The spectrum of a platinum hollow-cathode lamp containing neon carrier gas was recorded photographically and photoelectrically with a 10.7 m normal-incidence vacuum spectrograph. Wavelengths and intensities were determined for about 5600 lines in the region 1130–4330 Å. An atlas of the spectrum is given, with the spectral lines marked and their intensities, wavelengths, and classifications listed. Lines of impurity species are also identified. The uncertainty of the photographically measured wavelengths is estimated to be ± 0.0020 Å. The uncertainty of lines measured in the photoelectric scans is 0.01 Å for wavelengths shorter than 2030 Å and 0.02 Å for longer wavelengths. Ritz-type wavelengths are given for many of the classified lines of Pt II with uncertainties varying from ±0.0004 to ± 0.0025 Å. The uncertainty of the relative intensities is estimated to be about 20%. The deployment of the Hubble Space Telescope (HST) on April 24,1990, launched a new era in astronomy. With the HST, stars and other astronomical objects are being observed with unprecedented clarity. The improvement over ground-based telescopes is most significant in the ultraviolet region of the spectrum, where the earth’s atmosphere absorbs most of the radiation. Although the much-publicized spherical aberration in the HST’s primary mirror [6. Line-of-sight velocities of stellar objects can thus be determined to an accuracy of about 1 km/s. In order to achieve this accuracy, of course, an accurate wavelength scale must be established. This is accomplished by illuminating the spectrograph with an onboard platinum/neon hollow-cathode lamp during periods in which stellar observations are not being made [The region of observation of GHRS is 1100–3200 Å. In its echelle mode it has a resolving power of 90,000 and a wavelength accuracy of a few parts in 10ing made , who oriTo achieve the accuracy for which GHRS was designed, the calibration wavelengths must be accurate to about 0.002 Å. However, tests carried out in our laboratory in 1983 indicated that the best available wavelengths for Pt  had erroSome of the results of our work have appeared in two previous papers. In the first  we deterOur wavelengths for the Pt/Ne lamp are currently being used for calibration of GHRS as well as for wavelength calibration of the Faint Object Spectrograph on HST, which uses a Pt-Cr/Ne hollow-cathode lamp for both wavelength and radiometric calibration . Our datIn the present paper we present a comprehensive report of our observations of the Pt/Ne hollow-cathode lamp. For completeness we give a full account of the experimental work and data analysis. Some of this information has been given in our previous papers.Our results are presented in the form of an atlas of the spectrum emitted by a Pt/Ne hollow-cathode lamp in the region 1130–4330 Å. The atlas consists of plots of the spectrum accompanied by tables that include the wavelengths, wave numbers, intensities, and identifications or classifications where known for more than 5600 lines. We have attempted to provide the best available wavelength data, substituting values from the literature or calculated Ritz-type wavelengths where these are more accurate than our measurements.The line list developed in this work was communicated to J. Blaise and J.-F. Wyart of the Laboratoire Aimé Cotton, Orsay, France, who have used it to substantially extend the energy level analysis of Pt II. Based on our measurements they have located nearly 150 new Pt II levels. Their report on the analysis appears as a companion paper in the same issue of this journal . Blaise The data included in this atlas should be of use not only for astronomical spectroscopy but also for the calibration of general laboratory spectra obtained with medium to high resolution diffraction grating spectrographs. No other source provides such a dense and complete coverage of this spectral region with lines suitable for use as reference wavelengths. The Pt/Ne hollow cathode is easy to operate and is commercially available at moderate cost.Our observations were made with the 10.7 m normal-incidence vacuum spectrograph at the National Institute of Standards and Technology. Two different gratings were used, the first blazed at 1200 Å in first order and the second blazed at 3000 Å in first order. Both gratings were ruled with 1200 lines/mm. All measurements were made in the first order, the plate factor being 0.78 Å/mm. The slit width was 0.023 mm. With this slit width the resolving limit throughout the region of observation was about 0.020 Å. Photographic exposures were made on Kodak SWR plates.1Two different light sources were used. The first was a windowless, demountable hollow-cathode lamp having a solid copper cathode containing a helical platinum wire and some chips of silicon and germanium. The general design of the lamp was similar to that of Reader and Davis . In the The second source was a sealed hollow-cathode lamp similar to the one used by GHRS. It has a platinum hollow cathode with neon carrier gas and is sealed with a magnesium fluoride window. The lamp was manufactured by the Westinghouse Corporation (Model WL34045). It was connected to the spectrograph by a quick-disconnect flange. The cathode was located 215 mm from the slit. The lamp was operated with a 5000 Ω ballast resistor at a dc voltage of 310 V and a current of 20 mA. Exposure times ranged from 2 to 150 min.In the first phase of the wavelength reductions of the photographic data, the spectra of Pt observed with the demountable Pt-Cu lamp were measured with respect to lines of Cu II, Si I, Si II, Ge I, Ge II, Ne I, and Ne II to determine accurate wavelengths for a select group of Pt lines. Wavelengths for Cu II were Ritz values derived from the level values of Ross . WavelenIn the second phase of the reductions the spectra of all lines observed with the sealed Pt/Ne lamp were measured with respect to the above group of Pt lines, lines of Ne I and II, and lines of Pt I reported by Engleman . In the J value has been revised as a result of the work of Blaise and Wyart [Next, our values for lines of Pt II with known classifications were combined with values for classified lines of Pt II measured by Engleman by means of Fourier-transform spectroscopy to determine accurate values for 28 even and 72 odd energy levels of Pt II [nd Wyart . For tho6/s by decades.To determine the relative intensities of lines emitted by the Pt/Ne lamp and to observe lines weaker than those recorded on the photographic plates, we recorded the spectrum by translating an exit slit and photomultiplier tube along the focal curve of the 10.7 m vacuum spectrograph. The entrance and exit slit widths were 0.050 mm. The line intensities were measured by photon counting. Signals from the photomultiplier were amplified and processed by a discriminator and logarithmic ratemeter. The analog output signal from the ratemeter was sampled at 1 Hz by a computer, which digitized and stored the data. This acquisition rate corresponded to a wavelength interval of 0.0086 Å per sample. Prior to each scan the analog response of the ratemeter was calibrated by using a pulse generator to simulate the amplified pulse signal from the photomultiplier tube. The response of the ratemeter was digitized and recorded for pulse frequencies ranging from 10/s to 10The resolution limit for the scans was about 0.07 Å. The spectrum was scanned in overlapping 650 Å segments, each segment corresponding to a different rotational setting of the grating. Each scan lasted 20 h. Two scans were made for each region above 1685 Å, the first a normal scan and the second a scan at reduced sensitivity to record very intense lines that were saturated at normal recording conditions. The sensitivity was reduced by introducing a one decade offset in the logarithmic ratemeter. In addition, for the region above 2000 Å, the source intensity was attenuated by reflecting the lamp from an uncoated glass plate.Four different Pt/Ne lamps were used in the course of the experiments. Two lamps were used for the photographic exposures. One of these and two additional ones were used for the photoelectric scans. The longest use of any lamp was during the photoelectric scans, where one of the lamps was run for about 250 h. After this time the cavity of the cathode had become noticeably enlarged.The position and intensity of each spectral line in the photoelectric scans was determined by using a computer line-finding algorithm. First, the recorded signal at each point in the spectrum was converted to absolute counts/s by using the calibration information mentioned above. Then these data were scanned by the computer to locate peaks in the spectrum. The position of each peak was determined by calculating the quadratically smoothed first derivative of the data in the vicinity of the maximum intensity point and linearly interpolating the zero crossing of the derivative. The wavelength was then calculated by making a linear fit of wavelength versus position for the local spectral region, using as standards four lines accurately measured from the photographic observations on either side of the line to be determined.The intensities derived from the raw data for each scan were adjusted to produce a consistent set of values over the whole spectral region. First, using the measured intensities for lines of moderate strength in the overlapping regions of the various scans, a set of multiplicative factors was determined to bring the separate scans onto the same relative scale. Then the spectral response of the spectrograph/detector combination as a function of wavelength was calibrated by using accurate radiance values for about 80 lines of platinum measured by Klose  in a simThe number of lines observed by photon counting was much greater than observed photographically. Whereas the weakest photographic lines produced count rates of about 500 photons/s, lines having signals as low as about 10 photons/s could be observed photoelectrically. The most intense lines produced counts of about 2,000,000 photons/s. In all scans we observed a residual background count in excess of the photomultiplier dark count. This background was only a few counts/s at low wavelengths but increased to about 60 counts/s at the highest wavelengths. This increasing background is apparent in the atlas plots. The background count has been subtracted from the measured line intensities printed in the table so that the value reported accurately reflects the count rate due to the spectral line.The atlas is a series of tables and plots that provides a comprehensive description of the spectrum of the Pt/Ne hollow-cathode lamp in the region 1128–4333 Å. Each page of plots depicts a 32 Å section of the spectrum. Every spectral line for which a wavelength and intensity have been determined is indicated with a tic mark at the bottom of the plot. The wavelengths, wave numbers, and relative intensities for these lines are listed in the table on the page facing the plot.The wavelengths and intensities of Rowland ghosts (spurious lines caused by imperfections in the ruling of the grating) were predicted from the known properties of the gratings. Ghost lines are marked on the plots with a carat instead of a tic mark to distinguish them from true spectral lines. They are not listed in the table.Wavelengths of lines measured on our photographic plates, taken from the literature, or calculated from optimized Pt II energy levels are given to four decimal places. Lines measured in the photoelectric data only are given to two decimal places. Wavelengths below 2000 Å are given in vacuum; wavelengths above 2000 Å are given in air. For lines originally observed in vacuum, conversion of the wavelengths from vacuum to standard air was carried out by using the three-term formula of Peck and Reeder [Also listed in the table under the column heading CODE are the sources for wavelengths of various lines emitted by the Pt/Ne lamp that we have taken from the literature, mainly Pt I, Ne I, and Ne II. Most of these lines were used as wavelength standards. Literature values were also substituted for lines of impurity species such as H I, C I, O I, Si I, Al I, and Al II. The presence of additional impurity lines of Mg I, Mg II, Fe I, Cr I, Pd I, Rh I, Au I, Ag I, Ni I, Ca I, and Ca II were subsequently pointed out by J. Blaise. These lines are identified in the table. Literature values for their wavelengths have been substituted only for Ca II and Fe I.The intensity of impurity lines varies greatly from lamp to lamp. For example, we did not observe the intense Al I lines at 3944 and 3961 Å on our photographic plates. However, in a lower