VL30 Element Research Articles

Regulation of a rat VL30 element in human breast cancer cells in hypoxia and anoxia: role of HIF-1.

Novel approaches to cancer gene therapy currently exploit tumour hypoxia to achieve transcriptional targeting using oxygen-regulated enhancer elements called hypoxia response elements. The activity of such elements in hypoxic cells is directly dependent on upregulation of the hypoxia-inducible transcription factor-1 However tumours also contain areas of anoxia, which may be considered a more tumour-selective transcriptional stimulus than hypoxia for targeting gene therapy to tumours. Another element, from the rat virus-like retrotransposon, VL30 (termed the ‘secondary anoxia response element’) has been reported to be more highly inducible in rat fibroblasts under anoxia than hypoxia. To investigate anoxia as a potential transcriptional target in human tumours, we have examined secondary anoxia response element inducibility in two human breast cancer cell lines, MCF-7 and T47D, under anoxia, hypoxia and normoxia. In both cell types, the trimerised secondary anoxia response element showed greater inducibility in anoxia than hypoxia (1% and 0.5% O2). The anoxic response of the secondary anoxia response element was shown to be dependent on hypoxia-inducible transcription factor-1 and the presence of a hypoxia-inducible transcription binding site consensus (5′-ACGTG-3′). Mutational analysis demonstrated that the base immediately 5′ to this modulates the anoxic/hypoxic induction of the secondary anoxia response element, such that TACGTG>GACGTG>>CACGTG. A similar correlation was found for erythropoietin, phosphoglycerate kinase 1, and aldolase hypoxia response elements, which contain these respective 5′ flanking bases.British Journal of Cancer (2002) 87, 1173–1181. doi:10.1038/sj.bjc.6600576 www.bjcancer.com© 2002 Cancer Research UK

Conserved, Erythropoietin-Responsive VL30 Promoters Isolated from Erythroid Progenitor Cells

ABSTRACTVirus-like 30S (VL30) elements are endogenous retro-elements of the mouse retrotransposon family. These elements are transcriptionally responsive in a temporal and tissue-specific manner due to the U3 promoter region of the elements' long terminal repeat (LTR). We have analyzed VL30 promoters from erythroid progenitor cell lines (MEL 585S and ELM-I-1) that contrasted in their response to erythropoietin (epo). Through RT-PCR-generated cDNAs, VL30 promoters were identified and showed homology to the third and fourth U3 subgroups, with GATA-1, Jak2/STAT5, and B10 RRE sites. One clone (ELM5) showed 97% homology to BVL-1, a putative epo-responsive VL30 element. In addition, a novel U3 promoter (MEL/ELM CONSTIT) showed complete sequence homology between both cell lines. Ribonuclease protection confirmed that epo-induced VL30 promoters were activated in ELM-I-1 cells, whereas the conserved VL30 MEL-ELM CONSTIT VL30 promoter showed no enhanced expression in the epo-unresponsive MEL cells. Identification of these U3 promoters suggests that VL30s are conserved and can be transcriptionally activated in an epo-specific manner.

Molecular analysis of reverse mutations from nonagouti (a) to black-and-tan (a(t)) and white-bellied agouti (Aw) reveals alternative forms of agouti transcripts.

