H1 linker histone proteins are essential for the structural and functional integrity of chromatin and for the fidelity of additional epigenetic modifications. Deletion of H1c, H1d and H1e in mice leads to embryonic lethality by mid-gestation with a broad spectrum of developmental alterations. To elucidate the cellular and molecular mechanisms underlying H1 linker histone developmental functions, we analyzed embryonic stem cells (ESCs) depleted of H1c, H1d and H1e subtypes (H1-KO ESCs) by utilizing established ESC differentiation paradigms. Our study revealed that although H1-KO ESCs continued to express core pluripotency genes and the embryonic stem cell markers, alkaline phosphatase and SSEA1, they exhibited enhanced cell death during embryoid body formation and during specification of mesendoderm and neuroectoderm. In addition, we demonstrated deregulation in the developmental programs of cardiomyocyte, hepatic and pancreatic lineage elaboration. Moreover, ectopic neurogenesis and cardiomyogenesis occurred during endoderm-derived pancreatic but not hepatic differentiation. Furthermore, neural differentiation paradigms revealed selective impairments in the specification and maturation of glutamatergic and dopaminergic neurons with accelerated maturation of glial lineages. These impairments were associated with deregulation in the expression profiles of pro-neural genes in dorsal and ventral forebrain-derived neural stem cell species. Taken together, these experimental observations suggest that H1 linker histone proteins are critical for the specification, maturation and fidelity of organ-specific cellular lineages derived from the three cardinal germ layers. Eukaryotic DNA is packaged and maintained within highly regulated chromatin structures through association with various histone proteins. The fundamental unit of chromatin, the nucleosome, consists of ∼146 bp of DNA wrapped around an octamer of core histones, including H2A, H2B, H3 and H4 A. Immersus and Arabidopsis, respectively Mammals have at least eleven histone H1 variants, including somatic subtypes (H1a-H1e), testis-specific  and oocyte-specific (H1oo) variants, and differentiation-associated (H1.0) subtypes, each exhibiting differential temporally and spatially defined expression profiles and locus-specific gene interactions in vivo systems Embryonic stem cells (ESCs) have the capability of giving rise to embryoid bodies comprised of cells representing the three cardinal germ layers and are useful tools to study developmental programs, which are otherwise relatively experimentally inaccessible in In this study, we further examined the roles of H1 linker histone subtypes during early embryonic development. Utilizing established ESC culture paradigms, we analyzed the capacity of H1-KO ESCs to recapitulate early developmental events, including stem cell maintenance, the elaboration of the cardinal germ layers, and specification and maturation of organ-specific cellular lineages. Our studies demonstrate that H1 linker histones are essential for preserving the fidelity of mesoendodermal and neuroectodermal lineages, in part, by suppressing the generation of alternate cell fates.Two H1-KO ESC lines (F6 and F1) and two control ESC lines (F18 and ATCC) were employed in our study. As the experimental observations are consistent, we will focus on data derived from the H1-KO (F6) and CTL (F18) cell lines, hereby referred to as H1-KO and CTL cells respectively. The H1-KO ESCs have been shown to exhibit normal growth rates and appear morphologically undifferentiated in-vitro (DIV) EBs revealed that H1-KO EBs displayed statistically significant reductions in the expression patterns of Oct4 and Sox2 as compared to CTL EBs  to generate mesendodermal and neuroectodermal species, respectively The findings that H1-KO ESCs retained normal profiles of markers of pluripotency and ESC identity suggested that the occurrence of developmental deficiencies in H1-KO mouse embryos as early as E8.5 may have arisen during germ layer specification and incipient organ-specific lineage elaboration. To begin to characterize these putative early developmental defects, H1-KO ESCs were first cultured in suspension to form EBs, which partially recapitulate the early embryonic developmental milestones associated with formation of the three cardinal germ layers  [17], [1in vivo developmental defects and embryonic lethality previously observed. Ectoderm-derived neural differentiation was examined by utilizing an established ESC differentiation paradigm (EB/ITFS/CM), previously described by McKay et al. (1996) to generate neural stem cells (NSCs) and subsequently more differentiated neural lineage species FGF5  a mesodermal gene, Brachyury , dorsal patterning/arealization genes, Lhx2 , Pax6  and Emx2  as well as proneural/neuronal maturation genes Ngn1 , Ngn2 , and NeuroD1 . There was no significant change in the expression of Nodal  that is present in endodermal lineages or of Emx1,  a homeodomain transcription factor involved in dorsal patterning  and mesodermal, Brachyury  genes, ventral patterning/arealization genes, Lhx7/8  and Nkx2.1 , and proneural/neuronal maturation genes, Mash1 , Islet1  and NeuroD1 . The expression of Nodal  and Nkx2.2 , a homeodomain transcription factor involved in the elaboration of oligodendroglial cells, were upregulated whereas there was no significant change in the expression of other ventral patterning genes, including Lhx6 , Gsh1  and Gsh2   . Consist= 0.063) . These fin vivo spatial specification and maturation of neural lineage subtypes in vitro: dorsal telencephalic ; ventral telencephalic ; and ventral midbrain (dopaminergic neurons)  and alpha-fetoprotein (AFP), which begins as early as 9 DIV and persists until at least 20 DIV, followed by the expression of tyrosine aminotransferase (TAT) and glucose-6-phosphate (G6P) that are restricted to temporal intervals around 18 DIV We next investigated the integrity of endodermal induction and endoderm-derived tissue-specific differentiation utilizing specific paradigms to generate hepatic in vitro that includes sequential expression of early pancreatic progenitor genes, Pdx1, Islet-1, Insm1, followed by mature pancreatic genes, Insulin1/2, Glucagon and Somatostatin Mash1 , Neurogenin 2 , Neurogenin 3 , as well as NeuroD1  at early stages of pancreatic differentiation  at later differentiation stages (data not shown). In addition, we also observed ectopic cardiomyogenesis in one of the H1-KO ES cell lines  and H1-KO (F6) ESC lines were previously generated by Fan et. al., 4 cells/cm2, or 2) ESCs cultured as hanging drops 4 cells/cm2 on gelatin-coated tissue culture plates or poly-L-ornithine coated glass coverslips in N2B27 media for 48 hr (Pre-induction stage). After 48 hr, media is replaced with fresh N2B27 supplemented with either 500 nM retinoic acid (Sigma) or 200 ng/ml Wnt3A (R&D), and cells were cultured for a further 24–36 hr (Post-induction stage).Embryoid body differentiation was carried out by 1) plating ESCs onto non-adherent bacterial culture dishes at a density of 2–2.5×105 cells/cm2 in Complete Media (CM)  and 10 mg Insulin), supplemented with 1 mg/ml laminin  and 10 ng/ml FG2  to expand cells for 4–6 DIV. After expansion, specific neural differentiation was induced by removal of FGF2 and supplemented with the following growth factors for a further 7 DIV: 1) glutamatergic neurons; 1000 U/ml LIF, 2) astrocytes; 20 ng/ml BMP2+1000 U/ml LIF, 3) GABAergic neurons; 100 ng/ml SHH + 10 µg/ml BMP2, 4) dopaminergic neurons; 100 ng/ml+100 ng/ml FGF8, 5) oligodendrocytes; 100 ng/ml SHH+10 ng/ml PDGFα.The ITFS/CM neural differentiation includes selection and expansion of nestin-positive cells as previously described 4 cells/cm2 in DMEM/F12 supplemented with N2 and Neurobasal supplemented with B27 in a (1∶1) ratio . After 5–7 DIV, cells were dissociated with trypsin and re-plated onto glass cover slips coated with polyornithine/laminin and expansion of nestin-positive cells was performed using 10 ng/ml bFGF+1 µg/ml laminin. Induction of specific neural differentiation was performed as described in previously.The differentiation protocol was carried out as previously described with minor modifications −7 M Dexamethasone , 5 mg/ml insulin , 5 mg/ml transferrin , and 5 µg/ml sodium selenium . The media was changed daily. Cells were analyzed at 12 days and 20 days for early hepatic progenitors and mature hepatocytes, respectively.The hepatic differentiation paradigm was carried as previously described The pancreatic differentiation paradigm was carried out as previously described Cardiomyocyte differentiation was carried out as previously described Samples were fixed with 4% PFA and immunofluorescence analysis was carried out as previously described HPRT1). All RNA samples were normalized using fluorometric procedures and levels of cDNA input were quantified and normalized using the Qubit-Invitrogen fluorometric kit for single strand DNA . Finally, an additional normalization step was employed through quantifying the ROX fluorescence emission in each well by multiplex scanning after each PCR cycle. These additional normalization procedures significantly contributed to the achievement of a uniform, and highly replicable