Tadao Uchibayashi

DOMINANT ROLE OF E-CADHERIN IN THE PROGRESSION OF BLADDER CANCER

PURPOSE To elucidate factors with a role in the progression of bladder cancer. MATERIALS AND METHODS One invasive (T24) and two superficial (RT4 and KK47) human bladder cancer cell lines, which express metastasis-related genes, were used. Cells were intravenously inoculated into chick embryos to evaluate metastatic potential to the liver. An orthotopic model with severe combined immunodeficiency mice was also used to investigate both histological appearance and changes in metastasis-related gene expression. Finally, gene expression patterns in a clinical setting were compared between superficial and invasive bladder cancers. RESULTS In culture condition metastasis-related genes, including matrix metalloproteinases, urokinase-type plasminogen activator, and integrins alpha2 and alpha3 were continually expressed in T24 but only slightly or not at all in RT4 and KK47, respectively. The expression pattern of the metastasis-related genes in vitro reflected the characteristics of the original tumors. Liver metastasis in chick embryos was demonstrated not only with T24 cells, but also with RT4 cells in which enhanced expression of metastasis-related genes was induced. In the orthotopic model, histological appearances were in accordance with the characteristics of the original tumors, although enhanced gene expression was notable with RT4. Expression of E-cadherin by Western blotting was demonstrated only with RT4 under these experimental conditions. Furthermore, predominant E-cadherin mRNA expression was found in superficial and not in invasive human primary bladder cancers; expression of other genes was similar in the two groups. Dominant expression of E-cadherin in superficial tumors was confirmed by immunohistochemical analysis. CONCLUSIONS These results implicate loss of E-cadherin expression as a critical factor in facilitating the progression of bladder cancer.

EXPRESSION OF MESSENGER RNAS FOR MEMBRANE-TYPE 1, 2, AND 3 MATRIX METALLOPROTEINASES IN HUMAN RENAL CELL CARCINOMAS

PURPOSE Three different membrane-type matrix metalloproteinases (MT1, 2, and 3-MMP) that can activate proMMP-2 (progelatinase A) are thought to play an important role in invasion and metastasis by various human carcinomas. To further clarify this role, we examined mRNA expression of MT-MMPs in human renal cell carcinomas. MATERIALS AND METHODS mRNA was extracted from 25 clinical specimens of renal cell carcinoma and 23 specimens of normal renal parenchyma remote from the tumor. Reverse transcription polymerase chain reaction (RT-PCR) using specific primers was performed, and PCR products were hybridized to 32P-labeled internal probes and analyzed by a bioimage analyzer. RESULTS MT1, 2, and 3-MMP mRNA expression in carcinomas was significantly higher than in normal parenchyma. In terms of the pathologic stage, MT1-MMP mRNA expression in pT2 and pT3 tumors was significantly higher than those in pT1 tumors. Although the sample size was small, it was evident that MT3-MMP mRNA expression in clear cell subtype renal cell carcinomas was higher than in the group of tumors including the granular cell subtype. CONCLUSIONS These three MT-MMPs may play an important role in the pathogenesis of human renal cell carcinoma, and MT1-MMP in particular is important in invasion by carcinoma cells. It is interesting that the expression of MT3-MMP was higher in carcinomas, especially clear cell carcinoma, than in normal parenchyma, so that MT3-MMP may provide a clue an understanding of the molecular mechanism of carcinogenesis in human kidney.

Whole bladder wall photodynamic therapy for refractory carcinoma in situ of the bladder.

Whole bladder wall photodynamic therapy (PDT) with haematoporphyrin derivative and an argon dye laser as a light source was performed on 34 patients with refractory carcinoma in situ (CIS) of the bladder. Twenty-five of the 34 patients (73.5%) had achieved a complete response (CR) at 3 months after the treatment. The median follow-up for these CR patients is 49.3 months. Although recurrence within 2 years of follow-up occurred in 14 (77.8%) of the 18 CR patients followed to that point, since most of the recurrent tumours were superficial and low-grade papillary tumours, transurethral resection of the bladder tumours appeared to be sufficient. Of the total of 34 patients, ten were alive with bladder intact with a mean follow-up period of 64.0 months. Skin photosensitivity and transient decrease in bladder capacity were noted as adverse reactions, caused by retention of haematoporphyrin derivative in the skin and normal portion of the bladder. These data suggest that PDT can be an effective form of therapy for CIS of the bladder.

