Cell Proliferation In Primary Cultures Research Articles

S-Allyl-L-cysteine Promotes Cell Proliferation by Stimulating Growth Hormone Receptor/Janus Kinase 2/Phospholipase C Pathways and Promoting Insulin-Like Growth Factor Type-I Secretion in Primary Cultures of Adult Rat Hepatocytes.

The mechanism of insulin-like growth factor type-I (IGF-I) secretion stimulated by S-allyl-L-cysteine (SAC) was investigated as part of a study of SAC-induced DNA synthesis and cell proliferation in primary cultures of adult rat hepatocytes. When 10-6 M SAC was added to the culture, the amount of IGF-I in the medium was significantly increased at 10 min. The peak IGF-I level (140 pg/mL) was observed 20 min after SAC stimulation. The SAC-induced IGF-I secretion was completely suppressed by a selective Janus kinase 2 (JAK2) inhibitor (TG101209), a selective phospholipase C (PLC) inhibitor (U-73122), an intracellular Ca2+ chelating agent (BAPTA-AM), and a granule secretion inhibitor (somatostatin). On the other hand, 10-6 M SAC-stimulated hepatocytes showed increased intracellular Ca2+ concentration in a time-dependent manner from 0 to 10 min. Phosphorylation of SAC-induced JAK2 and IGF-I receptor tyrosine kinase (RTK) was completely suppressed by TG101209. In addition, U-73122, BAPTA-AM, and somatostatin did not suppress SAC-induced JAK2 phosphorylation, but significantly suppressed SAC-induced IGF-I RTK phosphorylation. Furthermore, binding of the monoclonal antibody against growth hormone (GH) to GH receptor was dose-dependently suppressed by SAC on immunofluorescence. These results showed that SAC promotes cell proliferation by stimulating GH receptor/JAK2/phospholipase C pathways and promoting autocrine secretion of IGF-I in primary cultures of adult rat hepatocytes.

Signal transduction mechanism for S-allyl-L-cysteine-induced cell proliferation in primary cultures of adult rat hepatocytes

Signal transduction mechanism for S-allyl-L-cysteine-induced cell proliferation in primary cultures of adult rat hepatocytes

Nampt (Visfatin) Influence on Proliferative Activity of Normal Rat Adrenocortical Cells and Human Adrenal Corticocarcinoma Nci-H295r Cells

Abstract Nampt (Nicotinamidephosphoribosyltransferase - also known as visfatin/PBEF) is the enzyme that regulates the NAD+ level, therefore influencing many metabolic pathways within the cells. As circulating cytokine, extracellular Nampt (eNampt) exerts pro-inflammatory, pro-chemotactic, pro-angiogenic and insulin-like effects; however the mechanism of eNampt action is still unclear.Earlier studies have shown that eNampt exerts a stimulating effect on the proliferation of many cancer cell lines. However, the effect of this cytokine on cell proliferation in primary culture is little known. Therefore, the aim of the study was to analyse the influence of eNampt on the proliferation of rat adrenocortical cells in primary culture and to investigate similar influence of eNampt on the line H295R of human adrenal corticocarcinoma cells. Proliferation of the examined cells was assessed using the RTCA (Real Time Cell Analyzer) method. The obtained results indicate that eNampt stimulates the proliferation of H295R cells, but does not change the proliferation of cultured rat adrenocortical cells. In primary culture of rat adrenocortical cells, Fk866 (specific Nampt inhibitor) does not modify the rate of proliferation of tested cells. In H295R cells the addition of Fk866 alone inhibits proliferative activity and stimulates apoptosis. Fk866 also inhibits the stimulating effect of eNampt on H295R cell proliferation.

