Radiation-sensitive Cell Lines Research Articles

A Radiosensitizing Inhibitor of HIF-1 alters the Optical Redox State of Human Lung Cancer Cells In Vitro

Treatment failure caused by a radiation-resistant cell phenotype remains an impediment to the success of radiation therapy in cancer. We recently showed that a radiation-resistant isogenic line of human A549 lung cancer cells had significantly elevated expression of hypoxia-inducible factor (HIF-1α), and increased glucose catabolism compared with the parental, radiation-sensitive cell line. The objective of this study was to investigate the longitudinal metabolic changes in radiation-resistant and sensitive A549 lung cancer cells after treatment with a combination of radiation therapy and YC-1, a potent HIF-1 inhibitor. Using label-free two-photon excited fluorescence microscopy, we determined changes in the optical redox ratio of FAD/(NADH and FAD) over a period of 24 hours following treatment with YC-1, radiation, and both radiation and YC-1. To complement the optical redox ratio, we also evaluated changes in mitochondrial organization, glucose uptake, reactive oxygen species (ROS), and reduced glutathione. We observed significant differences in the optical redox ratio of radiation-resistant and sensitive A549 cells in response to radiation or YC-1 treatment alone; however, combined treatment eliminated these differences. Our results demonstrate that the optical redox ratio can elucidate radiosensitization of previously radiation-resistant A549 cancer cells, and provide a method for evaluating treatment response in patient-derived tumor biopsies.

Expression of muitidrug resistance-associated proteins 4 protein in colorectal cancer cell lines with different radiosensitivity

Objective To screen the radiation-resistant and radiation-sensitive cell strains from 5 colorectal cancer cell lines and observe the muitidrug resistance-associated proteins 4 (MRP4) protein expression in two cell lines,and explore the relashionship between MRP4 protein expression and radiation sensitivity.Methods Different radiation doses (0,2,4,6 and 8 Gy) were used to irradiate colonrectal cancer cell lines LOVO,DLD-1,HT-29,HCT116 and RKO,and radiation sensitive and radiation resistant cell lines were screened.Immunohistochemistry was used to detect MRP4 protein expression in these two cell lines.Results Radiation sensitive and radiation resistant cell lines were LOVO and RKO separately.Their growth inhibition curve after the intervention was significantly different (P ＜0.01).For MRP4 protein expression in the radiation resistant RKO and sensitive LOVO cell lines,high expression and low expression rate was 71％ and 17％,and 29％ and 83％ separately with the difference being significant (P ＜0.01).Conclusion The low-expression of MRP4 protein in radiation-sensitive cell line LOVO and the high-expression of MRP4 protein in RKO radiation-resistant cell line reveals that MRP4 protein expression is not only related to chemotherapy resistance,but also radiotherapy resistance. Key words: Colorectal cancer; Radiosensitivity; Muitidrug resistance-associated proteins 4

The WST survival assay: an easy and reliable method to screen radiation-sensitive individuals

An easy, fast and reliable method was developed to screen hundreds of Epstein-Barr virus-transformed cell lines (lymphoblastoid cell lines, LCLs) for radiation sensitivity that were generated from lymphocytes isolated from young lung cancer patients. The WST-1 test explores the metabolic activity of the mitochondria as an indicator for the vital status of cells. Cell proliferation as well as indirect cell death can be quantified by this method on a large scale in microtiter plates. Cell survival was measured at 24- and 48-h post-irradiation with 10 Gy ((137)Cs source) by the WST-1 assay and Trypan blue staining. To set up the experimental screening conditions and to establish a positive and a negative control, an ATM-mutated cell line from a radiation-sensitive ATM patient and an ATM proficient cell line from a healthy brother were compared. An optimal differentiation between the two cell lines was demonstrated for 10 Gy and 24- and 48-h cell growth after irradiation. Upon screening 120 LCLs of young lung cancer patients under these conditions, 5 of them were found to be radiation sensitive to a high degree of statistical significance. The results have been confirmed by a second laboratory by means of Trypan blue testing. The WST-1 test represents an efficient and reliable method by means of screening for radiation-sensitive cell lines.

