Repair of potentially lethal X-ray damage in fibroblasts derived from patients with hereditary and D-deletion retinoblastoma

We examined X-ray induced potentially lethal damage repair (PLDR) in density inhibited plateau phase cultures of six fibroblast strains derived from patients with hereditary retinoblastoma and two patients with D-deletion retinoblastoma and compared them to three normal controls. PLD was measured in hereditary retinoblastoma (7 Gy exposure) and normal cells (7 and 9 Gy exposure) after 24 h repair time. PLD survival curves were performed at 2-9 Gy on six retinoblastoma and three normal control cell strains. Thus, PLDR was compared at equitoxic survival levels as well as after exposure to equal doses of radiation. Some retinoblastoma strains showed normal PLDR whereas others were possibly deficient. Implications of PLDR for susceptibility to radiation-induced and spontaneous tumours in hereditary retinoblastoma patients are discussed.     

Potentially lethal damage repair in cell lines of radioresistant human tumours and normal skin fibroblasts

Radiation cell survival data were obtained in vitro for three cell lines isolated from human tumours traditionally considered to be radioresistant--two melanomas and one osteosarcoma--as well as from a diploid skin fibroblast cell line. One melanoma cell line was much more radioresistant than the other, while the osteosarcoma and fibroblast cell lines were more radiosensitive than either. For cells growing exponentially, little potentially lethal damage repair (PLDR) could be demonstrated by comparing survival data for cells in which subculture was delayed by 6 h with those sub-cultured immediately after treatment. For the malignant cells in plateau phase, which in these cells might be better termed 'slowed growth phase', since an appreciable fraction of the cells are still cycling, a small amount of PLDR was observed, but not as much as reported by other investigators in the literature. The normal fibroblasts, which achieved a truer plateau phase in terms of noncycling cells, showed a significantly larger amount of PLDR than the tumour cells.     

Evidence for the repair of potentially lethal damage in irradiated bone marrow

Summary The effects on cell survival of maintaining bone marrow cells (CFU-S) in situ following irradiation and before assay by transplantation was investigated. When the CFU-S cells are maintained in situ following irradiation survival drops and plateaus at about 9 h post-irradiation. Evidence is presented that this decrease in survival may be due to potentially lethal damage repair (PLD) inhibition caused by post-irradiation in situ holding. This effect on PLD repair is different than that usually found in cells in vitro and in vivo tumors in that it mainly alters the shoulder rather than the slope of the survival curve of CFU-S cells. It is different than PLDR found in vivo for normal mammary and thyroid gland epithelial cells because in situ holding decreases rather than increases the survival of CFU-S cells. Evidence is also presented that the radiation survival curve for in situ bone marrow cells (CFU-S) may not have a shoulder.Supported in part by NIH, NCI grants P01 CA 19298 and P30 CA 14520Supported in part by an American Cancer Society Clinical Fellowship     

The relationship between radiosensitivity and repair of potentially lethal damage in human tumor cell lines with implications for radioresponsiveness

The relationship between intrinsic radiosensitivity and repair capacity was studied for 22 human tumor cell lines in vitro. The experimental material was taken from 19 published papers. Parameters from three radiobiological models were used to assess this relationship: the one-hit multitarget model (D0 and n), the linear-quadratic model (alpha and beta), and the mean inactivation dose (D). Data were obtained for cells in three stages: exponentially growing cells (exp), plateau-phase cells plated immediately after irradiation (ip), and plateau-phase cells plated after completion of PLD repair (dp). No significant difference was found between radiosensitivity of exp and ip cells. There was no correlation between repair capacity and intrinsic radiosensitivity assessed with plateau-phase cells plated immediately after irradiation. The correlation studies between intrinsic radiosensitivity or repair capacity and clinical responsiveness were achieved by assigning cell lines to one of three groups of decreasing in vivo radioresponsiveness: highly, medium, and poorly responsive. There was a significant correlation between radiosensitivity and radioresponsiveness, but no correlation between repair capacity and radioresponsiveness. The average repair capacity was about 0.6 Gy, in terms of D. Three parameters, the mean inactivation dose of exponentially growing cells, of plateau-phase cells plated immediately after irradiation, and of plateau-phase cells plated after completion of PLD repair, could be used equally to assess the relationship between in vitro data and radioresponsiveness. The present results are compared to those obtained in a similar study on a group of 48 nontransformed fibroblast cell strains.     

