Scaphander lignarius has been analyzed in the systematic search for novel bioactive compounds in Arctic marine organisms using bioassay guided fractionation. A number of highly cytotoxic fractions were shown to contain mainly polyunsaturated fatty acids (PUFAs). Selected PUFAs were isolated and identified using both liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR). It was shown that the opisthobranch contained unusual PUFAs such as several ω3 fatty acids and the ω7 heneicosa-5,8,11,14-tetraenoic acid (21:4 n-7) not isolated before. The organism was shown to be a very rich source of PUFAs and the activity of the isolated compounds against a range of human cancer cell lines  is further reported. The ω7 PUFA was significantly more cytotoxic in comparison with reference ω6 arachidonic and ω3 eicosapentaenoic acid. A noteworthy non-selective cytotoxicity against normal lung fibroblasts was also established. The paper contains isolation protocols in addition to cytotoxicity data of the isolated compounds. The potential of marine mollusks as a source for rare PUFAs is also discussed. The marine opisthobranch  The marine environment is highly competitive and being able to produce compounds providing an adaptive advantage is highly beneficial for any marine organism. Such compounds are fundamentally interesting to study but they also represent entities with significant potential for several industrial and medical applications as they have been chemically optimized during millions of years of evolution and selection . Marine Scaphander lignarius generated fractions with high cytotoxic activities  displayed in The active components in the fractions were shown to be a group of PUFAs, present in high concentrations. Marine mollusks and invertebrates are rich in lipids and they have been extensively studied over the years and comprehensively reviewed by Bergé and Barnathan . NumerouScaphander lignarius extract. Three representative PUFAs, arachidonic acid (20:4 n-6) (ARA), eicosapentaenoic acid (20:5 n-3) (EPA) and the rare heneicosa-5,8,11,14-tetraenoic acid (21:4 n-7) (HTA) were isolated and selected for dose response cytotoxicity studies against a set of human cancer cell lines and structure activity relationship (SAR) establishment. The current paper describes the isolation and cytotoxic bioactivities of these selected PUFAs against several human cancer cell lines. It represents the first study of the fatty acid composition of Scaphander lignarius and the first assessment of the cytotoxic properties of an oddly numbered ω7 PUFA.Any biological source able to generate large amounts of unusual, biologically active fatty acids is important to further study as their biological properties are poorly known . In the Scaphander lignarius in the current study has previously been described as an abundant Mediterranean mollusk  (MTS) assay. At the end of the exposure time, 10 µL Cell Titer 96® Aqueous One Solution Reagent  was added to each well, and the plates were incubated for 1 h before absorbance was measured using a DTX multimode detector  at 485 nm. Cells in RPMI-1640 medium were used as negative control, and cells treated with Triton® X-100 (Sigma-Aldrich) reagent was used as positive control. Relative cellular survival was determined by using the measured optical density (OD) and was calculated as follows: Cell survival (%) = (OD treated well − OD positive control)/(OD negative control − OD positive control) × 100.The effect of the PUFAs on cell viability was tested against three cancer cell lines: Human melanoma , human breast carcinoma , and human colon carcinoma . In addition, non-malignant lung fibroblasts  were used as toxicity control. Cell lines, suspended in RPMI-1640 or E-MEM medium (Biochrom AG) with 10% fetal bovine serum medium and 10 µg/mL gentamicin, were seeded in 96-well microtitre plates at 2,000 cells/well. The cell lines were incubated for 24 h before PUFAs (concentration range 6.25–100 µg/mL) were added and then incubated for 72 h at 37 °C in a humidified atmosphere of 5% COScaphander lignarius collected in Arctic waters has been investigated. The mollusk was rich in ARA, EPA, DHA and the unusual ω7 HTA. Selected PUFAs were isolated and their cytotoxic activity was evaluated against a range of adherent human cancer cell lines. HTA was shown to be a significantly more potent cytotoxic agent than ARA and EPA. The cytotoxicity was not selective and normal fibroblasts were equally sensitive to the increased cytotoxicity of HTA. Tumor selective delivery systems would have to be employed to benefit from the cytotoxic properties of HTA without collateral damage to surrounding tissue. This is the first study of the cytotoxic action of an ω7 PUFA isolated from a particularly rich marine source.The fatty acid composition in an extract of


Human endogenous retrovirus K (HERV-K) is the most intact retrovirus in the human genome. There are multiple full-length or near full-length HERV-K proviruses in it. To analyze which HERV-K proviruses give rise to viral transcripts in cancer cell lines and to test whether ionizing radiation can alter the levels of HERV-K transcripts, RT-PCR studies were undertaken using multiple human cancer cell lines. Primers from several positions in the viral genome were used and included pairs designed to cross splice junctions in viral RNAs. In the absence of ionizing radiation, transcripts were detected from multiple HERV-K proviruses in cell lines from human prostate, cervical, head and neck, or breast cancers, and the proviruses from which the transcripts originated varied among the different lines. Only one of 13 cell lines tested  failed to show HERV-K transcripts. Spliced RNAs detected included viral RNAs spliced as expected at the conventional viral splice sites, plus several alternatively spliced RNAs. Alternatively spliced transcripts arose from specific proviruses, and were detected in most of the cell lines used. Quantitative RT-PCR was performed to assess the effects of ionizing radiation. These analyses showed that HERV-K transcripts were elevated in four of twelve lines tested, specifically all three prostate cancer lines used and one breast cancer line. The increases were transient, peaking at 24 hours following a single dose of gamma-irradiation that ranged from 2.5 to 20 Gy, and returning to baseline levels by 72 hours. In summary, these studies showed that ionizing radiation can affect the levels of HERV-K transcripts in cells, and these effects vary among different cells. The changes in HERV-K transcript levels might affect multiple biological processes in cells, and future studies of the effects of ionizing radiation on HERV-K are worth pursuing. The effects of ionizing radiation (IR) on endogenous retroviruses (ERVs) are largely unknown. The DNA genomes of ERVs are integrated into the genome of their host species as a result of infections of germline cells over evolutionary time. To date, the effects of ionizing radiation on ERVs have been explored only in mice Ionizing radiation has been reported to increase the transcriptional activity of many viruses including CMV Endogenous retroviruses are present in the genome of every cell of an individual and can be targets for immune responses such as those against MERVs HERV-K is transcribed to various extents in several human diseases including cancer pro), polymerase (pol) were capable of detecting unspliced viral RNA, and envelope (env) gene primers were able to detect both unspliced and singly spliced env mRNAs. The expected size products were observed in 12 cell lines out of the 13 tested for the pro and pol amplicons and in 11 cell lines for the env amplicon  on RNA isolated from 13 cancer cell lines from cancer sites commonly treated with radiotherapy: prostate, uterine cervix, head & neck, and breast. RT-PCRs were performed using primer pairs from multiple parts of the viral genome shown in amplicon . Paralleomic DNA .GAPDH control amplification  for env mRNA and the first intron of rec mRNA is situated upstream of the 292 bp stretch, but the 5′SS for the second intron of rec mRNA is located within the 292 bp . The less prominent, approximately 800 bp product  correspoll lines . In addi137Cs source. Doses of γ-irradiation were varied at 0, 2.5, 5, 10 or 20 Gy, and cells at each dose were collected 1, 3, 8, 24, and 72 hours after irradiation. The primer pair used was designed in env, downstream of the 292 nucleotide deletion, so that it detected both unspliced RNA and singly-spliced env mRNA  was performed on RNA isolated from the 12 cell lines that showed HERV-K transcription. Cells at 50% confluence were irradiated in a single exposure to a env mRNA . Each exenv RNA at 24 hours were increased two- to eight-fold compared to unirradiated cells. Non-parametric, Wilcoxon-signed rank tests were performed to compare the measurements at each dose of gamma radiation with those at 0 Gy, and the P-values are presented in Most of the cell lines including the three cervical cancer lines tested, the three head and neck tumors, and three of the breast cancer lines showed no notable differences in the level of HERV-K transcripts at any dose of ionizing radiation and at any time point . HoweverOne of the four breast carcinoma cell lines (MCF-7) also showed an increase in HERV-K RNA levels following γ-irradiation . The kinIn summary, the levels of HERV-K RNAs were substantially