Serum-induced Cell Research Articles

Methoxyestradiols mediate the antimitogenic effects of 17beta-estradiol: direct evidence from catechol-O-methyltransferase-knockout mice.

Studies using pharmacological agents suggest but do not prove that the antimitogenic effects of estradiol are caused by conversion of estradiol to hydroxyestradiols (mediated by CYP450s) followed by methylation of hydroxyestradiols to methoxyestradiols (mediated by catechol-O-methyltransferase, COMT). To test this hypothesis more rigorously, we used aortic smooth muscle cells (SMCs) from mice lacking COMT (COMT-KO). Wild-type (WT) but not COMT-KO SMCs efficiently converted 2-hydroxyestradiol to 2-methoxyestradiol. Both WT and COMT-KO SMCs expressed estrogen receptors. Estradiol and 2-hydroxyestradiol concentration-dependently inhibited serum-induced DNA synthesis, cell numbers, and collagen synthesis in WT but not COMT-KO SMCs. 2-Methoxyestradiol inhibited DNA synthesis, cell numbers, and collagen synthesis in both WT and COMT-KO SMCs. These data provide strong evidence that the vascular antimitogenic effects of estradiol are estrogen receptor-independent and involve the sequential conversion of estradiol to hydroxyestradiols and then to methoxyestradiols.

Adrenomedullin is an autocrine/paracrine growth factor for rat vascular smooth muscle cells

Adrenomedullin is a potent vasodilator peptide secreted by vascular endothelial and smooth muscle cells. Adrenomedullin stimulates the proliferation of quiescent rat vascular smooth muscle cells (VSMCs) via p42/p44 ERK/MAP kinase activation. Recently, receptor-activity-modifying proteins (RAMPs) have been shown to transport calcitonin-receptor-like-receptor (CRLR) to the cell surface to present either as CGRP receptor or adrenomedullin receptor. We investigated whether adrenomedullin acts as an autocrine/paracrine growth factor for cultured rat VSMCs and whether coexpressions of RAMP isoform and CRLR may mediate p42/p44 ERK/MAP kinase activation by adrenomedullin. Adrenomedullin dose-dependently stimulated the proliferation of quiescent rat VSMCs, and this effect was inhibited by an adrenomedullin receptor antagonist, a MAP kinase kinase inhibitor and phosphatidylinositol 3-kinase inhibitors. Addition of either CGRP(8–37) or anti-adrenomedullin antibody to exponentially growing rat VSMCs inhibited the serum-induced cell proliferation, suggesting its role as an autocrine/paracrine growth factor. Cotransfection of RAMP2 or RAMP3 with CRLR into rat VSMCs potentiated activation of cAMP activity, but not of p42/p44 ERK/MAP kinase activity in response to adrenomedullin. Our results suggest that adrenomedullin is an autocrine/paracrine growth factor for rat VSMCs via p42/p44 ERK/MAP kinase and phosphatidylinositol 3-kinase pathways and that it is not mediated by human RAMP–CRLR receptors.

Quercetin exerts multiple inhibitory effects on vascular smooth muscle cells: role of ERK1/2, cell-cycle regulation, and matrix metalloproteinase-9

The French paradox has been attributed to the antioxidant properties of flavonoids present in the red wine. Quercetin, a bioflanoid present in the human diet, is known to inhibit angiotensin II-induced hypertrophy and serum-induced smooth muscle cell proliferation. However, it is not known whether quercetin exerts similar cardioprotective effects in cells treated with TNF-α. In this study, we investigated whether quercetin exerts the multiple suppressive effects on cytokine TNF-α-induced human aortic smooth muscle cells (HASMC). Treatment of quercetin showed potent inhibitory effects on the DNA synthesis of cultured HASMC in the presence of TNF-α. These inhibitory effects were associated with reduced extracellular signal-regulated kinase (ERK)1/2 activity and G1 cell-cycle arrest. Treatment of quercetin, which induced a cell-cycle block in G1-phase, induced down-regulation of cyclins and CDKs and up-regulation of the CDK inhibitor p21 expression, whereas up-regulation of p27 or p53 by quercetin was not observed. Because anti-atherogenic effects need not be limited to antiproliferation, we decided to examine whether quercetin exerted inhibitory effects on matrix metalloproteinase-9 (MMP-9) activity in TNF-α-induced HASMC. Quercetin inhibited TNF-α-induced MMP-9 secretion on HASMC in a dose-dependent manner. This inhibition was characterized by down-regulation of MMP-9, which was transcriptionally regulated at NF-κB site and activation protein-1 (AP-1) site in the MMP-9 promoter. These findings indicate the efficacy of quercetin in inhibiting cell proliferation, G1- to S-phase cell-cycle progress, and MMP-9 expression through the transcription factors NF-κB and AP-1 on TNF-α-induced HASMC.

