O. Sapora

RBE-LET relationships for cell inactivation and mutation induced by low energy protons in V79 cells: further results at the LNL facility.

PURPOSE RBE-LET relationships for cell inactivation and hprt mutation in V79 cells have been studied with mono-energetic low-energy proton beams at the radiobiological facility of the INFN-Laboratori Nazionali di Legnaro (LNL), Padova, Italy. MATERIALS AND METHODS V79 cells were irradiated in mono-layer on mylar coated stainless steel petri dishes, in air. Inactivation data were obtained at 7.7, 34.6 and 37.8 keV/microm and hprt mutation was studied at 7 7 and 37.8 keV/microm. Additional data were also collected for both the end points with the proton LET already considered in our previous publications, namely 11.0, 20.0 and 30.5 keV/microm. RESULTS A maximum in the RBE-LET relationship for cell inactivation was found at around 31 keV/microm, while the RBE for mutation induction increased continuously with LET. CONCLUSIONS The proton RBE-LET relationship for cell inactivation is shifted to lower LET values compared with that for heavier ions. For mutation induction, protons of LET equal to 7.7keV/microm gave an RBE value comparable with that obtained by helium ions of about 20 keV/microm. Mutagenicity and lethality caused by protons at low doses in the LET range 7.7-31 keV/microm were proportional, while the data at 37.8 keV/microm suggest that this may not hold at higher LET values.

RBE-LET Relationship for the Survival of V79 Cells Irradiated with Low Energy Protons

The survival of V79 Chinese hamster cells irradiated with proton beams with energies of 0.73, 0.84, 1.16, 1.70 and 3.36 MeV, corresponding to LET values, evaluated at the cell midplane, of 34.5, 30.4, 23.9, 17.8 and 10.6 keV/micron respectively, have been studied in the dose range 0.5-6.0 Gy. As a reference, the survival curve obtained with 200 kV X-rays was used. The initial shoulder, typical of survival curves obtained with sparsely ionizing radiation, decreases as the LET increases and completely disappears at 23.9 keV/micron. This value corresponds to the maximum of the RBE, expressed as the initial slope ratio. In the energy range we have considered, the RBEs for protons are higher than those reported for other ions of comparable LET and the RBE-LET relationship results shifted to lower LET values. Our data seem to indicate that the RBE-LET curve depends on the type of radiation and this could imply that LET is not a good reference for the dose-effectiveness relationship.

Bcl-2 overexpression and hypoxia synergistically act to modulate vascular endothelial growth factor expression and in vivo angiogenesis in a breast carcinoma line.

We have previously demonstrated that bcl‐2 overexpression enhances the metastatic potential of the MCF7 ADR human breast cancer cell line resistant to adriamycin by inducing metastasis‐associated properties. To further elucidate the relationship between bcl‐2 expression and the metastatic potential of the MCF7 ADR line, we evaluated whether bcl‐2 could be also involved in the modulation of the angiogenic phenotype. Four bcl‐2‐overex‐pressing clones, a control transfectant clone, and the MCF7 ADR parental line were used for in vitro and in vivo experiments. Bcl‐2 overexpression enhanced the synthesis of the hypoxia‐stimulated VEGF protein and mRNA. Northern blot analysis demonstrated an increased VEGF mRNA expression in bcl‐2‐overex‐pressing clones, and reverse transcription‐polymer‐ase chain reaction showed higher levels of the VEGF121 and VEGF165 mRNA isoforms, which are the most active in eliciting angiogenesis. When incorporated into matrigel, supernatants of bcl‐2‐trans‐fected cells cultured under hypoxic conditions induced an increased angiogenic response in C57BL/6 mice compared with that of control clone. Tumors from bcl‐2 transfectants demonstrated increased VEGF expression and neovascularization as compared to the parental line, whereas the apoptosis in in vivo xenografts was similar in control and bcl‐2 transfectants. The effect of bcl‐2 on angiogenesis was not mediated by p53 protein. These results demonstrate that bcl‐2 and hypoxia can act synergis‐tically to modulate VEGF expression and the in vivo angiogenic response in the MCF7 ADR line.—Biroccio, A., Candiloro, A., Mottolese, M., Sapora, O., Albini, A., Zupi, G., Del Bufalo, D. Bcl‐2 overexpression and hypoxia synergistically act to modulate vascular endothelial growth factor expression and in vivo angiogenesis in a breast carcinoma line. FASEB J. 14, 652–660 (2000)

