IsoTools: a flexible workflow for long-read transcriptome sequencing analysis.

MOTIVATION: Long-read transcriptome sequencing (LRTS) has the potential to enhance our understanding of alternative splicing and the complexity of this process requires the use of versatile computational tools, with the ability to accommodate various stages of the workflow with maximum flexibility. RESULTS: We introduce IsoTools, a Python-based LRTS analysis framework that offers a wide range of functionality for transcriptome reconstruction and quantification of transcripts. Furthermore, we integrate a graph-based method for identifying alternative splicing events and a statistical approach based on the beta-binomial distribution for detecting differential events. To demonstrate the effectiveness of our methods, we applied IsoTools to PacBio LRTS data of human hepatocytes treated with the histone deacetylase inhibitor valproic acid. Our results indicate that LRTS can provide valuable insights into alternative splicing, particularly in terms of complex and differential splicing patterns, in comparison to short-read RNA-seq. AVAILABILITY AND IMPLEMENTATION: IsoTools is available on GitHub and PyPI, and its documentation, including tutorials, CLI, and API references, can be found at https://isotools.readthedocs.io/.

Dynamic Interplay between the Transcriptome and Methylome in Response to Oxidative and Alkylating Stress.

In recent years, it has been shown that free radicals not only react directly with DNA but also regulate epigenetic processes such as DNA methylation, which may be relevant within the context of, for example, tumorigenesis. However, how these free radicals impact the epigenome remains unclear. We therefore investigated whether methyl and hydroxyl radicals, formed by tert-butyl hydroperoxide (TBH), change temporal DNA methylation patterns and how this interferes with genome-wide gene expression. At three time points, TBH-induced radicals in HepG2 cells were identified by electron spin resonance spectroscopy. Total 5-methylcytosine (5mC) levels were determined by liquid chromatography and tandem mass spectrometry and genome-wide changes in 5mC and gene expression by microarrays. Induced methylome changes rather represent an adaptive response to the oxidative stress-related reactions observed in the transcriptome. More specifically, we found that methyl radicals did not induce DNA methylation directly. An initial oxidative and alkylating stress-related response of the transcriptome during the early phase of TBH treatment was followed by an epigenetic response associated with cell survival signaling. Also, we identified genes of which the expression seems directly regulated by DNA methylation. This work suggests an important role of the methylome in counter-regulating primary oxidative and alkylating stress responses in the transcriptome to restore normal cell function. Altogether, the methylome may play an important role in counter-regulating primary oxidative and alkylating stress responses in the transcriptome presumably to restore normal cell function.

PERK/eIF2α signaling protects therapy resistant hypoxic cells through induction of glutathione synthesis and protection against ROS.

Hypoxia is a common feature of tumors and an important contributor to malignancy and treatment resistance. The ability of tumor cells to survive hypoxic stress is mediated in part by hypoxia-inducible factor (HIF)-dependent transcriptional responses. More severe hypoxia activates endoplasmatic reticulum stress responses, including the double-stranded RNA-activated protein kinase (PKR)-like endoplasmic reticulum kinase (PERK)/eukaryotic initiation factor 2α (eIF2α)-dependent arm of the unfolded protein response (UPR). Although several studies implicate important roles for HIF and UPR in adaption to hypoxia, their importance for hypoxic cells responsible for therapy resistance in tumors is unknown. By using isogenic models, we find that HIF and eIF2α signaling contribute to the survival of hypoxic cells in vitro and in vivo. However, the eIF2α-dependent arm of the UPR is uniquely required for the survival of a subset of hypoxic cells that determine tumor radioresistance. We demonstrate that eIF2α signaling induces uptake of cysteine, glutathione synthesis, and protection against reactive oxygen species produced during periods of cycling hypoxia. Together these data imply that eIF2α signaling is a critical contributor to the tolerance of therapy-resistant cells that arise as a consequence of transient changes in oxygenation in solid tumors and thus a therapeutic target in curative treatments for solid cancers.

