Evaluation of PET and laparoscopy in STagIng advanced gastric cancer: a multicenter prospective study (PLASTIC-study).

BACKGROUND: Initial staging of gastric cancer consists of computed tomography (CT) and gastroscopy. In locally advanced (cT3-4) gastric cancer, fluorodeoxyglucose positron emission tomography with CT (FDG-PET/CT or PET) and staging laparoscopy (SL) may have a role in staging, but evidence is scarce. The aim of this study is to evaluate the impact and cost-effectiveness of PET and SL in addition to initial staging in patients with locally advanced gastric cancer. METHODS: This prospective observational cohort study will include all patients with a surgically resectable, advanced gastric adenocarcinoma (cT3-4b, N0-3, M0), that are scheduled for treatment with curative intent after initial staging with gastroscopy and CT. The modalities to be investigated in this study is the addition of PET and SL. The primary outcome of this study is the proportion of patients in whom the PET or SL lead to a change in treatment strategy. Secondary outcome parameters are: diagnostic performance, morbidity and mortality, quality of life, and cost-effectiveness of these additional diagnostic modalities. The study recently started in August 2017 with a duration of 36 months. At least 239 patients need to be included in this study to demonstrate that the diagnostic modalities are break-even. Based on the annual number of gastrectomies in the participating centers, it is estimated that approximately 543 patients are included in this study. DISCUSSION: In this study, it is hypothesized that performing PET and SL for locally advanced gastric adenocarcinomas results in a change of treatment strategy in 27% of patients and an annual cost-reduction in the Netherlands of €916.438 in this patient group by reducing futile treatment. The results of this study may be applicable to all countries with comparable treatment algorithms and health care systems. TRIAL REGISTRATION: NCT03208621 . This trial was registered prospectively on June 30, 2017.

Systematic review: pharmacotherapy for high-output enterostomies or enteral fistulas.

BACKGROUND: High-output enterocutaneous fistula or enterostomies can cause intestinal failure. There is a wide variety of options in medical management of patients with high output. AIM: To systematically review the literature on available pharmacotherapy to reduce output and to propose an algorithm for standard of care. METHODS: Relevant databases were systematically reviewed to identify studies on pharmacotherapy for reduction in (high-) output enterostomies or fistula. Randomised controlled trials and within subjects controlled prospective trials were included. An algorithm for standard of care was generated based on the outcomes of the systematic review. RESULTS: Two studies on proton pump inhibitors, six on anti-motility agents, three on histamine receptor antagonists, one on an α2- receptor agonist and eight on somatostatin (analogues) were included. One study examined a proton pump inhibitor and a histamine receptor antagonist within the same patients. Overall, we found evidence for the following medical therapies to be effective: omeprazole, loperamide and codeine, ranitidine and cimetidine. On the basis of these outcomes and clinical experience, we proposed an algorithm for standard of care which consists of high-dose proton pump inhibitors combined with high-dose loperamide as the first step followed by addition of codeine in case of insufficient output reduction. So far, there is insufficient evidence for the standard use of somatostatin (analogues). CONCLUSIONS: The available evidence on the efficacy of medication to reduce enterostomy or enterocutaneous fistula output is hampered by low quality studies. We propose an algorithm for standard of care output reduction in these patients.

Intraoperative motive for performing a laparoscopic appendectomy on a postoperative histological proven normal appendix.

BACKGROUND: Diagnostic laparoscopy is the ultimate tool to evaluate the appendix. However, the intraoperative evaluation of the appendix is difficult, as the negative appendectomy rate remains 12%-18%. The aim of this study is to analyze the intraoperative motive for performing a laparoscopic appendectomy of an appendix that was proven to be noninflamed after histological examination. METHODS: In 2008 and 2009, in five hospitals, operation reports of all negative laparoscopic appendectomies were retrospectively analyzed in order to assess the intraoperative motive for removing the appendix. RESULTS: A total of 1,465 appendectomies were analyzed with an overall negative appendectomy rate of 9% (132/1,465). In 57% (841/1,465), a laparoscopic appendectomy was performed, with 9% (n = 75) negative appendectomies. In 51% of the negative appendectomies, the visual assessment of the appendix was decisive in performing the appendectomy. In 33%, the surgeon was in doubt whether the appendix was inflamed or normal. In 4%, the surgeon was aware he removed a healthy appendix, and in 9%, an appendectomy was performed for different reasons. CONCLUSION: In more than half of the microscopic healthy appendices, the surgeon was convinced of the diagnosis appendicitis during surgery. Intraoperative laparoscopic assessment of the appendix can be difficult.

