Activation of phosphatidylinositol 3-kinase in response to interleukin-1 leads to phosphorylation and activation of the NF-kappaB p65/RelA subunit.

The work of Reddy et al. (S. A. Reddy, J. A. Huang, and W. S. Liao, J. Biol. Chem. 272:29167-29173, 1997) reveals that phosphatidylinositol 3-kinase (PI3K) plays a role in transducing a signal from the occupied interleukin-1 (IL-1) receptor to nuclear factor kappaB (NF-kappaB), but the underlying mechanism remains to be determined. We have found that IL-1 stimulates interaction of the IL-1 receptor accessory protein with the p85 regulatory subunit of PI3K, leading to the activation of the p110 catalytic subunit. Specific PI3K inhibitors strongly inhibit both PI3K activation and NF-kappaB-dependent gene expression but have no effect on the IL-1-stimulated degradation of IkappaBalpha, the nuclear translocation of NF-kappaB, or the ability of NF-kappaB to bind to DNA. In contrast, PI3K inhibitors block the IL-1-stimulated phosphorylation of NF-kappaB itself, especially the p65/RelA subunit. Furthermore, by using a fusion protein containing the p65/RelA transactivation domain, we found that overexpression of the p110 catalytic subunit of PI3K induces p65/RelA-mediated transactivation and that the specific PI3K inhibitor LY294,002 represses this process. Additionally, the expression of a constitutively activated form of either p110 or the PI3K-activated protein kinase Akt also induces p65/RelA-mediated transactivation. Therefore, IL-1 stimulates the PI3K-dependent phosphorylation and transactivation of NF-kappaB, a process quite distinct from the liberation of NF-kappaB from its cytoplasmic inhibitor IkappaB.

Human papillomavirus 16 immortalization of normal human ectocervical epithelial cells alters retinoic acid regulation of cell growth and epidermal growth factor receptor expression.

Retinoids are potent regulators of epithelial cell growth and differentiation. Recently, they have been demonstrated to be effective in the treatment of preneoplastic cervical lesions in which human papillomavirus (HPV) is expressed. To better understand the mechanism of the antineoplastic effect of retinoic acid on HPV-positive cells, the effects of retinoic acid on both normal and HPV-immortalized human ectocervical epithelial cell growth, epidermal growth factor (EGF) receptor level, and EGF receptor function were investigated. Both HPV-immortalized cells (ECE16-1) and normal ectocervical cells (ECE cells) are growth stimulated by EGF. ECE16-1 but not normal ectocervical epithelial cells are growth inhibited by trans-retinoic acid which attenuates the stimulatory effect of EGF on ECE16-1 cell growth. Retinoic acid reduces both EGF binding and EGF receptor protein levels in ECE16-1 cells but not in normal ectocervical cells. The reduction in EGF receptor binding and receptor protein levels in ECE16-1 cells is not associated with the induced secretion of a soluble EGF receptor ligand, altered EGF receptor affinity, receptor internalization, or decreased receptor stability. Interestingly, the level of EGF receptors is consistently elevated in the ECE16-1 cell line as compared to normal ectocervical epithelial cells. Investigation of a second HPV-immortalized cell line (ECE16-D1) and two other HPV-positive cervical carcinoma cell lines revealed similar elevated EGF-binding capacity and regulation by retinoic acid. In contrast, two HPV-negative cervical carcinoma cell lines demonstrated various EGF-binding levels but demonstrated no significant loss of EGF binding following retinoic acid treatment. Other normal cells and an SV40 large T-antigen-immortalized foreskin keratinocyte cell line, KER-1, had EGF receptor levels similar to the normal ectocervical epithelial cells, and no regulation by retinoic acid was observed. These data indicate that HPV immortalization may increase EGF receptor levels in ectocervical cells, elevating their sensitivity to growth stimulation by EGF, and that retinoic acid can possibly attenuate this increased responsiveness to EGF.

Ras, but not Src, transformation of RIE-1 epithelial cells is dependent on activation of the mitogen-activated protein kinase cascade.

