Analysis of matrix metalloproteinase (MMP-8 and MMP-2) activity in gingival crevicular fluid from children with Down's syndrome.

BACKGROUND AND OBJECTIVE: High levels of colonization by periodontopathic bacteria and a high prevalence of chronic inflammatory periodontal disease have been reported in children with Down's syndrome. Matrix metalloproteinases (MMPs) are mediators of extracellular matrix degradation and remodelling, and are deeply involved in the course of periodontal disease. To clarify the relationship between Down's syndrome and periodontitis, we investigated levels of MMP-2 and MMP-8 in gingival crevicular fluid (GCF) and detection of periodontopathic bacteria from subgingival plaque. MATERIAL AND METHODS: Samples of GCF and plaque were isolated from central incisors. Levels of MMPs were evaluated by enzyme-linked immunosorbent assay, and periodontopathic bacteria were detected by polymerase chain reaction. RESULTS: Levels of MMP-2 and MMP-8 in Down's syndrome patients were higher than those in healthy control subjects. In the Down's syndrome group, increases in these MMPs were observed in GCF from patients with an oral hygiene index score of < 2 and in GCF from sites that were negative for bleeding on probing. The detection rate of periodontopathic bacteria in Down's syndrome patients was higher than that in the control subjects. Matrix metalloproteinase-2 levels in sites harbouring Porphyromonas gingivalis or Aggregatibacter (Actinobacillus) actinomycetemcomitans were lower than in those without these microorganisms. CONCLUSION: These results suggest an increase in MMP-2 and MMP-8 in Down's syndrome patients, regardless of whether inflammation of periodontal tissue is present or not.

Colonization pattern of periodontal bacteria in Japanese children and their mothers.

BACKGROUND AND OBJECTIVE: The purpose of this study was to determine the time of infection by anaerobic gram-negative rods associated with periodontal disease, and to clarify their transmission from mother to child. MATERIAL AND METHODS: Seventy-eight Japanese children (including 10 siblings), aged from 3 to 9 years, and 68 mothers, were enrolled in this study. Colonization by 11 periodontal bacterial species was determined using polymerase chain reaction amplification of samples of subgingival plaque obtained from the children and their mothers. RESULTS: The detection rates of Porphyromonas gingivalis, Tannerella forsythensis and Treponema denticola increased in children after the age of 6 years. We found a high consistency in colonization by P. gingivalis, T. denticola, Prevotella intermedia and Prevotella nigrescens in 9 of the 10 siblings. The average number of bacterial species in plaque samples harboring Fusobacterium nucleatum and/or Fusobacterium periodonticum was significantly greater than in those without, in both children and mothers. Kappa statistical analysis revealed that the detection of Capnocytophaga gingivalis, Capnocytophaga ochracea, Campylobacter rectus and T. denticola in children was consistent with that in the mother. CONCLUSION: Periodontal bacterial colonization in Japanese children increased with age and was associated with F. nucleatum and/or periodonticum, and the bacterial flora in children was similar to that in their mothers.

Cross-resistance to diverse drugs is associated with primary cisplatin resistance in ovarian cancer cell lines.

We have previously obtained, by exposure to near continuous increasing concentrations of cisplatin, a panel of human ovarian cancer cell lines that exhibit a wide range of primary resistance to the drug (9- to > 400-fold). These cells had strikingly increased (4- to 50-fold) levels of glutathione (GSH) as compared with the drug-sensitive cells of origin (A. K. Godwin et al., Proc. Natl. Acad. Sci. USA, 89: 3070-3074, 1992). Utilizing this panel of resistant cell lines, we evaluated cross-resistance to classical alkylating agents, natural product drugs, and irradiation. We observed that cross-resistance to carboplatin paralleled that of cisplatin, culminating in approximately 250-fold resistance. Similarly, melphalan cross-resistance continued to increase to > 400-fold and again paralleled the primary cisplatin resistance. Cell lines with low to very high levels of resistance to cisplatin are 8- to 850-fold resistant to the epipodophyllotoxin derivative etoposide. Cross-resistance is also observed for other natural product drugs, including Adriamycin (approximately 80-fold), mitoxantrone (approximately 440-fold), and taxol (approximately 40-fold). Cross-resistance to irradiation is, however, modest (< 2-fold). The cells with the greatest primary resistance to cisplatin most commonly had the highest cross-resistance to the other drugs examined. The cross-resistance to the natural product category drugs was found not to be mediated by the products of either the multidrug resistance 1 (MDR1) or multidrug resistance-associated protein (MRP) genes based on lack of coordinate increased expression or amplification of these genes as assessed by Northern and Southern blot analyses. Furthermore, verapamil failed to markedly increase drug sensitivity. Although there was no indication that these natural product drug efflux pumps were operative, we observed decreased doxorubicin accumulation in these cell lines cross-resistant to natural products. In addition, alternations in DNA topoisomerase II mRNA levels, which have been observed in a variety of human tumor cell lines selected in vitro for resistance to etoposide or teniposide, were not detected. Only intracellular levels of GSH correlated with cross-resistance to these diverse anticancer agents and partial loss of resistance was associated with a marked decrease in glutathione levels. In the absence of alternative mechanisms, we speculate that the very broad clinically relevant cross-resistance seen in this model system may, at least in part, be the direct result of GSH-mediated drug inactivation or may be due to a combination of GSH conjugation to drug and conjugate efflux mediated by the putative ATP-dependent glutathione S-conjugate export pump.