Involvement of endogenous transforming growth factor-α in signal transduction pathway for interleukin-1β-induced hepatocyte proliferation

We studied the effects of interleukin (IL)-1β on DNA synthesis and cell proliferation in primary cultures of adult rat hepatocytes in order to elucidate the mechanisms of its action. Hepatocyte parenchymal cells maintained in a serum-free, defined medium synthesized DNA and proliferated in the presence of IL-1β (3–30ng/ml), but not IL-1α (0.1–30ng/ml) in a time- and dose-dependent manner. Specific inhibitors of growth-related signal transducers, such as AG1478, LY294002, PD98059, and rapamycin, completely abolished IL-1β-stimulated hepatocyte DNA synthesis and proliferation. Western blot analysis showed that IL-1β significantly stimulated mitogen-activated protein (MAP) kinase activation within 10min. Addition of a monoclonal antibody against transforming growth factor (TGF)-α, but not a monoclonal antibody against insulin-like growth factor-I, to the culture dose-dependently inhibited IL-1β-induced hepatocyte mitogenesis. Culture medium TGF-α levels increased significantly within 3min in response to IL-1β from baseline levels. Peak TGF-α levels (33pg/ml) were reached at 10min after IL-1β stimulation. These results indicate that the proliferative mechanism of action of IL-1β is mediated through an increase in autocrine secretion of TGF-α from primary cultured hepatocytes. Secreted TGF-α, in turn, acts as a complete mitogen to induce hepatocyte mitogenesis through the receptor tyrosine kinase/phosphatidylinositol 3-kinase/MAP kinase/mammalian target of rapamycin pathway.

Role of D2 dopamine receptor in adrenal cortical cell proliferation and aldosterone-producing adenoma tumorigenesis

Aldosterone-producing adenoma (APA) and bilateral adrenal hyperplasia are the two characteristic types of primary aldosteronism. Dysregulation of adrenal cortical cell proliferation contributes to both diseases. We previously demonstrated that APA expressed less dopamine D2 receptor than the respective non-tumor tissue and might contribute to the overproduction of aldosterone. As activation of D2 receptor inhibits the proliferation of various cells, downregulation of D2 receptor in APA may play a role in the tumorigenesis of APA. In this study, we demonstrate that D2 receptor plays a role in angiotensin II (AII)-stimulated adrenal cortical cell proliferation. The D2 receptor agonist, bromocriptine, inhibited AII-stimulated cell proliferation in primary cultures of the normal human adrenal cortex and APA through attenuating AII-induced phosphorylation of PK-stimulated cyclin D1 protein expression and cell proliferation. D2 receptor also inhibited AII-induced ERK1/2 phosphorylation. Our results demonstrate that, in addition to inhibiting aldosterone synthesis/production, D2 receptor exerts an anti-proliferative effect in adrenal cortical and APA cells by attenuating PKCμ and ERK phosphorylation. The lower level of expression of D2 receptor in APA may augment cell proliferation and plays a crucial role in the tumorigenesis of APA. Our novel finding suggests a new therapeutic target for primary aldosteronism.

