schmitz1,2*. 8µM for 5-FU, 0. Effect of Simvastatin on Cetuximab Resistance in Human Colorectal Cancer With KRAS Mutations JNCI Journal of the National Cancer Institute , Apr 2011 Jeeyun Lee , Inkyoung Lee , Boram Han , Joon Oh Park , Jiryeon Jang , Chaehwa Park , Won Ki Kang. Sonic hedgehog pathway activation is associated with cetuximab resistance and EPHB3 receptor induction in colorectal cancer. Parental (KRAS wild-type) SW48 cells were engineered using rAAV-mediated homologous recombination to create a panel of clones harbouring different mutant KRAS variants at the endogenous locus, but otherwise shared the same. 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. Forster a , Craig P. In the present work, we have developed a robust internal control for methylation-specific ddPCR, the 4Plex. 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. Activity of regorafenib was evaluated in isogenic SW48 KRAS wild-type (WT) and mutant cells. Deals with the establishment, characterization, deposition, storage, and distribution of cell lines, management of a database, and holds related workshops, and conferences. Cells were grown in growth medium alone or growth medium with cetuximab (20 μg/ml) for 48 hours. 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. Expression of SLC25A22 was knocked down in KRAS mutant CRC cell lines (DLD1, HCT116, LOVO, SW480, SW620, and SW1116) and CRC cell lines without mutations in KRAS (CACO-2, COLO205, HT29, and SW48); cells were analyzed for colony formation, proliferation, glutaminolysis and aspartate synthesis, and apoptosis in Matrigel and polymerase chain. AML is a malignant disease in which hematopoietic precursors are arrested in an early stage of development. In SW48 cells, exon 2 mutations of the KRAS gene may influence antitumor effects of regorafenib. Unlike HRAS, the KRAS DNA coding sequence has a high frequency of rare codons, resulting in poor KRAS protein translation and expression (Lampson et al. SW48 is a human colon adenocarcinoma cell line expressing mutant G719S EGFR. Cell cycle profiles of SW48 KRAS WT and p. 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. cetuximab, whereas two cells (GEO and SW48) were highly sensitive. View Lab Report - KRAS Genotype Correlates with Proteasome from DEPARTMENT BIOL3704 at Mid America Nazarene University. The percentage of animals developing lymph node metastasis was higher in KRas G12V than in KRas G13D mice. 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. A similar but greater effect is also observed in murine pancreatic organoids expressing mutant oncogenic Kras compared with controls ( Figure 7 E). 6) Cetuximab+MEKi 30 (27. Selection of KRAS exon 2 wild-type for inclusion in the FIRE-3 trial as well as lack of testing for KRAS exon 3-4 and NRAS exon 2-4 in AIO KRK 0604 and RO91 may have contributed to this result. The human SW48 (catalogue number: HTL99020) (KRAS, NRAS, BRAF and PIK3CA WT), colon cancer cell line was obtained from IRCCS “Azienda Ospedaliera Universitaria San Martino-IST Istituto Nazionale per la Ricerca sul Cancro, Genova” Italy. 3 days following infection, protein lysates were prepared and probed with the indicated antibodies. Human colon cancer cells HT-29 (KRAS wt, BRAF V600E, TP53 R273H), RKO (KRAS wt, BRAF V600E, TP53 wt), and SW48 (KRAS wt, BRAF wt, TP53 wt), and normal human colon epithelial CCD841 CoN cells were purchased from American Type Culture Collection (ATCC; Rockville, MD). The in vitro data suggest that the SNU175 (KRAS A59T) cell line has a higher proliferative fraction in the face of cetuximab treatment versus the KRAS wild-type human CRC cell lines DiFi and SW48, but direct statistical comparisons of these groups treated with equivalent drug concentrations were not performed. Propidium iodide (PI) flow cytometry plots. Molecular mechanisms of resistance to anti EGFR based therapies in colorectal cancer. 