Epidermal growth factor dependence and TGF alpha autocrine growth regulation in primary rat tracheal epithelial cells.

We have examined dependence of primary rat tracheal epithelial (RTE) on exogenous epidermal growth factor (EGF) and determined whether a TGF alpha autocrine pathway is operating in these cells. Primary RTE cells plated in serum free media (SFM) without EGF and bovine pituitary factor (BPE) show little proliferation compared to cultures propagated in media containing EGF/BPE (CSFM). Removal of EGF/BPE shortly after plating, however, results in significant proliferation, although plateau cell densities are reduced and cell morphology is significantly altered compared to cells propagated in CSFM. Addition of EGF and/or BPE to cultures propagated in SFM minus EGF/BPE restores maximum cell density. The concentration of TGF alpha peptide in media conditioned by cells propagated without EGF/BPE is lower than the concentration in the media of CSFM cultures. TGF alpha mRNA and protein levels are also significantly lower in cells late in culture compared to logarithmically growing cells regardless of the presence or absence of EGF/BPE. The proliferation of primary RTE cells propagated without EGF/BPE is inhibited by neutralizing TGF alpha antiserum and by a tyrphostin compound that blocks TGF alpha/EGF receptor tyrosine kinase activity. These results indicate that primary RTE cells utilize TGF alpha as an autocrine growth factor and that the autocrine pathway is regulated as a function of growth state of the cells. However, this pathway does not provide growth autonomy to primary RTE cells, since cultures remain dependent on exogenous EGF/BPE for sustained proliferation.

Abnormalities of growth factor systems in transformed airway epithelial cells.

The role of the peptide growth factors transforming growth factor alpha (TGF alpha) and transforming growth factor beta (TGF beta) in the regulation of proliferation of normal and transformed airway epithelial cells was studied. Normal as well as transformed rat tracheal epithelial (RTE) cell cultures secrete similar amounts of TGF alpha during logarithmic growth. Once normal RTE cell cultures reach the plateau phase of growth, they down-regulate TGF alpha expression at the RNA and protein level; in contrast, transformed cells do not down-regulate TGF alpha. Using neutralizing TGF alpha antiserum and a tyrphostin TGF alpha/EGF receptor tyrosine kinase inhibitor, we show that the secreted TGF alpha is utilized by both normal and transformed cells as an autocrine mitogenic factor. Normal RTE cells are highly sensitive to the growth inhibitory effects of TGF beta, particularly during early phases of logarithmic growth. At late logarithmic and plateau phases of proliferation, cultures of normal RTE cells secrete large amounts of TGF beta. That the endogenous TGF beta is exerting growth inhibitory effects can be demonstrated by adding TGF beta antisera to the cultures which causes a burst of proliferation. Many transformed RTE cell lines exhibit a markedly reduced sensitivity to the growth inhibitory effects of TGF beta. However, the cells remain responsive to regulation of ECM genes by TGF beta. The transformed cell lines examined secrete less than one tenth the amount of TGF beta of normal cells. Our studies show that the autocrine TGF beta growth restraining mechanism is inoperative in many of the RTE cell transformants. We conclude, therefore, that alterations in TGF alpha and TGF beta regulation of cell proliferation are important factors contributing to the abnormal growth behavior of transformed RTE cells.

Temporal regulation of equine herpesvirus type 3 transcription.

