Reprogramming of glucocorticoid receptor function by hypoxia.

Here, we investigate the impact of hypoxia on the hepatic response of glucocorticoid receptor (GR) to dexamethasone (DEX) in mice via RNA-sequencing. Hypoxia causes three types of reprogramming of GR: (i) much weaker induction of classical GR-responsive genes by DEX in hypoxia, (ii) a number of genes is induced by DEX specifically in hypoxia, and (iii) hypoxia induces a group of genes via activation of the hypothalamic-pituitary-adrenal (HPA) axis. Transcriptional profiles are reflected by changed GR DNA-binding as measured by ChIP sequencing. The HPA axis is induced by hypothalamic HIF1α and HIF2α activation and leads to GR-dependent lipolysis and ketogenesis. Acute inflammation, induced by lipopolysaccharide, is prevented by DEX in normoxia but not during hypoxia, and this is attributed to HPA axis activation by hypoxia. We unfold new physiological pathways that have consequences for patients suffering from GC resistance.

Point mutation I634A in the glucocorticoid receptor causes embryonic lethality by reduced ligand binding.

The glucocorticoid (GC) receptor (GR) is essential for normal development and in the initiation of inflammation. Healthy GRdim/dim mice with reduced dimerization propensity due to a point mutation (A465T) at the dimer interface of the GR DNA-binding domain (DBD) (here GRD/D) have previously helped to define the functions of GR monomers and dimers. Since GRD/D retains residual dimerization capacity, here we generated the dimer-nullifying double mutant GRD+L/D+L mice, featuring an additional mutation (I634A) in the ligand-binding domain (LBD) of GR. These mice are perinatally lethal, as are GRL/L mice (these mice have the I634A mutation but not the A465T mutation), displaying improper lung and skin formation. Using embryonic fibroblasts, high and low doses of dexamethasone (Dex), nuclear translocation assays, RNAseq, dimerization assays, and ligand-binding assays (and Kd values), we found that the lethal phenotype in these mice is due to insufficient ligand binding. These data suggest there is some correlation between GR dimerization potential and ligand affinity. We conclude that even a mutation as subtle as I634A, at a position not directly involved in ligand interactions sensu stricto, can still influence ligand binding and have a lethal outcome.

TNF-α inhibits glucocorticoid receptor-induced gene expression by reshaping the GR nuclear cofactor profile.

Glucocorticoid resistance (GCR) is defined as an unresponsiveness to the therapeutic effects, including the antiinflammatory ones of glucocorticoids (GCs) and their receptor, the glucocorticoid receptor (GR). It is a problem in the management of inflammatory diseases and can be congenital as well as acquired. The strong proinflammatory cytokine TNF-alpha (TNF) induces an acute form of GCR, not only in mice, but also in several cell lines: e.g., in the hepatoma cell line BWTG3, as evidenced by impaired Dexamethasone (Dex)-stimulated direct GR-dependent gene up- and down-regulation. We report that TNF has a significant and broad impact on this transcriptional performance of GR, but no impact on nuclear translocation, dimerization, or DNA binding capacity of GR. Proteome-wide proximity-mapping (BioID), however, revealed that the GR interactome was strongly modulated by TNF. One GR cofactor that interacted significantly less with the receptor under GCR conditions is p300. NFκB activation and p300 knockdown both reduced direct transcriptional output of GR whereas p300 overexpression and NFκB inhibition reverted TNF-induced GCR, which is in support of a cofactor reshuffle model. This hypothesis was supported by FRET studies. This mechanism of GCR opens avenues for therapeutic interventions in GCR diseases.

Development and Validation of a Small Single-domain Antibody That Effectively Inhibits Matrix Metalloproteinase 8.

A detrimental role for matrix metalloproteinase 8 (MMP8) has been identified in several pathological conditions, e.g., lethal hepatitis and the systemic inflammatory response syndrome. Since matrix MMP8-deficient mice are protected in the above-mentioned diseases, specific MMP8 inhibitors could be of clinical value. However, targeting a specific matrix metalloproteinase remains challenging due to the strong structural homology of matrix metalloproteinases, which form a family of 25 members in mammals. Single-domain antibodies, called nanobodies, offer a range of possibilities toward therapy since they are easy to generate, express, produce, and modify, e.g., by linkage to nanobodies directed against other target molecules. Hence, we generated small MMP8-binding nanobodies, and established a proof-of-principle for developing nanobodies that inhibit matrix metalloproteinase activity. Also, we demonstrated for the first time the possibility of expressing nanobodies systemically by in vivo electroporation of the muscle and its relevance as a potential therapy in inflammatory diseases.

