Inhibitory effect of testosterone on gap junctional intercellular communication of human transitional cell carcinoma cell lines.

A dye transfer method was applied to investigate the effect of testosterone on gap junctional intercellular communication (IC) of two kinds of human transitional cell carcinoma cell lines, JTC-30 and JTC-32. When JTC-30 cells were cultured with testosterone at nontoxic concentrations (17-69 microM), a dose and time dependent inhibition of dye transfer was observed. More than 90% inhibition occurred after exposure to 69 microM testosterone for 96 h. The inhibition was reversed rapidly after testosterone deprivation. Similar results were obtained with JTC-32 cells. 17 beta-Estradiol showed no inhibitory effect on IC of both transitional cell carcinoma cell lines even at toxic levels. Testosterone exhibited no inhibitory effect on IC of human fibroblasts. The inhibitory effect of 5 alpha-dihydrotestosterone was almost similar to that of testosterone. At concentrations examined, cyproterone acetate influenced neither dye transfer nor the inhibitory effect of testosterone, suggesting a mechanism of testosterone action different from that of the known receptor system. Since blockage of IC has been indicated as one reliable evidence for tumor promotion, current results suggest that testosterone is a possible endogenous promoter of the bladder carcinoma and may therefore possibly play a role on the sexually different incidence of bladder carcinoma.

A dose-escalation and pharmacokinetic study of subcutaneously administered recombinant human interleukin 12 and its biological effects in Japanese patients with advanced malignancies.

A pilot dose-escalation study of recombinant human interleukin 12 (rhIL-12) was conducted in Japanese patients with advanced malignancies. Cohorts of three patients received escalating doses of rhIL-12 that increased from 50 to 300 ng/kg/day s.c. three times a week for 2 weeks followed by 1-week rest. The same dosage and schedule was repeated for two additional courses. Sixteen previously treated patients were registered, and 15 were evaluated. Common toxicities were fever and leukopenia; the abnormality of laboratory tests included elevations in aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, C-reactive protein, and beta2-microglobin. Dose-limiting toxicity was the grade 3 elevation of aminotransferases, and was observed in two of six patients at the 300-ng/kg dose level after the first course in one patient and after the third course in the other. Leukopenia was observed at all of the dose levels; two of six patients at 300 ng/kg experienced grade 3 leukopenia. Thus, 300 ng/kg was determined to be the maximum acceptable dose. Peak plasma levels of rhIL-12 decreased in the second courses, but the areas under the curve were almost the same in the first and second courses. Biological effects included increases of plasma levels of IFN-gamma, tumor necrosis factor-alpha, IL-6, IL-10, and neopterin. In two patients with renal cell carcinoma, complete response and partial response of metastatic tumors were observed with 50 and 300 ng/kg; the responses lasted for 5 and 3.5 months, respectively. Although immunological response to rhIL-12 varies depending on administration route and schedule and on patients' physiological conditions, the recommended dose for Phase II studies is 300 ng/kg s.c. three times a week for 2 weeks followed by 1-week rest.

Erythrocyte sodium-lithium countertransport activity as a marker of predisposition to hypertension and diabetic nephropathy in NIDDM.

OBJECTIVE: To evaluate the potentiality of erythrocyte sodium-lithium countertransport activity (SLC) as a marker of predisposition to hypertension and diabetic nephropathy in non-insulin-dependent diabetes mellitus (NIDDM). RESEARCH DESIGN AND METHODS: We examined 96 patients with NIDDM and 26 healthy control subjects. SLC and other data were compared among subgroups of the patients classified on the basis of hypertension, family history of hypertension, and stages of nephropathy. Data were also analyzed by stepwise multiple regression analyses. RESULTS: SLC was significantly higher in patients with hypertension than in those with normotension and significantly higher in patients with a positive family history of hypertension than in the negative group. Further analysis revealed that a family history of hypertension has independent influence on SLC, but hypertension itself does not. SLC was significantly higher in patients with macroalbuminuria than with microalbuminuria and higher in patients with microalbuminuria than with normalbuminuria. In stepwise multiple regression analyses, a family history of hypertension was the most important determinant of SLC, and SLC was the most important determinant of nephropathy. CONCLUSIONS: These data suggest that SLC strongly reflects a predisposition to hypertension and that it can be a useful marker of diabetic nephropathy in NIDDM.

Influence of TNF microsatellite polymorphisms (TNFa) on age-at-onset of insulin-dependent diabetes mellitus.

