Mechanisms that generate transcript diversity are of fundamental importance in eukaryotes. Although a large fraction of human protein-coding genes and lincRNAs produce more than one mRNA isoform each, the regulation of this phenomenon is still incompletely understood. Much progress has been made in deciphering the role of sequence-specific features as well as DNA-and RNA-binding proteins in alternative splicing. Recently, however, several experimental studies of individual genes have revealed a direct involvement of epigenetic factors in alternative splicing and transcription initiation. While histone modifications are generally correlated with overall gene expression levels, it remains unclear how histone modification enrichment affects relative isoform abundance. Therefore, we sought to investigate the associations between histone modifications and transcript diversity levels measured by the rates of transcription start-site switching and alternative splicing on a genome-wide scale across protein-coding genes and lincRNAs. We found that the relationship between enrichment levels of epigenetic marks and transcription start-site switching is similar for protein-coding genes and lincRNAs. Furthermore, we found associations between splicing rates and enrichment levels of H2az, H3K4me1, H3K4me2, H3K4me3, H3K9ac, H3K9me3, H3K27ac, H3K27me3, H3K36me3, H3K79me2, and H4K20me, marks traditionally associated with enhancers, transcription initiation, transcriptional repression, and others. These patterns were observed in both normal and cancer cell lines. Additionally, we developed a novel computational method that identified 840 epigenetically regulated candidate genes and predicted transcription start-site switching and alternative exon splicing with up to 92% accuracy based on epigenetic patterning alone. Our results suggest that the epigenetic regulation of transcript isoform diversity may be a relatively common genome-wide phenomenon representing an avenue of deregulation in tumor development. Traditionally, the regulation of gene expression was thought to be largely based on DNA and RNA sequence motifs. However, this dogma has recently been challenged as other factors, such as epigenetic patterning of the genome, have become better understood. Sparse but convincing experimental evidence suggests that the epigenetic background, in the form of histone modifications, acts as an additional layer of regulation determining how transcripts are processed. Here we developed a computational approach to investigate the genome-wide prevalence and the level of association between the enrichment of epigenetic marks and transcript diversity generated via alternative transcription start sites and splicing. We found that the role of epigenetic patterning in alternative transcription start-site switching is likely to be the same for all genes whereas the role of epigenetic patterns in splicing is likely gene-specific. Furthermore, we show that epigenetic data alone can be used to predict the inclusion pattern of an exon. These findings have significant implications for a better understanding of the regulation of transcript diversity in humans as well as the modifications arising during tumor development. Molecular processes such as alternative splicing and transcription start-site switching are primary drivers of transcript diversity. About 95% of the ∼23,000 human genes are estimated to produce more than one mRNA isoform The analysis of transcript diversity regulation has traditionally focused on splicing factors and RNA sequence features such as splicing enhancers and silencers While the connection of epigenetic regulation with overall gene expression has largely been elucidated Module S1 in Text S1).In addition to elucidating the epigenetic regulation of transcript diversity, further open questions remain. These questions pertain for instance to the genome-wide prevalence of epigenetic regulation of transcript diversity generated via alternative splicing or transcription start-site switching. Furthermore, it is unclear to what extent the involvement of epigenetic marks in the regulation of transcript diversity is gene-specific, ie. whether individual genes respond to different histone marks or whether there is a “universal” set of marks for alternative splicing. Several studies aimed at deciphering the association between histone modifications and alternative splicing on a genome-wide scale  and Here, we sought to perform a detailed study investigating the association between histone modification enrichments and the processes that influence isoform abundance – transcription start-site switching and splicing – on a genome-wide level  and Table 1http://genome.ucsc.edu/ENCODE/), of which six were normal  and three were cancer cell lines . For all nine cell lines, we obtained information of the genome-wide patterns of the following twelve histone marks: H3K4me1, H3K4me2, H3K4me3, H3K9ac, H3K9me1, H3K9me3, H4K20me1, H3K27ac, H3K27me3, H3K36me3, H3K79me2, H2az.We examined RNA-seq data from nine human cell lines   a genome-wide approach, and (ii) an exon-specific approach. The genome-wide method analyzes each cell line individually and investigates all exons with a given characteristic  at once, irrespective of the gene of origin. The exon-specific approach, in contrast, analyzes one exon at a time across multiple cell lines. The latter approach is able to identify candidate exons or genes with potential epigenetic regulation of transcription diversity and is analogous to an experimental setup in which each cell line represents an experimental condition (i.e. varying levels of histone modification enrichment) resulting in a particular exon inclusion or transcription start site outcome. The genome-wide approach requires a set of assumptions see  section;To assess the level of transcript diversity in the human genome, we analyzed RNA-seq data from nine human cell lines and quantified the abundance of specific mRNA isoforms for each protein-coding gene and lincRNA. We mapped and assembled the transcriptome of each cell line using the TopHat2 and Cufflinks2 softwares We hypothesized that, if histone modification enrichment patterns play a significant role in transcript diversity, then the levels of transcription start-site switching and splicing should correlate with the enrichment levels of certain histone modifications within each cell line analyzed. We therefore investigated the transcriptome-wide association between histone mark enrichment and TSSIR and SEIR within each cell line. To address the possibility that transcript diversity in cancer cell lines is regulated differently as compared to that in normal cell lines, we quantified the level of association in the normal cell lines first and then assessed the degree of similarity in this pattern between normal and cancer cell lines. To this end, we determined the expression profiles as well as histone modification enrichment for all annotated exons of protein-coding genes and lincRNAs in the normal cell lines Methods.The transcription profiles of the nine cell lines revealed that many protein-coding genes as well as lincRNAs undergo alternative splicing. Given the fact that transcription start-site switching occurs in a similar epigenetic background for protein coding genes as well as lincRNAs, we then sought to investigate whether splicing in protein-coding genes and lincRNAs is also associated with a similar set of histone marks. We found that splicing in protein-coding genes was most strongly positively correlated with the enrichment of H3K36me3 and negatively correlated with H3K4me2 and H3K4me3 Fig. 2A.We then aimed to investigate whether this pattern was consistent when taking into account exon number per gene, gene expression patterns, and genomic features such as simple repeats, microsatellites, and conserved elements. Controlling for these factors, the correlations between TSSIR and H3K4me2 as well as H3K9ac were very robust, varying for instance in the Gm12878 cell line between 0.35<ρ<0.37 for H3K4me2 (uncontrolled correlation ρ = 0.37) and between 0.35<ρ<0.38 for H3K9ac (uncontrolled correlation ρ = 0.37). Similarly, controlling for H3K9ac enrichment reduced the correlation between TSSIR and H3K4me2 by only 0.5%, and controlling for H3K4me2 enrichment reduced the correlation between TSSIR and H3K9ac by only 3.18%. These observations suggest that, while the interplay between transcript diversity and epigenetics probably involves many other factors, which might occlude the signal, the association between the SEIR and specific histone marks is genuine.CD45 showed that molecular interactions as far as 1 kb downstream of exon 5 affected its inclusion rate z0 kb = 0.09) but rose to much higher levels as close as 1 kb upstream and downstream of the spliced exon ; this level of correlation was also observed for distances of 2 kb and 5 kb upstream and downstream of the exon . Interestingly, a very different spatial pattern was observed for the histone mark H3K79me2, for which the correlation between TSSIR and histone enrichment upstream and at the exon locus was weak , but became much stronger at distances of 1–5 kb downstream of the exon . The spatial pattern of correlation between H3K4me1 enrichment and TSSIR for lincRNAs was less pronounced .Recently, a study examining the alternative splicing of 0 kb = 0.26. It remains unclear which factors drive the spatial distribution of H3K36me3; for example, Luco et al. showed that H3K36me3 interacts with the FGFR2 pre-mRNA via the MRG15/PTB chromatin-adaptor complex, which regulates the inclusion rates of alternatively spliced IIIb and IIIc exons The only spatial pattern evident for an association between histone enrichment and SEIR was observed for H3K36me3 Fig. 2A.Overall, our observations suggest that histone mark enrichment is associated with transcription start site exon inclusion and splicing and has a strong spatial signature. In addition to these analyses, we performed several control studies to establish that our results are genuine. First, our findings were robust even after controlling for gene expression, exon number, and genomic features such as simple repeats, microsatellites, and evolutionary conservation. Although the overall correlation between both TSSIR and SEIR and various histone modifications was moderate transcriptome-wide, the rapid change of correlation over short distances from exons and consistent patterns across multiple cell lines suggest an authentic relationship.p>0.89 across all −5 kb, −2 kb, −1 kb, 0 kb, 1 kb, 2 kb, and 5 kb regions). Similarly, the other comparisons between normal and cancer cells, for both protein-coding genes and lincRNAs, did not show significant differences either . These findings imply that the same histone modifications are associated with transcript diversity in both normal and cancer cells and that perturbation of the epigenetic environment via experimental manipulation in normal cells would potentially be informative of cancer cells.Since cells accumulate many genetic and epigenetic aberrations during tumorigenesis rd exon is always excluded (SEIR = 0) in all HPS4 isoforms in H1hesc, Helas3, Hsmm, Huvec, and Nhlf cells, but is only occasionally included (0.03<SEIR<0.15) in Gm12878, Hepg2, K562, and Nhek cells  resulting in a particular exon inclusion outcome. For example, the gene HPS4 (Hermansky-Pudlak syndrome gene 4) is expressed in all nine cell lines; its 3ek cells Fig. 3. Supplementary Dataset S1). Specifically, 399 and 473 genes displayed a significant association between splicing and transcription start-site switching, respectively. Note that a single gene can be significant for the association between epigenetic patterning and both splicing and transcription start-site switching. To understand whether obtaining 840 candidate genes was a result of chance, we performed 1000 permutations by randomly reassigning exon labels for TSSIR and SEIR while keeping the epigenetic background of a gene constant. Observing 840 candidate genes in total was significantly higher (p<0.001) as compared to what was expected by chance (Table S6 in Text S1) including the regulation of the response to stimulus and development process. Thirty three of these genes were cancer-associated genes (Supplementary Dataset S1) (http://www.sanger.ac.uk/genetics/CGP/Census/).We thus analyzed all exons across all cell lines in a similar fashion, first only taking into account histone enrichment at the exon locus. Given the TSSIR and SEIR values across cell lines, each exon may be constitutively excluded (TSSIR = 0 and SEIR = 0), occasionally excluded (TSSIR>0 and SEIR<1), or retained (TSSIR = 1 and SEIR = 1). We then directly compared the histone modification levels for the inclusion pattern of a given exon across all available cell lines. The three possible two-way comparisons are: i) cell lines in which a given exon is always excluded versus retained (TSSIR = 0 vs. TSSIR = 1 or SEIR = 0 vs. SEIR = 1), ii) cell lines in which a given exon is retained versus occasionally excluded (TSSIR = 0 vs. 0<TSSIR<1 or SEIR = 0 vs. 0<SEIR<1), and iii) cell lines in which a given exon is occasionally excluded versus retained (0<TSSIR<1 vs. TSSIR = 1 or 0<SEIR<1 vs. SEIR = 1). Unfortunately, since the number of cell lines with available histone modification was limited, the power of this test was low. Nonetheless, given our stringent criteria Methods,http://genome.ucsc.edu/ENCODE/downloads.html). We limited our predictions to the 840 candidate genes identified above, since the cell lines previously analyzed provided evidence for an involvement of epigenetic marks in transcript diversity for only 840 