Inactivation and Mutation Induction in V79 Cells by Low Energy Protons: Re-evaluation of the Results at the LNL Facility

During the upgrading of the radiobiological facility at the Laboratori Nazionali di Legnaro (LNL) we found that uncorrected values of the proton energy were used in the past. This circumstance prompted us to perform the re-evaluation of the physical parameters for all the proton beams used in our previous radiobiological investigations (Belli et al. 1987) and, subsequently, the re-evaluation of all our previous dose-response curves for inactivation and mutation induction (Belli et al. 1989, 1991). This re-evaluation leads to significant changes in the dose-response curves and in the RBE-LET relationships only at the two lowest energies (highest LET) used. These two points are not reliable for the identification of a peak in RBE-LET relationship for cell inactivation. In spite of that, the extent of the changes is not such as to modify the general conclusion previously drawn, pointing out that there is a LET range where protons are more effective than alpha-particles.

Evidence for an increase in water concentration in bilayers after oxidative damage of phospholipids induced by ionizing radiation

The two membrane fluorescent probes 2-dimethyl-amino-6-lauroyl-naphthalene (Laurdan) and 2-dimethylamino-6-propionyl-naphthalene (Prodan) have been used to study the molecular basis of the damage induced in phospholipid membranes by ionizing radiation. Laurdan and Prodan display a spectral sensitivity to the polarity of their environment, showing a red shift of both excitation and emission spectra with increase of the polarity of their environment. Owing to their chemical differences, the two probes are anchored in the membrane with different strengths. In aqueous environments Laurdan is not fluorescent while Prodan shows appreciable fluorescence. Laurdan and Prodan show an opposite response to oxidative damage produced in phospholipid bilayers by ionizing radiation. The results support the model recently developed of water penetration in the bilayer as a consequence of oxidative damage.

Proton irradiation facility for radiobiological studies at a 7 MV Van de Graaff accelerator

Abstract This paper describes the proton irradiation facility built up at the 7 MV Van de Graaff accelerator of the INFN-Laboratori Nazionali di Legnaro for radiobiological studies. The system mainly consists of two vacuum chambers, a fast tantalum shutter for intercepting the beam and a beam line containing the beam collimator arrangement and ending with a thin window for the beam extraction in air. Each chamber houses a Ta beam collimator, an Au scatterer foil for the broadening and uniformization of the proton beam over the cell samples and a solicon surface barrier detector for beam monitoring purposes. The test of the facility performance is discussed. With the double scattering system a counting rate in air as low as hundreds of protons per second and a fluence heterogeneity less than 5% within a circular field of 15 mm diameter were obtained. The first results of survival studies of Chinese hamster lung cells (V79-753B) irradiated with a 1.1 MeV proton beam are presented. For comparison, results obtained irradiating the cells with 200 kV X-rays are reported, too.

Relationships Between Cell Killing, Mutation Induction and DNA Damage in X-irradiated V79 Cells: The Influence of Oxygen and DMSO

The relationships between cell killing, mutation induction and DNA double (dsb) and single (ssb) strand breaks have been studied in V79 cells irradiated with X-rays under oxic and anoxic conditions in the presence and in the absence of dimethylsulphoxide (DMSO). Curvilinear relationships were found between all pairs of endpoints, except for dsb versus ssb. Statistical analysis of experimental data has shown that in the absence of DMSO there is evidence of good correlations between cell killing, mutation induction and dsb in oxic and anoxic conditions. However, when DMSO was present, no significant correlation was found. In the presence of oxygen DMSO always exerts a protective effect while in anoxia it is generally much less protective and induces a strong sensitization with respect to mutation induction. Possibly DMSO acts not only as a radical scavenger but also as an agent inducing chromatin relaxation and/or under anoxia, forming highly mutagenic short-term radicals. The present data suggest that lethal and mutational events are at least partially independent and not proportional to the initial number of DNA breaks. This may imply that either other kinds of lesions are involved in cell lethality and mutability, or dose-dependent repair mechanisms of dsb have to be considered.

