Glucose-, energy-metabolism and cell proliferation in tumors.
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Animals↗
Biomedical subjects
C Streffer
Publications and source records attributed to C Streffer.
Non-invasive infrared-spectroscopic measurements of intravasal oxygen content in xenotransplanted tumours on nude mice--correlation to growth rate.
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Adenocarcinoma↗
The question of threshold doses for radiation damage: malformations induced by radiation exposure of unicellular or multicellular preimplantation stages of the mouse.
Mouse embryos of the one-cell stage or the 32- to 64-cell stage were exposed to various X-ray doses (one-cell stage: 0.25-2 Gy; 32- to 64-cell stage: 1-3 Gy). It turned out that the shape of the dose-response curves is statistically compatible with the assumption derived from biological considerations that there is no threshold for radiation-induced malformations in the case of the exposure of one-cell embryos, whereas there is a threshold dose (close to 1 Gy) in the case of the exposure of 32- to 64-cell embryos.
Animals↗
DNA content as a predictor of clinical outcome in soft tissue sarcoma patients.
The prognostic relevance of cellular DNA content has been shown for a variety of human malignancies. However, only a few studies concerning soft tissue sarcomas have been published. Biopsies of 81 patients with soft tissue sarcomas, referred for primary or secondary surgery, were analysed by flow cytometry to determine cellular DNA content of tumours. Most patients (60/81) already had one or more local recurrences at the time of first presentation at Essen University. The median age of the patients was 45 years (range 14-79). 44 (54%) patients had euploid and 37 (46%) had aneuploid tumours. Age, sex, and tumour localisation (trunk versus extremity) were equally distributed between euploid and aneuploid sarcoma patients. The median follow-up was 69 months (range 9-312). The median survival time for euploid and aneuploid tumours was 84 and 30 months, respectively (P < 0.0005). In the univariate analysis, ploidy, S-phase percentage, localisation and tumour grading were significant predictors of survival, whereas in the multivariate analysis, only DNA content and tumour localisation were independent prognostic variables for survival.
Adolescent↗
Assessment of the proliferative activity and radiosensitivity of human tumours using the cytokinesis-block micronucleus assay.
We established an in vitro cytokinesis-block micronucleus assay of human tumours for estimation of the proportion of cells undergoing mitosis (the dividing fraction, DF), the time for the number of nuclei to double and the radiosensitivity in terms of the micronucleus frequency, based on a concept described previously. Under certain conditions, the nuclear number doubling time (NNDT) was considered to represent the potential doubling time. Tumour specimens obtained at surgery were disaggregated into single-cell suspensions and were directly cultured in the presence of cytochalasin B with or without irradiation. At various intervals, the percentage of multinucleate cells (the plateau value represented the DF), the average number of nuclei per cell and the number of micronuclei in binucleate cells were determined. DF and NNDT values were obtained in 58 of the 73 tumours investigated, and the micronucleus frequency was obtained in 54 of these 58 tumours. The DF ranged from 4.1% to 71% and the NNDT ranged from 3.1 to 83 days. A DF > or = 20% was associated with a higher recurrence rate in patients undergoing curative operation. A correlation was found between the NNDT and the time to relapse in patients with recurrent disease. The average number of micronuclei per binucleate cell at 2 Gy of irradiation (after subtraction of the value at 0 Gy) ranged from 0.052 to 0.35. Tumours which produced more micronuclei after irradiation showed a better response to radiotherapy. This assay can be readily performed on human tumours and appears to have promise as a predictive assay for radiation therapy.
Adenocarcinoma↗
Comet assay studies of radiation-induced DNA damage and repair in various tumour cell lines.
We used the 'comet assay' to compare the amount of radiation-induced DNA damage in three tumour cell lines (MeWo, PECA 4451 and PECA 4197) and the extent of DNA repair in two of these lines (MeWo and PECA 4197). Tumour cells were irradiated with X-rays (0.1-10 Gy), embedded in agarose on slides, lysed with sodium dodecyl sulphate and exposed to an electric field. DNA migrated within the agarose and formed comets whose length depended on the amount of DNA damage. When the cells were incubated at 37 degrees C for various time intervals before electrophoresis started, the comets shrank in the course of time, indicating repair of DNA damage. All three cell lines showed the same extent of DNA damage after radiation exposure, despite the fact that in the colony-forming assay MeWo and PECA 4451 were much more sensitive to radiation exposure than PECA 4197. The repair characteristics, however, were markedly different for MeWo and PECA 4197 cells. PECA 4197 cells showed a much faster restoration of the original shape of the cell nucleus than MeWo cells.
