Can widely used cell type markers predict the suitability of immortalized or primary mammary epithelial cell models?

BACKGROUND: Mammary cell cultures are convenient tools for in vitro studies of mammary gland biology. However, the heterogeneity of mammary cell types, e.g., glandular milk secretory epithelial or myoepithelial cells, often complicates the interpretation of cell-based data. The present study was undertaken to determine the relevance of bovine primary mammary epithelial cells isolated from American Holstein (bMEC US) or Swiss Holstein-Friesian (bMEC CH) cows, and of primary bovine mammary alveolar epithelial cells stably transfected with simian virus-40 (SV-40) large T-antigen (MAC-T) for in vitro analyses. This was evaluated by testing their expression pattern of cytokeratin (CK) 7, 18, 19, vimentin, and α-smooth muscle actin (α-SMA. RESULTS: The expression of the listed markers was assessed using real-time quantitative PCR, flow cytometry and immunofluorescence microscopy. Characteristic markers of the mesenchymal (vimentin), myoepithelial (α-SMA) and glandular secretory cells (CKs) showed differential expression among the studied cell cultures, partly depending on the analytical method used. The relative mRNA expression of vimentin, CK7 and CK19, respectively, was lower (P < 0.05) in immortalized than in primary mammary cell cultures. The stain index (based on flow cytometry) of CK7 and CK19 protein was lower (P < 0.05) in MAC-T than in bMECs, while the expression of α-SMA and CK18 showed an inverse pattern. Immunofluorescence microscopy analysis mostly confirmed the mRNA data, while partly disagreed with flow cytometry data (e.g., vimentin level in MAC-T). The differential expression of CK7 and CK19 allowed discriminating between immortal and primary mammary cultures. CONCLUSIONS: The expression of the selected widely used cell type markers in primary and immortalized MEC cells did not allow a clear preference between these two cell models for in vitro analyses studying aspects of milk composition. All tested cell models exhibited to a variable degree epithelial and mesenchymal features. Thus, based on their characterization with widely used cell markers, none of these cultures represent an unequivocal alveolar mammary epithelial cell model. For choosing the appropriate in vitro model additional properties such as the expression profile of specific proteins of interest (e.g., transporter proteins) should equally be taken into account.

Merkel Cell Polyomavirus Is Frequently Detected in Korean Patients with Merkel Cell Carcinoma

BACKGROUND: Merkel cell carcinoma (MCC) is an increasingly common neuroendocrine cancer of the skin. Merkel cell polyomavirus (MCPyV) is one of the causative agents of MCC. The prevalence of MCPyV in primary MCC and sun-exposed non-MCC tumors has been known to have different results depending on where it was investigated. OBJECTIVE: This study assesses the prevalence of MCPyV from primary MCC and sun-exposed non-MCC tumors in Korea. METHODS: A molecular pathology study was performed on 7 tissue specimens of MCC, 1 tissue specimen of metastatic small cell carcinoma of the lung, and 32 tissue specimens of non-MCC tumors occurring from sun-exposed areas [8 basal cell carcinomas (BCCs), 8 squamous cell carcinomas (SCCs), 8 actinic keratoses (AKs), and 8 seborrheic keratoses (SKs)]. All specimens were analyzed to determine the presence of MCPyV-DNA using both polymerase chain reaction (PCR) and real-time quantitative PCR. Immunohistochemistry with monoclonal antibody of MCPyV large T antigen (CM2B4) was also conducted. RESULTS: Using both PCR, MCPyV sequences were detected in six of seven MCC tissue specimens (85.7%). Five (71%) of seven MCC tumors were immunoreactive for CM2B4. All five immunoreactive cases were positive for MCPyV. However, there was no association of MCPyV with BCC, SCC, AK, and SK. CONCLUSION: Our results implicate that MCPyV may contribute to the pathogenesis of primary MCC, not of non-MCC skin tumors in Korea, and the persons with MCPyV infection are unusual in Korea compared to other areas.

