Tumour-derived plasma cell-free DNA in patients with head and neck cancer: A short review.

Head and neck squamous cell carcinoma is one of the leading causes of cancer mortality worldwide. The prognosis for patients with head and neck squamous cell carcinoma has not substantially improved during the last decades, despite numerous advancements in treatment options. Reliable markers for early tumour detection and treatment response, which complement clinical examinations, imaging techniques, and biopsies would be extremely useful. One fairly new and promising method is the analysis of tumour-derived cell-free DNA (ctDNA) in the plasma of cancer patients. First data indicate that this method may assist, in the future, in the early detection of head and neck squamous cell carcinoma, the real-time monitoring of the disease course, the therapy response, and the prediction of prognosis with direct therapeutic implications by determining the best therapeutic modality for patient care.

Testicular radiation dose after multimodal curative therapy for locally advanced rectal cancer. Influence on hormone levels, quality of life, and sexual functioning.

PURPOSE: The purpose of the current work was to prospectively measure the influence of testicular radiation dose on hormone levels, quality of life (QoL), and sexual functioning following multimodal therapy (neoadjuvant radiochemotherapy, surgery, and adjuvant chemotherapy) for rectal cancer. PATIENTS AND METHODS: From November 2007 to November 2009, 83 male patients were treated at the University of Goettingen with radiochemotherapy (RCT) for locally advanced rectal cancer [total dose 50.4 Gy, concomitant chemotherapy with two cycles of 5-fluorouracil (FU) or 5-FU and oxaliplatin]. Testicular radiation doses were analyzed and correlated with hormone levels [luteinizing hormone (LH), follicle stimulating hormone (FSH), total testosterone and free androgen index (FAI) serum levels], QoL, and sexual functioning, which were determined before and up to 1 year after RCT. RESULTS: Mean dose at the testes was 3.9 Gy (range 0.28-11.98 Gy). It was higher for tumors located < 6 cm from the anocutaneous line (p < 0.05). One year after therapy, testosterone, the testosterone/LH ratio, and the FAI/LH ratio were significantly decreased (3.5-3.0 µg/l, 0.9-0.4, 7.9-4.5, respectively) while LH and FSH (4.2-8.5 IU/l, 6.0-21.9 IU/l) were increased. QoL and sexual functioning were significantly impaired. However, there was no statistical correlation between testicular radiation dose and changes in hormone levels, QoL, or sexual functioning. CONCLUSION: Multimodal treatment for rectal cancer including RCT leads to hormone level changes and to impaired QoL and sexual functioning. However, because there was no apparent correlation between the analyzed parameters, QoL is probably also influenced by other factors, e.g., psychosocial aspects.

Irradiation leads to apoptosis of Kupffer cells by a Hsp27-dependant pathway followed by release of TNF-alpha.

In a previous publication, we were able to show that irradiation of Kupffer cells, the liver resident macrophages, leads to an increased TNF-alpha concentration in the culture medium. The pathomechanisms underlying this phenomenon, however, remained to be elucidated. Here, we show that following irradiation of Kupffer cells, the apoptosis rate increased drastically within 48 h. At the same time, the total TNF-alpha concentration in cell lysates of Kupffer cells attached to the culture plate decreased. However, normalization of the TNF-alpha concentration with respect to cell number revealed that TNF-alpha concentration per attached cell remained constant during the observation period. Western blot analysis showed that heat shock protein 27 (Hsp27) is strongly downregulated and bax is upregulated in irradiated Kupffer cells as compared to sham-irradiated cells. Overexpression of Hsp27 in Kupffer cells was shown to prevent the effect of irradiation on bax expression, apoptosis and, at the same time, on increase of TNF-alpha concentration in the Kupffer cell medium. We conclude that irradiation of Kupffer cells leads to apoptosis because of downregulation of Hsp27 and consecutive upregulation of bax expression. Furthermore, we suggest that apoptosis of Kupffer cells leads to an increase of TNF-alpha concentration in the culture medium which may be due to cell death rather than active release or synthesis.

Combining radiation with oxaliplatin: a review of experimental results.

Oxaliplatin is integrated in treatment strategies against a variety of cancers including chemoradiation protocols against gastrointestinal, especially rectal cancers. Solid biological data with respect to radiosensitizing activity of oxaliplatin are still rare. This review is based on in vitro and experimental in vivo data concerning the combination of oxaliplatin and radiation published until July 2007. Taking either cell viability or clonogenic survival as an endpoint all reported on oxaliplatin-induced radiosensitization, and enhancement ratios ranged from 1.1 to 2.2. In vivo, enhanced tumor growth delay after combined oxaliplatin and radiation treatment was also reported. Therefore, oxaliplatin should be considered a potent radiosensitizer, although the mechanisms causing radiosensitizing properties of oxaliplatin have not been studied in detail. Herein, they are discussed with respect to apoptosis induction, p53-related signalling, cell cycle control, and DNA-repair.

