Radiation-induced Impacts on Cell Adhesion and Its Cell Cycle Dependence

Recombinant retroviruses are currently used as gene delivery vehicles for the purpose of gene therapy. It is generally believed that the efficiency of retroviral transduction depends on the cell cycle status of the target cells. However, it has been reported that this is not the case for the transduction of human and murine fibroblasts, in contrast to other cell types such as lymphocytes. The predictions of a mathematical model that we constructed, offer an explanation of this contradiction, based on the dynamics of the underlying processes of target cell growth and the intracellular decay of retroviral vectors. The model suggests that the utility of synchronization experiments, that are usually employed to study cell cycle specificity, is severely limited when the time scales of the above kinetic events are comparable to each other. The predictions of the model also suggest the use of retroviral vectors as cell cycle markers, as an alternative way to detect cell cycle dependence of retroviral transduction. This method obviates the need for cell synchronization and therefore, it does not perturb the cell cycle or interfere with the life cycle of retroviral vectors. Moreover, it does not depend on the intracellular stability of retroviral vectors. Our results show that in contrast to previously reported results, transduction of murine fibroblasts is cell cycle dependent, and they are consistent with the current notion that mitosis is the phase that confers transduction susceptibility.

Oxalate-Induced and Cell-Cycle-Dependent Expression of Nuclear Pore Complex Oxalate Binding Protein gp210

Background: ALMS1 is a ubiquitous gene associated with Alström syndrome (ALMS). The main symptoms of ALMS affect multiple organs and tissues, generating at last, multi-organic fibrosis in the lungs, kidneys and liver. TGF-β is one of the main pathways implicated in fibrosis, controlling the cell cycle, apoptosis, cell migration, cell adhesion and epithelial-mesenchymal transition (EMT). Nevertheless, the role of ALMS1 gene in fibrosis generation and other implicated processes such as cell migration or cell adhesion via the TGF- β pathway has not been elucidated yet. Methods: Initially, we evaluated how depletion of ALMS1 affects different processes like apoptosis, cell cycle and mitochondrial activity in HeLa cells. Then, we performed proteomic profiling with TGF-β stimuli in HeLa ALMS1 -/- cells and validated the results by examining different EMT biomarkers using qPCR. The expression of these EMT biomarkers were also studied in hTERT-BJ-5ta ALMS1 -/-. Finally, we evaluated the SMAD3 and SMAD2 phosphorylation and cell migration capacity in both models. Results: Depletion of ALMS1 generated apoptosis resistance to thapsigargin (THAP) and C2-Ceramide (C2-C), and G2/M cell cycle arrest in HeLa cells. For mitochondrial activity, results did not show significant differences between ALMS1 +/+ and ALMS1 -/-. Proteomic results showed inhibition of downstream pathways regulated by TGF-β. The protein-coding genes (PCG) were associated with processes like focal adhesion or cell-substrate adherens junction in HeLa. SNAI1 showed an opposite pattern to what would be expected when activating the EMT in HeLa and BJ-5ta. Finally, in BJ-5ta model a reduced activation of SMAD3 but not SMAD2 were also observed. In HeLa model no alterations in the canonical TGF-β pathway were observed but both cell lines showed a reduction in migration capacity. Conclusion: ALMS1 has a role in controlling the cell cycle and the apoptosis processes. Moreover, the depletion of ALMS1 affects the signal transduction through the TGF-β and other processes like the cell migration and adhesion capacity.

