Flow Cytometric Analysis Of DNA Content Research Articles

Sodium Metabisulfite Suppresses Stoichiometry of Phenolic Compounds in Oil Palm Leaves for Flow Cytometric DNA Content Analysis

Sodium Metabisulfite Suppresses Stoichiometry of Phenolic Compounds in Oil Palm Leaves for Flow Cytometric DNA Content Analysis

Apoptin Overexpression Efficiently Amplified Cytotoxic Effects of PI3K Inhibition Using BKM120 in Lymphoblastic Leukemia Cell Lines.

Purpose: Although the complex structure of acute lymphoblastic leukemia (ALL) and involvement of diverse pathways in its pathogenesis have put an obstacle in the way of efficient treatments, identification of strategies to manipulate the genome of neoplastic cells has made the treatment prospective more optimistic. Methods: To evaluate whether the transduction of apoptin __a gene encoding a protein that participates in the induction of apoptosis__ could reduce the survival of leukemic cells, we generated recombinant lentivirus expressing apoptin, and then, MTT assay, flow cytometric analysis of DNA content, western blotting, and quantitative reverse transcription polymerase chain reaction (qRT-PCR) were applied. Results: Transduction of apoptin into different leukemic cells was coupled with the reduction in the viability and proliferative capacity of the cells. Among all tested cell lines, Nalm-6 and C8166 were more sensitive to the anti-leukemic property of apoptin. Moreover, we found that the transduction of apoptin in the indicated cell lines not only induced G2/M cell cycle arrest but also induced apoptotic cell death by altering the balance between pro- and anti-apoptotic target genes. The efficacy of apoptin transduction was not limited to these findings, as we reported for the first time that the overexpression of this gene could potentiate the anti-leukemic property of pan PI3K inhibitor BKM120. Conclusion: The results of this study showed that the transduction of apoptin into lymphoblastic leukemia cell lines induced cytotoxic effects and enhanced therapeutic value of PI3K inhibition; however, further investigations are demanded to ascertain the safety and the efficacy of apoptin transduction in patients with ALL.

Can Flow Cytometric DNA Content Analysis Become a Routine Procedure in Aquaculture?

Can Flow Cytometric DNA Content Analysis Become a Routine Procedure in Aquaculture?

Highly Selective Mitochondria-Targeting Amphiphilic Silicon(IV) Phthalocyanines with Axially Ligated Rhodamine B for Photodynamic Therapy

Two axially ligated rhodamine-Si(IV)-phthalocyanine (Rh-SiPc) conjugates, bearing one and two rhodamine B, were synthesized and their linear and two-photon photophysical, subcellular localization and photocytotoxic properties were studied. These Rh-SiPc conjugates exhibited an almost exclusive mitochondrial localizing property in human nasopharyngeal carcinoma (HK-1) cells and human cervical carcinoma (HeLa) cells. Strong photocytotoxic but low dark cytotoxic properties were also observed for the two Rh-SiPc conjugates toward the HK-1 cells. Using nuclei staining method and flow cytometric DNA content analysis, apoptotic cell death was induced by these conjugates upon photoactivation. This observation is consistent with their mitochondrial localization property. The observed properties of these conjugates qualify them as promising PDT agents.

High-resolution cell cycle and DNA ploidy analysis in tissue samples.

This unit describes an easy, rapid, and universal procedure to process fresh and nitrogen-frozen tissue specimens for high-resolution cell cycle and DNA ploidy analysis. Unlike other protocols, this procedure does not require treating tissues with enzymes, detergents, or other plasma membrane-lysing chemicals, but it achieves tissue dispersion by a simple two-step mechanical process that can be performed in ∼5 min. Resulting single-cell suspensions are fixed with ethanol, stained with propidium iodide, and subjected to flow cytometric DNA content analysis. The method can be applied without any alterations to all tissue types (except bones) derived from several species and results in highly reproducible cell cycle profiles of excellent resolution. The described protocol can be used to reliably and accurately detect subtle cell cycle and ploidy alterations in tissue specimens, including cell cycle arrest, aneuploidy, and apoptosis/necrosis-associated DNA fragmentation.

