Meningioma progression in mice triggered by Nf2 and Cdkn2ab inactivation.

Aggressive variants of meningiomas (WHO grade II and III) represent up to 30% of those tumors that are among the most common primary central nervous system tumors in adults. Currently, there is no effective treatment for grade-II and -III meningiomas, the main treatment remaining surgical excision. Genetic studies have highlighted two main events associated with meningioma progression: an increase of chromosomal instability in tumors with NF2 inactivation and homozygous deletions or point mutations of the CDKN2AB locus. In this study we demonstrated that in mice, in addition to bi-allelic Nf2 inactivation, homozygous and heterozygous Adenovirus Cre-mediated Cdkn2ab deletions lead to increased meningioma frequency (72% and 50%, respectively) with a shorter latency (3.5 and 7.8 months, respectively) compared with control cohorts and induce grade II/III meningioma progression with an incidence of 34% and 28%, respectively. Moreover, Cdkn2ab inactivation in arachnoidal cells was associated with decreased senescence compared with Nf2(-/-) and wild-type arachnoidal cells in vitro. We have established three mouse meningioma cell lines and generated a syngenic orthotopic meningioma mouse model with 50-100% grade-II/III meningiomas after reimplantation. Comparative genomic hybridization of four meningiomas from Cdkn2ab homozygous mice and three cell cultures revealed the absence of unbalanced chromosomal segments in tumors and several chromosome imbalances in cell cultures. In addition, we were able to detect meningiomas by using bioluminescence and to evaluate tumor vascular permeability by dynamic magnetic resonance imaging. These results show that Nf2 and Cdkn2ab cooperate to promote meningioma progression in mice. The short latency of tumor development and the ability to derive grade II/III meningioma cell cultures are key aspects of this model to promote its use in pre-clinical drug testing.

Identification of a progenitor cell of origin capable of generating diverse meningioma histological subtypes.

Meningiomas are among the most common primary central nervous system tumours in adults. Studies focused on the molecular basis for meningioma development are hampered by a lack of information with regard to the cell of origin for these brain tumours. Herein, we identify a prostaglandin D synthase-positive meningeal precursor as the cell of origin for murine meningioma, and show that neurofibromatosis type 2 (Nf2) inactivation in prostaglandin D2 synthase (PGDS) (+) primordial meningeal cells, before the formation of the three meningeal layers, accounts for the heterogeneity of meningioma histological subtypes. Using a unique PGDSCre strain, we define a critical embryonic and early postnatal developmental window in which biallelic Nf2 inactivation in PGDS (+) progenitor cells results in meningioma formation. Moreover, we identify differentially expressed markers that characterize the two major histological meningioma subtypes both in human and mouse tumours. Collectively, these findings establish the cell of origin for these common brain tumours as well as a susceptible developmental period in which signature genetic mutations culminate in meningioma formation.

Merlin regulates transmembrane receptor accumulation and signaling at the plasma membrane in primary mouse Schwann cells and in human schwannomas.

The NF2 gene product, merlin/schwannomin, is a cytoskeleton organizer with unique growth-inhibiting activity in specific cell types. A narrow spectrum of tumors is associated with NF2 deficiency, mainly schwannomas and meningiomas, suggesting cell-specific mechanisms of growth control. We have investigated merlin function in mouse Schwann cells (SCs). We found that merlin regulates contact inhibition of proliferation by limiting the delivery of several growth factor receptors at the plasma membrane of primary SCs. Notably, upon cell-to-cell contact, merlin downregulates the membrane levels of ErbB2 and ErbB3, thus inhibiting the activity of the downstream mitogenic signaling pathways protein kinase B and mitogen-activated protein kinase. Consequently, loss of merlin activity is associated with elevated levels of ErbB receptors in primary SCs. We also observed accumulation of growth factor receptors such as ErbB2 and 3, insulin-like growth factor 1 receptor and platelet-derived growth factor receptor in peripheral nerves of Nf2-mutant mice and in human NF2 schwannomas, suggesting that this mechanism could play an important role in tumorigenesis.

Urine diacetylspermine as a novel tumour maker for pancreatobiliary carcinomas.