The agouti gene regulates the differential production of eumelanin (black or brown) and phaeomelanin (yellow) pigment granules by melanocytes in the hair follicles of mice. The original nonagouti (a) allele, which confers a predominantly black coat color, has been shown to revert to two other more dominant agouti alleles, black-and-tan (a(t)) and white-bellied agouti (Aw), with an exceptionally high frequency. The a(t) and Aw alleles confer phenotypes in which the pigmentation is not uniformly distributed over the dorsal and ventral surfaces of the animal; in both cases the ventral surface of the animal is markedly lighter than the dorsal surface due to an increase in phaeomelanin production. To understand the unusually high reversion rate of a to a(t) or Aw, and to decipher the molecular events associated with the different pigmentation patterns associated with these three agouti alleles, we have characterized a, a(t) and Aw at the molecular level. Here, we report that insertions of 11, 6, and 0.6 kb are present at precisely the same position in the first intron of the agouti gene in a, a(t), and Aw, respectively. The a insertion consists of a 5.5-kb VL30 element that has incorporated 5.5 kb of additional sequence internally; this internal sequence is flanked by 526-bp direct repeats. The a(t) allele contains only the VL30 element and a single, internal 526-bp repeat. The Aw allele has only a solo VL30 LTR. Based on the comparison of the structure of the a(t) and Aw insertions, we propose that reverse mutations occur by excision of inserted sequences in a through homologous recombination, utilizing either the 526-bp direct repeats to generate a(t) or the VL30 LTRs to generate Aw. Moreover, the analysis of these three alleles has allowed us to identify additional exons of the agouti gene that give rise to alternatively processed forms of agouti mRNA. We demonstrate that the distinct insertions in a, a(t) and Aw cause pigmentation differences by selectively inactivating the expression of different forms of agouti transcripts.

A small and efficient dimerization/packaging signal of rat VL30 RNA and its use in murine leukemia virus-VL30-derived vectors for gene transfer.

Retroviral genomes consist of two identical RNA molecules associated at their 5' ends by the dimer linkage structure located in the packaging element (Psi or E) necessary for RNA dimerization in vitro and packaging in vivo. In murine leukemia virus (MLV)-derived vectors designed for gene transfer, the Psi + sequence of 600 nucleotides directs the packaging of recombinant RNAs into MLV virions produced by helper cells. By using in vitro RNA dimerization as a screening system, a sequence of rat VL30 RNA located next to the 5' end of the Harvey mouse sarcoma virus genome and as small as 67 nucleotides was found to form stable dimeric RNA. In addition, a purine-rich sequence located at the 5' end of this VL30 RNA seems to be critical for RNA dimerization. When this VL30 element was extended by 107 nucleotides at its 3' end and inserted into an MLV-derived vector lacking MLV Psi +, it directed the efficient encapsidation of recombinant RNAs into MLV virions. Because this VL30 packaging signal is smaller and more efficient in packaging recombinant RNAs than the MLV Psi + and does not contain gag or glyco-gag coding sequences, its use in MLV-derived vectors should render even more unlikely recombinations which could generate replication-competent viruses. Therefore, utilization of the rat VL30 packaging sequence should improve the biological safety of MLV vectors for human gene transfer.

Characterization of a unique enhancer element responsive to cyclic adenosine 3',5'-monophosphate and elevated calcium.

The VL30 family of defective retrovirus-like elements is abundantly transcribed in response to numerous transforming and proliferative stimuli. We have identified a novel enhancer element in the long terminal repeat of the transcriptionally active VL30 element RVL-3. The RVL-3 enhancer mediates a calcium-dependent induction of gene expression in response to treatment with either epidermal growth factor or the phorbol ester tumor promoter 12-O-tetradecanoylphorbol acetate. In this report we present in vivo and in vitro evidence indicating that the RVL-3 enhancer is also responsive to cAMP in the presence of elevated intracellular calcium. Proteins present in nuclear extracts obtained from Rat-1 fibroblasts bind specifically to a 20-basepair sequence within the RVL3 triple repeat. Competition binding studies and mutational analyses indicate that the cAMP responsiveness maps to the same region responsible for mediating the inductive response to epidermal growth factor and 12-O-tetradecanoylphorbol. The responsive sequence is different from previously described enhancer elements. This novel enhancer mediates transcription by multiple agonists and promotes a greater than additive increase in gene expression when more than one signal transduction pathway is stimulated simultaneously.

