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Biomedical subjects

C F Holinka

Publications and source records attributed to C F Holinka.

At least 37 records · Page 2Linked to original sources
PubMed1986

Progesterone receptors in two groups of endometrial carcinoma.

A previous study evaluated two types of endometrial carcinoma: one with, the other without, associated adenomatous hyperplasia (Groups 1 and 2, respectively). On the basis of histologic observations, the study concluded that Group 1 tumors were likely to be estrogen-dependent, whereas Group 2 tumors appeared to represent a hormone-independent type of cancer. The authors present quantitative biochemical data to support the proposition that Group 1 tumors are likely to be under the influence of estrogen. Progesterone receptor levels, known to be increased by estradiol, were significantly higher in Group 1 than in Group 2. Analysis of histologic variants revealed that some histologic components, such as mucinous adenocarcinoma, squamous components, and stromal foam cells, predominated in Group 1 and may, therefore, be indicative of estrogen action. Certain histologic features, however, such as papillary carcinoma, clear cells, and anaplastic carcinoma with giant tumor cells, were found exclusively in Group 2, thus suggesting a lack of estrogenic influence. These findings indicate that Group 1 and Group 2 endometrial carcinomas may constitute two different biologic entities and, thus, encourage further basic and clinical research to develop specific therapies for patients diagnosed to have endometrial cancer.

Aged↗
PubMed1986

Responses to estradiol in a human endometrial adenocarcinoma cell line (Ishikawa).

A human endometrial adenocarcinoma cell line (Ishikawa) has been found to be estrogen responsive. The growth stimulatory effects of estradiol (10(-8) M) could be clearly demonstrated when cell cultures containing the hormone were compared with the maximal cell density achieved in control cultures. The approx. 3-fold increase in cell density observed 2-3 weeks after plating, with frequent medium changes, could by blocked by a 100-fold molar excess of the antiestrogen trans-4-monohydroxytamoxifen. When added to hormone-free cultures that had reached a plateau level of cell numbers on day 14 after plating, estradiol (10(-8) M) caused the resumption of proliferation: after 6 days in the presence of the hormone, the cultures contained nearly twice the cell numbers of controls. Effects of estradiol on Ishikawa cells were also evident from the several-fold increases in the levels of specific progesterone binders provoked by the hormone at 10(-9)-10(-6) M concentrations. Cells injected into nude mice formed tumors which contained estrogen and progesterone binders. The availability of a fast-growing (doubling time approx. 30 h) endometrial cancer cell line responsive to estradiol at near physiologic levels will facilitate biochemical studies of hormonal effects on the human endometrium.

Adenocarcinoma↗
PubMed1986

Effects of estradiol on proliferation of endometrial adenocarcinoma cells (Ishikawa line).

Estradiol (E2) stimulates the proliferation of human endometrial adenocarcinoma cells of the Ishikawa line, which had been previously shown to respond to estrogen by increasing their levels of progesterone receptor and the specific activities of DNA polymerase alpha and alkaline phosphatase. Although E2 (10(-8) M) did not increase rates of proliferation during the initial logarithmic growth period of the cultures under the chosen experimental conditions (MEM with 15% charcoal-treated fetal bovine serum renewed every 2-3 days), it sustained cell proliferation after about day 10, when parallel control cultures had reached plateau cell densities. Cell proliferation in control cultures at plateau levels was resumed when the hormone was added. Growth rates of cultures containing E2 from the time of seeding and the proportion of quiescent cells, estimated by using a simple cell kinetic model, decreased steadily with time. Ornithine decarboxylase and DNA polymerase alpha activities, as well as estrogen receptor levels, also decreased with time in culture. Ishikawa cells formed colonies in soft agar; colony formation efficiencies were higher as the number of cells seeded was increased from 10,000 to 100,000 cells/6 cm dish, were not influenced by the addition of E2 to the medium (10(-9) to 10(-5) M) and were markedly reduced by difluoromethylornithine (10(-2) M), an effect that was counteracted by putrescine (25 X 10(-6) M).

Adenocarcinoma↗
PubMed1986

Effects of steroid hormones and antisteroids on alkaline phosphatase activity in human endometrial cancer cells (Ishikawa line).