Clinical features of ovarian cancer in Japanese women with germ-line mutations of BRCA1.

We analyzed the clinical features of 25 ovarian cancer patients who were associated with germ-line mutations of BRCA1 from four site-specific ovarian cancer families and seven breast-ovarian cancer families in Japan. The average age at diagnosis was 51.1 years (range, 38-77 years). Histological examination revealed 24 serous cyst adenocarcinomas in 25 patients. In 23 patients with clear clinical records, 3 patients had stage I disease, 17 had stage III disease, and 3 had stage IV disease. Thirteen patients with stage III disease who were treated with cisplatin-containing chemotherapy following tumor reduction surgery showed more favorable outcomes in both the survival rate and disease-free intervals, compared with age- and treatment course-matched controls (5-year survival rate, 0.786 versus 0.303; median disease-free interval, 91.43 versus 40.92 months; P < 0.05 for both, by logarithmic rank test). Our statistical model for the inheritance of susceptibility to ovarian cancer was derived from the analysis of 26 patients and 19 healthy carriers of 12 families. The expected lifetime risk of ovarian cancer is about 80% for women with mutations of BRCA1. These results suggest that the clinical outcome of ovarian cancer with germ-line mutations of BRCA1 appears to be more favorable than that with sporadic cases and that the disease penetrance among pedigrees with germ-line mutations of the BRCA1 gene is substantially high.

Irinotecan (CPT-11) combined with cisplatin in patients with refractory or recurrent ovarian cancer.

Irinotecan hydrochloride (CPT-11) is reportedly effective for the treatment of refractory or recurrent ovarian cancer. We investigated the antitumor efficacy and toxicity of combination therapy with CPT-11 and cisplatin in 25 patients (mean age 55 years, range 35-73 years) with refractory or recurrent ovarian cancer who had previously undergone platinum-based combination chemotherapy. Patients received two or more courses of treatment consisting of 50 or 60 mg/m2 of CPT-11 on days 1, 8 and 15 and 50 or 60 mg/m2 of cisplatin on day 1 administered intravenously. All patients were evaluable for the response and the toxicity profile. Complete responses were obtained in two (8.0%) patients and partial responses were obtained in eight (32.0%) patients, giving an overall response rate of 40% (10 of 25 patients) (95% CI 23.0-59.0%). The median duration of response was 5.5 months (range 2-27 months), the median time to tumor progression was 6 months (range 3-28 months) and the median overall survival was 12 months (range 3-39+ months). Grade 3 or 4 neutropenia, which was the most frequent and severe toxic effect, occurred in 36 (54.5%) of the 66 treatment courses and in 16 (64.0%) of 25 patients. The nadir of the leukocyte count occurred on days 18-19. Neutropenia was reversed by short-term administration of granulocyte colony-stimulating factor for 2-10 days. Less serious hematologic effects and non-hematologic effects, such as diarrhea, were also observed. This preliminary study showed that this regimen of CPT-11 and cisplatin was effective in patients with recurrent ovarian cancer.

Establishment and characterization of ovarian endometrioid adenocarcinoma cell line in nude mice and analyses of the immunohistochemical property among the original, recurred, and heterotransplanted tumor.

