DiFi Cells Research Articles

Protein Expression Signatures for Inhibition of Epidermal Growth Factor Receptor-mediated Signaling

Analysis of cellular signaling networks typically involves targeted measurements of phosphorylated protein intermediates. However, phosphoproteomic analyses usually require affinity enrichment of phosphopeptides and can be complicated by artifactual changes in phosphorylation caused by uncontrolled preanalytical variables, particularly in the analysis of tissue specimens. We asked whether changes in protein expression, which are more stable and easily analyzed, could reflect network stimulation and inhibition. We employed this approach to analyze stimulation and inhibition of the epidermal growth factor receptor (EGFR) by EGF and selective EGFR inhibitors. Shotgun analysis of proteomes from proliferating A431 cells, EGF-stimulated cells, and cells co-treated with the EGFR inhibitors cetuximab or gefitinib identified groups of differentially expressed proteins. Comparisons of these protein groups identified 13 proteins whose EGF-induced expression changes were reversed by both EGFR inhibitors. Targeted multiple reaction monitoring analysis verified differential expression of 12 of these proteins, which comprise a candidate EGFR inhibition signature. We then tested these 12 proteins by multiple reaction monitoring analysis in three other models: 1) a comparison of DiFi (EGFR inhibitor-sensitive) and HCT116 (EGFR-insensitive) cell lines, 2) in formalin-fixed, paraffin-embedded mouse xenograft DiFi and HCT116 tumors, and 3) in tissue biopsies from a patient with the gastric hyperproliferative disorder Ménétrier's disease who was treated with cetuximab. Of the proteins in the candidate signature, a core group, including c-Jun, Jagged-1, and Claudin 4, were decreased by EGFR inhibitors in all three models. Although the goal of these studies was not to validate a clinically useful EGFR inhibition signature, the results confirm the hypothesis that clinically used EGFR inhibitors generate characteristic protein expression changes. This work further outlines a prototypical approach to derive and test protein expression signatures for drug action on signaling networks.

Growth response of human colorectal tumour cell lines to treatment with afatinib (BIBW2992), an irreversible erbB family blocker, and its association with expression of HER family members

Despite the approval of the anti-epidermal growth factor receptor (EGFR) monoclonal antibodies (mAbs), cetuximab and panitumumab, for the treatment of colorectal cancer patients, there is currently no reliable predictive marker for response to therapy. In addition, the duration of response is often limited. Therefore, this study aimed to investigate the effect of afatinib, an irreversible erbB family blocker, as a single agent or in combination with cytotoxic drugs (5-fluorouracil, irinotecan and oxaliplatin) or mAb ICR62 on the proliferation of a panel of human colorectal tumour cell lines and the association between the expression levels of the EGFR family members and response to treatment. Of the cells examined, EGFR-overexpressing DiFi cells were the most sensitive to treatment with both afatinib (IC50=45 nM) and ICR62 (IC50=4.33 nM). Afatinib also inhibited the growth of other tumour cell lines with IC50 values which ranged from 0.33 µM (CCL-221) to 1.62 µM (HCT-116). A significant association was found between the co-expression of EGFR, human epidermal growth factor receptor (HER)-2 and HER-3 and response to treatment with afatinib (R=0.915, P=0.021). Treat-ment with afatinib and cytotoxic drugs was accompanied by an increase in the proportion of these cells in the sub-G0/G1 and in the S and G2/M phase of the cell cycle, respectively. We conclude that afatinib as monotherapy or in combination with other drugs shows activity in colorectal tumour cells and that determination of the co-expression of HER family members should be conducted in clinical trials using drugs targeting erbB signaling. This approach could lead to the identification of a specific subpopulation of cancer patients more likely to benefit from erbB-directed therapy.

Differentiation between the EGFR antibodies necitumumab, cetuximab, and panitumumab: In vitro biological and binding activities.

