Tyrphostin AG 1024 modulates radiosensitivity in human breast cancer cells.

Insulin-like growth factor-1 (IGF-1) plays an important growth-promoting effect by activating the PI3K/Akt signalling pathway, inhibiting apoptotic pathways and mediating mitogenic actions. Tyrphostin AG 1024, one selective inhibitor of IGF-1R, was used to evaluate effects on proliferation, radiosensitivity, and radiation-induced cell apoptosis in a human breast cancer cell line MCF-7. Exposure to Tyrphostin AG 1024 inhibited proliferation and induced apoptosis in a time-dependent manner, and the degree of growth inhibition for IC20 plus irradiation (4 Gy) was up to 50% compared to the control. Examination of Tyrphostin AG 1024 effects on radiation response demonstrated a marked enhancement in radiosensitivity and amplification of radiation-induced apoptosis. Western blot analysis indicated that Tyrphostin AG 1024-induced apoptosis was associated with a downregulation of expression of phospho-Akt1, increased expression of Bax, p53 and p21, and a decreased expression of bcl-2 expression, especially when combined with irradiation. To our knowledge, this is the first report showing that an IGF-1 inhibitor was able to markedly increase the response of tumour cells to ionizing radiation. These results suggest that Tyrphostin AG 1024 could be used as a potential therapeutic agent in combination with irradiation.

Dose-rate effect on radiation-induced DNA double-strand breaks in the human fibroblast HF19 cell line.

We measured DNA double-strand breaks (dsbs) immediately after exposure of a non-transformed human fibroblast cell line (HF19) to gamma-rays (0-40 Gy) at four dose-rates (10, 1, 0.1, and 0.01 Gy/min) at 37 degree C using clamped homogeneous electric field (CHEF) gel electrophoresis. The shape of the dose-response curves, which could be approximated by a straight line over the range 0-20 Gy for irradiation at 4 degree C, became curvilinear when irradiation was carried out at 37 degree C at 10, 1, 0.1, and 0.01 Gy/min and reached a plateau at 10 Gy after irradiation at 0.01 Gy/min. We present a mathematical analysis that predicts the results of irradiation at 37 degree C from dsb induction and repair data obtained at 4 degree C, followed by incubation for repair at 37 degree C. The model assumes that the rate of dsb rejoining changes continuously with repair time and that it is independent of dose and dose-rate in the range 10-40 Gy. The model also assumes a linear induction of dsb with dose at 4 degree C and dsb induction is independent of dose-rate and of temperature during irradiation. Independent measurements of dsb induction at 4 degree C and of repair rate accurately predict the dsb levels after irradiation at 37 degree C, during which both phenomena occur simultaneously.

Induction and rejoining of DNA double-strand breaks and interphase chromosome breaks after exposure to X rays in one normal and two hypersensitive human fibroblast cell lines.

The aim of this work was to measure simultaneously and in a quantitative manner double-strand breaks (DSBs), interphase chromosome breaks and cell lethality either immediately after irradiation, or at various times thereafter (up to 24 h), in cells of three nontransformed human fibroblast cell lines of widely different intrinsic radiosensitivity. We wished to assess initial damage, repair kinetics and residual damage at the DNA and the chromosome level, and to correlate these parameters with cell killing. We employed HF19 cells, a normal fibroblast cell line, AT2 cells, a radiosensitive cell line from a patient suffering from ataxia telangiectasia (AT), and 180BR cells, a radiosensitive cell line from a patient with no clinical symptoms of AT. AT2 and 180BR cells, in addition to being radiosensitive, also display a reduced ability to repair potentially lethal damage compared to HF19 cells. The yield of DSBs, as measured by pulsed-field gel electrophoresis, is similar in all three cell lines (slopes correspond to 1.6-1.7% Gy-1 of DNA-associated radioactivity released from the gel well into the lane). In contrast, residual DSBs measured 24 h after irradiation are almost zero for HF19 cells (0.1% confidence interval = 0-1.4%), but are 12.5% (+/- 2.3%) and 43.8% (+/- 1.2%) of those measured immediately after irradiation in AT2 and 180BR cells, respectively. Residual interphase chromosome breaks are 11.6% (+/- 1.6%), 29.7% (+/- 5.7%) and 41.4% (+/- 2.2%) of those measured immediately after irradiation in HF19, AT2 and 180BR cells, respectively. Neither the initial yield of DSBs nor that of excess interphase chromosome breaks can explain the differences in radiosensitivity between the three cell lines; however, there is a correlation between residual DSBs, rate of DSB rejoining at 24 h, residual interphase chromosome breaks on the one hand and cell survival on the other hand.

