BCR-ABL1 translocation occurs in chronic myeloid leukemia (CML) and in 25% of cases with acute lymphoblastic leukemia (ALL). The advent of tyrosine kinase inhibitors (TKI) has fundamentally changed the treatment of CML. However, TKI are not equally effective for treating ALL. Furthermore, de novo or secondary TKI-resistance is a significant problem in CML. We screened a panel of BCR-ABL1 positive ALL and CML cell lines to find models for imatinib-resistance.The BCR-ABL1 positive cell lines were resistant to imatinib-induced apoptosis . None of the resistant cell lines carried mutations in the kinase domain of BCR-ABL1 and all showed resistance to second generation TKI, nilotinib or dasatinib. STAT5, ERK1/2 and the ribosomal S6 protein (RPS6) are BCR-ABL1 downstream effectors, and all three proteins are dephosphorylated by imatinib in sensitive cell lines. TKI-resistant phosphorylation of RPS6, but responsiveness as regards JAK/STAT5 and ERK1/2 signalling were characteristic for resistant cell lines. PI3K pathway inhibitors effected dephosphorylation of RPS6 in imatinib-resistant cell lines suggesting that an oncogene other than BCR-ABL1 might be responsible for activation of the PI3K/AKT1/mTOR pathway, which would explain the TKI resistance of these cells. We show that the TKI-resistant cell line KCL-22 carries a PI3Kα E545G mutation, a site critical for the constitutive activation of the PI3K/AKT1 pathway. Apoptosis in TKI-resistant cells could be induced by inhibition of AKT1, but not of mTOR.Five of 19 BCR-ABL1 or other molecular aberrations previously indicted in the context of imatinib-resistance. These cell lines are unique as they dephosphorylate ERK1/2 and STAT5 after treatment with imatinib, while PI3K/AKT1/mTOR activity remains unaffected. Inhibition of AKT1 leads to apoptosis in the imatinib-resistant cell lines. In conclusion, Ph+ cell lines show a form of imatinib-resistance attributable to constitutive activation of the PI3K/AKT1 pathway. Mutations in PIK3CA, as observed in cell line KCL-22, or PI3K activating oncogenes may undelie TKI-resistance in these cell lines.We introduce five Philadelphia-chromosome positive cell lines as TKI-resistance models. None of these cell lines carries mutations in the kinase domain of  ABL1 on chromosome 9 with BCR on chromosome 21, is the hallmark of chronic myeloid leukemia (CML), but is also found in 20-30% of acute lymphoblastic leukemia (ALL) cases. The development of clinically applicable tyrosine kinase inhibitors (TKI) has fundamentally changed the treatment of patients with CML: imatinib mesylate induces hematologic remission in nearly all CML patients -thymidine and annexin-V/propidium iodide (PI) assays to find models for TKI-resistance studies , MHH-TALL1 (1 μM), NALM-1 (> 10 μM), SD-1 (> 10 μM), and SUP-B15 (2 μM)  are effective in most BCR-ABL1 mutated cases, all 5 imatinib-insensitive cell lines identified here were also resistant to nilotinib suggesting that resistance might not be caused by BCR-ABL1 mutations (Table Point mutations in the kinase domain of e of CML . AlthougIkaros is found in the majority of BCR-ABL1-positive ALL and of CML in progression to lymphoid blast crisis -thymidine uptake, which was paralleled by an increase in the percentage of G1-phase cells  proved to be imatinib-responsive, inhibition of these pathways did not affect the viability of cells. In contrast to imatinib, wortmannin (PI3K inhibitor), OSU-03102 (PDK1 inhibitor) and rapamycin (mTOR inhibitor) inhibited the PI3K/AKT1/mTOR pathway, suggesting that the TKI-resistance observed in the Ph+ cell lines might be caused by a PI3K-activating oncogene other than BCR-ABL1 itself  -thymidine incorporation data suggested that PI3Kα (PIK3CA), but not PI3K β or PI3Kγ play a role in the imatinib-resistance of the cell lines tested  or in the kinase domain (exon 21) of the gene -thymidine incorporation were executed as follows: 1.25 × 104 cells (in 100 μl) were seeded in triplicate in 96-well flat-bottom microtiter plates. Inhibitors were added as 2x concentrated solution in a 100 μl volume. For the last 3 h of the incubation period, 1 μCi [3H]-thymidine  was added to each well. Apoptotic cells were detected and quantified with the annexin-V/PI method using the TACS Annexin-V-FITC kit  according to the manufacturer's instructions. Binding of fluorescein isothiocyanate-labeled annexin-V and PI staining of the cells was determined by flow cytometry on the FACSCalibur . For cell cycle analysis, cells were fixed with 70% ethanol , washed with phosphate-buffered saline, and stained with PI (20 μg/ml). DNA content of the cells was determined by flow cytometry.Assays of -thymidine uptake analysis, MZ performed quantitative real-time PCR analysis and flow-cytometry, HGD provided cell lines and critically read the manuscript. All authors read and approved the final manuscript.HQ designed the study and wrote the manuscript, SE participated in data analysis, JR performed Western blot analysis and [Leibniz-Institute DSMZ - German Collection of Microorganisms and Cell Cultures, Braunschweig, GermanyIKZF1 in cell line NALM-1Deletion of . Quantitative genomic PCR confirmed loss of the genes IKZF1 and DDC, located between ELMO-1 and ADAM-22 at chromosome 7p12.2. The cytogenetically-verified diploid B-lymphoblastoid cell line NC-NC was used as reference, the repetitive element LINE1 was used as endogenous control. Cell lines SD-1, SUP-B15 and MHH-TALL1 did not show loss of IKZF1 according to quantitative PCR.Click here for filePhosphorylation levels of SRC, STAT5 and RPS6 in TKI-sensitive (JURL-MK2) and -resistant (SUP-B15) cell lines. Cell lines were treated for 3 h with the BCR-ABL1 and SRC kinase inhibitor dasatinib (20 nM) or with the SRC kinase inhibitor SU-6656 (2 μM) or control. Phosphorylation of the SRC kinases Fyn and Lyn, of STAT5 and RPS6 was determined by Western blot analysis.Click here for filePhosphorylation levels of RPS6 in TKI-sensitive and -resistant cell lines. Cell lines were treated for 3 h with imatinib (1 μM) and/or Akt inhibitor IV (1 μM). Phosphorylation of RPS6 was determined by Western blot analysis. Note that RPS6 was dephosphorylated with Akt inhibitor IV in both cell lines.Click here for filePhosphorylation levels of STAT5, ERK1/2 and RPS6 in TKI- resistant cell line SUP-B15. Cell line SUP-B15 was treated for 3 h with/without imatinib (1 μM), Raf kinase inhibitor I (100 nM), OSU-03012 (20 μM), wortmannin (1 μM) and rapamycin (10 nM). Phosphorylation of STAT5, ERK1/2 and RPS6 was determined by Western blot analysis. Note that RPS6 was dephosphorylated with all three inhibitors of the PI3K/mTOR pathway.Click here for file


The Anaplastic Lymphoma Kinase (ALK) is an orphan receptor tyrosine kinase, which undergoes post-translational N-linked glycosylation. The catalytic domain of ALK was originally identified in the t translocation that produces the unglycosylated oncogenic protein NPM-ALK, which occurs in Anaplastic Large Cell Lymphoma (ALCL). Recently, both germline and somatic activating missense mutations of ALK have been identified in neuroblastoma (NB), a pediatric cancer arising from neural crest cells. Moreover, we previously reported that ALK expression is significantly upregulated in advanced/metastatic NB. We hypothesized that ALK function may depend on N-linked glycosylation and that disruption of this post-translational modification would impair ALK activation, regardless the presence of either gene mutations or overexpression.We employed tunicamycin to inhibit N-linked glycosylation. The following ALK-positive NB cell lines were used: SH-SY5Y and KELLY (ALK mutation F1174L), UKF-NB3 (ALK mutation R1275Q) and NB1 (ALK amplification). As a control, we used the NB cell lines LA1-5S and NB5 (no ALK expression), and the ALCL cell line SU-DHL1 (NPM-ALK).Tunicamycin treatment of ALK-positive NB cells resulted in a hypoglycosylated ALK band and in decreased amounts of mature full size receptor. Concomitantly, we observed a marked reduction of mature ALK phosphorylation. On the contrary, tunicamycin had no effects on NPM-ALK phosphorylation in SU-DHL1 cells. Moreover, phosphorylation levels of ALK downstream effectors  were clearly impaired only in ALK mutated/amplified NB cell lines, whereas no significant reduction was observed in both ALK-negative and NPM-ALK-positive cell lines. Furthermore, inhibition of N-linked glycosylation considerably impaired cell viability only of ALK mutated/amplified NB cells. Finally, the cleavage of the Poly-ADP-ribose-polymerase (PARP) suggested that apoptotic pathways may be involved in cell death.In this study we showed that inhibition of N-linked glycosylation affects ALK phosphorylation and disrupts downstream pro-survival signaling, indicating that inhibition of this post-translational modification may be a promising therapeutic approach. However, as tunicamycin is not a likely candidate for clinical use other approaches to alter N-linked glycosylation need to be explored. Future studies will assess whether the efficacy in inhibiting ALK activity might be enhanced by the combination of ALK specific small molecule and N-linked glycosylation inhibitors. Neuroblastoma (NB) is a pediatric cancer that arises from neural crest cells committed to the adrenal medulla and the sympathetic nervous system. NB accounts for 7-10% of all childhood cancers and the disease prevalence is about one case in 7,000 live births . In addiALK is an orphan trans-membrane receptor tyrosine kinase (RTK), which belongs to the insulin receptor superfamily of RTKs. The catalytic domain of ALK was originally identified in the t chromosomal translocation that produces the unglycosylated oncogenic fusion protein NPM-ALK, which occurs in Anaplastic Large Cell Lymphoma (ALCL) . ALK is N-linked glycosylation is a highly regulated post-translational modification, which is involved in several biological processes such as protein folding and conformation, oligomerization, sorting, cell-cell interactions, and targeting of proteins to sub- or extra-cellular locations ,11. MoreTunicamycin is a specific inhibitor of N-linked glycosylation that blocks the first step of glycoprotein synthesis, thus inhibiting the synthesis of all N-linked glycoproteins. Interestingly, tunicamycin was shown to impair the function of several RTKs such as EGFR, ErbB2, ErbB3, and IGF-IR .We hypothesized that, as demonstrated for other RTKs , ALK funThe following ALK-positive NB cell lines were employed: SH-SY5Y and KELLY (F1174L ALK mutation), UKF-NB3 (R1275Q ALK mutation) and NB1 (ALK amplified). As a control, we included LA1-5S and NB5 NB cell lines that are both ALK-negative, and the ALCL derived cell line SU-DHL1, which harbors the fusion protein NPM-ALK. Human NB cell line SH-SY5Y was purchased from the Biological Bank and Cell Factory core facility  and the LA1-5S cell line was purchased from the European Collection of Cell Cultures (ECACC). UKF-NB3, NB1, NB5, KELLY and SU-DHL1 cell lines were kindly provided by the Pediatric Oncology Unit . All cell lines employed in this study have been rechecked for ALK mutations, alterations and expression levels of the protein. LA1-5S were grown in MEM/Ham's F12 1:1 supplemented with non-essential amino acids and 10% inactivated fetal bovine . All other cell lines were grown in RPMI 1,640 supplemented with 10% inactivated fetal bovine serum (Lonza). Tunicamycin was purchased from Sigma-Aldrich .A standard immunoblotting protocol was used to assay protein expression in cell lysates. Cells were washed twice with ice-cold PBS and lysed with 100-200 μl of lysis buffer , supplemented with protease and phosphatase inhibitor