High and low fluences of alpha-particles induce a G1 checkpoint in human diploid fibroblasts. (9/190)

The effects of exposure to high and very low fluence alpha-particles on the G1 checkpoint were investigated in human diploid fibroblasts irradiated and released from density-inhibited confluent cultures by the use of the cumulative labeling index method. Transient and permanent arrests in G1 occurred in fibroblast populations exposed to mean doses as low as 1 cGy, suggesting that nontraversed bystander cells may contribute to the low dose response. In cells exposed to high fluences, the G1 checkpoint is at least as extensive as in gamma-irradiated cells. In contrast to gamma-irradiated cells, neither repair of potentially lethal damage nor a reduction in the fraction of cells transiently or permanently arrested in G1 were observed in cells held in confluence for 6 h after alpha-particle irradiation. Studies with isogenic wild-type, p53-/-, and p21Waf1-/- mouse embryo fibroblasts exposed to either gamma or alpha-particle radiation revealed a total lack of G1 arrest in either p53-/- or p21waf1-/- cells, indicating that the G1 checkpoint in wild-type cells is p53-dependent and that p21Wf1 fully mediates the role of p53 in its induction. In contrast to human cells, mouse embryo fibroblasts do not undergo a permanent G1 arrest. Except under conditions favoring potentially lethal damage repair, a comparable expression pattern of p53, p21Waf1, and other cell cycle-regulated proteins (pRb, p34cdc2, and cyclin B1) was observed in alpha-particle or gamma-irradiated human fibroblasts.  (+info)

An alpha-particle emitting antibody ([213Bi]J591) for radioimmunotherapy of prostate cancer. (10/190)

A novel alpha-particle emitting monoclonal antibody construct targeting the external domain of prostate-specific membrane antigen (PSMA) was prepared and evaluated in vitro and in vivo. The chelating agent, N-[2-amino-3-(p-isothiocyanatophen-yl)propyl]-trans-cyclohexane-1, 2-diamine-N,N',N',N'',N''-pentaacetic acid, was appended to J591 monoclonal antibody to stably bind the 213Bi radiometal ion. Bismuth-213 is a short-lived (t 1/2 = 46 min) radionuclide that emits high energy alpha-particles with an effective range of 0.07-0.10 mm that are ideally suited to treating single-celled neoplasms and micrometastatic carcinomas. The LNCaP prostate cancer cell line had an estimated 180,000 molecules of PSMA per cell; J591 bound to PSMA with a 3-nM affinity. After binding, the radiolabeled construct-antigen complex was rapidly internalized into the cell, carrying the radiometal inside. [213Bi]J591 was specifically cytotoxic to LNCaP. The LD50 value of [213Bi]J591 was 220 nCi/ml at a specific activity of 6.4 Ci/g. The potency and specificity of [213Bi]J591 directed against LNCaP spheroids, an in vitro model for micrometastatic cancer, also was investigated. [213Bi]J591 effectively stopped growth of LNCaP spheroids relative to an equivalent dose of the irrelevant control [213Bi]HuM195 or unlabeled J591. Cytotoxicity experiments in vivo were carried out in an athymic nude mouse model with an i.m. xenograft of LNCaP cells. [213Bi]J591 was able to significantly improve (P < 0.0031) median tumor-free survival (54 days) in these experiments relative to treatment with irrelevant control [213Bi]HuM195 (33 days), or no treatment (31 days). Prostate-specific antigen (PSA) was also specifically reduced in treated animals. At day 51, mean PSA values were 104 ng/ml +/- 54 ng/ml (n = 4, untreated animals), 66 ng/ml +/- 16 ng/ml (n = 6, animals treated with [213Bi]HuM195), and 28 ng/ml +/- 22 ng/ml (n = 6, animals treated with [213Bi]J591). The reduction of PSA levels in mice treated with [213Bi]J591 relative to mice treated with [213Bi]HuM195 and untreated control animals was significant with P < 0.007 and P < 0.0136, respectively. In conclusion, a novel [213Bi]-radiolabeled J591 has been constructed that selectively delivers alpha-particles to prostate cancer cells for potent and specific killing in vitro and in vivo.  (+info)

Direct evidence for the participation of gap junction-mediated intercellular communication in the transmission of damage signals from alpha -particle irradiated to nonirradiated cells. (11/190)

