Deletion of podocyte Rho-associated, coiled-coil-containing protein kinase 2 protects mice from focal segmental glomerulosclerosis.

Focal segmental glomerulosclerosis (FSGS) shares podocyte damage as an essential pathological finding. Several mechanisms underlying podocyte injury have been proposed, but many important questions remain. Rho-associated, coiled-coil-containing protein kinase 2 (ROCK2) is a serine/threonine kinase responsible for a wide array of cellular functions. We found that ROCK2 is activated in podocytes of adriamycin (ADR)-induced FSGS mice and cultured podocytes stimulated with ADR. Conditional knockout mice in which the ROCK2 gene was selectively disrupted in podocytes (PR2KO) were resistant to albuminuria, glomerular sclerosis, and podocyte damage induced by ADR injection. In addition, pharmacological intervention for ROCK2 significantly ameliorated podocyte loss and kidney sclerosis in a murine model of FSGS by abrogating profibrotic factors. RNA sequencing of podocytes treated with a ROCK2 inhibitor proved that ROCK2 is a cyclic nucleotide signaling pathway regulator. Our study highlights the potential utility of ROCK2 inhibition as a therapeutic option for FSGS.

Mice with R2509C-RYR1 mutation exhibit dysfunctional Ca2+ dynamics in primary skeletal myocytes.

Type 1 ryanodine receptor (RYR1) is a Ca2+ release channel in the sarcoplasmic reticulum (SR) of the skeletal muscle and plays a critical role in excitation-contraction coupling. Mutations in RYR1 cause severe muscle diseases, such as malignant hyperthermia, a disorder of Ca2+-induced Ca2+ release (CICR) through RYR1 from the SR. We recently reported that volatile anesthetics induce malignant hyperthermia (MH)-like episodes through enhanced CICR in heterozygous R2509C-RYR1 mice. However, the characterization of Ca2+ dynamics has yet to be investigated in skeletal muscle cells from homozygous mice because these animals die in utero. In the present study, we generated primary cultured skeletal myocytes from R2509C-RYR1 mice. No differences in cellular morphology were detected between wild type (WT) and mutant myocytes. Spontaneous Ca2+ transients and cellular contractions occurred in WT and heterozygous myocytes, but not in homozygous myocytes. Electron microscopic observation revealed that the sarcomere length was shortened to ∼1.7 µm in homozygous myocytes, as compared to ∼2.2 and ∼2.3 µm in WT and heterozygous myocytes, respectively. Consistently, the resting intracellular Ca2+ concentration was higher in homozygous myocytes than in WT or heterozygous myocytes, which may be coupled with a reduced Ca2+ concentration in the SR. Finally, using infrared laser-based microheating, we found that heterozygous myocytes showed larger heat-induced Ca2+ transients than WT myocytes. Our findings suggest that the R2509C mutation in RYR1 causes dysfunctional Ca2+ dynamics in a mutant-gene dose-dependent manner in the skeletal muscles, in turn provoking MH-like episodes and embryonic lethality in heterozygous and homozygous mice, respectively.

Exogenous ANP Treatment Ameliorates Myocardial Insulin Resistance and Protects against Ischemia-Reperfusion Injury in Diet-Induced Obesity.

