EEF1A1) promoter in an antibody protein-expression vector which was integrated into the Chinese hamster ovary (CHO) cell line genome.Recombinant cell lines developed for therapeutic antibody production often suffer instability or lose recombinant protein expression during long-term culture. Heterogeneous gene expression among cell line subclones may result from epigenetic modifications of DNA or histones, the protein component of chromatin. We thus investigated in such cell lines, DNA methylation and the chromatin environment along the human eukaryotic translation elongation factor 1 alpha 1 (EEF1A1 promoter region. Each PT1-CHO cell line displayed specific epigenetic signatures or chromatin marks correlating with recombinant mRNA expression. The cell line with the lowest recombinant mRNA expression (PT1-1) was characterized by the highest nucleosome occupancy and displayed the lowest enrichment for histone marks associated with active transcription. In contrast, the cell line with the highest recombinant mRNA expression (PT1-55) exhibited the highest numbers of formaldehyde-assisted isolation of regulatory elements (FAIRE)-enriched regions, and was marked by enrichment for histone modifications H3K9ac and H3K9me3. Another cell line with the second highest recombinant mRNA transcription and the most stable protein expression (PT1-7) had the highest enrichments of the histone variants H3.3 and H2A.Z, and the histone modification H3K9ac. A further cell line (PT1-30) scored the highest enrichments for the bivalent marks H3K4me3 and H3K27me3. Finally, DNA methylation made a contribution, but only in the culture medium with reduced FCS or in a different expression vector.We analyzed four PT1-CHO cell lines which exhibited losses of protein expression at advanced passage number (>P35) growing in adherent conditions and in culture medium with 10 % FCS. These cell lines exhibited different integration sites and transgene copy numbers as determined by fluorescence in situ hybridization (FISH) and quantitative PCR (qPCR), respectively. By qRT-PCR, we analyzed the recombinant mRNA expression and correlated it with DNA methylation and with results from various approaches interrogating the chromatin landscape along the EEF1A1 promoter region can help predict recombinant mRNA expression, and thus may assist in selecting desirable clones during cell line development for protein production.Our results suggest that the chromatin state along the The online version of this article (doi:10.1186/s12896-016-0238-0) contains supplementary material, which is available to authorized users. Cell lines combining high-production and stability are important for recombinant protein production, notably of therapeutic antibodies. These antibodies are chiefly produced in Chinese hamster ovary (CHO) cells which combine several advantageous qualities, notably that these antibodies are compatible with humans and bioactive therein . HoweverChromatin is a complex nucleoprotein structure in which the DNA is packaged in the cell nucleus. At the chromatin level, different epigenetic events operate that can affect the integration sites of the protein-expression vector into the CHO genome. Thus, epigenetic events may contribute to the transcriptional repression of the transgene , 7. The A transgene in the CHO genome can also be influenced by the positioning of the nucleosome at the integration site. The nucleosome is the fundamental repeating chromatin subunit comprised of eight histones encompassed by circa 147 bp of DNA in 1.65 superhelical turns. The histone octamer itself comprises two copies each of histones H2A, H2B, H3, and H4. Nucleosome positioning is key to higher-order chromatin folding and transcriptional regulation –14. NuclEEF1A1) promoter sequence in four recombinant CHO cell lines which exhibited loss of productivity during long-term culture. We found each cell line exhibited chromatin marks highly associated with recombinant mRNA expression. Understanding chromatin environment in recombinant CHO cell lines should help facilitate selection of stable and productive cell lines for recombinant protein production.A number of studies have shown that aberrant DNA methylation , 18, 19 In this study, we investigated four PT1-CHO cell lines which exhibited attenuated recombinant protein production after a long-term culture (at passage > P35). These cell lines were developed for antibody production by transfecting CHO-K1 cells with a plasmid-expression-vector construct (designated PT1) carrying cDNA encoding heavy and light chains of a murine IgG2a antibody. As shown by fluorescence in situ hybridization (FISH) analysis, integration sites of the transgene may involve different chromosome regions and chromosomes Fig. . For insn = 22) determined at high passage (P55) in these PT1-CHO cell lines was found in 84–98 % in 100 analyzed metaphases . A difference with respect to light chain expression due to time point of measurements was also detected (*P = 0.0184), but not for the heavy chain. Overall, the highest expression was obtained for PT1-55, then PT1-7 and PT1-30, the lowest for PT1-1, and this relationship remained essentially constant, even in a total of n = 14 mRNA expression measurements (data not shown). We can deduce from this result that the poor protein productivity for PT1-1 was associated with the negligible recombinant mRNA expression of this subclone.Because the loss of recombinant protein expression in the PT1-CHO cell lines could primarily reflect the loss of recombinant mRNA expression, we measured the mRNA expression of each cell line by qRT-PCR in various passages i.e. P49 to P73) throughout the study. We designed PCR primers along the sequences encoding the heavy and light chains contained in the plasmid-expression-vector construct (PT1), carried out qRT-PCR for heavy and light chains using mRNA isolated from different time points and quantified recombinant mRNA expression of each   promoter contained in the PT1 expression vector. Using bioinformatic tools (see Methods), we annotated the 1261-bp EEF1A1 promoter and identified two CpG islands in the promoter region . Cloning of the PCR fragments and sequencing of clones to enable analysis of single molecules also confirmed low methylation, i.e. highest was 6.25 % found in PT1-1  and high passage (P22) at 10 % FCS, and under adherent culture conditions. Unlike the PT1 expression vector in which there are three copies of the EEF1A1 promoter, there is only one promoter copy in the VT2 vector (not shown). Under these conditions, we observed more CpG methylation in VT2-CHO cell lines at late than at early passage , but with reduced FCS (0.5 % instead of 10 %) in the culture medium. Thus, we observed higher methylation with 0.5 % FCS than 10 % FCS  and subsequent bisulfite sequencing of individual progeny DNA molecules [M.SssI unless the CpGs are blocked (or protected) by nucleosomes or DNA-binding proteins. Specific footprints can then be revealed contingent upon nucleosome positions and transcription factor binding sites on promoters . For prediction, we used the 1261-bp EEF1A1 promoter sequences, and analyzed the two predicted nucleosomes towards and nearest the transcription start site (TSS). For ease of scoring, these two nucleosomes were arbitrarily designated Nuc 853 (nt 853–999) and Nuc 1008 (nt 1008–1154). We next isolated chromatin from the PT1-CHO cell lines at high passage (P49), followed by a brief treatment with M.SssI and genomic DNA isolation. Subsequently, we undertook bisulfite sequencing of several clones from each cell line interrogating 18 CpG sites within two predicted nucleosomes nearest the TSS, and the same sites earlier analyzed during DNA methylation analysis. Control estimates of the methylation efficiency of M.SssI on the EEF1A1 promoter obtained from ‘naked’ genomic DNA of two PT1-CHO cell lines yielded average levels of 98 %  in PT1-7 than in PT1-55 , which is used to map proteins such as histones, transcription factors, and other chromatin-modifying complexes associated with specific regions of the genome. Briefly, chromatin is isolated, fragmented, and immunoprecipitated with antibodies specific to the protein or modification of interest. The purified ChIP-enriched DNA is then analyzed by quantitative-PCR, microarray technology, or sequencing [M.SssI chromatin mapping. We quantified the ChIP DNA and input DNA before performing qPCR, and then normalized results using percentage input relative to the canonical histone H2A. We then correlated the different ChIP enrichments in chromatin isolated at high passages (P52 – P70) to the respective recombinant mRNA expression of the four PT1-CHO cell lines.The encouraging results obtained with single-molecule mapping with quencing –26. Specn = 12 ChIP experiments with H2A. ChIP-PCR was undertaken using the four primer pairs on two predicted nucleosome positions along the human EEF1A1 promoter region. We thus showed significant differences in H2A enrichments (i.e. H2A nucleosome occupancy) among the four cell lines . Differences owing to the qPCR primer pair used (i.e. nucleosome) were not significant. Notably, we observed higher H2A enrichments for PT1-1 and PT1-30 than PT1-7 and PT1-55, with PT1-1 garnering the highest while PT1-55 was the lowest (e. g. t-test ***P < 0.0001 for PT1-1 vs. PT1-7 or PT1-55). Altogether, H2A enrichment negatively correlated with the recombinant mRNA expression, consistent with the findings previously obtained with the M.SssI chromatin mapping.That a tight chromatin conformation was associated with repression of recombinant mRNA expression or vice versa was confirmed by the ChIP results obtained with the canonical H2A antibody  and correlated positively with recombinant mRNA expression  coupled with qPCR with the same primers used in ChIP experiments. FAIRE identifies nucleosome-depleted regions; i.e. regions (= regulatory elements) bound by transcription factors or other regulatory proteins [P = 0.0114), but not on specific primers used. Crucially, PT1-55 exhibited the highest FAIRE-enrichment levels  used for antibody production, and which was integrated into the CHO genome. We analyzed four PT1-CHO cell lines which differed in the loss-of-protein expression seen after high passage (> P35) in adherent condition supplemented with 10 % FCS in the culture medium. We found that epigenetic signatures in the PT1-CHO cell lines correlated highly with recombinant mRNA expression. Furthermore, the lowest-producing cell line exhibited chromatin marks suggestive of tight chromatin, while the highest-producing line showed marks associated with open chromatin. Our results thus demonstrate that the mapping of chromatin structures can be useful in CHO cell line development and metabolic engineering.Epigenetic silencing of the recombinant gene can be listed among the prime causes leading to reduced recombinant protein production. Thus, we analyzed epigenetic modifications affecting chromatin structure that are associated with decreases or loss of recombinant mRNA expression during cell line development for recombinant protein production. Using a variety of approaches, we investigated the DNA methylation pattern and chromatin landscape around the human EEF1A1 promoter, but none of the PT1-CHO cell lines under the condition of 10 % FCS and high passage exhibited significant increases in CpG methylation to suggest that DNA methylation alone underlay reduced production. Nonetheless, our results also implied culture-context-dependent methylation of the EEF1A1 promoter. Higher methylation became evident in the same CHO cell line even at low passage if the FCS concentration of the culture medium was reduced from 10 to 0.5 %. Furthermore, when present in another vector construct, methylation along the EEF1A1 promoter in CHO cell lines was enhanced at higher passage numbers  are frequently methylated in unstable antibody-producing CHO cell lines , 19. We M.SssI (a CpG methylase) provided visualization of nucleosome occupancy on single EEF1A1 promoter molecules, which correlated well with the recombinant mRNA