BrdUrd-induced radiosensitization of two human tumour cell lines at iso-levels of incorporation.

Bromodeoxyuridine (BrdUrd)-induced radiosensitization of two different tumour cell lines was compared at equal levels of thymidine replacement. Human lung carcinoma cells (SW-1573) and human colorectal carcinoma cells (RKO) were grown for 48 h in the presence of respectively 1 microM BrdUrd and 4 microM of BrdUrd in order to obtain equal levels of BrdUrd into the DNA. In SW cells the level of thymidine replacement by BrdUrd was 6.7+/-0.5% and in RKO cells this was 7.1+/-0.8. Cell survival after irradiation with single doses up to 8 Gy, was determined with clonogenic assay. The magnitude of BrdUrd-induced radiosensitization was determined by analyzing radiation-dose survival curves with the linear-quadratic formula [S(D)/S(0)=exp-(alphaD+betaD2)]. In the SW cells BrdUrd radiosensitization led to a significant increase of the linear parameter, alpha, determining the initial slope of the survival curves, by a factor of about 2. In the RKO cells BrdUrd increased the value of alpha by a factor 1.4. This suggests that repair of potentially lethal damage (PLD) is inhibited. In both cell lines the quadratic term, beta, strongly influencing the high dose region of the survival curves, was not altered by sensitization by BrdUrd. The increase of alpha is of interest for clinical applications as BrdUrd sensitizes tumour cells after low doses of radiation.

Experimental autoimmune keratitis induced in rats by anti-cornea T-cell lines.

PURPOSE: Idiopathic inflammation of the cornea, keratitis, has been proposed to result from an autoimmune process, but thus far no convenient animal model of keratitis exists. An attempt was made to establish an animal model for keratitis, to investigate possible autoimmune mechanisms. METHODS: T-cell lines were established from lymph node cells removed from rats immunized with bovine corneal epithelium (BCE) extract. After restimulation in vitro with BCE or a specific corneal antigen, the cells were transferred by intraperitoneal injection into naive rats, rats subjected to total body irradiation, or rats in which only one eye was irradiated. RESULTS: Neither direct immunization with corneal antigens nor transfer of activated anti-corneal T-cells into naive rats gave any signs of keratitis. Irradiation alone did not induce corneal inflammation. Transfer of corneal-specific activated T cells into irradiated rats produced keratitis starting around day 4 and culminating around day 8. The disease was self-limiting and the severity dependent on the dose and site of radiation. Keratitis was characterized by corneal haze, conjunctival and episcleral hyperemia, episcleral hemorrhages, chemosis, corneal infiltrates, and vascularization. Immunohistochemistry showed T-cell and macrophage infiltration of epithelium and stroma in the affected corneas. CONCLUSIONS: Thus, keratitis may be produced by T cells reactive to corneal antigens, provided that the target tissue has been made susceptible by irradiation. The effectiveness of T-cell vaccination in preventing adoptive keratitis suggests that systemic as well as local tissue factors may regulate the disease process.

Correlation between cell reproductive death and chromosome aberrations assessed by FISH for low and high doses of radiation and sensitization by iodo-deoxyuridine in human SW-1573 cells.

PURPOSE: To study the relationship between cell reproductive death and exchange frequency in SW-1573 human lung tumour cells with and without incorporated iodo-deoxyuridine (IdUrd) following irradiation of plateau-phase cultures with y-rays. METHOD: Linear-quadratic (LQ) analysis was performed for the data on clonogenic survival and on the frequency of chromosomal exchanges studied with fluorescence in situ hybridization in chromosomes X and 2. RESULTS: Differences in the LQ parameters alpha and beta of both non-sensitized and sensitized chromosomes were found. In both chromosomes an increase in the number of chromosomal exchanges in IdUrd-radiosensitized cells compared with non-sensitized cells was observed. The alpha-enhancement factors of 1.7 and 1.9 for the X-chromosome and for chromosome 2, respectively, are similar. For the X-chromosome, the beta coefficient increased by a factor of 3.9 and for chromosome 2 by a factor of 1.4. After correction to a full genome equivalence, no significant difference in alpha was found between chromosomes X and 2 for both control and sensitized cells. In contrast, an almost 2.8 times higher beta was found for the sensitized X-chromosome compared to this value for chromosome 2. CONCLUSIONS: It can be concluded that the linear-quadratic analysis of dose-response relationships offers insights into the correlation between cell survival and induction of exchanges in non-sensitized and radiosensitized cells.