Author Correction: Hypoxia-mediated downregulation of miRNA biogenesis promotes tumour progression.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Taking advantage of tumor cell adaptations to hypoxia for developing new tumor markers and treatment strategies.

Cancer cells in hypoxic areas of solid tumors are to a large extent protected against the action of radiation as well as many chemotherapeutic drugs. There are, however, two different aspects of the problem caused by tumor hypoxia when cancer therapy is concerned: One is due to the chemical reactions that molecular oxygen enters into therapeutically targeted cells. This results in a direct chemical protection against therapy by the hypoxic microenvironment, which has little to do with cellular biological regulatory processes. This part of the protective effect of hypoxia has been known for more than half a century and has been studied extensively. However, in recent years there has been more focus on the other aspect of hypoxia, namely the effect of this microenvironmental condition on selecting cells with certain genetic prerequisites that are negative with respect to patient prognosis. There are adaptive mechanisms, where hypoxia induces regulatory cascades in cells resulting in a changed metabolism or changes in extracellular signaling. These processes may lead to changes in cellular intrinsic sensitivity to treatment irrespective of oxygenation and, furthermore, may also have consequences for tissue organization. Thus, the adaptive mechanisms induced by hypoxia itself may have a selective effect on cells, with a fine-tuned protection against damage and stress of many kinds. It therefore could be that the adaptive mechanisms may take advantage of for new tumor labeling/imaging and treatment strategies. One of the Achilles' heels of hypoxia research has always been the exact measurements of tissue oxygenation as well as the control of oxygenation in biological tumor models. Thus, development of technology that can ease this control is vital in order to study mechanisms and perform drug development under relevant conditions. An integrated EU Framework project 2004-2009, termed EUROXY, demonstrates several pathways involved in transcription and translation control of the hypoxic cell phenotype and evidence of cross-talk with responses to pH and redox changes. The carbonic anhydrase isoenzyme CA IX was selected for further studies due to its expression on the surface of many types of hypoxic tumors. The effort has led to marketable culture flasks with sensors and incubation equipment, and the synthesis of new drug candidates against new molecular targets. New labeling/imaging methods for cancer diagnosing and imaging of hypoxic cancer tissue are now being tested in xenograft models and are also in early clinical testing, while new potential anti-cancer drugs are undergoing tests using xenografted tumor cancers. The present article describes the above results in individual consortium partner presentations.

Phosphorylation of eIF2α promotes cell survival in response to benzo[a]pyrene exposure.

Cellular adaptation is important to cope with various stresses induced by altered environmental conditions. By controlling mRNA translation rates cells may adapt to stress to promote survival. Phosphorylation of eIF2α at serine 51 is one of the pathways controlling mRNA translation. Here we investigated the role of phosphorylated eIF2α during exposure to the environmental carcinogen benzo(a)pyrene (BaP). For our study we used mouse embryonic fibroblasts with a wild type eIF2α (MEF WT) and mouse embryonic fibroblasts with an eIF2α S51A knock-in mutation that cannot be phosphorylated. Here, we show that eIF2α phosphorylation occurs in MEF WT cells but not in MEF S51A cells. Survival of MEF S51A cells is profoundly reduced compared to MEF WT controls after BaP exposure. No differences in DNA damage or ROS production were observed between MEF WT and S51A cells. Disruption of eIF2α phosphorylation caused increased levels of apoptosis in response to BaP. This work demonstrates that eIF2α phosphorylation is important for reducing apoptosis and promoting cell survival in order to adapt to BaP exposure.

The mTOR target 4E-BP1 contributes to differential protein expression during normoxia and hypoxia through changes in mRNA translation efficiency.