expression, i.e. higher nucleosome occupancy meant lower mRNA expression. Nonetheless, there was high nucleosome occupancy, ranging from 62.5 to 86.7 %, in the PT1-CHO cell lines which had undergone long-term culture. Recently, a similar approach of nucleosome mapping in yeast PHO5 promoter with M.CviPI (a GpC methylase) in single cells revealed significant heterogeneity of nucleosome configurations within a population [The chromatin mapping with the DNA methyltransferase pulation . The celpulation .EEF1A1 promoter in affecting recombinant mRNA expression and productivity in the PT1-CHO cell lines.Histones form the protein core of chromatin around which DNA is wrapped. Various modifications to histones play a key role in epigenetic control of cellular activity . HistoneCertain histone variants and histone post-translational modifications (PTMs) have been considered variously as chromatin marks for transcriptional activation or repression. For example, the histone modification H3K4me3 is often associated with transcriptional activation , and thiA well-known PTM and a key determinant of complex chromatin states is the methylation of histone lysine residues. Histone methylation  of lysine residues is catalyzed by SET-domain containing proteins and plays a critical role in the regulation of gene expression, cell cycle, genome stability, and nuclear architecture (see reviews –38). HisM.SssI, ChIP with different histones and FAIRE on the EEF1A1 promoter contained in an expression vector integrated into the CHO genome, we found that each PT1-CHO cell line displayed a distinct epigenetic signature or chromatin marks indicative of recombinant mRNA expression. Altogether, these results suggest that the chromatin structure along the EEF1A1 promoter region can be predictive of recombinant mRNA expression in the PT1-CHO cell lines analyzed and together with culture conditions might contribute to the eventual loss of recombinant protein expression after long-term culture. These findings provide insights on how unstable CHO cell clones could be weeded out early in the generation process of stable-expressing cell clones and protein production might be better assured.After carrying out DNA methylation analysis, followed by single-molecule chromatin mapping with Briefly, adherent CHO-K1 cells  were transfected with a vector carrying cDNA of the heavy and light chain of a murine IgG2a antibody. After selection of transfected cells with 500 μg/mL Hygromycin B, a limiting dilution was performed to obtain the clonal cell lines, namely: PT1-1, PT1-7, PT1-30, and PT1-55.FISH was performed to determine integration sites of the plasmid-expression-vector construct (PT1) in the CHO genome. Cytogenetic harvesting and FISH were performed on short-term cultures (P10) according to protocols described previously , 51. Met6) were grown at 37 °C in T25 culture flasks containing 5 mL culture medium  supplemented with 8 mM GlutaMAX, 10 % FCS  and 50 μg/mL Hygromycin B (Sigma-Aldrich). After 48 h, 150 μL of 10 μg/mL colcemid (Sigma-Aldrich) was added for 2 h. For hypotonic treatment, cells were detached enzymatically with trypsin-EDTA . After washing, 5 mL pre-warmed 1 % sodium citrate was added and incubated for 10 min at 37 °C. Cells were fixed in 3 parts methanol : 1 part glacial acetic acid, and dropped onto ice-cold pre-cleaned microscope slides. Metaphase spreads were stained with DAPI for fluorescence microscopy. The chromosome numbers at passage P55 were determined from n =100 metaphases per cell line.We performed cytogenetic analysis on PT1-CHO cell lines using standard procedures. PT1-CHO cells (0.25 × 104) were grown in single-well chamber slides (Nunc-LabTek) in 3 mL medium as described above for 24 h at 37 °C. Cells were fixed in 3 parts methanol : 1 part glacial acetic acid and stained with DAPI for microscopy. Per cell line, n = 4000 cells were scored for MN, NBUD, and chromatin condensation/fragmentation, while mitotic aberrations determined in n = 100 ana/telophases. Results are presented as the mean of two passages (P55 and P72).To determine the frequencies for micronucleus (MN), nuclear bud (NBUD), ana/telophase aberrations, and chromatin condensation/fragmentation, PT1-CHO cells (6.6 × 10Vezt (Vezt-305-up/ Vezt-305-dw) in host line, at an early passage number (P5). Oligonucleotide primer sequences are given in Table The copy numbers of the transgene were determined by qPCR on the light chain (PT1-LC-Fwd-2/ PT1-LC-Rev-2), and a reference gene For stability study of the PT1 clonal cell lines, cells were cultivated for 22 passages in Ham’s F12 medium supplemented with 6 mM L-Glutamine and 500 μg/mL Hygromycin B and passaged every 3–4 days. Every third passage, supernatants were collected for titer determination. To determine the specific productivity, seeding and harvesting cell densities were determined. Specific productivity was calculated according to equations (1) and (2):IVCC = Integral of the viable cell concentrationXV1 = viable cell density (109/L) at time point t1CXV0 = viable cell density (109/L) at time point t0CqP = specific productivity (pg/cell/day)P1 = Titer (μg/mL) at time point t1CP0 = Titer (μg/mL) at time point t0C2O2. After the reaction was stopped with 1 M H2SO4, optical densities (OD) were measured at 490 nm with a SpectraMax 190 .Microtiter plates (96-well) were coated with a goat anti-mouse antibody  and incubated with cell culture supernates overnight at 8 °C. Purified mouse IgG2a κ, isotype control  served as a standard. Detection was performed after incubation with a horseradish-peroxidase conjugated goat anti-mouse IgG  followed by incubation with the substrate o-Phenyldiamindihydrochloride and H6 cells from each PT1-CHO cell line were grown in T75 culture flasks as described above  until about 100 % confluency (2–3 d) to obtain circa 1 × 107 cells. Cells were pelleted, and washed twice with 1 mL cold PBS. Cell pellets were used immediately or flash frozen in liquid nitrogen and stored at 80 °C for subsequent analysis.For epigenetic analysis, approximately 1 × 10EEF1A1 using bioinformatic tools such as: (1) nucleosome prediction by genomic sequence (http://genie.weizmann.ac.il/software/nucleoprediction.html) [http://nucleosome.stats.northwestern.edu/)[http://dna.ccs.tulane.edu/icm/) [EEF1A1 for putative binding sites of transcription factors using TRANSFAC (http://www.gene-regulation.com/pub/databases.html) or MatInspector . We annotated the EEF1A1 promoter using CLC Workbench (www.clcbio.com/products/clc-main-workbench).To determine epigenetic and transcriptional heterogeneity in the PT1-CHO cell lines, we used approaches and protocols, as previously described , 53. We on.html) –56; (2) ern.edu/); and (3)du/icm/) . We analWe analyzed recombinant mRNA essentially as described previously . We isolhttp://www.urogene.org/methprimer/index1.html) on the human eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) promoter contained in the PT1 vector (Table http://dna.leeds.ac.uk/cpgviewer/) or BISMA (http://services.ibc.uni-stuttgart.de/BDPC/BISMA/).We performed DNA methylation analysis as previously described . Brieflyor Table . We sequM.SssI treatment, micrococcal nuclease (MNase) digestion, CHIP, and FAIRE have been described earlier [EEF1A1 promoter in the PT1 vector (Table t-tests contained in GraphPad Prism. About 5 μg of antibody was used for each ChIP experiment. Histone antibodies were obtained from Abcam: H2A (ab18255), H2A.Z (ab18263), H3.3 (ab62642), H3K4me3 (ab1012), H3K27me3, (ab6002), H3K9me3 (ab4441), and H3K9ac3 (ab8898). The normal rabbit IgG antibody (sc-2027) used as a control was obtained from Santa Cruz Biotechnology .The protocols used for the various experiments on chromatin, i.e. chromatin isolation,  earlier . ChIP exor Table . These p


Furthermore, enhanced TKTL1 expression was put into context with resistance to chemotherapy and ionizing radiation. Here, a panel of human malign and benign cells, which cover a broad range of chemotherapy and radiation resistance as well as reliance on glucose metabolism, was analyzed 17 malign and three benign cell lines were characterized according to their expression of TKTL1 on the protein level with three commercially available anti-TKTL1 antibodies utilizing immunohistochemistry and Western blot, as well as on mRNA level with three published primer pairs for RT-qPCR. Furthermore, sensitivities to paclitaxel, cisplatin and ionizing radiation were assessed in cell survival assays. Glucose consumption and lactate production were quantified as surrogates for the “Warburg effect”.Considerable amounts of tktl1 mRNA and TKTL1 protein were detected only upon stable transfection of the human embryonic kidney cell line HEK293 with an expression plasmid for human TKTL1. Beyond that, weak expression of endogenous tktl1 mRNA was measured in the cell lines JAR and U251. Western blot analysis of JAR and U251 cells did not detect TKTL1 at the expected size of 65 kDa with all three antibodies specific for TKTL1 protein and immunohistochemical staining was observed with antibody JFC12T10 only. All other cell lines tested here revealed expression of tktl1 mRNA below detection limits and were negative for TKTL1 protein. However, in all cell lines including TKTL1-negative HEK293-control cells, antibody JFC12T10 detected multiple proteins with different molecular weights. Importantly, JAR and U251 did neither demonstrate an outstanding production of lactic acid nor increased resistance against chemotherapeutics or to ionizing radiation, respectively.in vitro. The presence of TKTL1 was unrelated to either the rate of glucose consumption/lactic acid production or resistance against chemo- and radiotherapy.Using RT-qPCR and three different antibodies we observed only exceptional occurrence of TKTL1 in a panel of malignant human cell lines The online version of this article (doi:10.1186/1471-2407-15-2) contains supplementary material, which is available to authorized users. TKT, together with the transaldolase enzyme, reversibly links the PPP to glycolysis [Transketolase TKT; EC: 2.2.1.1) is a thiamine-dependent key enzyme involved in the non-oxidative branch of the pentose phosphate pathway (PPP), which catalyzes the transfer of a 2-carbon fragment  as monolayers and harvested at 80-90% confluence using a cell-scraper (Biochrom) for further experiments. HEK293 cell transfectants stably producing full-length TKTL1 protein (293pCAG TKTL1) were used as positive control cells. HEK293 cells transfected with empty expression vector (293pCAG Δ) do not produce TKTL1 protein and were used as negative control cells. Both transfectants have been previously described in detail [The cell lines investigated herein were directly obtained from the companies given in Table 6 cells per cell line were harvested upon trypsinization, washed twice with PBS and fixed for 1 h in 4% PBS-buffered formalin at room temperature. All centrifugation steps were performed at 280x g at 4°C for 10 min. The pellet was then re-suspended in 150 μl PBS and mixed with the same quantity of pre-cooled 2% high-melting agarose . The mixture was cooled down on ice. The resulting clot was first transferred to a sample-embedding capsule and then to a 4% PBS-buffered formalin solution. The dehydration with graded alcohols and xylene, and the embedding into paraffin  were done automatically in parallel to tissue biopsy samples at the Institute of Pathology, University of Würzburg, in a Leica ASP200 S embedding unit. The resulting paraffinized cell clot samples were then set into paraffin blocks. Paraffin blocks with cell samples were cut into 2 μm thick sections and mounted up on aminopropylethoxysilane (APES)-coated slides. Slides for immunohistochemistry were rehydrated in descending concentrations of ethanol before being heated for antigen unmasking in 10 mmol/l sodium citrate buffer (pH 6.0) in a microwave oven at 600 W for 5 min. After rinsing in distilled water, inhibition of endoperoxidase was performed by incubating sections for 10 min in 3% H2O2 in methanol. Slides were washed in PBS and incubated with 1% human immunoglobulin  in PBS for 15 min to block FC-receptors [Approximately 1x10eceptors . Subsequeceptors , 40–43. 