Cell surface tagging and a suicide mechanism in a single chimeric human protein.

Many therapeutic uses of gene-modified cells could benefit from inclusion of a surface marker for immunoselecting transduced cells. Another desired feature is a failsafe mechanism to ablate engineered cells if required. We describe here a system that combines a cell surface tag and an inducible apoptosis mechanism in a single protein. Spencer et al. (Curr. Biol. 1996;6:839-847) described an inducible cell suicide gene containing a myristoylation sequence, the human protein FKBP12, and the intracellular domain of Fas. Cells expressing this protein apoptose on treatment with a cell-permeable chemical dimerizing agent that binds two FKBP domains and cross-links the chimeric Fas proteins. We modified this system by anchoring a Fas-FKBP construct to the membrane with the extracellular and transmembrane domains of the low-affinity nerve growth factor receptor (LNGFR), thereby uniting cell surface tagging with the inducible apoptosis mechanism. Cells retrovirally transduced with this construct apoptosed on exposure to a chemical dimerizer, AP1903 (Clackson et al., Proc. Natl. Acad. Sci. U.S.A. 1998;95:10437-10442). The LNGFR-tagged construct showed an unpredicted clear advantage over the myristoylation-anchored construct in its efficiency of signaling in HT1080 cells. This linked marker and failsafe mechanism may have particularly attractive safety properties for gene therapy. The use of gene-modified cells in basic research and clinical studies is enhanced by the use of a selectable surface marker for immunoselection of transduced cells. Another desired feature for gene and cell therapies is an inducible suicide system to eliminate transduced cells when necessary. Spencer et al. (Curr. Biol. 1996;6:839-847) described a potential failsafe mechanism whereby exposure of cells to a chemical dimerizing agent activates the Fas-mediated apoptotic pathway. In this system, the intracellular signaling domain of Fas is linked to one or more copies of the human protein FKBP12. Treatment of engineered cells with a cell-permeable chemical dimerizing agent that simultaneously binds to two FKBP domains cross-links the chimeric Fas protein and induces apoptosis. Here, we modify the system by anchoring a Fas-FKBP construct to the membrane with the extracellular domain of the low-affinity nerve growth factor receptor (LNGFR), to unite cell surface tagging of transduced cells with the inducible apoptosis mechanism. Cells retrovirally transduced with this construct undergo apoptosis on exposure to a chemical dimerizer, AP1903. A linked marker and failsafe mechanism may have particularly attractive safety properties for gene therapy.

Synthesis and activity of bivalent FKBP12 ligands for the regulated dimerization of proteins.

The total synthesis and in vitro activities of a series of chemical inducers of dimerization (CIDs) is described. The use of small-molecule CIDs to control the dimerization of engineered FKBP12-containing fusion proteins has been demonstrated to have broad utility in biological research as well as potential medical applications in gene and cell therapies. The facility and flexibility of preparation make this new class of wholly synthetic compounds exceptionally versatile tools for the study of intracellular signaling events mediated by protein-protein interactions or protein localization. While some congeners possess potency comparable to or better than the first generation natural product-derived CID, FK1012, structure-activity relationships are complex and underscore the need for application-specific compound optimizations.

Redesigning an FKBP-ligand interface to generate chemical dimerizers with novel specificity.

FKBP ligand homodimers can be used to activate signaling events inside cells and animals that have been engineered to express fusions between appropriate signaling domains and FKBP. However, use of these dimerizers in vivo is potentially limited by ligand binding to endogenous FKBP. We have designed ligands that bind specifically to a mutated FKBP over the wild-type protein by remodeling an FKBP-ligand interface to introduce a specificity binding pocket. A compound bearing an ethyl substituent in place of a carbonyl group exhibited sub-nanomolar affinity and 1,000-fold selectivity for a mutant FKBP with a compensating truncation of a phenylalanine residue. Structural and functional analysis of the new pocket showed that recognition is surprisingly relaxed, with the modified ligand only partially filling the engineered cavity. We incorporated the specificity pocket into a fusion protein containing FKBP and the intracellular domain of the Fas receptor. Cells expressing this modified chimeric protein potently underwent apoptosis in response to AP1903, a homodimer of the modified ligand, both in culture and when implanted into mice. Remodeled dimerizers such as AP1903 are ideal reagents for controlling the activities of cells that have been modified by gene therapy procedures, without interference from endogenous FKBP.

