Tamoxifen (Tam) is widely used in chemotherapy of estrogen receptor-positive breast cancer. It inhibits proliferation and induces apoptosis of breast cancer cells by estrogen receptor-dependent modulation of gene expression, but recent reports have shown that Tam (especially at pharmacological concentrations) has also rapid nongenomic effects. Here we studied the mechanisms by which Tam exerts rapid effects on breast cancer cell viability. In serum-free medium 5-7 microM Tam induced death of MCF-7 and MDA-MB-231 cells in a time-dependent manner in less than 60 min. This was associated with release of mitochondrial cytochrome c, a decrease of mitochondrial membrane potential and an increase in production of reactive oxygen species (ROS). This suggests that disruption of mitochondrial function has a primary role in the acute death response of the cells. Accordingly, bongkrekic acid, an inhibitor of mitochondrial permeability transition, was able to protect MCF-7 cells against Tam. Rapid cell death induction by Tam was not associated with immediate activation of caspase-9 or cleavage of poly (ADP-ribose) polymerase. It was not blocked by the caspase inhibitor z-Val-Ala-Asp-fluoromethylketone either. Diphenylene ionodium (DPI), an inhibitor of NADPH oxidase, was able to prevent Tam-induced cell death but not cytochrome c release, which suggests that ROS act distal to cytochrome c. The pure antiestrogen ICI 182780 (1 microM) could partly oppose the effect of Tam in estrogen receptor positive MCF-7 cells, but not in estrogen receptor negative MDA-MB-231 cells. Pre-culturing MCF-7 cells in the absence of 17beta-estradiol (E(2)) or in the presence of a low Tam concentration (1 microM) made the cells even more susceptible to rapid death induction by 5 or 7 microM Tam. This effect was associated with decreased levels of the anti-apoptotic proteins Bcl-X(L) and Bcl-2. In conclusion, our results demonstrate induction of a rapid mitochondrial cell death program in breast cancer cells at pharmacological concentrations of Tam, which are achievable in tumor tissue of Tam-treated breast cancer patients. These mechanisms may contribute to the ability of Tam therapy to induce death of breast cancer cells.

Tamoxifen (Tam) is widely used in chemotherapy of breast cancer. It inhibits proliferation and induces apoptosis of breast cancer cells by estrogen receptor (ER)-dependent modulation of gene expression. In addition, recent reports have shown that Tam also has nongenomic effects. We previously reported induction of a rapid mitochondrial death program in breast cancer cells at pharmacological concentrations of Tam. Here we studied the upstream signaling events leading to mitochondrial disruption by Tam. We observed that 5 mum Tam rapidly induced sustained activation of ERK1/2 in ER-positive breast cancer cell lines (MCF-7 and T47D) and that PD98059 (inhibitor of ERK activation) was able to protect MCF-7 cells against Tam-induced death. These data suggest that activation of ERK has a primary role in the acute death response of the cells. In addition, inhibition of epidermal growth factor receptor (EGFR) opposed both Tam-induced ERK1/2 phosphorylation and cell death, which suggests that EGFR-associated mechanisms are involved in Tam-induced death. ERK1/2 phosphorylation was associated with a prolonged nuclear localization of ERK1/2 as determined by fluorescence microscopy with ERK2-green fluorescent protein construct. 17beta-Estradiol was shown to exert a different kind of temporal pattern of ERK nuclear localization in comparison with Tam. Moreover, 17beta-estradiol was found to oppose the rapid effects of Tam in MCF-7 and T47D cells but not in MDA-MB-231 cells, which implies a role for estrogen receptors in the protective effect of estrogen. The pure antiestrogen ICI182780 could not, however, prevent Tam-induced ERK1/2 phosphorylation, suggesting that the Tam-induced rapid cell death is primarily ER-independent or mediated by ICI182780 insensitive nongenomic mechanisms.

