ISIS Report 20/04/09

Intercommunication via Circulating Nucleic Acids

DNA and RNA circulating in the bloodstream are offering golden opportunities for disease diagnosis and may play an important role in intercommunication between cells Dr. Mae-Wan Ho

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Nucleic acids (DNA and RNA) are genetic material generally thought to be confined within cells. But they were discovered circulating in the bloodstream in 1948. Since then, many diagnostic applications have been developed for cancers and other diseases based on these circulating nucleic acids [1]. The discovery of foetal DNA circulating in the mother’s bloodstream also opened up the possibility of non-invasive prenatal diagnosis and monitoring of many maternal pregnancy-associated disorders. However, the origins and functions of these nucleic acids remain obscure.

Mattias Belting and Anders Wittrup at Lund University in Sweden suggest that they may play an important role in intercellular communication and signalling [2]. Throughout the evolution of higher eukaryotes (organisms with nucleus in their cells), carrier mechanisms on cell membranes that allow viruses and other microbes to invade the cell, incorporate foreign genetic material, and cause diseases, have been conserved; that simply does not make sense unless those mechanisms have other functions. Recent evidence from different fields are pointing to the active trafficking of nucleic acids between cells that could make use of those mechanisms. Nucleic acid trafficking may be involved in intercellular signalling during development, in epigenetic remodelling, tissue regeneration and fine tuning of the adaptive immune system. It may also be involved in cancer development and immune surveillance.

RNA trafficking

Systemic RNAi

Ten years ago, it was found that double-stranded RNA (dsRNA) injected or fed to the lab nematode Caenorhabdities elegans triggered silencing of transcripts with complementary base sequences throughout the adult animal as well as its progeny. Several genes are involved in this systemic RNA interference (RNAi). One of them, sid-1 (systemic silencing deficient -1) encodes a protein that loops back and forth through the cell membrane 11 times to form a membrane pore for dsRNA transport across the membrane. Sid-1 homologues have been found in humans and other mammals, though not in the fruit fly Drosophila melanogaster, which appears to use other mechanisms for systemic RNAi.

RNAi is ubiquitous and essential for normal development and gene regulation [3, 4] (see Subverting the Genetic Text, SiS 24; Rewriting the Genetic Text in Human Brain Development, SiS 41). Release of RNAi species into the circulatory system will result in systemic RNAi responses. The RNAi response is amplified in C. elegans and in plants by RNA-dependent RNA polymerases

Tunnelling nanotubes transport between cells

Recently, tunnelling nanotubes (TNTs) have been discovered that can connect cells from several cell diameters apart, providing membrane continuity between the cells [2]. First found in cultured cells, TNTs have since been detected in the mammalian cornea. There are two kinds of TNTs, a thin <0.7 mm actin-containing tube that supports unidirectional movement of the plasma membrane constituents, including surface attached pathogens, and a wider, >0.7 mm microtubule-containing tube that supports bidirectional transport of vesicles and organelles such as endosomes (membrane-bound inclusion inside cells) and mitochondria. Endosomes may be of particular significance for the transmission of RNAi.

Plasmadesmata

Plasmadesmata are microscopic channels traversing the cells walls between plant cells, and are involved in direct cell-to-cell communication in plants. Shuttling of endogenous over-expressed RNA was demonstrated to occur through plasmadesmata. Plant viruses encode movement proteins that mediate infectious spread of viral nucleic acids via plasmadesmata. Endogenous plant proteins, including transcription factors, use the same pathway to traffic between cells. It has been shown that the protein encoded by the maize knotted1 homeobox could selectively transfer its own mRNA to surrounding cells through plasmodesmata.

Microvesicles and exosomes

Long-distance signalling with messenger RNA (mRNA) or micro RNA (miRNA, involved in RNA interference) may be achieved by packaging these RNA species into exocytotic (secretory) vesicles endowed with specific cell surface-targeting motifs. These small membrane vesicles, or exosomes (50-90nm in diameter), released by cells, may well have a role in intercellular communication. Embryonic stem cells secrete vesicles highly enriched in specific mRNAs, which can be transferred to and induce phenotypic changes in haematopoietic (blood) progenitor cells. Mast cells, involved in the body’s allergic response, secrete exosomes that contain a unique set of ~1 300 different mRNAs, some of which are translated in recipient cells. The exosomes also contain >100 different miRNAs that are promiscuous in binding and inactivating target mRNA, so the impact on gene expression in recipient cells might be quite extensive.

