BrdU Incorporation Analysis Research Articles

PI 3-kinase/AKT/FOXO1 signaling in smooth muscle cells protects against abdominal aortic aneurysm

Abstract Background Abdominal aortic aneurysms (AAA) are characterized by loss of vascular smooth muscle cells (SMCs). Growth factors induce proliferation and cell survival of SMCs. PI 3-kinase (PI3K) is an important downstream mediator in growth factor signaling. Objective This project is based on the hypothesis that PI3K-mediated SMC proliferation can compensate for the loss of SMCs in AAA and thus inhibit AAA progression. Therefore, we determined effects of reduced PI3K activity in SMCs on AAA progression, cell proliferation as well as vascular remodeling. Methods To investigate the role of PI3K, we analyzed mice and aortic SMCs harboring a smooth muscle-specific deficiency of the PI3K catalytic subunit p110α (SM-p110α-/-). AAA development in SM-p110α-/- mice and wild-type (WT) littermates was induced by perfusion of the infrarenal aortic segment with porcine pancreatic elastase (PPE). AAA diameter was determined by echocardiography. Structural changes in the aorta were identified by (immuno)histochemical staining. SMC proliferation was measured in vitro by BrdU incorporation or real-time cell analysis. p110α induced signal transduction pathways were characterized by Western blot and transcriptome analysis. Results PPE treatment led to a massive loss of SMCs in the compromised aortic segment after 3 days in both SM-p110α-/- and WT mice. The aortic diameter was enlarged after 28 days in all PPE-treated mice. However, the increase was significantly higher in SM-p110α-/- mice (70.2±10.8%, n=9) compared to WT animals (42.4±6.0%, n=10) (p<0.05). A similar effect was obtained in aged (untreated) mice: In 24-month-old SM-p110α-/- mice, abdominal aortic lumen diameter was increased by 42% compared to 3-month-old animals, but only by 15% in corresponding WT mice. PPE-treated SM-p110α-/- mice were characterized by reduced vascular remodeling with less PCNA-positive proliferating cells compared to WT animals. In addition, proliferation of aortic p110α-/- SMCs in serum-containing medium was impaired. Mechanistic analyses showed that PDGF- and insulin-induced activation of AKT as well as AKT-dependent phosphorylation and inactivation of the transcription factors FOXO-1, -3 and -4 were significantly reduced in p110α-/- SMCs compared to WT cells. Further analyses revealed that specific inhibition of FOXO1 by AS1842856 rescued PDGF or serum stimulated proliferation of p110α-/- SMCs. In addition, expression analysis of FOXOs in patients revealed significantly increased FOXO1 expression in AAA compared to aortic samples from healthy individuals. Conclusion Deficiency of the catalytic PI3K isoform p110α in SMCs promotes the development and progression of AAA. p110α/AKT-dependent inactivation of FOXO1 induces SMC proliferation, which can ameliorate aortic wall damage. As FOXO1 inhibitors and a recently developed p110α activator are available, the PI3K/AKT/FOXO1 signaling cascade may provide a therapeutic target to influence the stability of the aortic wall in AAA.

P53 activation enhances the sensitivity of non-small cell lung cancer to the combination of SH003 and docetaxel by inhibiting de novo pyrimidine synthesis

BackgroundIdentifying molecular biomarkers for predicting responses to anti-cancer drugs can enhance treatment precision and minimize side effects. This study investigated the novel cancer-targeting mechanism of combining SH003, an herbal medicine, with docetaxel in non-small cell lung cancer (NSCLC) cells. Also, the present study aimed to identify the genetic characteristics of cancer cells susceptible to this combination.MethodsCell viability was analyzed by WST-8 assay. Apoptosis induction, BrdU incorporation, and cell cycle analysis were performed using flow cytometry. Metabolites were measured by LC–MS/MS analysis. Real-time PCR and western blotting evaluated RNA and protein expression. DNA damage was quantified through immunofluorescence. cBioPortal and GEPIA data were utilized to explore the mutual co-occurrence of TP53 and UMPS and UMPS gene expression in NSCLC.ResultsThe combination treatment suppressed de novo pyrimidine nucleotide biosynthesis by reducing the expression of related enzymes. This blockade of pyrimidine metabolism led to DNA damage and subsequent apoptosis, revealing a novel mechanism for inducing lung cancer cell death with this combination. However, some lung cancer cells exhibited distinct responses to the combination treatment that inhibited pyrimidine metabolism. The differences in sensitivity in lung cancer cells were determined by the TP53 gene status. TP53 wild-type lung cancer cells were effectively inhibited by the combination treatment through p53 activation, while TP53 mutant- or null-type cells exhibited lower sensitivity.ConclusionsThis study, for the first time, established a link between cancer cell genetic features and treatment response to simultaneous SH003 and docetaxel treatment. It highlights the significance of p53 as a predictive factor for susceptibility to this combination treatment. These findings also suggest that p53 status could serve as a crucial criterion in selecting appropriate therapeutic strategies for targeting pyrimidine metabolism in lung cancer.

