Quantitative analysis of contribution of mild and moderate hyperthermia to thermal ablation and sensitization of irreversible electroporation of pancreatic cancer cells.

INTRODUCTION: Irreversible electroporation (IRE) is an ablation modality that applies short, high-voltage electric pulses to unresectable cancers. Although considered a non-thermal technique, temperatures do increase during IRE. This temperature rise sensitizes tumor cells for electroporation as well as inducing partial direct thermal ablation. AIM: To evaluate the extent to which mild and moderate hyperthermia enhance electroporation effects, and to establish and validate in a pilot study cell viability models (CVM) as function of both electroporation parameters and temperature in a relevant pancreatic cancer cell line. METHODS: Several IRE-protocols were applied at different well-controlled temperature levels (37 °C ≤ T ≤ 46 °C) to evaluate temperature dependent cell viability at enhanced temperatures in comparison to cell viability at T = 37 °C. A realistic sigmoid CVM function was used based on thermal damage probability with Arrhenius Equation and cumulative equivalent minutes at 43 °C (CEM43°C) as arguments, and fitted to the experimental data using "Non-linear-least-squares"-analysis. RESULTS: Mild (40 °C) and moderate (46 °C) hyperthermic temperatures boosted cell ablation with up to 30% and 95%, respectively, mainly around the IRE threshold Eth,50% electric-field strength that results in 50% cell viability. The CVM was successfully fitted to the experimental data. CONCLUSION: Both mild- and moderate hyperthermia significantly boost the electroporation effect at electric-field strengths neighboring Eth,50%. Inclusion of temperature in the newly developed CVM correctly predicted both temperature-dependent cell viability and thermal ablation for pancreatic cancer cells exposed to a relevant range of electric-field strengths/pulse parameters and mild moderate hyperthermic temperatures.

Molecular and biological rationale of hyperthermia as radio- and chemosensitizer.

Mild hyperthermia, local heating of the tumour up to temperatures <43 °C, has been clinically applied for almost four decades and has been proven to substantially enhance the effectiveness of both radiotherapy and chemotherapy in treatment of primary and recurrent tumours. Clinical results and mechanisms of action are discussed in this review, including the molecular and biological rationale of hyperthermia as radio- and chemosensitizer as established in in vitro and in vivo experiments. Proven mechanisms include inhibition of different DNA repair processes, (in)direct reduction of the hypoxic tumour cell fraction, enhanced drug uptake, increased perfusion and oxygen levels. All mechanisms show different dose effect relationships and different optimal scheduling with radiotherapy and chemotherapy. Therefore, obtaining the ideal multi-modality treatment still requires elucidation of more detailed data on dose, sequence, duration, and possible synergisms between modalities. A multidisciplinary approach with different modalities including hyperthermia might further increase anti-tumour effects and diminish normal tissue damage.

Outcome of a rabbit model for late irradiation effects in mandibular oral mucosa and bone: A pilot study.

BACKGROUND/AIM/OBJECTIVE: Late side effects of radiotherapy (RT) in the treatment for head and neck (HN) malignancies involve an inadequate healing response of the distressed tissue due to RT-induced hypovascularity. The aim of this study was to develop a pilot model in which vascular alterations associated with the onset of late irradiation (IR) injury could be measured in rabbit oral mucosa and mandibular bone. MATERIALS AND METHODS: Eight male New Zealand white rabbits were divided over four treatment groups. Group I-III received four fractions of RT (5.6 Gy, 6.5 Gy, and 8 Gy, respectively) and Group IV received 1 fraction of 30 Gy. Oral microcirculatory measurements were performed at baseline (before RT) and once a week during 11 consecutive weeks after RT assessing perfusion parameters, that is, total vessel density (TVD), perfused vessel density (PVD), proportion of perfused vessels (PPV), and microvascular flow index (MFI). Post-mortem histopathology specimens were analyzed. RESULTS: Five weeks after RT, TVD, and PVD in all groups showed a decrease of >10% compared to baseline, a significant difference was observed for Groups I, II, and IV (P<0.05). At T11, no lasting effect of decreased vessel density was observed. PPV and MFI remained unaltered at all-time points. Group IV showed a marked difference in scattered telangiectasia such as microangiopathies, histological necrosis, and loss of vasculature. CONCLUSION: No significant lasting effect in mucosal microcirculation density due to IR damage was detected. Observed changes in microcirculation vasculature and histology may align preliminary tissue transition towards clinical pathology in a very early state associated with late IR injury in the oral compartment. RELEVANCE FOR PATIENTS: Enhancing knowledge on the onset of late vascular IR injury in the HN region could help the development, monitoring, and timing of therapies that act on prevention, discontinuation, or repair of radiation pathology.

