Cellular signal transduction events as a function of linear energy transfer (LET).

In order to obtain a deeper insight into the molecular mechanism controlling the cellular response to high-linear energy transfer (LET) radiation, the number and size of pATM (S1981) and gamma-H2AX foci were compared in cultures of diploid human fibroblasts after exposure to charged particles of varying species, energy and LET at the NIRS-HIMAC-facility (Chiba, Japan). Particle LET ranged from 2.2 to 300 keV/mum, and a low fluence of 7.3 x 10(4) cm(-2) was chosen. Therefore, about 1 out of 7 nuclei was traversed by a particle. Doses and LET were verified with thermoluminescence detectors (LiF:Mg, Ti) evaluated according to the high temperature ratio method. Two hours after irradiation, fibroblasts were fixed and the subcellular distribution of pATM (S1981) and gamma-H2AX was visualised by immunofluorescence or histochemical staining using phosphorylation-specific antibodies. It was found that the number of pATM (S1981) foci per nucleus was higher after exposure to higher-LET particles. Irradiation with the two highest LET beams (Fe-ions, 197 and 300 keV/mum) gave a significant increase in the number of pATM foci, whereas ions with an LET lower than 30 keV/mum yielded similar numbers of pATM foci compared with unirradiated control samples. These data show that the early cellular response to high-LET radiation is modulated by the energy deposition of the particle. Therefore, the correlation between the microdosimetric aspect of energy deposition and biologic consequences at low radiation doses deserves further study.

Involvment of c-Abl in the radiation-induced inhibition of myoblast differentiation.

PURPOSE: While radiation-induced differentiation is common in many cell types it has been shown (Ikeda et al. 2000) that irradiation inhibits differentiation of murine myoblast cell line C2C12. To investigate the mechanisms responsible for this phenomenon we looked into the role of the non-receptor tyrosine kinase c-Abl. MATERIAL AND METHODS: C-Abl activity was determined using an immune-complex kinase assay and the functional role of c-Abl activity was assessed by using the kinase inhibitor STI 571 (Gleevec) and by transfecting a dominant negative c-Abl mutant (K290R). RESULTS: We found an up-regulation of c-Abl activity 6h after differentiation induction when compared to uninduced control cells. Exposure of C2C12 cells to ionizing radiation, which inhibits myotube formation, inhibited this up-regulation. Transfected dominant negative c-Abl as well as STI 571 treatment and ionizing radiation inhibited myotube formation but did not significantly suppress the expression of the muscle-specific transcription factor Myogenin or of the differentiation marker Myosin heavy chain. CONCLUSIONS: These data suggest that c-Abl is involved in the radiation-induced inhibition of myoblast differentiation and lead to the hypothesis that c-Abl plays a role in late events during myogenic differentiation probably in the process of myoblast fusion.

Protein kinase C isoforms in normal and transformed cells of the melanocytic lineage.

Enzymes belonging to the protein kinase C (PKC) family represent one of the major mediators of signal transduction in melanocytes. To identify PKC isoforms that may be associated with the process of malignant transformation and metastasis, we investigated the expression pattern of 11 different PKC isoforms (alpha, beta I, beta II, gamma, delta, epsilon, eta, theta, zeta, lambda, and iota) in melanoma lymph node metastases, in cell lines established from these metastases, in primary cell cultures from normal melanocytes, and in permanent cell lines established from spontaneously transformed melanocytes. PKC alpha, beta I, beta II, delta, epsilon, eta, zeta, lambda and iota were found to be expressed in total lysates from melanoma metastases. In permanent cell lines established from these metastases, the expression levels of PKC beta I, beta II, delta, epsilon, and eta were lower or undetectable when compared with initial expression in tumour lysates. In normal primary melanocyte cultures, the PKC isoforms beta II, delta, epsilon, eta and iota were undetectable. PKC gamma and theta isoforms were undetectable in all melanocytic cell types examined. PKC iota was the only isoform exclusively detected in tumour lysates, in spontaneously transformed melanoma cells and melanoma cell lines, but not in normal melanocytes, and may therefore be associated with the transformed phenotype in human melanoma in vitro and in vivo.

NF-kappaB1 (p50) homodimers contribute to transcription of the bcl-2 oncogene.

