Factors Prognostic for Survival in Japanese Patients Treated with Sunitinib as First-line Therapy for Metastatic Clear Cell Renal Cell Cancer.

BACKGROUND: Factors predictive of survival have been identified in Western patients with metastatic clear cell renal cell carcinoma (mCCRCC) treated with sunitinib. Less is known, however, about factors predictive of survival in Japanese patients. This study evaluated factors prognostic of survival in Japanese patients with mCCRCC treated with first-line sunitinib. MATERIALS AND METHODS: This retrospective study evaluated 46 consecutive Japanese mCCRCC patients treated with sunitinib as first line therapy. Clinical and biochemical markers associated with progression-free survival (PFS) were analyzed, with prognostic factors selected by uni- and multivariate Cox regression analyses. RESULTS: Univariate analysis showed that factors significantly associated with poor PFS included Memorial Sloan-Kettering Cancer Center poor risk scores, International Metastatic RCC Database Consortium poor risk and high (>0.5 mg/dl) serum C-reactive protein (CRP) concentrations (p<0.001 each). Multivariate analysis showed that high serum CRP was independently associated with poorer PFS (p=0.040). Six month disease control rate (complete response, partial response and stable disease) in response to sunitinib was significantly higher in patients with normal (≤0.5 mg/dl) than elevated baseline CRP (p<0.001). CONCLUSIONS: CRP is a significant independent predictor of PFS for Japanese patients with mCCRCC treated with first-line sunitinib. Pretreatment CRP concentration may be a useful biomarker predicting response to sunitinib treatment.

Prognostic significance of amino-acid transporter expression (LAT1, ASCT2, and xCT) in surgically resected tongue cancer.

BACKGROUND: Amino-acid transporters are necessary for the tumour cell growth and survival, and have a crucial role in the development and invasiveness of cancer cells. But, it remains unclear about the prognostic significance of L-type amino-acid transporter 1 (LAT1), system ASC amino-acid transporter-2 (ASCT2), and xCT expression in patients with tongue cancer. We conducted the clinicopathological study to investigate the protein expression of these amino-acid transporters in tongue cancer. METHODS: Eighty-five patients with surgically resected tongue cancer were evaluated. Tumour sections were stained by immunohistochemistry for LAT1, ASCT2, xCT, 4F2hc/CD98hc (4F2hc), Ki-67, and microvessel density (MVD) determined by CD34, and p53. RESULTS: L-type amino-acid transporter 1 and 4F2hc were highly expressed in 61% (52 out of 85) and 45% (38 out of 47), respectively. ASC amino-acid transporter-2 and xCT were positively expressed in 59% (50 out of 85) and 21% (18 out of 85), respectively. The expression of both LAT1 and ASCT2 was significantly associated with disease staging, lymph-node metastasis, lymphatic permeation, 4F2hc expression and cell proliferation (Ki-67). xCT expression indicated a significant association with advanced stage and tumour factor. By univariate analysis, disease staging, lymphatic permeation, vascular invasion, LAT1, ASCT2, 4F2hc, and Ki-67 had a significant relationship with overall survival. Multivariate analysis confirmed that LAT1 was an independent prognostic factor for predicting poor prognosis. CONCLUSIONS: L-type amino-acid transporter 1 and ASCT2 can serve as a significant prognostic factor for predicting worse outcome after surgical treatment and may have an important role in the development and aggressiveness of tongue cancer.

ASC amino-acid transporter 2 (ASCT2) as a novel prognostic marker in non-small cell lung cancer.

BACKGROUND: ASC amino-acid transporter 2 (ASCT2) is a major glutamine transporter that has an essential role in tumour growth and progression. Although ASCT2 is highly expressed in various cancer cells, the clinicopathological significance of its expression in non-small cell lung cancer (NSCLC) remains unclear. METHODS: One hundred and four patients with surgically resected NSCLC were evaluated as one institutional cohort. Tumour sections were stained by immunohistochemistry (IHC) for ASCT2, Ki-67, phospho-mTOR (mammalian target of rapamycin), and CD34 to assess the microvessel density. Two hundred and four patients with NSCLC were also validated by IHC from an independent cohort. RESULTS: ASC amino-acid transporter 2 was expressed in 66% of patients, and was closely correlated with disease stage, lymphatic permeation, vascular invasion, CD98, cell proliferation, angiogenesis, and mTOR phosphorylation, particularly in patients with adenocarcinoma (AC). Moreover, two independent cohorts confirmed that ASCT2 was an independent marker for poor outcome in AC patients. CONCLUSIONS: ASC amino-acid transporter 2 expression has a crucial role in the metastasis of pulmonary AC, and is a potential molecular marker for predicting poor prognosis after surgery.

