Kihei Kubo

Effect of radiation on the expression of carcinoembryonic antigen of human gastric adenocarcinoma cells

The changes of antigenic expression of cultured human gastric adenocarcinoma MKN45 cells caused by irradiation were investigated to elucidate the immune responses to localized irradiation. The expression of carcinoembryonic antigen (CEA) showed remarkable increases in the culture supernatant and on the surface of the membrane of irradiated cells. The expression of major histocompatibility complex Class I antigen on the membrane also was enhanced by irradiation. In addition, the irradiated cell groups, when analyzed using a CEA‐specific probe, showed remarkable increases in the CEA mRNA. These enhancements increased in the 10‐Gy and 15‐Gy irradiated populations compared with the 5‐Gy irradiated population. These results suggest that the enhancement of expression of CEA by radiation takes place at the CEA gene expression (mRNA) level but not at the protein level.

Highly sensitive assay of DNA abasic sites in mammalian cells-optimization of the aldehyde reactive probe method

We have recently developed a novel method for detection and quantitation of abasic (AP) sites in DNA, in which the biotinylated reagent, called the aldehyde reactive probe (ARP) specifically reacts with aldehyde groups of AP sites and biotin-tagged damage is detected by an ELISA-like assay. The present study has been carried out to improve the feasibility and the sensitivity of ARP assay. For immobilization of DNA, a protamine sulfate-coated plate was used instead of the conventional UV-irradiated plate to enhance DNA binding. As the result, the time for immobilization was shortened to 1 h without any loss of signal. The amount of [ 3 H]-labeled DNA bound to the plate was proportional to the DNA concentration employed. When DNA containing AP sites was treated with ARP in solution prior to coating the protamine-plate, the sensitivity of the assay was greatly increased. A linear relationship between the DNA concentration and the signal intensity was also observed. Thus, ∼0.1 fmol of AP sites (0.5 sites per 10 5 nt) could be detected in DNA isolated from HeLa cells after treatment with a sublethal dose (0.5 mM) of methylmethanesulfonate (MMS). Using this system, the number of total methylpurines generated by MMS in the cellular DNA was estimated after heat treatment, which converted methylated base lesions to AP sites. It was shown that the number of AP sites was about 140 sites per 10 4 nt with 25 mM MMS and 10% of total methylated bases were already released without heat depurination.

Increase in Endogenous Spleen Colonies without Recovery of Blood Cell Counts in Radioadaptive Survival Response in C57BL/6 Mice

Abstract Yonezawa, M., Horie, K., Kondo, H. and Kubo, K. Increase in Endogenous Spleen Colonies without Recovery of Blood Cell Counts in Radioadaptive Survival Response in C57BL/6 Mice. Radiat. Res. 161, 161–167 (2004). The radioadaptive survival response induced by a conditioning exposure to 0.45 Gy and measured as an increase in 30-day survival after mid-lethal X irradiation was studied in C57BL/6N mice. The acquired radioresistance appeared on day 9 after the conditioning exposure, reached a maximum on days 12–14, and disappeared on day 21. The conditioning exposure 14 days prior to the challenge exposure increased the number of endogenous spleen colonies (CFU-S) on days 12–13 after the exposure to 5 Gy. On day 12 after irradiation, the conditioning exposure also increased the number of endogenous CFU-S to about five times that seen in animals exposed to 4.25–6.75 Gy without preirradiation. The effect of the interval between the preirradiation and the challenge irradiation on the increase in endogenous CFU-S was also examined. A significant increase in endogenous CFU-S was observed when the interval was 14 days, but not 9 days. This result corresponded to the increase in survival observed on day 14 after the challenge irradiation. Radiation-inducted resistance to radiation-induced lethality in mice appears to be closely related to the marked recovery of endogenous CFU-S in the surviving hematopoietic stem cells that acquired radioresistance by preirradiation. Preirradiation enhanced the recovery of the numbers of erythrocytes, leukocytes and thrombocytes very slightly in mice exposed to a sublethal dose of 5 Gy, a dose that does not cause bone marrow death. There appears to be no correlation between the marked increase in endogenous CFU-S and the slight increase or no increase in peripheral blood cells induced by the radioadaptive response. The possible contribution by some factor, such as Il4 or Il11, that has been reported to protect irradiated animals without stimulating hematopoiesis is discussed.

