Tumor radiosensitizers--current status of development of various approaches: report of an International Atomic Energy Agency meeting.

PURPOSE: The International Atomic Energy Agency (IAEA) held a Technical Meeting of Consultants to (1) discuss a selection of relatively new agents, not those well-established in clinical practice, that operated through a variety of mechanisms to sensitize tumors to radiation and (2) to compare and contrast their tumor efficacy, normal tissue toxicity, and status of development regarding clinical application. The aim was to advise the IAEA as to which developing agent or class of agents would be worth promoting further, by supporting additional laboratory research or clinical trials, with the eventual goal of improving cancer control rates using radiotherapy, in developing countries in particular. RESULTS: The agents under discussion included a wide, but not complete, range of different types of drugs, and antibodies that interfered with molecules in cell signaling pathways. These were contrasted with new molecular antisense and gene therapy strategies. All the drugs discussed have previously been shown to act as tumor cell radiosensitizers or to kill hypoxic cells present in tumors. CONCLUSION: Specific recommendations were made for more preclinical studies with certain of the agents and for clinical trials that would be suitable for industrialized countries, as well as trials that were considered more appropriate for developing countries.

Role of 2'-2' difluorodeoxycytidine (gemcitabine)-induced cell cycle dysregulation in radio-enhancement of human head and neck squamous cell carcinomas.

BACKGROUND AND PURPOSE: To try to get a better insight on the interaction between dFdC and ionizing radiation at the cellular level, we examined in vitro the effect of dFdC on the cell cycle of two human head and neck squamous cell carcinoma cell lines (SQD9 and SCC61). PATIENTS AND METHODS: Experimental conditions yielding radio-enhancement were used. Confluent cells were incubated with dFdC (5 microM) for different incubation times, washed, pulse-labeled with BrdUrd (10 microM), fixed and then processed for flow cytometry analysis. Alternatively, cells preincubated or not with dFdC were irradiated (5Gy) in drug-free medium, incubated at 37 degrees C for various times and then processed for flow cytometry analysis. RESULTS: In both cell lines, dFdC incubated between 1 and 6 h induced a DNA synthesis inhibition with accumulation of cells in the G1-S boundary followed, when DNA reinitiated, by a synchronous progression of cells throughout the cycle. A slightly different kinetics was observed in the two cell lines. A weak correlation between dFdC radio-enhancement and distribution of cells in the cell cycle was observed. It was also observed that for longer dFdC incubation times, DNA synthesis could reinitiate while cells were still incubated with dFdC. This reinitiation could be correlated with a decrease in the intracellular dFdCTP pool to non-inhibitory levels. Finally in both cell lines, dFdC modified neither the importance nor the kinetics of the radiation-induced G1 delay. CONCLUSIONS: This study provides evidence that gemcitabine used at radio-enhancing concentration induces alteration of cell kinetics and cell redistribution throughout the cell cycle. This effect is cell line-dependent. However, the weak correlation between dFdC radio-enhancement and cell cycle distribution suggests that the cell cycle effect does not constitute the most important mechanism of interaction with ionizing radiation. Our study also indicated that in the two cell lines studied, a modulation of the G1-S checkpoint was not implicated in enhancement of radiation response by dFdC.

Role of deoxycytidine kinase (dCK) activity in gemcitabine's radioenhancement in mice and human cell lines in vitro.

BACKGROUND: Gemcitabine (dFdC, 2',2'-difluorodeoxycytidine) is a deoxycytidine nucleoside analog which has a marked effect on several enzymes involved in DNA synthesis and repair. Gemcitabine has been tested as a radiosensitizer in various biological models, and radiation dose modification factors (DMF) have been reported in the range between 1.1 and 2.4. Gemcitabine is a prodrug that requires intracellular activation by phosphorylation into its active triphosphate dFdCTP form. Deoxycytidine kinase (dCK) is the enzyme involved in the first phosphorylation cascade, and several observations have suggested that dCK was a limiting factor for the cytotoxic activity of gemcitabine. OBJECTIVE: In the present article, we investigated the relationship between dCK activity and gemcitabine's radiosensitization in four mice and two human cell lines. MATERIALS AND METHODS: Four mice and two human tumor cell lines were investigated. Radiosensitization was assessed on confluent cell incubated with 5 microM gemcitabine for 3 h prior to a single radiation dose. Enzymatic activity was assessed using deoxycytidine as substrate with (specific activity) or without (total activity) inhibition of thymidine kinase 2 activity. dCK protein level was assessed by immunoblotting using a rabbit anti-human dCK antibody. mRNA expression was assessed with Northern blot using beta-actin as internal control. RESULTS: Gemcitabine's radiosensitization was heterogeneous with DMF ranging from 0.8 to 1.5. A good correlation was observed between the specific dCK activity and the protein level or the mRNA expression indicating that in our cell systems no post-transcriptional or post-translational activation occurred. An excellent correlation (r = 0.99) was observed between the specific enzymatic activity and gemcitabine's radiosensitization. Cell lines that expressed a high enzymatic activity were the more radiosensitized by gemcitabine. This correlation holds when radiosensitization was plotted against the dCK mRNA expression and protein level. CONCLUSIONS: The present study has suggested the role of dCK activity in gemcitabine's radioenhancement in human and mice cell lines. The study suggests that determination of the enzymatic activity prior to a concurrent gemcitabine and radiotherapy treatment might represent a good predictive assay for tumor response. Such concept should deserve further testing in pre-clinical and clinical settings.