BACKGROUND: Serum carcinoembryonic antigen (highly specific) and carbohydrate antigen 19-9 (highly sensitive) have been used as tumour markers for pancreatobiliary cancers. A novel urine tumour marker, diacetylspermine, was compared with the two conventional serum tumour markers in 125 patients with pancreatobiliary diseases. RESULTS: When the diagnosis of benign or malignant condition was examined, the sensitivity of urine diacetylspermine (75%) was higher than that of serum carcinoembryonic antigen (44%; P=0.048) and the same as that of serum carbohydrate antigen 19-9 (75%). The specificity of urine diacetylspermine (81%) was lower than that of serum CEA (92%) and as high as that of serum carbohydrate antigen 19-9 (80%). The efficiency of urine diacetylspermine (79%) was higher than that of serum carcinoembryonic antigen (74%) and the same as that of serum carbohydrate antigen 19-9 (79%). CONCLUSION: These results suggest that urine diacetylspermine is a marker for pancreatobiliary carcinoma, which is as highly sensitive and specific as serum carbohydrate antigen 19-9.

Liver-targeted disruption of Apc in mice activates beta-catenin signaling and leads to hepatocellular carcinomas.

Although inappropriate activation of the Wnt/beta-catenin pathway has been implicated in the development of hepatocellular carcinoma (HCC), the role of this signaling in liver carcinogenesis remains unclear. To investigate this issue, we constructed a mutant mouse strain, Apc(lox/lox), in which exon 14 of the tumor-suppressor gene adenomatous polyposis coli (Apc) is flanked by loxP sequences. i.v. injection of adenovirus encoding Cre recombinase (AdCre) at high multiplicity [10(9) plaque-forming units (pfu) per mouse] inactivated the Apc gene in the liver and resulted in marked hepatomegaly, hepatocyte hyperplasia, and rapid mortality. beta-Catenin signaling activation was demonstrated by nuclear and cytoplasmic accumulation of beta-catenin in the hepatocytes and by the induction of beta-catenin target genes (glutamine synthetase, glutamate transporter 1, ornithine aminotransferase, and leukocyte cell-derived chemotaxin 2) in the liver. To test a long-term oncogenic effect, we inoculated mice with lower doses of AdCre (0.5 x 10(9) pfu per mouse), compatible with both survival and persistence of beta-catenin-activated cells. In these conditions, 67% of mice developed HCC. beta-Catenin signaling was strongly activated in these Apc-inactivated HCCs. The HCCs were well, moderately, or poorly differentiated. Indeed, their histological and molecular features mimicked human HCC. Thus, deletion of Apc in the liver provides a valuable model of human HCC, and, in this model, activation of the Wnt/beta-catenin pathway by invalidation of Apc is required for liver tumorigenesis.

UDP-sugar transporter implicated in glycosylation and processing of Notch.

Glycosylation modifies protein activities in various biological processes. Here, we report the functions of a novel UDP-sugar transporter (UST74C, an alternative name for Fringe connection (Frc)) localized to the Golgi apparatus in cellular signalling of Drosophila. Mutants in the frc gene exhibit phenotypes resembling wingless and Notch mutants. Both Fringe-dependent and Fringe-independent Notch pathways are affected, and both glycosylation and proteolytic maturation of Notch are defective in mutant larvae. The results suggest that changes in nucleotide-sugar levels can differently affect Wingless and two distinct aspects of Notch signalling.

[Transperitoneal laparoscopic radical prostatectomy with the Montsouris technique: experience in initial 5 cases].

PURPOSE: In 1998 Guillonneau et al reported feasible and safe technique for laparoscopic radical prostatectomy. Herein we review initial 5 cases with using the Montsouris technique. MATERIALS AND METHODS: Between January and April 2000, 5 patients underwent transperitoneal laparoscopic radical prostatectomy. Clinical stages were T1c in 2, T2a in 1 and T2b in 2 patients. Preoperative PSA levels and Gleason grades in needle biopsies ranged from 7.9 to 39 ng/ml and from 2 to 6, respectively. Under general anesthesia 5 to 6 trocars were introduced and the patient was placed in the exaggerated Trendelenburg position. In 2 patients bilateral obturator lymph nodes were dissected for frozen pathological examination. Antegrade prostatectomy was performed initiating with the transperitoneal dissection of seminal vesicles. A watertight vesicourethral anastomosis was made with 8 to 10 interrupted sutures. RESULTS: Operating time and blood loss ranged from 505 to 925 minutes and from 100 to 700 gm, respectively. There were no intraoperative complications and one postoperative complication of prolonged urinary leakage, which was spontaneously closed. In other 4 patients Foley catheters were removed on postoperative day 6 to 10. CONCLUSIONS: Laparoscopic radical prostatectomy provides better visualization, inducing meticulous surgical procedures and less blood loss. More sophisticated maneuver would be required in dissection between the prostate and the bladder neck.