e13030 Background: The monoclonal antibodies cetuximab (chimeric IgG1), necitumumab (human IgG1), and panitumumab (human IgG2) competitively inhibit ligand binding to epidermal growth factor receptor (EGFR) in domain 3. These antibodies block ligand induced EGFR activation and downstream molecular signaling. Beyond protein backbone differences, further differentiation of these EGFR antibodies is an important area of investigation. Methods: The effect on the in vitro proliferation of DiFi human colorectal cancer cells was assessed by ATP luminescence. The binding of the antibodies to the soluble extracellular domain of EGFR was evaluated by ELISA and surface plasmon resonance (SPR). Inhibition of ligand induced EGFR activation and downstream signaling was evaluated utilizing electrochemiluminescence method. Finally, binding kinetics was analyzed by SPR using a capture method. Results: The necitumumab concentration producing a half-maximal anti proliferative response (IC50) in DIFI cells was comparable to that of cetuximab. In contrast, a 1.6 fold greater concentration of panitumumab was required to produce the similar level of anti- proliferative response. This observation is supported by in vitro binding assays, which indicated that cetuximab and necitumumab binds to EGFR more effectively than panitumumab. Furthermore, the IC50 of nectitumab for the inhibition of EGFR phosphorylation was at least two fold greater than that of panitumumab. When EGFR binding kinetics were analyzed by SPR, panitumumab showed a much higher affinity (Kd) to EGFR than necitumumab and cetuximab, with a lower binding rate (kon) and a much slower dissociation rate (koff). Given the results in functional assays and the SPR data indicates that the high binding affinity (Kd) of panitumumab does not correlate with improved ability to block EGFR activation, downstream pathway activation and tumor cell proliferation. Conclusions: Although, as published, panitumumab has a higher affinity (Kd) than cetuximab and necitumumab, in vitro functional assays indicate that necitumumab and cetuximab have equal or greater ability to impact EGFR signaling and effect downstream activation of EGFR.

The role of epidermal growth factor receptor (EGFR)-directed recombinant dimeric IgA antibodies in tumor cell killing.

e13014 Background: The epidermal growth factor receptor (EGFR) is a validated target for personalized cancer therapy with IgG antibodies. IgA antibodies contribute to the humoral part of the mucosal immune system, but their immunotherapeutic potential has hardly been explored. Here, we describe the production, purification and functional evaluation of recombinant dimeric IgA antibodies against EGFR. Methods: Human J-chain-containing IgA was produced under serum-free conditions by transfecting non-adherent CHO-K1 cells expressing EGFR-specific monomeric 225-IgA (mIgA) with a vector coding for His-tagged human J-chain. Dimeric IgA (dIgA) was purified by two different affinity (anti-human-κ and anti-His-tag) and one size exclusion chromatography. The resulting homogenous preparation of highly pure IgA dimers was biochemically and functionally characterized. Results: Functional studies demonstrated dIgA to be at least as effective as mIgA in triggering antibody-dependent cellular cytotoxicity of A431 tumor cells by isolated monocytes (EC50 1.9/0.8 nM for mIgA/dIgA), by isolated PMN (EC50 5.6/3.7 nM for mIgA/dIgA) and in human whole blood 51chromium-release-assays (EC50 1.8/1.4 nM for mIgA/dIgA). Importantly, dimeric IgA was more effective in F(ab)-mediated mechanisms: inhibition of binding of FITC-labelled EGF to A431 cells and furthermore downmodulation of EGFR by dIgA was achieved at significantly lower concentrations than by mIgA (EC50 14.9/124.5 nM, 76.2/335.2 nM, respectively). In addition, growth of EGFR-expressing DiFi cells was inhibited more effectively by dimeric than by monomeric IgA (EC50 6.2/49.7 nM). Furthermore, only dimeric but not monomeric IgA or IgG was directionally transported via the pIgR through an epithelial cell monolayer in a transcytosis assay with polarized MDCK cells stably transfected with human polymeric immunoglobulin receptor (pIgR). Conclusions: These studies demonstrate that recombinant dimeric IgA antibodies against EGFR trigger a distinct repertoire of effector functions compared to monomeric IgA or IgG1 antibodies - suggesting that dimeric IgA antibodies may constitute an interesting antibody format for tumor therapy.

HGF Rescues Colorectal Cancer Cells from EGFR Inhibition via MET Activation

Cetuximab, an antibody targeting the epidermal growth factor receptor (EGFR), is active in colorectal cancer (CRC). However, response rates range from only 10% to 20%. Here, we investigate hepatocyte growth factor (HGF)-dependent mesenchymal-epithelial transition factor (MET) activation as a mediator of cetuximab resistance through signal diversification in CRC cell lines. DiFi, GEO, and LIM1215 cells were treated with varying concentrations and combinations of EGF, HGF, cetuximab, and PHA-665752 (a highly specific MET kinase inhibitor). Biological end points included proliferation, cell cycle arrest, and apoptosis. Proliferation was measured using WST-1 assays and synergy investigated via isobolograms. Expression and signaling were examined using immunoblotting. EGFR and MET are coexpressed in these CRC cell lines, and dual receptor activation synergistically increased proliferation. Cetuximab inhibited cell growth by 60%-80% with an associated dephosphorylation of EGFR, MAPK, and/or AKT. Addition of HGF to cetuximab-treated cells phosphorylated MET, but not EGFR or ErbB3, restimulated the MAPK and AKT pathways, restored cell proliferation, and rescued cells from G1 arrest and apoptosis. Importantly, this effect could be abrogated by inhibiting MET activation with PHA-665752 or by downregulating MET expression with RNAi. HGF-induced MET activation is a novel mechanism of cetuximab resistance in CRC. Inhibition of the HGF-MET pathway may improve response to EGFR inhibitors in CRC, and combination therapy should be further investigated.

