Heterogeneous X-ray survival characteristics of lymphocytes in prolymphocytic leukaemia: mathematical analysis distinguishing delayed cell death and true radioresistance.

The survival of non-dividing (G0) leukaemic lymphocytes in culture is generally too short for their radiosensitivity to be quantitatively assessed, since lethally X-irradiated cells may show a long delay before manifestations of cell death ("interphase death") are seen. Counts of surviving cells will therefore include both lethally-hit cells (apparent survivors), and real survivors which have not been lethally hit. Death rates of irradiated leukaemic and normal cells show great variation between individuals, so that comparisons of radiosensitivity between different cell populations based on surviving cell counts at a single time-point are invalid. In this study the supposed radioresistance of prolymphocytic leukaemia lymphocytes was examined in 6 patients with B-cell disease. Survival curves were plotted from serial observations made over several days after graded X-irradiation (0-1000 cGy). We attempted to interpret these radiation responses in terms of their dose dependence (intrinsic radiosensitivity) and time dependence (cell death rate) characteristics using the best-fitting of four mathematical models, all based on classical "single-hit" target theory. The apparent radioresistance shown in 4 cases could be explained by very slow death rates (T1/2 values 55-205 h) of cells proving otherwise radiosensitive (D37 values 38-123 cGy). Genuine radioresistance was found in only 1 case (actual D37 value above 2000 cGy). By ignoring delayed cell death in clinical assessments, pathological lymphocytes could be mistakenly categorised as resistant to elimination by radiotherapy.

Simplified quantitative estimation in vitro of lymphocyte radiosensitivity applied to patients with chronic lymphocytic leukaemia.

Lymphocyte survival changes observed at 1, 2 and 3 days as responses to 3 doses of ionising radiation in vitro (40, 100, and 500 cGy) are analysed by computer according to a simple (single cell population) mathematical model. Intrinsic radiosensitivity, the susceptibility to lethal injury, which is expressed as the D37 value (the radiation dose permitting 37% survival), is estimated separately from the kinetics of subsequent death of lethally-irradiated cells (expressed as their half-life, or t1/2 value). Among the 35 patients with B-cell CLL studied (15 were never treated), both parameters varied widely and independently of one another. t1/2 ranged from 9-200 h and above, D37 from 14-500 cGy or above. Twenty-three patients were deemed 'radiosensitive' (D37 below 110 cGy). D37 level did not correlate with treatment status, mode of treatment, clinical staging (Rai) or lymphocyte count. With some exceptions, D37 remained relatively constant for individual patients with increasing duration of disease or alterations in treatment status. The assay method may prove useful as an aid in predicting response to low-dose splenic irradiation (SI) in CLL.

The intrinsic radiosensitivity of lymphocytes in chronic lymphocytic leukaemia, quantitatively determined independently of cell death rate factors.

Survival curve shape for lymphocytes X-irradiated in vitro is governed by death rate as well as intrinsic radiosensitivity. We have resolved into these two components the survival curves obtained for CLL lymphocytes by use of a simple mathematical model. A multiple correlation coefficient comparing the predicted with the experimental survival curves was close to unity (0.954-0.999). For 14/18 patients with unequivocal B-cell CLL, the leukaemic (colchicine ultrasensitive) cells behaved as a homogeneous population (D37 0.32-1.28 Gy). This is similar to the more radiosensitive class of lymphocytes of normal blood (believed to include the B cells) and is some 4-fold less than the more radioresistant class (comprising most of the T cells). The lethally hit cells were homogeneous in death rate, which followed first order kinetics. The half-life (range 9-87 h) was, on average, some 50 per cent shorter than the more radiosensitive normal lymphocytes. The remaining four patients constituted a miscellaneous group. From one of these, it can be seen that an excessively slow death rate can give the misleading impression of radioresistance. It is hypothesized that the benefit afforded certain CLL patients treated with low-dose total body irradiation (TBI) or splenic irradiation (SI) may reside, partly, in the sparing of T lymphocytes of the helper type and in accompanying selective elimination (or functional inactivation) of those of the suppressor type.

Colchicine ultrasensitivity of lymphocytes in chronic lymphocytic leukaemia.

