Prostate cancer cell growth is modulated by adipocyte-cancer cell interaction.

OBJECTIVE: To assess whether adipocytes (mesenchymal stromal cells thought to affect the proliferation and differentiation of epithelial cells) affect the behaviour of prostate cancer cells in vitro, as prostate cancer metastasizes to the bone, which is an adipocyte-rich environment. MATERIALS AND METHODS: The human bone-metastatic prostate carcinoma cell line PC3 was cultured with or without adipocytes in a three-dimensional collagen gel matrix. Histological and immunohistochemical assays were used to evaluate the proliferation and differentiation of PC3 cells. The cytokine expression of this culture assembly was also examined by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The proliferation and differentiation of cancer cells were clearly changed on co-culture with adipocytes compared with the control cultures. The mean (range) bromodeoxyuridine (BrdU) indices estimated (according to uptake) to evaluate the growth of the cultured cells were 36.3 (8.55)% in the co-culture and 26.95 (10.50) in the control (P < 0.02). PC3 cells in co-culture formed larger clusters than in the control, at 16.0 (11.0) vs 14.0 (10.0), respectively (P < 0.01). Cancer cells also showed pleomorphism, varying from cuboidal to spindle-shaped. The expressions of vascular endothelial and platelet-derived growth factor were greater in co-culture than in the control. CONCLUSION: These findings suggest that adipocytes modulate the growth, morphology and cytokine expression of prostate cancer cells. This specific mesenchymal stromal cell type is important in the biological behaviour of prostate cancer cells.

Bladder cancer cell implantation in reconstructed bladder in vitro: a model of tumour recurrence.

OBJECTIVE: To evaluate the involvement of umbrella cells in tumour adhesion and growth, by examining whether human urinary bladder carcinoma cells (HUBCC) can grow on reconstructed urinary bladder mucosa in vitro, as the implantation of tumour cells after resection is thought to be a cause of bladder tumour recurrence. MATERIALS AND METHODS: Normal transitional epithelial cells isolated from porcine bladder were cultured on reconstructed lamina propria using fibroblasts in type I collagen gel. The urothelium thus reconstructed was artificially injured either by a scalpel or by dilute acid, after which transitional epithelial cells began to grow in a stratified fashion within a few days of culture. A HUBCC line (HT-1197) was seeded onto this impaired mucosa to determine whether the cells could become implanted. Cultured cells on the reconstructed mucosa were evaluated by histological observation of vertical paraffin sections. RESULTS: The inoculated transitional epithelial cells grew in a stratified fashion and closely resembled urothelium in vivo. The superficial cells that were in contact with the medium solution differentiated into umbrella cells. HUBCC were unable to adhere to reconstructed mucosa which had not been injured, but these cells could adhere to and become implanted on the reconstructed mucosa after it had been injured either by a scalpel or by dilute acid. After acid injury, only the surface-covering cells were removed sporadically, while the lower epithelial cell layer remained intact. The bladder cancer cells adhered to and proliferated within these stripped regions. CONCLUSION: These results suggest that the urothelium, especially umbrella cells, seems to be important in preventing the adhesion and growth of urinary bladder tumour cells.

Relation of vascular endothelial growth factor production to expression and regulation of hypoxia-inducible factor-1 alpha and hypoxia-inducible factor-2 alpha in human bladder tumors and cell lines.