CXCR4 Loss Reduces the Local Growth of Richter Syndrome Murine and Patient-Derived Xenograft Models and Impairs BCR and VLA-4 Activation

Richter Syndrome (RS) represents transformation of chronic lymphocytic leukemia (CLL) into an aggressive lymphoma and is considered the most important unmet clinical need in CLL because of the lack of effective treatments. An important feature of RS cells is the frequent acquisition of genetic lesions in cell cycle regulators, such as CDKN2A/CDKN2B and TP53, resulting in increased proliferative capacity of the transformed cells and reduced dependence on proliferative microenvironmental signals. However, interactions with the tumor microenvironment are still required to promote the expansion of the transformed cells, as evidenced by the delayed growth of murine TCL1-derived or patient-derived xenograft (PDX) RS models following BCR disruption or macrophage depletion (Chakraborty S et al. Blood 2021, Martines C et al. Blood 2022). To further define the relevance of individual microenvironmental signals in supporting the growth of RS cells in vivo, in this study we evaluated the effects of CRISPR/Cas9-mediated knockout of the chemokine receptor CXCR4, which plays a central role in regulating the homing and interaction with the tumor microenvironment of non-transformed CLL cells. CRISPR/Cas9 editing of CXCR4 was done in the murine TCL1-derived RS model TC1-355 TKO and in the RS-PDX model RS9737, which are both characterized by biallelic disruption of TP53, CDKN2A and CDKN2B. The edited cells, comprising a mixed population with more than 80% disrupted CXCR4 alleles, were then transplanted in the peritoneal cavity and subcutaneous tissue of recipient mice (n=3 C57BL/6 mice for TCL1-355 TKO, n=8 NSG mice for RS9737). Analysis of the mutant allele frequency several weeks after injection revealed a significant reduction in the proportion of cells with mutated CXCR4 in the spleens of the injected mice and to a lesser extent in the peritoneal cavity and subcutaneous tumors, suggesting that disruption of CXCR4 signaling affects not only the migration and homing of the malignant B cells but also their local growth. To further evaluate the latter possibility, we transplanted CXCR4 knockout and CXCR4 wild type RS9737 cells in separate flanks of NSG mice (n=8) and after 4 weeks investigated the percentage of proliferating cells in the tumors by in vivo BrdU incorporation assay. A significantly lower percentage of proliferating cells was observed in the CXCR4 knockout compared to CXCR4 wild type tumors (22.3 ± 2.1 vs 37 ± 1.4, respectively, p<0.001), further suggesting that loss of CXCR4 affects the proliferation of the tumor cells. Since CXCL12 stimulation had no effect on the in vitro proliferation rate of the RS cells, we next investigated whether the reduced in vivo proliferation could be an indirect effect. For this purpose, we investigated whether CXCR4 knockout would affect integrin VLA4 or BCR signaling, as both BCR and CXCR4 can mediate inside-out VLA4 activation and there are data suggesting a potential crosstalk between the BCR and CXCR4 pathways (Maity PC et al. Front Immunol. 2018). Analysis of BCR signaling in the TCL1-355 TKO model showed a significant reduction in anti-IgM induced activation of AKT, ERK and reduced calcium mobilization in the CXCR4 knockout cells despite increased surface IgM expression. However, no effect of CXCR4 knockout on BCR signaling was observed in RS9737 cells, whereas CXCL12-induced VLA4 activation and VCAM1 adhesion were impaired in both models by CXCR4 knockout. To investigate whether impaired VLA4 activation could be responsible for the reduced in vivo proliferation rate of CXCR4 knockout RS cells, we performed BrdU incorporation analysis of RS9737 CXCR4 knockout and CXCR4 wild type cells co-cultured with the human stromal cell line HS5 + CXCL12. Co-culture with HS5 + CXCL12 resulted in a significantly increased proliferation of RS9737 CXCR4 wild type cells compared to the unstimulated control condition (58.5% ± 0.9% vs 50.1% ± 4.3%, respectively, p=0.031), whereas no such difference was observed with RS9737 CXCR4 knockout cells (50.0% ± 1.5% vs 49.1% ± 0.5%, respectively, p=n.s.). Together, these data show that CXCR4 disruption results in negative selection and reduced growth of murine and PDX RS models in vivo and affects BCR and VLA-4 signaling, suggesting that the CXCR4 pathway could represent a therapeutic vulnerability in Richter Syndrome.

