Developmental processes involving symmetry-breaking of homogeneous cell populations into leaders and followers are found in many important contexts. Cells constrained by culture on two-dimensional scaffolds, as well as in three-dimensional shapes, appear to respond to convex curves with an increasing propensity to protrude, while concave curves in contrast appear to inhibit protrusion. This has interesting implications in terms of a potential positive feedback loop. This feedback may act in symmetry-breaking, through amplification of initial stochastic differences in cell shape, and also in collective migration, through reinforcing and directing the coherent movement of collectives. In this study, epithelial cells were cultured on two-dimensional micropatterns with variable curvatures to examine the effect of edge geometry and other variables on the likelihood of protrusions forming. This platform allowed the quantification of F-actin-based protrusions at the periphery of multicellular epithelial clusters, in segments defined by cluster edge curvature. The initial observations confirmed reports in the literature of preferential localisation of protrusions at more convex regions, and relative inhibition at more concave regions. A previously-published work has postulated a role for secreted modulators of motility, with the shape of a group of cells determining the concentration of diffusing morphogen each individual cell is exposed to. To test this hypothesis, a low-shear flow culture chamber was used to disrupt the putative gradients. Despite theoretical and empirical support for the sufficiency of the flow condition to disrupt autocrine signalling, micropatterned cells cultured under flow showed no significant differences from the control condition. These findings form the basis of a manuscript which has been accepted for publication by the Journal of Anatomy. The results of an Atomic Force Microscopy (AFM) study carried out by collaborators were suggestive of a role for cellular mechanotransduction in sensing and responding to micropattern curvature. Differential calcium channel mechanoactivation was hypothesised as being one potential mechanism underlying the response to curvature, given the known involvement of mechanosensitive ion channels in cellular responses to force and substrate stiffness, and the multiple roles of calcium in cellular motility. Artificially increasing cytosolic calcium levels with Ionomycin reduced protrusion rates at convex curves. However, treatment with BAPTA-AM to sequester intracellular calcium had no effect on protrusion rates. ROCK inhibitor, in contrast, increased protrusion rates at concave curves, and Blebbistatin increased protrusion rates globally. These results together are suggestive of differential control of myosin depending on local curvature: cyclic and driven by calcium-activation of MLCK in the convex regions (with lamellipodia undergoing protrusion-retraction cycles), versus sustained and controlled by ROCK in the concave regions (where lamellipodia are inhibited). The unexpected finding that protrusions at convex regions were resistant to the actin cytoskeleton-disrupting drug Cytochalasin D may point to a role for a tropomyosin isoform in defining the differing mechanical characteristics of the actin cytoskeleton in response to local curvature. In addition, the previously-noted lack of effect of BAPTA-AM treatment (which has been shown to interfere with dynamic microtubules) is suggestive of a role for stabilised microtubules in protrusions at convex regions. These indications of unique characteristics to the protrusions promoted by convex curvature give added support to the curvature-protrusion feedback model, and its relevance to tissue morphogenesis. In summary, this work provides evidence against a previously-published suggested mechanism for the curvature-protrusion feedback loop that is proposed to act during epithelial morphogenesis, and evidence in support of a role for a calcium-based mechanism in driving the initiation and maintenance of leader cells in migrating epithelial sheets. Further work is called for in characterising the protrusions promoted by convex curvature, and the mechanisms controlling them. This area is of significance in gaining greater understanding of tissue morphogenesis in pathogenesis and development, and of potential value in tissue engineering applications.