The effect of time interval between radiotherapy and hyperthermia on planned equivalent radiation dose.

PURPOSE: Thermoradiotherapy is an effective treatment for locally advanced cervical cancer. However, the optimal time interval between radiotherapy and hyperthermia, resulting in the highest therapeutic gain, remains unclear. This study aims to evaluate the effect of time interval on the therapeutic gain using biological treatment planning. METHODS: Radiotherapy and hyperthermia treatment plans were created for 15 cervical cancer patients. Biological modeling was used to calculate the equivalent radiation dose, that is, the radiation dose that results in the same biological effect as the thermoradiotherapy treatment, for different time intervals ranging from 0-4 h. Subsequently, the thermal enhancement ratio (TER, i.e. the ratio of the dose for the thermoradiotherapy and the radiotherapy-only plan) was calculated for the gross tumor volume (GTV) and the organs at risk (OARs: bladder, rectum, bowel), for each time interval. Finally, the therapeutic gain factor (TGF, i.e. TERGTV/TEROAR) was calculated for each OAR. RESULTS: The median TERGTV ranged from 1.05 to 1.16 for 4 h and 0 h time interval, respectively. Similarly, for bladder, rectum and bowel, TEROARs ranged from 1-1.03, 1-1.04 and 1-1.03, respectively. Radiosensitization in the OARs was much less than in the GTV, because temperatures were lower, fractionation sensitivity was higher (lower α/ß) and direct cytotoxicity was assumed negligible in normal tissue. TGFs for the three OARs were similar, and were highest (around 1.12) at 0 h time interval. CONCLUSION: This planning study indicates that the largest therapeutic gain for thermoradiotherapy in cervical cancer patients can be obtained when hyperthermia is delivered immediately before or after radiotherapy.

The alfa and beta of tumours: a review of parameters of the linear-quadratic model, derived from clinical radiotherapy studies.

BACKGROUND: Prediction of radiobiological response is a major challenge in radiotherapy. Of several radiobiological models, the linear-quadratic (LQ) model has been best validated by experimental and clinical data. Clinically, the LQ model is mainly used to estimate equivalent radiotherapy schedules (e.g. calculate the equivalent dose in 2 Gy fractions, EQD2), but increasingly also to predict tumour control probability (TCP) and normal tissue complication probability (NTCP) using logistic models. The selection of accurate LQ parameters α, ß and α/ß is pivotal for a reliable estimate of radiation response. The aim of this review is to provide an overview of published values for the LQ parameters of human tumours as a guideline for radiation oncologists and radiation researchers to select appropriate radiobiological parameter values for LQ modelling in clinical radiotherapy. METHODS AND MATERIALS: We performed a systematic literature search and found sixty-four clinical studies reporting α, ß and α/ß for tumours. Tumour site, histology, stage, number of patients, type of LQ model, radiation type, TCP model, clinical endpoint and radiobiological parameter estimates were extracted. Next, we stratified by tumour site and by tumour histology. Study heterogeneity was expressed by the I2 statistic, i.e. the percentage of variance in reported values not explained by chance. RESULTS: A large heterogeneity in LQ parameters was found within and between studies (I2 > 75%). For the same tumour site, differences in histology partially explain differences in the LQ parameters: epithelial tumours have higher α/ß values than adenocarcinomas. For tumour sites with different histologies, such as in oesophageal cancer, the α/ß estimates correlate well with histology. However, many other factors contribute to the study heterogeneity of LQ parameters, e.g. tumour stage, type of LQ model, TCP model and clinical endpoint (i.e. survival, tumour control and biochemical control). CONCLUSIONS: The value of LQ parameters for tumours as published in clinical radiotherapy studies depends on many clinical and methodological factors. Therefore, for clinical use of the LQ model, LQ parameters for tumour should be selected carefully, based on tumour site, histology and the applied LQ model. To account for uncertainties in LQ parameter estimates, exploring a range of values is recommended.

