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

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 ß deficiency on MPG-initiated BER efficiency and the expression levels of BER-related proteins in log-phase and growth-arrested (G(0)) mouse embryonic fibroblasts (MEFs). G(0) wild-type (WT) or POL ß-deficient (Pol ß-KO) cells showed greater resistance to methyl methanesulfonate than did log-phase cells, and repair of methylated bases was less efficient in the G(0) cells. Apex1 mRNA expression was significantly lower in Pol ß-KO or G(0) 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 ß-KO cells or either type of G(0) cells. Additionally, proliferating cell nuclear antigen protein levels were also reduced in log-phase Pol ß-KO cells or either type of G(0) cells. These results indicated that MPG-initiated BER functions mainly in proliferating cells, but less so in G(0) cells, and that POL ß may be involved in regulation of the amount of intracellular repair proteins.

AP endonuclease knockdown enhances methyl methanesulfonate hypersensitivity of DNA polymerase ß 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 ß (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.

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.

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.

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.

NEIL1 mRNA splicing variants are expressed in normal mouse organs.

Oxidized pyrimidines are mainly repaired by base excision repair, which is initiated by damage-specific DNA glycosylases. NEIL1, the mammalian homolog of Escherichia coli endonuclease VIII and a major DNA glycosylase, initiates repair of oxidized pyrimidines. Here, we investigated the expression of two putative variant mouse NEIL1 (mNEIL1) mRNAs--variant 1 ("Neil1 protein" mRNA; BC043297 in the NCBI database) and variant 2 ("unnamed protein" mRNA; AK040802 in the NCBI database)--in normal mouse organs. Reverse transcription-PCR showed that both mRNAs were expressed in total RNA samples from 9 organs. Immunoblot analysis of a nuclear extract from normal mouse liver revealed three bands corresponding to full-length mNEIL1 protein and the two predicted variant proteins. However, neither variant protein, which included an N-terminal enzymatic activity domain deduced from the mRNA variants, were enzymatically active under multiple reaction conditions when expressed as his-tagged recombinant proteins. Nevertheless, recombinant variant 1 protein influenced mNEIL1 activity, while recombinant variant 2 protein had no influence. These results suggest that mNEIL1 mRNA variants are expressed in a variety of organs in normal mice and that variant 1 protein may regulate mNEIL1 activity.

A novel monofunctional DNA glycosylase activity against thymine glycol in mouse cell nuclei.

Reactive oxygen species continuously oxidize DNA bases and threaten the genetic integrity. Thymine glycol (TG), one of the representative oxidized products, is repaired mainly by base excision repair (BER). In Escherichia coli, endonuclease III (Nth) and endonuclease VIII (Nei) are known to actively remove TG from DNA, and the homologs are well conserved in various organisms. These are bifunctional glycosylases, also associated with apurinic/apyrimidinic (AP) lyase activity. In the present study, a monofunctional TG-DNA glycosylase activity is shown to be one of the predominant nuclear activities present in some mouse tissues. By combining hypertonic extraction and column chromatography, we successfully separated the novel activity from majority of the bifunctional activities. Since it has been reported that mNTH1 may not be a dominant nuclear activity, the monofunctional glycosylase activity, together with mNEIL1, may be the major TG-DNA glycosylases in the mouse nucleus. The optimal reaction conditions for the monofunctional activity were found to be pH 7-8 and 100-150 mM KCl, and the activity was resistant to 20 mM EDTA. High monofunctional activity was detected in the spleen and stomach, while the level was significantly lower in the liver, suggesting that the contribution of the monofunctional activity is variable among organs.

Expression patterns of the BRCA1 splicing variants in canine normal tissues and mammary gland tumors.

Human BRCA1 is familial breast cancer susceptibility gene. Recently, decreased BRCA1 mRNA and protein expression has been identified in sporadic breast tumors. In the reported human BRCA1 splicing variants, delta11b lacks the majority of exon11 and is suspected to have a distinct function in normal tissues. The splicing variants display a variety of expression pattern in breast cancer samples. Although mammary gland tumor is important disease in dog, there are few reports for BRCA1 in the canine tumors. In this study, we examined the relative amounts of BRCA1 splicing variants mRNA in canine normal and mammary tumor samples by RT-PCR to investigate whether there is the altered expression of variant mRNAs in the canine tumor as reported in human. The exon11b-defecting RT-PCR products were observed in all the normal tissues examined and the nucleotide sequence was quite similar to that of human BRCA1 delta11b. In some tumor samples, we did not detect the products targeted for exon10-13 and exon14-15, while these products were observed in all the normal samples examined. Especially, the relative amounts of the exon11-defecting products were remarkably decreased in most of the tumors (11/16).

Expression patterns of the slit subfamily mRNA in canine malignant mammary tumors.

Slit, a secreted protein, functions as a chemorepellent factor in axon guidance and neuronal migration and as an inhibitor in leukocyte chemotaxis. In humans, slit2 protein attracts endothelial cells and promotes tube formation in the tumor angiogenic mechanism. In this study, we cloned a part of the canine slit subfamily and examined the expression of slit subfamily mRNAs in 3 normal canine mammary glands and 11 mammary tumor samples by RT-PCR. The cloned part of the slit gene sequences showed high similarity to those of the human, mouse, and rat. The mRNAs were expressed at low levels in the normal mammary gland. The expression levels of slit1 mRNA were low in both the normal and tumor tissues. In contrast, the expression of slit2 mRNA increased in most of the malignant mammary tumors, and an increase in slit3 mRNA expression was observed in 2 of the malignant mixed tumors. These results suggest that the expression of slit2 plays an important role in tumor angiogenesis in canine mammary gland tumors and that slit2 can be a putative marker for malignancy diagnosis of these tumors.