Repair Kinetics of Abasic Sites in Mammalian Cells Selectively Monitored by the Aldehyde Reactive Probe (ARP)

Human methylpurine N-glycosylase (MPG) activity was investigated by monitoring abasic (AP) sites resulting from removal of alkylated bases. The amount of AP sites in MMS-treated HeLa cells transiently increased at 3 h, then gradually decreased to 40% at 24 h. The presence of adenine, an inhibitor of AP endonucleases, in the repair incubation of MMS-treated cells induced moderate accumulation of AP sites, suggesting inhibition of the activities of MPG as well as AP endonucleases by adenine metabolites.

Utilization of CDKN1A/p21 gene for class discrimination of DNA damage-induced clastogenicity

The in vitro mammalian cytogenetic tests monitor chromosomal aberrations in cultured mammalian cells to test the mutagenicity of compounds. Although these tests are especially useful for evaluating the potential clastogenic effects of chemicals, false positives associated with excessive toxicity occur frequently. There is a growing demand for mechanism-based assays to confirm positive results from cytogenetic tests. We hypothesized that a toxicogenomic approach that is based on gene expression profiles could be used to investigate mechanisms of genotoxicity. Human lymphoblastoid TK6 cells were treated with each of eight different genotoxins that included six DNA damaging compounds-mitomycin C, methyl methanesulfonate, ethyl methanesulfonate, cisplatin, etoposide, hydroxyurea-and two compounds that do not damage DNA-colchicine and adenine. Cells were exposed to each compound for 4h, and Affymetrix U133A microarrays were then used to comprehensively examine gene expression. A statistical analysis was used to select biomarker candidates, and 103 probes met our statistical criteria. Expression of cyclin-dependent kinase inhibitor 1A (CDKN1A)/p21 was ranked highest for discriminating DNA-damaging compounds. To further characterize the biological significance of alterations in gene expression, functional network analysis was performed with the 103 selected probes. Interestingly, a CDKN1A-centered interactome was identified as the most significant network. Together, these findings indicated that DNA-damaging compounds often induced changes in the expression of a large number of these 103 probes and that upregulation of CDKN1A was a common key feature of DNA damage stimuli. The utility of CDKN1A as a biomarker for assessing the genotoxicity of drug candidates was further evaluated; specifically, quantitative RT-PCR was used to assess the effects of 14 additional compounds-including DNA damaging genotoxins and genotoxins that do not damage DNA and five newly-synthesized drug candidates-on CDKN1A expression. In these assays, DNA damage-positive clastogens were clearly separated from DNA damage-negative compounds based on CDKN1A expression. In conclusion, CDKN1A may be a valuable biomarker for identifying DNA damage-inducing clastogens and as a follow-up assay for mammalian cytogenetic tests.

Combinatorial Measurement of CDKN1A/p21 and KIF20A Expression for Discrimination of DNA Damage-Induced Clastogenicity

In vitro mammalian cytogenetic tests detect chromosomal aberrations and are used for testing the genotoxicity of compounds. This study aimed to identify a supportive genomic biomarker could minimize the risk of misjudgments and aid appropriate decision making in genotoxicity testing. Human lymphoblastoid TK6 cells were treated with each of six DNA damage-inducing genotoxins (clastogens) or two genotoxins that do not cause DNA damage. Cells were exposed to each compound for 4 h, and gene expression was comprehensively examined using Affymetrix U133A microarrays. Toxicogenomic analysis revealed characteristic alterations in the expression of genes included in cyclin-dependent kinase inhibitor 1A (CDKN1A/p21)-centered network. The majority of genes included in this network were upregulated on treatment with DNA damage-inducing clastogens. The network, however, also included kinesin family member 20A (KIF20A) downregulated by treatment with all the DNA damage-inducing clastogens. Downregulation of KIF20A expression was successfully confirmed using additional DNA damage-inducing clastogens. Our analysis also demonstrated that nucleic acid constituents falsely downregulated the expression of KIF20A, possibly via p16 activation, independently of the CDKN1A signaling pathway. Our results indicate the potential of KIF20A as a supportive biomarker for clastogenicity judgment and possible mechanisms involved in KIF20A downregulation in DNA damage and non-DNA damage signaling networks.