The TNF-alpha gene is located in the HLA region and has been implicated in the pathogenesis of Type I (insulin-dependent) diabetes mellitus (IDDM). We investigated the frequency of TNFa microsatellite alleles in 76 young-onset IDDM patients, 65 adult-onset IDDM patients, and 90 control subjects. We also examined the association of these TNFa alleles with HLA-DRB1 alleles, HLA-class I alleles, and TNF-alpha production. The frequency of the TNFa2 and TNFa9 alleles was increased in the young-onset IDDM patients compared to control subjects, but the increased frequency of TNFa2 was not significant after the correction for the number of comparisons was made. We did not find any association of TNFa2 or TNFa9 with any of the HLA-DRB1 alleles. In contrast, the frequency of the TNFa13 allele was decreased in both the young-onset and the adult-onset IDDM patients compared to the control subjects, but the difference lost significance after the correction was made in the adult-onset IDDM. The TNFa13 allele was strongly associated with DRB1*1502. Patients with TNFa2 or TNFa9 had greater TNF-alpha production, while those positive for TNFa13 had lower TNF-alpha production than patients with non-TNFa2, a9, and a13 alleles. These results suggest that TNFa polymorphisms are associated with age-at-onset of IDDM and influence the inflammatory process of pancreatic beta cell destruction in the development of IDDM.

Inactivation of calpain I and calpain II by specificity-oriented tripeptidyl chloromethyl ketones.

Three new tripeptidyl chloromethyl ketones, Leu-Leu-XCH2Cl, with X representing Phe, Tyr, or Lys, were synthesized and their potencies to inactivate calpains I and II were compared. They were designed to fulfil the specificity requirement of calpains established recently. When compared in terms of the dose for 50% inactivation, Leu-Leu-PheCH2Cl was the strongest inactivator, being 500-600 times more effective than tosyl-PheCH2Cl and 5-14 times more than N-[N-(L-3-trans-carboxyoxiran-2-carbonyl)-L-leucyl]agmatine (E-64). The potency toward calpain, either I or II, decreased in the order Phe greater than Tyr greater than Lys derivatives greater than E-64, whereas that toward papain was E-64 greater than Lys greater than Phe greater than Tyr derivatives. From the determined kinetic parameters, the Phe derivative was 18.3 and 16.6 times more effective than E-64 on calpains I and II, respectively. Likewise, the rate of the alkylation reaction by these chloromethyl ketones with calpain I was 2-4 times greater than that with calpain II. Leu-Leu-PheCH2Cl and its N-dansylated product should be useful for highly selective affinity labeling of calpains I and II.

Significant amount of multicatalytic proteinase identified on membrane from human erythrocyte.

Multicatalytic proteinase (MCP) was solubilized from human erythrocyte membrane with 0.1% Triton X-100 and purified to homogeneity using a combination of DEAE-cellulose, hydroxylapatite, and Ultrogel AcA34 chromatographies. This membranous MCP had similar properties to MCP purified in parallel from the cytosol. Both MCPs had a molecular mass of 570 kDa, were composed of apparently nine subunits of 22-36 kDa and had trypsin- and chymotrypsin-like activities. These activities were latent and required heating for the induction. However, slight differences were observed in the effects of reagents (DFP, monoiodoacetic acid, Mg2+, and Ca2+) between membranous and cytosolic MCP. The amount of MCP identified on membranes was estimated to be three-quarters or one-half of that found in the cytosol based on its trypsin- or chymotrypsin-like activity, respectively.

Cellular senescence of a human bladder carcinoma cell line (JTC-32) induced by a normal chromosome 11.

Human chromosome 11 is expected to carry tumor suppressor genes for a variety of human cancers, including bladder carcinoma. To examine the functional role of a putative tumor suppressor gene(s) on this chromosome in the development of bladder carcinoma, we performed microcell-mediated transfer of chromosome 11 into the bladder carcinoma cell line, JTC-32. Fifteen of 20 colonies formed by the transfer experiment showed a remarkable change in cell morphology. They flattened and ceased growing, or senesced, prior to 10 population doublings. The presence of transferred chromosome 11-derived fragments in the growth-arrested cells was confirmed by PCR-based polymorphism analyses. The remaining 5 microcell hybrid clones exhibited a parental cell-like morphology, and presumably escaped from senescence, which was accompanied by deletions and/or rearrangements of the transferred chromosome 11. On the other hand, a transferred normal chromosome 7 neither changed the cell morphology nor arrested the cell growth. These results support the hypothesis that chromosome 11 contains a gene or genes which restore the senescence program lost during the immortalization process of JTC-32 cells.

Combination chemotherapy with ifosfamide, 5-fluorouracil, etoposide and cisplatin for metastatic urothelial cancer.

