Effects of EGF and calcium on adult parenchymal hepatocyte proliferation

Adult rat hepatocytes were grown in serum-free medium containing 0.05-4 mM Ca++ and 40 ng/ml EGF. After 48 hours of cultivation the mitotic index and the percentage of second division metaphases were determined. The results demonstrated a maximum proliferation response to EGF at a Ca++ concentration of 0.4 mM. With lower and higher external Ca++ concentrations the fraction of cells undergoing more than one cell division decreased. At lower Ca++ concentrations this decrease appears to result from a reduced viability. In contrast, the low response to EGF at higher Ca++ concentrations--especially in the physiological range--may reflect the influence of Ca++ on the state of hepatocyte differentiation.     

Lead transport in IEC-6 intestinal epithelial cells

This study evaluated the use of IEC-6 cells as a model for studying lead (Pb) transport by intestinal epithelial cells (IECs) and examined potential transport mechanisms for Pb uptake and extrusion. Pb accumulation in IEC-6 cells exposed to 5 and 10 μM Pb for up to 60 min was time- and dose-dependent. Reduction of incubation temperature significantly reduced the total cellular Pb content of IEC-6 cells. Simultaneous exposure of cells to zinc (Zn) and Pb resulted in decreased total cellular Pb contents compared to total cellular Pb contents of cells exposed to Pb only. IEC-6 cells treated with ouabain (1 mM) or sodium azide (1 mM) and 5 μM Pb accumulated more Pb than cells exposed to Pb only. Cells treated withp-chloromercuribenzensulfonic acid (50 μM),p-chloromercuribenzoic acid (50 μM), or iodoacetimide (50 μM) accumulated less Pb than cells treated with Pb only. We conclude that Pb uptake by IEC-6 cells depends on the extracellular Pb concentration. Our data suggest that the mechanism of Pb uptake by IECs is complex, and that Pb transport in IEC-6 cells is time- and temperature-dependent, involves sulfhydryl groups, and is decreased by the presence of Zn. Extrusion of Pb is at least partially dependent on metabolic energy.     

RhoA stimulates IEC-6 cell proliferation by increasing polyamine-dependent Cdk2 activity

Although RhoA plays an important role in cell proliferation and in Ras transformation in fibroblasts and mammary epithelial cells, its role in intestinal epithelial cells (IEC) is unknown. In a previous study (Ray RM, Zimmerman BJ, McCormack SA, Patel TB, and Johnson LR. Am J Physiol Cell Physiol 276: C684-C691, 1999), we showed that polyamine depletion [dl-alpha-difluoromethylornithine (DFMO) treatment] strongly inhibits the proliferation of IEC. In this report, we examined the effect of RhoA on IEC-6 cell proliferation and whether polyamine depletion inhibits cell proliferation in the presence of constitutively active RhoA. Constitutively active RhoA and vector-transfected IEC-6 cell lines were grown in the presence or absence of DFMO, which causes polyamine depletion by inhibiting ornithine decarboxylase, the first rate-limiting step in polyamine synthesis. Constitutively active RhoA significantly increased the rate of cell proliferation. These cells also lost contact inhibition and formed conspicuous foci when they were fully confluent. Decreased p21Waf1/Cip1 expression and increased cyclin-dependent kinase (Cdk2) mRNA levels and activity accompanied the increased proliferation. The inhibition of p21Waf1/Cip1 was independent of p53. There was no activation of the Ras-Raf-MEK-ERK pathway in the RhoA-transfected cell line. Polyamine depletion totally prevented the effect of activated RhoA on IEC-6 cell proliferation, focus formation, and Cdk2 expression. The stability of mRNA and protein for Cdk2 and p21Waf1/Cip1 in V14-RhoA cells was not significantly different from that of vector-transfected cells. In conclusion, RhoA activation decreased p21Waf1/Cip1 expression and increased basal and serum-induced ornithine decarboxylase activity, Cdk2 expression, Cdk2 protein, and Cdk2 activity, leading to the stimulation of IEC proliferation and transformation. Polyamine depletion totally prevented RhoA's effect on proliferation by decreasing Cdk2 expression and activity.     

Influence of somatostatin and epidermal growth factor (EGF) on the proliferation of thyroid follicular cells in organ culture

The aim of the present study has been to examine the effects of various concentrations of somatostatin (SS), epidermal growth factor (EGF), as well as of interactions among SS, EGF and thyrotropin (TSH) in their influence upon the mitotic activity of thyroid follicular cells (TFC) in organ culture. The stathmokinetic method was employed. It was shown that: (1) SS, at the concentration of 10(-7) M, suppressed the mitogenic effect of TSH, as well as of TSH and EGF employed together, on TFC; (2) EGF, at the concentration of 10 and 100 ng/ml, increased the mean mitotic activity rate of TFC; (3) TSH and EGF revealed an additive action on TFC proliferation. The obtained results evidently suggest an antiproliferative effect of SS and mitogenic action of EGF on TFC in organ culture.     