Increasing evidence suggests natriuretic peptides (NPs) coordinate interorgan metabolic crosstalk. We recently reported exogenous ANP treatment ameliorated systemic insulin resistance by inducing adipose tissue browning and attenuating hepatic steatosis in diet-induced obesity (DIO). We herein investigated whether ANP treatment also ameliorates myocardial insulin resistance, leading to cardioprotection during ischemia-reperfusion injury (IRI) in DIO. Mice fed a high-fat diet (HFD) or normal-fat diet for 13 weeks were treated with or without ANP infusion subcutaneously for another 3 weeks. Left ventricular BNP expression was substantially reduced in HFD hearts. Intraperitoneal-insulin-administration-induced Akt phosphorylation was impaired in HFD hearts, which was restored by ANP treatment, suggesting that ANP treatment ameliorated myocardial insulin resistance. After ischemia-reperfusion using the Langendorff model, HFD impaired cardiac functional recovery with a corresponding increased infarct size. However, ANP treatment improved functional recovery and reduced injury while restoring impaired IRI-induced Akt phosphorylation in HFD hearts. Myocardial ultrastructural analyses showed increased peri-mitochondrial lipid droplets with concomitantly decreased ATGL and HSL phosphorylation levels in ANP-treated HFD, suggesting that ANP protects mitochondria from lipid overload by trapping lipids. Accordingly, ANP treatment attenuated mitochondria cristae disruption after IRI in HFD hearts. In summary, exogenous ANP treatment ameliorates myocardial insulin resistance and protects against IRI associated with mitochondrial ultrastructure modifications in DIO. Replenishing biologically active NPs substantially affects HFD hearts in which endogenous NP production is impaired.

Extended-synaptotagmin 1 engages in unconventional protein secretion mediated via SEC22B+ vesicle pathway in liver cancer.

Protein secretion in cancer cells defines tumor survival and progression by orchestrating the microenvironment. Studies suggest the occurrence of active secretion of cytosolic proteins in liver cancer and their involvement in tumorigenesis. Here, we investigated the identification of extended-synaptotagmin 1 (E-Syt1), an endoplasmic reticulum (ER)-bound protein, as a key mediator for cytosolic protein secretion at the ER-plasma membrane (PM) contact sites. Cytosolic proteins interacted with E-Syt1 on the ER, and then localized spatially inside SEC22B+ vesicles of liver cancer cells. Consequently, SEC22B on the vesicle tethered to the PM via Q-SNAREs (SNAP23, SNX3, and SNX4) for their secretion. Furthermore, inhibiting the interaction of protein kinase Cδ (PKCδ), a liver cancer-specific secretory cytosolic protein, with E-Syt1 by a PKCδ antibody, decreased in both PKCδ secretion and tumorigenicity. Results reveal the role of ER-PM contact sites in cytosolic protein secretion and provide a basis for ER-targeting therapy for liver cancer.

ROCK2-induced metabolic rewiring in diabetic podocytopathy.

Loss of podocytes is a common feature of diabetic renal injury and a key contributor to the development of albuminuria. We found that podocyte Rho associated coiled-coil containing protein kinase 2 (ROCK2) is activated in rodent models and patients with diabetes. Mice that lacked ROCK2 only in podocytes (PR2KO) were resistant to albuminuria, glomerular fibrosis, and podocyte loss in multiple animal models of diabetes (i.e., streptozotocin injection, db/db, and high-fat diet feeding). RNA-sequencing of ROCK2-null podocytes provided initial evidence suggesting ROCK2 as a regulator of cellular metabolism. In particular, ROCK2 serves as a suppressor of peroxisome proliferator-activated receptors α (PPARα), which rewires cellular programs to negatively control the transcription of genes involved in fatty acid oxidation and consequently induce podocyte apoptosis. These data establish ROCK2 as a nodal regulator of podocyte energy homeostasis and suggest this signaling pathway as a promising target for the treatment of diabetic podocytopathy.

Effect of cellular communication network factor 2/connective tissue growth factor on tube formation by endothelial cells derived from human periodontal ligaments.