Abstract A006: FGF-2 stimulates breast cancer growth activating ER and PR

Abstract Estrogen and progesterone receptors (ER and PR respectively) are prognostic markers and dictate therapeutic decisions in breast cancer patients. Although most of the evidence suggests that estrogens are the major etiological factors in breast cancer, there is experimental and epidemiological data that also points to the involvement of PR in mammary gland carcinogenesis and breast cancer growth. Both ER and PR are activated by their natural ligands or by ligand-independent pathways. Carcinoma-associated fibroblasts produce growth factors that stimulate epithelial cell proliferation. We have recently demonstrated that Fibroblast growth factor 2 (FGF-2) is able to activate PR and induce progestin-dependent tumor growth. In addition, we have demonstrated an interaction between ER and PR at the promoters of Cyclin D1 (CCND1) and MYC, which is necessary to mediate progestin-induced gene transcription and cell proliferation. The aim of this study was to evaluate the role of ER in FGF-2-induced cell proliferation. We used C4-HD and C4-HI tumors from the medroxyprogesterone acetate (MPA)-induced murine breast cancer model and two human cell lines, T47D and MCF-7. The C4-HD tumors only grow with the exogenous administration of progestins or FGF-2. High levels of activated ER expression were observed in C4-HD tumors growing in NOD/SCID female mice treated with FGF-2 (5 ug/day) or MPA (20 mg/depot). The inhibitor of FGF receptors (PD173074, 25 mg/kg) which inhibits C4-HI tumor growth and PR phosphorylation also reduced the expression and phosphorylation of ER. FGF-2 (50 ng/ml) stimulated cell proliferation of primary cultures of C4-HD and C4-HI tumor cells, and of T47D and MCF-7 cells (p<0.001). The antiestrogen ICI 182.780 (ICI, 1-100 nM), the FGFR inhibitors (PD 0.1 uM and BGJ398 1 nM), and siRNAs against ER, all blocked the FGF-2 stimulation (p<0.001) in the aforementioned models. An increase in pSer118 and pSer167 ER and in the nuclear interaction between ER with the coactivator AIB1 and with FOXA1, were observed in FGF-2-treated MCF-7 and T47D cells. Moreover, FGF-2 induced an increase in the expression of the estrogen responsive elements (ERE)-Luc reporter (p<0.001), that was blocked by ICI and PD (p<0.001). FGF-2 also mediated the up-regulation of two ER-regulated genes, MYC and pS2/TFF1 (p<0.001), which was inhibited by ICI (p<0.001). Finally, ER and FOXA1 were recruited to ERE sites at pS2/TFF1 promoter after FGF-2 incubation in MCF-7 cells (p<0.01). Our results indicate that FGF-2 mediates PR and ER activation in breast cancer cells, suggesting that the combination of antiestrogens, antiprogestins and FGF-2-signaling inhibitors may be an option in selected breast cancer patients. Citation Format: Tomás Guillardoy, María A. Gorostiaga, Claudia Lanari, Sebastián Giulianelli. FGF-2 stimulates breast cancer growth activating ER and PR. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A006.

Chitosan and platelet‐derived growth factor synergistically stimulate cell proliferation in gingival fibroblasts

Chitosan is a naturally derived polymer that may be applied in periodontal therapy for tissue-reconstruction purposes. Previous studies have shown that chitosan may stimulate tissue healing. However, reports exploring the cellular responses stimulated by chitosan are lacking. In the present study we analyzed whether chitosan may promote cell proliferation in primary cultures of human gingival fibroblasts. Chitosan particles were generated, and their size, zeta potential and morphology were characterized using transmission and scanning electron microscopy and zetasizer analysis. The biocompatibility of chitosan particles was analyzed using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cell-viability assay and by detecting the release of lactate dehydrogenase into the cell-culture medium. The total number of cells was estimated by staining with crystal violet followed by measurement of the absorbance at 560nm on a microplate reader. Cell proliferation was studied by detecting proliferating cell nuclear antigen protein levels, immunofluorescence for Ki67 and incorporation of 5'-bromo-2'-deoxyuridine. The sizes of the chitosan particles generated were in the micrometer and nanometer ranges. Cell viability was increased in the presence of chitosan. Moreover, the combination of chitosan and platelet-derived growth factor (PDGF-BB) potently stimulated cell viability, cell proliferation and activation of the ERK1/2 pathway involved in cell proliferation. The present study shows that chitosan is well tolerated by gingival fibroblasts and is able to stimulate cell proliferation through the ERK1/2 signaling pathway. A synergistic response between chitosan and growth factors (such as PDGF-BB) may stimulate cell proliferation in gingival fibroblasts exposed to this biomaterial.

Signal transduction pathway for l-ascorbic acid- and l-ascorbic acid 2-glucoside-induced DNA synthesis and cell proliferation in primary cultures of adult rat hepatocytes