candidate genes; attempting to predict exon inclusion based on epigenetic information for genes that are not epigenetically regulated would thus not be appropriate.We then aimed to predict exon inclusion patterns in an independent sample set. Specifically, given the histone enrichment levels and the inclusion pattern in the nine previously studied cell lines, we sought to determine, in independent cell lines, whether a given exon was always retained (SEIR = 1), always excluded (SEIR = 0), or occasionally excluded (0<SEIR<1) with regard to splicing or transcription start-site switching . These predictions were performed in the Hmec and Monocytes CD14 cell lines, for which more complete epigenetic information became available  for the original cell line set , and identified the SEIR of this exon in each cell line. All ETV1 isoforms in Gm12878 and Hepg2 cell lines lacked exon 5 (SEIR = 0) whereas some isoforms expressed in H1hesc, Hsmm, Huvec, K562, Nhek, and Nhlf cell lines contained exon 5 (SEIR range 0.43–0.69) Fig. 5A.In this study, we analyzed the association between transcription start-site switching, spliced exon inclusion rates and histone modification patterns across multiple normal and cancer cell lines for protein-coding genes and lincRNAs. Unlike previous studies We used two approaches to address this issue. The first approach correlated transcriptome-wide (ie “within cell line”) transcription start site inclusion rates and spliced exon inclusion rates with histone enrichment levels. The second approach investigated gene-specific associations between transcription start site inclusion rates, spliced exon inclusion rates and histone enrichment levels. The shortcomings and assumptions made by each method are discussed below. Overall, our study led to four main findings. (i) The role of epigenetic patterning in transcription start-site switching is likely to be common across the genome for both protein-coding genes as well as lincRNAs. (ii) The role of epigenetic patterns in splicing is likely gene-specific, with the exception of H3K36me3 (discussed below). (iii) Our gene-specific approach led to the identification of 840 candidate genes whose exon inclusion rates for transcription start-site switching and splicing were strongly associated with patterns of histone modifications. (iv) Lastly, histone modification data alone can be used to predict the inclusion pattern of an exon.Our first and second findings are based on the observation that both transcriptome-wide and gene-specific approaches identified a common set of histone marks that were associated with transcription start-site switching , whereas the results of these two methods differed for the case of splicing. Transcriptome-wide analysis for splicing showed a pronounced association of splicing inclusion rates with H3K36me3 whereas the gene-specific approach identified H2az, H3K4me1, H3K4me2, H3K4me3, H3K9ac, H3K9me3, H3K27ac, H3K27me3, H3K36me3, H3K79me2, and H4K20me1 as significantly associated marks. This discrepancy is likely a result of a bias by the transcriptome-wide approach to detect common genome-wide trends and the gene-specific approach to identify unique relationships for each exon.Observing both common and gene-specific histone marks associated with splicing is in line with the proposed models of epigenetic regulation of splicing: the kinetic model and the chromatin-adaptor model Interestingly, a large fraction of alternatively spliced exons are DNase I sensitive There are however shortcomings to both approaches. The transcriptome-wide method makes two assumptions that may be violated in cells. First, correlating transcription start site inclusion rates and spliced exon inclusion rates with histone mark enrichment assumes that (i) transcript diversity of all genes is associated with their epigenetic background, and additionally (ii) these rates are associated with the same histone modification. Likely, it is for these reasons that the correlations between exon inclusion rates and histone mark enrichment are rather moderate. As mentioned above, however, because of the rapid change of these correlations over short distances from exons and the consistent patterns across multiple cell lines, these associations suggest a genuine relationship. The shortcoming of the gene-specific approach lies in the low statistical power of eleven cell lines analyzed and the natural tendency to miss tissue specific exon behavior. This is particularly the case for lincRNAs, of which 30%, according to recent estimates, have tissue specific expression Since a large body of experimental data indicates that aberrant splicing of gene transcripts significantly contributes to many areas of cancer biology, including metabolism, apoptosis, cell cycle control, invasion and metastasis http://genome.ucsc.edu/ENCODE/), of which six were normal  and three were cancer cell lines . For all nine cell lines, we obtained information of the genome-wide patterns of the following twelve histone marks: H3K4me1, H3K4me2, H3K4me3, H3K9ac, H3K9me1, H3K9me3, H4K20me1, H3K27ac, H3K27me3, H3K36me3, H3K79me2, H2az   and used to calculate relative exon expression. Prior to mapping reads to the human genome (hg19), each fastq file was processed in the following way: quality score statistics at all nucleotide positions for all fastq files were obtained using a python script https://main.g2.bx.psu.edu/). All reads in each fastq file were trimmed at the same position, at which the second lowest quartile quality score dropped below 20. This procedure resulted in read lengths of about 50–70 bp, depending on the quality of the sequencing run. Multiple replicates were pooled and analyzed together. Read alignment was performed using the TopHat software package RNA-seq data (paired-end 75 nt reads) from nine human cell lines  was downloaded from the UCSC ENCODE database (i.e. exon coordinates), we directly counted the number of overlapping reads with a given genomic region to attain the raw signal. The minimum overlap between a read and a genomic location was set to one nucleotide.In order to assess the level of histone modifications across all exons, we analyzed pre-computed bam files from ENCODE ChIP-seq experiments Exons were grouped into four categories: transcription start site, internal, transcription end site, or overlapping exons. We then quantified the presence of each exon type. Only internal and transcription start site exons were used for further analysis. The relative presence of transcription start site exons (TSSIR – transcription start site inclusion rate) and spliced exons (SEIR – splicing exon inclusion rate) was calculated from the Cufflinks .gtf output file and reflects the fraction of all isoforms from a given gene that contain a given exon. The inclusion rates therefore have ranges of 0<TSSIR≤1 and 0≤SEIR≤1.p<0.0001. The level of enrichment was calculated as RPKM is defined as r represents the number of reads mapped to a given exon, R is the total number of reads mapped, and L defines the length of a given exon. RPKM therefore denotes the number of reads per kilobase of exon per million reads mapped.Using raw signal read counts of histone marks and reference samples (input DNA) for each cell line, we calculated the presence of histone mark enrichment using a Fisher's test statistic and considered enrichment significant Prior to further analysis, we filtered our exon set to contain only internal exons and exons of genes that express more than one isoform in at least one normal or cancer cell line. In order to avoid potential problems with mapping RNAseq reads to closely related genes, we further excluded genes with paralogs more than 95% identical on the DNA level to generate the final curated exon dataset. We then calculated the Spearman rank correlation  between TSSIR and SEIR and histone enrichment values, excluding all exons for which Fisher's test for histone enrichment was not significant (p>0.0001).To assess the spatial patterns of correlation between histone modifications and SEIR as well as TSSIR, we calculated the extent of histone enrichment inside 1 kb, 2 kb, and 5 kb blocks immediately upstream or downstream of exons. The upstream 1 kb, 2 kb, and 5 kb regions extended from the upstream exon coordinate a given distance whereas the downstream 1 kb, 2 kb, and 5 kb regions extended from the downstream exon coordinate for a given distance. The Spearman rank correlation was then determined between each upstream or downstream block and the corresponding exon TSSIR or SEIR.p using the Fisher transformation formula, To allow for direct comparisons between correlation coefficients of different cell lines and histone modifications, we transformed the Spearman p-values were corrected for false discovery rate (FDR) To identify genes with epigenetically regulated transcript diversity, we analyzed each exon in the context of the nine cell lines . We categorized the exon inclusion rate into three groups: SEIR = 0, 0<SEIR<1, and SEIR = 1. We followed the same approach for TSSIR. Next, we tested whether any histone modification displayed a statistically significant difference in its enrichment in any possible two-group comparison, given an exon's SEIR values across the nine cell lines. For example, if the pattern of SEIR values for a given exon allowed us to separate the nine cell lines into two groups that showed either SEIR = 0 or SEIR = 1, we used T-test to determine whether the respective histone modification enrichment among the two groups of cell lines was statistically different. All We limited our predictions of exon exclusion or retention in the Hmec and Monocytes CD14 cell lines to the 840 candidate genes that showed significant association between the TSSIR or SEIR and histone modification enrichment in the original set of nine human cell lines . For a given exon, we constructed a Euclidean distance matrix with the formerly identified set of histone modifications for all cell lines, including Hmec and Monocytes CD14. Next, we determined the three closest neighbors of Hmec and Monocytes CD14 from among the original set of nine cell lines . Because the exon inclusion rates for a given exon were known in the original set of nine cell lines, we separated these cell lines into three comparison groups: i) cell lines in which a given exon was always excluded versus retained (SEIR = 0 vs. SEIR = 1), ii) cell lines in which a given exon was retained versus occasionally excluded (SEIR = 0 vs. 0<SEIR<1), and iii) cell lines in which a given exon was occasionally excluded versus retained (0<SEIR<1 vs. SEIR = 1). The inclusion status - retained, occasionally excluded, or always excluded - of a given exon in the Hmec or Monocytes CD14 cell lines was then determined based on what comparison group the majority of the three closest neighbors belonged to. For example, if the majority of Hmec's three closest neighbors (based on the Euclidian distance matrix) belonged to the group SEIR = 1, then we would predict that particular exon in the Hmec cell line was always retained, ie. a given gene was expressing only those isoforms that included our exon of interest. We applied the same approach to transcription start site exons and their respective TSSIR values.EV represents the histone enrichment variance across normal cell lines, and E is the enrichment mean of normal cell lines . Regions lacking any enrichment were excluded from the subsequent quantile analysis. We identified those 100 kb blocks with the lowest quartile index of dispersion (iod) as epigenetically conserved regions. Next, we compared these epigenetically conserved regions in normal cell lines to each of the cancer cell lines  and calculated a Z statistic. In a given cancer cell line, genomic regions with the lowest quartile absolute value Z score were deemed “conserved” whereas regions with the highest quartile absolute value Z score were identified as “divergent”. This approach identified between 11,285 and 22,506 conserved and between 616 and 1410 divergent regions given a particular histone modification.Cancer cells often undergo dramatic epigenetic reprogramming http://www.sanger.ac.uk/genetics/CGP/Census/) for a curated list of known cancer associated genes. For each cancer cell line and histone modification, we then performed a hypergeometric test on the enrichment of cancer-associated genes in epigenetically divergent regions. Taking into consideration all cancer-related genes classified in the COSMIC database, we found that regions with a significant enrichment of H3K4me3 and H3K79me2 displayed an enrichment for cancer-associated genes. For example, regions that were aberrant in H3K4me3 in cancer cells as compared to normal cells were enriched for cancer-associated genes in the Helas3 and K562 cell lines . Similarly, aberrant regions for H3K79me2 were enriched for cancer-associated genes in the Hepg2 and K562 cell lines .To assess whether epigenetically perturbed regions in cancer cell lines harbored cancer-associated genes, we performed a gene enrichment analysis. We utilized the cancer gene census (COSMIC) maintained by the Welcome Trust Sanger Institute , occasionally excluded (TSSIR>0 and SEIR<1), or retained (TSSIR = 1 and SEIR = 1). The “SplicingOrTsss” column then differentiates whether a given comparison category corresponds to transcription start site switching or splicing. Note that column “Statistic” frequently contains value “-Inf”; this refers to the case for comparisons where one of the groups has an enrichment of 0, ie. no histone mark enrichment.(XLSX)Click here for additional data file.Text S1Supporting Materials. Includes: Module S1 in (DOC)Click here for additional data file.


