Primary solitary fibrous tumour of the prostate: A case report and literature review.

INTRODUCTION: Solitary fibrous tumour (SFT) is a rare mesenchymal tumour with intermediate malignant potential. Although this tumour arises in several sites, prostatic SFT is an extremely rare neoplasm and may prove confusing owing to the lack of clinical experience because of tumour rarity. The diagnosis may be further difficult because SFTs can manifest positive immunoreactivity for CD34 and progesterone receptor, which are known markers of prostatic stromal tumours. Herein, we describe a case of prostatic SFT that was difficult to differentiate from a prostatic stromal tumour of uncertain malignant potential because of positive immunoreactivity to CD34 and progesterone receptor. CASE REPORT: A 40-year-old Japanese man presented with lower abdominal pain. Computed tomography revealed a prostatic mass; furthermore, prostate core needle biopsy revealed proliferating bland spindle cells, without necrosis or prominent mitoses. Tumour cells were positive for CD34 and progesterone receptor on immunohistochemical analysis; thus, a prostatic stromal tumour of uncertain malignant potential was initially suspected. However, as the tumour cells showed positive immunoreactivity for STAT6, the final diagnosis was an SFT of the prostate. The patient underwent tumour resection, and at the 6-month postoperative follow-up, neither local recurrence nor distant metastasis occurred. CONCLUSION: For an accurate diagnosis of an SFT of the prostate, STAT6 immunohistochemistry should be conducted for all mesenchymal tumours of the prostate. When STAT6 immunohistochemical analysis is unfeasible, pathologists should be aware that the morphological and immunohistochemical characteristics of SFT variable from case to case and diagnose with combined analysis of several immunohistochemical markers.

Transduction of Oct6 or Oct9 gene concomitant with Myc family gene induced osteoblast-like phenotypic conversion in normal human fibroblasts.

INTRODUCTION: Osteoblasts play essential roles in bone formation and regeneration, while they have low proliferation potential. Recently we established a procedure to directly convert human fibroblasts into osteoblasts (dOBs). Transduction of Runx2 (R), Osterix (X), Oct3/4 (O) and L-myc (L) genes followed by culturing under osteogenic conditions induced normal human fibroblasts to express osteoblast-specific genes and produce calcified bone matrix both in vitro and in vivo Intriguingly, a combination of only two factors, Oct3/4 and L-myc, significantly induced osteoblast-like phenotype in fibroblasts, but the mechanisms underlying the direct conversion remains to be unveiled. MATERIALS AND METHODS: We examined which Oct family genes and Myc family genes are capable of inducing osteoblast-like phenotypic conversion. RESULTS: As result Oct3/4, Oct6 and Oct9, among other Oct family members, had the capability, while N-myc was the most effective Myc family gene. The Oct9 plus N-myc was the best combination to induce direct conversion of human fibroblasts into osteoblast-like cells. DISCUSSION: The present findings may greatly contribute to the elucidation of the roles of the Oct and Myc proteins in osteoblast direct reprogramming. The results may also lead to establishment of novel regenerative therapy for various bone resorption diseases.

A 3.0-kb deletion including an erythroid cell-specific regulatory element in intron 1 of the ABO blood group gene in an individual with the Bm phenotype.

We developed a sequence-specific primer PCR (SSP-PCR) for detection of a 5.8-kb deletion (B(m) 5.8) involving an erythroid cell-specific regulatory element in intron 1 of the ABO blood group gene. Using this SSP-PCR, we performed genetic analysis of 382 individuals with Bm or ABm. The 5.8-kb deletion was found in 380 individuals, and disruption of the GATA motif in the regulatory element was found in one individual. Furthermore, a novel 3.0-kb deletion involving the element (B(m) 3.0) was demonstrated in the remaining individual. Comparisons of single-nucleotide polymorphisms and microsatellites in intron 1 between B(m) 5.8 and B(m) 3.0 suggested that these deletions occurred independently.

Blood group B gene is barely expressed in in vitro erythroid culture of Bm-derived CD34+ cells without an erythroid cell-specific regulatory element.

