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Yin Yang 1 (YY1) is a transcription factor that regulates epigenetic pathways and protein modifications. CP2c is a transcription factor that functions as an oncogene to regulate cell proliferation. YY1 is known to interact with CP2c to suppress CP2c’s transcriptional activity. This study aimed to investigate YY1 and CP2c expression in breast cancer and prognostic implications. In this study, YY1 and CP2c expression was evaluated using immunohistochemical staining, Western blot and RT-PCR assays. Of 491 patients with primary breast cancer, 138 patients showed YY1 overexpression. Luminal subtype and early stage were associated with overexpression (p < 0.001). After a median follow-up of 68 months, YY1 overexpression was found to be associated with a better prognosis (disease-free survival rates of 92.0% vs. 79.2%, p = 0.014). In Cox proportional hazards model, YY1 overexpression functioned as an independent prognostic factor after adjustment of hormone receptor/HER2 status and tumor size (hazard ratio of 0.50, 95% CI 0.26–0.98, p = 0.042). Quantitative analysis of YY1 and CP2c protein expression in tumors revealed a negative correlation between them. In conclusion, YY1 overexpression is a favorable prognostic biomarker in patients with breast cancer, and it has a negative correlation with CP2c at the protein level.

Background YY1 is transcription factor that regulates differential epigenetic pathways and protein modifications. CP2c is transcription factor that functions as oncogene to regulate cancer cell proliferation. YY1 is known to interact with CP2c to suppress CP2c’s transcriptional activity. However, it is unknown whether YY1 has prognostic significance in breast cancer. Thus, we aimed to investigate YY1 and CP2c expression in breast cancer and prognostic implications. Methods Clinical information and tissues were obtained from 491 patients with invasive breast cancer. YY1 expression was evaluated using immunohistochemical staining and patients were divided into two groups according to H-score. Western blot and RT-PCR assays were used for quantifications of YY1 and CP2c mRNA and protein expression. Results Of 491 patients with breast cancer, 138 patients showed YY1 overexpression. Luminal subtype, and early stage were associated with overexpression. After follow-up of 68 months, YY1 overexpression was found to be associated with better prognosis. In Cox proportional hazards model, YY1 overexpression functioned as independent prognostic factor after adjustment of ER/HER2 status and tumor size. Quantitative analysis of YY1 and CP2c protein expression in tumor revealed negative correlation between them. Conclusions YY1 overexpression is a favorable prognostic biomarker in patients with breast cancer, and it has negative correlation with CP2c.

Transcription factor CP2c (also known as TFCP2, α-CP2, LSF, and LBP-1c) is involved in diverse ubiquitous and tissue/stage-specific cellular processes and in human malignancies such as cancer. Despite its importance, many fundamental regulatory mechanisms of CP2c are still unclear. Here, we uncover an unprecedented mechanism of CP2c degradation via a previously unidentified SUMO1/PSME3/20 S proteasome pathway and its biological meaning. CP2c is SUMOylated in a SUMO1-dependent way, and SUMOylated CP2c is degraded through the ubiquitin-independent PSME3 (also known as REGγ or PA28)/20 S proteasome system. SUMOylated PSME3 could also interact with CP2c to degrade CP2c via the 20 S proteasomal pathway. Moreover, precisely timed degradation of CP2c via the SUMO1/PSME3/20 S proteasome axis is required for accurate progression of the cell cycle. Therefore, we reveal a unique SUMO1-mediated uncanonical 20 S proteasome degradation mechanism via the SUMO1/PSME3 axis involving mutual SUMO-SIM interaction of CP2c and PSME3, providing previously unidentified mechanistic insights into the roles of dynamic degradation of CP2c in cell cycle progression.

Spermatogonial stem cells (SSCs) have stemness characteristics, including germ cell-specific imprints that allow them to form gametes. Spermatogenesis involves changes in gene expression such as a transition from expression of somatic to germ cell-specific genes, global repression of gene expression, meiotic sex chromosome inactivation, highly condensed packing of the nucleus with protamines, and morphogenesis. These step-by-step processes finally generate spermatozoa that are fertilization competent. Dynamic epigenetic modifications also confer totipotency to germ cells after fertilization. Primordial germ cells (PGCs) in embryos do not enter meiosis, remain in the proliferative stage, and are referred to as gonocytes, before entering quiescence. Gonocytes develop into SSCs at about 6 days after birth in rodents. Although chromatin structural modification by Polycomb is essential for gene silencing in mammals, and epigenetic changes are critical in spermatogenesis, a comprehensive understanding of transcriptional regulation is lacking. Recently, we evaluated the expression profiles of Yin Yang 1 (YY1) and CP2c in the gonads of E14.5 and 12-week-old mice. YY1 localizes at the nucleus and/or cytoplasm at specific stages of spermatogenesis, possibly by interaction with CP2c and YY1-interacting transcription factor. In the present article, we discuss the possible roles of YY1 and CP2c in spermatogenesis and stemness based on our results and a review of the relevant literature.

Stemness and epithelial–mesenchymal transition (EMT) are two fundamental characteristics of metastasis that are controlled by diverse regulatory factors, including transcription factors. Compared with other subtypes of breast cancer, basal-type or triple-negative breast cancer (TNBC) has high frequencies of tumor relapse. However, the role of alpha-globin transcription factor CP2 (TFCP2) has not been reported as an oncogenic driver in those breast cancers. Here, we show that TFCP2 is a potent factor essential for EMT, stemness, and metastasis in breast cancer. TFCP2 directly bound promoters of EGF and TGFα to regulate their expression and stimulate autocrine signaling via EGFR. These findings indicate that TFCP2 is a new antimetastatic target and reveal a novel regulatory mechanism in which a positive feedback loop comprising EGF/TGFα and AKT can control malignant breast cancer progression. Significance: TFCP2 is a new antimetastatic target that controls TNBC progression via a positive feedback loop between EGF/TGFα and the AKT signaling axis.

Background: YY1 is conserved transcriptional factor that highly expressed in various types of cancers. It regulates differential epigenetic pathways and protein modifications. However, there is still unknown whether YY1 overexpression has any prognostic significance in breast cancer patients. In this study, we evaluated YY1 expression levels using tissue microarrays and analyzed clinicopathologic characteristics with survival outcomes. Methods: Clinical information and tissue blocks were obtained retrospectively from 491 patients who underwent surgery at Hanyang University Hospital between 2002 and 2016. Immunohistochemical staining was performed using rabbit monoclonal antibody (ab-109237). YY1 expression was determined using H-score. Overexpression was defined as H-score ≥ 28.4 (mean value). Overall survival (OS) and disease-free survival (DFS) was analyzed by Kaplan Meier analysis. Results: Of 491 patients with primary breast cancer, 138 (28.1%) patients had YY1 overexpression. Luminal subtype (ER+/HER2-), early tumor stage, and low grade tumor were significantly associated with overexpression (P Citation Format: Chihwan Cha, Hosub Park, Chul Geun Kim, Min Sung Chung. Prognostic implication of Yin Yang 1 (YY1) overexpression in patients with primary breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-05-13.