OBJECTIVES: To clarify the role of cellular communication network factor 2/connective tissue growth factor (CCN2/CTGF) in periodontal tissue regeneration by investigating, the proliferative and tubulogenic responses of human endothelial cells obtained from the periodontal ligament to CCN2/CTGF. DESIGN: Endothelial cells were seeded on agar gel medium with or without 50 ng/mL recombinant CCN2/CTGF (rCCN2/CTGF) and cultured for 6 h. Cells were morphologically and phenotypically analyzed by immunofluorescent microscopy. A colorimetric assay was used to evaluate cell proliferation, and transmission electron microscopy (TEM) was used for ultrastructural analysis. RESULTS: The proliferation of endothelial cells was best promoted by rCCN2/CTGF at 50 ng/mL. In the control group, tube formation was not observed within 6 h. In contrast, endothelial cells seeded on the agar with 50 ng/mL rCCN2/CTGF clearly showed proliferation with network formation. Under a two-dimensional culture condition, a dense network of endothelial cells was not constructed on the plastic bottom. However, drastic morphological change was observed in the endothelial cells on the agar containing rCCN2/CTGF. The endothelial cells in the dense network were interconnected with each other and showed a tube-like structure. Tight junctions or adherens junctions were observed between the adjoining endothelial cells in the dense network. CONCLUSIONS: CCN2/CTGF was found to promote the proliferation and tubulogenesis of endothelial cells from the periodontal ligament. These results suggest that CCN2/CTGF may contribute to the regeneration of damaged periodontal tissue by activating the remaining endothelial cells.

Osteoblastic MC3T3-E1 cells on diamond-like carbon-coated silicon plates: Field emission scanning electron microscopy data.

Diamond-like carbon (DLC) is an amorphous form of carbon that contains aspects of both the diamond and graphite structures. It is composed of carbon and hydrogen, and owing to its texture, high mechanical hardness, chemical inertness, and optical transparency, DLC is widely used as a protective coating in the form of a thin film, which is applied to the surfaces of many materials. Recently, it has attracted attention as a biomedical material because of its high biocompatibility and stability [1,2]. DLC is particularly suitable to be embedded in the body owing to its low friction properties and selective cell surface attachment properties [3]. The material is currently being developed for the treatment of bone fractures [4]. However, unlike fibroblasts, the attachment of osteoblasts has not been extensively examined and no morphological data is available on how osteoblastic cells form contacts with the surface of biocompatible DLC-coated materials. Herein, such data were collected by coating DLC on the surface of silicon plates. The attachment of mouse cells to the DLC-coated plates was examined by colorimetric cell proliferation assay, and morphological observations were made using a field emission scanning electron microscope. Also, the flat cross section of the cell and plate was obtained by the ion milling method.

Gelatin Sponge as an Anchorage for Three-dimensional Culture of Colorectal Cancer Cells.

BACKGROUND: Compared to two-dimensional cultures, three-dimensional (3D) cultures have many advantages in cancer studies. Nevertheless, their implementation is unsatisfactory. This study aimed to develop an anchorage-dependent 3D culture model for colorectal cancer research. MATERIALS AND METHODS: Human HCT116, DLD-1 and SW620 colorectal cell lines were cultured in a gelatin sponge, and its applicability for morphological examination was studied. RESULTS: The resulting specimens were suitable for scanning electron microscopy, transmission electron microscopy, and immunohistochemical examination. HCT116 formed smaller structures and migrated through the pores of the sponge. DLD-1 formed larger structures with tight cell-to-cell adhesion. SW620 also formed large structures but small clustered cells tended to attach to the anchorage more favorably. Immunohistochemical staining demonstrated phosphorylated yes-associated protein (YAP) localized near the attachment site in HCT116 cells. CONCLUSION: Because the gelatin sponge provided suitable anchorage and the cultured cells formed distinguishable 3D structures, this method may be useful for further colorectal cancer research.

Thiamine treatment preserves cardiac function against ischemia injury via maintaining mitochondrial size and ATP levels.