We examined the effects of l-ascorbic acid and its analogues on DNA synthesis and cell proliferation. We also investigated the signal transduction pathways involved in the induction of mitogenesis by l-ascorbic acid and its analogues using primary cultures of adult rat hepatocytes. Following a 4-h serum-free cultivation, both l-ascorbic acid and its stable analogue, l-ascorbic acid 2-glucoside, time- and dose-dependently stimulated hepatocyte DNA synthesis and cell proliferation, with EC50 values of 6.46×10−8M and 3.34×10−8M, respectively. Dehydroascorbic acid (10−6M–10−5M) weakly stimulated hepatocyte mitogenesis, whereas isoascorbic acid (10−9M–10−5M) had no effect. Hepatocyte mitogenesis induced by l-ascorbic acid or l-ascorbic acid 2-glucoside was dose-dependently abolished by treatment with monoclonal antibodies against insulin-like growth factor (IGF)-I receptor, but not by treatment with monoclonal antibodies against insulin receptor or IGF-II receptor. Western blot analysis showed that both l-ascorbic acid and l-ascorbic acid 2-glucoside significantly stimulated IFG-I receptor tyrosine kinase activity within 3min, and mitogen-activated protein (MAP) kinase activity within 5min. These results demonstrate that both l-ascorbic acid and l-ascorbic acid 2-glucoside induce DNA synthesis and cell proliferation in primary cultures of adult rat hepatocytes by interacting with the IGF-I receptor site and by activating the receptor tyrosine kinase/MAP kinase pathway.

The Molecular Signature of Spermatogonial Stem/Progenitor Cells in the 6-Day-Old Mouse Testis1

To characterize the molecular phenotype of spermatogonial stem cells (SSCs), we examined genes that are differentially expressed in the stem/progenitor spermatogonia compared to nonstem spermatogonia. We isolated type A spermatogonia (stem and nonstem type A) from 6-day-old mice using sedimentation velocity at unit gravity and further selected the stem/progenitor cell subpopulation by magnetic activated cell sorting with an antibody to GDNF-receptor-alpha-1 (GFRA1). It has been previously shown that GFRA1 is expressed in SSCs and is required for their stemness. The purity of the isolated cells was approximately 95% to 99% as indicated by immunocytochemistry using anti-GFRA1. Comparison of GFRA1-positive and GFRA1-negative spermatogonia by microarray analysis revealed 99 known genes and 12 uncharacterized transcripts that are overexpressed in the former cell population with a >2-fold change. Interestingly, the highest level of overexpression was observed for Csf1r, encoding the receptor for macrophage colony-stimulating factor (M-CSF, official symbol CSF1), which has a well-established role in the regulation of myeloid progenitor cells. Analysis of our microarray data with a bioinformatics software program (Ingenuity Systems) revealed the potential role of various signaling pathways in stem/progenitor spermatogonia and suggested a common pathway for GFRA1 and CSF1R that may lead to their proliferation. Further investigation to test this hypothesis has shown that CSF1 promotes cell proliferation in primary cultures of the isolated type A spermatogonia and in the spermatogonial-derived stem cell line C18-4. Semiquantitative RT-PCR and immunohistochemistry confirmed the previously mentioned microarray data. Collectively, this study provides novel molecular signatures for stem/progenitor spermatogonia and demonstrates a role for CSF1/CSF1R signaling in regulating their proliferation.

RNA Interference Elucidates the Role of Focal Adhesion Kinase in HLA Class I-Mediated Focal Adhesion Complex Formation and Proliferation in Human Endothelial Cells

Ligation of class I molecules by anti-HLA Ab stimulates an intracellular signaling cascade resulting in endothelial cell (EC) survival and proliferation, and has been implicated in the process of chronic allograft rejection and transplant-associated vasculopathy. In this study, we used small interfering RNA blockade of focal adhesion kinase (FAK) protein to determine its role in class I-mediated organization of the actin cytoskeleton, cell survival, and cell proliferation in primary cultures of human aortic EC. Knockdown of FAK appreciably inhibited class I-mediated phosphorylation of Src at Tyr(418), p85 PI3K, and Akt at both Thr(308) and Ser(473) sites. FAK knockdown also reduced class I-mediated phosphorylation of paxillin at Try(118) and blocked class I-induced paxillin assembly into focal contacts. FAK small interfering RNA completely abrogated class I-mediated formation of actin stress fibers. Interestingly, FAK knockdown did not modify fibroblast growth factor receptor expression induced by class I ligation. However, FAK knockdown blocked HLA class I-stimulated cell cycle proliferation in the presence and absence of basic fibroblast growth factor. This study shows that FAK plays a critical role in class I-induced cell proliferation, cell survival, and focal adhesion assembly in EC and may promote the development of transplant-associated vasculopathy.