The root of Dipsacus asperoides C. Y. Cheng et T. M. Ai is traditionally used as an analgesic and anti-inflammatory agent to treat pain, rheumatoid arthritis, and bone fractures. However, neither its effects on osteoarthritis (OA) nor its effects on the arthritic cartilage tissue transcriptome have not been fully investigated. In this study, we used a rat model of monosodium iodoacetate- (MIA-) induced OA to investigate the therapeutic effects of a Dipsacus asperoides ethanolic extract (DAE, 200 mg/kg for 21 days). The study first assessed joint diameter, micro-CT scans, and histopathological analysis and then conducted gene expression profiling using RNA sequencing in articular cartilage tissue. We found that DAE treatment ameliorates OA disease phenotypes; it reduced the knee joint diameter and prevented changes in the structural and histological features of the joint, thereby showing that DAE has a protective effect against OA. Based on the results of gene expression profiling and subsequent pathway analysis, we found that several canonical pathways were linked to DAE treatment, including WNT/β-catenin signaling. Taken together, the present results suggest molecular mechanism, involving gene expression changes, by which DAE has a protective effect in a rat model of MIA-induced OA.

CP2c, also known as TFCP2, α-CP2, LSF, and LBP-1c, is a prototypic member of the transcription factor (TF) CP2 subfamily involved in diverse ubiquitous and tissue/stage-specific cellular processes and in human malignancies including cancer. Despite its importance, many fundamental regulatory mechanisms of CP2c are still unclear. Here, we uncover unprecedented structural and functional aspects of CP2c using DSP crosslinking and Western blot in addition to conventional methods. We found that a monomeric form of a CP2c homotetramer (tCP2c; [C4]) binds to the known CP2c-binding DNA motif (CNRG-N(5~6)-CNRG), whereas a dimeric form of a CP2c, CP2b, and PIAS1 heterohexamer ([C2B2P2]2) binds to the three consecutive CP2c half-sites or two staggered CP2c binding motifs, where the [C4] exerts a pioneering function for recruiting the [C2B2P2]2 to the target. All CP2c exists as a [C4], or as a [C2B2P2]2 or [C2B2P2]4 in the nucleus. Importantly, one additional cytosolic heterotetrameric CP2c and CP2a complex, ([C2A2]), exerts some homeostatic regulation of the nuclear complexes. These data indicate that these findings are essential for the transcriptional regulation of CP2c in cells within relevant timescales, providing clues not only for the transcriptional regulation mechanism by CP2c but also for future therapeutics targeting CP2c function.

Intrinsically disordered proteins exist as highly dynamic conformational ensembles of diverse forms. However, the majority of virtual screening only focuses on proteins with defined structures. This means that computer-aided drug discovery is restricted. As a breakthrough, understanding the structural characteristics of intrinsically disordered proteins and its application can open the gate for unrestricted drug discovery. First, we segmented the target disorder-to-order transition region into a series of overlapping 20-amino-acid-long peptides. Folding prediction generated diverse conformations of these peptides. Next, we applied molecular docking, new evaluation score function, and statistical analysis. This approach successfully distinguished known compounds and their corresponding binding regions. Especially, Myc proto-oncogene protein (MYC) inhibitor 10058F4 was well distinguished from others of the chemical compound library. We also studied differences between the two Methyl-CpG-binding domain protein 2 (MBD2) inhibitors (ABA (2-amino-N-[[(3S)-2,3-dihydro-1,4-benzodioxin-3-yl]methyl]-acetamide) and APC ((R)-(3-(2-Amino-acetylamino)-pyrrolidine-1-carboxylic acid tert-butyl ester))). Both compounds bind MBD2 through electrostatic interaction behind its p66&alpha, binding site. ABA is also able to bind p66&alpha, through electrostatic interaction behind its MBD2-binding site while APC-p66&alpha, binding was nonspecific. Therefore, structural heterogeneity mimicking of the disorder-to-order transition region at the peptide level and utilization of the new docking score function represent a useful approach that can efficiently discriminate compounds for expanded virtual screening toward intrinsically disordered proteins.

Chromatin remodeling, including histone modification, chromatin (un)folding, and nucleosome remodeling, is a significant transcriptional regulation mechanism. By these epigenetic modifications, transcription factors and their regulators are recruited to the promoters of target genes, and thus gene expression is controlled through either transcriptional activation or repression. The Mat1-mediated transcriptional repressor (MMTR)/DNA methyltransferase 1 (DNMT1)-associated protein (Dmap1) is a transcription corepressor involved in chromatin remodeling, cell cycle regulation, DNA double-strand break repair, and tumor suppression. The Tip60-p400 complex proteins, including MMTR/Dmap1, interact with the oncogene Myc in embryonic stem cells (ESCs). These proteins interplay with the stem cell-related proteome networks and regulate gene expressions. However, the detailed mechanisms of their functions are unknown. Here, we show that MMTR/Dmap1, along with other Tip60-p400 complex proteins, bind the promoters of differentiation commitment genes in mouse ESCs. Hence, MMTR/Dmap1 controls gene expression alterations during differentiation. Furthermore, we propose a novel mechanism of MMTR/Dmap1 function in early stage lineage commitment of mouse ESCs by crosstalk with the polycomb group (PcG) proteins. The complex controls histone mark bivalency and transcriptional poising of commitment genes. Taken together, our comprehensive findings will help better understand the MMTR/Dmap1-mediated transcriptional regulation in ESCs and other cell types.

The intrinsically disordered region of MBD2 is a promising target for treating cancer metastasis., Although intrinsically disordered protein regions (IDPRs) are commonly engaged in promiscuous protein-protein interactions (PPIs), using them as drug targets is challenging due to their extreme structural flexibility. We report a rational discovery of inhibitors targeting an IDPR of MBD2 that undergoes disorder-to-order transition upon PPI and is critical for the regulation of the Mi-2/NuRD chromatin remodeling complex (CRC). Computational biology was essential for identifying target site, searching for promising leads, and assessing their binding feasibility and off-target probability. Molecular action of selected leads inhibiting the targeted PPI of MBD2 was validated in vitro and in cell, followed by confirming their inhibitory effects on the epithelial-mesenchymal transition of various cancer cells. Identified lead compounds appeared to potently inhibit cancer metastasis in a murine xenograft tumor model. These results constitute a pioneering example of rationally discovered IDPR-targeting agents and suggest Mi-2/NuRD CRC and/or MBD2 as a promising target for treating cancer metastasis.

