Masaki Cho

Immunohistochemical Analysis of Midkine Expression in Human Prostate Carcinoma

Midkine (MK) is a growth/differentiation factor frequently expressed at high levels in some types of human malignancies. To investigate whether MK is a useful marker in prostate carcinogenesis, immunohistochemical analysis was performed on samples of both latent and clinical prostate cancers of various stages, as well as on specimens of normal gland and prostatic intraepithelial neoplasia (PIN). Of the 80 clinical cancers examined, 69 specimens (86.3%) were immunoreactive for MK, with metastatic lesions generally showing higher expression than the corresponding primaries; normal prostate tissues were negative or showed only weak staining. Midkine was also detected in 12 of 15 latent cancers (80%) and in 12 of 16 cases of PIN (75%). In sections of whole prostate, MK showed variable expression through tumorous sections, probably in reflection of heterogeneous cell populations. The results demonstrate the possible value of MK as a marker for early and latent disease, as well as for more advanced clinical stages of prostate cancer.

E-cadherin and α -, β - and γ -catenin expression in prostate cancers: correlation with tumour invasion

SummaryThe E-cadherin–catenin complex plays an important role in establishing and maintaining intercellular connections and morphogenesis and reduced expression of its constituent molecules is associated with invasion and metastasis. In the present study, we examined E-cadherin and α-, β- and γ-catenin levels in tumour tissues obtained by radical prostatectomy in order to investigate the relationship with histopathological tumour invasion. Immunohistochemical findings for 45 prostate cancer specimens demonstrated aberrant expression of each molecule to be associated with dedifferentiation and, in addition, alteration of staining patterns for the three types of catenin was significantly correlated with capsular but not lymphatic or vascular invasion. The data thus suggest that three types of catenin may be useful predictive markers for biological aggressiveness of prostate cancer.

Hypomethylation of the MN/CA9 promoter and upregulated MN/CA9 expression in human renal cell carcinoma

MN/CA9 is a cancer-related gene, frequently activated in human renal cell carcinomas (RCCs). To reveal the activation mechanism, we investigated the relationship between methylation status of the MN/CA9 promoter region and gene expression using 13 human RCCs, and examined the effect of in vitro CpG methylation on the MN/CA9 promoter activity using a human RCC cell line (SK-RC-44), expressing MN/CA9. MN/CA9 expression was evaluated by RT-PCR and observed in 10 of 13 RCCs (77%). A total of 9 out of 10 MN/CA9 -positive RCCs (90%) contained clear cell components. Methylation status of 6 CpGs in the MN/CA9 promoter region was decided by using the bisulfite genomic sequencing protocol. Out of 13 RCCs 9 (69%) showed partial hypomethylation of the CpG at –74 bp, while the other 4 RCCs and 3 normal kidney tissue samples showed complete methylation. Hypomethylation of the CpG at –74 bp was strongly correlated with MN/CA9 expression. Luciferase assay revealed that the MN/CA9 promoter activity was strongly suppressed by methylation of the CpG at –74 bp. These findings suggest that hypomethylation of the CpG at –74 bp in the MN/CA9 promoter region might play an important role in this gene activation of human RCC.

Aggressive angiomyxoma in the scrotum expressing androgen and progesterone receptors

Abstract  Aggressive angiomyxoma is a rare benign mesenchymal myxoid tumor that arises from the pelvic soft tissues and perineum in relatively young females. This tumor has the ability to infiltrate locally and has a high risk of local recurrence after extirpation, but no potential to metastasize. We report here a rare case of aggressive angiomyxoma that developed in the scrotum of a 47‐year‐old male. Immunostaining of the resected specimen revealed that the tumor cell nuclei stained strongly and diffusely for androgen receptors (80% of the tumor cells), and moderately and partly for progesterone receptors (20% of the tumor cells). However, staining was negative for estrogen receptors. It is highly suggested that the growth of aggressive angiomyxoma in males may depend on androgen manipulation, contrary to its frequent and close association with estrogen receptor expression, which has been reported in females.

Activation of the MN/CA9 gene is associated with hypomethylation in human renal cell carcinoma cell lines

The MN/CA9 (G250) gene expressed in the normal alimentary tract in a tissue‐specific manner is often activated in renal cell carcinomas. To cast light on the activation mechanism, we examined the methylation status of this gene in seven human renal cell carcinoma cell lines (SKRC‐01, ‐06, ‐10, ‐12, ‐14, ‐44, and ‐59) and three normal kidney tissue samples by using the bisulfite genomic sequencing protocol. CpG methylation was measured at seven locations in the MN/CA9 5′ region. MN/CA9 transcripts were detected by reverse transcription–polymerase chain reaction in five of the renal cell carcinoma cell lines (SKRC‐01, ‐06, ‐10, ‐44, and ‐59). These MN/CA9 positive cell lines showed hypomethylation, whereas the remaining two cell lines (SKRC‐12, and ‐14), and three normal kidney tissue samples without transcripts demonstrated hypermethylation. Treatment with the demethylating agent 5‐aza‐2′‐deoxycytidine resulted in activation of the MN/CA9 gene in the negative cell lines (SKRC‐12 and ‐14). These data suggest that hypomethylation in the 5′ region may have a major role in expression of the MN/CA9 gene in renal cell carcinoma cells. Mol. Carcinog. 27:184–189, 2000.

Genetic changes in prostate cancer.

