SIPA1L1/SPAR1 Interacts with the Neurabin Family of Proteins and is Involved in GPCR Signaling.

Signal-induced proliferation-associated 1 (SIPA1)-like 1 (SIPA1L1; also known as SPAR1) has been proposed to regulate synaptic functions that are important in maintaining normal neuronal activities, such as regulating spine growth and synaptic scaling, as a component of the PSD-95/NMDA-R-complex. However, its physiological role remains poorly understood. Here, we performed expression analyses using super-resolution microscopy (SRM) in mouse brain and demonstrated that SIPA1L1 is mainly localized to general submembranous regions in neurons, but surprisingly, not to PSD. Our screening for physiological interactors of SIPA1L1 in mouse brain identified spinophilin and neurabin-1, regulators of G-protein-coupled receptor (GPCR) signaling, but rejected PSD-95/NMDA-R-complex components. Furthermore, Sipa1l1-/- mice showed normal spine size distribution and NMDA-R-dependent synaptic plasticity. Nevertheless, Sipa1l1-/- mice showed aberrant responses to α2-adrenergic receptor (a spinophilin target) or adenosine A1 receptor (a neurabin-1 target) agonist stimulation, and striking behavioral anomalies, such as hyperactivity, enhanced anxiety, learning impairments, social interaction deficits, and enhanced epileptic seizure susceptibility. Male mice were used for all experiments. Our findings revealed unexpected properties of SIPA1L1, suggesting a possible association of SIPA1L1 deficiency with neuropsychiatric disorders related to dysregulated GPCR signaling, such as epilepsy, attention deficit hyperactivity disorder (ADHD), autism, or fragile X syndrome (FXS).SIGNIFICANCE STATEMENT Signal-induced proliferation-associated 1 (SIPA1)-like 1 (SIPA1L1) is thought to regulate essential synaptic functions as a component of the PSD-95/NMDA-R-complex. In our screening for physiological SIPA1L1-interactors, we identified G-protein-coupled receptor (GPCR)-signaling regulators. Moreover, SIPA1L1 knock-out (KO) mice showed striking behavioral anomalies, which may be relevant to GPCR signaling. Our findings revealed an unexpected role of SIPA1L1, which may open new avenues for research on neuropsychiatric disorders that involve dysregulated GPCR signaling. Another important aspect of this paper is that we showed effective methods for checking PSD association and identifying native protein interactors that are difficult to solubilize. These results may serve as a caution for future claims about interacting proteins and PSD proteins, which could eventually save time and resources for researchers and avoid confusion in the field.

Mutational inactivation of Apc in the intestinal epithelia compromises cellular organisation.

The adenomatous polyposis coli (Apc) protein regulates diverse effector pathways essential for tissue homeostasis. Truncating oncogenic mutations in Apc removing its Wnt pathway and microtubule regulatory domains drives intestinal epithelia tumorigenesis. Exuberant cell proliferation is one well-established consequence of oncogenic Wnt pathway activity; however, the contribution of other deregulated molecular circuits to tumorigenesis has not been fully examined. Using in vivo and organoid models of intestinal epithelial tumorigenesis we found that Wnt pathway activity controls intestinal epithelial villi and crypt structure, morphological features lost upon Apc inactivation. Although the Wnt pathway target gene c-Myc (also known as Myc) has critical roles in regulating cell proliferation and tumorigenesis, Apc specification of intestinal epithelial morphology is independent of the Wnt-responsive Myc-335 (also known as Rr21) regulatory element. We further demonstrate that Apc inactivation disrupts the microtubule cytoskeleton and consequently localisation of organelles without affecting the distribution of the actin cytoskeleton and associated components. Our data indicates the direct control over microtubule dynamics by Apc through an independent molecular circuit. Our study stratifies three independent Apc effector pathways in the intestinal epithelial controlling: (1) proliferation, (2) microtubule dynamics and (3) epithelial morphology.This article has an associated First Person interview with the first author of the paper.

Altered microbiota caused by disordered gut motility leads to an overactivation of intestinal immune system in APC1638T mice.

