The immortal DNA strand hypothesis was proposed in 1975 by John Cairns as a mechanism for adult stem cells to minimize mutations in their genomes. This hypothesis proposes that instead of segregating their DNA during mitosis
in a random manner, adult stem cells divide their DNA asymmetrically,
and retain a distinct template set of DNA strands (parental strands) in
each division. By retaining the same set of template DNA strands, adult
stem cells would pass mutations arising from errors in DNA replication on to non-stem cell daughters that soon terminally differentiate
(end mitotic divisions and become a functional cell). Passing on these
replication errors would allow adult stem cells to reduce their rate of
accumulation of mutations that could lead to serious genetic disorders such as cancer.
Although evidence for this mechanism exists, whether it is a mechanism acting in adult stem cells in vivo is still controversial.
Although evidence for this mechanism exists, whether it is a mechanism acting in adult stem cells in vivo is still controversial.
Methods
Two main assays are used to detect immortal DNA strand segregation: label-retention and label-release pulse/chase assays.
In the label-retention assay, the goal is to mark 'immortal' or parental DNA strands with a DNA label such as tritiated thymidine or bromodeoxyuridine (BrdU). These types of DNA labels will incorporate into the newly synthesized DNA of dividing cells during S phase.
A pulse of DNA label is given to adult stem cells under conditions
where they have not yet delineated an immortal DNA strand. During these
conditions, the adult stem cells are either dividing symmetrically
(thus with each division a new 'immortal' strand is determined and in
at least one of the stem cells the immortal DNA strand will be marked
with DNA label), or the adult stem cells have not yet been determined
(thus their precursors are dividing symmetrically, and once they
differentiate into adult stem cells and choose an 'immortal' strand, the
'immortal strand' will already have been marked). Experimentally, adult
stem cells are undergoing symmetric divisions during growth and after
wound healing, and are not yet determined at neonatal stages. Once the
immortal DNA strand is labelled and the adult stem cell has begun or
resumed asymmetric divisions, the DNA label is chased out. In symmetric
divisions (most mitotic
cells), DNA is segregating randomly and the DNA label will be diluted
out to levels below detection after five divisions. If, however, cells
are using an immortal DNA strand mechanism, then all the labeled DNA
will continue to co-segregate with the adult stem cell, and after five
(or more) divisions will still be detected within the adult stem cell.
These cells are sometimes called label-retaining cells (LRCs).
In the label-release assay, the goal is to mark the newly
synthesized DNA that is normally passed on to the daughter (non-stem)
cell. A pulse of DNA label is given to adult stem cells under
conditions where they are dividing asymmetrically. Under conditions of homeostasis,
adult stem cells should be dividing asymmetrically so that the same
number of adult stem cells is maintained in the tissue compartment.
After pulsing for long enough to label all the newly replicated DNA, the
DNA label is chased out (each DNA replication now incorporates
unlabeled nucleotides) and the adult stem cells are assayed for loss of
the DNA label after two cell divisions. If cells are using a random
segregation mechanism, then enough DNA label should remain in the cell
to be detected. If, however, the adult stem cells are using an immortal
DNA strand mechanism, they are obligated to retain the unlabeled
'immortal' DNA, and will release all the newly synthesized labeled DNA
to their differentiating daughter cells in two divisions.
Some scientists have combined the two approaches,
by first using one DNA label to label the immortal strands, allowing to
adult stem cells to begin dividing asymmetrically, and then using a
different DNA label to label the newly synthesized DNA. Thus, the adult
stem cells will retain one DNA label and release the other within two
divisions.
Evidence
Evidence for the immortal DNA strand hypothesis has been found in various systems. One of the earliest studies by Karl Lark et al. demonstrated co-segregation of DNA in the cells of plant root tips.
Plant root tips labeled with tritiated thymidine tended to segregate
their labeled DNA to the same daughter cell. Though not all the labeled
DNA segregated to the same daughter, the amount of thymidine-labeled
DNA seen in the daughter with less label corresponded to the amount that
would have arisen from sister-chromatid exchange. Later studies by Christopher Potten et al. (2002),
using pulse/chase experiments with tritiated thymidine, found long-term
label-retaining cells in the small intestinal crypts of neonatal mice.
These researchers hypothesized that long-term incorporation of tritiated
thymidine occurred because neonatal mice have undeveloped small
intestines, and that pulsing tritiated thymidine soon after the birth of
the mice allowed the 'immortal' DNA of adult stem cells to be labeled
during their formation. These long-term cells were demonstrated to be
actively cycling, as demonstrated by incorporation and release of BrdU.
