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The ontogeny of sulphated glycoconjugate-producing cells in the rat fundic gland has been studied using high iron diamine (HID), Alcian Blue (AB) at pH 1.0, high iron diamine in combination with Alcian Blue at pH 2.5 (HID-AB), cationic colloidal gold (CCG) at pH 1.0 under light microscopy and CCG (1.0), HID-thiocarbohydrazide (TCH)-silver proteinate (SP)-physical development (PD) under electron microscopy. From day 19.5 of gestation, sulphated glycoconjugate-producing cells were discernible under both light and electron microscopy. The development of such cells can be classified into four stages: (1) a prenatal period from day 19.5 of gestation extending to 0.5 days after birth; (2) 1 day to 2 weeks after birth; (3) 2 to 4 weeks after birth; and (4) the final period from 4 to 8 weeks after birth. Glycoconjugate-producing cells reached maturity by 4 weeks after birth. Our results indicated that glycoconjugate-producing cells were cells along the wall of foveolar lumen, but not those covering the gastric mucosa surface. Our results also suggested that the trans to transmost Golgi apparatus lamellae were the sites of sulphation in the developing rat stomach.

Effects of antineoplastic prostaglandins (PG) on human ovarian cancer cell growth were examined by using HR cells derived from ascites of a patient with serous cystadenocarcinoma of the ovary. With regard to inhibition of cancer cell proliferation in vitro, the effects of delta 7-PGA1 was most marked, followed by that of delta 12-PGJ2, PGJ2 and PGD2. When antineoplastic prostaglandins were administered to nude mice bearing HR cells, tumor growth in groups treated with PGJ2 and delta 12-PGJ2 alone was significantly inhibited 63 days after tumor inoculation, compared to that in an untreated group. Consequently, a significant prolongation of median survival was obtained with delta 12-PGJ2, compared to that in untreated groups and in groups with cisplatin alone. In addition, when prostaglandins were administered together with cisplatin, adjuvant inhibitory effects on the tumor growth were obtained 35, 56 and 63 days after tumor inoculation. Subsequently a significant prolongation of median survival was observed when cisplatin was combined with PGD2 or delta 7-PGA1, compared to the results in groups treated with PGD2 alone, delta 7-PGA1 alone or cisplatin alone. Combination of PGJ2 or delta 12-PGJ2 and cisplatin resulted in a significant decrease of hematocrit and body weight 63 days after tumor inoculation, suggesting a deterioration of the median survival. These results suggest that combination of PGD2 or delta 7-PGA1 with cisplatin may be of clinical use for ovarian cancer resistant to cisplatin.

The mechanism of cis-diamminedichloroplatinum (II) (CDDP) resistance was examined by using a CDDP-resistant (KFr) cell line established by continuous exposure of KF cells (derived from serous cystadenocarcinoma of the ovary) to escalating doses of CDDP. When KFr cells were incubated with 66.7 microM CDDP, the uptake of CDDP was significantly inhibited and the cellular content in the KFr cells was about a half of that in KF cells after incubation for 4 h. When the KF or KFr cells were incubated for 4 h with 100 microM CDDP, the release pattern of CDDP from KFr cells was similar to that from KF cells. In addition, the DNA histogram of both KF and KFr cells revealed that KF cells seemed to contain two clones of cell population and the KFr cells may have been selected by exposure to CDDP. At 3 h after intraperitoneal administration of 0.5 mg of CDDP per mouse to nude mice with KF or KFr tumor, the CDDP content in the KFr tumor was significantly lower than that in the KF tumor. In contrast, at 6 or 9 h after CDDP administration the CDDP content in the KFr tumor was significantly higher than that in the KF tumor. Furthermore, the KFr cells had cross-resistance to various CDDP analogues including carboplatin. It was shown that cellular uptakes of two CDDP analogues into KFr cells were significantly lower than those into KF cells.

