Simple fluctuation of Ca2+ elicits the complex circadian dynamics of cyclic AMP and cyclic GMP in Paramecium.

The circadian dynamics of cyclic adenosine 3',5'-monophosphate (cAMP) and cyclic guanosine 3',5'-monophosphate (cGMP) were simulated in Paramecium multimicronucleatum. The mathematical functions determined closely mimic the Ca2+ dependence of adenylate cyclase (AC) and guanylate cyclase (GC) activities as documented in P. tetraurelia. Patterns of cAMP concentration ([cAMP]), cGMP concentration ([cGMP ]), and the ratio [cGMP]/[cAMP] were calculated with respect to Ca2+ concentrations ([Ca2+]) fluctuating sinusoidally with a period of 24 hours at three different levels: low, medium, and high. The functions displayed varying patterns of [cAMP] characteristic for [Ca2+] fluctuating at each level, while patterns of [cGMP] and [cGMP]/[cAMP] almost paralleled [Ca2+] fluctuations. Similar patterns were observed for actual [cAMP] and [cGMP] measured during the light/dark cycle in P. multimicronucleatum, grown in axenic media additionally containing [Ca2+] at 25 (low), 100 (medium), or 400 (high) microM, respectively. The coincidence between simulated and measured fluctuations of [cAMP] and [cGMP] suggests that the circadian fluctuations of intracellular [Ca2+] primarily stimulate activities of AC and GC via their different degrees of Ca2+ dependence, which are ultimately responsible for the circadian spatiotemporal organization of various physiological functions in Paramecium.

[In vitro study on the effect of sex steroid and growth factor on growth and laminin, collagen IV, and tissue plasminogen activator production of normal endometrial cells and endometrial cancer cells in culture].

The mechanism of the invasion and proliferation of endometrial cancer is closely related to interactions between the endometrial glands and stroma. In this study, we examined the biological role of sex steroids (estradiol; E2, progesterone; P) and growth factors (epidermal growth factor; EGF, transforming growth factor-beta; TGF-beta) on cell growth and laminin, collagen IV and tissue plasminogen activator (t-PA) production of normal endometrial cells and endometrial cancer cells in culture. Normal endometrial gland cells and stromal cells, and endometrial cancer cell lines (Ishikawa, OMC-2) were used. E2, P, EGF and TGF-beta were added to the culture in physiological concentrations. The growth of normal endometrial gland cells was promoted by E2 and EGF, whereas that of Ishikawa cells and OMC-2 cells was promoted by EGF. E2 enhanced the effects of EGF in normal endometrial gland cells. The growth of normal endometrial stromal cells was not affected by them. OMC-2 was inhibited by anti-EGF receptor antibody. On the other hand, the production of laminin and collagen IV of these cultured cells was inhibited by EGF and promoted by TGF-beta, whereas that of t-PA was promoted by EGF and inhibited by TGF-beta. These results suggest that the growth of normal endometrial gland cells with estrogen receptor (ER) is controlled by both E2 and EGF, whereas that of endometrial cancer cells is affected only by EGF, and those cells without ER depend particularly on the autocrine growth mechanism of EGF.(ABSTRACT TRUNCATED AT 250 WORDS)

[Study on the management of dysplasia of the uterine cervix].

In this study, we investigated problems on the management of dysplasia on the basis of our clinical data obtained in the past ten years. In addition, we also examined the biological significance of protooncogene expression and human papilloma virus (HPV) infection in the initiation and promotion of dysplasia. Among 540 cases of dysplasia we have managed, 48.8% of the cases of mild, moderate and severe dysplasia which we followed up for more than 6 months regressed, and 24.1% of the cases progressed. The cure rate for laser therapy based on the follow up for 6 to 96 months was significantly high (97.9%) in 342 cases treated by the cone method, and low (89.5%) in 38 cases treated by the vaporization method. Preoperative histological findings confirmed 60.5% of postoperative findings, and several early cervical carcinomas were found in preoperative cases of dysplasia by laser conization. Among 28 cases (8.2%) of incomplete excision, 24 cases (85.7%) regressed spontaneously. On the other hand, the positive rate of immunostaining of epidermal growth factor receptor (EGF-R) and c-myc oncogene product (c-myc protein) increased from mild or moderate to severe dysplasia, and the positive rate for EGF-R was significantly high (80.0%) in the progressive group. The positive rate for HPV genome was quite low (16.0%) in dysplasia. Among them, EGF-R was most associated with the prognosis of dysplasia. These results suggest that laser conization should be performed for many cases of dysplasia because of the preoperative possibility of the existence of early cancer, and EGF-R is also useful in determining the necessity for therapy for dysplasia.

[In vitro studies on the expression of c-myc oncogene product in gynecological cultured cancer cells].

In this study, immunocytochemical and biochemical detection of c-myc protein in gynecological cultured cancer cells were performed together with gene expression of the cells. OMC-1 (cervical squamous carcinoma cell line), OMC-2 (endometrial adenocarcinoma cell line), OMC-3 (ovarian mucinous adenocarcinoma cell line) and OMC-4 (cervical adenocarcinoma cell line) were used. Immunocytochemically, c-myc protein was detected in both nuclei and cytoplasms of cultured cells when they were fixed in 95% ethanol, 10% formalin and 4% paraformaldehyde (PFA) including 10mM NaCl. However, it was detected in the nuclei of almost all of the cells in nuclei of the cells when they were fixed in 4% PFA including 1,000mM NaCl. Western blotting against a nuclear fraction of the cells demonstrated 66Kd protein in OMC-1 and 62Kd protein in OMC-2,3,4, respectively. They were completely absorbed by c-myc synthetic peptide. However, there was no reaction against the cytoplasmic fraction of the cells. Slot blot hybridization against the DNA of the cells demonstrated 15 times and 5 times c-myc gene amplification in OMC-2 and OMC-4, respectively. These results suggest that OMC-1,2,3,4 can be used as positive controls for the immunocyto- and histochemical detection of c-myc protein in clinical materials. However, it must be noted that the redistribution of c-myc nuclear protein into the cytoplasm may occur in the fixation process.