Src transformation of NIH3T3 mouse fibroblasts has been shown to be dependent on Ras function. Since we recently showed that the signaling pathways that mediate Ras transformation of RIE-1 rat intestinal epithelial cells are distinct from those that cause Ras transformation of fibroblasts, we utilized three approaches to determine if Src transformation of RIE-1 cells is dependent on Ras. First, although both Ras and Src cause upregulation of an epidermal growth factor (EGF) receptor-dependent autocrine growth loop, only Ras transformation required this activity. Second, whereas both Src and Ras caused upregulation of the p42 and p44 mitogen-activated protein kinases (MAPKs), only Ras transformation was blocked by the inhibition of MAPK activation by treatment with the PD 98059 MEK inhibitor. Third, treatment with the farnesyltransferase inhibitor FTI-277 blocked Ras, but not Src, transformation. Taken together, these observations suggest that Src transformation of RIE-1 cells is not dependent on Ras. Finally, we determined that Ras activation of Raf-independent pathways alone is sufficient to cause growth transformation of RIE-1 cells. Thus, both Ras and Src cause transformation of RIE-1 cells via pathways distinct from those required to cause transformation of NIH3T3 cells.

Ras-mediated suppression of TGFbetaRII expression in intestinal epithelial cells involves Raf-independent signaling.

Ras-transformed intestinal epithelial cells are resistant to the growth inhibitory actions of TGFbeta and have a marked decrease in expression of the TGFbeta type II receptor (TGFbetaRII). Rat intestinal epithelial cells (RIE) were stably transfected with activated Ras, Sos and Raf constructs and tested for expression of TGFbetaRII and sensitivity to growth inhibition by TGFbeta. The parental RIE line and the RIE-Raf cells were non-transformed in morphology and were sensitive to TGFbeta (70-90% inhibited). In contrast, the RIE-Ras and RIE-Sos lines were transformed, resistant to TGFbeta and expressed 5- to 10-fold decreased levels of the TGFbetaRII mRNA and protein. Cyclin D1 protein expression was repressed by TGFbeta treatment in parental RIE and RIE-Raf cells, whereas levels of cyclin D1 in RIE-Ras and RIE-Sos cells remained unchanged. Treatment of RIE-Ras cells with 25 microM farnesyl transferase inhibitor, FTI L739,749, for 48 hours restored expression of TGFbetaRII to levels equivalent to control cells. In addition, treatment of RIE-Ras cells for 48 hours with PD-98059, a specific MAPKK inhibitor, also increased expression of TGFbetaRII to control levels. Collectively these results suggest that downregulation of TGFbetaRII and loss of sensitivity to growth inhibition by TGFbeta in Ras-transformed intestinal epithelial cells is not mediated exclusively by the conventional Ras/Raf/MAPKK/MAPK pathway. However, activation of MAPK, perhaps by an alternate Ras effector pathway, appears to be necessary for Ras-mediated downregulation of TGFbetaRII.

Polycyclic aromatic hydrocarbons enhance terminal cell death of human ectocervical cells.

Polycyclic aromatic hydrocarbons (PAH) are a class of chemical carcinogens whose active metabolites form DNA adducts, resulting in specific mutational events. The tumor suppressor protein p53 is believed to play a pivotal role in the ability of cells to response to DNA damage, resulting in either cell cycle arrest in G1 or apoptosis under conditions of excessive damage. This growth inhibition is associated with the concomitant induction of p53 and enhanced terminal cell differentiation. In this study we evaluated the effects of PAH on cell growth, cell differentiation, xenobiotic metabolism, and DNA adduct levels in normal ectocervical epithelial cells (ECE) and compared them to cervical cells whose p53 have been inactivated either by binding to viral HPV E6 oncogene (ECE16-1) or by mutation (C33A). The PAH 3-methylcholanthrene (3MC) inhibited normal ECE and to a lesser extent ECE16-1 cell proliferation. Not only did the growth inhibition occur at lower concentrations in the normal cells but the extent of inhibition was also greater in normal as compared to immortalized cells. Benzanthracene (BA) had a minor effect on normal ECE cells with no effect on immortalized ECE16-1 cells. C33A cell growth was unaffected by 3MC and BA. Terminal cell death was enhanced only in normal ECE cells as evidenced by increased envelope formation and was paralleled by an increase in the level of p53 following 3MC treatment. The differentiation status of the 3MC-treated cells was similar to untreated cells as indicated by three independent markers of cell differentiation; transglutaminase, involucrin, keratin expression. There was no difference in the pattern or level of DNA adducts formed in normal and immortalized cells following 3MC treatment. In addition the basal level of metabolism of 14C-BaP to phenols, diols and quinnones was unaltered by pretreatment with either 3MC or BA. These results demonstrate that immortalized cervical cells are less sensitive to toxicant damage [i.e. cell proliferation and terminal differentiation], and as a result, immortalized cells proliferate in the presence of genotoxic damage and are at increased risk for mutations and cancer.

