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1 Section of Virology, Department of Medical Sciences, Uppsala University Hospital, Uppsala, Sweden
2 Department of Genetics and Pathology, Uppsala University, Uppsala, Sweden
3 Department of Surgery, Uppsala University Hospital, Uppsala, Sweden
Correspondence
J. Blomberg
jonas.blomberg{at}medsci.uu.se
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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These authors contributed equally to this work. 
A supplementary table showing synthetic control DNA templates is available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The presence of DNA from an alleged closely related retrovirus, human exogenous retrovirus (HMTV), and a retrovirus almost identical to HMTV, has been reported in human breast cancer and in primary biliary cirrhosis (Pogo & Holland, 1997; Holland & Pogo, 2004; Mant & Cason, 2004; Xu et al., 2004). Several reports indicate that HMTV exists, is associated preferentially with breast cancer and is not present in normal breast tissue. For example, Ford et al. (2004a) detected highly MMTV-like sequences, probably not of endogenous origin, in cancerous epithelial cells of female and male breast tumours and not in adjacent, non-malignant cells. This finding should not be confused with that of Yin et al. (1997), where high RNA expression of endogenous human MMTV-like sequences (HMLs), related more distantly to MMTV (Andersson et al., 1996), in one of 60 breast cancers was reported. Needless to say, the identification of an exogenous retrovirus causative of human breast cancer would have great clinical implications. To verify the HMTV reports, a prospective study using Swedish breast cancer patients and controls was performed. A real-time PCR, based on env of MMTV and the non-endogenous highly MMTV-like sequences reported from humans (here collectively called HMTV), was designed. It can detect one to ten copies of MMTV DNA per PCR. As in many other real-time PCRs, the threshold cycle number is correlated linearly with the logarithm of template DNA concentration from 101 to 106 copies per reaction. |
METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) as shown in Table 1. After consent, a blood sample was collected and, during surgery, the specimen was sent on ice to the Department of Pathology. Within 30 min, a piece of the tumour and normal breast parenchyma was cut out and stored at –70 °C. Further patient and tumour data are shown in Table 1. Thus, samples from both the tumour and control normal glandular tissue of the same breast, as well as plasma and white blood cells (WBCs), were taken from each patient. The control persons provided samples of the same kind, with the exception of a tumour sample. The study was approved by the Ethical Committee of the Faculty of Medicine of the University of Uppsala (permit no. 01/381).
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DNA from WBCs of 100 Swedish blood donors was also prepared. The DNA concentration of each sample was determined in a DyNA Quant fluorimeter (Amersham Biosciences).
cDNA synthesis.
cDNA was synthesized by using 5 µl 1x StrataScript buffer, 5 µl 0.1 M dithiothreitol, 0.8 µl 100 mM dNTP mix (Sigma), 2 µl random hexamers (265 ng µl–1; Pharmacia), 29.2 µl RNase-free H2O, 2 µl RNasin (40 U µl–1; Promega), 1 µl StrataScript reverse transcriptase (50 U µl–1) and 5 µl RNA (approx. 1 µg µl–1), at 25 °C for 10 min, 42 °C for 60 min and 90 °C for 5 min. Each cDNA reaction was run with and without reverse transcriptase.
Primers and probe for the real-time PCR method.
The primers and the probe were derived from the env region of published MMTV and the highly MMTV-like HMTV sequences reported from humans (GenBank accession nos AF346816
[GenBank]
, AF239172
[GenBank]
and M15122
[GenBank]
) (Fig. 1). The primers were as follows: ABL (5'-TAGTTCCCCATACAGAATTGTTTCGCT-3') and ABR (5'-TCATCACCAATATCTACAGGTAGCAGCAC-3'), and ABL1 (5'-TAGTCCCCCATACAGAATTGTTTCGCT-3') and ABR1 (5'-TCATCACCAATATCTACAGGTAGCAGTGAC-3'); and the probe, ABP [5'-6-FAM*ACTATGATCGCT*(TAMRA)GCATAGTCGTAGGCAGAAGAATCT-phosphate-3']. Positions of mismatch between MMTV and HMTV are underlined. Primers ABL and ABL1, and ABR and ABR1, respectively, were tested singly (i.e. ABL versus ABR and ABL1 versus ABR1, in separate tubes) and as a 1+1 mixture (ABL+ABL1 versus ABR+ABR1 in the same tube). All primers and probe were purchased from Thermo Hybaid. They were used at 125, 125 and 300 nM final concentrations, respectively, in a TaqMan universal master mix with uracil N-glycosylase (UNG; Applied Biosystems). The reaction volume was 50 µl, with 2 µl DNA sample. Real-time PCR was performed in a RotorGene thermocycler (Corbett Research) with a temperature profile of 50 °C for 600 s and 95 °C for 600 s, followed by 60 repeats of 95 °C for 25 s and 60 °C for 60 s. Fluorescence was measured during the latter period at 60 °C. The ability of the PCR to amplify MMTV env was tested by using DNA from the mouse strain C3H/HeJ, which contains one proviral MMTV copy per genome, as positive template (information from Jackson Laboratories). A dilution series of this DNA in six 10-fold steps, covering from 1000 to 0.01 proviral copies per PCR, was included in every PCR run. Negative-control samples without templates were also included. Synthetic control DNA templates of 140 bp were ordered from Thermo Hybaid (see Supplementary Table S1, available in JGV Online). Mismatches in the primers were covered by the two primer variants ABL and ABL1, and ABR and ABR1, respectively. The TaqMan real-time PCR has several built-in features, which minimize the risk of false-positive results due to amplicon carry-over: UNG was used to degrade any amplicon, which may contaminate the reaction mixture, and the measurements took place in a closed tube during the PCR.