[Bone marrow suppression in gynecological patients with malignant tumor treated with combination chemotherapy cyclophosphamide, pirarubicin and cisplatin].

We treated 14 patients with cyclophosphamide, adriamycin and cisplatin (CAP), and 16 patients with cyclophosphamide, Pirarubicin and cisplatin (CTP). Hematological changes in the peripheral blood were compared in the two groups to determine whether there was any difference in bone marrow suppression. 1) The lowest leukocyte counts were similar in the two groups. The leukocyte count reached its nadir at 11.6 +/- 2.1 days in the CTP group and at 15.1 +/- 2.8 days in the CAP group, a significant difference (p less than 0.01). 2) The leukocyte count recovered rapidly in the CTP group and was significantly higher (p less than 0.01) at 3 to 4 weeks than in the CAP group, and it returned to the pretreatment level in the CTP group in the fourth week. 3) The platelet count reached its lowest level in the second week in both groups. In the CTP group, it was significantly higher (p less than 0.01) than in the CAP group. 4) Reticulocyte count reached its lowest level in the first week in both groups, and then started increasing. 5) In the CTP group, a course of treatment was 28.0 +/- 2.3 days and it was 29.5 +/- 2.3 days in the CAP group. In 28% of the CPA group, it took 30 days or more for the leukocyte count to return to normal after one course, while none of the CTP group did the leukocyte count remain low for 30 days. These results show that CTP causes more rapid bone marrow suppression (particularly leukocytopenia) than CAP, does but that a recovery is more rapid with CTP. These CTP may be a better form of treatment than CAP.

[A study of the expression of class II antigen on uterine cervical cancer cells and cell lines].

An immunohistochemical method with monoclonal antibody to DR antigen was used to study class II antigens and HLA-DR antigen in 20 patients with uterine cervical cancer. Eight patients had various amounts of DR antigen. Regional infiltrating lymphocytes were also examined with anti-Leu 1, Leu 2a, Leu 3a, and Leu 10. Among the many infiltrating T cells, Leu 3a-positive cells were relatively predominant surrounding the cancer nests which contained DR antigen. Cervical cancer cell lines OMC-1 and OMC-4 both demonstrated DR antigen. Interferon (IFN)-gamma treatment enhanced the expression of DR antigen in OMC-1 and OMC-4. The DR antigen in OMC-1 and OMC-4 were capable of stimulating allogenic lymphocytes in mixed lymphocyte reaction (MLR), and their stimulatory activity was significantly enhanced by IFN-gamma treatment. These results demonstrated the expression of class II antigen, especially DR, on some cervical cancer cells and cell lines and showed that the DR antigen in uterine cancer cell lines can stimulate MLR.

[EGF receptor and effects of EGF on growth and tumor marker secretion in uterine cervical cancer cells].

Effects of EGF on proliferation and tumor marker secretion of cervical cancer cells are reported together with the characteristics of EGF receptors on the cells. TA-4 producing cell line (OMC-1) originating from cervical squamous cell carcinoma and CA-125 producing cell line (OMC-4) originating from cervical adenocarcinoma, were used. Scatchard plot of EGF binding to OMC-1 indicated a single class of binding sites with a dissociation constant (Kd) of 360pM, whereas that of OMC-4 was curvilinear suggesting two classes of binding sites with a Kd of 170pM and 510pM. The theoretical maximum number of binding sites of OMC-1 and OMC-4 was 2.4 X 10(4) and 1.6 X 10(5), respectively. Effects of EGF on growth were studied by monitoring cell number and the incorporation of 3H-thymidine into the DNA of the cells. OMC-1 was stimulated by EGF at low concentrations (0.01-0.1nM) and inhibited at higher concentrations. OMC-4 was not stimulated by EGF. The TA-4 secretion of OMC-1 was slightly stimulated by EGF at low concentrations (0.01-1nM) and significantly stimulated at high concentration (10nM). The CA-125 secretion of OMC-4 was not stimulated by EGF. These results suggest that there are some differences between cervical squamous cell carcinoma and adenocarcinoma in the mechanisms of regulation of proliferation and tumor marker secretion by EGF.

[Immunohistochemical studies on epidermal growth factor receptor in experimental squamous cell carcinoma of the uterine cervix of mice].

The tissue localization of epidermal growth factor receptor (EGF-R) in experimental squamous cell carcinoma of the mouse uterine cervix was examined immunohistochemically. Carcinoma was induced by the insertion of a 20-methylcholanthrene (MC)-impregnated thread into the cervical canal of the mouse. Tissue sections (of normal columnar epithelium, proliferation, atypical proliferation, early invasive carcinoma, invasive carcinoma and metastatic carcinoma) were stained by the avidin/biotin immunoperoxidase technique using anti-EGF-R monoclonal antibody. Normal columnar epithelium was negative for EGF-R, whereas proliferation was partly positive. The lesions of atypical proliferation and early invasive carcinoma had a positive staining for EGF-R. The staining for EGF-R declined in the lesion of invasive carcinoma. Metastatic carcinoma was not stained for EGF-R. These results suggest that EGF-R may play an important role in the early stage of carcinogenesis of the mouse uterine cervix induced by 20-MC.