7 Additional mutations ALK P1543S 50. A Multicenter Blinded Study Evaluating EGFR and KRAS Mutation Testing Methods in the Clinical NoneSmall Cell Lung Cancer SettingdIFCT/ERMETIC2 Project Part 1 Comparison of Testing Methods in 20 French Molecular Genetic National Cancer Institute Platforms. Bailey, 1,2 Le Zhan, 2,10 Dipen Maru, 3 Imad Shureiqi, 4 Curtis R. It is evident that KRAS detaches from plasma membrane and translocates to the cytoplasm when treated with simvastatin. To investigate the impact of different KRAS mutations on treatment with the tyrosine kinase inhibitor sunitinib in SW48 colorectal cancer cell line variants. 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. 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. This demonstrated that these two phenotypes were indeed related to Ras. PubMed Central. Subcutaneous xenografts (KRAS WT and G12C mutant variants) in NOD/SCID mice were analyzed to elucidate the effect of regorafenib treatment in vivo. isogenic human cell lines comprising mutated cancer alleles and process using the cell lines. Patrick Casey (Duke Univer-. 方法选择人源的细胞株 kras和braf野生的sw48,kras突变的 lovo ,kras突变且不表达表皮生长因子受体(egfr)的sw620,braf突变的ht29,实时荧光定量pcr(rt‐pcr)测定egfr基因转录水平,分别用5×. (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). Cell culture. 3 Of 130 cell lines with KRas variants, 35 showed KRas variant DNA read frequencies consistent with homozygous mutant K-ras status. KRAS mutations occur in one third of human cancers and cluster in several hotspots, with codons 12 and 13 being most commonly affected. , Report on the clinical utility of reflex testing of resected stage I through III lung adenocarcinomas for EGFR and KRAS mutatations. 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. Human colon cancer cells HT-29 (KRAS wt, BRAF V600E, TP53 R273H), RKO (KRAS wt, BRAF V600E, TP53 wt), and SW48 (KRAS wt, BRAF wt, TP53 wt), and normal human colon epithelial CCD841 CoN cells were purchased from American Type Culture Collection (ATCC; Rockville, MD). E: KRAS exon 2, p. Relative phosphorylation. We tested 3 clinically relevant oncogenic drivers of the RAS-RAF pathway; BRAF V600E, KRAS G12V and the kinase dead form of BRAF (D594A) which cooperates with KRAS G12V and CRAF. Results are normalized to growth of saline-treated cells and are represented as mean ± SD of at least two independent experiments. 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). Mice were randomized to IgG or cetuximab and treated twice weekly with 0. 5 weeks based on relative tumor growth rates. SW48 ARM Median survival (95% CI) in weeks Cetuximab 10 (7. Cell Culture Revival Rapidly thaw cells in a 37°C water bath. Animals were treated with bevacizumab and erlotinib, alone or in combination, and the influence on tumor growth, and the presence of activated EGFR. The percentage of animals developing lymph node metastasis was higher in KRas G12V than in KRas G13D mice. Medium was removed, and cells were washed with ice-cold tris-buffered saline. Methodology. Try again. Customer Service. In the present work, we have developed a robust internal control for methylation-specific ddPCR, the 4Plex. A Multicenter Blinded Study Evaluating EGFR and KRAS Mutation Testing Methods in the Clinical NoneSmall Cell Lung Cancer SettingdIFCT/ERMETIC2 Project Part 1 Comparison of Testing Methods in 20 French Molecular Genetic National Cancer Institute Platforms. Technologies) at 37°C in a humidified incubator with. Parise2, Edward Chu1,2 and John C. View Lab Report - KRAS Genotype Correlates with Proteasome from DEPARTMENT BIOL3704 at Mid America Nazarene University. 03 [Poloppin]μM % Survival Normalised to DMSO GI50 (μM) A B C Figure 2. Models for testing the efficacy of anti-cancer agents. f miR-193a-3p expression was decreased in the KRAS/BRAF-wild-type SW48 and DiFi cells, but not in the KRAS-mutant HCT8 cells and the BRAF-mutant LIM2405 cells 72 h after overexpression of mutant BRAF protein. 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. METHODS: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. It is well known that RASA1 and its downstream proteins play crucial roles in the regulation of cellular growth and differentiation, down-regulation of RASA1 increases cell proliferation. to KRAS status and to investigate the prognostic and predic-tive value of Cdk5 in representative patients cohorts. 