The transcription of equine herpesvirus type 3 (EHV-3; equine coital exanthema virus) has been examined and found to be temporally regulated into three classes: immediate early (IE), early (E), and late (L). Hybridization of in vivo 32PO4-labeled transcripts revealed that IE transcript(s) are derived exclusively from the inverted repeat segments (IRs) of the viral genome, while E and L transcripts are not restricted to any specific region of the genome. Northern blot analysis of EHV-3 IE RNA revealed a single transcript of approximately 5.7 kb (3.8 MDa). We have previously shown that transcription of equine herpesvirus type 1 (EHV-1) DNA is temporally regulated and produces a single 6 kb IE RNA which is derived from the IRs segments. In this paper, we show that the EHV-1 and EHV-3 IE RNA species are homologous, reflecting the colinearity of the genomes of these two related viruses. While four IE polypeptides are synthesized in EHV-1 infected cells in the presence of actinomycin D following the removal of a cycloheximide block, only one major IE polypeptide (180 kDa) is detectable in EHV-3 infected cells under these conditions. However, immunoprecipitation of EHV-3 infected cell extracts with polyvalent rabbit antisera to IE1 of EHV-1 revealed at least two other viral specific IE polypeptides.

Abnormalities in growth regulation of transformed rat tracheal epithelial cells.

We have examined the changes in growth regulation which take place during multistage transformation of rat tracheal epithelial (RTE) cells exposed to chemical carcinogens in vitro. Transformed RTE cells demonstrate: (1) altered negative growth factor sensitivity and (2) altered positive growth factor dependence and gene expression compared with primary RTE cells in culture. Primary RTE cells are sensitive to the growth inhibitory effects of retinoic acid and transforming growth factor beta (TGF beta). Enhanced growth variants of RTE cells, recognized 4-6 weeks after carcinogen treatment, lose sensitivity to the antiproliferative effects of retinoic acid. Likewise, many immortalized RTE cell lines lose responsiveness to growth inhibition by TGF beta. We also examined positive growth factor requirements and gene expression in primary and transformed RTE cells propagated in serum-free media. Many transformed cell lines lose dependence on epidermal growth factor in the media. These cell lines also overexpress TGF alpha mRNA and protein. Since TGF alpha acts at the epidermal growth factor receptor, these data suggest that TGF alpha plays an autocrine role in growth regulation of transformed RTE cells. Thus, alterations in both negative and positive growth factor regulation contribute to expression of the transformed phenotype in RTE cells.

Altered growth factor dependence and transforming growth factor gene expression in transformed rat tracheal epithelial cells.

The role of peptide growth factors in neoplastic progression of transformed rat tracheal epithelial (RTE) cells was assessed by examining growth factor requirements and expression of growth factor and growth factor receptor genes in normal and transformed RTE cells. Neoplastically transformed cell lines showed decreased requirements for bovine pituitary extract, insulin, and epidermal growth factor compared to normal primary RTE cells. Neoplastic RTE cell lines expressed significantly increased levels of transforming growth factor alpha (TGF alpha) RNA and secreted TGF alpha into the media, suggesting an autocrine role for this growth factor. Increased levels of TGF alpha RNA were also observed in the preneoplastic stages of the same cell lines, indicating that increased TGF alpha expression is an early event in the multistage process of neoplastic transformation of RTE cells. TGF beta transcripts were also overexpressed in neoplastically transformed cell lines. Our studies suggest that aberrant expression of growth factors may play an important role in the development and/or maintenance of the transformed phenotype in RTE cells.

Analysis of the in vitro translation products of the equine herpesvirus type 1 immediate early mRNA.

Equine herpesvirus type 1 (EHV-1) gene expression is coordinately regulated in an alpha, beta, gamma fashion. Viral alpha gene products include a 6.0-kb immediate early (IE) mRNA species (W. L. Gray et al., 1987, Virology 158, 79-87) and at least four closely related IE polypeptides (IEPs) (G.B. Caughman et al., 1985, Virology 145, 49-61). In this report, we describe results obtained from a series of in vitro translation experiments which were performed in an effort to characterize the IEPs and identify the mechanism by which individual IE protein species are generated. Our data indicate that a family of IEPs is generated in vitro from the 6.0-kb mRNA size class and that these IEPs correspond in overall size and antigenicity to those synthesized in infected cells. Using time-course/pulse-chase analyses, we show that production of three of the major IEPs [IE1' (193 kDa), IE3' (166 kDa), and IE4' (130 kDA)] occurs concomitantly, that none of these protein species can be chased completely into another, and that at least two additional minor species appear to be processed following synthesis. Finally, we show that the 6.0-kb mRNA species isolated during early or late stages of the infection cycle can be translated to yield all of the major IE proteins, indicating that production of the family of IEPs is not dependent upon accumulation of the IE mRNA which occurs during a cycloheximide blocked infection cycle. The implications of these findings are discussed as they relate to the origin and production of the IEPs both in vivo and in vitro.