A versatile synthetic dimerizer for the regulation of protein-protein interactions.

The use of low molecular weight organic compounds to induce dimerization or oligomerization of engineered proteins has wide-ranging utility in biological research as well as in gene and cell therapies. Chemically induced dimerization can be used to activate intracellular signal transduction pathways or to control the activity of a bipartite transcription factor. Dimerizer systems based on the natural products cyclosporin, FK506, rapamycin, and coumermycin have been described. However, owing to the complexity of these compounds, adjusting their binding or pharmacological properties by chemical modification is difficult. We have investigated several families of readily prepared, totally synthetic, cell-permeable dimerizers composed of ligands for human FKBP12. These molecules have significantly reduced complexity and greater adaptability than natural product dimers. We report here the efficacies of several of these new synthetic compounds in regulating two types of protein dimerization events inside engineered cells--induction of apoptosis through dimerization of engineered Fas proteins and regulation of transcription through dimerization of transcription factor fusion proteins. One dimerizer in particular, AP1510, proved to be exceptionally potent and versatile in all experimental contexts tested.

A humanized system for pharmacologic control of gene expression.

Gene therapy was originally conceived as a medical intervention to replace or correct defective genes in patients with inherited disorders. However, it may have much broader potential as an alternative delivery platform for protein therapeutics, such as cytokines, hormones, antibodies and novel engineered proteins. One key technical barrier to the widespread implementation of this form of therapy is the need for precise control over the level of protein production. A suitable system for pharmacologic control of therapeutic gene expression would permit precise titration of gene product dosage, intermittent or pulsatile treatment, and ready termination of therapy by withdrawal of the activating drug. We set out to design such a system with the following properties: (1) low baseline expression and high induction ratio; (2) positive control by an orally bioavailable small-molecule drug; (3) reduced potential for immune recognition through the exclusive use of human proteins; and (4) modularity to allow the independent optimization of each component using the tools of protein engineering. We report here the properties of this system and demonstrate its use to control circulating levels of human growth hormone in mice implanted with engineered human cells.

The cystic fibrosis transmembrane conductance regulator is a dual ATP and chloride channel.

The cystic fibrosis transmembrane conductance regulator (CFTR) belongs to a superfamily of proteins implicated in the transport of ions, proteins, and hydrophobic substances. Recent studies have demonstrated that CFTR is a protein kinase A-sensitive anion channel regulated by ATP. In the present study, patch-clamp techniques were used to assess the role of CFTR in the transport of Cl- and ATP. The stable transfection of mouse mammary carcinoma cells, C127i, with the cDNA for human CFTR resulted in the appearance of a diphenylamine-2-carboxylate-inhibitable Cl- channel, which was activated by cAMP under whole-cell and cell-attached conditions and by protein kinase A plus ATP under excised, inside-out conditions. CFTR expression was also associated with the electrodiffusional movement of ATP as indicated by the cAMP activation of ATP currents measured under whole-cell conditions. In excised, inside-out patches, it was demonstrated that ATP currents were mediated by ATP-conductive channels, which were also activated by protein kinase A and blocked by the Cl- channel blocker diphenylamine-2-carboxylate under excised, inside-out conditions. Single-channel currents observed in the presence of asymmetrical Cl-/ATP concentrations indicated that the same conductive pathway was responsible for both ATP and Cl- movement. Thus, CFTR is a multifunctional protein with more than one anion transport capability and may modify signal transduction pathways for Cl- or other secretory processes by the selective delivery of nucleotides to the extracellular domain.

