Independent mechanisms involved in suppression of the Moloney leukemia virus genome during differentiation of murine teratocarcinoma cells

Expression and DNA methylation of the Moloney murine leukemia virus (M-MuLV) genome were investigated in murine teratocarcinoma cells after virus infection. The newly acquired viral genome was devoid of methylation, yet its expression was repressed. The integrated viral genome in undifferentiated teratocarcinoma cells was methylated within 15 days after infection. Although 5-azacytidine decreased the level of DNA methylation, it did not activate M-MuLV in undifferentiated cells. Activation by 5-azacytidine occurred only in differentiated teratocarcinoma cells. Thus two independent mechanisms seem to regulate gene expression during the course of differentiation. The first mechanism operates in undifferentiated cells to block expression of M-MuLV and other exogeneously acquired viral genes, such as SV40 and polyoma virus, and does not depend on DNA methylation. The second mechanism relates only to differentiated cells and represses expression of genes in which DNA is methylated.     

Generation of glucocorticoid-responsive Moloney murine leukemia virus by insertion of regulatory sequences from murine mammary tumor virus into the long terminal repeat.

The glucocorticoid-regulatory sequences from the murine mammary tumor virus long terminal repeat (MMTV LTR) were introduced into the LTR of Moloney murine leukemia virus (M-MuLV) by recombinant DNA techniques. The site of insertion was in the M-MuLV LTR U3 region at -150 base pairs with respect to the RNA cap site. Infectious M-MuLVs carrying the altered LTRs (Mo + MMTV M-MuLVs) were recovered by transfection of proviral clones into NIH-3T3 cells. The Mo + MMTV M-MuLVs were hormonally responsive in that infection was 3 logs more efficient when performed in the presence of dexamethasone, irrespective of the orientation of the inserted MMTV sequences. However, even in the presence of hormone, the Mo + MMTV M-MuLVs were less infectious than wild-type M-MuLV. In contrast to the large effect on infectivity, dexamethasone induced virus-specific RNA levels in chronically Mo + MMTV M-MuLV-infected cells only two- to fourfold. Fusion plasmids between the altered LTRs and the bacterial chloramphenicol acetyltransferase gene allowed the investigation of LTR promoter strength by the transient chloramphenicol acetyltransferase expression assay. The chloramphenicol acetyltransferase assays indicated that the insertion of MMTV sequences into the M-MuLV LTR reduced promoter activity in the absence of glucocorticoids but that promoter activity could be induced two- to fivefold by dexamethasone. The Mo + MMTV M-MuLVs were also tested for the possibility that viral DNA synthesis or integration during initial infection was enhanced by dexamethasone. However, no significant difference was detected between cultures infected in the presence or absence of hormone. The insertion of MMTV sequences into an M-MuLV LTR deleted of its enhancer sequences did not yield infectious virus or active promoters, even in the presence of dexamethasone.     

Gene activation mediated by protein kinase C in human macrophage and teratocarcinoma cells expressing aminoglycoside phosphotransferase activity.

The bacterial neomycin phosphotransferase gene driven by the Moloney mouse leukemia virus long terminal repeat (LTR) or SV40 early region promoter was introduced into the human promonocyte-macrophage cell line, U937, and into the pluripotential human embryonic teratocarcinoma cell line, NT2/D1. Clonally derived cell lines capable of growing in 2-4 mg/ml of the aminoglycoside antibiotic, G418 (Geneticin), were established and transfected with pHIVCat, a plasmid expressing the bacterial chloramphenicol acetyl transferase (CAT) activity under the control of the human immunodeficiency virus (HIV-1) LTR. All of the G418 resistant (neo(r)) U937 cell lines and 10 of 14 neo(r) NT2/D1 cell lines exhibited reduced basal levels of CAT expression or impaired responses to activation of the HIV-1 LTR by phorbol 12-myristate 13-acetate (PMA) when compared to the parental lines. Other differences included inhibition of tat activation of the HIV-1 LTR and increased sensitivity of U937 cells to human tumor necrosis factor alpha. The expression of other eukaryotic promoters including the HTLV-1 LTR, SV40 ori sequences, and the human beta-actin gene promoter was similarly affected. However, differentiation of the neo(r) U937 cells into macrophages was neither delayed nor impaired. Because PMA is an activator of protein kinase C (PKC) and a potent inducer of HIV-1 directed gene expression, the amounts, sensitivity to G418, and cytosol to membrane translocation of this enzyme were determined in the wild type and neo(r) U937 cells. G418 at concentrations too low to affect cell growth (12-150 micrograms/ml) inhibited PMA-induced transactivation responses in wild type cells but did not inhibit PKC-dependent protein phosphorylation in vitro. PKC activities in the wild type and neo(r) cells were similar in absolute amounts and in the cytosol-membrane distribution of the enzyme. In contrast with wild type cells, however, all of the cytosolic Ca(2+)-phospholipid-dependent form of PKC disappeared from the neo(r) cells within 30 min after PMA induction. The results suggested that, depending upon the cell type, gene cotransfer using aminoglycoside resistance as a selectable marker may seriously perturb important cellular control mechanisms such as the PKC pathway leading to activation of gene expression.     

