Carbon ion and proton beam irradiation of a normal human TK6 lymphoblastoid cell line within a magnetic field of 1.0 tesla.

BACKGROUND: Considering the increasing simultaneous application of magnetic resonance imaging (MRI) for more precise photon radiotherapy, it will be likely for particle radiotherapy to adopt MRI for future image guiding. It will then be imperative to evaluate the potential biological effects of a magnetic field (MF) on particle irradiation. This study explores such effects on the highly radiosensitive TK6 lymphoblastoid human cell line. METHODS: The following three parameters were measured after irradiation with either carbon ion or proton beams using spread out Bragg peaks and applying different doses within a perpendicular 1.0 T MF: (1) cell survival fraction (14 days postirradiation), (2) treatment-specific apoptosis, which was determined through the measurement of population in the sub-G1 phase, and (3) cell cycle progression by means of flow cytometry. These were compared to the same parameters measured without an MF. RESULTS: The clonogenic assay in both treatment groups showed almost identical survival curves with overlapping error bars. The calculated α values with and without an MF were 2.18 (σ=0.245) and 2.17 (σ=0.234) for carbon ions and 1.08 (σ=0.138) and 1.13 (σ=0.0679) for protons, respectively. Similarly, the treatment-specific apoptosis and cell cycle progression showed almost identical curves with overlapping error bars. A two-sample, unpooled t-test analysis was implemented for comparison of all mean values and showed p-values >0.05. CONCLUSION: No statistically significant difference in biological response of the TK6 cells was observed when they were irradiated using spreadout Bragg peaks within a perpendicular 1.0 T MF as compared to those, which received the same dose without the MF. This should serve as another supporting piece of evidence toward the implementation of MRI in particle radiotherapy, though further research is necessary.

In vitro sensitivity of malignant melanoma cells lines to photon and heavy ion radiation.

BACKGROUND: The role of radiotherapy in malignant melanoma is still in discussion due to its relative resistance to radiation. In various literature, heavy ions show a higher relative biological effectiveness than photons. The aim of this work is to evaluate the radiotherapeutical effect from photons as well as heavy ions on malignant melanoma cells and to indicate the possible radiosensitivity based on its proliferation-inhibitory effect. METHODS: Two different cell lines of malignant melanoma, WM115 (primary tumor) and WM266-4 (metastatic site, skin) were used in this in vitro study. The cells were treated with photons or heavy ions (C12 and O16 ions). Cell-proliferation assay using hemocytometer was used for the quantitative and qualitative evaluation of cell growth. Furthermore, flow cytometry was also used to analyze the cell cycle distribution. RESULTS: Heavy ions compared to photons and between the two heavy ion modalities, O16 ions showed an improved suppression of cell growth in both cell lines. Furthermore, a G2/M arrest was detected in both cell lines after all radiotherapy modalities - with the arrest increasing with the dose applied. CONCLUSION: Heavy ions showed a greater inhibitory effect on cell proliferation compared to photons and an increased G2/M arrest. Therefore, C12 and O16 heavy ions might overcome the relative radioresistance of malignant melanoma to photons. Further research is warranted.

The influence of a magnetic field on photon beam radiotherapy in a normal human TK6 lymphoblastoid cell line.

BACKGROUND: The implementation of magnetic resonance imaging (MRI) guided radiotherapy (RT) continues to increase. Very limited in-vitro data on the interaction of ionizing radiation and magnetic fields (MF) have been published. In these experiments we focused on the radiation response in a MF of the TK6 human lymphoblastoid cells which are known to be highly radiosensitive due to efficient radiation-induced apoptosis. METHODS: Clonogenicity was determined 12-14 days after irradiation with 1-4 Gy 6 MV photons with or without a 1.0 Tesla MF. Furthermore, alterations in cell cycle distribution and rates of radiation induced apoptosis (FACS analysis of cells with sub-G1 DNA content) were analyzed. RESULTS: Clonogenic survival showed an exponential dose-dependence, and the radiation sensitivity parameter (α = 1.57/Gy) was in accordance with earlier reports. Upon comparing the clonogenic survival between the two groups, identical results within error bars were obtained. The survival fractions at 2 Gy were 9% (without MF) and 8.5% (with MF), respectively. CONCLUSION: A 1.0 Tesla MF does not affect the clonogenicity of TK6 cells irradiated with 1-4 Gy 6MV photons. This supports the use of MRI guided RT, however ongoing research on the interaction of MF and radiotherapy is warranted.

