Patterns in Swedish pharmacy students' performance and attitudes towards their education.

INTRODUCTION: For some time, our faculty have expressed concerns regarding an apparent decrease in pharmacy students' academic productivity and performance. This study aimed to elucidate present conditions and suggest suitable interventions to improve the pharmacy program. METHODS: Student cohorts starting the pharmacy program from 2009 to 2014 were followed with respect to performance in two courses (earlier and later). The students were segmented by entry qualifications, age, gender, etc. Eight students were further interviewed about their attitudes regarding their education. RESULTS AND CONCLUSIONS: Achievement in the earlier course fell sharply over time, despite basically unchanged entry grade levels, increasing the workload for both teachers and students. This decrease was greater for male students. In the later course, the overall achievement level was higher, possibly due to less successful students dropping out. Subgrouping of students revealed differences in study achievement depending on age, gender, study program entrance qualifications, and admission "ranking". In the interviews, students frequently stressed that connections to their future profession should be clearer and appear earlier in the program. Furthermore, students claimed that lectures with many attendees prevent peer learning and suggested that smaller groups be formed to foster cooperation and unity within the program. Remaining within their original cohort was viewed as very important by most students.

Loss of E-cadherin expression is not a prerequisite for c-erbB2-induced epithelial-mesenchymal transition.

Recent research into the mechanisms of tumour cell invasiveness has highlighted the parallels between carcinogenesis and epithelial-mesenchymal transition (EMT), originally described as a developmental transdifferentiation program but also implicated in fibrosis and cancer. In a model system for mammary carcinogenesis, we previously observed that induced signalling from a homodimer of the c-erbB2 (HER2) receptor tyrosine kinase in an initially non-malignant mammary cell line caused EMT where i) cell scattering occurred before downregulation of the cell-cell adhesion molecule E-cadherin and ii) the progress of EMT was dramatically delayed when cells were grown at high density. Here, we have further analysed these phenomena. Ectopic expression of E-cadherin concomitant with c-erbB2 signalling was unable to impede the progression of EMT, suggesting that E-cadherin downregulation is not required for EMT. Furthermore, fibroblast-like cells isolated after EMT induced in the presence or absence of ectopic E-cadherin expression showed highly similar morphology and vimentin expression. E-cadherin expressed in these fibroblastic cells had a subcellular localisation similar to that found in epithelial cells, but it exhibited a much weaker attachment to the cytoskeleton, suggesting cytoskeletal rearrangements as an important mechanism in EMT-associated cell scattering. We also investigated whether density-dependent inhibition of EMT is mediated by E-cadherin as a sensor for cell-cell contact, by expressing dominant-negative E-cadherin. While expression of this mutant weakened cell-cell adhesion, it failed to facilitate EMT at high cell densities. These results indicate that loss of E-cadherin expression is a consequence rather than a cause of c-erbB2-induced EMT and that density­dependent inhibition of EMT is not mediated by E-cadherin signalling.

Inhibition of NF-kappaB activation and chemokine expression by the leukocyte glycoprotein, CD43, in colon cancer cells.

CD43 is a heavily O-glycosylated type I trans-membrane protein, expressed at high levels on the surface of leukocytes. It is frequently overexpressed in early colon adenomas, but not in normal colon epithelial cells. To identify CD43 target genes, gene array analysis was performed using a tetracycline-inducible CD43 expression system in human colon adenocarcinoma SW480 cells. CD43 was demonstrated to down-regulate a variety of chemokine genes. Overexpression of CD43 suppressed constitutive as well as PMA-induced NF-kappaB activation and reduced the DNA binding of transcription factor p65 but not p50. Furthermore, a reduced NF-kappaB responsive promoter activity was observed and a decreased expression of proinflammatory chemokines MCP-1, IL-8 and GRO-alpha. These results suggest that overexpression of CD43 suppresses a subset of NF-kappaB target genes, partly via the inhibition of p65 transcriptional activity.

Characterization of integrin and anchorage dependence in mammary epithelial cells following c-erbB2-induced epithelial-mesenchymal transition.