Abstract The effect of mercury (Hg2+), a known nephrotoxicant, on intracellular free Ca2+ levels ([Ca2+]i) in Madin Darby canine kidney (MDCK) cells was explored. [Ca2+]i was measured by using the Ca2+-sensitive dye fura-2. Hg2+ increased [Ca2+]i in a concentration-dependent manner with an EC50 of 6 mM. The Ca2+ signal comprised a gradual increase. Removal of extracellular Ca2+ decreased the Hg2+-induced [Ca2+]i increase by 67%, suggesting that the Ca2+ signal was due to both extracellular Ca2+ influx and store Ca2+ release. In Ca2+-free medium, the Hg2+-induced [Ca2+]i increase was nearly abolished by pretreatment with 1 mM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), and conversely, pretreatment with Hg2+ abolished thapsigargin-induced Ca2+ increase. Hg2+-induced Ca2+ release was not altered by inhibition of phospholiase C but was potentiated by activation of protein kinase C. Overnight treatment with 1 mM Hg2+ did not alter cell proliferation rate, but 10 mM Hg2+ killed all cells. Collectively, this study shows that Hg2+ induced protein kinase C-regulated [Ca2+]i increases in renal tubular cells via releasing store Ca2+ from the endoplasmic reticulum in a manner independent of phospholipase C activity. Hg2+ also caused cytotoxicity at higher concentrations.

No description available.

The effect of 2,4,6-trimethyl-N-(meta-3-trifluoromethyl-phenyl)- benzenesulfonamide (m-3M3FBS), a presumed phospholipase C activator, on cytosolic free Ca2+ concentrations ([Ca2+]i) in Madin Darby canine kidney (MDCK) cells and OC2 human oral cancer cells was unclear. This study explored whether m-3M3FBS changed basal [Ca2+]i levels in suspended MDCK and OC2 cells by using fura-2 as a Ca2+-sensitive fluorescent dye. m-3M3FBS at concentrations between 0.1-20 £gM increased [Ca2+]i in a concentration-dependent manner in MDCK cells, however in OC2 cells, m-3M3FBS at concentrations between 10-60 £gM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signals were reduced partly by removing extracellular Ca2+ in the two cell types. m-3M3FBS-induced Ca2+ influx was inhibited by the store-operated Ca2+ channel blockers nifedipine, econazole and SK&F96365, and by the phospholipase A2 inhibitor aristolochic acid. In Ca2+-free medium, m-3M3FBS pretreatment abolished the [Ca2+]i rise induced by the endoplasmic reticulum Ca2+ pump inhibitors thapsigargin, cyclopiazonic acid or 2,5-di-tert-butylhydroquinone (BHQ). Conversely, pretreatment with thapsigargin, cyclopiazonic acid or BHQ partly reduced m-3M3FBS-induced [Ca2+]i rise. Inhibition of phospholipase C with U73122 did not alter m-3M3FBS-induced [Ca2+]i rise. Collectively, in MDCK and OC2 cells, m-3M3FBS induced [Ca2+]i rises by causing phospholipase C-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via store-operated Ca2+ channels and other unidentified Ca2+ channels. Additionally, 5-100 £gM of m-3M3FBS killed cells in a concentration-dependent manner in OC2 cells. The cytotoxic effect of m-3M3FBS was not reversed by prechelating cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane- N,N,N',N'-tetraacetic acid (BAPTA). Propidium iodide staining data suggest that m-3M3FBS (20 or 50 £gM) induced apoptosis in a Ca2+-independent manner. We were also interested in knowing whether BAPTA suppressed cell death during oxidative stress in MDCK cells. BAPTA loading altered tBHP (tert-butyl hydroperoxide) and H2O2-induced cell death in a concentration-dependent manner. This suggests that the cell death induced by tBHP and H2O2 appears to be Ca2+-dependent in MDCK cells. The tBHP and H2O2-induced cell death was not suppressed by 2 £gM U73122 (PLC inhibitor), 50 £gM zVAD-fmk (caspase inhibitor), 2 £gM cyclosporin A (a potent inhibitor of the MPTP), 20 £gM PD98059 (ERK inhibitor) or 2 £gM SP600125 (JNK inhibitor). This suggests that the tBHP and H2O2-induced MDCK cells death was not via the PLC, MPTP, caspase, ERK or JNK pathways. Propidium iodide staining, caspase-3 