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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 . 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 . 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.
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.
Recently, tunnelling nanotubes (TNTs) have been discovered that can connect
cells from several cell diameters apart, providing membrane continuity between
the cells . 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 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.
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  as well as germ cells  (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  (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 . 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 . 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 . 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 .
For example, plasma DNA concentrations in 102 patents with lung
cancer and 105 healthy individuals were compared using quantitative PCR analysis
. 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 . 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  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 . 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
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 .
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
. 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 , 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  (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
 (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 . 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 .
Meanwhile, research on the fascinating science of the dynamic