IntroductionThe rheumatoid arthritis (RA) synovium is characterised by the presence of an aggressive population of activated synovial fibroblasts (RASFs) that are prominently involved in the destruction of articular cartilage and bone. Accumulating evidence suggests that RASFs are relatively resistant to Fas-ligand (FasL)-induced apoptosis, but the data concerning tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) have been conflicting. Here, we hypothesise that the susceptibility of RASFs to receptor-mediated apoptosis depends on the proliferation status of these cells and therefore analysed the cell cycle dependency of FasL- and TRAIL-induced programmed cell death of RASFs in vitro.MethodsSynovial fibroblasts were isolated from patients with RA by enzymatic digestion and cultured under standard conditions. Cell cycle analysis was performed using flow cytometry and staining with propidium iodide. RASFs were synchronised or arrested in various phases of the cell cycle with 0.5 mM hydroxyurea or 2.5 μg/ml nocodazol and with foetal calf serum-free insulin-transferrin-sodium selenite supplemented medium. Apoptosis was induced by stimulation with 100 ng/ml FasL or 100 ng/ml TRAIL over 18 hours. The apoptotic response was measured using the Apo-ONE® Homogenous Caspase-3/7 Assay (Promega GmbH, Mannheim, Germany) and the Cell Death Detection (ELISAPlus) (enzyme-linked immunosorbent assay) (Roche Diagnostics GmbH, Mannheim, Germany). Staurosporin-treated cells (1 μg/ml) served as a positive control. Expression of Fas and TRAIL receptors (TRAILR1-4) was determined by fluorescence-activated cell sorting analysis.ResultsFreshly isolated RASFs showed only low proliferation in vitro, and the rate decreased further over time, particularly when RASFs became confluent. RASFs expressed Fas, TRAIL receptor-1, and TRAIL receptor-2, and the expression levels were independent of the cell cycle. However, the proliferation rate significantly influenced the susceptibility to FasL- and TRAIL-induced apoptosis. Specifically, proliferating RASFs were less sensitive to FasL- and TRAIL-induced apoptosis than RASFs with a decreased proliferation rate. Furthermore, RASFs that were synchronised in S phase or G2/M phase were less sensitive to TRAIL-induced apoptosis than synchronised RASFs in G0/G1 phase.ConclusionsOur data indicate that the susceptibility of RASFs to FasL- and TRAIL-induced apoptosis depends on the cell cycle. These results may explain some conflicting data on the ability of RASFs to undergo FasL- and TRAIL-mediated cell death and suggest that strategies to sensitise RASFs to apoptosis may include the targeting of cell cycle-regulating genes.

The clonogenic growth inhibition, the cell cycle dependence of N4-hexadecyl-1-beta-D-arabinofuranosylcytosine (NHAC) cytotoxicity and the capability to induce apoptosis in ara-C-sensitive and -resistant HL-60 cells were investigated and compared with arabinofuranosylcytosine (ara-C). In the clonogenic assay with sensitive HL-60 cells, ara-C was slightly more effective than a liposomal preparation of NHAC, whereas in the resistant cells, NHAC revealed its potency to overcome ara-C resistance, resulting in a 23-fold lower 50% inhibitory concentration compared with ara-C. Cell cycle dependent cytotoxicity and induction of apoptosis were studied by flow cytometry, using the bromodeoxyuridine-propidium iodide and terminal transferase method respectively. In contrast to ara-C, NHAC exerted no phase-specific toxicity at low concentrations (< 40 microM). At higher concentrations the S-phase-specific toxicity increased, probably resulting from ara-C formed from NHAC. NHAC induced apoptosis at higher drug concentrations than ara-C, however apoptosis appeared not to be limited to the S-phase cells. Apoptosis occurred in both cell lines within 2-4 h after drug exposure. These results give further evidence that NHAC exerts its cytotoxicity by different mechanisms of action than ara-C and might therefore be active in ara-C-resistant tumours.

The relation of radiation exposure (dose) with acute radiation syndrome (ARS) depends on many factors. In this overview, we reconsider (1) radiation exposure characteristics (e.g. radiation quality, fractionation, dose rate, partial/total body irradiation) and (2) biological processes (e.g. radiosensitivity, cell cycle dependency, oxygenation) affecting acute health effects after exposure. Furthermore we include evidence from recently published work that examined the relationship of absorbed dose and risk of clinically relevant ARS in persons exposed after a radiation accident. We introduce the concept of radiation-related bioindicators for effect prediction. Bioindicators are considered here to be factors that integrate multiple radiation exposure characteristics and cell- and molecular-based processes to improve clinical prediction in persons with ARS.

i-Motifs (iMs) are quadruplex nucleic acid conformations that form in cytosine-rich regions. Because of their acidic pH dependence, iMs were thought to form only in vitro. The recent development of an iM-selective antibody, iMab, has allowed iM detection in cells, which revealed their presence at gene promoters and their cell cycle dependence. However, recent evidence emerged which appeared to suggest that iMab recognizes C-rich sequences regardless of their iM conformation. To further investigate the selectivity of iMab, we examined the binding of iMab to C-rich sequences, using a combination of pull-down and western blot assays. Here, we observe that the composition of buffers used during binding and washing steps strongly influences the selectivity of antibody binding. In addition, we demonstrate by nuclear magnetic resonance that several of the previously reported C-rich sequences, which were not expected to form iMs, actually form intermolecular iMs which are selectively recognized by iMab. Our results highlight the specificity of the iMab antibody, emphasize the importance of avoiding in vitro artifacts by optimizing DNA concentrations, blocking and washing conditions, and confirm thatiMab is selectivenot only for intramolecular iMs but also for intermolecular iMs,whilenot affecting theiM conformation.