Hypoxia causes a rapid and sustained inhibition in mRNA translation that is characterized by both a transient phosphorylation of eukaryotic initiation factor 2-alpha (eIF2alpha) and by inhibition of the mRNA cap binding protein eIF4E via activation of two distinct inhibitory proteins, the mammalian target of rapamycin (mTOR) target 4E-BP1 and the eIF4E transporter 4E-T. Although the importance of eIF2alpha phosphorylation during hypoxia has been clearly demonstrated, there is little information on the potential relevance of eIF4E regulation. We generated HeLa cells stably expressing a short hairpin interfering RNA (shRNA) against 4E-BP1 and found that despite efficient knockdown, no significant changes occurred in the overall inhibition of mRNA translation during hypoxia. However, using a proteomics approach we identified seven proteins that were exclusively expressed in the 4E-BP1 knockdown cells during both normoxic and hypoxic conditions. Further investigation of the transcriptional and translational regulation of these genes by quantitative RT-PCR indicated that the loss of 4E-BP1 causes a significant increase in the rate of protein synthesis of S100 calcium-binding protein A4 (S100A4) and transgelin 2. These 4E-BP1 regulated proteins have previously been associated with tumor cell motility, invasion and metastasis and may thus contribute to an adverse tumor phenotype.

Radioprotective effects of ATP in human blood ex vivo.

Damage to healthy tissue is a major limitation of radiotherapy treatment of cancer patients, leading to several side effects and complications. Radiation-induced release of pro-inflammatory cytokines is thought to be partially responsible for the radiation-associated complications. The aim of the present study was to investigate the protective effects of extracellular ATP on markers of oxidative stress, radiation-induced inflammation and DNA damage in irradiated blood ex vivo. ATP inhibited radiation-induced TNF-alpha release and increased IL-10 release. The inhibitory effect of ATP on TNF- alpha release was completely reversed by adenosine 5'-O-thiomonophosphate, indicating a P2Y(11) mediated effect. Furthermore, ATP attenuated radiation-induced DNA damage immediate, 3 and 6h after irradiation. Our study indicates that ATP administration alleviates radiation-toxicity to blood cells, mainly by inhibiting radiation-induced inflammation and DNA damage.

Translational regulation is a key determinant of the cellular response to benzo[a]pyrene.

Translational control is a cellular response mechanism which initiates adaptation during various stress situations. Here, we investigated the role of translational control after benzo[a]pyrene (BaP) exposure in primary mouse hepatocytes. Translated mRNAs were separated and captured based on the number of associated ribosomes using sucrose gradients and subjected to RNA sequencing (RNAseq) to investigate translational changes. Furthermore, unseparated RNA (total RNA) was used for RNAseq to determine the transcriptional alterations. We showed that, after 24 h of exposure to 10 µM BaP, the number of genes altered by changes in mRNA translation was substantially higher compared with the number of genes altered by transcription. Although part of the BaP regulated genes were regulated by both transcription and translation, we identified genes that were uniquely regulated by mRNA translation. These mRNA transcripts encode proteins that are involved in biological processes that are not affected by transcriptional regulation. Al together this work identified a new layer of gene expression regulation that might contribute to BaP-induced carcinogenesis.

A cross-omics approach to investigate temporal gene expression regulation by 5-hydroxymethylcytosine via TBH-derived oxidative stress showed involvement of different regulatory kinases.

Regulation of DNA methylation plays a crucial role in biological processes and carcinogenesis. The formation of 5-hydroxymethylcytosine (5hmC) by oxidation of 5-methylcytosine (5mC) has been proposed as an intermediate of active demethylation. However, whether and how active demethylation is regulated by oxidative stress-related processes is not well understood. Here we investigated whether free oxygen radicals are capable of directly forming 5hmC and how this enhanced whole genome gene expression. We applied LC-MS/MS technology for the analysis of 5mC, 5hmC, 5-formylcytosine (5fC) and 5-hydroxymethyluracyl (5hmU) in HepG2 cells exposed to hydroxyl- and methyl radicals, formed by tert-butyl hydroperoxide (TBH) at multiple time points. We observed that TBH is able to induce a significant increase in 5hmC. A detailed evaluation of the hydroxymethylome using a combination of 5hmC-immunoprecipitation and microarrays resulted in the identification of highly dynamic modifications that appear to increase during prolonged oxidant exposure. Analyses of temporal gene expression changes in combination with network analysis revealed different subnetworks containing differentially expressed genes (DEGs) with differentially hydroxyl-methylated regions (DhMRs) in different regulatory kinases enriched with serine-threonine kinases. These serine-threonine kinases compromises MAPK14, RPSK6KA1, RIPK1, and PLK3 and were all previously identified as key-regulators in hepatocarcinogenesis and subject of study for chemotherapeutic interventions.