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.

Incidence of acute peripheral neurotoxicity after deep regional hyperthermia of the pelvis.

BACKGROUND: After observing rather severe acute neurotoxicity in a few patients following deep hyperthermia treatment for a pelvic tumour, we evaluated the incidence of neurotoxicity in all patients treated with deep hyperthermia of the pelvis between June 1990 and April 2004. MATERIALS AND METHODS: Hyperthermia treatment registrations and hospital charts of all 736 patients were reviewed. Differences between the incidence of neurotoxicity in subgroups of patients were evaluated by 2 x 2 exact tests. RESULTS: Grade 2 or 3 acute neurotoxicity occurred in 2.3% of patients, grade 3 in 0.7%. The duration of symptoms was longer than 3 months in 6 patients (0.8%). Neurological examination in 5 patients showed that the most commonly involved structures are the sacral and lower lumbar nerve roots and the sacral plexus. Acute neurotoxicity occurred only after November 1999 and only in patients treated for primary cervical cancer. Comparison of applied powers and achieved temperatures in patients developing neurotoxicity did not show differences between treatment sessions which resulted in neurotoxicity and sessions not resulting in neurotoxicity. CONCLUSION: Acute neurotoxicity following hyperthermia for pelvic tumours is a rare complication, but can result in symptoms affecting the activities of daily life. We found no patient, tumour or treatment characteristics predictive for a risk of neurotoxicity.

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.

Enhanced activity of deoxycytidine kinase after pulsed low dose rate and single dose gamma irradiation.

In both pulsed low dose rate (LDR) and single high dose radiation schedules, gemcitabine pretreatment sensitizes tumor cells to radiation. These radiosensitizing effects could be the result of decreased DNA repair. In this study, the effect of irradiation on the deoxycytidine kinase (dCK) needed for DNA repair was investigated. The activity of dCK, a deoxynucleoside analogue-activating enzyme was increased upon irradiation in both schedules. No change in dCK protein expression was observed that indicates a post-translational regulation. The benefit of this increased activity induced by irradiation should be further investigated in combination with deoxynucleoside analogues activated by this enzyme.

Results of streamlined regional ambulance transport and subsequent treatment of acute abdominal aortic aneurysms.

OBJECTIVE: To describe the triage of patients operated for non-ruptured and ruptured abdominal aortic aneurysms (AAAs) before the endovascular era. DESIGN: Retrospective single-centre cohort study. METHODS: All patients treated for an acute AAA between 1998 and 2001 and admitted to our hospital were evaluated in the emergency department for urgent AAA surgery. All time intervals, from the telephone call from the patient to the ambulance department, to the arrival of the patient in the operating theatre, were analysed. Intraoperative, hospital and 1-year survival were determined. RESULTS: 160 patients with an acute AAA were transported to our hospital. Mean (SD) age was 71 (8) years, and 138 (86%) were men. 34 (21%) of these patients had symptomatic, non-ruptured AAA (sAAA) and 126 patients had ruptured AAA (rAAA). All patients with sAAA and 98% of patients with rAAA were operated upon. For the patients with rAAA, median time from telephone call to arrival at the hospital was 43 min (interquartile range 33-53 min) and median time from arrival at the hospital to arrival at the operating room was 25 min (interquartile range 11-50 min). Intraoperative mortality was 0% for sAAA and 11% for rAAA (p = 0.042), and hospital mortality was 12% and 33%, respectively (p = 0.014). CONCLUSIONS: A multidisciplinary unified strategy resulted in a rapid throughput of patients with acute AAA. Rapid transport, diagnosis and surgery resulted in favourable hospital mortality. Despite the fact that nearly all the patients were operated upon, survival was favourable compared with published data.

Time course of enhanced activity of deoxycytidine kinase and thymidine kinase 1 and 2 in cultured human squamous lung carcinoma cells, SW-1573, induced by gamma-irradiation.