The bcl-2 proto-oncogene is frequently expressed in human cancer. Although bcl-2 was first cloned as the t(14;18) translocation breakpoint from human follicular B-cell lymphoma, it has become apparent that many cell types express bcl-2 because of transcriptional regulation. As such, several transcription factors have been demonstrated to activate expression of bcl-2, including NF-kappaB. We investigated the role of NF-kappaB1 (p50) homodimers in the expression of Bcl-2 in two murine B-cell lymphoma cell lines: LY-as, an apoptosis-proficient line with low Bcl-2 protein expression and no nuclear NF-kappaB activity, and LY-ar, a nonapoptotic line with constitutive p50 homodimer activity and 30 times more Bcl-2 protein expression than LY-as. We found that nuclear p50 homodimer activity correlated with Bcl-2 expression in these cell types and identified several sites within the bcl-2 5'-flanking region that p50 was capable of binding. In vitro transcription revealed that recombinant p50 enhanced the production of run-off transcripts from the bcl-2 P1 promoter. Additional in vitro transcription experiments suggested the sites by which p50 afforded this effect. We conclude that the p50 homodimer is capable of transcriptional activation of the bcl-2 gene and suggest that its nuclear activity contributes to the expression of bcl-2 in LY-ar cells.

Anthracycline-induced erythroid differentiation of K562 cells is inhibited by p28, a novel mammalian glutathione-binding stress protein.

When exposed to the anthracycline doxorubicin, K562 cells undergo differentiation which is characterized by arrested cell division, an increased mean cell diameter, and the production of hemoglobin. The influence of expression of p28, a low-molecular weight stress protein, on the differentiation of K562 cells was examined. Expression of p28 was modulated by transfection of K562 cells with expression vectors containing the murine p28 cDNA in either the sense or antisense orientation, or without the p28 cDNA. In K562 cells where p28 expression was either unaltered or downregulated, exposure to 40 nM Doxorubicin resulted in an arrest of cell division, the production of hemoglobin, and an increased cell diameter consistent with cells undergoing differentiation. K562 cells that overexpressed p28 continued to divide, had fewer hemoglobin-producing cells, had a smaller mean cell diameter and had a 5.5-fold increase in cell survival. Consistent with an inhibition of doxorubicin-induced erythroid differentiation, p28 may act by changes in redox regulation via the glutathione-binding activity of p28 and suggests a general role for p28 in cellular differentiation. Furthermore, p28 expression may be useful in predicting resistance to chemo- or radiation therapy in the treatment of leukemia and lymphoma.

Erythropoietin receptor expression in human melanoma cells.

Erythropoietin is well known for its role in the control of erythropoiesis, where it acts by binding to its cognate receptor (EpoR) on the surface of erythroid progenitor cells. Here we present the novel finding that the EpoR is also expressed in cells of the melanocytic lineage. It is expressed in transformed cell lines established from normal melanocytes and also in established human melanoma cell lines derived from melanoma metastases, but not in normal primary human melanocytes. The analysis of individual subclones isolated from spontaneously transformed melanocytes revealed that approximately 50% of all the clones examined expressed the EpoR. Further analysis of the individual growth characteristics of EpoR-positive and EpoR-negative clones indicated that, under standard cell culture conditions, expression of the receptor did not affect cell growth. Expression of this receptor is consequently most likely driven by an event that is associated with, but not absolutely required for, the transformed phenotype. While the definite function of this receptor in melanoma cells is still unknown and additional studies are required, our findings support the hypothesis that the EpoR may serve as a progression marker for human melanoma. This observation might be useful in the early diagnosis of melanoma.

Effects of betulinic acid alone and in combination with irradiation in human melanoma cells.