Regulation of Cited2 expression provides a functional link between translational and transcriptional responses during hypoxia.

BACKGROUND AND PURPOSE: Protein synthesis rates are greatly reduced under hypoxic conditions as a consequence of an overall inhibition of mRNA translation. Certain specific mRNA species have the ability to escape this general translational repression. At the cellular level this results in differential protein expression during hypoxic conditions. The objective of this study was to characterize the translational regulation of the postulated HIF-1alpha antagonist Cited2. MATERIALS AND METHODS: DU145 prostate carcinoma cells and mouse embryonic fibroblasts with a homozygous knock-in mutation for eIF2alpha (S51A) or wild-type eIF2alpha were exposed to severe hypoxia after which both total mRNA and efficiently translated mRNA were isolated. Quantitative RT-PCR was used to measure and compare changes in transcription (total mRNA) with changes in translation (efficiently translated mRNA fraction). RESULTS: We show using HIF-1alpha null MEF cells that transcriptional induction of Cited2 during hypoxia is dependent on HIF-1alpha. Although global mRNA translation is inhibited during hypoxia Cited2 mRNA remains efficiently translated. An evolutionary conserved upstream open reading frame (uORF) in the 5'UTR of Cited2 did not stimulate translation in an eIF2alpha dependent manner during hypoxia. CONCLUSIONS: Selective translation Cited2 by an eIF2alpha independent mechanism establishes a link between translation and HIF-1 dependent transcription during hypoxia.

Phosphorylation of eIF2alpha is required for mRNA translation inhibition and survival during moderate hypoxia.

BACKGROUND AND PURPOSE: Human tumors are characterized by temporal fluctuations in oxygen tension. The biological pathways that respond to the dynamic tumor microenvironment represent potential molecular targets for cancer therapy. Anoxic conditions result in eIF2alpha dependent inhibition of overall mRNA translation, differential gene expression, hypoxia tolerance and tumor growth. The signaling pathway which governs eIF2alpha phosphorylation has therefore emerged as a potential molecular target. In this study, we investigated the role of eIF2alpha in regulating mRNA translation and hypoxia tolerance during moderate hypoxia. Since other molecular pathways that regulate protein synthesis are frequently mutated in cancer, we also assessed mRNA translation in a panel of cell lines from different origins. MATERIALS AND METHODS: Immortalized human fibroblast, transformed mouse embryo fibroblasts (MEFs) and cells from six cancer cell lines were exposed to 0.2% or 0.0% oxygen. We assayed global mRNA translation efficiency by polysome analysis, as well as proliferation and clonogenic survival. The role of eIF2alpha was assessed in MEFs harboring a homozygous inactivating mutation (S51A) as well as in U373-MG cells overexpressing GADD34 (C-term) under a tetracycline-dependent promoter. The involvement of eIF4E regulation was investigated in HeLa cells stably expressing a short hairpin RNA (shRNA) targeting 4E-BP1. RESULTS: All cells investigated inhibited mRNA translation severely in response to anoxia and modestly in response to hypoxia. Two independent genetic cell models demonstrated that inhibition of mRNA translation in response to moderate hypoxia was dependent on eIF2alpha phosphorylation. Disruption of eIF2alpha phosphorylation caused sensitivity to hypoxia and anoxia. CONCLUSIONS: Disruption of eIF2alpha phosphorylation is a potential target for hypoxia-directed molecular cancer therapy.

Proteomic analysis of gene expression following hypoxia and reoxygenation reveals proteins involved in the recovery from endoplasmic reticulum and oxidative stress.

