Generation of a human induced pluripotent stem cell line (INEUi001-A) from an amyotrophic lateral sclerosis/frontotemporal dementia patient with a C9ORF72 G4C2 genotype of <2 (GGGGCCG) and 10 repeats.

Human induced pluripotent stem cell (hiPSC) line INEUi001-A was reprogrammed from peripheral blood mononuclear cells (PBMC) using the lentiviral-hSTEMCCA-loxP vector. PBMCs were obtained from a 75- year-old female ALS/FTD disease patient carrying a heterozygous deletion within the C9ORF72 hexanucleotide repeat region resulting in a GGGGCCG sequence (∼1.16 repeats). C9ORF72 genotype was maintained and stemness and pluripotency confirmed in INEUi001-A hiPSC line.

Human embryonic stem cells display a pronounced sensitivity to the cyclin dependent kinase inhibitor Roscovitine.

BACKGROUND: The essentially unlimited expansion potential and the pluripotency of human embryonic stem cells (hESCs) make them attractive for cell-based therapeutic purposes. Although hESCs can indefinitely proliferate in culture, unlike transformed cancer cells, they are endowed with a cell-intrinsic property termed mitochondrial priming that renders them highly sensitive to apoptotic stimuli. Thus, all attempts to broaden the insights into hESCs apoptosis may be helpful for establishing pro-survival strategies valuable for its in vitro culture and further use in clinical applications. Cyclin-dependent kinases (CDKs), a family of serine/threonine protein kinases originally identified as regulators of the eukaryotic cell cycle, can also regulate transcription and differentiation. Moreover, there are compelling data suggesting that its activities are involved in certain apoptotic programs in different cell types. Currently, it is not completely determined whether CDKs regulate apoptotic processes in rapidly proliferating and apoptosis-prone hESCs. In this study, to elucidate the effect of CDKs inhibition in hESCs we used Roscovitine (ROSC), a purine analogue that selectively inhibits the activities of these kinases. RESULTS: Inhibition of CDKs by ROSC triggers programmed cell death in hESCs but not in proliferating somatic cells (human fibroblasts). The apoptotic process encompasses caspase-9 and -3 activation followed by PARP cleavage. ROSC treatment also leads to p53 stabilization, which coincides with site-specific phosphorylation at serine 46 and decreased levels of Mdm2. Additionally, we observed a transcriptional induction of p53AIP1, a repression of pro-survival factor Mcl-1 and an up-regulation of pro-apoptotic BH3-only proteins NOXA and PUMA. Importantly, we found that the role of CDK2 inhibition appears to be at best accessory as an active CDK2 is not required to ensure hESCs survival. CONCLUSION: Our experimental data reveal that hESCs, contrary to fibroblasts, exhibit a pronounced sensitivity to ROSC.

p19INK4d is involved in the cellular senescence mechanism contributing to heterochromatin formation.

BACKGROUND: During evolution, organisms with renewable tissues have developed mechanisms to prevent tumorigenesis, including cellular senescence and apoptosis. Cellular senescence is characterized by a permanent cell cycle arrest triggered by both endogenous stress and exogenous stress. The p19INK4d, a member of the family of cyclin-dependent kinase inhibitors (INK4), plays an important role on cell cycle regulation and in the cellular DNA damage response. We hypothesize that p19INK4d is a potential factor involved in the onset and/or maintenance of the senescent state. METHODS: Senescence was confirmed by measuring the cell cycle arrest and the senescence-associated ß-galactosidase activity. Changes in p19INK4d expression and localization during senescence were determined by Western blot and immunofluorescence assays. Chromatin condensation was measured by microccocal nuclease digestion and histone salt extraction. RESULTS: The data presented here show for the first time that p19INK4d expression is up-regulated by different types of senescence. Changes in senescence-associated hallmarks were driven by modulation of p19 expression indicating a direct link between p19INK4d induction and the establishment of cellular senescence. Following a senescence stimulus, p19INK4d translocates to the nucleus and tightly associates with chromatin. Moreover, reduced levels of p19INK4d impair senescence-related global genomic heterochromatinization. Analysis of p19INK4d mRNA and protein levels in tissues from differently aged mice revealed an up-regulation of p19INK4d that correlates with age. CONCLUSION: We propose that p19INK4d participates in the cellular mechanisms that trigger senescence by contributing to chromatin compaction. GENERAL SIGNIFICANCE: This study provides novel insights into the dynamics process of cellular senescence, a central tumor suppressive mechanism.

