Reproductive Cell Death Research Articles

Radiosensitization by bromodeoxyuridine and hyperthermia: analysis of linear and quadratic parameters of radiation survival curves of two human tumor cell lines.

Sensitization by bromodeoxyuridine (BrdUrd) and hyperthermia (HT) on cell reproductive death induced by ionizing radiation was analyzed using the linear-quadratic [S(D)/S(0)=exp(-(alphaD + betaD2)]] model. Plateau-phase human lung tumor cells (SW-1573) and human colorectal carcinonoma cells (RKO) were treated with BrdUrd, radiation and HT. LQ-analysis was performed at iso-incubation dose and at iso-incorporation level of BrdUrd. and at iso-HT doses and iso-survival levels after HT. Clonogenic assays were performed 24 h after treatment to allow repair of potentially lethal damage (PLD). In SW cells BrdUrd. HT or the combination significantly increased the alpha-parameter (factor 2.0-5.7), without altering the beta-parameter. In RKO cells sensitization with BrdUrd increased both a (factor 1.4) and beta (factor 1.3) while HT only influenced beta (factor 2.1-4.0). The combination did not further increase the a and beta. The results indicate that BrdUrd has its main effect on the parameter alpha, dominant at clinically relevant radiation doses but that HT can affect both a and beta. The addition of BrdUrd and HT provides a method to enhance the efficacy of radiotherapy.

Radiation-induced Genomic Instability

Genomic instability induced by radiation is characterized by the increased rate of acquisition of genetic alter-ations in the progenies of irradiated cell.These changes encompass a diverse set of biological end points,such as kary-otypic abnormolities,gene mutation and amplification,and delayed reproductive cell death etc.Damages of some key genes play important roles in the initiation and transition of genomic instability.Epigenetic factors may influence the on-set of genomic instability.Loss of stability of the genome is one of the important aspects of radiation carcinogenesis.

Additive cytotoxic effect of cisplatin and X-irradiation on human glioma cell cultures derived from biopsy-tissue.

Investigation of the in vitro cytotoxic effect of X-rays, either alone or combined with cisplatin on early passage cell cultures derived from human glioblastoma multiforme biopsy tissue. Fresh tumour specimens from four patients were processed to cell cultures. The U373 glioma cell line was used as a reference. Early passage cell cultures were X-irradiated (0-8 Gy) either alone or in combination with cisplatin (0.5-1 microgram/ml). Cell survival was determined by either clonogenic assay or the colorimetric MTT assay. Survival curves were generated and mathematically analysed using the linear quadratic model, to obtain the radiosensitivity parameters alpha, beta, and SF2, i.e., the Surviving Fraction after 2 Gy. Two patient-derived glioma cell cultures and the U373 cell line showed rather high SF2 values of 0.61-0.72 in the clonogenic assay, indicating relative high radiation resistance. Cisplatin alone (1 microgram/ml) reduced cell survival by 10-30% (n = 4). When combined with irradiation, a clear additive cytotoxic effect of cisplatin was demonstrated by the unaltered value of the alpha-parameter for reproductive cell death. Cisplatin exerted an additive rather than radiosensitising cytotoxic effect in uncharacterised patient derived glioma cell cultures.

N–3 and n–6 polyunsaturated fatty acids induce cytostasis in human urothelial cells independent of p53 gene function

