Development of taxane resistance in a panel of human lung cancer cell lines

Using a selection process designed to reflect clinically relevant conditions, a panel of taxane-selected variants were developed to study further the mechanisms of resistance in lung cancer. Unlike continuous or pulse exposure to high concentrations of chemotherapeutic drugs which yield high resistance and often cross resistance, most variants developed here displayed low level resistance to the selecting drug with slight cross-resistance. Pulsing with taxol resulted in more highly resistant clones (up to 51.4-fold). Analysis of taxol and taxotere in the four major lung cancer cell types showed the taxanes to be more effective against NSCLC (with the exception of SKMES-taxane selected variants) than against the SCLC. Comparison of taxol and taxotere shows that taxol induces higher levels of resistance than taxotere. Further, in taxotere-selected cell lines, the cells are more resistant to taxol than taxotere, suggesting that taxotere may be a superior taxane from a clinical view. Taxol treatment resulted in increased cross-resistance to 5-FU in all classes of lung cancer except DMS-53. The high levels of Pgp in the DMS-53 and selected variant suggests this mechanism is not related to Pgp expression. Analysis of the Pgp and MRP-1 status by combination inhibitory assays and Western blotting showed no consistent relationship between expression of the membrane pumps Pgp or MRP-1 and resistance. However, where high level resistance was seen, the parent cell line expressed Pgp or MRP-1 and was accompanied by increased levels in the variants. Overall we found that the clinically relevant models used here are useful for investigating mechanisms of taxane resistance

Rolling cycle translation of circularized infinite open reading frames; fooling the ribosome

Poster Number 156 ROLLING CYCLE TRANSLATION OF CIRCULARIZED INFINITE OPEN READING FRAMES; FOOLING THE RIBOSOME Alan Costello, National Institute for Cellular Biotechnology, Dublin City University [email protected] Nga Lao, National Institute for Cellular Biotechnology, Dublin City University Niall Barron, National Institute for Bioprocessing Research and Training, University College Dublin Martin Clynes, National Institute for Cellular Biotechnology, Dublin City University Key Words: Circular RNA, Translation, RNA structure, Cell engineering, Chinese Hamster Ovary (CHO) cell Recent. Please click Additional Files below to see the full abstract

Differential protein expression following low temperature culture of suspension CHO-K1 cells

<p>Abstract</p> <p>Background</p> <p>To ensure maximal productivity of recombinant proteins (rP) during production culture it is typical to encourage an initial phase of rapid cell proliferation to achieve high biomass followed by a stationary phase where cellular energies are directed towards production of rP. During many such biphasic cultures, the initial phase of rapid cell growth at 37°C is followed by a growth arrest phase induced through reduction of the culture temperature. Low temperature induced growth arrest is associated with many positive phenotypes including increased productivity, sustained viability and an extended production phase, although the mechanisms regulating these phenotypes during mild hypothermia are poorly understood.</p> <p>Results</p> <p>In this study differential protein expression in suspension CHO-K1 cells was investigated following a reduction of the culture temperature from 37°C to 31°C in comparison to standard batch culture maintained at 37°C using 2D-DIGE (Fluorescence 2-D Difference Gel Electrophoresis) and mass spectrometry (MS). There is only limited proteomic analysis of suspension-grown CHO cells describing a direct comparison of temperature shifted versus non-temperature shifted cultures using 2D-DIGE. This investigation has enabled the identification of temperature-dependent as well as temperature-independent proteomic changes. 201 proteins were observed as differentially expressed following temperature shift, of which 118 were up regulated. Of the 53 proteins identified by MALDI-ToF MS, 23 were specifically differentially expressed upon reduction of the culture temperature and were found related to a variety of cellular functions such as regulation of growth (HNRPC), cap-independent translation (EIF4A), apoptosis (importin-α), the cytoskeleton (vimentin) and glycoprotein quality control (alpha glucosidase 2).</p> <p>Conclusion</p> <p>These results indicate the extent of the temperature response in CHO-K1 cells and suggest a number of key regulatory proteins and pathways that are involved in modulating the response of cells to mild hypothermia. Regulation of these identified proteins and pathways could be useful for future approaches to engineer CHO cells for improved recombinant protein production.</p

Bromodeoxyuridine induces keratin protein synthesis at a posttranscriptional level in human lung tumour cell lines