Murine erythroleukemia (MEL) cells are often employed as a model to dissect mechanisms of erythropoiesis and erythroleukemia in vitro. Here, an allograft model using MEL cells resulting in splenomegaly was established to develop a diagnostic model for isolation/quantification of metastatic cells, anti-cancer drug screening, and evaluation of the tumorigenic or metastatic potentials of molecules in vivo. In this animal model, circulating MEL cells from the blood stream were successfully isolated and quantified with an additional in vitro cultivation step. In terms of the molecular-pathological analysis, we were able to successfully evaluate the functional discrimination between methyl-CpG-binding domain 2 (Mbd2) and p66&alpha, in erythroid differentiation, and tumorigenic potential in spleen and blood stream of allograft model mice. In addition, we found that the number of circulating MEL cells in anti-cancer drug-treated mice was dose-dependently decreased. Our data demonstrate that the newly established allograft model is useful to dissect erythroleukemia pathologies and non-invasively provides valuable means for isolation of metastatic cells, screening of anti-cancer drugs, and evaluation of the tumorigenic potentials.

Maintaining stemness and permitting differentiation mediated by combinations of transcription factors (TFs) are key aspects of mammalian spermatogenesis. It has been established that yin yang 1 (YY1), a target factor of mammalian polycomb repressive complex 2 (PRC2) and a regulator of stemness, is involved in the stable maintenance of prophase stage spermatocytes. Recently, we have demonstrated that the TF CP2c partners with YY1 in some cells to antagonistically regulate the other protein's function. To date, the functional roles of YY1 and CP2c in spermatogonial stem cells and their derived germ cells remain unclear. Here, we investigated the expression of YY1 and CP2c in mouse gonocytes and germ cells using tissue immunohistochemical and immunofluorescence analyses. At E14.5, both YY1 and CP2c were stained in gonocytes and Sertoli cells in testicular cords, showing different proportion and density of immunoreactivity. However, in adult testes, YY1 was localized in the nuclei of spermatogonial stem cells and spermatocytes, but not in spermatozoa. It was also detected in spermatogonia and spermatids in a stage-specific manner during spermatogenic cycle. CP2c could be detected mostly in the cytoplasm of spermatocytes but not at all in spermatogonial stem cells, indicating mutually exclusive expression of CP2c and YY1. Interestingly, however, CP2c was stained in the cytoplasm and nucleus of spermatogonia at elongation and release stages, and co-localized with YY1 in the nucleus at grouping, maturation, and releasing stages. Neither YY1 nor CP2c was expressed in spermatozoa. Our data indicate that YY1 strongly localizes in the spermatogonial stem cells and co-localizes heterogeneously with CP2c to permit spermatogenesis, and also suggest that YY1 is essential for stemness of spermatogonial stem cells (SCs) whereas CP2c is critical for the commitment of spermatogonia and during the progression of spermatogonia to spermatids. This evaluation expands our understanding of the molecular mechanism of spermatogonia formation as well as spermatogenesis in general.

So-Cheong-Ryoung-Tang is a traditionally used herbal formula for the treatment of pulmonary diseases in China, Korea, and Japan. We investigated the protective effects of So-Cheong-Ryong-Tang water extract (SCWE) in cigarette smoke concentrate (CSC) stimulated human airway epithelial cell line NCI-H292 and mice exposed cigarette smoke (CS) and lipopolysaccharide (LPS). In the CSC-stimulated NCI-H292 cells, SCWE inhibited proinflammatory cytokines in a concentration-dependent manner, as evidenced by a reduction in their mRNA levels. Also, SCWE significant reduced inducible nitric oxide synthase (iNOS) expression and nuclear factor kappa B (NF-κB) phosphorylation in CSC-stimulated cells. The mice were exposed to CS for 1 h per day (a total of eight cigarettes per day) for 7 days and received LPS intranasally on day 5. The mice were administered a dose of SCWE (100 and 200 mg/kg) 1 h before CS exposure. In in vivo, SCWE decreased the inflammatory cell count and reduced the expression of the proinflammatory cytokines in the broncho-alveolar lavage fluid (BALF) compared with CS and LPS exposed mice. SCWE attenuated inflammatory cell infiltration in airway induced by CS and LPS exposure, and this decrease was accompanied by a reduction in the expression levels of iNOS and MMP-9 in lung tissue. The extract also inhibited the phosphorylation of inhibitor of kappa B alpha (IκBα) and NF-κB induced by CS and LPS exposure in lung tissue. These results suggest that SCWE may effectively inhibit airway inflammatory responses induced by CS and LPS exposure via the NF-κB pathway. Therefore, SCWE may be a potential treatment for airway inflammatory diseases, such as chronic obstructive pulmonary disease (COPD).

In the present study, necrosis inhibitor-5 (NecroX-5), a novel Cyclopentylamino carboxymethylthiazolylindole (NecroX) series compound was investigated for its protective role against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity in a zebrafish model of Parkinson’s disease (PD). MPTP-induced locomotor behavior was measured in zebrafish larvae and the protein expression level of tyrosine hydroxylase (TH) was estimated in zebrafish larva homogenates. MPTP (15 μM) induced a significant (p

During hematopoiesis, red blood cells originate from the hematopoietic stem cell reservoir. Although the regulation of erythropoiesis and globin expression has been intensively investigated, the underlining mechanisms are not fully understood, including the interplay between transcription factors and epigenetic factors. Here, we uncover that the Mbd2-free NuRD chromatin remodeling complex potentiates erythroid differentiation of proerythroblasts via managing functions of the CP2c complexes. We found that both Mbd2 and Mbd3 expression is downregulated during differentiation of MEL cells in vitro and in normal erythropoiesis in mouse bone marrow, and Mbd2 downregulation is crucial for erythropoiesis. In uninduced MEL cells, the Mbd2-NuRD complex is recruited to the promoter via Gata1/Fog1, and, via direct binding through p66α, it acts as a transcriptional inhibitor of the CP2c complexes, preventing their DNA binding and promoting degradation of the CP2c family proteins to suppress globin gene expression. Conversely, during erythropoiesis in vitro and in vivo, the Mbd2-free NuRD does not dissociate from the chromatin and acts as a transcriptional coactivator aiding the recruitment of the CP2c complexes to chromatin, and thereby leading to the induction of the active hemoglobin synthesis and erythroid differentiation. Our study highlights the regulation of erythroid differentiation by the Mbd2-CP2c loop.

