The retinoblastoma gene product in acute myeloid leukemia: a possible involvement in promyelocytic leukemia.

The retinoblastoma susceptibility gene in leukemia and lymphoma has been investigated using different approaches involving either gene or protein analysis. In this study, a novel method, which evaluates the functional status of the retinoblastoma gene product by a binding assay to an in vitro-translated viral oncoprotein, has been applied to leukemic cells from acute myeloid leukemia patients. One hundred twenty-two cases were considered, and 42 of them were also analyzed by Western blot. Results obtained with the two methods were comparable, with the exception of few cases, where the retinoblastoma protein appeared detectable but unable to bind to the viral oncoprotein. The retinoblastoma protein has been found defective mostly in the M3 promyelocytic subtype.

Energy metabolism of human LoVo colon carcinoma cells: correlation to drug resistance and influence of lonidamine.

The relationship between modification of energy metabolism and extent of drug resistance was investigated in two sublines (LoVoDX and LoVoDX10) from human LoVo colon carcinoma cells that exhibit different degrees of resistance to doxorubicin. Results indicated that the extent of alteration in energy metabolism strictly correlated with degree of resistance. In LoVoDX cells, only 14CO2 production was enhanced, whereas in the more resistant LoVoDX10 cells, both 14CO2 and aerobic lactate production were stimulated. The basal and glucose-supported efflux rate and the amount of drug extruded by LoVoDX10 cells were significantly higher than in the resistant LoVoDX cells. Because the expression of surface P-170 glycoprotein was similar in both cell lines, this phenomenon was attributed to increased efflux pump activity resulting from greater ATP availability. Inhibition of 14CO2 production, aerobic glycolysis, and clonogenic activity by lonidamine (LND) increased with enhancement of the energy metabolism. Moreover, LND, by affecting energy-yielding processes, reduced intracellular ATP content, lowered the energy supply to the ATP-driven efflux pump, and inhibited, almost completely, doxorubicin extrusion by resistant LoVo cells. These findings strongly suggest that LND, currently used in tumor therapy, reduces drug resistance by restoring the capacity to accumulate and retain drug of cells with the MDR phenotype that overexpress P-170.

Effects of lonidamine on the membrane electrical properties of Ehrlich ascites tumor cells.

The effects of lonidamine on membrane electrical properties of Ehrlich ascites tumor cells are investigated. Using a dielectric relaxation technique based on the Maxwell-Wagner effect and elaborated by a 'single-shell' fitting procedure, the data indicate that both membrane conductivity and membrane permittivity increase after treatment of these cells with lonidamine while the conductivity of the cytosol remains unchanged. Changes in membrane proteins and/or lipids are suggested which lead to altered membrane structure and/or function.

Study on the reference dose level in radiotherapy treatment planning.

PURPOSE: The reference dose level of the dose distribution in the tumor volume is studied. METHODS AND MATERIALS: The study is performed using a formula based on the Linear Quadratic (LQ) model. The calculated reference dose level to which the prescribed dose must be referred, for the eradication of a homogeneous tumor, is investigated by varying the dose distribution, that is, the dose volume histogram shape, its range, the prescribed total dose, the fraction size and the linear quadratic model parameters, alpha and beta. RESULTS: For all the simulated dose volume histograms the calculated reference dose level is lower than the mean dose level, depending on the range of dose variation and the considered tumor sensitivity. When the dose nonuniformity is not too great the reference dose level is very near to the mean dose level; when the inhomogeneity of dose distribution is high the reference level is clearly lower than the mean level but not necessarily equal to the minimum level in the tumor. For the dose volume histograms derived from the actual dose distributions obtained from a two tangential beams technique, a four beams technique and a moving beam technique, the reference levels are calculated and compared with the ICRU 29 reference point dose level. In two cases the reference levels are lower than the level at the ICRU 29 reference point. In the case of the four beams technique, the two levels are equal. CONCLUSION: These theoretical results show the possibility of administering the prescribed dose to a dose level higher than the minimum in the tumor, with the same value of Tumor Control Probability (TCP) as the one corresponding to a uniform tumor irradiation. The application of the proposed study can offer a general support to the choice of the reference dose level, based on the actual dose distribution in the tumor volume.

Rhein enhances the effect of adriamycin on mitochondrial respiration by increasing antibiotic-membrane interaction.

