Decrease In Plating Efficiency Research Articles

Review of radiation-induced bystander effects.

It is now apparent that the target for the biological effects of ionizing radiation (IR) is not solely the irradiated cell(s), but also includes the surrounding cells/tissue as well. Radiation-induced bystander effects (BSEs) are defined by the presence of the biological effects of radiation in cells that were not themselves in the field of irradiation. Decreased plating efficiency, increased sister chromatid exchanges, oncogenic transformation, among other endpoints have been used to describe the BSE. Two primary means have been established for the transmission of the bystander signal; one is mediated by gap-junction intracellular communication, and the other is initiated through the secretion of factors from irradiated cells. While the basis for these phenomena have been established in cell culture systems, there is also evidence for their presence in vivo. This in vivo effect may contribute to increased tumor cell killing, and may also play a role in the abscopal effects of radiation, where radiation responses are seen in areas separated from the irradiated tissue. Although the precise molecular components and mechanisms remain unknown, their discovery will shed new light on the role of the BSEs in radiation risk assessment, and clinical radiotherapy in the clinic.

Persistent oxidative stress and gene expression changes in radiation-induced genomic instability

In addition to the damage caused by the initial exposure to ionizing radiation, it is now known that ionizing radiation induces delayed DNA damage in cells by a process known as radiation-induced genomic instability. Characterized using many different endpoints including increased mutation, frequency, increased level of reactive oxygen species (ROS), and decreased plating efficiency, radiation-induced genomic instability is best characterized in the GM10115 system by chromosomal instability. While the cellular and molecular mechanism of induced instability is not known, we are investigating the hypothesis that clones exhibiting instability display an altered pattern of gene expression. An 1152 gene microarray chip was used to uncover potential gene expression changes in two sets of two unstable clones in comparison to an irradiated but chromosomally stable clone. One candidate gene, Cu/Zn superoxide dismutase 1 (SOD1), was under-expressed in the unstable clones by an average of 3.66-fold by array analysis. Given the high levels of ROS observed in unstable clones, we performed a detailed analysis of SOD1 in four chromosomally unstable cell lines. Northern and western blotting assays could not confirm the microarray data, showing a decrease in expression of SOD1 in only one of four unstable lines, and increases in average protein level in all four unstable clones relative to control. Average SOD1 activity was also elevated in three of four unstable clones tested. These results indicate that while increased ROS levels are characteristic of many of our clones showing instability, this is not due to under-expression of SOD1 as suggested by DNA microarray analysis.

Inhibition of DNA methylation and reactivation of silenced genes by zebularine.

Gene silencing by abnormal methylation of promoter regions of regulatory genes is commonly associated with cancer. Silenced tumor suppressor genes are obvious targets for reactivation by methylation inhibitors such as 5-azacytidine (5-Aza-CR) and 5-aza-2'-deoxycytidine (5-Aza-CdR). However, both compounds are chemically unstable and toxic and neither can be given orally. We characterized a new demethylating agent, zebularine [1-(beta-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one], which is a chemically stable cytidine analog. We tested the ability of zebularine to reactivate a silenced Neurospora crassa gene using a hygromycin gene reactivation assay. We then analyzed the ability of zebularine to inhibit DNA methylation in C3H 10T1/2 Cl8 (10T1/2) mouse embryo cells as assayed by induction of a myogenic phenotype and in T24 human bladder carcinoma cells, using the methylation-sensitive single nucleotide primer extension (Ms-SNuPE) assay. We also evaluated the effects of zebularine (administered orally or intraperitoneally) on growth of EJ6 human bladder carcinoma cells grown in BALB/c nu/nu mice (five mice per group) and the in vivo reactivation of a methylated p16 gene in these cells. All statistical tests were two-sided. In N. crassa, zebularine inhibited DNA methylation and reactivated a gene previously silenced by methylation. Zebularine induced the myogenic phenotype in 10T1/2 cells, which is a phenomenon unique to DNA methylation inhibitors. Zebularine reactivated a silenced p16 gene and demethylated its promoter region in T24 bladder carcinoma cells in vitro and in tumors grown in mice. Zebularine was only slightly cytotoxic to T24 cells in vitro (1 mM zebularine for 48 hours decreased plating efficiency by 17% [95% confidence interval (CI) = 12.8% to 21.2%]) and to tumor-bearing mice (average maximal weight change in mice treated with 1000 mg/kg zebularine = 11% [95% CI = 4% to 19%]). Compared with those in control mice, tumor volumes were statistically significantly reduced in mice treated with high-dose zebularine administered by intraperitoneal injection (P<.001) or by oral gavage (P<.001). Zebularine is a stable DNA demethylating agent and the first drug in its class able to reactivate an epigenetically silenced gene by oral administration.