Induction of reproductive cell death and chromosome aberrations in radioresistant tumour cells by carbon ions.

PURPOSE: To study the induction of reproductive cell death and chromosome aberrations in radioresistant tumour cells exposed to carbon ions in vitro. MATERIALS AND METHODS: X-ray-resistant colon carcinoma cells (WiDr) were used. Confluent G0/G1 cells were irradiated in vitro with graded doses of 100/200/400 MeV u(-1) carbon ions and carbon ions from the middle of a 1 cm extended Bragg peak, and 200 kV X-rays for comparison. Cells were harvested in their first post-irradiation division and aberrations were analysed either by the Giemsa/Hoechst 33258-staining technique or by the fluorescent in-situ hybridization technique involving whole chromosome hybridization and 4',6-diaminido-2-phenylidole (DAPI)-staining. Whole chromosome probes were used for chromosomes 2, 4 and 5, and the chromosome painting patterns were classified according published protocols. Reproductive cell survival was determined by a standard clonogenic assay. RESULTS: With respect to the induction of reproductive cell death and chromosome aberrations, carbon ions of different energies were more effective than 200 kV X-rays. As expected, irradiation in the extended Bragg peak was the most efficient mode. For cell killing, relative biological effectiveness increased with linear energy transfer up to 2.9. The frequencies of total dicentrics and excess acentric fragments as determined in Giemsa-stained cells were higher in cells irradiated with carbon ions than in cells with X-rays. For 100 MeV u(-1) ions, the dose dependence of apparently simple dicentrics as determined for chromosomes 2, 4 and 5 by single-colour fluorescent in-situ hybridization was linear up to 4 Gy, and linear-quadratic for excess acentric fragments and apparently simple translocations. After irradiation with D=4 Gy carbon ions with energy of 100 MeV u(-1) and from the extended Bragg peak, 12 and 54% of cells displayed complex exchanges, respectively. In contrast, after irradiation with D=4 Gy X-rays, only 1% of cells displayed complex aberrations. Hence, the number of cells with complex exchange aberrations increased strongly after irradiation with carbon ions. CONCLUSION: An increased biological efficiency of carbon ions could be confirmed in radioresistant tumour cells with respect to the induction of reproductive cell death and of unstable as well as stable chromosome aberrations. Relative biological effectiveness reached 2.9 for cell killing by carbon ions from the extended Bragg peak. The yields of apparently simple dicentrics as well as of total dicentrics, i.e. simple dicentrics plus dicentrics belonging to complex exchanges, evaluated in Giemsa-stained metaphases as observed in first post-irradiation mitoses were rather low. In contrast, apparently simple translocations displayed yields systematically higher than simple dicentrics in WiDr cells irradiated with either X-rays or 100 MeV u(-1) or Bragg peak carbon ions. Frequencies o f cells containing complex aberrations increased dramatically after carbon ion irradiation, reaching a maximum for ions from the extended Bragg peak.

Role of DNA-dependent protein kinase in the process of radiation-induced aberration formation.

PURPOSE: To further investigate the role of DNA-dependent protein kinase (DNA-PK) and ataxia-telangiectasia mutation (ATM) in the formation of chromosome aberrations, we analysed radiation-induced aberrations in M059J cells, complemented with the PRKDC (DNA-PK) gene by introducing a fragment of human chromosome 8 containing a copy of the human PRKDC gene. One hybrid cell line (M059J Fus1) displayed kinase activity and was radioresistant; the other hybrid cell line (M059J Fus9) showed no kinase activity and was radiosensitive. Both Fus1 and Fus9 cells have only a low ATM activity. Wortmannin, an inhibitor of DNA-PKCS and ATM, was added before irradiation in order to study the effect of DNA-PKCS--and ATM--inhibition on the formation of chromosome aberrations. Furthermore, aberration formation was studied in a lymphoblastoid ATM-deficient cell line AT-1 and in an ATM-proficient control. MATERIALS AND METHODS: Confluent cells were irradiated with 200 kV X-rays. Dicentrics, excess acentric fragments, chromatid/ isochromatid breaks and chromatid exchanges were scored in the absence and presence of wortmannin. RESULTS: In M059J-Fus1 cells and normal lymphoblastoid cells, only chromosome-type aberrations were observed independently of the presence of wortmannin. In DNA-PK-deficient Fus9 cells and in ATM-deficient AT-1 cells, an increasing proportion (30-80%) of cells containing chromatid-type aberrations was observed. This proportion increased with irradiation dose and with wortmannin addition. The aberration yields observed in the complemented M059J-Fus1 cell line were much lower than the corresponding yields observed in the deficient M059J and AT-1 cell lines. However, the low yields observed in the DNA-PK-proficient 'wild-type' cell line M059K were not completely restored. CONCLUSIONS: Since in M059J-Fus1 cells the radioresistant phenotype with respect to chromosome-type aberration formation was restored by the complementation of PRKDC, ATM expression determines the chromosomal radiosensitivity of M059J cells only to a minor extent. The increasing presence of chromatid-type aberrations in cells irradiated in G0/G1 phase as observed either in DNA-PK- or ATM-deficient cells definitely requires the lack of either kinase. Thus, the aberration spectrum observed is determined by the genetic profile of the respective cells and aberration class amplitudes can be modulated by the inhibition of either kinase.