Comparison was made between lymphocyte subsets in peripheral blood from patients with benign ovarian tumor and those with advanced ovarian carcinoma. In addition, changes of lymphocyte subsets of patients with ovarian carcinoma before and after operation were also examined. The percentage and absolute number of CD3-/HLA-DR+ (B cells) in peripheral blood from patients with advanced ovarian carcinoma were significantly lower than values from patients with benign ovarian tumor, whereas both percentage and absolute number of CD3-/HLA-DR- (null cells) cells in patients with advanced ovarian carcinoma were significantly higher. Although there was no significant difference in natural killer (NK) cell subsets (CD57+ CD16- and CD57+ CD16+ cells) between patients with benign ovarian tumor and ovarian carcinoma, the percentage and absolute number of CD57-/CD16+ (highly differentiated NK cells) cells in patients with ovarian carcinoma were significantly higher than those in patients with benign ovarian tumor. Both the absolute number and percentage of CD3+/HLA-DR+ (activated T cells) cells in ovarian cancer patients with minimal residual tumors after operation were significantly increased, compared to the levels before operation, while the values in the patients with large residual tumors were significantly decreased. In addition, the percentage and absolute number of CD3-/HLA-DR- (null cells) cells in the patients with minimal residual tumors were significantly decreased after operation, while values in the patients with large residual tumors remained unchanged before and after operation. The patients with minimal residual tumors after operation were characterized by a significant increase in the percentage of CD57- CD16+ (highly differentiated NK cells) cells. On the other hand, in the patients with large residual tumors no change of the NK cell subsets was observed before and after operation.

The ontogeny of pepsinogen C-producing cells in rat fundic glands was studied by means of light and electron microscopy using an antiserum raised against a synthetic peptide based on rat pepsinogen C. To confirm the immunocytochemistry results, the expression of rat pepsinogen C messenger RNA (mRNA) in the fundic gland was also examined by in situ hybridization using a digoxigenin-labeled RNA probe. In adult rats, pepsinogen C was produced by chief cells, mucous neck cells, and intermediate mucopeptic cells. Pepsinogen C-producing cells appeared in embryos as early as 18.5 days’ gestation. The development of these cells could be classified into four stages: (1) 18.5 days’ gestation to 0.5 days after birth; (2) 0.5 days to 2 weeks after birth; (3) 3–4 weeks after birth; (4) 4–8 weeks after birth. In embryos and young animals, pepsinogen C-producing cells were mucopeptic cells. By 4 weeks after birth, mucous neck cells could be distinguished morphologically. The maturation stages of the chief cells could be traced by electron microscopy along the longitudinal axis of the rat fundic gland by double-staining with anti-pepsinogen C antibody and periodic acid-thiocarbohydrazide-silver proteinate. Positive reactions for pepsinogen C and pepsinogen C mRNA expression were detected in mucous neck cells. Therefore, we conclude that mucous neck cells are precursor cells of chief cells. Mucous neck cells, intermediate cells, and chief cells are in the same differentiating cell lineage.

In the present study, we attempted to determine effects of calmodulin antagonists (W-7 and W-5) on cisplatin uptake by human ovarian cancer cells, using KF cells derived from serous cystadenocarcinoma of the ovary and cisplatin-resistant cells (KFr). The degree of cisplatin resistance of the KFr cells was about 3.7-fold higher than that of the parent KF cells, with regard to the concentration of cisplatin required for 50% inhibition of cell proliferation (IC50). When KF and KFr cells were incubated with 10 micrograms/ml cisplatin for 4 hr, cisplatin-content in the KF cells was significantly higher than that in the KFr cells. When KF cells were incubated in the presence of W-7 (but not W-5), cisplatin uptake significantly increased, compared to cells treated with cisplatin alone. On the other hand, when KFr cells were incubated in the presence of 5 micrograms/ml W-7 or W-5, cisplatin uptake was significantly higher than uptake by KFr cells treated with cisplatin alone, being comparable to that by KF cells treated with cisplatin alone. Such an increase in cisplatin uptake seemed to bring about adjuvant effects to cisplatin of KFr cell proliferation in vitro. The KF tumor grown in nude mice took up 24.8 ng/g dry wt of cisplatin 4 hr after intraperitoneal administration. When cisplatin was administered with calmodulin antagonists, cisplatin uptake by the KF and KFr tumors was significantly increased, compared to that after treatment with cisplatin alone. In particular, the cisplatin uptake by the KFr tumor was about 2.5-fold higher than that by the KFr tumor treated with cisplatin alone. These results suggest that coadministration of calmodulin antagonists and cisplatin may be of use in patients with refractory ovarian cancer.

