Heterologous Cell Types Research Articles

A family business: stem cell progeny join the niche to regulate homeostasis

Stem cell niches, the discrete microenvironments in which the stem cells reside, play a dominant part in regulating stem cell activity and behaviours. Recent studies suggest that committed stem cell progeny become indispensable components of the niche in a wide range of stem cell systems. These unexpected niche inhabitants provide versatile feedback signals to their stem cell parents. Together with other heterologous cell types that constitute the niche, they contribute to the dynamics of the microenvironment. As progeny are often located in close proximity to stem cell niches, similar feedback regulations may be the underlying principles shared by different stem cell systems.

Real-Time RT-PCR Analysis of Individual Osteolineage Cells within the Hematopoietic Stem Cell Niche

Abstract 2389Osteolineage cells (OLCs) have been shown to participate in a regulatory bone marrow microenvironment for the hematopoietic stem and progenitor cells (HSPCs) – the endosteal niche. Our previous experiments using live animal imaging have demonstrated that single transplanted HSPCs preferentially home in close proximity to the individual OLCs. We hypothesized that these HSPC-proximal cells represent a distinct subpopulation of OLCs, which is specifically involved in a non-cell autonomous regulation of HSPC quiescence and self-renewal.To test this hypothesis, we developed a novel experimental platform, which allows visualization of HSPC-OLC cell pairs in-vivo and retrieval of the individual OLCs for molecular analysis. We intravenously injected DiI labeled adult bone marrow-derived FACS-sorted Lin−Sca1+c-kit+CD34−Flk2− HSPCs into irradiated newborn collagen 2.3GFP mouse recipients; in this transgenic strain, the majority of the OLCs are labeled with green fluorescent protein (GFP). 48 hours later, we sacrificed the animals and obtained fresh unfixed sections of femoral trabecular bone. Using a combination of differential interference contrast fluorescent microscopy, in-situ enzymatic digestion and micromanipulation, we harvested individual GFP-positive OLCs located within 2 cell diameters (“niche” OLCs) or greater than 5 cell diameters (“control” OLCs) from single DiI-bright HSPCs.Following reverse transcription and cDNA amplification with 29 cycles of PCR, as per the single cell RNA-Seq protocol (Tang et al, Nature Protocols 2010), we performed real-time RT-PCR analysis of 31 samples – 15 niche cells and 16 controls - for the OLC signature genes (osteocalcin, osterix) and for the genes implicated in playing a functional role in the HSPC-OLC cell interaction (osteopontin, CXCL12, angiopoietin 1). Transcripts for GAPDH, collagen 1 and GFP served as positive controls for the amplification.As expected, all cells were positive for GFP and over 85% cells expressed collagen 1. Osteopontin and CXCL12 were expressed at a similar level and frequency in the niche and control OLCs. However, we found that angiopoietin 1 transcripts were detected exclusively in the niche OLCs (3/15 versus 0/16, p <0.05 by Chi-squared). Moreover, niche OLCs were enriched for the osterix-positive cells (7/15 versus 2/16, p <0.05 by Chi-squared) and expressed a lower level of osteocalcin, as normalized for GAPDH expression (1.13 vs. 0.97, p< 0.05 by t-test).Our results suggest that niche OLCs may have a distinct molecular signature and reside within a population of very immature OLCs, as evidenced by the osterix + osteocalcin low phenotype. Further unbiased transcriptome characterization of these cells using genome-wide RNA-Seq assay is therefore likely to provide more evidence in support of our hypothesis and reveal novel non-cell autonomous regulators of HSPC quiescence. To our knowledge, this approach represents the first attempt to define molecular heterogeneity in-vivo at a single cell level using the micro-anatomical relationship between two heterologous cell types. Disclosures:Scadden:Fate Therapeutics: Equity Ownership.

Piecing together the pigment-type switching puzzle

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TRPA1 and Cold Transduction: An Unresolved Issue?

