János Aradi

Immunodetection of human telomerase reverse-transcriptase (hTERT) re-appraised: nucleolin and telomerase cross paths

The involvement of telomerase in cellular immortalization and senescence has often been assessed by means of telomerase expression at the RNA level and quantification of telomerase activity by the telomeric repeat amplification protocol assay. However, these methods either neglected the existence of various telomerase splice variants, or ignored the nonconventional functions of telomerase independent of its ability to elongate and maintain telomere length. Immunodetection of telomerase is now being recognized as a necessary approach to precisely elucidate its roles in oncogenesis and senescence. A few antibodies directed against the catalytic subunit of the human telomerase (hTERT) are currently used but their specificity is not always demonstrated. A survey of the literature showed inconsistencies and led us to comparatively re-evaluate the most frequently used antibodies. Surprisingly, mass spectrometry, two-dimensional gel analysis and immunofluorescent experiments revealed that the most frequently used hTERT immunoprobe, a mouse monoclonal antibody that was claimed to be directed against an hTERT protein epitope, in fact recognizes nucleolin rather than telomerase. Our findings have interesting implications regarding the biology of nucleolin and telomerase in the context of pathophysiological investigations recently carried out.

Interleukin-2 family cytokines stimulate phosphorylation of the Pro-Ser-Pro motif of Stat5 transcription factors in human T cells: resistance to suppression of multiple serine kinase pathways.

Signal transducer and activator of transcription (Stat)5a and Stat5b are critical for normal immune function. Progression of T cells through G1‐S phase of cell cycle requires T cell receptor (TCR)‐ and/or cytokine‐inducible tyrosine phosphorylation of Stat5a/b. Stat5a/b may also, in a cell‐dependent manner, be constitutively or cytokine‐inducibly phosphorylated on a Pro‐Ser‐Pro (PSP) motif located within the transcriptional activation domain. Phosphorylation of the PSP motif is needed for maximal transcriptional activation by Stat5, at least in certain promoter contexts. The basal and cytokine‐inducible serine phosphorylation state of Stat5a/b has not been determined in T cells. Using primary human T cells and T lymphocytic cell lines coupled with novel phospho‐specific antibodies to this conserved phosphoserine motif in Stat5a or Stat5b, we report that: Stat5a and Stat5b were unphosphorylated on the PSP motif under basal conditions and became markedly phosphorylated in response to several T cell growth factor stimuli, including interleukin (IL)‐2, ‐7, ‐9, and ‐15 and phorbol ester 12‐myristate 13‐acetate but not TCR engagement; inducible Stat5a/b serine phosphorylation differed quantitatively and temporally; and Stat5a/b serine phosphorylation was, in contrast to inducible Stat3 serine phosphorylation, insensitive to inhibitors of mitogen‐activated protein kinase, phosphatidylinositol‐3 kinase, and mammalian target of rapamycin or deletion of Raf‐A, ‐B, or ‐C by antisense oligonucleotides. We conclude that IL‐2 family cytokines tightly control Stat5 serine phosphorylation through a kinase distinct from the Stat3 serine kinase.

Retinoid/arsenic combination therapy of promyelocytic leukemia: induction of telomerase-dependent cell death

Acute promyelocytic leukemia (APL) is efficiently treated with a cell differentiation inducer, all-trans retinoic acid (ATRA). However, a significant percentage of patients still develop resistance to this treatment. Recently, arsenic trioxide (As2O3), alone or in combination with ATRA, has been identified as an alternative therapy in patients with both ATRA-sensitive and ATRA-resistant APL. Previous investigations restricted the mechanism of this synergism to the modulation and/or degradation of PML-RARα oncoprotein through distinct pathways. In this study, using several ATRA maturation-resistant APL cell lines, we demonstrate in vitro that the success of ATRA/As2O3 treatment in APL pathology can be explained, at least in part, by a synergistic effect of these two drugs in triggering downregulation of telomerase efficient enough to cause telomere shortening and subsequent cell death. Such long-term low-dose combinatorial therapy strategies, developed also to avoid acute side effects, reinforce the notion that the antitelomerase strategy, based on a combination of active agents, should now be considered and evaluated not only in APL but also in other malignancies.

Epigenetic plasticity of hTERT gene promoter determines retinoid capacity to repress telomerase in maturation-resistant acute promyelocytic leukemia cells

The expression of hTERT gene, encoding the catalytic subunit of telomerase, is a feature of most cancer cells. Changes in the chromatin environment of its promoter and binding of transcriptional factors have been reported in differentiating cells when its transcription is repressed. However, it is not clear whether these changes are directly involved in this repression or only linked to differentiation. In a maturation-resistant acute promyelocytic leukemia (APL) cell line (NB4-LR1), we have previously identified a new pathway of retinoid-induced hTERT repression independent of differentiation. Using a variant of this cell line (NB4-LR1SFD), which resists to this repression, we show that although distinct patterns of histone modifications and transcription factor binding at the proximal domain of hTERT gene promoter could concur to modulate its expression, this region is not sufficient to the on/off switch of hTERT by retinoids. DNA methylation analysis of the hTERT promoter led to the identification of two distinct functional domains, a proximal one, fully unmethylated in both cell lines, and a distal one, significantly methylated in NB4-LR1SFD cells, whose methylation was further re-enforced by retinoid treatment. Interestingly, we showed that the binding to this distal domain of a known hTERT repressor, WT1, was defective only in NB4-LR1SFD cells. We propose that epigenetic modifications targeting this distal region could modulate the binding of hTERT repressors and account either for hTERT reactivation and resistance to retinoid-induced hTERT downregulation.