Non-steroidal anti-inflammatory drugs affect the methotrexate transport in IEC-6 cells

Methotrexate (MTX) is used not only for the cancer chemotherapy but also for the treatment of rheumatic disease, often together with non-steroidal anti-inflammatory drugs (NSAIDs). MTX is actively cotransported with H(+) in the small intestine, mediated by a reduced folate carrier (RFC). The coadministration of some NSAIDs with MTX to rats caused a decrease of MTX absorption through the small intestine. This may be due to the uncoupling effect of oxidative phosphorylation of the NSAIDs. The present study investigated whether flufenamic acid, diclofenac and indomethacin, NSAIDs, decreased ATP content of rat-derived intestinal epithelial cell line IEC-6 cells and affected the MTX transport in IEC-6 cells. The MTX uptake in IEC-6 cells was dependent on medium pH and maximum around pH 4.5-5.5. The MTX uptake was composed of a transport inhibited by 4, 4'-diisothiocyanostilbene-2, 2'-disulfonic acid (DIDS) and a non-saturable one. The DIDS-sensitive component in the MTX uptake showed a saturation kinetics (Michaelis-Menten constant (Km): 3.91 +/- 0.52 microM, Maximum velocity (Vmax): 94.66 +/- 6.56 pmol/mg protein/5 min). The cellular ATP content in IEC-6 cells decreased significantly at 30 min after the cells were started to incubate with the NSAIDs (250 microM flufenamic acid, 500 microM diclofenac and 500 microM indomethacin). The MTX uptake in IEC-6 cells in the presence of the NSAIDs decreased with the reduction of cellular ATP content and showed a good correlation with the ATP content (correlation coefficient: 0.982). Thus it seems likely that the ATP content in IEC-6 cells with the NSAIDs decreased due to the uncoupling effect of oxidative phosphorylation of the NSAIDs, resulting in the inhibition of the secondary active transport of MTX in IEC-6 cells. The present results also suggest that IEC-6 cells are useful to evaluate the drug interaction relating to this carrier system.     

Effects of TCDD on embryonic ureteric epithelial EGF receptor expression and cell proliferation

The potent toxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is teratogenic in mice, producing hydronephrosis and cleft palate. Because of the long half-life of TCDD, the urinary tract is exposed throughout development after a single dose on gestation day (GD) 10 or earlier. TCDD-induced hydronephrosis is a consequence of occlusion of the ureter by epithelial cells. Since embryonic growth factors and the epidermal growth factor (EGF) receptor are probably involved in regulation of embryonic cell proliferation, this study examines the effects of TCDD on expression of EGF receptors and proliferation of ureteric epithelial cells in vivo and in culture. After exposure to TCDD by gavage (12, 24, or 30 micrograms/kg on GD 10; 6 or 24 micrograms/kg on GD 12) the mean cell depth of the ureteric and bladder epithelia was increased. EGF receptors were detected immunohistochemically in sectioned urinary tracts. The expression of receptors decreased with advancing development in control ureteric epithelia. However, after TCDD exposure the level of EGF receptors failed to decline. The incorporation of 3H-TdR was observed in sections by autoradiography, and after exposure to TCDD more epithelial cells showed incorporation than was apparent in controls. Transmission electron microscopy (TEM) of embryonic ureters from fetuses exposed to TCDD in vivo showed no cytotoxicity in basal cells and the cells remained undifferentiated, as in controls. Ureters taken from GD 12 embryos and cultured with 1 x 10(-10)M TCDD showed ureteric epithelial hyperplasia without cytotoxicity, but at 1 x 10(-8)M TCDD evidence of cytotoxicity was observed by TEM. The levels of TCDD found in fetuses after in vivo exposure (204-307 pg/fetus, with 1-2 pg in the urinary tract) compare well with the in vitro level (32 pg/ml), which was most effective in producing hyperplasia of the epithelial cells. The present study correlates a TCDD-induced increase in cell depth with altered regulation of EGF receptors and excessive proliferation, both in vivo and in cultured embryonic ureters.     

