Prevalence and trends of sunscreen use and sunburn among Norwegian women.

BACKGROUND: Sunscreen is recommended to prevent sunburn and skin cancer. OBJECTIVES: To investigate sunscreen use in relation to demographic and phenotypic characteristics among women in Norway, as well as solar UV exposure, sunburn experience in different decades of life, and temporal trends in sunscreen use. METHODS: We used data from the Norwegian Women and Cancer Study, a large population-based prospective cohort study. Log-binomial regression was used to estimate the association between sunscreen use and personal characteristics. Results are presented as prevalence ratios (PRs) and 99% confidence intervals (CIs). RESULTS: The study sample consisted of 148,869 women, with a mean age, when answering the questionnaire, of 53 years (range 41-75). Sixty-five per cent of the women used sunscreen during the Easter holiday, 73% in northern latitudes and 87% in bathing vacations in southern latitudes. Sunscreen with sun protection factor (SPF) ≥ 15 was used by 25,156 (18%) at Easter, 18,118 (13%) in northern latitudes and 22,678 (30%) in southern latitudes. The prevalence of sunscreen use increased from 1997 to 2007, and this increase was associated with age. In 1997, 39% of women reported at least one sunburn per year in the recent decade, compared with 46% in 2007 (Ptrend = 0·001). Women who experienced at least four sunburns per year during adolescence reported more sunscreen use in adulthood (PREaster 1·54, 99% CI 1·30-1·83; PRnorthern latitudes 1·49, 99% CI 1·20-1·84; PRsouthern latitudes 1·37, 99% CI 1·14-1·65). CONCLUSIONS: The prevalence of sunscreen use increased from 1997 to 2007. However, this increase has not been accompanied by a decrease in sunburn. Moreover, use of sunscreen with the recommended SPF was not common among Norwegian women.

The effect of types I and III interferons on adrenocortical cells and its possible implications for autoimmune Addison's disease.

Autoimmune Addison's disease (AAD) is caused by selective destruction of the hormone-producing cells of the adrenal cortex. As yet, little is known about the potential role played by environmental factors in this process. Type I and/or type III interferons (IFNs) are signature responses to virus infections, and have also been implicated in the pathogenesis of autoimmune endocrine disorders such as type 1 diabetes and autoimmune thyroiditis. Transient development of AAD and exacerbation of established or subclinical disease, as well as the induction of autoantibodies associated with AAD, have been reported following therapeutic administration of type I IFNs. We therefore hypothesize that exposure to such IFNs could render the adrenal cortex susceptible to autoimmune attack in genetically predisposed individuals. In this study, we investigated possible immunopathological effects of type I and type III IFNs on adrenocortical cells in relation to AAD. Both types I and III IFNs exerted significant cytotoxicity on NCI-H295R adrenocortical carcinoma cells and potentiated IFN-γ- and polyinosine-polycytidylic acid [poly (I : C)]-induced chemokine secretion. Furthermore, we observed increased expression of human leucocyte antigen (HLA) class I molecules and up-regulation of 21-hydroxylase, the primary antigenic target in AAD. We propose that these combined effects could serve to initiate or aggravate an ongoing autoimmune response against the adrenal cortex in AAD.

lacZ-neoR transfected glioma cells in syngeneic rats: growth pattern and characterization of the host immune response against cells transplanted inside and outside the CNS.

