- Email: [email protected]
The content of DNA and RNA in microparticles released by Jurkat and HL-60 cells undergoing in vitro apoptosis
E XP E RI ME N TA L C E L L R E S EA RC H 315 ( 20 0 9) 76 0 –76 8
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / y e x c r
Research Article
The content of DNA and RNA in microparticles released by Jurkat and HL-60 cells undergoing in vitro apoptosis Charles F. Reich III, David S. Pisetsky⁎ Medical Research Service, 151G Durham VAMC, 508 Fulton Street, Durham, NC 27705, USA Division of Rheumatology and Immunology, Duke University Medical Center, Durham, NC 27705, USA
A R T I C L E I N F O R M AT I O N
AB ST R AC T
Article Chronology:
Microparticles are small membrane-bound vesicles that are released from apoptotic cells during
Received 3 July 2008
blebbing. These particles contain DNA and RNA and display important functional activities,
Revised version received
including immune system activation. Furthermore, nucleic acids inside the particle can be analyzed
9 December 2008
as biomarkers in a variety of disease states. To elucidate the nature of microparticle nucleic acids,
Accepted 11 December 2008
DNA and RNA released in microparticles from the Jurkat T and HL-60 promyelocytic cell lines
Available online 30 December 2008
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
Keywords:
particles, DNA showed laddering by gel electrophoresis and was present in a form that allowed
Microparticles
direct binding by a monoclonal anti-DNA antibody, suggesting antigen accessibility even without
Apoptosis
fixation. Analysis of RNA by gel electrophoresis showed intact 18s and 28s ribosomal RNA bands,
Necrosis
although lower molecular bands consistent with 28s ribosomal RNA degradation products were
DNA
also present. Particles also contained messenger RNA as shown by RT-PCR amplification of
RNA
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. © 2008 Elsevier Inc. All rights reserved.
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 [1–3]. ⁎ Corresponding author. E-mail address: [email protected] (D.S. Pisetsky). 0014-4827/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2008.12.014
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 [8–14]. In contrast to RNA, DNA can appear in both a particulate and non-particulate form [15].
E XP E RI ME N TA L C E L L R E S EA RC H 315 ( 20 0 9) 76 0 –76 8
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 [16–20]. 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 [21–32]. MPs can also modulate the host response to tumors, an effect that may also relate to their content of nucleic acids [24–28]. 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.
761
recentrifuged. This wash step was repeated three times. Following the final wash, the pellet was either frozen at −70 °C for the later extraction of DNA and RNA or resuspended in PBS to one tenth the volume of the original culture supernatant for flow cytometry.
Flow cytometry
Materials and methods
The measurement of MP numbers and nucleic acid content was performed with a FACScan flow cytometer using Cellquest software (BD Biosciences, San Jose CA). All data were accumulated in logarithmic mode. Noise was excluded by setting a side scatter threshold with sample buffer alone. Analysis was done using FLOWJO software (Tree Star, Ashland OR). To determine microparticle numbers, the sample flow rate was calibrated by weighing sample tubes before and after a timed run and using this measurement to calculate relative volume. For statistical analysis of MP number and PI-positive cell numbers, we used a one-tailed T-test, for unpaired samples and equal variance. For nucleic acid analysis, MPs, with or without previous fixation and permeabilization, were stained with propidium iodide (PI) (Sigma) at 10 μg/ml in PBS or with the HYB331-01 monoclonal anti-double stranded DNA antibody (Abcam, Cambridge, MA) or an isotype control (Sigma) followed by Fab'2 goat anti-mouse IgG PE (Sigma). In some experiments, MPs were fixed with paraformaldehyde and permeabilized with saponin. (BD Cytofix/Cytoperm, BD Biosciences, San Diego). After 20 min fixation, MPs were centrifuged at 16,000× g for 30 min followed by 2 washes with permeabilization buffer. For nuclease treatments, prior to PI staining, MPs were incubated for 30 min at 37 °C with 200 U/ml DNase-free RNase (Sigma) or 100 U/ml RNase-free DNase (Invitrogen) in permeabilization buffer supplemented with additional calcium and magnesium.
Cell culture and microparticle preparation
Nucleic acid extraction and analysis
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 growth were first pelleted at 400× g for 5 min and resuspended at a concentration of 1 × 107 cells/ml in fresh medium. The cells were then treated to induce apoptosis with either 1 μM staurosporine (STS, Sigma), 1 μg/ml camptothecin (Sigma) or irradiated with 100 mJ/cm2 of UV-B light using a calibrated light source (UVP, Upland, CA.). To induce necrosis, cells were treated with ethanol at a final concentration of 10% or cells were heated to 56 °C for 20 min. After these treatments, cells were cultured for an additional 18–24 h and then centrifuged at 400× g for 5 min to remove cells. Supernatant was removed to a fresh tube and recentrifuged again at 400× g. To prepare MPs for nucleic acid extraction, the supernatant following centrifugation was passed through a filter with a 1.2 μm pore size (Pall, Cornwall U.K.) to remove any remaining large cell debris. The resulting filtrate was centrifuged for 20 min at 35,000 rpm in a Beckman L7 centrifuge with a SW41 rotor (Beckman Coulter, Fullerton CA). The supernatant was decanted and the pellet resuspended in Dulbecco's PBS (Invitrogen) and
RNA for PCR and gel electrophoresis was extracted using an RNeasy Minikit (Qiagen, Valencia, CA). DNA for laddering analysis was extracted with an Apoptotic DNA Laddering Kit (Roche, Basel Switzerland). DNA electrophoresis was performed using a 1% agarose gel in TBE and stained with ethidium bromide. RNA was examined for size by 1.2% formaldehyde-agarose gel electrophoresis or, in the case of miRNA, electrophoresis through a denaturing 15% polyacrylamide/8 M urea gel. RNA was stained with ethidium bromide. Low molecular weight RNA was prepared using a Mirvana miRNA Isolation Kit (Ambion, Ausin, TX). The total amount of DNA and RNA in the microparticle preparations was determined spectrophotometrically. RT-PCR was performed with kits purchased from Qiagen following protocols supplied by the manufacturer using the following primers synthesized by IDT Technologies (Coralville IA): TCG GCA TGG GTC AGA AGG ATT CC, GTA CAT GGC TGG GGT GTT GAA GG for β-actin and GAA GTT GAA GGT CGG AGT CAA CG, CTT CTC ATG GTT CAC ACC CAT GAC G; GAPDH. Images of gels stained with ethidium bromide were photographed with a digital imaging system from Alpha Innotech (San Leandro, CA).
Results For these studies, we have used the Jurkat T cell and HL-60 promyelocytic cell lines as models because they are well
762
E XP E RI ME N TA L C E L L R E S EA RC H 315 ( 20 0 9) 76 0 –76 8
Fig. 1 – The production of microparticles by apoptotic and necrotic cells. Jurkat and HL-60 cells were treated to induce apoptosis (1 μm staurosporine, 100 mJ/cm2 UV-B, or 10 μg/ml camptothecin) or necrosis (10% ethanol or heated to 56 °C). After 24 h of incubation, MP numbers (A, B) and cell death (C, D) were determined by flow cytometry and PI staining. Results presented are the means and standard deviations of three separate experiments. Values differing statistically from controls (p < .05) are indicated.
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 differential centrifugation and characterized by flow cytometry using forward and side scatter to identify subcellular particles. The viability of cells in the cultures was measured by propidium iodide (PI) staining to assess the relationship between the extent of cell death and particle release. Fig. 1 (panels A and B) presents the number of MPs produced by the two cell lines using three inducers of apoptosis (staurosporine, 100 mJ/cm2 UV-B irradiation and camptothecin) and two inducers of necrosis (10% ethanol and heat); panels C and D present the percentage of PI-positive cells from the same cultures at the time that particles were prepared. As these results indicate, for Jurkat cells, treatment with STS caused the greatest release of particles. Cells treated with UV-B released much fewer particles while the number released after camptothecin was not statistically significant. In contrast to findings with Jurkat cells, particle release by HL-60 cells was similar following induction of apoptosis by STS, UV-B or camptothecin. For HL-60 cells, the extent of cell death was similar with all three inducers of apoptosis while, for Jurkat cells, STS was more effective than either UV-B or camptothecin. At the 24 h time point, essentially all Jurkat or HL-60 cells treated to induce necrosis were PI positive, but the number of particles released by either line
with necrosis was much lower than those observed with apoptosis. Together, these results indicate that MP release occurs prominently during apoptosis, although the extent of release may vary depending on cell type and inducing agent. To characterize MP nucleic acids, we first used staining with propidium iodide which can bind both DNA and RNA. We characterized unfixed and fixed MPs from STS-treated HL-60 and Jurkat cells and treated preparations with DNase and RNase to distinguish between the nucleic acids. Since PI binds DNA as well as RNA, enzyme treatment is needed to determine the type of nucleic acid present. As results in Fig. 2 indicate, PI can bind to unfixed MPs, with Jurkat MPs showing more binding than HL-60 MPs. For both cell lines, treatment with DNase and RNase decreased PI binding to MPs although, for Jurkat cells, RNase treatment caused a greater reduction in PI binding than DNase. In contrast to unfixed cells, fixed and permeabilized preparations showed greater binding of PI. While both RNase and DNase treatment diminished binding, DNase treatment had a greater effect. Together, results of these experiments suggest that some of the particle nucleic acid may be accessible on the outside of these structures; alternatively, some particles may be permeable to PI although these possibilities are not mutually exclusive. In these experiments, particles were isolated from cultures after 24 h, a time point at which cells are in late apoptosis, a stage when cells lose membrane integrity and PI can cross the plasma membrane to bind nucleic acids. As such, PI may be able to enter MPs just as it
E XP E RI ME N TA L C E L L R E S EA RC H 315 ( 20 0 9) 76 0 –76 8
Fig. 2 – Flow cytometry analysis of staining of microparticles with propidium iodide. Microparticles purified from the media of Jurkat or HL-60 cells treated overnight with 1 μM STS were incubated with 10 μM propidium iodide without fixation (A, B, E, F) or after fixation with paraformaldehyde and permeabilization with saponin (C, D, G, H). To assess the contribution of DNA and RNA to the binding of PI, microparticles were treated with 100 U/ml DNase (A, E, C, G) or 200 U/ml RNase (B, F, D, H) at 37 °C for 30 min.
