Cancer stem-like cells of ovarian clear cell carcinoma are enriched in the ALDH-high population associated with an accelerated scavenging system in reactive oxygen species.

OBJECTIVE: In ovarian cancer cases, recurrence after chemotherapy is frequently observed, suggesting the involvement of ovarian cancer stem-like cells (CSCs). The chemoresistance of ovarian clear cell carcinomas is particularly strong in comparison to other epithelial ovarian cancer subtypes. We investigated the relationship between a CSC marker, aldehyde dehydrogenase 1 (ALDH1), and clinical prognosis using ovarian clear cell carcinoma tissue samples. Furthermore, we investigated the antioxidant mechanism by which CSCs maintain a lower reactive oxygen species (ROS) level, which provides protection from chemotherapeutic agents. METHODS: Immunohistochemical staining was performed to examine the CSC markers (CD133, CD44, ALDH1) using ovarian clear cell carcinoma tissue samples (n=81). Clear cell carcinoma cell lines (KOC-7C, OVTOKO) are separated into the ALDH-high and ALDH-low populations by ALDEFLUOR assay and fluorescence-activated cell sorting (FACS). We compared the intracellular ROS level, mRNA level of the antioxidant enzymes and Nrf2 expression of the two populations. RESULTS: High ALDH1 expression levels are related to advanced stage in clear cell carcinoma cases. ALDH1 expression significantly reduced progression free survival. Other markers are not related to clinical stage and prognosis. ALDH-high cells contained a lower ROS level than ALDH-low cells. Antioxidant enzymes were upregulated in ALDH-high cells. ALDH-high cells showed increased expression of Nrf2, a key transcriptional factor of the antioxidant system. CONCLUSIONS: ALDH-positive CSCs might have increased Nrf2-induced antioxidant scavengers, which lower ROS level relevant to chemoresistance in ovarian clear cell carcinoma.

The recessive phenotype displayed by a dominant negative microphthalmia-associated transcription factor mutant is a result of impaired nucleation potential.

In the DNA binding domain of microphthalmia-associated transcription factor (MITF), four mutations are reported: mi, Mi wh, mi ew, and mi or. MITFs encoded by the mi, Mi wh, mi ew, and Mi or mutant alleles (mi-MITF, Mi wh-MITF, Mi ew-MITF, and Mi or-MITF, respectively) interfered with the DNA binding of wild-type MITF, TFE3, and another basic helix-loop-helix leucine zipper protein in vitro. Polyclonal antibody against MITF was produced and used for investigating the subcellular localization of mutant MITFs. Immunocytochemistry and immunoblotting revealed that more than 99% of wild-type MITF and Mi wh-MITF located in nuclei of transfected NIH 3T3 and 293T cells. In contrast, mi-MITF predominantly located in the cytoplasm of cells transfected with the corresponding plasmid. When the immunoglobulin G (IgG)-conjugated peptides representing a part of the DNA binding domain containing mi and Mi wh mutations were microinjected into the cytoplasm of NRK49F cells, wild-type peptide and Mi wh-type peptide-IgG conjugate localized in nuclei but mi-type peptide-IgG conjugate was detectable only in the cytoplasm. It was also demonstrated that the nuclear translocation potential of Mi or-MITF was normal but that Mi ew-MITF was impaired as well as mi-MITF. In cotransfection assay, a strong dominant negative effect of Mi wh-MITF against wild-type MITF-dependent transactivation system on tyrosinase promoter was observed, but mi-MITF had a small effect. However, by the conjugation of simian virus 40 large-T-antigen-derived nuclear localization signal to mi-MITF, the dominant negative effect was enhanced. Furthermore, we demonstrated that the interaction between wild-type MITF and mi-MITF occurred in the cytoplasm and that mi-MITF had an inhibitory effect on nuclear localization potential of wild-type MITF.

Role of DNA methylation for expression of novel stem cell marker CDCP1 in hematopoietic cells.