increased by ionizing radiation in a fraction of the cancer cell lines tested. The increases were transient and peaked at about 24 hours post-irradiation. This effect was observed in all three prostate cancer cell lines tested, suggesting that HERV-K in these cells may be particularly sensitive to ionizing irradiation.The principal findings of this study were that ionizing radiation increased the levels of HERV-K transcripts in some cancer cells, and that the effects varied among the lines tested. In particular, all three prostate cancer lines that were tested exhibited significantly increased levels of HERV-K RNAs, which peaked at about 24 hours post-exposure to a single dose of γ-irradiation and then subsided. A similar increase and subsequent decrease were also observed in one of three breast cancer lines. In the other cancer cell lines tested, the levels of viral RNAs did not appreciably change following γ-irradiation. The mechanisms by which γ-irradiation might alter the levels of HERV-K RNA in lines, particularly prostate cancer lines, are unclear, as is the basis for why it occurred only in certain cell lines. It is curious to note that a small fraction of prostate cancers have translocations of the ETV1 gene (a member of the Ets family of transcription factors) into HERV-K loci The increases in HERV-K RNA levels documented here following γ-irradiation raise at least three potential biological consequences. One is that it may affect immune responses against viral antigens. HERV-K proteins are known to be recognized by immune responses A second is that it might affect HERV-K proteins that may participate in oncogenesis. Expression of presumably high levels of at least two HERV-K proteins has been shown to be able to contribute to oncogenesis The third is that it might affect recombination among the HERV-K loci in the human genome. Although no single HERV-K provirus has yet been found in contemporary humans that encodes infectious HERV-K, all the components for viral infectivity exist among the multiple HERV-K proviruses in the human genome today This study extended the types of cancer cell lines for which the identification of specific analysis of HERV-K proviruses that encoded the detected transcripts has been undertaken. The key findings were that the specific proviruses varied among the different tumor types and among the specific cell lines derived from them, and that the most recently acquired proviruses in the human genome, those that formed after the human lineage diverged from that leading to chimpanzees, were abundant among those detected. The depth of sequencing employed here limited the detection of transcripts to those that were the relatively most abundant, and it is possible, probably likely, that deeper sequencing would identify additional proviruses. Indeed deeper analysis of HERV-K cDNAs performed by Flockerzi et al. The latter led to the frequent detection of alternative splice sites for several specific proviruses, but not for others , Fig. 5.In summary, the effects of ionizing radiation on HERV-K as well as the alternative splicing indicate that further study of these phenomena are warranted. HERV-K has been reported to be expressed in many types of human tumors 2 in a humidified 37°C incubator to 90% confluence.Three human prostate cancer cell lines , four cervix cancer cell lines , one head and neck cancer cell line (FaDu) and three breast cancer cell lines  were purchased from ATCC . UM-SCC-47 and UPCI-SCC-90 137Cs Irradiator) at 0, 2.5, 5, 10 or 20 Gy administered in a single dose. For each cell line, one subset was harvested for each of five timepoints . A total of three independent experiments were performed for each cell line.From each cell line, subsets at 50% confluence were treated with γ-irradiation . RNA was then subjected to DNAse I digestion (TURBO DNA-free kit - Applied Biosystems #AM1907) to remove any genomic DNA contamination. RNA quality and concentration were determined by evaluation of rRNA bands in agarose gel electrophoresis and by NanoDrop spectrophotometric analysis (Thermo Scientific).env splicing junction to identify the singly and doubly spliced variants of HERV-K transcript. Parallel controls were performed to detect GAPDH transcripts. Primer sequences were as follows:To identify the specific transcriptionally active HERV-K loci, 3 µg of each RNA was used for RT-PCR. Primers were designed in well-conserved segments of HERV-K genome to ensure amplification of as many loci as possible. A set of primers was designed across the viral 5′-AGGGAAAAACCGCCTTAGGG-3′1X-env(1)-Fwd: 5′-TGCGGGCAGCAATACTGCT-3′1X-env(2)-Fwd: 5′-CACCGCACTATTGGCCACA-3′1X-env-Rev: 5′-AGGGAAAAACCGCCTTAGGG-3′2X-rec(1)-Fwd: 5′-CAGATGAAGTTGCCATCCACCA-3′2X-rec(2)-Fwd: 5′-ACAAAACCGCCATCGTCATC-3′2X-rec-Rev: 5′-GGAGATGCAAAGAAAAGGGCCT-3′2X-np9-Fwd: 5′-ACAAAACCGCCATCGTCAT-3′2X-np9-Rev: 5′-AAACGAGCAAAGGGGCCA-3′pro-Fwd: 5′-TTTCCCTAGTCCCTTTCCTGGT-3′pro-Rev: 5′-CTGGTGCATGGAAGATTGGT-3′pol-Fwd: 5′-ACAAGCAAAACCTCTCCCCCA-3′pol-Rev: 5′-CATGGTAAGCGGGATGTCACT-3′env(1)-Fwd: 5′-ATGTCACTCAGGCCACGG-3′env(2)-Fwd: 5′-ACAAAACCGCCATCGTCATC-3′env-Rev: 5′-AGATCATCAGCAATGCCTCCT-3′hGAPDH-Fwd: 5′-AGTCTTCTGGGTGGCAGTG-3′hGAPDH-Rev: 20 primer for a final concentration of 2.5 µM and DEPC-treated water. After an initial denaturation step, 5 minutes at 65°C followed by 1 min at 4°C, 2 µL of 10X RT buffer, 4 µL of 25 mM MgCl2, 2 µL of 0.1 M DTT, 40 U RNaseOUT, and 200 U SuperScript III RT enzyme were added to reach a final volume of 20 µL. The RT elongation step was performed at 50°C for 50 minutes, followed by enzyme heat inactivation at 85°C for 5 minutes. After brief cooling of the sample at 4°C, digestion of residual RNA was performed with 2 U RNAse-H at 37°C for 20 minutes. Parallel experiments in which no RT enzyme was added were simultaneously carried out.The reverse transcription reactions (SuperScript III First-Strand Synthesis System - Invitrogen #18080-051) were performed using 3 µg RNA in an initial volume of 10 µL with 1 µL of dNTP mix 10 mM, Oligo(dT)PCR was performed with 4 µL of RT product, 200 nM primers 200, Platinum PCR SuperMix HiFi (Invitrogen) and nuclease-free water to a total volume of 20 µL. After a denaturation step at 94°C for 2 minutes, 30 cycles of denaturation-annealing-elongation were performed, followed by final elongation at 68°C for 7 minutes. Denaturation was performed at 94°C for 25 seconds, annealing for 25 seconds, and extension at 68°C for each experiment as follows: Detection of pro, pol and env amplicons  were perElectrophoresis of PCR products was performed in 1% agarose gels. PCR products obtained were purified (PCR purification - Qiagen), and recovered cDNA was sequenced. For shotgun subcloning analyses, PCR products were cloned (TOPO TA cloning - Invitrogen) into pCR4 plasmids using TOP10-DH5α competent cells and sequenced.Each sequence obtained, trimmed of any vector and primers sequences, was aligned to the human genome using BLAT software 20 primer, and DEPC-treated water. After an initial denaturation step of 5 minutes at 65°C followed by 1 min at 4°C, 2 µL of 10X RT buffer, 4 µL of 25 mM MgCl2, 2 µL of 0.1 M DTT, 40 U RNaseOUT, and 200 U SuperScript III RT enzyme were added to reach a final volume of 20 µL. The RT elongation step was performed at 50°C for 50 minutes, followed by enzyme heat inactivation at 85°C for 5 minutes. After brief cooling of the sample at 4°C, digestion of residual RNA was performed with 2 U RNAse-H at 37°C for 20 minutes. Parallel experiments with no RT enzyme were carried out simultaneously.To quantify HERV-K transcripts, 1 µg of RNA from each set was used for two-step, qRT-PCR (Superscript III first strand - Invitrogen) and PowerSybr Green (AppliedBiosystems). RT (SuperScript III First-Strand Synthesis System - Invitrogen #18080-051) of 1 µg RNA was performed in an initial volume of 10 µL with 1 µL of 10 mM dNTP mix, 1 µL of 50 µM Oligo(dT)1 µL of a 1∶10 dilution of the RT product was then added to 2x SYBR Green PCR Master Mix (AppliedBiosystems #4367659) and nuclease-free water to a final volume of 8 µL. Primers q-env-Fwd and q-env-Rev were designed in a well-conserved region of HERV-K genome common to unspliced and once-spliced transcript variants downstream of the Δ292 deletion characteristic of type-I HERV-Ks. Four housekeeping genes, human GAPDH (hGAPDH), human beta glucuronidase (hGUSB), human hydroxymethybilane synthase (hHMBS), and human phosphoglycerate kinase 1 (hPGK1) were used as endogenous controls for normalization. Primer sequences as follows:5′-TCACATGGTAAGCGGGATGTC-3′q-env-Fwd: 5′-CGCACTATTGGCCACACATTC-3′q-env-Rev: 5′-AGATCATCAGCAATGCCTCCT-3′hGAPDH-Fwd: 5′-AGTCTTCTGGGTGGCAGTG-3′hGAPDH-Rev: 5′-CGCGCCGACTTCTCTGACAA-3′hGUSB-Fwd: 5′-CCACACCCAGCCGACAAAA-3′hGUSB-Rev: 5′-CTTCCTCCTGGCTTCACCAT-3′hHMBS-Fwd: 5′-GGTTCCCACCACACTCTTCT-3′hHMBS-Rev: 5′-AGCGGGTCGTTATGAGAGT-3′hPGK1-Fwd: 5′-ACTACCGACTTGGCTCCAT-3′hPGK1-Rev: Three replicates per sample were used in 384-well plate PCR system (7900HT - AppliedBiosystems) with cycling conditions of 50°C for 2 minutes, 95°C for 10 minutes, 40 cycles of 95°C for 10 seconds, 60°C for 20 seconds and 72°C for 30 seconds, and three biological replicates were performed. Data analysis was performed with SDS 2.4 and DataAssist softwares to determine means and standard deviations and to normalize the levels of viral transcripts to those of the housekeeping genes (AppliedBiosystems).


