Regulation of progesterone receptor-mediated transcription by phosphorylation.

The progesterone receptor (PR) in the chicken oviduct is a phosphoprotein that regulates gene transcription in the presence of progesterone. Treatment with progesterone in vivo stimulates phosphorylation of the progesterone receptor. With transient transfection assays, the present work has tested whether phosphorylation participates in the regulation of PR-mediated transcription. Treatment with 8-bromo-cyclic adenosine monophosphate (8-Br cAMP), a stimulator of cAMP-dependent protein kinase [protein kinase A (PKA)], mimicked progesterone-dependent, receptor-mediated transcription in the absence of progesterone. Inhibition of PKA blocked hormone action. Treatment with okadaic acid, an inhibitor of protein phosphatases 1 and 2A, stimulated transcription in a manner similar to that of progesterone. These observations suggest that phosphorylation of the PR or other proteins in the transcription complex can modulate PR-mediated transcription in vivo.

Molecular cloning of the chicken progesterone receptor.

To define the functional domains of the progesterone receptor required for gene regulation, complementary DNA (cDNA) clones encoding the chicken progesterone receptor have been isolated from a chicken oviduct lambda gt11 cDNA expression library. Positive clones expressed antigenic determinants that cross-reacted with six monospecific antibodies derived from two independent sources. A 36-amino acid peptide sequence obtained by microsequencing of purified progesterone receptor was encoded by nucleotide sequences in the longest cDNA clone. Analysis of the amino acid sequence of the progesterone receptor deduced from the cDNA clones revealed a cysteine-rich region that was homologous to a region found in the estrogen and glucocorticoid receptors and to the avian erythroblastosis virus gag-erb-A fusion protein. Northern blot analysis with chicken progesterone receptor cDNA's indicated the existence of at least three messenger RNA species. These messages were found only in oviduct and could be induced by estrogens.

Independent sensitivity of human tumor cell lines to interferon and double-stranded RNA.

The antiproliferative effect of human interferons (IFNs) and double-stranded RNAs (dsRNAs) was measured in eight human tumor cell lines, five of which were derived from carcinomas of the bladder. Dose-response curves were generated for a 72-hr treatment period. The concentration of interferon or dsRNA necessary to inhibit tumor cell growth 50% compared to untreated cells was generated by linear regression analysis of the dose-response data. In the seven of eight cell lines in which a direct comparison could be made, IFN-beta was a more potent inhibitor than IFN-alpha. Polyriboinosinic acid X polyribocytidylic acid consistently gave an increased antiproliferative response compared to its mismatched analogue, rln X r(C12,U)n. Correlations could not be made between either IFN-alpha or IFN-beta and the dsRNA effect. No correlation was seen between IFN or dsRNA sensitivity and cell type, ability to bind IFN, growth rate, or tumorigenicity in nude mice. The antiproliferative effect of dsRNA was studied in the presence of antibodies against IFN-beta in HT1080 Cl 4, a cell line sensitive to both IFN and dsRNA, and A2182, a cell line relatively resistant to IFN-beta but sensitive to dsRNA. In both cell lines, the anti-IFN-beta antibodies inhibited the antiproliferative effect of the dsRNAs. After treatment with a concentration of dsRNA necessary to inhibit tumor cell growth 50% compared to untreated cells, a concentration of IFN-beta necessary to inhibit tumor cell growth 50% was induced in the HT1080 Cl 4 cells; however, only a low level of IFN-beta was detected in the culture medium of the A2182 cells.

In vivo sensitivity and resistance of chronic myelogenous leukemia cells to alpha-interferon: correlation with receptor binding and induction of 2',5'-oligoadenylate synthetase.