External ATP and its analogs activate the cystic fibrosis transmembrane conductance regulator by a cyclic AMP-independent mechanism.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel activated by protein kinase A and regulated by ATP in a complex manner. We have applied patch-clamp techniques to C127i mouse mammary carcinoma cells transfected with human CFTR to assess the role of external ATP in the modulation of CFTR function. Extracellular ATP was sufficient to activate non-rectifying, Cl(-)-selective whole-cell currents in CFTR-transfected, but not mock-transfected cells. The ATP-mediated activation of CFTR was independent of protein kinase A since channel activation by ATP was preserved in cells that were (a) depleted of intracellular ATP, (b) incubated with the cAMP antagonist Rp-cAMPS, or (c) exposed to the protein kinase A inhibitor, 5-24 amide. In each of these conditions, 8-Br-cAMP was no longer capable of activating CFTR. The possibility that the extracellular ATP activation of Cl- currents in CFTR-expressing C127i cells was mediated by a P2-type purinergic receptor was supported by studies in which the effect of external ATP on the Cl- currents was mimicked by the ATP analogs, ATP gamma S and beta,gamma-methylene ATP, but not the uridine nucleotide, UTP. Single-channel analysis of ATP-activated Cl -currents under both cell-attached and excised, inside-out patch-clamp configurations indicated that this channel is only present in CFTR-transfected cells and indistinguishable from CFTR. External ATP also activated ATP currents in CFTR-transfected cells, a novel function of CFTR. These findings are consistent with the presence of a purinergic receptor signal transduction mechanism in C127i cells whose activation by external ATP is linked to the activation of CFTR in a cAMP-independent manner. The data provide additional support for the use of ATP and its analogs as alternative therapies in cystic fibrosis.

Stoichiometry of recombinant cystic fibrosis transmembrane conductance regulator in epithelial cells and its functional reconstitution into cells in vitro.

We have generated several clones of Chinese hamster ovary, mouse epitheloid C127, and pig kidney epithelial LLCPK1 cells producing high levels of functional recombinant human cystic fibrosis transmembrane conductance regulator (CFTR). Processing of CFTR to the mature and fully glycosylated form in these cells is inefficient with only approximately 40% of all newly synthesized CFTR being converted to the mature form. Furthermore, expression of the most frequent mutant allele of the cystic fibrosis (CF) gene, the delta F508 mutant in these epithelial cells, indicated that it is biosynthetically arrested at the endoplasmic reticulum and fails to traffic to the plasma membrane. Using a combination of CFTR mutants and monoclonal antibodies, all the detectable recombinant CFTR in these cells was determined at least under the conditions used, to be present as a monomer. To demonstrate the feasibility of protein replacement therapy, we were able to effect the physical transfer of functional recombinant CFTR produced in Chinese hamster ovary cells to the plasma membranes of Ha3b fibroblasts, a cell line devoid of cAMP-stimulated chloride channels. Transfer of CFTR was mediated by the hemagglutinin viral fusion protein of influenza virus present on the Ha3b cells. Efficiency of transfer was up to 25% of the target cells, and CFTR chloride channel activity was detectable for up to 12 h post-fusion. Therefore, with the development of an appropriate formulation of fusogenic proteoliposome or virosome containing reconstituted purified CFTR, it should be feasible to introduce functional CFTR into CF-affected cells.