activity assay and cell morphology data suggest that tBHP and H2O2-induced cell death was necrosis, not via apoptosis, and the cell death appears to be caspase-independent and Ca2+-dependent. The effect of the antidepressant paroxetine on [Ca2+]i in OC2 human oral cancer cells is unclear. This study also explored whether paroxetine changed basal [Ca2+]i levels in suspended OC2 cells by using fura-2 as a Ca2+-sensitive fluorescent dye. Paroxetine at concentrations between 100-1000 £gM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced by 50% by removing extracellular Ca2+. Paroxetine-induced Ca2+ influx was inhibited by the store-operated Ca2+ channel blockers nifedipine, econazole and SK&F96365, the phospholipase A2 inhibitor aristolochic acid, and protein kinase C modulators. In Ca2+-free medium, pretreatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin abolished paroxetine¡Vinduced [Ca2+]i rise. Inhibition of PLC with U73122 did not alter paroxetine-induced [Ca2+]i rise. Paroxetine at 10-50 £gM induced cell death in a concentration-dependent manner. The death was not reversed when cytosolic Ca2+ was chelated with BAPTA. Propidium iodide staining suggests that apoptosis played a role in the death. Collectively, in OC2 cells, paroxetine induced [Ca2+]i rise by causing PLC-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via store-operated Ca2+ channels in a manner regulated by protein kinase C and phospholipase A2. Paroxetine also induced cell death in a Ca2+-independent manner.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Bothropoides erythromelas à responsÃvel por muitos acidentes no Nordeste do Brasil. O veneno desta serpente induz insuficiÃncia renal aguda. Rins isolados de ratos Wistar, pesando 250 a 300g, foram perfundidos durante 120 min com soluÃÃo Krebs-Henseleit contendo 6g% de albumina bovina. O veneno total de Bothropoides erythromelas foi estudado anteriormente (SOUSA, 2004) e utilizado neste estudo para posterior comparaÃÃo com os grupos tratados com as fraÃÃes PLA2 Lys 49 e Asp 49 do veneno. O veneno total (10mg/mL) e as fraÃÃes PLA2 Lys 49 e Asp 49 (5mg/mL) de B. erythromelas foram adicionados ao sistema 30 min apÃs o inÃcio de cada experimento. Os parÃmetros estudados incluÃram pressÃo de perfusÃo (PP), resistÃncia vascular renal (RVR), ritmo de filtraÃÃo glomerular (RFG), fluxo urinÃrio (FU), percentual de transporte tubular de sÃdio, potÃssio e cloreto (%TNa+, %TK+ e %TCl-), percentual de transporte proximal de sÃdio, potÃssio e cloreto (%pTNa+, %pTK+ e %pTCl-), excreÃÃo de sÃdio, potÃssio e cloreto (ENa+, EK+ e ECl-) e clearance osmÃtico (Cosm) (p< 0,05*). O grupo controle perfundido com albumina foi funcionalmente estÃvel por todos os 120 min. A infusÃo do veneno causou um aumento significante no FU, ENa+, ECl- e Cosm e uma diminuiÃÃo na PP, RVR, %TNa+, %TK+, %TCl-, %pTNa+, %pTK+ e %pTCl-. O RFG e a EK+ diminuÃram aos 60 min e aumentaram aos 90 e 120 min quando comparado com o grupo controle. A infusÃo de Lys 49 causou um aumento significante na PP, FU, ENa+, EK+ e Cosm e diminuiu o RFG e o %TNa+ quando comparada com o grupo controle. Lys 49 nÃo modificou os outros parÃmetros funcionais renais. A infusÃo de Asp 49 modificou apenas os parÃmetros funcionais renais %pTK+ (diminuiÃÃo) e EK+ (aumento) quando comparada ao grupo controle. Lys 49 apresentou um efeito similar ao veneno total nos parÃmetros FU, %TNa+, ENa+, EK+ e Cosm e Asp 49 nos parÃmetros %pTK+ e EK+. A anÃlise histolÃgica mostrou uma quantidade moderada de material proteinÃceo nos glomÃrulos e tÃbulos de rins perfundidos com o veneno, Lys 49 e Asp 49, bem como regiÃes focais de apoptose/necrose em rins perfundidos com Lys 49 e Asp 49. CÃlulas MDCK foram cultivadas em meio de cultura RPMI 1640 suplementado com 10% vv de soro bovino fetal e entÃo avaliadas na presenÃa do veneno total, Lys 49 e Asp 49 de B. erythromelas nas concentraÃÃes (100; 50; 25; 12,5; 6,25 e 3,125mg/mL). A