Adenomatous polyposis coli (APC) is recognized as an antioncogene related to familial adenomatous polyposis and colorectal cancers. However, APC is a large protein with multiple binding partners, indicating APC has diverse roles besides as a tumor suppressor. We have ever studied the roles of APC by using APC1638T/1638T (APC1638T) mice. Through those studies, we have noticed stools of APC1638T mice were smaller than those of APC+/+ mice and hypothesized there be a disturbance in fecal formation processes in APC1638T mice. The gut motility was morphologically analyzed by immunohistochemical staining of the Auerbach's plexus. Gut microbiota was analyzed by terminal restriction fragment length polymorphism (T-RFLP). IgA concentration in stools was determined by enzyme-linked immunosorbent assay (ELISA). As results, macroscopic findings suggestive of large intestinal dysmotility and microscopic findings of disorganization and inflammation of the plexus were obtained in APC1638T mice. An alteration of microbiota composition, especially increased Bacteroidetes population was observed. Increases in IgA positive cells and dendritic cells in the ileum with high fecal IgA concentration were also confirmed, suggesting over-activation of gut immunity. Our findings will contribute to our understanding of APC's functions in the gastrointestinal motility, and lead to a development of novel therapies for gut dysmotility-related diseases.

Decreased Podocyte Vesicle Transcytosis and Albuminuria in APC C-Terminal Deficiency Mice with Puromycin-Induced Nephrotic Syndrome.

In minimal change nephrotic syndrome, podocyte vesicle transport is enhanced. Adenomatous polyposis coli (APC) anchors microtubules to cell membranes and plays an important role in vesicle transport. To clarify the role of APC in vesicle transport in podocytes, nephrotic syndrome was induced by puromycin amino nucleoside (PAN) injection in mice expressing APC1638T lacking the C-terminal of microtubule-binding site (APC1638T mouse); this was examined in renal tissue changes. The kidney size and glomerular area of APC1638T mice were reduced (p = 0.014); however, the number of podocytes was same between wild-type (WT) mice and APC1638T mice. The ultrastructure of podocyte foot process was normal by electron microscopy. When nephrotic syndrome was induced, the kidneys of WT+PAN mice became swollen with many hyaline casts, whereas these changes were inhibited in the kidneys of APC1638T+PAN mice. Electron microscopy showed foot process effacement in both groups; however, APC1638T+PAN mice had fewer vesicles in the basal area of podocytes than WT+PAN mice. Cytoplasmic dynein-1, a motor protein for vesicle transport, and α-tubulin were significantly reduced in APC1638T+PAN mice associated with suppressed urinary albumin excretion compared to WT+PAN mice. In conclusion, APC1638T mice showed reduced albuminuria associated with suppressed podocyte vesicle transport when minimal change nephrotic syndrome was induced.

Circulating microRNA-92a-3p in colorectal cancer: a review.

Recent studies have found that microRNAs (miRNAs) are present in body fluids, including blood, cerebrospinal fluid, tears, saliva, breast milk, and urine in a stable form, and are called circulating miRNAs. Although their biological roles remain to be determined, circulating miRNAs are considered as mediators of intercellular communication like hormones and cytokines. Because circulating miRNAs can be collected in a non-invasive manner called as "liquid biopsy", they have also been studied as potential biomarkers for early detection, evaluation of therapeutic effects, and prediction of prognosis in various diseases, including cancers. In this review, we focus on the studies on circulating microRNA-92a-3p (miR-92a-3p) in colorectal cancer (CRC), considering their existence form, isolation methods, potential as biomarkers, and roles in CRC development and progression.

A further study on a disturbance of intestinal epithelial cell population and kinetics in APC1638T mice.

Adenomatous polyposis coli (APC), a well-known anti-oncogene, is considered to have multiple functions through its several binding domains. We have continuingly studied APC1638T/1638T mice (APC1638T mice) to elucidate the functions of APC other than tumor suppression. A distinctive feature of the APC1638T mice is they are tumor free and live as long as APC+/+ mice (WT mice). Previously, we found the length of crypt-villus axis in the jejunum was significantly elongated in APC1638T mice compared with that of WT mice. The populations of goblet cells, Paneth cells, and enteroendocrine cells were also disordered in APC1638T mice. Here, we further analyzed the intestinal dyshomeostasis in APC1638T mice, focusing on the proliferation and differentiation of intestinal stem cell (ISC) lineages, and apoptotic cell shedding at the villus tips. We found that the proliferation of ISC lineages was normally controlled; however, the shedding process of apoptosis cells was significantly delayed in the APC1638T mouse jejunum. Furthermore, the number of microfold cells (M cells) was significantly increased in the APC1638T mouse jejunum. Our data suggested both differentiation process of ISCs and turnover process of intestinal epithelia were disturbed in APC1638T mice, and that contributed to the villus elongation in the APC1638T mouse jejunum.

