Dose Dependent Effects on Cell Cycle Checkpoints and DNA Repair by Bendamustine

Bendamustine (BDM) is an active chemotherapeutic agent approved in the U. S. for treating chronic lymphocytic leukemia and non-Hodgkin lymphoma. Its chemical structure suggests it may have alkylator and anti-metabolite activities; however the precise mechanism of action is not well understood. Here we report the concentration-dependent effects of BDM on cell cycle, DNA damage, checkpoint response and cell death in HeLa cells. Low concentrations of BDM transiently arrested cells in G2, while a 4-fold higher concentration arrested cells in S phase. DNA damage at 50, but not 200 µM, was efficiently repaired after 48 h treatment, suggesting a difference in DNA repair efficiency at the two concentrations. Indeed, perturbing base-excision repair sensitized cells to lower concentrations of BDM. Timelapse studies of the checkpoint response to BDM showed that inhibiting Chk1 caused both the S- and G2-arrested cells to prematurely enter mitosis. However, whereas the cells arrested in G2 (low dose BDM) entered mitosis, segregated their chromosomes and divided normally, the S-phase arrested cells (high dose BDM) exhibited a highly aberrant mitosis, whereby EM images showed highly fragmented chromosomes. The vast majority of these cells died without ever exiting mitosis. Inhibiting the Chk1-dependent DNA damage checkpoint accelerated the time of killing by BDM. Our studies suggest that BDM may affect different biological processes depending on drug concentration. Sensitizing cells to killing by BDM can be achieved by inhibiting base-excision repair or disrupting the DNA damage checkpoint pathway

Concomitant use of tamoxifen with radiotherapy enhances subcutaneous breast fibrosis in hypersensitive patients

Concomitant use of adjuvant tamoxifen (TAM) and radiation therapy (RT) is not widely accepted. We aim to assess whether this treatment is associated with an increased risk of developing subcutaneous fibrosis after conservative or radical surgery in breast cancer patients. We analysed 147 women with breast cancer treated with adjuvant RT, and who were included in the KFS 00539-9-1997/SKL 00778-2-1999 prospective study aimed at evaluating the predictive value of CD4 and CD8 T-lymphocyte apoptosis for the development of radiation-induced late effects. TAM (20 mg day(-1)) with concomitant RT was prescribed in 90 hormone receptor-positive patients. There was a statistically significant difference in terms of complication-relapse-free survival (CRFS) rates at 3 years, 48% (95% CI 37.2-57.6%) vs 66% (95% CI 49.9-78.6%) and complication-free survival (CFS) rates at 2 years, 51% (95% CI 40-61%) vs 80% (95% CI 67-89%) in the TAM and no-TAM groups, respectively. In each of these groups, the CRFS rates were significantly lower for patients with low levels of CD8 radiation-induced apoptosis, 20% (95% CI 10-31.9%), 66% (95% CI 51.1-77.6%), and 79% (95% CI 55-90.9%) for CD8 </=16, 16-24, and >24%, respectively. Similar results were observed for the CFS rates. The concomitant use of TAM with RT is significantly associated with an increased incidence of grade 2 or greater subcutaneous fibrosis; therefore, caution is needed for radiosensitive patients

miR-124 and miR-137 inhibit proliferation of glioblastoma multiforme cells and induce differentiation of brain tumor stem cells

Glioblastoma multiforme (GBM) is an invariably fatal central nervous system tumor despite treatment with surgery, radiation, and chemotherapy. Further insights into the molecular and cellular mechanisms that drive GBM formation are required to improve patient outcome. MicroRNAs are emerging as important regulators of cellular differentiation and proliferation, and have been implicated in the etiology of a variety of cancers, yet the role of microRNAs in GBM remains poorly understood. In this study, we investigated the role of microRNAs in regulating the differentiation and proliferation of neural stem cells and glioblastoma-multiforme tumor cells.status: publishe

Integrated-boost IMRT or 3-D-CRT using FET-PET based auto-contoured target volume delineation for glioblastoma multiforme - a dosimetric comparison