anÃlise dos efeitos citotÃxicos em cÃlulas MDCK foi executada pelo mÃtodo MTT. O veneno promoveu efeito citotÃxico nas concentraÃÃes de 50 e 100mg/mL (IC50 =93,31mg/mL). Lys 49 promoveu efeito citotÃxico nas concentraÃÃes de 6,25; 12,5; 25; 50 e 100mg/mL (IC50 = 38,29mg/mL). Asp 49 promoveu efeito citotÃxico nas concentraÃÃes de 50 e 100mg/mL (IC50 = 158mg/mL). TambÃm foram mensurados os nÃveis de lactato desidrogenase (LDH) e nenhum aumento significante foi observado com veneno total e Asp 49. Lys 49 promoveu um aumento significante nos nÃveis de lactato desidrogenase apenas na concentraÃÃo de 100mg/mL. ApÃs o cultivo de cÃlulas MDCK com o veneno total, nas concentraÃÃes de 46,65 e 23,32mg/mL, foi realizada a reaÃÃo de polimerase em cadeia em tempo real para a avaliaÃÃo da expressÃo de genes prà (Caspase-3, Caspase-8 e Bax) e antiapoptÃticos (Bcl-XL e Mcl-1). NÃo foi realizada avaliaÃÃo da expressÃo de genes prà e antiapoptÃticos com Lys 49 e Asp 49. Na expressÃo de genes prÃ-apoptÃticos o veneno total promoveu um aumento da expressÃo de caspase-3 na concentraÃÃo de 23,32Âg/mL e de caspase-8 nas concentraÃÃes de 46,65 e 23,32Âg/mL, quando comparado com os controles positivo (DOXO) e negativo (PBS) e diminuiu a expressÃo de Bax em ambas as concentraÃÃes. Na expressÃo de genes antiapoptÃticos o veneno total promoveu induÃÃo significativa de Mcl-1 somente na concentraÃÃo de 46,65mg/mL e nÃo modificou a expressÃo de Bcl-XL, quando comparado com o controle negativo. O veneno e as fraÃÃes PLA2 Lys 49 e Asp 49 da serpente Bothropoides erythromelas à capaz de promover significativos efeitos sobre os parÃmetros de funÃÃo renal e sobre cÃlulas MDCK, com indicativo de morte celular por apoptose atravÃs da via extrÃnseca. / Bothropoides erythromelas is responsible for a great deal of snakebites in Northeastern from Brazil. The venom of this snake induces acute renal failure. Isolated kidneys from Wistar rats, weighting 250 to 300g, were perfused with Krebs-Henseleit solution containing 6g% of bovine serum albumin for 120 min. The whole venom of Bothropoides erythromelas been previously studied (SOUSA, 2004) and used in this study for comparison with the treated groups with the PLA2 fractions Lys 49 and Asp 49 of the venom. The whole venom (10mg/mL) and the fractions PLA2 Lys 49 and Asp 49 of B. erythromelas (5mg/mL) were added into the system 30 min after the beginning of each experiment. The parameters studied included perfusion pressure (PP), renal vascular resistance (RVR), glomerular filtration rate (GFR), urinary flow (UF), percent sodium, potassium and chloride tubular transport (%TNa+, %TK+ and %TCl-), percent sodium, potassium and chloride proximal transport (%pTNa+, %pTK+ and %pTCl-), sodium, potassium and chloride excretion (ENa+, EK+ e ECl-) and osmotic clearance (Cosm) (p< 0.05*). The control group perfused with albumin was functionally stable for over 120 min. The infusion of venom caused a significant increase in UF, ENa+, ECl- and Cosm and a decreased in PP, RVR, %TNa+, %TK+, %TCl-, %pTNa+, %pTK+ and %pTCl-. The GFR and the EK+ decreased at 60 min and increased at 90 and 120 min when compared with control group. The infusion of Lys 49 caused a significant increase in PP, UF, ENa+, EK+ and Cosm and decreased the GFR and the %TNa+ when compared with control group. Lys49 did not modify the others functional kidney parameters. The infusion of Asp 49 only modify the functional kidney parameters %pTK+ (decreased) and EK+ (increase) when compared with control group. Lys 49 showed a similar effect at whole venom in parameters UF, %TNa+, ENa+, EK+ and Cosm and Asp 49 in parameters %pTK+ and EK+. The histological analysis showed a mild amount of a proteinaceous substance in the renal tubules and glomeruli of kidneys perfused with the venom, Lys 49 and Asp 49, as well as focal areas of apoptosis/necrosis in perfused kidneys with Lys 49 e Asp 49. MDCK cells were cultured in RPMI 1640 medium