Tumor's multidrug resistance phenotype, namely, P-glycoprotein (p-gp), and the multidrug resistance related proteins (MRPs) expression have been suggested to influence both tracers' uptake. In the present study we set out to compare 99mTc-TF and 99mTc-MIBI uptake in high-grade glioma cell lines and to investigate the influence of gliomas p-gp expression on both tracers' uptake. We used four glioma cell lines . The expression of p-gp protein was evaluated by flow cytometry. Twenty μCi (7.4·105 Bq) of 99mTc-TF and 99mTc-MIBI were used. The radioactivity in the cellular lysate was measured with a dose calibrator. P-gp was significantly expressed only in the U251MG cell line (P < 0.001). In all gliomas cell lines  the 99mTc-TF uptake was significantly higher than 99mTc-sestamibi. The U251MG cell line, in which significant p-gp expression was documented, exhibited the strongest uptake difference. 99mTc-TF uptake was higher than 99mTc-MIBI in all studied high-grade glioma cell lines. Thus, 99mTc-TF may be superior to 99mTc-MIBI for glioma imaging in vivo. Both imaging , 4, neop imaging , assessm imaging , 7. Nevepe (MDR) –10. MDR pe (MDR) . Both 99se pumps , 9. In t2 atmosphere as described in detail elsewhere [The human glioma cell lines U251MG and A172 were obtained from Dr. W. K. Alfred Yung . U87MG and T98G were obtained from American Type Culture Collection . They were cultured in Dulbecco's modified Eagle's medium  supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin  and grown at 37°C in a 5% COlsewhere .μL PBS. Cells were kept on ice until being analyzed by flow cytometry within 1 h. Antibody specificity was controlled by staining of all cell lines with an isotype-matched control antibody . Flow cytometric analysis of p-gp stained cells was performed on a FACSCalibur (BD Bioscience) equipped with a standard argon laser for 488 nm excitation and with 530/30 nm band pass (FL1), 585/42 nm band pass (FL2), and 670 long pass (FL3) filters. Results were evaluated using CellQuest software .P-gp staining was accomplished using a PE-conjugated monoclonal antibody against human p-gp  in accordance with standard surface staining protocols. In brief, cells were stained for 30 min in the dark at room temperature (20° to 25°C). After completion of staining, cells were washed in PBS and resuspended in 500 99mTc-Tetrofosmin  and 99mTc-sestamibi  were prepared according to the manufacturer instructions. The radiochemical purity of each radiotracer was greater than 95%.3 cells were platted per each 4 cm plate. At the fourth day 10 μCi (3.7·105 Bq) (200 μL) of 99mTc-Tetrofosmin was added to the medium. We used four time points  for incubation with the tracer and then the medium was discarded. The cells were rapidly washed three times with phosphate buffered saline (PBS) at 4°C. Cells were then treated with 0.5 mL of trypsin. When the cells had detached from the bottom of the well (within 5 min), 1 mL of DMEM was added to stop the proteolytic action. Cell clumps were removed by repeated (at least 10-fold) pipetting of the trypsin/DMEM mixture. The cells were then harvested. The radioactivity in the cellular lysate was counted ten times with a gamma scintillation counter . Tracer's uptake was increased practically linearly with incubation times up to 30 min. Previous studies on 99mTc-MIBI have used an incubation period of 30 min [Initially, time activity curves for all cell lines were constructed to study the optimum time for the calculation of tracer activity. More specifically, about 5 × 10f 30 min , 14. The5 cells were platted per each 10 cm plate. At the fourth day 20 μCi (7.4·105 Bq) (200 μL) of each tracer was added to the medium. After 30 min of incubation with each tracer, the medium was discarded. The cells were scraped from the dishes. The radioactivity in the cellular lysate was counted with a dose calibrator  found to have linear response down to 15 kBq of technetium-99 (99mTc), the lowest studied activity The results were expressed as the percentage of the administered activity. All experiments were performed in duplicate and repeated three times.About 5 × 10D value, was used to compare binding of antibodies and of matched isotype controls. Differences were considered significant at P values less than 0.05.Unless otherwise stated, data are expressed as mean ± SD. The significance of differences between experimental conditions was determined using Mann-Whitney test. The Kolmogorov-Smirnov (KS) statistic, expressed as a P < 0.001) (P-gp was significantly expressed in the U251MG cell lines (< 0.001) . On the 99mTc-TF uptake, ranging between ~21% and 22% in the four studied cell lines (99mTc-MIBI in the four studied glioma cell lines (16% to 18%). In U251MG glioma cell the percentage of 99mTc-Tetrofosmin uptake was significantly higher than that of 99mTc-sestamibi . In U87MG there was also significant higher 99mTc-Tetrofosmin uptake . In A172 cell line the difference was also statistically significant . In T98G there was higher 99mTc-Tetrofosmin uptake compared to 99mTc-sestamibi and the difference was also statistically significant .The ll lines , was hig99mTc to that of 99mTc-MIBI. The results showed higher 99mTc-TF uptake, thus suggesting that 99mTc-TF could be superior to 99mTc-MIBI for glioma imaging. Apart from U251MG cell line, in which significant p-gp expression was documented, significant uptake difference was found in the other cell lines suggesting that other mechanisms may be implicated apart from p-gp expression.The present study compared the uptake in four high-grade glioma cell lines of the monovalent lipophilic cationic diphosphine TF labeled with 99mTc labeled compounds have been proven advantageous in tumor imaging over 201Tl due to higher number of photons of appropriate energy emitted by the human body per administered activity and lower radiation burden to both patient and members of the general public [99mTc-MIBI diffuses passively through the cell membrane and an estimated 95% of intracellular 99mTc-MIBI is localized in mitochondria because of the negatively charged mitochondrial membrane. 99mTc-TF enters viable cells mainly via passive transport, driven by the negative electric potential of the intact cell membrane, and it mostly localizes within the cytosol, with only a small fraction passing into the mitochondria [l public . Regardichondria . MDR1, MRPs, major vault protein (MVP) gene, the MGMT gene, and the Survivin gene [ MDR1 gene is the most extensively studied [Chemoresistance is a major obstacle for effective cancer treatment and can be present in a tumor at the time of initial diagnosis or can develop following treatment with chemotherapeutic agents . One mecvin gene . The previn gene , 16, 17. studied . This ge studied . MRPs ar studied . MRPs ha studied , 20.99mTc-TF was more lipophilic than MIBI and thus could enter easier than MIBI in glioma cells [Perek et al. studied the effect of glutathione (GSH) depletion on the chemosensitivity of human malignant glioma cell lines . None ofma cells .99mTc-TF uptake was not related to the MRP5 immunohistochemical expression; consequently, this might be another reason explaining the higher 99mTc-TF uptake in the studied glioma cell lines.In the present study U251MG cell line exhibited increased p-gp expression, in accordance with published data . Similar99mTc-TF uptake is higher than that of 99mTc-MIBI in all high-grade glioma cell lines studied. Thus, 99mTc-TF is anticipated to be a superior tracer to 99mTc-MIBI for gliomas imaging in vivo. Further studies are needed in order to elucidate the exact mechanisms involved in 99mTc-TF uptake.A limitation of the present study was the absence of positive and negative control cells lines in order to define MDR protein expression. Thus, we cannot rule out the existence of an experimental bias regarding the uptake of both radiotracers that could influence their relationship with P-gp protein expression. In conclusion, the present study showed that


Cisplatin is one of the most common chemotherapeutic drugs for non-small cell lung cancer (NSCLC). However, the response rate is limited because of drug resistance. Histone deacetylase inhibitors (HDACis), which can alter DNA accessibility by regulating chromatin structure and inducing apoptosis, exhibit a synergistic action with cisplatin. However, no biomarkers that can predict the efficacy of the combination of HDACis and cisplatin have been reported. Our study found that panobinostat, an HDAC inhibitor, increased the cisplatin sensitivity of several NSCLC cell lines with low ERCC1 expression but not those with high ERCC1 expression or gain-of-function (GOF) p53 mutation despite of ERCC1 expression level. ERCC1 knockdown increased the cisplatin sensitivity of NSCLC cell lines with high ERCC1 expression without GOF p53 mutations. In addition, in low ERCC1 expression NSCLC cell lines, knockdown of GOF mutant p53 enhanced cisplatin sensitivity. Further double knockdown of ERCC1 and GOF mutant p53 but not ERCC1 knockdown alone increased the cisplatin sensitivity of cells with both high ERCC1 expression and GOF p53 mutations. Therefore, this study demonstrated that ERCC1 expression combined with p53 mutation status may determine the efficacy of cisplatin and HDACi combined therapy and guide the development of future NSCLC therapies. Lung cancer is the leading cause of cancer death worldwide and non-small cell lung cancer (NSCLC) accounts for more than 85% of all lung cancer cases . AlthougEpigenetic alterations such as DNA methylation, mRNA regulation and other posttranslational changes in chromatin have emerged as novel targets in cancer treatment. Among them, the balance between histone transacetylases and deacetylases, which mediate the expression of tumor suppressor genes and oncogenes, makes these enzymes promising therapeutic targets . PreviouThe antitumor mechanisms of HDACis primarily include altering DNA accessibility by regulating chromatin structure and inducing apoptosis by up-regulating acetylated p53 and p21 , 9. PanoExcision repair cross-complementation group 1 (ERCC1) is a critical protein involved in nucleotide excision repair (NER), and ERCC1 expression reflects DNA repair capacity and clinical drug resistance, especially cisplatin resistance . Wild-tyLow/p53WT cell lines. H23 and H441, which harbor non-gain-of-function (GOF) p53 mutations, were classified as ERCC1Low/p53MU cell lines. H1299 and H2172, which do not express p53, were classified as ERCC1High/p53Null