Prediction of radiosensitivity in human bladder cell lines using nuclear chromatin phenotype.

BACKGROUND: Nuclear texture analysis measures phenotypic changes in chromatin distribution within a cell nucleus, while the alkaline Comet assay is a sensitive method for measuring the extent of DNA breakage in individual cells. The authors aim to use both methods to provide information about the sensitivity of cells to ionizing radiation. METHODS: The alkaline Comet assay was performed on six human bladder carcinoma cell lines and one human urothelial cell line exposed to gamma-radiation doses from 0 to 10 Gy. Nuclear chromatin texture analysis of 40 features was then performed in the same cell lines exposed to 0, 2, and 6 Gy to explore if nuclear phenotype was related to radiation sensitivity. RESULTS: Comet assay results demonstrated that the cell lines exhibited different levels of radiosensitivity and could be divided into a radiosensitive and a radioresistant group at >6 Gy. Using stepwise discriminant analysis, a subset of important nuclear texture features that best discriminated between sensitive and resistant cell lines were identified A classification function, defined using these features, correctly classified 81.75% of all cells into their radiosensitive or radioresistant groups based on their pretreatment chromatin phenotype. Posttreatment chromatin changes also varied between cell lines, with sensitive cell lines showing a relaxed chromatin conformation following radiation, whereas resistant cell lines exhibited chromatin condensation. CONCLUSIONS: The authors conclude that the alkaline Comet assay and nuclear texture methodologies may prove to be valuable aids in predicting the response of tumor cells to radiotherapy.     

Establishment and characterization of two fetal fibroblast cell lines from the yak

The objective of this work was to not only establish two fetal fibroblast cell lines from yak lung and ear tissue using a primary explant technique and cell cryogenic preservation technology but also check for their quality and biological characteristics. The cells showed typical morphologic characteristics of fibrous and long spindle appearance. Outgrowth of fibroblast-like cells from the lung and ear explants was around 2 and 3 d, and reaching 90% confluence level was in the ninth day and the thirteenth day, respectively. Biological analysis showed that the average viability of the lung fibroblast cells (ear fibroblast cells) was 97.5% (95.0%) before freezing and 91.0% (89.5%) after thawing. Analysis of the growth of the fifth passage culture revealed an ??S??-shaped growth curve with the population doubling times of 30 h for lung fibroblast cell line and 35 h for ear fibroblast cell line. Karyotyping indicated the chromosome number of yak was 2n?=?60, comprising 29 pairs of autosomes and one pair of sex chromosomes (XY). All somatic chromosomes were telocentric autosomes except that the two sex chromosomes were submetacentric. Assays for bacteria, fungi, and mycoplasmas were negative. Immunocytochemical staining showed that the cells were positive for the expression of vimentin and negative for the expression of cytokeratin. In conclusion, two yak fetal fibroblast cell lines (YFLF and YFEF) from lung and ear explants are successfully established in culture. It will not only preserve the genetic resources of yaks at the cellular level but also provide valuable materials for somatic cell cloning and transgenic research.     

Structural change in the cell center during fibroblast polarization and movement

In polarizing and migrating 3T3/Balb mouse fibroblasts, the centrioles are located between the nucleus and the leading edge of the cell. In cells within the monolayer and in migrating cells, the centrioles have a random orientation towards the substrate. In polarized cells, that still remain in the monolayer, one centriole may be perpendicular to the substrate plane in 70% of cases. Upon polarization and migration of fibroblasts, the number of microtubules, which radiate from the centriolar region, increases. These data support a hypothesis that the number of microtubules in the cell centre characterizes the rate of their renovation in the cytoplasm. It is concluded that the cell centre is strongly involved in polarization and migration of fibroblasts.     

Evidence for removal at different rates of O-ethyl pyrimidines and ethylphosphotriesters in two human fibroblast cell lines.