Activation of apoptotic signalling events in human embryonic stem cells upon Coxsackievirus B3 infection.

Human embryonic stem cells (hESCs) are self-renewing pluripotent cells that can differentiate to a wide range of specialized cells and hold great promise as models for human development and disease, as well as for drug discovery and cell-replacement therapies. Group B Coxsackie viruses (CVBs) produce acute myocarditis, pancreatitis, non-septic meningitis and encephalitis in neonates, children and young adults. Moreover, CVBs can produce spontaneous miscarriage after early embryo infection. It was reported that hESCs express CVBs receptors and are susceptible to CVB3 infection. Apoptosis is one of the hallmarks of CVBs infection although details regarding CVB3 involvement in the apoptotic processes remain elusive. In order to evaluate the mechanisms of cell death induced by CVB3 in these pluripotent cells, we infected HUES-5 (H5) and WA01 (H1) hESC lines with CVB3. After validating the maintenance of stemness in these hESC lines when grown as confluent monolayers in feeder-free conditions, we analysed several aspects of programmed cell death triggered by CVB3. In all cases, we detected chromatin condensation, DNA fragmentation and caspase-9 and 3 cleavages. Moreover, we observed the presence of cleaved PARP product which was preceded by the appearance of p17, the catalytically active fragment of caspase-3. Mitochondrial function assays revealed a MOI dependent decrease in cell viability at 24 h post-infection (pi). No appreciable modifications in Bcl-2, Bcl-X(L) and Bax protein levels were observed upon CVB3 infection during 5-24 h observation period. However, a marked decrease in pro-apoptotic Bad abundance was detected without changes in its mRNA levels. In this study we found that the hESCs are highly susceptible to CVB3 infection and display elevated apoptosis rates, thus emerging as suitable human non-transformed in vitro models to study CVB3-induced apoptosis and resulting relevant to understand CVBs pathogenesis.

E2F1-mediated upregulation of p19INK4d determines its periodic expression during cell cycle and regulates cellular proliferation.

BACKGROUND: A central aspect of development and disease is the control of cell proliferation through regulation of the mitotic cycle. Cell cycle progression and directionality requires an appropriate balance of positive and negative regulators whose expression must fluctuate in a coordinated manner. p19INK4d, a member of the INK4 family of CDK inhibitors, has a unique feature that distinguishes it from the remaining INK4 and makes it a likely candidate for contributing to the directionality of the cell cycle. p19INK4d mRNA and protein levels accumulate periodically during the cell cycle under normal conditions, a feature reminiscent of cyclins. METHODOLOGY/PRINCIPAL FINDINGS: In this paper, we demonstrate that p19INK4d is transcriptionally regulated by E2F1 through two response elements present in the p19INK4d promoter. Ablation of this regulation reduced p19 levels and restricted its expression during the cell cycle, reflecting the contribution of a transcriptional effect of E2F1 on p19 periodicity. The induction of p19INK4d is delayed during the cell cycle compared to that of cyclin E, temporally separating the induction of these proliferative and antiproliferative target genes. Specific inhibition of the E2F1-p19INK4d pathway using triplex-forming oligonucleotides that block E2F1 binding on p19 promoter, stimulated cell proliferation and increased the fraction of cells in S phase. CONCLUSIONS/SIGNIFICANCE: The results described here support a model of normal cell cycle progression in which, following phosphorylation of pRb, free E2F induces cyclin E, among other target genes. Once cyclinE/CDK2 takes over as the cell cycle driving kinase activity, the induction of p19 mediated by E2F1 leads to inhibition of the CDK4,6-containing complexes, bringing the G1 phase to an end. This regulatory mechanism constitutes a new negative feedback loop that terminates the G1 phase proliferative signal, contributing to the proper coordination of the cell cycle and provides an additional mechanism to limit E2F activity.

Human embryonic stem cells and derived contractile embryoid bodies are susceptible to Coxsakievirus B infection and respond to interferon Iß treatment.

