Biomedical subjects
J A Holt
Publications and source records attributed to J A Holt.
Squamous-cell carcinoma treatment.
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Estrogen regulation of steroidogenesis in rabbit luteal cells.
Estradiol is a potent modifier of gonadotropin-stimulated steroidogenesis. By the seventh day after ovulation, estradiol is the only agent required for the stimulation of progesterone synthesis by corpora lutea of superovulated pseudo-pregnant rabbits. To learn which control points in steroidogenesis are susceptible to regulation by estradiol alone, we have studied the production of pregnenolone, progesterone, and 20 alpha-hydroxy-4-pregnen-3-one by corpora lutea of estradiol-stimulated and estradiol-deprived pseudopregnant rabbits. In previous investigations, we learned that estradiol deprivation in vivo, on day 9 of pseudopregnancy, causes an abrupt cessation of progesterone and 20 alpha-hydroxy-4-pregnen-3-one production, which is associated with accumulation of cholesterol and cholesteryl ester in the luteal tissue. We now report that production of pregnenolone, measured as its concentration in serum, also decreases abruptly by 84% within 48 h when the estradiol stimulus is removed on day 9 of pseudopregnancy. In addition, short term incubations of luteal tissue demonstrate that corpora lutea from estradiol-deprived rabbits do not use stores of luteal intracellular cholesterol for production of pregnenolone and progestin. These findings suggest that upon estradiol deprivation, rabbit luteal cells lose their capacity for using stored cholesteryl ester, or cholesterol synthesized de novo, for the production of pregnenolone and progestins. We, therefore, tested the hypothesis that a blockade of steroidogenesis caused by estrogen deprivation occurs at the point of cytochrome P-450 cholesterol side-chain cleavage (P-450scc), a principal rate-limiting step in the conversion of cholesterol to hormonal steroid products. To this end, we assayed the P-450scc activity in mitochondria-rich fractions of corpora lutea from rabbits that were deprived of estradiol for 24 and 48 h beginning on day 9 after induction of superovulation. Surprisingly, withdrawal of the estradiol stimulus did not cause loss of luteal P-450scc activity, measured as the amount of aminoglutethimide-inhibitable conversion of 25-hydroxycholesterol to pregnenolone by mitochondria-rich preparations. From these results, we infer that the luteotropic action of estradiol is probably not effected at P-450scc in the rabbit corpus luteum, but, presumably, occurs at control points that regulate the availability of stored cholesterol and/or its movement to or within the mitochondria for conversion to pregnenolone.
Monoclonal antibody recognition of multiple forms of estrogen receptor tagged with [125I]methoxy-iodovinyl estradiol in ovarian carcinomas.
Based on previous studies of the properties of moxestrol, we hypothesized that a radiohalogenated analog of moxestrol, [125I]11 beta-methoxy-17 alpha-iodovinyl-estradiol [( 125I] MIVE2), should bind to the estrogen receptor (ER) in some ovarian adenocarcinomas (OVCA), thereby offering the potential for imaging and/or treatment of these cancers. We used monoclonal antibodies (H222, H226, and D547) against human breast cancer ER to identify the [125I]MIVE2-binding moiety in OVCA cytosols that is found on high salt sucrose gradients. After gel electrophoresis and western blotting, exposure of OVCA extracts to the ER antibodies, followed by exposure to goat antirat serum and then rat peroxidase antiperoxidase, demonstrated a moiety in OVCA that migrated indistinguishably from the ER in MCF-7 human breast cancer cells and from that in specimens of breast cancer tissue. Because few studies have demonstrated efficacy of hormone management for OVCA, we also wanted to learn whether ER exists in multiple forms in OVCA, in view of the possibility that some forms may be inactive in regulating growth-dependent cell functions while retaining estrogen-binding capacity. By incubating the monoclonal antibodies H222, H226, and D547, each of which recognizes a different region on the ER protein, with OVCA cytosol fractions, we demonstrated that ER in OVCA can exist in multiple forms, some of which fail to express an H226-recognized site and some of which fail to express a D547-recognized site. This observation indicates that a relationship may exist between the presence or absence of certain forms of ER in ovarian epithelial cancer and a patient's response to hormone therapy.
Persistence and distribution of estrogen receptor in advanced epithelial ovarian carcinoma after chemotherapy.
