Influence of poly(ADP-ribose) synthesis inhibitors on the repair of sublethal and potentially lethal damage in gamma-irradiated mammalian cells

The influence of poly(ADP-ribose) synthesis inhibitors on mammalian cell radiosensitivity was investigated. Four different inhibitors were studied: 3-methoxybenzamide, 3-aminobenzamide, 6-aminonicotinamide and nicotinamide. When exponentially growing or plateau phase cells are incubated before irradiation with non-toxic concentrations of these compounds, their radiosensitivity is enhanced except in the case of nicotinamide. The poly(ADP-ribose) inhibitors do not modify the repair of sublethal damage, but totally suppress the repair of potentially lethal damage, as shown by the survival of CHO cells and of a human osteosarcoma cell line.     

The potential value of the neutral comet assay and the expression of genes associated with DNA damage in assessing the radiosensitivity of tumor cells

The assessment of tumor radiosensitivity would be particularly useful in optimizing the radiation dose during radiotherapy. Therefore, the degree of correlation between radiation-induced DNA damage, as measured by the alkaline and the neutral comet assays, and the clonogenic survival of different human tumor cells was studied. Further, tumor radiosensitivity was compared with the expression of genes associated with the cellular response to radiation damage. Five different human tumor cell lines were chosen and the radiosensitivity of these cells was established by clonogenic assay. Alkaline and neutral comet assays were performed in γ-irradiated cells (2-8Gy; either acute or fractionated). Quantitative PCR was performed to evaluate the expression of DNA damage response genes in control and irradiated cells. The relative radiosensitivity of the cell lines assessed by the extent of DNA damage (neutral comet assay) immediately after irradiation (4Gy or 6Gy) was in agreement with radiosensitivity pattern obtained by the clonogenic assay. The survival fraction of irradiated cells showed a better correlation with the magnitude of DNA damage measured by the neutral comet assay (r=-0.9; P<0.05; 6Gy) than evaluated by alkaline comet assay (r=-0.73; P<0.05; 6Gy). Further, a significant correlation between the clonogenic survival and DNA damage was observed in cells exposed to fractionated doses of radiation. Of 15 genes investigated in the gene expression study, HSP70, KU80 and RAD51 all showed significant positive correlations (r=0.9; P<0.05) with tumor radiosensitivity. Our study clearly demonstrated that the neutral comet assay was better than alkaline comet assay for assessment of radiosensitivities of tumor cells after acute or fractionated doses of irradiation.     

Low-expression of E-cadherin in leukaemia cells causes loss of homophilic adhesion and promotes cell growth

E-cadherin (epithelial cadherin) belongs to the calcium-dependent adhesion molecule superfamily and is implicated in the interactions of haematopoietic progenitors and bone marrow stromal cells. Adhesion capacity to bone marrow stroma was impaired for leukaemia cells, suggesting that a breakdown of adhesive mechanisms governed by an adhesion molecule may exist in leukaemic microenvironment. We previously found that E-cadherin was low expressed in primary acute leukaemia cells compared with normal bone marrow mononuclear cells. In this study, we investigate the functional importance of low E-cadherin expression in leukaemia cell behaviours and investigate its effects in the abnormal interaction of leukaemic cells with stromal cells. After expression of E-cadherin was restored by a demethylating agent in leukaemia cells, E-cadherin-specific adhesion was enhanced. Additionally, siRNA (small interfering RNA)-mediated silencing of E-cadherin in Raji cells resulted in a reduction of cell homophilic adhesion and enhancement of cell proliferation and colony formation. These results suggest that low expression of E-cadherin contributes to the vigorous growth and transforming ability of leukaemic cells.     

