Blocking of transcription factor E2F/DP by dominant-negative mutants in a normal breast epithelial cell line efficiently inhibits apoptosis and induces tumor growth in scid mice

The transcription factor E2F is regulated during the cell cycle through interactions with the product of the retinoblastoma susceptibility gene and related proteins. It is thought that E2F-mediated gene regulation at the G1/S boundary and during S phase may be one of the rate-limiting steps in cell proliferation. It was reported that in vivo overexpression of E2F-1 in fibroblasts induces S phase entry and leads to apoptosis. This observation suggests that E2F plays a role in both cell cycle regulation and apoptosis. To further understand the role of E2F in cell cycle progression, cell death, and tumor development, we have blocked endogenous E2F activity in HBL-100 cells, derived from nonmalignant human breast epithelium, using dominant-negative mutants under the control of a tetracycline-dependent expression system. We have shown here that induction of dominant-negative mutants led to strong downregulation of transiently transfected E2F-dependent chloramphenicol acetyl transferase reporter constructs and of endogenous c-myc, which has been described as a target gene of the transcription factor E2F/DP. In addition, we have shown that blocking of E2F could efficiently protect from apoptosis induced by serum starvation within a period of 10 d, whereas control cells started to die after 24 h. Surprisingly, blocking of E2F did not alter the rate of proliferation or of DNA synthesis of these cells; this finding indicates that cell-cycle progression could be driven in an E2F-independent manner. In addition, we have been able to show that blocking of endogenous E2F in HBL-100 cells led to rapid induction of tumor growth in severe combined immunodeficiency mice. No tumor growth could be observed in mice that received mock-transfected clones or tetracycline to block expression of the E2F mutant constructs in vivo. Thus, it appears that E2F has a potential tumor-suppressive function under certain circumstances. Furthermore, we provide evidence that dysregulation of apoptosis may be an important step in tumorigenesis

Molecular Changes in Pre-Metastatic Lymph Nodes of Esophageal Cancer Patients

Lymph node metastasis indicates poor prognosis in esophageal cancer. To understand the underlying mechanisms, most studies so far focused on investigating the tumors themselves and/or invaded lymph nodes. However they neglected the potential events within the metastatic niche, which precede invasion. Here we report the first description of these regulations in patients on transcription level. We determined transcriptomic profiles of still metastasis-free regional lymph nodes for two patient groups: patients classified as pN1 (n = 9, metastatic nodes exist) or pN0 (n = 5, no metastatic nodes exist). All investigated lymph nodes, also those from pN1 patients, were still metastasis-free. The results show that regional lymph nodes of pN1 patients differ decisively from those of pN0 patients--even before metastasis has taken place. In the pN0 group distinct immune response patterns were observed. In contrast, lymph nodes of the pN1 group exhibited a clear profile of reduced immune response and reduced proliferation, but increased apoptosis, enhanced hypoplasia and morphological conversion processes. DKK1 was the most significant gene associated with the molecular mechanisms taking place in lymph nodes of patients suffering from metastasis (pN1). We assume that the two molecular profiles observed constitute different stages of a progressive disease. Finally we suggest that DKK1 might play an important role within the mechanisms leading to lymph node metastasis

Diffusion and jump-length distribution in liquid and amorphous Cu33_{33}Zr67_{67}

Using molecular dynamics simulation, we calculate the distribution of atomic jum ps in Cu$_{33}$Zr$_{67}$ in the liquid and glassy states. In both states the distribution of jump lengths can be described by a temperature independent exponential of the length and an effective activation energy plus a contribution of elastic displacements at short distances. Upon cooling the contribution of shorter jumps dominates. No indication of an enhanced probability to jump over a nearest neighbor distance was found. We find a smooth transition from flow in the liquid to jumps in the g lass. The correlation factor of the diffusion constant decreases with decreasing temperature, causing a drop of diffusion below the Arrhenius value, despite an apparent Arrhenius law for the jump probability