BACKGROUND AND PURPOSE: Human tumors are characterized by large variations in oxygen concentration and hypoxic tumors are associated with poor prognosis. In addition, tumors are subjected to periodic changes in oxygenation characterized by hypoxia followed by reoxygenation. Cellular adaptation to hypoxia is well documented, nevertheless little is known about adaptive mechanisms to reoxygenation. Here, we investigate the changes in protein expression during reoxygenation using proteomics. MATERIALS AND METHODS: HeLa cervix carcinoma cells were exposed to 4h of hypoxia (<0.01% O(2)) followed by 1h of reoxygenation. The cellular proteome was examined using 2D gel electrophoresis coupled with mass spectrometry. Validation and investigation of the underlying basis for induced protein expression was investigated using Western blot analysis and quantitative RT-PCR. RESULTS: We identified proteins involved in several cellular processes that are responsible for regulating RNA metabolism, protein synthesis and degradation, including ribosomal protein P0, VCP/p97 and FUSE binding protein 2. CONCLUSIONS: Our results suggest that these newly identified proteins function in pathways that may assist in the recovery of ER stress and protein synthesis during reoxygenation. These proteins may thus be important determinants of the behaviour and survival of tumor cells to transient hypoxic exposures.

Identification of essential transcription factors for adequate DNA damage response after benzo(a)pyrene and aflatoxin B1 exposure by combining transcriptomics with functional genomics.

DNA damage mediates widespread changes in transcription through activation or repression of transcription factors (TFs). However, the consequences of regulating specific TFs for the outcome of the DNA repair process remain incompletely understood. Here, we combined transcriptomics and TF binding prediction with functional genomics to identify TFs essential for adequate DNA repair in HepG2 liver cells after a non-cytotoxic dose of carcinogens benzo(a)pyrene (BaP) (2µM) and aflatoxin B1 (AFB1) (5µM). BaP and AFB1 induced a largely common transcriptional response, mediated by similar TFs. A lentiviral shRNA screen knocking down the top31 identified TFs, was performed to determine their effect on DNA repair by assessing phosphorylation of H2AX (γ-H2AX). In addition to the top candidate p53, we identified several other interesting TFs that modulated γ-H2AX after BaP and AFB1 treatment. Validation studies confirmed the role of p53 in reducing γ-H2AX formation and DNA breaks measured by COMET assay after BaP and AFB1 exposure. Expression of the cell cycle inhibitor p21 was profoundly impaired upon p53 knock-down. In addition, the expression of 2 genes involved in nucleotide exchange repair, DDB2 and XPC was significantly reduced in p53 knock-down cells. Although p63 knock-down affected DNA damage upon BaP treatment this was not associated with altered expression of DDB2 or XPC. Finally, knock-down of ARNT reduced γ-H2AX in response to BaP, which was associated with reduced CYP1A1 expression. Importantly, our results suggest a new role for ARNT and its dimerization partner AHR in the occurrence of H2AX phosphorylation after AFB1 treatment. These data show that modulation of TF activity impacts on the repair of BaP- and AFB1-induced DNA damage. Our study also demonstrates the potential of combining functional genomics with genome-wide expression analysis to identify yet unknown causal relationships, thereby aiding in the interpretation of complex biological systems.

Translational control of gene expression during hypoxia.

Poor oxygenation is a unique and prevalent feature of solid tumors associated with poor patient prognosis. In part, this is caused by a series of adaptive cellular responses that together have a large impact on gene expression and cell phenotype. HIF plays a key role in this response by activating a transcriptional program that stimulates genes involved in angiogenesis, cell metabolism, cell survival and cell invasion. Recently, hypoxia has also been shown to suppress protein synthesis through the regulation of the initiation step of mRNA translation. This appears to be a common feature of the cell in response to hypoxia and is mediated by two distinct pathways. The first occurs rapidly, is transient, and is associated with activation of the unfolded protein response (UPR) that occurs in response to endoplasmic reticulum (ER) stress. Translation inhibition during this initial phase is due to phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha) in a PERK dependent manner. Although this effect is transient, overall levels of translation remain low during hypoxia due to inhibition of a second eukaryotic initiation complex, eIF4F. This second mechanism is multi-factorial, but due at least in part to inhibition of the mTOR kinase. Although each of these pathways leads to a general inhibition in translation, the consequence at the individual gene level is highly variable. This is due to sequences in the 5' and 3' untranslated regions (UTRs) of mRNA that confer their ability to maintain, or even increase, translation efficiency in spite of the overall inhibition. Consequently, regulation of mRNA translation appears to be an important mediator of gene expression during hypoxia.

