A Step-by-Step Clinical Approach for the Management of Neuroendocrine Tumours.

Neuroendocrine tumours (NET) are rare neoplasms, but the incidence is permanently increasing. Most of the NETs are slow proliferating and clinically silent, and for that reason, they are often diagnosed at a stage with advanced disease. The complexity and diversity of the NET-biology require the treatment of patients in specialised centres to guarantee a qualified, multidisciplinary treatment planning. At our institution, we developed an interdisciplinary model for the assessment and treatment of NET. The aim was to adapt the guidelines to the clinical practice, exchange of current knowledge, and a tailored approach to the individual patient. In our team are included medical professionals from pathology, radiology, oncology, gastroenterology, oncological surgery, and nuclear medicine. In this paper, we describe step-by-step a procedural algorithm for the management of patients with neuroendocrine tumours, focusing on midgut-NETs in terms of therapy.

Control of the acute phase response. Demonstration of C-reactive protein synthesis and secretion by hepatocytes during acute inflammation in the rabbit.

To determine the cell of origin of C-reactive protein (CRP) and to cast light on the mechanisms leading to the acute phase response, we used an immunoenzymatic technique to visualize this protein in livers from rabbits at intervals after intramuscular injection of turpentine. CRP was detected only in hepatocytes. 8 h after turpentine injection, CRP was demonstrated in occasional periportal hepatocytes. With time, larger numbers of positive cells were detected successively in perilobular, midlobular, and centrilobular areas. On electron microscopy, CRP was detected in rough endoplasmic reticulum (RER), smooth endoplasmic reticulum (SER), and Golgi apparatus (GA). When colchicine was administered to inhibit cellular secretion of CRP, intensity of reaction and number of CRP-containing hepatocytes were substantially greater than without colchicine, but the sequence of intralobular distribution was similar. At peak serum response 38 h after turpentine injection, CRP could be demonstrated in most hepatocytes. Electron microscopic studies showed accumulation of CRP on membranes and lumina of RER, SER, GA, and in cytoplasmic vacuoles. These findings indicate that CRP is produced by progressively increasing numbers of hepatocytes after inflammatory stimulus and suggest that a mediator, acting initially in portal zones, is responsible for recruitment of cells to CRP production.

Heterogeneous lobular distribution of hepatocytes expressing acute-phase genes during the acute inflammatory reaction.

Functional heterogeneity in the lobule with regard to plasma protein synthesis is still debated. Therefore, we have localized in liver sections from normal rats and from rats with turpentine-induced AIR the mRNA and protein products of three genes with different alterations in their hepatic expression during an AIR: alpha 2M and alpha 1PI, two positively reacting acute-phase genes, and alpha 1I3, a negative acute-phase reactant. In normal liver, all hepatocytes expressed alpha 2M and alpha 1I3 mRNA, but a preferential expression of alpha 2M and alpha 1I3 mRNA and protein in the PP and ML zones was observed. During an AIR, the level of alpha 2M mRNA increased fourfold in the cytoplasm of PP and ML hepatocytes, while the level of cytoplasmic alpha 1I3 mRNA was decreased about fourfold in the same zones, with parallel variations in the expression of the corresponding proteins. In contrast, no significant modulation of the RNA and protein concentrations of both genes was detected in PV areas. alpha 1PI mRNA was expressed at the same levels in the three lobular zones in normal liver, but staining for the alpha 1PI protein was more intense in the PV zones. During the acute-phase response alpha 1PI mRNA levels were increased twofold in all three lobular zones, and alpha 1PI staining became homogeneous within the lobule. These results demonstrate that the location of a hepatocyte with the liver lobule can influence the expression of the three genes under study both at pre- and post-translational levels, in basal conditions, as well as during modulation of their expression during the inflammatory reaction.

Ultrastructural immunoperoxidase demonstration of autologous albumin in the alveolar capillary membrane and in the alveolar lining material in normal rats.

The location of autologous serum albumin within the alveolar-capillary membrane was studied in the rat under physiological conditions using antialbumin antibodies labeled with peroxidase. Albumin was detected in the lung interstitium, and in numerous pinocytic vesicles within endothelial cells and type I alveolar epithelial cells. The immunoreaction was also positive at the level of plasmalemmal membranes of both cell types and in the alveolar lining material.

Inhibition by colchicine of fibrinogen translocation in hepatocytes.