PMA decreases the proliferation of retinal cells in vitro: the involvement of acetylcholine and BDNF

Protein kinase C (PKC) is involved in several cell events including proliferation, survival and differentiation. The aim of this work was to investigate the role of PKC activation on retinal cells proliferation. We demonstrated that PKC activation by phorbol 12-myristate 13-acetate (PMA), a tumor promoter phorbol ester, is able to decrease retinal cells proliferation. This effect was mediated by M1 receptors and dependent on intracellular Ca(2+) increase, tyrosine kinase activity, phosphatidylinositol 3-kinase activity, polypeptide secretion and activation of TrkB receptors. The effect of PMA was not via activation of mitogen-activated protein (MAP) kinase. Carbamylcholine and brain derived neurotrophic factor were both able to decrease retinal cells proliferation to the same level as PMA did. Our results suggest that PKC activation leads to a decrease in retinal cells proliferation through the release of acetylcholine and brain derived neurotrophic factor in the culture, and activation of M1 and TrkB receptors, respectively.     

Influence of epidermal growth factor (EGF) on ruffling activity, pinocytosis and proliferation of cultivated human glia cells

Normal human glia cells in culture were studied with respect to ruffling activity, macro-pinocytosis and cell proliferation under standard culture conditions with 10% serum in the medium, in serum-free medium and after addition of epidermal growth factor (EGF) or serum to previously serum-free medium. Pinocytotic uptake of droplets of medium occurred only in relation to well developed ruffling membranes. Omitting the serum from the medium led to a drastic reduction in thymidine incorporation. The cells became slender under these conditions, and soon after the change of medium their ruffling activity and pinocytosis were almost completely abolished. Following the change to a medium containing 2 ng EGF/ml a rapid reappearance of ruffling and pinocytosis was observed. DNA synthesis, however, was not demonstrated until after 20 h, showing that ruffling and pinocytosis occurred before DNA synthesis had started. Thus EGF may initially induce conformational changes of the plasma membrane, resulting in its internalization due to formation of endocytotic vacuoles. The observed relationship between occurrence of well developed ruffling membranes, macro-pinocytosis and cell multiplication indicates that one of the functions of growth-promoting factors may be stimulation of plasma membrane turnover.     

Comparison of the efficacy of alpha-lactalbumin from equine, bovine, and human milk in the growth of intestinal IEC-6 cells

Native alpha-lactalbumins (α-LA) from equine, bovine, and human milk were not cytotoxic. However, after treatment with trifluoroethanol (TFE), all three α-LAs exhibited cytotoxicity. Toxic potencies were distinctly different among them. Equine α-LA was the most robust, bovine α-LA was moderate, and human α-LA was weak. There were no significant structural changes as between the native and the TFE-treated α-LAs.     

Effects of Interferon γ on growth,apoptosis, and MHC class II expression of immature rat intestinal crypt (IEC-6) cells

Intestinal epithelial cells and the mucosal immune cells in close proximity are thought to interact very closely. One well-established mechanism of this intercellular cross-talk is via the production of cytokines such as interferon gamma (IFNγ). The aim of this study was to analyze the effects of IFNγ on intestinal crypt epithelial cells. IEC-6 cells were cultured in the presence or absence of IFNγ to measure its effects on proliferation, cell cycle, apoptosis, and major histocompatibility complex (MHC) class II antigen expression. Even at very low doses (0.01 U/ml), IFNγ significantly inhibited IEC-6 cell proliferation, as demonstrated by reduced ³H-thymidine uptake, stable cell count, and complete arrest in the quiescent G₀/G₁ phase of the cell cycle. Incubation with supraphysiological doses of IFNγ (100–1,000 U/ml) did not induce apoptosis, as assessed by morphology and the TUNEL assay. IFNγ significantly induced de novo IEC-6 class II antigen expression. Tumor necrosis factor alpha (TNFα), which alone had no effect, synergistically enhanced this effect of IFNγ. MHC class II antigen expression was observed to be independent of cell cycle phase. Our results indicate that IFNγ alters immature crypt epithelial cell turnover and upregulates MHC class II expression. These alterations may be important in the pathogenesis of immune-mediated bowel disorders. J. Cell. Physiol. 176:120–126, 1998. © 1998 Wiley-Liss, Inc.     

Effects of insulin on cellular growth and proliferation

Insulin stimulates the growth and proliferation of a variety of cells in culture. The growth-stimulatory effects of insulin are observed in G_o/G_l arrested cells limited for serum growth factors or essential nutrients, and in cells growing in hormone-supplemented serum-free media. Some, but not all, of the effects of insulin on growth require superphysiological concentrations of insulin. The action of insulin on growth is synergistic with the action of other hormones and growth factors, including FGF, PDGF, PGF_2α and vasopressin. This observation, as well as other observations regarding the temporal sequence of action of growth factors, suggests that different growth factors act on different intracellular biochemical events. Several hypotheses have been proposed to explain the effect of insulin on cellular proliferation, including regulation of essential metabolic processes and interaction of insulin with receptors for insulin-like growth factors. Evidence supporting these various hypotheses is reviewed. In addition to the growth-stimulatory effect of insulin observed in cell culture, a number of clinical examples suggest that insulin is an important growth-regulating hormone during fetal development.     

