Cell Line LIM1215 Research Articles

Abstract 5770: Inhibition of KRAS G12D mutant with small molecules

Abstract Background: RAS mutations are occurring at a high rate in colorectal cancer, lung cancer, and pancreatic cancer. RAS is family of small GTPase proteins with important roles in cell growth, differentiation, proliferation, and survival. There are three types of RAS genes: HRAS, KRAS, and NRAS. The The RAS gene is the most commonly found oncogene in human cancer. KRAS exhibits a highest mutation rate compared to those of HRAS and NRAS in various cancer types. Methods: Colorectal cell line LIM1215, SW48 and NSCLC cell line NCI-H1975, NCI-H838 were used for MTT assays. Apoptosis assay was performed using the annexin V-FITC apoptosis detection kit (BioBud, Cat. LS-02-100). Colony forming assay was performed with 6-well culture plates by seeding 500 cells/well. The expression levels of proteins involved in Q2a-induced signaling pathway were determined by Western blot. In vivo study was performed with tumor xenografts. HT-29 cells were implanted into the right flank of 5-week-old BALB/c nude mice. Tumor size was determined by measuring the diameter (width, length) of the tumor every two days with a digital caliper. All group mice sacrificed at day 19. Results: Patients with cancer involving KRAS mutations are resistant to therapeutics that target epidermal growth factor receptor and show poor outcomes. In addition, effective treatments that target G12D in KRAS have not been developed. Thus, therapeutic agents targeting the G12D mutation in KRAS are needed. In this study, we identified the Q2a compound, a quinazoline-based derivative that specifically acts on the KRAS G12D mutation. This compound induced cell apoptosis by reducing phosphorylation of AKT in the PI3K pathway downstream of KRAS. Q2a compound specifically inhibited the growth of KRAS G12D mutant cells. In addition, cells with the KRAS G12D mutation were more sensitive to Q2a compound compared to the sensitivity of wild-type cells and those with other KRAS mutations, resulting in apoptosis. Treatment with the Q2a compound also decreased C-RAF phosphorylation in the MAPK pathway, which is downstream of KRAS. In KRAS G12D-mutated colorectal cancer xenografts, Q2a compound inhibited tumor production without significant liver toxicity. Conclusion: Q2a compounds show potential as therapeutic agents that target KRAS G12D mutations in colorectal and pancreatic cancers. Kyewords: Anti-Cancer Drug, KRAS G12D Mutant, apoptosis, colon cancerJi Yoon Lee, Dae-Hee Lee*Department of Marine Bio Food Science, Gangneung-Wonju national University Citation Format: Dae-Hee Lee, Ji Yoon Lee. Inhibition of KRAS G12D mutant with small molecules [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5770.

EGFR-targeted semiconducting polymer nanoparticles for photoacoustic imaging

Semiconducting polymer nanoparticles (SPN), formulated from organic semiconducting polymers and lipids, show promise as exogenous contrast agents for photoacoustic imaging (PAI). To fully realise the potential of this class of nanoparticles for imaging and therapeutic applications, a broad range of active targeting strategies, where ligands specific to receptors on the target cells are displayed on the SPN surface, are urgently needed. In addition, effective strategies for quantifying the level of surface modification are also needed to support development of ligand-targeted SPN. In this paper, we have developed methods to prepare SPN bearing peptides targeted to Epidermal Growth Factor Receptors (EGFR), which are overexpressed at the surface of a wide variety of cancer cell types. In addition to fully characterising these targeted nanoparticles by standard methods (UV–visible, photoacoustic absorption, dynamic light scattering, zeta potential and SEM), we have developed a powerful new NMR method to determine the degree of conjugation and the number of targeting peptides attached to the SPN. Preliminary in vitro experiments with the colorectal cancer cell line LIM1215 indicated that the EGFR-targeting peptide conjugated SPN were either ineffective in delivering the SPN to the cells, or that the targeting peptide itself destabilised the formulation. This in reinforces the need for effective characterisation techniques to measure the surface accessibility of targeting ligands attached to nanoparticles.

