Expression Of Env Gene Research Articles

Multiple RNA splicing and the presence of cryptic RNA splice donor and acceptor sites may contribute to low expression levels and poor immunogenicity of potential DNA vaccines containing the env gene of equine infectious anemia virus (EIAV)

The env gene is an excellent candidate for inclusion in any DNA-based vaccine approach against equine infectious anemia virus (EIAV). Unfortunately, this gene is subjected to mutational pressure in E. coli resulting in the introduction of stop codons at the 5′ terminus unless it is molecularly cloned using very-low-copy-number plasmid vectors. To overcome this problem, a mammalian expression vector was constructed based on the low-copy-number pLG338-30 plasmid. This permitted the production of full-length EIAV env gene clones (plcnCMV env) from which low-level expression of the viral surface unit glycoprotein (gp90) was detected following transfection into COS-1 cells. Although this suggested the nuclear export of complete env mRNA moieties at least two additional polypeptides of 29 and 20 kDa (probably Rev) were produced by alternative splicing events as demonstrated by the fact that their synthesis was prevented by mutational inactivation of EIAV env splice donor 3 (SD3) site. The plcnCMV env did not stimulate immune responses in mice or in horses, whereas an env construct containing an inactivated SD3 site (plcnCMVΔSD3) did induce weak humoral responses against gp90 in mice. This poor immunogenicty in vivo was probably not related to the inherent antigenicity of the proteins encoded by these constructs but to some fundamental properties of EIAV env gene expression. Attempts to modify one of these properties by mutational inactivation of known viral RNA splice sites resulted in activation of previously unidentified cryptic SD and slice acceptor sites.

A gene therapy model for retrovirus-induced disease with a viral env gene: expression-dependent resistance in immunosuppressed hosts.

At the initial stage of retroviral infection, virion envelope glycoprotein (env product) binds to cell surface receptors. Cells infected with retrovirus or into which the env gene was introduced, become resistant to superinfection by other retroviruses with the same receptor specificity, a phenomenon known as receptor interference. We have demonstrated previously that the introduction of an env gene from a truncated endogenous ecotropic murine leukemia virus (MuLV), the Fv-4 resistance (Fv-4r) gene, into the bone marrow hematopoietic cells of Fv-4 sensitive (Fv-4s) mice protected mice from ecotropic retrovirus-induced disease. Using the gene transfer system under the control of the retroviral vector and bone marrow transplantation (BMT), here we could show that the expression of an introduced Fv-4r gene in hematopoietic cells continued for more than 1 year after BMT. To determine the inhibitory mechanism of Fv-4r env gene expression against FLV-infection in this model system, peripheral blood mononuclear cells (PBMCs), or spleen cells from chimeras with various degrees of env-expression, were mixed with green fluorescence protein (GFP)-conjugated Friend MuLV envglycoprotein (GFP-Fr-ENV). The amount of GFP-Fr-ENV bound to these cells inversely correlated with the expression intensity of the transduced env gene indicating the receptor interference effect. Next, to see whether transduction of the Fv-4r gene would protect an immunosuppressed host from FLV-induced leukemogenesis, we generated immunocompromised chimeras by transplanting env-transduced bone marrow cells into a thymectomized host. These chimeras also resisted FLV-induced leukemogenesis, indicating that receptor interference-based gene therapy could become a therapeutic basis for immunodeficiency virus-induced diseases in vivo.

The sequence complementarity between HIV-1 5' splice site SD4 and U1 snRNA determines the steady-state level of an unstable env pre-mRNA.

HIV-1 env expression from certain subgenomic vectors requires the viral regulatory protein Rev, its target sequence RRE, and a 5' splice site upstream of the env open reading frame. To determine the role of this splice site in the 5'-splice-site-dependent Rev-mediated env gene expression, we have subjected the HIV-1 5' splice site, SD4, to a mutational analysis and have analyzed the effect of those mutations on env expression. The results demonstrate that the overall strength of hydrogen bonding between the 5' splice site, SD4, and the free 5' end of the U1 snRNA correlates with env expression efficiency, as long as env expression is suboptimal, and that a continuous stretch of 14 hydrogen bonds can lead to full env expression, as a result of stabilizing the pre-mRNA. The U1 snRNA-mediated stabilization is independent of functional splicing, as a mismatch in position +1 of the 5' splice site that led to loss of detectable amounts of spliced transcripts did not preclude stabilization and expression of the unspliced env mRNA, provided that Rev enables its nuclear export. The nucleotides capable of participating in U1 snRNA:pre-mRNA interaction include positions -3 to +8 of the 5' splice site and all 11 nt constituting the single-stranded 5' end of U1 snRNA. Moreover, env gene expression is significantly decreased upon the introduction of point mutations in several upstream GAR nucleotide motifs, which are mediating SF2/ASF responsiveness in an in vitro splicing assay. This suggests that the GAR sequences may play a role in stabilizing the pre-mRNA by sequestering U1 snRNP to SD4.

