Visual assessment of Ki67 using a 5-grade scale (Eye-5) is easy and practical to classify breast cancer subtypes with high reproducibility.

AIMS: Personalised breast cancer therapy requires pathological characterisation of tumours. The proliferative index, based on Ki67, is pivotal, but a standard method has not been established. Here we look for an easy and practical way to evaluate Ki67. METHODS: Immunohistochemical staining of estrogen receptors, progesterone receptors, HER2 and Ki67 (MIB-1) was performed on resected specimens from 406 primary invasive ductal carcinomas. Ki67 labelling index (LI) from manual counting was compared with visual assessment using a 5-grade scale (Eye-5). Next, 10 pathologists evaluated 100 samples with marked hot spots by using Eye-5. Another 100 samples without marking were also assessed by eight pathologists. One year later, two pathologists reviewed 222 cases with Eye-5. Prognosis was analysed among estrogen receptor-positive cases with postoperative endocrine therapy. RESULTS: Eye-5 showed good correlation to LI. All 136 cases of score 4-5 had LI >20% and all 56 cases of score 1 had LI<20%, which means that manual counting was not necessary for about half of the cases. Interobserver and intraobserver variability was low even when a hot spot was not fixed. Eye-5 also correlated with histological grade and lymph node metastasis. Combining Eye-5 and histological grade created a new algorism to predict LI, which allows 80% of all cases (74% of luminal cases) without manual counting. Cases of Eye-5 score 1-2 had significantly better survival than score 3-5. CONCLUSIONS: Visual assessment of Ki67 by a 5-grade scale (Eye-5) is fast, easy, and reliable with acceptably low interobserver and intraobserver variability. Eye-5 can replace LI in many luminal tumours, and is a strong candidate as a standard method of evaluating Ki67.

Toxicokinetics of dioxins and other organochlorine compounds in Japanese people: association with hepatic CYP1A2 expression levels.

Concentrations of persistent organochlorine compounds (OCs) including polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and polychlorinated biphenyls (PCBs) in the liver and adipose tissue of Japanese cadavers were measured, and their toxicokinetics were examined in association with hepatic cytochrome P450 (CYP) 1A protein expression levels. Total 2,3,7,8-tetrachlorodibenzo-p-dioxin toxic equivalents (TEQs) were 66±74 and 65±57 pg/g lipid weight (mean±S.D.) in the liver and adipose tissue, respectively. Total PCBs (sum of 62 congeners targeted), p,p'-dichlorodiphenyl-dichloroethylene (p,p'-DDE) and ß-hexachlorocyclohexane (ß-HCH) were detected at concentrations over 1 µg/g lipid in both tissues of some specimens. For most of the dioxin-like congeners, total PCBs, p,p'-DDE, oxychlordane, α- and ß-HCH, hexachlorobenzene (HCB), and tris(4-chlorophenyl)methane (TCPMe), age-dependent increases in concentrations were found in the adipose tissue of males. No such age-dependent trend was observed in the liver, suggesting that there are different mechanisms underlying the hepatic concentrations of OCs. Immunoblot analyses indicated detectable expression of hepatic CYP1A2 protein, whereas no CYP1A1 protein was detected. The CYP1A2 expression levels were positively correlated with concentrations (on wet weight basis) of 2,3,4,7,8-P5CDF, the dominant TEQ-contributed congeners in the liver, indicating the induction of this CYP. Hepatic CYP1A2 protein levels were strongly correlated with the liver to adipose concentration (L/A) ratios of PCDD/F congeners with more than 5 chlorine atoms. Together with higher concentrations of the congeners in the liver than in the adipose tissue, the observation on L/A ratios of highly chlorinated PCDD/Fs suggests that induced hepatic CYP1A2 protein is involved in their sequestration in this human population, as observed in model animals (rodents). Nonetheless, the magnitude of hepatic sequestration (L/A ratio) of PCDD/Fs in this human population was lower than in other mammals and birds, reported previously. This study emphasizes the fact that toxicokinetics of some OCs can be affected at least partly by CYP1A2 protein levels in humans. For the extrapolation of their toxicokinetics from model animals to humans, knowledge on the induction and sequestration potencies of CYP1A is necessary.