P860: THE DE NOVO DNA METHYLTRANSFERASE DNMT3B PLAYS AN IMPORTANT ROLE IN MM CELL GROWTH, CLONOGENICITY AND DRUG RESPONSE

Background: Multiple myeloma (MM) is an incurable plasma cell malignancy due to the development of drug resistance (DR). In about half of the MM patients, modifications are observed in epigenetic modifiers (epiplayers) at the time of diagnosis and this frequency is further increased at relapse, indicating an important role for epiplayers in MM cell DR. However, so far, only for two epiplayers, MMSET and EZH2, a clear role in MM cell DR development has been established. With the aim to identify new, clinically relevant epiplayers involved in MM progression and relapse, we recently compared the RNASeq data from matched newly diagnosed and relapsed patients from the MMRF CoMMpass study. We found that the epiplayer DNMT3B is significantly increased in relapsed patients, suggesting a possible role for DNMT3B in MM relapse. Aims: The aim of this study is to explore the role of DNMT3B in MM cell biology and drug response. Methods: Publicly available gene expression profiling data of three independent cohorts of newly diagnosed MM patients (GSE4581, E-MTAB-372, MMRF-COMMPASS), one cohort of relapsed patients (GSE9782), BM plasma cells (E-MTAB-372), primary MM cells (E- MTAB-372), and human MM cell lines (HMCLs; E-TABM-1088 and E-TABM-937) was used. DNMT3B specific targeting was achieved by using the DNMT3B specific inhibitor Nanaomycin A and genetic inhibition using doxycycline inducible shRNA against DNMT3B. Viability and apoptosis were assessed using a CellTiter-Glo assay and an AnnexinV/7AAD staining respectively. In addition, BrdU incorporation and cell cycle analysis was evaluated to assess cell proliferation. Clonogenic capacity was evaluated by a colony formation assay. Results: We found that DNMT3B mRNA levels increase during disease progression and high DNMT3B mRNA expression correlates with a worse disease outcome in both newly diagnosed and relapsed patients, indicating a role for DNMT3B in MM progression and DR. In line, Nanaomycin A treatment (up to 72h) led to a significant and dose-dependent decrease in cell viability and proliferation and increase in apoptosis in the XG-2, XG-7 and AMO-1 HMCLs. We also validated the anti-MM activity of Nanaomycin A on human primary MM cells. Since DNMT3B is thought to play a significant role in cancer cell stemness, we next evaluated the effect on clonogenicity. A significant and dose-dependent decrease in the number of colonies was observed when low doses of Nanaomycin A were added to AMO-1 (100 and 200 nM) and XG-2 (30 and 50 nM) cells on the day of plating (day 0), but not when added 7 days after plating (day 7). In contrast, treatment with a high Nanaomycin A concentration (800 nM) significantly reduced the number of colonies at both timepoints, indicating that high Nanaomycin A doses are cytotoxic whereas low doses impair the proliferation and clonogenicity of the MM cells. The anti-clonogenic effect of DNMT3B targeting was confirmed by DNMT3B knockdown in AMO-1 cells. Finally, combining Nanaomycin A (100 nM) with bortezomib (Bz, 4 nM) resulted in a significant decrease in colony formation compared to either Nanaomycin A or Bz treatment alone. Summary/Conclusion: Taken together, our findings indicate that DNMT3B is a novel promising target to overcome or delay relapse in MM. DNMT3B targeting impairs MM cell proliferation and clonogenicity and sensitizes the cells to the proteasome inhibitor Bz. In the near future, the anti-myeloma activity of DNMT3B targeting will be validated in vivo using the 5TMM murine MM models and the underlying mechanisms will be further determined.

ATF6α promotes prostate cancer progression by enhancing PLA2G4A-mediated arachidonic acid metabolism and protecting tumor cells against ferroptosis.

BackgroundDespite the clinical success of androgen receptor (AR)‐targeted therapies, prostate cancer (PCa) inevitably progresses to castration‐resistant prostate cancer (CRPC). Transcription factor 6 α (ATF6α), an effector of the unfolded protein response (UPR) that modulates the cellular response to endoplasmic reticulum (ER) stress, has been linked to tumor development, metastasis, and relapse. However, the role of ATF6α in CRPC remains unclear.MethodsThe effect of ATF6α on the CRPC‐like phenotype in PCa cells was evaluated by 3‐(4,5‐dimethylthiazol‐2‐yl)−5‐(3‐carb‐Oxymethoxyphenyl)−2‐(4‐sulfophenyl)−2H‐tetrazolium inner salt (MTS), 5‐Bromo‐2‐deoxyUridine (BrdU) incorporation analysis, and cell death assay. Mechanistically, bioinformatic analysis was utilized to evaluate the potential of PLA2G4A as the target of ATF6α. Moreover, Western blot analysis, real‐time polymerase chain reaction, chromatin immunoprecipitation, arachidonic acid (AA), and prostaglandin E2 (PGE2) assays were performed to identify the regulatory effect of ATF6α on PLA2G4A.ResultsIn this study, we found that the increase of ATF6α expression in response to androgen deprivation generates PCa cells with a CRPC‐like phenotype. PCa cells with high levels of ATF6α expression are resistant to ferroptosis, and genetic and pharmacological inhibition of ATF6α could, therefore, promote the ferroptotic death of tumor cells and delay PCa progression. Molecular analyses linked ATF6α regulation of ferroptosis to the PLA2G4A‐mediated release of AA and the resulting increase in PGE2 production, the latter of which acts as an antiferroptotic factor.ConclusionsThis study defines ATF6α as a novel antiferroptotic regulator that exacerbates PCa progression. In addition, our data establish ATF6α‐PLA2G4A signaling as an important pathological pathway in PCa, and targeting this pathway may be a novel treatment strategy.

