Observation of the selective staining of chromosomal DNA in dividing cells using a luminescent terbium(iii) complex

Article abstract of DOI:10.1039/b924679g

The emission intensity of a monocationic Tb or Eu(iii) complex of a bis(1-azaxanthone) ligand is enhanced in the presence of serum albumin; the lanthanide complex stains dividing cells, allowing visualisation of mitotic chromosomes. The Royal Society of Chemistry.

Full text of DOI:10.1039/b924679g

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Observation of the selective staining of chromosomal DNA in dividing
cells using a luminescent terbium(III) complexw
Ga-Lai Law,a Cornelia Man,b David Parker*a and James W. Waltona
Received (in Cambridge, UK) 24th November 2009, Accepted 16th January 2010
First published as an Advance Article on the web 2nd February 2010
DOI: 10.1039/b924679g
The emission intensity of a monocationic Tb or Eu(III) complex
of a bis(1-azaxanthone) ligand is enhanced in the presence of
serum albumin; the lanthanide complex stains dividing cells,
allowing visualisation of mitotic chromosomes.
strongly emissive; the Tb analogues stain the cell nucleus
selectively, allowing the monitoring of nuclear DNA changes
in dividing cells, as mitosis progresses.
Reaction of the nicotinic acid derivative, 1a,11 with POCl3
gave the 4-chloropyridine, 1b (56%). Treatment with sodium
phenoxide followed by cyclisation in polyphosphoric acid
afforded the 3-azaxanthone, 2a (50%). Benzylic bromination
(NBS, CCl4) allowed isolation of 2b. Ligands H2L1 and H2L2
were obtained by reaction of 2b and 3, respectively, with the
bis-tBu ester of 1,4,7,10-tetraazacyclododecane-1,7-diacetic
acid (DO2A), followed by deprotection with TFA (CH2Cl2,
20 1C). Complexation with Ln(OAc)3 (pH 6, H2O, 50 1C),
followed by ion-exchange (Dowex ClÀ resin), led to isolation
of the monocationic complexes. Measurements of excited state
lifetimes in H2O and D2O allowed an estimation of the metal
hydration state, Table 1. For [LnÁL1]+, both the Eu and Tb
centres are coordinated to one water molecule, whereas for
[LnÁL2]+, no coordinated waters are present,13 consistent with
the increased steric demand of the bound 1-azaxanthone
moiety.
Non-invasive live cell imaging is the key to obtain information
on cell division (mitosis). In current practice, membrane-
permeable organic dyes that specifically bind DNA are used
to visualise chromosomes in live cells.1 A typical example is the
fluorescent cationic dye, Hoechst 33342 (lexc 350 nm, lem 461 nm).
It permeates the outer and nuclear membranes of cells
non-selectively and exhibits a fluorescence enhancement when
bound to nuclear DNA. Care needs to be exercised in its usage
as higher concentrations and direct UV-phototoxicity can lead
to cellular damage.2,3
Out of all the different phases in the cell cycle, the ‘M’ phase
is most sensitive and has in-built stringent controls for
phototoxicity. Thus, a dividing cell will rapidly raise ‘red flags’
when it is damaged, resulting in the reversal to the ‘G-2’ state
or the activation of the spindle assembly checkpoint. The
consequence of checkpoint activation is a pause or a full arrest
in mitotic progression. The cell may also return to a ‘G-2’
like state and, in more extreme cases, undergo apoptosis
(programmed cell death).4
Emissive lanthanide complexes constitute an emerging class
of metal-based emissive probes that are being examined
actively at present for use in cellulo.5–8 Typically, these
lanthanide complexes incorporate a sensitising chromophore
that absorbs light efficiently (high e) and facilitates emission
from the proximate Ln(III) ion, following intramolecular
energy transfer. Here, we describe Eu and Tb(III) complexes
of two constitutionally isomeric dibasic ligands H2L1 and
H2L2 that possess two trans-related azaxanthone chromo-
phores. These chromophores are effective sensitising moieties
for lanthanide emission;9,10 the latter creates a greater steric
demand at the metal centre in the derived Ln(III) complexes,
[LnÁL1]+ and [LnÁL2]+. Furthermore, we report the surprising
behaviour of the monocationic luminescent lanthanide(III)
complexes [LnÁL2]+. The Eu and Tb complexes bind to serum
albumin changing the coordination environment and remain
Incremental addition of human serum albumin (HSA) to
[EuÁL1]+ led to a 20% reduction in the Eu emission lifetime
(0 to 0.5 mM) echoed by a reduction in the observed emission
intensity, with no change in spectral form. In contrast, parallel
experiments with [EuÁL2]+ revealed a dramatic change in the
Eu spectral emission profile, accompanied by a 50% increase
in the Eu excited state lifetime (Fig. 1 and ESIw). A similar
pattern of behaviour was observed in titrations with [TbÁL1]+
and [TbÁL2]+. In the latter case, the emission lifetime increased
by 65% following HSA addition, and in the former the overall
lifetime reduced by a factor of 90% and the emission intensity