Hypoxia is an important regulator of vascular endothelial growth factor (VEGF) expression, and VEGF is associated with poor prognosis in bladder cancer. To investigate further the mechanisms of VEGF regulation, we examined VEGF expression by mRNA and protein analysis in four human bladder cancer cell lines, showing a progression from well to poorly differentiated phenotypes under varying conditions of confluence and hypoxia (0.1% O(2)) and with chemical mimics of hypoxia. Hypoxia significantly increased VEGF protein expression in all cell lines, although this effect was dependent on the degree of confluence. The superficial bladder cancer cell line RT4 lost hypoxia inducibility at confluence, whereas inducibility was maintained in the invasive cell lines 253J and EJ28. This pattern of VEGF expression in the invasive cell lines correlated with the expression of the transcription factor hypoxia inducible factor-1 alpha (HIF-1 alpha) and with hypoxia-inducible factor-2 alpha (HIF-2 alpha) and in RT4 correlated with a marked reduction in HIF-1 alpha inducibility at confluence. Using the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor LY 294002, we show that this VEGF hypoxia-inducible pathway regulated by HIF-1 alpha is distinct from a PI 3-kinase-dependent pathway, which regulates basal amounts of VEGF, but does not affect inducibility. Both HIF-1 alpha and HIF-2 alpha protein and mRNA were up-regulated in primary human bladder tumors (n = 12) compared with normal bladder specimens (n = 4), with significant intertumor variation. These results suggest that components of the hypoxia response pathway, including HIF-1 alpha and HIF-2 alpha, are important cofactors in the regulation of VEGF in bladder cancer and are therapeutic targets in this disease.

Human bladder cancer invasion model using rat bladder in vitro and its use to test mechanisms and therapeutic inhibitors of invasion.

As well as being a passive support, the extracellular matrix also regulates key biological processes such as invasion, differentiation and angiogenesis. We have therefore developed an in vitro model of bladder cancer invasion using de-epithelialized rat bladder to allow for tumour cell-extracellular matrix interactions. Onto this we have seeded a panel of human bladder cancer cell lines (RT4, RT112, 253J and EJ28 (T24)) representing progression from well to poorly differentiated phenotypes and used as models of superficial to invasive bladder cancer. The better differentiated cell lines RT4 and RT112 reproducibly grew as stratified epithelium, whereas poorly differentiated EJ28 cells invaded across a broad front. Invasion was not simply related to proliferation rate, measured either as doubling time on plastic (non-invasive 253J and invasive EJ28 having the same doubling time) or by Ki-67 proliferation index within the model. We used the model to test the ability of 4 compounds that interfere with tumour cell-extracellular matrix interactions (suramin, N-acetylcysteine and the urokinase plasminogen activator pathway antagonists A5 compound and monoclonal antibody Mab 3936) to inhibit invasion. At non-toxic concentrations, all significantly inhibited invasion (P< 0.05), although to varying degrees, suramin and A5 almost completely and N-acetylcysteine the least. In conclusion, this model shows the urokinase system is important for bladder invasion and can be used to investigate other mechanisms of bladder cancer invasion and also for the testing of intravesical drugs.

Assessment of angiogenic factors the chick chorioallantoic membrane assay.

Angiogenesis is the growth of new vessels from existing vessels. It is important in the physiological processes of wound healing, embryogenesis, and the female menstrual cycle and involved in pathologies such as diabetic retinopathy and rheumatoid arthritis (1). There is now abundant evidence that tumors are angiogenesis dependent. Unless tumors can stimulate angiogenesis, and generate their own blood supply, they fail to grow larger than 2-3 mm(3). The angiogenic status of a tumor can be assessed directly using immunohistochem istry on pathology sections (see Chapter 13 by Kilic and Ergün in this volume and Chapter 7 by Ranieri and Gasparini and Chapter 8 by Turner and Harris in the companion volume) to count the number of blood vessels within a given area (microvessel density [MVD]). MVD is a prognostic indicator in a number of tumors including breast, bladder, and prostate (2). Angiogenic capacity can also be measured by assaying the various stimulatory and inhibitory factors that regulate angiogenesis. This can be done either by determining the mRNA level using ribonuclease protection (see Chapter 16 by Jones et al. in the companion volume) or the protein by either Western blotting (see Chapter 11 by Blancher and Jones in the companion volume) or with one of the commercially available enzyme-linked immunosorbent assay (ELISA) kits. Again, levels of these factors have been shown to be prognostic in a number of tumors.

RNA purification and quantification methods.