The role of long-chain polyunsaturated fatty acids (PUFA) in the etiopathology and treatment of cancer is poorly understood. We have studied the effects of n;-3 and n;-6 PUFA on the proliferation and survival of normal human uroepithelial (NHU) cells, cells with disabled p53 function after stable transfection with the human papillomavirus 16 (HPV16) E6 gene (HU-E6), and p53-disabled cells that had passed through crisis and acquired karyotypic abnormalities (HU-E6P). The n;-3 and n;-6 PUFA had distinct reversible antiproliferative and irreversible cytostatic effects according to concentration and exposure time. The reversible antiproliferative effect was partly due to the production of lipoxygenase metabolites. NHU and HU-E6 cells were equally sensitive to n;-3 and n;-6 PUFA, but HU-E6P cells were more resistant to both the antiproliferative and cytostatic effects. Cytostatic concentrations of n;-3 and n;-6 PUFA did not induce apoptosis, but caused permanent growth arrest ("interphase" or "reproductive" cell death) and mRNA levels for genes involved in cell cycle control (p21, p16, p27, cdk1, cdk2, and cdk4) were not altered. Neither n;-3 nor n;-6 PUFA promoted acquisition of karyotypic abnormalities in HU-E6 cells, suggesting that n;-3 and n;-6 PUFA do not cause genotoxic damage. In conclusion, our studies show that the antiproliferative and cytostatic effects of n;-3 and n;-6 PUFA are not dependent on p53 function and, further, that transformation results in a loss of sensitivity to n;-3 and n;-6 PUFA-mediated growth inhibition.

Relative biological effectiveness for cell-killing effect on various human cell lines irradiated with heavy-ion medical accelerator in Chiba (HIMAC) carbon-ion beams

Purpose: To clarify the relative biological effectiveness (RBE) values of various human cell lines for carbon-ion beams with 2 different linear energy transfer (LET) beams and to investigate the relationship between the cell-killing effect and the biophysical characters, such as the chromosome number and the area of the cell nucleus, using qualitatively different kinds of radiations. Methods and Materials: Sixteen different human cell lines were irradiated with carbon-ion beams, having 2 different LET values (LET ∞ = 13.3 and approximately 77 keV/μm), accelerated by the Heavy Ion Medical Accelerator in Chiba (HIMAC) at National Institute of Radiological Sciences in Japan. Cell-killing effect was detected as reproductive cell death using a colony-formation assay. The number of chromosomes was observed in a metaphase spread using the conventional method. The area of the cell nucleus was calculated as an ellipse on photographs using a micrometer. Results: The RBE values calculated by the D 10, which is determined as the dose (Gy) required to reduce the surviving fraction to 10%, relative to X-rays, range from 1.06 to 1.33 for 13-keV/μm-beam and from 2.00 to 3.01 for approximate 77-keV/μm-beam irradiation on each cell line. There was a good correlation in the D 10 values of each cell line between X-rays and carbon-ion beams. However, the D 10 values did not clearly depend on either the chromosome number or the area of the cell nuclei. Conclusion: The RBE values for HIMAC carbon-ion beams are consistent with previous reports using carbon-ion beams with the similar LET values, and the cellular radiosensitivity of different cell lines well correlate among different types of radiation.

Induction of apoptosis in KB cells by pingyangmycin.

Pingyangmycin (PYM; Bleomycin A(5)), an antitumour antibiotic is currently used during anticancer therapy. Previous experiments demonstrated that the therapeutic efficiency of PYM for treatment of malignant tumours is considered to be related to its ability to cause DNA strand breaks in vitro. However, very little is known about the interaction of PYM with the target cells, and it is still unclear how PYM enters the cells. In this study, cell death induced by PYM was studied in a human squamous cell carcinoma cell line (KB cells). In order to determine if cell death occurred by necrosis (reproductive cell death) or apoptosis (programmed cell death), KB cells were exposed to different concentrations of PYM and evaluated by biochemical and morphological criteria. Our results indicate that KB cells displayed an arrest in the G(2)-M phase of the cell cycle and became enlarged and polynucleated before dying at the low concentrations of PYM. In contrast, when cells were exposed to high concentrations of PYM, morphological changes identical to those usually associated with apoptosis were observed as well as internucleosomal digestion of genomic DNA. In conclusion, we demonstrate that PYM is able to induce two distinct modes of cell death depending on the doses of PYM.