Fourteen patients with chronic myelogenous leukemia were treated with partially pure leukocyte interferon (HuIFN alpha). The binding of recombinant leukocyte clone A IFN and the induction of 2',5'-oligoadenylate synthetase (2,5A) in peripheral blood cells were studied to determine whether they correlate with clinical response to IFN therapy. The mean pretherapy binding of radiolabeled recombinant leukocyte clone A IFN to peripheral blood cells was 0.053 +/- 0.02 (SE) fmol (53 +/- 20 amol)/10(6) cells and 0.049 +/- 0.015 fmol/10(6) cells in sensitive and resistant patients, respectively. Twenty-four h after the first HuIFN alpha dose, the binding of recombinant leukocyte clone A IFN decreased 3- to 8-fold in both sensitive and resistant patients. The activity of 2,5A synthetase was induced approximately 100-fold in sensitive patients from a pretherapy mean of 3 +/- 2 nmol/mg to a maximum of 317 +/- 184 nmol/mg during therapy. In contrast, 2,5A synthetase was induced from a pretherapy mean of 0.9 +/- 0.9 nmol/mg to only 6.7 +/- 4.9 nmol/mg in resistant patients. In two patients originally sensitive to HuIFN alpha who developed resistance to therapy, receptors were present in both sensitive and resistant disease stages and appeared to down regulate with therapy regardless of response. In these two patients, 2,5A synthetase was significantly induced with therapy in the sensitive stage but not in the resistant stage. This study shows that lack of clinical response to interferon therapy may coincide with failure to induce 2,5A synthetase activity. This suggests that resistance to alpha-interferon therapy may be mediated by events beyond receptor binding resulting in a failure to induce enzymes responsible for mediation of interferon antiproliferative effects.

High efficiency gene transfection by electroporation using a radio-frequency electric field.

In order to develop a safe and effective way to introduce exogenous genes into cells, we have experimented with a new method of electroporation which uses a radio-frequency (RF) electric field to permeabilize the cell membrane. This RF method has several advantages over the conventional electroporation method which uses a direct current (DC) field. We have shown that the RF electroporation method can be used to introduce marker genes into a wide variety of cell lines, including COS-M6, CV-1, CHO, 3T3 and hepatocytes, and is able to increase substantially the efficiency of gene transfection. (For example, the amount of DNA required for transfecting two million COS-M6 cells can be as low as 0.1 microgram). The transfection efficiency is shown to be affected by a number of factors, including cell type, field strength, pulse protocol and medium buffer. Because of its wide range of applications, high transfection efficiency and lack of harmful side-effect, the RF electroporation method would be particularly useful for introducing genes into human cells for gene therapy.

Structural organization and regulation of the chicken estrogen receptor.

We have cloned the chicken estrogen receptor (ER) from a chicken oviduct lambda gt11 library using the human ER cDNA sequence. This chicken ER sequence is virtually identical to the recently published sequence. One noteable difference is an amino acid change from glutamine to arginine located toward the central region of the sequence. The size of the ER protein predicted from the 589 amino acids is approximately 66,000 which fits well with the range of molecular weights previously published for the calf uterine and human ER (65,000-70,000). We observed the size of the chicken ER mRNA to be approximately 7.8 kilobases which is in agreement with the previously published size of 7.5 kilobases. In vivo secondary stimulation of chicken oviduct total RNA with diethylstilbestrol does not induce chicken ER mRNA. A time course following the chicken ER mRNA levels after secondary stimulation with diethylstilbestrol indicated a decrease in mRNA levels 8 h after DES administration. A similar study was performed using progesterone for the secondary stimulation. An increase in the chicken ER mRNA levels was observed 24 h after stimulation with progesterone. Two regions of very high homology were delineated by analyzing the sequence of this chicken ER cDNA and comparing it to the sequences of the human ER, human glucocorticoid, and chicken progesterone receptors and the P75-erbA fusion product of the avian erythroblastosis virus. The first concensus region is 72 amino acids in length and the second region of high homology is 62 amino acids long. Detailed comparisons of these regions for the steroid hormone receptors and v-erb A are presented. Possible functions for the individual regions of high homology are discussed.

Structure-function properties of the chicken progesterone receptor A synthesized from complementary deoxyribonucleic acid.