Correlation between radiosensitivity, percentage hypoxic cells and pO2 measurements in one rodent and two human tumor xenografts.

Computerized pO2 histography has been used to measure the intratumor pO2 in patients for the past few years, and there is now evidence that these tumors contain hypoxic cells. One of the major questions that remains to be answered is the relevance of such data to radiosensitivity. The present study looks for a correlation between intratumor pO2, the percentage of hypoxic cells in the tumor and the radiosensitization induced by carbogen and/or the oxygen carrier, perflubron emulsion. Two human tumor xenografts (HRT18, Na11+) and one rodent tumor (EMT6) were used. The radiosensitivity (clonogenic assay) and the oxygen tension (computerized pO2 histography) were measured. All experiments were performed under similar conditions. Carbogen increased tumor radiosensitivity; sensitization was greatest when 4 ml/kg perflubron emulsion was used in conjunction with carbogen. The pO2 distribution was shifted to higher pO2 values in the tumors whatever the treatment; the shift was greater for perflubron emulsion plus carbogen. The low pO2 values (< 0.4 kPa) were lost for the HRT18 cells. A correlation (EMT6, HRT18) or a link (Na11+) between the radiosensitization and the oxygen tension measurements was found for values below 1.07 or 1.33 kPa. A trend between the percentage of hypoxic cells and pO2 measurements was found taking into account pO2 measurements comprised between 0.27 and 0.67 kPa.

In vivo measurement of the potential doubling time by flowcytometry in oropharyngeal cancer treated by conventional radiotherapy.

PURPOSE: Experimental and clinical studies suggest that the pre-treatment potential doubling time could be predictive of tumor control in patients treated by conventional radiotherapy and could help to identify the rapidly growing tumors for which accelerated radiotherapy is required. METHODS AND MATERIALS: To test this hypothesis, we studied prospectively 48 patients with a squamous cell carcinoma of the oropharynx and treated by conventional radiotherapy (70 Gy/7 weeks). The duration of S phase, the labeling index and the potential doubling time were obtained by flowcytometry measurements of a tumor biopsy obtained after injection of 200 mg bromodeoxyuridine to the patient. RESULTS: Three parameters were significantly associated with an increased risk of relapse namely the tumors size (T4; p < 0.01), the nodal status (> or = N2; p < 0.05) and the site of the primary within the oropharynx (p = 0.08). The S phase, labeling index, DNA index and potential doubling time were not significantly associated with an increased risk of relapse. However when considering only the T2 subgroup of patients, high labeling indexes and short potential doubling time were associated with an increased risk of relapse: the mean pre-treatment potential doubling time of the tumors which relapsed was 3.21 versus 5.5 days when there was no evidence of local relapse (p < 0.05). The mean labeling index for the group of tumors associated with a tumor recurrence was 11.7% compared to 7.3% when there was no evidence of relapse (p = 0.02). CONCLUSION: Factors other than proliferation play a role in determining the outcome of oropharyngeal cancers treated by conventional radiotherapy. However there was a significant correlation between short potential doubling time, high labeling index and tumor recurrence in the T2 subgroup of patients. The finding of significance for potential doubling time and labeling index in the T2 subset of tumors may be a reflexion of the more homogeneous nature of these tumors with regard to prognostic variables.

Radiosensitivity, blood perfusion and tumour oxygenation after perflubron emulsion injection.

