Fluorescent DNA-binding Dye Hoechst Research Articles

Isolation of Cancer Stem Cells by Side Population Method.

The Hoechst side population (SP) method is a flow cytometry technique used to obtain stem cells based on the dye efflux properties of the ATP-binding cassette (ABC) transporters. The SP cells are characterized by their capability to efflux the fluorescent DNA-binding dye Hoechst 33342 through their ABC transporters and are enriched in stem cells, which are endowed with a self-renewal capacity and multilineage differentiation potential and express the stemness genes including ABC multidrug transporters. The protocols outlined in this book chapter describe the isolation method of the SP cells from human lung carcinoma cell lines by using Hoechst 33342. In addition, we refer to the propagation method of SP cells by successive rounds of fluorescence-activated cell sorting analysis for SP cells. These approaches will be helpful for the establishment of novel in vitro and in vivo models using cancer stem cells, which may play a key role during carcinogenesis and/or tumor progression.

Identification and gene expression analysis of the side population subclone in mantle cell lymphoma

Introduction: It has been increasingly understood that seemingly effective treatments merely remove the bulk of tumor cells, while more rare cancer stem cells reside. The treatment resistant cells soon repopulate and form new tumor mass. These are often selected by the exposure to therapy and thus increasingly resistant. One way of identifying such cells is through analysis of the side population (SP) in tumor masses. In this study, we investigated the presence of SP in twenty-five lymphoma cell lines, representing five different types of B-cell lymphomas. Methods: Cells were labelled with fluorescent DNA-binding dye Hoechst 33342, and SP cells were identified as a subpopulation of cells that have an elevated rate of Hoechst efflux. All samples were analyzed on a FACS Aria II (BD Bioscience, San Jose, CA, USA). Using cell sorting, both SP and non-SP cells could be isolated. The presence of SP was assessed in MCL patient samples and cell lines derived from MCL, FL, DLBCL, Burkitt's lymphoma (BL), and CLL. Subsequent gene expression analysis was performed using Affymetrix Human Transcriptome Array 2.0 (HTA 2.0; Affymetrix Inc, Santa Clara, CA, USA). Results: The presence of SP was limited to one mantle cell lymphoma (MCL) derived primary sample (P3) and cell line (REC-1). Downstream analysis involving in vitro cultivation of sorted SP shows that SP cells are able to grow and differentiate into daughter cells and non-SP cells. Of interest, the frequency of SP cells was enriched when wild type REC-1 cells were exposed and became resistant to chemotherapy. Moreover, a differential gene expression profile between SP cells and non-SP cells was revealed by comprehensive gene expression analysis showing deregulation of group of genes involved in cell proliferation, cell cycle regulation, cell migration, BCR signaling, cancer progression, and tumorigenesis. Of major importance, validation of gene expression results reveal that SP, compared to non-SP, exhibits significantly higher expression levels of CD44 and CD69, known to be involved in cancer stem cells and lymphocyte proliferation, respectively. Conclusion: We show that SP is found among MCL patients and that genes involved in several cell signaling pathways are deregulated in SP derived from both REC-1 and patient material. This indicates that REC-1 can serve as a tool for future screening efforts. Importantly, we show evidence that CD69 and CD44 are commonly upregulated in SP cells from MCL patient material and could have important functional roles in relapse and treatment resistance. Keywords: flow cytometry; mantle cell lymphoma (MCL)

MicroRNA-192-5p Promote the Proliferation and Metastasis of Hepatocellular Carcinoma Cell by Targeting SEMA3A.

Side population (SP) cells are a small subset of cells isolated from a cultured cancer cell line with characteristics similar to those of cancer stem cells, such as high metastatic and tumorigenic potentials. However, the molecular mechanisms remain unclear for the malignant properties of SP cells. In this study, SP cells were isolated by staining cultured HCCLM3 cells with fluorescent DNA-binding dye Hoechst 33342 and sorted by flow cytometry. The proportion of SP cells was 2.79%±0.19% in the HCCLM3 cell line. Compared with non-SP cells, SP cells possessed stronger capability of sphere formation and tumorigenicity, and expressed higher levels of CD133 and CD90. Then, we found that SP cells possessed 25 upregulated and 34 downregulated microRNAs with differences of >3-fold. As one of the upregulated microRNAs, miR-192-5p was computationally predicted to target semaphorin 3A (SEMA3A), a potent suppressor of tumor angiogenesis in various cancer models. Luciferase reporter assay showed that SEMA3A was a direct target of miR-192-5p. Overexpression of miR-192-5p promoted cell proliferation and metastasis targeting SEMA3A in HCCLM3 cells. Immunohistochemical staining revealed that SEMA3A expression was significantly reverse associated with metastasis in hepatocellular carcinoma tissues. The results indicate that miR-192-5p contributes to targeting SEMA3A in HCCLM3 cells, and this may be used as a target in targeted therapy and a marker for cancer behavior and prognosis.