BACKGROUND AND OBJECTIVES: Previously, a weak phenotype Am or Bm was assumed to be caused by a reduction of A or B gene expression in bone marrow cells, but not in mucus-secreting cells. However, ABO expression has not been examined in erythroid progenitor cells of Am or Bm individuals. MATERIALS AND METHODS: We carried out in vitro erythroid differentiation of CD34(+) cells from peripheral blood of a Bm individual harbouring a 3.0-kb deletion including an erythroid cell-specific regulatory element, named the +5.8-kb site, in intron 1 of the human ABO blood group gene. RESULTS: During the in vitro differentiation of CD34(+) cells from this Bm individual into erythroid cells, B-antigens were not detectable on the cultured cells by flow cytometric analysis, and allele-specific RT-PCR consistently detected the transcripts from the O allele, but not from the B allele. Moreover, chromatin immunoprecipitation assay demonstrated that both RUNX1 and GATA-2 or GATA-1 were bound to the +5.8-kb site in cultured erythroid cells expressing ABO. CONCLUSION: It is likely that the +5.8-kb site enhances transcription from the ABO promoter in erythroid cells through binding of RUNX1 and GATA-2 or GATA-1.

High pressure treatment under subfreezing temperature results in drastic inactivation of enveloped and non-enveloped viruses.

Some viruses are sensitive to high pressure. The freeze-pressure generation method (FPGM) applies pressure as high as 250 MPa on a substance, simply by freezing a pressure-resistant reservoir in which the substance is immersed in water. Here we examined whether the FPGM successfully inactivates herpes simplex virus type 1 (HSV-1), an enveloped DNA virus belonging to the human Herpesviridae, and encephalomyocarditis virus (EMCV), an envelope-free RNA virus belonging to the Picornaviridae. After the treatment, HSV-1 drastically reduced the ability to form plaque in Vero cells in vitro as well as to kill mice in vivo. EMCV that had been pressurized failed to proliferate in HeLa cells and induce interferon response. The results suggest that the FPGM provides a feasible procedure to inactivate a broad spectrum of viruses.

Germline and somatic mutations in the tyrosine kinase domain of the MET proto-oncogene in papillary renal carcinomas.

Hereditary papillary renal carcinoma (HPRC) is a recently recognized form of inherited kidney cancer characterized by a predisposition to develop multiple, bilateral papillary renal tumours. The pattern of inheritance of HPRC is consistent with autosomal dominant transmission with reduced penetrance. HPRC is histologically and genetically distinct from two other causes of inherited renal carcinoma, von Hippel-Lindau disease (VHL) and the chromosome translocation (3;8). Malignant papillary renal carcinomas are characterized by trisomy of chromosomes 7, 16 and 17, and in men, by loss of the Y chromosome. Inherited and sporadic clear cell renal carcinomas are characterized by inactivation of both copies of the VHL gene by mutation, and/or by hypermethylation. We found that the HPRC gene was located at chromosome 7q31.1-34 in a 27-centimorgan (cM) interval between D7S496 and D7S1837. We identified missense mutations located in the tyrosine kinase domain of the MET gene in the germline of affected members of HPRC families and in a subset of sporadic papillary renal carcinomas. Three mutations in the MET gene are located in codons that are homologous to those in c-kit and RET, proto-oncogenes that are targets of naturally-occurring mutations. The results suggest that missense mutations located in the MET proto-oncogene lead to constitutive activation of the MET protein and papillary renal carcinomas.

Degeneration patterns of the olfactory receptor genes in sea snakes.

The sense of smell relies on the diversity of olfactory receptor (OR) repertoires in vertebrates. It has been hypothesized that different types of ORs are required in terrestrial and marine environments. Here we show that viviparous sea snakes, which do not rely on a terrestrial environment, have significantly lost ORs compared with their terrestrial relatives, supporting the hypothesis. On the other hand, oviparous sea snakes, which rely on a terrestrial environment for laying eggs, still maintain their ORs, reflecting the importance of the terrestrial environment for them. Furthermore, we found one Colubroidea snake (including sea snakes and their terrestrial relatives)-specific OR subfamily which had diverged widely during snake evolution after the blind snake-Colubroidea snake split. Interestingly, no pseudogenes are included in this subfamily in sea snakes, and this subfamily seems to have been expanding rapidly even in an underwater environment. These findings suggest that the Colubroidea-specific ORs detect nonvolatile odorants.