Angiotensin-converting Enzyme 2–containing Small Extracellular Vesicles and Exomeres Bind the Severe Acute Respiratory Syndrome Coronavirus 2 Spike Protein

Angiotensin-converting Enzyme 2–containing Small Extracellular Vesicles and Exomeres Bind the Severe Acute Respiratory Syndrome Coronavirus 2 Spike Protein

The State of The Jak/Stat Pathway Affects the Sensitivity of TumorCells to Oncolytic Enteroviruses

A test of the sensitivity of seven colon cancer cell lines to a panel of 12 nonpathogenic human enteroviruses revealed significant differences in the ability of tumor cells to become infected and replicate different viral strains. Among the factors that can affect the sensitivity of cells to viruses are differences in the state of the mechanisms of antiviral protection, associated with a reaction to type I interferons. Using the two colon cancer cell lines CaCo2 and LIM1215 as a model, significant differences were revealed in the ability of cells to defend themselves against virus infection after 16 hours of treatment with 1000 units/mL of interferon-alpha. To study the effect of the state of the interferon response system, represented by the Jak/STAT signaling pathway, on the sensitivity of cells to different strains of enteroviruses, HEK293T cell lines were used. These are capable of supporting replication of each of the tested enteroviruses, as well as maintaining the ability to protect against viral infection after the treatment with interferon. Using the CRISPR/Cas9 system, HEK293T sublines with knockouts of the IFNAR1 and STAT2 genes were obtained. The sensitivity of control and knockout cells to infection with five strains of enteroviruses and the vesicular stomatitis virus was analyzed. It was noted that knockout of the IFNAR1 and STAT2 genes resulted in an increased sensitivity to all tested viruses. In knockout cells, the levels of reproduction of the vaccine derived of poliovirus type 1, Echoviruses 7 and 30, and Coxsackie viruses B5 and A7 were also significantly increased in comparison with the control HEK293T cells. Thus, deficiencies in the Jak/STAT signaling pathway in tumor cells lead to an overall increase in the sensitivity to oncolytic viruses.

Methods of selecting combination therapy for colorectal cancer patients: a patent evaluation of US20160025730A1

ABSTRACTIntroduction: Colorectal cancer (CRC) is the fourth most common cancer worldwide. Targeted therapy drugs (TTDs) are a valid treatment, epithelial growth factor receptor (EGFR) inhibitors being one of the most commonly used for CRC patients. However, this treatment is only useful for patients with wild-type KRAS (wtKRAS) and is effective only on about 40 to 60% of this subset due to the high plasticity of ErbB network.Areas covered: The invention proposes the use of ErbB protein levels and ErbB receptor dimer formation as biomarkers for selecting, predicting and monitoring CRC patients showing sensitivity to the action of EGFR inhibitors to benefit from the combination therapy of EGFR and HER2 inhibitors. The in vitro data on Lim1215 cells suggest the over-activation of HER3 signaling pathway in response to the use of EGFR inhibitors on monotherapy; the use of HER2 or HER3 or MEK inhibitors in combination with EGFR inhibitors reversed this activation.Expert opinion: To assess the clinical applicability of this invention, further studies are needed since the conclusions are derived solely based on the data obtained from only one CRC cell line (Lim1215). Furthermore, other biofactors/mutations should be considered to assure the potential benefits of the combination therapies proposed.

Construction of a pIX-modified Adenovirus Vector Able to Effectively Bind to Nanoantibodies for Targeting

Current targeting strategies for genetic vectors imply the creation of a specific vector for every targeted receptor, which is time-consuming and expensive. Therefore, the development of a universal vector system whose surface can specifically bind molecules to provide efficient targeting is of particular interest. In this study, we propose a new approach in creating targeted vectors based on the genome of human adenovirus serotype 5 carrying the modified gene of the capsid protein pIX (Ad5-EGFP-pIX-ER): recombinant pseudoadenoviral nanoparticles (RPANs). The surfaces of such RPANs are able to bind properly modified chimeric nanoantibodies that specifically recognize a particular target antigen (carcinoembryonic antigen (CEA)) with high affinity. The efficient binding of nanoantibodies (aCEA-RE) to the RPAN capsid surfaces has been demonstrated by ELISA. The ability of the constructed vector to deliver target genes has been confirmed by experiments with the tumor cell lines A549 and Lim1215 expressing CEA. It has been shown that Ad5-EGFP-pIX-ER carrying aCEA-RE on its surface penetrates into the tumor cell lines A549 and Lim1215 via the CAR-independent pathway three times more efficiently than unmodified RPAN and Ad5-EGFP-pIX-ER without nanoantibodies on the capsid surface. Thus, RPAN Ad5-EGFP-pIX-ER is a universal platform that may be useful for targeted gene delivery in specific cells due to "nanoantibody-modified RPAN" binding.