data results across the technical and biological replicates. Data collection and quality assessment were performed utilizing 7000 SDS 1.1 RQ Software . The statistical analysis was performed using the Pair Wise Fixed Reallocation Randomization test provided by the Relative Expression Software Tool (REST) developed by Corbett Research Harvesting of RNA from samples was carried out using TRI reagent  according to manufacturer's protocol. Quantification of total RNA concentration was determined using the Qubit RNA assay kit and Qubit 2.0 Fluorometer (Invitrogen). Single strand cDNA synthesis was performed using the High Capacity cDNA Reverse Transcription Kit  following the manufacturer's recommendations. TaqMan probes and SYBR Green primers were purchased from PE Applied Biosystems and Invitrogen service respectively See . We utilt-test. Unless otherwise indicated, statistically significant differences between samples were considered using a probability of at least <0.0001 and/or <0.001.All quantifications of different culture models reflect the analysis of a minimum of 10 randomly selected fields from three distinctive biological replicates. Cell counts were performed using a 40× objective. Statistical comparisons were evaluated based on the type of data studied: proportions were compared with Chi-square test and the means of samples with either Mann-Whitney U test or Figure S1H1-KO (F1) ESCs maintain embryonic stem cell and pluripotency markers while exhibiting increased numbers of TUNEL+ cells during EB formation. (A–G) CTL (ATCC) and H1-KO (F1) ESC lines maintained expression of the embryonic stem cell markers, ALP and SSEA1, as well as the pluripotency markers, Sox2, Oct4 and Nanog . At 4 DIV, H1-KO EBs exhibited statistically significant reductions in Sox2 and Oct4  and H1KO(F1), respectively with a p<0.001) (F) and an increase in the number of TUNEL+ cells  and H1-KO (F1), respectively, p<0.0001) (G). Scale bar = (A) 500 µm; (B) 200 µm and (C–G) 20 µm.(TIF)Click here for additional data file.Figure S2Deregulation of the induction and survival of mesendodermal and neuroectodermal cell types in H1-KO (F1) ESCs. (A) Representative images of immunofluorescence analysis of Brachyury+ mesendodermal progenitors after Wnt3A induction and the corresponding quantification of reduction in Brachyury+ cells in H1-KO (F1) cells  and H1-KO (F1), respectively). (B) Representative images of immunofluorescence analysis of SOX1+ neuroectodermal progenitors after RA induction and the corresponding quantification of SOX1+ cells in H1-KO (F1) cells  and H1-KO (F1) respectively). (C) Representative images of immunofluorescence analysis of TUNEL+ cells and quantification of TUNEL+ in different conditions  and H1-KO (F1) respectively). All error bars represent ±95% CI; *p<0.0001 unless otherwise noted. Scale bar = 20 µm.(TIF)Click here for additional data file.Figure S3Comparative expression profiles of developmental genes in dorsal and ventral neural progenitor cell types derived from CTL (ATCC) and H1-KO (F1) ESCs using the ITFS/CM paradigm. (A) QPCR expression analysis of stage-specific developmental genes in dorsal neural progenitor cell types at 7 DIV. (B) QPCR expression analysis of stage-specific developmental genes in ventral neural progenitor cell types at 7 DIV. All error bars represent ±95% CI.(TIF)Click here for additional data file.Figure S4Selective developmental deregulation of H1-KO (F1) ESC-derived dorsal forebrain-specific neural lineage elaboration using the ITFS/CM paradigm. (A) CTL cell lines displayed an increased number of nestin+ NSCs and β-tubulin III+ neuronal progenitor species compared to the H1-KO cell lines  and H1-KO (F1) respectively). (B) Following additional in vitro maturation, CTL (ATCC) cell lines exhibited higher numbers of NSCs and neuronal progenitor species than H1-KO (F1) cell lines  and H1-KO (F1) respectively). (C) CTL (ATCC) cell lines showed a robust complement of glutamatergic neurons while H1-KO (F1) cell lines displayed an absence of normal neuronal differentiation. (D) CTL (ATCC) and H1-KO (F1) cell lines showed similar profiles of GFAP+ astrocytes  and H1-KO (F1) respectively), but H1-KO (F1) gave rise to significantly decreased number of CD44+ progenitors  and H1-KO. (E) The elaboration of O4+ oligodendrocyte progenitors in H1-KO (F1) cell lines was accelerated compared to those of CTL (ATCC) cell lines which predominantly has NG2+ oligodendrocyte precursors  and H1-KO F1) respectively). All error bars represent ±95% CI, * p<0.05 unless otherwise noted. Scale bar = 100 µm.(TIF)Click here for additional data file.Figure S5Selective developmental deregulation of H1-KO (F1) ESC-derived ventral brain-specific neural lineage elaboration using the ITFS/CM paradigm. (A) Both CTL (ATCC) and H1-KO (F1) cell lines exhibited comparable profiles of GABA+ GABAergic neurons (n = 1184 1257 for CTL (ATCC) and H1-KO (F1) respectively). (B) H1-KO (F1) cell lines do not give rise to TH+ dopaminergic neurons as compared to CTL cell lines. (C) The elaboration of O4+ oligodendrocyte progenitors in H1-KO (F1) cell lines was accelerated compared to those of CTL cell lines  and H1-KO (F1) respectively). All error bars represent ±95% CI, * p<0.05 unless otherwise noted. Scale bar = 100 µm.(TIF)Click here for additional data file.Figure S6Selective developmental deregulation of H1-KO (F1) ESC-derived dorsal forebrain-specific neural lineage elaboration using the Neurobasal paradigm. (A) H1-KO (F1) cell lines generated significantly less nestin+ NSCs and β-tubulin III+ neuronal progenitor species as compared to CTL cell lines  and H1-KO (F1) respectively). (B) Following additional in vitro maturation, CTL cell lines generated higher numbers of nestin+ NSCs and β-tubulin III+ neuronal progenitor species than H1-KO cell lines  and H1-KO (F1) respectively). (C) H1-KO (F1) ESCs failed to give rise to glutamatergic neurons as compared to CTL ESCc. (D) H1-KO ESCs generated significantly lower number of CD44+ astrocytic precursors and GFAP+ astrocytes as compared to CTL ESCs  and H1-KO (F1) ESCs, respectively). (E) The elaboration of dorsally derived oligodendrocyte progenitors was low in both cell lines . All error bars represent ±95% CI, * p<0.05 unless otherwise noted. Scale bar = 100 µm.(TIF)Click here for additional data file.Figure S7Selective developmental deregulation of H1-KO (F1) ESC-derived ventral brain-specific neural lineage elaboration using the Neurobasal paradigm. (A) There was a modest reduction in the percentage of GABAergic neurons derived from in H1-KO cell lines as compared to the CTL cell lines  and H1-KO (F1) respectively). (B) H1-KO (F1) cell lines did not give rise to TH+ dopaminergic neurons as compared to CTL cell lines. (C) The elaboration of ventrally-derived oligodendrocyte progenitors was low in both cell lines  and H1-KO (F1) respectively). All error bars represent ±95% CI, * p<0.05 unless otherwise noted. Scale bar = 100 µm.(TIF)Click here for additional data file.Figure S8Comparative developmental gene expression profiles associated with cardiomyocyte and hepatic differentiation of CTL (ATCC) and H1-KO (F1) ESCs. (A) Cardiomyocyte differentiation. CTL cell lines displayed normal acquisition of cardiomyocyte marker genes FLK-1, MHC-α, MHC-β, and MLC-2V, whereas the expression of these genes was significantly developmentally deregulated in the H1-KO (F1) cell lines. (B) Hepatic differentiation. The developmental profiles of gene expression of TTR, AFP, TAT and G6P were similar between CTL and H1-KO cell lines.(TIF)Click here for additional data file.Figure S9Ectopic neurogenesis associated with pancreatic differentiation paradigms in H1-KO (F1) ESCs. (A) Gene expression analysis of pancreatic genes in early pancreatic progenitors and late mature pancreatic cells in H1-KO (F1) cell lines. (B) At 33DIV, H1-KO (F1) cell lines ectopically generated β-TubIII+ neurons. (C) Gene expression analysis of proneural genes in early pancreatic progenitors in H1-KO (F1) cell lines as compared to CTL (ATCC) cell lines. All error bars represent ±95% CI; *p<0.0001 unless otherwise noted. Scale bar = 20 µm.(TIF)Click here for additional data file.Figure S10Ectopic cardiomyogenesis associated with pancreatic differentiation paradigms in H1-KO (F1) ESCs. Representative images of immunofluorescence analysis of the expression of myosin heavy chain (MF-20) present selectively in H1-KO (F1) cell line as compared to the CTL  and H1-KO (F6) cell lines. Scale bar = 20 µm.(TIF)Click here for additional data file.Flow Chart S1Tissue specific differentiation paradigms.(TIF)Click here for additional data file.Flow Chart S2Experimental approach for generating neural lineage species (ITFS paradigm).(TIF)Click here for additional data file.Flow Chart S3Experimental approach for generating neural lineage species .(TIF)Click here for additional data file.Table S1Summary of data obtained from CTL (ATCC) and H1-KO (F1) lines.(XLSX)Click here for additional data file.Table S2List of TaqMan probes and SYBR Green primers utilized in this study. All TaqMan probes are listed with catalogue numbers from Applied Biosystems. All SYBR Green primers are listed with forward and reverse sequences.(DOCX)Click here for additional data file.