Protection against FIV challenge infection by genetic vaccination using minimalistic DNA constructs for FIV env gene and feline IL-12 expression.

To evaluate the efficacy of a genetic vaccination protocol based on minimalistic, immunogenic defined gene expression (MIDGE) vectors coding for domains of the feline immunodeficiency virus (FIV) env gene and feline IL-12. Three groups of four cats each were immunized three times within 6 weeks by the ballistic transfer of gold particles coated with MIDGE vectors. Group 1 received non-coated gold beads, groups 2 and 3 MIDGE vectors expressing FIV surface plus part of the transmembrane protein. In addition, group 3 received feline IL-12 DNA. All cats were challenged by intraperitoneal injection of 25 TCID50 of infectious FIV Z2. The following criteria were monitored: clinical signs, antibodies to transmembrane protein, antibodies to whole FIV, haematological parameters and kinetics of CD4 and CD8 cells, FIV proviral load (determined by quantitative polymerase chain reaction; PCR) and cytotoxic T lymphocyte (CTL) activity (in selected cats). None of the cats developed a detectable antibody response during immunizations. Four weeks after challenge exposure, all cats in group 1 (control) and group 2 (FIV surface-transmembrane protein) had seroconverted and showed a high proviral load until week 19 (end of experiment). In contrast, only one of four cats in group 3 (surface-transmembrane protein and IL-12) showed antibodies; it was provirus positive at reduced virus load. Short-lived CTL activity was found in two cats in group 3. Genetic vaccination using a MIDGE-based construct for the expression of the surface-transmembrane protein domain of FIV env and feline IL-12 DNA led to protection against homologous virus challenge in three out of four vaccinated cats.

Cytokine regulation of env gene expression of human endogenous retrovirus-R in human vascular endothelial cells.

To determine whether human endogenous retroviruses are implicated in the pathogenesis of inflammatory vascular diseases of unknown etiology, we examined mRNA expression of a human endogenous retrovirus, HERV-R, which has a long open reading frame in the env region, in cultured human vascular endothelial and smooth muscle cells stimulated in the presence of various cytokines. mRNA of HERV-R was always evident in these cells but not in fibroblastic cells. Levels of expression in vascular endothelial cells were significantly regulated by treatment with tumor necrosis factor-alpha, interleukin (IL)-1alpha, and IL-1beta as up-regulators and interferon-gamma as a down-regulator. These observations are interpreted to mean that HERV-R expression may be up- or down-regulated at sites of inflammation in vessels in vivo and hence may play a pathogenetic role in inflammatory vascular diseases in humans, perhaps similar to endogenous retroviruses in mouse models of polyarteritis nodosa in humans.

IL-12 Delivery from Recombinant Vaccinia Virus Attenuates the Vector and Enhances the Cellular Immune Response Against HIV-1 Env in a Dose-Dependent Manner

To develop vaccination strategies against HIV-1 infection aimed to specifically enhance the cell-mediated immunity (CMI), we have engineered vaccinia virus (VV) recombinants expressing HIV-1 Env (rVVenv) and murine IL-12 (rVVlucIL-12) genes or coexpressing both genes (rVVenvIL-12). In mice inoculated with rVVlucIL-12 there is a rapid clearance of the virus, and this correlates with the induction of high levels of IL-12 and IFN-gamma in serum and spleen early after infection. Enzyme-linked immunospot analysis of mice inoculated with rVVlucIL-12, revealed a nearly 2-fold increase in the number of specific anti-VV CD8+ T cells compared with that in mice given control rVV, and the serum Ab response was biased in favor of a Th1 response. An enhancement of about 2-fold in the number of anti-gp160 IFN-gamma-secreting CD8+ T cells was observed in mice inoculated with rVVenvIL-12, when a dose of 1 x 107 PFU/mouse was used, but this enhancement was not observed when mice were given 5 x 107 PFU. This variation with virus dosage was confirmed in mice immunized simultaneously with different multiplicities of rVV expressing singly the env or IL-12 genes. The highest specific CMI was obtained in mice coadministered a low dose (2 x 104 PFU) of rVVlucIL-12 and 1 x 107 PFU of rVVenv. Our findings provide evidence for specific enhancement of the CMI to HIV-1 Env by the differential expression of IL-12 and env genes delivered from VV recombinants. This approach can be of wide vaccination interest as a means to improve immune responses to other Ags.