Flow cytometric fluorescence pulse width analysis of etoposide-induced nuclear enlargement in HCT116 cells

Fluorescence pulse width can provide size information on the fluorescence-emitting particle, such as the nuclei of propidium iodide-stained cells. To analyze nuclear size in the present study, rather than perform the simple doublet discrimination approach usually employed in flow cytometric DNA content analyses, we assessed the pulse width of the propidium iodide fluorescence signal. The anti-cancer drug etoposide is reportedly cytostatic, can induce a strong G2/M arrest, and results in nuclear enlargement. Based on these characteristics, we used etoposide-treated HCT116 cells as our experimental model system. The fluorescence pulse widths (FL2-W) of etoposide-treated (10 μM, 48 h) cells were distributed at higher positions than those of vehicle control, so the peak FL2-W value of etoposide-treated cells appeared at 400 while those of vehicle control cells appeared at 200 and 270. These results were consistent with our microscopic observations. This etoposide-induced increase in FL2-W was more apparent in G2/M phase than other cell cycle phases, suggesting that etoposide-induced nuclear enlargement preferentially occurred in G2/M phase cells rather than in G0/G1 or S phase cells.Electronic supplementary materialThe online version of this article (doi:10.1007/s10529-010-0277-x) contains supplementary material, which is available to authorized users.

DRAQ5: Improved flow cytometric DNA content analysis and minimal residual disease detection in childhood malignancies

Background The majority of flow cytometric DNA content analyses are performed on whole peripheral blood, bone marrow or tumor samples containing significant numbers of non-malignant cells which hamper specific DNA content analysis. Simultaneous analysis of DNA content and immunophenotype greatly improves the specificity of DNA-ploidy measurements. Therefore, a three-color flow cytometric assay using DRAQ5 was developed and validated. Methods The results of DNA content analysis using DRAQ5 and propidium iodide were compared using peripheral blood samples of 15 healthy volunteers. The reproducibility of the multiparameter DRAQ5 assay was assayed in 10 analytical runs and the sensitivity was evaluated with addition experiments. Result Using DRAQ5, slightly wider CVs of the G0/G1 peak were obtained (average CV = 3.29%). When DRAQ5 staining and immunophenotyping were combined, the within- and between-run imprecision was 1.98% and 1.67%, respectively and the DNA content of 25 DNA-hyperdiploid tumor cells (DNA-index = 1.16) per 4.10 4 mononuclear cells (0.06%) could be determined. Conclusions The multiparameter DRAQ5 assay has a superior sensitivity and specificity compared to the propidium iodide based method. Since at least 25 DNA-hyperdiploid cells per 10 4 DNA-diploid mononuclear cells could be detected, multiparameter DRAQ5 DNA content analysis could be used to study minimal residual disease.

Flow cytometric DNA content analysis of neoplastic cells in haemolymph of the cockle Cerastoderma edule

Epizootiological outbreaks of disseminated neoplasia (DN) have been reported in association with mass mortalities in various bivalve species including the cockle Cerastoderma edule. A flow cytometric (FCM) procedure to study DNA content was successfully adapted and tested in haemolymph cells (haemocytes and neoplastic cells) of the cockle. The FCM results were similar to those obtained by histological analysis (DN diagnosis and haemolymph cell features). FCM analysis revealed differences in DNA content among normal haemocytes (diploid) and neoplastic cells. Four types of cells with abnormal DNA content were found in the haemolymph of affected animals: hypodiploid, hyperdiploid, triploid-sesploid and pentaploid. Our results suggest that the flow cytometric DNA content analysis can be applied to identify neoplastic cell types and to study the association between different cell types and the DN progression or remission in this edible and commercially important bivalve species.

Relationship between DNA Index and Cellular Drug Resistance in Children with Newly Diagnosed Acute Lymphoblastic Leukemia.