CYP450- and COMT-derived estradiol metabolites inhibit activity of human coronary artery SMCs.

The purpose of this study is to test the hypothesis that the inhibitory effects of estradiol in human coronary vascular smooth muscle cells are mediated via local conversion to methoxyestradiols via specific cytochrome P450s (CYP450s) and catechol-O-methyltransferase (COMT). The inhibitory effects of estradiol on serum-induced cell activity (DNA synthesis, cell number, collagen synthesis, and cell migration) were enhanced by 3-methylcholantherene, phenobarbital (broad-spectrum CYP450 inducers), and beta-naphthoflavone (CYP1A1/1A2 inducer) and were blocked by 1-aminobenzotriazole (broad-spectrum CYP450 inhibitor). Ellipticine, alpha-naphthoflavone (selective CYP1A1 inhibitors), and pyrene (selective CYP1B1 inhibitor), but not ketoconazole (selective CYP3A4 inhibitor) or furafylline (selective CYP1A2 inhibitor), abrogated the inhibitor effects of estradiol on cell activity, a profile consistent with a CYP1A1/CYP1B1-mediated mechanism. The inhibitory effects of estradiol were blocked by the COMT inhibitors OR486 and quercetin. The estrogen receptor antagonist ICI 182,780 blocked the inhibitory effects of estradiol, but only at concentrations that also blocked the metabolism of estradiol to hydroxyestradiols (precursors of methoxyestradiols). Western blot analysis revealed that coronary smooth muscle cells expressed CYP1A1 and CYP1B1. Moreover, these cells metabolized estradiol to hydroxyestradiols and methoxyestradiols, and the conversion of 2-hydroxyestradiol to 2-methoxyestradiol was blocked by OR486 and quercetin. These findings provide evidence that the inhibitory effects of estradiol on coronary smooth muscle cells are largely mediated via CYP1A1- and CYP1B1-derived hydroxyestradiols that are converted to methoxyestradiols by COMT.

BCL-x(L) and BCL2 delay Myc-induced cell cycle entry through elevation of p27 and inhibition of G1 cyclin-dependent kinases.

The anti-apoptotic molecules BCL-x(L) and BCL2 delay cell cycle entry from quiescence. We used serum induction and induction of a Myc-estrogen receptor fusion protein (MycER) in quiescent fibroblasts to investigate the mechanisms underlying the cell cycle activity of BCL-x(L) and BCL2. We demonstrate for the first time that BCL-xL and BCL2 delayed serum-induced and Myc-induced, but not E2F-induced, cell cycle entry. The cyclin-dependent kinase inhibitor p27 was elevated during serum deprivation and cell cycle entry in BCL-x(L) or BCL2-expressing NIH3T3 cells and a Rat1MycER cell line. Activation of cyclin-dependent kinase 2 (cdk2) and cyclin-dependent kinase 4 (cdk4) were delayed during progression to S phase, while the induction of cyclin D1 protein, as well as the levels of cyclin E, cdk2, and cdk4 were unaltered by BCL-x(L) or BCL2. Inhibition of cyclin/cdk activities in BCL-x(L) or BCL2 expressing cells was associated with excess p27 in the cyclin/cdk complexes. Neither BCL-x(L) nor BCL2 delayed S phase entry in cells deficient in p27, thus p27 is required for the cell cycle function of BCL-x(L) and BCL2. The cell cycle effects of BCL-x(L) and BCL2 were more profound in Myc-induced than in serum-induced cell cycle entry. Our results suggest that one possible mechanism by which BCL-x(L) and BCL2 delay cell cycle entry may be the inhibition of Myc activity through the elevation of p27.

The mechanism involved in the regulation of phospholipase Cgamma1 activity in cell migration.