Results shows that Cisplatin resistance pattern is characterized in spectrum with the alteration of conformation in secondary structure of proteins and a shift toward the high wave numbers of CH2 stretching vibration. The FTIR data set between 1000 and 3000 cm-1 could be consumed as biochemical typicality spectra among resistant and sensitive cell lines while correctly classified by PCA model. Our work supports the promise of PCA analysis of FTIR data as a powerful combined approach for the development of automated methods to recognize resistant to cisplatin in experimental cell lines. One of the advantages of this tool is to investigate the resistant percent of cancer cells .Such technique may bring new tool in cancer diagnosis and stage definition in cancerous tissues.Cisplatin is a common chemotherapeutic agent that used for treatment of many solid cancers. Rapid identification of chemotherapy resistance is very important and may lead to effective treatment plan. Spectroscopy techniques, such as infrared spectroscopy, which are sensitive to biochemical composition of samples, have shown potentials to discriminate tissues. Developing in Fourier transform infrared (FTIR) as a diagnostic tool support conventional technique in investigating cell phenotype. By this goal three different cell lines, two cisplatin resistant OV2008-DDP (C13) and A2780-CP ovarian cell lines and one cisplatin sensitive A2780 cell line were investigated by FTIR spectroscopy. Data were subjected to principle component analysis (PCA) to obtain FTIR pattern for cisplatin resistance. Using FTIR spectroscopy on these cells in the range of 400-4000 cm Cisplatin is a commonly chemotherapeutic agent that used for treatment of many cancer types including testicular, ovarian, cervical, head and neck, non-small cell lung and lymphoma. HoweverThere are many theories to explain platinum resistant activity . One of Cisplatin at plasma concentrations down regulates protein expression of copper transporter (Ctr1) in human ovarian carcinoma cell lines , 8. In tThere is an increasing interest in the use of FTIR technology in large number of different research. It has been used to investigate biochemical composition of cells  and the Molecular difference in sensitive and resistant cell lines by FTIR spectroscopy has not been studied broadly. In this research, we used a FTIR-based assay followed by PCA analysis to look for the resistant specific patterns. This effort demonstrated changes in biochemical index, which can be used for platinum resistant activity. Cell lines 2. The medium was replaced every two day.A2780 (human ovarian carcinoma-sensitive to cisplatin), A2780-CP and C13 (human ovarian carcinoma-resistant to cisplatin) cell lines were obtained from national cell bank of Iran . All cell lines were grown in RPMI-1640 medium and supplemented with 10% heat inactivated fetal bovine serum, antibiotics: penicillin, streptomycin . Cells were maintained at 37°C in humidified atmosphere containing 5% COCell preparation for spectroscopy 2 flasks with fresh medium to reach the logarithmic phase of growth curve. After that cell were washed twice in saline (0.9% NaCl), suspend and centrifuged at 1000 rpm for 5 min, then resuspended in saline to obtain a concentration of 1 × 105 cells. 10 μL of each cell suspension was placed on a zinc selenide sample carrier, which was dehydrated in a vacuum cabin (0.8 bar) for approximately 4 min. These plates were then used for FTIR spectroscopy.The following procedure was similarly applied for both of sensitive and resistant cell lines. Cells were trypsinized from the original flask and seeded in 25 cmFTIR spectroscopy -1 for every wave number between 400 and 4000 cm-1. These experimental conditions were kept constant for all the measurements. Each single spectrum was baseline corrected and then normalized in order to have the range spanning from 0 to 1.For FTIR studies, thin dried films of cell suspensions were used on the Zinc selenide window by using a WQF-510  spectrometer, equipped with a KBr beam splitter and a DLaTGS (deuterated Lantanide triglycine sulphate) detector. The whole system was continuously purged with N2 (99.999%). In each spectrum, 100 scans were collected at a resolution of 4 cmData analysisData set-1 have been used in this study as the dataset. Distribution of different FTIR spectra was equal for types of A2780, A2780-CP and C13 cell lines.A total of 45 FTIR spectrums between 1000-3000 cmPrinciple component analysisp original variables X =  to a new set of q predictor variables, T = , which are linear combinations of the original variables. Principle component analysis (PCA) is a well-known method of dimension reduction. The basic idea of PCA is to reduce the dimensionality of a data set, while retaining as much as possible the variation present in the original predictor variables. This is achieved by transforming the -1 have been used for input.In mathematical terms, PCA sequentially maximizes the variance of a linear combination of the original predictor variables . PCA werSpectrum alteration-1. The normalized FTIR spectra in this region showed alterations in different spectrum areas. Comparison between spectra showed at least five areas of variation: Spectral features of A2780, A2780-CP and C13 cell lines are shown in -1 which can be related to β-sheet secondary structure of amid I (-1 shifted toward the lower wave numbers. This may represent the possibility of molecular interaction in β-sheet conformation structure of ovarian sensitive cells.a) there is a peak about 1636 cm-1 is assigned to β-sheet secondary structure of amid II (-1 while there are two bands at 1530 and 1540 cm-1 in the sensitive cell line. There are positive shoulder peaks at 1540 cm-1 and a peak at 1530 cm-1 in C13. The bands at 1540 cm-1 are assigned to α-secondary structure of amid II (α-secondary and β-sheet structure of amid II in sensitive cell while in the C13 resistant cell the reduction of α-secondary structure of amid II was observed. The result shows α-secondary structure conversion to β-sheet structure conformation in the C13 resistant cell. b) The vibration band at 1530 cm amid II . In A278 amid II . This ca-1 are assigned to amid III (-1 shifted toward the lower wave numbers. This shows the addition of molecular interaction in amid III structure of ovarian sensitive cell. c) The vibration bands at 1240 cmamid III . In the -1 is assigned to glycoprotein (d) The vibration band at 1380 cmoprotein . There i-1 and 1330 cm-1 in the sensitive cell line while there aren’t in the ovarian human resistant cells. e) There are positive shoulder peaks at 1407 cm-1. The CH stretching region (3000-2800 cm-1) contains the asymmetric and symmetric membrane lipids .These result show, the lipid stretching region (2500-3000 cm-1) and protein band (Data of A2780, A2780-cp and C13 cell lines sorted into data sets composed of 45 variables. FTIR wave number from 1000 to 3000 cm-1 were analyzed with PCA resulting in the clustered shown in The data of the cell lines between 1000 and 3000 cm The cluster of points derived from the first two PC scores which summarized spectral features of three cell lines are shown in a 2-dimensional projection. PCA correctly classified 96% of all spectra using two PCs and thus provide a good separation for representing the variety of sensitive and resistant cell line spectra. The data of the resistant cells are in the central area of PCA projection while it could not be distinguished between resistant cells. There are similarities between the resistance cell lines of A2780-CP and C13 spectrum, which could not discriminate using this technique. Thus the molecular compositions of two different resistant cell lines which cause resistance to cisplatin might almost be similar.Loadings plots allow for identification of wave numbers and corresponding biochemical responsibility. -1 in resistant and sensitive cells correctly classified by PCA model This region of FTIR data could be consumed as “Biochemical typicality spectra” among resistant and sensitive cell lines. In this region amid I and amid II molecules band have higher difference in sensitive and resistant cells.Resistance to Chemotherapy is a serious hurdle in the treatment plan of cancer. That is why identification of resistant cells at the procedure of a patient treatment saves time and is very much critical for the therapy outcome. The FTIR spectra provide a unique molecular fingerprint that can be interpreted based on the macromolecular biochemistry of the cells. This study suggests that infrared spectroscopy and clustering of data with PCA may represent a simple and precise method for discrimination biochemical pattern of resistant in cell lines. One of the advantages of this tool is to investigate the resistant percent of cancer cells using FTIR. Such technique may bring new tool in cancer diagnosis and stage definition in cancerous tissues. The FTIR data set between 1000 and 3000 cm


While platinum-based chemotherapeutic agents are widely used to treat various solid tumors, the acquired platinum resistance is a major impediment in their successful treatment. Since enhanced DNA repair capacity is a major factor in conferring cisplatin resistance, targeting of DNA repair pathways is an effective stratagem for overcoming cisplatin resistance. This study was designed to delineate the role of nucleotide excision repair (NER), the principal mechanism for the removal of cisplatin-induced DNA intrastrand crosslinks, in cisplatin resistance and reveal the impact of DNA repair interference on cisplatin sensitivity in human ovarian cancer cells.We assessed the inherent NER efficiency of multiple matched pairs of cisplatin-sensitive and -resistant ovarian cancer cell lines and their expression of NER-related factors at mRNA and protein levels. Our results showed that only the cisplatin-resistant ovarian cancer cell line PEO4 possessed an increased NER capacity compared to its inherently NER-inefficient parental line PEO1. Several other cisplatin-resistant cell lines, including CP70, CDDP and 2008C13, exhibited a normal and parental cell-comparable NER capacity for removing cisplatin-induced DNA intrastrand cross-links (Pt-GG). Concomitant gene expression analysis revealed discordance in mRNA and protein levels of NER factors in various ovarian cancer cell lines and NER proteins level were unrelated to the cisplatin sensitivity of these cell lines. Although knockdown of NER factors was able to compromise the NER efficiency, it only caused a minimal effect on cisplatin sensitivity. On the contrary, downregulation of BRCA2, a critical protein for homologous recombination repair (HRR), significantly enhanced the efficacy of cisplatin in killing ovarian cancer cell line PEO4.Our studies indicate that the level of NER factors in ovarian cancer cell lines is neither a determinant of their NER capacity nor of the sensitivity to cisplatin, and suggest that manipulation of the HRR but not the NER factor expression provides an effective strategy for sensitizing cisplatin-resistant tumors to platinating agents. Since the introduction of inorganic platinum (Pt) drug molecule cisplatin into the clinic, platinum-based chemotherapy drugs have been in widespread use to treat various malignant tumors, including ovarian, testicular, head and neck, and lung cancers . It is gcis-Pt(NH3)2d(GpG) (Pt-GG), with Pt bound to two adjacent guanines, and cis-Pt(NH3)2d(ApG) (Pt-AG), in which the Pt is bound to adenine and an adjacent guanine. These lesions contribute to 90% of total damage introduced by cisplatin. Other DNA damage introduced by cisplatin includes 1, 3-intrastrand cross-links (5-10%) and interstrand cross-links (1-2%) pyrene Diol Epoxide (BPDE) and cisplatin-induced bulky adducts. The complete NER reaction involves several biochemical steps including damage recognition, dual incision, and gap-filling DNA synthesis . In humaEvidence for increased repair of platinum-induced DNA damage in resistant ovarian cancer cells has been demonstrated by many groups (see review ). Howeve2 in air. Sensitivity to cisplatin in these ovarian cancer cell lines following one hour treatment was assessed by growth inhibition assay using 96-well plates as described later. The IC50 of each cell line was shown in Table The experiments were performed with three different groups of human ovarian cancer cell lines, each group having one cisplatin-sensitive parental cell line and one or two cisplatin-resistant variants Table . The hum2 followed by incubation for varying times. UV-C light (254 nm) was delivered from a germicidal lamp at a dose rate of 0.5 J/m2/s, as measured by a UVX digital radiometer connected to a UVX-31 sensor For cisplatin treatment, cells were maintained in medium with the desired doses of cisplatin  for 1 h, and then washed with PBS and followed by incubation in fresh cisplatin-free medium for varying times post-treatment. For UV exposure, the cultures were washed with PBS and irradiated with UV at 10 J/mCells were pre-treated with hydroxyurea (HU) for 24 h to exclude cells from S phase , then UVThe HCR assay was performed to determine the DNA repair capacity of individual cell lines. For this study, the pCMV-Tag 2 expression control plasmid  was treated with cisplatin (1 or 10 μM) to introduce DNA damage into the plasmid DNA. Both the undamaged and the damaged pCMV-Tag 2 plasmid were then transfected into cells (0.5 μg/35-mm dish) using Lipofectamine 2000 transfection reagent (Invitrogen). As an internal control, the pGL4.73 plasmid , which carries a renilla luciferase gene, was also co-transfected into the cells. The cells were harvested 2 days after transfection, and both firefly and renilla luciferase activities were determined from the transfected cells using a Dual Luciferase Activity Detection System (Promega). The activity of firefly luciferase in each experiment was calculated as relative activity to the renilla luciferase activity to minimize the experimental variations. The ratio of luciferase activities in the same cell line for both undamaged and damaged plasmid was used to determine the DNA repair capacity of the host cells.c Protein Assay , equal amounts of proteins were loaded, separated on a polyacrylamide gel, and transferred to a nitrocellulose membrane. Protein bands were immuno-detected with appropriate antibodies, e.g., rabbit anti-XPC and anti-DDB2 antibodies generated in our laboratory . In addin vitro . Howeverin vitro . In the in vitro . In our in vitro, in vivo, and clinical studies have demonstrated that high expression of ERCC1 correlates with cisplatin resistance in multiple tumors [Among the basic NER proteins, ERCC1 is one of the most extensively studied NER factors. The e tumors  while kne tumors . Moreovee tumors . In addie tumors ,41. It ie tumors , indicatDNA repair proteins can be excellent candidate targets for the development of new therapies to overcome resistance to cancer therapy. Since NER is the major mechanism for removing cisplatin-induced DNA damage, NER factors are being extensively investigated for realizing this goal. However, the effect of down-regulation of NER proteins on cisplatin sensitivity has remained contradictory. For example, it was reported that transfection with antisense XPA RNA could sensitize human lung adenocarcinoma cells to cisplatin , whereasAs discussed above, cisplatin forms a variety of adducts including DNA intrastrand crosslinks and interstrand crosslinks. Given the cisplatin-induced DNA interstrand crosslinks are mainly repaired by HRR , our finOur studies indicate that enhanced NER capacity is not the underlying cause of acquired cisplatin resistance of ovarian cancer cell lines derived from cells that have already high NER efficiency. In contrast, if the cisplatin-sensitive cells have a deficient NER, its derivative resistant cells can exhibit enhanced NER efficiency. Nevertheless, the level of NER factors in ovarian cancer cell lines is neither a determinant of their NER capacity nor of the sensitivity to cisplatin. Interfering with NER pathway through downregulation of NER factors is not an effective means of enhancing cisplatin resistance in ovarian cancer cells. In contrast, the disruption of HR through downregulation of BRCA2 can efficiently sensitize the functional BRCA2-containing cisplatin-resistant ovarian cancer cells to cisplatin treatment. Overall study suggests that the manipulation of HRR but not NER factor expression can serve as an effective strategy for sensitizing cisplatin-resistant tumors to platinating agents.The authors declare that they have no competing interests.KM, CH, Q-EW and JT participated in the Immuno-slot blot and Western blot analysis. CH carried out cell survival analysis and siRNA transfection. Y-WH carried out Real time PCR. Q-EW and AAW conceived, designed, analyzed the data, and prepared manuscript. All authors read and approved the final manuscript.


