[Oncocytic tumours of the kidney-new differential diagnoses]. / Onkozytäre Tumoren der Niere ­ neue Differenzialdiagnosen.

BACKGROUND: Recent developments in differential diagnosis have led to new knowledge about oncocytic renal neoplasms. OBJECTIVES: Overview of differential diagnosis of oncocytic tumours. MATERIALS AND METHODS: We performed a literature search on oncocytic renal tumours and mapped known tumour types. Possible differential diagnoses are discussed. RESULTS: Besides the tumour types already acknowledged by the 2016 WHO classification, there is new evidence regarding the group of hard-to-classify oncocytic neoplasms. Findings point to immunohistochemical and molecular characteristics that may lead to the establishment of new entities in the future. In addition, important differential diagnosis can now be identified, facilitating specific therapies for oncocytic renal tumours. CONCLUSION: A correct diagnosis of oncocytic renal tumours not only improves prognostic assessment (and, if necessary, specific therapies) but is also clinically relevant regarding a possible association with hereditary tumour syndromes.

Pleomorphic lobular carcinoma of the breast: a comparison of cytopathological features with other lobular carcinoma variants.

OBJECTIVE: Pleomorphic lobular carcinoma (PLC) is a subtype of breast cancer with unique morphological features, but it remains controversial whether PLC should be considered an independent disease entity. The aim of this study was to illustrate cytopathological characteristics of PLC in comparison with other lobular carcinoma variants. METHODS: We investigated clinicopathological features of PLC (n = 11) compared with those of other variants of invasive lobular carcinoma (ILC, non-PLC) (n = 32). Histological variants of the non-PLC group consisted of classic (n = 25), solid (n = 2), alveolar (n = 1) and a tubulolobular type (n = 4). A review of cytological reports and fine needle aspiration (FNA) smear samples was performed for the PLC (n = 9) and non-PLC (n = 27) groups. RESULTS: Patients with PLC were older, and had a higher nuclear grade and a higher incidence of axillary lymph node metastasis and triple negative phenotype than non-PLC patients (P = 0.007, P < 0.001, P = 0.02 and P < 0.001, respectively). Cytological findings in PLC included medium- to large-sized nuclei, prominent nucleoli, a moderate-to-severe degree of pleomorphism, apocrine change and background necrosis, none of which were evident in the smears of the non-PLC group (P < 0.001, P = 0.002, P < 0.001, P < 0.001, and P = 0.03, respectively). Despite these differences, patients with PLC and non-PLC showed similar clinical outcomes in our follow-up period. CONCLUSIONS: Based on our results, a cytological diagnosis of PLC should be proposed if there are moderate- to large-sized nuclei, prominent nucleoli, a moderate-to severe degree of nuclear pleomorphism, apocrine change and necrosis in the background in FNA biopsy samples.

Mucinous carcinoma of the breast: a comparative study on cytohistological findings associated with neuroendocrine differentiation.

OBJECTIVE: Mucinous carcinoma (MCA) may show neuroendocrine differentiation (ND), but the cytological features characteristic of ND remains elusive. We compared fine needle aspiration (FNA) findings of MCA between cases with high and low degrees of ND. METHODS: Histological sections of 37 MCA cases were immunohistochemically evaluated for expression of chromogranin A and synaptophysin, and were graded as 0 to 3+ degrees of ND. They were divided into low ND (grade 0 and 1+) and high ND (grade 2+ and 3+) groups. Pre-operative FNA samples of each group were assessed for cytological features. RESULTS: The mean age of the high ND group (n = 18) was higher than the low ND group (n = 19, P = 0.01). In FNA samples of the high ND group, 17 cases showed moderate to severe degrees of discohesiveness, but low ND cases mainly showed no or only mild discohesiveness (P < 0.001). Nine of the low ND cases displayed overlapped, cohesive cell clusters, whereas, in the high ND cases, the cells were arranged in a loose, flat and monolayered pattern (P = 0.045). Fourteen of the high ND cases had round nuclei, but oval nuclei were predominant in the low ND cases (P = 0.027). The nuclei were eccentrically located in 12 of the high ND cases but were centrally located in 14 of the low ND cases (P = 0.01). CONCLUSIONS: Mucinous carcinoma with high ND may be diagnosed by the presence of discohesiveness, a flat, monolayered pattern, and round or eccentrically located nuclei. Features of ND in carcinomas in other organs, such as intracytoplasmic granules and coarse chromatin, may not be reliable cytological features of ND in MCA.

