NaCl Nuclear Extracts Research Articles

Specific receptors for thyroxine in nuclear non-histone proteins of rat thymus during early, medium and late period of life.

Specific binding of 125I-thyroxine to thymus nuclear non-histone proteins of rats aged 7, 14, 45, 80 and 365 days was studied. In 0.4 mol l-1 NaCl nuclear extract of thymocytes practically the same values of Ka were found in rats aged 7, 14, 45 and 80 days (5.72 +/- 0.11 X 10(9) l mol-1, 3.44 +/- 1.65 X 10(9) l mol-1, 5.00 +/- 1.09 X 10(9) l mol-1 and 5.55 +/- 0.95 X 10(9) l mol-1, respectively). As compared to groups of animals of 7, 45 and 80 days of age a marked diminution of Ka (= 1.30 +/- 0.25 X 10(9) l mol-1) with P less than 0.001, less than 0.02 and less than 0.01, respectively, was found in rats aged 365 days. The highest value representing maximum binding capacity (MBC) of 125I-thyroxine was noted in rats aged 45 days (MBC = 6.16 +/- 0.58 X 10(10) molecules per mg of protein) and in adult rats (80 days old) (MBC = 5.82 +/- 1.28 X 10(10) molecules mg-1). In rats aged 7 days a lower value for MBC (= 1.92 +/- 0.77 X 10(10) molecules mg-1; P less than 0.01) was observed as compared to the animals aged 45 days (see above). Similar decrease was found also in old rats aged 365 days (MBC = 3.46 +/- 0.69 X 10(10) molecules mg-1; P less than 0.02). The data quantify the affinity and the specific 125I-thyroxine binding capacity in nuclear non-histone proteins of rat thymus during early, adult and late period of life.

Cell cycle-dependent dynamic association of cyclin/Cdk complexes with human DNA replication proteins.

We have previously described the isolation of a replication competent (RC) complex from calf thymus, containing DNA polymerase alpha, DNA polymerase delta and replication factor C. Here, we describe the isolation of the RC complex from nuclear extracts of synchronized HeLa cells, which contains DNA replication proteins associated with cell-cycle regulation factors like cyclin A, cyclin B1, Cdk2 and Cdk1. In addition, it contains a kinase activity and DNA polymerase activities able to switch from a distributive to a processive mode of DNA synthesis, which is dependent on proliferating cell nuclear antigen. In vivo cross-linking of proteins to DNA in synchronized HeLa cells demonstrates the association of this complex to chromatin. We show a dynamic association of cyclins/Cdks with the RC complex during the cell cycle. Indeed, cyclin A and Cdk2 associated with the complex in S phase, and cyclin B1 and Cdk1 were present exclusively in G(2)/M phase, suggesting that the activity, as well the localization, of the RC complex might be regulated by specific cyclin/Cdk complexes.

Mechanism of increased sensitivity to etoposide in a mitomycin C-resistant human bladder cancer cell line.

The mechanism of increased sensitivity to etoposide (VP-16) in a human bladder cancer cell line (J82/MMC-2), which is >9-fold more resistant to mitomycin C (MMC) compared with parental cells (J82/WT), was investigated. Colony formation assays, following 1 hr drug exposure, revealed that about a 2.2-fold higher concentration of VP-16 was required to kill 50% of the J82/WT cell line compared with J82/MMC-2. The MTT assays, following continuous drug exposure, also showed that the J82/MMC-2 cell line was significantly more sensitive to VP-16 compared with J82/WT. Accumulation of VP-16 was significantly higher in the J82/MMC-2 cell line compared with J82/WT at every drug concentration tested. Likewise, intracellular VP-16 retention was significantly higher in the J82/MMC-2 cell line compared with J82/WT when drug uptake was measured as a function of varying incubation time and at a fixed VP-16 concentration. The efflux of VP-16 from the J82/MMC-2 cell line was equivalent to that from J82/WT. In agreement with the results of drug uptake studies, the levels of VP-16-induced protein-DNA complexes were markedly higher in the J82/MMC-2 cell line compared with J82/WT. The catalytic activity of topoisomerase II (topo II) in 0.35 M NaCl nuclear extract of J82/WT cells was equivalent to that of J82/MMC-2. The levels of topo II mRNA were also comparable in these cells. Our results suggest that the mechanism responsible for the collateral sensitivity of the J82/MMC-2 cell line to VP-16 may be attributable to a relatively higher drug accumulation in this cell line compared with parental cells.

