An amino-terminal truncated progesterone receptor isoform, PRc, enhances progestin-induced transcriptional activity.

Previously we reported the identification of two unique progesterone receptor (PR) messenger RNA transcripts that encode a smaller PR isoform, termed the C-receptor (PRc). These two PR transcripts encode a protein that is N-terminally truncated, so that it lacks the first zinc finger of the DNA binding domain, but still contains a complete hormone binding region with sequences for dimerization and nuclear localization. We also have demonstrated the existence of a 60-kDa progestin-specific binding protein in progestin target cells using a monoclonal antibody directed to the C-terminus of PRs, suggesting that these two novel transcripts generate a truncated form of PR. In this paper, we address the hypothesis that the C-receptor arises from the initiation of translation of a methionine C-terminal to the methionine start sites that generate the larger 94-kDa A and 116-kDa B human PR isoforms. The studies shown here support the postulate that another downstream in-frame methionine within the PR-coding region can serve as a translation initiation site for the generation of a third PR protein. A partial PR complementary DNA, lacking the translation start sites for B- and A-receptors was translated in vitro. The synthetic protein product bound [3H]progestins and unlabeled progestins. The antiprogestin RU486 also competed for this binding. Transfection of this partial PR complementary DNA into PR-negative HeLa cells resulted in progestin-specific binding activity. Because the third PR isoform lacks the first zinc finger of the DNA binding domain, but contains sequences for dimerization, we reasoned that the C-receptor isoform would be transcriptionally in-active and not bind DNA directly. Surprisingly, however, in the presence of A- and/or B-receptors, we found that C-receptors can modulate the transcriptional activity of A- and/or B-receptors using a reporter gene. These studies emphasize that multiple receptor isoforms may have distinct biological properties, and that the truncated C-receptor may play a role in explaining some of the pleiotropic effects of progestins.

Human progesterone receptor transformation and nuclear down-regulation are independent of phosphorylation.

We have studied the phosphorylation of progesterone receptors (PR) in T47Dco human breast cancer cells using a monoclonal antibody directed against human PR called AB-52. This antibody recognizes both the A- (Mr approximately 94,000) and B- (Mr approximately 120,000) hormone binding proteins of PR, and was used to immunoprecipitate phosphorylated receptors isolated from cells incubated in vivo with [32P]orthophosphate. The specific activity, or phosphorylation levels, relative to protein levels was quantified by combined immunoblotting and autoradiography followed by densitometry. We find that immunopurified untransformed hormone-free receptors, which have a characteristic triplet B, singlet A structure, are phosphoproteins with similar levels of phosphate incorporation in all protein bands. If PR are first transformed to the nuclear binding form by treatment of cells with progesterone, and then labeled with [32P]orthophosphate, the receptor proteins are additionally phosphorylated. These chromatin-bound hormone occupied receptors incorporate five to 10 times more labeled phosphate per total receptor protein than do PR from untreated cells during the same [32P]incubation time. The second round of phosphorylation may also account for mobility shifts of transformed A- and B-receptors observed in sodium dodecyl sulfate-polyacrylamide gels. Both untransformed and transformed species of A- and B-receptors are phosphorylated only on serine residues, and neither the extent of phosphorylation, nor the phosphoamino acids, are affected by treatment of the cells with epidermal growth factor or insulin. We previously reported that after hormone binding and transformation of receptors to the tight chromatin binding state, PR undergo processing, or nuclear down-regulation. AB-52 was used to compare PR protein and phosphorylation levels when cells were treated for 0.5-48 h with progesterone or the synthetic progestin R5020. Both agonists lead to hyperphosphorylation of nuclear PR before phosphorylation levels decrease, in parallel with the drop in protein levels as receptors down-regulate. Treatment of cells with RU 486, an antiprogestin, leads to PR transformation as determined by immunoblotting, but subsequent down-regulation does not occur. After transformation, chromatin-bound RU 486-occupied receptors become intensely phosphorylated however, with specific activities 15 times greater than those of untransformed PR. Since these receptors are phosphorylated but not processed, the hormone-induced nuclear phosphorylation of PR is unlikely to be a signal for receptor processing.(ABSTRACT TRUNCATED AT 400 WORDS)

The use and development of retroviral vectors to deliver cytokine genes for cancer therapy.

