Identification and characterization of the antigen recognized by the germ cell mAb TRA98 using a human comprehensive wet protein array.

TRA98 is a rat monoclonal antibody (mAb) which recognizes a specific antigen in the nuclei of germ cells. mAb TRA98 has been used to understand the mechanism of germ cell development and differentiation in many studies. In mice, the antigen recognized by mAb TRA98 or GCNA1 has been reported to be a GCNA gene product, but despite the demonstration of the immunoreactivity of this mAb in human testis and sperm in 1997, the antigen in humans remains unknown, as of date. To identify the human antigen recognized by mAb TRA98, a human comprehensive wet protein array was developed containing 19,446 proteins derived from human cDNAs. Using this array, it was found that the antigen of mAb TRA98 is not a GCNA gene product, but nuclear factor-κB activating protein (NKAP). In mice, mAb TRA98 recognized both the GCNA gene product and NKAP. Furthermore, conditional knockout of Nkap in mice revealed a phenotype of Sertoli cell-only syndrome. Although NKAP is a ubiquitously expressed protein, NKAP recognized by mAb TRA98 in mouse testis was SUMOylated. These results suggest that NKAP undergoes modifications, such as SUMOylation in the testis, and plays an important role in spermatogenesis.

Haprin-deficient spermatozoa are incapable of in vitro fertilization.

Haprin (TRIM36) is a ubiquitin-protein ligase that mediates ubiquitination and subsequent proteasomal degradation of target proteins. It is expressed in the testes in both mice and humans and is thought to be involved in spermiogenesis, the acrosome reaction, and fertilization. However, the functional role of Haprin is poorly understood. The aim of this study was to investigate the physiological role of Haprin in fertility. Homozygous haprin-deficient mice were generated and these mice, and their spermatozoa, were analyzed to detect morphological and fertility-related abnormalities. In these models, normal spermatogenesis was observed but sperm quality was reduced with haprin-deficient mice having poorer sperm morphology and motility than wild-type mice. Interestingly, haprin-deficient mice showed normal in vivo fertility but could not fertilize oocytes under standard in vitro fertilization conditions. In conclusion, this study demonstrated that Haprin deficiency causes morphological abnormalities in spermatozoa, indicating that Haprin is involved in spermiogenesis.

Two activators of in vitro fertilization in mice from licorice.

Systems for artificial insemination have been established in some animals. However, due to limited availability of sperm and oocytes, more effective treatment methodologies are required. Recently, it was demonstrated that the rate of in vitro fertilization (IVF) in mice was improved by adding a water extract of licorice (Glycyrrhiza uralensis), but not glycyrrhizic acid, to the artificial insemination culture medium. In this study, we examined licorice extract for active compounds using bioassay-guided separation. The results indicated that isoliquiritigenin and formononetin were the active molecules in licorice that contributed to the improved rate of IVF.

Involvement of trefoil factor family 2 in the enlargement of intestinal tumors in Apc(Min/+) mice.

It is assumed that tumor size may be associated with malignant tumor conversion. However, the molecules responsible for determination of tumor size are not well understood. We counted the number of intestinal tumors in 8, 12 and 30-week-old Apc(Min/+) mice and measured tumor sizes, respectively. Genes involved in determining tumor size were examined using microarray analysis. Cultured cells were then, transfected with a mammalian expression vector containing a candidate gene to examine the functional role of the gene. The effect of forced expression of candidate gene on cell growth was evaluated by measuring the doubling time of the cultured cells and the growth of grafted cells in nude mice. Unexpectedly, microarray analysis identified trefoil factor family 2 (Tff2) rather than growth related genes and/or oncogenes as a most variable gene. Overexpressing Tff2 in cultured cells reduced doubling time in vitro and rapidly increased xenograft tumor size in vivo. We found Tff2 as a novel important factor that to be able to enlarge an intestinal tumor size.

Intestinal Peyer's patches prevent tumorigenesis in Apc (Min/+) mice.

