Awt Konings

Reduction of cellular cisplatin resistance by hyperthermia--a review.

Resistance to cisplatin (cDDP) is a major limitation to its clinical effectiveness. Review of literature data indicates that cDDP resistance is a multifactorial phenomenon. This provides an explanation why attempts to reverse or circumvent resistance using cDDP-analogues or combination therapy with modulators of specific resistance mechanisms have had limited success so far. It therefore provides a rationale to use hyperthermia, an agent with pleiotropic effects on cells, in trying to modulate cDDP resistance. In this review the effects of hyperthermia on cDDP cytotoxicity and resistance as well as underlying mechanisms are discussed. Hyperthermia is found to be a powerful modulator of cDDP cytotoxicity, both in sensitive and resistant cells. Relatively high heat doses (60 min 43 degrees C) seem to specifically interfere with cDDP resistance. The mechanism of interaction has not been fully elucidated so far, but seems to consist of multiple (simultaneous) effects on drug accumulation, adduct-formation and -repair. This may explain why hyperthermia seems to be so effective in increasing cDDP cytotoxicity, irrespective of the presence of resistance mechanisms. Therefore, the combination of hyperthermia and cDDP deserves further attention.

Radiation dose-fractionation and dose-rate relationships for long-term repopulating hemopoietic stem cells in a murine bone marrow transplant model

Fractionated and low-dose-rate total-body irradiation (TBI) were compared with single-dose high-dose-rate TBI for induction of long-term hemopoietic chimerism in a murine syngeneic bone marrow transplantation model. At 5 months after TBI and bone marrow transplantation, the degree of stable blood chimerism was determined from the proportion of stem cell-derived donor (B6-Gpi-1a) and host (B6-Gpi-1b) blood erythrocytes. This end point was used to construct radiation dose-response curves for long-term donor marrow engraftment corresponding to ablation of primitive bone marrow stem cells of the host. Increasing dose fractionation and decreasing dose rate had the effect of restoring host hemopoiesis and required higher TBI doses for equal donor engraftment. Most of the dose recovery occurred within the first 6 h between fractions, consistent with the kinetics of sublethal damage repair. The late chimerism data were fitted to the linear-quadratic model using indirect and direct analysis for a fixed threshold response. Both analyses gave relatively low alpha/beta ratios (below 2 Gy), within the range normally seen in late-responding tissues. The dose-rate data gave a repair half-time of 2 h as estimated by the incomplete-repair model. These estimates contrast with the much higher alpha/beta values and lower repair half-times derived from acute hemopoietic failure as indicated by LD50/30, with the implication that separate target cell populations with differing radiosensitivities are involved in these two bone marrow end points.

Contrasting dose-rate effects of gamma-irradiation on rat salivary gland function.

The aim of this study was to investigate the effects of 60Co irradiation delivered at high (HDR) and low (LDR) dose-rates on rat salivary gland function. Total-body irradiation (TBI; total doses 7.5, 10 and 12.5 Gy) was applied from a 60Co source at dose-rates of 1 cGy/min (LDR) and 40 cGy/min (HDR) followed by syngeneic bone marrow rescue. Four days before and 1-30 days after TBI, submandibular and parotid saliva samples were collected in male albino Wistar rats using Lashley cups. Lag phase and flow rate were recorded, and [Na+] and [K+] were measured. The severity of salivary gland dysfunction for each dose-rate was dependent on total TBI dose in all parameters. LDR irradiation significantly enhanced the increase of lag phase, while it tended to further decrease flow rate during days 0-3. At later times the reverse effect was seen with significant LDR sparing in most cases. The changes in [Na+] and [K+] showed similar trends; LDR had an enhancing effect for early damage, while beyond day 3 it consistently produced less damage. From this dose-rate study it is concluded that the early postirradiation changes in salivary gland function are probably predominantly caused by irradiation damage to membrane structures and are less the result of reproductive failure. The later changes in salivary gland function are probably mainly dependent on repopulation of surviving stem cells.