Integral Laser Photodynamic Treatment of Refractory Multifocal Bladder Tumors

Integral photodynamic therapy with hematoporphyrin derivative was performed on 35 patients who had resistant transitional cell carcinoma of the bladder, mainly carcinoma in situ. The light source was an argon ion pumped dye laser (wavelength 630 nm.) using rhodamine B. Two types of laser light scattering diffuser developed at our department were used: a motor driven laser light scattering diffuser with computer regulation, and an endoscope modified light scattering diffuser tipped with a small quartz bulb containing a lipid nutritious solution as the scattering medium. The total energy density used was 10 to 30 J./cm.2. Of the 35 patients 24 (68.6%) achieved a complete response and 5 (14.3%) a partial response at 3 months. In 10 of the 24 patients there was no recurrence with an average tumor-free interval of 20.9 +/- 16.7 months, ranging from 5 to 60 months. Bladder capacity was decreased to approximately 150 ml. for 3 months after the integral photodynamic therapy without any evidence of hydronephrosis on excretory urograms, except for 2 patients who had a contracted bladder before photodynamic therapy. Integral photodynamic therapy may prove to be useful for the treatment of carcinoma in situ of the bladder.

Endoscopic Treatment of Vesicoureteral Reflux in Patients With Myelodysplasia

PURPOSE We assessed the usefulness of and indications for endoscopic treatment of vesicoureteral reflux in myelodysplasia patients. MATERIALS AND METHODS A total of 26 patients treated with intermittent catheterization was divided into 11 (16 ureters) with and 15 without vesicoureteral reflux. In 9 patients (13 ureters) endoscopic correction was performed with 3% atelo-collagen and without anesthesia at the outpatient clinic. In each ureter we obtained the sum of scores for 4 risk factors for upper urinary tract deterioration: bladder compliance less than 10 ml./cm. water, grade 2 to 3 bladder deformity, detrusor-sphincter dyssynergia and urethral closure pressure 50 cm. water or greater. RESULTS No reflux was demonstrated immediately after the initial collagen injection but cystography 3 to 6 months later showed recurrent reflux in 5 ureters (38%). Repeat injection cured the reflux, with results persisting for an average of 17 months. Mean risk factor score for patients without vesicoureteral reflux was significantly lower than that for patients with reflux. In patients treated with intermittent catheterization and anticholinergic agents the mean score for ureters with an increased or unchanged reflux grade was significantly greater than for those with a decreased grade. CONCLUSIONS Endoscopic treatment of reflux appears to be safe and useful in patients with myelodysplasia. The treatment is preferable in those with high risk factor scores due to the possibility of increased reflux grade in such patients.

The Role of Alkaline Phosphatase Isoenzymes as Tumor Markers for Testicular Germ Cell Tumors

The role of serum alkaline phosphatase as a tumor marker for testicular germ cell disease was investigated in 26 patients with testicular seminoma and 13 with nonseminomatous germ cell testis tumors. Placental alkaline phosphatase-like enzyme was elevated in 50% of the stage I seminoma patients and in all patients with stages II to III disease. In addition, liver (tissue unspecific) alkaline phosphatase was elevated in 10 and 83% of the patients, respectively. Lactic dehydrogenase and beta-human chorionic gonadotropin (beta-HCG) were detected in 50 to 60% of the patients with stage I seminoma. By combining placental alkaline phosphatase-like enzyme, lactic dehydrogenase and beta-HCG, 75% of the stage I and 100% of the stages II and III seminoma patients could be identified correctly. Placental alkaline phosphatase-like enzyme in serum also occurred with nonseminomatous germ cell tumor but less frequently, while liver alkaline phosphatase was not detected at all. Thus, placental alkaline phosphatase-like enzyme and liver alkaline phosphatase were predominantly determined in the serum of patients with seminoma. In studies of tumor tissues from 31 of these patients, those with normal serum placental alkaline phosphatase-like enzyme levels had significantly lower tissue placental alkaline phosphatase-like enzyme levels than patients with elevated serum levels (p less than 0.01). Seminoma tissues showed significantly higher levels of placental alkaline phosphatase-like enzyme and liver alkaline phosphatase than nonseminomatous germ cell tumors (p less than 0.01), explaining the infrequent elevation of serum placental alkaline phosphatase-like enzyme and liver alkaline phosphatase found in patients with nonseminomatous germ cell tumors.

A Combined Therapy of Hyperthermia and Tumor Necrosis Factor for Nude Mice Bearing KK-47 Bladder Cancer

A combined therapy of hyperthermia (43.5 C) and tumor necrosis factor (10(3) and 10(4) units) for the treatment of experimental bladder carcinoma KK-47 in athymic mice was studied. Briefly, mice were injected subcutaneously with 10(7) disaggregated cells. When the tumors were 250 mm3 in size, tumor necrosis factor was administered, either intravenously or by intratumor injection. Intravenous injection was 10(3) to 10(4) units tumor necrosis factor in the tail vein and intratumor was 10(3) to 10(4) units injected directly into the center of the tumor. Immediately following injection, the tumor bearing leg was placed in a 43.5 C water bath for 20 minutes. Tumor size was monitored once a week for seven weeks and the animals were divided into control, hyperthermia alone, tumor necrosis factor alone and combined therapy. Results of the study showed no significant difference in 10(3) units of tumor necrosis factor intravenously versus control but a significant regression in hyperthermia alone. Anti-tumor effects significantly increased in hyperthermia plus 10(3) units tumor necrosis factor versus hyperthermia alone. Similar results were seen with 10(4) units tumor necrosis factor intravenously though in the combination group of hyperthermia and tumor necrosis factor, eight mice of eight died one to three days following treatment. In those receiving intratumor injections, there was no difference between tumor necrosis factor or control. Tumor necrosis factor with hyperthermia had the approximate same characteristics as hyperthermia alone and therefore there was no synergistic finding. These results reflect on the suggestion that the combination therapy of hyperthermia and systemic administration of the proper dosage of tumor necrosis factor may produce synergistic anti-cancer effects in bladder cancer patients.