In this review, we describe technical advancements of retroviral vectors to address issues of safety, titer, and clinical scale manufacturing to produce high-quality retroviral vector preparations that have made direct intratumoral administration of cytokine encoding recombinant vectors a feasible cancer therapy in the clinic. We also review possible further advances in retroviral vector design, which may prove important in expanding these clinical applications.

Development of a flow microcalorimetry method for the assessment of surface properties of powders.

PURPOSE: This study describes the development of a microcalorimetric flow cell which allows powder surface energetics to be probed my means of measuring their interaction with water vapour. METHODS: A flow cell has been constructed and tested in an isothermal microcalorimeter to assess the interaction between water vapour and powder surfaces. The cell was constructed to mix two separate air streams (0% and 100% relative humidity respectively) to create any humidity at a standard flow rate. The powder sample was equilibrated in dry air and then exposed to sequential increments in humidity. RESULTS: Adsorption isotherms were constructed from the cumulative heat as a function of humidity. It was possible to differentiate between different samples of alpha-lactose monohydrate (which appeared identical by contact angle determination). It was also possible to measure adsorption to two different alkyl p-hydroxybenzoates which were hydrophobic and of low surface area. CONCLUSIONS: This technique offers a very sensitive and versatile method of obtaining a reliable indication of powder surface energetics and as such is a major advance in the field.

Phosphotryptic peptide analysis of human progesterone receptor. New phosphorylated sites formed in nuclei after hormone treatment.

Human progesterone receptors (PR) exist as two independent naturally occurring steroid-binding forms of approximately 120 kDa (B-receptors) and 94 kDa (A-receptors). Both are phosphorylated in hormone-untreated T47Dco breast cancer cells. Hormone treatment leads to receptor transformation and an increased phosphorylation state: the 32P-labeling intensity is 3-5 times higher after progestin treatment and 8-10 times higher after RU 486 treatment. Only serine residues are phosphorylated. To determine whether there are unique phosphorylation sites in transformed nuclear PR, we analyzed the phosphopeptides of untransformed and transformed A- and B-receptors by tryptic cleavage and reverse-phase high pressure liquid chromatography. Untransformed A- and B-receptors share at least five common phosphopeptides, and a sixth is unique to B. Following transformation by either R5020 or RU 486, A-receptors generate at least six and B-receptors seven phosphopeptides. Compared with untransformed PR, there are at least two different phosphopeptides in transformed nuclear PR. Cyanogen bromide cleavage of transformed nuclear A-receptors, which lack the proximal 165 amino-terminal residues of the 933 amino acid B-receptors, produces two large fragments of approximately 43 and 19 kDa. These fragments contain all of the 32P label and comprise amino acids 165-595. Cleavage of transformed B-receptors also produces peptides of 43 and 19 kDa plus an additional 36-kDa fragment corresponding to residues 1-165. No 32P-labeled low molecular mass peptides are detected. Thus, all the hormone-dependent phosphoserine residues produced in nuclei are located in the first 595 amino acids of human PR, representing the amino terminus and 28 residues of the DNA-binding domain.

Synthesis of human progesterone receptors in T47D cells. Nascent A- and B-receptors are active without a phosphorylation-dependent post-translational maturation step.

Human progesterone receptors (PR) are structurally complex. At basal states there are two forms: A-receptors of approximately 94 kDa and B-receptors which are triplets of approximately 114, 117, and 120 kDa. All the proteins bind hormone and are phosphorylated. By using PR-rich T47Dco human breast cancer cells, pulse-labeling with [35S]methionine, and receptor immunopurification with anti-PR monoclonal antibodies, we show that PR are synthesized as single B-proteins of 114 kDa and single A-proteins of 94 kDa. The mature B-triplets form 6-10 h later by post-translational phosphorylation at sites restricted to the B-proteins. This slow maturation is not required for PR activation to hormone binding states, however, since A- and B-receptors that are less than 15 min old respond to progestins by undergoing transformation and nuclear binding accompanied by a rapid secondary phosphorylation common to both proteins. These studies explain the complex structure of the mature human B-receptors and the transformed A- and B-receptors, and address issues dealing with A- and B-proreceptor synthesis and receptor activation rates.