The effect of 90% and/or 100% w/v perflubron (perfluorooctyl bromide (PFOB); Alliance Pharmaceutical Corp.) emulsions on radiosensitivity, tumour relative perfusion and oxygenation was studied using EMT6 tumours in nude mice. Perflubron (2-15 ml/kg) emulsion was injected. The mice inhaled carbogen for 30 min and 60 min prior to irradiation. The radiosensitizing effect of the 90% w/v emulsion was maximal at 4 ml/kg. The tumour relative perfusion diminished after injection of both 100% and 90% w/v emulsions in carbogen-breathing mice at a dose of 15 ml/kg. This drop could explain the lack of efficiency of these treatments at this high concentration. Lastly, tumour oxygenation was increased after administration of perflubron emulsion plus carbogen.

Predictive value of in vitro radiosensitivity parameters in head and neck cancers and cervical carcinomas: preliminary correlations with local control and overall survival.

PURPOSE: To determine whether in vitro radiosensitivity parameters are predictive of treatment outcome. METHODS AND MATERIALS: Biopsies were obtained from patients with head and neck cancers (57) and cervical carcinomas (20) and in vitro radiosensitivity parameters were obtained using the CAM plate assay. RESULTS: In most cases (75%) patients were treated with radiation alone. The median follow up was 461 days. When the whole group of head and neck cancers and cervical carcinomas was considered, patients with a SF2 value below 0.36 had a higher 2-year local control rate (93% versus 68%) and a higher 2-year survival rate (71% vs. 62%) than those with SF2 values above that threshold, but differences were not significant. These trends persisted when head and neck cancers were considered alone with a higher local control rate (86% vs. 67%) and a higher survival rate (75% vs. 52.5%) obtained for patients with a SF2 value below 0.36. When the alpha value was evaluated for the whole group of patients a significantly higher local control rate (80.5% vs. 40.5%) and overall survival rate (71% versus 37.5%) at 2 years were obtained for patients with alpha values above 0.07 Gy-1. When only the group of head and neck cancers was considered, local control rate was significantly higher (79% vs. 33%) but overall survival rate (65.5% vs. 33%) was not significantly higher for alpha values above 0.07 Gy-1. CONCLUSION: These results are encouraging but need to be confirmed with a larger number of patients with a longer follow-up.

The effect of the hypoxic cell drug SR-4233 alone or combined with the ionizing radiations on two human tumor cell lines having different radiosensitivity.

The influence of the hypoxic cell drug, SR-4233, alone and/or combined with ionizing radiations on the survival of two human cell lines having very different intrinsic radiosensitivity was analysed. The killing effect of SR-4233 in hypoxia was similar for both cell lines. Twenty microM SR-4233 under hypoxic conditions had a killing effect equivalent to 6.6 Gy for the poorly sensitive cell line (HT29) and equivalent to 3.9 Gy for the highly sensitive cell line (SW48). The effect of SR-4233 and ionizing radiations on these two cell lines was tested in vitro: the cells were incubated for one hour in hypoxia with or without 20 microM SR-4233 and then irradiated in air. The HT29 cells that survived treatment with SR-4233 are more radiosensitive than untreated cells. However, their radiosensitivity is similar to that of cells that have been given a dose of 6.6 Gy. This suggests that SR-4233 acts additively, rather than as a radiosensitiser. As SR-4233 acts selectively in hypoxia, these results appear encouraging for the treatment of poorly-radiosensitive human tumors.

[Measurement of the kinetics of cell proliferation of cancer of the oropharynx in vivo by the incorporation of bromodeoxyuridine and flow cytometry]. / Mesure de la cinétique de prolifération in vivo des cancers de l'oropharynx par incorporation de bromodesoxyuridine et cytométrie de flux.

Forty-six tumors from patients with oropharyngeal carcinoma were analysed by flow cytometry after injection of bromodeoxyuridine (Budr) for the labelling index, the duration of S phase and the potential doubling time (Tpot). The results show large variations in Tpot (from 2.6 to 16.7 days) among these tumors from the same site and with the same histology. The variations in Tpot were not significantly related to TNM status and differentiation grade. However, aneuploid tumors had statistically significant shorter Tpot. The predictive value of Tpot regarding the response to radiotherapy is currently under investigation.