In the rat, 8 h after intraperitoneal administration of colchicine, fibrinogen (detected by antirat fibrinogen antibodies labeled with peroxidase) accumulated in the lumina of the rough endoplasmic reticulum of the hepatocytes; 16 and 24 h after colchicine administration, fibrinogen was detected, respectively, in the lumina of the smooth endoplasmic reticulum and in the Golgi apparatus. The effect of colchicine on the cytoplasmic translocation of fibrinogen could be due to a direct action of the drug on the membranes of the endoplasmic reticulum or could be the indirect result of the disruptive action of the drug on the microtubules.

Cellular analysis by in situ hybridization and immunoperoxidase of alpha-fetoprotein and albumin gene expression in rat liver during the perinatal period.

To analyze at the cellular level the decrease in alpha-fetoprotein (AFP) gene expression during the early postnatal growth, we searched for AFP gene transcripts by in situ hybridization using a specific cDNA probe, and for the corresponding protein by immunocytochemistry, on rat liver sections at various times of the perinatal period. The relative number of mRNA sequences was evaluated by Northern blot analysis. Albumin (ALB) gene expression was studied simultaneously with the same techniques. In 17-19-d-old fetuses all hepatocytes express simultaneously, for both genes, the mRNAs and the corresponding proteins. During the first postnatal weeks, at a time when the global number of AFP mRNA molecules decreases, all hepatocytes still contain cytoplasmic transcripts and protein. A zonal heterogeneity in the level of AFP gene expression develops around the first week, a higher number of gene products being detected in perivenous than in periportal hepatocytes. This heterogeneity persists until the fourth week when AFP mRNA sequences and protein are barely detectable. All hepatocytes express the ALB gene after birth, but at around the second week, a periportal intensification of the in situ hybridization signal and immunostaining becomes apparent. Our data indicate that co-expression of the AFP and ALB genes by all hepatocytes is a normal step in liver ontogeny; the diminution of AFP gene expression after birth is not the result of the disappearance of specialized cell clones; and zonal quantitative differences in the level of AFP and ALB gene expression are observed within the maturing liver lobule.

Hedgehog inhibition prolongs survival in a genetically engineered mouse model of pancreatic cancer.

BACKGROUND AND AIMS: Pancreatic cancer is among the most dismal of human malignancies. Current therapeutic strategies are virtually ineffective in controlling advanced, metastatic disease. Recent evidence suggests that the Hedgehog signalling pathway is aberrantly reactivated in the majority of pancreatic cancers, and that Hedgehog blockade has the potential to prevent disease progression and metastatic spread. METHODS: Here it is shown that the Hedgehog pathway is activated in the Pdx1-Cre;LsL-Kras(G12D);Ink4a/Arf(lox/lox) transgenic mouse model of pancreatic cancer. The effect of Hedgehog pathway inhibition on survival was determined by continuous application of the small molecule cyclopamine, a smoothened antagonist. Microarray analysis was performed on non-malignant human pancreatic ductal cells overexpressing Gli1 in order to screen for downstream Hedgehog target genes likely to be involved in pancreatic cancer progression. RESULTS: Hedgehog inhibition with cyclopamine significantly prolonged median survival in the transgenic mouse model used here (67 vs 61 days; p = 0.026). In vitro data indicated that Hedgehog activation might at least in part be ascribed to oncogenic Kras signalling. Microarray analysis identified 26 potential Hedgehog target genes that had previously been found to be overexpressed in pancreatic cancer. Five of them, BIRC3, COL11A1, NNMT, PLAU and TGM2, had been described as upregulated in more than one global gene expression analysis before. CONCLUSION: This study provides another line of evidence that Hedgehog signalling is a valid target for the development of novel therapeutics for pancreatic cancer that might be worth evaluating soon in a clinical setting.

Expression of the zinc-finger transcription factor Snail in adrenocortical carcinoma is associated with decreased survival.

In this study, we evaluate whether Snail is expressed in adrenocortical cancer (ACC) and if its expression is related to patient outcome. One of the best known functions of the zinc-finger transcription factor Snail is to induce epithelial-to-mesenchymal transition (EMT). Increasing evidence suggests that EMT plays a pivotal role in tumour progression and metastatic spread. Snail and E-cadherin expression were assessed by immunohistochemistry in 26 resected ACCs and real-time quantitative RT-PCR expression analysis was performed. Data were correlated with clinical outcome and in particular with overall patient survival. Seventeen of 26 (65%) ACC tumour samples expressed Snail when assessed by immunohistochemistry. Snail expression was neither detected in normal adrenocortical tissue, nor in benign adrenocortical adenomas. Expression levels were confirmed on the mRNA level by Real-Time-PCR. Survival rates were significantly decreased in Snail-positive tumours compared to Snail-negative tumours: 10 out of 16 vs one out of eight patients succumbed to disease after a median follow up of 14.5 and 28.5 months, respectively (P=0.03). Patients with Snail-expressing ACCs presented in advanced disease (11 out of 12 vs 6 out of 14, P=0.01) and tend to develop distant metastases more frequently than patients with negative staining (7 out of 11 vs two out of eight, P=0.19). In conclusion, we describe for the first time that Snail is expressed in a large subset of ACCs. Furthermore, Snail expression is associated with decreased survival, advanced disease and higher risk of developing distant metastases.