Genome-wide analysis of DNA copy number alterations and gene expression in gastric cancer.

Genomic copy number aberrations (CNAs) are believed to play a major role in the development and progression of human cancers. Although many CNAs have been reported in gastric cancer, their genome-wide transcriptional consequences are poorly understood. In this study, to reveal the impact of CNAs on genome-wide expression in gastric cancer, we analysed 30 cases of gastric cancers for their CNAs by array comparative genomic hybridization (array CGH) and 24 of these 30 cases for their expression profiles by oligonucleotide-expression microarray. We found that with the application of laser microdissection, most CNAs were detected at higher frequency than in previous studies. Notably, gain at 20q13 was detected in almost all cases (97%), suggesting that this may play an important role in the pathogenesis of gastric cancer. By comparing the array CGH data with expression profiles of the same samples, we showed that both genomic amplification and deletion strongly influence the expression of genes in altered genomic regions. Furthermore, we identified 125 candidate genes, consisting of 114 up-regulated genes located in recurrent regions (>10%) of amplification and 11 down-regulated genes located in recurrent regions of deletion. Up-regulation of several candidate genes, such as CDC6, SEC61G, ANP32E, BYSL and FDFT1, was confirmed by immunohistochemistry. Interestingly, some candidate genes were localized at genomic loci adjacent to well-known genes such as EGFR, ERBB2 and SMAD4, and concordantly deregulated by genomic alterations. Based on these results, we propose that our list of candidate genes may contain novel genes involved in the pathogenesis of advanced gastric cancer.

Cellular proteins that bind the von Hippel-Lindau disease gene product: mapping of binding domains and the effect of missense mutations.

The von Hippel-Lindau disease (VHL) gene is a novel tumor suppressor gene that plays a role in the pathogenesis of renal cell carcinomas and hemangioblastomas of the central nervous system. To begin an evaluation of the biological functions of the VHL gene product (pVHL), we prepared bacterial fusion protein between glutathione S-transferase and wild-type or mutant pVHLs. The fusion proteins were used to identify cellular proteins that bind to pVHL in vitro. Monkey kidney cells transfected with wild-type or mutant VHL cDNAs were used to identify cellular proteins that bind to pVHL in vivo. Wild-type pVHL consistently bound two cellular proteins with apparent molecular masses of 10 and 14 kilodaltons that were designated p10 and p14, respectively. Mapping studies with a panel of VHL deletion mutant proteins demonstrated that p10 and p14 bound to a 32-amino acid peptide located in the carboxy terminal portion of pVHL. Missense mutation located within this 32-amino acid peptide abrogated the ability of the VHL protein to bind p10 and p14. Of 67 VHL families with identified germline mutations, 42 families had mutations predicted to affect the p10/p14-binding region. Maintenance of the integrity of the p10/p14-binding region appears to be essential for cellular growth regulation by pVHL.

Suppression of growth of renal carcinoma cells by the von Hippel-Lindau tumor suppressor gene.

Clear cell renal carcinomas are most frequently characterized by loss of function of both copies of the von Hippel-Lindau (VHL) disease gene, suggesting that the VHL gene product plays an important role in regulating renal cell proliferation. To directly assess the function of the VHL gene product, we transfected the wild-type VHL gene into two renal carcinoma cell lines that lacked normal expression of the gene. Expression of the wild-type VHL gene led to a dramatic suppression of growth in two renal carcinoma cell lines, A498 and UMRC6 in vitro, as measured by colony formation and direct cell counting. Transfection of a naturally occurring mutant VHL gene (nucleotide 713 G to A, Arg to Gln) did not lead to growth suppression of these renal carcinoma cells, nor did transfection of the wild-type VHL gene into two non-renal tumor cell lines that expressed the endogenous wild-type VHL gene. Expression constructs, which included the first ATG at nucleotide 214, were sufficient to produce the strongest growth suppression. These experiments provide direct evidence that the VHL gene product functions to suppress the growth of renal carcinoma cells and also provide a model for mapping the domains of the VHL protein important in suppressing tumor growth.

Direct and indirect effects of interferon on in vivo murine tumor cell growth.