TRPA1 was originally characterized as a cold-activated ion channel; however, since the initial description there has been some disagreement about its role in cold sensation. Here, we review the literature on the function of TRPA1 as a cold sensor. Many studies have demonstrated that TRPA1 can be activated by cold when expressed in heterologous systems, but its role as a cold sensor in native peripheral sensory neurons remains uncertain. We argue that TRPA1 is activated indirectly by cold via a background Ca2+ influx seen in cells during cooling. This background cold response is present in most heterologous cell types, but not in sensory neurons. Thus, we propose that cold sensitivity of TRPA1 is indirect and is dependent on cellular context.

Second and third generation voltage-sensitive fluorescent proteins for monitoring membrane potential

Over the last decade, optical neuroimaging methods have been enriched by engineered biosensors derived from fluorescent protein (FP) reporters fused to protein detectors that convert physiological signals into changes of intrinsic FP fluorescence. These FP-based indicators are genetically encoded, and hence targetable to specific cell populations within networks of heterologous cell types. Among this class of biosensors, the development of optical probes for membrane potential is both highly desirable and challenging. A suitable FP voltage sensor would indeed be a valuable tool for monitoring the activity of thousands of individual neurons simultaneously in a non-invasive manner. Previous prototypic genetically-encoded FP voltage indicators achieved a proof of principle but also highlighted several difficulties such as poor cell surface targeting and slow kinetics. Recently, we developed a new series of FRET-based Voltage-Sensitive Fluorescent Proteins (VSFPs), referred to as VSFP2s, with efficient targeting to the plasma membrane and high responsiveness to membrane potential signaling in excitable cells. In addition to these FRET-based voltage sensors, we also generated a third series of probes consisting of single FPs with response kinetics suitable for the optical imaging of fast neuronal signals. These newly available genetically-encoded reporters for membrane potential will be instrumental for future experimental approaches directed toward the understanding of neuronal network dynamics and information processing in the brain. Here, we review the development and current status of these novel fluorescent probes.

Metabolic Acidosis Increases Intracellular Calcium in Bone Cells Through Activation of the Proton Receptor OGR1

Metabolic acidosis increases urine Ca without increasing intestinal absorption, leading to bone Ca loss. It is unclear how bone cells detect the increase in proton concentration. To determine which G protein-coupled proton sensing receptors are expressed in bone, PCR was performed, and products were detected for OGR1, TDAG8, G2A, and GPR4. We tested the hypothesis that the G protein-coupled proton sensor, OGR1, is an H+-sensing receptor in bone. To determine whether acid-induced bone resorption involves OGR1, we incubated mouse calvariae in neutral pH (NTL) or acidic (MET) medium ± the OGR1 inhibitor CuCl2. CuCl2 decreased MET-induced Ca efflux. We used fluorescent imaging of perfused bone cells to determine whether MET increases Cai. Perfusion with MET induced a rapid, flow-independent, increase in Cai in individual bone cells. To determine whether transfection of OGR1 into a heterologous cell type would increase Cai in response to H+, we perfused Chinese hamster ovary (CHO) cells transfected with mouse OGR1 cDNA. Perfusion with MET induced a rapid increase in Cai in OGR1-transfected CHO cells. These data indicate that OGR1 induces an increase in Cai in response to MET and is a prime candidate for an osteoblast proton sensor.

Microfibril-associate glycoprotein-2 (MAGP-2) promotes angiogenic cell sprouting by blocking notch signaling in endothelial cells