Prognostic significance of L-type amino-acid transporter 1 expression in surgically resected pancreatic cancer.

BACKGROUND: The expression of L-type amino-acid transporter 1 (LAT1) is tumour-specific and has been shown to have essential roles in cell growth and survival. However, little is known regarding the clinical significance of LAT1 expression in pancreatic cancer. This study was conducted to determine the prognostic significance of LAT1 expression. METHODS: A total of 97 consecutive patients with surgically resected pathological stage I-IV pancreatic ductal adenocarcinoma were retrospectively reviewed. Tumour sections were stained by immunohistochemistry for LAT1, CD98, Ki-67 and vascular endothelial growth factor (VEGF), and microvessel density was determined by CD34 and p53. RESULTS: L-type amino-acid transporter 1 and CD98 were highly expressed in 52.6% (51/97) and 56.7% (55/97) of cases, respectively (P=0.568). The expression of LAT1 within pancreatic cancer cells was significantly associated with disease stage, tumour size, Ki-67, VEGF, CD34, p53 and CD98. L-type amino-acid transporter 1 expression was confirmed to be a significant prognostic factor for predicting poor outcome by multivariate analysis. CONCLUSION: L-type amino-acid transporter 1 expression is a promising pathological marker for the prediction of outcome in patients with pancreatic cancer.

Pathogenic GLUT9 mutations causing renal hypouricemia type 2 (RHUC2).

Renal hypouricemia (MIM 220150) is an inherited disorder characterized by low serum uric acid levels and has severe complications such as exercise-induced acute renal failure and urolithiasis. We have previously reported that URAT1/SLC22A12 encodes a renal urate-anion exchanger and that its mutations cause renal hypouricemia type 1 (RHUC1). With the large health-examination database of the Japan Maritime Self-Defense Force, we found two missense mutations (R198C and R380W) of GLUT9/SLC2A9 in hypouricemia patients. R198C and R380W occur in highly conserved amino acid motifs in the "sugar transport proteins signatures" that are observed in GLUT family transporters. The corresponding mutations in GLUT1 (R153C and R333W) are known to cause GLUT1 deficiency syndrome because arginine residues in this motif are reportedly important as the determinants of the membrane topology of human GLUT1. Therefore, on the basis of membrane topology, the same may be true of GLUT9. GLUT9 mutants showed markedly reduced urate transport in oocyte expression studies, which would be the result of the loss of positive charges in those conserved amino acid motifs. Together with previous reports on GLUT9 localization, our findings suggest that these GLUT9 mutations cause renal hypouricemia type 2 (RHUC2) by their decreased urate reabsorption on both sides of the renal proximal tubule cells. However, a previously reported GLUT9 mutation, P412R, was unlikely to be pathogenic. These findings also enable us to propose a physiological model of the renal urate reabsorption via GLUT9 and URAT1 and can lead to a promising therapeutic target for gout and related cardiovascular diseases.

Establishment of a human hepatocyte line (OUMS-29) having CYP 1A1 and 1A2 activities from fetal liver tissue by transfection of SV40 LT.