Single high dose rate irradiation of 4 Gy in SW-1573 cells, derived from non-small cell lung cancer, led to increased activities of deoxycytidine kinase (dCK) and thymidine kinase 1 and 2 (TK1 and 2). The activity of dCK increased by approximately 30% between 1 and 5 h after irradiation, after which the activity returned to the level of control cells by 8 h after irradiation. TK1 activity also increased by 30-50% between 1 and 6 h after irradiation. The decline to normal levels of dCK concurred with a further increase in the activity of TK1, 8 h after irradiation. TK2 activity was below control levels during the first 4 h after irradiation but rose 3-fold at 8 and 16 h after treatment. The activities of TK1 and TK2 had returned to approximate control levels 24 h after irradiation. The observation that mitochondrial TK2 activity increased to a very high level after irradiation may indicate that the activity of this enzyme is not only important for the damage to mitochondrial DNA, the increased activity may also be instrumental for repair of damage to nuclear DNA. We presume that the increase in activity of TK1 after irradiation is limited to cells in S-phase. Recruitment of cells into S-phase, to replace cells killed by irradiation, is probably too slow to offer an explanation for the enhanced activity of TK1 8 h after irradiation. The increase in activity of both dCK, and TK1 and 2 might be involved in an adaptive response of the cells to radiation by facilitation of DNA repair. The expression of protein kinase C (PKC) decreased during the first 5 h after irradiation. At 5 h after irradiation the level of expression had decreased by >50%. The decrease in PKC expression is concurrent with the increase in dCK activity. This suggests a role of PKC in the signal transduction from DNA damage to the increase in activity of enzymes instrumental in DNA repair.

Endocrine intervention during irradiation does not prevent damage to the thyroid gland.

Radiation to the head-neck region may damage the thyroid gland, leading to hypothyroidism or thyroid carcinoma. Outcomes of radiation protection by lowering plasma thyroid-stimulating hormone (TSH) have thus far been ambiguous. Our aim was to evaluate the radioprotective effect of inhibiting the thyroid gland's activity during x-radiation. For this purpose, of 80 5-week old Wistar rats, 64 received cervical irradiation with 15 Gy (single dose). During irradiation, endocrine intervention was done, using thyroxine (T(4)), T(4) plus iodine, or iodine alone compared to placebo. During the endocrine interventions and follow-up, TSH and T(4) concentrations were measured periodically. Histologic examination of thyroid, pituitary gland, or the hypothalamus and any suspect lymph nodes, lungs, and liver was performed after 6 and 54 weeks. It was found that during the endocrine intervention, plasma levels of TSH were lower in rats given T(4) and higher in rats given iodine. After 6 and 54 weeks, no significant reduction in hypothyroidism or thyroid carcinoma was found between the different groups of rats given any endocrine intervention or no intervention. In conclusion, the administration of T(4), iodine or the combination during x-irradiation does not protect against radiation-induced thyroid damage.

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.

Effects of hyperthermia on the central nervous system: what was learnt from animal studies?

Animal studies show that nervous tissue is sensitive to heat. Although inter-species variations may play a role, the data indicate that the maximum heat dose without obvious complications after localized hyperthermia in regions of the central nervous system (CNS) lies in the range of 40-60 min at 42-42.5 degrees C or 10-30 min at 43 degrees C. Expression of thermotolerance after a 'conditioning' heat dose was clearly observed in the spinal cord of rodents and the thermotolerance ratio's (ratio between heat doses with and without conditioning required to obtain a certain defined effect) were high, approximately 2. The thermotolerant state of CNS is shown to protect also against other types of injury as well: pre-treatment of rats with hyperthermia protected against spinal cord ischemic injury. During the rather long period required for temperature elevation which is inherent to WBH, some degree of thermotolerance may develop. The correlation between thermotolerance and hsp70 induction in CNS is obvious. Heat, at least if applied shortly after X-rays, enhances the response of nervous tissue to radiation. Data on the combined effects of X-ray irradiation and hyperthermia on rodent spinal cord clearly show that the radiation response can be enhanced with a factor of 1.1-1.3. There are no clear experimental data indicating an increase in adverse effects specific to the CNS after localized or whole body hyperthermia as a result of combined treatment with chemotherapy.

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.

The effect of cervical X-irradiation on activity index of thyrocytes and plasma TSH: a pre-clinical model for radiation-induced thyroid damage.