Bowman-Birk protease inhibitor activates DNA-dependent protein kinase and reduces formation of radiation-induced dicentric chromosomes.

PURPOSE: To test a stimulatory effect of the radioprotector Bowman Birk protease inhibitor (BBI) upon DNA repair processes. MATERIALS AND METHODS: An effect of BBI upon DNA repair was investigated by quantification of radiation-induced dicentric chromosomes. Sensitivity to ionizing radiation was determined by clonogenic survival assay. Quantification of activity of the DNA-dependent kinase was performed by immunoprecipitation and phosphorylation of a TP53-derived peptide. RESULTS: The formation of radiation-induced dicentric chromosomes was reduced significantly after pretreatment of cells with BBI. By using a cell line with an inducible expression of a mutated TP53, it was shown that the BBI-mediated reduction of dicentric chromosome formation depended on the presence of wild-type TP53. To get further insights into the molecular mode of action of BBI, activity of the DNA-dependent protein kinase (DNA-PK) was quantified. BBI treatment resulted in a stimulation of basal (DNA-PK) activity. In SCID mouse fibroblasts deficient in DNA-PK activity, BBI failed to reduce the amount of radiation-induced dicentric chromosomes and the radioprotective effect was absent. Likewise, cells expressing mt.TP53 did not show radioprotection by BBI. CONCLUSIONS: It was observed that BBI exerts its radioprotective effect by a reduction of incorrect DNA repair, resulting in a reduced amount of dicentric chromosomes. This effect on the fidelity of DNA repair is TP53 dependent and correlated with induction of DNA-PK activity.

Effect of keratinocyte growth factor on the proliferation, clonogenic capacity and colony size of human epithelial tumour cells in vitro.

PURPOSE: The effect of recombinant human keratinocyte growth factor (rHuKGF) on the proliferation, clonogenic capacity and colony size of low-passage human epithelial tumour cells was tested in vitro. MATERIALS AND METHODS: Five tumour cell cultures derived from head and neck squamous cell carcinomas, three cultures derived from pleural effusions of carcinomas of different origin and normal human nasal epithelial cells were analysed in passages 2-4. Expression of FGF7 and its receptor (FGFR2) were determined by the RNase protection assay. Cells were incubated with rHuKGF (10-200 ng ml(-1)) 3 days before or immediately after plating for clonal growth in serum-depleted media. To determine cellular radiosensitivity, single doses of 1-8 Gy X-rays were applied. Colony formation as well as colony size, reflecting the number of cell divisions, was determined after 10-15 days of growth in rHuKGF-treated and control cells. RESULTS: Normal nasal epithelial cells showed a two- to threefold increase in the number of cell divisions due to rHuKGF-treatment. In tumour cell cultures, significant stimulation of proliferation occurred in only one of eight samples. Tumour cells expressed FGF7 mRNA and protein, and low levels of FGFR2 mRNA. The addition of rHuKGF to the medium of the tumour cell cultures influenced neither radiation-induced impairment of proliferation nor clonogenic cell survival. CONCLUSION: rHuKGF has been shown to ameliorate the radiation tolerance of normal epithelia. The minimum in vitro tumour cell response to rHuKGF compared with normal epithelial cells suggests a potential for selective protection of normal epithelia during radiotherapy. The low FGFR2 expression as well as the FGF7 expression in the tumour cells may contribute to their resistance to rHuKGF treatment.

Role of DNA-PK in the process of aberration formation as studied in irradiated human glioblastoma cell lines M059K and M059J.