Mechanism of increased sensitivity to etoposide in a mitomycin C‐resisitant human bladder cancer cell line

The mechanism of increased sensitivity to etoposide (VP-16) in a human bladder cancer cell line (J82/MMC-2), which is >9-fold more resistant to mitomycin C (MMC) compared with parental cells (J82/WT), was investigated. Colony formation assays, following 1 hr drug exposure, revealed that about a 2.2-fold higher concentration of VP-16 was required to kill 50% of the J82/WT cell line compared with J82/MMC-2. The MTT assays, following continuous drug exposure, also showed that the J82/MMC-2 cell line was significantly more sensitive to VP-16 compared with J82/WT. Accumulation of VP-16 was significantly higher in the J82/MMC-2 cell line compared with J82/WT at every drug concentration tested. Likewise, intracellular VP-16 retention was significantly higher in the J82/MMC-2 cell line compared with J82/WT when drug uptake was measured as a function of varying incubation time and at a fixed VP-16 concentration. The efflux of VP-16 from the J82/MMC-2 cell line was equivalent to that from J82/WT. In agreement with the results of drug uptake studies, the levels of VP-16-induced protein-DNA complexes were markedly higher in the J82/MMC-2 cell line compared with J82/WT. The catalytic activity of topoisomerase II (topo II) in 0.35 M NaCl nuclear extract of J82/WT cells was equivalent to that of J82/MMC-2. The levels of topo II mRNA were also comparable in these cells. Our results suggest that the mechanism responsible for the collateral sensitivity of the J82/MMC-2 cell line to VP-16 may be attributable to a relatively higher drug accumulation in this cell line compared with parental cells. Int. J. Cancer 70:612–618. © 1997 Wiley-Liss Inc.

Mapping of DNA topoisomerase II poisons (etoposide, clerocidin) and catalytic inhibitors (aclarubicin, ICRF-187) to four distinct steps in the topoisomerase II catalytic cycle

The complex catalytic cycle of topoisomerase II is the target of important antitumor agents. Topoisomerase II poisons, such as etoposide and daunorubicin, inhibit the resealing of DNA breaks created by the enzyme. This enzyme-coupled cell kill is susceptible to pharmacological regulation by drugs interfering with other steps in the enzyme's catalytic cycle (i.e. so-called catalytic inhibitors). From in vitro studies, it appears that there are 2 distinct sites in the cycle at which a complete antagonism of the toxicity of topoisomerase II poisons can be obtained. The first is the inhibition of the enzyme's binding to its DNA substrate as seen with intercalating drugs such as chloroquine and aclarubicin; a second, more specific, interaction is elicited by bisdioxopiperazines, which are thought to lock the homodimeric topoisomerase II in the form of a closed bracelet surrounding the DNA at the postreligation step. To investigate these in vitro findings in the more complex whole cell system, we studied enzyme-DNA binding in Western blots of 0.35 M NaCl nuclear extracts from human small cell lung cancer OC-NYH cells incubated with the bisdioxopiperazine ICRF-187 and aclarubicin. With ICRF-187, we found a reversible ATP dependent decrease in the extractable levels of both the α and the β isoforms of topoisomerase II. In contrast to ICRF-187, aclarubicin increased the amount of extractable enzyme from cells. Further, when using the terpenoid clerocidin, which differs from conventional topoisomerase II poisons by forming a salt- and heat-stable inhibition of DNA resealing, no antagonism was found by ICRF-187 on formation of DNA strand breaks and cytotoxicity. However, aclarubicin, which interferes early in the topoisomerase II catalytic cycle, was able to antagonize DNA breaks and cytotoxicity caused by clerocidin. The results indicate 4 different steps in the topoisomerase II cycle that can be uncoupled in the cell by different drug types: etoposide and clerocidin cause reversible and irreversible inhibition of DNA resealing, respectively, and DNA intercalating agents, such as aclarubicin, inhibit binding of topoisomerase II enzyme to its DNA substrate. Finally, bisdioxopiperazines as ICRF-187 partake in an energy dependent inappropriate binding of topoisomerase II to DNA after the resealing step. This knowledge may enable the design of rational combinations of topoisomerase II poisons and catalytic inhibitors to enhance the efficacy of anticancer therapy.

