C. Van Bree

Hyperthermia, cisplatin and radiation trimodality treatment: a promising cancer treatment? A review from preclinical studies to clinical application.

This review discusses available clinical and experimental data and the underlying mechanisms involved in trimodality treatment consisting of hyperthermia, cisplatin and radiotherapy. The results of phase I/II clinical trials show that trimodality treatment is effective and feasible in various cancer types and sites with tolerable toxicity. Based on these results, phase III trials have been launched to investigate whether significant differences in treatment outcome exist between trimodality and standard treatment. In view of the clinical interest, it is surprising to find so few preclinical studies on trimodality treatment. Although little information is available on the doses of the modalities and the treatment sequence resulting in the largest degree of synergistic interaction, the results from in vivo and in vitro preclinical studies support the use of trimodality treatment for cancer patients. Animal studies show an improvement in treatment outcome after trimodality treatment compared with mono- and bimodality treatment. Studies in different human tumour cell lines show that a synergistic interaction can be obtained between hyperthermia, cisplatin and radiation and that this interaction is more likely to occur in cell lines which are more sensitive to cisplatin.

Inactivation of p53 and of pRb protects human colorectal carcinoma cells against hyperthermia-induced cytotoxicity and apoptosis

Abstract Cell-cycle checkpoints are thought to govern the cellular response to external stimuli. The involvement of the p53 tumour-suppressor protein and the retinoblastoma protein (pRb) in the cell-cycle checkpoint in G1 phase is well established. However, little is known about the importance of these G1 checkpoint regulators in hyperthermia-induced cytotoxicity. Such information is relevant because of the clinical application of hyperthermia in combination with chemotherapy or with radiotherapy. The effects of p53 or pRb inactivation were studied in a well-established isogenic system using the human colorectal carcinoma cell line (RKO). The cells were treated with clinically relevant heat doses (60 min at 40–43°C). Cell survival, cell-cycle redistribution and induction of apoptosis were investigated. Survival of the p53-inactivated transfectants was higher than that of the wild-type p53 cells. The pRb-inactivated transfectants showed an intermediate sensitivity to hyperthermia. All transfectants showed G2 arrest after hyperthermia and the appearance of a sub-G1 population. The induction of apoptosis was inhibited in p53-inactivated and pRb-inactivated transfectants. These results suggest that p53 and/or pRb status may be an important determinant of the clinical response to hyperthermia.

Importance of TP53 and RB in the repair of potentially lethal damage and induction of color junctions after exposure to ionizing radiation

Abstract Franken, N. A. P., van Bree, C., ten Cate, R., van Oven, C. H. and Haveman, J. Importance of TP53 and RB in the Repair of Potentially Lethal Damage and Induction of Color Junctions after Exposure to Ionizing Radiation. Radiat. Res. 158, 707–714 (2002). Repair of potentially lethal damage (PLD) was investigated in cells with functional G1-phase arrest with wild-type TP53 and wild-type RB and in cells in which G1-phase arrest was abrogated by inactivation of TP53 or RB. Confluent cultures of cells were plated for clonogenic survival assay either immediately or 24 h after irradiation. Induction of color junctions, an exchange between a painted and unpainted chromosome, was studied in chromosomes 18 and 19 after irradiation with 4 Gy γ rays. Significant repair of PLD was found in cells carrying both wild-type TP53 and wild-type RB. In cells in which TP53 or RB was inactivated, the survival curves from immediately plated and delayed-plated cells were not significantly different. The numbers of radiation-induced color junctions in chromosomes 18 and 19 were similar in all cell lines. From this study we conclude that a functional G1-phase arrest is important for repair of PLD and that TP53 and RB do not affect the frequencies of induction of color junctions in chromosome 18 or 19.

Chapter 19 Antibodies to neuropeptides as alternatives for peptide receptor antagonists in studies on the physiological actions of neuropeptides

Publisher Summary This chapter describes the characteristics of antibodies, which are crucial for blocking the biological activity of neuropeptides, and on the basis of the mechanism of action explains apparent contradictory results. The results presented clearly demonstrate that the binding capacity of an antibody is not a relevant predictor of its biological activity. What counts in passive immunization studies is whether the antibody concentration present in the compartment involved in signal transfer is sufficiently high to bind a significant proportion of the peptide within the signaling time. Thus, based on present knowledge, passive immunization is likely to interfere with (neuro) hormonal and slow nonsynaptic forms of transmission, whereas interference with fast forms of neurotransmission is unlikely. The large number of studies in which biological effects have been recorded after intracerebral administration of antibodies to neuropeptides, thus suggest that nonsynaptic forms of peptidergic signal transfer represent a major type of communication of peptidergic neurons in the mammalian brain.