Proteogenomics is the field of integrating data from mass spectrometry-based shotgun proteomics, and phosphoproteomics into next-generation RNA and DNA sequencing data analysis pipelines that promises new insights into cancer biology and therapeutic targeting. As well as analyses of clinical samples for disease phenotype association analysis, the application of proteogenomics to model systems also has considerable potential. A Clinical Proteomic Tumor Analysis Consortium (CPTAC) proteogenomic analysis prioritized dihydropyrimidinase-like-3 (DPYSL3) as a multi-level (RNA/Protein/Phosphoprotein) expression outlier specific to the Claudin-Low (CLOW) subset of triple negative breast cancers. A Pubmed informatics tool indicated a paucity of data in the context of breast cancer which further prioritized DPYSL3 for study. DPYSL3 was identified as a protein that is regulated during neuronal differentiation in the cerebral cortex and in neuronal cell lines and plays a role in regulating neurite outgrowth somehow through an association with vesicles in the growth cone. In addition, DPYSL3 expression has been observed in several malignant tumors, including prostate cancer, pancreatic cancer, gastric cancer and neuroblastoma. DPYSL3 is reported to play a role in cell migration and metastasis suppression in prostate cancer. However, in pancreatic cancer, DPYSL3 is positively associated with liver metastasis and poor outcome. DPYSL3 knock-down in DPYSL3 (+) CLOW cell lines demonstrated reduced proliferation, yet enhanced motility and increased expression of Epithelial to Mesenchymal Transition (EMT) markers suggesting that DPYSL3 is a multi-functional signaling modulator. Slower proliferation in DPYSL3 (-) CLOW cells was associated with accumulation of multi-nucleated cells indicating a mitotic defect that was associated with a collapse of the vimentin (VIM) microfilament networkinduced by VIM hyperphosphorylation. On the other hand, DPYSL3 suppressed the expression of EMT regulators TWIST and SNAIL and opposed p21 activated kinase 2 (PAK2) dependent migration, but these EMT regulators in turn induced DPYSL3 expression, suggesting DPYSL3 participates in negative feedback in EMT. Cell migration in DPYSL3 (-) cells correlated with increased phosphorylation of PAK2 on Ser20 and was sensitive to PAK2 siRNA and pharmacological PAK inhibition.Immunoprecipitation and mass spectrometry-based proteomics or western blotting strongly suggests that PAKs interact such that DPYSL3 may function as a direct negative regulator of PAK family kinases. Thus, a PAK inhibitor could potentially mitigate increase migration as an adverse effect of DPYSL3 suppression. In conclusion, DPYSL3 is a remarkable multifunctional signaling scaffold that should be examined further to provide insights into the stem cell-like state of claudin-low breast cancers, particularly in terms of their cell cycle dependencies, migratory activity and capacity for EMT. Citation Format: Matsunuma R, Chan DW, Kim B-J, Singh P, Han A, Saltzman A, Cheng C, Lei JT, Sahin E, Leng M, Fan C, Perou CM, Malovannaya A, Ellis MJ. DPYSL3 modulates mitosis, migration and epithelial to mesenchymal transition in claudin-low breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-08-01.

Spectrins are ubiquitous scaffolding components of the membrane skeleton that organize and stabilize microdomains on both the plasma membrane and the intracellular organelles. By way of their numerous interactions with diverse protein families, they are implicated in various cellular functions. Using small interfering RNA strategy in the WM-266 cell line derived from human melanoma, we found that alphaII-spectrin deficiency is associated with a defect in cell proliferation, which is related to a cell cycle arrest at the G1 phase (first gap phase), as evaluated by DNA analysis and Rb phosphorylation. These observations coincided with elevated expression of the cyclin-dependent kinase inhibitor, p21Cip. Concomitantly, spectrin loss impaired cell adhesion and spreading. These cell adhesion defects were associated with modifications of the actin cytoskeleton, such as loss of stress fibers, alterations of focal adhesions, and modified expression of some integrins. Our results provide novel insights into spectrin functions by demonstrating the involvement of alphaII-spectrin in cell cycle regulation and actin organization.

