trans E1 component requirements for maximal replication of E1-defective recombinant adenovirus.

Strategies that enable E1-defective recombinant adenoviruses to selectively undergo replication in neoplastic tissue may be useful for future investigations or therapies of malignancies. A growing body of evidence suggests that some molecular alterations commonly associated with malignancies, such as p53 mutations, can modify the specific E1 requirements for replication of human serotype adenoviruses. In the studies reported here, a panel of human non-small cell lung cancer cell lines with previously defined p53 status were characterized for basal interleukin-6 (IL-6) and bcl-2 content because previous studies have indicated both proteins can functionally substitute for the replication requirements provided by native E1 viral proteins. Cell lines were infected with E1-defective adenovirus 5 and simultaneously transfected with different combinations of E1 plasmids, or a bcl-2 expression plasmid, and adenovirus present in the cells was quantified 6 days later. These assays demonstrated that E1A with both 19- and 55-kDa E1B-encoding plasmids were required for maximal adenoviral replication, independent of the varying p53/IL-6/basal bcl-2 phenotypes of the host cell lines. E1A was required for maximal replication enablement, independent of the basal IL-6 content of these cell lines, and exogenous IL-6 also did not obviate the E1A requirement. Interestingly, the bcl-2 expression plasmid did not consistently substitute for the 19-kDa expression plasmid in the context of this replication complementation assay. These results suggest that (1) basal levels of IL-6 greater than that present in these cell lines are necessary for functional replacement of the E1A replication function and (2) bcl-2 does not predictably substitute for the 19-kDa E1B replication function in the context of trans complementation.

Amplification of recombinant adenoviral transgene products occurs by inhibition of histone deacetylase.

n-Butyrate (butyrate) has been shown to amplify transgene expression in cells infected with E1-defective adenoviruses. The present studies were undertaken in order to better define the actions of butyrate in the context of adenovirus gene expression, and to attempt to elucidate the mechanism by which butyrate mediates the transgene amplification. It was found that butyrate amplified viral transgene expression over a concentration range of 0.5-5 mM, and that the amplification required an exposure of 12-24 hr for maximal effect. Western blot analysis of representative viral proteins showed that butyrate treatment amplified DNA-binding protein, but not fiber protein. A transient adenoviral replication system suggested that butyrate had a modest inhibitory effect on replication of the E1-defective adenovirus. Use of a specific inhibitor of histone deacetylase, trichostatin A (TSA), reproduced the amplification of the viral transgene product achieved with the butyrate. In contrast, adenoviral transgene expression could not be amplified by TSA treatment in a cell line known to have a TSA-resistant histone deacetylase. Butyrate amplified steady-state gene expression of the viral transgene, but had no detectable effects on either DNA-binding protein or fiber steady-state gene expression. Nuclear run-off experiments showed that both butyrate and TSA caused an increase in the viral transgene transcription. It was concluded that inhibitors of histone deacetylase amplify adenoviral transgene expression at the transcriptional level.

E1A RNA transcripts amplify adenovirus-mediated tumor reduction.

Previous work by this group has established that E1-defective, recombinant adenoviruses can be replication-enabled by the codelivery of a plasmid encoding the deleted E1 functions, a strategy now designated conditional replication-enablement system for adenovirus (CRESA). In the studies reported here, the original replication-enabling plasmid was replaced by two separate plasmids that encoded the necessary E1A and E1B functions, respectively. An RNA transcript encoding the requisite E1A functions was shown to substitute functionally for the E1A plasmid without significant loss of new adenovirus production in in vitro experiments. No replication competent adenovirus was detectable in the cells treated with the plasmids, or the RNA and plasmid combinations. Subcutaneous human tumor nodules containing a fraction of cells cotransduced with the replication-enabling RNA + DNA and an adenovirus containing a herpes simplex virus thymidine kinase (HSVtk) expression cassette were reduced to a greater extent than control nodules containing the same fraction of cells cotransduced with the virus and an irrelevant plasmid. These experiments show that an E1-defective adenovirus can be conditionally replication-enabled by an RNA transcript encoding the required E1 functions, and that the replication-enablement is sufficient to produce an augmentation of an adenovirus-mediated therapeutic effect in vivo.