We studied the susceptibility of human embryonic stem cells and derived contractile embryoid bodies from WAO9, HUES-5 and HUES-16 cell lines to Coxsackievirus B infection. After validating stem cell-like properties and cardiac phenotype, Coxsackievirus B receptors CAR and DAF, as well as type I interferon receptors were detected in all cell lines and differentiation stages studied. Real-time PCR analysis showed that CAR mRNA levels were 3.4-fold higher in undifferentiated cells, while DAF transcript levels were 2.78-fold more abundant in differentiated cultures (P<0.05). All cell lines were susceptible to Coxsackievirus serotypes B1-5 infection as shown by RT-PCR detection of viral RNA, immunofluorescence detection of viral protein and infectivity titration of cell culture supernatants resulting in cell death. Supernatants infectivity titers 24-48 h post-infection ranged from 105-106 plaque forming units (PFU)/ml, the highest titers were detected in undifferentiated cells. Cell viability detected by a colorimetric assay, showed inverse correlation with infectivity titers of cell culture supernatants. Treatment with 100 U of interferon Iß significantly reduced viral replication and associated cell death during a 24-48 h observation period, as detected by reduced infectivity titers in the supernatants and increased cell viability by a colorimetric assay, respectively. We propose human embryonic stem cell and derived contractile embryoid bodies as a valid model to study cardiac Coxsackievirus B infection.

Transcriptional upregulation of p19INK4d upon diverse genotoxic stress is critical for optimal DNA damage response.

p19INK4d promotes survival of several cell lines after UV irradiation due to enhanced DNA repair, independently of CDK4 inhibition. To further understand the action of p19INK4d in the cellular response to DNA damage, we aimed to elucidate whether this novel regulator plays a role only in mechanisms triggered by UV or participates in diverse mechanisms initiated by different genotoxics. We found that p19INK4d is induced in cells injured with cisplatin or beta-amyloid peptide as robustly as with UV. The mentioned genotoxics transcriptionally activate p19INK4d expression as demonstrated by run-on assay without influencing its mRNA stability and with partial requirement of protein synthesis. It is not currently known whether DNA damage-inducible genes are turned on by the DNA damage itself or by the consequences of that damage. Experiments carried out in cells transfected with distinct damaged DNA structures revealed that the damage itself is not responsible for the observed up-regulation. It is also not known whether the increased expression of DNA-damage-inducible genes is related to immediate protective responses such as DNA repair or to more delayed responses such as cell cycle arrest or apoptosis. We found that ectopic expression of p19INK4d improves DNA repair ability and protects neuroblastoma cells from apoptosis caused by cisplatin or beta-amyloid peptide. Using clonal cell lines where p19INK4d levels can be modified at will, we show that p19INK4d expression correlates with increased survival and clonogenicity. The results presented here, prompted us to suggest that p19INK4d displays an important role in an early stage of cellular DNA damage response.

INK4 proteins, a family of mammalian CDK inhibitors with novel biological functions.

The cyclin D-Cdk4-6/INK4/Rb/E2F pathway plays a key role in controlling cell growth by integrating multiple mitogenic and antimitogenic stimuli. The members of INK4 family, comprising p16(INK4a), p15(INK4b), p18(INK4c), and p19(INK4d), block the progression of the cell cycle by binding to either Cdk4 or Cdk6 and inhibiting the action of cyclin D. These INK4 proteins share a similar structure dominated by several ankyrin repeats. Although they appear to be structurally redundant and equally potent as inhibitors, the INK4 family members are differentially expressed during mouse development. The striking diversity in the pattern of expression of INK4 genes suggested that this family of cell cycle inhibitors might have cell lineage-specific or tissue-specific functions. The INK4 proteins are commonly lost or inactivated by mutations in diverse types of cancer, and they represent established or candidate tumor suppressors. Apart from their capacity to arrest cells in the G1-phase of the cell cycle they have been shown to participate in an increasing number of cellular processes. Given their emerging roles in fundamental physiological as well as pathological processes, it is interesting to explore the diverse roles for the individual INK4 family members in different functions other than cell cycle regulation. Extensive studies, over the past few years, uncover the involvement of INK4 proteins in senescence, apoptosis, DNA repair, and multistep oncogenesis. We will focus the discussion here on these unexpected issues.