Activation of the enzyme phospholipase C (PLC) leads to the formation of second messengers inositol 1,4,5-trisphosphate and diacylglycerol. Tyrosine kinase receptors activate this reaction through PLCgamma isoenzymes. PLCgamma activity involves its activation with, and phosphorylation by, receptor tyrosine kinases. Recently, it has been shown that phosphoinositide 3-kinase (PI 3-K) may regulate PLCgamma activity through the interaction of the PI 3-K product phosphatidylinositol 3,4,5-trisphosphate (PtdIns-3,4,5-P(3)) and the PLCgamma pleckstrin homology (PH) domain. In an effort to understand the signalling pathway that involves PI 3-K regulation of PLCgamma, we found that EGF induces a PI 3-K-dependent translocation of PLCgamma1 at the leading edge of migrating cells in a wound healing assay. Similarly, the isolated PH, but not the Src-homology (SH) domains, N-SH2 or SH3, of PLCgamma1, translocates at the leading edge. Our experiments also showed that stable PH PLCgamma1 expression blocks epidermal growth factor (EGF)- and serum-induced cell motility and increases cell adhesion in MDA-MB-231 cells. This may suggest that influence of PI 3-K on PLCgamma1 could be relevant in cell migration, where PLCgamma1 seems to play a key role by modulating a series of events involved in actin polymerization.

Human serum-induced expression of E-selectin on porcine aortic endothelial cells in vitro is totally complement mediated.

Whereas complement is a key mediator of hyperacute xenograft rejection, its role in acute vascular rejection (AVR) is a matter of controversy. AVR is associated with de novo synthesis of endothelial cell-derived inflammatory mediators, including the leukocyte-recruiting adhesion molecule E-selectin. Here we investigate the role and mechanism of complement in human serum-induced porcine endothelial cell activation. An in vitro xenotransplantation method was designed using porcine aortic endothelial cells stimulated with human serum in microculture wells. E-selectin expression was measured by cell-enzyme immunoassay. Complement inhibitors acting at different levels in the cascade were investigated for their effect on E-selectin expression. E-selectin was strongly induced by normal human serum but not by heat-inactivated serum. Compstatin, a synthetic C3 inhibitor, markedly reduced human serum-induced E-selectin expression. Purified C1-inhibitor suppressed E-selectin induction completely, indicating activation through the classical or lectin pathway. Furthermore, a monoclonal antibody (mAb) that inhibits cleavage of C5 or another mAb that blocks the function of C7, completely inhibited the expression of serum-induced E-selectin, consistent with the terminal C5b-9 complement complex being the mediator of the endothelial cell activation. Inhibition of the alternative pathway using a novel antifactor D mAb did not reduce E-selectin expression. Human serum-induced expression of porcine E-selectin is totally complement dependent, induced by a C1-inhibitor regulated pathway and mediated through the terminal complement complex. The data may have implications for therapeutic strategies, particularly of C1-inhibitor and anti-C5 mAb, to protect against endothelial cell activation and subsequent AVR of porcine xenografts.

Catalytic oligodeoxynucleotides define a key regulatory role for early growth response factor-1 in the porcine model of coronary in-stent restenosis.

Early growth response factor-1 (Egr-1) controls the expression of a growing number of genes involved in the pathogenesis of atherosclerosis and postangioplasty restenosis. Egr-1 is activated by diverse proatherogenic stimuli. As such, this transcription factor represents a key molecular target in efforts to control vascular lesion formation in humans. In this study, we have generated DNAzymes targeting specific sequences in human EGR-1 mRNA. These molecules cleave in vitro transcribed EGR-1 mRNA efficiently at preselected sites, inhibit EGR-1 protein expression in human aortic smooth muscle cells, block serum-inducible cell proliferation, and abrogate cellular regrowth after mechanical injury in vitro. These DNAzymes also selectively inhibit EGR-1 expression and proliferation of porcine arterial smooth muscle cells and reduce intimal thickening after stenting pig coronary arteries in vivo. These findings demonstrate that endoluminally delivered DNAzymes targeting EGR-1 may serve as inhibitors of in-stent restenosis.

