Thomas H. Finlay

The Effect of Hypoxia on Human Trophoblast in Culture: Morphology, Glucose Transport and Metabolism

The response to hypoxia of trophoblast isolated from term placenta and maintained in culture was studied. Trophoblast exposed to normoxic (PO2 120-130 mmHg) or hypoxic (PO2 12-14 mmHg) conditions were examined by electron microscopy. After 48 h, the cytoplasm of the hypoxic cells was more electron-dense with increased numbers of mitochondria, lysosomes and vacuoles. Compared to normoxic cells, the surface microvilli of the hypoxic cells were sparse, short and unevenly distributed. [3H]thymidine incorporation by both hypoxic and normoxic trophoblast fell rapidly and equivalently after 2 days in culture. The percentage of cells with the proliferation-associated nuclear antigen, Ki 67, also decreased, but remained higher in hypoxic cells suggesting that hypoxia retarded completion of the cell cycle (normoxia, 10.80 +/- 2.51 s.e.; hypoxia, 19.87 +/- 2.73, P < 0.01). Glucose consumption was elevated in hypoxia (3.73 +/- 1.07 s.e. mumol/10(6) cells/24 h) as compared to normoxia (1.46 +/- 0.83, P = 0.01). Although lactate production was consistently higher in hypoxia, the difference was not statistically significant (hypoxia 5.38 +/- 1.54 mumol/10(6) cells/24 h versus normoxia, 1.52 +/- 0.29, P = 0.07). After 48 h, uptake of [3H]2-deoxglucose ([3H]2DG) by hypoxic cells was reduced to 12 per cent +/- 4.3 s.e. of that in normoxic cells; return to normoxia resulted in recovery within 10 min. Lineweaver-Burk plots of [3H]2DG uptake indicated high affinity (KM 2.2 +/- 0.4 x 10(-4) M) and low affinity transporters (KM 4.5 +/- 1.6 x 10(-3) M). Northern blot analysis identified mRNA for GLUT1 and GLUT3. In hypoxia, steady-state GLUT1 and GLUT3 mRNA were approximately three- and 10-fold higher than in normoxia respectively. Inhibitors of oxidative metabolism of glucose increased the uptake of [3H]2DG within 2 h, whereas hypoxia reduced uptake. Hence, trophoblast in culture survives in extreme hypoxia, but manifests striking changes in morphology and in glucose metabolism and transport. Completion of cell cycle appears to be retarded.

New methods for the preparation of biospecific adsorbents and immobilized enzymes utilizing trichloro-s-triazine☆

Abstract Methods for preparing biospecific adsorbents and immobilized enzymes utilizing Sepharose CL as a support and trichloro-s-triazine as the linking agent are described. The difficulties encountered during conventional aqueous and mixed aqueous-phase reactions of trichloro-s-triazine with insoluble polyols, particularly reagent hydrolysis, are avoided by performing the activation reactions in anhydrous organic phase and replacing the second chlorine on the triazine ring by an aromatic amine. Ligands can be coupled to the activated support in either aqueous or organic phase. The methods have been applied to the attachment of a number of different enzymes, proteins, and small-molecule ligands to Sepharose. The superiority of the triazine linkage to the cyanogen bromide linkage is demonstrated.

The effect of hypoxia on term trophoblast: Hormone synthesis and release

Isolated trophoblast in culture remained viable when exposed to severe hypoxia (Po2 12-14 mmHg) for at least 72 h as indicated by trypan blue exclusion and the synthesis and secretion of metabolically labelled proteins. However, release of hCG, hPL, progesterone and estradiol was reduced to < 10 per cent when compared to trophoblast in normoxia (Po2 120-130 mmHg). hCG mRNA was also reduced demonstrating interruption of synthesis at transcription. Acute exposure to hypoxia (2 h) suppressed progesterone release but not hCG, whereas inhibitors of oxidative phosphorylation suppressed hCG release but not progesterone. hCG release increases progressively during culture in normoxia, peaking at 72 h. Exposure of trophoblast to hypoxia for 48 h after 24, 48 and 72 h in normoxia interrupted this progression but did not suppress hCG release. Progesterone release, in contrast, was reduced by hypoxia. Exogenous dibutyryl cAMP increased hCG and progesterone release by normoxic trophoblast but not by hypoxic cells. Trophoblast returned to normoxia after 24 h in hypoxia increased hCG and progesterone release, suggesting early recovery. Conservation of oxygen and ATP by reducing hormone synthesis may contribute to survival of trophoblast in hypoxia.