Dichloroacetate induces tumor-specific radiosensitivity in vitro but attenuates radiation-induced tumor growth delay in vivo.

BACKGROUND: Inhibition of pyruvate dehydrogenase kinase (PDK) by dichloroacetate (DCA) can shift tumor cell metabolism from anaerobic glycolysis to glucose oxidation, with activation of mitochondrial activity and chemotherapy-dependent apoptosis. In radiotherapy, DCA could thus potentially enhance the frequently moderate apoptotic response of cancer cells that results from their mitochondrial dysfunction. The aim of this study was to investigate tumor-specific radiosensitization by DCA in vitro and in a human tumor xenograft mouse model in vivo. MATERIALS AND METHODS: The interaction of DCA with photon beam radiation was investigated in the human tumor cell lines WIDR (colorectal) and LN18 (glioma), as well as in the human normal tissue cell lines HUVEC (endothelial), MRC5 (lung fibroblasts) and TK6 (lymphoblastoid). Apoptosis induction in vitro was assessed by DAPI staining and sub-G1 flow cytometry; cell survival was quantified by clonogenic assay. The effect of DCA in vivo was investigated in WIDR xenograft tumors growing subcutaneously on BALB/c-nu/nu mice, with and without fractionated irradiation. Histological examination included TUNEL and Ki67 staining for apoptosis and proliferation, respectively, as well as pinomidazole labeling for hypoxia. RESULTS: DCA treatment led to decreased clonogenic survival and increased specific apoptosis rates in tumor cell lines (LN18, WIDR) but not in normal tissue cells (HUVEC, MRC5, TK6). However, this significant tumor-specific radiosensitization by DCA in vitro was not reflected by the situation in vivo: The growth suppression of WIDR xenograft tumors after irradiation was reduced upon additional DCA treatment (reflected by Ki67 expression levels), although early tumor cell apoptosis rates were significantly increased by DCA. This apparently paradoxical effect was accompanied by a marked DCA-dependent induction of hypoxia in tumor-tissue. CONCLUSION: DCA induced tumor-specific radiosensitization in vitro but not in vivo. DCA also induced development of hypoxia in tumor tissue in vivo. Further investigations relating to the interplay between tumor cell metabolism and tumor microenvironment are necessary to explain the limited success of metabolic targeting in radiotherapy.

Suberoylanilide hydroxamic acid affects γH2AX expression in osteosarcoma, atypical teratoid rhabdoid tumor and normal tissue cell lines after irradiation.

PURPOSE: Osteosarcoma and atypical teratoid rhabdoid tumors are tumor entities with varying response to common standard therapy protocols. Histone acetylation affects chromatin structure and gene expression which are considered to influence radiation sensitivity. The aim of this study was to investigate the effect of the combination therapy with the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) and irradiation on atypical teratoid rhabdoid tumors and osteosarcoma compared to normal tissue cell lines. METHODS: Clonogenic assay was used to determine cell survival. DNA double-strand breaks (DSB) were examined by pulsed-field electrophoresis (PFGE) as well as by γH2AX immunostaining involving flow cytometry, fluorescence microscopy, and immunoblot analysis. RESULTS: SAHA lead to an increased radiosensitivity in tumor but not in normal tissue cell lines. γH2AX expression as an indicator for DSB was significantly increased when SAHA was applied 24 h before irradiation to the sarcoma cell cultures. In contrast, γH2AX expression in the normal tissue cell lines was significantly reduced when irradiation was combined with SAHA. Analysis of initial DNA fragmentation and fragment rejoining by PFGE, however, did not reveal differences in response to the SAHA pretreatment for either cell type. CONCLUSION: SAHA increases radiosensitivity in tumor but not normal tissue cell lines. The increased H2AX phosphorylation status of the SAHA-treated tumor cells post irradiation likely reflects its delayed dephosphorylation within the DNA damage signal decay rather than chromatin acetylation-dependent differences in the overall efficacy of DSB induction and rejoining. The results support the hypothesis that combining SAHA with irradiation may provide a promising strategy in the treatment of solid tumors.

