A novel multiplex RT-PCR system detects human ... - Springer Link

Arch Virol (2005) 150: 177–184 DOI 10.1007/s00705-004-0378-8

A novel multiplex RT-PCR system detects human endogenous retrovirus-K in breast cancer Brief Report H. Davari Ejthadi1 , J. H. Martin1 , J. Junying1 , D. A. Roden1 , M. Lahiri1 , P. Warren1 , P. G. Murray2 , and P. N. Nelson1 1 Molecular

Immunology and Oncology Research Groups, Division of Biomedical Sciences, University of Wolverhampton, Wolverhampton, U.K. 2 Department of Pathology, Cancer Studies, University of Birmingham, Birmingham, U.K.

Received November 28, 2003; accepted May 28, 2004 c Springer-Verlag 2004 Published online September 21, 2004

Summary. Human endogenous retrovirus HERV-K like-sequences have been implicated in certain cancers. We developed a novel multiplex RT-PCR system for HERV-K that yielded a 533 bp product together with a smaller sized product (319 bp) of the house keeping gene, histidyl tRNA synthetase (HtRNAS). The latter spanned an intron that also served to validate target cDNA. PCR amplicons of HERV-K and HtRNAS were visualised using a gel documentation system and the pixel intensity used to derive semi-quantitative levels of viral expression. Our data showed that HERV-K10 was significantly elevated in MCF-7 cells treated with estrogen. Interestingly, HERV-K expression was higher in MCF-7 cells selected with adriamycin. RT-PCR combined with Southern blotting also detected HERV-K from breast cancer tissue using laser capture microscopy. This study highlights the presence of HERV-K in the breast cancer cell lines MCF-7 and MCF-7ADR and confirms HERV-K10 transcripts in the cell line T47D. We believe this study to be a novel approach in determining levels of HERV-K expression and for detecting this virus in cancer cell lines and tissues. ∗ Human endogenous retroviruses (HERVs) constitute about 8% of the human genome and have structural similarity to present day exogenous retroviruses e.g. HIV-1 (human immunodeficiency virus) and HTLV-I (human T cell leukaemia virus). Currently over 22 HERV families have been identified and have been

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classified principally as Class I (type C related viruses) or Class II (types A, B and D) related viruses [26]. Whilst many appear to be defective through the accumulation of deletions, mutations, and termination signals, a limited number of HERVs have the potential to produce viral products [10] and indeed produce viral-like particles [12, 22]. Furthermore some HERV families e.g. HERV-K have been implicated in certain autoimmune diseases [14, 15] and cancers [2]. Evidence in the literature suggests that the HERV-K family may play a role in the aetiology of human breast cancer [19, 23]. It has also been shown that the T47D human mammary carcinoma cell line produces retroviral particles with reverse transcriptase activity [21]. Furthermore HERV-K like sequences from T47D cells [18] and reverse transcriptase activity [21] were increased by steroid hormone treatment. Additional evidence for the involvement of HERVs in breast cancer has recently been supported by the demonstration of type 1 HERV-env RNA expression in breast cancer tissue but not in normal breast tissue [28] and the presence of type 2 HERV-K env transcripts in human breast cancers [29]. At present there is a need for PCR systems, both quantitative/semi-quantitative, for detecting HERVs [6]. A previous study has adopted a stable house keeping gene to produce an index of transcriptional levels of HERVs [15]. In this paper we report the use of a novel multiplex reverse transcriptase polymerase chain reaction system (RT-PCR) to determine and semi-quantify levels of HERV-K transcripts in breast cancer cell lines and breast cancer tissue obtained using laser capture microscopy. Furthermore we have investigated HERV-K mRNA expression in the multi-drug resistant cell line MCF-7-ADR as compared to the parent MCF-7 cell line. The multiplex RT-PCR system may serve as a useful technique to investigate the role of HERVs in breast cancer. RT-PCR was performed using a modification of established techniques [15]. In brief, RNA was extracted using TRI reagent (Sigma, Poole, UK) and included a DNase (Promega, UK) step. cDNA was generated using random hexamers employing a cDNA kit (Promega, Southampton, UK) and PCR performed on a Touch Down Thermal Cycler (Hybaid, Middlesex, UK) using primers for HERVK, accession number M14123, (sense: 5 -GCAAGTAGCCTATCAATACTGC-3 , (nt 1729 to 1751) antisense: 5 -GCAGCCCTATTTCTTCGGACC-3 , (nt 2241 to 2261)) and intron spanning primers for histidyl tRNA synthetase (HtRNAS) (sense: 5 -CTTCAGGGAGAGCGCGTGCG-3 antisense: 5 -CCTTCAGGTCAT AGATAAGC-3 ) with the following parameters: initially 94 ◦ C, 3 min; then 30 cycles: 1 min 94 ◦ C, 1 min 59 ◦ C, 2 min 72 ◦ C and final extension 10 min 72 ◦ C. Products were visualised using a Gel Documentaion system (BioGene). Similarly a 5 -biotinylated HERV-K probe, position 1931–1950 (5 -AAGGAGATACTG AGGCATGG-3 ) was used for southern blotting. Here, amplicons were resolved by 2% agarose in Tris-Borate-EDTA 2 and transferred onto a nylon membrane (Hybond-N,Amersham-Phaarmacia Biotech,Amersham, UK). Hybridization was performed using 5% Denhardt’s solution, 0.5% SDS, 5 × SSC using established methods. Products were then revealed using an ECL system (Amersham-Pharmacia Biotech) according to manufacturers instructions. For DNA sequencing, PCR products were purified using a GFX DNA purification kit (Amersham Pharmacia

