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Differential Effects of Retroviral Long Terminal Repeats on Interleukin-3 Gene Expression and Autocrine Transformation X-Y Wang1 and JA Mccubrey1,2

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Differential Effects of Retroviral Long Terminal Repeats on Interleukin-3 Gene Expression and Autocrine Transformation X-Y Wang1 and JA Mccubrey1,2 Leukemia (1997) 11, 1711–1725 1997 Stockton Press All rights reserved 0887-6924/97 $12.00 Differential effects of retroviral long terminal repeats on interleukin-3 gene expression and autocrine transformation X-Y Wang1 and JA McCubrey1,2 1Department of Microbiology and Immunology and 2Leo Jenkins Cancer Center, East Carolina University School of Medicine, Greenville, North Carolina 27858, USA Previously we documented the transposition of an intracister- gration include: promoter insertion,4,5 enhancer insertion,6,7 nal A particle (IAP) provirus to the interleukin 3 (IL-3) locus inactivation of a gene,8,9 and disruption of a mRNA stability which resulted in autocrine transformation. In the present 10 study, the effects of different long terminal repeats (LTRs) on region. IL-3 gene expression and autocrine transformation were inves- Tumors induced by insertional mutagenesis mechanisms tigated. LTRs from defective IAPs, and replication competent have also been observed after retrotransposon transpositions, Moloney murine leukemia virus (MoMuLV), human T cell leuke- such as murine intracisternal A particles (IAPs).11–13 Murine mia (HTLV), and immunodeficiency (HIV) viruses, were inserted IAPs are defective retroviruses which have deletions in their ′ 5 of the IL-3 promoter region, and their transforming abilities env genes, thus they do not appear to leave the cells. There determined. Addition of the lymphocyte specific (LS) IAP-LTR are approximately 1000 copies of IAP proviral elements in a to the germline IL-3 (gIL3) gene, the IAP-LTR present in the pre- 14 viously described transposition, resulted in a modified IL-3 haploid murine genome. Most IAP transpositions were dis- gene that only infrequently transformed IL-3-dependent cells. covered because they affected the function of genes at the In contrast, addition of plasmacytoma (PC) IAP-LTRs to the target sites which resulted in traits that were readily detected, gIL3 gene, which were isolated from IAPs expressed in plasma- eg ability of the cells to grow in the absence of a previously cytomas, resulted in modified IL-3 genes that transformed IL-3- required growth factor.11–13 In addition to IAP elements, six dependent cells more readily. The MoMuLV-LTR and the TCRd enhancer also stimulated high levels of IL-3 expression and other families of murine retrotransposons have been autocrine transformation. In contrast, the HTLV-I, HTLV-II and described. They are VL30, ETn, GLN, MuRRS, MrRVY, and 15 HIV LTRs did not induce significant IL-3 synthesis or autocrine MYS. The transposed elements range from a full length retro- transformation. Consistent with these results, higher levels of transposon or just a part of a LTR sequence. The transposed CAT expression were observed in cells transiently transfected retrotransposons, including IAP elements, can act as inser- with PC-IAP-LTR or a TCR enhancer compared with LS-IAP and tional mutagens as was described above for chronic retro- HTLV LTRs. In summary, the rank order for the effects of differ- ent LTRs on IL-3 expression and cell transformation is: TCRd- viruses. IAP proviral insertions alter the expression of many enhancer < MoMuLV-LTR . PC-IAP-LTRs À LS-IAP-LTR À different classes of genes, including: protooncogenes, devel- HTLV-LTRs < HIV-LTR. These results indicate that the LS-IAP- opmental, growth factor and receptor genes.11–13,16–19 LTR is very weak at inducing IL-3 gene transcription and Like other retroviral proviruses, IAP elements contain LTRs additional genetic mutations may be necessary for LS-IAPs to at both the 5′ and 3′ flanking regions. These regulatory LTR induce autocrine transformation of hematopoietic cells. In con- sequences consist of U3, R, and U5 regions. Several protein trast, the enhancers contained in PC-IAP-LTRs and TCR enhancers may be more effective in inducing abnormal gene binding sites within the U3 region have been described. The expression and malignant transformation. important sites with regard to the following studies are: a sim- Keywords: IL-3; autocrine transformation; retroviral LTRs ian virus 40 core enhancer sequence (Enh1), a transcriptional enhancer Enh2 binding site, and a cyclic AMP response element or the homologous ATF binding site (ATF/CRE).20–25 Introduction By the interaction of specific proteins to these and other regu- latory sequences contained within the LTRs, IAP expression Replication-competent retroviral genomes contain gag, pol can be controlled by alterations in methylation status, onco- 14,24–26 and env coding sequences which encode the functional pro- gene expression and hormonal modulations. IAP gene teins that are required for viral replication.1 A long terminal expression and particle formation have