DNA trafficking

While it is possible to identify the potential sources and functions of some circulating RNA species from living cells, it is not clear that DNA is actively secreted by living cells. Exosomes do not contain DNA, yet horizontal transfer of DNA does occur between somatic cells [2] as well as germ cells [5] (Epigenetic Inheritance through Sperm Cells, the Lamarckian Dimension in Evolution, SiS 42). In fact, DNA, like RNA, is so readily taken up that it has been widely exploited in ‘gene therapy’, while our regulators continue to ignore the potential hazards of the ever inicreasing range of genetically modified nucleic acids released into the environment [6] (Slipping through the regulatory net, ISIS/TWN publication).

DNA is known to be released in apoptotic bodies, membrane-bound vesicles containing fragmented DNA resulting from programmed cell death. And these apoptotic bodies can be phagocytosed (engulfed) and transported into the nucleus of recipient cells for expression and integration into the genome.

Co-cultivation of cell lines containing integrated copies of Epstein-Barr virus resulted in the rapid uptake and transfer of EBV-DNA as well as genomic DNA to the nucleus of the phagocytosing cells [7]. This is an efficient mode of gene transfer, as fluorescent in situ hybridisation (FISH) analysis of bovine aortic endothelial cells showed uptake of apoptotic DNA in the nuclei of 15 percent of the phagocytosing cells. Once transferred, expression of the EBV-encoded genes was detected at protein and mRNA levels. Apoptotic bodies derived from tumour cells induce foci (centres of malignancy) in p53-deficient fibroblast cultures in vitro and tumours in animals. Whole chromosomes or fragments are transferred by this phagocytosis pathway and integrated into the genome [8]. Horizontal gene transfer between cells may be important during tumour progression.

A further mechanism of horizontal DNA transfer has been suggested by studies in autoimmune disease [2]. The antimicrobial peptide LL-37, which is widely expressed in epithelia, bone marrow, and the genitourinary tract of human, forms stable complexes with DNA and translocates extracellular DNA to the nucleus. LL-37-mediated delivery of self DNA may be an early event in autoimmune disease. The ability of LL-37 to transfer DNA across the plasma membrane is a shared property within the growing family of ‘cell-penetrating peptides’. Among them, the Antennapedia homeobox peptide and the HIV-Tat transduction domain, are endowed with the ability to mediate efficient uptake of macromolecules into a wide variety of mammalian cells.  

Circulating nucleic acids in health and disease

Circulating DNA in cancer patients has many characteristics in common with the DNA of their tumours, and is suspected of being derived from apoptotic bodies from cancer cells. Furthermore, elevated concentration per se appears indicative of disease states, whether it is cancer, systemic lupus erthyematosus, rheumatoid arthritis, glomerulonephritis, pancreatic, hepatitis, inflammatory bowel disease, etc [9].

For example, plasma DNA concentrations in 102 patents with lung cancer and 105 healthy individuals were compared using quantitative PCR analysis [10]. The median plasma DNA concentrations for healthy and cancer groups were 10.4 and 22.6 ng/ml respectively (p<0,0001). Elevated DNA levels were also detected in patients with either stage I or II disease.In another study, plasma DNA from 121 women - 61 with breast cancer, 33 control patients with benign breast diseases and 27 healthy controls – were compared [11]. The median level was 65 ng/ml in breast cancer patients, significantly different from that in controls, 22 ng/ml and healthy controls, 13 ng/ml.

There is current debate as to whether circulating DNA is solely derived from dead cells [12] or whether they are actively secreted by living cells [9, 13].

Maniesh van der Vaart and Piet Pretorius at North-West University, Potchefstroom, South Africa, point out that the DNA circulating in healthy individuals simply do not have the characteristics of DNA from apoptotic or other dead cells [13]. In cell cultures with no dead cells, DNA is nevertheless actively secreted into the medium until a certain external concentration is reached. Replacing the medium leads to further secretion until the external equilibrium concentration is restored. Van der Vaart and Pretorius argue that in a healthy organism, most if not all the DNA from dead cells would be cleared immediately by phagocytes and other cells near at hand, and broken down in intracellular lysosomes. Instead, living cells maintain a low equilibrium level of circulating DNA by secretion. The secreted DNA is cleared from circulation, some probably taken up by other cells and incorporated into their genome, although the authors themselves have not suggested this possibility (see below). In disease states, however, the rate of cell death is such that it overwhelms the capacity of the normal phagocytic cells to take up and destroy the DNA, which therefore leaks into general circulation. In cancer states, there will be additional sources of DNA excreted by living cancer cells.