Sulindac modulates secreted protein expression from LIM1215 colon carcinoma cells prior to apoptosis

Colorectal cancer (CRC) is a major cause of mortality in Western populations. Growing evidence from human and rodent studies indicate that nonsteroidal anti-inflammatory drugs (NSAIDs) cause regression of existing colon tumors and act as effective chemopreventive agents in sporadic colon tumor formation. Although much is known about the action of the NSAID sulindac, especially its role in inducing apoptosis, mechanisms underlying these effects is poorly understood. In previous secretome-based proteomic studies using 2D-DIGE/MS and cytokine arrays we identified over 150 proteins released from the CRC cell line LIM1215 whose expression levels were dysregulated by treatment with 1mM sulindac over 16h; many of these proteins are implicated in molecular and cellular functions such as cell proliferation, differentiation, adhesion, angiogenesis and apoptosis (Ji et al., Proteomics Clin. Appl. 2009, 3, 433–451). We have extended these studies and describe here an improved protein/peptide separation strategy that facilitated the identification of 987 proteins and peptides released from LIM1215 cells following 1mM sulindac treatment for 8h preceding the onset of apoptosis. This peptidome separation strategy involved fractional centrifugal ultrafiltration of concentrated cell culture media (CM) using nominal molecular weight membrane filters (NMWL 30K, 3K and 1K). Proteins isolated in the >30K and 3–30K fractions were electrophoretically separated by SDS-PAGE and endogenous peptides in the 1–3K membrane filter were fractioned by RP-HPLC; isolated proteins and peptides were identified by nanoLC-MS–MS. Collectively, our data show that LIM1215 cells treated with 1mM sulindac for 8h secrete decreased levels of proteins associated with extracellular matrix remodeling (e.g., collagens, perlecan, syndecans, filamins, dyneins, metalloproteinases and endopeptidases), cell adhesion (e.g., cadherins, integrins, laminins) and mucosal maintenance (e.g., glycoprotein 340 and mucins 5AC, 6, and 13). A salient finding of this study was the increased proteolysis of cell surface proteins following treatment with sulindac for 8h (40% higher than from untreated LIM1215 cells); several of these endogenous peptides contained C-terminal amino acids from transmembrane domains indicative of regulated intramembrane proteolysis (RIP). Taken together these results indicate that during the early-stage onset of sulindac-induced apoptosis (evidenced by increased annexin V binding, dephosphorylation of focal adhesion kinase (FAK), and cleavage of caspase-3), 1mM sulindac treatment of LIM1215 cells results in decreased expression of secreted proteins implicated in ECM remodeling, mucosal maintenance and cell–cell-adhesion. This article is part of a Special Issue entitled: An Updated Secretome.

Detection of cadherin-17 in human colon cancer LIM1215 cell secretome and tumour xenograft-derived interstitial fluid and plasma

Colorectal cancer (CRC), one of the most prevalent cancers in the western world, is treatable if detected early. However, 70% of CRC is detected at an advanced stage. This is largely due to the inadequacy of current faecal occult blood screening testing and costs involved in conducting population-based colonoscopy, the ‘gold standard’ for CRC detection. Another biomarker for CRC, carcinoembryonic antigen, while useful for monitoring CRC recurrence, is ineffective, lacking the specificity required early detection of CRC. For these reasons there is a need for more effective blood-based markers for early CRC detection. In this study we targeted glycoproteins secreted from the human colon carcinoma cell line LIM1215 as a source of potential CRC biomarkers. Secreted candidate glycoproteins were confirmed by MS and validated by Western blot analysis of tissue/tumour interstitial fluid (Tif) from LIM1215 xenograft tumours grown in immunocompromised mice. Overall, 39 glycoproteins were identified in LIM1215 culture media (CCM) and 5 glycoproteins in LIM1215 tumour xenograft Tif; of these, cadherin-17 (CDH17), galectin-3 binding protein (LGALS3BP), and tyrosine-protein kinase-like 7 (PTK7) were identified in both CM and glycosylation motifs. Swiss-Prot was used to annotate Tif. Many of the glycoproteins identified in this study (e.g., AREG, DSG2, EFNA1, EFNA3, EFNA4, EPHB4, ST14, and TIMP1) have been reported to be implicated in CRC biology. Interestingly, the cadherin-17 ectodomain, but not full length cadherin-17, was identified in CM, Tif and plasma derived from mice bearing the LIM1215 xenograft tumour. To our knowledge, this is the first report of the cadherin-17 ectodomain in plasma. In this study, we report for the first time that the presence of full-length cadherin-17 in exosomes released into the CM. This article is part of a Special Issue entitled: An Updated Secretome.

