miRNA- and Cell Line-Specific Constraints on Precursor miRNA Processing of Stably Transfected Pancreatic Cancer and Other Mammalian Cells.

MicroRNAs (miRNAs) regulate approximately one-third of all human genes. The dysregulation of miRNAs has been implicated in the development of numerous human diseases, including cancers. In our investigation focusing on altering specific miRNA expression in human pancreatic cancer cells, we encountered an interesting finding. While two expression vector designs effectively enhanced miR-708 levels, they were unable to elevate mature forms of miR-29b, -1290, -2467, and -6831 in pancreatic cancer cell lines. This finding was also observed in a panel of other non-pancreatic cancer cell lines, suggesting that miRNA processing efficiency was cell line specific. Using a step-by-step approach in each step of miRNA processing, we ruled out alternative strand selection by the RISC complex and transcriptional interference at the primary miRNA (pri-miRNA) level. DROSHA processing and pri-miRNA export from the nucleus also appeared to be occurring normally. We observed precursor (pre-miRNA) accumulation only in cell lines where mature miRNA expression was not achieved, suggesting that the block was occurring at the pre-miRNA stage. To further confirm this, synthetic pre-miRNA mimics that bypass DICER processing were processed into mature miRNAs in all cases. This study has demonstrated the distinct behaviours of different miRNAs with the same vector in the same cell line, the same miRNA between the two vector designs, and with the same miRNA across different cell lines. We identified a stable vector pre-miRNA processing block. Our findings on the structural and sequence differences between successful and non-successful vector designs could help to inform future chimeric miRNA design strategies and act as a guide to other researchers on the intricate processing dynamics that can impact vector efficiency. Our research confirms the potential of miRNA mimics to surmount some of these complexities.

Dual-Hit Strategy for Therapeutic Targeting of Pancreatic Cancer in Patient-Derived Xenograft Tumors.

PURPOSE: Paracrine activation of pro-fibrotic hedgehog (HH) signaling in pancreatic ductal adenocarcinoma (PDAC) results in stromal amplification that compromises tumor drug delivery, efficacy, and patient survival. Interdiction of HH-mediated tumor-stroma crosstalk with smoothened (SMO) inhibitors (SHHi) "primes" PDAC patient-derived xenograft (PDX) tumors for increased drug delivery by transiently increasing vascular patency/permeability, and thereby macromolecule delivery. However, patient tumor isolates vary in their responsiveness, and responders show co-induction of epithelial-mesenchymal transition (EMT). We aimed to identify the signal derangements responsible for EMT induction and reverse them and devise approaches to stratify SHHi-responsive tumors noninvasively based on clinically-quantifiable parameters. EXPERIMENTAL DESIGN: Animals underwent diffusion-weighted magnetic resonance (DW-MR) imaging for measurement of intratumor diffusivity. In parallel, tissue-level deposition of nanoparticle probes was quantified as a marker of vascular permeability/perfusion. Transcriptomic and bioinformatic analysis was employed to investigate SHHi-induced gene reprogramming and identify key "nodes" responsible for EMT induction. RESULTS: Multiple patient tumor isolates responded to short-term SHH inhibitor exposure with increased vascular patency and permeability, with proportionate increases in tumor diffusivity. Nonresponding PDXs did not. SHHi-treated tumors showed elevated FGF drive and distinctly higher nuclear localization of fibroblast growth factor receptor (FGFR1) in EMT-polarized tumor cells. Pan-FGFR inhibitor NVP-BGJ398 (Infigratinib) reversed the SHHi-induced EMT marker expression and nuclear FGFR1 accumulation without compromising the enhanced permeability effect. CONCLUSIONS: This dual-hit strategy of SMO and FGFR inhibition provides a clinically-translatable approach to compromise the profound impermeability of PDAC tumors. Furthermore, clinical deployment of DW-MR imaging could fulfill the essential clinical-translational requirement for patient stratification.

Alteration in Levels of Specific miRNAs and Their Potential Protein Targets between Human Pancreatic Cancer Samples, Adjacent Normal Tissue, and Xenografts Derived from These Tumors.