The impact of hepatocyte-specific deletion of hypoxia-inducible factors on the development of polymicrobial sepsis with focus on GR and PPARα function.

Introduction: Polymicrobial sepsis causes acute anorexia (loss of appetite), leading to lipolysis in white adipose tissue and proteolysis in muscle, and thus release of free fatty acids (FFAs), glycerol and gluconeogenic amino acids. Since hepatic peroxisome proliferator-activated receptor alpha (PPARα) and glucocorticoid receptor (GR) quickly lose function in sepsis, these metabolites accumulate (causing toxicity) and fail to yield energy-rich molecules such as ketone bodies (KBs) and glucose. The mechanism of PPARα and GR dysfunction is not known. Methods & results: We investigated the hypothesis that hypoxia and/or activation of hypoxia inducible factors (HIFs) might play a role in these issues with PPARα and GR. After cecal ligation and puncture (CLP) in mice, leading to lethal polymicrobial sepsis, bulk liver RNA sequencing illustrated the induction of the genes encoding HIF1α and HIF2α, and an enrichment of HIF-dependent gene signatures. Therefore, we generated hepatocyte-specific knock-out mice for HIF1α, HIF2α or both, and a new HRE-luciferase reporter mouse line. After CLP, these HRE-luciferase reporter mice show signals in several tissues, including the liver. Hydrodynamic injection of an HRE-luciferase reporter plasmid also led to (liver-specific) signals in hypoxia and CLP. Despite these encouraging data, however, hepatocyte-specific HIF1α and/or HIF2α knock-out mice suggest that survival after CLP was not dependent on the hepatocyte-specific presence of HIF proteins, which was supported by measuring blood levels of glucose, FFAs, and KBs. The HIF proteins were also irrelevant in the CLP-induced glucocorticoid resistance, but we found indications that the absence of HIF1α in hepatocytes causes less inactivation of PPARα transcriptional function. Conclusion: We conclude that HIF1α and HIF2α are activated in hepatocytes in sepsis, but their contribution to the mechanisms leading to lethality are minimal.

Mutations in the histone methyltransferase Ezh2 drive context-dependent leukemia in Xenopus tropicalis.

CRISPR-mediated simultaneous targeting of candidate tumor suppressor genes in Xenopus tropicalis allows fast functional assessment of co-driver genes for various solid tumors. Genotyping of tumors that emerge in the mosaic mutant animals rapidly exposes the gene mutations under positive selection for tumor establishment. However, applying this simple approach to the blood lineage has not been attempted. Multiple hematologic malignancies have mutations in EZH2, encoding the catalytic subunit of the Polycomb Repressive Complex 2. Interestingly, EZH2 can act as an oncogene or a tumor suppressor, depending on cellular context and disease stage. We show here that mosaic CRISPR/Cas9 mediated ezh2 disruption in the blood lineage resulted in early and penetrant acute myeloid leukemia (AML) induction. While animals were co-targeted with an sgRNA that induces notch1 gain-of-function mutations, sequencing of leukemias revealed positive selection towards biallelic ezh2 mutations regardless of notch1 mutational status. Co-targeting dnm2, recurrently mutated in T/ETP-ALL, induced a switch from myeloid towards acute T-cell leukemia. Both myeloid and T-cell leukemias engrafted in immunocompromised hosts. These data underline the potential of Xenopus tropicalis for modeling human leukemia, where mosaic gene disruption, combined with deep amplicon sequencing of the targeted genomic regions, can rapidly and efficiently expose co-operating driver gene mutations.

ZBTB32 performs crosstalk with the glucocorticoid receptor and is crucial in glucocorticoid responses to starvation.

The hypothalamic-pituitary-adrenal (HPA) axis forms a complex neuroendocrine system that regulates the body's response to stress such as starvation. In contrast with the glucocorticoid receptor (GR), Zinc finger and BTB domain containing 32 (ZBTB32) is a transcription factor with poorly described functional relevance in physiology. This study shows that ZBTB32 is essential for the production of glucocorticoids (GCs) in response to starvation, since ZBTB32-/- mice fail to increase their GC production in the absence of nutrients. In terms of mechanism, GR-mediated upregulation of adrenal Scarb1 gene expression was absent in ZBTB32-/- mice, implicating defective cholesterol import as the cause of the poor GC synthesis. These lower GC levels are further associated with aberrations in the metabolic adaptation to starvation, which could explain the progressive weight gain of ZBTB32-/- mice. In conclusion, ZBTB32 performs a crosstalk with the GR in the metabolic adaptation to starvation via regulation of adrenal GC production.