The rat glioma cell lines BT4C and BT4Cn were stably transfected with the bacterial lacZ-neomycin resistance (neoR) gene construct. Both transfected (BT4ClacZ and BT4CnlacZ) and untransfected cell lines were injected intracerebrally and subcutaneously into rats. Survival time, morphology, growth rate and immunological properties of the tumors were studied. Survival time was significantly prolonged after intracerebral implantation of the transfected cell lines. No similar response was found in nude rats, indicating an immunological response towards the lacZ-neoR-transfected cells in immunocompetent animals. Morphological observations showed that the lacZ-neoR-transfected gliomas were smaller and had a distinct boundary with the normal brain tissue, whereas the parental cell lines revealed a more diffuse growth pattern. Immunostaining showed a higher proportion of immunocompetent cells infiltrating the lacZ-neoR-transfected tumors. After s.c. injection, the lacZ-neoR-transfected BT4C cell line had a prolonged lag phase before assuming a growth rate similar to that of the parental cells. The BT4CnvlacZ tumors initially grew fastest, but then disappeared within 3 weeks. A similar response was observed with mock-transfected tumor cells. A (3)HTdR-incorporation assay on spleen cells from rats transplanted s.c. with BT4CnvlacZ cells showed a 10-fold increase in cell activation as compared with rats with BT4Cn tumors. A humoral response towards the transfected cells was verified by Western-blot analyses.

Role of extracellular matrix in tumor invasion: migration of glioma cells along fibronectin-positive mesenchymal cell processes.

OBJECTIVE: The major morbidity of glioma lies in its infiltrative growth. One of the major patterns of this invasive growth is the formation of Scherer's secondary structures associated with the blood vessels and the leptomeninges. To better understand the role of extracellular matrix (ECM) in glioma invasion, we investigated in vitro the interaction between glioma cells and the meningeal mesenchymal tissue from the brain. As an aid to this study, ECM in glioma cell line spheroids was compared with that in primary fetal brain aggregates. METHODS: To study the expression of ECM, four glioma cell lines (U-87 MG, U-251 MG, AN1/lac-z, and HF-66) and primary cells from fetal rat brain were grown as spheroids and monolayers. To sudy the role of ECM in glioma invasion, spheroids from the glioma cell lines were grown over established cultures of fetal meningeal and mesenchymal tissue. Expression of fibronectin, laminin, tenascin, collagen VI, and chondroitin sulfate proteoglycan was studied by immunofluorescence. RESULTS: Expression of ECM by the spheroids was variable. U-87 MG expressed most of the ECM components robustly, whereas AN1/lac-z expressed them all weakly. Fetal rat brain aggregates produced minimal ECM. In cocultures of glioma spheroids and fetal meningeal mesenchymal tissue, individual cells from the glioma spheroids that expressed least fibronectin (AN1/lac-z and U-251 MG) migrated along the fibronectin-positive mesenchymal cells in the culture dish. Cells from the other two lines (U-87 MG and HF-66) that expressed fibronectin strongly did not demonstrate such behavior. None of the other ECM components showed a similar association; mesenchymal cells did not express laminin as strongly as fibronectin, and glioma cells were not observed to align with the laminin-positive structures. CONCLUSION: This study suggests that fibronectin may play a key role in intracerebral invasion of glioma cells.

Stimulation of extracellular matrix components in the normal brain by invading glioma cells.

Malignant gliomas are characterized by an extensive invasion of tumor cells into the normal brain parenchyma. A substantial amount of data indicates that cell movement in general is regulated by specific interactions between extracellular matrix components and specific cell-surface receptors. In the present work, multicellular spheroids from 4 human glioma cell lines (U-373Mg, A-172Mg, U-251Mg and HF-66) were confronted with normal rat brain cell aggregates in vitro, which resulted in a progressive invasion of tumor cells into the brain aggregates. The co-cultures were then sectioned and immuno-stained for specific extracellular matrix components (laminin, fibronectin and collagen type IV) and for specific cell-surface receptors which bind to these components (integrins beta1, beta4, alpha3, alpha6). In addition, flow-cytometric measurements and Northern blot analyses showed expression of several different integrins within the cell lines. The alpha3 subunit was expressed strongly in all cell lines. Whereas the beta1 subunit was expressed weakly in exponentially growing monolayer cultures, it showed a pronounced expression in multicellular spheroids, indicating that the integrin expression may vary depending on the micro-environment within a tumor. Furthermore, normal brain tissue was able to produce laminin when confronted with the glioma cells, which also was observed for fibronectin and collagen type IV. The relevance of our observations to the in vivo situation was investigated further by immuno-staining 5 human glioma biopsy samples for laminin. In some areas of the tumors, specific deposits of laminin were observed. In conclusion, we have shown that normal brain tissue has the ability to produce extracellular matrix components, such as laminin, collagen type IV and fibronectin, when confronted with invading glioma cells. Our results show that the glioma cells express specific integrins which can interact with these extracellular matrix components. Such interactions may facilitate tumor cell migration and invasion.