Quantitative and in vivo activity of adenoviral-producing cells made by cotransduction of a replication-defective adenovirus and a replication-enabling plasmid.

Achieving limited recombinant viral replication may provide a means of amplifying viral-mediated gene transfer in vivo. We have previously shown that cotransduction of an E1-defective adenovirus with a plasmid containing the deleted E1 functions into prostate carcinoma cells resulted in E1-defective virus production by those cells. The studies described here have extended these findings to more firmly establish the capacity of the trans complementation approach to achieve amplification of recombinant viral transgene expression. The recombinant virus used for all the studies was AdCMV-luc which contained a luciferase expression cassette; the replication-enabling plasmid, pE1, encoded the E1 functions deleted from AdCMV-luc. Quantitative in vitro studies with the HeLa cell line showed that for each plaque forming unit of AdCMV-luc originally exposed to the cells, 0.54 x 10(3) new replication-defective viruses were detected in supernatants and lysates over the following 4 days. Multiple cell lines were shown to support new virus production following cotransduction of AdCMV-luc and pE1. Small numbers of replication-competent viruses were detected in the lysates and supernatants from the cotransduced cells such that for every 10(5) replication-defective viruses approximately two replication-competent viruses were produced. Tumor nodules produced by engrafting a mixture of AdCMV-luc/pE1-cotransduced HeLa cells with uninfected HeLa cells yielded much higher levels of luciferase expression than control tumors containing mixtures of cells infected with AdCMV-luc alone. In total, these results demonstrate new virus production by cells receiving a replication-defective adenovirus and a replication-enabling plasmid are capable of amplifying recombinant viral transgene expression in vivo.

Supernatant rescue assay vs. polymerase chain reaction for detection of wild type adenovirus-contaminating recombinant adenovirus stocks.

The certification of recombinant adenoviruses prepared for clinical use requires the exclusion of contaminating, replication-competent adenovirus (wild type virus). Polymerase chain reaction (PCR)-based detection assays have been developed that detect the presence of viral sequences present only in wild type adenoviruses. As an alternative, this report describes a novel bioassay, designated the 'supernatant rescue assay', that detected minimal amounts of wild type virus mixed with high numbers of recombinant adenoviruses. This assay is based on the observation that minimal numbers of wild type adenovirus can be rescued from the supernatants of cells exposed to high multiplicities of infection (mois) of recombinant virus mixed with a known, minimal number of wild type virions. This assay was highly reproducible and routinely detected the presence of a single wild type virus mixed within 10(9) recombinant viruses. Under the experimental conditions employed, the supernatant rescue assay was significantly more sensitive than all three different PCR-detection assays.

Effects of transforming growth factor-beta on human fetal adrenal steroid production.

Transforming growth factor-beta (TGF-beta) was found to inhibit basal and ACTH-stimulated steroid production by cultured human fetal adrenal cells. The inhibitory effects of TGF-beta were both time and dose-dependent. Inhibition of basal dehydroepiandrosterone sulfate (DS) production usually was noted only after 3 or more days of treatment with > or = 0.1 ng TGF-beta/ml. The inhibitory effects of 1 ng/ml TGF-beta on ACTH-stimulated DS production were more striking than those on cortisol production by both fetal zone and neocortical cells. TGF-beta also was found to interfere with DS and cortisol production by fetal zone cells in response to forskolin and dibutyryl cAMP. TGF-beta interfered with ACTH stimulation of cytochrome P450(17) alpha mRNA in fetal zone and neocortex cells. These results are suggestive that TGF-beta differentially inhibits DS and cortisol production by human fetal adrenal cells and that the site of TGF-beta action on steroidogenesis may be distal to the generation of cAMP. Such results, along with those of others, are suggestive that TGF-beta may play an autocrine/paracrine role in the human adrenal.

Four-day duration of tumor promoter exposure required to transform JB6 promotion-sensitive cells to anchorage independence.