Cell cycle inhibitor, p19INK4d, promotes cell survival and decreases chromosomal aberrations after genotoxic insult due to enhanced DNA repair.

Genome integrity and cell proliferation and survival are regulated by an intricate network of pathways that includes cell cycle checkpoints, DNA repair and recombination, and programmed cell death. It makes sense that there should be a coordinated regulation of these different processes, but the components of such mechanisms remain unknown. In this report, we demonstrate that p19INK4d expression enhances cell survival under genotoxic conditions. By using p19INK4d-overexpressing clones, we demonstrated that p19INK4d expression correlates with the cellular resistance to UV treatment with increased DNA repair activity against UV-induced lesions. On the contrary, cells transfected with p19INK4d antisense cDNA show reduced ability to repair DNA damage and increased sensitivity to genotoxic insult when compared with their p19INK4d-overexpressing counterparts. Consistent with these findings, our studies also show that p19INK4d-overexpressing cells present not only a minor accumulation of UV-induced chromosomal aberrations but a lower frequency of spontaneous chromosome abnormalities than p19INK4d-antisense cells. Lastly, we suggest that p19INK4d effects are dissociated from its role as CDK4/6 inhibitor. The results presented herein support a crucial role for p19INK4d in regulating genomic stability and overall cell viability under conditions of genotoxic stress. We propose that p19INK4d would belong to a protein network that would integrate DNA repair, apoptotic and checkpoint mechanisms in order to maintain the genomic integrity.

Induction of p19INK4d in response to ultraviolet light improves DNA repair and confers resistance to apoptosis in neuroblastoma cells.

The genetic instability driving tumorigenesis is fueled by DNA damage and by errors made by the DNA replication. Upon DNA damage the cell organizes an integrated response not only by the classical DNA repair mechanisms but also involving mechanisms of replication, transcription, chromatin structure dynamics, cell cycle progression, and apoptosis. In the present study, we investigated the role of p19INK4d in the response driven by neuroblastoma cells against DNA injury caused by UV irradiation. We show that p19INK4d is the only INK4 protein whose expression is induced by UV light in neuroblastoma cells. Furthermore, p19INK4d translocation from cytoplasm to nucleus is observed after UV irradiation. Ectopic expression of p19INK4d clearly reduces the UV-induced apoptosis as well as enhances the cellular ability to repair the damaged DNA. It is clearly shown that DNA repair is the main target of p19INK4d effect and that diminished apoptosis is a downstream event. Importantly, experiments performed with CDK4 mutants suggest that these p19INK4d effects would be independent of its role as a cell cycle checkpoint gene. The results presented herein uncover a new role of p19INK4d as regulator of DNA-damage-induced apoptosis and suggest that it protects cells from undergoing apoptosis by allowing a more efficient DNA repair. We propose that, in addition to its role as cell cycle inhibitor, p19INK4d is involved in maintenance of DNA integrity and, therefore, would contribute to cancer prevention.

Repression of 5-aminolevulinate synthase gene by the potent tumor promoter, TPA, involves multiple signal transduction pathways.

The potent tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA) induces activator protein-1 (AP-1) transcription factors, early response genes involved in a diverse set of transcriptional regulatory processes, and protein kinase C (PKC) activity. This work was designed to explore the signal transduction pathways involved in TPA regulation of 5-aminolevulinate synthase (ALAS) gene expression, the mitochondrial matrix enzyme that catalyzes the first and rate-limiting step of heme biosynthesis. We have previously reported that TPA causes repression of ALAS gene, but the signaling pathways mediating this effect remain elusive. The present study investigates the role of different cascades often implicated in the propagation of phorbol ester signaling. To explore this, we combined the transient overexpression of regulatory proteins involved in these pathways and the use of small cell permeant inhibitors in human hepatoma HepG2 cells. In these experimental conditions, we analyzed TPA action upon endogenous ALAS mRNA levels, as well as the promoter activity of a fusion reporter construct, harboring the TPA-responsive region of ALAS gene driving chloramphenicol acetyl transferase gene expression. We demonstrated that the participation of alpha isoform of PKC, phosphatidylinositol 3-kinase (PI3K), extracellular-signal regulated kinase (ERK1/2), and c-Jun N-terminal kinase (JNK) is crucial for the end point response. Remarkably, in this case, ERK activation is achieved in a Ras/Raf/MEK-independent manner. We also propose that p90RSK would be a convergent point between PI3K and ERK pathways. Furthermore, we elucidated the crosstalk among the components of the cascades taking part in TPA-mediated ALAS repression. Finally, by overexpression of a constitutively active p90RSK and the coactivator, cAMP-response element protein (CREB)-binding protein (CBP), we reinforced our previous model, that implies competition between AP-1 and CREB for CBP.

