G(2)-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)-p hase cells increases as a consequence of heat treatment. On the basis of ce ll cycle distributions alone, however, it is difficult to say whether the t wo results are related. In particular, comparison is complicated by the fac t that the accompanying changes in the S-phase transition are different. Th ese changes play a minor role after irradiation but constitute by far the m ost important cell cycle effect after heat treatment. Two-parameter flow cy tometry 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-conjugate d antibody against BrdU; DNA was quantified after staining with propidium i odide. 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 c ells in the G(1) compartment (after they had gone through mitosis), the del ay in G(2) phase could be determined. The duration of the G(2)/M phase in c ontrol cells was about 6 h. This was increased to 12, 13 and 16 h after irr adiation (4 Gy X rays), heat treatment (1 h at 43 degreesC), and a combinat ion 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 p hase were still observable at this time. As expected, the radiation-induced G(2)-phase block was almost completely removed by incubating the cells wit h 5 or 10 mM caffeine. In the case of hyperthermia alone or in combination with radiation, however, caffeine was somewhat less effective. This does no t 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 even ts. The long delay in S phase after heat treatment may lead to a loss of su sceptibility to caffeine by the time the cells move into the G(2) phase. (C ) 2001 by Radiation Research Society.