Cellular FLICE-inhibitory protein (cFLIP, cflara) is a regulator of death receptor (DR)-induced apoptosis and NF-κB activation. cFLIP is known to prevent activation of the caspase cascade by binding to FADD/caspase-8. Up-regulated cFLIP has been identified in many tumor types, and therefore restoring apoptosis by silencing cFLIP may be one of the more potent strategies in cancer therapeutics. The zebrafish cFLIP gene, cflara, has 2 death effector domains (DEDs) and a single caspase-like domain. Expression of cflara was detected in the zebrafish embryo by RT-PCR and whole-mount in situ hybridization. To study the in vivo function of cflara, we generated a cflara knockout mutant zebrafish using transcription activator-like effector nucleases (TALENs). Frame shift mutation is caused by a 10-bp deletion in the first DED domain. By inbreeding the F1 generation, a homozygous mutant fish was produced and confirmed by PCR. Knockout of cflara leads to abnormal heart development and embryonic lethality in mice. However, mutant zebrafish did not show any differences from wild type in heartbeat rate, survival rate or development. Zebrafish have analogues of both cflara and cflarb. Quantitative PCR showed that cflarb mRNA levels of mutant zebrafish were higher than those in the wild type. In a chemical exposure experiment, mutant zebrafish larvae showed a longer survival rate compared with wild type after CoCl2 treatment. However, no significant difference was observed from cisplatin treatment. This data suggests that cflarb may contribute to normal development and causes a difference in chemical resistance.

Spermatogonial stem cells (SSCs) are adult stem cells that transmit genetic information from the parent to the next generation (progeny) in males, and thus, SSCs have used in germline-modification for generating transgenic animals. In this study, we demonstrated the feasibility of transgenic sperm production by employing an effective busulfan treatment method to prepare recipient pigs for the transplantation of genetically modified donor porcine SSCs. We purified SSCs from pig testis cells by sequentially employing Laminin-coated dishes and culture dishes. The purified cells were transduced with lentivirus expressing enhanced green fluorescent protein (eGFP) at a multiplicity of infection (MOI) of 6 for 9 h. eGFP transduced pig SSCs were then transplanted into the seminiferous tubules of 12 to 16-week-old recipients born to busulfan-treated sows. We obtained six recipient pigs after transplantation and maintained them for more than 6 months. The collected viable spermatozoa from 2 out of 6 recipients were positive for eGFP gene expression in polymerase chain reaction. eGFP-expressing spermatozoa appeared morphologically normal under the microscope. When spermatozoa from these recipients were used for intra cytoplasmic sperm injection, eGFP expression could be detected in the embryos. Furthermore, eGFP colonies were derived from donor-transduced SSCs observed in the recipients’ testes. In summary, we demonstrated the successful production of functional-transgenic spermatozoa by transplantation of porcine SSCs where the transgenic was transduced by employing the lentiviral vector system.

The structure and function of the Antarctic marine diatom Chaetoceros neogracile antifreeze protein (Cn-AFP), as well as its expression levels and characteristics of the ice-binding site, were analyzed in the present study. In silico analysis revealed that the Cn-AFP promoter contains both light- and temperature-responsive elements. Northern and Western blot analyses demonstrated that both Cn-AFP transcript and protein expression were strongly and rapidly stimulated by freezing, as well as temperature and high light stress. Immunogold labeling revealed that Cn-AFP is preferentially localized to the intracellular space near the chloroplast membrane. Recombinant Cn-AFP had clear antifreeze activity. Protein-folding simulation was used to predict the putative ice-binding sites in Cn-AFP, and site-directed mutagenesis of the Cn-AFP b-face confirmed their identification.

Requiem (REQ/DPF2) was originally identified as an apoptosis-inducing protein in mouse myeloid cells and belongs to the novel Kruppel-type zinc finger d4-protein family of proteins, which includes neuro-d4 (DPF1) and cer-d4 (DPF3). Interestingly, when a portion of the REQ messenger ribonucleic acid (mRNA) 3' untranslated region (3'UTR), referred to as G8, was overexpressed in K562 cells, beta-globin expression was induced, suggesting that the 3'UTR of REQ mRNA plays a physiological role. Here, we present evidence that the REQ mRNA 3'UTR, along with its trans-acting factor, Staufen1 (STAU1), is able to reduce the level of REQ mRNA via STAU1-mediated mRNA decay (SMD). By screening a complementary deoxyribonucleic acid (cDNA) expression library with an RNA-ligand binding assay, we identified STAU1 as an interactor of the REQ mRNA 3'UTR. Specifically, we provide evidence that STAU1 binds to putative 30-nucleotide stem-loop-structured RNA sequences within the G8 region, which we term the protein binding site core; this binding triggers the degradation of REQ mRNA and thus regulates translation. Furthermore, we demonstrate that siRNA-mediated silencing of either STAU1 or UPF1 increases the abundance of cellular REQ mRNA and, consequently, the REQ protein, indicating that REQ mRNA is a target of SMD.

Undifferentiated germ cells have the capacity to develop into sperm capable of fertilizing oocytes and contributing genetic material to subsequent generations. The most primitive prepubertal undifferentiated germ cells include gonocytes and undifferentiated spermatogonia, including spermatogonial stem cells (SSC). Gonocytes, present in the testis at birth, differentiate into SSC, which maintain spermatogenesis for the remainder of the male's life. Because of their capacity to contribute to lifelong spermatogenesis, undifferentiated germ cells are attractive targets for genetic modification to produce transgenic animals, including cattle. To maximize the efficiency of genetic modification of bovine gonocytes and SSC, effective enrichment techniques need to be developed. Selection of bovine gonocytes using differential plating was improved 8-fold (P < 0.001) when using a combination of extracellular matrix proteins, including laminin, fibronectin, collagen type IV, and gelatin, compared to using laminin and gelatin alone. Selected cells labeled with PKH26 formed colonies of donor-derived germ cells after transplantation into recipient mouse testes, indicating putative stem cell function. Significantly more colonies (P < 0.001) per 1 × 10(5) viable transplanted cells were formed from isolated nonadherent cells (203 ± 23.2) compared to adherent (20 ± 2.7) or Percoll (45.5 ± 4.5) selected cells. After selection, some gonocytes were transduced using a lentiviral vector containing the transgene for the enhanced green fluorescent protein. Transduction efficiency was 17%. Collectively, these data demonstrate effective methods for the selection and genetic modification of bovine undifferentiated germ cells.