cocktails and PMSF (phenylmethylsulfonyl fluoride) (Sigma-Aldrich). Lysates were vortexed and incubated on ice for 10 min twice and then cleared by spinning at 20,000 × g for 10 min at 4°C. Proteins were separated by 7.5%-10% SDS-PAGE gels and immunoblotted according to standard Western blotting procedures using primary antibodies for ALK, AKT, ERK1/2, STAT3 and related phosphoproteins , and either α-tubulin  or GAPDH  as a control for loading of equal amounts of cell lysates. Goat anti-rabbit and goat anti-mouse IgG HRP conjugated secondary antibodies were from Jackson Immunoresearch Inc  and Santa Cruz Biotechnologies Inc , respectively. Blots were developed with Amersham's ECL .Quantification of the western blot bands was performed by ImageJ .Six replicates of SH-SY5Y, KELLY, UKF-NB3, NB1 and SU-DHL1  and of LA1-5S  were plated in 96-well plates with 100 μl RPMI or MEM + Ham's F12 medium additioned with 10% fetal bovine serum. After 24 h cells were treated with decreasing concentrations of tunicamycin (500 nM - 200 nM - 100 nM - 50 nM - 25 nM), whereas no treatment was done as positive proliferation control. At 48 h, 25 μl of 2 mg/ml MTT (Sigma-Aldrich) was added and plates incubated for 4 h at 37°C and centrifuged 10 min at 1,500 rpm. Finally, 150 μl of DMSO were added to each well and absorbance was read at 560 nm.Ethanol fixed cells were incubated with 5 μl annexin-V FITC conjugate (Invitrogen) for 10 min at room temperature in the dark. Thereafter, cells were centrifuged, resuspended in 190 μl binding buffer and additioned with 10 μl propidium iodide (PI) (Sigma-Aldrich). Cells were analyzed immediately for Annexin-V-FITC and PI binding, using a FACS (fluorescence activated cell sorting) flow cytometer (CyAn™ Dako). Dot plots and histograms were analyzed by the Dako CyAn - Summit 4.3 software. Annexin-V and PI positive cells were considered apoptotic. Annexin-V and PI negative cells were considered viable.2 incubator at 37°C and 5% CO2 saturation. Dynamic cell proliferation was monitored in real-time from the time of plating until the end of the experiment. Electrode impedance at the bottom of each well was displayed as cell index, which is a direct measure of cell adhesion and it is a measure that indicates a change in cell behavior due to an increase or decrease in cell number, cell adhesion or cell morphology. Adherent NB cell lines were firstly tested to determine the optimal number of cells to be plated, and cells were allowed to reach a cell index of at least 0.5-1 before treatment. Exponentially growing cells were treated in duplicate with decreasing concentrations of tunicamycin . No treatment was used as positive proliferation control. Cell index values were acquired in real-time for at least 48 h after treatment. Cell index curves were calculated for every tunicamycin treatment and plotted on a graph by Excel.SH-SY5Y, KELLY, NB1, LA1-5S , and UKF-NB3  cells were seeded in E-Plates 16  in appropriate culture media to a final volume of 200 μl and maintained in a COTo investigate the effect that inhibition of N-linked glycosylation could have on ALK phosphorylation and downstream signaling we treated cells with 500 nM tunicamycin, a concentration previously employed in other cell lines of different origin , in timeWestern blot analysis of protein levels showed the accumulation of a hypoglycosylated ALK band 180 kDa) and a concomitant decreased of the amount of mature full size receptor (220 kDa) . Tunicamycin treatment did not affect AKT, ERK1/2 and STAT3 expression , which monitors cell adhesion in real-time Figure . As a reFinally, cell lines were analyzed by flow cytometry after double staining with annexin-V and PI. After 48 h of treatment with 500 nM tunicamycin, SH-SY5Y, KELLY, UKF-NB3, and NB1 cells showed a cell viability reduction of 27%, 48%, 42%, and 71%, respectively, whereas no significant variation was observed in both LA1-5S and SU-DHL1 cells Figure . Moreove50 greatly lower than the tunicamycin concentration used in the first set of experiments, we employed 50 nM tunicamycin in ALK-positive NB cell lines. As a result, we observed similar effects on ALK and its effectors in NB1 and UKF-NB3 . Cells were exposed to 500 nM tunicamycin for 48 h. We did not detect cleavage fragments in any of the analyzed caspases. On the contrary, we observed cleavage fragments of PARP in all ALK-positive NB cell lines but not in control cell lines Figure . CleavagIn the present study, we addressed the question of whether ALK function may depend on N-linked glycosylation. Inhibition of ALK activity in NB cell lines has already been approached by using specific small molecule ALK inhibitors, such as PF-2341066 , and morin vivo and increase radiosensitivity of gliomas, suggesting that targeting N-linked glycosylation may combine favorably with other classes of EGFR inhibitors to reduce both oncogenic signaling and the mechanisms of therapeutic resistance [In 2008, Contessa and colleagues reported that inhibition of N-linked glycosylation reduced RTK  signaling through AKT and radiosensitized tumor cells in glioma and pancreatic adenocarcinoma cell lines. In comparison, experiments carried out in nontransformed fibroblastic cells showed neither a reduction in RTK dependent signaling nor an enhancement in radiosensitivity . Afterwasistance . Moreovesistance .Similarly, our results indicated that N-linked glycosylation has a pivotal role also in ALK activation, as described for EGFR and other RTKs . TunicamInhibition of N-linked glycosylation also impaired cell proliferation only of ALK-positive NB cells. Particularly, UKF-NB3 and NB1 cells showed high sensitivity to tunicamycin by MTT assay, whereas SH-SY5Y and KELLY cells were less sensitive. However, cell viability assays carried out by flow cytometry and real-time monitoring of cell adhesion showed a marked cell growth reduction for all ALK-positive NB cell lines, including SH-SY5Y and KELLY. Finally, the cleavage of the Poly-ADP-ribose-polymerase (PARP) was observed only in ALK-positive NB cell lines, suggesting that apoptotic pathways may be involved in cell death.In this study we showed that the inhibition of N-linked glycosylation has the capability to affect ALK phosphorylation and disrupt pro-survival signaling associated to ALK, indicating that inhibition of this post-translational modification may be a promising therapeutic approach for ALK-depending NB patients. Moreover, the contributions of coexpressed growth factor receptors to developing resistance to therapeutic RTK inhibitors have been shown, confirming that inhibition of multiple RTKs may be of therapeutic benefit. Thus, as tunicamycin acts on all N-linked glycoproteins it is plausible to suppose that, differently from specific small molecule inhibitors, disruption of N-linked glycosylation should reduce a reliance on networks of RTKs that may overlap in effects with ALK.Since tunicamycin is not a likely candidate for clinical use our results are to be taken as a proof-of-concept and other approaches to alter N-linked glycosylation need to be explored. Future studies will assess whether the efficacy in inhibiting ALK activity might be enhanced by the combination of ALK specific small molecule and N-linked glycosylation inhibitors.The authors declare that they have no competing interests.FDG designed and performed experiments, interpreted results. MDM performed experiments. LP performed experiments and critically revised the manuscript. RL critically revised the manuscript. GPT critically revised the manuscript. LL conceived of the study, designed experiments, interpreted results, drafted and critically revised the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/11/525/prepub


Virus can replicate in several common cell lines, sometimes without cytopathic effect. Given the potential for laboratory-associated severe acute respiratory syndrome–associated coronavirus (SARS-CoV) infections, we must know which cell lines are susceptible to the virus. We investigated 21 cell lines routinely used for virus isolation or research. After infection with SARS-CoV, cells were observed for cytopathic effects, and quantitative real-time polymerase chain reaction was used to measure ongoing viral replication. An indirect immunofluorescence assay was also used as a confirmatory test. The study identified 10 new cell lines capable of supporting the replication of SARS-CoV and confirmed the susceptibility of 4 cell lines previously reported. This study shows that SARS-CoV can be isolated in several cell lines commonly used for diagnostic or research purposes. It also shows that SARS-CoV can achieve high titers in several cell lines, sometimes in the absence of specific cytopathic effects. Cells were tested for virus-specific antigens with an indirect immunofluorescence assay 11 days after infection with SARS-CoV if no CPE was observed (or when CPE developed that involved at least 75% of the cell monolayer). Eleven days after infection, cell lines negative for indicators of viral replication were blind-passaged twice for 7 days by adding 100 μL culture supernatant to the cells in question as well as to the highly susceptible Vero E6 cells. During these passages, cells were observed for SARS-CoV–specific CPE, and after the second passage, supernatants were collected for virus detection and quantification by PCR.260 U/mL random hexamers (Roche Diagnostics), 0.17 μmol/L deoxynucleoside triphosphates (Roche Diagnostics), and 7.5 U AMV-RT enzyme . After incubation at 42°C for 30 min, then 100°C for 10 min, cDNA products were stored at 4°C until analyzed by PCR.A 300-μL volume of lysis buffer containing guanidinium thiocyanate and Triton X-100  was added to 200 μL supernatant from cell cultures that had either been infected with SARS-CoV or were mock-infected. These samples were removed from the PC4 laboratory to a PC2 laboratory, where they underwent nucleic acid extraction with a MagNA Pure LC Total Nucleic Acid Isolation Kit with a MagNA Pure LC automated extraction robot (Roche Diagnostics). A 10-μL volume of eluate was treated for 10 min at 65°C and added to 12 μL reverse transcription master mix containing 5.2 A10.Real-time PCR that amplified an 81-bp fragment of the nucleoprotein gene was used to detect and quantify SARS-CoV by reference to a cycle threshold (Ct). The assay used ABI-7000 Prism instrumentation  with primers and probes designed with the associated Primer Express software. The forward primer was SARNP-F: 5´-CCC AGA TGG TAC TTC TAT TAC CTA GGA-3´. The reverse primer was SARNP-R: 5´-CCA TAC GAT GCC TTC TTT GTT AG-3´. The probe was SARNP-P: 6FAM 5´-AAG CTT CAC TTC CCT ACG G-3´ with 3´ MGB. For real-time PCR, 5 μL template cDNA was added to ABI TaqMan Universal PCR Master Mix (Applied Biosystems) containing 0.9 μmol/L each primer and 0.2 μmol/L probe in a total volume of 45 μL. The cycling conditions were as follows: 2 min at 50°C, 10 min at 95°C, then 45 cycles of 15 s at 95°C and 1 min at 60°C. Reference to a standard curve (not shown) demonstrated that negative changes in Ct values of 3.6 represented increases in virus titer of 1.0 log6 50% tissue culture infectious doses (results not shown). A 10-μL volume of diluted convalescent-phase serum from a SARS-CoV–infected patient was added to the fixed cells followed by incubation at 37°C for 30 min in a humidified chamber. The slides were washed twice with phosphate-buffered saline (PBS), dried, and each cell spot overlaid with 10 μL anti-human fluorescein isothiocyanate–conjugated secondary antibody  for 30 min at 37°C. The slides were washed twice with PBS before they were mounted with cover slips. Virus-specific