Hyperthermia, adriamycin transport, and cytotoxicity in drug-sensitive and -resistant Chinese hamster ovary cells

Adriamycin cytotoxicity and membrane permeability to Adriamycin were studied at elevated temperatures in a drug-sensitive Chinese hamster ovary cell line and in a pleiotropic drug-resistant mutant to determine whether hyperthermia can overcome this form of acquired drug resistance. In drug-sensitive cells Adriamycin cytotoxicity, measured by colony survival studies, increased at temperatures as low as 38 degrees C, and at 43 degrees C, the combined effect of Adriamycin and hyperthermia exceeded the predicted additive effect by a factor of 10. There was a marked increase in the rate of [14C]Adriamycin uptake between 37 degrees C and 45 degrees C. Although the rate of Adriamycin efflux was also increased, intracellular drug levels at equilibrium were higher at elevated temperatures. The magnitude of the increase in intracellular drug levels at elevated temperatures was insufficient to account for the larger increase in cytotoxicity observed. We were unable to increase membrane permeability to Adriamycin or to increase Adriamycin cytotoxicity in the drug-resistant Chinese hamster ovary cell line by the use of hyperthermia; however, the drug-resistant cells were not cross-resistant to hyperthermia. Therefore, heat may be effective against residual tumor cells which are resistant to chemotherapy.     

Temperature dependence of adriamycin, cis-diamminedichloroplatinum, bleomycin, and 1,3-bis(2-chloroethyl)-1-nitrosourea cytotoxicity in vitro

We have examined the effect of mildly hypothermic temperatures (22-32 degrees) on the cytotoxicity of Adriamycin, cis-diamminedichloroplatinum, bleomycin, and 1,3-bis(2-chloroethyl)-1-nitrosourea in Chinese hamster ovary cells in vitro. Over a dose range of Adriamycin, cell killing at 30 degrees was reduced by 1 to 3 orders of magnitude as compared to that at 37 degrees. cis-Diamminedichloroplatinum was also less cytotoxic at 30 degrees (0.4 to 1.2 orders of magnitude) than at 37 degrees. For bleomycin and 1,3-bis(2-chloroethyl)-1-nitrosourea, the reduction in cytotoxicity at 30 degrees in comparison to 37 degrees was less marked. All drugs were more toxic at 42.4-43 degrees than at 37 degrees. Precooling of cells for 2 hr at 30 degrees did not alter the cell killing caused by these drugs at elevated temperatures. These results suggest that a more selective anticancer effect might result if some chemotherapeutic drugs were administered during whole-body hypothermia and regional-local hyperthermia of tumor masses.     

Effect of heat on the cytotoxicity and interaction with DNA of a series of platinum complexes

The effect of elevated temperature on the cytotoxicity and interaction with DNA of a series of platinum(II) complexes was examined. CDDP showed greater enhancement in cell killing with heat than the other platinum(II) complexes. There were approximately 2 decades enhancement in cell killing by 10 microM CDDP at 42 degrees C compared to 37 degrees C. The other potential cross-linking agents also showed increasing cytotoxicity with increasing temperature. K2PtCl4 (500 microM) killed about 15 times more cells at 43 degrees C than at 37 degrees C and KPt(NH3)Cl3 (500 microM) killed about 18 times more cells at 43 degrees C than at 37 degrees C. The cytotoxicity of the triammine and tetraammine complexes was less influenced by temperature. There was no significant difference in the cytotoxicity of [Pt(NH3)3Cl]Cl at any of the temperatures examined. The cytotoxicity of [Pt(NH3)4]Cl2 (500 microM) was increased about 7-fold at 43 degrees C compared to 37 degrees C, but the total cell killing by this complex at 43 degrees C was less than 1 log. Carboplatin (250 microM) was about 5 times more toxic at 42 degrees C and killed about 2.5 decades more cells at 43 degrees C than at 37 degrees C. Although there was little enhancement in the cytotoxicity of trans-Pt(NH3)2Cl2 at 42 degrees C compared to 37 degrees C trans-Pt(NH3)2Cl2 (500 microM) was about 7 times more cytotoxic than at 37 degrees C. The interaction of the various drug/temperature treatments with supercoiled pBR322 plasmid DNA was examined to assess the effect of heat on the reaction of these agents with DNA. At 42 degrees C, CDDP was able to gradually alter the gel electrophoretic mobility of the plasmid DNA to near that of the linear form. This change also occurred at 37 degrees C but at a much slower rate. Carboplatin effected similar changes in the superhelical pBR322 DNA, and the effect of temperature appeared to increase the rate of the reaction. Trans-Pt(NH3)2Cl2 also interacted with the supercoiled DNA, but at a slower rate than CDDP even under hyperthermic conditions. These results indicate that neutral platinum complexes capable of cross-linking DNA interact positively with temperature elevation to increase cytotoxicity, and, that of the platinum complexes that meet these criteria, the effect of hyperthermia is greatest with CDDP.     