The bio-sensor technologies to detect cancer cells have been attracted much attention because detecting the presence of a cancer in a human blood at an early stage can extend the human lifetime. To diagnose a cancer in human normal blood cells, electrical biosensors such as carbon nanotube field-effective transistor, AlGaN/GaN HEMT, reduced graphene oxide TFT, Si nanowire FET, PEDOT:PSS conducting paper, and ZnO nano-rods have been intensively studied because they detect and diagnose the type and amount of cancer cell by estimating the electrical charge amount of the cancer cell in the human blood. However, they have not demonstrated for detecting and diagnosing a cancer cell in circulating cancer stem cell (CCSC). In our study, thus, we cultured breast cancer cell (BCC) line, MDA-MB-231, and designed cancer detecting system in which the detection devices are implemented with a separation device to separate the cancer cell from mixed solution of cancer cell line and normal blood by utilizing both size and deformability of cancer cell. In particular, we designed a real time enumeration image analysis tool to acquire morphology data of breast cancer cell line for image-based cell analysis. For detecting the cancer cell via determining the threshold voltage shift (∆Vth) of field effect transistor (FET), an In-Ga-Zn-Oxide (IGZO) based FET implemented with Au nano-particles (NPs) was fabricated. For the experimental process, the 10-μL solution of separated cancer cell lines connected with the cancer-anti-body (CD-44) was dropped on the IGZO FET channel with Au NPs where a linker (8-Mercaptooctanoic acid) was attached on Au NPs. Consequently, we successfully demonstrated the separation of cancer cell from normal blood sample and after dropping cancer cell on the detection device the ∆Vth value was shifted negative direction. Furthermore, we observed the fluorescent staining image of cancer cell separated from normal blood sample. We report the separation and detection system which enables to separate and detect cancer cell. Figure 1

The separation of circulating tumor cells (CTCs) from the peripheral blood is an important issue that has been highlighted because of their high clinical potential. However, techniques that depend solely on tumor-specific surface molecules or just the larger size of CTCs are limited by tumor heterogeneity. Here, we present a slanted weir microfluidic device that utilizes the size and deformability of CTCs to separate them from the unprocessed whole blood. By testing its ability using a highly invasive breast cancer cell line, our device achieved a 97% separation efficiency, while showing an 8-log depletion of erythrocytes and 5.6-log depletion of leukocytes. We also developed an image analysis tool that was able to characterize the various morphologies and differing deformability of the separating cells. From the results, we believe our system possesses a high potential for liquid biopsy, aiding future cancer research.

This work was supported by Basic Science Research Program (2010-00252250 to C. G. K.), National Research Foundation (NRF), Ministry of Education, Science and Technology (MEST), Republic of Korea. Converging Research Center Program (2013K000283 to C. G. K.), Ministry of Science, ICT & Future Planning (MSIFP), Republic of Korea.

Spermatogonial stem cells (SSCs) are the foundation of spermatogenesis throughout postnatal life in male and have the ability to transmit genetic information to the subsequent generation. In this study, we have optimized the transduction efficiency of SSCs using a lentiviral vector by considering different multiplicity of infection (MOI), duration of infection, presence or absence of feeder layer and polycationic agents. We tested MOI of 5, 10 or 20 and infection duration of 6, 9 or 12 h respectively. After infection, cells were cultured for 1 week and as a result, the number of transduced SSCs increased significantly for MOI of 5 and 10 with 6 h of infection. When the same condition (MOI of 5 with 6 hours) was applied in presence or absence of STO feeder layer and infected SSCs were cultured for 3 weeks on the STO feeder layer, a significant increase in the number of transduced cells was observed for without the feeder layer during infection. We subsequently studied the effects of polycationic agents, polybrene and dioctadecylamidoglycyl spermine (DOGS), on the transduction efficiency. Compared with the polybrene treatment, the recovery rate of the transduced SSCs was significantly higher for the DOGS treatment. Therefore, our optimization study could contribute to the enhancement of germ-line modification of SSCs using lentiviral vectors and in generation of transgenic animals.

The β-like globin genes are transcribed in a developmental stage specific fashion in erythroid cells. The specific transcription of globin genes is conferred by the locus control region (LCR), but the chromatin structure of the LCR in the human adult β-globin locus transcribing the δ- and β-globin genes is not clear. Here, we employed hybrid MEL cells that contain a human chromosome 11. The δ- and β-globin genes were highly transcribed in hybrid MEL/ch11 cells after transcriptional induction. LCR HS3 and HS2 were strongly occupied by erythroid specific transcriptional activators and co-factors in the induced locus. These HSs, but not HS4 and HS1, were in close proximity with the active globin genes as revealed by high resolution 3C experiments. The active features at HS3 were markedly established after transcriptional induction, while HS2 was in a relatively active conformation before the induction. Unexpectedly, HS1 did not show notable active features except histone hyperacetylation. Taken together, the LCR of the human β-globin locus transcribing the adult δ- and β-globin genes has HS specific chromatin structure. The structure at each HS, which is different from the locus transcribing the fetal globin genes, might relate to its role in transcribing the adult genes.

Protein kinase Cδ (PKCδ) plays a significant role in the regulation of growth, apoptosis, and differentiation in a diversity of cell types. We investigated the effect of PKCδ on Notch1 intracellular domain (NICD)-mediated transcription with Notch transcription reporter constructs. The results indicate that co-expression of PKCδ down-regulated NICD-dependent transcription. Co-expression of a dominant negative PKCδ (K376R) variant lacking kinase activity was also able to downregulate NICD-dependent transcription, suggesting that PKCδ exerts its inhibitory effect via a kinase-independent mechanism(s). Interestingly, expression of PKCδ as well as K376R induced NICD up-regulation by inhibiting proteasome-mediated degradation of NICD, indicating that NICD protein quantity is not proportional to its transcriptional activity. When the subcellular distribution of NICD was investigated by both subcellular fractionation and immunocytochemistry, it was found that PKCδ and K376R effectively impaired proper nuclear localization of NICD, possibly via a physical association between NICD and PKCδ, which was confirmed by co-immunoprecipitation experiments. Chromatin immunoprecipitation assays revealed that both PKCδ and K376R inhibit the association of NICD with the promoter region of its target gene, Hes1. Furthermore, silencing of PKCδ resulted in increased NICD nuclear localization and NICD transcriptional activity in MCF-7 cells. PKCδ silencing-induced increase in anti-apoptotic survivin could not rescue apoptosis induced by doxorubicin. The data herein indicate that PKCδ can induce down-regulation of NICD transcriptional activity via a kinase-independent inhibition of NICD nuclear targeting and dissociation of NICD from target gene promoters.

GATA-1 and NF-E2 are erythroid specific activators that bind to the β-globin locus. To explore the roles of these activators in transcription of the human fetal stage specific γ-globin genes, we reduced GATA-1 and p45/NF-E2 using shRNA in erythroid K562 cells. GATA-1 or p45/NF-E2 knockdown inhibited the transcription of the γ-globin genes, hypersensitive site (HS) formation in the LCR and chromatin loop formation of the β-globin locus, but histone acetylation across the locus was decreased only in the case of GATA-1 knockdown. In p45/NF-E2 knockdown cells, GATA-1 binding was maintained at the LCR HSs and γ-globin promoter, but NF-E2 binding at the LCR HSs was reduced by GATA-1 knockdown regardless of the amount of p45/NF-E2 in K562 cells. These results indicate that histone acetylation is dependent on GATA-1 binding, but the binding of GATA-1 is not sufficient for the γ-globin transcription, HS formation and chromatin loop formation and NF-E2 is required. This idea is supported by the distinctive binding pattern of CBP and Brg1 in the β-globin locus. Furthermore GATA-1-dependent loop formation between HS5 and 3′HS1 suggests correlation between histone modifications and chromatin looping.

