G2 arrest, binucleation, and single-parameter DNA flow cytometric analysis

One important facet of flow cytometry involves the effects of pharmacological agents on cell cycle progression. Comparative G2 fraction perturbations were examined: effects of sodium butyrate on articular chondrocytes, effects of an antineoplastic agent (SOAZ) and an antirheumatic drug (D-penicillamine) on HeLa cells. Even though DNA flow cytometric analysis detects preferentially an induction of G2 arrest, the mode of action of these agents on the cell cycle is different. Sodium butyrate and D-penicillamine lead to an increase of binucleate cells due to cytokinesis perturbation. Because of similar fluorescence intensity, distinguishing G2 from binucleate GO/1 cells is not easily possible using DNA content measurement and reflects a failure of flow cytometry in the detection of binucleate cells. Rapid cell cycle analysis of single cells should contribute greatly to the study of pharmacological interactions, but DNA flow cytometric measurements obtained from cultured cells exposed to certain agents must be cautiously interpreted because those may interact on cytokinesis and induce artefacts in histogram interpretation.     

Demonstration of endothelin (ET) receptors on cultured rabbit chondrocytes and stimulation of DNA synthesis and calcium influx by ET-1 via its receptors

Endothelin (ET) receptors on chondrocytes were demonstrated using cultured rabbit costal chondrocytes. After crosslinking the receptors on the cells with ¹²⁵ I-ET-1, two major bands of 43 kDa and 46 kDa were separated by SDS-PAGE. Scatchard analysis demonstrated two classes of ET receptors with K_d values of 1 × 10^?10 M and 5 × 10^?9 M. The numbers of high- and low- affinity receptors were 1 × 10⁴ and 2 × 10⁵ per cell, respectively. The binding of ET-1 to chondrocytes was increased by treatment with PTH, DBcAMP, TGF-β1, IL-1β, RA and EGF. ET-1 stimulated DNA synthesis in cultured rabbit chondrocytes. ET-1 also stimulated calcium incorporation through the cell membrane of chondrocytes. These findings indicate that ET-1 has a physiological effect on chondrocytes via its receptors on the cells.     

Mammalian cell fusion. VI. Regulation of mitosis in binucleate HeLa cells.

In two different cell fusion experiments a synchronized population of HeLa cells, prelabeled with ³H-TdR, was fused with an unlabeled one using inactivated Sendai virus. In the first experiment, HeLa cells in early G2 phase which were exposed to either 4 °C, cycloheximide, actinomycin D or X-irradiation were fused separately with untreated and more advanced G2 cells. A comparison of the rates of mitotic accumulation (in the presence of Colcemid) for the various classes of mono- and binucleate cells revealed that the hybrid (binucleate) cells were intermediate between those of the advanced and the retarded parental types indicating that the chromosome condensing factors of the advanced component were diluted as a result of such fusion. The manner in which the retarding effects of actinomycin D and cycloheximide were reversed in the hybrid cells suggested that proteins had a major role as chromosome condensing factors in the G2 mitotic transition. In the second experiment, when S phase HeLa cells were fused with those in G2, the resulting heterophasic (S/G2) binucleate cells reached mitosis at about the same time as the homophasic (S/S) cells of the lagging parent indicating a complete dominance of the S over the G2 with regard to their progress towards mitosis. However, the addition of Mg²⁺ (2 × 10^?2 M of MgCl₂) to the medium helped the G2 nuclei to enter mitosis asynchronously, which consequently induced premature chromosome condensation (PCC) in the S phase component. These data suggested that in the heterophasic (S/G2) binucleate cells the S phase component caused decondensation of the G2 chromatin thus blocking it from entering into mitosis. This effect which did not appear to be dose-dependent could be neutralized and the G2 nuclei relieved from this repression by an external supply of Mg²⁺ ions.     