Cadmium induces mitochondria-dependent apoptosis of normal human hepatocytes.

The heavy metal cadmium, an environmental pollutant, has been widely demonstrated to be toxic, in particular for liver. In murines, cadmium induces apoptosis of hepatocytes and hepatomas. In human cells, apoptosis induced by cadmium has been exclusively demonstrated in tumoral cell lines. Nothing was known in normal liver, in vitro or in vivo. In the present study, we examined the effects of cadmium in nonmalignant human hepatocytes. For that purpose, we investigated whether cadmium was able to induce apoptosis of normal human hepatocytes (NHH) in primary culture and of a SV40-immortalized human hepatocyte (IHH) cell line. Treatment of IHH and NHH with cadmium induced the presence of a sub-G(1) population at 10 and 100 micromol/L, respectively. DAPI staining of both cell types treated with cadmium 100 micromol/L revealed the induction of nuclear apoptotic bodies, supporting the hypothesis of apoptosis. In IHH and NHH, cadmium 100 micromol/L induced PARP cleavage into a 85 kDa fragment. In order to investigate the involvement of mitochondria in cadmium-induced apoptosis, we measured the mitochondrial membrane potential (Delta(Psim)). We observed that in IHH and NHH, cadmium 100 micromol/L induced a decrease of Delta(Psim). As expected, cadmium under the same conditions enhanced caspase-9 and caspase-3 activities. In addition, cadmium from 1 to 100 micromol/L induced the expression of p53 and phosphorylation of its Ser15 in IHH and NHH. In conclusion, we showed in this study that human hepatocytes were sensitive to cadmium and apoptosis induced at concentrations suggested in the literature to inhibit p53 DNA-binding and DNA repair.

[Lutetium-177-PSMA radioligand therapy : Consensus within the framework of GKV-funded care between the university hospitals in Aachen, Bonn, Düsseldorf, Essen, and Cologne and the MDK Nordrhein]. / Lutetium-177-PSMA-Radioligandentherapie : Konsensus im Rahmen der GKV-finanzierten Versorgung zwischen den Hochschulkliniken in Aachen, Bonn, Düsseldorf, Essen und Köln und dem MDK Nordrhein.

In the last 3 years, Lutetium-177 prostate-specific membrane antigen radioligand therapy (Lu-177-PSMA-RLT) has received increasing attention in nuclear medicine as a new form of treatment for castration-resistant metastatic prostate cancer. This therapy combines the radionuclide Lutetium-177, which has been therapeutically used in nuclear medicine for many years, with a molecular target of the transmembrane prostate-specific membrane antigen expressed by prostate cancer cells. Since there are no prospective randomized studies on Lu-177-PSMA-RLT and the question of reimbursement has repeatedly been the subject of review by the MDK Nordrhein (Medischenische Dienst der Krankenversicherung), there was a desire because of the increasing number of patients being treated to clarify under which circumstances Lu-177-PSMA-RLT can be reimbursed by German statutory health insurance. The goals of this article are to help treating physicians understand how this new therapy option works, to integrate it in the overall therapy concept for castration-resistant metastatic prostate cancer, and, above all, to use Lu-177-PSMA-RLT-based on the current data-at the right place in the therapy sequence of castration-resistant metastatic prostate cancer.

Immunoperoxidase localization of bile salts in rat liver cells. Evidence for a role of the Golgi apparatus in bile salt transport.