Detection of endonuclease III- and 8-oxoguanine glycosylase-sensitive base modifications in gamma-irradiated DNA and cells by the aldehyde reactive probe (ARP) assay.

Ionizing radiation generates diverse DNA lesions that differentially induce cell death and mutations. In the present study, calf thymus DNA (400 microg/ml) and HeLa cells were irradiated by (60)Co gamma-rays, and abasic (AP) sites and endonuclease (Endo)III- and 8-oxoguanine glycosylase (hOGG1)-sensitive base modifications in DNA were quantitated by the aldehyde reactive probe (ARP) assay. The irradiation of calf thymus DNA in phosphate buffer generated 91 Endo III- and 100 hOGG1-sensitive base modifications and 110 AP sites per 10(6) base pairs (bp) per Gy. The yield of the lesions in Tris buffer was 41- to 91-fold lower than that in phosphate, demonstrating a radioprotective effect of Tris. The HeLa cell chromosomal DNA contained 12 Endo III- and 3.8 hOGG1-sensitive base modifications and less than 1 AP sites per 10(6) bp as endogenous damage, and their level was increased by irradiation. The yields of the damage at 1 Gy (roughly equivalent to the lethal dose of HeLa cells [1.6-1.8 Gy]) were 0.13 Endo III, 0.091 hOGG1, and 0.065 AP sites per 10(6) bp, showing that irradiation with a lethal dose brought about only a marginal increase in base damage relative to an endogenous one. A comparison of the present data with those reported for DNA strand breaks supports the primary importance of double-strand breaks and clustered lesions as lethal damages formed by ionizing radiation.

Increase in endogenous spleen colonies without recovery of blood cell counts in radioadaptive survival response in C57BL/6 mice.

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 roles of hSMUG1 assessed by damage specificity and cellular activity.

Single-strand-selective monofunctional uracil-DNA glycosylase (SMUG1) was previously identified as a putative backup enzyme of major mammalian uracil-DNA glycosylase (UDG). However, the subsequent studies have shown conflicting results about the substrate specificity of SMUG1. In the present study, to clarify the repair role of SMUG1, we determined the damage specificity of purified human SMUG1 (hSMUG1) and its contribution to repair of oxidized bases in HeLa cell extracts.

Generation of large homozygous chromosomal segments by mitotic recombination during lymphomagenesis in F1 hybrid mice.

The loss of heterozygosity (LOH) has been reported in numerous neoplasms in both human and animals, and has often been observed in chromosomal regions, which contain tumor-suppressor genes. We previously found frequent LOH on chromosomes 4, 12 and 19 in radiation-induced lymphomas from (BALB/cHeA x STS/A)F1 hybrid mice by allelotype analysis at polymorphic microsatellite loci. In this study, to elucidate the nature of allelic losses, we refined the loss regions on chromosomes 4, 12 and 19 of the tumors from the F1 mice and then analyzed them cytogenetically. The results represent evidence of a wide range of allelic losses owing to mitotic recombination on chromosomes 4 and 19 in the tumors, possibly reflecting functional losses of putative tumor-suppressor genes. It is suggested that the generation of these large homozygous chromosomal segments probably containing the affected genes is one of the genetic alterations responsible for tumorigenesis.

Detection of NO-induced DNA lesions by the modified aldehyde reactive probe (ARP) assay.

When DNA is exposed to NO or HNO2, oxanine (Oxa) is formed as a major guanine lesion. For highly sensitive detection of Oxa using ARP, a probe molecule for DNA damage detection, the reactivity of ARP to Oxa was examined. Oxa site-specifically embedded in an oligonucleotide reacted with ARP but it took relatively long time (ca. 24 h) to completely convert Oxa to an ARP-labeled form.

p53 dependency of radio-adaptive responses in endogenous spleen colonies and peripheral blood-cell counts in C57BL mice.

Radio-adaptive responses at a conditioning X-ray dose of 0.45 Gy and a challenging dose of 5.0 Gy on hematopoietic indices were studied in C57BL mice with p53 (Trp53) wild, heterogenous and knockout allele. The conditioning irradiation, given 2 weeks before the challenging irradiation, induced radio-adaptive responses observed as a recovery of the peripheral blood-cell counts of leukocytes, thrombocytes and erythrocytes on day 14 after challenging irradiation in C57BL mice of the wild-type p53(+/+). The pre-irradiation also increased the endogenous spleen colonies (endo-CFU-S) on day 12 and the spleen weight on day 14. On the contrary, the knockout p53(-/-) mice gave no such radio-adaptive response. The heterogenous p53(+/-) mice gave an intermediate response. The radio-adaptive response in hematopoiesis at a challenge dose of 5.0 Gy seems to be a p53-dependent phenomenon. The possible role of induction in radio-resistance through the reduction of p53-drived apoptosis in hematopoietic stem cells in pre-irradiated mice is discussed.