α-1 Adrenergic receptor antagonist doxazosin reverses hepatic stellate cells activation via induction of senescence

BackgroundActivated hepatic stellate cells (aHSCs) are the main effector cells during liver fibrogenesis. α-1 adrenergic antagonist doxazosin (DX) was shown to be anti-fibrotic in an in vivo model of liver fibrosis (LF), but the mechanism remains to be elucidated. Recent studies suggest that reversion of LF can be achieved by inducing cellular senescence characterized by irreversible cell-cycle arrest and acquisition of the senescence-associated secretory phenotype (SASP). AimTo elucidate the mechanism of the anti-fibrotic effect of DX and determine whether it induces senescence. MethodsPrimary culture-activated rat HSCs were used. mRNA and protein expression were measured by qPCR and Western blot, respectively. Cell proliferation was assessed by BrdU incorporation and xCelligence analysis. TGF-β was used for maximal HSC activation. Norepinephrine (NE), PMA and m-3M3FBS were used to activate alpha-1 adrenergic signaling. ResultsExpression of Col1α1 was significantly decreased by DX (10 μmol/L) at mRNA (−30 %) and protein level (−50 %) in TGF-β treated aHSCs. DX significantly reduced aHSCs proliferation and increased expression of senescence and SASP markers. PMA and m-3M3FBS reversed the effect of DX on senescence markers. ConclusionDoxazosin reverses the fibrogenic phenotype of aHSCs and induces the senescence phenotype.

MicroRNA-3666 Suppresses Cell Growth in Head and Neck Squamous Cell Carcinoma Through Inhibition of PFKFB3-Mediated Warburg Effect.

PurposeMicroRNA-3666 (miR-3666) is aberrantly expressed and plays critical roles in numerous human tumors. However, the expression pattern, biological role, and mechanisms of action of miR-3666 in head and neck squamous cell carcinoma (HNSCC) remain unknown. Therefore, we attempted to determine the expression status and function of miR-3666 in HNSCC and to explore the underlying mechanisms in detail.MethodsIn this study, quantitative real-time polymerase chain reaction was carried out to measure the expression of miR-3666 HNSCC tissues. A series of experiments, including a Cell Counting Kit-8 assay, colony formation assay, BrdU incorporation and apoptosis analysis, were applied to test whether miR-3666 affects the growth of HNSCC cells. Glucose uptake and lactate production measurements and extracellular acidification and oxygen consumption rate assays were conducted to determine the effect of miR-3666 on glycolysis.ResultsWe found that miR-3666 showed a decreased expression in HNSCC tissues. Further functional studies demonstrated that miR-3666 inhibited the growth of HNSCC cells by suppressing cell proliferation and promoting apoptosis. Bioinformatics analysis and luciferase reporter assays identified phosphofructokinase-2/fructose-2,6-bisphosphatase 3 (PFKFB3), a key enzyme regulating glycolysis, as a direct target of miR-3666. Through inhibition of PFKFB3, miR-3666 decreased glycolysis in HNSCC cells by reducing the production of F2,6BP. Importantly, glycolysis suppression caused by miR-3666 was found to be required for its inhibitory effect on HNSCC cell growth.ConclusionOur data suggest that miR-3666 functions as a tumor suppressor by decreasing the rate of glycolysis through inhibition of PFKFB3 activity, and this miRNA may present a potential candidate for HNSCC therapy.

Platelet Lysate as source of growth factors for Bone Marrow derived Mesenchymal Stromal cells clinical expansion: a study of starting platelet concentration dose-dependent effects