Immortalization of the SD rats' dental follicle cell with simian virus 40 large tumor antigen gene / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To construct SD rat immortalized dental follicle cells (rDFC) induced by simian virus 40 large tumor antigen (SV40Tag) gene to provide a reliable cell source for periodontal tissue engineering research.</p><p><b>METHODS</b>The rDFC was isolated by tissue mass method combined with enzyme digestion method and evaluated by immunohistochemistry. Cell293 were transfected with plasmid pSSR69/pAmpho containing SV40Tag gene by mediating liposome. Normal rDFC were infected with virus-contained supernate and the successfully transfected cell lines were screened with hygromycin, and positive clones were cultured. While non-transfected cells served as negative controls, the cell morphology was observed, the proliferation characteristics was evaluated by calculating cell population. The expression of SV40Tag gene and telomerase in cells was detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting respectively. The biological property of immortalized rDFC was assessed with calculating formation rate of flat cloning, soft agar colony formation test and tumor-forming test.</p><p><b>RESULTS</b>Morphology of immortalized rDFC was not different from that of normal rDFC. The RT-PCR results of SV40Tag revealed amplification band at 357 bp, while no band was seen in the normal cells. The expression of telomerase in immortalized rDFC was higher than that in normal rDFC. The two groups had no significant difference in growth curves, but the immortalized rDFC exhibited stronger proliferative activity. No significant differences of formation rate in flat cloning were observed between the immortalized rDFC [34% (33/96)] and normal rDFC at passage four [22% (21/96)] (χ(2) = 3.71, P > 0.05). No cell cloning was seen in soft agar and the tumor formation was not observed in nude mice.</p><p><b>CONCLUSIONS</b>The rDFC induced by SV40Tag gene could be cultured and passaged in vitro, which retained the stable proliferation and differentiation characteristics and could be used for periodontal tissue engineering research.</p>

Utilidad clínica de los marcadores tumorales / Clinical utility of tumor markers

Los marcadores tumorales, también denominados marcadores biológicos o biomarcadores, se definen como moléculas, sustancias o procesos que se alteran cualitativa o cuantitativamente como resultado de una condición precancerosa o un cáncer, detectables mediante una prueba de laboratorio en sangre, en líquidos orgánicos o en tejidos. La naturaleza de los marcadores tumorales es muy variable: va desde ácido nucleico, ADN o ARN, una proteína o un péptido, hasta procesos complejos como un anticuerpo, la apoptosis, la amilogénesis y la proliferación. Desde el punto de vista de su origen, los marcadores tumorales se producen por el tumor mismo, como la gonadotropina coriónica en el coriocarcinoma, o como respuesta a la lesión tumoral en el tejido circundante, como el antígeno carcinoembrionario en el cáncer de mama. No hay un marcador tumoral ideal, definido como aquel con una sensibilidad y especificidad del 100. Los marcadores tumorales pueden ser utilizados para el cribado en población con riesgo de presentar un cáncer para su detección precoz con enfermedad confinada y potencialmente curable, como parte del diagnóstico, en el diagnóstico diferencial, como prueba de valor pronóstico y predictivo, como herramienta para evaluar el tratamiento administrado, y para la detección de las recaídas cuando éstas se presentan y el paciente tiene una nueva oportunidad de tratamiento, antes de que las manifestaciones clínicas reaparezcan. En este módulo se analizan los principales marcadores tumorales disponibles en el medio, como el antígeno carcinoembrionario, la alfafetoproteína, el antígeno específico de próstata, el CA 15-3, el CA 125, el CA 19-9, el Cyfra 21-1, la gonadotropina coriónica, la calcitonina, la ferritina, la beta 2 microglobulina, entre otros marcadores. Además, se hará referencia a marcadores subrogados de cáncer, como la presencia de la infección por Helicobacter pylori y el virus del papiloma humano.