DNA Damage↗
[The effects of low radiation doses: carcinogenesis].
In the low dose range such radiation risks, cf. the induction of cancer, have to be considered for which the lack of a threshold dose is assumed. The most important epidemiological data for cancer risk after irradiation come from the survivors in Hiroshima and Nagasaki. In the dose range below 100 mSv no increase of cancer rate can be observed in the total population with all age groups. Therefore the risk has to be estimated in the low dose range by extrapolation. Experimental studies of the mechanism of carcinogenesis have to clarify the question of a threshold dose. The variability of individual radiosensitivity is of great significance. After radiation exposures certain patterns of damage have been observed on the DNA. These phenomena may open the possibility to better recognize radiation-induced tumors in the low dose range.
Dose-Response Relationship, Radiation↗
Determination of potential doubling times in human melanoma cell cultures subjected to irradiation and/or hyperthermia by flow cytometry.
The proliferation of human melanoma cells in vitro after irradiation and/or hyperthermia was studied by means of two-parameter flow cytometry. Cultures were incubated with BrdU for 30 min and fixed either immediately or after a delay of several hours. Cells having synthesized DNA were identified with the help of an antibody against BrdU. DNA was stained quantitatively with propidium iodide. In this way the distribution of cells in the phases of cell cycle could be determined and the movement of labeled cells through the phases of the cycle could be analyzed. Experiments in which the cell cycle distribution was studied at 4-h intervals after treatment showed the following: (1) Irradiation (4 Gy X rays) causes the expected G2 block with a maximum after 12-16 h. The proportion of S-phase cells decreases continually during the first 48 h after treatment. (2) Hyperthermia (1 h, 43 degrees C) alone or in combination with irradiation causes a delay in S phase. The cells begin to move into G2 phase only after 12-16 h and accumulate there to some extent. From the progression of labeled cells through the cycle, the duration of S phase could be determined. Experiments and calculations of this kind were done 0, 24 and 48 h after treatment. The duration of S phase was increased only moderately (by 4 h) after irradiation, but a delay of about 30 h occurred after hyperthermia (alone or in combination with X rays). Smaller delays (up to 9 h) were observed 24 and 48 h after treatment. Two different methods were used to calculate potential doubling times. Both of them gave similar results, but a comparison with the actual population doubling times (determined by cell counting) showed that reasonable estimates could be achieved only for the untreated controls. With cultures subjected to irradiation and/or hyperthermia serious discrepancies were observed. This does not seem to be due to technical problems inasmuch as we are dealing with a whole set of data produced under well-defined in vitro conditions (in contrast to the clinical situation, where potential doubling times have to be estimated from single samples). Our results certainly do not encourage the extension of the method (which was originally intended for the prediction of unperturbed tumor growth) to a post-treatment setting.
Cell Cycle↗
Relation between rate of cell proliferation and formation of micronuclei after combined treatment with X-rays and caffeine.
We studied the effects of caffeine (2 mM), X-rays (1 Gy) and the combination of both agents on cell proliferation and formation of micronuclei in the early stages of preimplantation mouse embryos in vitro. Two-cell embryos were exposed to the agents shortly before division to the 4-cell stage. Proliferation and micronucleus production was monitored every 2 h in the 4- and 8-cell stages. A rather peculiar pattern of micronucleus formation after radiation exposure alone was observed for 8-cell embryos: those embryos that were the first to enter the 8-cell stage showed two to three times higher numbers of micronuclei per cell when compared with those embryos that entered the 8-cell stage some hours later. Studies of the kinetics of cell proliferation and of micronucleus formation in 4- and 8-cell embryos and exposure to caffeine revealed that this result could be explained by two factors: a slight asynchrony in the developmental stage at the time of exposure and the length of the interval being available for repair processes. When caffeine was present, a third factor had to be taken into consideration: direct inhibition of repair by caffeine.
Animals↗
Radiation induced micronuclei in subpopulations of human lymphocytes.
The micronucleus expression in T-helper, T-suppressor and B lymphocytes of the peripheral blood was studied after in vitro exposure to high (2.5 Gy and 5 Gy) and low (0.5 Gy and 1 Gy) doses of ionizing radiation. Investigations were carried out by combining the micronucleus assay with immunofluorescence staining using subpopulation specific antibodies. While in the higher dose range B cell proliferation was inhibited nearly completely-so that micronuclei could not be expressed-we found after exposure to lower doses that B cells were the lymphocyte subpopulation which was most sensitive to micronucleus induction. Among the T cell population, the T-suppressor subset revealed a higher yield of micronuclei than T-helper cells, whereas with regard to the effect of radiation on proliferative ability, T-helper cells reacted more sensitivity than the T-suppressor lymphocytes. Our studies provide insight into the effect of radiation exposure on the micronucleus expression of lymphocyte subpopulations and new information which may be useful for the further development of biological dosimetry.