Comparison of laparoscopic and open gastrectomy for malignant disease.

UNLABELLED: This study compares the outcome of a series of totally laparoscopic cases with that of matched open controls for the treatment of malignant gastric disease. Laparoscopic techniques can follow oncologic principles and obtain adequate margins. Short-term follow-up evaluation shows no difference in survival rates between the two approaches. BACKGROUND: Few studies have examined a totally laparoscopic approach to gastrectomy for malignancy. This is the first study to compare the outcome of a series of totally laparoscopic cases with matched open surgeries for gastric cancer. METHODS: A retrospective case-matched study was performed comparing open and laparoscopic partial gastrectomies for cancer. A total of 25 cases (12 laparoscopic and 13 open) were matched for age and indication for surgery. Stage, extent of lymphadenectomy, and survival at 18 months were examined. The intraoperative and postoperative details were compared. RESULTS: The stages ranged from I to IV, with no statistical difference between the two groups. All resected margins in the laparoscopic group were free of tumor. The extent of lymphadenectomy did not differ. Follow-up assessment at 18 months showed no difference in survival. CONCLUSIONS: Laparoscopic gastrectomy for malignancy is a viable alternative to open surgery. Laparoscopic techniques can obtain adequate margins and follow oncologic principles. Short-term follow-up evaluation shows no difference in survival rates between the two approaches.

Syndrome of inappropriate secretion of antidiuretic hormone following laparoscopic inguinal hernia repair.

Syndrome of inappropriate secretion of antidiuretic hormone (SIADH) after cardiac surgery and traditional open abdominal surgery has been reported. This disorder also has been associated with minor operative procedures with the patient under local anesthesia. However, SIADH after laparoscopic surgery is not well documented in the literature. We report a case of SIADH after laparoscopic inguinal hernia repair in an elderly woman.

Dose-dependent differential effects of low and pulsed dose-rate brachytherapy in a radioresistant syngenic rat prostate tumour model.

PURPOSE: To study the response of the Dunning prostate carcinoma (R3327-AT1 subline) to continuous low dose-rate (CLDR) and pulsed dose-rate (PDR) brachytherapy. MATERIALS AND METHODS: After subcutaneous tumour transplantation into the thigh of the Copenhagen rat, doses of 0, 20, 30, 40 and 50 Gy were applied to the tumour surface (tumour diameter 9+/-1mm). Eight animals were irradiated per dose group and exposure condition. Interstitial PDR ((192)Ir source, 37 GBq) and CLDR ((192)Ir seed, 150 MBq) brachytherapy were carried out with 0.75 Gy/pulse h(-1) and a dose-rate of 0.75Gyh(-1), respectively. Treatment response was assessed in terms of growth delay expressed as the time (T(5)) required for each tumour to reach five times the initial tumour volume. RESULTS: The median T(5) times for the CLDR groups (in the order: control, 20, 30, 40, 50 Gy) were 12 (12), 54.5 (21), 64.5 (31), 85.5 (51), and 65 (47.5) days. Values after PDR brachytherapy are given in parentheses and resulted in a significantly impaired tumour growth delay (log-rank test) in the 20Gy (p =0.006) and 30 Gy (p =0.036) groups. No significant difference was found in the 40-50 Gy dose range. CONCLUSIONS: In contrast to previous results and predictions of biological models we observed dose-dependent differential effects of PDR and CLDR brachytherapy with reduced efficacy of PDR in the lower dose range.

p53, apoptosis and radiosensitivity--experimental and clinical data.

The tumor suppressor gene p53 is the most frequently mutated gene found in human tumors. The p53 protein is a central regulator of the cell cycle and of the induction of apoptosis after genotoxic stress, e.g. due to radiation or drugs. p53 functions mainly through transcriptional control triggered by DNA damage and influences multiple response pathways. Because of this important role in the response of cells to radiation, the interrelation between p53 status and radiosensitivity has found wide interest. Experimental and clinical data are discussed to explain the mechanism by which p53 influences the cellular response to radiation and to demonstrate the clinical relevance.

