Increased cyclooxygenase-2 expression is correlated with suppressed antitumor immunity in cervical adenocarcinomas.

Cyclooxygenase-2 (COX-2) inhibition suppressed the growth of various tumors. The augmentation of antitumor immunity by increasing cytotoxic lymphocytes may be an important mechanism for COX-2 inhibition. Among cervical cancers, adenocarcinomas present more aggressive behavior and overexpressed COX-2. The expression of COX-2 and the CD8+ lymphocyte infiltrations were evaluated in this study by immunohistochemistry. We studied COX-2 expression and CD8+ lymphocyte infiltration in 55 women with cervical adenocarcinomas. COX-2 expression and tumor stromal CD8+ lymphocytes were evaluated by semiquantified methods. Tumor intraepithelial lymphocytes were counted under microscopic field of x200. Correlations between these data and other clinicopathologic features were investigated. Thirty-seven out of 55 (67.3%) cervical adenocarcinomas significantly expressed COX-2. Patients who died within 5 years showed higher percentage of COX-2 expression than survivors (100% vs 58.1%, P < 0.05). Victims also showed lesser intraepithelial CD8+ lymphocyte counts than survived patients (3.4 vs 26.4, P < 0.05). COX-2 expression and tumor intraepithelial lymphocyte count were reversely correlated with each other (correlation index: -0.38, P < 0.01). Up-regulated COX-2 expression and lesser tumor intraepithelial CD8+ lymphocyte count are poor prognostic indicators for cervical adenocarcinoma patients. COX-2 may play an important role in the suppression of host antitumor immunity in cervical adenocarcinomas.

C-erbB-2 or mutant Ha-ras induced malignant transformation of immortalized human ovarian surface epithelial cells in vitro.

Ovarian cancer is believed to develop from the ovarian surface epithelium through the accumulation of aberrations of oncogenes and/or tumor suppressor genes. However, it is unclear how the gene abnormalities are involved in ovarian carcinogenesis. To elucidate the process, we transfected genes reported to show their abnormalities in human ovarian cancers into human ovarian surface epithelial cells. Immortalization of the cells was achieved by the transfection of SV40 large T antigen (LT) and human telomerase reverse transcriptase (hTERT); however, the resultant cells showed no tumorigenesis. Additional transfection of either c-erbB-2 or mutant Ha-ras into the immortalized cells showed the anchorage-independent growth and tumorigenesis in mice with the incidence of 50% and 40%, respectively. Histologically, all the tumours were undifferentiated. In association with the tumorigenesis, the cells expressing c-erbB-2 or mutant Ha-ras demonstrated increased vascular endothelial growth factor secretion under hypoxia and enhanced resistance to apoptosis compared with the immortalized cells. Collectively, the introduction of either c-erbB-2 or mutant Ha-ras in the cells, which were efficiently immortalized by the transfection of LT and hTERT, showed tumorigenicity, suggesting that c-erbB-2 or mutant Ha-ras genes might be involved in ovarian carcinogenesis.

Combination effect of adenovirus-mediated pro-apoptotic bax gene transfer with cisplatin or paclitaxel treatment in ovarian cancer cell lines.

To develop a novel therapeutic strategy for ovarian cancer, we constructed a recombinant adenovirus which highly expresses pro-apoptotic Bax protein and examined its therapeutic effect on a series of ovarian cancer cell lines: A2780, A2780/cDDP, OVCAR-3 and SK-OV-3. A recombinant adenovirus carrying the Bax-alpha gene (AxCALNKYbax) induced high expression of the Bax-alpha protein in all the cell lines. The cytotoxic effect of Bax was observed in three ovarian cancer cell lines: the per cent reduction in the number of cells was 40.0% for cisplatin-sensitive A2780, 50.0% for cisplatin-resistant A2780/cDDP, and 64.8% for marginally cisplatin-resistant OVCAR-3. In contrast, it was only 12.3% for cisplatin-resistant SK-OV-3. Cisplatin-resistant A2780/cDDP had a p53 mutation and exhibited attenuated Bax induction after cisplatin treatment, which may explain why supplementation of Bax was effective in this chemoresistant ovarian cancer. Combination with cisplatin or paclitaxel enhanced the cytotoxic effect of Bax induction in all but one cell line including cisplatin-resistant A2780/cDDP. It appears that adenovirus-mediated Bax induction, with or without combination with conventional chemotherapy, useful strategy for the treatment of ovarian cancer.

