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Complement C4b-binding protein (C4BP) is a plasma protein synthesized in the liver and plays a regulatory role in the host defense complement system. We have previously reported that mRNAs of the C4BP α chain (C4BPα) are expressed at significant levels in the guinea pig and mouse epididymis in an androgen-dependent manner. Here, we analyze the murine C4bpagene and show that epididymal and liver C4BPα mRNAs are generated from a single-copy gene and that the epididymal C4BPα mRNAs are transcribed from novel transcription start sites located approximately 100 base pairs downstream from those used in the liver. Furthermore, in an immunohistochemical study using rabbit anti-mouse C4BP antiserum, we demonstrated that C4BP is localized in the stereocilia and Golgi apparatus of the epididymal epithelial cells and the surfaces of spermatozoa in the lumen in the region from the distal caput to the cauda but not in the proximal caput region. Indirect immunofluorescence of the isolated spermatozoa demonstrated that C4BP is localized preferentially on the head region of the spermatozoa, and immunoelectron microscopy located C4BP on the plasma membrane and the outer acrosomal membrane. These results indicate that epididymal C4BP is synthesized in the epithelial cells and secreted into the lumen in a region-restricted manner and is taken up to the sperm membranes on passage through the epididymis. Many epididymal proteins are secreted from the epithelial cells in a region-specific and androgen-dependent manner and are considered to contribute to sperm maturation. Our findings suggest a novel function of C4BP as one such epididymal secretory protein.

Renal cell cancer is a unique solid tumor that occasionally shows spontaneous regression even at an advanced stage, of which the underlying mechanism is not well understood. To investigate a potential role of the pro-apoptotic molecule caspase-1 in the growth regulation of renal cell cancer, we created transfectants expressing exogenous caspase-1 from a murine renal cancer cell line, Renca. Overexpression of caspase-1 did not affect the growth of Renca cells in vitro at the exponential phase but induced apoptotic cell death at 50% to 75% confluence, whereas control cells underwent apoptosis only after reaching 100% confluence. When implanted into the flank of a syngeneic BALB/c mouse, caspase-1-overexpressing Renca cells did not effectively establish growth as a solid tumor, forming a measurable tumor in only 7 of 11 (64%) animals, whereas control cells formed a tumor in 6 of 6 (100%) animals. The growth of tumors from caspase-1-overexpressing cells slowed down markedly after the tumors reached 5 to 10 mm in diameter, and histological examination of such tumors revealed numerous apoptotic cells positively stained by TUNEL assay. Interestingly, endogenous caspase-1 was not detected in the tumors from control cells, which re-expressed caspase-1 when they were re-cultured and exposed to a demethylation reagent, 5-aza-2'-deoxycytidine. Furthermore, treatment of a human renal cancer cell line, ACHN, with 5-aza-2'-deoxycytidine also caused recovery of caspase-1 expression, which was not detected before treatment. These data suggest that silencing of caspase-1 through DNA methylation may be involved in the oncogenesis of some renal cell cancers growing as a solid tumor. © 2001 Wiley-Liss, Inc.

Our aim was to determine the distribution of α1L‐adrenoceptors in canine prostate by an autoradiographic technique using [3H]JTH‐601 (an α1L‐adrenoceptor antagonist) and [3H]JTH‐601‐G1 (an active metabolite of JTH‐601).

Our aim was to determine the distribution of α1L-adrenoceptors in canine prostate by an autoradiographic technique using [3H]JTH-601 (an α1L-adrenoceptor antagonist) and [3H]JTH-601-G1 (an active metabolite of JTH-601).Prostates were removed from three male beagle dogs. Several slices of the specimens were incubated with 5 nmof [3H]JTH-601, [3H]JTH-601-G1 and [3H]tamsulosin (an α1A-adrenoceptor antagonist). For macroscopic autoradiography, visualization was performed using an imaging plate and image-analyser. To examine microscopic localization of binding sites, preparations were exposed, developed and fixed.Specific binding of [3H]JTH-601 and [3H]JTH-601-G1 was observed diffusely throughout the entire interstitium on macroscopic autoradiography. Specific binding of [3H]tamsulosin was also recognized although the binding was weaker than that of [3H]JTH-601. On microscopic autoradiograms, the grains of each ligand were mainly distributed on smooth muscle.These results indicate morphologically that specific binding sites of JTH-601 and JTH-601-G1 exist in canine prostate, suggesting the distribution of α1L-adrenoceptors in this tissue, in addition to α1A-adrenoceptors.