Nuclear proteins interacting with DNA and tubulin. Study of the interaction of the High Mobility Group protein 1 with tubulin

Fractionation of a 0.2 M NaCl nuclear extract from rat liver cells by both tubulin and DNA affinity chromatography steps allowed us to find three polypeptides interacting in vitro with both DNA and tubulin. A 22 kDa polypeptide was identified as a proteolytic fragment of High Mobility Group proteins 1 or 2 (HMG 1 or 2). Purified rat liver HMG 1 immobilized on nitrocellulose was found to bind radioiodinated dimeric tubulin through its central B domain. The C domain of HMG 1 appeared to play a negative role in this association process. Soluble HMG 1 depleted of its C-terminal domain interacted with tubulin immobilized on an agarose gel and with microtubules formed from purified tubulin. In contrast, undigested HMG 1 did not interact with tubulin in these conditions. The modification of HMG 1 with amine by 1-ethyl-3-(dimethylaminopropyl)carbodiimide which caused the neutralization of the C domain carboxyl groups restored the ability of HMG 1 to interact with microtubules. These results show that: (a) HMG 1, through its central B domain, binds to both assembled and non-assembled tubulin in vitro and (b) the C-terminal domain of HMG 1 exerts a negative regulatory action on the interaction.

Altered subcellular distribution of topoisomerase IIα in a drug-resistant human small cell lung cancer cell line : Feldhoff PW, Mirski SEL, Cole SPC, Sullivan DM. James Graham Brown Cancer Center, University of Louisville, 529 South Jackson Street, Louisville, KY 40292. Cancer Res 1994;54: 756–762

A drug-resistant human small cell lung cancer cell line, H209/V6, selected in the presence of increasing concentrations of 9-(4,6-O-ethylidene-beta-D-glucopyranosyl)-4'-demethylepipodophylloto xin (VP-16) from parental H209 cells, is 22-, 9-, and 4-fold resistant to VP-16, 4'-(9-acridinyl-amino)methanesulfon-m-anisidide, and doxorubicin, respectively, but not cross-resistant to 1,4-dihydroxy-5,8-bis((2-[(2-hydroxyethyl)amino] ethyl]-amino)-9,10-anthracenedione. These cells do not overexpress P-glycoprotein or the multidrug resistance-associated protein. Immunoblotting demonstrates that H209 cells contain the M(r) 170,000 isoform of topoisomerase II (topo II), while H209/V6 cells have a M(r) 160,000 enzyme but none of the M(r) 170,000 isoform. The cell lines have equal amounts of topo II beta. The H209/V6 cells have a 5-fold decrease in total immunoreactive topo II alpha. The catalytic and VP-16-induced DNA cleavage activities of the topo II present in 0.35 M NaCl nuclear extracts are decreased 2- to 3-fold in the drug-resistant cell line. This decrease in enzymatic activity is not consistent with either the 22-fold VP-16 resistance of the H209/V6 cell line or the approximately 5-fold decrease in immunoreactive topo II alpha in the cells. The M(r) 160,000 isoform from the H209/V6 cell line and the M(r) 170,000 enzyme from the parental cell line were purified so that the enzymatic activity of the 2 isoforms could be evaluated. The catalytic activities of the purified isoforms were found to be very similar. The drug-induced DNA cleavage activity of the M(r) 160,000 enzyme was reduced compared to the M(r) 170,000 enzyme. However, as with the nuclear extracts, the differences in enzymatic activity of the purified enzymes are considerably less than the level of drug resistance. Investigations of the subcellular localization of topo II by immunocytochemical techniques and cytoplasm/nuclear fractionation studies demonstrated that the M(r) 160,000 topo II alpha-related enzyme is primarily localized in the cytoplasm, while the M(r) 170,000 topo II alpha enzyme and topo II beta are located in the nucleus. These data imply that the deleted sequence in the M(r) 160,000 enzyme is not necessary for catalytic activity but is required to facilitate nuclear localization.