6 cells each were lysed in pre-cooled RIPA buffer  containing phosphatase inhibitors , proteinase inhibitors  and 2,5 mmol/l Dithiothreitol (DTT) reducing agent (Sigma-Aldrich). The mixture was incubated for 30 min on ice, combined with a thorough shaking every 10 min. Cell lysates were clarified of cell debris by centrifugation at 14,000x g for 5 min through a QIAshredder spin column assembly  at 4°C. Protein concentration was determined using the Bradford method [2O2[For protein extraction, 1x10d method  and coomthod [2O2 with sub6 cells each were disintegrated in Trizol reagent . Total RNA was extracted from Trizol as recommended by the manufacturer. RNA integrity was verified using the Experion automated electrophoresis station  and the RNA concentration was measured at 260 nm. For first strand cDNA synthesis, 1 μg total RNA was employed using the iScriptcDNA synthesis kit from BioRad. The cDNA synthesis of 1:2 diluted cDNA was performed by heating at 25°C for 5 min, at 42°C for 30 min, and at 85°C for 5 min.1x10TKTL1 primer pairs were used: primer pair 1 (150 bp), forward: TAACACCATGACGCCTACTGC, reverse: CATCCTAACAAGCTTTCGCTG [primer pair 2 (80 bp), forward: AAGCCTTTGGGTGGAAC ACTTA, reverse: CTGAGAAGCCTGCCAGAATACC [primer pair 3 (151 bp), forward: GGTATCTGTTGGTGACGATGGT, reverse: GCACATCCCCTTGGCATTGG [peptidyl propyl isomerase A , forward: TGTCCATGGCAAATGCTGGACCC, reverse: GCGCTCCAT GGCCTCCAC AA (140 bp); β-actin (access. No. NM_001101), forward: CCTTGCCATC CTAAAAGCC, reverse: CACGAAAGCAATGCTATCAC (96 bp). qPCR reactions were performed on a CFX96 real-time PCR system (Bio-Rad) operated by CFX Manager Software (version 3.0). The cycler protocol was 5 min at 95°C, 40 cycles of 15 sec at 95°C, 60 sec at 60°C, and 5 min at 72°C. Gene of interest expression was normalized to the reference genes PPIA and β-actin [Quantitative PCR (qPCR) was performed with MESA Green qPCRMasterMix Kit for SYBR Green containing Meteor Taq hotstart polymerase . Three sets of TTTCGCTG primer paAGAATACC ; primer GGCATTGG . TKTL1 p β-actin . Fold ex β-actin . Post-am5 cells/ml in 48-well flat bottom tissue plates  with culture medium and were grown for 24 h. Afterwards, medium was completely replaced, and glucose consumption and lactate production at 21% oxygen were analyzed 24 h later. Cell-free supernatant was snap frozen for analysis and cell numbers were calculated with the crystal violet assay [Cells were seeded at 1x1050 values.Adherently growing cell lines were seeded at 5,000 cells per 100 μl of cell culture medium in a 96 well flat bottom tissue culture plate  and grown for 24 h prior to treatment with chemotherapeutic drugs. Then, 100 μl cell culture medium containing paclitaxel or cisplatin (both from Sigma-Aldrich) were added, resulting in final drug concentrations ranging from 1% to 200% of the reported peak plasma concentrations achievable in patients . The max2. Medium was completely replaced after 60 min to start with fresh growth conditions. In addition, cell medium was replaced on day 4 and day 8. Ten days after irradiation, the CCK-8 assay was performed as described above.Cell lines were seeded at 100 cells in 200 μl cell culture medium in a 96 well flat bottom tissue culture plate  24 h prior to radiation. Graded X-irradiation  was then performed at room temperature using a 6 MV linear accelerator  at a dose rate of 3 Gy/min. Control cells were treated in a similar way, but without irradiation. Each radiation dose was applied in triplicates. 20 min after irradiation cells were cultivated at 37°C in a humidified incubator at 5% COSo far, TKTL1 protein expression was described primarily in paraffin-embedded tumor tissue. Hence, in order to produce comparable datasets, paraffin-embedded cell pellets of all selected cell lines (n = 20) served to analyze the expression of TKTL1 protein by immunohistochemistry. The cell lines assessed in the present study are summarized in Table Immunohistochemical staining with JFC12T10 and Sigma Prestige antibodies showed a strong cytoplasmatic staining in HEK293-TKTL1 transfectants, with an additional nuclear staining by the Sigma Prestige polyclonal antibody. JFC12T10, but not the Sigma Prestige antibody also stained the cytoplasm of TKTL1-negative HEK293-control cells. The staining intensity of JFC12T10 in HEK293-control and HEK293-TKTL1 transfectants was comparable indicating an unspecific staining pattern of JFC12T10 in HEK293-control transfectants  and published previously [Quantitative RT-PCR (RT-qPCR) was performed with three different primer pairs specific for the three eviously , 28, 35.High expression of TKTL1 was put into context of enhanced glucose consumption within the pentose-phosphate pathway, resulting in increased lactic acid production . In orde50 values were calculated , whereas U251 and MCF-7 were resistant to paclitaxel and Mel2A, and 23132/87 and HepG2 were resistant to cisplatin (IC50 > 35 nmol/ml). JAR cells displayed intermediate and high sensitivity towards placlitaxel and cisplatin, respectively  were subjected to treatment with paclitaxel and cisplatin in concentrations ranging from 0.2-31.8 nmol/ml and 0.1-25.4 nmol/ml, respectively. Representative results of dose–response curves after treatment with paclitaxel and cisplatin are shown  characterized by weak endogenous tktl1 mRNA expression (JAR and U251). Immunohistochemical proof of TKTL1 protein in cytoplasm, the expected location of TKTL1 as a protein which is supposed to be instrumental in the pentose-phosphate pathway, was successful in these two cell lines with JFC12T10. A very faint staining was seen for JAR cells while U251 cells were negative using Sigma Prestige polyclonal antibody, thus confirming negative Western blot results. All other cell lines demonstrated much lower tktl1 mRNA expression levels and no detection of TKTL1 protein in cell lysates by anti-TKTL1 antibodies 1C10 and Sigma Prestige, but multiple detections of proteins with apparent molecular weights above or below the calculated size of TKTL1 by anti-TKTL1 antibody JFC12T10. A relation of glucose consumption, lactic acid production and chemo- and radioresistance with expression levels of TKTL1 was not observed in any cell line tested in the present study. JAR and U251 cells did not show outstanding “aggressiveness” as measured by their resistance to both chemotherapeutic drugs and radiation or their ability to produce lactate  has gained increased attention due to several studies showing a TKTL1-positive staining of malignant tumor tissue of different origins , 43, 51 In this study, we tested three commercially available anti-TKTL1 antibodies for Western blot and immunohistochemistry staining as well as three primer pairs used in previously published work for the detection of tktl1 mRNA by RT-qPCR , 28, 35.in vitro.HeLa cells , colorecb anti-TKTL1 antibody clone JFC12T10, first described in 2005 [E. coli with putative smaller cleavage products. Furthermore, JFC12T10 showed a unique expression pattern of five different tumor cell lines with a predominant protein band at 75 kDa by Western blot analysis [Interestingly, the vast majority of studies based on antibody-based techniques published so far used the commercially available mouse monoclonal IgG2 in 2005 . JFC12T1analysis . This inanalysis .The second anti-TKTL1 antibody used for Western blot analysis was the monoclonal antibody clone 1C10 that detected a distinct single TKTL1 protein with approximately 65 kDa in HEK293-TKTL1 transfectants, without the additional protein bands seen with JFC12T10. Also, polyclonal Sigma Prestige antibody did not recognize additional proteins with different molecular weights in any of the cell lines. The marginal signals in a few cell lines seen with monoclonal 1C10 antibody most probably represents unspecific background, which was eventually similar in quality but inferior in quantity as compared to JFC12T10. Thus, monoclonal 1C10 and polyclonal Sigma Prestige antibody detect exogenously expressed TKTL1 in a highly specific manner and as a protein of expectedly 65.4 kDa.However, using a rabbit polyclonal antibody (Gene Tex), Li et al. detected endogenous TKTL1 expression in MCF-7 and HeLa cells by Western blot . This reSimilarly, immunohistochemistry analyses presented here support a rather rare expression of TKTL1 protein in cancer and normal cell lines. In 2 out of 3 benign and 8 out of 17 malign cell lines tested in the present study, TKTL1 protein was detectable in the cytoplasm with the two antibodies JFC12T10 and SigmaPrestige polyclonal antibody by immunohistochemistry. For only one of these positively stained cell lines (JAR) a weak expression of tktl1 mRNA was detectable. Therefore JAR appears to be positive for endogenous TKTL1 expression by RT-qPCR and immunohistochemistry, however failed to be detected by Western blot. The other cell line expressing tktl1 mRNA, U251, was positive in immunohistochemistry with the JFC12T10 antibody only and also showed no specific signal in Western blot.in vitro (Warburg effect) nor greater resistance against taxane and cisplatin or ionizing radiation than cells without clear TKTL1 detection. These findings are inconsistent with the hypothesis of TKTL1 being a predictor of an increased “Warburg effect” and special robustness of tumor cells against chemotherapy and radiation in vitro.In summary, two cell lines  were found positive for both endogenous mRNA expression as well as TKTL1 protein by immunohistochemistry with one or two different anti-TKTL1 antibodies. Intriguingly, these cell lines did neither exhibit an extraordinary production of lactic acid at 21% oxygen in vitro. TKTL1 expression emerged as a rather rare event in cultured cell lines. The two out of 17 human cancer cell lines JAR and U251, positive for tktl1 mRNA and TKTL1 protein by immunohistochemistry and Western blot for at least one antibody showed neither an outstanding production of lactic acid nor increased resistance against the chemotherapeutic drugs paclitaxel and cisplatin or to ionizing radiation, respectively, compared to the other cell lines.In this study we have demonstrated the necessity to carefully prove TKTL1 expression on mRNA and protein levels with multiple analytic tools  to identify cells with robust endogenous TKTL1 expression Additional file 1: Figure S1: Long time exposure of Western Blot. To make the WB results comparable, we exposed the x-Ray film to an extent, that the positive control cells (HEK293-TKTL1) resulted in a comparable intensity of band signal at 65.4 kDa. The negative HEK293 control cells as well as the tktl1 mRNA positive cell lines JAR and U251 were also shown. (PPT 10 MB)5 cells each. Summary of three independent experiments. (DOC 40 KB)Additional file 2: Table S1: Concentrations of glucose and lactate. Mean and standard deviation is shown for glucose consumption and lactate production of cell lines analyzed after 24 h of culture calculated per 10