Alkaline phosphatase activity in human endometrial cancer cells of the estrogen-responsive Ishikawa line was markedly stimulated (3-20-fold in 4 days) by estrogens, 5 alpha-dihydrotestosterone, and dehydroepiandrosterone but not by testosterone, medroxyprogesterone acetate, glucocorticoids, several peptide hormones, prostaglandins, or growth factors. Maximum responses to estradiol were obtained at concentrations between 10(-9) and 10(-7) M; at 10(-8) M estradiol, the highest activity was reached 48-72 h after addition of the hormone. A linear relationship between enzyme activity at 48 h and the length of exposure to the hormone was observed. Dibutyryl cyclic guanosine 3':5'-monophosphate, but not dibutyryl cyclic adenosine 3':5'-monophosphate enhanced alkaline phosphatase activity and acted synergistically with estradiol. trans-4-Monohydroxytamoxifen completely antagonized the stimulatory effect of estradiol and had no agonistic activity. Dihydrotestosterone and dehydroepiandrosterone appear to exert their effects, at least in part, by interacting with estrogen receptors, since the simultaneous presence in the medium of monohydroxytamoxifen abolished their influence on alkaline phosphatase activity. The specific antiandrogen monohydroxyflutamide partially antagonized the effect of these hormones, suggesting that their action involved androgenic mechanisms as well. Exposure to elevated temperature and to specific inhibitors identified alkaline phosphatase of Ishikawa cells as a placental-type isoenzyme, thus contrasting with the nonplacental type found in glandular epithelial cells of normal endometrium and in another human endometrial cancer cell line, HEC-50. This study extends our previous observations of estrogen responsiveness in the Ishikawa cell line. In addition to the previously reported stimulatory effects on growth and progesterone receptor levels, we are now describing the stimulation by estrogens and C19 steroids of an enzyme, alkaline phosphatase, which can be used as a convenient end point to examine mechanisms of hormonal action.

Alkaline Phosphatase↗
PubMed1985

Ornithine decarboxylase activity in human endometrium and endometrial cancer cells.

Ornithine decarboxylase (ODC) activities were significantly higher in proliferative endometrium during the estrogen-dominated follicular phase of the menstrual cycle than in secretory endometrium after the formation of the progesterone-secreting corpus luteum. The enzymatic activity was increased about fivefold by renewal of the medium during incubations of endometrial fragments or isolated endometrial glands. Endometrial adenocarcinoma cells (HEC-1, HEC-50), both in monolayers and suspension, also responded to medium renewal by increasing ODC activity about 10-fold after 4 h, with subsequent reduction to control levels after 7 h. These effects were blocked by actinomycin D and cycloheximide. Endometrial stromal cells exhibited highly variable ODC activities at different passages. Difluoromethylornithine (DFMO) and sodium molybdate had marked antiproliferative effects in HEC-50 cultures, reducing cell numbers to 10 to 20% of control values 11 d after plating and inhibiting ODC activity by approximately 80% on Day 7. The antiproliferative effect of DFMO, but not that of molybdate, was reversed by 10 microM putrescine, the product of ODC activity. In contrast to DFMO, molybdate had no effect on ODC activity of cell homogenates. Molybdate did not elicit antizyme formation in HEC-50 cells under conditions in which putrescine did. These results indicate that ODC activity, present in both epithelial and stromal cells, as shown analytically and also by autoradiography after labeling with [3H]DFMO, may be related to cell proliferation in vivo and that proliferation of human endometrial cancer cells in culture can be arrested by DFMO and by molybdate.

Adenocarcinoma↗
PubMed1984

Diamine oxidase activity in human decidua and endometrium.

The activity of diamine oxidase, the enzyme that catalyzes the oxidative deamination of putrescine, spermidine, histamine, and other substances involved in the regulation of cell proliferation and immune responses, increases up to 1000-fold in blood of pregnant women. In vivo experiments have shown that progestins stimulate diamine oxidase in the rodent uterus. We examined diamine oxidase activity in human endometrium at different phases of the menstrual cycle and in decidua from first-trimester pregnancies. Enzyme activity was also assayed in tissues and media of cultured human endometrium and decidua in the presence of estradiol or medroxyprogesterone acetate. Enzyme activity in first-trimester decidual tissues (286 +/- 86 mU of diamine oxidase per milligram of DNA; mean +/- SEM) was considerably higher (p less than 0.001) than that in endometrium (2.6 +/- 1.6), and the activity increased from 6 to 17 weeks of pregnancy. No significant differences were detected in proliferative and secretory endometrium. Activities in media of endometrium in organ culture generally were below detectability. Decidua secreted large amounts of the enzyme into the medium (160 to 600 mU per milligram of DNA per day), greatly exceeding the initial tissue activities. Bovine serum, when added to culture media, caused a marked increase in diamine oxidase levels, whereas estradiol (10(-8) mol/L) or medroxyprogesterone acetate (10(-6) mol/L) did not. Kinetic analysis showed a Michaelis constant of 7.4 X 10(-6) mol/L with putrescine as the substrate. Spermidine and histamine were competitive inhibitors of putrescine with an inhibition constant of 1.1 X 10(-4) and 3.5 X 10(-6) mol/L, respectively. These results demonstrate synthesis and export of diamine oxidase by decidua in organ culture, stimulation of activity by serum factors, and competition of histamine, spermidine, and putrescine for the enzyme.