763
764
E XP E RI ME N TA L C E L L R E S EA RC H 315 ( 20 0 9) 76 0 –76 8
Fig. 3 – Flow cytometry analysis of anti-DNA antibody binding to microparticles. MPs were purified from the media of Jurkat and HL-60 cells treated overnight with 1 μM STS. These particles, either untreated (A, C) or fixed and permeabilized (B, D) were incubated with an anti-dsDNA monoclonal antibody or an isotype control followed by Fab'2 goat anti-mouse IgG PE conjugate. To confirm the specificity of the binding, MPs were preincubated with 100 U/ml DNase for 30 min at 37 °C. FACS plots are presented for MPs incubated with the isotype control and conjugate, anti-DNA and conjugate and anti-DNA and conjugate following treatment with DNase.
can enter a late apoptotic cell. Of note, by combining both treatments, PI binding could almost be completely reduced (data not shown). These results indicate that MPs contain DNA and RNA in an accessible form. To verify this observation by an alternative approach, we used a monoclonal anti-dsDNA antibody to stain both fixed and unfixed MPs with and without treatment with DNase. The results of these experiments are presented in Fig. 3. These data indicate that both unfixed and fixed and permeabilized MPs stain with a monoclonal antibody to dsDNA, with the extent of binding greater with fixed MPs. These results are consistent with the display of DNA in an accessible form as well as an inaccessible compartment that can be revealed after fixation and permeabilization. The nucleic acids from STS-treated Jurkat MPs were next analyzed by molecular approaches. As shown in Table 1, apoptotic cells showed significant loss of DNA and RNA compared to untreated cells, although, for isolated MPs, the amount of RNA as a percentage of the total was greater than that of DNA. Fig. 4 shows an analysis of DNA purified from STS-treated Jurkat particles. As the data indicate, the DNA in the particle, like that in the apoptotic cell, is cleaved, and shows laddering.
We next assessed the size of total RNA present in Jurkat and HL-60 MPs in comparison to that of intact and apoptotic cells. As data in Fig. 5 indicate, MPs contain bands consistent with 28s and 18s ribosomal RNA as well as lower size molecular species. Furthermore, there are abundant bands that are consistent in size with 28s degradation products previously reported for apoptotic cells [29–34]. Nevertheless, despite similarity in the RNA from apoptotic cells and MPs, differences were observed in the species present
Table 1
RNA DNA
Apoptotic cells
MPs
39.88 ± 2.54% 30.66 ± 4.64%
0.65 ± .36% 0.05 ± .01%
Purified RNA and DNA from the cellular and microparticle fractions of cultures of Jurkat cells treated with 1 μm STS for 24 h. Values are expressed as a percentage of the amount purified from the same number of untreated Jurkat cells. Means and standard deviations are from three separate experiments.
E XP E RI ME N TA L C E L L R E S EA RC H 315 ( 20 0 9) 76 0 –76 8
765
Fig. 6 – Messenger RNA content of microparticles. Using primers for β-actin (A–C) and GAPDH (D–F), RT-PCR was performed on RNA isolated from untreated Jurkat cells (A, D), Jurkat cells treated with 1 μM staurosporine for 24 h (B, E) and microparticles derived from treated Jurkat cells (C, F).
Fig. 4 – Molecular analysis of microparticle DNA. DNA was extracted from cells or microparticles and analyzed on a 1% agarose gel in TBE buffer. DNA was visualized by staining with ethidium bromide. The gel indicates DNA from untreated Jurkat cells (lane B); Jurkat cells treated overnight with 1 μM staurosporine (lane C); microparticles isolated from the supernatant of Jurkat cells cultured overnight with 1 μM staurosporine (lane D). Lanes A and E are molecular weight markers.
depending on the inducing agent and the cell type. These findings suggest that, while MPs commonly contain species of degraded RNA, the process of RNA degradation and translocation into particles may vary depending on cell type and apoptosis pathway.
The nature of other RNA species was then investigated. While the gel analysis showed primarily ribosomal RNA and its degradation products, other investigators have shown the presence of mRNA in the MP fraction [6,7]. We therefore used PCR to determine whether the Jurkat MPs also contained mRNA; in these experiments, we tested for GAPDH and β-actin. Fig. 6 presents these data and shows that both mRNA can be amplified, indicating molecules that are sufficiently intact to allow amplification using the primers tested. Finally, we investigated the presence of low molecular weight species in the size range of microRNA. Fig. 7 presents gel analysis of low molecular weight RNA from untreated Jurkat cells, apoptotic cells and MPs. As these findings show, MPs as well as cells have species consistent in size with miRNA, indicating the possible presence in particles of RNA with regulatory as well as coding properties.
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
Fig. 5 – Gel electrophoresis analysis of microparticle RNA isolated from apoptotic Jurkat and HL-60 cells. 1.2% FA gel electrophoresis analysis was performed with RNA isolated from untreated cells, cells induced to undergo apoptosis for 24 h by various inducers of apoptosis and from MPs released by apoptotic cells. 1.5 μg of purified RNA was loaded in each well. (A) molecular weight markers; (B) untreated control cells; (C) STS-treated cells; (D) MPs from STS- treated cells; (E) UV-B treated cells; (F) MPs from UV-B treated cells; (G) camptothecin-treated cells; and (H) MPs from camptothecin-treated cells.