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Biotech, Amersham, UK) and sequences by Alta Bioscience, Birmingham, UK. Statistical evaluation was performed on the ratio of pixel intensities of HERV-K to HtRNAS amplicons using a Wilcoxon (Mann-Whitney) rank-sum test. The following human breast cancer cell lines were used to investigate HERV expression: MCF-7 adenocarcinoma [24], MCF-7-ADR a variant of the MCF-7 cell line which has been selected for resistance to adriamycin [27] and the ductal carcinoma T47D cell line [9]. Cell lines were maintained in RPMI-1640 medium (T47D cell line) or EMEM medium (MCF-7 and MCF-7-ADR) supplemented with foetal calf serum (10%), penicillin (50 IU/ml), streptomycin (50 µg/ml), Lglutamine (300 µg/ml) and 1% non essential amino acids (EMEM only). They were routinely maintained as monolayers in tissue culture flasks and were grown at 37 ◦ C in 5% CO2 in a humidified atmosphere. Where relevant, cells were treated with 1 µM β-oestradiol (Sigma-Aldrich Ltd., Poole, UK) in T75 Sarstedt Ltd. tissue culture flasks (Leicester, UK) and left to incubate overnight. Cells were harvested using Trypsin/EDTA purchased from GIBCO-BRL (Uxbridge, Middlesex, UK). B-cell lines BJAB and AG876 were kindly provided by Dr. PG Murray. MCF-7 and MCF7-ADR cells were also incubated with 0.01 µM tamoxifen (Sigma-Aldrich Ltd., Poole, UK) where appropriate. Breast cancer tumour type was assessed using standard criteria [16, 20]. Only frozen sections were used for laser capture microscopy. Clusters of tumour cells (4 cases, classification: no special type) were captured by laser microdissection of haematoxylin and eosin stained tissue sections using the PixCell II® LCM System (Arcturus Engineering, Mountain View CA, U.S.A.) and pooled. We have previously established that the pulsed laser facility enables the collection of thousands of tumour cells in less than 30 min [13]. RNA was extracted using a NucleoSpin RNA II kit (Macherey-Nagel, UK) that included a DNase step. CDNA and RT-PCR procedures were then carried out as stated previously. RT-PCR employing a multiplex system i.e. incorporating primers for a house keeping gene and HERV-K successfully amplified amplicons of 319 bp and 533 bp respectively from the cell line MCF-7 (Fig. 1). The Pixel intensity of HERV-K and the housekeeping gene was then obtained using a Biogene gel documentation system (employing Phoretix software) to provide a ratio of signal intensity.

Fig. 1. RT-PCR analysis of HERVK (533 bp) and histidyl tRNA synthetase (319 bp). 1: DNA ladder. 2: MCF-7 control. 3: MCF-7 + 1 µM estradiol. 4: MCF-7 + 0.01 µM tamoxifen. 5: MCF-7 1 µM estradiol + 0.01 µM tamoxifen. 6: blank

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Fig. 2. Analysis of HERV-K expression in breast cancer cell lines MCF-7, MCF-7-ADR, T47D and B cell lines BJAB and AG876