低能超声对人肺癌细胞的体外杀伤作用

目的 探讨低能超声对人肺癌细胞的影响。方法 用低能量连续波超声处理小细胞肺癌细胞系ＳＭ、腺癌细胞系Ａ２及人肺纤维母细胞ＦＢ,用胎盘蓝染料排斥试验及克隆存活试验判定效果,结果 低能量（０．８Ｗ／ｃｍ＾２）超声３分钟分各种人肺癌细胞及人肺纤维母细胞有杀伤作用（Ｐ〈０．０５）,１０分钟可杀死全部细胞（Ｐ〈０．００１）,细胞浓度越高,杀伤作用越低,超声１０分钟后含细胞悬液的试管内温度未见显著上升,声强０．８Ｗ／ｃｍ＾２的超声使阿霉对肺癌细胞ＳＭ的杀伤作用增加１００倍（Ｐ〈０．００１）,结论 低能连续波超声可杀伤肺癌细胞,并能显著增加阿霉素对肺癌细胞的细胞毒性。     

Interaction between the kinetics of thermotolerance and effect of cis-diamminedichloroplatinum(II) or bleomycin given at 37 or 43 degrees C.

The interaction between the cytotoxic effect of bleomycin (BLM) or cis-diamminedichloroplatinum(II) (cis-DDP) and the kinetics of thermotolerance was studied in cultured Chinese hamster ovary (CHO) cells. Pre-heated cells were treated with cis-DDP or BLM at 37 or 43 degrees C for various times after heating. Pre-heating enhanced cis-DDP cytotoxicity given immediately after heating, but this enhancement decreased within 24 h to an additive level. Cell survival following the initial heating and the second treatment of 'cis-DDP at 43 degrees C was minimal when cis-DDP at 43 degrees C was given immediately after the initial heating, but became higher with increasing treatment interval and reached 'less than additive' level when the treatment interval was extended to more than 24 h. This alteration in cell survival appeared to follow the kinetics of thermotolerance. The interaction between BLM treatment and the kinetics of thermotolerance was similar to that of cis-DDP. However, pre-heating enhanced BLM cytotoxicity much less extensively than cis-DDP cytotoxicity. These results indicate that: (a) pre-heating of cells enhanced drug-toxicity when the drug was given shortly after heating, but the magnitude of this enhancement depended on the drug; (b) pre-heating did not influence the cytotoxicity of drugs given at 37 degrees C; and (c) pre-heating decreased the magnitude of thermal sensitization of drug cytotoxicity. The magnitude of the decrease in thermal sensitization appeared to be parallel to the kinetics of thermotolerance. In this study it was also demonstrated that pre-treatment of CHO cells by cis-DDP or BLM did not alter sensitivity to subsequent drug treatment, hyperthermia or thermochemotherapy.     