Analysis of gene expression within tumors is frequently performed on the messenger RNA (mRNA). This chapter first describes the purification of mRNA from either tissues or cell culture and then describes the quantification by ribonuclease protection assay.Having initially purified the cellular RNA the technique of ribonuclease protection relies on generation of a radioactive probe of antisense messenger RNA that is homologous to, and therefore capable of hybridizing with, the complementary strand of endogenous sense mRNA. A cocktail of single-strand-specific ribonucleases is then added to degrade any regions of the probe not protected by hybridization. Double-stranded mRNA, which is resistant to ribonuclease degradation, is then separated by polyacrylamide gel electro-phoresis (a technique already described in Chapter 11 by Blancher and Jones) and subsequently detected by autoradiography.Premature contamination with ribonucleases that are both stable and active is one of the main reasons for the failure of these techniques. Contamination with exogenous ribonucleases is avoided by the following (1) Gloves should be wornskin is a plentiful source of ribonucleases. (2) Plasticware not touched by ungloved hands can be considered ribonuclease free. All glassware should be baked at 250'300°C for at least 4 h or rinsed with chloroform before use. (3) All solutions should be made up with water treated overnight with diethylpyrocarbonate (DEPC) which inactivates ribonucleases by covalent modification.

Thymidine phosphorylase induces carcinoma cell oxidative stress and promotes secretion of angiogenic factors.

Thymidine phosphorylase (TP) (E.C. 2.4.2.4), also known as platelet-derived endothelial cell growth factor, is a potent angiogenic factor. The expression of TP correlates with poor prognosis in a range of tumor types. 2-Deoxy-D-ribose-1-phosphate, a product of thymidine catabolism by TP, is a strongly reducing sugar that generates oxygen radical species during the early stages of protein glycation. We show that thymidine induces oxidative stress in TP-overexpressing carcinoma cells, promoting secretion of the stress-induced angiogenic factors vascular endothelial growth factor and interleukin-8, and inducing matrix metalloproteinase-1. Our findings outline a putative mechanism for TP-induced angiogenesis and identify novel targets for intervention.

Angiogenesis and lymphangiogenesis in stage 1 germ cell tumours of the testis.

OBJECTIVE: To determine whether angiogenesis can be used as an additional prognostic indicator in patients with stage 1 germ cell tumours of the testis. PATIENTS AND METHODS: Paraffin sections were assessed immunohistochemically from 51 patients with clinical stage 1 germ cell tumours of the testis (28 seminoma, 23 teratoma) treated by orchidectomy and surveillance only. Sections were analysed for microvascular density (MVD), and expression of the angiogenic factors vascular endothelial growth factor (VEGF) and thymidine phosphorylase (TP). In addition, in the seminoma cases the presence of mRNA for the lymphangiogenic factor VEGF-C was examined by in situ hybridization, and its corresponding receptor VEGFR-3 by immunohistochemistry. RESULTS: Teratoma specimens had a significantly higher mean (range) MVD (85, 26-163; P < 0.01) than both seminoma (37, 16-91) and four normal specimens (26, 18-30). Teratoma specimens also had significantly higher VEGF expression than both seminoma and normal specimens (P < 0.01). Despite these differences between groups, and indeed individual tumours, there was no significant correlation between MVD and VEGF, or between either MVD or VEGF and relapse-free survival. TP expression was significantly greater in tumours than in normal specimens (P < 0.02) but with very little inter-tumour variation. VEGF-C mRNA and VEGFR-3 protein were detected in a third to a half of cases, with expression mostly around endothelial vessels. CONCLUSIONS: The marked differences between normal testis and tumours implicate angiogenesis in the biology of germ cell tumours of the testis. In addition, the detection of factors involved in lymphangiogenesis in some seminomas, tumours which initially metastasize primarily to lymph nodes, indicate that although not prognostic in this study, further studies are warranted in both these areas in the search for further prognostic indicators and therapeutic targets.

Elevated serum vascular endothelial growth factor in patients with hormone-escaped prostate cancer.

