c-Myc overexpression sensitises colon cancer cells to camptothecin-induced apoptosis

The proto-oncogene c-Myc is overexpressed in 70% of colorectal tumours and can modulate proliferation and apoptosis after cytotoxic insult. Using an isogenic cell system, we demonstrate that c-Myc overexpression in colon carcinoma LoVo cells resulted in sensitisation to camptothecin-induced apoptosis, thus identifying c-Myc as a potential marker predicting response of colorectal tumour cells to camptothecin. Both camptothecin exposure and c-Myc overexpression in LoVo cells resulted in elevation of p53 protein levels, suggesting a role of p53 in the c-Myc-imposed sensitisation to the apoptotic effects of camptothecin. This was confirmed by the ability of PFT-alpha, a specific inhibitor of p53, to attenuate camptothecin-induced apoptosis. p53 can induce the expression of p21(Waf1/Cip1), an antiproliferative protein that can facilitate DNA repair and drug resistance. Importantly, although camptothecin treatment markedly increased p21(Waf1/Cip1) levels in parental LoVo cells, this effect was abrogated in c-Myc-overexpressing derivatives. Targeted inactivation of p21(Waf1/Cip1) in HCT116 colon cancer cells resulted in significantly increased levels of apoptosis following treatment with camptothecin, demonstrating the importance of p21(Waf1/Cip1) in the response to this agent. Finally, cDNA microarray analysis was used to identify genes that are modulated in expression by c-Myc upregulation that could serve as additional markers predicting response to camptothecin. Thirty-four sequences were altered in expression over four-fold in two isogenic c-Myc-overexpressing clones compared to parental LoVo cells. Moreover, the expression of 10 of these genes was confirmed to be significantly correlated with response to camptothecin in a panel of 30 colorectal cancer cell lines

p53 Transactivation and the Impact of Mutations, Cofactors and Small Molecules Using a Simplified Yeast-Based Screening System

The p53 tumor suppressor, which is altered in most cancers, is a sequence-specific transcription factor that is able to modulate the expression of many target genes and influence a variety of cellular pathways. Inactivation of the p53 pathway in cancer frequently occurs through the expression of mutant p53 protein. In tumors that retain wild type p53, the pathway can be altered by upstream modulators, particularly the p53 negative regulators MDM2 and MDM4. promoter, ii) single copy, chromosomally located p53-responsive and control luminescence reporters, iii) enhanced chemical uptake using modified ABC-transporters, iv) small-volume formats for treatment and dual-luciferase assays, and v) opportunities to co-express p53 with other cofactor proteins. This robust system can distinguish different levels of expression of WT and mutant p53 as well as interactions with MDM2 or 53BP1.We found that the small molecules Nutlin and RITA could both relieve the MDM2-dependent inhibition of WT p53 transactivation function, while only RITA could impact p53/53BP1 functional interactions. PRIMA-1 was ineffective in modifying the transactivation capacity of WT p53 and missense p53 mutations. This dual-luciferase assay can, therefore, provide a high-throughput assessment tool for investigating a matrix of factors that can influence the p53 network, including the effectiveness of newly developed small molecules, on WT and tumor-associated p53 mutants as well as interacting proteins

Autophagy Impairment Induces Premature Senescence in Primary Human Fibroblasts

BACKGROUND:Recent studies have demonstrated that activation of autophagy increases the lifespan of organisms from yeast to flies. In contrast to the lifespan extension effect in lower organisms, it has been reported that overexpression of unc-51-like kinase 3 (ULK3), the mammalian homolog of autophagy-specific gene 1 (ATG1), induces premature senescence in human fibroblasts. Therefore, we assessed whether the activation of autophagy would genuinely induce premature senescence in human cells. METHODOLOGY/PRINCIPAL FINDINGS:Depletion of ATG7, ATG12, or lysosomal-associated membrane protein 2 (Lamp2) by transfecting siRNA or infecting cells with a virus containing gene-specific shRNA resulted in a senescence-like state in two strains of primary human fibroblasts. Prematurely senescent cells induced by autophagy impairment exhibited the senescent phenotypes, similar to the replicatively senescent cells, such as increased senescence associated β-galactosidase (SA-β-gal) activity, reactive oxygen species (ROS) generation, and accumulation of lipofuscin. In addition, expression levels of ribosomal protein S6 kinase1 (S6K1), p-S6K1, p-S6, and eukaryotic translation initiation factor 4E (eIF4E) binding protein 1 (4E-BP1) in the mammalian target of rapamycin (mTOR) pathway and beclin-1, ATG7, ATG12-ATG5 conjugate, and the sequestosome 1 (SQSTM1/p62) monomer in the autophagy pathway were decreased in both the replicatively and the autophagy impairment-induced prematurely senescent cells. Furthermore, it was found that ROS scavenging by N-acetylcysteine (NAC) and inhibition of p53 activation by pifithrin-α or knockdown of p53 using siRNA, respectively, delayed autophagy impairment-induced premature senescence and restored the expression levels of components in the mTOR and autophagy pathways. CONCLUSION:Taken together, we concluded that autophagy impairment induces premature senescence through a ROS- and p53-dependent manner in primary human fibroblasts