Signalling from the proto-oncogene c-erbB2 in mammary epithelial cells has earlier been shown to result in epithelial-mesenchymal transition (EMT) giving rise to fibroblast-like cells, and acquisition of anchorage-independent growth (AIG) usually determined by growth capacity in soft agar. In this study, we have analysed AIG associated with c-erbB2-induced EMT in a human mammary epithelial cell line. Intriguingly, cells capable of growth in soft agar were shown to be dependent on the function of beta(1) integrin extracellular matrix receptors for growth in collagen. We therefore tested the hypothesis that apparent AIG was due to deposition of extracellular matrix in the agar. Although the fibroblastic cells had strongly upregulated expression of the fibronectin receptor subunit integrin alpha(5) andabundant fibronectin fibrils, these properties did not have a positive correlation with AIG. Furthermore, antibody blocking of integrin alpha(5) and beta(1) failed to inhibit AIG. These results indicate that the anchorage-independent cells are not dependent on connection to extracellular matrix, but instead may be subject to a growth-inhibitory effect from the collagen in the absence of integrin signalling. This notion was supported by the finding that integrin blocking of the fibroblastic cells in fibrin was without effect on proliferation.

c-erbB2-induced epithelial-mesenchymal transition in mammary epithelial cells is suppressed by cell-cell contact and initiated prior to E-cadherin downregulation.

Prolonged signalling from the growth factor receptor subunit and proto-oncogene c-erbB2 has been shown to cause epithelial-mesenchymal transition (EMT) in mammary epithelial cells. Using a system where c-erbB2 homodimer signalling can be induced in human mammary epithelial cells, we characterised the properties of c-erbB2-induced EMT. The cells resulting from this transdifferentiation showed a pronounced and stable fibroblastic phenotype with spindle-like morphology, homogeneous high expression of vimentin, N-cadherin, and integrin alpha5 as well as loss of E-cadherin and desmoplakin. However, the rate at which EMT occurred was very slow compared to other reported systems, as complete conversion was not seen until after 12-15 weeks of c-erbB2 signalling. This time delay was however not due to the presence of long-lived intermediate cell types as measured by expression of combinations of markers. By studying morphological time-courses of individual colonies of epithelial cells subjected to c-erbB2 signalling, we could conclude that apart from EMT, c-erbB2 could also cause transition to very large cells retaining cell-cell contact but with little or no proliferative capacity. EMT preferentially occurred in small colonies, suggesting that extensive cell-cell contact inhibits EMT. When testing this conclusion by exposing cells to c-erbB2 signalling at different cell densities, we concluded that only cells kept at low density would undergo EMT. High cell density also prevented the proliferative decrease associated with prolonged c-erbB2 signalling. Immunofluorescence microscopy revealed that disruption of cell-cell contact was preceded by partial relocalisation of beta-catenin to the cytoplasm whereas downregulation of E-cadherin appeared to occur after initiation of cell scattering.

PKB mediates c-erbB2-induced epithelial beta1 integrin conformational inactivation through Rho-independent F-actin rearrangements.

Signalling from the growth factor receptor subunit and proto-oncogene c-erbB2 has been shown to inhibit the adhesive function of the collagen receptor integrin alpha(2)beta(1) in human mammary epithelial cells. This anti-adhesive effect is mediated by the MAP ERK kinase 1/2 (MEK1/2) and protein kinase B (PKB) pathways. Here, we show that both pathways mediate suppression of matrix adhesion by causing the extracellular domain of the beta(1) integrin subunit to adopt an inactive conformation. The conformational switch was also dependent on rapid and extensive actin depolymerisation. While neither activation nor inhibition of the Rho GTPase affected this rearrangement, Rho was found to be activated by c-erbB2 and to be necessary for conformation-dependent integrin inactivation and, apparently by a different mechanism, a delayed re-formation of stress fibers which did not restore integrin function. Interestingly, the initial actin depolymerisation as well as its effects on integrin function was shown to be mediated by PKB. These results demonstrate how oncogenic growth factor signalling inhibits matrix adhesion by multiple pathways converging on integrin conformation and how Rho signalling can profoundly influence integrin activation in a cytoskeleton-independent manner.

