Plating Efficiency Of Cells Research Articles

The role of programmed cell death in the toxicity of the mutagens, ethyl methanesulfonate and N-ethyl- N′-nitrosourea, in AHH-1 human lymphoblastoid cells

In order to determine the pathway for cell death in alkylating agent-exposed human lymphoblastoid cells, AHH-1 cells were exposed to either ethyl methanesulfonate (EMS) or ethyl nitrosourea (ENU) and the effect on relative cell growth and plating efficiency quantified. Flow cytometric (FCM) assays were utilized to quantify cell viability and to determine if cell death occurred through necrosis or apoptosis. As expected, exposure to the simple ethylating agents resulted in concentration-dependent decreases in plating efficiencies at each time interval after exposure (Days 0,2, 3 and 7). EMS exposure did not significantly affect the relative cell growth, in contrast to ENU exposure, which inhibited cell growth. The FCM viability assay, based on light scatter characteristics, revealed that exposure to either alkylating agent resulted in a significant reduction in the percentage of viable cells. The results of the FCM dye-exclusion assays revealed that while necrosis occurred in EMS- and ENU-exposed cells, the primary manner of cell death was apoptosis. AHH-1 cells were stained with propidium iodide and fluorescein diacetate, the population of cells sorted electronically and the cell type (necrotic, apoptotic or viable) confirmed morphologically. Our results clearly indicate that exposure to EMS or ENU results in the movement of AHH-1 cells into the pathway for apoptosis and cell death.

UROKINASE-TYPE PLASMINOGEN-ACTIVATOR (UPA), A PROTEASE WITH CYTOKINE-LIKE ACTIVITY IN HUMAN HL-60 LEUKEMIC-CELL LINE

HL-60, a cell line originating from a human myeloid leukemia, expresses urokinase-type plasminogen activator (uPA) on the mRNA level and secretes the protein into the culture supernatant. Additionally, uPA receptors (uPA-R) could be detected in HL-60 on both the mRNA and the protein level, whereas the lymphoblastic cell line Raji studied in parallel was uPA-R negative. The cell lines were further studied in their clonal growth in methylcellulose under the influence of rhuPA (rhpro-uPA). The growth of Raji cells was not influenced by rhuPA. Colony and cluster formation of HL-60 was not reproducibly affected by rhuPA in concentrations between 1-100 ng/ml. In some experiments however, there were higher numbers of colonies in the HL-60 cultures incubated with rhuPA which was due to a cluster-to-colony shift. Furthermore, the HL-60 colonies in the rhuPA incubated plates always showed morphological alterations including an adherent basis indicating functional differentiation. This assumption was further supported by the observation that the secondary plating efficiency (PE2) of HL-60 cells taken from single colonies of uPA-incubated plates decreased significantly when compared with PE2 of cells from colonies grown without the presence of uPA. In conclusion, the intact uPA molecule functions like an autocrine cytokine for a human leukemic cell line, which in addition to its effects in tumor invasion makes it an interesting target molecule for further studies on tumor suppression.

Effects of (+)-, (−)- and (±)-indenestrols A and B on microtubule distribution and cytotoxicity in Chinese hamster V79 cells

We have reported that (+)-, (−)- and (±)-indenestrols A and B (IA and IB respectively) inhibit the polymerization of microtubule proteins isolated from porcine brain in vitro. In this study, the effects of (+)-, (−)- and (±)-IA and IB on the relative plating efficiency, chromosome number and cellular microtubular architecture of Chinese hamster V79 cells, detected with a fluorescent anti-tubulin antibody, were investigated. The results indicated that the effect of (±)-IA was similar to that of diethylstilbestrol and that of (±)-IB was greater than that of (±)-IA. We also determined the effects of the optically active IA and IB isomers and found that the rank order of cytotoxic activity of the IA and IB series was: (−)-IA>(±)-IA>(+)-IA and (±)- IB≧(−)- IB>(+)- IB . Furthermore, we studied the intracellular disturbance of microtubule formation induced by these compounds and found that (−)-IA had by far the greatest disruptive effect.

Clonal heterogeneity in delayed decrease of plating efficiency of irradiated HeLa cells.

