Oncogenic rearrangements of the ret proto-oncogene in thyroid tumors induced after exposure to ionizing radiation

A high frequency (approximately 60%) of ret rearrangements in Chernobyl papillary thyroid carcinomas (PTC) has been reported recently. The data suggested that the radiation exposure may be a direct inducer of activating rearrangements in the ret gene. In our study, we have analyzed for the presence of RET/PTC oncogenes using the RT-PCR, XL-PCR, Southern blot and direct sequencing techniques, 39 human thyroid tumors from patients who had received external radiation for benign or malignant conditions. As controls, we studied 39 'spontaneous' tumors. Our results indicate that: 1) the overall frequency of ret rearrangements was 84% in papillary carcinomas (16/19) and 45% (9/20) in follicular adenomas; 2) in contrast with the results obtained in the Chernobyl tumors, the most frequently observed chimeric gene was RET/PTC1; and 3) all the tumors were negative for RET/PTC2. In the 'spontaneous' tumors, only the papillary carcinomas presented a ret rearrangement (15%: 3/20). Our data confirm the crucial role played by the ret proto-oncogene activating rearrangements in the development of radiation-associated thyroid tumors, and show, for the first time, the presence of RET/PTC genes in follicular adenomas appeared after external irradiation.     

Database and software for the analysis of mutations at the human hprt gene

A computerized database containing DNA sequence information regarding human HPRT mutants has been created. The database itself is in the dBASE format and contains information on about 1500 mutants. In addition, an IBM PC compatible software package to analyze the information in the database has been developed. Both the database and software are freely available via the Internet.     

Genome wide spontaneous mutation in human cells determined by the spectrum of mutations in hprt cDNA genes

We have studied spontaneous mutagenesis in five hprt cDNA genes integrated at five different genomic positions in a human lymphoblastoid cell line (TK6). The spectra of 40 mutants from each position were combined to obtain a mutation spectrum of the overall genome. This collection of mutants was used to assess the contribution of several mutagenic processes to spontaneous mutagenesis. Deletions and single base pair changes account for the majority of the mutants and arise in approximately equal amounts (43 and 41%, respectively). The majority of the deletions and insertions are < 5 bp and are likely to be caused by template-directed misalignment (slippage) during replication. To account for frameshifts at non-iterated sites we propose a slightly different template-directed replication error model. A considerable amount of the observed base pair changes can also be explained by this last model, but several other processes leading to base pair changes such as depurination, deamination or spontaneously arising DNA damage are likely to contribute as well. We have compared this spectrum with mutation spectra in the endogenous hprt genes using published mutation data. It is shown that in the endogenous genes the contribution of base pair substitutions is much larger (71%) than in the hprt cDNA integrates and that deletions are less frequently observed (20%). The mutation rates of the integrated hprt cDNA genes show a mean increase of 30-fold as compared with the endogenous hprt gene. This results in a 60-fold increase of the absolute rate of deletion in the hprt cDNA genes and in a 15-fold increase of the base pair substitution rate. Replication errors such as slippage or the mechanism proposed in this study probably account to a large extent for this increase.     

Analysis of DNA damage recovery processes in the adaptive response to ionizing radiation in human lymphocytes

Size-dependent structural patterns in the conductive bronchial tree of four species of myomorph rodents of different body weight were determined by lung casts. The lungs of the harvest mouse, Micromys minutus, body weight 5-7 g, the house mouse, Mus musculus, body weight 35-45 g, the brown rat, Rattus norvegicus, body weight 200-400 g, and the African giant pouched rat, Cricetomys gambianus, body weight 1,200-1,800 g, were inflated to 20 cm H2O, frozen, freeze-dried, hardened, and filled with silicone rubber. The casts were pruned, and branching pattern, diameter, and volume of the conductive bronchial tree were determined using a binocular magnifier. All four species have four lobes on the right lung and an undivided left lung, and the central bronchial tree on either side shows an identical monopodial branching pattern. Although the ramification of the central conductive bronchi is not size-dependent, the diameter and volume are. The diameter of the left main bronchus equals 1.24% of body length in Micromys and 0.6% in Cricetomys, and the conductive bronchial tree makes up 13% of the total lung volume in Micromys and 6% in Cricetomys. Relatively wider airways and a decline in airway resistance with declining body mass in small mammals compared to large ones result in a high ventilatory dead space, which is compensated for by a higher breathing frequency.     

