Growth inhibition by STI571 in combination with radiation in human chronic myelogenous leukemia K562 cells.

Altered radiation responses by STI571 (Imatinib, Glivec), a specific inhibitor of the tyrosine kinase activity of Bcr-Abl, was assessed in K562 chronic myelogenous leukemia cells using growth inhibition and colony formation assays. Flow cytometry, Western blotting, and microscope observation were used to determine cell cycle redistribution, erythroid differentiation, apoptosis, necrosis, senescence, and expression and phosphorylation of effectors downstream from Bcr-Abl as endpoints. STI571 (> or =24-h contact) retarded the growth of K562 cells and elicited reduction in the G(2)-phase content due to an efficient arrest in early S phase rather than to the disruption of the G(2) checkpoint as confirmed by analysis of Lyn and CDK1 phosphorylation. STI571 brought about the inhibitory dephosphorylation of Bcr-Abl and STAT5, but the expression of DNA-PKcs and Rad51 was unaffected and the interaction between radiation and STI571 was strictly additive with regard to induction of apoptosis. Overall STI571 interacted cooperatively with radiation to retard the growth of K562 cells but did not affect intrinsic radiosensitivity. However, STI571 and radiation acted antagonistically with each other with regard to induction of senescence and erythroid differentiation.

Hypothermic modulation of doxorubicin, cisplatin and radiation cytotoxicity in vitro.

BACKGROUND: The influence of hypothermia on doxorubicin, cisplatin and radiation cytotoxicity was investigated in vitro. MATERIALS AND METHODS: A human glioma cell line (251MG) in early exponential growth was exposed to doxorubicin or cisplatin at various concentrations for 4 hours, or X-irradiation at 28 degrees C or 37 degrees C. The cells continued growing in multi-well plates at 37 degrees C and were counted every third day until the end of the logarithmic phase, on day 13. RESULTS: Exposure to doxorubicin 0.05-0.5 microg/ml or cisplatin 1-10 microg/ml caused a dose-dependent inhibition of cell growth with a significantly reduced toxicity when exposed at 28 degrees C as compared to 37 degrees C. Irradiation with 4 Gy also resulted in less toxicity during hypothermia. Chlorpromazine 0.01-10 microg/ml, used to induce hypothermia in vivo (1), neither influenced, cellular growth itself nor interacted with doxorubicin, cisplatin or irradiation. CONCLUSION: Moderate hypothermia (28 degrees C) appears to protect against the cellular insult of doxorubicin, cisplatin and ionising irradiation and their consequences.

G2-phase delays after irradiation and/or heat treatment as assessed by two-parameter flow cytometry.

Similar to what has been observed after irradiation, the fraction of G(2)-phase cells increases as a consequence of heat treatment. On the basis of cell cycle distributions alone, however, it is difficult to say whether the two results are related. In particular, comparison is complicated by the fact that the accompanying changes in the S-phase transition are different. These changes play a minor role after irradiation but constitute by far the most important cell cycle effect after heat treatment. Two-parameter flow cytometry was used here to study the proliferation of human melanoma cells in vitro. Cultures were pulse-labeled with BrdU after irradiation and/or heat treatment and were fixed either immediately or after a delay of up to 36 h. DNA-synthesizing cells were identified with the help of an FITC-conjugated antibody against BrdU; DNA was quantified after staining with propidium iodide. In this way, the cell cycle distribution could be determined and the progression through the cell cycle could be analyzed. From the movement of labeled cells through the cycle, in particular the appearance of labeled cells in the G(1) compartment (after they had gone through mitosis), the delay in G(2) phase could be determined. The duration of the G(2)/M phase in control cells was about 6 h. This was increased to 12, 13 and 16 h after irradiation (4 Gy X rays), heat treatment (1 h at 43 degrees C), and a combination of the two, respectively. In all these cases, the G(2)-phase block was completely overcome within 48 h after treatment, whereas changes in the S phase were still observable at this time. As expected, the radiation-induced G(2)-phase block was almost completely removed by incubating the cells with 5 or 10 mM caffeine. In the case of hyperthermia alone or in combination with radiation, however, caffeine was somewhat less effective. This does not mean, however, that the mechanisms involved are necessarily different. It can also be seen as a result of the differences in the time course of events. The long delay in S phase after heat treatment may lead to a loss of susceptibility to caffeine by the time the cells move into the G(2) phase.