KRAS or BRAF mutation has been shown to induce apoptosis, a timely result given CRC entities harboring such mutations are in need of new therapies. Since CRC comprises heterogeneous tumors with predominant hypoxic components, we investigated effects of an inhibitor of anti-apoptotic Bcl-2 family proteins (ABT-737) in combination with an mTOR inhibitor (AZD8055)—collectively referred to as combo-Rx, in hypoxic CRC cell lines.The use of targeted agents to impel dual inhibition of anti-apoptotic mechanisms and mTOR-mediated pro-survival signaling in colorectal carcinoma (CRC) cell lines with Cell viability measures, expression of proteins implicated in apoptosis and MAPK/PI3K-AKT/mTOR pathway signaling, and profiling of composite kinase activities were undertaken in a panel of 14 cell lines.KRAS/PIK3CA-mutant HCT-116 and HCT-15 cell lines (both with low endogenous expression of the anti-apoptotic Mcl-1 protein and showing augmented inhibition of viability following the addition of ABT-737 to AZD8055) responded to combo-Rx by induction of apoptosis but with the simultaneous strong Mcl-1 up-regulation and activation of MAPK/PI3K-conducted signaling. In contrast, in hypoxic KRAS-mutant LoVo (devoid of PIK3CA mutation), BRAF/PIK3CA-mutant RKO, and wild-type Colo320DM cell lines , combo-Rx did not elicit apoptotic or pro-survival responses.In hypoxic conditions, combo-Rx suppressed viability of 13 of the cell lines, albeit ABT-737 did not significantly potentiate the inhibitory effect of single-agent AZD8055 in six of the models. Hypoxic KRAS/PIK3CA-mutant CRC cell lines resulted in pro-survival responses in parallel with the intended anti-proliferative effects, a finding that should be of note if considering combinatory targeting of multiple pathways in this CRC entity.The concurrent inhibition of anti-apoptotic proteins and mTOR-mediated signaling in hypoxic The online version of this article (doi:10.1186/s12885-016-2600-y) contains supplementary material, which is available to authorized users. KRAS, BRAF, and PIK3CA commonly result in constitutive activation of cellular signaling mediated by mitogen-activated protein kinases (MAPK) and phosphatidylinositol 3-kinase–protein kinase B (PI3K–AKT) [Colorectal carcinoma (CRC), like most solid malignancies, comprises heterogeneous tumors with predominant hypoxic components. The adaptive tissue responses to hypoxic stress involve increased resistance to apoptosis (programmed cell death) as well as altered DNA damage repair and mutation rates, and thereby genomic instability –3, ultimI3K–AKT) , 5. ThesI3K–AKT)  and makeKRAS or BRAF mutation [There is however evidence of crosstalk between the mTOR-conducted signaling and other signaling pathways which will allow tumor cells to escape mTOR-inhibitory therapy , 8. Targmutation , a timelmutation , 11. Ovemutation –14. Intemutation –17.Since no information is available regarding the concurrent inhibition of anti-apoptotic proteins and mTOR-mediated pro-survival signaling under CRC tumor hypoxia, we investigated response to treatment with ABT-737 and AZD8055, in this report referred to as combo-Rx, in a panel of hypoxic CRC cell lines harboring various typical mutations.KRAS, BRAF, and PIK3CA by Ion Torrent PGM™ sequencing, and mutation profiles were in agreement to already published data [2) or hypoxic (0.2 % O2) conditions, the latter obtained using the hypoxic chamber Invivo2 300 . The mTOR inhibitor AZD8055, the PI3K/mTOR inhibitor BEZ235, the Bcl-2 family protein inhibitor ABT-737, and the pan-caspase inhibitor Z-VAD  were dissolved in dimethyl sulfoxide (Sigma-Aldrich). Control cells received the vehicle.Fourteen human CRC cell lines  were first determined for mutations in hed data –20. All ®AQueous One Solution Reagent according to the manufacturer’s instructions . Absorbance was measured using Varioscan . Values were corrected for background absorbance, and values for treated cells are reported as percentage cell viability to corresponding control cell values. Presented results are from between three and seven independent experiments, each plated at least in triplicate.Depending on the cell line, 12,000-20,000 cells were seeded per well in 96-well Costar plates . Cells were given ABT-737 or AZD8055, separately or combined, in increasing concentrations , the combination of ABT-737 and BEZ235 (10 μM of both compounds), or vehicle. When expedient, the cells were pre-treated for 45 min with Z-VAD (20 or 50 μM). Cell viability was determined after 24 or 72 h by adding CellTiter 96Cells were seeded in Nuncleon T25 flasks  and were treated as indicated, and protein lysates from both floating and adherent cells were harvested as previously described . Equal aMcl-1 expression was inhibited using short hairpin (sh)RNA , and control cells were generated using non-target sequence . The manufacturer’s instructions were followed apart from extending the lentiviral incubation period to 48 h.Cells were seeded in Nuncleon T25 flasks and treated as indicated for up to 72 h. When expedient, the cells were pre-treated for 45 min with Z-VAD. Phase-contrast images were processed at the start of experiment and further after 24, 48, and 72 h by Olympus IX81 .http://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-3870/) by accession number E-MTAB-3870. Background signals were subtracted, and negative signal intensities were managed by subtracting the 1 % quantile of all data and setting the remaining signal intensities less than 1 to the value of 1. Data were log2-transformed before mean signal intensity of three replicates that were analyzed for each experimental condition was calculated for each peptide substrate. The resulting data from each type of treatment was compared to the relevant control (vehicle-treated cells) for assessment of increase or decrease in substrate phosphorylation level. Substrates associated with PI3K-AKT and/or MAPK pathways were retrieved from PathCards (http://pathcards.genecards.org/), applying the super-pathway definitions ‘PI3K-AKT signaling pathway’ and ‘MAPK signaling pathway’.The Tyrosine Kinase PamChip® Array technology  enables profiling of composite tissue kinase activities . The arrt-test. p-values less than 0.05 were considered statistically significant. In the assessment of combination effects on cell viability, we chose to evade calculations based on median-effect equation for multiple drug interactions because the ABT-737 single-agent effects did not appear with typical dose-response curves.Differences between groups were analyzed using two-tailed Student’s First, individual effects of the Bcl-2 family protein inhibitor ABT-737 and the mTOR inhibitor AZD8055 on cell viability under hypoxia and normoxia at 24 h were examined  but in both of the wild-type models .Since Faber and co-workers found that the combination of ABT-263, a structurally related compound to ABT-737, with AZD8055 at concentrations of 50–500 nM for 72 h caused apoptosis in CRC cell lines with mutation , we nextKRAS-mutant and one of three wild-type cell lines but in none of five BRAF-mutant models. The findings in the two first-mentioned groups but not in BRAF-mutant cell lines were generally in agreement with previously reported data [Based on the two initial sets of results, the highest tested concentration (10 μM) of both compounds was chosen for further experiments. First, we investigated viability in 14 CRC cell lines under normoxic conditions . The addition of ABT-737 significantly potentiated inhibitory effects of AZD8055 in four of six ted data .KRAS-mutant, three of five BRAF-mutant, and two of three wild-type ones), ABT-737 did not significantly potentiate the inhibitory effect of single-agent AZD8055. Three of the 14 cell lines, two sensitive and one resistant to the additional effect of ABT-737, had PIK3CA mutation. Similarly, treatment with the PI3K/mTOR inhibitor BEZ235, which has demonstrated anti-proliferative effects in various models [Specifically, we were interested in elucidating inhibitory effects of combo-Rx under hypoxic conditions in the 14 cell lines  and the BRAF/PIK3CA-mutant RKO and wild-type Colo320DM cell lines   Fig. . Specifi37) Fig. . As show37) Fig. . On incr37) Fig. . NeverthTo explore the notion of non-apoptotic mechanisms of combo-Rx effects in RKO and Colo320DM cell lines, the three hypoxic cell lines were treated for 72 h and inspected at 24-h intervals  and LoVo cells . The five cell lines were assessed for induction of apoptosis, as indicated by cleavage of caspase-3, and for pro-survival signaling by phosphorylation of ERK1/2 (p-ERK1/2) of the MAPK pathway, of AKT (p-AKT(Ser473)) of the PI3K-AKT pathway, and of S6 (p-S6) down-stream of mTOR, and by expression of the anti-apoptotic protein Mcl-1  inhibited Mcl-1 expression in the majority of the cell lines. Although levels of caspase-3 were also lower under hypoxic compared to normoxic conditions in both cell lines that were sensitive to the additional effect of ABT-737 to ABT8055 (HCT-116 and HCT-15), cleaved caspase-3 was observed after 24 h of hypoxic combo-Rx. As expected, this apoptotic feature could not be detected in the three resistant cell lines . Next, under normoxia, ABT-737 treatment caused Mcl-1 up-regulation and p-S6 induction as a consequence, and these responses were counteracted by the addition of AZD8055 . These observations were in agreement with previously reported data [PIK3CA mutation) demonstrated a further strong induction of Mcl-1 and p-S6 and also of p-ERK1/2. In contrast, hypoxic combo-Rx did not cause any uniform pattern of regulation of these selected pro-survival factors in the resistant cell lines. Specifically, in the KRAS-mutant LoVo cell line (devoid of PIK3CA mutation), the addition of AZD8055 counteracted the effects of ABT-737 on Mcl-1 and p-S6, exactly like under normoxia. The hypoxic BRAF/PIK3CA-mutant RKO cells showed Mcl-1 and p-S6 responses similar to but not as strong as those of the HCT-116 and HCT-15 models. And in the wild-type Colo320 cell line, hypoxic combo-Rx did not cause Mcl-1 alteration nor was p-S6 detected. In summary, the results highlighted the possibility of dual apoptotic and pro-survival responses in KRAS/PIK3CA-mutant CRC cells responding under hypoxic conditions with augmented inhibition of viability to the addition of ABT-737 to AZD8055.Of initial note, 24 h of hypoxic incubation in itself  of a limited number of substrates. In contrast, kinase activity of HCC2998 cells, whether hypoxic or normoxic, was predominantly repressed by all treatment conditions, while Colo320DM kinase activity was basically unchanged.As depicted in Fig. , the combination treatment) suppressed viability of 13 of the cell lines, albeit ABT-737 did not significantly potentiate the inhibitory effect of single-agent AZD8055 in six of the models. On further mechanistic investigations, the hypoxic KRAS/PIK3CA-mutant HCT-116 and HCT-15 cell lines (both with low endogenous expression of the anti-apoptotic Mcl-1 protein and showing augmented inhibition of viability following the addition of ABT-737 to AZD8055) responded to combo-Rx by induction of apoptosis  and with the simultaneous strong Mcl-1 up-regulation and activation of MAPK/PI3K-conducted signaling. A ubiquitous activation of hypoxic kinase signaling by combo-Rx was also confirmed in the HCT-116 cells. In contrast, in hypoxic KRAS-mutant LoVo, BRAF/PIK3CA-mutant RKO, and wild-type Colo320DM cell lines , combo-Rx did not elicit apoptotic or pro-survival responses. Collectively, this data revealed complex responses to the concurrent inhibition of anti-apoptotic proteins and mTOR-mediated signaling in hypoxic CRC cell lines, where pro-survival responses were elicited in parallel with the intended anti-proliferative effects in KRAS/PIK3CA-mutant entities in particular, a finding that should be of note if considering the combinatory targeting of multiple pathways in CRC