Bystander Me45 Melanoma Cells Increase Damaging Effect in UVC-irradiated Cells.

The aim of our study was to investigate the possible mechanism(s) of the bystander effect induced by UVC light in malignant melanoma Me45 cells that were co-incubated with irradiated cells of the same line. We have found that the UVC band effectively generated apoptosis, premature senescence, single and double DNA strand breaks and reduced clonogenic survival of bystander cells. However, in the feedback response, the bystander cells intensified damage in directly irradiated cells, especially seen at the level of apoptosis and survival of clonogenic cells. Pretreatment of bystander cells with inhibitor of inducible nitric oxide synthase blocks this signaling. It seems that the mediators of this phenomenon produced and secreted by neighboring cells are superoxide, nitric oxide and TGF-ß. The reverse deleterious effect caused by cells not exposed to UVC in directly exposed cells is opposed to the protective/rescue effect exerted by the bystander cells in the case of ionizing radiation known in the literature. Whether this opposite adverse effect is a feature of only Me45 melanoma cells or whether it is a general phenomenon occurring between cells of other types exposed to ultraviolet radiation requires further research.

Modulation by neighboring cells of the responses and fate of melanoma cells irradiated with UVA.

UVA radiation, which accounts for about 95% of the solar spectrum, contributes to and may be the etiological factor of skin cancers of which malignant melanoma is the most aggressive. UVA causes oxidative stress in various types of cells in the skin, keratinocyte, melanocytes, and fibroblasts, which is responsible for its cytotoxic effect. Here we used a transwell system to explore how the responses of melanoma cells to a low dose of UVA (20kJ/m2, ~10% of the minimal erythema dose) are influenced by neighboring co-cultured melanoma cells or fibroblasts. This dose had a low toxicity for melanoma cells, but after irradiation, co-culture with non-irradiated melanoma cells caused a strong decline in their viability and an increased frequency of apoptosis, whereas co-culture with fibroblast exerted a protective effect on irradiated melanoma cells. At the same time, the presence of non-irradiated cells, especially fibroblasts, decreased the level of UVA-induced reactive oxygen and nitrogen species. Interleukins efficiently produced by fibroblasts seem to be main players in these effects. Our studies reveal that coexistence of fibroblasts with melanoma cells may strongly modulate the direct action and may change bystander effects exerted by UVA light. Similar modulation of the effect of UVA on melanoma cells in vivo by bystander-like signaling from neighboring cells would have consequences for the development of malignant melanoma.

The different radiation response and radiation-induced bystander effects in colorectal carcinoma cells differing in p53 status.

Radiation-induced bystander effect, appearing as different biological changes in cells that are not directly exposed to ionizing radiation but are under the influence of molecular signals secreted by irradiated neighbors, have recently attracted considerable interest due to their possible implication for radiotherapy. However, various cells present diverse radiosensitivity and bystander responses that depend, inter alia, on genetic status including TP53, the gene controlling the cell cycle, DNA repair and apoptosis. Here we compared the ionizing radiation and bystander responses of human colorectal carcinoma HCT116 cells with wild type or knockout TP53 using a transwell co-culture system. The viability of exposed to X-rays (0-8 Gy) and bystander cells of both lines showed a roughly comparable decline with increasing dose. The frequency of micronuclei was also comparable at lower doses but at higher increased considerably, especially in bystander TP53-/- cells. Moreover, the TP53-/- cells showed a significantly elevated frequency of apoptosis, while TP53+/+ counterparts expressed high level of senescence. The cross-matched experiments where irradiated cells of one line were co-cultured with non-irradiated cells of opposite line show that both cell lines were also able to induce bystander effects in their counterparts, however different endpoints revealed with different strength. Potential mediators of bystander effects, IL-6 and IL-8, were also generated differently in both lines. The knockout cells secreted IL-6 at lower doses whereas wild type cells only at higher doses. Secretion of IL-8 by TP53-/- control cells was many times lower than that by TP53+/+ but increased significantly after irradiation. Transcription of the NFκBIA was induced in irradiated TP53+/+ mainly, but in bystanders a higher level was observed in TP53-/- cells, suggesting that TP53 is required for induction of NFκB pathway after irradiation but another mechanism of activation must operate in bystander cells.

Connecting radiation-induced bystander effects and senescence to improve radiation response prediction.