Laparoscopic vs open gastrectomy. A retrospective review.

BACKGROUND: The totally laparoscopic approach to partial gastrectomy had not been compared previously with results of the open technique. This study compares the results of a series of laparoscopic cases with matched open cases. METHODS: A retrospective case-matched study was performed in 36 patients (18 laparoscopic surgeries, 18 open surgeries). Each laparoscopic case was matched for patient age and indication for surgery. The intraoperative and postoperative details of the two groups were compared. RESULTS: Laparoscopic surgery resulted in less blood loss, although operative time was increased. Nasogastric tubes were less likely to be used after laparoscopic surgery, and patients in the laparoscopic group had an earlier return to normal bowel function than those in the open group. Length of hospital stay was 2 days shorter in the laparoscopic group. CONCLUSIONS: The totally laparoscopic approach to partial gastrectomy is an excellent alternative to the more traditional open approach. It results in a more rapid return of intestinal function and a shorter hospital stay.

[The search for therapeutic gain in radiation oncology]. / Auf der Suche nach der Therapeutischen Breite in der Radioonkologie.

The Search for Therapeutic Gain in Radiation Oncology Novel strategies in radiation oncology aim at increasing the therapeutic gain, i.e., to decrease side effects while maintaining cure rates, or to increase cure rates at the same level of complications. Over the years, physical and biological strategies have been developed to achieve this goal. The physical development led to the possibility of precise, computer-controlled beam application by using modern imaging techniques and three-dimensional treatment planning. Improved patient immobilization methods allow minimal safety distances, resulting in steep dose gradients when used together with isocentric multi-field techniques. These predominantly stereotactic irradiation techniques yield therapeutic gain towards the tumor surrounding normal tissue. A critical issue that determines the tolerance of radiation therapy are structures at risk within the target volume. Fractionation is a reliable method to exploit the differential potential for recovery of radiation-induced DNA damage in normal tissues. Radiogenetic strategies aim at the sensibilization of tumor cells by targeting specific characteristics like mutations of p53. The reverse idea, gene-therapeutic radioprotection of normal tissue, is under investigation.

Induction of telomerase activity by irradiation in human lymphoblasts.

Neuhof, D., Ruess, A., Wenz, F. and Weber, K. J. Induction of Telomerase Activity by Irradiation in Human Lymphoblasts. Radiat. Res. 155, 693-697 (2001). Telomerase activity is a radiation-inducible function, which suggests a role of this enzyme in DNA damage processing. Since the tumor suppressor TP53 plays a central role in the regulation of the cellular response to DNA damage, our study explored the ability of ionizing radiation to change telomerase activity and telomere length in two closely related human lymphoblast cell lines with different TP53 status. TK6 cells (wild-type TP53) and WTK1 cells (mutated TP53) were exposed to different doses of X rays, and telomerase activity was measured by PCR ELISA at different times after irradiation. A dose-dependent increase in telomerase activity was observed. One hour after irradiation with 4 Gy, TK6 and WTK1 cells showed an approximately 2.5-fold increase; for lower doses (0.1 to 1 Gy), telomerase induction was seen only in TK6 cells. Telomerase induction was observed by 0.5 h after irradiation, with a further increase up to 24 h. Irradiated TK6 and WTK1 cells had longer telomeres (+1.3 kb) than unirradiated cells 14 days after exposure. Our data demonstrate a dose-dependent induction of telomerase activity and lengthening of telomeres by ionizing radiation in human lymphoblasts. Induction of telomerase activity by radiation does not generally appear to be controlled by the TP53-dependent DNA damage response pathway. However, for low doses, induction of telomerase requires wild-type TP53.

Overexpression of the DNA-binding domain of poly(ADP-ribose) polymerase inhibits rejoining of ionizing radiation-induced DNA double-strand breaks.