Calcium/calmodulin-dependent protein kinase II-δ isoform regulation of vascular smooth muscle cell proliferation

There is accumulating evidence that Ca(2+)-dependent signaling pathways regulate proliferation and migration of vascular smooth muscle (VSM) cells, contributing to the intimal accumulation of VSM that is a hallmark of many vascular diseases. In this study we investigated the role of the multifunctional serine/threonine kinase, calmodulin (CaM)-dependent protein kinase II (CaMKII), as a mediator of Ca(2+) signals regulating VSM cell proliferation. Differentiated VSM cells acutely isolated from rat aortic media express primarily CaMKIIgamma gene products, whereas passaged primary cultures of de-differentiated VSM cells express primarily CaMKIIdelta(2), a splice variant of the delta gene. Experiments examining the time course of CaMKII isoform modulation revealed the process was rapid in onset following initial dispersion and primary culture of aortic VSM with a significant increase in CaMKIIdelta(2) protein and a significant decrease in CaMKIIgamma protein within 30 h, coinciding with the onset of DNA synthesis and cell proliferation. Attenuating the initial upregulation of CaMKIIdelta(2) in primary cultured cells using small-interfering RNA (siRNA) resulted in decreased serum-stimulated DNA synthesis and cell proliferation in primary culture. In passaged VSM cells, suppression of CaMKIIdelta(2) activity by overexpression of a kinase-negative mutant, or suppression of endogenous CaMKII content using multiple siRNAs, significantly attenuated serum-stimulated DNA synthesis and cell proliferation. Cell cycle analysis following either inhibitory approach indicated decreased proportion of cells in G1, an increase in proportion of cells in G2/M, and an increase in polyploidy, corresponding with accumulation of multinucleated cells. These results indicate that CaMKIIdelta(2) is specifically induced during modulation of VSM cells to the synthetic phenotypic and is a positive regulator of serum-stimulated proliferation.

Obestatin‐mediated proliferation of human retinal pigment epithelial cells: Regulatory mechanisms

In this work, we have evaluated the effect of the new discovered peptide obestatin on cell proliferation in primary cultures of human retinal epithelial cells (hRPE cells). The results showed that this peptide induced, in a dose-dependent manner, cell proliferation by MEK/ERK 1/2 phosphorylation. A sequential analysis of the obestatin transmembrane signaling pathway showed that the ERK 1/2 activity is partially blocked after preincubation of the cells with pertussis toxin (PTX), as well as by wortmannin (an inhibitor of PI3K), claphostin C (an inhibitor of PKC), and PP2 (which inhibits the non receptor tyrosine kinase Src). Upon administration of obestatin, the intracellular levels of phospho-PKCepsilon-, theta-, and micro-isoenzymes rise with different time courses, from which PKCepsilon might be responsible for ERK 1/2 response. Based on the experimental data, a signaling pathway involving the consecutive activation of Gi, PI3K, novel PKC (probably PKCepsilon), and Src for ERK 1/2 activation is proposed. These results incorporate a new mitogenic factor to the group of factors that regulate proliferation of hRPE cells.

Effects of TGF‐β and glucocorticoids on map kinase phosphorylation, IL‐6/IL‐11 secretion and cell proliferation in primary cultures of human lung fibroblasts