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) that are cetuximab-sensitive, as well as in their derived cells with acquired resistance to cetuximab (GEO-CR and SW48-CR). The lack of testing in these two studies might cause a small negative bias on outcome of patients with unmutated tumors. To determine the frequency of WT K-ras allele loss in human tumor cell lines, we assessed the K-ras mutant allele frequency in a panel of 624 cell lines which had been subjected to exome-capture and deep sequencing. Horizon DNA standards eliminate the variability associated with patient-derived reference standards, and avoid the hassle of sourcing, characterizing, and documenting your own cell line mixes. 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. We had a strong interest in this mutation and wanted to evaluate whether our hypothesis and experimental model could be applied to p. Synergistic interaction between CENP-E inhibitor GSK923295 cell lines WT WT 35G>T Modest Modest Synergy SW620 WT WT WT Synergy Synergy Synergy SW48 1799T>A 3140A>G WT Synergy Synergy Synergy RKO WT 1633G>A 38G>A Synergy Synergy Modest HCT15 WT 3140A>G 38G>A Synergy Synergy HCT116 BRAF PIK3CA KRAS BLISS EOHSA CI Cell Line WT WT 35G>T Modest Modest Synergy SW620 WT WT WT Synergy Synergy. TET1 in CRCs Ichimura N et al. endogenous locus, a colorectal cancer cell line SW48 which evolvedintheabsenceofmutantRas,andnontransformedbut immortalizedHMECs(15). However the method to categorize WT vs. 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. 8µM for 5-FU, 0. Cell Culture Revival Rapidly thaw cells in a 37°C water bath. Subcutaneous xenografts (KRAS WT and G12C mutant variants) in NOD/SCID mice were analyzed to elucidate the effect of regorafenib treatment in vivo. v Differential Activity of the KRAS Oncogene by Method of Activation: Implications for Signaling and Therapeutic Intervention By Nathan Ihle B. Cancer mutations and targeted therapies in cells, mice and patients. In contrast, in KRAS-mutant CRC cells (DLD-1G13D and SW480G12V), KRAS knocked-down by KRAS-siRNA led to ERCC1 upregulation and increased oxaliplatin resistance. 過去の報告(Cancer Res 2008;68(6):1953-61)に基づき、Kras wildな感受性群(LIM1215、CAC02、SW948)、非感受性群(RKO、SW48、HT29)に分類し、スタンフォード大学が運営するGenePatternというマイクロアレイ解析ソフトを使用し、cetuximab感受性を反映したheat mapおよびgene signatureを. To confirm that KRAS mutation impacts response to ixazomib, we used SW48 isogenic colon cancer cell lines, in which a KRAS-G13D mutation was introduced into KRAS-WT SW48 cells to generate stable SW48-KRAS-G13D cells. KRAS G12V or G13D and chemotherapy in cellular models. by SW48 cells (Figure 2B), suggesting the anti-tumor effect of cetuximab against tumors harboring EGFR G719S mutant. 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. 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. • Top-down of KRAS from colorectal cancer cell lines, patient samples, and SW48 PAR, SW48 G13D, SW48 G12D, NCI -H1792, Subjects 1-6 249915. Although SW48-KRAS mut and SW620 cells also had increased LGR5 expression , KRAS mut was dominant in activating the embryonic SC-like program. Molecular and Cellular Pathobiology Colorectal Cancer Cell Lines Are Representative Models of the Main Molecular Subtypes of Primary Cancer Dmitri Mouradov1,2,4, Clare Sloggett5, Robert N. 6) Cetuximab+MEKi 30 (27. To confirm this observation, we evaluated the role of the G13D allele using a panel of SW48 isogenic cell lines harboring a KRAS G12C, KRAS G12D, or KRAS G13D mutation. Interacts (via N-terminus) with RGS14 (via RBD domains); the interaction mediates the formation of a ternary complex with RAF1, a ternary complex inhibited by GNAI1 (By similarity). Then we screen for each line KRAS mutations in codons 12 and 13 and BRAF mutations at codon 600 per pyrosquen lacing. (2002) employed a somatic cell gene targeting strategy to show that β‐catenin expression was essential for clonal growth of the APC mutant and β‐catenin mutant colorectal cancer lines, DLD-1 and SW48, respectively. sw48 [sw-48] (atcc ® ccl-231 ™) Organism: Homo sapiens , human / Tissue: colon / Disease: Dukes' type C, grade IV, colorectal adenocarcinoma. Schettere,. Methods: 