In vivo radioadaptive response: a review of studies relevant to radiation-induced cancer risk.

Radioadaptive response (RAR) describes phenomena where small conditioning doses of ionizing radiation (IR) reduce detrimental effects of subsequent higher IR doses. Current radiation protection regulations do not include RAR because of the large variability in expression among individuals and uncertainties of the mechanism. However, RAR should be regarded as an indispensable factor for estimation and control of individual IR sensitivity. In this article, RAR studies relevant to individual cancer risk are reviewed. Using various stains of mice, carcinogenic RAR has been demonstrated. Consistently much in vivo evidence for RAR with end points of DNA and chromosome damage is reported. Most in vivo RAR studies revealed efficient induction of RAR by chronic or repeated low-dose priming irradiation. Chronic IR-induced RAR was observed also in human individuals after environmental, occupational, and nuclear accident radiation exposure. These observations may be associated with an intrinsically distinct feature of in vivo experimental systems that mainly consist of nonproliferating mature cells. Alternatively, induction of RAR by gap junction-mediated bystander effects suggests that multicellular systems comprising densely communicating cells may be capable of responding to long-lasting low-dose-rate priming irradiation. Regulation by endocrine factors is also a plausible mechanism for RAR at an individual level. Emerging evidence suggests that glucocorticoids, known as stress hormones, participate in in vivo RAR induction following long-term low-dose-rate exposure to IR.

Adaptive response in embryogenesis: vi. Comparative microarray analysis of gene expressions in mouse fetuses.

PURPOSE: Exposure of sublethal doses of ionizing radiation can induce protective mechanisms against a subsequent higher dose irradiation. This phenomenon, called radiation-induced adaptive response (AR), has been described in a wide range of biological models. We previously demonstrated the existence of AR in mice during late organogenesis. In this study, we investigated molecular mechanisms underlying AR in this model. MATERIALS AND METHODS: Using DNA microarrays, we performed a global analysis of transcriptome regulations in adapted and non-adapted cells collected from whole mouse fetuses, after in utero exposure to priming irradiation. RESULTS: We identified AR-specific gene modulations. Our results suggested the involvement of signal transduction and Tumor protein (p53)-related pathways in the induction of AR. CONCLUSIONS: Our results are in agreement with previous investigations showing that AR could be dependant on p53 activity. The observed gene modulations may also have possible consequences for subsequent developmental process of the fetus. This is the first report of AR-specific modulations at the molecular level in utero, which could serve as a basis for subsequent studies aimed at understanding AR in this model and possible long-term effects.

Identification of the female-determining region of the W chromosome in Bombyx mori.

The W chromosome of the silkworm Bombyx mori is devoid of functional genes, except for the putative female-determining gene (Fem). To localize Fem, we investigated the presence of W-specific DNA markers on strains in which an autosomal fragment containing dominant marker genes was attached to the W chromosome. We produced new W-chromosomal fragments from the existing Zebra-W strain (T(W;3)Ze chromosome) by X-irradiation, and then carried out deletion mapping of these and sex-limited yellow cocoon strains (T(W;2)Y-Chu, -Abe and -Ban types) from different Japanese stock centers. Of 12 RAPD markers identified in the normal W chromosomes of most silkworm strains in Japan, the newly irradiated W(B-YL-YS)Ze chromosome contained three, the T(W;2)Y-Chu chromosome contained six, and the T(W;2)Y-Abe and -Ban chromosomes contained only one (W-Rikishi). To investigate the ability of the reduced W-chromosome translocation fragments to form heterochromatin bodies, which are found in nuclei of normal adult female sucking stomachs, we examined cells of the normal type p50 strain and the T(W;2)Y-Chu and -Abe strains. A single sex heterochromatin body was found in nuclei of p50 females, whereas we detected only small sex heterochromatin bodies in the T(W;2)Y-Chu strain and no sex heterochromatin body in the T(W;2)Y-Abe strain. Since adult females of all strains were normal and fertile, we conclude that only extremely limited region, containing the W-Rikishi RAPD sequence of the W chromosome, is required to determine femaleness. Based on a comparison of the normal W-chromosome and 7 translocation and W-deletion strains we present a map of Fem relative to the 12 W-specific RAPD markers.

Isolation and characterization of sex chromosome rearrangements generating male muscle dystrophy and female abnormal oogenesis in the silkworm, Bombyx mori.