Development and utilization of the rat lymphocyte hprt mutation assay

Much of the recent progress in the field of genetic toxicology has come from an increased understanding of the molecular and cellular biology of the mammalian organism. Most prominent has been the ability to detect and quantify somatic mutation and relate the nature of the mutation to the specific type of chemical damage. Building upon the foundation of the human lymphocyte hypoxanthine guanine phosphoribosyl transferase (hprt) system, and later, the mouse hprt system, methods for the detection and quantification of hprt mutations in rat lymphocytes were developed. These methods are described in this report as is the ongoing validation of the assay. Additionally, the characterization of the recovered mutants and a comparison of the mutation spectrum in the rat lymphocyte system to the spectrum in cancer genes, such as H-ras and p53, and the spectrum in transgenic systems, such as lacI, are included. The development of the rat lymphocyte hprt system and validation of the assay at the molecular level, provide an effective and reliable measure of genetic damage in an in vivo system which is readily comparable to measurement of genetic damage in the human.     

Databases and software for the analysis of mutations in the human p53 gene, the human hprt gene and the lacZ gene in transgenic rodents

We have created databases and software applications for the analysis of DNA mutations in the human p53 gene, the human hprt gene and the rodent transgenic lacZ locus. The databases themselves are stand-alone dBase files and the software for analysis of the databases runs on IBM- compatible computers. The software created for these databases permits filtering, ordering, report generation and display of information in the database. In addition, a significant number of routines have been developed for the analysis of single base substitutions. One method of obtaining the databases and software is via the World Wide Web (WWW). Open home page http://sunsite.unc.edu/dnam/mainpage.ht ml with a WWW browser. Alternatively, the databases and programs are available via public ftp from anonymous@sunsite.unc.edu. There is no password required to enter the system. The databases and software are found in subdirectory pub/academic/biology/dna-mutations. Two other programs are available at the WWW site, a program for comparison of mutational spectra and a program for entry of mutational data into a relational database.     

Correlation of UV-induced mutational spectra and the in vitro damage distribution at the human hprt gene

We have determined the in vitro DNA damage distribution induced by 254 mm UV in the human hprt gene. The sequence-specific nature of the DNA damage for both main classes of UV-induced photoproducts, i.e., cyclobutane pyrimidine dimers (CPDs) and the pyrimidine <6-4> pyrimidone photoproducts (64PyPy), was evaluated. Utilizing an automated DNA sequencer plus auxiliary software, semi-automated analyses were performed for peak quantitation and retention-time to sequence-position correlation. 64PyPy were predominantly formed at 5'-YTC-3' sites (p < 0.02; where Y = C,T). The effect of the 3'flanking nucleotide on the 64PyPy formation at 5'-TC-3' sites was 64PyPy at 5'-TCT-3' sites were induced at lower frequencies compared to 5'-TCM-3' sites (where M = A or C; p < 0.03). No effect of flanking nucleotides was detected for CPDs recovered at 5'-TT-3' sites. Sites of mutations in the hprt gene were compared to the sites of DNA damage. Two regions of frequently mutated nucleotides corresponded to sites of high deposition of damage. The two sites either had a high frequency of CPDs or 64PyPy, which implicated both types of photoproducts as premutagenic lesions.     

Rejoining of DNA single- and double-strand breaks in human white blood cells exposed to ionizing radiation

PURPOSE: To characterize inter- and intra-individual differences in X-ray-induced DNA strand break rejoining kinetics in human peripheral white blood cells (WBC) obtained from 10 healthy volunteers. MATERIALS AND METHODS: The alkaline and neutral versions of the comet assay were used to measure the rate of rejoining of predominantly single-strand breaks (ssb) following exposure to 8 Gy and double-strand breaks (dsb) following 75 Gy. RESULTS: All cells within a population responded in a similar fashion to induction of ssb and dsb; however, a subset of the WBC appeared to rejoin ssb more rapidly. For the 10 individuals examined, the percentage of ssb rejoined by the rapid component(s) was 47 +/- 16% and the rejoining half-time for the slow component was 1.3 +/- 0.4 h. By 24 h after 8 Gy, 4.9 +/- 3.8% of the initial ssb remained. For dsb rejoining, 58 +/- 11% of the initial damage was still present 4h after 75 Gy and by 24 h 32% of the initial level of damage was still detected. Heavily damaged cells present 24 h after 75 Gy varied from 4% to 50% and were excluded from the analysis of repair rates. CONCLUSIONS: Inter-individual variability exceeded intra-individual variability for 2 of 4 endpoints examined for ssb repair, but not for dsb repair. It was concluded that DNA damage measured using the comet assay could identify a range in the X-ray repair responses of WBC from different normal individuals. Whether these differences correlate with differences in cell killing by radiation remains to be determined.     