We cloned two sublines (S1 and R1) of murine Meth A fibrosarcoma cells with respect to their sensitivity to a murine alpha/beta-interferon (IFN) preparation. The growth of S1 cells was suppressed and that of R1 cells was hardly affected by IFN in vitro. This was also the case with cells enclosed in cell-impermeable diffusion chambers in peritoneal cavities. Nevertheless, IFN suppressed the growth of not only S1 cells but also R1 cells in mice inoculated i.p. with these cells, and the survival rates of both S1 cell recipients and R1 cell recipients were markedly improved. S1 cells were observed microscopically to be injured by the direct effect of IFN in vitro and in vivo, but R1 cells in in vitro culture with IFN and those surviving in vivo in the presence of IFN appeared to proliferate well. In the peritoneal cavity of R1 recipients treated daily with IFN, the recruitment of macrophages was enhanced in comparison with untreated R1 recipients. Adherent peritoneal exudate cells obtained from IFN-treated, R1-bearing mice were highly suppressive for the in vitro growth of not only R1 cells but also allogeneic and human cells. The role of macrophages in the indirect effect of IFN on tumor cell growth is discussed.

Tumor degeneration by human embryonic fibroblasts and its enhancement by interferon.

KB cells from a human nasopharyngeal tumor were cocultivated with human embryonic fibroblasts (HF 8101 cells); 7 to 14 days after incubation, "spongy degeneration"-like changes developed in the target cell-growing area. These changes developed in other target cells [HeLa cells from human cervical cancer, human hepatoma cells (PLC/PRF/5), and human amnion FL cells] cocultured with several kinds of human embryonic fibroblasts [HF 8101 cells, HF 8103 cells, and HEL cells]; however, HF 8101 cells did not cause degenerative changes in murine L929 cells. The degenerative changes were enhanced by treatment with human leukocyte interferon or human fibroblast interferon at a dose of 1,000 or 10,000 IU/ml, but there was no significant difference in the enhancing effect between human leukocyte and human fibroblast interferons. Mouse L929 interferon did not enhance the degenerative changes in KB cells caused by HF 8101 cells. It was concluded that human fibroblasts caused the degenerative changes in the human tumor cells and the continuous cell line and that the changes were enhanced by treatment with either human leukocyte interferon or human fibroblast interferon.

Frequent somatic mutations and loss of heterozygosity of the von Hippel-Lindau tumor suppressor gene in primary human renal cell carcinomas.

We analyzed 47 primary sporadic human renal cell carcinomas (39 clear cell and 8 non-clear cell) for mutations of the von Hippel-Lindau (VHL) tumor suppressor gene using the polymerase chain reaction and single strand conformational polymorphism analysis of DNA. All of the positive cases in single strand conformational polymorphism analyses were further characterized by direct sequencing. Somatic mutations were detected in 22 (56%) of 39 clear cell renal carcinomas including 15 deletions, 3 insertions, 3 missense mutations, and 1 nonsense mutation. Nineteen of these mutations predicted to produce truncation of the VHL protein. These mutations mainly occurred in the last one-third region of exons 1, 2, and 3. In addition, loss of heterozygosity of the VHL gene was observed in 16 (84%) of 19 informative clear cell renal carcinomas. No somatic mutations were detected in 8 non-clear cell carcinomas. These results show that the VHL tumor suppressor gene is one of the major tumor suppressor genes in human renal cell carcinomas, especially in the clear cell subtype renal cell carcinoma. Clear cell carcinoma might be distinguished from other pathological types of renal cell carcinomas by molecular genetic techniques.

Epigenetic inactivation of the RASSF1A 3p21.3 tumor suppressor gene in both clear cell and papillary renal cell carcinoma.