Cloning of an SNF2/SWI2-related protein that binds specifically to the SPH motifs of the SV40 enhancer and to the HIV-1 promoter.

We have isolated a human cDNA clone encoding HIP116, a protein that binds to the SPH repeats of the SV40 enhancer and to the TATA/inhibitor region of the human immunodeficiency virus (HIV)-1 promoter. The predicted HIP116 protein is related to the yeast SNF2/SWI2 transcription factor and to other members of this extended family and contains seven domains similar to those found in the vaccinia NTP1 ATPase. Interestingly, HIP116 also contains a C3HC4 zinc-binding motif (RING finger) interspersed between the ATPase motifs in an arrangement similar to that found in the yeast RAD5 and RAD16 proteins. The HIP116 amino terminus is unique among the members of this family, and houses a specific DNA-binding domain. Antiserum raised against HIP116 recognizes a 116-kDa nuclear protein in Western blots and specifically supershifts SV40 and HIV-1 protein-DNA complexes in gel shift experiments. The binding site for HIP116 on the SV40 enhancer directly overlaps the site for TEF-1, and like TEF-1, binding of HIP116 to the SV40 enhancer is destroyed by mutations that inhibit SPH enhancer activity in vivo. Purified fractions of HIP116 display strong ATPase activity that is preferentially stimulated by SPH DNA and can be inhibited specifically by antibodies to HIP116. These findings suggest that HIP116 might affect transcription, directly or indirectly, by acting as a DNA binding site-specific ATPase.

Histone acetyltransferases regulate HIV-1 enhancer activity in vitro.

Specific inhibitors of histone deacetylase, such as trichostatin A (TSA) and trapoxin (TPX), are potent inducers of HIV-1 transcription in latently infected T-cell lines. Activation of the integrated HIV-1 promoter is accompanied by the loss or rearrangement of a positioned nucleosome (nuc-1) near the viral RNA start site. Here we show that TSA strongly induces HIV-1 transcription on chromatin in vitro, concomitant with an enhancer factor-assisted increase in the level of acetylated histone H4. TSA treatment, however, did not detectably alter enhancer factor binding or the positioning of nuc-1 on the majority of the chromatin templates indicating that protein acetylation and chromatin remodeling may be limiting steps that occur only on transcriptionally competent templates, or that remodeling of nuc-1 requires additional factors. To assess the number of active chromatin templates in vitro, transcription was limited to a single round with low levels of the detergent Sarkosyl. Remarkably, HIV-1 transcription on chromatin was found to arise from a small number of active templates that can each support nearly 100 rounds of transcription, and TSA increased the number of active templates in each round. In contrast, transcription on naked DNA was limited to only a few rounds and was not responsive to TSA. We conclude that HIV-1 enhancer complexes greatly facilitate transcription reinitiation on chromatin in vitro, and act at a limiting step to promote the acetylation of histones or other transcription factors required for HIV-1 enhancer activity.

Distinct roles for P-CREB and LEF-1 in TCR alpha enhancer assembly and activation on chromatin templates in vitro.