Radiation-induced genomic instability and delayed activation of p53

Recent studies have described that radiation induces genomic instability, which manifests in the progeny of surviving cells as a persistent decrease in plating efficiency (delayed reproductive death), increased chromosome instability, and increased mutation rate. Despite extensive studies into the process of delayed phenotypes, very little is known about the mechanism of genomic instability. Delayed chromosome instability is characterized by dicentric chromosomes, chromatid gaps and breaks, indicating that delayed DNA breakage is induced in the surviving cells several generations after irradiation. To determine the possibility, we have constructed a reporter plasmid containing the p53-responsible promoter and the β-galactosidase (β-gal) gene, and transfected the plasmid into human fibrosarcoma cells containing the wild-type p53 gene. The cells were irradiated with various doses of X-rays and plated to form the primary colonies. They were collected to perform the secondary colony formation, and we found that the expression of β-gal was frequently detected in the secondary colonies, derived from the primary colonies surviving X-rays. These observations indicate that p53 is frequently activated in the progeny of surviving cells, indicating that delayed DNA damage is responsible for the expression of delayed phenotypes.

Effects of HsRad51 Overexpression on Cell Proliferation, Cell Cycle Progression, and Apoptosis

Expression of the DNA repair and recombination protein human Rad51 (HsRad51) is increased in transformed cells and in cancer cell lines. In order to study the effects of acute HsRad51 ectopic overexpression on cell proliferation, cell cycle progression, and apoptosis, we generated clones of the human fibrosarcoma cell line HT1080 carrying a HsRad51 transgene under a repressible promoter. The HsRad51-overexpressing cells showed decreased plating efficiency and growth rate in a dose-dependent manner with regard to the degree of overexpression. An accumulation of HsRad51-overexpressing cells in G2 was observed following release of cells after synchronization with double thymidine block. Moreover, the fraction of apoptotic cells measured by annexin V-FACS increased with the time of HsRad51 overexpression. In the light of these observations, sustained increased levels of HsRad51 may contribute to tumor progression by causing a selection for cells tolerant to the growth-suppressive and apoptosis-inducing effects of acute HsRad51 overexpression.

Persistent genomic instability in the yeast Saccharomyces cerevisiae induced by ionizing radiation and DNA-damaging agents.

A "hypermutable" genome is a common characteristic of cancer cells, and it may contribute to the progressive accumulation of mutations required for the development of cancer. It has been reported that mammalian cells surviving exposure to gamma radiation display several highly persistent genomic instability phenotypes which may reflect a hypermutability similar to that seen in cancer. These phenotypes include an increased mutation frequency and a decreased plating efficiency, and they continue to be observed many generations after the radiation exposure. The underlying causes of this genomic instability have not been fully determined. We show here that exposure to gamma radiation and other DNA-damaging treatments induces a similar genomic instability in the yeast Saccharomyces cerevisiae. A dose-dependent increase in intrachromosomal recombination was observed in cultures derived from cells surviving gamma irradiation as many as 50 generations after the exposure. Increased forward mutation frequencies and low colony-forming efficiencies were also observed. Persistently elevated recombination frequencies in haploid cells were dominant after these cells were mated to nonirradiated partners, and the elevated recombination phenotype was also observed after treatment with the DNA-damaging agents ultraviolet light, hydrogen peroxide, and ethyl methanesulfonate. Radiation-induced genomic instability in yeast may represent a convenient model for the hypermutability observed in cancer cells.