In deletion-mapping of W-specific RAPD (W-RAPD) markers and putative female determinant gene (Fem), we used X-ray irradiation to break the translocation-carrying W chromosome (W( Ze )). We succeeded in obtaining a fragment of the W( Ze ) chromosome designated as Ze (W), having 3 of 12 W-RAPD markers (W-Bonsai, W-Yukemuri-S, W-Yukemuri-L). Inheritance of the Ze (W) fragment by males indicates that it does not include the Fem gene. On the basis of these results, we determined the relative positions of W-Yukemuri-S and W-Yukemuri-L, and we narrowed down the region where Fem gene is located. In addition to the Ze (W) fragment, the Z chromosome was also broken into a large fragment (Z(1)) having the +( sch ) (1-21.5) and a small fragment (Z(2)) having the +( od ) (1-49.6). Moreover, a new chromosomal fragment (Ze (W)Z(2)) was generated by a fusion event between the Ze (W) and the Z(2) fragments. We analyzed the genetic behavior of the Z(1) fragment and the Ze (W)Z(2) fragment during male (Z/Z(1) Ze (W)Z(2)) and female (Z(1) Ze (W)Z(2)/W) meiosis using phenotypic markers. It was observed that the Z(1) fragment and the Z or the W chromosomes separate without fail. On the other hand, non-disjunction between the Ze (W)Z(2) fragment and the Z chromosome and also between the Ze (W)Z(2) fragment and the W chromosome occurred. Furthermore, the females (2A: Z/Ze (W)Z(2)/W) and males (2A: Z/Z(1)) resulting from non-disjunction between the Ze (W)Z(2) fragment and the W chromosome had phenotypic defects: namely, females exhibited abnormal oogenesis and males were flapless due to abnormal indirect flight muscle structure. These results suggest that Z(2) region of the Z chromosome contains dose-sensitive gene(s), which are involved in oogenesis and indirect flight muscle development.

Accumulation of spermidine/spermine N1-acetyltransferase and alternatively spliced mRNAs as a delayed response of HeLa S3 cells following X-ray irradiation.

PURPOSE: A key enzyme of polyamine catabolism, spermidine/spermine N(1)-acetyltransferase (SSAT), is responsive to antiproliferative agents. The role of SSAT in cellular responses to X-ray irradiation was examined. MATERIALS AND METHODS: Exponentially growing HeLa S3 cells were irradiated by X-rays, and mRNA levels for SSAT were measured as a function of post-irradiation time through Northern hybridization. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect alternatively spliced SSAT mRNAs. The intracellular polyamine content was measured by the o-phthalaldehyde method and the enzymatic activity of SSAT by the increased amount of acetylated spermidine after incubation. RESULTS: Not only SSAT mRNA, but also an alternatively spliced mRNA accumulated at the initial stage of growth inhibition after the first or second replication of irradiated cells. The maximum fold increase relative to the level of non-irradiated cells was 3.0-3.5 for both transcripts after 5-Gy irradiation. On the other hand, the mRNA of ornithine decarboxylase, a key enzyme of polyamine synthesis, was little influenced by X-ray treatment. Enzymatic activity of SSAT and the acetylspermidine level were elevated after X-ray irradiation. CONCLUSIONS: Activation of SSAT and the induction of alternatively spliced mRNA of the SSAT gene play an important role in regulating growth inhibition and cell death after X-ray irradiation.

Regulation of the catalase gene promoter by Sp1, CCAAT-recognizing factors, and a WT1/Egr-related factor in hydrogen peroxide-resistant HP100 cells.

Reactive oxygen species play a critical role in the onset of apoptosis induced by various extracellular stimuli, including ionizing radiation. Therefore active regulation of reactive oxygen species-metabolizing enzymes may be one response to an apoptotic stimulus. In this regard, HP100 cells, H(2)O(2)-resistant variants derived from human leukemia HL60 cells, display an interesting phenotype in which the activity of catalase is constitutively high, whereas its mRNA is reduced after X-ray irradiation. In the present study, we investigated the molecular mechanisms underlying this phenomenon. By combining analyses from nuclear run-on, reporter gene transient transfection, genomic footprinting, site-directed mutagenesis, electrophoretic mobility shift analysis, and Western blotting experiments, we found that constitutively elevated catalase expression is strongly regulated at the transcriptional level by both Sp1 and CCAAT-recognizing factors and that much higher levels of nuclear Sp1 and NF-Y are present in HP100 nuclei as compared with HL60 nuclei. In addition, we demonstrated an X-ray-inducible association of a WT1/Egr-related factor with an overlapping Sp1/Egr-1 recognition sequence located within the core promoter of the catalase gene. This association may lead to inactivation of the promoter by disturbing or competing with the transactivating ability of Sp1.

Sequence of 16S rRNA gene of rat-origin cilia-associated respiratory (CAR) bacillus SMR strain.

