Multiple Sources of Contamination in Samples from Patients Reported to Have XMRV Infection

Xenotropic murine leukemia virus (MLV)-related retrovirus (XMRV) was reported to be associated with prostate cancer by Urisman, et al. in 2006 and chronic fatigue syndrome (CFS) by Lombardi, et al. in 2009. To investigate this association, we independently evaluated plasma samples from 4 patients with CFS reported by Lombardi, et al. to have XMRV infection and from 5 healthy controls reported to be XMRV uninfected. We also analyzed viral sequences obtained from supernatants of cell cultures found to contain XMRV after coculture with 9 clinical samples from 8 patients. A qPCR assay capable of distinguishing XMRV from endogenous MLVs showed that the viral sequences detected in the CFS patient plasma behaved like endogenous MLVs and not XMRV. Single-genome sequences (N = 89) from CFS patient plasma were indistinguishable from endogenous MLVs found in the mouse genome that are distinct from XMRV. By contrast, XMRV sequences were detected by qPCR in 2 of the 5 plasma samples from healthy controls (sequencing of the qPCR product confirmed XMRV not MLV). Single-genome sequences (N = 234) from the 9 culture supernatants reportedly positive for XMRV were indistinguishable from XMRV sequences obtained from 22Rv1 and XMRV-contaminated 293T cell-lines. These results indicate that MLV DNA detected in the plasma samples from CFS patients evaluated in this study was from contaminating mouse genomic DNA and that XMRV detected in plasma samples from healthy controls and in cultures of patient samples was due to cross-contamination with XMRV (virus or nucleic acid)

Failure to Confirm XMRV/MLVs in the Blood of Patients with Chronic Fatigue Syndrome: A Multi-Laboratory Study

Murine leukemia viruses (MLV), including xenotropic-MLV-related virus (XMRV), have been controversially linked to chronic fatigue syndrome (CFS). To explore this issue in greater depth, we compiled coded replicate samples of blood from 15 subjects previously reported to be XMRV/ MLV-positive (14 with CFS) and from 15 healthy donors previously determined to be negative for the viruses. These samples were distributed in a blinded fashion to nine laboratories which performed assays designed to detect XMRV/MLV nucleic acid, virus replication, and antibody. Only two laboratories reported evidence of XMRV/MLVs; however, replicate sample results showed disagreement and reactivity was similar among CFS subjects and negative controls. These results indicate that current assays do not reproducibly detect XMRV/MLV in blood samples and that blood donor screening is not warranted

The role of autophagy in the cross-talk between epithelial-mesenchymal transitioned tumor cells and cancer stem-like cells

Epithelial-mesenchymal transition (EMT) and cancer stem-like cells (CSC) are becoming highly relevant targets in anticancer drug discovery. A large body of evidence suggests that epithelial-mesenchymal transitioned tumor cells (EMT tumor cells) and CSCs have similar functions. There is also an overlap regarding the stimuli that can induce the generation of EMT tumor cells and CSCs. Moreover, direct evidence has been brought that EMT can give rise to CSCs. It is unclear however, whether EMT tumor cells should be considered CSCs or if they have to undergo further changes. In this article we summarize available evidence suggesting that, indeed, additional programs must be engaged and we propose that macroautophagy (hereafter, autophagy) represents a key trait distinguishing CSCs from EMT tumor cells. Thus, CSCs have often been reported to be in an autophagic state and blockade of autophagy inhibits CSCs. On the other hand, there is ample evidence showing that EMT and autophagy are distinct events. CSCs, however, represent, by themselves, a heterogeneous population. Thus, CSCs have been distinguished in predominantly noncycling and cycling CSCs, the latter representing CSCs that self-renew and replenish the pool of differentiated tumor cells. We now suggest that the non-cycling CSC subpopulation is in an autophagic state. We propose also two models to explain the relationship between EMT tumor cells and these two major CSC subpopulations: a branching model in which EMT tumor cells can give rise to cycling or non-cycling CSCs, respectively, and a hierarchical model in which EMT tumor cells are first induced to become autophagic CSCs and, subsequently, cycling CSCs. Finally, we address the therapeutic consequences of these insights

Ku Regulates the Non-Homologous End Joining Pathway Choice of DNA Double-Strand Break Repair in Human Somatic Cells

The repair of DNA double-strand breaks (DSBs) is critical for the maintenance of genomic integrity and viability for all organisms. Mammals have evolved at least two genetically discrete ways to mediate DNA DSB repair: homologous recombination (HR) and non-homologous end joining (NHEJ). In mammalian cells, most DSBs are preferentially repaired by NHEJ. Recent work has demonstrated that NHEJ consists of at least two sub-pathways—the main Ku heterodimer-dependent or “classic” NHEJ (C-NHEJ) pathway and an “alternative” NHEJ (A-NHEJ) pathway, which usually generates microhomology-mediated signatures at repair junctions. In our study, recombinant adeno-associated virus knockout vectors were utilized to construct a series of isogenic human somatic cell lines deficient in the core C-NHEJ factors (Ku, DNA-PKcs, XLF, and LIGIV), and the resulting cell lines were characterized for their ability to carry out DNA DSB repair. The absence of DNA-PKcs, XLF, or LIGIV resulted in cell lines that were profoundly impaired in DNA DSB repair activity. Unexpectedly, Ku86-null cells showed wild-type levels of DNA DSB repair activity that was dominated by microhomology joining events indicative of A-NHEJ. Importantly, A-NHEJ DNA DSB repair activity could also be efficiently de-repressed in LIGIV-null and DNA-PKcs-null cells by subsequently reducing the level of Ku70. These studies demonstrate that in human cells C-NHEJ is the major DNA DSB repair pathway and they show that Ku is the critical C-NHEJ factor that regulates DNA NHEJ DSB pathway choice