Thiamine (vitamin B1) is necessary for energy production, especially in the heart. Recent studies have demonstrated that thiamine supplementation for cardiac diseases is beneficial. However, the detailed mechanisms underlying thiamine-preserved cardiac function have not been elucidated. To this end, we conducted a functional analysis, metabolome analysis, and electron microscopic analysis to unveil the mechanisms of preserved cardiac function through supplementation with thiamine for ischemic cardiac disease. Male Sprague-Dawley rats (around 10 wk old) were used. Following pretreatment with or without thiamine pyrophosphate (TPP; 300 µM), hearts were exposed to ischemia (40 min of global ischemia followed by 60 min of reperfusion). We measured the left ventricle developed pressure (LVDP) throughout the protocol. The LVDP during reperfusion in the TPP-treated heart was significantly higher than that in the untreated heart. Metabolome analysis was performed using capillary electrophoresis-time-of-flight mass spectrometry, and it revealed that the TPP-treated heart retained higher adenosine triphosphate (ATP) levels compared with the untreated heart after ischemia. The metabolic pathway showed that there was a significant increase in fumaric acid and malic acid from the tricarboxylic acid cycle following ischemia. Electron microscope analysis revealed that the mitochondria size in the TPP-treated heart was larger than that in the untreated heart. Mitochondrial fission in the TPP-treated heart was also inhibited, which was confirmed by a decrease in the phosphorylation level of DRP1 (fission related protein). TPP treatment for cardiac ischemia preserved ATP levels probably as a result of maintaining larger mitochondria by inhibiting fission, thereby allowing the TPP-treated heart to preserve contractility performance during reperfusion.NEW & NOTEWORTHY We found that treatment with thiamine can have a protective effect on myocardial ischemia. Thiamine likely mediates mitochondrial fission through the inhibition of DRP1 phosphorylation and the preservation of larger-sized mitochondria and ATP concentration, leading to higher cardiac contractility performance during the subsequent reperfusion state.

Unconventional Secretion of PKCδ Exerts Tumorigenic Function via Stimulation of ERK1/2 Signaling in Liver Cancer.

Expression of human protein kinase C delta (PKCδ) protein has been linked to many types of cancers. PKCδ is known to be a multifunctional PKC family member and has been rigorously studied as an intracellular signaling molecule. Here we show that PKCδ is a secretory protein that regulates cell growth of liver cancer. Full-length PKCδ was secreted to the extracellular space in living liver cancer cells under normal cell culture conditions and in xenograft mouse models. Patients with liver cancer showed higher levels of serum PKCδ than patients with chronic hepatitis or liver cirrhosis or healthy individuals. In liver cancer cells, PKCδ secretion was executed in an endoplasmic reticulum (ER)-Golgi-independent manner, and the inactivation status of cytosolic PKCδ was required for its secretion. Furthermore, colocalization studies showed that extracellular PKCδ was anchored on the cell surface of liver cancer cells via association with glypican 3, a liver cancer-related heparan sulfate proteoglycan. Addition of exogenous PKCδ activated IGF-1 receptor (IGF1R) activation and subsequently enhanced activation of ERK1/2, which led to accelerated cell growth in liver cancer cells. Conversely, treatment with anti-PKCδ antibody attenuated activation of both IGF1R and ERK1/2 and reduced cell proliferation and spheroid formation of liver cancer cells and tumor growth in xenograft mouse models. This study demonstrates the presence of PKCδ at the extracellular space and the function of PKCδ as a growth factor and provides a rationale for the extracellular PKCδ-targeting therapy of liver cancer. SIGNIFICANCE: PKCδ secretion from liver cancer cells behaves as a humoral growth factor that contributes to cell growth via activation of proliferative signaling molecules, which may be potential diagnostic or therapeutic targets.

Ex Vivo Gene Therapy Treats Bone Complications of Mucopolysaccharidosis Type II Mouse Models through Bone Remodeling Reactivation.

Mucopolysaccharidosis type II is a disease caused by organ accumulation of glycosaminoglycans due to iduronate 2-sulfatase deficiency. This study investigated the pathophysiology of the bone complications associated with mucopolysaccharidosis II and the effect of lentivirus-mediated gene therapy of hematopoietic stem cells on bone lesions of mucopolysaccharidosis type II mouse models in comparison with enzyme replacement therapy. Bone volume, density, strength, and trabecular number were significantly higher in the untreated mucopolysaccharidosis type II mice than in wild-type mice. Accumulation of glycosaminoglycans caused reduced bone metabolism. Specifically, persistent high serum iduronate 2-sulfatase levels and release of glycosaminoglycans from osteoblasts and osteoclasts in mucopolysaccharidosis type II mice that had undergone gene therapy reactivated bone lineage remodeling, subsequently reducing bone mineral density, strength, and trabecular number to a similar degree as that observed in wild-type mice. Bone formation, resorption parameters, and mineral density in the diaphysis edge did not appear to have been affected by the irradiation administered as a pre-treatment for gene therapy. Hence, the therapeutic effect of gene therapy on the bone complications of mucopolysaccharidosis type II mice possibly outweighed that of enzyme replacement therapy in many aspects.