Effect of topoisomerase II inhibitors on hyperthermic cytotoxicity

Exposure of HeLa or Chinese hamster ovary cells to drugs (novobiocin, nalidixic acid, or oxolinic acid) which inhibit the nuclear enzyme topoisomerase II resulted in a sensitization of both cell lines to hyperthermic heating at 41 and 45 degrees C. Exposure to 0.5 mg/ml novobiocin decreased the reciprocal slope (T0) of the survival curve of HeLa cells heated at 41 and 45 degrees C by a factor of 7.5 and 2.4, respectively. Exposure to 0.5 mg/ml novobiocin decreased the T0 of the survival curve of Chinese hamster ovary cells heated at 41 and 45 degrees C by a factor of 9.8 and 1.8, respectively. Exposure of HeLa cells to 0.5 mg/ml novobiocin delayed thermotolerance development for 1.5 h and depressed by a factor of 27 the survival of cells heated at 45 degrees C once thermotolerance had developed. Coincident with the sensitization to heat-induced cytotoxicity, an enhancement of a heat-induced increase in the total protein mass co-isolating with the nuclei or nuclear matrices from heated cells was observed. A log-linear correlation was found between the reduction in cell survival and the relative nuclear matrix protein mass increase in cells heated at 41 or 45 degrees C in the presence or absence of these drugs. The results are consistent with the notion that exposure to these drugs disrupts the cell's capacity to regulate nuclear structure and composition, and thus enhances heat-induced cytotoxicity.     

Temperature dependence studies of adriamycin uptake and cytotoxicity

In order to learn whether a direct relationship exists between cellular uptake and cytotoxicity of Adriamycin, we have compared the temperature dependencies of these two processes in L1210 cells. We find that the equilibrium concentration of drug taken inside the cells varies smoothly with temperature between 37 degrees C and 0 degree C. Even at 0 degree C, however, there is still measurable uptake of the drug into cells. The cytotoxicity index (cloning in soft agar), on the other hand, does not parallel the uptake temperature dependence. Cytotoxicity rapidly diminishes as the temperature of drug exposure is lowered; at all temperatures below about 20 degrees C, Adriamycin is not active. In contrast, other cytotoxic anticancer drugs like mitomycin C, bleomycin, and ARK 73-21 (a platinum analogue) retain cytotoxic potency at low temperatures. The inability of Adriamycin to kill cells at low temperature persists even at very high drug concentrations where substantial quantities of drug enter the cells. The low temperature impotence is not a result of inoperative enzymes which could metabolize Adriamycin to an alkylating species or electron donor to oxygen, since NADH and NADPH dependent reductase activities show linear Arrhenius behavior with no indication of low temperature inactivity. Using purified L1210 plasma membranes with bound Adriamycin as a fluorescence polarization probe, we find evidence of a phase change in the cell surface occurring at the same temperature as the loss of biological activity (approximately equal to 20 degrees C). We conclude that Adriamycin induced cytotoxicity is not dictated solely by uptake, in apparent contradiction with mechanisms requiring an intracellular target. Moreover, the loss of cytotoxicity below 20 degrees C appears to be linked to a structural change in the cell surface membrane, supporting a role other than transport for this membrane in transducing Adriamycin action.     

Sensitization of rat 9L gliosarcoma cells to low dose rate irradiation by long duration 41 degrees C hyperthermia

Modification of survival by long duration, 41 degrees C hyperthermia in combination with low dose rate radiation (0.5 Gy/h) was determined in rat 9L gliosarcoma cells. Cells were exposed to radiation in a manner that simulated continuous irradiation at a dose rate relevant to clinical brachytherapy. High dose rate X-irradiation was fractionated in 1.0-Gy fractions at 2-h intervals (FLDRI). Previous studies had demonstrated that 9L cells exposed to FLDRI with these parameters have survival characteristics that are equivalent to continuous low dose rate irradiation. Cells exposed to 41 degrees C throughout FLDRI were sensitized significantly (thermal enhancement ratio of 2.07) compared with cells irradiated at 37 degrees C. Incubation for 24 h at 41 degrees C before and/or after FLDRI at either 37 degrees C or 41 degrees C did not increase the slope of the radiation survival curves but did reduce the shoulder. Similarly, heating at 43 degrees C for 30 or 60 min before and/or after irradiation at 0.5 Gy/h also did not enhance cell sensitivity. Survival of cells after irradiation at high dose rate (60 Gy/h) was independent of the temperature during irradiation. Preheat at 41 degrees C for 24 h did not sensitize cells to high dose rate irradiation by increasing the slope of the survival curve, although a loss of shoulder was observed. Sensitization of cells heated at 43 degrees C for 30 or 60 min before high dose rate irradiation was expressed as classical slope modification. Our results demonstrate that 41 degrees C heating during FLDRI greatly sensitizes cells to radiation-induced killing for exposure durations up to 36 h. Heating 9L cells at 41 degrees C or 43 degrees C adjacent to FLDRI at 0.5 Gy/h resulted in no additional enhancement of terminal sensitivity, although shoulder modification was observed. The sensitization by simultaneous heating described above occurred even though thermotolerance developed during extended incubation at 41 degrees C. These in vitro data demonstrate that simultaneous protracted heating at modest temperatures could greatly enhance the cytotoxic effects of low dose rate interstitial irradiation and could be of significance in clinical application.     