This approach (based triplicates) enabled a semi-quantitative analysis of HERV-K over HtRNAS expression. DNA sequencing of amplicons from the T47D cell line confirmed homology to HERV-K10. Our initial results confirmed the presence of HERV-K mRNA in the T47D human breast cancer cell line as previously reported in Ref. [21]. Subsequently, the RT-PCR system demonstrated that HERV-K mRNA was also detectable in the MCF-7 breast cancer cell line (Fig. 1). Furthermore mRNA transcripts were significantly increased (p = 0.02) in MCF-7 cells cultured with 1 µM estradiol (Fig. 2) but this effect was abrogated (p = 0.239) following addition of 0.01 µM tamoxifen to the estradiol treated cells. In contrast to MCF-7 cells, treatment of MCF-7-ADR (multi-drug resistant) cells with estradiol did not increase mRNA transcripts (p = 0.27). Interestingly, control levels of HERV-K expression was higher in MCF-7-ADR cells compared to the parent cell line. Comparison between two B cell lines, BJAB (control) and AG876 (EBV infected) also showed an increase in expression of HERV-K that was borderline in terms of statistical difference (p = 0.05). Four cases of breast cancer were subjected to laser capture microscopy and samples amplified using RT-PCR. Preliminary analysis of these cases produced very weak amplicons (revealed by gel analysis) although three bands were detected by Southern blotting using a chemiluminescent probe (Fig. 3). Case 2 showed a very weak band that was considered equivocal for the presence of HERV-K. The detection of MMTV (mouse mammary tumour virus) in murine mammary tumours has led to the suggestion of a similar virus being present in human breast cancers. Over the past decade, a number of reports have highlighted human endogenous retroviruses as potential aetiological agents of human cancers. Although the precise role(s) of HERVs in the carcinogenic process has not yet been fully elucidated, there are a number of studies that support the involvement of HERVs in the malignant process through the expression of HERV mRNA, functional proteins


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Fig. 3. Southern blotting of HERV-K from breast cancer cases 1–4 (classification: no specific type). Amplicon size 533 bp

or retroviral-like particles [22]. HERVs may also be associated with the generation of new promoters or the activation of proto-oncogenes. Over-expression is also a common mechanism through which proto-oncogenes become activated, leading to subsequent neoplastic transformation. Interestingly in an MNU induced rat mammary carcinoma, the insertion of an HERV-like element into the intron of c-Ha-ras was responsible for a ten-fold increase in expression [1]. The HERV-K family is one of the most biologically active endogenous viruses and has been implicated in autoimmune diseases and cancers, most notably in breast cancer [17]. We have previously [15] highlighted the use of intron-spanning oligonucleotide primers of a house-keeping gene, histidyl tRNA synthetase (HtRNAS) to validate extracted RNA/DNA. Furthermore, this gene appears to be stable before and after stimulation of cell lines thereby permitting the semi-quantitative comparison of a desired mRNA product and remains consistent with varying dilutions of template [15]. In optimising PCR conditions, both HtRNAS and HERV-K10 were amplified from cell lines tested producing amplicons of expected sizes 319 bp and 533 bp respectively. In the current study, the presence of HERVK10 was confirmed by sequencing and Southern Blotting and was evident in T47D and MCF-7 cell lines. In a previous study, using T47D cells, the housekeeping gene GDPH (glyceraldehyde 3-phosphate dehydrogenase) was used in a separate PCR reaction to assess HERV gene expression, with estrogen and progesterone being employed to maximise HERV-K mRNA expression [21]. We have confirmed the role of estradiol in elevating HERV-K expression in T47D (although the elevated level in our study did not reach statistical significance) and have demonstrated that estradiol also causes a statistically significant increase in HERV-K expression in MCF-7 cells. As expected, HERV-K was not modulated when MCF-7 cells were co-cultured with estrogen in the presence of tamoxifen highlighting the role of antagonist and the hormone responsive element within HERV LTRs to regulate mRNA expression. Preliminary data showed that HERV-K transcripts were detected using RT-PCR from cells isolated from breast cancer biopsies using laser capture microscopy. This data is therefore consistent with recent reports of type 1 and type 2 HERV-K env expression in breast cancer tissue [28, 29]. Clearly whilst many studies have used cell lines, further work using tissues of varying grade and stage need to be investigated. Clearly it would be of interest to address the levels of HERV-K10 in an alternative ER negative breast cancer cell line (e.g. MDA-MB-231; [3]).