Regulation of CLOCK and MOP4 by Nuclear Hormone Receptors in the Vasculature: A Humoral Mechanism to Reset a Peripheral Clock

Circadian clock genes are expressed in the suprachiasmatic nucleus and in peripheral tissues to regulate cyclically physiological processes. Synchronization of peripheral oscillators is thought to involve humoral signals, but the mechanisms by which these are mediated and integrated are poorly understood. We report a hormone-dependent interaction of the nuclear receptors, RARα and RXRα, with CLOCK and MOP4. These interactions negatively regulate CLOCK/MOP4:BMAL1-mediated transcriptional activation of clock gene expression in vascular cells. MOP4 exhibits a robust rhythm in the vasculature, and retinoic acid can phase shift Per2 mRNA rhythmicity in vivo and in serum-induced smooth muscle cells in vitro, providing a molecular mechanism for hormonal control of clock gene expression. We propose that circadian or periodic availability of nuclear hormones may play a critical role in resetting a peripheral vascular clock.

A peptide analogue of thrombin receptor-activating peptide inhibits thrombin and thrombin-receptor-activating peptide-induced vascular smooth muscle cell proliferation.

The serine protease thrombin, in addition to its pivotal role in the coagulation cascade, plays an important role in the development of atherosclerosis and restenosis by inducing smooth cell proliferation. Thrombin exerts its cellular effects mainly by cleaving its own receptor, leaving a new NH2-terminus that can act as a tethered ligand to activate the thrombin receptor. Peptides derived from the new NH2-terminus are able to fully activate thrombin receptor and mimic cellular effects of thrombin. Peptides with structural similarities to the tethered ligand have been tested for their ability to prevent thrombin- and tethered ligand-induced platelet aggregation and thrombus formation. We synthesized a peptide with multiple alanine substitutions in both critical and noncritical residues of tethered ligand that specifically inhibited platelet aggregation induced by thrombin and thrombin receptor-activating peptide and prevented thrombus formation in a rabbit thrombosis model. In the present study we demonstrate that this peptide inhibited only thrombin- and tethered ligand-induced human vascular smooth muscle cell proliferation as determined by (3H)-thymidine incorporation and has no effect on platelet-derived growth factor and serum-induced smooth muscle cell proliferation. The inhibitory effect of this peptide is dependent on the concentration of the antagonist used and length of preincubation time. The possible mechanism by which this peptide exerts its inhibitory effect may by desensitizing the thrombin receptor. The results of the present study suggest that apart from being antithrombotic, tethered ligand antagonist peptides can also act as antiatherosclerotic or antirestenotic agents.

WISP-2: A Serum-Inducible Gene Differentially Expressed in Human Normal Breast Epithelial Cells and in MCF-7 Breast Tumor Cells

WISP-2 is a Wnt-1-induced signaling protein identified as a member of CCN growth factor family. A role for this molecule during tumorigenesis is suspected but remains unproven. Here we show that WISP-2 expression was undetectable, or minimally detectable, in nontransformed human mammary epithelial cells, but was overexpressed in MCF-7 cells. Expression of WISP-2 in MCF-7 cells was modulated by serum and correlated with the serum-induced MCF-7 tumor cell proliferation, suggesting that WISP-2 is serum responsive and may be a positive regulator of tumor cell proliferation.

Identification of Rho GTPase-dependent Sites in the Dbl Homology Domain of Oncogenic Dbl That Are Required for Transformation

The Dbl family guanine-nucleotide exchange factors (GEFs) for Rho GTPases share the structural array of a Dbl homology (DH) domain in tandem with a Pleckstrin homology (PH) domain. For oncogenic Dbl, the DH domain is responsible for the GEF activity, and the DH-PH module constitutes the minimum structural unit required for cellular transformation. To understand the structure-function relationship of the DH domain, we have investigated the role of specific residues of the DH domain of Dbl in interaction with Rho GTPases and in Dbl-induced transformation. Alanine substitution mutagenesis identified a panel of DH mutants made in the alpha1, alpha6, and alpha9 regions and the PH junction site that suffer complete or partial loss of GEF activity toward Cdc42 and RhoA. Kinetic and binding analysis of these mutants revealed that although most displayed decreased k(cat) values in the GEF reaction, the substrate binding activities of T506A and R634A were significantly reduced. E502A, Q633A, and N673A/D674A, on the other hand, retained the binding capability to the Rho GTPases but lost the GEF catalytic activity. In general, the in vitro GEF activity of the DH mutants correlated with the in vivo Cdc42- and RhoA-activating potential, and the GEF catalytic efficiency mirrored the transforming activity in NIH 3T3 cells. Moreover, the N673A/D674A mutant exhibited a potent dominant-negative effect on serum-induced cell growth and caused retraction of actin structures. These studies identify important sites of the DH domain involved in binding or catalysis of Rho proteins and demonstrate that maintaining a threshold of GEF catalytic activity, in addition to the Rho GTPase binding activity, is essential for efficient transformation by oncogenic Dbl.