Renal cell carcinoma (RCC), the most common adult kidney neoplasm, is histopathologically heterogeneous, with most sporadic RCCs ( approximately 80%) classified as clear cell (CC) tumors. Chromosome 3p allele loss is the most frequent genetic alteration in RCC but is associated specifically with sporadic and hereditary forms of clear cell RCC (CC-RCC) and is not a feature of non-CC-RCC, such as papillary (chromophilic) RCC. The VHL tumor suppressor gene (TSG) maps to chromosome 3p25, and somatic inactivation of the VHL gene occurs in up to 70% of CC-RCC tumors and cell lines. However, VHL inactivation is not sufficient for CC-RCC tumorigenesis, and inactivation of 3p12-p21 TSG(s) appears to be necessary in CC-RCC irrespective of VHL gene inactivation status. Recently, we demonstrated that the candidate 3p21 TSG, RASSF1A, is hypermethylated in most small cell lung cancers. We have now investigated the role of RASSF1A inactivation in primary RCC tumors. RASSF1A promoter methylation was detected in 23% (32 of 138) of primary CC-RCC tumors. In CC-RCC cell lines, RASSF1A methylation was associated with silencing of RASSF1A expression and restoration of expression after treatment with 5'-azacytidine. The frequency of RASSF1A methylation was similar in CC-RCC with and without VHL gene inactivation (24% versus 21%), and there was no association between epigenetic silencing of the RASSF1A and VHL TSGs, because 0 of 6 tumors with VHL hypermethylation had RASSF1A methylation, and VHL was not methylated in 26 CC-RCCs with RASSF1A methylation. Although 3p allele loss has been reported rarely in papillary RCC, we identified RASSF1A methylation in 44% (12 of 27) of papillary RCCs analyzed. Thus: (a) inactivation of RASSF1A is a frequent event in both CC-RCC and papillary RCC tumors; (b) there is no relationship between epigenetic silencing of RASSF1A and VHL inactivation status in CC-RCC. Fifty-four CC-RCCs analyzed for RASSF1A methylation were informative for 3p21 allele loss, and 20% (7 of 35) with 3p21 allele loss demonstrated RASSF1A methylation. All informative CC-RCCs with 3p21 allele loss and no RASSF1A methylation also demonstrated allele losses at other regions of 3p so that tumorigenesis in these cases may result from: (a) haploinsufficiency of RASSF1A; (b) inactivation of other 3p21 TSGs; or (c) inactivation of 3p TSGs from outside of 3p21. RASSF1A is the first TSG to be inactivated frequently in both papillary and CC-RCCs. The finding of frequent epigenetic inactivation of RASSF1A in papillary RCCs despite previous studies reporting infrequent 3p21 allele loss in this tumor type illustrates how the systematic identification of all major human cancer genes will require detailed analysis of the cancer genome and epigenome.

Tumor suppressor protein VHL is induced at high cell density and mediates contact inhibition of cell growth.

In spite of the general recognition of von Hippel-Lindau (VHL) as a tumor suppressor gene, the physiological and pathological importance of VHL protein in cell growth regulation and tumorigenesis remains unclear. Here we show that in normal human renal proximal tubule epithelial cells (RPTEC), the steady-state amount of VHL protein is strictly regulated by cell density. The cellular VHL content is more than 100-fold higher in dense cultures than in sparse cultures. The increase in VHL protein at high cell density was also observed for NIH3T3 fibroblasts, suggesting the generality of the phenomenon. The growth rates of renal cell carcinoma cells lacking an intact VHL gene and their derivatives with wild-type or mutant VHL expression vector do not differ significantly when they are growing in log-phase. Importantly, however, there is a difference when they reach confluency: cells lacking wild-type VHL grew continuously, while cells expressing exogenous VHL protein showed relatively limited cell growth. Using an ecdysone-inducible VHL expressing cell line, we also show that the growth inhibition at high cell density can be released by attenuating the VHL expression. Taken together, we propose that VHL protein functions as a growth suppressor at high cell density, and this might be the basis of the tumor suppressor function of VHL.

Differential interleukin 12 responsiveness for interferon gamma production in advanced stages of cancer patients correlates with performance status.