Accumulation of β-amyloid protein (Aβ) is one of the most important pathological features of Alzheimer’s disease. Although Aβ induces neurodegeneration in the cortex and hippocampus through several molecular mechanisms, few studies have evaluated the modulation of transcription factors during Aβ-induced neurotoxicity. Therefore, in this study, we investigated the transcriptional activity of transcription factor CP2 in neuronal damage mediated by Aβ (Aβ1–42 and Aβ25–35). An unbiased motif search of the transferrin promoter region showed that CP2 binds to the transferrin promoter, an iron-regulating protein, and regulates transferrin transcription. Ectopic expression of CP2 led to increased transferrin expression at both the mRNA and protein levels, whereas knockdown of CP2 down-regulated transferrin mRNA and protein expression. Moreover, CP2 trans-activated transcription of a transferrin reporter gene. An electrophoretic mobility shift assay and a chromatin immunoprecipitation assay showed that CP2 binds to the transferrin promoter region. Furthermore, the binding affinity of CP2 to the transferrin promoter was regulated by Aβ, as Aβ (Aβ1–42 and Aβ25–35) markedly increased the binding affinity of CP2 for the transferrin promoter. Taken together, these results suggest that CP2 contributes to the pathogenesis of Alzheimer’s disease by inducing transferrin expression via up-regulating its transcription.

Gonocytes are long-lived primary germ cells that reside in the center of seminiferous cords until differentiation into spermatogonia that drive spermatogenesis. In pigs, gonocytes have research value in the production of transgenic offspring through germline modification and transplantation. However, the rarity of pig gonocytes has raised the need for an efficient isolation method. Therefore, in this study we use components of extracellular matrix, laminin, fibronectin, and collagen type IV and their derivative, gelatin, to establish a negative selection system for functionally viable gonocytes in neonatal pig. We then demonstrate functional analysis with genetic modification using lentiviral transduction and successfully transplant the donor gonocytes, which colonized the seminiferous tubules of the recipient mouse. The most effective selection method was established by sequential use of laminin and gelatin, in which the purity of gonocytes was 80% and the recovery rate of gonocytes was 78%. The selected gonocytes were labeled with fluorescent dye PKH26 and transplanted into busulfan-treated immunodeficient mouse testes. The fluorescent gonocytes colonized the recipient testes, and the resultant germ cell colonies were visible up to 4 mo after transplantation. When gonocytes were transplanted after transduction with an enhanced green fluorescent protein marker gene using lentiviral vectors, the transduced germ cell colonies were visible up to 6 mo and displayed an estimated transduction efficiency of 11.1%. These results can be applied and extended to isolate and enrich gonocytes of other species for in vitro and in vivo studies and to assist in genetic modification of male germline stem cells of livestock species.

Data presented here extends our previous observations on α-globin transcriptional regulation by the CP2 and PIAS1 proteins. Using RNAi knockdown, we have now shown that CP2b, CP2c and PIAS1 are each necessary for synergistic activation of endogenous α-globin gene expression in differentiating MEL cells. In this system, truncated PIAS1 mutants lacking the ring finger domain recruited CP2c to the nucleus, as did wild-type PIAS1, demonstrating that this is a sumoylation-independent process. In vitro, recombinant CP2c, CP2b and PIAS1 bound DNA as a stable CBP (CP2c/CP2b/PIAS1) complex. Following PIAS1 knockdown in MEL cells, however, the association of endogenous CP2c and CP2b with the α-globin promoter simultaneously decreased. By mapping the CP2b- and CP2c-binding domains on PIAS1, and the PIAS1-binding domains on CP2b and CP2c, we found that two regions of PIAS1 that interact with CP2c/CP2b are required for its co-activator function. We propose that CP2c, CP2b, and PIAS1 form a hexametric complex with two units each of CP2c, CP2b, and PIAS1, in which PIAS1 serves as a clamp between two CP2 proteins, while CP2c binds directly to the target DNA and CP2b mediates strong transactivation.

We examined the role of zebrafish ( Danio rerio ) Jak2a, a homolog of mammalian Jak2, in the developing embryo by injecting in vitro synthesized Jak2a shRNA into zebrafish zygotes. Blood circulation was suppressed in Jak2a shRNA-injected embryos from 24 hours post fertilization (hpf) and all embryos died with enlarged pericardium, shortened body lengths, and defects in some vasculature within 8 days post fertilization. O-dianisidine staining of red blood cells revealed normal blood island formation with no circulating red blood cells. As in Jak2 −/− transgenic mice, expression of definitive Ba1 globin was significantly reduced in Jak2a knockdown embryos at 36 hpf, whereas expression of other hematopoietic markers, primitive be1 globin, gata-1, and scl, were unaffected. More importantly, blood vessel formation was disturbed in Jak2a knockdown embryos as revealed by alkaline phosphatase staining at 72 hpf. Thus, our data indicate that zebrafish Jak2a is important in both definitive hematopoiesis and blood vessel formation.

GATA-1 is an erythroid activator that binds beta-globin gene promoters and DNase I hypersensitive sites (HSs) of the beta-globin locus control region (LCR). We investigated the direct role of GATA-1 interaction at the LCR HS2 enhancer by mutating its binding sites within minichromosomes in erythroid cells. Loss of GATA-1 in HS2 did not compromise interaction of NF-E2, a second activator that binds to HS2, nor was DNase I hypersensitivity at HS2 or the promoter of a linked epsilon-globin gene altered. Reduction of NF-E2 using RNAi confirmed the overall importance of this activator in establishing LCR HSs. However, recruitment of the histone acetyltransferase CBP and RNA pol II to HS2 was diminished by GATA-1 loss. Transcription of epsilon-globin was severely compromised with loss of RNA pol II from the transcription start site and reduction of H3 acetylation and H3K4 di- and tri-methylation in coding sequences. In contrast, widespread detection of H3K4 mono-methylation was unaffected by loss of GATA-1 in HS2. These results support the idea that GATA-1 interaction in HS2 has a prominent and direct role in co-activator and pol II recruitment conferring active histone tail modifications and transcription activation to a target gene but that it does not, by itself, play a major role in establishing DNase I hypersensitivity.