Recovery from potentially lethal damage and recruitment time of noncycling clonogenic cells in 9L confluent monolayers and spheroids

Cells that have been grown as multicell tumor spheroids exhibit radioresistance compared to the same cells grown in monolayers. Comparison of potentially lethal damage (PLD) repair and its kinetics was made between 9L cells grown as spheroids and confluent monolayers. Survival curves of cells plated immediately after irradiation showed the typical radioresistance associated with spheroid culture compared to plateau-phase monolayers. The dose-modification factor for spheroid cell survival is 1.44. Postirradiation incubations in normal phosphate-buffered saline (PBS), conditioned media, or 0.5 M NaCl in PBS reduced the differences in radiosensitivity between the two culture conditions. Postirradiation treatment in PBS or conditioned medium promoted repair of potentially lethal damage, and 0.5 M NaCl prevented the removal of PLD and allowed the fixation of damage resulting in lower survival. Survival of spheroid and monolayer cells after hypertonic NaCl treatment was identical. NaCl treatment reduced Do more than it did the shoulder (Dq) of the survival curve. PLD repair kinetics measured after postirradiation incubation in PBS followed by hypertonic NaCl treatment was the same for spheroids and for plateau-phase monolayers. The kinetics of PLD repair indicates a biphasic phenomenon. There is an initial fast component with a repair half-time of 7.9 min and a slow component with a repair half-time of 56.6 min. Most of the damage (59%) is repaired slowly. Since the repair capacity and kinetics are the same for spheroids and monolayers, the radioresistance of spheroids cannot be explained on this basis. Evidence indicates that the time to return from a Go (noncycling G1 cells) state to a proliferative state (recruitment) for cells from confluent monolayers and from spheroids after dissociation by protease treatment may be the most important determinant of the degree of PLD repair that occurs. Growth curves and flow cytometry cell cycle analysis indicate that spheroid cells have a lag period for reentry into a proliferative state. Since plating efficiency remains high and unchanging during this period, one cannot account for the delay on the basis of the existence of a large fraction of Go cells which are not potentially clonogenic. The cell cycle progression begins in 6-8 h for monolayer cells and in 14-15 h for spheroids. It is hypothesized that the slower reentry of spheroid cells into a cycling phase allows more time for repair than for the rapidly proliferating monolayer cells.     

Changes in repair of potentially lethal damage with culture age in EMT6 cells.

We have investigated the changes in the amplitude of repair of potentially lethal damage (PLD) in EMT6 cells with increasing culture age and determined the delay necessary to achieve this repair. This experimental system presents all intermediaries between the exponential growth type and a plateau with a cell turnover nearly nil. The radiosensitivity was studied by the colony method. When the percentage of surviving cells was tested immediately after irradiation it was observed that their radiosensitivity increased with culture age. This percentage fell only slightly when the cells were tested for viability 6 hours after irradiation. Therefore, the amplitude of repair increases with culture age. Repair was found to terminate 1, 1.75, 3 and 6 hours after irradiation of cultures aged respectively 2, 4, 6 and 9 days. The delay and the amplitude of repair did not vary significantly for cultures of 9, 11 and 13 days.     

Repair of potentially lethal radiation damage in confluent and non-confluent cultures of human hybrid cells

The repair of potentially lethal damage (PLDR) in a gamma-irradiated human hybrid cell line (skin fibroblast X HeLa) and its tumourigenic segregant has been studied as a function of cell density at the time of irradiation and during the postirradiation repair period. The data show that PLDR occurs in both non-confluent and confluent cultures of both cell lines. Furthermore, there is evidence that the extent of PLDR is dependent on cell density and that cell-cell contact may be an important factor in this regard.     