766
E XP E RI ME N TA L C E L L R E S EA RC H 315 ( 20 0 9) 76 0 –76 8
Fig. 7 – Analysis of low molecular weight RNA species. In these experiments, RNA was extracted from untreated Jurkat cells (A, B), Jurkat cells treated with 1 μM STS for 24 h (C, D) and microparticles isolated from the media of STS-treated Jurkats (E, F). RNA was then analyzed on a 15% Urea-TBE acrylamide gel electrophoresis as described in Materials and methods. Rows A, C, and E are the high molecular weight fractions. Rows B, D and F are the low molecular weight fractions. Row G is a molecular weight standard. which are consistent in size with degraded ribosomal RNA or possibly microRNA. While the MPs contain intact ribosomal RNA, smaller species, discrete in size, are also present, occurring in amounts greater than those found in the apoptotic cells. These bands are consistent in size with 28s ribosomal RNA degradation products [29–34]. While there were some differences in the extent and pattern of RNA degradation in the Jurkat and HL-60 cells and MPs, these results nevertheless indicate the incorporation of diverse nuclear and cytoplasmic components into particles as the cell breaks down during apoptotic cell death. As shown in studies of Halicka et al. [15], DNA and RNA appear to leave the cell separately with RNA appearing predominantly in a particulate form while DNA appears in the extracellular space in both a soluble and particulate form. Our results are consistent with these observations. Thus, although MPs contain both DNA and RNA, the percentage of total cellular RNA in the MP fraction is greater than the percentage of total cellular DNA. For RNA, the environment of particles may be crucial for its presence and detection because of the sensitivity of RNA to nuclease digestion. In particles, however, RNA appears to be at least partially protected from nuclease digestion and circulates in the blood as a long-lived remnant of cell damage that can be measured by molecular and genomic approaches. Indeed, levels of RNA as well as DNA are elevated in the blood in many clinical situations characterized by accentuated levels of cell death [6,8]. In this regard, because of the presence of both DNase and RNase in the blood, the nucleic acids inside the particle may be subject to degradation and therefore may decline over time. Since the particle membrane may become permeable because of events during apoptosis, it may allow entrance of blood nucleases which can affect the content of both DNA and RNA. Of the RNA in MPs, ribosomal RNA species are the most abundant and are readily detected by gel analysis. While both 18s and 28s RNA bands appear prominently in the MPs, a series of degradation products is also evident. Several studies have demon-
strated the cleavage of 28s RNA during apoptosis [29–34]. Indeed, preliminary studies by Northern blot analysis indicate that at least some of the observed bands correspond to 28s degradation products. This degradation process may not be linked to cleavage of DNA, although degradation of both types of nucleic acid may represent a mechanism to limit the replicative or transcriptional potential of cells [29–37]. In the context of infection, a strong driving force for apoptosis, degradation of cellular nucleic acids may limit the production of viruses, for example. While the enzymes responsible for DNA degradation have been well defined, the basis of RNA degradation has received less attention. A comparison of the RNA in MPs with that in the growing or apoptotic cells indicates an enrichment in the apparent degradation products of 28s RNA. Thus, while these products appear prominently on gels of the MP RNA, they are present in relatively lower amounts in the cells themselves. These findings are consistent with previous observations indicating that RNA degradation in the S49neo cell line undergoing apoptosis occurs primarily in shrunken cells in contrast to those cells that maintain their volume [32]. It is of interest that, in the prior study of King et al., the shrunken cells contained only the degraded 28s RNA fragment. The difference among these studies may reflect the cell lines used and the analysis in our study of MPs as opposed to shrunken cells. Nevertheless, both studies indicate clearly that degradation products of rRNA may occur preferentially (or primarily) in shrunken cells (apoptotic bodies) or MPs. Two mechanisms could account for these findings. The first posits a selective incorporation of the degradation products into the MPs. This selectivity may relate to the timing of events during apoptosis and the physical disposition of the degradation products. Thus, the amount of degradation product should increase as apoptosis occurs. If this degradation occurs near the site of MP release, the degradation products may have a greater content in MPs than other RNA species. A second mechanism for an increased content of RNA cleavage products concerns degradation within the particle itself. If the enzymes involved in RNA cleavage accumulate in particles along with RNA, degradation could proceed after the release of MPs from cells. Furthermore, degradation inside the particle could also result from the presence of RNase in the serum that enters particles since, as noted above, particles may become permeable because of membrane changes during apoptosis. In these experiments, we have isolated MPs late in culture to obtain adequate amounts of material for study. At the time of isolation, most cells have undergone apoptosis and are in a stage of late apoptosis or secondary necrosis, when the biochemical events leading to death have already occurred. Since the time courses of both intra- and extracellular DNA degradation are both unknown, we cannot distinguish between the mechanisms responsible for RNA breakdown. Nevertheless, the appearance of degradation products in MPs may be important in identifying MPs as a product of apoptotic cells as opposed to activated cells, the other source of MPs. In addition to rRNA, MPs contain mRNA in a form that can be amplified by genomic techniques and, indeed, may be analyzable by performance of microarrays. In the setting of malignancy, MP mRNA can provide a biomarker in the blood for the presence of particular tumor types, especially for differentiated cell types that may have a uniquely expressed message (e.g., tyrosinase). The presence of such an mRNA in the blood can allow screening for a tumor as well as longitudinal assessment for progression of a tumor or response to therapy [7–14]. In the setting of pregnancy,
E XP E RI ME N TA L C E L L R E S EA RC H 315 ( 20 0 9) 76 0 –76 8
extracellular mRNA provides a source of material for assessing fetal gene expression [38,39], although the conclusion about transcriptional events would differ depending upon whether the MPs were released from apoptotic or necrotic cells. As in the case of rRNA, the mechanisms for the incorporation of mRNA into MPs are unknown. Limited data suggest that the mRNA in the MPs may differ from that in the total cell. Such differences could occur because of differential synthesis or degradation of mRNA during apoptosis as well as regional differences in the localization of mRNA in the cell [40–43]. In this regard, models for the functional properties of MPs have raised the possibility that these structures can transfer genetic information from one cell to another and that the incorporated RNA functions after uptake by another cell [25–28]. As such, MPs can trigger responses in another cell type because of the transferred RNA. A similar mechanism has been observed for exosomes which represent a type of particle derived from the multivesicular body. In addition to internalized nucleic acids, MPs display both RNA and DNA in an accessible form. The presence of this material can be demonstrated by staining of unfixed MP preparations with PI, using treatment with RNase or DNase to show specificity. In these experiments, MPs were derived from apoptotic cells which, at the time of particle preparation, were PI positive. It is therefore not surprising that the MPs are also PI positive although binding of PI could result from an interaction at the surface or access to an internalized source of nucleic acid. In this scenario, differences in the extent of nucleic acid degradation by nucleases may reflect size differences between RNase and DNase and their ability to reach internal sources. This mechanism may also occur with serum nucleases as well as those added exogenously in experimental situations. While unfixed cells could bind PI, the extent of binding was increased in cells that had been fixed and permeabilized. This finding suggests that MPs released from cells may not all be permeable to PI or that fixation and permeabilization may allow greater access of the dye to nucleic acid. In the case of DNA, the binding of anti-DNA antibodies provides other evidence for the presence of DNA on the MP surface or otherwise readily accessible site. In this study, we did not have available a specific anti-RNA antibody although we could show that antibodies to RNA-containing snRNP antigens also bound to the MP surface (preliminary observations). The relationship between the RNA in the snRNPs with RNA that we detected by PI staining is speculative although the results are consistent with interior and exterior nucleic acids. Since DNA and RNA are present on the surface of MPs, they therefore are capable of binding anti-nuclear antibodies in the setting of diseases such as systemic lupus erythematosus [44]. The consequences of such binding are many and include activation of the complement system, deposition in the tissue, and altered clearance or phagocytosis. In addition, the presence of antibody could modify the cellular responses to MPs in a manner analogous to the effects of antibody to nuclear antigens released by dead and dying cells. As shown in studies on SLE immune complexes, antibodies to nuclear components can dramatically enhance the response to such material by effects on Fc receptors [21–23]. While MPs are much larger than ICs, they nevertheless may undergo functional changes when antibody is bound to their surface. Studies are therefore in progress to investigate further the nature of the nucleic acids in MPs and their functional properties both alone and in the presence of anti-nuclear antibodies.
767
Acknowledgments This work was supported by a VA Merit Review grant and a grant from the Lupus Foundation of America.
REFERENCES
[1] M.J. VanWijk, E. VanBavel, A. Sturk, R. Nieuwland, Microparticles in cardiovascular diseases, Cardiovasc. Res. 59 (2003) 277–287. [2] L.L. Horstman, W. Jy, J.J. Jimenez, C. Bidot, Y.S. Ahn, New horizons in the analysis of circulating cell-derived microparticles, Keio J. Med. 53 (2004) 210–230. [3] J.H. Distler, D.S. Pisetsky, L.C. Huber, J.R. Kalden, S. Gay, O. Distler, Microparticles as regulators of inflammation: novel players of cellular crosstalk in the rheumatic diseases, Arthritis Rheum. 52 (2005) 3337–3348. [4] J.D. Lane, V.J. Allan, P.G. Woodman, Active relocation of chromatin and endoplasmic reticulum into blebs in late apoptotic cells, J. Cell. Sci. 118 (2005) 4059–4071. [5] M. Schiller, I. Bekeredjian-Ding, P. Heyder, N. Blank, A.D. Ho, H.M. Lorenz, Autoantigens are translocated into small apoptotic bodies during early stages of apoptosis, Cell Death Differ. 15 (2008) 183–191. [6] D.O. Hasselmann, G. Rappl, W. Tilgen, U. Reinhold, Extracellular tyrosinase mRNA within apoptotic bodies is protected from degradation in human serum, Clin. Chem. 47 (2001) 1488–1489. [7] E.K. Ng, N.B. Tsui, N.Y. Lam, R.W. Chiu, S.C. Yu, S.C. Wong, E.S. Lo, T.H. Rainer, P.J. Johnson, Y.M. Lo, Presence of filterable and nonfilterable mRNA in the plasma of cancer patients and healthy individuals, Clin. Chem. 48 (2002) 1212–1217. [8] T. El-Hefnawy, S. Raja, L. Kelly, W.L. Bigbee, J.M. Kirkwood, J.D. Luketich, T.E. Godfrey, Characterization of amplifiable, circulating RNA in plasma and its potential as a tool for cancer diagnostics, Clin. Chem. 50 (2004) 564–573. [9] M.S. Kopreski, F.A. Benko, L.W. Kwak, C.D. Gocke, Detection of tumor messenger RNA in the serum of patients with malignant melanoma, Clin. Cancer Res. 5 (1999) 1961–1965. [10] K.W. Lo, Y.M. Lo, S.F. Leung, Y.S. Tsang, L.Y. Chan, P.J. Johnson, N.M. Hjelm, J.C. Lee, D.P. Huang, Analysis of cell-free Epstein–Barr virus associated RNA in the plasma of patients with nasopharyngeal carcinoma, Clin. Chem. 45 (1999) 1292–1294. [11] G. Rappl, D.O. Hasselmann, M. Rossler, S. Ugurel, W. Tilgen, U. Reinhold, Detection of tumor-associated circulating mRNA in patients with disseminated malignant melanoma, Ann. N.Y. Acad. Sci. 945 (2001) 189–191. [12] F. Dasi, S. Lledo, E. Garcia-Granero, R. Ripoll, M. Marugan, M. Tormo, J. Garcia-Conde, S.F. Alino, Real-time quantification in plasma of human telomerase reverse transcriptase (hTERT) mRNA: a simple blood test to monitor disease in cancer patients, Lab. Invest. 81 (2001) 767–769. [13] S.C. Wong, S.F. Lo, M.T. Cheung, K.O. Ng, C.W. Tse, B.S. Lai, K.C. Lee, Y.M. Lo, Quantification of plasma beta-catenin mRNA in colorectal cancer and adenoma patients, Clin. Cancer Res. 10 (2004) 1613–1617. [14] E. Sueoka, N. Sueoka, K. Iwanaga, A. Sato, K. Suga, S. Hayashi, K. Nagasawa, K. Nakachi, Detection of plasma hnRNP B1 mRNA, a new cancer biomarker, in lung cancer patients by quantitative real-time polymerase chain reaction, Lung Cancer 48 (2005) 77–83. [15] H.D. Halicka, E. Bedner, Z. Darzynkiewicz, Segregation of RNA and separate packaging of DNA and RNA in apoptotic bodies during apoptosis, Exp. Cell Res. 260 (2000) 248–256. [16] H. Wagner, The immunobiology of the TLR9 subfamily, Trends Immunol. 25 (2004) 381–386. [17] M. Schroder, A.G. Bowie, TLR3 in antiviral immunity: key player or bystander? Trends Immunol. 26 (2005) 462–468.