Effects of sodium butyrate on growth and cell-cycle kinetics of cultured rabbit articular chondrocytes

The sodium salt of n-butyric acid was found to inhibit the growth of asynchronous cultures of rabbit articular chondrocytes. This inhibitory effect was dose-dependent between 1 mM and 5 mM, reversible, and accompanied by volume enhancement and modification of cellular morphology. Flow-cytometric analysis showed that drug exposure led to a slowing-down of the cell-cycle progression; after 1 day's exposure, cells accumulated in G1, and after 2 or 3 days' treatment, in G2, without a blockage in M; the increase of cells in G2 was in fact due to an enhancement of binculeated cells. The treated cells had an increased RNA content. Articular chondrocytes seem to be target cells for sodium butyrate and therefore it represents a valuable biological tool for studying the mechanisms of their growth regulation.     

Reversible arrest of Chinese hamster V79 cells in G2 by dibutytyl AMP.

Mouse L cells 929 were cloned in supplemented Eagle's minimal medium enriched with lactalbumin and yeast extract and buffered with HEPES. Multiplication was followed photographically in single clones from the 8-cell stage through 6–7 days. Addition of the folic acid analogue methotrexate (amethopterin) in 5 × 10^?6 M concentration slowed growth only after two cell generations; 10^?4 M uridine had no effect on growth except when combined with methotrexate. The two agents together blocked cell division quickly and symptoms of thymine-less death developed in few days. The cells could be rescued before 48 h by removal of the inhibitors, or by addition of folic acid or thymidine. The combination of methotrexate with uridine blocks DNA synthesis in Tetrahymena by inhibition of thymidylate synthesis and of thymidine uptake from the complex medium. Apparently the same mechanisms operate in L cells grown in a complex medium containing thymidine.     

In Vitro Evaluation of the Effects of Electromagnetic Fields used for Bone Healing

The use of therapeutic electromagnetic fields (EMF) for bone healing has positive clinical effects but may have adverse biologic effects. For this reason, EMF exposure has been repeatedly investigated to exclude the possibility of genotoxic effects and tumour risk. This paper describes the effects of EMFs on cell cultures. We analyzed the effects of EMF (28 gauss, 75 Hz) on growth and metabolic activities in four different cell types: L929 fibro-blasts, osteoblast-like HOS/TE85 cells, human lymphocytes, and rabbit chondrocytes. We found no cytotoxic or mutagenic effects on cultures exposed to EMF compared with unexposed controls. Results of cell proliferation showed a statistically significant increase for all cultures exposed to EMF with respect to controls (L929 +45%, p = 0.002; HOS/TE85 +32%, p = 0.001; chondrocytes +40%, p = 0.0003; lymphocytes +39%, p = 0.0002). Biochemical and enzymatic tests gave different results, depending on cell types: all tested values were increased after EMF exposure, even if only some of them reached statistical significance (total proteins: HOS/TE85 p = 0.004, chondrocytes p = 0.003; alkaline phosphatase: L929p = 0.0003, HOS/RE85 p = 0.0001, chondrocytes p = 0.009, lymphocytes p = 0.006; lactate dehydrogenase: chondrocytes p = 0.0002, lymphocytes p = 0.0005). Biochemical and enzymatic tests and cell proliferation results suggest a more active metabolism in cartilage and bone cells after EMF exposure. These effects could be relevant for bone healing in clinical practice.     

Cytotoxic effects of dexamethasone restricted to noncycling,early G1-phase cells of L1210 leukemia

The cytostatic and cytolytic effects of dexamethasone were studied as functions of cell cycle position in mouse L1210 leukemia cells. To this end, the cells were separated according to size by sedimentation at unit gravity in a specially designed sedimentation chamber. The fractions were analyzed by radioautography and flow cytophotometry. The size-distributions obtained by 1g sedimentation coincided with cell-cycle age distribution. With increasing fraction number, samples highly enriched in G1, S, and G2/M cells, respectively were obtained: the smallest cells being in early G1 and the largest in mitosis. In the presence of dexamethasone (10^?6-10^?5 M), growth slowed down after a few cell cycles and the cells accumulated in early G1 phase. Lytic cell kill by continued exposure to the drug was confined to the fractions containing the small, early G1-phase cells. These fractions were also enriched in noncycling cells that were not labeled by prolonged exposure to ³H-thymidine. After removal of dexamethasone, the cells in S and G2/M phase completed cell cycle traverse but were retarded again in the G1 and early S phase of the next division cycle. The data suggest a memory effect for previous drug exposure. It is concluded that the cytostatic and cytolytic effects of dexamethasone are separate, though not unrelated events. Cytolysis is confined to the noncycling cells that in untreated populations can exit from the dividing compartment during a transitional phase of about 60 minutes subsequent to mitotic division. The cytostatic effects potentiate cytolysis by accumulating the cells in the early G1 phase and thus increasing the probability of their transit to the G0 compartment, sensitive for drug-mediated cytolysis.     