Human parechoviruses (HPeVs) are among the most frequently detected picornaviruses in humans. HPeVs are usually associated with mild gastrointestinal and respiratory symptoms with the exception of HPeV3 which causes neonatal sepsis and CNS infection. Previous studies showed various results in culturing different HPeV genotypes, inducing only a low cytopathic effect (CPE).In vitro growth characteristics of the different HPeV genotypes in a range of 10 different cell lines are scored with CPE and measured in the supernatant by real time PCR. In the optimal cell line for each genotype a standard neutralization assay with the available HPeV antibodies (Abs) was performed and scored by CPE and measured by real time PCR.All six HPeV types were able to replicate on the RD99, A549, and Vero cell lines. HPeV1 was the only genotype able to replicate on all cell lines. Most efficient growth of HPeV1, 2, 4, 5, and 6 was shown on the HT29 cell line, while HPeV3 was unable to replicate on HT29. In all cases viral replication could be measured by real time PCR before CPE appeared. The polyclonal Abs available against HPeV1, 2, 4 and 5 all showed neutralization of their respective genotype after 7 days with inhibition of >60% in real time PCR and full inhibition of CPE, although cross-neutralization is shown. Replication of HPeV3 could only be inhibited by 12% by the anti-HPeV3 (aHPeV3) Ab and no inhibition of CPE was shown after 7 days.When replication is monitored by PCR, growth of HPeV genotypes 1 to 6 is supported by most of the cell lines tested, where viral replication is measured before appearance of CPE. A combination of HT29 and Vero cells would therefore support replication of all culturable HPeV types, so viral replication could be detected by PCR within 3 days for all genotypes.In addition, we showed efficient neutralization for HPeV1, 2, 4, 5, while cross- neutralization was shown between these types, indicating possible common neutralizing epitopes. For HPeV3 no efficient (cross-) neutralization was shown, indicating different neutralizing epitopes for HPeV3 compared to the other HPeV genotypes. Picornaviridae family. HPeVs are associated with a wide range of clinical manifestations ranging from gastrointestinal and respiratory symptoms to more severe symptoms like central nervous system (CNS) infections and neonatal sepsis [. Because HPeV detection with cell culture is laborious and limited to HPeV1-6, PCRs targeting the 5’UTR are commonly used to diagnose HPeV infection [Human Parechoviruses are single stranded RNA viruses belonging to the l sepsis -4. Nowadl sepsis -10, HPeVl sepsis ,11,12. Fl sepsis . For HPenfection . Since tin vitro growth characteristics of different HPeV genotypes by measuring viral RNA in the supernatant of a range of cell lines by real time PCR. In addition, neutralization capacities of available type-specific antibodies is tested in in vitro cell culture.Although cell culture is a commonly used method in diagnostics and research, HPeVs are difficult to culture for many laboratories . HPeVs culture characteristics have never been completely elucidated in cell culture experiments. In this study we investigated Virus growth characteristics of HPeV1 to 6 were determined on HT29, Caco-2, A549, RD99, Hep-2, Vero, BGM, LLCMK2, SK-N-SH, and SH-SY-5Y by infection with a fixed MOI (0.001). Replication was monitored in the culture supernatant at day 0, 1, 3, 7 and 10 by quantitative RT-PCR, and CPE was scored at the same time points.All six HPeV types were able to replicate on the RD99, A549, and Vero cell lines  neutralization. More research needs to be done to elucidate the differences between HPeV3 and the other genotypes.For virus culture, the following cell lines were used: human colon carcinoma (HT29), human colon adenocarcinoma (Caco-2), human lung carcinoma , rhabdomyosarcoma (RD99), epidermoid carcinoma of the larynx (Hep-2), African green monkey kidney (Vero), buffalo green monkey kidney , rhesus monkey kidney , and human neuroblastomas . The cells were maintained in Eagle’s Minimum Essential Medium (EMEM) supplemented with L-glutamic acid (0.2X), non essential amino acid (1X), streptomycin (0.1 μg/ml) and ampicillin (0.1 μg/ml). For HT29, A549, RD99, Hep-2, Vero, LLCMK2, and BGM the medium was supplemented with 8% heat-inactivated Fetal Calf Serum (FCS) and for the Caco-2 cell line with 20% heat-inactivated FCS. The human neuroblastoma cell lines were cultured in Dulbecco’s MEM and supplemented with 10% heat-inactivated FCS, L-glutamic acid (0.2X), non essential amino acid (1X), streptomycin (0.1 μg/ml) and ampicillin (0.1 μg/ml).The following HPeV strains were used as prototypes: HPeV1A Harris, HPeV2-751312, HPeV3-150237, HPeV4-251176, HPeV5-552322 and HPeV6-550389 . HPeV1-HThe anti-HPeV (aHPeV) Abs against HPeV1 (Harris) and 2 (Williamson) were obtained from a rabbit Ab pool prepared at the RIVM. The aHPeV3 (A308-99) Ab was a kind gift from Dr. Shimizu, National Institute of Infectious Diseases, Tokyo, Japan, prepared as pooled guinea pig serum . The aHPMonolayers of the different cell lines were cultured in 24 wells plates (Cellstar) with 1 ml medium and incubated at 37°C, 5% CO2. At day 0 the cell lines with a confluence of ~80% were infected with HPeV at a multiplicity of infection (MOI) of 0.001 in a volume of 200 μl culture medium for two hours, after which the non absorbed virus was removed and replaced with 1 ml maintenance medium  and incubated for 10 days. The low MOI is chosen to elucidate the entire process of the infection cycle of HPeV in cell culture. Twenty μl culture supernatant was removed for RNA extraction and quantitative RT-PCR detection at days 0, 1, 3, 7 and 10. The supernatant was extracted by automatic extraction using the total nucleic acid isolation kit with the MagnaPure LC instrument® (Roche Diagnostics). The RNA was eluted in 50 μl elution buffer and reverse transcribed as described previously . Five μlAbs were mixed with the different HPeV1 to 6 virus suspension containing 100 TCID50/50 μl. Ab dilutions of aHPeV1, aHPeV2, aHPeV3, aHPeV4 and 5 (1:100) were used for end-point neutralization of 100TCID50 HPeV1 to 6. Mixtures were incubated at 37°C for 1 hr, and were used to inoculate HT29 cells  and Vero cells (HPeV3) on a 96-wells plate (200 μl). Virus, cell and Ab controls were included as positive and negative control. The cells were examined for the appearance of CPE every 24hrs for 3 and 7 days and amount of viral copies were defined by real time PCR.The authors declare that they have no competing interests.BW, KB, KW contributed to the study design. BW, SM, SJ contributed to acquisition of data, and analysis and interpretation of the data. BW, KB, KW have been involved in preparing the manuscript. All authors read and approved the final manuscript.Table S2. Neutralization of HPeV1 to 6 by polyclonal Abs, percentage of inhibition measured by real time PCR at day 3 post infection. Neutralization of HPeV1 to 6 by polyclonal Abs, read out of CPE at day 3 post infection. Click here for file


Many cancers contain cell subpopulations that display characteristics of stem cells. Because these cancer stem cells (CSCs) appear to provide resistance to chemo-radiation therapy, development of therapeutic agents that target CSCs is essential. Curcumin is a phytochemical agent that is currently used in clinical trials to test its effectiveness against cancer. However, the effect of curcumin on CSCs is not well established. The current study evaluated curcumin-induced cell death in six cancer cell lines derived from human esophageal squamous cell carcinomas. Moreover, these cell lines and the ones established from cells that survived curcumin treatments were characterized.Cell loss was assayed after TE-1, TE-8, KY-5, KY-10, YES-1, and YES-2 cells were exposed to 20–80 μM curcumin for 30 hrs. Cell lines surviving 40 or 60 μM curcumin were established from these six original lines. The stem cell markers aldehyde dehydrogenase-1A1 (ALDH1A1) and CD44 as well as NF-κB were used to compare CSC-like subpopulations within and among the original lines as well as the curcumin-surviving lines. YES-2 was tested for tumorsphere-forming capabilities. Finally, the surviving lines were treated with 40 and 60 μM curcumin to determine whether their sensitivity was different from the original lines.The cell loss after curcumin treatment increased in a dose-dependent manner in all cell lines. The percentage of cells remaining after 60 μM curcumin treatment varied from 10.9% to 36.3% across the six lines. The cell lines were heterogeneous with respect to ALDH1A1, NF-κB and CD44 expression. KY-5 and YES-1 were the least sensitive and had the highest number of stem-like cells whereas TE-1 had the lowest. The curcumin-surviving lines showed a significant loss in the high staining ALDH1A1 and CD44 cell populations. Tumorspheres formed from YES-2 but were small and rare in the YES-2 surviving line. The curcumin-surviving lines showed a small but significant decrease in sensitivity to curcumin when compared with the original lines.Our results suggest that curcumin not only eliminates cancer cells but also targets CSCs. Therefore, curcumin may be an effective compound for treating esophageal and possibly other cancers in which CSCs can cause tumor recurrence. Curcumin longa) [Esophageal cancer is the eighth most common cancer worldwide and the sixth most common cause of death among cancers . Of the n longa) -8. Curcun longa) , human mn longa) , and lunn longa)  without n longa) ,13. Curcn longa) ). Therefn longa)  or in con longa)  and radin longa) . Althougn longa) ,19, curcOne possible reason for the poor prognosis of ESCC is the presence of cancer stem cells (CSCs) in the tumor ,21. It iCSCs have high expression of aldehyde dehydrogenase (ALDH) that can be used as a potential marker for identifying and isolating CSCs -35. ALDHAnother marker for CSCs is CD44 (cluster of differentiation 44). CD44 is an integral cell membrane glycoprotein involved in cell-cell interaction . CD44 haAn additional property of cancer stem cells (CSCs) is the ability to grow as non-adherent spheres when cultured in serum-free medium supplemented with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) -50. ReceIn this study, curcumin-surviving cell lines were established from six human ESCC lines. The CSC markers ALDH1A1 and