Leukemic cell ploidy is known risk factor in childhood leukemia. Hypodiploidy (DNA index < 1.00 or chromosome number <46) was shown as adverse prognostic factor in many study. The cause of poor prognosis is not known. Cellular drug resistance might provide important information on this subject. In this study, we want to evaluate LC50 (lethal concentration to 50% of cells) values of 7 different drugs in 34 newly diagnosed patient and compare those of hypodiploid with nonhypodiploid patients. DNA index was determined by flowcytometric analysis of cellular DNA content and cell cycle distribution with Coulter EPICS XL-MCL system on propidium iodide stained cells. DNA index was defined as the modal DNA content of leukemic cells compared with that of reference normal lymphocytes. Patients were divided in hypodiploid (DNA index < 1.00) and nonhypodiploid (DNA index ≥ 1.00)We carried out in vitro tests with a 4-day culture and a methyl-thiazol- tetrazolium (MTT) assay on bone morrow samples of patients to get LC50 values for 7 different classes of drugs(PRD, prednisolon; DEXA, dexamethasone; VCR, vincristine; ASP, l-asparaginase; ADR, adriamisin; DNR, daunorubisin; ARA-C, cytosine arabinoside) commonly used in treatment of ALL. Eigth patients (23.5%) were belong to hypodiploid group whereas 26 patiens (76.5%) were included in nonhypodiploid group. Sex and age distribution were not different between groups. MTT test were succesfully performed in 7 out 8 hypodiploid patient for each drug. Median LC50 values for PRD and ARA-C were similar. Median LC50 values of hypodiploid group for DEXA, VCR, ASP and ADR were 5.4, 3.5, 5.9 and 3.4 fold higher than those of nonhypodiploid group and were found to be statistically significant. Only the median LC50 value of hypodiploid group for DNR were lower than that of the nonhypodiploid group (p <0.046).In conclusion, cellular drug resistance might provide important information in relation to poor prognosis of hypodiploid ALL patients. It might also help to individualize the treatment according to drug resistance profiles. Further studies, large study groups and long-term follow-up are needed to confirm these results.

Solid tumor DNA content analysis.

Flow cytometric DNA content analysis provides a rapid and reliable analysis of DNA ploidy and the proliferative fraction of tumor specimens. Both archival and fresh specimens are suitable for this technique. This information can be used in conjunction with traditional prognostic factors in the biological assessment of certain carcinomas. Among these tumors are early stages of breast, colon, lung, and bladder carcinomas.

Red oil A5 inhibits proliferation and induces apoptosis in pancreatic cancer cells.

To study the effect of red oil A5 on pancreatic cancer cells and its possible mechanisms. Effect of different concentrations of red oil A5 on proliferation of three pancreatic cancer cell lines, AsPC-1, MiaPaCa-2 and S2013, was measured by (3)H-methyl thymidine incorporation. Time-dependent effects of 1:32 000 red oil A5 on proliferation of three pancreatic cancer cell lines, were also measured by (3)H-methyl thymidine incorporation, and Time-course effects of 1:32 000 red oil A5 on cell number. The cells were counted by Z1-Coulter Counter. Flow-cytometric analysis of cellular DNA content in the control and red oil A5 treated AsPC-1, MiaPaCa-2 and S2013 cells, were stained with propidium iodide. TUNEL assay of red oil A5-induced pancreatic cancer cell apoptosis was performed. Western blotting of the cytochrome c protein in AsPC-1, MiaPaCa-2 and S2013 cells treated 24 hours with 1:32 000 red oil A5 was performed. Proteins in cytosolic fraction and in mitochondria fraction were extracted. Proteins extracted from each sample were electrophoresed on SDS-PAGE gels and then were transferred to nitrocellulose membranes. Cytochrome c was identified using a monoclonal cytochrome c antibody. Western blotting of the caspase-3 protein in AsPC-1, MiaPaCa-2 and S2013 cells treated with 1:32 000 red oil A5 for 24 hours was carried out. Proteins in whole cellular lysates were electrophoresed on SDS-PAGE gels and then transferred to nitrocellulose membranes. Caspase-3 was identified using a specific antibody. Western blotting of poly-ADP ribose polymerase (PARP) protein in AsPC-1, MiaPaCa-2 and S2013 cells treated with 1:32 000 red oil A5 for 24 hours was performed. Proteins in whole cellular lysates were separated by electrophoresis on SDS-PAGE gels and then transferred to nitrocellulose membranes. PARP was identified by using a monoclonal antibody. Red oil A5 caused dose- and time-dependent inhibition of pancreatic cancer cell proliferation. Propidium iodide DNA staining showed an increase of the sub-G0/G1 cell population. The DNA fragmentation induced by red oil A5 in these three cell lines was confirmed by the TUNEL assay. Furthermore, Western blotting analysis indicated that cytochrome c was released from mitochondria to cytosol during apoptosis, and caspase-3 was activated following red oil A5 treatment which was measured by procaspase-3 cleavage and PARP cleavage. These findings show that red oil A5 has potent anti-proliferative effects on human pancreatic cancer cells with induction of apoptosis in vitro.