wavelength exposure using a different lamp the normally less intense lines at 3082 and 3092 Å did appear. For this reason we have given no intensities for the impurity lines.The energy level designations for classified lines of Pt I and II correspond to the integer parts of the level energies and are given with the even parity level first. Classifications and wavelengths for Pt I lines with CODES D and E were taken from Engleman . Pt I liThe intensities in the atlas are a uniform set of relative values covering the entire region of observation. For lines that were blended on the photoelectric scans but resolved or nearly resolved on the photographic exposures, the intensities were estimated visually from the photographic plates by comparison with nearby well-resolved lines. In a few places a real spectral line is blended with a grating ghost. This is noted with an M in the CODE column in the table. The intensities measured for such lines are probably affected by the presence of the ghost. As mentioned, the spectral sensitivity of the spectrometer and detector combination was taken into account by using the accurate radiance values of Klose  for abouOur relative intensities for lines emitted by the Pt/Ne lamp are potentially useful for calibration of the spectral response of spectrographic systems in other laboratories. In general, the values are sufficiently reliable to provide a good semi-quantitative calibration. Of course the accuracy that can be obtained is limited by the degree to which other Pt/Ne lamps might vary from those we used. We found only small variations in the relative intensities of lines in our lamps, all of which were purchased separately over a 5 year period. Nevertheless, it is not certain that other lamps would exhibit identical properties. In particular, comparison of lines in the 1130–1300 Å region with lines in higher wavelength regions could be affected by variation in the low wavelength transmission of the magnesium fluoride windows of different lamps. Since we used only a small number of lamps and did not scan each lamp over the entire spectral region, we can make no definitive statement regarding lamp to lamp variation. Further investigation would be needed to evaluate the importance of such systematic variations.The standard deviation of our polynomial fits for the Cu II reference lines in the Pt/Cu lamp was typically 0.0010 Å.The standard deviation of our polynomial fits for the Pt lines used as internal standards for measurements in the Pt/Ne lamp was typically 0.0015 Å.A comparison of a group of about 100 lines measured by different operators on different plates and taken with different grating rotations in the region 1470–1520 Å showed an average deviation of 0.0001 Å and an rms difference of 0.0014 Å. In general, our separate measurements of the wavelengths of individual lines agreed to about this level of accuracy.A comparison of the wavelengths of 37 lines of Pt II in the region 2247–3700 Å that were measured in this work and independently by Engleman [For the 508 lines of Pt II whose wavelengths can be calculated from the optimized level values, the rms difference between the calculated and observed wavelengths is about 0.0015 Å.A comparison of our measured wavelengths for impurity lines appearing in the Pt/Ne lamp with standard wavelengths for these lines shows an average deviation of 0.0003 Å and an rms difference of 0.0015 Å.Our estimate of the uncertainty of the photographically measured wavelengths is based on several considerations:Based on these comparisons we estimate an uncertainty of ± 0.0020 Å for the wavelengths measured photographically.As mentioned above, the wavelengths of classified lines of Pt II in the atlas which have numbers in the CODE column are those derived from the optimized level values. The uncertainties of these wavelengths are taken to be the square root of the sum of the squares of the uncertainties of the combining levels as given by Reader, Acquista, Sansonetti, and Engleman . They arThe uncertainties of the photoelectrically measured lines were estimated by comparing the measured wavelengths of Pt II lines observed only in the photoelectric scans with calculated Ritz wavelengths for the same lines. The standard deviation of the differences was about 0.006 Ä for lines below 2030 Å and about 0.015 Å for lines at longer wavelengths. Based on these comparisons we estimate the uncertainty to be ±0.01 Å for lines below 2030 Å and ±0.02 Å for lines above 2030 Å.The uncertainties of lines whose wavelengths have been taken from the literature are discussed in some detail in the notes to the atlas. Most of these uncertainties are less than 0.001 Å and virtually all are less than 0.002 Å.The cathodes of the lamps used in this work and with GHRS contain isotopes of Pt in their natural abundances. Some lines of Pt I and II show appreciable isotope and magnetic hyperfine structure (hfs). At the resolution of our spectrograph  almost all Pt lines appear sharp and symmetric. A few lines show evidence of unresolved structure and appear wide, hazy, or asymmetric on the photographic plates. These lines are noted  adjacent to their intensities in the atlas. Lines showing partially resolved structure are noted in the atlas as being complex (C). A few hyperfine patterns occurred in the photographic data as three fully resolved features and were measured as separate lines.5 or less, the existence of hfs in some lines should present no problem in using the present list of Pt lines for wavelength calibration. To achieve the highest accuracy, lines with notations indicating detectable unresolved structure should not be used. For instruments with resolving limits significantly below 0.02 Å, structure may be observed in many additional Pt lines, and our present wavelength list may not be adequate for calibration purposes. Thus, for calibration of spectrographs having much higher resolution, it may be desirable to develop calibration wavelengths based on a lamp whose cathode contains a single even isotope of Pt.For GHRS and other instruments with resolving power of 10


Reversing multidrug resistance (MDR) has been an important goal for clinical and investigational oncologists. In the last few decades, significant effort has been made to search for inhibitors to reverse MDR by targeting ATP-binding cassette (ABC) transporters  directly, but these efforts have achieved little clinical success. Protein kinases play important roles in many aspects of tumor cell growth and survival. Combinations of kinase inhibitors and chemotherapeutics have been observed to overcome cancer drug resistance in certain circumstances.We screened a kinase specific inhibitor compound library in human osteosarcoma MDR cell lines to identify inhibitors that were capable of reversing chemoresistance to doxorubicin and paclitaxel.MR and KHOSR2. We identified A-770041 as one of the most effective MDR reversing agents when combined with doxorubicin or paclitaxel. A-770041 is a potent Src family kinase (Lck and Src) inhibitor. Western blot analysis revealed A-770041 inhibits both Src and Lck activation and expression. Inhibition of Src expression in U-2OSMR and KHOSR2 cell lines using lentiviral shRNA also resulted in increased doxorubicin and paclitaxel drug sensitivity. A-770041 increases the intracellular drug accumulation as demonstrated by calcein AM assay.We identified 18 small molecules that significantly increase chemotherapy drug-induced cell death in human osteosarcoma MDR cell lines U-2OSThese results indicate that small molecule inhibitor A-770041 may function to reverse ABCB1/Pgp-mediated chemotherapy drug resistance. Combination of Src family kinase inhibitor with regular chemotherapy drug could be clinically effective in MDR osteosarcoma.The online version of this article (doi:10.1186/1471-2407-14-681) contains supplementary material, which is available to authorized users. Osteosarcoma, the most common primary malignant tumor of bone, is typically treated with surgery and adjuvant chemotherapy , 2. ChemThe human kinome contains approximately 600-protein kinases that mediate phosphorylation of proteins at an estimated 250,000 sites , 14. MulBased on these observations, it is anticipated that kinases represent a class of potential therapeutic targets in chemotherapy resistant osteosarcoma. We hypothesized that inhibiting of the expression and activation of certain kinases may enhance the efficacy of chemotherapy and/or reverse MDR. In order to identify the most suitable kinase candidates as therapeutic targets in drug resistant osteosarcoma patients, we screened a well-annotated kinase inhibitor focused library comprised of 3,000 commercially available known kinase inhibitors, as well as novel, ATP competitive kinase inhibitors targeting either active or inactive kinase conformations . We systBRFV600E, KRAS, c-Myc, etc.) 308), pmTOR, CDK11, survivin, and BcL-XMR and KHOSR2 were seeded at 8 × 103 cells/per well in a 96-well plate for 24 hours before being treated with 0.1 μM doxorubicin plus different concentrations of A-770041 for 48 hours. The cells were then lysed by adding 10 μl 10% NP-40 per well, and the manufacturer’s instructions for the apoptosis assay were followed. Apoptosis was also evaluated by Western blot using whole-cell lysates immunoblotted with specific antibodies to PARP  and its cleavage products.Apoptosis was evaluated by a caspase-cleaved keratin 18 based quantification kit, the M30-Apoptosense ELISA assay, as per manufacturer’s instructions . The ELISA apoptosis detects a 21-kDa fragment of cytokeratin 18 that is only revealed after caspase cleavage of the protein. Osteosarcoma MDR cells of U-2OSR2 were plated onto 96-well plates at a density of 4 × 103 cells/well in a volume of 100 μl RPMI1640 medium and grown for 24 h. Different concentrations  of A-770041 were added for one hour. Calcein AM (Invitrogen) was diluted to 1 μM in culture medium, and then 50 μl of calcein AM was added to each microplate well containing 100 μl of the culture medium. After 30 minutes of exposure to calcein AM at 37°C, the medium was removed by aspiration, and cells were counterstained with Hoechst 33342 (1 μg/ml in culture medium) for 2 min. The intracellular accumulation of calcein AM was then visualized and quantified on a Nikon Eclipse Ti-U fluorescence microscope equipped with a SPOT RT digital camera.Osteosarcoma MDR cells of KHOSStatistical analyses were performed using the GraphPad PRISM5 software from GraphPad Software, Inc . The student t test was used to analyze the differences between two groups. Results are expressed as mean ± SD and P < 0.05 was considered statistically significant.MR and KHOSR2 , kinases in cell cycle regulations such as PLK1 (GSK461364), CDK (GP74514A), and kinases in cell stress responses such as JNK (JNK-IN-X). Further studies validated A-770041, a compound previously reported as a highly potent pan-Src family kinase inhibitor [After screening 3,000 compounds from a preselected, kinase-based small molecule library, we identified 18 small molecule compounds that can significantly increase chemotherapy drug-induced cell death in human osteosarcoma cell lines U-2OSnhibitor –33, as tMR or KHOSR2 cell lines to paclitaxel, doxorubicin, or A-770041 alone, or the combination of paclitaxel or doxorubicin with A-770041 in complete cell culture medium for 96 hours, the relative numbers of viable cells were determined by MTT assay. Incubation of both U-2OSMR and KHOSR2 cell lines with nonlethal concentrations of A-770041 (0.5 μM) was found to increase paclitaxel and doxorubicin drug sensitivities and reverse drug resistance . The results showed that Src down-regulation by shRNA partially recovered sensitivity to paclitaxel and doxorubicin .The potential reversal of MDR by A-770041 was further evaluated in KHOSs Figure . The senMR, KHOSR2 or U-2OSTR cell lines were incubated either with a range of concentrations  of A-770041 for 48 hours. Western blot analysis revealed that A-770041 inhibits both Src and Lck activation and expression in osteosarcoma MDR cells, but has less or no effect on other kinases such as pAKT, pmTOR or CDK11  and A-770041  for 48 hours, after which the extent of apoptosis was measured. While doxorubicin only led to insignificant apoptosis at this low concentration, its combination with A-770041 was found to dramatically increase the apoptosis in the resistant U-2OSMR or KHOSR2 cells as demonstrated by M30-Apoptosense ELISA assay .Reversing Pgp-mediated MDR will result in an intracellular accumulation of chemotherapeutics, which can be achieved by inhibiting Pgp function. Therefore, we examined the effect of