Effect of hypoxia and acidosis on the cytotoxicity of four platinum complexes at normal and hyperthermic temperatures

The cytotoxicities of cis-diamminedichloroplatinum(II) (CDDP) and of three recently developed dichloro complexes of bivalent platinum with radiosensitizing ligands [(1,2-diamino-4-nitrobenzene)dichloroplatinum(II) (Plato), trans-bis(2-amino-5-nitrothiazole)dichloroplatinum(II) (Plant), and trans-bis(2-nitroimidazole)dichloroplatinum(II) (NIPt)] were evaluated at 37 degrees C, 42 degrees C, and 43 degrees C at normal pH, at pH 6.45, and under normally oxygenated and hypoxic conditions in EMT6 cells in vitro. For CDDP, marked hyperthermic sensitization to the drug was evident in normally oxygenated cells, but hypoxic cells showed essentially no sensitization to the cytotoxicity of CDDP at elevated temperature at normal pH. Low pH further increased the cytotoxicity of CDDP toward normally oxygenated but not hypoxic cells at 37 degrees C and 42 degrees C. At 43 degrees C, however, low pH increased the cytotoxicity of CDDP toward both normally oxygenated and hypoxic cells, restoring nearly the full sensitizing effect of hyperthermia on CDDP cytotoxicity in the hypoxic cells. Plato was much more cytotoxic toward hypoxic than normally oxygenated cells under all culture conditions. At normal pH, hyperthermia increased the cytotoxicity of Plato in both hypoxic and normally oxygenated cells. At low pH, however, the cytotoxicity of Plato was inhibited at all temperatures and in both normally oxygenated and hypoxic cells. Plant was also more toxic to both normally oxygenated and hypoxic cells at elevated temperatures at normal pH. In contrast to Plato, however, Plant became much more cytotoxic toward hypoxic cells and showed increased cytotoxicity in normally oxygenated cells at low pH. Hyperthermia, however, did not further increase the rate of cell killing by Plant at low pH. NIPt, at the concentrations tested, was essentially nontoxic to cells at normal pH at 37 degrees C. Hyperthermia significantly increased the killing of hypoxic cells by NIPt under both normal and low pH conditions, but little cytotoxicity was noted for NIPt in normally oxygenated cells under any culture conditions. These results demonstrate that pH and the level of oxygenation of cells significantly affect the cytotoxicity of drugs at both normal and elevated temperatures. This sort of investigation may help delineate optimum drugs for use against environmentally determined subpopulations of cells within tumors.     

Selective enhancement of the cytotoxicity of the bleomycin derivative, peplomycin, by local anesthetics alone and combined with hyperthermia

The effect of local anesthetics alone and combined with hyperthermia on the cytotoxic effect of the bleomycin derivative, peplomycin, was studied in FM3A and HeLa cells. Noncytotoxic doses of the local anesthetics procaine, lidocaine, butacaine, tetracaine, and dibucaine enhanced peplomycin cytotoxicity. This enhancement correlated with the reported anesthetic potency of these agents. Combination of lidocaine (3 to 6 mM) and moderate hyperthermia (40 and 41 degrees) greatly enhanced peplomycin cytotoxicity, although these doses of lidocaine alone were ineffective at 37 degrees, and the temperatures alone enhanced the cytotoxicity only slightly. Cell sensitization to peplomycin cytotoxicity induced by lidocaine combined with 41 degrees hyperthermia produced a decrease in cell survival that depended on the dose of lidocaine and the dose and duration of peplomycin treatment. Lidocaine at 37 or 41 degrees did not enhance the cytotoxicity of Adriamycin, mitomycin C, and cis-diamminedichloroplatinum(II), suggesting a unique interaction with peplomycin. The enhancing effect of lidocaine on peplomycin-induced cell killing was found to increase as pH increased within the range of 7.0 to 8.0.     