MiR-99a directly targets the mTOR signalling pathway in breast cancer side population cells.

miR-99a has been reported to function as a tumour suppressor in breast cancer. However, its role in the regulation of breast cancer stem cell (CSC) phenotype has up to now remained unknown. In this study, we isolated the side population (SP) cells by staining cultured MCF-7 and MDA-MB-231 cells with fluorescent DNA-binding dye Hoechst 33342, then by flow cytometric sorting. Next, we detected expression of miR-99a in the SP cells compared to non-SP cells using real-time PCR, and explored effects of miR-99a on the CSC phenotype of the breast cancer cells, including sphere formation, self-renewal, tumourigenicity and cell migratory capability. We found that expression of miR-99a was down-regulated in the SP cells compared to non-SP cells. Restoration of expression of miR-99a inhibited cell migration and invasion, reduced sphere formation of breast SP cells in vitro, and suppressed tumour growth in vivo. Finally, bioinformatic prediction suggested the oncogene, mammalian target of rapamycin (mTOR) - a downstream effector of the PI3K/AKT signalling pathway, was a target gene of miR-99a in SP cells. Further, quantitative RT-PCR and western blot assays identified that overexpression of miR-99a suppressed expression of mTOR and its downstream gene, HIF-1α. Collectively, these data suggest that miR-99a reversed the breast cancer malignant CSC phenotype, probably by targeting the mTOR signalling pathway.

Nicotine induces apoptosis in bovine coronary artery endothelial cells

Tobacco smoking is a major risk factor for coronary heart disease. Human and animal in vivo studies have suggested for years that nicotine impairs endothelial function, both acutely and chronically. However, the direct cytotoxic effects of nicotine on coronary artery endothelial cells remain unclear. The present study tested the hypothesis that nicotine inhibits nitric oxide (NO) synthesis and increases apoptosis in coronary artery endothelial cells resulting in endothelial dysfunction. Bovine coronary artery endothelial cells (BCAECs) of the fifth passage at 80% confluence were treated with control medium or medium with nicotine for 24 and 48 h. NO (measured as NOx of NO, NO2−, and NO3−) in the medium was assayed by the chemiluminescence method. Protein levels of eNOS in the cell lysates were determined by Western blotting analysis. Fluorescent DNA-binding dye Hoechst 33258 was used to define nuclear chromatin morphology as a quantitative index of apoptosis. Nicotine (1 μM) significantly decreased basal NOx release from BCAECs after 24 and 48 h of treatment. This was accompanied by a significant decrease in eNOS protein levels in BCAECs at 48 h. Apoptotic cells showed condensed, coalesced, and segmented nuclei. Nicotine (0.3, 1, 3 μM) treatment of BCAECs for 48 h produced a concentration-dependent increase in apoptotic cell death. The results suggest that chronic nicotine exposure causes a direct cytotoxic effect on coronary artery endothelial cells by increasing apoptosis. Inhibition of NO synthesis may play an important role in the nicotine-induced apoptosis.

Isolation and characterization of side population cells.

The protocol for isolation of side population (SP) cells was originally established for murine bone marrow hematopoietic stem cells (HSCs), but it has also been adapted for other species and tissues. This purification strategy offers a simple and reproducible strategy to obtain a highly homogeneous population of HSCs. The method is based on the differential efflux of the fluorescent DNA-binding dye Hoechst 33342 from stem cells relative to nonstem cells. The protocols outlined in this chapter describe the isolation of murine SP cells from both bone marrow and skeletal muscle using the fluorescent DNA-binding dye Hoechst 33342. In these tissues, the SP cells that are isolated are HSCs.

Induction of cell death by saponin and antigen delivery.