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. (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. The Caco2, RKO and SW48 cell lines, which harbour wild-type KRAS, were used for comparative purposes. SW48, DLD-1 KRAS wild-type cell lines and DLD-1 xenograft models exposed to cetuximab, oxaliplatin, or oxaliplatin + cetuximab (control [saline]; n = 3 mice per treatment group) were used. References for E3LZ10. Leibovitz, et al. 1158/1078-0432. I discovered a set of cell lines that were resistant to the ATP-competitive mTOR inhibitor PP242 and identified a defect in inhibition of 4E-BP1 phosphorylation as giving rise to. was effective as a single agent or in combination with zoledronic acid (ZOL) in human CRC cell lines with different KRAS sta-tus. knocked down in KRAS mutant CRC cell lines (DLD1, HCT116, LOVO, SW480, SW620, and SW1116) and CRC cell lines without mutations in KRAS (CACO-2, COLO205, HT29, and SW48); cells were analyzed for colony formation, proliferation, gluta-minolysis and aspartate synthesis, and apoptosis in Matrigel and polymerase chain reaction array analyses. When unstimulated NK cells were used as effectors, SW48 lysis increased from 38% without cetuximab pre-coating to 50% with cetuximab pre-coating (E/T ratio of 50:1, p<0. Kulemann, Birte; Liss, Andrew S; Warshaw, Andrew L; Seifert, Sindy; Bronsert, Peter. 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. Split at 70-80% confluency, approximately 1:3-1:6. Cell Culture. profound synthetic lethaleffects, namelyamedianZscoreacross thep. Centrifuge cells and seed into a 25cm2 flask containing pre-warmed media. 2) Cetuximab+PI3Ki 19 (14. 0 EGFR G719S 33. Tan, Cong; Du, Xiang. KRAS exon 2 mutations influence activity of regorafenib in an SW48-based disease model of colorectal cancer. 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. The KRAS oncogene is mutated in approximately 35%-45% of colorectal cancer. Our interests include the mechanisms by which cells respond to external signals that regulate their proliferation and survival, in particular the signalling pathways along which information is transferred leading from cell surface receptors to events in the cell nucleus, and how these are altered during the process of malignant transformation. Consequently, dual targeting of RSK and Akt efficiently inhibited cell proliferation in KRAS(G13D)-mutated HCT116 and KRAS wild-type SW48 cells. 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). 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. 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. To determine the frequency of WT K-ras allele loss in human tumor cell lines, we assessed the K-ras mutant allele frequency in a panel of 624 cell lines which had been subjected to exome-capture and deep sequencing. (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). growth assays. The average cell number was measured by determining ATP content in four replicate wells. lines are sorted and colored according to mutations in the BRAF/KRAS, PIK3CA/PTEN and TP53 cancer genes. Is the SW480 cell line mutated on K-RAS? If yes, what large intestine adenocarcinoma cell line can I use with a non mutated K-Ras? SW48 and CACO-2 are both KRAS wild type. Because the glycine (G)-to-aspartate (D) transition mutation is the most frequent codon 13 mutation in colorectal cancer, 5 we studied the association of this p. All cell lines were supple-mented with 10% fetal bovine serum (Life Technologies) and antibiotic–antimycotic reagent (Life Technologies) at 37 Cina humidified incubator with 5% CO 2. 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. therapies exist. It was isolated from the tissue of a 51-year-old Caucasian male (blood group A, Rh+) as was SW480 ( ATCC CCL-228). 8% before knockdown), SW48 yielded proliferation rate of 46. The lack of testing in these two studies might cause a small negative bias on outcome of patients with unmutated tumors. Tricarico, Rossella; Bet, Paola; Ciambotti, Benedetta; Di G. We used both in vitro (the HCT116, SW48, SW620 and HT29 colon adenocarcinoma cell lines and four SN-38 resistant HCT-116 and SW48 clones) and in vivo models (nude mice xenografted with SN-38 resistant HCT116 cells) to test the efficacy of sorafenib alone or in combination with irinotecan, or its active. SW48, RKO, and HT29 cells. It has been suggested that the position and type of amino. 