Correlation of micronucleus and apoptosis assays with reproductive cell death can be improved by considering other modes of death

Purpose : To evaluate, using 10 cell lines, the already shown improved correlation between the sum of micronucleated plus apoptotic cells and reproductive cell death. Materials and methods : The cell lines were X-irradiated with doses selected to obtain comparable survival levels. Micronucleated and apoptotic cells as well as abnormal cells (multinucleated and necrotic cells) were counted over several days. Apoptosis was also confirmed by gel electrophoresis. Results : The data confirmed the already shown improved relationship over the solely performed micronucleus or apoptosis assays with reproductive cell death and radiation dose. However, even this approach revealed a saturation after irradiation in certain cell lines. Including other modes of cell death such as mitotic failure or necrosis could eliminate this effect. Conclusion : Multiple parameters should be considered when evaluating the use of a predictive assay for ionizing radiation-induced cell killing or biological dosimetry.

Radiobiological studies on the 65MeV therapeutic proton beam at Nice using human tumour cells

Purpose : To determine the relative biological effectiveness (RBE) for initial and delayed inactivation of cells by a modulated proton beam suitable for the treatment of tumours of the eye, within the spread-out Bragg peak and in its distal declining edge. Materials and methods : Human tumour SCC25 cells were irradiated with the 65 MeV proton beam at the Cyclotron Medicyc in Nice. Perspex plates of different thickness were used to simulate five positions along the beam line: 2 mm corresponding to the entrance beam; 15.6 and 25mm in the spread-out Bragg peak; 27.2 and 27.8mm for the distal edge. At each position clonogenic survival of the irradiated cells and of their progeny were determined at various dose values. 60 Co γ-rays were used as reference radiation. Results : RBE values evaluated at the survival level given by 2 Gy of γ-rays increased with increasing depth from close to 1.0 at the proximal to about 1.2 at the distal part of the peak. Within the declining edge it reached the value of about 1.4 at 27.2 and about 2 at 27.8 mm. For the progeny of irradiated cells, the RBE value ranged from 1.0 to 1.1 within the spread-out Bragg peak and then increased up to a value of 2.0 at the last position. The dose-effect curves for the progeny always had a larger shoulder than for the irradiated progenitors, their α parameters being lower by a factor of about 4 and their β parameters always being higher. The alpha/beta ratio was about 50Gy for the progenitors and about 6Gy for their progeny. The incidence of delayed eÚects increased with dose and with the depth within the beam. Conclusions : RBE values for the inactivation of cells irradiated in the spread-out Bragg peak are compatible with the value currently assumed in clinical applications. In the distal declining edge of the beam, the RBE values increased significantly to an extent that may be of concern when the region of the treatment volume is close to sensitive tissues. The yield of delayed reproductive cell death was significant at each position along the beam line.

Influence of dose-rate, post-irradiation incubation time and growth factors on interphase cell death by apoptosis and clonogenic survival of human peripheral lymphocytes

Purpose: To investigate the effects of dose-rate, post-irradiation incubation time and growth factors on radiation-induced interphase cell death by apoptosis and reproductive cell death in human peripheral lymphocytes. Materials and methods : Lymphocytes in G0-phase were exposed in vitro to 1-3Gy 137Cs gamma-radiation at a high- (HDR, 45Gy/h) or a low dose-rate (LDR, 0.024Gy/h). HDR exposures were performed either on day 1 (HDR1) simultaneously with the start of the 3Gy LDR exposure, or on day 6 (HDR6) when the LDR exposures ended. Apoptosis was studied at different times after irradiation by measuring (1) cellular membrane integrity, (2) morphological changes and (3) cell size reduction. Clonogenic survival was analysed for cells plated directly after irradiation (LDR, HDR6, HDR1 day 1) or after 5 days post-irradiation incubation (HDR1 day 6). Results: A significant decrease in reproductive cell death was observed after 3Gy LDR exposure as compared with the HDR1 exposure for cells plated day 6. For the lower doses applied, the dose-rate effect could not be statistically verified. A decrease in apoptosis for all three doses applied (i.e. 1, 2 and 3Gy) was observed when the LDR exposures were compared with the HDR1 analysed day 6, although not of statistical significance. Radiation-induced apoptosis was e fficiently counteracted by growth factors up to 24-48h after 3Gy HDR exposure. The prevention of radiation-induced cell death by growth factors was dependent on dose and post-irradiation time in G0. When the growth factors were added after a prolonged post-irradiation incubation in G0 (HDR1 cells plated day 6), a significant increase in reproductive cell death was found (3Gy) as compared with HDR protocols where the growth factors were added directly after irradiation (HDR1 plated day 1 and HDR6). Conclusions: A dose-rate effect on radiation-induced apoptosis was indicated but not statistically verified. A significant dose-rate effect on reproductive cell death was observed. This dose-rate effect was, however, inverted when growth factors were added directly after the HDR irradiations.