Recombinant Dimeric IgA Antibodies as Tumor-Specific Agents.

AbstractAbstract 1488Dimeric IgA antibodies contribute significantly to the humoral part of the mucosal immune system. However, their potential as immunotherapeutic agent has hardly been explored. Here, we describe the production, purification and functional evaluation of recombinant dimeric IgA, exemplarily directed against the epidermal growth factor receptor (EGF-R). Human J-chain-containing IgA was produced under serum-free conditions by transfecting non-adherent CHO-K1 cells expressing EGF-R specific monomeric IgA with a vector coding for the His-tagged human J-chain. For purification of J-chain-containing dimeric IgA two different affinity (anti-human-kappa and anti-His-tag) and one size exclusion chromatography were combined, resulting in a homogenous preparation of highly pure IgA dimers, as determined by gel electrophoresis under denaturing and native conditions using silver stain, Coomassie blue or Western blots for protein detection, respectively. Functional studies demonstrated dimeric IgA (dIgA) to be at least as effective as monomeric IgA (mIgA) in triggering ADCC of A431 tumor cells by isolated monocytes (EC50 0.35 and 0.30 μg/ml for mIgA and dIgA, 0.23 μg/ml for the corresponding IgG1; maximal killing 51.2 vs 43.3 vs 56.3 %), by isolated PMN (EC50 0.96, 1.20 and 0.8 μg/ml for mIgA, dIgA and IgG1; maximal killing 52.6 vs 50.6 vs 19.9 %) and in human whole blood assays (EC50 0.32 and 0.32 μg/ml for mIgA and dIgA; maximal killing 7.9 vs 12.5 %; IgG1: maximal killing 0.7 %, EC50 not determinable). Importantly, dimeric IgA was more effective in F(ab)-mediated mechanisms: inhibition of binding of FITC-labelled EGF to A431 cells by dimeric IgA was achieved at significantly lower concentrations than by monomeric IgA (EC50 7.1 vs 18.1 μg/ml, respectively). In addition, growth of EGF-R expressing DiFi colon carcinoma cells was inhibited at significantly lower concentrations by dimeric than by monomeric IgA (EC50 1.6 vs 3.7 μg/ml). Both IgA isoforms and their IgG1 variant were similarly effective in triggering apoptosis of DiFi cells, as analyzed by PARP cleavage. Furthermore, only dimeric but not monomeric IgA or IgG was directionally transported by the polymeric immunoglobulin receptor (pIgR) through an epithelial cell monolayer, as analyzed by an experimental transcytosis assay with polarized MDCK cells stably transfected with human pIgR. Together, these studies demonstrate that recombinant dimeric IgA antibodies recruit a distinct repertoire of effector functions compared to monomeric IgA or IgG1 antibodies, trigger important biological functions and constitute an interesting antibody format for future tumor therapies. Disclosures:van de Winkel:Genmab: Employment, Membership on an entity's Board of Directors or advisory committees. Valerius:Genmab: Consultancy, Research Funding.

Epidermal Growth Factor Receptor (EGFR) Ubiquitination as a Mechanism of Acquired Resistance Escaping Treatment by the Anti-EGFR Monoclonal Antibody Cetuximab