The effect of the combination of Adriamycin (ADM) with rhein (RH), an anti-inflammatory drug, on the electron flow through site III and IV of the respiratory chain of rat liver mitochondria was investigated. RH, even at high concentrations, does not inhibit either duroquinol (DHQ) oxidation or cytochrome oxidase activity both of which are decreased by ADM in a dose-dependent manner. The analysis of interaction, performed with the isobolar method, shows a strong synergistic effect that cannot be ascribed to increased permeability of the mitochondrial membranes brought about by RH. The mechanism by which RH potentiates the effect of ADM on DHQ oxidation and cytochrome oxidase activity is most likely to be changes induced in the physical status of the inner mitochondrial membrane such as to permit low ADM concentrations to bind and segregate enough cardiolipin to inhibit electron transport through complex III and IV.

Glycolysis and growth rate in normal and in hexokinase-transfected NIH-3T3 cells.

The glycolytic enzyme hexokinase plays a key role in regulating cell energy metabolism. Its activity has been associated with cell growth rate and, notably, with neoplastic transformation. NIH-3T3 cells were transfected with a tumor hexokinase cDNA. The transfected cells showed increased hexokinase amount and activity, mainly located in the particulate cellular fraction, increased glycolytic rate evaluated as lactate production, and, finally, enhanced growth rate. These data may suggest that high hexokinase activity might be not merely the consequence of peculiar metabolic demands by actively replicating normal or cancer cells, but also a modification able per se to drive, at least partially, a more intense mitotic activity.

Thermal behavior of a human glioma cell line and its response to combinations of hyperthermia and lonidamine.

The effect of hyperthermia and lonidamine, alone and in combination, on the clonogenic activity of a human glioma cell line was investigated. The time-temperature relationship of asynchronous, exponentially growing cells was defined in the range of 40-45 degrees C. All survival curves were exponential and an Arrhenius plot for heat killing was linear over the temperature range tested, with an activation energy of 192 Kcal/mol. The survival curve of lonidamine-treated cells was also exponential after an initial shoulder. The analysis of the interaction between lonidamine and hyperthermia, performed by the isobolar method, demonstrated an additivity of response so that the effectiveness of the combined treatment was the result of two independent effects. Lonidamine inhibits the neoplastic growth mainly through an ATP depletion, but the thermal killing was not mediated by the drug-induced changes in the energy status of the cell. The effectiveness of the combined treatment was strongly influenced by the schedule of administration. In fact, the sequence lonidamine-->hyperthermia made the cells less sensitive to heat so that the pre-established end-point, i.e. 30% survival, was never achieved whichever combination was used. This "drug-induced heat resistance" was not associated with the induction of heat shock proteins, but rather with modification of cell cycle. On the contrary, showing a purely additive effect, the sequence hyperthermia-->lonidamine allowed achievement of the pre-established cell killing (70%), with exposure times (1-2 hr) and with a temperature (42 degrees C) generally accepted as clinically achievable. Therefore, also considering its low systemic toxicity, lonidamine may be useful in reducing the side effects of hyperthermia.

Thermal behavior of human melanoma cell line in vitro and enhancement of hyperthermic response by bupivacaine.

The effect of hyperthermia and bupivacaine, alone and in combination, on the clonogenic activity of a human melanoma cell line was investigated. The time-temperature relationship of exponentially growing cells was defined in the range of 41-45 degrees C. All the survival curves were exponential and the Arrhenius plot was linear over the temperature range tested. The survival curve of bupivacaine-treated cells was also exponential after an initial shoulder. Bupivacaine affected cell survival mainly through an ATP depletion because of deep alterations of mitochondria, essentially due to changes in the physical state of membrane lipids. The analysis of the interaction between hyperthermia and bupivacaine, performed with an isobolar method, demonstrated a synergism of response at all combinations tested, but only with simultaneous exposure. Such a response did not depend on an impairment of the energy-yielding processes, but may be ascribed to combined effects of both agents on cell structure and function. The hyperthermic enhancement achieved by low bupivacaine concentrations allowed to achieve a preestablished cell killing with a reduced exposure time (e.g., 50 min) and with a temperature (42 degrees C) generally accepted as clinically achievable. Therefore, a combined modality in which local treatment with bupivacaine was coupled to local heating could result in high local damage with reduced systemic complications.

Interaction of cytotoxic agents: a rule-based system for computer-assisted cell survival analysis.

The actual effectiveness of environmental noxious agents or anticancer drugs can be fully determined only by knowing if the effects (in the present case, the cytotoxic effects) induced by a given agent are enhanced by exposure to another (or other) agent(s). Given a certain combination of agents, it is possible to distinguish three types of interaction: (a) zero interaction or additivity; (b) positive interaction or synergism; and (c) negative interaction or antagonism. In this work, the methodological problems involved in evaluating the type and level of interaction between biologically active agents are discussed and an "intelligent" approach to the problem is proposed. In particular, a prototype of a computer-assisted rule based system, named CISA (Cytotoxic Interaction and Survival Analysis), designed in a KES environment (Knowledge Engineering System) and implemented on a personal computer, is described. By constructing isoboles based on experimental cell survival data and taking into account the relative confidence intervals, the system can indicate the appropriate combinations of dosages to be tested and finally determine the type and level of interaction. The system, which represents an attempt to administer "intelligently" the experimental data, is therefore able to identify the best strategy of analysis, to carry out the data processing and to offer suggestions to the investigator about the usefulness of the data and the planning of further experiments.

