Expression of proliferating cell nuclear antigen and deoxyribonucleic acid value in renal cell carcinoma: correlation with different histopathological parameters and patient survival.

Forty-six human renal cell carcinoma tissues obtained from radical nephrectomy, fixed in 10% formaldehyde solution and embedded in paraffin for histopathological examination were used for immunohistochemical staining of proliferating cell nuclear antigen (PCNA) using a monoclonal antibody PC10, and 37 of the 46 were also used for flow cytometric DNA ploidy analysis. PCNA-positive rates were compared with different histopathological parameters and patient survival. The relationships between the DNA ploidy and PCNA-positive rates and patient survival were also determined. Statistically significant differences in PCNA-positive rates were observed in different histopathological grades and stages of renal cell carcinoma. No relationship was observed between the PCNA-positive rate and DNA ploidy. For all cases analyzed, patients whose tumors had PCNA-positive rates of less than 10% survived statistically longer than those with tumors with PCNA-positive rates of 10% or more (p < 0.02). When patients were stratified by histopathological stage-I and grade-1 tumors, however, there was no significant difference between the PCNA-positive rate and survival. No difference in survival was observed according to DNA ploidy. The PCNA-positive rates showed a close relation to the different histopathological grades and stages of renal cell carcinoma. For all cases analyzed, high PCNA-positive rates showed poor prognosis. But for patients with histopathological stage-I and grade-1 tumors, the PCNA-positive rates and DNA ploidy did not provide independent prognostic information.

Cytokine-mediated antitumor effect of bacillus Calmette-Guérin on tumor cells in vitro.

Intravesical instillation therapy of bacillus Calmette-Guérin (BCG) is a useful modality for recurrent superficial transitional-cell carcinoma (TCC) of the urinary bladder. The mechanism of BCG effect has not yet been well characterized. BCG was tested in vitro for cytokine-mediated antiproliferative activity against T24 and KK47 cells (cell lines established from human TCC of the urinary bladder), and ACHN cells (cell line established from human renal cell carcinoma) using a modified human tumor clonogenic assay. Continuous exposure of cells to BCG at concentrations of more than 5 micrograms/ml in the presence of peripheral blood mononuclear cells (PBMC) consisting of a mixture of 5 x 10(4) monocytes/dish and 5 x 10(5) lymphocytes/dish, obtained from healthy donors, significantly inhibited colony formation of T24 and ACHN cells in comparison with growth inhibition in the absence of PBMC (P < 0.05). Slightly inhibited colony formation was observed with KK47 cells under the same conditions. At the same time various cytokines were measured in supernatants when BCG and the same conditioned PBMC were co-cultured. Tumor necrosis factor alpha (TNF alpha) and interleukin-1 beta (IL-1 beta) were detected at markedly high levels at 24 h, and interferon gamma (IFN gamma) was detected at 120 h. IL-2 and macrophage-colony-stimulating factor were not detected. Neutralizing anti-TNF alpha monoclonal antibody significantly reduced the anti-proliferative activity of ACHN cells, and anti-IFN gamma antibody reduced that of T24 cells. The results obtained suggest that cytokines mediated by BCG play an important role in the antitumor activity of BCG and that the sensitivity of bladder cancer cells to the cytokines induced by BCG may differ considerably.

Direct and indirect effects of recombinant human granulocyte-colony stimulating factor on in vitro colony formation of human bladder cancer cells.

Although the present experimental use of recombinant human granulocyte-colony-stimulating factor (rG-CSF) has been proven to alleviate the myelosuppression induced by antitumor chemotherapy, it is also believed to stimulate growth of some nonhematopoietic tumor cells. We investigated both the direct and indirect effects of rG-CSF on in vitro colony formation of human bladder cancer cell lines using a modified human tumor clonogenic assay. Peripheral blood mononuclear cells (PBMC) were used as feeder cells (a mixture of 5 x 10(4) monocytes/dish and 5 x 10(5) lymphocytes/dish obtained from healthy donors). Human bladder cancer cell lines KK-47, TCCSUP and T24, all derived from human transitional-cell carcinomas, were incubated continuously with various concentrations of rG-CSF ranging from 0.01 ng/ml to 10 ng/ml both with and without PBMC for 7-21 days. The concentrations of rG-CSF used were chosen as being in the range of achievable serum concentrations in patients treated with rG-CSF. At the end of incubation, colonies were counted under an inverted phase-contrast microscope, and an increase in the number of colonies in comparison with the control was used to evaluate the effects of rG-CSF. Results were expressed as a percentage of controls. rG-CSF in the upper layer at concentrations ranging from 0.1 ng/ml to 10 ng/ml stimulated the colony formation of all the cancer cell lines tested in the absence of PBMC in the feeder layer, whereas cells with PBMC in the feeder layer were significantly stimulated more than those without PBMC in the feeder layer (P < 0.05) up to a certain concentration, which varied from cell line to cell line. At higher concentrations of rG-CSF, no further stimulation but, on the contrary, a decrease in colony formation was observed in cells with PBMC in the feeder layer in all the cell lines tested. Colony formation in KK-47 and T24 cell lines was significantly inhibited at 5 ng/ml and/or 10 ng/ml rG-CSF compared with cells without PBMC in the feeder layer. Our results suggest that rG-CSF may have both direct and indirect stimulatory effects on the growth of human bladder cancer cell lines in vitro. The results obtained also raise the possibility of adverse effects of rG-CSF in bladder cancer patients whose malignant cells may be directly and indirectly stimulated by this factor while it is being used clinically to alleviate the myelosuppression induced by antitumor chemotherapy.