Generation of Human Renal Vesicles in Mouse Organ Niche Using Nephron Progenitor Cell Replacement System.

Animal fetuses may be used for the regeneration of human organs. We have previously generated a transgenic mouse model that allows diphtheria toxin (DT)-induced ablation of Six2-positive nephron progenitor cells (NPCs). Elimination of existing native host NPCs enables their replacement with donor NPCs, which can generate neo-nephrons. However, this system cannot be applied to human NPCs, because DT induces apoptosis in human cells. Therefore, the present study presents a transgenic mouse model for the ablation of NPCs using tamoxifen, which does not affect human cells. Using this system, we successfully regenerate interspecies neo-nephrons, which exhibit urine-producing abilities, from transplanted rat NPCs in a mouse host. Transplantation of human induced pluripotent stem cell (iPSC)-derived NPCs results in differentiation into renal vesicles, which connect to the ureteric bud of the host. Thus, we demonstrate the possibility of the regeneration of human kidneys derived from human iPSC-derived NPCs via NPC replacement.

Osteogenic cocktail induces calcifications in human breast cancer cell line via placental alkaline phosphatase expression.

Breast cancer is frequently characterized by calcifications in mammography. The mechanism for calcifications in breast cancer is not completely known. Understanding this mechanism will improve diagnostic accuracy. Herein, we demonstrated that calcifications occur and that alkaline phosphatase enzyme activity increases in MDA-MB-231 cells cultured using an osteogenic cocktail-containing medium. Microarray transcript analysis showed that the PI3K-Akt signaling pathway was significantly involved, with recruitment of placental alkaline phosphatase. Calcifications and alkaline phosphatase enzyme activity were suppressed by silencing placental alkaline phosphatase using a small interfering RNA. Inhibition of the PI3K-Akt signaling pathway suppressed phospho-c-Jun and placental alkaline phosphatase and resulted in absence of calcifications. These findings reveal that breast cancer cells acquire alkaline phosphatase enzyme activity via placental alkaline phosphatase expression and suggest that breast calcification formation is closely associated with the PI3K-Akt signaling pathway.

Novel homozygous CLN3 missense variant in isolated retinal dystrophy: A case report and electron microscopic findings.

BACKGROUND: Biallelic CLN3 gene variants have been found in either juvenile-onset neuronal ceroid lipofuscinosis (JNCL) or isolated retinal dystrophy. It has been reported that most JNCL patients carry a common 1.02-kb deletion variant homozygously. Clinical characteristics of patients with biallelic CLN3 missense variants are not well elucidated. METHODS: We described a 26-year-old Japanese male patient with isolated retinal dystrophy. Whole-exome sequencing (WES) and transmission electron microscopy (TEM) were performed. RESULTS: Whole-exome sequencing identified a novel homozygous CLN3 missense variant [c.482C>T; p.(Ser161Leu)]. Ophthalmoscopy revealed retinal degeneration and macular atrophy, and later attenuated retinal vessels. Severely reduced responses were observed in both rod and cone electroretinograms. In TEM of the patient's lymphocytes, fingerprint profiles, which are specific findings in CLN3-associated JNCL, were observed in 16/624 (2.56%) lymphocytes of the patient, who has never exhibited neurological signs during the 13-year follow-up period. CONCLUSION: Our results indicated that this novel CLN3 missense variant is associated with teenage-onset isolated retinal dystrophy. This is the first report of any patient with CLN3-associated disorder in the Japanese population. Although fingerprint profiles have never been reported in CLN3-associated isolated retinal dystrophy, these profiles were observed, albeit infrequently, suggesting that frequency of the fingerprint profiles might depend on variant types.