immunofluorescence was read by using an Axioskop UV microscope . The final results for the indirect immunofluorescence assay, as shown in the Table, were based on the observations of 2 independent readers.Cells were collected 11 days after infection if no CPE was observed by microscopy or on the day they developed CPE involving at least 75% of the cell monolayer. Cells were manually scraped from monolayers into 1 mL culture medium, then subjected to 50 kGy gamma radiation before being spotted onto a slide, air dried, and fixed in acetone for 10 min. Earlier testing showed that this dose of gamma radiation reduced the titer of SARS-CoV by at least 10Susceptibilities to SARS-CoV of the cell lines we investigated are shown in the SARS-CoV replication in BGM, CV-1, FRhK, LLC-Mk2, MA-104, pCMK, RK-13, and Vero cell lines produced a CPE as early as day 4 after inoculation, with evidence of high levels of virus-specific RNA established by quantitative PCR. CPE was focal, with cell rounding and a refractivity that was soon followed by cell detachment, and CPE quickly spread to involve the entire cell monolayer . In cont10/mL above the input virus for these cells. The results for HeLa-T, Hep-2, and MDCK cells, representing cell lines that do not support SARS-CoV replication, are shown in –After the SARS epidemic ended, several cases have occurred as a direct or indirect result of breaches in laboratory biosafety , is highly expressed in kidney tissue (Generally, close agreement was seen between our results and those previously reported . NeitherGiven that primate kidney–derived cell lines are particularly susceptible to infection with SARS-CoV and virus has been isolated from the kidney of an infected human patient (This study has shown that SARS-CoV can be isolated in several cell lines commonly used for diagnostic and research purposes and highlights that the virus can achieve high titers in some cell lines, sometimes in the absence of CPE. These findings are particularly relevant to laboratory scientists undertaking virus-isolation procedures on specimens collected from patients with atypical respiratory disease or in research laboratories where the possibility of simultaneously handling more than 1 virus exists.


Genomic integrity of human pluripotent stem cell (hPSC) lines requires routine monitoring. We report here that novel karyotyping assay, utilizing bead-bound bacterial artificial chromosome probes, provides a fast and easy tool for detection of chromosomal abnormalities in hPSC lines. The analysis can be performed from low amounts of DNA isolated from whole cell pools with simple data analysis interface. The method enables routine screening of stem cell lines in a cost-efficient high-throughput manner. ► We report a novel high-throughput karyotyping assay for human pluripotent cells. ► This assay provides fast and easy tool for detection of chromosomal abnormalities. ► The analysis can be run from low DNA amounts with simple data analysis interface. ► The results are in good concordance with the data from G-banding and SNP arrays. ► The assay enables routine screening of stem cell lines in a cost-efficient manner. Primary human embryonic stem cell (hESC) lines, as well as induced pluripotent stem cell (iPSC) lines, provide a valuable resource for developmental studies, disease modeling and development of regenerative therapies. Long term culture of hESC is associated with the accumulation of karyotypic abnormalities . Based oConventional karyotyping has been most commonly used to monitor the genomic status of the cells. The method enables early detection of abnormalities in heterogeneous populations. However, the analysis is usually limited to less than 50 mitosis instead of the whole cell population. Furthermore, conventional karyotyping is costly, slow and laborious hampering the frequency of the analysis. While array based methods enable high resolution analysis of the whole cell population, they are also expensive, slow and require specialized expertise for data analysis and interpretation. Furthermore, the complexity of the array data can be difficult to interpret, as for instance it is not clear which of the small scale aberrations detected in hESCs have been present already in the original blastocyst from which the cells have been derived. For routine and frequent monitoring of the hESC and iPSC karyotypes, fast, simple and inexpensive method would be highly beneficial for laboratories producing, maintaining and utilizing hESCs or iPSCs.KaryoLite™ BoBs™, developed by PerkinElmer, is a novel improved method for fast and easy molecular karyotyping based on BACs-on-Beads™ technology . The assTo compare the novel method to conventional G-banding and array based methods, we reanalyzed seven different hESC lines from our previous detailed genomic analysis  by NarvaTo compare the novel method to conventional G-banding and array based methods, we reanalyzed seven different hESC lines from our previous detailed genomic analysis  by NarvaAs expected, the analysis results did not indicate any large scale genomic abnormalities in the karyotypically normal hESC lines  . For theThese results highlight that the novel KaryoLite™ BoBs™ assay provides a valuable tool for routine screening of the recurrent and random chromosomal abnormalities observed in hESC lines. The method is most likely to be applicable for monitoring of iPSC lines as well, although these were not included in this study. The analysis with KaryoLite™ BoBs™ can be carried out in a 96-well format enabling scaling up the sample numbers. The data analysis interface is simple to use and the results are clear and easy to interpret. The sensitivity of the method may also enable detection of large scale karyotypic aberrations in mosaic populations. The benefit of the method in comparison to conventional karyotyping also includes examination of the whole cell population rather than fraction of the cells  providing improved representation of the genomic integrity of the whole cell population . The metThese results highlight that the novel KaryoLite™ BoBs™ assay provides a valuable tool for routine screening of the recurrent and random chromosomal abnormalities observed in hESC lines. The method is most likely to be applicable for monitoring of iPSC lines as well, although these were not included in this study. The analysis with KaryoLite™ BoBs™ can be carried out in a 96-well format enabling scaling up the sample numbers. The data analysis interface is simple to use and the results are clear and easy to interpret. The sensitivity of the method may also enable detection of large scale karyotypic aberrations in mosaic populations. The benefit of the method in comparison to conventional karyotyping also includes examination of the whole cell population rather than fraction of the cells  providing improved representation of the genomic integrity of the whole cell population (Supplementary Table 1). The method is not suitable for detecting balanced translocations or with current probe selection sensitive enough for small scale changes. However, most of the recurrent genomic changes in pluripotent cells involve whole chromosomes or chromosome arms . Also foWorkflow of the Karyolite Assay.Comparison of the three methods used to detect karyotypic abnormalities in this study.The following are the supplementary related to this article.http://dx.doi.org/10.1016/j.scr.2012.06.008.Supplementary related to this article can be found online at


Obtaining a germ cell line is one of the most important steps in developing a transgenic or knockout mouse with a targeted mutated gene of interest. A common problem with this technology is that embryonic stem (ES) cells often lack, or are extremely inefficient at, germ line transmission.in situ hybridization (FISH). Chromosomal abnormalities occurred in 44 (45%) out of the 97 specimens analyzed: 31 specimens had trisomy 8 or mosaic trisomy 8, eight specimens had partial trisomy 8 resulting from unbalanced translocations, and five specimens had other chromosomal anomalies.To determine whether chromosomal anomalies are correlated with inefficient ES cell germ line transmission, we examined 97 constructed ES cell lines using conventional cytogenetic analysis, and fluorescence Our data suggest that chromosomal analysis is an important tool for improving the yield and quality of gene targeting experiments. Also, its sub-ES cell lines showed various karyotypic abnormalities.On average, the ES cells used for this study had undergone at least fifteen to twenty passages. For this reason, we conclude that extended culture Of the 11 sub-ES cell lines, two were normal, four were trisomy 8, one was mosaic trisomy 8, two were double trisomy with chromosomes 8 and 11 and one was derivative chromosome 14 resulting from an unbalanced translocation between chromosomes 8 and 14. The most interesting finding is that one of the sub-ES cell lines showed very complex numerical chromosome abnormalities, including the most common numerical abnormalities found in our study: 40,X,-Y,+8/40,X,-Y,+11/41,X,-Y,+6,+8/41,X,-Y,+6,+11/42,XY,+6,+8/42,XY,+6,+11/40, XY. We were not able to follow up on whether or not this cell line affected other ES cell lines.We have performed chromosomal analysis of 97 constructed ES cell lines. Chromosomal anomalies were seen in 44(45%) out of the specimens analyzed. We found trisomy 8 to be a common anomaly in the sub-ES cell lines identified from the original mouse strains. It is not uncommon that ES cells with trisomy 8 over-proliferate. This should serve as a warning for collaborative researchers attempting to maximize their limited resources by growing more and more ES cells before sharing their clones. Our findings indicate that before the injection of ES cells into blastocysts, karyotype analysis by conventional methods including FISH analysis is needed in order to ensure genome stability.All the experiments were performed on the established cell lines obtained from mice. No ethical issues is applied to this study.Ninety-seven ES cell lines obtained from various research teams at the University of Oklahoma Health Sciences Center and the Oklahoma Medical Research Foundation between 2000 and 2006. The ES cell lines were of multiple origins, obtained either from commercial or academic sources, with different gene constructs. We were able to examine the AB2.2, CJ7, GSI-1 and J1 ES cell lines. Some cell lines originated from one single source and were expanded in different laboratories for at least another five to ten passages (1:3 or 1:4 splits) in culture under varying conditions. Thus, the cells used for this study had all undergone at least fifteen to twenty passages.The standard procedures for harvesting, making slides and staining the cells were followed . ES cellThe whole chromosome painting probes (WCP) for chromosomes 8, 10, 14 and 17 were purchased from a commercial source  for the ES cell lines with complex chromosomal abnormalities. FISH analysis was performed according to the manufacturer’s instructions.in situ hybridization; PBS: Phosphate buffered saline; KCl: Potassium chloride; WCP: Whole chromosome painting probes.ES: Embryonic stem; FISH: Fluorescence The authors declare that they have no competing interests.YK collected and/or assembly of data analysis and interpretation. JL carried data analysis and interpretation. LX provided of study material. JM participated in final approval of manuscript. SL conceived of the study concept and design, and participated in data analysis and interpretation, and final approval of manuscript. All authors read and approved the final manuscript.