The distal enhancer of the T-cell receptor (TCR) alpha chain gene has become a paradigm for studies of the assembly and activity of architectural enhancer complexes. Here we have reconstituted regulated TCR alpha enhancer activity in vitro on chromatin templates using purified T-cell transcription factors (LEF-1, AML1, and Ets-1) and the cyclic AMP-responsive transcription factor CREB. When added in combination, these factors activate the TCR alpha enhancer in a highly synergistic manner. Alternatively, the enhancer could also be activated in vitro by high levels of either CREB or a complex containing all of the T-cell proteins (LEF-1, AML1, and Ets-1). Phosphorylation of CREB by protein kinase A enhanced transcription 10-fold in vitro, and this effect was abolished by a point mutation affecting the CREB PKA phosphorylation site (Ser-133). Interestingly, LEF-1 strongly enhanced the binding of the AML1/Ets-1 complex on chromatin, but not nonchromatin, templates. A LEF-1 mutant containing only the HMG DNA-binding domain was sufficient to form a higher-order complex with AML1/Ets-1, but exhibited only partial activity in transcription. We conclude that the T cell-enriched proteins assemble on the enhancer independently of CREB and function synergistically with CREB to activate the TCR alpha enhancer in a chromatin environment.

Activation of the HIV-1 enhancer by the LEF-1 HMG protein on nucleosome-assembled DNA in vitro.

Lymphoid enhancer-binding factor 1 (LEF-1) is a regulatory high mobility group (HMG) protein that activates the T cell receptor alpha (TCR alpha) enhancer in a context-restricted manner in T cells. In this paper we demonstrate that the distal region of the human immunodeficiency virus-1 (HIV-1) enhancer, which contains DNA-binding sites for LEF-1 and Ets-1, also provides a functional context for activation by LEF-1. First, we show that mutations in the LEF-1-binding site inhibit the activity of multimerized copies of the HIV-1 enhancer in Jurkat T cells, and that LEF-1/GAL4 can activate a GAL4-substituted HIV-1 enhancer 80- to 100-fold in vivo. Second, recombinant LEF-1 is shown to activate HIV-1 transcription on chromatin-assembled DNA in vitro. By using a nucleosome-assembly system derived from Drosophila embryos, we find that the packaging of DNA into chromatin in vitro strongly represses HIV-1 transcription and that repression can be counteracted efficiently by preincubation of the DNA with LEF-1 (or LEF-1 and Ets-1) supplemented with fractions containing the promoter-binding protein, Sp1. Addition of TFE-3, which binds to an E-box motif upstream of the LEF-1 and Ets-1 sites, further augments transcription in this system. Individually or collectively, none of the three enhancer-binding proteins (LEF-1, Ets-1, and TFE-3) could activate transcription in the absence of Sp1. A truncation mutant of LEF-1 (HMG-88), which contains the HMG box but lacks the trans-activation domain, did not activate transcription from nucleosomal DNA, indicating that bending of DNA by the HMG domain is not sufficient to activate transcription in vitro. We conclude that transcription activation by LEF-1 in vitro is a chromatin-dependent process that requires a functional trans-activation domain in addition to the HMG domain.

NF-kappa B-mediated chromatin reconfiguration and transcriptional activation of the HIV-1 enhancer in vitro.

NF-kappa B is a potent inducible transcription factor that regulates many genes in activated T cells. In this report we examined the ability of different subunits of NF-kappa B to enhance HIV-1 transcription in vitro with chromatin templates. We find that the p65 subunit of NF-kappa B is a strong transcriptional activator of nucleosome-assembled HIV-1 DNA, whereas p50 does not activate transcription, and that p65 activates transcription synergistically with Sp1 and distal HIV-1 enhancer-binding factors (LEF-1, Ets-1, and TFE-3). These effects were observed with chromatin, but not with nonchromatin templates. Furthermore, binding of either p50 or p65 with Sp1 induces rearrangement of the chromatin to a structure that resembles the one reported previously for integrated HIV-1 proviral DNA in vivo. These results suggest that p50 and Sp1 contribute to the establishment of the nucleosomal arrangement of the uninduced provirus in resting T cells, and that p65 activates transcription by recruitment of the RNA polymerase II transcriptional machinery to the chromatin-repressed basal promoter.

Immunologic analysis of human breast cancer progesterone receptors. 2. Structure, phosphorylation, and processing.