The chicken progesterone receptor (PR) cDNA has been cloned and sequenced in our laboratory. Functional receptor A was synthesized from cDNA in two independent systems, by transient transfection of receptor-negative COS M6 cells and by in vitro transcription and translation. These receptors exhibited DNA and hormone binding properties similar to the native PR from oviduct. The ability of receptor to induce target gene transcription was measured by cotransfection of receptor-negative CV-1 cells with expression vectors containing the receptor A cDNA and a progesterone-inducible promotor linked to the chloramphenicol acetyl transferase (CAT) gene. In these assays, receptor A produced hormone-dependent induction of CAT activity. In order to define the functional domains of receptor A, expression constructs coding for C-terminal deletion proteins were prepared. Deletion of the C-terminus resulted in loss of hormone binding activity as well as a loss of CAT induction. However, when 290 amino acids were removed from the C-terminus, this severely truncated receptor protein produced hormone-independent target gene activation. Mutant receptor proteins which retained the highly conserved cysteine-rich (C1) region were able to bind to DNA-cellulose, although removal of 290 amino acids from the C-terminus resulted in reduced affinity for DNA. Deletion of part or all of the C1 region resulted in loss of both DNA-binding and transcriptional activation capacities. These results confirm that C1 functions in DNA binding and transcriptional activation and that hormone binding activity can be localized to the C-terminal half of the protein.

Polymorphism, genetic stability, and autosomal location of trimeric nucleoside phosphorylase in Peromyscus eremicus cell lines.

Nucleoside phosphorylase (NP: EC 2.4.2.1) has not been demonstrated to be an extensively polymorphic enzyme locus in mammals. We have studied NP electrophoretically in five independently derived cell lines established from Peromyscus eremicus as well as in various tissues of a sixth animal. Four different NP phenotypes involving three different alleles were resolved. The data suggest that (1) the enzyme is trimeric and its genetic locus is polymorphic in P. eremicus, (2) heterozygous enzyme phenotypes are stable during long-term culture, and (3) the enzyme locus is autosomal in Peromyscus.

Nucleotide metabolism in Drosophila melanogaster salivary glands during temperature and dinitrophenol-induced puffing.

When Drosophila melanogaster salivary glands are exposed to temperature or dinitrophenol (DNP) treatments, their nucleotide metabolism is altered such that the amount of cellular ATP decreases, whereas the amount of ADP increases. Since both treatments also elicit specific puffing patterns on the salivary gland chromosome, it is suggested that specific loci on the chromosome are activated to form puffs when the efficiency of respiration decreases. The possible relationship between the formation of these puffs and the intramitochondrial metabolism is discussed.

Down-regulation of peripheral blood cell interferon receptors in chronic myelogenous leukemia patients undergoing human interferon (HuIFN alpha) therapy.

Our interest in studying interferon (IFN) receptor activity in peripheral blood cells (PBCs) from patients with chronic myelogenous leukemia (CML) receiving therapeutic doses of partially purified leukocyte IFN (IFN alpha) stems from a need for more adequate monitoring of IFN therapy. The binding of 35S-labelled recombinant DNA-derived leukocyte clone A IFN (35S-rIFN alpha A) to PBCs from 8 patients with CML was determined before and during IFN alpha treatment. The patients' mean pretherapy binding level (0.049 femtomoles of bound 35S-rIFN alpha A) was in the range of values obtained from 4 normal donors (mean of 0.054 femtomoles bound). Within 24 hr of the first IFN alpha dose, the mean femtomoles bound decreased 10-fold and remained low during the course of IFN alpha treatment. In I/I patient, we demonstrated that this decreased binding was due to a loss in number of IFN receptors. The apparent number of receptors after 5 doses of IFN alpha decreased from approximately 600 receptors per cell at pretherapy to approximately 75 receptors per cell, with no difference in the dissociation constants (1.13 X 10(-10)M, 0.968 X 10(-10)M, before and during treatment, respectively). In 4/4 patients, we demonstrated indirectly that the decreased binding was not due to receptor saturation as a result of residual circulating IFN alpha. In 3/3 patients, we demonstrated a gradual recovery of binding capacity after incubating the patients' PBCs at 37 degrees C. Within 2-7 days in vivo recovery of binding, comparable to pretherapy levels, was observed in 3/3 patients whose IFN alpha therapy was discontinued. Combining all these data, we conclude that in both responding and nonresponding patients with CML, IFN alpha exposure induces decreased binding of labelled IFN when a single recombinant DNA-derived IFN species is used. We feel the supporting data indicate that the decreased binding capacity may be due to receptor down-regulation. In the limited number of patients studied thus far, there was no correlation between clinical hematologic response and occurrence of down-regulation, however, down-regulation of cell surface receptors may be required to sustain a biological effect. Further studies of both the kinetics of down-regulation and activation of key enzyme systems are required to fully evaluate the relevance of these findings.