Impact of receptor downregulation on clearance of two human EGFs with different receptor binding activity.

Human epidermal growth factor [hEGF(1-53)] has been thought to be cleared mainly via an EGF receptor (EGFR) endocytosis pathway. Pretreatment of rats with hEGF(1-53) has been shown previously to cause a dramatic reduction in clearance of the peptide contributable to EGFR downregulation. The impact of receptor downregulation has raised concerns for rational design of dosage regimen for this potential wound-healing therapeutic peptide. However, following a similar protocol, we could not reproduce the dramatic reduction in clearance reported previously mediated by an i.v. bolus acute dose. As EGFR downregulation may be sensitive to the length of exposure and to the activation of the receptor tyrosine kinase activity, two other pretreatment protocols were also evaluated: a 4-h i.v. infusion (prolonged exposure) of the peptide and an i.v. bolus of a potent synthetic kinase inhibitor pretreatment were evaluated for effects on clearance. However, neither pretreatment affected the peptide's clearance profile. Further, no effects on clearance and other kinetic parameters were observed for any pretreatment paradigms with a truncated analogue hEGF (1-48), whose EGF receptor binding activity is much weaker but plasma clearance is much higher than hEGF (1-53). In addition, a study in a second rat strain showed no difference in clearance profile of hEGF-(1-53) following pretreatment. Results of the present investigation suggest that receptor binding does not have a direct relationship with plasma clearance, and that the EGF clearance mechanisms is highly refractory with EGF receptors possibly recovering rapidly from downregulation through the recycling process.

Tamoxifen regulation of ectocervical cell differentiation.

OBJECTIVE: To determine the effects of tamoxifen on the growth and differentiation of normal human cervical cells and compare those effects with those of a synthetic estrogen, diethylstilbestrol (DES). In addition, the effects of these compounds on immortalized cervical cells and cervical tumor cells were ascertained. METHODS: Growth curves were used to determine the effects on cell proliferation. The expression of several proteins was used to determine the effects on cell differentiation. Binding assays and Western analysis were used to determine estrogen receptor levels. RESULTS: Both tamoxifen and DES inhibited the proliferation of normal cervical cells. This growth inhibition was coincident with an increase in cell differentiation as determined by cornified envelope formation. The increase in envelope number was not accompanied by an increase in involucrin or cornifin, two protein precursors of the envelope. The activity of transglutaminase, which enzymatically incorporates precursor proteins into the envelope, was not stimulated following treatment. Diethylstilbestrol did not alter the growth or differentiation of the human papillomavirus 16-immortalized cell line ECE16-1 or any of the cervical tumor cell lines. Of all the immortalized or cancer cell lines, only Caski cells were growth inhibited by tamoxifen. Normal ectocervical epithelial cells and Caski cells expressed the high-affinity 56-kDa estrogen receptor, but 3H-estradiol binding was not detected in cell extracts from either ME180 or ECE16-1 cells. Nevertheless, extracts from ME180 cells contained an immunoreactive band at the appropriate molecular weight for the estrogen receptor. CONCLUSIONS: These results suggest that tamoxifen and DES act similarly in normal cervical cells to promote cervical cell differentiation. However, because Caski cell growth was inhibited by tamoxifen and not DES, the effects of tamoxifen in these cells may be mediated by non-estrogen receptor mechanisms.

Human cervical cancer. Retinoids, interferon and human papillomavirus.

Our studies highlight the importance of dietary vitamin A (retinol) and other retinoids in maintaining normal cervical cell function and in inhibiting the growth of cervical tumors. Based on our results we conclude that 1) HPV 16-immortalization enhances cervical cell sensitivity to retinoids, 2) cytokeratin expression may be useful as a marker for evaluating the success of retinoid therapy in vivo, 3) retinoids do not necessarily act to inhibit proliferation of HPV-immortalized cervical cells via effects on HPV E6 and E7 RNA levels and 4) retinoids may act to inhibit cervical proliferation by "suppressing" the activity of the EGF and IGF signalling pathways. Based on these and other results, it is worth considering the possibility that vitamin A or related retinoids could be administered therapeutically, early in the neoplastic process (either systemically or locally), to inhibit the progress of the disease. These results also suggest that combined interferon/retinoid therapy may provide an enhanced beneficial effect to reduce cervical tumor size due to the fact that each agent is inhibiting cervical cell proliferation via distinct, but reinforcing, pathways (i.e., IFN gamma reduces E6/E7 expression, RA inhibits the function of the EGF and IGF1 signalling pathways).