The potent carcinogen, ethylnitrosourea, has been shown to ethylate oxygens, in preference to nitrogens, in the DNA of cultured cells. We have now studied the removal of seven ethyl derivatives in replicating cells. The following findings are reported. 1) The absolute amounts of 02-EtT, 04-EtT and 02-EtC are decreased in cellular DNA after correction for cell growth. However the rate of decrease diminishes after approximately 20 hr and after more than two cell doublings 20--40% of each derivative persists. This decrease is presumed to be due to enzymes since these derivatives are stable in isolated DNA. 2) The amount of ethyl phosphotriesters remains almost unchanged during 72 hr of cell culture. 3) The unstable purine derivatives, 7-EtG and 3-EtA, are both removed from cellular DNA with a rate faster than can be accounted for by the lability of the glycosyl bond. 4) Both GM 637 fibroblasts and Xeroderma pigmentosum fibroblasts (12-RO) (XP-12) have similar ability to remove ethyl products, except for O6-ethyl G which persists to a greater extent in XP12 cells. 5) The implications of the in vivo persistence of ethylated bases is discussed in regard to recent demonstrations that O2-EtT, O4-ET, O2-EtC and O6-EtG are all mutagenic.     

Increased radiosensitivity in cells of two human cell lines treated with bystander medium from irradiated repair-deficient cells

Radiation-induced bystander factors have been shown to be more toxic if they are from medium harvested from irradiated repair-deficient cells. The aim of this study was to test the hypothesis that the radiosensitivity of repair-proficient cells can be increased by exposing them to medium-borne factors harvested from sensitive cells and vice versa. Cells from a mismatch repair (MMR)-deficient cell line (Raji 10) with a sensitive response to radiation or the wild-type parent cell line were irradiated to 0.5 Gy gamma rays and then monitored for growth rate in their own medium or in the alternative conditioned medium. In other experiments, cells or conditioned medium were added to reporter cells (HPV-G, which are relatively sensitive keratinocytes, or highly radioresistant HT29 cells). The subsequent responses of the two cell lines to a 0.5-Gy dose of (60)Co gamma rays were measured. The results show that prior exposure of resistant cells to medium from irradiated sensitive cells reduced the clonogenic survival of the subsequently irradiated resistant cells. The reverse is also true. Measurement of the apoptosis index and BCL2 expression confirmed that the harvested medium was capable of modulating apoptosis after irradiation. This may have important applications in tumor therapy and also in the understanding of mechanisms involved in induction of adaptive responses.     

The cytogenetic damage, radiosensitivity and adaptive response in children living in the different districts of Moscow

The spontaneous level of blood lymphocytes with micronuclei (MN), the sensitivity to 1.0 Gy irradiation and adaptive response (AR) after adaptive irradiation with a dose of 0.05 Gy 5 hr later have been studied in children population living in different districts of Moscow. It was shown that spontaneous frequency of cells with MN, the sensitivity to 1.0 Gy acute irradiation and the AR manifestation have significant differences in samples taken from children living in different districts. The individual variability is significant also. In each group of children the individuals with the enhanced radiosensitivity after adaptive irradiation have been observed. In conformance with the data of radioecological inspection the radiation situation in different Moscow districts is quite safe on overage but in some districts the spontaneous level of lymphocytes with MN, and radiosensitivity after 0.05 Gy irradiation were enhanced, the AR was not found.     

Structural rearrangements of tubulin and actin during the cell cycle of the yeast Saccharomyces