Human ovarian surface epithelial (OSE) cells express LH/hCG receptors, and hCG inhibits apoptosis of OSE cells via up-regulation of insulin-like growth factor-1.

Gonadotropins including luteinizing hormone (LH) or human chorionic gonadotropin (hCG) have been implicated as playing an important role in the development of epithelial ovarian carcinomas, most of which are believed to originate from the ovarian surface epithelium (OSE). To address this issue, we examined the expression of LH/hCG receptors and the influence of hCG on cell proliferation and on the apoptosis of cultured human OSE cells. RT-PCR and binding assay revealed that OSE cells express the LH/hCG receptor mRNA and have specific binding activity for hCG. Treatment with hCG stimulated the proliferation of OSE cells in a dose-dependent manner. In addition, hCG treatment inhibited the apoptosis of OSE cells induced by serum deprivation. Among the apoptosis-related genes, hCG treatment did not change the mRNA levels of bcl-2, bax and IGF-1 receptor but significantly increased that of IGF-1. Treatment with IGF-1 alone also suppressed the apoptosis of OSE cells, and treatment by hCG along with neutralization antibody against IGF-1 receptor reversed the anti-apoptotic effect of hCG. Accordingly, LH/hCG signaling followed by up-regulation of IGF-1 is involved in the inhibition of apoptosis of OSE cells, the possible histogenetic origin of epithelial ovarian carcinomas.

Comparison of human cytomegalovirus DNA polymerase activity for ganciclovir-resistant and -sensitive clinical strains.

Previously, we found that a ganciclovir (GCV)-resistant clinical human cytomegalovirus (HCMV) isolate had an amino acid substitution at codon 501 (Leu --> Phe) in the delta-region C of the DNA polymerase gene. DNA polymerases have now been (partially) purified from both the GCV-resistant and sensitive parental strains and the activity of DNA polymerase and 3'-5' exonuclease compared. With respect to DNA polymerase activity, the Michaelis constant (Km) and maximum velocity (Vmax) of the GCV-resistant strain for the DNA template were lower than those of the GCV-sensitive strain. With respect to 3'-5' exonuclease activity, the Km and Vmax of the GCV-resistant strain for the DNA substrate in the presence of ammonium sulfate were lower than those of the GCV-sensitive strain, while being similar in the absence of ammonium sulfate. Although the polymerase activity of the two strains showed almost the same sensitivity for the different polymerase inhibitors, the 3'-5' exonuclease activity of the GCV-resistant strain was more resistant to these inhibitors than that of the GCV-sensitive strain.

Adenocarcinoma of the uterine cervix with choriocarcinomatous and hepatoid differentiation: report of a case.

A case of adenocarcinoma of the uterine cervix that showed choriocarcinomatous and hepatoid differentiation was encountered in a 65-year-old woman. She presented with genital bleeding and had multiple metastatic nodules in the lungs. At operation, a large, hemorrhagic, and necrotic tumor was found in the uterine cervix. The major portion of the tumor consisted of typical choriocarcinoma admixed with minor areas of hepatoid carcinoma and endocervical adenocarcinoma. Human chorionic gonadotropin and alpha-fetoprotein were detected in tumor cells in the choriocarcinomatous and hepatoid areas, respectively. The patient died of pulmonary metastasis 4 months after the operation. The coexistence of choriocarcinomatous and hepatoid carcinoma in an endocervical adenocarcinoma has not been reported previously. Both heterotopic components were probably derived from aberrant differentiation (or neometaplasia) of the somatic epithelial cells of the endocervical adenocarcinoma.

