Tomosumi Ikeda

Expression of survivin does not predict survival in patients with transitional cell carcinoma of the upper urinary tract

Abstract. The members of the IAP (inhibitors of apoptosis) family, which includes survivin, have recently emerged as modulators of an evolutionarily conserved step in apoptosis. Survivin is present during embryonic and fetal development, but it is downregulated in normal adult tissues. However, it becomes re-expressed in a variety of cancers. We investigated the prognostic importance of the expression of survivin in transitional cell carcinoma of the upper urinary tract (TCC-UUT). In 126 cases of TCC-UUT, we examined its expression (using immunohistochemistry), and also its relationship with the expressions of bcl-2 oncoprotein, p53 oncoprotein, and proliferating cell nuclear antigen (PCNA) immunoreactivity, clinicopathologic parameters, and clinical outcome. A positive expression of survivin was recognized in 12.7% of samples, a granular pattern being apparent within the cytoplasm of tumor cells. Survivin expression did not correlate with clinicopathologic findings, bcl-2 oncoprotein expression, p53 oncoprotein expression, PCNA index, or prognosis. In the normal urothelium, its expression was not detected. In conclusion, the expression of survivin does not predict prognosis in TCC-UUT.

E-cadherin expression in upper-urinary-tract carcinoma

Carcinoma of the upper urinary tract is a relatively rare neoplasm, and few studies have dealt with clinicopathological findings and prognosis in a large number of cases. The purpose of our investigation was to look for a possible relation between E‐cadherin (E‐CD) immunoreactivity and clinicopathologic findings or clinical outcome in transitional‐cell carcinoma of the upper urinary tract (TCC‐UUT). To this end, we investigated E‐CD immunoreactivity in 154 cases of TCC‐UUT. E‐CD immunoreactivity was recognized as being of “normal” pattern in 29.2% of samples. The relationship between E‐CD immunoreactivity and clinicopathologic findings was significant for stage, grade and pattern of growth. The 5‐year disease‐free rate for 147 cases and 5‐year overall survival rate for 154 cases were 55.7% and 71.5%, respectively. A univariate analysis of survival revealed that stage, grade, pattern of growth and E‐CD immunoreactivity all had a significant effect on disease‐free and overall survival rates. In the final models of multivariate analysis, however, we found that, for disease‐free survival and for overall survival, only stage was a factor for progression or prognosis. Detection of E‐CD immunoreactivity appears to be of limited value in deciding the prognosis of TCC‐UUT. Int. J. Cancer 74:446–449, 1997.

Gene transfer of human hepatocyte growth factor into rat skin wounds mediated by liposomes coated with the sendai virus (hemagglutinating virus of Japan).

Hepatocyte growth factor (HGF) regulates cell growth, cell motility, and morphogenesis in various types of cells, including epithelial and endothelial cells, indicating that it probably promotes epithelial repair and neovascularization during wound healing. To better understand the effects of HGF on wound healing, we performed human HGF-gene transfer into skin wounds in rats. The rat HGF mRNA levels, and human and rat HGF protein concentrations in the wounds in HGF gene-transfer rats were significantly elevated at 3 days, 3 to 14 days, and 3 and 14 days after gene transfer, respectively. An expression of human HGF mRNA and protein was revealed in squamous cells in the epidermis, in endothelial cells and smooth muscle cells in blood vessels, and in fibroblasts in granulation tissues at 3, 7, and 14 days after gene transfer in HGF gene-transfer rats. The wound lesion area in HGF gene-transfer rats was significantly less than that in control rats from 3 to 7 days after gene transfer. The re-epithelialization rate, microvessel counts in granulation tissues, proliferating cell nuclear antigen index of fibroblasts in granulation tissues, and the proliferating cell nuclear antigen index in the epidermis of HGF gene-transfer rats were significantly increased at 3 and 7 days after gene transfer. Semiquantitative reverse transcriptase-polymerase chain reaction revealed that the expression levels of transforming growth factor-beta1 and Colalpha2(I) mRNAs in the wounds of HGF gene-transfer rats were significantly decreased at 7 and 14 days, respectively. The hydroxyproline concentration in the wound was significantly less in HGF gene-transfer rats than in control rats at 3 days after gene transfer. These results suggest that HGF gene transfer into a skin wound may aid re-epithelialization and neovascularization in the early phase of wound healing, and that HGF may play a role in modulating cutaneous wound healing.

