Apoptotic cell death triggered by camptothecin or teniposide. The cell cycle specificity and effects of ionizing radiation.

We have previously observed that the DNA topoisomerase I inhibitor camptothecin (CAM), or DNA topoisomerase II inhibitors teniposide (TEN) and amsacrine (m-AMSA) trigger endonucleolytic activity in myelogenous (HL-60 or KG1), but not lymphocytic (MOLT-4) leukaemic cell lines. DNA degradation and other signs of apoptotic death were seen as early as 2-4 h after cell exposure to these inhibitors. Cells replicating DNA (S phase) were selectively sensitive whereas cells in G1 were resistant; the sensitivity of G2 or M cells could not be assessed in these studies. The present studies were aimed at revealing whether DNA repair replication induced by ionizing radiation can sensitize the cells, and to probe the sensitivity of cells arrested in G2 or M, to these inhibitors. The data show that gamma-irradiation (0.5-15 Gy) of HL-60 cells does not alter their pattern of sensitivity, i.e. G1 cells, although engaged in DNA repair replication, still remain resistant to CAM compared with the S phase cells. Likewise, irradiation of MOLT-4 cells also does not render them sensitive to either CAM or TEN, regardless of their position in the cell cycle. Irradiation, however, by slowing the rate of cell progression through S, increased the proportion of S phase cells, and thus made the whole cell population more sensitive to CAM. HL-60 cells arrested in G2 either by irradiation or treatments with Hoechst 33342 or doxorubicin appear to be more resistant to CAM relative to S phase cells. Also resistant are cells arrested in M by vinblastine. The data suggest that some factor(s) exist exclusively in S phase cells, which precondition them to respond to the inhibitors of DNA topoisomerases by rapid activation of endogenous nuclease(s) and subsequent death by apoptosis. HL-60 cells in G1, G2 or M, or MOLT-4 cells, regardless of the phase of the cycle, appear to be protected from such a mechanism, and even induction of DNA repair replication cannot initiate DNA degradation in response to DNA topoisomerase inhibitors. These data, together with the evidence in the literature that topoisomerase I may be involved in DNA repair, suggest that a combination of these inhibitors with treatments that synchronize cells in the S phase and/or recruit quiescent cells to proliferation, including radiation, may be of value in the clinic.

Relationship between mitotic delay and the minimum dose rate of X irradiation required to stop cell proliferation.

When cells are subjected to irradiation, their progression through the cell cycle can be arrested. If the arrested cells are subjected to additional damage, their period of arrest is prolonged. Under continuous low-dose-rate irradiation, the cumulative nature of arrest time leads to a geometric increase in the arrest time as a function of the dose rate. Above a certain cell-line-specific dose rate, the arrest duration becomes infinite and cell proliferation ceases. We find that the lowest dose rate (critical dose rate) required to stop cell proliferation during continuous irradiation is the reciprocal of the mitotic delay per gray of high-dose-rate irradiation. The calculated critical dose rates for X rays agree with those measured by Mitchell et al. (Radiat. Res. 79, 537-551, 1979), provided that the critical dose rate is identified with the minimum dose rate which allows no more than one population doubling. This specific identification of the critical dose rate is explained on the basis of cell cycle kinetics. A kinetic mechanism for cell cycle arrest and recovery leads to the critical dose rate as the reciprocal sensitivity for G2 arrest.

Relationships between mitotic delay and the dose rate of X radiation.

Upon exposure of cells to radiation delivered at a continuous low dose rate, cell proliferation may be sustained with the cells exhibiting a constant doubling time that is independent of the total dose. The doubling time or mitotic delay under these conditions has been shown to depend on the dose rate in HeLa, V79 and P388F cells (Mitchell et al., Radiat. Res. 79, 520-536, 1979; Fox and Gilbert, Int. J. Radiat. Biol. 11, 339-347, 1966). Reanalysis of the data for these particular cell lines shows that there is a threshold dose rate for mitotic delay, and that above the threshold there is a linear relationship between the length of mitotic delay and the logarithm of the dose rate which is referred to as the dose-rate response. We have observed the same relationships for L5178Y (LY)-R and LY-S cells exposed to low-dose-rate radiation. The threshold dose rates for LY-R, LY-S and P388F cells are similar (0.01-0.02 Gy/h) and are much lower than for V79 and HeLa cells. The slope of the dose-rate response curve is the greatest for HeLa cells, followed in order by LY-S, V79 and P388F cells, and finally by LY-R cells. The slopes for HeLa and LY-R cells differ by a factor of 35.

Hyperthermia-induced intracellular ionic level changes in tumor cells.

The intracellular content of potassium (K) ions in P815 cells decreases when the media pH is lowered, and it increases when media pH is raised. The determination of the ion content therefore requires accurate control of the medium pH. The K ion content measured both by the flame emission method and by the K analog. 86Rb, exhibits a decline when the cells are incubated at 43 degrees C at a fixed pH chosen between 7.4 and 6.7. The chloride content also decreases while the sodium content does not change by a significant amount. Under the same hyperthermic conditions the intracellular pH decreases by a fraction of a pH unit. Simultaneously, the cell water volume increases by 20%, as measured by tritiated water. In the final analysis, hyperthermia produces an apparent deficit in the cellular osmolarity. A possible explanation is given.