Osteopontin-mediated enhanced hyaluronan binding induces multidrug resistance in mesothelioma cells.

Malignant pleural mesothelioma (MPM) is resistant to chemotherapy and thus shows a dismal prognosis. Osteopontin (OPN), a secreted noncollagenous and phosphoprotein, is suggested to be involved in the pathogenesis of MPM. However, the precise role of OPN, especially in the multidrug resistance of MPM, remains to be elucidated. We therefore established stable transfectants (ACC-MESO-1/OPN), which constitutively express OPN, to determine its role in the chemoresistance observed in MPM. The introduction of the OPN gene provides MPM cells with upregulated multidrug resistance through the mechanism of enhanced hyaluronate (HA) binding. The expression of CD44 variant isoforms, which inhibit HA binding, significantly decreased in ACC-MESO-1/OPN cells in comparison to control transfectants. Interestingly, the inhibition of the HA-CD44 interaction abrogated multidrug resistance in the ACC-MESO-1/OPN, thus suggesting the involvement of the surviving signal emanating from the HA-CD44 interaction. An enhanced level of the p-Akt in ACC-MESO-1/OPN cells was observed, and was diminished by CD44 siRNA. Inhibition of the Akt phosphorylation increased in number of the cells underwent apoptosis induced by NVB, VP-16 and GEM. Collectively, these results indicate that OPN is strongly involved in multidrug resistance by enhancing the CD44 binding to HA.

Dysregulated expression of P1 and P2 promoter-driven hepatocyte nuclear factor-4alpha in the pathogenesis of human cancer.

Hepatocyte nuclear factor-4alpha (HNF4alpha) exists in multiple isoforms that are generated by alternative promoter (P1 and P2) usage and splicing. Here we establish monoclonal antibodies (mAbs) for detecting P1 and P2 promoter-driven HNF4alpha, and evaluate their expression in normal adult human tissues and surgically resected carcinomas of different origins. Using immunohistochemical analysis, we demonstrate that, while P1 promoter-driven HNF4alpha is expressed in hepatocytes, small intestine, colon, kidney and epididymis, P2 promoter-driven HNF4alpha is expressed in bile duct, pancreas, stomach, small intestine, colon and epididymis. Altered expression patterns of P1 and P2 promoter-driven HNF4alpha were observed in gastric, hepatocellular and colorectal carcinomas. HNF4alpha was expressed in lung metastases from renal cell, hepatocellular and colorectal carcinoma but was not observed in lung tumours. The P1 and P2 promoter-driven HNF4alpha expression pattern of tumour metastases correlated with the primary site of origin. P1 promoter-driven HNF4alpha was also found in intestinal metaplasia of the stomach. These data provide evidence for the tissue distribution of P1 and P2 promoter-driven HNF4alpha at the protein level and suggest that HNF4alpha may be a novel diagnostic marker for metastases of unknown primary. We propose that the dysregulation of alternative promoter usage of HNF4alpha is associated with the pathogenesis of certain cancers.

Reverse cholesterol transport and cholesterol efflux in atherosclerosis.