Measurement and analysis of the impact of time-interval, temperature and radiation dose on tumour cell survival and its application in thermoradiotherapy plan evaluation.

PURPOSE: Biological modelling of thermoradiotherapy may further improve patient selection and treatment plan optimisation, but requires a model that describes the biological effect as a function of variables that affect treatment outcome (e.g. temperature, radiation dose). This study aimed to establish such a model and its parameters. Additionally, a clinical example was presented to illustrate the application. METHODS: Cell survival assays were performed at various combinations of radiation dose (0-8 Gy), temperature (37-42 °C), time interval (0-4 h) and treatment sequence (radiotherapy before/after hyperthermia) for two cervical cancer cell lines (SiHa and HeLa). An extended linear-quadratic model was fitted to the data using maximum likelihood estimation. As an example application, a thermoradiotherapy plan (23 × 2 Gy + weekly hyperthermia) was compared with a radiotherapy-only plan (23 × 2 Gy) for a cervical cancer patient. The equivalent uniform radiation dose (EUD) in the tumour, including confidence intervals, was estimated using the SiHa parameters. Additionally, the difference in tumour control probability (TCP) was estimated. RESULTS: Our model described the dependency of cell survival on dose, temperature and time interval well for both SiHa and HeLa data (R2=0.90 and R2=0.91, respectively), making it suitable for biological modelling. In the patient example, the thermoradiotherapy plan showed an increase in EUD of 9.8 Gy that was robust (95% CI: 7.7-14.3 Gy) against propagation of the uncertainty in radiobiological parameters. This corresponded to a 20% (95% CI: 15-29%) increase in TCP. CONCLUSIONS: This study presents a model that describes the cell survival as a function of radiation dose, temperature and time interval, which is essential for biological modelling of thermoradiotherapy treatments.

Targeting therapy-resistant cancer stem cells by hyperthermia.

Eradication of all malignant cells is the ultimate but challenging goal of anti-cancer treatment; most traditional clinically-available approaches fail because there are cells in a tumour that either escape therapy or become therapy-resistant. A subpopulation of cancer cells, the cancer stem cells (CSCs), is considered to be of particular significance for tumour initiation, progression and metastasis. CSCs are considered in particular to be therapy-resistant and may drive disease recurrence, which positions CSCs in the focus of anti-cancer research, but successful CSC-targeting therapies are limited. Here, we argue that hyperthermia - a therapeutic approach based on local heating of a tumour - is potentially beneficial for targeting CSCs in solid tumours. First, hyperthermia has been described to target cells in hypoxic and nutrient-deprived tumour areas where CSCs reside and ionising radiation and chemotherapy are least effective. Second, hyperthermia can modify factors that are essential for tumour survival and growth, such as the microenvironment, immune responses, vascularisation and oxygen supply. Third, hyperthermia targets multiple DNA repair pathways, which are generally upregulated in CSCs and protect them from DNA-damaging agents. Addition of hyperthermia to the therapeutic armamentarium of oncologists may thus be a promising strategy to eliminate therapy-escaping and -resistant CSCs.

3D radiobiological evaluation of combined radiotherapy and hyperthermia treatments.