Background & Aim Background In the last decades the replacement of fetal bovine serum (FBS)with human Platelet Lysate (hPL) for ATMPs clinical expansion has been for a long time investigated to overcome FBS related issues.Despite several studies confirm hPL safety and efficiency in Mesenchymal Stromal Cell(MSC)expansion, there are still some gaps in the knowledge of hPL as cell culture supplement, starting from composition and quality release criteria. Aim of the study As growth factors are released after thrombocytes lysis during hPL production, starting platelet concentration may affect hPL quality.The aim of this study was to investigate hPL starting platelet concentration effects on bone marrow derived MSC(BM-MSC). Methods, Results & Conclusion Material and methods MSC were isolated from the bone marrow of 7 donors and cultured from p1 to p7 in 4 different conditions: DMEM 10% FBS and DMEM 5% hPL varying starting platelet concentration.Particularly hPL was produced according to standardized protocol by in-hospital Transfusion Service, in three different starting platelet concentrations (sPLTC): high sPLTC(4 × 109 PLTS/ml), medium(2 × 109 PLTS/ml) and low(1 × 109 PLTS/ml).The study focused on analysis of parameters that are mostly affected by hPL such as proliferation, immunophenotype, telomeric length, differentiation and cell senescence. Results and Conclusions Evaluation of proliferation indexes(PDT and PD) underlined dose dependent effects of sPLTC (figure 1), also confirmed by flow cytometry cell cycle analysis of BrdU incorporation. Immunophenotype seems not to be affected by sPLTC, as classical MSC markers meet ISCT criteria (table 1). Differences were instead detected by analyzing mean fluorescence intensity (MFI) in a dose dependent manner (figure 2), both for intrinsic and extrinsic parameters. Differentiation potential seems to be unaffected by different sPLTC as all cells batches differentiated into osteoblasts, adipocytes and chondrocytes. On the contrary senescence and relative telomeric length RTL (detected by SA-β-GAL activity and PNA-FITC flow cytometry) are strongly affected by sPLTC, in a dose dependent manner. Particularly high sPLTC results in increased senescent cells associated with decreased RTL (figure 3). In conclusion our data showed that sPLTC affects some BM MSCs properties, underlying its importance during hPL preparation. According to this study we suggest a medium sPLTC for hPL preparation, as best compromise between increase in proliferation index and effects on senescence.

Uptake and tumor-suppressive pathways of exosome-associated GKN1 protein in gastric epithelial cells.

Gastrokine 1 (GKN1) is a stomach-specific tumor suppressor that is secreted into extracellular space as an exosomal cargo protein. The objective of this study was to investigate the uptake and tumor-suppressive pathways of exosome-associated GKN1 protein in gastric epithelial cells. Immunofluorescent and Western blot analysis were used to investigate gastric-specific uptake of HFE-145-derived exosomes. Binding affinity of HFE-145 derived exosomes with integrin proteins was examined using protein microarray chip. Tumor suppressor activities of exosome-carrying GKN1 protein were analyzed using transwell co-culture, MTT assay, BrdU incorporation, immunoprecipitation, and Western blot analysis. HFE-145-derived exosomes were internalized only into HFE-145 gastric epithelial cells and gastric cancer cells. Gastric-specific uptake of stomach-derived exosomes required integrin α6 and αX proteins. Clathrin and macropinocytosis increased the uptake of exosomes into gastric epithelial cells, whereas caveolin inhibited the uptake of exosomes. Transwell co-culture of AGS cells with HFE-145 cells markedly inhibited viability and proliferation of AGS cells. Following uptake of HFE-145-derived exosomes in recipient cells, GKN1 protein bound to HRas and inhibited the binding of HRas to b-Raf and c-Raf which subsequently downregulated HRas/Raf/MEK/ERK signaling pathways in AGS, MKN1 cells, and MKN1-derived xenograft tumor tissues. In addition, exosomal GKN1 protein suppressed both migration and invasion of gastric cancer cells by inhibiting epithelial-mesenchymal transition. Gastric-specific uptake of exosomes derived from gastric epithelial cells requires integrin α6 and αX proteins in both gastric epithelial cells and exosomes. Exosomal GKN1 protein inhibits gastric carcinogenesis by downregulating HRas/Raf/MEK/ERK signaling pathways.

Quantitative proteomic analysis identifies novel regulators of methotrexate resistance in choriocarcinoma

ObjectiveAlthough methotrexate (MTX) is commonly used for the treatment of choriocarcinoma, chemoresistance to MTX may occur in a considerable fraction of patients. Further understanding on the mechanisms of MTX resistance would help to develop more effective therapy for choriocarcinoma. MethodsQuantitative proteomic approach involving TMT labeling and LC-MS/MS was used to identify MTX resistance-related proteomic profiles in choriocarcinoma cell models. Pathway and process enrichment analysis were conducted to identify MTX resistance-related biological processes/molecular pathways. CCK-8 viability assay, clonogenic survival assay, and BrdU incorporation analysis were used to examine the chemosensitivity to MTX in choriocarcinoma cells. ResultsIn total, 5704 protein groups were identified, among which 4997 proteins were quantified. Bioinformatic analysis revealed that multiple biological processes/molecular pathways might be associated with MTX resistance in JEG3/JEG3/MTX cell systems. DPP4 and METTL7A were selected for further investigation. Increased expression of DPP4 or METTL7A was observed in MTX-resistant cancer cell lines and choriocarcinoma tissues. Knockdown of DPP4 or METTL7A significantly decreased cell viability, impaired clonogenesis, and increased apoptosis after MTX treatment in JEG3/MTX and JAR/MTX cells; while over-expression of DPP4 or METTL7A promoted cell viability and reduced apoptosis following exposure to MTX in JEG3, JAR and BEWO cells. Further, DPP4 and METTL7A differentially activated prosurvival signaling pathways including PI3K/AKT, ERK1/2 and STAT3, and attenuated the accumulation of reactive oxygen species (ROS) in choriocarcinoma cell lines. ConclusionsDPP4 and METTL7A might promote MTX resistance through activating pro-survival signaling pathways and attenuating the accumulation of ROS in choriocarcinoma cells. Targeting DPP4 and METTL7A might be useful to sensitize choriocarcinoma cells to MTX-based chemotherapy.