Reverse cholesterol transport (RCT) is a pathway by which accumulated cholesterol is transported from the vessel wall to the liver for excretion, thus preventing atherosclerosis. Major constituents of RCT include acceptors such as high-density lipoprotein (HDL) and apolipoprotein A-I (apoA-I), and enzymes such as lecithin:cholesterol acyltransferase (LCAT), phospholipid transfer protein (PLTP), hepatic lipase (HL) and cholesterol ester transfer protein (CETP). A critical part of RCT is cholesterol efflux, in which accumulated cholesterol is removed from macrophages in the subintima of the vessel wall by ATP-binding membrane cassette transporter A1 (ABCA1) or by other mechanisms, including passive diffusion, scavenger receptor B1 (SR-B1), caveolins and sterol 27-hydroxylase, and collected by HDL and apoA-I. Esterified cholesterol in the HDL is then delivered to the liver for excretion. In patients with mutated ABCA1 genes, RCT and cholesterol efflux are impaired and atherosclerosis is increased. In studies with transgenic mice, disruption of ABCA1 genes can induce atherosclerosis. Levels of HDL are inversely correlated with incidences of cardiovascular disease. Supplementation with HDL or apoA-I can reverse atherosclerosis by accelerating RCT and cholesterol efflux. On the other hand, pro-inflammatory factors such as interferon-gamma (IFN-gamma), endotoxin, tumour necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1beta), can be atherogenic by impairing RCT and cholesterol efflux, according to in vitro studies. RCT and cholesterol efflux play a major role in anti-atherogenesis, and modification of these processes may provide new therapeutic approaches to cardiovascular disease. Further research on new modifying factors for RCT and cholesterol efflux is warranted.

Salivary cystatins influence ingestion of capsaicin-containing diets in the rat.

Dietary capsaicin consumed by rats over several days induces cystatin-like substances in submandibular saliva. Yet the physiological role of these salivary proteins has not been thoroughly investigated. Salivary cystatins in the rat submandibular glands are known to be induced by chronic treatment with the sympathetic beta-agonist, isoproterenol. In the present study, the possible roles of the salivary proteins on food intake were examined by comparing consumption of a capsaicin-adulterated (0.05%) diet in rats with and without isoproterenol pretreatment (0.1 and 5.0 mg/kg, 5 days). Electrophoretic analysis performed prior to feeding trials revealed that the group pretreated with 5 mg/kg isoproterenol had large amounts of cystatin in the saliva compared with the group pretreated with 0.1 mg/kg isoproterenol and control group. The group treated with 5 mg/kg isoproterenol showed greater consumption of the capsaicin-adulterated diet than the other groups until the 3rd day of trials. Bilateral removal of the submandibular and sublingual glands neutralized the effects of isoproterenol. Induction of salivary cystatins by isoproterenol treatment was not mimicked by systemic and intragastric administration of capsaicin. These results suggest that cystatins are included in the salivary proteins induced by capsaicin and that they contribute to enhanced ingestion of the capsaicin diet. Induction of salivary cystatins may be triggered by irritation of the oral mucosa by capsaicin.

Vascular endothelial growth factor enhances glomerular capillary repair and accelerates resolution of experimentally induced glomerulonephritis.

Vascular endothelial growth factor (VEGF) regulates angiogenesis through endothelial cell proliferation and plays an important role in capillary repair in damaged glomeruli. We tested the hypothesis that VEGF might be beneficial in rats with severe glomerular injury in glomerulonephritis (GN) based on its angiogenic and vascular remodeling properties. Acute GN with severe glomerular destruction was induced in rats by injection of anti-Thy-1.1 antibody (day 0) and Habu-snake venom (day 1). Rats were intraperitoneally injected with recombinant human VEGF(165) (10 microg/100 g body wt/day) or vehicle from day 2 to day 9, and monitored changes in glomerular capillaries, development of glomerular inflammation, and progression to glomerular sclerosis after acute glomerular destruction in both groups. Rats that received anti-Thy-1.1 antibody and Habu-snake venom showed severe mesangiolysis and marked destruction of capillary network on day 2. VEGF was expressed on glomerular epithelial cells, proliferating mesangial cells, and some infiltrating leukocytes, and VEGF(165) protein levels increased in damaged glomeruli during day 5 to day 7. Normal, damaged, and regenerating glomerular endothelial cells expressed VEGF receptor flk-1. However, endothelial cell proliferation and capillary repair was rare in vehicle-treated rats with severe glomerular damage, which progressed to global sclerosis and chronic renal failure by week 8. In contrast, in the VEGF-treated group, VEGF(165) significantly enhanced endothelial cell proliferation and capillary repair in glomeruli by day 9 (proliferating endothelial cells: VEGF(165), 4.3 +/- 1.1; control, 2.2 +/- 0.9 cells on day 7, P < 0.001; and glomerular capillaries: VEGF(165), 24.6 +/- 4.8; control, 16.9 +/- 3.4 capillaries on day 7, P < 0.01). Thereafter, damaged glomeruli gradually recovered after development of capillary network by week 8, and significant improvement of renal function was evident in the VEGF-treated group during week 8 (creatinine: VEGF(165), 0.3 +/- 0.1; control, 2.6 +/- 0.9 mg/dl, P < 0.001; proteinuria: VEGF(165), 54 +/- 15; control, 318 +/- 60 mg/day, P < 0.001). We conclude that the beneficial effect of VEGF(165) in severe glomerular injury in GN emphasizes the importance of capillary repair in the resolution of GN, and may allow the design of new therapeutic strategies against severe GN.