PIK3CA mutations. In telomerase-immortalized normal urothelial cells (TERT-NHUC), ectopic expression of mutant PIK3CA induces PI3K pathway activation, cell proliferation and cell migration. However, it is not clear whether advanced UC tumors are PIK3CA-dependent and whether PI3K pathway inhibition is a good therapeutic option in such cases.Many urothelial carcinomas (UC) contain activating We used retrovirus-mediated delivery of shRNA to knock down mutant PIK3CA in UC cell lines and assessed effects on pathway activation, cell proliferation, migration and tumorigenicity. The effect of the class I PI3K inhibitor GDC-0941 was assessed in a panel of UC cell lines with a range of known molecular alterations in the PI3K pathway.in vivo tumor growth of cells with PIK3CA mutations. Sensitivity to GDC-0941 was dependent on hotspot PIK3CA mutation status. Cells with rare PIK3CA mutations and co-occurring TSC1 or PTEN mutations were less sensitive. Furthermore, downstream PI3K pathway alterations in TSC1 or PTEN or co-occurring AKT1 and RAS gene mutations were associated with GDC-0941 resistance.Specific knockdown of PIK3CA inhibited proliferation, migration, anchorage-independent growth and PIK3CA is a potent oncogenic driver in many UC cell lines and may represent a valuable therapeutic target in advanced bladder cancer.Mutant The online version of this article (doi:10.1186/s12885-016-2570-0) contains supplementary material, which is available to authorized users. Advanced urothelial carcinoma (UC) of the bladder has a poor prognosis. At presentation, 15–30 % of UC patients are diagnosed with muscle-invasive tumors, and these carry a 5-year risk of death ranging from 33 to 73 % , 3, but PIK3CA, [AKT1 [PTEN [PIK3R1 [TSC1 and TSC2 [TSC1 or mTOR mutations [TSC1 mutations have shown responses [PIK3CA mutation. Taken together, it is clear that a thorough understanding of the signaling events initiated by the PI3K pathway is required in order to maximize clinical benefit.The phosphatidylinositol 3-kinase (PI3K) signaling pathway plays a critical role in regulation of cell metabolism, migration, proliferation and survival  and mutaPIK3CA, , 9 and AA, [AKT1 , and inaT1 [PTEN , 12, PIK [PIK3R1 , TSC1 anand TSC2 . Assessmand TSC2 . These tutations , 17. In utations , and indesponses . A potenesponses . Inhibitesponses , 21. ImpPIK3CA represent the most frequent PI3K pathway mutations in this cancer type, including 12–20 % of muscle-invasive tumors [PIK3CA or loss of PTEN function are related to sensitivity to inhibitors of class I PI3K and that mutations in RAS genes are associated with resistance , thoughPreviously we examined the effect of ectopic expression of mutant PIK3CA in telomerase-immortalized normal human urothelial cells (TERT-NHUC) and showed that this induces cell proliferation and migration . In blad2. TERT-NHUC [2). All cells were tested routinely for mycoplasma whilst in culture and before freezing by PCR using PCR Mycoplasma Test Kit I/C (PromoKine PK-CA91-1048) according to the manufacturer’s protocol.Cell lines with known PI3K pathway mutation status were chosen /2; where a is the smaller and b is the larger diameter of the tumor) up to day 43. Tumor volume is shown in mm3.Xenografts were established in mice by subcutaneous inoculation of VM-CUB-3 cells with PIK3CA knockdown and control cells (NS shRNA knockdown). Pure strain male BALB/cOlaHsd-Fox1escribed . Each ceIn Situ Apoptosis Detection Kit; Chemicon) and analyzed as described previously [Tumors were formalin-fixed and embedded in paraffin wax. Sections were stained with haematoxylin and eosin, anti-human Ki-67 proliferation-associated antibody (Dako) and for apoptosis using the terminal deoxynucleotidyl transferase–mediated dUTP nick-end labelling (TUNEL) assay , was used to treat bladder cancer cell lines and TERT-NHUC. The dose range chosen was based on previous studies that report ICpatients , 41. CelCell cycle and apoptosis analysis of cells cultured with 1 μM GDC-0941 or DMSO only for 48 h was evaluated by flow cytometry as described . All ass50 data was analyzed using the Fisher exact test  and Student’s t test was used to calculate significance of sensitivity of cells to GDC-0941 relative to PIK3CA wildtype and mutant status. Analysis of variance (ANOVA) and Student’s t-test  were also used and P values were adjusted for multiple testing using the Bonferroni method. P < 0.05 was accepted as significant. All tests were conducted using Prism software, except for the Fisher exact test, which was conducted in R for Mac OS X 3.2.1.Tumor growth was analysed using the Mann-Whitney U-test. Drug ICPIK3CA , the most commonly mutated codon of PIK3CA in UC, and no known additional PI3K pathway aberrations  were designed and validated. These and a non-specific shRNA (NS) and empty vector (control) were retrovirally transduced into the UC cell lines VM-CUB-3, BFTC909 and 253J, all of which have mutations in codon E545 of  and KD2 P < 0.05). shRNA KD1 expression had the most profound effect in all 3 cell lines, compatible with it’s more significant effect on PIK3CA expression , with cells expressing KD1 shRNA showing the greatest reduction    Fig. . VM-CUB-01) Fig. . No signin vivo in nude mice [3) was measured from day 5 to day 43. p110α-KD xenografts showed significantly reduced growth rate compared with controls  .VM-CUB-3 cells can produce tumors ude mice . We test05) Fig. . StudentP < 0.05)  Sections were stained for Ki67 and TUNEL to assess effects on proliferation and apoptosis, respectively. A significant decrease in the proliferative index of p110α-KD tumors was found  Fig.  but signPIK3CA, PIK3R1, AKT1, TSC1, PTEN and the three RAS genes [PIK3CA, PIK3CB and PIK3CD were available for these and for normal human urothelial cells . In 13 of 17 lines, expression of PIK3CA was >2-fold higher than in normal urothelial cells. Six of these also showed >2-fold upregulation of PIK3CB. Expression of PIK3CD was >2-fold lower than in normal urothelial cells in all but two cell lines , on a panel of UC cell lines and TERT-NHUC. We selected a panel of 17 UC cell lines with known mutation status for AS genes , 13, 42 50) to this compound were within the same range as seen in other cancer cell types [PIK3CA showed IC50 values from 0.4 μM to 1.25 μM  or NRAS Q61R (HT-1197) mutations. Overall cell lines with wild-type PIK3CA were significantly less sensitive to GDC-0941 treatment than mutant PIK3CA cell lines . Figure PIK3CA mutation status . The two cell lines with PIK3CA mutation but minimal response to the drug contained either homozygous deletion of PTEN (J82) or mutation of TSC1 (639V). Furthermore, unlike the more GDC-0941-sensitive cell lines that harbor hotspot PIK3CA mutations, these two lines contain rare PIK3CA mutations (P124L and A1066V), which have been shown previously to be 5 times less potent than hotspot mutants in activating the PI3K pathway [Cells were cultured in 0-2.0 μM GDC-0941 for 72 h and cell viability measured relative to untreated controls. Sensitivity values  than cell lines with mutant PIK3CA. Three wildtype PIK3CA cell lines showed similar sensitivities to GDC-0941 treatment as TERT-NHUC. These cell lines harbored a single AKT1 (MGH-U3) or TSC1 (97-1) mutation or both PTEN and RAS mutations (UM-UC3). Four wildtype PIK3CA cell lines  showed varying degrees of GDC-0941 resistance (<10 % viability reduction at 2 μM), with RT4 (TSC1 mutation) showing extreme resistance. Of these, DSH1 and LUCC1 have loss of PTEN, KU-19-19 has mutant AKT1 and NRAS mutations. Therefore, downstream PI3K pathway alterations in TSC1 or PTEN or co-occurring AKT1 and RAS gene mutations were associated with GDC-0941 resistance.TERT-NHUC showed a higher ICPTEN deleted) exhibited similar levels of pAKT reduction (3 %). Treatment of DSH1 (PTEN deleted) reduced AKT phosphorylation to 24 % of untreated levels. However, treatment of the resistant cell line KU-19-19 had little effect on AKT phosphorylation levels. This may be due to the presence of two mutations in AKT1 (E17K and E49K), which was shown to increase AKT activity in comparison to the single mutant E17K found in the sensitive MGHU3 cell line [Phosphorylation of AKT at Ser473 was examined in cell lines treated for 1 h with GDC-0941. Drug treatment reduced AKT phosphorylation to 0.5–7 % of that in untreated controls in all sensitive cell lines Fig. . Of the ell line . Interesell line .50 values up to 1.2 μM, and TERT-NHUC, were assessed for effects on cell cycle distribution and apoptosis. An increase in the proportion of cells in G1 accompanied by a decrease in S phase was observed in all cell lines except 253J and HT-1197 after 48 h of drug exposure, with the least effect in TCCSUP . 253J and HT-1197 showed an increased fraction of cells in G2/M   Fig.  & b.Fig.PIK3CA are found in bladder tumors of all grades and stages. Whilst these are more common in tumors of low grade and stage (26–34 %) they are also found at significant frequency (12–20 %) in advanced UC (> stage T2) [Activating mutations of tage T2) , 9, 22  or mutation of TSC1 (639V), suggesting that co-existing PTEN or TSC1 mutations confer some resistance to PI3K inhibition. Cell lines with wildtype PIK3CA were less sensitive or lacked sensitivity to GDC-0941 completely, in agreement with studies in breast cancer, multiple myeloma, lung cancer, and endometrial cancer, where PIK3CA mutations are also frequent [PIK3CA mutations (J82 and 639V) or those with PTEN and RAS gene or TSC1 mutations (UMUC3 and RT4) had IC50 values above 2 μM (classed as resistant).The class IA PI3K inhibitor GDC-0941 greatly reduced cell viability of UC cell lines with hotspot l models , 41, 51.frequent , 26, 51.frequent . HotspotPTEN mutation on sensitivity to class I PI3K inhibition. For example, in non-small cell lung carcinoma, endometrioid and breast carcinoma cell lines, PTEN loss of expression or mutation was associated with sensitivity to GDC-0941 [PTEN, AKT1 and TSC1 mutant UC cell lines were less sensitive than those with PIK3CA mutations, and in the case for RT4 (TSC1 mutant), resistant to PI3K inhibition.As reported in several other tumor types, additional information on PI3K pathway mutational status is needed to correctly predict the response to PI3K inhibition. There have been conflicting reports of the effect of GDC-0941 , 26, 51 GDC-0941 , PTEN loPIK3CA and NRAS mutations, was sensitive to GDC-0941 but other lines with RAS mutations co-occurring with PTEN or AKT1 mutations were less sensitive. This is likely to be related to relative dependence on RAS-MAPK and PI3K pathways in individual cases. Recent observations have highlighted the effect of context-dependent crosstalk on MEK signaling associated with inhibition of PI3K in breast cancer [PIK3CA [Similarly, in some studies RAS mutation has been reported to predict resistance to PI3K inhibition and in others to have no impact on sensitivity , 52. Hert cancer , 54. Int [PIK3CA . As manyIn this study, shRNA knockdown of PIK3CA in three cell lines clearly demonstrated dependence on this p110 isoform. However, as many of the cell lines examined expressed PIK3CB, we cannot rule out that the effects of GDC-0941 on PIK3CB may have contributed to the observed effects of this inhibitor. As p110α-specific drugs e.g. NVP-BYL719 , are nowTSC1, this implies that only the mTOR branch of the pathway is active in this case, as shown previously [PIK3CA mutant cancers rely on effectors other than AKT, such as PDK1 and its substrate SGK3 [Levels of pAKT (Ser473) were decreased by GDC-0941 treatment, independent of whether a cell line was sensitive or resistant, as previously reported , 27, 56.eviously . As manyate SGK3 , phosphoThe effect of GDC-0941 in the majority of sensitive bladder cancer cells was via induction of both G1 cell cycle arrest and some apoptosis. Thus in some contexts, single agent GDC-0941 may exert cytotoxic and cytostatic effects in UC as seen in other tumor models , 27, 58.NRAS mutations. The first in human phase I clinical study of GDC-0941 in patients with advanced solid tumors has recently been completed and showed promising results with minimal toxicities at doses sufficient to decrease PI3K pathway activation [PIK3CA mutations and that mutation can act as a biomarker for patient selection. Additional preclinical work is warranted to assess the impact of dual mTOR-PI3K and MEK-PI3K inhibition on a large panel of UC cell lines with a range of PI3K/AKT and MEK/ERK pathway genetic alterations to fully understand the context for optimal therapy of PI3K pathway-dependent bladder tumors. Currently, trials of GDC-0941 in combination with other agents suggested by preclinical studies, such as paclitaxel and inhibitors of MEK1 and EGFR [Our observations indicate a good therapeutic window for PI3K inhibitors in some bladder cancers that harbor PI3K hotspot mutations, as well as those with co-existing tivation . In UC, and EGFR –61, are KD, knockdown; NS, non-specific; PI, propidium iodide; PI3K, phosphatidylinositol 3-kinase; TCC, transitional cell carcinoma; TERT-NHUC, telomerase-immortalized normal urothelial cells; UC, urothelial carcinoma.