Saponin is the major component in the formation of immune stimulating complex (ISCOM), a potent adjuvant able to induce both humoral and cellular immune reactions. The immunogenicity induced by saponin, however, has been unclear. The objective of this study was to investigate the apoptotic and necrotic effects induced by saponin in ELA mouse lymphoma cells, expected to be a possible mechanism of the cytotoxic T-lymphocyte (CTL) effect elicited by the ISCOM. EL4 cells were treated with saponin, and viability of the cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase release assays. Fluorescence microscopy was used to detect the morphological changes by staining saponin-treated cells with Hoechst 33342. Extent of apoptosis and necrosis was determined by Annexin V-FITC/propidium iodide staining, followed by flow cytometric analysis. Dendritic cells were cultured with either saponin-protein complexes or saponin-treated cells and analyzed by flow cytometry. Treatment of EL4 cells with saponin resulted in concentration-dependent cytotoxicity and the appearance of the hypodiploid DNA peak. Cells treated with saponin showed highly condensed chromatin when stained with fluorescent DNA-binding dye Hoechst 33342. Analysis of EL4 cells by flow cytometry after Annexin V/propidium iodide staining demonstrated that saponin induced both apoptosis and necrosis. Pretreatment of EL4 cells with zVAD-fmk, a broad-range caspase inhibitor, did not prevent cell death induced by saponin, indicating the non-caspase-dependent cell death. Dendritic cells were shown to phagocytose both the antigen-saponin complexes and the saponin-induced dead cells. Results obtained in this study demonstrated that saponin induced both apoptosis and necrosis in ELA cells. These events are critical for antigen processing and presentation.

Side-population cells from different precursor compartments.

The rapid efflux of the fluorescent DNA-binding dye Hoechst 33342 identifies a rare, so-called side population (SP), which rapidly expels the dye, can reconstitute the bone marrow (BM) of lethally irradiated mice, and has proven negative for most lineage markers including CD34. Because SP cells from human cell sources, such as mobilized peripheral blood [apheresis products (AP)], cord blood (CB), or BM have not been extensively characterized to date, we sought to analyze SP cells from various cell sources. We detected murine SP cells with a median frequency of 0.04% (n = 23) and a 52-fold colony-forming units (CFU) increase compared to unsorted cells (p = 0.028). The median frequency of human SP cells was 0.02% (n = 90), with highest numbers in donor AP, and lower in CB and BM. Human SP cells were mostly CD34(-) and lineage marker-negative. These showed no enrichment in CFU before expansion; however, they displayed a CFU increase after 5-7 days of cytokine-supported suspension culture (10.7-fold at day 5, 7.2-fold at day 7; n = 17) that was significant compared to both input (day 0) SP and to non-SP cells before and after expansion (p < 0.05). SP cells demonstrated a significant long-term culture-initiating cell (LTC-IC) increase of 167-fold (n = 17) as compared to non-SP cells (p = 0.002), with the highest numbers from AP specimens. We conclude that human primitive hematopoietic cells can be isolated via Hoechst staining and that SP cells of various human sources show substantial differences and represent a rare CD34(-) population with stem cell potential.

Low concentrations of glutamate induce apoptosis in cultured neurons: Implications for amyotrophic lateral sclerosis

Evidence is accumulating that excessive glutamate concentration in the extracellular space is neurotoxic and plays a role in amyotrophic lateral sclerosis (ALS). However, the published results on glutamate levels in cerebrospinal fluid (CSF) and on glutamate-mediated toxicity of CSF in ALS disease remain controversial. In this report, we studied CSF from patients with sporadic ALS and controls to determine glutamate concentrations, and then analyzed the neurotoxic effect of glutamate at the concentrations present in CSF from ALS patients on cultured cortical neuronal cells. Our study shows that glutamate, at the concentrations found in CSF from ALS patients (5.8 μM), diminished cell viability and increased apoptosis determined by the fluorescent DNA-binding dye Hoechst 33342 as well as by Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP Nick End-Labeling (TUNEL) reaction in cultured neuronal cells. However, glutamate concentrations as those found in CSF from controls (2.8 μM or below) did not induce any effect. Both significant glutamate-induced effects were inhibited in the presence of NBQX (2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline-2,3-dione), an α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate-sensitive glutamate receptor antagonist. These results demonstrate that AMPA/kainate receptors are involved in the glutamate-mediated neurotoxic effects on cultured neurons, according to reports that implicate these receptors in ALS disease. We conclude that the glutamate-mediated neuronal apoptosis through AMPA/kainate receptors could occur in ALS patients who have elevated CSF glutamate concentration.

