Cellular effects of long wavelength UV light (UVA) in mammalian cells.

UVA should receive significant consideration as a human health risk as it is a large proportion of the solar spectrum that reaches the earth's surface and because of its ability to penetrate human skin. It is only relatively recently that this has been recognized and this previously under-researched part of the UV spectrum is becoming increasingly well characterized at doses that are quite low in relation to those experienced by humans. Absorption of UVA in a cell leads to the production of reactive oxygen and nitrogen species that can damage major biomolecules including DNA and membrane lipids. Various types of damage induced in these molecules lead to significant biological effects including cytotoxicity, mutations and alterations in cell signalling pathways. Longer-term effects such as persistent genomic instability and bystander effects have also been observed following UVA treatment of mammalian cells and, as with ionizing radiation, this changes some of the fundamental thinking around tissue effects of irradiation. Antioxidants have been assessed extensively for their ability to protect against the biological effects of UVA and a number have been shown to be successful at least in-vitro, for example vitamin E and epigallocatechin-3-gallate. Other potential targets for protection are suggested through the increased understanding of some of the signalling mechanisms activated following treatment, for example the inhibition of NADPH oxidase is seen to reduce a bystander effect. The search for appropriate and successful photoprotective agents remains an important area of research.

Assessing radiation-associated mutational risk to the germline: repetitive DNA sequences as mutational targets and biomarkers.

This review assesses recent data on mutational risk to the germline after radiation exposure obtained by molecular analysis of tandemly repeated DNA loci (TRDLs): minisatellites in humans and expanded simple tandem repeats in mice. Some studies, particularly those including exposure to internal emitters, indicate that TRDL mutation can be used as a marker of human radiation exposure; most human studies, however, are negative. Although mouse studies have suggested that TRDL mutation analysis may be more widely applicable in biomonitoring, there are important differences between the structure of mouse and human TRDLs. Mutational mechanisms probably differ between the two species, and so care should be taken in predicting effects in humans from mouse data. In mice and humans, TRDL mutations are largely untargeted with only limited evidence of dose dependence. Transgenerational mutation has been observed in mice but not in humans, but the mechanisms driving such mutation transmission are unknown. Some minisatellite variants are associated with human diseases and may affect gene transcription, but causal relationships have not yet been established. It is concluded that at present the TRDL mutation data do not warrant a dramatic revision of germline or cancer risk estimates for radiation.

Clinical and cellular ionizing radiation sensitivity in a patient with xeroderma pigmentosum.

XP14BR is a cell line derived from a xeroderma pigmentosum (XP) patient from complementation group C. The patient was unusual in presenting with an angiosarcoma of the scalp, treated by surgical excision and radiotherapy. Following 38 Gy in 19 fractions with 6 MEV electrons, a severe desquamation and necrosis of the underlying bone ensued, and death followed 4 years later. The cell line was correspondingly hypersensitive to the lethal effects of gamma irradiation. We had previously shown that this sensitivity could be discriminated from that seen in ataxia-telangiectasia (A-T). The cellular response to ultraviolet radiation below 280 nm (UVC) was characteristic of XP cells, indicating the second instance, in our experience, of dual cellular UVC and ionizing radiation hypersensitivity in XP. We then set out to evaluate any defects in repair of ionizing radiation damage and to verify any direct contribution of the XPC gene. The cells were defective in repair of a fraction of double strand breaks, with a pattern reminiscent of A-T. The cell line was immortalized with the vector pSV3neo and the XPC cDNA transfected in to correct the defect. The progeny derived from this transfection showed the presence of the XPC gene product, as measured by immunoblotting. A considerable restoration of normal UVC, but not ionizing radiation, sensitivity was observed amongst the clones. This differential correction of cellular sensitivity is strong evidence for the presence of a defective radiosensitivity gene, distinct from XPC, which is responsible for the clinical hypersensitivity to ionizing radiation. It is important to resolve how widespread ionizing radiation sensitivity is amongst XP patients.

Use of the tetrazolium assay in measuring the response of human tumor cells to ionizing radiation.

