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International Journal of Molecular... Jul 2023Despite the decreasing trend in mortality from colorectal cancer, this disease still remains the third most common cause of death from cancer. In the present study, we...
Despite the decreasing trend in mortality from colorectal cancer, this disease still remains the third most common cause of death from cancer. In the present study, we investigated the antiproliferative and pro-apoptotic effects of (2,3,4)-2-tridecylpyrrolidine-3,4-diol hydrochloride on colon cancer cells (Caco-2 and HCT116). The antiproliferative effect and IC values were determined by the MTT and BrdU assays. Flow cytometry, qRT-PCR and Western blot were used to study the cellular and molecular mechanisms involved in the induction of apoptotic pathways. Colon cancer cell migration was monitored by the scratch assay. Concentration-dependent cytotoxic and antiproliferative effects on both cell lines, with IC values of 3.2 ± 0.1 μmol/L (MTT) vs. 6.46 ± 2.84 μmol/L (BrdU) for HCT116 and 2.17 ± 1.5 μmol/L (MTT) vs. 1.59 ± 0.72 μmol/L (BrdU), for Caco-2 were observed. The results showed that tridecylpyrrolidine-induced apoptosis was associated with the externalization of phosphatidylserine, reduced mitochondrial membrane potential (MMP) accompanied by the activation of casp-3/7, the cleavage of PARP and casp-8, the overexpression of TNF-α and FasL and the dysregulation of Bcl-2 family proteins. Inhibition of the migration of treated cells across the wound area was detected. Taken together, our data show that the anticancer effects of tridecylpyrrolidine analogues in colon cancer cells are mediated by antiproliferative activity, the induction of both extrinsic and intrinsic apoptotic pathways and the inhibition of cell migration.
Topics: Humans; Bromodeoxyuridine; Caco-2 Cells; Apoptosis; Signal Transduction; Colonic Neoplasms; Cell Proliferation; Cell Line, Tumor; Membrane Potential, Mitochondrial
PubMed: 37511455
DOI: 10.3390/ijms241411696 -
Methods in Molecular Biology (Clifton,... 2018Cellular quiescence is a key component of hematopoietic stem cell (HSC) homeostasis; therefore, a reliable method to measure HSC cell division is critical in many...
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.
Topics: Bromodeoxyuridine; Cell Cycle; Cell Division; Cell Proliferation; Flow Cytometry; Hematopoietic Stem Cells; Homeostasis; Humans
PubMed: 29030815
DOI: 10.1007/978-1-4939-7371-2_7 -
Journal of Virology Oct 1967When simian virus 40 (SV40)-transformed mouse kidney cells (mKS) were grown in the presence of susceptible indicator cells, SV40 was readily recovered from: (i) 15...
When simian virus 40 (SV40)-transformed mouse kidney cells (mKS) were grown in the presence of susceptible indicator cells, SV40 was readily recovered from: (i) 15 transformed cell lines, (ii) transformed cells subcultured 45 times over a 7-month period in medium containing antiviral serum and bromodeoxyuridine (dBU), (iii) 45 of 46 clonal lines isolated in the presence of antiviral serum, (iv) 19 of 19 secondary clones isolated from two clonal lines, and (v) dBU-resistant transformed cell lines. dBU-resistant SV40-transformed mouse kidney cell lines were selected and shown to contain the T antigen and to have normal levels of thymidylate kinase and deoxyribonucleic acid (DNA) polymerase, but to be deficient in thymidine (dT) kinase. Radioautographic and biochemical experiments demonstrated that very little (3)H-dT was incorporated into DNA of dBU-resistant cells during a 6-hr labeling period. After infection of dT kinase-deficient mKS cells with vaccinia virus, high levels of dT kinase were induced. The properties of SV40 recovered from dBU-sensitive and dBU-resistant cells were studied. SV40 recovered from transformed cells was shown to express in CV-1 cells at least six functions characteristic of parental virus: synthesis of capsid antigen, synthesis of T antigen, synthesis of viral DNA, induction of dT kinase, induction of DNA polymerase, and induction of host cell DNA synthesis. In addition, SV40 recovered from the transformed cells induced T antigen, dT kinase, deoxycytidylate deaminase, thymidylate kinase, and DNA polymerase in abortively infected mouse kidney cultures, and the virus was also capable of transforming primary cultures of mouse kidney cells.
Topics: Animals; Antigens; Bromodeoxyuridine; Cell Line; Cell Transformation, Neoplastic; Culture Techniques; Cytopathogenic Effect, Viral; Haplorhini; Immune Sera; Kidney; Mice; Neoplasms; Simian virus 40; Tritium
PubMed: 4316241
DOI: 10.1128/JVI.1.5.968-979.1967 -
The Journal of Histochemistry and... Mar 2022Epithelial proliferation in the rat mammary gland is recommended in regulatory guidelines as an endpoint for assessment of the in vivo carcinogenic potential of insulin...