Effects of insulin-like growth factor-II on proliferation and differentiation of ovarian granulosa cells.

The effects of insulin-like growth factor-II (IGF-II) on the proliferation and differentiation of ovarian granulosa cells were studied in cultured human and porcine granulosa cells. IGF-II significantly increased basal progesterone secretion in granulosa cells at concentrations of 1-100 ng/ml. A stimulatory effect was also observed in gonadotropin-stimulated porcine granulosa cells treated with IGF-II. The secretion of estradiol by basal and gonadotropin-stimulated porcine granulosa cells was also significantly increased by adding IGF-II. IGF-II led to dose-dependent increases in [3H]thymidine incorporation into DNA and in the number of granulosa cells. To further characterize the cellular mechanisms underlying the stimulatory effects of IGF-II on the proliferation and differentiation of granulosa cells, we investigated the intermediary roles of cyclic AMP and intracellular Ca2+ concentration ([Ca2+]i). Treatment with 100 ng/ml IGF-II produced a significant increase in the basal accumulation of cyclic AMP in porcine granulosa cells. However, no change of [Ca2+]i by IGF-II was noted. IGF-II produced effects in accumulation that were similar to those of IGF-I. Our findings suggest that IGF-II may be a general stimulator in the proliferation and differentiation of granulosa cells, and that cyclic AMP may be a second messenger for the effects of IGF-II in ovarian granulosa cells.     

Transforming growth factor-beta enhances secretory component and major histocompatibility complex class I antigen expression on rat IEC-6 intestinal epithelial cells.

Transforming growth factor-beta (TGF-beta) has been implicated as having a role in inflammatory responses by inducing cellular infiltration and the release of inflammatory cytokines. In this study, the IEC-6 rat intestinal epithelial cell line was used as a model to assess the effect of TGF-beta 1 on the expression of various plasma membrane determinants. TGF-beta 1 induced a dose-dependent increase in the percentage of cells expressing surface secretory component (SC) and class I major histocompatibility (MHC) antigens. However, the expression of class II MHC was unaffected. In contrast, epidermal growth factor had no effect on any of the surface proteins studied. The TGF-beta 1-enhanced expression of SC was accompanied by an enhanced binding of polymeric, but not monomeric, immunoglobulin A (IgA). Preincubation of the TGF-beta 1-treated cells with an anti-human beta-galactosyltransferase (beta-GT) antiserum did not block the binding of the anti-SC antibody, indicating that the TGF-beta-induced increase in SC staining was due to SC expression and not the polymeric immunoglobulin-binding enzyme, beta-GT. These results indicate that TGF-beta 1 may be important in immune functions involving intestinal epithelial cells by enhancing the expression of surface class I MHC antigens and SC, a protein responsible for the transport of polymeric IgA into the intestinal lumen.     

EGFR expression and EGF stimulation of proliferation in human liver carcinoma cells

Epidermal growth factor receptor (EGFR) gene expression and growth stimulation of EGF on human hepatoma cells of cell lines BEL-7404 and SMMC-7721 were studied. 125I-EGF binding assay was used to measure the binding characteristics and the amounts of EGFR on these cells. The binding time course and the binding competition assay showed that the binding of 125I-EGF to 7404 cells was saturable and specific. Scatchard analysis of EGF binding curve indicated that 7404 and 7721 cells expressed approximately 1.1 x 10(5) and 0.7 x 10(5) EGFRs per cell with binding affinity (Kd) 2.1 nM and 1.8 nM respectively. Northern hybridization and immunoblotting analysis showed the EGFR gene expression products in 7404 and 7721 cells were 5.6 Kb mRNA and 170 Kilo-dalton glycoprotein. Anchorage-dependent growth of 7404 and 7721 cells was stimulated in the presence of nanogram quantities of EGF in medium containing 10% calf serum or 0.5% calf serum. The factors in serum appeared to act synergitically in stimulating of cell proliferation. EGF also stimulated the anchorage-independent growth of 7404 and 7721 cells in soft agar. The results suggest that EGFR is actively expressed in human hepatoma 7404 and 7721 cells and EGF may be one of the mitogens needed for the growth of hepatoma cells.     