The mechanisms of intracellular transport of bile acids from the sinusoidal pole to the canalicular pole of the hepatocyte are poorly understood. There is physiological and autoradiographic evidence for a vesicular pathway. The purpose of this study was to determine the localization of natural bile acids in the liver using antibodies against cholic acid conjugates and ursodeoxycholic acid. An indirect immunoperoxidase technique was used on rat liver sections fixed either with paraformaldehyde (PF) and saponin, a membrane-permeabilizing agent that allows penetration of antibodies into the cell, or with PF alone. Retention of taurocholate in the liver after tissue processing was 26 +/- SD 15% of the bile acid initially present. When sections fixed with PF and saponin were incubated with the antibody against cholic acid conjugates, a granular cytoplasmic staining was observed by light microscopy in all hepatocytes. By electron microscopy, strong electron-dense deposits were observed mostly on vesicles of the Golgi apparatus (GA) and, sometimes, in the smooth endoplasmic reticulum (SER). After taurocholate infusion, the intensity of the reaction increased. When the liver was fixed with PF alone, almost no reaction was visible on light microscopy, but on electron microscopy the label was localized on the hepatocyte plasma membrane, mainly on the bile canalicular domain and to a lesser extent on the sinusoidal domain. With the antibody against ursodeoxycholic acid, no staining was observed in three of four livers, and a slight staining was observed in one. However, after infusion of ursodeoxycholic acid, staining of GA and SER vesicles was observed when the liver was fixed with PF and saponin. With PF alone, the reaction was intense on the canalicular membrane. These results support the view that, within the limits of the method, vesicles from the GA and possibly vesicles of the SER are involved in the intracellular transport of bile acids before canalicular secretion.

[Apoptosis of granulosa cells as a predictive marker of in vitro fertilization success?]. / L'apoptose des cellules de la granulosa peut-elle être considérée comme un marqueur prédictif du succès de la fécondation in vitro?

During in vitro fertilization (IVF) morphological criteria are the only means usable today to select embryos before their uterine transfer in order to obtain pregnancy with the best chances of success. Since several years many attempts have been made to find more functional means. Quantification of apoptosis of granulosa cells has been proposed for this purpose. The aim of this review is to take stock of our knowledge on apoptosis and its mechanisms in granulosa cells and to analyse how quantification of these apoptotic cells could be a reliable and predictive marker of success for an attempt of an IVF in terms of pregnancy.

Cellular expression of alpha-fetoprotein gene and its relation to albumin gene expression during rat azo-dye hepatocarcinogenesis.

During hepatocarcinogenesis, alpha-fetoprotein (AFP) synthesis may be dramatically increased while albumin synthesis is frequently decreased. Therefore, a reciprocal modulation between both gene expressions has been hypothesized. In this work, we combined in situ hybridization and immunoperoxidase on parallel liver tissue sections in order to analyze at the cellular level, AFP gene expression and its relation to ALB gene expression in both early and neoplastic lesions induced by 3MeDAB in the rat. In early lesions, cell populations were heterogenous as regards AFP expression. High levels of AFP transcripts were detected both in oval type cells and in a subset of basophilic hepatocytes within preneoplastic lesions. In these two highly AFP-expressing cell populations, significant levels of ALB transcripts were concomitantly detected. In the majority of altered hepatocytes, no AFP expression was detected while the level of ALB expression was decreased. In neoplastic lesions, AFP expression was strikingly heterogenous and independent from the degree of morphological differentiation. No evidence of reciprocal modulation with ALB gene expression could be assessed. In both preneoplastic and neoplastic lesions, a few altered hepatocytes displayed significant levels of AFP transcripts while no corresponding protein could be detected; such a discrepancy was not observed for ALB. This work shows that during 3MeDAB hepatocarcinogenesis, AFP gene activation occurs in heterogenous cell populations and according to different cellular patterns. Our observations lend no support to the hypothesis of a reciprocal modulation between AFP and ALB gene expressions during rat azo-dye hepatocarcinogenesis.

Analysis of hepatocyte plasma membrane polarity during rat azo dye hepatocarcinogenesis using monoclonal antibodies directed against domain-associated antigens.

While carcinogenesis is known to induce various alterations in epithelial cell polarity, little information is available about the fate of plasma membrane polarity during the neoplastic process. Using three monoclonal antibody-defined antigens as markers of the three plasma membrane domains of rat hepatocytes, the sinusoidal, the lateral, and the canalicular, we demonstrated by immunohistochemical techniques that changes in hepatocyte plasma membrane polarity occur at every stage of 3'-methyldimethylaminoazobenzene-induced hepatocarcinogenesis. At the preneoplastic stage, the division of hepatocyte plasma membrane into three distinct domains was retained despite rearrangements in hepatocytic architecture, characterized by the disorganization of hepatocytic plates and the formation of pseudoacinar structures. The most striking change was the distribution of the canalicular-associated antigen over the entire plasma membrane in disorganized plates. At the neoplastic stage, changes in plasma membrane polarity depended on the degree of morphological differentiation of neoplastic cells. Poorly differentiated cells inconstantly expressed the monoclonal antibody-defined antigens and showed no evidence of plasma membrane polarization. Differentiated cells constantly expressed the three antigens, and their plasma membrane was divided into antigenically distinct domains. The changes in hepatocyte plasma membrane polarity demonstrated in situ during 3'-methyldimethylaminoazobenzene hepatocarcinogenesis may therefore be compared with situations known to occur in vitro, in cultured epithelial cells presenting varying degrees of polarization. Our observations suggest that these alterations are of relevance to the in vivo biology of cancer.