Increased chromosome exchange frequencies in iodo-deoxyuridine-sensitized human SW-1573 cells after gamma-irradiation.

The induction of chromosome exchanges was investigated in SW-1573 human lung tumour cells radiosensitized with iododeoxyuridine (IdUrd) and irradiated with gamma-rays. Following treatment chromosome 2 and X were analyzed using fluorescence in situ hybridization (FISH) with chromosome-specific DNA libraries. The yield of chromosome exchanges involving chromosome 2 was higher than those involving chromosome-X. On the basis of the DNA content the relative involvement of the X-chromosome in exchange frequencies after 2 Gy was much higher than of chromosome 2. After 4 Gy the relative involvement of both chromosomes in exchanges is approximately equal. After radiosensitization, increased chromosome exchange frequencies are observed in both studied chromosomes. For the total chromosome exchange frequencies the sensitizer enhancement ratio (SER) at 2 Gy is 1.8 and 1.3 for chromosome 2 and X respectively. The SER at 4 Gy for total exchange frequencies is 1.6 and 1.9 chromosome 2 and X respectively. For reciprocal exchanges at 2 Gy higher SER values and at 4 Gy lower SER values were observed for both chromosomes.

Hyperthermia and incorporation of halogenated pyrimidines: radiosensitization in cultured rodent and human tumor cells.

PURPOSE: To investigate the possible benefit of hyperthermia (HT) in combination with radiosensitization by halogenated pyrimidines (HPs) in rodent as well as in human tumor cells. METHODS AND MATERIALS: Exponentially growing rodent cells, radiosensitive R-1 and MOS cells and radioresistant RUC-II and V79 cells, and human SW1573 cells, were exposed to 0, 1, 2, and 4 microM of chloro- (CldUrd), bromo- (BrdUrd), or iodo-deoxyuridine (IdUrd) in the culture medium. Survival after irradiation with gamma-rays from a 137Cs source and/or hyperthermic treatment (HT, 60 min at 42 degrees C) was determined by clonogenic assay. Linear-quadratic analyses of the radiation survival curves were performed to assess sensitization in the dose range 1 to 3 Gy relevant to radiotherapy. RESULTS: The incorporation of HPs sensitized all cell lines to HT and resulted in radiosensitization dependent on the percentage of thymidine replacement. At equal levels of thymidine replacement, IdUrd was the most potent radiosensitizer. HT further increased radiation-induced lethality of cells that had incorporated HPs. Linear-quadratic analyses showed that HT further increased the linear parameter of the LQ formula while the quadratic parameter was not significantly changed. CONCLUSION: The combination of HT and HPs act additively in increasing the radiosensitivity of rodent tumor cell lines with varying radiosensitivities as well as of a human tumor cell line. In particular, the ratio of the linear parameter to the quadratic parameter, relevant for fractionation effects in radiotherapy, was increased.

Modification of potentially lethal damage in irradiated Chinese hamster V79 cells after incorporation of halogenated pyrimidines.