Reduced levels of topoisomerase II alpha and II beta in a multidrug-resistant lung-cancer cell line.

We have previously shown that the doxorubicin-selected multidrug-resistant small-cell lung-cancer cell line H69AR is resistant to VP-16-induced single-strand DNA breaks as compared with its parental H69 cell line. Levels of immunoreactive topoisomerase II alpha are also reduced in H69AR cells. In the present study, we found that cleaved complex formation in the presence of VP-16 was decreased in H69AR cells as compared with H69 cells. In addition, the resistant cells contained lower levels of both topoisomerase II alpha and topoisomerase II beta protein and mRNA. However, these changes were not accompanied by a decrease in the P4-unknotting (strand-passing) activity of 0.67 M NaCl nuclear extracts of H69AR cells, nor was there any difference in VP-16 inhibition of unknotting activity in the H69 and H69AR nuclear extracts. These data suggest that reduced levels of topoisomerase II alpha and II beta may contribute to the resistance of H69AR cells to VP-16 and other drugs that target these isoenzymes.

Evidence for the presence of complex high-molecular mass N-linked oligosaccharides in intranuclear glycoproteins from HeLa cells.

Nonhistone proteins were extracted in 0.4 M NaCl from membrane-depleted nuclei of HeLa cells grown in the presence or the absence of [5,6-3H]fucose. Control experiments strongly suggest that most extracted proteins were indeed nuclear components. Several proteins, present in the 0.4 M NaCl nuclear extract, with M(r) ranging from 35,000 to 115,000 were identified on Western blots as fucosylated glycoproteins owing to their binding to the fucose-specific lectin, Ulex europeus agglutinin I. Results of experiments involving mild alkaline treatment and peptide N-glycosidase F digestion showed that the carbohydrate moieties of these fucosylated nuclear glycoproteins were N-linked to the polypeptide backbone. Analysis of the N-glycans revealed the presence of two populations of sialylated oligosaccharides on the basis of their relative molecular masses. The sensitivity of the high-M(r) oligosaccharides to endo-beta-galactosidase and their incorporation of [3H]glucosamine suggest that they could contain repeating N-acetyllactosamine units. [3H]Fucose incorporated into nuclei was confined to the nucleoli, as judged by autoradiography of sections cut through cells grown in the presence of [3H]fucose. Electron microscopy autoradiography showed that the fibrillar centers were never labeled, while silver grains were observed on the dense and the granular components of nucleoli. Taking into account of these data most nuclear fucosylated glycoproteins extracted in 0.4 M NaCl might be nucleolar ribonucleoproteins.

Characterization of 3,5,3′-triiodothyronine receptors in primary cultures of hepatocytes and neurons from chick embryo