OBJECTIVE: To investigate the role of serum vascular endothelial growth factor (VEGF) in the assessment of patients with prostate cancer. Patients, subjects and methods Serum from 78 men was assayed for VEGF using a commercially available enzyme-linked immunosorbent assay kit. Forty-eight patients had a histopathological diagnosis of prostate cancer (16 local disease, 32 metastatic), nine had benign prostatic hyperplasia (BPH) and 21 were healthy controls. RESULTS: The mean serum VEGF level was significantly higher in patients with hormone-escaped prostate cancer than in all other groups (P

Case of mesothelioma of the tunica vaginalis testis with characteristic findings on ultrasonography and magnetic resonance imaging.

A case of mesothelioma of the right tunica vaginalis testis in a 32-year-old man is reported. Trans-scrotal ultrasonography revealed hydrocele and multiple nodular masses measuring 1.0-4.5 cm in size attached to the parietal vaginal layer. Magnetic resonance imaging demonstrated more clearly nodular masses with irregular surfaces lined on the hydrocele cavity. Histologic diagnosis of the tumor when orchiectomized was mesothelioma. The patient has been free of disease for approximately 3 years since the treatment.

Epstein-Barr virus-associated gastric carcinoma in Mexico: analysis of 135 consecutive gastrectomies in two hospitals.

Epstein-Barr virus (EBV) has been implicated in the genesis of gastric carcinoma. The presence of clonal episomal viral forms in the nuclei of neoplastic gastric epithelial cells suggests that viral infection occurs before the development of gastric carcinoma. Mexico is a country at high risk for gastric cancer-it is the second cause of death among patients who die of cancer in that country. A series of 135 consecutive non-selected gastrectomies from two hospitals in Mexico City were analyzed to search for EBV in gastric carcinomas. EBV-encoded small non-polyadenylated RNA (EBER) in situ hybridization was performed on 5-microm paraffin-embedded tissue sections. Age, gender, anatomical site, histological type, and invasiveness of gastric carcinomas were obtained from the records in the corresponding Departments of Pathology. Eleven (8.15%) of the 135 cases were EBER-1-positive gastric carcinomas. Six occurred in males and five in females. In three women, the neoplasia was localized in the antrum. Five of the 11 cases were lymphoepithelioma-like carcinomas and, in two of them, an unusual foreign body-type inflammation was observed. Environmental factors could influence the distinctive pathologic features of EBV-associated gastric carcinoma in the Mexican population.

Novel human PDCD4 (H731) gene expressed in proliferative cells is expressed in the small duct epithelial cells of the breast as revealed by an anti-H731 antibody.

The novel gene H731 (approved name: PDCD4 (programmed cell death 4)) has been isolated as an antigen gene of the monoclonal antibody Pr-28 which recognized a nuclear antigen in proliferating cells. The gene is homologous to the mouse gene (MA-3/Pdcd4/A7-1) which was associated with apoptosis and was shown to suppress tumor promoter-induced neoplastic transformation. A polyclonal antibody against H731-protein derived from an extract of Escherichia coli transformed with an H731 expression plasmid was prepared, and the H731-protein expression in human normal and tumor cells using the antibody was studied. The staining patterns of asynchronous cultures of human normal fibroblasts (MRC-5) were heterogeneous but the antigen was accumulated in the nuclei at the G0 phase. On the contrary, the antigen was overproduced and localized in the cytoplasm during the cell cycle in tumor cell lines. Immunohistological studies revealed that the H731-protein was highly expressed in bladder carcinoma and breast carcinoma tissues compared with the normal tissues so far tested. These results indicated that expression of the H731-protein was up-regulated or induced in the proliferative cells. Immunohistological studies also revealed that the protein was abundantly expressed in the small duct epithelial cells of the normal mammary gland.

Relationship between ambient sulfur dioxide levels and neonatal mortality near the Mt. Sakurajima volcano in Japan.