PURPOSE: To assess the influence of trans-dominant inhibition of poly(ADP-ribosyl)ation on the rejoining kinetics of radiation-induced DNA double-strand breaks (DSB). MATERIALS AND METHODS: Stable transfectants of the SV40-transformed hamster cell line CO60 were used: COM3 cells contain a construct to overexpress the poly(ADP-ribose) polymerase (PARP-1) DNA-binding domain (DBD) when induced by dexamethasone, as well as a construct for the constitutive overexpression of the human glucocorticoid receptor (Hg0). COR3 are control cells containing only the Hg0 plasmid. DSB induction and rejoining in X-irradiated cells was assessed by DNA pulsed-field electrophoresis. RESULTS: DSB induction was identical in both cell lines and independent of the presence of dexamethasone. DSB rejoining kinetics was independent of dexamethasone in COR3 cells and identical to COM3 cells without dexamethasone. However, in COM3 cells treated with dexamethasone to induce PARP-1 DBD overexpression, the fast component of the rejoining kinetic was largely reduced, and residual fragmentation increased concomitant with the increased damage fraction in slow rejoining. CONCLUSIONS: The results indicate that inhibition of cellular PARP-1 does not affect the rate-limiting step of either fast or slow DSB rejoining. Rather, it appears that absence of poly(ADP-ribosyl)ation due to dominant negative PARP-1 expression induces a shift from rapid to slow DSB rejoining and by this mechanism PARP inhibition may increase the risk of repair failures.

Measurement of hypoxia using the comet assay correlates with preirradiation microelectrode pO2 histography in R3327-AT rodent tumors.

Polarographic determination of tumor oxygenation by Eppendorf histography is currently under investigation as a possible predictor of radiotherapy outcome. Alternatively, the alkaline comet assay has been proposed as a radiobiological approach for the detection of hypoxia in clinical tumor samples. Direct comparisons of these methods are scarce. One earlier study with different murine tumors could not establish a correlation, whereas a weak correlation was reported for a variety of human tumors. Considering the different end points and spatial resolution of the two methods, a direct comparison for a single tumor entity appeared desirable. Anaplastic R3327-AT Dunning prostate tumors were grown on Copenhagen rats to volumes of 1-6 cm(3). Eppendorf histography (100-200 readings in 5 parallel tracks) for 8 different tumors revealed various degrees of oxygenation, with median pO(2) values ranging from 1.1 to 23 mmHg. Within 5 min after an acute exposure to 8 Gy (60)Co gamma rays, tumors were excised from killed animals and rapidly cooled to limit repair, and a single cell suspension was prepared for use with the comet assay. The resulting comet moment distributions did not exhibit two subpopulations (one hypoxic and the other aerobic), and a hypoxic fraction could not be calculated. Instead, the average comet moment distribution was taken as a parameter of overall strand break induction. Corresponding experiments with tumor cells grown in vitro allowed us to derive the relationship between the oxygen enhancement ratio (OER) for the average comet moment and oxygen partial pressure (Howard-Flanders and Alper formula). The validity of this relationship was inferred for cells exposed in situ, and the convolution of a pO(2) distribution with the formula of Howard-Flanders and Alper yielded an array of expected OER values for each tumor. The average expected OER correlated well with the average comet moment (r = 0.89, P < 0.01), and the in situ comet moment distributions could be predicted from the Eppendorf data when 50% repair was taken into account, assuming a 5-min damage half-life. The findings confirm the potential of interstitial polarography to reflect radiobiologically relevant intracellular oxygenation, but also underscore the confounding influence of differences in repair that may occur when cells are prepared from irradiated tissues for use with the comet assay.

Radiochemotherapy with paclitaxel: synchronization effects and the role of p53.