Growth inhibition by rhein and lonidamine of human glioma cells in vitro.

The effect of Rhein (RH) and Lonidamine (LND) on the clonogenic activity of cultured human glioma cells has been evaluated. Both these drugs decrease the survival fraction, but their effect is strictly related to the duration of exposure. A brief exposure, i.e. 4 hours, even at the highest drug concentrations does not induce any significant decrease in the survival which, on the contrary, is strongly affected by 24 and 48 hours of exposure. The reason for this behaviour lies in the mechanism of action of these drugs which do not interfere with replicative processes, but selectively affect the energy metabolism of the neoplastic cell. The validity of currently employed screening tests to evaluate the antitumoral activity of non anti-mitotic drugs is also discussed.

A simulation model for tumor growth controlled by therapeutic agents.

Results obtained in a controlled tumor growth depend on the experimental model used, the agent employed (drugs, ionizing radiations, heat), aw well as the administration scheduling and the host environment. Empirical search and treatment optimization are related to a discrete number of agent combinations and fractioning, and system parameters and dependencies are strictly connected to biological assumptions. A simplified model for the evaluation of the rate of change of tumor volume at the time of cytotoxic agent administration is proposed here, as well as a computer program to perform the simulation of tumor growth controlled by therapeutic agents.

A treatment planning system for stereotactic radiotherapy.

Stereotactic radiotherapy to treat neoplastic lesions or artero-venus malformations in the brain may be accomplished with a linear accelerator by performing several non-coplanar arcs of irradiation with a highly collimated beam focused on a fixed point. This paper introduces a system to perform treatment planning. It is based on a Personal Computer and allows the acquisition, reconstruction and visualization of the target volume, within the brain, from CT (Computerized Tomography) or MR (Magnetic Resonance) images, and then it permits calculation and visualization of a 3-D (three-dimensional) dose distribution due to small photon beams. The performances of the system and its use in a practical case are described.

A dielectric relaxation study on the effects of the antitumor drugs Lonidamine and Rhein on the membrane electrical properties of Ehrlich ascites tumor cells.

The effects of the anti-tumor drugs lonidamine and rhein on the plasma membrane electrical properties of Ehrlich ascites tumor cells were investigated and compared. Dielectric relaxation measurements in the radiowave frequency range (10(4) to 10(8) Hz) as well as at higher frequencies (10(4) to 10(9) Hz) were performed and the data elaborated using a "single-shell" fitting procedure. The results obtained in both frequency ranges indicate that membrane conductivity and membrane permittivity are altered by 200 microM Lonidamine while 150 microM rhein induces only very slight variations in these two plasma membrane parameters. The usefulness of the dielectric relaxation technique described here in evaluating antitumor drugs and improving clinical protocols is discussed.

A stochastic model of the acquisition of drug resistance during antineoplastic chemotherapy.

A stochastic model is proposed to simulate the phenomenon of acquisition of genetic resistance by a tumor cell population treated with antineoplastic cytotoxic agents. In the model, stochastic differential equations are numerically integrated to simulate the expected response after treatment with two different agents. The model takes into account the initial number of cells, the sequence of the agents, the time intervals between administrations, the birth and death rates of the untreated cells, the probabilities of mutations to resistance, and the induced cell-killing kinetics. Satisfactory demonstration runs of the model indicate that it could represent a useful tool in verifying the results of experimental and clinical chemotherapy courses and planning treatment strategies.

Estimate of normal tissue damage in treatment planning for stereotactic radiotherapy.

A personal computer (PC) system was developed to perform treatment planning for radiosurgery and stereotactic radiotherapy. These techniques of irradiation of the brain may be accomplished with a linear accelerator by performing several non-coplanar arcs of a highly collimated beam focused at a fixed point. The PC system allows the acquisition, reconstruction and the visualization of the target volume from CT or MR images, and then it permits to calculate a three-dimensional (3-D) dose distribution due to small photon beams and to visualize it. The software calculates not only total dose distribution, administered fractionated or in single fraction, but also the NTD2 (normalized total dose) predicted to have a biological effect equivalent to the single irradiation. The choice of the best technique is supported by the dose volume histograms (DVH) calculation and by an estimate of complication probability to the brain normal tissue (NTCP). The algorithm for NTCP calculation is based on two models: the linear quadratic and the logistic. A comparison of three different dose calculations for a typical cerebral target volume is presented to demonstrate the system performances.