Comparison of endogenous TP53 genomic status with clonogenicity and different modes of cell death after X irradiation

Although extensive data indicate that the tumor suppressor TP53 modifies the radiation responses of human and rodent cells, the exact relationship between TP53 and radiation responsiveness remains controversial. To elucidate the relevance of endogenous TP53 genomic status to radiosensitivity in a cell-type-independent manner, different cells of 10 human tumor cell lines with different tissues of origin were examined for TP53 status. The TP53 status was compared with radiation-related cell survival parameters (D(q), D(0), SF2) and with the mode of cell death. Different modes of cell death were examined by measuring radiation-induced micronucleation, apoptosis and abnormal cells. Alterations of the TP53 gene were detected in eight cell lines. No splicing mutation was found. Five cell lines showed codon 68 polymorphism. Codon 72 alterations were found in four cell lines. "Hot spot" alterations were detected in only two of 10 cell lines. Although the cells differed widely in survival parameters (D(q), D(0), SF2) and modes of cell death (micronucleation/apoptosis/abnormal cells) after irradiation, significant cell-type-independent correlations were obtained between the multiple cell death parameter micronucleation/apoptosis/abnormal cells and SF2 (P < 0.001) and D(q) (P = 0.003). Moreover, cells with a wild-type TP53 gene were more resistant to X rays than cells with a mutated TP53 gene or cells that were TP53-deficient. The alterations within exons 5-10 of the TP53 correlated with a enhanced radiosensitivity. For the first time, we demonstrated a correlation between endogenous genetic alterations within exons 5-10 of TP53 and radiation-related cell survival and cell death. This indicates a new molecular relevance of TP53 status to intrinsic cellular radiosensitivity.     

Modulation of haematopoietic progenitor development by FLT-3 ligand.

The Flt-3 receptor is expressed in primitive haematopoietic cells and its ligand exerts proliferative effects on these cells in vitro in synergy with other cytokines. To increase our knowledge of the functional properties of the human Flt-3 ligand (FL) as relating to in vitro expansion of haematopoietic stem cells, the effects on murine haematopoiesis of FL alone or in combination with other growth factors were studied. Analysis of Flk-2/Flt-3 mRNA expression indicated that Flk-2/Flt-3 was preferentially expressed in primitive haematopoietic cell populations. To examine the expression of the Flk-2/Flt-3 receptor on megakaryocyte progenitors (CFU-Meg), Flk-2/Flt-3 positive and negative CD34(+)populations were separated from human bone marrow and cultured in a plasma clot culture system. CFU-Meg colonies were found in the Flk-2/Flt-3 negative fraction. Myeloid (CFU-GM) derived colonies appeared in the presence of FL alone. Neither FL+IL-3 nor FL+IL-3+IL-6 had any effect on the generation of megakaryocyte colonies (CFU-MK), due to the lack of FL receptor expression on megakaryocyte progenitors. Bone marrow cells remaining after 5-fluorouracil (5-FU) treatment of mice represent a very primitive population of progenitors enriched for reconstituting stem cells. This cell population expressed FL receptors, as revealed by RT-PCR analysis. Addition of FL alone did not enhance the replication of such cells in liquid cultures as compared to controls. However, a significantly greater generation of myeloid progenitors (CFU-GM) in clonogenic assays was observed in the presence of FL+IL-3, FL+GM-CSF or FL+CSF-1. In addition, the effects of FL on in vitro expansion of murine haematopoietic stem cells were studied using lineage-negative (lin(-)) Sca-1 positive (Sca-1(+)) c-kit positive (c-kit(+)) marrow cells from 5-FU treated mice. FL enhanced the survival of primitive murine lin(-)Sca-1(+)c-kit(+)cells. FL and IL-6 were able to significantly expand murine progenitor stem cells in vitro and promote their survival. These studies strongly suggest that FL significantly and selectively enhanced the generation of myeloid progenitors in vitro and increased myeloid progenitor responsiveness to later acting growth factors. In addition, FL synergized with IL-6 to support in vitro expansion of haematopoietic progenitors and promoted the survival of lin(-)Sca-1(+)c-kit(+)cells.     

The evaluation of the role of recovery from potentially lethal and sublethal damages in the RBE of densely ionizing radiation

The new data confirming the relations between RBE and recovery of cells are presented. The quantitative evaluation of the contribution of potentially lethal and sublethal damage recovery in radiosensitivity of cells of various origin after exposure to low- and high-LET ionizing radiation was carried out. The conclusion about the greater contribution of potentially lethal damage recovery in the magnitude of RBE in comparison with sublethal damage recovery was made.     