treatment.A number of recent findings , 16, 17 KRAS or BRAF mutation to undergo apoptosis, a timely result as such CRC entities are refractory to current targeted therapies. In vitro studies have shown promising treatment effects of both types of agents, but concerns have been raised with regard to lacking therapeutic efficacy of mTOR inhibitors in solid tumors [As recently shown , when cod tumors , 25. Botd tumors , 16, andd tumors , 27.KRAS-mutant HCT-116, HCT-15, and LoVo models and the BRAF/PIK3CA-mutant RKO and wild-type Colo320DM cells), combo-Rx under hypoxic conditions caused dual phenotypic responses in terms of concurrent apoptotic and pro-survival effects in the HCT-116 and HCT-15 cell lines, as demonstrated through the specific examination of the anti-apoptotic Mcl-1 protein, microscopy of cell cultures, and both targeted and comprehensive analysis of kinase signaling. Importantly, both of these cell lines also harbor PIK3CA mutation. These findings suggest that CRC cancers with co-occurring KRAS and PIK3CA mutations, which is not a frequent entity [Invariably, CRC comprises heterogeneous tumors with predominant hypoxic components , which iKRAS-mutant HCC2998 cell line, which was sensitive to combo-Rx for viability but devoid of PIK3CA mutation, and the resistant wild-type Colo320DM model, showed repressed or unchanged global kinase activities to the experimental perturbations. Again, the findings indicate a particular susceptibility of KRAS/PIK3CA-mutant CRC entities to unfavorable responses to the combined inhibition of anti-apoptotic proteins and mTOR signaling. Of final note, in the context of interpreting the ex vivo kinase substrate data, some important considerations should be kept in mind [The MAPK and PI3K-AKT signaling pathways merge at the mTOR complex, which promotes cell survival through phosphorylation of S6 and the resulting increase in Mcl-1 protein translation . Intrigu in mind . One is KRAS and PIK3CA mutations revealed complex responses of pro-survival effects elicited in parallel with apoptosis. Recognizing the obvious limitation that CRC cell lines do not fully reflect the heterogeneity of this disease with regard to mutation profiles or the dynamic nature of tumor oxygenation status, more extensive preclinical studies are required for further elucidation of responses to this kind of combination treatment. Still, from a clinical perspective, and particularly with tumor hypoxia posting a demand for more tailored therapies [To conclude, under hypoxia, which is an important feature of CRC tumors and a main mechanism of therapy resistance, the apparently rational approach of combining ABT-737 for inhibition of anti-apoptotic proteins with the mTOR inhibitor AZD8055  in CRC cherapies , cautionCAIX: Carbonic anhydrase IX; CRC: Colorectal carcinoma; ERK1/2: Mitogen-activated protein kinase 3/1; HIF-1α: Hypoxia-inducible factor type 1α; MAPK: Mitogen-activated protein kinases; mTOR: Mechanistic target of rapamycin; PI3K-AKT: Phosphatidylinositol 3-kinase–protein kinase B; S6: Ribosomal protein S6 kinase beta-2; sh: Short hairpin


Spheroid based culture methods are gaining prominence to elucidate the role of the microenvironment in liver carcinogenesis. Additionally, the phenomenon of epithelial-mesenchymal transition also plays an important role in determining the metastatic potential of liver cancer. Tumor spheroids are thus important models to understand the basic biology of liver cancer.We cultured, characterized and examined the formation of compact 3-D micro-tumor spheroids in five hepatocellular carcinoma (HCC) cell lines, each with differing TP53 mutational status (wt vs mutant vs null). Spheroid viability and death was systematically measured over a course of a 10 day growth period using various assays. We also examined the TP53 and E-cadherin (CDH1) mRNA and protein expression status in each cell line of the 2-D and 3-D cell models.A novel finding of our study was the identification of variable 3-D spheroid morphology in individual cell lines, ranging from large and compact, to small and unstable spheroid morphologies. The observed morphological differences between the spheroids were robust and consistent over the duration of spheroid culture growth of 10 days in a repeatable manner. Highly variable CDH1 expression was identified depending on the TP53 mutational status of the individual HCC cell line, which may explain the variable spheroid morphology. We observed consistent patterns of TP53 and CDH1 expression in both 2-D and 3-D culture models.In conclusion, we show that 3-D spheroids are a useful model to determine the morphological growth characteristics of cell lines which are not immediately apparent in routine 2-D culture methods. 3-D culture methods may provide a better alternative to study the process of epithelial-mesenchymal transition (EMT) which is important in the process of liver cancer metastasis. The incidence of hepatocellular carcinoma (HCC) has been steadily increasing in the past decade. HCC is currently one of the fastest growing cancers in the United States. The outcomes in cases of HCC are dismal, with a 5 year survival of less than 8 % for stage III and above , 2. GlobTraditional research techniques of cancer biology utilize two-dimensional, monolayer cell cultures grown in flasks. However, the role of the spatial cell signaling cues of the tumor microenvironment in the formation and metastasis of tumors, including HCC, is being increasingly appreciated , 6. PrevThe role of adhesion molecules such as integrins and E-cadherin (CDH1), in intra-cellular cancer cell signal transduction have been elucidated previously, including HCC –13. The In the current study we describe the growth and characterization of tumor spheroids from HCC cell lines. We have optimized the growth conditions of five HCC cell line spheroids  that are used in our lab. The initial choice of cell lines was driven by the TP53 mutational status of in each cell line. TP53 gene somatic mutation was observed in HCC in approximately ~21 % of clinical cases in one study . In gene® Basement Membrane Extract or Methylcellulose (MC) at 0.30 % in the initial trials. The protocol for preparation of MC stock was adapted and modified from [Hanging drop plates (HDPs) suspend tumor cells in a droplet of media which encourages cell–cell interactions in three dimensions resulting in the formation of a spheroid . We elecied from . MC and ® cell proliferation assay (b) CellTiter-Glo® luminescent assay and (c) LDH cytotoxicity assay.We sought to establish the spheroid viability of the HCC cell lines throughout the 10 day culture period. We used three, commercially available assays to establish the viability of the tumor cells in these spheroids: (a) alamarBlue® is a widely-used cell proliferation indicator which works by estimating the reductive and oxidative components of the media in which the cells are being cultured. As the tumor spheroid cells grow, innate metabolic activity results in a chemical reduction of alamarBlue®. Continued growth maintains a reduced environment while inhibition of growth leads to an oxidized environment. HCC lines produced absorbance values which were between 95 % and just over 200 % greater compared to control wells containing media only  and the TP53-mutant cell line SNU-475 (124 %) and the lowest reductions in the TP53-null cell line Hep3B and the remaining TP53-mutant, SNU-387 . It is important to note that the alamarBlue® assay measures the redox nature of the “cell media” of the spheroids and not the viability of the cells themselves. Our current results indicate the presence of a “growth state” in all the cell lines at the end of 10 days of spheroid culture used in this study.AlamarBlue® assay as a complement to alamarBlue®. The CellTiter-Glo® assay is a highly sensitive assay which determines the number of viable cells based on a quantitation of the ATP present in the cells using a luciferase based reaction. The amount of ATP present in the cells indicates their metabolic activity. The HCC cell lines in the current study generate different sized spheroids . Similar to the CellTiter-Glo® assay, we normalized the LDH values by obtaining measurements from a single average volume (~15 uL) of the media in each of the HCC cell lines. We observe a progressive increase in LDH values in the initial 3–5 days in all the cell lines  enzyme assay. Cells undergoing active remodeling or cell death release lactate-dehydrogenase enzyme into the culture media which can then be measured using a plate reader set-up. The amount of LDH measured is indicative of the extent of cell death in the measured sample. The LDH assay (cell death measurement) is complementary to the CellTiter-Glones Fig.  indicati® and LDH values simultaneously (phase 1). Days 3–5 constitute phase 2, which is characterized by consolidation of the spheroid growth, while days 5–10 (phase 3) are indicated by progressive decline in tumor spheroid viability in all of our cancer cell lines. Day 5 post seeding appears to be the most stable with regards to spheroid metabolic activity and the stability of the spheroid size. Based on the results of these assays, we conclude that the days 4–5 post-seeding is the appropriate window period to conduct functional assays of the tumor spheroids in all the 5 HCC cell lines.Overall, these results appear to indicate 3 distinct phases of tumor spheroid growth formation. In the first 72 h after seeding, there is extensive growth and remodeling resulting in the increase of the CellTiter-GloOne of our initial observations during the testing phase of the spheroid growth protocol was the distinct differences of the spheroid morphology we noted between the five HCC cell lines included in this study. These 3-D morphological structure differences are not immediately apparent in regular 2-D flask cell culture. Our initial observations regarding cell line specific spheroid morphology appeared to be consistent and repeatable. In order to assess these differences more rigorously, we measured multiple replicates eight of the individual spheroids for the duration of growth (10 days) and have reported the mean diameters (µm).Qualitatively, we observe distinct differences in the shape and appearance of the individual spheroids depending on the TP53 status  gene expression status in these HCC cell lines. Cell–cell junctions play a very important role in the modulation of the tumor microenvironment . There aWe performed immunofluorescence (IF) staining based on the protocol described in the methods for TP53 and CDH1 proteins. All the cell lines showed TP53 and CDH1 staining, with variable intensities . We conducted RT-PCR assays on cells grown in 2-D (cell culture flasks) as well as 3-D (tumor spheroids from HDP). We observe a similar pattern of RT-PCR results for the TP53 gene expression as observed for the IF staining. SNU-475 shows the highest amount of TP53 relative expression which is statistically significant compared to the wild-type cell lines of HepG2 and C3A see Fig. . As expeFor the CDH1 gene, we observe a similar pattern of gene expression as the IF staining. The TP53-null cell line of Hep3B shows the maximum expression quantitatively, while the TP53-wt cell lines (HepG2 and C3A) show an intermediate expression and the TP53-mutant cell lines (SNU-387 and SNU-475) show the least amount of expression in the cells. Interestingly, we observe a higher level of relative expression in the TP53-null cell line, Hep3B, in the 3-D tumor spheroids compared to the 2-D cell culture. This was confirmed by repeating the experiment multiple times. This overexpression of CDH1 may explain the relatively smaller size of Hep3B spheroids as well as the high degree of compactness  has been shown to play an important role in the pathogenesis of liver cancer by influencing the local growth and formation of distant metastasis within these tumors . The phe®, CellTiter-Glo® and LDH) we have shown that all of our cell spheroid lines undergo a high amount of cell remodeling during the initial spheroid formation process within the first 72 h after seeding. We have identified three distinct phases of growth during the spheroid formation in all the cell lines in this study. Based on these results, we feel days 4–5 after seeding of cells is ideal for functional spheroid analysis.In our study we have demonstrated a feasible and consistent system for culturing hepatocellular carcinoma cell spheroids in five different cell lines  using the hanging drop method. We have characterized in detail, the necessary growth conditions for these cell lines as well as the growth viability over the duration of the growth (10 days). An extended growth period beyond 10 days is indeed feasible and may be of interest to understand issues related to hypoxia and growth necrosis in tumor spheroids. Through the cell viability assays . Individual HCC cell lines were chosen based on TP53 mutation status: HepG2 and its clonal derivative, C3A, express wild type TP53, Hep3B is TP53-null and the SNU-387 and SNU-475 cell lines express mutant TP53 protein. Cell stocks were maintained in T-75 flasks under standard culture conditions , in either EMEM  or RPMI-1640 , supplemented with 10 % fetal bovine serum .