Epithelial proliferation in the rat mammary gland is recommended in regulatory guidelines as an endpoint for assessment of the in vivo carcinogenic potential of insulin analogues. Epithelial proliferation is traditionally assessed by immunohistochemical staining of a proliferation marker, for example, 5-bromo-2'-deoxyuridine (BrdU) or Ki67, followed by labor-intensive manual counting of positive and negative cells. The aim of this study was to develop and validate an approach for image analysis based on artificial intelligence, which can be used for quantification of proliferation in rat mammary gland, independent of the choice of proliferation marker. Furthermore, the aim was to compare the markers BrdU, Ki67, and phosphorylated histone H3 (PHH3). A sequence of image analysis applications were developed, which allowed for quantification of proliferative activity in the mammary gland epithelium. These endpoints agreed well with manually counted labeling indices, with correlation coefficients in the range ≈0.92-0.93. In addition, all three proliferation markers were significantly correlated and could detect the variation in epithelial proliferation during the estrous cycle. In conclusion, image analysis can be used to quantify epithelial proliferation in the rat mammary gland and thereby replace time-consuming manual counting. Furthermore, BrdU, Ki67, and PHH3 can be used interchangeably to assess proliferation.
Topics: Animals; Artificial Intelligence; Biomarkers; Bromodeoxyuridine; Cell Proliferation; Epithelium; Female; Histones; Immunohistochemistry; Ki-67 Antigen; Mammary Glands, Animal; Rats; Rats, Sprague-Dawley
PubMed: 35057663
DOI: 10.1369/00221554221075327 -
International Journal of Molecular... Dec 2022Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder and warrants further study as well as timely treatment. Additionally, the mechanisms of the...
Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder and warrants further study as well as timely treatment. Additionally, the mechanisms of the brain's intrinsic defense against chronic injury are not yet fully understood. Herein, we examined the response of the main neurogenic niches to amyloid exposure and the associated changes in structure and synaptic activity. Flow cytometry of Nestin-, Vimentin-, Nestin/Vimentin-, NeuN-, GFAP-, NeuN/GFAP-, NSE-, BrdU-, Wnt-, BrdU/Wnt-, VEGF-, Sox14-, VEGF/Sox14-, Sox10-, Sox2-, Sox10/Sox2-, Bax-, and Bcl-xL-positive cells was performed in the subventricular zone (SVZ), hippocampus, and cerebral cortex of rat brains on 90th day after intracerebroventricular (i.c.v.) single injection of a fraction of β-amyloid (Aβ) (1-42). The relative structural changes in these areas and disruptions to synaptic activity in the entorhinal cortex-hippocampus circuit were also evaluated. Our flow analyses revealed a reduction in the numbers of Nestin-, Vimentin-, and Nestin/Vimentin-positive cells in neurogenic niches and the olfactory bulb. These changes were accompanied by an increased number of BrdU-positive cells in the hippocampus and SVZ. The latter changes were strongly correlated with changes in the numbers of VEGF- and VEGF/Sox14-positive cells. The morphological changes were characterized by significant neural loss, a characteristic shift in entorhinal cortex-hippocampus circuit activity, and decreased spontaneous alternation in a behavioral test. We conclude that although an injection of Aβ (1-42) induced stem cell proliferation and triggered neurogenesis at a certain stage, this process was incomplete and led to neural stem cell immaturity. We propose the idea of enhancing adult neurogenesis as a promising strategy for preventing dementia at healthy elderly people andpeople at high risk for developing AD, or treating patients diagnosed with AD.
Topics: Animals; Rats; Vascular Endothelial Growth Factor A; Neurogenesis; Amyloid beta-Peptides; Brain; Hippocampus; Alzheimer Disease; Bromodeoxyuridine; Amyloidogenic Proteins
PubMed: 36499771
DOI: 10.3390/ijms232315444 -
Stem Cells (Dayton, Ohio) Dec 2008Bromodeoxyuridine (BrdU) is a halogenated pyrimidine that incorporates into newly synthesized DNA during the S phase. BrdU is used ubiquitously in cell birthdating...