Abstract 705: Crosstalk with stromal fibroblasts contributes to EGFR inhibitor resistance in colorectal cancer cells

Abstract The mechanism for resistance to EGFR targeted therapies in colorectal cancer (CRC) with intact KRAS is still unknown. Host stromal cells contribute to tumorigenicity; however their effect on the sensitivity to EGFR targeted therapies has still to be determined. We hypothesized that crosstalk between stromal fibroblasts and CRC cancer cells contributes to cetuximab resistance via activation of the HGF/MET axis. Primary CRC associated fibroblasts (CAFs) were isolated and expanded from five surgical specimens. Fibroblasts were enriched by magnetic activated cell sorting (MACS®) and activated fibroblast lineage was confirmed by alpha smooth muscle actin (SMA) immunofluorescence. Supernatant HGF content and cellular mRNA expression levels in primary CRC CAFs, an embryonic lung fibroblast cell line (MRC-5), and a normal colon fibroblast cell line (CCD18) were determined by ELISA and quantitative RT-PCR. The effect of fibroblasts or HGF (75ng/ml) on the CRC cell lines LIM1215 and DiFi treated with cetuximab (5nM) for 72 hours was determined by co-culture using Transwells and WST-1 cell proliferation assays. Alterations in proliferation were compared by ANOVA. Mean fibroblast HGF secretion (8.3 ng/ml) and relative mRNA expression (2.0) could be augmented by co-culture with conditioned media (CM) from the CRC cell LIM1215 (14.1 ng/ml and 4.1, respectively) (Table). Further, co-culture of CRC cells with HGF, CCD18 cells, or primary CAFs reversed cetuximab-induced growth inhibition. LIM1215 proliferation was inhibited by cetuximab (55.3 ± 14.4%) which could be reversed by co-culture with either HGF (93.1 ± 10.2%) or CCD18 cells (107.2 ± 10.8%) (p<0.01 for both). Stromal-tumor interactions may play an important role in CRC resistance to cetuximab via the HGF/MET axis. Inhibition of both EGFR and MET may be necessary to realize the full potential of EGFR targeted therapies in CRC.Supernatant HGF levels and relative mRNA expression in fibroblasts treated with LIM1215 CM LIM1215 (Control)MRC5CCD18CAF3CAF4CAF5CAF6HGF (ng/ml) Ctrl<0.11.217.310.97.24.08.9LIM1215-CM<0.10.840.015.08.45.015.5 HGF mRNA Ctrl00.81.82.63.01.6 LIM1215-CM01.07.65.44.91.8 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 705.

Proteomics Analysis of A33 Immunoaffinity-purified Exosomes Released from the Human Colon Tumor Cell Line LIM1215 Reveals a Tissue-specific Protein Signature