CHOLESTEROL PROTECTS THE PHOSPHOLIPID BILAYER FROM OXIDATIVE DAMAGE

The measurement of fluorescence lifetime distribution of 1,6-diphenyl-1,3,5-hexatriene is used for the detection of oxidative damage produced in phospholipid membranes by ionizing radiation. The recently developed method is based on the linear relationship between the width of the probe lifetime distribution and the logarithm of the dose. The molecular origin of the damage resides in the production of hydroperoxide residues at the level of acyl chains double bonds. A chemiluminescence assay was used to quantitate the amount of produced hydroperoxides. Consequences of the produced damages include an increased disorder in the upper portion of the bilayer, accompanied by the penetration of water molecules. In the presence of the physiological concentration of cholesterol in phopholipid bilayers, the amount of hydroperoxides produced by ionizing radiation is dramatically reduced. The packing effect of cholesterol in phopholipid bilayers is well recognized, as well as its influence on the reduction of water concentration in the bilayer. The dramatic reduction of hydroperoxides concentration observed when irradiation is performed in the presence of cholesterol probably originates from a steric hindrance to the radical chain reaction through the unsaturated lipids due to the presence of cholesterol.

DNA fragmentation in V79 cells irradiated with light ions as measured by pulsed-field gel electrophoresis. I. Experimental results.

Purpose : To compare the results on DNA fragmentation induced in Chinese hamster V79 cells by various doses of γ-rays and low-energy protons and helium-4 ions. Materials and methods : V79 cells were irradiated as monolayers with monoenergetic protons and helium-4 ions; γ-rays were used as the reference radiation. DNA double-strand breaks were evaluated by calibrated pulsed-field gel electrophoresis using conditions covering the range 5.7 Mbp-23.1 kbp. Results : The fragment-counting method gave double-strand breaks yields and the relative biological effectiveness higher than those obtained by the fraction of activity released method. The frequency distribution of fragments showed that protons and helium ions induced more fragments below the Mbp region than did γ-rays at the same dose. The distributions for both the irradiated and non-irradiated samples clearly appeared to be non-random. Conclusion : Differences were observed in the yield and spatial correlation, at a molecular size scale characteristic of loop dimensions, of the double-strand breaks induced by γ-rays and by light ions. These effects may have a role in the observed different cell response to these radiations.

Alterations in erythrocyte membrane lipids induced by low doses of ionizing radiation as revealed by 1,6-diphenyl-1,3,5-hexatriene fluorescence lifetime

Damage in membrane lipids induced by low doses of ionizing radiation in the presence of oxygen has been detected in rabbit erythrocyte ghosts labelled with 1,6-diphenyl-1,3,5-hexatriene (DPH). Multifrequency phase and modulation fluorometry was used to measure DPH fluorescence lifetime. This technique is particularly suited for the observation of heterogeneous fluorescence decays. DPH decay in erythrocyte membranes is described by a two-component continuous distribution of lifetimes. The value of the distribution width of the long-lived component is found to be affected by radiation-induced membrane lipid damage at doses as low as 0.5 Gy, well within the dose range used to measure cell survival. The width of the DPH lifetime distribution decreases when the ghosts are irradiated in the presence of oxygen. Such a decrease is a linear function of the logarithm of the dose. After a dose of 110 Gy and above, the fractional intensity of the short-lived component of the DPH decay increases linearly, indicating severe membrane damage. Experiments performed in the absence of oxygen do not show any change in the fluorescence parameters up to a dose of 550 Gy. The molecular identification of the produced damage has not been accomplished, but the necessity of oxygen to observe the damage suggests that hydroperoxides and lipids crosslinks are produced.