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Biomedical subjects

M Poot

Publications and source records attributed to M Poot.

At least 37 records · Page 2Linked to original sources
PubMed1997

Verapamil inhibition of enzymatic product efflux leads to improved detection of beta-galactosidase activity in lacZ-transfected cells.

The beta-galactosidase activity encoded by the lacZ gene of Escherichia coli is widely used to monitor successful expression of transfected genes. Fluorogenic substrates allow detection of enzyme activity in viable cells, which, subsequently, can be selected for further study on the basis of fluorescence emission. We analyzed three fluorogenic substrates (FDG, C12FDG, and CMFDG), all of which incorporate fluorescein as their fluorophore, regarding intensity of fluorescent signal and selectivity toward the transfected beta-galactosidase activity vs. lysosomal enzyme activity. Among these substrates, 5-chloromethylfluoresecein di-beta-D-galactopyranoside (CMFDG) showed the strongest selectivity toward the lacZ-encoded enzyme activity. An attempt to improve this selectivity by alkalinization of the lysosomal pH with chloroquine, such that the endogenous enzyme would be exposed to a suboptimal pH, led to significant cell death. In contrast, inhibition of dye efflux with verapamil enhanced the selectivity of CMFDG toward the lacZ-encoded enzyme activity by approximately threefold. Incubation with probenecid, on the other hand, showed little effect.

3T3 Cells↗
PubMed1997

Detection of apoptosis in live cells by MitoTracker red CMXRos and SYTO dye flow cytometry.

We characterized the ability of six SYTO nucleic acid stains and a mitochondrial stain to resolve by flow cytometry camptothecin-induced apoptotic and non-apoptotic cells. Staining live human lymphoid B-cells showed such resolution with SYTO 11, 12, 13, 14, and 16 dyes. H9, HL-60, and Jurkat cells did not show resolution with the SYTO 12 dye, but did with the others. SYTO 15 dye did not show resolution with any cell type. RNase A treatment of fixed lymphoid B-cells strongly reduced fluorescence after staining with SYTO 12 dye; the other SYTO dyes showed little or no RNase A sensitivity. Reduced SYTO 12 fluorescence may reflect RNA breakdown during apoptosis, while decreased fluorescence of the other SYTO dyes in apoptotic cells may be due to chromosomal alterations during apoptosis. In all cell types tested, clear resolution between apoptotic and non-apoptotic cells was observed with the MitoTracker Red dye CMXRos. In double-staining experiments, cells exhibiting reduced SYTO 11 fluorescence were the same as those showing decreased CMXRos fluorescence. We conclude that changes in nucleic acid stability or conformation may vary during apoptosis from one cell type to another, but mitochondrial demise may be common to all apoptotic pathways.

Antineoplastic Agents, Phytogenic↗
PubMed1997

An apoptosis-inducing genotoxin differentiates heterozygotic carriers for Werner helicase mutations from wild-type and homozygous mutants.

Immortalized B lymphocytes from Werner syndrome subjects are shown to be hypersensitive to 4-nitroquinoline-1-oxide (4NQO), supporting earlier work on T lymphocytes. We also show that B cell lines from clinically normal heterozygous carriers exhibit sensitivities to this genotoxic agent, which are intermediate to those of wild-type and homozygous mutants. 4NQO is shown to induce an apoptotic response. These data encourage research on DNA repair with such cell lines and raise the question of an enhanced sensitivity of the relatively prevalent heterozygous carriers to certain environmental genotoxic agents.

4-Nitroquinoline-1-oxide↗
PubMed1997

Highly sensitive and specific detection of P-glycoprotein function for haematological and solid tumour cells using a novel nucleic acid stain.