We have detected the presence of nuclear 3,5,3′-triiodothyronine (T 3) receptors in primary cultures of chick embryo hepatocytes and neurons. Hepatocytes were isolated from livers of embryos of 12, 16 and 19 days by treatment with 0.2% collagenase and hyaluronidase. They were plated at a density of 3–4 × 10 5 35 -mm petri dish in Ham's F-10 medium containing fetal calf serum, tryptose phosphate, and antibiotics. Cells were used for the binding assay at Day 3 of culture. Neurons from 8-day-old embryo brains were cultured in a serum-free medium at a density of 1.2 × 10 6 cells/35-mm petri dish and used for the binding assay after 7 days of culture. Biological activity of hepatocytes was determined by measuring insulin binding, inositol phosphate formation, and 5′-monodeiodinase activity. Neurons or glial cells in culture were identified by immunostaining with anti-neurofilaments and anti-glial fibrillary acidic protein antisera. Binding assay was performed with isolated nuclei and 0.4 M NaCl nuclear extracts. With the latter preparation, the Scatchard analysis showed, in both cells, a single, high-affinity, low-capacity T 3 receptor. In the hepatocytes of 12-, 16-, and 19-day-old embryos association constants ( K a ) were, respectively, 0.93 ± 0.02, 0.74 ± 0.03, and 0.56 ± 0.04 n M −1, whereas the maximal binding capacities (MBC) were 2.26 ± 0.2, 2.72 ± 0.33, and 1.83 ± 0.19 fmol/μg DNA (mean ± SE, n = 3). In neurons K a was 1.25 ± 0.53 n M −1 and MBC 0.59 ± 0.14 fmol/μg DNA ( n = 3). The receptor had a sedimentation coefficient of 3.4 S, an estimated M r of 59 kDa, and the following relative affinity for thyroid hormone analogues: TRIAC l-T 3 > l-T 4. These data indicate that cultured hepatocytes and neurons of chick embryo contained T3 receptors with properties similar to those described in intact tissues from this and other species. Only the MBC of neurons was 50% lower than that observed in whole brain of embryo, but was comparable to values observed in cultured neurons from other species.

Mitoxantrone resistance in HL-60 leukemia cells: reduced nuclear topoisomerase II catalytic activity and drug-induced DNA cleavage in association with reduced expression of the topoisomerase II beta isoform.

Mitoxantrone-resistant variants of the human HL-60 leukemia cell line are cross-resistant to several natural product and synthetic antineoplastic agents. The resistant cells (HL-60/MX2) retain sensitivity to the Vinca alkaloids vincristine and vinblastine, drugs that are typically associated with the classical multidrug resistance phenotype. Mitoxantrone accumulation and retention are equivalent in the sensitive and resistant cell types, suggesting that mitoxantrone resistance in HL-60/MX2 cells might be associated with an alteration in the type II DNA topoisomerases. We discovered that topoisomerase II catalytic activity in 1.0 M NaCl nuclear extracts from the HL-60/MX2 variant, as measured by the decatenation of Crithidia fasciculata kinetoplast DNA, was reduced 4- to 5-fold compared to that in the parental HL-60 cells. Total cellular topoisomerase II activity in HL-60/MX2 cells was only 50% lower than that in HL-60 cells, however, because the "cytosolic fraction" of the HL-60/MX2 nuclear preparation contained high levels of decatenating activity. Antisera to calf thymus topoisomerase II defined a distinctive immunoreactive pattern of topoisomerase II proteins in crude nuclear extracts from the HL-60/MX2 cells. Both alpha (170 kDa) and beta (180 kDa) forms of topoisomerase II were detected in the HL-60 cell extracts, but only the alpha form was detected in extracts from HL-60/MX2 cells. This finding was associated with the appearance of a new 160-kDa immunoreactive species in nuclear extracts from HL-60/MX2 but not HL-60 cells. Studies were designed to minimize the proteolytic degradation of the topoisomerase II enzymes by extraction of whole cells with hot SDS.(ABSTRACT TRUNCATED AT 250 WORDS)

