Torbjörn Hartman

Rejoining of DNA double-strand breaks induced by accelerated nitrogen ions

Rejoining of radiation-induced DNA double-strand breaks (dsb) was measured in cultured cells with pulsed-field gel electrophoresis after radiation doses in the range of 5-30 Gy. Human glioma, U-343MG and Chinese hamster, V79, cells were irradiated with either accelerated nitrogen ions of high linear energy transfer, LET approximately 125 keV/ microns, or photons from 60Co. The induction frequencies of dsb were similar for the two radiation qualities with a relative biological effectiveness, RBE, of 0.90 and 0.89 for the human and hamster cell lines respectively. The biphasic rejoining kinetics differed significantly between the two radiation qualities when studied in the human glioma cells. The difference was seen within the first hour after irradiation and after 6 h there were considerable differences in both the total amount of unrejoined dsb and the fraction of dsb rejoined during the slow phase. When rejoining was analysed 20-22 h after irradiation, the nitrogen ions gave 2.5-2.9 times more residual dsb than the gamma photons. The results for the hamster V79 cells were, up to 2h after irradiation, similar, but the difference between the two radiation qualities was less accentuated. In summary, similar initial yields of dsb after exposure of cells to high or low LET resulted in both radiation quality and cell type-dependent differences when the rejoining of these breaks were compared.

Chemistry and biology of some low molecular weight boron compounds for Boron Neutron Capture Therapy

Boronated DNA targeting agents are especially attractive candidatesfor BNCT because they may deliver boron-10 tothe nuclei of tumor cells. Numerous boron-containing analogshave been synthesized and some have shown promisingresults in initial biological tests. One of themost challenging tasks in this special field ofresearch remains the finding of suitable targeting strategiesfor the selective delivery of boron rich DNA-intercalator/alkylatorto tumor cells. Synthetic and biological studies ofboron compounds suitable for DNA-binding are reviewed.The amino acid p-boronophenylalanine (BPA) is presently ofconsiderable clinical interest. Other boronated amino acids mightalso be candidates for BNCT either per se,as part of part of tumor-seeking peptides orconjugated to targeting macromolecules. A large number ofboronated L- and D-amino acids with varying liphophicilityand sterical requirements are now available for evaluation.Recent synthetic and biological studies of aromatic boronoaminoacids, carboranylamino acids and carboranyl amines are alsoreviewed.

Strategy for boron neutron capture therapy against tumor cells with over-expression of the epidermal growth factor-receptor.

PURPOSE Gliomas, squamous carcinomas and different adenocarcinomas from breast, colon and prostate might have an increased number of epidermal growth factor (EGF) receptors. The receptors are, in these cases, candidates for binding of receptor specific toxic conjugates that might inactivate cellular proliferation. The purpose of this study was to evaluate whether it is reasonable to try ligand-dextran based conjugates for therapy. METHODS AND MATERIALS EGF or TGF alpha were conjugated to dextran and binding, internalization, retention and degradation of eight types of such conjugates were analyzed in EGF-receptor amplified glioma cells. The conjugates were labelled with radioactive nuclides to allow detection and two of the conjugates were carrying boron in the form of carboranyl amino acids or aminoalkyl-carboranes. Comparative binding tests, applying 125I-EGF, were made with cultured breast, colon and prostate adenocarcinoma, glioma and squamous carcinoma cells. Some introductory tests to label with 76Br for positron emission tomography and with 131I for radionuclide therapy were also made. RESULTS The dextran part of the conjugates did not prevent receptor specific binding. The amount of receptor specific binding varied between the different types of conjugates and between the tested cell types. The dextran part improved intracellular retention and radioactive nuclides were retained for at least 20-24 h. The therapeutical effect improved when 131I was attached to EGF-dextran instead of native EGF. CONCLUSION The improved cellular retention of the ligand-dextran conjugates is an important property since it gives extended exposure time when radionuclides are applied and flexibility in the choice of time for application of neutrons in boron neutron capture therapy (BNCT). It is possible that ligand-dextran mediated BNCT might allow, if the applied neutron fields covers rather wide areas around the primary tumor, locally spread cells that otherwise would escape treatment to be inactivated.

Radiation doses to the cell nucleus in single cells and cells in micrometastases in targeted therapy with 131I labeled ligands or antibodies

PURPOSE The aim of this study was to theoretically investigate how the radiation dose to cell nuclei depends on the subcellular position of (131)I. The influence of the size of the cells and crossfire irradiation in clusters of cells was also studied. METHODS AND MATERIAL Using data describing the dose rate around a point source of (131)I, we calculated the dose distributions inside and around cell models of different sizes. The assumed positions of (131)I were on the cellular or nuclear membrane, in the cytoplasm, in the nucleus, or spread in the whole cell. The mean doses to the nucleus of the targeted cell and to the nuclei of its neighbors were calculated using the dose distributions. RESULTS The dose distributions inside a single targeted cell showed very different distribution profiles depending on the subcellular position of the (131)I. Targeting the nucleus instead of the cellular membrane could increase the dose to the nucleus 10-fold. Crossfire irradiation can be the major contributor to the nuclear dose in clusters of more than six cells. CONCLUSIONS Dosimetry without microscopic considerations is inadequate for targeted radionuclide therapy of disseminated or clustering tumor cells exposed to (131)I. Therapeutic doses could be achieved, even in single cells, when (131)I was positioned near, or inside the cell nucleus, or when the clusters were large enough.