Use of ionizing radiation to prevent restenosis in interventional cardiology

It is well known that the expression of carbohydrate chains on cell changes during the development and progression of carcinoma. Some carbohydrate chains have been used in clinical practice as tumor markers. Some, such as sialyl Lewis a (CA 19.9), sialyl Lewis X, and Tn, are known to function as adhesion molecules. However, their role in the development of hematogenous metastasis is still controversial. In this paper, many investigations concerning this issue are reviewed. Clinical trials for treatment of patients with advanced cancer using carbohydrate chains are also mentioned.     

Radioresistant MTp53-expressing rat embryo cell transformants exhibit increased DNA-dsb rejoining during exposure to ionizing radiation

Recent data suggest that aberrant function of the wild type p53 protein (WTp53) may alter cellular survival following DNA damage through cellular pathways involving apoptosis and cell-cycle checkpoints, but little is known concerning it's possible role in DNA repair. In the present study, the ionizing radiation sensitivity was determined for a series of rat embryo fibroblast (REF) cell lines transfected with an activated form of the H-ras oncogene alone, or in combination with a variety of missense-mutant p53 (MTp53) alleles. Transformed REF clones which expressed exogenous MTp53 and p21ras proteins (CLASS II clones) were generally radioresistant in culture as determined by higher values for the surviving fraction after 2 Gy (SF2 value) and the radiation dose required to reduce survival to a fraction of 0.1 (D10 value), compared either to transformed REF clones expressing p21ras protein alone (CLASS I clones), or to non-transfected REF control cell lines expressing baseline endogenous levels of p21ras and WTp53 protein. The increased radioresistance observed in the CLASS II clones (following both HDR- and LDR-irradiation), was significantly correlated with increased expression of MTp53 protein, and a decreased radiation-induced G1 arrest response. The variability observed in clonogenic radiosensitivity among REF clones was not explained by differential radiation-induced apoptosis. Using the Comet assay performed after continuous low dose-rate (LDR)-irradiation, MTp53-expressing REF clones were also found to be more proficient at the rejoining of DNA double-strand breaks (DNA-dsb), compared to WTp53-expressing REF clones. These results suggest that an enhanced DNA and cellular repair capacity may, in part, explain the increased radiation survival observed in some MTp53-expressing transformed fibroblasts and tumours.     

Is there reliable experimental evidence for a chromosomal "fingerprint" of exposure to densely ionizing radiation?

BACKGROUND: As an inhibitor of the reuptake of serotonin and norepinephrine in the spinal cord, the mechanism of action of tramadol resembles that of nefopam, which has been used in the treatment of postanesthetic shivering. METHODS: In a randomized, placebo-controlled, double-blind study, we assessed the effects of tramadol (0.5 mg.kg-1, 1 mg.kg-1 and 2 mg.kg-1 i.v.) or normal saline on shivering after a standardized general anesthesia in 40 adult patients, ASA physical status I or II (group 1), and in 64 adult patients regardless of the foregoing general anesthesia and ASA physical status (group 2). RESULTS: Tramadol 1 mg.kg-1 or more abolished shivering completely 5 min after treatment in all patients of groups 1 and 2. In group 1, the three dosages of tramadol were not statistically different in lowering the severity and prevalence of postanesthetic shivering. Tramadol 0.5 mg.kg-1 was significantly slower than tramadol 1 or 2 mg.kg-1 in tempering the severity as well as lowering the prevalence of postanesthetic shivering in group 2. CONCLUSION: Tramadol's distinct features in the treatment of shivering reside in its high safety profile and weak sedative properties, particularly in patients with poor cardiorespiratory reserve, in outpatients and on recurrence of shivering.     