PURPOSE: Currently, clinical decisions regarding thermoradiotherapy treatments are based on clinical experience. Quantification of the radiosensitising effect of hyperthermia allows comparison of different treatment strategies, and can support clinical decision-making regarding the optimal treatment. The software presented here enables biological evaluation of thermoradiotherapy plans through calculation of equivalent 3D dose distributions. METHODS: Our in-house developed software (X-Term) uses an extended version of the linear-quadratic model to calculate equivalent radiation dose, i.e. the radiation dose yielding the same effect as the thermoradiotherapy treatment. Separate sets of model parameters can be assigned to each delineated structure, allowing tissue specific modelling of hyperthermic radiosensitisation. After calculation, the equivalent radiation dose can be evaluated according to conventional radiotherapy planning criteria. The procedure is illustrated using two realistic examples. First, for a previously irradiated patient, normal tissue dose for a radiotherapy and thermoradiotherapy plan (with equal predicted tumour control) is compared. Second, tumour control probability (TCP) is assessed for two (otherwise identical) thermoradiotherapy schedules with different time intervals between radiotherapy and hyperthermia. RESULTS: The examples demonstrate that our software can be used for individualised treatment decisions (first example) and treatment optimisation (second example) in thermoradiotherapy. In the first example, clinically acceptable doses to the bowel were exceeded for the conventional plan, and a substantial reduction of this excess was predicted for the thermoradiotherapy plan. In the second example, the thermoradiotherapy schedule with long time interval was shown to result in a substantially lower TCP. CONCLUSIONS: Using biological modelling, our software can facilitate the evaluation of thermoradiotherapy plans and support individualised treatment decisions.

Biological modelling of the radiation dose escalation effect of regional hyperthermia in cervical cancer.

BACKGROUND: Locoregional hyperthermia combined with radiotherapy significantly improves locoregional control and overall survival for cervical tumors compared to radiotherapy alone. In this study biological modelling is applied to quantify the effect of radiosensitization for three cervical cancer patients to evaluate the improvement in equivalent dose for the combination treatment with radiotherapy and hyperthermia. METHODS: The Linear-Quadratic (LQ) model extended with temperature-dependent LQ-parameters α and ß was used to model radiosensitization by hyperthermia and to calculate the conventional radiation dose that is equivalent in biological effect to the combined radiotherapy and hyperthermia treatment. External beam radiotherapy planning was performed based on a prescription dose of 46Gy in 23 fractions of 2Gy. Hyperthermia treatment using the AMC-4 system was simulated based on the actual optimized system settings used during treatment. RESULTS: The simulated hyperthermia treatments for the 3 patients yielded a T50 of 40.1 °C, 40.5 °C, 41.1 °C and a T90 of 39.2 °C, 39.7 °C, 40.4 °C, respectively. The combined radiotherapy and hyperthermia treatment resulted in a D95 of 52.5Gy, 55.5Gy, 56.9Gy in the GTV, a dose escalation of 7.3-11.9Gy compared to radiotherapy alone (D95 = 45.0-45.5Gy). CONCLUSIONS: This study applied biological modelling to evaluate radiosensitization by hyperthermia as a radiation-dose escalation for cervical cancer patients. This model is very useful to compare the effectiveness of different treatment schedules for combined radiotherapy and hyperthermia treatments and to guide the design of clinical studies on dose escalation using hyperthermia in a multi-modality setting.

[Medical and biological consequences of nuclear disasters]. / Medische en biologische gevolgen van kernrampen: medische stralingsrisico's overdreven; psychologische risico's onderschat.

Medical risks of radiation exaggerated; psychological risks underestimated. The discussion about atomic energy has become topical again following the nuclear accident in Fukushima. There is some argument about the gravity of medical and biological consequences of prolonged exposure to radiation. The risk of cancer following a low dose of radiation is usually estimated by linear extrapolation of the incidence of cancer among survivors of the atomic bombs dropped on Hiroshima and Nagasaki in 1945. The radiobiological linear-quadratic model (LQ-model) gives a more accurate description of observed data, is radiobiologically more plausible and is better supported by experimental and clinical data. On the basis of this model there is less risk of cancer being induced following radiation exposure. The gravest consequence of Chernobyl and Fukushima is not the medical and biological damage, but the psychological and economical impact on rescue workers and former inhabitants.

Chromatin mobility is increased at sites of DNA double-strand breaks.