Erythropoietin regulates vascular smooth muscle cell apoptosis by a phosphatidylinositol 3 kinase-dependent pathway

Recent studies have shown that several cytokines could induce apoptosis in vascular smooth muscle cells (VSMCs) via the induction of nitric oxide (NO). In the present study, we explored whether human recombinant erythropoietin (rHuEPO) has a modulatory effect of apoptosis on interleukin-1beta (IL-1beta) or NO donor sodium nitroprusside (SNP)-induced apoptosis in rat cultured VSMCs. The quantitation of apoptosis among VSMCs was assessed by nuclear morphological analysis with fluorescent DNA-binding dye Hoechst 33258. Apoptotic changes were also confirmed by the detection of DNA fragmentation. The expression of EPO receptor (EpoR), cellular protein tyrosine phosphorylation, including EpoR and Janus kinase (JAK) 2, and the association of p85 subunit of phosphatidylinositol 3 kinase (PI3-kinase) to tyrosine-phosphorylated proteins, including EpoR, were explored by using Western blotting analysis combined in part with immunoprecipitation. rHuEPO inhibited the apoptosis induced by IL-1beta or SNP in a dose- and time-dependent manner. The anti-apoptotic effects of rHuEPO were diminished in the presence of a tyrosine kinase (TK) inhibitor genistein or anti-EpoR antibody. After stimulation with rHuEPO, EpoR and JAK 2 were tyrosine phosphorylated, and p85 subunits were associated with EpoR. Also, rHuEPO induced phosphorylation of Akt through a PI3-kinase-dependent pathway. The phosphorylation of Akt and the anti-apoptotic effects of rHuEPO were diminished in the presence of a PI3-kinase inhibitor, wortmannin. Our present study demonstrates that rHuEPO inhibites IL-1beta or SNP-induced VSMC apoptosis. The TK-dependent pathway, particularly the PI3-kinase-dependent pathway, seems to be critical to the countervailing effect of rHuEPO on IL-1beta and SNP-induced VSMC apoptosis.

Axonal damage induced by cerebrospinal fluid from patients with relapsing-remitting multiple sclerosis

The importance of axonal damage in multiple sclerosis (MS) has been recently stressed in proton magnetic resonance spectroscopy and pathological studies, but the exact mechanism producing this damage is unknown. The aim of our study was to ascertain whether soluble mediators present in the cerebrospinal fluid (CSF) of patients with relapsing-remitting MS could induce neuron injury in culture. Different biochemical and cytochemical parameters were determined in primary embryonal rat neuron cultures following 8 days of exposure to CSF. Cytotoxic activity was evaluated with a blue formazan production colorimetric assay. Morphological and immunocytochemical studies performed with antibodies against β-tubulin revealed neuritic fragmentation, axonal damage and cellular shrinkage indicating apoptosis. Detection of apoptosis was carried out using the fluorescent DNA-binding dye Hoechst 33342, as well as by a Terminal deoxynucleotidyl transferase-mediated dUTP Nick End-Labeling assay. We observed that soluble factors in CSF from patients with “aggressive” MS i.e, those with poor recovery after relapses, induced neurite breakdown and neuronal apoptosis in cultures. Neuron injury is not related with blood-brain barrier dysfunction nor with IgG index. Interestingly, CSF from patients with “non-aggressive” MS i.e., relapsing-remitting patients with a good recovery after relapses, did not induce any damage. In conclusion, we report that CSF from patients with aggressive MS bears soluble mediators that induce axonal damage and apoptosis of neurons in culture. These mediators can be present during the first attack of the disease, and the neuronal damage caused could be related to the functional deficit of these MS patients.

Induction of apoptosis by cerebrospinal fluid from patients with primary-progressive multiple sclerosis in cultured neurons

We have studied the noxious effect of cerebrospinal fluids (CSF) from patients with primary-progressive multiple sclerosis (MS) on cultured neurons. Cells were exposed to CSF for 8 days and the possible neuronal damage was determined. Morphological studies with phase-contrast microscopy showed cellular shrinkage indicating apoptosis. CSF-induced apoptosis as evidenced by the fluorescent DNA-binding dye Hoechst 33342, as well as by the TUNEL-reaction, was only present in primary-progressive MS patients with a worsening disease. This neuron injury did not correlate with blood–brain barrier dysfunction nor with intrathecal IgG synthesis. On the contrary, CSF from either stable primary-progressive or other non-inflammatory neurological diseases, did not induce any culture damage. Undetectable or low similar tumor necrosis factor-alpha (TNF- α) levels (range to 8.7 pg/ml) were found in the CSFs tested regardless they damage cultures or not. These results suggest that soluble factors, other than TNF- α, molecules transudated from blood or IgG, present in the CSF of active primary-progressive patients with MS induce neuronal apoptosis.