Adjuvant effects of prostaglandin D2 to cisplatin on tumor growth were studied by using nude mice bearing HR cells derived from human ovarian carcinoma. Combinations of 0.2 or 0.4 microgram/ml cisplatin and 0.05 or 0.1 microgram/ml prostaglandin D2, which did not affect the HR cell proliferation alone, resulted in a significant inhibition of cell proliferation. In addition, tumor take of HR cells by nude mice in groups treated with a combination of cisplatin and prostaglandin D2 was inhibited. Although there was no significant difference between tumor volumes in mice treated with prostaglandin D2 alone or cisplatin alone and untreated mice, when cisplatin was administered with 1 mg/kg prostaglandin D2 the tumor volume was significantly smaller on days 21 and 35, compared to that of untreated mice. When cisplatin and 2 or 4 mg/kg prostaglandin D2 were combined, the tumor growth was significantly inhibited after day 21, compared not only to that of untreated mice but also of mice treated with cisplatin alone or prostaglandin D2 alone. Such a combination therapy by cisplatin and prostaglandin D2 seemed to result in prevention by prostaglandin D2 of immunological suppression which may be induced by cisplatin. Thus, such a combination therapy brought about a significant prolongation to the survival time.

The present study was designed to elucidate the effect of cimetidine on CD4 and CD8 positive cells, interleukin-2 (IL-2) production and IL-2 receptor expression in peripheral blood lymphocytes (PBL) from patients with advanced ovarian carcinoma during the course of combination chemotherapy. The absolute number of CD8 (but not CD4) positive cells in PBL from patients with advanced ovarian carcinoma before surgery was significantly higher than that with benign ovarian tumor, while the IL-2 productivity was significantly lower. However, the IL-2 receptor expression was comparable to that with benign ovarian tumor. When a combination chemotherapy consisting of cisplatin, adriamycin and cyclophosphamide was given to these ovarian cancer patients, the IL-2 production was markedly depressed. If cimetidine was given with the combination chemotherapy, the inhibition of IL-2 production by chemotherapy was significantly diminished with a significant increase of CD4 positive cells. On the other hand, the IL-2 receptor expression was not affected by this treatment. When treatment with cimetidine was initiated after completion of chemotherapy, the depressed IL-2 production was restored to the level of control patients with benign ovarian tumor. The restoration seemed to result from an increase in proportion of CD4 positive cells. However, the expression of IL-2 receptor remained unchanged even if cimetidine was given.

The effect of intraperitoneal instillations of interleukin-2 (IL-2) and/or lymphokine-activated killer (LAK) cells on the ascites formation and the survival time was examined using nude mice as a model, with malignant ascites produced by intraperitoneal inoculation of human ovarian cancer cells derived from ascites of a patient with serous cystadenocarcinoma of the ovary. Twenty-eight days after tumor inoculation, all nude mice in the untreated group and in the group treated with spleen cells alone formed ascites. Two of ten nude mice treated with IL-2 alone after tumor inoculation survived without forming ascites during the experimental period. On the other hand, all nude mice treated with LAK cells alone had formed ascites 14 days after tumor inoculation. When LAK cells and IL-2 were combined, five of ten mice survived without forming ascites during the experimental period. The survival time of the group treated with IL-2 alone was significantly prolonged compared to the groups that received medium alone, spleen cells alone and LAK cells alone. When administration of LAK cells was followed by IL-2, the survival time was further prolonged.