768
E XP E RI ME N TA L C E L L R E S EA RC H 315 ( 20 0 9) 76 0 –76 8
[18] E. Meylan, J. Tschopp, Toll-like receptors and RNA helicases: two parallel ways to trigger antiviral responses, Mol. Cell 22 (2006) 561–569. [19] E. Meylan, J. Tschopp, M. Karin, Intracellular pattern recognition receptors in the host response, Nature 442 (2006) 39–44. [20] G. Trinchieri, A. Sher, Cooperation of Toll-like receptor signals in innate immune defence, Nat. Rev. Immunol. 7 (2007) 179–190. [21] T. Lovgren, M.L. Eloranta, U. Bave, G.V. Alm, L. Ronnblom, Induction of interferon-alpha production in plasmacytoid dendritic cells by immune complexes containing nucleic acid released by necrotic or late apoptotic cells and lupus IgG, Arthritis Rheum. 50 (2004) 1861–1872. [22] T.K. Means, E. Latz, F. Hayashi, M.R. Murali, D.T. Golenbock, A.D. Luster, Human lupus autoantibody–DNA complexes activate DCs through cooperation of CD32 and TLR9, J. Clin. Invest. 115 (2005) 407–417. [23] J. Tian, A.M. Avalos, S.Y. Mao, B. Chen, K. Senthil, H. Wu, P. Parroche, S. Drabic, D. Golenbock, C. Sirois, J. Hua, L.L. An, L. Audoly, G. La Rosa, A. Bierhaus, P. Naworth, A. Marshak-Rothstein, M.K. Crow, K.A. Fitzgerald, E. Latz, P.A. Kiener, A.J. Coyle, Toll-like receptor 9-dependent activation by DNA-containing immune complexes is mediated by HMGB1 and RAGE, Nat. Immunol. 8 (2007) 487–496. [24] L.E. Graves, E.V. Ariztia, J.R. Navari, H.J. Matzel, M.S. Stack, D.A. Fishman, Proinvasive properties of ovarian cancer ascites-derived membrane vesicles, Cancer Res. 64 (2004) 7045–7049. [25] J. Ratajczak, M. Wysoczynski, F. Hayek, A. Janowska-Wieczorek, M.Z. Ratajczak, Membrane-derived microvesicles: important and underappreciated mediators of cell-to-cell communication, Leukemia 20 (2006) 1487–1495. [26] M. Baj-Krzyworzeka, R. Szatanek, K. Weglarczyk, J. Baran, B. Urbanowicz, P. Branski, M.Z. Ratajczak, M. Zembala, Tumour-derived microvesicles carry several surface determinants and mRNA of tumour cells and transfer some of these determinants to monocytes, Cancer Immunol. Immunother. 55 (2006) 808–818. [27] M. Baj-Krzyworzeka, R. Szatanek, K. Weglarczyk, J. Baran, M. Zembala, Tumour-derived microvesicles modulate biological activity of human monocytes, Immunol. Lett. 113 (2007) 76–82. [28] H. Valadi, K. Ekstrom, A. Bossios, M. Sjostrand, J.J. Lee, J.O. Lotvall, Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells, Nat. Cell Biol. 9 (2007) 654–659. [29] G. Houge, S.O. Doskeland, R. Boe, M. Lanotte, Selective cleavage of 28S rRNA variable regions V3 and V13 in myeloid leukemia cell apoptosis, FEBS Lett. 315 (1993) 16–20. [30] G. Houge, B. Robaye, T.S. Eikhom, J. Golstein, G. Mellgren, B.T. Gjertsen, M. Lanotte, S.O. Doskeland, Fine mapping of 28S rRNA sites specifically cleaved in cells undergoing apoptosis, Mol. Cell. Biol. 15 (1995) 2051–2062.
[31] A. Samali, B. Gilje, S.O. Doskeland, T.G. Cotter, G. Houge, The ability to cleave 28S ribosomal RNA during apoptosis is a cell-type dependent trait unrelated to DNA fragmentation, Cell Death Differ. 4 (1997) 289–293. [32] K.L. King, C.M. Jewell, C.D. Bortner, J.A. Cidlowski, 28S ribosome degradation in lymphoid cell apoptosis: evidence for caspase and Bcl-2-dependent and -independent pathways, Cell Death Differ. 7 (2000) 994–1001. [33] W.G. Degen, G.J. Pruijn, J.M. Raats, W.J. van Venrooij, Caspase-dependent cleavage of nucleic acids, Cell Death Differ. 7 (2000) 616–627. [34] T.X. Hoat, H. Nakayashiki, Y. Tosa, S. Mayama, Specific cleavage of ribosomal RNA and mRNA during victorin-induced apoptotic cell death in oat, Plant J. 46 (2006) 922–933. [35] D.R. Crawford, R.J. Lauzon, Y. Wang, J.E. Mazurkiewicz, G.P. Schools, K.J. Davies, 16S mitochondrial ribosomal RNA degradation is associated with apoptosis, Free Radic. Biol. Med. 22 (1997) 1295–1300. [36] K. Samejima, W.C. Earnshaw, Trashing the genome: the role of nucleases during apoptosis, Nat. Rev. Mol. Cell Biol. 6 (2005) 677–688. [37] P.T. Sobol, K.L. Mossman, ICP0 prevents RNase L-independent rRNA cleavage in herpes simplex virus type 1-infected cells, J. Virol. 80 (2006) 218–225. [38] E.K. Ng, N.B. Tsui, T.K. Lau, T.N. Leung, R.W. Chiu, N.S. Panesar, L.C. Lit, K.W. Chan, Y.M. Lo, mRNA of placental origin is readily detectable in maternal plasma, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 4748–4753. [39] P.B. Larrabee, K.L. Johnson, C. Lai, J. Ordovas, J.M. Cowan, U. Tantravahi, D.W. Bianchi, Global gene expression analysis of the living human fetus using cell-free messenger RNA in amniotic fluid, JAMA 293 (2005) 836–842. [40] J. Fan, X. Yang, W. Wang, W.H. Wood III, K.G. Becker, M. Gorospe, Global analysis of stress-regulated mRNA turnover by using cDNA arrays, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 10611–10616. [41] D.J. Taxman, J.P. MacKeigan, C. Clements, D.T. Bergstralh, J.P. Ting, Transcriptional profiling of targets for combination therapy of lung carcinoma with paclitaxel and mitogen-activated protein/ extracellular signal-regulated kinase kinase inhibitor, Cancer Res. 63 (2003) 5095–5104. [42] X. Lu, W.E. Burgan, M.A. Cerra, E.Y. Chuang, M.H. Tsai, P.J. Tofilon, K. Camphausen, Transcriptional signature of flavopiridol-induced tumor cell death, Mol. Cancer Ther. 3 (2004) 861–872. [43] X. Lu, L. de la Pena, C. Barker, K. Camphausen, P.J. Tofilon, Radiation-induced changes in gene expression involve recruitment of existing messenger RNAs to and away from polysomes, Cancer Res. 66 (2006) 1052–1061. [44] M. Radic, T. Marion, M. Monestier, Nucleosomes are exposed at the cell surface in apoptosis, J. Immunol. 172 (2004) 6692–6700.