Three human tumor cell lines of widely differing radiosensitivity were used to examine the characteristics of the 3-[4,5-dimethyl(thiazol-2-yl)-3,5-diphery]tetradium bromide (MTT) assay and to select suitable conditions for its use in assessing the response of cells to ionizing radiation. The optimal concentration of MTT and the time of incubation of the cells with MTT were individualized for each cell line. The relationship between absorbance and cell number was not linear over the wide range of cell numbers that were used. A calibration curve of absorbance against cell number for each cell line was therefore used. Using the assay to quantify metabolically viable cells, growth curves of irradiated and unirradiated cells were constructed on days 0-14 after irradiation. Accurate surviving fractions could be calculated only when cells were in exponential growth. Using this modification to its interpretation, the MTT assay was able to provide a reproducible measure of survival, which compared well with clonogenic cell survival measurements. However, the necessity to optimize conditions of the MTT assay for each cell line severely limits its usefulness in determining the radiosensitivity of cells in primary human tumor cultures.

DNA strand break rejoining defect in xrs-6 is complemented by transfection with the human Ku80 gene.

The radiosensitive mutant xrs-6, derived from Chinese hamster ovary cell line CHO-K1, has been demonstrated to be defective in DNA double-strand break repair and also in its proficiency to undergo V(D)J recombination. Recent work has provided both genetic and biochemical evidence that the M(r) 80,000 subunit of the Ku protein is able to complement the radiosensitivity and the V(D)J recombination defect in the xrs-6 mutant. We demonstrate here that complementation of the radiosensitive phenotype in xrs-6 cells by the introduction of Ku80 cDNA is accompanied by the concomitant restoration of DNA double-strand break rejoining proficiency to almost that of the parental CHO-K1 cells, as measured both by neutral single-cell microgel electrophoresis (Comet) technique and by pulsed-field gel electrophoresis. These results provide further biochemical evidence for the involvement of the Ku protein in the repair of DNA double-strand breaks.

Enhanced experimental metastatic capacity of a murine melanoma following pre-treatment with anticancer drugs.

The effect of in vitro pretreatment of B16 murine melanoma cells with various cancer chemotherapeutic agents on their subsequent experimental metastatic capacity has been examined. Methotrexate, cytosine arabinoside, 5-azacytidine and aphidicolin all produced significant increases in the number of lung nodules formed following the i.v. injection of tumour cells. This effect was not seen with melphalan or 5-fluorouracil. Since the doses of melphalan and 5-fluorouracil used produced similar levels of cell kill to cytosine arabinoside and aphidicolin it appears that a cytotoxic effect was not sufficient to produce increased lung nodule formation. Analysis of the perturbations in the cell cycle induced by the drugs did not reveal any consistent differences between those drugs which enhanced experimental metastasis and those which did not. The precise mechanisms underlying this phenomenon remain to be elucidated but the results are consistent with the possibility that anticancer agents may play a role in assisting tumour progression.

Residual DNA damage: what is left over and how does this determine cell fate?

Radiation-induced DNA damage may remain unrepaired for a number of reasons: it may be too severe, it may be in inaccessible parts of the genome, it may be induced at critical points in the cell cycle or it may be converted into large DNA deletions. This residual damage is likely to be responsible for cell death either by physical restriction of replication or transcription or by metabolic disruption due to loss of function of critical genes. Although residual damage is important, cells may differ in their ability to tolerate it, which may be a factor that determines the relative radiosensitivity of a given cell population.

Measurement of radiation survival using the MTT assay.

There is increasing interest in the development of rapid assays of radiosensitivity which can be used on clinical specimens. Unfortunately, the measurement of radiation survival using clonogenic assays, which are the established standard, can be difficult and time consuming. We have used the MTT assay to measure the radiation survival of four lymphoblastoid cell lines with low plating efficiencies. We measured surviving fractions both when the irradiated cells had regained exponential growth and when the non-irradiated cells had undergone four or more doublings. The results were compared to surviving fractions measured by clonogenic assay. We found both methods could be used successfully to rank the cell lines in order of radiosensitivity. However, cells exposed to the higher radiation doses in the MTT assay did not always regain exponential growth, limiting the dose range for which the assay was useful. We also found the best correlation between the two assays was sometimes obtained by using the MTT surviving fractions from different days for different radiation dose levels. Thus, although the MTT assay can be used to measure radiation survival in relation to other cell lines, its use can be complicated by restrictions on radiation dose ranges and difficulties with data interpretation.

High frequency of double drug resistance in the B16 melanoma cell line.