PURPOSE: The participation of various DNA double-strand break repair mechanisms in the formation of chromosome aberrations is not yet fully understood. To investigate particularly the role of non-homologous end-joining, we analysed the formation of radiation-induced aberrations in a DNA-protein kinase (PK(CS))-proficient cell line M059K and in a deficient cell line M059J. MATERIALS AND METHODS: Plateau-phase M059K and M059J cells were irradiated with low doses of X-rays. Chromosome aberrations were determined as genomic yields of dicentric chromosomes and excess acentric fragments, scored in Giemsa-stained metaphases, and as partial yields of reciprocal translocations and total visible complex exchanges (complex aberrations) for chromosomes 4, 7 and 11 using the FISH method. M059K cells were also analysed in the presence of 50 micro m wortmannin, a DNA-PK inhibitor. RESULTS: DNA-PK-deficient cells showed a higher yield of simple stable and unstable and of complex aberrations in comparison with DNA-PK-proficient cells. The largest differences were observed for excess acentric fragments and for complex aberrations. DNA-PK inhibition by wortmannin in M059-K cells resulted in increased aberration yield in the same qualitative and quantitative manner as in M059-J cells. CONCLUSIONS: The results obtained with DNA-PK-deficient M059J cells and with DNA-PK-proficient M059K cells treated with wortmannin, an inhibitor of DNA-PK and ATM, suggest that the elimination of DNA-PK-dependent non-homologous end-joining can recruit a slow, error-prone repair process, which is DNA-PK independent and favours the increased formation of chromosome aberrations. The nature of this pathway and the way of its participation in aberration formation need further elucidation.

In vitro response of human dermal fibroblasts to X-irradiation: relationship between radiation-induced clonogenic cell death, chromosome aberrations and markers of proliferative senescence or differentiation.

PURPOSE: To analyse the relationship between radiation-induced clonogenic cell death, chromosome aberrations and markers of proliferative senescence or differentiation. MATERIALS AND METHODS: Plateau-phase human dermal fibroblasts from 18 donors were irradiated with graded doses of 1-6 Gy 200kV X-rays. Cell survival was determined by a colony-forming assay. Markers of differentiation or senescence were: spontaneous and radiation-induced clonal differentiation, which was determined morphologically and by the cellular potential to proliferate in clonal culture, also single-cell beta-galactosidase (beta-gal) staining at pH 6.0; and the secretion of transforming growth factor-beta (TGF-beta1) into the culture medium. Chromosome aberrations were determined as genomic yields of dicentric chromosomes and the excess acentric fragments, scored in Giemsa-stained metaphases, and as partial yields of reciprocal translocations for chromosomes 4, 7 and 9 using the FISH method. RESULTS: A broad spread was found in the shapes of the survival curves, with SF2 ranging from 0.041+/-0.015 to 0.63+/-0.05. Radiation-induced clonal differentiation as well as the secretion of TGF-beta1 was elevated in radiosensitive samples. With respect to chromosome aberrations, a significant correlation was found between clonogenic survival and radiation-induced excess acentric fragments. CONCLUSIONS: In the fibroblast cell system, in vitro radiosensitivity is determined not only by processes directly involved in DNA-damage recognition and repair, but also by intracellular signalling cascades, which will lead to differentiation processes.

Reciprocal translocations in patients with testicular seminoma before and after radiotherapy.

PURPOSE: The evaluation of radiation-induced chromosomal translocations in peripheral lymphocytes using fluorescent in situ hybridization is a promising method for retrospective dosimetry after a radiation accident. We evaluated the genomic frequency of chromosomal translocations in patients with testicular seminoma who received adjuvant radiotherapy to the retroperitoneal lymph nodes, to evaluate the time-effect relationship of radiation-induced stable aberrations after partial body irradiation. METHODS: In 13 patients, peripheral lymphocytes could be evaluated before radiotherapy and at several time points after radiotherapy. In 17 additional patients, lymphocyte samples were obtained after radiotherapy. Thirteen healthy men served as age-matched controls for the aberration frequency before radiotherapy. Fluorescent in situ hybridization was performed using whole chromosome probes against chromosomes No. 4, No. 6, and No. 7. RESULTS: Nearly all patients displayed an increased spontaneous rate of genomic translocations (F(G)) before radiotherapy compared to age-matched, healthy men. The difference was significant in the paired ranks test (p < 0.0001). After adjuvant radiotherapy, the F(G) increased 2- to 8-fold in individual patients. Within 20 months after radiotherapy, the F(G) returned to pretherapeutic levels. CONCLUSIONS: The frequency of genomic translocations after partial body irradiation is time dependent. A persistence of chromosomal aberrations, which is to be expected after total body irradiation, could not be observed. It is likely that the dose and the volume of the irradiated bone marrow are playing a role in the persistence of stable chromosomal aberrations. Patients with testicular seminoma displayed an increased frequency of spontaneous genomic translocations before the initiation of radiotherapy. This chromosomal instability might be related to the known increased rate of secondary cancers in this patient group.