Radiosensitization of exponentially growing and plateau phase Chinese hamster V79 cells by incorporation of halogenated pyrimidines (HP) was investigated for different culture conditions that influenced repair. For this purpose cells were grown for 72 h with 0, 1, 2 and 4 microM of chloro-(CldUrd), bromo- (BrdUrd) or iodo-deoxyuridine (IdUrd) and were subsequently irradiated with gamma-rays from a 197Cs source, either in exponential growth or in plateau-phase. Cell survival after irradiation was determined by clonogenic assay. In exponentially growing cultures thymidine-replacement in the DNA of the cells after incubation with 4 microM of CldUrd, BrdUrd and IdUrd was 22.3, 32.7 and 12.7%, respectively. In plateau-phase cultures the percentage thymidine replacement in the DNA of the cells after incubation during growth with 4 microM CldUrd, BrdUrd and IdUrd was 27.5, 33.8 and 10.7%, respectively. Linear-quadratic analyses of the radiation survival curves were performed. In exponentially growing cells a marked increase by a factor 2-3 of the value of alpha was obtained. The beta term significantly increased only in cells which were grown in the presence of BrdUrd and which were trypsinized and replated immediately after irradiation. In plateau-phase cells which were trypsinized and plated immediately after irradiation both alpha and beta increased up to a factor 2-3 with increasing incorporation of halogenated pyrimidines. In plateau phase cells which were allowed to repair potentially lethal damage (PLD) for 6 h and subsequently trypsinized and plated, alpha increased by a factor 3-4. In these latter conditions changes in beta were smaller. In exponentially growing cells in which repair was allowed after irradiation by plating prior to the treatment, the alpha values decreased for all the HP drugs tested as compared to the alpha of cells plated immediately after irradiation. In contrast, delay of plating for plateau phase cells yielded increased alpha values not only when compared with the alpha of plateau phase cells plated immediately after treatment but also when compared with the alpha value of radiosensitized exponentially growing cells. The increase of alpha might be interpreted as an enhancement in the expression of PLD. The larger contribution of fixation of PLD might be due to initial DNA damage and/or to inhibition of PLD repair resulting from incorporation of HP. The increase of beta might be attributed to enhanced interaction or to fixation of sublethal damage (SLD). In view of clinical applications of HP it is of interest that sensitization is not abolished in plateau-phase cells.

Local hyperthermia enhances the effect of cis-diamminedichloro-platinum(II) on nonirradiated and preirradiated rat solid tumors.

PURPOSE: We have investigated differences in the efficacy of combined treatment with cis-diamminedichloroplatinum(II) (cDDP) and local hyperthermia (HT) in nonirradiated and preirradiated experimental tumors. METHODS AND MATERIALS: Survival of R-1 rhabdomyosarcoma cells was assessed after treatment with various cDDP-concentrations at 37 degrees C and 42 degrees C in vitro. Rats bearing R-1 rhabdomyosarcomas of 190 mm3 (SE 15 mm3) were treated with cDDP (6 mg/kg i.p.), HT (1 h at 43 degrees C), or cDDP+HT (45 min interval) without preirradiation or at day 16 after the first dose of fractionated irradiation. Fractionated irradiation consisted of four daily doses of 5 Gy of x-rays each and tumor volumes had regrown to their original volume at the time of treatment. Experimental endpoint was tumor growth delay (TGD). RESULTS: Hyperthermia-enhanced cDDP cytotoxicity in vitro by a factor of about 5. Treatment with cDDP or HT alone resulted in a similar TGD in non- and preirradiated tumors (7.2 vs. 7.4 days and 1.1 vs. 0.9 days, respectively). In non- as well as in preirradiated tumors, HT given in combination with cDDP significantly enhanced the effect of cDDP, prolonging the TGD (11.1 days (p = 0.0001) and 16.2 days (p < 0.0001), respectively) corresponding to a TGD-enhancement of 1.54 and 2.19, respectively. The TGD after cDDP+HT in preirradiated tumors was significantly longer than in nonirradiated tumors (p = 0.0003). CONCLUSIONS: In this tumor model, HT enhanced the antitumor effect of cDDP. Previous radiation treatment did not reduce the HT-enhanced effect of cDDP. Combined cDDP and HT may be useful in the treatment of nonirradiated tumors as well as previously irradiated tumors.

Hyperthermia-enhanced effectiveness of mitoxantrone in an experimental rat tumour.

The influence of local hyperthermia (HT) on Mitoxantrone (MITOX) effectiveness was studied in an experimental rat tumour. R-1 rhabdomyosarcomas were treated with MITOX (5 mg/kg ip), HT (43 degrees C for 1 h) or combinations applied at various time intervals up to 24 h. Tumour growth delay and tumour cell clonogenicity were assessed in correlation with the pharmacokinetics in blood plasma and with MITOX-concentrations in tumour tissue. Combined treatments were more effective than expected on the basis of simple addition of effects of single treatments. With increasing time intervals between treatments up to 8 h, an increase in effectiveness was observed. Unfortunately, treatment with an 8-h interval resulted in a high mortality: 80% of the rats died with 5-10 days after treatment. Treatment with a 3-h interval between MITOX and HT was the most effective combination resulting in the highest therapeutic ratio. Even local tumour controls (14/18 rats) were observed. These enhanced effects were associated with a higher MITOX-concentration in the fraction of intact cells recovered from tumours. However, no differences were observed in MITOX-concentration in total tumour tissue nor in plasma concentrations. In conclusion, timing between MITOX and HT is important for drug availability, for interaction of the two modalities to increase damage in tumour cells and for limiting the toxicity to normal tissues.