Morphological and functional abnormalities of hippocampus in APC1638T/1638T mice.

In the present study, we examined morphology and function of hippocampus in the APC1638T/1638T mouse. Expression levels of the APC mRNA and protein were both identical in the hippocampus of the APC+/+ and APC1638T/1638T mice. The dentate gyrus of the APC1638T/1638T hippocampus was thicker, and has more densely-populated granule cells in the APC1638T/1638T mouse hippocampus. Immunoelectron microscopy revealed co-localization of APC with alpha-amino-3- hydroxy-5-methyl- isoxazole-4-propionate receptor (AMPA-R) and with PSD-95 at post-synapse in the APC+/+ hippocampus, while APC1638T was co-localized with neither AMPA-R nor PSD-95 in the APC1638T/1638T hippocampus. By immunoprecipitation assay, full-length APC expressed in the APC +/+ mouse was co-immunoprecipitated with AMPA-R and PSD-95. In contrast, APC1638T expressed in the APC1638T/1638T mouse was not co-immunoprecipitated with AMPA-R and PSD-95. In the hippocampal CA1 region of the APC1638T/1638T mouse, c-Fos expression after electric foot shock was decreased compared with the APC+/+ mouse. The present study showed some abnormalities on morphology of the hippocampus caused by a truncated APC (APC1638T). Also, our findings suggest that failure in APC binding to AMPA-R and PSD-95 may bring about less activities of hippocampal neurons in the APC1638T/1638T mouse.

Extracellular Vesicles Containing MicroRNA-92a-3p Facilitate Partial Endothelial-Mesenchymal Transition and Angiogenesis in Endothelial Cells.

Extracellular vesicles (EVs) are nanometer-sized membranous vesicles used for primitive cell-to-cell communication. We previously reported that colon cancer-derived EVs contain abundant miR-92a-3p and have a pro-angiogenic function. We previously identified Dickkopf-3 (Dkk-3) as a direct target of miR-92a-3p; however, the pro-angiogenic function of miR-92a-3p cannot only be attributed to downregulation of Dkk-3. Therefore, the complete molecular mechanism by which miR-92a-3p exerts pro-angiogenic effects is still unclear. Here, we comprehensively analyzed the gene sets affected by ectopic expression of miR-92a-3p in endothelial cells to elucidate processes underlying EV-induced angiogenesis. We found that the ectopic expression of miR-92a-3p upregulated cell cycle- and mitosis-related gene expression and downregulated adhesion-related gene expression in endothelial cells. We also identified a novel target gene of miR-92a-3p, claudin-11. Claudin-11 belongs to the claudin gene family, which encodes essential components expressed at tight junctions (TJs). Disruption of TJs with a concomitant loss of claudin expression is a significant event in the process of epithelial-to-mesenchymal transition. Our findings have unveiled a new EV-mediated mechanism for tumor angiogenesis through the induction of partial endothelial-to-mesenchymal transition in endothelial cells.

BRI2 as an anti-Alzheimer gene.

There are several theories regarding the etiologies of Alzheimer disease (AD). Considering that all genes responsible for familial AD are amyloid protein precursor (APP) or APP metabolizing enzymes, surely aberrant APP metabolism is crucial to pathogenesis of AD. BRI2, a type II transmembrane protein, binds APP and inhibits all α, ß, and γ pathways of APP proteolysis. Crossing AD model mice with BRI2 transgenic or BRI2 knockout mice confirmed that BRI2 is an anti-Alzheimer gene. Mutations of BRI2 are known to cause rare familial dementias in human. Analysis of knock-in mice harboring the disease mutation revealed the memory defect in the mice, attributable to loss of protective function of BRI2. Further studies are needed to decipher this anti-Alzheimer mechanism of BRI2 to develop a novel therapeutic application for AD. In this review, after describing basic assumptions in AD study, we focus on BRI2 as an anti-Alzheimer gene.

Validation and application of a novel APC antibody in western blotting, immunoprecipitation, and immunohistochemistry.