Abstract: The CD95/CD95 ligand (CD95L) system plays an important role in the induction of lymphoid apoptosis and has been implicated in the suppression of immune responses. In this system, two murine CD95L-transfected renca clones and a control renca clone transfected only with the vector were implanted into the subcapsule of the left kidney of Balb/c and Balb/c nude mice. Both CD95L-expressing and control renca clones formed macroscopic tumors in all of the Balb/c and Balb/c nude hosts 14 days after implantation. Growth of tumors of murine CD95L-transfected renca cells was significantly better than that of control renca cells in Balb/c mice, while the growth advantage of CD95L transfectants was not observed in Balb/c nude mice. Lymphocytes underwent apoptosis mainly in the periphery of the CD95L-expressing tumors but not in control tumors grown in Balb/c mice, while lymphocytes undergoing apoptosis were not observed in CD95L-expressing tumors or in control tumors grown in Balb/c nude mice. Neutrophilic recruitment was rarely observed in CD95L-expressing or control tumors. CD95L expressed on renca cells possibly suppressed immune responses against renca tumors by inducing apoptosis of the infiltrating lymphocytes. However, CD95L-expressing renca cells did not form tumors in the renal subcapsule of allogeneic C3H/HeJ mice.

PurposeWe compared the affinities of a new alpha1-adrenoceptor (AR) antagonist, JTH-601 with those of several alpha1-AR antagonists in human prostates and arteries.ResultsIn the functional study, noradrenaline produced concentration-dependent contractions in human prostates and mesenteric arteries. The pA2/pKB values for the antagonists in the human prostate were 9.78 for tamsulosin, 8.84 for JTH-601, 8.39 for WB4101, 8.23 for prazosin, 8.12 for JTH-601-G1 (a main metabolite of JTH-601 in human) and 6.57 for BMY7378. Compared these affinities with those in the mesenteric artery, only JTH-601 and JTH-601-G1 exhibited unique uroselectivity, showing 10- to 20-fold higher affinity for the human prostate than for mesenteric artery. The affinity profile of these antagonists suggested that the noradrenaline-induced contractions in the human prostate and the mesenteric artery were mediated by the alpha1L-AR and alpha1B-AR, respectively. In the competition binding study, the pharmacological profiles of the antagonists against [(3) H]-prazosin were examined in the human prostate and aorta. The resulting pKi values for JTH-601 and JTH-601-G1 were also approximately 10- to 20-fold higher for the human prostate than for the human aorta.ConclusionThese results have suggested that JTH-601 and JTH-601-G1 are unique uroselective alpha1-AR antagonists that show higher affinity for the human prostate than for the human arteries.

1This study was intended to quantify the amounts of the α1‐adrenoceptor subtype mRNAs in human vas deferens, and demonstrate the receptor subtype responsible for the vas contraction.2The RNase protection assay showed that the mean total amount of α1amRNA was 7.4±2.2 pg/5 μg of poly (A)+RNA (97.0% of the total α1mRNA) in the epididymal portion (E‐vas) and 4.9±0.8 pg/5 μg of poly (A)+RNA (96.3% of the total) in the pelvic portion (P‐vas). The E‐vas showed a tendency to have a greater α1amRNA abundance than the P‐vas (P=0.11). The α1band α1dmRNAs were absent or of extremely low abundance.3By an in situhybridization, the α1aand α1dmRNAs were recognized in the smooth muscle cells of the E‐vas and the P‐vas, and the distribution pattern the same in both tissues. The α1bmRNA positive site was scarcely detectable in both vas portions.4In a functional study, l‐phenylephrine produced concentration‐dependent contraction in the E‐vas (Emax=2.24±0.70 g; pD2=5.32±0.09) and the P‐vas (Emax=2.46±0.46 g; pD2=5.07±0.12). KMD‐3213, a novel α1A‐adrenoceptor‐selective antagonist, caused parallel rightward shifts of the concentration–response curves for l‐phenylephrine. Apparent pKBvalues were 9.90±0.16 for the E‐vas and 9.71±0.17 for the P‐vas. There was no significant difference in Emax, pD2or pKBestimates between the two portions.5We have found that α1amRNA is predominant in the human vas deferens, and confirmed that contraction of this organ is mediated by the α1A‐adrenoceptor.