Dzyaloshinsky-Moriya Spin Canting in the LTT Phase of La2-x-yEuySrxCuO4

The Cu spin magnetism in La2-x-yEuySrxCuO4 (x<=0.17; y<=0.2) has been studied by means of magnetization measurements up to 14 T. Our results clearly show that in the antiferromagnetic phase Dzyaloshinsky-Moriya (DM)superexchange causes Cu spin canting not only in the LTO phase but also in the LTLO and LTT phases. In La1.8Eu0.2CuO4 the canted DM-moment is about 50% larger than in pure La2CuO4 which we attribute to the larger octahedral tilt angle. We also find clear evidence that the size of the DM-moment does not change significantly at the structural transition at T_LT from LTO to LTLO and LTT. The most important change induced by the transition is a significant reduction of the magnetic coupling between the CuO2 planes. As a consequence, the spin-flip transition of the canted Cu spins which is observed in the LTO phase for magnetic field perpendicular to the CuO2 planes disappears in the LTT phase. The shape of the magnetization curves changes from the well known spin-flip type to a weak-ferromagnet type. However, no spontaneous weak ferromagnetism is observed even at very low temperatures, which seems to indicate that the interlayer decoupling in our samples is not perfect. Nonetheless, a small fraction (<15%) of the DM-moments can be remanently magnetized throughout the entire antiferromagnetically ordered LTT/LTLO phase, i.e. for T<T_LT and x<0.02. It appears that the remanent DM-moment is perpendicular to the CuO2 planes. For magnetic field parallel to the CuO2 planes we find that the critical field of the spin-flop transition decreases in the LTLO phase, which might indicate a competition between different in-plane anisotropies. To study the Cu spin magnetism in La2-x-yEuySrxCuO4, a careful analysis of the Van Vleck paramagnetism of the Eu3+ ions was performed.Comment: 22 pages, 27 figure

Acquired resistance to DZNep-mediated apoptosis is associated with copy number gains of AHCY in a B-cell lymphoma model

BackgroundEnhancer of zeste homolog 2 (EZH2) is considered an important driver of tumor development and progression by its histone modifying capabilities. Inhibition of EZH2 activity is thought to be a potent treatment option for eligible cancer patients with an aberrant EZH2 expression profile, thus the indirect EZH2 inhibitor 3-Deazaneplanocin A (DZNep) is currently under evaluation for its clinical utility. Although DZNep blocks proliferation and induces apoptosis in different tumor types including lymphomas, acquired resistance to DZNep may limit its clinical application.MethodsTo investigate possible mechanisms of acquired DZNep resistance in B-cell lymphomas, we generated a DZNep-resistant clone from a previously DZNep-sensitive B-cell lymphoma cell line by long-term treatment with increasing concentrations of DZNep (ranging from 200 to 2000nM) and compared the molecular profiles of resistant and wild-type clones. This comparison was done using molecular techniques such as flow cytometry, copy number variation assay (OncoScan and TaqMan assays), fluorescence in situ hybridization, Western blot, immunohistochemistry and metabolomics analysis.ResultsWhole exome sequencing did not indicate the acquisition of biologically meaningful single nucleotide variants. Analysis of copy number alterations, however, demonstrated among other acquired imbalances an amplification (about 30 times) of the S-adenosyl-L-homocysteine hydrolase (AHCY) gene in the resistant clone. AHCY is a direct target of DZNep and is critically involved in the biological methylation process, where it catalyzes the reversible hydrolysis of S-adenosyl-L-homocysteine to L-homocysteine and adenosine. The amplification of the AHCY gene is paralleled by strong overexpression of AHCY at both the transcriptional and protein level, and persists upon culturing the resistant clone in a DZNep-free medium.ConclusionsThis study reveals one possible molecular mechanism how B-cell lymphomas can acquire resistance to DZNep, and proposes AHCY as a potential biomarker for investigation during the administration of EZH2-targeted therapy with DZNep

Catchment classification:hydrological analysis of catchment behavior through process-based modeling along a climate gradient

Catchment classification is an efficient method to synthesize our understanding of how climate variability and catchment characteristics interact to define hydrological response. One way to accomplish catchment classification is to empirically relate climate and catchment characteristics to hydrologic behavior and to quantify the skill of predicting hydrologic response based on the combination of climate and catchment characteristics. Here we present results using an alternative approach that uses our current level of hydrological understanding, expressed in the form of a process-based model, to interrogate how climate and catchment characteristics interact to produce observed hydrologic response. The model uses topographic, geomorphologic, soil and vegetation information at the catchment scale and conditions parameter values using readily available data on precipitation, temperature and streamflow. It is applicable to a wide range of catchments in different climate settings. We have developed a step-by-step procedure to analyze the observed hydrologic response and to assign parameter values related to specific components of the model. We applied this procedure to 12 catchments across a climate gradient east of the Rocky Mountains, USA. We show that the model is capable of reproducing the observed hydrologic behavior measured through hydrologic signatures chosen at different temporal scales. Next, we analyze the dominant time scales of catchment response and their dimensionless ratios with respect to climate and observable landscape features in an attempt to explain hydrologic partitioning. We find that only a limited number of model parameters can be related to observable landscape features. However, several climate-model time scales, and the associated dimensionless numbers, show scaling relationships with respect to the investigated hydrological signatures (runoff coefficient, baseflow index, and slope of the flow duration curve). Moreover, some dimensionless numbers vary systematically across the climate gradient, possibly as a result of systematic co-variation of climate, vegetation and soil related time scales. If such co-variation can be shown to be robust across many catchments along different climate gradients, it opens perspective for model parameterization in ungauged catchments as well as prediction of hydrologic response in a rapidly changing environment