Peyer's patches are nodules that play a central role in intestinal immunity. Few studies demonstrate the relationship between the number of Peyer's patches and intestinal polyps. Here we identify a statistically significant inverse correlation between the quantity of Peyer's patches and of the development of intestinal polyps in Apc (Min/+) mice, which are a useful model to clarify the role of Peyer's patches in intestinal tumorigenesis. Using this model, we increased the number of Peyer's patches using 0.1% and 1% corn husk arabinoxylan through feed. Intestinal polyp formation significantly decreased, concomitant with an increase in Peyer's patches development (n = 12/group). In Aly (-/-) Apc (Min/+) mice (negative control; no Peyer's patches) there was no change in the amount of intestinal polyps (n = 10/group). Immune reaction following corn husk arabinoxylan treatment was measured by cytokine array. Increasing the number of Peyer's patches decreased interleukin-17 production, which showed a dose dependent correlation with transcription factor/lymphoid enhancer-binding factor. This study identified a relationship between levels of Peyer's patches and intestinal polyp formation, partly explained by the involvement of interleukin-17 production and ß-catenin signaling in Apc (Min/+) mice.

The expression of trefoil factor family member 2 in increased at an acidic pH.

Trefoil factor family member 2 (Tff2) is significantly involved in intestinal tumor growth in ApcMin/+ mice, which can be used as a human colon cancer model. TFF2, which encodes TFF2 (spasmolytic protein 1) is highly expressed in human cancer tissues, including the pancreas, colon and bile ducts, as well as in normal gastric and duodenum tissues. By contrast, TFF2 exhibits low expression levels in other normal tissues, including the small and large intestine. Furthermore, TFF2 expression has not been detected in DLD-1 cells, a cell line derived from human colon cancer. What induces TFF2 expression in normal and tumor cells is still unknown. Highly malignant tumor tissues are characterized by higher temperatures and lower pH (6.2-6.9) than in normal tissues, where normal pH ranges from 7.2 to 7.4. This microenvironment exacerbates malignancy by promoting the acquisition of cell death resistance, drug resistance and immune escape. Therefore, the present study examined how TFF2 expression is affected in cultured cells that imitate the tumor tissue microenvironment. The incubation temperature was increased from 37 to 40°C, but no expression of TFF2 was induced. Subsequently, a culture solution with an acidic pH was prepared to simulate the Warburg effect in tumors. TFF2 expression was increased by 42.8- and 5.8-fold in cells cultured in acidic medium at pH 6.5 and 6.8 compared with at pH 7.4, respectively, as determined using the relative quantification method following quantitative polymerase chain reaction. The present study also analyzed fluctuations in the expression levels of genes other than TFF2, under acidic conditions. Acidic conditions upregulated the expression of genes related to cell membranes and glycoproteins, based on the Database for Annotation, Visualization, and Integrated Discovery. In conclusion, TFF2 was highly expressed under acidic conditions, implying that it may have an important function in protecting the plasma membrane from acidic environments in both normal and cancer cells. These findings warrant further investigation of TFF2 as a target of cancer therapy and diagnosis.

OAZ-t/OAZ3 is essential for rigid connection of sperm tails to heads in mouse.

Polyamines are known to play important roles in the proliferation and differentiation of many types of cells. Although considerable amounts of polyamines are synthesized and stored in the testes, their roles remain unknown. Ornithine decarboxylase antizymes (OAZs) control the intracellular concentration of polyamines in a feedback manner. OAZ1 and OAZ2 are expressed ubiquitously, whereas OAZ-t/OAZ3 is expressed specifically in germline cells during spermiogenesis. OAZ-t reportedly binds to ornithine decarboxylase (ODC) and inactivates ODC activity. In a prior study, polyamines were capable of inducing a frameshift at the frameshift sequence of OAZ-t mRNA, resulting in the translation of OAZ-t. To investigate the physiological role of OAZ-t, we generated OAZ-t-disrupted mutant mice. Homozygous OAZ-t mutant males were infertile, although the polyamine concentrations of epididymides and testes were normal in these mice, and females were fertile. Sperm were successfully recovered from the epididymides of the mutant mice, but the heads and tails of the sperm cells were easily separated in culture medium during incubation. Results indicated that OAZ-t is essential for the formation of a rigid junction between the head and tail during spermatogenesis. The detached tails and heads were alive, and most of the headless tails showed straight forward movement. Although the tailless sperm failed to acrosome-react, the heads were capable of fertilizing eggs via intracytoplasmic sperm injection. OAZ-t likely plays a key role in haploid germ cell differentiation via the local concentration of polyamines.