Use of silicone fluids in studies of cellular amino acids.

In numerous cellular studies, cells labeled with radioisotopes have been separated from the labeling medium by an aqueous solution in order to determine the quantity of internalized labels; however, the aqueous wash tends to remove significant labeling from the cells. Therefore, in order to preserve all of the internalized labels, non-aqueous medium such as silicone fluids may be used. The termination of the labeling is achieved in the silicone method when, upon centrifugation, the cells separate from the medium and enter the silicone fluid to sediment to the tube bottom. This sedimentation of cells placed above a layer of silicone fluid exhibits a critical dependence on the centrifugal force, and gives rise to an uncertainty of only 2 s in determining the time of separation of cells from the medium using General Electric F-50 silicone fluid and a modified Beckman J2-21 centrifuge. It is therefore possible to determine the kinetics of incorporation of labeled amino acids into intracellular pools and proteins. In particular, since this silicone wash method determines the size of the total pool and the aqueous wash method determines the size of the acid-extractable pool, the simultaneous measurements of the size of both pools leads to the determination of the kinetics of labeling of the free amino acid pool. Among many possible applications and extensions of these methods, the studies of formation of intracellular pools and relations among different pools of transported molecules, such as water and amino acids, appear promising.

Comparison of isoeffect relationships in radiotherapy.

Irradiation affects numerous physiological processes within cells and tissues and can lead to damage or death. If the damage is not too severe, cells have the ability to repair and regenerate. Many small injuries are repaired more easily than ones causing extensive damage and, consequently, tissues typically respond differently to one large dose of radiation than to many small doses, separated in time. In the radiotherapy of tumors, the choice of the fractionation regimen of dose over time is therefore as crucial as the total radiation dose. The interdependence between total dose, fractionation regimen, and radiation effect has been described mathematically with various isoeffect relationships. These relationships appear to be fundamentally distinct and have been considered unrelated; some even claim that one class of isoeffect relationships is appropriate whereas other relationships are rather useless. We examine how alternative isoeffect models relate to each other and test the reliability of estimating parameter values of one model from the other.

Mobilization of amino acids in tumor cells by hyperthermia.

When p815 mastocytoma cells are heated to 43 degrees C from 37 degrees C, pools of individual amino acids and the total amino acid pool increase significantly, the latter reaching a new plateau within 20 minutes. The increase in the total amino acid pool may reach as much as 75 mOsm per liter of cell water or 26% of the cellular osmolarity. The source of this excess amino acid is not due to an accelerated degradation of proteins, but either to the amino acids left behind by slowed protein synthesis or to the accelerated transport of extracellular amino acids into the cell. The excess amino acid should increase the osmolarity and the cell volume. Evidence suggests that the cell volume increase triggers the osmoregulatory process to reduce the contents of major ions.

Phase transitions in phosphatidylcholine dispersion observed with an interference refractometer.

An interferometer is used to measure the refractive index change accompanying the crystal-to-liquid-crystal phase transition in the dispersion of phosphatidylcholines. Two separate methods of obtaining the refractive index change are employed: the first method analyzes the intensity transmitted through a spatial filter and the second method utilizes a piezoeletric crystal-based electronic compensator. The results of the two methods agree well. The accuracy of the apparatus (6 X 10(-6)) permitted us to use a very dilute sample to detect the phase change. Only a fraction of a milligram of dry lecithin is needed to observe the change. The result confirms conclusively that the major reason for the turbidity change at the transition temperature is the alteration in the refractive index of the lipid membranes. The fractional change in the refractive index does not agree well with the fractional change in the density of lipid molecules in vesicles.

Swelling of multicellular spheroids induced by hyperthermia.

EMT6 multicellular spheroids invariably swell by 10 to 50 per cent after incubation at 43 to 45 degrees C for 1 h. Both scanning electron and optical microscopy reveal morphological alterations particularly in the outer region of the spheroids. While the control cells are contiguous to one another and tightly held to the spheroid body, the heated spheroids exhibit partially disrupted contacts among cells. Measurements of intercellular volume and water volume of spheroids with labelled water and inulin show that changes in the spheroid volume are not due to an increase in cell volume, but that they can be explained by a 60-100 per cent increase in the intercellular space within a spheroid. Continuous observation of individual spheroids heated to 43-45 degrees C shows loss of adhesion of cells in the outer region and even detachment of a few surface cells. This 'melting' of the spheroid surface appears to result from a disorder in the extracellular material. Treatment with cell swelling agents such as hypotonic solution, ouabain, excess extracellular potassium ions, or ionophore nigericin, K+/H+ exchanger, each separately causes the spheroids to swell at the control temperature. On the other hand, A23187, Ca2+ ionophore, causes shrinkage of the spheroids. Thus, under hyperthermia, the volume of spheroids increases due to the disruption in the cell organization in their outer region.