supplemented with 10% vv fetal bovine serum and then assessed in the presence of the whole venom, Lys 49 and Asp 49 of B. erythromelas in the concentrations (100; 50; 25; 12.5; 6.25 and 3.125mg/mL). The analysis of cytotoxic effects on MDCK cells was performed by MTT method. The venom promoted cytotoxic effect in the concentrations of 50 and 100mg/mL (IC50 =93.31mg/mL). Lys 49 promoted cytotoxic effect in the concentrations of 6.25; 12.5; 25; 50 and 100 mg/mL (IC50 = 38.29mg/mL). Asp 49 promoted cytotoxic effect in the concentrations of 50 and 100 mg/mL (IC50 = 158mg/mL). Also the levels of lactic dehydrogenase (LDH) were measured and no significant increase was observed with whole venom and Asp 49. Lys 49 promoted a significant increase in the levels of LDH only in the concentration of 100mg/mL. After culture of MDCK cells with the whole venom, at concentrations of 46.65 and 23.32Âg/mL, was performed the real time polymerase chain reaction for evaluation of pro (Caspase-3, Caspase-8 and Bax) and antiapoptotic (Bcl-XL and Mcl-1) genes expression. The evaluation of pro and antiapoptotic genes expression with Lys 49 e Asp 49 did not realized. In the expression of pro-apoptotic genes the whole venom caused increase of caspase-3 at concentration of 23.32Âg/mL and of caspase-8 at concentrations of 46.65 and 23.32Âg/mL, when compared with negative (PBS) and positive (DOXO) controls and decreased the expression of Bax in both concentrations. In the expression of anti-apoptotic genes the whole venom caused significant induction of Mcl-1 only at a concentration of 46.65Âg/mL and did not modify the expression of Bcl-XL, when compared with the negative control. The venom and the fractions PLA2 Lys 49 e Asp 49 of Bothropoides erythromelas is able to promote significant effects on renal function parameters and on MDCK cells, with indications of cell death by apoptosis through the extrinsic pathway.

O presente trabalho visa contribuir para o esclarecimento da seqüência de eventos intracelulares produzidos pelas PKCs a e e, na modulação do pHi, via NHE1. Os estudos foram realizados em células MDCK e as medidas de pHi efetuadas por microscopia de fluorescência. A expressão das PKCs a e e, bem como do NHE1 foi investigada por western blot, utilizando anticorpos específicos para cada proteína. Os estudos foram realizados na situação controle ou na vigência de PMA ou ANG II, AVP e /ou inibidores específicos para cada receptor hormonal ou isoforma de PKC. Nossos resultados indicam que PMA (10-7 M) estimula a recuperação do pHi, por modular a atividade das PKCs a e e. ANG II e AVP em concentrações fisiológicas estimulam a recuperação do pHi após sobrecarga ácida, concomitante com o aumento da fosforilação da PKC a. Em concentração elevada, ambos hormônios não alteram estes parâmetros. O efeito de ANG II ou de AVP depende da interação de cada hormônio com receptores específicos para modular as vias de sinalização celular envolvidas com o aumento dos níveis de diacilglicerol, cálcio citosólico e AMPc. / The purpose of this work was to investigate the signaling events of PKCs a e e on the NHE1 activity. The effect of phorbol 12-myristate 13-acetate (PMA), angiotensin II (ANG II) or arginine vasopressin (AVP) on the intracellular pH (pHi) was investigated in MDCK cells by using the fluorescence microscopy and fluorescent probe BCECF/AM. The NHE1 or PKCs a e e expression was examined by western blot and specific antibodies. Our results indicate that PMA (10-7 M) or low concentration of ANG II and AVP induced a significant increase of pH recovery rate and PKC a expression, after intracellular acidification with NH4Cl pulse. ANG II or AVP did not change the PKC a expression. However, in right concentration, both hormones did not change these parameter. In conclusion, the effect of ANG II and AVP on NHE1 activity, depend of specifics membrane receptors and cellular signaling of intracellular calcium, DAG and PKCs a and e.