For the last two decades radiation-induced bystander effects (RIBEs) have attracted significant attention due to their possible implications for radiotherapy. However, despite extensive research, the molecular pathways associated with RIBEs are still not completely known. In the current study we investigated the role of senescence in the bystander response. Irradiated (2, 4, 6 and 8 Gy) human colorectal carcinoma cells (HCT116) with p53(+/+) (wild-type) or p53(-/-) (knockout) gene were co-incubated with nonirradiated cells of the same type. Clonogenic and senescence assays were used for both irradiated and co-incubated bystander cell populations. We also performed additional measurements on the number of remaining cells after the whole co-incubation period. For radiation doses larger than 2 Gy we observed much larger fractions of senescent cells in p53-positive populations compared to their p53-negative counterparts (15.81% vs. 3.63% in the irradiated population; 2.89% vs. 1.05% in the bystander population; 8 Gy; P < 0.05). Statistically significant differences between cell lines in the clonogenic cell surviving fraction were observed for doses higher than 4 Gy (1.61% for p53(+/+) vs. 0.19% for p53(-/-) in irradiated population; 3.57% for +/+ vs. 50.39% for -/- in bystander population; 8 Gy; P < 0.05). Our main finding was that the number of senescent cells in the irradiated population correlated strongly with the clonogenic cell surviving fraction (R = -0.98, P < 0.001) and the number of senescent cells (R = 0.97, P < 0.001) in the bystander population. We also extended the standard linear-quadratic radiation response model by incorporating the influence of the signals released by the senescent cells, which accurately described the radiation response in the bystander population. Our findings suggest that radiation-induced senescence might be a key player in RIBE, i.e., the strength of RIBE depends on the amount of radiation-induced senescence.

Induction of bystander effects by UVA, UVB, and UVC radiation in human fibroblasts and the implication of reactive oxygen species.

Radiation-induced bystander effects are various types of responses displayed by nonirradiated cells induced by signals transmitted from neighboring irradiated cells. This phenomenon has been well studied after ionizing radiation, but data on bystander effects after UV radiation are limited and so far have been reported mainly after UVA and UVB radiation. The studies described here were aimed at comparing the responses of human dermal fibroblasts exposed directly to UV (A, B, or C wavelength range) and searching for bystander effects induced in unexposed cells using a transwell co-incubation system. Cell survival and apoptosis were used as a measure of radiation effects. Additionally, induction of senescence in UV-exposed and bystander cells was evaluated. Reactive oxygen species (ROS), superoxide radical anions, and nitric oxide inside the cells and secretion of interleukins 6 and 8 (IL-6 and IL-8) into the medium were assayed and evaluated as potential mediators of bystander effects. All three regions of ultraviolet radiation induced bystander effects in unexposed cells, as shown by a diminution of survival and an increase in apoptosis, but the pattern of response to direct exposure and the bystander effects differed depending on the UV spectrum. Although UVA and UVB were more effective than UVC in generation of apoptosis in bystander cells, UVC induced senescence both in irradiated cells and in neighbors. The level of cellular ROS increased significantly shortly after UVA and UVB exposure, suggesting that the bystander effects may be mediated by ROS generated in cells by UV radiation. Interestingly, UVC was more effective at generation of ROS in bystanders than in directly exposed cells and induced a high yield of superoxide in exposed and bystander cells, which, however, was only weakly associated with impairment of mitochondrial membrane potential. Increasing concentration of IL-6 but not IL-8 after exposure to each of the three bands of UV points to its role as a mediator in the bystander effect. Nitric oxide appeared to play a minor role as a mediator of bystander effects in our experiments. The results demonstrating an increase in intracellular oxidation, not only in directly UV-exposed but also in neighboring cells, and generation of proinflammatory cytokines, processes entailing cell damage (decreased viability, apoptosis, senescence), suggest that all bands of UV radiation carry a potential hazard for human health, not only due to direct mechanisms, but also due to bystander effects.

The influence of XPD, APE1, XRCC1, and NBS1 polymorphic variants on DNA repair in cells exposed to X-rays.