Herein, we describe the global comparison of miRNAs in human pancreatic cancer tumors, adjacent normal tissue, and matched patient-derived xenograft models using microarray screening. RNA was extracted from seven tumor, five adjacent normal, and eight FI PDX tumor samples and analyzed by Affymetrix GeneChip miRNA 4.0 array. A transcriptome analysis console (TAC) was used to generate comparative lists of up- and downregulated miRNAs for the comparisons, tumor vs. normal and F1 PDX vs. tumor. Particular attention was paid to miRNAs that were changed in the same direction in both comparisons. We identified the involvement in pancreatic tumor tissue of several miRNAs, including miR4534, miR3154, and miR4742, not previously highlighted as being involved in this type of cancer. Investigation in the parallel mRNA and protein lists from the same samples allowed the elimination of proteins where altered expression correlated with corresponding mRNA levels and was thus less likely to be miRNA regulated. Using the remaining differential expression protein lists for proteins predicted to be targeted for differentially expressed miRNA on our list, we were able to tentatively ascribe specific protein changes to individual miRNA. Particularly interesting target proteins for miRs 615-3p, 2467-3p, 4742-5p, 509-5p, and 605-3p were identified. Prominent among the protein targets are enzymes involved in aldehyde metabolism and membrane transport and trafficking. These results may help to uncover vulnerabilities that could enable novel approaches to treating pancreatic cancer.

FOLFIRINOX Pharmacodynamic Interactions in 2D and 3D Pancreatic Cancer Cell Cultures.

The multi-drug combination regime, FOLFIRINOX, is a standard of care chemotherapeutic therapy for pancreatic cancer patients. However, systematic evaluation of potential pharmacodynamic interactions among multi-drug therapy has not been reported previously. Here, pharmacodynamic interactions of the FOLFIRINOX agents (5-fluorouracil (5-FU), oxaliplatin (Oxa) and SN-38, the active metabolite of irinotecan) were assessed across a panel of primary and established pancreatic cancer cells. Inhibition of cell proliferation was quantified for each drug, alone and in combination, to obtain quantitative, drug-specific interaction parameters and assess the nature of drug interactions. The experimental data were analysed assuming Bliss independent interactions, and nonlinear regression model fitting was conducted in SAS. Estimates of the drug interaction term, psi (ψ), revealed that the Oxa/SN-38 combination appeared synergistic in PANC-1 (ψ = 0.6, 95% CI = 0.4, 0.9) and modestly synergistic, close to additive, in MIAPaCa-2 (ψ = 0.8, 95% CI = 0.6, 1.0) in 2D assays. The triple combination was strongly synergistic in MIAPaCa-2 (ψ = 0.2, 95% CI = 0.1, 0.3) and modestly synergistic/borderline additive in PANC-1 2D (ψ = 0.8, 95% CI = 0.6, 1.0). The triple combination showed antagonistic interactions in the primary PIN-127 and 3D PANC-1 model (ψ > 1). Quantitative pharmacodynamic interactions have not been described for the FOLFIRINOX regimen; this analysis suggests a complex interplay among the three chemotherapeutic agents. Extension of this pharmacodynamic analysis approach to clinical/translational studies of the FOLFIRINOX combination could reveal additional pharmacodynamic interactions and guide further refinement of this regimen to achieve optimal clinical responses.

Label-Free Quantitative Proteomics Analysis of Adriamycin Selected Multidrug Resistant Human Lung Cancer Cells.

The development of drug resistance in lung cancer is a major clinical challenge, leading to a 5-year survival rate of only 18%. Therefore, unravelling the mechanisms of drug resistance and developing novel therapeutic strategies is of crucial importance. This study systematically explores the novel biomarkers of drug resistance using a lung cancer model (DLKP) with a series of drug-resistant variants. In-depth label-free quantitative mass spectrometry-based proteomics and gene ontology analysis shows that parental DLKP cells significantly differ from drug-resistant variants, and the cellular proteome changes even among the drug-resistant subpopulations. Overall, ABC transporter proteins and lipid metabolism were determined to play a significant role in the formation of drug resistance in DKLP cells. A series of membrane-related proteins such as HMOX1, TMB1, EPHX2 and NEU1 were identified to be correlated with levels of drug resistance in the DLKP subpopulations. The study also showed enrichment in biological processes and molecular functions such as drug metabolism, cellular response to the drug and drug binding. In gene ontology analysis, 18 proteins were determined to be positively or negatively correlated with resistance levels. Overall, 34 proteins which potentially have a therapeutic and diagnostic value were identified.

Yeast Mannan-Rich Fraction Modulates Endogenous Reactive Oxygen Species Generation and Antibiotic Sensitivity in Resistant E. coli.