DNA double-strand breaks (DSBs) can efficiently kill cancer cells, but they can also produce unwanted chromosome rearrangements when DNA ends from different DSBs are erroneously joined. Movement of DSB-containing chromatin domains might facilitate these DSB interactions and promote the formation of chromosome rearrangements. Therefore, we analyzed the mobility of chromatin domains containing DSBs, marked by the fluorescently tagged DSB marker 53BP1, in living mammalian cells and compared it with the mobility of undamaged chromatin on a time-scale relevant for DSB repair. We found that chromatin domains containing DSBs are substantially more mobile than intact chromatin, and are capable of roaming a more than twofold larger area of the cell nucleus. Moreover, this increased DSB mobility, but not the mobility of undamaged chromatin, can be reduced by agents that affect higher-order chromatin organization.

Transient inhibition of Calyculin A induced premature chromosome condensation by hyperthermia.

The analysis of chromosomal aberrations by premature chromosome condensation (PCC) induced by Calyculin A (Cal) is feasible in tumor biopsies from patients and has the potential to predict sensitivity to radiotherapy. As hyperthermia (HT) improves radiotherapy outcome in certain tumor sites, it was investigated whether PCC induction is still possible after temperatures reached in the clinic. Human cervical carcinoma (CaSki) and lung carcinoma (SW-1573) cells were incubated with Cal to induce PCC immediately after 1 h treatment at temperatures ranging from 41 degrees C to 43 degrees C and after recovery for up to 24 h after treatment with 43 degrees C. Levels of phosphorylated Cdc2 (at the Tyr15 residue), histone H3 (at the Ser10 residue) and Cyclin B1 were investigated by immunoblotting. The amount of cells positive for phosphorylated histone H3 was determined by flow cytometry. Temperatures > or =42.5 degrees C inhibited the induction of PCC by Cal, while recovery of PCC-induction was observed at >20 h after treatment in both cell lines. The phosphorylation status of Cdc2 as well as of histone H3 in cells treated with Cal directly after HT at 43 degrees C was similar to that of cells treated with Cal alone or treated with Cal 24 h after HT at 43 degrees C. HT alone did not affect the levels of phosphorylated Cdc2, while phosphorylation levels of histone H3 were increased as compared with control status of these two proteins. Phosphorylated and total Cyclin B1 levels were not influenced by any of the treatments. Flow cytometric analysis confirmed that HT at 43 degrees C did not interfere with phosphorylation of histone H3. Our data indicate that HT transiently inhibits PCC induction by Cal in a temperature-dependent manner. Therefore, an interval of at least 24 h after HT should be applied before taking tumor biopsies for karyogram analysis of patients treated with temperatures above 42.5 degrees C.

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.

Gadolinium enhances the sensitivity of SW-1573 cells for thermal neutron irradiation.

Gadolinium neutron capture therapy (Gd-NCT) is an experimental cancer treatment based on the physical principal that neutron capture by gadolinium-157 ensures the release of focal high-dose radiation, such as gamma-rays and electrons. Survival and induction of chromosomal aberrations of human SW-1573 cells was studied after thermal neutron irradiation without and with gadolinium. After neutron irradiation with Gd-DTPA, an alpha-enhancement factor of 2.3 was obtained compared to thermal neutron irradiation alone. Gd-DTPA could not radioenhance the cells for gamma-ray irradiation. Induction of colour junctions and chromosome fragments by thermal neutron irradiation and Gd-NCT were studied using PCC-FISH. Correlations (r2-value) between survival and chromosome aberrations ranged from 0.81 to 0.94 for colour junctions and from 0.78 to 0.98 for chromosome fragments of chromosomes 18 and 2 respectively. Thermal neutron irradiation with or without gadolinium induced more chromosome aberrations than gamma-ray irradiation. After correction for chromosome length it appeared that both chromosomes were equally sensitive to radiation. It is concluded that Gd-NCT at a non-toxic concentration of gadolinium is effective in inducing cell death and chromosome aberrations in in vitro cell cultures.

Effects of cisplatin and gamma-irradiation on cell survival, the induction of chromosomal aberrations and apoptosis in SW-1573 cells.