A-770041 on the uptake and efflux of Calcein AM, a substrate of Pgp in KHOSs Figure . A-77004Protein kinases play important roles in the many aspects of cancer including in maintaining or supporting the drug resistance phenotype , 22, 23.MR and KHOSR2, having defined overexpression of the major MDR transporters, ABCB1/Pgp. These osteosarcoma resistant cell lines have been extensively characterized with stable MDR phenotype in previous studies [MR and KHOSR2 MDR cell lines. Knockdown of Src with lentiviral shRNA also increased drug sensitivity to transporter substrate anticancer drugs paclitaxel and doxorubicin, suggesting that blocking the Src pathway might overcome MDR in osteosarcoma. We then evaluated A-770041’s potential synergistic cytotoxic effect when used in combination with paclitaxel and doxorubicin. The data showed that A-770041 is highly synergistic with both paclitaxel and doxorubicin in these MDR cell lines. As demonstrated by Western analysis, part of these effects was potentially through decreased expression of Src and Lck.Cell-based screening of a preselected kinase-based small molecule compound library identified A-770041 as one of the most effective drug resistance reversing agents in two osteosarcoma MDR cell line models, U-2OS studies , 29. MTTSrc is a nonreceptor tyrosine kinase encoded by the Src proto-oncogene. It is one of the eight members  of the Src family of kinases. Both overexpression and overactivation of Src have been shown to aid in the development of osteosarcoma by promoting key oncogenic mechanisms such as cell proliferation, adhesion, invasion, and resistance to apoptosis induced by chemotherapy drugs , 40, 41.R2 in a dose-dependent manner. These results indicated that A-770041 can also inhibit Pgp efflux function via interaction with Pgp. Drugs interacting with MDR transporter proteins like Pgp may be useful not only for the reversal of cancer drug resistance, but also for increasing the absorption or the brain entry of various pharmacological agents [The interaction of kinase inhibitor with ABC transporter Pgp has been documented in several studies. For example, gefitinib (Iressa), a selective EGFR tyrosine kinase inhibitor, is used for the treatment of lung cancer. It has been found that clinically achievable levels of gefitinib moderately reversed the Pgp-mediated resistance to paclitaxel and docetaxel in Pgp overexpressing cells . Gefitinl agents .in vivo research is warranted to understand the implication of A-770041 in overcoming drug resistance.In summary, we found that A-770041 could increase the sensitivity of drug-resistant osteosarcoma cells to various cytotoxic chemotherapeutic agents . These studies provided a proof of principle that A-770041 is active against osteosarcoma cell lines with known resistance to conventional anticancer agents, as well as primary tumor cells from osteosarcoma patients. Further Kinases play an essential role in cancer cell growth, survival and drug resistance, and these enzymes are actively investigated by the pharmaceutical industry to create molecular therapies.In this study, a cell-based screening assay using the MDR osteosarcoma cell line was used to screen a kinase specific inhibitor compound library and identify small-molecule compounds capable of reversing MDR. We identified A-770041, a potent Src family kinase (Lck and Src) inhibitor, as one of the most effective MDR reversing agents when combined with doxorubicin or paclitaxel. Because osteosarcoma is characterized by frequent relapse that is associated with the development of MDR, these findings provide support for further development of A-770041 or its derivates as candidate inhibitors of MDR in osteosarcoma treatment.Identification of 18 kinase inhibitors that reverse drug resistance in osteosarcoma MDR cell lines.(XLSX 12 KB)Additional file 1:


Data from a comparative proteomic analysis of three human breast epithelial cell lines are presented. M13SV1 cells and their tumorigenic derivatives M13SV1-R2-2 and M13SV1-R2-N1 were used. Proteomic data were obtained using 2-dimensional gel electrophoresis and subsequent identification of proteins by MALDI-TOF mass spectrometry. In a second experiment, the three cell lines were treated with different concentrations of the estrogenic compounds β-estradiol or genistein and alterations in protein expression were monitored by 2-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. Presented data provide a comprehensive overview of proteomic differences between the three cell lines and their response to estrogenic stimulation. Specifications TableValue of the data•Differences between M13SV1 human breast epithelial cells and their tumorigenic variants M13SV1-R2-2 and M13SV1-R2-N1 were identified by comprehensive proteomic analysis•Quantitative proteomic responses of the three cell lines to the estrogenic compounds genistein and β-estradiol were identified•Data can help to elucidate biological mechanisms of tumorigenicity and the molecular response of breast cancer cells to estrogenic substances.1Proteomic profiles of the three cell lines M13SV1 human breast epithelial cells p values. −5 µM, 10−3 µM, 1 µM; termed “E1-E3”). A legend with explanations is contained in the last tab, termed “explanations”. Please see also Data are presented in form of an Excel spreadsheet (supplementary file) containing information about the identity of the proteins, fold difference in abundance between different cell lines or different treatments, and corresponding 22.12 in Michigan-State-University-1 (MSU-1) medium , supplemented with 5% charcoal-stripped fetal calf serum and antibiotics . Cells were treated at ~80% confluency with different concentrations of genistein or β-estradiol for 24 h . A cutoff of │log2 ratio│≥0.3 for up- or downregulation of individual protein spots was applied.Data analysis and statistical evaluation of 2D gels using the software R has been described in detail in a previous paper 2.4Robot-assisted spot picking and tryptic in-gel digestion methodology has been described in detail in a previous paper 2.5Mass spectrometric analyses were used for protein identification, whereas protein quantification was based on spot intensity evaluation of 2D gel electrophoresis . Protein identification by mass spectrometry has been described in detail in a previous paper


The use of temozolomide (TMZ) has improved the prognosis for glioblastoma multiforme patients. However, TMZ resistance may be one of the main reasons why treatment fails. Although this resistance has frequently been linked to the expression of O6-methylguanine-DNA methyltransferase (MGMT) it seems that this enzyme is not the only molecular mechanism that may account for the appearance of drug resistance in glioblastoma multiforme patients as the mismatch repair (MMR) complex, P-glycoprotein, and/or the presence of cancer stem cells may also be implicated.Four nervous system tumor cell lines were used to analyze the modulation of MGMT expression and MGMT promoter methylation by TMZ treatment. Furthermore, 5-aza-2’-deoxycytidine was used to demethylate the MGMT promoter and O(6)-benzylguanine to block GMT activity. In addition, MMR complex and P-glycoprotein expression were studied before and after TMZ exposure and correlated with MGMT expression. Finally, the effect of TMZ exposure on CD133 expression was analyzed.50 showed a high MMR complex expression, whereas cell lines with high MGMT expression and high TMZ IC50 did not express the MMR complex. In addition, modulation of MGMT expression in A172 and LN229 cell lines was accompanied by a significant increase in the TMZ IC50, whereas no differences were observed in SF268 and SK-N-SH cell lines. In contrast, P-glycoprotein and CD133 was found to be unrelated to TMZ resistance in these cell lines.Our results showed two clearly differentiated groups of tumor cells characterized by low (A172 and LN229) and high (SF268 and SK-N-SH) basal MGMT expression. Interestingly, cell lines with no MGMT expression and low TMZ ICThese results may be relevant in understanding the phenomenon of TMZ resistance, especially in glioblastoma multiforme patients laking MGMT expression, and may also aid in the design of new therapeutic strategies to improve the efficacy of TMZ in glioblastoma multiforme patients. Glioblastoma multiforme (GBM), the most common astrocytic tumor, representing about 65% of all adult nervous system tumors, is characterized by a high aggressiveness, with an average survival period of less than 15 months . CurrentTemozolomide (TMZ), a second-generation imidazotetrazine lipophilic prodrug, has improved the prognosis for GBM patients because it can cross the BBB and induce glioblastoma cell death by introducing alkyl groups into DNA . TemozolTwo candidates, namely O6-methlyguanine-DNA-methyltransferase (MGMT) and the mismatch repair (MMR) system, have been associated with ineffective GBM therapy, although their relationship is not yet clear. The MGMT repair protein protects the cellular genome from the mutagenic effects of alkylating agents such as TMZ by removing the O6-alkylguanine DNA adduct. This adduct is transferred from the alkyl group to one of its own cysteine residues and normal guanine is restored , thereby50 presented high MMR complex levels. In contrast, cell lines with a high MGMT expression and a high IC50 against TMZ did not express the MMR complex. In addition, neither P-gp nor CD133 expression appeared to play a relevant role in the TMZ resistance phenomenon of these cell lines. Thus, the status of the MMR complex could be related to MGMT activity in GBM patients as both are related to TMZ resistance. This connection could have clinical importance in terms of explaining TMZ resistance in GBM patients and, therefore, the differences in their responses to treatment.The aim of this study was to analyze the relevance of MGMT, the MMR system and P-gp in the development of resistance against TMZ in tumor cell lines A172, LN229, SF268 and SK-N-SH. Interestingly, we have demonstrated a significant correlation between MGMT and MMR complex expression such that cell lines with no MGMT expression and a low TMZ IC2.A172, LN229  and SF268  human glioblastoma cell lines and the SK-N-SH human neuroblastoma cell line (from American Type Culture Collection: HTB-11™) were grown in Dulbecco’s Modified Eagle’s Medium , supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin and streptomycin). Cells were maintained in monolayer culture at 37°C under a humidified atmosphere containing 5% COTemozolomide (TMZ), 5-aza-2’-deoxycytidine (5Aza) and O(6)-benzylguanine (O6-BG) were purchased from Sigma-Aldrich. Aliquots of TMZ dissolved in DMSO (20 mg/ml) and prepared in serum-free culture medium (10 mg/ml) were protected from light and stored at –20°C. O6-BG stocks were dissolved in methanol (10 mg/ml) and stored at room temperature.50 dose. Cell lines exposed to the first and second TMZ cycle  were subsequently subjected to further studies at the IC50 for TMZ. 5-Aza was used in de-methylation studies at a concentration of a 30 μM for A172 and LN229 and 10 μM for SF268 and SK-N-SH. In addition, SF268 and SK-N-SH cell lines were exposed to 30 μM O6-BG prior to TMZ treatment.Temozolomide treatment of all tumor cell lines comprised a double cycle (3 days of drug exposure followed by 3 days without drug) using the previously determined ICCell lines exposed to TMZ (with or without 5-Aza or O6-BG pre-treatment) were grown in 24-well plates (Sigma) under standard culture conditions for 6 days. Cytotoxicity was determined using the sulphorhodamine-B (SRB) method. Briefly, the cells were fixed with 10% trichloroacetic acid for 20 min at 4°C then washed three times with water. After 24 hours, cells were stained for 30 min at room temperature with 0.4% SRB dissolved in 1% acetic acid and then washed three times with 1% acetic acid. The plates were air-dried and the dye solubilized with 300 ml/well of 10 mM Tris base (pH 10.5) for 10 min on a shaker. The optical density of each well was measured spectrophotometrically using a Titertek multiscan colorimeter  at 492 nm.QIAamp DNA Mini Kit  in accordance with the manufacturer's standard recommendations. Thus, 2 μg of DNA from each cell line was denatured, modified, and purified using the EpiTect Bisulfite kit . The MGMT promoter CpG islands methylation status of different cell lines was based on chemical modification of unmethylated cytosine with bisulfite to uracil. Methylation-specific PCRs (MSP) were performed using specific primers for either methylated or unmethylated DNA in the MGMT promoter. Primer sequences for MGMT were 5'-TTTGTGTTTTGATGTTTGTAGGTTTTTGT-3' (forward