In vitro holding and PLD-repair: II. A flow cytometric and electron microscopic analysis of some mammalian cell lines

Summary The repair of potentially lethal damage (PLDR) in mammalian cells is expected to be better in quiescent cultures since PLD is supposedly fixed during cycle progression. Plateau phase cultures, therefore, serve as models because of assumed mitotic quiescence. Four established cell lines (V79, CHO, L5178Y and HELA) and one euploid cell strain IMR-90 have been analysed by flow cytometry and electron microscopy to address questions on quiescence in the plateau phase and the effect of holding (induction of quiescence by nutrient privation). In contrast to commonly held views, our results indicate that the quiescent fraction in cultures from transformed cells is exceedingly low (1% or less). Plateau phase cultures of transformed cells are constantly turning over. Euploid cells like the IMR-90 show true quiescence in the plateau phase. Holding causes typical cytopathological changes. These changes have been ultrastructurely characterised. Resistant sub-populations of cells can be selected out under holding-conditions. Such selected cells show completely different radiobiological characteristics, which raise questions on the interpretation of data on PLDR.Dedicated to Prof. Dr. Johannes Meißner, Borstel on the occasion of his 75th birthday     

Abnormal sensitivity of some Cockayne's syndrome cell strains to UV- and gamma-rays. Association with a reduced ability to repair potentially lethal damage

Cockayne's syndrome (CS) is a rare autosomal recessive genetic disease characterized by mental and physical retardation, microcephaly, dwarfism, retinitis pigmentosa and a hypersensitivity to sunlight. Cells originating from patients also exhibit, in vitro, a hypersensitivity to UV radiation. Using a colony assay in vitro, we studied the sensitivity of 5 CS cell strains (GM739, BOR, CS697, CS698 and KA) and two normal ones (HF19 and GP) to UV- and gamma-irradiation. The 5 CS strains appear to be UV-hypersensitive but the sensitivity varies widely from one strain to another. Hypersensitivity to gamma-rays has been reported for 4 out of the 5 CS cell strains investigated. However, these CS cell strains are less sensitive to gamma-rays than are ataxia telangiectasia cells. The KA cell strain exhibits a normal response to gamma-irradiation. Repair of potentially lethal damage (PLD) after UV- and gamma-irradiation was investigated by using unfed plateau-cell cultures. Under these conditions, control cells show a great capacity to repair PLD (10- to 30-fold survival increase at 1% survival level). The two CS strains (GM739 and BOR), which are hypersensitive to both UV- and gamma-irradiation, exhibit no or only little PLD repair after treatment. In contrast, the normal response of KA cells to gamma-rays is associated with a normal PLD repair capability. This latter cell strain exhibits an intermediate sensitivity to UV and shows an intermediate PLD repair capacity. The response of CS cell strains after gamma-irradiation suggests a genetic heterogeneity. Three complementation groups are described in CS cells when dealing with UV radiosensitivity. However, variations in gamma-ray sensitivity are reported for cells within the same UV complementation group.     

Radiation response of haematopoietic cell lines of human origin

Six human haematopoietic cell lines, five of leukaemic origin, including cells with myeloid, lymphoid and undifferentiated phenotype have been studied with respect to radiation response. The intrinsic radiosensitivity of the cells varied widely, the D0s ranging from 0.53 to 1.39 Gy. Five of the cell lines showed some capacity to accumulate sublethal damage; in three of these, enhanced survival was demonstrated in split-dose experiments. One cell line (HL-60) was anomalous in that although little accumulation of sublethal damage was demonstrable, survival was enhanced by fractionation of the dose. Five of the six cell lines studied were of leukaemic origin. The results support the belief that, in contrast to the almost constant radiosensitivity of normal haematopoietic cell progenitors, leukaemic cell progenitors may show a wide range of radiosensitivites.     