Mutated in colorectal cancer, a putative tumor suppressor for serrated colorectal cancer, selectively represses beta-catenin-dependent transcription.

Mutated in colorectal cancer (MCC) was originally identified as a candidate gene for familial adenomatous polyposis (FAP) but further study identified adenomatous polyposis coli (APC) as responsible for FAP and the physiologic/pathologic roles of MCC remained poorly understood. Recently, MCC promoter methylation was discovered as a frequent early event in a distinct subset of precursor lesions and colorectal cancer (CRC) associated with the serrated CRC pathway. Here we provide the first evidence of the biological significance of MCC loss in CRC and the molecular pathways involved. We show MCC expression is dramatically decreased in many CRC cell lines and the distinct subset of sporadic CRC characterized by the CpG island methylator phenotype and BRAF(V600E) mutation due to promoter methylation as reported previously. Importantly, we find MCC interacts with beta-catenin and that reexpression of MCC in CRC cells specifically inhibits Wnt signaling, beta-catenin/T-cell factor/lymphoid-enhancer factor-dependent transcription and cellular proliferation even in the presence of oncogenic mutant APC. We also show that MCC is localized in the nucleus and identify two functional nuclear localization signals. Taken together, MCC is a nuclear, beta-catenin-interacting protein that can act as a potential tumor suppressor in the serrated CRC pathway by inhibiting Wnt/beta-catenin signal transduction.

Calcium modulates cornified envelope formation, involucrin content, and transglutaminase activity in cultured human ectocervical epithelial cells.

The entrance to the cervical canal, the ectocervix, is covered by a stratified squamous epithelium. In the present study we demonstrate that the extracellular calcium levels regulate the differentiation of cultured human ectocervical epithelial cells (ECE cells). ECE cells growing in serum-free medium containing 0.09 mM calcium remain as individual cells that do not spread on the culture dish or form cornified envelopes. Increasing the extracellular calcium concentration results in a dose-dependent increase in ECE cell differentiation that is manifest by an increase in cornified envelope (superficial cell) formation. These morphological changes are accompanied by a twofold increase in the levels of involucrin, a precursor of the cornified envelope. The extent of increase in involucrin mRNA is similar to that of involucrin protein, suggesting that the regulation is at the level of involucrin mRNA synthesis and/or stability. Transglutaminase, a calcium-activated enzyme responsible for assembly of the covalent cross-links that form the cornified envelope, increases to a similar extent to the increase in cornified envelope formation, suggesting that in ECE cells, activation of transglutaminase may be the rate-limiting step in envelope assembly. These results and the finding of a calcium gradient in other stratifying epithelia in vivo, indicate that the calcium is likely to be an important regulator of ectocervical epithelial cell differentiation in vivo.

Transforming growth factor beta regulation of epidermal growth factor receptor in ectocervical epithelial cells.

Transforming growth factor beta (TGF beta) is a pluripotent modulator of cell function and an important suppressor of cervical epithelial cell proliferation. In the present study, we examine the effects of TGF beta 1 on the level and activity of the epidermal growth factor receptor (EGFR) in HPV-16 immortalized cervical epithelial cells. In ECE16-1 cells, increased EGFR levels are observed within 24 h after initiation of TGF beta 1 treatment and levels continue to increase with time. This increase is correlated with a TGF beta 1-dependent decrease in proliferation rate. Scatchard analysis indicates that the population of EGFR sites induced by TGF beta 1 have a low affinity for EGF (Kd = 4.08 nM) compared to the receptors present prior to TGF beta 1 treatment (Kd = 0.3 and 1.6 nM). TGF beta 1 treatment also reduces EGFR kinase autophosphorylation activity. Cell cycle studies indicate that TGF beta 1-treated cells arrest in the G1 phase of the cell cycle and that regulation of EGFR level was independent of cell cycle stage in both TGF beta 1-treated and untreated cells. However, EGFR level was related to the G1 phase time. Parallel studies indicate that a TGF beta 1-dependent increase in p53 level is also correlated with increased time spent in G1. These results suggest that TGF beta 1 inhibition of ECE16-1 cell proliferation may act both by the replacement of high affinity/high kinase activity EGFR sites with low affinity/low kinase activity EGFR sites and a p53-mediated cell cycle arrest.