After we have conducted this work, it has been reported that one the cell lines used in our study (KU-7) was cross-contaminated with HeLa-cells. Nevertheless, the authors are very confident with our findings since all the experimental results were conducted with different cell lines in parallel with those obtained using KU-7. For instance, the baseline of PPARg and EGFR and the dose-response to PPARg agonists and EGFR inhibitor shown in figure 1, is represented in a panel of 9 bladder cancer cell lines. The effect of the combined therapy on proliferation, on EGFR downstream signaling, on p21 expression, on the induction of PPARg and CEBPb expression in response to different concentrations of Gefitinib,  respectively, were performed with the urothelial carcinoma cell line UM-UC13 as well.With regards to the in vivo experiment, similar results were obtained with theUM-UC13 cell line where tumor weights were only significantly reduced in the combination therapy as compared to the control group . There was no significant reduction in tumor weights with either drug alone when compared to placebo.Finally, one of our main results that shows that the efficacy of combination therapy can be improved in a schedule-specific manner was conducted with three different urothelial cell lines, UM-UC3, UM-UC6 and UM-UC13. Therefore, the report about the cross-contamination of KU-7 does not affect our findings. Our analysis is robust and dependent on several cell lines where KU7 is just one element of the overall experimental evidence.


Listeria monocytogenes is a gram-positive, foodborne bacterium responsible for disease in humans and animals. Listeriolysin O (LLO) is a required virulence factor for the pathogenic effects of L. monocytogenes. Bioinformatics revealed conserved putative epitopes of LLO that could be used to develop monoclonal antibodies against LLO. Continuous and discontinuous epitopes were located by using four different B-cell prediction algorithms. Three-dimensional molecular models were generated to more precisely characterize the predicted antigenicity of LLO. Domain 4 was predicted to contain five of eleven continuous epitopes. A large portion of domain 4 was also predicted to comprise discontinuous immunogenic epitopes. Domain 4 of LLO may serve as an immunogen for eliciting monoclonal antibodies that can be used to study the pathogenesis of L. monocytogenes as well as develop an inexpensive assay. Listeria monocytogenes, a gram-positive pathogenic bacterium [L. monocytogenes is the only species in the genus Listeria that can infect humans [L. monocytogenes that is incapable of secreting LLO (Δhly) is not pathogenic [Listeriosis is a foodborne illness caused by infection with acterium . L. monot humans . After tt humans . L. monothogenic . Listerithogenic . LLO is thogenic . LLO alsthogenic , as wellthogenic   have many functions. They can be used to study structure/function of a protein, pathogenesis of an organism, and/or quantitative analysis of a protein , 12. To vailable –14. Sitehttp://www.biocompare.com/pfu/110627/soids/371711/Assay_Kit/listeriolysin_O and http://www.mybiosource.com/datasheet.php?products_id=705426) [There are two companies that manufacture ELISAs (BioCompare and MyBioSource) to detect LLO (=705426) , 16. In http://www.ncbi.nlm.nih.gov/protein/) to acquire the Listeria proteins listeriolysin O (LLO) (NP_463733) and ivanolysin O (P31831) [http://www.rcsb.org) at http://www.ncbi.nlm.nih.gov/blast/Blast.cgi. Suilysin (pdb: 3HVN) [In this study we used the NCBI GenBank Protein database (P31831) . The acc31 . The http://www.ebi.ac.uk/Tools/msa/clustalw2/ [http://www.ebi.ac.uk/Tools/services/web/toolform.ebi?tool=emboss_needle&context=protein [Pairwise sequence alignments of LLO were generated using the ClustalW2 Multiple Sequence Alignment at ustalw2/ . The pur=protein .http://zhanglab.ccmb.med.umich.edu/I-TASSER/ [To generate a molecular model of LLO, we used the molecular modeling program I-TASSER -TASSER/ , 22. We -TASSER/ , 23, 24.-TASSER/ . The C-shttp://polyview.cchmc.org/polyview3d.html) [Amino acids 60–525 of LLO were used to predict a three-dimensional model since these residues correspond to amino acids 32–242 and 245–499 in SLY. Molecular models were prepared in different orientations using POLYVIEW 3D  also uses physicochemical properties to predict B-cell epitopes similar to BepiPred [Important properties for predicting B-cell epitopes are flexibility, hydrophilicity, and linear epitope predictions. We analyzed the linear epitope predictions, flexibility, and hydrophilicity of LLO using the BepiPred linear epitope prediction , KarplusBepiPred .Listeria species revealed that it is highly conserved with only six polymorphic sites (L. seeligeri) and ivanolysin O (L. ivanovii), respectively. In contrast, NCBI-BLASTn searches against Listeria species L. grayi, L. innocua, L. marthii genomes (members of the genus Listeria that have their genomes sequenced) yielded no LLO homologs. The genomes of species L. fleischmannii and L. rocourtiae have not been sequenced.An alignment of all completely sequenced amino acid coding sequences of LLO from several ic sites . Howeveric sites . We usedSince there is no LLO crystal structure, we created a molecular model of LLO to visualize the locations of the predicted B-cell epitopes . The SLYWe used amino acids 60–525 of LLO to predict a three-dimensional model. In contrast, we used amino acids 28–529 to analyze the immunogenicity of LLO since the latter amino acids comprise the full-length mature protein . Three dWe also evaluated discontinuous epitopes in the LLO molecular model that we created. ElliPro predicted three discontinuous epitopes with PI scores higher than our cutoff: two in domain 4 and one in domain 1 . DiscontIn terms of antibody recognition, changing one amino acid in an antibody epitope can dramatically decrease the antigen-antibody interaction . Four ofImmunogenic epitopes are accessible on the protein surface, located in flexible regions, and often overlap . Our preIn some instances, when a protein has multiple overlapping B-cell epitopes it is referred to as immunodominant . We predBioinformatics has been used for many purposes, such as vaccine design, characterization of novel genes, and the discovery of novel viruses –39. ReceSeveral characteristics make domain 4 an attractive immunogenic candidate. Domain 4 is the only continuous domain in LLO . Domain L. seeligeri and ivanolysin O expressed by L. ivanovii. However, L. seeligeri does not infect humans, and L. ivanovii is not pathogenic [L. seeligeri, it is unlikely that the latter toxins would be present in contaminated food products consumed by humans. Thus, even if they readily cross-react with LLO L. seeligeri and ivanolysin O, antibodies capable of detecting LLO in an ELISA would not be likely to produce a false positive in food screening or clinical tests. Previous molecular models of LLO used PFO as a template [Bioinformatics of LLO demonstrates that its closest homologs are the LLO protein of thogenic . Therefotemplate , 42. OurL. monocytogenes as well as develop a diagnostic test for LLO detection. This analysis is important because it is focusing our antibody development efforts on domain 4 as an immunogen.Bioinformatics of LLO predicted that most of the epitopes deemed likely to be immunogenic were located in domain 4. Furthermore, since it is the only domain in LLO that is continuous, we believe that it has a high probability of eliciting antibodies that could be used to study the pathogenesis of


It is currently in use in combination with cisplatin against malignant pleural mesothelioma and non-squamous non-small cell lung cancer with favourable results. By real-time RT-PCR gene expression assays and restoration viability assays we demonstrated that Pemetrexed targets folate-dependent enzymes involved in de novo biosynthesis of purines differently depending on the intrinsic genetic characteristics of the tumour. These differences did not, however, interfere either with the initial response to the drug or with the activation of apoptotic pathways. In addition, these genetic fingerprints can differentiate two groups of tumours: those capable of developing resistance to antifolate, and not capable. These results may be useful to employ targets gene expression as resistance markers, a valuable tool for identifying patients likely to receive combination therapy to prevent the development of resistance.Although treatment options for cancer patients are increasing every year, the drug resistance problem remains very present. It is very difficult to find a drug that acts equally on tumours of the same histology as the individual's genetic characteristics often determine the response to treatment. Furthermore, tumours that initially respond to anti-tumour therapy are able to adapt and develop resistance to the drug, while others do not. In addition, this usually implies resistance development to agents to which the cells have not been exposed, a phenomenon called cross-resistance or multidrug resistance. Given this situation, it has been suggested that the most appropriate treatment would be able to act in parallel on multiple pathways constitutively altered in tumour cells. Pemetrexed is a multitargeted antifolate that exerts its activity against folate-dependent enzymes involved in  There is increasing awareness of the importance of genetic background in individual susceptibility to cancer treatments. In human populations, multiple genetic parameters have been associated with response or resistance to chemotherapeutic agents: SNPs, gene copy number or gene expression. Interindividual variation in biological responses to anticancer drug could be a result of internal and external alterations. On one hand, the intrinsic characteristics of each tumour enable them to be sensitive or refractory to the agent employed. This is the case of inherent drug resistance to some antifolates where alterations of the reduced folate carrier (RFC) role results in the impaired drug uptake. In addition, cancer treatments are often administered cyclically to allow recovery of the patient. But this scheme also makes possible the tumour cell recovery and adaptation. The treatments do not always manage to eradicate all malignant cells, enabling tumour cells to adapt their genetic characteristics in order to achieve a survival advantage Both inherent and acquired resistance has become a major challenge for the oncologist. That is why combined agents and drugs having multiple targets are being used aimed to simultaneously act on multiple constitutively modified pathways that confer a survival advantage to tumor cells, and on key factors weakening the malignant cells to prevent the new resistance development.de novo biosynthesis of purines and pyrimidines. MTA acts as a potent inhibitor of thymidylate synthase (TS), and this enzyme is defined as its primary target Pemetrexed  is a multitargeted antifolate which inhibits folate-dependent enzymes involved in The adequate definition of MTA targets is a relevant point to better understand clinical responses to the drug and explore potential mechanisms of resistance development.A375, Hs294T, HT144 and MeWo human melanoma cell lines and the Calu-3 NSCLC cell line were obtained from the American Type Culture Collection . A375, HT144 and Hs294T were maintained in DMEM cell culture medium supplemented with 10% FBS  as recommended by the ATCC. MeWo melanoma and Calu-3 lung adenocarcinoma cell lines were maintained in Eagle Minimum Essential Medium  with 2 mM L-glutamine  and Earle's BSS adjusted to contain 1.5 g/L sodium