Vascular endothelial growth factor expression and vascular density as prognostic markers of survival in patients with low-grade astrocytoma.

UNLABELLED: It has long been recognized that some patients with low-grade astrocytoma may survive for many years, whereas in others the disease follows a more malignant course resulting in a short survival time, usually due to malignant transformation into higher-grade tumors. OBJECT: The aim of this study was to investigate angiogenesis in the initial biopsy specimen of tumor tissue as a biological marker to identify patients with low-grade astrocytoma who are at high risk of malignant tumor transformation or death. METHODS: Tumor tissue was studied in 74 consecutively treated adult patients in whom a diagnosis of diffuse supratentorial hemispheric histologically proven fibrillary low-grade astrocytoma was made and who underwent surgery between January 1972 and January 1994. Studies were conducted using monoclonal antibodies to the antigens of the proliferation-associated Ki-67 (MIB-1), factor VIII, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF). The overall 5-year survival rate for the entire patient population was 65%, with a median survival time of 7.5 years. The total mean follow-up period was 6.1 years. All tumors showed a low proliferative potential at the time of the initial operation, as demonstrated by an MIB-1 labeling index of less than 1.5%. Patients with more than seven microvessels in tumor tissue (29 cases) had a shorter survival time (mean 3.8 years) than those with seven or fewer microvessels (mean survival 11.2 years). This difference in survival times was significant by univariate (p = 0.001) and stepwise multivariate analyses (p < 0.001). Tumors with a larger number of microvessels also had a greater chance of undergoing malignant transformation (p = 0.001). Similarly, significant staining for VEGF was correlated with shorter survival times when using univariate (p = 0.003) and multivariate (p = 0.008) analyses and with a greater chance of malignant transformation (p = 0.002). Patients with tumors staining positive for VEGF (39 individuals) had a median survival time of 5.3 years, and those with tumors negative for VEGF (35 patients) had a median survival time of 11.2 years. No association was observed between bFGF, EGF, and survival or malignant transformation. The stepwise multivariate analysis included histological and clinical variables simultaneously. CONCLUSIONS: The authors have shown that microvessel density and VEGF levels are independent prognostic markers of survival in fibrillary low-grade astrocytoma. This finding leads them to propose that fibrillary diffuse low-grade astrocytoma is not a single pathological entity but is composed of a spectrum of tumors with differing propensities to undergo malignant transformation that is at least partly based on their inherent angiogenic potential.

Hippocampal connexin 43 expression in human complex partial seizure disorder.

An increase in the cellular production of gap junction proteins and increased numbers of gap junctions in the neuronoglial syncytium of an epileptic focus have been proposed as a possible mechanism underlying synchronization of discharge. To study this issue, both Northern and Western blot analyses of the gap junction protein connexin 43 mRNA and protein abundance were performed on hippocampal tissue resected from patients presenting with a complex partial seizure disorder arising from the medial temporal area and the hippocampus in particular. Samples from 15 patients with medically intractable seizures were compared to those from 5 nonepileptic patients requiring temporal lobectomy in life-threatening situations. Six of the 15 epileptic patients underwent noninvasive electrographic recording, whereas the remaining 9 patients required intracerebral electrodes for extraoperative recording and therefore showed a more discrete focality than the noninvasive recordings. A decline in the mean levels of connexin 43 mRNA expressed predominantly in astrocytes was noted in the epileptic patient groups, particularly for those cases requiring intracranial electrode placement where ictal onset was more clearly established to be intrahippocampal. Quantitation of connexin 43 protein in both epileptogenic and nonepileptogenic hippocampal tissues showed no significant differences in expression. Although mean values for mRNA showed a decline, clinical outcomes postoperatively showed no correlation with either mRNA or protein expression individually in our epileptic population. The findings indicate that there is effectively no upregulation of mRNA and no increased production of connexin 43 protein in response to the development of epileptogenicity. Rather it appears the influence of gap junctions as a substrate of epileptogenicity in any mechanism(s) underlying synchrony or electrical propagation may be a function of the dynamic state (open versus closed) of the membrane-bound gap junction.