Differential response of normal and HPV immortalized ectocervical epithelial cells to B[a]P.

Cigarette smoking has been established as a risk factor for the development of cervical cancer. Polycyclic aromatic hydrocarbons such as benzo[a]pyrene (B[a]P), which are present in cigarette smoke, might account for this increased risk. The effects of B[a]P on cell growth, aryl hydrocarbon hydroxylase, DNA adducts and p53 levels was measured in cervical cells. Since 90% of cervical preneoplastic lesions are positive for the human papillomavirus (HPV) we compared the effects of these chemicals in normal ectocervical epithelial cells (ECE) and human papillomavirus 16 (HPV16) immortalized ectocervical epithelial cells (ECE16-1). Exposure of normal ECE and HPV immortalized ECE16-1 cells to B[a]P inhibited cell proliferation. Inhibition occurred at 20-fold lower concentrations in the normal ECE cells compared to ECE16-1 cells. The proliferation of cervical cells which express mutated p53 was unaffected by B[a]P. Neither cervical stromal cells nor endometrial stromal cells were affected by these compounds. The effects of B[a]P on normal ECE cell proliferation correlated with increased terminal differentiation as measured by increased envelope formation. In contrast, B[a]P exposure did not induce envelope formation in immortalized ECE16-1 cells or in cervical tumor cells. Pretreatment of both ECE and ECE16-1 cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin, which induces P450 expression and activity, did not alter B[a]P metabolism in either normal or immortalized cells. Furthermore, equivalent levels of DNA adducts were formed by B[a]P in ECE and ECE16-1 cells. Neither the extent of adduct formation nor the rate of their removal differed in normal and immortalized cervical cells. Therefore, the diminished growth inhibition of the ECE16-1 cells as compared to normal ECE cells by B[a]P is not due to changes in cytochrome P450 of the 1A family metabolism or DNA adduct number. Furthermore, analysis of the p53 levels in both normal and ECE16-1 cells revealed that p53 levels are higher in normal versus immortalized ectocervical cells, and p53 is induced in both cell types following B[a]P treatment. Thus reduced p53 levels in ECE16-1 cells may contribute to a lack of growth suppression following B[a]P treatment. These results demonstrate that HPV16 immortalization diminishes ectocervical epithelial cell responsiveness to toxicant damage (i.e. decreased cell proliferation and increased terminal differentiation). As a result, ECE16-1 cells that sustain genotoxic damage which leads to DNA adduct formation continue to proliferate and may be at increased risk for mutations and further progression towards a fully transformed phenotype.

Transcriptional regulation of the EGF receptor promoter by HPV16 and retinoic acid in human ectocervical epithelial cells.

We have previously demonstrated that human papillomavirus 16 (HPV16)-immortalized human ectocervical epithelial cells and cells derived from tumors which express HPV16 oncogenes express high levels of epidermal growth factor receptor (EGFR) compared to normal cervical cells. We have also shown that proliferation of these cells is inhibited by retinoid treatment. We have hypothesized that the retinoid inhibition of cell proliferation may be due to the retinoid-dependent reduction in EGFR level. In this study we examine the regulation of EGFR expression in cervical cells with emphasis on two aspects: (1) the mechanism of retinoid-dependent suppression of EGFR levels in HPV16-positive cells and (2) the mechanism of EGFR upregulation by HPV16. EGFR levels were found to be elevated 5-, 3. 7-, and 1.25-fold in the HPV16-immortalized ECE16-1, ECE16-D1, and ECE16-D2 cells, respectively, compared to normal cervical cells. Treatment of ECE16-1 and ECE16-D1 cells with retinoic acid suppresses proliferation, EGFR level, EGFR mRNA level, and EGFR promoter activity. The reduction in EGFR promoter activity appears to account for the reduction in EGFR protein and mRNA levels. In contrast, retinoic acid does not affect cell growth or EGFR level in ECE16-D2 cells or normal cervical cells. To study the mechanisms regulating EGFR expression in HPV16-positive cells, normal ECE cells were cotransfected with an EGFR promoter reporter plasmid and an expression plasmid encoding the HPV16 E6/E7 open reading frames. In the presence of E6/E7, EGFR promoter activity was increased by 2- to 3-fold, suggesting that the E6/E7 proteins are directly or indirectly responsible for the increased EGFR level and that the EGFR promoter contains the DNA elements necessary to mediate this response. Nevertheless expression of E6/E7 proteins did not confer retinoic acid regulation, as EGFR promoter activity remained elevated in normal cells cotransfected with pHPVE6/E7 and treated with retinoic acid. These results suggest that human papillomavirus and retinoic acid regulate EGFR levels by independent effects on the EGFR promoter.

