In vitro study on the schedule-dependency of the interaction between pemetrexed, gemcitabine and irradiation in non-small cell lung cancer and head and neck cancer cells.

BACKGROUND: Based on their different mechanisms of action, non-overlapping side effects and radiosensitising potential, combining the antimetabolites pemetrexed (multitargeted antifolate, MTA) and gemcitabine (2',2'-difluorodeoxycytidine, dFdC) with irradiation (RT) seems promising. This in vitro study, for the first time, presents the triple combination of MTA, dFdC and irradiation using various treatment schedules. METHODS: The cytotoxicity, radiosensitising potential and cell cycle effect of MTA were investigated in A549 (NSCLC) and CAL-27 (SCCHN) cells. Using simultaneous or sequential exposure schedules, the cytotoxicity and radiosensitising effect of 24 h MTA combined with 1 h or 24 h dFdC were analysed. RESULTS: Including a time interval between MTA exposure and irradiation seemed favourable to MTA immediately preceding or following radiotherapy. MTA induced a significant S phase accumulation that persisted for more than 8 h after drug removal. Among different MTA/dFdC combinations tested, the highest synergistic interaction was produced by 24 h MTA followed by 1 h dFdC. Combined with irradiation, this schedule showed a clear radiosensitising effect. CONCLUSIONS: Results from our in vitro model suggest that the sequence 24 h MTA --> 1 h dFdC --> RT is the most rational design and would, after confirmation in an in vivo setting, possibly provide the greatest benefit in the clinic.

Comparative study of the radiosensitizing and cell cycle effects of vinflunine and vinorelbine, in vitro.

BACKGROUND: Vinca alkaloids are an important class of anticancer agents and semisynthetic vinca alkaloids are developed to improve the therapeutic index of this class of drugs. In the present study, a direct comparison was made between vinflunine and vinorelbine regarding their radiosensitizing and cell cycle effects. METHODS: Four human tumour cell lines were tested under identical experimental conditions, using equitoxic concentrations of vinflunine and vinorelbine. RESULTS: Vinflunine and vinorelbine induced a comparable radiosensitizing effect (p-value never below 0.01) when cells were incubated for 24 h immediately prior to radiation. Regarding the cell cycle effects, a statistically significant concentration-dependent G2/M block was seen after 24 h incubation with vinorelbine in all tested cell lines. Similar results, with small cell line-related differences, were observed with vinflunine. CONCLUSION: The radiosensitizing effects of both semisynthetic vinca alkaloids were comparable (not statistically different) and nearly always cell line-specific and concentration-dependent. The cell cycle effects could be related to the observed radiosensitizing effects. Considering the more favourable toxicity profile of vinflunine, this agent might be more promising than vinorelbine for chemoradiation studies in the clinic.

Further mechanistic unravelling of the influence of the cell cycle effects on the radiosensitising mechanism of vinflunine, in vitro.

PURPOSE: Vinflunine is an innovative microtubule inhibitor belonging to the vinca alkaloid class that possesses radiosensitising properties, which could lead to promising activity in chemoradiation studies in the clinic. METHOD: In the current study, different incubation times with vinflunine, immediately before radiation and different time intervals between vinflunine treatment and radiation were investigated, in vitro, using four different human tumour cell lines differing in cell type and p53 status. Results were correlated with the cell cycle distribution at the moment of radiation, in order to elucidate the role of cell cycle perturbations caused by vinflunine on its radiosensitising effect. RESULTS: Radiosensitisation was observed in all cell lines, and maximal radiosensitisation was both cell line- and schedule-dependent. The cell cycle distributions were cell line-dependent also, and when correlated with the observed radiosensitising effects could explain many (but not all) of the radiosensitising properties of vinflunine. CONCLUSION: The cell cycle perturbations caused by vinflunine may definitely have an impact on its radiosensitising potential, but other factors must play a role because of some unaccountable differences between cell cycle distribution and the radiosensitising potential.

The relation between deoxycytidine kinase activity and the radiosensitising effect of gemcitabine in eight different human tumour cell lines.