Inhibition of membrane redox activity by rhein and adriamycin in human glioma cells.

The effect of the combination of adriamycin (ADM) with the anti-inflammatory drug rhein (RH) on the membrane redox activity in human glioma cells was investigated. RH, although less effective than ADM, inhibits ferricyanide reduction by human glioma cells in a dose-dependent manner as well as ferricyanide-induced proton release. The inhibition of the plasma membrane redox system might represent a further mechanism by which RH, other than ATP depletion, affects cell survival. The analysis of the interaction between ADM and RH, performed with the isobolar method, demonstrates a strong synergic response, probably due to an effect on different sites of action. The synergism of the ADM-RH association allows us to achieve a pre-established extent of inhibition with ADM concentrations much lower than with ADM alone. RH might, therefore, represent a very useful tool to improve the therapeutic index of ADM and to lower its general toxicity.

Thermosensitization, heat shock protein synthesis and development of thermotolerance in M-14 human tumor cells subjected to step-down heating.

M-14 human tumor cells have been subjected to two regimens of step-down heating (SDH) consisting of a conditioning treatment at 42 degrees C for 1 h or at 44.5 degrees C for 20 min, immediately followed by heating at 40 degrees C. Both conditioning treatments thermosensitize the cells towards the subsequent heating at 40 degrees C; the thermosensitization ratio is 6.4 for cells conditioned at 42 degrees C for 1 h and 32.3 for cells conditioned at 44.5 degrees C for 20 min. The overall protein synthetic activity is reduced to 32.7% or 18.4% of control values following 1 h at 42 degrees C and 20 min at 44.5 degrees C, respectively; this inhibition is followed by a full recovery of the synthetic activity during the subsequent exposure at 40 degrees C. SDH-treated cells synthetize four heat shock proteins, with approximate molecular weights of 28, 64, 70 and 90 kDa. The pattern of HSPs induction observed in SDH-treated cells is similar to that found in cells subjected to single hyperthermic exposures. Cells subjected to the SDH sequence 42 degrees C/1 h-->40 degrees C/4 h develop thermotolerance, as indicated by a reduced sensitivity to further hyperthermic challenges.

A simple method to calculate the influence of dose inhomogeneity and fractionation in normal tissue complication probability evaluation.

PURPOSE: Since volumetric dose distributions are available with 3-dimensional radiotherapy treatment planning they can be used in statistical evaluation of response to radiation. This report presents a method to calculate the influence of dose inhomogeneity and fractionation in normal tissue complication probability evaluation. METHODS: The mathematical expression for the calculation of normal tissue complication probability has been derived combining the Lyman model with the histogram reduction method of Kutcher et al. [14] and using the normalized total dose (NTD) instead of the total dose. RESULTS: The fitting of published tolerance data, in case of homogeneous or partial brain irradiation, has been considered. For the same total or partial volume homogeneous irradiation of the brain, curves of normal tissue complication probability have been calculated with fraction size of 1.5 Gy and of 3 Gy instead of 2 Gy, to show the influence of fraction size. The influence of dose distribution inhomogeneity and alpha/beta value has also been simulated: considering alpha/beta = 1.6 Gy or alpha/beta = 4.1 Gy for kidney clinical nephritis, the calculated curves of normal tissue complication probability are shown. CONCLUSION: Combining NTD calculations and histogram reduction techniques, normal tissue complication probability can be estimated taking into account the most relevant contributing factors, including the volume effect.

Ideal dose level in treatment planning optimization.

The biological response of the tumor is expressed in terms of tumor control probability (TCP) and its dependence on the inhomogeneous dose distribution throughout the tumor volume is studied. The ideal dose level to which the prescribed dose must be referred is derived, by employing a formula based on the linear quadratic model. To administer the prescribed dose to the ideal dose level renders the tumor control probability equal to that one corresponding to a uniform irradiation of the tumor. For the normal tissue irradiated a normal tissue complication probability index (NTCPI) is also defined and calculated. The comparison between NTCPIs of competing plans supports the optimization. In general the resulting ideal dose level is lower than the mean dose level, but not necessarily equal to the minimum in the tumor. This result shows the possibility of administering the prescribed dose to a dose level higher than the minimum, maintaining the tumor control probability at a good level and consequently lowering the complications to the normal tissue. The method offers a general support for the choice of the reference dose level and of the better technique. An example of application of the method is shown.