cell lines. Similarly, H1975 was classified as a ERCC1Low/p53GOF cell line, and PC14 was classified as a ERCC1High/p53GOF cell line. The information on the 8 cell lines is listed in abbreviated form based on ERCC1 expression level and p53 status in First, we examined the expression levels of ERCC1 in 8 NSCLS cell lines: A549, PC14, NCI-H23, HCI-H441, HCC827, NCI-H1299, NCI-H1975 and NCI-H2172. ERCC1 expression in these cell lines was analyzed by reverse transcription (RT)-qPCR Fig.  and west50 of approximately 12 μM. However, the other 7 cell lines all exhibited resistance to cisplatin independent of their ERCC1 expression level. The IC50 of these 7 lines was higher than 50 μM. There was no obvious relationship between ERCC1 expression and the effect of cisplatin in these cells.Next, we examined the cisplatin sensitivity of the NSCLC cells. We treated all 8 cell lines with different doses of cisplatin from 0 to 100 μM for 48 hours, and cell proliferation was measured using Cell Titer-Glo. As shown in Fig. Panobinostat was selected to treat the NSCLC cells because of its high potency. In our previous study, panobinostat itself did not kill tumor cells but sensitized cisplatin below 20 nM after 48 hours of treatment (data not shown). Here, we treated the NSCLC cells with panobinostat at 10 nM. After 48 hours of treatment, cell viability was measured, and 10 nM panobinostat had a less than 5% inhibition rate in all cancer cell lines Fig. .Low/p53WT, as well as in H23  and H23 cells (p53MU/ERCC1Low), but not in H1975 (p53GOF/ERCC1Low) or PC14 (p53GOF/ERCC1High) cells. Flow cytometry data confirmed a significant increase in apoptosis from 3.6% to 57.5% in A549 cells after combined treatment  cells (Low/p53MU) cells (Low/p53GOF) cells  cells  and NCI-U) cells , p53 KD lls Fig.  showed slls Fig. . Howeverlls Fig. . Considells Fig.  and flowlls Fig.  both shoHigh/p53Null) cells  cells . The celMany novel drugs, such as anti-angiogenesis monoclonal antibodies (mAbs), molecular tyrosine kinase inhibitors (TKIs) and immune checkpoint inhibitors have emerged in NSCLC treatment over the last 10 years. However, platinum-based regimens are still preferred in NSCLC adjuvant treatment and are first-line therapy in advanced NSCLC. Almost all regimens eventually fail due to the development of drug resistance. Therefore, enhancing drug sensitivity, reversing drug resistance and prolonging the time to drug resistance are very important research goals.Cisplatin is a chemotherapeutic drug that binds to and crosslinks DNA, leading to the induction of cancer cell apoptosis. The anticancer effect of cisplatin is increased by HDACis, and the underlying mechanisms have been investigated since 2003. Many mechanisms contribute to the synergistic effect of combined treatment , 23–26. Furthermore, some studies have found that up-regulation of apoptotic proteins such as p53, Bim and caspase 3 and down-regulation of glutathione (GSH) enhance cisplatin-induced cytotoxicity in oral squamous cell carcinoma (OSCC) cells. Since vorinostat and romidepsin were approved for use in cutaneous T-cell lymphoma (CTLC) by the US FDA, several similar drugs have been evaluated in NSCLC treatment in recent years . For exaThe identification of biomarkers is one of the most important aspects of studying a new therapeutic class. It is necessary to identify which patients may benefit from a particular therapy. However, to date, predictable biomarkers for selecting patients or predicting responses to HDACi treatment remain undetermined. Some studies have reported that histone 3 and 4 hyperacetylation (because an accumulation of acetylated histones is a direct downstream consequence of HDAC inhibition) and HR23B (a protein that transports ubiquitinated cargo to proteasomes) are biomarkers for sensitivity to HDACi-induced apoptosis , 29. HowNull cell lines but not PC14 (ERCC1High/p53GOF) became sensitive to cisplatin and panobinostat combination treatment. These results indicated that ERCC1 can be used as an effective biomarker for combination therapy with HDACis and cisplatin, and the mechanism may be related to the DNA repair capability determined by the ERCC1 level. However, the data also implied that GOF p53 mutations might contribute to resistance to panobinostat and cisplatin combination treatment.Although the association between cisplatin sensitivity and ERCC1 expression level was not as strong in our eight NSCLC cell lines as in previous reports, the present study found that ERCC1 expression had a very clear predictive role with regard to the efficacy of panobinostat and cisplatin combination therapy. Notably, we used 10 nM panobinostat, which is a concentration that is not typically employed in cancer treatment but is used as a sensitizer to cisplatin to avoid the toxicity of non-specific HDACis. Improved cytotoxicity was only observed in the four NSCLC cell lines with low ERCC1 expression, with the exception of the H1975 cell line, which also contains a GOF p53 mutation. None of the other three cell lines with high ERCC1 expression levels showed increased sensitivity to panobinostat and cisplatin combination treatment. Next, we knocked down ERCC1 in these three cell lines with high ERCC1 expression. The two p53Low/p53GOF) and PC14 (ERCC1High/p53GOF) cell lines. After KD of GOF mutant p53, the H1975 cells became sensitive, whereas the PC14 cells remained resistant to cisplatin and panobinostat combination treatment. However, it is very interesting that double KD of ERCC1 and GOF mutant p53 in the PC14 cell line significantly increased the sensitivity to combination panobinostat and cisplatin treatment. These results demonstrated that GOF p53 mutation as well as a high ERCC1 expression level plays important roles in the resistance of NSCLC cell lines to panobinostat and cisplatin combination treatment. Therefore, based on these findings, combined screening for GOF p53 mutations and ERCC1 expression level could be more valuable than either individual biomarker in predicting the efficacy of panobinostat and cisplatin combination treatment.Our above results suggest that p53 mutation status is very important in the response of NSCLC cell lines to panobinostat and cisplatin combination treatment. It has also been reported that GOF mutant p53 can lead to drug resistance by inhibiting cancer cell apoptosis , 31. ThuWT and the two p53MU cell lines, which all showed increased sensitivity to combination treatment. However, in the two p53GOF cell lines (H1975 and PC14) that were still resistant to combination treatment, no p21 or PARP was induced. Our study indicated that HDACis could significantly improve the cytotoxicity of cisplatin only in low ERCC1-expressing NSCLC cell lines without GOF p53 mutations based not only on chromatin structure alterations but also on p53-induced cell apoptosis. We confirmed that high expression levels of ERCC1 and GOF p53 mutants can contribute to the resistance of NSCLC cell lines to the above treatment, and combined screening for these two biomarkers might have great potential value in future clinical practice.HDACis not only loosen the structure of chromosomes to allow DNA-damaging drugs such as cisplatin to come into contact with the DNA strands more easily, but they also up-regulate apoptotic proteins such as p53 and p21. Our western blotting showed that the p21 and PARP levels were increased in the two p532.Human lung cancer cell lines A549 (ATCC Cat#: CCL-185), NCI-H23 (ATCC Cat#: CRL-5800), NCI-H1299 (ATCC Cat#: CRL-5803), NCI-H441 (ATCC Cat#: HTB-174), HCC827 (ATCC Cat#: CRL-2868), NCI-H1975 (ATCC Cat#: CRL-5908) and NCI-H2172 (ATCC Cat#: CRL-5930) were obtained from American Type Culture Collection (ATCC) and cultured as instructed. The human lung cancer PC14 cell line was kindly provided by Mr. Yu . The cancer cells were maintained in Roswell Park Memorial Institute (RPMI)1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin . Cells were maintained under a standard gas atmosphere of humidified air/5% COCell growth was measured using CellTiter-Glo (Promega). The cells were seeded in 96-well plates in 100 μl of medium with different drug doses. Cisplatin  and panobinostat  were dissolved in dimethyl sulfoxide (DMSO). After the cells were cultured for 48 hours, 100 μl of CellTiter-Glo reagent was added to each well to measure cell growth according to the manufacturer's instructions.TM RNAiMAX (Invitrogen), and the cells were assayed 3 days after transfection.The shRNA against p53 was obtained from Sigma (Clone ID: NM_000546.4-887s21c1). The following sequence was used: CCGGCACCATCCACTACAACTA CATCTCGAGATGTAGTTGTAGTGGATGGTGTTTTTG. Two independent siRNAs were used to KD ERCC1. The respective sequences were as follows: si-Q1, 5′-CAGCATGCGAATTCTGGGCAA-3′; si-Q2, 5′-TCGGGTGGTCGCCAAATACAA-3′; Ctrl-siRNA, siGLO RISC-Free Control siRNA (Dharmacon). The siRNAs were transfected using LipofectamineRNA was extracted using an RNeasy Mini Kit (Qiagen). The RNA quality was confirmed by Nanodrop spectrophotometry. Reverse transcription was performed according to the manufacturer's instructions using an RT Kit (Invitrogen). Real-time PCR analysis was performed on an ABI Prism 7900 Sequence Detection System using the SYBR Green PCR Master Mix . The relative expression of each gene was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The primers used for the quantitative RT-PCR are shown in Western blot was performed as described previously. Samples were collected directly in 1X NuPAGE LDS sample buffer with 1X sample-reducing buffer (Invitrogen) and denatured at 95°C for 5 min followed by a centrifugation at 13200 rpm for 5 min. The supernatant was electrophoresed on a 4–12% Tris-HCl gel and transferred to nitrocellulose membranes (Invitrogen). After blocking with Superblock T20 blocking buffer (Thermo Scientific), the membranes were incubated with a primary antibody overnight at 4°C and then with a secondary antibody conjugated with alkaline phosphatase for 1 hour each at room temperature; the signal was detected using a chemiluminescence method. The following primary antibodies were used: anti-p53 ; anti-phospho-p53 ; anti-GAPDH ; anti-ERCC1 ; and anti-PARP .Cells were plated in 6-cm dishes and exposed to compounds with or without p53 and ERCC1 KD. After 2 days of drug exposure, the cells were collected and stained using propidium iodide solution and Annexin V (BD). The cells were quantified and analyzed by flow cytometry on a fluorescence-activated cell scan cytometer.