BACKGROUND: Gemcitabine (dFdC) is an active antitumour agent with radiosensitising properties, shown both in preclinical and clinical studies. In the present study, the relation between deoxycytidine kinase (dCK) activity and the radiosensitising effect of gemcitabine was investigated in eight different human tumour cell lines. METHODS: Tumour cells were treated with dFdC (0-100 nM) for 24 h prior to radiotherapy (RT) (gamma-Co60, 0-6 Gy, room temperature). Cell survival was determined 7, 8, or 9 days after RT by the sulforhodamine B test. dCK activity of the cells was determined by an enzyme activity assay. RESULTS: A clear concentration-dependent radiosensitising effect of dFdC was observed in all cell lines. The degree of radiosensitisation was also cell line dependent and seemed to correlate with the sensitivity of the cell line to the cytotoxic effect of dFdC. The dCK activity of our cell lines varied considerably and differed up to three fold from 5 to 15 pmol/h/mg protein between the tested cell lines. In this range dCK activity was only weakly related to radiosensitisation (correlation coefficient 0.62, p = 0.11). CONCLUSION: Gemcitabine needs to be metabolised to the active nucleotide in order to radiosensitise the cells. Since dFdCTP accumulation and incorporation into DNA are concentration dependent, the degree of radiosensitisation seems to be related to the extent of dFdCTP incorporated into DNA required to inhibit DNA repair. The activity of dCK does not seem to be the most important factor, but is clearly a major factor. Other partners of the intracellular metabolism of gemcitabine in relation to the cell cycle effects and DNA repair could be more responsible for the radiosensitising effect than dCK activity.

The radiosensitising effect of difluorodeoxyuridine, a metabolite of gemcitabine, in vitro.

PURPOSE: Gemcitabine is an active antitumour agent with radiosensitising properties. Gemcitabine is rapidly metabolised, intracellularly as well as extracellularly, by deoxycytidine deaminase to difluorodeoxyuridine (dFdU), a compound with little antitumour activity. However, plasma concentrations are maintained for a prolonged period (>24 h) at levels known to cause growth inhibition. This is the first study that investigates the radiosensitising potential of dFdU in vitro. METHODS: ECV304 and H292, human cancer cells, were treated with different concentrations dFdU (0-100 microM) during 24 h before radiation treatment (RT). The schedule dependency of the radiosensitising effect was studied by varying the interval between dFdU and radiation treatment. In addition, the cell cycle effect of dFdU was investigated with flow cytometry, and the induction of apoptosis under radiosensitising conditions was determined by Annexin V staining and caspase 3 cleavage. RESULTS: dFdU caused a clear concentration-dependent radiosensitising effect in both ECV304 and H292 cells. Dose enhancement factor (DEF) increased with an increasing concentration of dFdU: DEFs were 1.10, 1.60 and 2.17 after treatment with 10, 25 and 50 microM dFdU, respectively, in ECV304 cells and 1.08, 1.31 and 1.60 after treatment with 25, 50 and 100 microM, respectively, in H292 cells. DEFs decreased with an increasing interval of 0-24 h between dFdU treatment and radiation. Under radiosensitising conditions, the combination dFdU and radiation resulted in an increased induction of apoptosis. In addition, the cell cycle effect of dFdU, an arrest at the early S phase, is comparable with the cell cycle effect of gemcitabine. CONCLUSIONS: dFdU, the main metabolite of gemcitabine, causes a concentration- and schedule- dependent radiosensitising effect in vitro. Since the metabolite is present in plasma for a long period (>24 h) after treatment with gemcitabine, it might be partly responsible for the interaction between radiotherapy and gemcitabine. This observation might have important consequences for the optimal schedules of the combination gemcitabine and radiation therapy.

Cell cycle effects of vinflunine, the most recent promising Vinca alkaloid, and its interaction with radiation, in vitro.

PURPOSE: Vinflunine (VFL) is a novel third generation Vinca alkaloid with superior antitumour activity in preclinical models and an anticipated more favourable toxicity profile compared to the other Vinca alkaloids. METHOD: We investigate the radiosensitising properties of VFL and its cell cycle effects in four human tumour cell lines (ECV304, MCF-7, H292, and CAL-27). The sulforhodamine B test was used to determine cell survival, and cell cycle analysis was performed by flow cytometry. Radiosensitisation (RS) was represented by dose enhancement factors (DEFs). RESULTS: Twenty-four hours treatment with VFL before radiation caused dose-dependent RS in all cell lines. This was most pronounced in ECV304 cells with RS already at VFL concentrations that reduced cell survival by 10% (IC10). DEFs ranged from 1.57 to 2.29 in the different cell lines. A concentration-dependent G2/M block was observed (starting at 4 h of incubation). After maximal G2/M blockade cells started cycling again, mainly by mitosis, while a small portion of cells started a polyploid cell cycle. Also drug removal immediately caused recycling of cells and induction of a polyploid cell population. The polyploid cell population was most impressively noticeable after prolonged incubation with VFL (48 h), in particular in CAL-27 and ECV304. This was never observed in a tested normal fibroblast cell line (Fi 360). The fate of these cells is of particular interest, but yet uncertain. CONCLUSION: VFL has radiosensitising potential. The exact role of the cell cycle effects of VFL in its radiosensitising mechanism is still not fully elucidated and requires further study.