Correlation between cell reproductive death and chromosome aberrations assessed by FISH for low and high doses of radiation and sensitization by iodo-deoxyuridine in human SW-1573 cells

Purpose : To study the relationship between cell reproductive death and exchange frequency in SW-1573 human lung tumour cells with and without incorporated iodo-deoxyuridine (IdUrd) following irradiation of plateau-phase cultures with gamma -rays. Method : Linear-quadratic (LQ) analysis was performed for the data on clonogenicsurvival and on the frequency of chromosomal exchanges studied with fluorescence in situ hybridization in chromosomes X and 2. Results : Differences in the LQ parameters alpha and beta of both nonsensitized and sensitized chromosomes were found. In both chromosomes an increase in the number of chromosomal exchanges in IdUrd-radiosensitized cells compared with nonsensitized cells was observed. The alpha -enhancement factors of 1.7 and 1.9 for the X-chromosome and for chromosome 2, respectively, are similar. For the X-chromosome, the beta coefficient increased by a factor of 3.9 and for chromosome 2 by a factor of 1.4. After correction to a full genome equivalence, no significant difference in alpha was found between chromosomes X and 2 for both control and sensitized cells. In contrast, an almost 2.8 times higher beta was found for the sensitized X-chromosome compared to this value for chromosome 2. Conclusions : It can be concluded that the linear-quadratic analysis of dose-response relationships offers insights into the correlation between cell survival and induction of exchanges in nonsensitized and radiosensitized cells.

Penclomedine-induced DNA fragmentation and p53 accumulation correlate with reproductive cell death in colorectal carcinoma cells with altered p53 status.

Penclomedine, a synthetic pyridine derivative, has documented antitumor activity and is being investigated in clinical trials. Its mechanism of action is unknown although it may be metabolized to a free radical, DNA-reactive species. We previously reported that telomerase positive colorectal carcinoma (RKO) cells with abrogated p53 function were more sensitive to penclomedine than were telomerase positive cells with wild-type p53. The present study demonstrates that significant differences in DNA fragmentation in response to penclomedine were observed in RKO cells lacking functional p53 compared with RKO cells with normal p53 function. No differences in DNA fragmentation in response to ionizing radiation were seen in RKO cells with normal or abrogated p53 function. RKO cells with functional p53 respond to penclomedine treatment with a dose-dependent increase in p53 protein levels. However, RKO cells with abrogated p53 function did not show any such change in p53 protein levels. Further, p53-independent increase of p21 was observed, although the significance of this response remains uncertain. These studies suggest that penclomedine may have a therapeutic advantage in killing cells that have abrogated p53 function.