Polymorphism of genes coding for proteins which participate in DNA repair may predispose to or protect against development of cancer. Here we studied how common polymorphisms of the genes XPD (Asp312Asn and Lys751Gln), APE1 (Asp148Glu), XRCC1 (Arg399Gln), and NBS1 (Gln185Glu) influence DNA repair and other responses after X-irradiation of lymphocytes from colon carcinoma patients. Genotypes with polymorphic Asp148Glu APE1 and Asp312Asn XPD showed a significantly higher level of DNA incisions immediately after irradiation (p=0.049 and p=0.047 respectively) and Asp312Asn XPD showed a significantly increased capacity to repair of DNA strand breaks as measured 180min after irradiation by comet assays (p=0.004). In contrast, it was the wild type XRCC1 genotype which was associated with a lower level of DNA breaks after irradiation (p=0.014, at 180min after irradiation) and polymorphism of NBS1 did not correlate with any changes in DNA breaks or repair capacity. To confirm the influence of XPD polymorphism on repair, we established stably-transfected HCT116 (colon carcinoma) cells which over-expressed the wild-type or variant XPD protein. Cells over-expressing Asp312Asn XPD showed a higher level of DNA breaks shortly after irradiation and more efficient repair than cells over-expressing the wild-type gene XPD312Asp, and an earlier inhibition of cell cycle transit but faster recovery from this inhibition. Polymorphisms in DNA repair genes therefore influence not only DNA repair capacity but also cell proliferation, and may serve as markers of individual repair capacity and susceptibility to environmental and occupational carcinogens.

Bystander effect induced by UV radiation; why should we be interested?

The bystander effect, whose essence is an interaction of cells directly subjected to radiation with adjacent non-subjected cells, via molecular signals, is an important component of ionizing radiation action. However, knowledge of the bystander effect in the case of ultraviolet (UV) radiation is quite limited. Reactive oxygen and nitrogen species generated by UV in exposed cells induce bystander effects in non-exposed cells, such as reduction in clonogenic cell survival and delayed cell death, oxidative DNA damage and gene mutations, induction of micronuclei, lipid peroxidation and apoptosis. Although the bystander effect after UV radiation has been recognized in cell culture systems, its occurrence in vivo has not been studied. However, solar UV radiation, which is the main source of UV in the environment, may induce in human dermal tissue an inflammatory response and immune suppression, events which can be considered as bystander effects of UV radiation. The oxidative damage to DNA, genomic instability and the inflammatory response may lead to carcinogenesis. UV radiation is considered one of the important etiologic factors for skin cancers, basal- and squamous-cell carcinomas and malignant melanoma. Based on the mechanisms of actions it seems that the UV-induced bystander effect can have some impact on skin damage (carcinogenesis?), and probably on cells of other tissues. The paper reviews the existing data about the UV-induced bystander effect and discusses a possible implication of this phenomenon for health risk. 

Bystander normal human fibroblasts reduce damage response in radiation targeted cancer cells through intercellular ROS level modulation.

The radiation-induced bystander effect is a well-established phenomenon which results in damage in non-irradiated cells in response to signaling from irradiated cells. Since communication between irradiated and bystander cells could be reciprocal, we examined the mutual bystander response between irradiated cells and co-cultured with them non-irradiated recipients. Using a transwell culture system, irradiated human melanoma (Me45) cells were co-cultured with non-irradiated Me45 cells or normal human dermal fibroblasts (NHDF) and vice versa. The frequency of micronuclei and of apoptosis, ROS level, and mitochondrial membrane potential were used as the endpoints. Irradiated Me45 and NHDF cells induced conventional bystander effects detected as modest increases of the frequency of micronuclei and apoptosis in both recipient neighbors; the increase of apoptosis was especially high in NHDF cells co-cultured with irradiated Me45 cells. However, the frequencies of micronuclei and apoptosis in irradiated Me45 cells co-cultured with NHDF cells were significantly reduced in comparison with those cultured alone. This protective effect was not observed when irradiated melanomas were co-cultured with non-irradiated cells of the same line, or when irradiated NHDF fibroblasts were co-cultured with bystander melanomas. The increase of micronuclei and apoptosis in irradiated Me45 cells was paralleled by an increase in the level of intracellular reactive oxygen species (ROS), which was reduced significantly when they were co-cultured for 24h with NHDF cells. A small but significant elevation of ROS level in NHDF cells shortly after irradiation was also reduced by co-culture with non-irradiated NHDF cells. We propose that in response to signals from irradiated cells, non-irradiated NHDF cells trigger rescue signals, whose nature remains to be elucidated, which modify the redox status in irradiated cells. This inverse bystander effect may potentially have implications in clinical radiotherapy.

Ionizing radiation-induced bystander effects, potential targets for modulation of radiotherapy.