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Multidrug resistance is a major problem in breast cancer chemotherapy where initial chemotherapeutic treatment is often followed by relapse, with tumour recurrence exhibiting an acquired resistance to a set of previously used anticancer drugs. Multiple mechanisms have been identified including over-expression of pglycoprotein [7], changes in glutathione and glutathione transferase biochemical pathways [25], DNA repair pathways [4] and drug sensitivity genes [8]. Despite our current knowledge it is likely that other mechanisms may also be involved in drug resistance. In the present study, it was apparent that the basal level of HERV-K was elevated in MCF-7-ADR cells. This phenomenon was consistent with selectivity of the cells with adriamycin. Adriamycin may have modified gene expression, as it has been shown in other studies to upregulate: CD95 gene expression [11], c-myc expression [5] and hepatitis B virus X gene [30]. Whilst the significance of HERV-K expression may remain elusive it might be plausible that enhanced HERV expression may highlight a multi-drug resistant phenotype. Interestingly HERV-K expression was enhanced in AG876 cells as compared to BJAB. Since HtRNAS signals were equivalent from both cell lines there remains the possibility that increased expression of HERV-K was a constitutive phenomenon in AG876 cells or due to the presence of the transforming virus EBV (Epstein-Barr virus). The latter argument was consistent with the recent report of HERV-K18 (a variant of HERV-K) transactivation by potential superantigen motifs present on the EpsteinBarr virus EBV. Furthermore the herpes virus CMV (genus Cytomegalovirus) has previously been reported to transactivate HERV-K10 and HERV-RTVL-H [15]. Hence the role of helper viruses in the activation of HERVs and the subsequent production of viral proteins may be of relevance to the pathological involvement of these viruses in certain cancers. In the current study our results confirm the presence of HERV-K mRNA transcripts in human breast cancer cell lines and indicate higher expression of HERV-K mRNA in the adriamycin selected multidrug resistant cell line. This adds to the body of evidence supporting enhanced expression of several different genes by adriamycin and suggests the possibility of an involvement of HERVs in breast cancer in response to this chemotherapeutic drug. Further investigation of the role of HERVs in breast cancer is therefore warranted.

Acknowledgements This work was funded through a University of Wolverhampton PhD studentship.

References 1. Bera TK, Tsukamoto T, Panda DK, Huang T, Guzman RC, Hwang S-I, Nandi S (1998) Defective retrovirus insertion activates c-Ha-ras proto-oncogene in an MNU-induced rat mammary carcinoma. Biochem Biophys Res Commun 248: 835–840 2. Boller K, Konig H, Sauter M, Mueller-Lantzsch N, Lower R, Lower J, Kurth R (1993) Evidence that HERV-K is the endogenous retrovirus sequence that codes for the human teratocarcinoma-derived retrovirus HTDV. Virology 196: 349–353


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3. Cailleau R, Young R, Olive M, Reeves WJ (1974) Breast tumour cell lines from pleural effusions. JNCI 53: 661–674 4. Calsou P, Salles B (1993) Role of DNA repair in the mechanisms of cell resistance to alkylating agents and cisplatin. Cancer Chemother Pharmacol 32: 85–89 5. Chen J, Yuan YW, Zhang JR, Zhou DY (2002) Up-regulation of c-myc expression in MCF-7/Adr human breast cancer cells and its association with resistance against doxorubicin. Di Yi Jun Yi Da Xue Xue Bao 22: 124–126 6. deParseval N, Lazar V, Casella J-F, Benit L, Heidmann T (2003) Survey of human genes of retroviral origin: identification and transcriptome of the genes with coding capacity for complete envelope proteins. J Virol 77: 10414–10422 7. Endicott JA, Ling V (1989) The biochemistry of p-glycoprotein-mediated multidrug resistance. Annu Rev Biochem 58: 137–171 8. Gudkov AV (1996) Drug sensitivity genes: identification and analysis using the genetic suppressor element approach. In: Gupta S, Tsuruo T (eds) Multidrug resistance in cancer cells. John Wiley and Sons, Chichester, UK, pp 193–215 9. Keydar I, Chen L, Karby S, Weiss FR, Delarea J, Radu M, Chaitcik S, Brenner HJ (1979) Establishment and characterisation of a cell line of human breast carcinoma origin. Eur J Cancer 15: 659–670 10. Keydar I, Hono T, Nayak E, Sweet R, Simoni F, Weiss F, Karby S, Meas-Tejada R, Spiegelman S (1984) Properties of retrovirus-like particles produced by a human breast carcinoma cell line: immunological relationship with mouse mammary tumour virus proteins. Proc Natl Acad Sci USA 81: 4188–4192 11. Lorenzo E, Ruiz-Ruiz C, Quesada AJ, Hernandez G, Rodriguez A, Lopez-Rivas A, Redondo JM (2002) Doxorubicin induces apoptosis and CD95 gene expression in human primary endothelial cells through a p53-dependent mechanism. J Biol Chem 277: 10883–10892 12. Lower R, Boller K, Hasenmaier B, Korbmacher C, Muller-Lantzsch N, Lower J, Kurth R (1993) Identification of human endogenous retroviruses with complex mRNA expression and particle formation. Proc Natl Acad Sci USA 90: 4480–4484 13. Dutton A, Lopes V, Murray PG (2003) Laser capture microdissection: techniques and applications in the molecular analysis of the cancer cell. In: Crocker J, Murray PG (eds) Molecular biology in cellular pathology. John Wiley & Sons Ltd., Chichester, UK, pp 213–231 14. Nelson PN (1995) Retroviruses in rheumatic diseases. Ann Rheum Dis 55: 441–442 15. Nelson PN, Lever AML, Smith S, Pitman R, Murray P, Perera SA, Westwood OMR, Hay FC, Ejtehadi HD, Booth JC (1999) Molecular investigations implicate human endogenous retroviruses as mediators of anti-retroviral antibodies in autoimmune rheumatic disease. Immunol Invest 28: 277–289 16. Nelson PN, Astley J, Warren P (2003) Monoclonal Antibodies: the generation and application of ‘tools of the trade’ for diagnostics and therapy. In: Crocker J, Murray PG (eds) Histopathology. John Wiley & Sons Ltd., Chichester, UK, pp 329–349 17. Nelson PN, Davari Ejtehadi H, Carnegie P, Roden D, Martin J, Rowland-Jones S, Hooley P, Warren P, Astley SJ, Murray PG (2003) Demystified. Human endogenous retroviruses. J Clin Path Mol Pathol 56: 11–18 18. Ono M, Kawakami M, Ushikubo H (1987) Stimulation of expression of the human endogenous retrovirus genome by female steroid hormones in human breast cancer cell line T47D. J Virol 61: 2059–2062 19. Ono M, Yasunaga T, Miyata T, Ushikubo H (1986) Nucleotide sequence of human endogenous retrovirus genome related to the mouse mammary tumor virus genome. J Virol 60: 589–598