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. 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) that are cetuximab-sensitive, as well as in their derived cells with acquired resistance to cetuximab (GEO-CR and SW48-CR). We have used rAAV‐mediated homologous recombination to generate suites of SW48 and LIM1215 colorectal cancer cells which harbor one of 7 different K‐Ras G12 or G13 mutations. The Student's t test was used to analyze statistical differences. isogenic human cell lines comprising mutated cancer alleles and process using the cell lines. Isogenic SW48 KRAS wt, G12A, G12C, G12D, G12R, G12S, G12 V, and G13D cells were evaluated for ERK phosphorylation with and without EGF stimulation. Although SW48-KRAS mut and SW620 cells also had increased LGR5 expression , KRAS mut was dominant in activating the embryonic SC-like program. A Multicenter Blinded Study Evaluating EGFR and KRAS Mutation Testing Methods in the Clinical Non–Small Cell Lung Cancer Setting—IFCT/ERMETIC2 Project Part 1 Comparison of Testing Methods in 20 French Molecular Genetic National Cancer Institute Platforms. 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. PubMed Central. Transfer contents into a tube containing pre-warmed media. SW48 is a human colon adenocarcinoma cell line expressing mutant G719S EGFR. SW48 andSW48-KRAS-G13Dand SW48-KRAS-G12Vcellswereobtainedfrom HorizonDis-coveryLtd. KRAS WT SW-48 (SW48 [SW48] (ATCC ® CCL231TM)) and KRAS-Mut CRC cells SW480 ([SW480] (ATCC ® CCL228TM)) colorectal cancer cell lines (CRC cell lines) were obtained from American Type Culture Collection (ATCC). KRAS遺伝子変異を持ったがんを標的とした新規のアルキル化剤の開発について 平成27年4月27日 は、 1 論文発表の概要 (1)研究論文 ¡:Inhibition of KRAS mutant using a novel DNA-alkylating Pyrrole-Imidazole polyamide conjugate targeting Codon 12 Mutant DNA. A, Proliferation of wild-type and KRAS mutated SW48 cells was assessed in the presence of cetuximab. 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. Here we report the comparative and nucleotide-dependent interactomes of KRas4a and KRas4b. mutations in codon 12 or 13 of KRAS and 19 contained the V600E BRAF mutation. Language Label Description Also known as; English: SW48 KRAS (G12V/+) cell line. Differences in the signaling functions of the two KRas proteins are poorly understood. 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 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. KRAS mutation testing in metastatic colorectal cancer. , Report on the clinical utility of reflex testing of resected stage I through III lung adenocarcinomas for EGFR and KRAS mutatations. Human colon cancer cells HT-29 (KRAS wt, BRAF V600E, TP53 R273H), RKO (KRAS wt, BRAF V600E, TP53 wt), and SW48 (KRAS wt, BRAF wt, TP53 wt), and normal human colon epithelial CCD841 CoN cells were purchased from American Type Culture Collection (ATCC; Rockville, MD). , The presence of KRAS mutations and loss of PTEN expression were not associated with impaired response to cetuximab-based. Patrick Casey (Duke Univer-. Corresponding C911 siRNA pools were included as rescue controls. Mol Cancer. Among the cells with wild‐type KRAS, SW48 cells showed the highest EGFR and p‐EGFR expression levels. Expression of the genes EPDR1 and ZNF518B was negligible in the Caco2, RKO and SW48 cell lines, which possess wild-type KRAS, while the HCT116, DLD1 and D-Mut1 cell lines, harbouring the G13D mutation, expressed these genes. Statistical tests were two-sided. 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. KRAS exon 2 mutations influence activity of regorafenib in an SW48-based disease model of colorectal cancer. Cell viability assays showed that two compounds, Y11 and Y17 ( Figure 1 B), had a significantly different core structure than the parent compound, 3W2R ligand. We also generated SW48 cells stably transfected with different K-RAS mutation. Viability assays and the apoptotic analysis of cells show that SW48 cells maintain high cell viability and low apoptotic signal for 24 hours in serum-free media, which is not different than that obtained on the. Another study demonstrated CDX2 as a “lineage-survival” oncogene which supports the proliferation and survival of colorectal cancers cells when deregulated. 