The prophylactic use of thio-tepa and urokinase in transitional cell carcinoma of the bladder: a preliminary report.

The prevention of recurrences of bladder cancer was attemped in 48 patients by means of the combined intravesical instillation of thio-tepa and urokinase and in 28 patients through the instillation of thio-tepa alone. The recurrence rates of both therapies for the postoperative 18 months were 7.9 and 32.6 per cent, respectively, indicating a significant drop in the recurrence rate in the group subjected to the combined therapy. No significant difference was found between the 2 instillation groups in terms of the blood transmission of 32-P thio-tepa. Serious leukopenia was found in 2 of the 48 patients receiving the combined instillation therapy but we concluded that this was not attributable to the use of urokinase.

Studies of effects of anticancer agents in combination with/without hyperthermia on metastasized human bladder cancer cells in chick embryos using the polymerase chain reaction technique

Cultivated T24 cells derived from a human bladder cancer were inoculated into the chorioallantoic membrane vein of chick embryos. Hyperthermic treatment was performed following injection of anticancer agents 3 days after the inoculation of the T24 cells. DNA samples were obtained from the livers of the chick embryos, and the polymerase chain reaction technique was used to amplify a DNA fragment specific to the human β-globin gene. The Southern hybridization method was used to evaluate the inhibitory effects of anticancer agents in combination with/without hyperthermia on T24 cells metastasized to the liver. The hyperthermia exerted an inhibitory effect on the growth of the T24 cells in the livers of the chick embryos, and this was dependent on the thermal dose. The antitumor effects of hyperthermia performed at 42.5° C for 20 min and at 43.0° C for 10 min were evidenced by 69.2% an 82.0% inhibition of the growth of the metastasized T24 cells, respectively, as compared with the growth of untreated T24 cell. Hyperthermia performed at 42.5° C for 10 min alone produced 26.7% tumor growth inhibition, and these conditions for hyperthermia were subsequently used as a criterion for evaluating the effects of its combination with various anticancer agents. Adriamycin (20 μg/egg) alone, mitomycin C (10 μg/egg) alone, carboplatin (10 μg/egg) alone, and cisplatin (10 μg/egg) alone produced 13.5%, 58.9%, 27.3%, and 29.1% tumor growth inhibition, respectively. Adriamycin and mitomycin C applied in combination with hyperthermia showed additive inhibitory effects on the growth of the metastasized T24 cells in this chick embryo model.

A HUMAN SEMINOMA XENOGRAFT MODEL WITH REGIONAL LYMPH NODE METASTASIS

PURPOSE To establish a seminoma orthotopic model with lymph node metastasis to investigate the factors related to the lymphophilic behavior of seminoma cells. MATERIALS AND METHODS Testicular seminoma xenografts were established by the inoculation of small fragments from subcutaneous (s.c.) xenografts that had previously been established in severe combined immunodeficient (SCID) mice with a supraclavicular lymph node metastasis from a human seminoma. Hematologic dissemination of tumor cells was analyzed by polymerase chain reaction (PCR) amplification of the human beta-globin gene. Xenograft messenger RNA levels of metastasis-related genes were examined by reverse transcription (RT)-PCR. RESULTS Testicular seminoma xenografts grew in 32/32 (100%) of the inoculated mice, of which 15 mice (47%) developed macroscopic metastasis to the renal hilar lymph node. Circulating tumor cells and tumor cell shedding in the lung and liver were detectable by PCR assay in 25/32 (78%), 32/32 (100%), and 27/32 (84%) mice, respectively, although metastatic foci were not histologically evident in these organs. Increased expression of matrix metalloproteinase-2 (MMP-2), membrane-type 3 matrix metalloproteinase (MT3-MMP) and vascular endothelial growth factor (VEGF), and reduction in expression of plasminogen activator inhibitor-2 (PAI-2) were demonstrated by RT-PCR assay in the testicular xenografts as compared with the s.c. xenografts. CONCLUSIONS This model mimics the lymphophilic behavior of seminoma and may help in elucidating the molecular mechanism of tumor spread via the lymphatics.