A novel UDP-sugar, UDP-3-ketoglucosamine or UDP-4-ketoglucosamine, from bovine heart muscle reduces metmyoglobin with NAD(P)H.

3-Ketoglucose and similar ketosugars have been identified in microorganisms only and little is known about their functions. UDP-sugars are widely found as an intermediate in sugar metabolism in living organisms. Yet what role UDP-sugars play, or whether they play a direct role in metabolism, is still unknown. UDP-sugars were isolated and purified from bovine heart muscle, and a UDP-sugar fraction capable of NAD(P)H-dependent catalytic reduction of metmyoglobin was detected. Subsequent identification revealed that the active UDP-sugar was UDP-3- or UDP-4-ketoglucosamine. These compounds were purified from bovine cardiac muscle by ultrafiltration, anion-exchange column chromatography and reverse-phase chromatography. They were further characterized by determination of their chemical reducing activity, by comparison with synthetic UDP-3- or UDP-4-ketoglucosamine standards using high-performance liquid chromatography, by estimation of molecular mass using fast atom bombardment mass spectrometry, and by Fourier transform infrared microspectroscopy and electron probe microanalysis. The results suggest that UDP-3- or UDP-4-ketoglucosamine reduces metmyoglobin in bovine cardiac muscle. It is important that the reducing activity displayed by this ketosugar is not the effect of UDP-3- or UDP-4-ketoglucosamine alone but depends on NAD(P)H. In other words, this action of UDP-3- or UDP-4-ketoglucosamine is catalytic.

Hormonal regulation in the production of macrophage colony-stimulating factor and transforming growth factor-beta by human endometrial stromal cells in culture.

The effects of gonadal steroids on the secretion and gene expression of macrophage colony-stimulating factor (M-CSF) and on the secretion of transforming growth factor (TGF)-beta 1 and TGF-beta 2 by human endometrial stromal cells (ESCs) were examined by an in vitro system of ESC differentiation (decidualization). M-CSF production by ESCs was dose-dependently enhanced by the addition of progesterone or testosterone, while estradiol treatment had no effect. TGF-beta 2 secretion by ESCs was inhibited by progesterone, estradiol and testosterone treatment, and on the contrary, slight enhancement by estradiol was observed in TGF-beta 1 secretion. These findings indicate that human ESCs produce cytokines of M-CSF and TGF-beta s, which are important for the growth and differentiation of the peri-implantation embryo as well as local immune cells under direct control of gonadal steroidal actions, and suggest a novel network between endocrine and immune systems in the human endometrium.

Progesterone enhances macrophage colony-stimulating factor production in human endometrial stromal cells in vitro.

Increasing evidence suggests that macrophage colony-stimulating factor (M-CSF) is produced in the uterine endometrium and that it plays an important role in the reproductive process. In the present study, using an in vitro decidualization model and human endometrium, we investigated M-CSF messenger RNA (mRNA) expression in human endometrial stromal cells (ESC) by Northern blotting and in situ hybridization. The secreted M-CSF in the culture medium of ESC was measured by enzyme-linked immunosorbent assay. ESC were cultured in the presence of progesterone (P) or estrogen. After a 9-day culture with P, when in vitro decidualization was confirmed by the production of PRL, M-CSF mRNA and protein levels were 3.1 +/- 0.5- and 3.2 +/- 0.8-fold (mean +/- SEM) higher, respectively, than those in cultures without P (P < 0.01). The P-induced increase was dose dependent. On the other hand, estrogen did not increase M-CSF mRNA expression. M-CSF mRNA expression in the first trimester deciduae that expressed PRL mRNA was higher than that in the endometria. By in situ hybridization, ESC as well as epithelial cells were shown to express M-CSF both in vitro and in vivo. These findings indicate that human ESC (decidua cells) express M-CSF mRNA and suggest that they secrete M-CSF in a P-dependent manner during the process of decidualization.