Mannan-rich fraction (MRF) isolated from Saccharomyces cerevisiae has been studied for its beneficial impact on animal intestinal health. Herein, we examined how MRF affected the formation of reactive oxygen species (ROS), impacting antibiotic susceptibility in resistant Escherichia coli through the modulation of bacterial metabolism. The role of MRF in effecting proteomic change was examined using a proteomics-based approach. The results showed that MRF, when combined with bactericidal antibiotic treatment, increased ROS production in resistant E. coli by 59.29 ± 4.03% compared to the control (p ≤ 0.05). We further examined the effect of MRF alone and in combination with antibiotic treatment on E. coli growth and explored how MRF potentiates bacterial susceptibility to antibiotics via proteomic changes in key metabolic pathways. Herein we demonstrated that MRF supplementation in the growth media of ampicillin-resistant E. coli had a significant impact on the normal translational control of the central metabolic pathways, including those involved in the glycolysis-TCA cycle (p ≤ 0.05).

Mapping the molecular basis for growth related phenotypes in industrial producer CHO cell lines using differential proteomic analysis.

BACKGROUND: The ability to achieve high peak viable cell density earlier in CHO cell culture and maintain an extended cell viability throughout the production process is highly desirable to increase recombinant protein yields, reduce host cell impurities for downstream processing and reduce the cost of goods. In this study we implemented label-free LC-MS/MS proteomic profiling of IgG4 producing CHO cell lines throughout the duration of the cell culture to identify differentially expressed (DE) proteins and intracellular pathways associated with the high peak viable cell density (VCD) and extended culture VCD phenotypes. RESULTS: We identified key pathways in DNA replication, mitotic cell cycle and evasion of p53 mediated apoptosis in high peak VCD clonally derived cell lines (CDCLs). ER to Golgi vesicle mediated transport was found to be highly expressed in extended culture VCD CDCLs while networks involving endocytosis and oxidative stress response were significantly downregulated. CONCLUSION: This investigation highlights key pathways for targeted engineering to generate desirable CHO cell phenotypes for biotherapeutic production.

Differential expression of miRNAs and functional role of mir-200a in high and low productivity CHO cells expressing an Fc fusion protein.

OBJECTIVES: We used miRNA and proteomic profiling to understand intracellular pathways that contribute to high and low specific productivity (Qp) phenotypes in CHO clonally derived cell lines (CDCLs) from the same cell line generation project. RESULTS: Differentially expressed (DE) miRNAs were identified which are predicted to target several proteins associated with protein folding. MiR-200a was found to have a number of predicted targets associated with the unfolded protein response (UPR) which were shown to have decreased expression in high Qp CDCLs and have no detected change at the mRNA level. MiR-200a overexpression in a CHO CDCL was found to increase recombinant protein titer by 1.2 fold and Qp by 1.8 fold. CONCLUSION: These results may suggest a role for miR-200a in post-transcriptional regulation of the UPR, presenting miR-200a as a potential target for engineering industrially attractive CHO cell phenotypes.

Transfection of miR-31* boosts oxidative phosphorylation metabolism in the mitochondria and enhances recombinant protein production in Chinese hamster ovary cells.

MicroRNAs are increasingly being used to enhance relevant pathways of interest during CHO cell line development and to optimise biopharmaceutical production processes. Previous studies have demonstrated that genetic manipulation of microRNAs has led to the development of highly productive phenotypes by increasing cell density through modifying the cell cycle, extending the culture lifespan by delaying apoptotic mechanisms, or improving the energetic flux by targeting mitochondrial metabolism. Re-programming mitochondrial metabolism has arisen as a potential area of interest due to the potential to decrease the Warburg effect and increase cell specific productivity with significant impact on the manufacture of recombinant therapeutic proteins. In this study, we have demonstrated a role for miR-31* to enhance specific productivity in CHO cells by boosting oxidative phosphorylation in the mitochondria. A detailed analysis of the mitochondrial metabolism revealed that miR-31* transfection increases basal oxygen consumption and spare respiratory capacity that leads to an increase in ATP production. Additionally, a proteomic analysis unveiled a number of potential targets involved in fatty acid metabolism and the TCA cycle, both implicated in mitochondrial metabolism. This data demonstrates a potential role for miR-31* to reprogramme the mitochondrial energetic metabolism and increase recombinant protein production in CHO cells.