Keratin intermediate filaments are formed in epithelial cells in a cell- and tissue-specific manner, but much remains unknown regarding the mechanisms which control the synthesis of these proteins. We examined the effect of the differentiation modulation agent, bromodeoxyuridine (BrdU), on two human keratin-negative (by immunocytochemistry) lung cell lines, DLKP and H82, and showed immunohistochemically that treatment with 10 µM BrdU over 7 days induced K8 and K18 protein synthesis in both lines. Immunoprecipitation and Western blot analyses revealed low levels of K8 and K18 proteins in untreated cell homogenates. These levels increased following treatment with BrdU for 7 days. K8 and K18 mRNAs were detected by Northern blot and reverse transcriptase polymerase chain reaction analyses in both lines before BrdU treatment, but no increase in mRNA levels was observed in either cell line over 21 days of treatment. This suggests, firstly, that keratin synthesis is normally blocked at a posttranscriptional level in DLKP and H82 cells, and secondly, that BrdU can reverse this block. A549 is a human lung cell line which contains K8 and K18 proteins. Treatment with BrdU increased K8 and K18 protein levels in these cells. No corresponding increase in K8 mRNA levels occurred, while an apparent increase in K18 mRNA levels was detected. HL-60 is a leukaemic cell line of haematopoietic rather than epithelial lineage which contains K8 and K18 mRNA transcripts prior to BrdU treatment, but does not contain keratin proteins. Again, K8 and K18 mRNA levels remained unchanged during BrdU treatment. However, neither K8 nor K18 proteins were detected following treatment, although BrdU is known to alter expression of other genes in HL-60 cells. BrdU thus appears to act at a posttranscriptional level and in an epithelial-specific manner to reverse a block in keratin synthesis in keratinnegative lung cancer cells and increase synthesis in keratin-positive lung cancer cells. This may represent a regulatory step in early lung development or a mechanism whereby tumour cells downregulate expression of a differentiated phenotype

Bromodeoxyuridine induces keratin protein synthesis at a posttranscriptional level in human lung tumour cell lines

Keratin intermediate filaments are formed in epithelial cells in a cell- and tissue-specific manner, but much remains unknown regarding the mechanisms which control the synthesis of these proteins. We examined the effect of the differentiation modulation agent, bromodeoxyuridine (BrdU), on two human keratin-negative (by immunocytochemistry) lung cell lines, DLKP and H82, and showed immunohistochemically that treatment with 10 µM BrdU over 7 days induced K8 and K18 protein synthesis in both lines. Immunoprecipitation and Western blot analyses revealed low levels of K8 and K18 proteins in untreated cell homogenates. These levels increased following treatment with BrdU for 7 days. K8 and K18 mRNAs were detected by Northern blot and reverse transcriptase polymerase chain reaction analyses in both lines before BrdU treatment, but no increase in mRNA levels was observed in either cell line over 21 days of treatment. This suggests, firstly, that keratin synthesis is normally blocked at a posttranscriptional level in DLKP and H82 cells, and secondly, that BrdU can reverse this block. A549 is a human lung cell line which contains K8 and K18 proteins. Treatment with BrdU increased K8 and K18 protein levels in these cells. No corresponding increase in K8 mRNA levels occurred, while an apparent increase in K18 mRNA levels was detected. HL-60 is a leukaemic cell line of haematopoietic rather than epithelial lineage which contains K8 and K18 mRNA transcripts prior to BrdU treatment, but does not contain keratin proteins. Again, K8 and K18 mRNA levels remained unchanged during BrdU treatment. However, neither K8 nor K18 proteins were detected following treatment, although BrdU is known to alter expression of other genes in HL-60 cells. BrdU thus appears to act at a posttranscriptional level and in an epithelial-specific manner to reverse a block in keratin synthesis in keratinnegative lung cancer cells and increase synthesis in keratin-positive lung cancer cells. This may represent a regulatory step in early lung development or a mechanism whereby tumour cells downregulate expression of a differentiated phenotype

Demodex-Associated Bacillus Proteins Induce an Aberrant Wound Healing Response in a Corneal Epithelial Cell Line: Possible Implications for Corneal Ulcer Formation in Ocular Rosacea