Nonviral HVJ (hemagglutinating virus of Japan) liposome-mediated retrograde gene transfer of human hepatocyte growth factor into rat nervous system promotes functional and histological recovery of the crushed nerve.

Hepatocyte growth factor (HGF) is well known to be involved in many biological functions, such as organ regeneration and angiogenesis, and to exert neurotrophic effects on motor, sensory, and parasympathetic neurons. In this study, we gave repeated intramuscular injections of the human HGF gene, using nonviral HVJ (hemagglutinating virus of Japan) liposome method, to examine whether transfection of the rat nervous system with this gene is able to exert neurotrophic effects facilitating recovery of a crushed nerve. The expression of HGF protein and HGF mRNA indicated that gene transfer into the nervous system did occur via retrograde axonal transport. At 4 weeks after crush, electrophysiological examination of the crushed nerve showed a significantly shorter mean latency and a significantly greater mean maximum M-wave amplitude with repeated injections of HGF gene. Furthermore, histological findings showed that the mean diameter of the axons, the axon number and the axon population were significantly larger in the group with repeated injections of HGF gene. The above results show that repeated human HGF gene transfer into the rat nervous system is able to promote crushed-nerve recovery, both electrophysiologically and histologically, and suggest that HGF gene transfer has potential for the treatment of crushed nerve.

Tissue factor is associated with the nonbacterial thrombotic endocarditis induced by a hypobaric hypoxic environment in rats

Abstract High-altitude hypoxia causes a hypercoagulable state. In our previous study on the blood coagulation system in rats, nonbacterial thrombotic endocarditis (NBTE) developed after 4–12 weeks’ exposure to the equivalent of 5500 m in altitude. We hypothesized that TF (tissue factor)-producing cells in the cardiac valves might be induced by the hypobaric hypoxic environment (HHE) and then trigger NBTE. A total of 170 male Wistar rats were housed in a chamber at the equivalent of 5500 m altitude for 1–12 weeks. We measured TF activity in the plasma and studied morphological changes in the mitral valves using immunohistochemical and immunoelectrical methods for TF protein and in situ hybridization for TF mRNA. After 4 weeks or more of exposure to HHE, 28 of the 56 surviving rats had developed NBTE. After 4–8 weeks’ exposure to HHE, the plasma TF activity level was significantly higher than in control rats. There was a significant correlation between plasma TF activity and the incidence of NBTE. After 1 weeks’ exposure to HHE, immunoreactivity for TF protein was detected in foamy macrophages and stromal cells in the cardiac valves. In rats with NBTE, TF protein was present in foamy macrophages and spindle stromal cells and focally present in the extracellular matrix. TF mRNA was detected in some foamy macrophages within the thrombus, TF protein was localized to the rough endoplasmic reticulum and plasma membrane of many macrophages, some fibroblasts, and a few endocardial cells. TF is associated with the pathogenesis of the NBTE induced by exposure to HHE. The accumulation of TF-producing macrophages during exposure to HHE may be responsible for initiating thrombus formation.