Cells exposed to ionizing radiation show DNA damage, apoptosis, chromosomal aberrations or increased mutation frequency and for a long time it was generally accepted that these effects resulted from ionization of cell structures and the action of reactive oxygen species formed by water radiolysis. In the last few years, however, it has appeared that cells exposed to ionizing radiation and other genotoxic agents can release signals that induce very similar effects in non-targeted neighboring cells, phenomena known as bystander effects. These signals are transmitted to the neighboring non-hit cells by intercellular gap-junction communication or are released outside the cell, in the case of cultured cells into the medium. The signaling is mutual, and irradiated cells can also receive signals from non-irradiated neighbors. Most experiments show a decrease in survival of unirradiated bystander cells, but some studies of the influence of unirradiated or low dose-irradiated cells on those irradiated with higher doses show that intercellular bystander signaling can also increase the survival of irradiated cell populations. In the last few years, communication between irradiated and non-irradiated cells has attracted interest in many studies as a possible target for modulation of radiotherapy. Understanding the mechanisms underlying bystander effects is important for radiation risk assessment and for evaluation of protocols for cancer radiotherapy. In this review we describe different aspects of ionizing radiation-induced bystander effects: experimental examples, types of DNA damage, situations in vivo, and their possible role in adaptive response to irradiation, and we discuss their possible significance for anticancer therapy.

[Radiation-induced bystander effect: the important part of ionizing radiation response. Potential clinical implications]. / Popromienny efekt sasiedztwa, wazny element odpowiedzi na promieniowanie jonizujace - potencjalne implikacje kliniczne.

It has long been a central radiobiological dogma that the damaging effects of ionizing radiation, such as cell death, cytogenetic changes, apoptosis, mutagenesis, and carcinogenesis, are the results of the direct ionization of cell structures, particularly DNA, or indirect damage via water radiolysis products. However, several years ago attention turned to a third mechanism of radiation, termed the "bystander effect" or "radiation-induced bystander effect" (RIBE). This is induced by agents and signals emitted by directly irradiated cells and manifests as a lowering of survival, cytogenetic damage, apoptosis enhancement, and biochemical changes in neighboring non-irradiated cells. The bystander effect is mainly observed in in vitro experiments using very low doses of alpha particles (range; mGy, cGy), but also after conventional irradiation (X-rays, gamma rays) at low as well as conventional doses. The mechanisms responsible for the bystander effect are complex and still poorly understood. It is believed that molecular signals released from irradiated cells induce different signaling ways in non-irradiated neighboring cells, leading to the observed events. The molecular signals may be transmitted through gap junction intercellular communication and through a medium transfer mechanism. The nature of these transmitted factors are diverse, and still not definitely established. It seems that RIBE may have important clinical implications for health risk associated with radiation exposure. Potentially, this effect may have important implications in the creation of whole-body or localized side effects in tissues beyond the irradiation field and also in low-dose radiological and radioisotope diagnostics. Factors emitted by irradiated cells may result in the risk of genetic instability, mutations, and second primary cancer induction. They might also have their own part in inducing and extending post-radiation side effects in normal tissue. The bystander effect may be a potentially harmful or a useful event in radiotherapy. The elevation of damage to tumor cells not directly hit by radiation or the initiation of tumor cell differentiation may increase the therapeutic ratio. If, however, molecular species secreted by irradiated tumor cells in vivo damage neighboring normal cells (epithelial and endothelial cells, fibroblasts, or lymphocytes), the bystander effect would be harmful and could lead to increased side effects in normal tissue. This is especially important in modern radiotherapy, as 3D conformal radiation therapy (3D-CRT) and intensity-modulated radiation therapy (IMRT) are aimed at diminishing the radiation dose in normal tissues. Recent in vivo studies on animals indicate that bystander effects may appear in organs and tissues remote from the irradiated field and the extension of tissue damage seems to be tissue-type dependent. However, recent experimental results indicate that non-irradiated cells that are neighbors of irradiated cells may diminish radiation damage in the radiation-focused cells. Less is known about the bystander effect during fractionated irradiation. Thus the clinical implications of the bystander effect and its possible modification for radiotherapeutic usefulness is still under debate.

X-irradiation and bystander effects induce similar changes of transcript profiles in most functional pathways in human melanoma cells.

