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Biomedical subjects

D A Freeman

Publications and source records attributed to D A Freeman.

82 records · Page 5Linked to original sources
PubMed1983

The low-density lipoprotein pathway of cultured Leydig tumor cells. Utilization of low-density lipoprotein-derived cholesterol for steroidogenesis.

We have previously reported that low-density lipoprotein (LDL) enhances and prolongs steroidogenesis in human choriogonadotropin (CG)-stimulated Leydig tumor cells (MA-10). The studies described herein elucidate the mechanisms by which LDL increases human CG stimulated steroidogenesis. Our results show that the MA-10 cells express the classic LDL pathway. LDL is bound to specific surface binding sites which are regulated by the level of intracellular cholesterol. The cellular processing of bound LDL is temperature-dependent and is inhibited by blocking lysosomal function. By using an LDL derivative in which the core cholesteryl esters have been replaced with [3H]cholesteryl linoleate, we show that LDL cholesterol is rapidly utilized for steroid hormone synthesis. The utilization of LDL cholesterol quantitatively accounts for the LDL-induced augmentation of steroidogenesis. We also show that the addition of LDL to human CG-stimulated MA-10 cells maintains cellular free and esterified cholesterol levels and increases progesterone biosynthesis. The addition of LDL does not, however, affect the cellular utilization of preexisting cholesterol stores for steroidogenesis.

Binding Sites↗
PubMed1983

Inhibition of steroidogenesis in cultured Leydig tumor cells by 22-amino-23,24-bisnor-5-cholen-3 beta-O1 and (20R) 20-phenyl-5-pregnene-3 beta,20-diol.

Using the cholesterol side-chain cleavage enzyme purified from bovine adrenals, we have previously shown that 22-amino-23,24-bisnor-5-cholen-3-beta-ol (22-ABC) and (20R) 20-phenyl-5-pregnene-3 beta, 20 diol (20-PPD) are potent competitive inhibitors of this enzyme. The studies presented herein were designed to characterize the effects of these new inhibitors on steroid production by intact cells. Using cultured Leydig tumor cells, we compared the effects of 22-ABC, 20-PPD, and aminoglutethimide (a well-known inhibitor of the cholesterol side-chain cleavage enzyme) on hormone-stimulated steroidogenesis. Our results show that these compounds inhibit steroid production in a dose-dependent manner and that 20-PPD and 22-ABC are about 100 and 4 times more active, respectively, than aminoglutethimide. In these cells, the inhibitory effects of the new compounds seem to be localized exclusively at the conversion of cholesterol to pregnenolone.

Aminoglutethimide↗
PubMed1982

Studies on the source of cholesterol used for steroid biosynthesis in cultured Leydig tumor cells.

The studies presented herein were designed to determine the source(s) of cholesterol used for steroid biosynthesis in a clonal strain of cultured Leydig tumor cells (designated MA-10). Our results show that, when the cells are placed in medium devoid of extracellular cholesterol and acutely stimulated with human choriogonadotropin, most of the cholesterol used for steroid biosynthesis is derived from intracellular stores. Upon prolonged stimulation, when the intracellular stores are depleted, the cells meet the enhanced demand for cholesterol by increasing de novo synthesis. The presence of low density lipoprotein has little or no effect on the amount of steroid synthesized during acute stimulation, but it enhances the amount of steroid produced during prolonged stimulation by directly providing cholesterol substrate to the cells.

Acetates↗
PubMed1982

Desensitization of steroidogenesis in cultured Leydig tumor cells: role of cholesterol.

We have reported previously that the human choriogonadotropin (hCG) receptors of cultured Leydig tumor cells can be down-regulated with hCG or mouse epidermal growth factor (mEGF) and that such down-regulation results in a loss of the steroidogenic response of the cells to hCG. Exposure of the cells to hCG, but not to mEGF, also resulted in a decrease in steroidogenic responses to cholera toxin and cAMP. The results presented herein show that the hCG-induced loss of steroidogenic response to cAMP is due to the depletion of intracellular cholesterol and that this depletion can be prevented by the addition of low density lipoprotein. Our results also show that after exposure of the cells to mEGF or hCG their steroidogenic response to hCG is limited by the number of hCG receptors, regardless of the presence of low density lipoprotein.

Animals↗
PubMed1981

Desensitization to gonadotropins in cultured Leydig tumor cells involves loss of gonadotropin receptors and decreased capacity for steroidogenesis.

The ability of human choriogonadotropin (hCG) to regulate its receptors and target cell responses has been studied in a clonal strain of cultured Leydig tumor cells (MA-10). Exposure of the MA-10 cells to hCG results in decrease in hCG binding activity which is dependent on time and the concentration of hCG. This decrease is due to a change in the number of receptors rather than in the affinity of the receptors, and it is accompanied by a corresponding reduction in the ability of hCG to stimulate steroidogenesis. Exposure of the MA-10 cells to hCG also resulted in a reduction of the steroidogenic responses to cholera toxin and 8-Br-adenosine cyclic 3',5'-monophosphate. The hCG-induced loss of steroidogenic responses to these stimuli seems to be due to the stimulation of steroidogenesis rather than to the decrease in hCG receptors because it also can be induced when steroidogenesis is stimulated with cholera toxin or 8-Br-adenosine 3',5'-monophosphate under conditions such that the number of hCG receptors is not reduced.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1975

Evaluation of six fluorescent protein stains for use in flow microfluorometry.