Microsatellite instability correlates with normal expression of cyclin E in hepatocellular carcinomas.

It has been reported that microsatellite instability (MSI) strongly correlates with carcinogenesis and cancer progression. In the present study, we studied the incidence of MSI at 5 polymorphic microsatellite markers (D5S406, D13S153, D16S402, D17S796, and poly(A) tract BAT26), the expression of G1 cyclins (cyclin A, cyclin D and cyclin E), and Ki-67 labeling index in 30 surgically resected hepatocellular carcinomas (HCCs) and their adjacent non-cancerous tissues. The results of analysis showed that 43% of HCCs exhibited MSI in one locus, 10% in two loci, and 3% in three loci. Overexpressions of cyclin E and cyclin A were observed in 57% and 83% of HCCs, respectively. MSI in HCCs, however, correlated with normal expressions of cyclin E and cyclin A and with a low labeling index of Ki-67. Thus, patients with HCCs exhibiting MSI at these 5 markers may have less involvement of G1/S disregulation and may have better prognosis than other patients with HCC.

Enhanced expression of cyclin E and cyclin A in human hepatocellular carcinomas.

BACKGROUND: Disruption of the G1/S check point leads to uncontrolled cell growth, resulting in the development of cancers. MATERIALS AND METHODS: Cell cycle regulatory molecules were investigated in 33 surgically resected hepatocellular carcinoma (HCC) samples from 30 patients by Western blotting. RESULTS: Enhanced expressions of cyclin E and cyclin A were detected at frequencies of 18/33 and 26/33 in HCCs, respectively, as compared with their neighboring noncancerous tissues. The enhanced expression of cyclin E, but not that of cyclin A, correlated with hyperphosphorylation of pRb and high frequency of Ki-67-positive cells. Thus, the HCCs with enhanced cyclin E expression probably contain a relatively large number of proliferating cancer cells. CONCLUSIONS: The degree of cyclin E expression can be used as a prognostic parameter of HCC. In addition, cyclin E may become a molecular target in the treatment of HCCs.

Different expression of positive and negative regulators of hepatocyte growth in growing and shrinking hepatic lobes after portal vein branch ligation in rats.

Portal vein branch embolization is often performed before hepatectomy to prevent postoperative liver failure. It is, however, still not clear how the embolized lobe shrinks and the non-embolized lobe proliferates in counterbalance. We investigated the expression of positive and negative regulators of hepatocyte growth to clarify the mechanisms of liver growth and atrophy in a rat portal vein ligation (PVL) model compared with partial hepatectomy (PH). A significant increase in DNA synthesis within the non-ligated lobe reached a peak at 36 h, a delay of 12 h as compared with PH, while no increase occurred in the ligated lobe. Expression of hepatocyte growth factor mRNA remarkably increased in the non-ligated growing lobe between 6 and 24 h, but was only slightly elevated in the ligated shrinking lobe. Contrarily, negative regulators of hepatocyte proliferation, such as TGF-beta1 and IL-1beta, were strongly expressed in the ligated shrinking lobe. Thus, the changes of portal venous flow and/or pressure caused by PVL may contribute to induction of different kinds of growth factors between the ischemic and non-ischemic lobes; these factors possibly regulate liver regeneration and atrophy after PVL.

Yeast functional assay of the p53 gene status in 11 cell lines and 26 surgical specimens of human hepatocellular carcinoma.