Impaired NHEJ function in multiple myeloma

Multiple myeloma (MM) is characterized by multiple chromosomal aberrations. To assess the contribution of DNA repair to this phenotype, ionizing radiation was used to induce DNA double strand breaks in three MM cell lines. Clonogenic survival assays showed U266 (SF4 = 15.3 + 6.4%) and RPMI 8226 (SF4 = 12.6.0 + 1.7%) were radiation sensitive while OPM2 was resistant (SF4 = 78.9 + 4.1%). Addition of the DNA-PK inhibitor NU7026 showed the expected suppression in radiation survival in OPM2 but increased survival in both radiation sensitive cell lines. To examine non-homologous end joining (NHEJ) repair in these lines, the ability of protein extracts to support in vitro DNA repair was measured. Among the three MM cell lines analyzed, RPMI 8226 demonstrated impaired blunt ended DNA ligation using a ligation-mediated PCR technique. In a bacterial based functional assay to rejoin a DNA break within the β-galactosidase gene, RPMI 8226 demonstrated a 4-fold reduction in rejoining fidelity compared to U266, with OPM2 showing an intermediate capacity. Ionizing radiation induced a robust γ-H2AX response in OPM2 but only a modest increase in each radiation sensitive cell line perhaps related to the high level of γ-H2AX in freshly plated cells. Examination of γ-H2AX foci in RPMI 8226 cells confirmed data from Western blots where a significant number of foci were present in freshly plated untreated cells which diminished over 24 h of culture. Based on the clonogenic survival and functional repair assays, all three cell lines exhibited corrupt NHEJ repair. We conclude that suppression of aberrant NHEJ function using the DNA-PK inhibitor NU7026 may facilitate access of DNA ends to an intact homologous recombination repair pathway, paradoxically increasing survival after irradiation. These data provide insight into the deregulation of DNA repair at the site of DNA breaks in MM that may underpin the characteristic genomic instability of this disease.

Transcriptional response to ionizing radiation in human radiation sensitive cell lines

Background and purpose Radiation is a common treatment of cancer, but some patients show severe side effects when exposed to small doses of radiation. The aim of this study was to explore the underlying cause of radiation sensitivity in a group of radiation sensitive patients. Materials and methods Lymphoblastoid cell lines from 5 normal individuals, 4 Ataxia Telangiectasia (AT), and 12 non-AT radiation sensitive (RS) patients were irradiated. RNA was isolated before and after radiation and hybridized to 15k cDNA microarrays and gene expression was recorded. Results and conclusion The RS cell lines showed an expression phenotype different from both the AT and normal cell lines. Six of the RS cell lines had a distinct expression profile before radiation. This implies that the RS patients are a heterogeneous group, but that six of the patients may have a common cause of radiation sensitivity.

Gene expression studies in radiation-sensitive cell lines

Gene expression studies in radiation-sensitive cell lines

Survivin enhances radiation resistance in primary human glioblastoma cells via caspase-independent mechanisms

The observed radioresistance of human glioblastoma multiforme (GBM) poses a major challenge, which, if overcome, may lead to significant advances in the management of this patient population. There is accumulating evidence from correlative studies that Survivin expression is associated with increased malignant potential of human gliomas. The purpose of this study was to investigate whether Survivin plays a direct role in mediating radiation resistance in primary human glioma cell lines, and, if so, investigating the underlying mechanisms. Our panel of GBM cell lines included two that were relatively radiation resistant (GM20 and GM21) and two that were more radiation sensitive (GM22 and GM23), which demonstrated differential levels of Survivin expression between the two groups. Through the use of adenoviral vectors containing either dominant-negative (pAd-S(T34A)) or wild-type Suvrivin (pAd-S(WT)), we were able to inactivate or overexpress Survivin, respectively. Our findings suggest that Survivin plays a critical role in mediating radiation resistance in primary GBM cells, in part through suppression of apoptotic cell death via a caspase-independent manner. We have identified novel mechanisms by which Survivin may enhance tumor cell survival upon radiation exposure such as regulation of double-strand DNA break repair and tumor cell metabolism, which were most evident in the radiation-resistant cell lines. These differences in Survivin function both in radiation-resistant vs radiation-sensitive cell lines and in the presence vs absence of radiation exposure warrant further investigation and highlight potentially important mechanisms of radiation resistance in these tumors.

Genome-wide screening for radiation response factors in head and neck cancer.

Radiation therapy is an integral part of the treatment of head and neck cancer. Factors predicting radiation response are ill defined. The aim of this study was to identify genetic aberrations associated with radiation response in cell lines derived from head and neck squamous cell carcinomas (HNSCC) using comparative genomic hybridization (CGH) for genome-wide screening. Five cell lines derived from HNSCC were subjected to a single course of radiation (400 cGy) in parallel with a similarly handled, untreated control. Cellular response to radiation was determined on posttreatment days 1, 2, 3, 4, and 5 using a cell viability assay (MTT assay). Radiation response was defined as 35% or greater decrease in cell survival relative to control. Tumor doubling time was determined by cell counts performed at day 0 and 1 for each cell line. All experiments were done in quadruplicate. CGH analysis was performed by differentially labeling DNA from tumor and normal tissue with fluorescent agents. The labeled DNAs were simultaneously hybridized to normal metaphase chromosomes. Image analysis for fluorescence intensity along the entire length of each metaphase chromosome allowed generation of a color ratio, which was used to detect copy number changes. Radioresistance was identified in two of five cell lines. The tumor doubling time was not a predictor of radiation response. CGH identified a complex pattern of aberrations, with gain of 3q common to all cell lines. The number of genetic aberration was higher in radiation-sensitive cell lines than in radiation-resistant ones. No recurrent aberrations were unique to the radiation-resistant cell lines. Recurrent gains at 7p and 17q and losses at 5q, 7q, and 18q were unique to the radiation-sensitive cell lines. The number of aberrations identified by CGH analysis may be a predictor of radiation response. A large study of primary tumors is warranted to confirm this association and identify specific genetic aberrations associated with radiation response.