The 16S rRNA gene of the SMR strain of cilia-associated respiratory (CAR) bacillus, which was isolated from a spontaneously infected rat at our institute, was sequenced. Its 1,521 nucleotides were determined. On the basis of the results of the sequence analysis, the SMR strain was found to be most closely related to members of the Flavobacter/Flexibacter group. This sequence was compared with the previously determined 16S rRNA gene sequences (rat-origin: three; mouse-origin: one; rabbit-origin: one) of CAR bacillus isolates. The SMR strain showed the highest sequence similarity (99.9%) to the rat-origin CARB-NIH strain (Schoeb et al., 1993), and it was concluded that the strains are identical.

Interspecific comparison in the frequency of concerted evolution at the polyubiquitin gene locus.

The polyubiquitin gene, encoding tandemly repeated multiple ubiquitins, constitutes a uniquitin gene subfamily. It has been demonstrated that polyubiquitin genes are subject to concerted evolution; namely, the individual ubiquitin coding units contained within a polyubiquitin gene are more similar to one another than they are to the ubiquitin coding units in the orthologous gene from other species. However there has been no comprehensive study on the concerted evolution of polyubiquitin genes in a wide range of species, because the relationships (orthologous or paralogous) among multiple polyubiquitin genes from different species have not been extensively analyzed yet. In this report, we present the results of analyzing the nucleotide sequence of polyubiquitin genes of mammals, available in the DDBJ/EMBL/GenBank nucleotide sequence databases, in which we found that there are two groups of polyubiquitin genes in an orthologous relationship. Based on this result, we analyzed the concerted evolution of the polyubiquitin gene in various species and compared the frequency of concerted evolutionary events interspecifically by taking into consideration that the rate of synonymous substitution at the polyubiquitin gene locus may vary depending on species. We found that the concerted evolutionary events in polyubiquitin genes have been more frequent in rats and Chinese hamsters than those in humans, cows, and sheep. The guinea pig polyubiquitin gene was an intermediate example. The frequency of concerted evolution in the mouse gene was unexpectedly low compared to that of other rodent genes.

Significant increases in the steady states of putrescine and spermidine/spermine N1-acetyltransferase mRNA in HeLa cells accompanied by growth arrest.

Polyamines are intrinsic polycations which play critical roles in cell proliferation. Ornithine decarbolylase (ODC) catalyzes the first step of polyamine biosynthesis converting ornithine to putrescine. In addition to polyamine degradation, spermidine/spermine acetyltransferase (SSAT) regulates interconversion pathway of spermine and spermidine to putrescine. We quantified the polyamines and mRNAs of ODC and SSAT in HeLa S3 cells at various stages during exponential and plateau phases of culturing. Unexpectedly, putrescine and SSAT mRNA levels increased remarkably at the plateau phase, in contrast to the decrease of ODC mRNA level. It will be suggested that the putrescine has a novel function linked to the arrest of cell growth in which the SSAT-mediated pathway producing putrescine takes part.

Higher frequency of concerted evolutionary events in rodents than in man at the polyubiquitin gene VNTR locus.

The polyubiquitin gene is an evolutionarily conserved eukaryotic gene, encoding tandemly repeated multiple ubiquitins, and is considered to be subject to concerted evolution. Here, we present the nucleotide sequences of new alleles of the polyubiquitin gene UbC in humans and CHUB2 in Chinese hamster, which encode a different number of ubiquitin units from those of previously reported genes. And we analyze the concerted evolution of these genes on the basis of their orthologous relationship. That the mean of the synonymous sequence difference Ks which is defined as the number of synonymous substitution relative to the total number of synonymous sites, within the UbC and CHUB2 genes (0.192 +/- 0.096) is significantly less than Ks between these genes (0.602 +/- 0.057) provides direct evidence for concerted evolution. Moreover, it also appears that concerted evolutionary events have been much more frequent in CHUB2 than in UbC, because Ks within CHUB2 (0.022 +/- 0.018) is much less than that within UbC (0.362 +/- 0.192). By a numerical simulation, postulating that the major mechanism of concerted evolution in polyubiquitin genes is unequal crossing over, we estimated the frequency of concerted evolutionary events of CHUB2 at 3.3 x 10(-5) per year and that of UbC at no more than 5.0 x 10(-7) per year.

Comparison of the 5' upstream region of the evolutionarily equivalent polyubiquitin gene of humans and Chinese hamsters.