Anchorage-independent myelomonocytic cells acquire adherence within minutes of differentiation stimuli, such as the proteolytically inactive N-terminal fragment of urokinase binding to its cognate glycosylphosphatidylinositol (GPI)-anchored receptor. Here, we report that urokinase-treated differentiating U937 monocyte-like cells exhibit a rapid and transient inhibition of p56/59(hck) and p55(fgr) whereas no changes in the activity of other Src family kinases, such as p53/56(lyn) and p59(fyn) were observed. U937 transfectants expressing a kinase-defective (Lys267 to Met) p56/59(hck) variant exhibit enhanced adhesiveness and a marked F-actin redistribution in thin protruding structures. Conversely, urokinase as well as expression of wild-type or constitutively active (Tyr499 to Phe) p56/59(hck) stimulates the directional migration of uninduced U937 cells. Accordingly, expression of constitutively active or kinase inactive p56/59(hck) selectively prevents urokinase receptor-dependent induction of either adhesion or motility, indicating that a specific activation state of p56/59(hck) is required for each cell response. In conclusion, modulation of the intracellular p56/59(hck) tyrosine kinase activity switches cell motility towards adherence, providing a mutually exclusive mechanism to regulate these properties during monocyte/macrophage differentiation in vivo.

Mechanistic target of rapamycin (mTOR) signaling is typically increased in hepatocellular carcinoma (HCC). A panel of HCC cell lines (HepG2, Hep3B and HuH6) was exposed to various concentrations of the mTOR inhibitors, everolimus and temsirolimus, in order to investigate their effects on cell growth, clonal formation, cell cycle progression, and adhesion and chemotactic migration using MTT and clonal cell growth assays, fluorometric detection of cell cycle phases and a Boyden chamber assay. In addition, integrinαandβ adhesion receptors were analyzed by flow cytometry and blocking studies using function blocking monoclonal antibodies were conducted to explore functional relevance. The results demonstrated that everolimus and temsirolimus significantly suppressed HCC cell growth and clonal formation, at 0.1 or 1nM (depending on the cell line). In addition, the number of cells in G0/G1 phase was increased in response to drug treatment, whereas the number of G2/M phase cells was decreased. Drug treatment also considerably suppressed HCC cell adhesion to immobilized collagen. Integrin profiling revealed strong expression of integrin α1, α2, α6 and β1 subtypes; and integrin α1 was upregulated in response to mTOR inhibition. Suppression of integrin α1 did not affect cell growth; however, it did significantly decrease adhesion and chemotaxis, with the influence on adhesion being greater than that on motility. Due to a positive association between integrin α1 expression and the extent of adhesion, whereby reduced receptor expression was correlated to decreased cell adhesion, it may be hypothesized that the adhesion‑blocking effects of mTOR inhibitors are not associated with mechanical contact inhibition of the α1 receptor but with integrin α1‑dependent suppression of oncogenic signaling, thus preventing tumor cell‑matrix interaction.

A chalcone-syringaldehyde hybrid inhibits triple-negative breast cancer cell proliferation and migration by inhibiting CKAP2-mediated FAK and STAT3 phosphorylation

Novel therapies for multiple myeloma.

22P - Therapeutic potential of connective tissue growth factor (CTGF) in triple-negative breast cancer

The transformable mouse embryo fibroblast cell line C3H10T 1/2 C18 has been employed to study the induction by the carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) of morphological transformation and mutation to ouabain resistance throughout the cell cycle. Cells were synchronized by means of isoleucine deprivation for 24 hr and initiated DNA synthesis with a high degree of synchrony 7.5 hr after release of the isoleucine block. At various intervals throughout the cell cycle cultures were treated with MNNG at 1.0 microgram/ml and the induction of cytotoxicity, morphological transformation, and ouabain-resistant colonies was determined. All three phenomena exhibited marked cell-cycle phase dependency. Maximal induction of transformation occurred in cultured treated 7.5 hr after release from isoleucine deprivation, when the cells were at the G1/S boundary. In contrast, induction of ouabain-resistant colonies was at a minimum at the time of maximal induction of transformation, and peak induction of ouabain resistance did not occur until 16-18 hr after release from the isoleucine block, when cells were in late S phase. A close correlation was observed between the induction of cytotoxicity and of ouabain-resistant mutants. The results suggest that differences exist in the production or cellular processing of the various early lesions.