An estrogen-dependent esterase activity in MCF-7 cells

The presence of steroidal esters in hormonally sensitive tissues lends importance to the esterases which convert the biologically inactive adducts to the parent potent forms. Accordingly, esterase-activities were studied in a human breast cancer model--the MCF-7 cell line. Tritiated estradiol esters- estradiol-17-acetate (EA), estradiol-17-valerate (EV) and estradiol-17-stearate (ES) were tested systematically, but 3 beta-ol esters of androgens, and phorbol diesters were also investigated. All compounds tested, except the phorbol diesters were hydrolyzed either when added to growing cultures or to the 28,000 g supernate of homogenized MCF-7 cells. Among the estrogens, the relative rates of hydrolysis were EA greater than EV greater than ES. The esterase for EA was different as it was not inhibited by saturating concentrations of EV or ES, and unlike the others its activity was stimulated by the addition of estradiol to the culture medium. The antiestrogen keoxifene,[(6-Hydroxy-2-(4-hydroxyphenyl)benzo less than b greater than thien-3-yl greater than less than 4- less than 2-(1-piperidinyl)ethoxy greater than phenyl greater than methanone], negated the stimulatory effect. Other major classes of steroids did not influence EA esterase activity. Results of inhibition experiments indicated that the esterases are of the serine active-site types. The significance of the estrogen-dependent esterase activity can be assessed when the natural substrate(s) for the enzyme is elucidated.

Estradiol esters can replace 17β-estradiol in the stimulation of DNA and esterase synthesis by MCF-7 cells: A possible role for the estrogen-sensitive MCF-7 cell esterase

In this communication we extend our earlier observations on estrogen-sensitive carboxyl esterases in MCF-7 human breast cancer cells able to hydrolyze esters of estradiol. Using either estradiol acetate or p-nitrophenyl hexanoate as substrates, esterase activity was found to increase 2-3-fold in MCF-7 cells maintained in the presence of 10(-8) M estradiol. Following sucrose density centrifugation, over 85% of total esterase activity was found in the cytoplasmic fraction. No esterase activity was found in spent media from growing cells. By size exclusion chromatography, estradiol acetate esterase activity exhibited a mol. wt of 45-50 kDa. Attempts to demonstrate incorporation of [3H]estradiol into estradiol fatty acid esters by the above MCF-7 cell line (203P) were unsuccessful, although, such incorporation could be demonstrated in two other MCF-7 cell sublines. Incubation of the 203P cells with 10 nM [3H]estradiol in the presence of 0.5 mM radioinert estradiol acetate resulted in the incorporation of 35 +/- 12% of the label into the estradiol acetate in 10 min. In the absence of radioinert estradiol acetate, no incorporation was observed. When MCF-7 cells were incubated with [3H]estradiol in the presence of a large excess of radioinert estradiol valerate, label was found only in estradiol valerate. Similarly, when the incubation was carried out in the presence of a mixture of radioinert estradiol acetate and valerate, label was incorporated into both esters. We conclude that the apparent formation of radiolabeled estradiol esters by MCF-7 cells incubated under the above conditions, results at least in part, from an esterase-catalyzed exchange reaction. Under conditions where no ester hydrolysis could be detected in the absence of cells, valerate and stearate esters of estradiol were found to be as effective as unesterified estradiol in stimulating esterase synthesis and the incorporation of [3H]thymidine into DNA. These results are consistent with a model in which an intracellular esterase in MCF-7 cells can generate estradiol from an exogenous lipoidal steroid and elicit an estrogen response.

Hypoxia Stimulates ecNOS mRNA Expression by Differentiated Human Trophoblasts

Cytotrophoblasts isolated from normal human placenta cultured under normoxic conditions (20% O2, pO2 = 130 mmHg) for 48-72 h differentiate to a form which expresses high levels of hCG and which morphologically resembles syncytiotrophoblast. We had previously shown that hypoxia (0-1% O2, pO2 = 12-14 mmHg) blocks this differentiation process, although trophoblasts exposed to hypoxia for up to 96 h were completely viable. In this article we showed that trophoblast responds to hypoxia by expressing the hypoxia-sensitive DNA binding protein HIF-1. We also showed that in trophoblast cultured under normoxic conditions, expression of endothelial cell nitric oxide synthase (ecNOS) mRNA increases with time, reaching a maximum in 48-72 h. However, in trophoblast maintained under hypoxic conditions for 48 h (after an initial 24 h in normoxia), expression of ecNOS mRNA is greatly reduced. These observations are consistent with the expression of ecNOS by syncytiotrophoblast but not by cytotrophoblast. In contrast, exposure of differentiated trophoblasts to hypoxia for 24 h (after 48-72 h in normoxia) significantly stimulates expression of ecNoS mRNA over that of cells maintained continuously in normoxia. These results suggest that in differentiated trophoblast hypoxia can stimulate ecNOS expression.

