Gene expression profile of human lymphocytes exposed to (211)At alpha particles.

In this study, the Whole Human Genome 44K DNA microarray assay was used for the first time to obtain gene expression profiles in human peripheral blood lymphocytes 2 h after exposure (in suspension) to 6.78 MeV mean energy alpha particles from extracellular (211)At. Lymphocytes were exposed to fluences of 0.3-9.6 x 10(6) alpha particles/cm(2) [corresponding to mean absorbed alpha-particle doses (D(alpha)) of 0.05-1.60 Gy] over 30 min. Significantly modulated expression was identified in 338 early-response genes. Up-regulated expression was evident in 183 early-response genes, while the remaining 155 were down-regulated. Over half of the up-regulated genes and 40% of the down-regulated genes had a known biological process related primarily to cell growth and maintenance and cell communication. Genes associated with cell death were found only in the up-regulated genes and those with development only in the down-regulated genes. Eight selected early-response genes that displayed a sustained up- or down-regulation (CD36, HSPA2, MS4A6A, NFIL3, IL1F9, IRX5, RASL11B and SULT1B1) were further validated in alpha-particle-irradiated lymphocytes of two human individuals using the TaqMan(R) RT-qPCR technique. The results confirmed the observed microarray gene expression patterns. The expression modulation profiles of IL1F9, IRX5, RASL11B and SULT1B1 genes demonstrated similar trends in the two individuals studied. However, no significant linear correlation between increasing relative gene expression and the alpha-particle dose was evident. The results suggest the possibility that a panel of genes that react to alpha-particle radiation does exist and that they merit further study in a greater number of individuals to determine their possible value regarding alpha-particle biodosimetry.

Early gene expression in human lymphocytes after gamma-irradiation-a genetic pattern with potential for biodosimetry.

PURPOSE: Identification of early radiation response genes (ERG) in human lymphocytes after gamma-irradiation by using the whole-human-genome DNA-microarrays and the evaluation of their possible role in rapid radiation biodosimetry by applying real-time quantitative polymerase chain reaction (RT-qPCR) methodology for validation in a small group of human individuals. MATERIALS AND METHODS: Whole blood from a healthy human donor was exposed at 37 degrees C to 137Cs gamma-radiations (absorbed dose: 1-4 Gy). Fifteen minutes following irradiation the lymphocytes were isolated from the blood (for 2 h at 20 degrees C) and their gene expression was investigated using the DNA-microarrays. Subsequently, 14 genes were selected and validated using the TaqMan probes based upon the RT-qPCR assay within a group of 6 human donors. RESULTS: A dose-related relative change in quantitative gene expression using the DNA-microarray assay was demonstrated in 24 of 102 genes. Up-regulation of expression was observed in 15 genes: CD69 (CD69 molecule), CDKN1A (cyclin-dependent kinase inhibitor 1A), EGR1 (early growth response 1), EGR4 (early growth response 4), FLJ35725 (chromosome 4 ORF 23), hCG2041177 (hCG - human Celera Genome), hCG1643466.2, IFN-gamma (interferon-gamma), ISG20L (interferon stimulated exonuclease gene 20 kDa - like 1), c-JUN (jun oncogene), MDM2 (mouse double minute 2), MUC5B (mucine), PLK2 (polo-like kinase 2), RND1 (rho-family GTPase 1) and TNFSF9 (tumour necrosis factor superfamily member 9). Down-regulation of expression was found in the remaining nine genes: GRIK3 (glutamate receptor ionotropic kainate 3), hCG1985174, hCG1998530, hCG2038519, OCLN (occludin), RPL10A (ribosomal protein L10a), SERHL2 (serine hydrolase-like 2), SGK3 (serum/glucocorticoid regulated kinase 3) and STARD13 (START domain containing 13). CONCLUSION: A significant correlation between absorbed radiation dose and change in relative gene expression was particularly evident for EGR1, EGR4, IFN-gamma, c-JUN and TNFSF9 (p < or = 0.05). Results warrant the further investigation of these ERG as potential biodosimetric markers.

Biodistribution and pharmacokinetics of variously sized molecular radiolabelled polyethyleneiminomethyl phosphonic acid as a selective bone seeker for therapy in the normal primate model.