Retinoid regulation of human ectocervical epithelial cell transglutaminase activity and keratin gene expression.

Cornified envelope formation, the level of transglutaminase activity and the pattern of cytokeratin gene expression are important biochemical markers of cervical epithelial cell differentiation in vivo. In the present study we examine the effects of retinoid treatment on transglutaminase (TG) activity and keratin gene expression in cultured human ectocervical epithelial cells (ECE cells). All-trans-retinoic acid (RA) and a synthetic retinoid, Ro 13-6298, suppress TG activity by 85-90% with half-maximal inhibition at 0.1 nM Ro 13-6298 or 1 nM RA. In contrast, the predominant circulating retinoid, retinol, does not inhibit TG activity. The level of type I transglutaminase protein, measured using a type I TG-specific antibody, decreases in parallel with the decrease in activity as does the level of the TG RNA transcript. Cytokeratin K16 decreases more than 20-fold while the level of K7, K8 and K19 increase 5 to 10-fold in the presence of 10 nM RA. Studies using cDNAs encoding K5, K13, K16 and K19 indicate that the RNA transcript levels change in parallel with the change in keratin protein production. Thus, all-trans-retinoic acid suppresses ectocervical epithelial cell differentiation in vitro, a result that suggests an in vivo role for retinoids in regulating cervical cell differentiation.

A raf-independent epidermal growth factor receptor autocrine loop is necessary for Ras transformation of rat intestinal epithelial cells.

We recently have shown that activated Ras, but not Raf, causes transformation of intestinal (RIE-1, IEC-6) epithelial cells, whereas both activated Ras and Raf transform NIH 3T3 fibroblasts (Oldham, S. M., Clark, G. J., Gangarosa, L. M., Coffey, R. J., and Der, C. J. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 6924-6928). The observations that conditioned medium from Ras-, but not Raf-, transfected RIE-1 cells, as well as exogenous transforming growth factor alpha (TGFalpha), promoted morphological transformation of parental RIE-1 cells prompted us to identify epidermal growth factor (EGF) receptor (EGFR) ligands produced by Ras-transformed RIE-1 cells responsible for this autocrine effect. Since studies in fibroblasts have shown that v-Src is transforming, we also determined if v-Src could transform RIE-1 cells. H- or K-Ras-transformed cells secreted significant amounts of TGFalpha protein, and mRNA transcripts for TGFalpha, amphiregulin (AR), and heparin-binding EGF-like growth factor (HB-EGF) were induced. Like Ras, v-Src caused morphological and growth transformation of parental RIE-1 cells. However, TGFalpha protein was not secreted by RIE-1 cells stably expressing v-Src or activated Raf, and only minor increases in EGFR ligand mRNA expression were detected in these cells. A selective EGFR tyrosine kinase inhibitor PD153035 attenuated the Ras-, but not Src-, transformed phenotype. Taken together, these observations provide a mechanistic and biochemical basis for the ability of activated Ras, but not activated Raf, to cause transformation of RIE-1 cells. Finally, we suggest that an EGFR-dependent mechanism is necessary for Ras, but not Src, transformation of these intestinal epithelial cells.

Development and validation of two solid-phase enzyme immunoassays (ELISA) for quantitation of human epidermal growth factors (hEGFs).

