Keiji Suzuki

Growth of persistent foci of DNA damage checkpoint factors is essential for amplification of G1 checkpoint signaling

Several DNA damage checkpoint factors form nuclear foci in response to ionizing radiation (IR). Although the number of the initial foci decreases concomitantly with DNA double-strand break repair, some fraction of foci persists. To date, the physiological role of the persistent foci has been poorly understood. Here we examined foci of Ser1981-phosphorylated ATM in normal human diploid cells exposed to 1Gy of X-rays. While the initial foci size was approximately 0.6microm, the one or two of persistent focus (foci) grew, whose diameter reached 1.6microm or more in diameter at 24h after IR. All of the grown persistent foci of phosphorylated ATM colocalized with the persistent foci of Ser139-phosphorylated histone H2AX, MDC1, 53BP1, and NBS1, which also grew similarly. When G0-synchronized normal human cells were released immediately after 1Gy of X-rays and incubated for 24h, the grown large phosphorylated ATM foci (> or =1.6microm) were rarely (av. 0.9%) observed in S phase cells, while smaller foci (<1.6microm) were frequently (av. 45.9%) found. We observed significant phosphorylation of p53 at Ser15 in cells with a single grown phosphorylated ATM focus. Furthermore, persistent inhibition of foci growth of phosphorylated ATM by an ATM inhibitor, KU55933, completely abrogated p53 phosphorylation. Defective growth of the persistent IR-induced foci was observed in primary fibroblasts derived from ataxia-telangiectasia (AT) and Nijmegen breakage syndrome (NBS) patients, which were abnormal in IR-induced G1 checkpoint. These results indicate that the growth of the persistent foci of the DNA damage checkpoint factors plays a pivotal role in G1 arrest, which amplifies G1 checkpoint signals sufficiently for phosphorylating p53 in cells with a limited number of remaining foci.

Different involvement of radical species in irradiated and bystander cells

Purpose: To examine whether nitric oxide (NO) and other radical species are involved in radiation-induced bystander effects in normal human fibroblasts. Materials and methods: Bystander effects were modeled by co-culture of non-irradiated cells with X-irradiated cells, and induction levels of micronuclei in co-cultured non-irradiated cells were examined. Three types of radical scavenger, 2-(4-carboxyphenyl)-4,4,5,5- tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO), dimethylsulfoxide (DMSO) and ascorbic acid phosphoric ester magnesium salt (APM), were used to discover which types of radicals are involved in bystander responses. Results: When irradiated cells were treated with c-PTIO, known to be an NO scavenger, the induction of micronuclei in non-irradiated bystander cells was suppressed. On the other hand, bystander effects were most effectively suppressed when non-irradiated bystander cells were treated with ascorbic acid, known to be a scavenger of long lived radicals. Conclusion: These results suggest that NO participates in bystander signal formation in irradiated cells but not in bystander cells that are receiving bystander signals.

Foci formation of P53-binding protein 1 in thyroid tumors: activation of genomic instability during thyroid carcinogenesis.

Defective DNA damage response (DDR) can result in genomic instability (GIN) and lead to the transformation into cancer. P53‐binding protein 1 (53BP1) belongs to a family of evolutionarily conserved DDR proteins. Because 53BP1 molecules localize at the sites of DNA double strand breaks (DSBs) and rapidly form nuclear foci, the presence of 53BP1 foci can be considered as a cytologic marker for endogenous DSBs reflecting GIN. Although it has been proposed that GIN has a crucial role in the progression of thyroid neoplasms, the significance of GIN during thyroid tumorigenesis remains unclear, particularly in patients. We analyzed, therefore, the level of GIN, as detected with immunofluorescence of 53BP1, in 40 cases of resected thyroid tissues. This study demonstrated a number of nuclear 53BP1 foci in thyroid cancers, suggesting a constitutive activation of DDR in thyroid cancer cells. Because follicular adenoma also showed a few 53BP1 nuclear foci, GIN might be induced at a precancerous stage of thyroid tumorigenesis. Furthermore, high‐grade thyroid cancers prominently exhibited an intense and heterogeneous nuclear staining of 53BP1 immunoreactivity, which was also observed in radiation‐associated cancers and in mouse colonic crypts as a delayed response to a high dose ionizing radiation, suggesting increased GIN with progression of cancer. Thus, the present study demonstrated a difference in the staining pattern of 53BP1 during thyroid carcinogenesis. We propose that immunofluorescence analysis of 53BP1 expression can be a useful tool to estimate the level of GIN and, simultaneously, the malignant potency of human thyroid tumors.