Location of adult and fetal aldolases A, B, and C by immunoperoxidase technique in LF fast-growing rat hepatomas.

The resurgence of aldolase isozymes in cancerous tissues is a well-known but poorly understood phenomenon. This resurgence poses the problem of whether or not adult and fetal aldolase isozymes are produced by the same cells. For clarification of this question, the immunoperoxidase technique was used to locate aldolases A, B, and C in one type of fast-growing hepatoma, the LF hepatoma and, by comparison, in normal adult liver. Under optical microscopy, aldolases A and C were located in the cytoplasm of almost all of the cancerous cells. An isozyme antigenically identical with aldolase B was also demonstrated to be present in almost all of the cells, but the reaction indicating the presence of this isozyme was weaker. In normal adult liver, only aldolases A and B were demonstrated to be present in almost all the hepatocytes. Under electron microscopy in LF hepatoma, the three isozymes were found to be present mainly in the cytoplasm. These facts suggest that the three types of aldolase are very probably present in the same cells at the same time, and they provide indirect arguments leading us to think that the resurgence of fetal aldolase isozymes in cancer is not the consequence of cellular selection but is due to a disturbance at the gene control level.

Ultrastructural aspects of the liver perisinusoidal space in diabetic patients with and without microangiopathy.

To determine whether abnormalities of the perisinusoidal space of Disse are present in the liver of diabetic patients with microangiopathy, an ultrastructural stereologic study of the space of Disse was performed in six insulin-treated diabetics with severe performed in six insulin-treated diabetics with severe proliferative retinopathy and six insulin-treated diabetics with normal fluorescein angiography, six patients with familial unconjugated hyperbilirubinemia were studied as controls. No patient had clinical and/or biochemical hepatic abnormalities and none suffered from any of the pathologic conditions known to be associated with collagenization of the perisinusoidal space. In control patients, the space of Disse of liver sinusoids contained occasional small deposits of collagen fibers. The relative volume of these fibers per unit of sinusoid represented 2.63 +/- 0.82%. In all diabetic patients with retinopathy, marked deposition of collagen fibers within the perisinusoidal space was constantly observed, a finding confirmed by ultrastructural stereologic analysis which showed that the relative volume of collagen fibers per unit of sinusoid represented 7.33 +/- 1.44% and differed significantly from control patient values (P less than 0.001). On the contrary, the relative volume of collagen fibers within the space of Disse in diabetic patients without retinopathy (3.95 +/- 2.96%) did not differ significantly from control patient values. These findings demonstrate that collagenization of the space of Disse is positively correlated with the presence of diabetic microangiopathy. Ultrastructural examination of the liver sinusoids might constitute a sensitive and useful approach for detecting the early changes of the microcirculation in diabetic patients.

Calcium-binding properties and ATPase activities of rat liver plasma membranes.

Plasma membranes from rat liver purified according to the procedure of Neville bind calcium ions by a concentration-dependent, saturable process with at least two classes of binding sites. The higher affinity sites bind 45 nmol calcium/mg membrane protein with a K(D) of 3 microM. Adrenalectomy increases the number of the higher affinity sites and the corresponding K(D). Plasma membranes exhibit a (Na(+)-K(+))-independent-Mg(2+)-ATPase activity which is not activated by calcium between 0.1 microM and 10 mM CaCl(2). Calcium can, with less efficiency, substitute for magnesium as a cofactor for the (Na(+)-K(+))-independent ATPase. Both Mg(2+)- and Ca(2+)-ATPase activities are identical with respect to pH dependence, nucleotide specificity and sensitivity to inhibitors. But when calcium is substituted for magnesium, there is no detectable membrane phosphorylation from [gamma-(32)P] ATP as it is found in the presence of magnesium. The existence of high affinity binding sites for calcium in liver plasma membranes is compatible with a regulatory role of this ion in membrane enzymic mechanisms or in hormone actions. Plasma membranes obtained by the procedure of Neville are devoid of any Ca(2+)-activated-Mg(2+)-ATPase activity indicating the absence of the classical energy-dependent calcium ion transport. These results would suggest that the overall calcium-extruding activity of the liver cell is mediated by a mechanism involving no direct ATP hydrolysis at the membrane level.