Interaction of platinum complexes of thiazin and xanthene dyes with hyperthermia

Summary In an attempt to develop platinum-containing drugs for use with hyperthermia that would be relatively nontoxic at 37° C but would become very cytotoxic at 42° or 43° C, several nuclear dyes were complexed to the tetrachloroplatinum(II) dianion (PtCl₄) at a ratio of 2:1. The cytotoxicity of PtCl₄ complexes of three thiazin dyes (thionin, azure B, and methylene blue), the xanthene dye pyronin Y, and the thiazole dye thioflavin was examined in exponentially growing euoxic and hypoxic EMT6 cells in vitro at 37°, 42°, and 43° C and at pH 7.40 and 6.45. Of the thiazin dye complexes, the cytotoxicity of Pt(methylene blue)₂ was most enhanced at hyperthermic temperatures. Both Pt(pyronin Y)₂ and Pt(thioflavin)₂ also became markedly more cytotoxic at 42° and 43° C at pH 6.45 vs pH 7.40. In vivo tumor excision assays in the FSaIIC fibrosarcoma showed that with each of the thiazin dye-platinum complexes, hyperthermia enhanced cell kill [most effectively on Pt(methylene blue)₂] but was not dose-modifying. For both Pt(pyronin Y)₂ and Pt(thioflavin)₂, however, administration of 43° C, 30-min hyperthermia to the tumor immediately after i.p. drug injection was dose-modifying. Tumor growth delay studies in the FSaIIC tumor system demonstrated that, as with the in vitro studies, Pt(pyronin Y)₂ and Pt(methylene blue)₂ were most enhanced by hyperthermia [tumor growth delay increased by 4.8- and 3.0-fold, respectively, vs only 1.3-fold for cisplatin (CDDP)]. Examination of intracellular platinum levels after exposure of EMT6 cells to 25 μM of drug for 1 h at 37° and 42° C and at pH 7.40 and 6.45 showed that each platinum-dye complex achieved platinum levels that were 100–600 times higher at 37° C and pH 7.40 than those obtained using CDDP. The platinum levels for each drug dropped markedly when exposure took place at pH 6.45. Exposure at 42°C only moderately increased platinum levels in cells exposed to these drugs. Thus, for several of these drugs the level of cytotoxicity observed was in great part independent of the intracellular platinum levels achieved. Pt(pyronin Y)₂ is an effective drug for use with hyperthermia, and further studies using this combination with and without radiation are under way. This work was supported by NCI grants ROI-CA47379 and ROI-CA36508     

Hyperthermic enhancement of rhodamine 123 cytotoxicity in B16 mouse melanoma cells in vitro

The effect of elevated temperature on cytotoxicity of rhodamine 123 (R123) was tested in vitro on B16 mouse melanoma cells. Simultaneous 1-h exposure to R123 and hyperthermia (43 degrees C for 1 h) resulted in marked enhancement of R123 cytotoxicity. Thermal enhancement of R123 cytotoxicity occurred at temperatures as low as 38 degrees C. Heat treatment (43 degrees C for 1 h) given immediately before or after R123 exposure (37 degrees C for 1 h) yielded no significant increase in cytotoxicity over that expected for strict additivity. The effects of heat on two mechanisms reported to be associated with R123 cytotoxicity were evaluated: (a) target inactivation by R123; and (b) R123 intracellular accumulation. Hyperthermia caused an increased rate of target inactivation by R123 and also caused an increased net intracellular accumulation of R123. This indicates that at least two mechanisms are responsible for the synergistic cytotoxicity of R123 and hyperthermia.     

