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

1800 Vol. 8, 1800–1807, June 2002 Clinical Cancer Research

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.

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 numbers 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.

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 amplification 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. The sec- For further sequence analysis, the research programs PSORT II ond exon of np9 was then ligated into the BamH1 site of the from Swiss Prot and Scan Prosite were used. pATH-10 vector (9). The cORF plasmids pCEP4-cORF and Tumor Samples. Tumor biopsies were collected from pEGFP-wtcORF have been described previously (7, 10). four institutions (Women and Children’s Clinic University of Cell Culture, Transfection, and Infection. All cells Saarland Medical School, Homburg; the Gerhard-Domagk- Institute of Pathology, University of Muenster, Muenster; the were maintained at 37°Canda5%CO2 atmosphere. The generation of stable cORF expressing Raji cell line (Raji- Urology Clinic of the Philipps-University Marburg, Marburg; wtcORF) has been described (7). The stable np9 expressing and and the Institute for Pathology of the Clinic Bamberg, Bamberg, a matching vector-only expressing cell line (Raji-np9 and Raji- Germany). pCEP-4, respectively) were developed by transfecting Raji cells with the pCEP-4-np9 or pCEP-4 vector and by subsequent RESULTS selection with 300 ␮g/ml hygromycin B. Cell lines 293gp-np9 RT-PCR Analysis. During further investigations on the and 293gp-pLRNL were generated by stable transfection with role of HERV-K cORF in GCT development, the RNA expres- plasmids pLRNL-np9 or pLRNL, followed by selection with sion patterns of the env gene was analyzed by RT-PCR in tumor 400 ␮g/ml G418. The production of the retroviral vectors and biopsy samples and in transformed cell lines. Apart from the infection of Rat-1 cells was performed as described elsewhere 473-bp fragment specific for the cORF transcript and a variety (7). Rat-1-np9 and Rat-1-pLRNL express the np9 transgene and of PCR products not consistently detected in all samples, we the pLRNL empty vector, respectively. found a 256-bp fragment in three of three seminoma biopsies, of Antibodies, Western Blot Analysis, and Immunofluo- which one is documented in Fig. 2. This prompted us to study rescence Studies. To raise polyclonal antibodies, the second this variant in more detail. exon of np9 was bacterially expressed from the pATH-np9–2e Sequence Analysis of the Novel np9 Gene. GenBank construct. Rabbits were immunized with the protein as described searches exhibited 100% homology of the 256-bp fragment with previously (11). Raji-np9 cells, Raji-wtcORF cells, and untrans- the HERV-K env sequence from HERV-K101 published by fected Raji cells were treated with 20 ␮g/ml TPA for 48 h to Barbulescu et al. (1). This fragment, shown in Fig. 1A, is used induce the expression of the recombinant proteins. Western blot for all following studies and is designated np9. Further RT-PCR analysis was performed with the cellular extracts following studies revealed the presence of two other variants of the np9 standard procedures (7). Immunofluorescence studies were car- sequence that differ from np9 in up to four nt positions, giving ried out as described elsewhere (12). Subcellular-localization Assay. 293gp cells were grown to ϳ20% density on glass cover slips and were then incubated with a calcium phosphate coprecipitate that included 2 ␮gof 4 The HERV sequences and their respective GenBank accession num- either the empty vector or the pEGFP-np9 or the pEGFP- bers are as follows: HERV-K10, M14123; HERV-K101, AF164609; HERV-K102, AF164610; HERV-K103, AF164611; HERV-K104, wtcORF vector construct. Cells were fixated in paraformalde- AF164612; HERV-K108, AF164614; HERV-K109, AF164615; HERV- hyde (4% in PBS) at 24 h after transfection and the DNA was KII (Chr. 3q13), AB047240; HERV-K (Chr. 22q11) AC007326; and stained with 200 ng/ml DAPI in methanol to visualize nuclei. HERV-K (HLM-2.HOM), AF074086.