® Hanging drop plates . After trypsinization, cells from culture flasks were centrifuged, resuspended and counted using a Countess Cell Counter . Cells were then seeded into 96-Well Perfecta3D® HDPs  at a concentration of 6000 cells/well. Cells were supplemented with 1.5 % methylcellulose (MC) stock solution to aid in spheroid formation. 0.75 g of methylcellulose powder  were added to 50 mL sterile Phosphate Buffered Saline  and autoclaved. The resulting solution was centrifuged  and the supernatant was saved and aliquoted. 0.30 % Cultrex® Basement Membrane Extract  was also tested in the initial trials but discontinued in favor of MC. Suspended hanging drops contained a total volume of 40 µL according to company recommendations; 32 µL of the cell suspension contained the appropriate media (EMEM or RPMI). The remaining 8 µL was comprised of 1.5 % MC solution, resulting in a final concentration of 0.30 % MC per well. In later trials, the final concentration of MC was increased to 1 % for SNU-387 and SNU-475 with the expectation that it would improve spheroid compactness due to the increased viscosity of the medium. Small volume media droplets are prone to evaporation, so HDP humidity control was achieved by filling the plate reservoirs with a 0.5 % solution of LMP Agarose in PBS (autoclaved). This mixture created a resilient, gelatinous wall, preventing excessive evaporation. Media was changed by aspirating a small volume of the droplet (~ 5–10 µL) and adding between 10–20 µL of fresh media, resulting in a final volume of between 40–50 µL on alternate days. The spheroids were imaged using a Celestron Digital Microscope Imager  at consecutive 24 h intervals for a duration of 10 days. To preserve the integrity of the sterile-HDP environment, spheroids were imaged from above with a 2.5×, wide-angle objective without removing the plate cover. Differences in spheroid diameter and shape were quantified using the open source software, ImageJ (NIH).HCC spheroids were formed using the hanging drop method with the aid of Perfecta3D® kit from AbDSerotec . alamarBlue® reagent was added to appropriate wells within the HDP at 10 % of the total volume of the droplet (4 µL in most cases). Reagent was also added to control wells containing media only (no spheroid) as a negative control. Cells were then incubated under standard conditions for 4 h. Absorbance values were obtained at 535 and 595 nm using a Perkin Elmer 1420-040 Victor 3 V plate reader. Reduction related to growth causes the redox indicator to change from oxidized  form to reduced  form detected by the plate reader.To measure cell proliferation rates, we used a simple, one-step alamarBlue®  which uses ATP and a luciferase-based substrate to indicate cell viability. Spheroids from each line were collected from the HDP into small volume eppendorf tubes at 1, 3, 5, 7 and 10 days incubation. CellTiter-Glo® substrate was added by volume to each tube according to the manufacturer’s recommendations. The resulting solution was vortexed thoroughly to aid in cell lysis. After 15 min incubation at room temperature, 120 µL of solution was transferred from each tube into a LUMNITRAC 200 white-walled 96-well plate . Luminescence was measured using a Perkin Elmer Victor 3 V plate reader.We used an alternative method, CellTiter-GloTo assess the cell death in the spheroid cultures, media samples of 15 µL were extracted in triplicate from each cell line  at 1, 3, 5, 7 and 10 day time-points of spheroid growth. Each sample was transferred from the HDP into a LUMNITRAC 200 white-walled 96-well plate . LDH reagent was added according to manufacturer instructions. All samples were allowed to incubate for 30 min at room temperature before adding the reaction stop-solution. Sample absorbance was then measured at 490 and 575 nm using a Perkin Elmer Victor 3 V plate reader.d-lysine coated coverslips  and incubated overnight in 6-well culture plates. Cells were washed with PBS and then fixed for 30 min in 4 % formalin . Cells were later incubated with D0-1 TP53 antibody  or anti E-Cadherin antibody , washed and then followed immediately by Alexa Fluor® 488 secondary antibody . Negative controls included both the addition of primary or secondary antibody alone. Coverslips were then washed thoroughly, coated with FluoroGel  and fixed onto standard microscope slides. Slides were imaged at 63× using a Zeiss Axioskop.Liver cancer cells were seeded onto circular, poly-® RNA Mini Kit . To maximize the amount of RNA from 3-D spheroids, each HCC cell line was seeded into 16 wells within the HDP. All spheroids were processed with the PureLink® kit on day 4 after seeding. After the initial elution, a clean-up step using the RNeasy Mini Kit  was performed to improve RNA quality. cDNA was synthesized using SuperScript® II Reverse Transcriptase  according to manufacturer instructions. We elected to use custom oligo-sequences purchased from Integrated DNA Technologies  for TP53, CDH1 gene and the TATA Binding Protein (TBP) housekeeping control. qRT-PCR was performed in a Roche LightCycler® 96  and analyzed using the comparative cycle threshold method (2−∆∆CT) to quantify gene expression. The following RT-PCR probes were used for gene expression analysis in all of the samples examined in this study. TP53-alpha, Exon 9/10, Forward:5′-aaccactggatggagaatatttcac-3′, Reverse:5′-cagctctcggaacatctcgaa-3′, Probe:FAM-tcagatccgtgggcgtgagcg-VIC, CDH1, Exon6/7, Forward: 5′-ctgaggatggtgtaagcgatg-3′, Reverse:5′-gtctgtcatggaaggtgctc-3′, Probe: FAM-agacgcggacgatgatgtgaacac-VIC, Housekeeping gene, TBP (TATA Binding Protein), Forward:5′-cacgaaccacggcactgatt-3′, Reverse:5′-ttttcttgctgccagtctggac-3′, Probe: FAM-tgtgcacaggagccaagagtgaaga-VIC.RNA was isolated from both 2-D cell culture and 3-D spheroids using the PureLinkWhole protein lysates from both 2-D and 3-D cultures were extracted using RIPA buffer under standard conditions. Protein concentrations were determined using the detergent-compatible, BCA Protein Assay Kit . Protein lysates were separated using SDS-PAGE electrophoresis and transferred to nitrocellulose membranes for probing. The monoclonal mouse antibody, DO-1  was used to probe full-length p53 protein, while M168 antibody  was used to probe E-cadherin. The blots were then washed and incubated with a goat anti-mouse-HRP conjugate . After probing, blots were treated with either Amersham ECL Detection Reagent  or SuperSignal™ West Femto Maximum Sensitivity Substrate  and imaged using a Bio-Rad ChemiDoc™. Blots were then stripped and re-probed with GAPDH primary antibody  as a positive loading control.


Rhipicephalus (Boophilus) microplus is an economically important parasite of livestock. Effective control of ticks using acaricides is threatened by the emergence of resistance to many existing compounds. Several continuous R. microplus cell lines have been established and provide an under-utilised resource for studies into acaricide targets and potential genetic mutations associated with resistance. As a first step to genetic studies using these resources, this study aimed to determine the presence or absence of two genes and their transcripts that have been linked with acaricide function in cattle ticks: β-adrenergic octopamine receptor  and ATP-binding cassette B10  in six R. microplus cell lines, five other Rhipicephalus spp. cell lines and three cell lines representing other tick genera .The cattle tick βAOR gene and transcripts in DNA and RNA extracted from the tick cell lines, followed by capillary sequencing of amplicons. Quantitative real-time PCR (qPCR) was performed to determine the levels of expression of ABCB10.End-point polymerase chain reaction (PCR) was used for detection of the βAOR gene expression was detected in all Rhipicephalus spp. cell lines. We observed a second amplicon of approximately 220 bp for the βAOR gene in the R. microplus cell line BME/CTVM6, derived from acaricide-resistant ticks. Sequencing of this transcript variant identified a 36 bp insertion in the βAOR gene, leading to a 12-amino acid insertion (LLKTLALVTIIS) in the first transmembrane domain of the protein. In addition, nine synonymous SNPs were also discovered in R. appendiculatus, R. evertsi and R. sanguineus cell lines. Some of these SNPs appear to be unique to each species, providing potential tools for differentiating the tick species. The BME/CTVM6 cell line had significantly higher ABCB10 (P = 0.002) expression than the other R. microplus cell lines.βAOR gene and demonstrated a higher ABCB10 expression level in the BME/CTVM6 cell line, indicating that tick cell lines provide a useful experimental tool for acaricide resistance studies and further elucidation of tick genetics.The present study has identified a new The online version of this article (doi:10.1186/s13071-016-1708-x) contains supplementary material, which is available to authorized users. Rhipicephalus (Boophilus) microplus, also known as the cattle tick or southern cattle tick, is a parasite of major global economic importance. Sales-Junior et al. [R. microplus, making it one of the most prevalent of bovine parasites. Rhipicephalus microplus causes significant economic losses; direct losses include damage to the hide from tick attachment, anaemia and in severe cases death [Babesia bigemina, Babesia bovis and Anaplasma marginale, all of which cause production losses and can be fatal [Rhipicephalus appendiculatus is an important tick of cattle in eastern Africa, where it transmits Theileria parva [Rhipicephalus sanguineus is a globally widespread tick that is commonly found on dogs [Amblyomma variegatum infests cattle throughout sub-Saharan Africa and transmits Ehrlichia ruminantium, the causal agent of heartwater [Ixodes ricinus is the most widely distributed tick species in northern, western and central Europe and is of human health significance as the vector of Lyme disease (Borrelia burgdorferi) as well as being an important vector of several diseases of cattle and sheep [Hyalomma anatolicum infests cattle in North Africa, the Middle East and Asia and transmits Theileria annulata, of economic significance in the region [r et al.  estimatees death . More imbe fatal . Rhipiceia parva . Rhipice on dogs . Amblyomartwater . Ixodes nd sheep , 7. Hyale region .R. microplus acts together with high fecundity to result in rapid selection for development of acaricide resistance. There are several mechanisms by which acaricide resistance has evolved; those most commonly noted are target site modification and metabolic resistance [para sodium channel, the target of synthetic pyrethroids (SP) [The short, one-host life-cycle of sistance –10. Targids (SP) , 12. Metids (SP) .