Tumor cell invasion and angiogenesis in the central nervous system.

Tumor cell growth and invasion within the CNS imply complex interactions among malignant cells, neural cells, and endothelial cells. Various in vitro assays have been developed to study tumor cell invasion that includes the use of cocultures between tumor spheroids and organotypic cultures of normal brain tissue. Furthermore, various animal models have been developed to study biologic characteristics of brain tumors. At present, there is evidence that several growth factors are involved in both endothelial and tumor cell proliferation, whereas the interrelationship between glioma growth and invasion is less well established. It is also emerging that the process of invasion is characterized by dynamic interactions between the extracellular matrix, proteases, and specific cell surface receptors. The dissemination of tumor cells within the CNS also involves a passive component where single tumor cells may follow specific pathways mediated by the constant flow of cerebrospinal fluid.

Role of high molecular weight extracellular matrix proteins in glioma cell migration.

Malignant human gliomas are characterized by an uncontrolled cell proliferation and infiltrative growth within the brain. Complete surgical removal is difficult due to disseminated tumour cells, and the fundamental mechanisms responsible for this spread are poorly understood. An extensive tumour cell movement along blood vessels is frequently observed and this may be due to specific interactions between tumour cell surface receptors and specific extracellular matrix (ECM) components present in conjunction with vascular elements. In order to investigate the influence of ECM on glioma cell migration, three different human glioma cell lines (U-373 MG, A-172 MG and HF-66) were exposed to known ECM components of the basement membrane (laminin, fibronectin and collagen type IV). Cell migration from multicellular spheroids was studied, using a custom-made medium which was prepared by removing the high molecular weight protein fraction (>100 kDa) from newborn calf serum by ultrafiltration. To this medium, the specific ECM components were added. For two of the cell lines (A-172 MG and U-373 MG), laminin was the most potent stimulator of glioma cell migration; the effect of laminin exceeded that evoked by ordinary serum-supplemented medium. For the HF-66 cell line, fibronectin was the most potent stimulator of migration. Western blot analysis showed that the A-172 MG and HF-66 cell lines expressed low amounts of laminin compared with U-373 MG, which showed extensive intrinsic synthesis of this ligand. U-373 MG was the only cell line that migrated in pure filtered medium. The cells stimulated by fibronectin expressed a different morphology from those stimulated by laminin suggesting that specific ECM-receptor binding may activate different cytoskeletal components within the cells. Furthermore, it was shown that there was no difference in the amount of protein synthesis between cells grown in filtered medium and in filtered medium supplemented with different ECM components. This suggests that ECM-induced cell migration is not dependent on a high level of protein synthesis. It is also shown that alpha3 integrin, which is a receptor-subunit for laminin, fibronectin and collagen type IV, was highly expressed in all cell lines. This study indicates that glioma cells need serum proteins with a molecular weight >100 kDa to migrate in vitro, and that laminin and fibronectin play an important role in this process.

The first immunoglobulin-like neural cell adhesion molecule (NCAM) domain is involved in double-reciprocal interaction with the second immunoglobulin-like NCAM domain and in heparin binding.