Fig. 3 Expression of recombinant Np9 protein. A, Western blot analysis of total cellular protein prepared from Raji cells stably transfected with pCEP4-cORF (Raji-cORF) or pCEP4-np9 (Raji- np9), and from nontransfected Raji cells, with the use of the polyclonal ␣-Np9 antiserum at 1:100 is shown. Expression of the Np9 protein was enhanced by treatment of cultures with 20 ng/ml TPA. B, immunofluorescence of recombinant Np9 in Raji-np9 cells. Cells were stained 48 h after TPA treatment with (1) preserum or (2) and (3) with ␣-Np9 antiserum, (3) Np9 expressing Raji cell in more detail. C, fluorescence of 293gp cells transiently transfected to produce the hybrid proteins EGFP-np9, EGFP-cORF, or EGFP alone. Cell nuclei were stained with DAPI as control. DAPI stained relevant nuclei are marked with an arrow.

rise to three amino acid changes (Fig. 1A). The variants show 100% homology to proviruses located on chromosome 3q13 [HERV-KII (Chr. 3q13); see Ref 13] and 22q11 [HERV-K (Chr. 22q11)], respectively (Fig. 1A). Importantly, all np9 genes originate from HERV-K sequences of type 1, which are not able to express intact cORF because of the type 1-specific deletion of the 292 bp (see Ref. 8; Fig. 1B). The expression of np9 from provirus type 1 is the result of the presence of an alternative splice donor site, produced through the replacement at nt positions 6495 and 6496 of HERV-K101 of A and G, present in type 2 viruses, for a T and A in type 1 (see Ref. 14; Fig. 1C). Further differences between cORF and the 256 bp np9 sequences are outlined in Fig. 1A. Translation reveals that the first exon of np9 encodes the N-terminal on aa 15 of the 87 aa that constitute the first exon of cORF. The second exon of np9, beginning at nt position 8118 in the HERV-K101 sequence, contains 178 bp of coding sequence. The COOH-terminal 59 aa of Np9 are derived from the third (non-env, non-cORF) reading frame. The deduced Np9 protein has thus a predicted size of 74 aa and a predicted molecular mass of 8.7 kDa. By motif analysis, three putative NLSs were identified (see Fig. 1A, light boxes). The putative NLSs I and III were identified by the PSORT II search program from Swiss Prot; NLS II was predicted on the basis of a minimal consensus sequence, defined by Garcia-Bustos et al. (15). The presence of these motifs suggested a nuclear localization of the HERV-K protein Np9. Furthermore, a putative casein kinase II-phosphorylation site was identified between nt positions 8460 and 8475 by the Scan Prosite research program Fig. 4 Growth and colony formation analyses. A, initially, 2 ϫ 104 of (Fig. 1A). the indicated cells were seeded and the number of live cells was determined at various time points with a CASY cell counter. B, for Detection of Np9 by Western Blot Analysis and Immu- standard colony formation analysis, 103 live Rat 1-pLNRL-np9 (1), Rat nofluorescence Studies. To show expression of the recombi- 1-pLRNL (2), or parental Rat-1 cells (3) were seeded and colonies were nant Np9 protein in a human cell system, a stable np9 expressing stained with crystal violet after 7 days. Raji-cell-line (Raji-np9) with the pCEP4-np9 construct was generated. Raji cells were chosen because they express recombinant HERV-K proteins at high levels (see Ref. 7 and unpub- Cell Proliferation and Colony Formation. Stably con- lished observations). Furthermore, the levels of transgene ex- trol-transfected 293gp or Raji cells expressing either Np9 or pression from pCEP4 can be enhanced by TPA (16). Western control vector were subjected to growth analysis. There was no blot analysis showed expression of a protein of ϳ9 kDa in cell significant difference in the population doubling time between extracts from Raji-np9 cells (Fig. 3A). Furthermore, we were the cell lines expressing Np9 or the control plasmid over a time able to detect the recombinant Np9 protein in TPA-treated course of 5 days (Fig. 4A). Furthermore, to study whether the Raji-np9 cells by indirect immunofluorescence (Fig. 3B). No presence of Np9 has a biological relevance in proliferation and signal was detected in Raji-cORF cells transfected to stably survival, standard colony formation assays were carried out with produce recombinant cORF (see Ref. 7; Fig. 3A), or in Raji-np9 infected Rat-1 cells. Likewise, no differences were observed without TPA-induction (data not shown), indicating that Np9 is between the colony formation capability of cells expressing Np9 normally expressed at very low levels. or the empty vector (Fig. 4B), suggesting that Np9 expression Subcellular-localization Analysis. We then wanted to confers no simple growth and/or survival advantage on cells in study the subcellular localization of recombinant Np9. In a first vitro. set of experiments, a hybrid protein consisting of full-length Np9 Expression in Tumor Biopsies and Transformed