Mechanism of hyperthermic potentiation of cisplatin action in cisplatin-sensitive and -resistant tumour cells

In this study, the mechanism(s) by which heat increases cis-diamminedichloroplatinum (cisplatin, cDDP) sensitivity in cDDP-sensitive and -resistant cell lines of murine as well as human origin were investigated. Heating cells at 43 degrees C during cDDP exposure was found to increase drug accumulation significantly in the cDDP-resistant cell lines but had little effect on drug accumulation in the cDDP-sensitive cell lines. DNA adduct formation, however, was significantly increased in all cell lines studied. Furthermore, ongoing formation of platinum (Pt)-DNA adducts after the end of cDDP treatment was enhanced and/or adduct removal was decreased in heated cells, resulting in relatively more DNA damage remaining at 24 h after the end of cDDP exposure. Correlation plots with survival revealed weak correlations with cellular Pt accumulation (r2 = 0.59) and initial Pt-DNA adduct formation (r2 = 0.64). Strong correlations, however, were found with Pt-DNA adducts at 6 h (r2 = 0.97) and 24 h (r2 = 0.89) after the incubation with the drug. In conclusion, the mechanism by which heat sensitizes cells for cDDP action seems to be the sum of multiple factors, which comprise heat effects on accumulation, adduct formation and adduct processing. This mechanism did not seem to differ between cDDP-sensitive and -resistant cells, emphasizing the potential of hyperthermia to reduce cDDP resistance.

Radiation-Induced Cell Proliferation in the Parotid and Submandibular Glands of the Rat

Repopulation of tissues with cells at damaged sites is an important feature in the recovery of radiation-induced tissue injury. To obtain insight into the regenerative process in salivary gland tissue, proliferative activity was measured as a function of time in the different epithelial cell compartments of rat parotid and submandibular glands after local X irradiation with a single dose of 15 Gy. Bromodeoxyuridine-labeling indices were determined before and 10 h and 1, 3, 6, 10, 15, 20 and 30 days after irradiation. In both glands, X irradiation caused cell death and cell cycle delay manifested during the first day. Three days after irradiation, cell proliferation started in the intercalated duct. Six days after irradiation, proliferation was also observed in acinar and granular convoluted tubule cells. The striated ducts showed proliferative activity starting at day 6 (parotid) and day 10 (submandibular), respectively. The results of this study suggest that after 15 Gy of X rays repopulation takes place in all cell compartments. From the present study it cannot deduce if these cells are originating solely from progenitor cells residing in the intercalated duct or if cells of the other compartments are also stimulated. Proliferative activity was found to be higher in the intercalated duct compartment of the parotid gland than of the submandibular gland, which may be related to the suggested greater radiosensitivity and thus a greater demand for cell replenishment in the parotid gland.

Correlation between slowly repairable double-strand breaks and thermal radiosensitization in the human HeLa S3 cell line

The effect of heat on double-strand breaks (dsb) repair was compared with thermal radiosensitization using HeLa S3 cells. Cells were exposed to a combined treatment of X-irradiation followed by heat (44 degrees C, 0.5 h) separated by time intervals up to 8 h. DNA dsb were measured by PFGE and survival by the colony forming assay. In non-heated HeLa S3 cells repair of dsb was biphasic with the majority of breaks being repaired fast with a half-time of 14 min and only a minority were repaired slowly with a half-time of 130 min. Heat applied immediately after irradiation was found to cause an increase in both half-times but mainly to result in an increased fraction of slowly repairable dsb. The latter effect was shown to result from the formation of additional dsb. The number of additional dsb declined when irradiation and heat were separated by an interval at 37 degrees C with a half-time of 120 +/- 30 min. This half-time was similar to the half-time of 100 +/- 20 min found for the loss of thermal radiosensitization studied for the same protocol. Both processes were recently found also to correlate in CHO cells but occurred much faster in rodent cells than in the human HeLa S3 cells used in the current study. These results show that in human cells, unlike previously suggested on the basis of rodent cells, thermal radiosensitization is still a substantial contributor to the killing efficacy of a combined treatment even when irradiation and heat are separated by a time internal of 4 h.

ON THE ROLE OF HSP72 IN HEAT-INDUCED INTRANUCLEAR PROTEIN AGGREGATION

Heat treatment of cells results in an increased protein content of nuclei and nuclear matrices when isolated after the heat treatment. This increase of TX-100 insoluble protein is interpreted as being the result of protein denaturation and subsequent aggregation. After the heat treatment cells can (partly) recover from these aggregates. Recent data suggest that heat shock proteins (hsps) might be involved in the recovery (disaggregation) from these heat-induced insoluble protein complexes. In this report, the role of hsp72 in the process of aggregation and disaggregation was investigated using: non-tolerant rat-1 cells, thermotolerant rat-1 cells (rat-1 TT), and transfected rat-1 cells constitutively expressing the human inducible hsp72 gene (HR-24 cells). After heating the various cells, it was observed that the expression of the human hsp72 confers heat resistance (43-45 degrees C). Heat-induced intranuclear protein aggregation was less in HR and rat-1 TT cells as compared to nontolerant rat-1 cells. After heat treatments leading to the same initial intranuclear protein aggregation, rat-1 TT cells recovered more rapidly from these aggregates, while HR cells recovered at the same rate as nontolerant rat-1 cells. Our data suggest that increased levels of hsp72 can confer heat resistance at the level of initial (nuclear) heat damage. Elevated levels of hsp72 alone, however, do not enable cells to recover more rapidly from heat-induced intranuclear protein aggregates.