bicarbonate, 0.1 mM sodium pyruvate and 10% FBS as described by the ATCC. MTA  was dissolved in phosphate buffered saline (PBS) at 2 mg/ml and stored at –80°C until use.2. As previously described For each cell line, 10,000 cells were plated in 96-well plate and allowed to adhere for 24 h at 37°C and 5% COThe mRNA was extracted from cell lines with the RNeasy Plus Mini Kit . Gene expression levels were measured in each sample by one-step real-time RT-PCR using human validated TaqMan Gene Expression Assays: GARFT (Hs00531926_m1), AICART (Hs00269671_m1), TS (Hs00426586_m1), FPGS (Hs00191956_m1), DHFR (Hs00758822_s1) and RFC (Hs00161870_m1) and the human RPLPO was used as endogenous control, all of them from Applied Biosystems. Three replicates were run for each sample in a 96-well and 200 ng RNA were used for each reaction. Results were analysed using the ΔΔCt method.In each case, 500,000 4% formalin-fixed cells were washed twice in 1×PBS and permeabilized with ice-cold 70% ethanol for 30 min. Cells were labelled with 25 µg/ml in 1×PBS of anti-PAkt at S473  for 30 min on ice. Cells were washed with 1×PBS and incubated with secondary antibody anti-rabbit-Alexa 488  for 30 min on ice and in the dark. Finally, cells were washed with 1×PBS and 5,000 cells were analyzed on a Cytomics FC 500 MPL cytometer (Beckman Coulter).MTA-treated and untreated human melanoma cells were harvested by trypsinization. Total cell lysate was isolated by resuspending pelleted cells on ice cold Igepal-Triton lysis buffer  containing protease and phosphatase inhibitors  followed by 30 minutes incubation on ice and vortexed every 10 min. Aliquots of lysate were resuspended in Laemmli sample buffer (Bio-Rad Laboratories), resolved by SDS-PAGE and transferred to PVDF membrane with the iBlot® Gel Transfer Device as described by the manufacturer (Invitrogen Life Technologies). The following antibodies were used in immunoblotting: anti-actin (1∶200) from Sigma, Ph-4E-BP1 at Thr37/46  mAB and horseradish peroxidase (HRP)-conjugated secondary antibodies .6 cells. After 24 hours, the medium was changed, and 20% of their respective IC50 of MTA was added. Thereafter, the medium was changed every 2 days, and when cell reached 80% confluence the MTA concentration was doubled. This was repeated gradually increasing the MTA concentration (up to 1.7 µM in the last cycle). If cell confluence fell to less than 20%, the medium was changed to drug-free medium or medium with the previous dosage. Resultant cell lines were designated as A375-M, Hs294T-M and MeWo-M.Tumour cells were plated at a density of 1.5×10XTT cell viability assays revealed that MTA-induced growth inhibition was not completely blocked by the preformed purine Hx in all cell lines , but incThe restoration of the purine biosynthesis was not, however, so homogeneous, responses to the assay varying between the cell lines studied . IncludiOn the other hand, either no effect or a mild increase in growth inhibition was obtained for HT144 and Calu-3. In these cases, the failure to restore the pathway suggests that MTA disrupts the pathway by targeting an enzyme downstream of AICA, i.e. AICART, regardless of its effect on GARFT.In order to assess a possible correlation between enzyme expression and the secondary target, the expression level of MTA-related enzymes as well as reduced folate carrier (RFC) and the folylpolyglutamate synthase enzyme (FPGS) were determined by real-time RT-PCR. Relative quantification of gene expression patterns by the ΔΔCt method revealed consistent differences between the six genes . The levOf the melanoma cell lines studied, the lowest level of expression of the main genes coding for MTA targets was found in HT144. This expression pattern also occurs in the NSCLC cell line Calu-3 where there is also a significantly lower expression of the main MTA target, TS.Given the patterns of expression obtained, two analyzes are possible. If we restrict the data to those of melanoma cells, it seems possible to distinguish two distinct groups: firstly, the lines  with high levels of expression of major drug targets TS, DHFR, and AICART GARFT and on the other hand, those lines (HT144) with low levels of expression of said enzymes.If we include in this analysis the NSCLC cell line, we can still clearly distinguishing these two groups, but it is possible to classify them based on a single marker: TS expression.Notably, both groups coincide with the groups identified in the previous restoration pathway assay, based on AICART or GARFT inhibition.Thus, there appears to be a clear correlation between expression levels of TS inhibition and AICART where: firstly, in cells with low TS, MTA inhibits AICART, while in those cells with high levels TS, MTA does not act on AICART, but on GARFT.The blockade of AICART by MTA can be expected to induce an accumulation of ZMP causing the inactivation of the mTOR signalling pathway through AMPK and Akt regulation, because, among other functions, Akt acts as regulator of the mTOR pathway and its inactivation represses the mTOR complex. Despite AICART inhibition not occurring in all cell lines, flow cytometry analysis revealed decreased levels of phosphorylated Akt in all cell lines after MTA treatment . This MTin vivo and in vitro. Results showed that after MTA exposure, p-4EBP1 protein levels fell ; this is a well known downstream substrate of mTOR which is widely used as indicator of mTOR activity both els fell , pointinels fell .in vitro as described in the Material and Methods section. After 3 months, we obtained MTA-resistant cells able to grow in the presence of 1.7 µM of MTA. Resistance was maintained over several freeze/thaw cycles and was transferred genetically to the subsequent generations. MTA-resistant cell lines were designated as A375-M, Hs294T-M and MeWo-M. On the other hand, we were unable to establish HT144-M (melanoma) or Calu3-M (NSCLC) cell lines; indeed, cells from these lines did not seem to develop resistance to MTA, pointing to a connection between MTA sensitivity, TS expression and/or AICART inhibition ; on the other hand, cell lines that can not . And anew, these two groups coincide perfectly with the two groups identified based on the antifolate secondary targets, and based on the expression levels of TS.Given these results, we can suspect that there is a relationship between the TS expression levels and the ability to acquire resistance to the antifolate: those cells with high TS expression levels are able to develop resistance to the drug after prolonged exposures, while those cells with low TS expression levels are unable to develop resistance to the drug. It is likely that this inability is because in these cells MTA acts on AICART, representing a new point of attack.Expression levels of MTA-related enzymes and the RFC in MTA-resistant cell lines were assessed by real-time RT-PCR and analysed by the ΔΔCt method, normalizing samples against RPLPO as the endogenous housekeeping gene and comparing results with those for the corresponding sensitive cell lines. TS levels increased in two of the three MTA-resistant cell lines, A375-M and Hs294T-M, while this enzyme seemed not to be particularly relevant in MeWo-M, where the most important changes were a rise in RFC levels and a strong down-regulation of the other three targets of MTA: AICART, DHFR and GARFT . In contMTA is a relatively new antifolate that is being used in clinical oncology in combination with cisplatin for the treatment of mesothelioma and non-squamous non-small cell lung cancer with encouraging results showing a improved safety profile when administered with vitamin B12 and folic acid Results of the present study show that the basic biochemistry of MTA seems to be common to all tumour cells despite small variations that do not affect its cytotoxicity The importance of which enzyme is the secondary target stems from the fact that AICA is phosphorylated by adenosine kinase to AICA ribotide, also known as ZMP, which mimics the effects of AMP in activating AMPK Therefore, AICA-mediated AMPK activation enables the inhibition of proliferation and cell cycle progression, via inhibition of both the TSC2/mTOR and PI3K/Akt pathways, and this prompts the up-regulation of p21, p27 and p53, resulting in cell cycle arrest In this study, differences in terms of the secondary targets of MTA did not seem to have impact on the inhibition of Akt phosphorylation, given that Akt phosphorylation decreased in all cell lines after MTA exposure. A similar observation was made in a previous study exploring the effect of the antifolate Methotrexate (MTX) on various human cancers. On the one hand, MTX was able to inhibit AICART, causing an increase in ZMP levels which potently sensitized tumour cells to apoptosis. At the same time, however, the inhibition of GARFT also induced an AICA riboside-induced ZMP accumulation, suggesting that the effects of GARFT inhibition may induce a more complex cascade related to the purine synthesis pathway Previous research We propose that MTA prompted the inhibition of TS, DHFR, GARFT and/or AICART and, consequently, induced oxidative stress and DNA damage leading to the up-regulation of p53 and cell cycle arrest In order to correlate differences in MTA secondary targets with the expression of related enzymes, basal mRNA expression levels of MTA-related enzymes  were assessed by one-step real-time RT-PCR. Interestingly, cell lines whose secondary target was AICART  had significantly lower TS expression. TS expression has been widely studied as a prognostic factor in MTA-treated tumours and has previously been related to the sensitivity of tumour cells to antifolates Likewise, this TS low expression seems to be a characteristic feature of those cell lines capable of acquiring drug resistance after exposure. As a result of our attempts to develop MTA-resistant cell lines, we obtained firstly, three well established resistant cell lines that were named: A375-M, Hs294T-M and MeWo-M; and secondly, a failure to develop resistance in HT144 or Calu-3 cell lines with the employed method. Assuming that the intrinsic genetic characteristics of the cells causing the tumour are what define the innate ability of the tumour to resist treatment, drug resistance has a significant hereditary component. Consistent with the aforementioned scenario, the intrinsic characteristics of the lines that are capable of developing resistance to MTA appeared to differ from those lines unable to develop such resistance, in terms of whether low basal expression of enzymes related to MTA was correlated with inhibition of AICART. This correlation was observed in both HT144 and Calu-3 and, moreover, these were the lines that were the most sensitive to MTA, in the sense that they were unable to develop drug resistance. It is therefore feasible that the inherent characteristics of each cell type have a significant role and, in this case, the element acted on is AICART. This is a positive factor, the action on AICART strengthening the effect of the antifolate by bringing a new point at which to block the synthesis of nucleotides and strength of activation of the AMPK route.Current treatment regimens are cyclical, and are based on allowing the patient's recovery from the effects caused by such aggressive treatment. However, these schemes also enable the adaptation of the cells to the agent to which the tumour is being treated thus favouring a resistance due to prolonged exposure to the drug. The genetic changes that tumour cells acquire during treatment serve to nurture and help develop resistance that is specific to each drug. Some genetic changes attributable to drug exposure were found in these new resistant strains . Specifically, the acquired changes were fundamentally an increased expression of TS, the main target of MTA, in A375-M and Hs294T-M, and a decreased expression of secondary targets in the case of MeWo-M. TS expression has previously been associated with sensitivity to MTA and is one of the main determinants of acquired resistance to antifolates in other tumour types such as lung cancer. In addition, patients with this disease that show low levels of TS expression have a better prognosis in vitro studies suggest it.It should be kept into account that the expression of DHFR, TS and GARFT are correlated with each other; furthermore, there is a link between TS and p53 status, as described in colon cancer. It has been found that patients with p53-wt have lower levels of TS expression and therefore a better prognosis; on the contrary, patients whose p53 is mutated (p53-mt) exhibit elevated levels of TS V600E, LKB1, p53, CDKN2A, PTEN and NF1 (We can conclude that MTA exerts its effects on a wide spectrum of tumours, independent of the presence of mutations in BRAF and NF1 . That MT