βAOR) and resistance to amitraz in R. microplus ticks in Australia [Octopamine receptors have been shown from studies in insects to be the target for formamidines such as amitraz, leading to the hypothesis that mutations in octopamine receptors would confer resistance to formamidines . There australia . A non-sR. microplus [R. microplus ABC subtype B10 (RmABCB10) in two different ivermectin-resistant strains. The importance of increased ABCB10 expression through metabolic processes was subsequently demonstrated in an ivermectin resistance-induced R. microplus cell line [ABCB10 plays a role in resistance to other classes of acaricides.ATP-binding cassette (ABC) transporters are a superfamily of transmembrane proteins, consisting of up to 8 subfamilies  found inicroplus . They tricroplus . An assoicroplus . These aell line  but it iOver 40 continuous tick cell lines derived from species of worldwide economic importance are maintained in the Tick Cell Biobank at The Pirbright Institute . The estR. microplus continuous cell lines for future genomic and transcriptomic studies on resistance to acaricides. In this study we wished to confirm that two genes that have been strongly associated with resistance (one that operates via target site modification and one that operates via metabolic resistance mechanisms) are expressed by tick cells in vitro, to identify new variants of these genes if any were present, and to determine whether there is variation in their expression level. These two genes were selected for this study because (i) amitraz and macrocyclic lactones (including ivermectin) are globally the most widely used acaricides at present, (ii) we have well-established protocols for working with these genes, and (iii) they represent two of the main mechanisms of resistance-detoxification (ABCB10) and target-site insensitivity (βAOR). The first objective was to partially sequence the βAOR gene for single nucleotide polymorphisms (SNPs) that were reported previously by Corley et al. [ABCB10 in the Rhipicephalus spp., in particular the R. microplus, cell lines. The study design did not allow a robust determination of the relationship between genotype or expression level and the resistance status of the population of ticks from which the continuous cell lines were developed.The purpose of the present study was to assess the suitability of six y et al. . The secR. microplus, three other Rhipicephalus spp., A. variegatum, H. anatolicum and I. ricinus, were provided by the Tick Cell Biobank at The Pirbright Institute. The cell lines, passage level(s) tested, species and instar of origin, geographic origin and acaricide resistance status of the parent ticks (if known) are listed in Table rcf for 5 min, most of the supernatant medium was discarded, leaving only 3 ml of medium to re-suspend the cells. The cells were then divided into 3 aliquots in 1.5 ml microtubes and centrifuged at 900 rcf for 5 min. The supernatants were then discarded and the cell pellets frozen at −80 °C for nucleic acid extraction.Fourteen tick cell lines, derived from Total RNA was extracted from cell pellets using the Qiagen miRNeasy Mini Kit (#217004) with on-column DNase digestion following the manufacturer’s protocol for extraction from cells. Genomic DNA (gDNA) was extracted from cell pellets using the Qiagen QiaAMP Mini Kit (#51304) following the manufacturer’s protocol. The RNA and DNA concentrations were measured spectrophotometrically using a Nanodrop ND-1000 and quality was confirmed at absorbance 260/280 nm to be ≥ 1.95.First strand complementary DNA (cDNA) was synthesised using 800 ng total RNA with TaqMan Reverse Transcription Reagents (Life Technologies #N8080234) in a 20 μl reaction volume according to the manufacturer’s instructions. This was performed in a Life Technologies SimpliAmp™ Thermal Cycler using the recommended cycling conditions. The cDNA was then diluted with 30 μl of RNase-free water, before any subsequent experiments.2), dNTPs (0.2 mM), forward primer (0.5 μM), reverse primer (0.5 μM), Taq polymerase (5 U/μl), RNase-free H2O and 2 μl of cDNA or 20 ng of gDNA. The primers and PCR conditions used are shown in Table ABCB10 and the housekeeping gene β-actin (ActB) using cDNA of all cell lines was done to check for presence of transcript before proceeding to quantitative PCR (qPCR). Thermal cycling conditions were initial activation at 95 °C for 5 min; 35 cycles of 95 °C for 1 min, annealing temperature (Table Each reaction was set up using a HotStar Taq Plus DNA Polymerase kit Qiagen #203603) in a 20 μl volume containing final concentrations of 1× buffer  according to the manufacturer’s instructions and reconstituted in 40 μl of RNase-free water. For cell line BME/CTVM5, only the amplicon from cDNA was sequenced because the other two genomic fragments that were amplified were not transcribed. Cleaned amplicons were checked by 1.5 % agarose gel electrophoresis before sequencing. Sequencing reactions were set up using HPLC-purified primers and a BigDye® Terminator v3.1 Cycle Sequencing Kit (Life Technologies #4337455) in a 20 μl volume containing a final concentration of 3.2 pmol of forward or reverse primer, 0.5 μl of BigDye reaction mix, 3.5 μl of 5× sequencing buffer and 10 μl of cleaned amplicon.Sequencing reactions were cleaned using an Agencourt CleanSEQ Dye Terminator Removal Kit (#A29151) according to the manufacturer’s instructions, and trace signal was detected through capillary electrophoresis on a 3130XL Genetic Analyzer. CLC Genomics Workbench 7.5.2 was used to manually check the trace results, generate alignments and the amino acid sequence used for domain prediction and 2-dimensional representation of protein structure.ABCB10 and ActB expression were determined in a 10 μl reaction volume containing 1X Brilliant III Ultra-Fast SYBR Green Q-PCR Master Mix (Agilent Technologies #600882), 0.03 μM of ROX reference dye, 0.5 μM forward and reverse primers  revealed that the transcript variant in BME/CTVM6 and the 220 bp gene variant in BME/CTVM5 contained a 36 bp insertion at position 190 from the start codon  of the don Fig. , not predon Fig. , which adon Fig. . A discoAttempts to sequence the additional 245 bp amplicon from BME/CTVM5 were unsuccessful despite successful gel extraction of the product , KU836739 (BME/CTVM2), KU836740 (BME/CTVM5), KU836741 (BME/CTVM6), KU836742 (BME/CTVM23), KU836743 (BME/CTVM30), KU836744 (RAN/CTVM3), KU836745 (RA243), KU836746 (REN/CTVM32), KU836747 (RML-RSE) and KU836748 (RSE/PILS35).ActB, expression of the ABCB10 gene in BME/CTVM6 was significantly higher  = 8.1, P = 0.002), by approximately seven-fold, compared to expression in all the other cell lines  resistant to multiple acaricides. This insertion introduced 12 aa into the first transmembrane domain of the seven-transmembrane protein structure, resulting in a longer extracellular domain. The βAOR protein is the predicted target for formamidines [Acaricide resistance in AOR gene . We demoy et al.  identifiamidines ; hence, R. microplus strain as BME/CTVM6, has potentially three different βAOR genes, only one of which was transcribed. The transcribed gene is completely homologous to the published βAOR gene JN974909 [βAOR gene, present at both low and high passage levels, only one seems to have been transcribed. As the two cell lines were each derived from different pooled egg batches laid by over 50 female ticks, a possible explanation for this phenomenon is that BME/CTVM5 and BME/CTVM6 were derived from a population of ticks in which resistance was developing but in which the resistance-conferring allelic variants were not fixed at 100 % in the population. The establishment of the two cell lines would likely have been subject to normal stochastic effects on founder populations and subsequent genetic drift, leading to fixation of the initially more frequent allele over time after establishment of the culture. If this is the case, it highlights the potential value of the heterogeneous cell culture model for studies on the evolution of acaricide resistance, and draws attention to aspects of the system that must be taken into consideration in the design of experiments. We hypothesise that the βAOR gene was first duplicated in the base population. Gene duplication or multiple gene copy number has been linked to evolutionary adaptation in the parasitic trematode Fasciola hepatica [Anopheles gambiae [R. microplus [The BME/CTVM5 cell line, derived from the same JN974909 . One of hepatica , the mos gambiae  and R. microplus , 29.ABCB10 in particular, and ivermectin resistance. BME/CTVM6 demonstrated approximately seven-fold higher ABCB10 expression when compared to BmVIII-SCC, a cell line derived from acaricide-susceptible ticks . The importance of ABCB10 expression level in ivermectin resistance was demonstrated in a R. microplus [I. scapularis cell line, another ABC transporter gene, ABCB8, was upregulated [ABCB10 expression compared to BmVIII-SCC. In order to verify if these increased gene expression levels translate to protein expression levels, further work will be necessary.Pohl et al.  reportedicroplus  cell linegulated . BME/CTVβAOR gene and a higher level of ABCB10 expression compared to the other R. microplus cell lines, whereas the BME/CTVM5 cell line, derived from the same strain, did not show these characteristics. It is also notable that both BME/CTVM5 and BME/CTVM6 were established over 30 years ago, prior to the recent heavy and widespread use of macrocyclic lactone products and the documented emergence of ivermectin resistance. It is possible that ABCB10 played a role in resistance to acaricides other than ivermectins, but it is unlikely that any mutation in βAOR would lead to resistance to macrocyclic lactones, as the macrocyclic lactones are not known to bind to βAOR. The fact that the same tick strain has returned quite different results in terms of the expression of a detoxifying gene and the genotype of a target gene clearly demonstrates the importance of initial stochastic effects at the time of the establishment of the cell culture.It is interesting that the BME/CTVM6 cell line derived from an acaricide-resistant strain has both a different R. appendiculatus, R. evertsi and R. sanguineus cell lines. Some of these SNPs appear to be unique to individual species. These SNPs provide a potential tool for identifying the tick species; however, they need to be verified with R. appendiculatus, R. evertsi and R. sanguineus ticks from different regions of the world and with known acaricide resistance status.Nine synonymous SNPs were found in βAOR gene in gDNA extracted from the Amblyomma, Hyalomma and Ixodes spp. cell lines could be due to insufficient sequence similarity between the tick genera, such that the primers designed for R. microplus did not bind to the gene in DNA or RNA extracted from these cell lines. A search for adrenergic octopamine receptor genes in Ixodes scapularis (https://www.vectorbase.org/organisms/ixodes-scapularis and http://www.ncbi.nlm.nih.gov/nuccore) found nothing, but there were numerous putative G-protein coupled octopamine receptors. Alternatively, the βAOR gene may be absent from these species or genera. It is more likely that the annotation for β-adrenergic-like octopamine receptor is incomplete.Failure to detect the R. microplus cell lines have proven to be useful tools for investigating tick genetics in relation to acaricide resistance as evidenced from the findings of this study and other reports [The  reports , 21, 22.The present study has identified a new βAOR gene and demonstrated a higher ABCB10 expression level in the BME/CTVM6 cell line, indicating that tick cell lines provide a useful experimental tool for acaricide resistance studies and further elucidation of tick genetics.aa, amino acid; ABCB10, ATP-binding cassette B10; ANOVA, analysis of variance; bp, base-pair; cDNA, complementary deoxyribonucleic acid; gDNA, genomic deoxyribonucleic acid; NTC, non-template control; PCR, polymerase chain reaction; qPCR, real-time quantitative PCR; βAOR, β-adrenergic octopamine receptor; SNP, single nucleotide polymorphism


