Spreds, inhibitors of the Ras/ERK signal transduction, are dysregulated in human hepatocellular carcinoma and linked to the malignant phenotype of tumors.

Aberrant activation of the Ras/Raf-1/extracellular-regulated kinase (ERK) pathway has been shown to be involved in the progression of human hepatocellular carcinoma (HCC). However, the mechanism of dysregulation of ERK activation is poorly understood. Recently, we identified Sprouty-related protein with Ena/vasodilator-stimulated phosphoprotein homology-1 domain (Spred) as a physiological inhibitor of the Ras/Raf-1/ERK pathway. In this study, we found that the expression levels of Spred-1 and -2 in human HCC tissue were frequently decreased, comparing with those in adjacent non-tumorous tissue. Moreover, Spred expression levels in HCC tissue were inversely correlated with the incidence of tumor invasion and metastasis. Forced expression of Spred-1 inhibited HCC cell proliferation in vitro and in vivo, which was associated with reduced ERK activation. Spred-1 overexpression also reduced the secretion of matrix metalloproteinase-9 (MMP-9) and MMP-2, which play important roles in tumor invasion and metastasis. In addition, Spred-1 inhibited growth factor-mediated HCC cell motility. These data indicate that the reduction of Spred expression in HCC is one of the causes of the acquisition of malignant features. Thus, Spred could be not only a novel prognostic factor but also a new therapeutic target for human HCC.

Regulation by human chorionic gonadotropin of protein kinases in rabbit endometrium.

The effect of human chorionic gonadotropin (CG) on protein kinase levels has been studied in rabbit endometrium. A good correlation was observed between alterations of total protein kinase activity and DNA content in the tissue. The level of type I cyclic AMP-dependent protein kinase had slightly increased by 6 h after human CG treatment, and then decreased later. In contrast, the type II enzyme gradually increased after 3 days. The increase of the activity at 7 days was mainly due to that of type II enzyme, and the ratio of type II to type I enzyme increased. The activity pattern at the later stage closely resembled that of treatment with progesterone.

Purification and characterization of myosin light-chain kinase from porcine myometrium and its phosphorylation and modulation by cyclic AMP-dependent protein kinase.

Myosin light-chain kinase was purified from porcine myometrium to apparent homogeneity at about 262-fold with an Mr of 130 000 as determined by SDS-polyacrylamide gel electrophoresis and a sedimentation coefficient of 4.5 S. The approximate content of the soluble myosin light-chain kinase was estimated to be about 0.85 microM. The purified enzyme exhibited strict substrate specificity only for 20-kDa myosin light chain and Ka values of 0.6 nM and 0.3 microM for calmodulin and Ca2+, respectively. The enzyme was phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase, which resulted in a decrease in the affinity for calmodulin of 4-7-fold without effect on the Vmax. The maximal amount of phosphate incorporated into the enzyme was 0.5-0.8 and 1.0-1.4 mol per mol of the enzyme in the presence and absence of Ca2+ and calmodulin, respectively. In the presence of a subsaturating concentration of calmodulin, the enzyme showed a lower sensitivity for Ca2+ by phosphorylation.

Steroidogenesis in cerebral metastatic chorionepithelioma tissue in vitro.

Homogenates of cerebral metastatic chorionepithelioma tissue were incubated with labelled dehydroepiandrosterone, pregnenolone or 20alpha-hydroxypregn-4-en-3-one. The metabolites of dehydroepiandrosterone which were isolated and identified were androstenedione, testosterone, oestrone, and oestradiol; no oestriol was detected. The only metabolite of pregnenolone and 20alpha-hydroxypregn-4-en-3-one isolated and identified was progesterone. No conversion of C-21 to C-19 steroids occurred in the metastatic chorionepithelioma tissue.

Effect of intravenous or intra-amniotic injection of dehydroepiandrosterone sulphate on oestrogen levels in urine and serum in late pregnancy with live anencephalic foetuses.