Cetuximab is an epidermal growth factor receptor (EGFR)-blocking antibody that has been approved for treatment of patients with metastatic colorectal cancer. In this study, we investigated biochemical changes in signaling pathways of a cetuximab-resistant subline of DiFi colorectal cancer cells (DiFi5) that was developed by exposing the parental sensitive cells to subeffective doses of cetuximab over an extended period of time. Compared with parental DiFi cells that express high levels of EGFR and in which cetuximab induces apoptosis, the cetuximab-resistant DiFi5 cells showed markedly lower protein levels of EGFR, an increased association of EGFR with Cbl, and an increased ubiquitination of EGFR. DiFi5 cells also had a markedly higher level of Src-Y416 phosphorylation both at baseline and on EGF stimulation. Although EGFR levels were low, DiFi5 cells responded to EGF stimulation with robust phosphorylation of EGFR on Y845 and strong phosphorylation of Akt and extracellular signal-regulated kinase, comparable to those of parental cells. Most importantly, inhibition of Src kinase activity with PP2 reversed the resistance of DiFi5 cells to cetuximab-induced apoptosis without affecting the levels of EGFR in the cells. Our results indicate that colorectal cancer cells may develop acquired resistance to cetuximab via altering EGFR levels through promotion of EGFR ubiquitination and degradation and using Src kinase-mediated cell signaling to bypass their dependency on EGFR for cell growth and survival.

Novel tricyclic azepine derivatives: Biological evaluation of pyrimido[4,5- b]-1,4-benzoxazepines, thiazepines, and diazepines as inhibitors of the epidermal growth factor receptor tyrosine kinase

Novel tricyclic derivatives containing an oxazepine, thiazepine, or diazepine ring were studied for their EGFR tyrosine kinase inhibitory activity. While the oxazepines were in general more potent than thiazepines, the diazepines displayed somewhat different structure–activity relationships. Moreover, the diazepines, in contrast to the oxazepines, showed appreciable inhibitory activity against the KDR tyrosine kinase. Furthermore, both oxazepines and diazepines demonstrated significant ability to inhibit autophosphorylation of EGFR in DiFi cells (generally, IC 50 values in the single-digit micromolar to submicromolar range).

Responses of Human Colorectal Tumor Cells to Treatment with the Anti–Epidermal Growth Factor Receptor Monoclonal Antibody ICR62 Used Alone and in Combination with the EGFR Tyrosine Kinase Inhibitor Gefitinib

The anti-epidermal growth factor receptor (EGFR) monoclonal antibody cetuximab has been approved for the treatment of patients with metastatic colorectal cancer. However, there is currently no reliable marker for response to therapy with the EGFR inhibitors. In this study, we investigated the sensitivity of 10 human colorectal tumor cell lines (DiFi, CCL218, CCL221, CCL225, CCL227, CCL228, CCL231, CCL235, CCL244, and HCT-116) to treatment with our anti-EGFR monoclonal antibody, ICR62, and/or the EGFR tyrosine kinase inhibitor, gefitinib. Of the cells examined, only DiFi contained high levels of constitutively active EGFR and were highly sensitive to treatment with both ICR62 (IC(50) = 0.52 nmol/L) and gefitinib (IC(50) = 27.5 nmol/L). In contrast, the growth of other tumor cell lines, which contained low levels of the EGFR, HER-2, and pAkt but comparable or even higher basal levels of phosphorylated mitogen-activated protein kinase (pMAPK), were relatively resistant to treatment with both inhibitors. Both ICR62 and gefitinib induced EGFR down-regulation, reduced the basal levels of pEGFR at five known tyrosine residues, pMAPK, and pAkt, and increased the sub-G(1) population in DiFi cells. However, treatment with a combination of ICR62 and gefitinib neither sensitized colorectal tumor cells that were insensitive to treatment with the single agent nor enhanced the growth-inhibitory effect of the single agent in DiFi cells. These results indicate that basal levels of pMAPK and pAkt are not good indicators of response to the EGFR inhibitors in colorectal cancer cells and dual targeting of the EGFR by a combination of ICR62 and gefitinib is not superior to treatment with a single agent.

Irreversible pan-ErbB tyrosine kinase inhibitor CI-1033 induces caspase-independent apoptosis in colorectal cancer DiFi cell line

The epidermal growth factor receptor (EGFR) is overexpressed in the majority of colorectal carcinomas and represents a target for therapeutic interventions with signal transduction inhibitors. We investigated the ability of CI-1033 to induce apoptosis and inhibition of proliferation in the colorectal cancer cell lines DiFi and Caco-2, which both express high levels of EGFR. While in Caco-2 cells CI-1033 treatment at a concentration 0.1 microM for 72 hours demonstrated only antiproliferative (53.7 +/- 4.3%) but no pro-apoptotic effects, treatment of DiFi cells resulted in a reduced proliferation rate (31.4 +/- 3.1%) and in apoptosis (44.2 +/- 8.9%). In order to define proteins involved in the regulation of apoptosis, we aimed to determine differences in the proteome profile of both cell lines before and after treatment with CI-1033. Cellular proteins were analyzed by 2-D gel electrophoresis followed by computational image analysis and mass spectrometry. Our data show that DiFi cells differ from Caco-2 cells in nine significantly upregulated proteins, and their potential role in apoptosis is described. We demonstrate that induction of apoptosis was triggered via caspase-independent pathways. Overexpression of leukocyte elastase inhibitor (LEI) and translocation of cathepsin D to the cytosol accompanied by upregulation of other defined proteins resulted in Bax-independent AIF translocation from mitochondria into the nucleus and apoptosis. Definition of these proteins can pave the way for functional studies and contribute to a better understanding of the effects of CI-1033 and the pathways of caspase-independent cell death.