Changes in myosin heavy chain and its localization in rat heart in association with hypobaric hypoxia-induced pulmonary hypertension

Experimental pulmonary hypertension induced in a hypobaric hypoxic environment (HHE) is characterized by structural remodelling of the heart. In rat cardiac ventricles, pressure and volume overload are well known to be associated with changes in cardiac myosin heavy chain (MHC) isoforms. To study the effects of HHE on the MHC profile in the ventricles, 83 male Wistar rats were housed in a chamber at the equivalent of 5500 m altitude for 1–8 weeks. Pulmonary arterial pressure, right ventricular free wall (RVFW) weight, the ratio of RVFW weight over body weight (BW), the ratio of left ventricular free wall (LVFW) weight over BW, and myocyte diameter in both ventricles showed significant increases after 1 week, 2 weeks, 1 week, 6 weeks, and 4 weeks of HHE, respectively. Semi‐quantitative reverse transcriptase–polymerase chain reaction revealed that β‐MHC mRNA expression was increased significantly in both ventricles at 6 and 8 weeks of HHE, whereas α‐MHC mRNA expression was decreased significantly at 6 and 8 weeks of HHE in the right ventricle (RV) and at 6 weeks of HHE in the left ventricle (LV). The percentage of myosin containing the β‐MHC isoform was increased significantly at 4–8 weeks of HHE in RV and at 6 weeks of HHE in LV. In situ hybridization showed that the area of strong staining for β‐MHC mRNA was increased in both ventricles at 8 weeks of HHE, and showed a decrease from RVFW to cardiac septum, and from cardiac septum to LVFW. These results suggest that HHE has a significant effect on the expression of both MHC mRNA and protein in the heart, particularly in RV. These changes may reflect a role for cardiac MHC in the response to pulmonary hypertension in HHE. Copyright

Expression of Telomerase Catalytic Subunit (hTERT) mRNA Does Not Predict Survival in Patients with Transitional Cell Carcinoma of the Upper Urinary Tract

Telomerase is a ribonucleoprotein enzyme that synthesizes telomeric repeats onto chromosomal ends using a segment of its RNA component as a template. Its activity has become an established indicator of the diagnosis, biological behavior, and prognosis of several tumors. However, few studies have investigated the diagnostic and prognostic importance of the expression of telomerase catalytic subunit (hTERT) mRNA in transitional cell carcinoma of the upper urinary tract (TCC-UUT). We investigated the expression of hTERT mRNA using in situ hybridization in 125 cases of TCC-UUT, and also its relation with the expression of telomerase RNA component (hTERC), proliferating cell nuclear antigen (PCNA) immunoreactivity, clinicopathologic parameters, and clinical outcome. A positive expression of hTERT mRNA was recognized in 93.6% of the samples and was apparent within the cytoplasm of tumor cells. In the normal urothelium examined in a few cases, its expression was barely detected. hTERT mRNA scores showed a significant association with hTERC score. However, no relationship was found between the expression of hTERT mRNA and clinicopathologic findings, PCNA index, or prognosis. These results suggest that the expression of hTERT mRNA does not predict prognosis in TCC-UUT.

Expression of C-type natriuretic peptide during development of rat lung

C-type natriuretic peptide (CNP), recently found to be secreted from vascular endothelial cells, is now viewed as a novel endothelium-derived relaxing peptide. However, the distribution and expression of CNP during cardiopulmonary development is unclear. To follow changes in the expression of CNP during lung development, we examined rat embryos and neonates using Northern blot analysis and in situ hybridization for CNP mRNA and radioimmunoassay and immunohistochemistry for CNP protein. A substantial expression of CNP mRNA was first detected on postnatal day 2, and it thereafter remained fairly steady. The level of CNP protein also increased rapidly after postnatal day 1, reaching a settled level on postnatal day 4. CNP protein and mRNA were detected in the endothelium and smooth muscle cells of blood vessels and in bronchial airway and alveolar epithelia. Immunoreactivity for CNP protein in the endothelium of blood vessels increased to an intense level after the saccular stage. These results suggest that the changes in CNP levels may be related to the occurrence of pulmonary vasodilation after birth.C-type natriuretic peptide (CNP), recently found to be secreted from vascular endothelial cells, is now viewed as a novel endothelium-derived relaxing peptide. However, the distribution and expression of CNP during cardiopulmonary development is unclear. To follow changes in the expression of CNP during lung development, we examined rat embryos and neonates using Northern blot analysis and in situ hybridization for CNP mRNA and radioimmunoassay and immunohistochemistry for CNP protein. A substantial expression of CNP mRNA was first detected on postnatal day 2, and it thereafter remained fairly steady. The level of CNP protein also increased rapidly after postnatal day 1, reaching a settled level on postnatal day 4. CNP protein and mRNA were detected in the endothelium and smooth muscle cells of blood vessels and in bronchial airway and alveolar epithelia. Immunoreactivity for CNP protein in the endothelium of blood vessels increased to an intense level after the saccular stage. These results suggest that the changes in CNP levels may be related to the occurrence of pulmonary vasodilation after birth.