In vivo optical imaging of MMP2 immuno protein antibody: tumor uptake is associated with MMP2 activity.

Matrix metalloproteinase-2 (MMP2) is important in tumorigenesis, angiogenesis and tumor invasion. In this study, we investigated if the Cy5-tagged small immuno protein targeting the catalytic domain of human MMP2 (aMMP2-SIP) detects MMP2 in tumors non-invasively. For this purpose, we generated MMP2 expressing (empty vector EV) and knock-down (KD) HT1080, U373 and U87 cells, which were injected subcutaneously in the lateral flank of NMRI-nu mice. Optical imaging (Optix MX2) performed at 0.5, 2, 4, 8, 24 and 48 hour post injection (h.p.i.) of Cy5 tagged aMMP2-SIP, indicated significantly lower tumor to background ratios at both 24 (P = 0.0090) and 48 h.p.i. (P < 0.0001) for the U87 MMP2-KD compared to control tumors. No differences were found for HT1080 and U373 models. U87 MMP2-KD tumors had significantly lower MMP2 activity (P < 0.0001) than EV tumors as determined by gelatin zymography in tumor sections and lysates, while no differences were observed between EV and MMP2-KD in HT1080 and U373. In line with these data, only U87 MMP2-KD tumors had a reduced tumor growth compared to control tumors (P = 0.0053). aMMP2-SIP uptake correlates with MMP2 activity and might therefore be a potential non-invasive imaging biomarker for the evaluation of MMP2 activity in tumors.

Quantitative analysis of ChIP-seq data uncovers dynamic and sustained H3K4me3 and H3K27me3 modulation in cancer cells under hypoxia.

BACKGROUND: A comprehensive assessment of the epigenetic dynamics in cancer cells is the key to understanding the molecular mechanisms underlying cancer and to improving cancer diagnostics, prognostics and treatment. By combining genome-wide ChIP-seq epigenomics and microarray transcriptomics, we studied the effects of oxygen deprivation and subsequent reoxygenation on histone 3 trimethylation of lysine 4 (H3K4me3) and lysine 27 (H3K27me3) in a breast cancer cell line, serving as a model for abnormal oxygenation in solid tumors. A priori, epigenetic markings and gene expression levels not only are expected to vary greatly between hypoxic and normoxic conditions, but also display a large degree of heterogeneity across the cell population. Where traditionally ChIP-seq data are often treated as dichotomous data, the model and experiment here necessitate a quantitative, data-driven analysis of both datasets. RESULTS: We first identified genomic regions with sustained epigenetic markings, which provided a sample-specific reference enabling quantitative ChIP-seq data analysis. Sustained H3K27me3 marking was located around centromeres and intergenic regions, while sustained H3K4me3 marking is associated with genes involved in RNA binding, translation and protein transport and localization. Dynamic marking with both H3K4me3 and H3K27me3 (hypoxia-induced bivalency) was found in CpG-rich regions at loci encoding factors that control developmental processes, congruent with observations in embryonic stem cells. CONCLUSIONS: In silico-identified epigenetically sustained and dynamic genomic regions were confirmed through ChIP-PCR in vitro, and obtained results are corroborated by published data and current insights regarding epigenetic regulation.

The hypoxic proteome is influenced by gene-specific changes in mRNA translation.