To study the function of the first immunoglobulin (Ig)-like domain of the neural cell adhesion molecule (NCAM), it was produced as a recombinant fusion protein in a bacterial expression system and as a recombinant protein in a eukaryotic expression system of the yeast Pichia pastoris. For comparison, other NCAM domains were also produced as fusion proteins. By means of surface plasmon resonance analysis, it was shown that the first Ig-like NCAM domain binds the second Ig-like NCAM domain with a dissociation constant 5.5 +/- 1.6 x 10(-5) M. Furthermore, it was found that the first Ig-like domain binds heparin. It was also demonstrated that the second Ig-like NCAM domain binds heparin and that both domains bind collagen type I via heparin but not collagen type I directly.

Effect of NCAM-transfection on growth and invasion of a human cancer cell line.

A cDNA encoding the human transmembrane 140 kDa isoform of the neural cell adhesion molecule (NCAM) was transfected into the highly invasive MDA-MB-231 human breast cancer cell line. Transfectants with a homogeneous expression of NCAM showed a restricted capacity for penetration of an artificial basement membrane. However, when injected into nude mice, both control and NCAM-expressing cell lines produced equally invasive tumors. Tumors generated from NCAM-transfected cells were heterogeneous, containing NCAM-positive as well as NCAM-negative areas, indicating the existence of host factors capable of modulating NCAM expression in vivo. In nude mice, NCAM-transfected cells developed tumors with longer latency periods and slower growth rates than tumors induced by NCAM-negative control cells, implying that NCAM may be involved not only in adhesive and motile behavior of tumor cells but also in their growth regulation. There was no indication of differences in cell proliferative characteristics between the different NCAM-transfected and the control transfected cells as determined by flow cytometric DNA analysis, suggesting an increased cell loss as the reason for decreased in vivo growth rate of the NCAM-transfected cells. The fact that NCAM expression influences growth regulation attributes a pivotal role to this cell adhesion molecule during ontogenesis and tumor development.

Neuronatin mRNA in PC12 cells: downregulation by nerve growth factor.

Neuronatin was recently cloned from neonatal rat brain (Biochem, Biophys. Res. Commun., 201 (1994) 1227-1234). In subsequent studies, we noted neuronatin mRNA was brain-specific and that there were two alternatively spliced forms, alpha and beta (Brain Res., 690 (1995) 92-98). Furthermore, on sequencing the human neuronatin gene, it was determined that the alpha-form was encoded by three exons, and the beta-form was encoded by the first and third exons only (Genomics, 33 (1996) 292-297). The middle exon was spliced out in the beta-form. The human neuronatin gene is located in single copy of chromosome 20q 11.2-12 (Brain Res., 723 (1996) 8-22). These studies called for an understanding of the function of this gene. Therefore, we studied the expression of neuronatin in PC12 cells, an established model of neuronal growth and differentiation. Neuronatin mRNA expression was found to be abundant in undifferentiated PC12 cells. Treatment with nerve growth factor (NGF), resulting in neuronal differentiation, was associated with a downregulation of neuronatin mRNA expression. Removal of NGF was associated with a return of neuronatin mRNA levels towards baseline. These effects appear to be specific for NGF as they were not seen with transforming growth factor, epidermal growth factor, 12-O-tetradecanoylphorbol-13-acetate or dexamethasone. Although, basic fibroblast growth factor also reduced neuronatin mRNA levels, the effect was less pronounced than with NGF. The NGF-induced decreased in neuronatin mRNA occurred even in the presence of protein and RNA syntheses inhibitors. Of the two spliced forms, only the alpha-form was expressed in PC12 cells. In conclusion, we report the presence of neuronatin mRNA in PC12 cells, and that NGF downregulates its expressions. These findings provide a basis for investigating the role of neuronatin in neuronal growth and differentiation.

Functional characterization of NCAM fibronectin type III domains: demonstration of modulatory effects of the proline-rich sequence encoded by alternatively spliced exons a and AAG.