[Role of ABC Transporters in Cancer Development and Malignant Alteration].

ATP-binding cassette (ABC) transporters, which comprise the largest gene-family in humans, are membrane proteins that transport various substrates, depending on ATP hydrolysis. Among these transporters, several include ABCB1 (P-glycoprotein), identified here for the first time in humans, which exports anti-cancer drugs from cancer cells, thus participating in multidrug resistance (MDR). ABC transporters also export drugs, in general, from the human body, therefore affecting overall pharmacokinetics. We have contributed, here, to a better understanding of the role of these exporter proteins in two aspects. First, we have cloned the human ABCC2 gene and identified mutations in hereditary hyperbilirubinemia patients, demonstrating the role of ABCC2 as a xenobiotic export pump. Second, we also found an unexpected role of ABCB1 in cancer, in that it promotes tumor initiation independently of the MDR phenomenon, which was further confirmed by a chemoprevention experiment using verapamil, an ABCB1 inhibitor. In this review, I discuss the role of ABC transporters, both in biodefense against xenobiotics and in cancer development and malignant alterations, based on our results as well as the studies of others.

Inhibitory Effect of the HASPIN Inhibitor CHR-6494 on BxPC-3-Luc, A Luciferase-Expressing Pancreatic Cancer Cell Line.

HASPIN acts in chromosome segregation via histone phosphorylation. Recently, HASPIN inhibitors have been shown to suppress growth of various cancer cells. Pancreatic cancer has no symptom in the early stages and may progress before detection. So, the 5-year survival rate is low. Here, we reported that administration of the HASPIN inhibitor, CHR-6494, to mice bearing pancreatic BxPC-3-Luc cancer cells significantly suppressed growth of BxPC-3-Luc cells. CHR-6494 might be a useful agent for treating pancreatic cancer.

Evaluation of the antiproliferative effects of the HASPIN inhibitor CHR-6494 in breast cancer cell lines.

HASPIN is a serine/threonine kinase that regulates mitosis by phosphorylating histone H3 at threonine 3. The expression levels of HASPIN in various cancers are associated with tumor malignancy and poor survival, suggesting that HASPIN inhibition may suppress cancer growth. As HASPIN mRNA levels are elevated in human breast cancer tissues compared with adjacent normal tissues, we examined the growth-suppressive effects of CHR-6494, a potent HASPIN inhibitor, in breast cancer cell lines in vitro and in vivo. We found that HASPIN was expressed in breast cancer cells of all molecular subtypes, as well as in immortalized mammary epithelial cells. HASPIN expression levels appeared to be correlated with the cell growth rate but not the molecular subtype of breast cancer. CHR-6494 exhibited potent antiproliferative effects against breast cancer cell lines and immortalized mammary epithelial cells in vitro, but failed to inhibit the growth of MDA-MB-231 xenografted tumors under conditions that have significant effects in a colorectal cancer model. These results imply that CHR-6494 does have antiproliferative effects in some situations, and further drug screening efforts are anticipated to identify more potent and selective HASPIN inhibition for use as an anticancer agent in breast cancer patients.

Gene knockout and metabolome analysis of carnitine/organic cation transporter OCTN1.

PURPOSE: Solute carrier OCTN1 (SLC22A4) is an orphan transporter, the physiologically important substrate of which is still unidentified. The aim of the present study was to examine physiological roles of OCTN1. METHODS: We first constructed octn1 gene knockout (octn1 ( -/- )) mice. Metabolome analysis was then performed to identify substrates in vivo. The possible association of the substrate identified with diseased conditions was further examined. RESULTS: The metabolome analysis of blood and several organs indicated complete deficiency of a naturally occurring potent antioxidant ergothioneine in octn1 ( -/- ) mice among 112 metabolites examined. Pharmacokinetic analyses after oral administration revealed the highest distribution to small intestines and extensive renal reabsorption of [(3)H]ergothioneine, both of which were much reduced in octn1 ( -/- ) mice. The octn1 ( -/- ) mice exhibited greater susceptibility to intestinal inflammation under the ischemia and reperfusion model. The blood ergothioneine concentration was also much reduced in Japanese patients with Crohn's disease, compared with healthy volunteers and patients with another inflammatory bowel disease, ulcerative colitis. CONCLUSIONS: These results indicate that OCTN1 plays a pivotal role for maintenance of systemic and intestinal exposure of ergothioneine, which could be important for protective effects against intestinal tissue injuries, providing a possible diagnostic tool to distinguish the inflammatory bowel diseases.