In order to establish that the tumor burden in nude mice bearing a xenografted human ovarian tumor can be assessed by measuring plasma levels of alpha-hydroxybutyrate dehydrogenase (HBD) (E.C. 1.1.1.30), the plasma HBD and the tumor volume were measured every week after inoculation of a human ovarian carcinoma cell line designated HR. About 3 weeks after tumor inoculation, all nude mice had a palpable tumor while elevated levels of HBD were observed in a half of the mice at that time. Thereafter the HBD levels rose with increasing tumor volume. There was a quantitative linear relationship between the tumor weight and HBD activity per mouse. When 40 micrograms of cisplatin (about 2 mg/kg) was administered i.p. every week for 5 weeks 2 weeks after inoculation, plasma levels of HBD were significantly decreased on day 27 compared to those in untreated nude mice. Decreases in the HBD levels during treatment with cisplatin occurred 7 days prior to changes in tumor volumes. Further, removal of local tumor mass resulted in a marked decrease in HBD levels on day 1 after surgery and the HBD levels in nude mice which had no recurrent tumor fell into the normal range 8 days after surgery.

Effects of N-(6-aminohexyl)-1-naphthalenesulfonamide (W-5) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) on antitumor activity of 1-(2-tetrahydrofuryl)-5-fluorouracil (ftorafur, FT) and a mixture of FT and uracil (UFT) were determined in nude mice bearing human ovarian carcinoma (KF cells). All nude mice developed palpable tumor 17 days after KF cell inoculation unless treated. A combination of UFT and calmodulin antagonists (W-5 and W-7) resulted in a delay in tumor formation in nude mice. One of 10 nude mice treated with a combination of UFT and W-5 and 3 of 10 nude mice treated with UFT and W-7 did not develop tumors during the experimental period. Tumor volumes in groups treated with the combinations of UFT and calmodulin antagonists were significantly smaller than those in the untreated group after 31 days of tumor inoculation. In addition, tumor volumes in a group treated with UFT plus W-7 were significantly smaller than not only those in the untreated group but also those in groups treated with UFT alone, FT alone, W-5 alone, or W-7 alone. The longest median survival time was also observed in the group treated with UFT plus W-7. 5-Fluorouracil (5-FU) content of the tumors of groups treated with FT plus W-5 or W-7 was similar to that of the group treated with FT alone. On the other hand, 5-FU content of a group treated with UFT plus W-7 was about twofold higher than that of a group treated with UFT alone. These results suggest that a synergistic effect of W-7 and UFT on inhibition of tumor growth and prolongation of survival time may result from accumulation of 5-FU in the tumor.

The present study was designed to elucidate the mechanism of resistance to cisplatin. A cisplatin-resistant cell line (KFr) was established from KF cells derived from human serous cystadenocarcinoma of the ovary. The DNA histogram revealed an increase of S-phase cells and a decrease of G1-phase cells in cultured KFr cells, compared to that in cultured KF cells. Although the cisplatin content in the KF cells incubated with cisplatin at 10 micrograms/ml increased in a time-dependent manner, that in the KFr cells remained unchanged during the experimental period. When 0.5 mg of cisplatin was administered ip to nude mice with KF or KFr tumor, the cisplatin content in the KFr tumor was significantly lower than that in the KF tumor. The KFr cells showed a cross-resistance to L-phenylalanine mustard, while no cross-resistance to vincristine or 5-fluorouracil was observed. These findings suggest that the mechanism of cisplatin resistance in the KFr cells involves a decrease of cisplatin accumulation in the tumor cells.