Characterization of equine herpesvirus type 1 immediate early proteins.

EHV-1 immediate early (IE) gene expression in lytic infection results in the production of four high mol wt immediate early polypeptides (IEPs), designated IE1, IE2, IE2, and IE4; however, IE transcription is limited to the synthesis of a single 6-kb mRNA. Together, these findings raised questions as to whether the four IEPs were related products of the same gene. In the present study the IEPs were characterized with respect to their structural similarities, antigenic relatedness, and postsynthetic modifications. IE1 was the most abundant IEP, in that it accounted for approximately 80% of the IEP-incorporated radiolabel in infected rabbit kidney cells labeled under IE conditions with [35S]methionine or 14C-labeled amino acid mixtures. IE1 also was the major phosphorylated species. Limited proteolytic digestion of isolated radiolabeled IEP bands with Staph V8 protease yielded virtually identical fragment profiles in SDS-PAGE, as did digestions with chymotrypsin and N-chlorosuccinimide. Monospecific rabbit antisera raised against each of the four isolated IEPs reacted with all the IEP species in immunoblotting assays. Pulse-chase experiments indicated that all the IEPs were detectable immediately after a 15-min pulse and that several alterations in the IEP profile occurred during subsequent chase periods. Thus, the EHV-1 IEPs are closely related structurally and antigenically and appeared to be either produced simultaneously or processed to yield the individual forms immediately.

Characterization and mapping of equine herpesvirus type 1 immediate early, early, and late transcripts.

Northern blot analysis was used to characterize and map equine herpesvirus type 1 (EHV-1) immediate early (IE), early, and late transcripts. Genomic EHV-1 DNA and cloned EHV-1 restriction endonuclease fragments, representing the entire genome, were 32P-labeled and hybridized to immobilized total cell RNA isolated from EHV-1 infected rabbit kidney cells incubated in the presence or absence of metabolic inhibitors. A single 6.0 kilobase (kb) IE transcript mapped to viral inverted repeat sequences. Approximately 41-45 early transcripts ranging in size from 0.8 to 6.4 kb and 18-20 late transcripts ranging in size from 0.8 to 10.0 kb were identified. These findings demonstrate that EHV-1 gene expression is regulated at the level of transcription, although regulation at the level of translation is also possible. The results provide a basis for examining alterations in viral gene expression in EHV-1 oncogenically transformed and persistently infected cells.

Regulation of equine herpesvirus type 1 gene expression: characterization of immediate early, early, and late transcription.

The regulation of equine herpesvirus type 1 (EHV-1) transcription was examined in infected rabbit kidney cells using metabolic inhibitors. In order to map EHV-1 immediate early, early, and late transcripts, viral RNA was 32P-labeled in vivo and hybridized to EHV-1 DNA restriction fragments immobilized on nitrocellulose filters. Immediate early viral RNA was mapped to one region of the viral genome within the inverted repeat DNA sequences (map units 0.78-0.83 and 0.95-1.0). Northern blot hybridization analysis using a 32P-labeled cloned DNA probe from this region identified a single immediate early viral transcript (approximately 6 kb). Transcription of early and late genes was not restricted to any specific region on the viral genome as indicated by the ability of 32P-labeled early and late RNA to hybridize to EHV-1 restriction endonuclease fragments from both the long and short components of EHV-1 DNA. Additional experiments performed without the use of metabolic inhibitors confirmed that EHV-1 transcription is temporally regulated. The characterization of EHV-1 transcription during productive infection will serve as a reference for the analysis of viral transcripts in oncogenically transformed and persistently infected cells.

