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Insertional mutagenesis is an important risk with all genetically modified cell therapies, including chimeric antigen receptor (CAR)-T cell therapy used for hematological malignancies. Here we describe a new tagmentation-assisted PCR (tag-PCR) system that can determine the integration sites of transgenes without using restriction enzyme digestion (which can potentially bias the detection) and allows library preparation in fewer steps than with other methods. Using this system, we compared the integration sites of CD19-specific CAR genes in final T cell products generated by retrovirus-based and lentivirus-based gene transfer and by the piggyBac transposon system. The piggyBac system demonstrated lower preference than the retroviral system for integration near transcriptional start sites and CpG islands and higher preference than the lentiviral system for integration into genomic safe harbors. Integration into or near proto-oncogenes was similar in all three systems. Tag-PCR mapping is a useful technique for assessing the risk of insertional mutagenesis.
Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.
To examine the interplay between tumor cells and the microenvironment during early breast cancer metastasis, we developed a technique for ex vivo imaging of murine tissue explants using two-photon microscopy. Cancer cells in the liver and the lung were compared by imaging both organs at specific time points after the injection of the same polyomavirus middle T-initiated murine mammary tumor cell line. Extravasation was greatly reduced in the lung compared with the liver, with 56% of tumor cells in the liver having extravasated by 24 hours, compared with only 22% of tumor cells in the lung that have extravasated. In the liver, imaged cells continually transitioned from an intravascular location to an extravascular site, whereas in the lung, extravasation rates slowed after 6 hours. Within the liver microenvironment, the average size of the imaged micrometastatic lesions increased 4-fold between days 5 and 12. Histologic analysis of these lesions determined that by day 12, the micrometastases were heterogeneous, consisting of both tumor cells and von Willebrand factor-positive endothelial cells. Further analysis with intravenously administered lectin indicated that vessels within the micrometastatic tumor foci were patent by day 12. These data present the use of two-photon microscopy to directly compare extravasation times in metastatic sites using the same tumor cell line and highlight the differences in early events and metastatic patterns between two important secondary sites of breast cancer progression with implications for future therapy.
The genetic variants underlying the strong heritable component of prostate cancer remain largely unknown. Genome-wide association studies of prostate cancer have yielded several variants that have significantly replicated across studies, predominantly in cases unselected for family history of prostate cancer. Additional candidate gene variants have also been proposed, many evaluated within familial prostate cancer study populations. Such variants hold great potential value for risk stratification, particularly for early-onset or aggressive prostate cancer, given the comorbidities associated with current therapies. Here, we investigate a Caucasian study population of 523 independent familial prostate cancer cases and 523 age-matched controls without a personal or family history of prostate cancer. We replicate identified associations at genome-wide association study loci 8q24, 11q13, and 2p15 (P = 2.9 x 10(-4) to P = 4.7 x 10(-5)), showing study population power. We also find evidence to support reported associations at candidate genes RNASEL, EZH2, and NKX3-1 (P = 0.031 to P = 0.0085). We further explore a set of candidate genes related to RNASEL and to its role in retroviral restriction, identifying nominal associations at XPR1 and RBM9. The effects at 8q24 seem more pronounced for those diagnosed at an early age, whereas at 2p15 and RNASEL the effects were more pronounced at a later age. However, these trends did not reach statistical significance. The effects at 2p15 were statistically significantly more pronounced for those diagnosed with aggressive disease.
Five X-linked severe combined immunodeficiency patients (SCID-X1) successfully treated with autologous bone marrow stem cells infected ex vivo with an IL2RG-containing retrovirus subsequently developed T-cell leukemia and four contained insertional mutations at LMO2. Genetic evidence also suggests a role for IL2RG in tumor formation, although this remains controversial. Here, we show that the genes and signaling pathways deregulated in murine leukemias with retroviral insertions at Lmo2 are similar to those deregulated in human leukemias with high LMO2 expression and are highly predictive of the leukemias induced in SCID-X1 patients. We also provide additional evidence supporting the notion that IL2RG and LMO2 cooperate in leukemia induction but are not sufficient and require additional cooperating mutations. The highly concordant nature of the genetic events giving rise to mouse and human leukemias with mutations at Lmo2 are an encouraging sign to those wanting to use mice to model human cancer and may help in designing safer methods for retroviral gene therapy.
We review different uses of the retroviral mutagenesis technology as the tool to manipulate the zebrafish genome. In addition to serving as a mutagen in a phenotype-driven forward mutagenesis screen as it was originally adapted for, retroviral insertional mutagenesis can also be exploited in reverse genetic approaches, delivering enhancer- and gene-trap vectors for the purpose of examining gene expression patterns and mutagenesis, making sensitized mutants amenable for chemical and genetic modifier screens, and producing gain-of-function mutations by epigenetically overexpressing the retroviral-inserted genes. From a technology point of view, we also summarize the recent advances in the high-throughput cloning of retroviral integration sites, a pivotal step toward identifying mutations. Lastly, we point to some potential directions that retroviral mutagenesis might take from the lessons of studying other model organisms.