PURPOSE. The aim of the work presented here was to establish the response of a corneal epithelial cell line (hTCEpi) to protein extracted from a bacterium (Bacillus oleronius) previously isolated from a Demodex mite from a rosacea patient. METHODS. The response of the corneal epithelial cell line to Bacillus proteins was measured in terms of alterations in cell migration and invasiveness. Changes in the expression of metalloproteinase genes and proteins were also assessed. RESULTS. The results indicated increased cell migration (14.5- fold, P ¼ 0.001) as measured using 8-lm PET inserts (BD Falcon) in a transwell assay and invasiveness (1.7-fold, P ¼ 0.003) as measured using 8-lm Matrigel (BD Biocoat) invasion inserts in a 24-well plate assay format, following exposure to the Bacillus proteins. Cells exposed to the Bacillus protein showed a dose-dependent increase in expression of genes coding for matrix metalloprotease (MMP)-3 (61-fold) and MPP-9 (301-fold). This dose-dependent increase in gene expression was also reflected in elevated levels of MMP-9 protein (1.34- fold, P ¼ 0.033) and increased matrix metalloprotease activity (1.96-fold, P¼0.043) being present in the culture supernatant. Cells also displayed reduced levels of b-integrin (1.25-fold, P ¼ 0.01), indicative of increased motility and elevated levels of vinculin (2.7-fold, P ¼ 0.0009), suggesting altered motility. CONCLUSIONS. The results indicate that exposure of corneal epithelial cells to Bacillus proteins results in an aberrant wound healing response as visualized using a scratch wound assay. These results suggest a possible link between the high density of Demodex mites on the eyelashes of ocular rosacea patients and the development of corneal ulcers. (Invest Ophthalmol Vis Sci. 2012;53:3250–3259) DOI:10.1167/ iovs.11-929

Demodex-Associated Bacillus Proteins Induce an Aberrant Wound Healing Response in a Corneal Epithelial Cell Line: Possible Implications for Corneal Ulcer Formation in Ocular Rosacea

PURPOSE. The aim of the work presented here was to establish the response of a corneal epithelial cell line (hTCEpi) to protein extracted from a bacterium (Bacillus oleronius) previously isolated from a Demodex mite from a rosacea patient. METHODS. The response of the corneal epithelial cell line to Bacillus proteins was measured in terms of alterations in cell migration and invasiveness. Changes in the expression of metalloproteinase genes and proteins were also assessed. RESULTS. The results indicated increased cell migration (14.5- fold, P ¼ 0.001) as measured using 8-lm PET inserts (BD Falcon) in a transwell assay and invasiveness (1.7-fold, P ¼ 0.003) as measured using 8-lm Matrigel (BD Biocoat) invasion inserts in a 24-well plate assay format, following exposure to the Bacillus proteins. Cells exposed to the Bacillus protein showed a dose-dependent increase in expression of genes coding for matrix metalloprotease (MMP)-3 (61-fold) and MPP-9 (301-fold). This dose-dependent increase in gene expression was also reflected in elevated levels of MMP-9 protein (1.34- fold, P ¼ 0.033) and increased matrix metalloprotease activity (1.96-fold, P¼0.043) being present in the culture supernatant. Cells also displayed reduced levels of b-integrin (1.25-fold, P ¼ 0.01), indicative of increased motility and elevated levels of vinculin (2.7-fold, P ¼ 0.0009), suggesting altered motility. CONCLUSIONS. The results indicate that exposure of corneal epithelial cells to Bacillus proteins results in an aberrant wound healing response as visualized using a scratch wound assay. These results suggest a possible link between the high density of Demodex mites on the eyelashes of ocular rosacea patients and the development of corneal ulcers. (Invest Ophthalmol Vis Sci. 2012;53:3250–3259) DOI:10.1167/ iovs.11-929

miR-CATCH identifies biologically active miRNA regulators of the pro-survival gene XIAP, in Chinese hamster ovary cells