Long-lasting effects of lindane on mouse spermatogenesis induced by in utero exposure

Long-lasting effects on mouse spermatogenesis induced by prenatal exposure to the insecticide lindane have been investigated by conventional reproductive endpoints complemented by the flow cytometric (FCM) DNA content analysis of testis cells and by the Sperm Chromatin Structure Assay (SCSA). Two lindane dose levels, 15 and 25 mg/kg bw, and diethylstilboestrol (DES, 10 μg/kg bw) as positive control, were administered daily by gavage to pregnant CD1 mice on gestation days (GD) 9–16. Reproductive endpoints were evaluated on F1 male mice on postnatal day (PND) 60; additionally, animals treated with lindane 25 mg/kg per day and DES were examined on PND 100 to evaluate the possible reversibility of the effects. On PND 60, lindane and DES caused a reduction in the sperm head count and concentration, with recovery in older lindane 25 mg/kg per day animals (PND 100). By contrast, the DES group exhibited a greater reduction in the sperm head count on PND 100 than on PND 60. Changes in biochemical parameters in the testes, lactate dehydrogenase-C 4 (LDH-C 4), and sorbitol dehydrogenase (SDH) activities, were also observed in adult treated F1 mice. Furthermore on PND 60, the FCM analysis revealed changes in the pattern of testicular germ cell distribution, especially in the haploid subcompartment, in the lindane 25 mg/kg per day group. A dose-dependent increase in chromatin abnormalities of the epididymal sperm was also shown by SCSA. These changes recovered on PND 100. Preliminary qualitative examination did not reveal any significant difference in the structure of testicular tissue; however, there were suggestions of a moderate increase in number and size of Leydig cells in both DES- and lindane-treated animals. The partial reversibility of these effects and the lack of structural modification of the testicular tissue as evidenced by histopathologic assessment suggest a functional impairment of sperm production and maturation, possibly associated with changes induced by lindane on factors affecting intratesticular steroidogenesis.

Assessment of Age-Related Polyploidy in Quercus Robur L. Somatic Embryos and Regenerated Plants Using DNA Flow Cytometry

Flow cytometric analysis with 4,6-diamidino-2-phenylindole (DAPI) staining was used to screen for chromosomal changes in Quercus robur during in vitro culture. The initiated cell lines (1992 until 1999) were maintained via secondary embryogenesis on P24 medium with 0.9 μM 6-benzylaminopurine (BAP) in regular subculture intervals of 6 weeks. Regenerated plants established in the greenhouse and in vitro plantlets derived from encapsulated somatic embryos were screened. The embryogenic cell lines were characterized as individual clones by isoenzyme analysis. Flow cytometric relative DNA content analysis of the first screening period revealed that somaclonal variation in form of tetraploidy occurred in two out of 26 tested somatic embryo clones (Alt and Jung). These two clones lost their ability to convert into plantlets. Intraspecific relative DNA content variation including technical variation was below 3 %. In the second screening period, however, 3 out of 37 clones (Alt, E4.31H9 and P3.27H) contained tetraploid cells leading to the assumption that the frequency of tetraploidy seems to be correlated with the duration of in vitro culture. No chromosomal differences were detected in regenerated plants. However, tetraploidy occurred in 8 % of the tested clones over a culture period of 7 years.