Short Communication: Salt Extends the Upper Temperature Limit for Growth of Lactococcus lactis ssp. cremoris on Solid M17 Medium

We have determined conditions for plating of the Lactococcus lactis ssp. cremoris laboratory strain MG1363 on solid M17 broth at 38°C, which is required for the optimal use of the pGhost plasmids. The addition of 1% NaCl (or KCl, potassium acetate, or sucrose at 170mM) to M17 agar plates results in extension of the upper temperature limit for growth from 37 to 40°C; no decrease in plating efficiency was detected from 30 to 39°C.

Two distinct mechanisms for inhibition of cell growth in human prostate carcinoma cells with antioxidant enzyme imbalance

The purpose of the present study was to determine whether manganese superoxide dismutase (MnSOD) overexpression in DU145 human prostate carcinoma cells affected cell reduction-oxidation state (cell redox) and to correlate changes in cell redox status with cell cycle progression and plating efficiency. One MnSOD-overexpressing cell line had no change in other antioxidant enzymes (AEs) (nonadapted clone), whereas a second MnSOD-overexpressing cell line studied had an increase in catalase (CAT) activity (adapted clone). Correlation of biochemical studies with cell cycle studies suggested that heteroploidy observed in the nonadapted MnSOD-overexpressing cell line may be due to increased intracellular peroxides with resultant disruption of the microtubule network, while a decreased mitotic rate was associated with decreased ATP levels in mitosis. In contrast, the decrease in cell growth in the adapted cell line was demonstrated to be due to a decrease in plating efficiency. Our results demonstrate complex effects of AE imbalance on cell growth of DU145 prostate cancer cells.

Expression of a kinase‐deficient IGF‐I‐R suppresses tumorigenicity of rhabdomyosarcoma cells constitutively expressing a wild type IGF‐I‐R

Previous results have shown that the insulin-like growth factor type I receptor (IGF-I-R) plays a critical role in the control of rhabdomyosarcoma (RMS) growth. The purpose of this study was to investigate whether a mutated IGF-I-R, when expressed in RMS cells, may interfere with the function of the endogenous wild-type IGF-I-R. We also examined whether the expression of a mutated IGF-I-R may induce phenotypic changes in RMS cells. We used here the mutated IGF-I-R with a lysine to arginine residue 1003 substitution, called IGF-I-KR, which carries a mutation in the ATP-binding domain of the intracellular β subunit, while the extracellular, ligand binding α subunit remains unchanged. We observed that the expression of this mutated IGF-I-KR markedly decreased the response of RMS cells to stimulation with IGF-I. While stimulation with IGF-I increases the autophosphorylation of IGF-I-R in the parent cells, stimulation with IGF-I failed to produce a comparable increase in autophosphorylation in the cells expressing the mutated IGF-I-KR. We also observed a decreased plating efficiency of cells expressing the mutated IGF-I-KR. Consistently, a decrease of RMS growth in vivo was observed in an animal model. Our data suggest that the IGF/IGF-I-R signaling pathway may be inhibited by expressing a mutated IGF-I-KR and that such a mutant gene could be utilized in developing novel therapeutic strategies to suppress RMS growth. Int. J. Cancer 76:223–227, 1998. Published 1998 Wiley-Liss, Inc.1 This article is a US Government work and, as such, is in the public domain in the United States of America.

Expression of a kinase-deficient IGF-I-R suppresses tumorigenicity of rhabdomyosarcoma cells constitutively expressing a wild type IGF-I-R.