Nebrodeolysin,a novel hemolytic protein from mushroom Pleurotus nebrodensis with apoptosis-inducing and anti-HIV-1 effects

A novel hemolysin was isolated from the edible mushroom Pleurotus nebrodensis by ion exchange and gel filtration chromatography on DEAE-Sepharose and Sephacryl S-100. The hemolysin from Pleurotus nebrodensis hemolysin (nebrodeolysin) is a monomeric protein with a molecular weight of approximately 27 kDa as determined by gel filtration and SDS-PAGE. Nebrodeolysin exhibited remarkable hemolytic activity towards rabbit erythrocytes and caused efflux of potassium ions from erythrocytes. Subsequently, this hemolysin showed strong cytotoxicity against Lu-04, Bre-04, HepG2, L929, and HeLa cells. It was also found that this hemolysin induced apoptosis in L929 and HeLa cells as evidenced by microscopic observations and DNA ladder, respectively. Moreover, this hemolysin was shown to possess anti-HIV-1 activity in CEM cell culture.     

Influence of DL-beta-hydroxybutyric acid on cell proliferation and calcium influx

Poly(hydroxybutyrate-co-hydroxyhexanoate) (PHBHHx), a member of the polyhydroxyalkanoate family of biopolyesters, has superior mechanical properties and biocompatibilities that enable it to meet diverse biomedical requirements. The main component of PHBHHx is DL-beta-hydroxybutyric acid (HB), a ketone body that is also produced in vivo. The effects of HB treatment on murine fibroblast L929 cells, human umbilical vein endothelial cells, and rabbit articular cartilages were investigated. HB (0.005-0.10 g/L) promoted cell proliferation for each cell line. Cell cycle analysis indicated that HB had a stimulatory effect on DNA synthesis. Flow cytometric analysis of L929 cells revealed changes in the [Ca2+]i for different stages of the cell cycle. In L929 cells, HB (0.02 g/L) stimulated a rapid increase in the concentration of cytosolic calcium that was blocked by verapamil and diltiazem, inhibitors of L-type Ca2+ channels. Finally, verapamil inhibited HB-induced L929 cell proliferation. Collectively, these results indicated that HB had a stimulatory effect on cell cycle progression that is mediated by a signaling pathway dependent upon increases in [Ca2+]i. This trophic effect may underlie the good biocompatibility observed for PHBHHx.     

Flow cytometric detection of transforming growth factor-beta expression in rabbit articular chondrocytes (RAC) in culture--association with S-phase traverse.

We have previously shown that TGF-beta 1 decreased the entry of G0/G1-synchronized rabbit articular chondrocytes (RAC) into S-phase, whereas it enhanced the proliferation rate of actively dividing cells (asynchronous or S-phase-synchronized cells). The growth proliferative effect was accompanied by both increased DNA replication rate and G2/M delay. Since TGF-beta mRNA has been detected in chondrocytes, it was of interest to study the expression of the factor in correlation with the cell cycle of RAC. Using cytofluorometric analysis of both DNA content and TGF-beta protein level, we demonstrated that S-phase-synchronized RAC constitutively expressed TGF-beta, whereas G0/G1-synchronized cells only display very low levels of the factor. The data showed that the expression of TGF-beta is correlated with S-phase traverse since it increases with the percentage of cells in S-phase (less than 27% in G0/G1 to 70% in S-phase-synchronized cells). Moreover, exposure of RAC to TGF-beta 1 (1 ng/ml) for 24 h increased the percentage of positive cells, independently of the number of cells in S-phase, indicating that the factor may up-regulate its own expression. All together, these data suggest that TGF-beta could play a role in initiating the proliferation of articular chondrocytes during the early events of osteoarthritis and might take a part in the repair of cartilage matrix.     