Progress in our understanding of the contribution of P-glycoprotein (P-gp)-mediated resistance to chemotherapy failure in haematological as well as solid tumours has been hampered by the lack of highly sensitive, reliable methods for the detection of P-gp function in fresh human tumour cells. The present study identifies the novel nucleic acid stain SYTO16 as a highly sensitive and specific dye to assess P-gp function. The effect of P-gp is expressed here as the ratio of dye fluorescence (RF) from cells incubated with dye with or without 2 microM of the P-gp inhibitor PSC 833. Using flow cytometric analysis, an RF of 0.9 was found for SYTO16 in the KB3-1 (P-gp-) and 1.6 in KB8 (P-gp+) cells. Three types of patients' cells were studied: (1) in haematopoietic CD34+ cells, which are known to express P-gp, the RF was 6.0 for SYTO16 compared with 2.5 for rhodamine 123 and 1.3 for daunorubicin (mean of five individuals); (2) in acute myeloid leukaemia cells, the RF for SYTO16 was 1.0 in P-gp- and 4.5 in P-gp+ samples; (3) for the first time, we have quantitated P-gp function in fresh human solid tumour (sarcoma) cells. We found, in a P-gp+ leiomyosarcoma, an RF of 16 for SYTO16 and 2.7 for daunorubicin. This means that complete inhibition of P-gp function in these sarcoma cells would lead to an increase of daunorubicin accumulation with 170% compared with 30% in the CD34+ cells. Next, we showed that SYTO16 could be fixed in nuclei by 3.6% formaldehyde treatment, allowing quantification of the nuclear fluorescence on cytospins by laser scanning microscopy. In conclusion, SYTO16 proved to have a combination of favourable properties: it can be excited at 488 nm and has large fluorescence enhancement upon binding to nucleic acids, allowing the use of low, nontoxic (< 10 nM) concentrations. Because the RF for SYTO16 is much higher than for daunorubicin, it can be applied for the determination of P-gp function in relatively small numbers of low-P-gp-expressing tumour cells by laser scanning microscopy. Individual sarcomas were found to have high P-gp function compared with CD34+ cells. This assay may be used to select patients for P-gp modulation protocols.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Mutagenic activity of ambient oxygen and mitomycin C in Fanconi's anaemia cells.

Cellular evidence suggests that Fanconi's anaemia (FA) might be a condition of increased oxygen sensitivity. In order to test this hypothesis, a common shuttle vector assay with the plasmid pZ189 was utilized. We transfected intact, circular plasmid into FA and control lymphoblast and fibroblast host cells maintained at 5 and 20% O2 (v/v). In parallel experiments, host cells were exposed to different concentrations of mitomycin C (MMC), a cross-linking agent towards which FA cells are known to be hypersensitive. Baseline mutation frequencies at 20% oxygen were significantly higher in plasmids passaged through FA lymphoblasts or FA fibroblasts in comparison with passage through the corresponding control cells. Lowering the oxygen concentration during the 48 h transfection period to 5% resulted in a significant decrease of mutation frequencies in plasmids passaged through FA cells. Sequence analysis of plasmids recovered from FA lymphoblasts revealed a mutation hot spot (22% of point mutations with G:C to A:T base substitutions) at base 117 of the supF tRNA gene. This hot spot was present only at 20% oxygen. 59% of the base changes at the hot spot and 39% of the changes elsewhere in the supF gene were C to T transitions (the corresponding figures are 0 and 27% at 5% oxygen), the most common type of base change induced by oxygen. The mutation spectrum observed suggests a role for 8-hydroxydeoxyguanosine in G:C to A:T base substitutions: at 20% oxygen, FA cells displayed 4 times as many G:C to T:A transversions than FA cells kept at 5% O2. In MMC treated cells the decrease in plasmid survival is dose dependent and more pronounced in FA than control cells. Mutation analysis shows similar rates of deletions for both control and FA cells. However, FA cells generate a specific type of deletion whose breakpoint involves an indirect repeat that corresponds to a heptamer signal sequence commonly seen at recombination sites. Together our data provide compelling evidence that the genetic defect in FA causes oxygen sensitivity and recombinational types of DNA lesions following exposure to MMC.

