Direct duplication 12p11.21–p13.31 mediated by segmental duplications: a new recurrent rearrangement?

We describe the characterization of an interstitial duplication of 12p, dup(12)(p11.21p13.31), by array-CGH and FISH in a patient with mental retardation and dysmorphic features. The sequence analysis of the breakpoints revealed the presence of homologous low copy repeats (LCRs) flanking the duplication region, thus suggesting that they have mediated the rearrangement. Pip-maker analysis showed that a third cluster of homologous LCRs lie distally to the two mediating the 12p duplication. We hypothesize that this duplication might be a new recurrent rearrangement and that, thanks to the different orientations of the homologous regions lying within each cluster, the three clusters are responsible for at least some of the several 12p aneuploidies reported in the literature such as direct and inverted duplications, deletions and supernumerary analphoid chromosomes. Moreover, we excluded that polymorphic inversions between these three clusters are present in the normal population.Manuela De Gregori, Tiziano Pramparo contributed equally to this paper.     

Growth arrest and induction of apoptosis in breast cancer cells by antisense depletion of protein kinase A-RIα subunit: p53-independent mechanism of action

The enhanced expression of the RI subunit of cyclic AMP-dependent protein kinase type 1 (PKA-I) has been correlated with cancer cell growth. We have investigated the effects of sequence-specific inhibition of RI gene expression on the growth of MCF-7 human breast cancer cells. We report that RI antisense treatment results in a reduction in RI expression at both mRNA and protein levels and inhibition of cell growth. The growth inhibition was accompanied by changes in cell morphology, cleavage of poly(ADP-ribose) polymerase (PARP) and appearance of apoptotic nuclei. In addition, bcl-2 protein level was reduced and p53 expression increased in growth arrested cells. Interestingly, RI antisense inhibited cell viability and induced apoptosis in the absence of p53, suggesting that these actions of RI antisense are exerted independent of p53. In contrast, two- and four-base mismatched control oligonucleotides had no effect on either cell growth or morphology. These results demonstrate that the RI antisense, which efficiently depletes the growth stimulatory molecule RI, induces cell differentiation and apoptosis, providing a new approach to combat breast cancer cell growth.     

Regulation of autophagy by p38α MAPK

Autophagy is induced in mammalian cells by nutrient deprivation, which acts through repression of the protein kinase mammalian target of rapamycin (mTOR) and may involve other unknown mechanisms. Mitogen-activated protein kinases (MAPKs), and in particular p38 MAPK, are implicated in amino acid signalling. Furthermore, the extracellular signal-regulated kinase (ERK) and p38 regulate autophagy in response to various stimuli. However, the molecular mechanisms of p38 regulation of autophagy are still widely unknown. Our recent data suggest that p38α MAPK negatively regulates the interaction of mAtg9 and a novel mAtg9 binding partner, p38IP, to control the levels of autophagy induced in response to starvation.     

Inhibition of glutamate racemase by substrate–product analogues

d-Glutamate is an essential biosynthetic building block of the peptidoglycans that encapsulate the bacterial cell wall. Glutamate racemase catalyzes the reversible formation of d-glutamate from l-glutamate and, hence, the enzyme is a potential therapeutic target. We show that the novel cyclic substrate–product analogue (R,S)-1-hydroxy-1-oxo-4-amino-4-carboxyphosphorinane is a modest, partial noncompetitive inhibitor of glutamate racemase from Fusobacterium nucleatum (FnGR), a pathogen responsible, in part, for periodontal disease and colorectal cancer (K_i = 3.1 ± 0.6 mM, cf. K_m = 1.41 ± 0.06 mM). The cyclic substrate–product analogue (R,S)-4-amino-4-carboxy-1,1-dioxotetrahydro-thiopyran was a weak inhibitor, giving only ∼30% inhibition at a concentration of 40 mM. The related cyclic substrate–product analogue 1,1-dioxo-tetrahydrothiopyran-4-one was a cooperative mixed-type inhibitor of FnGR (K_i = 18.4 ± 1.2 mM), while linear analogues were only weak inhibitors of the enzyme. For glutamate racemase, mimicking the structure of both enantiomeric substrates (substrate–product analogues) serves as a useful design strategy for developing inhibitors. The new cyclic compounds developed in the present study may serve as potential lead compounds for further development.     

Inhibition of EGF expression and NF-κB activity by treatment with quercetin leads to suppression of angiogenesis in nasopharyngeal carcinoma

The present study was performed to investigate the effect of quercetin on nasopharyngeal carcinoma (NPC) angiogenesis. The real-time RT-PCR and enzyme-linked immunosorbent assays (ELISA) were performed to analyze the expression levels of vascular endothelial growth factor (VEGF) in nasopharyngeal carcinoma cell lines prior to and after the quercetin treatment. Effect of quercetin on the rate of cell proliferation was measured by MTT assay. It was observed that quercetin treatment at a concentration of 10 mg/mL reduced the rate of NPC039 cell viability to 36% compared to control after 24 h. The expression of VEGF and activity of NF-κB was also markedly reduced. The ability of tube formation in HUVECs was inhibited significantly on exposure to quercetin compared to the untreated cells. Therefore, quercetin plays an important role in the inhibition of NPC039 nasopharyngeal carcinoma and can be of therapeutic importance.     

