LIM1215 Cells Research Articles

Design, construction, and in vitro analysis of A33scFv::CDy, a recombinant fusion protein for antibody-directed enzyme prodrug therapy in colon cancer

Antibody-directed enzyme-prodrug therapy (ADEPT) aims at improving the specificity of conventional chemotherapy by employing artificial antibody-enzyme constructs to convert a non-toxic prodrug into a cytotoxic agent specifically localized to the tumor site. The gpA33 antigen is a promising target for ADEPT in colon cancer, as it is expressed by >95% of human colon cancers, but is absent in all non-gastrointestinal tissues. We designed a recombinant fusion construct of a phage display-generated anti-gpA33 single chain fragment, A33scFv, with cytosine deaminase from yeast (CDy), which converts 5-fluorocytosine (5-FC) into 5-fluorouracil (5-FU). The resulting construct, A33scFv::CDy, was overexpressed in Pichia pastoris and secreted into culture supernatant. The fusion protein was purified by affinity chromatography on protein L. Silver-staining after SDS-polyacrylamide gel electrophoresis confirmed molecular mass and purity. Antibody binding and specificity were quantified by flow cytometry. The complete ADEPT system was applied in vitro on gpA33-positive LIM1215 cells, assessing cell survival by a fluorescein diacetate assay. Cytotoxicity of the prodrug 5-FC after A33scFv::CDy binding was equimolar to that of 5-FU, and this effect depended specifically on both antibody and enzyme function. These results demonstrate bifunctional activity of the heterogeneous Pichia-produced A33scFv::CDy fusion protein and proof of principle for the ADEPT system proposed herein.

Protoporphyrin IX Interacts with Wild-type p53 Protein in Vitro and Induces Cell Death of Human Colon Cancer Cells in a p53-dependent and -independent Manner

Photodynamic therapy (PDT) of cancer is an alternative treatment for tumors resistant to chemo- and radiotherapy. It induces cancer cell death mainly through generation of reactive oxygen species by a laser light-activated photosensitizer. It has been suggested that the p53 tumor suppressor protein sensitizes some human cancer cells to PDT. However, there is still no direct evidence for this. We have demonstrated here for the first time that the photosensitizer protoporphyrin IX (PpIX) binds to p53 and disrupts the interaction between p53 tumor suppressor protein and its negative regulator HDM2 in vitro and in cells. Moreover, HCT116 colon cancer cells exhibited a p53-dependent sensitivity to PpIX in a dose-dependent manner, as was demonstrated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and fluorescence-activated cell sorter (FACS) analysis of cell cycle profiles. We have also observed induction of p53 target pro-apoptotic genes, e.g. puma (p53-up-regulated modulator of apoptosis), and bak in PpIX-treated cells. In addition, p53-independent growth suppression by PpIX was detected in p53-negative cells. PDT treatment (2 J/cm2) of HCT116 cells induced p53-dependent activation of pro-apoptotic gene expression followed by growth suppression and induction of apoptosis.

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.

Stimulation of colorectal cancer cell line growth by ET-1 and its inhibition by ETA antagonists

BACKGROUNDThe vasoactive peptide endothelin 1 (ET-1) acts via two receptors, endothelin receptors A (ETA) and B (ETB). ET-1 is overexpressed by human cancers in vivo and in vitro and may...

Role of urokinase and its receptor in basal and stimulated colonic epithelial cell migration in vitro

BACKGROUNDMigration of colonic epithelial cells is important for mucosal repair following injury. The urokinase (u-PA) system regulates migration in other cell types.AIMTo examine the role of u-PA and its receptor...

Short-chain fatty acids reduce expression of specific protein kinase C isoforms in human colonic epithelial cells.

LIM1215 colon cancer cells were used as a model of human colonic epithelium to examine the effects of butyrate on protein kinase C (PKC) activity and isoform expression. On Western blot analysis, LIM1215 cells express the PKC isoforms alpha, beta, epsilon, zeta, and lambda, but not gamma, straight theta, or micro. Treatment with 2 mM butyrate for 48 h reduced cellular PKC activity up to 50% and specifically reduced the expression of PKC alpha and PKC epsilon. Similar results were obtained using Caco-2 colon cancer cells. These effects were neither a consequence of the induction of differentiation itself nor the result of direct or indirect activation of PKC. Although dependent on gene transcription and protein synthesis, the effect was not due to a reduction in the synthesis of PKC protein. Butyrate's effect was independent of its beta-oxidation but was mimicked, at least in part, by trichostatin A, an inhibitor of histone deacetylase.