Binding of methylmercury(II) by HeLa S3 suspension-culture cells: Intracellular methylmercury levels and their effect on DNA replication and protein synthesis

HeLa S3 cells were exposed to varied concentrations of methylmercury over varied periods of time and its binding by the cells was studied using ²⁰³Hg-labeled methylmercuric chloride as radioactive marker. Also studied was the effect of cell-bound methylmercury on DNA replication and protein synthesis and on the growth rate of the cells. The results show that methylmercury binding is a rapid process, with much of the organomercurial bound within the the first 60 min of incubation, and that considerable quantities of organic mercury become affixed to the cells. The amounts of bound methylmercury, [CH₃Hg(II)]_bound, given in mol/cell, range from 2 × 10^?16 (at 1 h of incubation and at 1 μM CH₃Hg(II) in the medium) to almost 4 × 10^?14 (at 24 h of incubation and at 100 μM CH₃Hg(II) in the medium). A [CH₃Hg(II)]_bound value of about 30 × 10^?16 mol/cell appears to be the threshold below which cells display a normal growth pattern and below which metabolic events such as DNA replication or protein synthesis are affected only to a minor degree but above which major changes in cell metabolism and cell growth take place. Methylmercury binding by the cells is tight so that only 20% of the bound material is released from the cells over a 3-h incubation period when the cells are placed into fresh, methylmercury-free growth medium. Analysis of the binding data in terms of binding to identical and completely independent sites yields an association constant K of 7.92 × 10⁴ l/mol and for the maximum concentration of cellular binding sites the value 2.40 × 10^?14 mol/cell or 1.45 × 10¹⁰ sites/cell. Evidence is presented which shows that cellular sulfhydryl groups do not suffice to provide all the sites taken up by methylmercury and that binding, in all likelihood, involves basic nitrogen, too. The levels of cell-bound methylmercury are such that binding to HeLa DNA and HeLa chromatin, for instance, can readily take place. Methylmercury binding data obtained by using the technique of particle-induced X-ray emission (PIXE) are in good agreement with the data obtained via isotope dilution.     

Cordycepin Analogs of 2–5 a as Activators of RNase L: Study of the Structural Requirements for RNase L Activation

Abstract

Enzymatically and chemically synthesized cordycepin analogs of 2–5A^? trimer and tetramer were found to be biologically active as protein synthesis inhibitors in intact cultured human fibroblast and murine L929 cells ^1,2. In rabbit reticulocyte lysates, the cordycepin tetramer analog of 2–5A inhibits protein synthesis through binding to and activation of RNase L³. Our present results using L929 cell extracts provide direct evidence that the cordycepin analogs of 2–5A can bind to and activate RNase L.     

Synthesis and antiproliferative evaluation of 4-anilino-n-methoxyfuro[2,3-b]quinoline derivatives (n=6, 7). Part 5

A series of 27 differently substituted 4-anilinofuro[2,3-b]quinolines were synthesized and evaluated for their antiproliferative activities against the HeLa, SKHep1, SAS, AGS, A549, and CE81T cell lines, cancers commonly found in Asian countries. Among the compounds tested, 1-{4-[(3-chloro-7-methoxyfuro[2,3-b]quinolin-4-yl)amino]phenyl}ethanone (1) was the most potent, with IC(50) values of 3.1, 3.0, and 4.2 microM, resp., against the growth of HeLa, SKHep, and CE81T cells. Compound 1 was, thus, further evaluated by flow cytometry to evaluate its effect on the cell-cycle distribution of HeLa cells. Our results indicated that 1 readily induces cell-cycle arrest in the G2/M phase, followed by DNA fragmentation and, ultimately, cell death.     

TGF-beta-induced G2/M delay in proliferating rabbit articular chondrocytes is associated with an enhancement of replication rate and a cAMP decrease: possible involvement of pertussis toxin-sensitive pathway.