Anoxia, wound healing, VL30 elements, and the molecular basis of malignant conversion

Although VL30 retrotransposable elements have been associated with certain cancers for nearly twenty years, because of their expression in rodent malignancies and recombination into murine sarcoma viruses, their causative role, if any, in cancer has been uncertain and enigmatic. Recent findings suggest loss of normal transcriptional control of specific VL30 element expression may make a critical contribution to tumor progression at a step associated with malignant conversion, by bringing into play a cellular program normally involved in wound healing. This program, the fibroblast anoxic response system, includes an adaptation to glycolytic metabolism, secretion of metalloproteinases, and activation of an endonuclease. While appropriate for facilitating debris removal during wound healing, loss of control of this program in a cell which has already progressed to the benign neoplastic state has the potential to simultaneously produce the invasiveness and genomic instability characteristic of malignancy. Examination of tumors and tumor derived cell lines has confirmed that key aspects of this system are in fact activated in cancer.

Identification of a novel enhancer element mediating calcium-dependent induction of gene expression in response to either epidermal growth factor or activation of protein kinase C.

The VL30 family of defective murine retroviruses consists of 100 to 200 members, of which fewer than 5% appear to be transcriptionally active. A genomic clone of the transcriptionally active VL30 element RVL-3 was identified and sequenced. Genetic analysis indicated that a triple-repeat sequence within the RVL-3 long terminal repeat is capable of functioning as an inducible enhancer element responding to a variety of agonists. In Rat-1 fibroblasts, the ability of the RVL-3 enhancer to mediate induction of gene expression from a heterologous promoter in response to either epidermal growth factor (EGF) or phorbol ester treatment required coelevation of intracellular calcium. Two CArG boxes present in the triple-repeat sequence appeared to exert a negative effect on gene expression, as mutation of these sequences elevated the basal level of expression observed without altering the fold induction in response to either EGF or protein kinase C activation. In the presence of these CArG elements, mutation of AP-1-like sites adjacent to the CArG elements significantly inhibited the ability of either EGF or phorbol esters to induce gene expression. The effect of mutating these AP-1-like sites was relieved by simultaneous mutation of the CArG sites, indicating that interactions among these sites modulate RVL-3 expression. Mutational analysis and gel mobility shift experiments have identified a third sequence within the VL30 triple-repeat element that is required for the induction of gene expression and serves as a binding site for nuclear proteins. Sequence comparisons indicate that this enhancer element has not been described previously.

Identification of a novel enhancer element mediating calcium-dependent induction of gene expression in response to either epidermal growth factor or activation of protein kinase C.

The VL30 family of defective murine retroviruses consists of 100 to 200 members, of which fewer than 5% appear to be transcriptionally active. A genomic clone of the transcriptionally active VL30 element RVL-3 was identified and sequenced. Genetic analysis indicated that a triple-repeat sequence within the RVL-3 long terminal repeat is capable of functioning as an inducible enhancer element responding to a variety of agonists. In Rat-1 fibroblasts, the ability of the RVL-3 enhancer to mediate induction of gene expression from a heterologous promoter in response to either epidermal growth factor (EGF) or phorbol ester treatment required coelevation of intracellular calcium. Two CArG boxes present in the triple-repeat sequence appeared to exert a negative effect on gene expression, as mutation of these sequences elevated the basal level of expression observed without altering the fold induction in response to either EGF or protein kinase C activation. In the presence of these CArG elements, mutation of AP-1-like sites adjacent to the CArG elements significantly inhibited the ability of either EGF or phorbol esters to induce gene expression. The effect of mutating these AP-1-like sites was relieved by simultaneous mutation of the CArG sites, indicating that interactions among these sites modulate RVL-3 expression. Mutational analysis and gel mobility shift experiments have identified a third sequence within the VL30 triple-repeat element that is required for the induction of gene expression and serves as a binding site for nuclear proteins. Sequence comparisons indicate that this enhancer element has not been described previously.

Modulation of RNA expression by intracellular calcium. Existence of a threshold calcium concentration for induction of VL30 RNA by epidermal growth factor, endothelin, and protein kinase C.