Combined glucocorticoid resistance and hyperlactatemia contributes to lethal shock in sepsis.

Sepsis is a potentially lethal syndrome resulting from a maladaptive response to infection. Upon infection, glucocorticoids are produced as a part of the compensatory response to tolerate sepsis. This tolerance is, however, mitigated in sepsis due to a quickly induced glucocorticoid resistance at the level of the glucocorticoid receptor. Here, we show that defects in the glucocorticoid receptor signaling pathway aggravate sepsis pathophysiology by lowering lactate clearance and sensitizing mice to lactate-induced toxicity. The latter is exerted via an uncontrolled production of vascular endothelial growth factor, resulting in vascular leakage and collapse with severe hypotension, organ damage, and death, all being typical features of a lethal form of sepsis. In conclusion, sepsis leads to glucocorticoid receptor failure and hyperlactatemia, which collectively leads to a lethal vascular collapse.

Serum N-glycome biomarker for monitoring development of DENA-induced hepatocellular carcinoma in rat.

BACKGROUND: There is a demand for serum markers for the routine assessment of the progression of liver cancer. We previously found that serum N-linked sugar chains are altered in hepatocellular carcinoma (HCC). Here, we studied glycomic alterations during development of HCC in a rat model. RESULTS: Rat HCC was induced by the hepatocarcinogen, diethylnitrosamine (DENA). N-glycans were profiled using the DSA-FACE technique developed in our laboratory.In comparison with control rats, DENA rats showed a gradual but significant increase in two glycans (R5a and R5b) in serum total N-glycans during progression of liver cirrhosis and cancer, and a decrease in a biantennary glycan (P5). The log of the ratio of R5a to P1 (NGA2F) and R5b to P1 [log(R5a/P1) and log(R5b/P1)] were significantly (p < 0.0001) elevated in HCC rats, but not in rats with cirrhosis or fibrosis or in control rats. We thus propose a GlycoTest model using the above-mentioned serum glycan markers to monitor the progression of cirrhosis and HCC in the DENA-treated rat model. When DENA-treated rats were subsequently treated with farnesylthiosalicyclic acid, an anticancer drug, progression to HCC was prevented and GlycoTest markers (P5, R5a and R5b) reverted towards non-DENA levels, and the HCC-specific markers, log(R5a/P1) and log(R5b/P1), normalized completely. CONCLUSIONS: We found an increase in core-alpha-1,6-fucosylated glycoproteins in serum and liver of rats with HCC, which demonstrates that fucosylation is altered during progression of HCC. Our GlycoTest model can be used to monitor progression of HCC and to follow up treatment of liver tumors in the DENA rat. This GlycoTest model is particularly important because a rapid non-invasive diagnostic procedure for tumour progression in this rat model would greatly facilitate the search for anticancer drugs.

Alteration of N-glycome in diethylnitrosamine-induced hepatocellular carcinoma mice: a non-invasive monitoring tool for liver cancer.

BACKGROUND AND AIMS: There is a demand for serum markers that can routinely assess the progression of liver cancer. DENA (diethylnitrosamine), a hepatocarcinogen, is commonly used in an experimental mouse model to induce liver cancer that closely mimics a subclass of human hepatocellular carcinoma (HCC). However, blood monitoring of the progression of HCC in mouse model has not yet been achieved. In this report, we studied glycomics during the development of mouse HCC induced by DENA. METHODS: Mouse HCC was induced by DENA. Serum N-glycans were profiled using the sequencer assisted-Fluorophore-assisted carbohydrate electrophoresis technique developed in our laboratory. Possible alteration in the transcription of genes relevant to the synthesis of the changed glycans was analysed by real-time polymerase chain reaction. RESULTS: In comparison with the control mice that received the same volume of saline, a tri-antennary glycan (peak 8) and a biantennary glycan (peak 4) in serum total glycans of DENA mice increased gradually but significantly during progression of liver cancer, whereas a core-fucosylated biantennary glycan (peak 6) decreased. Expression of alpha-1,6-fucosyltransferase 8 (Fut8), which is responsible for core fucosylation, decreased in the liver of DENA mice compared with that of age-matched control mice. Likewise, the expression level of Mgat4a, which is responsible for tri-antennary, significantly increased in the liver of DENA mice (P<0.001). CONCLUSIONS: The changes of N-glycan levels in the serum could be used as a biomarker to monitor the progress of HCC and to follow up the treatment of liver tumours in this DENA mouse model.