Overexpression of aldehyde reductase protects PC12 cells from the cytotoxicity of methylglyoxal or 3-deoxyglucosone.

The glycation reaction (Maillard reaction) plays a major role in diabetic complications, since some reaction intermediates are responsible for the modification and cross-linking of long-lived proteins, resulting, in turn, in a deterioration of normal cell function. The reaction intermediates include methylglyoxal (MG) and 3-deoxyglucosone (3-DG), both of which are cytotoxic dicarbonyl compounds and are elevated during hyperglycemia. Aldehyde reductase (ALR) catalyzes the reduction of both compounds. To examine the intracellular role of ALR in the diabetic complications of neural cells, its gene was overexpressed in rat pheochromocytoma PC12 cells, which normally express a low level of ALR. Western blot analysis showed that ALR protein in the ALR gene-transfected cells was more than twice as much as in the control cells. In the parental cells, cytotoxicity, including apoptotic cell death, which was determined by fluorescent microscopy using the fluorescent DNA binding dye Hoechst 33258, was observed at 100 microM MG. In the ALR gene-transfected cells, the cytotoxicity of both MG and 3-DG and apoptotic cell death were decreased. This suggests that intracellular ALR protects neural cells from the cytotoxicity of 3-DG or MG, and that neural cells, which normally express a low level of ALR, might be susceptible to diabetic complications caused by intermediate products of the Maillard reaction, such as 3-DG and MG.

Dye efflux studies suggest that hematopoietic stem cells expressing low or undetectable levels of CD34 antigen exist in multiple species.

We previously described a method for isolating murine hematopoietic stem cells capable of reconstituting lethally irradiated recipients, which depends solely on dual-wavelength flow cytometric analysis of murine bone marrow cells stained with the fluorescent DNA-binding dye Hoechst 33342. This method, which appears to rely on the differential ability of stem cells to efflux the Hoechst dye, defines an extremely small and homogeneous population of cells (termed SP cells). We show here that dual-wavelength analysis of Hoechst dye-stained human, rhesus and miniature swine bone marrow cells reveals a small, distinct population of cells that efflux the dye in a manner identical to murine SP cells. Like the murine SP cells, both human and rhesus SP cells are primarily CD34-negative and lineage marker-negative. In vitro culture studies demonstrated that rhesus SP cells are highly enriched for long-term culture-initiating cells (LTC-ICs), an indicator of primitive hematopoietic cells, and have the capacity for differentiation into T cells. Although rhesus SP cells do not initially possess any hematopoietic colony-forming capability, they acquire the ability to form colonies after long-term culture on bone marrow stroma, coincident with their conversion to a CD34-positive phenotype. These studies suggest the existence of a hitherto unrecognized population of hematopoietic stem cells that lack the CD34 surface marker classically associated with primitive hematopoietic cells.

Isolation and genetic characterization of temperature-sensitive mutants of vaccinia virus WR

One hundred temperature-sensitive mutants of vaccinia virus WR were isolated from virus that had been mutagenized with 5-bromodeoxyuridine or N-methyl-N'-nitro-N-nitrosoguanidine. A rapid screening procedure based on the ability of vaccinia virus to form plaques under liquid overlay medium was used to identify potential mutants among randomly picked plaque isolates or plaques preselected for their small size after temperature shift-up. The preselection technique resulted in a sixfold increase in the number of successful mutant isolations relative to the number of plaques picked. All of the mutants had efficiencies of plating at 39.5 degrees C relative to that at 33 degrees C of 10(-4) or less, and 33 of 40 produced 10% or less of the amount of virus at the nonpermissive temperature (39.5 degrees C) relative to that at the permissive temperature (33 degrees C). Experiments with the fluorescent DNA binding dye Hoechst 33258 demonstrated that 6 of the 100 mutants failed to form characteristic cytoplasmic DNA factories at 39.5 degrees C. To facilitate the functional grouping of such a large number of mutants, a rapid infectious center assay was developed. Thirty of the mutants were assigned to 16 or 17 complementation-recombination groups by using this assay. Recombination experiments have allowed the construction of a genetic map representing 22 mutants in 12 of these groups.