Multiple Autocrine Factors Including an Extracellular Matrix Protein are Required for the Proliferation and Spreading of Human Colon Carcinoma Cells In Vitro

The human colon carcinoma cell line LIM1215 proliferates and changes morphology (spread) in a cell density-dependent manner in response to epidermal growth factor (EGF). At high density, production of autocrine transforming growth factor-a enables the cells to proliferate and spread in the absence of exogenous EGF or serum. At low cell density (> 1 × 104/cm2) EGF alone fails to elicit a mitogenic or morphological response and requires the presence of conditioned medium (derived from high cell density serum-free culture of the same cells) to exert its effects. This synergy between EGF and LIM 1215 conditioned medium was investigated further. Using a low cell density assay and fractionated LIM1215 conditioned medium, we show that EGF-mediated mitogenic and morphological responses are separable. These responses are dependent on the synergistic action of a low molecular weight autocrine survival factor and an extracellular matrix-like spreading factor(s) secreted into the culture medium respectively. We find that under low cell density, serum-free conditions, EGF alone is insufficient to rescue LIM1215 from rapid apoptotic death. Catalase or LIM1215 autocrine survival factor prevent the death of LIM1215 cells and restore their proliferative (but not morphological) response to EGF, suggesting that cell death under these conditions may be the result of oxidative stress. Combination of EGF, partially purified autocrine survival and spreading factors induced proliferation and spreading of low density LIM1215 cells similar to that observed with EGF and unfractionated conditioned medium. GRGDS peptides strongly inhibited the spreading of LIM1215 cells in the presence of EGF and the partially purified autocrine spreading factor, demonstrating that integriu receptors are involved in the spreading process. Comparison of the spreading response of LIM 1215 and Colo 526 cells on ASF and various adhesion proteins indicate that ASF is not collagen-I, collagen-IV, fibronectin or vitronectin. Taken together, these results support the concept that the autonomous growth of colon carcinoma cells in vitro is dependent on the synergistic interaction between several autocrine systems.

In vivo real-time analysis of intraperitoneal radiolabeled tumor cell movement during laparoscopy.

A porcine model has been developed to allow the real-time imaging of radiolabeled tumor cell movement throughout the peritoneal cavity, both at rest and during carbon dioxide insufflation. Fifteen 30-kg domestic white female pigs were used. Under anesthesia, 15 to 20 million radiolabeled human colorectal tumor cells (LIM1215) were introduced into the peritoneal cavity under laparoscopic vision into the pelvis. Radiolabeled tumor cell movement was examined by using a 25-cm-diameter, low-energy mobile gamma camera with high resolution collimator. Tumor cell movement and distribution during two hours without insufflation was examined in four pigs. Then tumor cell movement and distribution during two hours with CO2 insufflation was examined in four pigs. In a further four pigs, tumor cells were then mixed with blood and injected into the peritoneal cavity and the effect of no insufflation vs. insufflation was noted. A further three pigs were examined with manipulation of the intra-abdominal contents after injection of LIM1215 cells into the peritoneal cavity. Venting insufflating CO2 was filtered for tumor cells. Widespread intraperitoneal distribution of tumor cells from the pelvis was identified both with CO2 insufflation of the peritoneal cavity and without insufflation. Tumor cells dispersed throughout the peritoneal cavity at a slower rate without carbon dioxide insufflation. There was a differential rate of tumor cell movement to the left upper quadrant and right upper quadrant with insufflation and without insufflation. Blood within the peritoneal cavity and an extended contact of the laparoscopic trocars with the peritoneal cavity in this setting increased contamination of the trocars and trocar sites with tumor cells. Tumor cells were identified on laparoscopic instruments in all experiments. No evidence of aerosolization of tumor cells was found. Tumor cells move throughout the peritoneal cavity both at rest and during CO2 insufflation. The pattern of tumor cell dispersion differs with CO2 insufflation. The presence of blood and extended contact of trocars with peritoneal contents are a major factor in trocar and trocar site tumor cell contamination.

P53-dependent effects of RAS oncogene on chromosome stability and cell cycle checkpoints.