Asynaptic behavior of X and Y chromosomes in the Virginia oppossum and the southern pygmy mouse.

The meiotic behavior of silver-stained X and Y chromosomes of the Virginia opossum, Didelphis virginiana, and the southern pygmy mouse, Baiomys musculus, was studied by light microscopy. While the sex chromosomes of these two species differ in both size and morphology, their meiotic behavior is very similar. In both species a typical sex vesicle is formed during pachytene, but unlike most other mammalian species thus far studied, a synaptonemal complex does not form between the X and Y chromosomes. During pachytene, variable modes of association of the ends of the sex chromosomes (both heterologous and autologous) are sometimes observed. During diplotene, diakinesis, and metaphase I, the X and Y rarely appear to be associated. In some pachytene nuclei of B. musculus, the single axis of the biarmed X chromosome folds back upon itself, and the telomeres form a close association. While synaptic pairing segments of the sex chromosomes of the Virginia opossum may be displaced by telomeric heterochromatin on the X, true pairing regions between the X and Y of the pygmy mouse may be lacking.

Leukocyte interferon conjugated to beta-D-galactosidase for receptor studies: a preliminary report.

In this report, we describe a simple procedure for labeling leukocyte interferon (IFN) with the enzyme beta-D-galactosidase (beta-gal), through a low-molecular-weight linking agent. The labeling procedure did not appear to alter the properties of the enzyme, the antiviral activity of the recombinant DNA-derived human clone A leukocyte IFN (rIFN alpha A), or the ability of the IFN to bind to Daudi cells. The binding of the putative IFN-enzyme complex to Daudi cells could be blocked by excess unlabeled IFN or by monoclonal antibody to IFN receptors. Our preliminary results indicate that enzyme labeling of IFN can result in a biologically active agent that has potential for use in the quantitation of circulating IFN and specific receptor proteins. In addition, this labeling procedure may be potentially useful with other immunomodulatory proteins.

Efferent connections of the medial prefrontal cortex in the rabbit.

The different cytoarchitectonic regions of the medial prefrontal cortex (mPFC) have recently been shown to play divergent roles in associative learning in rabbits. To determine if these subareas of the mPFC, including areas 24 (anterior cingulate cortex), 25 (infralimbic cortex), and 32 (prelimbic cortex) have differential efferent connections with other cortical and subcortical areas in the rabbit, anterograde and retrograde tracing experiments were performed using the Phaseolus vulgaris leukoagglutinin (PHA-L), and horseradish peroxidase (HRP) techniques. All three areas showed local dorsal-ventral projections into each of the other areas, and a contralateral projection to the homologous area on the other side of the brain. All three also revealed a trajectory through the striatum, resulting in heavy innervation of the caudate nucleus, the claustrum, and a lighter projection to the agranular insular cortex. The thalamic projections of areas 24 and 32 were similar, but not identical, with projections to the mediodorsal nucleus (MD) and all of the midline nuclei. However, the primary thalamic projections from area 25 were to the intralaminar and midline nuclei. All three areas also projected to the ventromedial and to a lesser extent to the ventral posterior thalamic nuclei. Projections were also observed in the lateral hypothalamus, in an area just lateral to the descending limb of the fornix. Amygdala projections from areas 32 and 24 were primarily to the lateral, basolateral and basomedial nuclei, but area 25 also projected to the central nucleus. All three areas also showed projections to the midbrain periaqueductal central gray, median raphe nucleus, ventral tegmental area, substantia nigra, locus coeruleus and pontine nuclei. However, only areas 24 and the more dorsal portions of area 32 projected to the superior colliculus. Area 25 and the ventral portions of area 32 also showed a bilateral projection to the parabrachial nuclei and dorsal and ventral medulla. The dorsal portions of area 32, and all of area 24 were, however, devoid of these projections. It is suggested that these differential projections are responsible for the diverse roles that the cytoarchitectonic subfields of the mPFC have been demonstrated to play in associative learning.