Dehydroepiandrosterone sulphate (DHAS) was injected intravenously or intra-amniotically into eight volunteers carrying live anencephalic foetuses (including one microcephalic foetus). Urinary and unconjugated serum oestrone, oestradiol and oestriol were measured before and after DHAS administration. In seven pregnant women with live anencephalic foetuses the urinary excretion of oestriol was very low, and the ratio of oestriol to oestrone + oestradiol was much less than that during normal pregnancy. Increases of urinary oestrone and oestradiol but no significant change in the ratio of oestriol to oestrone + oestradiol were observed 24 h after i.v. administration of DHAS to five patients. In three patients, between 1 and 12 h after i.v. administration of DHAS (100-200 mg), the concentrations of serum oestrone, oestradiol and oestriol increased to 13-5, 6-8 and 3-1 times the control values, respectively. After injection of DHAS (200 mg) intra-amniotically into two patients, the urinary excretion of all three oestrogens increased much more on day 2 than on day 1, and the ratio of urinary oestriol to oestrone + oestradiol rose greatly. On the other hand, the concentrations of unconjugated serum oestrogens in these patients increased progressively between 1 and 12 h or more after DHAS administration, and the maximal level of serum oestriol was 9-8 times the control value while those of oestrone and oestradiol were 4-6 times and 5-0 times the control values, respectively. These results suggest that in late human pregnancy DHAS in the circulation of the mother is converted to oestriol largely via the phenolic pathway (DHAS leads to oestrone leads to oestriol), whereas DHAS circulating within the foeto-placental compartment is converted to oestriol via both the phenolic and the neutral intermediates.

Plasma levels of human chorionic somatomammotrophin, progesterone, unconjugated oestradiol and oestriol, and alpha-foetoprotein in patients with hydatidiform moles.

Human chorionic somatomammotrophin (HCS), progesterone, and unconjugated oestradiol and oestriol were measured in the plasma of 13 patients with intact hydatidiform moles from week 9 to 19 of pregnancy and in 89 normal women from week 5 to 20 of pregnancy. Plasma alpha-foetoprotein (AFP) was also measured in 9 out of 13 patients and in 23 of the normal women from week 13 to 20 of pregnancy. All the compounds were measured by radioimmunoassay. The plasma HCS concentration in 35 samples from 13 patients with hydatidiform moles ranged from 10 to 910 ng/ml; this was lower than that in normal pregnancy of corresponding duration in eight patients; within the normal range in four patients and high in one patient. The plasma progesterone concentration ranged from 17-5 to 79-2 ng/ml; it was higher than that in normal pregnancy in eight patients and within the normal range in five patients. The plasma unconjugated oestradiol concentration ranged from 1-82 to 8-10 ng/ml; it was higher than normal in six patients and within the normal range in seven patients. The plasma unconjugated oestriol concentration ranged from 0-168 to 1-37 ng/ml, the levels at 15-19 weeks of gestation being significantly lower than those in normal pregnancy at this time (P less than 0-005). Plasma AFP was not detectable in the nine patients (less than 10 ng/ml) whereas it ranged from 10 to 80 ng/ml in 18 out of 23 women in week 13-20 of normal pregnancy. The present results suggest that both plasma oestriol and AFP could be helpful in the diagnosis of hydatidiform mole at about 12-14 weeks though diagnosis could not be made with absolute certainty.

Hormone treatments and pregnancy alter myosin light chain kinase and calmodulin levels in rabbit myometrium.

Myosin light chain kinase activity and the amount of calmodulin were measured in rabbit myometrium after the rabbits had been treated with oestrogen, progesterone and human chorionic gonadotrophin, and also during pregnancy. Injections of oestrogen and progesterone produced significant increases of of 2.8- and 2.1-fold in the enzyme activity/mg protein and of 1.4- and 1.2-fold in the calmodulin content/mg protein respectively. The increases seen with oestrogen treatment were inhibited by cycloheximide administration, suggesting that protein synthesis is involved in the process. Calmodulin increased about twofold in the particulate fraction under conditions of oestrogen and progesterone treatments and the raised level of calmodulin detected in the cytosol fraction was not due to translocation from the particulate fraction. During pregnancy the specific enzyme activity significantly increased in comparison with the values seen in samples from the non-treated rabbits. These findings suggest that myosin light chain kinase and calmodulin in the myometrium are under hormonal regulation and may play a role in expulsion of the fetus at the latter phase of pregnancy.

Growth stimulation by androgens, glucocorticoids or fibroblast growth factors and the blocking of the stimulated growth by antibody against basic fibroblast growth factor in protein-free culture of Shionogi carcinoma 115 cells.