Enhanced sensitivity to topoisomerase inhibitors in synchronous CHO cells pre-treated with 5-azacytidine

Multidrug combination has been shown to be very useful to improve antitumor activity as well as to reduce the toxicity of different anti-cancer drugs. We have evaluated the interaction between the hypomethylating agent 5-azacytidine and the topoisomerase I and topoisomerase II inhibitors Camptothecin (CPT) and 4′-(9-acridinylamino) methanesulfon- m-anisidide ( m-AMSA) respectively, based on the hypothesis that through the alteration of chromosome replication timing following DNA hypomethylation, the number of replication forks in early S phase might increase, so enhancing the probability of a collision between a blocked cleavable complex (DNA-topo I-CPT or DNA-topo II- m-AMSA) and a replication fork. We have tested the capacity of CPT and m-AMSA to induce chromosomal aberrations as well as reproductive cell death in synchronous cultured Chinese hamster ovary cells after a pretreatment with 5-azacytidine with positive results.

Lack of ceramide generation in TF-1 human myeloid leukemic cells resistant to ionizing radiation.

The mechanism(s) by which ionizing radiation (IR) induces cell death is of fundamental importance in understanding cell sensitivity and resistance. Here we evaluated the response to IR of two subclones of the autonomous human erythromyeloblastic cell line TF-1: TF-1-34 (which expresses CD34) and TF-1-33 (which lacks CD34). In clonogenic assays, TF-1-34 cells were found to be relatively less sensitive to IR compared to TF-1-33 cells based on the D0 determination (3.01 vs 1.56 Gy). Furthermore, after IR at 12 Gy, TF-1-33 cell viability decreased by approximately 50% within 24 h, whereas TF-1-34 cell growth was unaffected during this time. Gradual loss of TF-1-34 cell viability was observed only after 48 h. Morphological and molecular analysis revealed that TF-1-33 cells died of apoptosis, and TF-1-34 cells of delayed reproductive cell death. While IR produced comparable amounts of DNA double strand breaks (DSB) in both cell lines, TF-1-34 retained DSB much longer than TF-1-33 suggesting that radioresistance and the defective apoptotic response of TF-1-34 cells was not related to a higher DNA repair capacity. However, the lack of an apoptotic response in TF-1-34 was correlated to the absence of a sphingomyelin (SM)-ceramide (CER) signaling pathway. Indeed, IR triggered in TF-1-33 cells but not in TF-1-34, SM hydrolysis (25% at 12 Gy) and CER generation (>50%) through the activation of neutral but not acid sphingomyelinase. Synthetic cell permeate CER induced apoptosis in both TF-1-33 and TF-1-34 cells. This study indicates that alterations of the SM-CER signaling pathway can significantly influence the cell death process as well as the survival of acute myeloid leukemia cells after IR exposure.

Chromosomal instability, reproductive cell death and apoptosis induced by O6-methylguanine in Mex −, Mex + and methylation-tolerant mismatch repair compromised cells: facts and models

O 6-Methylguanine ( O 6-MeG) is induced in DNA by methylating environmental carcinogens and various cytostatic drugs. It is repaired by O 6-methylguanine-DNA methyltransferase (MGMT). If not repaired prior to replication, the lesion generates gene mutations and leads to cell death, sister chromatid exchanges (SCEs), chromosomal aberrations and malignant transformation. To address the question of how O 6-MeG is transformed into genotoxic effects, isogenic Chinese hamster cell lines either not expressing MGMT (phenotypically Mex −), expressing MGMT (Mex +) or exhibiting the tolerance phenotype (Mex −, methylation resistant) were compared as to their clastogenic response. Mex − cells were more sensitive than Mex + cells to N-methyl- N′-nitro- N-nitrosoguanidine (MNNG)-induced chromosomal breakage, with marked differences in sensitivity depending on recovery time. At early recovery time, when cells out of the first post-treatment mitosis were scored, aberration frequency was about 40% reduced in Mex + as compared to Mex − cells. At later stages of recovery when cells out of the second post-treatment mitosis were analyzed, the frequency of aberrations increased strongly in Mex − cells whereas it dropped to nearly control level in Mex + cells. From this we conclude that, in the first post-treatment replication cycle of Mex − cells, only a minor part of aberrations (<40%) was due to O 6-MeG whereas, in the second post-treatment replication cycle, the major part of aberrations (>90%) was caused by the lesion. Thus, O 6-MeG is a potent clastogenic DNA damage that needs two DNA replication cycles in order to be transformed with high efficiency into aberrations. The same holds true for sister chromatid exchanges (SCEs). MNNG is highly potent in inducing SCEs in Mex − cells in the second replication cycle after alkylation. Under these conditions, SCE induction is nearly completely prevented by the expression of MGMT. This is opposed to SCE induction in the first post-treatment replication cycle, where higher doses of MNNG were required to induce SCEs and no protective effect of MGMT was observed. This indicates that SCEs induced in the first replication cycle after alkylation are due to other lesions than O 6-MeG. In methylation tolerant cells, which are characterized by impaired G–T mismatch binding and MSH2 expression, aberration frequency induced by MNNG was weakly reduced in the first and strongly reduced in the second post-treatment mitoses, as compared to CHO wild-type cells. The results indicate that mismatch repair of O 6-MeG–T mispairs is decisively involved in O 6-MeG born chromosomal instability and recombination. We also show that Mex + and methylation tolerant cells are more resistant than Mex − cells with regard to induction of apoptosis, indicating O 6-MeG to be also an apoptosis-inducing lesion. The data are discussed as to the mechanism of cytotoxicity, aberration and SCE formation in cells treated with a methylating agent.