Base Sequence↗
PubMed1997

Bacterial viability and antibiotic susceptibility testing with SYTOX green nucleic acid stain.

A fluorescent nucleic acid stain that does not penetrate living cells was used to assess the integrity of the plasma membranes of bacteria. SYTOX Green nucleic acid stain is an unsymmetrical cyanine dye with three positive charges that is completely excluded from live eukaryotic and prokaryotic cells. Binding of SYTOX Green stain to nucleic acids resulted in a > 500-fold enhancement in fluorescence emission (absorption and emission maxima at 502 and 523 nm, respectively), rendering bacteria with compromised plasma membranes brightly green fluorescent. SYTOX Green stain is readily excited by the 488-nm line of the argon ion laser. The fluorescence signal from membrane-compromised bacteria labeled with SYTOX Green stain was typically > 10-fold brighter than that from intact organisms. Bacterial suspensions labeled with SYTOX Green stain emitted green fluorescence in proportion to the fraction of permeabilized cells in the population, which was quantified by microscopy, fluorometry, or flow cytometry. Flow cytometric and fluorometric approaches were used to quantify the effect of beta-lactam antibiotics on the cell membrane integrity of Escherichia coli. Detection and discrimination of live and permeabilized cells labeled with SYTOX Green stain by flow cytometry were markedly improved over those by propidium iodide-based tests. These studies showed that bacterial labeling with SYTOX Green stain is an effective alternative to conventional methods for measuring bacterial viability and antibiotic susceptibility.

Ampicillin Resistance↗
PubMed1996

Cell cycle defect in connection with oxygen and iron sensitivity in Fanconi anemia lymphoblastoid cells.

Fanconi anemia (FA) is an autosomal recessive disorder involving progressive pancytopenia, skeletal malformations, and a predisposition to leukemia. The in vitro growth of FA fibroblasts is impaired, due to a defective G2 phase traverse of the cell cycle. Analyzing the cell cycle of lymphoid cell lines (LCLs) obtained from peripheral blood of FA patients by transformation with Epstein-Barr virus, we found a similar G2 phase defect, which was dependent upon the oxygen concentration. In addition, FA cells exhibited hypersensitivity toward cis-dichlorodiammineplatinum and mitomycin C, and moderate sensitivity toward trans-dichlorodiammineplatinum. FA cells, however, showed no elevated sensitivity toward paraquat, an intracellular generator of superoxide radicals, or cumene hydroperoxide, a model organic peroxide. Chelating iron with low concentrations of o-phenanthrolin improved cell proliferation and G2 phase transit of FA cells at 20% oxygen, but little at 5% oxygen. LCL cultures from healthy subjects were inhibited in their proliferation rate at all concentrations of o-phenanthrolin. Exposure to excess iron, on the other hand, was very toxic to FA cells at 20%, but less toxic at 5% oxygen. In conclusion, the FA mutation leads to a cell cycle defect, which is expressed in cultures of lymphoid cells from FA patients, and involves hypersensitivity toward bifunctional alkylating agents, oxygen, and iron.

Alkylating Agents↗
PubMed1996

Probenicid inhibition of fluorescence extrusion after MCB-staining of rat-1 fibroblasts.

The intracellular fluorescence level of cells stained continuously with monochlorobimane was monitored by flow cytometry in order to assess the initial rate of glutatione to monochlorobimane conjugation as a measure of glutathione S-transferase activity. In addition to a rapid initial increase and a plateau level, a decline in fluorescence intensity was found upon prolonged flow cytometric monitoring. Exposure to probenicid, an inhibitor of an ATP-dependent organic anion pump, prevented this decrease. Incubation with vanadate and verapamil was without effect. Thus, extrusion of fluorescentglutathione-conjugate perturbs the proportionality between initial glutathione level and monochlorobimane-dependent fluorescence intensity. Monitoring by flow cytometry the decrease in monochlorobimane-dependent fluorescence may be useful to detect multidrug resistant cells.