Insertion of sequences into the 3' untranslated region of a replication-competent spleen necrosis virus vector disrupts env gene expression.

The genomes of all replication-competent retroviruses contain cis-acting elements that regulate gene expression. However, the identities of many of these elements remain to be characterized. Inserting sequences into the 3' untranslated region of a replication-competent spleen necrosis virus (SNV) vector disrupted its ability to replicate. Inserts varying in sequences and sizes (0.4-kb to 1.6-kb) all resulted in this defect. Genetic compensation experiments and immunostaining revealed that env gene expression was deficient. Northern analysis indicated the presence of spliced viral mRNA of the correct size although at a reduced level compared to a wildtype vector. It is likely that the block in env expression occurs at a post-transcriptional step. These results suggest that the function of a cis-acting element distinct from the constitutive transport element is disrupted by the inserted sequences into the 3' untranslated region of SNV.

Distinct domains in herpes simplex virus type 1 US11 protein mediate post-transcriptional transactivation of human T-lymphotropic virus type I envelope glycoprotein gene expression and specific binding to the Rex responsive element.

Herpes simplex virus type 1 (HSV- 1) US11 protein is an RNA-binding protein which is able to mediate post-transcriptional transactivation of human T-lymphotropic virus type I (HTLV-I) envelope glycoprotein gene expression by interacting with the Rex responsive element (XRE) located at the 3' end of the env mRNA. In view of this functional activity, and because US11 protein is capable of substituting for HTLV-I Rex protein, it was hypothesized that US11 protein should exhibit at least two functional domains, an RNA-binding domain for specific interaction with the target RNA, and an effector domain involved in transport and translation of this mRNA. Recombinant US11 wild-type and deleted proteins were tested for their ability (i) to bind to the XRE and to HSV-1 UL34 RNA, the natural target of US11 protein, and (ii) to transactivate HTLV-I env gene expression. The C-terminal half of US11 protein, consisting of 20-24 XPR repeats, was necessary and sufficient to mediate RNA-binding with a high affinity and specificity. Structure prediction analyses showed the likely conformation of this domain to be that of a polyproline type II helix. Localized within the first 40 amino acids of the N-terminal region of US11 protein was the effector domain, deletion of which created US11(delta1-40), a trans-dominant negative mutant. These results demonstrate structural differences between US11 protein and proteins like Rex and Rev, despite their functional similarities.

Identification and Purification of Cellular Proteins That Specifically Interact with the RNA Constitutive Transport Elements from Retrovirus D

Human immunodeficiency virus (HIV) encodes a transacting protein, Rev, which interacts with an RNA element (RRE) to mediate nuclear export of unspliced viral mRNA. Recently, the RNA constitutive transport elements (CTE) from Mason-Pfizer monkey virus (MPMV) and simian retrovirus type I (SRV-1) were shown to render Rev-independent expression of gag, pol, or env genes in subgenomic constructs of HIV-1 and to support replication of HIV-1 mutants lacking RRE and Rev. Since CTEs act incis,in the absence of any viral regulatory proteins, it is widely believed that they interact directly with the cellular export machinery by means of RNA–protein interaction. In this report, Electrophoretic mobility shift and UV-crosslinking assays were carried out to identify nuclear proteins that interact specifically with CTEs from both MPMV and SRV-1. Two of the four proteins (65 and 40 kDa, respectively) that bound CTE RNA did not interact in the same assays with RNA of a nonfunctional CTE mutant generated by site-directed mutagenesis. Both proteins have been partially purified. The nature of these proteins and their roles in RNA intracellular trafficking are currently under investigation.