Repair of Potentially Lethal Damage does not Depend on Functional TP53 in Human Glioblastoma Cells

Abstract van Bree, C., Franken, N. A. P., Rodermond, H. M., Stalpers, L. J. A. and Haveman, J. Repair of Potentially Lethal Damage does not Depend on Functional TP53 in Human Glioblastoma Cells. Radiat. Res. 161, 511–516 (2004). The functionality of G1-phase arrest was investigated in relation to repair of potentially lethal damage (PLD) in human glioblastoma Gli-06 cells. Confluent cultures were irradiated and plated for clonogenic survival either immediately or 24 h after γ irradiation. Bivariate flow cytometry was performed to assess the distribution over the cell cycle. Levels of TP53 and CDKN1A protein were assessed with Western blotting and levels of CDKN1A mRNA with RT-PCR. Confluence significantly reduced the number of proliferating cells. Marked PLD repair was found in the absence of an intact G1 arrest. No accumulation of TP53 was observed, and the protein was smaller than the wild-type TP53 of RKO cells. No increased expression of CDKN1A at the mRNA or protein levels was found in Gli-06 cells. The TP53 of Gli-06 cells was unable to transactivate the CDKN1A gene. From this study, it is evident that PLD repair may be present without a functional TP53 or G1 arrest.

Wild-type p53-function is not required for hyperthermia-enhanced cytotoxicity of cisplatin

The objective was to test the hypothesis that wild-type p53-function is required for the enhancement of the cytotoxicity of cis-diammine-dichloroplatinum(II) (cDDP) cytotoxicity by hyperthermia (HT). Human colorectal carcinoma cells (RKO) with wild-type p53-function and transfectants with HPV16-E6 or with a dominant negative mutant p53 were used. Cells were treated with HT (60 min at 41 degrees C, 43 degrees C, 45 degrees C: HT41, HT43, HT45). with various doses of cDDP alone or with a combined treatment, simultaneously applied. Survival was determined by clonogenic assays. Levels and localization of p53 were analysed with immunocytochemistry and Western blotting. The extent of HT41-enhanced cytotoxicity of cDDP was similar in all cell lines studied. Immunocytochemistry of wild-type p53 cells showed that p53 is transferred to the nucleus within 5 h after HT43, whilst after HT41 no significant effects were observed. Cell fractionation experiments of wild-type p53 cells showed that, immediately after HT43/HT45, nuclear p53-levels increased as compared to controls, but could not be extracted from the matrix. The extractability was restored 3-5 h after treatment. No significant differences in p53-levels were observed after HT41. These results indicate that, although HT43/HT45 might shortly inactivate p53-function, probably by protein aggregation to the nuclear matrix, the HT-enhanced cDDP-cytotoxicity does not depend on p53-function.

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.

Differential Response to Radiation of TP53-Inactivated Cells by Overexpression of Dominant-Negative Mutant TP53 or HPVE6

Abstract Franken, N. A. P., van Bree, C. and Haveman, J. Differential Response to Radiation of TP53-Inactivated Cells by Overexpression of Dominant-Negative Mutant TP53 or HPVE6. Radiat. Res. 161, 504–510 (2004). The inactivation of TP53 by transfection of a dominant- negative mutated TP53 (MP53.13 cells) was compared with inactivation of TP53 by transfection with the HPV E6 gene (RC10.1 cells) with respect to PLD repair, G1-phase arrest, and induction of color junctions. Functional G1 arrest was demonstrated in parental (RKO) cells with wild-type TP53, while in RC10.1 cells the G1 arrest was eliminated. In MP53.13 cells an intermediate G1 arrest was found. Functionality of endogenous TP53 was confirmed in RKO and MP53.13 cells by accumulation of TP53 protein and its downstream target CDKN1A (p21). Radiation survival of MP53.13 cells was higher than that of RKO cells, and PLD repair was found in RKO cells and MP53.13 cells but not in RC10.1 cells. Both with and without irradiation, the number of color junctions was 50 to 80% higher in MP53.13 cells than in RKO and RC10.1 cells. In the MP53.13 cells, the genetic instability appears to lead to more aberrations and to radioresistance. In spite of the presence of an excess of mutated TP53, wild- type TP53 functions appear to be affected only partly or not at all.

Chromosome aberrations detected by FISH and correlation with cell survival after irradiation at various dose-rates and after bromodeoxyuridine radiosensitization

Purpose : To determine whether measurement of chromosome aberrations by fluorescence in situ hybridization (FISH) predicts cell survival after irradiation at different dose-rates and after radiosensitization by bromodeoxyurdine (BrdU) in a lung carcinoma cell line. Materials and methods : The human lung carcinoma cell line SW1573 was irradiated at high dose-rate (HDR: 0.8 Gy min -1) or at pulsed low dose-rate (p-LDR: average dose-rate 1 Gy h -1) with or without radiosensitization by bromodeoxyuridine (BrdU). Cell survival was determined by clonogenic assay. Chromosome aberrations (colour junctions) were measured by whole-chromosome FISH of chromosome 2 and 18 and were scored according to the PAINT method. Results : Clear radiosensitization by BrdU was observed both after HDR and p-LDR irradiation. Chromosome 18 was more radiosensitive than chromosome 2. There was a good correlation between induction of colour junctions and cell survival both after HDR and p-LDR irradiation and after radiosensitization by BrdU. Conclusions : Determination of chromosome aberrations by FISH can predict cell survival after different dose-rates and after radiosensitization by BrdU

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.