Processing of mutant cystic fibrosis transmembrane conductance regulator is temperature-sensitive.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a plasma membrane Cl- channel regulated by cyclic AMP-dependent phosphorylation and by intracellular ATP. Mutations in CFTR cause cystic fibrosis partly through loss of cAMP-regulated Cl- permeability from the plasma membrane of affected epithelia. The most common mutation in cystic fibrosis is deletion of phenylalanine at residue 508 (CFTR delta F508) (ref. 10). Studies on the biosynthesis and localization of CFTR delta F508 indicate that the mutant protein is not processed correctly and, as a result, is not delivered to the plasma membrane. These conclusions are consistent with earlier functional studies which failed to detect cAMP-stimulated Cl- channels in cells expressing CFTR delta F508 (refs 16, 17). Chloride channel activity was detected, however, when CFTR delta F508 was expressed in Xenopus oocytes, Vero cells and Sf9 insect cells. Because oocytes and Sf9 cells are typically maintained at lower temperatures than mammalian cells, and because processing of nascent proteins can be sensitive to temperature, we tested the effect of temperature on the processing of CFTR delta F508. Here we show that the processing of CFTR delta F508 reverts towards that of wild-type as the incubation temperature is reduced. When the processing defect is corrected, cAMP-regulated Cl- channels appear in the plasma membrane. These results reconcile previous contradictory observations and suggest that the mutant most commonly associated with cystic fibrosis is temperature-sensitive.

Intracellular protein trafficking defects in human disease.

Secretory proteins and integral membrane proteins travel through the secretory pathway to a variety of destinations. Their targets are often specified by signals in the amino acid sequence or signals added post-translationally. The KDEL sequence that retains soluble proteins in the endoplasmic reticulum and the mannose 6-phosphate group of lysosomal enzymes are well-characterized examples of targeting signals; other signals are less well understood. Given the complexity and importance of the intracellular trafficking pathways, it is perhaps not surprising that mutations that affect the trafficking of proteins are associated with some human genetic diseases.

Intracellular degradation of unassembled asialoglycoprotein receptor subunits: a pre-Golgi, nonlysosomal endoproteolytic cleavage.

The human asialoglycoprotein receptor is a heterooligomer of the two homologous subunits H1 and H2. As occurs for other oligomeric receptors, not all of the newly made subunits are assembled in the RER into oligomers and some of each chain is degraded. We studied the degradation of the unassembled H2 subunit in fibroblasts that only express H2 (45,000 mol wt) and degrade all of it. After a 30 min lag, H2 is degraded with a half-life of 30 min. We identified a 35-kD intermediate in H2 degradation; it is the COOH-terminal, exoplasmic domain of H2. After a 90-min chase, all remaining intact H2 and the 35-kD fragment were endoglycosidase H sensitive, suggesting that the cleavage generating the 35-kD intermediate occurs without translocation to the medial Golgi compartment. Treatment of cells with leupeptin, chloroquine, or NH4Cl did not affect H2 degradation. Monensin slowed but did not block degradation. Incubation at 18-20 degrees C slowed the degradation dramatically and caused an increase in intracellular H2, suggesting that a membrane trafficking event occurs before H2 is degraded. Immunofluorescence microscopy of cells with or without an 18 degrees C preincubation showed a colocalization of H2 with the ER and not with the Golgi complex. We conclude that H2 is not degraded in lysosomes and never reaches the medial Golgi compartment in an intact form, but rather degradation is initiated in a pre-Golgi compartment, possibly part of the ER. The 35-kD fragment of H2 may define an initial proteolytic cleavage in the ER.

Specific mRNA destabilization in Dictyostelium discoideum requires RNA synthesis.

We tested the effects of inhibitors of protein and RNA synthesis on the disaggregation-mediated destabilization of prespore mRNAs in Dictyostelium discoideum. Incubating disaggregated cells with daunomycin to inhibit RNA synthesis prevented the loss of prespore mRNAs, whereas the inhibitor decreased or did not affect levels of the common mRNAs CZ22 and actin. Protein synthesis inhibitors varied in their effects. Cycloheximide, which inhibited protein synthesis almost completely, prevented the loss of the prespore mRNAs, but puromycin, which inhibited protein synthesis less well, did not. These results indicate that the process of specific mRNA destabilization requires the synthesis of RNA and possibly of protein.

Regulation of prolactin production and cell growth by estradiol: difference in sensitivity to estradiol occurs at level of messenger ribonucleic acid accumulation.