The clonogenic potential of progeny of irradiated HeLa cells was studied at different times after single doses of 4-12 Gy. The dose-dependent decrease in plating efficiency that was observed resembled the effect termed "delayed lethal mutation" by Seymour et al. (1986). The effect decreased with time after irradiation. Individual clones of irradiated and non-irradiated cells were isolated, expanded and replated 5 weeks after irradiation, i.e., after between 200,000 and 1,000,000 progeny had formed from the individual parent cell. The plating efficiency of progeny of unirradiated cells did not vary much, whereas clonal progeny of irradiated cells had plating efficiencies ranging from 3% to 76%. The plating efficiency was not related to the cell number in the original clone.

Different HSP70 expression and cell survival during adaptive responses of 3T3 and transformed 3T3 cells to osmotic stress

Responses both to hyperosmotic stress and to heat shock were compared in 3T3 cells, spontaneously transformed cells (ST3T3) and simian virus 40-transformed cells (SV3T3). Cell adaptation to these stresses was measured in terms of surviving cell viability and plating efficiency, while their induced synthesis of stress proteins was monitored in terms of the presence of mRNA for HSP70, the pattern of polypeptides synthesised and the accumulation of HSP70 detectable by monoclonal antibodies. All three types of cells responded similarly to heat shock in their expression of HSP70 and showed no clear differences in ability to recover. In contrast, both ST3T3 and SV3T3 cells adapted more poorly and much more slowly to hyperosmotic stress (0.5 osM incubation) than did normal 3T3 cells. This different pattern of adaptation to hyperosmotic stress was parallelled by the cells' different expression of a stress protein that could not be distinguished from the heat-induced HSP70 by any of the methods listed above. In view of these findings it seems possible that hyperosmotic treatment might be useful in selectively affecting the survival of tumour cells.

The toxicity of antibiotics to protoplast cultures of Triticum aestivum L.

A cell suspension culture was established from an immature embryo-derived callus culture of Triticum aestivum cv Fundulea. This culture provided high yields of protoplasts which, at low plating densities (2.5–5.0 × 10 4/ml), could reestablish cell cultures. Low density cultures were required to measure reproducibly responses to a range of antibiotics. Cell division and plating frequencies were used to evaluate phytotoxic responses. The aminoglycoside antibiotics hygromycin, G418, bekanamycin and kanamycin were extremely toxic to the wheat cells (50% inhibition at 15 μg/ml). The least toxic antibiotics were vancomycin, spectinomycin and cefotaxime. Vancomycin (1000–2000 μg/ml) enhanced cell division and plating efficiencies two-fold indicating its potential for establishing protoplast cultures of recalcitrant species.

Minimal growth requirements of mature T lymphocytes: interleukin (IL)-1 and IL-6 increase growth rate but not plating efficiency of CD4 cells stimulated with anti-CD3 and IL-2.

We show that interleukin (IL)-2 is necessary and sufficient for the proliferation of both CD4 and CD8 subsets of peripheral murine T cells activated by plastic-bound anti-CD3 monoclonal antibodies (mAb). The frequency of proliferating cells (f) was 0.32 for CD4 cells and 0.63 for CD8 cells. These frequencies were not increased by the addition of IL-1 or IL-6, alone or in combination. These cytokines were unable to induce responsiveness to IL-2 in T cells confirming that they cannot substitute for the signal delivered via the TcR/CD3 complex. On the other hand, IL-1 and IL-6 increase the growth rate of CD4 cells. The addition of IL-6 significantly lowered the mean doubling time (dt) of CD4 cells (dt: 26 h vs. 38 h in the presence of IL-2 alone, p less than 0.01), while the addition of IL-1, ineffective by itself, combined with IL-6 further increased the growth rate of CD4 cells (dt: 23 h, p less than 0.001). The growth rate of CD8 cells stimulated with anti-CD3 and IL-2, was markedly faster than that of CD4 cells (dt: 18 h vs. 38 h, p less than 0.001) and was not significantly influenced by addition of IL-1 and/or IL-6.

The source of the growth-promoting factor(s) affects the plating efficiency of Paracoccidioides brasiliensis.

We studied the influence of the growth factor (GF) source, concentration and production time on the plating efficiency of Paracoccidioides brasiliensis yeast cells. The highest plating efficiencies were achieved when the GF was derived from a fast growing P. brasiliensis isolate which was not homologous to the plated samples.

Inhibition of cellular transformation by triphenylmethane: a novel chemopreventive agent.