Recent advances in molecular biology have allowed us to understand that it is the accumulation of genetic alterations which leads to each step of tumor genesis. What the specific alterations may be, however, often varies with each neoplasm. Prostate cancer is somewhat unique in its presentation to the pathologist of a bewildering array of histologies difficult to assign to diagnostic categories and contributing to misinterpretations of underlying molecular events. As with any malignancy, It is of utmost importance to thoroughly analyze and record the genetic aberrations found in prostate cancer with the objective of correlation to the pathology and natural history of the disease. Multiple ontogeny and tumor suppressor genes have been investigated in both clinical and latent cancers using conventional mutational analyses. To probe deeper Into these genes and to uncover novel molecular events, genomic tumor DNA were examined using restriction landmark genomic scanning (RLGS), a method which allows the Identification and comparison of specific genetic alterations within large segments and multiple samples of DNA at a time. This article reviews what has been identified based on numerous molecular studies, focusing on the genetic alterations peculiar to human prostate cancer.

Expression of pepsinogen II with androgen and estrogen receptors in human prostate carcinoma

The expression of pepsinogen II (PG II), an aspartyl proteinase usually involved in the digestion of proteins in the stomach, was immunohistochemically investigated in conjunction with androgen (AR) and estrogen receptor (ER) status in prostate adenocarcinomas. Of a total of 38 samples obtained from radical prostatectomies, 23 tumors (60.5%) were positive for PG II and there was a significant positive correlation to the expression of AR but not to ER. Cells positive for PG II were localized mainly to the peripheral zones of tumorous glands which, in normal prostate, are negative, and in areas also expressing AR. In addition, a significant correlation between AR and ER was detected in the prostate carcinomas examined, which suggests a hormone‐dependent status. On the basis of these results, PG II expression might be closely related to hormonal alterations associated with the development of prostate tumors.

Formation of 8-hydroxydeoxyguanosine in rat kidney DNA after administration of N-ethyl-N-hydroxyethylnitrosamine

N-Ethyl-N-hydroxyethylnitrosamine (EHEN) is known to induce renal and liver tumors in rodents. Recent reports have indicated the formation of 8-hydroxydeoxyguanosine (8-OHdG), an oxidative DNA product, induced by various carcinogens. In the present study, to examine whether oxygen radicals are involved in tumorigenesis induced by EHEN, we investigated the formation and localization of 8-OHdG in kidney, liver and lung of rats. The effects of reduced glutathione (GSH) and diethylmaleate on these responses were also studied. Multiple doses of EHEN administrations (250, 500 or 750 mg/kg, i.p.) resulted in a significant elevation of the 8-OHdG level in kidney DNA in a dose-dependent manner and the formation of 8-OHdG reached the maximal level at 1-2 h after EHEN injection and recovered to the control level at 4 h. On the other hand, no increase in the 8-OHdG level was observed in the DNA of liver and lung. Combined pre- and post-treatment of rats with 2 x 800 mg/kg of GSH i.p. inhibited the elevation of the 8-OHdG level induced by EHEN. Pre-treatment with 0.3 ml/kg of diethylmaleate i.p. increased the formation of 8-OHdG. In the immunohistochemical examinations of rats treated with EHEN (750 mg/kg, i.p.), nuclear expression of 8-OHdG was detected in the epithelial cells of renal cortex, while no induction was observed in liver and lung. These findings suggest that the formation of 8-OHdG by active oxygen species may be an important factor in the initiation of EHEN-induced kidney carcinogenesis.

Effect of Hexachloro-1,3-butadiene on Renal Carcinogenesis in Male Rats Pretreated with N-Ethyl-N-hydroxyethylnitrosamine

Hexachloro-1,3-butadiene (HCBD) is a potent nephrotoxicant that selectively damages the straight portion (pars recta) of the proximal tubule in the rat. To determine its effects on carcinogenesis, HCBD was administered for 30 wk at a concentration of 0.1% by weight in basal diet to male Wistar rats previously given 0.1% Af-ethyl-W-hydroxyethylnitrosamine (EHEN) in the drinking water for 2 wk. The combined treatment resulted in a significantly higher incidence of renal cell tumors than when EHEN was administered alone. This chronic exposure and a short course of a 0.2% HCBD diet for 3 wk caused marked increase in the numbers of bromo-deoxyuridine-incorporating cells or proliferating cell nuclear antigen-positive cells in the outer stripe of the kidney. The ability of HCBD to promote EHEN-initiated renal tumorigenesis in rats thus appears to be associated intimately with linked nephropathy and subsequent cell proliferation.

Effect of polyphenon-60 on the development of renal cell tumors in rats treated with N-ethyl-N hydroxyethylnitrosamine

Green tea consumed as a beverage in Asia contains polyphenols, which contain about a 15% mixture of catechins. The present paper reports the effect of polyphenon-60 (60% pure catechin) on the development of renal cell neoplasms in Wistar rats pretreated with N-ethyl-N-hydroxyethylnitrosamine (EHEN): 0.1% polyphenon-60 in block diet was given over a period of 30 weeks while EHEN was given in drinking water for 2 weeks. The results appears to show a tendency for green tea catechins (GTC) to decrease the incidence of renal cell tumors greater than 3 mm in diameter in Wistar rats but not tumors that are less than 3 mm in diameter. Polyphenon-60 did not affect EHEN initiation in the kidneys of rats. It is postulated that free radicals induced by EHEN may be suppressed by GTC, resulting in a lowering of the tendency for tumor growth.