Np9 and the EGFP fused to the NH2 terminus of Np9 was used Cell Lines. To study the expression of np9 in human and in transient transfections of human 293gp cells. The fact that primate cells, a panel of established cell lines, tumor biopsies, EGFP-Np9 is located predominantly in the nucleus, and that it and lymphocytes from leukemic and normal individuals, as well preferentially stains structures of unknown identity within the as normal, nontransformed cells were screened for np9 mRNA nucleus, is shown in Fig. 3C. A similar nuclear-staining pattern expression by RT-PCR analysis. Examples for the obtained is generated by EGFP-cORF. In contrast, EGFP alone stains the results are shown in Fig. 2. Transcripts from the gag and cORF cell uniformly. Thus, EGFP-Np9 is a nuclear protein. The nu- reading frames were analyzed in parallel (Fig. 1A). RNA quality clear localization of recombinant Np9 was confirmed by immu- and amplification efficiency were monitored by amplification of nofluorescence on Raji-np9 cells with the polyclonal anti-Np9 a fragment from the housekeeping gene GAPDH. The HERV- antibody (Fig. 3B). K-negative cell line B95–8 (a B-cell line) derived from a New

Table 1 RNA expression of cORF, np9, and gag in normal cells, Table 1 Continued tumor cell lines, and tumor tissues cORF np9 gag cORF np9 gag Ovarian carcinoma Transformed cell lines 1 ϪϪ Ϫ GCT cell lines 2 ϪϪ Ϫ Tera 1 ϩϩϩ ⌺ 0/2 0/2 0/2 Pa 1 Ϫϩϩ ⌺ Healthy tissues and immortal 1/2 2/2 2/2 (nontransformed) cells Mammary carcinoma cell lines Ϫϩϩ Lymphocytes 0/10 0/10 9/10 MCF7 Fibroblasts ϪϪ ϩ MDA-MB 231 Ϫϩϩ ϪϪ Ϫ Ϫϩϩ Gut T47D Placenta ϪϪ Ϫ SKBR ϩϩϩ ϪϪ ϩ ⌺ Stomach 1/4 4/4 4/4 KH 5109 ϪϪ ϩ B-cell lines KH 5109-175b ϪϪ ϩ BL41 Ϫϩϩ ϪϪ ϩ ϪϪϩ 041 HL60 b ϪϪ ϩ Ϫϩϩ 041-175 Raji EREB ϪϪ ϩ JMML Ϫϩϩ ϪϪ ϩ ⌺ EREB stimulated 0/4 3/4 4/4 ⌺ 0/20 0/20 17/20 B95-8a ϪϪϪ Other cell lines a B95-8 is a HERV-K negative cell derived from a New World HCT 116 Ϫϩϩmonkey. 293gp Ϫϩϩ b Immortal cell types expressing the p53 mutant 175H. Saos ϪϩϪ Hep3B Ϫϩϩ Hela Ϫϩϩ ⌺ 0/5 5/5 4/5 World monkey served as negative control. The results of these Tumor tissues studies are summarized in Table 1. Np9 expressed from Germ-cell tumors HERV-K type 1 proviruses was detected in Ͼ90% of all trans- 1 ϪϪϩformed cell lines (n ϭ 15). Likewise, gag