The KEAP1-NRF2 pathway regulates cellular redox homeostasis by transcriptional induction of genes associated with antioxidant synthesis and detoxification in response to oxidative stress. Previously, we reported that KEAP1 mutation elicits constitutive NRF2 activation and resistance to cisplatin (CDDP) and dacarbazine (DTIC) in human melanomas. The present study was conducted to clarify whether an HSP90 inhibitor, 17-AAG, efficiently eliminates melanoma with KEAP1 mutation, as the NRF2 target gene, NQO1, is a key enzyme in 17-AAG bioactivation. In melanoma and non-small cell lung carcinoma cell lines with or without KEAP1 mutations, NQO1 expression and 17-AAG sensitivity are inversely correlated. NQO1 is highly expressed in normal melanocytes and in several melanoma cell lines despite the presence of wild-type KEAP1, and the NQO1 expression is dependent on NRF2 activation. Because either CDDP or DTIC produces reactive oxygen species that activate NRF2, we determined whether these agents would sensitize NQO1-low melanoma cells to 17-AAG. Synergistic cytotoxicity of the 17-AAG and CDDP combination was detected in four out of five NQO1-low cell lines, but not in the cell line with KEAP1 mutation. These data indicate that 17-AAG could be a potential chemotherapeutic agent for melanoma with KEAP1 mutation or NQO1 expression. NRF2 is an oxidative stress-activated transcription factor that regulates transcription of a subset of genes including those encoding enzymes involved in antioxidant synthesis and detoxification ,2. UnderThe present study was conducted to investigate whether melanoma and NSCLC cell lines harboring KEAP1 mutation would be sensitive to 17-AAG. NQO1 was found to be highly expressed in normal melanocytes and several melanoma cell lines, irrespective of the presence of wild-type KEAP1, and they were also 17-AAG-sensitive in comparison with NQO1-low cell lines.2 in RPMI 1640  supplemented with 10% fetal bovine serum (FBS), non-essential amino acids (NEAA) and penicillin-streptomycin (Invitrogen). Two human melanoma cell lines (GAK and HMY-I) were obtained from the Japanese Collection of Research Bioresources  and maintained in F-12 HAM  and DMEM (Invitrogen), respectively, supplemented with 10% FBS, NEAA and antibiotics. Three human melanoma cell lines  were obtained from American Tissue Culture Collection  and maintained according to the culture method of the ATCC. Nine non-small cell lung cancer cell lines  were obtained from the ATCC. All NSCLC cell lines were maintained in RPMI supplemented with 10% FBS, NEAA and antibiotics. Normal human epithelial melanocytes, neonatal  and normal human dermal fibroblasts, and neonatal skin fibroblasts (NHDF-neo) were obtained from Invitrogen and from Lonza , respectively, and maintained in accordance with the supplier’s instructions.Four human melanoma cell lines  were obtained from the Cell Resource Center for Biomedical Research, Tohoku University . Two human melanoma cell lines (A7 and MM-AN) were kindly provided by Dr. M.C. Mihm . The cells were maintained at 37°C under 5% COTo establish immortalized melanocytes, a human TERT expression vector was constructed by transferring the insert of pBABE-neo-hTERT  into the multicloning site of pLVSIN-neo . Transfection and packaging were carried out using the Lenti-X HTX Packaging system  in accordance with the manufacturer’s instructions. HEMn-LP cells were infected and selected in the presence of 800 μg/ml G418 for a week.Silencer® Select siRNAs against NRF2  and negative control siRNA (Cat# 4390844), Lipofectamine RNAiMAX Transfection Reagent, and Opti-MEM were obtained from Life Technologies . Cells were treated with 10 nM siRNA and 7.5 μl RNAiMAX in Opti-MEM in a 6-well plate format in accordance with the manufacturer’s instructions.17-(Allylamino)-17-demethoxygeldanamycin (17-AAG), the NQO1 inhibitor ES936, cis-diammineplatinum (II) dichloride (CDDP), and dacarbazine (DTIC) were obtained from Sigma Aldrich. 17-AAG and ES936 were prepared at a stock concentration of 10 mM in dimethyl sulfoxide. CDDP was directly dissolved in culture medium. DTIC was prepared as a 0.1 M stock in 0.1 M HCl.The antibody against NRF2 was obtained from Abcam . The antibody against NQO1 was from Cell Signaling Technology . α-tubulin antibody was from Sigma Aldrich (T5168). GAPDH antibody was from Covance . HRP-linked anti-mouse IgG and anti-rabbit IgG were from GE Healthcare .Trypsinized cells were inoculated into 96-well plates at a density of 3,000 cell/well for 24 h. For dose-response analysis, cells were treated with two-fold serial dilutions of 17-AAG (10 μM to 1 nM), CDDP (1 mM to 0.1 μM) or DTIC (5 mM to 1 μM) for 72 h. NQO1 was inhibited by pretreatment with 100 nM ES936 or vehicle for 30 min, and then an equal volume of 2× concentrated 17-AAG with 100 nM ES936 was added . For com3VO4, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with complete protease inhibitor cocktail  and 0.5 mM PMSF. Protein concentration was measured with a BCA Protein Assay Kit . Total protein lysates of human adult normal tissues  were obtained from BioChain Institute . Protein samples were separated on SDS-PAGE gel and then transferred onto polyvinylidene difluoride transfer membranes . The membranes were blocked with 5% BSA (Sigma Aldrich) in 0.1% Tween-20/PBS for NQO1 or with 5% non-fat dried milk  in 0.1% Tween-20/PBS for other antibodies. The membranes were then immunoreacted with an appropriate primary antibody overnight at 4°C and with HRP-conjugated secondary antibodies  for 1 h at room temperature. Signals were detected with ECL prime detection reagents  and ChemiDoc XRS . Densitometric analysis of each protein signal was carried out by ImageJ.Cells were washed twice with cold PBS, harvested by scraping, and lysed in RIPA buffer  in accordance with the manufacturer’s instructions. RNA concentration was measured by NanoDrop (Thermo Scientific) and an equal amount of extracted RNA was reverse-transcribed by SuperScript® III First-Strand Synthesis SuperMix (Invitrogen). cDNAs of NRF2, NQO1 or GAPDH were quantified by real-time PCR  using TaqMan® Gene Expression MasterMix and TaqMan® Gene Expression Assays for NRF2 (Hs00975961_g1), NQO1 (Hs02512143_s1), and GAPDH (Hs02758991_g1).Genomic DNA was extracted using a PureLink® Genomic DNA Mini Kit (Invitrogen) and quantified by NanoDrop (Thermo Scientific). Equal amounts of genomic DNA were mixed with TaqMan® Genotyping Master Mix supplemented with TaqMan® Copy Number Reference Assay RNase P (Life Technologies) and TaqMan® Copy Number designed to the 1st exon of NQO1 (Hs03028502_cn) or 3' downstream of the NQO1 gene (Hs05457080_cn). Each copy number was quantified by real-time PCR . NQO1 copy numbers were normalized to the average of normal melanocyte samples as 2 copies.50 was calculated. In both melanoma and NSCLC, IC50 values for cell lines with KEAP1 mutation and high NQO1 expression were lower than those of cell lines with low NQO1 expression , but not in cells pretreated with the NQO1 inhibitor   .NQO1 expression in normal melanocytes was compared with that in normal human dermal fibroblasts. Although NQO1 was detectable in fibroblasts from a neonatal donor, signals in melanocytes were more abundant than those in fibroblasts . Among n90, a synergistic effect of the 17-AAG and CDDP combination was detected in four out of five NQO1-low cell lines Click here for additional data file.S1 Figp = 0.0697).(A) Genomic DNA was extracted from melanocytes and melanoma cell lines, and NQO1 gene copy number was determined using two probes against Exon 1 and the 3' downstream region of the NQO1 gene. Copy number was normalized against normal melanocytes as two copies. The data were expressed as mean±S.D. of three independent experiments (n = 3). (B) NQO1 gene copy number and NQO1 protein abundance were inversely correlated (r = −0.5722) but not to a significant degree ((EPS)Click here for additional data file.


There has been considerable progress in the management of acute lymphoblastic leukemia (ALL) but further improvement is needed to increase long-term survival. The thiopurine agent 6-mercaptopurine (6-MP) used for ALL maintenance therapy has a key influence on clinical outcomes and relapse prevention. Genetic inheritance in thiopurine metabolism plays a major role in interindividual clinical response variability to thiopurines; however, most cases of thiopurine resistance remain unexplained.We used lymphoblastoid cell lines (LCLs) from healthy donors, selected for their extreme thiopurine susceptibility. Thiopurine metabolism was characterized by the determination of TPMT and HPRT activity. We performed genome-wide expression profiling in resistant and sensitive cell lines with the goal of elucidating the mechanisms of thiopurine resistance.P = 0.024) in resistant compared to sensitive cell lines, although there was no difference in HPRT activity. We identified a 32-gene transcriptomic signature that predicts thiopurine resistance. This signature includes the GTPBP4 gene coding for a GTP-binding protein that interacts with p53. A comprehensive pathway analysis of the genes differentially expressed between resistant and sensitive cell lines indicated a role for cell cycle and DNA mismatch repair system in thiopurine resistance. It also revealed overexpression of the ATM/p53/p21 pathway, which is activated in response to DNA damage and induces cell cycle arrest in thiopurine resistant LCLs. Furthermore, overexpression of the p53 target gene TNFRSF10D or the negative cell cycle regulator CCNG2 induces cell cycle arrest and may also contribute to thiopurine resistance. ARHGDIA under-expression in resistant cell lines may constitute a novel molecular mechanism contributing to thiopurine resistance based on Rac1 inhibition induced apoptosis and in relation with thiopurine pharmacodynamics.We determined a higher TPMT activity  contains supplementary material, which is available to authorized users. Approximately 6,000 patients are diagnosed with acute lymphoblastic leukemia (ALL) each year in the USA, including about two-thirds who are younger than 20 years, making ALL the most common malignancy in children and adolescents . ClinicaThe treatment of ALL relies on combination chemotherapy. One of the cornerstone drugs for both intensification and maintenance therapy is the thiopurine agent 6-mercaptopurine (6-MP) . EffectiS-methyltransferase (TPMT), hypoxanthine phosphoribosyltransferase (HPRT), and inosine triphosphate pyrophosphatase (ITPA). These studies have shed light on the toxicity of thiopurine agents but have provided little information on thiopurine resistance [Genetic inheritance in thiopurine metabolism plays a major role in the interindividual variability that characterizes the clinical response to thiopurine agents ,8. Extensistance -10. To dsistance -16.Transcriptomic analysis is a powerful tool for characterizing susceptibility and resistance to drugs ,18. Thisin vitro growth inhibition assays. We then used these transcriptomic profiles to identify genes predicting thiopurine resistance and relevant 6-MP metabolic pathways.Here, we investigated the molecular basis of thiopurine resistance by exploring the whole-genome basal transcriptomic profiles involved in 6-MP resistant and sensitive phenotypes of LCLs originating from unrelated healthy individuals and selected by We screened 53 LCLs originating from consenting unrelated healthy adults and obtained via a collaboration program with the National Laboratory for the Genetics of Israeli Populations (NLGIP), Tel-Aviv