Shionogi carcinoma 115 (SC115) has been accepted for 20 years as an androgen-responsive mouse mammary tumor. We have established an androgen-dependent cloned cell line (SC-3) from a SC115 tumor. In a serum-free medium, testosterone (T) or fibroblast growth factors (FGFs) markedly stimulate the growth of SC-3 cells, and the T-induced growth was shown to be mediated through FGF-like peptide(s) in an autocrine mechanism. Since we used the serum-free culture including 0.1% bovine serum albumin (BSA), a partially serum-containing condition, putative roles of BSA- or serum-borne growth factors in growth stimulation of autocrine production of FGF-like peptide(s) could not be excluded. This paper reports findings performed in a protein-free medium including plating [Ham's F-12:MEM (1:1; v/v)]. In the protein-free culture, the growth of SC-3 cells was significantly stimulated by the addition of greater than or equal to 10(-10) M T (up to 20-fold), greater than or equal to 10(-7) M dexamethasone (Dex; up to 7-fold) or greater than or equal to 1 ng/ml basic (b) or acidic FGF (up to 10-fold); other various growth factors had no such effects. Furthermore, DNA synthesis of SC-3 cells induced by T, Dex or bFGF was similarly and markedly inhibited by bFGF neutralizing antibody IgG. Therefore, the present findings seem to demonstrate that androgens or high levels of glucocorticoids induce the production and secretion of FGF-like peptide(s) from SC-3 cells for their growth even in the absence of additional support by other factors.

Eccrine adenocarcinoma of the vulva producing isolated alpha-subunit of glycoprotein hormones.

A rare case of adenocarcinoma of the sweat glands of the vulva producing isolated alpha-subunit of glycoprotein hormones is reported. By an immunohistochemical method, eccrine gland adenocarcinoma tissues obtained from the right vulva and from its metastatic lesion in the right subclavicular lymph nodes were found to react with human chorionic gonadotropin (hCG), alpha-hCG but not beta-hCG. The concentrations of alpha-subunit in the serum and urine were markedly elevated to 6000 ng/mL and 55,000 ng/mL, respectively, just before the death of the patient.

Digital subtraction angiography for the evaluation of persistent uterine trophoblastic disease.

In three patients with persistent uterine trophoblastic disease, follow-up after primary chemotherapy was undertaken by digital subtraction angiography. It was found that the angiographic appearance of the intramural uterine lesions was comparable with that obtained by conventional pelvic angiography. Digital subtraction angiography may be applicable as an easy, safe, and valuable diagnostic method for gestational trophoblastic disease in place of conventional pelvic angiography because it is a relatively noninvasive procedure that can be performed on outpatients.

Endocrine profile of an ovarian androblastoma.

The endocrine profile of a 16-year-old girl with an androblastoma of the left ovary is presented. Calculated ratios of steroid hormones between the intraoperative peripheral vein blood, the left ovarian vein blood, and the left ovarian tumor fluid were progesterone, 1:10.2:39.5; 17-hydroxyprogesterone, 1:18.7:64.7; dehydroepiandrosterone (DHEA), 1:10.4:35.6; androstenedione (A), 1:24.4:92.3; testosterone (T), 1:18.6:69.2; and estradiol (E2), 1:11.0:26.3. The peripheral levels of hormones before left salpingo-oophorectomy were T, 7.5 ng/ml; DHEA, 19.9 ng/ml; A, 12.3 ng/ml; and cortisol, 11.4 micrograms/dl. Corresponding levels at 14 days after surgery were (0.75 ng/ml; 5.84 ng/ml; 1.94 ng/ml; and 15.6 micrograms/dl, respectively. Preoperatively, an elevated basal level of luteinizing hormone (LH) and a normal basal level of follice-stimulating hormone (FSH) (high LH:FSH ratio) were found. These data suggest that 1) androgens from the androblastoma are responsible for virilization despite aromatizing enzyme activities within normal limits, and 2) both the delta 5 and delta 4 pathways are involved in the biosynthesis of androgens, with that of delta 5 being predominant.

In vivo and in vitro studies on the production of placental proteins (human chorionic gonadotropin, human placental lactogen, and pregnancy-specific beta 1-glycoprotein) in an adrenal choriocarcinoma.

The ectopic production of placental proteins (human chorionic gonadotropin [hCG], human placental lactogen, and pregnancy-specific beta 1-glycoprotein) by an adrenal choriocarcinoma was investigated experimentally in vivo and in vitro. By an immunohistochemical method, the choriocarcinoma tissues obtained from the right adrenalectomy were found to react with hCG, human placental lactogen, and pregnancy-specific beta 1-glycoprotein antibodies. The concentrations of hCG-beta, human placental lactogen, and pregnancy-specific beta 1-glycoprotein in the tumor fluid were 1480, 100, and 47 ng/mL, respectively. On incubation of the tumor slices in vitro, the concentration of hCG-beta in the incubation medium increased markedly with time. Serial sections of the removed uterus and right ovary did not reveal any primary trophoblastic lesions. The present tumor responded well to double chemotherapy with actinomycin D and methotrexate, resulting in a decrease of the level of serum hCG-beta to less than 10 ng/mL after four courses of the chemotherapy.