It has been previously reported that, in comparison with normal lymphocytes, the lymphocytes in chronic lymphocytic leukaemia (CLL) are ultrasensitive in culture to the cytocidal action of colchicine. In this report the results of 240 colchicine studies in 87 patients with CLL are presented and analysed in terms of the diagnostic, clinical and haematological significance of colchicine ultrasensitivity (CUS) in CLL. All patients with CLL showed very significantly increased CUS of lymphocytes. The CUS did not vary with clinical and haematological status or time and only very rarely with treatment. There was no correlation between % CUS and subsequent course or survival. The results in CLL and in other forms of lymphocytosis and in non-Hodgkin's lymphoma are compared. It is concluded that the test is of considerable diagnostic value, particularly in identifying low-count and treated CLL and in the exclusion of non-CLL lymphocytosis. It appears to have no prognostic value but may have future application in monitoring treatment which selectively eliminates the abnormal cell population.

The fatty acyl chain composition of human normal and leukaemic lymphocytes and its modulation by specialised hydrogenation.

Thirty species of fatty acyl chain have been quantitatively identified in human normal peripheral blood lymphocytes (four donors) and lymphocytes circulating in eight patients with chronic lymphocytic leukaemia (CLL). Towards the aim of influencing cell behaviour by lowering membrane fluidity, reaction conditions for catalytic hydrogenation at physiological temperature and pH have been established that effect reduction of the unsaturated species, and preferentially the polyunsaturated forms, but this has not yet been accomplished without killing the cells. That saturation of ethylenic linkages per se is the cause of death is indicated by separate findings showing that the lymphocytes are capable of withstanding hydrogen gas at the requisite high pressure (9 atm.) or exposure alone to the rhodium catalyst [chlorotris (sodium diphenylphosphinobenzene-m-sulphonate)-rhodium(I) tetrahydrate]. It remains feasible that future use of these two agents in combination under milder conditions to produce much lower degrees of hydrogenation than those reported here will permit the cells to survive. Concerning fatty acyl chain composition, the lymphocytes from most of the patients exhibited an inversion in the level of palmitic and stearic acid. A consistently abnormal pattern exhibited by the patients was a rise in oleic acid and a fall in arachidonic acid content. This same alteration has been demonstrated elsewhere in transformed/neoplastic cell types and hence it could well represent phenotypic expression in the CLL lymphocyte of malignant change. Fatty acyl chain composition remained unchanged in lymphocytes reconstituted after cryopreservation in liquid nitrogen.

A mathematical model resolving normal human blood lymphocyte population x-ray survival curves into six components: radiosensitivity, death rate and size of two responding subpopulations.

The analysis was based on observations of survival decrease as a function of dose (range 0-5 Gy (= 500 rad) and time after irradiation in vitro. Since lymphocyte survival is also sensitive to culture conditions the effects of radiation were examined daily up to 3 days only, while survival of control cells remained ca. 90 per cent. The time-dependent changes were resolved as the death rates (first-order governed) of lethally-hit cells (apparent survivors), so rendering these distinguishable from the morphologically identical, true (ultimate) survivors. For 12 blood donors the estimated dose permitting 37 per cent ultimate survival (D37 value) averaged 0.72 +/- 0.18 (SD) Gy for the more radiosensitive lymphocyte fraction and 2.50 +/- 0.67 Gy for the less radiosensitive, each fraction proving homogeneously radiosensitive and the latter identifying substantially in kind with T-type (E-rosetting lymphocytes). The half-life of lethally-hit members of either fraction varied widely among the donors (ranges, 25-104 hours and 11-40 hours, respectively). Survival curves reconstructed by summating the numerical estimates of the six parameters according to the theoretical model closely matched those observed experimentally (range in multiple correlation coefficient, 0.9709-0.9994 for all donors). This signified the absence of any additional, totally radioresistant cell fraction.

Plasma membrane cholesterol regulates human lymphocyte cytotoxic function.