Mutations activating the function of ras proto-oncogenes are often observed in human tumors. Their oncogenic potential is mainly due to permanent stimulation of cellular proliferation and dramatic changes in morphogenic reactions of the cell. To learn more on the role of ras activation in cancerogenesis we studied its effects on chromosome stability and cell cycle checkpoints. Since the ability of ras oncogenes to cause cell transformation may be dependent on activity of the p53 tumor-suppressor the cells with different p53 state were analysed. Ectopic expression of N-ras(asp12) caused in p53-deficient MDAH041 cell line an augmentation in the number of chromosome breaks in mitogenic cells, significant increase in the frequency of metaphases showing chromosome endoreduplication and accumulation of polyploid cells. Similar effects were induced by different exogenous ras genes (N-ras(asp12), H-ras(leu12), N-ras proto-oncogene) in Rat1 and Rat2 cells which have a defect in p53-upstream pathways. In contrast, in REF52 and human LIM1215 cells showing ras-induced p53 up-regulation, ras expression caused only slight increase in the number of chromosome breaks and did not enhance the frequency of endoreduplication and polyploidy. Inactivation in these cells of p53 function by transduction of dominant-negative C-terminal p53 fragment (genetic suppressor element #22, GSE22) or mutant p53s significantly increased the frequency of both spontaneous and ras-induced karyotypic changes. In concordance with these observations we have found that expression of ras oncogene caused in p53-defective cells further mitigation of ethyl-metansulphonate-induced G1 and G2 cell cycle arrest, but did not abrogate G1 and G2 cell cycle checkpoints in cells with normal p53 function. These data indicate that along with stimulation of cell proliferation and morphological transformation ras activation can contribute to cancerogenesis by increasing genetic instability.

Short-chain fatty acids promote the migration of colonic epithelial cells in vitro

BACKGROUND & AIMS: Repair of colonic epithelial erosions requires cell migration. This study aimed to examine the effects of physiologically relevant short-chain fatty acids on migration in colonic epithelial cell lines.METHODS: Butyrate, propionate, and acetate were added to confluent monolayers of LIM1215 colon cancer cells after wounding. Migration in circular wounds was assessed after 24 hours.RESULTS: The migration of LIM1215 cells was stimulated in a concentration-dependent manner by all short-chain fatty acids. In four experiments, 2 mmol/L butyrate, 8 mmol/L propionate, and 16 mmol/L acetate induced 112.6% +/- 6.7%, 98.5% +/- 5.4%, and 63.4% +/- 7.2% (mean +/- SEM) stimulation above control migration, respectively. Their effects were additive at submaximal concentrations and reversible. Butyrate also stimulated migration in two other colon cancer cell lines, Caco-2 and LIM2405. However, butyrate failed to stimulate the migration of nongastrointestinal and nonepithelial cell lines. The stimulatory effect of butyrate required protein and RNA synthesis but was independent of cell proliferation, presence of serum, beta-oxidation, transforming growth factor beta, intracellular acidification, and substratum composition.CONCLUSIONS: In wounded in vitro models of colonic epithelium, short-chain fatty acids promote cell migration. If such an effect occurs in vivo, it would have ramifications for the biology and pathobiology of the colonic mucosa.(Gastroenterology 1997 Aug;113(2):487-96)

Purification and Characterization of a Novel Restricted Antigen Expressed by Normal and Transformed Human Colonic Epithelium

A cell surface antigen that is expressed by normal and 95% of transformed colonic epithelium and is recognized by the monoclonal antibody A33 (Welt, S., Divgi, C. R., Real, F. X., Yeh, S. D., Garin-Chesa, P., Finstad, C. L., Sakamoto, J., Cohen, A., Sigurdson, E. R., Kemeny, N., Carswell, E. A., Oettgen, H. F., and Old, L. J. (1990) J. Clin. Oncol. 8, 1894-1906) has been purified to homogeneity from the human colonic carcinoma cell line LIM1215. The A33 protein was purified from Triton X-114 extracts of LIM1215 cells under nondenaturing conditions. These extracts were applied sequentially to Green-Sepharose HE-4BD, Mono-Q HR 10/10, Superose 12 HR 10/30, and micropreparative Brownlee Aquapore RP 300. The purification was monitored by biosensor analysis using surface plasmon resonance detection with a F(ab')2 fragment of the humanized A33 monoclonal antibody immobilized on the sensor surface and Western blot analysis following SDS-polyacrylamide gel electrophoresis (PAGE) under nonreducing conditions using humanized A33 monoclonal antibody. The purified A33 antigen has a Mr on SDS-PAGE of 43,000 under nonreducing conditions. By contrast, the purified protein displayed a Mr of approximately 180,000 under native conditions on both size exclusion chromatography and native PAGE, possibly due to the formation of a homotetramer. N-terminal amino acid sequence analysis of the purified protein identified 34 amino acid residues of a unique sequence: ISVETPQDVLRASQGKSVTLPXTYHTSXXXREGLIQWD. A polyclonal antibody was raised against a synthetic peptide corresponding to residues 2-20 of this sequence. The antipeptide serum recognized the purified protein using Western blot analysis under both nonreducing (Mr 43,000) and reducing (Mr 49,000) conditions.