Progesterone-dependent secretion of macrophage colony-stimulating factor by human endometrial stromal cells of nonpregnant uterus in culture.

Recent studies have suggested that macrophages colony-stimulating factor (M-CSF), a hematopoietic glycoprotein essential to the proliferation and differentiation of mononuclear phagocytes and their progenitor cells, is also involved in the reproductive process in mice and humans. In this study, we examined, by enzyme-linked immunosorbent assay, the supernatants of stromal cell-enriched fraction (SF) of human nonpregnant endometrium for the presence of M-CSF during culture with progesterone (P) or estrogen. The bioactivity of M-CSF was assessed in a colony-forming assay of murine bone marrow cells. In addition, the M-CSF level in the culture supernatant of SF further purified by subculture, of epithelial cell-enriched fraction purified from human endometrium, and of peripheral blood lymphocytes, including about 10% monocytes, was examined with or without P, because SF is contaminated by epithelial cells and macrophages, both of which are suggested to secrete M-CSF. During 2-week culture, the level of M-CSF in the supernatants of SF cultured with P was markedly higher than that of control culture and estrogen-treated culture on any day tested, except for the first 2 days. P had a dose-dependent effect on M-CSF production by SF. Estrogen also enhanced M-CSF production by SF, but did not show dose dependency. The SF culture supernatants showed a colony-forming activity that was completely blocked by neutralizing anti-M-CSF antibody. SF subcultured three times, which was confirmed to be of more than 99% purity, secreted M-CSF in a P-dependent manner. M-CSF was also detected in the culture supernatants of epithelial cell-enriched fraction and peripheral blood lymphocytes, but P-dependent M-CSF production was not shown in these cultures. These results suggest that human endometrial stromal cells themselves can secrete bioactive M-CSF in a P-dependent manner in vitro, indicating that the M-CSF reported to be present in human endometrium is secreted in part by stromal cells and may play a role in the regulation of endometrial function under P control.

Androgens induce prolactin production by human endometrial stromal cells in vitro.

Although there is a significant quantity of androgens in the endometrium, the function of these hormones has not been clarified, except for being estrogen precursors. Human endometrial stromal cells (ESC) were cultured in the presence of testosterone (T) and 5 alpha-dihydrotestosterone. Following culture, prolactin (PRL), a biochemical marker of stromal cell differentiation (decidualization) which is produced by ESC, was examined. T induced PRL production in a time- and dose-dependent manner, as reported previously for progesterone (P) stimulation. In addition, 5 alpha-dihydrotestosterone, which cannot be converted to estrogens, similarly induced PRL production. T in combination with P enhanced PRL production in cultured ESC significantly more than either P or T stimulation alone. A specific androgen receptor blocker, flutamide, when added to cultures containing T, inhibited PRL production in a dose-dependent manner, but did not affect the production of PRL induced by P. These results indicate that in vitro PRL production by human ESC is induced not only by P, but also by androgens through specific receptors and further suggest that androgens play an important role in human endometrial differentiation.

[Conditions for low-intensity voluntary wheel running in rats and its chronic effects on health indexes].