Prolactin, Estradiol and Testosterone Differentially Impact Human Hippocampal Neurogenesis in an In Vitro Model

Previous studies have indicated that sex hormones such as prolactin, estradiol and testosterone may play a role in the modulation of adult hippocampal neurogenesis (AHN) in rodents and non-human primates, but so far there has been no investigation of their impact on human hippocampal neurogenesis. Here, we quantify the expression levels of the relevant receptors in human post-mortem hippocampal tissue and a human hippocampal progenitor cell (HPC) line. Secondly, we investigate how these hormones modulate hippocampal neurogenesis using a human in vitro cellular model. Human female HPCs were cultured with biologically relevant concentrations of either prolactin, estradiol or testosterone. Bromodeoxyuridine (BrdU) incorporation, immunocytochemistry (ICC) and high-throughput analyses were used to quantify markers determining cell fate after HPCs were either maintained in a proliferative state or allowed to differentiate in the presence of these hormones. In proliferating cells, estrogen and testosterone increased cell density but had no clear effect on markers of proliferation or cell death to account for this. In differentiating cells, a 3-day treatment of prolactin elicited a transient effect, whereby it increased the proportion of microtubule-associated protein 2 (MAP2)-positive and Doublecortin (DCX)-positive cells, but this effect was not apparent after 7-days. At this timepoint we instead observe a decrease in proliferation. Overall, our study demonstrates relatively minor, and possibly short-term effects of sex hormones on hippocampal neurogenesis in human cells. Further work will be needed to understand if our results differ to previous animal research due to species-specific differences, or whether it relates to limitations of our in vitro model.

Deactivation of ATP-Binding Cassette Transporters ABCB1 and ABCC1 Does Not Influence Post-ischemic Neurological Deficits, Secondary Neurodegeneration and Neurogenesis, but Induces Subtle Microglial Morphological Changes.

ATP-binding cassette (ABC) transporters prevent the access of pharmacological compounds to the ischemic brain, thereby impeding the efficacy of stroke therapies. ABC transporters can be deactivated by selective inhibitors, which potently increase the brain accumulation of drugs. Concerns have been raised that long-term ABC transporter deactivation may promote neuronal degeneration and, under conditions of ischemic stroke, compromise neurological recovery. To elucidate this issue, we exposed male C57BL/6 mice to transient intraluminal middle cerebral artery occlusion (MCAO) and examined the effects of the selective ABCB1 inhibitor tariquidar (8 mg/kg/day) or ABCC1 inhibitor MK-571 (10 mg/kg/day), which were administered alone or in combination with each other over up to 28 days, on neurological recovery and brain injury. Mice were sacrificed after 14, 28, or 56 days. The Clark score, RotaRod, tight rope, and open field tests revealed reproducible motor-coordination deficits in mice exposed to intraluminal MCAO, which were not influenced by ABCB1, ABCC1, or combined ABCB1 and ABCC1 deactivation. Brain volume, striatum volume, and corpus callosum thickness were not altered by ABCB1, ABCC1 or ABCB1, and ABCC1 inhibitors. Similarly, neuronal survival and reactive astrogliosis, evaluated by NeuN and GFAP immunohistochemistry in the ischemic striatum, were unchanged. Iba1 immunohistochemistry revealed no changes of the overall density of activated microglia in the ischemic striatum of ABC transporter inhibitor treated mice, but subtle changes of microglial morphology, that is, reduced microglial cell volume by ABCB1 deactivation after 14 and 28 days and reduced microglial ramification by ABCB1, ABCC1 and combined ABCB1 and ABCC1 deactivation after 56 days. Endogenous neurogenesis, assessed by BrdU incorporation analysis, was not influenced by ABCB1, ABCC1 or combined ABCB1 and ABCC1 deactivation. Taken together, this study could not detect any exacerbation of neurological deficits or brain injury after long-term ABC transporter deactivation in this preclinical stroke model.

Proliferative Activity of Colorectal Cancer Cells with Different Levels of CD133 Expression.

We studied proliferative activity of colorectal cancer cells with different expression level of CD133 molecule associated with cancer stem cells phenotype. Analysis of BrdU incorporation into Caco-2 and HT-29 cell lines showed that the percentage of cells in the DNA synthesis phase in the CD133+/high population is higher than in CD133-/low population. The expression of proliferation marker Ki-67 and the percentage of Ki-67+ cells were also higher in the CD133+/high population. Colorimetric analysis with crystal violet dye showed that the number of cells after 10-days culturing was higher in the CD133+/high population in both cell lines. These findings suggest that cells with high level of CD133 expression are characterized by higher proliferative activity, which can contribute to the tumor progression.