PURPOSE: We have studied the interaction of paclitaxel (Taxol) and radiation in V79 cells and human lymphoblasts with special emphasis on cell cycle effects and the role of p53. MATERIAL AND METHODS: V79 cells in log- and plateau-phase and human lymphoblasts (p53wt TK6 and p53mut WTK1) were used. Paclitaxel was given for 2 hours. Survival was determined using clonogenic assays. Cell cycle analysis was done using DNA flow cytometry. RESULTS: In V79 cells there was a dose dependent delay of colony formation after paclitaxel. The LD50 was about 0.4 microM with a 2-hour exposure. In exponentially growing cells, there was an accumulation of 40% of cells in G2/M 6 hours after paclitaxel. The dose modification factor was about 3.9 when radiation was given 6 hours after 0.3 microM paclitaxel for 2 hours. Synchronization experiments using serum starvation and induction showed that synchronization was not sufficient to induce a comparable dose modification factor. Human lymphoblasts with mutated p53 (WTK1, LD50 = 75 microM) were more resistant to paclitaxel than wild type p53 cells (TK6, LD50 = 25 microM). CONCLUSION: The radiosensitization induced by paclitaxel was critically dependent on the timing of irradiation and chemotherapy, although synchronization alone was not sufficient to explain the dose modification. Lymphoblasts with mutated p53 were less sensitive than wild type p53 cells.

Radiation induced chromosome aberrations and clonogenic survival in human lymphoblastoid cell lines with different p53 status.

PURPOSE: To better understand the relation of radiation induced chromosome aberrations and clonogenic survival in cells with different p53 status. MATERIALS AND METHODS: The human lymphoblasts TK6 and WTK1 were derived from the same donor, but differ in radiosensitivity, p53 status and kinetics of apoptosis. TK6 cells have wild type p53 (p53wt), whereas WTK1 cells have a mutated, non-functional p53 (p53mut). Additionally, a HPV16 E6 transfected TK6 cell line (TK6E6), which is also negative for p53 function (p53neg), was studied. The cells were irradiated, incubated with colcemid, hypotonically lysed and fixed. After staining with Giemsa, asymmetric chromosomal exchange type aberrations were counted in 50 mitoses each per dose point (0 to 4 Gy). Clonogenic survival was determined using the microtiter plate assay. All experiments were performed in triplicate. RESULTS: WTK1 (p53mut) show a higher spontaneous frequency of chromosome aberrations than TK6 (p53wt). No significant differences were noted in radiation induced aberration frequency. TK6E6 (p53neg) show comparable aberration frequencies like TK6. However, the dose required to reduce survival to 10% (D10) was about 2 Gy for TK6 and TK6E6, whereas the D10 for WTK1 was approximately 3 Gy. CONCLUSION: The p53 status influences the radiosensitivity in this lymphoblast cell system showing a high rate of radiation induced apoptosis. Cells with p53mut (WTK1), survive with a damaged genome, because they do not undergo apoptosis to loose their clonogenicity. There was no difference between the p53wt (TK6) and p53neg cells (TK6E6) suggesting a suppression of radiation induced apoptosis by p53mut.

Acute and late toxicity, tumour control and intrinsic radiosensitivity of primary fibroblasts in vitro of patients with advanced head and neck cancer after concomitant boost radiochemotherapy.

BACKGROUND AND PURPOSE: The existence of hereditary factors influencing the cellular response to ionising radiation has led to the hypothesis that the inter-patient variability of clinical radiation reactions may, at least in part, be attributable to an individual, or intrinsic, radiosensitivity. Considerable effort has been spent in the development of test systems that would determine individual radiosensitivity before or early during radiotherapy to possibly predict treatment outcome, but the results are still conflicting. The present explorative study was therefore aimed at the detection of associations between acute and late radiation effects, tumour control and in vitro radiosensitivity of primary normal tissue fibroblasts. PATIENTS AND METHODS: Sixty-eight patients with squamous cell carcinoma of the head and neck (93% UICC stage IV) were treated with a simultaneous concomitant boost radiochemotherapy with Carboplatin as part of a prospective non-randomised multicenter study at the University of Heidelberg. Primary fibroblasts were obtained from skin biopsies prior to treatment from 25 unselected patients of this study and the SF2 was determined using the colony forming assay and high dose-rate irradiation. The median follow-up was 21 months (range 2.5-81 months). RESULTS: The locoregional control rate at three years was 32%. No significant association between acute (mucosa reaction grade 1 or 2 vs. grade 3 and 4), late radiation effects (subcutaneous fibrosis, osteonecrosis, larynx oedema), locoregional tumour control and SF2 of primary fibroblasts was found using Cox proportional hazards regression analysis, log-rank test and Mann-Whitney U-test. Although a steep dose-response relationship was observed for the radiation-induced severe larynx oedema, Cox proportional hazards regression analysis could not fully explain the occurrence of severe radiation-induced larynx oedema with the dose to the larynx (P = 0.09). In the subgroup of twenty-five patients, where the SF2 was determined, bivariate analysis revealed about the same non-significant influence of the dose to the larynx on the larynx oedema (P = 0.1) and no influence of the SF2 (P = 0.5). CONCLUSIONS: In our study of patients with advanced cancer of the head and neck, neither the normal fibroblast SF2 nor the severity of acute radiation effects were able to predict late radiation effects or locoregional tumour control.