The effect of exposure to elevated temperatures on membrane permeability to adriamycin in Chinese hamster ovary cells in vitro

The effect of environmental temperature on plasma membrane permeability to Adriamycin was studied in Chinese hamster ovary (CHO) cells in vitro using flow cytometry. Initial rates of uptake increased steadily between 23 degrees C and 47 degrees C and there was a greater than 2-fold increase in permeability between 37 degrees C and 47 degrees C. The increase in permeability with increasing temperature was greater than expected based on the model of passive drug influx. Adriamycin uptake was also measured at 37 degrees C following previous exposure of the cells to elevated temperatures. Twenty-minute preexposures to temperatures above 41 degrees C caused a significant decrease in membrane permeability, which fell to approximately 60% of control levels after exposure to 45 degrees C. Longer periods of pre-exposure to temperatures as low as 40 degrees C were also shown to decrease membrane permeability to Adriamycin subsequently measured at 37 degrees C. The state of decreased permeability to Adriamycin induced by hyperthermia persisted for at least 2 h. The thermally induced decrease in membrane permeability to Adriamycin is of potential importance to the design of optimal schedules for thermochemotherapy.     

Temperature effect on mitoxantrone cytotoxicity in Chinese hamster cells in vitro

The effect of heat on 1,4-dihydroxy-5,8-bis[2-[(2-hydroxyethyl)amino]ethylamino]-9, 10-anthracenedione dihydrochloride (DHAD; mitoxantrone, NSC 301739) cytotoxicity was studied in V79 Chinese hamster cells. An overnight exposure to the drug at 40 degrees C enhanced drug damage in chromosome aberrations, culture growth, and cellular reproductive integrity. Preincubation of cells overnight in medium containing no drug at this temperature also showed some enhancement in subsequent DHAD lethality (at 37 degrees C as well as 43 degrees C). Short exposures (1 h) to DHAD at 43 degrees C was more damaging than were exposures at 37 degrees C. This was also true for cells in the plateau phase of culture growth. As compared with exponentially growing cells, plateau-phase cells were more resistant to DHAD.     

Response of EMT6 multicellular tumour spheroids to hyperthermia and cytotoxic drugs

The response of multicellular tumour spheroids of the EMT6 cell line to combinations of hyperthermia and Bleomycin (BLM) or Adriamycin (ADM) has been investigated. Using this model system, we have demonstrated enhanced BLM cytotoxicity at 43 degrees C and also heat-induced drug tolerance to BLM at 43 degrees C. ADM cytotoxicity was not significantly increased after 43 degrees C x 1 h but after 6 h at 42 degrees C greatly enhanced cell-killing was evident. These results are discussed in relation to our previous data for EMT6 cells growing either as monolayer cultures in vitro or as solid tumours in mice.     

Enhancement of hyperthermia-induced apoptosis by non-thermal effects of ultrasound

To determine the effect of ultrasound on hyperthermia-induced apoptosis, we exposed U937 cells (in air-saturated suspension) to continuous 1 MHz ultrasound at intensities 0.5 or 1.0 W/cm(2), considered non-thermal and sub-threshold for inertial cavitation, while at 44.0 degrees C for 10 min. We found that 0.5 W/cm(2), in combination with hyperthermia, synergistically induced apoptosis. On the other hand, 1.0 W/cm(2) in combination with hyperthermia showed an augmented instant cell lysis but no significant change in the ratio of apoptosis. This result might be useful when apoptosis induction is desired over instant cell killing in cancer therapy.     

The interaction of thermal tolerance with drug cytotoxicity in vitro.

The effect of preheating EMT6 cells in vitro on their response to cytotoxic agents of either 43 degrees C or 37 degrees C has been investigated. Preheating for 3 h at 40 degrees C produced measurable protection (thermal tolerance) to subsequent treatment for 1 h at 43 degrees C. This preheat treatment was further found to reduce cell killing by BLM and BCNU (drug tolerance) present during 1 h at 43 degrees C. In contrast, no such heat-induced drug tolerance was seen with ADR. An additional effect with ADR was the apparent elimination of heat-induced thermal tolerance at toxic drug doses. However, preheating under these conditions had no effect on the subsequent cytotoxicity of any of these drugs at 37 degrees C. Also, preheating for 1 h at 43 degrees C was found to sensitize cells to BLM and BCNU toxicity at 37 degrees C but to protect against ADR toxicity. The results are discussed in relation to known mechanisms of cell killing by heat and of thermal tolerance.     