In order to characterize the functions of the two fibronectin type III (F3) homology domains of the neural cell adhesion molecule (NCAM), we investigated the effects of two variants, expressed as fusion proteins, of the NCAM-F3 domains on attachment and spreading of NCAM-expressing fibroblasts, cerebellar cell aggregation and fiber formation, and on growth cones. The two fusion proteins were different with regard to a short proline-rich insert of six amino acids between the two F3 domains. Immobilized NCAM-F3 fusion proteins were found to mediate attachment of both transmembrane and lipid-anchored NCAM expressing fibroblasts. Also NCAM-negative cells adhered to the NCAM-F3 substratum, although to a lesser extent, implying the possibility of a heterophilic ligand to NCAM-F3 domains on the surface of fibroblasts. Cellular spreading on NCAM-F3 substratum was selectively increased in fibroblasts expressing transmembrane NCAM, and only the NCAM-F3 fusion protein lacking the proline-rich insert was able to elicit this effect. Primary cultures of mouse cerebellum were strongly inhibited with regard to formation of cellular aggregates and fibers, when incubated in the presence of either of the two NCAM-F3 fusion proteins, the fusion protein with the proline-rich insert being the more effective one. Finally, the morphology of growth cones from rat cerebellar granule cells changed significantly when grown on NCAM-F3 substrata as revealed by computer-assisted image analysis. Thus, our data indicate that the NCAM-F3 domain are involved in cell-cell adhesion, and that insertion of the proline-rich sequence has a modulatory effect on NCAM-F3 domain functions.

Stimulation of glioma-cell migration by laminin and inhibition by anti-alpha3 and anti-beta1 integrin antibodies.

An induction of laminin in the confrontation zone between tumor cells and normal brain tissue has been observed in our model systems in vivo and in vitro. In order to study the effects of ECM components on glioma-cell migration and invasion, we have used 2 lacZ-transfected glioma cell lines, AN1/lacZ and U-251 /lacZ. Cell migration from multicellular spheroids was studied using different types of media: DMEM with 10% serum, Ultra Culture medium, and filtrated DMEM with serum in which the protein fraction > 100 kDa had been removed by ultrafiltration. Laminin, fibronectin and collagen type-IV were individually added to the different media, and cell migration from the spheroids was studied. The results show that cell migration in both cell lines, was stimulated by laminin and fibronectin. Collagen type-IV stimulated only cell migration of U-251/lacZ cells. Scanning electron microscopy revealed an extensive change in cell shape as a result of laminin stimulation. Flowcytometric studies showed that both AN1/lacZ and U-251/lacZ strongly express the alpha3 beta1 integrin receptor, which can bind to several ECM components (laminin, fibronectin, collagen). Immunofluorescence microscopy demonstrated that the same integrin sub-units were expressed in multicellular spheroids. When monoclonal antibodies to alpha3 and beta1 were added to the laminin-stimulated cultures, cell migration was significantly reduced. This indicates that the alpha3 beta1 integrin receptor plays an important role during glioma-cell migration.

The lac-z reporter gene: a tool for in vitro studies of malignant glioma cell invasion.

A reporter gene (lac-z) was introduced into rat (BT4C) and human (D-54 MG) proliferating glioma cell lines by means of liposomal transfection. Lac-z-transfected glioma cells were first cultured as multicellular spheroids and then confronted with fetal brain aggregates. After various intervals the lac-z reporter gene product, bacterial beta-galactosidase, was histochemically detected in the cocultures. beta-Galactosidase was only detected in the glioma cells which showed an intense blue staining, which made them easily distinguishable from fetal tissue. Both glioma cell lines showed a clear pattern of migration and increasing invasion with time as the tumor cells infiltrated and destroyed the brain aggregates. Spheroid growth curves showed no significant differences between transfected and nontransfected cell lines. Likewise, flow cytometry measurements revealed no significant changes in ploidy between transfected and nontransfected rat glioma cells. In comparison, a shift in ploidy was observed in the human glioma cells after lac-z transfection. Stable integration of the lac-z gene into tumor cells was verified by Southern blot analysis. The results indicate that transfection of the lac-z reporter gene into glioma cells lines does not affect their growth or invasion potential in vitro. The lac-z reporter gene can thus be exploited to facilitate visualization of single migrating tumor cells and quantification of tumor invasion in in vitro coculture systems.