We investigated the role of intracellular [Ca2+] in mediating the independent signal transduction pathways leading to induction of VL30 RNA expression by multiple agonists in the Rat-1-derived RVL-3 cell line. This cell line contains a single integrated VL30 element, and displays a rapid transcriptional activation of VL30 following stimulation by epidermal growth factor, endothelin, or the phorbol ester tumor promoter 12-O-tetradecanoylphorbol acetate (Rodland, K. D., Brown, A. M. C., and Magun, B. E. (1987) M. Cell. Biol. 7, 2296-2298). Neither epidermal growth factor nor endothelin is dependent upon protein kinase C for activation of VL30 expression, as both of these agonists induce normal levels of VL30 RNA expression, even in cells which have been severely depleted of protein kinase C following chronic 12-O-tetradecanoylphorbol acetate exposure. Induction of VL30 RNA expression by either endothelin or 12-O-tetradecanoylphorbol acetate was blocked by concomitant exposure of RVL-3 cells to the intracellular Ca2(+)-chelating agent 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid at a concentration sufficient to buffer intracellular [Ca2+] below 200 nM, and VL30 RNA was induced by the application of the Ca2+ ionophore A23187 in the absence of agonist. Normal levels of VL30 expression in response to epidermal growth factor were observed at 165 nM [Ca2+], but were significantly inhibited at 115 nM [Ca2+]. Both the protein kinase C-dependent and protein kinase C-independent pathways leading to VL30 transcription were dependent upon the presence of an intracellular [Ca2+] exceeding 115 nM. The dependence upon intracellular Ca2+ transients for transcriptional induction by endothelin appears to be a characteristic of VL30 expression, as 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid treatment did not prevent the endothelin-induced transcription of the protooncogenes c-jun and c-fos.

Normal Fibroblasts Responding to Anoxia Exhibit Features of the Malignant Phenotype

Cancer has two fundamental features: neoplasia, representing the aberrant expression of a normal cell proliferation response, and malignancy, an ability to penetrate normal tissue boundaries. Although the role of oncogenes in neoplastic transformation is becoming clearer, malignancy remains far less well understood. Normal rat fibroblasts exhibit a staged response to anoxia which, if expressed in an uncontrolled fashion, may contribute vital aspects to the malignant phenotype. The response begins with induction of retrotransposon-like VL30 element transcription, progresses through induction of several intracellular proteins, and is followed by secretion of three major proteins including the protease cathepsin L. The induced VL30 element RNA encodes a 61-kDa secretory protein of unknown function. The response of fibroblasts to anoxia is evidently not a survival response. Instead, the response represents a close match to the role of fibroblasts during the early stages of wound healing where they are active under near-anoxic conditions. Malignant cancer cells are known to exhibit several of the characteristics we find induced in fibroblasts by anoxia. Conversion to the malignant phenotype may represent coordinate loss of control of this normal cellular response.

Retrotransposon-like VL30 elements are efficiently induced in anoxic rat fibroblasts

VL30 elements are a multigene family within the class of retroviruses and retrotransposons. We have characterized the response of normal rat fibroblasts to anoxia, in which endogenous VL30 element expression is strongly induced. Optimal induction up to 500-fold occurs under complete anoxia, although a lesser response is seen under atmospheres up to 2% oxygen. Phorbol esters and diacylglycerol, which induce mouse VL30 RNA approximately eightfold, show no effect on the rat VL30 system. The hypoxic conditions optimal for rat VL30 induction represent a mild cellular stress, with no reduction in cell viability during the induction period. Although the precise physiological role of this fibroblast response to temporary anoxia is unknown, it may occur during wound healing. The induction of VL30 by anoxia provides a unique model system wherein a member of the mammalian retrovirus/retrotransposon family is highly responsive to a common physiological signal.

