Retention of estrogen receptors in vitro requires limited estradiol exposure in vivo.

Maintenance of functional estrogen receptors in culture has been accomplished in chick oviduct cells by manipulating the estrogen exposure before tissue dissociation. Tissue from chicks pre-treated with daily 17-beta-estradiol injections for 2 weeks or with 2 weekly diethylstilbestrol implants can be established in culture using a variety of enzymes. Tissue from animals with chronic estrogen stimulation must be withdrawn from hormone in culture at least 4 days before the digestion procedure. When tissue is digested using collagenase and pancreatin buffered by bovine serum albumin (Fraction V), large quantities of virtually fibroblast-free cultures can be established. The estrogen and progesterone receptors remain intact at normal levels using this procedure. The receptors have maintained biological function as evidenced by two hormone-dependent measurements. The first was an increase in the amount of ovalbumin mRNA transcribed in response to estrogen supplementation of the cultures compared to cultures with no estrogen. The second function was an increase in ovalbumin protein secreted into the medium upon estrogen stimulation. The protein increment demonstrated that the hormone-induced levels of mRNA were functional and capable of being translated.

A similar 5'-flanking region is required for estrogen and progesterone induction of ovalbumin gene expression.

We have previously transferred an ovalbumin-beta-globin fusion gene (ovalglobin) into primary cultures of chick oviduct cells and demonstrated that an ovalbumin gene 5'-flanking sequence between -221 and -95 is necessary for progesterone-mediated transcriptional induction (Dean, D. C., Knoll, B. J., Riser, M. E., and O'Malley, B. W. (1983) Nature (Lond.) 305, 551-554). Here we compare 5'-flanking sequences required for induction of the ovalglobin gene by 17 beta-estradiol and progesterone. The early gene of simian virus 40 was inserted into the same plasmid as the ovalbumin fusion gene to serve as an internal control. Since transcription of the viral early gene was unaffected by the presence of steroid hormone or deletions in the ovalbumin gene 5'-flanking region, the level of its transcripts could be monitored as a reference standard for ovalglobin transcription. Ovalglobin transcripts initiated principally from the ovalbumin cap site in the presence or absence of progesterone and 17 beta-estradiol. Deletion of 5'-flanking sequences to -197 had little effect on the induction with either hormone, while successive deletions to -180, -161, and -143 resulted in a gradual decrease in the level of induction. Deletion to -95 eliminated the induction. The results of this study indicate that DNA control elements for regulation of the ovalbumin gene by estrogen and progesterone either overlap directly or are clustered in close proximity in the 5'-flanking region near the ovalbumin gene promoter.

A 5'-flanking sequence essential for progesterone regulation of an ovalbumin fusion gene.

Ovalbumin gene transcripts are not detectable in unstimulated chick oviducts but comprise about half of oviduct cell transcripts after steroid hormone induction. This seems to result mostly from an increased level of transcription. When steroid hormones enter the cytoplasm of target cells they bind to specific receptors and the steroid-receptor complex accumulates in the nucleus. Presumably this complex then binds in a sequence- or conformation-specific way near the regulated gene and enhances transcription. Several recent studies have shown that steroid hormone receptors bind preferentially to the 5'-flanking region of steroid-responsive genes in vitro. Transcription of cloned genes for alpha 2u globulin, growth hormone, mouse mammary tumour virus and lysozyme can be induced in vivo by steroid hormones after transfer to cells containing steroid hormone receptors. In some of these studies, 5'-flanking regions were shown to be important for steroid regulation. We have now constructed a hybrid gene containing the ovalbumin gene promoter fused to the chicken adult beta-globin gene and transferred it into primary cultures of chicken oviduct cells. We show that progesterone-mediated induction of transcription in untransformed oviduct cells depends on an ovalbumin gene flanking sequence between positions -95 and -222.

The chorionic gonadotropin alpha-subunit gene is on human chromosome 18 in JEG cells.

