Reed-Sternberg cells in Hodgkin's lymphoma present features of cellular senescence.

Hodgkin's Lymphoma (HL) is one of the most prevailing malignancies in young adults. Reed-Sternberg (RS) cells in HL have distinctive large cell morphology, are characteristic of the disease and their presence is essential for diagnosis. Enlarged cells are one of the hallmarks of senescence, but whether RS cells are senescent has not been previously investigated. Here we show that RS cells have characteristics of senescent cells; RS cells in HL biopsies specifically express the senescence markers and cell cycle inhibitors p21Cip1 and p16INK4a and are negative for the proliferation marker Ki-67, suggesting that these cells have ceased to proliferate. Moreover, the RS-like cells in HL lines, stained specifically for senescence-associated ß-galactosidase (SA-ß-gal). Oxidative stress promoted senescence in these cells as demonstrated by their staining for p21Cip1, p16INK4a, p53 and γH2AX. Senescent cells produce copious amounts of inflammatory cytokines termed 'senescence-associated secretory phenotype' (SASP), primarily regulated by Nuclear Factor κB (NF-κB). Indeed, we show that NF-κB activity and NF-κB-dependent cytokines production (e.g., IL-6, TNF-α, GM-CSF) were elevated in RS-like cells. Furthermore, NF-κB inhibitors, JSH-23 and curcumin reduced IL-6 secretion from RS-like cells. Thus, defining RS cells as senescent offers new insights on the origin of the proinflammatory microenvironment in HL.

Oxidative stress protection by novel telomerase activators in mesenchymal stem cells derived from healthy and diseased individuals.

Human Bone Marrow Mesenchymal Stem cells (hMSC) are a promising candidate for cytotherapy. However, the therapeutic potential is limited since the therapy requires ex-vivo cell culturing in which deterioration in cellular viability and aging is observed with time.Telomerase ribonucleoprotein complex re-elongates telomeres and therefore promotes genomic integrity, proliferation and lifespan. Recently we showed that increasing telomerase reverse transcriptase (TERT) expression by novel compound confers resistance from apoptosis induced by oxidative stress. Here we investigated the possibility that a controlled induction of human TERT (hTERT) levels by chemical compounds (AGS-499 and AGS-500) might improve the functionality of hMSC derived from healthy and neurodegenerative diseased individuals. We demonstrate that AGS treatments of hMSC increased telomerase activity and hTERT levels in a time and dose dependent manner. Prolonged treatments with the compounds increased the average telomeres length, without altering population doublings (PD) or inducing chromosomal aberrations. AGS treatments of hMSC protected the cells from apoptosis and DNA damages induced by H2O2, and from the toxicity induced by long term exposure to DMSO. These AGS effects were shown to be mediated by telomerase since they were not observed when TERT was depleted from hMSC or in mouse embryonic stem cells derived from TERT knockout mice. Furthermore, AGS compounds did not alter the functionality of hMSC as examined by their ability to differentiate into various lineages in the presence of the compounds. These results suggest that pharmaceutical increase of telomerase may confer a beneficial therapeutic advantage in regenerative medicine when hMSC therapy is applied.

Intracellular stores maintain stable cytosolic Ca(2+) gradients in epithelial cells by active Ca(2+) redistribution.

A stable localized region of high calcium concentration near the plasma membrane has been postulated to exist as an outcome of prolonged calcium influx and to play a crucial role in regulation of cellular life. However, the mechanism supporting this phenomenon is a perplexing problem. We show here that a sustained localized region of high cytosolic Ca(2+) concentration is formed near the plasma membrane. Calcium influx, calcium uptake by intracellular stores and calcium release from the stores are essential for this phenomenon. Our results strongly suggest that the mechanism of formation of stable calcium gradient near the plasma membrane involves a process of active redistribution-uptake of entering calcium into intracellular stores and its release from the stores toward the plasma membrane.

Role of calcium and calmodulin in ciliary stimulation induced by acetylcholine.