Transforming growth factor-beta1 (TGF-beta1) is crucially involved in the fibrotic events characterizing interstitial lung diseases (ILDs), as well as in the airway remodeling process typical of asthma. Within such a context, the aim of our study was to investigate, in primary cultures of normal and fibrotic human lung fibroblasts (HLFs), the effects of TGF-beta1 on mitogen-activated protein kinase (MAPK) phosphorylation, cell proliferation, and production of interleukins 6 (IL-6) and 11 (IL-11), in the presence or absence of a pretreatment with budesonide (BUD). MAPK phosphorylation was detected by Western blotting, cell viability and proliferation were evaluated using Trypan blue staining and [(3)H]-thymidine incorporation assay, respectively, and the release of IL-6 and IL-11 into cell culture supernatants was assessed by ELISA. TGF-beta1 (10 ng/ml) significantly stimulated MAPK phosphorylation (P < 0.01), and also enhanced cell proliferation as well as the secretion of both IL-6 and IL-11, which reached the highest increases at the 72nd h of cell exposure to this growth factor. All such effects were prevented by BUD (10(-8) M) and, with the exception of IL-6 release, also by a mixture of MAPK inhibitors. Therefore, our findings suggest that the fibrotic action exerted by TGF-beta1 in the lung is mediated at least in part by MAPK activation and by an increased synthesis of the profibrogenic cytokines IL-6 and IL-11; all these effects appear to be prevented by corticosteroids via inhibition of MAPK phosphorylation.

The Role of 5αR Inhibitors in the Early Growth of Pca Cells

Aim of this study is to investigate the efficacy of Finasteride (MK906), a selective 5α reductase type 2(5αR2) inhibitor, and of MK386, a selective 5α reductase type 1 (5αR1), on the cellular proliferation of primary cell cultures derived from patients with prostatic diseases. Methods We evaluated the effects of Finasteride and MK386 in 30 primary cultures obtained from prostatic carcinoma (PCa), 6 from high grade intraepithelial neoplasia (HGPIN) and 10 from benign prostatic hyperplasia (BPH). Results Primary cultures require testosterone (T) for optimal growth and both 5αR inhibitors decreased cell proliferation of primary cultures of PCa cells. IC50 values of Finasteride were lower when compared to those of MK386. In particular, Finasteride was more active in cultures derived from Gleason 2–6 PCa, compared to those derived from Gleason 7–10 PCa. On the contrary, we observed higher effectiveness of MK386 in Gleason 7–10 PCa derived cultures compared to those derived from Gleason 2–6 PCa tissues. The growth of PCa primary cultures was down-modulated by MK386 through a direct inhibition of the epithelial 5αR1, whereas the anti-proliferative activity of Finasteride seems to be mediated by stromal cells present in the primary cultures. Conclusions We found biological evidence for an important role of 5αR inhibitors in the early growth of PCa cells and for the necessity of combining anti-hormonal treatment for a sustained inhibition of PCa through the inhibition of stromal cell function even in advanced and androgen-resistant tumor progression phases.

Estrogen Receptor-α Agonists Promote Angiogenesis in Human Myometrial Microvascular Endothelial Cells

The relative role of the two estrogen receptors, ERalpha and ERbeta, in mediating angiogenic responses in adult human endothelium is unknown. The aim of this study was to determine whether novel ERalpha-selective agonists, propyl pyrazole triol (PPT) and the tetrahydrochrysene (R,R-THC), up-regulate the expression of vascular endothelial growth factor receptor-2 (VEGFR-2), and promote VEGF-stimulated endothelial cell proliferation in primary cultures of adult female microvascular endothelial cells co-expressing endogenous ERalpha and ERbeta. Confluent primary cultures of microvascular endothelial cells isolated from human myometrium were incubated with 17beta-estradiol (1 and 10 nM), PPT (10 nM to 3 microM), or R,R-THC (10 nM to 3 microM) for 18 hours and VEGFR-2 expression measured by biotin-VEGF165 binding and flow cytometry. Endothelial cell proliferation was assessed in microvascular endothelial cells after incubation with 17beta-estradiol (10 nM), PPT (100 nM), and R,R-THC (100 nM) for 6 days using a tetrazolium-based bioassay. Both PPT and R,R-THC increased VEGFR-2 expression on myometrial microvascular endothelial cells in a dose-dependent manner, reaching a maximum at 1 microM. Approximately 40% of myometrial microvascular endothelial cell isolates only express ERbeta and do not express ERalpha, and in these neither PPT, R,R-THC, nor 17beta-estradiol increased VEGF binding. PPT- or R,R-THC-stimulated increase in VEGF binding was significantly different between ERalpha+ and ERalpha- microvascular endothelial cell samples (P < .001 and P < .05, respectively). PPT, R,R-THC, and 17beta-estradiol significantly augmented VEGF-stimulated microvascular endothelial cell proliferation in ERalpha+ (P < .05), but not in ERalpha- samples. This angiogenic effect of 17beta-estradiol on adult female microvascular endothelial cells is mediated by ERalpha, rather than ERbeta.