Chromosome changes caused by alterations of p53 expression

It has been proposed that p53 tumor-suppressor plays a key role in maintaining genome integrity in mammalian cells. We analyzed karyotype alterations in human and murine cell sublines expressing various exogenous human mutant (His175, Trp248, His273) or wild-type (wt) p53 cDNAs. In human pseudodiploid LIM1215 cells that contain two endogenous wt-p53 gene alleles, p53 mutants caused both an increase in the frequency of chromosome breaks and an emergence of hyperdiploid cells. Murine T 12 − / − and 10(1) fibroblasts lacking endogenous p53 expression have very unstable karyotypes and show a strong tendency to increase their ploidy levels during growth in culture. Transduction of a wt-p53 construct into p53-deficient cells inhibited an accumulation of highly polyploid cell variants. Transduction of mutant p53 did not show such an effect. Modification of endogenous and exogenous p53 expression by caffeine, which interferes with normal induction of p53 in response to DNA damage, showed no correlation between the induction of chromosome breaks and heteroploidy. We conclude that the caffeine- or mutant p53-induced increase in the frequency of chromosomal breaks in dividing LIM1215 cells is assonated with inactivation of wt-p53 function(s) responsible for control of G 1 checkpoint and/or DNA repair, while numerical chromosome changes in these cells may be a result of elimination or modification of a separate p53 function, or due to gain-of-function activities of p53 mutants. p53 modifications may therefore cause chromosome instability by different pathways: (1) through changes in the system(s) preventing proliferation of cells with genomic alterations; and (2) by increasing the probability of events, such as chromosome non-disjunction and/or endoreduplication that can lead to chromosome gains.

Cell-specific effects of RAS oncogene and protein kinase C agonist TPA on P-glycoprotein function

We compared the influence of exogenous N- ras oncogene and treatment with PKC agonist 12- O-tetradecanoylphorbol-13-acetate (TPA) on P-glycoprotein (Pgp) function in various human, rat and dog cell lines. Two approaches were used: (a) flow cytometry analysis of Rhodamine 123 (Rh123) exclusion; and (b) sensitivity to cytotoxic action of colchicine. We have found that in Rat1 fibroblasts, rat IAR2 epithelial cells and rat McA RH 7777 (hepatoma), ras activates Pgp function, while in MDCK (dog kidney), K562 (human chronic myelogenous leukaemia) and LIM1215 (human colon carcinoma) cells it either has no effect or even acts in opposite direction. TPA-induced Pgp function shows dissimilar pattern of cell specificity. It is assumed that PKC and ras oncogene regulate mdr1 gene expression through at least partially distinct signalling pathways.

Contrasting effects of butyrate on the expression of phenotypic markers of differentiation in neoplastic and non-neoplastic colonic epithelial cells in vitro.

The in vitro effect of butyrate on expression of differentiation markers in colonic epithelial cells was assessed in the colon cancer cell line, LIM1215 and in epithelial cells isolated from a surgically resected histologically normal colon. Markers used to assess cell differentiation were: net glycoprotein synthesis ([3H]-glucosamine uptake) expressed relative to net protein synthesis ([14C]-leucine uptake), and the expression of the brush border glycoproteins (alkaline phosphatase and carcino-embryonic antigen) in cell homogenates calculated relative to cellular protein content. In response to 24 h exposure to 1 mmol/L butyrate, all markers significantly increased in LIM1215 cells whereas they all significantly decreased in isolated colonic epithelial cells under identical culture conditions. Similar effects were seen at butyrate concentrations of up to 4 mmol/L. Butyrate suppressed proliferation of LIM1215 cells but had no consistent effect on [3H]-thymidine uptake by, or DNA content of, normal epithelial cells. Additional experiments found no evidence of a toxic effect of butyrate at those concentrations nor of an alteration of cell responsiveness to butyrate due to the isolation process itself. In contrast to its differentiative effect on neoplastic cells, butyrate reduces the expression of phenotypic markers of differentiation in vitro in colonic epithelial cells from non-neoplastic mucosa.