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / y e x c r
Research Article
The content of DNA and RNA in microparticles released by Jurkat and HL-60 cells undergoing in vitro apoptosis Charles F. Reich III, David S. Pisetsky⁎ Medical Research Service, 151G Durham VAMC, 508 Fulton Street, Durham, NC 27705, USA Division of Rheumatology and Immunology, Duke University Medical Center, Durham, NC 27705, USA
A R T I C L E I N F O R M AT I O N
AB ST R AC T
Article Chronology:
Microparticles are small membrane-bound vesicles that are released from apoptotic cells during
Received 3 July 2008
blebbing. These particles contain DNA and RNA and display important functional activities,
Revised version received
including immune system activation. Furthermore, nucleic acids inside the particle can be analyzed
9 December 2008
as biomarkers in a variety of disease states. To elucidate the nature of microparticle nucleic acids,
Accepted 11 December 2008
DNA and RNA released in microparticles from the Jurkat T and HL-60 promyelocytic cell lines
Available online 30 December 2008
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
Keywords:
particles, DNA showed laddering by gel electrophoresis and was present in a form that allowed
Microparticles
direct binding by a monoclonal anti-DNA antibody, suggesting antigen accessibility even without
Apoptosis
fixation. Analysis of RNA by gel electrophoresis showed intact 18s and 28s ribosomal RNA bands,
Necrosis
although lower molecular bands consistent with 28s ribosomal RNA degradation products were
DNA
also present. Particles also contained messenger RNA as shown by RT-PCR amplification of
RNA
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. © 2008 Elsevier Inc. All rights reserved.
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 [1–3]. ⁎ Corresponding author. E-mail address: [email protected] (D.S. Pisetsky). 0014-4827/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2008.12.014
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 [8–14]. In contrast to RNA, DNA can appear in both a particulate and non-particulate form [15].
E XP E RI ME N TA L C E L L R E S EA RC H 315 ( 20 0 9) 76 0 –76 8
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 [16–20]. 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 [21–32]. MPs can also modulate the host response to tumors, an effect that may also relate to their content of nucleic acids [24–28]. 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.
761
recentrifuged. This wash step was repeated three times. Following the final wash, the pellet was either frozen at −70 °C for the later extraction of DNA and RNA or resuspended in PBS to one tenth the volume of the original culture supernatant for flow cytometry.
Flow cytometry
Materials and methods
The measurement of MP numbers and nucleic acid content was performed with a FACScan flow cytometer using Cellquest software (BD Biosciences, San Jose CA). All data were accumulated in logarithmic mode. Noise was excluded by setting a side scatter threshold with sample buffer alone. Analysis was done using FLOWJO software (Tree Star, Ashland OR). To determine microparticle numbers, the sample flow rate was calibrated by weighing sample tubes before and after a timed run and using this measurement to calculate relative volume. For statistical analysis of MP number and PI-positive cell numbers, we used a one-tailed T-test, for unpaired samples and equal variance. For nucleic acid analysis, MPs, with or without previous fixation and permeabilization, were stained with propidium iodide (PI) (Sigma) at 10 μg/ml in PBS or with the HYB331-01 monoclonal anti-double stranded DNA antibody (Abcam, Cambridge, MA) or an isotype control (Sigma) followed by Fab'2 goat anti-mouse IgG PE (Sigma). In some experiments, MPs were fixed with paraformaldehyde and permeabilized with saponin. (BD Cytofix/Cytoperm, BD Biosciences, San Diego). After 20 min fixation, MPs were centrifuged at 16,000× g for 30 min followed by 2 washes with permeabilization buffer. For nuclease treatments, prior to PI staining, MPs were incubated for 30 min at 37 °C with 200 U/ml DNase-free RNase (Sigma) or 100 U/ml RNase-free DNase (Invitrogen) in permeabilization buffer supplemented with additional calcium and magnesium.
Cell culture and microparticle preparation
Nucleic acid extraction and analysis
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 growth were first pelleted at 400× g for 5 min and resuspended at a concentration of 1 × 107 cells/ml in fresh medium. The cells were then treated to induce apoptosis with either 1 μM staurosporine (STS, Sigma), 1 μg/ml camptothecin (Sigma) or irradiated with 100 mJ/cm2 of UV-B light using a calibrated light source (UVP, Upland, CA.). To induce necrosis, cells were treated with ethanol at a final concentration of 10% or cells were heated to 56 °C for 20 min. After these treatments, cells were cultured for an additional 18–24 h and then centrifuged at 400× g for 5 min to remove cells. Supernatant was removed to a fresh tube and recentrifuged again at 400× g. To prepare MPs for nucleic acid extraction, the supernatant following centrifugation was passed through a filter with a 1.2 μm pore size (Pall, Cornwall U.K.) to remove any remaining large cell debris. The resulting filtrate was centrifuged for 20 min at 35,000 rpm in a Beckman L7 centrifuge with a SW41 rotor (Beckman Coulter, Fullerton CA). The supernatant was decanted and the pellet resuspended in Dulbecco's PBS (Invitrogen) and
RNA for PCR and gel electrophoresis was extracted using an RNeasy Minikit (Qiagen, Valencia, CA). DNA for laddering analysis was extracted with an Apoptotic DNA Laddering Kit (Roche, Basel Switzerland). DNA electrophoresis was performed using a 1% agarose gel in TBE and stained with ethidium bromide. RNA was examined for size by 1.2% formaldehyde-agarose gel electrophoresis or, in the case of miRNA, electrophoresis through a denaturing 15% polyacrylamide/8 M urea gel. RNA was stained with ethidium bromide. Low molecular weight RNA was prepared using a Mirvana miRNA Isolation Kit (Ambion, Ausin, TX). The total amount of DNA and RNA in the microparticle preparations was determined spectrophotometrically. RT-PCR was performed with kits purchased from Qiagen following protocols supplied by the manufacturer using the following primers synthesized by IDT Technologies (Coralville IA): TCG GCA TGG GTC AGA AGG ATT CC, GTA CAT GGC TGG GGT GTT GAA GG for β-actin and GAA GTT GAA GGT CGG AGT CAA CG, CTT CTC ATG GTT CAC ACC CAT GAC G; GAPDH. Images of gels stained with ethidium bromide were photographed with a digital imaging system from Alpha Innotech (San Leandro, CA).
Results For these studies, we have used the Jurkat T cell and HL-60 promyelocytic cell lines as models because they are well
762
E XP E RI ME N TA L C E L L R E S EA RC H 315 ( 20 0 9) 76 0 –76 8
Fig. 1 – The production of microparticles by apoptotic and necrotic cells. Jurkat and HL-60 cells were treated to induce apoptosis (1 μm staurosporine, 100 mJ/cm2 UV-B, or 10 μg/ml camptothecin) or necrosis (10% ethanol or heated to 56 °C). After 24 h of incubation, MP numbers (A, B) and cell death (C, D) were determined by flow cytometry and PI staining. Results presented are the means and standard deviations of three separate experiments. Values differing statistically from controls (p < .05) are indicated.
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 differential centrifugation and characterized by flow cytometry using forward and side scatter to identify subcellular particles. The viability of cells in the cultures was measured by propidium iodide (PI) staining to assess the relationship between the extent of cell death and particle release. Fig. 1 (panels A and B) presents the number of MPs produced by the two cell lines using three inducers of apoptosis (staurosporine, 100 mJ/cm2 UV-B irradiation and camptothecin) and two inducers of necrosis (10% ethanol and heat); panels C and D present the percentage of PI-positive cells from the same cultures at the time that particles were prepared. As these results indicate, for Jurkat cells, treatment with STS caused the greatest release of particles. Cells treated with UV-B released much fewer particles while the number released after camptothecin was not statistically significant. In contrast to findings with Jurkat cells, particle release by HL-60 cells was similar following induction of apoptosis by STS, UV-B or camptothecin. For HL-60 cells, the extent of cell death was similar with all three inducers of apoptosis while, for Jurkat cells, STS was more effective than either UV-B or camptothecin. At the 24 h time point, essentially all Jurkat or HL-60 cells treated to induce necrosis were PI positive, but the number of particles released by either line
with necrosis was much lower than those observed with apoptosis. Together, these results indicate that MP release occurs prominently during apoptosis, although the extent of release may vary depending on cell type and inducing agent. To characterize MP nucleic acids, we first used staining with propidium iodide which can bind both DNA and RNA. We characterized unfixed and fixed MPs from STS-treated HL-60 and Jurkat cells and treated preparations with DNase and RNase to distinguish between the nucleic acids. Since PI binds DNA as well as RNA, enzyme treatment is needed to determine the type of nucleic acid present. As results in Fig. 2 indicate, PI can bind to unfixed MPs, with Jurkat MPs showing more binding than HL-60 MPs. For both cell lines, treatment with DNase and RNase decreased PI binding to MPs although, for Jurkat cells, RNase treatment caused a greater reduction in PI binding than DNase. In contrast to unfixed cells, fixed and permeabilized preparations showed greater binding of PI. While both RNase and DNase treatment diminished binding, DNase treatment had a greater effect. Together, results of these experiments suggest that some of the particle nucleic acid may be accessible on the outside of these structures; alternatively, some particles may be permeable to PI although these possibilities are not mutually exclusive. In these experiments, particles were isolated from cultures after 24 h, a time point at which cells are in late apoptosis, a stage when cells lose membrane integrity and PI can cross the plasma membrane to bind nucleic acids. As such, PI may be able to enter MPs just as it
E XP E RI ME N TA L C E L L R E S EA RC H 315 ( 20 0 9) 76 0 –76 8
Fig. 2 – Flow cytometry analysis of staining of microparticles with propidium iodide. Microparticles purified from the media of Jurkat or HL-60 cells treated overnight with 1 μM STS were incubated with 10 μM propidium iodide without fixation (A, B, E, F) or after fixation with paraformaldehyde and permeabilization with saponin (C, D, G, H). To assess the contribution of DNA and RNA to the binding of PI, microparticles were treated with 100 U/ml DNase (A, E, C, G) or 200 U/ml RNase (B, F, D, H) at 37 °C for 30 min.