Previous results have shown that the insulin-like growth factor type I receptor (IGF-I-R) plays a critical role in the control of rhabdomyosarcoma (RMS) growth. The purpose of this study was to investigate whether a mutated IGF-I-R, when expressed in RMS cells, may interfere with the function of the endogenous wild-type IGF-I-R. We also examined whether the expression of a mutated IGF-I-R may induce phenotypic changes in RMS cells. We used here the mutated IGF-I-R with a lysine to arginine residue 1003 substitution, called IGF-I-KR, which carries a mutation in the ATP-binding domain of the intracellular beta subunit, while the extracellular, ligand binding alpha subunit remains unchanged. We observed that the expression of this mutated IGF-I-KR markedly decreased the response of RMS cells to stimulation with IGF-I. While stimulation with IGF-I increases the autophosphorylation of IGF-I-R in the parent cells, stimulation with IGF-I failed to produce a comparable increase in autophosphorylation in the cells expressing the mutated IGF-I-KR. We also observed a decreased plating efficiency of cells expressing the mutated IGF-I-KR. Consistently, a decrease of RMS growth in vivo was observed in an animal model. Our data suggest that the IGF/IGF-I-R signaling pathway may be inhibited by expressing a mutated IGF-I-KR and that such a mutant gene could be utilized in developing novel therapeutic strategies to suppress RMS growth. 1998.

SEEKING POTATO CLONES WITH ELEVATED TUBER METHIONINE USING PROTOPLAST REGENERATION PROTOCOLS

`Russet Burbank' leaf protoplasts were cultured in cell layer (CL) media containing the amino acid analog, ethionine (ETN), at concentrations ranging from 0 to 9 μm. A decrease in plating efficiency was observed when ETN concentrations in CL exceeded 3 μm. Calli that developed were placed on C media containing ETN concentrations up to 200 μm. Regenerated plants were grown to maturity in a growth chamber and resulting tubers were analyzed for free methionine. Selected protoclones produced tubers whose free methionine content exceeded that of the `Russet Burbank' control by ≈3-fold. Methodology used will be discussed.

The effect of tirapazamine (SR-4233) alone or combined with chemotherapeutic agents on xenografted human tumours.

Recent data have shown that the in vitro and in vivo cytotoxicity of bioreductive drugs could be significantly increased when combined with chemotherapy drugs such as cisplatinum, depending on the timing of administration. The aim of this study was to define the toxicity (animal lethality) and the activity (growth delay assay, excision assay) of a bioreductive drug, tirapazamine, alone and combined with chemotherapy agents (5-FU, VP16, bleo, DTIC and c-DDP) on nude mice bearing xenografted human tumours: a rectal carcinoma (HRT18) and a melanoma (Na11+). Animal lethality was markedly increased when tirapazamine at the lethal dose 10% was combined with the other drugs. For the HRT18 tumour the combination of tirapazamine and bleomycin significantly increased the delay of regrowth compared with bleomycin alone (P = 0.04) and was more cytotoxic than tirapazamine alone (P = 0.04). For the Na11+ tumours the combination of tirapazamine with VP16 significantly increased tumour doubling time compared with the controls (P = 0.001) or VP16 alone. The combination of tirapazamine and VP16 was more cytotoxic than VP16 alone (P = 0.0001). When compared with c-DDP or tirapazamine alone, there was a significant decrease in plating efficiency when tirapazamine and c-DDP were given at the same time (P = 0.04), but not when tirapazamine was given 3 h before c-DDP. In conclusion, tirapazamine was shown to be cytotoxic against clonogenic human tumour cells. Its efficacy in vivo may depend on its combination with already active chemotherapy drugs on the tumour model used. The timing of administration may be less important than previously thought.

Growth and pigmentation in genetically related Cloudman S91 melanoma cell lines treated with 3‐isobutyl‐1‐methyl‐xanthine and β‐melanocyte‐stimulating hormone