Exosomes are 40-100-nm-diameter nanovesicles of endocytic origin that are released from diverse cell types. To better understand the biological role of exosomes and to avoid confounding data arising from proteinaceous contaminants, it is important to work with highly purified material. Here, we describe an immunoaffinity capture method using the colon epithelial cell-specific A33 antibody to purify colorectal cancer cell (LIM1215)-derived exosomes. LC-MS/MS revealed 394 unique exosomal proteins of which 112 proteins (28%) contained signal peptides and a significant enrichment of proteins containing coiled coil, RAS, and MIRO domains. A comparative protein profiling analysis of LIM1215-, murine mast cell-, and human urine-derived exosomes revealed a subset of proteins common to all exosomes such as endosomal sorting complex required for transport (ESCRT) proteins, tetraspanins, signaling, trafficking, and cytoskeletal proteins. A conspicuous finding of this comparative analysis was the presence of host cell-specific (LIM1215 exosome) proteins such as A33, cadherin-17, carcinoembryonic antigen, epithelial cell surface antigen (EpCAM), proliferating cell nuclear antigen, epidermal growth factor receptor, mucin 13, misshapen-like kinase 1, keratin 18, mitogen-activated protein kinase 4, claudins (1, 3, and 7), centrosomal protein 55 kDa, and ephrin-B1 and -B2. Furthermore, we report the presence of the enzyme phospholipid scramblase implicated in transbilayer lipid distribution membrane remodeling. The LIM1215-specific exosomal proteins identified in this study may provide insights into colon cancer biology and potential diagnostic biomarkers.

Secretome‐based proteomics reveals sulindac‐modulated proteins released from colon cancer cells

Although experiments in rodents and human population-based studies have demonstrated the efficacy of nonsteroidal anti-inflammatory drugs (NSAIDs) such as sulindac in colorectal cancer (CRC) prevention, a detailed knowledge of the underlying mechanism of action of this drug is limited. To better understand the chemopreventitive effects of sulindac, especially early sulindac-induced apoptotic events, we used the CRC cell line LIM1215 as an experimental model, focusing on proteins secreted into the LIM1215 culture medium - i.e., the secretome. This subproteome comprises both soluble-secreted proteins and exosomes (30-100 nm diameter membrane vesicles released by several cell types). Selected secretome proteins whose expression levels were dysregulated by 1 mM sulindac treatment over 16 h were analyzed using 2-D DIGE, cytokine array, Western blotting, and MS. Overall, 150 secreted proteins were identified, many of which are implicated in molecular and cellular functions such as cell proliferation, differentiation, adhesion, invasion, angiogenesis, metastasis, and apoptosis. Our secretome-based proteomic studies have identified several secreted modulators of sulindac-induced apoptosis action (e.g., Mac-2 binding protein, Alix, 14-3-3 isoforms, profilin-1, calumenin/Cab45 precursors, and the angiogenic/tumor growth factors interleukin 8 (IL-8) and growth related oncogene (GRO-α)) that are likely to improve our understanding of the chemopreventitive action of this NSAID in CRC.

Mechanisms of endothelin 1-stimulated proliferation in colorectal cancer cell lines

The peptide endothelin (ET) 1 promotes proliferation in a number of epithelial cancers. The aim of this study was to identify the mechanism of ET-1-stimulated proliferation in colorectal cancer cells in vitro. The effects of ET-1 on colorectal cancer cell lines HT29, LIM1215 and SW620 were studied. Cells were cultured with ET-1 plus antagonists/inhibitors to ET(A) or ET(B) receptors, G protein subtypes, phosphoinositide 3-kinase (PI3K) or protein kinase C (PKC). DNA replication and apoptosis were investigated by 5-bromo-2'-deoxyuridine incorporation and Annexin V staining. Transactivation of the epidermal growth factor (EGF) receptor was investigated by blockade of the receptor in the presence of ET-1, measurement of levels of phosphorylated EGF receptor in the presence of ET-1, and comparing the effects of ET-1 and EGF on cell proliferation. ET-1 significantly stimulated growth of all cell lines via ET(A) receptors. ET-1 stimulated DNA replication, not apoptosis. ET-1-stimulated growth was inhibited by antagonism of pertussis toxin-sensitive G proteins, PI3K and PKC. Inhibition of the EGF receptor reduced the effect of ET-1. ET-1 increased levels of phosphorylated EGF receptor via the ET(A) receptor. ET-1 increased DNA replication in colorectal cancer cells via the ET(A) receptor. This mitogenic action was mediated via pertussis toxin-sensitive G proteins, PI3K, PKC and transactivation of the EGF receptor.