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H. D. Ejthadi et al.: Multiplex RT-PCR system

20. Page DL, Anderson TJ (1987) Diagnostic histopathology of the breast. Churchill Livingstone, Edinburgh 21. Patience C, Simpson GR, Colletta AA, Welch HM, Weiss RA, Boyd MT (1996) Human endogenous retrovirus expression and reverse transcriptase activity in the T47D mammary carcinoma cell line. J Virol 70: 2654–2657 22. Sauter M, Schommer S, Kremmer E, Remberger K, Dolken G, Lemm I, Buck M, Best B, Neumann-Hafelin D, Mueller-Lantzsch N (1995) Human endogenous retrovirus K10: expression of gag protein and detection of antibodies in patients with seminomas. J Virol 69: 414–421 23. Seifarth W, Baust C, Murr A, Skladny H, Krieg-Schneider F, Blusch J, Werner T, Hehlmann R, Leib-Mosch C (1988) Proviral structure, chromosomal location, and expression of HERV-K-T47D, a novel human endogenous retrovirus derived from T47D particles. J Virol 72: 8384–8391 24. Soule HD, Vazguez J, Long A, Albert S, Brennan M (1973) A human cell line from a pleural effusion derived from a breast carcinoma. JNCI 51: 1409–1416 25. Tew KD (1994) Glutathione-associated enzymes in anticancer drug resistance. Cancer Res 54: 4313–4321 26. Tristem M (2000) Identification and characterisation of novel human endogenous retrovirus families by phylogenetic screening of the human genome mapping project database. J Virol 74: 3715–3730 27. Vickers PJ, Dickson RB, Shoemaker R, Cowan KH (1998) A multidrug-resistant MCF7 human breast cancer cell line which exhibits cross-resistance to antiestrogens and hormone-independent tumor growth in vivo. Mol Endocrinol 2: 886–892 28. Wang-Johanning F, Frost AR, Johanning GL, Khazaeli MB, LoBuglio AF, Shaw DR, Strong TV (2001) Expression of human endogenous retrovirus K envelope transcripts in human breast cancer. Clin Cancer Res 7: 1553–1560 29. Wang-Johanning F, Frost AR, Jian B, Epp L, Lu DW, Johanning GL (2003) Quantitation of HERV-K env gene expression and splicing in human breast cancer. Oncogene 22: 1528–1535 30. Yun C, Lee JH, Wang JH, Seong JK, Oh SH,Yu DY, Cho H (2002) Expression of hepatitis B virus X (HBx) gene is up-regulated by adriamycin at the post-transcriptional level. Biochem Biophys Res Commun 296: 1157–1163 Author’s address: Dr. J. H. Martin, Division of Biomedical Sciences, School of Applied Sciences, University of Wolverhampton, Wulfruna Street, Wolverhampton WV1 1SB, U.K.; e-mail: [email protected]