Effects of docetaxel in combination with radiation on human head and neck cancer cells (ZMK-1) and cervical squamous cell carcinoma cells (CaSki ).

The purpose of this study was to determine, as we did for paclitaxel, the cytotoxic and radiosensitizing potential of docetaxel in human head and neck cancer cells (ZMK-1), and in cervical squamous cell carcinoma cells (CaSki). ZMK-1 cells were incubated with docetaxel for 3, 9 or 24 hr before irradiation and 24 hr after irradiation. CaSki cells were incubated with docetaxel 24 hr before and after irradiation. For ZMK-1 cells, the docetaxel concentrations (0.7, 0.7 and 0.35 nM) were determined to obtain approximately equivalent cell survival at the different incubation times (3, 9 and 24 hr, respectively). For CaSki cells, the necessary concentration of docetaxel was 0.07 nM. Radiation doses were given from 0 to 7 Gy. Cell survival was measured by a standard clonogenic assay after a 9-day incubation. Flow cytometry was used to measure the capacity of docetaxel to accumulate cells in the G2/M phase of the cell cycle. We observed a weak accumulation of cells in the G2/M phase for the ZMK-1 cells and a pronounced accumulation for CaSki cells. For docetaxel incubation before irradiation, the isoeffect enhancement ratios for ZMK-1 cells determined at the 37% survival level were 1.18, 2.01, and 2.40 for pre-incubation at 3, 9 and 24 hr, respectively; for CaSki cells the ratio was 1.44. For a docetaxel incubation of 24 hr after irradiation, the isoeffect enhancement ratios determined at the 37% survival level were 1.54 and 1.17 for the ZMK-1, and CaSki cells, respectively. A radiosensitizing effect of docetaxel could be demonstrated unambiguously in the two cell lines used. In contrast to our previously published results with paclitaxel, docetaxel seems to be a better radiosensitizer than paclitaxel.

Radiosensitizing effect of natural and recombinant beta-interferons in a human lung carcinoma in vitro.

PURPOSE: The enhancing effect of natural interferon-beta (n-IFN-beta) or recombinant interferon-beta (r-IFN-beta) on radiation damage in tumor cells has been evaluated in several sudies. It is not clear whether the different forms of IFN-beta available today are equally efficient in modulating the intrinsic radiosensitivity of tumor cells. The purpose of this study was to compare the radiosensitizing effect of n-IFN-beta, r-IFN-beta1a, and r-IFN-beta1b in one cancer cell line. METHODS: The A549 lung-cancer cell line was grown as a monolayer culture and incubated for 24 h with n-IFN-beta (Fiblaferon), r-IFN-beta1a (Betaserin), or r-IFN-beta1b (Betaferon). Thereafter, the cultures were irradiated with single graded doses of 0, 1, 2, 4, and 6 Gy. Cellular survival was counted in a colony-forming assay at 10 days after treatment. Survival curves were established using the linear quadratic model. Statistical comparison of the survival data was performed using Student's t-test for each dose point. Interactions of IFN-beta and radiation were evaluated using isobologram analysis. RESULTS: All three types of IFN-beta enhanced the radiation sensitivity of A549 cells in a similar way as shown in the alteration of the survival curves and the isobologram analysis. The isoeffective concentration of r-IFN-beta1b was 2.7-fold higher than that of r-IFN-beta1a or n-IFN-beta. All three interferons increased the alpha-component of the survival curves in a concentration-dependent way, suggesting an influence on repair of radiation damage. The maximal sensitizing enhancement ratio (SER) obtained with n-IFN-beta or r-IFN-beta1a at 3000 IU/ml was 1.66 and 1.51, respectively. The highest SER, obtained with r-IFN-beta1b, at 8000 IU/ml was 1.93. CONCLUSIONS: All three interferons tested can equally modify the intrinsic radiosensitivity of A549 cells. The isoeffective concentration of r-IFN-beta1b is 2.7-fold that of n-IFN-beta or r-IFN-beta1a.

[In vitro study of a paclitaxel-radiotherapy combination on a human epidermoid tumor cell line]. / Etude in vitro de l'association paclitaxel-radiothérapie sur une lignée cellulaire tumorale épidermoïde humaine.