It has generally been considered that important biological effects of ionizing radiation arise as a direct consequence of DNA damage occurring in irradiated cells. We have examined this hypothesis by exposing cells to very low fluences of alpha-particles, similar to those emitted by radon gas, such that as few as 1% of the cells in a population are traversed by a particle and thus receive any radiation exposure. By using the endpoints of changes in gene expression and induction of DNA damage, we show that nonirradiated "bystander" cells participate in the overall response of confluent density-inhibited populations of cultured fibroblast and epithelial cells. By in situ immunofluorescence techniques and the use of cells genetically compromised in their ability to perform gap junction intercellular communication, we present direct evidence for the involvement of connexin43-mediated intercellular communication in the transmission of damage signals to nonirradiated cells. Induction of the stress-inducible p21(Waf1) protein in aggregates of neighboring cells far exceeding the fraction of cells whose nucleus has been traversed occurred in gap junction-competent cells only. These changes in p21(Waf1) expression correlated with both the induction of DNA damage (as measured by micronucleus formation) as well as increased Ser-15 phosphorylation of p53.  (+info)

Direct evidence for a bystander effect of ionizing radiation in primary human fibroblasts. (12/190)

Bystander responses underlie some of the current efforts to develop gene therapy approaches for cancer treatment. Similarly, they may have a role in strategies to treat tumours with targeted radioisotopes. In this study we show direct evidence for the production of a radiation-induced bystander response in primary human fibroblasts. We utilize a novel approach of using a charged-particle microbeam, which allows individual cells within a population to be selected and targeted with counted charged particles. Individual primary human fibroblasts within a population of 600-800 cells were targeted with between 1 and 15 helium ions (effectively, alpha-particles). The charged particles were delivered through the centre of the nucleus with an accuracy of +/- 2 micrometer and a detection and counting efficiency of greater than 99%. When scored 3 days later, even though only a single cell had been targeted, typically an additional 80-100 damaged cells were observed in the surviving population of about 5000 cells. The yield of damaged cells was independent of the number of charged particles delivered to the targeted cell. Similar results of a 2-3-fold increase in the background level of damage present in the population were observed whether 1 or 4 cells were targeted within the dish. Also, when 200 cells within one quadrant of the dish were exposed to radiation, there was a 2-3-fold increase in the damage level in an unexposed quadrant of the dish. This effect was independent of the presence of serum in the culture medium and was only observed when a cell was targeted, but not when only the medium was exposed, confirming that a cell-mediated response is involved.  (+info)

Response of LNCaP spheroids after treatment with an alpha-particle emitter (213Bi)-labeled anti-prostate-specific membrane antigen antibody (J591). (13/190)

A theoretical drawback to alpha-particle therapy with 213Bi is the short range of the particle track coupled with the short half-life of the radionuclide, thereby potentially limiting effective cytotoxicity to rapidly accessible, disseminated individual tumor cells (e.g., as in leukemia). In this work, a prostate carcinoma spheroid model was used to evaluate the feasibility of targeting micrometastatic clusters of tumor cells using 213Bi-labeled anti-prostate-specific membrane antigen (PSMA) antibody, J591. In prostate cancer, vascular dissemination of tumor cells or tumor cell clusters to the marrow constitutes an important step in the progression of this disease to widespread skeletal involvement, an incurable state. Such prevascularized clusters are ideal targets for radiolabeled antibodies because the barriers to antibody penetration that are associated with the capillary basal lamina have not yet formed. Beta- and gamma-emitting radionuclides such as 131I, which are widely used in radioimmunotherapy, are not expected to be effective when targeting single cells or small cell clusters. This is because the range of the emissions is one to two orders of magnitude greater than the target size, and the energy deposited per traversal is insufficient to produce any significant radiobiological effect. Spheroids of the prostate cancer cell line, LNCaP-LN3, were used as a model of prevascularized micrometastases; their response to an anti-PSMA antibody, J591, radiolabeled with the alpha-particle emitter 213Bi (T(1/2), 45.6 min.) has been measured. The time course of spheroid volume reductions was found to be sensitive to the initial spheroid volume. J591 labeled with 0.9 MBq/ml 213Bi resulted in a 3-log reduction in spheroid volume on day 33, relative to control, for spheroids with an initial diameter of 130 microm; 1.8 MBq/ml were required to achieve a similar response for spheroids with an initial diameter of 180 microm. Equivalent spheroid responses were observed after 12 Gy of acute external beam photon irradiation. Monte Carlo-based microdosimetric analyses of the 213Bi decay distribution in individual spheroids of 130-microm diameter yielded an average alpha-particle dose of 3.7 Gy to the spheroids, resulting in a relative biological effectiveness factor of 3.2 over photon irradiation. The activity concentrations used in the experiments were clinically relevant, and this work supports the possibility of using 213Bi-labeled antibodies not only for disseminated single tumor cells, as found in patients with leukemia, but also for micrometastatic tumor deposits up to 180 microm in diameter (1200 cells).  (+info)