Inhibition of GST－π Expression by Retrovirus－mediated Antisense RNA Transfection

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Increased expression of interleukin-1β in triglyceride-induced macrophage cell death is mediated by p38 MAP kinase

Triglycerides (TG) are implicated in the development of atherosclerosis through formation of foam cells and induction of macrophage cell death. In this study, we report that addition of exogenous TG induced cell death in phorbol 12-myristate 13-acetate-differentiated THP-1 human macrophages. TG treatment induced a dramatic decrease in interleukin-1β (IL-1β) mRNA expression in a dose- and time-dependent manner. The expression of granulocyte macrophage colony-stimulating factor and platelet endothelial cell adhesion molecule remained unchanged. To identify signaling pathways involved in TG-induced downregulation of IL-1β, we added p38 MAPK, protein kinase C (PKC) or c-Raf1 specific inhibitors. We found that inhibition of p38 MAPK alleviated the TG-induced downregulation of IL-1β, whereas inhibition of PKC and c-Raf1 had no effect. This is the first report showing decreased IL-1β expression during TG-induced cell death in a human macrophage line. Our results suggest that downregulation of IL-1β expression by TG-treated macrophages may play a role during atherogenesis.     

Is G1 arrest in plant seeds induced by a p53-related pathway?

In mammals, p53 is crucial for inducing the genes that lead to G1 arrest following DNA damage, enabling DNA repair. However, the possibility that such a system exists in plants has attracted little attention. Even though some plant cDNA sequences with partial homology to p53 have been reported recently, there has been little analysis of how these molecules might relate to DNA damage. The lack of investigation into whether a DNA-damage-induced, p53-mediated G1-arrest pathway might exist in plants is remarkable given that plant DNA, like that of all organisms, is continually under the threat of attack.     

Loss of Ssq1 leads to mitochondrial dysfunction,activation of autophagy and cell cycle arrest due to iron overload triggered by mitochondrial iron–sulfur cluster assembly defects in Candida albicans

Iron–sulfur clusters perform essential functions in enzymatic catalysis and homeostatic regulation. Here we for the first time identified Ssq1 as an essential component for iron–sulfur cluster assembly in Candida albicans. Ssq1 played an important role in cell growth. Shutting off SSQ1 led to accumulation of intracellular iron, especially in mitochondria, and disorder of intracellular iron regulation. In tetO-SSQ1, iron overloading triggered the oxidative damage of mitochondrial function. Surprisingly, disruption of SSQ1 activated autophagic pathway. The mitochondrial dysfunction was further aggravated when CCZ1 (which is essential for autophagy) and SSQ1 was simultaneously deleted, suggesting that autophagy played a critical role in maintenance of mitochondrial function in tetO-SSQ1. In addition, double deletion of SSQ1 and CCZ1 further elevated cellular iron levels in comparison with tetO-SSQ1, indicating that autophagy participated in maintenance of iron homeostasis. Furthermore, we found that loss of SSQ1 led to increasing protein expression of Rnr1 and redistribution of Rnr2 from the nucleus to cytoplasm, and further resulted in cell cycle arrest. The results implied that cell cycle arrest was caused by activating the checkpoint pathway because of impairing the iron–sulfur cluster assembly in tetO-SSQ1. Shutting off SSQ1 led to a significant defect in filamentous development. Interestingly, the tetO-SSQ1ccz1Δ/Δ growth was inhibited on hyphae-inducing solid media. Both tetO-SSQ1 and tetO-SSQ1ccz1Δ/Δ exhibited extremely attenuated virulence, indicating that Ssq1 might provide a promising target for antifungal drugs development. In summary, our findings provide new insights into the understanding of iron–sulfur cluster assembly-related gene in C. albicans.     

The enhancement of stability of p53 in MTBP induced p53–MDM2 regulatory network

We have modeled an MTBP-MDM2–p53 regulatory network by integrating p53–MDM2 autoregulatory model (Proctor and Gray, 2008) with the effect of a cellular protein MTBP (MDM2 binding protein) which is allowed to bind with MDM2 (Brady et al., 2005). We study this model to investigate the activation of p53 and MDM2 steady state levels induced by MTBP protein under different stress conditions. Our simulation results in three approaches namely deterministic, Chemical Langevin equation and stochastic simulation of Master equation show a clear transition from damped limit cycle oscillation to fixed point oscillation during a certain time period with constant stress condition in the cell. This transition is the signature of transition of p53 and MDM2 levels from activated state to stabilized steady state levels. We present various phase diagrams to show the transition between unstable and stable states of p53 and MDM2 concentration levels and also their possible relations among critical value of the parameters at which the respective protein level reach stable steady states. In the stochastic approach, the dynamics of the proteins become noise induced process depending on the system size. We found that this noise enhances the stability of the p53 steady state level.     