primer) and 5'-AACTCCACACTCTTCCAAAAACAAAACA-3' (reverse primer) for the unmethylated (UM) reaction and 5'-TTTCGACGTTCTAGGTTTTCGC-3' (forward primer) and 5'-GCACTCTTCCGAAAACGAAACG-3' (reverse primer) for the methylated (M) reaction. Agarose electrophoresis visualization by ethidium bromide and UV illumination was performed after PCR.DNA was extracted from culture cells using the The high-resolution MGMT methylation analysis of bisulfite samples was performed using high-sensitive SYBR® Green  at the Center for Genomics and Oncological Research . The reaction was conducted using an Eco Real-Time PCR System  and data were analyzed using the Eco Real-Time PCR System v4.0 software. Methylated EpiTect Control DNA, methylated and unmethylated EpiTect Control DNA,  were used for the methylation curve, with methylated-unmethylated ratios of 0, 0.25, 0.5, 0.75, and 1. All samples and the methylation curve were analyzed using a pair of primers for the specific region.5'- TCACGGCCAGTCCTCCGGAG -3' and Rw 5'- GTTCCCCGTGCCGGCTCTTC -3'. PCR was performed under classical conditions with a melting temperature of 58°C. Amplified products were separated by electrophoresis on a 3% agarose gel and visualized under UV illumination. In addition, cDNA was amplified by real-time PCR in 96-well plates using an Applied Biosystem 7500 system . The total reaction volume of 20 μl contained 20 ng cDNA, 1xTAqMan Universal PCR Mastermix , and 1x TaqMan Gene Expression assay . The PCR was run at 50°C for 2 min, 95°C for 10 min, and 40 cycles of 95°C for 15 s and 60°C for 60 s. Each sample was analyzed using the TaqMan Gene Expression Assays MGMT , ABCB1  as well as an endogenous control GADPH . For MMR genes . Reverse transcription-PCR was performed with 1.5 μg of isolated total RNA and synthesized to cDNA in a 20 μl reaction system using reverse transcriptase (Promega) with oligo-dT primers according to the manufacturer’s instructions. cDNA was used to determine MGMT expression before and after 5Aza treatment with the following primers: Fw MR genes , real-tiCells were washed twice with phosphate-buffered saline (PBS) and lysed with a lysis buffer . Protein concentration was determined using Bradford Reagent (Bio-Rad) after sonication. Thus, 25 μg of protein was electrophoretically separated by 12% SDS-PAGE and transferred to nitrocellulose membranes. These membranes were blocked for 30 min at room temperature in 5% (w/v) milk powder in PBS containing 0.1% Tween 20, co-incubated overnight at 4°C with the primary antibodies , washed three times with 0.1% Tween 20 in PBS, and incubated for 1 h with a horseradish peroxidase-conjugated (HRP) goat anti-mouse secondary antibody 1:2500 (Santa Cruz Biotechnology). Proteins were visualized using the ECL system  in the LAS-4000 mini equipment. Further analysis, as well as image processing and quantification of the bands, was performed using the program ImageQuant Las-4000. MGMT expression was normalized relative to the β-actin level of the tumor.50 doses for 120 hour), harvested, and fixed in 70% (v/v) cold ethanol for 20 minutes. They were then pelleted, washed once with PBS and resuspended in propidium iodide (PI) solution  for 30 min in the dark. Data were collected and analyzed using the Cellfit program with a FACScan flow cytometer . In addition, CD133 marker for cancer stem cells were also studied by FACScan. Cells were removed from culture using a nonenzymatic cell dissociation solution  and washed with PBS. Approximately 2 × 105 cells were incubated with primary CD133 antibody directly coupled to phycoerythrin  for 15 minutes in the dark at room temperature. Isotypic controls were used to establish the right gating.The cell-cycle distribution was determined by flow cytometry. Thus cells were treated with TMZ . The level of statistical significance was set at p < 0.05 for all tests. Experimental data were expressed as mean ± standard deviation (SD) and the results compared using Student’s t-test.50 for TMZ in different cell lines gave values ranging from 14.1 to 234.6 μM that fell into two clearly differentiated groups , which included A172 (14.1 ± 1.1 μM) and LN229 cells (14.5 ± 1.1 μM), and those with high IC50 values (> 100 μM), which included SF268 (147.2 ± 2.1 μM) and SK-N-SH cells (234.6 ± 2.3 μM). Analysis of the modulation of TMZ toxicity using two cycles of treatment demonstrated that the TMZ IC50 increased significantly in cell lines with a low basal IC50, whereas no modulation was observed in cell lines with a high basal TMZ IC50  and 5.5 times  higher, than their respective basal values  showed a high percentage of promoter methylation, reaching 100% in the LN229 line and 75–100% in the A172 cell line. No significant changes were detected in either of these lines after TMZ treatment. In contrast, a lower percentage of promoter methylation was detected in untreated SF268 (50–75%) and SK-N-SH (75%) cell lines . After TMZ treatment, SK-N-SH showed a significant decrease to 0–25% after the second cycle of TMZ. However, no change in promoter methylation percentage was observed for the SF268 cell line.The MGMT promoter CpG island methylation percentage was determined before and after treatment of each cell line with TMZ . The A1750 values), whereas no MGMT expression was observed in the A172 and LN229 cell lines  whereas, no significant variation in TMZ IC50 was observed for SF268 and SK-N-SH cell lines . As shown in Real-time PCR was used to determine the expression of five genes for the MMR system  in tumor cell lines. The highest level of MMR subunit expression was observed in A172 and LN229 cell lines, whereas such expression was practically undetectable in SF268 and SK-N-SH lines . After T50. As shown in 50 after verapamil treatment (p = 0.3875), whereas cell lines with high levels of MGMT expression (SF268 and SK-N-SH) showed a significant increase in the value of TMZ IC50 .P-glycoprotein expression was determined by Western blot for all tumor cell lines. The resistant human colorectal cancer cell line HCT-15 which endogenously expresses of P-gp, was used as a control. As shown in In order to estimate the percentage of cancer stem cells in the cancer cell population after performing two TMZ cycles, the expression of the cancer stem cell marker CD133 was evaluated. Our results showed a low percentage of CD133 positive (CD133+) population in all cell lines under basal conditions  . Interesin vivo studies have demonstrated TMZ treatment to be more effective when combined with a methylated MGMT gene promoter that induces protein silencing [Althought the use of TMZ represented a moderate improvement in the prognosis of GBM patients , the development of drug resistance is one of the main causes of treatment failure . To the ilencing ,23,24. Hilencing  concludeilencing  determinilencing , indicatin vitro results reveal a significant association between MGMT expression in tumor cells and TMZ resistance. Indeed, SF268 and SK-N-SH cell lines, which are characterized by high levels of MGMT mRNA and protein expression, showed a pronounced resistance to TMZ , whereas A172 and LN229 cell lines which exhibit no MGMT expression, both have low TMZ IC50 values (14.1 ± 1.1 μM and 14.5 ± 1.1 μM respectively). Similar results were observed by Gaspar et al. [50 values of between 10 and 20 μmol/L for A172 and LN229 cell lines, thereby establishing a direct correlation between MGMT methylation and TMZ sensitivity. Subsequent studies by Yoshino et al. [50 concentrations (< 100 μM), whereas high MGMT expression in T98G and U-138MG cell lines was associated with high IC50 values (> 350 μM). Interestingly, cell lines that do not express MGMT showed an exponential increase of TMZ resistance after two TMZ treatment cycles. Thus, the LN229 cell line, which has a low basal TMZ IC50 (14.5 ± 1.1 μM), has exhibited a TMZ IC50 of 109.4 ± 1.9 and 547.4 ± 2.6 μM after the first and second TMZ cycles, respectively. Conversely, cell lines with high levels of MGMT expression, such as SF268 and SK-N-SH, maintained their basal TMZ IC50 despite two TMZ cycles . This phenomenon has also been described in the generation of resistant GBM cell lines [Our r et al. , who obtll lines ,31.The negative correlation between MGMT mRNA and protein levels in some cell lines such as SF268 and SK-N-SH supports the results of some authors ,33 who d50 in tumor cells, we also analyzed the modulation of the cell cycle as this drug, like most DNA-damaging chemotherapeutic agents, induces cell cycle arrest [After determining TMZ ICe arrest . Our res50 concentrations experienced a high percentage of promoter methylation, whereas this percentage was much lower in cell lines with a high TMZ IC50. However, no significant changes in promoter methylation were detected in either the A172 or LN229 cell lines after TMZ treatment. Further to this, only small changes were noted in the SF268 cell line. SK-N-SH cells, with a methylation range of 75–100%, were the only cell line with a gradual loss of MGMT promoter methylation, decreasing to 25–50% after the first TMZ cycle and then 0–25% after the second. As stated above, it is difficult to correlate methylation status with MGMT expression. In fact, To determine the relationship between MGMT promoter gene methylation, MGMT expression, and TMZ treatment, we analyzed the methylation status of the promoter in all tumor cell lines before and after TMZ treatment. It is clear that cell lines with low TMZ IC50 for A172 and LN229 cell lines  suffered a significant increase (from 14 ± 1.1 μM to 66 ± 1.6 μM p = < 0.0001), whereas no modulation of TMZ IC50 was observed for the SF268 and SK-N-SH lines, which have a high basal MGMT expression. Treatment with O6-BG, which is capable of blocking MGMT action [50. Our results support the findings of Von Bueren et al. [In this context, experiments were performed with 5-Aza and O6-BG. Treatment with 5-Aza, which can demethylate the MGMT promoter , increasT action , decreasn et al. , who obsn et al. , who sug50 in the GBM A172 cell line. In addition, Yip et al. [50 had a very low level of MMR expression, whereas cell lines with no MGMT expression and a low TMZ IC50 were characterized by high MMR expression levels. In addition, TMZ exposure induced a general decrease in MMR expression in all tumor cells, with this decrease being most pronounced in the A172 cell line (more than 80%). Although TMZ treatment increased expression of MSH3, MSH6, and PMS2 in SF268 and SK-N-SH cell lines, the significant decrease in MLH1 and MSH2 expression detected could explain the observed deficiency in MMR function. It is known that MLH1 protein binds the MMR complex with to DNA after mismatch recognition [50 in SF268 and SK-N-SH cell lines could be related to a compensatory mechanism in which the decrease in MGMT expression (which increases the cell's sensitivity to TMZ) is accompanied by a decrease in the levels of MMR complex, thereby further increasing the cell’s resistance to TMZ. This could refute the classical concepts in which TMZ resistance is only mediated by MGMT expression since a blockade of MGMT does not affect the sensitivity to TMZ. Maxwell et al. [The MMR complex is involved in repairing DNA damage caused by alkylating agents. Deficiencies in the expression of MSH2, MSH6, and PMS2, which form part of the MMR complex, have been linked to modulation of TMZ resistance independently of MGMT . Indeed,p et al.  concludeognition  and thatognition . Interesognition . Thus, tl et al.  showed tl et al.  demonstrl et al. . This fal et al. . On the 50 were observed for A172 and LN229 cell lines (p = 0.3875). In contrast, treatment with verapamil significantly increased the TMZ IC50 values for SF268 and SK-N-SH . This seemingly contradictory result can be explained by verapamil's ability to inhibit membrane calcium transporters [50 modulation. Thus, P-gp does not appear to be related to TMZ resistance in GBM patients.Although P-gp has recently been associated with nervous system tumors ,43, our sporters . In cellsporters , thus mein vivo studies have demonstrated that the presence of CSCs (CD133 positive) in GBM correlated with chemoradioresistance and a poor prognosis [Finally, the theory in which cytotoxic treatment selects resistant CSCs could explain post-therapy cancer drug resistance. Although CSCs may be detected in different tumors, including GBM, by the presence of CD133 , the valrognosis , whereasrognosis  recentlyrognosis . Some aurognosis . In thisrognosis  and A172rognosis . Our resThe molecular mechanisms mediating temozolomide resistance, one of the leading causes of treatment failure in GBM patients, are unclear. As such, we used nervous system tumor cell lines exposed to TMZ treatment to analyze the relevance of MGMT, the MMR system, and the ABC transporter in the TMZ resistance phenomenon. Our results demonstrate that MGMT does not play a key role in TMZ resistance in either A172 or LN229 cell lines (both with low MGMT expression levels), and nor does it have an essential part in either SF268 or SK-N-SH lines, both of which have high levels of MGMT expression. It is worth noting, however, that the degree of MGMT expression correlates with expression of the MMR complex, which is also modulated by TMZ. This may have a bearing on TMZ resistance and could explain the failure of TMZ treatment in tumor cells with no MGMT expression. In contrast, the ABC-transport protein P-gp does not appear to participate in TMZ drug resistance. Furthermore, we have demonstrated that TMZ treatment produces a significant change in CD133+ cell percentages in GMB cell lines. Further studies into CD133+ population characteristics in GMB cell lines are necessary to determine the population's relevance to drug resistance and its differences with respect to the negative CD133 cell population. Thus, therapeutic strategies that restore the expression of the MMR system could lead to possible routes to improve the efficacy of TMZ therapy in GBM patients.S1 FigA) Real-time PCR analysis of CD133 expression in tumor cell lines. The A549 cell line was used as a positive control. B) Modulation of CD133 expression in tumor cell lines by TMZ treatment. B: basal cells, 1C: first TMZ cycle; 2C: second TMZ cycle. All data represent the mean value ± SD of triplicate cultures.(TIF)Click here for additional data file.


