J. Haveman

Clonogenic assay of cells in vitro.

Clonogenic assay or colony formation assay is an in vitro cell survival assay based on the ability of a single cell to grow into a colony. The colony is defined to consist of at least 50 cells. The assay essentially tests every cell in the population for its ability to undergo “unlimited” division. Clonogenic assay is the method of choice to determine cell reproductive death after treatment with ionizing radiation, but can also be used to determine the effectiveness of other cytotoxic agents. Only a fraction of seeded cells retains the capacity to produce colonies. Before or after treatment, cells are seeded out in appropriate dilutions to form colonies in 1–3 weeks. Colonies are fixed with glutaraldehyde (6.0% v/v), stained with crystal violet (0.5% w/v) and counted using a stereomicroscope. A method for the analysis of radiation dose–survival curves is included.

Analysis of Gene Expression Using Gene Sets Discriminates Cancer Patients with and without Late Radiation Toxicity

Background Radiation is an effective anti-cancer therapy but leads to severe late radiation toxicity in 5%–10% of patients. Assuming that genetic susceptibility impacts this risk, we hypothesized that the cellular response of normal tissue to X-rays could discriminate patients with and without late radiation toxicity. Methods and Findings Prostate carcinoma patients without evidence of cancer 2 y after curative radiotherapy were recruited in the study. Blood samples of 21 patients with severe late complications from radiation and 17 patients without symptoms were collected. Stimulated peripheral lymphocytes were mock-irradiated or irradiated with 2-Gy X-rays. The 24-h radiation response was analyzed by gene expression profiling and used for classification. Classification was performed either on the expression of separate genes or, to augment the classification power, on gene sets consisting of genes grouped together based on function or cellular colocalization. X-ray irradiation altered the expression of radio-responsive genes in both groups. This response was variable across individuals, and the expression of the most significant radio-responsive genes was unlinked to radiation toxicity. The classifier based on the radiation response of separate genes correctly classified 63% of the patients. The classifier based on affected gene sets improved correct classification to 86%, although on the individual level only 21/38 (55%) patients were classified with high certainty. The majority of the discriminative genes and gene sets belonged to the ubiquitin, apoptosis, and stress signaling networks. The apoptotic response appeared more pronounced in patients that did not develop toxicity. In an independent set of 12 patients, the toxicity status of eight was predicted correctly by the gene set classifier. Conclusions Gene expression profiling succeeded to some extent in discriminating groups of patients with and without severe late radiotherapy toxicity. Moreover, the discriminative power was enhanced by assessment of functionally or structurally related gene sets. While prediction of individual response requires improvement, this study is a step forward in predicting susceptibility to late radiation toxicity.

Effect of hyperthermia on the central nervous system: A review

Experimental data show that nervous tissue is sensitive to heat. Animal data indicate that the maximum tolerated heat dose after local hyperthermia of the central nervous system (CNS) lies in the range of 40-60 min at 42-42 x 5 degrees C or 10-30 min at 43 degrees C. No conclusions concerning the heat sensitivity of nervous tissue can be derived from clinical studies using localized hyperthermia. The choice whether or not to exceed the critical heat dose, as derived from laboratory studies, in clinical practice is very much dependent on the clinical situation such as the anatomical site and volume of the tissue involved, and prior therapy. Data on clinical application of whole body hyperthermia (WBH) show that nervous tissue can withstand a slightly higher heat dose than after localized heating, which might be the result of developing thermal resistance during treatment. Expression of thermotolerance was observed in the spinal cord of laboratory animals. After WBH in man at a maximum between 40 and 43 degrees C for 6 h-30 min CNS complications were reported, but other complications seemed to be more life-threatening. Most studies indicate that impairment of the CNS after WBH was not due to direct heat injury to the brain or spinal cord, but was secondary as a result of physiological changes. Heat, at least if applied shortly after X-rays, enhances the response of nervous tissue to radiation. Neurotoxicity of chemotherapeutic drugs does not seem to be a limiting complication in hyperthermia if combined with chemotherapy, but only few data are available. The limited clinical experience shows that safe hyperthermic treatment of CNS malignancies or tumours located close to the CNS seems feasible under appropriate technical conditions with adequate thermometry and taking the sensitivity of the surrounding normal nervous tissue into account.