A phase I study of fludarabine combined with radiotherapy in patients with intermediate to locally advanced head and neck squamous cell carcinoma.

BACKGROUND AND PURPOSE: Fludarabine, 9-beta-D-arabinofuranosyl-2-fluoroadenine, is an adenine nucleoside analogue that has significant activity in hematological malignancies and has shown promising activity in combination with radiation in preclinical solid tumor models. In this framework, we designed two phase I trials (one conducted at M.D. Anderson Cancer Center in Houston, and the other conducted in two Belgian hospitals) exploring concurrent fludarabine and radiotherapy in patients with intermediate to locally advanced head and neck squamous cell carcinomas (HNSCC). MATERIALS AND METHODS: Fludarabine was administered i.v. daily 3-4 h before the last 10 fractions of a standard radiation fractionation regimen (70 Gy in 7 weeks). The main objective of the trials was to determine the maximum tolerated dose (MTD) of fludarabine in this particular setting. Twenty-eight patients with stage T2-T4, any N, M0 were included in the study. Fludarabine doses started at 7.5 mg/m(2) per day (3 mg/m(2) per day in Houston) and increased by steps of 2.5 mg/m(2) per day (3 mg/m(2) per day in Houston). RESULTS: The addition of fludarabine at increasing doses to radiation did not result in increased intensity or duration of skin (18% grade 3 dermatitis) or mucosal (60% grade 3 mucositis) radiotoxicity compared to what was expected for radiation alone. At a daily dose of 17.5 mg/m(2), two patients out of five (40%) developed a grade 4 neutropenia, of whom one developed a neutropenic fever. This dose was set as the MTD. All patients developed a fludarabine dose-dependant lymphocytopenia. The plasma F-ara-A concentration peaked after the 30-min infusion in a dose-dependent fashion and reached an average peak concentration of approximately 2 microM for doses of 15 mg/m(2) and higher. CONCLUSIONS: This study demonstrates that fludarabine can be safely administered concurrently with radiation at a daily dose of 15 mg/m(2) during the final 2 weeks of radiotherapy. A phase II trial will be required to establish the potential role of concurrent fludarabine and radiotherapy in the treatment of moderately to locally advanced HNSCC.

The radioenhancement of two human head and neck squamous cell carcinomas by 2'-2' difluorodeoxycytidine (gemcitabine; dFdC) is mediated by an increase in radiation-induced residual chromosome aberrations but not residual DNA DSBs.

PURPOSE: The present study aimed at investigating if 2'-2' difluorodeoxycytidine (dFdC) radioenhancement was mediated by an effect on induction and/or repair of radiation-induced DNA DSBs and chromosome aberrations in cells with different intrinsic radiosensitivity. METHODS: Confluent human head and neck squamous cell carcinoma cell lines designated SCC61 and SQD9 were treated with 5 microM dFdC for 3 or 24 h prior to irradiation. DNA DSBs induction and repair were analyzed by PFGE. Radiation-induced chromosome aberrations were examined with a FISH technique. RESULTS: In both cell lines, dFdC did not modify radiation-induced DNA DSBs in a dose range between 0 and 40 Gy. After a single dose of 40 Gy, dFdC affected neither the kinetic of repair nor the residual amount of DNA DSBs up to 4 h after irradiation. Whereas dFdC did not increase the induction of chromosome aberrations, after a single dose of 5 Gy, the percentage of aberrant cells and the number of aberrations per aberrant cells were significantly higher in combination with dFdC. CONCLUSION: Our data suggest that under experimental conditions yielding substantial radioenhancement, dFdC decreases the repair of genomic lesions inducing secondary chromosome breaks but has no effect on DNA DSBs repair as measured by PFGE.