A Fully Human Recombinant IgG-like Bispecific Antibody to Both the Epidermal Growth Factor Receptor and the Insulin-like Growth Factor Receptor for Enhanced Antitumor Activity

Both the epidermal growth factor receptor (EGFR) and the insulin-like growth factor receptor (IGFR) have been implicated in the tumorigenesis of a variety of cancers. Here we propose that simultaneous targeting of both receptors with a bispecific antibody would lead to enhanced antitumor activity. To this end, we produced a recombinant human IgG-like bispecific antibody, a Di-diabody, using the variable regions from two antagonistic antibodies: IMC-11F8 to EGFR and IMC-A12 to IGFR. The Di-diabody binds to both EGFR and IGFR and effectively blocked both EGF- and IGF-stimulated receptor activation and tumor cell proliferation. The Di-diabody also inherited the biological properties from both of its parent antibodies; it triggers rapid and significant IGFR internalization and degradation and mediates effective antibody-dependent cellular cytotoxicity in a variety of tumor cells. Finally, the Di-diabody strongly inhibited the growth of two different human tumor xenografts in vivo. Our results underscore the benefits of simultaneous targeting of two tumor targets with bispecific antibodies.

The monoclonal antibody 225 activates caspase-8 and induces apoptosis through a tumor necrosis factor receptor family-independent pathway.

We previously reported that the anti-epidermal growth factor (EGF) receptor monoclonal antibody (mAb) 225 induces DiFi colon cancer cells to undergo apoptosis, and this apoptosis was accompanied by activation of the two apoptosis initiation caspases, caspase-8 and caspase-9. In the current study, we found that pretreatment of DiFi cells with the caspase-8-specific inhibitor z-IETD-fmk but not pretreatment with the caspase-9-specific inhibitor z-LEHD-fmk inhibited mAb 225-induced apoptosis, indicating that caspase-8 plays an essential role in initiating mAb 225-induced apoptosis. Because caspase-8 is activated primarily by the members of the tumor necrosis factor (TNF) receptor family, such as Fas, TNF receptor-1 (TNFR1), or receptors for TNF-related apoptosis-inducing ligand (TRAIL), we investigated whether mAb 225 activated caspase-8 by regulating one or more of these known pathways. Exposure of DiFi cells to TNFalpha or TRAIL activated caspase-8 and induced apoptosis in the cells. A TNFR1-antagonistic mAb or a TRAIL decoy receptor inhibited the activation of caspase-8 and the subsequent apoptosis induced by TNFalpha or TRAIL, respectively, in the cells. However, neither the TNFR1-antagonistic mAb nor the TRAIL decoy receptor inhibited mAb 225-induced activation of caspase-8 and apoptosis in DiFi cells. DiFi cells express detectable level of Fas but are not sensitive to the treatment by the Fas-agonistic mAb CH-11. A Fas-antagonistic mAb (ZB-4) inhibited the Fas-agonistic mAb CH-11-induced caspase-8 activation and apoptosis in Jurkat T-leukemic cells (used as positive control), but had no effect on mAb 225-induced activation of caspase-8 and apoptosis in DiFi cells. Taken together, our results suggest that mAb 225 does not interact with or regulate these known death receptor pathways. An exploration is therefore warranted for a novel mechanism by which mAb 225 activates caspase-8 and triggers apoptosis in DiFi cells.

Poly(ethylene glycol)-conjugated anti-EGF receptor antibody C225 with radiometal chelator attached to the termini of polymer chains.