Angiogenesis is highly sensitive to the composition of the vascular microenvironment, however, our understanding of the structural and matricellular components of the vascular microenvironment that regulate angiogenesis and the molecular mechanisms by which these molecules function remains incomplete. Our previous results described a novel pro-angiogenic activity for Microfibril-Associated Glycoprotein-2 (MAGP-2), but did not address the molecular mechanism(s) by which this is accomplished. We now demonstrate that MAGP-2 promotes angiogenic cell sprouting by antagonizing Notch signaling pathways in endothelial cells. MAGP-2 decreased basal and Jagged1 induced expression from the Notch sensitive Hes-1 promoter in ECs, and blocked Jagged1 stimulated Notch1 receptor processing in transiently transfected 293T cells. Interestingly, inhibition of Notch signaling by MAGP-2 seems to be restricted to ECs since MAGP-2 increased Hes-1 promoter activity and Notch1 receptor processing in heterologous cell types. Importantly, constitutive activation of the Notch signaling pathway blocked the ability of MAGP-2 to promote angiogenic cell sprouting, as well as morphological changes associated with angiogenesis. Collectively, these observations indicate that MAGP-2 promotes angiogenic cell spouting in vitro by antagonizing Notch signaling pathways in ECs.

Demonstration of Multipotent Stem Cells in the Adult Human Endometrium by In Vitro Chondrogenesis

Stem cells are defined by their unique capacity for self-renewal and multilineage differentiation. Stem cells have been obtained from multiple extramedullary tissues. Recently, a population of progenitor cells have been identified in the endometrium. However, multilineage differentiation of endometrial stem cells has not been reported. Endometrial tissue was obtained from reproductive-aged women undergoing surgery for benign disease, from which monolayer endometrial stromal cell (ESC), myometrial, fibroid, fallopian tube, and uterosacral ligament tissue cultures were generated. Once confluent, cells were trypsinized and centrifuged in conical tubes to form a cell pellet. Cell pellets were cultured in a defined chondrogenic media (CM) containing dexamethasone and transforming growth factor (TGF)-beta2 or TGF-beta 3 for 3 to 21 days. Samples were analyzed for markers of human articular cartilage, including sulfated glycosaminoglycans and expression of type II collagen. ESC pellets cultured in CM were found to contain cells that resemble chondrocytes. These cells expressed sulfated glycosaminoglycans and type II collagen typical of human articular cartilage. Myometrial, fibroid, fallopian tube, and uterosacral ligament cells were unable to undergo chondrogenic differentiation using the pellet culture method. Cells derived from the endometrium were able to differentiate into a heterologous cell type: chondrocytes, thus demonstrating the presence of multipotent stem cells. Endometrium is a potential source of multipotent stem cells.

A Sertoli Cell-Specific Knockout of Connexin43 Prevents Initiation of Spermatogenesis

The predominant testicular gap junctional protein connexin43 (cx43) is located between neighboring Sertoli cells (SCs) and between SCs and germ cells. It is assumed to be involved in testicular development, cell differentiation, initiation, and maintenance of spermatogenesis with alterations of its expression being correlated with various testicular disorders. Because total disruption of the cx43 gene leads to perinatal death, we generated a conditional cx43 knockout (KO) mouse using the Cre/loxP recombination system, which lacks the cx43 gene solely in SCs (SCCx43KO), to evaluate the SC-specific functions of cx43 on spermatogenesis in vivo. Adult SCCx43KO(-/-) mice showed normal testis descent and development of the urogenital tract, but testis size and weight were drastically lower compared with heterozygous and wild-type littermates. Histological analysis and quantitation of mRNA expression of germ cell-specific marker genes revealed a significant reduction in the number of spermatogonia but increased SC numbers/tubule with only a few tubules left showing normal spermatogenesis. Thus, SC-specific deletion of cx43 mostly resulted in an arrest of spermatogenesis at the level of spermatogonia or SC-only syndrome and in intratubular SC clusters. Our data demonstrate for the first time that cx43 expression in SCs is an absolute requirement for normal testicular development and spermatogenesis.

Immune response to stem cells and strategies to induce tolerance

Although recent progress in cardiovascular tissue engineering has generated great expectations for the exploitation of stem cells to restore cardiac form and function, the prospects of a common mass-produced cell resource for clinically viable engineered tissues and organs remain problematic. The refinement of stem cell culture protocols to increase induction of the cardiomyocyte phenotype and the assembly of transplantable vascularized tissue are areas of intense current research, but the problem of immune rejection of heterologous cell type poses perhaps the most significant hurdle to overcome. This article focuses on the potential advantages and problems encountered with various stem cell sources for reconstruction of the damaged or failing myocardium or heart valves and also discusses the need for integrating advances in developmental and stem cell biology, immunology and tissue engineering to achieve the full potential of cardiac tissue engineering. The ultimate goal is to produce 'off-the-shelf' cells and tissues capable of inducing specific immune tolerance.

