Research ArticleThe content of DNA and RNA in microparticles released by Jurkat and HL-60 cells undergoing in vitro apoptosis
Introduction
Microparticles (MPs) are small membrane-bound particles that circulate in the blood and play an important role in a variety of critical processes including inflammation, thrombosis and tumor invasion. These particles are 0.1–1.0 μm in size and arise from activated and dying cells by a blebbing process that incorporates membrane, cytoplasmic and nuclear constituents. The phenotype and composition of particles varies by cell of origin, with membrane markers allowing their identification and quantification by flow cytometry. In view of the origin of MPs, their levels in the blood are elevated in many different disease states, providing a measure of events in pathogenesis at the level of specific tissue or organs [1], [2], [3].
In addition to their content of membrane and cytoplasmic components, MPs contain both DNA and RNA [4], [5]. Indeed, MPs appear to be a major source of RNA circulating in the blood, with the membrane structure shielding this molecule from digestion by blood nucleases [6], [7]. As shown in studies on patients with malignancy, MPs in the blood can contain mRNA from the tumor in a form that can be analyzed by genomic techniques. Thus, in patients with melanoma, for example, tyrosinase mRNA in the blood represents a tumor-specific marker that can be quantitated by PCR techniques; similar results have been obtained with other cancers including breast and lung cancer [8], [9], [10], [11], [12], [13], [14]. In contrast to RNA, DNA can appear in both a particulate and non-particulate form [15].
While circulating nucleic acids can provide markers of both diagnostic and prognostic significance, they may also be important as immune activators. Depending on the structure and mode of interaction with immune cells, DNA and RNA can both trigger responses via the Toll-like receptors and induce cytokines such as IFN-α among others [16], [17], [18], [19], [20]. As such, in in vitro studies, this activation can involve TLR 3 (double stranded RNA), TLR 7 (single stranded RNA) and TLR9 (DNA). In diseases such as systemic lupus erythematosus, nucleic acids provide a major driving force for autoimmunity, although the role of MPs in this activation has not been established [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32]. MPs can also modulate the host response to tumors, an effect that may also relate to their content of nucleic acids [24], [25], [26], [27], [28].
Because of the importance of MP nucleic acids as a source of material for genomic studies as well as their potential as immune activators, we have initiated studies to characterize the properties of DNA and RNA found in MPs released in vitro from tumor cell lines. For this purpose, we have used the Jurkat T cell leukemia and the HL-60 promyelocytic cell lines as models and characterized the DNA and RNA present in the particles from cells undergoing apoptosis. The results of these studies indicate that MPs contain a spectrum of cellular DNA and RNA molecules that include both ribosomal and messenger RNA as well as low molecular weight species. Furthermore, our studies indicate that MPs contain DNA in a form that allows antibody binding. Together with other studies on MP composition, these studies highlight the importance of these particles as carriers of cellular nucleic acids and their potential utility as blood biomarkers of critical events during disease pathogenesis.
Section snippets
Cell culture and microparticle preparation
The Jurkat human T cell and HL-60 promyelocytic cell lines were obtained from the Duke University Comprehensive Cancer Center Cell Culture Facility and tested negative for mycoplasma contamination by Gen-Probe nucleic acid hybridization (Gen-Probe, San Diego, CA). Cells were cultured in RPMI 1640 medium (Invitrogen, Carlsbad CA) supplemented with 10% fetal bovine serum (HyClone, Logan, Utah) and 20 μg/ml gentamicin (Sigma, St. Louis MO) at 37 °C in 5% CO2/95% air. Cells in logarithmic cell
Results
For these studies, we have used the Jurkat T cell and HL-60 promyelocytic cell lines as models because they are well characterized tumor systems that can be induced to undergo activation or death by chemical agents. We have focused on apoptosis because this form of cell death is a major source of MP release; for comparison with another death form, we also assessed particle release during necrosis induced in vitro by ethanol or heat treatment. For these experiments, MPs were isolated by
Discussion
Results presented herein provide new insights into the composition of cellular MPs and demonstrate an abundance of nucleic acid components. Thus, using a combination of molecular and flow cytometric approaches, we have demonstrated the presence of DNA and RNA in accessible forms that could be on the interior as well as exterior of MPs. Of the RNA species, both intact ribosomal and messenger RNA are present, along with smaller species of RNA which are consistent in size with degraded ribosomal
Acknowledgments
This work was supported by a VA Merit Review grant and a grant from the Lupus Foundation of America.
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