Inhibition of murine prostate tumor growth and activation of immunoregulatory cells with recombinant canarypox viruses.

BACKGROUND: Immunization with modified tumor cells carrying recombinant immunomodulatory genes is being explored as cancer immunotherapy. In this study, we examine whether canarypox ALVAC viruses carrying immunostimulatory cytokine genes (granulocyte-macrophage colony-stimulating factor, interleukin 2, interleukin 12, and tumor necrosis factor-alpha) can induce antitumor immunity (to rechallenge) in the RM-1 model of a highly aggressive, weakly immunogenic murine prostate cancer. METHODS: For antitumor activity studies, RM-1 murine prostate cancer cells were infected with the parental ALVAC virus or one or two recombinant ALVAC-cytokine viruses and then injected into male C57BL/6 mice. For rechallenge studies, other mice were first given an injection subcutaneously with irradiated (nonproliferating) recombinant ALVAC-infected RM-1 cells and then (10 days later) with untreated RM-1 cells. For the determination of which immune cells were required for antitumor activity, mice were immunodepleted of CD4, CD8, or natural killer (NK) NK1.1 cells with the corresponding monoclonal antibodies and were then given an injection of ALVAC-cytokine-infected RM-1 cells. For all experiments, tumor outgrowth and animal survival were monitored. RESULTS: After subcutaneous injection into mice, RM-1 cells infected with one (except ALVAC-interleukin 2) or two ALVAC-cytokine recombinants had statistically significantly greater antitumor activity than RM-1 cells infected with parental ALVAC (P<.001 for all; two-sided test). The antitumor activity of RM-1 cells infected with any two ALVAC-cytokine recombinants was greater than, but not statistically significantly different from, that of RM-1 cells infected with any one ALVAC-cytokine recombinant. NK1.1 cells were necessary for antitumor activity, but tumor-specific CD4(+) regulatory T cells were also induced that inhibited CD8(+) RM-1-specific cytotoxic T cells, resulting in the lack of immunity to a rechallenge by RM-1 cells. DISCUSSION: Canarypox viruses can transfer immunostimulatory cytokine genes into RM-1 prostate cancer cells. When such cells were injected into mice, the cytokines induced an antitumor response against this highly aggressive, weakly immunogenic tumor. This response, however, did not protect the mouse against a rechallenge with RM-1 cells because suppressor CD4(+) T cells were induced that inhibited tumor-specific CD8(+) cytotoxic T cells.

Increased expression of UDP-galactose transporter messenger RNA in human colon cancer tissues and its implication in synthesis of Thomsen-Friedenreich antigen and sialyl Lewis A/X determinants.

A series of human nucleotide sugar transporters of the Golgi apparatus was recently cloned, including the transporters for UDP-galactose (UDP-Gal), UDP-N-acetylglucosamine (UDP-GlcNAc) and CMP-sialic acid (CMP-SA). We have examined the mRNA expression of these three transporters in human colon cancer tissues by reverse transcription-PCR analysis and compared it with that in nonmalignant colonic mucosa prepared from the same patients. The amount of mRNA for UDP-Gal transporter was significantly increased in colon cancer tissues compared with nonmalignant mucosa tissues (P = 0.035; n = 20). The increase was more prominent in patients with advanced colorectal cancer of Dukes' stages C and D, in which the amount of UDP-Gal transporter mRNA in cancer tissues showed on average about a 3.6-fold increase over the paired nonmalignant mucosa (statistically significant at P = 0.004; n = 14). The mRNA content of the other two transporters showed no significant difference between the paired cancer and normal tissues. When UDP-Gal transporter cDNA was stably transfected to cultured human colon cancer cells, the expression of Thomsen-Friedenreich (TF) antigen and of sialyl Lewis A (NeuAcalpha2-->3Galbeta1-->3[Fucalpha1-->4]GlcNAcbeta1-->R) and sialyl Lewis X (NeuAcalpha2-->3Galbeta1-->4[Fucalpha1-->3]GlcNAcbeta1-->R) determinants was significantly induced on transfectant cells, which resulted in markedly enhanced cell adhesion to vascular E-selectin. These findings suggest that the increase of UDP-Gal transporter mRNA is involved in the enhanced expression of cancer-associated carbohydrate determinants such as TF and sialyl Lewis A/X antigens in colon cancers.

Molecular characterization of human UDP-glucuronic acid/UDP-N-acetylgalactosamine transporter, a novel nucleotide sugar transporter with dual substrate specificity.