been most often docu- repeat (LTR) flanks the coding sequences at both the 5′ and mented in actively dividing cells, such as, preimplantation 14 3′ ends of the retroviral provirus. Retroviruses which induce embryos, immature thymocytes, or in tumor cells. IAP tumors are called oncogenic or transforming retroviruses. expression has been less frequently observed in normal mouse Acute oncogenic retroviruses, which contain an oncogene, tissues. Organs and cells which express low levels of IAPs cause cancer after a short period of time post-infection of a include: spleen, muscle, pancreas, ovary, thymus, placenta, 14,27 susceptible host, ranging from one to a few months. Chronic macrophage and fibroblast cells. IAP expression is under oncogenic retroviruses do not normally encode oncogenes, genetic control in non-neoplastic cells. This results in differ- and a malignancy only occurs many months or years post- ences in the amount of IAP RNAs detected in certain cells as infection. Chronic oncogenic retroviruses can cause cancer well as the proportion of genomic and subgenomic mRNA 27–29 via proviral insertional activation of flanking cellular genes.2,3 transcripts. The novel insertion of the viral sequences into host cell DNAs Murine plasmacytoma cells (B plasma immunoglobulin- can have profound effects on the regulation of the adjacent secreting tumor cells) generally express higher levels of IAP 14 genes and lead to malignant transformation. Mechanisms mRNA transcripts than normal activated B or T lymphocytes. which result in abnormal gene expression after proviral inte- Moreover, there are sequence differences between the LTR cDNAs isolated from the plasmacytoma cells and normal lym- phocytes.14,26 These observations gave rise to the nomencla- Correspondence: JA McCubrey ture plasmacytoma (PC) IAP and lymphocyte specific (LS) Received 11 March 1997; accepted 6 June 1997 IAP.26 Furthermore, there are two classes of PC-IAPs (PCI-IAP Effects of LTRs on IL-3 expression X-Y Wang and JA McCubrey 1712 and PCII-IAP), whose DNA sequences differ primarily in the Materials and methods Enh2 domain. The PC-IAP and LS-IAP LTR sequences vary considerably in the Enh1, Enh2, and ATF/CRE DNA Cell culture sequences.26 In addition, the LTRs present in PC-IAPs are dif- ferentially hypomethylated when compared to the IAP-LTRs 26 Cells were maintained in a humidified 5% CO2 incubator with expressed in the normal lymphocytes. These variations in Dulbecco’s modified Eagle’s medium (DMEM) containing 5% DNA sequences and methylation status in the enhancer iron-supplemented bovine calf serum (CS; Hyclone, Logan, regions of the PC and LS IAP-LTRs have been proposed to UT, USA). In all experiments, medium (DMEM) contained 5% 26 result in different patterns of expression of these IAPs. CS and is referred to as DMEM. The IL-3-dependent lymphoid The type I human T cell leukemia virus (HTLV-I) is the pri- line, FL5.12,59,60 and the factor-dependent myeloid FDC-P161 mary cause of adult T cell leukemia/lymphoma (ATLL).30,31 cell line were maintained in DMEM supplemented with clari- Infection by HTLV-I normally results in a slow onset of clinical fied 20% supernatant prepared from the WEHI-3B cell line disease which manifests itself after a long latency period rang- (WEHI conditioned medium, WCM) which served as a source ing from 20 to 30 years.32,33 HTLV-I infection usually targets of IL-3. CD4+ T cells and is capable of transforming these cells.30,31 The viral protein product p40tax has been shown to immor- talize T cells34,35 and transform rat fibroblasts.36,37 This protein Genetic modifications of the germline IL-3 gene by is essential for the transforming effects induced by HTLV-I adding enhancers infection. Tax exerts its effects by transactivation of both viral38–41 and cellular genes, including cytokines (IL-2) and cytokine receptor (IL-2 receptor) genes.42–45 HTLV-II was orig- The germline IL-3 gene (gIL3) was subcloned into the 61 46,47 pSV2neo expression vector as described (Figure 1). To facili- inally isolated from patients with hairy cell leukemia. ′ HTLV-II is closely related to HTLV-I, and also encodes a tax tate further manipulations, a 5 end unique EcoRI site was cre- ated which resulted in p5′EcoRI-gIL3.62 Subsequent manipu- gene.48–50 Although HTLV-II has been shown to associate with lations were performed on the p5′EcoRI-gIL3 construct. The a variety of lymphoproliferative disorders, there is no evidence lymphocyte-specific (LS) IAP-LTR,13 and the Moloney murine indicating a direct contribution of HTLV-II in lymphoprolifer- leukemia virus (MoMuLV)-LTR63 sequences were inserted at ative malignancies.51–54 Moreover, the function of the Tax the 5′ end of IL-3 gene by using the unique EcoRI site of protein in HTLV-II is different from that in HTLV-I.55 p5′EcoRI-gIL3 (Figure 1). The plasmacytoma (PC) IAP-LTRs Human immunodeficiency virus type 1 (HIV-1) is the (PCI-IAP, PCII-IAP),26 which were provided by Dr KK Lueders etiologic agent of acquired immunodeficiency syndrome 56,57 (NIH, Bethesda, MD, USA), were cleaved from (AIDS).
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