The host protein TRIM5alpha inhibits retroviral infection at an early post-penetration stage by targeting the incoming viral capsid. While the detailed mechanism of restriction remains unclear, recent studies have implicated the activity of cellular proteasomes in the restriction of retroviral reverse transcription imposed by TRIM5alpha. Here, we show that TRIM5alpha is rapidly degraded upon encounter of a restriction-susceptible retroviral core. Inoculation of TRIM5alpha-expressing human 293T cells with a saturating level of HIV-1 particles resulted in accelerated degradation of the HIV-1-restrictive rhesus macaque TRIM5alpha protein but not the nonrestrictive human TRIM5alpha protein. Exposure of cells to HIV-1 also destabilized the owl monkey restriction factor TRIMCyp; this was prevented by addition of the inhibitor cyclosporin A and was not observed with an HIV-1 virus containing a mutation in the capsid protein that relieves restriction by TRIMCyp IVHIV. Likewise, human TRIM5alpha was rapidly degraded upon encounter of the restriction-sensitive N-tropic murine leukemia virus (N-MLV) but not the unrestricted B-MLV. Pretreatment of cells with proteasome inhibitors prevented the HIV-1-induced loss of both rhesus macaque TRIM5alpha and TRIMCyp proteins. We also detected degradation of endogenous TRIM5alpha in rhesus macaque cells following HIV-1 infection. We conclude that engagement of a restriction-sensitive retrovirus core results in TRIM5alpha degradation by a proteasome-dependent mechanism.
Reduced levels of the cyclin-dependent kinase inhibitor p27(Kip1) connote poor prognosis in cancer. In human Burkitt lymphoma and in precancerous B cells and lymphomas arising in Emu-Myc transgenic mice, p27(Kip1) expression is markedly reduced. We show that the transcription of the Cks1 component of the SCF(Skp2) complex that is necessary for p27(Kip1) ubiquitylation and degradation is induced by Myc. Further, Cks1 expression is elevated in precancerous Emu-Myc B cells, and high levels of Cks1 are also a hallmark of Emu-Myc lymphoma and of human Burkitt lymphoma. Finally, loss of Cks1 in Emu-Myc B cells elevates p27(Kip1) levels, reduces proliferation and markedly delays lymphoma development and dissemination of disease. Therefore, Myc suppresses p27(Kip1) expression, accelerates cell proliferation and promotes tumorigenesis at least in part through its ability to selectively induce Cks1.
We investigated whether TGF-beta induced by anticancer therapies accelerates tumor progression. Using the MMTV/PyVmT transgenic model of metastatic breast cancer, we show that administration of ionizing radiation or doxorubicin caused increased circulating levels of TGF-beta1 as well as increased circulating tumor cells and lung metastases. These effects were abrogated by administration of a neutralizing pan-TGF-beta antibody. Circulating polyomavirus middle T antigen-expressing tumor cells did not grow ex vivo in the presence of the TGF-beta antibody, suggesting autocrine TGF-beta is a survival signal in these cells. Radiation failed to enhance lung metastases in mice bearing tumors that lack the type II TGF-beta receptor, suggesting that the increase in metastases was due, at least in part, to a direct effect of TGF-beta on the cancer cells. These data implicate TGF-beta induced by anticancer therapy as a pro-metastatic signal in tumor cells and provide a rationale for the simultaneous use of these therapies in combination with TGF-beta inhibitors.
The CXC chemokine receptor 4 (CXCR4) contributes to the metastasis of human breast cancer cells. The CXCR4 COOH-terminal domain (CTD) seems to play a major role in regulating receptor desensitization and down-regulation. We expressed either wild-type CXCR4 (CXCR4-WT) or CTD-truncated CXCR4 (CXCR4-DeltaCTD) in MCF-7 human mammary carcinoma cells to determine whether the CTD is involved in CXCR4-modulated proliferation of mammary carcinoma cells. CXCR4-WT-transduced MCF-7 cells (MCF-7/CXCR4-WT cells) do not differ from vector-transduced MCF-7 control cells in morphology or growth rate. However, CXCR4-DeltaCTD-transduced MCF-7 cells (MCF-7/CXCR4-DeltaCTD cells) exhibit a higher growth rate and altered morphology, potentially indicating an epithelial-to-mesenchymal transition. Furthermore, extracellular signal-regulated kinase (ERK) activation and cell motility are increased in these cells. Ligand induces receptor association with beta-arrestin for both CXCR4-WT and CXCR4-DeltaCTD in these MCF-7 cells. Overexpressed CXCR4-WT localizes predominantly to the cell surface in unstimulated cells, whereas a significant portion of overexpressed CXCR4-DeltaCTD resides intracellularly in recycling endosomes. Analysis with human oligomicroarray, Western blot, and immunohistochemistry showed that E-cadherin and Zonula occludens are down-regulated in MCF-7/CXCR4-DeltaCTD cells. The array analysis also indicates that mesenchymal marker proteins and certain growth factor receptors are up-regulated in MCF-7/CXCR4-DeltaCTD cells. These observations suggest that (a) the overexpression of CXCR4-DeltaCTD leads to a gain-of-function of CXCR4-mediated signaling and (b) the CTD of CXCR4-WT may perform a feedback repressor function in this signaling pathway. These data will contribute to our understanding of how CXCR4-DeltaCTD may promote progression of breast tumors to metastatic lesions.