Adenomatous polyposis coli (APC) is a large protein with multiple binding partners, suggesting diverse functions besides its well-known role in the destruction of ß-catenin. To elucidate these complex functions, it is crucial to evaluate the precise subcellular distribution of APC within a cell and tissue. However, most of the commercially available anti-APC antibodies can only be used for limited applications, resulting in the use of independently generated antibodies. This has led to various discrepancies between studies as a common antibody has not been established. In this study, we generated an antibody against the c-terminal domain of human APC, designated APC-C antibody, and evaluated its specificity and application in various immunological methods. Our data indicate that this novel APC-C antibody is a specific and versatile antibody that can be used in western blotting, immunoprecipitation, immunocytochemistry, and immunohistochemistry. Widespread use of this APC antibody will help enhance our understanding of APC's function in both normal and cancer cell biology.

A disturbance of intestinal epithelial cell population and kinetics in APC1638T mice.

The adenomatous polyposis coli (APC) is a multifunctional protein as well as a tumor suppressor. To determine the functions of the C-terminal domain of APC, we explored APC 1638T/1638T (APC1638T) mice that express a truncated APC lacking the C-terminal domain. The APC1638T mice were tumor free and exhibited growth retardation. In the present study, we compared small intestinal crypt-villus cells homeostasis in APC +/+ (WT) mice and APC1638T mice. The body weight of APC1638T mice was significantly smaller than that of WT mice at all ages. The length of small intestine of APC1638T mice was significantly shorter than that of WT mice. The crypt-villus axis was significantly elongated, and the number of intestinal epithelial cells also increased in APC1638T mice compared with those in WT mice. However, the number of intestinal epithelial cells per 100 µm of villi was not different between WT and APC1638T mice. Migration and proliferation of intestinal epithelial cells in APC1638T mice were faster than that in WT mice. The population of Goblet cells, Paneth cells, and enteroendocrine cells was significantly altered in APC1638T mice. These results indicate that C-terminal domain of APC has a role in the regulation of intestinal epithelium homeostasis.

The osteocyte plays multiple roles in bone remodeling and mineral homeostasis.

Osteocytes are the most abundant cells in bone and are the major orchestrators of bone remodeling and mineral homeostasis. They possess a specialized cellular morphology and a unique molecular feature. Osteocytes are a stellate shape with numerous long, slender dendritic processes. The osteocyte cell body resides in the bone matrix of the lacuna and the dendritic processes extend within the canaliculi to adjacent osteocytes and other cells on the bone surface. Osteocytes form extensive intercellular network to sense and respond to environmental mechanical stimulus by the lacunar-canalicular system and gap junction. Osteocytes are long-lived bone cells. They can undergo apoptosis, which may have specific regulatory effects on osteoclastic bone resorption. Osteocytes can secrete several molecules, including sclerostin, receptor activator of nuclear factor κB ligand and fibroblast growth factor 23 to regulate osteoblastic bone formation, osteoclastic bone resorption and mineral homeostasis. A deeper understanding of the complex mechanisms that mediate the control of osteoblast and osteoclast function by osteocytes may identify new osteocyte-derived molecules as potential pharmacological targets for treating osteoporosis and other skeletal diseases.

Mitochondrial fractions located in the cytoplasmic and peridroplet areas of white adipocytes have distinct roles.

The role of mitochondria in white adipocytes (WAs) has not been fully explored. A recent study revealed that brown adipocytes contain functionally distinct mitochondrial fractions, cytoplasmic mitochondria, and peridroplet mitochondria. However, it is not known whether such a functional division of mitochondria exists in WA. Herein, we observed that mitochondria could be imaged and mitochondrial DNA and protein detected in pellets obtained from the cytoplasmic layer and oil layer of WAs after centrifugation. The mitochondria in each fraction were designated as cytoplasmic mitochondria (CMw) and peridroplet mitochondria (PDMw) in WAs, respectively. CMw had higher ß-oxidation activity than PDMw, and PDMw was associated with diacylglycerol acyltransferase 2. Therefore, CMw may be involved in ß-oxidation and PDMw in droplet expansion in WAs.

Identification of DNA-dependent protein kinase catalytic subunit as a novel interaction partner of lymphocyte enhancer factor 1.