Methotrexate (MTX) and N-(phosphonacetyl)-L-aspartate (PALA) are two agents to which cellular resistance can be conferred by gene amplification, but they do not generally show cross resistance. However, combined treatment with these two agents produced drug resistant cells in the B16 melanoma cell line at a much higher frequency than would be expected if resistance to the two agents was totally independent. An isolated doubly resistant clone, B16-F1 MP, showed a high frequency of resistance to pyrazofurin and ouabain, which are also agents to which resistance can be conferred by gene amplification. Thus MTX combined with PALA selected cells with an 'amplificator' phenotype (an increased ability to amplify parts of the genome). These B16-F1 MP cells had a decreased ability to form experimental lung metastases compared with the parent line but this difference was not found in baby hamster kidney cells with the amplificator phenotype. The mechanism underlying drug resistance may need to be considered when designing combination chemotherapy.

Molecular assays of radiation-induced DNA damage.

There is a need for assays of DNA damage in many areas of laboratory research applied to radiation therapy, in order to understand the molecular processes involved in cell killing by ionising radiation and to predict in vivo response. Assays exist which measure many types of DNA damage following ionising radiation. From studies of the dose-response relationships for different types of damage, the double-strand break (dsb) has been shown to be the most significant lesion. Assays for DNA dsb have been of low sensitivity, such that supralethal doses of radiation had to be used in order to study dsb induction or repair. New assays, such as pulsed-field gel electrophoresis, are sensitive to dsb in a dose range relevant to cell survival. In addition, these assays can assess the distribution of dsb in different parts of the genome and determine heterogeneity of damage induction and repair. Assays which measure the effects of strand breaks on DNA complexed with nuclear matrix can reveal features of chromatin organisation and their influence on cellular radiosensitivity.

Differences in the level of DNA double-strand breaks in human tumour cell lines following low dose-rate irradiation.

It is now well accepted that differences exist in the intrinsic radiosensitivity of human tumour cells although the molecular basis of this is still unclear. Current evidence suggests that of the lesions induced in DNA by ionising radiation, double-strand breaks (DSB) are the most closely linked to cell death. In this study, levels of DSB were measured by neutral filter elution under conditions of both repair inhibition and maximum recovery and compared with clonogenic survival curves for high (HDR) and low dose-rate (LDR) irradiation in human carcinoma lines of differing radiosensitivity. Four human lung carcinoma lines were used, two small-cell (SCLC; HC12 and HX149) and two non-small cell lines (NSCLC; HX147A7 and HX148G7). Cell survival was measured by soft agar and monolayer colony-forming assays as appropriate and a large variation in sensitivity of the cell lines was seen (alpha values of 0.06 to 0.56 Gy-1). We have previously reported that the damage induced at high dose rate does vary in these cell lines but not in a way which correlates with their cell survival response [5]. Following irradiation to 15 Gy at low dose rate essentially no DSBs were detected in any of the four lines but at 70 Gy the more sensitive SCLC showed more residual damage than in the more radioresistant NSCLC lines. The prime determinant of the difference between the LDR and HDR damage curves is likely to be repair occurring during irradiation.(ABSTRACT TRUNCATED AT 250 WORDS)

The repair fidelity of restriction enzyme-induced double strand breaks in plasmid DNA correlates with radioresistance in human tumor cell lines.

PURPOSE: The accuracy of DNA repair may play a role in determining the cytotoxic effect of ionizing radiation. Repair, as measured by DNA strand breakage, often shows little difference between tumor cell lines of widely different radiosensitivity. The mechanism by which DNA fragments are rejoined is poorly understood. This study used plasmid transfection as a probe to assess the balance between correct repair and misrepair. METHODS AND MATERIALS: Using techniques described, a double-strand break was introduced into a coding sequence of circular plasmid DNA using a restriction endonuclease as a model for a radiation-induced double-strand break; it was then transfected as a linear molecule into human tumor cells, and the subsequent cell-mediated restoration of the coding sequence, evidenced by intact gene function, was documented. The plasmid used in these experiments, pPMH16, is known to integrate into genomic DNA. Gene function was tested by the ability to grow colonies in selection media. The plasmid also contains a second selectable marker gene that was used to identify transfected cells, before the function of the damaged gene was tested. The proportion of transfected cells that had correctly restored the damaged gene gave a measure of repair fidelity. RESULTS: A general trend for sensitive cells to show lower repair fidelity relative to resistant cells was observed. The type of double-strand cleavage of the plasmid (staggered or blunt) made little difference to the measured repair fidelity, in contrast to published studies in which restriction-enzyme breaks had been introduced into DNA within chromatin. Specific comparison of parent lines and their radiosensitive clones showed significant differences in repair fidelity for a relatively small change in radiation response, which was in line with the overall correlation. These same pairs have previously been shown to have no difference in the loss of DNA fragmentation with time after irradiation, and Southern analysis had confirmed the integrated plasmid copy number was similar in the cell lines compared. The number of intact copies of the damaged gene relative to the undamaged gene mirrored the observed repair fidelity. However, in one cell line out of the 10 studied, an exception to the observed trend was found. In a comparison of two equally radioresistant bladder cancer cell lines, large differences in repair fidelity were observed. Again, no difference in the integrated copy number was found, and the damaged gene was highly rearranged or deleted in the cell line with low repair fidelity. CONCLUSION: These studies have shown repair fidelity to correlate closely with radiosensitivity, including the comparison of genetically related lines. It is suggested that repair fidelity can be, but is not invariably, a measure of correct repair relative to misrepair, resulting from the processing of double-strand breaks and, hence, the response to ionizing radiation.