Long-term observation of a Japanese mucolipidosis IV patient with a novel homozygous p.F313del variant of MCOLN1.

Mucolipidosis type IV (MLIV) is an autosomal recessively inherited lysosomal storage disorder characterized by progressive psychomotor delay and retinal degeneration that is associated with biallelic variants in the MCOLN1 gene. The gene, which is expressed in late endosomes and lysosomes of various tissue cells, encodes the transient receptor potential channel mucolipin 1 consisting of six transmembrane domains. Here, we described 14-year follow-up observation of a 4-year-old Japanese male MLIV patient with a novel homozygous in-frame deletion variant p.(F313del), which was identified by whole-exome sequencing analysis. Neurological examination revealed progressive psychomotor delay, and atrophy of the corpus callosum and cerebellum was observed on brain magnetic resonance images. Ophthalmologically, corneal clouding has remained unchanged during the follow-up period, whereas optic nerve pallor and retinal degenerative changes exhibited progressive disease courses. Light-adapted electroretinography was non-recordable. Transmission electron microscopy of granulocytes revealed characteristic concentric multiple lamellar structures and an electron-dense inclusion in lysosomes. The in-frame deletion variant was located within the second transmembrane domain, which is of putative functional importance for channel properties.

Clinical Course and Electron Microscopic Findings in Lymphocytes of Patients with DRAM2-Associated Retinopathy.

DRAM2-associated retinopathy is a rare inherited retinal dystrophy, and its outcome has not been determined. A single retinal involvement by a mutation of the DRAM2 gene is unexplained. We found three unrelated patients with a disease-causing DRAM2 variant in a biallelic state from 1555 Japanese individuals of 1314 families with inherited retinal dystrophy. We reviewed their medical records and examined their peripheral lymphocytes by transmission electron microscopy (TEM). Patient 1 was a 38-year-old woman who complained of night blindness and reduced vision. She developed macular degeneration at age 43 years. Patients 2 and 3 were a man and a woman both of whom noticed night blindness in their 30s. Both had a degeneration in the macula and midperiphery in their 40s, which progressed to a diffuse retinal degeneration in their 60s when their vision was reduced to hand motions. Three novel DRAM2 variants were identified. TEM of the lymphocytes of Patients 1 and 2 showed abnormal structures in 40.6% and 0.3% of the peripheral lymphocytes, respectively. We concluded that the DRAM2-associated retinopathy of our patients was a progressive rod-cone dystrophy, and the visual outcome was poor. The systemic effect of DRAM2 mutations may be compensable and have variations.

Establishment and characterization of human gingival squamous cell carcinoma cell line NOCS-1.

The NOCS-1 cell line was established from the left gingiva tumor in an 86-year-old Japanese man. Histopathological diagnosis of the original tumor was well-differentiated squamous cell carcinoma. NOCS-1 cells were adhesive epithelial cells with neoplastic or pleomorphic features and grew without contact inhibition. It has been subcultured 70 times during the past 26 months. From passage 3, melanin-containing cells began to be observed in the NOCS-1 cell line. The plating efficiencies were 25% and 23%, doubling times were 29 and 26 h, and saturation densities were 6.9 × 104/cm2 and 8.7 × 104/cm2, at passage 12 and 30, respectively. When NOCS-1 cells were xenotransplanted subcutaneously into SCID mice, they produced tumors that histopathologically resembled the original tumor. In addition, NOCS-1-XG cells derived from the xenotransplanted tumor were similar to NOCS-1 cells. We believe that this cell line may be a valuable tool to develop immunotherapy and chemotherapy regimens.

Platelets play an essential role in murine lung development through Clec-2/podoplanin interaction.