An ideal radiopharmaceutical for the treatment of neoplastic and inflammatory (benign) bone disease would be a radiolabelled compound that predominantly accumulates in bone lesions with limited access to normal bone and other organs. Neoplastic tissue's abnormal blood supply (increased permeability) and lack of lymphatics will selectively accumulate radiolabelled macromolecules. This enhanced permeability and retention effect forms the basis of this study, using various molecular sizes of the radiolabelled macromolecule polyethyleneiminomethyl phosphonic acid (PEI-MP) for increased selectivity of the bone-seeking radiopharmaceutical. PEI-MP was synthesized by condensation of polyethyleneimine, phosphonic acid and formaldehyde, followed by fractionation into different molecular sizes by membrane ultrafiltration. Labelling efficiency to 99mTc (as radiotracer) was approximately 99% with complexes stable for 24 h. The pharmacokinetics and biodistribution of various 99mTc-PEI-MP fractions were investigated using 4 experimental baboons (Papio ursinus) per fraction. Scintigraphy was performed on the baboons under general anaesthesia of pentobarbital i.v. After an i.v. bolus of 99mTc-PEI-MP (approximately 185 MBq) both dynamic studies (30 x 1 min frames), and static studies (2 min acquisition every hour for 4 h) were done, as well as blood samples and urine collected. From the results macromolecules with sizes ranging between 30-300 kDa were characterized by excessive liver (21%-57% retained activity) and kidney (40% retained activity) uptake and accompanying long residing times (t1/2 up to 24 h). The percentage bone uptake averaged at 8% for these particles excluding sizes 100-300 kDa where very little bone uptake was seen (< 1%). In this case the blood clearance was also slow (t1/2 approximately 2 h). The fraction size 10-30 kDa had comparatively low accumulation and short residence times in the liver and kidneys (resp. 20%, t1/2 = 22 +/- 4 min; 17.5%, t1/2 = 20 +/- 3 min) and although the bone uptake of 18% in this case was high, it is still low for a bone-seeking agent. These particles cleared the blood with t1/2 = 25 +/- 2 min and seemed suitable for labelling with a therapeutic radioisotopic agent.

Response of high mobility group proteins of human kidney T1 and murine L 929 cell lines to heat shock.

High mobility group (HMG) proteins in human kidney T1 and murine L 929 cells have been investigated after exposure to heat shock at 41 degrees C and their influence on the organizational change of chromatin under heat shock condition has been examined. Results reveal that the two cell lines show differential response of the HMG proteins 1 & 2 and 14 & 17 to heat shock. Neither T1 nor L 929 cells show significant differences in response to heat shock with respect to the binding affinities of HMG proteins 1 & 2 or 14 & 17 to DNA, as revealed by DNase I sensitivity and chromatin reconstitution assays. Furthermore, the HMG proteins of both the non-heat shocked and the heat shocked T1 and L 929 cells can recover their chromatin activity following reconstitution. These findings suggest that although the HMG proteins might undergo some change in response to heat shock, their inherent potential of reassociation with DNA is still retained.

211At-alpha-dose dependence of poly-ADP-ribosylation of human glioblastoma cells in vitro. Suitability in cancer therapy?

AIM: It was intended to test the biological response (poly-ADP-ribosylation of cellular proteins) of alpha-particles from extracellular 211At for enhanced damage to human glioblastoma cells in vitro and to discuss its suitability for potential application in therapy of high-grade gliomas. MATERIALS AND METHODS: Confluent cultures of human glioblastoma cells were exposed to different doses of alpha-radiations from homogeneously distributed extracellular 211At. Cellular poly-ADP-ribosylation of all proteins including histones was monitored since it is an indirect but sensitive indicator of chromatin damage and putative repair in both normal and malignant mammalian cells. RESULTS: A significant diminution (average 85.6%) in poly-ADP-ribosylation of total cellular proteins relative to that for non-irradiated glioblastoma cells was observed following 0.025 to 1.0 Gy alpha-radiations. In the dose range of 0.0025 to 0.01 Gy there was an increase with a maximum value of approximately 119.0% at 0.0025 Gy. Below 0.0025 Gy no change in poly-ADP-ribosylation was observed. CONCLUSIONS: Level of cellular poly-ADP-ribosylation of proteins at 0.025 to 1.0 Gy of alpha-radiation dose from 211At appears to cause enhanced damage by creating molecular conditions which are not conducive to repair of DNA damages in human glioblastoma cells in vitro. Therefore, it is assumed that clinical application of 211At at least in this dose range might enhance clinical efficacy in radiotherapy of cancer.

Arecoline-induced changes of poly-ADP-ribosylation of cellular proteins and its influence on chromatin organization.

Arecoline, the major alkaloid of betel nut (Areca catechu L.) and a suspected carcinogen, has been implicated in human cancers of various sites. A considerable portion of the world's population is constantly exposed to arecoline due to the habit of masticating betel nuts. The present work relates to the study of early molecular events following chronic arecoline exposure at a dose of 10 microg/ml to Swiss albino mice. Poly-ADP-ribosylation of all cellular proteins, histones and poly-ADP-ribose polymerase were studied in bone marrow and spleen cells and correlated with the organizational status of the chromatin. While most proteins showed lowering of their poly-ADP-ribosylation following arecoline treatment, only histone protein H1 in spleen cells and H2B in bone marrow cells exhibited an increase. The chromatin of both the tissues was progressively relaxed upon arecoline exposure. The implications of these changes have been discussed regarding the process of initiation of arecoline-induced carcinogenesis.