Immortalized human hepatocytes that can retain functions of drug-metabolizing enzymes would be useful for medical and pharmacological studies and for constructing an artificial liver. The aim of this study was to establish immortalized human hepatocyte lines having differentiated liver-specific functions. pSVneo deoxyribonucleic acid, which contains large and small T genes in the early region of simian virus 40, was introduced into hepatocytes that had been obtained from the liver of a 21-wk-old fetus. Neomycin-resistant immortalized colonies were cloned and expanded to mass cultures to examine hepatic functions. Cells were cultured in a chemically defined serum-free medium, ASF104, which contains no peptides other than recombinant human transferrin and insulin. As a result, an immortal human hepatocyte cell line (OUMS-29) having liver-specific functions was established from one of the 13 clones. Expression of CYP 1A1 and 1A2 messenger ribonucleic acid by the cells was induced by treatment with benz[a]pyrene, 3-methylcholanthrene, and benz[a]anthracene. OUMS-29 cells had both the polycyclic aromatic hydrocarbon receptor (AhR) and AhR nuclear translocator. Consequently 7-ethoxyresorufin deethylase activity of the cells was induced time- and dose-dependently by these polycyclic aromatic hydrocarbons. This cell line is expected to be instrumental as an alternative method in animal experiments for studying hepatocarcinogenesis, drug metabolisms of liver cells, and hepatic toxicology.

Developments in bioartificial liver research: concepts, performance, and applications.

As an alternative to liver transplantation, numerous researchers have been working toward the goal of development of a fully functional artificial liver. In recent years, artificial liver support systems have been advocated as interim treatments for patients awaiting hepatocyte replacement therapy or liver transplantation; so-called "bridging" treatments. It is recognized that an effective artificial liver system requires: (1) a viable and highly functional hepatocyte cell line, (2) a suitable bioreactor environment and peripheral control systems, and (3) an effective extracorporeal circulatory system to incorporate an artificial liver system. Conventional systems have, however, suffered from various drawbacks, including incompatibility of cell cultures derived from non-human cells, insufficient cell proliferation, rapid deterioration of cellular function due to an impoverished cellular environment, and lack of system scalability. A newly established artificial liver system overcomes many of these problems and demonstrates a long-term capacity to maintain multiple liver-specific functions, such as protein synthesis, enzyme activity, and drug metabolism, both quantitatively and qualitatively. The present review provides an overview of the concepts underpinning artificial liver systems, the performance of presently available systems and the practical applications of available systems and those in development.

Two SecG molecules present in a single protein translocation machinery are functional even after crosslinking.

SecG, a membrane component of the protein translocation apparatus of Escherichia coli, undergoes membrane topology inversion, which is coupled to the membrane insertion and deinsertion cycle of SecA. Eighteen SecG derivatives possessing a single cysteine residue at various positions were constructed and expressed in a secG null mutant. All the SecG-Cys derivatives retained the SecG function, and stimulated protein translocation both in vivo and in vitro. Inverted membrane vesicles containing a SecG-Cys derivative were labeled with a membrane-permeable or -impermeable sulfhydryl reagent before or after solubilization with a detergent. The accessibility of these reagents to the cysteine residue of each derivative determined the topological arrangement of SecG in the membrane. Derivatives having the cysteine residue in the periplasmic region each existed as a homodimer crosslinked through disulfide bonds, indicating that two SecG molecules closely co-exist in a single translocation machinery. The crosslinking did not abolish the SecG function and the crosslinked SecG dimer underwent topology inversion upon protein translocation.

Involvement of insulin-like growth factor binding protein-3 in the retinoic acid receptor-alpha-mediated inhibition of hepatocellular carcinoma cell proliferation.

We examined the relationship between the expression of retinoic acid receptor-alpha (RAR-alpha) and upregulation of insulin-like growth factor binding protein-3 (IGFBP-3) in the retinoid-induced inhibition of hepatocellular carcinoma (HCC) cell proliferation. HCC cell lines showed a marked expression of RAR-alpha, whereas the expression levels of RAR-beta and RAR-gamma were relatively lower. An RAR-alpha agonist significantly inhibited the HCC cell proliferation both in vitro and in vivo. The RAR-alpha expression closely related to the upregulation of IGFBP-3 as compared with RAR-beta or RAR-alpha expressions. RAR-alpha agonist would be beneficial to inhibit the growth of HCC.

Retinol esterification activity contributes to retinol transport in stellate cells.