Inhibition of human telomerase by oligonucleotide chimeras, composed of an antisense moiety and a chemically modified homo‐oligonucleotide

Most tumor cells attain their immortality by reactivating telomerase. We report here the telomerase inhibitory potential of chimeric oligonucleotides composed of a 13mer antisense sequence targeting the telomerase RNA template region and a (s4dU) n moiety at its 3′ or 5′‐end. The increase of the thiolated chain length enhances the telomerase inhibitory potential, but decreases specificity, indicated by HIV reverse transcriptase inhibition. Chimeras with 5′ (s4dU) n s were more potent inhibitors than the antisense alone or the 3′ modified ones. Cy5‐labeled (s4dU)4AS and (s4dU)8AS proved the internalization of the oligonucleotides, raising the possibility to be tested as cellular anti‐telomerase agents.

(s4dU)35: a novel, highly potent oligonucleotide inhibitor of the human immunodeficiency virus type 1 reverse transcriptase

Oligodeoxycytidylates were converted to s4dUMP‐containing oligomers by treatment with liquid H2S. The inhibitory potency of the modified oligonucleotides on human immunodeficiency virus type 1 reverse transcriptase depended on the chain length and on the percentage of modification. The most potent reverse transcriptase inhibitor was (s4dU)35. The inhibitory pattern was competitive, when either poly(A)·(dT)16 or poly(C)·(dG)16 was used as template‐primer (variable substrate), suggesting that the free enzyme interacts with (s4dU)35. The K i values were 3.0 and 2.2 nM in the presence of poly(A)·(dT)16 and poly(C)·(dG)16, respectively.

A challenging epigenetic message: telomerase activity is associated with complex changes in lifestyle

As an outcome of The 2009 Nobel Prize in Physiology or Medicine, a connection has been highlighted between the length of telomeres and epigenetic effects, such as intensive changes in lifestyle and nutrition as well as behavioural and psychological factors. In this review, the various elements of molecular, cell biological, nutritional and lifestyle changes are introduced and discussed.

4-Thio-uridylate (UD29) interferes with the function of protein –SH and inhibits HIV replication in vitro

In this short communication, it is shown that 4-thio-uridylate (s(4)UMP, designated as UD29) inhibits glyceraldehyde 3-phosphate dehydrogenase (GAPDH), suggesting that the enol-form of the thiolated nucleotide may interfere with the function of the essential -SH group in the active center of the enzyme. Since HIV entry requires thiol/disulfide exchange processes, this activity prompted us to study the anti-HIV activity of the nucleotide. Indeed, UD29 inhibited the replication of HIV-1(IIIB) in the MT-4 cell line and HIV-1(Ada-M) in peripheral blood mononuclear cells (PBMC). Furthermore, UD29 was not toxic in PBMCs in vitro or in mice when the compound was administered intravenously.

In vitro and in vivo activity of 4-thio-uridylate against JY cells, a model for human acute lymphoid leukemia.

We have previously reported the in vitro anti-proliferative effect of 4-thio-uridylate (s(4)UMP) on OCM-1 uveal melanoma cells. Here, we assessed the efficacy of s(4)UMP on JY cells. Treatment of JY cells with s(4)UMP suppressed their colony forming activity and induced apoptosis; healthy human bone marrow granulocyte-macrophage progenitor cells were 14-fold less sensitive to the nucleotide. In vivo effectiveness of s(4)UMP was determined using xenograft SCID mouse model. s(4)UMP decreased the cell number and colony forming activity of the total cell content of the femur of SCID mice transplanted with JY cells without affecting the bone marrow of healthy mice. These results suggest that s(4)UMP alone or in combination with other clinically approved anti-leukemic remedies should be further explored as a potential novel therapeutic agent.

2009 Nobel Prize in Medicine and an interesting message: telomerase activity is associated with lifestyle

A 2009. evi orvosi Nobel-dijat a telomerak es a telomeraz enzim felfedezeseert es az ezekkel kapcsolatos uttorő kutatasokert harom, Amerikaban dolgozo tudos kapta megosztva. Nem regota az is ismert, hogy bizonyos eletmod- es taplalkozasbeli valtozasok (Ornish-fele eletmodvaltozas), magatartasbeli, pszichologiai tenyezők, valamint a telomerarovidules kozott bizonyitott kapcsolat letezik. Az osszefoglaloban ezen osszefuggesek molekularis, sejtbiologiai, eletmodbeli es magatartasi vonatkozasait, valamint ezek lehetseges osszefuggeseit mutatjak be.The 2009 Nobel Prize in Physiology and Medicine was awarded to three scientists for their pioneer research on telomeres – and the enzyme that forms them – telomerase. Their work highlighted the considerable connection between the length of telomeres and intensive changes in lifestyle and nutrition (Ornish method) as well as behavioral and psychological factors. In this review the various elements of molecular, cell biological, nutritional and lifestyle changes are introduced and discussed.The 2009 Nobel Prize in Physiology and Medicine was awarded to three scientists for their pioneer research on telomeres - and the enzyme that forms them - telomerase. Their work highlighted the considerable connection between the length of telomeres and intensive changes in lifestyle and nutrition (Ornish method) as well as behavioral and psychological factors. In this review the various elements of molecular, cell biological, nutritional and lifestyle changes are introduced and discussed.