Estrogen receptor in tumor cytosol extracts from primary ovarian adenocarcinomas and/or from their metastatic foci was measured for determination of whether or not estrogen receptor content is preserved after chemotherapy. Tumor tissue was analyzed from 36 patients who had not received chemotherapy and from 16 patients after varying periods of chemotherapy. The percentages of relatively estrogen receptor-rich tumors (with more than 35 fmol estrogen receptor per milligram soluble protein, or with more than 1200 fmol estrogen receptor per gram tissue wet weight) were similar in the two groups (31 and 37%). In three of four patients who had specimens analyzed both before and after chemotherapy, there was little difference between the levels of estrogen receptor in the initial sample and in samples taken after ten to 32 months of chemotherapy. In one patient, a decrease in the amount of receptor after chemotherapy appeared to be related to the sampling technique used. There was no discernible effect of treatment on the prevalence or amount of estrogen receptor in metastases. No correlation was observed between estrogen receptor levels and the histologic type or grade of the tumor or the patients' survival. The data suggest that chemotherapy has little effect on measurable receptor levels in ovarian epithelial carcinomas. When present in one tumor site, receptor can be expected to be present in a majority of other sites in the same patient.
Immunocytochemical identification of estrogen receptor in ovarian carcinomas. Localization with monoclonal estrophilin antibodies compared with biochemical assays.
Estrogen receptor (estrophilin) has been identified in ovarian carcinomas by a variety of physicochemical methods. Since these methods require disruption of the tissue, they do not provide any anatomic information about the cellular distribution and location of receptor. The authors have used monoclonal estrophilin antibodies and an indirect immunoperoxidase technique to study the immunocytochemical localization of estrogen receptor in 43 tissue samples of ovarian carcinoma from 27 patients. The immunocytochemical findings were compared with the results of conventional estrogen receptor assays of cytosolic and nuclear extracts prepared from adjacent pieces of ovarian carcinoma. Exclusively nuclear localization of estrogen receptor was observed with the immunocytochemical technique in all of the 25 tumor samples which had a cytosolic estrogen receptor content, determined by either the dextran-coated charcoal or hydroxylapatite techniques, greater than 700 fmoles/gm wet weight of tissue. Only 3 of 16 tumor samples with cytosolic estrophilin concentrations of less than 700 fmoles/gm wet weight displayed nuclear staining for estrogen receptor; two of these three were metastases from receptor-rich primaries. Specific cytoplasmic staining for estrogen receptor was not observed. These results indicate that many ovarian carcinomas have estrogen receptor, predominantly localized in the nucleus, which is similar to tissues of the female genital tract (vagina, cervix, endometrium, fallopian tube) and breast carcinoma.
Estradiol-induced changes in progesterone secretion by rabbit corpora lutea are associated with quantitative ultrastructural changes in luteal cells.
In this study, we examined changes in luteal cell structure that accompany estradiol-altered progesterone production by the rabbit corpus luteum. To stimulate progesterone production, polydimethylsiloxane (Silastic) capsules containing 17 beta-estradiol were inserted sc into superovulated New Zealand White rabbits. Luteal progesterone production, assessed by measurement of progesterone in peripheral serum, was high after the estradiol-filled capsules were inserted, declined within 24 h after the capsules were removed, and increased within 32 h after reinsertion of the capsules. Stereological analyses at the light microscopic level revealed that the number of luteal cells and the volume of an average luteal cell did not differ significantly between estradiol-stimulated and estradiol-deprived rabbits over the time period employed. In contrast, stereological analyses at the electron microscopic level demonstrated that the surface areas of smooth endoplasmic reticulum and inner mitochondrial membrane declined with estradiol deprivation, but were restored by reimposition of estradiol stimulation. These changes in the surface area per cell of smooth endoplasmic reticulum and inner mitochondrial membrane were strongly correlated (r = 0.94 and r = 0.88, respectively) with changes in progesterone concentrations in peripheral serum. Changes in the surface area of lipid droplets per luteal cell also occurred, but were inversely correlated (r = -0.87) with progesterone levels. No significant changes were seen in the surface areas per cell of outer mitochondrial membrane or rough endoplasmic reticulum. These results demonstrate that estradiol stimulation and deprivation cause reversible quantitative changes in the rabbit luteal cell organelles known to be directly involved in progesterone biosynthesis. This leads to the conclusion that the steroidogenic activity of the luteal cell is tightly coupled to its subcellular structure.