was produced in ϪϪϪ 2 Ͼ90% of the cell lines. In contrast, cORF expressed from 3 ϩϩϩ 4 ϪϩϩHERV-K type 2 was detected in only 13% of the cell lines; it 5 ϩϩϩwas present in only one teratocarcinoma and one mammary 6 ϪϪϪcarcinoma line. Remarkable differences between the presence of 7 ϪϪϩ ϪϪϩnp9 and cORF were observed in primary tumor samples. Al- 8 Ͼ ⌺ 2/8 3/8 6/8 though np9 could be detected in 45% of the samples, the Mammary carcinoma presence of the transcript was not equally distributed among the 1 Ϫϩϩtumor types. The transcript was most frequently found in mam- 2 ϩϩϩmary carcinomas (52%; n ϭ 21), whereas it was less frequently ϪϩϪ 3 present in germ-cell tumors (37%; n ϭ 8) and leukemia blood 4 ϪϪϪ 5 ϪϪϪlymphocytes (33%; n ϭ 6). Both ovarian carcinomas tested 6 ϩϩϩnegative. Of further interest might be the relative frequencies of 7 ϪϩϩcORF and np9 in the tumor biopsies. Although similar numbers Ϫϩϩ 8 of germ-cell tumors express cORF and np9, mammary carcino- 9 Ϫϩϩ 10 ϪϪϪmas expressing np9 are more frequent than mammary carcino- 11 ϪϪϪmas expressing cORF by a factor of five to six. This is in accord 12 ϪϩϪwith findings by Etkind et al. (17), demonstrating a 10-fold 13 ϪϪϪincrease in the quantity of full-length transcripts from provirus ϪϪϩ 14 type 1 relative to type 2 in the breast carcinoma cell line T47D, 15 Ϫϩϩ 16 ϪϪϪand by Wang-Johanning et al. (18), detecting exclusively type 17 Ϫϩϩ1-specific env transcripts in breast carcinomas. Grading of the 18 ϪϪϪmammary carcinomas according to the Tumor-Node-Metastasis ϪϪϪ 19 classification (Union International Contre Cancer) failed to 20 ϪϪϪ 21 ϪϩϪshow any correlation between tumor grade and np9 expression ⌺ 2/21 11/21 9/21 (data not shown). Finally and most importantly, none of the Leukemia blood lymphocytes tested healthy tissues (n ϭ 14; lymphocytes and fibroblasts, and AML ϪϪϪsamples from the gut, placenta, and stomach) expressed np9 at ϪϪϩ ALL 1 detectable quantities. The normal diploid human fibroblasts ALL 2 Ϫϩϩ ALL 3 ϪϩϪKH5109 and nontransformed derivatives designed to express ALL 4 ϪϪϪthe dominant-negative mutant 175H of the p53 tumor suppres- ALL 5 ϪϪϩsor also failed to test positive, as did immortal nontransformed ⌺ 0/6 2/6 3/6 human 041 fibroblasts lacking wild-type p53 and their 175H-