Several biological barriers, including significant liver uptake, limit the clinical application of radiolabeled antibodies in radioimmunoscintigraphy. Here, a general approach is described for radiolabeling of monoclonal antibodies conjugated with poly(ethylene glycol) (PEG). This strategy is demonstrated with C225, a monoclonal antibody directed against epidermal growth factor (EGF) receptor. We synthesized a heterofunctional PEG with one end attached to a radiometal chelator, diethylenetriaminepentaacetic acid (DTPA), and the other end to a protected thiol group, S-acetylthioacetate. After a deprotection step, the resulting DTPA-PEG-SH was conjugated to maleimide-activated C225 to yield DTPA-PEG-C225 conjugate. Characterization of DTPA-PEG-C225 with immunoprecipitation and Western blot analysis revealed that the conjugate was biologically active in binding to the EGF receptor in A431 cells. Competitive EGF receptor binding assay in MDA-MB-468 cells showed that DTPA-PEG-C225, with up to 60% of the amino groups in C225 substituted, retained 66% of C225's binding affinity. Moreover, DTPA-PEG-C225 with increasing degrees of NH(2) substitution from 20% to 70% retained the activity of C225 to induce apoptosis in DiFi cells. More importantly, DTPA-PEG-C225 demonstrated less nonspecific interaction than DTPA-C225. Pharmacokinetic analysis using (111)In-labeled compounds revealed narrower steady-state distribution of (111)In-DTPA-PEG-C225 than (111)In-DTPA-C225, probably due to reduced nonspecific binding of PEG-modified antibody to tissues. The terminal half-life (t(1/2,)(gamma)) of (111)In-DTPA-PEG-C225, 21.1 h, was shorter than that of (111)In-DTPA-C225, 52.9 h. These data suggest that (111)In-DTPA-PEG-C225 may provide better imaging characteristics than (111)In-DTPA-C225, and that using PEG as a linker between the monoclonal antibody and DTPA may be a promising strategy in optimizing the imaging characteristics of immunoscintigraphic agents.

Fibroblast growth factor and insulin-like growth factor differentially modulate the apoptosis and G1 arrest induced by anti-epidermal growth factor receptor monoclonal antibody

DiFi human colon carcinoma cells are stimulated by the transforming growth factor-alpha (TGF-alpha)/epidermal growth factor (EGF) receptor autocrine loop. Exposure of DiFi cells to monoclonal antibody (mAb) 225, which blocks ligand-induced activation of the EGF receptor, induces G1 arrest and subsequent cell death via apoptosis. We investigated the signal pathways by which basic fibroblast growth factor (bFGF) and insulin-like growth factor-1 (IGF-1) modulate mAb 225-induced G1 arrest and apoptosis in DiFi cells. Both bFGF and IGF-1 activated the mitogen-activated protein kinase (MAPK) kinase (MEK) pathway in DiFi cells. Additionally, IGF-1 activated the phosphoinositide 3-kinase (PI-3K)/Akt pathway. Both bFGF and IGF-1 inhibited mAb 225-induced apoptosis; however, bFGF provided sustained protection against apoptosis, while the protection by IGF-1 was only temporary. Also, bFGF reversed the mAb 225-induced increase in the p27(Kip1) level, inhibition of cyclin-dependent kinase-2 (CDK-2) activity, dephosphorylation of the retinoblastoma (Rb) protein and the resultant G1 arrest of the cells. In contrast, IGF-1 did not reverse such effects by mAb 225. The prevention of mAb 225-induced G1 arrest and apoptosis in DiFi cells by bFGF was sensitive to the MEK/MAPK inhibitor PD98059 but not to the PI-3K inhibitor LY294002. In contrast, inhibition of apoptosis by IGF-1 in DiFi cells was sensitive only to LY294002 and not to PD98059. These results further our understanding of how mAb 225 induces apoptosis in DiFi cells.

Butyric acid induces apoptosis by up-regulating bax expression via Stimulation of the c-Jun N-Terminal Kinase/Activation Protein-1 pathway in human colon cancer cells

The colonic epithelial cells near the top of the crypt have been shown to undergo apoptosis. Because butyric acid (BA) is the major short-chain fatty acid produced by fermentation of dietary fiber in the large bowel, it may be an important regulator of apoptosis in colorectal cancer. We investigated which signaling pathway is triggered by BA to undergo apoptosis in human colorectal cancer cells. Human DiFi and FET colorectal cells were treated with BA to undergo apoptosis and were assayed for activation of c-Jun N-terminal kinase (JNK), transcription factor activation protein 1 (AP1) and NF-kappaB, and the proapoptotic molecule Bax. The contribution of specific pathways was assessed by examining the effects of dominant-negative mutants of JNK/AP1 or NF-kappaB on BA-induced Bax expression and apoptosis. BA-mediated DNA fragmentation and Bax induction were preceded by early stimulation of JNK, and the DNA-binding activities of AP1 and NF-kappaB. BA-induced enhancement of DNA fragmentation and stimulation of Bax promoter activity were blocked by the expression of dominant-negative mutants of JNK1 or AP1 but not NF-kappaB. These findings suggest that apoptosis triggered by BA involves transcriptional stimulation of the Bax gene via activation of the JNK/AP1 pathway in colonic epithelial cells.