Rearrangements and insertions in the Moloney murine leukemia virus long terminal repeat alter biological properties in vivo and in vitro.

The effects of rearrangement and insertion of sequences in the Moloney murine leukemia virus (M-MuLV) long terminal repeat (LTR) were investigated. The alterations were made by recombinant DNA manipulations on a plasmid subclone containing an M-MuLV LTR. Promoter activity of altered LTRs was measured by fusion to the bacterial chloramphenicol acetyltransferase gene, followed by transient expression assay in NIH 3T3 cells. M-MuLV proviral organizations containing the altered LTRs were also generated, and infectious virus was recovered by transfection. Infectivity of the resulting virus was quantified by XC plaque assay, and pathogenicity was determined by inoculating neonatal NIH Swiss mice. Inversion of sequences in the U3 region containing the tandemly repeated enhancer sequences (-150 to -353 base pairs [bp]) reduced promoter activity approximately fivefold in the transient-expression assays. Infectious virus containing the inverted sequences (Mo- M-MuLV) showed a 20-fold reduction in relative infectivity compared with wild-type M-MuLV, but the virus still induced thymus-derived lymphoblastic lymphoma or leukemia in mice, with essentially the same kinetics as for wild-type M-MuLV. We previously derived an M-MuLV which carried inserted enhancer sequences from the F101 strain of polyomavirus (Mo + PyF101 M-MuLV) and showed that this virus is nonleukemogenic. In Mo + PyF101 M-MuLV, the PyF101 sequences were inserted between the M-MuLV promoter and the M-MuLV enhancers (at -150 bp). A new LTR was generated in which the PyF101 sequences were inserted to the 5' side of the M-MuLV enhancers (at -353 bp, PyF101 + Mo M-MuLV). The PyF101 + Mo LTR exhibited promoter activity similar (40 to 50%) to that of wild-type M-MuLV, and infectious PyF101 + Mo M-MuLV had high infectivity on NIH 3T3 cells (50% of wild type). In contrast to the nonleukemogenic Mo + PyF101 M-MuLV, PyF101 + Mo M-MuLV induced leukemia with kinetics similar to that of wild-type M-MuLV. Thus, the position of the PyF101 sequences relative to the M-MuLV LTR affected the biological behavior of the molecular construct. Furthermore, PyF101 + Mo M-MuLV induced a different spectrum of neoplastic disease. In comparison with wild-type M-MuLV, which induces a characteristic thymus-derived lymphoblastic lymphoma with extremely high frequency, PyF101 + Mo M-MuLV was capable of inducing both acute myeloid leukemia or thymus-derived lymphoblastic lymphoma, or both. Tumor DNA from both the PyF101 + Mo- and Mo- M-MuLV-inoculated animals contained recombinant proviruses with LTRs that differed from the initially inoculated virus.     

Construction of cell lines that regulate by temperature the amplification and expression of influenza virus non-structural protein genes.

Monkey cell lines have been transformed with a mixture of plasmids pSV2neo and pSLVa232N, a derivative of plasmid pSLVa232 (Portela et al., 1985b). Plasmid pSLVa232N contained the influenza virus genes encoding non-structural proteins under the control of the SV40 late promoter in pSLts1 vector that includes the SV40 ori and the tsA209 T-antigen gene. At restrictive temperature, plasmid sequences remained stably integrated in the cell genome, but upon temperature shift-down, defined circular DNA molecules were generated and amplified up to 2000-5000 copies/cell. Restriction analysis, Southern blot hybridization and partial sequencing indicate that one such episome, pC5, was derived from the integrated plasmid sequences by a homologous recombination event that led to deletion of the pBR322 sequences included in pSLVa232N. Concomitant with gene amplification, an induction of 20-65-fold in the expression of NS1 and NS2 proteins was observed after temperature shift-down. Thus, gene cloning into vector pSLts1 and transformation at restrictive temperature of cells permissive for SV40 DNA replication, appears to be a useful strategy for the controlled amplification and expression of cloned genes.     