Transmission of genomic instability from a single irradiated human chromosome to the progeny of unirradiated cells.

Abstract Mukaida, N., Kodama, S., Suzuki, K., Oshimura, M. and Watanabe, M. Transmission of Genomic Instability from a Single Irradiated Human Chromosome to the Progeny of Unirradiated Cells. Radiat. Res. 167, 675–681 (2007). Ionizing radiation can induce chromosome instability that is transmitted over many generations after irradiation in the progeny of surviving cells, but it remains unclear why this instability can be transmitted to the progeny. To acquire knowledge about the transmissible nature of genomic instability, we transferred an irradiated human chromosome into unirradiated mouse recipient cells by microcell fusion and examined the stability of the transferred human chromosome in the microcell hybrids. The transferred chromosome was stable in all six microcell hybrids in which an unirradiated human chromosome had been introduced. In contrast, the transferred chromosome was unstable in four out of five microcell hybrids in which an irradiated human chromosome had been introduced. The aberrations included changes in the irradiated chromosome itself and rearrangements with recipient mouse chromosomes. Thus the present study demonstrates that genomic instability can be transmitted to the progeny of unirradiated cells by a chromosome exposed to ionizing radiation, implying that the instability is caused by the irradiated chromosome itself and also that the instability is induced by the nontargeted effect of radiation.

Alteration of p53-binding protein 1 expression during skin carcinogenesis: association with genomic instability.

Epidermal cells are the first cells to be exposed to environmental genotoxic agents such as ultraviolet and ionizing radiations, which induce DNA double strand breaks (DSB) and activate DNA damage response (DDR) to maintain genomic integrity. Defective DDR can result in genomic instability (GIN) which is considered to be a central aspect of any carcinogenic process. P53‐binding protein 1 (53BP1) belongs to a family of evolutionarily conserved DDR proteins. Because 53BP1 molecules localize at the sites of DSB and rapidly form nuclear foci, the presence of 53BP1 nuclear foci can be considered as a cytological marker for endogenous DSB reflecting GIN. The levels of GIN were analyzed by immunofluorescence studies of 53BP1 in 56 skin tumors that included 20 seborrheic keratosis, eight actinic keratosis, nine Bowens disease, nine squamous cell carcinoma, and 10 basal cell carcinoma. This study demonstrated a number of nuclear 53BP1 foci in human skin tumorigenesis, suggesting a constitutive activation of DDR in skin cancer cells. Because actinic keratosis showed a high DDR type of 53BP1 immunoreactivity, GIN seems to be induced at the precancerous stage. Furthermore, invasive cancers exhibited a high level of intense, abnormal 53BP1 nuclear staining with nuclear accumulation of p53, suggesting a disruption of DDR leading to a high level of GIN in cancer cells. The results of this study suggest that GIN has a crucial role in the progression of skin carcinogenesis. The detection of 53BP1 expression by immunofluorescence can be a useful histological marker to estimate the malignant potential of human skin tumors. (Cancer Sci 2008; 99: 946–951)

The contribution of radiation-induced large deletion of the genome to chromosomal instability.