Primary angioleiomyoma of the iliac bone: clinical pathological study of one case with flow cytometric DNA content and S-phase fraction analysis

We report on a primary angioleiomyoma of the right iliac bone in a 28-year-old woman. To our knowledge this is the 16th reported case of a primary leiomyoma of bone, the 9th reported primary osseous angioleiomyoma and the first description of a primary angioleiomyoma located in the iliac bone. The problems of differentiating primary leiomyoma of bone from primary or metastatic leiomyosarcoma of bone are stressed. A literature review of primary leiomyomas of bone is presented.

Progression of cell cycle monitored by dielectric spectroscopy and flow-cytometric analysis of DNA content.

A dielectric method has already been developed for the real-time monitoring of cell cycle progression in synchronized cell culture (Asami et al., 1999). This method, in combination with DNA content analysis by fluorescence-activated cell sorting (FACS), was applied to the synchronized cell culture of a CDC28-13th mutant (Saccharomyces cerevisiae). In synchronous cell growth, relative permittivity epsilon (or dielectric constant) for the culture broth showed cyclic changes at low frequencies below 0.5 MHz, being correlated to phases in the cell cycle that were simultaneously determined by FACS. The epsilon increased in the period from S phase to G(2) phase and decreased between M and G(1) phases. Peaks in these cyclic changes of epsilon indicated the time when daughter cells segregated from mother cells.

Induction of apoptosis by N-(4-hydroxyphenyl)retinamide in glioma cells.

N-(4-hydroxyphenyl)retinamide (fenretinide) is a synthetic retinoid with anticancer properties. We investigated the effects of fenretinide on the growth of glioma cells. Four glioma cell lines (C6, 9L, Med3 and U87) were treated with fenretinide. Cell viability and independent growth was determined by MTS assay and soft agar assay, respectively. The induction of apoptosis was evaluated by microscopic examination, flow cytometric DNA content analysis, and in situ TdT methods. Fenretinide markedly reduced cell viability of all the glioma cell lines examined at a range of concentrations from 1 to 10 microM. In all cell lines examined, fenretinide also induced morphological changes consistent with apoptosis, including cellular shrinkage, chromatin condensation, and nuclear fragmentation. Flow cytometric analysis also revealed an apoptotic pattern of the DNA content, and in situ detection of apoptosis showed increased incorporation of digoxigenin-nucleotide triphosphate in fenretinide-treated glioma cells. These findings indicate that fenretinide inhibits the growth of glioma cells via the induction of apoptosis, suggesting potential clinical use of fenretinide for treatment of glioma patients.

The Clinical Value of Flow Cytometric DNA Content Analysis in Patientswith Soft Tissue Sarcomas

Purpose. The purpose of this study was to evaluate: (1) the correlation between grade and ploidy or S-phase fraction (SPF), (2) the prognostic value of DNA flow cytometric study in soft tissue sarcomas. Patients /Methods. In all, 47 tissue samples from soft tissue sarcoma patients, surgically treated in the same center, were included. Flow cytometric analyses were performed according to a modified version of the original method of Hedley et al. Results. DNA ploidy status could be determined in 44 samples out of 47 (success rate 94%). Of these 44, S-phase fraction could be calculated in 34 samples (77%). In the study group as a whole, aneuploidy was significantly correlated with high grade. Survival analyses were carried out in 21 patients with soft tissue sarcoma, all surgically treated in the same center, without chemotherapy or radiotherapy. In univariate analyses, DNA ploidy was found to be a significant factor for overall survival (OAS) and metastasis-free survival MFS. Mean OAS for aneuploid tumors and diploid tumors were 35 and 65 months (p=0.034), and mean MFS 23 and 61 months, respectively (p=0.005) . Discussion.There is a relation between histological grade and ploidy in soft tissue sarcomas. It appears that low-grade tumors are generally diploid, whereas high-grade tumors tend to be aneuploid. In a subgroup of patients treated only with surgery, DNA ploidy was found to be an important factor for predicting OAS and MFS.