The authors retract this publication due to concerns about the cell lines employed in the study.The study reports experiments looking at pathways involved in anoikis resistance in human adenoid cystic carcinoma; the experiments involved the use of the cell lines ACCM and ACC2. After the publication of the article, a reader raised concerns that these two cell lines may not originate from adenoid cystic carcinoma. The authors have completed a short tandem repeat analysis on the cell lines and this has revealed cross-contamination with other human cells, which compromises the relevance of the work to human adenoid cystic carcinoma, and thus the conclusions of the study.In the light of this, the authors retract this publication.


Treatment of MDA-MB-231 and ACHN xenografts with trametinib in combination with rapamycin resulted in significant growth inhibition in both but without an apparent effect on EMT. Future studies are needed to determine whether EMT status is predictive of sensitivity to rapalogs and to determine whether combination therapy with EMT modulating agents can enhance antitumor effects of PI3K/mTOR inhibitors.Rapamycin analogues have antitumor efficacy in several tumor types, however few patients demonstrate tumor regression. Thus, there is a pressing need for markers of intrinsic response/resistance and rational combination therapies. We hypothesized that epithelial-to-mesenchymal transition (EMT) confers rapamycin resistance. We found that the epithelial marker E-cadherin protein is higher in rapamycin sensitive (RS) cells and mesenchymal breast cancer cell lines selected by transcriptional EMT signatures are less sensitive to rapamycin. MCF7 cells, transfected with constitutively active mutant Snail, had increased rapamycin resistance (RR) compared to cells transfected with wild-type Snail. Conversely, we transfected two RR mesenchymal cell lines—ACHN and MDA-MB-231—with miR-200b/c or ZEB1 siRNA to promote mesenchymal-to-epithelial transition. This induced E-cadherin expression in both cell lines, and ACHN demonstrated a significant increase in RS. Treatment of ACHN and MDA-MB-231 with trametinib modulated EMT in ACHN cells  The PI3K/mTOR pathway performs essential functions for maintaining the malignant phenotype including controlling cell growth, metabolism, and autophagy , 2. RapaThe epithelial to mesenchymal transition (EMT) is defined by the loss of intracellular links along with the gain of migratory and invasive abilities . PlasticBRAF V600 mutant melanoma in combination with dabrafenib and that is effective for inhibiting growth in triple negative breast cancer cell lines in vitro [Type 3 EMT is involved in cancer progression and metastasis, and thus it is a potential mechanism of attaining the malignant phenotype . This phin vitro . Histonein vitro .in vitro and in mouse xenografts.Despite the cross-talk between EMT programming and the mTOR pathway, the relationship between rapamycin sensitivity in immortalized cancer cells lines and markers of EMT has not been previously investigated. We performed a functional proteomic screen with reverse phase protein array (RPPA) to determine biomarkers associated with sensitivity and resistance to rapamycin, and we found and association with EMT and rapamycin resistance. We hypothesized that the mesenchymal status of cancer cells imparts resistance to rapamycin. Thus, we proposed to modulate EMT in immortalized cancer cell lines and determine whether alterations in EMT biomarkers correlated with sensitivity to rapamycin both 50 of rapamycin greater than 100 nM were classified as resistant and 31 with IC50s less than 100 nM as sensitive [To determine sensitivity to rapamycin in immortalized cancer cell lines, sulforhodamine B (SRB) assay was performed to classify cell lines as resistant or sensitive. Twelve cell lines with an ICensitive . Reverse10GI50 of rapamycin were significantly lower in the epithelial group than the mesenchymal group . The median Log10GI50 of temsirolimus was lower in the epithelial group than the mesenchymal group, but this difference did not achieve statistical significance. We also classified the breast cancer cell lines with 261 probe sets mapping to the 125 gene symbols of a EMT signature described Gröger et al. [10GI50 of rapamycin was significantly lower in the epithelial group than that in the mesenchymal group with this classification as well , and two mutant Snails (Snail-2SA and Snail-6SA) . Snail-2To test whether modulation of EMT to a more epithelial phenotype could increase rapamycin sensitivity in RR mesenchymal cell lines, we targeted the EMT program through transfection of miR-200b/c mimics and siRNA knockdown of ZEB1. We selected to attempt EMT modulation in two RR cell lines: the triple-negative breast carcinoma cell line, MDA-MB-231, and the renal cell carcinoma cell line, ACHN.Both cell lines demonstrated increased E-cadherin expression with miR-200b/c transfection Figure . DespiteSimilarly, siRNA against ZEB1 was successful in abolishing ZEB1 expression in both ACHN and MDA-MB-231 compared to non-targeting siRNA, with concurrent increased expression of E-cadherin in both cell lines Figure . SimilarTherefore, miR-200b/c transfection and ZEB1 siRNA knockdown modulated EMT in both ACHN and MDA-MB-231 but only increased the sensitivity of ACHN to rapamycin.in vivo, vorinostat alone or in combination with rapamycin was administered to MDA-MB-231 xenograft bearing mice for 38 days. Although the tumor volume in combination treatment group was slightly smaller than the single drug groups, there was no significant difference among the groups ; therefore vorinostat appeared to be unable to reverse EMT in vivo in this model.As histone deacetylase (HDAC) inhibitors, have been reported to reverse EMT , 24, we s Figure . NotablyIn addition, vorinostat alone or in combination with two different doses of rapamycin was administered to ACHN bearing mice for 19 days. Compared to control, mean tumor volume decreased only in rapamycin 4 mg/kg group and was smaller in combination groups. There was no statistically significant difference among groups.Previous studies have reported that MEK inhibition modulates EMT . We thusin vivo, effects of the combination of trametinib and rapamycin compared to single agent therapy was tested. Trametinib alone or in combination with rapamycin for 14 days caused tumor regression in MDA-MB-231 xenografts  transformed with V12H-Ras oncogene and rendered oncogenic , 26. HMLin vitro and in mouse xenografts. However, some, but not all mechanisms of EMT modulation resulted in increased sensitivity to rapamycin in cancer cell lines in vitro and in vivo.We found an association with EMT and rapamycin resistance on a functional proteomic screen. Further, we demonstrated that mesenchymal cell lines selected by an EMT signature are more resistant to rapamycin. We thus hypothesized that the mesenchymal status of cancer cells, exhibited through absence of E-cadherin expression and presence of vimentin expression, correlated with resistance to rapamycin both The rapamycin analog temsirolimus is approved by the Food and Drug Administration for the treatment of advanced renal cell carcinoma, and the rapamycin analog everolimus is FDA-approved for the treatment of pancreatic neuroendocrine tumors, renal cell carcinoma, sub-ependymal giant cell astrocytoma associated with tuberous sclerosis, and hormone-receptor positive breast cancer (in combination with exemestane). There has been intensive study on mechanism of action and mechanisms of intrinsic sensitivity and resistance to rapamycin, but largely, biomarkers predictive for response to rapalogs have not been identified. Although both somatic TSC1 mutation  and mTORThere were differences in expression and phosphorylation of MAPK and Akt/mTOR pathway markers in MCF7 Snail-WT and Snail-6SA cell lines. Rapamycin was reported to activate MAPK pathway through S6K-PI3K-Ras feedback loop in various models , 35. MAPin vitro findings, metaplastic breast cancer, a mesenchymal tumor type, has previously been reported to be especially responsive to a temsirolimus containing therapy regimen; 42% objective response rate was reported for temsirolimus, bevacizumab and liposomal doxorubicin [EMT has been associated with chemoresistance to several chemotherapeutic agents including paclitaxel , oxaliplorubicin . Whetherin vitro, ACHN demonstrated increased E-cadherin expression and increased sensitivity to rapamycin. In addition, vorinostat and trametinib treatments induced E-cadherin expression. However, in vivo neither of the drugs increased E-cadherin expression, and rapamycin and vorinostat combination had no effect on tumor growth. Thus, in vitro findings were not capitulated with in vivo results. Notably our results differ than those reported in epidermoid squamous cell carcinoma xenografts, where vorinostat both reversed EMT and inhibited tumor growth [3) whereas the other model introduced an early treatment protocol that started on day 3 [A variety of preclinical approaches has been proposed to reverse EMT. In our experiments, in each mechanism of EMT modulation r growth . In addion day 3 . HoweverWe observed an increase in E-cadherin expression in MDA-MB-231 by miR200b/c transfection and ZEB1 knockdown, however, there was no growth inhibition. MDA-MB-231 has BRAF and RAS mutations that may render MDA-MB-231 resistant to rapamycin regardless of EMT status. This was further supported by our findings with HMLER cell line, and in the presence of RAS mutation, reverting EMT by trametinib treatment did not induce sensitivity to rapamycin. In ACHN cell line, E-cadherin was increased after miR200b/c or ZEB1 transfections and trametinib treatment, which may have been adequate to alter EMT and its crosstalk with PI3K/mTOR signaling, ultimately to result in increased sensitivity to rapamycin. Simultaneous targeting of Ras/Raf/MEK/Erk and PI3K/Akt/mTOR pathways has already been proposed as an approach to overcome resistance to Raf-i or MEK inhibitors , 43. In Future studies are needed to determine optimal agents for reversion of EMT modulation and to determine the effect of combinations of these agents with PI3K/Akt/mTOR inhibitors.2. MCF7 cell lines containing Snail-WT, Snail-2SA or Snail-6SA were created as previously described [ACHN, BT-474, BT-483, MCF7, MDA-MB-231, MDA-MB-453, and ZR75–1 cell lines were obtained from the American Type Culture Collection. MDA-MB-435 and NCI/ADR-RES cells were obtained from the National Cancer Institute. Cell lines were passaged for less than six months following resuscitation, and thus were not tested for characterization. ATCC utilizes Short Tandem Repeat (STR) profiling to verify cell line identity. Cells were cultured in DMEM/F12 supplemented with 10% fetal bovine serum at 37°C and humidified 5% COin vivo experiments, 0.5% hydroxypropylmethylcellulose (Sigma) and 0.2% Tween-80 (Sigma) in distilled water (pH 8.0) was used as oral gavage vehicle as previously described [in vitro and in vivo