Telomeres in ICF syndrome cells are vulnerable to DNA damage due to elevated DNA:RNA hybrids.

DNA:RNA hybrids, nucleic acid structures with diverse physiological functions, can disrupt genome integrity when dysregulated. Human telomeres were shown to form hybrids with the lncRNA TERRA, yet the formation and distribution of these hybrids among telomeres, their regulation and their cellular effects remain elusive. Here we predict and confirm in several human cell types that DNA:RNA hybrids form at many subtelomeric and telomeric regions. We demonstrate that ICF syndrome cells, which exhibit short telomeres and elevated TERRA levels, are enriched for hybrids at telomeric regions throughout the cell cycle. Telomeric hybrids are associated with high levels of DNA damage at chromosome ends in ICF cells, which are significantly reduced with overexpression of RNase H1. Our findings suggest that abnormally high TERRA levels in ICF syndrome lead to accumulation of telomeric hybrids that, in turn, can result in telomeric dysfunction.

Mutations in STN1 cause Coats plus syndrome and are associated with genomic and telomere defects.

The analysis of individuals with telomere defects may shed light on the delicate interplay of factors controlling genome stability, premature aging, and cancer. We herein describe two Coats plus patients with telomere and genomic defects; both harbor distinct, novel mutations in STN1, a member of the human CTC1-STN1-TEN1 (CST) complex, thus linking this gene for the first time to a human telomeropathy. We characterized the patients' phenotype, recapitulated it in a zebrafish model and rescued cellular and clinical aspects by the ectopic expression of wild-type STN1 or by thalidomide treatment. Interestingly, a significant lengthy control of the gastrointestinal bleeding in one of our patients was achieved by thalidomide treatment, exemplifying a successful bed-to-bench-and-back approach.

Induced pluripotent stem cells as a model for telomeric abnormalities in ICF type I syndrome.

Human telomeric regions are packaged as constitutive heterochromatin, characterized by extensive subtelomeric DNA methylation and specific histone modifications. ICF (immunodeficiency, centromeric instability, facial anomalies) type I patients carry mutations in DNA methyltransferase 3B (DNMT3B) that methylates de novo repetitive sequences during early embryonic development. ICF type I patient fibroblasts display hypomethylated subtelomeres, abnormally short telomeres and premature senescence. In order to study the molecular mechanism by which the failure to de novo methylate subtelomeres results in accelerated telomere shortening, we generated induced pluripotent stem cells (iPSCs) from 3 ICF type I patients. Telomeres were elongated in ICF-iPSCs during reprogramming, and the senescence phenotype was abolished despite sustained subtelomeric hypomethylation and high TERRA levels. Fibroblast-like cells (FLs) isolated from differentiated ICF-iPSCs maintained abnormally high TERRA levels, and telomeres in these cells shortened at an accelerated rate, leading to early senescence, thus recapitulating the telomeric phenotype of the parental fibroblasts. These findings demonstrate that the abnormal telomere phenotype associated with subtelomeric hypomethylation is overridden in cells expressing telomerase, therefore excluding telomerase inhibition by TERRA as a central mechanism responsible for telomere shortening in ICF syndrome. The data in the current study lend support to the use of ICF-iPSCs for modeling of phenotypic and molecular defects in ICF syndrome and for unraveling the mechanism whereby subtelomeric hypomethylation is linked to accelerated telomeric loss in this syndrome.

Role of the plasma membrane leaflets in drug uptake and multidrug resistance.

The present study aimed to investigate the role played by the leaflets of the plasma membrane in the uptake of drugs into cells and in their extrusion by P-glycoprotein and multidrug resistance-associated protein 1. Drug accumulation was monitored by fluorescence resonance energy transfer from trimethylammonium-diphenyl-hexatriene (TMA-DPH) located at the outer leaflet to a rhodamine analog. Uptake of dye into cells whose mitochondria had been inactivated was displayed as two phases of TMA-DPH fluorescence quenching. The initial phase comprised a rapid drop in fluorescence that was neither affected by cooling the cells on ice, nor by activity of mitochondria or ABC transporters. This phase reflects the association of dye with the outer leaflet of the plasma membrane. The subsequent phase of TMA-DPH fluorescence quenching occurred in drug-sensitive cell lines with a half-life in the range 20-40 s. The second phase of fluorescence quenching was abolished by incubation of the cells on ice and was transiently inhibited in cells with active mitochondria. Thus, the second phase of fluorescence quenching reflects the accumulation of dye in the cytoplasmic leaflet of the plasma membrane, presumably as a result of flip-flop of dye across the plasma membrane and slow diffusion from the inner leaflet into the cells. Whereas activity of P-glycoprotein prevented the second phase of fluorescence quenching, the activity of multidrug resistance-associated protein 1 had no effect on this phase. Thus, P-glycoprotein appears to pump rhodamines from the cytoplasmic leaflet either to the outer leaflet or to the outer medium.