Estradiol-induced changes in rabbit luteal cell progestin production and cholesterol and cholesterol ester content.
Estradiol deprivation in vivo causes accumulation of cholesterol and cholesterol ester in the corpora lutea of pseudo-pregnant rabbits within 24 hours. These accumulations occur concomitantly with an abrupt cessation of progestin production. Retreatment with estradiol restores progestin production and can block further increases in cholesterol accumulation (per mg total tissue protein). Ample quantities of precursor cholesterol ester and cholesterol substrate are present in corpora lutea when progestin falls because of estradiol deprivation. We conclude therefore that the gonadotropic action of estradiol is, in part, because of its stimulation of cholesterol conversion to progestins.
Sulfhydryl sensitivity and [125I]-16 alpha-IODO-17 beta-estradiol binding of estrogen receptor in ovarian epithelial carcinomas.
We find that estrophilin from either the cytosolic or nuclear fractions of ovarian epithelial carcinomas (OVCA) binds irreversibly to controlled-pore glass beads (CPG). The CPG-adsorbed estrophilin releases [3H]-estradiol at 4 degrees C in the presence of 1.25 mM AgNO3; estradiol binding capacity from the nuclear fraction is restored by 10 mM dithiothreitol. The number of available estradiol binding sites in cytosol that are sensitive to AgNO3 correlate with (a) the number of estradiol-inhibitable binding sites found in the 8S region in low-salt sucrose gradients (r = 0.9) but not with (b) estradiol-inhibitable binding in the 4S region (r = 0.3) and thus, as expected, only poorly with (c) estimates of available cytoplasmic estrogen binding sites using a standard dextran-coated charcoal analysis (r = 0.7). Total estrogen binding extracted from the nuclear fraction with 0.5 M KCl and adsorbed to hydroxylapatite agreed with the sulfhydryl blocking reagent sensitive moiety (r = 0.9). Estrophilin from OVCA cytosol or nuclear fractions bound [125I]-16 alpha-iodo-estradiol indistinguishably from [3H]-estradiol. The two forms of radiolabelled-estradiol yielded equivalent data that demonstrated a shift in the estrogen binding moiety from the 8S region to the 4-5S region when 0.5 M KCl was added to gradient analyses of OVCA cytosols. From these observations we conclude that the estrophilin found in OVCA is similar to that found in normal and cancerous tissues of other female reproductive organs and that this estrophilin can bind a biologically active radiohalogenated estrogen potentially useful for imaging or treating these tumors.
Cancer, a disease of defective glucose metabolism.
The cancer cell appears to contain two non phosphorylating glycolytic pathways in addition to phosphorylating glycolysis and the aerobic citric acid and phosphogluconate cycles. It is hypothesised that these pathways are all interconnected and in the static adult cell non phosphorylating glycolysis is in a state of controlled inactivity. Cancer occurs when one or both non phosphorylating glycolytic pathways escape from control thereby producing an autonomous flow of energy and mitosis. Both pathways appear dependent upon glutathione to convert glucose to lactic acid. The pathways can be differentiated by their responses to ionising and 434MHz non ionising radiation, hyperthermia and biochemical environmental changes in vivo.
The fundamental chemistry of life. An attempt to define and identify the basic reaction responsible for life's creation and evolution.
All natural growth follows exponential characteristics which vary from a simple exponential equation (non-solid cancer, bacteria) to complex Gompertzian functions describing solid cancer and multicellular organisms. Like all chemical processes the reagents (energy sources of food) react during life to produce vital energy, but in addition also create the next generation of life. This latter reaction is unique in that a simple proportional increase in the reagents creates an exponential increase in products: it is the sole invariable criterion of all life. The target of combined ionising and non-ionising radiations in cancer cells appears to be identical with this fundamental exponential chemical reaction. Identification of this target as a system of anaerobic glycolysis suggests that life's first reaction is a unique one whereby a simple proportional increase in available glucose causes an exponential proportional increase in energy which is available solely for reproduction.
Binding of [3H] monohydroxytamoxifen in ovarian carcinoma.
A monoclonal antibody (D-547) against human breast cancer oestrogen receptor was used to demonstrate that the oestrogen receptor in ovarian epithelial carcinomata binds [3H]monohydroxytamoxifen. This binding was observed in sedimentation velocity analyses of both cytosol and nuclear compartments. Binding of [3H]monohydroxytamoxifen to the antibody-recognized moiety could be inhibited by diethylstilboestrol or oestradiol, but not by androgen, progestin or corticoid, indicating oestrogen specificity for the binding site. The sedimentation coefficient of the antibody-recognized [3H]monohydroxytamoxifen binding moiety was similar to that when 16 alpha-[125I]iodo-oestradiol was used as the ligand.
