Our study demonstrates that prostate cancer cell lines appear to have a propensity for infection by murine gammaretroviruses. We propose that this tendency may be due to the fact that most of the established prostate cancer cell lines were created by passage through immunocompromised mice. In fact, Bxv
-1 is present in SCID and nude
mice and tumor cells passaged through these animals can become infected with xenotropic MLVs 
. Although the infection of human cell lines with murine gammaretroviruses has been previously described in the literature, what makes our current study particularly important is that prior to this study it was not known that multiple commonly used prostate cancer cell lines are contaminated with MLVs. In fact, we discovered during the course of our study that other cell lines maintained in the laboratory (DU145, LNCaP) have apparently become infected with XMRV in the laboratory.
In concordance with the data provided in the current study, in a recent study by Hue et. al.
, which screened 411 human tumor cell lines from the Catalogue Of Somatic Mutations In Cancer (COSMIC) collection, EKVX was found to be positive for a xenotropic MLV-related virus by direct sequencing of viral gag
. Furthermore, the Hue et. al.
study tested all but 14 of the cell lines examined in the current study (with the exception of LAPC4, LNCaP, PrEC, VCaP, CWR22Rv1, RWPE, abl, PrSC, C4-2B, 957 E/h, PacMet UT1, MDA-PAC2b, DLD-1 and Hep3B) and likewise found them to be negative for MLV. Although the authors of the study did not attempt to obtain a full-length sequence of the virus contaminating EKVX or to demonstrate that the virus is replication competent, partial pol
sequences from the Hue et. al.
study (GenBank Accession #FR670589 and FR670597, respectively) match at 100% similarity to the near full-length viral sequence obtained in the present study (GenBank Accession #JF908817). We have now demonstrated that the virus contained in the EKVX cell line is replication competent (), clusters in phylogenetic analysis with known xenotropic MLVs (, Supporting Figure S3
) and is 99% similar to a retrovirus previously isolated from the DG-75 B-lymphoblastoid cell line. Like all of the prostate cancer cell lines found to contain MLVs in the present study, the EKVX cell line was established by xenograft passage in nude
There are several reported examples of retroviral infection modifying the biological properties of human cell lines. For example, the in vivo
immunogenicity of cell lines has been shown to be strongly altered following retroviral infection after a single passage in nude
. Likewise, xenotropic MLVs acquired in cell lines used in HIV research after in vivo
passage in immunocompromised mice were shown to alter the biological properties of HIV-1 
. Studies have also shown that MLV tends to integrate near transcription start sites (within 5 kb upstream or downstream) of actively transcribed genes 
. Interestingly, two of the seven viral integration sites identified for VCaP are within 5 kb of a transcription start site (). An integration site located on Chromosome 1 is located approximately 2 kb downstream of the transcription start site of the EFCAB2 (EF-hand calcium binding domain 2) gene. Likewise, an integration site identified on Chromosome 7 is located approximately 1.4 kb upstream of LFNG (O-fucosylpeptide 3-beta-N-acetylglucosaminyltransferase), a gene involved in Notch signaling. It is not known what confounding effects the infection of commonly used prostate cancer cell lines with replication competent murine gammaretroviruses may have on experimental outcomes. Interestingly, it has been reported that XMRV proteins are more abundant in cell culture media of CWR22Rv1 cells than any human protein 
. For reasons such as this, we suggest that cancer cell lines should undergo routine screening for contaminating MLVs, much like the current practice of routine testing of cultured cells for mycoplasma.