3T3 Cells↗
PubMed1996

Analysis of mitochondrial morphology and function with novel fixable fluorescent stains.

Investigation of mitochondrial morphology and function has been hampered because photostable, mitochondrion-specific stains that are retained in fixed, permeabilized cells have not been available. We found that in live cell preparations, the CMXRos and H2-CMXRos dyes were more photostable than rhodamine 123. In addition, fluorescence and morphology of mitochondria stained with the CMXRos and CMXRos-H2 dyes were preserved even after formaldehyde fixation and acetone permeabilization. Using epifluorescence microscopy, we showed that CMXRos and H2-CMXRos dye fluorescence fully co-localized with antibodies to subunit I of cytochrome c oxidase, indicating that the dyes specifically stain mitochondria. Confocal microscopy of these mitochondria yielded colored banding patterns, suggesting that these dyes and the mitochondrial enzyme localize to different suborganellar regions. Therefore, these stains provide powerful tools for detailed analysis of mitochondrial fine structure. We also used poisons that decrease mitochondrial membrane potential and an inhibitor of respiration complex II to show by flow cytometry that the fluorescence intensity of CMXRos and H2-CMXRos dye staining responds to changes in mitochondrial membrane potential and function. Hence, CMXRos has the potential to monitor changes in mitochondrial function. In addition, CMXRos staining was used in conjunction with spectrally distinct fluorescent probes for the cell nucleus and the microtubule network to concomitantly evaluate multiple features of cell morphology.

3T3 Cells↗
PubMed1995

Heterogeneity of bromodeoxyuridine sensitivity of cultured cells from melanoma metastases.

Continuously growing cell cultures, testing positive for tyrosine activity, were derived from two brain and three lymph-node metastases of five patients with malignant melanoma. These cell cultures were analyzed regarding their proliferation rate with continuous bromodeoxyuridine (BrdUrd) labeling followed by bivariate Hoechst 33258/ethidium bromide flow cytometry. Melanoma cell cultures are more sensitive toward BrdUrd in comparison to human diploid fibroblast cultures: 50% growth inhibition at 360 +/- 130 microM BrdUrd (range: 130-520; n = 11) vs. 650 +/- 50 microM BrdUrd (n = 3) for fibroblasts. Moreover, BrdUrd sensitivity in melanoma cells is oxygen dependent: 50% growth inhibition at 200 +/- 55 microM (range: 65-400 microM) for 20% oxygen vs. 360 +/- 130 microM BrdUrd for 5% oxygen. The cell cycle kinetic mechanisms of BrdUrd-induced growth inhibition is accumulation of cells in the G2 phase. Cultures from a single metastasis showed up to a 3-fold variation in BrdUrd sensitivity. In one of the brain metastases two populations of different ploidy level (pseudotriploid vs. pseudotetraploid) and BrdUrd sensitivity could be resolved. Thus, continuous BrdUrd labeling followed by bivariate Hoechst 33258/ethidium bromide flow cytometry is a powerful tool to detect heterogeneity in proliferative capacity and drug sensitivity of cell populations within one tumor biopsy.

Bisbenzimidazole↗
PubMed1995

Significance of conventional and new prognostic factors for locally confined renal cell carcinoma.