Ionizing radiation damage to DNA: molecular aspects

Radioresistant tumor cells are found in tumor specimens from patients in whom radiotherapy has failed or whose tumors have recurred after therapy. This suggests that inherent cellular radioresistance may in part underlie the failure of radiotherapy, and therefore determination of the presence of resistant cells within a tumor might be a useful predictor of response to radiation therapy. Most standard clonogenic assays of radiation response are time-consuming, and alternative assays of radiation response are being sought. In an earlier publication (J. L. Schwartz et al., Int. J. Radiat. Oncol. Biol. Phys. 15, 907-912, 1988), we reported that radioresistant human tumor cells rejoin DNA double-strand breaks, as measured by DNA neutral filter elution (pH 9.6), faster than more sensitive cell lines. To determine whether DNA elution might have potential as a rapid predictive assay, we examined the relationship between the rate of DNA double-strand break rejoining and radiosensitivity in nine first-passage-after-explant squamous cell carcinomas under conditions that minimized the influence of nontumor and nonclonogenic cells. The frequency of DNA double-strand breaks measured 1 h after irradiation with 100 Gy 60Co gamma rays was used as an estimate of relative rejoining rate. This number is a reflection of both the initial DNA double-strand break frequency and the amount of repair that occurs in 1 h. The relative break frequency was compared to radiosensitivity as measured by standard clonogenic survival assays in later passages (p3-p14) of these same cells. A significant relationship (r = 0.61, P less than 0.01) was found between break frequency measured in first-passage cells and radiosensitivity measured in later passages, suggesting that the neutral elution assay as described here has some promise as a relatively rapid assay of the radiosensitivity of human tumor cells.     

Induction of the blood–brain barrier marker neurothelin/HT7 in endothelial cells by a variety of tumors in chick embryos

Neurothelin/HT7, a transmembrane glycoprotein of the immunoglobulin superfamily, is a marker of blood–brain barrier (BBB)-forming endothelial cells. We have studied the expression of neurothelin in tumors grown on the chorioallantoic membrane (CAM) of chick embryos. We inoculated each 3–5×10⁶ rat C6 glioma, rat 10AS pancreatic carcinoma, human A375 melanoma, and human mammary duct adenoma cells on the CAM of 10-day-old chick embryos. The tumors were harvested on day 17. All four tumor cell lines formed solid tumors which were supplied by vessels of CAM origin. Foci of bleeding were regularly observed within the tumors. All four tumors induced the expression of neurothelin/HT7 (but not of glucose transporter-1) in tumor endothelial cells, whereas expression in adjacent endothelial cells of normal CAM did not occur. Confocal laser scanning microscopy revealed that the pattern of neurothelin expression in tumor endothelial cells was different from that in normal central nervous system (CNS) endothelium, but the relative molecular weight of neurothelin, studied by western blot analysis, was the same in brain and in tumors. It has been shown that, with increasing malignancy, vessels of CNS tumors lose their morphological characteristics, and BBB markers such as the glucose transporter-1 are downregulated. Our results show that, in contrast, the BBB marker, neurothelin, is expressed de novo in tumor endothelial cells. Potential common functions of neurothelin in endothelial cells of the CNS and tumors are discussed. Accepted: 6 December 1999     

PLD repair in rat rhabdomyosarcoma tumor cells irradiated in vivo and in vitro with high-LET and low-LET radiation