CDCP1, a novel stem cell marker, is expressed in hematopoietic cell line K562 but not in Jurkat. When CDCP1 promoter was transfected exogenously, Jurkat showed comparable promoter activity with K562, suggesting that the factor to enhance transcription was present but interfered to function in Jurkat. The reporter assay and si-RNA-mediated knockdown experiment revealed that zfp67, a zinc-finger protein, enhanced CDCP1 transcription. Amount of zfp67 in Jurkat was comparable with K562, but chromatin immunoprecipitation showed that zfp67 bound to CDCP1 promoter in K562 but not in Jurkat. There are CpG sequences around the promoter of CDCP1, which were heavily methylated in Jurkat but not in K562. Addition of demethylating reagent to Jurkat induced CDCP1 expression, and increased the zfp67 binding to CDCP1 promoter. Among normal hematopoietic cells such as CD34+CD38- cells, lymphocytes and granulocytes, inverse correlation between proportion of methylated CpG sequences and CDCP1 expression level was found. Demethylation of CpG sequences in lymphocytes, in which CpG sequences were heavily methylated, induced CDCP1 expression and its expression level further increased through zfp67 overexpression. The methylation of DNA appeared to regulate the cell-type-specific expression of CDCP1 through the control of interaction between chromatin DNA and transcription factors.

Deficiency in WT1-targeting microRNA-125a leads to myeloid malignancies and urogenital abnormalities.

The Wilms' tumor gene WT1 is overexpressed in leukemia and solid tumors and has an oncogenic role in leukemogenesis and tumorigenesis. However, precise regulatory mechanisms of WT1 overexpression remain undetermined. In the present study, microRNA-125a (miR-125a) was identified as a miRNA that suppressed WT1 expression via binding to the WT1-3'UTR. MiR-125a knockout mice overexpressed WT1, developed myeloproliferative disorder (MPD) characterized by expansion of myeloid cells in bone marrow (BM), spleen and peripheral blood, and displayed urogenital abnormalities. Silencing of WT1 expression in hematopoietic stem/progenitor cells of miR-125a knockout MPD mice by short-hairpin RNA inhibited myeloid colony formation in vitro. Furthermore, the incidence and severity of MPD were lower in miR-125a (-/-) mice than in miR-125a (+/-) mice, indicating the operation of compensatory mechanisms for the complete loss of miR-125a. To elucidate the compensatory mechanisms, miRNA array was performed. MiR-486 was occasionally induced in compete loss of miR-125a and inhibited WT1 expression instead of miR-125a, resulting in the cancellation of MPD occurrence. These results showed for the first time the post-transcriptional regulatory mechanisms of WT1 by both miR-125a and miR-486 and should contribute to the elucidation of mechanisms of normal hematopoiesis and kidney development.

Synergy of PEBP2/CBF with mi transcription factor (MITF) for transactivation of mouse mast cell protease 6 gene.

The mi locus encodes a member of the basic - helix - loop - helix - leucine zipper (bHLH-Zip) protein family of transcription factors (hereafter called MITF). Although the bHLH-Zip family transcription factors generally recognize and bind CANNTG motifs, the expression of mouse mast cell protease 6 (MMCP-6) gene is regulated by MITF through the GACCTG motif in the promoter region. The GACCTG motif was partly overlapped the TGTGGTC sequence, which was bound by polyomavirus enhancer binding protein 2 (PEBP2). In the present study, the effect of PEBP2 on the expression of MMCP-6 gene was examined. PEBP2 that is composed of alpha and beta subunits was expressed by mast cell lines and cultured mast cells derived from spleen. The overexpression of dominant negative PEBP2 cDNA reduced the expression of MMCP-6. Moreover, the simultaneous transfection of the plasmid containing MITF cDNA and the plasmid containing PEBP2 cDNA increased the MMCP-6 promoter activity. For the synergistic action of PEBP2 and MITF, the intact GACCTG and TGTGGTC motifs were prerequisite. The PEBP2alphaB1 mutant which lacked the region downstream from the Runt domain did not bind MITF and lost the synergistic function. These results indicated that PEBP2 and MITF synergistically transactivated the MMCP-6 gene and that the region downstream from the Runt domain of PEBP2alphaB1 was essential for the physical and functional interactions with MITF.