Combined Pap and HPV testing in primary screening for cervical abnormalities: should HPV detection be delayed until age 35?

In 2003, the United States Food and Drug Administration has approved the Hybrid Capture 2 assay for use with a Pap test to adjunctively screen women of 30 years and older for the presence of high-risk human papillomavirus (HR-HPV) infection. Although the predictive power of a negative test is strong, the number of false-positives may still be high. We investigated HPV prevalence in relation to age in a group of 2293 women, aged between 20 and 50, with normal cytology. Overall HR-HPV prevalence was 6.9% (95%CI=5.9-8.0%). Regression analysis using 5-year intervals showed that the HR-HPV prevalence did not significantly decline up to age 34, whereas it declined significantly after age 35. This would suggest that postponing HPV detection in primary screening from age 30 to 35 would result in a decrease of almost 50% of the number of women with normal cytology and a transient HPV infection. However, larger scale studies are required to confirm this finding.

Unraveling the mechanism of radiosensitization by gemcitabine: the role of TP53.

Gemcitabine has excellent radiosensitizing properties, as shown in both preclinical and clinical studies. Radiosensitization correlated with the early S-phase block of gemcitabine. In the present study, we investigated the role of TP53 in the radiosensitizing effect of gemcitabine. Isogenic A549 cells differing in TP53 status were treated with gemcitabine during the 24 h prior to irradiation. Cell survival was determined 7 days after irradiation by the sulforhodamine B test. In addition, cell cycle perturbation was determined by flow cytometry and TP53 expression by Western blot analysis. Gemcitabine caused a concentration-dependent radiosensitizing effect in all cell lines. Transformed A549 cells were less sensitive to the cytotoxic effect of gemcitabine. The cell cycle arrest early in the S phase was dependent on the drug dose but was comparable in the different cell lines and was not related to functional TP53. Using isogenic cell lines, we have shown that neither TP53 status nor the transfection procedure influenced the radiosensitizing effect of gemcitabine. Since both the radiosensitizing effect at equitoxic concentrations and the cell cycle effect of gemcitabine were independent of TP53 expression, it is likely that TP53 protein does not play a crucial role in the radiosensitizing mechanism of gemcitabine.

Cell cycle effect of gemcitabine and its role in the radiosensitizing mechanism in vitro.

PURPOSE: The mechanism of radiosensitization by gemcitabine is still unclear. It has been hypothesized that the accumulation of cells in early S phase may play a role in enhancing radiosensitivity. METHODS AND MATERIALS: The schedule dependency of the radiosensitizing effect was studied in ECV304, human bladder cancer cells, and H292, human lung cancer cells, by varying the incubation time and time interval between gemcitabine and radiation treatment. To determine the role of cell cycle perturbations in the radiosensitization, the influence of gemcitabine on the cell cycle at the moment of radiation was investigated by flow cytometry. RESULTS: The radiosensitizing effect increased with a longer incubation period: Dose enhancement factors varied from 1.30 to 2.82 in ECV304 and from 1.04 to 1.78 in H292 after treatment during 8-32 h, respectively. Radiosensitization decreased with an increasing interval: Dose enhancement factors varied from 2.26 to 1.49 in ECV304 and from 1.45 to 1.11 in H292 after an interval 0-24 h, respectively. Cells were blocked in the early S phase of the cell cycle by gemcitabine. The highest percentage S-phase cells was observed after treatment with the schedules that resulted in the highest radiosensitizing effect. CONCLUSIONS: We observed a clear schedule-dependent radiosensitization by gemcitabine. Our findings demonstrated a correlation between gemcitabine-induced early S-phase block and the radiosensitizing effect.

Comparison of the sulforhodamine B assay and the clonogenic assay for in vitro chemoradiation studies.