The structural abnormalities of the p53 gene have frequently been detected in hepatocellular carcinomas (HCCs). To ascertain whether or not functional inactivation of this gene also occurs in HCCs, the transactivation of p53 gene products in 11 HCC cell lines maintained in our laboratory and 26 HCC surgical specimens was examined by yeast functional assay (functional analysis of separated alleles in yeast: FASAY), which determines the functional status of the individual p53 alleles. The p53 gene product was inactivated in 8 of 11 (72.7%) HCC cell lines and in 12 of 26 (46.2%) HCC surgical specimens. The inactivation frequency of the gene was 37.5%, 36.4%, and 71.4% in well, moderately, and poorly differentiated HCCs, respectively. In HCC surgical specimens larger than 5 cm in diameter, the inactivation frequency was 83.3% while in those smaller than 2. 5 cm, it was 14.3%. These results show that functional inactivation of p53 gene products often occurs in HCCs and that the inactivation frequency of the gene in HCCs is well correlated with differentiation degree and tumor size, suggesting that the inactivation of p53 gene products plays a role in progression from well to poorly differentiated HCC.

Enhanced activity of cyclin A-associated kinase in immortalized human fibroblasts.

Deregulation of cell cycle regulatory mechanisms leads to disruption of normal control of cell growth and may be associated with neoplastic transformation. To determine whether immortalized human cells have alterations in their cell cycle regulatory mechanisms, we analyzed cell cycle regulatory proteins in 2 immortalized human fibroblast cell lines, KMST-6 and OUMS-24/P6X, established by repeated irradiation with (60)Co gamma rays alone or mutant p53 gene transfection plus X-ray irradiation, respectively. Both the immortalized cell lines had markedly enhanced activity of cyclin A-associated kinase as compared with their normal counterparts. The high activity of cyclin A-associated kinase was well correlated with the increased expression of cyclin A mRNA and its protein. In addition, the immortalized cell lines showed significantly reduced amounts of p21(Cip1/Waf1/Sdi1), a potent inhibitor of cyclin dependent kinases. Furthermore, among the pRb family of proteins, p107 and p130, were hyperphosphorylated in both the immortalized cell lines, suggesting possible participation in upregulation of cyclin A associated kinase activity. These changes represent some important characteristics of immortalized cells.

Association of angiotensin-converting enzyme gene polymorphism and renal pathology in Japanese children with IgA nephropathy.

Polymorphism of the gene that codes for angiotensin I-converting enzyme (ACE) is associated with increased severity of immunoglobulin A (IgA) nephropathy in adult patients. We evaluated the relationship between the polymorphism of ACE genotypes and the pathological and clinical findings in Japanese children with IgA nephropathy. Patients with moderate/diffuse mesangial proliferation, glomerular sclerosis and tubulointerstitial damage showed a significant increase of the D/D type compared to those who had mild/focal mesangial proliferation, without glomerular sclerosis or tubulointerstitial damage (p < 0.05). Proteinuria at the first renal biopsy was significantly higher in the former group compared with the latter group except glomerular sclerosis (p < 0.01). IgA nephropathy patients with tubulointerstitial damage also showed an increased serum creatinine level compared to patients without the damage (p < 0.03). We conclude that ACE gene polymorphism may be correlated with the prognosis of IgA nephropathy in Japanese children.

Maintenance of near-diploid karyotype of PA-1 human ovarian teratocarcinoma cells due to death of polyploid cells by chromosome fragmentation/pulverization.

Chromosome instability (polyploidy or aneuploidy) is one of the characteristics of malignant tumors. Human teratocarcinoma cell line PA-1, which was established more than 10 years ago, consists of a majority of near-diploid cells and a minority of polyploid cells, indicating that it is karyologically very stable. In the present study we investigated this genomic stability from the view point of cytogenetics. Cleavages and breaks in the chromosome were found in the metaphase of PA-1 polyploid cells, accompanied by the formation of polynucleosomal DNA fragments. These findings were absent in the near-diploid cells. In addition, polyploid cells did not show colony-formation ability by in situ analysis of cytogenetics in each colony. Thus, the maintenance of the near-diploid karyotype in PA-1 cells may be due to a blockage in the M-phase of the polyploid cells by functional mitotic checkpoints, if any, leading to cell death due to inability to enter the next cell cycle.