Detection of JC Virus T-Ag in Early Gastric Cancer

BACKGROUND: JC virus (JCV) is a polyomavirus that commonly infects humans and can cause progressive multifocal leukoencephalopathy in immunocompromised patients. Recently, many reports have documented detection of JCV in gastrointestinal tract cancers. We investigated the presence of JCV in gastric adenocarcinoma, adenoma, and non-neoplastic gastric mucosa. METHODS: We selected paraffin-embedded tissue from endoscopic mucosal resections performed from January 2007 to September 2008. DNA was extracted from the paraffin-embedded specimens of 30 adenocarcinomas, 20 adenomas of the stomach, and 20 non-neoplastic gastric mucosa. Polymerase chain reaction amplifications were performed using gene-specific primers to detect the JCV gene sequences, and immunohistochemical staining was performed to detect the T-antigen (T-Ag) protein. RESULTS: The T-Ag sequence was detected in nine of 30 gastric cancers (30%), two of 20 adenomas (10%), and eight of 20 non-neoplastic gastric mucosa specimens (40%). T-Ag protein expression was found in five of 30 gastric cancers (16.7%) and one of 20 non-neoplastic gastric mucosa specimens (5%), whereas no expression was observed in any of the adenomas. CONCLUSIONS: Although we could not detect a correlation between JCV and gastric cancer, we demonstrated the presence of JCV T-Ag expression in human gastric cancers. These findings suggest a possible role for JCV in gastric carcinogenesis.

T Types, emm Genotypes and Antibiotic Resistance of Streptococcus pyogenes Isolated fromSchool Children in Jinju, 2006 / 대한임상미생물학회지

BACKGROUND: Streptococcus pyogenes is the most common cause of bacterial pharyngitis. T antigens and emm genotypes are essential markers for an epidemiological study of S. pyogenes. Macrolide resistance of S. pyogenes is a serious obstracle to successfully treating a sore throat. METHODS: One-hundred forty-seven strains of S. pyogenes isolated from healthy school children in 2006 were subjected to T typing and emm genotyping. A disk diffusion method was applied for several antibiotics. A double disk diffusion test was performed to evaluate the phenotype distribution of macrolide resistance. RESULTS: Among T antigens and emm genotypes, T11 (19.7%) and emm78 (16.7%), respectively, were the most common in 2006. Both T5/27/44 (2.3%) and emm44/61 (9.1%) declined to a great extent from about 29% in 2004. The rate of resistance to antibiotics were 11.6% to erythromycin, 4.8% to clindamycin, 21.8% to tetracycline, and 7.5% to ofloxacin. M and cMLSB phenotypes were 52.9% and 41.2% respectively. CONCLUSION: T typing and emm genotyping proved a dynamic change in their distribution in 2006 compared to the results of 2004. Erythromycin and clindamycin resistance remained low as in 2004, whereas ofloxacin resistance increased slightly. M and cMLSB phenotypes were equivalent in 2006, whereas cMLSB was predominant in 2004.

Detection of SV40 Large T Antigen in Malignant Lymphomas

BACKGROUND: The association of simian virus 40 (SV40) with certain types of human cancers, including malignant lymphomas, has been a topic of interest for some time. Although the virus is distributed worldwide, its incidences vary according to the specific types of tumors, and the epidemiological areas. The aim of this study was to investigate the frequency of SV40 in malignant lymphomas among Korean patients. METHODS: One hundred seventy three cases of malignant lymphomas were evaluated by immunohistochemical staining for SV40 large T antigen (TAg), using an extremely sensitive, tyramide based, catalyzed signal amplification method. RESULTS: From 158 non-Hodgkin's lymphomas, including 115 diffuse large B-cell lymphomas, and 15 Hodgkin's lymphomas, none of the cases were positive for SV40 TAg. CONCLUSIONS: SV40 does not appear to be related to the pathogenesis of malignant lymphomas among Koreans.