Teratogenic potency of valproate analogues evaluated by quantitative estimation of cellular morphology in vitro.

To develop a simple prescreening system for teratogenicity testing, a novel in vitro assay was established using computer assisted microscopy allowing automatic delineation of contours of stained cells and thereby quantitative determination of cellular morphology. The effects of valproic acid (VPA) and analogues with high as well as low teratogenic activities-(as previously determined in vivo)-were used as probes for study of the discrimination power of the in vitro model. VPA, a teratogenic analogue (+/-)-4-en-VPA, and a non-teratogenic analogue (E)-2-en-VPA, as well as the purified (S)- and (R)-enantiomers of 4-yn-VPA (teratogenic and non-teratogenic, respectively), were tested for their effects on cellular morphology of cloned mouse fibroblastoid L-cell lines, neuroblastoma N2a cells, and rat glioma BT4Cn cells, and were found to induce varying increases in cellular area: Furthermore, it was demonstrated that under the chosen conditions the increase in area correlated statistically significantly with the teratogenic potency of the employed compounds. Setting the cellular area of mouse L-cells to 100% under control conditions, the most pronounced effect was observed for (S)-4-yn-VPA (211%, P = < 0.001) followed by VPA (186%, P < 0.001), 4-en-VPA (169%, P < 0.001) and non-teratogenic 2-en-VPA (137%, P < 0.005) and (R)-4-yn-VPA (105%). This effect was independent of the choice of substrata, since it was observed on L-cells grown on plastic, fibronectin, laminin and Matrigel. However, when VPA-treated cells were exposed to an arginyl-glycyl-aspartate (RGD)-containing peptide to test whether VPA treatment was able to modulate RGD-dependent integrin interactions with components of the extracellular matrix, hardly any effect could be observed, whereas control cells readily detached from the substratum, indicating a changed substrate adhesion of the VPA-treated cells. The data thus indicate that measurement of cellular area may serve as a simple in vitro test in the early pre-screening evaluation of teratogenicity of novel therapeutic agents.

Migratory patterns of lac-z transfected human glioma cells in the rat brain.

Malignant brain tumors are characterized by extensive tumor-cell infiltration into the normal brain tissue. The present work describes the migratory behavior of human glioma cells transplanted into the adult rat brain with the aim of exploiting the extent of active cell migration and passive cell displacement within the central nervous system. To detect every transplanted tumor cell, a stably bacterial beta-galactosidase (lac-z) transfected human glioma cell line was used. To distinguish between an active cell migration process and passive cell displacement, rat brains were also implanted with inert fluorescent polystyrene microspheres and the distribution of tumor cells and microspheres was studied 1 hr and 3 days after implantation. One hour after implantation the tumor cells were strictly localized at the implantation site. However, 3 days after implantation, both tumor cells and microspheres showed an extensive distribution within the brain. Confirming earlier neuropathological and experimental studies, it is shown that the lac-z-transfected glioma cells had the capacity to move within the Virchow-Robin and subarachnoid spaces. However, since fluorescent microspheres were also found in these areas, this spread of tumor cells may be primarily mediated by the extensive cerebrospinal fluid flow that exists within the brain. Three days after implantation, the glioma cells also showed an active migration over the corpus callosum. In comparison, the fluorescent microspheres showed only limited spread along the callosal body. It is concluded that the bacterial lac-z gene can be stably transfected into human glioma cells and, since every tumor cell can be visualized within the brain, this model provides a tool for studying the mechanisms behind tumor-cell invasion of the brain.