The goal of this work was to elucidate the molecular events underlying stimulation of ciliary beat frequency (CBF) induced by acetylcholine (ACh) in frog esophagus epithelium. ACh induces a profound increase in CBF and in intracellular Ca(2+) concentration ([Ca(2+)](i)) through M(1) and M(3) muscarinic receptors. The [Ca(2+)](i) slowly decays to the basal level, while CBF stabilizes at an elevated level. These results suggest that ACh triggers Ca(2+)-correlated and -uncorrelated modes of ciliary stimulation. ACh response is abolished by the phospholipase C (PLC) inhibitor U-73122 and by depletion of intracellular Ca(2+) stores but is unaffected by reduction of extracellular Ca(2+) concentration and by blockers of Ca(2+) influx. Therefore, ACh activates PLC and mobilizes Ca(2+) solely from intracellular stores. The calmodulin inhibitors W-7 and calmidazolium attenuate the ACh-induced increase in [Ca(2+)](i) but completely abolish the elevation in CBF. Therefore, elevation of [Ca(2+)](i) is necessary for CBF enhancement but does not lead directly to it. The combined effect of Ca(2+) elevation and of additional factors, presumably mobilized by Ca(2+)-calmodulin, results in a robust CBF enhancement.

Feasibility of a sustained steep Ca(2+)Gradient in the cytosol of electrically non-excitable cells.

In electrically non-excitable cells the predominant mode of calcium signaling is a biphasic rise in cytosolic calcium concentration. It results from Ca(2+)release from intracellular stores, followed by Ca(2+)influx across the plasma membrane. It has been hypothesized that prolonged calcium influx may result in a sustained local elevation of the cytosolic calcium concentration near the plasma membrane. The mathematical model presented here evaluates the cytosolic concentration of Ca(2+)as a function of time and distance from the plasma membrane. It consists of cytoplasmic calcium stores and a plasma membrane, both equipped with calcium channels and pumps, and an immobile cytoplasmic calcium buffer. The model has verified quantitatively the feasibility of a stable Ca(2+)gradient in the cytosol with high values of Ca(2+)concentration near the plasma membrane and evaluated its properties as a function of different cellular parameters. The formation of the gradient does not require special distribution of the intracellular contents, channels and pumps. However, it requires buffering of the cytosolic calcium by the intracellular stores and that the rate of calcium release from the stores near the plasma membrane be higher than in other parts of the cell. We suggest that this model can provide an adequate description of the elevated calcium plateau generally observed in electrically non-excitable cells.

PKA induces Ca2+ release and enhances ciliary beat frequency in a Ca2+-dependent and -independent manner.

The intent of this work was to evaluate the role of cAMP in regulation of ciliary activity in frog mucociliary epithelium and to examine the possibility of cross talk between the cAMP- and Ca2+-dependent pathways in that regulation. Forskolin and dibutyryl cAMP induced strong transient intracellular Ca2+ concentration ([Ca2+]i) elevation and strong ciliary beat frequency enhancement with prolonged stabilization at an elevated plateau. The response was not affected by reduction of extracellular Ca2+ concentration. The elevation in [Ca2+]i was canceled by pretreatment with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM, thapsigargin, and a phospholipase C inhibitor, U-73122. Under those experimental conditions, forskolin raised the beat frequency to a moderately elevated plateau, whereas the initial strong rise in frequency was completely abolished. All effects were canceled by H-89, a selective protein kinase A (PKA) inhibitor. The results suggest a dual role for PKA in ciliary regulation. PKA releases Ca2+ from intracellular stores, strongly activating ciliary beating, and, concurrently, produces moderate prolonged enhancement of the beat frequency by a Ca2+-independent mechanism.

Protein kinase C induced calcium influx and sustained enhancement of ciliary beating by extracellular ATP.

The major purpose of this work was to determine protein kinase C (PKC) influence on ciliary beat frequency (CBF) and to assess participation of PKC in purinergic ciliary stimulation. The experiments were performed by simultaneous measurement of [Ca2+]i and CBF on tissue culture of frog esophagus epithelium. The PKC activators TPA and DiC8 produced significant elevation of [Ca2+]i and strong frequency enhancement. The calcium elevation was inhibited by lowering the extracellular calcium level, or by La3+, but was unaffected by verapamil and the phospholipase C inhibitor U-73122, suggesting that Ca2+ influx was via non-voltage-operated calcium channels. The inhibition of [Ca2+]i elevation resulted in corresponding inhibition of CBF enhancement. The effect of TPA was blocked by the selective PKC inhibitors chelerythrine, calphostin C, and GF109203X, and by the enzyme downregulation. The downregulation of PKC, or the enzyme inhibitors did not affect the immediate response to extracellular ATP but caused rapid decay of initially stimulated [Ca2+]i and CBF to the basal level. These results suggest that PKC produces CBF enhancement via activation of calcium influx through non-voltage-operated calcium channels. This calcium influx seems to be responsible for the duration of ciliary stimulation produced by the extracellular ATP.