Sequences of herpes simplex virus type 1 that inhibit formation of stable TK+ transformants.

We have identified two regions of the herpes simplex virus type 1 (HSV-1) genome that inhibit DNA-mediated transformation of thymidine kinase-less L (Ltk-) cells by the cloned HSV-1 tk gene. When plasmids containing the EcoRI fragments EK or JK were mixed at 30 fmol/ml with the tk gene and transfected into Ltk- cells, the frequency of transformation was inhibited 80 to more than 90% relative to the control. Of the remaining 10 EcoRI fragments of the HSV-1 genome, 8 were inactive and 2 were weakly active. A 6.1-kilobase PstI subclone between 0.743 and 0.782 map units was isolated from pEK. This clone, pEK-P3P4, exhibited antitransformation activity toward HSV-1 tk and also the bacterial genes gpt and neo. pEK-P3P4 contains the alpha 27 gene, and restriction endonuclease inactivation and subcloning studies established that alpha 27 alone did not inhibit transformation. However, alpha 27 plus sequences both upstream and downstream of alpha 27 did inhibit transformation. In addition, alpha 0 or alpha 4 could substitute for alpha 27 in effecting antitransformation with these sequences. Therefore, an alpha gene and two additional loci in pEK-P3P4 are required for antitransformation. A second antitransforming locus in the reiterated sequences common to EK and JK and distinct from those in pEK-P3P4 was also identified but not characterized in detail. How antitransformation may be an expression of regulation of viral and host cell gene expression is discussed.     

Exogenous delta-crystallin gene expression as probe for differentiation of teratocarcinoma stem cells

We developed an experimental system in which differentiation of teratocarcinoma stem cell is probed by expression of stably introduced exogenous genes. We used chicken delta-crystallin gene (delta gene) and its derivative (Mo delta gene) driven by long terminal repeat (LTR) promoter of Moloney murine leukemia virus (Mo-MuLV). Neither of the genes was expressed in the undifferentiated condition. Differentiation to primitive endoderm induced by retinoic acid (RA) led to expression of delta but not Mo delta, while differentiation to more advanced endodermal cells by RA plus dibutyryl cAMP elicited Mo delta expression in addition to delta. These results are interpreted as a consequence of differential activation/suppression of gene expression through enhancer elements associated with the genes.     

Transformation of the cells of transplantable teratocarcinoma CBA9H6 by a bacterial dihydrofolate reductase gene

A plasmid containing the bacterial gene of methotrexate-resistant dihydrofolate reductase (dhfr), under the control of early SV40 promoter, was introduced into murine teratocarcinoma CBA9H6 cells. From the whole pool of teratocarcinoma cells, which survived after transient methotrexate selection in vivo, the individual cells were isolated to give rise to 15 clones of tumors. Six of the 15 clones displayed nucleotide sequences of the original vector containing pBR322 sequences and the early SV40 promoter region; however, the bacterial dhfr gene was absent from the transformant clones. Possible causes of the loss of introduced dhfr gene from teratocarcinoma cells under non-selective conditions are discussed.     

Stable Expression of a Foreign Gene, Delivered by Gene Gun, in the Muscle of Rainbow Trout Oncorhynchus mykiss

We report the efficient delivery of a foreign gene into muscle of rainbow trout Oncorhynchus mykiss with a gene gun. The foreign gene was a reporter gene, chloramphenicol acetyltransferase (CAT). Two CAT-containing plasmids were used: pCMV-CAT, which contains cytomegalovirus immediate early promoter, and pSV2-CAT, which contains the simian virus 40 early promoter. All plasmids were introduced by particle bombardment using a gene gun. During the 90-day sampling period following bombardment, CAT was strongly and stably expressed in the muscle of all the fish bombarded with pCMV-CAT and pSV2-CAT. No CAT expression was detected in the blood samples until 90 days after introduction, when it was found in only one fish from the pCMV-CAT group and one from the pSV2-CAT group. The stable and long-term expression of plasmid DNA in muscle makes muscle an attractive target tissue for the introduction of viral DNA for the purpose of DNA vaccination. Received June 5, 1999; accepted November 2, 1999.