O presente trabalho visa contribuir para o esclarecimento da seqüência de eventos intracelulares produzidos pelas PKCs a e e, na modulação do pHi, via NHE1. Os estudos foram realizados em células MDCK e as medidas de pHi efetuadas por microscopia de fluorescência. A expressão das PKCs a e e, bem como do NHE1 foi investigada por western blot, utilizando anticorpos específicos para cada proteína. Os estudos foram realizados na situação controle ou na vigência de PMA ou ANG II, AVP e /ou inibidores específicos para cada receptor hormonal ou isoforma de PKC. Nossos resultados indicam que PMA (10-7 M) estimula a recuperação do pHi, por modular a atividade das PKCs a e e. ANG II e AVP em concentrações fisiológicas estimulam a recuperação do pHi após sobrecarga ácida, concomitante com o aumento da fosforilação da PKC a. Em concentração elevada, ambos hormônios não alteram estes parâmetros. O efeito de ANG II ou de AVP depende da interação de cada hormônio com receptores específicos para modular as vias de sinalização celular envolvidas com o aumento dos níveis de diacilglicerol, cálcio citosólico e AMPc. / The purpose of this work was to investigate the signaling events of PKCs a e e on the NHE1 activity. The effect of phorbol 12-myristate 13-acetate (PMA), angiotensin II (ANG II) or arginine vasopressin (AVP) on the intracellular pH (pHi) was investigated in MDCK cells by using the fluorescence microscopy and fluorescent probe BCECF/AM. The NHE1 or PKCs a e e expression was examined by western blot and specific antibodies. Our results indicate that PMA (10-7 M) or low concentration of ANG II and AVP induced a significant increase of pH recovery rate and PKC a expression, after intracellular acidification with NH4Cl pulse. ANG II or AVP did not change the PKC a expression. However, in right concentration, both hormones did not change these parameter. In conclusion, the effect of ANG II and AVP on NHE1 activity, depend of specifics membrane receptors and cellular signaling of intracellular calcium, DAG and PKCs a and e.