A novel radioresistant mechanism of galectin-1 mediated by H-Ras-dependent pathways in cervical cancer cells

Galectin-1 is a lectin recognized by galactoside-containing glycoproteins, and is involved in cancer progression and metastasis. The role of galectin-1 in radiosensitivity has not previously been investigated. Therefore, this study tests whether galectin-1 is involved in the radiosensitivity mediated by the H-Ras signaling pathway using cervical carcinoma cell lines. A knockdown of galectin-1 expression in HeLa cells decreased clonogenic survival following irradiation. The clonogenic survival increased in both HeLa and C33A cells with galectin-1 overexpression. The overexpression or knockdown of galectin-1 did not alter radiosensitivity, whereas H-Ras was silenced in both cell lines. Whereas K-Ras was knocked down, galectin-1 restored the radiosensitivity in HeLa cells and C33A cells. The knockdown of galectin-1 increased the high-dose radiation-induced cell death of HeLa cells transfected by constitutively active H-Ras. The knockdown of galectin-1 inhibited the radiation-induced phosphorylation of Raf-1 and ERK in HeLa cells. Overexpression of galectin-1 enhanced the phosphorylation of Raf-1 and ERK in C33A cells following irradiation. Galectin-1 decreased the DNA damage detected using comet assay and γ-H2AX in both cells following irradiation. These findings suggest that galectin-1 mediates radioresistance through the H-Ras-dependent pathway involved in DNA damage repair.     

Generation time of leukaemic blast progenitor cells

Previous studies have indicated that the generation time of human leukaemic cells is longer than that of normal haematopoietic cells. We have employed a modification of the thymidine (TdR)-suicide technique to measure directly the generation time of leukaemic progenitor cells capable of colony formation. The results obtained with two human leukaemic cell lines (KG-1 and HL-60) and with blast progenitor cells from two patients with acute myelogenous leukaemia indicate generation times ranging from 9 X 0-22 X 0 hr and S-phase durations ranging from 5 X 5-8 X 0 hr. Using the same technique, the generation time of normal bone marrow CFU-c was determined to be 9-11 hr. These findings suggest that the proliferation rate of human leukaemic blast progenitor cells is similar to that of normal haematopoietic stem cells.     

The relationship of DNA and chromosome damage to survival of synchronized X-irradiated L5178Y cells. I. Initial damage

We investigated the role of initial DNA and chromosome damage in determining the radiosensitivity difference between the variant murine leukemic lymphoblast cell lines L5178Y-S (sensitive) and L5178Y-R (resistant) and the difference in cell cycle-dependent variations in radiosensitivity of L5178Y-S cells. We measured initial DNA damage (by the neutral filter elution method) and chromosome damage (by the premature chromosome condensation method) and compared them with survival (measured by cloning) for both cell lines synchronized in G1 or G2 phase of the cell cycle (by centrifugal elutriation) and irradiated with low doses of X rays (up to 10 Gy). The initial yield of DNA and chromosome damage in G2 L5178Y-S cells was almost twice that in G1 L5178Y-S cells and G1 or G2 L5178Y-R cells. In all cases DNA damage expressed as relative elution corresponded with chromosome damage (breaks in G1 chromosomes, breaks and gaps in G2 chromosomes). Also we found that the initial DNA and chromosome damage did not determine cell age-dependent radiosensitivity variations in L5178Y-S cells, as there was less initial damage in the more sensitive G1 phase than in the G2 phase. L5178Y-R cells showed only small changes in survival or initial yield of DNA and chromosome damage throughout the cell cycle. Because survival and initial damage in sensitive and resistant cells irradiated in G2 phase correlated, the difference in radiosensitivity between L5178Y-S and L5178Y-R cells might be determined by initial damage in G2 phase only.     

