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

AbstractWe 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 24-48h 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 Iβ 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

Human embryonic stem cells and derived contractile embryoid bodies are susceptible to Coxsakievirus B infection and respond to interferon Iβ 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 (P5-106 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 Iβ 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.Instituto de Biotecnologia y Biologia MolecularFacultad de Ciencias Exacta

Human embryonic stem cells and derived contractile embryoid bodies are susceptible to Coxsakievirus B infection and respond to interferon Iβ 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 (P5-106 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 Iβ 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.Instituto de Biotecnologia y Biologia MolecularFacultad de Ciencias Exacta

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

E2F1 increases the transcriptional activity of p19INK4d.

<p><b>A</b>. BHK-ER-E2F1 cells were treated with 4-OHT for 8 h and subjected to nuclear run-on assay. Transcription rates of the indicated genes were normalized to that of β-tubulin. Results are representative of two independent experiments. <b>B–D</b>. Indicated cells were cotransfected with p19CAT (4.4 µg) and pCEFL-β-galactosidase (5 µg) reporter plasmids. CAT activity was determined 48 h after transfection and normalized to β-galactosidase activity. BHK-21 cells were transfected with reporter plasmids and 1 or 3 µg of E2F1 expression vector and grown in medium containing 10% or 1% FBS (<b>B</b>). BHK-ER-E2F1 cells were transfected with reporter plasmids, grown in medium containing 10% or 1% FBS, and treated with 4-OHT as indicated (<b>C</b>). BHK-ER-E2F1 were transfected with wild-type or mutant pRB expression vectors (6 µg) or wild-type or mutant E2F DO (100 nM) for 24 h. Cells were transfected with reporter plasmids for another 24 h before 4-OHT treatment (<b>D</b>). In panels <b>B</b>, <b>C</b> and <b>D</b> values are the average ± SD of three independent experiments, each performed in triplicates.</p

E2F1 sequentially induces cyclin E and p19 during the cell cycle.

<p><b>A</b>. WI-38 cells were synchronized by serum deprivation. Total RNA was extracted at the indicated times following serum restoration and subjected to northern blot analysis. <b>B</b>. EMSA was performed using oligonucleotides corresponding to the E2F-C site or the E2F consensus sequence (CS) as radiolabeled probes. WI-38 nuclear extracts (N.E.) were incubated with probes alone or in the presence of 20-, 50-, 100-, 200-, or 500-fold molar excess of the indicated unlabeled competitors. Relative quantification of DNA-protein complexes is shown (<i>bottom panel</i>). <b>C</b> and <b>D</b>. Synchronized BHK-ER-E2F1 cells were untreated (<b>C</b>) or treated with 4-OHT (<b>D</b>) for 3 h before cells were stimulated to re-enter the cycle. Total RNA was extracted at indicated time points and subjected to northern blot analysis. Results are representative of at least two independent experiments.</p

p19INK4d is induced by E2F1, 2 and 3.

<p><b>A</b>. BHK-21 cells were cotransfected with pCMV, E2Fs and DP1 expression vectors (0.5–2.5 µg) and pBabe-Puro (0.5 µg), with total DNA amounts normalized with pCMV. Total RNA from puromycin-resistant cells was extracted and subjected to northern blot analysis. <b>B</b>. HEK-293 cells were cotransfected with E2F1 and DP1 expression vectors (2 µg) and cell lysates (100 µg) were immunoblotted. <b>C</b>. BHK-ER-E2F1 cells were transfected as indicated with pCMV, wild-type or mutant pRB expression vectors (2.5 µg) or wild-type or mutant E2F DO (100 nM), and pBabe-Puro (0.5 µg). After 24 h, cells were treated with 4-OHT for another 24 h. Total RNA was extracted from puromycin-resistant cells and subjected to northern blot analysis. Results showed in Figures are representative of at least two independent experiments.</p