A tightly regulated high level expression vector that utilizes a thermosensitive lac repressor: production of the human T cell receptor Vβ5.3 in Escherichia coli

A series of vectors has been constructed to express the human T cell receptor Vβ5.3 under the control of the hybrid trc promoter in Escherichia coli. Transcriptional induction of the trc promoter was achieved chemically by using isopropyl β- d-thiogalactopyranoside (IPTG) in a bacterial strain that harbors the lacI q gene, or thermally by using the mutant laclts gene that encodes a temperature-sensitive lac repressor [Bukrinsky et al. (1988) Multicopy expression vector based on temperature-regulated lac repressor: expression of human immunodeficiency virus env gene in Escherichia coli. Gene 70, 415–417]. Several of the plasmids tested also contain the E. coli heat-stable enterotoxin II (STII) signal sequence for protein secretion. In addition, the gene 10 leader sequence from bacteriophage T7 and a minicistron localized upstream of the Vβ5.3 coding sequence were tested for their potential effect on protein production. These elements increased protein yield two-fold when transcription was induced by IPTG, but had no detectable effect on protein yield when transcription was induced thermally. The highest protein yield was obtained when Vβ5.3 was expressed either from plasmid pKB containing the STII signal in strain LJ24, or from plasmid pKBi that lacks the signal sequence, in the protease deficient strain SG21173 ( lon, htpR, clp). Both plasmids contain the laclts gene, the trc promoter, the two transcription terminators of the rrnB operon, and a tetracycline selection marker. Production of Vβ5.3 using pKBi-Vβ5.3 in strain SG21173 in a 5-liter fermenter under controlled growth conditions yielded over 25 mg Vβ5.3/liter culture. Conversion of the laclts to the lacI qts gene yielded vector pKBiq-Vβ5.3 which exhibits complete repression of the trc promoter at 30°C. This stringent regulation of the thermally inducible trc promoter, the elimination of IPTG, the inclusion of the tetracycline resistance gene, and the high level of protein yield should render this expression system broadly useful for the high level production of heterologous proteins in E. coli, for both basic research and human therapeutic applications.

Expression of HIV env gene in a human T cell line for a rapid and quantifiable cell fusion assay.

Human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins present at the surface of infected cells are known to mediate fusion with CD4-positive target cells. In this study we have developed a novel Env-expressing cell line for investigating the fusion process in a biologically significant system. Cell surface expression of the HIV-1 env gene, isolated from the highly fusogenic strain SF33, was obtained in the CD4-negative T cell line A2.01. To render the system versatile and efficient, HIV-1 regulatory proteins Tat and Rev were supplied in trans. The presence of Env at the cell surface was shown by cytofluorometry and immunofluorescence and precursor processing of gp160 to gp120/gp41 was demonstrated by Western blot. The fusion capacity of A2.01-Env cells was assessed by coculture with CD4-positive T lymphocytes or the fusion indicator cell line, HeLa-CD4-LTR-beta-Gal. By coincubation with CD4-positive T cells such as SupT1, A2.01-Env cells were observed to mediate rapidly numerous well-defined syncytia in a reproducible fashion. By expressing Tat, they also had the capacity to trans-activate the LTR-linked reporter beta-Gal gene following fusion with HeLa-CD4-LTR-beta-Gal cells. The fusion-inhibiting anti-CD4 monoclonal antibodies Q425 and Q428 were used to block specifically Env-mediated fusion with CD4-positive cells and to demonstrate application of this system to the search for potential fusion-blocking agents. Our system thus offers a biologically significant model for studying fusion events with the advantages of being rapid, reproducible and versatile.

Examination of TAR-independent Trans activation by human immunodeficiency virus type 1 Tat in human glial cells.

Astrocytic glial cells derived from central nervous system (CNS) can support human immunodeficiency virus type 1 (HIV-1) replication in cell culture, may be infected in tissue culture, and are thought to be a large HIV-1 reservoir in vivo. The Tat protein of HIV-1 interacts with a cis-acting target sequence referred to as TAR. However, Tat can also stimulate gene expression directed from some heterologous promoters and, in certain circumstances, an HIV-1 long terminal repeat (LTR) that lacks the TAR element. Therefore, we attempted to investigate Tat trans activation of HIV-1 LTR in the astrocytic glial cells. Using transfection of LTR-reporter gene constructs and HIV-1 proviral constructs, we demonstrate TAR-dependent replication in astrocytic cells. We also examined the expression of HIV-1 env gene from an LTR that lacks TAR element. In a previous study (Kim and Panganiban: J Virol 67:3739-3747, 1993), we observed that env expression is trans activated only by the full-length Tat protein through a TAR-independent manner in HeLa cells. However, in astrocytic glial cells, the trans activation of env expression from the LTR-lacking TAR element was mediated by the first exon peptide of Tat as well as the full-length Tat peptide through a post-transcriptional mechanism rather than a transcriptional one. This result suggests that cell type-specific factor(s) is involved in the TAR-independent Tat responsiveness.