Cell cholesterol is believed to be confined mainly to the plasma membrane. Treatment here of human peripheral blood lymphocytes with cholesterol-free and cholesterol-containing liposomes to effect, respectively, decreases or increases in cholesterol content measureable by chemical analysis, markedly altered effector functions of the cells. Depletion of cholesterol evoked inhibition of spontaneous and phytohemagglutinin-dependent lymphocyte cytotoxicity against allogeneic target cells. Opposite effects resulted from cholesterol enrichment, with PHA-dependent and antibody-dependent cytotoxicities increasing significantly. Treatment, instead, with the known inhibitor of cholesterol biosynthesis, 25-hydroxycholesterol, had suppressive effects like those resulting from lowering the cholesterol level physically by liposome treatment. Our data suggest that the plasma membrane cholesterol content of different categories of lymphocytes in man is both essential and regulatory for their cytotoxic function.

Lateral pressures in biomembranes estimated from the dynamics of fluorescent probes.

A theoretical model is proposed which states that the time-independent fluorescence anisotropy of the rod-shaped molecule diphenylhexatriene incorporated into lipid bilayers is a direct result of forces constraining the diphenyl-hexatriene molecule. These forces are postulated as equating with the lateral pressure operating within the bilayer independently of the probe molecule. Insertion into the model of experimental observations (recorded in the literature) on anisotropy of diphenylhexatriene in lipid bilayers as a function of temperature yielded values of lateral pressure, which decreased with temperature, and sharply at the temperature defining the transition from gel phase to fluid phase. The values so predicted for the mid-point of the transition and for the entirely fluid phase, respectively, compared favourably with estimates of the lateral pressures in these physical states, that have been reported elsewhere and arrived at either from theories describing lipid chain behaviour or from lipid monolayer compression experiments. Previously documented effects on anisotropy induced by incorporation of cholesterol into fluid lipid bilayers have been interpreted as reflections of rises in intramembranal lateral pressure.

The modulation of membrane fluidity by hydrogenation processes. IV. Homogenous catalysis of liposomes using a water-soluble catalyst.

A water-soluble homogeneous catalyst has been applied to the hydrogenation of the double bonds within phospholipid liposomes in water. When the catalyst is added to an aqueous system, containing liposomes of unsaturated phospholipids, it partitions between the water and the liposomes. The structure of the liposomes as indicated by electron microscopy is not affected at low concentrations of the catalyst. The permeability properties of the liposomes are also unaffected. In the presence of hydrogen gas the unsaturated bonds of the lipids are readily hydrogenated. Potential applications of water-soluble homogeneous catalysts using liposome systems are described.

Lipid phase transitions in model biomembranes. The effect of ions on phosphatidylcholine bilayers.

Differential scanning calorimetry has been used to study the endothermic phase behaviour of some model biomembranes (i.e. phosphatidylcholine-water systes) in the presence of a wide range of alkaline, alkaline earth and heavy metal salts. Studies and comparisons were made of both cation and anion effects. Shifts occur in the temperatures of both the pre-transition and main transition endotherms. The observed shifts are smaller than those which have been reported for charged lipids, and no evidence has been found for the formation of specific complexes. Electron microscopic studies on freez-fractured dispersions of phosphatidylcholine-water-salt systems show that with some salts the typical rippled surface observed with L-alpha-dimyristoyl phosphatidylcholine, when in the gel state, is replaced by a smooth surface.

Investigation of lipid + water systems. Part 6. Proton magnetic resonance in ordered lyotropic mesophases.

P.m.r. measurements have been made on macroscopically ordered samples of the smectic mesophase in two 1-monoglyceride + water systems and the 1-amino-octane + water system. Three doublets have been observed, one from hydroxyl protons, and the remaining two from methylene protons in two sections of the alkyl chain undergoing different types of molecular motion. The protons of the glyceryl residue also contribute to one of the alkyl chain doublets. The magnitudes of the doublet splittings have been measured at a number of different temperatures and compositions in each system. Some correlations have been made between the splittings obtained and the type of head group and the length and conformation of the alkyl chain. The effects of temperature change have been interpreted in terms of the increasing occurrence of gauche configurations in segments of the alkyl chain. Correlations have been made between degrees of order obtained from p.m.r. and d.m.r. spectra.