Studies were undertaken to evaluate the fundamental conditions for a low-intensity voluntary wheel running model in rats and its chronic effects on health indexes. Male Fischer rats (SPF) 5 weeks of age were housed in individual sedentary conditions or in individual wheel-cage units which allowed free access to voluntary wheel running for 8 months. Voluntary running averaged 640 +/- 198 m/day, reached a peak (965m) at the 2nd month and waned over time, reaching a plateau after the 6th month (about 400-500m). Exercising rats consumed more food (+23%), but exhibited decreased body weight gains (-9%), suggesting a remarkable lowering of fat. A lowering effect on resting blood pressure (-5%) was also recognized. In addition, preventive effects on oxygen toxicity and effective bactericidal activity of neutrophils and pulmonary alveolar macrophages (PAM) were suggested. Although the amount of exercise in this study was the smallest of the other preceding ones conducted with a voluntary wheel running model, many potential health benefits were recognized. Such health promoting and protective effects by low-intensity voluntary exercise and the harmfulness of forced exercise in rats have been reported in researches on cancer, lowering fat and hypertension. Therefore it is important to set up conditions for low-intensity voluntary running. It was also demonstrated by this study that strictly controlled environmental conditions, such as room temperature and humidity, a 12-hr light-dark cycle and prevention of infection and psychological stress to rats, as well as using male rats, which are more inactive, were important factors to establish this model.

Dipeptidyl peptidase IV as a differentiation marker of the human endometrial glandular cells.

To investigate the involvement of membrane-bound peptidases in the human endometrial function, we examined the expression of dipeptidyl peptidase (DPP) IV and its enzyme activity. Immunohistological studies revealed that DPP IV was detected on human endometrial glandular cells and endometrial surface epithelium, but not on endometrial stromal cells or decidual cells in the first trimester of pregnancy. DPP IV expression on glandular cells and surface epithelium was weak in the proliferative phase, began to increase gradually in the early secretory phase, and was strong in mid-to late secretory phase and in the first trimester of pregnancy. DPP IV enzyme activity was detected histochemically in glandular cells and surface epithelium in the mid-secretory phase, and became stronger in the late secretory phase, but was rarely detected in the proliferative phase and early secretory phase. During the first trimester of pregnancy DPP IV enzyme activity in glandular cells and surface epithelium was slightly weaker than in the late secretory phase. Endometrial stromal cells and decidual cells, however, had no detectable DPP IV enzyme activity at any time throughout the menstrual cycle or during the first trimester of pregnancy. These findings indicate that DPP IV is a differentiation marker for glandular cells and surface epithelium and that active DPP IV is present in both areas during the peri-implantation period and thereafter.

Expression of aminopeptidase N and neutral endopeptidase on the endometrial stromal cells in endometriosis and adenomyosis.

Indirect immunofluorescence staining revealed that endometrial stromal cells (ESC) in the ectopic endometrium of patients with endometriosis or adenomyosis expressed aminopeptidase N/cluster of differentiation (CD) 13 antigen and neutral endopeptidase/CD10 antigen, both of which are expressed on ESC in the normal endometrium throughout the menstrual cycle. Thus, ESC in the ectopic endometrium resembled ESC in the normal endometrium not only morphologically but also antigenically. Both peptidase antigens may be useful markers for the histological diagnosis of endometriosis and adenomyosis.

Suppression of natural killer cell activity by sera from patients with endometriosis.

OBJECTIVE: We determined the effect of sera from patients who have endometriosis on natural killer cell activity. STUDY DESIGN: The natural killer cell activity of lymphocytes from healthy volunteers was examined after incubation with sera from patients who had endometriosis or from controls, with K562 cells used as targets. RESULTS: Lymphocytes treated with sera from patients who had endometriosis expressed significantly lower levels of cytotoxicity compared with lymphocytes treated with control sera. This suppression of cytotoxicity was dose dependent, and the degree of suppression was proportional to the incubation time of the effector cells with the sera. Decreased cytotoxicity after serum treatment was also observed with sera from patients who had been treated with danazol. CONCLUSIONS: These findings show that humoral factors that can inhibit natural killer cell activity in vitro are present in the peripheral blood of patients who have endometriosis; moreover, they suggest that the suppressed natural killer cell activity may allow the development of endometrial cells at ectopic sites.

Is there any effect of volcanic eruptions of Mount Sakurajima on canine lungs exposed naturally?--Morphometric analysis of intrapulmonary particulate deposit amount and histopathological investigations.