The Transduction Channel TRPM5 Is Gated by Intracellular Calcium in Taste Cells

Bitter, sweet, and umami tastants are detected by G-protein-coupled receptors that signal through a common second-messenger cascade involving gustducin, phospholipase C beta2, and the transient receptor potential M5 (TRPM5) ion channel. The mechanism by which phosphoinositide signaling activates TRPM5 has been studied in heterologous cell types with contradictory results. To resolve this issue and understand the role of TRPM5 in taste signaling, we took advantage of mice in which the TRPM5 promoter drives expression of green fluorescent protein and mice that carry a targeted deletion of the TRPM5 gene to unequivocally identify TRPM5-dependent currents in taste receptor cells. Our results show that brief elevation of intracellular inositol trisphosphate or Ca2+ is sufficient to gate TRPM5-dependent currents in intact taste cells, but only intracellular Ca2+ is able to activate TRPM5-dependent currents in excised patches. Detailed study in excised patches showed that TRPM5 forms a nonselective cation channel that is half-activated by 8 microM Ca2+ and that desensitizes in response to prolonged exposure to intracellular Ca2+. In addition to channels encoded by the TRPM5 gene, we found that taste cells have a second type of Ca2+-activated nonselective cation channel that is less sensitive to intracellular Ca2+. These data constrain proposed models for taste transduction and suggest a link between receptor signaling and membrane potential in taste cells.

Extracellular Acid Block and Acid-enhanced Inactivation of the Ca2+-activated Cation Channel TRPM5 Involve Residues in the S3-S4 and S5-S6 Extracellular Domains

TRPM5, a member of the superfamily of transient receptor potential ion channels, is essential for the detection of bitter, sweet, and amino acid tastes. In heterologous cell types it forms a nonselective cation channel that is activated by intracellular Ca(2+). TRPM5 is likely to be part of the taste transduction cascade, and regulators of TRPM5 are likely to affect taste sensation. In this report we show that TRPM5, but not the related channel TRPM4b, is potently blocked by extracellular acidification. External acidification has two effects, a fast reversible block of the current (IC(50) pH = 6.2) and a slower irreversible enhancement of current inactivation. Mutation of a single Glu residue in the S3-S4 linker and a His residue in the pore region each reduced sensitivity of TRPM5 currents to fast acid block (IC(50) pH = 5.8 for both), and the double mutant was nearly insensitive to acidic pH (IC(50) pH = 5.0). Prolonged exposure to acidic pH enhanced inactivation of TRPM5 currents, and mutant channels that were less sensitive to acid block were also less sensitive to acid-enhanced inactivation, suggesting an intimate association between the two processes. These processes are, however, distinct because the pore mutant H896N, which has normal sensitivity to acid block, shows significant recovery from acid-enhanced inactivation. These data show that extracellular acidification acts through specific residues on TRPM5 to block conduction through two distinct but related mechanisms and suggest a possible interaction between extracellular pH and activation and adaptation of bitter, sweet, and amino acid taste transduction.

The Minor Capsid Protein L2 Contributes to Two Steps in the Human Papillomavirus Type 31 Life Cycle