The MCF7 (ER+/HER2-), T47D (ER+/HER2-), BT474 (ER+/HER2+) and SKBR3 (ER-/HER2+) breast cancer cell lines are widely used in breast cancer research as paradigms of the luminal and HER2 phenotypes. Although they have been subjected to cytogenetic analysis, their chromosomal abnormalities have not been carefully characterized, and their differential cytogenetic profiles have not yet been established. In addition, techniques such as comparative genomic hybridization (CGH), microarray-based CGH and multiplex ligation-dependent probe amplification (MLPA) have described specific regions of gains, losses and amplifications of these cell lines; however, these techniques cannot detect balanced chromosomal rearrangements  or low frequency mosaicism.in situ hybridization, M-FISH). We detected previously unreported chromosomal changes and determined the content and frequency of chromosomal markers. MCF7 and T47D (ER+/HER2-) cells showed a less complex chromosomal make up, with more numerical than structural alterations, compared to BT474 and SKBR3 (HER2+) cells, which harbored the highest frequency of numerical and structural aberrations. Karyotype heterogeneity and clonality were determined by comparing all metaphases within and between the four cell lines by hierarchical clustering. The latter analysis identified five main clusters. One of these clusters was characterized by numerical chromosomal abnormalities common to all cell lines, and the other four clusters encompassed cell-specific chromosomal abnormalities. T47D and BT474 cells shared the most chromosomal abnormalities, some of which were shared with SKBR3 cells. MCF7 cells showed a chromosomal pattern that was markedly different from those of the other cell lines.A range of 19 to 26 metaphases of the MCF7, T47D, BT474 and SKBR3 cell lines was studied using conventional (G-banding) and molecular cytogenetic techniques (multi-color fluorescence Our study provides a comprehensive and specific characterization of complex chromosomal aberrations of MCF7, T47D, BT474 and SKBR3 cell lines.The chromosomal pattern of ER+/HER2- cells is less complex than that of ER+/HER2+ and ER-/HER2+ cells. These chromosomal abnormalities could influence the biologic and pharmacologic response of cells. Finally, although gene expression profiling and aCGH studies have classified these four cell lines as luminal, our results suggest that they are heterogeneous at the cytogenetic level. The MCF7, T47D, BT474 and SKBR3 breast cancer cell lines are commonly used in experimental studies of cellular function, and much of the current knowledge of molecular alterations in breast cancer has been obtained from these cell lines -4.Whole-genome studies using microarray expression analyses have identified distinct subtypes of breast carcinomas  based on the expression of approximately 500 genes  . These mAs determined by immunohistochemistry, the receptor profile classifies MCF7 and T47D cells (ER+/PR+/HER2-) as belonging to the luminal A subtype, BT474 cells (ER+/PR+/HER2+) as luminal B and SKBR3 cells (ER-/HER2+) as HER2 ,10. Howein situ hybridization (M-FISH) [Different works have assayed the DNA genetic profile of these cell lines using comparative genomic hybridization (CGH) and multiplex ligation-dependent probe amplification (MLPA) to describe many different copy number alterations -13. With(M-FISH) ,12-16. H(M-FISH) . Thus, tIn the present study, we evaluated structural and numerical alterations on a large number of metaphases of MCF7, T47D, BT474 and SKBR3 breast cancer cell lines using a combination of G-banding and M-FISH. This allowed us to analyze cell clonality within each cell line and to thoroughly compare the cytogenetic of the cell lines by clustering analysis.Between 19 and 26 metaphases with good chromosome dispersion and morphology were analyzed for each cell line to define the structural and numerical alterations, and 100 metaphases/cell line were analyzed to determine the level of ploidy. The rate and type of chromosomal abnormalities for each cell line are shown in Figure The cytogenetic analysis performed on 26 metaphases of MCF7 cells demonstrated a modal number hypertriploid to hypotetraploid (4n+/-) (76 to 88 chromosomes). Each chromosome harbored either a numerical or structural aberration, which accounted for 58 different rearrangements . Polyploidy was observed in 2% of the cells. Numerical alterations were present in all chromosomes; losses were more frequent than gains  constituted cluster 3 (green bar), and very rare alterations (ranging from 0 in metaphases M_21 and M_26 to 5 in metaphases M_13 and M_22) constituted cluster 4 (purple bar).In the T47D cells, 24 metaphases were examined. The modal number was near triploidy (3n+/-) (57 and 66 chromosomes). T47D cells had 52 different chromosomal alterations  Figure . PolyploAs in the MCF7 cells, the types of chromosomal alterations were almost homogeneously distributed among the 24 metaphases of T47D cells, as demonstrated by hierarchical clustering  (from 65 to 106 chromosomes) and showed 35 numerical and 36 structural aberrations : in cluster 1 (red bar), both numerical and structural alterations were present in almost all cells. Only three structural alterations were reproduced in all metaphases, namely der(6)tq25;q31), der(11)t and der  (76 to 83 chromosomes) karyotype. Polyploidy was observed in 19% of all cells. SKBR3 cells had 29 numerical and 33 structural aberrations , T47D (ER+/HER2-), BT474 (ER+/HER2+) and SKBR3 (ER-/HER2+) cell lines are widely used in breast cancer research as paradigms of the luminal and HER2 immunophenotypes ,10. Alth-, T47D  and that the chromosomal structural alterations were cell-type specific, with the exception of two derivative chromosomes that were shared by the BT474 and SKBR3 HER2+ cell lines.c-MYC on 8q24; HRAS, CD151, CTSD on 11p15; CCND1 on 11q13 [TOP2A on 17q21. Moreover, in HER2+ cells and carcinomas, rearrangements of chromosome 17 are more frequent than is polysomy. Pathologists must consider this observation for when diagnosing the HER2 amplification in interphase nuclei of breast carcinomas, which uses a ratio between HER2 copies and chromosome 17 centromere signals [The HER2+ cell lines BT474 and SKBR3 showed the highest frequency of numerical and structural aberrations in comparison with the HER2- cell lines MCF7 and T47D. Polyploidy, which was more frequent in HER2+ than in HER2- cells, has been correlated with short survival, drug resistance and metastasis . In addion 11q13 -24; and  signals ,26.. Conversely, T47D cells (ER+/HER2-) and BT474 cells share similarities in the chromosome profile, and both have some chromosomal similarities with SKBR3 cells. For example, T47D and BT474 cells share numerical alterations, such as losses of chromosome 6 and gains of chromosomes 11 and 20, but they have no structural abnormalities in common.Among ER + cells, MCF7 cells are cytogenetically different than both T47D (ER+/HER2-) and BT474 (ER+/HER2+) cells and are characterized by a specific subset of complex structural alterations, which are listed in the cluster analysis comparison of the four cell lines , T47D (ER+/HER2-), BT474 (ER+/HER2+) and SKBR3 (ER-/HER2+) were obtained from the American Type Culture Collection  in March 2010. Short tandem repeat (STR) analysis is routinely performed by ATCC during both accessioning and culture replenishment to avoid distributing misidentified cell lines to the scientific community. When received by our lab, these cell lines were expanded, and 3 vials were immediately frozen. Cells obtained from these stocks were used for the experiments. The cell lines were further authenticated based on the expression of epithelial markers (keratins 8 and 18) and the presence of specific receptors  using quantitative PCR (qPCR) and immunohistochemical analysis. The expression status of ERα and HER2 was further confirmed by western blot.The human breast cancer cell lines MCF7 , while BT474 cells were cultured in DMEM medium (Sigma). Culture media were supplemented with 10% fetal bovine serum (FBS) (Sigma), antibiotic-antimycotic solution (1X) (Sigma) and L-glutamine 2 mM) . The cultures were maintained in an incubator at 37°C and 5% CO2 and were determined to be free of contamination with mycoplasma by PCR assay. Cell line characteristics and culture conditions are further described in supplemental information . Briefly, colcemid solution (0.03 μg/ml) (Sigma) was added to cultures 2.5 hours (h) before cell harvesting; cells were then treated with hypotonic solution, fixed three times with Carnoy’s fixative (3:1 methanol to acetic acid) and spread on glass.Glass slides were baked at 70°C for 24 h, incubated in HCl and placed in 2xSSC buffer before treatment with Wright’s stain. Image acquisition and subsequent karyotyping of metaphases were performed using a Nikon microscope with the cytogenetic software CytoVision System . Chromosome aberrations were described according to the International System for Human Cytogenetic Nomenclature (ISCN) 2013 .M-FISH was performed with the aim of identifying complex chromosomal rearrangements. The probe cocktail containing 24 differentially labeled chromosome-specific painting probes  was denatured and hybridized to denatured tumor metaphase chromosomes according to the manufacturer’s protocol for the Human Multicolor FISH kit (MetaSystems). Briefly, the slides were incubated at 70°C in saline solution (2xSSC), denatured in NaOH, dehydrated in ethanol series, air-dried, covered with 10 μl of probe cocktail (denatured) and hybridized for two days at 37°C. The slides were then washed with post-hybridization buffers, dehydrated in ethanol series and counter-stained with 10 μl of DAPI/antifade. The signal detection and analysis of subsequent metaphases used the Metafer system and Metasytems’ ISIS software .http://www.bioconductor.org) for the R statistical language. A Euclidean distance was used to calculate the matrix of distances, and clusters were built using Ward’s method.The first cluster analysis was performed to assess the chromosomal heterogeneity of each cell line by considering the type and frequency of chromosomal alterations within metaphases. Each alteration was computed as present or absent within the karyotype of different metaphases. In the second cluster analysis, the frequency (%) of each chromosomal alteration was compared among the four cell lines. Hierarchical clustering was performed using package gplots from the Bioconductor project   and locaClick here for fileCharacteristics of breast cancer cell lines. Data obtained from ATCC.Click here for file