Expression of p27Kip1 Protein in Transitional Cell Carcinoma of the Upper Urinary Tract

The expression of p27Kip1, a negative regulator of the cell cycle, has been reported to correlate with the biological behavior and prognosis of several tumors. However, its prognostic importance in transitional cell carcinoma of the upper urinary tract (TCC-UUT) has not previously been investigated. We investigated p27Kip1 protein expression using immunohistochemistry in 132 cases of TCC-UUT and also its relation to proliferating cell nuclear antigen (PCNA) immunoreactivity, p53 oncoprotein immunoreactivity, clinicopathologic parameters, and clinical outcome. A positive expression of p27Kip1 protein was recognized in 94.7% of the samples and was apparent within tumor nuclei. In the normal urothelium, its expression was identified in all cell layers. A positive expression of p53 oncoprotein was recognized in 27.2% of the patients. The PCNA index was 7.4 to 93.1% (mean, 66.4%). Examination of the relationships between the expression of p27Kip1 protein and clinicopathologic findings, PCNA index, and the expression of p53 oncoprotein revealed that the expression of p27Kip1 protein decreased significantly with stage and grade. In a univariate analysis of disease-free and overall survival rates, no correlation was found between the expression of p27Kip1 protein and prognosis. The expression of p27Kip1 protein appears to be of little or no value in informing the prognosis in TCC-UUT.

Mucin histochemistry in primary adenocarcinoma of the urinary bladder (of urachal or vesicular origin) and metastatic adenocarcinoma originating in the colorectum.

In order to evaluate the mucin histochemistry of primary adenocarcinomas (PA) of the urinary bladder and metastatic adenocarcinoma (MA) originating in the colorectum, 52 PA and nine MA were examined. It was determined that the percentage of cases in which more than 25% of the tumor was stained by each of the following: (i) Alcian blue pH 2.5 periodic acid–Schiff (AB‐PAS); (ii) high iron diamine‐AB (HID‐AB); (iii) periodic acid‐sodium borohydride‐potassium hydroxide‐PAS (PA‐SB‐PH‐PAS); (iv) galactose oxidase– Schiff (GOS); and (v) paradoxical concanavalin A stain (PCS). For PA, the values obtained were: 75% of cases (blue, AB‐PAS), 85% (magenta, AB‐PAS), 71% (black, HID‐AB), 75% (blue, HID‐AB), 0% (PA‐SB‐PH‐PAS), 19% (GOS), 8% (class II concanavalin A (Con A)‐reactive mucin)), and 0% (class III Con A‐reactive mucin). For MA, the corresponding values were 33, 22, 0, 11, 0, 0, 11, and 0%, respectively. A higher percentage of PA than MA cases showed staining in AB‐PAS for acidic and neutral mucins, in HID‐AB for sialo‐ and sulfomucins, and in GOS for terminal β‐galactose and β‐N‐acetylgalactosamine. PA and MA were significantly different in terms of both frequency of staining with AB‐PAS and frequency of staining with HID‐AB. However, the overlap was such that in practice, it might be difficult, if not impossible, to use mucin histochemistry to inform a differential diagnosis. In view of the differences in AB‐PAS and HID‐AB positivity between PA and MA, we speculate that MA (originating in the colorectum) may have undergone structural distortion affecting the production and/or secretion of neutral mucins and acidic mucins (sialo‐ and sulfomucins) during metastasis or invasion.