X-ray induced changes in immunostaining of proliferating cell nuclear antigen (PCNA) in V79 hamster fibroblasts.

BACKGROUND: Proliferating cell nuclear antigen (PCNA) ist a 36 kD protein that is involved in DNA-replication and -repair. For V79 hamster cells, a mutated p53 and a so-called "adaptive response", an improved radiation tolerance after pre-irradiation with low X-ray doses hours before definitive irradiation with higher doses have been reported. To better understand the role of PCNA after photon irradiation in vivo, using flow cytometry, we studied the immunochemical PCNA-staining in V79 cells after irradiation with 6-MeV photons with and without serum depletion and with and without low-dose pre-irradiation under different growth conditions. MATERIAL AND METHODS: Using V79 hamster cells, BrdUrd incorporation, total and DNA-bound PCNA were measured for exponential cells and for confluent cells at different times (up to 14 days) after reaching confluence. Cells were either grown with medium containing 10% fetal calf serum (FCS) or 0.5% FCS. Six days after reaching confluence, cells were irradiated with 1 Gy (and 8 Gy for non-serum-depleted cells) (6-MV photons, 2 Gy/min). Then, immunochemical PCNA-staining was measured by flow cytometry at 0, 30, 60 and 120 min after irradiation. For studying the adaptive response, exponentially growing cells and cells that were 6 days in confluence were pretreated with 0.01 Gy, reincubated for 5 h and then definitively treated with 1 Gy and harvested and processed as described above. RESULTS: Four days after reaching confluence, DNA-bound PCNA and BrdUrd content were reduced to a minimum of < 15% positive cells while total PCNA remained essentially unchanged. After irradiation with 1 Gy 6 days after reaching confluence, cells grown with 10% FCS showed a moderate but distinct transient increase in DNA-bound PCNA at 30 min after irradiation. After irradiation with 8 Gy, there was no clear increase at 30 min but a more distinct decrease at 60 min, implying that the increase might occur earlier in the time course at higher doses. Total cellular PCNA and BrdUrd uptake were constant during the first 2 hours after irradiation. In cells that were kept with serum depleted medium for 6 days after reaching confluence, total PCNA was reduced and no changes in either DNA-bound PCNA or BrdUrd-uptake were observed after irradiation. When cells were primed with a dose of 0.01 Gy 5 h before subsequent treatment with 1 Gy, neither for exponentially growing cells nor for those in confluence a significant difference in the detected amount of PCNA (total and DNA-bound) or BrdUrd was observed when compared to cells treated without a priming dose. CONCLUSIONS: The moderate X-ray induced DNA association of PCNA is indicative for ongoing DNA repair but appears to require serum stimuli. However, this p53-independent pathway involving PCNA does not seem to be the most relevant for survival in these rodent cells that tolerate much residual damage. Furthermore, no adaptive response for DNA-association of PCNA could be detected in V79 cells.

[In vitro studies of PDR brachytherapy]. / In-vitro-Untersuchungen zur PDR-Brachytherapie.