Neither MafA/L-Maf nor MafB is essential for lens development in mice.

The importance of the large Maf transcription factor family has been investigated in lens development in the chick, Xenopus and mammals. Previously we reported that c-maf-deficient mice exhibit severe defects in lens fibre cells. Here, we report the roles of other large Mafs, MafA/L-Maf and MafB, during mouse lens development. MafA/L-Maf and MafB were expressed in lens epithelial cells and fibre cells at E12.5 but had largely disappeared from the lens at E18.5. The lens of mafA-, mafB-deficient and mafA::mafB double-deficient mice developed normally. In c-maf-deficient mice, the pattern of expression of MafA and MafB differed from their expression in wild-type mice. Moreover, the expression of crystallin genes was unchanged in mafA-, mafB- and mafA::mafB double-deficient lens. These results indicate that c-Maf alone is essential for lens development, and that MafA/L-Maf and MafB are dispensable in mice.

Typing of the nt343C>T (R95X) allele of the C9 gene by PCR-SSP and the allele frequency of R95X in five ethnic populations.

One of the alleles which leads to ninth component of complement deficiency (C9D) is R95X (nt343C>T), which is present in most cases of C9D in Japan. In this study, we carried out nt343C>T typing by the method of polymerase chain reaction with sequence-specific primers (PCR-SSP), and showed the frequency of the R95X allele in German, Italian, Thai, Korean and Chinese populations. We did not find the R95X allele in the German or Italian populations. The allele frequency of R95X in the three Asian populations is as follows: Thais 0.019, Koreans 0.008, and Chinese 0.002. As the allele frequency in the Japanese population is 0.036, the results provide supporting evidence that the R95X is an allele characteristic of Japanese.

Haplotypes in the 5'-upstream region, exons 3 and 4, and introns 2 and 3 of the ABO blood group gene.

We previously described two haplotypes named the ABORR*L-associated and ABORR*S-associated haplotypes in the 5'-upstream region of the ABO blood group gene. Here we studied polymorphisms in exons (Exs) 3 and 4 and introns (Ints) 2 and 3 of the ABO gene, and analyzed the haplotypes in those Exs, Ints, and the 5'-upstream region. Two haplotypes (at Int2nt108-Int2nt362-Int2nt369-Int2nt539-Ex3nt106-Int3nt1178-Int3nt1357-Ex4nt188-Ex4nt189) were deduced to be (1) A-C-C-C-T-C-T-A-T, which was linked with ABORR*L and ABO*O(A), and (2) A-C-C-C-G-T-C-G-C, G-C-C-C-G-T-C-G-C, and A-T-G-A-G-T-C-G-C, which were linked with ABORR*S and the other common ABO alleles. This finding also shows the existence of two major lineages of the Japanese ABO alleles.

Rare alleles of the ABO blood group system in two European populations.

We performed genotyping of the ABO system in Italian and Israeli population samples. The nucleotides at 11 positions, nts 261, 297, 467, 526, 646, 681, 703, 796, 802, 803 and 1060, were analyzed by PCR-RFLP, PCR-SSP and PCR direct sequencing methods. We found three rare ABO alleles besides the common alleles (*)A1(Pro) (=(*)A101), (*)A2(Leu) (=(*)A201), (*)B (=(*)B101), (*)O(T) (=(*)O01), (*)O(A) (=(*)O02) and (*)O2 (=(*)O03), but did not detect ( *)A1(Leu) (=( *)A102) which is a common allele in Asians. The rare alleles were tentatively named (*)Ov1, (*)Ov2, and (*)Bv. As ( *)Bv has been found in two Japanese individuals and (*)O2 is not a rare ABO allele in Europeans, not only (*)O2 but also the (*)Ov1 and (*)Ov2 alleles may be characteristic of European populations.