The JB6 mouse epidermal cell lines have been developed to study promotion of neoplastic transformation in vitro. Treatment of JB6 cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) at 1 to 100 ng/ml results in the irreversible acquisition of tumorigenicity in nude mice and anchorage-independent growth. Among the biochemical responses which occur during TPA treatment is a decrease in procollagen synthesis. During a study of the possible role of H2O2 in the process of promotion, it was observed that catalase purchased commercially would inhibit TPA promotion as well as the reduction of collagen synthesis in a dose-dependent manner. A highly purified catalase preparation failed to demonstrate the TPA blocking activity, suggesting that this activity was due to a contaminating factor. We have separated the TPA blocking factor from the catalase itself using concanavalin A affinity chromatography. The factor is a Mr 60,000 glycoprotein showing TPA hydrolase activity. The enzyme, which is similar to a murine liver-derived TPA hydrolase, produces a single phorbol product from TPA that has been identified as phorbol-13-acetate. TPA hydrolase was used to terminate TPA action in soft agar. This made it possible to establish that approximately 4 days of exposure to phorbol esters are required for promotion of transformation in the JB6 model system.

Purification of an angiotensin II binding protein by using antibodies to a peptide encoded by angiotensin II complementary RNA.

We have generated a monospecific antibody to a synthetic peptide encoded by an RNA complementary to the mRNA for angiotensin II (AII) and determined whether this antibody recognizes the AII receptor. We demonstrate that the antibody competes specifically with 125I-labeled AII for the same binding site on rat adrenal membranes. Furthermore, we show that this antibody inhibits the secretion of aldosterone from cultured rat adrenal cells, suggesting that the antibody recognizes the biologically relevant AII receptor. Finally, we demonstrate that antibody to the complementary peptide can be used to immunoaffinity-purify a protein of Mr 66,000 that specifically binds radiolabeled AII.

Antibodies to the binding site of the receptor for luteinizing hormone-releasing hormone (LHRH): generation with a synthetic decapeptide encoded by an RNA complementary to LHRH mRNA.

A molecular recognition code has been hypothesized to exist in which ligands and their binding sites are encoded on complementary segments of genomic DNA. We have tested this hypothesis by generating a rabbit antibody to a synthetic decapeptide (complementary peptide) encoded by an RNA complementary to the mRNA for luteinizing hormone-releasing hormone (LHRH) and determining whether this antibody recognizes the LHRH receptor. When the antibody was used for immunoperoxidase staining of enzymatically dispersed rat anterior pituitary cells, only those that contained and secreted luteinizing hormone (i.e., the gonadotropes) were recognized. This staining could be abolished by preincubation with the complementary peptide or with an LHRH agonist, suggesting that the antibody is specific to the complementary peptide and is directed at the binding site of the receptor. Further evidence that the antibody recognizes the LHRH receptor was obtained in immunoblot experiments on solubilized receptors from pituitary glands. Immunoperoxidase staining with the antibody revealed two bands at 60 kDa and 51 kDa, which are values similar to those previously obtained for the LHRH receptor in photoaffinity-labeling experiments. The staining of these bands was inhibited by preincubation with the complementary peptide or an LHRH agonist. The antibody as well as the complementary peptide to LHRH also suppressed LHRH-stimulated luteinizing hormone release in a quantitative reverse hemolytic plaque assay, presumably by binding to the LHRH receptor and by binding LHRH, respectively. These findings suggest that the synthetic decapeptide whose sequence is specified by the complementary RNA to LHRH mRNA is sufficiently similar to an LHRH binding site that the peptide not only binds LHRH but was also recognized by the immune system as such a site. These findings provide strong support for the hypothesis that recognition molecules are encoded by complementary segments of genomic DNA.

Opiate receptor mediated effects of IFN-alpha and lymphocyte derived endorphin-like peptides.

Viral infection of lymphocytes induces the synthesis of interferon (IFN)-alpha and immunoreactive corticotropin (irACTH). We have previously shown the irACTH to be antigenically, structurally, and functionally related (if not identical) to pituitary ACTH. Virus infected lymphocytes also synthesize in endorphin as measured by immunofluorescence. The endorphin-like activity was found to be associated directly with IFN-alpha as well as other, lower molecular weight peptide(s) having no antiviral activity. Crude IFN-alpha at 1000 U/ml exhibited opiate receptor binding activity by inhibiting 47.2% of 3H dihydromorphine (4 X 10(-9) M) binding to mouse brain tissue. Homogeneous HuIFN-alpha (10(8.3) U/mg) also bound to opiate receptors but required 10 times the antiviral activity than crude IFN-alpha for a 50% inhibition of dihydromorphine binding. When injected intracerebrally both crude and homogeneous IFN-alpha induced transient analgesia in mice that was reversible and preventable by the opiate antagonist naloxone. The low molecular weight (less than 10 Kd) ir endorphins purified from the IFN-alpha had no antiviral activity, but may represent the majority of the opiate receptor binding material in the infected lymphocyte culture fluid. These data seem to indicate that the opiate-like side effects of exogenous IFN administration may be due to the IFN-alpha molecule binding to opiate receptors and also may be due to the associated low molecular weight endorphin-like moieties synthesized by lymphocytes.