Because radiotherapy in the head and neck region is necessary in the treatment of childhood cancer, possibilities to prevent damage to the thyroid gland must be explored. We developed a model in which radiation-induced effects can be investigated in a way that these effects can be quantified, using thyroid dysmorphology and plasma TSH. Thirty-five Wistar rats, 5 weeks old, were X-irradiated on the cervical region, with a single dose varying from 0 to 20 Gy. After 6 weeks, TSH, T4 and T3 were determined, and thyroid glands were processed for histological examination by two independent pathologists. A histological classification scale was developed, using follicular size, colloid density and cell height of thyrocytes to measure hyperplasia and hypertrophy. By the sum of these scores, a cell-activity index was calculated, which was related to plasma TSH concentration. Numbers of PAS-positive droplets and epithelial desquamation were also counted. Inter-observer reliability was assessed. Good to very good reliability was found for scores of follicular size, colloid density and cell height. Significant increase of cell-activity index was found after 10, 15 and 20 Gy. The plasma TSH concentration was positively correlated to the cell-activity index, increasing with radiation-doses up to 15 Gy. The number of desquamated cells was significantly increased after radiation doses >10 Gy, with moderate reliability. In conclusion, this model using cell-activity index of thyrocytes together with plasma thyrotropin concentrations and desquamation of cells can be used for interpretation and future (pre-clinical) studies of prevention of radiation-induced thyroid damage.

Cellular response to pulsed low-dose rate irradiation in X-ray sensitive hamster mutant cell lines.

The role of DNA repair mechanisms in the cellular response to low dose rate (LDR) irradiation was studied with the aim to gain insight in the process of sublethal damage (SLD) repair. Chinese hamster cell lines mutated in either DNA single strand break (ssb) repair or DNA double strand break (dsb) repair by non homologous end joining (NHEJ) and homologous recombination (HR), or showing an AT-like phenotype, were irradiated in plateau-phase either at high dose rate (HDR, 3.3 Gy/min) or at pulsed low dose rate (p-LDR, average 1 Gy/h). Cell survival after irradiation was assessed using the clonogenic assay. A change in sensitivity when the dose rate was decreased was observed for all parental cell lines and the DNA ssb repair mutant. No difference in cell survival after p-LDR versus. HDR irradiation was observed for the two NHEJ mutants, the AT-like mutant and the HR mutant. Based on these results we conclude that single strand break repair does not play a role in the dose rate effect. The AT like protein, functional NHEJ and XRCC3 are required for the dose rate effect.

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.

Effects of hyperthermia on the peripheral nervous system: a review.

The present paper overviews the current knowledge about effects of hyperthermia at temperatures used in clinical oncology on the peripheral nervous system. From the experimental studies it may be concluded that the heat sensitivity of the nerve is determined by the sensitivity of the nerve vasculature. These studies show that in order to avoid induction of severe neuropathy, application of heat to the peripheral nerves should not be in excess of doses of 30 min at 44 degrees C or equivalent. Using modern equipment for application of loco-regional hyperthermia the incidence of even mild neurological complications is very low. In hyperthermic isolated limb perfusion (HILP) neurotoxicity is an often-mentioned side effect, this is in spite of the fact that in all studies a relatively mild hyperthermic temperature is used that, based on the experimental studies, should be well tolerated by the nerves and other normal tissues in the limbs. It seems that the neurotoxicity observed after HILP results from thermal enhancement of drug toxicity, very probably combined with effects of a high tourniquet pressure that is used to isolate the blood flow in the leg. Whole body hyperthermia (WBH), using anesthesia and appropriate monitoring to avoid cardiovascular stress is at present considered a safe procedure. Still in the recent past cases of neuropathy after treatment have been described. When chemotherapy, and notably cisplatin, is administered before or during hyperthermia there are several clinical and experimental observations that indicate a limited tolerance of the peripheral nervous tissue in such case. Also previous radiotherapy may limit the tolerance of nerves to hyperthermia, notably when radiation is applied with a large field size. Experimental studies show that combined treatment with radiation and heat leads to enhancement of effects of radiation (enhancement ratio approximately 1.5 at 60 min at 44 degrees C). A clear contraindication for the application of hyperthermia in patients is the presence of a neurodegenerative disease, such as multiple sclerosis. Vigilance is also required in the treatment of diabetic patients with hyperthermia, this based on experimental animal studies, but so far no clear clinical data are available.

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.