The gene for the alpha subunit of human chorionic gonadotropin (hCG) has been tentatively assigned to human chromosome 18. This localization was accomplished through the use of Southern blot analysis. A full-length cDNA probe for the hCG alpha subunit and DNA isolated from a series of somatic hybrids between mouse and human cells were utilized to make this assignment. In addition, in situ hybridization with normal human peripheral blood lymphocytes as a source of human chromosomes and with the same cDNA probe confirmed this result. The presence of human chromosome 18 was required for the detection of DNA fragments characteristic of the alpha-hCG gene. These results are consistent with our previous observation that human chromosomes 10 and 18 are required for the production of hCG in cultured cells.

Regulated expression of the chicken ovalbumin gene in a human estrogen-responsive cell line.

To study the regulation of expression of the chicken ovalbumin gene by steroid hormones, the entire ovalbumin gene and its flanking sequences were cloned together with the bacterial gene for xanthine-guanine phosphoribosyltransferase in plasmid pBR322. This recombinant plasmid was linearized and used to transform an estrogen-responsive breast carcinoma cell line (MCF-7) which was shown to possess estrogen receptors and to be estrogen responsive. Transformants were selected by their ability to grow in a medium containing mycophenolic acid and xanthine. The entire ovalbumin gene was integrated into high molecular weight DNA within all transformants analyzed and it retained its original sequence organization. Ovalbumin mRNA and protein were identified from these transformant cells and they were found to be indistinguishable from the authentic counterparts. An 8- to 10-fold increase in the amount of ovalbumin mRNA was observed to be present in cells cultured in 10(-8)M estradiol. We also constructed a hybrid gene containing the 5'-flanking sequence and the first exon of the ovalbumin gene which was linked to the xanthine-guanine phosphoribosyltransferase gene such that expression of this bacterial gene would be promoted and regulated by the chicken sequences. After introduction of this hybrid gene into MCF-7 cells, we observed that the survival of the transformed cells in our selection medium was highly dependent on the presence of estradiol. Our results indicated that the chicken ovalbumin sequence was expressed properly and was regulated to some extent by estradiol in this heterologous system.

Pepsin can be used to subculture viable mammary epithelial cells.

Normal mouse mammary epithelial cells in primary culture can be passaged as viable single cells using 0.5 to 1.0 mg/ml pepsin in Hanks' salt solution. After 5 min the pepsin treatment preferentially removes fibroblasts, leaving a monolayer of purified epithelial cells that can be removed by pipetting and transferred to new culture vessels or injected into animals.

Increased expression of one of two adenosine deaminase alleles in a human choriocarcinoma cell line following selection with adenine nucleosides.

JEG-3 is a human choriocarcinoma cell line characterized by low levels of adenosine deaminase expression. For the purpose of studying adenosine deaminase gene regulation in the JEG-3 cells, we attempted to select variant cells having increased adenosine deaminase expression. This was accomplished by selecting cells for resistance to the cytotoxic adenosine analogs 9-beta-D-arabinofuranosyl adenine (ara-A) or 9-beta-D-xylofuranosyl adenine (xyl-A), both of which could presumably be detoxified by the action of adenosine deaminase. Single step high dose selection was ineffective in obtaining cells with increased adenosine deaminase. However, multistep selection using either ara-A or xyl-A resulted in cell populations with increased adenosine deaminase activity. Removal of selective pressure resulted in decreased adenosine deaminase levels. Subclones of xyl-A-resistant cells belonged to one of three phenotypic classes characterized by either elevated adenosine deaminase levels, decreased adenosine kinase levels, or both of these features. One subclone (A3-1A7) with unaltered adenosine kinase expression showed a 20-fold increase in adenosine deaminase expression. Further selection of this subclone for increasing xyl-A resistance resulted in an additional 2-fold increase in adenosine deaminase expression, followed by loss of adenosine kinase expression. These adenosine kinase-deficient cells showed no subsequent increase in adenosine deaminase expression in response to further xyl-A selection pressure. These results confirmed that xyl-A toxicity was mediated through its phosphorylated form and indicated that resistance may result from increased adenosine deaminase levels and/or adenosine kinase deficiency. The increased adenosine deaminase expression of the A3-1A7 subclone was exclusively in the ADA 2 allelic form. However, cell fusion experiments between A3-1A7 cells and mouse C1-1D cells established the existence of functional copies of both ADA 1 and ADA 2 allelic genes in the A3-1A7 cells. The increased expression of only one of the two functional ADA alleles, the requirement for a stepwise selection protocol to obtain cells with increased adenosine deaminase, and the instability of the adenosine deaminase phenotype in the absence of selective pressure suggest that the alteration of adenosine deaminase phenotype in the drug-resistant cells was the result of adenosine deaminase gene amplification.