The 5' upstream region of the Chinese hamster polyubiquitin gene CHUB2 was determined, and compared to that of the evolutionarily equivalent polyubiquitin gene UbC of humans. The 5' upstream region of the CHUB2 gene is distinct from that of the UbC gene in containing fewer recognition sequences for binding of transcription factors, which are quite sparsely distributed in this region. It seemed probable that the absence of AP-1 sites in the promoter of the CHUB2 gene was likely to be responsible for the very dissimilar regulation of the two genes by UV light and TPA, despite the fact that these genes are evolutionarily equivalent.

Heterogeneous structure of the polyubiquitin gene UbC of HeLa S3 cells.

The nucleotide sequence of the polyubiquitin gene UbC of HeLa S3 cells and its upstream region was determined and characterized. Recognition sequences for the transcription factors HSF, NF kappa B, AP-1(c-jun), NF-IL6 and Sp1 were found in the upstream control region, a result consistent with the observation of a distinct regulatory response for the UbC gene compared with that of another polyubiquitin gene UbB. Employing a PCR procedure to amplify the entire coding region from genomic DNA, we found a heterogeneity in the repeat number (eight and nine repeats) of the ubiquitin coding units, which resulted from an apparent deletion of either the seventh or the eighth unit in the predominant nine-ubiquitin-unit coding gene. In addition, by comparison with the nucleotide sequence of the UbC gene of human leukocytes previously determined, we found a significant number of nucleotide discrepancies. However, these discrepancies could be substantially reduced by realigning the units so that the first and second ubiquitin units of the sequence determined here are translocated to the boundary between the eighth and the ninth units.

Expression of the Bombyx mori beta-tubulin-encoding gene in testis.

We have isolated a beta-tubulin-encoding cDNA clone of Bombyx mori from testes and determined the nucleotide sequence. Northern analyses showed that its expression is testis-specific and most active in the pupal stage.

The human gene encoding the largest subunit of RNA polymerase II.

The nucleotide sequence of a large portion of the human RNA polymerase II large subunit (RpII LS)-encoding gene and its whole gene structure were determined. The RpIILS gene consists of 29 exons. The sequence of the 5' flanking region is highly conserved as compared with that of the mouse RpIILS and contains several SP1-binding sites, a CCAAT sequence and a sequence homologous to a heat-shock element. In addition, several inverted repeats and palindrome sequences were involved in the 5' upstream region. Those suggest that the 5' flanking domain of RpIILS would be highly structured which may be responsible for transcriptional regulation.

Novel structure of a Chinese hamster polyubiquitin gene.

We isolated a polyubiquitin gene, CHUB2, from the V79 Chinese hamster genomic library, and determined its complete structure. Based on sequence homology to the human polyubiquitin gene UbC in the 5' and 3' untranslated region, the CHUB2 gene was characterized as the V79 Chinese hamster equivalent to the human UbC gene. However, the overall coding region structure of the CHUB2 gene was altered from the consensus structure of polyubiquitin genes, with the last ubiquitin coding unit being followed by 161 bp of partially deleted and mutated ubiquitin-like sequence. Although a similarly deleted and mutated polyubiquitin gene was recently reported in a partially sequenced cDNA of mouse (Finch et al. (1992) Cell Growth Differ. 3, 269-278), the present study describes the complete sequence of a polyubiquitin gene containing this unusual structure for the first time, and suggests that this structure is conserved in rodents. By employing both Southern and Northern analysis with a probe specific to the CHUB2 gene, it was found that a second, closely related gene is present in the Chinese hamster genome, and that both loci are transcriptionally active in V79 cells. The two genes, and their respective transcripts, differ in size because of variation in the relative number of repeating ubiquitin coding units.

Evolutionarily conserved structure of the 3' non-translated region of a Chinese hamster polyubiquitin gene.

From a V79 Chinese hamster genomic library, we isolated a clone containing a polyubiquitin gene (designated as CHUB1), and determined its nucleotide sequence. The coding region of the CHUB1 gene consisted of five direct repeats of the ubiquitin unit with no spacer, followed by a single tyrosine residue. Northern hybridization analysis with a synthesized probe specific to the 3' non-translated region of the CHUB1 gene revealed that it codes for a 1.8 kb mRNA. An evident homology to the human polyubiquitin gene UbB and the chicken UbI gene was observed in the region corresponding to the full extent of the mature mRNA sequence, suggesting that these three genes belong to a common polyubiquitin gene subfamily, and that the sequence in the 3' non-translated region of the CHUB1 gene is unique to this subfamily.

Induced accumulation of polyubiquitin gene transcripts in HeLa cells after UV-irradiation and TPA-treatment.