The use of DNA double-strand break quantification in radiotherapy.

DNA double-strand breaks (DSB) are an important direct consequence of treating cells with ionizingradiation. A variety of evidence points toward DSBs being the key damage type linked to radiation-induced lethality. In particular, the link between DSB and chromosome breakage, which in turn closely correlates with cell death in some cell types, is strongly supportive of this concept. There has been much interest in the possibility of using measures of strand breaks as a pretreatment test of radiation response. This has largely been in the context of assessing inherent cellular sensitivity through damage induction or repair parameters. A number of studies have produced hopeful results, but overall there has been no parameter that can reliably predict radiosensitivity. This may be due to the inadequacies of the assays, but it is more likely to reflect the fact that the radiosensitivity of cells is dictated by a whole series of events; alterations in many of these can alter the overall response. In addition, it is now recognized that cell-signalling pathways form an essential part of the cellular response to damage, and these can be triggered by damage other than DSB. It is therefore possible that while DSBs are clearly important--and they may be the single most important lesion in some types--other damage types may be significant triggers of cell death pathways after ionizing radiation treatment.

Heterogeneity of radiosensitivity in a human glioma cell line.

Sixteen clones were isolated from an early-passage human glioma cell line (IN859) and have been found to show variation in several biological characteristics including DNA content, modal chromosome number, and morphology. In addition, heterogeneity of radiosensitivity was detected: the doses that gave a surviving fraction of 0.01 varied by a factor of approximately 1.5. The most sensitive (clone 6) and the most resistant (clone 9) clones were selected for further study; their surviving fractions at 2Gy (SF2) were 0.37 and 0.64, respectively. When compared at a fixed radiation dose the sensitive clone surprisingly demonstrated greater split-dose recovery than the resistant clone; it also showed greater low dose-rate sparing.

Clonal variation of DNA repair in a human glioma cell line.

Clonal heterogeneity in response to ionizing radiation was found for a human glioma cell line, IN859. We have investigated the most sensitive clone, the most resistant clone and the parent line for differences in DNA repair fidelity using the method of plasmid reconstitution. Significant differences in repair fidelity were found between the two clones, and between the sensitive clone and the parent line. The resistant clone and the parent line showed the greater repair fidelity. A comparison of two different restriction enzymes, which cleave the plasmid with blunt or cohesive-ended double-strand breaks, did not reveal differences in repair fidelity. Equal numbers of plasmids were integrated in each cell line, but the sensitive clone showed a higher frequency of misrepair of cleaved plasmid. Misrepair was characterized by partial or complete loss of sequence at the site of plasmid cleavage. We conclude that the radiosensitive clone exhibits increased misrepair.

Radiosensitivity, recovery and dose-rate effect in three human glioma cell lines.

The results of radiotherapy in the treatment of high-grade gliomas are disappointing. In this study three recently established cell lines from high-grade human gliomas have been found to exhibit a sensitivity that is at the resistant end of the spectrum of radiosensitivities seen in human tumour cells generally. The results support the view that inherent cellular radioresistance may be an important cause of failure in this disease. All three cell lines showed an increase in survival when the radiation dose rate was reduced. In split-dose experiments, recovery was found to increase with dose in a manner consistent with the predictions of the linear-quadratic equation.