Parameters of linear-quadratic radiation dose-effect relationships: dependence on LET and mechanisms of reproductive cell death

An analysis of mammalian cell radiation-dose survival curves, based on the linear-quadratic formalism, is shown to yield insights in the various components of damage that contribute to cell reproductive death. RBE -LET relationships of single-track lethal damage, sublethal damage, potentially lethal damage and DNA double-strand breaks are compared. Singletrack lethal damage is derived to be composed of two components: (1) damage that remains unrepaired in an interval between irradiation and assay for proliferative capacity, with a very strong dependence on LET, and (2) potentially lethal damage that is only weakly dependent on LET, similar to sublethal damage and DNA double-strand breaks. The results of this analysis lead to new interpretations of published experimental results and to suggestions for applications in radiotherapy.

Genomic Instability Induced by Ionizing Radiation

Genomic instability is characterized by the increased rate of acquisition of alterations in the mammalian genome. These changes encompass a diverse set of biological end points including karyotypic abnormalities, gene mutation and amplification, cellular transformation, clonal heterogeneity and delayed reproductive cell death. The loss of stability of the genome is becoming accepted as one of the most important aspects of carcinogenesis, and the numerous genetic changes associated with the cancer cell implicate genomic stability as contributing to the neoplastic phenotype. Multiple metabolic pathways govern the accurate duplication and distribution of DNA to progeny cells; other pathways maintain the integrity of the information encoded by DNA and regulate the expression of genes during growth and development. For each of these functions, there is a normal baseline frequency at which errors occur, leading to spontaneous mutations and other genomic anomalies. This review summarizes the current status of knowledge about radiation-induced genomic instability. Those events and processes likely to be involved in the initiation and perpetuation of the unstable phenotype, the potential role of epigenetic factors in influencing the onset of genomic instability, and the delayed effects of cellular exposure to ionizing radiation are discussed.

Radiation-enhanced differentiation of erythroid progenitor cells and its relation to reproductive cell death.

Terminally differentiated cells usually do not divide and are, thus, reproductively dead. To elucidate the significance of radiation-enhanced differentiation to reproductive cell death, murine erythroid progenitor cells were gamma-irradiated in plasma clot cultures and the development of haemoglobinized clones was studied thereafter. If irradiation occurred when the cells had resumed proliferation, the total numbers of haemoglobinized clones and, in parallel, the numbers of newly haemoglobinized clones were elevated above control levels 6-24 h after 10-30 Gy and 24-48 h after 1 Gy respectively. Thereafter, clone numbers decreased below controls. This decrease was faster with the newly haemoglobinized clones, indicating that both the accumulation of haemoglobinized clones and fast exhaustion of the pool of more primitive precursors in the cultures are due to accelerated differentiation. The haemoglobinized clones appearing after irradiation were reduced in size without indication of direct cell death. We conclude that the reproductive cell death occurring in our system is due to enhancement of differentiation. Enhancement of differentiation is expressed by omission of cell cycles normally passed through by the cell progeny before terminal differentiation is reached. Dependence of differentiation enhancement on the presence of cycling cells at the time of irradiation indicates involvement of growth of essential cytoplasmic constituents during mitotic delay as observed in other cell systems.