Characterization of calcium-mobilizing, purinergic P2Y(2) receptors in human ovarian cancer cells.

In human ovarian EFO-21 and EFO-27 carcinoma cells, extracellular ATP induced a concentration-dependent rise in intracellular calcium concentration ([Ca(2+)](i)), suggesting the expression of a purinoreceptor. ATP and UTP were equipotent in generating [Ca(2+)](i) signals, followed by ATP-gamma-S and ADP, whereas beta, gamma-ATP, 2 methyl 1 thio-ATP, 3'-o-(4-benzoyl) benzoyl-ATP, AMP, and adenosine were ineffective. This pharmacological profile suggested the presence of the P2Y(2) subtype in both cell types, and this was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) analysis using P2Y(2) primers. ATP-induced [Ca(2+)](i) signals were composed of two phases: an early and extracellular calcium-independent phase, followed by a sustained plateau phase that was dependent on capacitative calcium influx. In addition to the rise in the [Ca(2+)](i), a time- and concentration-dependent increase in phosphatidylethanol accumulation was observed in ATP-stimulated cells, indicating an increase in phospholipase D activity. RT-PCR analysis identified the expression of a transcript for the phospholipase D-1 subtype of this enzyme. Activation of these receptors by a slowly degradable analogue, ATP-gamma-S, attenuated basal and fetal calf serum-induced cell proliferation in a time- and concentration-dependent manner. These results indicate that ATP may act as an extracellular messenger in controlling the ovarian epithelial cell cycle through P2Y(2) receptors.

Nuclear localization of the type 1 parathyroid hormone/parathyroid hormone-related peptide receptor in MC3T3-E1 cells: association with serum-induced cell proliferation

We have recently demonstrated that the receptor for parathyroid hormone (PTH) and PTH-related peptide (PTHrP), PTHR, can be localized to the nucleus of cells within the liver, kidney, uterus, gut, and ovary of the rat. We set out to determine the localization of the PTHR in cultured osteoblast-like cells. MC3T3-E1, ROS 17/2.8, UMR106, and SaOS-2 cells were cultured in alpha-modified eagle medium containing 15% fetal calf serum under standard conditions. Untreated cells were grown on glass coverslips to 75–95% confluence and fixed in 1% paraformaldehyde. For experiments designed to examine cells synchronized by serum starvation, cells were grown on glass coverslips, starved of serum for 46 h, and then fixed at 2-h intervals for a total of 26 h after the addition of serum to the medium. Parallel sets of cells were pulsed with [ 3H]thymidine to track the DNA duplication interval. The PTHR was localized by immunocytochemistry using a primary antibody raised against a portion of the N-terminal extracellular domain of the PTHR. The results presented herein indicate that the PTHR attains a nuclear localization in each cell line examined. In UMR106 cells, PTHR immunoreactivity was restricted to the nucleolus. After cell synchronization, MC3T3-E1 cells double approximately 24 h after the addition of serum. Immunocytochemistry for the PTHR in these cells showed that the receptor staining is initially diffuse for the first 6 h, then becomes more perinuclear in distribution by 12–16 h. Nuclear localization of the receptor is achieved approximately 16–20 h after the addition of serum and remains there throughout the mitotic phase. Intense staining of mitotic and postmitotic cells was observed. No change in cell proliferation kinetics was observed in MC3T3-E1 cells cultured in the presence of 25 nM PTH(1–34). These data suggest an important role for the PTHR in the nucleus of MC3T3-E1 cells at the time of DNA synthesis and mitosis.

Butyrate inhibits proliferation-induced proliferating cell nuclear antigen expression (PCNA) in rat vascular smooth muscle cells.