The genomic DNA organisation and evolution of a retrovirus-transmissible family of mouse (VL30) genetic elements

The sequence organisation of endogenous VL30 elements in the mouse genome was investigated by using a cloned representative of a retrovirus-transmissible VL30 cDNA. The majority of dispersed VL30 sequences could be assigned to a proviral-like structure 5.2–5.3 kbp long and bounded by long terminal repeats (LTRs). The existence of a hierarchy of evolutionarily conserved elements was rather limited and sequence heterogeneity between different elements was randomly distributed. However, the retrovirus-transmissible class of VL30 element was found to represent a distinct minority subgroup distinguishable by restriction sites and size (4.6–4.9 kbp long). Analysis of sequence conservation showed that VL30 elements display a more rapid turnover than endogenous murine leukaemia virus-related proviral sequences, and that VL30 LTRs show the most limited evolutionary distribution. Although discrete subsets of VL30 unique sequence were conserved in different rodents, the location of conserved regions was found to be variable, arguing against the presence of a functionally conserved protein coding region. These observations support the hypothesis that high frequency recombination, probably occurring during reverse transcription and the accompanying processes of duplicative transposition and amplification, have been a major determinant in the mode of evolution of the VL30 gene family.

Harvey sarcoma virus genome contains no extensive sequences unrelated to those of other retroviruses except ras.

The Harvey murine sarcoma virus genome contains two rat-derived sets of genetic information recombined with the Moloney mouse leukemia virus. The rat sequences represent a ras oncogene and a rat VL30 element. The VL30 sequences have several discrete regions of similarity with retroviral sequences which were detected by searching a protein database for similarities with predicted polypeptide sequences from the VL30 regions. On the 5' side, the most similar sequences were those of feline sarcoma viruses; on the 3' side, murine leukemia viruses were the most similar. Some of the regions of similarity could also be detected directly by searching a nucleic acid sequence database with the viral DNA sequences. The most extensive region of similarity was that which corresponded to the endonuclease in the pol gene of a murine leukemia virus. The majority of the rat-derived sequences present in the Harvey sarcoma virus genome can now be attributed exclusively to ras or retrovirus- or retrotransposon-related sequences.

Independent transcriptional regulation of a single VL30 element by epidermal growth factor and activators of protein kinase C.

A single VL30 element present in the RVL-3 cell line was transcriptionally induced by both epidermal growth factor (EGF) and the protein kinase C (pkC) activators 12-O-tetradecanoylphorbol-13-acetate (TPA) and sn-1,2-dioctanoylglycerol within 5 min of stimulation. Following TPA-induced depletion of protein kinase C activity, EGF stimulation of VL30 transcription and accumulation was unaffected while TPA effects were inhibited, implying that EGF and TPA act by separable pathways.

Independent transcriptional regulation of a single VL30 element by epidermal growth factor and activators of protein kinase C.

A single VL30 element present in the RVL-3 cell line was transcriptionally induced by both epidermal growth factor (EGF) and the protein kinase C (pkC) activators 12-O-tetradecanoylphorbol-13-acetate (TPA) and sn-1,2-dioctanoylglycerol within 5 min of stimulation. Following TPA-induced depletion of protein kinase C activity, EGF stimulation of VL30 transcription and accumulation was unaffected while TPA effects were inhibited, implying that EGF and TPA act by separable pathways.

Isolation of an integrated provirus of Moloney murine leukemia virus with long terminal repeats in inverted orientation: integration utilizing two U3 sequences.

We have previously described the construction of a mutant of Moloney murine leukemia virus, in594-2, which carries a 2-base-pair insertion in the U5 region of the genome and is partially defective in forming the integrated proviral DNA. We have now recovered a cloned copy of an unusual provirus from rat cells infected with this mutant. The viral genome is flanked by long terminal repeats in inverted orientation, with U3 sequences joined to cellular DNA at both of the outer edges. In addition, the provirus is a recombinant, containing a segment of a VL30 element in inverted orientation in place of the Moloney murine leukemia virus env region. The recovery of this provirus indicates that two U3 regions can be used for viral integration and suggests that there may be no absolute requirement in the reaction for those U5 sequences outside the 13-base-pair inverted repeats.