04µM for Ox, 25 µg/mL for Cet, and C6-Cer concentrations ranged from 5 to 10 µM). Therefore, the A59T mutation was. Bevacizumab-erlotinib as maintenance therapy in metastatic colorectal cancer. The other sample was a KRAS exon 3 mutation (A59E), which is a mutation outside the detectable range of the RASKET KIT. ABT-263也称为Navitoclax是一种有效且口服生物可利用的Bcl-2家族抑制剂(Bcl-2,Bcl-xL和Bcl-w的Ki. Isogenic SW48 KRAS wt, G12A, G12C, G12D, G12R, G12S, G12 V, and G13D cells were evaluated for ERK phosphorylation with and without EGF stimulation. Asimismo, se propone una correlación significativa entre los casos de CCR con mutación en el codón 12 de gen KRAS y el estado de hipermetilación de este gen, por lo que se sugiere que, además de considerarse importante en el desarrollo del CCR, también podría ser útil como un biomarcador de metilación en este tipo de cáncer 39. The in silico results were experimentally validated by quantitative real?time PCR. The sensitivity of irinotecan and 5FU had not changed in the paired CRC cells. The results indicated that TRIM29 is highly expressed in SW620 cells and weakly expressed in RKO cells (Fig. Giacomini a,c , Stephanie Huang a ,. 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. Calcium for dogs and cats. 5 mg) 3 times a week for 2 wk once tumors had reached a mean tumor volume of 140 mm 3. The NRAS gene belongs to a class of genes known as oncogenes. The expression of KRAS protein as a predicted downstream target for miR-193a was studied by immunohistochemistry. Shackelford, Rodney E. It has been suggested that the position and type of amino. to KRAS status and to investigate the prognostic and predic-tive value of Cdk5 in representative patients cohorts. Methods: Cells were incubated with IC50 (0. SW48 is a human colon adenocarcinoma cell line expressing mutant G719S EGFR. PI3K mutational profiling. Cells were grown in growth medium alone or growth medium with cetuximab (20 μg/ml) for 48 hours. Characterization of KRAS and BRAF mutational status in the KM12 cell line was performed in a total reaction volume of 50 μl containing 1X AmpliTaq Gold 360 Master Mix (Life Technologies, Paisley, UK), 1 μM each primer and 100 ng of genomic DNA template using primer couples and conditions reported in Table S3. Isogenic SW48 KRas (G12D/þ) cells (RRID:CVCL_LC92) were obtained (10/08/14) from Horizon Discovery, where they were authenticated by gDNA and cDNA genotyping. We have used rAAV‐mediated homologous recombination to generate suites of SW48 and LIM1215 colorectal cancer cells which harbor one of 7 different K‐Ras G12 or G13 mutations. This enables the detection of low levels of protein, while also using less sample. Sonic hedgehog pathway activation is associated with cetuximab resistance and EPHB3 receptor induction in colorectal cancer. Error: Network error: undefined. 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. 39 With the exception of a KRAS mutation detected in. 1) MEKi 25 (21. 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. PubMed Central. Cell Culture Revival Rapidly thaw cells in a 37°C water bath. However, monolayer morphology is not the natural appearance of all the established cell lines. Box plot of the percentage of nonamplificable (NA) NSCLC paraffin-embedded samples recovered by alternative techniques of the 74 tested. The KRAS gene encodes two isoforms, KRas4a and KRas4b. Corresponding C911 siRNA pools were included as rescue controls. 0 EGFR G719S 33. 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. Six sections were analysed using each of the DNA extraction kits. Kaja C and for mutation hotspots in KRAS in contrast to SW48 and CL-11. 