Inhibition of c-Myc Oncoprotein Limits the Growth of Human Melanoma Cells by Inducing Cellular Crisis

Abstract Here, we show that inhibition of c-Myc causes a proliferative arrest of M14 melanoma cells through cellular crisis, evident by the increase in size, multiple nuclei, vacuolated cytoplasm, induction of senescence-associated β-galactosidase activity and massive apoptosis. The c-Myc-induced crisis is associated with decreased human telomerase reverse transcriptase expression, telomerase activity, progressive telomere shortening, glutathione (GSH), depletion and, increased production of reactive oxygen species. Treatment of control cells with l-buthionine sulfoximine decreases GSH to levels of c-Myc low expressing cells, but it does not modify the growth kinetic of the cells. Surprisingly, when GSH is increased in the c-Myc low expressing cells by treatment with N-acetyl-l-cysteine, cells escape crisis. To test the hypothesis that both oxidative stress and telomerase dysfunction are involved in the c-Myc-dependent crisis, we directly inhibited telomerase function and glutathione levels. Inactivation of telomerase, by expression of a catalytically inactive, dominant negative form of reverse transcriptase, reduces cellular lifespan by inducing telomere shortening. Treatment of cells with l-buthionine sulfoximine decreases GSH content and accelerates cell crisis. Analysis of telomere status demonstrated that oxidative stress affects c-Myc-induced crisis by increasing telomere dysfunction. Our results demonstrate that inhibition of c-Myc oncoprotein induces cellular crisis through cooperation between telomerase dysfunction and oxidative stress

relA over-expression reduces tumorigenicity and activates apoptosis in human cancer cells

We previously demonstrated that bcl-2 over-expression increases the malignant behaviour of the MCF7 ADR human breast cancer cell line and enhances nuclear factor-kappa B (NF-k B) transcriptional activity. Here, we investigated the direct effect of increased NF-k B activity on the tumorigenicity of MCF7 ADR cells by over-expressing the NF-k B subunit relA/p65. Surprisingly, our results demonstrated that over-expression of relA determines a considerable reduction of the tumorigenic ability in nude mice as indicated by the tumour take and the median time of tumour appearance. In vitro studies also evidenced a reduced cell proliferation and the activation of the apoptotic programme after relA over-expression. Apoptosis was associated with the production of reactive oxygen species, and the cleavage of the specific substrate Poly-ADP-ribose-polymerrase. Our data indicate that there is no general role for NF-k B in the regulation of apoptosis and tumorigenicity. In fact, even though inhibiting NF-k B activity has been reported to be lethal to tumour cells, our findings clearly suggest that an over-induction of nuclear NF-k B activity may produce the same effect. © 2001 Cancer Research Campaign http://www.bjcancer.co

AKTIP/Ft1, a new shelterin-interacting factor required for telomere maintenance

Telomeres are nucleoprotein complexes that protect the ends of linear chromosomes from incomplete replication, degradation and detection as DNA breaks. Mammalian telomeres are protected by shelterin, a multiprotein complex that binds the TTAGGG telomeric repeats and recruits a series of additional factors that are essential for telomere function. Although many shelterin-associated proteins have been so far identified, the inventory of shelterin-interacting factors required for telomere maintenance is still largely incomplete. Here, we characterize AKTIP/Ft1 (humanAKTIP and mouse Ft1 are orthologous), a novel mammalian shelterin-bound factoridentified on the basis of its homology with the Drosophila telomere protein Pendolino. AKTIP/Ft1 shares homology with the E2 variant ubiquitin-conjugating (UEV) enzymes and has been previously implicated in the control of apoptosis and in vesicle trafficking. RNAi-mediated depletion of AKTIP results in formation of telomere disfunction foci (TIFs). Consistent with these results, AKTIP interacts with telomeric DNA and binds the shelterin components TRF1 and TRF2 both in vivo and in vitro. Analysis of AKTIP- depleted human primary fibroblasts showed that they are defective in PCNA recruiting and arrest in the S phase due to the activation of the intra S checkpoint. Accordingly, AKTIP physically interacts with PCNA and the RPA70 DNA replication factor. Ft1-depleted p53-/- MEFs did not arrest in the S phase but displayed significant increases in multiple telomeric signals (MTS) and sister telomere associations (STAs), two hallmarks of defective telomere replication. In addition, we found an epistatic relation for MST formation between Ft1 and TRF1, which has been previously shown to be required for replication fork progression through telomeric DNA. Ch-IP experiments further suggested that in AKTIP-depleted cells undergoing the S phase, TRF1 is less tightly bound to telomeric DNA than in controls. Thus, our results collectively suggest that AKTIP/Ft1 works in concert with TRF1 to facilitate telomeric DNA replication

Glutathione transferases and glutathionylated hemoglobin in workers exposed to low doses of 1,3-butadiene.

We evaluated glutathione transferase (GST) activities and the levels of glutathionylated hemoglobin in the RBCof 42 workers exposed to 1,3-butadiene in a petrochemical plant, using 43 workers not exposed to 1,3-butadiene and 82 foresters as internal and external controls, respectively. Median 1,3-butadiene exposure levels were 1.5, 0.4, and 0.1 Mg/m3 in 1,3-butadieneexposed workers, in workers not directly exposed to 1,3-butadiene, and in foresters, respectively. In addition, we determined in the peripheral blood lymphocytes of the same individuals the presence of GST polymorphic genes GSTT1 and GSTM1 and the distribution of GSTP1 allelic variants. Comparing the mean values observed in petrochemical workers with those of control foresters, we found a marked decrease of GST enzymatic activity and a significant increase of glutathionylated hemoglobin in the petrochemical workers. A weak but significant negative correlation was found between levels of 1,3-butadiene exposure and GST activity, whereas a positive correlation was found between 1,3-butadiene exposure and glutathionylated hemoglobin. A negative correlation was also observed between GST activity and glutathionylated hemoglobin. No influence of confounders was observed. Using a multiple linear regression model, up to 50.6% and 41.9% of the variability observed in glutathionylated hemoglobin and GST activity, respectively, were explained by 1,3- butadiene exposure, working setting, and GSTT1 genotype. These results indicate that occupational exposure to 1,3-butadiene induces an oxidative stress that impairs the GST balance in RBC, and suggest that GST activity and glutathionylated hemoglobin could be recommended as promising biomarkers of effect in petrochemical workers