The establishment and characterization of a new human endometrioid adenocarcinoma cell line (KOC-8e) derived from the malignant ascites is described. The original tumor at the initial operation showed positive in TPA, and SLX immunohistochemically. By contrast, recurred tumor showed positive for CA125, TPA, SLX, MUC-1, and p53. In addition, the heterotransplanted tumor showed positive for CA125, TPA, SLX, MUG-1, and p53. The DNA index was similar in the primary, recurred, and nude mouse tumor ranging between 1.93 and 2.05. The tumor growths were suppressed dose dependently by CDDP and CPT-11. CA125 showed useful as a tumor marker of this tumor, however, the nude mice had detectable tumor without elevation of CA125 after low dose application of CDDP and CPT-11. Thus, clinical determination of chemotherapeutic effect and residual tumor cannot be made only by CA125. KOC-8e cells could be useful to study histological analysis and chemosensitivity.

Expression of matrix metalloproteinase-2 (mmp-2) in cervical neoplasia - immunohistochemical and in-situ hybridization studies.

The expression of matrix metalloproteinase-2 (MMP-2, type IV collagenase, 72 kDa) in 47 cervical precancerous and cancerous lesions were localised by in situ hybridization and immunohistochemical studies. MMP-2 mRNA was present in the stromal cells surrounding invasive cells in the majority of squamous cell carcinomas and adenocarcinomas. In contrast, MMP-2 immunoreactivity was found in the epithelial cells. Our results indicate that there may be some interaction between carcinoma cells and stromal cells to produce MMP-2 which may contribute to the destruction of the extracellular matrix in invasive cervical carcinomas.

Overexpression of the tumor-suppressor gene p53 in human ovarian tumor-tissues and its correlation with clinical stage.

An extensive series of histological sections from various types of benign, borderline, and malignant human ovarian tumors were examined for expression of the p53 protein by immunohistochemical staining, using a new anti-p53 mouse monoclonal antibody. Positive staining for p53 was detected in 34% of all malignant ovarian cancer tissues. A high rate of positivity was observed in serous cystadenocarcinoma (59.7%). None of the borderline or benign tumors showed any reaction. No relation was observed between clinical stage, and the frequency of p53 positivity. Mutation of the p53 gene with overexpression of the mutant protein appears to be a specific genetic change in human ovarian cancer.

Treatment of gynecological malignancies with a combination of cisplatin, adriamycin and ifosfamide.

Histogenetic similarities in the female genital system suggest that a group of cancers from the coelomic epithelium may have a common sensitivity to cytotoxic treatment. A total of 24 patients with evaluable gynecologic cancer were treated with a new combination regimen consisting of cisplatin (50 mg/m2), Adriamycin (50 mg/m2), and ifosfamide (1 g/m2 X 5 days) (PAI) given in five courses at 4-week intervals. The tumors included ten cervical, four endometrial, seven ovarian, and three peritoneal cancers. In all, 20 of our patients had recurrent disease and had previously received other cytotoxic treatment, including radiation (11 cases), cisplatin-containing chemotherapy (8 cases), or both (1 case). As a schedule modification, PAI plus bleomycin (20 mg/m2, on days 1 and 8) was recommended for tumors containing squamous components. According to the response criteria of the International Union Against Cancer (UICC), a 96% response rate was obtained (cervical, 9/10; endometrial, 4/4; ovarian, 7/7; peritoneal, 3/3). Of 23 responders 8 (35%) achieved a complete remission. The dose-limiting side effect of the PAI regimen was hematologic toxicity: grade 4 leukopenia was observed in 92% of patients, and grade 4 thrombocytopenia was seen in 17%. However, the myelosuppression reversed spontaneously within 3 weeks, and none of the patients was incapable of completing the planned treatment courses. The results suggest that PAI combination chemotherapy is effective and can be used in the management of patients with gynecologic malignancies derived from the coelomic epithelium (müllerian tumor group).

Combination of CPT-11 with cisplatin as first-line chemotherapy in advanced epithelial ovarian cancer.

As CPT-11 was shown to be efficacious in recurrent ovarian cancer, a phase II trial has been undertaken at a recommended dose determined in the phase I trials of combination therapy of CPT-11 with cisplatin (CDDP). As first-line chemotherapy, 60 mg/m(2) of CPT-11 (on days 1, 8, and 15) and 60 mg/m(2) of CDDP (on day 1) were intravenously administered to patients with epithelial ovarian cancer with residual lesions larger than 2 cm, and patients who underwent exploratory laparotomy. Case 1 and 2 achieved CR and PR after completion of the first and second courses, respectively. After the third course when CA125 values turned negative, secondary cytoreductive surgery was performed in both cases, and the tumor was completely extirpated. Dose limiting toxicity was neutropenia, which was managed by administration of granulocyte stimulating factor or by skipping the administration of CPT-11. In Case 2, the number of platelets decreased with repetition of the courses. Grade 3 or worse diarrhea was not observed. The combination therapy of CPT-11 with CDDP is considered to be safe and efficacious in treatment of epithelial ovarian cancer.

