Smoking is Associated with Hypermethylation of the APC 1A Promoter in Colorectal Cancer: the ColoCare Study

Smoking tobacco is a known risk factor for the development of colorectal cancer, and for mortality associated with the disease. While smoking has been reported to be associated with changes in DNA methylation in blood and in lung tumour tissues, there has been scant investigation of how epigenetic factors may be implicated in the increased risk of developing colorectal cancer. To identify epigenetic changes associated with smoking behaviours, we performed epigenome-wide analysis of DNA methylation in colorectal tumours from 36 never smokers, 47 former smokers and 13 active smokers, and adjacent mucosa from 49 never smokers, 64 former smokers and 18 active smokers. Our analyses identified 15 CpG sites within the APC 1A promoter that were significantly hypermethylated and 14 CpG loci within the NFATC1 gene body that were significantly hypomethylated (pLIS<1x10-5) in tumours of active smokers. The APC 1A promoter was hypermethylated in 7 of 36 tumours from never smokers (19%), 12 of 47 tumours from former smokers (26%), and 8 of 13 tumours from active smokers (62%). Promoter hypermethylation was positively associated with duration of smoking (Spearman rank correlation, =0.26, p=0.03) and was confined to tumours, with hypermethylation never observed in adjacent mucosa. Further analysis of adjacent mucosa revealed significant hypomethylation of four loci associated with the TNXB gene in tissue from active smokers. Our findings provide exploratory evidence for hypermethylation of the key tumour suppressor gene APC being implicated in smoking-associated colorectal carcinogenesis. Further work is required to establish the validity of our observations in independent cohorts

Polybrene Inhibits Human Mesenchymal Stem Cell Proliferation during Lentiviral Transduction

Human mesenchymal stem cells (hMSCs) can be engineered to express specific genes, either for their use in cell-based therapies or to track them in vivo over long periods of time. To obtain long-term expression of these genes, a lentivirus- or retrovirus-mediated cell transduction is often used. However, given that the efficiency with these viruses is typically low in primary cells, additives such as polybrene are always used for efficient viral transduction. Unfortunately, as presented here, exposure to polybrene alone at commonly used concentratons (1–8 µg/mL) negatively impacts hMSC proliferation in a dose-dependent manner as measured by CyQUANT, EdU incorporation, and cell cycle analysis. This inhibition of proliferation was observable in culture even 3 weeks after exposure. Culturing the cells in the presence of FGF-2, a potent mitogen, did not abrogate this negative effect of polybrene. In fact, the normally sharp increase in hMSC proliferation that occurs during the first days of exposure to FGF-2 was absent at 4 µg/mL or higher concentrations of polybrene. Similarly, the effect of stimulating cell proliferation under simulated hypoxic conditions was also decreased when cells were exposed to polybrene, though overall proliferation rates were higher. The negative influence of polybrene was, however, reduced when the cells were exposed to polybrene for a shorter period of time (6 hr vs 24 hr). Thus, careful evaluation should be done when using polybrene to aid in lentiviral transduction of human MSCs or other primary cells, especially when cell number is critical

LEDGF/p75-Independent HIV-1 Replication Demonstrates a Role for HRP-2 and Remains Sensitive to Inhibition by LEDGINs

Lens epithelium–derived growth factor (LEDGF/p75) is a cellular cofactor of HIV-1 integrase (IN) that interacts with IN through its IN binding domain (IBD) and tethers the viral pre-integration complex to the host cell chromatin. Here we report the generation of a human somatic LEDGF/p75 knockout cell line that allows the study of spreading HIV-1 infection in the absence of LEDGF/p75. By homologous recombination the exons encoding the LEDGF/p75 IBD (exons 11 to 14) were knocked out. In the absence of LEDGF/p75 replication of laboratory HIV-1 strains was severely delayed while clinical HIV-1 isolates were replication-defective. The residual replication was predominantly mediated by the Hepatoma-derived growth factor related protein 2 (HRP-2), the only cellular protein besides LEDGF/p75 that contains an IBD. Importantly, the recently described IN-LEDGF/p75 inhibitors (LEDGINs) remained active even in the absence of LEDGF/p75 by blocking the interaction with the IBD of HRP-2. These results further support the potential of LEDGINs as allosteric integrase inhibitors