Monoclonal antibody recognition of estrogen receptor in rabbit corpus luteum.
We demonstrated previously by administering [3H]estradiol and using autoradiography that, in vivo, the exogenous estradiol that will sustain progesterone production is sequestered to nuclei of the luteal cells in corpora lutea of pseudopregnant rabbits. Our objective in the present experiments was to use an immunological method to demonstrate that estrogen receptor of the rabbit corpus luteum is associated with this uptake. For this purpose, we prepared nuclear extracts from corpora lutea removed from anesthetized pseudopregnant rabbits 10 min after arterial infusion of 125I-estradiol. These extracts were then analyzed by sucrose density gradient centrifugation in the presence and absence of monoclonal antibody against human breast cancer estrogen receptor. We found that the 125I-estradiol-binding component extracted with 0.5 M KCl from the nuclear pellet sediments in the 3-4S region in high-salt gradients; in the presence of monoclonal antibody (D-547 Sp2 gamma), the entire estradiol-binding component is shifted to the 8-9S region, indicating the formation of immunocomplex with the 125I-estradiol-receptor moiety. The principal estradiol-binding component that can be observed in the 3-4S region of high-salt gradients of cytosol of rabbit corpora lutea reacts with this antibody regardless of whether 125I- or [3H]estradiol is given in vivo or in vitro. In both cytosol and nuclear extracts of luteal tissue, unlabeled diethylstibestrol totally inhibits the binding of radiolabeled estrogen to this antibody-shifted 125I- or [3H]estradiol-binding moiety; neither progesterone nor testosterone inhibit binding of radiolabeled estrogen to the antibody-shifted moiety. From these findings we conclude that sequestering of estradiol by the rabbit corpus luteum in vivo is accomplished by the steroid-specific estrogen receptor that shares characteristics of the estrogen receptor found in human breast cancers, including a) antigenic features and b) the capacity to translocate estrogens from the cytosol to the nuclear fraction.
Limitations of qualitative serum beta-HCG assays in the diagnosis of ectopic pregnancy.
Seventy-four patients had ectopic pregnancies proven by surgery. Three of them had a serum beta-HCG test for pregnancy that was reported as negative (less than 25-35 mIU/ml). We sought a threshold for positive in the serum beta-HCG test that would maximize its usefulness in diagnosing ectopic pregnancy. Sera from 52 of the patients were available for reanalysis. Quantitative values of beta-HCG were determined. Lowering the threshold for positive from 25-35 to 10 mIU/ml might increase the test's sensitivity without sacrificing specificity but would still not ensure detection of all ectopic pregnancies. Of 445 cases of ectopic pregnancy described in the literature, 6 had serum beta-HCG values reported as negative. In the published reports and in our own cases, clinical histories and histologies indicated that a nonviable ectopic pregnancy can be expected to have an associated serum concentration of beta-HCG that may be below the sensitivity of detection even with current, commercially available quantitative tests.
Specific antibodies and immunoglobulins in the oviductal fluid of the rhesus monkey.
Levels of specific antibodies against model antigens, immunoglobulins G and A and also albumin, in oviductal fluid were studied in the rhesus monkey during the periovulatory period. Animals were systemically or intravaginally immunized against T4 coliphages. Attempts to induce ovulation were made with human menopausal gonadotropin (hMG) and human chorionic gonadotropin (hCG) monitored by radioimmunoassay of serum estrogen and progesterone. Collection of tubal fluid over 6 to 13 days was accomplished by surgical cannulation using a refrigerated extracorporeal collection device for each side. The results indicated the following: (1) The levels of specific antibodies against T4 coliphage and immunoglobulins (IgG, IgA) in the oviduct fluid averaged approximately one tenth of the serum values and showed a characteristic decrease and subsequent increase by a factor of 4 to 5 during and following treatment with hMG/hCG. The nadir was observed on the first or second day after hCG injection. (2) This pattern was similar in both ovulatory or nonovulatory cycles; therefore, these changes seem to be associated with the changes in serum estrogen levels. (3) There was a striking difference in serum and tubal fluid antibody levels after systemic versus after vaginal immunization by a factor of 10(3) and 10(4); however, the patterns in tubal fluid under treatment with hMG/hCG were very similar. (4) Specific antibodies in oviductal fluid and serum were mainly of the IgG class. (5) A concomitant change of total protein and albumin in oviduct fluid was also observed. The presence of sperm agglutination antibody in oviductal fluid was demonstrated in two monkeys after systemic immunization with homologous spermatozoa. The sperm antibody titers showed a similar pattern of change after hMG/hCG treatment.