We examined the association between neonatal mortality and ambient sulfur dioxide (SO2) levels in the neighborhood of Mt. Sakurajima, Yamashita public health district of Kagoshima City, during the period between 1978 and 1988. The analysis using Poisson regression models showed that the monthly average level of SO2 was positively associated with the neonatal mortality (P = 0.002). When the SO2 levels were categorized into four groups to estimate the relative risk (RR) of neonatal mortality using the lowest exposure category as a reference, the RR increased with elevated exposure levels (P for trend < 0.001) and was the highest in the group with the highest level of exposure (RR = 2.2, 95% confidence interval; 1.2-4.1). Other than SO2, we also examined the number of eruptions, the amount of ashfall, and the average level of suspended particulate matter. None of these factors was associated with neonatal mortality. Although the present study suggests that increase in SO2 levels has had an adverse effect on neonatal mortality in the neighborhood of Mt. Sakurajima, it is difficult to determine the source of the SO2. Further studies are necessary to elucidate the mechanisms of the excess neonatal mortality probably associated with the volcanic SO2 levels.

An Assessment of Amniotic Fluid Index Among Japanese (A Longitudinal Study)

>Objective: To obtain a longitudinal gestational reference range for the amniotic fluid index (AFI) among Japanese women and to compare the study with those of previous reports.Methods: A total of 739 measurements of the AFI of 96 Japanese women with normal pregnancies were analyzed. The criteria were singleton pregnancies between 20 and 40 weeks, without fetal anomalies, diabetes mellitus, hypertension, and other maternal complications. Logarithmic transformation was used to obtain the predicted mean AFI values with 95% confidence intervals at each gestational week.Results: The AFI rose from 20 weeks reaching its peak at 30 weeks. After the peak, it declined toward 40 weeks. Comparison of this study with previously published reports revealed differences in the mean AFI values.Conclusions: We obtained the gestational age-specific value of AFI in normal pregnancy for Japanese women.

Reconstruction of prostatic acinus-like structure from ventral and dorsolateral prostatic epithelial cells of the rat in three-dimensional collagen gel matrix culture.

PURPOSE: The study was carried out to reconstruct a prostatic acinus-like structure from prostatic epithelial cells in a new culture system which can provide a more physiological condition than conventional cell culture methods. MATERIALS AND METHODS: Prostatic epithelial cells were isolated from ventral and dorsolateral prostates of the rat and were separately cultured in three-dimensional collagen gel matrix. The cultured cells were observed by photo-microscopy and transmission electron microscopy. Differentiation and proliferation of the cultured cells were examined by immunohistochemistry using PAP and PSA kits and a bromodeoxy-uridine (BrdU) kit. The cell area of acinus-like structures in the transverse sections was measured by computer with the Interaktive Bild-Analyse System. RESULTS: Dissociated prostatic epithelial cells were organized into three-dimensional spherical or branching cellular aggregates in collagen gel matrix. Through cell proliferation and differentiation, the cellular aggregates formed prostatic acinus-like structures, which consisted of a small intercellular lumen enclosed by one layer of cuboidal epithelial cells. The epithelial cells were connected together by junctional complexes, had microvilli at the luminal surface, and the basal side was surrounded by a distinct basal lamina. In the lumina of acinus-like structures, secretory products were often found. CONCLUSIONS: Prostatic acinus-like structures were reconstructed from dissociated prostatic epithelial cells in three-dimensional collagen gel matrix culture. The reconstructed acinus-like structures are similar to prostatic acini in vivo in structure and function. This three-dimensional collagen gel matrix culture model may provide a useful means for investigating prostatic diseases and influences that hormones and growth factors exert on prostatic epithelial cells.

[EBV-related stomach cancer and HLA].

Recently, the involvement of EB virus in gastric cancer has drawn world-wide attention. In Japan, it is suspected that EB-virus is related to 5-10% of stomach cancer. In this paper, we briefly reviewed EBV-related immune responses and HLA distributions in EBV-related diseases. A brief description on our recent studies on the comparison of HLA distributions in EB-virus positive and negative stomach cancers was also given.

MDA-MDMA concentrations in urine specimens.