Altered serum N-glycomics in chronic hepatitis B patients.

BACKGROUND: We previously reported on serum N-glycans as markers for the diagnosis of cirrhosis in patients with chronic hepatitis C infection. Our present study aimed to evaluate the use of serum glycan markers for the diagnosis of liver fibrosis in patients with chronic hepatitis B infection. METHODS: Patients with hepatitis B virus (HBV) infection (n=173) were diagnosed by clinical laboratory analysis and histological examination. Liver fibrosis was staged using Ishak score. N-glycan profiles of serum proteins were determined by DNA sequencer-based carbohydrate analytical profiling. RESULTS: We found that in HBV patients, like in hepatitis C virus patients, several serum N-glycans were altered during the development of liver fibrosis. We found higher levels of total agalactosylated biantennary glycans in fibrosis patients with HBV infection than in healthy controls. The biantennary (NA2) and the triantennary (NA3) N-glycans decreased significantly (P<0.001) with increased severity of fibrosis. The diagnostic power of serum glycan marker (GlycoFibroTest) [area under the curve (AUC)=0.735) was similar to that of FibroTest (AUC=0.740) for discriminating between moderate and advanced fibrosis (F3-F6) from non- or mild fibrosis (F0-F2). However, GlycoFibroTest (AUC=0.740) was slightly better than FibroTest (AUC=0.696) for distinguishing fibrotic patients (F1 or more) from non-fibrotic patients (F0). CONCLUSIONS: The assay for serum glycan profiling showed satisfactory reproducibility and is a non-invasive blood test for the diagnosis of liver fibrosis. The changes of N-glycan level in serum can be used to monitor or follow-up the progress of fibrosis using specific N-glycan markers.

Zinc inhibits lethal inflammatory shock by preventing microbe-induced interferon signature in intestinal epithelium.

The cytokine TNF drives inflammatory diseases, e.g., Crohn's disease. In a mouse model of TNF-induced systemic inflammatory response syndrome (SIRS), severe impact on intestinal epithelial cells (IECs) is observed. Zinc confers complete protection in this model. We found that zinc no longer protects in animals which lack glucocorticoids (GCs), or express mutant versions of their receptor GR in IECs, nor in mice which lack gut microbiota. RNA-seq studies in IECs showed that zinc caused reduction in expression of constitutive (STAT1-induced) interferon-stimulated response (ISRE) genes and interferon regulatory factor (IRF) genes. Since some of these genes are involved in TNF-induced cell death in intestinal crypt Paneth cells, and since zinc has direct effects on the composition of the gut microbiota (such as several Staphylococcus species) and on TNF-induced Paneth cell death, we postulate a new zinc-related anti-inflammatory mechanism. Zinc modulates the gut microbiota, causing less induction of ISRE/IRF genes in crypt cells, less TNF-induced necroptosis in Paneth cells, and less fatal evasion of gut bacteria into the system.

Fishing for lectins from diverse sequence libraries by yeast surface display - an exploratory study.

The establishment of a robust technology platform for the expression cloning of carbohydrate-binding proteins remains a key challenge in glycomics. Here we explore the utility of using yeast surface display (YSD) technology in the interaction-based lectin cloning from complete cDNA libraries. This should pave the way for more detailed studies of protein-carbohydrate interactions. To evaluate the performance of this system, lectins representing three different subfamilies (galectins, siglecs, and C-type lectins) were successfully displayed on the surface of Saccharomyces cerevisiae and Pichia pastoris as a-agglutinin and/or alpha-agglutinin fusions. The predicted carbohydrate-binding activity could be detected for three out of five lectins tested (galectin-1, galectin-3, and siaoadhesin). For galectin-4 and E-selectin, no specific carbohydrate-binding activity could be detected. We also demonstrate that proteins with carbohydrate affinity can be specifically isolated from complex metazoan cDNA libraries through multiple rounds of FACS sorting, employing multivalent, fluorescent-labeled polyacrylamide-based glycoconjugates.

N-glycomic changes in hepatocellular carcinoma patients with liver cirrhosis induced by hepatitis B virus.