Many advanced human tumors, including hepatocellular carcinomas (HCC) are auxotrophic for arginine due to down-regulation of argininosuccinate synthetase (ASS1), the rate-limiting enzyme in arginine synthesis. The arginine-lowering agent PEGylated arginine deiminase (ADI-PEG 20) has shown efficacy as a monotherapy in clinical trials for treating arginine-auxotrophic tumors and is currently being evaluated in combination with cisplatin in other cancer types. Epigenetic silencing via methylation of the ASS1 promoter has been previously demonstrated in other cancer types, and a reciprocal relationship between ASS1 expression and cisplatin resistance has also been observed in ovarian cancer. However, the mechanism of ASS1 down-regulation, as well as the correlation with cisplatin resistance has not been explored in HCC. The present study investigates ADI-PEG 20 and cisplatin sensitivities in relation to ASS1 expression in HCC. In addition, we show how this biomarker is regulated by cisplatin alone and in combination with ADI-PEG 20.ASS1 protein expression in both untreated and drug treated human HCC cell lines was assessed by western blot. The correlation between ASS1 protein levels, ADI-PEG 20 sensitivity and cisplatin resistance in these cell lines was established using a luminescence-based cell viability assay. Epigenetic regulation of ASS1 was analyzed by bisulfite conversion and methylation-specific PCR.A good correlation between absence of ASS1 protein expression, ASS1 promoter methylation, sensitivity to ADI-PEG 20 and resistance to cisplatin in HCC cell lines was observed. In addition, cisplatin treatment down-regulated ASS1 protein expression in select HCC cell lines. While, at clinically relevant concentrations, the combination of ADI-PEG 20 and cisplatin restored ASS1 protein levels in most of the cell lines studied.ASS1 silencing in HCC cell lines is associated with simultaneous cisplatin resistance and ADI-PEG 20 sensitivity which suggests a promising combination therapeutic strategy for the management of HCC. Arginine, a semi-essential amino acid in humans, is critical for the growth of human cancers . TumoralThe significance for ASS1 loss in cancer is currently unclear; however, several groups have revealed that reduced expression of ASS1 is a predictive biomarker for the development of metastasis and is associated with a worse clinical outcome –25. EpigHCC is one of the most common cancers in the world, especially in Asia and Africa . CisplatPrevious work has shown that the sensitivity of HCC cell lines to ADI-PEG 20 is due to the absence of ASS1 . However2.The following human HCC cell lines were obtained from Dr. Yuh-Shan Jou at the Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan: Sk-Hep1, Huh7, Tong, HCC36, Hep3B, Malhavu, PLC5 and Huh6. The human HCC cell lines HepG2, SNU398 and SNU182 were from American Type Culture Collection . A2780 is an ovarian cancer cell line (cisplatin sensitive) derived from a patient prior to treatment and A2780CR is a cisplatin-resistant cell line that was developed by exposure of the parent A2780 cell line to increasing concentrations of cisplatin. Both A2780 and A2780CR cell lines were obtained from Sigma-Aldrich . The following cells were grown in Dulbecco’s Modified Eagle Medium (DMEM)  containing 10% heat-inactivated fetal bovine serum , 1% L-glutamine (Life Technologies) and 1% non-essential amino acids : Sk-Hep1, Huh7, Tong, HCC36, Hep3B, Malhavu, PLC5, Huh6 and HepG2. SNU398, SNU182 and the ovarian cell lines were maintained in RPMI 1640 (Lonza) with 10% heat-inactivated FBS and 1% L-glutamine. All cells were sub-cultured two times a week using trypsin/EDTA (Life Technologies) and were grown at 37°C in 5% CO50) values for ADI-PEG 20 and cisplatin (Sigma-Aldrich) were determined using the CellTiter-Glo (CTG) luminescent cell viability assay . Cells  were plated in 100 μL medium/well in 96-well black micro-clear plates . Following overnight incubation at 37°C and 5% CO2, cells were exposed to a range of drug concentrations from a 50X plate (2 μL/well). Each concentration of drug was added to duplicate wells. After 72 h incubation, 25 μL/well of CTG reagent was added directly to the medium and the plates were shaken for 5 min, resulting in cell lysis and the generation of a luminescent signal proportional to the amount of ATP present. The luminescence values were read on a SpectraMax M3 microplate reader  and converted to a percent cell viability that was calculated relative to the viability in corresponding matched DMSO-treated cells, which was designated as 100% viable. IC50 values  were obtained from nonlinear regression analysis of concentration-effect curves using GraphPad Prism version 6.0 software .Cell viability , with added protease inhibitor cocktail  and PMSF (Sigma-Aldrich). Total lysate protein was quantified using a Coomassie Plus (Bradford) Protein Assay Reagent . Cell extracts (20 μg) were run on NuPage 4-12% Bis-Tris Gels (Life Technologies) and then transferred to PVDF membranes (Life Technologies). The membranes were blocked in TBST buffer  containing 5% Blotting-Grade Blocker  for 2 h at room temperature and then probed using a mouse monoclonal antibody to ASS1  at a dilution of 1:500. GAPDH was used as a loading control for each western blot, so the membranes were cut and also probed with a rabbit polyclonal antibody to GAPDH  at a dilution of 1:10,000. The blots were incubated with both primary antibodies overnight at 4°C on a rocker. After washing with TBST buffer, the membranes were incubated with secondary antibodies: goat anti-mouse for ASS1   and goat anti-rabbit for GAPDH (Santa Cruz Biotechnology)  and incubated at room temperature for 1 h. The secondary antibodies were detected using either the SuperSignal West Pico (GAPDH) or Femto (ASS1) Chemiluminescent Substrate (Pierce) and blots were read on a Bio-Rad ChemiDox XRS + System. ASS1 and GAPDH levels were quantified using Image Lab Software .For ASS1 protein determination after cisplatin treatments or for ADI-PEG 20 and cisplatin combination analysis, the same procedure was used with the following modifications. Cells were plated in two identical 96-well plates: one for cell viability and/or normalization for cell numbers between wells  and one for lysis (ASS1 detection). After 72 h drug treatments, lysates were made and probed for protein analysis. For ASS1 and GAPDH detection, media was removed and each well of the microplate was washed with 100 μL of PBS buffer (Gibco by Life Technologies). NuPage LDS sample buffer  containing 50 mM DTT was then added to each well and the plate was wrapped in parafilm and frozen at -80°C for at least one hour to ensure lysis. After lysis, the samples in each well were spun and then used for immunoblot analysis. To account for the different number of viable cells in each well of the microplate, samples were normalized using the relative luminescence values for each corresponding well of the identical microplate.The EZ DNA Methylation-Direct Kit  was used to perform complete DNA bisulfite conversion directly from the human cell lines. This process converts unmethylated cytosine residues to uracil while methylated cytosine residues remain unchanged. In general, 10,000-40,000 cells (~60-250 ng genomic DNA) were used as starting material for each cell line. Methylation-specific PCR (MSP) of a 188 bp fragment located between 300 and 500 bp downstream of the transcription start site (TSS) was then performed to determine the methylation status of the ASS1 promoter. Bisulfite-modified DNA (150 ng) was used as a template for PCR reactions with primers specific for methylated (M) or unmethylated (UM) sequences. Primer sequences are: (1) M forward: 5′-TTTTTTTCGTTGTTTATTTTTTAGTC-3′; (2) M reverse: 5′-CTAAAATCCGATACCAAACGTT-3′; (3) UM forward: 5′-TTTT TGTTGTTTATTTTTTAGTTGA-3′ and (4) UM reverse: 5′-AACCTAAAATCCAATACCAAACATT-3′. Primers were purchased from IDT Technologies . PCR conditions for the methylated primers were as follows: 8 cycles of 95°C for 2 min, 54.8°C for 30 sec and 72°C for 30 sec were followed by 32 cycles of 95°C for 30 sec, 54.8°C for 30 sec and 72°C for 30 sec, then a final extension at 72°C for 10 min. The PCR conditions for the unmethylated primers were identical, except the annealing temperature was 48°C instead of 54.8°C. The HotStarTaq d-Tect polymerase  was used for the PCR reactions. The PCR samples were run on 2% pre-cast agarose E-gels (Life Technologies) containing a fluorescent stain for visualization. The human methylated and non-methylated DNA set (Zymo Research) were used as negative and positive controls for bisulfite conversion efficiency, and MSP was similarly performed using a set of control primers designed to amplify non-methylated, methylated and mixed methylation copies of the death-associated protein kinase 1 gene (DAPK1), with an expected size of 274 bp.t-test. A p value < 0.05 was set as a level of statistical significance. In determining statistical significance for ASS1 protein levels after cisplatin treatments or for ADI-PEG 20 and cisplatin combination analysis, each drug concentration was compared to the untreated, or zero drug, sample to attain a p value for that particular drug concentration.GraphPad Prism version 6.0 was used to test results for statistical significance. Differences in ASS1 levels between groups were analyzed using an unpaired two-tailed 50 values around 1 nM , while this drug only kills 50% of the ASS1-medium HepG2 cells at the same concentration. In contrast, the ASS1-high line Malhavu is completely resistant to ADI-PEG 20.To study the relationship between ASS1 expression and sensitivity to ADI-PEG 20 and cisplatin, we first screened 11 human HCC cell lines for this protein. The western blot shown in Figure 50 value above 30 μM . In addition, cisplatin IC50 values have been obtained for multiple cell lines from three other cancer types  and no such trend is observed, at least in the cell lines tested (data not shown). Thus, this reciprocal relationship between ASS1 expression and cisplatin may be exclusive to HCC and ovarian cancer. In summary, our data thus far indicate that ADI-PEG 20 is effective at killing ASS1-negative HCC cells, while cisplatin is effective at killing ASS1-positive HCC cells. This observation suggests that their combination should be an efficacious treatment for heterogeneous cell populations that exist in tumors.A reciprocal relationship between ASS1 expression and platinum resistance has been previously observed in ovarian cancer , 36. IntEpigenetic silencing via promoter CpG methylation in cell lines lacking ASS1 expression has been demonstrated in multiple cancer types , 26, 27.50 values for cisplatin in the HepG2 and HCC36 cell lines are 4.7 μM and is 2.9 μM, respectively. Thus, the ASS1 levels drop by approximately 50% relative to zero drug at this concentration of cisplatin in the HepG2 cell line and 40% in the HCC36 cells  of the three cell lines we chose to investigate. Because cisplatin is more potent in this