Effect of hypoxia and acidosis on the cytotoxicity of six metal(ligand)4(rhodamine-123)2 complexes at normal and hyperthermic temperatures.

Several analogues of PtCl4(Rh-123)2 in which the metal may be Pt or Pd and the coordinated ligand may be -Cl, -CN or -NO2 were prepared and tested in cell culture with EMT-6 cells at normal (37 degrees C) and hyperthermic (42 degrees C and 43 degrees C) temperatures and various environmental conditions (normally oxygenated vs. hypoxic and pH 7.40 vs. pH 6.45). Pd is a much more reactive metal than Pt, while -CN and -NO2 are more tightly bound ligands than is -Cl. The goal of these studies was to define the complex with the least cytotoxicity at 37 degrees C and the greatest enhancement in cytotoxicity under hyperthermic conditions. The Pt complexes Pt(CN)4(Rh-123)2 and Pt(NO2)4(Rh-123)2 were much less cytotoxic than PtCl4(Rh-123)2 under both normothermic and hyperthermic conditions. The Pd complexes were, in general, more cytotoxic than the corresponding Pt complexes. The level of metal (Pt or Pd) in the cells did not appear to be a major factor in the level of cytotoxicity obtained. Complexes which were not cytotoxic at 37 degrees C regardless of oxygenation level or pH did not become cytotoxic at hyperthermic temperatures. In conclusion, the optimal members of this series were the complexes with chloro ligands, indicating that aquation is probably a necessary step in the cytotoxic mechanism and cytotoxicity at 37 degrees C was necessary to obtain cytotoxicity at higher temperatures.     

Adaptation to low pH modifies thermal and thermo-chemical responses of mammalian cells

The thermal and thermochemical survival responses of mammalian cells growing routinely at pH 7.4 are affected by extracellular pH. The cells show an increase in sensitivity when heated at pH values below about pH 7.0. The possibility that this sensitivity at low pH can be modified by maintaining the cells at pH 6.8 ('EP-2' cells) or pH 6.5 ('EP-1' cells) was examined. When Chinese hamster cells (HA-1) were exposed to 42 degrees C at pH 6.5, 6.8 and 7.4, a dramatic increase in sensitivity was seen at pH 6.5. EP-2 cells similarly exposed showed considerably less difference at the three pH values. Variations in survival due to these pH changes were almost eliminated when the EP-1 cells were exposed to 42 degrees C. HA-1 cells tended to develop less thermotolerance rates and did so possibly at a reduced rate at pH 6.8 when compared to pH 7.4; the reduced pH had only a minor effect on the ability of EP-2 cells to develop thermotolerance. The effects of pH adaptation on the pH dependence of drug cytotoxicity at 37 degrees C or at 43 degrees C were also compared. Adaptation also modified drug cytotoxicity except that cell killing by BCNU (as a function of pH) was similar for both HA-1 and EP-2 cells at both temperatures. EP-2 cells were more sensitive to MMS treatment at 43 degrees C and more resistant at 37 degrees C than were HA-1 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Laser flow cytometric studies on the intracellular accumulation of anthracyclines when combined with heat

The effects of heat on intracellular accumulation of anthracyclines were investigated by laser flow cytometry analysis. Sarcoma-180 cells were exposed to Adriamycin (ADM), epirubicin (EPIR), daunomycin (DM), THP-Adriamycin (THP), ME-2303 (ME) and KRN-8602 (KRN) at 37°C and at higher temperatures. There was a dose-dependent increase in the fluorescence intensity of all drugs at 37°C, but heat varied the fluorescence intensity of each drug. At 43°C the cellular fluorescence of ADM and EPIR increased by approximately 200%, but for DM the increase was 110–130%. The cellular fluorescence of THP and ME was little affected by heat, and heat reduced that of KRN to 80–90%. Each drug showed was unique in the relationship between drug exposure time and the fluorescence intensity at 37°C and 43°C. Cytotoxicity determined by the MTT assay was enhanced with heat in the cases of ADM and EPIR, but not with DM, THP, ME, or KRN. Thus, ADM and EPIR are expected to show enhanced antitumor activities when given in combination with hyperthermia.