Arterial injury-induced vascular smooth muscle cell (VSMC) proliferation in intima is the important etiologic factor in vascular proliferative disorders such as atherosclerosis, hypertension and restenosis after balloon angioplasty. Butyrate, a naturally occurring short chain fatty acid, is produced by bacterial fermentation of dietary fiber and by mammary glands of certain mammals. Studies have shown that butyrate at millimolar concentrations, which are physiological, induces growth arrest, differentiation and apoptosis. We examined the effect of physiological concentrations of butyrate on rat VSMC proliferation and proliferation-induced PCNA expression to determine anti-atherogenic potential of butyrate. Butyrate concentrations, closer to physiological range, exhibited antiproliferative effects on both serum-induced proliferation of serum-starved quiescent VSMCs and actively proliferating non-confluent VSMCs. Treatment of serum-starved quiescent VSMCs with 1-8 mmol/l concentration of butyrate caused a concentration-dependent decrease in serum-induced VSMC proliferation and cell proliferation-associated increase in total cellular proteins and RNA levels. Similarly, exposure of actively growing VSMCs to 5 mmol/l butyrate resulted in the inhibition of cell proliferation and proliferation-induced increase in cellular proteins and RNA levels. Furthermore, cellular morphology was significantly altered. Analysis of cell cycle regulatory proteins indicated that levels of PCNA, an excellent marker for cell proliferation, was significantly altered by butyrate both in actively proliferating and serum-induced quiescent VSMCs. These observations suggest that butyrate exhibits potential antiatherogenic capability by inhibiting VSMC proliferation and proliferation-associated increase in PCNA expression and thus merits further investigations regarding therapeutic significance of butyrate in vascular proliferative disorders.

Antiangiogenesis efficacy of nitric oxide donors.

Angiogenesis is a complex process involving endothelial cell migration, proliferation, invasion, and tube formation. Inhibition of these processes might have implications in various angiogenesis-mediated disorders. Because nitric oxide (NO) is known to play a key role in various vascular diseases, the present study was undertaken to determine the role of NO in angiogenesis-mediated processes using the NO donor, S-nitroso N-acetyl penicillamine (SNAP) and S-nitroso N-acetyl glutathione (SNAG). The antiangiogenic efficacy of these NO donors was examined using in vivo and in vitro model systems. The in vitro studies demonstrated the ability of SNAP to inhibit cytokine fibroblast growth factor (FGF2)-stimulated tube formation and serum-induced cell proliferation. The inhibitory effect on cell proliferation by SNAP concentrations above the millimolar range was associated with significant shifts in the concentration of NO metabolites. Furthermore, using the mouse Matrigel implant model and the chick chorioallantoic membrane (CAM) models, SNAP demonstrated maximal inhibitory efficacy (85-95% inhibition) of cytokine (FGF2)-induced neovascularization in both in vivo models. SNAP and SNAG resulted in 85% inhibition of FGF2-induced neovascularization in the mouse Matrigel model when given at 5 mg/kg/day infusion in minipumps during 14 days and 87% inhibition of angiogenesis induced by FGF2 in the CAM when administered a single dose of 50 microg. Thus, NO donors might be a useful tool for the inhibition of angiogenesis associated with human tumor growth, or neovascular, ocular, and inflammatory diseases.

Evidence for enhanced rates of complement activation in serum from patients with newly diagnosed insulin-dependent diabetes mellitus exposed to rat islet cells and complement-dependent induction of islet cell apoptosis.

In this paper we report the concentration of terminal complement complexes (TCCs, SC5b-9, an index of complement activation) in newly diagnosed insulin-dependent diabetes mellitus (IDDM) patient serum and normal human serum. In the nine patients studied, levels of serum soluble TCCs were approximately 1.6-fold higher than in sera obtained from normal control individuals. On incubation of rat islet cells with diluted serum (10%, v/v, concentration), complement activation was increased at a significantly faster rate and the total TCC concentration was significantly higher in culture medium containing IDDM patient serum than in medium containing control serum. The concentration of anti-(glutamic acid decarboxylase) autoantibodies in newly diagnosed IDDM patient serum was on average 60-fold higher than in normal human control serum. IDDM patient serum (10%, v/v) induced apoptosis in islet cells, as determined by islet cell density changes and DNA fragmentation patterns. However, serum from IDDM patients was not able to induce apoptosis of the cells when complement components (C1q and C3) or antibodies were depleted. In addition, glutamine and the potent antioxidant 1-pyrrolidinecarbodithioic acid partially reversed cell death induced by IDDM patient serum in a concentration-dependent manner. The ATP concentration in islet cells incubated for 24 h in the presence of diluted IDDM patient serum was reduced to 4.4% of that observed in islet cells incubated in fetal calf serum or 7.3% of that observed in islet cells incubated in normal human serum. On the basis of these observations, we suggest that the pathway of IDDM patient serum-induced islet cell apoptosis may involve antibody-dependent complement activation, free radical generation and a precipitous fall in ATP levels.