The monoclonal antibody MRG-1 has been evaluated for the immunohistochemical detection of the type 3 chain of blood group A in human normal bladder epithelium and bladder tumours. Light microscope examination of paraffin sections demonstrated that this antigen was present in normal epithelium and superficial bladder tumour in patients with blood group A or AB, but was absent in the invasive type of bladder tumour. In normal epithelium, the plasma membrane was positive for this antigen, and the cytoplasm was diffusely stained. In superficial transitional cell carcinoma, the plasma membrane was negative, whereas the cytoplasm was intensely stained in the perinuclear region. This pattern was different from that observed for type 1 and 2 group A antigen, which was recognized mainly at the plasma membrane. However, in superficial transitional cell carcinoma, the staining was also seen on the plasma membrane. The pattern of the localization of this antigen in this carcinoma was influenced by the treatment of organic solvents. Electron microscopical observations confirmed that this antigen was localized on the plasma membrane and also in the Golgi apparatus of the superficial tumour. These results proved that the type 3 chain of blood group A is present in human bladder epithelium and low grade tumours in correspondence with the blood type, but disappears in tumours with high malignant potential. However, its expression is independent of the expressions of the other subtypes which have been studied. Furthermore, the changes in the staining pattern caused by pretreatment with organic solvents suggested possible differences in the microenvironment of the glycolipids containing this type of sugar chain.

Fourteen specimens of human hypertrophied prostate were evaluated for the distribution of α1adrenoceptors using autoradiography with a computerized image analysis system. The hypertrophied prostatic specimens, obtained at open prostatectomy, were dissected vertically to the urethra, and sectioned at 10 μm. They were immersed in 1 nM of specific α1ligand, [3H]tamsulosin chloride ([3H]tamsulosin) and exposed to autoradiographic film. The images were analysed by a computerized image analysis system. The total binding of [3H]tamsulosin in the whole section (n= 14) was 0.82 ± 0.21 (mean ± se) nCi mg−1. The autographic data were correlated with data obtained in a membrane-binding assay. The prostatic tissue studied was divided into urethral, glandular and stromal zones, the latter two zones being further divided into the inner and outer areas. The total binding of [3H]tamsulosin in the urethral zone (n= 7) was 0.65 ± 0.32 nCi mg−1. The glandular zone contained significantly more abundant α1adrenoceptors than the stromal zone and their densities (glandular vs stromal) were 1.15 ± 0.19 nCi mg−1(n= 14) vs 0.72 ± 0.15 nCi mg−1(n= 14), respectively (p< 0.05). The data from the whole section were not affected by prostatic weight. This method described enabled the distribution of the receptors in different sites to be evaluated both morphologically and quantitatively.

BackgroundPreoperative endocrine therapy has been suggested to improve surgical radicality and/or patient prognosis in prostate cancer.

We have investigated whether the Fas-mediated cell death pathway is functional in renal cell carcinoma. The expression of Fas in surgical specimens and cell lines of renal cell carcinoma was examined. Fas expression was positive in six out of 18 tumors measured by flow cytometry and was prominent in advanced tumors. Three out of the six Fas-positive tumors had already metastasized at the time of surgery. A significant correlation was found between the tumor volume and the percentage of Fas-positive cells in a tumor (r= 0.70, P=0.0007). Fas-positive tumors were larger than Fas-negative tumors [mean tumor volume (ml) ± SD, Fas(+), 265.6 ± 136.8; Fas(–), 65.8 ± 80.9, P= 0.0012]. All human renal carcinoma cell lines tested (ACHN, Caki-1, SMKT-R-2, SMKT-R-3 and SMKT-R-4) expressed Fas abundantly, as Fas-positive cells accounted for >50% in all cell lines by flow cytometry. Treatment with anti-Fas antibody caused apoptosis in Fas-positive renal cell carcinoma cell lines. However, the effectiveness of apoptosis induction in individual cell lines was not correlated with the level of Fas expressed. These data suggest that Fas targeting may be a therapeutic option for treatment of advanced renal cell carcinoma which is refractory to either chemotherapy or irradiation.