To determine the genetic contribution to leukocyte endothelial adhesion.Leukocyte endothelial adhesion was assessed through a novel cell-based assay using human lymphoblastoid cell lines. A high-throughput screening method was developed to evaluate the inter-individual variability in leukocyte endothelial adhesion using lymphoblastoid cell lines derived from different donors. To assess heritability, ninety-two lymphoblastoid cell lines derived from twenty-three monozygotic twin pairs and twenty-three sibling pairs were compared. These lymphoblastoid cell lines were plated with the endothelial cell line EA.hy926 and labeled with Calcein AM dye. Fluorescence was assessed to determine endothelial cell adhesion to each lymphoblastoid cell line. Intra-pair similarity was determined for monozygotic twins and siblings using Pearson pairwise correlation coefficients.A leukocyte endothelial adhesion assay for lymphoblastoid cell lines was developed and optimized . A higher adhesion correlation was found between the twins than that between the siblings. Intra-pair similarity for leukocyte endothelial adhesion in monozygotic twins was 0.60 compared to 0.25 in the siblings. The extent to which these differences are attributable to underlying genetic factors was quantified and the heritability of leukocyte endothelial adhesion was calculated to be 69.66% .There is a heritable component to leukocyte endothelial adhesion. Underlying genetic predisposition plays a significant role in inter-individual variability of leukocyte endothelial adhesion. Leukocyte endothelial adhesion has been implicated as a key disease process in many conditions of major public health import such as multiple sclerosis, atherosclerosis, inflammatory bowel disease, diabetic retinopathy, malignancy, pulmonary edema, rheumatoid arthritis, and stroke Corroborating lines of evidence suggest that genetic variability contributes to differences in leukocyte endothelial adhesion. For instance, it has been shown that the expression of cell surface adhesion molecules on endothelial cells varies between individuals To explore the hypothesis that inter-individual genetic variability contributes to differences in leukocyte endothelial adhesion we assessed adhesion in monozygotic twins and compared them to matched sibling pairs. We quantified the extent to which these differences are attributable to underlying genetic factors Participants gave written informed consent. The University of Illinois at Chicago Institutional Review Board approved all protocols and informed consent documents in accordance with federal regulations and the principles expressed in the Declaration of Helsinki.http://ccr.coriell.org/) (Ninety–two lymphoblastoid cell lines (twenty-three monozygotic twin pairs and twenty-three sibling pairs) were purchased from Coriell Institute for Medical Research (ll.org/) . The min5 cells/mL and incubated with 2 µM Calcein AM for 30 min at 37°C. The cells were collected and subjected to three washes with PBS  to remove the free Calcein AM. Calcein AM labeled cells were added at a density of 50,000 cells/well on top of confluent monolayer cultures of EA.hy926 cells in 96-well plates and incubated for 30 min at 37°C. Nonadherant cells were removed by an optimized automated wash protocol adapted using the EP3 liquid handling system. Calcein AM fluorescence in the labeled cells was assessed by the high content imager Acumen at an excitation and emission wavelength of 485/535 nm before and after each wash. Endothelial adhesion for each cell line was determined by measuring the total area of fluorescence in relative fluorescence units (RFU) of each well following the third wash. The (%) remaining RFU/Input was determined by the following formula: [Total area of fluorescence (RFU) following third wash / Total area of fluorescence (RFU) before wash] ×100. A standard curve for lymphoblastoid cell lines, peripheral blood mononuclear cells, and the control cell lines  was generated using known concentrations of respective cell lines and fitting to a four parametric logistic curve , and cultured to confluency for 48 hours. On the day of the assay, lymphoblastoid cell lines or peripheral blood mononuclear cells were suspended in RPMI 1640 with 10% FBS at a concentration of 8×10ic curve . The % aA mean, standard deviation, and coefficient of variation (CV) of leukocyte endothelial adhesion for each lymphoblastoid cell line was calculated. Plates included positive and negative controls for quality control. Positive controls were a Trisomy 21 lymphoblastoid cell line that also provided a reference for the control of inter-plate variability. Ramos, a Burkitt lymphoma cell line, served as an additional positive control. The negative controls were EA.hy926 cells alone. A Z′-factor and CV for each plate was determined. A composite Z′-factor was determined to assess the overall robustness of the assay using the equation Intra-pair similarity was determined for monozygotic twins and siblings using the interclass correlation coefficient (ICC). Heritability was estimated by using the twinan90 function in the R package We developed and optimized a leukocyte endothelial adhesion assay for lymphoblastoid cell lines . Specifically, we have successfully converted this assay onto a 96-well plate platform amenable to robotic screening. The advantage of this format is that it affords a high degree of consistency and repeatability between runs . A 96-weThe assay demonstrates a large dynamic range, with close to a 7-fold difference, evident between the control cell lines, namely the low adhesive Burkitt lymphoma Ramos line and the highly adhesive Trisomy 21 lymphoblastoid cell lines . SpecifiUsing twenty unrelated lymphoblastoid cell lines generated from normal subjects, we measured their leukocyte endothelial adhesion in order to estimate the inter-individual variability obtained through our high-throughput assay. We were interested to assess the extent to which genetic factors contributed to inter-individual differences in leukocyte endothelial adhesion. We performed a heritability study for leukocyte endothelial adhesion utilizing lymphoblastoid cell lines derived from identical twins and those derived from matching sibling pairs. The hypothesis was that there would be greater correlation between the adhesion of the identical twins than that of the sibling pairs. Summarized subject data are reported in It is certainly possible, though, that confounding factors contribute to differences in leukocyte endothelial adhesion between lymphoblastoid cell lines. In The leukocyte endothelial adhesion assay we developed is able to sensitively and reproducibly detect differences in leukocyte adhesion to endothelial cell monolayers. This novel high-throughput assay was able for the first time to reveal the extent to which the important biological property of leukocyte endothelial adhesion is impacted by genetic variation.Lymphoblastoid cell lines have evolved into a powerful model system for functional population-based studies in humans. Lymphoblastoid cell lines are transformed cell lines derived from a subject's circulating B-lymphocytes. They were originally generated as a perpetual resource of a specific subject's DNA. Given the associated difficulties with maintenance, storage, collection, and the control of confounding variables in clinical samples, they have evolved into a popular model system In this study, we sought to explore the feasibility of using lymphoblastoid cell lines to assess the heritability of leukocyte endothelial adhesion. It was first necessary to create a high-throughput assay for leukocyte endothelial adhesion that would enable us to compare the adhesion of multiple different cell lines. We developed a robust assay (Z′-factor = 0.67). The excellent Z′-factor suggests that the variability introduced by the washing step in other high-throughput assays was not an issue in our assay To our knowledge the present paper is the first study to determine the heritability of leukocyte endothelial adhesion. Current approaches have the power to reveal the genetic network underlying this trait. For these genetic tools to work, though, there must be a heritable basis to leukocyte endothelial adhesion. A substantial body of literature implicates genetic variation as a basis for differential leukocyte adhesion The use of lymphoblastoid cell lines is a potential limitation of this study, though. An issue regarding the use of lymphoblastoid cell lines is the extent to which they serve as relevant surrogates for different primary tissues. Previous work has demonstrated the broad translatability of studies with lymphoblastoid cell lines A persistent concern about the usefulness of lymphoblastoid cell lines is that changes associated with the Epstein-Barr virus transformation reduce their value and relevance as a model. The virus transformation of primary white blood cells does change some of the cellular properties within the lymphoblastoid cell lines Adhesion in lymphoblastoid cell lines demonstrates similar variation observed in primary leukocytes. In our experiments, we observed, that the Burkitt lymphoma cell lines show the least endothelial adhesion while Trisomy 21 lymphoblastoid cells bind the highest compared to the lymphoblastoid cell lines derived from 92 normal subjects. A study comparing the expression of adhesion molecules as determined by flow cytometry in lymphoblastoid cell lines generated from normal peripheral blood mononuclear cells and Burkitt lymphoma cell lines demonstrated that the normal lymphoblastoid cell lines express higher levels of integrins, including integrin alpha 4 beta 1, leukocyte function associated antigen LFA-1, and integrin alpha 5 beta 3 as well as enhanced expression of the IgG superfamily adhesion molecules, like ICAM-1, and leukocyte function associated antigen type 3, and, glycoproteins like L-selectin and CD34 Another potential limitation is the use of an immortalized endothelial cell line. EA.hy926 is one of the best-characterized and most frequently used endothelial cell lines There are several strengths of this study. The assay was implemented on a high- throughput platform with very little intra-individual or inter-plate variability. Access to a large cohort of monozygotic twin and sibling pair lines enabled us to confidently identify leukocyte endothelial adhesion as a heritable trait. Heritability is the best statistic for the representation of the genetic contribution to phenotypic variance In summary, there is a major heritable component to leukocyte endothelial adhesion. Genetic predisposition appears to play a significant role in inter-individual variability of leukocyte endothelial adhesion. As such, future genetic studies should be able to identify those key molecular elements that regulate this important cellular trait.Figure S1Standard curves for cell lines. Four parametric logistic curves of (A) Trisomy 21 lymphoblastoid cell line (B) Ramos Burkitt lymphoma (C) GM14581 a reperesentative twin lymphoblastoid cell line (D) U937 a monocytic leukemic cell line (E) and peripheral blood mononuclear cells (PBMC) were generated using known concentration Calcein AM labeled cells. On the x-axis is the known concentration of cells lines and on the Y- Axis are the relative fluorescence units. The standard curve generated was used to deduce the adherent cells that remained following washes.(TIFF)Click here for additional data file.Table S1Subject Characteristics.(PDF)Click here for additional data file.