Studies on the interaction of heparin with thrombin, antithrombin, and other plasma proteins

Abstract The interaction of heparin with plasma proteins was studied by gel chromatography using elution profile analysis to determine binding constants. A commercial heparin preparation was found to bind 100- to 200-fold more tightly to antithrombin than to either albumin or fibrinogen. With tri- or tetrapeptide nitroanilides as substrates, this heparin preparation proved to be an apparent mixed competitive inhibitor of thrombin and other serine proteases with K i s of approximately 10 −5 m . At 3 × 10 −8 m , it caused 50% loss in the activity of human thrombin on fibrinogen. However, at 10 −5 m it did not inhibit the esterase activity of human thrombin on N -tosyl arginine methyl ester. These observations suggest that in the presence of heparin the size of the substrate able to bind at the thrombin-active site is restricted. When this commercial heparin was chromatographed on immobilized antithrombin, thrombin, albumin, and fibrinogen, measurable binding occurred only to the antithrombin and thrombin affinity materials. The heparin purified by chromatography on immobilized antithrombin had a greater affinity for antithrombin and a greater anticoagulant activity than unfractionated heparin but the same K i for thrombin (using a peptide nitroanilide substrate). Heparin purified on immobilized thrombin also had the same K i for thrombin as unfractionated heparin but had a lower affinity for antithrombin and a lower anticoagulant activity. These findings indicate that heparin binds to antithrombin with greater specificity than it does to thrombin.

Synthesis of α 1 -Antichymotrypsin and α 1 -Antitrypsin by Human Trophoblast

ABSTRACT: α1-Antichymotrypsin (α1-ACHY) and α1-an-titrypsin (α1-AT) are closely related glycoprotein protease inhibitors, present in plasma and other extracellular fluids, that neutralize proteases released by leukocytes in response to trauma and inflammatory stimuli. Both inhibitors are synthesized primarily by hepatocytes, although lower levels of synthesis by monocytes and breast and intestinal epithelial cells have been demonstrated. Recently, the immunohistochemical localization of α1-AT and α1-ACHY in intrauterine and extrauterine human trophoblastic tissue has been reported. In the present study, we have sought to determine whether human trophoblast is also able to synthesize α1-AT and α1-ACHY. Messenger RNA for both inhibitors was found by Northern blotting in chorionic villi obtained from first trimester and term placenta. Substantial differences in messenger levels for both inhibitors among individual placentas were noted. α1-ACHY and α1-AT messenger was also present in trophoblast cells in primary culture. Synthesis of α1-AT and α1-ACHY protein was demonstrated by SDS-PAGE after immunoprecipitation of [35S]-labeled α1-AT and α1-ACHY from conditioned media of trophoblast cells in culture metabolically labeled with [35SJ-methionine. It is of some interest that the Mr of the α1-AT and α1-ACHY secreted by trophoblast were 50 000 and 49 000, respectively, compared with 54 000 and 68 000 for these proteins in plasma (or secreted by HepG2 human hepatoma and MCF-7 human breast cancer cells). After enzymatic deglycosylation, the Mr of the α1-AT and α1-ACHY secreted by trophoblast and HepG2 cells were all approximately 46 000, suggesting incomplete glycosylation of the inhibitors released by trophoblast.

cDNA cloning of esterase 1, the major esterase activity in mouse plasma.

We report here the cloning of a partial cDNA for Esterase 1, the major esterase activity in mouse plasma. A 470 base pair insert was isolated from a lambda gt11 cDNA library constructed from mouse liver poly A+ RNA, and identified by hybrid selected translation. We show that the sexual dimorphism displayed in the plasma levels of this protein is caused by a difference at the level of transcription. In addition, RFLP data using mouse recombinant inbred strains mapped this clone at the Es-1 locus on mouse chromosome 8.

Synthesis of alpha 1-antichymotrypsin and alpha 1-antitrypsin by human trophoblast.

ABSTRACT: α1-Antichymotrypsin (α1-ACHY) and α1-an-titrypsin (α1-AT) are closely related glycoprotein protease inhibitors, present in plasma and other extracellular fluids, that neutralize proteases released by leukocytes in response to trauma and inflammatory stimuli. Both inhibitors are synthesized primarily by hepatocytes, although lower levels of synthesis by monocytes and breast and intestinal epithelial cells have been demonstrated. Recently, the immunohistochemical localization of α1-AT and α1-ACHY in intrauterine and extrauterine human trophoblastic tissue has been reported. In the present study, we have sought to determine whether human trophoblast is also able to synthesize α1-AT and α1-ACHY. Messenger RNA for both inhibitors was found by Northern blotting in chorionic villi obtained from first trimester and term placenta. Substantial differences in messenger levels for both inhibitors among individual placentas were noted. α1-ACHY and α1-AT messenger was also present in trophoblast cells in primary culture. Synthesis of α1-AT and α1-ACHY protein was demonstrated by SDS-PAGE after immunoprecipitation of [35S]-labeled α1-AT and α1-ACHY from conditioned media of trophoblast cells in culture metabolically labeled with [35SJ-methionine. It is of some interest that the Mr of the α1-AT and α1-ACHY secreted by trophoblast were 50 000 and 49 000, respectively, compared with 54 000 and 68 000 for these proteins in plasma (or secreted by HepG2 human hepatoma and MCF-7 human breast cancer cells). After enzymatic deglycosylation, the Mr of the α1-AT and α1-ACHY secreted by trophoblast and HepG2 cells were all approximately 46 000, suggesting incomplete glycosylation of the inhibitors released by trophoblast.