CD44, curcumin toxicity, and tumorsphere formation were examined. When the surviving cell lines were compared with the original untreated lines, the results indicated that curcumin produced a permanent reduction in the CSC-like subpopulation of these cells.2. When the cells reached near confluency, they were washed with phosphate-buffered saline (PBS) once and incubated with trypsin  for two min. After incubation, the cells were washed once with complete DMEM to inactivate trypsin. The cells were then collected by centrifugation at 800 x g, resuspended in complete DMEM and plated into new culture dishes.The six human esophageal squamous cancer cell lines  were obtained from various sources -56. The 5 cells/plate) in complete DMEM and incubated at 37°C. When the cells reached near confluency, approximately 70-75%, the cultures were photographed using a phase-contrast microscope (Olympus CK40) equipped with a color digital camera (Kodak M575).To examine the morphology of the ESCC cell lines, each cultured line was trypsinized, washed and counted using a hemocytometer . The cells were then plated into 100-mm dishes  was dissolved in dimethyl sulfoxide (DMSO) to make a stock solution of 20 mM curcumin. For each experiment, stock solution was diluted to the final concentrations  in complete DMEM. Twenty-four hours after plating, the wells were washed with PBS once, and 1 ml of each curcumin dosage was added (4 wells/dosage) and incubated for 30 hrs. Four other wells received complete DMEM or complete DMEM with 0.2% DMSO as controls. The inhibition of cell growth in response to curcumin treatment was assayed with the crystal violet staining assay according to a standard protocol [To determine the effect of different doses of curcumin on the ESCC lines, each cell line was plated in 24-well plates (Corning) at a density of 5×10protocol . Briefly5 cells/dish) with complete DMEM and incubated at 37°C until reaching near confluency, approximately 80%. After incubation, the medium was removed and the cells were incubated in complete DMEM containing either 40 or 60 μM curcumin dissolved in 0.2% DMSO for 30 hrs. After incubation, the medium-containing curcumin was removed and replaced with complete DMEM to allow cells that survived the treatment to grow. After the surviving cells formed colonies , the colonies were counted, trypsinized, and passaged. The cell lines that developed were designated the curcumin-surviving cell lines.To select for a curcumin-surviving cell subpopulation, each of the ESCC cell lines was cultured in 100-mm dishes (1×104 cells/ well) in complete DMEM and incubated at 37°C. Twenty-four hours after plating, wells were treated with 40 or 60 μM curcumin and incubated for 30 hrs (4 wells/dosage). The inhibition of cell growth in response to curcumin treatment was assayed with the crystal violet staining assay as described above.To compare the effect of curcumin on the original cell lines and the curcumin-surviving cell lines, the curcumin-surviving lines were cultured in 24-well plates  at 1:100 dilution in PBS for 30 min. The cover slips were then incubated with a primary antibody for 2 hrs at room temperature, after which the cover slips were washed three times with PBS and incubated with a secondary antibody for 30 min. For ALDH1A1 staining, a rabbit anti-human ALDH1A1 antibody  at 1:100 dilution and a goat anti-rabbit antibody conjugated with horseradish peroxidase  at 1:1000 dilution were used. For CD44 staining, a mouse monoclonal anti-human CD44 antibody  at 1:10 dilution and a goat anti-mouse horseradish peroxidase-conjugated antibody  at 1:40 dilution were used. The cells were then reacted with ImmunPACT DAB  for 10 min. The cover slips were washed three times with water, dehydrated in an ethanol series, cleared with Citrosolve , and mounted on glass slides with Permount . Control coverslips did not receive the primary antibody but were otherwise treated identically.The images of cell cultures immunostained for ALDH1A1 or CD44 were analyzed with ImageJ (NIH) and OriginLab (OriginLab) software. To determine the intensity of staining, all cells were imaged with a 12-bit digital camera  and a Zeiss Axiophot microscope. The images were thresholded identically using a pixel intensity (brightness) value of 500 analog-to-digital units (ADUs). For counting cells, ImageJ was used to identify cells within a normal size range (500–4000 pixels in area) that did not touch the edge of the image.4 cells/well) and incubated for 24 hrs at 37°C. After incubation, the wells were washed once with PBS, fixed with methanol for 5 min, and then washed twice with PBS. Anti-p65 at 1:50 dilution  and anti-ALDH1A1 at 1:500 dilution  were prepared in complete DMEM. The wells were incubated with 100 μl of each antibody (3 wells/antibody dilution) for 2 hrs at 37°C. After incubation, the wells were washed with PBS three times and incubated with 100 μl/well of β-galactosidase-conjugated goat anti-rabbit secondary antibody  for 2 hrs at 37°C. After incubation, the wells were washed with PBS three times and 100 μl/well of substrate solution  was added and incubated at 37°C for one hour in darkness. The development of yellow color indicated a positive reaction. The antigen-antibody reactions were measured by determining the optical density (O.D.) at 410 nm using a microwell plate reader .The level of RelA (p65), a subunit of transcription factor NF-κB, and ALDH1A1 was examined in both curcumin-surviving cells and original cell lines. ELISA was performed as described previously . Briefly2 incubator for a week and then formation of free-floating spheres was evaluated. The experiment was performed in triplicate.To examine the ability of the cell line to form spheres, YES-2 and YES-2S cell lines were cultured in stem cell medium. Cells were maintained as a monolayer in complete DMEM until they were nearly confluent. Cells were trypsinized, collected, and washed two times to remove serum, then suspended in serum-free DMEM/F12 supplemented with penicillin (100 units/ml), streptomycin (100 μg/ml), 20 ng/ml human epidermal growth factor (EGF), 20 ng/ml human basic fibroblast growth factor (bFGF), and B27 supplement . The cells were subsequently cultured in ultra-low attachment 60-mm plates  at a density of 10.000 cells/ plate. Plates were incubated at 37°C in a humidified 5% COt-test. For comparing more than two groups, one-way analysis of variance (ANOVA) was used followed by the Scheffe multiple comparisons test with a significance of p< 0.05. All errors are shown as standard deviation (SD).For statistical analysis OriginPro 7.5 (OriginLab) was used. Statistical comparisons between two groups were made using Student’s The characteristics of patients, tumors, and esophageal cell lines derived from each tumor are listed in Table To assess the sensitivity of the six human ESCC lines to curcumin, the cell lines  were plated in 24-well plates and maintained in culture for 24 hrs and then exposed to 20, 30, 40, 60, and 80 μM curcumin and to 0.2% DMSO as a control for 30 hrs. The resulting cell density was determined by a standard crystal violet assay. The cell density remaining after the treatment is shown in the dose–response curves  curcumin. The number of surviving colonies formed after 60 μM curcumin was 0, 8, 17, 19, 25, and too many colonies to count for YES-2, TE-1, TE-8, KY-10, YES-1, and KY-5, respectively. The KY-5 cell line had the highest number of surviving colonies, whereas the YES-2 cell line did not form any colonies after 60 μM curcumin but formed 15 colonies after treatment with 40 μM curcumin.According to the dose–response results, 60 μM curcumin was chosen to select for the curcumin-surviving cell line subpopulations, indicated by “S”. To establish this subpopulation, 1×10To further distinguish between the original ESCC cell lines and the curcumin-surviving lines, we measured NF-κB, ALDH1A1, and CD44 levels. We also examined the effect of curcumin on these lines and their ability to form tumorspheres.The ALDH1A1 and NF-κB expression levels were measured in the original ESCC lines and the curcumin-surviving lines using ELISA. As shown in Figure The results also showed that the curcumin-surviving lines had lower ALDH1A1 levels when compared with the original cell lines, except for TE-1 Figure A. This dALDH1A1 immunostaining was examined in the six original ESCC lines by measuring the staining intensity of individual cells. As shown in Figure Within all of the six original lines, there were two subpopulations, each with its own distinctive peak in the histogram, (high and low ALDH1A1-staining subpopulations), as shown in Figure Like the original cell lines, there was a significant high-staining subpopulation within each surviving line except TE-1S and TE-8S, as shown in Figures Interestingly, each of the curcumin-surviving cell lines showed a significant decrease in the percentage of cells in the high-staining subpopulation when compared to their corresponding original line . This decline is visible as a loss of nearly all of the cells in the high-staining subpopulations  to form three-dimensional spheres in serum-free medium containing EGF and bFGF on low-attachment plates was examined. This procedure has been reported by others to identify stem cells -61. UndeTo compare the effect of curcumin on the ESCC lines and curcumin-surviving lines, the curcumin-surviving cell lines were passaged four times and treated with 40 or 60 μM curcumin. The resulting cell density was determined by standard crystal violet assay and the percentage of cells remaining after treatment was calculated for each line Figure . This exEsophageal cancer is a highly aggressive malignant disease resulting in low patient survival. Because of the poor success rate of standard therapies, innovative approaches for treatment have been tested, and two studies have examined whether curcumin could be a potential candidate for esophageal cancer treatment. In one study, Hartojo et al.  examinedWhen the cell lines were treated with curcumin 20–80 μM) for 30 hrs there was a reduction in cell number in all lines in a dose-dependent manner, consistent with studies using breast, lung, and colorectal cancer cell lines , and es0–80 μM fThis is the first study to characterize curcumin-surviving cell lines. All six curcumin-surviving cell lines had lower levels of ALDH1A1 when compared with each