cell line than the others, there is significant cell death at the higher concentrations of drug, and we found it challenging to use luminescence values to compare the total ASS1 and GAPDH protein at both 15 μM and 30 μM cisplatin with that in the lower concentration wells. For this reason, we have only quantified ASS1 to a concentration of 7.5 μM in the SNU182 cell line , suggesting the mechanism of cisplatin down-regulation during these acute drug treatments is not epigenetic-based.We have previously demonstrated a reciprocal relationship between ASS1 expression and cisplatin resistance in our HCC cell lines. Therefore, we next investigated whether treatment with cisplatin could down-regulate the ASS1 expression in three representative ASS1-positive HCC cell lines: HepG2, HCC36 and SNU182. Cells were treated in 96-well microplates with increasing cisplatin concentrations for 72 h, and luminescence values, indicative of cell viability, were then used to load similar amounts of protein across all of the cisplatin-treated wells. ASS1 protein expression was subsequently determined by immunoblot analysis and normalized to GAPDH protein at each cisplatin concentration. Figure 50 value. The same experiment was performed with the HCC36 cell line and we observe a similar trend . Thus, for the HCC36 cell line, the ASS1 levels will also be determined by the ADI-PEG 20 at a cisplatin concentration around its IC50 value.The data thus far demonstrate a correlation between low ASS1 expression, resistance to cisplatin and sensitivity to ADI-PEG 20. Such a relationship provides a situation where one drug drives efficacy in an ASS1-negative cell line (ADI-PEG 20) and the other drives efficacy in an ASS1-high cell line (cisplatin), suggesting a favorable combination drug treatment. Our results demonstrate that cisplatin down-regulates ASS1 in three representative HCC cell lines. Thus, we wanted to determine the effect of the addition of ADI-PEG 20 to cisplatin on the ASS1 levels in these same cell lines. To determine the ASS1 expression during combination drug treatment, cells were simultaneously treated with cisplatin and ADI-PEG 20 in the same wells of a microplate, and then analyzed for ASS1 protein expression. Two concentrations of cisplatin that are known to reduce the ASS1 protein expression were chosen (5 μM and 7.5 μM) and increasing concentrations of ADI-PEG 20 were simultaneously added to the same wells. As expected, for HepG2 cells, treatment at both cisplatin concentrations alone causes a decline in ASS1 protein levels by approximately 50%, which agrees with our previous results Figure A. Figured Figure B. In agr50 value for cisplatin than the other 2 cell lines , indicating that cisplatin will dictate ASS1 levels.The last cell line we investigated was SNU182, and surprisingly, we observe a different outcome. At both cisplatin concentrations, the ASS1 levels do not return, even at the highest concentration of ADI-PEG 20 Figure C. The daThe future for the treatment of arginine auxotrophic cancers lies in combination therapies. Several ADI-PEG 20 and cisplatin combination trials are planned. Therefore, understanding the correlation between ASS1 expression and cisplatin and ADI-PEG 20 sensitivities, as well as how ASS1 is regulated by both drugs could provide valuable information for trial design. For the first time, we have shown that there is a reciprocal relationship between ASS1 expression and cisplatin resistance in several human HCC cell lines. We have observed that resistance is specific to cisplatin, as sensitivity to other platinums and chemotherapeutic agents are unaffected by ASS1 expression. In addition, methylation of the ASS1 promoter does associate with sensitivity to ADI-PEG 20, and in HCC, also corresponds with cisplatin resistance, as previously demonstrated in ovarian cancer . These fOur novel data also indicate that cisplatin down-regulates ASS1 protein expression in three HCC cell lines. How exactly cisplatin is affecting ASS1 levels during these acute treatments is currently unknown. Previously published studies indicate that ASS1 regulation occurs at the transcriptional level –40. For 50 value increase for cisplatin in HepG2CR over the parental cell line after only one month of drug exposure. Once we acquire a more permanent resistant phenotype, we will determine the methylation status of HepG2CR and perform other analyses to understand the mechanisms of acquired cisplatin resistance in HCC.Interestingly, there is suggestion that acquired resistance to cytotoxic agents occurs predominantly via epigenetic events , 45. A sSeveral ADI-PEG 20 combination trials are ongoing or planned, including a combination with cisplatin for metastatic melanoma, ovarian cancer and other solid tumors, docetaxel for prostate and non-small cell lung cancer (NSCLC), doxorubicin for breast cancer, and cisplatin and pemetrexed for NSCLC and malignant pleural mesothelioma –25. ThesOur data support the rationale of combining cisplatin and ADI-PEG 20 in the clinical treatment of HCC. We believe these two drugs will be complementary in a clinically relevant heterogeneous tumor. Furthermore, in HCC, sensitivity to ADI-PEG 20 may be superior in cases that have relapsed after cisplatin-based chemotherapy. Extending beyond HCC to other cancers, our results suggest that in the combination setting, a patient does not necessarily need to have an ASS1-deficient tumor to reap benefit from an ADI-PEG 20 and cisplatin drug treatment.


SLC26A4, which encodes pendrin, are responsible for hearing loss with an enlarged vestibular aqueduct and Pendred syndrome. The most prevalent mutation in East Asia is p.H723R (His723Arg), which leads to defects in protein folding and cell-surface expression. Here we show that H723R-pendrin can be rescued to the cell surface by an HSP70 co-chaperone DNAJC14-dependent unconventional trafficking pathway. Blockade of ER-to-Golgi transport or activation of ER stress signals induced Golgi-independent cell-surface expression of H723R-pendrin and restored its cell-surface Cl−/HCO3− exchange activity. Proteomic and short interfering RNA screenings with subsequent molecular analyses showed that Hsc70 and DNAJC14 are required for the unconventional trafficking of H723R-pendrin. Moreover, DNAJC14 upregulation was able to induce the unconventional cell-surface expression of H723R-pendrin. These results indicate that Hsc70 and DNAJC14 play central roles in ER stress-associated unconventional protein secretion and are potential therapeutic targets for diseases such as Pendred syndrome, which arise from transport defects of misfolded proteins.Mutations in  Mutations in pendrin, a plasma membrane transporter, lead to Pendred syndrome, which is associated with hearing loss. Here, Jung et al. show that cell-surface expression of a mutated form of pendrin can be restored by blocking ER-to-Golgi traffic and triggering a DNAJC14 dependent unconventional secretion pathway. SLC26A4 gene is a transmembrane protein that transports monovalent anions, such as Cl−, I−, HCO3−, and formate in the inner ear, thyroid follicles and renal cortical collecting ductsSLC26A4 cause non-syndromic recessive deafness with an enlarged vestibular aqueduct  and Pendred syndrome , and are considered to be the most common cause of hereditary hearing loss in East Asia and the second most common cause worldwide234568−/ HCO3− or Cl−/I− exchange activity at the cell surface is lost.Pendrin encoded by the , MIM 60091) and PHSPA8), a member of heat shock 70 kDa proteins (HSP70s), is one of the premier chaperones involved in this process. Although Hsc70 is a cytosolic chaperone, because most membrane proteins have large cytosolic domains, Hsc70 binds to and censors the folding status of substrate membrane proteins that are synthesized in the ER and transported to the cell surface via the conventional ER-to-Golgi secretion pathway. For example, Hsc70 has been shown to promote folding of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel and also to be involved in the ERAD of the folding-deficient Phe508-deleted (ΔF508) CFTR, the most common cystic fibrosis-causing mutation worldwideThe folding of secretory and transmembrane proteins is monitored by the ER quality control (ERQC) systems. During ERAD, non-native or misfolded proteins that do not pass ERQC are retrotranslocated to the cytoplasm and degraded by the ubiquitin–proteasome machineryThe HSP70 chaperones do not function alone. In fact, co-chaperones of Hsc70, such as Hdj-2 and CHIP, have been shown to participate in the folding, biogenesis and degradation of CFTR1315− channel activity at the cell surfaceRecently, it was found that ΔF508-CFTR can be expressed on the cell surface via an unconventional secretion pathway under specific cellular conditionsHere we report that the clinically important, folding-deficient membrane protein H723R-pendrin can be rescued to the cell surface by the unconventional protein secretion pathway. More importantly, the HSP70 co-chaperone DNAJC14 plays an essential role in allowing H723R-pendrin to escape ERAD and be expressed on the cell surface. These results provide insights into the mechanism of ER stress-associated unconventional protein secretion. Furthermore, identification of DNAJC14 as a factor targeting entry of a folding-deficient membrane protein into the unconventional protein secretion pathway will have an impact on the development of therapeutic strategies for diseases arising from transport defects of misfolded proteins.Pendrin is a glycosylated membrane protein that undergoes ER core-glycosylation  and Sar1 (a component of COPII), or by overexpression of syntaxin 5 (required for the fusion of COPII transport vesicles with the acceptor Golgi membrane)Similar to the case of ΔF508-CFTR−/HCO3− exchange activity was examined in a clone of HEK 293 cells that has minimal intrinsic Cl−/HCO3− exchange activity , but not H723R-pendrin . Notably, blockade of the ER-to-Golgi transport by Arf1-Q71L significantly increased the H723R-pendrin-mediated Cl−/HCO3− exchange activity to 0.060±0.011 ΔpH unit min−1 , which corresponded to 26% of the WT-pendrin activity. Taken together, these results indicate that ER-to-Golgi blockade or ER stress induces the functional cell-surface expression of core-glycosylated H723R-pendrin via a GRASP-independent route that bypasses the Golgi.To determine whether the core-glycosylated H723R-pendrin transported to the cell surface was functional, the pendrin-dependent Clactivity . The Cl−To identify the components involved in the unconventional cell-surface transport of core-glycosylated pendrin, we first examined the binding partners of H723R-pendrin using tandem affinity purification (TAP) with mass spectrometryWe then further examined the role of Hsc70 in the unconventional secretion of core-glycosylated pendrin. Immunoprecipitation results revealed that both core-glycosylated WT-pendrin  and H723In the canonical model of the ATP-dependent binding and release cycle of HSP70 substrates, J proteins