4 clonal sublines of Cloudman S91 melanoma cells, S91/mel, S91/I3, S91/6 and S91/amel, were evaluated for changes in growth, pigment content and plating efficiency during and after treatment with a cyclic-AMP phosphodiesterase inhibitor-melanin-stimulating agent, 3-isobutyl-1-methyl-xanthine (IBMX) plus beta-melanocyte stimulating hormone (beta-MSH) or IBMX alone. After combined treatment, increases in melanin content on day 3 were 48, 27, 11, and 2 pg/cell in the four cell lines respectively. In each case IBMX alone was less effective than IBMX plus beta-MSH. Doubling time increased and plating efficiency decreased with increased melanization. The increases in doubling time and decreases in plating efficiency were cell line dependent. The greatest rate of increase in doubling time and decrease in plating efficiency as a function of melanin content were seen in S91/amel, which produced the least pigment. The lowest rates of increase/decrease were seen in S91/mel, which produced the most pigment. Melanin pigment induced in the cells was classified as eumelanin by EPR determination. The differential response to induction of pigmentation makes these cell lines suitable models for comparative studies on the role of melanin in pigment cell biology.

Borrelia burgdorferi mutant lacking Osp: biological and immunological characterization

All Borrelia burgdorferi sensu lato isolates characterized to date have one or a combination of several major outer surface proteins (Osps). Mutants of B. burgdorferi lacking Osps were selected with polyclonal or monoclonal antibodies at a frequency of 10(-6) to 10(-5). One mutant that lacked OspA, -B, -C, and -D was further characterized. It was distinguished from the OspA+B+ cells by its (i) autoaggregation and slower growth rate, (ii) decreased plating efficiency on solid medium, (iii) serum and complement sensitivity, and (iv) diminished capacity to adhere to human umbilical vein endothelial cells. The Osp-less mutant was unable to evoke a detectable immune response after intradermal live cell immunization even though mutant survived in mouse skin for the same duration as wild-type cells. Polyclonal mouse serum raised against Osp-less cells inhibited growth of the mutant but not of wild-type cells, an indication that other antigens are present on the surface of the Osp-less mutant. Two types of monoclonal antibodies (MAbs) with growth-inhibiting properties for mutant cells were identified. The first type bound to a 13-kDa surface protein of B. burgdorferi sensu stricto and of B. afzelii. The MIC of the Fab fragment of one MAb of this type was 0.2 micrograms/ml. The second type of MAb to the Osp-less mutant did not bind to B. burgdorferi components by Western blotting (immunoblotting) but did not bind to unfixed, viable cells in immunofluorescence and growth inhibition assays. These studies revealed possible functions Osp proteins in borrelias, specifically serum resistance, and indicated that in the absence of Osp proteins, other antigens are expressed or become accessible at the cell surface.

Xenobiotic metabolizing enzyme activities and viability are well preserved in EDTA-isolated rat liver parenchymal cells after cryopreservation.

Rat liver parenchymal cells (PC) were isolated by EDTA perfusion and were purified by a subsequent Percoll centrifugation. The isolated PC had a viability of 95%, as judged by trypan blue exclusion. Freshly isolated PC were cryopreserved with an optimized protocol in a computer-controlled freezer. After thawing, the PC still retained a viability of 89%. The activities of representative xenobiotic metabolizing enzymes were compared between freshly isolated and cryopreserved PC after thawing. The cytochrome P450 content and the cytochrome P450 2C11 isoenzyme activity, determined by hydroxylation of testosterone in intact cells, were not affected by the cryopreservation. The following phase II enzyme activities were also well maintained after cryopreservation: Phenol sulfotransferase (92%), 1-naphthol UDP-glucuronosyl transferase (95%), soluble epoxide hydrolase (87%), and glutathione S-transferase (88%), determined with broad spectrum substrate 1-chloro-2,4-dinitrobenzene. However, there was a significant decrease in plating efficiency between freshly isolated (86%) and cryopreserved (57%) PC when they were cultured. The initial quality of the freshly isolated PC is decisive for the success of cryopreservation. These results support the use of cryopreserved PC in pharmacology and toxicology with the aim to reduce the number of experimental animals used.

Plant Regeneration from Protoplast-Derived Somatic Embryos of Asparagus officinalis L.