Results are reported of studies to measure the extent of recovery of potentially lethal damage (PLD) in rat rhabdomyosarcoma tumor cells after irradiation both in vivo and in vitro with either high-LET or low-LET radiation. Stationary-phase cultures were found to exhibit repair of PLD following irradiation in vitro either with low-LET X rays or with high-LET neon ions in the extended-peak ionization region. Following a 9-Gy dose of 225-kVp X rays or a 3.5-Gy dose of peak neon ions, both of which reduced the initial cell survival to 6-8%, the maximum PLD recovery factors were 3.4 and 1.6, respectively. In contrast, the standard tumor excision assay procedure failed to reveal any recovery from PLD in tumors irradiated in situ with either X rays or peak neon ions. PLD repair by the in vivo tumor cells could be observed, however, when the excision assay procedure was altered by the addition of a known PLD repair inhibitor beta-arabinofuranosyladenine (beta-ara-A). When a noncytotoxic 50 microM concentration of beta-ara-A was added to the excised tumor cells immediately following a 14.5-Gy in situ dose of X rays, cell survival in the inhibitor-treated cells was lower than in the untreated cells (0.018 compared to 0.056), resulting in a PLD repair inhibition factor of 3.1. Delaying the addition of beta-ara-A for 1, 2, or 3 h following tumor excision reduced the PLD repair inhibition factor to 1.6, 1.5, and 0.9, respectively. Following tumor irradiation in situ with neon ions in the extended-peak ionization region (median LET = 145 keV/micron), less PLD repair was observed than after X irradiation. For 5.8 Gy of peak neon ions, the PLD repair inhibition factors were 2.1, 1.5, 1.3, and 1.1 at 0, 1, 2, and 3 h, respectively. We interpret the absence of measurable PLD repair using the standard tumor excision assay procedure as resulting from undetectable repair occurring during the long interval (about 2 h) required for the cell dissociation and plating procedures. We conclude that at least for our tumor system, PLD repair does occur after irradiation of tumors in situ, even though it is not detectable using the standard tumor excision assay procedure. Thus a failure to measure such repair by this assay in a given tumor system does not necessarily mean the cells are incapable of PLD repair.     

Initial slope of radiation survival curves is characteristic of the origin of primary and established cultures of human tumor cells and fibroblasts

The published survival curves of 110 human tumor cell lines and 147 nontransformed human fibroblast strains have been reanalyzed using three different statistical methods: the single hit multitarget model, the linear-quadratic model, and the mean inactivation dose. The 110 tumor cell lines were classified in two ways: (a) into three categories defined by clinical radiocurability criteria, and (b) into seven categories based on histopathology. The 147 fibroblast strains were divided into eight genetic groups. Differences in the radiosensitivities of both the tumor cell and fibroblast groups could be demonstrated only by parameters that describe the slopes of the initial part of the survival curves. The capacity of the survival level to identify significant differences between groups was dose dependent over the range 1 to 6 Gy. This relationship showed a bell-shaped curve with a maximum at 1.5 Gy for the tumor cell lines and 3 Gy for the fibroblasts. Values for intrinsic radiosensitivity for a number of groups of tumors have also been obtained by primary culture of tumor cells. These values are strictly comparable to those obtained by clonogenic methods. This confirms that intrinsic radiosensitivity is a determinant of the response of tumor cells to radiotherapy and suggests that tissue culture methods may be used as a predictive assay.     

Interaction between radiation and drug damage in mammalian cells. III. The effect of adriamycin and actinomycin-D on the repair of potentially lethal radiation damage.

We have studied the effects of actinomycin-D (AMD) and Adriamycin (ADRM) on the repair of radiation damage in Chinese hamster cells (V79) in plateau phase growth. Suppression of potentially lethal damage repair (PLDR) was observed in the presence of non-toxic levels of AMD and minimally toxic levels of ADRM. The suppression of PLDR by AMD persisted as long as the drug was present. Removal of AMD was followed by prompt repair of potentially lethal injury suggesting that suppression of PLDR by AMD was not accompanied by fixation of injury to a non-repairable state. On the other hand, irradiated cells exposed to ADRM eventually repair potentially lethal injury in the presence of drug after an initial delay. AMD, but not ADRM, inhibited repair of sublethal radiation damage.     