Lovastatin induces apoptosis in malignant mesothelioma cells.

Malignant mesothelioma causes profound morbidity and nearly universal mortality that is refractory to conventional treatment with aggressive surgery, radiotherapy, or chemotherapy. We report that pharmacologic concentrations of lovastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitor, induced apoptosis in human malignant mesothelioma cell lines. Mesothelioma cell viability was decreased in a dose-dependent manner by lovastatin (5 to 30 microM). These effects were not reversed by exogenous growth factors or cholesterol, but were reversed by addition of 100 microM mevalonate, confirming that lovastatin affected mesothelioma viability by inhibiting mevalonate synthesis. Lovastatin appeared to decrease mesothelioma viability by inducing apoptosis, as indicated by morphologic changes, histologic evidence of nuclear condensation and degeneration, and flow-cytometric analysis of DNA content. Lovastatin's effects on cell viability were partially reversed in the presence of farnesol, and treatment of mesothelioma cells with a specific farnesyl-protein transferase (FTP) inhibitor decreased cell viability and induced morphologic changes indistinguishable from those caused by lovastatin. In addition, lovastatin-treated cells showed translocation of ras guanosine triphosphate (GTP)-binding proteins from membrane to cytosolic fractions on Western blots, suggesting that lovastatin's effects on mesothelioma were mediated in part by disrupting acylation of GTP-binding proteins. Thus, lovastatin is a commercially available and clinically well-tolerated agent that reduces viability and induces apoptosis of mesothelioma cells, and may provide the basis for adjunctive treatments of patients with mesothelioma.

Induction of apoptosis by a short-chain neuropeptide analog in small cell lung cancer

Small cell lung cancer (SCLC) cells express a variety of neuropeptides which act as autocrine growth factors. Although several neuropeptide analogs have been reported to antagonize SCLC proliferation, the development of these compounds has been limited by their low potency and the cytostatic nature of their effects. In the present study we evaluated the cytotoxic activity of four short-chain substance P analogs (NY3460, NY3238[-pHOPA], NY3238[Phe 1], NY3238[Lys 5]) against a panel of five SCLC cell lines. NY3460 was the most potent compound in all five SCLC cell lines (IC 50 = 2.8–3.7 μ M) as assessed by a MTT growth inhibitory assay. NY3238[Phe 1] was also relatively active in all cell lines (IC 50 = 3.5–11.2 μ M), while NY3238[Lys 5] and NY3238[-pHOPA] were substantially less active. NY3460 was the only agent to induce an increase in the percentage of cells with subdiploid DNA content suggestive of apoptosis by flow cytometric DNA content analysis. The induction of apoptosis was confirmed by fluorescent microscopy in NCI-H69, NCI-H82, NCI-H446, and NCI-H510 cells after exposure to 5.0 μ M NY3460 for 48 h. These findings suggest that NY3460 is a relatively potent cytotoxic inhibitor of SCLC growth, and that short-chain neuropeptide analogs deserve further evaluation as anti-SCLC agents.

Solid tumor DNA ploidy analysis.

Flow cytometric DNA content analysis of human neoplasia provides quantitative information on DNA ploidy, clonal DNA heterogeneity, and proliferative activity. These submicroscopic features are important for the biological and clinical evaluation of tumors as demonstrated in clinical studies of a wide spectrum of human lymphoreticular and solid malignancies (, , , , , , , , , , , , , , , , , , , , , , , ). Although valuable, such information should not be used in isolation from other conventional diagnostic and biological parameters in the risk assessment of cancer patients.