Recovery from sublethal and potentially lethal damage in an X-ray-sensitive CHO cell

It has been suggested that DNA strand breaks are the molecular lesions responsible for radiation-induced lethality and that their repair is the basis for the recovery of irradiated cells from sublethal and potentially lethal damage. EM9 is a Chinese hamster ovary cell line that is hypersensitive to killing by X rays and has been reported to have a defect in the rate of rejoining of DNA single-strand breaks. To establish the importance of DNA strand-break repair in cellular recovery from sublethal and potentially lethal X-ray damage, those two parameters, recovery from sublethal and potentially lethal damage, were studied in EM9 cells as well as in EM9's parental repair-proficient strain, AA8. As previously reported, EM9 is the more radiosensitive cell line, having a D0 of 0.98 Gy compared to a D0 of 1.56 Gy for AA8 cells. DNA alkaline elution studies suggest that EM9 cells repair DNA single-strand breaks at a slower rate than AA8 cells. Neutral elution analysis suggests that EM9 cells also repair DNA double-strand breaks more slowly than AA8 cells. All of these data are consistent with the hypothesis that DNA strand-break ligation is defective in EM9 cells and that this defect accounts for increased radiosensitivity. The kinetics and magnitude of recovery from sublethal and potentially lethal damage, however, were similar for both EM9 and AA8 cells. Six-hour recovery ratios for sublethal damage repair were found to be 2.47 for AA8 cells and 1.31 for EM9 cells. Twenty-four-hour recovery ratios for potentially lethal damage repair were 3.2 for AA8 and 3.3 for EM9 cells. Both measurements were made at approximately equitoxic doses. Thus, the defect in EM9 cells that confers radiosensitivity and affects DNA strand-break rejoining does not affect sublethal damage repair or potentially lethal damage repair.     

Response of cultured normal human mammary epithelial cells to X rays

The effect of X rays on the reproductive death of cultured normal human mammary epithelial cells was examined. Techniques were developed for isolating and culturing normal human mammary epithelial cells which provide sufficient cells at second passage for radiation studies, and an efficient clonogenic assay suitable for measuring radiation survival curves. It was found that the survival curves for epithelial cells from normal breast tissue were exponential and had D0 values of about 109-148 rad for 225 kVp X rays. No consistent change in cell radiosensitivity with the age of donor was observed, and no sublethal damage repair in these cells could be detected with the split-dose technique.     

Recovery capacity of glial progenitors after in vivo fission-neutron or X irradiation: age dependence, fractionation and low-dose-rate irradiations

Previous experiments on the radiosensitivity of O-2A glial progenitors determined for single-dose fission-neutron and X irradiation showed log-linear survival curves, suggesting a lack of accumulation of recovery of sublethal damage. In the present study, we addressed this question and further characterized the radiobiological properties of these glial stem cells by investigating the recovery capacity of glial stem cells using either fractionated or protracted whole-body irradiation. Irradiations were performed on newborn, 2-week-old or 12-week-old rats. Fractionated irradiations (four fractions) were performed with 24-h intervals, followed by cell isolations 16- 24 h after the last irradiation. Single-dose irradiations were followed by cell isolation 16-24 h after irradiation or delayed cell isolation (4 days after irradiation) of the O-2A progenitor cells from either spinal cord (newborns) or optic nerve (2- and 12-week-old rats). Results for neonatal progenitor cell survival show effect ratios for both fractionated fission-neutron and X irradiation of the order of 1.8 when compared with single-dose irradiation. A similar ratio was found after single-dose irradiation combined with delayed plating. Comparable results were observed for juvenile and adult optic nerve progenitors, with effect ratios of the order of 1.2. The present investigation clearly shows that fractionated irradiation regimens using X rays or fission neutrons and CNS tissue from rats of various ages results in an increase in O-2A progenitor cell survival while repair is virtually absent. This recovery of the progenitor pool after irradiation can be observed at all ages but is greatest in the neonatal spinal cord and can probably be attributed to repopulation.     