Identification of anti-α-enolase autoantibody as a novel serum marker for endometriosis.

Diagnosis of endometriosis needs invasive maneuvers. New serum marker that possesses both high sensitivity and high specificity has long been desired. To establish novel serum marker for endometriosis, serum autoantibodies (autoAbs) were investigated using proteomic approach. AutoAbs in sera of endometriotic patients and healthy controls were analyzed using a mesothelial cell line, 2-DE and Western blotting. Proteins in reacted spots were identified using MALDI TOF-MS with MASCOT analysis. ELISAs were established using recombinant proteins and autoAb-titers were estimated in sera of endometriotic patients, disease and healthy controls. Several autoAbs were identified. Anti-α-enolase (Eno1)-autoAb levels in endometriotic patients were significantly elevated compared with both healthy and disease controls. Sensitivity and specificity of serum anti-Eno1-autoAb was nearly comparable to serum CA125. When anti-Eno1-autoAb and CA125 assays were combined, diagnostic sensitivity and accuracy improved. Serum anti-Eno1-autoAb can be a new serum endometriotic marker and it is useful as a supplement assay for CA125. This study validates further clinical evaluation of this novel marker.

Myasthenia gravis experimentally induced with muscle-specific kinase.

Here we present the first evidence that muscle-specific kinase (MuSK) antigen can cause myasthenia in animals. MuSK is expressed at the postsynaptic membranes of neuromuscular junctions (NMJ) and forms complexes with acetylcholine receptors (AChR) and rapsyn. MuSK is activated by agrin, which is released from motoneurons, and induces AChR clustering and subsequent formation of NMJ in embryos. Notably, autoantibodies against MuSK were found in a proportion of patients with generalized myasthenia gravis (MG) but without the characteristic AChR autoantibodies. However, MuSK autoantibodies had no known pathogenic potential, and animals immunized with purified MuSK proteins did not develop MG in former studies. In contrast, we have now injected rabbits with MuSK ectodomain protein in vivo and evoked a MG-like muscle weakness with a reduction of AChR clustering at the NMJ. Our results showed that MuSK is required for maintenance of synapses and that interference with that function by MuSK antibodies causes myasthenic weakness. In vitro, AChR clustering in myotubes is induced by agrin and agrin-independent inducers, which do not activate MuSK. Neither the receptor nor the activation mechanisms of AChR clustering induced by agrin-independent inducers has been identified with certainty, but MuSK autoantibodies in myasthenic animals inhibited both agrin and agrin-independent AChR clustering. MuSK plays multiple roles in pre-patterning of the postsynaptic membrane before innervation and formation of NMJ in embryos. Some of these mechanisms may also participate in the maintenance of mature NMJ. This model system would provide new knowledge about the molecular pathogenesis of MG and MuSK functions in mature NMJ.

Serum autoantibody to sideroflexin 3 as a novel tumor marker for oral squamous cell carcinoma.

The purpose of this study is to establish a tumor marker that can be applied for the early detection and follow-up of oral cancer patients. Employing the proteomic approach using MALDI TOF-MS, 2-DE, patient's sera and culturing cell lines, the serum autoantibodies (autoAbs) were screened and the serum levels were estimated by ELISA. Targeting the tumor cell invasion into the surrounding stromal tissues, MRC-5 human fibroblasts were employed as the target cells and a mitochondrial membrane protein, sideroflexin 3 (SFXN3), was identified. The serum anti-SFXN3-autoAb levels elevated in patients with the oral squamous cell carcinoma significantly: with 77% sensitivity and 89% specificity against control samples. The serum anti-SFXN3-autoAb levels were mildly correlated with the primary tumor sizes, however, the levels were slightly highly elevated in T1 early cancer. An immunohistochemical analysis revealed that the SFXN3 protein is expressed in the stromal fibroblasts between the caner nests and also in the basal layer of the squamous epithelium. Changes in the serum anti-SFXN3-autoAb levels after therapy correlated with the clinical tumor burden. These findings demonstrated that the serum anti-SFXN3-autoAb is worthy of clinical evaluation as a potentially of the novel tumor maker for the early detection of oral squamous cell carcinoma.