Hyperthermia enhances tumor uptake and antitumor efficacy of thermostable liposomal daunorubicin in a rat solid tumor.

The combination of local hyperthermia (HT) with thermostable liposomal daunorubicin (DaunoXome, DX) was investigated to assess targeted drug delivery to experimental tumors. Female Wag/Rij rats bearing solid R-1 rhabdomyosarcomas received i.v. injections of 10 or 15 mg/kg of DX or free-Daunorubicin (f-Dau). After a 1-h interval, HT (60 min at 43 degrees C) was applied. Pharmacokinetics were studied in relation to tumor growth time (TGT), i.e., the time for tumors to reach their original volumes. Pharmacokinetic studies revealed that DX accumulation in tumor tissue was similar to f-Dau. A 5.4-fold increase (P = 0.0084) in tumor drug delivery was observed when DX was combined with HT, whereas liposomes remained stable. For f-Dau, HT had an additional effect on TGT at both drug doses tested (9.6 and 6.2 days, respectively, for 10 mg/kg, P = 0.0092; 17.7 and 13.7, respectively, for 15 mg/kg, P = 0.0431). For DX, HT significantly enhanced TGT of DX in the lower dose (17.1 and 6.4 days, respectively, P = 0.0005), whereas tumors did not regrow at day 25 after DX + HT in the higher dose. Unfortunately, after this time interval, the animals died of late toxicity, probably not related to HT. These results indicate that HT promotes extravasation of DX into tumor tissue and enhances its effectiveness. The finding that HT-induced drug release from the liposomes was not responsible for enhanced antitumor activity provides a rationale for further investigation of thermostable liposomes in conjunction with HT.

Local hyperthermic treatment does not enhance mitoxantrone effectiveness for responses of a rat solid tumour regrowing after irradiation.

Tumours regrowing after irradiation may respond differently to chemo-hyperthermia as compared to non- irradiated tumours. In this study, the efficacy of combined treatment of previously irradiated tumors with mitoxantrone and local hyperthermia (HT) was investigated. Rat R-1 tumours were irradiated with dose fractions of 5Gy X-rays applied on 4 consecutive days. Animals were retreated with mitoxantrone (5mg/kg i.p.), HT (1 h at 43 degrees C) or mitoxantrone + HT (3-h interval) on day 9 after the start of irradiation when tumour volumes were decreasing, or on day 16 when tumour volumes were increasing again. Pharmacokinetics were studied in relation to tumor cell survival and tumour growth delay. No Ht=induced changes in the pharmacokinetics of mitoxantrone were observed. The data on clonogenic survival correlated well with these findings and combined treatment were not more effective than mitoxantrone alone. In the treatment schedule applied, HT did not induce pharmacokinetic changes in irradiated tumours leading to an enhanced cytotoxicity of mitoxantrone. The HT- enhanced effectiveness of the drug observed in non- irradiated tumours is much less in pre-irradiated tumours. Responses of regrowing tumours to combined chemo- hyperthermia depend in a complex way on the stage of regrowth and on the treatment schedule.

Enhancement of the effectiveness of methotrexate for the treatment of solid tumours by application of local hyperthermia.