0 NOTCH1 P668S 50. endogenous mutant KRas, we analyzed the signaling and biological properties of a small panel of isogenic cell lines. FXR and small interfering KRAS (siKRAS) expression in human colon cancer cell lines and the predicted role of FXR in colon cancer development. The invention refers to an in vitro method for monitoring the response and resistance to a treatment of a patient suffering from an EGFR expressing tumor, with an anti-cancer agent against said tumor, the method comprising: determining the level of pEGFR in a sample taken from the patient selected from a blood sample, a blood derived sample, a secretome sample and a secretome derived sample. Animals were treated with bevacizumab and erlotinib, alone or in combination, and the influence on tumor growth, and the presence of activated EGFR. Of the 45 tumor samples, only one sample (2. The percentage of animals developing lymph node metastasis was higher in KRas G12V than in KRas G13D mice. Error: Network error: undefined. 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). Transfer contents into a tube containing pre-warmed media. It is evident that KRAS detaches from plasma membrane and translocates to the cytoplasm when treated with simvastatin. Of 656 tumours for which the other molecular alterations, i. 腫瘍細胞株コレクションより厳選した細胞株セットです。がん研究,関連因子スクリーニング,バイオマーカー,情報伝達経路の解析,および標的物質の開発などにおいて非常に有用なツールです。. cell line Gene Sets. 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. We’re committed to accelerating cancer research by pioneering improvements that may impact tumor detection and. It is evident that KRAS detaches from plasma membrane and translocates to the cytoplasm when treated with simvastatin. 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. Lung Cancer and the EGFR G719C Mutation. Try again. We had a strong interest in this mutation and wanted to evaluate whether our hypothesis and experimental model could be applied to p. For invitro use,ixazomibwasformulated inDMSOand diluted inmedia todesired concentration. RESEARCH ARTICLE KRAS Genotype Correlates with Proteasom Inhibitor Ixazomib. The line was derived from a metastasis. Cells may experience a lag period of approximately 1 week before stable growth is. 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). 3 Of 130 cell lines with KRas variants, 35 showed KRas variant DNA read frequencies consistent with homozygous mutant K-ras status. Effect of Simvastatin on Cetuximab Resistance in Human Colorectal Cancer With KRAS Mutations JNCI Journal of the National Cancer Institute , Apr 2011 Jeeyun Lee , Inkyoung Lee , Boram Han , Joon Oh Park , Jiryeon Jang , Chaehwa Park , Won Ki Kang. G12 mutant celllines (KRASWT/G12D,KRASWT/G12S and KRASWT/G12V)of -2(approximatelyequal toap<0. lines are sorted and colored according to mutations in the BRAF/KRAS, PIK3CA/PTEN and TP53 cancer genes. However the method to categorize WT vs. 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). 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 KRAS gene is frequently mutated in human cancer, especially in pancreatic, lung, and colorectal tumors. 5C, left) and DLD1-based (Fig. E: KRAS exon 2, p. SW48 andSW48-KRAS-G13Dand SW48-KRAS-G12Vcellswereobtainedfrom HorizonDis-coveryLtd. Citation: Davis SL, Robertson KM, Pitts TM, Tentler JJ, Bradshaw-Pierce EL, Klauck PJ, Bagby SM, Hyatt SL, Selby HM, Spreafico A, Ecsedy JA, Arcaroli JJ, Messersmith WA, Tan AC and Eckhardt SG (2015) Combined inhibition of MEK and Aurora A kinase in KRAS/PIK3CA double-mutant colorectal cancer models. Corning ® T-75 flasks (catalog #430641) are recommended for subculturing this product. Cell culture. 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. Patrick Casey (Duke Univer-. International audienceDespite recent advances in the treatment of colorectal cancer (CRC), tumor resistance is a frequent cause of chemotherapy failure. This material will help you understand: • the basics of lung cancer • the role of the EGFR gene in lung cancer • if there are any drugs that might work better if you have certain changes in the EGFR gene. Bevacizumab-erlotinib as maintenance therapy in metastatic colorectal cancer. 