High molecular weight plasma proteins induce apoptosis and Fas/FasL expression in human proximal tubular cells

In proteinuria, proximal tubular epithelial cells (PTECs) are exposed to abnormally high protein concentrations, eventually leading to tubular atrophy and end-stage renal disease. The mode of cell death leading to tubular atrophy in proteinuria has not been fully established. This study examines the role of protein overload on apoptosis, necrosis and cell proliferation in primary cultures of human PTECs using plasma protein fractions representative of selective and non-selective proteinuria. The involvement of the Fas/Fas ligand (FasL) system was also investigated. Plasma was collected from healthy volunteers and fractionated into albumin-rich (30-100 kDa), high molecular weight (100-440 kDa) and combined (30-440 kDa) fractions. PTECs were exposed to 10 mg/ml of the protein fractions for 24, 48 and 72 h. Apoptosis was measured using fluorescein isothiocyanate (FITC)-annexinV and TUNEL. Necrosis was measured using propidium iodide, metabolic activity by MTT and cell proliferation by bromodeoxyuridine incorporation. Fas and FasL expression was analysed by western blotting. Exposure to the 100-440 and 30-440 kDa fractions produced significant increases in apoptosis at all time points, whereas PTECs exposed to the 30-100 kDa fraction were not significantly different from control cells. There were no changes in the rates of necrosis as a result of protein loading. A significant reduction in metabolic activity was observed in PTECs exposed to the 100-440 and 30-440 kDa fractions, but not to the 30-100 kDa fraction. Cell proliferation was significantly reduced by 24 h in cells exposed to the 100-440 and 30-440 kDa fractions. By 48 and 72 h, all the three fractions had inhibited cell proliferation. PTECs exposed to the 100-440 and the 30-440 kDa fractions showed a significant upregulation in the expression of Fas and FasL. Overall, the high molecular weight fraction was more 'toxic' than the albumin-rich or combined fraction. Increased apoptosis and decreased cell proliferation are the major mechanisms of cell death in human PTECs in response to protein overload. These effects may be mediated at least in part by overexpression of the Fas/FasL system. The severity of such changes is largely determined by the high molecular weight fraction (100-440 kDa) rather than the albumin-rich fraction.

Effects of pathophysiological concentrations of albumin on NHE3 activity and cell proliferation in primary cultures of human proximal tubule cells.

The progression of renal disease correlates strongly with hypertension and the degree of proteinuria, suggesting a link between excessive Na+ reabsorption and exposure of the proximal tubule to protein. The present study investigated the effects of albumin on cell growth and Na+ uptake in primary cultures of human proximal tubule cells (PTC). Albumin (1.0 mg/ml) increased cell proliferation to 134.1 +/- 11.8% (P < 0.001) of control levels with no change in levels of apoptosis. Exposure to 0.1 and 1.0 mg/ml albumin increased total 22Na+ uptake to 119.1 +/- 6.3% (P = 0.005) and 115.6 +/- 5.3% (P < 0.006) of control levels, respectively, because of an increase in Na+/H+ exchanger isoform 3 (NHE3) activity. This was associated with an increase in NHE3 mRNA to 161.1 +/- 15.1% (P < 0.005) of control levels in response to 0.1 mg/ml albumin. Using confocal microscopy with a novel antibody raised against the predicted extracellular NH2 terminus of human NHE3, we observed in nonpermeabilized cells that exposure of PTC to albumin (0.1 and 1.0 mg/ml) increased NHE3 at the cell surface to 115.4 +/- 2.7% (P < 0.0005) and 122.4 +/- 3.7% (P < 0.0001) of control levels, respectively. This effect was paralleled by significant increases in NHE3 in the subplasmalemmal region as measured in permeabilized cells. These albumin-induced increases in expression and activity of NHE3 in PTC suggest a possible mechanism for Na+ retention in response to proteinuria.