Platelets participate in not only thrombosis and hemostasis but also other pathophysiological processes, including tumor metastasis and inflammation. However, the putative role of platelets in the development of solid organs has not yet been described. Here, we report that platelets regulate lung development through the interaction between the platelet-activation receptor, C-type lectin-like receptor-2 (Clec-2; encoded by Clec1b), and its ligand, podoplanin, a membrane protein. Clec-2 deletion in mouse platelets led to lung malformation, which caused respiratory failure and neonatal lethality. In these embryos, α-smooth muscle actin-positive alveolar duct myofibroblasts (adMYFs) were almost absent in the primary alveolar septa, which resulted in loss of alveolar elastic fibers and lung malformation. Our data suggest that the lack of adMYFs is caused by abnormal differentiation of lung mesothelial cells (luMCs), the major progenitor of adMYFs. In the developing lung, podoplanin expression is detected in alveolar epithelial cells (AECs), luMCs, and lymphatic endothelial cells (LECs). LEC-specific podoplanin knockout mice showed neonatal lethality and Clec1b-/--like lung developmental abnormalities. Notably, these Clec1b-/--like lung abnormalities were also observed after thrombocytopenia or transforming growth factor-ß depletion in fetuses. We propose that the interaction between Clec-2 on platelets and podoplanin on LECs stimulates adMYF differentiation of luMCs through transforming growth factor-ß signaling, thus regulating normal lung development.

Establishment and characterization of a squamous cell carcinoma cell line, designated hZK-1, derived from a metastatic lymph node tumor of the tongue.

The hZK-1 cell line was successfully established from the metastatic foci of a lymph node of an 82-year-old Japanese woman with squamous cell carcinoma of the tongue. The pathological diagnosis of the tumor was moderately to well-differentiated squamous cell carcinoma. The hZK-1 cells were angular in shape, and had neoplastic and pleomorphic features. Adjacent hZK-1 cells were joined by desmosomes and well-developed microvilli, and many free ribosomes were observed in the cytoplasm. The doubling time of the hZK-1 cells was approximately 36, 33, and 29 h at the 10th, 20th, and 30th passages, respectively. The cell line was shown to be triploid, with a chromosomal distribution of 75-80. Immunocytochemical staining of the hZK-1 cells revealed cytokeratin (CK) 17-, Ki67-, and p53-positive staining, and negative staining for CK13. The hZK-1 cells were negative for human papillomavirus (HPV)-16 or-18 infection. Grafting was not successful when the hZK-1 cells were transplanted into the subcutis of SCID mice. The hZK-1 cells (2 × 106 cells/3 ml of growth medium) secreted vascular endothelial growth factor (VEGF) that reached a concentration of 2.6 ng/ml media after 3 days of culture. Hypoxia enhanced cellular HIF-1α expression and VEGF secretion in hZK-1 cells. The HIF-1α inhibitor YC-1 partially inhibited hypoxia-induced VEGF secretion in ZK-1 cells. The reverse transcription-polymerase chain reaction (RT-PCR) results revealed that the expression of CK17, Ki67, and p53 was elevated in the hZK-1 cells. hZK-1 cells were not sensitive to CDDP, TXT, 5-FU, or a mixture of these three anti-tumor agents.

Establishment and characterization of a clear cell carcinoma cell line, designated NOCC, derived from human ovary.

A cell line, designated NOCC, was established from the ascites of a patient with clear cell adenocarcinoma of the ovary. The cell line has been grown without interruption and continuously propagated by serial passaging (more than 76 times) over 7 years. The cells are spherical to polygonal-shaped, display neoplastic, and pleomorphic features, and grow in a jigsaw puzzle-like pattern while forming monolayers without contact inhibition. The cells proliferate rapidly, but are easily floated as a cell sheet. The population doubling time is about 29 h. The number of chromosomes ranges from 60 to 83. The modal number of chromosomes is 70-74 at the 30th passage. NOCC cells secreted 750.5 ng/ml of VEGF over 3 days of culture. Hypoxia inducible factor-1α (HIF-1α) is a primary regulator of VEGF under hypoxic conditions. NOCC cells were not sensitive to the anticancer drugs BEV, DOX, GEM, ETP, CDDP, or TXT. The graft of NOCC cells to a scid mouse displayed similar histological aspects to the original tumor. Both the NOCC cells and the graft of the NOCC cells gave a positive PAS reaction.