Retinoic Acid Receptor Gene Expression in Human Skin

Human skin exhibits a characteristic, pleiotypic response to topical retinoic acid. In attempting to understand this response at the molecular level, we have used fast protein liquid chromatography (FPLC) and RNA blot hybridization to characterize the expression of the nuclear retinoic acid receptor (RAR) alpha, beta, and gamma genes in adult human epidermis. Size exclusion FPLC of 0.6 M NaCl nuclear extracts prepared from keratome biopsies revealed two peaks of specific [3H] retinoic acid (RA) binding at Mr 45 and 18 kDa, in agreement with the expected sizes of RAR and cellular RA binding protein. Blot hybridization analysis of total RNA extracted from keratome biopsies revealed that RAR-gamma was the predominant RAR species expressed in human epidermis, as RAR-alpha transcripts were detectable only at low levels and RAR-beta transcripts were undetectable. RAR transcripts were not induced by topical treatment with 0.1% RA cream under occlusion for 4 h or 4 d. Moreover, there was no significant difference in RAR-gamma transcript levels in normal and psoriatic epidermis. RAR-gamma transcripts were constitutively expressed not only in cultured human keratinocytes, but also in human dermal and lung fibroblasts. RAR-beta was induced by RA in dermal fibroblasts, but not in keratinocytes. RA induced IL-1 beta transcripts in keratinocytes rapidly (2 to 4 h) and at low concentrations (3 x 10(-10) M), consistent with activation of the IL-1 beta gene via RAR. These results demonstrate constitutive expression of RAR-gamma in human epidermis, and suggest that RAR-gamma is a molecular target of RA action in adult human skin.

Altered catalytic activity of and DNA cleavage by DNA topoisomerase II from human leukemic cells selected for resistance to VM-26.

The simultaneous development of resistance to the cytotoxic effects of several classes of natural product anticancer drugs, after exposure to only one of these agents, is referred to as multiple drug resistance (MDR). At least two distinct mechanisms for MDR have been postulated: that associated with P-glycoprotein and that thought to be due to an alteration in DNA topoisomerase II activity (at-MDR). We describe studies with two sublines of human leukemic CCRF-CEM cells approximately 50-fold resistant (CEM/VM-1) and approximately 140-fold resistant (CEM/VM-1-5) to VM-26, a drug known to interfere with DNA topoisomerase II activity. Each of these lines is cross-resistant to other drugs known to affect topoisomerase II but not cross-resistant to vinblastine, an inhibitor of mitotic spindle formation. We found little difference in the amount of immunoreactive DNA topoisomerase II in 1.0 M NaCl nuclear extracts of the two resistant and parental cell lines. However, topoisomerase II in nuclear extracts of the resistant sublines is altered in both catalytic activity (unknotting) of and DNA cleavage by this enzyme. Also, the rate at which catenation occurs is 20-30-fold slower with the CEM/VM-1-5 preparations. The effect of VM-26 on both strand passing and DNA cleavage is inversely related to the degree of primary resistance of each cell line. Our data support the hypothesis that at-MDR is due to an alteration in topoisomerase II or in a factor modulating its activity.

Expression of Human Cytomegalovirus Immediate Early Antigens Is Responsible for a Novel Chromatin Structure of Infected Human Embryonic Lung Cells

Whereas human embryonic lung (HEL) cells displayed chromatin fibers composed of a repeat of conventional nucleosomes of 15 nm in diameter, human cytomegalovirus (HCMV) infection induced transient appearance of a novel chromatin structure composed of a repeat of large ellipsoids of 45-65 nm X 15-30 nm with linkers of 50-60 nm long and 6-7 nm thick. Essentially the same change in chromatin structure could be induced when uninfected HEL cell nuclei were incubated in vitro with a 0.4 M NaCl nuclear extract from HCMV-infected HEL cells expressing immediate early antigens (IEA's) or with a similar nuclear extract from NIH/3T3 cells constitutively expressing HCMV IEA's. The latter cell line was established by transformation of the mouse cells with a plasmid carrying the HCMV major immediate early and immediate early 2 genes. These results together with those of control experiments suggest that the expression of IEA's is directly or indirectly responsible for the appearance of the novel chromatin structure in HCMV-infected HEL cells.