Recently, betulinic acid was identified as a highly selective inhibitor of human melanoma growth and was reported to induce apoptosis in these cells. We have investigated the growth-inhibitory properties of this compound alone and in combination with ionizing radiation in a panel of established human melanoma cell lines as well as in normal human melanocytes. Betulinic acid strongly and consistently suppressed the growth and colony-forming ability of all human melanoma cell lines investigated. In combination with ionizing radiation the effect of betulinic acid on growth inhibition was additive in colony-forming assays. Betulinic acid also induced apoptosis in human melanoma cells as demonstrated by Annexin V binding and by the emergence of cells with apoptotic morphology. The growth-inhibitory action of betulinic acid was more pronounced in human melanoma cell lines than in normal human melanocytes. Notably, despite the induction of apoptosis, analysis of the expression of Bcl-2 family members in betulinic-acid-treated cells revealed that expression of the anti-apoptotic protein Mcl-1 was induced. Furthermore, the antiproliferative action of betulinic acid seemed to be independent of the p53 status. The properties of betulinic acid make it an interesting candidate, not only as a single agent but also in combination with radiotherapy. We conclude that the strictly additive mode of growth inhibition in combination with irradiation suggests that the two treatment modalities may function by inducing different cell death pathways or by affecting different target cell populations.

The cloning and characterization of a new stress response protein. A mammalian member of a family of theta class glutathione s-transferase-like proteins.

Using differential display, a cDNA fragment was identified as being overexpressed in a mouse lymphoma cell line that had gained resistance to cell death after exposure to a variety of agents used in cancer therapy. The full-length cDNA of 1.1 kb that was cloned contained an open reading frame coding for a previously unidentified 28-kDa mammalian protein, p28. p28 showed significant homologies to a large family of stress response proteins that contain a glutathione S-transferase (GST) domain. In correspondence with the sequence homology, p28 was found to bind glutathione; however, GST or glutathione peroxidase activity could not be demonstrated. Northern analysis of the mRNA of this protein showed abundant expression in mouse heart and liver tissues, whereas anti-p28 antibody binding identified p28 expression in mouse 3T3 cells and early passage mouse embryo fibroblasts. Subcellular protein fractionation revealed p28 localization in the cytoplasm, but with thermal stress p28 relocated to the nuclear fraction of cellular proteins. Based on sequence homology and protein activity we conclude that p28 acts as a small stress response protein, likely involved in cellular redox homeostasis, and belongs to a family of GST-like proteins related to class theta GSTs.

Enhancement of mitomycin C efficiency by vitamin C, E-acetate and beta-carotene under irradiation. A study in vitro.

Using E.coli bacteria (AB 1157) and leukemia cells (HL 60) as a model for in vitro studies it was established that the efficiency of mitomycin C (MMC) can be influenced in the presence of antioxidant vitamins. This synergistic effect of the vitamins C, E-acetate and beta-carotene on MMC activity is rather strong for E.coli bacteria under irradiation (15 and 50 Gy) in the presence of air. Vitamin C contributes more efficiently to the MMC-activity in leukemia cells than the other two vitamins. The effect is explained by a cascade electron transfer process from the vitamins to MMC, where vitamin C is acting as a major electron source. These results might be of importance in cancer therapy.

Radioresistant cell strain of human fibrosarcoma cells obtained after long-term exposure to x-rays.

A radioresistant cell strain from human fibrosarcoma HT 1080 has been obtained after prolonged exposure to x-rays for 7 months (2 Gy per day, 5 days per week). This new strain, HT1080R, differs from HT1080 in a significantly increased ability of clonogenical survival, with coefficient alpha decreasing from 0.161 to 0.123 Gy(-1) and coefficient beta decreasing from 0.0950 to 0.0565 Gy(-2). Furthermore, the radioresistance of HT1080R proved to be stable in long-term passaged cultures as well as in frozen samples. Differences between the two cell lines are also observed in the G-banded karyotype; the new cell line shows monosomy of chromosome 17 and loss of 5p+ and 11q+ present in the parental cells. These data suggest that the radioresistance may have been caused by radiation-induced cell mutation and that the resistant cells may have been selected by repeated irradiations. In order to characterize this new strain, the ability of the cells to rejoin DNA double-strand breaks, the cell cycle distribution and the amount of apoptosis after irradiation have been estimated; however, no differences are observed between these two cell strains. Although the mechanism of the elevated radioresistance remains unknown, this pair of cell strains can provide a new model system for further investigations with regard to the mechanisms of cellular radioresistance. The results also show that any type of irradiation similar to the schedules used in radiotherapy can lead to the formation and selection of more radioresistant cell clones in vitro, a phenomenon with possible implications for radiotherapy.