The mechanisms of retinol transport and accumulation in hepatic stellate cells (HSC) remain to be elucidated. Our previous studies suggested that retinol esterification activity, particularly lecithin:retinol acyltransferase (LRAT) activity, in liver retinoid metabolism is important to elucidate the relationship between retinol uptake by HSC and the esterification of retinol. In the present study, using a human HSC-like cell line, LI90, we demonstrated that retinol esterification activity of LI90 cells is similar to that of primary cultures of rat HSC and higher than that of a human hepatoma cell line. Further, since progesterone or diphospho-lauroyl-phosphatidylcholine increased retinol esterification activity of LI90 cells, it is likely that LRAT contributes to retinol esterification in LI90. We examined retinol esterification in LI90 cells and clearance of retinol from culture medium. The percentages of both retinol and esterified retinol in LI90 cells increased in a manner dependent on retinol concentration in medium, whereas that of retinol in medium decreased. The percentages of esterified and unesterified retinol in LI90 cells and of retinol in medium were linearly dependent on the logarithm of the initial concentration of retinol in the medium. These results suggest that retinol esterification activity contributes to retinol uptake by HSC and maintenance of non-toxic retinol levels in plasma.

Nuclear translocation of Fos is stimulated by interaction with Jun through the leucine zipper.

Jun and Fos, b-ZIP transcription factors, form a heterodimer and bind to DNA enhancer elements, thereby regulating the expression of target genes. The present study was undertaken to investigate the molecular mechanism underlying nuclear translocation of the Jun/Fos complex. For this purpose, normal rat kidney cells were microinjected with a DNA expression vector containing wild-type or mutant c- or v-jun together with c- or v-fos, followed by detection of the subcellular localization of Jun or Fos by immunofluorescence staining. The nuclear accumulation of Fos was markedly enhanced by the presence of wildtype Jun, but not by Jun mutants lacking nuclear targeting or zipper dimerization functions, implying that Jun and Fos mutually interact via their leucine zippers and translocate from the cytoplasm to the nucleus using the markedly stronger nuclear localization signal of Jun.

Invasiveness of hepatocellular carcinoma cell lines: contribution of membrane-type 1 matrix metalloproteinase.

Intrahepatic metastasis is one of the malignant features of hepatocellular carcinoma (HCC). Matrix metalloproteinases (MMPs) and urokinase-type plasminogen activator (u-PA)/plasmin, are known to be associated with the invasive properties of various types of tumor cells. In this study, we examined which proteinases play a role in the metastatic invasion of human HCC cell lines. JHH-5 and JHH-6 cells constitutively expressed mRNAs for both membrane-type 1 matrix metalloproteinase (MT1-MMP) and u-PA and invaded through reconstituted MATRIGEL in vitro, whereas JHH-7 cells expressed u-PA mRNA but not MT1-MMP and did not invade. However, hepatocyte growth factor (HGF) induced MT1-MMP expression on the surface of JHH-7 cells and markedly increased invasiveness of JHH-7 in a concentration-dependent manner. Moreover, cleavage activity for pro-MMP-2 was induced in HGF-treated JHH-7 cells. MMP inhibitor, rather than serine proteinase inhibitor, potently inhibited HCC cell invasion. Intrahepatic injection of HCC cell lines into athymic nude mice caused visible intrahepatic metastases in vivo. Moreover, JHH-7 tumors showed expression of MT1-MMP mRNA, while in vitro cultured JHH-7 cells did not. These findings suggest that MT1-MMP plays an important role in the invasive properties of HCC cells, and that HGF modifies the invasive properties of noninvasive HCC cells.

Treatment of metastatic nonseminomatous germ cell tumors of the testis: significance of the international consensus prognostic classification as a prognostic factor-based staging system.

BACKGROUND: We reviewed treatment results in patients with metastatic nonseminomatous germ cell tumors of the testis and examined the significance of the International Consensus Prognostic Classification to make appropriate risk-based decisions concerning induction chemotherapy. METHODS: We divided 37 patients treated with platinum-based combination chemotherapy into good, intermediate, and poor prognostic groups utilizing the International Consensus Prognostic Classification. The data was analyzed for both overall survival and progression-free survival among the 3 prognostic groups. RESULTS: Among the 37 patients, 10 died (8 of progressive disease, 1 of pneumonia during induction chemotherapy and 1 of cyclophosphamide-induced hemorrhagic cardiomyolitis during salvage chemotherapy). The survivors were followed for 6 to 1 84 months from the beginning of induction chemotherapy (median, 80 months). Five of the 37 patients (14%) were classified as having a good prognosis, 1 8 (48%) as intermediate, and 14 (38%) as having a poor prognosis. The patients in the poor prognostic group had a 5-year overall survival of only 40%, while those in the good and intermediate groups had 5-year overall survivals of 100% and 94%, respectively. When we applied the International Consensus Prognostic Classification to patients with advanced disease classified by the Indiana University Staging System, these patients could be clearly divided into good-risk and poor-risk groups. CONCLUSIONS: The International Consensus Prognostic Classification is easily applicable and accurate for risk assessment in patients with metastatic nonseminomatous germ cell tumors of the testis. This classification will now be widely used in general oncology practices and for clinical trials in these patients.