Diversity of nuclear phorbol ester tumor promoter receptors in mouse liver: Evidence for two classes of binding sites

Summary Two classes of phorbol ester binding sites have been demonstrated in the nuclear fraction of liver from female CD-1 mice. Equilibrium binding studies of 0.40 M NaCl nuclear extracts yielded a sigmoidal saturation curve which was resolved by Hill plot analysis into two components. However, when nuclei were extracted in 0.24 M NaCl, the 12-0-tetradecanoylphorbol-13-acetate saturation curve of the extract was biphasic in shape consisting of both high and low affinity binding sites termed Class I and II receptors. When the 0.24 M NaCl extracted nuclear pellet was extracted further in 0.40 M NaCl, saturation analysis of the extract revealed only the low affinity binding site (Class II). This is the first study to identify the existence of phorbol ester receptors in liver nuclei.

Poly(adenosine diphosphate ribose) polymerase activity in neuronal and glial nuclei from bovine cerebrum.

Two different preparations isolated from beef cerebrum have been used to compare the polyadenosine diphosphate ribose (polyADPR) polymerase activities in neuronal and glial nuclei: (1) nuclear suspensions (with or without DNase I treatment), and (2) 1 M NaCl nuclear extracts (soluble enzyme). The DNAse I treatment of nuclei and the solubilization of polyADPR polymerase by 1 M NaCl enhances the polyADPR polymerase activity. The polyADPR polymerase activity is similar in neuronal and glial nuclear suspensions, while the neuronal soluble enzyme activity is significantly higher than that of the glial soluble enzyme. Evidence is presented that the difference in soluble enzyme activities is not due to the effects of DNA or degrading enzymes. Some activating factor(s) seem to be present in neuronal soluble extracts, while both inhibiting and activating factor(s) seem to be present in glial soluble extracts.

Cytosol and nuclear [3H]oestradiol binding in the foetal tissues of guinea pig.

The formation of [3H]oestradiol macromolecule complexes was studied in vivo and in vitro in the kidney, lung and liver of intact foetal guinea pig. Specific binding of [3H]oestradiol was demonstrated in the cytosol and nuclear fractions of foetal kidney. Intensive binding was found in the cytosol of foetal lung but most of the bound radioactive material (70 %) was [3H]oestrone. Some binding was found in the cytosol of foetal liver but not in the nucleus of this tissue. In the in vivo experiments, the binding of radioactive material to plasma proteins was studied: 22 % of the plasma radioactivity was bound of which 67 % was identified as oestrone sulphate. Oestradiol sulphate represented 7% and unconjugated oestradiol only 0.6 % (0.1 % of the total plasma radioactivity). On the other hand, 2-3 % of the foetal plasma radioactivity was found as unbound [3H]oestradiol. In the kidney, the formation of [3H]oestradiol complexes in the cytosol fraction does not depend on temperature while nuclear [3H]oestradiol complexes increase with increasing temperature. Maximal formation of [3H]oestradiol complexes in the cytosol fraction and the 0.1 M TRIS and 0.3 M NaCl nuclear extracts was reached after 15 min but binding in the 1 M NaCl nuclear extract continued to increase up to 30 min. After incubation of purified nuclei of foetal kidney with 1.1 x 10(-7) M [3H]oestradiol, specific binding was found in the different nucelar fractions. Specific binding was also detected in isolated nuclei previously extracted by 0.1 M TRIS and 0.3 M NaCl before being incubated with 1.1 x 10(-7) M [3H]oestradiol. The Kd of binding of [3H]oestradiol in the renal cytosol fraction is 2.5 x 10(-10) M with n = 4.5 x 10(-14) moles/mg protein. Incubation of isolated 1 M NaCl nuclear extract from foetal kidney with [3H]oestradiol gives a Kd of 3.3 x 10(-10) M with n = 2.5 x 10(-14) moles/mg protein. It is concluded that the nuclear complexes in the foetal kidney could be formed either through an intermediate cytosol complex or that the "two step" mechanism could take place in the nucleus. Furthermore, direct binding of [3H]oestradiol with high affinity was observed in the 1 M NaCl nuclear extract.