Investigations were performed to assess the influence of hyperthermia on the pharmacokinetics of a chemotherapeutic drug and on the effectiveness of combined treatments for induction of tumour cell death and growth delay of experimental tumours. Treatments consisted of methotrexate (MTX, 20 mg/kg ip), hyperthermia at 43 degrees C during 60 min (HT60) or 90 min (HT90) and combined chemo-hyperthermia using various time intervals up to 24 h. The results indicate that, for MTX + HT90, concentrations in excess of 0.02 mg/kg are maintained in tumour tissue during at least 22 days, whereas after the other single and combined treatments, the concentration decreased below this level within 5-8 days. The combinations of MTX + HT90 also were more effective with respect to tumour growth delay, 26-28 days, and frequency of partial remissions, 75-100%, as compared to the other treatments: 7-12 days and 0-28% respectively. These observations correlate well with cell survival data. It is concluded that hyperthermia can enhance the effectiveness of MTX and that variation of time-intervals between administration of MTX and hyperthermia as well as the duration of the hyperthermic treatment have a great influence on tumour responses. Unfortunately, also toxic effects were induced distantly from the site of local hyperthermic treatment by the combination of MTX + HT90 which was most effective with respect to tumour eradication.

Cytidine triphosphate (CTP) synthetase activity during cell cycle progression in normal and malignant T-lymphocytic cells.

The role of cytidine triphosphate (CTP) synthetase (EC 6.3.4.2.) in the pyrimidine ribonucleotide metabolism of MOLT-3 human T-ALL cell line cells and normal human T lymphocytes during the cell cycle traverse was studied. Highly pure G1-phase samples and samples enriched in S-phase cells were obtained by counterflow centrifugation. The activity of CTP synthetase in situ, measured in pulse-chase experiments, was similar in the G1-phase and S-phase MOLT-3 cells. In contrast, in S-phase T lymphocytes, an increased activity of CTP synthetase was observed compared with G1-phase T lymphocytes. Nevertheless, the MOLT-3 samples showed an increased activity of CTP synthetase in comparison with either G1-phase or S-phase enriched samples of normal T lymphocytes. Therefore, the increased activity of CTP synthetase of MOLT-3 cells is a cell cycle-independent feature, whereas among normal T lymphocytes, the increase in activity of CTP synthetase that arises after a growth stimulus is more prominent in the S-phase.

Viscum album preparations inhibit DNA methylation by preventing DNA methyltransferase-induced methyl group transfer

Interactions between viscum album (Iscador) and DNA were studies by amplification of specific human pepsinogen A gene promoter sequences by polymerase chain reaction (PCR). Gel retardation assays showed no binding of Viscum album to these promoter sequences. Incubation of plasmid constructs consisting of human pepsigen A promoter fragments coupled to thechloramphenicol acetyl-transferase (CAT) reporter gene with Iscador had no effect on transcriptional activity. Promoter sequences incubated with Iscador became insensitive to methylation by specific DNA methyltransferases by destruction of DNA methyltransferase activity

Elutriation of T-lymphoblastic cells and cell cycle analysis of fractions.

Elutriation of human lymphoblastic cells has to be performed at a flow rate of 13 ml/min at a temperature of 10 degrees C to retain a high cell viability. Cell cycle analysis of elutriated cells on the basis of their DNA contents is inaccurate. The combined detection of BrdUrd incorporated into the DNA and analysis of the DNA content offers an alternative. After elutriation, fractions of cells with increased percentages of either G1 or S phase cells can be selected on the basis of the median cell size as established by a coulter counter channelyzer.

Pharmacokinetics and biological responses after treatment of the rat R-1 rhabdomyosarcoma with methotrexate.

Time relationships of drug concentrations in tissue of a transplantable rat rhabdomyosarcoma and of tumour responses up to 120 hr after treatment with methotrexate (MTX) were analysed and compared. MTX was shown to be retained within the tumour in a substantial concentration for several days, although no evidence of MTX polyglutamation was obtained. The response data confirm that MTX is active in the tumour for up to at least 3 days after injection. Within the first day after MTX treatment the nucleotide pools are only partly depleted. This indicates that the inhibition of DNA synthesis is still incomplete at the time when salvage precursors in increasing amounts are becoming available from decaying cells. From flow cytometric analysis of cell-cycle progression it is concluded that subsequent cohorts arriving in early S-phase were retarded, but not inhibited, in their progression through the S phase. At 3 days after MTX treatment the mean rate of cell-cycle progression as well as the relative clonogenic capacity were maximally reduced to 30% and 1% of control values, respectively. From 3 to 5 days the rate of cell-cycle progression was gradually restored, whereas from day 5 onwards the clonogenic capacity increased at a high rate corresponding to the proliferation rate of exponentially growing rhabdomyosarcoma cells in culture. However, a continuous reduction of cell recovery lasting for at least 12 days after treatment contributed to an 8-day delay in tumour volume growth.