Adult↗
Chromosomal damage in preimplantation mouse embryos and its development through the cell cycle.
Cytogenetic damage is usually studied in the first metaphase after exposure to ionizing radiation although it is well known that further chromosomal aberrations are also expressed during later mitotic divisions (Bauchinger et al., 1986; Lloyd et al., 1992). For such investigations the preimplantation mouse embryo is very well suited. If irradiation takes place in the 1-cell stage it can easily be followed whether the cells of the embryo have reached the first, second or third mitosis after the exposure. Furthermore, the duration of the various cell cycle phases is well known and therefore radiation effects on the cell cycle, e.g., the G2 block, can be investigated (Streffer and Molls, 1987). Studies on these two phenomena will be reported here.
Animals↗
Induction of quiescent S-phase cells by irradiation and/or hyperthermia. I. Time and dose dependence.
DNA synthetic activity and DNA content of individual cells can be determined simultaneously by means of two-parameter flow cytometry. We used this method to study the effects of irradiation and/or hyperthermia on the proliferation of human melanoma cells in vitro. In untreated cultures, most of the cells with an S-phase DNA content showed incorporation of BrdU, but a small percentage did not. The fraction of these quiescent S-phase cells increased after irradiation (up to 8 Gy X-rays) and/or hyperthermia (up to 6 h at 42 degrees C or up to 2 h at 43 degrees C). Four days after the treatment up to 50% of the S-phase cells did not incorporate BrdU. There was a clear dose dependence for irradiation and hyperthermia alone or in combination. Generally, the combined effects seemed to be additive. Possible pitfalls of the technique used were taken into consideration. Our main practical conclusion is that single-parameter measurements of DNA content are insufficient to characterize the proliferative status of cell populations, especially after irradiation and/or hyperthermia, because a large part of those cells identified as being in S-phase may be quiescent.
Dose-Response Relationship, Radiation↗
Induction of quiescent S-phase cells by irradiation and/or hyperthermia. II. Correlation with colony forming ability.
Quiescent S-phase cells, i.e. cells with an S-phase DNA content that do not show BrdU incorporation, can be induced in a dose-dependent manner by irradiation and/or hyperthermia (Zölzer et al. 1992). As they begin to appear only 48-72 h after treatment, they do not seem to be related to early cell cycle disturbances, but rather to late events involved with cell death. We therefore determined colony forming ability under the same conditions, and tried to correlate the two parameters. Although, in general, higher frequencies of unlabelled S-phase cells were associated with lower survival, there were interesting differences. At the same level of survival, for instance, quiescent cells were induced more efficiently by hyperthermia than by irradiation. Additional experiments with split dose protocols showed that while cell killing was reduced by fractionation, the frequency of unlabelled S-phase cells increased. This further corroborates our conclusion that there is no simple relationship between the two parameters. Quiescence in S-phase is not just an expression of cell death irrespective of the treatment by which it may have been caused.
Humans↗
Is there an adaptive response in spleen lymphocytes of C57B1/6 mice as assessed by chromosomal aberrations?
In previous publications we have shown that no adaptive response could be induced in preimplantation embryos and spleen lymphocytes of Heiligenberger mice, a strain inbred at our institute (Wojcik et al., Int. J. Radiat. Biol. 62, 177-186, 1992; Müller et al., Int. J. Radiat. Biol. 62, 169-175, 1992). Because of data indicating that some humans may be genetically deficient in the ability to express an adaptive response, we have speculated that the cells of the Heiligenberger strain may lack an adaptive response. To check this, experiments have been done with spleen lymphocytes of the C57B1/6 mice. Initial results indicated the presence of an adaptive response in some individual C57B1/6 mice. However, an analysis of aberration scores of parallel lymphocyte cultures revealed a high intraindividual variability, indicating that the adaptive response observed initially was a reflection of this variability rather than of induced radiation resistance. This conclusion is confirmed further by the lack of such variability in parallel cultures of lymphocytes from Heiligenberger mice.
Adaptation, Physiological↗
Combined morphological approaches in the study of network formation in tumor angiogenesis.