A novel human nucleotide sugar transporter (NST) which transports both UDP-glucuronic acid (UDP-GlcA) and UDP-N-acetylgalactosamine (UDP-GalNAc) has been identified, cloned and characterized. The strategy for the identification of the novel NST involved a search of the expressed sequence tags database for genes related to the human UDP-galactose transporter-related isozyme 1, followed by heterologous expression of a candidate gene (hUGTrel7) in Saccharomyces cerevisiae and biochemical analyses. Significantly more UDP-GlcA and UDP-GalNAc were translocated from the reaction medium into the lumen of microsomes prepared from the hUGTrel7-expressing yeast cells than into the control microsomes from cells not expressing hUGTrel7. The possibility that this transporter participates in glucuronidation and/or chondroitin sulfate biosynthesis is discussed.

Substrate recognition by UDP-galactose and CMP-sialic acid transporters. Different sets of transmembrane helices are utilized for the specific recognition of UDP-galactose and CMP-sialic acid.

Human UDP-galactose transporter (hUGT1) and CMP-sialic acid transporter (hCST) are related Golgi membrane proteins with 10 transmembrane helices. We have constructed chimeras between these proteins in order to identify submolecular regions responsible for the determination of substrate specificity. To assess the UGT and CST activities, chimeric cDNAs were transiently expressed in either UGT-deficient mutant Lec8 cells or CST-deficient mutant Lec2 cells, and the binding of plant lectins, GS-II or PNA, respectively, to these cells was examined. During the course of analysis of various chimeric transporters, we found that chimeras whose submolecular regions contained helices 1, 8, 9, and 10, and helices 2, 3, and 7 derived from hUGT1 and hCST sequences, respectively, exhibited both UGT and CST activities. The dual substrate specificity for UDP-galactose and CMP-sialic acid of one such representative chimera was directly confirmed by in vitro measurement of the nucleotide sugar transport activity using a heterologous expression system in the yeast Saccharomyces cerevisiae. These findings indicated that the regions which are critical for determining the substrate specificity of UGT and CST resided in different submolecular sites in the two transporters, and that these different determinants could be present within one protein without interfering with each other's function.

Automatic color-temperature compensator with guest-host liquid-crystal filter for video image sensing.

A color-temperature compensating system with an electrically controllable liquid-crystal filter and a color sensor mounted on a video camera has been developed for color image sensing. The filter contains two guest-host liquid-crystal devices with dichroic dyes that have strong light absorption for shorter-wavelength light; two devices are necessary because of the spectral difference between the sun and an incandescent lamp as light sources. The filter's absorption is continuously controlled by the voltage applied to the filter. Because the filter is driven according to spectral information about the illumination detected by the color sensor, the color balance of the video image to be sensed can be compensated automatically and rapidly. This is especially useful for video image shooting in which a video camera experiences changes in illumination color temperatures.

Future of urologic laparoscopy.

Laparoscopy has become increasingly popular in urology, reducing the invasiveness of treatment and shortening the period of convalescence. Adrenalectomy is one of the procedures most widely accepted for urologic laparoscopy. During the twenty-first century, most urologic operations will likely be performed laparoscopically, benefiting from advancements in medical engineering. The development of new tissue approximation methods, tissue retrieval systems, microlaparoscopic instruments, sensor technology, and telerobotics, as well as virtual reality, will contribute to the establishment of safe, easy laparoscopic urologic surgery.

Functional analysis of the individual oligosaccharide chains of sendai virus fusion protein.

The roles of N-linked glycosylation in the intracellular transport and fusion activity of the Sendai virus fusion (F) protein were studied. Each of three potential glycosylation motifs (designated g1, g2, and g3) in the F protein was mutated separately or in combination with the other sites. When the mutant F proteins were transiently expressed in COS cells, they showed significant changes in electrophoretic mobility, indicating that all three motifs in the F protein are glycosylated. Glycosylation-defective mutants which lacked the g2-oligosaccharide chain showed decreased immunoreactivity with a monoclonal antibody specific for the native conformation and were inefficiently transported to the cell surface. Such mutants, with the exception of a double mutant lacking g1 and g2-oligosaccharide chains, were also not able to induce syncytia formation when cells expressing them plus the hemagglutinin-neuraminidase protein were treated with trypsin. Mutations at the other glycosylation sites did not significantly affect the immunoreactivity with the monoclonal antibody or the efficiency of intracellular transport of the F protein. These results indicate that the N-linked oligosaccharide chain attached at g2 is important for efficient intracellular transport and for the fusion activity of the F protein.