BACKGROUND AND PURPOSE: Hypoxia causes a rapid reduction in mRNA translation efficiency. This inhibition does not affect all mRNA species to the same extent and can therefore contribute significantly to hypoxia-induced differential protein expression. Our aim in this study was to characterize changes in gene expression during acute hypoxia and evaluate the contribution of regulation via mRNA translation on these changes. For each gene, the contribution of changes in mRNA abundance versus mRNA translation was determined. MATERIALS AND METHODS: DU145 prostate carcinoma cells were exposed to 4h of hypoxia (<0.02% O2). Efficiently translated mRNAs were isolated by sedimentation through a sucrose gradient. Affymetrix microarray technology was used to evaluate both the transcriptional and translational contribution to gene expression. Results were validated by quantitative PCR. RESULTS: One hundred and twenty genes were more than 4-fold upregulated by hypoxia in the efficiently translated fraction of mRNA, in comparison to only 76 genes at the level of transcription. Of the 50 genes demonstrating the largest changes in translation, 11 were found to be more than 2-fold over represented in the translated fraction in comparison to their overall transcriptional level. The gene with the highest translational contribution to its induction was CITED-2, which is a negative regulator of HIF-1 transcriptional activity. CONCLUSIONS: Gene-specific regulation of mRNA translation contributes significantly to differential gene expression during hypoxia.

Canonical autophagy does not contribute to cellular radioresistance.

BACKGROUND: (Pre)clinical studies indicate that autophagy inhibition increases response to anti-cancer therapies. Although promising, due to contradicting reports, it remains unclear if radiation therapy changes autophagy activity and if autophagy inhibition changes the cellular intrinsic radiosensitivity. Discrepancies may result from different assays and models through off-target effects and influencing other signaling routes. In this study, we directly compared the effects of genetic and pharmacological inhibition of autophagy after irradiation in human cancer cell lines. MATERIALS AND METHODS: Changes in autophagy activity after ionizing radiation (IR) were assessed by flux analysis in eight cell lines. Clonogenic survival, DNA damage (COMET-assay) and H2AX phosphorylation were assessed after chloroquine or 3-methyladenine pretreatment and after ATG7 or LC3b knockdown. RESULTS: IR failed to induce autophagy and chloroquine failed to change intrinsic radiosensitivity of cells. Interestingly, 3-methyladenine and ATG7- or LC3b-deficiency sensitized cancer cells to irradiation. Surprisingly, the radiosensitizing effect of 3-methyladenine was also observed in ATG7 and LC3b deficient cells and was associated with attenuated γ-H2AX formation and DNA damage repair. CONCLUSION: Our data demonstrate that the anti-tumor effects of chloroquine are independent of changes in intrinsic radioresistance. Furthermore, ATG7 and LC3b support radioresistance independent of canonical autophagy that involves lysosomal degradation.

Construction and diversification of yeast cell surface displayed libraries by yeast mating: application to the affinity maturation of Fab antibody fragments.

Yeast display is a powerful technology for the affinity maturation of human antibody fragments. However, the technology thus far has been limited by the size of antibody libraries that can be generated, as using current transformation protocols libraries of only between 10(6) and 10(7) are typically possible. We have recently shown that Fab antibodies can be displayed on the cell surface of Saccharomyces cerevisiae [van den Beucken, T., Pieters, H., Steukers, M., van der Vaart, M., Ladner, R.C., Hoogenboom, H.R., Hufton, S.E., 2003. Affinity maturation of Fab antibody fragments by fluorescent-activated cell sorting of yeast-displayed libraries. FEBS Lett. 546, 288-294]. This discovery and the knowledge that Fab antibodies are heterodimeric suggest that independent repertoires of heavy chain (HC) and light chain (LC) can be constructed in haploid yeast strains of opposite mating type. These separate repertoires can then be combined by highly efficient yeast mating. Using this approach, we have rapidly generated a naive human Fab yeast display library of over 10(9) clones. In addition, utilizing error-prone polymerase chain reaction, we have diversified Fab sequences and generated combinatorial and hierarchical chain shuffled libraries with complexities of up to 5 x 10(9) clones. These libraries have been selected for higher affinity using a repeating process of mating-driven chain shuffling and flow cytometric sorting.