Contribution of organic anion transporting polypeptide to uptake of its possible substrates into rat hepatocytes.

Organic anion transporting polypeptide (oatp1) has been cloned from rat liver as one of the transporters responsible for the hepatic uptake of ligands, and its substrate specificity has been determined. However, the contribution of oatp1 to the Na+-independent uptake of ligands into rat hepatocytes remains to be investigated. In the present study, we determined the contribution of oatp1 and examined the uptake of ligands into primary cultured hepatocytes (cultured for 4 h) and into COS-7 cells transiently expressing oatp1 and normalized using estradiol-17beta-D-glucuronide as a reference compound. Western blot analysis indicated that oatp1 was less extensively glycosylated in transfected COS-7 cells, and the expression level in transfectant was one-seventh that in rat liver. The Km values for the uptake of estradiol-17beta-D-glucuronide were similar for cultured hepatocytes and oatp1-transfected COS-7 cells (Km = 12.3 versus 20.4 microM), although the Vmax value for oatp1-transfected COS-7 cells was one-seventh that for cultured hepatocytes (Vmax = 1.30 versus 0.175 nmol/min/mg protein). The contribution of oatp1 to the Na+-independent uptake of taurocholic acid and cholic acid into rat hepatocytes was more than 50 to 60%, whereas the corresponding values for the sulfate-conjugates of estrone and 6-hydroxy-5, 7-dimethyl-2-methylamino-4-(3-pyridylmethyl)benzothiazole were 20 to 30%. In addition, the analysis indicated that the contribution of oatp1 to the Na+-independent uptake of several ligands [glucuronide-conjugate of 6-hydroxy-5, 7-dimethyl2-methylamino-4-(3-pyridylmethyl)benzothiazole, ibuprofen, pravastatin, ouabain, and 2,4-dinitrophenyl-S-glutathione] was minimal. Collectively, the transfected COS-7 cells may be used to quantitatively predict oatp1 activity in hepatocytes after correction of its expressed amount. It is also suggested that multiple transport mechanisms are responsible for the Na+-independent uptake of organic anions into hepatocytes.

Molecular and functional identification of sodium ion-dependent, high affinity human carnitine transporter OCTN2.

Primary carnitine deficiency, because of a defect of the tissue plasma membrane carnitine transporters, causes critical symptoms. However, the transporter has not been molecularly identified. In this study, we screened a human kidney cDNA library and assembled a cDNA-encoding OCTN2 as a homologue of the organic cation transporter OCTN1, and then we examined the function of OCTN2 as a carnitine transporter. OCTN2-cDNA encodes a polypeptide of 557 amino acids with 75.8% similarity to OCTN1. Northern blot analysis showed that OCTN2 is strongly expressed in kidney, skeletal muscle, heart, and placenta in adult humans. When OCTN2 was expressed in HEK293 cells, uptake of L-[3H]carnitine was strongly enhanced in a sodium-dependent manner with Km value of 4.34 microM, whereas typical substrates for previously known organic cation transporters, tetraethylammonium and guanidine, were not good substitutes. OCTN2-mediated L-[3H]carnitine transport was inhibited by the D-isomer, acetyl-D,L-carnitine, and gamma-butyrobetaine with high affinity and by glycinebetaine with lower affinity, whereas choline, beta-hydroxybutyric acid, gamma-aminobutyric acid, lysine, and taurine were not inhibitory. Because the observed tissue distribution of OCTN2 is consistent with the reported distribution of carnitine transport activity and the functional characteristics of OCTN2 coincide with those reported for plasma membrane carnitine transport, we conclude that OCTN2 is a physiologically important, high affinity sodium-carnitine cotransporter in humans.

Contribution of sodium taurocholate co-transporting polypeptide to the uptake of its possible substrates into rat hepatocytes.