BACKGROUND: The prognosis of patients with locally confined renal cell carcinoma is variable. To improve the prognostic knowledge and select patients at high risk, additional prognostic parameters are needed. METHODS: The significance with respect to survival and tumor recurrence of "classic" and "new" prognostic parameters has been examined by following 41 patients with locally confined renal cell carcinoma after nephrectomy (mean follow-up, 5.2 years). The significance of histologic grade, tumor stage, Ki-67 index, proliferating cell nuclear antigen index, 3H-thymidine labeling index, tumor ploidy status, and tumor growth after xenotransplantation into nude mice (GAX range) was tested using the Kaplan-Meier plots by the log rank test or Tarone's test and also by the Cox multiple hazard regression analysis. RESULTS: Tumor stage (P < 0.0025), histologic grade (P < 0.005), Ki-67 index (P < 0.006), and GAX range (P < 0.00004) were found to be significant prognostic parameters for survival and tumor recurrence using single-factor analysis. Applying the multivariate analysis, the combination of the "new" factors, GAX range and Ki-67 index, resulted in even a higher prognostic relevance than the combination of the "classic" prognostic factors, tumor stage and histologic grade. The calculated prognostic index based on the results of the Cox analysis, which, except for stage and grade, included the Ki-67 index, was shown to be highly correlated with survival (P = 0.00002) and tumor recurrence (P = 0.0004). Its prognostic validity was studied with the receiver operating characteristics procedure and was found to be considerably superior to that of the two conventional prognosticators. CONCLUSIONS: The additional determination of the Ki-67 labeling index increases the prognostic assessment of patients with locally confined renal cell carcinoma.

Animals↗
PubMed1995

De novo synthesis of glutathione is required for both entry into and progression through the cell cycle.

To study the putative role of de novo synthesis of glutathione (GSH) in the regulation of the cell cycle, we exposed NIH-3T3 cells to buthionine sulfoximine (BSO) and analysed cell cycle kinetics with continuous bromodeoxyuridine (BrdU) labeling and bivariate Hoechst 33258/ethidium bromide flow cytometry. Treating quiescent cells, which themselves had a low GSH content, with BSO did not affect subsequent entry into and progression through the cell cycle. Adding BSO during serum stimulation, however, provoked a dose-dependent inhibition of cell growth and a delayed increase in GSH level. The cell kinetic mechanism underlying BSO-induced growth inhibition is a diminished entry into the cell cycle and a permanent arrest in the S and G2 phase of the cell cycle. Our results are consistent with the hypothesis that GSH de novo synthesis is required for cell activation and proper S and G2 phase transit.

3T3 Cells↗
PubMed1995

Distinct patterns of cell cycle disturbance elicited by compounds interfering with DNA topoisomerase I and II activity.

DNA topoisomerases are enzymes governing the multitude of conformational changes DNA undergoes during the cell cycle. Several compounds are likely to interfere with specific steps of the catalytic cycle of these enzymes. Camptothecin arrests the activity of DNA topoisomerase I by provoking the formation of a single-stranded DNA break with the enzyme molecule covalently attached to the DNA. Exposure to m-AMSA arrests DNA topoisomerase II by the formation of a ternary complex involving the drug, the enzyme, and DNA carrying a double-stranded break. Netropsin, distamycin A, and berenil inhibit DNA topoisomerase-mediated relaxation of supercoiled DNA by an as-yet unknown mechanism. Here, we analyze the cell cycle kinetic effects of exposure to camptothecin, m-AMSA, netropsin, distamycin A, and berenil by using continuous bromodeoxyuridine labeling followed by bivariate Hoechst 33258/ethidium bromide flow cytometry. Camptothecin elicits an accumulation of cells in all compartments of the cell cycle, while exposure to m-AMSA leads mainly to retention of cells in the G0/G1 compartment and to accumulation in the G2 phase. Neither camptothecin nor m-AMSA shows a synergism with bromodeoxyuridine incorporation into the DNA. These results point toward distinct functions of the two DNA topoisomerases in the process of cell cycle traverse. The compounds binding to the minor groove of DNA interfere with all phases of the cell cycle, but with a relative emphasis on the G2 phase. Neither camptothecin nor m-AMSA exhibits a synergistic effect in combination with berenil. Hence, at the level of perturbed cell cycle kinetics a distinction can be made between compounds provoking an abortive inhibition of the catalytic cycle of DNA topoisomerases (e.g., camptothecin, m-AMSA) and those interfering with the activity of the enzyme by a distinct mechanism.