Urine specimens collected from active-duty U.S. Army personnel were submitted for analysis to the Tripler Army Medical Center, Forensic Toxicology Drug Testing Laboratory as part of the random drug testing program. During an 18-month drug-screening period, 34 specimens tested positive for amphetamines with the Roche Abuscreen Radioimmunoassay for Methamphetamine (High Specificity); based on gas chromatographic-mass spectrometric (GC-MS) analysis, the presence of 3,4-methylenedioxymethamphetamine (MDMA) was suspected. These samples were subsequently submitted to the Division of Forensic Toxicology, Office of the Armed Forces Medical Examiner, Armed Forces Institute of Pathology for further testing. All 34 samples screened positive using both the Abbott TDx Amphetamine/ Methamphetamine II assay and the Amphetamine class assay. Confirmation and quantitation by GC-MS revealed the presence of both MDMA and 3,4-methylenedioxyamphetamine (MDA) in all samples. The MDMA concentrations ranged from 0.38 to 96.2 mg/L (mean, 13.4 mg/L) and the MDA concentrations ranged from 0.15 to 8.6 mg/L (mean, 1.6 mg/L). The mean ratio of MDA, the N-demethylation metabolite of MDMA, to MDMA was 0.15, similar to the ratio of amphetamine, the N-demethylation metabolite of methamphetamine, to methamphetamine of 0.10. The presence of MDA in urine specimens at a concentration approximately 10-15% that of the MDMA present is consistent with MDMA metabolism, which may be indicative of the use of MDMA only, as compared with the combined use of both drugs.

Characterization of a unique T-cell clone established from a patient with HAM/TSP which recognized HTLV-I-infected T-cell antigens as well as spinal cord tissue antigens.

Five T-cell clones reactive to autologous HTLV-I-infected T-cells (KODA-TV) were established from peripheral blood lymphocytes of a HAM/TSP patient (KODA) by the limiting dilution method. All the clones showed CD3+, CD4+ and CD25+ surface markers and expressed alpha beta+ T-cell receptors to recognize KODA-TV antigens. One of the five T-cell clones (KODA-408) was infected with HTLV-I but the remaining four clones (KODA-400, 404, 405 and 409) were free of HTLV-I infection. KODA-408 recognized both KODA-TV and spinal cord antigens, the latter being extracted from autopsy tissues of a HTLV-I seronegative donor. KODA-408 did not recognize either alloantigens of peripheral blood mononuclear cells extracted from unrelated HTLV-I seronegative donors or purified human myelin basic protein. KODA-408 T-cell clone produced a considerable amount of TNF-alpha, IFN-gamma, and IL-6. The CDR3 motif of KODA-408 T-cell receptor showed a unique sequence CASSAGQS of v beta 8-D beta-J beta 1.5. These results indicated that HAM/TSP CD4+ T-cells were polyclonally activated by HTLV-I infection and antigenic stimulation. The T-cell repertoire shaped by HTLV-I infection included T-cells which recognized HTLV-I-infected T-cell antigens as well as spinal cord antigen in particular.

Ethnic segregation of HTLV-I and HTLV-II carriers among South American native Indians.

To investigate the genetic background of human T-cell leukemia virus type I (HTLV-I) and II (HTLV-II) carriers among South American native Indians, we analyzed HLA DRB1*-DQB1* haplotypes of the virus carriers from Andes highlands and Orinoco lowlands by the PCR-RFLP genotyping method. It was revealed that the HTLV-I-carrying Andes natives had one of the 5 HLA haplotypes: DRB1*-DQB1* 0403-0302, 0802-0402, 0901-0303, 1406-0302 and 0407-0302, and that the Orinoco HTLV-II carriers had one of the 3 HLA haplotypes: DRB1*-DQB1* 1402-0301, 1602-0301 and 0404-0302. The HLA haplotypes of Andes HTLV-I carriers and Orinoco HTLV-II carriers were mutually exclusive. The haplotypes associated with HTLV-I carriers were commonly found among the Andes Indians and Japanese, which is the known HTLV-I endemic population, while the haplotypes associated with HTLV-II carriers were specifically found among the Orinoco Indians and North American Indians, among whom HTLV-II is endemic. These results suggested that HLA haplotypes might be ethnically segregated among South American natives and might be involved in the susceptibility to HTLV-I and HTLV-II infections.