Relative sensitivities of repair-deficient mammalian cells for clonogenic survival after alpha-particle irradiation

The clonogenic survival of cells of the radiation-sensitive hamster cell lines irs1, irs2, irs3 and xrs5, representing different DNA repair pathways, was compared to that of their parent lines after alpha-particle irradiation. Measurements of nuclear area were made to calculate the probability of surviving a single alpha-particle traversal, the average number of lethal lesions per track and per unit dose, along with the "intrinsic radiosensitivity" of these cells, allowing for the potential of multiple lethal lesions per traversal. For all cell lines studied, alpha particles were found to be more biologically effective per unit absorbed dose than X rays at inducing cell inactivation. The repair-deficient cells showed an enhanced sensitivity to alpha particles compared to their parent line, but the degree of enhancement was less than for X rays. The reduction in additional sensitivity for alpha-particle irradiation was shown not to be due predominantly to differences in cell geometry limiting the probability of a cell nucleus being traversed. The results suggest that both the nonhomologous end-joining pathway and to a lesser extent the homologous recombination repair pathway play a role in successful repair of alpha-particle-induced damage, although a large proportion of damage is not repaired by either pathway.     

The effects of hyperthermia and ionizing radiation in normal and ataxia telangiectasia human fibroblast lines

The effects of 45 degrees C hyperthermia and gamma radiation have been studied in three normal human fibroblast lines (GM38, GM730, WI38) and compared to the effects in two lines derived from patients with the hereditary disease ataxia telangiectasia (AT3BI, AT5BI). All lines, both normal and gamma-sensitive AT, showed a similar resistance to killing by heat alone, suggesting that the defect responsible for the increased radiation sensitivity in AT lines does not confer increased heat sensitivity. Shouldered survival curves were obtained in each case indicating the ability to accumulate sublethal heat damage. All normal and AT cell lines exhibited increased resistance to the lethal effects of heat in response to a thermal stress, indicating that the defect that causes radiosensitivity in AT cell lines does not prevent the induction of thermotolerance. Heat (45 degrees C, 30 min) was shown to increase the sensitivity of the normal cell lines to killing by gamma radiation. The thermal enhancement ratios obtained ranged from about 2.5 to 3.0. The same heat treatment, however, produced very little increase in the radiation sensitivity of the AT cells. Thermal enhancement ratios of about 1.2 were obtained in these lines. We hypothesize that, in normal cells, this heat treatment inactivates the process which is already defective in AT lines, and that this process may be required for the proper rejoining of double-strand breaks produced during the repair of other radiation-induced lesions.     

Differential sensitization of human tumor cells by ara-A to X irradiation and its relationship to inherent radioresponse.

We have previously shown that pretreatment of plateau-phase cultures of human tumor cells with ara-A can markedly sensitize them to the cytotoxic effects of X irradiation; the degree of sensitization varied in two different cell lines. The present study was undertaken to determine whether variability in radiosensitization by ara-A occurs at random in human tumor cell lines or if it is related to their intrinsic radiosensitivity (human tumor radioresponse). The interaction between ara-A and X irradiation was examined in plateau-phase cultures of early-passage tumor cell lines of varying radioresponse (D0 range 0.85-3.15 Gy) subcultured immediately after irradiation to measure survival. In six of the eight cell lines studied, pretreatment with ara-A greatly enhanced the lethal effects of X irradiation in a concentration-dependent fashion. Little or no effect was observed in the two radiosensitive cell lines. When ara-A sensitization was plotted as a function of D10 or D, a linear relationship was observed. These data suggest that pretreatment with ara-A is effective in sensitizing radiation-resistant human tumor cells to the lethal effects of X rays, and that this phenomenon may be dependent upon inherent tumor cell radiosensitivity.     