experiments), polyethylene glycol (vehicle for vorinostat for in vivo experiments), and G418 were purchased from Sigma.Rapamycin and vorinostat were purchased from LC Laboratories, Inc. Trametinib was purchased from Selleck Chemicals. For escribed . DMSO (vCells were washed with cold PBS and lysed in 100 mM Tris-HCl (pH 6.8), 4% SDS, and 20% glycerol, and then protein were separated by SDS-PAGE. The protein was transferred to a 0.2 μm nitrocellulose membrane (Bio-Rad Laboratories). Membranes were blocked with 0.1% casein in TBS. Immunoblotting was performed with the following antibodies: caveolin-1, pan-cytokeratin, E-cadherin, Erk 1/2, p-Erk 1/2 (Thr202/Tyr204), MEK1/2, p-MEK1/2 (S217/221), p-S6 (Ser240/244), p-S6 (Ser235/236), S6, p-Akt T308, p-Akt S473, Akt, p90RSK S380, α-tubulin , fibronectin, ERα, smad3 (Epitomics), Snail, vimentin (Abcam), actin (Sigma), and ZEB1 (Bethyl). The immunoblots were visualized using the Odyssey IR imaging system and software (Li-Cor Biosciences).2 of mean expression values.Reverse phase protein array (RPPA) was performed at the MD Anderson Cancer Center Functional Proteomics RPPA Core Facility as described previously –46, and The silencing of ZEB1 with small interfering RNA (siRNA) was performed using DharmaFECT 1 transfection reagent and siGENOME SMARTpool Human ZEB1 siRNA  or siGENOME Non-targeting siRNA Pool #2 (Dharmacon). Cells were harvested after 72 hours and lysates obtained for western blotting or trypsinized and plated for growth assay experiments.NeoFX transfection reagent (Ambion). Cells were harvested after 72 hours and lysates obtained for western blotting or trypsinized and plated for growth assay experiments.The transient transfection of miR-200b (CCAGCUCGGGCAGCCGUGGCCAUCUUACUGGG CAGCAUUGGAUGGAGUCAGGUCUCUAAUACUG CCUGGUAAUGAUGACGGCGGAGCCCUGCACG) and miR-200c (CCCUCGUCUUACCCAGCAGUGUUU GGGUGCGGUUGGGAGUCUCUAAUACUGCCGGG UAAUGAUGGAGG) in a 50:50 ratio or Negative Control miRNA #1 was performed using reverse transfection method and siPORT 50) and combination index (CI) were determined from dose-response curves as previously described [50 cutoff value of 100 nM.For rapamycin antiproliferative activity, cells were plated in triplicate in 96-well plates at densities of 500 to 5, 000 cells per well depending on growth characteristics of each cell line. Cell growth was measured at 5 days (baseline rapamycin sensitivity classification) and 4 days (miR-200 transfection and ZEB1 knockdown) using sulforhodamine B (SRB) assay as previously described . The medescribed . Cells w6) and MDA-MB-231 (1 × 107) cells, mixed with Matrigel (BD Biosciences), were inoculated into the mammary fat pads of six-week-old female athymic nude (nu/nu) mice . In trametinib-rapamycin study, after tumors formed, MDA-MB-231 xenografts were randomized into 4 groups  weekly/oral gavage vehicle daily, DMSO IP weekly/trametinib 0.3 mg/kg body weight oral gavage daily, rapamycin 1 mg/kg IP weekly/oral gavage vehicle daily, or rapamycin 1 mg/kg IP weekly/trametinib 0.3 mg/kg body weight oral gavage daily, n = 7–8). After tumors formed, ACHN xenografts were randomized into 5 groups . In vorinostat-rapamycin study, after tumors formed, MDA-MB-231 xenografts were randomized into 4 groups . After tumors formed, ACHN xenografts were randomized into 6 groups . The tumor growth was followed by caliper measurements and tumor volumes were calculated as previously described [All animal studies were conducted according to the guidelines of the American Association of Laboratory Animal Care under a protocol approved by the MD Anderson Animal Care and Use Committee. ACHN (6.67 × 10escribed . Mice wet-test. Growth inhibition with rapamycin (SRB data) for miR-200 transfection and ZEB1 knockdown was compared to respective control using 2-Way ANOVA for treatment v. rapamycin/DMSO followed by Bonferroni multiple comparison tests. For trametinib-rapamycin and vorinostat-rapamycin in vivo experiments, tumor volume at the last day and percent change in tumor volume from day 0 to the last day were both compared among all treatment groups by 1-way ANOVA followed by Tukey multiple comparison test or Kruskal Wallis test followed by Dunn's multiple comparison test. All statistical tests used a significance level of 5%.The cell line RPPA slide data was analyzed for differences in expression between RS and RR cell lines using linear mixed effects model with fixed effect of sensitivity and time point, and random effect of cell line. To account for multiple testing, we estimated the false discovery rates (FDR) of the F-tests of the cell line sensitivity effect using beta-uniform mixture model. All results shown in bar graphs and line graphs are presented as means ± SE. Growth inhibition (SRB data) of MCF7 Snail-WT v. MCF7 Snail-6SA following rapamycin treatment was compared with a Student's 50 values on Log10 scale of all cell lines available in GI50 data from Daemen et al. for rapamycin, everolimus or temsirolimus respectively [50 and sensitivity information for the compounds of interest. For the Byers EMT signature, probe set ID was used as the identifier. There are 48 probe sets in Daemens's Affymetrix U133A data mapping to Byers' 97 probe sets signature from Affymetrix U133plus2 data. For Gröger's signature, gene symbol was used as identifier and multiple probe sets for the same gene symbols were extracted when available. We conducted similar analysis for Byers' and Gröger's EMT signatures on the probe set level. Two-way hierarchical clustering was conducted to cluster Affymetrix probe sets corresponding to the EMT signature genes using Pearson correlation distance metric and cluster cell lines using Euclidean distance metric with the Ward's linkage rule. The cell lines were grouped into epithelial (green bar) and mesenchymal (red bar) groups at the first major branching of the dendrogram. Wilcoxon rank sum tests were used to compare the Log10GI50 values between epithelial and mesenchymal cell lines.For the analysis of association between breast cancer cell line sensitivity and EMT signature, we defined the cell line sensitivity by using the average of GIectively . We extr


Problem-based learning (PBL) is well established in medical education and beyond, and continues to be developed and explored. Challenges include how to connect the somewhat abstract nature of classroom-based PBL with clinical practice and how to maintain learner engagement in the process of PBL over time.A study was conducted to investigate the efficacy of decision-PBL (D-PBL), a variant form of PBL that replaces linear PBL cases with virtual patients. These Web-based interactive cases provided learners with a series of patient management pathways. Learners were encouraged to consider and discuss courses of action, take their chosen management pathway, and experience the consequences of their decisions. A Web-based application was essential to allow scenarios to respond dynamically to learners’ decisions, to deliver the scenarios to multiple PBL classrooms in the same timeframe, and to record centrally the paths taken by the PBL groups.A randomized controlled trial in crossover design was run involving all learners (N=81) in the second year of the graduate entry stream for the undergraduate medicine program at St George’s University of London. Learners were randomized to study groups; half engaged in a D-PBL activity whereas the other half had a traditional linear PBL activity on the same subject material. Groups alternated D-PBL and linear PBL over the semester. The measure was mean cohort performance on specific face-to-face exam questions at the end of the semester.D-PBL groups performed better than linear PBL groups on questions related to D-PBL with the difference being statistically significant for all questions. Differences between the exam performances of the 2 groups were not statistically significant for the questions not related to D-PBL. The effect sizes for D-PBL–related questions were large and positive (>0.6) except for 1 question that showed a medium positive effect size. The effect sizes for questions not related to D-PBL were all small (≤0.3) with a mix of positive and negative values.The efficacy of D-PBL was indicated by improved exam performance for learners who had D-PBL compared to those who had linear PBL. This suggests that the use of D-PBL leads to better midterm learning outcomes than linear PBL, at least for learners with prior experience with linear PBL. On the basis of tutor and student feedback, St George’s University of London and the University of Nicosia, Cyprus have replaced paper PBL cases for midstage undergraduate teaching with D-PBL virtual patients, and 6 more institutions in the ePBLnet partnership will be implementing D-PBL in Autumn 2015. The early years of medical training have seen a progressive move away from curricula organized around single-discipline bioscience material and the use of passive forms of instruction . ProblemThere are a number of educational technologies that share certain characteristics with PBL. For instance, virtual patients are on-screen learning resources that typically present a clinical problem for learners to solve or manage, and in doing so involve aspects of both PBL and simulation . AlthougSt George’s University of London (SGUL) replaced paper PBL cases for midstage undergraduate teaching with decision-PBL (D-PBL) cases. These are Web-based branching virtual patients that require learners to discuss, debate, and make patient management decisions to negotiate the case . This paPBL involves a combination of students working in small groups with a facilitator and independent research. Each group works through a predefined patient case, discussing the information provided and implied, exploring possible diagnoses, suggesting investigations and treatments, and identifying the research they need to undertake to be able to resolve the case . LearnerPBL has been deployed in many different contexts  and althThere are other teaching modalities in medical education that employ cases, including simulation, case-based learning, and virtual patients, reflecting the role of the patient case as “the primary, vicarious means of shaping clinical judgment for new learners and experienced practitioners alike” . SimulatThe study presented here explored the efficacy of D-PBL in terms of student exam performance. Our working hypothesis was that D-PBL experiences would lead to better exam performance than linear PBL experiences. A randomized controlled trial in crossover design was run to compare aggregate learner performance in those learners who had D-PBL with those who had linear PBL with supplementary PBL “triggers” to compensate for the absence of D-PBL. The trial analyzed students’ performance on questions related to D-PBL decision points compared with their performance on questions not related to D-PBL. For each comparison cohort the learning activity was different, but the learning objectives were the same.SGUL is located in South London in the United Kingdom and runs a multiple stream medical education program. The undergraduate entry stream undertake a 5-year program and the graduate entry stream undertake a 4-year program. SGUL has employed PBL since the 1990s  and at tA project was initiated in 2007 to rewrite the second year graduate entry paper PBL cases as branching virtual patients (D-PBL) and to modify the facilitation model to stimulate debate around predefined choices, and their consequences, at key points in the D-PBL virtual patient case . The objA D-PBL virtual patient case is designed as a series of interconnected nodes, each of which is presented as a Web page. Each node represents a step in the unfolding of the case. Some nodes are connected in chains; others have multiple nodes linked to them that allow learners to choose which path they will follow. Learners can only take 1 path through a case and they need to deal with the consequences of their decisions as the D-PBL case unfolds. The small-group facilitator encourages learners to consider the options and to debate different courses of action at each D-PBL decision point. Tutors report increased student engagement at option points and students express the value of simulating the experience of managing cases in real life, including the stresses that can lead to poor decisions ,23.The use of a Web-based virtual patient delivery system was essential to creating and running D-PBL activities. The virtual patient cases were to be delivered to multiple PBL classrooms in the same time frame, the scenarios were required to respond to the choices of different learner groups dynamically and independently, and the