Desensitization and cyclic AMP turnover in S49 cells.

Measurements of cAMP accumulation and turnover have been used to quantitate desensitization of S49 wild type and variant cells to epinephrine stimulation. The extent of desensitization varied with the cell type and increased with temperature. Two traditional methods for detecting desensitization (the existence of a peak in the time course of accumulation or a diminution of the response to an agonist after prior exposure to the agent) did not always demonstrate its existence. That is, those methods always underestimated the degree of desensitization, sometimes to the extent of obscuring the phenomenon altogether.

The A and B forms of the chicken progesterone receptor arise by alternate initiation of translation of a unique mRNA.

In order to establish the origin of the A and B proteins of the chicken progesterone receptor we have expressed its cDNA in vivo in heterologous cells and in vitro in reticulocyte cell lysates. The A and B proteins were expressed from a single cDNA both in heterologous receptor negative cells and in a cell-free system. Both proteins bind progesterone and are indistinguishable from chick oviduct authentic A and B proteins in terms of size, immunoreactivity and hormone binding properties. Truncated mRNA's which lack the receptor B protein translation signal are capable of generating the receptor A protein by initiation of translation at a second internal start site. We conclude from these data that the chicken progesterone receptor A and B proteins arise most likely by alternate initiation of translation from a single mRNA transcript.

Structure-function relationships of the chicken progesterone receptor.

To define the functional domains of the chicken progesterone receptor (CPR) and to establish the origin of the receptor A and B proteins, we have cloned and sequenced the complete cDNA encoding the CPR. The receptor A and B proteins arise by alternate initiation of translation from a single mRNA transcript. Using deletion mutants in the receptor A cDNA, we have determined that the DNA binding and gene activator regions reside in the central portion of the protein, while the hormone binding domain is located in the C-terminal region. The target gene activator site of the receptor is modulated by interaction with the hormone binding domain.

Mutational analysis of the chicken progesterone receptor.

Oligonucleotide-directed site mutagenesis was used to prepare a series of chicken progesterone receptor deletion mutants in an attempt to elucidate structure-function relationships of the receptor. These mutants spanned the entire 659-amino acid coding region of the A form of the receptor. The ability of these mutants to bind progesterone was analyzed following in vitro transcription and translation. Results obtained indicate that a large portion of the protein ranging from amino acid 420 to the extreme carboxyl terminus is necessary to maintain the protein in a conformation which is capable of binding hormone. Following transient cotransfection of mutant receptor proteins into CV-1 cells along with a reporter gene containing an authentic GRE/PRE (PRE-TK-CAT), our results indicated that any deletion throughout the entire molecule results in a decrease in transcriptional activation. Most of these decreases result from an inability of the mutant receptor proteins to bind DNA or hormone. However, two areas of the receptor have been identified which are unrelated to either DNA or hormone binding but markedly affect the ability of the receptor to transactivate target genes.

Antibodies to chromosome 21 coded cell surface components block binding of human alpha interferon but not gamma interferon to human cells.

Antisera raised against a human X mouse hybrid cell line containing human chromosome 21 as its only human chromosome, block induction of an antiviral state by human alpha interferon (IFN-alpha), block induction of (2'-5')oligoisoadenylate synthetase [2'-5')A synthetase), and block binding of 125I-labeled and 35S-labeled recombinant, human IFN-alpha A, but not 125I-labeled IFN-gamma, to cell surface receptors. The data presented clearly demonstrate that the cell surface receptors for IFN-alpha and IFN-gamma are different, and provide independent evidence of the role of a chromosome 21 coded cell surface molecule in the pathway to the generation of the antiviral state.