High density culture of immortalized liver endothelial cells in the radial-flow bioreactor in the development of an artificial liver.

Liver endothelial cells are important components of the tissue along the hepatic sinusoid. They are responsible for microcirculation in the liver and scavenger functions. It would therefore be important to include these cells in any hybrid type of artificial liver in addition to hepatocytes. However, it is difficult to culture these cells in vitro. The development of a liver endothelial cell line, which maintains the characteristics of the primary culture, would thus be of great benefit in the development of an artificial liver. In the present study we established immortalized liver endothelial cells from the liver of an H-2Kb-tsA58 transgenic mouse, which harbors the SV40 TAg gene. Hepatic sinusoidal cells isolated from H-2Kb-tsA58 mouse proliferated in the presence of gamma-interferon at 33 degrees C. Four clones were established, out of which clone M1 had the highest amounts of PGI2 production, as well as plasminogen activator activity and internalized acetylated low density lipoprotein. On culture dishes the M1 cells grew individually and spread. Sieve plates on the cell surface were not readily visible, but small pores were detected under electron microscopic observation. These results suggest that M1 clone cells originated from liver endothelial cells. Moreover it was possible to culture the immortalized liver endothelial cells in a radial-flow bioreactor for 5 days, with a maximum 6-keto prostaglandin F1alpha production of 25 microg per day. This suggests that immortalized liver endothelial cells and a radial-flow bioreactor can prove useful tools in the development an artificial liver.

Massive culture of human liver cancer cells in a newly developed radial flow bioreactor system: ultrafine structure of functionally enhanced hepatocarcinoma cell lines.

With a view to initiating clinical trials, cell morphology and function for a newly developed artificial liver support system employing highly functional human liver cell line, FLC-7, cultured in a radial flow bioreactor were compared to cells grown in a conventional monolayer culture. The radial flow bioreactor consists of a vertically extended cylindrical matrix comprised of porous glass bead microcarriers through which liquid medium flows from the periphery in toward the central axis generating a beneficial concentration gradient of oxygen and nutrients, while preventing excessive shear stresses or buildup of waste products. The three-dimensional culture system supports high-density (1.1 x 10(8) cells/ml-matrix), large scale cultures (4.4 x 10(10) cells/400 ml-bioreactor) with long-term viability. Scanning and transmission electron microscopy (SEM and TEM) revealed that cells cultured in a monolayer system were flattened and extended with numerous cytoplasmic projections. Cells in the three-dimensional culture were spherical and covered with microvillilike processes resembling liver cells in vivo. The cells were solidly attached on the surfaces and within the pores of the microcarriers in highly dense colonies. The spherical cells remained in close contact with adjacent cells, while circulation of liquid medium flowed freely through spaces between cells. FLC-7 cells produced albumin at a rate of 6.41 micrograms/24 h/10(6) cells. Alpha-fetoprotein (AFP) production dropped nearly threefold in comparison to monolayer cultures. Results demonstrated that the new artificial liver support systems (ALSS) provides a superior three-dimensional culture environment that allows cells to perform at naturally functioning levels.

5-fluorouracil and low-dose recombinant interferon-alpha-2a in patients with hormone-refractory adenocarcinoma of the prostate.