Estimation of clonogenic cell fraction in primary cultures derived from human squamous cell carcinomas.

Tumor clonogenic cell content is believed to play an important role in the outcome of radiotherapy. However, there is no proven method to assess the number of clonogens in human tumors accurately. All currently available assays employ in vitro plating efficiency or in vivo TD50 (the average number of cells needed to induce tumors in 50% of injected mice) to estimate the tumor clonogenic ability. In this study, a monolayer mass primary culture system was used to estimate the clonogenic cell fraction in human tumors. For this purpose, 25 growth curves were performed for 25 tumor specimens derived from 21 head and neck and 4 cervical squamous cell carcinomas. The exponential portion of each growth curve was extrapolated through the ordinate (day 0) to estimate the clonogenic cell fraction; this method is only an estimate because it assumes no lag phase before exponential growth of clonogenic cells. The mean clonogenic cell fraction, expressed as clonogens/tumor cells inoculated, was relatively low (mean: 0.71%, range: 0.11-9.28), and the variation was wide (coefficient of variation = 148%). On the other hand, the doubling time of the growing population was 1.46 days and exhibited a very narrow range (0.98-2.24, coefficient of variation = 24%). The mean and range of clonogenic cell fraction were found to be in agreement with published values of soft agar colony forming efficiencies in both murine and human tumors. However, further investigation is necessary to determine how accurately this method measures the relative clonogenic cell content in human tumors. Clinical correlations between clonogenic cell fraction values and the response to radiotherapy are still too early to determine.

The relationship between radiosensitivity and repair of potentially lethal damage in human tumor cell lines with implications for radioresponsiveness.

The relationship between intrinsic radiosensitivity and repair capacity was studied for 22 human tumor cell lines in vitro. The experimental material was taken from 19 published papers. Parameters from three radiobiological models were used to assess this relationship: the one-hit multitarget model (D0 and n), the linear-quadratic model (alpha and beta), and the mean inactivation dose (D). Data were obtained for cells in three stages: exponentially growing cells (exp), plateau-phase cells plated immediately after irradiation (ip), and plateau-phase cells plated after completion of PLD repair (dp). No significant difference was found between radiosensitivity of exp and ip cells. There was no correlation between repair capacity and intrinsic radiosensitivity assessed with plateau-phase cells plated immediately after irradiation. The correlation studies between intrinsic radiosensitivity or repair capacity and clinical responsiveness were achieved by assigning cell lines to one of three groups of decreasing in vivo radioresponsiveness: highly, medium, and poorly responsive. There was a significant correlation between radiosensitivity and radioresponsiveness, but no correlation between repair capacity and radioresponsiveness. The average repair capacity was about 0.6 Gy, in terms of D. Three parameters, the mean inactivation dose of exponentially growing cells, of plateau-phase cells plated immediately after irradiation, and of plateau-phase cells plated after completion of PLD repair, could be used equally to assess the relationship between in vitro data and radioresponsiveness. The present results are compared to those obtained in a similar study on a group of 48 nontransformed fibroblast cell strains.

Growth rate, kinetics of tumor cell proliferation and long-term outcome in human breast cancer.

Thymidine labelling index (LI) was prospectively assayed in vitro in a series of 128 consecutive patients with breast cancer. The follow-up was longer than 15 years for all patients. The distribution of LI was log normal and patients were subdivided into 3 groups (patients with LI = m +/- sigma, lower than m - sigma and greater than m + sigma where m is the geometrical mean). The incidence of relapse and death remained significantly lower in the group with a low LI than in the 2 other groups, whereas the difference between the 2 other groups faded away when follow-up exceeded 10 years. Multivariate analyses show that LI is one of the two most important independent prognostic indicators for relapse or death, the other being histological grading. Data concerning 2,648 patients treated at our Institute, prior to the introduction of chemotherapy into treatment protocols, have been used to investigate the influence of tumor growth rate on the probability of distant dissemination. A model of the natural history was used, in order to generate metastasis appearance curves, in 3 subgroups of patients, according to the value of the tumor doubling time (TDT). Our results show that after a follow-up exceeding 8 years, there is no longer any difference between the subgroups of patients with rapid or intermediate growth rate, whereas after 25 years large and highly significant differences in relapse and survival between the slow-growing tumors and the other two subgroups still remain. These two sets of data concur to show that tumor growth rate or proliferation rate correlates with the probability of metastatic dissemination.