Transcriptional regulation of osteopontin gene in vivo by PEBP2alphaA/CBFA1 and ETS1 in the skeletal tissues.

Osteopontin (Opn) and polyoma enhancer-binding protein (PEBP) 2alphaA/core binding factor (CBFA) 1 have been suggested to play important roles in ossification. The overlapping localization of opn and PEBP2alphaA/CBFA1 mRNA, and the marked decrease of opn mRNA expression in PEBP2alphaA knockout mice, indicated that the transcription of opn gene was controlled by PEBP2alphaA. In the present study, we determined the direct regulation of PEBP2alphaA on the opn promoter activity. Opn promoter activity was markedly enhanced by PEBP2alphaA and ETS1 in a synergistic manner. The synergistic effect was diminished when either the PEBP2alphaA or ETS1 binding site was mutated, or the spatial arrangement of these sites was mutated by a 4-nt insertion. The distance between these sites was important for transactivation but not protein-DNA binding. The direct interaction between PEBP2alphaA and ETS1 was depended on protein-DNA binding. These results suggested that the specific spatial arrangement of both sites and direct interaction between PEBP2alphaA and ETS1, were essential for promoter function. Furthermore, endogenous opn mRNA was decreased with the introduction of dominant negative PEBP2alphaA to MC3T3/E1 cells expressing endogenous PEBP2alphaA, ETS1 and opn. These findings suggest that PEBP2alphaA and ETS1 cooperate in vivo to regulate expression of the opn gene in the skeletal tissue. Cell type-specific regulation of Opn gene expression will also be discussed.

Arl4c expression in colorectal and lung cancers promotes tumorigenesis and may represent a novel therapeutic target.

We recently demonstrated that expression of ADP-ribosylation factor (ARF)-like 4c (Arl4c) induced by a combination of Wnt/ß-catenin and epidermal growth factor/Ras signaling in normal epithelial cells grown in three-dimensional culture promotes cellular migration and proliferation, resulting in formation of tube-like structures, suggesting the involvement of Arl4c in epithelial morphogenesis. It is conceivable that there could be a common mechanism between epithelial morphogenesis and carcinogenesis. Therefore the current study was conducted to investigate whether Arl4c might be involved in tumorigenesis. Immunohistochemical analyses of tissue specimens obtained from colorectal and lung cancer patients revealed that Arl4c was not observed in non-tumor regions but was strongly expressed at high frequencies in tumor lesions. Inhibition of Wnt/ß-catenin or Ras/mitogen-activated protein kinase signaling reduced Arl4c mRNA levels in HCT116 colorectal cancer cells and A549 lung cancer cells. Knockdown of Arl4c inhibited Rac activity and also prevented nuclear localization of yes-associated protein (YAP)/transcriptional co-activator with PDZ-binding motif (TAZ) in these cancer cells. Arl4c-depleted cancer cells consistently showed decreased migration, invasion and proliferation capabilities both in vitro and in vivo. Furthermore, direct injection of Arl4c small interfering RNA (siRNA) into HCT116 cell-derived tumors (in vivo treatment with siRNA) inhibited tumor growth in immunodeficient mice. These results suggest that Arl4c is involved in tumorigenesis and might represent a novel therapeutic target for suppressing proliferation and invasion of colorectal and lung cancer cells.

Evaluation of PSF1 as a prognostic biomarker for prostate cancer.