The role of ligands in the effect of exposure to ionizing radiation on Ca2(+)-ATPase and Mg2(+)-ATPase

Over the past year, a number of advances have been made in the large-scale purification of macromolecules, particularly proteins. Although refinements to individual unit operations have occurred, especially in improving the speed of operation and performance of large-scale chromatographic media, a major research thrust has been the development of processes in which steps are combined or eliminated to improve operability and reduce cost.     

The health status of children up to 14 years old living in an area of long-term post-accident exposure to low doses of ionizing radiation

An assessment is submitted of morbidity rates and physical development of children aged under 14, residing in the territories being monitored after the Chernobyl Power Plant accident. A high level of disharmony in physical development of the children examined was recordable, as was an excess in morbidity of both general and separate classes of disease entities among the pediatric population having been victims of the Chernobyl accident, as compared to that in relatively "clean" areas and in Ukraine as a whole.     

No detectable bioeffects following acute exposure to high peak power ultra-wide band electromagnetic radiation in rats

A wide range assessment of the possible bioeffects of an acute exposure to high peak power ultra-wide band (UWB) electromagnetic radiation was performed in rats. The UWB-exposure consisted of 2 min of pulsed (frequency: 60 Hz, pulse width: 5-10 ns) UWB (bandwidth: 0.25-2.50 GHz) electromagnetic radiation. Rats were examined using one of the following: 1) a functional observational battery (FOB); 2) a swimming performance test; 3) a complete panel of blood chemistries; or 4) determination of the expression of the c-fos protein in immunohistologically-stained sections of the brain. No significant differences were found between UWB- or sham-exposed rats on any of the measured parameters.     

Sensitivity of human prostatic carcinoma cell lines to low dose rate radiation exposure

PURPOSE: Low dose rate radioemitters, such as 125I, 103Pd, and 89Sr, have been used both for local and systemic treatment of prostate cancer. Most normal cells exposed to ionizing radiation characteristically activate cell cycle checkpoints, resulting in cell cycle arrest at the G1/S and G2/M transition points. Cancer cells are typically quite sensitive to radiation killing late in the G2 phase of the replicative cell cycle. Furthermore, most cancer cells accumulating at the G2/M transition point as a result of low dose rate radiation exposure appear to become sensitive to further low dose rate irradiation. For this reason, protracted exposure of cancer cells to low dose rate radiation has been proposed to result in increased cancer cell killing as compared with brief exposures of cancer cells to high dose rate radiation. Since many human prostatic carcinomas contain somatic genome alterations targeting genes which affect the cell cycle and radiation-associated cell cycle checkpoints, we evaluated the effects of low dose rate radiation exposure on the cell cycle and on clonogenic survival for various human prostatic carcinoma cell lines. MATERIALS AND METHODS: Human prostatic carcinoma cells from the LNCaP, DU 145, PC-3, PPC-1, and TSU-Pr1 cell lines were exposed to low dose rate (0.25 Gy/hour) or high dose rate (60 Gy/hour) radiation in vitro and then assessed for radiation cytotoxicity by clonogenic survival assay. Cell cycle perturbations following protracted exposure to low dose rate radiation were evaluated using flow cytometry. RESULTS: For LNCaP cells, low dose rate radiation exposure resulted in an accumulation of cells at both the G1/S and the G2/M cell cycle transition points. For DU 145, PC-3, PPC-1, and TSU-Pr1 cells, treatment with low dose rate radiation triggered G2/M cell cycle arrest, but not G1/S arrest. Unexpectedly, the cell cycle redistribution pattern phenotypes observed, G1/S and G2/M cell cycle arrest versus G2/M arrest alone, appeared to have little effect on low dose rate radiation survival. Furthermore, while PC-3, PPC-1, and TSU-Pr1 cells exhibited increased cytotoxic sensitivity to low dose rate versus fractionated high dose rate radiation treatment, DU 145 and LNCaP cells did not. CONCLUSIONS: Radiation-associated pertubations in replicative cell cycle progression were not dominant determinants of low dose rate radiation killing efficacy in human prostate cancer cell lines in vitro.