The distribution of actin and tubulin during the cell cycle of the budding yeast Saccharomyces was mapped by immunofluorescence using fixed cells from which the walls had been removed by digestion. The intranuclear mitotic spindle was shown clearly by staining with a monoclonal antitubulin; the presence of extensive bundles of cytoplasmic microtubules is reported. In cells containing short spindles still entirely within the mother cells, one of the bundles of cytoplasmic microtubules nearly always extended to (or into) the bud. Two independent reagents (anti-yeast actin and fluorescent phalloidin) revealed an unusual distribution of actin: it was present as a set of cortical dots or patches and also as distinct fibers that were presumably bundles of actin filaments. Double labeling showed that at no stage in the cell cycle do the distributions of actin and tubulin coincide for any significant length, and, in particular, that the mitotic spindle did not stain detectably for actin. However, both microtubule and actin staining patterns change in a characteristic way during the cell cycle. In particular, the actin dots clustered in rings about the bases of very small buds and at the sites on unbudded cells at which bud emergence was apparently imminent. Later in the budding cycle, the actin dots were present largely in the buds and, in many strains, primarily at the tips of these buds. At about the time of cytokinesis the actin dots clustered in the neck region between the separating cells. These aspects of actin distribution suggest that it may have a role in the localized deposition of new cell wall material.     

Differential response of two derivatives of the BHK21 hamster cell to thymidine

Attempts to synchronize the BHK21 hamster cell C-13 and its polyoma-transformed derivative P-183 with excess thymidine resulted in the observation that the parent cell line could be readily synchronized but the transformed derivative could not. Differences in the growth pattern indicate that excess thymidine (10 mM) stops progress of the virus-transformed derivative at all stages in the life cycle rather than exclusively in S. The data are suggestive but do not establish that the difference is a result of the presence of the virus genome.     

Heat-induced modulation of lamin B content in two different cell lines

Previous work in our laboratory indicates that the nuclear matrix protein lamin B is a "prompt" heat shock protein, which increases significantly when human U-1 melanoma and HeLa cells are exposed to 45.5 degrees C for 5-40 min. Using Western blotting, we found that the lamin B content in U-1 and HeLa cells increased to a greater extent during post-heat incubation at 37 degrees C than during the heat dose itself. When HeLa cells were heated at 45.5 degrees C for 30 min, and then incubated at 37 degrees C for up to 7 h, lamin B content was increased significantly (1.69-fold maximum increase at 3 h) compared to unincubated heated cells. Also, thermotolerant HeLa cells showed a greater increase (up to 1.72-fold) in lamin B content during subsequent heating compared to nontolerant cells. The increase in lamin B content in thermotolerant cells, or when heated cells were incubated at 37 degrees C, was also observed in U-1 cells. HeLa cells heated in the presence of glycerol (a heat protector) showed a 1.21-1.72-fold increase in lamin B content compared to cells heated for 10-30 min without glycerol. In contrast, lamin B content decreased 1.23-1.85-fold when cells were heated for 10-30 min in the presence of procaine (a heat sensitizer) compared to cells heated without procaine. These data suggest that lamin B may play an important role in the heat shock response, and that modulation of lamin B content by heat sensitizers or protectors may play a role in regulation of heat sensitivity.     

Activities of curcumin-related compounds in two cell lines persistently infected with different prion strains

BackgroundCultured cell lines infected with prions produce an abnormal isoform of the prion protein (PrP^Sc). In this study, two types of cells persistently infected with prion were treated with curcumin-related compounds. We found that the compounds behave differently in neuroblastoma neuro-2a (N2a) cells infected with different prion strains.MethodsCurcumin and related compounds were applied to the two types of persistently prion infected cells to analyze the different activities of the compounds.ResultsIn ScN2a cells, which were infected with the Rocky Mountain Laboratory prion strain, two of the six compounds significantly reduced the PrP^Sc level in a dose-dependent manner. On the other hand, in N167 cells, effective suppression of the total amount of PrP^Sc was not observed; instead, two other compounds promoted the formation of covalently linked PrP^Sc dimers.ConclusionsChemometric analysis was used to determine the factors that contributed to the different effects of the six compounds. It showed that the ability to form hydrogen bonds, such as phenolic hydroxyl groups, and hydrophobic molecular properties predominantly contributed to the reduction of the PrP^Sc level in the ScN2a cells and the dimer formation of PrP^Sc in the N167 cells, respectively.General significanceThe extracted information can be used to delineate the differences among prion strains and to design compounds that are directed toward their respective activities.