763
764
E XP E RI ME N TA L C E L L R E S EA RC H 315 ( 20 0 9) 76 0 –76 8
Fig. 3 – Flow cytometry analysis of anti-DNA antibody binding to microparticles. MPs were purified from the media of Jurkat and HL-60 cells treated overnight with 1 μM STS. These particles, either untreated (A, C) or fixed and permeabilized (B, D) were incubated with an anti-dsDNA monoclonal antibody or an isotype control followed by Fab'2 goat anti-mouse IgG PE conjugate. To confirm the specificity of the binding, MPs were preincubated with 100 U/ml DNase for 30 min at 37 °C. FACS plots are presented for MPs incubated with the isotype control and conjugate, anti-DNA and conjugate and anti-DNA and conjugate following treatment with DNase.
can enter a late apoptotic cell. Of note, by combining both treatments, PI binding could almost be completely reduced (data not shown). These results indicate that MPs contain DNA and RNA in an accessible form. To verify this observation by an alternative approach, we used a monoclonal anti-dsDNA antibody to stain both fixed and unfixed MPs with and without treatment with DNase. The results of these experiments are presented in Fig. 3. These data indicate that both unfixed and fixed and permeabilized MPs stain with a monoclonal antibody to dsDNA, with the extent of binding greater with fixed MPs. These results are consistent with the display of DNA in an accessible form as well as an inaccessible compartment that can be revealed after fixation and permeabilization. The nucleic acids from STS-treated Jurkat MPs were next analyzed by molecular approaches. As shown in Table 1, apoptotic cells showed significant loss of DNA and RNA compared to untreated cells, although, for isolated MPs, the amount of RNA as a percentage of the total was greater than that of DNA. Fig. 4 shows an analysis of DNA purified from STS-treated Jurkat particles. As the data indicate, the DNA in the particle, like that in the apoptotic cell, is cleaved, and shows laddering.
We next assessed the size of total RNA present in Jurkat and HL-60 MPs in comparison to that of intact and apoptotic cells. As data in Fig. 5 indicate, MPs contain bands consistent with 28s and 18s ribosomal RNA as well as lower size molecular species. Furthermore, there are abundant bands that are consistent in size with 28s degradation products previously reported for apoptotic cells [29–34]. Nevertheless, despite similarity in the RNA from apoptotic cells and MPs, differences were observed in the species present
Table 1
RNA DNA
Apoptotic cells
MPs
39.88 ± 2.54% 30.66 ± 4.64%
0.65 ± .36% 0.05 ± .01%
Purified RNA and DNA from the cellular and microparticle fractions of cultures of Jurkat cells treated with 1 μm STS for 24 h. Values are expressed as a percentage of the amount purified from the same number of untreated Jurkat cells. Means and standard deviations are from three separate experiments.
E XP E RI ME N TA L C E L L R E S EA RC H 315 ( 20 0 9) 76 0 –76 8
765
Fig. 6 – Messenger RNA content of microparticles. Using primers for β-actin (A–C) and GAPDH (D–F), RT-PCR was performed on RNA isolated from untreated Jurkat cells (A, D), Jurkat cells treated with 1 μM staurosporine for 24 h (B, E) and microparticles derived from treated Jurkat cells (C, F).
Fig. 4 – Molecular analysis of microparticle DNA. DNA was extracted from cells or microparticles and analyzed on a 1% agarose gel in TBE buffer. DNA was visualized by staining with ethidium bromide. The gel indicates DNA from untreated Jurkat cells (lane B); Jurkat cells treated overnight with 1 μM staurosporine (lane C); microparticles isolated from the supernatant of Jurkat cells cultured overnight with 1 μM staurosporine (lane D). Lanes A and E are molecular weight markers.
depending on the inducing agent and the cell type. These findings suggest that, while MPs commonly contain species of degraded RNA, the process of RNA degradation and translocation into particles may vary depending on cell type and apoptosis pathway.
The nature of other RNA species was then investigated. While the gel analysis showed primarily ribosomal RNA and its degradation products, other investigators have shown the presence of mRNA in the MP fraction [6,7]. We therefore used PCR to determine whether the Jurkat MPs also contained mRNA; in these experiments, we tested for GAPDH and β-actin. Fig. 6 presents these data and shows that both mRNA can be amplified, indicating molecules that are sufficiently intact to allow amplification using the primers tested. Finally, we investigated the presence of low molecular weight species in the size range of microRNA. Fig. 7 presents gel analysis of low molecular weight RNA from untreated Jurkat cells, apoptotic cells and MPs. As these findings show, MPs as well as cells have species consistent in size with miRNA, indicating the possible presence in particles of RNA with regulatory as well as coding properties.
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
Fig. 5 – Gel electrophoresis analysis of microparticle RNA isolated from apoptotic Jurkat and HL-60 cells. 1.2% FA gel electrophoresis analysis was performed with RNA isolated from untreated cells, cells induced to undergo apoptosis for 24 h by various inducers of apoptosis and from MPs released by apoptotic cells. 1.5 μg of purified RNA was loaded in each well. (A) molecular weight markers; (B) untreated control cells; (C) STS-treated cells; (D) MPs from STS- treated cells; (E) UV-B treated cells; (F) MPs from UV-B treated cells; (G) camptothecin-treated cells; and (H) MPs from camptothecin-treated cells.