PURPOSE: To investigate the activity of combination chemotherapy with ifosfamide, 5-fluorouracil, etoposide and cisplatin in patients with metastatic urothelial cancer. METHODS: A group of 29 patients were treated with 2000 mg/m2 ifosfamide, 750 mg/m2 5-fluorouracil, 100 mg/m2 etoposide and 20 mg/m2 cisplatin. All four drugs were given intravenously on days 1 through 3 and the treatment was repeated every 3 weeks. Of the 29 patients, 14 had lymph node metastasis alone, and 15 had visceral lesions. RESULTS: An objective response was achieved in 17 patients (59%). There was no difference in response rates according to metastatic site including osseous lesions, which responded well in four of six patients. The 3-year survival rate for all patients was 16% with four patients who had undergone salvage surgery being alive with no evidence of disease 15 to 61 months after initiation of the treatment. A good performance status, lymph node metastasis alone and administration of chemotherapy at the full dosage had a significantly favorable impact on patient survival. Bone marrow toxicity was significant and one patient died of treatment-related sepsis. CONCLUSIONS: Ifosfamide, 5-fluorouracil, etoposide and cisplatin combination chemotherapy appeared to be active in the treatment of metastatic urothelial cancer. Although bone marrow toxicity was significant, the treatment was well tolerated by the majority of the patients. Further study may be warranted.

Intravesical combination chemotherapy with mitomycin C and doxorubicin for superficial bladder cancer: a randomized trial of maintenance versus no maintenance following a complete response.

Between November 1986 and April 1989, 101 patients with superficial bladder cancer were treated with intravesical instillations of mitomycin C on day 1 and doxorubicin on day 2 of each week for 5 consecutive weeks. Of 61 complete responders, 23 patients with carcinoma in situ and 28 with papillary cancer were randomly assigned to a non-maintenance group or to a group receiving maintenance therapy consisting of monthly instillations of the same drugs for 12 months. The 2-year non-recurrence rate calculated for patients with carcinoma in situ was significantly better in the maintenance group than in the non-maintenance group. A similar tendency was observed for patients with papillary cancer, although the difference was not significant. Side effects were considerable, with moderate to severe bladder irritation occurring in approximately half of the patients. In addition to our previous findings, the present results indicate that this intravesical combination chemotherapy is effective in eliminating superficial bladder cancers and that since the effect is not durable, even in complete responders, maintenance therapy is necessary to reduce subsequent tumor recurrence.

Sequential instillation therapy with mitomycin C and adriamycin for superficial bladder tumors.

From October 1983 to September 1985, 84 patients with superficial bladder tumor (Ta, Tl, Tis) were treated with sequential instillation of mitomycin C (MMC) and adriamycin (ADM). Doses of 20 mg MMC on day 1 and 40 mg ADM on day 2 were instilled into the bladder and retained for at least 2 h; this was repeated once a week for 5 consecutive weeks. Patients who achieved complete response (CR), were randomized and underwent prophylactic treatment taking the form of either intermittent instillation of MMC or daily oral administration of 5-fluorouracil. Of 79 evaluable patients, 72 (91%) had received prior treatment for superficial bladder tumors, 69 (87%) had high-grade tumors, and 18 (23%) had non-papillary Tis. The overall response rate was 68%, made up of CR in 43 patients (54%) and partial response (PR) in 11 (14%). Patients with either five or more tumors or tumors larger than 1 cm showed a significantly lower response rate than those with fewer than five tumors and tumors smaller than 1 cm, respectively. There was no correlation between tumor growth pattern, tumor grade and response rate, though non-papillary Tis appeared to respond better than papillary tumors. A history of prior instillation therapy or of toxicity to this treatment had no significant influence on the response rate. Although no systemic toxicity was observed, 62 patients (74%) experienced cystitis and the treatment had to be discontinued within 4 weeks in 13 of 33 cases with severe symptoms. The preliminary conclusion of prophylactic treatment was that intermittent instillation of MMC was superior to 5-FU medication in reducing the recurrence rate for at least 2 years after the treatment.

The calpain-calpastatin system in hematopoietic cells.

Calpain I requires low Ca2+ for activation and calpain II requires high Ca2+. It was generally accepted that erythrocytes contain calpain I and calpastatin, but no calpain II. We have recently found, however, that nucleated chicken erythrocytes contain both calpains I and II in addition to calpastatin. The finding is significant in rectifying the previous view that the chicken has only one molecular species of calpain, whereas mammals have two. Another erroneous view which prevailed previously was that polymorphonuclear (PMN) cells contain only one calpain species. We could also recently demonstrate that pig PMN cells do contain both calpains I and II. The cloning of cDNAs for calpastatin enabled us to utilize them as the probes in studying the expression of calpastatin in various hematopoietic cell-line cells. We found that several T cells infected with human retrovirus HTLV-I markedly increased the production of calpastatin, which could be measured both by calpain-inhibition assay and by Western blot analysis, but the level of mRNA for calpastatin did not significantly change when compared with noninfected T cells. The increase in calpastatin protein always parallels with the expression of interleukin 2 receptor protein by the HTLV-I-infected T cells, although the biological implication of such phenomena is almost entirely unknown yet.