831 Effects of ionizing irradiation and adrenergic stimulation on gene expression pattern in rat submandibular glands

Radiotherapy administrated to patients with head and neck malignancies often results in severe xerostomia. We evaluated the expression of early response proto-oncogenes (c-fos and jun-B), tissue specific genes (proline rich protein (PRP) and kallikrein), and proteolysis linked ubiquitin gene following exposure to 15 Gy irradiation and/or adrenergic stimulation of the rat submandibular gland. We observed that the expression of the genes whose regulation is associated with DNA damage (i.e.jun-B and c-fos) was enhanced by irradiation or the combination of irradiation and isoproterenol administration. In contrast, the expression of genes associated with the routine functional integrity of the cell (i.e. kallikrein. ubiquitin and PRP) was uneffected. These findings, in addition to delayed gland dysfunction, leaves us to believe that irradiation induced injury to the submandibular glands is to be attributed to reproductive stem cell death which may be obliterated in some part in a clinical setting.

442 Characteristics of irradiation induced cell death mode of a human salivary cell line

Irradiation induced injury to the major salivary glands is a known complication of radiotherapy. We used HSG cell line, which is derived from an irradiated human submandibular gland, as an <i>in vitro</i> model for the studying this damage. Ninety-six h post irradiation of HSG cells at log phase stage of growth the LD₅₀ was found to be 5–7.5 Gy, with a cell death rate of 75%, resulting from 10–20 Gy. In contrast cells at the confluent stage of growth demonstrated radioresistancy. However, irradiation latent damage was again observed when these irradiated cells were reinduced into log phase growth. Incubation of the HSG cells with INF-g for 2–24 h partially minimized the irradiation induced injury but incubation with TNF-a, Interleukin-6 or heat shock protein did not reduce the damage. In summary, irradiation induced damage to HSG cells is in accordance with the delayed reproductive cell death mode.

Effect of Cell Cycle Stage, Dose Rate and Repair of Sublethal Damage on Radiation-Induced Apoptosis in F9 Teratocarcinoma Cells

There are at least two different modes of cell death after treatment with ionizing radiation. The first is a failure to undergo sustained cell division despite metabolic survival, and we refer to this end point as "classical reproductive cell death." The second is a process that results in loss of cell integrity. This second category includes cellular necrosis as well as apoptosis. Earlier studies in our laboratory showed that the predominant mechanism of cell death for irradiated F9 cells is apoptosis, and there is no indication that these cells die by necrosis. We have therefore used cells of this cell line to reassess basic radiobiological principles with respect to apoptosis. Classical reproductive cell death was determined by staining colonies derived from irradiated cells and scoring colonies of less than 50 cells as reproductively dead and colonies of more than 50 cells as survivors. Cells that failed to produce either type of colony (detached from the plate or disintegrated) were scored as having undergone apoptosis. Using these criteria we found that the fraction of the radiation-killed F9 cells that died by apoptosis did not vary when cells were irradiated at different stages of the cell cycle despite large variations in overall survival. This suggests that the factors that influence radiation sensitivity throughout the cell cycle have an equal impact on apoptosis and classical reproductive cell death. There was no difference in cell survival between split doses and single doses of X rays, suggesting that sublethal damage repair is not a factor in radiation-induced apoptosis of F9 cells. Apoptosis was not affected by changes in dose rate in the range of 0.038-4.96 Gy/min.