Pk Wierenga

HYPERTHERMIA, INTRACELLULAR FREE CALCIUM AND CALCIUM IONOPHORES

It is shown that heat-induced increase of intracellular calcium does not correlate with hyperthermic cell killing. Six different cell lines were investigated; in four (EAT, HeLa S3, L5178Y-R and L5178Y-S) heat treatments killing 90% of the cells did not affect the levels of intracellular free calcium ([Ca2+]i). In one cell line (3T3) a heat-induced increase in [Ca2+]i was observed. LM cells showed a heat-induced increase of the ratio of the fluorescent signals, but this may be explained by Fura-2 leakage out of the cells. Calcium ionophores are used to address the question whether rises in [Ca2+]i might cause cell killing. To investigate the existence of sensitization to Ca2+ toxicity by heat, ionophore treatments are combined with hyperthermia. Both ionophores used, A23187 and ionomycin, cause cell killing corresponding with increases in [Ca2+]i at 37 degrees C in EAT cells. In HeLa S3 cells, substantial increases in [Ca2+]i due to the action of ionomycin were observed without corresponding cell killing. This indicates the presence of a threshold concentration of [Ca2+]i in HeLa S3 cells before the treatment becomes toxic. Both ionophores show synergism with hyperthermia for cell killing as well as at the level of increased [Ca2+]i. The synergistic action may be explained as thermal enhancement of calcium toxicity.

Effect of a hyperthermic treatment on the pluripotent haemopoietic stem cell in normal and anaemic mice

Up to now, the hyperthermic sensitivity of pluripotent haemopoietic stem cells is unknown, and the few existing data from reports in the literature are conflicting. There are two main drawbacks in the set-up of those studies: (1) only CFU-S day 9 results were presented, whereas it is questionable if this assay gives a true reflection of the pluripotent stem cell, and (2) no attention has been paid to heat effects on the seeding efficiency, i.e. the amount of stem cells which will lodge in the spleen. The present study focused on the procedural differences and compared the results of a hyperthermic treatment (60 min, 42 degrees C) on the stem cells, assayed with the CFU-S day 9 and the CFU-S day 12 method, using the following three stem cell suspensions, all differing in their proliferative activity: bone marrow from normal mice and bone marrow and spleen cells from anaemic mice. Furthermore, we investigated the seeding efficiency before and after heat treatment. Resting stem cells, assayed with the CFU-S day 12 method, turned out to be resistant to hyperthermia as compared with the active cycling stem cells, while with the CFU-S day 9 assay the stem showed the same thermosensitivity in the two bone marrow suspensions. The active cycling stem cells do not significantly differ in thermosensitivity, in CFU-S day 9 and day 12 assays, although there is a difference between bone marrow and spleen. Hyperthermia appears to influence the seeding efficiency for spleen CFU-S; an increase of 1.73 was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

THERMAL SENSITIVITY OF THE MURINE CFU-S-12 - ROLE OF ENVIRONMENTAL CELLS

The hyperthermic sensitivity of the CFU-S-12 in bone marrow from normal and anaemic mice was determined. The terminal slope of the survival curves, demonstrated by the T0 values, does not significantly differ in the resting and active cycling stem cells. In the active cycling stem cells the initial shoulder region was less dominant compared with the resting stem cells. The difference in heat sensitivity between resting and active proliferating CFU-S-12 might be explained by a difference in the accumulation of damage before lethality becomes manifest. The difference in heat sensitivity appears to be independent of the environmental accessory cells, demonstrated by a similar hyperthermic effect of the purified stem cells from bone marrow and spleen and the stem cells in the total cell suspensions. Therefore the heat sensitivity of the haemopoietic stem cell is not mediated by a release of injurious substances from environmental heat-damaged cells. The heat treatment does not result in a selection of macroscopic detectable colonies 12 days after inoculation, as is demonstrated by the same morphology of the spleen colonies from the stem cells before and after the hyperthermic treatment.