Prior studies, which have relied upon the use of pseudovirions generated in heterologous cell types, have led to sometimes conflicting conclusions regarding the role of the minor capsid protein of papillomaviruses, L2, in the viral life cycle. In this study we carry out analyses with true virus particles assembled in the natural host cell to assess L2's role in the viral infectious life cycle. For these studies we used the organotypic (raft) culture system to recapitulate the full viral life cycle of the high-risk human papillomavirus HPV31, which was either wild type or mutant for L2. After transfection, the L2 mutant HPV31 genome was able to establish itself as a nuclear plasmid in proliferating populations of poorly differentiated (basal-like) human keratinocytes and to amplify its genome to high copy number, support late viral gene expression, and cause formation of virus particles in human keratinocytes that had been induced to undergo terminal differentiation. These results indicate that aspects of both the nonproductive and productive phases of the viral life cycle occur normally in the absence of functional L2. However, upon the analysis of the virus particles generated, we found an approximate 10-fold reduction in the amount of viral DNA encapsidated into L2-deficient virions. Furthermore, there was an over-100-fold reduction in the infectivity of L2-deficient virus. Because the latter deficiency cannot be accounted for solely by the 10-fold decrease in encapsidation, we conclude that L2 contributes to at least two steps in the production of infectious virus.

Interaction of maize Opaque-2 and the transcriptional co-activators GCN5 and ADA2, in the modulation of transcriptional activity

Maize Opaque-2 (ZmO2), a bZip class transcription factor has been shown to activate the transcription of a series of genes expressed in the maturation phase of endosperm development. Activation requires the presence of one or more enhancer binding sites, which confer the propensity for activation by ZmO2 on heterologous promoters and in heterologous plant cell types, such as tobacco mesophyll protoplasts. The region of ZmO2 required for conferring transcriptional activation has been localised to a stretch of acidic residues in the N-terminal portion of the ZmO2 sequence, which is conserved between O2-related bZip factor sequences. Previously we identified the maize homologues of yeast transcriptional co-activators GCN5 and ADA2 that are implicated in nucleosome modification and transcription. In the present study we have shown that transcriptional modulation by ZmO2 involves the intranuclear interaction of ZmO2 with ZmADA2 and ZmGCN5. Förster resonance energy transfer (FRET) based techniques have enabled us to estimate the intracellular site of these intermolecular interactions. As a functional readout of these intranuclear interactions, we used the ZmO2 responsive maize b-32 promoter to drive the beta-glucuronidase (GUS) in the presence and absence of ZmGCN5 and ZmADA2. Our results suggest that the likely recruitment of ZmADA2 and ZmGCN5 modulates the transactivation of b-32 promoter by ZmO2 and that there may be a competition between ZmGCN5 and ZmO2 for binding to the amino-terminal of ZmADA2. The results may be taken as a paradigm for other processes of transcriptional modulation in planta involving acidic activation domains.

Endothelial cell signaling during conducted vasomotor responses.

ACh and KCl stimulate vasomotor responses that spread rapidly and bidirectionally along arteriole walls, most likely via spread of electric current or Ca2+ through gap junctions. We examined these possibilities with isolated, cannulated, and perfused hamster cheek pouch arterioles (50- to 80-microm resting diameter). After intraluminal loading of 2 microM fluo 3 to measure Ca2+ or 1 microM di-8-ANEPPS to measure membrane potential, photometric techniques were used to selectively measure changes in intracellular Ca2+ concentration ([Ca2+]i) or membrane potential in endothelial cells. Activation of the endothelium by micropipette application of ACh (10-4 M, 1.0-s pulse) to a short segment of arteriole (100-200 microm) increased endothelial cell [Ca2+]i and caused hyperpolarization at the site of stimulation. This response was followed rapidly by vasodilation of the entire arteriole ( approximately 2-mm length). Change in membrane potential always preceded dilation, both at the site of stimulation and at distant sites along the arteriole. In contrast, an increase in endothelial cell [Ca2+]i was observed only at the application site. Micropipette application of KCl, which can depolarize both smooth muscle and endothelial cells (250 mM, 2.5-s pulse), also caused a rapid, spreading response consisting of depolarization followed by vasoconstriction. With KCl stimulation, in addition to changes in membrane potential, increases in endothelial cell [Ca2+]i were observed at distant sites not directly exposed to KCl. The rapid longitudinal spread of both hyperpolarizing and depolarizing responses support electrical coupling as the mode of signal transmission along the arteriolar length. In addition, the relatively short distance between heterologous cell types enables the superimposed radial Ca2+ signaling between smooth muscle and endothelial cells to modulate vasomotor responses.