Cross-fire doses from β-emitting radionuclides in targeted radiotherapy. A theoretical study based on experimentally measured tumor characteristics

A mathematical model based upon histological findings of cell cluster distributions in primary breast cancers and lymph node metastases was developed. The model is unique because it accounts for tumor cell cluster formations within both primary tumors and metastases. The importance of inter-cell cluster cross-fire radiation dose for beta-emitting radionuclides of different energies was studied. The cell clusters were simulated as spheres with 15, 25 and 50 microm radii having a homogeneous radioactivity distribution. The self-dose as well as the dose distribution around the spheres was calculated for seven radionuclides, (90)Y, (188)Re, (32)P, (186)Re, (159)Gd, (131)I and (177)Lu using the GEANT4 Monte Carlo code. Generally, the self-dose was decreasing with increasing energy of the emitted beta particles. An exception was (188)Re which, compared to (32)P, had higher beta energy as well as higher self-dose. This was due to the higher emission of conversion and Auger electrons in the (188)Re-decay. When the cell clusters had a mean distance that was shorter than the maximum range of beta-particles, then the inter-cluster cross-fire radiation contributed significantly to the absorbed dose. Thus, high-energy beta-particles may, in spite of a low self-dose to single clusters, still be favorable to use due to the contribution of inter-cluster cross-fire radiation.

Formation and repair of clustered damaged DNA sites in high LET irradiated cells

Purpose: Radiation with high linear energy transfer (LET) produces clustering of DNA double-strand breaks (DSB) as well as non-DSB lesions. Heat-labile sites (HLS) are non-DSB lesions in irradiated cells that may convert into DSB at elevated temperature during preparation of naked DNA for electrophoretic assays and here we studied the initial formation and repair of these clustered damaged sites after irradiation with high LET ions. Materials and methods: Induction and repair of DSB were studied in normal human skin fibroblast (GM5758) after irradiation with accelerated carbon and nitrogen ions at an LET of 125 eV/nm. DNA fragmentation was analyzed by pulsed-field gel electrophoresis (PFGE) and by varying the lysis condition we could differentiate between prompt DSB and heat-released DSB. Results: Before repair (t = 0 h), the 125 eV/nm ions produced a significant fraction of heat-released DSB, which appeared clustered on DNA fragments with sizes of 1 Mbp or less. These heat-released DSB increased the total number of DSB by 30–40%. This increase is similar to what has been found in low-LET irradiated cells, suggesting that the relative biological effectiveness (RBE) for DSB induction will not be largely affected by the lysis temperature. After 1–2 hours repair, a large fraction of DSB was still unrejoined but there was essentially no heat-released DSB present. Conclusions: These results suggest that high LET radiation, as low LET gamma radiation, induces a significant fraction of heat-labile sites which can be converted into DSB, and these heat-released DSB may affect both induction yields and estimates of repair.

In vitro determination of toxicity, binding, retention, subcellular distribution and biological efficacy of the boron neutron capture agent DAC-1

In boron neutron capture therapy (BNCT), 10B is delivered selectively to the tumour cells and the nuclide then forms high-LET radiation (4He2+ and 7Li3+) upon neutron capture. Today much research is focused on development of a variety of boron compounds aimed for BNCT. The compounds must be thoroughly analysed in preclinical tests regarding basic characteristics such as binding and subcellular distribution to enable accurate estimations of dose-modifying factors. DAC-1,2-[2-(3-amino-propyl)-1,2-dicarba-closo-dodecaboran (12)-1-yl-methoxy]- 1,3-propanediol was synthesized at our laboratories and the human colon carcinoma cells LS-174T were used as an in vitro model. The boron compound showed a remarkable intracellular accumulation, 20-100 times higher than the boron content in the culture medium, in cultured cells and was not removed by extensive washes. Approximately half of the boron taken up also remained within the cells for at least 4 days. The DAC-1 compound alone was not toxic at boron concentrations below 2.5 micrograms B/g. The intracellular distribution of the boron compound was investigated by subcellular fractionation experiments and low pH treatments. It is possible that DAC-1 binds to some intracellular molecules or to membranes connected with organelles in the cytoplasm or even to the inside of the outer cell membrane. Another possibility is that the compound, due to the somewhat lipophilic properties, is embedded in the membranes. Thermal neutron irradiations were carried out at the Brookhaven Medical Research Reactor (BMRR). At a survival level of 0.1, DAC-1 + thermal neutrons were about 10.5 times more effective in cell inactivation than the thermal neutrons alone. Monte Carlo calculations gave a mean value of the 10B-dependent specific energy, the dose, of 0.22 Gy. The total physical dose during irradiation of DAC-1-containing cells with a neutron fluence of 0.18 x 10(12) n/cm2 was 0.39 Gy. The dose-modifying factor, at survival level 0.1, when comparing irradiation with thermal neutrons with and without DAC-1 was 3.4, while the dose-modifying factor when comparing neutron irradiations of cells with DAC-1 and irradiation of the cells with 60Co-gamma was 7.3. The results are encouraging and in vivo tests of tissue distributions and tumour uptake should now be carried out.