Lymphocyte enhancer factor 1 (LEF1), a member of the LEF/T-cell-specific factor (TCF) family of the high mobility group domain transcription factors, acts downstream in canonical Wnt signaling. Aberrant transactivation of LEF1 contributes to the tumorigenesis of colonic neoplasms, sebaceous skin tumors, and lymphoblastic leukemia. LEF1-associated proteins are crucial for regulating its transcriptional activity. In this study, glutathione-S-transferase pull-down assay and mass spectrometry enabled identification of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) as a novel interaction partner for LEF1. The interaction between LEF1 and DNA-PKcs was confirmed using in vivo co-immunoprecipitation. Furthermore, double immunofluorescence observations showed that LEF1 and DNA-PKcs colocalized in the nuclei of colon adenocarcinoma cell lines. Identification of the interaction between LEF1 and DNA-PKcs may provide clues for a novel therapy for cancer treatment as well as for understanding LEF1-mediated transcriptional regulation.

Colocalization of APC and PSD-95 in the nerve fiber as well as in the post-synapse of matured neurons.

Adenomatous polyposis coli protein (APC) is highly expressed in the nervous tissue, but its function there is not yet known. We previously found that the microtubule-bundling activity of APC is stimulated by its interaction with PSD-95, a neuronal scaffolding protein, in cultured COS-7 cells. In the present study, we investigated the distribution and localization of the intrinsic APC and PSD-95 in both cultured rat hippocampal neurons and in the rat cerebellum by immunofluorescence and immunoelectron microscopy. In cultured neurons, most of the PSD-95 immunofluorescence puncta were colocalized with APC, and the APC and PSD-95 immunogolds were colocalized in the nerve fibers as well as in the postsynaptic site, but not in the presynaptic site. In the molecular layer of the rat cerebellum, colocalization of APC and PSD-95 was also detected in the nerve fibers and the postsynaptic site, but not in the presynaptic site. Based on these results, we conclude that APC and PSD-95 colocalize and bind to form a protein complex in nerve fibers as well as in postsynaptic sites in matured neurons, suggesting the involvement of the APC/PSD-95 complex in the microtubule functions within the nerve fibers and the synapse functions at the postsynaptic site.

Some fine-structural findings on the thyroid gland in Apc1638T/1638T mice that express a C-terminus lacking truncated Apc.

Adenomatous polyposis coli (Apc) is a multifunctional protein as well as a tumor suppressor. To determine the functions of the C-terminal domain of Apc, we examined Apc(1638T/1638T) mice that express a truncated Apc lacking the C-terminal domain. The Apc(1638T/1638T) mice were tumor free and exhibited growth retardation. We recently reported abnormalities in thyroid morphology and functions of Apc(1638T/1638T) mice, although the mechanisms underlying these abnormalities are not known. In the present study, we further compared thyroid gland morphology in Apc(1638T/1638T) and Apc(+/+) mice. The diameters of thyroid follicles in the left and right lobes of the same thyroid gland of Apc(1638T/1638T) mice were significantly different whereas the Apc(+/+) mice showed no significant differences in thyroid follicle diameter between these lobes. To assess the secretory activities of thyroid follicular cells, we performed double-immunostaining of thyroglobulin, a major secretory protein of these cells, and the rough endoplasmic reticulum (rER) marker calreticulin. In the Apc(1638T/1638T) follicular epithelial cells, thyroglobulin was mostly colocalized with calreticulin whereas in the Apc(+/+) follicular epithelial cells, a significant amount of the cytoplasmic thyroglobulin did not colocalize with calreticulin. In addition, in thyroid-stimulating hormone (TSH)-treated Apc(1638T/1638T) mice, electron microscopic analysis indicated less frequent pseudopod formation at the apical surface of the thyroid follicular cells than in Apc(+/+) mice, indicating that reuptake of colloid droplets containing iodized thyroglobulin is less active. These results imply defects in intracellular thyroglobulin transport and in pseudopod formation in the follicular epithelial cells of Apc(1638T/1638T) mice and suggest suppressed secretory activities of these cells.

Calreticulin-2 is localized in the lumen of the endoplasmic reticulum but is not a Ca2+ -binding protein.