<p>Abstract</p> <p>Background</p> <p>Biological brain tumor imaging using O-(2-[¹⁸F]fluoroethyl)-L-tyrosine (FET)-PET combined with inverse treatment planning for locally restricted dose escalation in patients with glioblastoma multiforme seems to be a promising approach.</p> <p>The aim of this study was to compare inverse with forward treatment planning for an integrated boost dose application in patients suffering from a glioblastoma multiforme, while biological target volumes are based on FET-PET and MRI data sets.</p> <p>Methods</p> <p>In 16 glioblastoma patients an intensity-modulated radiotherapy technique comprising an integrated boost (IB-IMRT) and a 3-dimensional conventional radiotherapy (3D-CRT) technique were generated for dosimetric comparison. FET-PET, MRI and treatment planning CT (P-CT) were co-registrated. The integrated boost volume (PTV1) was auto-contoured using a cut-off tumor-to-brain ratio (TBR) of ≥ 1.6 from FET-PET. PTV2 delineation was MRI-based. The total dose was prescribed to 72 and 60 Gy for PTV1 and PTV2, using daily fractions of 2.4 and 2 Gy.</p> <p>Results</p> <p>After auto-contouring of PTV1 a marked target shape complexity had an impact on the dosimetric outcome. Patients with 3-4 PTV1 subvolumes vs. a single volume revealed a significant decrease in mean dose (67.7 vs. 70.6 Gy). From convex to complex shaped PTV1 mean doses decreased from 71.3 Gy to 67.7 Gy. The homogeneity and conformity for PTV1 and PTV2 was significantly improved with IB-IMRT. With the use of IB-IMRT the minimum dose within PTV1 (61.1 vs. 57.4 Gy) and PTV2 (51.4 vs. 40.9 Gy) increased significantly, and the mean EUD for PTV2 was improved (59.9 vs. 55.3 Gy, p < 0.01). The EUD for PTV1 was only slightly improved (68.3 vs. 67.3 Gy). The EUD for the brain was equal with both planning techniques.</p> <p>Conclusion</p> <p>In the presented planning study the integrated boost concept based on inversely planned IB-IMRT is feasible. The FET-PET-based automatically contoured PTV1 can lead to very complex geometric configurations, limiting the achievable mean dose in the boost volume. With IB-IMRT a better homogeneity and conformity, compared to 3D-CRT, could be achieved.</p

CDK2 and PKA Mediated-Sequential Phosphorylation Is Critical for p19INK4d Function in the DNA Damage Response

DNA damage triggers a phosphorylation-based signaling cascade known as the DNA damage response. p19INK4d, a member of the INK4 family of CDK4/6 inhibitors, has been reported to participate in the DNA damage response promoting DNA repair and cell survival. Here, we provide mechanistic insight into the activation mechanism of p19INK4d linked to the response to DNA damage. Results showed that p19INK4d becomes phosphorylated following UV radiation, β-amyloid peptide and cisplatin treatments. ATM-Chk2/ATR-Chk1 signaling pathways were found to be differentially involved in p19INK4d phosphorylation depending on the type of DNA damage. Two sequential phosphorylation events at serine 76 and threonine 141 were identified using p19INK4d single-point mutants in metabolic labeling assays with 32P-orthophosphate. CDK2 and PKA were found to participate in p19INK4d phosphorylation process and that they would mediate serine 76 and threonine 141 modifications respectively. Nuclear translocation of p19INK4d induced by DNA damage was shown to be dependent on serine 76 phosphorylation. Most importantly, both phosphorylation sites were found to be crucial for p19INK4d function in DNA repair and cell survival. In contrast, serine 76 and threonine 141 were dispensable for CDK4/6 inhibition highlighting the independence of p19INK4d functions, in agreement with our previous findings. These results constitute the first description of the activation mechanism of p19INK4d in response to genotoxic stress and demonstrate the functional relevance of this activation following DNA damage

Activation of Akt by the Bacterial Inositol Phosphatase, SopB, is Wortmannin Insensitive