The emerging role of cellular post-translational modifications in modulating growth and productivity of recombinant Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells are one of the most commonly used host cell lines used for the production human therapeutic proteins. Much research over the past two decades has focussed on improving the growth, titre and cell specific productivity of CHO cells and in turn lowering the costs associated with production of recombinant proteins. CHO cell engineering has become of particular interest in recent years following the publication of the CHO cell genome and the availability of data relating to the proteome, transcriptome and metabolome of CHO cells. However, data relating to the cellular post-translational modification (PTMs) which can affect the functionality of CHO cellular proteins has only begun to be presented in recent years. PTMs are important to many cellular processes and can further alter proteins by increasing the complexity of proteins and their interactions. In this review, we describe the research presented from CHO cells to date related on three of the most important PTMs; glycosylation, phosphorylation and ubiquitination.

Copper toxicity of inflection point in human intestinal cell line Caco-2 dissected: influence of temporal expression patterns.

We previously described a non-monotonic dose response curve at low copper concentrations where 3.125 µM CuSO4 (the early inflection point) was more toxic than 25 µM CuSO4 in Caco-2 cells. We employed global proteomics to investigate this observation. The altered expression levels of a small number of proteins displaying a temporal response may provide the best indication of the underlying mechanism; more well-known copper response proteins including the metal binding metallothioneins (MT1X, MT1F, MT2A) and antioxidant response proteins including Heme oxygenase were upregulated to a similar level in both copper concentrations and so are less likely to underpin this phenomenon.The temporal response proteins include Granulins, AN1-type zinc finger protein 2A (ZFAND2A), and the heat shock proteins (HSPA6 and HSPA1B). Granulins were decreased after 4 h only in 25 µM CuSO4 but from 24 h, were decreased in both copper concentrations to a similar level. Induction of ZFAND2A and increases in HSPA6 and HSPA1B were observed at 24 h only in 25 µM CuSO4 but were present at 48 h in both copper conditions. The early expression of ZFAND2A, HSPs, and higher levels of α-crystallin B (CRYAB) correlated with lower levels of misfolded proteins in 25 µM CuSO4 compared to 3.125 µM CuSO4 at 48 h. These results suggest that 3.125 µM CuSO4 at early time points was unable to activate the plethora of stress responses invoked by the higher copper concentration, paradoxically exposing the Caco-2 cells to higher levels of misfolded proteins and greater proteotoxic stress.

Polypyridyl-Based Copper Phenanthrene Complexes: Combining Stability with Enhanced DNA Recognition.

We report a series of copper(II) artificial metallo-nucleases (AMNs) and demonstrate their DNA damaging properties and in-vitro cytotoxicity against human-derived pancreatic cancer cells. The compounds combine a tris-chelating polypyridyl ligand, di-(2-pycolyl)amine (DPA), and a DNA intercalating phenanthrene unit. Their general formula is Cu-DPA-N,N' (where N,N'=1,10-phenanthroline (Phen), dipyridoquinoxaline (DPQ) or dipyridophenazine (DPPZ)). Characterisation was achieved by X-ray crystallography and continuous-wave EPR (cw-EPR), hyperfine sublevel correlation (HYSCORE) and Davies electron-nuclear double resonance (ENDOR) spectroscopies. The presence of the DPA ligand enhances solution stability and facilitates enhanced DNA recognition with apparent binding constants (Kapp ) rising from 105 to 107 m-1 with increasing extent of planar phenanthrene. Cu-DPA-DPPZ, the complex with greatest DNA binding and intercalation effects, recognises the minor groove of guanine-cytosine (G-C) rich sequences. Oxidative DNA damage also occurs in the minor groove and can be inhibited by superoxide and hydroxyl radical trapping agents. The complexes, particularly Cu-DPA-DPPZ, display promising anticancer activity against human pancreatic tumour cells with in-vitro results surpassing the clinical platinum(II) drug oxaliplatin.

Development of whole-cell and cell-free biosensors for the detection and differentiation of organic and inorganic forms of copper.

The modern world has seen exposure of bacterial communities to toxic metals at selective levels. This manifests itself both intentionally, through medicines and un-intentionally through waste streams. There is growing concern that selective exposure to metals may be linked to microbial resistance to antibiotics. For a microbe to become resistant to a specific metal it must first come in contact with it. The transition metal copper has the ability to enter bacterial cells without need for a copper specific uptake mechanism. Copper is commonly used as an antimicrobial in the healthcare industry, consumer products and as a growth promoter of livestock in the agricultural sector. Here we report a study into the uptake of different organic and inorganic sources of copper. A whole-cell bacterial biosensor was developed to quantify the specific uptake of copper from various sources. Furthermore, a cell-free sensor was utilized to investigate the response to copper sources when uptake is eliminated as a factor. The data within suggest inorganic copper to have greatly reduced uptake compared to organic sources and that there is significant difference between copper oxides, Cu2O and CuO.