Fas/Fas-Ligand pathway is impaired in patients with type 2 diabetes. Influence of hypertension and insulin resistance.

OBJECTIVES: In type 2 diabetic patients with no cardiac history or symptoms, 1) to evaluate whether the soluble forms of Fas (sFas) and Fas-ligand (sFasL), involved in apoptosis, may be markers of silent coronary disease or related to hypertension or microangiopathic complications; 2) to examine the effect of short-term glycemic control on sFas and sFasL. METHODS: (1) sFas and sFasL were measured with the ELISA method in 44 asymptomatic diabetic patients, 33 with hypertension, and with a normal myocardial scintigraphy (n=14), with silent myocardial ischemia (SMI) and without (n=15) or with (n=15) significant coronary stenoses; and in 14 controls; (2) sFas and sFasL were measured in 15 poorly controlled diabetic patients before and after 7 days of CSII treatment. RESULTS: (1) sFas and sFasL differed in the four groups of patients (p=0.003 each). sFas was significantly higher in the patients with SMI without (p=0.035) and with coronary stenoses (p=0.002) than in the control group. sFasL was lower in the three groups of diabetic patients (p<0.05 each) than in control group. In the diabetic population, sFas correlated positively with hypertension (p=0.021), and sFasL negatively with hypertension (p=0.027) and HOMA index in the non-insulin treated patients (p=0.049); (2) sFas did not differ before or after CSII, and there was a marginal decrease in sFasL. CONCLUSION: Fas-mediated apoptosis is involved in type 2 diabetes and might be associated with hypertension and/or its vascular consequences. sFasL might be affected by insulin resistance. sFas and sFasL are not effective markers of SMI.

Characterization of rat hepatocyte plasma membrane domains by monoclonal antibodies.

The hepatocyte plasma membrane consists of three morphologically and functionally distinct domains, the sinusoidal, the lateral and the canalicular. To study the distribution of antigenic determinants among these domains, we prepared monoclonal antibodies by immunizing mice with a crude, plasma membrane-enriched liver fraction. Four monoclonal antibodies were obtained that recognized various parts of the rat hepatocyte plasma membrane when tested by indirect immunofluorescence and immunoperoxidase assay performed on formaldehyde-fixed liver tissue. Each antibody gave a different staining pattern when analyzed by light and electron microscopy. A59 exclusively labelled the part of the sinusoidal membrane facing the sinusoids. A39 mainly labelled the sinusoidal membrane. B1 mainly labelled the lateral membrane, while the labelling by B10 was almost completely limited to the canalicular membrane. Immunoblotting showed that the antibody B1 recognized an antigen of approximately 100 kilodaltons and that B10 recognized an antigen of approximately 125 to 130 kilodaltons. These antibodies allow us to distinguish the three domains of the hepatocyte plasma membrane.

Microtubule disruption interferes with the structural and functional integrity of the apical pole in primary cultures of rat hepatocytes.

The effect of microtubule disruption on the development and maintenance of cell polarity was studied in rat hepatocytes cultured as primary monolayers in the presence of colchicine or nocodazole. Addition of colchicine immediately after plating did not inhibit the generation of bile canaliculi (the apical pole) after 1 day of culture, as judged by electron microscopic examination, and did not allow penetration of Ruthenium Red through the tight junctions. However, the bile canaliculi developed in the presence of colchicine or nocodazole were not fully normal since they were not able to concentrate fluorescein diacetate in their lumina, and did not enrich with proteins of the apical plasma membrane domain, as control cells did. When the drugs were added after 1 or 2 days of culture, the new bile canaliculi appeared to be unaffected when examined by electron microscopy, but many of them did not concentrate fluorescein and were not enriched with apical membrane proteins within 4 to 24 h after drug addition. Whenever the drugs were added, the proteins that would normally concentrate on the membrane of the bile canaliculi accumulated intracellularly in endocytic vesicles after 2 to 4 h of drug treatment, and in vacuoles resembling lysosomes when the drugs were maintained for 24 h or more. These results show that microtubule disruption does not inhibit the structural reconstitution of bile canaliculi, but impairs their normal function and the transport of proteins of the apical plasma membrane domain.