In vitro Culture of Hepatitis C Virus (HCV) Using Immortalized Hepatocyte / 대한소화기학회지

BACKGROUND/AIMS: It is essential to develop an in vitro culture model of primary hepatocytes for the study of hepatocellular function and the pathogenesis of hepatitis C virus (HCV) infection. In this study, we have established the immortalized primary human hepatocyte (IPHH) and performed in vitro culture of HCV derived from human patient. METHODS: Primary human hepatocytes were isolated from surgically resected liver tissue and then were immortalized by transfection with the SV40 large T antigen. The characterization of the IPHH during culture was analyzed by immunocytochemistry, RT-PCR, Western blot, ELISA, and soft agar assay. Next, sera and/or liver tissue homogenates from surgically resected liver tissues of patients with HCV infection were inoculated for the culture of HCV in IPHH. After HCV RNA extraction from IPHH and culture media, positive or negative stranded HCV RNA was examined by specific nest RT-PCR. RESULTS: IPHH expressed liver-associated proteins but did not express alpha-fetoprotein. Also IPHH showed ammonia removal activity. With regard to its malignant potential, colony formation in soft agar assay was not observed. Next, positive and negative stranded HCV RNAs in IPHH infected with patient's sera plus liver tissue homogenates were clearly detected whereas those in IPHH infected with only patient's sera were not detected. CONCLUSIONS: These results demonstrated the phenotypic characteristics of IPHH and the feasibility in vitro culture system of HCV infected human samples. This system might be useful for study of pathogenesis of HCV infection or hepatocyte-based applications.

Relationship between the malignant mesothelioma and simian virus 40 in China: a study of 17 cases / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate whether simian virus 40 (SV40) was related to patients of malignant mesothelioma in China.</p><p><b>METHODS</b>Paraffin-embeded samples of 17 patients with malignant mesothelioma were collected. After isolation of DNA from paraffin blocks, polymerase chain reaction (PCR) analyses were performed using three different sets of primer for detection of SV40 large T antigen gene. These samples were also immunohistochemically evaluated for expression of SV40 TAg protein with two different anti-SV40 Tag (Pab101 and Ab-2).</p><p><b>RESULTS</b>Only one of the three primer pairs successfully amplified SV40 genome in three malignant mesothelioma samples. No immunopositive staining for SV40 TAg was found in any of the samples.</p><p><b>CONCLUSIONS</b>The study shows that malignant mesothelioma in China may be independent of SV40 infection.</p>

Association of T Antigens with emm Genotypes of Group A Streptococci / 대한임상미생물학회지

BACKGROUND: T typing has been used as a screening test for epidemiologic studies of group A streptococci (GAS) infections or carriers, and M typing has been performed for virulence studies. However, M typing is difficult to perform in routine laboratories. Recently, genotyping of the emm gene, which encodes the M protein, has become available. We investigated which T antigen is closely associated with a certain emmgenotype. METHODS: GAS were collected from the children in Jinju who were asymptomatic carriers (N=349) or had acute pharyngitis (N=122) during the 3 year-period from 2002 through 2004. T typing was performed by a slide aggulutination, and emmgenotyping by PCR and DNA sequencing. RESULTS: More than 90% of T1, T3, T6, T12, T25, and T5/27/44 antigens were associated with emm1, emm3, emm6, emm12 and 22, emm75, and emm44/61 genotypes, respectively; however, other T antigens, such as T2, T4, T7, T11, and B3264, were not associated with any particular emm genotypes. CONCLUSION: Several T antigens are so closely associated with particular emm genotypes that one could predict emmgenotypes based on the result of T typing.

Distribution of T types and emm Genotypes of Streptococcus pyogenes in Jinju, 2004 / 대한진단검사의학회지

BACKGROUND: T antigens and emm genotypes are useful markers for epidemiologic investigation of Streptococcus pyogenes infections. Epidemiologic studies of S. pyogenes were performed on a large scale in Jinju. This was the third study being carried out in the same area over the past 10 years. METHODS: A total of 328 S. pyogenes were isolated from throat cultures obtained from asymptomatic schoolchildren in the Jinju area in 2004. T typing was performed by a slide agglutination, and emm genotyping by PCR and DNA sequencing. We compared the results of this study with those of the previous ones performed in 1995 and 2002. RESULTS: T5/27/44 were the most prevalent, accounting for 29.6% of all isolates; T12 and T6 were 13.4% and 10.7%, respectively, and T nontypeable was 3.4%. The emm44/61 type was the most prevalent accounting for 29.3%, and emm6 and emm1 were 11.6% and 9.8%, respectively. CONCLUSIONS: Newly recognized T5/27/44 and emm44/61 were the most prevalent, accounting for about 30% of all isolates, while T12 and emm12 were significantly decreased in 2004 compared to the results of previous years. This study demonstrated divergent features of S. pyogenes epidemiology over the past 10 years in the Jinju area.