We have used a monoclonal antibody (MAb) directed against chick oviduct progesterone receptors (PR), that cross-reacts with human PR, to analyze PR structure and phosphorylation. This MAb, designated PR-6, interacts only with B receptors (Mr 120,000) of T47D human breast cancer cells; it has no affinity for A receptors (Mr 94,000) or for proteolytic fragments from either protein. The antibody immunoprecipitates native B receptors and was used to study the structure of native untransformed 8S and transformed 4S receptors, using sucrose density gradient analysis, photoaffinity labeling, and gel electrophoresis. On molybdate-containing low-salt gradients, PR-6 complexes with 8S B receptors, causing their shift to the bottom of the gradient while A receptors remain at 8 S. Therefore, A and B receptors form separate 8S complexes, and we conclude that A and B do not dimerize in the holoreceptor. Similar gradient studies using salt-containing, molybdate-free buffers show that there are two forms of salt-transformed 4S receptors, comprising either A proteins or B proteins, suggesting that A and B are also not linked to one another in transformed PR. The independence of A- and B-receptor complexes was confirmed by the finding that purified, transformed B receptors bind well to DNA-cellulose. Since PR-6 cross-reacts with nuclear PR, it was used to analyze nuclear PR processing--a down-regulation step associated with receptor loss as measured by hormone binding. Insoluble nuclear receptors and soluble cytosol receptors were measured by immunoblotting following treatment of T47D cells for 5 min to 48 h with either R5020 or progesterone. From 8 to 48 h after R5020 treatment, immunoassayable receptors decreased in nuclei and were not recovered in cytosols. Nuclear receptors also decreased after progesterone treatment but replenished in cytosols between 8 and 24 h after the start of treatment. Thus, processing involves a true loss of nuclear receptor protein, and not just loss of hormone binding activity, and occurs after progesterone or R5020 treatment. This loss is chronic, however, only in R5020-treated cells. Additional studies focused on the covalent modifications of receptors. We previously described shifts in apparent molecular weight of nuclear PR following R5020 treatment using in situ photoaffinity labeling. To show whether these shifts can be explained by receptor phosphorylation, untreated cells and hormone-treated cells were metabolically labeled with [32P]orthophosphate, and the B receptors were isolated by immunoprecipitation with PR-6 and analyzed by sodium dodecyl sulfate (SDS) gel electrophoresis.(ABSTRACT TRUNCATED AT 400 WORDS)

VSV-G pseudotyped lentiviral vector particles produced in human cells are inactivated by human serum.

Lentiviral vectors transduce dividing and postmitotic cells and thus are being developed toward therapies for many diseases affecting diverse tissues. One essential requirement for efficacy will be that vector particles are resistant to inactivation by human serum complement. Most animal studies with lentiviral vectors have utilized VSV-G pseudotyped envelopes. Here we demonstrate that VSV-G pseudotyped HIV and FIV vectors produced in human cells are inactivated by human serum complement, suggesting that alternative envelopes may be required for therapeutic efficacy for many clinical applications of lentiviral vectors.

Generation of retroviral packaging and producer cell lines for large-scale vector production and clinical application: improved safety and high titer.

For many applications, human clinical therapies using retroviral vectors still require many technological improvements in key areas of vector design and production. These improvements include higher unprocessed manufacturing titers, complement-resistant vectors, and minimized potential to generate replication-competent retrovirus (RCR). To address these issues, we have developed a panel of human packaging cell lines (PCLs) with reduced homology between retroviral vector and packaging components. These reduced-homology PCLs allowed for the use of a novel high multiplicity of transduction ("high m.o. t.") method to introduce multiple copies of provector within vector-producing cell lines (VPCLs), resulting in high-titer vector without the generation of RCR. In a distinct approach to increase vector yields, we integrated manufacturing parameters into screening strategies and clone selection for large-scale vector production. Collectively, these improvements have resulted in the development of diverse VPCLs with unprocessed titers exceeding 2 x 10(7) CFU/ml. Using this technology, human Factor VIII VPCLs yielding titers as high as 2 x 10(8) CFU/ml unprocessed supernatant were generated. These cell lines produce complement-resistant vector particles (N. J. DePolo et al., J. Virol. 73: 6708-6714, 1999) and provide the basis for an ongoing Factor VIII gene therapy clinical trial.