Expression of sequences homologous to HTLV-I tax gene in the labial salivary glands of Japanese patients with Sjögren's syndrome.

To address the question of whether the human T cell leukemia virus type I (HTLV-I) gene is associated with the etiology of Sjögren's syndrome (SS). RNA expression of HTLV-I gag, pol, env, and tax genes in labial salivary glands (LSGs) from SS patients who were seronegative for antibodies to HTLV-I was examined using reverse transcription and polymerase chain reaction techniques. The HTLV-I tax gene, but not the HTLV-I gag, pol, or env genes, was detected in LSG samples from 4 of 14 patients (29%). The nucleotide sequences of the HTLV-I pXIV region in these 4 patients' LSGs showed 100% homology to the HTLV-I pXIV gene from the MT-2 cell line. These findings suggest that products encoding sequences homologous to the HTLV-I pXIV gene in SS patients' LSGs might be candidates for self-antigen and/or lead to activation of autoreactive T lymphocytes through trans-acting transcriptional activation.

Requirement of the Human-Immunodeficiency-Virus Type-1 env Gene Sequence for Tar-Independent trans Activation by Tat from the Major Immediate-Early Promoter of Murine Cytomegalovirus

Previously it has been demonstrated that the human immunodeficiency virus type 1 (HIV-1) Tat protein mediates induction of the HIV-1 env expression through a TAR-independent manner in heterologous and homologous promoter systems (Kim and Risser, 1993, J. Virol. 67, 239; Kim and Panganiban, 1993, J. Virol. 67, 3739). To further explore the transactivation of HIV-1 env gene, I examined expression of the env, the baterial CAT, and the firefly luciferase genes from a heterologous promoter, the major immediate-early promoter (MIEP) of murine cytomegalovirus (MCMV). Here we show that Tat augments gene expression from the MCMV MIEP only when linked to the env gene, Surprisingly, in contrast to the expression from an HIV-1 LTR lacking the TAR element, TAR-independent transactivation of env gene expression from MCMV MIEP did not require the full length Tar protein. In addition, deletion of the previously identified cis-acting Tat-responsive element in env did not affect Tat transactivation of the env gene expression. Thus, there are multiple distinct elements that mediate Tat responsiveness in the absence TAR.

Identification of spliced RNA species of Drosophila melanogaster gypsy retrotransposon New evidence for retroviral nature of the gypsy element

We have identified a novel RNA species of Drosophila melanogaster gypsy retrotransposon that is ca. 2 kb in length and corresponds to the third open reading frame (ORF3) of the gypsy element. This RNA is generated by splicing of the primary gypsy transcript, as is the case for retroviral env gene expression. Therefore, the striking resemblance between gypsy and retroviruses has now been extended by this study to the expression strategies of these retroelements. The primary structure of spliced RNA was determined, and its analysis shows that both gypsy subfamilies (6K and 7K) apparently are able to encode functionally active ORF3 translation products.

Expression of env gene of bovine leukemia virus in rodent cells.

The BamHI-BamHI fragment of the env gene of bovine leukemia virus (BLV) cloned in pMMEx expression vector was transfected into Chinese hamster cells. Monoclonal antibodies (MAbs) directed against both conformational and sequential epitopes of gp51 of BLV recognized viral polypeptides expressed in hamster cells in Western blotting and enzyme-linked immunosorbent assay.