Overexpression of hTERT and c-erbB-2 are correlated in ovarian epithelial cancer / 부인종양

OBJECTIVE: It is still unclear how the abnormal hTERT expression is involved in the process of ovarian carcinogenesis. A recent report demonstrated that the introduction of c-erbB-2 could efficiently induce tumorigenicity of cells with the transfection of SV40 large T antigen and hTERT. It is designed to find correlation between overexpression of hTERT and c-erbB-2 in ovarian carcinogenesis. METHODS: Using immunohistochemistry, we tested whether overexpression of hTERT and c-erbB-2 were associated in ovarian cancer. Immunohistochemical staining of hTERT and c-erbB-2 was done in 63 cases of ovarian cancer. Overexpression of hTERT and c-erbB-2 were correlated to clinicopathological variables. RESULTS: Overexpression of hTERT was found in 7 (11.1%) of cases, whereas overexpression of c-erbB2 was founded in 3 (4.8%) of cases. It was found that overexpression of hTERT and c-erbB-2 were significantly correlated (p=0.03). Neither overexpression of hTERT nor that of c-erbB-2 was associated with any of clinicopathological variables, such as stage, grade, and histology. CONCLUSION: Although the significant correlation between hTERT and c-erbB-2 was found, the low frequency of overexpression of hTERT and c-erbB-2 suggests that cooperation of hTERT and c-erbB-2 may be minor mechanism of ovarian carcinogenesis.

Overexpression of hTERT and c-erbB-2 are correlated in ovarian epithelial cancer / 부인종양

OBJECTIVE: It is still unclear how the abnormal hTERT expression is involved in the process of ovarian carcinogenesis. A recent report demonstrated that the introduction of c-erbB-2 could efficiently induce tumorigenicity of cells with the transfection of SV40 large T antigen and hTERT. It is designed to find correlation between overexpression of hTERT and c-erbB-2 in ovarian carcinogenesis. METHODS: Using immunohistochemistry, we tested whether overexpression of hTERT and c-erbB-2 were associated in ovarian cancer. Immunohistochemical staining of hTERT and c-erbB-2 was done in 63 cases of ovarian cancer. Overexpression of hTERT and c-erbB-2 were correlated to clinicopathological variables. RESULTS: Overexpression of hTERT was found in 7 (11.1%) of cases, whereas overexpression of c-erbB2 was founded in 3 (4.8%) of cases. It was found that overexpression of hTERT and c-erbB-2 were significantly correlated (p=0.03). Neither overexpression of hTERT nor that of c-erbB-2 was associated with any of clinicopathological variables, such as stage, grade, and histology. CONCLUSION: Although the significant correlation between hTERT and c-erbB-2 was found, the low frequency of overexpression of hTERT and c-erbB-2 suggests that cooperation of hTERT and c-erbB-2 may be minor mechanism of ovarian carcinogenesis.

Establishment of Human Bone Marrow-Derived Mesenchymal Stem Cell Lines with SV40 Large T Antigen and Their Differentiation to Chondrocytes / 대한소아혈액종양학회지

PURPOSE: Mesenchymal stem cells (MSC) can be isolated from bone marrow (BM) and when systemically administrated to different species, they undergo site-specific differentiation. In this study, we isolated MSC from human BM and generated a continuously growing colony of cell lines (SNU-hMSC) with SV40 large T antigen. The purposes of this study are to identify whether SNU-hMSC have the characteristics of MSC and their possibility of chondrogenic differentiation. METHODS: MSC were mobilized from BM and cultured in DMEM-LG media for 2 weeks. We obtained SNU-hMSC, by introducing a viral vector of SV40 large T antigen and culturing it in the selected media for 6 months. We identified specific cell markers of MSC via FACS analysis and analyzed expression of cytokines, chemokines and receptors by RT-PCR. To stimulate the proliferation of the cells, we processed the media with FGF, BMP-2 and IL-6. The each medium's cell counts were counted in day 7 and day 14. To differentiate SNU-hMSC, they were cultured in chondrogenic media. After 2 weeks, chondrogenic differentiation was evaluated with safranin-O staining and the expression of COMP, aggrecan and SOX-9. RESULTS: SNU-hMSC exhibited MSC markers. When the IL-6, BMP-2 and FGF were added to each medium, the cell numbers were significantly increased as compared with control. In the study of differentiation, SNU-hMSC exhibited strong safranin-O staining, and chondrogenic gene expression was observed. CONCLUSION: SNU-hMSC expressed markers and cytokines identical with MSC. SNU-hMSC maintained multipotency of differentiation.