Hyperthermia enhances the cytotoxicity and platinum-DNA adduct formation of lobaplatin and oxaliplatin in cultured SW 1573 cells

The cytotoxicity of cisplatin and cisplatin-DNA adduct formation in vitro and in vivo is clearly enhanced by hyperthermia. We investigated whether cytotoxicity and platinum-DNA adduct formation of two promising new third-generation platinum derivatives, lobaplatin [1,2-diamminomethylcyclobutane platinum(II) lactate] and oxaliplatin [oxalato-1,2-diaminocyclohexane platinum(II)], are also enhanced by hyperthermia. Cisplatin was used for comparison. SW 1573 cells were incubated with cisplatin, lobaplatin or oxaliplatin at different concentrations for 1 h at 37°, 41° and 43°C. The reproductive capacity of cells was determined by cloning experiments. Immunocytochemical detection of platinum-DNA adducts was performed with the rabbit antiserum NKI-A59. At 37°C, cisplatin was the most cytotoxic, followed by oxaliplatin and lobaplatin. Hyperthermia clearly enhanced the cytotoxicity of cisplatin, lobaplatin and oxaliplatin. There was no further increase in cytotoxicity at 43°C compared to 41°C for cisplatin and oxaliplatin. A further increase in cytotoxicity at 43°C was observed for lobaplatin. At 43°C thermal enhancement was higher for lobaplatin than for oxaliplatin, with the reverse pattern at 41°C. For both drugs, thermal enhancement of cytotoxicity was lower than observed for cisplatin. Immunocytochemical detection of platinum-DNA adducts was feasible for all the drugs. Adduct formation was enhanced at 43°C for cisplatin, lobaplatin and oxaliplatin with a relative increase of 410%, 170% and 180%. These results seem to confirm that an increase in platinum-DNA adduct formation is involved in the in vitro thermal enhancement of cytotoxicity. The observed thermal enhancement of cytotoxicity of lobaplatin and oxaliplatin in vitro warrants further in vivo investigations.

Comparison of RBE values of high-LET α-particles for the induction of DNA-DSBs, chromosome aberrations and cell reproductive death

BackgroundVarious types of radiation effects in mammalian cells have been studied with the aim to predict the radiosensitivity of tumours and normal tissues, e.g. DNA double strand breaks (DSB), chromosome aberrations and cell reproductive inactivation. However, variation in correlations with clinical results has reduced general application. An additional type of information is required for the increasing application of high-LET radiation in cancer therapy: the Relative Biological Effectiveness (RBE) for effects in tumours and normal tissues. Relevant information on RBE values might be derived from studies on cells in culture.MethodsTo evaluate relationships between DNA-DSB, chromosome aberrations and the clinically most relevant effect of cell reproductive death, for ionizing radiations of different LET, dose-effect relationships were determined for the induction of these effects in cultured SW-1573 cells irradiated with gamma-rays from a Cs-137 source or with α-particles from an Am-241 source. RBE values were derived for these effects. Ionizing radiation induced foci (IRIF) of DNA repair related proteins, indicative of DSB, were assessed by counting gamma-H2AX foci. Chromosome aberration frequencies were determined by scoring fragments and translocations using premature chromosome condensation. Cell survival was measured by colony formation assay. Analysis of dose-effect relations was based on the linear-quadratic model.ResultsOur results show that, although both investigated radiation types induce similar numbers of IRIF per absorbed dose, only a small fraction of the DSB induced by the low-LET gamma-rays result in chromosome rearrangements and cell reproductive death, while this fraction is considerably enhanced for the high-LET alpha-radiation. Calculated RBE values derived for the linear components of dose-effect relations for gamma-H2AX foci, cell reproductive death, chromosome fragments and colour junctions are 1.0 ± 0.3, 14.7 ± 5.1, 15.3 ± 5.9 and 13.3 ± 6.0 respectively.ConclusionsThese results indicate that RBE values for IRIF (DNA-DSB) induction provide little valid information on other biologically-relevant end points in cells exposed to high-LET radiations. Furthermore, the RBE values for the induction of the two types of chromosome aberrations are similar to those established for cell reproductive death. This suggests that assays of these aberrations might yield relevant information on the biological effectiveness in high-LET radiotherapy.