As one of the Na+-dependent transporters responsible for the hepatic uptake of ligands, sodium taurocholate (TC) co-transporting polypeptide (NTCP) has been cloned from rat liver and its substrate specificity has been clarified by examining the inhibition of TC uptake mediated by NTCP. The contribution of NTCP to the Na+-dependent uptake of ligands into rat hepatocytes, however, still needs to be clarified. To determine the contribution of NTCP, we examined the uptake of ligands into primary cultured hepatocytes (cultured for 4 h) and into COS-7 cells, transiently expressing NTCP, and normalized the uptake of ligands with TC as a reference compound. Western Blot analysis indicated that NTCP was glycosylated much less extensively in the transfected COS-7 cells, although the expression level was comparable for the cultured hepatocytes and transfectant. Kinetic parameters for the Na+-dependent uptake of TC were similar for the cultured hepatocytes and NTCP-transfected COS-7 cells (Km = 17.7 vs. 17.4 microM; Vmax = 1.63 vs. 1.45 nmol/min/mg protein). Glycocholic acid and cholic acid were taken up by NTCP-transfected COS-7 cells. The contribution of NTCP to the Na+-dependent uptake of glycocholic acid into rat hepatocytes was approximately 80%, whereas that of cholic acid was 40%. In addition, the analysis indicated that the contribution of NTCP to the Na+-dependent uptake of several ligands (ouabain, ibuprofen, glutathione-conjugate of bromosulfophthalein, glucuronide- and sulfate-conjugates of 6-hydroxy-5, 7-dimethyl-2-methylamino-4-(3-pyridylmethyl) benzothiazole) was negligible. Thus, this is a convenient method to determine the contribution of NTCP to the uptake of ligands into hepatocytes. It is also suggested that multiple transport mechanisms are responsible for the Na+-dependent uptake of organic anions into hepatocytes.

Transforming growth factor-beta 1 stimulates or inhibits cell growth via down- or up-regulation of p21/Waf1.

Transforming growth factor-beta (TGF-beta) regulates cell proliferation positively or negatively. The mitoinhibition by TGF-beta has been attributed to induction of cyclin-dependent kinase (CDK) inhibitors, such as p15/ Ink4B, p27/Kip1, and p21/Waf1 also known as Cip1 and Sdi1. However, the biological process by which TGF-beta exerts the stimulatory effects on cell growth remains poorly understood. Here we report that TGF-beta 1 stimulates DNA synthesis of IMR-90 human embryonic lung fibroblasts but inhibits that of HuCCT1 human cholangiocarcinoma cells, via down- or up-regulation of p21/Waf1, respectively. TGF-beta 1 markedly suppresses IMR-90 cells to express two different kinds of the p21/Waf1 gene transcription factors, the p53 tumor suppressor and the interferon regulatory factor-1 (IRF-1). This is followed by a marked decrease in expression of p21/Waf1 in a manner consistent with the timing of activation of cyclin E-associated kinase, which normally accompanies the G1-S transition in the cell cycle. Contrarily, TGF-beta 1-induced inhibition of DNA synthesis in HuCCT1 cells is preceded by IRF-1-dependent but p53-independent up-regulation of p21/Waf1 expression followed by inactivation of cyclin E-associated kinase. Thus the cell growth stimulation or inhibition by TGF-beta 1 are mediated by the down- or up-regulation of p21/ Waf1, respectively.

Ubiquitous presence of cellular proteins that specifically bind to the 3' terminal region of hepatitis C virus.

The 3' terminal region (3'-X tail) of hepatitis C virus (HCV) genomic RNA forms a stable stem-loop structure. The 3'-X tail consists of 98 nucleotides (nt) that are highly conserved among the HCV strains and supposed to function as a cis-acting region for replication of negative strand RNA and/or viral encapsidation. In the present study, by UV cross-linking assay we found two kinds of cellular proteins of approximately 87 and 130 kDa, which specifically bind to the full-length 3'-X tail (nt 1 to 98), but not the 3'- or 5'-truncated 3'-X tail, consisting of nt 1 to 50 or nt 51 to 98, respectively. These proteins were detected in human cell lines such as hepatic tumor cell lines and a T-lymphocyte cell line and also in a human embryonic lung fibroblast cell strain. In addition, human hepatocellular carcinoma tissues expressed these proteins regardless of infection or uninfection of HCV. Furthermore, these proteins were also detected in normal human tissues derived from the lung, heart, kidney, stomach, intestine, and colon. Thus, these cellular proteins, which are ubiquitously present in human tissues, might be involved in viral replication and/or encapsidation.