Induction of apoptosis and activation of the caspase cascade by anti-EGF receptor monoclonal antibodies in DiFi human colon cancer cells do not involve the c-jun N-terminal kinase activity.

We previously reported that exposure of DiFi human colon cancer cells to the anti-epidermal growth factor (EGF) receptor monoclonal antibody (mAb) 225 resulted in apoptosis, but the mechanisms remain to be elucidated. In the present study, we investigated the effects of a panel of four anti-EGF receptor mAbs, each of which binds to different epitopes of the EGF receptor in DiFi cells, on the induction of apoptosis. We found that each of these mAbs induced apoptosis in DiFi cells. Exposure of DiFi cells to mAb 225 activated the initiation caspase-8, which was detectable between 8 and 16 h after exposure of the cells to the antibody. There was also an activation of the initiation caspase-9, which lagged a few hours behind the activation of caspase-8. Exposure of DiFi cells to mAb 225 also activated the execution caspase-3, which was accompanied temporally by evidence of cleavage of a well-characterized caspase-3 substrate, poly(ADP)-ribosepolymerase (PARP). Pre-exposure of the cells to the caspase-3-specific inhibitor DEVD-CHO partially reduced the mAb 225-induced PARP cleavage and apoptosis, whereas pre-exposure of the cells to the caspase pan-inhibitor z-VAD-fmk completely inhibited mAb 225-induced apoptosis. Caspases-3, -8 and -9 were not activated in the cell lines in which mAb 225 only induced G1 phase arrest of the cell cycle. In contrast to the apoptosis of DiFi cells induced by ultraviolet irradiation, which strongly activated the c- jun N-terminal kinase-1 (JNK1) and the caspase cascade, mAb 225-induced apoptosis and activation of the caspase cascade in DiFi cells were not associated with activation of JNK1. © 2000 Cancer Research Campaign

Coordinate up-regulation of low-density lipoprotein receptor and cyclo-oxygenase-2 gene expression in human colorectal cells and in colorectal adenocarcinoma biopsies.

Many colorectal cancers have high levels of cyclo-oxygenase 2 (COX-2), an enzyme that metabolizes the essential fatty acids into prostaglandins. Since the low-density lipoprotein receptor (LDLr) is involved in the uptake of essential fatty acids, we studied the effect of LDL on growth and gene regulation in colorectal cancer cells. DiFi cells grown in lipoprotein-deficient sera (LPDS) grew more slowly than cells with LDL. LDLr antibody caused significant inhibition of tumor cell growth but did not affect controls. In addition, LDL uptake did not change in the presence of excess LDL, suggesting that ldlr mRNA lacks normal feedback regulation in some colorectal cancers. Analysis of the ldlr mRNA showed that excess LDL in the medium did not cause down-regulation of the message even after 24 hr. The second portion of the study examined the mRNA expression of ldlr and its co-regulation with cox-2 in normal and tumor specimens from patients with colorectal adenocarcinomas. The ratio of tumor:paired normal mucosa of mRNA expression of ldlr and of cox-2 was measured in specimens taken during colonoscopy. ldlr and cox-2 transcripts were apparent in 11 of 11 carcinomas. There was significant coordinate up-regulation both of ldlr and of cox-2 in 6 of 11 (55%) tumors compared with normal colonic mucosa. There was no up-regulation of cox-2 without concomitant up-regulation of ldlr. These data suggest that the LDLr is abnormally regulated in some colorectal tumors and may play a role in the up-regulation of cox-2.

Nuclear targeting of Bax during apoptosis in human colorectal cancer cells.