Effects of Local Hyperthermia on the Motor Function of the Rat Sciatic Nerve

The effect of local heat treatment of the sciatic nerve was assessed using the toe-spreading test, which mainly assesses the motor function of the sciatic nerve. A 5 mm long segment of the nerve was heated at temperatures from 42.0 to 45.0 degrees C in vivo using a brass thermode. Hyperthermia led to a decrease in spreading of the toes. Recovery from functional loss took place in all cases, and this recovery was completed in 4 weeks. A 50 per cent functional loss in 50 per cent of the treated animals was observed after 58, 32 and 12 min of heating at 43.0, 44.0 and 45.0 degrees C respectively.

Hyperthermic injury versus crush injury in the rat sciatic nerve: A comparative functional, histopathological and morphometrical study

Functional and morphological changes of the rat sciatic nerve after local hyperthermia (30 min, 45 degrees C) and crush treatment were compared. After hyperthermic injury nerve function loss developed in a time period of about 7 h. Nerve crush led to an immediate loss of nerve function. Nerve function loss was assessed by a motor and a sensory function test. Recovery from function loss took place in both treatment groups and was complete in 4-5 weeks. Early (within 8 h post-treatment) histopathological changes in the nerve after heating included edema, possible blood stasis and changes in the blood vessel wall, like swelling of the media. During this period some axonal changes were observed. Immediate after crushing axons were severely damaged, while many blood vessels remained normal. Within one week after both treatments, degeneration of axons and myelin was observed at the site and distal from the site of the lesion (Wallerian degeneration). Three weeks after treatment a major part of the axons had regenerated and remyelinated. Vascular changes at the site of lesion could still be observed in the heat-treated nerves. Twelve weeks after both treatments, blood vessels appeared to be normal again. Morphometrical analysis of the treated nerves confirmed the histological observations. Three and 12 weeks after treatment average axon diameters were significant smaller and average myelin sheaths were significant thinner compared to untreated nerves. These parameters did not differ significantly when the two treatment groups were compared.

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.

The pH of the cytoplasm as an important factor in the survival of in vitro cultured malignant cells after hyperthermia. Effects of carbonylcyanide 3-chlorophenylhydrazone

Abstract The effects of heat treatments at 43°C (hyperthermia), were studied on in vitro cultured cells, derived from a murine mammary carcinoma, at different pH values of the medium in the absence and presence of the proton conducting drug carbonylcyanide- 3 -chlorophenylhydrazone (CCCP). The survival of these cells after hyperthermia was always optimal at a pH of the medium between 7.75 and 8.0 . At this optimal pH the presence of CCCP hardly influenced the survival for treatment times up to 90 min , whereas there was a large effect of CCCP at lower pH values, pH 6.5–7.5 . At higher pH values, 8.0–9.0 CCCP only had effect after at least 1 hr heating. For exposure times up to 3 hr CCCP only had a small effect at normal temperature, 37°C , except at very low pH values, below 6.5 , where cell survival was impaired, even without CCCP. The large effect of CCCP in the pH range 6.5–7.5 strongly suggests that the survival after hyperthermia is mainly determined by the cytoplasm pH . Changes in the pH of the cell medium may affect survival only after modifying the cytoplasm pH . In the pH range 6.5–8.0 the cells are apparently able to maintain the optimal pH value inside, close to 8.0 . The capacity to control pH is impaired by the addition of CCCP. Outside the range 6.5–8.0 the capacity to control pH appears to be insufficient or absent. Moreover it is presumed that there is a progressive deregulation of the pH control mechanism during a 43°C heat treatment. After 2 hr treatment at 43°C the cells apparently are no longer capable of maintaining their optimal cytoplasm pH .

Lack of thermal enhancement for taxanes in vitro

The taxanes represent a new class of clinical chemotherapeutic agents. A series of in vitro studies were independently of each other initiated in two different institutes (Amsterdam and Madison) to test the hypothesis that hyperthermia might enhance the cytotoxicity of taxanes. Clonogenic capacity experiments (Amsterdam) included the exposure of R1- and SW 1573-cells to 1, 4, or 24 h of paclitaxel with heat 43 degrees C x 60 min in the last hour of drug treatment or at 24, 48 as well as 72 h post drug treatment. Survival assay experiments (Madison) included the exposure of L-929-cells to paclitaxel and docetaxel for 24 h with heat 41.8 degrees C x 60 min the first or last hour of drug treatment as well as 24 and 48 h post treatment. No thermal enhancement of cytotoxicity for the taxanes was observed in these human and murine cell lines, with congruent data in both institutes. In addition, high performance liquid chromatography studies at 41.8 degrees C and 43 degrees C demonstrated paclitaxel and docetaxel were heat stable.