PURPOSE: Paclitaxel is an agent which stabilizes microtubules, and has been shown to block different cells in the G2/M phase of the cell cycle and thus to modulate their radioresponsiveness. We investigated the radiosensitizing potential of paclitaxel in human head and neck cancer cells. MATERIALS AND METHODS: ZMK-1 cells were incubated with paclitaxel for 3, 9, or 24 h before or during 24 h after irradiation. Paclitaxel concentrations of 70 nM, 7 nM, and 0.7 nM were chosen to obtain equivalent toxicity at the different incubation times: 3 h, 9 h, and 24 h, respectively. Radiation doses ranged from 0 to 8 Gy using 60Co source. Cell survival was measured by a standard clonogenic assay after a 9-day incubation. Flow cytometry was used to measure the capacity of paclitaxel to accumulate cells in the G2/M phase. RESULTS: Paclitaxel alone possessed cytotoxicity dependent on time and concentration. There was a total of 40% of cells accumulated in G2/M after 24-36 h. When combined with radiation, the 9 h preincubation resulted in a radiosensitization. The 3 h pre-incubation as well as the 24 h post-incubation resulted in an infra-additive effect. CONCLUSION: In our cells a radiosensitizing effect of paclitaxel could not be demonstrated unambiguously. The blockage of the cells in the G2/M phase is not the only mechanism to explain the potential radiosensitization of paclitaxel.

The combined effect of interferon beta and radiation on five human tumor cell lines and embryonal lung fibroblasts.

PURPOSE: The combined effect of natural Interferon-beta (n-IFN-beta) and ionizing radiation was tested in vitro on 5 different tumor cell lines and 1 embryonal lung fibroblast cell line. MATERIALS AND METHODS: The following cell lines were used: A549 (lung cancer), MCF-7 (breast cancer), CaSki (cervical cancer), WiDr (colon cancer), ZMK-1 (head and neck cancer), and MRC-5 (embryonal lung fibroblast line). Cells were incubated with n-IFN-beta (30 I.U./ml to 3000 I.U./ml) 24 h before irradiation. Irradiation was given as single dose between 1 and 6 Gy. Cell survival was evaluated using a standard colony-forming assay. RESULTS: Incubation with n-IFN-beta enhanced the effect of radiation in all tumor cell lines tested. The maximum sensitizing enhancement ratios (SER) at the 37% survival level were: 1.66 for A549 cells, 1.47 for CaSki cells, 1.56 for MCF-7 cells, 1.40 for WiDr cells, and 1.57 for ZMK-1 cells. In the nonneoplastic MRC-5 cell line, no radiosensitizing effect of n-IFN-beta could be demonstrated. The linear quadratic fit of the survival curves showed an increase of the alpha-component for all tumor cell lines treated with n-IFN-beta. CONCLUSIONS: IFN-beta enhanced the effect of radiation in the tumor cell lines, but not in the nonmalignant lung fibroblasts. The increase of the alpha component in the survival curves indicates that impaired radiation repair or the accumulation of sublethal damage might play a role for the radiosensitizing effect of n-IFN-beta.

Effects of paclitaxel in combination with radiation on human head and neck cancer cells (ZMK-1), cervical squamous cell carcinoma (CaSki), and breast adenocarcinoma cells (MCF-7).

BACKGROUND AND PURPOSE: The anticancer drug paclitaxel, a natural product from Taxus brevifolia, is a microtubule-stabilising agent, which has been shown to block different cells in the G2/M phase of the cell cycle and so modulate their radioresponsiveness. We investigated the radiosensitizing potential of paclitaxel in human head and neck cancer cells (ZMK-1), in cervical squamous cell carcinoma cells (CaSki) and in breast adenocarcinoma cells (MCF-7). METHODS: ZMK-1 cells were incubated with paclitaxel for 3, 9, or 24 h before irradiation. ZMK-1-, CaSki- and MCF-7 cells were incubated with paclitaxel for 24 h after irradiation. The paclitaxel concentration (70 nM, 7 nM, 0.7 nM) was chosen to obtain equivalent toxicity at the different incubation times (3 h, 9 h, 24 h respectively). Radiation doses were from 0 to 8 Gy. Cell survival was measured by a standard clonogenic assay after a 9-day incubation. Flow cytometry was used to measure the capacity of paclitaxel to cause accumulation of cells in the G2/M phase of the cell cycle. RESULTS: Paclitaxel alone was cytotoxic in a time- and concentration-dependent manner. Up to 36% of the ZMK-1 cells accumulated in G2/M after treatment for 24-36 h. If the cells were incubated with paclitaxel before irradiation the isoeffect enhancement ratios for ZMK-1 cells, determined at the 37% survival level, were 0.81, 1.48 and 1.15 for 3-h, 9-h, and 24-h pre-incubations respectively. For a paclitaxel incubation of 24 h after irradiation, the isoeffect enhancement ratios, determined at the 37% survival level, were 0.72, 0.76 and 1.2 for the ZMK-1. CaSki, and MCF-7 cells respectively. CONCLUSION: In the three cell lines no radiosensitizing effect of paclitaxel could be demonstrated unambiguously. The use of asynchronized cells or the support of cellular repair mechanisms while the cells are blocked in G2/M could partly explain the results.