HASPIN kinase inhibitor CHR-6494 suppresses intestinal polyp development, cachexia, and hypogonadism in Apcmin/+ mice.

HASPIN has been identified as a nuclear Ser/Thr kinase specifically expressed in haploid germ cells. HASPIN kinase inhibitors were recently isolated, and their antitumor activity reported. Colorectal cancer occurs with high incidence worldwide. In this study, we examined whether HASPIN inhibitor CHR-6494 suppresses cancer progression in Apc mice, a familial colon tumor disease model. Mice were treated by intraperitoneal injection of CHR-6494 for 50 days. Following the treatment period, intestinal polyps were counted and testosterone and spermatogenesis levels were observed. Intraperitoneal administration of CHR-6494 significantly inhibited intestinal polyp development and recovered body weight in Apc mice. Although spermatogenesis was inhibited with increasing age in Apc mice, CHR-6494 significantly improved blood testosterone levels and spermatogenesis. Our results suggest that HASPIN inhibitors may be useful as anti-cancer agents and for the treatment of hypogonadism in colorectal cancer patients.

Unique alternative translation from two open reading frames on Acpin1 mRNA yields an acrosomal protein and a salivary-gland-specific protein.

OBJECTIVE: To examine the expression profiles of the proteins translated from Acpin1 mRNA in germ cells. METHODS: Northern and western blotting of various tissues and immunohistochemical analysis of germ cells were carried out in a mouse model. RESULTS: ACPIN1 protein was transcribed from the longer, 3' open reading frame (ORF) of Acpin1. An alternative-splicing variant, Acpin1vs, contained only the smaller, 5' ORF of the full-length Acpin1 gene. Its gene product, SAGSIN1, was expressed specifically in salivary glands. Retrotransposed regions of Acpin1 homology were also detected in various chromosomes, and intronless paralogous genes on the X chromosome were expressed in the testis and other tissues. The genomic structure of Acpin1 is highly conserved in mammals. CONCLUSION: The two ORFs on the Acpin1 mRNA are independently translated in differentiated cells. Analysis of gene Acpin1 might clarify the molecular mechanism of spermatogenesis.

Genetic Polymorphisms within The Intronless ACTL7A and ACTL7B Genes Encoding Spermatogenesis-Specific Actin-Like Proteins in Japanese Males.

Actins play essential roles in cellular morphogenesis. In mice, the T-actin1 and 2 genes, which encode actin-like proteins, are specifically expressed in haploid germ cells. Both T-ACTIN1/ACTLB and T-ACTIN2/ACTL7A have also been cloned and studied. The orthologous genes in humans are present on chromosome 9q31.3 as intronless genes. Defects of germ cell-specific genes can introduce infertility without somatic function impairment. We determined T-ACTIN1 and 2, specifically expressed in the testis using reverse-transcription polymerase chain reaction (RT-PCR). To examine whether genetic polymorphisms of the T-ACTIN1 and 2 genes are associated with male infertility, we screened for T-ACTIN1 and 2 polymorphisms by direct sequencing of DNA from 282 sterile and 89 fertile Japanese men. We identified five and six single nucleotide polymorphisms (SNPs) in the T-ACTIN1 and 2 regions of the sterile and fertile subjects respectively. Among these genetic polymorphisms was a novel SNP that was not in the National Center for Biotechnology Information SNP database. Although we could not determine whether these SNPs cause infertility, the prevalence of these genetic polymorphisms may be useful for analyzing polymorphisms in future largescale genetic analyses.