In order to see whether any effect of inhalation of volcanic ash and gases from Mt. Sakurajima on canine lungs is observed or not, we examined the amount of intrapulmonary particulate deposits (IPD) and histopathological changes. Twenty-five abandoned or stray dogs (group A) in the areas affected enormously by volcanic ash and gases were examined in comparison with 13 abandoned or stray dogs (group B) in the area scarcely influenced. The amount of IPD was measured by using an image analyzer combined with a microscope. Age-associated increase of IPD values was noted, but mean IPD values were not different between groups A and B. Incidence of goblet cell hyperplasia was not different between the two groups. In none of the cases examined, squamous metaplasia of respiratory epithelia, pulmonary fibrosis, silicotic nodules, emphysematous change, or histopathological findings, which are indicative of bronchial asthma, were observed. In conclusion, obvious effect of volcanic eruption on canine lungs was not observed through both the measurement of IPD value and the histopathological evaluation.

The expression and localization of mRNA for colony-stimulating factor (CSF)-1 in human term placenta.

A 4-kb mRNA for colony-stimulating factor 1 (CSF-1) was detected in normal human placenta at term by Northern blot analysis. In-situ hybridization revealed that the mRNA for CSF-1 was localized in the mesenchymal cells of the chorionic villous stroma, but not in the trophoblasts or capillary epithelial cells. Because there are significant numbers of tissue macrophages (Hofbauer cells) in the placental stroma and because the receptor for CSF-1 (the c-fms proto-oncogene product) is known to be expressed by trophoblasts, our results suggest that CSF-1 produced by placental stromal cells may act as a growth and survival factor for human placental macrophages and trophoblasts.

Human endometrial stromal cells and decidual cells express cluster of differentiation (CD) 13 antigen/aminopeptidase N and CD10 antigen/neutral endopeptidase.

With specific monoclonal antibodies, we found that human endometrial stromal cells and decidual cells express two function-related surface antigens. Indirect immunofluorescence staining revealed that both endometrial stromal cells and decidual cells during the first trimester of pregnancy expressed cluster of differentiation (CD) 13 antigen and CD10 antigen, which are identical to aminopeptidase N and neutral endopeptidase, respectively. By flow cytometric analysis, CD13 antigen was detected on 82-93% of the examined cells, and CD10 antigen was detected on 75-93% of the examined cells in endometrial stromal cell-enriched preparations. Furthermore, peptidase activity was detected in these cell preparations by an assay based on the hydrolysis of alanine-p-nitroanilide into p-nitroaniline and alanine.

Is there any effect of volcanic eruptions of Mount Sakurajima on human lungs?--histopathological investigation and measurement of intrapulmonary particulate deposits amounts.

In order to investigate effects of volcanic eruptions of Mt. Sakurajima on human lungs, an amount of intrapulmonary particulate deposits (IPD) and histopathological changes were evaluated, using autopsied lungs of deceased residents of Kagoshima (the area affected most by the volcanic eruptions of Mt. Sakurajima, n = 66) in comparison with those of Saitama (n = 73). The amount of IPD was measured by macroscopic image analysis and by alkaline extraction. Correlation was seen in the IPD values obtained by these two methods (Spearman's correlation coefficient: 0.525). The IPD value measured through chemical digestion, which increased with age, was lower in the residents of Kagoshima (17.0 +/- 6.3 mg/g) than in those of Saitama (26.8 +/- 10.6 mg/g). This difference of IPD value between Kagoshima and Saitama residents was also observed in male, female, smoker and nonsmoker subgroups. The incidence of pulmonary emphysema was not different between Kagoshima and Saitama cases. No silicotic nodules were observed in any cases. But, the incidence of bronchial goblet cell hyperplasia and squamous metaplasia in Kagoshima was higher than that in Saitama in male cases and smoker cases. In Kagoshima cases, the incidence of squamous metaplasia was significantly higher in smokers than in nonsmokers. The synergistic influence of volcanic eruptions and cigarette smoking on human airways was suggested.