Reversible inhibition of bovine parvovirus DNA replication by aphidicolin and L-canavanine.

The replication of the autonomous parvovirus, bovine parvovirus (BPV), has been studied in virus-infected cells. Gel electrophoresis was used to determine the effect of aphidicolin, a specific inhibitor of DNA polymerase alpha, and L-canavanine, an inhibitor of protein synthesis, on viral DNA replication. Synchronized cell cultures were infected with 32P-labelled or unlabelled BPV in the presence or absence of aphidicolin and L-canavanine. Cells were harvested at various times post-infection, and DNA was electrophoresed and blotted. When aphidicolin was added to cells at the time of infection, then removed 8 h later, BPV replicative form DNA (RF) synthesis began within 2 h after its removal. This preceded the peak of cellular DNA synthesis by 2 h, unlike an uninhibited infection, when viral RF synthesis follows the peak of S phase by 2 to 4 h. Furthermore, if aphidicolin was added at any point during the replication cycle, BPV DNA synthesis stopped. This effect was shown to be completely reversible and indicated that aphidicolin did not disrupt the replication apparatus required for viral DNA synthesis. L-Canavanine inhibited synthesis of the virus-specific proteins NP-1 and VP3 and synthesis of BPV DNA. Upon removal of L-canavanine, viral protein synthesis was detected by 30 min followed by viral DNA synthesis. These results indicate that a specific S phase function other than cellular DNA synthesis is required for initiation of BPV DNA synthesis, that DNA polymerase alpha plays a major role in BPV DNA replication in vivo, and that these inhibitors can be used to inhibit reversibly various stages of BPV DNA replication.

Aphidicolin inhibition of the production of replicative-form DNA during bovine parvovirus infection.

Since parvoviruses apparently do not possess a DNA polymerase activity, one or more of the host cell DNA polymerases must be responsible for replicating the single-stranded DNA genome. We have focused on determining which polymerase, alpha, beta, or gamma (pol alpha, pol beta, or pol gamma, respectively), is responsible for the first step in bovine parvoviral DNA replication: conversion of the single-stranded DNA genome to a parental replicative form (RF). In this study, we used aphidicolin, a specific inhibitor of DNA pol alpha, to assay for the requirement of pol alpha activity in parental RF formation in vivo. Synchronized cell cultures were infected with bovine parvovirus with or without aphidicolin, and the products of viral replication were separated on agarose gels and identified by Southern blot analysis. We found that complete inhibition of viral DNA synthesis resulted when 20 microM aphidicolin was present throughout the infection. In addition, viral DNA synthesis was inhibited by as little as 1 microM aphidicolin, whereas lower concentrations (0.1 and 0.01 microM) resulted in partial inhibition of the replication process. Using 32P-labeled bovine parvovirus as the input virus we differentiated parental RF from daughter RF and progeny DNA synthesis. We conclude that DNA pol alpha is required for the production of RF during bovine parvovirus replication in vivo and that this requirement is most likely for the conversion of bovine parvovirus input single-stranded DNA to parental RF. These results do not rule out a possible role for DNA pol gamma in the first step, nor do they rule out a role for pol alpha or pol gamma in later stages of the replication cycle.

Purified DNA polymerase gamma replicates bovine parvovirus DNA to a unit-length product.

DNA polymerase gamma, purified from fetal bovine liver, replicated virion single-stranded DNA from bovine parvovirus to a unit-length double-stranded DNA molecule. This product was not nicked and was covalently linked to the 3' hairpin primer. The reaction was inhibited by dideoxythymidine 5'-triphosphate, but was unaffected by ATP or aphidicolin. Double-stranded viral DNA was not a functional template for purified DNA polymerase gamma.