BACKGROUND: Partner of SLD5 1 (PSF1) is an evolutionarily conserved DNA replication factor. Previous studies have suggested that transcriptional activity of the PSF1 gene correlated with malignancy of cancer cells. The objective of the current study was to evaluate the relationship between PSF1 expression and the clinical features of prostate cancer. METHODS: We determined the expression of PSF1 in 120 needle biopsy samples of prostate cancer by immunohistochemistry. We divided patients into PSF1-positive or -negative groups and analyzed the relationships between the expression of PSF1, the Gleason score, PSA level, TNM classification and prognosis. RESULTS: Our results showed that the PSF1 expression correlated significantly with PSA values at diagnosis (P=0.0028), with tumor grade (P<0.0001), and with clinical stage (P=0.0005). Moreover, the PSF1 expression correlated significantly with overall survival (hazard ratio (HR) 5.5; 95% confidence interval (CI) 2.17-15.8; P=0.003) and progression-free survival in 99 consecutive patients with prostate cancer. Noteworthy, the prognosis of PSF1-positive cases was also worse in patients with a Gleason score of 8-10 (HR 3.7; 95% CI 1.28-13.43; P=0.0143). Limitations include that this study had a retrospective design, that patients in the study were heterogeneous and included those with early and advanced cancer, and that small tumor fragments may not be representative of the entire carcinoma. CONCLUSIONS: PSF1 is expressed in high-grade prostate cancer and may be a useful biomarker to identify patients with a poor prognosis at the time of diagnosis.

PKC gamma gene expression is delayed in postnatal central nervous system of mi/mi mice.

In the central nervous system (CNS), the expression of protein kinase C (PKC) genes is strictly controlled by the developmental stage. We have examined the expression of PKC genes (cPKC alpha, beta, gamma, and nPKC delta, epsilon) in the process of the postnatal development in normal (+/+) C57BL/6 and microphthalmic (mi/mi) C57BL/6 mouse brains by Northern blotting and in situ hybridization. By Northern blotting, the expression level of cPKC gamma mRNA in mi/mi mice was significantly lower than that in +/+ littermates at d 9, 13, and 17. By in situ hybridization analysis, cPKC gamma mRNA-positive cells were detected in hippocampal and Purkinje cells in +/+ and mi/mi mice, but the magnitude of the signals in mi/mi mice was lower than that of +/+ mice, and the number of positive cells was smaller, whereas other isozymes (cPKC alpha, beta, and nPKC delta, epsilon) showed no significant difference between normal and mi/mi mice. The neuronal morphometric analysis by anti-P400 antibody revealed the same number and expression level of P400 protein in cerebellar Purkinje cells compared with +/+ mice. These results indicate that the deficiency of mi gene product causes the delayed expression of the cPKC gamma gene.

Expression and localization of gamma-aminobutyric acid A (GABAA) receptor alpha 1 subunit and L-glutamate decarboxylase (GAD) mRNAs in rat retina: an analysis by in situ hybridization.

The localization of the mRNAs encoding gamma-aminobutyric acidA receptor alpha 1 subunit (GABAA alpha 1) and L-glutamate decarboxylase (GAD) was elucidated in the rat retina by in situ hybridization. Soma diameter analysis of signal positive cells in the ganglion cell layer demonstrated that a subpopulation including alpha-cells of retinal ganglion cells expressed GABAA alpha 1 mRNA and a subpopulation of ganglion cells smaller than alpha-cells expressed GAD mRNA.

Localization of mRNA for c-kit receptor and its ligand in the brain of adult rats: an analysis using in situ hybridization histochemistry.

Localization of mRNA for the c-kit receptor and its ligand (Sl factor) in the brain of adult rats was studied using in situ hybridization histochemistry. The mRNA for the c-kit receptor was detected in the forebrain, the lower brain stem and the cerebellum. In the forebrain, the c-kit mRNA signals were detected in the olfactory bulb, the caudate-putamen, throughout the superficial cortex, the accumbens nucleus, the nucleus of vertical limb diagonal band, the bed nucleus of anterior commissure, Ammon's horn, the entopeduncular nucleus, the subthalamic nucleus, the dorsal raphe nucleus, the parasubiculum, the presubiculum, the ventricular nucleus of lateral lemniscus, and the entorhinal cortex. In the lower brain stem, the signals were detected in the inferior colliculus, the spinal vestibular nucleus, the spinal tract nucleus of trigeminal nerve, and the pyramidal tract. In the cerebellum, the signals were detected in the molecular layer of the cortex and cerebellar nuclei. By contrast, the signals of mRNA for Sl factor were detected in the forebrain and the cerebellum. In the forebrain, the signals were detected in the olfactory bulb, the endopiriform nucleus, the septohippocampal nucleus, the habenular nuclei, and most of the thalamic nuclei. In the cerebellum, the signals were detected in Purkinje cells. Several pairs of structures were found in which mRNA of either the c-kit receptor or the Sl factor was expressed and between which the synaptic connection had been reported, suggesting that the interaction between the c-kit receptor and the Sl factor may play some roles in the development of such synaptic connections.