Radiation rendered more cytotoxic by fludarabine monophosphate in a human oropharynx carcinoma cell-line than in fetal lung fibroblasts.

PURPOSE: Fludarabine monophosphate (fludarabine-P) is a relatively new drug in the treatment of different haematological diseases. The mechanism of action also implies a possible role of this drug as a radiosensitizer. Up to now no in vitro investigations dealing with radiosensitizing effects of fludarabine-P in carcinoma cell lines and fibroblasts have been published. The aim of our studies was to analyse the cytotoxic and radiosensitizing effects of different dosages and application schedules of fludarabine-P in a human squamous carcinoma cell line of the oropharynx (ZMK-1) and of fetal lung fibroblasts (MRC-5) in vitro. Possible mechanisms of interaction of fludarabine-P and radiation were investigated. METHODS: ZMK-1 and MRC-5 cells were cultured under standard conditions with different concentrations of fludarabine-P in combination with escalating doses of radiation. Cytotoxic effects were measured by colony-forming assays. Induction and rejoining of radiation-induced DNA double-strand breaks after incubation with fludarabine-P were measured using constant-field gel electrophoresis. Incubation times for rejoining varied from 0 h to 24 h. RESULTS: Fludarabine-P showed a radiosensitizing activity in ZMK-1 tumour cells and MRC-5 fibroblasts. The observed effects depended on the concentration and the incubation time. The largest effect was demonstrable for an incubation of 5 days, which started shortly before irradiation, whereas an incubation solely before irradiation did not have a clear effect on the cellular survival. The sensitizer enhancement ratio, at the 10% survival level, in the ZMK-1 cells was 2.2 in comparison to 1.6 in MRC-5 cells. The analysis of the interaction of fludarabine-P and ionising radiation by means of the isobologram approach, revealed an overadditive effect in the tumour cell line and an additive effect in the lung fibroblasts. Fludarabine-P did not modify the rejoining of radiation-induced DNA double-strand breaks in either cell line. CONCLUSIONS: We conclude that fludarabine-P in clinically attainable doses is a strong radiosensitizer in ZMK-1 cells and has a lower activity in the MRC-5 fibroblasts in vitro. The radiosensitization of fludarabine-P seems to be over additive in the malignant cells and additive in normal fetal fibroblasts. This would indicate that fludarabine-P might enhance the therapeutic ratio of radiation. Further investigations are warranted to identify the potential of this drug as a radiosensitizer in vivo and to elucidate the mechanism of interaction of the drug and radiation.

Influence of clodronate on breast cancer cells in vitro.

UNLABELLED: One of the major therapies of bone metastasis is administration of clodronate. But the influence of clodronate alone on tumour cells is not quite clear. Radiotherapy and administration of clodronate increasingly are used in combination. The influence of clodronate on radiosensitivity of tumour cells is not known. METHODS: We used MDA-MB-435S and MCF-7 cells (breast cancer) in vitro and exposed the cells to clodronate in different concentrations and different short application times. In a second experiment we added graded doses of radiotherapy to the cell cultures. All experiments were done under standard conditions of the colony test. RESULTS: At different concentrations and different incubation times clodronate is able to reduce the cell survival of MDA-MB-435S cells, but not of MCF-7 cells. Even very low concentrations of clodronate in the cell culture medium are sufficient to reduce the tumour cell survival of MDA-MB-435S down to 59%. This reduction is time and concentration dependent. Using irradiation, clodronate has definitively no influence on the radiosensitivity of MDA-MB-435S cells in vitro, but the shoulder of the survival curve of MCF-7 cells is markedly reduced, demonstrating reduced repair of sublethal radiation damage.

Improved treatment of medullary thyroid cancer in a nude mouse model by combined radioimmunochemotherapy: doxorubicin potentiates the therapeutic efficacy of radiolabeled antibodies in a radioresistant tumor type.