Cellular dose conversion factors for alpha-particle--emitting radionuclides of interest in radionuclide therapy. (14/190)

alpha-Particle--emitting radionuclides are of increasing interest in radionuclide therapy. The decay scheme of alpha-emitting radionuclides typically includes a chain of unstable progeny. It is generally assumed that alpha-particle emission by the parent radionuclide will break the chemical bond with its carrier molecule and that the resulting daughter atom will no longer be associated with the carrier molecule. If the daughter is very short lived, it will not have enough time to be carried any significant distance from the site of parent decay and a cellular, absorbed dose estimate must consider the energy deposited by the daughter as well as the parent. Depending on the site of parent decay and the expected removal rate of daughter atoms from this site, the contribution of emissions from longer-lived daughters may also be warranted. In this study, dose conversion factors (DCFs) for cellular dimensions that incorporate the fate of daughter radionuclides were derived for (225)Ac, (213)Bi, (211)At, and (223)Ra, the alpha-particle--emitting radionuclides of interest in radionuclide therapy. METHODS: The dose contribution of daughter radionuclides at the site of parent decay was made dependent on a cutoff time parameter, which was used to estimate the fraction of daughter decays expected at the site of parent decay. Previously tabulated S values (cell-surface to nucleus and cell-surface to cell) for each daughter in the decay scheme were scaled by this fraction and a sum over all daughters was performed to yield a cutoff time--dependent set of corresponding DCF values for each radionuclide. RESULTS: DCF values for the absorbed dose to the nuclear or cellular volume from cell-surface decays are presented as a function of the cutoff time for 4 different cellular and nuclear dimensions. CONCLUSION: In contrast to the cellular S values that account only for parent decay, the DCF values provided in this study make it possible to easily include the contribution of daughter decays in cellular alpha-particle emitter dose calculations.  (+info)

Relationship between LET and RBE values for Escherichia coli determined using carbon ion beams from the TIARA cyclotron and HIMAC synchrotron.(15/190)

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High-level production of alpha-particle-emitting (211)At and preparation of (211)At-labeled antibodies for clinical use. (16/190)

In vitro and in vivo studies in human glioma models suggest that the antitenascin monoclonal antibody 81C6 labeled with the 7.2-h-half-life alpha-particle emitter (211)At might be a valuable endoradiotherapeutic agent for the treatment of brain tumors. The purpose of this study was to develop methods for the production of high levels of (211)At and the radiosynthesis of clinically useful amounts of (211)At-labeled human/mouse chimeric 81C6 antibody. METHODS: (211)At was produced through the (209)Bi(alpha, 2n)(211)At reaction using an internal target system and purified by a dry distillation process. Antibody labeling was accomplished by first synthesizing N-succinimidyl 3-[(211)At]astatobenzoate from the corresponding tri-n-butyl tin precursor and reacting it with the antibody in pH 8.5 borate buffer. Quality control procedures consisted of methanol precipitation, size-exclusion high-performance liquid chromatography (HPLC), and pyrogen and sterility assays, as well as determination of the immunoreactive fraction by a rapid procedure using a recombinant tenascin fragment coupled to magnetic beads. RESULTS: A total of 16 antibody labeling runs were performed. Using beam currents of 50-60 microA alpha-particles and irradiation times of 1.5-4.5 h, the mean (211)At production yield was 27.75 +/- 2.59 MBq/microA.h, and the maximum level of (211)At produced was 6.59 GBq after a 4-h irradiation at 55 microA. The decay-corrected distillation yield was 67% +/- 16%. The yield for the coupling of the (211)At-labeled active ester to the antibody was 76% +/- 8%. The fraction of (211)At activity that eluted with a retention time corresponding to intact IgG on HPLC was 96.0% +/- 2.5%. All preparations had a pyrogen level of <0.125 EU/mL and were determined to be sterile. The mean immunoreactive fraction for these 16 preparations was 83.3% +/- 5.3%. Radiolysis did not interfere with labeling chemistry or the quality of the labeled antibody product. CONCLUSION: These results show that it is feasible to produce clinically relevant activities of (211)At-labeled antibodies and have permitted the initiation of a phase I trial of (211)At-labeled chimeric 81C6 administered directly into the tumor resection cavities of brain tumor patients.  (+info)