Role of specific membrane and genetic changes in the mechanism of tumour promotion. Studies with promoter-resistant variants.

Several cell culture model systems in current use for studying tumour promotion mechanism are reviewed briefly. The conclusions that can be drawn from studies with the JB6 mouse epidermal system are summarized. Promoter-induced mitogenic stimulation, epidermal growth factor receptor binding and stimulated hexose transport are apparently not required for promotion of neoplastic transformation in JB6 cells by phorbol esters and other promoters. Phorbol ester receptor binding (or protein kinase C activation) and switched-off collagen synthesis may be required, but definitive proof is not available. Decreased cell surface trisialoganglioside synthesis and one or more genes that determine promotion sensitivity appear to distinguish sensitive from resistant cells and to be required for promotion of neoplastic transformation in JB6 cells.

Regulation of lymphokine (gamma-interferon) production by corticotropin.

We have shown that corticotropin (ACTH), alpha-endorphin, and enkephalins can regulate antibody responses, which suggested a role for neuropeptides in a regulatory circuit between the immune and neuroendocrine systems. ACTH and structurally related peptides were examined here for regulation of mitogen induction of the lymphokine gamma-interferon (IFN gamma) in C57BL/6 mouse spleen cell cultures. Synthetic ACTH1-39 and a porcine pituitary extract containing ACTH activity were potent suppressors of the IFN gamma response. Synthetic ACTH1-39 suppressed the response by approximately 62% at 1 to 3 microM, whereas the porcine extract suppressed by greater than 90% at 1 to 3 microM ACTH. The greater potency of the pituitary extract was shown to be due to the presence of an additional peptide of Mr 2100 that was reactive with antibodies to the N-terminal region of ACTH (ACTH1-13), possessed potent anti-cellular activity against L cells and various transformed cells, but lacked ACTH biologic activity. The anti-cellular peptide suppressed the IFN gamma response by greater than 99% at 0.05 microM. The ACTH1-39 cleavage products, alpha-melanocyte stimulating hormone (alpha MSH; acetylated and amidated ACTH1-13), and corticotropin-like intermediate lobe peptide (CLIP; ACTH18-39) had no effect on IFN gamma production. ACTH1-24, like ACTH1-39, has full steroidogenesis activity but also had no effect on IFN gamma production, which suggests a dissociation of the immunoregulatory and steroidogenic properties of ACTH1-39. ACTH1-39, and possibly also the anti-cellular 2100 Mr peptide, is initially synthesized as the precursor polyprotein pro-opiomelanocortin (POMC). Enzymatic processing of POMC, first to the active ACTH1-39 or the anti-cellular peptide and then to the inactive smaller peptides, probably plays an important role in regulation of lymphokine and antibody production by ACTH and ACTH-related neuropeptides. This is consistent with the recent demonstration of the production of ACTH-like peptides by lymphocytes.

Tumor-promoting phorbol esters inhibit procollagen synthesis at a pretranslational level in JB-6 mouse epidermal cells.

Collagen synthesis was inhibited in JB-6 mouse epidermal cells after exposure to 12-O-tetradecanoylphorbol-13-acetate under conditions leading to irreversible neoplastic transformation. In vitro translation and hybridization studies demonstrated a dramatic decrease in collagen mRNA in 12-O-tetradecanoylphorbol-13-acetate-treated cells, suggesting that the inhibition of collagen synthesis in response to 12-O-tetradecanoylphorbol-13-acetate is due to regulation at a pretranslational level.

A corticotropin (ACTH) related peptide with cytotoxic activity.