Flow microfluorometric (FMF) analysis of stained cells has provided protein distribution histograms for large populations of cells. Spectral data and staining protocols were evaluated for six fluorescent protein dyes suggested for staining cells in liquid suspension. The requirements for dyes and/or staining protocol included minimal cell clumping and cell loss, near-optimal dye excitation at existing laser wavelengths, and tenacity of the dye/protein interaction. These criteria were best satisfied by fluorescein isothiocyanate (FITC) and rhodamine B isothiocyanate (RITC). Both fluorescamine and 8-aniline-1-naphthalene sulfonic acid (ANSA) showed potential applicability for use in systems where excitation wavelengths in the ultraviolet range are available. Protein staining with fluorescamine was extremely rapid. Brilliant sulfaflavine and 1-dimethyl-aminonaphthalene-5-sulfonyl chloride (DANSYL) were found unsatisfactory in these studies, since the former dye tended to diffuse from the cells, while the latter induced excessive cell clumping and cell loss. These techniques have application to immunofluorescence analysis and can also be profitably employed in dual-parameter analysis systems in connection with double-staining techniques for simultaneous DNA and protein analysis.

Animals↗
PubMedDate not supplied

Serum levels of squamous cell carcinoma antigen and ovarian carcinoma antigen (CA 125) in patients with benign and malignant diseases of the uterine cervix.

In the present study we evaluated the clinical usefulness of the tumor antigens, squamous cell carcinoma antigen (SCC) and ovarian carcinoma antigen (CA 125), in populations of patients with benign and malignant cervical disease. SCC and CA 125 levels were determined in the serum of 59 patients with invasive carcinoma of the uterine cervix and in 21 patients with benign cervical diseases. Before treatment of cervical cancer, SCC levels were elevated in 63% of the patients with squamous cell cancer while all 5 patients with adenocarcinoma had normal levels. CA 125 levels were elevated in 21% of the patients with cervical squamous cell cancer and in 3 of the 5 cases of adenocarcinoma of the cervix. In patients with benign cervical diseases, only 1 had a positive SCC level and none were positive for CA 125. No correlation was found between SCC levels and histological differentiation or clinical stage. In positive patients, serial SCC determinations correlated with the clinical course in 72.2%. Increasing levels were always associated with progression and increased on average 3 months before there was clinical evidence for disease progression. It is concluded from these studies that SCC levels are a useful marker for cervical cancer progression and recurrence. Levels of CA 125 were more likely to be elevated in patients with adenocarcinoma than squamous cell carcinoma, but when elevated in these latter patients, it also tended to predict tumor recurrence.

Adenocarcinoma↗
PubMedDate not supplied

Varying susceptibility of different low density lipoproteins to oxidative modification.

The oxidative modification of low density lipoprotein (LDL) may provide a crucial link between plasma LDL and the atherosclerotic lesion. The studies presented herein define time-dependent modifications of LDL constituents caused by CuSO4-catalyzed oxidation. Measurement of the cholesterol content of oxidized LDL by the cholesterol esterase-oxidase assay was found to be inaccurate. The enzymatic assay detected oxysterols as well as cholesterol and thus substantially overestimated the actual cholesterol content. Alteration of electrophoretic mobility and conversion of sterols into oxysterols increased in a parallel, time-dependent manner. Lipid peroxidation, judged by the thiobarbituric acid-reacting substances assay, increased early to maximal values but was not linearly related to either electrophoretic mobility or to oxysterol formation. Neither electrophoretic mobility nor oxysterol formation varied much between repeated oxidative modifications of any given LDL preparation but varied markedly among LDLs from different normolipidemic individuals, suggesting that LDL particles contain some factor conferring susceptibility or resistance to oxidation. Indeed, LDL preparations from different individuals have varying susceptibilities to oxidative modification as evidenced by the three indexes used. The major oxysterol generated was 7-ketocholesterol. Macrophage modification of LDL also resulted in the generation of oxysterols. Thus, measurement of oxysterols may afford an additional index of the oxidative modification of LDL. Since incubation of macrophages with oxidized LDL but not native LDL resulted in the accumulation of oxysterols, this could account for some of the toxic and metabolic effects of oxidized LDL on cells.

Adult↗
PubMedDate not supplied

Plasma membrane steroidogenic cholesterol: the relative importance of membrane internalization rate and cholesterol extraction rate of internalized membrane.

Most cholesterol destined to become steroid hormone passes through the plasma membrane. Using MA-10 Leydig tumor cells as a model system, we determined that plasma membrane cholesterol disappeared 2.5-fold faster in cAMP-stimulated cells. Stimulation of MA-10 cells increased internalization so that 1.4-fold more membrane cycled through the cell per unit time. Increased internalization accounted for 26% of the cholesterol actually lost from cycling membrane. The remaining 74% was accounted for by increasing the extraction of cholesterol from internalized membrane.

Animals↗