Whereas in advanced metastatic medullary thyroid cancer (MTC), a variety of chemotherapeutic regimens have achieved only limited success clinically, more recently, radioimmunotherapy (RIT) with 131I-labeled anti-carcinoembryonic antigen (CEA) monoclonal antibodies (MAbs) has shown promising results. The aims of this study were to compare, in an animal model, the therapeutic efficacy of RIT to clinically used "standard" chemotherapeutic regimens and to evaluate whether combination strategies of both modalities may be feasible and may help to improve therapeutic results in this rather radioresistant tumor type. Nude mice, bearing s.c. xenografts of the human MTC cell line, TT, were treated either with the 131I-labeled anti-CEA MAb, F023C5 IgG, or were administered chemotherapeutic regimens that had shown promising results in patients with metastatic MTC (doxorubicin and cisplatinum monotherapy, combinations of both agents, and a 5-fluorouracil/dacarbazine/streptozotocin scheme). Control groups were left untreated or were injected with an irrelevant radiolabeled antibody at equitoxic dose levels. The maximum tolerated dose (MTD) of each agent was determined. Combinations of chemotherapy and RIT were evaluated as well. Toxicity and tumor growth were monitored at weekly intervals. From the chemotherapeutic agents and schemes tested, doxorubicin monotherapy was the most effective; combination therapies did not result in an increased antitumor efficacy, but they did result in more severe toxicity. At equitoxic doses, no significant difference was found between the therapeutic efficacy of doxorubicin and that of RIT. Myelotoxicity was dose limiting with radiolabeled MAbs (MTD, 600 microCi), as well as with chemotherapeutic regimens containing alkylating agents (cisplatinum, dacarbazine, or streptozotocin). At its MTD (200 microg), doxorubicin caused only mild myelotoxicity, and despite signs of cardiac toxicity, gastrointestinal side effects were dose limiting. Accordingly, bone marrow transplantation (BMT) enabled dose intensification with RIT (MTD with BMT, 1100 microCi), which led to further increased antitumor efficacy, whereas BMT was unable to increase the MTD of doxorubicin. Due to the complementarity of toxic side effects but an anticipated synergism of antitumor efficacy, combinations of RIT with doxorubicin were tested. Administrations of 500 microCi of 131I-labeled anti-CEA and, 48 h later, 200 microg of doxorubicin (i.e., 83 and 100% of the respective single-agent MTDs), were the highest doses that did not result in an increased lethality; with bone marrow support, 1000 microCi of 131I-labeled anti-CEA could be combined with 200 microg of doxorubicin (i.e., 90 and 100% of the individual MTDs). Therapeutic results of this combined radioimmunochemotherapy were superior to equitoxic monotherapy with either agent, and indication for synergistic antitumor effects is given. At its respective MTD, radioimmunochemotherapy led to a 36% cure rate if it was given without bone marrow support and to a 85% permanent cure rate if it was given with bone marrow support. The animal model, as presented in this study, seems to be useful for the preclinical testing of therapeutic agents for the systemic treatment of MTC. At equitoxic doses, RIT with radiolabeled anti-CEA antibodies seems to be equally as effective as chemotherapy with doxorubicin. Combination of RIT and doxorubicin chemotherapy seems to have synergistic therapeutic efficacy, which may be due to a radiosensitizing effect of doxorubicin.

Differences in the yields of dicentrics and reciprocal translocations observed in the chromosomes of irradiated human skin fibroblasts and blood lymphocytes from the same healthy individuals.

Plateau-phase human dermal fibroblasts and whole blood G0 lymphocytes obtained from the same three healthy donors were irradiated with different doses of 200 kV X rays. The genomic yields of dicentrics were evaluated in the first postirradiation mitosis. Fluorescence in situ hybridization was used to "paint" the chosen whole chromosomes. The yields of reciprocal translocations were scored for all donors for chromosome 4, and also for the third donor for chromosomes 7 and 9. The amounts of reciprocal translocations were scored in the first as well as in later (4th or 5th) postirradiation cell divisions. The yields of dicentrics involving the painted chromosomes were scored for one donor as well. The yields of dicentrics observed in the lymphocytes and in the skin fibroblasts from the same individual were significantly different. Yields of dicentrics were much lower in fibroblasts than in lymphocytes. This was observed for all doses up to 4 Gy for all three donors studied. Fibroblasts from two individuals were also irradiated with 6 Gy. The interindividual variability was similar in both cell types. The yields of reciprocal translocations in lymphocytes and in fibroblasts were similar. The yields of reciprocal translocations were practically constant when comparing the first mitotic divisions and later divisions. The observed ratio of translocations:dicentrics was higher in fibroblasts than in lymphocytes, where it was about 1 for one donor and about 2 for the other two donors. Significant differences between the yields of dicentrics and translocations as well as between the shapes of their dose responses were observed in lymphocytes and fibroblasts from the same individual. Our experimental data suggest that repair processes with different efficiencies or different repair processes might be active in cells of different types of tissue.