Salmonella enterica uses effector proteins translocated by a Type III Secretion System to invade epithelial cells. One of the invasion-associated effectors, SopB, is an inositol phosphatase that mediates sustained activation of the pro-survival kinase Akt in infected cells. Canonical activation of Akt involves membrane translocation and phosphorylation and is dependent on phosphatidyl inositide 3 kinase (PI3K). Here we have investigated these two distinct processes in Salmonella infected HeLa cells. Firstly, we found that SopB-dependent membrane translocation and phosphorylation of Akt are insensitive to the PI3K inhibitor wortmannin. Similarly, depletion of the PI3K regulatory subunits p85α and p85ß by RNAi had no inhibitory effect on SopB-dependent Akt phosphorylation. Nevertheless, SopB-dependent phosphorylation does depend on the Akt kinases, PDK1 and rictor-mTOR. Membrane translocation assays revealed a dependence on SopB for Akt recruitment to Salmonella ruffles and suggest that this is mediated by phosphoinositide (3,4) P2 rather than phosphoinositide (3,4,5) P3. Altogether these data demonstrate that Salmonella activates Akt via a wortmannin insensitive mechanism that is likely a class I PI3K-independent process that incorporates some essential elements of the canonical pathway

Platelet Activating Factor Blocks Interkinetic Nuclear Migration in Retinal Progenitors through an Arrest of the Cell Cycle at the S/G2 Transition

Nuclear migration is regulated by the LIS1 protein, which is the regulatory subunit of platelet activating factor (PAF) acetyl-hydrolase, an enzyme complex that inactivates the lipid mediator PAF. Among other functions, PAF modulates cell proliferation, but its effects upon mechanisms of the cell cycle are unknown. Here we show that PAF inhibited interkinetic nuclear migration (IKNM) in retinal proliferating progenitors. The lipid did not, however, affect the velocity of nuclear migration in cells that escaped IKNM blockade. The effect depended on the PAF receptor, Erk and p38 pathways and Chk1. PAF induced no cell death, nor a reduction in nucleotide incorporation, which rules out an intra-S checkpoint. Notwithstanding, the expected increase in cyclin B1 content during G2-phase was prevented in the proliferating cells. We conclude that PAF blocks interkinetic nuclear migration in retinal progenitor cells through an unusual arrest of the cell cycle at the transition from S to G2 phases. These data suggest the operation, in the developing retina, of a checkpoint that monitors the transition from S to G2 phases of the cell cycle

Diffuse glioma growth: a guerilla war

In contrast to almost all other brain tumors, diffuse gliomas infiltrate extensively in the neuropil. This growth pattern is a major factor in therapeutic failure. Diffuse infiltrative glioma cells show some similarities with guerilla warriors. Histopathologically, the tumor cells tend to invade individually or in small groups in between the dense network of neuronal and glial cell processes. Meanwhile, in large areas of diffuse gliomas the tumor cells abuse pre-existent “supply lines” for oxygen and nutrients rather than constructing their own. Radiological visualization of the invasive front of diffuse gliomas is difficult. Although the knowledge about migration of (tumor)cells is rapidly increasing, the exact molecular mechanisms underlying infiltration of glioma cells in the neuropil have not yet been elucidated. As the efficacy of conventional methods to fight diffuse infiltrative glioma cells is limited, a more targeted (“search & destroy”) tactic may be needed for these tumors. Hopefully, the study of original human glioma tissue and of genotypically and phenotypically relevant glioma models will soon provide information about the Achilles heel of diffuse infiltrative glioma cells that can be used for more effective therapeutic strategies

Functional interdependence of BRD4 and DOT1L in MLL leukemia.

Targeted therapies against disruptor of telomeric silencing 1-like (DOT1L) and bromodomain-containing protein 4 (BRD4) are currently being evaluated in clinical trials. However, the mechanisms by which BRD4 and DOT1L regulate leukemogenic transcription programs remain unclear. Using quantitative proteomics, chemoproteomics and biochemical fractionation, we found that native BRD4 and DOT1L exist in separate protein complexes. Genetic disruption or small-molecule inhibition of BRD4 and DOT1L showed marked synergistic activity against MLL leukemia cell lines, primary human leukemia cells and mouse leukemia models. Mechanistically, we found a previously unrecognized functional collaboration between DOT1L and BRD4 that is especially important at highly transcribed genes in proximity to superenhancers. DOT1L, via dimethylated histone H3 K79, facilitates histone H4 acetylation, which in turn regulates the binding of BRD4 to chromatin. These data provide new insights into the regulation of transcription and specify a molecular framework for therapeutic intervention in this disease with poor prognosis