A Novel Gene from the Human Endogenous Retrovirus K Expressed in Transformed Cells1

1800 Vol. 8, 1800–1807, June 2002 Clinical Cancer Research A Novel Gene from the Human Endogenous Retrovirus K Expressed in Transformed Cells1 Vivienne Armbruester, Marlies Sauter, INTRODUCTION Ellen Krautkraemer, Eckart Meese, Retroviral proviruses were inserted into the germ line of human ancestors 40 million years ago (1) and are the origin of Anya Kleiman, Barbara Best, Klaus Roemer, and 3 2 the present HERV sequences. Data provided by the Human Nikolaus Mueller-Lantzsch Genome Sequencing Project show that up to 8% of the human Institut fu¨r Mikrobiologie und Hygiene, Abteilung Virologie, Geb. 47 genome consists of retroviral sequences (2), most of which are [V. A., M. S., E. K., B. B., N. M-L.], and Institut fu¨r Humangenetik, defective because of the accumulation of mutations. The only Geb. 60 [E. M.], Universita¨tskliniken des Saarlandes, Homburg/Saar D-66421, Germany; and Blokhin Cancer Research Center, Russian HERV family still encoding all essential retroviral proteins is ϳ Academy for Medical Science, Moscow 115478, Russia [A. K.] HERV-K (HML-2). Although most of the 30 copies are mu- tated, this family contains the most intact HERV discovered thus far, the HERV-K (HML-2.HOM) provirus localized on chro- ABSTRACT mosome 7 (3). Recently we provided evidence for a link be- Purpose: We investigated the expression of human en- tween HERV-K expression and the development of GCTs in dogenous retrovirus K (HERV-K) transcripts in various humans (4). Individuals with GCTs produce serum antibodies tumor tissues and transformed cell lines. against the viral Gag and Env proteins, and the gag and env Experimental Design: We performed reverse transcrip- genes are expressed in these tumors, but not in other tumor types tion-PCR analysis to examine expression of env reading (5). When studying the transforming potential of HERV-K frame transcripts in mammary carcinoma biopsies, germ- sequences, we found that cORF, a protein encoded by the central open reading frame within the env-gene (6), can support cell tumor samples, ovarian carcinomas, and lymphocytes of tumor growth in nude mice and associates with PLZF (7). PLZF leukemic patients, as well as in a variety of transformed cell has been documented to be critical for spermatogenesis in mice. lines. The novel np9 gene was analyzed by sequencing. Ex- Furthermore, abnormal spermatogenesis is thought to predis- pression of the recombinant Np9 protein was shown by pose humans for the development of GCT. In this context, it was Western blot analysis and immunofluorescence studies with interesting to ask whether other splice variants of the HERV-K polyclonal Np9-specific antibodies. Subcellular localization env sequences may be associated with tumors. Here we describe was determined with a Np9-enhanced-green fluorescence the identification of a novel HERV-K gene, termed np9, that is protein fusion protein, and the effects of Np9 on cell prolif- transcribed exclusively from the HERV-K type 1 provirus be- eration and survival were studied in growth and standard cause of a type 1-specific splice donor site. In contrast, cORF is colony formation assays. transcribed exclusively from type 2 proviruses and includes a Results: We have identified a novel gene, np9, within the 292-bp sequence that is deleted in type 1 (8). In this report, we HERV-K env-reading frame that gives rise to a 9-kDa pro- show that np9 is significantly expressed in tumor tissues and tein localized predominantly in the cell nucleus. np9 tran- transformed cell lines, but not in normal cells. script results from a novel, HERV-K type 1-specific splice donor site and is expressed in various tumor tissues and MATERIALS AND METHODS transformed cell lines but not in normal, nontransformed RNA Preparation and RT-PCR Analysis. For RT-PCR, cells. total RNA from tissues or cell lines was extracted with RNA clean Conclusion: The highly specific expression of np9 in (Hybaids-AGS, Heidelberg, Germany), according to the manufac- tumor tissue suggests that the protein may possess a function turer’s protocol. To remove residual DNA, a DNase I treatment in tumorigenesis. was carried out. The RT with Superscript II (Life Technologies, Inc.) was performed on 5 ␮g RNA and 25 pmol of random primers. To amplify env reading-frame transcripts, the following primers were used: A, 5Ј-ATGAACCCATCGGAGATGCAA-3Ј; and B, 5Ј-ACAGAATCTCAAGGCAGAAG-3Ј (underlined in Fig. 1A). Received 11/8/01; revised 3/6/02; accepted 3/7/02. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 3 The abbreviations used are: HERV, human endogenous retrovirus; 1 This work was supported by the Deutsche Forschungsgesellschaft GCT, germ cell tumor; PLZF, promyelocytic leukemia zinc finger (SFB 399). protein; RT-PCR, reverse transcription-PCR; Np9, nuclear-protein of 9 2 To whom requests for reprints should be addressed, at Institut fu¨r kDa; EGFP, enhanced green fluorescent protein; TPA, 12-O-tetradeca- Mikrobiologie und Hygiene, Abt. Virologie, Geb. 47, Universita¨tsklini- noyl phorbol-13 acetate; DAPI, 4Ј,6-diamidino-2-phenylindole; NLS, ken des Saarlandes, Homburg/Saar, Germany. Phone: 49-6841- nuclear localization signal; nt, nucleotide(s); aa, amino acid(s); LTR, 1623936; Fax: 49-6841-1623980; E-mail: [email protected]. long terminal repeat. Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2002 American Association for Cancer Research. Clinical Cancer Research 1801 Fig. 1 Alignment of np9 and cORF. A,nt sequences of np9 and cORF were aligned, beginning with the first exon at nt position 6451 in the HERV-K (HML-2.HOM) sequence. The positions of the PCR primers A and B (see “Materials and Methods”) are underlined. The deduced amino acid sequences are shown. Single nt and resulting aa residue substitutions between np9, the two np9 variants V1 and V2 that have been allo- cated to HERV-K sequences on chromosome 3q13 (13) and 22q11, respectively, and cORF are depicted. SD1, novel splice donor site present in np9 and variants; SD2, splice donor site for cORF. The 292-bp deletion present in HERV-K type 1 is indicated. SA, splice acceptor site. Note that SD1 combined with SA gives rise to a frame shift in np9 exon 2. NLS, putative NLSs. CKII, putative casein kinase II phosphorylation site. B, schematic representation of the cORF and np9 splice variants. cORF and np9 originate from HERV-K type II and type I proviruses that differ in their env genes by a 292-bp deletion (del). The splice donor (SD) and acceptor (SA) sites are indicated. The num- bers within the bars depict the reading frames. LTRs are depicted. C, alignment of HERV-K sequences between nt positions 6488 and 6501 in the HERV-K101 sequence (1), documenting the novel type 1-specific splice donor site requiring G and T at positions 6494 and 6495. Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2002 American Association for Cancer Research. 1802 A Novel HERV-K Gene For gag fragments, the following primers were used: C, 5Ј-GGC- CATCAGAGTCTAAACCACG-3Ј; and D, 5Ј-GCAGCCCTATT- TCTTCGGACC-3Ј. As a control, a 192-bp fragment from the GAPDH cDNA was amplified with the following primers: E, 5Ј-AGTCCAGTGAGCTTCCCGTTCAGCA-3Ј; and F, 5Ј-TGG- Fig. 2 Expression of np9 RNA in different cell types and mammary TATCGTGGAAGGACTCATGAC-3Ј. PCR cycling conditions carcinoma, as well as seminoma biopsies. Total RNA was prepared and subjected to RT-PCR analysis with primer pair A and B (see “Materials were as follows: 3 min at 94°C; 30 cycles of 50 s at 94°C, 50 s at and Methods”). Samples were separated on a 3% agarose gel. Primary 58°C, and 3 min at 72°C; and 10 min at 72°C. human lymphocytes with (2) and without RT (3), transformed B cells Plasmids. The plasmid pGEM-np9 was constructed with with (4) and without RT (5), Tera-1 teratocarcinoma cells with (6) and the pGEM-T vector kit (Promega) for Taq polymerase amplified without RT (7), mammary carcinoma biopsy with (8) and without RT (9), seminoma biopsy without (10) and with RT (11), and seminoma PCR products. The np9 fragment was amplified with primers A GAPDH control without (12) and with RT (13). The major fragment of and B and ligated into the pGEM-T vector. Plasmids pCEP4- 473 bp is cORF and of 256 bp is np9. np9, pLRNL-np9, and pEGFP-np9 were constructed by ampli- fication of np9 with the following primers: G, 5Ј-CGCGCggatc- cATGAACCCATCGGAGATGCAA-3Ј; and H, 5Ј-CGCGCg- gatccAACAGAATCTCAAGGCAGAAG-3Ј. Primers G and H Intracellular localization of EGFP fusion proteins was studied were derived from primers A and B (see Fig. 1A) to provide by fluorescence microscopy. BamH1 restriction sites, represented by lowercase letters in Sequence Analysis. The plasmid pGEM-np9 was se- primers G and H. The amplified fragment was digested with quenced with the SequiTherm EXCEL II kit (Biozym) with T7 BamH1 and ligated into the BamH1 sites of pCEP4 (Invitrogen), and SP6 primers from the pGEM-T vector kit (Promega) ac- pLRNL (7), and pEGFP-C1 (Clontech), respectively. For the cording to the manufacture’s protocol. GenBank searches were performed with the National Center for Biotechnology Informa- generation of the pATH-np9–2e construct, the second exon of 4 np9 was amplified from pCEP4-np9 with the following primers: tion program for Nucleotide-GenBank research, the alignment I, 5Ј-CGCGCggatccGAGATGTCTGCAGGTGTAC-3Ј; and K, of np9 and cORF was performed by the BCM Search Launcher. 5Ј-AACAggatccCAAGGCAGAAGAATTTTTC-3Ј.

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