Significance of angiotensin sensitivity test for prediction of pregnancy-induced hypertension.

In 48 normotensive women with epidemiologic high-risk factors for pregnancy-induced hypertension, the angiotensin sensitivity test was performed serially between 26 and 32 weeks of gestation. If an effective pressor dose of less than 12 ng/kg per minute was considered to be a positive test result, 20 subjects were positive at 30 weeks of gestation and destined to develop pregnancy-induced hypertension. Of ten subjects with a false-positive test result, seven patients developed proteinuria and/or clinically significant edema without apparent hypertension, and only three subjects remained normal throughout their pregnancy. Twenty-eight subjects with negative test results had uneventful pregnancies. Before 30 weeks' gestation, it was difficult to identify all patients destined for pregnancy-induced hypertension. Although high false-positive test results were detected, these results suggest that an angiotensin sensitivity test at 30 weeks' gestation represents an appropriate means of identifying women who remain normal throughout pregnancy. Careful follow-up should be undertaken in all patients with positive test results.

The effects of A23187 and phorbol ester on the phosphorylation of proteins in rat anterior pituitary cells.

The effects of A23187 and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on the phosphorylation of proteins in normal rat anterior pituitary cells were compared. A23187 rapidly activated the phosphorylation of at least 5 proteins (45, 47, 53, 54 and 58 kDa), which reached the maximal level in 2-10 min, and decreased gradually thereafter. In contrast, TPA activated the phosphorylation of at least 6 proteins (45, 62, 64, 72, 76 and 82 kDa), which were mostly distinguished from those activated by A23187. TPA-induced response was elicited more slowly, reached a plateau after about 10 min, and was sustained thereafter. These results suggest that the protein phosphorylation stimulated by A23187 and TPA is conducted by different mechanisms.

Action of tamoxifen on folliculogenesis in the menstrual cycle of infertile patients.

Daily estimations of follicle-stimulating hormone, luteinizing hormone, prolactin, estradiol, and progesterone were made in the serum of eight infertile patients from day 1 through the follicular phase during menstrual cycles before and after tamoxifen therapy. Tamoxifen therapy was found to shorten the follicular phase from 15.4 +/- 0.8 days (mean +/- standard error of the mean) to 14.0 +/- 0.6 days (difference not significant) and to lengthen the luteal phase from 12.8 +/- 0.4 days to 14.1 +/- 0.8 days (P less than 0.05). The mean estradiol concentration in the eight patients during tamoxifen treatment cycles rose on day 8 (3 days after starting tamoxifen treatment) and increased significantly (P less than 0.05) from day 10 to midcycle. The integrated follicular phase estradiol concentration in the tamoxifen treatment cycle increased to 2450.1 +/- 208.1 pg/ml/cycle, and was significantly higher (P less than 0.025) than that in the nontreatment cycle. In contrast, the concentrations of follicle-stimulating hormone, luteinizing hormone, and prolactin during the follicular phase and at the midcycle peak of tamoxifen treatment cycles were not significantly different from those of the nontreatment cycle. These results suggest that the mechanism of tamoxifen in improving folliculogenesis may involve a direct action on the ovary without intervention of the hypothalamic-pituitary system.

Glycogen metabolism in vesicles of hydatidiform mole in vitro.

Glycogen content, glycogen synthetase, and glycogen phosphorylase were studied in placental tissue of normal pregnancy and in vesicles of hydatidiform mole. The glycogen content of placental tissue of normal pregnancy decreased significantly with increased gestational age: 6 to 10 weeks, 716.6 +/- 55.7 mg/100 gm wet weight (mean +/- standard error of the mean); 15 to 20 weeks, 216.1 +/- 11.2 mg/100 gm; and 37 to 41 weeks, 176.1 +/- 18.1 mg/100 gm. The decrease in placental glycogen content was accompanied by a corresponding decrease in the placental glycogen synthetase enzyme levels, whereas no remarkable change was found in the glycogen phosphorylase enzyme levels. The glycogen content of hydatidiform mole tissue from 10 patients (13 to 20 weeks of gestation) was 507.0 +/- 58.0 mg/100 gm and was significantly (P less than 0.005) higher than that of normal placental tissue with a corresponding period of gestation. A possible cause of this phenomenon may be the marked decrease in the glycogen phosphorylase enzyme level in hydatidiform mole tissue, which was about one-third that of the normal placental tissue.