HIV infection of non-dividing cells: a divisive problem

Understanding how lentiviruses can infect terminally differentiated, non-dividing cells has proven a very complex and controversial problem. It is, however, a problem worth investigating, for it is central to HIV-1 transmission and AIDS pathogenesis. Here I shall attempt to summarise what is our current understanding for HIV-1 infection of non-dividing cells. In some cases I shall also attempt to make sense of controversies in the field and advance one or two modest proposals

Role of PSIP1/LEDGF/p75 in lentiviral infectivity and integration targeting

To replicate, lentiviruses such as HIV must integrate DNA copies of their RNA genomes into host cell chromosomes. Lentiviral integration is favored in active transcription units, which allows efficient viral gene expression after integration, but the mechanisms directing integration targeting are incompletely understood. A cellular protein, PSIP1/LEDGF/p75, binds tightly to the lentiviral-encoded integrase protein (IN), and has been reported to be important for HIV infectivity and integration targeting.Here we report studies of lentiviral integration targeting in 1) human cells with intensified RNAi knockdowns of PSIP1/LEDGF/p75, and 2) murine cells with homozygous gene trap mutations in the PSIP1/LEDGF/p75 locus. Infections with vectors derived from equine infections anemia virus (EIAV) and HIV were compared. Integration acceptor sites were analyzed by DNA bar coding and pyrosequencing.In both PSIP1/LEDGF/p75-depleted cell lines, reductions were seen in lentiviral infectivity compared to controls. For the human cells, integration was reduced in transcription units in the knockdowns, and this reduction was greater than in our previous studies of human cells less completely depleted for PSIP1/LEDGF/p75. For the homozygous mutant mouse cells, similar reductions in integration in transcription units were seen, paralleling a previous study of a different mutant mouse line. Integration did not become random, however-integration in transcription units in both cell types was still favored, though to a reduced degree. New trends also appeared, including favored integration near CpG islands. In addition, we carried out a bioinformatic study of 15 HIV integration site data sets in different cell types, which showed that the frequency of integration in transcription units was correlated with the cell-type specific levels of PSIP1/LEDGF/p75 expression

Loss of Tumor-Initiating Cell Activity in Cyclophosphamide-Treated Breast Xenografts1

Cancer stem cells (CSCs) are a subpopulation of tumor cells with preferential tumor-initiating capacity and have been purported to be resistant to chemotherapy. It has been shown that breast CSC are, on average, enriched in patient tumors after combination neoadjuvant chemotherapy including docetaxel, doxorubicin, and cyclophosphamide (CPA). Here, we investigate the resistance of breast CSC to CPA alone in a xenograft model. CPA treatment led to a 48% reduction in tumor volume during a 2-week period. Cells bearing the CD44+ CD24- phenotype were reduced by 90% (2.5% to 0.24%) in CPA-treated tumors, whereas cells with aldehyde dehydrogenase activity were reduced by 64% (4.7% to 1.7%). A subsequent functional analysis showed that CPA-treated tumors were impaired in their ability to form tumors, indicating loss of functional tumor-initiating activity. These results are consistent with a CSC phenotype that is sensitive to CPA and indicate that some patient CSC may not display the expected resistance to therapy. Deciphering the mechanism for this difference may lead to therapies to counteract resistance

Ablation of Breast Cancer Stem Cells with Radiation1

Tumor radioresistance leads to recurrence after radiation therapy. The radioresistant phenotype has been hypothesized to reside in the cancer stem cell (CSC) component of breast and other tumors and is considered to be an inherent property of CSC. In this study, we assessed the radiation resistance of breast CSCs using early passaged, patient-derived xenografts from two separate patients. We found a patient-derived tumor in which the CSC population was rapidly depleted 2 weeks after treatment with radiation, based on CD44+ CD24- lin- phenotype and aldehyde dehydrogenase 1 immunofluorescence, suggesting sensitivity to radiotherapy. The reduction in CSCs according to phenotypic markers was accompanied by a decrease in functional CSC activity measured by tumor sphere frequency and the ability to form tumors in mice. In contrast, another patient tumor sample displayed enrichment of CSC after irradiation, signifying radioresistance, in agreement with others. CSC response to radiation did not correlate with the level of reactive oxygen species in CSC versus non-CSC. These findings demonstrate that not all breast tumor CSCs are radioresistant and suggest a mechanism for the observed variability in breast cancer local recurrence