Effect of transforming growth factor beta on proliferation of L6 and embryonic porcine myogenic cells

We have examined the effect of Transforming Growth Factor (TGF) beta on proliferation of L6 and embryonic porcine myogenic cells. Proliferation of L6 cells was suppressed by both TGF beta-1 and TGF beta-2 in a dose-dependent manner. Half-maximal suppression of proliferation occurred at .036 ng TGF beta-1/ml and .06 ng TGF beta-2/ml. Maximal inhibition (60% suppression of proliferation for TGF beta-1 and 52% for TGF beta-2) occurred between .1 and .3 ng/ml for each growth factor. Suppression of proliferation was completely abolished in the presence of an anti-TGF beta antibody that inhibited the biological activity of TGF beta-1 and TGF beta-2. When we evaluated the effect of TGF beta-1 on proliferation of embryonic porcine myogenic cells we obtained results which were very similar to those obtained for L6 cells. Insulin-like growth factor (IGF)-I stimulated proliferation of L6 cells in a dose-dependent manner in serum-free, defined medium. However as little as .02 ng TGF beta-1/ml detectably suppressed this stimulation and .3 ng TGF beta-1/ml caused a 60% reduction in cell number in cultures treated with 30 ng IGF-l/ml. Thus TGF beta-1 significantly suppressed IGF-I-stimulated proliferation of L6 cells.     

Tyrphostins inhibit epidermal growth factor (EGF)-receptor tyrosine kinase activity in living cells and EGF-stimulated cell proliferation

Synthetic compounds called tyrphostins were examined for their effects on cells which are mitogenically responsive to epidermal growth factor (EGF). We studied in detail the effects of two tyrphostins on EGF binding, tyrosine phosphorylation in intact cells, EGF-receptor internalization, and mitogenesis. These compounds inhibited EGF-stimulated [3H]thymidine incorporation in a specific manner and the degree of selectivity varied. Both compounds inhibited EGF-stimulated receptor autophosphorylation and tyrosine phosphorylation of endogenous substrates in intact cells at doses that correlated with the IC50 for [3H] thymidine incorporation. These results are consistent with the notion that tyrosine phosphorylation is a crucial signal in transduction of the mitogenic message delivered by EGF. The compound RG50864 demonstrated specificity at inhibiting EGF-stimulated cell growth compared with stimulation with either platelet-derived growth factor or serum. For both compounds RG50864 and RG50810, long term exposure (16 h) of cells to tyrphostins was required for optimal inhibition because of the instability and slow action of these compounds. Tyrphostins did not alter cell surface display of EGF-receptor, EGF binding or EGF-induced internalization, degradation, and down-regulation of EGF receptors. These novel synthetic inhibitors, specific for EGF-receptor kinase, offer a new method to inhibit EGF-stimulated cell proliferation which may be useful in treating specific pathological conditions involving cellular proliferation, including different types of cancers.     

Effects of insulin-like growth factor 1 and testosterone on the proliferation of antlerogenic cells in vitro.

Androgen hormones and growth factors are implicated in pedicle formation and antler transformation in deer. The potential to form a pedicle and an antler is only found in the antlerogenic periosteum (AP) overlying the presumptive antler growth region. Histological studies (Li and Suttie, '94) showed that AP consists of an inner cellular layer and an outer fibrous layer. Pedicle and antler are mainly derived from the cellular layer cells of the AP. Ossification takes place in four stages: intramembranous (IMO), transitional (OPC), pedicle endochondral (pECO) and antler endochondral (aECO). However, the precise mechanism whereby androgen hormones and growth factors control pedicle and antler formation is unknown. The aim of this study was to use cell culture techniques to investigate how testosterone and IGF1 affects the proliferation of antlerogenic cells from the four ossification stages of pedicle/antler in vitro. The results showed that in serum-free medium IGF1 stimulated the proliferation of antlerogenic cells from all four ossification stages in a dose-dependent manner. In contrast, testosterone alone did not show any mitogenic effects on these antlerogenic cells. However, in the presence of IGF1, testosterone increased proliferation of the antlerogenic cells from the IMO and the OPC stages (pedicle tissue), and reduced proliferation of the antlerogenic cells from transformation point (TP) and aECO stages (antler tissue). Therefore, the results from the present in vitro study support the in vivo findings that androgen hormones stimulate pedicle formation but inhibit antler growth. The change in the mitogenic effects of testosterone on antlerogenic cells from positive to negative occurs approximately at the change in ossification type from OPC to pECO. Therefore, these results reinforce the hypothesis that the transformation from a pedicle to an antler takes place at the time when the ossification type changes from OPC to pECO rather than at the time when the pedicle grows to its full species-specific height.