Amine Oxidase (Copper-Containing)↗
PubMed1984

Histological evaluation of in vitro responses of endometrial adenocarcinoma to progestins and their relation to progesterone receptor levels.

In vitro responsiveness of endometrial adenocarcinoma to progestins was evaluated histologically by incubation of tissue fragments in medium containing 10(-6) M medroxyprogesterone acetate. In a series of 19 experiments, formation of sub- and supranuclear vacuoles, which reflects accumulation of glycogen in response to medroxyprogesterone acetate added to the medium, was observed in well-preserved glandular epithelial cells only when the level of cytosolic progesterone receptor was above 300 fmol/mg protein. Previously, we have reported similar results obtained in in vivo experiments. The present findings suggest that simple organ culture and histological procedures can be used to identify specimens of endometrial cancer that have functional progesterone receptors and are capable of responding to progestins. They also indicate that levels of progesterone receptor required to obtain responses to progestins are considerably higher than those necessary for analytical detection and that therefore the quantity and not merely the detectability of progesterone receptors must be taken into consideration for the prediction of responses to progestins. In addition, in vitro responses to progestins may indicate the presence in endometrial cancer tissue of functional estrogen receptors and potential responsiveness to antiestrogens, since estrogen stimulation appears to be needed for the synthesis of progesterone receptors.

Adenocarcinoma↗
PubMed1982

Aging and uterine growth during implantation in C57BL/6J mice.

Uterine growth during implantation was compared in C57BL/6J mice aged 3-7 mo vs. 11-12 mo. All mice had given birth to at least one previous litter. Older mice had smaller uteri during early gestation (3 to 10 days post coitum) as measured by DNA content, although wet weight and protein were generally similar in both age groups. However, there were no age effects on uterine growth during implantation, as measured by the increments of DNA, as well as protein and wet weight per implantation site (decidual swelling) on days 6-10. These data are discussed in terms of the major increase in fetal death and resorptions in older mice subsequently observed by day 12-13. Larger age-related impairment of the artificially-induced decidual response is also considered. We conclude that impairments of the artificially induced decidual response do not predict the extent of decidual responses during pregnancy in aging mice.

Aging↗
PubMed1980

Peroxidase activity in glands and stroma of human endometrium.

Peroxidase activity was measured in specimens of human endometrium taken at different days of the menstrual cycle. High specific activities were more frequently found in secretory endometrium than in proliterative tissue, but the variance at each phase of the cycle was large and differences were statistically not significant. The results obtained do not support the hypothesis that peroxidase activity is determined by the levels of circulating estrogens, as is apparently true in rats. Extremely high levels of peroxidase activity were noticed in some, but not all, specimens of endometrial adenocarcinoma. Endometrial glands were separated from the stroma after collagenase digestion of normal and abnormal endometrial glands were separated from the stroma after collagenase digestion of normal and abnormal endometrium, and peroxidase activity was measured in each fraction. The enzyme was found to be preferentially localized in the stroma, in contrast to the reported findings of predominantly epithelial localization in the rat.

Adenocarcinoma↗
PubMed1980

In vivo uptake of estrone sulfate by rabbit uterus.

The capability of rabbit uterus and liver to hydrolyze estrone sulfate (E1S) to estrone (E1) was demonstrated in vitro by superfusion of tissue slices with mixtures of [3H]-E1S and [14C]E1. However, capillary barriers or binding factors may prevent the utilization of the blood-borne estrogen sulfate. To evaluate the extent of uptake of circulating E1S by uterine cells, a mixture of [3H]E1S and [14C]E1 was injected into the abdominal aorta of anesthetized rabbits. Shortly after the injection, samples of blood, uterine tissue, and liver were simultaneously taken. Isotope ratios in E1 isolated from these samples were measured and compared. The 3H to 14C ratios in E1 isolated from the uterus and from plasma were similar. It was concluded from these results that blood-borne E1S was not taken up directly by the uterus, since, otherwise, uterine E1 would be expected to have a higher 3H to 14C ratio than plasma E1. The isotope ratios in hepatic E1 were higher than those in E1 isolated from plasma or uterus, probably as a result of direct uptake of [3H]E1S by the liver.

Animals↗