University, Israel ,26. Six The authors state that they have obtained appropriate institutional review board approval for the collection of these samples in accordance with local legislation, or have followed the ethical principles outlined in the Declaration of Helsinki for experimental investigations. In addition, informed written consent has been obtained from the healthy donors and patients, or their guardians, involved in this study.Roswell Park Memorial Institute medium, L-glutamine, and antibiotics for cell culture were purchased from Life Technologies , fetal bovine serum from GE Healthcare , and Falcon cell culture materials from Fisher Scientific . Drugs  were purchased from Sigma-Aldrich . Stock solutions  were prepared in 0.1 N sodium hydroxide and diluted in phosphate-buffered saline (PBS) to working solutions containing at least 25-fold the final tested concentrations.50) and allowed optimal LCL classification [Drug concentrations for assessing growth inhibition were 2 μM for 6-MP, 5 μM for azathioprine, and 0.5 μM for 6-TG. These concentrations were close to the mean half-maximal inhibitory concentration (ICfication . The celfication .Nt) was estimated in PBS control wells using MTS reagent, as described above, and calibration curve. The basal cell growth rate (r) was calculated using the following formula appropriate for the usual exponential kinetics of cell growth after a defined time (t): Nt = N0.2tr.Basal cell growth rates were estimated for each cell line during phenotyping experiments. After 72 h of the proliferation assay, cell density , respectively, according to the manufacturer’s instructions. DNA was quantified using an ND-1000 spectrophotometer . RNA quality and quantity were assessed using the 2100-Bioanalyzer .After validation of RNA quality (RIN score ≥8), 50 ng of total RNA was reverse-transcribed using the Ovation PicoSL WTA System V2 , according to the manufacturer’s instructions. Biotin-labelled cDNA was then hybridized to GeneChip® Human Gene 2.0ST microarrays  at 45°C for 17 h. The microarrays were washed on the fluidic station FS450 according to the specific manufacturer’s protocols and scanned using the GCS3000 7G (Affymetrix). The scans were then analyzed with Expression Console software (Affymetrix) to obtain raw data (.cel files) and metrics for quality controls. Examination of these quality-control metrics and of raw-data distribution showed no outlier samples.Data were normalized using the Robust Multi-array Average (RMA) algorithm in R software with the custom chip description file (CDF) version 17.0.0 . Data arDifferentially expressed gene enrichment analysis was carried out using the DAVID bioinformatics resources (NIH), based on gene ontology (GO) biological processes, and Ingenuity Pathways Analysis  -31. To iGUSB/GAPDH/RPL13A/B2M). The RT-qPCR assay is detailed in the Additional file Microfluidic-based quantitative PCR assay was performed to validate the differential microarray expression patterns of the molecular signature genes. Transcripts were quantified for 40 genes of interest and four reference genes  according to the manufacturer’s instructions. Briefly, luminescence measured in the microplate wells was related to intracellular ATP after the addition of luciferase and D-luciferin. Intracellular ATP concentrations were calculated using a calibration curve.RPPH1 gene) as an internal reference, using the VIC® dye-labeled TAMRA™ probe and sequence-specific forward and reverse primers (Applied assay ID 4403328). The mtDNA copy number assay examined a 72-bp fragment at the ND2 locus multiplexed with a 90-bp fragment assay from NRF1 gene as an internal reference. Final concentrations for EBV primers, EBV probe, mtDNA primers, mtDNA probe, NRF1 primers and NRF1 probe were 90 nM, 250 nM, 30 nM, 250 nM, 900 nM, and 250 nM, respectively. RT-qPCR TaqMan® reactions were performed in 384-well plates  using 10 ng of DNA template  and 5 μL of TaqMan® Genotyping Master Mix, according to the manufacturer’s protocol. Equal efficiency of amplification was observed for each assay in the multiplex reaction.Copy number variations (CNVs) of Epstein-Barr virus (EBV) and mitochondrial (mt) DNA were determined in the LCL DNA samples using TaqMan® CNV Assays . Custom TaqMan® assays were designed using Primer 3; primer and probe sequences are reported in Additional file -ΔΔCt method [in-situ hybridization to have integrated EBV copies in the diploid genome [Gene CNVs were determined using the previously described 2t method . The absd genome . The relTPMT activity was assessed using the previously described reverse-phase high-performance liquid chromatography (HPLC) method therefore adapted for lymphocyte pellets . The pel2, whose production is monitored directly at 340 nm using a microplate reader spectrophotometer [HPRT activity was measured as the rate of inosine monophosphate (IMP) production, using the Precice® kit . Briefly, as described elsewhere, the assay is based on the effect of IMP-dehydrogenase, which catalyzes the irreversible oxidation of IMP to xanthosine monophosphate while simultaneously reducing NAD to NADHotometer . Assay rt test. Correlations were assessed using the Spearman rs test.Data are described as mean ± standard error (SEM). To compare 6-MP resistant and sensitive cell lines, we used the non-parametric Mann–Whitney t test and computed fold-changes for the expression of each gene in thiopurine resistant versus sensitive cell lines. Genes associated with P values <0.01 were selected for functional bioinformatics analyses using DAVID and Ingenuity®. Statistical analyses were carried out using Partek® Genomics Suite™  and Prism 5.0 .Gene expression values were analyzed using unsupervised hierarchical clustering and principal component analysis (PCA). Then, to identify differentially expressed genes, we performed the parametric Student rs = 0.95 and rs = 0.81, respectively; P <0.0001)  . Moreover, TPMT activity was about 44% higher in resistant compared to sensitive cell lines . None of the other study parameters  differed significantly between 6-MP resistant and sensitive cell lines . The remaining 32 genes were validated for the transcriptomic signature using GUSB as the reference gene  .For identifying the most relevant genes that were differentially expressed in 6-MP resistant compared to sensitive cell lines, we performed PAM. This shrunken centroid algorithm identified 40 genomic loci that sorted the cell lines between resistant and sensitive, with a cross-validation error rate of 0.09  led to 119 terms each containing at least five genes. After a Benjamini correction adjusted at a significance level (0.01/119), we found 10 GO terms, including 122 single genes, involved in the phenotype difference  or inhibited  in resistant cell lines (P <0.01). These regulators targeted 128 of the differentially expressed genes (Table P <0.01)  inhibitor, also called p21WAF1/CIP1, which binds to and inhibits the cyclin-CDK2 or -CDK4 complexes, preventing the phosphorylation of critical CDK substrates and blocking cell cycle progression [CDKN1A acts as a negative regulator of cell cycle progression at G1. More specifically, when located in the nucleus, p21WAF1/CIP1 controls the cell cycle and DNA replication, whereas cytoplasmic p21WAF1/CIP1 has been implicated in apoptosis inhibition [WAF1/CIP1 levels, related to phosphatidylinositol 3-kinase (PI3K) pathway inhibition, induced chemoresistance by causing a cell cycle delay [WAF1/CIP1 [WAF1/CIP1 expression as a major factor in resistance to promising anticancer drugs acting within the cell cycle [WAF1/CIP1, through which it mediates the p53-dependent cell cycle G1-phase arrest in response to a variety of stress stimuli. We found upregulation of p53 target genes and of ATM, a p53 upstream regulator, which is activated in response to DNA damage, to be related with thiopurine resistance. Moreover, p21WAF1/CIP1 mediates NUPR1-induced chemoresistance, and our analysis predicted NUPR1 activation in resistant cell lines [Transcriptomic analysis identified 210 genes that were significantly upregulated or downregulated in resistant cell lines. Our GO analysis of these genes showed that 10 GO terms were enriched in these genes, including five related with the cell cycle, most notably the M phase. Ingenuity® pathway analysis predicted gression . Thus, Chibition . A studyle delay . MoreoveAF1/CIP1 . A recenll cycle . The tumll lines .TNFRSF10D, which encodes tumor necrosis factor-related apoptosis-inducing ligand receptor 4 (TRAILR4) and whose overexpression was a component of the transcriptomic signature identified in our study. Increased TNFRSF10D expression has been found to be associated with chemoresistance [Another p53 target gene is sistance . Taken iMYC inhibition, associated with cell cycle repression, was predicted in resistant cell lines [Furthermore, a study of the genome-scale protein-interaction profile of p53 showed that GTPBP4 was a p53 interactor involved in 60S ribosome biogenesis . This null lines .RFC3 and POLDIP2, both known to interact with the MMR system and cell replication. Thus, MMR deficiency and, possibly, the expression levels of RFC3 and POLDIP2, may help to predict thiopurine resistance. Furthermore, a study based on MOLT-4 cell lines also suspected the role of alterations in the MMR system in the resistance phenotype to 6-MP [CCNG2 under-expression was a risk factor for treatment-related myeloid leukemia (t-ML) [CCNG2 expression may enhance cell cycle progression and contribute to failure of the cells to respond to DNA-damage stimuli that would otherwise promote exit from the cell cycle; subsequently, the proliferation of cells carrying misrepaired DNA may lead to leukemic transformation [CCNG2 expression that might stop the cell cycle at the G1/S phase, preventing the MMR system from promoting thiopurine susceptibility and thereby contributing to thiopurine resistance.Our canonical pathway analysis identified 18 significant processes that were differentially expressed between 6-MP resistant and sensitive cell lines, among which at least five were related to DNA repair in response to damage, including ‘Role of CHK proteins in cell cycle checkpoint control’ and ‘cell cycle: G2/M DNA damage checkpoint regulation’. Many anticancer drugs acting as antimetabolites require involvement of the DNA mismatch repair (MMR) system to exert their cellular responses . The pri to 6-MP . Moreove to 6-MP . In a sta (t-ML) . CCNG2, a (t-ML) . Alteratormation . ConversARHGDIA, encoding for Rho-GDP dissociation inhibitor alpha (RhoGDIα) in resistant cell lines. RhoGDIα regulates and sequesters in cytoplasm inactive GDP-bound forms of RhoGTPase, including Rac1, a molecular target inhibited by thiopurine nucleotides [ARHGDIA expression may therefore increase the amount of potentially active Rac1, preventing effective Rac1 inhibition by the thiopurine nucleotide 6-thioguanosine triphosphate (6-TGTP). RhoGDIα thinly regulates RhoGTPase activation, involved in cellular processes and contributing to tumor invasion and metastasis [via Rac1 inhibition in lymphocytes [Finally, we found under-expression of leotides . Thus, Rtastasis ,58. Moretastasis . This meIn vitro biological noise may limit the usefulness of LCLs as a pharmacogenomics research tool [The transcriptomic content of LCLs includes many genes from diverse cellular pathways and has proven valuable for studying genome-wide individual differences in alternative mRNA splicing . The relrch tool . Severalrch tool ,25,61,62rch tool . As discrch tool . Thus, oIn conclusion, our study using an LCL-based model identified a transcriptomic signature of thiopurine resistance. We used a well-established pharmacogenomics approach involving transcriptomic profiling of basal mRNA in a cell-line model, taking advantage of the extra power afforded by analyzing extreme phenotype cell lines ,65. Thus