Polybrominated diphenyl ethers and persistent organochlorines in Japanese human adipose tissues.

The present study determined concentrations of polybrominated diphenyl ethers (PBDEs) and persistent organochlorines (OCs) in Japanese human adipose tissues collected during 2003-2004. Concentrations of PBDEs in adipose tissues were 1-2 orders of magnitude lower than those of OCs. However, observed PBDE congener levels in this study were relatively higher than those in Japanese human adipose tissues collected during 2000 reported previously, while OC levels were comparable to those in specimens collected during 1999 reported by our group. In addition, no age-dependent accumulation of PBDEs was observed, while OC levels except chlordane compounds increased with age. These results indicate recent human exposure to PBDEs in Japan. Among PBDE congeners accumulated in Japanese adipose tissues, BDE-153 was dominant, but this trend was different from those in human milk (BDE-47) and blood (BDE-209) reported previously in Japan, implying the congener-specific kinetics in human bodies. The significant positive correlations between PBDEs and OCs were observed in Japanese adipose tissues, indicating the similar exposure route of these contaminants for Japanese citizens, probably via fish intake.

Differences in lectin-binding properties between the common mucosal epithelium and follicle-associated epithelium in the rabbit small intestine.

Differences in sugar distribution between the villous epithelium and follicle-associated epithelium (FAE) were compared using lectins in the rabbit small intestine. In every portion, villous columnar epithelial cells primarily exhibited a positive reaction to the GalNAc, GlcNAc, galactose, and oligosaccharide. In the ileal Peyer's patch (PP), whereas microvillous epithelial cells exhibited positive reactions, M cells tended to be negative. The villous epithelial reaction to the fucose group was negative, but M cells and microvillous epithelial cells showed a positive to the fucose. No epithelium had a positive reaction to the mannose and glucose. The variety of lectin-binding properties of villous epithelial cells and M cells may reflect specificity for the recognizing luminal substances such as antigenic molecules and bacterial elements.

A mitotic kinase TOPK enhances Cdk1/cyclin B1-dependent phosphorylation of PRC1 and promotes cytokinesis.

A MAPKK-like mitotic kinase, TOPK, implies the formation of mitotic spindles and spindle midzone and accomplishing cytokinesis, however, its underlying mechanism remains unclear. A microtubule bundling protein, PRC1, plays a pivotal role in the formation of mitotic spindles and spindle midzone. Because of their functional resemblance, we attempted to clarify the links between these two molecules. TOPK supported mitotic advance via the cdk1/cyclin B1-dependent phosphorylation of PRC1. TOPK induced the phosphorylation of PRC1 at T481 in vivo, however, TOPK did not phosphorylate PRC1 in vitro. TOPK induced the phosphorylation of PRC1 at T481 only when the cdk1/cyclin B1 existed simultaneously in vitro. Both the enzymatic activity of TOPK and association competence of TOPK with PRC1 were mandatory for this phosphorylation. TOPK binds to cdk1/cyclin B1, microtubules and PRC1 via its unique region near the C terminus. TOPK co-localized closely with cdk1 throughout the cell cycle in vivo. Collectively, these data indicate that TOPK, which makes a kinase-substrate complex with cdk1/cyclin B1 and PRC1 on microtubules during mitosis, enhances the cdk1/cyclin B1-dependent phosphorylation of PRC1 and thereby strongly promotes cytokinesis.

Human scribble accumulates in colorectal neoplasia in association with an altered distribution of beta-catenin.