Hepatic nuclear factor 3 and nuclear factor 1 regulate 5-aminolevulinate synthase gene expression and are involved in insulin repression.

Although the negative regulation of gene expression by insulin has been widely studied, the transcription factors responsible for the insulin effect are still unknown. The purpose of this work was to explore the molecular mechanisms involved in the insulin repression of the 5-aminolevulinate synthase (ALAS) gene. Deletion analysis of the 5'-regulatory region allowed us to identify an insulin-responsive region located at -459 to -354 bp. This fragment contains a highly homologous insulin-responsive (IRE) sequence. By transient transfection assays, we determined that hepatic nuclear factor 3 (HNF3) and nuclear factor 1 (NF1) are necessary for an appropriate expression of the ALAS gene. Insulin overrides the HNF3beta or HNF3beta plus NF1-mediated stimulation of ALAS transcriptional activity. Electrophoretic mobility shift assay and Southwestern blotting indicate that HNF3 binds to the ALAS promoter. Mutational analysis of this region revealed that IRE disruption abrogates insulin action, whereas mutation of the HNF3 element maintains hormone responsiveness. This dissociation between HNF3 binding and insulin action suggests that HNF3beta is not the sole physiologic mediator of insulin-induced transcriptional repression. Furthermore, Southwestern blotting assay shows that at least two polypeptides other than HNF3beta can bind to ALAS promoter and that this binding is dependent on the integrity of the IRE. We propose a model in which insulin exerts its negative effect through the disturbance of HNF3beta binding or transactivation potential, probably due to specific phosphorylation of this transcription factor by Akt. In this regard, results obtained from transfection experiments using kinase inhibitors support this hypothesis. Due to this event, NF1 would lose accessibility to the promoter. The posttranslational modification of HNF3 would allow the binding of a protein complex that recognizes the core IRE. These results provide a potential mechanism for the insulin-mediated repression of IRE-containing promoters.

Inhibitory effect of AP-1 complex on 5-aminolevulinate synthase gene expression through sequestration of cAMP-response element protein (CRE)-binding protein (CBP) coactivator.

Activation protein-1 (AP-1) transcription factors are early response genes involved in a diverse set of transcriptional regulatory processes. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) is often used to induce AP-1 activity. The purpose of this work was to explore the molecular mechanisms involved in the TPA regulation of ubiquitous 5-aminolevulinate synthase (ALAS) gene expression, the first and rate-controlling step of the heme biosynthesis. Previous analysis of the 5'-flanking sequence of ALAS revealed the existence of two cAMP-response elements (CRE) required for basal and cAMP-stimulated expression. The fragment -833 to +42 in the 5'-flanking region of rat ALAS gene was subcloned into a chloramphenicol acetyltransferase (CAT) reporter vector. The expression vector pALAS/CAT produced a significant CAT activity in transiently transfected HepG2 human hepatoma cells, which was repressed by TPA. Sequence and deletion analysis detected a TPA response element (TRE), located between -261 and -255 (TRE-ALAS), that was critical for TPA regulation. We demonstrated that c-Fos, c-Jun, and JunD are involved in TPA inhibitory effect due to their ability to bind TRE-ALAS, evidenced by supershift analysis and their capacity to repress promoter activity in transfection assays. Repression of ALAS promoter activity by TPA treatment or Fos/Jun overexpression was largely relieved when CRE protein-binding protein or p300 was ectopically expressed. When the TRE site was placed in a different context with respect to CRE sites, it appeared to act as a transcriptional enhancer. We propose that the decrease in ALAS basal activity observed in the presence of TPA may reflect a lower ability of this promoter to assemble the productive pre-initiation complex due to CRE protein-binding protein sequestration. We also suggest that the transcriptional properties of this AP-1 site would depend on a spatial-disposition-dependent manner with respect to the CRE sites and to the transcription initiation site.