PURPOSE: Since there is a growing interest in preclinical research on interactions between radiation and cytotoxic agents, this study focused on the development of an alternative to the very laborious clonogenic assay (CA). METHODS: The colorimetric sulforhodamine B (SRB) assay was compared to the clonogenic assay for radiosensitivity testing in two lung cancer cell lines (A549, H292), one colon cancer cell line (HT-29) and one breast cancer cell line (MCF-7). In addition, the combination of the radiosensitizing agent gemcitabine and radiation was investigated with both assays. RESULTS: The dose-response curves obtained with the SRB assay and the CA were very similar up to 6 Gy. The radiosensitivity parameters (SF(2), alpha, beta, MID and ID(50)) obtained from the SRB assay and the CA were not significantly different between H292, A549 and MCF-7 cells. The radiation dose-response curves for A549 and H292 cells pretreated with 4 n M gemcitabine for 24 h clearly showed a radiosensitizing effect with both assays. The dose-enhancement factors obtained with the SRB assay and the CA were 1.80 and 1.76, respectively, for A549 cells, and 1.52 and 1.41 for H292 cells. CONCLUSIONS: The SRB assay was shown to be as useful as the more traditional CA for research on chemotherapy/radiotherapy interactions in cell lines with moderate radiosensitivity. This assay will be used for more extensive in vitro research on radiosensitizing compounds in these cell lines.

Retention of the in vitro radiosensitizing potential of gemcitabine under anoxic conditions, in p53 wild-type and p53-deficient non-small-cell lung carcinoma cells.

PURPOSE: Whereas radiosensitization by gemcitabine is well studied under normal oxygen conditions, little is known about its radiosensitizing potential under reduced oxygen conditions. Therefore, the present study evaluated the impact of anoxia on gemcitabine-mediated radiosensitization. METHODS AND MATERIALS: The clonogenic assay was performed in three isogenic A549 cell lines differing in p53 status (24 h, 0-15 nM gemcitabine, 0-8 Gy irradiation, normoxia vs. anoxia). Using radiosensitizing conditions, cells were collected for cell cycle analysis and apoptosis detection. RESULTS: Whereas wild-type p53 A549-LXSN cells were more sensitive to radiation than p53-deficient A549-E6 cells, both cell lines showed similar radiosensitization by gemcitabine under normoxia and anoxia. Independent of p53 functionality, gemcitabine was able to overcome anoxia-induced G(0/1) arrest and established an (early) S phase block in normoxic and anoxic cells. The percentage early and late apoptotic/necrotic cells increased with the gemcitabine/radiation combination, with a significant difference between A549-LXSN and A549-E6. CONCLUSIONS: This study is the first to show that gemcitabine retains its radiosensitizing potential under low oxygen conditions. Although radiosensitization was observed in both p53 wild-type and p53-deficient cells, p53 status might influence induction of apoptosis after gemcitabine/radiation treatment, whereas no effect on cell cycle progression was noticed.

Counting clonogenic assays from normoxic and anoxic irradiation experiments manually or by using densitometric software.

The clonogenic assay is the method of choice to determine cell reproductive death after in vitro irradiation treatment. Traditionally, colony quantification has been performed by manual counting, a very laborious, time-consuming and rather subjective task. In this study, we compared manual counting by two skilled investigators with automated counting using the freely available ClonoCounter program. Five human tumour cell lines were irradiated under normoxia (21% O(2)) or anoxia (<0.1% O(2)), after 24 h or 6 h anoxic preincubation. Colonies were quantified manually or using the ClonoCounter software. A positive correlation between the absolute number of colonies counted manually and automatically was shown. Though there was a general trend of underpredicting the absolute number of cell colonies when counting automatically, survival curves were very similar, and in none of the cell lines were significant differences in radiobiological parameters such as mean inactivation dose, surviving fraction at 2 Gy and oxygen enhancement ratio detected. Our results suggest that the ClonoCounter provides sufficient reliability to be implemented for counting human tumour colonies in in vitro irradiation experiments. In contrast to several previously reported computer-aided colony-counting methods, it is a freely available program, requiring only minimal instrument costs.

Chemoradiation interactions under reduced oxygen conditions: Cellular characteristics of an in vitro model.

Hypoxic tumour regions often contain viable cells that are more resistant to chemotherapy and/or radiotherapy, making it of key importance to analyse new combination treatments under both normoxic and hypoxic conditions. In this study, the impact of moderate hypoxia and anoxia on cellular characteristics was investigated in isogenic A549 cells differing in p53 status. VEGF expression, doubling time, cell cycle distribution, induction of apoptosis and p53 protein expression were evaluated. Radiation survival curves yielded an oxygen enhancement ratio of 1.16-1.67. In conclusion, an in vitro hypoxia model that will be highly useful to analyse chemoradiation interactions is presented.