Control of human immunodeficiency virus type-1 protease activity in insect cells expressing Gag-Pol rescues assembly of immature but not mature virus-like particles

Expression of human immunodeficiency virus type 1 (HIV-1) Gag protein in insect cells using baculovirus vectors leads to the abundant production of virus-like particles (VLPs) that represent the immature form of the virus. When Gag-Pol is included, however, VLP production is abolished, a result attributed to premature protease activation degrading the intracellular pool of Gag precursor before particle assembly can occur. As large-scale synthesis of mature noninfectious VLPs would be useful, we have sought to control HIV protease activity in insect cells to give a balance of Gag and Gag-Pol that is compatible with mature particle formation. We show here that intermediate levels of protease activity in insect cells can be attained through site-directed mutagenesis of the protease and through antiprotease drug treatment. However, despite Gag cleavage patterns that mimicked those seen in mammalian cells, VLP synthesis exhibited an essentially all-or-none response in which VLP synthesis occurred but was immature or failed completely. Our data are consistent with a requirement for specific cellular factors in addition to the correct ratio of Gag and Gag-Pol for assembly of mature retrovirus particles in heterologous cell types.

The use of synthetic genes for the expression of ciliate proteins in heterologous systems

The common fish parasite, Ichthyophthirius multifiliis, expresses abundant glycosylated phosphatidylinositol (GPI)-anchored membrane proteins known as immobilization antigens, or i-antigens. These proteins are targets of the host immune response, and have been identified as potential candidates for recombinant subunit vaccine development. Nevertheless, because Ichthyophthirius utilizes a non-standard genetic code, expression of the corresponding gene products, either as subunit antigens in conventional protein expression systems, or as vector-encoded antigens in the case of DNA vaccines, is far from straightforward. To overcome this problem, we utilized ‘assembly polymerase chain reaction’ to manufacture synthetic versions of two genes (designated IAG52A[G5/CC] and IAG52B[G5/CC]) encoding ∼52/55 kDa i-antigens from parasite strain G5. This approach made it possible to eliminate unwanted stop codons and substitute the preferred codon usage of channel catfish for the native sequences of the genes. To determine whether the synthetic alleles could be expressed in cells that use the standard genetic code, we introduced IAG52A[G5/CC] into a variety of heterologous cell types and tested for expression either by immunofluorescence light microscopy or Western blotting. When cloned downstream of appropriate promoters, IAG52A[G5/CC] was expressed in Escherichia coli, mammalian COS-7 cells, and channel catfish where it elicited antigen-specific immune responses. Interestingly, the localization pattern of the corresponding gene product in COS-7 cells indicated that while the protein was correctly folded, it was not present on the cell membrane, suggesting that the signal peptides required for GPI-anchor addition differ in ciliate and mammalian systems. Construction of synthetic alleles should have practical utility in the development of vaccines against Ichthyophthirius, and at the same time, provide a general method for the expression of ciliate genes in heterologous systems.

The bystander effect mediated by the new murine gammaherpesvirus 72--thymidine kinase/5'-fluoro-2'-deoxyuridine (MHV72-TK/5-FUdR) system in vitro.