The research group headed by Howard Urnovitz, CEO of Chronix Biomedical GmbH, Goettingen, Germany, used high through-put parallel DNA sequencing technologies to profiles of total circulating DNA from the serum of 51 healthy humans (27 females and 24 males) and compared them with the genomes from 4 of the subjects, as well as from genomic DNA sequences from public databases [14].

They obtained 4.5 x 10⁵ sequences (7.5 x 10⁷ nucleotides).Of these, 97 percent were genomic and 3 percent foreign, with 0.16 percent from bacterial genomes, 0.02 percent from viruses and 0.01 percent from fungi. On the whole, the profile of circulating DNA resembled genomic DNA with the following exceptions. Chromosome 19 sequences are under-represented; chromosome 19 contains most genes and has the highest amount of Alu elements, a subclass of primate-specific short interspersed nuclear elements (SINEs) that spreads around the genome by retro-transposition. Alu sequences, however, are over-represented, accounting for 11.4 + 0.4 percent in circulating DNA samples compared to 8.5 + 0.8 percent in the genomic samples; while L1 and L2 long interspersed nuclear elements (LINEs) were under-represented, accounting for 19 percent in serum DNA samples compared with 22.8 percent in genomic samples. Also notable were the relatively large individual variations of circulating DNA for coding sequences, which ranged from 0.78 to 1.4 times genomic sequences; untranslated regulatory sequences, ranging from 0.58 to 1.3 times genomic sequences, and pseudogenes (relict genes previously believed to be no longer active) ranging from 0.85 to 1.15 times genomic sequences.  The researchers conclude that non-specific release (due to cell death) is not the sole origin for circulating DNA.

Nevertheless the role of the actively secreted DNA from living cells is unknown. It has been suggested that circulating DNA takes part in homologous recombination with genomic DNA, and that this process can correct mutations as well as induce genetic changes, with the external DNA fragments serving as reference molecules [15].

Recently, a substantial literature has come to light on the transforming power of blood transfusions that are most likely due to circulating nucleic acids [16] (see Darwin’s Theory of Pangenesis, the Hidden History of Genetics, & the Dangers of GMOs, SiS 42).

The RNA species circulating in health are part of the vast regulatory RNA network discovered within a few years since the human genome was announced [3]. Some 97-98 percent of the transcripts do not code for proteins, and the job of mediating between DNA and proteins is the centre stage of molecular life. On account of widespread mechanisms such as alternative splicing, trans-splicing, and RNA editing [4], many RNA species cannot be easily mapped to the genome. The picture gets even more complex in disease states; though it may offer more diagnostic opportunities.  

Some of the most impressive diagnostic applications of circulating nucleic acids have been based on RNA, and was pioneered by Howard Urnovitz (see above). We first featured his work in 2003 [17] (Dynamic Genomics, SiS 19) when he reported unique RNA markers in patients with Gulf War Syndrome and other chronic diseases. Two years later, he had developed the first Living Test for Mad Cow Disease [18] (SiS 28), again based on unique gene markers that were in 100 percent of cows with BSE as well as in 100 percent of asymptomatic high-risk animals (housed with the affected) 6 month before they became ill.

This diagnostic work has continued, and has been confirmed recently using high through-put DNA sequencing [19]. Disease-specific patterns in circulating DNA were found in elk and cattle during a 25 month experiment in which they were infected orally with chronic wasting disease material. Infection-specific sequences were found as early as 11 months in elk at least three months before the appearance of the first clinical signs and at least 4 months before clinical signs in cattle. Some of the patterns identified contain transcription factor binding sites linked to endogenous retroviral integration, suggesting that retroviruses may be connected to Mad Cow disease. Thus, predictive diagnosis is now available [20].

Meanwhile, research on the fascinating science of the dynamic genome languishes.

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