Competition between innate multidrug resistance and intracellular binding of rhodamine dyes.

The present study aimed to elucidate the contribution of the intracellular binding of drugs to multidrug resistance. For this purpose, uptake of rhodamines was studied in cells whose mitochondria had been uncoupled with carbonyl cyanide m-chlorophenylhydrazone. Surprisingly, in a variety of drug-untreated cells, presumed to be sensitive to multidrug resistance-type drugs, rhodamines were excluded from entering the cells. Thus, the amount of rhodamine 123 taken up into parental untreated K562 cells was less than the amount bound to the cell exterior. Rhodamine uptake was prevented by an active efflux pump. The efflux was inhibited by 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl) and MK571 and, to a lesser extent, by ATP depletion, indomethacin, probenecid and vanadate. All the inhibitors, apart from NBD-Cl, are known to modulate multidrug resistance-associated protein (MRP) 1. Because MRP1 was expressed in all the cell lines tested and the efflux of rhodamines in MRP1 over-expressing cells was abolished by NBD-Cl, it appears that rhodamines are excluded from these cells by MRP1. On the other hand, the uptake of rhodamines into cells respiring with their coupled mitochondria demonstrated diminished sensitivity to NBD-Cl and MK571. Thus, active pumping into the mitochondria allowed enhanced uptake into the cells, overcoming the innate resistance. The innate resistance provided by MRP1 to cells prevents rhodamine dyes, and possibly drugs such as doxorubicin, from achieving equilibration of their concentration in the cytoplasm with their concentration in the external medium. The protection provided to multidrug resistance cells by ABC transporters has to overcome competition by passive uptake of the drugs and binding/uptake of the drugs into intracellular targets.

Modulation of P-glycoprotein-mediated multidrug resistance by acceleration of passive drug permeation across the plasma membrane.

The drug concentration inside multidrug-resistant cells is the outcome of competition between the active export of drugs by drug efflux pumps, such as P-glycoprotein (Pgp), and the passive permeation of drugs across the plasma membrane. Thus, reversal of multidrug resistance (MDR) can occur either by inhibition of the efflux pumps or by acceleration of the drug permeation. Among the hundreds of established modulators of Pgp-mediated MDR, there are numerous surface-active agents potentially capable of accelerating drug transbilayer movement. The aim of the present study was to determine whether these agents modulate MDR by interfering with the active efflux of drugs or by allowing for accelerated passive permeation across the plasma membrane. Whereas Pluronic P85, Tween-20, Triton X-100 and Cremophor EL modulated MDR by inhibition of Pgp-mediated efflux, with no appreciable effect on transbilayer movement of drugs, the anesthetics chloroform, benzyl alcohol, diethyl ether and propofol modulated MDR by accelerating transbilayer movement of drugs, with no concomitant inhibition of Pgp-mediated efflux. At higher concentrations than those required for modulation, the anesthetics accelerated the passive permeation to such an extent that it was not possible to estimate Pgp activity. The capacity of the surface-active agents to accelerate passive drug transbilayer movement was not correlated with their fluidizing characteristics, measured as fluorescence anisotropy of 1-(4-trimethylammonium)-6-phenyl-1,3,5-hexatriene. This compound is located among the headgroups of the phospholipids and does not reflect the fluidity in the lipid core of the membranes where the limiting step of drug permeation, namely drug flip-flop, occurs.

Transport of anthracyclines and mitoxantrone across membranes by a flip-flop mechanism.

The objectives of the present work are to characterize the transport of mitoxantrone and three anthracyclines in terms of binding to the membrane surface, flip-flop across the lipid core of the membrane, and release into the medium. Mitoxantrone and anthracyclines are positively charged amphipathic molecules, and as such are located at the surface of membranes among the headgroups of the phospholipids. Therefore, their transport across membranes occurs by a flip-flop mechanism, rather than by diffusion down a continuous concentration gradient located in the lipid core of the membrane. Flip-flop rates have been estimated with liposomes labeled at their surface with 7-nitrobenzo-2-oxa-1,3-diazol-4-yl (NBD) moiety attached to the headgroup of phosphatidylethanolamine. Flip-flop of mitoxantrone, doxorubicin, daunorubicin, and idarubicin occurred with half-lives of 6, 0.7, 0.15, and 0.1min, respectively. Partition of the drugs into the membrane occurred with lipid phase/aqueous medium coefficients of 230,000, 8600, 23,000, and 40,000 for mitoxantrone, doxorubicin, daunorubicin, and idarubicin, respectively, which are much higher than their corresponding octanol/aqueous medium values. There was no direct correlation between the lipophilicity of the drugs and their lipid phase/aqueous medium partition coefficient or their flip-flop rate. Mitoxantrone exhibited the highest affinity toward liposome membranes, but the slowest flip-flop across the lipid core of the membranes. Simulation of drug uptake into liposomes revealed that transmembrane movement of the mitoxantrone and anthracyclines is determined by their flip-flop rate and affinity toward membranes.