and nucleotide exchange factors (NEFs) are key regulators of HSP70 functionTo further substantiate our observations, we evaluated the surface expression of pendrin using immunofluorescence analysis. In permeabilized control cells , WT-pendNext, we examined whether DNAJC14 upregulation can rescue the transport defects caused by the H723R-pendrin mutation. Control experiments with WT-pendrin indicated that neither overexpression of Hsc70 and DNAJC14 nor inhibition of Hsc70-ATPase activity by Hsc70-K71M affected the conventional surface transport of mature WT-pendrin . In cont−/HCO3− exchange activity of H723R-expressing PANC-1 cells from 0.039±0.015 ΔpH unit min−1 to 0.201±0.034 ΔpH unit min−1 (P<0.01)  . Measure-pendrin . Co-exprThe immunostaining results were consistent with the surface biotinylation and functional data. DNAJC14, alone or together with Hsc70, induced the cell-surface expression of H723R-pendrin in non-permeabilized cells. In addition, Hsc70-K71M blocked the DNAJC14-mediated effect and DNAJC14-H471Q was not effective in inducing surface trafficking of H723R-pendrin . Then, wMost signal peptide-containing proteins, including transmembrane proteins, are synthesized in the ER and reach the plasma membrane by following the well-documented Golgi-mediated secretion pathway. However, recent evidence suggests that many cytosolic, nuclear, secretory and membrane proteins reach the cell surface by unconventional secretion pathwaysHsc70 is a constitutively expressed molecular chaperone and achieves functional diversity based on specific cooperation with co-chaperones. Hsc70 facilitates biogenesis and stabilization of CFTR in cooperation with Hdj1 (DNAJB1), Hdj2 (DNAJA1) and HOP (Hsp-organizing protein)13272930312 and COOH termini and major functional domains, including the J domain, are exposed to the cytosolAn important finding of the present study is the novel role of DNAJC14 in regulating the function of Hsc70. DNAJC14 was known to modulate the cell-surface trafficking of dopamine D1 receptor, the SNARE complex-mediated lysosomal trafficking, and the specific membrane targeting of yellow fever virus replication proteins2433Interestingly, conditions that induced unconventional cell-surface expression of CFTR and pendrin did not evoke unconventional secretion of the transferrin receptor , indicatn=11). These findings may raise the possibility that accumulation of H723R-pendrin in the ER evokes nonspecific leakage of protein to the cell surface. However, several lines of evidence indicate that this is not the case and that a specific regulatory pathway is involved in the ER-to-Golgi blockade-induced cell-surface expression of H723R-pendrin. First, inhibition of Hsc70 or DNAJC14, which did not affect the cytosolic levels of H723R-pendrin, abolished the unconventional secretion of H723R-pendrin /streptomycin (50 μg ml−1). The mammalian expression plasmids for WT- and H723R-pendrin were subcloned into pcDNA3.1(+) (Invitrogen) and TAP expression vectors  from pCMV-myc-human pendrin described previouslyPANC-1 and HEK 293 cells were maintained in Dulbecco's modified Eagle medium-HG  supplemented with 10% fetal bovine serum and penicillin . Apoptozole was purchased from Millipore . Anti-pendrin , anti-HA, anti-Myc, anti-aldolase and anti-Flag antibodies were from Santa Cruz Biotechnology ; anti-pendrin , anti-calnexin, anti-giantin, anti-Lamp1, anti-CHMP2B, anti-ATG7 and anti-Hsc70 were from Abcam ; anti-EEA1 was from BD Biosciences; anti-CD9 was from LSBio ; anti-DNAJC14 was from Novus ; anti-Rab7 was from Cell Signaling Technologies ; and anti-CFTR antibody (M3A7) was from Millipore. Detailed information for primary and secondary antibodies used in this study is summarized in N-glycosidase F , protein samples were incubated with PNGase F (500 U per reaction) in solutions containing 1% Triton X-100 for 2 h.For immunoprecipitation, cell lysates were mixed with the appropriate antibodies and incubated overnight at 4 °C in a lysis buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% (v/v) Triton X-100, and complete protease inhibitor mixture . Immune complexes were precipitated using mixed protein A/G beads  and then washed three times with lysis buffer before elution with 2 × sodium dodecyl sulfate (SDS) sample buffer. Protein samples were separated by SDS-polyacrylamide gel electrophoresis (PAGE). The separated proteins were transferred to a nitrocellulose membrane and blotted with appropriate primary and secondary antibodies. Protein bands were detected by enhanced chemiluminescence, and the densities of the bands were measured using imaging software . For surface biotinylation assay, the surface proteins were biotinylated with sulfo-NHS-SS-biotin (Pierce) for 30 min before cell lysis. Cells were washed with quenching buffer containing 1% bovine serum albumin (BSA) and then washed three times with phosphate-buffered saline (PBS). After cell lysis, the lysates were incubated overnight at 4 °C with 10% NeutrAvidin beads (Pierce). NeutrAvidin-bound biotinylated proteins were centrifuged and washed three times with PBS and then eluted in 2 × sample buffer. The next steps of the biotinylation assay were identical to those for the immunoblotting procedure described above. For the digestion of glycosylated pendrin by Peptide-−1. For tandem mass spectrometry, the full mass scan range mode was an m/z of 450–2,000 Da. After determination of the charge states of an ion using zoom scans, product ion spectra were acquired in MS/MS mode with a relative collision energy of 55%. The individual MS/MS spectra were processed using the TurboSEQUEST software . The generated peak list files were used to query either the MSDB or NCBI database using the MASCOT program (http://www.matrixscience.com). Modifications of methionine and cysteine  with a peptide mass tolerance of 2 Da, MS/MS ion mass tolerance of 1 Da, allowance of missed cleavage of 1, and charge states of +1, +2, and +3, were all taken into account. Only significant hits as defined by MASCOT probability analysis were considered initially.TAP was performed using TAP expression vector with two tandem affinity tags, a streptavidin-binding peptide and a calmodulin-binding peptide (Stratagene). Cell lysates were applied to streptavidin resin, and unbound proteins were washed three times with streptavidin-binding buffer according to the manufacturer's protocol. Eluted protein complexes were applied to calmodulin resin and contaminants were removed by washing them three times with calmodulin-binding buffer. Tagged proteins and interacting partners were eluted with elution buffer, and eluted proteins were separated by SDS–PAGE. Protein bands of interest were excised and digested with trypsin using the following protocolPANC-1 cells were cultured on coverslips and immunostained 1 day after transfection. Cells grown on coverslips were fixed and permeabilized by incubation in cold ethanol and acetone (1:1) for 8 min at −20 °C. Nonspecific binding sites were blocked by incubation with 0.2 ml PBS containing 5% horse serum, 1% BSA and 0.1% gelatin (blocking medium) for 1 h at room temperature. After the blocking step, cells were stained by incubating with appropriate primary antibodies and then treated with fluorophore-tagged secondary antibodies. Some cells were treated with primary and secondary antibodies after fixation in 4% formaldehyde without permeabilization to stain pendrin on the cell surface. Fluorescence images were obtained with a Zeiss LSM 780 confocal microscope . Morphometric analysis of the captured confocal images was performed using the MetaMorph microscopy analysis software . For the quantification of the images under each condition, 24-bit confocal images containing red, green and blue components were converted into three 8-bit mono-channel images. The colocalization of DNAJC14 and each marker protein was analysed using the colocalization module of the MetaMorph Apps package. For the analyses of the surface expression of pendrin in non-permeabilized cells, pixels above the threshold level of 30 were defined as surface pendrin positive.t) method with GAPDH as the control as follows: ΔCt=Ct(GAPDH)−Ct(target gene). The fold-change in gene expression normalized to GAPDH and relative to the control sample was calculated as 2−ΔΔCt.Quantitative real-time PCR (qPCR) was performed to examine the efficiency of siRNA treatments  and to qi in HEK 293 and PANC-1 cells were performed with the pH-sensitive fluorescent probe 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). After a 24–48-h transfection with plasmids encoding WT or H723R pendrin, cells were incubated with 2 μM BCECF acetoxy-methylester for 5 min and then perfused with a HCO3−-buffered solution . BCECF fluorescence was recorded at the excitation wavelengths of 490 and 440 nm at a resolution of 2/ s−1 on a recording setup . Cl−/HCO3− exchange activities were estimated from the initial rate of pHi increase as a result of Cl− removal in the HCO3−-containing buffer (25 mM HCO3− with 5% CO2). pHi calibration was done with standard pH solutions containing 150 mM KCl and 5 μM nigericin (Sigma-Aldrich). The intrinsic buffer capacity (βi) was calculated by measuring ΔpHi in response to 5–40 mM NH4Cl pulses in Na+-free solutions. Because the βi values were not significantly affected by transfection with plasmids encoding WT-pendrin, H723R-pendrin, Hsc70 or DNAJC14, the Cl−/HCO3− exchange activities were expressed as ΔpH unit min−1 without compensating for the buffer capacity.Measurements of pH−/I− exchange activity of pendrin was measured with the halide-sensitive fluorescent sensor yellow fluorescent protein (YFP)-H148Q/I152L/F46L .Supplementary Figures 1-10 and Supplementary Table 1-4