766
E XP E RI ME N TA L C E L L R E S EA RC H 315 ( 20 0 9) 76 0 –76 8
Fig. 7 – Analysis of low molecular weight RNA species. In these experiments, RNA was extracted from untreated Jurkat cells (A, B), Jurkat cells treated with 1 μM STS for 24 h (C, D) and microparticles isolated from the media of STS-treated Jurkats (E, F). RNA was then analyzed on a 15% Urea-TBE acrylamide gel electrophoresis as described in Materials and methods. Rows A, C, and E are the high molecular weight fractions. Rows B, D and F are the low molecular weight fractions. Row G is a molecular weight standard. which are consistent in size with degraded ribosomal RNA or possibly microRNA. While the MPs contain intact ribosomal RNA, smaller species, discrete in size, are also present, occurring in amounts greater than those found in the apoptotic cells. These bands are consistent in size with 28s ribosomal RNA degradation products [29–34]. While there were some differences in the extent and pattern of RNA degradation in the Jurkat and HL-60 cells and MPs, these results nevertheless indicate the incorporation of diverse nuclear and cytoplasmic components into particles as the cell breaks down during apoptotic cell death. As shown in studies of Halicka et al. [15], DNA and RNA appear to leave the cell separately with RNA appearing predominantly in a particulate form while DNA appears in the extracellular space in both a soluble and particulate form. Our results are consistent with these observations. Thus, although MPs contain both DNA and RNA, the percentage of total cellular RNA in the MP fraction is greater than the percentage of total cellular DNA. For RNA, the environment of particles may be crucial for its presence and detection because of the sensitivity of RNA to nuclease digestion. In particles, however, RNA appears to be at least partially protected from nuclease digestion and circulates in the blood as a long-lived remnant of cell damage that can be measured by molecular and genomic approaches. Indeed, levels of RNA as well as DNA are elevated in the blood in many clinical situations characterized by accentuated levels of cell death [6,8]. In this regard, because of the presence of both DNase and RNase in the blood, the nucleic acids inside the particle may be subject to degradation and therefore may decline over time. Since the particle membrane may become permeable because of events during apoptosis, it may allow entrance of blood nucleases which can affect the content of both DNA and RNA. Of the RNA in MPs, ribosomal RNA species are the most abundant and are readily detected by gel analysis. While both 18s and 28s RNA bands appear prominently in the MPs, a series of degradation products is also evident. Several studies have demon-
strated the cleavage of 28s RNA during apoptosis [29–34]. Indeed, preliminary studies by Northern blot analysis indicate that at least some of the observed bands correspond to 28s degradation products. This degradation process may not be linked to cleavage of DNA, although degradation of both types of nucleic acid may represent a mechanism to limit the replicative or transcriptional potential of cells [29–37]. In the context of infection, a strong driving force for apoptosis, degradation of cellular nucleic acids may limit the production of viruses, for example. While the enzymes responsible for DNA degradation have been well defined, the basis of RNA degradation has received less attention. A comparison of the RNA in MPs with that in the growing or apoptotic cells indicates an enrichment in the apparent degradation products of 28s RNA. Thus, while these products appear prominently on gels of the MP RNA, they are present in relatively lower amounts in the cells themselves. These findings are consistent with previous observations indicating that RNA degradation in the S49neo cell line undergoing apoptosis occurs primarily in shrunken cells in contrast to those cells that maintain their volume [32]. It is of interest that, in the prior study of King et al., the shrunken cells contained only the degraded 28s RNA fragment. The difference among these studies may reflect the cell lines used and the analysis in our study of MPs as opposed to shrunken cells. Nevertheless, both studies indicate clearly that degradation products of rRNA may occur preferentially (or primarily) in shrunken cells (apoptotic bodies) or MPs. Two mechanisms could account for these findings. The first posits a selective incorporation of the degradation products into the MPs. This selectivity may relate to the timing of events during apoptosis and the physical disposition of the degradation products. Thus, the amount of degradation product should increase as apoptosis occurs. If this degradation occurs near the site of MP release, the degradation products may have a greater content in MPs than other RNA species. A second mechanism for an increased content of RNA cleavage products concerns degradation within the particle itself. If the enzymes involved in RNA cleavage accumulate in particles along with RNA, degradation could proceed after the release of MPs from cells. Furthermore, degradation inside the particle could also result from the presence of RNase in the serum that enters particles since, as noted above, particles may become permeable because of membrane changes during apoptosis. In these experiments, we have isolated MPs late in culture to obtain adequate amounts of material for study. At the time of isolation, most cells have undergone apoptosis and are in a stage of late apoptosis or secondary necrosis, when the biochemical events leading to death have already occurred. Since the time courses of both intra- and extracellular DNA degradation are both unknown, we cannot distinguish between the mechanisms responsible for RNA breakdown. Nevertheless, the appearance of degradation products in MPs may be important in identifying MPs as a product of apoptotic cells as opposed to activated cells, the other source of MPs. In addition to rRNA, MPs contain mRNA in a form that can be amplified by genomic techniques and, indeed, may be analyzable by performance of microarrays. In the setting of malignancy, MP mRNA can provide a biomarker in the blood for the presence of particular tumor types, especially for differentiated cell types that may have a uniquely expressed message (e.g., tyrosinase). The presence of such an mRNA in the blood can allow screening for a tumor as well as longitudinal assessment for progression of a tumor or response to therapy [7–14]. In the setting of pregnancy,
E XP E RI ME N TA L C E L L R E S EA RC H 315 ( 20 0 9) 76 0 –76 8
extracellular mRNA provides a source of material for assessing fetal gene expression [38,39], although the conclusion about transcriptional events would differ depending upon whether the MPs were released from apoptotic or necrotic cells. As in the case of rRNA, the mechanisms for the incorporation of mRNA into MPs are unknown. Limited data suggest that the mRNA in the MPs may differ from that in the total cell. Such differences could occur because of differential synthesis or degradation of mRNA during apoptosis as well as regional differences in the localization of mRNA in the cell [40–43]. In this regard, models for the functional properties of MPs have raised the possibility that these structures can transfer genetic information from one cell to another and that the incorporated RNA functions after uptake by another cell [25–28]. As such, MPs can trigger responses in another cell type because of the transferred RNA. A similar mechanism has been observed for exosomes which represent a type of particle derived from the multivesicular body. In addition to internalized nucleic acids, MPs display both RNA and DNA in an accessible form. The presence of this material can be demonstrated by staining of unfixed MP preparations with PI, using treatment with RNase or DNase to show specificity. In these experiments, MPs were derived from apoptotic cells which, at the time of particle preparation, were PI positive. It is therefore not surprising that the MPs are also PI positive although binding of PI could result from an interaction at the surface or access to an internalized source of nucleic acid. In this scenario, differences in the extent of nucleic acid degradation by nucleases may reflect size differences between RNase and DNase and their ability to reach internal sources. This mechanism may also occur with serum nucleases as well as those added exogenously in experimental situations. While unfixed cells could bind PI, the extent of binding was increased in cells that had been fixed and permeabilized. This finding suggests that MPs released from cells may not all be permeable to PI or that fixation and permeabilization may allow greater access of the dye to nucleic acid. In the case of DNA, the binding of anti-DNA antibodies provides other evidence for the presence of DNA on the MP surface or otherwise readily accessible site. In this study, we did not have available a specific anti-RNA antibody although we could show that antibodies to RNA-containing snRNP antigens also bound to the MP surface (preliminary observations). The relationship between the RNA in the snRNPs with RNA that we detected by PI staining is speculative although the results are consistent with interior and exterior nucleic acids. Since DNA and RNA are present on the surface of MPs, they therefore are capable of binding anti-nuclear antibodies in the setting of diseases such as systemic lupus erythematosus [44]. The consequences of such binding are many and include activation of the complement system, deposition in the tissue, and altered clearance or phagocytosis. In addition, the presence of antibody could modify the cellular responses to MPs in a manner analogous to the effects of antibody to nuclear antigens released by dead and dying cells. As shown in studies on SLE immune complexes, antibodies to nuclear components can dramatically enhance the response to such material by effects on Fc receptors [21–23]. While MPs are much larger than ICs, they nevertheless may undergo functional changes when antibody is bound to their surface. Studies are therefore in progress to investigate further the nature of the nucleic acids in MPs and their functional properties both alone and in the presence of anti-nuclear antibodies.
767
Acknowledgments This work was supported by a VA Merit Review grant and a grant from the Lupus Foundation of America.
REFERENCES
[1] M.J. VanWijk, E. VanBavel, A. Sturk, R. Nieuwland, Microparticles in cardiovascular diseases, Cardiovasc. Res. 59 (2003) 277–287. [2] L.L. Horstman, W. Jy, J.J. Jimenez, C. Bidot, Y.S. Ahn, New horizons in the analysis of circulating cell-derived microparticles, Keio J. Med. 53 (2004) 210–230. [3] J.H. Distler, D.S. Pisetsky, L.C. Huber, J.R. Kalden, S. Gay, O. Distler, Microparticles as regulators of inflammation: novel players of cellular crosstalk in the rheumatic diseases, Arthritis Rheum. 52 (2005) 3337–3348. [4] J.D. Lane, V.J. Allan, P.G. Woodman, Active relocation of chromatin and endoplasmic reticulum into blebs in late apoptotic cells, J. Cell. Sci. 118 (2005) 4059–4071. [5] M. Schiller, I. Bekeredjian-Ding, P. Heyder, N. Blank, A.D. Ho, H.M. Lorenz, Autoantigens are translocated into small apoptotic bodies during early stages of apoptosis, Cell Death Differ. 15 (2008) 183–191. [6] D.O. Hasselmann, G. Rappl, W. Tilgen, U. Reinhold, Extracellular tyrosinase mRNA within apoptotic bodies is protected from degradation in human serum, Clin. Chem. 47 (2001) 1488–1489. [7] E.K. Ng, N.B. Tsui, N.Y. Lam, R.W. Chiu, S.C. Yu, S.C. Wong, E.S. Lo, T.H. Rainer, P.J. Johnson, Y.M. Lo, Presence of filterable and nonfilterable mRNA in the plasma of cancer patients and healthy individuals, Clin. Chem. 48 (2002) 1212–1217. [8] T. El-Hefnawy, S. Raja, L. Kelly, W.L. Bigbee, J.M. Kirkwood, J.D. Luketich, T.E. Godfrey, Characterization of amplifiable, circulating RNA in plasma and its potential as a tool for cancer diagnostics, Clin. Chem. 50 (2004) 564–573. [9] M.S. Kopreski, F.A. Benko, L.W. Kwak, C.D. Gocke, Detection of tumor messenger RNA in the serum of patients with malignant melanoma, Clin. Cancer Res. 5 (1999) 1961–1965. [10] K.W. Lo, Y.M. Lo, S.F. Leung, Y.S. Tsang, L.Y. Chan, P.J. Johnson, N.M. Hjelm, J.C. Lee, D.P. Huang, Analysis of cell-free Epstein–Barr virus associated RNA in the plasma of patients with nasopharyngeal carcinoma, Clin. Chem. 45 (1999) 1292–1294. [11] G. Rappl, D.O. Hasselmann, M. Rossler, S. Ugurel, W. Tilgen, U. Reinhold, Detection of tumor-associated circulating mRNA in patients with disseminated malignant melanoma, Ann. N.Y. Acad. Sci. 945 (2001) 189–191. [12] F. Dasi, S. Lledo, E. Garcia-Granero, R. Ripoll, M. Marugan, M. Tormo, J. Garcia-Conde, S.F. Alino, Real-time quantification in plasma of human telomerase reverse transcriptase (hTERT) mRNA: a simple blood test to monitor disease in cancer patients, Lab. Invest. 81 (2001) 767–769. [13] S.C. Wong, S.F. Lo, M.T. Cheung, K.O. Ng, C.W. Tse, B.S. Lai, K.C. Lee, Y.M. Lo, Quantification of plasma beta-catenin mRNA in colorectal cancer and adenoma patients, Clin. Cancer Res. 10 (2004) 1613–1617. [14] E. Sueoka, N. Sueoka, K. Iwanaga, A. Sato, K. Suga, S. Hayashi, K. Nagasawa, K. Nakachi, Detection of plasma hnRNP B1 mRNA, a new cancer biomarker, in lung cancer patients by quantitative real-time polymerase chain reaction, Lung Cancer 48 (2005) 77–83. [15] H.D. Halicka, E. Bedner, Z. Darzynkiewicz, Segregation of RNA and separate packaging of DNA and RNA in apoptotic bodies during apoptosis, Exp. Cell Res. 260 (2000) 248–256. [16] H. Wagner, The immunobiology of the TLR9 subfamily, Trends Immunol. 25 (2004) 381–386. [17] M. Schroder, A.G. Bowie, TLR3 in antiviral immunity: key player or bystander? Trends Immunol. 26 (2005) 462–468.
