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Overview
·
Asymmetric cell divisions and stem cell biology ·
Genetic instability, aging and cancer ·
Replication and repair of G-rich DNA and telomere
repeats ·
Fluorescence in situ hybridization and flow
cytometry Asymmetric
cell divisions are required for the development of all multi-cellular
organisms. Many mechanisms are known to help secure differences between
daughter cells following a parental
cell division. We are interested in exploring the possibility that differences
in parental cells before they
divide contribute to differences between daughter cells as well. For example,
it seems possible that chromatin differences between sister chromatids
co-regulate the expression of specific genes as predicted by the silent sister hypothesis (1). In order to test this hypothesis it is
necessary to reliably distinguish between sister chromatids. We recently
described a novel approach to identify and study sister chromatids (2). Current studies focus on testing the silent sister hypothesis using cells
that divide asymmetrically and cells in which asymmetry is not obvious such
as self-renewing normal and malignant (stem) cells. Accurate
replication of DNA and faithful segregation of chromosomes are essential for
all forms of life. In multi-cellular organisms, the control of these
processes differs markedly between various cell types and between organisms (3). For example, the number of cell divisions is
tightly controlled in adult blood-forming stem cells, whereas embryonic stem
cells are essentially immortal. Furthermore, most adult stem cells are
typically quiescent, whereas embryonic stem cells turnover rapidly. The
number of cell divisions that adult stem cells can undergo seems much more
tightly controlled in humans than in mice, perhaps because tumor growth poses
a greater risk to reproductive fitness in humans (with a longer lifespan and
a larger body mass) than in mice (3). We
previously engineered murine ES cells (4) with
specific defects in the replication of chromosome ends (telomeres).
Surprisingly, such cells appear to grow normally until differentiation is
induced. In order to study the molecular mechanisms that underpin these
observations, we are using these and other ES cells expressing fluorescent
reporter constructs before and after various genetic manipulations. The goal
of these studies is to characterize the pathways involved in cell cycle
checkpoints, telomere maintenance and DNA damage responses in ES cells before
and after induction of differentiation. In
all vertebrates telomeres are characterized by TTAGGG repeats and associated
proteins. A minimum number of telomere repeats is needed at each end in order
to distinguish a normal end form a double strand break and protect chromosome
ends from fusion and degradation. Critically short or truncated telomeres
trigger a DNA damage response that can cause cell cycle arrest or cell death.
Telomeric DNA is lost with each round of cell division via several distinct
pathways (5). Lost telomere repeats are normally
replenished by the enzyme telomerase, a reverse transcriptase which can add
telomere repeats to the 3 end of chromosomes using an RNA template.
Telomerase levels appear to be limiting in normal human blood forming stem
cells (6). We have developed novel tools to
measure the telomere length in individual cells and individual chromosomes (7, 8) and these quantitative fluorescence in situ
hybridization techniques are used to address questions about the role of
telomeres in normal aging, tumor progression and specific genetic,
hematological and immune disorders. Based on our
studies in C.elegans (9) we are partcilarly interested in guanine-rich
DNA capable of forming higher order structures known guanine quadruplex (G4)
DNA. We have developed a panel of monoclonal antibodies against different G4
DNA structures to study on the role of G4 DNA in the regulation of gene
expression and telomere biology. References: 1.
Lansdorp PM. Immortal strands? Give me
a break. Cell 129: 1244-1247, 2007. 2.
Falconer
E, Chavez EA, Henderson A, Poon SSS, McKinney S, Brown L, Huntsman DG & Lansdorp PM. Identification of sister
chromatids by DNA template strand sequences.
Nature (Epub ahead of print on 3. Lansdorp
PM.
Telomeres and disease. EMBO J 28:
2532-2540, 2009. 4. Ding H, Schertzer M, Wu X,
Gersteinsen M, Selig S, Kammori M, Pourvali R, Poon S, Vulto I, Chavez E, Tam
PPL, Nagy A & Lansdorp PM.
Regulation of murine telomere length by Rtel: An essential gene encoding a
helicase-like protein. Cell 117:873-886, 2004. 5.
Lansdorp PM. Major Cutbacks at chromosome
ends. Trends Biochem Sci 30:388-395, 2005. 6.
Aubert
G & Lansdorp P. Telomeres and
aging. Physiol Rev 82:557-579, 2008. 7.
Lansdorp PM, Verwoerd NP, van de Rijke
FM, Dragowska V, Little M-T, Dirks RW, Raap AK & Tanke HJ. Heterogeneity
in telomere length of human chromosomes. Hum Mol Genet 5:685-691, 1996. 8.
Baerlocher
GM, Vulto I, de Jong G & Lansdorp
PM. Flow cytometry and FISH to measure the average length of telomeres
(flow FISH). Nat Protoc 1: 2365-2376, 2006. 9.
Cheung
I, Schertzer M, Rose A & Lansdorp
PM. Disruption of dog-1 in Caenorhabditis elegans triggers deletions
upstream of guanine-rich DNA. Nat Genet 31:405-409, 2002. |
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Terry
Fox Laboratory |
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