Clinical significance of "cannibalism" in urinary cytology of bladder cancer.

OBJECTIVE: To investigate the cytologic characteristics and clinical significance of "cannibalism" (cytophagocytosis), a characteristic histologic finding associated with malignancy, found in urinary cytology samples during the diagnosis of bladder cancer. STUDY DESIGN: The subjects were 252 patients with bladder cancer initially treated from 1981 to 1991. For their pretreatment urine samples, membrane filtration was performed to determine the presence and amount of cannibalism, a tumor cell within a tumor cell. The urinary cytologic findings were then correlated with the histologic findings of primary bladder cancer. RESULTS: Only cells from urinary cytology-positive specimens demonstrated cannibalism. The positive rate of cannibalism was significantly higher in grade 3 (25%) than grade 1 (0%) or 2 (8%) superficial papillary cancer (P < .01) and also higher in muscle-invading bladder cancer (57%) than grade 3 superficial papillary cancer (P < .01). The rate did not differ significantly between muscle-invading cancer and superficial nonpapillary cancer (44%). In 198 patients with superficial bladder cancer, progression was observed in 19 (10%) during a mean follow-up of 72 months. All of them showed positive urinary cytology results in pretreatment samples, and among the patients with positive urinary cytology, the progression rate was significantly higher when cannibalism was present than when it was absent (38% vs. 17%, P < .05). Moreover, in high grade cases, cannibalism had significant predictive value for progression. By multivariate analysis, cannibalism was an independent factor for prediction of progression, as were tumor grade and stage. CONCLUSION: Cannibalism in urinary cytology appears to be an indicator of both the anaplastic grade and invasiveness of transitional cell carcinoma of the bladder. Furthermore, cannibalism may provide a reliable predictor of progression of superficial bladder cancer.

[Sequential intravesical chemotherapy with mitomycin C and adriamycin for superficial bladder tumor (preliminary report)].

Intravesical chemotherapy with sequential instillation of mitomycin C and adriamycin was carried out on 19 patients with superficial bladder cancer (Ta, T1 and Tis). Twenty milligram of mitomycin C on day 1 and 40 mg of adriamycin on day 2 were instilled into the bladder, this treatment being repeated weekly for 5 consecutive weeks if there were no serious side effects. The following results were obtained. Of 16 evaluable patients, 10 patients (63%) achieved complete response and 2 patients achieved partial response, 4 patients showing no response. In patients with high grade tumor, especially with carcinoma in situ, a high CR rate (6/7 or 86%) was achieved. Chemical cystitis occurred in 10 out of 19 (53%) patients. In 5 of the 6 patients who suffered from severe cystitis symptoms, treatment was discontinued. However, the symptoms resolved within 4 weeks in all patients.

[Evaluation of intercellular communication of human prostatic epithelium].

Intercellular communication (IC) among epithelial and/or interstitial cells of human prostate was evaluated. IC was measured by the dye transfer method. In brief, fluorescent lucifer yellow CH was microinjected into an individual cell and communication was measured by scoring the number of surrounding fluorescent cells. Cells used here were outgrowth cells from small tips of tissues of various prostatic diseases and established prostatic carcinoma cell line PC-3. To identify the epithelial cells, keratin staining was done. In prostatitis and benign prostatic hyperplasia, epithelial cells grew in five of six cases cultured and extent of maximum IC was from 18 to 46 cells (mean = 26 cells) at 4 to 6 culture days. Three specimens of separate cases cultured for more than one month had a maximum IC of more than 100 cells. In prostatic carcinoma, epithelial cells grew in 7 of 10 cases cultured and extent of maximum IC was from 6 to 38 cells (mean = 18 cells) at 3 to 6 days. The extent of IC among prostatic carcinoma cells was lower than that of benign prostatic diseases, though not significantly, this indicates the possibility that benign cells were present among the malignant cells in the specimens examined above. Prostatic carcinoma cells cultured from metastatic lesions of bone (PC-3) and lymph nodes had maximum IC of 3 to 5 cells. IC was very limited or absent between benign prostatic epithelium and PC-3 cells. No IC was present between prostatic epithelial and interstitial cells. Interstitial cells prepared from prostatic benign and malignant diseases similarly revealed a high extent of IC.(ABSTRACT TRUNCATED AT 250 WORDS)

[Two cases of urachal cyst].