Unirradiated cells which neighbor cells exposed to ionizing radiation (IR) show responses termed bystander effects, including DNA damage, chromosomal instability, mutation, and apoptosis. We used genome-wide microarrays to compare the change in transcript profiles in Me45 (human melanoma) cells grown in culture medium from irradiated cells (irradiation conditioned medium, ICM) with those which occurred after IR, sampling after more than one division cycle to detect long-term changes which could be relevant in radiotherapy. Transcripts of >10,000 genes showed an increased or decreased level in both conditions using the criterion of a >+/-10% change, and >85% of these were common to growth in ICM and after IR. When these genes were grouped into metabolic pathways according to the Kyoto Encyclopedia of Genes and Genomes (KEGG), significant differences (p<0.01) were seen between the numbers of up- and down-regulated transcripts in certain groups after both ICM and IR, particularly in the groups neuroactive ligand-receptor interactions, oxidative phosphorylation, cytokine-cytokine receptor interactions, proteasomes, and ribosomes. Quantitative RT-PCR assays of transcripts of selected genes in these pathways confirmed the similar effects of growth in ICM and IR. We conclude that factors transmitted from irradiated cells can influence transcript levels in bystander cells, and that these changes persist for more than one cell cycle consistent with the long-term transmissible effects seen in progeny cells, revealing new facets of the IR-induced bystander effect.

[Dose rate-dependent cellular and molecular effects of ionizing radiation]. / Wplyw mocy dawki na komórkowe, biochemiczne i molekularne efekty promieniowania jonizujacego.

The aim of radiation therapy is to kill tumor cells while minimizing damage to normal cells. The ultimate effect of radiation can be apoptotic or necrotic cell death as well as cytogenetic damage resulting in genetic instability and/or cell death. The destructive effects of radiation arise from direct and indirect ionization events leading to peroxidation of macromolecules, especially those present in lipid-rich membrane structures as well as chromatin lipids. Lipid peroxidative end-products may damage DNA and proteins. A characteristic feature of radiation-induced peroxidation is an inverse dose-rate effect (IDRE), defined as an increase in the degree of oxidation(at constant absorbed dose) accompanying a lower dose rate. On the other hand, a low dose rate can lead to the accumulation of cells in G2, the radiosensitive phase of the cell cycle since cell cycle control points are not sensitive to low dose rates. Radiation dose rate may potentially be the main factor improving radiotherapy efficacy as well as affecting the intensity of normal tissue and whole-body side effects. A better understanding of dose rate-dependent biological effects may lead to improved therapeutic intervention and limit normal tissue reaction. The study reviews basic biological effects that depend on the dose rate of ionizing radiation.

[Mechanisms of metastasis and molecular markers of malignant tumor progression. I. Colorectal cancer]. / Mechanizmy przerzutowania i molekularne markery progresji nowotworów zlosliwych. I. Rak jelita grubego.

The ability of neoplastic cells to dissemination from a primary tumor to lymphatic nodes and to adjacent and distant tissues and organs is an inseparable feature of malignant tumors and the main cause of failure in their treatment. Metastasis formation is a multistage process which includes proteolysis, the motility and migration of cells, proliferation, and neoangiogenesis. In the first step, the cells released from the primary tumor have to penetrate to the blood or lymphatic vessels (intravasation), the road which dissemination follows. Circulating cells can then migrate through the walls of vessels to surrounding tissues (extravasation) where they settle, proliferate, and induce angiogenesis, creating metastases. Indispensable in the process of intra- and extravasation is the activation of proteolytic enzymes capable of degrading the extracellular matrix (ECM) surrounding the endothelium or creating the basement membrane of epithelial tissue in different organs. In this stage, the activation of proteolytic enzymes, such as proteinases of the plasmin system, serine proteinases, and matrix metalloproteinases (MMPs), is necessary. Simultaneously, changes occur in the expression of many superficial glycoproteins and factors responsible for cell adhesion (integrins) and intercellular communication (cadherins). Neoangiogenesis is connected with the expression of many markers of this process, among them vascular endothelial growth factor (VEGF), endoglin (CD105), a transmembranous glycoprotein which is a component of the receptor for transforming growth factor beta (TGFbeta), as well as neuropilin (NRP), the co-receptor for VEGF. Conventionally, the prognosis of neoplastic disease and its treatment are based mainly on exact clinical and histopathological staging. This prognosis could, however, be improved by measuring the molecular and cellular markers which play key roles in tumor progression. Understanding the cellular processes responsible for tumor dissemination can be useful not only in the diagnosis and prognosis of treatment results, but also in developing targeted drugs, selectively directed towards those factors responsible for tumor invasiveness, as well as in creating new therapeutic strategies permitting the use of such drugs. In the present review the authors concentrate mainly on one tumor type, colorectal carcinoma, in which distant metastases, predominantly to the liver, are the main cause of failure, in spite of surgical curing of the primary tumor.