Selective overproduction of adenosine deaminase in cultured mouse cells.

The objective of this work was to isolate cultured mouse cells with amplified adenosine deaminase genes. Such cell lines should be very useful in an effort to obtain the protein and nucleic acid probes required to study adenosine deaminase gene structure and regulation. Since adenosine deaminase expression is not required for growth of cells in culture, the first step necessary to isolate adenosine deaminase gene amplification mutants was to devise selective conditions in which adenosine deaminase activity was required for survival. This was accomplished by developing a new selection system, termed 11AAU, which selected simultaneously for adenosine deaminase and adenosine kinase. The 11AAU selection medium consists of alanosine (0.05 mM) to block de novo AMP biosynthesis, adenosine (1.1 mM) to provide a salvage route for AMP biosynthesis via the adenosine kinase reaction, and uridine (1.0 mM) to alleviate the block in UMP biosynthesis caused by adenosine at the concentration employed. Because adenosine is highly cytotoxic at 1.1 mM, adenosine deaminase expression is required to detoxify excess adenosine by converting it to inosine. We used 11AAU selection in conjunction with stepwise selection for increasing resistance to deoxycoformycin, an adenosine deaminase inhibitor, to obtain highly drug-resistant cells with a 6000-fold increase in adenosine deaminase activity. Adenosine deaminase accounted for approximately 50% of the soluble protein in highly drug-resistant lines and was indistinguishable from that in the parent as judged by isoelectric focusing, electrophoretic mobility on starch gels, and by deoxycoformycin binding studies. Increased adenosine deaminase was also correlated with the presence of numerous double-minutes, cytogenetic structures indicating the presence of amplified DNA. Growth in the absence of selection was accompanied with the loss of double-minutes and a ten-fold decline in adenosine deaminase levels. Based on the stepwise selection protocol employed, the instability of the phenotype, and the presence of double-minutes, we believe that the increased adenosine deaminase is most likely the result of amplification of adenosine deaminase genes.

Human insulinoma hybrids produce proinsulin-like material.

We have established somatic cell hybrids by fusing cells from two human insulinomas with an established murine cell line LMTK- Cl1D. After selection of the hybrids, the media were analyzed and found to contain insulin and human C-peptide immunoreactive material. The newly synthesized material was further characterized by pulse labeling and immunoprecipitation, and shown in five hybrid lines on Sephadex G-50 chromatography to have a size similar to proinsulin. The hybrids produced the apparent proinsulin-like material for up to 7 mo. Chromosome composition of the hybrids was determined by isozyme analysis and banding techniques. Chromosome 11, which previously has been assigned the insulin gene using cDNA probes, was identified in the hybrids producing proinsulin-like material. However, the retention of this chromosome did not always assure the production of hormone. This independent technique has confirmed the localization of the insulin gene to chromosome 11 and offers the opportunity of studying insulin processing and developing continuous insulin-producing cell lines.

Naturally soluble component(s) that confer(s) guanine nucleotide and fluoride sensitivity to adenylate cyclase.