Induction of bone formation in rat osteoprogenitor cell culture by sera of climacteric women before and after hormone replacement therapy

Objective: To evaluate the effect of hormone replacement therapy (HRT) on growth and differentiation of cultured osteoprogenitor cells. Design: Prospective clinical study. Setting: Outpatients in a menopause clinic. Patient(s): Women with climacteric symptoms. Intervention(s): Daily oral conjugated estrogen, 0.625 mg, and medroxyprogesterone acetate, 2.5 mg, for 7–12 months. Bone density measurement before HRT and blood sampling before and after HRT. Main Outcome Measure(s): Sera of climacteric women were added to the culture of rat osteoprogenitor cells, and indices of cell proliferation and differentiation (alkaline phosphatase activity and mineralization) were measured before and after HRT. Result(s): Sera after HRT significantly decreased cell counts but not alkaline phosphatase activity or mineralization as compared with sera before HRT. However, mineralization induced in the bioassay by both sera showed a positive correlation ( r = 0.56) with E 2 levels before treatment and a negative correlation ( r = −0.6181) with time in menopause of serum donors. The change in mineralization showed a significant correlation with hip bone mineral density z scores ( r = −0.67) but not with spine z scores ( r = −0.1915), whereas the change in cell count correlated with spine bone mineral density z scores ( r = 0.49) only. Conclusion(s): Changes in serum-induced cell proliferation and mineralization may be helpful in studying the response to HRT in climacteric women. Serum-induced mineralization is more efficient in diagnosing osteopenia than in monitoring HRT effects.

Evidence for Repressional Role of an Inverted CCAAT Box in Cell Cycle-dependent Transcription of the Human DNA Topoisomerase IIα Gene

Expression of DNA topoisomerase IIalpha (topo IIalpha) is cell cycle-regulated at both the transcriptional and the post-transcriptional levels. In order to identify cis-acting elements responsible for transcriptional regulation during the cell cycle, we investigated NIH/3T3 cells stably transfected with luciferase reporter plasmids containing various lengths of the human topo IIalpha gene promoter. Serum-deprived cells expressed low levels of luciferase, and following serum-induced cell cycle re-entry luciferase levels were gradually elevated 2-fold. During S phase, a steep 3-fold increase in luciferase activity was seen, reaching its maximum approximately 22 h after serum addition. This pattern was observed with both a full-length (nucleotides (nt) -295 to +90] and a deletion (nt -90 to +90) promoter construct. In contrast, when testing a deletion construct (nt -51 to +90) lacking the first inverted CCAAT box (ICB1) the S phase-specific induction was absent. Mutation of ICB1 revealed that it had a repressive character, since luciferase levels rose rapidly to maximal levels immediately following serum addition. Furthermore, electrophoretic mobility shift assays demonstrated a marked decrease in ICB1 binding activity following serum addition. Together, this suggests a role of ICB1 in cell cycle-dependent repression of topo IIalpha transcription.

Overexpression of p21Waf-1 in Vascular Smooth Muscle Cells: Regulation of Proliferation, Differentiation, and Cell Size

Cyclin-dependent kinase inhibitor p21Waf-1 is recognized as a negative regulator of cell cycle progression, and it possibly mediates cell differentiation and apoptosis. To understand the role of p21Waf-1 in phenotypic modulation of vascular smooth muscle cells (SMC), we induced the overexpression of p21Waf-1 in cultured rat SMC. The recombinant adenovirus vector encoding p21Waf-1 (AdvCMVp21) was constructed by homologous recombination and the vector encoding β-galactosidase (AdvCMVLacZ) was used as an experimental control. Administration of AdvCMVp21 suppressed serum-induced proliferation and cell cycle progression; however, the number of quiescent cells and the population of TUNEL-positive cells were not altered. Overexpression of p21Waf-1 did not affect the expression of contractile proteins and the availability of an endogenous growth factor signal p21Waf-1 may regulate cell cycle progression in SMC without affecting the apoptotic process and cell differentiation. Furthermore, the longitudinal diameter of AdvCMVp21 infected cells was increased compared with that of AdvCMVLacZ infected cells. Total protein content was also increased in AdvCMVp21 infected cells. Responses to the serum stimulation, proliferation and total protein synthesis may be independently regulated. Thus, the suppression of cell cycle progression by p21Waf-1 resulted in cellular hypertrophy of SMC.