Loss of heterozygosity induced by a chromosomal double-strand break.

The repair of chromosomal double-strand breaks (DSBs) is necessary for genomic integrity in all organisms. Genetic consequences of misrepair include chromosomal loss, deletion, and duplication resulting in loss of heterozygosity (LOH), a common finding in human solid tumors. Although work with radiation-sensitive cell lines suggests that mammalian cells primarily rejoin DSBs by nonhomologous mechanisms, alternative mechanisms that are implicated in chromosomal LOH, such as allelic recombination, may also occur. We have examined chromosomal DSB repair between homologs in a gene targeted mammalian cell line at the retinoblastoma (Rb) locus. We have found that allelic recombinational repair occurs in mammalian cells and is increased at least two orders of magnitude by the induction of a chromosomal DSB. One consequence of allelic recombination is LOH at the Rb locus. Some of the repair events also resulted in other types of genetic instability, including deletions and duplications. We speculate that mammalian cells may have developed efficient nonhomologous DSB repair processes to bypass allelic recombination and the potential for reduction to homozygosity.

Mammalian endo-exonuclease activity and its level in various radiation sensitive cell lines.

The levels of endo-exonuclease in various mammalian cell lines were examined. While the expression of the endo-exonuclease during cell growth behaved exactly the same as the pattern observed in lower eukaryotes, the amount of activity was found to be reduced in the radiosensitive Chinese hamster ovary (CHO) xrs-5 and various human AT, AT-5 and NE-1 cells when compared to the radionormal CHO K1 and human HeLa cell lines. The reduced endo-exonuclease activity in these cells was due to a decreased amount of protein as demonstrated with the immuno-blot method. The results presented here suggest that endo-exonuclease may be one of the many proteins whose expression is regulated by genes coding for xrs-5 in CHO and AT in humans.

Postirradiation exposure to hypotonic saline shows normal damage processing in radiation-sensitive cell lines.

Three pairs of cell lines (one human and two Chinese hamster ovary (CHO) cell lines) each comprising a cell line with a normal radiation response and a radiation-sensitive mutant, were evaluated for recovery of potentially lethal damage (PLDR) and recovery of sublethal damage (SLDR). In all cases, the normal cell lines (GM1522, human; AA8-4 and K1, CHO) exhibited capacity for PLDR and SLDR was also normal in the two CHO lines. For the mutants (GM3395, human AT; V3 and 5-11, CHO) there was no ability for PLDR and SLDR was also absent in the two CHO cell lines. Postirradiation exposure to hypotonic NaCl solutions resulted in fixation of radiation damage in all the cell lines. This form of damage is repaired if left unperturbed after irradiation. This shows that cells have a large capacity for repair of this form of damage which accounts for much greater changes in survival than those observed in conventional PLDR experiments. These data show that the mutant cell lines retained their capacity to repair the damage which was susceptible to postirradiation fixation by anisotonic salt solutions. In addition, initial (i.e. preirradiation) DNA polymerase activities were measured in the four CHO cell lines; they were not correlated to radiation sensitivity.

Sublethal damage repair in squamous cell carcinoma cell lines.

Squamous cell carcinoma (SCC) cell lines recently established from head and neck tumors were studied for their capability of repairing sublethal radiation induced damage (SLDR). Total doses of 1.25, 2.50, 5.00, and 7.50 Gy were used either as a single dose or split in two equal fractions with a 24 h interval. Cell survival was determined using a 96-well plate clonogenic assay based on limiting dilution. Survival data was fitted by linear quadratic model, and the area under the survival curve (AUC) was obtained with numerical integration. The amount of SLDR was expressed as AUC-ratio comparing survivals from split dose vs single dose experiments. SLDR capacity was observed to vary markedly between individual cell lines (AUC-ratios 1.0-1.5). The relatively radiation-sensitive cell lines had a tendency toward higher SLDR proficiency (correlation coefficient 0.85). The differences in repair capacity could not be explained by the differences in doubling times of the cell lines. The inverse relationship between SLDR capacity and inherent radiosensitivity could explain the poor radiocurability of the sensitive donor tumors. Two of the most resistant cell lines were found SLDR deficient. This is a novel finding, since SLDR has been previously reported in all studied cells.

Induction, repair and biological relevance of radiation-induced DNA lesions in eukaryotic cells

This report summarizes data on the induction, repair and biological relevance of five types of radiation-induced DNA lesions for which repair kinetic studies have been performed in eukaryotic cells by various laboratories. These lesions are: DNA-protein crosslinks, base damage, single-strand breaks, double-strand breaks and bulky lesions (clustered base damage in the nm-range). The influence of various factors, such as oxia/anoxia, linear energy transfer of the radiation used, incubation medium, cell cycle stage, thiol content, hyperthermia, on the induction and repair of these lesions is described. Radiation-sensitive cell lines are also included.