paths taken by the individual groups were tracked and logged centrally. The latter would permit later analysis and research into the paths that groups took to better understand the effectiveness of the option steps and to iteratively improve the cases.To minimize the impact on the curriculum as a whole, D-PBL was designed to only differ from traditional linear PBL in a few key areas. D-PBL (like PBL) involved small-group (6 to 8) learners, face-to-face sessions at the start and end of the week, independent research and study between face-to-face sessions, scaffolding by a case with triggers based around problems, and 1 case worked on per week. The differences were the replacement of static paper cases with dynamic Web-based virtual patient cases and the periodic debates and decision making when alternative paths were presented (within face-to-face sessions).The 5-week Life Protection module  in the second-year SGUL graduate entry stream was selected as the context for the study. All 5 cases within the Life Protection module were rewritten both as linear PBL cases and as D-PBL cases using the same virtual patient software; the only difference being the addition of different routes through the case for the D-PBL versions.The faculty committee for each module reviewed the completed D-PBL cases to make sure that the modified cases still fell within the range of the existing learning objectives . Then the linear PBL cases were supplemented by trigger questions that would cover the same area of learning emphasized by the options. For example, in the anaphylaxis case shown in In this way, the study sought to ensure that the D-PBL provided different learning opportunities rather than additional learning opportunities. The development of the D-PBL cases has been discussed more fully elsewhere , althougA preliminary analysis of D-PBL in 2007 compared student engagement between learners in paired cohorts who had either a linear version of the case (without options) or a branched version . Cohorts alternated between branched and linear over the first 4 weeks, with a fifth case delivered in the branched version to both cohorts. It was noted that students had performed markedly better than in previous years to a question that was related to 1 of the D-PBL decision points in the week 5 case. However, the D-PBL deployment was not designed as an experiment and there was no control group or any other means to systematically assess the impact of using D-PBL on summative assessment in this first year of using D-PBL. Therefore, a study was developed and run in the following academic year (2008) analyzing the exam performance of students with questions that had been previously encountered in either a D-PBL or linear PBL format.All 81 students  in the graduate entry class in 2008 were entered in to the study. The SGUL Teaching and Learning Committee reviewed the study protocol, noting that the study did not require further ethical review because the early test of the system had not raised any serious concerns and the intervention was restricted to relatively small changes in the execution of PBL sessions.Students were randomly allocated to 11 tutorial groups (4 groups of 8 and 7 groups of 7) by the SGUL Registry as part of its standard protocol for student assignments. The 11 PBL groups were separated into 2 cohorts: C1 with 5 PBL groups and C2 with 6 PBL groups. During weeks 1, 3, and 5, cohort C1 ran linear PBL cases and C2 ran the branching D-PBL cases. During weeks 2 and 4, C1 ran branching D-PBL cases and C2 ran linear PBL cases. Blinding to the intervention was not possible; both the participants and the investigators were aware which style of case they received. The material in both groups of questions was encountered in both D-PBL and linear PBL cases. The difference was that D-PBL cases required a learner to select a choice that may have had negative consequences, whereas there were no alternative paths or consequences in the linear PBL case. The flow of participants through the study is illustrated in the Consolidated Standards of Reporting Trials (CONSORT) diagram in Two weeks after the closure of the Life Protection module, the students were given a Short-Answer Question Summative Module Exam. Of the 81 students in the study, 80 took the face-to-face examination. Within the short-answer questions section of the exam, 5 questions were chosen which were significantly linked to individual options and consequences points within the relevant D-PBL case. This represented our test set of questions. Another 5 questions were deemed to have no relevance to options within the cases, and these represented a comparison set. Questions were not identified to the students as relating to the study or D-PBL within the exam. For each question, the relevant subject specialist submitted criteria for assessment and a marking scheme to the module team for review. The subject specialist had no role in the creation of the relevant option point. The module team reviewed each item and then estimated the proportion of minimally competent examinees that would correctly answer the item.Scripts were marked in each case by the relevant subject specialist and reviewed by another marker. Maximum points for each SAQ varied from 10 to 12 marks. The Module Organizing Team ratified the validity of these 2 sets of questions and rejected 1 question for which the study team agreed did not fit cleanly into either of the 2 question categories. This reduced the comparison set to 4 questions. The examination office passed the final results to the study team who added tags to indicate which groups and cohorts they were in. The mapping between the type of PBL case, the exam questions, and the 2 cohorts are shown in U test using SPSS v21 . Tests were run to evaluate these hypotheses: (1) learners who had not encountered key material related to D-PBL decisions would score lower, on average, than those who had and (2) learners who had worked with D-PBL would score the same, on average, than those who had not on material not related to D-PBL decisions. Effect sizes were calculated for the 2 cohorts using Cohen’s d.Because the exam results for the 9 questions were found to follow a nonnormal distribution, statistical significance was tested comparing the results for each question between the D-PBL and linear PBL cohorts as independent samples employing a Mann-Whitney P<.05 rejects the null hypothesis. The 80 learners in 2 cohorts were exposed to either a branching case D-PBL or linear PBL case each week  and positive except for question 2, which had a medium effect size (0.6-0.3) and was positive. The effect sizes for the questions not related to D-PBL were all small (<0.3) except for question 8, which had a medium effect size. Overall, there was a mix of positive and negative values.The results confirmed the study hypothesis that learner performance was higher for those who had learned with D-PBL than for those who had learned with linear PBL on questions related to D-PBL experiences and there were no significant differences between D-PBL and linear PBL cohort performance on questions not related to D-PBL experiences. This suggests that the use of D-PBL leads to better midterm learning outcomes than linear PBL. However, we should clarify a number of contextual factors that underpin this assertion.Firstly, participants were midstage undergraduate learners with some limited clinical exposure. Our background hypothesis was that the D-PBL approach suited learners who already have some PBL experience and who had enough clinical knowledge to work with the patient management challenges that D-PBL involves. Secondly, it should be made clear that the intervention was the whole activity, involving the use of particular artifacts  in a particular way (facilitated debate and discussion at decision points) within an otherwise traditional small-group PBL setting. Research is continuing into the conduct of D-PBL activities and the experiences of those involved. It is also important to note that the Web-based D-PBL did not detract from the process of discussion, enquiry, and problem solving in PBL. Options only appeared after the preceding steps in the case had been discussed fully. Moreover, there was no need for students to find the “correct” path through a case (or to be guided in doing so) because all paths were designed to engender equivalent learning opportunities and the scenario itself provided excellent feedback in the context of the patient. If the students take poor options, the changes in the scenario are sufficient guidance to encourage students to review their choices and re-evaluate new optional routes for continuation. Therefore, careful scenario construction and testing were essential and all case writers were trained with a formal set of guidelines on scenario construction.Because of the specificity of the activity, the type of virtual patient used within it, and the provisional nature of these current findings, no assertions can be made regarding the efficacy of virtual patients in general. We acknowledge the importance of the activity that is constructed around a virtual patient , somethiThere are a number of limitations to this study. Firstly, only the 1 class iteration has been tested. Although these findings are considered strong enough to justify publication at this stage, at least to encourage others to explore the D-PBL approach, further replication studies are planned to test the efficacy of D-PBL using a wider range of learning and curriculum contexts. Secondly, an experimental approach was taken to investigate the efficacy of the D-PBL activity at the single-item level. Although this has proved useful, there is much work to be done in exploring the nature of the activity and the ways in which students experience it. Finally, it is acknowledged that the cognitive aspects of D-PBL and the links between students’ actions in small-group settings have not been explored. The nature of learning that follows from these actions and its retention and application also need further exploration.Although our work is a hybrid of PBL and virtual patient instructional models, there are connections with studies that explore the importance of scaffolding and learner agency in PBL  as well Before this study was completed, SGUL decided to implement D-PBL in both its graduate and undergraduate (school-leaver) curricula in the common transitional year between campus-based learning and clinical attachments. This decision was based solely on tutor and student feedback. D-PBL is now running in both SGUL and the University of Nicosia, Cyprus (UNic), and in 2015 it will be implemented in the curricula of 6 further institutions in the ePBLnet consortium, a European Commission-funded program  implemenWith successful implementation of the D-PBL model, our attention has turned to where the decision-making capabilities of virtual patients in PBL can be further improved. There are 3 Web-based developments in this issue that describe alternative approaches, each of which has the potential to add to the model we describe here. Kononowicz et al  describeAntoniou et al  considerSalminen et al  took a dAll 3 of these interventions have been received favorably by students. The model we have described in this study has relatively low resource implications and further work is needed to establish which interventions can be widely introduced to achieve improved pedagogic value at reasonable cost.This study investigated the efficacy of D-PBL, a variant form of Web-based PBL that replaced linear PBL cases with branched virtual patients to present medical learners with alternative patient management decisions and having made a decision to deal with the consequences. Learners were encouraged to consider and discuss courses of action before taking them and to explore the consequences of their actions once they had been taken. Efficacy was measured in exam performance after a semester of weekly D-PBL sessions. It was found that D-PBL led to statistically significant improvement in student performance on key questions linked to the D-PBL process.If the promise of our findings are borne out and D-PBL proves to be a more efficacious way of structuring learning, at least for students who have already had a year or more of traditional PBL, then this has the possibility of being a major contribution to medical education. Although our findings are provisional pending further studies in and around the D-PBL model, we propose Web-based D-PBL as a candidate activity model for improving medical education through the inclusion of structured debate and decision making in small-group learning.