BACKGROUND: Calculations on the basis of the LQ-model have been focussed on the possible radiobiological equivalence between common continuous low dose rate irradiation (CLDR) and a superfractionated irradiation (PDR = pulsed dose rate) provided that the same total dose will be prescribed in the same overall time as with the low doserate. A clinically usable fractionation scheme for brachytherapy was recommended by Brenner and Hall and should replace the classical CLDR brachytherapy with line sources with an afterloading technique using a stepping source. The hypothes is that LDR equivalency can be achieved by superfractionation was tested by means of in vitro experiments on V79 cells in monolayer and spheroid cultures as well as on HeLa monolayers. MATERIALS AND METHODS: Simulating the clinical situation in PDR brachytherapy, fractionation experiments were carried out in the dose rate gradient of afterloading sources. Different dose levels were produced with the same number of fractions in the same overall incubation time. The fractionation schedules which were to be compared with a CLDR reference curve were: 40 x 0.47 Gy, 20 x 0.94 Gy, 10 x 1.88 Gy, 5 x 3.76 Gy, 2 x 9.4 Gy given in a period of 20 h and 1 x 18.8 Gy as a "single dose" exposition. As measured by flow cytometry, the influence of the dose rate in the pulse on cell survival and on cell cycle distribution under superfractionation was examined on V79 cells. RESULTS: V79 spheroids as a model for a slowly growing tumor, reacted according to the radiobiological calculations, as a CLDR equivalency was achieved with increasing fractionation. Rapidly growing V79 monolayer cells showed an inverse fractionation effect. A superfractionated irradiation with pulses of 0.94 Gy/h respectively 0.47 Gy/0.5 h was significantly more effective than the CLDR irradiation. This inverse fractionation effect in log-phase V79 cells could be attributed to the accumulation of cycling cells in the radiosensitive G2/M phase (G2 block) during protected exposure which was drastically more pronounced for the pulsed scheme. HeLa cells were rather insensitive to changes of fractionation. Superfractionation as well as hypofractionation yielded CLDR equivalent survival curves. CONCLUSIONS: The fractionation scheme, derived from the PDR theory to achieve CLDR equivalent effects, is valid for many cell lines, however not for all. Proliferation and dose rate dependend cell cycle effects modify predictions derived from the sublethal damage recovery model and can influence acute irradiation effects significantly. Dose rate sensitivity and rapid proliferation favour cell cycle effects and substantiate, applied to the clinical situation, the possibility of a higher effectiveness of the pulsed irradiation on rapidly growing tumors.

Radiosensitizing potential of gemcitabine (2',2'-difluoro-2'-deoxycytidine) within the cell cycle in vitro.

PURPOSE: Gemcitabine (2',2'-difluorodeoxycytidine; dFdCyd) is a new deoxycitidine analog which exhibits substantial activity against solid tumors and radiosensitizing properties in vitro. To examine cell cycle-specific effects of a combined treatment with gemcitabine and radiation, the in vitro clonogenic survival of two different cell lines was measured for cells from log-phase culture, G1 and S-phase cells. METHODS AND MATERIALS: Chinese hamster (V79) and human colon carcinoma (Widr) cells were exposed to different radiation doses and for different points of time relative to gemcitabine treatment (2 h). Experiments were also carried out with different cell-cycle populations obtained after mitotic selection (V79) or after serum stimulation of plateau-phase cells (Widr). The resulting survival curves were analyzed according to the LQ model, and mean inactivation doses (MID) and the cell cycle-specific enhancement ratios (ER) were calculated from the survival curve parameters. RESULTS: Effectiveness of combined treatment of log-phase cells was greatest when cells were irradiated at the end of the gemcitabine exposure [ER: 1.28 (V79), 1.24 (Widr)]. For later times after the removal of the drug, radiosensitization declined, approaching independent toxicity. From the time course of interactive-type damage decay half-life values of 75 min (V79) and 92 min (Widr) were derived. Gemcitabine did not radiosensitize G1 Widr cells or V79 cells from the G1/S border, but substantial radiosensitization was observed for the S-phase cell preparations [ER: 1.45 (V79-lateS), 1.57 (Widr)]. CONCLUSIONS: Treatment of cells with gemcitabine immediately before irradiation eliminates, or at least greatly reduces, the variation in radiosensitivity during the cell cycle that is manifested by radioresistance during S phase. This reversal of S-phase radioresistance could imply that gemcitabine interferes with the potentially lethal damage repair/fixation pathway. Other approaches have been taken to overcome S-phase radioresistance, such as hyperthermia or densely ionizing radiation, and combined treatments with dFdCyd could prove of value to complement such efforts.