Antibody-recognized [125I]estradiol-receptor complex in ovarian epithelial carcinoma.
Monoclonal antibody against human breast cancer estrogen receptor was used to demonstrate binding of a gamma- and Auger electron-emitting estrogen to the estrogen receptor in ovarian epithelial carcinomas. When cytosols of estrogen receptor-rich ovarian adenocarcinomas were analyzed on sucrose gradients containing 0.4 M KCl, the presence of monoclonal antibody against breast cancer estrogen receptor caused a binding peak for [125I]- and [3H]estradiol to shift from the 4S to the 8-9S region, indicating antibody complex formation with ovarian adenocarcinoma estrogen receptor. The antibody-shifted peak of 8-9S [125I]- and [3H]estradiol binding was totally inhibited by a 100-fold molar excess of diethylstilbestrol (DES), but not by testosterone or progesterone, indicating a preference for estrogen binding by the antibody-shifted estrogen receptor. When estrogen receptor from the nuclear fraction of ovarian adenocarcinomas was incubated with [125I]estradiol at 1C, in the presence of 0.4 M NaSCN to facilitate exchange with endogenous ligand, binding occurred that was inhibitable by DES and restricted to the 4S region. Under these conditions the nuclear estrogen receptor was also shifted to the 8-9S region by the presence of the monoclonal antibody against estrogen receptor.
Site of estrogen action in rabbit ovarian tissue.
In superovulated or naturally mated pseudopregnant rabbits, an abrupt premature decline of progesterone production by the corpus luteum occurs after removal of polydimethylsiloxane capsules filled with 17 beta-estradiol. Reinsertion of the estradiol-filled capsules stimulates a striking restoration of progesterone production. We have given [3H]estradiol by arterial infusion to anesthetized rabbits to determine by cellular autoradiography whether the exogenous estradiol localizes to luteal tissues responsible for progesterone production. Autoradiography with both freeze-dried and touch-thaw mounted preparations demonstrated that tritium activity localized to the nuclei of luteal cells and that such uptake could be blocked by radioinert diethylstilbestrol. We extracted the tritium activity sequestered in vivo by the corpora lutea and cocrystallized the extract with 14C-labeled and radiostable 17 beta-estradiol to confirm its presence (greater than 90%) as [3H]estradiol. In addition, we were able to demonstrate that radiostable diethylstilbestrol blocked ovarian uptake of 16 alpha-125I-17 beta-estradiol in vivo. In concert, these observations provide compelling evidence that the exogenous estradiol responsible for restoring progesterone production may act through an estrogen-specific mechanism directly in the luteal cells.
Nuclear progestin receptor-like component in human cervical tissue.
To study progestin action in human glandular cervix, we extracted the nuclear pellet with 0.5 M KCl and immediately adsorbed a progestin-binding receptor-like component to hydroxylapatite. Binding of 2 nM [3H]R5020 at 4 degrees C for 18 h, in the presence or absence of 100-fold molar excess progesterone and other steroids, was used to measure the total extractable and adsorbed progestin-binding component. With this assay, the amounts of progestin-binding component in the nuclear-rich fraction in specimens from women in the follicular and luteal phases of the ovarian cycle were 930 +/- 143 and 995 +/- 148 fmol/g wet wt (+/- SE), respectively; total cytoplasmic progestin receptor was 2,989 +/- 812 and 1,861 +/- 188 fmol/g wet wt (+/- SE) in the follicular and luteal phases, respectively. The progestin-binding component extracted from the nuclear pellet has a sedimentation velocity coefficient in the 3.5 S region when measured in high salt on 5-20% linear sucrose density gradients that contain 10% glycerol. The progestin binding measured by the hydroxylapatite-exchange assay with [3H]R5020 and radio-insert progesterone is high affinity (Kd 2.6 X 10(-10) M) and heat labile and appears specific for progestins and progesterone target tissues.