Gene expression profiling of copper-resistant Caco-2 clones.

The Caco-2 cell line is composed of a heterogeneous mix of cells; isolation of individual clonal populations from this mix allows for specific mechanisms and phenotypes to be further explored. Previously we exposed Caco-2 cells to inorganic copper sulphate or organic copper proteinate to generate resistant variant populations. Here we describe the isolation and characterisation of clonal subpopulations from these resistant variants to organic (clone Or1, Or2, Or3) or inorganic (clone In1 and In2) copper. The clones show considerable homogeneity in response to Cu-induced toxicity and heterogeneity in morphology with variations in level of cross-resistance to other metals and doxorubicin. Population growth was reduced for Cu-resistant clones In2 and Or3 in selective pressure relative to parental Caco-2 cells. Gene expression analysis identified 4026 total (2102 unique and 1924 shared) differentially expressed genes including those involved in the MAP Kinase and Rap1 signalling pathways, and in the focal adhesion and ECM-receptor contact pathways. Gene expression changes common to all clones included upregulation of ANXA13 and GPx2. Our analysis additionally identified differential expression of multiple genes specific to copper proteinate exposure (including overexpressed UPK1B) in isolated clones Or1, Or2 and Or3 and CuSO4 exposure (including decreased AIFM2 expression) in isolated clones In1 and In2. The adaptive transcriptional responses established in this study indicate a cohort of genes, which may be involved in copper resistance regulation and chronic copper exposure.

Proteomic analysis of pancreatic ductal adenocarcinoma.

INTRODUCTION: Pancreatic ductal adenocarcinoma (PDAC), which represents approximately 80% of all pancreatic cancers, is a highly aggressive malignant disease and one of the most lethal among all cancers. Overall, the 5-year survival rate among all pancreatic cancer patients is less than 9%; these rates have shown little change over the past 30 years. A more comprehensive understanding of the molecular mechanisms underlying this complex disease is crucial to the development of new diagnostic tools for early detection and disease monitoring, as well as to identify new and more effective therapeutics to improve patient outcomes. AREA COVERED: We summarize recent advances in proteomic strategies and mass spectrometry to identify new biomarkers for early detection and monitoring of disease progression, predict response to therapy, and to identify novel proteins that have the potential to be 'druggable' therapeutic targets. An overview of proteomic studies that have been conducted to further our mechanistic understanding of metastasis and chemotherapy resistance in PDAC disease progression will also be discussed. EXPERT COMMENTARY: The results from these PDAC proteomic studies on a variety of PDAC sample types (e.g., blood, tissue, cell lines, exosomes, etc.) provide great promise of having a significant clinical impact and improving patient outcomes.

Proteomic Analysis of Cell Lines and Primary Tumors in Pancreatic Cancer Identifies Proteins Expressed Only In Vitro and Only In Vivo.

OBJECTIVES: A limited repertoire of good pancreatic ductal adenocarcinoma (PDAC) models is one of the main barriers in developing effective new PDAC treatments. We aimed to characterize 6 commonly used PDAC cell lines and compare them with PDAC patient tumor samples using proteomics. METHODS: Proteomic methods were used to generate an extensive catalog of proteins from 10 PDAC surgical specimens, 9 biopsies of adjacent normal tissue, and 6 PDAC cell lines. Protein lists were interrogated to determine what extent the proteome of the cell lines reflects the proteome of primary pancreatic tumors. RESULTS: We identified 7973 proteins from the cell lines, 5680 proteins from the tumor tissues, and 4943 proteins from the adjacent normal tissues. We identified 324 proteins unique to the cell lines, some of which may play a role in survival of cells in culture. Conversely, a list of 63 proteins expressed only in the patient samples, whose expression is lost in culture, may place limitations on the degree to which these model systems reflect tumor biology in vivo. CONCLUSIONS: Our work offers a catalog of proteins detected in each of the PDAC cell lines, providing a useful guide for researchers seeking model systems for PDAC functional studies.