Treatment of the rat R-1 rhabdomyosarcoma with methotrexate and radiation; effects of timing on cell survival and tumour growth delay.

The interaction of radiation (10 Gy 300-kV X-rays) and methotrexate (MTX; 3 x 10 mg/kg at 3.5-h intervals) was investigated with respect to effects on cell survival and tumour regrowth of the transplantable rat R-1 rhabdomyosarcoma. The treatment with MTX alone caused acceptable toxicity and no lethality. On day 3 after treatment with MTX alone a maximum decrease in the fraction of clonogenic cells was observed, which is in accordance with data on MTX concentrations in tumour tissue, indicating that MTX is active in the tumour for at least 3 days after injection. The clonogenic capacity after combined treatments, i.e. MTX before or after radiation, was assessed 3 days after the MTX administration. The fractions of clonogenic cells determined after combined therapy with intervals of up to 4 days were not significantly different from those expected on the basis of simple addition of the effects from individual treatments. However, the excess growth delay was positive at specific intervals (6-8 days after X-rays plus MTX and 5-6 days after MTX plus X-rays), whereas negative excess delays were observed when the two treatments were separated by less than 3 days. It is concluded that expectations with respect to clinical application of the combination must be modest in view of the short duration of favourable intervals and the observed absence of synergistic effects with respect to cell killing. The discrepancy between the two assays indicates that erroneous conclusions can be obtained if one endpoint only is assessed.

Effect of Viscum album preparations on gastric chief cells and DNA methylation by N-methyl-N-nitro-N-nitrosoguanidine

The protective effect of extracts of mistletoe Viscum album (Iscador) with reference to carcinogenesis was tested on in vitro cultured porcine gastric chief cells. Putative protection against in vitro methylation of lambda DNA by N-methyl-N-nitro-N-nitrosoguanidine (MNNG) was studied with restriction endonucleases. Preparations of Iscador from mistletoe grown on oak as host tree had a high cytotoxic effect on gastric chief cells, with an LC50 after 24hrs that ranged between 0.01 and 0.03 mg/ml. At 0.01 mg/ml, Iscador was also found to protect lambda DNA from methylation or destruction by MNNG. During the course of this study a high variability was found both in cytotoxicity and protection rate against methylation which we attribute to batch to batch differences. Thus the LC50 for Iscador MH86L12 was 0.005 mg/ml, while for MH87 D24 it was 0.05 mg/ml after 24hrs. The Iscador batch W frf 50 mg/ml, which was made from a fresh plant extract at the Hiscia Institute, showed less variability. Results are therefore given for this batch of Iscador only

Differences in immunological susceptibility to cadmium toxicity between two rat strains as demonstrated with cell biological methods. Effect of cadmium on DNA synthesis of thymus lymphocytes.

When 2 inbred rat strains, the Brown-Norway rat and the Lewis rat were exposed to the same amount of CdCl2 for 15 days, a completely different immunological reaction pattern could be demonstrated. Despite the same amount of intrathymic cadmium in both strains, the Brown-Norway rat showed a significant decrease in thymocytes in the S-phase and a significant increase of thymocytes in the G2 phase and mitosis, in contrast with findings in the Lewis rats. A new method for estimating subtle forms of thymus atrophy showed a slight decrease in the number of the smallest thymocytes in the Brown-Norway rat after exposure to cadmium, in contrast with that in the Lewis rat. Evidence is presented that the approximately 1.7 times larger number of thymocytes/mg thymus in the Lewis rat, compared to the Brown-Norway rat, as well as the approximately 2.5 times lower proliferation rate of the thymocytes, and an approximately 1.5 times higher metallothionein content of the thymus medulla epithelial cells in the Lewis rat, might be responsible for the observed difference in toxicity. The zinc content of the thymus was not significantly decreased by exposure to CdCl2, and did not differ significantly between both strains.