Metabolic reprogramming is commonly found in cancer but it is poorly understood in melanoma. Recent works ,2 providWe studied new cell lines extracted from sporadic metastatic melanomas hmel1, M3, Mba72) and primary melanomas , genotyped for genes involved in melanoma development compared to control melanoma cell lines  wt for MC1R and BRAF genes. Hmel1, hmel9 and hmel11 have already been described in Zanna et al., 2011 , Mba72 a. We evalThe HBL and LND1 cell lines, wt for BRAF, highly express PGC1alpha while hmel1, hmel9, hmel11, Mba72, M3, presenting BRAF mutations at the V600 residue, show a downregulation of this gene. MITF expression levels were more abundant in HBL and LND1 cell lines with respect to the other cell lines harbouring BRAF mutations. There is a direct correspondence between PGC1alpha and MITF levels: higher levels of PGC1alpha are associated with an enhanced MITF quantity. The analysis of cAMP levels in our melanoma cell lines showed a similar trend, being higher in wt BRAF cell lines compared to the other cell lines.Our data confirm the key role of BRAF mutations, MITF and cAMP levels in melanoma biology, suggesting a very important association with the transcriptional co-activator PGC1alpha, involved in energy metabolism and in mitochondrial biogenesis but also in various physiological stimuli that are reprogrammed in melanoma cells. These data support the divergent pathways hypothesis for melanoma, which may require a reappraisal of targeted cancer prevention and target therapeutic activities.


The antimetabolite 6-thioguanine (6-TG) has been used to treat both human and canine lymphoid malignancies. 6-TG has been shown to be epigenetically active as a demethylating agent in a human lymphoma cell line, causing downregulation of DNA methyltransferase 1 (DNMT1) through ubiquitin-targeted degradation. Zebularine (Zeb), a similar cytidine analog, also has demethylating activity as well as oral bioavailability. The hypothesis of the present study was that 6-TG and Zeb would cause downregulation of DNMT1 and globally demethylate the genomic DNA of canine lymphoma cells. The secondary hypothesis was that these agents would cause a dose-dependent decrease in cell proliferation in canine lymphoma cells. Canine CLGL-90 malignant T cells and CLL 17–7 cells were incubated in modified RPMI media. They were treated with 6-TG, Zeb, or control media at biologically relevant concentrations.Following treatment with each agent, DNMT1 protein and global DNA methylation were significantly decreased. A dose-dependent decrease in cell survival was also observed, with apoptosis being the primary mode of cell death in the CLGL-90 cell line.These results confirm the demethylating action of 6-TG and Zeb in canine cells which is similar to that shown in human cell lines. Confirmation of this mechanism supports the clinical application of these compounds as demethylating drugs in veterinary patients. Lymphoma is the most commonly diagnosed hemo-lymphatic neoplasm of the dog . Little in vitro evaluation of its effect on global methylation on canine lymphoma cell lines. Zebularine (Zeb), a similar antimetabolite, also has been used in vitro and in vivo [Demethylating agents have previously been used for human and canine hematopoietic malignancies -15, with in vivo -24 and hin vivo [Two cell lines, a canine large T cell leukemia line CLGL-90 and a canine B cell line CLL 17–7  were groin vivo ,26. CellProtein was extracted from the same samples using MPER reagent (Thermo Fisher). Protein concentrations were measured (Nanodrop spectrophotometer) and samples were stored in −20°C. Equal amounts of protein (approximately 30 ug) extracted from the cell lines were separated by SDS–PAGE and blotted onto a PVDF membrane as described previously . MembranHpaII or MspI. HpaII is unable to cleave DNA at its restriction site (CCGG) when cytosine is methylated, as it is a methylation-sensitive restriction endonuclease. However, its isochizomer MspI is able to cleave the restriction site regardless of cytosine methylation status. As DNA becomes hypomethylated, HpaII cleaves the strands more completely, resulting in smaller restriction fragments. This results in a change in intensity of the bands on DNA gel electrophoresis optical analysis. DNA (200 ng) was incubated with HpaII (New England BioLabs) or MspI (New England BioLabs) along with supplied NE Buffer and nuclease-free water for one hour at 37°C. After incubation, samples were electrophoresed on a 1% agarose gel at approximately 100 V/h. Comparison of the restriction enzymes’ (HpaII vs. MspI) banding patterns was used to determine alterations in the global methylation status of the DNA. Methylation score values (MSVs) were calculated for the samples [DNA was extracted using a DNA extraction kit . DNA concentrations were measured  and samples were immediately used or stored at −20°C. Global methylation was analyzed using restriction landmark genomic scanning (RLGS) as previously described . RLGS pr samples . The gelCLGL-90 cells  were groApoptosis assay was performed using Apo3/7 HTS assay kit according to the manufacturer’s  instructions. 15,000 cells were loaded per well in replicates of six for each treatment  in 50 μl media, followed by addition of the apo HTS assay reagent containing quenched (z-DEVD)2-R110 dye substrate (50 μl). This was incubated for 1 h at 37°C. Active caspase 3/7 R110 cleaved the free dye, which excites at 488 nm, and the emission at 520 nm was read using a fluorescent plate reader.For cell survival analysis, treatment groups were compared by ANOVA  with Holm-Sidak pair-wise comparison. One-way ANOVA was also used to compare methylation status. P-values less than or equal to 0.05 were considered significant.The described experiments were conducted using existing, immortalized canine cell lines. No humans or vertebrate animals were used in the described studies.To determine whether DNMT1 expression is reduced by 6-TG and Zeb treatment of canine cells, western blot analysis was performed. DNMT1 was reduced when treated with 6-TG and Zeb in both cell lines  or MspI (methylation unrestricted). Analysis of the resulting electrophoresis gels demonstrated significant decreases (P < 0.001) in global methylation following treatment with 6-TG and Zeb , 1.5, 3.0, 6.0 μM 6-TG or 50, 100, 200 μM Zeb at time-points 0 and 24 hours. Cells were counted at 24 and 48 hours. There was a decrease in cell viability compared to control following exposure to Zeb at the highest concentration (P = 0.026)   activation, which could lead to cell cycle arrest of tumor cells. It is also unclear whether proto-oncogenes could become activated, promoting cells to enter the cell cycle. A potential pit-fall of this treatment could be tumor promotion if previously silenced pro-survival, anti-apoptotic, or other “aggressive” genes are awakened.in vitro [In vivo effects of Zeb in dogs have yet to be published, although preliminary evaluation of in vivo canine safety has been reported .Because 6-TG is a purine antimetabolite, with activity against lymphoproliferative diseases ,28, we iin vitro . Howeverin vitro  with a sSMAD1, in the family of transforming growth factor-beta, was shown to be hypermethylated and silenced in the resistant lymphoma cell lines. Treatment with decitabine caused reactivation of SMAD1, and re-sensitization to doxorubicin in vitro. This treatment (decitabine priming followed by standard chemotherapy) was then administered to human patients, and SMAD1 demethylation was shown in lymph node biopsies. The phase I clinical trial was safely administered and showed encouraging complete responses [Our group has begun to investigate the strategy of priming with demethylation therapy immediately prior to chemotherapy in the relapse-resistant setting. A similar approach has been investigated in a mouse model of human diffuse large B cell lymphoma, giving decitabine prior to doxorubicin and CHOP chemotherapy for synergistic effect . In immuesponses .in vitro. The cell lines selected are traditional canine lymphoma cell lines, but were recently re-characterized as lymphoid leukemia cell lines [ABCB1 gene associated with multi-drug resistance was hypomethylated in the drug-resistant cell lines [While this research is encouraging for future canine lymphoma treatment, it does have its limitations. All work reported was ll lines . Our groll lines . This fiWhile our research investigates canine lymphoproliferative disorders, human clinical trials have shown significant clinical responses in patients with other, resistant myeloproliferative disorders (MPD) . While tin vivo research to determine whether these compounds could improve response to therapy in dogs with resistant lymphoma.These results confirm the demethylating action of 6-TG and Zeb in canine cells, confirm a similar demethylating mechanism as that shown in human cell lines, and support the need for