Supernatant fractions (300,000 x g, 60 min) from homogenates of rat liver, heart, and skeletal muscle, dog liver, and rabbit liver prepared without detergent in the homogenization medium (referred to as S300) are shown to contain an activity that restores Mg2+-dependent fluoride- and guanine nucleotide-stimulated cyclizing activity to the adenylate cyclase system [ATP pyrophosphate-lyase (cyclizing); EC 4.6.1.1] in cyc- S49 murine lymphoma cell membranes. Approximately 25% of the total cyc- reconstituting activity in the above tissues is present in S300. Reconstituting activity is proportional to S300, is sensitive to trypsin, is protected against heat inactivation by guanine nucleotide, and has a sedimentation coefficient of 5.3 in both H2O and 2H2O linear sucrose density gradients. Treatment with cholera toxin and NAD+ results in reconstitution of cyc- adenylate cyclase with enhanced activity in the presence of GTP. Reconstituion with S300 is stable, as seen in cyc- membranes after washing. All of these properties of S300 are similar to those of membrane-derived cyc- reconstituting activity. It is concluded that cell cytoplasm contains a naturally soluble protein or mixture of proteins having guanine nucleotide regulatory component activity of adenylate cyclase.

A human trophoblastic isozyme (lactate dehydrogenase-Z) associated with choriocarcinoma.

A unique electrophoretic form of lactate dehydrogenase (LDH-Z), formerly observed in a choriocarcinoma cell line (JEG-3) and first-trimester placenta, has been shown to be the same as that produced in hydatidiform mole and term placenta. We have also observed LDH-Z in second-trimester placenta, choriocarcinoma metastasized to the liver, and five of five additional independently derived (from different patients) choriocarcinoma cell lines. The only exception to the production of LDH-Z in a choriocarcinoma was in the cell line BEWO, which was established from the same tumor as JEG-3. Since BEWO has been in culture for over 200 passages more than any of the independently derived lines, its lack of LDH-Z is viewed as being consistent with the loss of certain expressional characteristics upon such long-term culture. Analysis of JEG-3 subclones revealed the expression of LDH-Z to be independent of the products of the LDH-A genetic locus. Present data do not allow us to determine whether LDH-Z is the product of a newly discovered LDH locus or is a modified form of the product of the LDH-B locus. LDH-Z has not been observed in other human tissues, nor have we observed it in homogenates prepared from over 60 cell lines established from a wide variety of human neoplasms. We therefore conclude that LDH-Z is an isozyme associated with human choriocarcinoma and is indicative of the trophoblastic origin of the cells.

Ovalbumin is synthesized in mouse cells transformed with the natural chicken ovalbumin gene.

The entire chicken ovalbumin gene, accompanied by genomic DNA sequences flanking both termini of the gene and three copies of the herpes simplex virus thymidine kinase gene, has been cloned in plasmid pBR322. This recombinant plasmid was linearized and used to transform thymidine kinase-deficient mouse cells. Thymidine kinase-positive transformants were selected by their ability to grow in the hypoxanthin/aminopterin/thymidine (HAT) medium. The entire ovalbumin gene integrated into high molecular weight DNA within all the transformants and retained its original sequence organization. In all of the transformants examined, a protein identified as chicken ovalbumin by immunoreactivity was detected within the cells. It is estimated that between 1000 and 100,000 molecules of chicken ovalbumin were produced per mouse cell in each of these transformants. Our results demonstrate that the mouse cellular machinery can be utilized to accurately express genetic information encoded in a cloned gene from a different eukaryotic organism into its specific protein product.

Necessity for two human chromosomes for human chorionic gonadotropin production in human-mouse hybrids.

Through a series of human-mouse hybrids we have identified that two human chromosomes, 10 and 18, must be present for production of the pregnancy protein hormone human chorionic gonadotropin (hCG). Human choriocarcinoma cells producing hCG were hybridized to mouse cells. From 49 independent clones three hybrid clones continued to produce whole hCG. Chromosomal analysis was done on the 3 producer clones and 5 nonproducer clones. The additional 41 nonproducer clones were genetically characterized by isozymes. Only when chromosomes 10 and 18 were present in a clone would the whole hCG molecule be produced. Clones with only 10 or only 18 did not produce hormone. Nine subclones of a producer clone confirmed this observation. Three subclones retaining both 10 and 18 continued to produce hCG. This study demonstrated the need to use cellular chromosome data and population enzyme data to identify two chromosomes necessary for hCG production in heterogeneous human-mouse hybrids.