N-glycosylation sites together with the natural O-glycosylation site in Thr106 [N- and O-glycosylated protein shows higher apparent molecular mass and longer plasmatic half-life compared to the non-glycosylated IFN-alpha produced in bacterial systems and used for clinical applications. As a consequence, the correct glycosylation of our modified cytokine is very important for its in vivo activity. For this reason, it is of great relevance the evaluation of different mammalian host cells for its production. While hamster-derived CHO cells are widely used for large scale production of recombinant therapeutic glycoproteins, human HEK cells are a promising system because they are easy to grow and transfect [IFN alpha is an important cytokine of the immune system. It has the ability to interfere with virus replication exerting antiviral activity. Moreover, it displays antiproliferative activity and can profoundly modulate the immune response. IFN4N  is an IFN-alpha2b mutein developed in our laboratory using glycoengineering strategies. This molecule contains 4 potential 6cell-1.day-1, which represents an 8-fold increment compared to the parental line. The HEK-IFN4N cell line was resistant to 200 ug/ml of puromycin and showed a 15-fold increment in the specific productivity compared to the parental line, reaching a value of 1,490 ± 332 ng.106cell-1.day-1. In both cases, complete culture death was achieved at higher puromycin concentrations. The specific productivity of IFN4N of HEK 293T cell line duplicated the value obtained for the CHO-K1 cell line, and it was achieved at a lower concentration of puromycin, making the selection process shorter  in a RQflex® Reflectometer (Merck Millipore). Levels of amonium in the culture supernatant were determined by the Berthelot reaction.Both cell lines were cloned using the limiting dilution method, and after 15 days of culture more than 100 clones were screened. To achieve the characterization and study both cell lines as recombinant protein expression hosts, the 6 best producer clones were isolated and amplified. The adherent clones were grown for 7 days in order to construct their growth curves. Cell density and viability were determined every 24 h by trypan-blue exclusion method and the culture supernatant was collected to determine IFN4N and metabolites concentration. The IFN4N production was assessed employing a sandwich ELISA assay developed in our laboratory. Glucose consumption and lactate production were evaluated using specific Reflectoquant5-1.5.106 cell.ml-1) than HEK clones (between 6.105-9.105 cell.ml-1), probably because of space limitation and higher glucose consumption, since average qgluc of HEK clones was higher (see Table -1).As shown in Table CHO and HEK cells were genetically modified to produce IFN4N by using lentiviruses as a tool for the IFN4N gene transfer. Since both cell lines expressed high levels of IFN4N, 6 clones were amplified for an intensive characterization. Culture and production properties of both groups of clones were very different. On the one hand, CHO clones were easy to maintain in culture for a long period of time, reaching higher cell densities than HEK clones. On the other hand, the best specific productivity of IFN4N was achieved employing HEK cells. The behavior of CHO and HEK cells at large scale production should be analyzed in order to select the proper system for the cytokine's production.Wide differences have been observed between the glycosylation profile of the same recombinant therapeutic protein produced in CHO and HEK systems . Conside


In the present study, we screened newly synthesized antiviral aminopyrazoloquinoline derivatives for cytotoxic potential in human normal and breast cancer cell lines using apoptosis as biomarker. These derivatives and the well known antiviral drug, acyclovir, were incubated with the normal  and cancer  cell lines at 10, 50 and 100 μM for 72 h at 37°C. Both the parent compounds and their sugar derivatives were found to be differentially cytotoxic in various cell lines. MCF-7 cells were more or less completely resistant to all these compounds while MDA-MB-231 cells were significantly killed by apoptosis. The methoxy derivative of aminopyrazoloquinoline (compound 3) was found to be the most cytotoxic in the normal breast epithelial cell lines (MCF-10A and MCF-12A) and MDA-MB-231 cell lines at 100 μM killing over 90% of the cells with up to 80% apoptosis. Interestingly MCF-7 cells showed only up to 50% killing at 100 μM dose with less than 20% apoptosis. Acyclovir did not cause any cytotoxicity, apoptosis or cell cycle arrest in any of the cells lines at the doses tested. Our results suggest that the newly synthesized antiviral compounds have an associated risk of being cytotoxic compared to the acyclovir. Acyclovir had been the drug of choice for several decades; however, the emergence of acyclovir-resistant Herpes Simplex Virus (HSV) isolate in the immunocompromised patients and organ or bone marrow recipients has led to the discovery of new antiviral compounds -5, 9, 10In order to provide the new generation of antiviral drugs with the least possible cytotoxicity, we evaluated their cytotoxic potential using apoptosis in various human normal and breast carcinoma cell lines. Apoptosis is a well-established and recognized biomarker to screen the new compounds for cytotoxicity , 11-13. Since the inception of apoptosis in 1842 and its revival almost 30 years ago, numerous publications using apoptosis have appeared in the literature . Apoptos+) normal MCF-12A cells showed about 60% apoptosis; however, the ER+ malignant MCF-7 cells tolerated this compound very well. Other biomolecular factors such as deficiencies of caspase-3 in MCF-7, BCL2 in MCF-12A, p16 in MCF-10A and presence of mutant p53 in MDA-MB-231 may be playing significant roles in cell-specific differential responses. However, the resistance of caspase-3 deficient MCF-7 cell lines  supplemented with cholera toxin (100 μg/ml), epidermal growth factor (20 μg/ml), insulin (10 μg/ml), hydrocortisone (0.5 μg/ml) and 5% calf serum. For apoptosis and cell viability assays, the cells were treated with the test compounds  in triplicate and incubated in the CO1 apoptotic population was analyzed . The results were analyzed using Cell Quest Pro software and represented as percentage of normal and apoptotic cells at various stages. Simultaneously, treated cells were also stained for nuclear DNA and sub-Ganalyzed .t test and the value p<0.05 was considered significant.The values represent mean ± SD (n=3). The statistical significance was determined by the student


None of the nine original cell linesshowed any cross resistance between CDDP andtaxanes, but one of the CDDP-resistant celllines, RPMI2650CR, demonstrated hypersensitivityto both taxanes when compared to the parentalcell line, RPMI2650. Furthermore, RPMI2650CRexhibited increased expression of BRCA1. Thesedata suggest that (i) taxanes are a goodcandidate for a second-line therapeutic drug forHNSCC patients with acquired CDDP resistanceand (ii) BRCA1 can be a candidate marker forpredicting an inverse CDDP/taxane sensitivityphenotype in HNSCC.Recently, an inverserelationship between resistance toplatinum-based chemotherapeutic agents andtaxanes has been implicated in breast andovarian cancers, and a possible pivotal role forBRCA1 has also been suggested. Because However, acquired resistance to CDDP is one of the major problems in the clinical management of such patients -2,5-diphenyl-tetrazo-lium bromide (MTT)/PBS and incubated for one hour. After the incubation, the MTT solution was removed, and the cells were suspended in 100% ethanol. Absorption was measured at 590 nm using a Versamax microplate reader . Independent triplicate assays were performed. All values represent the mean ± standard deviation (SD) from 3 sets of independent cultures.A total of 5 × 105 cells were harvested, and protein concentrations in total cell lysates were measured using a DC protein assay kit . A 40 μg aliquot of the protein was subjected to immunoblotting using a 10–20% polyacrylamide gradient gel . Antibodies used were rabbit anti-BRCA1 monoclonal antibody , mouse anti-β actin monoclonal antibody , and horseradish peroxidase conjugated anti-mouse immunoglobulin antibodies . For blocking conditions and concentrations of antibodies, we followed the manufacturer's recommendations. Signals were visualized by reaction with ECL Detection Reagent  and digitally processed using LAS 1000 Plus with a Science Lab 99 Image Gauge .A total of 2 × 10t-test, and the significant difference level was established at P < 0.05.The statistical differences in cell viability between the parental and CDDP-resistant cell lines were determined with student's IGF2 as one of the candidate genes for acquired CDDP resistance in HNSCC by cDNA microarray analysis, and the stably IGF2-expressing RPMI2650 cells did show resistance to CDDP similar to that observed in RPMI2650CR [In a previous study, we successfully established CDDP resistant cell lines from all of the HNSCC cell lines we attempted . These rMI2650CR . IGF2-expressing RPMI2650 was also used to determine the taxane sensitivity, but it showed a sensitivity similar to that of parental RPMI to both taxanes (data not shown), indicating that IGF2 is not involved in the hypersensitivity to taxanes. Although the molecular mechanisms behind the inverse correlation of chemosensitivity between taxane and CDDP are not well understood, it can be proposed that taxanes are good candidates as a second-line chemotherapeutic choice for HNSCC patients with acquired chemoresistance to CDDP. Stordal et al. reported an inverse correlation of chemosensitivity between taxane and CDDP . TherefoSeveral studies have suggested an association between taxane sensitivity and BRCA1 expression , so we tAcquired CDDP resistance is often observed in patients with HNSCC, and taxanes are used as the second-line chemotherapeutic drug for HNSCC. We observed that an acquired CDDP-resistant HNSCC cell line showed higher sensitivity to taxane by two orders of magnitude than its parental cell line. Prominent upregulation of BRCA1 was also observed in this CDDP-resistant/taxane-sensitive cell line. Expression status of BRCA1 can be a biomarker to forejudge the taxane sensitivity to patients with acquired CDDP-resistant HNSCC.