Triphenylmethane (TPM) was found to inhibit 3-methyl-cholanthrene-induced neoplastic transformation of 10T1/2 cells in a dose-dependent manner (ED50 = 2.8 microM). This activity was independent of any effect on intercellular communication and did not appear to be directly related to the general antioxidant properties of TPM as measured by cellular thiobarbituric acid-reactive substances. Triphenylmethanol (TPMOL) and diphenylmethane also inhibited transformation (ED50 = 6.9 and 90 microM respectively). TPM had no effect on the proliferation of exponentially growing cells. At higher concentrations TPM and its analogues enhanced plating efficiency of cells indicating no significant toxicity for these compounds at levels up to 50 microM. The inhibitory effects of TPM on transformation were reversible when TPM was removed from the medium. While TPM had no effect on the growth of fully transformed cell lines, it was able to inhibit the growth of 1/3 neoplastic foci in the presence (but not absence) of 10T1/2 cells. TPM was found to stimulate protein kinase C (PKC) activity for both crude C3H10T1/2 cytosolic PKC and purified PKC obtained from rat brain. The ability of TPM to stimulate PKC activity appeared to be dependent on [CaCl2] and the order of reagent addition in the assay. Tamoxifen, a structurally related compound to TPM, was also found to enhance PKC activity over the same concentration range but was less potent than TPM. The biological effects of TPM and related compounds indicate that they function in a manner distinct from other highly unsaturated transformation inhibitors such as carotenoids and retinoids. The inability of triphenylene to inhibit transformation suggests that a reactive methyl carbon may be essential for activity.

Radiosensitivity testing of human primary brain tumor specimens

The inherent radiosensitivity of early passage cells derived from 22 patients with tumors of glial origin has been determined using a clonogenic assay system. The mean (±SD) surviving fraction at 2 Gy was 0.37 ± 0.22 (range = 0.02–0.87). No correlation between inherent radiosensitivity and tumor cell plating efficiency or intracellular glutathione was observed. Tumor cells that were both resistant to nitrosoureas and expressed the Mer+ phenotype did not differ significantly in their radiosensitivity as compared to cells that were repair deficient (Mer−) and sensitive to nitrosoureas. Initial clinical follow-up suggests that factors in addition to inherent tumor cell radiosensitivity, such as performance status and age, continue to be the most important determinants of the response of patients with primary brain tumors to radiotherapy.

Microwave Exposure Alters the Expression of 2-5A-Dependent RNase

The effects of 2.45-GHz continuous-wave microwaves (SAR = 130 mW/g) on the expression of the interferon-regulated enzymes 2'-5'-oligoadenylate (2-5A) synthetase(s) and 2-5A-dependent endoribonuclease (RNase L) were studied in murine L929 cells. Cells growing as monolayers were removed from the substratum and placed in suspension culture for a 4-h sham or microwave exposure. The cells were returned to monolayer growth for 18 h, and then harvested and assayed to determine the amount of RNase L protein (via [32P]2-5A binding) and the specific activities of RNase L and 2-5A synthetase. Binding of radioactive 2-5A to RNase L for sham- and microwave-exposed samples was 14.5 and 36.4% above control, respectively (the microwave-exposed bound 19.0% more probe than the sham-exposed). The increases in 2-5A binding were accompanied by corresponding elevations of RNase L specific activity. In contrast, sham or microwave irradiation produced no alterations in 2-5A synthetase specific activity. No detectable differences were noted in the postexposure cell viability, plating efficiency, or proliferation rate. Also, there were no detectable differences in cell viability or plating efficiency between controls and cultures irradiated for 2 h when the temperature was simultaneously increased to above normal physiological limits (39 to 45 degrees C). The SAR (130 mW/g) and the power density (95 mW/cm2) used for the greater part of this study were nearly 20 times higher than the ANSI limit of 8 mW/g and 5 mW/cm2 for any 1 g of exposed human tissue.

Different Metastasis Patterns of a Human Melanoma Cell Line in Nude Mice and Rats:Influence of Microenvironment

The metastatic capacity of intravenously injected human FEMX-I melanoma cells in athymic nude mice and rats was compared. Young rats given 1 x 10(6) ascites tumor cells all died of lung tumors with a life span of 50 +/- 10 days (mean +/- SD). In contrast, in accordance with previous findings, only extrapulmonary metastases developed in mice. This host-dependent difference in metastasis pattern permitted studies on the role of factors that may influence the organ specificity of metastases. The tissue distribution of 125I-labeled FEMX-I cells did not differ in the two nude species during the first 12 hours after cell injection. The plating efficiency of FEMX-I cells in soft agar was increased by the addition of conditioned medium prepared from rat lungs, resulting also in a significant increase in colony size. In contrast, conditioned medium prepared from mouse lungs reduced the clonogenic capacity of the FEMX-I cells in a dose-dependent manner. Conditioned media prepared from rat and mouse liver, kidney, and spleen tissues either inhibited or had no effect on colony formation. The results suggest that the unexpected differential metastatic patterns observed in vivo may reflect differences in the presence of growth-modulating paracrine factors in the host lungs.