Bromodeoxyuridine (BrdU) is a halogenated pyrimidine that incorporates into newly synthesized DNA during the S phase. BrdU is used ubiquitously in cell birthdating studies and as a means of measuring the proliferative index of various cell populations. In the absence of secondary stressors, BrdU is thought to incorporate relatively benignly into replicating DNA chains. However, we report here that a single, low-dose pulse of BrdU exerts a profound and sustained antiproliferative effect in cultured murine stem and progenitor cells. This is accompanied by altered terminal differentiation, cell morphology, and protein expression consistent with the induction of senescence. There is no evidence of a significant increase in spontaneous cell death; however, cells are rendered resistant to chemically induced apoptosis. Finally, we show that a brief in vivo BrdU regimen reduces the proliferative potential of subsequently isolated subependymal zone neurosphere-forming cells. We conclude, therefore, that BrdU treatment induces a senescence pathway that causes a progressive decline in the replication of rapidly dividing stem/progenitor cells, suggesting a novel and uncharacterized effect of BrdU. This finding is significant in that BrdU-incorporating neural stem/progenitor cells and their progeny should not be expected to behave normally with respect to proliferative potential and downstream functional parameters. This effect highlights the need for caution when results based on long-term BrdU tracking over multiple rounds of replication are interpreted. Conversely, the reliable induction of senescence in stem/progenitor cells in vitro and in vivo may yield a novel platform for molecular studies designed to address multiple aspects of aging and neurogenesis.
Topics: Animals; Apoptosis; Astrocytes; Bromodeoxyuridine; Cell Proliferation; Cells, Cultured; Cellular Senescence; Mice; Mice, Inbred C57BL; Neurons; Stem Cells; Time Factors; beta-Galactosidase
PubMed: 18802036
DOI: 10.1634/stemcells.2008-0299 -
Anales Del Sistema Sanitario de Navarra Aug 2018
Topics: Aged; Antimetabolites, Antineoplastic; Antiviral Agents; Bromodeoxyuridine; Diagnostic Errors; Drug Interactions; Fatal Outcome; Fluorouracil; Herpes Zoster; Humans; Male
PubMed: 29943764
DOI: 10.23938/ASSN.0297 -
Pharmacological Reports : PR Oct 2022Breast cancer (BC) is the most common malignancy and the leading cause of cancer-related death in women worldwide. Sirtuin inhibitors (SIRTi), belonging to the histone...
BACKGROUND
Breast cancer (BC) is the most common malignancy and the leading cause of cancer-related death in women worldwide. Sirtuin inhibitors (SIRTi), belonging to the histone deacetylase inhibitors group (HDIs), are potent epigenetic drugs that have been investigated for therapeutic use in different clinical disorders, including hematological malignancies and solid tumors.
METHODS
The influence of cambinol (CAM; SIRTi) used individually or in combination with standard chemotherapeutic paclitaxel (PAX) on viability (MTT assay), proliferation (BrdU assay), induction of apoptosis and cell cycle arrest (FACS analysis) was determined in MCF7 luminal and MDA-MB-231 triple-negative breast cancer (TNBC) cells. The types of pharmacological drug-drug interaction between CAM and PAX were determined by an exact and rigorous pharmacodynamic method-an isobolography, to determine the presence of synergism, addition or antagonism between analyzed drugs using a variety of fixed-dose ratios.
RESULTS
The combination of CAM and PAX at a fixed ratio of 1:1 exerted additive interaction in the viability of MCF7 and MDA-MB-231 BC cells. Both active agents used separately reduced viability and proliferation of BC cells as well as induced apoptosis and cell cycle arrest. These effects were much more evident in MCF7 than in MDA-MB-231 BC cells. Additionally, CAM combined with PAX increased anti-cancer activity compared to PAX used alone.
CONCLUSION
CAM might be considered a potential therapeutic agent individually or in combined therapy with PAX against luminal or TNBC.
Topics: Humans; Female; Triple Negative Breast Neoplasms; Paclitaxel; Histone Deacetylase Inhibitors; Breast Neoplasms; Sirtuins; Bromodeoxyuridine; Cell Line, Tumor; Cell Proliferation; Xenograft Model Antitumor Assays; Apoptosis
PubMed: 35900723
DOI: 10.1007/s43440-022-00393-w -
Journal of Bacteriology Oct 2016We tested pairwise combinations of classical base analog mutagens in Escherichia coli to study possible mutagen synergies. We examined the cytidine analogs zebularine...
UNLABELLED
We tested pairwise combinations of classical base analog mutagens in Escherichia coli to study possible mutagen synergies. We examined the cytidine analogs zebularine (ZEB) and 5-azacytidine (5AZ), the adenine analog 2-aminopurine (2AP), and the uridine/thymidine analog 5-bromodeoxyuridine (5BrdU). We detected a striking synergy with the 2AP plus ZEB combination, resulting in hypermutability, a 35-fold increase in mutation frequency (to 53,000 × 10(-8)) in the rpoB gene over that with either mutagen alone. A weak synergy was also detected with 2AP plus 5AZ and with 5BrdU plus ZEB. The pairing of 2AP and 5BrdU resulted in suppression, lowering the mutation frequency of 5BrdU alone by 6.5-fold. Sequencing the mutations from the 2AP plus ZEB combination showed the predominance of two new hot spots for A·T→G·C transitions that are not well represented in either single mutagen spectrum, and one of which is not found even in the spectrum of a mismatch repair-deficient strain. The strong synergy between 2AP and ZEB could be explained by changes in the dinucleoside triphosphate (dNTP) pools.