Heparin treatment mitigates radiation-induced oral mucositis in mice by interplaying with repopulation processes

PurposeTo investigate the mechanistic background of the muco-protective effect of systemic heparin treatment on the development of radiation-induced oral mucositis in mice.Materials and methodsFractionated irradiation was given to the snouts of male C3H/Neu mice over 2 weeks (10 × 3 Gy), either alone or in combination with daily subcutaneous application of unfractionated or low molecular weight heparin (40 or 200 I.U./mouse, respectively). Over this course of 14 days, groups of mice (n = 3) were sacrificed every second day, their tongues excised and processed for histological analysis. The epithelial radiation response with and without heparin treatment was evaluated in terms of tissue morphology, proliferation and expression of cell contact molecules.ResultsSystemic treatment with heparins significantly reduced the cellular effects of irradiation to the oral epithelium. Heparin treated animals showed significantly higher total epithelial cell numbers and thickness throughout the study course. Bromodeoxyuridine (BrdU) incorporation analyses revealed that markedly more epithelial cells retained their proliferative capacity in the beginning of the first treatment week, but the proliferation of the mucosa was not stimulated during the rest of the study course. The expression of the adherens junction protein β‑catenin was slightly elevated in heparin treated animals, on day 2 the increase was statistically significant. The expression of e‑cadherin and occludin was mostly unaffected by the concomitant heparin treatment.ConclusionThe findings of this study indicate an interplay of additional heparin treatment with the repopulation processes, leading to an earlier onset of this adaptive radiation response in oral mucosa. Importantly, we could demonstrate that the protective potential of heparin did not rely on stimulation of normal tissue proliferation. Since both heparin preparations are already approved for clinical use, they are considered as promising candidates for future clinical studies.

Quantitative Proteomic Analysis Identifies MAPK15 as a Potential Regulator of Radioresistance in Nasopharyngeal Carcinoma Cells.

Since resistance to radiotherapy remains refractory for the clinical management of nasopharyngeal cancer (NPC), further understanding the mechanisms of radioresistance is necessary in order to develop more effective NPC treatment and improve prognosis. In this study, an integrated quantitative proteomic approach involving tandem mass tag labeling and liquid chromatograph-mass spectrometer was used to identify proteins potentially responsible for the radioresistance of NPC. The differential radiosensitivity in NPC model cells was examined through clonogenic survival assay, CCK-8 viability assay, and BrdU incorporation analysis. Apoptosis of NPC cells after exposure to irradiation was detected using caspase-3 colorimetric assay. Intracellular reactive oxygen species (ROS) was detected by a dichlorofluorescin diacetate fluorescent probe. In total, 5,946 protein groups were identified, among which 5,185 proteins were quantified. KEGG pathway analysis and protein-protein interaction enrichment analysis revealed robust activation of multiple biological processes/pathways in radioresistant CNE2-IR cells. Knockdown of MAPK15, one up-regulated protein kinase in CNE2-IR cells, significantly impaired clonogenic survival, decreased cell viability and increased cell apoptosis following exposure to irradiation, while over-expression of MAPK15 promoted cell survival, induced radioresistance and reduced apoptosis in NPC cell lines CNE1, CNE2, and HONE1. MAPK15 might regulate radioresistance through attenuating ROS accumulation and promoting DNA damage repair after exposure to irradiation in NPC cells. Quantitative proteomic analysis revealed enormous metabolic processes/signaling networks were potentially involved in the radioresistance of NPC cells. MAPK15 might be a novel potential regulator of radioresistance in NPC cells, and targeting MAPK15 might be useful in sensitizing NPC cells to radiotherapy.

Chidamide-induced ROS accumulation and miR-129-3p-dependent cell cycle arrest in non-small lung cancer cells

BackgroundEpigenetic therapy is a promising popular treatment modality for various cancers. Histone modification and miRNA should not be underestimated in lung cancer. This study aimed to investigate whether chidamide, a histone deacetylase inhibitor (HDACi), which inhibits telomerase activity and induces cell cycle arrest, influences ROS and miRNA production in non-small cell lung cancer (NSCLC) cells. MethodsH1355 and A549 were treated with chidamide. The analysis of DNA content was measured by FACSCalibur equipped with a 488 nm laser. H1355 cells were transfected with miR-129-3p mimic by Lipofectamine2000. Telomerase activity was performed on the telomeric repeat amplification protocol (TRAP) assay. Detection of thymidylate synthase (TS), p21, p53, pRB, and β-actin, were performed by western blot analysis. ResultsOur data showed that expression of TS, p21, and pRB were altered in the presence of chidamide by PCR and western blot. Using BrdU-incorporation analysis, we found that chidamide induced G1 arrest through the regulation of the TS gene by miR-129-3p. Chidamide was shown to suppress telomerase activity in the TRAP assay and reduced the expression of human telomerase reverse transcriptase (hTERT) by PCR and q-PCR in H1355 and A549 cells. Chidamide increased the generation of reactive oxygen species (ROS) by flow cytometry. N-acetyl cysteine (NAC), a ROS scavenger, attenuated chidamide-induced telomerase activity inhibition. ConclusionChidamide repressed telomerase activity through ROS accumulation and cell cycle arrest by miR-129-3p upregulation in both H1355 and A549 cells. This is the first study to demonstrate that chidamide induces miR-129-3p upregulation and ROS accumulation, leading to cell cycle arrest.