BACKGROUND: The effectiveness of a chemotherapy regimen including 5-fluorouracil (5-FU) and recombinant interferon-alpha-2a (rIFN-alpha-2a) was evaluated in hormone-refractory prostate cancer patients. METHODS: Patients received a continuous intravenous infusion of 5-FU at 600 mg/m2/day for 5 days (D1-D5), followed by a bolus injection of 5-FU on D15 and D22. Patients received intramuscular injection of rIFN-alpha-2a at 3 million IU on D1, D3, D5, D15, and D22. This schedule was repeated every 4 weeks. RESULTS: Between 1993 and 1995, 23 patients with hormone refractory prostate cancer were enrolled in this study. Two of five patients with nodal disease exhibited partial responses according to the NPCP criteria. Fourteen of 17 patients with bone disease showed stable disease. Of 21 patients assessible for response, 9 patients had a decrease in the PSA level greater than 50% of baseline. Bone pain disappeared partially or completely in 8 of 14 patients with this symptom at entry. The median overall survival was 18 months. The associate toxicity was well tolerable. CONCLUSIONS: Combination chemotherapy of 5-FU and low dose rIFN-alpha-2a in patients with hormone-refractory prostate cancer proved feasible, and with acceptable toxicity.

Full-length complementary DNA of hepatitis C virus genome from an infectious blood sample.

We constructed a full-length complementary DNA (cDNA) clone of hepatitis C virus (HCV) from a blood sample of an HCV carrier. The blood from the carrier was eventually transfused to a patient who later developed typical posttransfusion hepatitis C. It was also shown to be infectious to chimpanzees. We obtained 12 overlapping cDNA fragments altogether, covering the entire HCV genome. By subcloning and sequencing, clones considered to constitute the major population were selected. We could also detect 98 base pairs of extra sequences at the 3' end of the genome. After confirming the overlapping sequences, we combined the fragments to make a full-length cDNA. The HCV population in the donor was heterogeneous, as determined by their nucleotide sequences of the hypervariable region in envelope protein, but a few virus clones were selected in the recipient after transmission. The similar convergence of the virus population was previously observed when the same blood sample was injected into a chimpanzee. Interestingly, virus clones isolated during the acute phase in the recipient and the chimpanzee had sequences in the hypervariable region identical to that of the full-length cDNA clone. The full-length cDNA clone of HCV constructed in this study may originate from infectious virus clones.

Mother-to-child transmission of a hepatitis C virus variant with an insertional mutation in its hypervariable region.

BACKGROUND/AIMS: We have analyzed the molecular basis of mother-to-child transmission of hepatitis C virus. METHODS/RESULTS: Healthy pregnant women were screened for anti-HCV antibody and babies born to hepatitis C virus carrier mothers were prospectively investigated. Among the 35 pairs studied, the hepatitis C virus genome was detectable in only one baby, who did not show any significant symptoms of hepatitis. The viral load in the blood of the mother was one of the highest of the 35, and the population of the hepatitis C virus genome was heterogeneous. Furthermore, she was found to have a mixed infection with type 1a and type 1b hepatitis C virus. However, the hepatitis C virus genome obtained from the baby was only from type 1b, less heterogeneous and composed of the clones which were detected in the blood of the mother. The selected hepatitis C virus had a 12-nucleotide insertion in the amino-terminus of the E2 hypervariable region of the genome. CONCLUSIONS: The incidence of mother-to-child transmission of hepatitis C virus from carrier mothers was shown by this prospective study to be low. The presence of selection pressure during transmission was suggested. The biological significance of the virus with 12-nucleotide insertion has to be determined.

[Effective administration of recombinant granulocyte colony-stimulating factor to prevent granulocytopenia due to combination chemotherapy for testicular germ cell tumors].

A prospective randomized study on the administration of recombinant granulocyte colony stimulating factor (rG-CSF) was conducted on 15 patients with testicular germ cell tumors. The clinical stagings of all patients except one were minimal to moderate extent according to the Indiana University staging system. Combination chemotherapy using bleomycin, etoposide and cisplatinum (BEP) was performed as the initial treatment on the eligible patients. rG-CSF was administered by two different methods; 1) routine administration on the 6th day after BEP chemotherapy (group A), and 2) the same method, but after granulocytopenia of 1,500/mm3 had developed (group B). The administration of rG-CSF in group A significantly reduced the severity of leucocytopenia and also the incidence of stomatitis compared with group B. Although rG-CSF produced no significant side effects, the thrombocytopenia was prominent in the group A patients (not significant). BEP chemotherapy itself is an easily-tolerable and well established method for treating young adult patients. The method used in group B seems to be suitable in situations where thrombocytopenia and cost effectiveness.