Amsacrine↗
PubMed1994

Interleukin 7 as interleukin 9 drives phytohemagglutinin-activated T cells through several cell cycles; no synergism between interleukin 7, interleukin 9 and interleukin 4.

The effects of the interleukins IL-7 and IL-9 on cell cycle progression were investigated by conventional [3H]thymidine incorporation and by the bivariate BrdU/Hoechst technique. Both IL-7 and IL-9 drive phytohemagglutinin-activated T cells through more than one cell cycle, but IL-7 was more potent on cell cycle progression than IL-9. Neither synergistic nor inhibitory effects were seen between various combinations of the lymphokines IL-7, IL-9 and IL-4 compared to each lymphokine alone. When T cells are activated with phytohemagglutinin for 3 days, all or most IL-4 responsive cells respond to IL-7 as well, whereas only a part of IL-7 responders are IL-4 responders. In contrast, when T cells are activated with phytohemagglutinin for 7 days, the quantitative data of the cell cycle distribution suggest that the population of IL-7 responders is at least an overlapping, if not a real subset of the population of the IL-4 responders.

Cell Cycle↗
PubMed1994

Hypermutable ligation of plasmid DNA ends in cells from patients with Werner syndrome.

Werner Syndrome is a rare autosomal recessive disorder characterized by an increased cancer risk and by symptoms suggestive of premature aging. Cells from these patients demonstrate a typical pattern of chromosomal instability and a spontaneous hypermutability with a high rate of unusually large deletions. We have studied the in vivo DNA ligation in three lymphoblast cell lines from Werner syndrome patients and three from normal donors. In our host cell ligation assay we transfected linearized plasmid pZ189 and measured the amount of plasmid DNA ends rejoined by these host cells as the ability of the recovered plasmid to transform bacteria. A mutagenesis marker gene close to the ligation site allowed screening for mutations. Subsequent mutation analysis provided information about the accuracy of the ligation process. The cells from Werner syndrome patients were as effective as normal cells in ligating DNA ends. However, mutation analysis revealed that the three Werner syndrome cell lines introduced 2.4-4.6 times more mutations (p < 0.001) than the normal cell lines during ligation of the DNA ends: the mutation rates were 69.4, 97.2, and 58.7%, as compared to 23.6, 21.7, and 24.4% in the normal cell lines. These increased mutation frequencies in plasmids ligated during passage through Werner syndrome cells were mainly due to a significant (p < 0.001) increase in deletions. This error-prone DNA ligation might be responsible for the spontaneous hypermutability and the genomic instability in Werner syndrome cells and related to the apparently accelerated aging and high cancer risk in affected patients.

Cell Line↗
PubMed1994

Cell cycle disturbance in relation to micronucleus formation induced by the carcinogenic estrogen diethylstilbestrol.

In addition to its tumor-promoting activity in hormone-receptive tissue, the carcinogenic estrogen diethylstilbestrol (DES) has been found to induce cell transformation, aneuploidy and micronucleus formation in mammalian cells. The majority of these micronuclei contained whole chromosomes and were formed during mitosis. Here a possible relationship between a disturbance in cell cycle progression and micronucleus formation is investigated by exposing Syrian hamster embryo (SHE) cells to DES. Continuous bromodeoxyuridine labeling followed by bivariate Hoechst 33258/ethidium bromide flow cytometry was employed for analysis of cell cycle transit and related to the time course of micronucleus formation. Treatment of SHE cells with DES resulted in delayed and impaired cell activation (exit from the G0/G1 phase), impaired S-phase transit and, mainly, G2-phase traverse. Cells forming micronuclei, on the other hand, were predominantly in G2 phase during DES treatment. These results suggest that impairment of S and G2 transit may involve a process ultimately leading to micronucleus formation.

Animals↗