Irradiated HeLa Research Articles

Investigation on the Direct and Bystander Effects in HeLa Cells Exposed to Very Low α-Radiation Using Electrical Impedance Measurement.

The impact of radiation-induced bystander effect (RIBE) is still not well understood in radiotherapy. RIBEs are biological effects expressed by nonirradiated cells near or far from the irradiated cells. Most radiological studies on cancer cells have been based on biochemical characterization. However, biophysical investigation with label-free techniques to analyze and compare the direct irradiation effect and RIBE has lagged. In this work, we employed an electrical cell-indium tin oxide (ITO) substrate impedance system (ECIIS) as a bioimpedance sensor to evaluate the HeLa cells’ response. The bioimpedance of untreated/nonirradiated HeLa (N-HeLa) cells, α-particle (Am-241)-irradiated HeLa (I-HeLa) cells, and bystander HeLa (B-HeLa) cells exposed to media from I-HeLa cells was monitored with a sampling interval of 8 s over a period of 24 h. Also, we imaged the cells at times where impedance changes were observed. Different radiation doses (0.5 cGy, 1.2 cGy, and 1.7 cGy) were used to investigate I-HeLa and B-HeLa cells’ radiation-dose-dependence. By analyzing the changes in absolute impedance and cell size/number with time, compared to N-HeLa cells, B-HeLa cells mimicked the I-HeLa cells’ damage and modification of proliferation rate. Contrary to the irradiated cells, the bystander cells’ damage rate and proliferation rate enhancements have an inverse radiation-dose-response. Also, we report multiple RIBEs in HeLa cells in a single measurement and provide crucial insights into the RIBE mechanism without any labeling procedure. Unambiguously, our results have shown that the time-dependent control of RIBE is important during α-radiation-based radiotherapy of HeLa cells.

Radiosensitizing and anti-proliferative effects of resveratrol in two human cervical tumor cell lines

Resveratrol is a polyphenol isolated from the skins of grapes that has been shown to significantly alter the cellular physiology of tumor cells, as well as block the process of initiation and progression. At least one mechanism for the intracellular actions of resveratrol involves the suppression of prostaglandin (PG) biosynthesis. The involvement of PGs and other eicosanoids in the development of human cancer is well established. PGs are synthesized from arachidonic acid via the cyclooxygenase pathway and have multiple physiological and pathological functions. In addition, evidence has arisen suggesting that PGs may be implicated in the cytotoxic and/or cytoprotective response of tumor cells to ionizing radiation (IR). As such, we hypothesized that tumor cells may exhibit changes in the cellular response to IR following exposure to resveratrol, a naturally occuring compound that inhibits cyclooxygenase-1 (COX-1) activity. Thus, clonogenic cell survival assays were performed using irradiated HeLa and SiHa cells pretreated with resveratrol prior to IR exposure, and resulted in enhanced tumor cell killing by IR in a dose-dependent manner. Further analysis of COX-1 inhibition indicated that resveratrol pretreatment: (1), inhibited cell division as assayed by growth curves; and (2), induced an early S phase cell cycle checkpoint arrest, as demonstrated by fluorescence-activated cell sorting, as well as bromodeoxyuridine pulse-chase analysis. These results suggest that resveratrol alters both cell cycle progression and the cytotoxic response to IR in two cervical tumor cell lines.

Kinetics of induction of reactive oxygen species during the post-irradiation expression of neoplastic transformation in vitro

Purpose : To examine whether the delayed expression of cell death and neoplastic foci in irradiated HeLa ×human skin fibroblast human hybrid cells correlates with the presence of reactive oxygen species (ROS) in the progeny of the irradiated cells. Material and methods : HeLa ×human skin fibroblast human hybrid cells were irradiated and plated as for assay of neoplastic transformation. At regular time intervals during the post-irradiation expression period of 21 days, samples were harvested and assessed for the presence of oxyradical activity by measuring the oxidation of dichlorohydrofluorescein (DCFH) to dichlorofluorescein (DCF) using flow cytometry. This reagent principally detects hydrogen peroxide. The kinetics of production of ROS were compared with those previously measured using the identical experimental protocol for the expression of both cell death and neoplastic foci. Results : The kinetics of production of ROS closely matched those for the onset of delayed cell death and delayed expression of neoplastic transformation. Conclusions : The induction of ROS is associated with delayed cell death and delayed neoplastic transformation in irradiated HeLa ×human skin fibroblast human hybrid cells.