PPARα agonists stimulate progastrin production in human colorectal carcinoma cells

The three subtypes of peroxisome proliferator activated-receptors (PPARα, δ and γ) control the storage and metabolism of fatty acids. Treatment of rats with the PPARα ligand ciprofibrate increases serum gastrin concentrations, and several lines of evidence suggest that non-amidated gastrins act as growth factors for the colonic mucosa. The aim of the present study was to investigate the expression of PPARs and the effect of PPAR ligands on gastrin production and cell proliferation in human colorectal carcinoma (CRC) cell lines. mRNAs for all three PPAR subtypes were detected by PCR in all CRC cell lines tested. The concentrations of progastrin, but not of glycine-extended or amidated gastrin, measured by radioimmunoassay in LIM 1899 conditioned media and cell extracts were significantly increased by treatment with the PPARα ligand clofibrate. Similar increases in progastrin were seen following treatment with the PPARα ligands ciprofibrate and fenofibrate, but not with bezafibrate, gemfibrozil or Wy 14643. The PPARγ agonist rosiglitazone had no significant effect on progastrin production. The PPARα ligand clofibrate also stimulated proliferation of the LIM 1899 cell line. We conclude that some PPARα ligands increase progastrin production by the human CRC cell line LIM 1899, and that clofibrate increases proliferation of LIM 1899 cells. These studies have revealed a relationship between PPARs and gastrin, two regulatory molecules implicated in the pathogenesis of CRC.

Strategies for the purification of laminin-10 for studies on colon cancer metastasis.

Signals from the epidermal growth factor (EGF) receptor family are thought to combine with integrin-dependent adhesion to laminins to contribute to disease progression and metastasis in cancer. To date, little is known about the mechanisms by which these signals interact. Recently, we have shown that the colon cancer cell line LIM1215 secretes and adheres to laminin-10 through multiple integrin receptors, and that EGF stimulates spreading and migration of these cells on the same substrate. Additionally laminin-10/11 has been shown by immunohistochemistry to be present at the invasive edge of moderately differentiated colon cancers. To enable detailed structure-function studies to be undertaken, it is important to be able to rapidly obtain highly purified native laminin-10 from bulk biological samples in reasonable yield. The development of a multidimensional micropurification scheme to achieve this is presented and compared with other reported methods for the purification of laminins.

A new method for determining the status of p53 in tumor cell lines of different origin.

The tumor suppressor p53 is the most frequently mutated gene in human tumors. In response to DNA damage, aberrant growth signals, or chemotherapeutic drugs, p53 is stabilized and induces apoptosis and/or cell cycle arrest. While the mechanisms of p53-dependent apoptosis are not well understood, p53-dependent cycle arrest is primary mediated by the CDK inhibitor p21. p53 is a transcriptional activator and it is not surprising that a majority of p53 mutations occur in the core DNA binding domain and affect DNA binding and transactivation of p53 targets in tumors. We used the capability of p53 to activate transcription for developing a new assay that permits rapid determination of the status of p53 in cancer cell lines of different origin. Our strategy involved using a retrovirus containing a p53-regulated lacZ reporter gene that was introduced into colon and breast tumor cell lines to determine p53 status. Simple staining for beta-galactosidase allowed us to confirm that the colon cancer cell lines LIM1215 and HCT116, as well as the breast cancer cell line MCF7. have wild-type p53, and the colon cancer cell line Caco-2 as well as breast cancer cell lines MDA-MB-435 and MDA-MB-231 have mutant p53. This method may be applied to novel cell lines of any origin with unknown status of p53.

Continuous free-flow electrophoresis separation of cytosolic proteins from the human colon carcinoma cell line LIM 1215: a non two-dimensional gel electrophoresis-based proteome analysis strategy.