[Cardiotoxic consequences of ionizing radiation and anthracyclines]. / Kardiotoksyczne nastepstwa promieniowania jonizujacego i antracyklin.

The mechanisms of the toxic side effects of radiation and anthracyclines are generally based on free radical reactions. Target molecules for free radicals are not only DNA and proteins, but also membrane structures abundant in phospholipids. Cardiomyocyte membrane lipid peroxidation may consequently lead to structural and functional damage. The dynamics of oxidative injury result from the oxidative stress status on the one hand and lowered antioxidant defense on the other. Heart muscle is especially vulnerable to the oxidative activity of free radicals generated by both radiation and cytostatics because of its low antioxidant defense. The toxic effects of radiation and cytostatics are a serious clinical problem connected with the physiologically or pathologically reduced antioxidant defense of normal tissues involved in the therapy. The additive, even synergistic, effects of the polychemotherapy usually used before, during, and after radiation often disguise postradiation effects on cardiac muscle.

Spectroscopic properties and photodynamic effects of new lipophilic porphyrin derivatives: efficacy, localisation and cell death pathways.

Photodynamic therapy (PDT) and photodynamic diagnostics (PDD) of cancer are based on the use of non-toxic dyes (photosensitisers) in combination with harmless visible light. This paper reports physicochemical properties, cell uptake, localisation as well as photodynamic efficiency of two novel lipophilic porphyrin derivatives, suitable for use as PDT sensitisers. Both compounds are characterised by high quantum yield of singlet oxygen generation which was measured by time-resolved phosphorescence. Photodynamic in vitro studies were conducted on three cancer cell lines. Results of cell survival tests showed negligible dark cytotoxicity but high phototoxicity. The results also indicate that cell death is dependent on energy dose and time following light exposure. Using confocal laser scanning microscopy both compounds were found to localise in the cytoplasm around the nucleus of the tumour cells. The mode of cell death was evaluated based on the morphological changes after differential staining. In summary, good photostability, high quantum yield of singlet oxygen and biological effectiveness indicate that the examined lipophilic porphyrin derivatives offer quite interesting prospects of photodynamic therapy application.

Protective effect of local temporary ischemia depends on applied dose of radiation.

The aim of this study was to verify hypothesis that protective effect of local temporary ischemia depends on dose of radiation. 56 male WAG-strain rats were used. Total body irradiation with 3 x 3 and 3 x 5 Gy was performed. Local temporary ischemia was induced by clamping the tail base. The biochemical parameters were the thiobarbituric acid-reactive substances (TBA-RS). In bone marrow smears the polychromatic erythrocyte (PCE) numbers were counted and the numbers of micronucleated PCEs were analyzed. In small intestines the numbers of crypts were calculated. The levels of TBA-RS in the serum of the animals irradiated with a 3 x 3 Gy dose were significantly different (P < 0.002). Also in animals irradiated with a dose of 3 x 3 Gy the numbers of intestinal crypts were different (P < 0.05). In animals irradiated with dose 3 x 5 Gy, for analyzed parameters differences did not achieve statistical significance. Local temporary ischaemia provides general protection against radiation damage for lower dose. This protective effect disappeared after applications of a higher dose of radiation.

Cell proliferative activity estimated by histone H2B mRNA level correlates with cytogenetic damage induced by radiation in human glioblastoma cell lines.

We studied the relationship between proliferative activity and radiation-induced DNA damage in human malignant gliomas in vitro. Nine human glioblastoma established cell lines were gamma-irradiated (60Co) over a dose range of 0-10 Gy. H2B and H4 histone mRNA level was assessed with quantitative RT-PCR technique (TaqMan) and histone labeling index (HLI) with in situ hybridization to define proliferation rate, while cytochalasin-block micronucleus assay was performed to measure cytogenetic damage. Micronucleus frequency correlated with H2B mRNA level (Spearman's R up to 0.82 at 8 Gy), HLI, nuclear division index (NDI) and percentage of binucleated cells (%BNC). There was a high correlation between H2B mRNA level and NDI (R = 0.80) as well as %BNC and HLI (R = 0.72). Histone H2B and H4 mRNA level (not significant), HLI, NDI, and %BNC (significant) were higher in cell lines sensitive to DNA damage. Proliferative activity correlates with radiation-induced DNA damage in human glioma cell lines. Histone H2B mRNA level and HLI may be a useful molecular predictor of the tumour response to radiation treatment in gliomas of the same histological grade, however the risk of potentially more rapid tumour-cell repopulation must be considered. Presumed protective activity of histones against radiation-induced DNA damage was not confirmed at the transcript level.

