Abstract. Cellular uptake of [3H]thymidine ([3H]TdR) and incorporation into DNA of Ehrlich ascites tumour cells were studied in relation to the cell cycle by measuring the activity in the acid‐soluble and insoluble parts of the cell material. Cells were synchronized at various stages of the cell cycle using centrifugal elutriation. The degree of synchrony of the various cell fractions was measured by flow‐cytofluorometric DNA analysis. From the cellular uptake, the TdR triphosphate (dTTP) concentration of a mean cell in an unseparated cell population was calculated to be 20 × 10‐18 mol/cell. The pool activity of G1 cells was unmeasurable but rose to maximum values at the border of the G1‐S phase. It decreased again during G2. The [3H]TdR incorporation into DNA was low during early S phase, reached a maximum value at two‐thirds of the S phase and decreased again during late S phase. These changes in DNA synthesis were not due to changes in the dTTP pool being a limiting factor. During maximum DNA synthesis, 10%× min‐1 of the dTTP pool was utilized, at which time the pool size also decreased by about 30%. Changes in pool size during the cell cycle have to be taken into account when the results of incorporation of radioactive TdR into DNA are discussed.

Twenty-three patients undergoing pelvic exenteration for primary and recurrent gynecological malignancies from 1976 to 1994 are reported. Fifteen patients underwent total pelvic exenteration, 3 underwent anterior exenteration, and 5 underwent a posterior procedure. Eight patients had exenteration as their primary treatment (primary group), and 15 underwent exenteration as secondary treatment (recurrent group). In the primary group, two patients developed recurrence and died of it at 6 and 20 months after operation. Five patients are still being followed up and are alive without disease. Four of these 5 patients have survived more than 5 years. In the recurrent group, 12 patients were followed up and three died of complications during the early years. Seven patients died of cancer with the mean survival time of 16.6 months. The mean age, average operating time, and mean blood loss in the primary and recurrent groups were 57 vs. 53 years, 8 hours and 20 min vs. 8 hours and 10 min, and 4,120 vs. 4,190 ml, respectively. The overall cumulative 5-year survival rate was 34.7%, being 68.6% in the primary group and 16.7% in the recurrent group. It is noteworthy that the 5-year survival rate was 51.3% in the patients who had surgical margins free of disease. In conclusion, pelvic exenteration should be considered an acceptable therapeutic option when appropriately selected.

Abstract. Comparison of the results of immunohistochemical expression, such as proliferating cell nuclear antigen (PCNA) in archival material of tumours, with the clinical course is extremely valuable in determining the biological malignant potential of newly detected tumours. To obtain stable and reproducible results of immunohistochemical expression of the PCNA of tumours, we studied the optimal conditions of fNation, processing and staining of samples using animal‐implanted MBT‐2 cells derived from chemical‐induced mouse bladder carcinoma and PC10, a monoclonal antibody for PCNA. The intensity of staining and PCNA positive rates were stable and reproducible when resected specimens from the tumours were covered with gauze wetted with physiological saline at room temperature before fixation for less than 12 h, fixation in formaldehyde was less than 48 h, and paraffin‐embedded sections were dried for less than 1 h. The most clear staining of PCNA positive nuclei was observed when 10% neutral buffered formaldehyde was used as a fixative. The PCNA positive rates obtained under these conditions was compared with the bromodeoxyuridine (BrdUrd) labelling indices. Although the average PCNA positive rate was significantly higher than the BrdUrd labelling index (P≪0.01), a significant correlation between PCNA positive rates and BrdUrd labelling indices was observed. In order to study the cell cycle related expression of PCNA, Ehrlich ascites tumour cells were separated by centrifugal elutriation. PCNA positive nuclei were observed in all phases of the cell cycle including G,. Occurrence of PCNA positive G1 cells was expected at a half‐life of the PCNA‐protein of 20 h and a tumour cell doubling time of about 24h. Thus, the percentage of PCNA positive nuclei in a conventionally paraffin‐embedded specimen of a tumour reflects both the growth fraction and the doubling time of the tumour and it may be a useful parameter of the biological malignant potential of tumours.

Inhibition of the salvage DNA synthesis was studied in ascites tumour cells up to 240 min at moderately increased temperatures of 39 degrees C, 40.5 degrees C and 43 degrees C alone or in combination with adriamycin (ADR). Hyperthermia and ADR acted additively on salvage DNA synthesis. The [3H]thymidine (TdR) incorporation was strongly related to the cellular uptake of [3H]TdR. In order to evaluate the significance of the decrease in [3H]TdR uptake for the incorporation into DNA, the thymidine kinase (TK) has also been studied. TK activity started to decrease at a temperature of 39 degrees C, probably due to inhibition of translation and/or transcription of the enzyme, and was almost completely inhibited at 43 degrees C. ADR did not affect TK activity. Inhibition of DNA synthesis by aphidicolin neither decreased the ability of the cell to take up [3H]TdR nor the ability to affect TK activity. We concluded that moderate increases in temperature alone, or in combination with ADR, inhibit salvage DNA synthesis by inhibition of TdR uptake, possibly due to inhibition of phosphorylation; ADR, on the contrary, inhibits TdR uptake by other mechanisms such as changes in membrane function.

The purpose of this study was to investigate the possibility that healing processes following repeated injury by nonphysiological dialysate play a significant role in the pathogenesis of peritoneal ultrastructural alteration, mediated by the production of growth factors and extracellular matrix proteins (ECM). To test a possible mechanism for peritoneal membrane alteration, we investigated whether chemically injured peritoneal mesothelial cells and fibroblasts upregulate their production of growth factors and ECM as a consequence of the healing process. Using 1 N NaOH, circular wounds of uniform surface area were made in monolayers of subconfluent rat peritoneal mesothelial cells (RPMC) and peritoneal fibroblasts (RPFB). At 0, 24, and 72 h after wounding, changes in mRNA expression of transforming growth factor-beta 1 (TGF-Beta1), b-FGF, HGF, VEGF, and FN were semiquantified by reverse transcription-polymerase chain reaction. Nonwounded monolayers of RPMC and RPFB were used as controls with mRNA expression being determined at the same times. For RPMC, TGF-Beta1, HGF, b-FGF, and FN mRNA gradually increased up to 72 h postwounding to 1.5-fold, 1.6-fold, 1.3-fold, and 2.1-fold of the control levels, respectively. A significant increase was only observed for TGF-Beta1, while VEGF showed the least change with time. For RPFB, HGF, b-FGF, VEGF, and FN mRNA expression were slightly suppressed compared to control levels up to 72 h postwounding. TGF-Beta1, however, increased markedly above control expression levels by the end of the wound healing process. The production of profibrotic growth factors by mesothelial cells in response to injury may represent a mechanism whereby fibroblast activation, resulting in fibroblast hyperplasia and excessive extracellular matrix accumulation, culminates in alteration of the peritoneal membrane ultrastructure.