Aim: The aims were to describe the development of a modified national online OSCE during COVID-19 and assess related student feedback. Material and methods: The modified online OSCE comprising of eight question entities was organised simultaneously in all four dental institutes of Finland using the Moodle virtual learning environment. All fourth-year students (n = 179) attended the examination online at home. Student feedback was collected via an anonymous questionnaire with multiple-choice questions and open-ended questions concerning attitudes towards the modified online OSCE, as well as content and usability of the question entities in the examination. Means and standard deviations were calculated for multiple-choice questions. Content analysis was used for open-ended questions.Results: Of 179 students, 119 (66%) consented to the study. Students experienced they had received adequate information (mean 3.8; SD 1.2), had a positive attitude before the examination (4.0; 1.0) and found the practice test useful (3.7; 1.1) (range 1-5). Technical implementation (2.7; 0.7) and the difficulty of the questions (2.9; 0.6) (range 1-4) were found to be good. The teaching students received during their studies was sufficient (3.2; 0.5) (range 1-4). Content (mean 3.2; 0.4) and usability (2.9; 0.4) of the question entities were good (range 1-4). The themes arising from open-ended questions were importance and practicality of the topic (in questions) in relation to the work of a dentist and gratitude for the rapid conversion of the OSCE into an online examination despite COVID-19. The themes arising from negative experiences included difficulties in completing the examination within the time allocated, and dissatisfaction with the model answers provided after the examination. Conclusion:The positive student feedback towards the modified online OSCE encourages including an online examination to complement the traditional OSCE.

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Transfection with either of the ERs was able to render the U2OS cells sensitive to E 2 . Weshow that E 2 opposed etoposide-induced apoptosis and that the effect was mediated via both ER isotypes. The ER isotype selective agonists propyl-pyrazole-triol (PPT) and diarylpropionitrile (DPN) had the same effect in U2OS/ERα and U2OS/ER cells, respectively. Osp also opposed apoptosis at least in U2OS/ERα cells. Tam and Ral were not able to protect against etoposide-induced cell death. In order to evaluate the protective effects of E 2 and Osp upon etoposide challenge we studied the expression of two E 2 -regulated, osteoblast-produced cytokines, IL-6 and OPG in E 2 and SERM-treated U2OS/ERα and U2OS/ER cells. Etoposide strongly increased expression of IL-6 and decreased that of OPG.E 2 opposed IL-6 increase only in U2OS/ERα cells and OPG decrease primarily in ER cells.Osp opposed the effect of etoposide on OPG primarily in U2OS/ER cells but interestingly, it had little effect on IL-6 expression. E2, PPT, DNP and Osp also inhibited etoposideinduced death and cytokine changes in SAOS-2 osteosarcoma cells expressing endogenous ERα and ER. Collectively, our results suggest that the osteoblast protective antiapoptotic effects of E 2 are mediated by both ERα and ER but those of Osp primarily by ER. In addition, E 2 and Osp opposed the etoposide-induced increase of IL-6 and decrease of OPG which changes would increase osteoclastic activity. These antiresorptive effects of E 2 andPage 3 of 53 A c c e p t e d M a n u s c r i p t 2 Osp upon etoposide challenge differed from each other and they seemed to be differentially mediated in ERα and ER expressing osteoblast-derived U2OS cells.

We evaluated associations between changes in dental anxiety and tobacco use, adjusted for general anxiety and depressive symptoms. The FinnBrain Birth Cohort Study data, collected at gestational weeks 14 and 34 and at 3 months postpartum, were used. Questionnaires included the Modified Dental Anxiety Scale (MDAS), the Edinburgh Postnatal Depression Scale (EPDS), and the anxiety subscale of the Symptom Checklist-90 (SCL). Smoking was categorized as "stable non-smoking", "started smoking", "quit smoking", and "stable smoking". Changes in smoking and dental anxiety were evaluated "during pregnancy" (i.e., from gestational week 14 to gestational week 34) in 2442 women and 1346 men and "after pregnancy" (i.e., from gestational week 34 to 3 months postpartum) in 2008 women and 1095 men. Changes were evaluated in three smoking categories (stable non-smoking, fluctuating, and stable smoking), using data from all three time-points (1979 women and 1049 men). Modeling used repeated measures analysis of covariance. Stable smoking mothers had statistically significantly higher levels of dental anxiety (mean MDAS 12.3-12.6) than non-smoking mothers (mean MDAS 10.1-10.7) or mothers who smoked at some point during pregnancy (mean MDAS 10.8-11.5). A similar tendency was observed in fathers. However, no systematic change in dental anxiety by changes in smoking habits was observed. Those smoking during pregnancy and with high dental anxiety may need special support for smoking cessation.