Whole-Mount BrdU Staining with Fluorescence In Situ Hybridization in Planarians.

Detection of cell proliferation based on the incorporation of 5'-bromo-2'-deoxyuridine (BrdU) has become a standard approach for studying stem cell and progenitor cell populations in developing and adult tissue. In this chapter, we describe three BrdU administration methods for planarians and a staining protocol combining BrdU detection with whole-mount fluorescence in situ hybridization (FISH). Collectively, these protocols enable the combined analysis of BrdU-incorporation and endogenous gene expression, as for example during lineage tracing applications.

Detecting Hematopoietic Stem Cell Proliferation Using BrdU Incorporation.

Cellular quiescence is a key component of hematopoietic stem cell (HSC) homeostasis; therefore, a reliable method to measure HSC cell division is critical in many studies. However, measuring the proliferation rate of largely quiescent and rare populations of cells can be challenging. Bromo-deoxyuridine (BrdU) incorporation into replicating DNA is a commonly used and highly reproducible method to detect cell division history. Here, we describe a protocol for BrdU incorporation analysis in hematopoietic stem and progenitor cells that can provide a sensitive measure of cell division even in rare cell populations. In combination with flow cytometry, this method can be generalized to analyze other cell populations and other tissues as identified by cell surface markers.

Flow Cytometry Analysis of Cell Cycle and Specific Cell Synchronization with Butyrate.

Synchronized cells have been invaluable in many kinds of cell cycle and cell proliferation studies. Butyrate induces cell cycle arrest and apoptosis in MDBK cells. We explore the possibility of using butyrate-blocked cells to obtain synchronized cells and we characterize the properties of butyrate-induced cell cycle arrest. The site of growth inhibition and cell cycle arrest was analyzed using 5-bromo-2'-deoxyuridine (BrdU) incorporation and flow cytometry analyses. Exposure of MDBK cells to 10 mM butyrate caused growth inhibition and cell cycle arrest in a reversible manner. Butyrate affected the cell cycle at a specific point both immediately after mitosis and at a very early stage of the G1 phase. After release from butyrate arrest, MDBK cells underwent synchronous cycles of DNA synthesis and transited through the S phase. It takes at least 8 h for butyrate-induced G1-synchronized cells to begin the progression into the S phase. One cycle of cell division for MDBK cells is about 20 h. By combining BrdU incorporation and DNA content analysis, not only can the overlapping of different cell populations be eliminated, but the frequency and nature of individual cells that have synthesized DNA can be determined.

Interleukin-9 promotes cell survival and drug resistance in diffuse large B-cell lymphoma.

BackgroundInterleukin-9 (IL-9) was discovered as a helper T cell growth factor. It has long been recognized as an important regulator in allergic inflammation. Recent years it was discovered to induce cell growth and differentiation of multiple transformed cells. However, its oncogenic activities in B-cell lymphomas have not been reported in detail.MethodsSerum levels of IL-9 in DLBCL patients were quantified by ELISA, and its clinical significance was analysed. The expression of IL-9 receptor (IL-9R) was investigated in lymphoma cell lines by RT-PCR and western blot, respectively. In DLBCL cell lines LY1 and LY8, IL-9R genes were knocked down by RNA interference and stable transfected cells were selected with puromycin. Normal and final siIL-9R (and siControl) LY1 and LY8 cells were treated with IL-9 alone and in synergy with chemotherapeutic drugs. Cell proliferation and apoptosis were assessed by Brdu incorporation and flow cytometric analysis. The mRNA of apoptosis regulation genes were measured with real-time PCR.ResultsElevated serum levels of IL-9 were detected in DLBCL patients (24/30) compared to healthy controls (0/15). Positive expression of IL-9 (defined as a serum level ≥1 pg/ml) was correlated with lower serum albumin levels and high international prognostic index (IPI) scores. IL-9R was expressed in both mRNA and protein levels in the five lymphoma cell lines, including LY1, LY8, MINO, SP53 and Jurkat. In vitro studies showed that IL-9 directly induced proliferation and inhibited apoptosis in LY1 and LY8 cells. It protects LY1 and LY8 cells from prednisolone induced apoptosis, and promotes their proliferation that were inhibited by rituximab, vincristine and prednisolone. Its molecular mechanism may be concerned with upregulating expression of p21CIP1 gene. Knock-down of IL-9R gene could reverse the effects of IL-9 on LY1 and LY8 cells.ConclusionsIL-9 is associated with clinical features of DLBCL patients. It promotes survival of DLBCL cells and reduces the sensitivities of tumor cells to chemotherapeutic drugs via upregulation of p21CIP1 genes.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-016-0374-3) contains supplementary material, which is available to authorized users.