IMPORTANCE
Although mutagens have been widely studied, the mutagenic effects of combinations of mutagens have not been fully researched. Here, we show that certain pairwise combinations of base analog mutagens display synergy or suppression. In particular, the combination of 2-aminopurine and zebularine, analogs of adenine and cytidine, respectively, shows a 35-fold increased mutation frequency compared with that of either mutagen alone. Understanding the mechanism of synergy can lead to increased understanding of mutagenic processes. As combinations of base analogs are used in certain chemotherapy regimens, including those involving ZEB and 5AZ, these results indicate that testing the mutagenicity of all drug combinations is prudent.
Topics: Azacitidine; Base Pairing; Bromodeoxyuridine; Cytidine; Drug Synergism; Escherichia coli; Escherichia coli Proteins; Mutagens; Mutation
PubMed: 27457718
DOI: 10.1128/JB.00391-16 -
Sichuan Da Xue Xue Bao. Yi Xue Ban =... Sep 2023To investigate the effect of photobiomodulation (PBM) on hippocampal neurogenesis, cognitive function, and inflammatory injury in rats with chronic cerebral...
OBJECTIVE
To investigate the effect of photobiomodulation (PBM) on hippocampal neurogenesis, cognitive function, and inflammatory injury in rats with chronic cerebral hypoperfusion.
METHODS
Bilateral ovariectomy (OVX) was performed on female Sprague-Dawley (SD) rats. One week later, the rats were randomly assigned to three groups, Sham surgery (or Sham) group, bilateral common carotid artery occlusion (BCCAO) group, and PBM intervention (or BCCAO+PBM) group. There were 8 rats in each group. In the BCCAO group, chronic cerebral hyporeperfusion was induced by permanent ligation of bilateral common carotid arteries and no PBM was given. Rats in the Sham group underwent the same surgical procedure except for the occlusion of the two carotids arteries and no PBM was given. In addition to the BCCAO surgery, rats in the BCCAO+PBM group received 808 nm laser therapy (5 min each time at a laser dose of 20 mW/cm ) of the frontal cortex every other day for 1 month. Between 86 and 90 days after BCCAO, Morris water maze (MWM) was used to observe the spatial learning and memory function of the rats. The rats were sacrificed on day 90 and immunofluorescence staining and Western blot were performed thereafter. Immunofluorescence staining was used to determine the expression of 5-bromodeoxyuracil nucleoside (BrdU), a cell proliferation marker, glial fibrillary acidic protein (GFAP), an astrocyte marker, doublecortin (DCX), a specific marker of newborn neuron precursor cells, NeuN, a marker of mature neurons, and Iba1, a microglia marker, in the hippocampal dentate gyrus (DG) region. Western blot was performed to analyze the protein expressions of inflammasome components, NLRP3, ASC, cleaved caspase-1, and Iba1 in the hippocampus.
RESULTS
In the latency trial of MWM test, BCCAO+PBM rats spent shorter periods of time finding the underwater platform than the BCCAO rats did. In the probe trial, after the platform that was original placed in a quadrant was removed, the BCCAO+PBM rats spent longer periods of time exploring the quadrant than the BCCAO animals did ( <0.05). Compared with BCCAO rats, BCCAO+PBM rats showed significant decrease in the immunofluorescence intensities of GFAP and Iba1 ( <0.01). PBM intervention significantly increased the number of BrdU-positive cells in the hippocampal DG region compared with those of Sham and BCCAO groups ( <0.05). Furthermore, the number of NeuN positive cells showed no significant difference among the three groups, while in BCCAO+PBM group, the number of DCX-positive cells was significantly increased ( <0.001) and the number of DCX /NeuN co-located cells was significantly increased compared to that of the BCCAO group ( <0.001). Compared with those of the BCCAO group, Western blot results showed that the protein expression levels of Iba1, NLRP3, and cleaved caspase-1 in the BCCAO+PBM group were significantly decreased ( <0.05), while the ASC protein expression level showed no significant difference.
CONCLUSION
PBM can effectively improve the spatial learning and memory function in rats with chronic cerebral hypoperfusion, inhibit the activation of glial cells, reduce inflammatory damage mediated by NLRP3 inflammasome, and promote the regeneration of endogenous neural stem cells in the hippocampal DG region of rats.
Topics: Rats; Female; Animals; Rats, Sprague-Dawley; Inflammasomes; NLR Family, Pyrin Domain-Containing 3 Protein; Bromodeoxyuridine; Brain Ischemia; Cognition; Anti-Inflammatory Agents; Hippocampus; Maze Learning; Neurogenesis; Caspases
PubMed: 37866954
DOI: 10.12182/20230960202