Cell-cycle progression during continuous low dose rate irradiation of a human bladder carcinoma cell line.

At very low radiation dose rates, the proliferation of mammalian cells continues unaffected but as the dose rate is increased there comes a point at which it is interrupted. The dose rate at which this happens is often thought to be a significant factor in the effects of brachytherapy: it may determine the range from an implanted source at which cell-cycle redistribution and repopulation effects will occur. By means of mitotic counts and DNA flow cytometry, we have examined the dose rate effect in a human bladder carcinoma cell line (MGH-U1). Irradiation at dose rate 0.1 cGy/min had little or no effect on cell-cycle progression. Suppression of mitosis and arrest of cells in G2 was observed at 0.4 cGy/min and above. Surprisingly, the duration of mitotic arrest showed little dose rate dependence; it was followed by an overshoot of cells in mitosis after 24-39 h of irradiation. An even more pronounced overshoot of cells in G2 occurred and persisted throughout the irradiation period. The cell kinetic data indicate that after the temporary block in cell-cycle progression, cell proliferation continued at all dose rates up to 1.4 cGy/min. We have evaluated these results in the light of previous studies in this department of the dose rate effect for cell survival in the MGH-U1 cell line. After 24 h irradiation at 1.4 cGy/min the surviving fraction was below 10(-2), also after 30 h at 1.0 cGy/min. When cell-cycle blockade is considerable, so is the level of cell killing. Flow-cytometric data therefore are dominated by the properties of cells that are doomed to die.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of cell attachment matrix (CAM) coated plates for primary culture of human tumour biopsies.

Baker et al. [Cancer Res. 46: 1263-1274, 1986] developed an adhesive tumour cell culture system (ATCCS) using a culture surface coated with a cell attachment matrix (CAM). The ability of CAM-coated plates to support the growth of cells from human tumour biopsies has been evaluated. Successful growth was obtained in 9/22 samples (41%), but fibroblasts, rather than tumour cells, grew in the majority. Comparison of CAM with other surfaces showed that CAM was no better for establishing tumour cell growth than the presence of feeder cells or an alternative attachment factor vitronectin.

Relationship between DNA damage, rejoining and cell killing by radiation in mammalian cells.

The prevailing hypothesis on the mechanism of radiation-induced cell killing identifies the genetic material deoxyribonucleic acid (DNA) as the most important subcellular target at biologically relevant doses. In this review we present new data and summarize the role of the DNA double-strand breaks (dsb) induced by ionizing radiation and DNA dsb rejoining as determinants of cellular radiosensitivity. When cells were irradiated at high dose-rate, two molecular end-points were identified which often correlated with radiosensitivity: (1) the apparent number of DNA dsb induced per Gy per DNA unit and (2) the half-time of the fast component of the DNA dsb rejoining kinetics. These two molecular determinants, not mutually exclusive, may be linked through a common factor such as the conformation of DNA.

Variation in sensitizing effect of caffeine in human tumour cell lines after gamma-irradiation.

BACKGROUND AND PURPOSE: We have investigated whether the protective role of the G2 checkpoint has increasing importance when the p53-dependent G1 checkpoint is inactivated. MATERIALS AND METHODS: We have studied the differential effect of caffeine by clonogenic assays and flow cytometry in three human tumour cell lines with different functionality of p53 protein. RESULTS: The radiosensitizing effect of caffeine (2 mM) expressed itself as a significant decrease in surviving fraction at 2 Gy and a significant increase in alpha-values in RT112 and TE671, both with non-functional p53. However, no radiosensitizing effect was seen in cells with a normal p53 function (MCF-7 BUS). Two millimoles of caffeine also caused important changes in the cell cycle progression after irradiation. MCF-7 BUS showed a G1 arrest after irradiation and an early G2 arrest but those cells that reached the second G2 did not arrest significantly. In contrast, TE671 exhibited radiosensitization by caffeine, no G1 arrest, a G2 arrest in those cells irradiated in G2, no significant accumulation in the second G2 but an overall delay in release from the first cell cycle, which could be abrogated by caffeine. RT112 was similar to TE671 except that the emphasis in a G2 arrest was shifted from the block in cells irradiated in G2 to those irradiated at other cell cycle phases. CONCLUSION: The data presented confirm that p53 status can be a significant determinant of the efficacy of caffeine as radiosensitizer in these tumour cell lines, and document the importance of the G2 checkpoint in this effect.