Multiple bystander effect of irradiated megacolonies of melanoma cells on non-irradiated neighbours.

The multicellular megacolonies of human melanoma Me45 line growing on one part of the bottom of culture flasks were irradiated with 5 Gy (60Co), whereas megacolonies growing on the second part of the bottom were shielded. The bystander effect of radiation-traversed cells on non-traversed cells was studied during postradiation co-cultivation. Activity of superoxide dismutase (Mn and CuZn subunits), glutathione peroxidase (GSH-Pox) and malondialdehyde (MDA) concentration as a biochemical markers of bystander effect were monitored for a period of 72 h. The DNA damage was measured by the comet assay. Micronucleus induction, mitotic index and cellular death as apoptosis or necrosis were simultaneously estimated, based on morphologic criteria. The bystander effect of irradiated cells on their neighbours was observed as a slight increase of MDA concentration, comparable decrease of GSH-Pox activity, and some fluctuation of mitochondrial and cytoplasmic isoenzymes of SOD. DNA strand breaks and rejoining measured by comet assay as mean tail length, demonstrated clearly the bystander effect for nontraversed radiation cells, additionally verified by tail moment. There was also a significant increase of micronucleation and apoptosis generated by radiation traversed cells in shielded neighbours. Furthermore, significantly higher increase of necrosis in shielded neighbour cells compared to radiation traversed cells was observed. Proliferative activity showed a suppression in both, radiation traversed and shielded neighbour cells in all measured time points. The behaviour of used parameters points to the radical nature of modificators secreted by radiation traversed cells inducing bystander toxic damage in shielded neighbour cells.

Apoptosis and clonogenic survival in three tumour cell lines exposed to gamma rays or chemical genotoxic agents.

We compared the extent to which apoptosis is induced and clonogenicity reduced in three tumour cell lines - the human melanoma Me45 and promyelocytic leukaemia HL-60, and the rat rhabdomyosarcoma R1 - after exposure to the anticancer drugs etoposide and cis-platinum or to gamma radiation; each induces different types of DNA damage. Cells which readily underwent apoptosis did not necessarily show a correlated loss of clonogenicity; for example, Me45 cells showed the highest sensitivity to all three agents in clonogenic assays but much lower levels of apoptotic cells than R1 or HL-60 cells. These results show that the efficiency of the eradication of clonogenic cells by genotoxic agents does not solely depend on the induction of apoptotic processes, and suggest that the induction of apoptosis and suppression of clonogenicity are independent processes.

Radiation-induced micronucleus frequency in peripheral blood lymphocytes is correlated with normal tissue damage in patients with cervical carcinoma undergoing radiotherapy.

In an effort to find a test to predict the response of normal tissue to radiotherapy, the lymphocyte micronucleus assay was used on blood samples from patients with cervical carcinoma. Peripheral blood samples from 55 patients with advanced-stage (II B-IV B) cervical carcinoma were obtained before radiotherapy. The patients were treated with external-beam radiotherapy followed by high-dose-rate brachytherapy. Acute and late normal tissue reactions were scored and correlated with the micronucleus frequency in lymphocytes after irradiation with 4 Gy in vitro. Great interindividual variability was observed in the radiation-induced lymphocyte micronucleus frequency, especially at 4 Gy. The mean number of micronuclei per 100 binucleated cells in cells irradiated with 4 Gy in vitro was significantly higher in samples from patients who suffered from acute and/or late normal tissue reactions than in those from patients with no reactions (51.0 +/- 17.7 and 29.6 +/- 10.1, respectively). A significant correlation was also found between the micronucleus frequency at 4 Gy and the severity of acute reactions and late reactions. However, the overlap between the micronucleus frequencies of patients with high-grade late normal tissue reactions and low-grade reactions is too great to recommend the micronucleus assay in its present form for routine clinical application.