transduced derivatives. Like np9, cORF was not detectably increased risk for testicular germ-cell cancers (28). CORF and produced in nontransformed cells and tissues. In stark contrast, Env share the N-terminal 87 aa residues, and both share their the gag transcript was detected not only in the transformed cells N-terminal 15 aa with the novel HERV-K protein Np9 reported and tumor samples but also in Ͼ80% of the normal, nontrans- here. However, the domain in CORF that makes contact with formed cells. Thus, np9 expression is closely correlated with PLZF (aa 21–87) is not present in Np9. It is thus unlikely that transformation, more closely than cORF expression, and might Np9 interacts with PLZF. serve as a marker for transformation. Although it has been reported that breast carcinomas contain exclusively HERV-K type 1-specific env transcripts (18), we were not able to find sequence motifs in the LTRs that were DISCUSSION specific for type 1 or type 2. However, sequence differences in HERV sequences are frequently transcribed in several nor- LTRs have indeed been linked to tissue specificity of the ex- mal tissues (19). Recent work has indicated that syncytin, a pression of HERV-K (13). They could not, however, be asso- product of the env gene of the defective HERV-W, may have an ciated with a particular type of provirus. This suggests that important function in human placental morphogenesis through sequences in the proximity of individual LTRs, and thus the the mediation of the fusion of the placental cytotrophoblasts (20, position of LTRs in the genome, play an important role for the 21). Several cellular genes are specifically regulated by HERV- tissue-specific expression of the provirus types. Such sequences LTRs, although thus far no LTR of the HERV-K class could be may constitute regulatory motifs for transcription factors or linked to the transcription initiation of cellular sequences. One methylation signals (29, 30). The sensitive regulation of the reason for the conspicuous absence of HERV-K-regulated cel- LTRs may also explain why HERV-K viral transcripts are lular genes may be that HERV-K-LTRs seem both to be regu- detectable with PCR methods, whereas, in contrast, the respec- lated highly specifically by cell-type and to be dependent on the tive proteins including the Np9 protein described here are dif- state of differentiation (22). ficult to detect. As of yet, we were not able to detect native Np9 Besides affecting normal cell physiology, endogenous ret- protein in any cell type, neither by Western blotting nor by roviral genes may contribute to tumorigenesis. Early hints point- immunofluorescence or immunoprecipitation; however, even ing in this direction came from the observation that certain the native Env and cORF proteins have thus far been detected in infectious retroviral proteins and related polypeptides may be only one cell type (6, 31). It is furthermore conceivable that immunosuppressive and anti-inflammatory. For example, the posttranscriptional regulatory mechanisms exist that influence retroviral Env p15E protein can inhibit interleukin 1-mediated the HERV-K protein levels. Whether Np9 has a function in signal transduction and protein kinase C activity (23, 24) and transformed mammary epithelial cells is the subject of further was documented to be able to negatively affect monocyte che- investigations. At present, there are no known sequence motifs motactic responses and lymphocyte blastogenesis in human in Np9, apart from the nuclear localization signals and CKII cells in vitro (25). Env p15E is homologous to the transmem- phosphorylation site, that would allow the identification of brane region of the Env protein against which serum antibodies putative protein domains and speculations about the function of are produced highly specifically in patients with germ-cell tu- this protein. mors. The env region of HERV-K (HLM-2.HOM) may thus positively influence the maintenance and progression of such ACKNOWLEDGMENTS tumors via the inhibition of an effective immune response. We are grateful to H. Herbst, M. Holla¨nder, T. Ka¨lble, and M. Seitz HERV-K message, proteins and antibodies directed against for providing tumor material and to J. Mayer for critically reading the them are very frequently observed in patients with GCT (4, 12) manuscript. and occasionally in breast cancer patients and in HIV- or cyto- megalovirus-infected individuals (26), but not or only very REFERENCES rarely in healthy individuals. 1. Barbulescu, M., Turner, G., Seaman, M. I., Deinard, A. S., Kenneth, Further hints at the implication of endogenous retroviral K. K., and Lenz J. Many human endogenous retrovirus K (HERV-K) genes and proteins in tumorigenesis were found in the murine proviruses are unique to humans. Curr. 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Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2002 American Association for Cancer Research. A Novel Gene from the Human Endogenous Retrovirus K Expressed in Transformed Cells

Vivienne Armbruester, Marlies Sauter, Ellen Krautkraemer, et al.

Clin Cancer Res 2002;8:1800-1807.

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