corresponding original line, based on ELISA and immunocytochemistry. ALDH1A1 is a stem cell marker that offers cellular protection against cytotoxic drugs ,41,64-66Across the six lines, levels of ALDH1A1 and NF-κB were inversely correlated with sensitivity to curcumin, as exemplified by KY-5 versus TE-1 Figure . BecauseThis study examined p65, a subunit of the NF-κB transcription factor, in the ESCC lines. The results support use of NF-κB as a marker for cancer stem cells because NF-κB and ALDH1A1 levels were strongly correlated. NF-κB levels decreased among the three surviving cell lines that had the highest initial levels of NF-κB  according to ELISA. Additionally, the other three lines showed the same (KY-10) or more NF-κB expression (TE-1 & TE-8) in the surviving lines, and this result was confirmed using immunocytochemistry with the same three lines . The increase in NF-κB but not ALDH1A1 in two lines could result from the differences between the functions of these markers. For example, ALDH1A1 is an enzyme, whereas NF-κB is a cell signaling molecule and transcription factor that serves in multiple cellular pathways. This difference suggests that ALDH1A1 may be a better indicator of stem-like cells.Whereas most studies measure the activity of the NF-κB signaling pathway, such as nuclear entry of p65, we determined the level of p65 in the ESCC lines. Several studies have shown that the level of NF-κB in cells is correlated with aggressiveness ,69, lackInterestingly, YES-2 differed from the other lines: Although it had high NF-κB and ALDH1A1 expression, it showed more sensitivity to curcumin and was the only line that had no survivors after the 60 μM curcumin treatment. It is not clear why the YES-2 cell line behaved differently, and it should be investigated further. Notably, YES-2 is the only one of the six lines derived from the tumor that was reported to have metastasized in the patient .Expression of CD44, a stem cell marker, was examined in two of the lines, YES-1 and YES-2, and in their curcumin-surviving lines. These original cell lines were chosen because they had high ALDH1A1 and NF-kB expression. As observed with ALDH1A1, two subpopulations of cells were recognized in these lines, one with high and one with low CD44 immunostaining. The intensity was two times greater in the high-staining cells than in the low-staining cells. However, the percentage of highly-stained cells was less then what was observed in the ALDH1A1 experiment. For YES-1 and YES-2 the percentage of highly stained cells was 20% and 36.2% for ALDH1A1, respectively, and 4.82% and 12.11% for CD44. One way to explain why the high-staining CD44 subpopulation was smaller than the high-staining ALDH1A1 subpopulation is that the first group may contain mostly CSCs, whereas the second group may include progenitor cells derived from CSCs, which are not fully differentiated but continue to express substantial amounts of ALDH1A1 .It is generally believed that CSCs are more numerous in tumors after chemo- and radiation therapy because they are more resistant than non-stem cells, which along with their greater aggressiveness makes CSCs a likely source of tumor recurrence -77. In cMultiple studies have demonstrated that invasion and metastasis are mediated by a cellular component that displays high ALDH1 activity ,78, suggIf the sensitivity of each line to curcumin depends, at least in part, on its ratio of CSCs to non-CSCs, then the loss of CSCs in the surviving cell lines, as shown by the immunostaining for stem cell markers, suggests that curcumin sensitivity decreases as the proportion of CSCs declines. However, when comparing across the original six lines, a decrease in sensitivity to curcumin was correlated with increasing stem-like characteristics, as epitomized by KY-5. One explanation for this apparent contradiction is that the range of sensitivity to curcumin detected across the lines could depend more on genetic differences among the lines than on sensitivity of CSCs to curcumin. In support of this explanation, the surviving lines appear in roughly the same order of sensitivity to curcumin as the original lines, despite having lost most or nearly all of their CSCs, as identified by the number of cells with high ALDH1A1 or CD44 levels.The specific mechanism causing the reduction in stem cells by curcumin was not examined in this study, but we can consider two possibilities. First, the curcumin may have selectively destroyed the CSCs. Second, curcumin may have caused the CSCs to differentiate more frequently into non-CSCs, similar to what has been described in glioma and PCC4 embryonal carcinoma cells ,80. The In agreement with both of these possible mechanisms, researchers have shown that curcumin can modulate or eliminate a variety of cellular targets in cancer cells ,83. AlthThe present study demonstrates that curcumin reduces the number of ESCC cells in a dose-dependent manner. By comparing the six ESCC lines the results showed that ESCC cells vary considerably based on CSC properties and sensitivity to curcumin. This study is the first to establish and characterize curcumin-surviving subpopulations among cancer cell lines. By comparing the original ESCC lines and the curcumin–surviving lines we found that the curcumin-surviving cell lines contain fewer stem-like cells than the original lines. These data support clinical applications for curcumin as a chemotherapeutic agent against cancers, particularly ESCC, rather than only as a preventative dietary supplement. Contrary to other chemotherapeutic agents, we predict that the cells that comprise a recurrent esophageal tumor after curcumin treatment would contain fewer CSCs than the original tumor. Because CSCs are considered aggressive and more metastatic, a tumor developing after curcumin treatment may be managed more easily than traditionally treated cancers.ESCC: Esophageal squamous cell carcinoma; CSCs: Cancer stem cells; ALDH1A1: Aldehyde dehydrogenase-1A1; CD44: Cluster of differentiation 44; NF-κB: Nuclear factor kappa-light-chain-enhancer of activated B cells.The authors have no conflicts of interest.All three authors contributed equally to the research and manuscript preparation. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6882/12/195/prepub


Ferula szowitsiana root, which proved to be the most active, was chosen for further phytochemical studies. The major compounds of the most potent acetone extract were isolated. They were identified as chimgin and chimganin, two known monoterpenoids, by spectroscopic means. Their cytotoxic activity was evaluated in three cell lines. The results show that these compounds are responsible, at least in part, for the cytotoxic activity of this plant.Methanolic extract of 15 Iranian medicinal plants were prepared and tested for their cytotoxic activities against three cancer cell lines  and one normal cell line (MDBK). Some plants showed cytotoxic activities. The extract of  Cancer is a general term applied to a series of malignant diseases which may affect many different parts of the body. If the process is not arrested, it may progress until it causes the death of the organism. Cancer Drug discovery from medicinal plants has played an important role in the treatment of cancer and, indeed, most new clinical applications of plant secondary metabolites and their derivatives over the last half century have been applied toward combating cancer5.Traditional medicine over the years has proved to be an invaluable guide in drug discovery and Iran has a long history in this field and is a great source of new bioactive compounds.7.in vitro antifungal activity against 19 fungal strains using CDCl3. Electron Impact mass spectra were obtained using a Finnigan MAT-EI-TSQ at 70 eV. Melting points were determined on a Reichert-jung apparatus and UV spectra were taken on a Shimadzu UV-3100 spectro-photometer.All solvents were purchased from Merck, Germany. NMR spectra were recorded on a Bruker Ultra shild NMR spectrometer , harvested by tripsinization, plated (104 cell/well) in 96-well plates, and incubated for 24 h at 37°C in the incubator. Afterwards, they were exposed to different concentrations of plant extracts and incubated for further 72h followed by MTT  assay at 570 nm(50) on normal cells to cancer cells.The cytotoxicity bioassay was performed against three cancer cell lines  and one normal cell line (MDBK). Cells were grown in monolayer cultures in Dulbecco’s Modified Eagle’s Medium (DMEM) and RPMI 1640 supplemented with 5% FBS (Gibco), 100 U/ml penicillin, 10 µg/ml streptomycin, and maintained at 37°C in a 5% COat 570 nm. ViabiliF. szowitsiana (40 g) which were collected from the Kalaleh region in Golestan province, at the height of 500-700 m, were extracted with methanol over night. After filtration, the solvent was evaporated to dryness under reduced pressure at 40°C. The residue was extracted by acetone. The dried acetone extract  was fractioned by column chromatography using Silica gel 60 eluting first with hexane, followed by a gradient of hexane CHCl3 up to 100% CHCl3 and CHCl3 -acetone up to 15% acetone. Eight fractions were obtained. Fraction 3 was separated by Preparative Thin Layer Chromatography (PTLC) over silica gel, using CH2 Cl2 : EtOAc (9.5:0.5) as mobile phase to obtain 175 mg compound 1 (Chimganin). Fraction 5 was crystallized from EtOH twice and afforded 500 mg of compound 2 (Chimgin).The ground roots of 1 HNMR  : δ 7.70 , 7.60 , 6.98 , 5.12 , 3.96 , 2.50 , 1.16 , 2.16 , 1.45 , 1.84 , 1.34 , 1.76 , 1.13 , 0.95 , 0.94 .13 C NMR : 167.13 (C-7’),150.28 (C-4’), 146.59 (C-3’), 124.36 (C-6’), 114.51 (C-5’), 123.40 (C-1’), 112.23 (C-2’), 80.75 (C-2), 56.46 (OMe), 49.50 (C-7), 48.27 (C-1), 45.42 (C-4), 37.34 (C-3), 28.52 (C-5), 27.54 (C-6), 19.92 (C-8), 19.34 (C-9), 14.05 (C-10). EIMS (70 eV) m/z (rel.int.): 304 (M+) (15), 136 (10), 121 (100), 118 (35), 93 (20).Colorless crystals, m.p.80-82 °C (lit. 85 °C);White needles, m.p. 150-152 °C (lit. 154-155 °C).1 H NMR  :δ 7.92 , 7.92 , 6.88 , 6.88 , 5.05 , 2.45 , 1.09 , 2.13 , 1.40 , 1.77 , 1.31 , 1.71 , 0.95 , 0.90 , 0.89 .13 CNMR : 167.34 (C-7’), 161.99 (C-4’), 132.01 (C-6’), 132.01 (C-2’), 115.73 (C-3’), 115.73 (C-5’), 122.44 (C-1’), 80.38 (C-2), 48.22 (C-1), 49.44 (C-7), 45.41 (C-4), 37.33 (C-3), 28.49 (C-5), 27.79 (C-6), 20.12 (C-8), 19.30 (C-9), 13.10 (C-10). EIMS (70 eV) m/z (rel.int): 274(M+) (15), 