The content of DNA and RNA in microparticles released by Jurkat and HL-60 cells undergoing in vitro apoptosis

Abstract

Microparticles are small membrane-bound vesicles that are released from apoptotic cells during blebbing. These particles contain DNA and RNA and display important functional activities, including immune system activation. Furthermore, nucleic acids inside the particle can be analyzed as biomarkers in a variety of disease states. To elucidate the nature of microparticle nucleic acids, DNA and RNA released in microparticles from the Jurkat T and HL-60 promyelocytic cell lines undergoing apoptosis in vitro were studied. Microparticles were isolated from culture media by differential centrifugation and characterized by flow cytometry and molecular approaches. In these particles, DNA showed laddering by gel electrophoresis and was present in a form that allowed direct binding by a monoclonal anti-DNA antibody, suggesting antigen accessibility even without fixation. Analysis of RNA by gel electrophoresis showed intact 18s and 28s ribosomal RNA bands, although lower molecular bands consistent with 28s ribosomal RNA degradation products were also present. Particles also contained messenger RNA as shown by RT-PCR amplification of sequences for β-actin and GAPDH. In addition, gel electrophoresis showed the presence of low molecular weight RNA in the size range of microRNA. Together, these results indicate that microparticles from apoptotic Jurkat and HL-60 cells contain diverse nucleic acid species, indicating translocation of both nuclear and cytoplasmic DNA and RNA as particle release occurs during death.

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.