Oxytocin stimulates proliferation of human osteoblast-like cells

Oxytocin receptors have recently been demonstrated in human osteoblast-like (hOB) cells. In this study, oxytocin 100–1000 pmol/l increased cell proliferation of primary cultures of hOB cells, measured by [ 3 H ]thymidine incorporation, ( P<0.01). In human osteosarcoma cell-line (SaOS-2), oxytocin 100 pmol/l increased cell proliferation (measured by [ 3 H ]thymidine incorporation and a commercially available kit) and protein synthesis ([ 3 H ]proline incorporation) ( P<0.05). The increase in cell proliferation was abolished when SaOS-2 cells were incubated with an oxytocin antagonist and oxytocin. Oxytocin 100 pmol/l decreased interleukin-6 (IL-6) production of the hOB cells (23.4±1.96 versus 33.4±2.65 pg/well; P<0.001). These findings indicate that oxytocin may affect bone metabolism in humans.

Relationship of p21WAF1/CIP1/SDI1 to cell proliferation in primary cultures of adrenocortical cells

p21(WAF1/CIP1/SDI1) was originally described as a protein expressed at high levels in senescent human fibroblasts. We have studied the expression of p21 in adrenocortical cells, p21 is not expressed under most circumstances in the intact adrenal gland in vivo, except when the gland is damaged. When human and bovine adrenocortical cells are isolated and placed in both short-term and long-term culture, p21 levels are much higher. These levels did not show a large increase when the cells senesce after long-term proliferation. Thus, these observations raise the question of whether the elevated p21 in primary cultures of adrenocortical cells is caused by damage or whether p21 is elevated because the cells are dividing rather than quiescent, because it has been reported that p21 levels peak in G1 and G2 in dividing cells. In the present experiments on bovine and human adrenocortical cells in primary culture, labeling techniques that correlated nuclear p21 with measures of cell proliferation (bromodeoxyuridine incorporation and nuclear Ki-67 antigen) supported the hypothesis that p21 is associated with cell division and not with damage. This is consistent with recent data showing that, when adrenocortical cells are transplanted into immunodeficient mice, p21 is associated with healthy dividing cells in the transplant, p21 is not a unique marker for senescence, and more studies are required both to clarify its role in cell biology and to determine molecular features which characterize the senescent state of cells both in vitro and in vivo.

Peptide-Bound Methionine as Methionine Sources for Protein Accretion and Cell Proliferation in Primary Cultures of Ovine Skeletal Muscle ,

Ruminants have high concentrations of peptide-bound amino acids in the circulation. Earlier studies in our laboratory showed that a myogenic cell line (C2C12) developed from mouse skeletal muscle and a mammary epithelial cell line (MAC-T) developed from bovine mammary epithelial tissues are able to utilize peptides as amino acid sources. In this study, primary cultures of ovine myogenic satellite cells were evaluated for their ability to use peptide-bound methionine as a source of methionine for protein accretion and cell proliferation. The basal medium contained methionine-free Dulbecco’s modified Eagle’s medium supplemented with 6% desalted fetal bovine serum. Treatment media included the basal medium supplemented with no methionine, methionine or one of 22 methionine-containing peptides. No protein or DNA accretion was observed in the presence of basal medium alone. Growth responses to all of the peptides were obtained, with protein and DNA accretion ranging from 49 to 107% and from 45 to 144% of the corresponding methionine response, respectively. These results indicate that the ovine myogenic satellite cells possess the ability to utilize methionine-containing peptides as methionine sources for protein accretion and cell proliferation.