This study was undertaken to gain more insight into the mechanism whereby TGF-beta influences the cell cycle progression of cultured rabbit articular chondrocytes. Using proliferating chondrocytes in fetal calf serum-containing medium, we have previously shown that TGF-beta induced a recruitment of cells at the end of the S phase (G2/M) observed 24 h after addition. The delayed cells may then be released, producing a proliferative effect at 48 h, provided a substantial amount of FCS (10%) is present in the medium. Otherwise, in low level of serum (2% FCS, for example), only inhibition of cell proliferation is observed. In chondrocytes synchronized in S phase by a thymidine block, we investigated here the time-course incorporation of [3H]-thymidine into DNA, the cell cycle traverse by flow cytofluorometric study of DNA content, the expression of PCNA (Proliferating Cell Nuclear Antigen), and cAMP levels. The data demonstrate that TGF-beta provoked a decrease of cAMP content (0.5-1 h) followed by an enhancement of the DNA synthesis rate (4 h) which was detectable through cytofluorometric analysis and [3H]-thymidine labeling and correlated with the PCNA expression. In contrast, addition of cAMP analogues to the cultures resulted in an inhibition of replication rate. We also showed that pertussis toxin produced a decrease of the DNA synthesis rate, in a transient manner and only in the presence of TGF-beta. All these results suggest that TGF-beta may accelerate the replication process of cyclized chondrocytes, making then accumulate at the G2/M boundary, via a mechanism that could involve the adenylate cyclase activity and a Gi-protein. The factor might be responsible for producing a pool of cells having already replicated their DNA and therefore capable of re-entering the cell cycle without delay. This cell population could serve as a tissue reserve able to induce a mitosis wave when necessary--for example, in the repair of tissue damage.     

Mechanisms of arsenic trioxide induced apoptosis of human cervical cancer HeLa cells and protection by Bcl-2

It was recently reported that arsenic trioxide (As²O³) can induce complete remission in patients with acute promyelocytic leukemia (APL). In this present article, the biological effect of (As²O³) on human cervical cancer HeLa cells and HeLa cells overexpressing Bcl-2 is studied. By MTT and colony forming ability assays, morphology alteration, flow cytometric analysis, DNA gel electrophoresis andin situ cell death detection (TUNEL), it was found that As²O³ inhibited the growth of HeLa cells and induced G2/M arrest and apoptosis of the cells. RT-PCR, Northern blot, Western blot analysis revealed that As²O³ induced HeLa cell apoptosis possibly via decreasing the expression of c-myc and viral genes. HeLa cells overexpressing Bcl-2 partly resist As²O³ induced apoptosis, which might be relative to preventing the cells from As²O³ caused G2/M block, downregulation of c-myc gene expression and inhibition of viral gene expression was also noted. However, it was found that As²O³ at a high concentration could also induce apoptosis of HeLa cells overexpressing Bcl-2 possibly mainly via downregulating Bcl-2 expression and slightly inhibiting viral gene expression.     

In vitro and in vivo effects of an anti-mouse endoglin (CD105)–immunotoxin on the early stages of mouse B16MEL4A5 melanoma tumours

TGF-beta superfamily co-receptors are emerging as targets for cancer therapy, acting both directly on cells and indirectly on the tumour neovasculature. Endoglin (CD105), an accessory component of the TGF-beta receptor complex, is expressed in certain melanoma cell lines and the endothelial cells of tumour neovessels. Targeting endoglin with immunotoxins is an attractive approach for actively suppressing the blood supply to tumours. Here, we report evidence indicating that endoglin is expressed in mouse melanoma B16MEL4A5 and mouse fibroblast L929 cell lines. We prepared an immunotoxin to target endoglin by coupling the rat anti-mouse MJ7/18 (IgG2a) monoclonal antibody (mAb) to the non-toxic type 2 ribosome-inactivating protein nigrin b (Ngb) with N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP) as a linker with a molar nigrin b at a MJ7/18 stoichiometry of 2:1. The MJ7-Ngb immunotoxin generated killed both cell lines, with IC₅₀ values of 4.2 × 10^?9 M for B16MEL4A5 and 7.7 × 10^?11 M for L929 cells. For in vivo assays of the immunotoxin, B16MEL4A5 cells were injected subcutaneously into the right flanks of 6-week-old C57BL/6 J mice. When the animals developed palpable solid tumours, they were subjected to treatment with the immunotoxin. While treatment with either MJ7/18 mAb or Ngb did not affect tumour development, treatment with the immunotoxin completely and steadily blocked tumour growth up to 7 days, after which some tumours re-grew. Thus, vascular-targeting therapy with this anti-vascular immunotoxin could promote the destruction of newly created tumour vessels at early stages of B16MEL4A5 tumour development and readily accessible CD105+ B16MEL4A5 melanoma cells.     