UNLABELLED: We evaluated the use of blood serum N-glycan fingerprinting as a tool for the diagnosis of hepatocellular carcinoma (HCC) in patients with cirrhosis induced by hepatitis B virus (HBV). A group of 450 HBV-infected patients with liver fibrosis or cirrhosis with or without HCC were studied. HCC was diagnosed by alpha-fetoprotein (AFP) analysis, ultrasonography, and/or computed tomography and was studied histologically. N-glycan profiles of serum proteins were determined with DNA sequencer-based carbohydrate analytical profiling technology. In this study, we found that a branch alpha(1,3)-fucosylated triantennary glycan was more abundant in patients with HCC than in patients with cirrhosis, patients with fibrosis, and healthy blood donors, whereas a bisecting core alpha(1,6)-fucosylated biantennary glycan was elevated in patients with cirrhosis. The concentration of these 2 glycans and the log ratio of peak 9 to peak 7 (renamed the GlycoHCCTest) were associated with the tumor stage. Moreover, for screening patients with HCC from patients with cirrhosis, the overall sensitivity and specificity of the GlycoHCCTest were very similar to those of AFP. CONCLUSION: This study indicates that a branch alpha(1,3)-fucosylated glycan is associated with the development of HCC. The serum N-glycan profile is a promising noninvasive method for detecting HCC in patients with cirrhosis and could be a valuable supplement to AFP in the diagnosis of HCC in HBV-infected patients with liver cirrhosis. Its use for the screening, follow-up, and management of patients with cirrhosis and HCC should be evaluated further.

Over-expression of heat shock protein 70 in mice is associated with growth retardation, tumor formation, and early death.

Experiments in lower organisms, such as worms and flies, indicate that the molecular chaperone protein heat shock protein 70 (HSP70) is a longevity factor. In contrast, we demonstrate here that mice overexpressing HSP70 display growth retardation and early death. HSP70 transgenic mice displayed increased levels of serum corticosterone and weaker expression and activity of the glucocorticoid receptor in the liver. Serum insulin-like growth factor-1 (IGF-1) concentrations in the transgenic mice were 50% lower than in the control mice, leading to growth retardation. HSP70 transgenic mice showed decreased expression of Casp9, which encodes caspase-9, and increased expression of the anti-apoptotic Bcl-2 gene, indicating that apoptosis is suppressed. Consequently, most of the transgenic animals died before the age of 18 months from tumors in their lungs and lymph nodes. We suggest that the proinflammatory and antiapoptotic effects of HSP70 might be responsible for the growth retardation, tumor formation, and early death observed in the HSP70 transgenic mice.

N-glycomic changes in serum proteins during human aging.

N-glycan profiling of the human serum glycoproteins including immunoglobulin fraction on different age groups of healthy persons shows substantial changes with increasing age in three major N-glycan structures. In individuals more than 40-50 years of age, there is an increase in under-galactosylated glycans and a decrease in the core alpha-1,6-fucosylated bi-galactosylated biantennary structure. These three glycan structures are also substantially changed in a Werner syndrome patient, to a level comparable or even more pronounced than those observed in a healthy Italian centenarian population. These data show that the glycosylation machineries in both liver cells and B-cells are affected in a similar way by the aging process despite their highly different nature. The observed changes in the glycan structures are indicative that biosynthetic processes are at the basis of the changes, possibly together with changes in serum clearing of glycan-altered proteins. Our data suggest that measurement of the N-glycan level changes could provide a noninvasive surrogate marker for general health or for age-related disease progression, and for monitoring the improvement of health after therapy.

N-glycome Profile Levels Relate to Silent Brain Infarcts in a Cohort of Hypertensives.

BACKGROUND: Silent brain infarcts (SBIs) are highly prevalent in the aged population and relate to the occurrence of further stroke and dementia. Serum N-glycome levels have been previously associated with aging and they might be related as well to the presence of SBIs and age-related white matter hyperintensities. METHODS AND RESULTS: We determined the serum N-glycome profile in a cohort study comprising 972 subjects and evaluated the relationship between N-glycome levels and the presence and number of SBIs and with age-related white matter hyperintensities grades, assessed by brain magnetic resonance imaging. Decreasing concentrations of bigalacto core-α-1,6-fucosylated biantennary glycan and increasing concentrations of branching α-1,3-fucosylated triantennary glycan remained as independent predictors of SBIs (odds ratio 0.4, 95% CI 0.3-0.7 and odds ratio 1.8, 95% CI 1-3.2, respectively), after controlling for the presence of age and classic vascular risk factors. A similar pattern was found to be related to an increasing number of SBIs and white matter hyperintensities grade. CONCLUSIONS: N-glycome levels might be potentially useful as biomarkers for the presence of silent cerebrovascular disease.