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; Medstrand & Blomberg, 1993; Yin et al., 1997; Andersson et al., 2005). Amplification mixtures (25 µl) contained approximately 100 ng template DNA, 12.5 µl 2x TaqMan Master Mix with UNG (Applied Biosystems), 200 nM forward and reverse primers and 200 nM Histone TaqMan probe. The thermal conditions comprised 2 min UNG treatment followed by 10 min polymerase activation at 95 °C, followed by 55 cycles at 95 °C for 15 s and 54 °C for 60 s. The primers were: Histone forward (HisFw), 5'-CTCTACTGGAGGGGTGAAGAA-3'; Histone reverse (HisRev), 5'-TGCCTCCTGCAAAGCACCGATA-3'; and Histone probe (HisProbe), 5'-CTCTGGAAGCGCAGATCTGTTTTAAAGTCCTG-3'. Reactions were run on a Rotor-Gene 2000 Real-time thermocycler (Corbett Research). Serial dilutions of Histone 3.3 plasmids containing 106–100 copies per PCR were used in the experiment as quantitative standards. A control for PCR inhibition was to run all DNA samples, and two samples containing water only, in the presence of (‘spiked with’) 10 proviral copies of C3H/HeJ DNA per reaction.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Recently, several research groups claim to have detected HMTV/MMTV-specific (distinctive from human endogenous retrovirus) env sequences in humans (Wang et al., 1995; Etkind et al., 2000; Liu et al., 2001; Ford et al., 2004b). The human genome contains a large variety of sequences related to MMTV, i.e. HMLs (Andersson et al., 1999). These are much more evolutionarily distant from MMTV than is the alleged HMTV (Andersson et al., 1999). HMTV/MMTV has also been reported recently in primary biliary cirrhosis (Xu et al., 2004). In the present study, the endogenous betaretroviral sequences were distinguished from HMTV by PCR primers located in the env region of the MMTV provirus, as this region is clearly distinct in nucleotide sequence from that of the HMLs (Wang et al., 1995).
The aim was to search for DNA of an exogenous MMTV-related virus (here referred to as HMTV). Despite having a very sensitive technique, we did not detect HMTV DNA in human breast cancer tissue. The PCR could detect not only MMTV, but also minor HMTV sequence variants in synthetic DNA form, with good efficiency. This diminishes the likelihood of a false-negative outcome. Our inability to detect the virus indicates that the concentration of a putative HMTV DNA is nil or very low in human malignant and non-malignant breast tissue. The negative outcome of this study deviates from some, but is concordant with other (reviewed by Mant & Cason, 2004), reports on HMTV sequences in human breast cancer. We conclude that HMTV DNA is not present in Swedish breast cancer samples at concentrations higher than one copy per 103 cells. If there is an HMTV at very low concentration in human breast cancers, it is unlikely to cause tumours in the same fashion as MMTV, i.e. by cis activation of onc genes by enhancement from adjacent proviruses, which requires a highly replicating virus and generates high viral DNA concentrations. The possibility that previous reports on HMTV in human breast cancers may have been due to unspecific (e.g. due to plasmid or amplimer contamination) PCR amplification should be considered.
Conclusion
A highly sensitive, env-based HMTV and MMTV detection technique, independent of previous ones, was developed. HMTV DNA was not detected in breast cancer or controls, due either to low concentration or to lack of it.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Andersson, M. L., Lindeskog, M., Medstrand, P., Westley, B., May, F. & Blomberg, J. (1999). Diversity of human endogenous retrovirus class II-like sequences. J Gen Virol 80, 255–260.[Abstract]
Andersson, A. C., Yun, Z., Sperber, G. O., Larsson, E. & Blomberg, J. (2005). ERV3 and related sequences in humans: structure and RNA expression. J Virol 79, 9270–9284.
Bittner, J. J. (1936). Some possible effects of nursing on the mammary gland tumor incidence in mice. Science 84, 162
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Ford, C. E., Faedo, M., Crouch, R., Lawson, J. S. & Rawlinson, W. D. (2004b). Progression from normal breast pathology to breast cancer is associated with increasing prevalence of mouse mammary tumor virus-like sequences in men and women. Cancer Res 64, 4755–4759.
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Yin, H., Medstrand, P., Andersson, M. L., Borg, A., Olsson, H. & Blomberg, J. (1997). Transcription of human endogenous retroviral sequences related to mouse mammary tumor virus in human breast and placenta: similar pattern in most malignant and nonmalignant breast tissues. AIDS Res Hum Retroviruses 13, 507–516.[Medline]
Received 6 December 2006;
accepted 12 February 2007.
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