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. Taken together, we. KRAS codon 11 is located in the region of mutation detection probes immobilized KRAS exon 2 color-coded beads, so that the PCR amplification product with the codon 11 mutation did not bind even to G12C beads. Found in a complex with at least BRAF, HRAS, MAP2K1, MAPK3 and RGS14. Parental (KRAS wild-type) SW48 cells were engineered using rAAV-mediated homologous recombination to create a panel of clones harbouring different mutant KRAS variants at the endogenous locus, but otherwise shared the same. Evaluation of KRAS G13D CRC cell lines that have been transduced with NF1. We used both in vitro (the HCT116, SW48, SW620 and HT29 colon adenocarcinoma cell lines and four SN-38 resistant HCT-116 and SW48 clones) and in vivo models (nude mice xenografted with SN-38 resistant HCT116 cells) to test the efficacy of sorafenib alone or in combination with irinotecan, or its active. 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. Patient-derived CRC cells with WT Kras (CRC102 and CRC103) were obtained and maintained as described previously (Gao et al. fast, sensitive analysis of activated Ras GTPase. Defects in KRAS are the cause of Noonan syndrome 3 (NS3) [MIM:609942]. Cell Culture Revival Rapidly thaw cells in a 37°C water bath. The encoded protein can promote tumor formation by targeting tumor suppressor proteins, such as p53, for proteasomal degradation. 5 mg) 3 times a week for 2 wk once tumors had reached a mean tumor volume of 140 mm 3. Patrick Casey (Duke Univer-. 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. HCQ produces antitumor activity as a single agent in both genetically engineered mouse models and patient-derived xenografts of KRAS-mutant pancreas cancer. to KRAS status and to investigate the prognostic and predic-tive value of Cdk5 in representative patients cohorts. The influence of afatinib/gefitinib on cell viability and cell cycle was evaluated in isogenic SW48 KRAS wild-type/mutant cells. routinely used irinotecan-based chemotherapy. Medium was removed, and cells were washed with ice-cold tris-buffered saline. 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. The human colon cancer cell lines SW48 and HCT116 were purchased from the American Type Culture Collection (ATCC; Rockville, MD, uSA), and Dainippon Pharma (Osaka, Japan), respectively. G12C (H358). GeneArt Engineered Cell Models for cancer research Prevail with tools designed to help unravel the complexities of cancer cells Scientific advancements happening now can potentially advance our pursuit of life without cancer. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country. Mol Cancer. 3 days following infection, protein lysates were prepared and probed with the indicated antibodies. Asimismo, se propone una correlación significativa entre los casos de CCR con mutación en el codón 12 de gen KRAS y el estado de hipermetilación de este gen, por lo que se sugiere que, además de considerarse importante en el desarrollo del CCR, también podría ser útil como un biomarcador de metilación en este tipo de cáncer 39. The heterogeneity of human cancers is a major obstacle to developing therapies. Unlike HRAS, the KRAS DNA coding sequence has a high frequency of rare codons, resulting in poor KRAS protein translation and expression (Lampson et al. 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 invention refers to an in vitro method for monitoring the response and resistance to a treatment of a patient suffering from an EGFR expressing tumor, with an anti-cancer agent against said tumor, the method comprising: determining the level of pEGFR in a sample taken from the patient selected from a blood sample, a blood derived sample, a secretome sample and a secretome derived sample. Cell culture. cetuximab, whereas two cells (GEO and SW48) were highly sensitive. High resolution melting analysis of KRAS , BRAF and PIK3CA in KRASexon 2 wild-type metastatic colorectal cancer. Jorissen1,2,4, Christopher G. 5 EGFR L858R 12. He has obtained his PhD in the field of Molecular Pathology in School of Medicine, Griffith University. Genetic alterations in the KRAS and PIK3CA pathways are determinant of tumor cells' response to everolimus. Parise2, Edward Chu1,2 and John C.