Signal transduction during apoptosis; implications for cancer therapy.

Programmed cell death is a fundamental aspect of organismal development and stasis through its role in the maintenance of the balance between cell growth and cell death. If the balance is tipped, e.g. by unregulated cell growth, the result can be cancer. If tipped the other direction, e.g. by dysregulated cell death, and the result can again be cancer. The concept of dysregulated cell death, which until recently was not considered by oncologists, has forced a shift in the paradigm of cancer development. Along with this shift in thinking comes the likelihood that radiation and chemotherapy, both major modalities of cancer therapy, can benefit from strategies that modulate programmed cell death. One form of programmed cell death that is distinguished by its morphological features is called apoptosis. This form of programmed cell death is an energy dependent biochemically regulated process that is contingent upon a set of factors: the initial stimulatory event or in some cases a cellular insult, the organism, the cell type, the cellular environment, and other factors. This biochemical process is the result of the expression of a number of genes. In this review, the roles of several genes and gene families considered to be critical to the signal transduction cascade of apoptosis are described. Growth factors and cytokines are also discussed in the context of their interaction with these genes. We also discuss how these genes and their protein products are being used as prognostic indicators for cancer and cancer therapy and/or how they are the focus of strategies that either cause apoptosis or alter a cancer cell's propensity to initiate apoptosis when insulted by chemotherapy agents or radiation in the course of cancer therapy.

Determination of radiation-induced DNA strand breaks in individual cells by non-radioactive labelling of 3' OH ends.

To quantify DNA strand breaks generated by ionizing radiation in single cells with preserved morphology, the number of radiation-induced 3' OH ends of nuclear DNA was determined by enzymatic labelling. Confluent CHO-K1 cells were irradiated with doses up to 100 Gy. After fixation and permeabilization of the cell monolayer the nuclear DNA was labelled with Digoxigenin-11-dUTP using terminal transferase. The incorporated nucleotide was detected with an anti-Digoxigenin antibody conjugated with alkaline phosphatase. The phosphatase bound was quantified by a colour reaction and the integrated optical densities of the cell nuclei were measured. For doses ranging from 20 to 100 Gy a linear relationship between dose and labelling signal was obtained. Repair experiments showed a fast component of repair with a half-time of about 14 min, followed by a slower decline to background values, which were reached after 6-8 h. This method allows the measurement of radiation-induced DNA strand breaks in morphologically preserved single cells in a reproducible way, which may be of importance in the prediction of tumour response in radiotherapy.

Determination of the radiation sensitivity of the stromal cells in the murine long-term bone marrow culture by measuring the induction and rejoining of interphase chromosome breaks.

PURPOSE: To determine the radiosensitivity of bone marrow stromal cells, the rate of interphase chromosome breakage and rejoining of stromal cells in the murine long term bone marrow culture and of human skin fibroblasts were compared. METHODS AND MATERIALS: The cells were irradiated with doses up to 6 Gy and repair times up to 6 hr were investigated. After induction of premature chromosome condensation by fusing the cells with mitotic HeLa cells, the number of interphase chromosome fragments was counted. RESULTS: The number of radiation induced breaks was found to be not significantly different for both cell types with 6.16 +/- 0.26 breaks per Gray for the fibroblasts and 5.96 +/- 0.20 breaks per Gray for the stromal cells. A significant difference was observed in the repair rate. The fibroblasts rejoined 39.6% of the breaks induced initially during the first hour after irradiation and 5.6 +/- 1.84 breaks remained unrejoined after 6 hr, while the stromal cells were able to rejoin 63.2% in 1 hr and had 2.05 +/- 0.07 breaks unrejoined after 6 hr. CONCLUSION: If the well substantiated assumption is made, that the capacity to repair DNA double strand breaks or interphase chromosome breaks is correlated with the cellular radiosensitivity, this finding indicate, that murine bone marrow stromal cells are more radioresistant than human skin fibroblasts.

[Representation of texture parameters of B-mode sonograms in image form]. / Darstellung der Texturparameter von B-Mode-Sonogrammen in bildmässiger Form.