Homeostasis in colonic epithelial cells is regulated by the balance between proliferative activity and cell loss by apoptosis. Because epithelial cells at the apex of colonic crypts undergo apoptosis and proliferative activity is usually restricted to the base of the crypts, it has been proposed that the limited availability of growth factor-signals at the upper portions of the crypts may trigger apoptosis. In the present studies, we investigate the mechanism of apoptosis mediated by growth factor deprivation in colorectal carcinoma cells by delineating the possible involvement of Bax and its subcellular localization. We report that inhibition of epidermal growth factor receptor (EGFR) tyrosine kinase activity and downregulation of EGFR by anti-EGFR mAb 225 induces apoptosis in human colorectal carcinoma DiFi and FET cells. Induction of apoptosis was preceded by enhanced expression of newly synthesized Bax protein, and required protein synthesis. In the mAb 225-treated cells, Bax was redistributed from the cytosol to the nucleus and subsequently, to the nuclear membranes. The observed induction of Bax expression by mAb 225 was not associated with p53 induction. However, mAb 225 treatment also triggered relocalization of p53 from the cytosol to a nuclear membrane-bound form. Induction of Bax and its redistribution to the nucleus of DiFi cells during apoptosis was also demonstrated in response to butyrate, a physiological relevant molecule in colonic epithelial cells as it is the principal short-chain fatty acid produced by bacterial fermentation of dietary fiber in colonic epithelium. Using immunofluorescence and confocal microscopy, we observed that Bax is predominantly localized in the cytosol, but during apoptosis it is localized both inside and along the nuclear membrane. Taken together, these findings suggest that apoptosis induced by growth factor-deprivation or butyrate may involve the subcellular redistribution of Bax in human colorectal carcinoma cells.

Induction of cyclo-oxygenase-2 mRNA by prostaglandin E2 in human prostatic carcinoma cells.

Prostaglandins are synthesized from arachidonic acid by the enzyme cyclo-oxygenase. There are two isoforms of cyclooxygenases: COX-1 (a constitutive form) and COX-2 (an inducible form). COX-2 has recently been categorized as an immediate-early gene and is associated with cellular growth and differentiation. The purpose of this study was to investigate the effects of exogenous dimethylprostaglandin E2 (dmPGE2) on prostate cancer cell growth. Results of these experiments demonstrate that administration of dmPGE2 to growing PC-3 cells significantly increased cellular proliferation (as measured by the cell number), total DNA content and endogenous PGE2 concentration. DmPGE2 also increased the steady-state mRNA levels of its own inducible synthesizing enzyme, COX-2, as well as cellular growth to levels similar to those seen with fetal calf serum and phorbol ester. The same results were observed in other human cancer cell types, such as the androgen-dependent LNCaP cells, breast cancer MDA-MB-134 cells and human colorectal carcinoma DiFi cells. In PC-3 cells, the dmPGE2 regulation of the COX-2 mRNA levels was both time dependent, with maximum stimulation seen 2 h after addition, and dose dependent on dmPGE2 concentration, with maximum stimulation seen at 5 microg ml(-1). The non-steroidal anti-inflammatory drug flurbiprofen (5 microM), in the presence of exogenous dmPGE2, inhibited the up-regulation of COX-2 mRNA and PC-3 cell growth. Taken together, these data suggest that PGE2 has a specific role in the maintenance of human cancer cell growth and that the activation of COX-2 expression depends primarily upon newly synthesized PGE2, perhaps resulting from changes in local cellular PGE2 concentrations.

Cis-acting elements required for expression of the nonspecific cross- reacting antigen gene in colorectal carcinoma

BACKGROUND & AIMS: The nonspecific cross-reacting antigen (NCA) is a cell adhesion molecule, and the messenger RNA for NCA is overexpressed in 92% of colorectal carcinomas. The aim of this study was to determine the cis-acting elements that may be responsible for the expression of NCA.METHODS: Deletion mutants of the 5' flanking sequence and first intron were ligated into chloramphenicol acetyltransferase expression vectors, transfected into Chinese hamster ovary (CHO), DiFi, and HT-29 human colorectal carcinoma cells, and BxPC-3 and MDAPanc-28 human pancreatic carcinoma cells. The amount of acetylated chloramphenicol was determined to show the presence and activity of cis-acting sequences.RESULTS: The 5' flanking sequence functions as a promoter in all of cell lines and contains negative regulatory and enhancer sequences. The minimal promoter is active in Chinese hamster ovary and HT-29, though not in MDAPanc-28 cells. The first intron contains a silencer capable of suppressing a heterologous promoter.CONCLUSIONS: The results show cis-acting sequences within and 5' to the NCA gene, which appear to play a role in the expression of this gene in malignant tissues. Some of these sequences function in a cell type-specific manner. Further studies of these elements may provide insight into the mechanisms of the abnormal growth patterns of malignant cells.(Gastroenterology 1997 Mar;112(3):776-82)