Since these cells were cycling but continued to contain the BrdU
label in their DNA, the researchers reasoned that they must be
segregating their DNA using an immortal DNA strand mechanism. Joshua
Merok et al. from the lab of James Sherley engineered mammalian cells with an inducible p53 gene that controls asymmetric divisions.
BrdU pulse/chase experiments with these cells demonstrated that
chromosomes segregated non-randomly only when the cells were induced to
divide asymmetrically like adult stem cells. These asymmetrically
dividing cells provide an in vitro model for demonstration and investigation of immortal strand mechanisms.
Scientists have strived to demonstrate that this immortal DNA strand mechanism exists in vivo
in other types of adult stem cells. In 1996 Nik Zeps published the
first paper demonstrating label retaining cells were present in the
mouse mammary gland
and this was confirmed in 2005 by Gilbert Smith who also published
evidence that a subset of mouse mammary epithelial cells could retain
DNA label and release DNA label in a manner consistent with the immortal
DNA strand mechanism.
Soon after, scientists from the laboratory of Derek van der Kooy showed
that mice have neural stem cells that are BrdU-retaining and continue
to be mitotically active.
Asymmetric segregation of DNA was shown using real-time imaging of
cells in culture. In 2006, scientists in the lab of Shahragim Tajbakhsh
presented evidence that muscle satellite cells, which are proposed to be adult stem cells of the skeletal muscle
compartment, exhibited asymmetric segregation of BrdU-labelled DNA when
put into culture. They also had evidence that demonstrated BrdU
release kinetics consistent with an immortal DNA strand mechanism were
operating in vivo, using juvenile mice and mice with muscle regeneration induced by freezing.
These experiments supporting the immortal strand hypothesis,
however, are not conclusive. While the Lark experiments demonstrated
co-segregation, the co-segregation may have been an artifact of
radiation from the tritium. Although Potten identified the cycling,
label-retaining cells as adult stem cells, these cells are difficult to
identify unequivocally as adult stem cells. While the engineered cells
provide an elegant model for co-segregation of chromosomes, studies with
these cells were done in vitro with engineered cells. Some features may not be present in vivo or may be absent in vitro. In May 2007 evidence in support of the Immortal DNA Strand theory was discovered by Michael Conboy et al.,
using the muscle stem/satellite cell model during tissue regeneration,
where there is tremendous cell division during a relatively brief period
of time. Using two BrdU analogs to label template and newly
synthesized DNA strands, they saw that about half of the dividing cells
in regenerating muscle sort the older "Immortal" DNA to one daughter
cell and the younger DNA to the other. In keeping with the stem cell
hypothesis, the more undifferentiated daughter typically inherited the
chromatids with the older DNA, while the more differentiated daughter
inherited the younger DNA.
Experimental evidence against the immortal strand hypothesis is
sparse. In one study, researchers incorporated tritiated thymidine into
dividing murine epidermal basal cells.
They followed the release of tritiated thymidine after various chase
periods, but the pattern of release was not consistent with the immortal
strand hypothesis. Although they found label-retaining cells, they
were not within the putative stem cell compartment. With increasing
lengths of time for the chase periods, these label-retaining cells were
located farther from the putative stem cell compartment, suggesting that
the label-retaining cells had moved. However, finding conclusive
evidence against the immortal strand hypothesis has proven difficult.
Further models
After Cairns first proposed the immortal DNA strand mechanism, the theory has undergone several refinements.
In 2002, he proposed that in addition to using immortal DNA
strand mechanisms to segregate DNA, when the immortal DNA strands of
adult stem cells undergo damage, they will choose to die (apoptose)
rather than use DNA repair mechanisms that are normally used in non-stem
cells.
Emmanuel David Tannenbaum and James Sherley developed a quantitative model describing how repair of point mutations might differ in adult stem cells.
They found that in adult stem cells, repair was most efficient if they
used an immortal DNA strand mechanism for segregating DNA, rather than a
random segregation mechanism. This method would be beneficial because
it avoids wrongly fixing DNA mutations in both DNA strands and
propagating the mutation.
Mechanisms
The
complete proof of a concept generally requires a plausible mechanism
that could mediate the effect. Although controversial, there is a
suggestion that this could be provided by the Dynein Motor. This paper is accompanied by a comment summarizing the findings and background.
However, this work has highly respected biologists among its
detractors as exemplified by a further comment on a paper by the same
authors from 2006. The authors have rebutted the criticism.