Molecular cloning and functional characterization of cynomolgus monkey multidrug resistance-associated protein 2 (MRP2).

The monkey is an important experimental model in the pharmacological evaluation of new drugs. We isolated monkey multidrug resistance-associated protein 2 (MRP2) cDNA to examine expression profiles among various tissues and measured ATPase activity to assess substrate specificity. The amino acid sequence encoded by monkey MRP2 cDNA was very similar (96% identity) to the reported human MRP2 cDNA (GenBank accession no. NM_000392). The tissue distribution of MRP2 in monkeys was partially different from that in humans. We found relatively high expression of MRP2 in the monkey kidney and small intestine using Northern blotting. Substrate specificity was compared between human and monkey MRP2. The affinity of 17beta-estradiol 17-(beta-d-glucuronide), methotrexate, vinblastine, and probenecid to monkey MRP2 was higher than that to human MRP2. Functional and expression differences between human and monkey MRP2 should be incorporated into the evaluation of candidate drugs.

Regulatory polymorphisms of multidrug resistance 1 (MDR1) gene are associated with the development of childhood acute lymphoblastic leukemia.

The aim of this study is to determine whether the polymorphisms of the MDR1 gene are associated with the development of childhood acute lymphoblastic leukemia (ALL). The MDR1 gene polymorphisms, -2352 G>A, -934A>G, -692T>C (5' regulatory region) and 3435C>T (exon 26), were examined in 157 ALL patients and 96 healthy children. The amounts of MDR1 mRNA were quantified in 54 healthy individuals using normal peripheral blood mononuclear cells to evaluate the effect of each polymorphism on the gene expression. The frequency of the G/G genotype of the -2352 G>A was significantly higher in ALL than in controls (74/109 versus 52/96, p=0.04). The frequency of the T/T genotype of the 3435C>T was also significantly higher in ALL (29/118 versus 10/96, p=0.006). In a haplotype analysis using the 5' regulatory sites, the frequency of a certain haplotype was higher in ALL than in controls (59/90 versus 42/88, p=0.048). When the -2352G>A was examined in different age groups, patients aged six or older were found to have the G/G genotype more frequently than the controls (42/51 versus 52/96, p=0.0014), while no difference was observed in the younger age group. The amounts of MDR1 mRNA were significantly higher in either G/G or G/A genotype of the -2352 G>A than in A/A genotype (p=0.04). The present study suggests that the genetic background of MDR1 may be associated with the development of childhood ALL, possibly due to a quantitative change in the MDR1 gene resulting from genetic polymorphisms.

Single nucleotide polymorphisms in ABCC2 and ABCB1 genes and their clinical impact in physiology and drug response.

Among the ABC proteins, some members including ABCB1, ABCC1, ABCC2 and ABCG2 are believed to contribute to multidrug resistance of cancer chemotherapy. In addition, the broad substrate-specificity and apical localization of the ABCB1 and ABCC2 in mucosal epithelium of intestine and hepatocyte give them a protective role against xenobiotics. The inter-individual variations in activity and expression levels of ABCB1 and ABCC2, thus, might affect on drug response and response to toxic substrates. In this review, I focus on (1) physiological and toxicological relevance of ABCB1 and ABCC2, and on (2) genetic variations of ABCB1 and ABCC2 genes and their association with biochemical function, expression level and tumor incidence.

The association of death-associated protein kinase hypermethylation with early recurrence in superficial bladder cancers.