153 (10) 136 (37), 121 (100), 93 (50).The cytotoxicity of two isolated compounds was evaluated against MCF-7, HepG2 and MDBK cell lines by the MTT assay.F. szowitsiana (yield 22.4%) showed a promising antiproliferative activity. It showed IC50 values less than 100 µg/ml in all evaluated cell lines and in 3 cell lines out of 4 it showed the highest cytotoxicity. Further-more, Perovskia abrotanoides exhibited moderate cytotoxicity against three cell lines, whereas Buxus hyrcana and Parrotia persica where only active against one cell line. According to the results of the bioassay, F. szowitsiana was selected for further studies in order to identify the active compounds.The cytotoxic activity of 15 different medicinal plants of Iran against MCF-7, HepG2, WEHI and MDBK cell lines was evaluated. The results of this screening are summarized in 1H NMR of compound 1 revealed the occurrence of a 1,2,4- trisubstituted aromatic system. In addition it showed characteristic signals of a bornyl moiety in which H-2 (exo) appears as a doublet of triplets  due to a “W” coupling to the coplanar H-6 (exo). By compH-6 (exo), it was H-6 (exo). The cyte curves .F. szowitsiana showed the most potent cytotoxicity against all of them.This study aimed to investigate the cytotoxic activity of 15 Iranian traditional medicinal plants against three cancer cell lines and one normal cell line. Some plant’s methanolic extracts showed low or no cytotoxicity against the cell lines, whereas F. szowitsiana. Both bornyl esters have been isolated before from different Russian Ferula species(F. szowitsiana. Previous investigations revealed the occurrence of prenylated coumarin derivatives in the roots of F. szowitsiana (50 value of 12.3 µM(Bioassay-guided fractionation led to the isolation of two monotepenoids from the root extract of a species–12, but witsiana . One of f 12.3 µM.50 values compared to the extract and were just slightly less active than tamoxifen which was used as positive control. Thus we conclude that these two compounds are responsible, at least in part, for the observed cytotoxicity of the extract of F. szowitsiana.Both compounds, Chimganin and Chimgin, showed significant cytotoxic effects with lower IC


B-cell Chronic Lymphocytic Leukemia (CLL) is the most common leukemia diagnosed in adults in the western world. Multiple Myeloma (MM), a plasma cell neoplasm based in bone-marrow, comprises about 1% of all malignant tumours. These are two incurable disorders with treatments that focus on controlling the disease and symptoms rather than eradicating it. Synthetic analogues of Retinoic Acid (RA) are currently used extensively for the treatment of skin disorders such as acne vulgaris and psoriasis, as well certain forms of cancer namely Acute Promyelocytic Leukemia. Preclinical studies have also garnered support for their chemopreventive potential in many more cancers, especially haematological malignancies.The purpose of this study was to illustrate the roles of Fenretinide (4HPR) and Acitretin in CLL and MM and establish whether the two retinoic acid derivatives may prove to be prospective treatments for these malignancies. Fenretinide (4HPR) is a drug studied in a variety of cancers and has shown to induce apoptosis through increased levels of Reactive Oxygen Species (ROS) and activation of caspase-8, 9 and 3. Acitretin is currently used to treat psoriasis but little is known about its effect on cancer. Therefore, its mechanism of action is unknown.For the purpose of this study, 5 MM cell lines  and primary cells isolated from whole blood of CLL patients were used. 4HPR significantly decreased cell viability in 4 out of 5 MM cell lines tested at 5, 10, 20μM, whereas Acitretin only had an effect at concentrations higher than 50uM in 2 MM cell lines. CLL cells were highly sensitive to 4HPR at all concentrations used, but were only sensitive to Acitretin up until 10μM. 4HPR and Acitretin also significantly decreased NCI-H929 and CLL cells migration capabilities. Additionally, western blot analysis on RPMI-8226 cells showed that 4HPR and Acitretin negatively affected the expression of the cell cycle markers cyclin D2 and phospho-Rb, the anti-apoptosis marker Mcl-1, and increased cleavage and therefore activation of the pro-apoptotic marker PARP. This suggests that the two inhibitors could affect both cell proliferation and apoptosis.Having conveyed promising cytotoxic and chemoattractant properties with both CLL and MM cells, 4HPR and Acitretin have proven to be promising therapeutic candidates for MM and CLL treatment.


Aneuploidy, a karyotype deviating from multiples of a haploid chromosome set, affects the physiology of eukaryotes. In humans, aneuploidy is linked to pathological defects such as developmental abnormalities, mental retardation or cancer, but the underlying mechanisms remain elusive. There are many different types and origins of aneuploidy, but whether there is a uniform cellular response to aneuploidy in human cells has not been addressed so far.Here we evaluate the transcription profiles of eleven trisomic and tetrasomic cell lines and two cell lines with complex aneuploid karyotypes. We identify a characteristic aneuploidy response pattern defined by upregulation of genes linked to endoplasmic reticulum, Golgi apparatus and lysosomes, and downregulation of DNA replication, transcription as well as ribosomes. Strikingly, complex aneuploidy elicits the same transcriptional changes as trisomy. To uncover the triggers of the response, we compared the profiles with transcription changes in human cells subjected to stress conditions. Interestingly, we found an overlap only with the response to treatment with the autophagy inhibitor bafilomycin A1. Finally, we identified 23 genes whose expression is significantly altered in all aneuploids and which may thus serve as aneuploidy markers.Our analysis shows that despite the variability in chromosome content, aneuploidy triggers uniform transcriptional response in human cells. A common response independent of the type of aneuploidy might be exploited as a novel target for cancer therapy. Moreover, the potential aneuploidy markers identified in our analysis might represent novel biomarkers to assess the malignant potential of a tumor. Aneuploidy, or a change in cellular chromosome numbers, has profound effects on the physiology of all eukaryotic cells analyzed to date . AneuploArabidopsis thaliana plant cells, mouse cell lines with Robertsonian translocations that lead to trisomies, and human cells from patients with trisomy syndromes [The exact mechanisms underlying the detrimental effects of aneuploidy remain unclear, but it has been convincingly shown that they are caused by the expression of the extra genes on the supernumerary chromosome . In mostyndromes . Additioyndromes . These syndromes . MoreoveTo obtain a comprehensive insight into the changes specific for human cells, we evaluated in detail multiple tri- and tetrasomic cell lines as well as model complex aneuploid human cell lines with hypotetraploid karyotypes more similar to cancer cells. Remarkably, we found that the cellular responses to complex multichromosomal aneuploidy and trisomy or tetrasomy of one single chromosome closely correlate. As the identified pathway pattern resembles the cellular stress response, we compared the aneuploidy response to the transcriptional changes in human cells subjected to various stress conditions. Additionally, we have also identified 18 genes that were upregulated and 5 genes downregulated in all analyzed aneuploid cell lines and might thus serve as markers of aneuploidy. This is the first study which compares a variety of different human cell lines with aneuploidy of different types and origins. By uncovering a uniform aneuploidy response pattern our results outline the cellular consequences of an abnormal karyotype in human cells.de novo human aneuploid cell lines that were derived from diploid and chromosomally stable cells by a micronuclei-mediated chromosome transfer [Recently, we established transfer . We perftransfer . To identransfer . Additiotransfer . As expeOur analysis identified a specific pattern of pathways that are altered in all compared model trisomic and tetrasomic cell lines Table . Among tde novo created trisomies and tetrasomies elicit a nearly identical pattern of pathway changes regardless of the identity of the supernumerary chromosome and the cell line.Interestingly, DLD1 with trisomy of chromosome 7 shows only partial overlap with the other aneuploid cell lines: pathways related to splicing and to DNA and RNA metabolism were similarly downregulated, and vacuole was upregulated, whereas additional changes in the transcription profile differed from the other trisomic and tetrasomic cell lines  showed only a partial aneuploidy response pattern , a set of transcriptional changes, common in a broad range of human aneuploid model cell lines. This, for the first time, shows that complex aneuploidy, which is frequently found in cancer cells, exhibits the same transcriptional pathway changes as simple trisomy and tetrasomy. A general response to aneuploidy, as identified here in a variety of aneuploid model cell lines, might serve as a novel therapeutic target in cancer treatment. Further, we found 23 genes consistently deregulated in our model aneuploid cell lines. A confirmation of these markers in aneuploid cells might open new strategies for identifying aneuploid tumors. Since the fraction of aneuploid cells in tumor correlates with malignant potential and poor prognosis in cancer, simple and reliable biomarkers for aneuploidy may help for determining the appropriate cancer therapy.Following model aneuploid cell lines were used for data analysis: parental cell line HCT116 (human colon carcinoma cell line): HCT116 3/3 (trisomy 3), HCT116 5/4 (tetrasomy 5) ; parentaThe HCT116- and RPE1- derived tri- and tetrasomic cell lines have been described previously . The posMaterial for transcription analysis was obtained at the earliest possible time point, after sufficient amount of cells was achieved, approximately 25 generations after the chromosome transfer or tetraploidization, respectively. Similar approach was taken for the analysis of trisomy 8 HE35-derived clones . The mRNFreshly cultured cells that carry H2B-GFP to fluorescently label the chromosomes were seeded 24 h before the