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Animals↗
Prospective flow cytometric analysis of head and neck carcinomas. Prognostic relevance of DNA-content and S-fraction.
Flow cytometric data were obtained from 142 primary squamous cell carcinomas of the oral cavity or the oropharynx. Aneuploidy was found in 36.8% of the tumours. The DNA indices showed a significant correlation with the S-fraction, tumour size and evidence of suspicious lymphnodes. There was no clear correspondence between the S-fraction and the tumour stage. Tumour size, histopathologically-positive lymphnodes and the mode of treatment were significantly correlated with the survival rates. In contrast, there was no clear correlation between flow cytometric data and the prognosis of the whole group as well as several clinical subgroups. In 40 patients who received preoperative irradiation, DNA indices and S-fractions were compared before and after the preoperative treatment. In 12 of 14 aneuploid tumours irradiation led to a decrease in the DNA indices into the range of euploidy. Only 2 tumours remained aneuploid. After irradiation 26 of 37 tumours showed a decrease in S-fraction, 11 tumours showed an increase. Loss of aneuploidy after irradiation was associated with a histologically-proven increasing devitalization of tumour cells, decrease in S-fraction corresponded to a tendency to a better prognosis.
Adult↗
Radiosensitization efficacy of KU-2285, RP-170 and etanidazole at low radiation doses: assessment by in vitro cytokinesis-block micronucleus assay.
Since the cytokinesis-block micronucleus assay is very sensitive at low radiation doses, we used it to investigate the in vitro sensitizing effects of two new hypoxic cell sensitizers (KU-2285, a fluorinated 2-nitroimidazole and RP-170, a 2-nitroimidazole nucleoside analogue) at 1-3 Gy in comparison with etanidazole. Exponentially growing EMT6 cells were treated with the drugs under aerobic or hypoxic conditions for 40 min prior to and during irradiation, after which the drugs were removed and cytochalasin B (2 micrograms/ml) was added to the medium. The number of micronuclei in binucleate cells was counted after 42 h of culture. Under aerobic conditions the three compounds at 5 mM had no sensitizing effect. Under hypoxic conditions the sensitizer enhancement ratio (SER) at 5 mM was 3.8 for KU-2285, 3.2 for RP-170, and 2.3 for etanidazole, while the oxygen enhancement ratio was 2.9. When the cells were pretreated under hypoxic conditions with drugs at 5 mM but then irradiated under aerobic conditions, KU-2285 and RP-170 had a sensitizing effect whereas etanidazole did not. The sensitizers were also tested at 0.5 and 1 mM, and the SER values were compared with those obtained at high doses (15-30 Gy) using a colony assay. The SER at low doses was higher than that at high doses for 1 and 5 mM KU-2285 and 5 mM RP-170, while the SERs were similar for all concentrations of etanidazole and the lower concentrations of KU-2285 and RP-170. These results might suggest the potential usefulness of KU-2285 and RP-170 in clinical radiotherapy.
Animals↗
Effects of caffeine on protein phosphorylation and cell cycle progression in X-irradiated two-cell mouse embryos.
The G2 phase/mitosis transition in cleavage-stage mouse embryos is correlated with an increased phosphorylation of a defined set of proteins at 46, 35, 30, and 29 kDa. Cell cleavage and the associated changes in protein phosphorylation are delayed after X-irradiation. To understand the mechanism of the caffeine-induced uncoupling of mitosis and the cellular reactions to DNA-damaging agents, we have studied the effects of caffeine treatment on cell cycle progression and protein phosphorylation in two-cell mouse embryos after X-irradiation. Caffeine alone had no effect on timing of and changes in phosphorylation associated with the embryonic cell cycle. In combination with X-rays, however, caffeine was able to override the radiation induced G2 block and restored the normal timing of these phosphorylation changes after X-irradiation. However, new additional changes in protein phosphorylation appeared after the combined treatment. Isobutylmethylxanthine (IBMX), a substance chemically related to caffeine but a more specific inhibitor of the phosphodiesterase that breaks down cyclic AMP, reduced the radiation induced G2 block from 4 to 5 h to about 1 h and restored the cell cycle associated changes in protein phosphorylation. However, the same new changes which appeared after the combined treatment of caffeine and X-rays were observed after the combination of IBMX and X-irradiation. IBMX specific changes in protein phosphorylation were detected in both the single and the combined treatment. These results indicate a similar action of caffeine and IBMX in overriding the radiation induced G2 block in two-cell mouse embryos.
Animals↗