Uptake, toxicity and radiation effects of the boron compounds DAAC-1 and DAC-1 in cultured human glioma cells

PURPOSE To study the uptake, toxicity and radiation effects in vitro of a diol-amino acid-carborane (DAAC-1) and make comparisons with the previously studied diol-amine-carborane (DAC-1). MATERIALS AND METHODS Toxicity and radiation effects were studied with clonogenic survival, uptake by measuring the cellular boron content and the subcellular distribution was investigated after organelle separation with centrifugation. The studied cell line was human glioma U343. RESULTS DAAC-1 showed an accumulation of 1-1.5 times, compared with the culture medium, and was non-toxic up to 47 microg boron/ml. The accumulation of DAC-1 was about 90 times, but toxic effects were detectable already at the concentration 5 microg boron/ml. None of the compounds was localized in the cell nucleus. Following irradiation with thermal neutrons, DAC-1 was about 2.5 times more effective than DAAC-1 and about 4.9 times more effective than neutrons alone, at the survival level 0.2. The dose modifying factors, when compared with the neutron beam alone, were for both DAAC-1 and DAC-1 about 1.5 and about 5 when compared with 60Co-gamma-radiation. CONCLUSIONS DAAC-1 was less toxic than DAC-1 but gave less accumulation of boron. Both substances gave significant boron-dependent cell inactivation when the test cells were exposed to thermal neutrons.

Dose Enhancement in Fast Neutron Tumour Therapy Due to Neutron Captures in 10B

High energy neutrons, applied in fast neutron tumour therapy, lose energy when passing through tissue and are at the end of their trajectories captured in nitrogen, hydrogen or other normally occurring elements. If the tissue contains 10B, which has a very high cross section for capture of thermal neutrons, then disintegration products of this process, helium and lithium ions, give a dose enhancement which, if the boron is targeted to tumour cells, may be beneficial. The dose enhancement was in the present study calculated as a function of the 10B concentration in the cells and as a function of different thermal neutron fluencies. If the tumour cells contained 10 or 100 microns 10B/g the average dose enhancement was about 20 or 200 mGy respectively. This was obtained with the thermal neutron fluency 2.0 x 10(10) n/cm2. The relative biological effectiveness of the neutron capture process is unknown but assuming the factor 2, these doses correspond to 0.04 or 0.4 CGE (cobolt-60 gray equivalent) respectively, which could directly be compared to the 2-3 Gy of low-LET radiation that is daily applied in conventional radiotherapy. However, if thermal or epithermal neutron fields are directly applied to the patients a hundred times higher thermal neutron fluency can be used. This gives, in the cases with 10 or 100 micrograms 10B/g, about a hundred times higher average doses so that 2-20 Gy, corresponding to about 4-40 CGE, can be given to the patients. Thus, a successful targeting with high amounts of 10B in the tumour cells gives a significant dose enhancement when applied in fast neutron therapy but it is then more reasonable to treat the patient directly with thermal or epithermal neutrons since the average dose enhancement in the latter case is about a hundred times higher and curable doses might be obtained by the tumour specific capture processes alone.

Boron Neutron Capture Therapy Against Tumor Cells with Overexpression of the EGF-Receptor

The binary nature of boron neutron capture therapy, BNCT, is an advantage because the tumor-seeking substance can be activated at any chosen time and because the neutron field can be delivered to selected areas so that exposure of critical healthy organs, which might contain significant amounts of boron, can be avoided. The tumor selective action should work in spite of the fact that tumor cells often have an infiltrative growth pattern being mixed with populations of normal cells. The targeting principle should be based on well-characterized properties of the tumor cells such as appearence of tumor-associated antigens or overexpression of receptors. The targeting agent could be antibodies, antibody-fragments or receptor ligands. Presently, mainly monoclonal antibodies are considered as targeting substances but it has been claimed that current approaches are limited by low uptake in the tumors studied. Thus, it seems necessary to also consider other principles such as growth factor mediated targeting (1–4).