Poly-ADP-ribosylation of histone proteins of human kidney T1-cells in vitro following gamma-irradiation.

Poly-ADP-ribosylation of cellular proteins is involved with radiation induced damage and its repair. It has been observed that suspension of human kidney T1-cells in vitro attained elevated levels of poly-ADP-ribosylation due to experimental manipulations necessary for preparation of single cell suspension from monolayer cell cultures. These cells in suspension were exposed to various doses of gamma-rays with or without subsequent repair incubation. The PADPR of histones H3, H1 and H2B increased with increasing dose of radiation and decreased after 90 min or repair incubation. Concomitant with these changes, the affinity of histones to DNA in chromatin reduced immediately after irradiation. Normal affinity was reestablished after post-irradiation repair incubation. The results indicate that induction of poly-ADP-ribosylation of histone proteins by radiation and by manipulations to prepare single cell suspension involved different cellular components.

Uptake of ethylenediamine tetramethylene phosphonic acid in normal bone after multiple applications. A non-human primate study.

Palliation of bone pain in patients with bone metastases has previously been evaluated using 153Sm (samarium) complexed to bone seeking ethylenediamine tetramethylene phosphonic acid (CAS 1429-50-1, EDTMP). Repeated application of the radioligand as needed was found progressively less effective. This study questions whether EDTMP exerts a blocking function, limiting access to bone or osseous tumours with successive administration. The pharmacokinetics and biodistribution of 153Sm-EDTMP in the normal experimental baboon (n = 6) during three successive applications (6 weekly) each with two different concentrations of EDTMP (0.7 and 1.4 mg/kg b.wt.) were investigated using bone scintigraphy. 153Sm-EDTMP (111 MBq) was injected in each case and monitored for 5 h. Curves of tracer kinetics and bone to background uptake were obtained, also blood and cumulative urine curves. Comparisons were statistically assessed in each group between successive applications and between EDTMP concentrations. Partial blocking with the low EDTMP concentration reached statistical significance after the third application. The first application of the high EDTMP concentration yielded lower uptake in the bone than did low EDTMP pointing to blocking by the high concentration, but not seen with repeated applications. Continual application of high concentration EDTMP could lead to a reduced level of calcium in serum and increased parathyroid hormone levels which might trigger osteoblastic activity and bone remodelling. This would partially affect the blocking which was thus more obvious at the low EDTMP concentration.

Neutrons affect ADP-ribosylation of proteins in human kidney T1-cells in vitro.

ADP-ribosylation (ADPR) of proteins has been shown to be involved with a variety of cellular responses in which chromatin organization and functions are affected. In order to look into this response, human kidney T1-cells were exposed in vitro to various doses up to 3 Gy of 6 MeV neutrons and compared with the effect caused by gamma photons. Whereas in case of neutrons the maximal inhibition of ADPR was reversed at 0.37 Gy, that in case of gamma-rays occurred at 1.5 Gy. For the reversal of inhibition of ADPR of proteins in T1-cells, neutrons were about 4-fold more efficient as compared to gamma rays.

Change of ADP-ribosylation in human kidney T1-cells by various external stimuli.

Human kidney T1-cells in culture respond to various externally applied stresses, such as mechanical manipulations, trypsinization combined with agitation and centrifugation, Gamma-irradiation, and hyperthermia, by increasing the ADP-ribosylation (ADPR) of proteins in cytoplasm and nuclei. On the other hand, a strong static magnetic field or gravitational force reduces ADPR of proteins. These responses are easily detected in cells in monolayer, yet are masked when the conventional assay of ADPR is used on cells in suspension. The multitude of stimuli for either increasing or depressing ADPR of proteins including histones suggests a common pathway of triggering ADPR in which cellular membranes may be involved.

125Iodine decay in DNA: a discussion of its effectiveness for the breaking of DNA strands.

125I incorporated in DNA is known to be exceptionally toxic. Values of D0 range from about 40 to about 90 decays for survival of mammalian cells. The effectiveness of 125I in DNA with respect to the induction of breaks of the DNA strands, however, appears to be comparatively low. The numbers of strand breaks per energy deposited in subnuclear cellular structures such as DNA is smaller for a disintegration of 125I than for gamma-rays. The difference in effectiveness diminishes with increasing mass of the considered sensitive volume. The apparent inefficiency of 125I-decay may, on one hand, result from a waste of local energy deposition. On the other hand, it may be caused by a multitude of local strand breaks (clusters) induced by 125I-decay which are measured as one break only by the conventionally applied techniques of strand break measurement. The apparent inefficiency of 125I may be evidence furthermore for the importance of considering not only the DNA as the sensitive target but with equal pertinence the gross sensitive volume, i.e. the whole cell nucleus. Further, for drawing meaningful comparisons, it may be necessary to take into consideration the microdosimetric event size distributions for the critical targets.