The effect of the oxic cell sensitizer, beta-ara A on human fibroblasts in plateau and in exponential growth phases.

Before an oxic cell sensitizer such as beta-ara A (a DNA-dependent DNA polymerase inhibitor) can be used in cancer treatment, it is essential to know both the influence of this type of drug on certain critical normal tissues and the role of proliferation kinetics in the radiosensitizing capacity. The biological system chosen for this in vitro study was the human fibroblast cell line HF19. Cells were studied in plateau phase and in the exponential growth phase. Cells were incubated with beta-ara A for 7 hr (1 hr before and 6 hr after irradiation). beta-ara A was extremely toxic to growing cells (concentrations ranging from 200 to 1000 microM), but no detectable effect was found on plateau-phase cells (up to 4000 microM). However, for a given drug concentration, the radiosensitizing effect (Sensitizing Enhancement Ratio SER) was very similar for growing and plateau phase cells (SER measured with Ds ratio was about 1.7 for a concentration of 500 microM). The enhancement ratio depended on the radiation dose; it was relatively higher for low doses. This can be explained by a differential effect of the drug on the alpha and beta components of the survival curve. Only the alpha component was increased.

The initial slope of human tumor cell survival curves: its modification by the oxic cell sensitizer beta-arabinofuranosyladenine.

The initial slope of the survival curve, which is a characteristic of each tumor cell line, varies with the histological group of the tumor. It is one of the factors on which clinical radioresponsiveness depends. The DNA dependant DNA polymerase inhibitor beta-ara A acts as an oxic cell sensitizer. This study was carried out on human tumor cell lines to look for a correlation between the degree of radiosensitization induced by beta-ara A and the radiosensitivity of a given cell line. Six human tumor cell lines with different radiosensitivities were used (the survival rate at 2 Gy and D ranged from 20 to 73% and from 1.2 to 3.2 Gy, respectively). beta-ara A had a major toxic effect on all cell lines but this varied greatly from one cell line to another and was concentration dependant; this toxic effect was taken into account when calculating the surviving fractions. For all cell lines, beta-ara A acted as an oxic radiosensitizer and the radiosensitization was concentration dependant. Analysis of the survival curves of the 6 cell lines using the linear quadratic model showed that concentrations of beta-ara A between 200 and 1000 microM induced an increase in the linear component while the quadratic component underwent no systematic change. The sensitizing enhancement ratio (SER) measured from the Ds ratios, varied greatly from one line to another. For example, at a concentration of 500 microM, the extreme values of Ds ratios were 1.5 and 2.6. The radiosensitization is greater, the higher the radiosensitivity of the cell line studied during exponential growth. The results do not favor the use of beta-ara A in the treatment of intrinsically radioresistant human tumors.

Is the proportion of hypoxic cells in a solid tumour dependent on the host (mouse or rat)?

Hypoxic cell fraction was measured in a human tumour xenografted on two different animal species: mouse and rat. These results suggest that, while the Na11 tumour response to radiation depends on the host (nude mouse, nude rat), the proportion of hypoxic cells tested with the growth delay assay is independent of the host.

Initial slope of radiation survival curves is characteristic of the origin of primary and established cultures of human tumor cells and fibroblasts.