Alpha-adrenoceptors have been considered to play an important role in the regulation of the voiding force. In order to determine the density and localization of the alpha-adrenoceptors in the prostate, binding assays for alpha-adrenoceptors were performed with [3H]prazosin and [3H] yohimbine in membrane preparations from enucleated hyperplastic prostate tissues. Furthermore, autoradiographic analysis of alpha-adrenoceptors in the sliced tissue specimens from benign prostatic hypertrophy was performed. Specific bindings of both ligands were saturable and of high affinity in membrane preparations, and Scatchard analyses indicated Bmax = 104fmole/mg. protein, Kd = 0.488nM for [3H]prazosin, and Bmax = 41fmole/mg. protein, Kd = 1.83nM for [3H] yohimbine. Bmax and Kd for [3H]prazosin were greater in the adenoma than in the submucosal tissue of the prostatiac urethra. No relationship was noted between the size of enucleated prostate and the density of alpha-adrenoceptors. Image analysis of autoradiograms using [3H]prazosin showed specific binding sites could not be clearly demonstrated, but only [125I]HEAT slightly exposed specific binding sites in the prostate.

Transmission (TEM) and scanning electron microscopic (SEM) observations were performed on well-differentiated tumours and chronic cystitis in the human urinary bladder. SEM showed that the pleomorphic microvilli were present not only on the luminal surface of the tumour but also on the surface of inflammatory mucosa. The ultrastructure of six tumours and 5 cases of chronic cystitis was evaluated morphometrically. Bladder tumour and inflammatory mucosa were divided into several layers, namely outermost cells (S), subsurface cells just beneath these (S1), subsurface cells of 2 or 3 layers below (S23), intermediate cells of 2 or 3 layers above the basal cells (123), intermediate cells just above the basal cells (I1) and basal cells (Ba). Areas of nucleus, cytoplasm and cytoplasmic organelles, numbers of nucleoli, nuclear bodies, mitochondria and lysosomes together with irregularity of the cell and nucleus were estimated according to the methods of Weibel. A multi-variate analysis of variance on these variables showed that the above subdivision of layers was necessary for the comparison of tumour and inflammation. Discriminant analysis showed various differences between tumour and inflammatory mucosa. The results indicated that the Ba layer is the most effective site for differentiating the tumour from inflammation. Ba cells with large and irregular cytoplasm with an enlarged Golgi area, accompanied by many vacuolar structures, may be indicative of tumour rather than inflammation.

Three cases of inverted papilloma of the urinary bladder were studied by transmission electron microscopy. Scanning electron microscopic observation was made in one of these. The surfaces of the outermost tumour cells were covered with short stubby microvilli. Multiple bud like proliferations of the tumour cells were compatible with a trabecular type of inverted papilloma. The tumour cells of the trabeculum mimicked the intermediate and basal cells of the epithelium which covered the surface. Microcysts are believed to be formed by epithelial migration into pits, creating an epithelial inversion, and do not represent central necrosis. Ultrastructure suggests that inverted papilloma is a very well differentiated tumour.

Tumor regression in experimental systems has been linked to the activities of Th1 cells. It is, therefore, conceivable that Th2 cells interrupt the expression of tumor immunity since interleukin-4 (IL-4) and IL-10 inhibit the generation of Th1 from precursors and modulate the competence of antigen-presenting cells to activate this lymphocyte subpopulation. Naive murine renal cell carcinoma (renca) cells (1×105) were implanted into the subcapsule of the left kidney of Balb/c and Balb/c nude mice at 6 – 8 weeks of age. After 14 days, Th2 cytokine (IL-4 and IL-10) mRNAs as well as transforming growth factor β1 mRNA, assessed by reverse transcriptase/polymerase chain rea ction were upregulated in the spleen of hosts upon naive renca tumor acceptance, while Th1 cytokine (IL-2 and interferon γ) mRNAs were almost undetectable. In the renca tumor, IL-10 mRNA was detected but IL-2, interferon γ, and IL-4 were not. Intraperitoneal administration of anti-(mouse IL-4) mAb (11B11) reduced the renca tumor size ( P= 0.018) and prolonged host survival (P= 0.03), but did not reduce the acceptance rate of the tumor (P= 0.18). However, prior depletion of CD4+or CD8+cells with monoclonal antibodies abrogated the antitumor effects of anti-IL-4 mAb. In addition, the significant antitumor effect of anti-IL-4 mAb was not observed in Balb/c nude hosts. Renca cells we re transfected with the mammalian expression vector pCAGGS containing murine IL-4 cDNA or vector alone, then stable IL-4 transfectants (RencaL or RencaH, low- or high-IL-4-producing respectively) and control renca cells (RencaC) were obtained. RencaL cell s, RencaH cells, or RencaC cells (1×105each) were implanted into the subcapsule of the left kidney of Balb/c, Balb/c nude, and allogenic C3H/HeJ mice, then tumor formation was evaluated 14 days later. When RencaH cells were innoculated into syngeneic Balb/c hosts, tumor volume was marginally suppressed (P= 0.03) and tumors tended to be rejected (P= 0.06) compared with RencaC cells. However, those effects were not observed in Balb/c nude mice. RencaC, RencaL, and RencaH cells were not accepted by allogeneic C3H mice with or without FK506 administration or donor-specific trans fusion. The administration of anti-(mouse IL-4) mAb to Balb/c mice significantly suppressed renca tumor growth by a CD4+and CD8+T-cell-dependent mechanism. By contrast, relatively high levels of IL-4 production by renca cells and T cells seemed to be required to induce the rejection and growth suppression of IL-4-producing renca cells in syngeneic hosts.