DR5-targeted, chemotherapeutic drug-loaded nanoparticles induce apoptosis and tumor regression in pancreatic cancer in vivo models.

Pancreatic cancer is usually advanced and drug resistant at diagnosis. A potential therapeutic approach outlined here uses nanoparticle (NP)-based drug carriers, which have unique properties that enhance intra-tumor drug exposure and reduce systemic toxicity of encapsulated drugs. Here we report that patients whose pancreatic cancers express elevated levels of Death Receptor 5 (DR5) and its downstream regulators/effectors FLIP, Caspase-8, and FADD had particularly poor prognoses. To take advantage of elevated expression of this pathway, we designed drug-loaded NPs with a surface-conjugated αDR5 antibody (AMG 655). Binding and clustering of the DR5 is a prerequisite for efficient apoptosis initiation, and the αDR5-NPs were indeed found to activate apoptosis in multiple pancreatic cancer models, whereas the free antibody did not. The extent of apoptosis induced by αDR5-NPs was enhanced by down-regulating FLIP, a key modulator of death receptor-mediated activation of caspase-8. Moreover, the DNA topoisomerase-1 inhibitor camptothecin (CPT) down-regulated FLIP in pancreatic cancer models and enhanced apoptosis induced by αDR5-NPs. CPT-loaded αDR5-NPs significantly increased apoptosis and decreased cell viability in vitro in a caspase-8- and FADD-dependent manner consistent with their expected mechanism-of-action. Importantly, CPT-loaded αDR5-NPs markedly reduced tumor growth rates in vivo in established pancreatic tumor models, inducing regressions in one model. These proof-of-concept studies indicate that αDR5-NPs loaded with agents that downregulate or inhibit FLIP are promising candidate agents for the treatment of pancreatic cancer.

Subphysiological temperature induces pervasive alternative splicing in Chinese hamster ovary cells.

RNA sequencing (RNASeq) has been widely used to associate alterations in Chinese hamster ovary (CHO) cell gene expression with bioprocess phenotypes; however, alternative messenger RNA (mRNA) splicing, has thus far, received little attention. In this study, we utilized RNASeq for transcriptomic analysis of a monoclonal antibody (mAb) producing CHO K1 cell line subjected to a temperature shift. More than 2,465 instances of differential splicing were observed 24 hr after the reduction of cell culture temperature. A total of 1,197 of these alternative splicing events were identified in genes where no changes in abundance were detected by standard differential expression analysis. Ten examples of alternative splicing were selected for independent validation using quantitative polymerase chain reaction in the mAb-producing CHO K1 cell line used for RNASeq and a further two CHO K1 cell lines. This analysis provided evidence that exon skipping and mutually exclusive splicing events occur in genes linked to the cellular response to changes in temperature and mitochondrial function. While further work is required to determine the impact of these changes in mRNA sequence on cellular phenotype, this study demonstrates that alternative splicing analysis can be utilized to gain a deeper understanding of post-transcriptional regulation in CHO cells during biopharmaceutical production.

Altered gene expression in CHO cells following polyamine starvation.

AIM: To investigate the impact of polyamine deprivation on the transcriptome of CHO cells RESULTS: Polyamines play a central but poorly-understood role in cell proliferation. Most studies to date have utilised chemical inhibitors to probe polyamine function. Here we exploit the fact that CHO cells grown in serum-free medium have an absolute requirement for putrescine supplementation due to their deficiency in activity of the enzyme arginase. A gene expression microarray (Affymetrix) analysis of CHO-K1 cells starved of polyamines for 3 days showed that cessation of growth, associated with increased G1/S transition and inhibition of M/G1 transition was accompanied by increased mRNA levels of mitotic complex checkpoint genes (Mad2l1, Tkk, Bub1b) and in the transition of G1- to S-phase (such as Skp2 and Tfdp1). mRNAs associated with DNA homologous recombination and repair (including Fanconi's anaemia-related genes) and with RNA splicing were consistently increased. Alterations in mRNA levels for genes related to protein processing in the ER, to ER stress, and to p53-related and apoptosis pathways were also observed. mRNAs showing highest levels of fold-change included several which code for membrane-localised proteins and receptors (Thbs1, Tfrc1, Ackr3, Extl1). CONCLUSIONS: Growth-arrest induced by polyamine deprivation was associated with significant alterations in levels of mRNAs associated with cell cycle progression, DNA repair, RNA splicing, ER trafficking and membrane signalling as well as p53 and apoptosis-related pathways.