Hypersensitivity of mouse NEIL1-knockdown cells to hydrogen peroxide during S phase

Oxidative base damage occurs spontaneously due to reactive oxygen species generated as byproducts of respiration and other pathological processes in mammalian cells. Many oxidized bases are mutagenic and/or toxic, and most are repaired through the base excision repair pathway. Human endonuclease VIII-like protein 1 (hNEIL1) is thought to play an important role during the S phase of the cell cycle by removing oxidized bases in DNA replication fork-like (bubble) structures, and the protein level of hNEIL1 is increased in S phase. Compared with hNEIL1, there is relatively little information on the properties of the mouse ortholog mNEIL1. Since mouse cell nuclei lack endonuclease III-like protein (NTH) activity, in contrast to human cell nuclei, mNEIL1 is a major DNA glycosylase for repair of oxidized pyrimidines in mouse nuclei. In this study, we made mNEIL1-knockdown cells using an shRNA expression vector and examined the cell cycle-related variation in hydrogen peroxide (H2O2) sensitivity. Hypersensitivity to H2O2 caused by mNEIL1 knockdown was more significant in S phase than in G1 phase, suggesting that mNEIL1 has an important role during S phase, similarly to hNEIL1.

Abundance of BER-related proteins depends on cell proliferation status and the presence of DNA polymerase β.

In mammalian cells, murine N-methylpurine DNA glycosylase (MPG) removes bases damaged spontaneously or by chemical agents through the process called base excision repair (BER). In this study, we investigated the influence of POL β deficiency on MPG-initiated BER efficiency and the expression levels of BER-related proteins in log-phase and growth-arrested (G0) mouse embryonic fibroblasts (MEFs). G0 wild-type (WT) or POL β–deficient (Pol β–KO) cells showed greater resistance to methyl methanesulfonate than did log-phase cells, and repair of methylated bases was less efficient in the G0 cells. Apex1 mRNA expression was significantly lower in Pol β–KO or G0 WT MEFs than in log-phase WT MEFs. Moreover, although Mpg mRNA levels did not differ significantly among cell types, MPG protein levels were significantly higher in log-phase WT cells than in log-phase Pol β–KO cells or either type of G0 cells. Additionally, proliferating cell nuclear antigen protein levels were also reduced in log-phase Pol β–KO cells or either type of G0 cells. These results indicated that MPG-initiated BER functions mainly in proliferating cells, but less so in G0 cells, and that POL β may be involved in regulation of the amount of intracellular repair proteins.

Cloning and expression analysis of prohibitin mRNA in canine mammary tumors

Prohibitin is an antiproliferative protein that is a product of a putative tumor suppressor gene. However, there is little information on prohibitins in companion animals. In this study, we cloned canine prohibitin mRNA using RT-PCR and 3′-RACE (Rapid Amplification of cDNA Ends). The sequence was well conserved compared with those of other mammals, including human. The deduced amino acid sequence translated from the open reading frame completely corresponded to the human sequence. Canine prohibitin mRNA was expressed in all normal mammary and tumor samples examined. These results suggest that this protein plays a vital role in cell growth mechanisms and may be related to the occurrence of canine mammary tumors.

AP endonuclease knockdown enhances methyl methanesulfonate hypersensitivity of DNA polymerase β knockout mouse embryonic fibroblasts.

Apurinic/apyrimidinic (AP) endonuclease (Apex) is required for base excision repair (BER), which is the major mechanism of repair for small DNA lesions such as alkylated bases. Apex incises the DNA strand at an AP site to leave 3′-OH and 5′-deoxyribose phosphate (5′-dRp) termini. DNA polymerase β (PolB) plays a dominant role in single nucleotide (Sn-) BER by incorporating a nucleotide and removing 5′-dRp. Methyl methanesulfonate (MMS)-induced damage is repaired by Sn-BER, and thus mouse embryonic fibroblasts (MEFs) deficient in PolB show significantly increased sensitivity to MMS. However, the survival curve for PolB-knockout MEFs (PolBKOs) has a shoulder, and increased sensitivity is only apparent at relatively high MMS concentrations. In this study, we prepared Apex-knockdown/PolB-knockout MEFs (AKDBKOs) to examine whether BER is related to the apparent resistance of PolBKOs at low MMS concentrations. The viability of PolBKOs immediately after MMS treatment was significantly lower than that of wild-type MEFs, but there was essentially no effect of Apex-knockdown on cell viability in the presence or absence of PolB. In contrast, relative counts of MEFs after repair were decreased by Apex knockdown. Parental PolBKOs showed especially high sensitivity at >1.5 mM MMS, suggesting that PolBKOs have another repair mechanism in addition to PolB-dependent Sn-BER, and that the back-up mechanism is unable to repair damage induced by high MMS concentrations. Interestingly, AKDBKOs were hypersensitive to MMS in a relative cell growth assay, suggesting that MMS-induced damage in PolB-knockout MEFs is repaired by Apex-dependent repair mechanisms, presumably including long-patch BER.