We studied production of, absorption of and response to interleukin 2 (IL-2) by peripheral blood mononuclear cells (PBMC) from 66 patients with gynecologic malignancies, in addition to measurement of the OKT 4/OKT 8 cell ratio. Patients with benign tumor served as controls. The OKT 4/OKT 8 cell ratio in patients with advanced (but not early) gynecologic malignancies was significantly lower than that in patients with benign tumor. PBMC from advanced cancer patients activated with phytohemagglutinin (PHA) had significantly lower IL-2 production compared to that from patients with benign tumors, while significant changes in their ability to respond to IL-2 and to absorb IL-2 were not observed. Absolute numbers of OKT 8 positive cells in PBMC of patients with good prognosis were significantly decreased after surgery and chemotherapy, while those of OKT 4 positive cells remained unchanged. Although IL-2 production in PBMC of patients with good prognosis was significantly elevated after chemotherapy, that in PBMC of patients with poor prognosis declined to about a half of pre-operative levels. The ability of PBMC to respond to IL-2 was significantly elevated not only in patients with good prognosis but also in patients with poor prognosis after termination of chemotherapy. On the other hand, the ability of PBMC to absorb IL-2 remained unchanged during the course of treatment. These findings may contribute to the understanding of tumor-induced immune suppression.

Direct inhibitory effects of calmodulin inhibitors on human ovarian cancer cell proliferation in vitro were examined. All calmodulin inhibitors used in this study inhibited proliferation of HR cells derived from human serous cystadenocarcinoma of the ovary in a dose-dependent manner. The degree of inhibition by melittin and W-5 was most marked and was followed by that of W-7, R24571, and bepridil. HR cell proliferation was dosedependently stimulated by epidermal growth factor (EGF). The stimulatory effects seemed to be a result of the binding of EGF to HR cells. Binding of EGF was significantly inhibited by W-5, R24571, and melittin. These results suggest that inhibition by calmodulin inhibitors of cancer cell proliferation may result in part from the interference in the binding of EGF to the cells.

A cisplatin-resistant cell line was established by use of KF-1 cells derived from human serous cystadenocarcinoma of the ovary. This resistant cell line, designated "KFr," was capable of proliferating in the presence of 1.0 micrograms cisplatin/ml. It had doubling times of 24.8 and 27.2 hours in the presence of 0.5 and 1.0 micrograms cisplatin/ml, respectively. The KFr cells had lactate dehydrogenase (LDH) activities about fourfold higher than those of the parent KF-1 cells. Analysis of the isozymes of the KFr cells revealed that they had a faint LDH-3 band in addition to LDH-4, the only stained band in the parent KF-1 cells. The morphologic characteristics of the KFr cells were an enlarged nucleus and prominent nucleoli, unlike the nucleus and nucleoli of the parent KF-1 cells. The degree of resistance to cisplatin of the KFr cells was about twenty-fold higher than that of the KF-1 cells, with regard to the concentrations of cisplatin required for 50% inhibition of cell proliferation. However, when 5 microM calmodulin antagonists [N-(6-aminohexyl)-1-naphthalenesulfonamide or N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide] were added in the presence of concentrations of cisplatin that hardly inhibited cell proliferation, the KFr cell proliferation was markedly inhibited. These results suggest that cisplatin resistance can be overcome by calmodulin antagonists.

Direct inhibitory effects of neuroendocrine hormones on human ovarian cancer cell proliferation in vitro were examined. Except for melatonin, all neuroendocrine hormones used in this study inhibited proliferation of KF cells derived from human serous cystadenocarcinoma of the ovary in a dose-dependent manner. The degree of inhibition of prostaglandin D 2 was most marked being comparable to that of 5-fluorouracil and followed by β-endorphin, α-endorphin, and Met-enkephalin. More than 200 μ M melatonin stimulated the cell proliferation. The inhibitory effects by endorphins were partially reversible by 20 μ M naloxone. From analyses of uptakes of radiolabeled precursors by the KF cell, endorphins were considered to inhibit protein and RNA syntheses but not DNA synthesis. These results suggest that neuroendocrine hormones may play an important role in regulation of tumor growth.