Steroid hormone receptors in fetal guinea-pig kidney

Cytosol and nuclear subcellular fractions isolated from fetal kidney of guinea pigs (35–55 days of gestation) form steroid macromolecule complexes both “ in vivo” and “ in vitro” with [ 3H]-aldosterone or [ 3H]-estradiol but not with [ 3H]-progesterone. d-Aldosterone competes in the formation of the cytosol and nuclear [ 3H]-aldosterone complexes but estradiol has no effect. Estradiol competes in the formation of the [ 3H]-estradiol complexes of both the cytosol and the nuclear extracts but d-aldosterone has no effect, suggesting the presence of two different receptors for these hormones in the fetal kidney during this period of development. In the [ 3H]-estradiol experiments 30 min after administration of this hormone to the fetus or 15 min after incubation at 37°C with the cell suspensions of the fetal kidney, the bulk of the [ 3H]-complexes (60–70%) is located in the nucleus; of this, 60% is found in the chromatin fraction. 62–95 % of the radioactivity in the complexes consists of non-metabolized estradiol. In addition to estradiol, estrone and estriol were found to compete for binding sites albeit less intensively, whereas testosterone and cortisol had no effect. Ultracentrifugation in a sucrose density gradient (in 0.5 M NaCl) of the 0.3 M NaCl and 1 M NaCl nuclear extracts yielded a component with a sedimentation coefficient of 3.7 S in which estradiol had a significant competitive effect. Experiments carried out with the [ 3H]-estradiol cytosol-complex obtained at 4°C and then reincubated with the purified nuclei at 4° or 37°C show that the quantity of [ 3H]-estradiol found in the different nuclear extracts was 2.5–3 times greater at 37°C than at 4°C. By the application of the Scatchard method, the presence of two binding sites was established, the first with a dissociation constant of 2.5 × 10 −10 M and the second with a K d of 7.7 × 10 −9 M. When [ 3H]-estrone was administered subcutaneously “ in situ” to the fetus, it was noted that most of the radioactive material in the 3H-macromolecule complexes of the cytosol and nuclear extracts of the kidney is [ 3H]-estradiol. It is concluded that during this period of fetal development (35–55 days of gestation): estradiol receptors are present in the fetal kidney; in the interconversion of estrone ai estradiol in the same fetal tissue the reaction proceeds predominantly in the formation of estradiol.

Mineralocorticosteroid receptors in the foetal compartment

Mineralocorticosteroid receptors have been found in the foetal kidney of the guinea pig (at 25–40 days of gestation) in experiments carried out both in vivo and in vitro. More than 50% of the total d-aldosterone receptors were found in the nucleus. 20% of the total nuclear radio-activity was extracted by the 0.1 M Tris-HCl solution and 50% by the 1 M NaCl -0.01 M Tris solution, suggesting that at this period of the foetal evolution the aldosterone receptors are localized principally in the chromatin fraction. The formation of these [ 3H]-aldosteronc-macro-molecule complexes is very rapid, maximum values being found at 4 min of incubation at 37°C. The nuclear receptors, but not those of the cytosol fraction, are temperature-dependent, d-Aldosterone and deoxycorticosterone compete with the [ 3H]-aldosterone complex. On the other hand, d-aldosterone has no effect on the [ 3H]-aldosterone-macromolecule complex if reincubation is carried out with the nuclear extracts (at 4°C) after extraction. Also, the 0.1 M Tris and 0.01 M Tris-1 M NaCl nuclear extracts do not form [ 3H]-aldosterone complexes after incubation (separately or combined) at 4°C or 37°C, suggesting that the configuration necessary for the formation of the [ 3H]-aldosterone complexes is altered by the extraction procedure. Incubation of the purified nucleus produces more [ 3H]-aldosterone-macromolecule complexes (per mg of protein) than incubation with the total cell or with the crude nuclear fraction. These data suggest that unbound aldosterone can cross the nuclear membrane and form the aldosterone complexes without the participation of a cytosol intermediate.