The conventional approach for analyzing the protein complement of a genome involves the combination of two-dimensional gel electrophoresis (2-DE) and mass spectrometric based protein identification technologies. While 2-DE is a powerful separation technique, it is severely limited by the insolubility of certain classes of proteins (e.g. hydrophobic membrane proteins), as well as the amount of protein that can be processed. Here, we describe a simple procedure for resolving complex mixtures of proteins that involves a combination of free flow electrophoresis (FFE), a liquid-based isoelectric focussing (IEF) method, and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Resolved proteins were identified by peptide fragment sequencing using capillary column reversed-phase high performance liquid chromatography (RP-HPLC)/mass spectrometry (MS). An initial demonstration of the method was performed using digitonin/ethylenediaminetetraacetic acid EDTA extracted cytosolic proteins from the human colon carcinoma cell line, LIM 1215. Cytosolic proteins were separated by liquid-based IEF (pH range 3-10) into 96 fractions, and each FFE fraction was further fractionated by SDS-PAGE. Selected protein bands were excised from the SDS-PAGE gel, digested in situ with trypsin, and subsequently identified by on-line RP-HPLC/electrospray-ionization ion trap MS. Our results indicate that FFE is: (i) an extremely powerful liquid-based IEF method for resolving proteins; (ii) not limited by the amount of sample that can be loaded onto the instrument; and (iii) capable of fractionating intact protein complexes (a potentially powerful tool for cell-mapping proteomics). An up-to-date list of cytosolic proteins from the human colorectal carcinoma cell line LIM 1215 can be found in the Joint Protein Structure Laboratory (JPSL) proteome database. This information will provide an invaluable resource for future proteomics-based biological studies of colon cancer. The JPSL proteome database can be accessed through the World Wide Web (WWW) network (http://www.ludwig.edu.au/jpsl/jpslhome.html).

Continuous free-flow electrophoresis separation of cytosolic proteins from the human colon carcinoma cell line LIM 1215: A non two-dimensional gel electrophoresis-based proteome analysis strategy

The conventional approach for analyzing the protein complement of a genome involves the combination of two-dimensional gel electrophoresis (2-DE) and mass spectrometric based protein identification technologies. While 2-DE is a powerful separation technique, it is severely limited by the insolubility of certain classes of proteins (e.g. hydrophobic membrane proteins), as well as the amount of protein that can be processed. Here, we describe a simple procedure for resolving complex mixtures of proteins that involves a combination of free flow electrophoresis (FFE), a liquid-based isoelectric focussing (IEF) method, and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Resolved proteins were identified by peptide fragment sequencing using capillary column reversed-phase high performance liquid chromatography (RP-HPLC)/mass spectrometry (MS). An initial demonstration of the method was performed using digitonin/ethylenediaminetetraacetic acid EDTA extracted cytosolic proteins from the human colon carcinoma cell line, LIM 1215. Cytosolic proteins were separated by liquid-based IEF (pH range 3–10) into 96 fractions, and each FFE fraction was further fractionated by SDS-PAGE. Selected protein bands were excised from the SDS-PAGE gel, digested in situ with trypsin, and subsequently identified by on-line RP-HPLC/electrospray-ionization ion trap MS. Our results indicate that FFE is: (i) an extremely powerful liquid-based IEF method for resolving proteins; (ii) not limited by the amount of sample that can be loaded onto the instrument; and (iii) capable of fractionating intact protein complexes (a potentially powerful tool for cell-mapping proteomics). An up-to-date list of cytosolic proteins from the human colorectal carcinoma cell line LIM 1215 can be found in the Joint Protein Structure Laboratory (JPSL) proteome database. This information will provide an invaluable resource for future proteomics-based biological studies of colon cancer. The JPSL proteome database can be accessed through the World Wide Web (WWW) network (http://www.ludwig.edu.au/jpsl/jpslhome.html).

Laminin-10 Mediates Basal and EGF-Stimulated Motility of Human Colon Carcinoma Cells via α3β1 and α6β4 Integrins

Signals from the epidermal growth factor (EGF) receptor and integrin-dependent adhesion to laminin contribute to the progression and metastasis of colonic tumors. However, little is know about the mechanisms by which these signals cooperate. Recently, we have reported that the colon cancer cell line LIM1215 secretes and adhere to autocrine laminin-10 via multiple integrin receptors and that EGF stimulates spreading of these cells on the same substrate. In this report, we investigate the effect of EGF and laminin-10 on colon cancer cell migration in vitro. EGF stimulates migration of LIM1215 cells in a wound healing assay. The response to EGF is inhibited by anti-EGF receptor antibody 528, the EGF receptor kinase inhibitor AG-1478, or the MAP kinase kinase inhibitor PD98059 but not the PI3-K inhibitor wortmannin. Using Transwell migration chambers, we demonstrate that laminin-10 but not collagen-I, collagen-IV, or a commercial preparation of human placental laminin is a potent motility factor for LIM1215 cells. The migration response to laminin-10 is increased upon stimulation of the cells with EGF and correlates with the up-regulation of α6β4 integrin expression as measured by analysis of Triton X-100-soluble cellular extracts. The results from integrin inhibition experiments indicate that basal migration on laminin-10 is mediated by α3β1 but not α2β1 nor α6β4 integrins. Alpha3 blocking antibodies also inhibited EGF-stimulated chemokinetic migration of LIM1215 cells on laminin-10. However, in contrast to unstimulated cells, α6 or β4 integrin-blocking antibodies inhibited the migration of EGF-stimulated cells by up to 50%. Taken together, these results support the cooperative role of EGF receptor and laminin-10 on colon cancer cell motility and suggest a critical role for both the α3β1 and the α6β4 integrins in this process.