The loss of epithelial polarity and tissue architecture is a diagnostic feature of malignant tumors. In Drosophila, genetic studies identified 3 neoplastic tumor suppressor genes (nTSGs), and a loss of nTSGs has been shown to result in a disruption of apical-basal polarity and neoplastic growth in epithelial cells. Scribble is one type of the Drosophila nTSGs, which encodes a membrane-associated cytoplasmic protein containing the multi-PDZ domain. In contrast to Drosophila scribble, the oncogenic roles of its mammalian homologues have not yet been established. We herein immunohistochemically examined the distributions of hScrib protein in human colorectal neoplasia using affinity-purified antibody. In 50 cases of colorectal adenomas and adenocarcinomas, the accumulation of hScrib protein was commonly observed in comparison with the adjacent normal epithelia. Furthermore, the overexpression and distribution of hScrib was observed to extensively overlap with the cytoplasmic accumulation of beta-catenin. Like beta-catenin, the intense immunoreactivity of hScrib was often observed in small adenomas, thus, suggesting that hScrib could be involved in an early step of colon carcinogenesis. Five corresponding liver metastases showed a comparable immunoreactivity for anti-hScrib in comparison with their primary sites. In an immunofluorescence analysis on cultured cell lines, the loss of membranous staining of hScrib was observed according to the cytoplasmic translocation of beta-catenin. We herein demonstrate that the accumulation of hScrib protein might therefore be involved in colon carcinogenesis while also providing a possible link between hScrib and beta-catenin.

A Small Ras-like protein Ray/Rab1c modulates the p53-regulating activity of PRPK.

PRPK phosphorylates serine-15 residue of p53 and enhances transcriptional activity. PRPK possesses a bipartite nuclear localization signal and localizes in nucleus when over-expressed in cells. However, intrinsic PRPK localizes mainly in the cytosol in situ. While studying the mechanisms in the distribution of intrinsic PRPK, we identified a PRPK binding protein, an ubiquitously expressed Small Ras-like GTPase, Rab1c, also named Ray or Rab35. The over-expressed Ray was distributed in the nucleus, cytosol, and cell membrane. Both Ray wild type and GTP-restrictively binding mutant Ray-Q67L, but not guanine nucleotide unstable binding mutant Ray-N120I, partially distributed the over-expressed PRPK to the cytosol and also suppressed the PRPK-induced p53-transcriptional activity profoundly. A Small Ras-like GTPase protein Ray was thus indicated to modulate p53 transcriptional activity of PRPK.

Induction of myasthenia by immunization against muscle-specific kinase.

Muscle-specific kinase (MuSK) is critical for the synaptic clustering of nicotinic acetylcholine receptors (AChRs) and plays multiple roles in the organization and maintenance of neuromuscular junctions (NMJs). MuSK is activated by agrin, which is released from motoneurons, and induces AChR clustering at the postsynaptic membrane. Although autoantibodies against the ectodomain of MuSK have been found in a proportion of patients with generalized myasthenia gravis (MG), it is unclear whether MuSK autoantibodies are the causative agent of generalized MG. In the present study, rabbits immunized with MuSK ectodomain protein manifested MG-like muscle weakness with a reduction of AChR clustering at the NMJs. The autoantibodies activated MuSK and blocked AChR clustering induced by agrin or by mediators that do not activate MuSK. Thus MuSK autoantibodies rigorously inhibit AChR clustering mediated by multiple pathways, an outcome that broadens our general comprehension of the pathogenesis of MG.

Expression and phosphorylation of TOPK during spermatogenesis.

Among normal organs and tissues, the MAPKK-like mitotic protein kinase TOPK is expressed exclusively in the testis. We analyzed the expression and phosphorylation of TOPK to address the functional role of this kinase during spermatogenesis. TOPK protein is expressed mainly in the cytosol of spermatocytes and spermatids, but not in spermatids and spermatogonia in situ. TOPK-Thr-9, a cdk1/cyclin B target residue, was specifically phosphorylated during mitotic and meiotic phases, while TOPK-Thr-198, a key amino acid for the ATP pocket, was constantly phosphorylated irrespective of the cell cycle. These data indicate that spermatogenic germ cells with vital proliferation activity express TOPK. As TOPK-Thr-9 was phosphorylated during both mitosis and meiosis, TOPK was indicted to play a role in cytokinesis and/or chromosomal segregation but not in DNA replication.