To investigate the potential of murine gamma-herpesvirus 72 thymidine kinase (MHV-72-TK) to act as a suicide gene, we used a mammalian expression vector on rat fibroblastoid cells deficient in the cellular TK gene. Substrate specificity was assessed in vitro in cells with stable expression of MHV-72-TK. The Herpes simplex virus 1-TK (HSV-1-TK) was used as a reference suicide gene. Unlike HSV-1-TK modified cells, which were sensitive to ganciclovir (GCV) (IC50=9.7 microM), cells modified by MHV-72-TK did not show sensitivity to this drug. The use of 3'-azido-3'-deoxythymidine (AZT) and (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) did not affect the growth of cells expressing either MHV-72-TK or HSV-1-TK in the range of concentration used for AZT (0-375 microM) and for BVDU (0-50 microM). In contrast, 5'-fluoro-2'-deoxyuridine (5-FUdR) was extremely cytotoxic and effectively killed MHV-72-TK expressing cells (IC50 value 2.1 microM). This value was 16 times lower than that required to kill cells expressing HSV1-TK. To test whether the bystander effect between two heterologous cell types could be mediated by the MHV-72-TK/5-FUdR system in vitro, cells expressing MHV-72-TK were co-cultured with the tumour fibroblastoid cell line NAD for 48 hours before the drug (10.8 microM) was added. The cell mixtures contained various ratios of cells expressing MHV-72-TK (0 to 50% of total cells). Only 1% of MHV-72-TK-expressing cells were needed to enhance mouse tumour cell killing and to decrease the survival rate to 25.6%. The bystander effect was more pronounced when 10% of cells expressing MHV-72-TK were used, decreasing survival to 17.4%. In parallel, the same concentration of 5-FUdR dose only marginally inhibited tumour cell growth in the absence of exogenous TK activity (84% survival). These results demonstrate the efficiency of MHV-72-TK as a suicide gene when 5-FUdR is used as a prodrug. When sequenced, MHV-72-TK proved to be identical to MHV-68 strain TK.

The pathology of nephroblastoma

This article summarises the approach to specimen processing, reviews the pathological features and briefly discusses the clinical features and genetics of nephroblastomas. The pathological assessment provides essential information necessary for determining further management, therefore great care should be taken to optimise processing and prevent artefacts. The classical nephroblastoma is triphasic, consisting of blastema, epithelium and stroma. However, monophasic and biphasic forms are not uncommon. Heterologous cell types and varying degrees of cellular differentiation are frequent findings which sometimes contribute to diagnostic difficulties. Cystic partially differentiated nephroblastoma has specific pathological and behavioural characteristics and therefore must be distinguished from nephroblastomas with cystic areas. The presence of anaplasia must be further qualified as either focal or diffuse because diffuse anaplasia is an indicator of adverse prognosis. Although nephroblastomas may be regarded as one of the successes in oncology, there is still much knowledge that remains to be gained.

Identification of a portable repression domain and an E1A-responsive activation domain in Pax4: a possible role of Pax4 as a transcriptional repressor in the pancreas.

Pax4 is a paired-domain (PD)-containing transcription factor which plays a crucial role in pancreatic beta/delta-cell development. In this study, we characterized the DNA-binding and transactivation properties of mouse Pax4. Repetitive rounds of PCR-based selection led to identification of the optimal DNA-binding sequences for the PD of Pax4. In agreement with the conservation of the optimal binding sequences among the Pax family transcription factors, Pax4 could bind to the potential binding sites for Pax6, another member of the Pax family also involved in endocrine pancreas development. The overexpression of Pax4 in HIT-T15 cells dose dependently inhibited the basal transcriptional activity as well as Pax6-induced activity. Detailed domain mapping analyses using GAL4-Pax4 chimeras revealed that the C-terminal region of Pax4 contains both activation and repression domains. The activation domain was active in the embryonic kidney-derived 293/293T cells and embryonal carcinoma-derived F9 cells, containing adenoviral E1A protein or E1A-like activity, respectively but was inactive or very weakly active in other cells including those of pancreatic beta- and alpha-cell origin. Indeed, the exogenous overexpression of type 13S E1A in heterologous cell types could convert the activation domain to an active one. On the other hand, the repression domain was active regardless of the cell type. When the repression domain was linked to the transactivation domain of a heterologous transcription factor, PDX-1, it could completely abolish the transactivation potential of PDX-1. These observations suggest a primary role of Pax4 as a transcriptional repressor whose function may involve the competitive inhibition of Pax6 function. The identification of the E1A-responsive transactivation domain, however, indicates that the function of Pax4 is subject to posttranslational regulation, providing further support for the complexity of mechanisms that regulate pancreas development.