Immortalized hepatocytes established with a temperature-sensitive SV40 T antigen could be independent of T antigen / 대한내과학회지

BACKGROUND: Conditionally immortalized hepatocytes (CIH) can be cultured almost indefinitely at permissive temperatures (33 degrees C), but they undergo apoptosis at nonpermissive temperatures (37~39 degrees C) by the release of p53 through inactivation of T antigen, which is called T antigen dependency. This study was aimed at examining if T antigen-independent clones can develop from CIH. METHODS: CIH established with a temperature-sensitive T antigen (WA1) were cultured continuously at 39 degrees C. Three clones (W39B, W39C, and W39J) survived at this temperature and was subject to following analyses: the morphology, growth, apoptosis, the expression of T antigen and p53, telomerase, and the T antigen gene sequence. RESULTS: WA1 proliferated at 33 degrees C with the population doubling time of 30.8 +/- 1.7 hours, but they underwent cell death at 39 degrees C. However, T antigen-independent clones (W39B, W39C, and W39C) proliferated at 39 degrees C without undergoing apoptosis, suggesting they lost the temperature-sensitive characteristics. WA1 expressed the T antigen at 33 degrees C, but not at 39 degrees C, and this temperature-sensitive pattern was maintained in T antigen-independent clones. In p53 expression, however, T antigen-independent clones revealed a different pattern. p53 was detected even at 39 degrees C where it normally would not be detected. Telomerase was activated in all the analyzed cell lines. A temperature-sensitive point mutation at nucleotide position 3505 of the WA1 was retained in all T antigen-independent clones. CONCLUSION: CIH can lost temperature-sensitive characteristics and acquire an ability to proliferate at nonpermissive temperatures. These changes might be related to the change of p53 rather than the change of T antigen itself in these cell lines.

A Experimental Model of Bladder Cancer Using Nonviral Vector Expressing Uroplakin II Promoter and SV40 T Antigen / 대한비뇨기과학회지

PURPOSE: The goal of this study was to constructing a nonviral vector, expressing the chimeric gene of the SV40 T antigen and mouse uroplakin II promoter (UPII promotor), which was uniquely expressed in the urothelium, to aid in the treatment of bladder cancer by the creation of a tumor that will express itself in the bladder only, but that will have no effect on the other urothelium. MATERIALS AND METHODS: 36 female C3H/He mice, weighing 20-25grams, were used in this study. A UPII-GFP-liposome complex was installed into the bladder, with Enhanced Green Fluorescent Protein (EGFP), expressing the bladder mucosa, and analyzed via fluorescent microscopy. A UPII- SV40T-liposome complex was then administered into the bladders of the mice, and the bladder and ureter examined, grossly and microscopically, at 1, 2, 3 and 4 weeks, to find transitional cell carcinomas specific to the bladder, the degree of bladder cancer development, and whether the development was from superficial to deep tumors, as well as tumor metastasis. RESULTS: The expression of EGFP was found in all four mice after 2 days. No development of tumors was evident in any mice. However, of the 6 mice sacrificed 28 days after bladder instillation, urothelial dysplasia was evident in 4. There was no evidence of transient cell carcinomas in the ureter or renal pelvis in any of the mice, or of distant metastasis during the term of the study. CONCLUSIONS: This model of bladder cancer seems to take longer than other models for cancer formation as the carcinogen affects the DNA of urothelial cell for the formation of bladder cancer. However, our bladder cancer model was better than others due to its similarity for the processes of normal bladder cancer formation.

