Introduction of real patients into problem-based learning in preclinical first-year anatomy curriculum.

BACKGROUND: Early patient contacts are considered important in medical education. AIMS: We studied the influence of a real patient trigger on study motivation and learning in problem-based study groups of first-year medical and dentistry students. METHODS: 156 eligible students were allocated into 17 groups. Six randomly selected groups received both the real patient and paper trigger, and 11 groups received only the paper trigger. The immediate and later effects of the trigger were assessed with qualitative and quantitative questionnaires and exam scores. The tutors answered questionnaires concerning learning outcomes. RESULTS: The students reported that the real patient trigger significantly improved their study motivation, understanding of the learning objectives and confidence in future patient encounters. The real patient trigger was considered significantly more interesting than the paper case. No statistically significant difference was observed in the exam scores. The tutors observed that groups with poor previous performance gained better results in study sessions. CONCLUSIONS: Real patient triggers motivate students to learn basic medical sciences. Ways to present real patients to students should be considered in medical curricula from early on.

Podosome-like structures of non-invasive carcinoma cells are replaced in epithelial-mesenchymal transition by actin comet-embedded invadopodia.

Podosomes and invadopodia are actin-based structures at the ventral cell membrane, which have a role in cell adhesion, migration and invasion. Little is known about the differences and dynamics underlying these structures. We studied podosome-like structures of oral squamous carcinoma cells and invadopodia of their invasive variant that has undergone a spontaneous epithelial-mesenchymal transition (EMT). In 3D imaging, podosomes were relatively large structures that enlarged in time, whereas invadopodia of invasive cells remained small, but were more numerous, degraded more extracellular matrix (ECM) and were morphologically strikingly different from podosomes. In live-cell imaging, highly dynamic, invadopodia-embedded actin tails were frequently released and rocketed through the cytoplasm. Resembling invadopodia, we found new club-ended cell extensions in EMT-experienced cells, which contained actin, cortactin, vinculin and MT1-matrix metalloproteinase. These dynamic cell extensions degraded ECM and, in field emission scanning electron microscopy, protruded from the dorsal cell membrane. Plectin, alphaII-spectrin, talin and focal adhesion kinase immunoreactivities were detected in podosome rings, whereas they were absent from invadopodia. Tensin potentially replaced talin in invadopodia. Integrin alpha(3)beta(1) surrounded both podosomes and invadopodia, whereas integrin alpha(v)beta(5) localized only to invadopodia heads. Pacsin 2, in conjunction with filamin A, was detected early in podosomes, whereas pacsin 2 was not found in invadopodia and filamin A showed delayed accumulation. Fluorescence recovery after photobleaching indicated faster reorganization of actin, cortactin and filamin A in podosomes compared to invadopodia. In conclusion, EMT affects the invasion machinery of oral squamous carcinoma cells. Non-invasive squamous carcinoma cells constitutively organize podosomes, whereas invasive cells form invadopodia. The club-ended cell extensions, or externalized invadopodia, are involved in ECM degradation and maintenance of contact to adhesion substrate and surrounding cells during invasion.

Laminin isoforms in human embryonic stem cells: synthesis, receptor usage and growth support.

To reveal the functional intrinsic niche of human embryonic stem cells (hESC) we examined the production of basement membrane (BM) proteins and the presence of their receptors in feeder-free cell culture conditions. In addition, we investigated binding of hESCs to purified human BM proteins and identified the receptors mediating these contacts. Also, we tested whether purified human laminin (Lm) isoforms have a role in hESC self-renewal and growth in short-term cultures. The results show that hESCs synthesize Lm alpha(1) and Lm alpha(5) chains together with Lm beta(1) and gamma(1) chains suggesting the production of Lms-111 and -511 into the culture medium and deposits on cells. hESCs contain functionally important integrin (Int) subunits, Int beta(1), alpha(3), alpha(6), alpha(5), beta(5) and alpha(V), as well as the Lm alpha(5) receptor, Lutheran (Lu) glycoprotein and its truncated form, basal cell adhesion molecule (B-CAM). In cell adhesion experiments, Int beta(1) was crucial for adhesion to most of the purified human BM proteins. Lu/B-CAM mediated adhesion to Lm-511 together with Int alpha(3)beta(1), and was essential for the adhesion of hESCs to embryonic feeder cells. Adhesion to Lm-411 was mediated by Int alpha(6)beta(1). Lm-511 supported hESC growth in defined medium equally well as Matrigel. These results provide consequential information of the biological role of BM in hESCs, warranting further investigation of BM biology of human pluripotent stem cells.

Epithelial-mesenchymal transition downregulates laminin alpha5 chain and upregulates laminin alpha4 chain in oral squamous carcinoma cells.

Basement membranes maintain the epithelial phenotype and prevent invasion and metastasis. We hypothesized that expression of basement membrane laminins might be regulated by epithelial-mesenchymal transition (EMT), hallmark of cancer progression. As EMT is mediated by transcription factor Snail, we used oral squamous carcinoma cells obtained from a primary tumor (43A), from its EMT-experienced recurrence (43B) and Snail-transfected 43A cells (43A-SNA) displaying full EMT, as a model to study laminins and their receptors. Northern blotting, immunofluorescence, and immunoprecipitation showed a gradual loss of laminin-511 and its receptor Lutheran from 43A to 43B and 43A-SNA cells. In contrast, neoexpression of laminin alpha4 mRNA was found congruent with synthesis of laminin-411. Chromatin immunoprecipitation disclosed direct binding of Snail to regions upstream of laminin alpha5 and alpha4 genes. Immunofluorescence and immunoprecipitation showed a switch from hemidesmosomal integrin alpha(6)beta(4) to alpha(6)beta(1) and neoexpression of alpha(1)beta(1) in 43A-SNA cells, and upregulation of integrin-linked kinase in both 43B and 43A-SNA cells. The cells adhered potently to laminin-511 and fibronectin, whereas adhesion to laminin-411 was minimal. In contrast, laminin-411 inhibited cell adhesion to other extracellular matrix proteins. In conclusion, EMT induces a switch from laminin-511 to laminin-411 expression, which may be directly controlled by Snail. Concomitant changes take place in laminin- and collagen-binding receptors. Laminin-411 reduces adhesion to laminin-511 and fibronectin, suggesting that tumor cells could utilize laminin-411 in their invasive behavior.