Solar UV radiation: differential effectiveness of UVB subcomponents in causing cell death, micronucleus induction and delayed expression of heritable damage in human hybrid cells

Purpose : To determine the effectiveness of two UV spectra with different UVB components for cell kill and micronucleus induction in irradiated human HeLa ×skin fibroblast (CGL1) hybrid cells and their progeny. To determine the presence of reactive oxygen species (ROS) in the progeny of the irradiated cells at various post-irradiation times and their relationship with induced delayed biological effects. Material and methods : A commercial solar ultraviolet simulator was used. Two different filters were employed: the first transmitted radiation with λ>284nm and the second radiation with λ>293nm. The resulting spectra have different UVB components (λbetween 284 and 320nm, 19 W/m 2, and between 293 and 320nm, 13 W/m 2) and the same UVA component (λbetween 320 and 400nm, 135 W/m 2) . CGL1 cells were irradiated with various doses. Clonogenic survival and micronucleus formation were scored in the irradiated cells and their progeny. ROS were detected by incubation of cultures at various post-irradiation times with dichlorodihydrofluorescein diacetate followed by flow cytometric measurement of the final product, dichlorofluorescein. Results : The biological effectiveness of the λ>284nm spectrum was higher by a factor of 3 compared to the λ>293nm spectrum for cell kill, and by a factor of 5 for micronucleus induction. No delayed cell death or micronucleus formation was found in the progeny of cells exposed to λ>293nm, while a large and dose-dependent effect was found in the progeny of cells exposed to λ>284nm for both of these endpoints. ROS levels above those in unirradiated controls were found only in the progeny of cells exposed to the λ>284nm spectrum. Conclusions : The spectrum with λ>284nm was more effective than that with λ>293nm for induction of cell kill and micronucleus formation in the directly irradiated cells as well as induction of delayed effects in the progeny in the form of delayed reproductive death and micronucleus formation. The presence of ROS in the progeny of the irradiated cells may be the cause of the delayed effects.

Regulation of DNA Replication in Irradiated Cells by Trans-Acting Factors

We compared DNA replication activity in cytoplasmic extracts prepared from irradiated and nonirradiated HeLa cells using a simian virus 40 (SV40)-based in vitro replication assay. The assay measures semi-conservative DNA replication in a plasmid carrying the SV40 origin of replication and requires SV40 T antigen as the sole noncellular protein. The plasmid DNA used in the replication reaction is never exposed to radiation. We find that replication of plasmid DNA is significantly reduced when cytoplasmic extracts from irradiated cells are used. Since plasmid replication proceeds to completion in extracts from irradiated cells, the observed reduction in the over-all replication activity is probably due to a reduction in the efficiency of initiation events. The degree of inhibition of DNA replication after exposure to 10, 30 and 50 Gy X rays as measured in vitro using this assay is similar to that measured in intact cells immediately before processing for extract preparation. These observations are compatible with the induction or activation by ionizing radiation of a factor(s) that inhibits in trans DNA replication. The results contribute to our understanding of the mechanism(s) developed by the cells to regulate DNA replication when exposed to clastogenic agents. Such processes may be of significance in the restoration of DNA integrity, and may define yet another checkpoint operating during S at the level of clusters of replicons.

Radiation-Induced Association of β-Glucuronidase with Purified Nuclei from Irradiated MOLT-4 and HeLa Cells

Beta-glucuronidase, a lysosomal marker enzyme, associates with purified nuclei from HeLa and MOLT-4 cell lines in a radiation dose-dependent manner, up to 300 cGy in MOLT-4 cells, and 1000 cGy in HeLa cells. In MOLT-4 cells (200-cGy exposure), there is a significant increase in beta-glucuronidase activity detected in the nuclear fraction 24 h postirradiation with a maximum association occurring at 72 h. In HeLa cells (1000-cGy exposure), a significant association is first detected 24 h postirradiation with a maximum association at 48 h. The association is not the result of nonspecific contamination occurring during nuclei purification since nuclei from irradiated cells show no greater levels of plasma membrane marker and mitochondrial marker than controls. The nature of the association remains unclear, but activity is not removed by detergents used in the nuclei isolation procedure, and incubation of the nuclei with EDTA reverses the association only modestly. Exposure of nuclei from irradiated cells to anisotonic buffers also results in only a small decrease in beta-glucuronidase activity associated with the nuclei. These observations suggest that lysosomal hydrolases become intimately associated with the nuclei of irradiated cells.

Intracellular radiation of HeLa cells by tritiated thymidine. I. Lactic acid production and exponential growth.

SummaryThe production of lactic acid by irradiated and unirradiated HeLa cells, grown as monolayer, was investigated. As long as exponential growth was maintained, the lactic acid production was proportional to the increase in cell number, and the production per hour and per cell was constant. When tritiated thymidine was added to the medium, a significant increase of 23 per cent of the lactic acid production per hour and per cell was found for the highest doses. The findings are discussed with regard to the proposed equations and related to the calculation methods of other workers.

Temperature dependence of thymidine uptake and ability of thymidine incorporated during unscheduled DNA synthesis in irradiated and unirradiated HeLa cells.

Temperature dependence of thymidine uptake and ability of thymidine incorporated during unscheduled DNA synthesis in irradiated and unirradiated HeLa cells.