Thymic carcinoma originating from the mid-posterior mediastinum.

An unusual thymic carcinoma in a 74-year-old woman is described. Initial chest CT revealed a mass at the mid-posterior mediastinum. Transbronchial fine needle biopsy of the mass failed to provide a definite diagnosis. The mass was treated as a malignant mediastinal tumour, and chemoradiotherapy was performed as initial treatment. The patient died 5 years after receiving primary treatment. The results of postmortem microscopic examination, including immunohistochemical study with CD5 antibody, were consistent with thymic carcinoma. This case is interesting in that the mid-posterior mediastinum is the site where thymic carcinoma is least likely to originate.

The nude rat as an orthotopic model for cervical cancer.

OBJECTIVE: The purposes of this study were to establish intracervical tumors of the nude rat as an orthotopic experimental model for human cervical cancer and to preliminary evaluate the effects of the adenoviral vector, Ad5CMV-p53, on orthotopic cervical tumor size. METHODS: Human cervical cancer SiHa and ME-180 cells were injected into the cervix of the nude rat. Four days later, 1 x 10(9) plaque forming units (PFU) of Ad5CMV-p53 were injected into the cervix. The rats were later sacrificed to determine cervical tumor size. RESULTS: Eight of ten nude rats developed SiHa cell tumors; all ten nude rats developed ME-180 cell tumors. Four of ten SiHa cell tumors metastasized to the pelvic cavity; no ME-180 cell tumors did. The growth of Ad5CMV-p53-infected cells was greatly suppressed. The ad5CMV-p53 treatment significantly reduced both cell tumor volumes in nude rat cervixes. CONCLUSION: The nude rat cervix grows tumors similar to human cervical cancer tumors and makes an excellent experimental model. Transfection of cervical cancer cells with the wild-type p53 gene via Ad5CMV-p53 is a potential therapeutic approach to cervical cancer.

AIP1/WDR1 supports mitotic cell rounding.

The actin cytoskeleton plays a fundamental role in configuring cell shapes and movements. Actin interacting protein 1 (AIP1)/tryptophan-aspartate-repeat protein 1 (WDR1) induces actin severing and disassembly cooperating with ADF/cofilin. We found that mitotic cell flattening but not rounding was manifested by suppression of AIP1/WDR1 in cells. This mitotic cell flattening was not due to any changes in phosphorylation and distribution of cofilin in cells. We carried out a direct observation of actin filament severing/disassembly assay and found that phosphorylated cofilin still somewhat severs/disassembles actin filaments and that AIP1/WDR1 effaces this in vitro. We suggest that the phosphorylation of ADF/cofilin will be insufficient to completely inhibit actin turnover during mitosis, and that AIP1/WDR1 could abort the severing/disassembly activity somewhat still carried out due to phosphorylated ADF/cofilin. This mechanism could be required to induce cell morphologic changes, especially mitotic cell rounding.

Characterization of a MAPKK-like protein kinase TOPK.

A MAPKK-like protein kinase TOPK expresses in a wide range of proliferating cells and tissues such as cancer cells and testis. However, details of this kinase are still uncovered. We investigated the intracellular distribution of TOPK and its association with cdk1/cyclin B and microtubules. In interphase cells, TOPK expresses in cytosol and nucleus without any significant association with microtubule networks. During mitosis, TOPK-Thr-9 was phosphorylated by cdk1/cyclin B and TOPK significantly associates with mitotic spindles. When TOPK expression was suppressed, formation of spindle midzone was thinned and dimmed and cytokinesis was disturbed. We propose that TOPK plays a role in the formation of spindle midzone and in cytokinesis.