In vivo treatment with the drug combination of mice bearing JeKo-1 xenografts (MCL) had a marked antitumor effect with tumor regressions observed at non-toxic doses (best T/C%=0.54%). Gene expression profiling suggested effect on genes involved in apoptosis. The strong synergism observed by combining Chk1 and Wee1 inhibitors in preclinical models of MCL provides the rationale for testing this combination in the clinical setting.Mantle cell lymphoma (MCL) is an aggressive, incurable disease, characterized by a deregulated cell cycle. Chk1 and Wee1 are main regulators of cell cycle progression and recent data on solid tumors suggest that simultaneous inhibition of these proteins has a strong synergistic cytotoxic effect. The effects of a Chk1 inhibitor (PF-00477736) and a Wee1 inhibitor (MK-1775) have been herein investigated in a large panel of mature B-cell lymphoma cell lines. We found that MCL cells were the most sensitive to the Chk1 inhibitor PF-00477736 and Wee1 inhibitor MK-1775 as single agents. Possible involvement of the translocation t in Chk1 inhibitor sensitivity was hypothesized. The combined inhibition of Chk1 and Wee1 was strongly synergistic in MCL cells, leading to deregulation of the cell cycle, with increased activity of CDK2 and CDK1, and activation of apoptosis.  CCND1 gene, encoding cyclin D1, is virtually present in all the cases [CDKN2A, CDK4, TP53 and ATM, involved in cell cycle regulation and DNA damage response [Mantle cell lymphoma (MCL) is one of the most common lymphoma subtypes, accounting for 5–10% of all non-Hodgkin's lymphomas , 3. MCL response , 8. In nresponse , 10. Theresponse –13. Chk1response , 14, 15.response , 12, 16.response , 17–19. response  and combresponse –22. Littin vivo. We provide the first preclinical evidence of Chk1 and Wee1 inhibitors as new therapeutic approach in MCL, which warrants investigation in a clinical setting.We herein performed a cytotoxic screening in 35 B-cell lymphoma cell lines with Chk1 and Wee1 inhibitors. MCL were the lymphoma cell lines most sensitive to Chk1 and to a lesser extent to Wee1 inhibition. Chk1 and Wee1 inhibitors were then combined leading to a strong synergistic cytotoxic effect, affecting cell cycle and apoptosis. The combined treatment also resulted in strong antitumor activity in MCL xenografts We investigated the effects of Chk1 and Wee1 inhibition in a large panel of lymphoma cell lines: 35 mature B-cell lymphoma cell lines comprising ten MCL and 25 DLBCL cell lines (seven ABC-DLBCL and 18 GCB-DLBCL), by treating them with specific Chk1 and Wee1 inhibitors, respectively PF-00477736 and MK-1775. MCL cell lines were significantly more sensitive to PF-0047736 and slightly more sensitive to MK-1775 as compared to DLBCL cell lines Figure . The medTo identify the biologic features determining the highest sensitivity to Chk1 inhibition in lymphomas, we compared the baseline gene expression profiling of 21 of the most sensitive cell lines to PF-00477736 (IC50 < 25 nM) versus the five most resistant cell lines (IC50 > 150 nM). The gene expression profiles of sensitive cell lines appeared significantly enriched of gene-sets involved in cell proliferation . This waSince cell proliferation signatures were associated with a higher sensitivity to Chk1-inhibition and since MCL is the only lymphoma bearing the t11;14) [In order to better elucidate if the high activity of the CDK4/6-cyclin D1 complex is involved in the sensitivity to such inhibitor, we performed a combined treatment of PF-00477736 with a selective inhibitor of the CDK4/6-cyclin D1 complex (PD-0332991)  in JeKo-The effect of the combined treatment with Chk1 and Wee1 inhibitors was then evaluated in all the ten MCL cell lines. The isobologram in Figure We performed FACS analysis in JeKo-1 cells treated for 24 and 48 hrs with the two drugs either individually or combined at concentrations not toxic as single agents. No cell cycle perturbation was observed after the single drugs, while combined treatment caused slight accumulation of cells in S phase with a DNA content between 2N and 4N starting from 24 hrs  and persisting up to 48 hrs after treatment  Figure . In sampin vivo in nude mice bearing JeKo-1-MCL cell line. Oral MK-1775 (30 mg/kg twice a day) and i.p. PF-00477736 (10 mg/kg once daily) were given for 16 consecutive days alone or combined. In addition, considering the striking cytotoxic activity in vitro, we decided to treat a group of mice with the combination at half the doses used as single agents (15 and 5 mg/kg respectively). All these treatments caused no significant body weight loss   Figure , and the) Figure –3. BasedI Figure .in vitro data of induction of apoptosis by the combination.These genes were validated by real time PCR, confirming their increased expression compared to all the other groups Figure . Additioin vitro and in vivo data collected on the synergistic activity of combining Chk1 and Wee1 inhibitors in solid tumors [In recent years the development of Chk1 and Wee1 inhibitors has emerged as an effective strategy to potentiate the cytotoxic effect of chemotherapeutic drugs , 26, 27.d tumors , 20–22 wd tumors , 20. Litd tumors . Chk1 ind tumors , and Weed tumors . Moreoved tumors , 32. Recd tumors .Here, we have explored the effects of Chk1 and Wee1 inhibitors as single agents in a wide panel of B-cell lymphomas. Our data clearly showed that, among all the lymphoma cell lines, MCL cell lines are significantly more sensitive to the Chk1 inhibitor PF-00477736 and, even though to a lesser extent, more sensitive to the Wee1 inhibitor MK-1775. In addition, MCL cell lines were 10 and 6 times more sensitive to Chk1 and Wee1 inhibitors, respectively, than different epithelial carcinoma cell lines . Due to Among MCL models, only one cell line (REC-1) showed a clear resistance to PF-00477736. Although the precise biological mechanisms of this resistance have still to be elucidated, when compared to other MCL cell lines REC-1 appears more resistant to several drugs  . Here, mCCND1 gene, leading to increased activity of the CDK4/6-cyclin D1 complex, which by phosphorylating Rb induces the release of E2F1 transcription factor and the consequent progression of cells into S phase. In MCL and MM with the translocation the control of G1-S transition is further impaired by the increased activity of the CDK2-cyclin E complex related to the CDK4-cyclin D1 dependent sequestration of its inhibitor p27kip and by the frequent presence of CDK4 amplification and p16INK4 inactivation [in vitro synergism also translated to the in vivo setting. Tumor regressions were observed only with the combination of Chk1 and Wee1 inhibitors and not with the single agents. The outstanding tumor growth inhibition (best T/C%: 0.54%) occurred at half of the doses used in our previous in vivo experiment in ovarian cancer xenografts [in vivo, but it was not observed at the end of the second cycle of combination treatment after which a lower effect of the combination was observed. Gene expression profiling also showed an effect on genes involved in apoptosis: four genes involved in apoptosis were found to be up-regulated only during the first cycle of combined treatment. C-JUN transcription factor is implicated in several functions including apoptosis induction in various experimental systems such as multiple myeloma [We did not observe any correlation between sensitivity to Chk1 inhibitor and the presence of a Myc-gene expression signature, suggesting that the high sensitivity to this class of compounds described in mouse models of Myc-driven lymphomas  might detivation , 36. Taktivation . The sentivation . In additivation , 38. Thenografts . The antnografts , 22. The myeloma ; the pos myeloma ; NFKBIA  myeloma , 42.in vitro and in an in vivo setting. The crucial non redundant role played by both proteins in control of DNA replication initiation and progression may explain why the drugs combination was highly effective in MCL cells, which are characterized by cell cycle deregulation. As a whole, our data provide strong preclinical evidence for the translation of Chk1 and Wee1 inhibitor combination in the clinical setting, hopefully providing a new therapeutic approach to treat MCL patients.In conclusion, MCL cell lines are considerably more sensitive to Chk1 and Wee1 inhibitors as single agents than other lymphoma cell lines and epithelial tumor cell lines. Our experimental data suggested a correlation of the t chromosomal translocation with Chk1 inhibitor sensitivity. The drugs combination presented a strong synergism at very low concentrations both in an A total of 40 human established cell lines were used: ten cell lines derived from MCL , seven  from activated B-cell like diffuse large B-cell lymphoma (ABC-DLBCL), 18  from germinal center B (GCB) cell DLBCL, and five  from multiple myeloma (MM). They were maintained in RPMI supplemented with 1% glutamine, 1% penicillin/streptomycin and 10% fetal bovine serum (FBS). Details on cell lines and drugs are specified in Cell lines were seeded in the experimental setting of 384 well plate and treated with ten growing concentrations of each drug (ranging between 0.05 nM and 100 μM) and seven replicates each concentration, using an automated liquid handling system , connected to a WinPREP for Janus software, with which it is possible to set up ad hoc programs for the seeding and treatments. MTS assay, performed 72 hrs after treatment, was used to measure cell proliferation using a plate reader . For validation step the experimental setting of 96 well plates was used. The IC50s of the two compounds for each cell line were calculated by Calcusyn Software. A non parametric Mann Whitney t-test statistical analysis was performed to compare median values. To obtain the response of cell lines to the combination of PF-00477736 with MK-1775, cells were treated simultaneously with growing concentrations of the two drugs. Results were examined by isobologram analysis with Calcusyn Software (Biosoft), to calculate the efficacy  of the experimental points , JeKo-1 and REC-1 cells were treated as already described .Caspase-3 activity was measured by enzymatic assay using a fluorogenic substrate for caspase-3, Ac-DEVD-AMC  as already described .To detect DNA by FACS, cells were fixed 24 and 48 hrs after PF-00477736 and MK-1775 treatments either singly or combined and processed as already described . PercentDNA fragmentation in JeKo-1 cells, either untreated or treated with PF-00477736, MK-1775 or the combination was detected by the TdT-mediated dUTP nick-end labeling technique (TUNEL), following a procedure already described .http://www.ncbi.nlm.nih.gov/geo) database. Real time PCR was performed as described in Total RNA was extracted from cell lines using TRIzol reagent (Invitrogen) and purified using RNeasy total RNA Isolation kit (Quiagen), and with the SV-total RNA kit according to manufacturer's instructions , for xenografts tumor fragments (homogenised in RNA lysis buffer in ice with an Ultra-Turrax), quantified by NanoDrop spectrophotometer. Gene expression profiling was done using the HumanHT-12 v4 Expression BeadChip , and data were first extracted with the Illumina GenomeStudio software and then imported in R and quantile normalized, as previously performed . Functio7 cells per mouse). When tumors grew, animals were sacrificed and tumor fragments were implanted subcutaneously in sixty mice. When tumors reached approximately 150 mg, animals were randomized (seven or eight per group) to receive PF-00477736 i.p., 10 mg/kg, MK-1775 orally twice a day, 30 mg/kg or the combination of both at these dosages or at half doses, for 16 days (COMBO I). A second cycle of treatment was given to the high dose combination group for 11 days, after 5 days treatment withdraw, when tumors started to re-grow (COMBO II). Animals were sacrificed when tumor weight reached the 10% of their body weight. Pharmacodynamic studies are described in Five-week-old female NCr-nu/nu mice were obtained from Harlan S.p.a Italy and maintained under specific pathogen-free conditions. Procedures involving animals and their care were conducted in conformity with institutional guidelines, in compliance with national and international laws and policies and in line with Guidelines for the welfare and use of animals in cancer research . Exponen