Correlation between PLD repair capacity and the survival curve of human fibroblasts in exponential growth phase: analysis in terms of several parameters

Published data on the in vitro radiosensitivity of 46 nontransformed fibroblasts of different genetic origins studied in plateau phase with immediate or delayed plating were used to investigate to what extent potentially lethal damage repair capacity is related to intrinsic radiosensitivity (i.e., irradiated in exponential growth phase). While most of the survival curve analysis is conducted in terms of D0, Dq, and the mean inactivation dose D, some of the data are also discussed in terms of the linear-quadratic model parameter alpha. Using D it is shown that: (i) the radiosensitivity of human fibroblasts in exponential growth phase does not significantly differ from that of plateau-phase fibroblasts with immediate plating; (ii) the radiosensitivity of plateau-phase cells with delayed plating is correlated to the radiosensitivity of cells with immediate plating: the more radioresistant the cell strain in exponential growth phase, the higher its repair capacity; (iii) the repair capacity of the cell strains is related to their genetic origin. In conclusion, we suggest that the survival curve of growing cells depends on the repair capacity of the cells.     

Breast Cancer Stem Cell-Like Cells Are More Sensitive to Ionizing Radiation than Non-Stem Cells: Role of ATM

There are contradictory observations about the different radiosensitivities of cancer stem cells and cancer non-stem cells. To resolve these contradictory observations, we studied radiosensitivities by employing breast cancer stem cell (CSC)-like MDA-MB231 and MDA-MB453 cells as well as their corresponding non-stem cells. CSC-like cells proliferate without differentiating and have characteristics of tumor-initiating cells [1]. These cells were exposed to γ-rays (1.25–8.75 Gy) and survival curves were determined by colony formation. A final slope, D₀, of the survival curve for each cell line was determined to measure radiosensitivity. The D₀ of CSC-like and non-stem MDA-MB-453 cells were 1.16 Gy and 1.55 Gy, respectively. Similar results were observed in MDA-MB-231 cells (0.94 Gy vs. 1.56 Gy). After determination of radiosensitivity, we investigated intrinsic cellular determinants which influence radiosensitivity including cell cycle distribution, free-radical scavengers and DNA repair. We observed that even though cell cycle status and antioxidant content may contribute to differential radiosensitivity, differential DNA repair capacity may be a greater determinant of radiosensitivity. Unlike non-stem cells, CSC-like cells have little/no sublethal damage repair, a low intracellular level of ataxia telangiectasia mutated (ATM) and delay of γ-H2AX foci removal (DNA strand break repair). These results suggest that low DNA repair capacity is responsible for the high radiosensitivity of these CSC-like cells.     

Inhibition of checkpoint kinase 1 sensitizes lung cancer brain metastases to radiotherapy

The most important therapeutic tool in brain metastasis is radiation therapy. However, resistance to radiation is a possible cause of recurrence or treatment failure. Recently, signal pathways about DNA damage checkpoints after irradiation have been noticed. We investigated the radiosensitivity can be enhanced with treatment of Chk1 inhibitor, AZD7762 in lung cancer cell lines and xenograft models of lung cancer brain metastasis. Clonogenic survival assays showed enhancement of radiosensitivity with AZD7762 after irradiation of various doses. AZD7762 increased ATR/ATM-mediated Chk1 phosphorylation and stabilized Cdc25A, suppressed cyclin A expression in lung cancer cell lines. In xenograft models of lung cancer (PC14PE6) brain metastasis, AZD7762 significantly prolonged the median survival time in response to radiation. Depletion of Chk1 using shRNA also showed an enhancement of sensitivity to radiation in PC14PE6 cells. The results of this study support that Chk1 can be a good target for enhancement of radiosensitivity.     

Promotion of haematopoietic activity in embryonic stem cells by the aorta-gonad-mesonephros microenvironment

We investigated whether the in vitro differentiation of ES cells into haematopoietic progenitors could be enhanced by exposure to the aorta-gonadal-mesonephros (AGM) microenvironment that is involved in the generation of haematopoietic stem cells (HSC) during embryonic development. We established a co-culture system that combines the requirements for primary organ culture and differentiating ES cells and showed that exposure of differentiating ES cells to the primary AGM region results in a significant increase in the number of ES-derived haematopoietic progenitors. Co-culture of ES cells on the AM20-1B4 stromal cell line derived from the AGM region also increases haematopoietic activity. We conclude that factors promoting the haematopoietic activity of differentiating ES cells present in primary AGM explants are partially retained in the AM20.1B4 stromal cell line and that these factors are likely to be different to those required for adult HSC maintenance.