17 beta-Estradiol increases the growth rate of GH4C1 cells with a half-maximally effective concentration (EC50) that is about 10-fold less than the EC50 for the stimulation of PRL production. We have examined the effects of five other estrogens: estriol, estrone, 17 alpha-estradiol, and the metabolism-resistant analogs ethynyl estradiol and moxestrol. All were full agonists for both effects, and all were more potent for the stimulation of cell growth than for stimulation of PRL production. The order of analog potency for both biological effects was the same as the order of potency for inhibiting saturable [3H]estradiol binding to intact cells. Therefore, both biological effects appear to be mediated through the same receptor, and metabolism of 17 beta-estradiol is unlikely to account for the difference in the concentrations required to elicit the two effects. We selected two estrogen-responsive clones from a cDNA library made from GH4C1 cells. The clones were chosen because they were induced at the estrogen concentrations that stimulate growth. Estradiol caused maximal stimulation of the mRNAs corresponding to the two recombinant clones at 10(-10) M, a concentration over 10-fold lower than that required for maximal stimulation of PRL mRNA. These data indicate that a difference in sensitivity to estrogen occurs at the level of mRNA accumulation as well as at the level of the biological responses.

Antiestrogens are partial estrogen agonists for prolactin production in primary pituitary cultures.

The antiestrogens Ly-117018 and tamoxifen increased prolactin production about 2-fold in primary cultures of male rat anterior pituitary cells. The dose-response relationship was biphasic; 10(-10) M Ly-117018 and 10(-7) M tamoxifen caused maximal stimulation, but higher concentrations caused no stimulation and completely antagonized the 5-fold stimulation caused by estrogen. The calmodulin antagonists, trifluoperazine, pimozide and W7 also prevented estrogen induction of prolactin production. Increasing concentrations of estradiol reversed inhibition by tamoxifen but not by pimozide. These results indicate that, in normal pituitary cells, estrogen antagonists may behave as partial agonists at low concentrations and as full antagonists at higher concentrations (tamoxifen, prolactin, calmodulin, 17 beta-estradiol).

Characterization of antiestrogen stimulation of cell number and prolactin production.

The antiestrogens LY117018 and tamoxifen increased prolactin production about 4-fold and cell number about 2.5-fold in the pituitary tumor cell line, GH4C1; these increases were 30-40% of the maximal effects of estradiol. The antiestrogens competed with binding of [3H]estradiol, and LY117018 was more active than tamoxifen in biological activities and binding activity. The antiestrogens inhibited stimulation caused by 10(-10) M estradiol; the inhibition could be overcome by increased estradiol concentrations. Tamoxifen and LY117018 increased the amount of prolactin mRNA per cell. These antiestrogens behave as partial agonists in the GH4C1 cells, but have two unusual features. Estrogens are approximately 10-fold more potent in stimulating cell number than in stimulating prolactin production, but the antiestrogens showed the same dose-response for both effects. The partial agonist activity was biphasic and at higher concentrations the antiestrogens showed more antagonist activity (GH4C1 cells, 17 beta-estradiol, tamoxifen).

17 beta-Estradiol has a biphasic effect on gh cell growth.

Estrogen causes mammotroph and pituitary tumor growth in vivo; we therefore studied its effect on growth of GH cells, a clonal line of rat pituitary tumor cells. Medium supplemented with gelding serum enabled us to detect effects of estradiol which are masked by the higher estrogen concentrations of commercially available horse serum. GH cells were treated with estradiol for 7 days. Cell number was affected in a biphasic fashion: estradiol at 10(-11) M and 3 X 10(-11) M increased cell number 6- to 13-fold. Concentrations between 3 X 10(-11) M and 10(-9) M caused a dose-dependent decrease from the maximal cell number. The half maximal concentration for this inhibitory effect was about 10(-10) M. Prolactin production was stimulated 3-to 11-fold by estradiol, also with a half-maximal concentration of 10(-10) M. We conclude that estradiol has at least 3 distinguishable effects on GH cells: stimulation of growth at concentrations below 3 X 10(-11) M, and inhibition of growth at higher concentrations, as well as the well known stimulation of prolactin production.