Transperitoneal anterior laparoscopic adrenalectomy: the easiest technique.

Four laparoscopic methods have been developed to approach the adrenal gland: anterior or lateral transperitoneal approaches, and lateral or posterior retroperitoneal approaches. The advantages and disadvantages of these methods were reviewed during a workshop held by the Japanese Society of Endourology and ESWL in 1997. The transperitoneal anterior approach, when combined with a lateral approach for the left side, is the easiest for small adrenal tumors, and discloses the adrenal vein early in the procedure. For large tumors over 5 cm in diameter, the transperitoneal lateral approach is the most feasible bilaterally. Regardless of the methods of approach, laparoscopic adrenalectomy has already become the standard procedure for adrenal tumors, because it minimizes the operative morbidity and postoperative hospital stay.

Conditional biallelic Nf2 mutation in the mouse promotes manifestations of human neurofibromatosis type 2.

Hemizygosity for the NF2 gene in humans causes a syndromic susceptibility to schwannoma development. However, Nf2 hemizygous mice do not develop schwannomas but mainly osteosarcomas. In the tumors of both species, the second Nf2 allele is inactivated. We report that conditional homozygous Nf2 knockout mice with Cre-mediated excision of Nf2 exon 2 in Schwann cells showed characteristics of neurofibromatosis type 2. These included schwannomas, Schwann cell hyperplasia, cataract, and osseous metaplasia. Thus, the tumor suppressor function of Nf2, here revealed in murine Schwann cells, was concealed in hemizygous Nf2 mice because of insufficient rate of second allele inactivation in this cell compartment. The finding of this conserved function documents the relevance of the present approach to model the human disease.

[A case of synchronous ipsilateral renal cell carcinoma and ureteral transitional cell carcinoma].

A 62-year-old man was admitted to our hospital with the chief complaint of right flank pain. Abdominal computed tomographic scan revealed a right hydronephrosis and intrapelvic tumor. Ultrasound revealed a renal mass lesion. Ultrasound guided renal biopsy and laparotomy of intrapelvic tumor was performed. The histopathological diagnosis was renal cell carcinoma and ureteral transitional cell carcinoma.

Thrombopoietin stimulates murine lineage negative, Sca-1+, C-Kit+, CD34- cells: comparative study with stem cell factor or interleukin-3.

It has recently been reported that human thrombopoietin (TPO) acts on early hematopoietic progenitor cells. Consequently, we investigated the effects of TPO on murine hematopoietic progenitor cells using lineage negative (Lin-), Sca-1+, c-Kit+ marrow cells from 5-fluorouracil-treated mice. One hundred enriched cells were cultured in suspension with various single cytokines for 5 days. When cultured with the single cytokines as stem cell factor (SCF), TPO, or interleukin (IL)-3, these cells were maintained or had increased by day 5, whereas only a few cells survived in cultures with granulocyte colony stimulating factor, IL-11, or IL-6. We extended the study in serum-free or serum-containing suspension cultures with SCF or TPO. Anti-TPO antibodies did not inhibit the effects of SCF on enriched cells but did inhibit the effects of TPO on those cells. We further examined the effects of TPO, SCF, and IL-3 on other populations of murine hematopoietic progenitor cells. Either TPO or SCF as a single cytokine could maintain murine Lin-, Sca-1+, c-Kit+, CD34- marrow cells, which are the most dormant cells. In addition, IL-3 increased Lin-, Sca-1-, c-Kit+ cells more than did SCF and TPO but did not stimulate Lin-, Sca-1+, c-Kit+, CD34- cells more. These results indicate that TPO as well as SCF may be key regulators in the proliferation of murine hematopoietic early progenitor cells.

[Adenocarcinoma of the ileal segment with transitional cell carcinoma of the bladder following ileocytoplasty: a case report].

A 67 year-old woman visited our hospital complaining of pollakisuria. She had undergone left nephrectomy and augmentation ileocystoplasty for tuberculous bladder atrophy 40 years previously. She underwent a total cystectomy and tubeless ureterocutaneostomy with a preoperative diagnosis of muscle-invading transitional cell carcinoma of the bladder. The pathological diagnosis was adenocarcinoma of the ileal segment and transitional cell carcinoma of the original bladder. This is the first case report of adenocarcinoma of the ileal segment and transitional cell carcinoma of the original bladder among 22 patients suffering from bladder cancer after ileocystoplasty.