Membrane Expression of HIV Envelope Glycoproteins Triggers Apoptosis in CD4 Cells

The cytopathic effect of HIV-1 and HIV-2 in CD4+ lymphocytes has been shown to be associated with apoptosis or programmed cell death. Using different experimental conditions, we demonstrate here that apoptosis is triggered by cell membrane expression of the mature HIV envelope glycoproteins, gp120-gp41 complex, and their interaction with CD4 receptor molecules. Viral entry alone did not induce apoptosis but virus replication was required in order to produce the gp120-gp41 complex. Indeed, expression of the HIV env gene alone in the CD4+ T cell line (CEM) was sufficient for the induction of apoptosis. In general, syncytium formation and apoptosis induction were closely associated as both events require functional envelope glycoproteins and CD4 molecules. Nevertheless, apoptosis but not syncytium formation was suppressed by a monoclonal antibody against CD4 that does not affect gp120 binding. Furthermore, single-cell killing by apoptosis was observed in infected cell cultures treated with a monoclonal antibody against gp41, which completely abolishes the formation of syncytia. These results indicate that apoptosis is not the consequence of toxic effects induced by the formation of syncytia but is triggered by the HIV envelope glycoproteins. Therefore, cell death during HIV infection in CD4+ lymphocyte cultures is due to a specific event triggered by the gp120-gp41 heterodimer complex programming death in metabolically active cells.

The full-length Tat protein is required for TAR-independent, posttranscriptional trans activation of human immunodeficiency virus type 1 env gene expression

Tat is a protein that dramatically increases the expression of all genes expressed from the human immunodeficiency virus type 1 (HIV-1) long terminal repeat through interaction with a cis-acting target sequence referred to as TAR (for trans-acting responsive region). The tat gene is divided into two coding exons which, when translated, result in the synthesis of an 86-amino-acid protein. However, the 72-amino-acid segment encoded by the first coding exon of tat is sufficient to encode a fully active Tat protein in known assays. We examined expression of the env gene from an LTR that lacks TAR (designated dTAR-env). Surprisingly, only the full-length Tat peptide trans activated expression of the env gene from dTAR-env. Comparison of RNA and protein expression of the env gene in the presence of Tat indicated that the mechanism of trans activation is posttranscriptional rather than transcriptional. To test whether the TAR-independent Tat function is specific to the HIV-1 env gene, we analyzed expression of heterologous genes from the long terminal repeat lacking TAR. These heterologous genes were not trans activated by Tat in the absence of a TAR element, which suggests that the second-exon peptide of Tat has a sequence-specific role in TAR-independent trans activation of the HIV-1 env gene. Analysis of a mutant in the 5' end of the env gene was used to identify a cis-acting sequence required for Tat responsiveness.

Integration is essential for efficient gene expression of human immunodeficiency virus type 1

A mutant of human immunodeficiency virus type 1 which carries a frameshift insertion in the integrase/endonuclease region of pol gene was constructed in vitro. Upon transfection into cells, although this mutant exhibited a normal phenotype with respect to expression of gag, pol, and env genes and to generation of progeny virions, no replication-competent virus in CD4-positive cells emerged. An assay for the single-step replication of a defective viral genome dependent on trans complementation by rev protein was established and used to monitor the early phase of viral infection process. Viral clones with a mutation in the vif, vpr, or vpu gene displayed no abnormality in the early phase. In contrast, the integrase mutant did not direct a marker gene expression after infection. Together with an observation that the mutant lacked the ability to integrate, these results indicated that the integration was required for efficient viral gene expression and productive infection of human immunodeficiency virus type 1.

Capture-RT-PCR assay of mRNA in breast tumors:int-2 and HERV-Kenv mRNA

Gene expression at the mRNA level is likely to be a rapidly responding intermediate biomarker for use as a surrogate endpoint in Phase II clinical trials of breast cancer. Using a technology called “capture-RT-PCR,” we have been able to show ectopic int-2 mRNA expression in 7/9 breast tumors, including two fibroadenomas. The level of expression varied, high expression being observed in three tumors. Frequent expression of int-2 in breast tumors is contrary to published reports, possibly reflecting differences in technology. int-2 expression in fibroadenomas suggests that the marker may precede malignancy. Conversely, if fibroadenomas are not premalignant, int-2 expression may be gratuitous. These alternatives need to be tested in a clinical trial. Human endogenous retrovirus K (HERV-K) env gene expression was detected in only 1/7 breast tumors. Expression of this retroviral gene is more restricted than int-2 expression. c-myc was expressed in all 10 tumors studied, albeit at widely varying levels. Expression of prad1 and erbB-2 are under study. Gene expression will be compared with gene amplification. It is anticipated that a capture-RT-PCR assay simultaneously signifying levels of expression of several oncogenes/anti-oncogenes will be a most informative surrogate endpoint biomarker. In particular, chaotropic salt methods that simplify sample preparation and mRNA isolation and reduce these combined steps to a 10 minute procedure will be presented.