The origins of replication are associated with nuclear matrices or are found in close proximity to matrix attachment regions (MARs). In this report, fish MARs were cloned into an autonomously replicating sequence (ARS) cloning vector and were screened for ARS elements in Saccharomyces cerevisiae. Sixteen clones were isolated that were able to grow on the selective plates. In particular, an ARS905 that shows high efficiency among them was selected for this study. Southern hybridization indicated the autonomous replication of the transformation vector containing the ARS905 element. DNA sequences analysis showed that the ARS905 contained two ARS consensus sequences as well as MAR motifs, such as AT tracts, ORI patterns, and ATC tracts. In vitro matrix binding analysis, major matrix binding activity and ARS function coincided in a subfragment of the ARS905. To analyze the effects of ARS905 on expression of a reporter gene, an ARS905(E1158) with ARS activity was inserted into pBaEGFP(+) containing mud loach β-actin promoter, EGFP as a reporter gene, and SV40 poly(A) signal. The pBaEGFP(+)-ARS905(E1158) was transfected into a fish cell line, CHSE-214. The intensity of EGFP transfected cells was a 7-fold of the control at 11 days post-transfection. These results indicate that ARS905 enhances the expression of the EGFP gene and that it should be as a component of expression vectors in further fish biotechnological studies.

A transcription corepressor, MAT1-mediated transcriptional repressor (MMTR), was found in mouse embryonic stem cell lines. MMTR orthologs (DMAP1) are found in a wide variety of life forms from yeasts to humans. MMTR down-regulation in differentiating mouse embryonic stem cells in vitro resulted in activation of many unrelated genes, suggesting its role as a general transcriptional repressor. In luciferase reporter assays, the transcriptional repression activity resided at amino acids 221 to 468. Histone deacetylase 1 (HDAC1) interacts with MMTR both in vitro and in vivo and also interacts with MMTR in the nucleus. Interestingly, MMTR activity was only partially rescued by competition with dominant-negative HDAC1(H141A) or by treatment with an HDAC inhibitor, trichostatin A (TSA). To identify the protein responsible for HDAC1-independent MMTR activity, we performed a yeast two-hybrid screen with the full-length MMTR coding sequence as bait and found MAT1. MAT1 is an assembly/targeting factor for cyclin-dependent kinase-activating kinase which constitutes a subcomplex of TFIIH. The coiled-coil domain in the middle of MAT1 was confirmed to interact with the C-terminal half of MMTR, and the MMTR-mediated transcriptional repression activity was completely restored by MAT1 in the presence of TSA. Moreover, intact MMTR was required to inhibit phosphorylation of the C-terminal domain in the RNA polymerase II largest subunit by TFIIH kinase in vitro. Taken together, these data strongly suggest that MMTR is part of the basic cellular machinery for a wide range of transcriptional regulation via interaction with TFIIH and HDAC.

Although ubiquitously expressed, the transcriptional factor CP2 also exhibits some tissue- or stage-specific activation toward certain genes such as globin in red blood cells and interleukin-4 in T helper cells. Because this specificity may be achieved by interaction with other proteins, we screened a peptide display library and identified four consensus motifs in numerous CP2-binding peptides: HXPR, PHL, ASR and PXHXH. Protein-database searching revealed that RE-1 silencing factor (REST), Yin-Yang1 (YY1) and five other proteins have one or two of these CP2-binding motifs. Glutathione S-transferase pull-down and coimmunoprecipitation assays showed that two HXPR motif-containing proteins REST and YY1 indeed were able to bind CP2. Importantly, this binding to CP2 was almost abolished when a double amino acid substitution was made on the HXPR sequence of REST and YY1 proteins. The suppressing effect of YY1 on CP2's transcriptional activity was lost by this point mutation on the HXPR sequence of YY1 and reduced by an HXPR-containing peptide, further supporting the interaction between CP2 and YY1 via the HXPR sequence. Mapping the sites on CP2 for interaction with the four distinct CP2-binding motifs revealed at least three different regions on CP2. This suggests that CP2 recognizes several distinct binding motifs by virtue of employing different regions, thus being able to interact with and regulate many cellular partners.

Although basic fibroblast growth factor (FGF2) is generally included in the media for maintenance of human embryonic stem cells (hESCs), the action of FGF2 in these cells has not been well defined. Here, we determined the roles of FGF2 in maintaining hESC self-renewal. Withdrawal of FGF2 from the media led to acquisition of typical differentiated characteristics in hESCs. In the presence of FGF2, which is normally required for proliferation in an undifferentiated state, inhibition of phosphatidylinositol 3-kinase (PI3K)/Akt/PKB signal stimulated differentiation and attenuated the expression of extracellular matrix (ECM) molecules. We suggest that FGF2 maintains hESC self-renewal by supporting stable expression of ECM molecules through activation of the PI3K/Akt/PKB pathway.

To elucidate the function of PLCgamma1, we have investigated the proteins that bind to its SH (Src homology) domain. Immunoscreening was performed with purified antisera specific for SH223 (two SH2 and one SH3)-binding proteins. Several immunoreactive clones were identified as putative binding proteins and one of them was identified as synapsin IIb. We demonstrate a stable association between PLCgamma1 and synapsin IIb, which binds the carboxyl terminal SH2 and SH3 domains of the enzyme and inhibits it.

Previous reports have indicated that extracellular matrices (ECMs) affect the developmental fate of human embryonic stem cells (hESCs). Specially, type IV collagen and laminin, which belong to a group of macromolecular proteins with a substantial proportion of ECMs, are known to influence the proliferation and differentiation of hES cells. In this study, we evaluated the effects of type IV collagen and laminin in freezing medium on the survival and differentiation rates of hES cells after slow freezing and rapid thawing. The addition of type IV collagen (1 microg/ml) to the freezing medium significantly increased the survival rate of hES cells after thawing compared with that of a control group. The spontaneous differentiation rates of groups treated with type IV collagen (1 microg/ml) or laminin (1 microg/ml) were significantly lower than those of the control group. Frozen-thawed hES cells have currently been cultured for more than 70 passages and retain key properties of hES cells such as morphological characteristics, normal karyotype, marker expression (alkaline phosphatase, SSEA-1, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, Rex-1, and Oct-4), basement membrane-related gene expression, and the potential to differentiate into derivatives of all three germ layers. This new slow freezing method by ECM treatment is a reliable and effective cryopreservation method for pluripotent hES cells.

Adipose tissue is an important endocrine regulator of glucose metabolism and energy homeostasis. Researches have focused on this tissue not only as a target for pharmacotherapy of obesity and insulin resistance but also as an endocrine tissue with leptin secretion and high insulin sensitivity. Brown adipose tissue (BAT) additionally plays a unique role in thermoregulation through the mitochondrial uncoupling protein 1 (UCP1), which uncouples oxidative phosphorylation. As a genetic tissue ablation model of BAT, we made transgenic mice expressing herpes simplex virus thymidine kinase (HSV-TK) driven by the brown adipocyte- specific UCP1 minimal regulatory element. The HSV-TK transgene was expressed specifically in BAT and more than 35% increase of apoptosis was induced by ganciclovir (GCV) treatment. Nevertheless, the expression level was not high enough to induce BAT ablation in GCV-treated adult mice. Importantly, however, we found that brown adipocytes in the periphery of interscapular BAT were transformed into white adipocyte-like unilocular cells. These cells express white adipocyte-specific leptin protein but are different in the ultrastructure of mitochondria from classical white adipocytes. Our data indicates that atrophy of BAT causes transformation into white adipocyte-like cells in the adult mouse and also suggests that further molecular understanding of adipocyte plasticity using our transgenic mouse model might be beneficial for the development of anti-obesity/anti-diabetic therapies.