Linum scabrellum Planchon (Linaceae) were analyzed. Using NMR spectra and mass spectrometry analyses of the extract we identified eight main constituents: oleic acid (1), octadecenoic acid (2), stigmasterol (3), α-amyrin (4), pinoresinol (5), 6 methoxypodophyllotoxin (6), coniferin (7), and 6-methoxypodophyllotoxin-7-O-β-D-glucopyranoside (8). By using the sulforhodamine B assay, an important cytotoxic activity against four human cancer cell lines, HF6 colon (IC50 = 0.57 μg/mL), MCF7 breast (IC50 = 0.56 μg/mL), PC3 prostate (IC50 = 1.60 μg/mL), and SiHa cervical (IC50 = 1.54 μg/mL), as well as toward the normal fibroblasts line HFS-30 IC50 = 1.02 μg/mL was demonstrated. Compound 6 (6-methoxypodophyllotoxin) was responsible for the cytotoxic activity exhibiting an IC50 value range of 0.0632 to 2.7433 µg/mL against the tested cell lines. Cell cycle studies with compound 6 exhibited a cell arrest in G2/M of the prostate PC3 cancer cell line. Microtubule disruption studies demonstrated that compound 6 inhibited the polymerization of tubulin through its binding to the colchicine site (binding constant Kb = 7.6 × 106 M−1). A dose-response apoptotic effect was also observed. This work constitutes the first investigation reporting the chemical composition of L. scabrellum and the first study determining the mechanism of action of compound 6.The cytotoxic activity and the chemical composition of the dichloromethane/methanol root extract of  Linum of the Linaceae family includes more than 200 globally distributed species. The phytochemical profiles that have been reported for the species of Linum suggest the presence of some lignan-type secondary metabolites 24D  −82.3° , and had the formula C20H22O6 derived from its positive FABMS [M+H]+ ion at m/z 359. IR : 3210, 2870, 1600, 1465 1050. UV λmax (nm): 232, and 280. 1H NMR  δH: 6.89 , 6.88 , 6.81 , 4.73 , 3.09 , 4.24 , 3.87 , 3.90 . 13C NMR  δC: 132.90 , 108.57 , 146.68 , 145.22 , 114.24 , 118.96 , 85.86 , 54.16 , 71.66 . On the basis of the spectral data, compound 5 was identified as (-)-pinoresinol + ion peak at m/z 445 with molecular formula C23H24O9. IR : 3220, 1768, 1600. UV λmax (nm): 272, and 282. 1H NMR  δH: 6.30 , 5.03 , 2.88 , 4.06 , 4.64 , 6.44 , 4.53 , 2.75 , 3.78 , 3.81 , 3.78 , 5.95 . 13C NMR  δC: 124.93 (C-1), 137.04 (C-2), 104.36 (C-3), 149.45 (C-4), 132.84 (C-5), 141.57 (C-6), 70.52 (C-7), 39.01 (C-8), 71.92 (C-9), 134.71 (C-1′), 108.09 (C-2′), 152.61 (C-3′), 134.93 (C-4′), 152.61 C-5′), 108.09 (C-6′), 45.11 (C-7′), 44.53 (C-8′), 174.44 (C-9′), 56.18 (C3′-OMe), 60.77 (C4′-OMe), 56.18 (C5′-OMe), 59.93 (C6-OMe), 101.37 (-O-CH2-O-). On the basis of the spectral data, compound 6 was identified as 6-methoxypodophyllotoxin . Positive FABMS m/z 342 [M + H]+, with molecular formula C16H22O8. IR : 3020, 1060, 940. UV λmax (nm): 256 and 292. [α]24D  −66.3 . 1H NMR  δH: 7.07 , 6.89 , 7.09 , 6.52 , 6.29 , 4.19 , 4.87 , 4.23 , 4.22 , 4.21 , 4.05 , 3.54 , 3.70 , 3.79 . 13C NMR  δC: 150.01 (C-1), 146.54 (C-2), 110.24 (C-3), 132.36 (C-4), 117.11 (C-5), 119.23 (C-6), 128.88 (C-1′), 128.87 (C-2′), 62.36 (C-3′), 55.53 (C2-OMe), 101.69 (C-1′′), 73.92 (C-2′′), 77.07 (C-3′′), 70.69 (C-4′′), 76.93 (C-5′′), 61.82 (C-6′′). On the basis of spectral data, compound 7 was identified as phenylpropane glycoside coniferin (7) . The positive FABMS spectrum showed the [M + H]+ ion peak at m/z 607, with molecular formula C29H34O14. IR : 3020, 1746, 1620, 1050, 920. UV λmax (nm): 276 and 284. 1H NMR  δH: 6.28 , 5.48 , 2.90 , 4.23 , 4.71 , 6.31 , 4.51 , 3.25 , 3.72 , 3.99 , 3.68 , 6.02 , 4.54 , 3.28 , 3.35 , 3.41 , 3.43 , 3.76 , 3.84 . 13C NMR  δC: 124.26 (C-1), 136.28 (C-2), 105.74 (C-3), 149.68 (C-4), 135.89 (C-5), 140.26 (C-6), 72.78 (C-7), 39.87 (C-8), 72.39 (C-9), 136.73 (C-1′), 108.58 (C-2′), 152.89 (C-3′), 136.54 (C-4′), 152.89 (C-5′), 108.58 (C-6′), 45.86 (C-7′), 46.23 (C-8′), 176.67 (C-9′), 56.24 , 60.76 (C4′-OMe), 60.76 (C6-OMe), 102.34 (-O-CH2-O-), 100.46 (C-1′′), 74.87 (C-2′′), 77.85 (C-3′′), 71.67 (C-4′′), 78.13 (C-5′′), 63.08 (C-6′′). On the basis of spectral data, compound 8 was identified as 6-methoxypodophyllotoxin 7-O-β-D-glucopyranoside [From the fraction F-6, compoundhttp://dx.doi.org/10.1155/2015/298463).The obtained data were compared with the literature –43 , while compound 6 (6MPTOX) showed an important cytotoxic activity with IC50 values ranging from 0.0632 to 2.7433 μg/mL against the cancer tested cell lines  and PTOX (0.0024 μM) were used as negative and positive controls, respectively. μM induced a similar effect on G2/M cell arrest with 39.8% and 41.1% of G2/M cell population, respectively. These results suggest that 6MPTOX efficiently arrest cells at G2/M phase.The effect of 6MPTOX in the cell cycle progression of PC3 cells was determined at four concentrations: 0.011 Histone H3 phosphorylated at serine 10 has long been used to identify mitotic cells nuclei in culture cell lines . The proα/β tubulin binding.Our results showed that 6MPTOX induced a mitotic cell arrest by the observed high mitotic index and the G2/M cell arrest, suggesting a similar effect as that displayed by PTOX or Taxol. In order to determine the mechanism of action of 6MPTOX, we analyzed the effect of microtubule polymerization by 6MPTOX through disruption of α-tubulin was observed in PC3 cells by confocal microscopy. Microtubules are in dynamic equilibrium with tubulin dimers as tubulin is polymerized into microtubules and depolymerized as free tubulin. This dynamic equilibrium is targeted by microtubule disrupting agents , which iThese results suggest that 6MPTOX as well as PTOX act as microtubule-destabilizing agents, while cells treated with DMSO (0.02%) demonstrated a normal and intact tubulin organization .α/β-tubulin as does PTOX, 6MPTOX was assayed for its ability to displace MTC from its binding at the colchicine site of α/β-tubulin [Kb = 7.26 × 106 M−1 by displacement of MTC in the colchicine binding site. MTC has Kb = 4.7 × 105 M−1 [To corroborate whether 6MPTOX binds to -tubulin , 55.  Fi 105 M−1 , which iμM did not show any effect on cell death. When the concentration was increased to 0.002 μM some apoptotic bodies were observed , and necrosis . Apoptosobserved ; with 0.necrosis . Three cnecrosis ; H2O2 foc bodies , and a nc bodies . The eff L. scabrellum was established. Eight compounds including lignans, terpenes, and fatty acids were isolated and identified. This study demonstrated the cytotoxic activity of 6MPTOX toward four selected human carcinomas and against a normal fibroblast cell line. Biochemical and biological experiments showed that 6MPTOX specifically arrested PC3 cells in their G2/M phase as well as a higher mitotic index. Moreover, at its lowest concentration it induces apoptosis as shown by an increase in its subG1 cell population and in stained apoptotic cells. Necrotic cell death was observed at higher concentrations. Data presented in this study show that 6MPTOX binds at the colchicine binding site of tubulin with a Kb = 7.26 × 106 M−1, causing disruption of tubulin polymerization. These results showed that 6MPTOX inhibit tubulin polymerization and arrest cells in G2/M as a mechanism of cytotoxic activity in PC3 cells. This is the first report describing the mechanism of action of 6MPTOX.For the first time, the metabolic content of the Mexican speciesSupplementary Material provides all the spectroscopic data of isolated compounds (NMR and MS spectra).