Changes in levels of mRNAs of transforming growth factor (TGF)-beta1, -beta2, -beta3, TGF-beta type II receptor and sulfated glycoprotein-2 during apoptosis of mouse uterine epithelium.

To examine the roles played by transforming growth factors (TGF)-beta1, -beta2, -beta3, and TGF-beta type II receptors in the induction of apoptosis in the mouse uterine epithelium after estrogen deprivation, we investigated the expression of their mRNAs and the mRNA of sulfated glycoprotein-2 (SGP-2). Pellets containing 100 microg estradiol-17beta (E2) were implanted into ovariectomized mice and removed four days later. Apoptotic indices (percentage of apoptotic cells) of both luminal and glandular epithelia increased after E2 pellets were removed, but administration of progesterone (P), 5alpha-dihydrotestosterone (DHT), or continued implantation of E2 pellets suppressed this increase. Levels of mRNAs of TGF-beta1, -beta2, and -beta3, and SGP-2 did not increase after estrogen deprivation. However, estrogen deprivation caused a gradual increase in the level of TGF-beta type II receptor mRNA, and its level increased about six-fold six days later. Moreover, E2, P, and DHT markedly decreased the level of TGF-beta type II receptor mRNA. In situ hybridization demonstrated that mRNAs of TGF-beta1, -beta2, -beta3 and TGF-beta type II receptor were localized to the epithelium. Exogenous administration of TGF-beta1 into the uterine stroma induced apoptosis in the epithelium, a finding that suggests that signals produced by TGF-betas can induce apoptosis. Therefore, the present results suggest that increased sensitivity of uterine epithelial cells to TGF-betas, as demonstrated by an increase in TGF-beta type II receptor mRNA, is involved in the induction of apoptosis after estrogen deprivation, although signals produced by TGF-betas do not appear sufficient to induce apoptosis.

mi-transcription factor as a regulator of mast cell differentiation.

Masts cells are progeny of the hematopoietic stem cell. For the differentiation of mast cells, a transcription factor encoded by the mouse mi locus (MITF) plays an important role. The expression of many genes encoding proteins that are essential for the function of mast cells is regulated by MITF. Because various mutant mice are available at the mi locus and because cultured mast cells are easily obtained from the spleen of these mutant mice, this system may be a good model for studying the regulation of hematopoietic cell differentiation by a transcription factor.

Role of mast cell histamine in the formation of rat paw edema: a microdialysis study.

We determined the endogenous histamine concentration in the subplantar space of rat hind paws using an in vivo microdialysis technique. A microdialysis probe was implanted into the rat hind paw and the histamine content in dialysates was measured by high performance liquid chromatography-fluorometry. In wild type (+/+) rats, the histamine output (basal level 25.7 +/- 0.9 pmol/ml) increased 115-, 199- and 426-fold rapidly after subplantar injection of compound 48/80 at doses of 0.5, 5 and 50 microg/paw, respectively. In genetically mast cell-deficient (Ws/Ws) rats, the basal level of histamine was one third of that obtained from +/+ rats, and was not increased by compound 48/80 injection. With this treatment, marked, dose dependent, but relatively gradual development of the paw edema was found in +/+ rats. However, no edema formation was observed in Ws/Ws rats. Histological observations showed neither mast cells nor edema to be present in the paw skin of Ws/Ws rats. These findings indicate the critical role of histamine as a trigger for the development of edema in vivo. In addition, Ws/Ws rats will provide important information as to the roles of mast cells in the inflammatory response.

Spatial expression of genes encoding c-kit receptors and their ligands in mouse cerebellum as revealed by in situ hybridization.