BINDING OF [99mTc]CHONDROITIN SULFATE TO SCAVENGER RECEPTORS ON HUMAN CHONDROCYTES AS COMPARED TO BINDING OF OXIDIZED [125I]LDL ON HUMAN MACROPHAGES

ABSTRACT

Chondroitin sulfate (CS) used for treatment of osteoarthritis exerts distinct effects on human articular chondrocytes in vitro. We performed a binding analysis with ^99mTc-labeled CS (Condrosulf, a commercial CS preparation containing calcium stearate) and cultured human chondrocytes in order to evaluate the presence of specific receptors. Saturation binding at 37°C for 2?h revealed the presence of high-affinity binding sites for CS with a K_d of 2.3 × 10^?9?mol/L and a B_max of 5.0 × 10⁸. Extensive dialysis of Chondrosulf led to a decrease of the binding affinity by 52.5 ± 19.5% and of the number of CS binding sites/cell by 62.0 ± 14.0%, demonstrating that the additive present in the Condrosulf preparation enhances CS binding. The nature of the binding site is not yet known but evidence exists in the literature that the scavenger receptor CD36, thoroughly investigated on macrophages, is also found on chondrocytes and might be involved in CS binding. Therefore, we undertook a comparative binding study with human monocytes and labelled LDL and oxidized LDL, the latter being a postulated atherogenic agent in atherosclerosis. For [¹²⁵I]-LDL binding we found a K_d of 0.45 × 10^?8?mol/L and a B_max of 0.14 × 10⁶ on quiescent monocytes and for [¹²⁵I]-(ox)LDL binding a K_d of 1.8 × 10^?8?mol/L and a B_max of 1.3 × 10⁶ using LPS-activated monocytes. These data are comparable to the binding affinity found for lipoprotein–proteoglycan-complexes and hence are an indication but not a proof that CD36 is involved in CS binding to human chondrocytes.     

The comparative cell cycle and metabolic effects of chemical treatments on root tip meristems. III. Chlorsulfuron

Intact and excised cultured pea roots (Pisum sativum L. cv Alaska) were treated with chlorsulfuron at concentrations ranging from 2.8 ×10^?4 M to 2.8×10^?6 M. At all concentrations this chemical was demonstrated to inhibit the progression of cells from G₂ to mitosis (M) and secondarily from G₁ to DNA synthesis (S). The S and M phases were not directly affected, but the transition steps into those phases were inhibited. Total protein synthesis was unaffected by treatment of intact roots with 2.8×10^?6 M chlorsulfuron. RNA synthesis was inhibited by 43% over a 24-h treatment period. It is hypothesized that chlorsulfuron inhibits cell cycle progression by blocking the G₂ and G₁ transition points through inhibition of cell cycle specific RNA synthesis.     

Antiproliferative activity of chloroformic extract of Persian Shallot, Allium hirtifolium, on tumor cell lines

Allium hirtifolim (Persian Shallot) belongs to Allium genus (Alliaceae family). We investigated the in vitro effects of chloroformic extract of A. hirtifolium and its Allicin on the proliferation of HeLa (cervical cancer), MCF7 (human, caucasion, breast, adenocarcinoma) and L929 (mouse, C3H/An, connective) cell lines. Our results showed that components of A. hirtifolium might inhibit proliferation of tumor cell lines. This inhibition in HeLa and MCF-7 cells was dose-dependent. The presence of Allicin was evaluated by TLC method in bulbs and the extract of A. hirtifolium was analyzed by HPLC. MTT test was performed 24, 48 and 72 h after cell culture. A significant decrease in cell lines was observed in HeLa and MCF-7 as compared to L929 cell lines. DNA fragmentation analysis revealed a large number of apoptotic cells in treated HeLa and MCF-7 cell groups, but no effects in L929 cells. Therefore A. hirtifolium might be a candidate for tumor suppression.