The study of the retrovirus, xenotropic murine leukemia virus-related virus, or XMRV, appears online in the journal Science.
The study traces the lineage of the XMRV virus back through several generations of laboratory-cultured prostate cancer cells. The researchers found the virus to be virtually undetectable in the earliest tumors propagated in laboratory mice, but teeming in abundance after many such mouse passages. In the process, they establish that XMRV derives from two parent retroviruses present in the mice.
“For a retrovirus to cause cancer, almost every cancer cell should harbor the virus. When we found the virus was not in the early passages of the tumor cells, but plentiful in the later ones, it made us suspicious that XMRV was not the cause of the original tumor,” said study coauthor Hsing-Jien Kung, professor and deputy director of the UC Davis Cancer Center. Kung is an expert on retroviruses and the biology of prostate cancer cells.
The study is one of two that refute connections between XMRV and human disease that appear in the journal Science. The second study does not find XMRV in chronic fatigue syndrome patients, including many individuals found to be infected in an earlier survey.
In 2006, scientists reported finding XMRV in 27 percent of human prostate cancer tumors, and in 2009, in the blood of 67 percent of chronic fatigue syndrome patients. Because retroviruses are known to cause a number of human diseases, including AIDS and adult T-cell leukemia, these results made XMRV a potential source of both disorders. However, other studies failed to find a correlation between these diseases and the virus.
“We became interested in finding out whether this virus was really a factor in these diseases and how that came about,” said study coauthor Clifford Tepper, associate research biochemist in the Department of Biochemistry and Molecular Medicine.
Kung had worked with some of the first prostate cancer tumors to be transplanted into and propagated in laboratory mice, as well as with subsequent generations of these tumors, which had been made into cell lines. Such mouse passages are a common method of studying cancer treatments considered too experimental for human use. Kung and Tepper had many of these cells and tumors in their freezers, and kept meticulous records that allowed them to recreate the timeline for when various cell lines and tumor cultures were made.
For the study, the scientists compared viral loads in both early and later generations of these cells. They found that the first seven generations of passaged cells had one to three copies of XMRV-like sequence per 100 cells, while later passages had roughly 60 to 70 copies. More extreme, passaged cells that had been transformed into cell culture lines had on the order of 2,000 to 3,000 copies of viral genomes.
At the same time, two previously undescribed viruses in the mouse lines used to passage the prostate cancer tumors were identified. Both microbes were sequenced, and the two genomes placed side by side.
“When we put them next to each other, they fit together like a puzzle. We knew immediately that they had generated XMRV. It was one of those eureka moments that comes along only a few times in a career,” said study coauthor Vinay Pathak, head of the viral mutation section of the National Cancer Institute, who is a UC Davis alumnus.
The two viruses had shuffled their genetic data in a process called recombination to generate XMRV. A recombination event can be envisioned as a train traveling along parallel railroad tracks, switching back and forth at will between the two. The scientists calculated that six track shifts would have been necessary to generate the XMRV sequence.
The XMRV sequences found in the cell lines are virtually identical. This indicates they all developed recently from a single recombination event.
“If the virus were to be generated again in other instances, its genetic sequence should diverge. It’s unlikely to happen again and again with perfect sequence fidelity. The fact that the virus sequences are so similar is the main reason we suspect that what’s been reported has been due to contamination of laboratory equipment or reagents,” Kung said.
The researchers calculate that the odds of generating an identical XMRV sequence a second time are roughly 1.3 x 10-16 — vanishingly low.
Based on the dates that the cell passages occurred, “we knew it could only have been generated between 1993 and 1996. This virus is so new it could not possibly be in such a broad segment of the human population,” Kung said.
By discounting one potential source of prostate cancer and chronic fatigue syndrome, the study will help scientists focus their efforts on more viable origins for these diseases.
“If you’re going to invest research dollars in a virus, you want to make sure it’s really there,” Tepper said.
The finding also speaks to the importance of avoiding laboratory contamination, the most likely cause of the associations between XMRV, prostate cancer and chronic fatigue syndrome.
“Contamination in the lab is a serious issue. Hopefully, when people look for evidence of new viruses in diseases of unknown origin they will take note of this work and rule out contamination before establishing an association between a virus and any human disease,” Pathak said.
Other study authors are Tobias Paprotka, Krista A. Delviks-Frankenberry, Wei-Shau Hu and Matthew J. Fivash, Jr., the National Cancer Institute; Oya Cingöz and John M. Coffin, Tufts University; and Anthony Martinez, UC Davis.
The study was supported in part by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research.
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