There are three species of ubiquitin gene transcripts in HeLa cells, termed UbA (approximately 0.7 kb), UbB (approximately 1.1 kb) and UbC (approximately 2.5 kb). In the present report, the UbC transcript was shown to accumulate up to 2.5-fold after irradiation with UV light or treatment with the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA). The kinetic analysis indicated that the induced accumulation of UbC was rapid and transient; maximal accumulation of UbC was induced at 2.5 h after UV irradiation or 3 h after TPA treatment. Inhibition of a de novo protein synthesis by cycloheximide did not repress the induction of UbC after treatment with UV light and TPA. On the other hand, induction of UbA and UbB, in most cases, was not observed. UV-inducibility of human ubiquitin conjugating enzyme, E2(17k), was also tested. E2(17k) is a protein with high sequence similarity to the product of yeast DNA repair gene, RAD6. While the RAD6 gene has been reported to be inducible by UV light, no change in E2(17k) gene transcript was observed after UV irradiation.

Essential factors determining codon usage in ubiquitin genes.

Ubiquitin is ubiquitous in all eukaryotes and its amino acid sequence shows extreme conservation. Ubiquitin genes comprise direct repeats of the ubiquitin coding unit with no spacers. The nucleotide sequences coding for 13 ubiquitin genes from 11 species reported so far have been compiled and analyzed. The G + C content of codon third base reveals a positive linear correlation with the genome G + C content of the corresponding species. The slope strongly suggests that the overall G + C content of codons of polyubiquitin genes clearly reflects the genome G + C content by AT/GC substitutions at the codon third position. The G + C content of ubiquitin codon third base also shows a positive linear correlation with the overall G + C content of coding regions of compiled genes, indicating the codon choices among synonymous codons reflect the average codon usage pattern of corresponding species. On the other hand, the monoubiquitin gene, which is different from the polyubiquitin gene in gene organization, gene expression, and function of the encoding protein, shows a different codon usage pattern compared with that of the polyubiquitin gene. From comparisons of the levels of synonymous substitutions among ubiquitin repeats and the homology of the amino acid sequence of the tail of monomeric ubiquitin genes, we propose that the molecular evolution of ubiquitin genes occurred as follows: Plural primitive ubiquitin sequences were dispersed on genome in ancestral eukaryotes. Some of them situated in a particular environment fused with the tail sequence to produce monomeric ubiquitin genes that were maintained across species. After divergence of species, polyubiquitin genes were formed by duplication of the other primitive ubiquitin sequences on different chromosomes. Differences in the environments in which ubiquitin genes are embedded reflect the differences in codon choice and in gene expression pattern between poly- and monomeric ubiquitin genes.

Interaction function gamma(x) for Chinese hamster cells treated with hypertonic phosphate-buffered saline after irradiation.

The repair of potentially lethal damage (PLD) in stationary-phase V79 Chinese hamster cells, which was expressible by a postirradiation treatment with hypertonic (0.5 M NaCl) phosphate-buffered saline (PBS), was analyzed within the framework of the theory of dual radiation action. The interaction function gamma(x) was estimated for cells permitted to repair PLD for various intervals of time. The experimental data indicated that 50-60% of the lethal lesions produced at the time of irradiation were repaired in 120 min. The repair of PLD was implicitly involved in the probability of the interaction of sublesions. That is, g(x,trep) was defined as the probability that two sublesions separated by distance x interact to produce a lethal lesion which will not be repaired until the fixation by treatment with hypertonic PBS at time trep after irradiation. It is concluded that the time dependence of the repair of PLD is not independent of the interaction distance x. Three conclusions are drawn: (1) The repair of a lesion produced by a long distance interaction is not detectable by postirradiation treatment with hypertonic PBS. (2) A lesion produced by a short distance interaction is rapidly repaired in about 20 min. (3) A lesion produced by the interaction of sublesions separated by a distance of about 100 nm is repaired slowly.

Estimation of interaction function gamma(x) with sparsely ionizing radiation.

The interaction function gamma(chi), which was introduced in the theory of dual radiation action as the probability that two energy transfers separated by distance chi combine with each other to produce a lesion, was estimated with sparsely ionizing radiation (60Co gamma rays and 40 kV X rays). Gamma(chi) was deduced on the assumption that the sensitive matrix is made up of small spherical flocculi distributed over the cell nucleus. The diameter of a flocculus was estimated at (4.0-11.2) X 10(-8) m when the diameter of the cell nucleus d was assumed to be 5 microns, and (4.0-11.4) X 10(-8) m when d was assumed to be 10 microns. It seems reasonable to hypothesize that the flocculus corresponds to the linker DNA in the chromatin structure of DNA, because the size of the linker DNA as a target (about 40 nm) is consistent with the diameter of flocculi obtained in this study.