CDKN1A expression was higher in DLBCL cells that, to undergo apoptosis, required a much higher amount of drug than that necessary to induce only an anti-proliferative effect.We investigated the pre-clinical activities of two novel histone deacetylase inhibitors (HDACi), ITF-A and ITF-B, in a large panel of pre-clinical lymphoma models. The two compounds showed a dose-dependent anti-proliferative activity in the majority of cell lines. Gene expression profiling (GEP) of diffuse large B-cell lymphoma (DLBCL) cells treated with the compounds showed a modulation of genes involved in chromatin structure, cell cycle progression, apoptosis, B-cell signaling, and genes encoding metallothioneins. Cell lines showed differences between the concentrations of ITF-A and ITF-B needed to cause anti-proliferative or cytotoxic activity, and cell cycle and apoptosis genes appeared implicated in determining the type of response. In particular, CDKN1A levels appeared to be important in determining the gap between HDACi concentrations causing cell cycle arrest and those that lead to cell death.In conclusion, the two novel HDACi ITF-A and ITF-B demonstrated anti-proliferative activity across different mature B-cell lymphoma cell lines. Basal  Despite the important advances made in the treatment of B-cell lymphomas, particularly with the addition of the monoclonal antibody Rituximab to standard chemotherapy, significant numbers of these neoplasms are still refractory to currently used therapies , 2. The The family of histone deacetylases (HDACs) is divided into four classes: class I HDACs are predominantly localized in the nucleus and comprise HDAC1, 2, 3 and 8; class II HDACs, subdivided into class IIa HDAC4, 5, 7, 9) and class IIb HDAC6 and 10), shuttle between the nucleus and cytoplasm; class III HDACs comprise Sirtuins 1-7 and are mainly localised in the nucleus and mitochondrion. Differently from the zinc-dependent HDACs in the other three classes, Sirtuins require NAD+ to catalyse deacetylation. HDAC class IV only contains HDAC11, a nuclear HDAC [, 7, 9 an and 10, CDKN1A, the gene encoding the cyclin dependent kinase inhibitor, p21WAF1/CIP1 [BCL2, BCL2L1, STAT1, STAT3 and the antioxidant genes TXN (thioredoxin), SOD2, GSR (glutathione reductase) has been reported to correlate with resistance to HDACi [CDKN1A expression following HDACi treatment is also associated with resistance to HDACi-mediated cytotoxicity and cells that fail to upregulate CDKN1A/p21WAF1/CIP1 following HDACi treatment readily undergo apoptosis [HDAC inhibitors (HDACi) function by interfering in the interaction between HDACs and their substrates , 10-13. AF1/CIP1 , 13, 18.AF1/CIP1 , 20. Celto HDACi , 23. Indpoptosis , 20.Here, we assessed the preclinical activities and the mechanism of action of two novel HDACi, in over 30 lymphoma cell lines.We assessed the anti-proliferative activities of two new HDACi, here termed ITF-A and ITF-B, using the MTT cell viability assay in 24 diffuse large B-cell lymphoma (DLBCL) cell lines, 5 mantle cell lymphoma (MCL) cell lines and 3 splenic marginal zone lymphoma (SMZL) cell lines. ITF-A and ITF-B presented dose-dependent anti-proliferative activities with median IC50 (50% Inhibitory Concentration) values of 12nM for ITF-A  and 34nM for ITF-B . The majority of IC50 values were below 20nM and 50nM for ITF-A and ITF-B, respectively Figure . In addiWe also assessed the ability of ITF-B and ITF-A to inhibit HDAC activity. The DLBCL cell lines DoHH2 and TMD8 treated with ITF-B or ITF-A showed increases in nuclear acetylated histone H3 and cytoplasmic acetylated α-tubulin levels Figure . Thus, IFollowing the demonstration of the anti-proliferative effects of ITF-A and ITF-B in lymphoma cell lines, we performed cell cycle and apoptosis analyses. We treated cells with fixed doses of 20 nM ITF-A or 50 nM ITF-B for 72 hours. Only the MCL cell line JeKo-1 exhibited a marked induction of apoptosis, as determined by flow cytometry analysis of Annexin V-stained cells Figure , also suCDKN1A, CDKN2D and CDKN2C. Among the top 100 most up-regulated genes, genes encoding histones, tubulin genes and metallothionein genes were frequently represented  supported the GEP results  experiments on two DLBCL cell lines: DoHH2 (GCB-DLBCL) and TMD8 (ABC-DLBCL). Cells were treated for 8 hours with 100nM of ITF-A, 200nM of ITF-B or DMSO. Treatment with either HDACi affected diverse processes in both cell lines as determined by Gene Set Enrichment Analysis (GSEA). Similar gene sets were enriched in both cell lines, suggesting that the two HDACi similarly modulate specific processes as well as the expression of multiple genes in DLBCL cells Figures . Commonlresented  and 6. D and BTK  and 6. Q results . Western results .CDKN1A, CDKN1B, CDKN2A) were more highly expressed in the high LC50/GI50 group  and those inducing cell death (LC50 doses), while other cell lines presented large differences between these values. We were therefore interested in determining if distinct genetic features characterized these different cell lines. We focused on 23 DLBCL cell lines for which we had drug response data as well as baseline GEP data , to prevent histology-specific genetic signatures from masking signatures specifically associated with response. For each of these 23 cell lines, LC50 values were divided by GI50 values to obtain LC50/GI50 values. For each HDACi, the 23 DLBCL cell lines were assigned to one of two groups using the median LC50/GI50 value for each HDACi as a cutoff. For ITF-B, GSEA showed the enrichment of multiple cell cycle associated gene sets in the group of cell lines with high LC50/GI50 values i.e. with LC50/GI50 values greater than the median . Genes ap Figure . Comparip Figure . These oBCL2 and BCL2L1) has been associated with resistance to HDACi [BCL2L1 was positively correlated with IC50 values for ITF-A , but not with IC50 values for ITF-B . Expression of BCL2L1 was not associated with LC50 values for either of the two HDACi. BCL2 expression exhibited no correlation with IC50 or LC50 values. Additionally, when considering only DLBCL cell lines we did not observe any association between BCL2 translocation status and IC50 values  and thioredoxin (TXN) have also been associated with resistance to HDACi treatment [SOD2 and GSR levels correlated with LC50 values for both HDACi suggesting that they may be specifically associated with the cytotoxic effects of ITF-A and ITF-B. For SOD2, the correlation between expression and LC50 values for ITF-B and ITF-A respectively, were r2 = 0.569, p-value = 0.001 and r2 = 0.542, p-value = 0.002). For GSR, the values were r2 = 0.444, p-value = 0.013 and r2 = 0.419, p-value = 0.020 for ITF-B and ITF-A, respectively . EP300 expression negatively correlated with LC50 values for both HDACi .We then looked at the association between the anti-proliferation responses of all 32 lymphoma cell lines used in this study and genetic or biological features previously reported as being associated with resistance to HDACi . The oveto HDACi . Express0 values . Increasreatment . SOD2 anectively . OverexpCL cells , which fIn this study, we have demonstrated the anti-proliferative activities of two novel HDACi in a panel of 32 lymphoma cell lines. ITF-A and ITF-B are hydroxamic acid HDACi. These novel HDACi target classes I, IIb and IV HDACs. Both HDACi showed the ability to inhibit the proliferation of lymphoma cell lines of different histologies  without any association between potency and lymphoma histology, also when comparing GCB-DLBCL and ABC-DLBCL. For both HDACi, IC50 values were in the nanomolar range with most cell lines displaying IC50 values below the thresholds of 20nM for ITF-A and below 50nM for ITF-B. These values compare favourably with other HDACi currently in the literature , 17. We MYC, CCND2 and NFKB1 likely comprised genes that were targeted for down-regulation following HDACi treatment, probably via mechanisms that specifically reduce the binding of acetylated histones and specific transcription factors to the regulatory regions of these genes [In order to better understand the mechanisms of action of the two HDACi, we performed GEP on two DLBCL cell lines that had been treated with each HDACi. We observed the modulation of multiple transcripts and consequently, of numerous signaling pathways and biological processes following the 8 hours treatment. These processes included those regulating cell cycle progression and apoptosis, although these were not the most frequently altered processes. The top 100 up-regulated genes following treatment primarily comprised histone, tubulin and metallothionein genes. Metallothioneins comprised 8/100 (8%) of the top 100 up-regulated genes. Metallothioneins are a class of heavy-metal binding proteins that also scavenge free radicals. HDACi treatment has been shown to up-regulate their expression in certain cell types, and they may be involved in drug resistance , 28. These genes .CDKN1A was more highly expressed in the high LC50/GI50 group than in the low LC50/GI50 group, suggesting that high basal expression of CDKN1A might protect cells from HDACi-mediated cytotoxicity. This observation is important considering the critical role of CDKN1A in the anti-proliferation response to HDACi treatment [WAF1/CIP1 following treatment with belinostat and do not undergo apoptosis whereas sensitive cell lines only transiently up-regulated CDKN1A/p21WAF1/CIP1 and underwent apoptosis [WAF1/CIP1 as well as the duration of its induction following HDACi treatment [The anti-proliferation response to HDACi can either result from cell cycle arrest or apoptosis . It is nreatment , 18, 31.reatment , 13, 18.reatment  and indereatment . Recentlpoptosis . This toreatment  are impoBCL2 and BCL2L1 has been reported to confer resistance to HDACi [BCL2 and IC50 or LC50 values for the 32 cell lines used in this study. Expression of BCL2L1 was only positively correlated with IC50 values for ITF-A. Notably, the expression of the antioxidant genes SOD2 and GSR correlated positively with LC50 values for both HDACi. The role of antioxidant genes in conferring resistance to HDACi-induced cytotoxicity has been reported [SOD2 and GSR correlate strongly with LC50 values, suggesting that their higher expression specifically impedes a cytotoxic response.The expression levels of several genes have been shown to impact on the response to HDACi treatment. Among these, overexpression of the anti-apoptotic genes to HDACi , 23, 33.reported , 34 and CREBBP and EP300 transcripts in all 32 cell lines as factors other than mutation or genomic aberrations may reduce the expression of these genes [CREBBP expression did not correlate with IC50 values for either HDACi, but positively correlated with LC50 values  for ITF-B. Conversely, EP300 levels showed a negative correlation with LC50 values for both HDACi.Recent discoveries of the prevalence of inactivating mutations in the acetyltransferase genes EP300 and CREBBP in lymphomas have highlighted the crucial role of epigenetics in the pathobiology of lymphomas, including especially DLBCL . As HDACse genes . CREBBP CDKN1A levels appear to be important in determining the gap between HDACi concentrations that cause cell cycle arrest and those that cause cell death. The importance of baseline CDKN1A levels in mediating cytotoxicity at doses similar to or much higher than the GI50 has not been previously reported. These data may be useful in identifying cells that would benefit from combination approaches using HDACi and agents that overcome cell cycle arrest.In conclusion, we have demonstrated the robust anti-proliferative activity of two novel HDACi, ITF-B and ITF-A. Basal Established human cell lines derived from DLBCL, MCL and SMZL were cultured according to the recommended conditions. All media were supplemented with fetal bovine serum (10%), Penicillin-Streptomycin-Neomycin  and L-glutamine (1%). Two novel HDACi were used: ITF-A and ITF-B .2. DMSO alone was added to negative control (untreated) cells. Wells containing medium only were included on each plate and served as blanks for absorbance readings. MTT; 3--2, 5-diphenyltetrazoliumbromide  was prepared as a 5 mg/ml stock in PBS and filter-sterilized. MTT solution (22μL) was added to each well and tissue culture plates were incubated at 37ºC for 4 hours. Cells were then lysed with 25% SDS lysis buffer and absorbance was read at 570 nm using an AD340 plate reader . For each cell line and biological replicate, a “time zero” (t0) plate was seeded with the appropriate number of cells per well and processed immediately to allow a time zero density to be determined. The IC50 (50% Inhibitory Concentration), GI50 (50% Growth Inhibition), LC50 , and TGI  were calculated from absorbance values obtained from the treatment plates alone (IC50) or from the treatment plates and time zero plates . The doses corresponding to the IC50, GI50, LC50 and TGI were estimated by fitting a sigmoidal model through the dose response curve using the R statistical package (www.r-project.org). Cell proliferation, apoptosis and cell cycle were evaluated on cells treated with DMSO or different doses of ITF-B and ITF-A. For cell cycle analyses, the percentages of cells in G1, S and G2/M phases of the cell cycle were determined using the Watson Pragmatic model and the FlowJo software .Cells were seeded in 96-well plates (non-tissue culture treated) at a density of 10,000 cells for all cell lines except for VAL and JeKo-1, which were seeded at 20,000 cells per well. Each HDACi was dissolved in dimethyl sulphoxide (DMSO). For treatment of cells, compounds were serially diluted in the appropriate tissue culture medium, at a range of 7.8 – 500 nM and added to cells (in five replicates). Cells were incubated for 72 hours at 37ºC, 5% COProtein extractions, SDS-PAGE and immunoblotting were performed as previously described . The antCells were fixed in 2% paraformaldehyde (PFA) and embedded in paraffin. Antigen retrieval was performed with Tris-EDTA at pH 9 for 30 minutes at 98°C. A monoclonal antibody against phospho-STAT3  was applied. Reactions were developed with the Ultravision Quanto Detection System .RNA was extracted using TRI-reagent . Quantitative Real-time Polymerase chain reaction (qPCR) was performed as previously described  (primer Gene Expression Profiling (GEP) was performed using the HumanHT-12 v4 Expression BeadChip , as described . Data pr