Shedding and gamma-secretase-mediated intramembrane proteolysis of the mucin-type molecule CD43.

CD43 is a transmembrane molecule that contains a 123-aminoacids-long cytoplasmic tail and a highly O-glycosylated extracellular domain of mucin type. Endogenous CD43 expressed in COLO 205, K562 and Jurkat cells revealed a membrane-associated, 20 kDa CD43-specific cytoplasmic tail fragment (CD43-CTF) upon inhibition of gamma-secretase. This fragment was formed by an extracellular cleavage, as it was not accumulated after treating cells with 1,10-phenanthroline, a metalloprotease inhibitor. When CD43 was transfected into HEK-293 cells expressing dominant-negative PS1 (presenilin-1), the CD43-CTF was accumulated, but not in cells with wild-type PS1. Owing to its accumulation in the presence of a non-functional PS variant, it may thus be a novel gamma-secretase substrate. This CTF is formed by an extracellular cleavage close to the membrane, is a fragment that can be concluded to be a substrate for gamma-secretase. However, the intracellular gamma-secretase product has not been possible to detect, suggesting a quick processing of this product. During normal growth the CTF was not found without gamma-secretase inhibition, but when the cells (COLO 205) were very confluent the fragment could be detected. The intracellular domain of CD43 has previously been shown to contain a functional nuclear localization signal, and has been suggested to be involved in gene activation. From this and the present results, a novel way to explain how mucin-type molecules may transduce intracellular signals can be proposed.

CD43 has a functional NLS, interacts with beta-catenin, and affects gene expression.

CD43 is a transmembrane molecule with a highly O-glycosylated extracellular domain of mucin type. It is a normal constituent of leukocytes and found in colon adenoma, but not in normal colon epithelia. Here it is shown that the cytoplasmic tail of CD43 contains a functional bipartite nuclear localization signal directing it to the nucleus. The intracellular domain of CD43 interacts with beta-catenin and causes an upregulation of the beta-catenin target genes c-MYC and CyclinD1. The present results suggest that CD43 can be involved in nuclear signaling and via beta-catenin interaction be involved in cell proliferation.

c-erbB2-induced disruption of matrix adhesion and morphogenesis reveals a novel role for protein kinase B as a negative regulator of alpha(2)beta(1) integrin function.

Overexpression of the growth factor receptor subunit c-erbB2, leading to its ligand-independent homodimerization and activation, has been implicated in the pathogenesis of mammary carcinoma. Here, we have examined the effects of c-erbB2 on the adhesive properties of a mammary epithelial cell line, HB2/tnz34, in which c-erbB2 homodimerization can be induced by means of a transfected hybrid "trk-neu" construct. trk-neu consists of the extracellular domain of the trkA nerve growth factor (NGF) receptor fused to the transmembrane and cytoplasmic domains of c-erbB2, allowing NGF-induced c-erbB2 homodimer signaling. Both spreading and adhesion on collagen surfaces were impaired on c-erbB2 activation in HB2/tnz34 cells. Antibody-mediated stimulation of alpha(2)beta(1) integrin function restored adhesion, suggesting a direct role for c-erbB2 in integrin inactivation. Using pharmacological inhibitors and transient transfections, we identified signaling pathways required for suppression of integrin function by c-erbB2. Among these was the MEK-ERK pathway, previously implicated in integrin inactivation. However, we could also show that downstream of phosphoinositide-3-kinase (PI3K), protein kinase B (PKB) acted as a previously unknown, potent inhibitor of integrin function and mediator of the disruptive effects of c-erbB2 on adhesion and morphogenesis. The integrin-linked kinase, previously identified as a PKB coactivator, was also found to be required for integrin inactivation by c-erbB2. In addition, the PI3K-dependent mTOR/S6 kinase pathway was shown to mediate c-erbB2-induced inhibition of adhesion (but not spreading) independently of PKB. Overexpression of MEK1 or PKB suppressed adhesion without requirement for c-erbB2 activation, suggesting that these two pathways partake in integrin inhibition by targeting common downstream effectors. These results demonstrate a major novel role for PI3K and PKB in regulation of integrin function.