PURPOSE: The purpose of the present investigation was to develop and validate two separate enzyme-linked immunosorbent assays (ELISA) for quantitation of exogenous human epidermal growth factor (hEGF1-53) and its truncated fragment (hEGF1-48) in rat plasma. METHODS: The present assay systems were based on the sandwiching of the antigen between a monoclonal mouse anti-hEGF1-53 antibody, pre-coated on a 96-well polystyrene plate, and a polyclonal rabbit anti-hEGF1-48 antibody, which is then detected with a peroxidase-labeled goat anti-rabbit antibody. RESULTS: The calibration curves for hEGF1-48 and hEGF1-53 in plasma were validated over a concentration range of 7.8-250 and 62.5-1000 pg/ml, respectively. Determined from replicate assays of hEGF1-48 quality control samples, the intra-assay precision and accuracy were < or = 8.8% RSD and within +/- 9.8%; and the inter-assay precision and accuracy were < or = 14.8% RSD and within +/- 9.7% RE, respectively. Determined from replicate assays of hEGF1-53 quality control samples, the intra-assay precision and accuracy were < or = 10.0% RSD and within +/- 8.5%; and the inter-assay precision and accuracy were < or = 10.0% RSD and within +/- 5.7% RE, respectively. The limit of quantitation of the hEGF1-48 and hEGF1-53 assay using 200 microliters plasma per well is 7.8 and 62.5 pg/ml, respectively. These two ELISA methods are specific to hEGFs and do not cross-react with mouse EGF or other growth factors (TGF alpha, TGF beta, PDGF, and FGF) or lymphokines (IL1 beta and TNF alpha). These validated methods have been routinely applied to assay of plasma samples from various pharmacokinetic studies in rats receiving intravenous hEGFs. Both assay methods were also adapted to assay endogenous hEGFs in biological fluids of different animal species. CONCLUSIONS: Two sensitive ELISA methods have been validated for quantitation of hEGF1-53 and hEGF1-48 in rat plasma. Their utility has been demonstrated in the application of assaying immunoreactive concentration of exogenous and endogenous epidermal growth factors.

Pharmacological inhibition of Ras-transformed epithelial cell growth is linked to down-regulation of epidermal growth factor-related peptides.

BACKGROUND & AIMS: Posttranslational farnesylation is required for Ras activation. Farnesyl transferase inhibitors (FTIs) selectively block protein farnesylation and reduce the growth of many Ras-transformed cells in vitro and in vivo. Activated Ras transforms rat intestinal epithelial (RIE-1) cells by a mechanism distinct from NIH 3T3 fibroblasts in that an epidermal growth factor receptor (EGFR) autocrine loop contributes significantly to the Ras-transformed RIE-1 phenotype. METHODS: The ability of FTIs to block growth of Ras-transformed RIE-1 cells was evaluated, and these results were correlated with decreased EGFR ligand production. RESULTS: FTI L744,832 caused a selective, dose-dependent, reversible blockade in proliferation of H-Ras-transformed RIE-1 cells, whereas control cell lines, K-Ras-transformed cells, and activated raf-transfected RIE cells were unaffected. The growth-inhibitory effects of L744,832 correlated with loss of farnesylated H-Ras protein and a marked reduction in transforming growth factor (TGF)-alpha and amphiregulin expression. Inhibition of proliferation of H-Ras RIE-1 cells by L744,832 was overcome by exogenous TGF-alpha, and enhanced growth inhibition was achieved by EGFR blockade in combination with L744,832. + CONCLUSIONS: These data suggest that one mechanism by which FTIs inhibit growth of H-Ras-transformed epithelial cells is by reducing Ras-induced EGFR ligand production.

A role for NF-kappa B in the induction of beta-R1 by interferon-beta.

Previous experiments have suggested that induction of the beta-R1 gene by interferon (IFN)-beta required transcription factor ISGF-3 (IFN-stimulated gene factor-3) and an additional component. We now provide evidence that nuclear factor-kappaB (NF-kappaB) can serve as this component. Site-directed mutagenesis of an NF-kappaB binding site in the beta-R1 promoter or over-expression of an IkappaBalpha super-repressor abrogated IFN-beta-mediated induction of a beta-R1 promoter-reporter. IFN-beta treatment did not augment abundance of NF-kappaB but did lead to phosphorylation of the p65 NF-kappaB subunit. It is proposed that IFN-beta-mediated enhancement of the transactivation competence of NF-kappaB components is required for inducible transcription of the beta-R1 promoter. These results provide a novel insight into the role of NF-kappaB in the transcriptional response to IFN-beta.