The published survival curves of 110 human tumor cell lines and 147 nontransformed human fibroblast strains have been reanalyzed using three different statistical methods: the single hit multitarget model, the linear-quadratic model, and the mean inactivation dose. The 110 tumor cell lines were classified in two ways: (a) into three categories defined by clinical radiocurability criteria, and (b) into seven categories based on histopathology. The 147 fibroblast strains were divided into eight genetic groups. Differences in the radiosensitivities of both the tumor cell and fibroblast groups could be demonstrated only by parameters that describe the slopes of the initial part of the survival curves. The capacity of the survival level to identify significant differences between groups was dose dependent over the range 1 to 6 Gy. This relationship showed a bell-shaped curve with a maximum at 1.5 Gy for the tumor cell lines and 3 Gy for the fibroblasts. Values for intrinsic radiosensitivity for a number of groups of tumors have also been obtained by primary culture of tumor cells. These values are strictly comparable to those obtained by clonogenic methods. This confirms that intrinsic radiosensitivity is a determinant of the response of tumor cells to radiotherapy and suggests that tissue culture methods may be used as a predictive assay.

Distribution of radiation sensitivities for human tumor cells of specific histological types: comparison of in vitro to in vivo data.

The radiosensitivities of human tumor cell lines, grouped into 6 histological categories, have been studied using data from the published literature. The parameters alpha, beta, n, D0, D, and the surviving fraction to 2 Gy (S2) and 8 Gy (S8) were calculated. Only the two parameters mainly derived from the initial part of the survival curve, alpha and D, together with S2, provided data which were correlated with the clinical radioresponsiveness of each histological group. Thus, there are intracellular factors which influence clinical radioresponsiveness whose relative importance varies from one histological cell type to another. The value of D gave the most precise characterization of the average group radiosensitivity. It was possible to compare the in vivo radiosensitivities of non-severely hypoxic cells with those of tumor cells irradiated in vitro for 7 tumor lines grown as xenografts in mice. The average radiosensitivity was 1.9 times less in vivo than in vitro. This difference indicates that, in addition to the intrinsic factors of radioresistance demonstrated in vitro, and independently of severe hypoxia, there are other factors which specifically reduce radiosensitivity in vivo.

Radiosensitizing and cytotoxic properties of misonidazole on glutathione synthetase deficient human fibroblasts.

The cytotoxic and radiosensitizing effects of misonidazole have been studied on glutathione synthetase deficient fibroblasts and on their controls. At any concentration from 0.1 to 4 mM, deficient cells are more sensitive to the cytotoxic effect of misonidazole than the control cells. The differential effect between the two cell strain concerns both the shoulder and the slope of the survival curve, thus suggesting that NPSH play a role in the determination of misonidazole cytotoxicity. Like oxygen, misonidazole clearly sensitizes deficient cells to a lesser extent than control cells. For both cell strains, the maximum sensitizing effect of misonidazole is very close to that of oxygen (1.5 and 1.5 for deficient cells, 2.8 and 2.9 for control cells, respectively). The sensitizing effect of misonidazole appears in the same concentration range for both cell strains, with a maximal effect at lower concentrations for deficient cells.

Detection in situ of gamma-ray-induced DNA strand breaks in single cells: enzymatic labelling of free 3'-OH ends.

We report a procedure allowing the detection and counting of free 3'-OH DNA strand extremities in single cells in situ. Terminal transferase (TdT) catalysed the incorporation of 3H-dGMP into fixed nuclei of human colonic adenocarcinoma cells (HT29), using free 3'-OH ends as initiator. Radioactivity was detected by autoradiography and determined quantitatively with a rapid image-processing system for grain counting. The initiator activity for TdT increases with the dose of gamma-rays in the dose range 2.5-20 Gy.

Reduced PLD repair ability in glutathione synthetase deficient human fibroblasts after UV irradiation.

Using a human cell strain deficient in glutathione synthetase and a related control, the role of glutathione in repair mechanisms has been investigated. UV light has been used in order to avoid the interaction between thiols and free radicals. When potentially lethal damage repair is completed, deficient cells in plateau phase exhibit smaller surviving fractions than do control cells. The ratio of surviving fractions in control/deficient cells is about 2 for the same radiation dose. These results indicate that thiols and especially GSH are involved in repair mechanisms.