768
E XP E RI ME N TA L C E L L R E S EA RC H 315 ( 20 0 9) 76 0 –76 8
[18] E. Meylan, J. Tschopp, Toll-like receptors and RNA helicases: two parallel ways to trigger antiviral responses, Mol. Cell 22 (2006) 561–569. [19] E. Meylan, J. Tschopp, M. Karin, Intracellular pattern recognition receptors in the host response, Nature 442 (2006) 39–44. [20] G. Trinchieri, A. Sher, Cooperation of Toll-like receptor signals in innate immune defence, Nat. Rev. Immunol. 7 (2007) 179–190. [21] T. Lovgren, M.L. Eloranta, U. Bave, G.V. Alm, L. Ronnblom, Induction of interferon-alpha production in plasmacytoid dendritic cells by immune complexes containing nucleic acid released by necrotic or late apoptotic cells and lupus IgG, Arthritis Rheum. 50 (2004) 1861–1872. [22] T.K. Means, E. Latz, F. Hayashi, M.R. Murali, D.T. Golenbock, A.D. Luster, Human lupus autoantibody–DNA complexes activate DCs through cooperation of CD32 and TLR9, J. Clin. Invest. 115 (2005) 407–417. [23] J. Tian, A.M. Avalos, S.Y. Mao, B. Chen, K. Senthil, H. Wu, P. Parroche, S. Drabic, D. Golenbock, C. Sirois, J. Hua, L.L. An, L. Audoly, G. La Rosa, A. Bierhaus, P. Naworth, A. Marshak-Rothstein, M.K. Crow, K.A. Fitzgerald, E. Latz, P.A. Kiener, A.J. Coyle, Toll-like receptor 9-dependent activation by DNA-containing immune complexes is mediated by HMGB1 and RAGE, Nat. Immunol. 8 (2007) 487–496. [24] L.E. Graves, E.V. Ariztia, J.R. Navari, H.J. Matzel, M.S. Stack, D.A. Fishman, Proinvasive properties of ovarian cancer ascites-derived membrane vesicles, Cancer Res. 64 (2004) 7045–7049. [25] J. Ratajczak, M. Wysoczynski, F. Hayek, A. Janowska-Wieczorek, M.Z. Ratajczak, Membrane-derived microvesicles: important and underappreciated mediators of cell-to-cell communication, Leukemia 20 (2006) 1487–1495. [26] M. Baj-Krzyworzeka, R. Szatanek, K. Weglarczyk, J. Baran, B. Urbanowicz, P. Branski, M.Z. Ratajczak, M. Zembala, Tumour-derived microvesicles carry several surface determinants and mRNA of tumour cells and transfer some of these determinants to monocytes, Cancer Immunol. Immunother. 55 (2006) 808–818. [27] M. Baj-Krzyworzeka, R. Szatanek, K. Weglarczyk, J. Baran, M. Zembala, Tumour-derived microvesicles modulate biological activity of human monocytes, Immunol. Lett. 113 (2007) 76–82. [28] H. Valadi, K. Ekstrom, A. Bossios, M. Sjostrand, J.J. Lee, J.O. Lotvall, Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells, Nat. Cell Biol. 9 (2007) 654–659. [29] G. Houge, S.O. Doskeland, R. Boe, M. Lanotte, Selective cleavage of 28S rRNA variable regions V3 and V13 in myeloid leukemia cell apoptosis, FEBS Lett. 315 (1993) 16–20. [30] G. Houge, B. Robaye, T.S. Eikhom, J. Golstein, G. Mellgren, B.T. Gjertsen, M. Lanotte, S.O. Doskeland, Fine mapping of 28S rRNA sites specifically cleaved in cells undergoing apoptosis, Mol. Cell. Biol. 15 (1995) 2051–2062.
[31] A. Samali, B. Gilje, S.O. Doskeland, T.G. Cotter, G. Houge, The ability to cleave 28S ribosomal RNA during apoptosis is a cell-type dependent trait unrelated to DNA fragmentation, Cell Death Differ. 4 (1997) 289–293. [32] K.L. King, C.M. Jewell, C.D. Bortner, J.A. Cidlowski, 28S ribosome degradation in lymphoid cell apoptosis: evidence for caspase and Bcl-2-dependent and -independent pathways, Cell Death Differ. 7 (2000) 994–1001. [33] W.G. Degen, G.J. Pruijn, J.M. Raats, W.J. van Venrooij, Caspase-dependent cleavage of nucleic acids, Cell Death Differ. 7 (2000) 616–627. [34] T.X. Hoat, H. Nakayashiki, Y. Tosa, S. Mayama, Specific cleavage of ribosomal RNA and mRNA during victorin-induced apoptotic cell death in oat, Plant J. 46 (2006) 922–933. [35] D.R. Crawford, R.J. Lauzon, Y. Wang, J.E. Mazurkiewicz, G.P. Schools, K.J. Davies, 16S mitochondrial ribosomal RNA degradation is associated with apoptosis, Free Radic. Biol. Med. 22 (1997) 1295–1300. [36] K. Samejima, W.C. Earnshaw, Trashing the genome: the role of nucleases during apoptosis, Nat. Rev. Mol. Cell Biol. 6 (2005) 677–688. [37] P.T. Sobol, K.L. Mossman, ICP0 prevents RNase L-independent rRNA cleavage in herpes simplex virus type 1-infected cells, J. Virol. 80 (2006) 218–225. [38] E.K. Ng, N.B. Tsui, T.K. Lau, T.N. Leung, R.W. Chiu, N.S. Panesar, L.C. Lit, K.W. Chan, Y.M. Lo, mRNA of placental origin is readily detectable in maternal plasma, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 4748–4753. [39] P.B. Larrabee, K.L. Johnson, C. Lai, J. Ordovas, J.M. Cowan, U. Tantravahi, D.W. Bianchi, Global gene expression analysis of the living human fetus using cell-free messenger RNA in amniotic fluid, JAMA 293 (2005) 836–842. [40] J. Fan, X. Yang, W. Wang, W.H. Wood III, K.G. Becker, M. Gorospe, Global analysis of stress-regulated mRNA turnover by using cDNA arrays, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 10611–10616. [41] D.J. Taxman, J.P. MacKeigan, C. Clements, D.T. Bergstralh, J.P. Ting, Transcriptional profiling of targets for combination therapy of lung carcinoma with paclitaxel and mitogen-activated protein/ extracellular signal-regulated kinase kinase inhibitor, Cancer Res. 63 (2003) 5095–5104. [42] X. Lu, W.E. Burgan, M.A. Cerra, E.Y. Chuang, M.H. Tsai, P.J. Tofilon, K. Camphausen, Transcriptional signature of flavopiridol-induced tumor cell death, Mol. Cancer Ther. 3 (2004) 861–872. [43] X. Lu, L. de la Pena, C. Barker, K. Camphausen, P.J. Tofilon, Radiation-induced changes in gene expression involve recruitment of existing messenger RNAs to and away from polysomes, Cancer Res. 66 (2006) 1052–1061. [44] M. Radic, T. Marion, M. Monestier, Nucleosomes are exposed at the cell surface in apoptosis, J. Immunol. 172 (2004) 6692–6700.