Tamoxifen in the treatment of infertility associated with luteal phase deficiency.

A group of 17 patients with suspected luteal phase deficiency was treated with tamoxifen. Tamoxifen therapy was found to lengthen the luteal phase in all patients and resulted in pregnancy in 6 of 17 patients. The integrated luteal phase progesterone (P) concentration in the nontreatment cycle of seven patients was significantly lower (P less than 0.01) than that of five normal women. Therapy with tamoxifen increased the P concentration to 186.0 +/- 24.4 ng/ml/cycle (mean +/- standard error of the mean), i.e., twice that of the control cycle. The mean estradiol (E2) concentration at the midcycle peak was about twice that observed during the nontreatment cycle. The glycogen content of the endometrial tissue at the midluteal phase in the tamoxifen cycle was significantly higher (P less than 0.025) than that of endometrial tissue in the nontreatment cycle, indicating improvement of the endometrial function.

A graduated regimen of clomiphene citrate: its correlation to glycogen content of the endometrium and serum levels of estradiol and progesterone in infertile patients at the midluteal phase.

The glycogen content, glycogen synthetase level, and glycogen phosphorylase level were studied in endometrial samples obtained from 14 infertile patients during the midluteal phase before and after clomiphene citrate (Clomid, Shionogi & Company, Ltd., Osaka, Japan) treatment, simultaneously with measurement of the serum concentrations of estradiol and progesterone. Increase in the endometrial glycogen content in the clomiphene cycle was accompanied by a corresponding increase of the dosage of clomiphene. Also, the midluteal concentrations of estradiol and progesterone in the clomiphene cycle were significantly higher (P less than 0.005 and P less than 0.005, respectively) than those in the nontreatment cycle. Clomiphene therapy at 50 mg/day resulted in pregnancy in three of ten patients, while clomiphene at 100 mg/day resulted in pregnancy in three of six patients. These results suggest a fair correlation between the dosage of clomiphene and the improvement of endometrial function in infertile patients following stimulated ovarian steroidogenesis.

Effect of progestogen on glycogen metabolism in the endometrium of infertile patients during the menstrual cycle.

The glycogen content and glycogen synthetase and glycogen phosphorylase levels were studied in endometrial samples obtained from 19 normal and 37 infertile patients during the menstrual cycle before and after administration of progestogen. Each of the above groups received the progestogen Lyndiol (lynestrenol, 5.0 mg, and mestranol, 0.15 mg) daily for 7 days during the follicular phase of the menstrual cycle. In both groups an increase in the endometrial glycogen deposition and an increase in glycogen synthetase enzyme levels were seen. When the administration of progestogen was started on day 7 after ovulation, during the luteal phase, the glycogen content of endometrial tissue from infertile patients increased significantly; no change was found in endometrial samples from normal patients. No difference was found in the serum progesterone levels of normal and infertile patients in the midsecretory phase of menstrual cycle, and Lyndiol reduced the serum level of progesterone to approximately that found during the follicular phase in untreated normal women. These studies suggest that the proliferative endometrium of infertile patients may be less stimulated by ovarian estrogen than is normal endometrium, whereas endometrial tissue obtained from both groups during the luteal phase responded similarly to progesterone in glycogen synthesis and storage.

Glycogen estimation by a rapid enzymic method in very small samples of human endometrium: glycogen content in the endometrium of infertile patients during the menstrual cycle.

An enzymic method using alpha-glucosidases was adapted for measuring glycogen in very small samples (3 mg) of human endometrium. The method is useful as a clinical test of the physiologic function of human endometrium. The glycogen content of the endometrium of normal and infertile patients was measured during the menstrual cycle. The maximal content in both groups was observed between the 16th and 23rd days of the cycle, but the glycogen content of the infertile group was significantly lower (P less than 0.005). These results confirm the reports of others. Endometrial glycogen and urinary pregnanediol levels in 32 infertile patients were measured on day 7 after ovulation. The glycogen content of the endometrium of 21 of these patients, who showed normal excretion of urinary pregnanediol (greater than or equal to 2 mg/day), was significantly higher than that of the other 11 patients who showed low excretion of urinary pregnanediol (less than 2 mg/day) (P less than 0.005). This finding suggests that there is a high correlation between the function of the corpus luteum and endometrial glycogen deposition.