Detection of JC Virus in the Brains of Korean Glioblastoma Multiforme Patients

OBJECTIVE: The ubiquitous human polyomavirus, JC virus(JCV) is the etiologic agent of the fatal demyelinating central nervous system(CNS) disease, progressive multifocal leukoencephalopathy(PML). Recent studies have reported the detection of the JCV in samples derived from several type of human neural tumors and suggested the possible association of JCV with CNS tumors. Here we report for the first time, the presence of JCV in Korean glioblastoma multiforme(GM) patients. METHODS: Two Korean GM patients were assayed for JCV. To detect JCV, we performed immunohistochemical analysis using anti-JCV and anti-glial fibrillary acidic protein(GFAP) serum and polymerase chain reaction(PCR) using primers. RESULTS: JCV antigen was detected in cytoplasm abundantly in cells of this tumor case. Also, GFAP immunoreactivity was predominantly observed in cytoplasm of the cells that were morphologically bizarred appearing astrocytes in GM. In addition, both of the large T antigen gene and the VP1 gene were detected and this result correspond with previous result of immunohistochemistry. CONCLUSION: Although it is not certain that GM is associated with the JCV, we are attempted to elucidate the possible implication of JCV in the tumorigenesis of certain human malignant gliomas.

Role of Transforming Growth Factor beta Induced by Podocyte in the Pathogenesis of Diabetic Nephropathy / 대한신장학회잡지

BACKGROUND: According to the development of methods in podocyte cell culture several studies for the role of podocyte in the progression of glomerulosclerosis have been recently reported. But there is few report for the regulation of TGFbeta1 synthesis and type IV collagen production in podocyte in diabetic nephropathy. We investigated the effects of high glucose and TGFbeta1 in culture medium on TGFbeta1 and type IV collagen production and whether their production is dependent on protein kinase C (PKC) pathway in cultured mouse podocyte cell line. METHODS: Conditionally immortalized mouse podocytes with a temperature-sensitive variant of SV40 large T antigen were cultivated. To propagate podocytes, cells were cultivated at 33 degrees C and treated with gamma-interferon (permissive condition). And to induce differentiation, podocytes were changed at 37 degrees C and deprived of gamma-interferon (non-permissive condition). The effects of high glucose and TGFbeta1 in culture media on procollagen alpha1 (PCalpha1 (IV)) and their relationships to PKC pathway were examined. The mRNA expression and protein production of TGFbeta1 and PCalpha1 (IV) were assayed by reverse transcription - polymerase chain reaction (RT-PCR) and western analysis. RESULTS: Compared with normal glucose (NG, 5.5 mM), high glucose exposure (HG, 15, 30 mM) increased the mRNA expression and protein production of TGFbeta1 and PCalpha1 (IV), but without a statistic significance: TGFbeta1 (after 6 hours: 69.62+/-9.00 vs 83.48+/-7.82 vs 74.49+/-24.73, after 24 hours: 65.06+/-20.55 vs 68.01+/-24.35 vs 94.23+/-13.14), PCalpha1 (IV) (after 6 hours: 109.94+/-10.43 vs 102.00+/-6.68 vs 138.65+/-39.83, after 24 hours: 105.88+/-9.53 vs 83.95+/-1.12 vs 109.14+/-3.29, after 72 hours: 99.18+/-5.30 vs 92.93+/-6.33 vs 109.25+/-4.11). TGFbeta1 significantly decreased the expression of PCalpha1 (IV). Calphostin C treatment further stimulated the increase of PCalpha1 (IV) production induced by HG and inhibited the decreased mRNA expression of PCalpha1 (IV) induced by TGFbeta1 administration. CONCLUSION: We suggest that TGFbeta1 have an important role in podocyte in the pathogenesis of diabetic nephropathy. The HG-induced increases of procollagen alpha1 type IV collagen seems to be negatively regulated by TGFbeta1 and PKC pathway and possibly another pathway will positively regulate the production of PCalpha1 (IV), and these pathways may have a different effect on collagen synthesis dependent on the renal cell type.