Case 1: A lower abdominal large painful mass was recognized by palpation, CT scan and ultrasonography in a 64-year-old house wife. Urine cytology was negative. The mass at the dome of bladder was covered with normal epithelium cystoscopically. Aspiration cytology of the lower abdominal mass demonstrated no malignancy and total excision of urachal remnant with a portion of bladder wall was carried out. Histologically, the mass was an urachal cyst with granulomatous change infected with C group beta-streptococcus. Case 2: A 46 year-old male engineer complained of asymptomatic hematuria. Cystoscopic examination revealed a small bleeding lesion at the dome of bladder. Urine cytology was negative. CT scan and ultrasonography revealed a tiny cystic mass lesion with irregular density. Biopsy or aspiration cytology appeared difficult because of the size and localization of the mass. En bloc segmental resection of urachal remnant was carried out. Since intraoperative rapid histological examination of the specimen confirmed no malignancies, dissection of pelvic lymph node was not performed. Urachal cysts presented above were suspicious of malignant degeneration from findings of imaging examination. Either preoperative or intraoperative histological examination in such cases appears to be indispensable to avoid unnecessary extensive operation as well as to perform radical operation required for malignant lesions.

[Treatment methods as a prognostic factor in eight patients with urachal carcinoma].

Between 1960 and 1982, 8 patients with urachal carcinoma underwent segmental resection of the bladder or en bloc resection, and their five-year survival rate was 50%. One patient each with well, moderate and poorly differentiated adenocarcinoma and one patient with transitional cell carcinoma, died of cancer from 6 months to 2 years and 2 months after operation (mean duration: 1 year and 3 months). The patient with well differentiated adenocarcinoma underwent en bloc resection and was recognized to have peritoneal involvement of the tumor at the operation. The remaining three patients were diagnosed to have tumors confined to their bladder and urachal remnant and were treated with segmental resection of the bladder. Two patients each, with well and moderately differentiated carcinoma confined to their bladder and urachal remnant, were treated with en bloc resection and have been surviving from 8 years and 5 months to 24 years and 10 months (mean duration: 13 years and 7 months) postoperatively as of Dec. 1987. Therefore, patients with well and moderately differentiated adenocarcinomas confined to the bladder and the urachal remnant could be expected to survive longer by en bloc resection.

[Intrapelvic cyst presenting with urinary retention: a case report].

A case of intrapelvic cyst is presented. A 74-year-old woman was admitted with the chief complaint of urinary retention. Intrapelvic cyst was found by computed tomography scan during evaluation for urinary retention. Surgical extirpation was performed and pathological examination revealed paraovarian cyst.

[Intravesical bacillus Calmette-Guerin instillation therapy of recurrent superficial bladder carcinomas resistant to intravesical anti-cancer chemotherapy].

Tokyo strain bacillus Calmette-Guerin (BCG) was instilled in a dose of 80 mg in 40 ml normal saline to the bladder of 8 patients with recurrent superficial bladder carcinoma (Ta, Tl, Tis) resistant to mitomycin C and/or adriamycin intravesical instillation chemotherapy. Instillation was performed weekly for 6 weeks and 3 to 4 weeks after the last instillation, the response was assessed by cystoscopy and urine cytology. Patients who achieved complete response underwent monthly maintenance instillation for a year and inspection with cystoscopy and urine cytology every 3 months. Of the 7 patients who underwent therapeutic instillation, 3 (43%) achieved complete response, and 2 partial response. Two patients with no response had carcinoma in situ of grade 3 anaplasia. Two of the 3 complete responders were free of disease for 11 and 12 months, but another 1 developed intravesical recurrence 13 months later. One patient underwent prophylactic instillation and remained free of disease for 23 months. Side effects included frequency, urgency and pain on urination which were tolerable with anti-analgesics. Two patients underwent total cystectomy because of tumor progression and had typical lesions of prostatic tuberculosis.

[Clinical effectiveness of short-term administration of norfloxacin in acute urethritis].

Thirty patients with acute urethritis were treated with short-term administration of norfloxacin. The drug was administered randomly in two regimens: Two 400 mg oral doses for a single day or three 200 mg oral doses for three days. For gonorrheal urethritis, the efficacy rates of one and three-day administrations were 75 and 90%, respectively. For non-gonorrheal urethritis, they were 75% each. No adverse effects were observed. The results indicate that short-term administration of norfloxacin is useful in the treatment of acute urethritis, especially for gonorrheal urethritis.