A new human male diploid cell strain, TIG-7: its age-related changes and comparison with a matched female TIG-1 cell strain

A new human diploid cell strain, TIG-7, which has the male karyotype, was established and characterized. Isozyme and histocompatibility typing of the cell strain was performed. The average in vitro life span of the cells is 73 population doublings. Changes in cell volume, doubling time, saturation density, the efficiency of cell attachment, plating efficiency, and relative DNA content were examined during in vitro cellular aging. Hydrocortisone slightly prolongs the life span of the cell strain when the hormone is administered to the cultures during middle passages. The age-related changes in the parameters of TIG-7 are not appreciably different from those of the previously established TIG-1 cell strain. These results show that this cell strain is useful for research on cellular aging; further profit is anticipated from research using a combination of these two sexually different cell strains.

Plant regeneration from protoplasts isolated from seedling cotyledons of Stylosanthes guianensis, S. macrocephala and S. scabra

Plant regeneration from protoplasts is a prerequisite to the production of modified plants using somatic hybridization and transformation. Whole plant regeneration was achieved from protoplasts isolated from seedling cotyledons of Stylosanthes guianensis, S. macrocephala and S. scabra, three economically important species of this tropical forage legume genus. The effects of both protoplast density and protoplast culture method on cell division and plating efficiency are presented.

Maintenance on extracellular matrix and expression of heparanase activity by human ovarian carcinoma cells from biopsy specimens

A routine procedure has been developed for the isolation and maintenance in culture of human ovarian carcinoma cells derived from biopsy specimens. Cell attachment, plating efficiency and initial outgrowth were greatly improved by seeding the cells on a basement-membrane-like extracellular matrix (ECM) deposited by cultured corneal endothelial cells. These effects were most significant in serum-free conditions which markedly reduced the rate of cell attachment and growth on regular tissue culture plastic. In 60-80% of the cases and regardless of the patient's age, cells cultured on ECM in the absence of serum divided actively and formed a tightly packed epithelial cell monolayer. Fibroblast overgrowth and cell detachment often occurred on ECM in the presence of serum. Incubation of the human ovarian carcinoma cells with sulfate-labelled ECM, resulted in the release of heparan sulfate degradation fragments, 4- to 7-fold smaller than intact heparan sulfate side chains. This degradation was brought about by endoglycosidase (heparanase) activity expressed to a higher extent by cells that were first maintained in primary cultures as compared with cell aggregates taken directly from the biopsy specimen. In most cases, cells derived from metastatic tumors expressed a higher heparanase activity than cells from the primary ovarian tumor. This result corroborates previous studies, performed with cell lines, on the possible involvement of heparanase in tumor cell invasion and metastasis.

Spermidine and morphogenesis in single cell cultures of Sideritis angustifolia lag.

Single cells from hypocotyl-derived callus of Sideritis angustifolia were evaluated for morphogenesis when cultured in either solidifier or liquid Murashige and Skoog medium supplemented with various concentrations of spermidine, naphthaeneacetic acid (NAA), benzyladenine (BA) and dicyclohexylamine (DCHA). Spermidine (Spd) did not replace the hormonal requirements for cell division and callus formation, but affected growth induced by NAA or BA. This polyamine increased the plating efficiency of cells cultured in the presence of NAA (0.05, 0.54 and 5.40 μM) or BA (0.40, 4.40 and 8.80 μM). In contrast, Spd reduced growth induced by 10.80 μM NAA, optimal auxin concentration for callus induction. Likewise, DCHA reduced the number of cell-derived calli obtained with 5.40 μM NAA. Data from suspension cultured cells respectively substantiated either the promoter or inhibitory effects of Spd and DCHA on cell proliferation induced by 5.40 μM NAA. To promote organogenesis, cell-derived calli were transferred to various regeneration media. Caulogenesis was only observed in those calli initially grown in the presence of Spd.

The differential effects of the indazole-carboxylic acid derivative, tolnidamine, on Sertoli cell protein secretion.