Localization of NCAM on NCAM-B-expressing cells with inhibited migration in collagen.

The extracellular matrix is a key element in neuronal development and tumour invasion, providing a substratum which sustains the adhesion and migration of cells. In order to study interactions between the neural cell adhesion molecule (NCAM) and collagen, we transfected mouse L cells with cDNA encoding the human transmembrane NCAM isoform of 140 kDa (NCAM-B). An L-cell/collagen type I system was used to study the influence of NCAM expression on in vitro invasion. We here report that migration of NCAM-expressing cells in collagen was inhibited compared to that of NCAM-negative cells transfected with the empty vector. Immunofluorescence confocal laser scanning microscopy (CLSM) and immunogold electron microscopy using anti-human NCAM antibodies demonstrated a heterogeneous distribution of NCAM on the plasma membrane of transfected L cells grown on collagen. NCAM was preferentially located at the surface of broad cytoplasmic protrusions and slender extensions, some of which were facing the collagen. This was in contrast to the homogeneous surface distribution of NCAM on cells grown on plastic. These data suggest that NCAM and collagen type I interact, and that this might lead to the migration inhibition of NCAM-expressing cells.

Characterization of N-cadherin messenger RNA and polypeptide expression in rat.

The cell adhesion molecule N-cadherin is a member of the cadherin gene superfamily. The protein is involved in morphogenetic processes, including neurite extension. In this study, N-cadherin mRNA and polypeptide expression were investigated in rat brain, liver, muscle, heart, kidney and lung during postnatal development and aging. Six synthetic oligonucleotide probes covering different parts of mouse N-cadherin cDNA all hybridized to 5.2, 4.3-4.4 and 3.5 kb mRNAs in rat tissues. The mRNA pattern differed between tissues and, furthermore, the amount of N-cadherin mRNA and polypeptides in brain, liver and heart was higher than in muscle, kidney and lung. N-cadherin expression decreased slightly during early postnatal development in all tissues, whereas no changes in N-cadherin expression were observed during aging. Antibodies against a fusion protein containing the transmembrane and cytoplasmic sequence of chick N-cadherin were produced. These antibodies, termed anti-N-cad-cyt, were compared to the R-156 antibodies which recognize the 24 C-terminal amino acids of N-cadherin and which have been shown to react with a broad spectrum of cadherins. Using these two antibodies, it was shown that the 130 kDa N-cadherin polypeptide was subject to calcium-dependent cleavage of the cytoplasmic domain. Conversely, in the absence of calcium the polypeptide was cleaved extracellularly, producing two C-terminal fragments of 85 and 95 kDa. A 122 kDa polypeptide was recognized by both antibodies and may be either an alternatively spliced form of N-cadherin or a closely related cadherin.

Expression and localization of N- and E-cadherin in the human testis and epididymis.

Cellular interactions in the testis and epididymis are an important prerequisite for spermatogenesis and sperm maturation, and involve a well-developed complex of intercellular junctions. Cadherins are cell surface proteins which mediate intercellular Ca(2+)-dependent adhesion and are believed to be fundamentally important for maintaining multicellular structures. In the present study we report the expression of a 135 kDa N-cadherin polypeptide in the human seminiferous epithelium by immunoblotting. The presence of N-cadherin was demonstrated by immunohistochemistry on the surface of spermatogonia and primary spermatocytes, and possibly also around some early spermatids, whereas late spermatids were always negative. Endothelial cells also stained for N-cadherin, whereas peritubular cells and Leydig cells did not. No expression of E-cadherin could be demonstrated in the human testis. In the human epididymis E-cadherin, but not N-cadherin, was expressed and localized to the surface of the principal epithelial cells as shown by immunohistochemistry. These observations indicate that cadherins play an important role in the organization of the seminiferous and epididymal epithelium.