Abstract Toyokuni, H., Maruo, A., Suzuki, K. and Watanabe, M. The Contribution of Radiation-Induced Large Deletion of the Genome to Chromosomal Instability. Radiat. Res. 171, 198–203 (2009). Ionizing radiation is known to induce genomic instability that is transmitted across many generations of the progeny of surviving cells. However, the mechanism underlying the initiation, perpetuation and manifestation of radiation-induced genomic instability remains unclear. We expect that large radiation-induced deletions destabilize the structure of chromatin and that this destabilization is transmitted across many generations and plays a role in the perpetuation of genomic instability. Therefore, in this study, we examined the relationship between deletion size and the frequency of delayed chromosomal aberrations in SV40-immortalized normal human fibroblast (GM638) cells. GM638 cells were irradiated with 3 Gy of X rays, and chromosomal aberrations were analyzed in clones derived after irradiation. To determine the size of each deletion, we isolated mutants of the HPRT gene from the X-irradiated cell population and examined the genes around the HPRT locus, which is located in the q-arm of chromosome X. The results indicated that X chromosomes with large (>0.5 Mb) deletions have a higher probability of exhibiting delayed chromosomal aberrations and that these aberrations were induced more frequently in q-arms than in p-arms. Because no induction of X-chromosomal instability was observed in clones that lacked such large deletions, the present findings suggest that chromosomes with large radiation-induced deletions can be genomically unstable.

Reciprocal Paracrine Interactions Between Normal Human Epithelial and Mesenchymal Cells Protect Cellular DNA from Radiation-Induced Damage

PURPOSEnTo explore whether interactions between normal epithelial and mesenchymal cells can modulate the extent of radiation-induced DNA damage in one or both types of cells.nnnMETHODS AND MATERIALSnHuman primary thyrocytes (PT), diploid fibroblasts BJ, MRC-5, and WI-38, normal human mammary epithelial cells (HMEC), and endothelial human umbilical cord vein endothelial cells (HUV-EC-C), cultured either individually or in co-cultures or after conditioned medium transfer, were irradiated with 0.25 to 5 Gy of gamma-rays and assayed for the extent of DNA damage.nnnRESULTSnThe number of gamma-H2AX foci in co-cultures of PT and BJ fibroblasts was approximately 25% lower than in individual cultures at 1 Gy in both types of cells. Reciprocal conditioned medium transfer to individual cultures before irradiation resulted in approximately a 35% reduction of the number gamma-H2AX foci at 1 Gy in both types of cells, demonstrating the role of paracrine soluble factors. The DNA-protected state of cells was achieved within 15 min after conditioned medium transfer; it was reproducible and reciprocal in several lines of epithelial cells and fibroblasts, fibroblasts, and endothelial cells but not in epithelial and endothelial cells. Unlike normal cells, human epithelial cancer cells failed to establish DNA-protected states in fibroblasts and vice versa.nnnCONCLUSIONSnThe results imply the existence of a network of reciprocal interactions between normal epithelial and some types of mesenchymal cells mediated by soluble factors that act in a paracrine manner to protect DNA from genotoxic stress.

Correlation of malignant phenotypes of human tumour cell lines with augmented expression of Hsp72 protein measured by laser scanning cytometry

Augmented expression of members of the heat shock protein 70 (Hsp70) family are frequently observed in various human cancers. In this study, we examined applicability of laser scanning cytometer (LSC) to evaluate the level of Hsp72, which is the member constitutively expressed and significantly induced after heat shock, in human tumour cell lines. The relative nuclear content of Hsp72 measured by LSC correlated well with the relative intracellular content determined by Western blotting (Ru2009=u20090.906). Furthermore, there was a close relationship between the relative nuclear content of Hsp72 measured by LSC and the colony-forming ability in soft agar, one of the malignant characteristics of tumour cells (Ru2009=u20090.880). These results indicate that LSC measurement is useful for predicting the degree of malignancy of cancer cells, as it is reliable, faster than Western blotting and more objective and quantitative than visual measurements.