False negative results from using common PCR reagents.

BACKGROUND: The sensitivity of the PCR reaction makes it ideal for use when identifying potentially novel viral infections in human disease. Unfortunately, this same sensitivity also leaves this popular technique open to potential contamination with previously amplified PCR products, or "carry-over" contamination. PCR product carry-over contamination can be prevented with uracil-DNA-glycosylase (UNG), and it is for this reason that it is commonly included in many commercial PCR master-mixes. While testing the sensitivity of PCR assays to detect murine DNA contamination in human tissue samples, we inadvertently discovered that the use of this common PCR reagent may lead to the production of false-negative PCR results. FINDINGS: We show here that contamination with minute quantities of UNG-digested PCR product or any negative control PCR reactions containing primer-dimers regardless of UNG presence can completely block amplification from as much as 60 ng of legitimate target DNA. CONCLUSIONS: These findings could potentially explain discrepant results from laboratories attempting to amplify MLV-related viruses including XMRV from human samples, as none of the published reports used internal-tube controls for amplification. The potential for false negative results needs to be considered and carefully controlled in PCR experiments, especially when the target copy number may be low - just as the potential for false positive results already is.

Negative regulation of C/EBPbeta1 by sumoylation in breast cancer cells.

Sumoylation is a post-translational modification that is oftentimes deregulated in diseases such as cancer. Transcription factors are frequent targets of sumoylation and modification by SUMO can affect subcellular localization, transcriptional activity, and stability of the target protein. C/EBPbeta1 is one such transcription factor that is modified by SUMO-2/3. Non-sumoylated C/EBPbeta1, p52-C/EBPbeta1, is expressed in normal mammary epithelial cells but not breast cancer cell lines and plays a role in oncogene-induced senescence, a tumor suppressive mechanism. Although p52-C/EBPbeta1 is not observed via immunoblot in breast cancer cell lines, higher molecular weight bands are observed when breast cancer cell lines are subjected to immunoblot analysis with a C/EBPbeta1-specific antibody. We show that exogenously expressed C/EBPbeta1 is sumoylated in breast cancer cells, and that the higher molecular weight bands we observe in anti-C/EBPbeta1 immunoblots of breast cancer cell lines is sumoylated C/EBPbeta1. Phosphorylation oftentimes enhances sumoylation, and phosphorylation cascades are activated in breast cancer cells. We demonstrate that phosphorylation of C/EBPbeta1Thr235 by Erk-2 enhances sumoylation of C/EBPbeta1 in vitro. In addition, sumoylated C/EBPbeta1 is phosphorylated on Thr235 and mutation of Thr235 to alanine leads to a decrease in sumoylation of C/EBPbeta1. Finally, using a C/EBPbeta1-SUMO fusion protein we show that constitutive sumoylation of C/EBPbeta1 completely blocks its capability to induce senescence in WI38 fibroblasts expressing hTERT. Thus, sumolylation of C/EBPbeta1 in breast cancer cells may be a mechanism to circumvent oncogene-induced senescence.

C/EBPß's role in determining Ras-induced senescence or transformation.

Introduction of activated Ras into normal cells leads to senescence, a tumor suppressive mechanism, whereas expression of this oncogene in many immortalized cell lines leads to transformation. Studying the signaling differences in cells that undergo Ras-induced senescence versus Ras transformation may shed light on potential therapeutic targets in the treatment of cancer. C/EBPß is a transcription factor necessary for both Ras-induced senescence and Ras transformation. Three isoforms of this transcription factor exist due to alternative translation initation at three in frame ATGs. C/EBPß1 is the isoform responsible for oncogene-induced senescence, and this isoform is degraded by the proteosome during Ras transformation. Phosphorylation of C/EBPß1 on Thr235 by Cdk2 is necessary, but not sufficient, for degradation of C/EBPß1. Proteasomal degradation of C/EBPß1 may represent a mechanism to evade senescence. In contrast, C/EBPß2 is expressed in breast cancer cells and is involved in proliferation, supporting a role for this isoform in Ras transformation. We propose here that one potential signaling difference in Ras-induced senescence versus Ras transformation is that Ras signals through different C/EBPß isoforms (C/EBPß1 versus C/EBPß2) during these processes.