Calreticulin (CRT)-1 is a major Ca(2+)-buffering protein in the lumen of the endoplasmic reticulum. Human and murine CRT-2 was isolated in 2002, but the subcellular localization and function is still unclear. Here, we studied the intracellular localization and function of CRT-2 with hemagglutinin-tagged (HA-) human CRT-2. Western blotting revealed HA-CRT-2 as a single band at 50 kDa. Using immunofluorescence microscopy of cultured fibroblasts and epithelial cells transfected with HA-CRT-2 cDNA, labeling for HA-CRT-2 was seen as a reticular network with a nuclear envelope pattern that colocalized with calnexin and protein disulfide isomerase. Immunoelectron microscopy confirmed that HA-CRT-2 was localized in the lumen of the endoplasmic reticulum. Stains-all staining, a method to detect Ca(2+)-binding proteins, could not stain the immunoprecipitate of HA-CRT-2, although HA-CRT-1 immunoprecipitate was stained blue. These results indicate that the molecular weight of the non-tagged CRT-2 on SDS-PAGE is 49 kDa, and that CRT-2, as well as CRT-1, is localized in the lumen of the endoplasmic reticulum, but that CRT-2 capacity for Ca(2+)-binding may be absent or much lower than that of CRT-1.

The C-terminal domain of the adenomatous polyposis coli (Apc) protein is involved in thyroid morphogenesis and function.

Adenomatous polyposis coli (APC) is a multifunctional protein as well as a tumor suppressor. To determine the functions of the C-terminal domain of Apc, we have investigated Apc ( 1638T/1638T ) mice, which express a truncated Apc that lacks the C-terminal domain. Apc ( 1638T/1638T ) mice are tumor free and exhibit growth retardation. In the present study, we analyzed the morphology and functions of the thyroid gland in Apc ( 1638T/1638T ) mice. There was no significant difference in the basal concentration of serum thyroid hormones between Apc ( 1638T/1638T ) and Apc (+/+) mice. Thyroid follicle size was significantly larger in Apc ( 1638T/1638T ) mice than in Apc (+/+) mice. The extent of serum T4 elevation following exogenous thyroid-stimulating hormone (TSH) injection was lower in Apc ( 1638T/1638T ) mice than in Apc (+/+) mice. TSH also induced a greater reduction in thyroid follicle size in Apc ( 1638T/1638T ) mice than in Apc (+/+) mice. Analyses using immunohistochemistry and electron microscopy indicated that follicular epithelial cells in Apc ( 1638T/1638T ) mice had an enlarged rough endoplasmic reticulum of irregular shape. These results suggest that the C-terminal domain of Apc is involved in thyroid morphology and function.

Expression of vascular endothelial growth factor and presence of angiovascular cells in tissues from different thyroid disorders.

BACKGROUND: Vascular endothelial growth factor (VEGF) is involved in tumor angiogenesis and other pathophysiological processes. MATERIALS AND METHODS: We studied the localization of VEGF in human thyroid tissues to clarify its involvement in proliferative processes in a variety of thyroid disorders. Immunohistochemical analysis using purified rabbit polyclonal anti-human VEGF or anti-human CD34 antibody and a streptavidin-biotin peroxidase complex detection system was performed on 58 tissue specimens from 53 patients with different thyroid disorders and 5 normal thyroid glands. RESULTS: Vascular endothelial growth factor was not detected in normal thyroid follicular cells. However, some thyroid tumor cells expressed VEGF in the cytoplasm (papillary carcinoma, 10/18; follicular carcinoma, 1/3; medullary carcinoma, 2/2; follicular adenoma, 3/11; adenomatous goiter, 2/4). In benign follicular adenoma and adenomatous goiter, weak expression of VEGF was found in small areas of the tumor, whereas in malignant thyroid tumors, it was strongly expressed in many cells. However, VEGF was not expressed in anaplastic carcinoma, malignant lymphoma, or Graves' disease. Angiovascular cells stained with CD34 antibody in tissues from different thyroid disorders reflected statistically significant differences in papillary carcinoma, follicular adenoma, and Graves' disease compared with normal thyroids, and such cells showed a trend toward increases in medullary carcinoma and adenomatous goiter. In contrast, low vascularity was observed in anaplastic carcinoma, malignant lymphoma, and follicular carcinoma. CONCLUSIONS: Because VEGF probably functions as a hypoxia-inducible angiogenic factor, overexpression of this mediator, concomitant with hypervascularity, may be induced more strongly in malignant thyroid tumors, which need more oxygen to proliferate, than in benign follicular tumors. However, neither VEGF nor CD34 was expressed in anaplastic thyroid carcinoma, which is an extremely poorly differentiated malignant tumor. CD34 but not VEGF was expressed in the hyperplastic thyroid tissues of Graves' disease composed of nontransformed cells. Thus, the expression of VEGF concomitant with CD34 is suggested to reflect both the transformation and differentiation state of malignant tumors.