Interleukin 12 (IL-12) has been shown to exhibit potent antitumor activity in murine tumor models through various mechanisms including the capacity to stimulate IFN-gamma production by T cells and natural killer cells. The aim of the present study was to examine the efficacy of IL-12 in inducing IFN-gamma secretion in cancer patients. A comparison was made between healthy individuals who served as controls and cancer patients for IFN-gamma production induced after the stimulation of whole blood samples with 1000 pg/ml IL-12. Samples from all healthy individuals showed positive IL-12 responsiveness. Approximately half of the samples from patients displayed levels of IFN-gamma production comparable to those observed for controls, whereas the rest of the samples exhibited almost-null responses. The incidences for reduced capacity of IFN-gamma production and null IL-12 responsiveness in cancer patients at all cancer stages or at a given advanced stage (stage IV) increased along with performance status. However, these correlated with neither the number of lymphocytes contained in the blood samples nor the tumor types. When peripheral blood mononuclear cells were isolated from patient blood samples showing null/marginal responses, and their responsiveness was examined, 7 of 13 samples exhibited positive responses. Whereas enhanced tumor necrosis factor alpha production was also observed in some patients after IL-12 stimulation, the elevation of tumor necrosis factor alpha was induced only in blood samples that showed IL-12-stimulated IFN-gamma production. These observations indicate that a remarkable difference exists in IL-12 reactivity among cancer patients, and that differential IL-12 responsiveness depends largely on performance status.

A bioassay for serum interferon based on induction of 2'5'-oligoadenylate synthetase activity.

We have developed a bioassay for interferons (IFN) based on measuring the amounts of 2',5' oligoadenylate synthetase (2-5AS) induced in cells of the THP-1 monocyte line in response to IFN. The assay can be completed in 20 h, gives reproducible results, and is at least 50 times more sensitive to IFN-alpha than conventional cytopathic effect inhibition antiviral assays. It is, respectively, less and much less sensitive to IFN-beta and IFN-gamma. The presence of preexisting 2-5AS activity in a sample does not influence the results. We have used this assay to measure very low levels (0.1-0.5 IU/ml) of endogenously formed IFN-alpha in serum samples from patients with various diseases and also to measure the residual small amounts of IFN-alpha still present in the serum as late as 48 h after an i.m. injection of 3 million IU, which is appreciably later than in previous methods. Thus, our highly sensitive assay offers considerable advantages, not least in relation to the clinical use of IFN.

Determination of interferon-alpha-producing capacity in whole blood cultures from patients with various diseases and from healthy persons.

To assess the clinical value of determination of the interferon (IFN)-producing capacity of patients, IFN production induced by Sendai virus (HVJ) in vitro was measured in cell cultures of whole blood from patients with various diseases. IFN production in patients with lung cancer, myelodysplastic syndromes, noninsulin-dependent diabetes mellitus, pulmonary tuberculosis, and asymptomatic HIV-1 infection was lower than that in healthy persons. Furthermore, periodic measurements of IFN production revealed decreasing IFN producing capacities in patients with lung cancer with progression of the tumor stage. However, increased IFN-producing capacities were observed in patients with tuberculosis after standard therapy. Further experiments showed that the main type of IFN induced in whole blood cultures was IFN-alpha, and decreased IFN production in patients did not result from a decreased number of leukocytes but rather from an impairment of cellular IFN production. The evaluation of IFN production in whole blood cell cultures may be a feasible method of assessing the impaired immune status.

Interferon-alpha treatment leads to accumulation of virus particles on the surface of cells persistently infected with the human immunodeficiency virus type 1.

The effect of interferon-alpha (IFN-alpha) on virus replication in cells acutely infected with human immunodeficiency virus type 1 (HIV-1) and virus production from cells persistently infected with HIV-1 was studied. In both cell systems, significant suppression was observed. However, this suppression was not due to protein synthesis of the major viral proteins. Electron microscopy revealed the accumulation of intact virus particles on the cell surface of the cells treated with IFN-alpha. Thus, IFN-alpha might suppress the release stage of the particle from infected cells.

Practical method for production of monoclonal antibody to human IgG allotype G1M F and its applications in ELISAs and dot immunobinding.

An anti-G1M F monoclonal antibody was produced by immunization of mice with a single dose of F(ab')2 fragments of normal IgG1-enriched IgG and subsequent fusion of their lymph node cells with P3U1 myeloma cells. Antibody specificity was tested by an ELISA in microtiter plates coated with allotype positive or negative IgG. The usefulness of the antibody as a G1M F typing reagent in inhibition and direct immobilization-type ELISAs and dot immunobinding was demonstrated by re-typing of 100 GM-allotyping control sera. The advantages and disadvantages of these assay methods are discussed.