Abstract The epithelial cells form barriers that compartmentalize the organs. Carcinomas are cancers stemming from epithelial cells and are the most common cancer type. The aim of this study was to understand the differentiation and malignant transformation of epithelial Madin-Darby canine kidney (MDCK) cells and to analyse the electrophysiological parameters which regulate their transport capacity. Emphasis was placed on comparing different culture environments, both in 2D and 3D. First, the effects of drugs or basal extracellular fluid composition on MDCK cell, cyst and lumen volumes were analysed using time-lapse microscopy. The results showed that MDCK cells were capable of both water secretion and reabsorption. The cells were able to perform these functions in a hyperpolarizing or depolarizing environment; change in osmolality of basal fluid was not required. Taken together, these results validate MDCK cells as a good basic model for studying kidney function. Next, the aim was to analyse the effect of 2D and 3D culture environments on the gene expression of untransformed MDCK and temperature sensitive ts-Src -transformed MDCK cells and the changes a single oncogene can induce. Microarray analysis revealed a decrease in the expression of survivin, an inhibitor of apoptosis protein, when switching the untransformed cells from 2D environment to 3D. This downregulation of survivin occurs in adult tissues as well, indicating that the cells grown in 3D are closer to the in vivo state than 2D cells. Src oncogene induced disintegration of cell junctions, but did not downregulate E-cadherin expression. The last part was to study further the factors controlling survivin expression and its significance to cell survival. MDCK cells grown in 3D did not suffer apoptosis if the cells remained in contact with the extracellular matrix. If MDCK cells were denied of ECM contacts they were more susceptible to apoptosis than survivin-expressing ts-Src MDCK cells. Finally, if cells were denied of cell-cell junctions, cells lacking survivin suffered apoptosis even though they had proper cell-matrix contacts. Taken together, these results highlighted the importance of cellular contacts to the cells: MDCK cells needed ECM contacts to differentiate and cell-cell contacts to avoid apoptosis. / Tiivistelmä Epiteelisolut ovat erikoistuneet toimimaan rajapintana elimen ja ympäristön välillä. Ihmisten yleisin syöpä on epiteelisoluista alkunsa saanut karsinooma. Tämän tutkimuksen tarkoituksena oli ymmärtää Madin-Darby-koiran munuaisen solujen (MDCK) erilaistumista ja pahanlaatuistumista sekä analysoida sähköfysiologisia tekijöitä, jotka säätelevät näiden solujen kuljetustoimintaa. Erityisenä kiinnostuksen kohteena oli erilaisten kasvuympäristöjen vertailu. Farmakologisten aineiden tai basaalisen, solunulkopuolisen nesteen koostumuksen vaikutusta MDCK-solujen, -kystan sekä luumenin kokoon tutkittiin valomikroskooppisten aikasarjojen avulla. Tulokset osoittivat MDCK-solujen olevan kykeneviä sekä veden eritykseen että absorptioon, niin hyperpolarisoivassa kuin depolarisoivassakin ympäristössä. Basaalisen nesteen osmolaliteetin muutosta ei tarvittu. Nämä tulokset osoittavat MDCK-solujen olevan hyvä munuaisen tutkimuksen perusmalli. Seuraavaksi analysoitiin kaksi- ja kolmiulotteisten (2D ja 3D) viljely-ympäristöjen vaikutusta ei-transformoitujen MDCK-solujen ja lämpötilaherkkien ts-Src-transformoitujen MDCK-solujen geenien ilmentymiseen sekä yhden onkogeenin aktivoimisen aikaansaamia muutoksia. Microarray-analyysi osoitti apoptoosin estäjän, surviviinin, ilmentymisen vähenemisen, kun kasvuympäristö vaihdettiin 2D-ympäristöstä 3D-ympäristöön. Koska surviviinin väheneminen on normaali tapahtuma aikuisissa kudoksissa, voitiin todeta, että 3D-ympäristössä kasvatetut solut ovat lähempänä luonnonmukaista olotilaa kuin 2D-ympäristössä kasvaneet. Src-onkogeeni sai aikaan soluliitosten hajoamisen, mutta ei vähentänyt E-kadheriinin ilmentymistä. Tutkimuksen viimeinen osa keskittyi surviviinin ilmentymistä säätelevien tekijöiden analysoimiseen ja surviviinin merkitykseen solujen eloonjäämiselle. 3D-ympäristössä kasvaneet MDCK-solut eivät kärsineet apoptoosista edellyttäen, että solut pysyivät kosketuksissa soluväliaineeseen. Jos solut irtautuivat soluväliaineesta, ne päätyivät herkemmin apoptoosiin kuin surviviinia ilmentävät ts-Src MDCK-solut. Mikäli solujen väliset liitokset pakotettiin avautumaan, solut joutuivat apoptoosiin, vaikka ne olivat kosketuksissa soluväliaineeseen. Yhteenvetona nämä tulokset korostavat solujen kontaktien merkitystä: MDCK-solut tarvitsevat soluväliainekontakteja erilaistumiseen ja solujen välisiä kontakteja välttyäkseen apoptoosilta.

No description available.

No description available.