Lutheran blood group antigen as a receptor for alpha5 laminins in gingival epithelia.

BACKGROUND: Lutheran blood group glycoprotein (Lu) is a transmembrane receptor of the immunoglobulin superfamily. Lu serves as a receptor for alpha5 laminins (Lm). The Lm alpha5 chain is a constituent of Lms-511 and -521. Lm-511 is found in most human basement membranes (BMs) and also is detected in BM of gingival epithelia. Recent studies indicated that Lu mediates cell adhesion to Lms-511/521 independently or in concert with integrins. This study focused on the expression of Lu in gingival epithelia and on cultured immortalized gingival keratinocytes. The role of Lu and alpha(3) and beta(1) integrin subunits in the adhesion of oral epithelial cells to Lms-511/521 was also studied. METHODS: Immunofluorescence microscopy was used to study the expression of Lu in gingival tissues and in cultured gingival keratinocytes. Immunoprecipitation of radioactively metabolically labeled cells was used to detect Lu. Cell adhesion to Lm-511/521 preparation and to pure Lm-511 was studied in quantitative cell adhesion experiments. Morphological adhesion assays were carried out for visualization of the morphology and adhesion structure formation of the adhering cells. RESULTS: Immunofluorescence studies on gingiva showed complete coalignment of Lu on basal epithelial cells with the BM Lm alpha5 chain. A surface-confined, punctate immunoreaction for Lu was detected throughout cell surfaces on cultured gingival cells. Immunoprecipitation showed a broad polypeptide with molecular weight 85,000. In quantitative cell adhesion assays, the adhesion of cells to both Lm alpha5 preparations was diminished with monoclonal antibodies (MAbs) against integrin alpha(3) and even more effectively with MAbs against the beta(1) subunit. When the adhesion sites were blocked with soluble recombinant Lu (sol-Lu), the adhesion of gingival epithelial cells to Lms-511/521 was reduced significantly, and sol-Lu increased the inhibition with integrin alpha(3) antibody. Lm-511 did not induce lamellipodia or focal contacts in cultured gingival keratinocytes. CONCLUSIONS: Lu was in coalignment with Lm alpha5 chain in gingival epithelia. Lu also seemed to have a role in gingival epithelial cell adhesion together with integrin alpha(3)beta(1).

Snail-dependent and -independent epithelial-mesenchymal transition in oral squamous carcinoma cells.

Disappearance of E-cadherin is a milestone for epithelial-mesenchymal transition (EMT), found both in carcinomas and in some fibrotic diseases. We have studied the mechanisms of EMT in oral squamous cell carcinoma (SCC) cells isolated from primary tumor (43A) and its recurrent tumor (43B). Whereas the cells from primary carcinoma displayed a typical phenotype of squamous epithelial cells including E-cadherin and laminin-332 (laminin-5), cells from recurrent tumor expressed characteristics of dedifferentiated, EMT-experienced tumors. 43B cells expressed E-cadherin repressors ZEB-1/deltaEF1 and especially ZEB-2/SIP1, which therefore appear as candidates for endogenous EMT in these cells. Differences between endogenous and exogenous EMT were assessed by transfecting 43A cells with SNAIL cDNA. SNAIL-transfected cells showed complete EMT phenotype with fibroblastoid appearance, vimentin filaments, E-cadherin/N-cadherin switch, lack of hemidesmosomes and, as a new feature of EMT, lack of laminin-332 synthesis. Upregulation of ZEB-1 and ZEB-2 was evident in these cells, suggesting that SNAIL can regulate these E-cadherin repressors. New monoclonal antibodies against SNAIL showed nuclear immunoreactivity not only in the SNAIL-transfected cells but also in carcinoma cells lacking production of Lm-332 and showing signs of EMT. These results suggest that changes in the epithelial cell differentiation program and EMT in SCC cells can result from the interplay among several E-cadherin repressors; however, SNAIL alone is able to accomplish a complete EMT.

Snail1 transcriptional repressor binds to its own promoter and controls its expression.

The product of Snail1 gene is a transcriptional repressor of E-cadherin expression and an inductor of the epithelial-mesenchymal transition in several epithelial tumour cell lines. Transcription of Snail1 is induced when epithelial cells are forced to acquire a mesenchymal phenotype. In this work we demonstrate that Snail1 protein limits its own expression: Snail1 binds to an E-box present in its promoter (at -146 with respect to the transcription start) and represses its activity. Therefore, mutation of the E-box increases Snail1 transcription in epithelial and mesenchymal cells. Evidence of binding of ectopic or endogenous Snail1 to its own promoter was obtained by chromatin immunoprecipitation (ChIP) experiments. Studies performed expressing different forms of Snail1 under the control of its own promoter demonstrate that disruption of the regulatory loop increases the cellular levels of Snail protein. These results indicate that expression of Snail1 gene can be regulated by its product and evidence the existence of a fine-tuning feed-back mechanism of regulation of Snail1 transcription.