The indazole-carboxylic acid derivative tolnidamine (TOL) has marked antispermatogenic activity in rats. Previous morphological and biochemical studies indicate that Sertoli cells are one of the targets of this compound. The aim of this study was to assess the effect of TOL on the in vitro secretory functions of primary Sertoli cell-enriched cultures prepared from rats of different ages by monitoring the changes of three known Sertoli cell proteins, androgen binding protein (rABP), transferrin (rTF), and testibumin (rTB). The addition of TOL at the beginning of the culture period reduced the plating efficiency of Sertoli cells; however, TOL did not induce a significant change in cell number if it was added 24 h after plating of the cells. Sertoli cell-enriched cultures prepared from tests of 10-day-old rats were highly sensitive to TOL as evidenced by a marked inhibition in secretions of rABP, rTB, and rTF in all experiments. In cultures prepared from 15- and 20-day-old rats, TOL had no apparent effect on rABP secretion, but reduced rTF and increased rTB secretion. Thus, TOL has a differential effect on the secretion of individual proteins in Sertoli cells cultured from rats between 10 and 20 days of age. This phenomenon is presumably a consequence of the progressive maturation of Sertoli cells in the seminiferous epithelium.

Hyperproliferation of conjunctival fibroblasts from patients with cicatricial pemphigoid.

The characteristic conjunctival scarring in cicatricial pemphigoid is a consequence of subepithelial fibrosis. The fibroblast is the cellular element responsible for fibrosis. To increase the understanding of the pathogenesis of abnormal fibrosis in cicatricial pemphigoid, the growth characteristics of conjunctival fibroblasts, from untreated patients with cicatricial pemphigoid (n = 9) and normal controls (n = 6), were studied in tissue culture. The latent period until fibroblast outgrowth began from the conjunctival explant was determined, as were the plating efficiency and doubling time of cells from first-passage cultures. Outgrowths of fibroblasts from patients with cicatricial pemphigoid appeared significantly sooner than from controls, 8.1 +/- 3.8 vs 19.3 +/- 6.4 (mean +/- SD) days. While there was no significant difference in the plating efficiency between fibroblasts from cicatricial pemphigoid (mean +/- SD, 116.4% +/- 44.6%) and those from controls (71.0% +/- 39.4%), the doubling time was significantly faster for cicatricial pemphigoid than for controls, 26.5 +/- 8.5 vs 50.7 +/- 7.8 hours. Thus, conjunctival fibroblasts from patients with cicatricial pemphigoid are hyperproliferative in tissue culture when compared with normal controls. Therefore, scarring, which characterizes cicatricial pemphigoid, may be due, in part, to excessive fibroblast proliferation.

The effects of dexamethasone on C6 astrocytoma radiosensitivity.

Brain-tumor patients often undergo radiation therapy while receiving corticosteroids for the treatment of cerebral edema. Studies have demonstrated that dexamethasone is radioprotective in a number of cell lines. The C6 astrocytoma cell line is well established in vitro and is modulated by dexamethasone treatment. It has therefore been hypothesized that dexamethasone-treated C6 astrocytoma cells would be more resistant to radiation-induced damage. The present study was carried out to assess this hypothesis using both the in vitro C6 astrocytoma monolayer and three-dimensional multicellular spheroid models. Dexamethasone was inhibitory to the C6 astrocytoma cells in the monolayer preparation, increasing their doubling time by 13%. In the spheroid cultures, dexamethasone treatment decreased the number of cells per spheroid by 46%. Dexamethasone did not affect the plating efficiency of either the cells from the monolayer experiment or those dissociated from spheroids, however, suggesting that the inhibitory effect was not tumoricidal. At a clinical concentration (1.94 x 10(-5) M), dexamethasone did not significantly influence plating efficiency of irradiated C6 astrocytoma cells in monolayer or three-dimensional spheroid cultures.

Effect of hypertonic medium on human cell growth: III. Changes in cell kinetics of EUE cells

The effects of hypertonicity on cell kinetics of EUE cells in culture have been investigated. After 4 days of growth in a hypertonic medium, the plating efficiency of EUE cells was reduced and cell growth was significantly slowed. Flow cytometric measurements of DNA content in synchronized cells, as well as flow cytometric determinations of DNA content and bromodeoxyuridine incorporation in asynchronous cells, also showed that the cell cycle is slowed in a hypertonic medium. In addition, the fraction of cycling cells is smaller and their progression through the S phase slower than in an isotonic medium.