We performed RNase protection assays and in situhybridization to investigate the ratio of the three α1‐adrenoceptor subtype mRNAs, α1a, α1b and α1d, in human proximal urethra, and their localization in urethral cross‐sections. As revealed by the RNase protection assays, α1a was the predominant subtype mRNA in both male and female urethral samples. α1d mRNA was detected only in the female sample, and α1b mRNA was not detected in any of the samples tested. The ratio of the abundance of the subtype mRNAs, α1a : α1b : α1d, was 100 : 0 : 0 in the male urethra and 90 : 0 : 10 in the female urethra.In situhybridization studies showed no significant differences in the cross‐sectional distribution of α1‐adrenoceptor subtype mRNAs between male and female urethras. Intense α1a staining was observed in the smooth muscle of the urethra, but α1b and α1d staining was much less intense.Of the three cloned α1 subtypes, α1a is the most likely to be responsible for the contraction of the human urethra. Owing to the side effects of nonselective α1drugs, α1‐selective drugs may be clinically superior to nonselective drugs for the treatment of urethral disorders.

Adrenergic stimulation induces contraction of hypertrophied prostatic tissue via the α1adrenoceptor, and the results of pharmacological studies suggested the existence of adrenoceptor subtypes. Recently three subtypes (α1a, α1b, and α1d) were cloned. Using probes for these subtypes, we demonstrated their expression in the tissues of ten cases of benign prostatic hypertrophy, usingin situhybridization. To determine the ratio between these subtypes, an RNase protection assay was also performed in three cases. Expression of the α1aand α1dadrenoceptors was diffuse in the smooth muscles of the interstitium, but was absent in glandular epithelial cells. On the contrary, the α1badrenoceptor was hardly detectable. The RNase protection assay confirmed the absence of the α1badrenoceptor, the ratio of α1aand α1dbeing 4∶1. These results supported the idea that the differences in prostatic contractile response to several adrenergic drugs are based on the affinities of these drugs for the different subtypes.

A 61‐year‐old woman with acute urinary retention was found to have a carcinosarcoma in the region of the urethra. Evaluation of the computed tomogram suggested a urethral tumor, which was resected by a transperineal approach. She received local radiotherapy after surgery, and is alive at 1 ‐year follow‐up with a tumor metastasis to the pelvis.

A hypospadiac boy with a hypoplastic penis and an apparent 46,XX karyotype in blood and testis cultures is described. Exploratory laparotomy and bilateral gonadal biopsy revealed the presence of 2 testes in the right and 1 in the left hemiscrotum, each of which only showed hypoplastic testicular tissues histologically. Uncultured testis smears showed Y chromatin in approximately 20% of the cells. Also, the Southern blot and polymerase chain reaction analyses detected a weak but distinct signal of Y chromosome-derived deoxyribonucleic acid sequences in the perineal skin but not in the blood lymphocytes. The results indicated that the boy had a small proportion of Y chromosome-containing cells in the form of mosaicism in limited tissues, such as the testes and perineal skin. This finding may have implications in the genesis of testes in some cases of XX patients, and true hermaphrodites or male pseudohermaphrodites with an apparent 46,XX karyotype. To our knowledge, this appears to be the first case of polyorchidism with an identified chromosome abnormality.