Human embryonic stem (hES) cells have been traditionally cultured on primary mouse embryonic fibroblasts (PMEFs). However, though STO cells have some advantages over PMEFs and human embryonic fibroblasts (hEFs) as feeder cells, they have never been used as feeder cells to establish hES cell lines. In this study, three hES cell lines (Miz-hES1, Miz-hES2, and Miz-hES3) were established from inner cell masses (ICM), using STO as feeder cells. The three hES cell lines had normal karyotypes and expressed high levels of alkaline phosphatase (AP), cell surface markers (SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81), and transcription factor Oct-4. After culture on STO cells for 2 yr, hES cells maintained the potential to form derivatives of all three embryonic germ layers. Our results show that STO feeder cells have the potential to support the establishment and maintenance of hES cell lines. In addition, our results suggest that laminin may play an important role in maintaining the undifferentiated proliferation of hES cells.

As a part of our effort to obtain a strong regulatory element for the construction of an autogenic mud loach transgenic vector, we isolated a genomic clone that contains an open reading frame encoding the beta-actin gene, then examined the transcriptional activity of the upstream sequences, including the first intron, in transiently transfected cell lines. It showed that the upstream region has substantially strong transcriptional activity, and that both the proximal promoter and distal region of intron 1 play a crucial role in the activity. A similar result, based on fish growth, was obtained with the expression vectors containing the growth hormone gene of mud loach. These were driven by the regulatory region of the mud loach beta-actin gene with various mutations, and were directly transferred into the trunk muscle of fish using an electrostimulation-mediated method. Fish weights were monitored over the next 4 weeks. These data suggest that the proximal promoter and the first intron enhancer of the mud loach beta-actin gene are useful for autogenic mud loach transgenesis.

Triploidy was induced in two selected lines of autotransgenic mud Ioach Misgurnus mizolepis containing no heterospecific genetic material. Cytological evaluations, including cellular DNA content, chromosome count, and erythrocyte measurement, proved the successful induction of triploidy. Patterns of Southern blot hybridization and tissue distribution of GH mRNA in autotransgenic triploids were similar to those of diploid autotransgenics. The autotransgenic triploids also displayed growth acceleration 22–25 times that of nontransgenic diploids, although the acceleration was relatively modest when compared to their diploid transgenic counterparts (more than a 30-fold increase). Significantly improved feed conversion was observed in both autotransgenic diploids and triploids, ranging from 1.8 to 2.0 times that in nontransgenics. Autotransgenic triploids were sterile at gonadal level, evidenced by significantly retarded and abnormal gametogenesis. With the persistence of accelerated growth and enhanced feed conversion efficiency, the autotransgenic triploid mud loach may offer economic benefits to aquaculture. Total absence of heterospecific genetic material and sterility may also allay public concern regarding food safety and environmental risk of crossbreeding between transgenics and conspecific populations.

Transgenic mud loaches (Misgurnus mizolepis), in which the entire transgene originated from the same species, have been generated by microinjecting the mud loach growth hormone (mlGH) gene fused to the mud loach beta-actin promoter. Out of 4,100 eggs injected, 7.5% fish derived from the injected eggs showed dramatically accelerated growth, with a maximum of 35-fold faster growth than their non-transgenic siblings. Many fast-growing transgenic individuals showed extraordinary gigantism: their body weight and total length (largest fish attained to 413 g and 41.5 cm) were larger and longer than even those of 12-year-old normal broodstock (maximum size reached to 89 g and 28 cm). Of 46 transgenic founders tested, 30 individuals transmitted the transgene to next generation with a wide range of germ-line transmission frequencies ranging from 2% to 33%. The growth performance of the subsequent generation (F1) was also dramatically accelerated up to 35-fold, although the levels of enhanced growth were variable among transgenic lines. Three transgenic germ-lines up to F4 were established, showing the expected Mendelian inheritance of the transgene. Expression of GH mRNA in many tissues was detected by RT-PCR analyses. The time required to attain marketable size (10 g) in these transgenic lines was only 30-50 days after fertilization, while at least 6 months in non-transgenic fish. Besides growth enhancement, significantly improved feed-conversion efficiency up to 1.9-fold was also observed.

BACKGROUND The p16INK4A gene encodes 2 cell cycle regulator proteins, p16 and p14ARF, by alternative splicing. This genetic locus also contains another cell cycle regulator gene, p15INK4B, which encodes p15. The inactivation of the p16 protein has been demonstrated in some hepatocellular carcinomas (HCCs); however, the inactivation of the other 2 cell regulator proteins and their inactivation patterns are not well characterized. METHODS To characterize the role of the above 3 cell cycle regulator proteins in HCCs, the authors examined the genomic status of the p16INK4A and p15INK4B genes and their RNA products in 20 HCC tissues and 7 human HCC cell lines. Homozygous deletions in each exon of p16INK4A and p15INK4B were evaluated by comparative multiplex polymerase chain reaction (PCR), and the methylation status of the p16INK4A and p15INK4B promoter region was analyzed by methylation specific PCR. RESULTS Homozygous deletions were found in 6 of 20 HCCs (30%) and 2 of 7 HCC cell lines (29%). In 20 HCCs, the frequency of homozygous deletions was 20% in exon 1 of p15INK4B, 20% in exon 2 of p15INK4B, 10% in exon 1β of p16INK4A, 25% in exon 1α of p16INK4A, 15% in exon 2 of p16INK4A, and 15% in exon 3 of p16INK4A. The authors found hypermethylation of the p16INK4A promoter region in 7 HCCs (35%) and 3 HCC cell lines (43%). The overall frequency of p16 alterations in HCCs, including hypermethylation and homozygous deletions, was 60% (12 of 20 cases). According to reverse transcriptase–PCR analysis, the absence of RNA expression was most frequent in p16 (11 of 20 cases, 55%) and less frequent in p15 (7 of 20 cases, 35%) and p14ARF (5 of 20 cases, 25%). CONCLUSIONS Among the 3 cell cycle regulator proteins encoded at the 9p21 genetic locus, inactivation of p16 is the most frequent event in HCCs in which promoter hypermethylation and homozygous deletions are the common mechanisms. Cancer 2000;89:60–8. © 2000 American Cancer Society.