Mechanisms for bladder carcinogenesis and the development of recurrentbladder cancer remain unclear. Aberrant methylation of the 5' CpG island is thought to play an important role in the inactivation of the tumor suppressor genes in cancer. To study whether specific or bulk hypermethylation predicts intrabladder recurrence, we determined the frequency of aberrant promoter hypermethylation of seven genes, hMLH1, O(6)-methylguanine-DNA-methyltransferase (MGMT), p16, Von Hippel-Lindau (VHL), death-associated protein kinase (DAP-kinase), glutathione S-transferase P1 (GST-P1) and E-cadherin in 55 superficial bladder cancers and 5 normal urothelial epithelia by methylation-specific PCR (MSP). These patients of superficial bladder cancer had been followed prospectively by cystoscopy. Simultaneous hypermethylation of three genes or more among the seven genes was observed in 2 (7%) of 30 patients in the nonrecurrence group and 7 (28%) of 25 patients in the recurrence group. There was a significant concordance between the number of methylated genes and the development of recurrence (P = 0.012). In particular, the recurrence rate for 24 months was 88% for hypermethylation of DAP-kinase and 28% for nonmethylation of DAP-kinase. Hypermethylation of DAP-kinase is, therefore, a strong indicator of the superficial bladder cancer associated with a high recurrence rate (P < 0.001; hazards ratio, 7.01). Our results suggest that hypermethylation of DAP-kinase might be a useful prognostic marker for disease recurrence in superficial bladder cancers.

Cyclosporin-A enhances docetaxel-induced apoptosis through inhibition of nuclear factor-kappaB activation in human gastric carcinoma cells.

PURPOSE: We sought to determine whether cyclosporin-A (CsA) enhances docetaxel [Taxotere (TXT)]- induced apoptosis in human gastric carcinoma cells, and, if so, to determine the relation between this apoptosis and nuclear factor-kappaB (NF-kappaB) activation. EXPERIMENTAL DESIGN: Two human gastric carcinoma cell lines (GCTM-1 and MK-1), a human embryonic pulmonary fibroblast cell line, and human umbilical vein endothelial cells were used as drug targets. Apoptotic cell death was verified morphologically by nuclear fragmentation assay with Hoechst staining. Electrophoretic mobility shift assays were performed to check for nuclear translocation of NF-kappaB. The therapeutic effects of a combination of TXT and CsA were assessed in a mouse peritoneal dissemination model. RESULTS: A combination of CsA (5 micro M) and TXT (10 nM) significantly enhanced apoptotic cell death in both carcinoma cell lines but not in nonmalignant cell lines in comparison with the single-agent treatment alone. This effect was not related to drug uptake, efflux, or MDR1 expression. These effects were also observed in freshly obtained TXT-resistant gastric carcinoma cells isolated from a patient with malignant ascites. TXT alone induced NF-kappaB activation in both carcinoma cell types, and this activation was suppressed by CsA. A combination of TXT and NF-kappaB decoy, a well-known NF-kappaB inhibitor, also enhanced apoptotic cell death in the carcinoma cells. A combination of CsA and TXT significantly suppressed peritoneal dissemination in vivo relative to the single-agent effect. CONCLUSIONS: Treatment with CsA and TXT in combination may be an effective therapeutic strategy for patients with gastric carcinoma.

Genetic polymorphism at the 5' regulatory region of multidrug resistance 1 (MDR1) and its association with interindividual variation of expression level in the colon.

The multidrug resistance 1 (MDR1) is a key molecule in determining not only the resistance of cancer cells to anticancer agents but also the disposition of a variety of drugs in intestinal and other tissues. However, the mechanism underlying interindividual variations in levels of MDR1 activity and expression in various tissues remains unclear. We analyzed the nucleotide sequence polymorphisms in the 5' upstream regulatory region of the gene spanning 4 kb from the transcriptional start site of MDR1 and tried to identify any associations between polymorphisms and MDR1 expression. Within that region, we identified eight single nucleotide polymorphisms (SNPs) in the region in the Japanese population. Of the SNPs identified, -2410T>C, -1910T>C, and 692T>C were in perfect linkage disequilibrium. In normal colorectal mucosa, diplotypes at the region showed more significant association with the expression level of MDR1 mRNA than each SNP did. In an in vitro reporter assay, transcription activity of the minor-type construct carrying haplotypes 2 and 3 was significantly lower than that of the major-type construct carrying haplotype 1. We next identified two DNA binding proteins: one protein bound to the nucleotide sequence carrying -692T but not to that carrying -692C and another bound to the nucleotide sequence carrying -2352G but three times weaker than that carrying -2352A. This suggested the significance of SNP at -692 and -2352 of MDR1 in variable expression in the colon interindividually. This is the first report connecting SNPs and interindividual variety of MDR1 expression rationally.