Colon Cancer Cells Adhesion and Spreading on Autocrine Laminin-10 Is Mediated by Multiple Integrin Receptors and Modulated by EGF Receptor Stimulation

Epidermal growth factor (EGF) receptor ligands such as EGF and transforming growth factor-α (TGF-α) play an important role in controlling the proliferation, survival, morphology, and motility of colonic epithelial cells. There is also increasing evidence that growth factors and extracellular matrix (ECM) proteins cooperate to regulate these cellular processes. We have reported previously that autocrine TGF-α and an unidentified ECM protein in the serum-free conditioned medium of the human colon carcinoma cell line LIM1215 synergize to induce spreading of these cells in low-density cultures. We have now purified the ECM protein secreted by LIM1215 cells and show that it synergizes with EGF to induce spreading of LIM1215 cells and other human cell lines from the colon and other tissues. The purified ECM migrated as a single protein band with an apparent molecular mass of approximately 800 kDa on SDS–PAGE under nonreducing conditions and, under reducing conditions, as three protein bands of approximately 360, 210, and 200 kDa. Immunoblotting experiments and mass spectrometry analysis of tryptic digests on the purified protein identified the 360-, 210-, and 200-kDa protein bands as laminin α5, β1, and γ1 chains, respectively, indicating that LIM1215 cells secrete laminin-10 (α5β1γ1). In serum-free medium, LIM1215 cells adhere to laminin-10 primarily via α2β1 and α3β1 integrin receptors. EGF-induced spreading of LIM1215 cells on laminin-10 is partially inhibited by pretreatment of the cells with blocking antibodies directed against integrin α3 or β1 but not α2, α6, or β4 subunits. Spreading is almost completely inhibited by blocking α3 + α2, α3 + α6, or β1 + β4 integrin chains and results in cell death. Increased spreading in the presence of EGF correlates with up-regulation of α6β4 integrins in these cells after exposure to EGF. These results indicate that colon cancer cells attach and spread on laminin-10 via multiple integrin receptors and suggest a critical role for α3β1 integrins in the spreading response. Together, our results support the concept that the adhesive properties of colon cancer cells are modulated by autocrine production of TGF-α and laminin-10 and autocrine induction of appropriate integrins.

Intracellular Zinc Depletion Induces Caspase Activation and p21Waf1/Cip1Cleavage in Human Epithelial Cell Lines

To better understand the mechanisms by which zinc deficiency induces epithelial cell death, studies were done of the effects of intracellular zinc depletion induced by the zinc chelator TPEN on apoptosis-related events in human malignant epithelial cell lines LIM1215 (colonic), NCI-H292 (bronchial), and A549 (alveolar type II). In TPEN-treated cells, depletion of zinc was followed by activation of caspase-3 (as demonstrated by enzymatic assay and Western blotting), DNA fragmentation, and morphologic changes. Increase in caspase-3 activity began 12 h after addition of TPEN, suggesting that zinc may suppress a step just before the activation of this caspase. Caspase-6, a mediator of caspase-3 processing, also increased, but later than caspase-3. Effects of TPEN on apoptosis were completely prevented by exogenous ZnSO4 and partially prevented by peptide caspase inhibitors. A critical substrate of caspase-3 may be the cell cycle regulator p21Waf1/Cip1, which was rapidly cleaved in TPEN-treated cells to a 15-kDa fragment before further degradation.