[Endopyelotomy for ureteropelvic junction obstruction: results and complications].

We treated eleven cases of primary ureteropelvic junction obstruction with percutaneous endopyeltomy. Endopyelotomy was successful in nine of the eleven cases. Complications during and after endopyelotomy occurred in three cases, two of which had pyelonephritis and the other severe postoperative hemorrhage requiring blood transfusion due to incision of a posterior crossing vessel. In this last case, the kidney was supplied by three arteries. The lower segmental artery passed behind the ureter. We emphasized that angiography should be performed to rule out the presence of a posterior crossing vessel before endopyelotomy, when it is doubtful if an extrinsic cause of ureteropelvic junction obstruction is present.

[Analysis of proliferating cell nuclear antigen and nucleolar organizer region in testicular tumors].

Immunohistochemical staining using the monoclonal antibody of proliferating cell nuclear antigen (PCNA) and argyrophilic nucleolar organizer region associated protein (AgNOR) staining were performed on 28 paraffin-embedded testicular tumors, and their correlation was assessed according to histological type, clinical staging and tumor markers. PCNA positive rates and AgNOR numbers in nuclei were 57.9%, 4.38 in seminomas, 63.4%, 5.79 in embryonal carcinomas, 37.7%, 2.63 in teratomas, 61.3%, 4.43 in yolk sac tumors, 79.5%, 7.12 in choriocarcinomas, and 20.9%, 3.03 in cells of the normal seminiferous tubule, respectively. In pure seminomas, the PCNA positive rates and AgNOR numbers of the stage II to III group (72.6%, 4.75) were higher than those of the stage I group (47.8%, 3.67), but there was no significant difference between the two groups. There was a significant correlation between PCNA positive rate and AgNOR number in all specimens (r = 0.76, p < 0.002). These findings indicate that PCNA and AgNOR are suitable indicators for proliferative activity of testicular tumors.

[Expression of proliferating cell nuclear antigen in superficial bladder cancer--application to paraffin-embedded tissue sections].

It is important to know the proliferating ability and the malignant potential of each tumor. We studied 56 cases of pTa to pT1 superficial bladder tumors using immunohistochemical staining for proliferating cell nuclear antigen (PCNA), and compared the results with the clinical course of each patient. We obtained the following results. 1) We detected the PCNA positive nuclei in all cases, and the PCNA positive rates varied within a range of 1.1-77.5% with a mean of 34.0%. 2) The PCNA positive rate showed no correlation with age, sex, duration of paraffin-embedded, or pathological stage, but showed a significant correlation with the number of tumors, pathological grade of malignancy, or non-recurrence rate. PCNA positive rate of Grade 1 cases (n = 19, 15.6%: mean) was significantly lower than those of Grade 2 cases (n = 27, 39.9%) or Grade 3 cases (n = 10, 53.1%) (P < 0.01). The recurrence rate of the cases with PCNA positive rates of more than 34% (n = 24) was significantly higher than that of the cases with a PCNA positive rate of less than 34% (n = 32) (P < 0.05). In conclusion, the method of counting the rate of PCNA positive nuclei is considered to be very useful because of its applicability to paraffin-embedded tissue sections and the simple and rapid techniques. Our results in bladder cancer tissues suggest that this method may also be useful for investigating the proliferating ability and the malignant potential of tumors in general.

Organic anion transport study in mutant rats with autosomal recessive conjugated hyperbilirubinemia.

The EHBR is a mutant rat strain with congenital conjugated hyperbilirubinemia bred from a Sprague-Dawley rat. Transport of conjugated bilirubin, indocyanine green, and tetrabromosulfophtalein from liver to bile is severely impaired in these rats. Serum bilirubin amounts to 6.0 +/- 0.05 mg/dl (n = 4) in adult rats, with 97% conjugates. The bile flow is reduced to about 65% of the control group, whereas total bile acid in 10-min bile samples is similar. Liver histology of 10 week-old rats revealed neither intracellular pigmentation nor architectural abnormalities.

Preparation of [35S]sulfobromophthalein of high specific activity.

Study of the hepatocyte transport mechanism of organic anions such as bilirubin and sulfobromophthalein has been limited by the relatively low specific activities of these ligands. [3H]Bilirubin and [35S]sulfobromophthalein have been available with specific activities of only approximately 100 mCi/mmol. We now report a relatively simple procedure to prepare [35S]sulfobromophthalein at a specific activity of approximately 3000 mCi/mmol. This compound is radiochemically pure and serves as a tracer for authentic sulfobromophthalein as judged by chromatography, hepatocyte uptake, metabolism, and biliary excretion. Use of this material as a photoaffinity probe and as a transported ligand may permit dissection and understanding of its transport mechanism.