Histone H2AX phosphorylation as a predictor of radiosensitivity and target for radiotherapy

Based on the role of phosphorylation of the histone H2A variant H2AX in recruitment of DNA repair and checkpoint proteins to the sites of DNA damage, we have investigated gammaH2AX as a reporter of tumor radiosensitivity and a potential target to enhance the effectiveness of radiation therapy. Clinically relevant ionizing radiation (IR) doses induced similar patterns of gammaH2AX focus formation or immunoreactivity in radiosensitive and radioresistant human tumor cell lines and xenografted tumors. However, radiosensitive tumor cells and xenografts retained gammaH2AX for a greater duration than radioresistant cells and tumors. These results suggest that persistence of gammaH2AX after IR may predict tumor response to radiotherapy. We synthesized peptide mimics of the H2AX carboxyl-terminal tail to test whether antagonizing H2AX function affects tumor cell survival following IR. The peptides did not alter the viability of unirradiated tumor cells, but both blocked induction of gammaH2AX foci by IR and enhanced cell death in irradiated radioresistant tumor cells. These results suggest that H2AX is a potential molecular target to enhance the effects of radiotherapy.     

Metformin Radiosensitizes p53-Deficient Colorectal Cancer Cells through Induction of G2/M Arrest and Inhibition of DNA Repair Proteins

The present study addressed whether the combination of metformin and ionizing radiation (IR) would show enhanced antitumor effects in radioresistant p53-deficient colorectal cancer cells, focusing on repair pathways for IR-induced DNA damage. Metformin caused a higher reduction in clonogenic survival as well as greater radiosensitization and inhibition of tumor growth of p53^-/- than of p53^+/+ colorectal cancer cells and xenografts. Metformin combined with IR induced accumulation of tumor cells in the G2/M phase and delayed the repair of IR-induced DNA damage. In addition, this combination significantly decreased levels of p53-related homologous recombination (HR) repair compared with IR alone, especially in p53^-/- colorectal cancer cells and tumors. In conclusion, metformin enhanced radiosensitivity by inducing G2/M arrest and reducing the expression of DNA repair proteins even in radioresistant HCT116 p53^-/- colorectal cancer cells and tumors. Our study provides a scientific rationale for the clinical use of metformin as a radiosensitizer in patients with p53-deficient colorectal tumors, which are often resistant to radiotherapy.     

Ectopically hTERT expressing adult human mesenchymal stem cells are less radiosensitive than their telomerase negative counterpart

During the past several years increasing evidence indicating that the proliferation capacity of mammalian cells is highly radiosensitive, regardless of the species and the tissue of origin of the cells, has accumulated. It has also been shown that normal bone marrow cells of mice have a similar radiosensitivity to other mammalian cells so far tested. In this study, we investigated the genetic effects of ionizing radiation (2.5-15 Gy) on normal human mesenchymal stem cells and their telomerised counterpart hMSC-telo1. We evaluated overall genomic integrity, DNA damage/repair by applying a fluorescence-detected alkaline DNA unwinding assay together with Western blot analyses for phosphorylated H2AX and Q-FISH was applied for investigation of telomeric damage. Our results indicate that hMSC and TERT-immortalized hMSCs can cope with relatively high doses of gamma-rays and that overall DNA repair is similar in the two cell lines. The telomeres were extensively destroyed after irradiation in both cell types suggesting that telomere caps are especially sensitive to radiation. The TERT-immortalized hMSCs showed higher stability at telomeric regions than primary hMSCs indicating that cells with long telomeres and high telomerase activity have the advantage of re-establishing the telomeric caps.     

Molecular aspects of individual radiosensitivity

Radiation therapy is a clinical treatment modality where ionizing radiation is used to treat patients with malignant neoplasms. The goal is to deliver a measured dose of radiation to a defined volume with minimal damage to surrounding normal tissue, resulting in eradication of the tumor. Radiotherapy is generally given in divided doses or fractionated. Molecular biology methods have enhanced our ability to investigate the response of cells to ionizing radiation. These methods can be applied to tissue-culture systems or to biopsies from patients both to develop a quick and easy way to predict the radiosensitivity of a patient and to understand how cells respond to stress produced by ionizing radiation. In this review we will mainly explain two major mechanisms involved in human individual radiosensitivity: the DNA-damage repair defect mechanism and the DNA-repair signaling via cell cycle checkpoint defect.