Normal tissue toxicity after radiotherapy shows variability between patients, indicating inter-individual differences in radiosensitivity. Genetic variation probably contributes to these differences. The aim of the present study was to determine if two cell lines, one radiosensitive (RS) and another radioresistant (RR), showed differences in DNA repair capacity, cell viability, cell cycle progression and, in turn, if this response could be characterised by a differential gene expression profile at different post-irradiation times. After irradiation, the RS cell line showed a slower rate of γ-H2AX foci disappearance, a higher frequency of incomplete chromosomal aberrations, a reduced cell viability and a longer disturbance of the cell cycle when compared to the RR cell line. Moreover, a greater and prolonged transcriptional response after irradiation was induced in the RS cell line. Functional analysis showed that 24 h after irradiation genes involved in “DNA damage response”, “direct p53 effectors” and apoptosis were still differentially up-regulated in the RS cell line but not in the RR cell line. The two cell lines showed different response to IR and can be distinguished with cell-based assays and differential gene expression analysis. The results emphasise the importance to identify biomarkers of radiosensitivity for tailoring individualized radiotherapy protocols. To test if differences existed in the rate of γ-H2AX disappearance with post-irradiation time between both cell lines, data were fitted to a one phase decay non-linear regression curve  probes, chromosome aberrations were scored after 2 Gy of irradiation . On the other hand, the frequency of complete chromosome elements (CCE) was higher for the RR cell line, but without statistical significance.Using pancentromeric and pantelomeric fluorescent adiation . As can p < 0.001, z-test). A significant viability reduction between 4 and 24 h and between 24 and 48 h after irradiation was also observed in the RS cell line  but only between 4 and 24 h after irradiation in the RR cell line .Results of cell viability after 2 Gy are presented in Cell cycle distribution was similar in both cell lines before irradiation . HoweverThe expression of genes involved in radiation response was evaluated by QuantSeq methodology at different time points up to 14 days post-irradiation. The results obtained for both cell lines on irradiated versus sham-irradiated samples at 4 and 24 h and 14 days after irradiation are available in 35The analysis showed differences between the RS and the RR cell line in the number of differentially expressed (DE) genes following 2 Gy irradiation . At 4 h Gene enrichment analysis using the ToppGene platform was performed. The set of differentially up-regulated genes for both cell lines 4 and 24 h after irradiation was enriched in pathways and biological processes known to be involved in radiation response, such as DNA damage response, cell cycle regulation and apoptosis , 4, , , In the RS cell line 4 h after irradiation the most enriched pathways were “direct p53 effectors” and “DNA damage response” . Up to 2Regarding the differentially up-regulated genes of the RR cell line, the most enriched pathways were “direct p53 effectors” and “DNA damage response”, but only 4 h after irradiation . In relaTo identify differences in up-regulated genes between both cell lines at different times after irradiation, Venn diagram analysis were performed for “DNA damage response”, “direct p53 effectors” and apoptosis . As can The tolerance of normal tissue is the limiting factor in the radiation dose applied for cancer treatments5937ATM  or BRCA1/2 are characterised by extreme radiosensitivity3940842344445The differences in radiation sensitivity are often associated with a reduced ability to efficiently repair DSBs and/or activate the DNA damage response. For example, patients carrying mutations at One of the initials steps in the DNA damage response is the phosphorylation of the histone H2AX. The RS cell line showed significantly higher foci counts and a slower rate of foci disappearance than the RR cell line. This is in agreement with studies on tumour cell lines, where a slower rate of γ-H2AX foci disappearance in RS cell lines has been described, when compared with RR tumour cell lines19ex vivo irradiated lymphocytes from clinically radiosensitive patients showed higher frequencies of exchange-type aberrations and deletions than the non-radiosensitive patientsThe analysis of chromosome aberrations is a well established method to assess the damage produced by IR. In the present study, a significantly higher frequency of ICE in the RS cell line was observed in comparison with the RR cell line, indicating that more chromosome breaks failed to be rejoined in the RS cell line. It has been described that Cell death assays have the potential to evaluate individual radiosensitivity1485051BCRA1 mutated heterozygotes showed a higher G2 delay but also a significantly higher frequency of G2 chromatid aberrations than normal controls, indicating that more damaged cells were able to pass the G2/M transitionCell cycle arrest in G2/M-phase was strongly induced by IR in the RS cell line compared to the RR cell line. This is in agreement with the results from other authors on LCLs after irradiation245054Regarding gene expression, from the 12,349 genes analysed, less than 100 genes for each analysis were found to be DE. Moreover, a significant enrichment was observed for pathways or biological processes related to the response to IR. These differential gene expression profiles clearly demonstrate the capability of the QuantSeq methodology to discriminate between irradiated and unirradiated samples, but also to determine the changes in gene expression in relation to the time after exposure and the differences in the response between the RS and the RR cell line.The different number of DE genes between the RS and the RR cell line, 4 and 24 h after irradiation, indicates that the RS cell line was transcriptionally more active than the RR cell line in response to IR. This is in agreement with previous results where more differentially regulated proteins were detected in the RS cell line than in the RR one after irradiation22242527585960RRM2B, CDKN1A and MDM2) were common to the three categories. RRM2B is involved in cell cycle regulation and DNA repair, and its expression is p53-dependent2259CDKN1A expression is also p53-dependent and plays a key role in the negative regulation of cell cycle both at G1 and G2/M in response to DNA damage induced by IR2122275859602224252763Analysis of differential gene expression profiles at 24 h after irradiation revealed that no genes related to “DNA damage response”, “direct p53 effectors” and apoptosis were up-regulated in the RR cell line, whereas four, 12 and 15 genes were still DE in the RS cell line, respectively . From th222425273058596062CDKN1A and MDM2 are also involved in “negative regulation of cell cycle”, a biological process significantly enriched only in the RS cell line 4 and 24 h after irradiation , causing a longer disturbance of the cell cycle in comparison with the RR cell line. These characteristics were probably responsible for the reduced viability after irradiation observed in the RS cell line when compared with the RR cell line. Gene expression signatures corroborated these results, given the sustained expression of genes related with “DNA damage response”, “negative regulation of cell cycle” and apoptosis, which are still necessary up to 24 h after IR in the RS cell line, but not in the RR one.Bayerische Landesärztekammer, Nr 99196, on January the 26th in the year 2000. Characterisation of the two cell lines by M-FISH was carried out before irradiation and no chromosome aberrations were observed. Their radiosensitivity was previously tested344445The two cell lines used in the present study were generated by immortalisation of B-lymphocytes by Epstein-Barr-virus (EBV) infection of peripheral blood leukocytes obtained from lung cancer patients. The methods were carried out in “accordance” with approved guidelines and regulations at the Helmholtz Institution Munich and the Federal Office of Radiation Protection, Neuherberg. Informed consent was obtained from all subjects. The study and protocols were approved by the Ethical Committee of the 2 atmosphere, in Roswell Park Memorial Institute 1640 (RPMI-1640) medium  supplemented with 15% fetal bovine serum , L-glutamine 2 mM (Biowest) and penicillin/streptomycin  (Biowest).These LCLs were grown in suspension, at 37 °C in a 5% CO137Cs-irradiator , at a dose rate from 4.87 to 4.84 Gy/min due to decay of source.The LCL cultures were irradiated in exponential growth phase at 1 and 2 Gy to study the kinetics of γ-H2AX foci and at 2 Gy for peptide nucleic acid (PNA) FISH analysis, cell death measurements, cell cycle analysis and RNA-seq. Sham-irradiated control cultures were also used in all analyses. Samples were irradiated with a To assess the kinetics of γ-H2AX foci induction and disappearance following IR, cell cultures were maintained at 37 °C after irradiation for 10 and 30 min and for 2, 4, 8 and 24 h. After that, cell cultures were incubated on ice until processing. Immunostaining and automated microscopic analysis of γ-H2AX foci were performed as previously describedNormality of foci distribution was tested with Kolmogorov-Smirnov with Lilliefors correction. Because compliance with normal distribution was not observed, nonparametric tests were used. Wilcoxon matched-pairs signed rank test was performed to assess if medians of signed ranks of the two replicas of γ-H2AX kinetics were different. Mann-Whitney test was used to compare foci counts.After irradiation, cell lines were cultured at 37 °C for 24 h. Colcemid  was added 4 h before harvesting at a final concentration of 0.3 μg/mL. Harvesting was carried out by the standard hypotonic treatment (KCl 0.075 M at 37 °C for 5 min) followed by fixation with Carnoy’s solution .FISH was performed according to a protocol previously describedTo determine the percentage of first division mitoses, a parallel culture was carried out with the same conditions but adding bromodeoxyuridine  at a final concentration of 12 μg/mL following irradiation. The percentage of first division metaphases at 24 h post-irradiation was higher than 95% for both the RS and the RR cell line.For the FISH analyses only metaphases containing 46 centromeric signals and 92 telomeric signals were considered. Centromeric signals allowed detecting unequivocally dicentrics, rings and acentric fragments, and furthermore, telomeric signals allowed determining the completeness of each chromosome piece. Chromosomes lacking a telomeric signal at one or both ends were recorded as chr(+/−) or chr(−/−), respectively. The same criterion was applied for dicentric chromosomes [dic(+/−) and dic(−/−)] and acentric fragments [ace(+/−)]. Acentric fragments without telomeric signals were classified as interstitial deletions (ID) and centric ring chromosomes were classified as R. For comparisons, the aberrant chromosome pieces were grouped as CCE , those with telomeric signals at both ends, or as ICE , those without telomeric signals at one or both endsTo