N-glycomic changes in serum proteins in type 2 diabetes mellitus correlate with complications and with metabolic syndrome parameters.

BACKGROUND: Glycosylation, i.e the enzymatic addition of oligosaccharides (or glycans) to proteins and lipids, known as glycosylation, is one of the most common co-/posttranslational modifications of proteins. Many important biological roles of glycoproteins are modulated by N-linked oligosaccharides. As glucose levels can affect the pathways leading to glycosylation of proteins, we investigated whether metabolic syndrome (MS) and type 2 diabetes mellitus (T2DM), pathological conditions characterized by altered glucose levels, are associated with specific modifications in serum N-glycome. METHODS: We enrolled in the study 562 patients with Type 2 Diabetes Mellitus (T2DM) (mean age 65.6±8.2 years) and 599 healthy control subjects (CTRs) (mean age, 58.5±12.4 years). N-glycome was evaluated in serum glycoproteins. RESULTS: We found significant changes in N-glycan composition in the sera of T2DM patients. In particular, α(1,6)-linked arm monogalactosylated, core-fucosylated diantennary N-glycans (NG1(6)A2F) were significantly reduced in T2DM compared with CTR subjects. Importantly, they were equally reduced in diabetic patients with and without complications (P<0.001) compared with CTRs. Macro vascular-complications were found to be related with decreased levels of NG1(6)A2F. In addition, NG1(6)A2F and NG1(3)A2F, identifying, respectively, monogalactosylated N-glycans with α(1,6)- and α(1,3)-antennary galactosylation, resulted strongly correlated with most MS parameters. The plasmatic levels of these two glycans were lower in T2DM as compared to healthy controls, and even lower in patients with complications and MS, that is the extreme "unhealthy" phenotype (T2DM+ with MS). CONCLUSIONS: Imbalance of glycosyltransferases, glycosidases and sugar nucleotide donor levels is able to cause the structural changes evidenced by our findings. Serum N-glycan profiles are thus sensitive to the presence of diabetes and MS. Serum N-glycan levels could therefore provide a non-invasive alternative marker for T2DM and MS.

Expression of the human ferritin light chain in a frataxin mutant yeast affects ageing and cell death.

Ferritin is one of the major eukaryotic proteins involved in regulating iron metabolism and maintaining iron homeostasis. However, Saccaromyces cerevisiae is an exception, possessing no ferritin and using other means to store excess iron. The only potential iron storage protein identified in yeast so far is the homologue of human frataxin (YFH1p). In this study, we found that dysfunction of yeast frataxin shortens mean lifespan by 49% compared to the WT control. Interestingly, the human ferritin L gene can, at least partially, complement the function of yeast frataxin, extending lifespan and protecting cells from death induced by oxidative stress or excess iron. Our findings indicate that ferritin L can perform functions in yeast that are similar to its functions in mammals, and suggest that common mechanisms may exist for preventing iron and oxidative damage in single- and multi-cellular eukaryotic organisms. Clearly, elucidation of the function of human ferritin in yeast would help in gaining a better understanding the molecular basis of iron storage diseases.

A high-throughput screening system for genes extending life-span.

We developed a high-throughput functional genomic screening system that allows identification of genes prolonging life-span in the baker's yeast Saccharomyces cerevisiae. The method is based on isolating yeast mother cells with extended number of cell divisions as indicated by the increased number of bud scars on their surface. Fluorescently labelled Wheat Germ Agglutinin was used for specific staining of chitin, a major component of bud scars. Screening of a human HepG2 cDNA expression library in yeast resulted in the isolation of 12 yeast transformants with a potentially prolonged life-span. The transgene in one of the lines was identified as ferritin light chain (FTL) and studied in more detail. Yeast cells containing FTL showed an enhanced iron and H(2)O(2) resistance, a reduced cell death rate and an increased number of cell divisions. Overexpression of FTL in the nematode Caenorhabditis elegans resulted in a life-span increase of 8% confirming our yeast observations in a multicellular organism. Our data demonstrate that this method permits a fast screening of libraries for hunting genes involved in ageing processes.