Dyads of G-Quadruplex Ligands Triggering DNA Damage Response and Tumour Cell Growth Inhibition at Subnanomolar Concentration

Naphthalene diimide (NDI) dyads exhibiting a different substitution pattern and linker length have been synthesised and evaluated as G-quadruplex (G4) ligands, by investigating their cytotoxicity in selected cell lines. The dyads with the long C7 linker exhibit extremely low IC50 values, below 10\u2005nm, on different cancer cell lines. Contrary, the dyads with the shorter C4 linker were much less effective, with IC values increasing up to 1\u2005\u3bcm. Among the three dyads with the longest linker, small differences in the IC50 values emerge, suggesting that the linker length plays a more important role than the substitution pattern. We have further shown that the dyads are able to induce cellular DNA damage response, which is not limited to the telomeric regions and is likely the origin of their cytotoxicity. Both absorption titration and dynamic light scattering of the most cytotoxic dyads in the presence of hTel22 highlight their ability to induce effective G4 aggregation, acting as non-covalent cross-linking agents

Proteome-based plasma biomarkers for Alzheimer's disease

Alzheimer's disease is a common and devastating disease for which there is no readily available biomarker to aid diagnosis or to monitor disease progression. Biomarkers have been sought in CSF but no previous study has used two-dimensional gel electrophoresis coupled with mass spectrometry to seek biomarkers in peripheral tissue. We performed a case-control study of plasma using this proteomics approach to identify proteins that differ in the disease state relative to aged controls. For discovery-phase proteomics analysis, 50 people with Alzheimer's dementia were recruited through secondary services and 50 normal elderly controls through primary care. For validation purposes a total of 511 subjects with Alzheimer's disease and other neurodegenerative diseases and normal elderly controls were examined. Image analysis of the protein distribution of the gels alone identifies disease cases with 56% sensitivity and 80% specificity. Mass spectrometric analysis of the changes observed in two-dimensional electrophoresis identified a number of proteins previously implicated in the disease pathology, including complement factor H (CFH) precursor and α-2-macroglobulin (α- 2M). Using semi-quantitative immunoblotting, the elevation of CFH and α- 2M was shown to be specific for Alzheimer's disease and to correlate with disease severity although alternative assays would be necessary to improve sensitivity and specificity. These findings suggest that blood may be a rich source for biomarkers of Alzheimer's disease and that CFH, together with other proteins such as α- 2M may be a specific markers of this illness. © 2006 The Author(s).link_to_subscribed_fulltex

Telomerase activity, apoptosis and cell cycle progression in ataxia telangiectasia lymphocytes expressing TCL1

Individuals affected by ataxia telangiectasia (AT) have a marked susceptibility to cancer. Ataxia telangiectasia cells, in addition to defects in cell cycle checkpoints, show dysfunction of apoptosis and of telomeres, which are both thought to have a role in the progression of malignancy. In 1-5% of patients with AT, clonal expansion of T lymphocytes carrying t(14;14) chromosomal translocation, deregulating TCL1 gene(s), has been described. While it is known that these cells can progress with time to a frank leukaemia, the molecular pathway leading to tumorigenesis has not yet been fully investigated. In this study, we compared AT clonal cells, representing 88% of the entire T lymphocytes (AT94-1) and expressing TCL1 oncogene (ATM- TCL1 +), cell cycle progression to T lymphocytes of AT patients without TCL1 expression (ATM- TCL1-) by analysing their spontaneous apoptosis rate, spontaneous telomerase activity and telomere instability. We show that in ATM- TCL1+ lymphocytes, apoptosis rate and cell cycle progression are restored back to a rate comparable with that observed in normal lymphocytes while telomere dysfunction is maintained. © 2003 Cancer Research UK

Estrogen receptor-α recruits P-TEFb to overcome transcriptional pausing in intron 1 of the MYB gene

The MYB proto-oncogene is expressed in most estrogen receptor-positive (ERα +) breast tumors and cell lines. Expression of MYB is controlled, in breast cancer and other cell types, by a transcriptional pausing mechanism involving an attenuation site located ∼1.7kb downstream from the transcription start site. In breast cancer cells, ligand-bound ERα binds close to, and drives transcription beyond this attenuation site, allowing synthesis of complete transcripts. However, little is known, in general, about the factors involved in relieving transcriptional attenuation, or specifically how ERα coordinates such factors to promote transcriptional elongation. Using cyclin dependent kinase 9 (CDK9) inhibitors, reporter gene assays and measurements of total and intronic MYB transcription, we show that functionally active CDK9 is required for estrogen-dependent transcriptional elongation. We further show by ChIP and co-immunoprecipitation studies that the P-TEFb complex (CDK9/CyclinT1) is recruited to the attenuation region by ligand-bound ERα, resulting in increased RNA polymerase II Ser-2 phosphorylation. These data provide new insights into MYB regulation, and given the critical roles of MYB in tumorigenesis, suggest targeting MYB elongation as potential therapeutic strategy