compare the frequencies obtained in the FISH study between the RS and the RR cell line, Mann-Whitney test was applied.To measure cell death after IR, the Annexin-V-FLUOS Staining Kit  was used following the manufacturer’s instructions. The cell lines were cultured at 37 °C for 4, 24 and 48 h after irradiation. Viable, apoptotic and necrotic (or late apoptotic) cells were scored by eye with an epifluorescence microscope BX51  using a 50× Plan objective.Two independent experiments were performed analysing a minimum of 1000 cells for each particular experimental condition. To evaluate the radiation-induced increase of cell death, the obtained values after IR were normalized to those from sham-irradiated cells. A z-test was used to compare if the difference between proportions of viable and total cells in the RS and the RR cell line were significant.After 2 Gy irradiation cells were incubated at 37 °C for 4, 12, 24 and 48 h. Sham-irradiated controls were also used. Cells were centrifuged at 250 g for 5 min, washed in phosphate buffered saline and fixed in 70% ethanol at least for 2 h at −20 °C. Then, cells were incubated for 15 min at 37 °C with a staining solution containing propidium iodide (10 μg/mL) , 0.1% TritonX100 (Sigma-Aldrich) and DNase-free RNase A (100 μg/mL) (Sigma-Aldrich) dissolved in 1× PBS. Cell cycle analysis was performed using a BD FACSCanto flow cytometer and BD FACSDiva software v7.0 . At least 10,000 cells per replica were analysed and two replicas were performed at each time point.To study the differential gene expression, irradiated (2 Gy) and sham-irradiated cell cultures of the RS and the RR cell line were incubated at 37 °C for 4 and 24 h and 14 days. After that, total RNA extraction was carried out with the SPLIT RNA Extraction kit  according to the manufacturer’s instructions. RNA concentration and purity for each sample were verified with a Nanodrop ND-100 spectrophotometer . The ratios of absorbance 260/280 nm and 260/230 nm were ∼2 for all the samples, and RNA concentration was ∼200 ng/μL per sample. RNA integrity was determined with Agilent 2100 Bioanalyzer . RNA Integrity Number (RIN) values close to 10 were obtained, indicative of high quality RNA samples. Pure RNA samples were stored at −80 °C until cDNA libraries were prepared.QuantSeq technology has been used as alternative to microarrays and conventional RNA-seq, in order to measure gene expression. cDNA libraries for each sample were generated at Functional Genomics Core  from 500 ng of total RNA using the Quantseq 3′ mRNA-Seq Library Prep kit for Illumina (Lexogen) following the manufacturer’s instructionsDuring library amplification (12 cycles of PCR), standard external barcodes were ligated to allow for multiplex sequencing. After PCR amplification, the libraries were size-selected with Agencourt AMPure XP magnetic beads . Libraries were quantified by QuBIT (Life Technologies) and their average size of ∼250 bp was determined using a High Sensitivity DNA chip on an Agilent 2100 Bioanalyzer (Agilent Technologies).Libraries were then sequenced (50 bp single read) by the NGS unit of Campus Science Support Facilities GmbH  using an Illumina Hiseq2500 sequencer. Quality control analyses on the resulting bam sequencing files were performed using FastQC tool .www.lexogen.com/quantseq-data-analysis). Most sequences were originated from the last exon and the 3′ untranslated region (UTR). Sequence reads were aligned onto the human genome reference sequence (hg19) using the TopHat2 splice-junction mapper, version 2.1.0Due to non-specific hybridisation of the random primer used to synthesise the second strand of cDNA, a trimming of the first nucleotides of the reads was performed before sequence alignment in order to improve the percentage of mapped reads  directly linking the number of reads mapped to a gene to its expression65https://bioconductor.org), count files from htseq-count were loaded into R and normalised using the default method for edgeR . To consider the multiple factors of our experimental design , a matrix was designed. Dispersion values were estimated using the Cox-Reid profile-adjusted likelihood method. Then, the generalised linear model likelihood ratio test was used to determine differential expression. Fold-changes were reported as the log (base 2) of normalised count abundance of the irradiated samples divided by count abundance for the sham-irradiated samples. P values reported for each gene were then corrected for multiple comparisons using Benjamini & Hochberg (B&H) false discovery rate (FDR)The package edgeR version 3.10.2 was used to identify DE geneshttps://toppgene.cchmc.org)To identify pathways and biological processes affected in response to IR, gene list enrichment analysis was performed using ToppGene suite (Other platforms such as DAVID and PANTHER (data not shown), as well as GOseq package (version 1.20.0)How to cite this article: Borràs-Fresneda, M. et al. Differences in DNA Repair Capacity, Cell Death and Transcriptional Response after Irradiation between a Radiosensitive and a Radioresistant Cell Line. Sci. Rep. 6, 27043; doi: 10.1038/srep27043 (2016).


INK4A, or overexpressed constitutive active cyclin D1/CDK2. In all cases, the replicative senescence barrier was bypassed by transduction of c-Myc. Cells from all resulting immortal lines exhibited normal karyotypes. Immunofluorescence, flow cytometry, and gene expression analyses of lineage-specific markers were used to categorize the intrinsic subtypes of the immortalized lines. Bypassing stasis with p16 shRNA in young strains generated cell lines that were invariably basal-like, but the lines examined from older strains exhibited some luminal features such as keratin 19 and estrogen receptor expression. Overexpression of cyclin D1/CDK2 resulted in keratin 19 positive, luminal-like cell lines from both young and old strains, and the lines examined from older strains exhibited estrogen receptor expression. Thus age and the method of bypassing stasis independently influence the subtype of immortalized human mammary epithelial cells.Based on molecular features, breast cancers are grouped into intrinsic subtypes that have different prognoses and therapeutic response profiles. With increasing age, breast cancer incidence increases, with hormone receptor-positive and other luminal-like subtype tumors comprising a majority of cases. It is not known at what stage of tumor progression subtype specification occurs, nor how the process of aging affects the intrinsic subtype. We examined subtype markers in immortalized human mammary epithelial cell lines established following exposure of primary cultured cell strains to a two-step immortalization protocol that targets the two main barriers to immortality: stasis (stress-associated senescence) and replicative senescence. Cell lines derived from epithelial cells obtained from non-tumorous pre- and post-menopausal breast surgery tissues were compared. Additionally, comparisons were made between lines generated using two different genetic interventions to bypass stasis: transduction of either an shRNA that down-regulated p16 Different patterns of gene and protein expression in breast tumors have led to the delineation of at least four intrinsic subtypes of breast cancers: HER2-related, luminal A, luminal B, and basal-like  from women aged 21, 19, 66, and 91 years, respectively, and were cultured as pre-stasis strains  was obtained from Greg Hannon. The pBabe-hygro-myc was used to transduce cells with c-Myc. The WPI-Cyclin D1/CDK2-GFP retrovirus was obtained from Mark Jackson. Retroviral containing supernatants were collected in M87A medium and transductions were performed as described  at −20°C for 15 min, blocked with PBS, 5% normal goat serum, 0.1% Triton X-100, and incubated with K14  and K19  overnight at 4°C, then visualized with fluorescent secondary antibodies (Invitrogen), and incubated for 2 h at room temperature. For 3-D cultures, HMEC were fixed in 4% paraformaldehyde for 30 min, blocked with PBS, 5% normal goat serum, 0.1% Triton X-100, and incubated with β-Catenin  and Estrogen Receptor-α  overnight at 4°C, then with secondaries for 2 h at room temp.CD227-FITC , CD10-PE , CD10-APC , CD227-PE  were added to cells in media for 25 min on ice, washed in PBS and analyzed with a FACSCalibur (Becton Dickinson).INK4 (BD Pharmigen #551154 Mouse 1:1000), Cyclin D1 , beta-actin , and visualized with goat anti mouse IgG Fc (HRP) conjugate .The following antibodies were used: p16Subconfluent cultures were harvested for RNA 24 h following feeding. Total RNA was harvested using TRIzol and purified using the miRNeasy Kit (Qiagen). RNA labeling and hybridization to Affymetrix Human Gene 1.0 ST Arrays, was performed according to the manufacturer's protocols. Microarray data were analyzed in R programming environment using the limma package.To determine whether chronological age and early events in cancer progression are determinants of the intrinsic subtype of immortal HMEC, a two step procedure that does not cause gross genomic changes 14 and CALLA/CD10 are proteins expressed primarily in myoepithelia, and K19 and Sialomucin-1/CD227 in luminal epithelia  protein was evaluated in the p16sMY and D1MY cell lines derived from the young strain 240L and the older strain 122L. ERα is expressed by a subset of luminal cells in the normal mammary gland, and is used to categorize the luminal subtypes of breast cancers. The p16sMY and D1MY lines derived from 240L and 122L were cultured for 14 days embedded in 3-D Matrigel. ERα was not detected in either the p16sMY or D1MY line derived from 240L Figure , but it Altogether these data indicate that these non-clonal lines are heterogeneous, containing a mixture of lineages. However, both IF and FACS-based immune-phenotypes show an age-dependent increased expression of luminal markers in the lines that bypassed stasis with p16sh. Lines that bypassed stasis with cyclin D1/CDK2 overexpression generated a more luminal phenotype independent of age.Two sets of genes, specifically expressed in luminal or myoepithelial/basal cells, were identified based on microarray data from FACS enriched CD10+/CD227− myoepithelial and CD10−/CD227+ luminal epithelial 240L and 122L passage 4 pre-stasis cells using a cutoff of 2 fold gene expression enrichment in respective cell lineage relative to the other. The expression of these two lineage-specific sets of genes was then analyzed in the immortal p16sMY and D1MY cell lines, as well as in the post-stasis direct precursors to the immortal lines, which had been transduced with either p16 shRNA or cyclin D1 only.Comparison of gene expression in p16sh vs. D1 post-stasis cells showed that basal genes were expressed more in p16sh cells, whereas luminal genes were expressed more in D1 cells Figure . The trein vivo—they are caricatures of normal at the best. Indeed, every cell line had some level of heterogeneity, with varying distributions of cells representing the luminal and myoepithelial lineages. Here we have shown that expression of lineage-related markers in immortalized HMEC is influenced by both chronological age and the method by which the normal HMEC escape an initial tumor-suppressive senescence barrier, stress-associated stasis. A comparison of HMEC from young (<30 years) vs. older (>60 years) women showed that increased age biased toward generation of immortalized lines with greater expression of luminal phenotypes. A comparison of immortalized lines generated by using either p16sh or cyclin D1/CDK2 to bypass stasis showed a bias toward a luminal phenotype when cyclin D1/CDK2 was utilized, independent of age. Advanced age combined with cyclin D1-mediated bypass of stasis generated a line that was qualitatively the most luminal, exhibiting expression of K19, CD227, ERα, and other luminal transcripts. While a younger strain transduced with cyclin D1 also gave rise to a K19 and CD227-expressing luminal line, it did not produce ERα. In contrast, the younger lines that bypassed stasis with p16sh were qualitatively very basal-like . These results are based on our currently small number of cell lines, due in part to the length of time required to generate and examine each line. As more immortal cell lines are generated using this methodology the relative importance of age and stasis bypass in the etiology of intrinsic subtypes will become more obvious. Using a phase diagram we have expressed our hypothesis that the relative luminal vs. basal phenotype of immortalized HMEC is influenced by both chronological age and the method of stasis bypass pyrene  mutant p53, cyclin D1, K-ras, and myristolated PI3K, or (ii) SV40 and K-ras, then were sorted into EpCAM+ or CD10+ subpopulations prior to orthotopic injection into mouse hosts (Keller et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.