p38δ Regulates p53 to Control p21Cip1 Expression in Human Epidermal Keratinocytes

PKCδ suppresses keratinocyte proliferation via a mechanism that involves increased expression of p21^Cip1. However, the signaling mechanism that mediates this regulation is not well understood. Our present studies suggest that PKCδ activates p38δ leading to increased p21^Cip1 promoter activity and p21^Cip1 mRNA/protein expression. We further show that exogenously expressed p38δ increases p21^Cip1 mRNA and protein and that p38δ knockdown or expression of dominant-negative p38 attenuates this increase. Moreover, p53 is an intermediary in this regulation, as p38δ expression increases p53 mRNA, protein, and promoter activity, and p53 knockdown attenuates the activation. We demonstrate a direct interaction of p38δ with PKCδ and MEK3 and show that exogenous agents that suppress keratinocyte proliferation activate this pathway. We confirm the importance of this regulation using a stratified epidermal equivalent model, which mimics in vivo-like keratinocyte differentiation. In this model, PKCδ or p38δ knockdown results in reduced p53 and p21^Cip1 levels and enhanced cell proliferation. We propose that PKCδ activates a MEKK1/MEK3/p38δ MAPK cascade to increase p53 levels and p53 drives p21^Cip1 gene expression.     

TGF-β1 induces endothelial cell apoptosis by shifting VEGF activation of p38(MAPK) from the prosurvival p38β to proapoptotic p38α

TGF-β1 and VEGF, both angiogenesis inducers, have opposing effects on vascular endothelial cells. TGF-β1 induces apoptosis; VEGF induces survival. We have previously shown that TGF-β1 induces endothelial cell expression of VEGF, which mediates TGF-β1 induction of apoptosis through activation of p38 mitogen-activated protein kinase (MAPK). Because VEGF activates p38(MAPK) but protects the cells from apoptosis, this finding suggested that TGF-β1 converts p38(MAPK) signaling from prosurvival to proapoptotic. Four isoforms of p38(MAPK) -α, β, γ, and δ-have been identified. Therefore, we hypothesized that different p38(MAPK) isoforms control endothelial cell apoptosis or survival, and that TGF-β1 directs VEGF activation of p38(MAPK) from a prosurvival to a proapoptotic isoform. Here, we report that cultured endothelial cells express p38α, β, and γ. VEGF activates p38β, whereas TGF-β1 activates p38α. TGF-β1 treatment rapidly induces p38α activation and apoptosis. Subsequently, p38α activation is downregulated, p38β is activated, and the surviving cells become refractory to TGF-β1 induction of apoptosis and proliferate. Gene silencing of p38α blocks TGF-β1 induction of apoptosis, whereas downregulation of p38β or p38γ expression results in massive apoptosis. Thus, in endothelial cells p38α mediates apoptotic signaling, whereas p38β and p38γ transduce survival signaling. TGF-β1 activation of p38α is mediated by VEGF, which in the absence of TGF-β1 activates p38β. Therefore, these results show that TGF-β1 induces endothelial cell apoptosis by shifting VEGF signaling from the prosurvival p38β to the proapoptotic p38α.     

C-terminal region of human p53 attenuates buffalo p53 N-terminal-specific transactivation of p21 promoter by modulating tetramerization of the protein

Here, we have studied in p53 null H1299 lung carcinoma cells, the dominant-negative effect of human p53 (h-p53) on buffalo p53 (b-p53) induced nuclear transactivation-dependent function. Recently, we have isolated and cloned the full-length cDNA of buffalo p53. Buffalo and human p53 proteins exhibit a high degree of structural and functional similarities. In transiently transfected H1299 cell line b-p53 appeared to be more sensitive to Mdm2-mediated degradation as compared to h-p53, although its ability to transactivate p21 promoter was stronger than that of the human counterpart. This higher transactivation ability of b-p53 was lost in the presence of h-p53 suggesting, a dominant-negative effect of h-p53 on b-p53’s transactivation of p21 promoter. Both human and buffalo p53 proteins could hetero-oligomerize but the b-p53 could tetramerize much faster than the h-p53. A chimeric cDNA construct of human p53 was made where the 1–260 bp N-terminus was replaced with buffalo p53 counterpart and expressed in H1299 cell line. The tetramerization ability of the chimeric p53 protein was comparable to that of h-p53. Properties of b-p53 like stronger p21 transactivation and super sensitivity to Mdm2 mediated degradation were lacking in the chimeric protein. Thus, it is suggested that faster ability of tetramerization as well as higher transactivation property of buffalo p53 is determined by the interplay of N- and C-terminal domains through macromolecular interactions.