Novel germ-cell tumor-cell line established from a testicular feminization syndrome.

We analyzed a novel cell line established from a testicular feminization syndrome. The original tumor was an endodermal sinus tumor with seminoma. The doubling time, the saturation density and the mitotic index were 36.1 hours, 3.65x10(4) cells/cm2 and 4.3%, respectively. The mode number of chromosomes was 102. Results of the MTT assay showed that the cell line had an IC50/PPC of less than 0.1 for methotrexate and actinomycin D. The effects of methotrexate were schedule dependent; the effects of actinomycin D were both schedule and dose dependent.

Study of angiotensin II-induced hypertensive chemotherapy for ovarian cancer in rats.

We investigated the usefulness of angiotensin II (AT II)-induced hypertensive chemotherapy for adenocarcinoma of the ovary, which was induced with dimethylbenzantracene (DMBA) in rats. Ovaries with DMBA-induced cancer had poor blood flow, while normal ovaries had abundant brood flow when blood pressure was normal. The brood flow of the normal ovaries was suppressed by AT II-induced hypertension, while the blood flow in ovarian cancer was more than doubled. Tissue levels of the anticancer drug cisplatin (CDDP) were almost the same in the normal ovary in the presence or absence of induced hypertension. However, the CDDP level in ovarian cancer tissue was significantly higher with AT II-induced hypertension than with normotension. Comparison of the ratio of the enlarged tumor to the primary tumor and the histopathologic antitumor effects, showed that AT II-induced hypertension treatment with CDDP was highly effective. Angiotensin II-induced hypertensive chemotherapy for adenocarcinoma of the ovary in rat was highly effective in comparison with normotension.

Intrasplenic autograft of testicular tissue in rats.

Intrasplenic heterotransplantation of infantile testis is a conventional method to produce testicular tumors in adult castrated rodents. To avoid possible immunological interference in the tumorigenic process in allografted testis, the parenchyma of testis of Wistar strain rats aged 7 weeks was injected into the spleen of the same rat after bilateral gonadectomy. In 17 of 20 animals testicular neoplasms, resembling gonadal stromal cell tumors in human, developed from the intrasplenic gonadal tissue in the 14th month following transplantation. This autografting technique is feasible to induce testicular neoplasms in granulosa cell tumor category in high incidence, and thus deserves further attention.

Intrasplenic grafting of ovarian tissue containing 7, 12-dimethylbenz[a]anthracene.

To produce malignant granulosa cell tumors, ovarian tissue containing 7,12-dimethylbenz[a]anthracene (DMBA) was autografted in the spleen of 20 castrated rats. Adenocarcinoma developed in 8 rats within 6 months from the transplantation. Granulosa cell tumor arose from the intrasplenic graft, in 6 rats at the 14th month. The neoplastic cells were diffusely arranged in sheet showing focal luteinization. In 3 cases extrasplenic spreads were found on the peritoneal surface. Since malignant ovarian tumors seldom develop by intrasplenic grafting alone, the DMBA treatment may contribute to the occurrence of malignant granulosa cell tumor.

Granulosa cell tumor arising in the ovary irradiated 22 years earlier for endometrial stromal sarcoma.

We encountered a rare case of metachronous tumors in a 57-year-old Japanese housewife who presented with an ovarian granulosa cell tumor 22 years after receiving treatment for endometrial stromal sarcoma. Treatment consisted of a total abdominal hysterectomy and left salpingo-oophorectomy followed by 40 Gy whole abdominal irradiation. Since the second tumor occurred in the ovary that had been previously irradiated to treat the first metachtonous neoplasm, the present case can help to improve our understanding of the histogenesis of granulosa cell tumors.

Relation between CA125 levels and second-look operation for ovarian cancer.

We investigated the relationship between the serum level of CA125 before a second-look operation (SLO) and SLO findings in 196 patients with adenocarcinoma of the ovary. SLO findings were positive in 38 (19.4%) of 196 patients. The positive rate tended to increase with the clinical stage, but SLO findings were positive even in a patient with stage Ia disease. The pre-SLO serum level of CA125 was positive in 11 patients before SLO, SLO findings were positive in 8 of these 11 patients. The highest diagnostic accuracy (37.9%) for the pre-SLO serum level of CA125 was obtained at a cut-off value of 11 U/ml. Our findings suggest that a positive pre-SLO serum level of CA125 does not necessarily indicate tumor positivity. In addition, we suggest that the cut-off value for prediction of SLO findings should be below 35 U/ml, which is a commonly used cut-off value.