Summary A method for regenerating plants from embryogenic calli derived from protoplasts of asparagus is described. The protoplasts were isolated from calli of a selected crown of the genotype G-203 of Asparagus officinalis L. Optimum protoplast yield was obtained from calli of 8 to 10 d of age after subculture following enzymatic digestion. Of the isolated protoplasts 80 % to 90 % were viable. The highest plating efficiency (12.45 %) was obtained when cells were cultured at a density of 1 × 10 5 protoplasts/mL in 1/2 strength of Murashige and Skoog (MS) medium with 1 mg/L NAA, 0.5 mg/L zeatin, 0.5mg/L folic acid, 0.05 mg/L biotin, 1 g/L glutamine, 0.2 g/L casein hydrolysate, 0.6 M glucose, 0.1 M mannitol, and 0.1 % (w/v) Gelrite. A remarkable decrease in plating efficiency was recorded in the presence of both spermine and spermidine in the culture medium. Colonies were visible after 30 d and grew consistently into slimy and friable embryogenic calli after transferring them onto MS medium containing 1 mg/L 2,4-D, 3 % (w/v) sucrose and 0.2 % (w/v) Gelrite. Somatic embryos were induced from such calli when cultured on 1/2 MS medium with only 1.% (w/v) sucrose devoid of any growth hormones. After transplanting on 1/2 MS medium with 1 % (w/v) sucrose and 1 mg/L GA3, 40–45 % of embryos germinated normally and grew into plantlets within 30 d. These plantlets from protoplast-derived somatic embryos were diploid revealing 2n = 20 chromosomes.

Clonal heterogeneity in the progeny of HeLa cells which survive X-irradiation.

Clones of HeLa cells that had survived 9 Gy X-rays were investigated for plating efficiency, radiosensitivity, the proportion of giant cells, and chromosome numbers. A significant decrease in plating efficiency was observed that persisted for > 20 population doublings. The reduced plating efficiency was associated with an increased proportion of giant cells in the colonies. There was pronounced clonal heterogeneity in the progeny of irradiated surviving cells with respect to plating efficiency, proportion of giant cells, and chromosome numbers. The radiosensitivity of progeny from irradiated cells did not change compared with controls.

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.

Development and initial characterization of a mitomycin C-resistant colon cancer cell line variant

Resistance may limit the clinical usefulness of a variety of chemotherapeutic drugs, including mitomycin C (MMC). In order to study resistance to MMC, a variant of the HT-29 human colon cell line was isolated by exposure to repeated doses of MMC. The 95% inhibitory concentration of MMC for this isolate (HT-29R13) was found to be approximately twice that for the parent line. The level of resistance did not increase with additional drug exposure, and resistance was stable for at least 6 months in the absence of drug exposure. HT-29R13 cells exhibit cross-resistance to melphalan and 5-FU but not to doxorubicin, cis-platinum, or etoposide. HT-29R13 cells are characterized by slightly decreased plating efficiency and slightly increased total protein compared with the parent line. This model of stable, low-level MMC resistance with an unusual cross-resistance pattern may prove useful for the study and characterization of MMC resistance mechanisms.

A potential pitfall in the use of electroporation: cellular radiosensitization by pulsed high-voltage electric fields.

Chinese hamster ovary cells have been exposed to high-voltage electric fields causing electroporation (EP) and the interaction between EP and radiation-induced cell lethality investigated. There was a voltage-dependent decrease in plating efficiency, assessed immediately following EP, and cell viability, assessed at 24 h. A linear decrease was seen for both. These decreases were accompanied by a voltage-dependent increase in cell volume, assessed immediately following EP. A good correlation between increases in cell volume and decreases in plating efficiency was seen (r = -0.91). The application of electric fields immediately prior to, or following, irradiation led to a radiosensitization of the cells. This radiosensitization still occurred when a 6 h interval was left between radiation and EP but was lost when cells were irradiated 24 h prior to EP. When cells were irradiated following EP, the radiosensitization was lost with a 1 h interval between the two treatments. These results suggest that, when studying the combined cellular effects of EP of macromolecules and radiation, care should be taken that sufficient time has elapsed between the two modalities to prevent the radiosensitization of cells.