PURPOSE: Cisplatin was found to radiosensitize SW-1573 cells by inhibition of PLDR. Therefore, it was investigated whether cisplatin combined with gamma-radiation leads to an increase in the number of chromosomal aberrations or apoptotic cells compared with radiation alone. METHODS: Confluent cultures of the human lung carcinoma cell line SW-1573 were treated with 1 microM cisplatin for 1 h, 4 Gy gamma-radiation, or a combination of both. Cell survival was studied by the clonogenic assay. Aberrations were analysed by FISH in prematurely condensed chromosomes (PCC) and the induction of apoptosis by counting fragmented nuclei. RESULTS: A radiosensitizing effect of cisplatin on cell survival was observed if time for PLDR was allowed. An increased number of chromosomal fragments were observed immediately after irradiation compared with 24 h after irradiation whereas color junctions are only formed 24 h after irradiation. No increase in chromosomal aberrations was found after combined treatment, but a significantly enhanced number of fragmented nuclei were observed when confluent cultures were replated after allowing PLDR. CONCLUSION: The inhibition of PLDR by cisplatin in delayed plated SW-1573 cells did not increase chromosomal aberrations, but increased the induction of apoptosis.

Effect of hyperthermia on uptake and cytotoxicity of cisplatin in cultured murine mammary carcinoma cells.

The cytotoxicity of cisplatin, applied alone or in combination with hyperthermia, to mouse mammary adenocarcinoma cells (M8013S) was studied with the cells either treated in medium [Eagle's minimum essential medium (MEM), supplemented with 10% foetal bovine serum, 100 IU/ml penicillin, 200 mM glutamine and 0.35 g/l NaHCO(3)] or in Hank's balanced salt solution (HBSS) without serum. To study the role of platinum uptake by the M8013 cells in cytotoxicity, uptake was determined under conditions similar to those used in the survival experiments. Our results show that hyperthermia (30 min at 43 degrees C) enhances the toxicity of cisplatin. Enhanced toxicity by heat treatment is not observed with the cells in HBSS. The thermal enhancement of effects of cisplatin to cells in MEM with serum is clearly related to an enhanced uptake of cisplatin. A novel observation is that in order to obtain a considerable thermal enhancement of the cytotoxic effect of cisplatin, the exposure of the cells to the drug is required not only during the hyperthermic treatment but the exposure has to be maintained for at least 2 h after hyperthermia. These same conditions are also required for enhanced uptake of cisplatin. The present results may indicate that cisplatin has to be bound to some serum component in order to facilitate an 'active' uptake. Hyperthermia leads to a considerable intracellular accumulation of cisplatin, relative to the extracellular concentration. This accumulation takes place during exposure to cisplatin but after heat treatment.

Cellular response of X-ray sensitive hamster mutant cell lines to gemcitabine, cisplatin and 5-fluorouracil.

Five mutant Chinese hamster cell lines deficient in DNA repair with the corresponding parental cell lines were used to determine their sensitivity to cisplatin, 5-fluorouracil and gemcitabine. The mutations in the cell lines led to defective single strand break repair (EM-C11), defective recombination mediated repair (irs1SF), defective double strand break repair (XR-V15B, a Ku-80 mutant and CR-C1, a DNA-PKcs mutant) and an AT-like mutation (VC-4). All mutant cell lines had an impaired doubling time during exponential growth and an increased sensitivity to X-irradiation. We may conclude that for cisplatin-induced cytotoxicity the homologous recombination-associated DNA repair plays an important role in the repair of the cisplatin induced lesions, confirming previous results. In 5-FU and gemcitabine induced toxicity to cells, repair processes involved with radiation-induced damage were not implicated. This is in striking contrast to the role of cisplatin in radiosensitization where inhibition of the NHEJ pathway is implicated, and to the role of gemcitabine in sensitization where specific interference with the HR pathway is implicated.

Repair of potentially lethal damage does not depend on functional TP53 in human glioblastoma cells.

The functionality of G(1)-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 gamma 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 G(1) 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 G(1) arrest.

Differential response to radiation of TP53-inactivated cells by overexpression of dominant-negative mutant TP53 or HPVE6.

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, G(1)-phase arrest, and induction of color junctions. Functional G(1) arrest was demonstrated in parental (RKO) cells with wild-type TP53, while in RC10.1 cells the G(1) arrest was eliminated. In MP53.13 cells an intermediate G(1) 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.