HEAT-INDUCED NUCLEAR-PROTEIN BINDING AND ITS RELATION TO THERMAL CYTOTOXICITY

When nuclei were isolated from exponentially growing HeLa S3 cells immediately after a treatment with hyperthermia and/or procaine-HCl, an increase in nuclear protein binding was observed. The extent of this increase, however, did not correlate with cell survival under all conditions of the various treatments. For example, an increase up to 40 per cent in nuclear protein binding as a result of procaine treatment did not lead to a decrease of survival, while a 40 percent increase of nuclear protein binding as a result of hyperthermia corresponded with over 90 per cent cell killing. In addition the extent of heat-induced enhancement of nuclear protein content was approximately equal for thermotolerant and heated control cells, or for cells heated in the presence of procaine. The rate of decay in nuclear protein binding upon post-heat incubations at 37 degrees C of the cells, however, was enhanced in tolerant cells and retarded in cells heated in the presence of procaine as compared to heated control cells. These results show that, in spite of suggestions in other reports, neither the initial rate of enhanced protein binding nor the extent of the protein bound to the nucleus seems a reliable measure for heat toxicity. The capacity of the cell to reverse this heat-induced protein binding must be considered.

Radiosensitivity of Normal and Polyunsaturated Fatty Acid Supplemented Fibroblasts after Depletion of Glutathione

Mouse fibroblast LM cells have been modified with respect to their phospholipid composition in all subcellular fractions, including the nuclear membrane. The content of polyunsaturated fatty acids (PUFA) was significantly increased but no difference in cell survival after X-irradiation could be observed between the normal and PUFA enriched cells. It is concluded that the radiosensitive PUFAs in the membranes are well protected against radiation damage. This protection of the PUFA cells could not be caused by vitamin E, because this membrane protector was not present in these fibroblasts. The content of glutathione (GSH) was about the same in the normal and the modified cells. Reduction of the cellular GSH content to less than 5 per cent of that for non-treated cells did not alter cellular survival after radiation of either normal or PUFA enriched cells under oxic or anoxic conditions. The radiosensitive lipids present in the membranes of the PUFA enriched cells proved to be vulnerable to radiation-induced lipid peroxidation when extracted from the cells and reconstituted into liposomes, indicating that the fatty acids per se are peroxidizable. It is concluded that the lipids in the membranes of mammalian cells are not the principal target in radiation-induced reproductive death, and that no generalization is permitted with respect to glutathione, as being the major hydrogen donating species in mammalian cells responsible for the repair of those target molecules responsible for cell survival after radiation.

The Role of Secretory Granules in Radiation-Induced Dysfunction of Rat Salivary Glands

To investigate the possible role of secretory granules in radiation-induced salivary gland dysfunction, rats were pretreated with isoproterenol (5 mg/kg intraperitoneally) to degranulate salivary gland acini. At maximal depletion, salivary glands were locally irradiated with a single dose of 15 Gy of X rays. Parotid and submandibular/sublingual saliva samples were collected before and 1-10 days after irradiation. The lag phase, flow rate, concentrations of potassium and sodium, and amylase secretion were determined. Sham-treated, isoproterenol-treated and irradiated animals provided reference data. In the parotid gland, but not in the submandibular gland, protection against radiation-induced changes in flow rate and composition of saliva occurred after pretreatment with isoproterenol. Combining morphological data from a previous study with data from the current study, it is suggested that improvement of parotid gland function is attributed predominantly to a proliferative stimulus on acinar cells by isoproterenol and not to its degranulation effect. After pretreatment with isoproterenol, an earlier expression of radiation-induced acinar cell damage leading to death was observed, followed by a faster tissue recovery. Thus the proliferative stimulus on acinar cells may accelerate the unmasking of latent lethal damage, resulting in the earlier replacement of dead cells by new, functionally intact cells.