Genetic engineering of mammalian cells, in particular Chinese hamster ovary (CHO) cells, is of critical interest to the biopharmaceutical industry as a means to further boost the yields of therapeutic proteins. Complimentary to already in place advanced bioprocesses, stable overexpression of the pro-survival X-linked inhibitor of apoptosis (XIAP) is one example of the successful manipulation of CHO cell genetics resulting in prolonged culture survival, ultimately increasing recombinant protein productivity. However, saturation or burdening of the cells translational machinery can occur in instances of forced expression of a trans-gene thereby achieving the anticipated cellular phenotype without the associated improvement in productivity. Ribosomal footprint sequencing has demonstrated that ~15% of an IgG-producing CHO cell translatome is occupied by the Neomycin selection marker. microRNAs (miRNAs) have the ability to fine tune endogenous gene expression thereby achieving elevated gene levels without the excess that could negatively impact global gene expression. Additionally, not only does a single miRNA have the capacity to regulate multiple mRNA transcripts simultaneously but individual mRNAs can be regulated by a multitude of miRNAs at the post-transcriptional level. This can facilitate the maximal translation of an endogenous gene without surpassing the superphysiological threshold associated with diminished productivity. The promiscuous nature of miRNA represented by the variety of binding patterns associated with mRNA targeting limits the predictability of high confidence miRNA regulators of attractive engineering candidates. This results in a lengthy list of falsely predicted in-silico miRNA regulators for a single gene. We explored the identification of direct miRNA regulators of the pro-survival endogenous XIAP gene in CHO-K1 cells by using a miR-CATCH1 protocol. A biotin-tagged antisense DNA oligonucleotide was designed for an exposed predicted secondary structure loop of endogenous CHO XIAP. This mRNA anchor resulted in the pulldown of XIAP and all associated RNA/protein complexes thereby enriching for all bound miRNAs. Two miRNAs were chosen out of the 14 miRNAs identified for further validation, miR-124-3p and miR-19b-3p. Transient transfection of mimics for both resulted in the diminished translation of endogenous CHO XIAP protein whereas their inhibition increased XIAP protein levels (Fig. 1). Please click Additional Files below to see the full abstract

Purification and Identification of a 7.6-kDa Protein in Media Conditioned by Superinvasive Cancer Cells

Background: Selection of the human drug sensitive and invasive cell line (MDA-MB-435S-F) with the chemotherapeutic agent paclitaxel, resulted in the development of drug resistant cell lines displaying enhanced invasion-related characteristics. Materials and Methods: Serum-free conditioned media from the human cancer drug-sensitive and invasive cell line (MDA-MB-435S-F) and its paclitaxel-resistant superinvasive variant (MDA-MB-435S-F/Taxol10p4pSI) were analyzed using Surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). Results: A differentially expressed protein was observed at 7.6 kDa, which was 4-fold upregulated in MDA-MB-435S-F/Taxol10p4pSI. The differentially expressed protein was identified using matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF MS), as a fragment of bovine transferrin. The transferrin receptor was also found to be overexpressed in the superinvasive cell line. Conclusion: Cleavage of serum proteins such as transferrin could provide a valuable source of markers for malignant tumours and could also play a role in aspects of cancer pathogenesis, such as tumour cachexia

Identification of pancreatic cancer invasion-related proteins by proteomic analysis

Background – Markers of pancreatic cancer invasion were investigated in two clonal populations of the cell line, MiaPaCa-2, Clone #3 (high invasion) and Clone #8 (low invasion) using proteomic profiling of an in vitro model of pancreatic cancer. Materials and methods – Using 2D-DIGE followed by MALDI-TOF MS, two clonal sub-populations of the pancreatic cancer cell line, MiaPaCa-2 with high and low invasive capacities were incubated on matrigel 24 hours prior to analysis to stimulate cell-ECM contact and mimic in vivo interaction with the basement membrane. Results - Sixty proteins were identified as being differentially expressed (>1.2 fold change and p ≤ 0.05) between Clone #3 and Clone #8. Proteins found to have higher abundance levels in the highly invasive Clone #3 compared to the low invasive Clone #8 include members of the chaperone activity proteins and cytoskeleton constituents whereas metabolism-associated and catalytic proteins had lower abundance levels. Differential protein expression levels of ALDH1A1, VIM, STIP1 and KRT18 and GAPDH were confirmed by immunoblot. Using RNAi technology, STIP1 knockdown significantly reduced invasion and proliferation of the highly invasive Clone #3. Knockdown of another target, VIM by siRNA in Clone #3 cells also resulted in decreased invasion abilities of Clone #3. Elevated expression of STIP1 was observed in pancreatic tumour tissue compared to normal pancreas, whereas ALDH1A1 stained at lower levels in pancreatic tumours, as detected by immunohistochemistry. Conclusion - Identification of targets which play a role in the highly invasive phenotype of pancreatic cancer may help to understand the biological behaviour, the rapid progression of this cancer and may be of importance in the development of new therapeutic strategies for pancreatic cancer