The possibility is exemplified of visualising important parameters of the statistical texture describing the echogenicity of ultrasound B-mode scans of the liver as a composite image. The two relevant parameters are gray level and standard deviation of gray levels. These were selected because they offer the best criterion to differentiate between tissues. In a composite image, the mean gray level is represented by the luminosity information, whereas the other main parameter of the gray-level histogram, the standard deviation of gray levels, is represented by the colour information. These composite images show texture parameters of the whole scan and allow an efficient documentation of the same.

[An object analytical procedure for determining the area of the thyroid gland parenchyma accumulating nuclides in the scintigram]. / Ein objektanalytisches Verfahren zur Bestimmung der Fläche des nuklidspeichernden Schilddrüsenparenchyms im Szintigramm.

An algorithm for object isolation was developed to determine the area of the thyroid in scintigraphic images, and its volume calculated therefrom so that operator-induced variations, common if the usual manual technique is used, could be avoided. The object isolation is performed for every possible threshold value. The resulting object isolation curves give a reliable and reproducible thyroid area. The method may be used routinely except in cases of blocked thyroid uptake or of multiple autonomous adenomas.

[The effect of 5-fluorouracil on the radiosensitivity of granulocytopoietic stem cells in the bone marrow and the blood]. / Wirkung von 5-Fluorouracil auf die Strahlensensibilität von granulozytopoetischen Stammzellen des Knochenmarks und des Blutes.

The radiation tolerance of haematopoietic stem-cells, originating from blood and bone marrow, after exposure to 5-Fluorouracil (5-FU) was investigated. The CFU-C Colony Formation Test was used to determine the surviving fraction of cryopreserved mononuclear bone marrow and blood cells, after incubation in culture medium containing 1 ng/ml 5-FU for 24 hours. With the same assay system we determined the radiation tolerance of peripheral CFU-C from three patients before and after 5-FU radio-chemotherapy (1000 mg/m2/24 h per day for four days), while the influence of the small radiotherapeutic treatment volume was neglected. Neither in the in vitro experiments nor in the patients we found a significant variation of the dose-survival curves with or without 5-FU. So it is concluded, that the enhanced myelodepression of 5-FU combined with radiation may be due to an additive effect or by effects on the microenvironment or on the pluripotent stem cell system.

[Chemo- and radiosensitivity testing of tumor tissue cultured in vitro. Development of a fiber culture system for cell harvesting]. / Chemo- und Strahlensensibilitätstestung von in vitro gezüchtetem Tumorgewebe. Entwicklung eines Erntefaserkreislaufes für die Zellentnahme.

Chemo- and radiosensitivity-testing of tumour cells should be performed in vitro under in vivo-like conditions. One problem is to grow cells to tissue-like cell densities in vitro. A hollow fiber reactor was prepared for cell growth to high densities. In order to obtain cells during and after sensitivity testing for quantitating cell damage, a harvesting fiber system was developed which can be inserted in the nutritional hollow fiber system. These hollow fibers have a diameter of 300 microns and a wall thickness of 10 microns. Small holes between 10 and 100 microns can be cut into the fibers for cell harvesting by electric sparks. Up to 60 holes per mm fiber length can be produced.

[A retrospective study of the results of postoperative radiotherapy of hypernephroma]. / Retrospektive Untersuchung der Ergebnisse der postoperativen Radiotherapie bei Hypernephromen.

Retrospectively the patients were analyzed, postoperatively irradiated because of a hypernephroidal kidney carcinoma during the years 1978 to 1988. With a total number of 44 patients 12 were in stage I (Robson), 13 in stage II, 17 in stage III and 2 in stage IV. The probabilities for a tumor-free survival of five years were 81%, 59% and 30% for the stages I to III. The local recurrence rate was 7%, caused by exclusion of clinically negative lymph-nodes from irradiation field. In addition to survival probabilities the complication rate of radiotherapy is analyzed too. To this additionally to analysis of symptoms of a possible side-effect the nuclear medical investigation of function of the remaining kidney was done in 9 selected tumor-free patients being irradiated in different techniques. A normal function was found in all cases. No severe side-effects can be shown in irradiated patients. Consequently the postoperative radiotherapy in hypernephroma is a supportive therapy of advanced tumor stages without severe side-effects. Further and greater planned analyses are necessary to comprehend prognostic factors.