Expression of mRNA for c-kit receptors and their ligands was examined in the cerebellum of mice by in situ hybridization technique. The c-kit receptors were expressed in the molecular layer of the cerebellum and the ligands for the c-kit receptors were detected at the boundary of molecular and granular layers. The expression of the c-kit ligands was not detectable in the cerebellum of lurcher (Lc/+) mutant mice that lack Purkinje cells, indicating the cells expressing the c-kit ligands were Purkinje cells. The cells expressing c-kit receptors decreased but were present in the cerebellum of Lc/+ mice. The c-kit mRNA-positive cells appeared to represent basket cells and stellate cells from their appearance and location. Since neurons in the molecular layer construct suppressive neurojunctions with Purkinje cells, the present result suggests that c-kit receptors and their ligands may play an important role for the construction of their junctions.

Characterization of c-kit-positive neurons in the dorsal root ganglion of mouse.

Previously, we showed that c-kit receptor tyrosine kinase is expressed by a subpopulation of dorsal root ganglion (DRG) neurons, and that the ligand for the c-kit receptor, stem cell factor (SCF), induces the neurite outgrowth and supports the survival of these neurons in culture [16]. However, it is unknown which class of DRG neurons express c-kit receptor and which factor regulates differentiation and survival of c-kit-positive neurons. In the present study, we attempted to characterize c-kit positive neurons in the mouse DRG. The c-kit-positive neurons were small or medium in size, and 44% of these neurons contained substance P. Central fibers of the c-kit-positive neurons terminated in laminae I and II of the gray matter of the spinal cord. These results suggest that c-kit-positive neurons in the DRG belong to a functional subpopulation. The c-kit receptor protein was presented on the membrane of processes and growth cones in neurons. When DRG cells of embryonic day 15.5 or 17.5 were cultured, the survival of c-kit-positive neurons was supported by SCF, nerve growth factor (NGF) or leukemia inhibitory factor. SCF and NGF synergistically supported the survival of c-kit-positive neurons at submaximal concentrations. c-kit-positive DRG neurons from neonatal mice survived without addition of any factor in culture, suggesting that the requirement for trophic support in c-kit-positive neurons changes during development.

Demonstration of retinal cells expressing messenger RNAs of the c-kit receptor and its ligand.

We found cells expressing c-kit receptor and its ligand (Sl factor, SLF) in the retina of rats and mice. c-kit messenger RNA (mRNA) was detected in a limited number of cells in the inner nuclear layer of rats and mice by in situ hybridization. The c-kit-expressing cells were assumed to be a subpopulation of the amacrine cells on the basis of their size and distribution pattern. c-kit protein-containing cells were demonstrated in the mouse retina by using ACK2 monoclonal antibody against the extracellular domain of the mouse c-kit receptor. The c-kit protein was detected in cell bodies and processes of the presumed amacrine cells. Hybridization signals for SLF mRNA were observed in the retinal ganglion cells. The results suggest that the c-kit receptor and its ligand may play some roles in the formation of junctions between the amacrine and retinal ganglion cells.

Possible involvement of c-kit receptor and its ligand in increase of mast cells in neurofibroma tissues.

Many mast cells are present in the tumor tissues of neurofibromatosis 1. We investigated the mechanism of the mast cell increase. Since the stem cell factor (SCF) induces development of mast cells and since the receptor of SCF is encoded by the c-kit gene, we examined the expression of SCF mRNA and c-kit mRNA in neurofibroma tissues. In situ hybridization demonstrated strong expression of c-kit messenger RNA in mast cells in the neurofibroma, but the expression of SCF mRNA was not demonstrable by in situ hybridization in either neurofibroma tissues or control normal skin tissues. When RNA extracted from neurofibroma tissues or normal skin tissues was reverse transcribed and then amplified by the polymerase chain reaction, the amount of SCF cDNA was greater in neurofibroma tissues than in normal skin tissues. The results suggest that SCF and the c-kit receptor are associated with the increase of mast cells in neurofibroma tissues.