experiment. Time laps movies were taken by imaging asynchronous cells in a 10 min or 4 min interval for 72 or 48 h, respectively. The time in interphase was measured as the time from nuclear envelope reformation to nuclear envelope break down.GGGGGAAGCAGGACTAGATG, reverse: CCTGAAGCTCCGGGTAGTC, RAB27B - forward: TGCGGGACAAGAGCGGTTCCG, reverse: GCCAGTTCCCGAGCTTGCCGTT, HOXB5 - forward: TCCACAAATCAAGCCCTCCA, reverse: GTCCGGGCCATTTGGATAAC, GLRX – forward: AACGGTGCCTCGAGTCTTTA, reverse: CCTATGAGATCTGTGGTTACTGC.Total RNA was extracted with the RNeasy Mini Kit (Qiagen), treated with DNAse  and subsequently transcribed into cDNA . Specific primers were designed using PrimerBlast; the sequences are listed below. Quantitative PCR was conducted using the Light Cycler 480 System (Roche Diagnostics) with the KAPA SYBR FAST master mix optimized for Roche Light Cycler 480 (KapaBiosystems). Absolute quantification with an external standard was performed and negative non-template controls were tested in all experiments. The specificity of the primer product amplification was confirmed in each run by melting curve analysis. mRNA expression was normalized to the control gene coding for ribosomal protein L30 (RPL30) and fold change to corresponding diploid mRNA expression was calculated. Primers: COL13A1 – forward: http://www.ncbi.nlm.nih.gov/geo/) accessible through GEO Series accession number GSE47830 and GSE47836.Genome-wide expression profiling of HCT116- and RPE1- derived aneuploid cells lines was conducted in three replicates by IMGM laboratories GmbH  as previously described . cDNA wahttp://www.ncbi.nlm.nih.gov/geo/). HCT116 cell line grown under high and low glucose conditions: accession number GSE31084; HCT116 after stress treatment: accession number GSE3176; HCT116 cell line treated with actinomycin D: with GSE12459, human embryonic diploid and trisomic cells: accession number GSE28076.Microarray data of trisomic and diploid colorectal cancer cell lines DLD1  were kinBioinformatics analysis of the microarray data was performed using Perseus (1.2.6.16) as part of the MaxQuant Software Package  and R ont-test was performed to verify the statistical significance of the fold change in mRNA expression between the signal intensities of aneuploid and diploid cell line. To correct the test statistics for multiple comparisons, false discovery rate control of the p-values was applied. Both local and frequent FDR were calculated with the “fdrtool” package [The Student’s  package . For fur package , KEGG pat-test followed by a false discovery rate correction in the Benjamini-Hochberg procedure (FDR cut off 0.02). Expression values for significant enriched annotations were summarized in an annotation score from -1 to 1 to represent levels of up- or downregulation. Significant and enriched annotations were summarized in larger, general categories for visualization purposes. 2-dimensional annotations enrichments of two cell lines were plotted in R. Highly up- or downregulated pathways were cross validated by submitting the fold change cut off datasets to the annotation enrichment tool DAVID [Pathway enrichment analysis was conducted in Perseus using the 2-dimensional annotation enrichment tool . Therebyol DAVID .Commonly used R packages were “lattice” , “genefihttp://www.ncbi.nlm.nih.gov/geo/) with the accession numbers GSE47830 and GSE47836.The data sets supporting the results of this article are available in the NCBI Gene Expression Omnibus repository (The authors declare that they have no competing interests.ZS initiated the study. AYK, SS and VP generated the cell lines and prepared the samples for analysis. MD performed the bioinformatics analysis and the quantitative RT PCR. MD, AYK, GS and ZS discussed and designed the data analysis strategies. ZS wrote the manuscript. All authors read and discussed the final manuscript.Figure S2. Time in interphase of the HCT116*- derived cell lines. Figure S3. Significantly altered pathways in response to stress stimuli compared to the ARP. Significantly altered pathways in aneuploid model cell lines. Click here for fileDataset 1 Gene expression data of HCT116 and RPE1 derived aneuploid cell lines compared to the diploid cell lines.Click here for fileDataset 2 Data derived from the 2- dimensional annotation analysis.Click here for file


The non-long terminal repeat (non-LTR) retrotransposons, long interspersed element-1 (LINE-1) and Alu are currently active retroelements in humans. We, and others, have observed that different populations of HeLa cells from different laboratories support retrotransposition of LINE-1 and Alu to varying degrees. We therefore tested whether individual cell clones of HeLa and HCT116 cell lines supported different levels of LINE-1 and Alu retrotransposition, and whether these variations were stable upon re-cloning.Standard retrotransposition tissue culture assays were used to measure a cell’s ability to support LINE-1 and Alu retrotransposition in clonal HeLa and HCT116 cell lines. We observed that both LINE-1 and Alu retrotransposition exhibited clonal variation in HeLa cells, with certain HeLa cell clones supporting high levels of LINE-1 and Alu retrotransposition and other cell clones being essentially retrotransposition-dead. This clonal variation was similarly observed in HCT116 cells, although possibly not to the same extent. These patterns of clonal variation are relatively consistent upon re-cloning.Observations of the variability of LINE-1 and Alu retrotransposition in different populations of the same cell line are supported by our results that indicate in some cell types, individual cell clones can have dramatically differing capacity for retrotransposition. The mixed populations of cells commonly used in laboratories have often been passaged for many generations and accumulated significant genetic and epigenetic diversity. Our results suggest that the clonal variability observed by our cloning experiments may lead to a homogenization of retrotransposition capacity, with the resulting mixed population of cells being composed of individual variants having either increased or decreased retrotransposition potential compared to the starting population. Long interspersed element-1 (LINE-1) and Alu retrotransposons make up nearly one half of the DNA content of the human genome -4. In orHeLa and HCT116 cells were obtained from ATCC . HeLa and HCT116 cells were maintained in minimum essential medium (MEM)  supplemented with 10% fetal bovine serum , non-essential amino acids and sodium pyruvate (Invitrogen). HeLa and HCT116 cell lines were cloned by limiting dilution in a 96-well plate format and multiple clones were tested for LINE-1 and Alu retrotransposition using previously described reporter assays .2 flask. One day after seeding, cells were transfected using Lipofectamine (Invitrogen) according to the manufacturer’s protocol, with either 1 μg JM101/L1.3 [TET[Briefly, one million cells were seeded per 75 cmL1.3 [TET  crystal violet solution and counted using an automated cell counter . Retrotransposition colony counts were normalized to pIRES colonies for each experiment to correct for differences in transfection efficiency. For each independent experiment, the clone with the highest transfection efficiency, as determined by pIRES colony number, was set as 100% and each other clone was adjusted by the percentage that that clone differed from the clone with the highest transfection efficiency for that experiment. The mean number of pIRES colonies across all experiments was 1,050 (SD = 332). Data are presented for LINE-1 and Alu retrotransposition. Statistical significance was performed using GraphPad Prism software  and one-way ANOVA with Tukey’s post-test to compare means.Because many cancer cell lines have relatively unstable genomes, we hypothesized that their genome variation could lead to altered abilities to support retrotransposition. To determine whether there was clonal variability in the potential for HeLa clones to support LINE-1 retrotransposition, we performed LINE-1 retrotransposition assays using eight individual HeLa cell clones and the parental population of HeLa from which they originated. The results of the LINE-1 retrotransposition assays in HeLa clones confirm that some cell clones are nearly incapable of supporting LINE-1 retrotransposition (HeLa clone 1), while other cell clones are particularly amenable to LINE-1 retrotransposition events (HeLa clone 7). LINE-1 retrotransposition rates in HeLa clone 7 (mean = 699 colonies) were significantly increased compared to those in HeLa clone 1 (mean = 5 colonies)  was significantly elevated (252-fold) compared to the ability of HeLa clone 1 to support Alu retrotransposition (mean = 1 colony). Additionally, remaining HeLa subclones were fairly consistent in their ability to retrotranspose Alu, showing fairly modest rates of retrotransposition. None of the individual HeLa clones supported Alu retrotransposition as well as the parental population, suggesting that there was even more heterogeneity that was not sampled in this study  to obtain HeLa subclones 1A, 1B, 1C, 1D and 7A, 7B, 7C and 7D. We also subcloned two HCT116 clones (clones 5 and 6) to obtain HCT116 subclones 5A, 5B, 5C and 6A, 6B and 6C. We then performed the same LINE-1 retrotransposition assay as above on the HeLa and HCT116 subclones and the parental populations of cells. The LINE-1 retrotransposition differences seen in the re-cloned HeLa clones  was consistent with the observed difference in these two clones prior to re-cloning  andANOVA: Analysis of variance; FBS: Fetal bovine serum; LINE-1: Long interspersed element-1; non-LTR: Non-long terminal repeat; MEM: Minimum essential medium; SD: Standard deviation.The authors declare that they have no competing interests.VAS and ZJF designed and performed experiments. VAS wrote the manuscript. PLD conceived and designed experiments and edited the manuscript. All authors read and approved the final manuscript.Representative flask images of HeLa and HCT116 clones. (A) Alu and LINE-1 retrotransposition and pIRES colony formation in HeLa clone 7. (B) Alu and LINE-1 retrotransposition and pIRES colony formation in HeLa clone 1. (C) Alu and LINE-1 retrotransposition and pIRES colony formation in HCT116 clone 7. (D) Alu and LINE-1 retrotransposition and pIRES colony formation in HCT116 clone 3.Click here for file