Measurement of polarization amplitudes and CP asymmetries in B-0 -> phi K*(892)(0)

An angular analysis of the decay B (0) -> phi K (*)(892)(0) is reported based on a pp collision data sample, corresponding to an integrated luminosity of 1.0 fb(-1), collected at a centre-of-mass energy of root S = 7 TeV with the LHCb detector. The P-wave amplitudes and phases are measured with a greater precision than by previous experiments, and confirm about equal amounts of longitudinal and transverse polarization. The S-wave K+ pi(-) and K+ K- contributions are taken into account and found to be significant. A comparison of the B (0) -> phi K (*)(892)(0) and results shows no evidence for direct CP violation in the rate asymmetry, in the triple-product asymmetries or in the polarization amplitudes and phases

Search for the rare decay K0S→μ+μ−

A search for the decay K0S→μ+μ− is performed, based on a data sample of 1.0 fb−1 of pp collisions at √<span style="text-decoration:overline">s</span>=7 TeV collected by the LHCb experiment at the Large Hadron Collider. The observed number of candidates is consistent with the background-only hypothesis, yielding an upper limit of B(K0S→μ+μ−) < 11(9) × 10−9 at 95 (90)% confidence level. This limit is a factor of thirty below the previous measurement

Search for the rare decays Bs -> mu+ mu- and B0 -> mu+ mu-

A search for the decays Bs -> mu+ mu- and B0 -> mu+ mu- is performed with 0.37 fb^-1 of pp collisions at sqrt{s} = 7 TeV collected by the LHCb experiment in 2011. The upper limits on the branching fractions are BR (Bs -> mu+ mu-) < 1.6 x 10^-8 and BR(B0 -> mu+ mu-) < 3.6 x 10^-9 at 95% confidence level. A combination of these results with the LHCb limits obtained with the 2010 dataset leads to BR (Bs -> mu+ mu-) mu+ mu-) < 3.2 x 10^-9 at 95% confidence level.Comment: 6+19 pages, 9 figures; minor changes; matches version accepted in Phys. Lett.

Study of eta-eta ' mixing from measurement of B-(s)(0) -&gt; J/psi eta((')) decay rates

A study of B and Bs meson decays into J/ψ η and J/ψ η′ final states is performed using a data set of proton-proton collisions at centre-of-mass energies of 7 and 8 TeV, collected by the LCHb experiment and corresponding to 3.0 fb−1 of integrated luminosity. The decay B0 → J/ψ η′ is observed for the first time. The following ratios of branching fractions are measured: B(B0→J/ψη′)B(B0s→ J/ψη′)=(2.28±0.65 (stat)±0.10 (syst)±0.13 (fs/fd))×10−2,B(B0→ J/ψη)B(B0s→ J/ψη)=(1.85±0.61 (stat)±0.09 (syst)±0.11 (fs/fd))×10−2, where the third uncertainty is related to the present knowledge of fs/fd, the ratio between the probabilities for a b quark to form a Bs or a B0 meson. The branching fraction ratios are used to determine the parameters of η − η′ meson mixing. In addition, the first evidence for the decay Bs → ψ(2S)η′ is reported, and the relative branching fraction is measured, B(B0s→ ψ(2S)η′)B(B0s→ J/ψη′)=(38.7±9.0 (stat)±1.3 (syst)±0.9(B))×10−2, where the third uncertainty is due to the limited knowledge of the branching fractions of J/ψ and ψ(2S) mesons

Measurement of indirect CP asymmetries in D-0 -&gt; K-K+ and D-0 -&gt; pi(-)pi(+) decays using semileptonic B decays

Time-dependent $CP$ asymmetries in the decay rates of the singly Cabibbo-suppressed decays $D^0\rightarrow K^-K^+$ and $D^0\rightarrow \pi^-\pi^+$ are measured in $pp$ collision data corresponding to an integrated luminosity of 3.0 fb$^{-1}$ collected by the LHCb experiment. The $D^0$ mesons are produced in semileptonic $b$-hadron decays, where the charge of the accompanying muon is used to determine the initial state as $D^0$ or $\bar{D}^0$. The asymmetries in effective lifetimes between $D^0$ and $\bar{D}^0$ decays, which are sensitive to indirect $CP$ violation, are determined to be \begin{align*} A_{\Gamma}(K^-K^+) = (-0.134 \pm 0.077 \; {}^{+0.026}_{-0.034})\% \, A_{\Gamma}(\pi^-\pi^+) = (-0.092\pm 0.145 \; {}^{+0.025}_{-0.033})\% \, \end{align*} where the first uncertainties are statistical and the second systematic. This result is in agreement with previous measurements and with the hypothesis of no indirect $CP$ violation in $D^0$ decays.Comment: 20 pages, 4 figure