Macrophage colony-stimulating factor as a marker of ovarian carcinoma.

Macrophage colony stimulating factor (M-CSF) is a cytokine which stimulates the proliferation and differentiation of phagocytic cells. We evaluated the usefulness of M-CSF as a serum tumor marker for ovarian malignancies and also assessed M-CSF production by tumor cells and the role of an autocrine system in such M-CSF production. The findings obtained were as follows: i) Serum M-CSF was a useful marker for malignant ovarian tumors. ii) M-CSF was a marker for both epithelial stromal tumors and for germ cell tumors. It was also a marker for dysgerminoma, for which no specific tumor marker is currently available. iii) The value of combined assays employing M-CSF was confirmed. iv) M-CSF production was demonstrated in various malignant ovarian tumor cell lines, but the presence of an autocrine system for M-CSF was not confirmed.

Induction of apoptosis in ovarian carcinoma cell line by glucocorticoids, and sex steroid hormones.

We investigated the interactions in the KOC-2s human ovarian cancer cells on the effect of glucocorticoids, and sex steroid hormones in ovarian carcinomas. At 10(-8) M to 10(-5) M, dexamethasone (Dex) decreased the number of cells by 75-80% (p<0.001). At 10(-8) M and 10(-7) M, hydrocortisone (HC) decreased the number by 50% (p<0.01); at 10(-6) M and 10(-5) M, the decrease in number of cells was 65%. The E-2 decrease in number was not statistically significant. Progesterone (PG) showed at 10(-8) to 10(-6) M an increase in number of cells, however, at 10(-5) M it was decreased by 70% with a significant difference (p<0.001). Dex (10(-8)-10(-5) M), HC (10(-8)-10(-5) M) and PG (10(-5) M) produced internucleosomal cleavage of DNA into fragments with multiples of 180 to 200 bp. The TNF-alpha with addition of Dex (10(-8)-10(-5) M) and HC (10(-8)-10(-5) M) was increased after 24 h, 48 h (p<0.001); however, gradually decrease after 72 h. When PG (10(-8)-10(-5) M) was added, PG (10(-5) M) increased the secretion of TNF-alpha after 72 h. Our findings demonstrate that glucocorticoids, and PG directly induce apoptotic DNA fragmentation of KOC-2s cells. However, the secretion of TNF-alpha and expression of Fas antigen were totally different in these substances. These data provide a basis for future studies on the mechanisms of apoptotic effect of glucocorticoids, and PG and the therapeutic effects of these substances.

Ovarian tumor complicated by a colo-ovarian fistula.

Although a fistula is a rare complication of ovarian tumor, we encountered four patients with cole-ovarian fistulas in three years. The first case was demonstrated by following barium enema by the extravasation of barium from the sigmoid colon, and by a gas-containing lesion in the tumor observed on computed tomography. Mature cystic teratoma was the pathological diagnosis in two cases. A mixed germ cell tumor and a serous cystadenoma of low malignant potential were diagnosed in the other two cases. The etiology and differential diagnosis of cole-ovarian fistula are reviewed.

Case of leiomyomatosis peritonealis disseminata treated with a gn-rh analog.

We report a rare case of leiomyomatosis peritonealis disseminata (LPD) which we attempted to treat by administering the Gn-RH superagonist, buserelin. A 48-year-old woman presented with a 6-month history of hypermenorrhea. Hysterectomy revealed a benign leiomyoma. Light and electron microscopic studies revealed that the tumors were composed of well- or poorly-differentiated smooth muscle cells. Their clinical appearance together with the mitotic figures found in tumor sections suggested an uncertain malignant potential. In the immunohistochemical study of intermediate filaments, antidesmin and anti-actin antibody reacted with tumor cells. Tumor re-growth in the pelvic cavity led to a second operation. The administration of the Gn-RH analogue, 900 mu g daily for two months, decreased the tumor size. The average final volume was 68.7% of the original. However, the tumors recurred during the next four weeks. The combined administration of the Gn-RH analogue and etoposide (25 mg x 2/day) did not prevent recurrence after a third operation. The serum level of immune-suppressive acid protein (IAP) was useful in monitoring the clinical course of this patient, finally diagnosed as having LPD.