Quiescence in S-phase and G1 arrest induced by irradiation and/or hyperthermia in six human tumour cell lines of different p53 status.

PURPOSE: Quiescent S-phase cells, i.e. cells with a DNA content intermediate between G1 and G2 that nevertheless do not synthesize DNA have been previously observed in human melanoma cells exposed to radiation and/or hyperthermia. This phenomenon has now been studied in more detail comparing six human tumour cell lines of different p53 status and thus different cell-cycle checkpoint control. MATERIALS AND METHODS: Two melanoma (Be11, MeWo), two squamous carcinoma (4197, 4451) and two glioma (EA14, U87) cell lines were used. Changes in the cell-cycle distribution after treatment were studied using two-parameter flow cytometry in order to measure DNA content and BrdU incorporation simultaneously. RESULTS: The fraction of unlabelled cells in the S-phase compartment was determined at daily intervals after treatment. Only background levels of such cells were seen in three of the cell lines (Be11, 4197, EA14). With the other three cell lines (MeWo, 4451, U87) we observed a time- and dose-dependent increase: a few days after treatment up to 20% of all cells did not incorporate BrdU. It is interesting to note that Bell, 4197 and EA14 are p53 wild-types and show a G1 block of several hours after irradiation and/or hyperthermia, while MeWo and 4451 are p53 mutants unable to exhibit such a delay, and U87 in spite of being a p53 wild-type has a reduced ability to do so. CONCLUSIONS: The MeWo, 4451 and U87 cell lines have less time available for the repair of DNA damage before entering into the S-phase, which leads to problems during replication and causes some kind of interphase death. Radiation-induced apoptosis does not seem to be involved here, as it is not unequivocally correlated with the induction of a G1 block or with p53 status.

Blood irradiation for intraoperative autotransfusion in cancer surgery: demonstration of efficient elimination of contaminating tumor cells.

BACKGROUND: Intraoperative blood salvage is contraindicated in cancer surgery because of contaminating tumor cells and the risk of systemic dissemination. On the basis of the radiosensitivity of cancer cells, irradiation of salvaged blood with 50 Gy is proposed as a way to allow return of salvaged blood. STUDY DESIGN AND METHODS: Elimination of tumor cells by blood irradiation was studied in vitro with cells from 10 cell lines and from 14 tumor preparations after their addition to red cells in high numbers, or with blood shed during cancer surgery. Before and after gamma radiation, tumor cells were isolated by density gradient centrifugation and tested for their proliferative capacity in a cell colony assay. DNA metabolism was analyzed by incorporation of 5' bromodesoxyuridine. RESULTS: Survival curves of cells from various tumors confirmed D0 (the dose required to reduce the fraction of surviving cells to 37 percent of the original value) values in the range of 1.2 to 2.2 Gy. After irradiation of tumor cell-contaminated blood with 50 Gy, no cell colony formation was observed, which indicates a reduction rate exceeding 10 log. Irradiated cancer cells showed viability, but no residual DNA metabolism. CONCLUSION: The level of inactivation by a 50-Gy dose far exceeds that needed to inactivate the number of proliferating tumor cells observed or expected in wound blood. These results provide the experimental basis for the clinical application of blood irradiation for intraoperative blood salvage in cancer surgery.

Effects of ultraviolet-B and PUVA on ornithine decarboxylase activity, DNA synthesis, and protein kinase C activity in mouse skin.

Ultraviolet-B and PUVA share several biological events with phorbol ester tumor promoters. The effects of ultraviolet-B irradiation and topical PUVA treatment on ornithine decarboxylase activity, DNA synthesis, and protein kinase C activity, which are known to be induced or activated by phorbol ester tumor promoter, were investigated in hairless mouse skin. Ornithine decarboxylase activity was remarkably enhanced by ultraviolet-B and PUVA. Although PUVA did not affect DNA synthesis significantly, ultraviolet-B stimulated epidermal DNA synthesis approximately 5-fold over control values at 48 h. However, unexpectedly, neither cytosolic nor membrane-bound protein kinase C activity showed any change during the 2 h after either treatment. These results suggest that the protein kinase C system is not involved in the initial signal transduction system of ultraviolet-B or PUVA, unlike the case with phorbol ester tumor promoter.

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.

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.