A commercially available preparation of corticotropin was found to have potent cytotoxic activity for several established cell lines. Neither synthetic corticotropin nor alpha melanocyte stimulating hormone demonstrated this cytotoxic activity. Gel filtration allowed separation of a 2100 dalton cytotoxic peptide from the actual corticotropin present in the commercially prepared material. The structural relatedness of the cytotoxic peptide to corticotropin was demonstrated by neutralization with antisera to alpha melanocyte stimulating hormone. These studies indicate the existence of a newly identified ACTH related peptide with cytotoxic activity.

Studies on the mechanism of retinoid-induced adhesion of spontaneously transformed mouse fibroblasts.

Transformed cells (Balb/c, 3T12-3), induced to increase their adhesion to the substrate by treatment with retinoic acid, display higher incorporation of (2-(3)H)-mannose into both lipids and glycoproteins than untreated controls. Stimulation of (2-(3)H)-mannose incorporation into manno-lipids is evident 8 hr after exposing the cells to retinoic acid, and stimulation of tritiated mannose incorporation into glycoproteins occurs slightly later. SDS-PAGE of (2-(3)H)-mannose labelled glycoproteins indicates that both retinoic acid and retinol treatments stimulate the incorporation of the radiolabelled sugar into a glycoprotein with subunit MW 180,000 (Gp 180) and, to a lesser extent, into other glycoproteins. 3H-leucine incorporation into a protein banding at the same position as the 3H-mannose labelled Gp 180 does not appear to be affected by retinoid treatment. A retinoic acid induced increase in the amount of Gp 180 can also be shown by lactoperoxidase catalyzed radioiodination of cultured 3T12 cells, and controlled trypsin digestion experiments indicate that Gp 180 is a component of the cell surface. On the contrary, the increased cell adhesion to the substrate induced by retinoic acid is not accompanied, in this system, by an increase in the amount of fibronectin, as judged by iodination of the cell surface components followed by SDS-PAGE.

Inhibition by tumor-promoting phorbol esters of procollagen synthesis in promotable JB6 mouse epidermal cells.

The JB6 mouse epidermal cell line has been developed to study promotion of neoplastic transformation in vitro. Treatment of JB6 cells with 12-O-tetradecanoylphorbol 13-acetate (TPA) or other tumor promoters resulted in the irreversible acquisition by JB6 cells of tumorigenicity in nude mice and anchorage-independent growth in soft agar. As previously reported, one of the biochemical responses that occurs during TPA treatment is a greater than 75% reduction in a mannose-labeled glycoprotein with an apparent molecular weight of 180,000. This TPA-sensitive glycoprotein has now been identified on the basis of collagenase and pepsin sensitivity as procollagen pro-alpha 1(I) chain. [3H]proline labeling also demonstrated a parallel decrease in the 150,000 procollagen pro-alpha 2(I) component. Two nonphorbol promoters, mezerein and epidermal growth factor, were also active in decreasing procollagen synthesis. Promotion-sensitive and promotion-resistant clonal derivatives of JB6 showed similar basal levels of collagen synthesis as well as similar degrees of TPA-dependent procollagen loss indicating that the collagen decrease may be necessary but is not sufficient to produce the promotion response. Comparison of chemically transformed mouse epidermal cell lines with paried nontransformants suggests the reduced procollagen synthesis is a stably acquired phenotypic change and may therefore be involved in maintenance of the transformed phenotype as well as in its induction.

Phorbol ester-induced anchorage independence and its antagonism by retinoic acid correlates with altered expression of specific glycoproteins.

Since tumor promoting phorbol esters produce a variety of glycoprotein synthesis changes and since retinoids act both as antipromoters and modulators of glycoprotein synthesis, we sought to ascertain whether specific changes in glycoprotein synthesis might be targets both for the promoting action of phorbol esters and for the antipromoting actin of retinoids. In this report we present evidence that tumor promoting but not nonpromoting phorbol esters produce decreased levels of 180,000 and 150,000 mol, wt, glycoproteins in mouse JB-6 cells which are promotable to tumor cell phenotype by phorbol esters. These relatively specific decreases are blocked by an antipromoting concentration of retinoic acid, thus suggesting that decreases in 180K and 150K glycoproteins may play a role in promotion of transformation.