a Department of Chemistry, Durham University, South Road, Durham,
UK DH1 3LE. E-mail: david.parker@dur.ac.uk
b Department of Applied Biology and Chemical Technology,
The Hong Kong Polytechnic University, Hunghom, Hong Kong,
China
w Electronic supplementary information (ESI) available: Ligand and
complex synthesis and emission spectra; spectral titrations of lantha-
nide complexes with human serum albumin; experimental details of
cell culture and microscopy. See DOI: 10.1039/b924679g
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Table 1 Radiative rate constants (k/msÀ1) and quantum yields for
much more sensitive to a quenching process that may occur
via exciplex formation between the chromophore and the
electron rich CG base pairs. Consistent with this idea, addition
of poly(dAdT) to either [TbL1(H2O)]+ or [TbÁL2]+ led to a
20% smaller reduction in the intensity of the lanthanide
emission.
lanthanide complexes (295 K, pH 5.5)a
Complex
fem
kH O/msÀ1
kD O/msÀ1
qb
2
2
[EuÁL2]Cl
[TbÁL2]Cl
[EuÁL1]Cl
[TbÁL1]Cl
18
36
10
34
1.02
0.43
1.79
0.66
0.65
0.36
0.64
0.36
0.14
0.05
1.1
1.2
The complexes [TbÁL2]+ and [TbÁL1]+ were examined in cell
uptake experiments using HeLa cells, observing cellular
staining by luminescence microscopy. Using relatively high
concentrations of added complex in the medium (range 10 to
100 mM), the pattern of staining was consistent with an
endosomal/lysosomal distribution. However, at much lower
added concentrations of complex (e.g. o1 mM [TbÁL2]+ or
[TbÁL1]+, 5 to 30 min incubation) less than 10% of the
observed cells were stained. The pattern of staining and
the nature of the nuclear localisation profile (Fig. 2) were
consistent with preferential labelling of those cells undergoing
division in the ‘M’ phase, where membrane integrity is some-
what compromised. This hypothesis was supported by
co-staining experiments using [TbÁL2]+ and Hoechst 33342
(each 0.5 mM in medium, 2 h incubation; lexc 350 nm). The
latter stained all cells observed but the Tb complex only
stained selected cells, including those in mitosis. Cells at each
stage of the mitotic cycle were discerned, e.g. after nuclear
envelope breakdown at prophase, prometaphase, metaphase
and at telophase, with chromosomal DNA stained
selectively.1,2
a
Measurements were made as reported in ref. 12, with 10% errors on
b
k and 20% errors on f values. Values of q (Æ20%) are derived using
methods in ref. 13.
fell by a factor of twow (zero to 0.7 mM HSA). The key
observation in these titrations is the change in spectral form
with [EuÁL2]+, following protein addition. This can only occur
if there is a change in the metal coordination environment.14
The large increase in the DJ = 2 transitions (610–620 nm,
Fig. 1) and the change in the DJ = 1 manifold splitting are
consistent with dissociation of one sensitiser, replacing the
neutral N donor by a polarisable charged oxygen donor
derived from the protein, e.g. an Asp or Glu side chain
carboxylate group.5a,14
The variation of lanthanide emission intensity and lifetime
was also studied in a series of titrations of [LnÁLn]+ (n = 1,2;
Ln = Eu, Tb) with calf thymus DNA (42% CG base pairs),
adding the DNA to the complex, up to a ratio of 4 base pairs
per complex. No changes (Æ10%) in emission lifetime and
intensity were observed for [EuÁL1]+ and [EuÁL2]+. Parallel
changes in absorption and excitation spectra were monitored
and no significant change occurred in the wavelength or
absorbance of the azaxanthone chromophore band at
336 nm. Addition of DNA to each Tb complex also caused
neither a spectral shift nor any hypochromism in the
azaxanthone chromophore as observed by absorption and
excitation spectroscopy, consistent with a binding interaction
that is not primarily intercalative in nature. In each case, the
emission lifetime and intensity were both reduced by about
50% at 1 base pair per complex. Previous studies examining
the behaviour of various lanthanide(III) complexes in the
presence of several nucleic acids have revealed charge transfer
quenching of lanthanide emission,12,15 with Tb complexes
A separate experiment at higher resolution attempted to
follow the fate of a single cell with time (5 min intervals),
following incubation of the complex in the cell growth medium
for 3 h (50 nM, [TbÁL2]+). Changes in the packaging and
assembly of chromatin from prometaphase to metaphase were
clearly discerned (Fig. 3), although the study was prematurely
halted, after 1 h prior to the onset of anaphase. This is most
likely due to phototoxicity, associated with the intensity of
Fig. 1 Variation of europium emission spectral form for [EuÁL2]Cl
(20 mM) with added human serum albumin (lexc 340 nm, 298 K,
pH 7.4, 10 mM HEPES, 10 mM NaCl). The inset shows the
concomitant increase in the Eu emission lifetime, showing the fit (line)
to the observed data for log K = 5.25 (Æ0.05); a parallel titration with
[EuÁ(H2O)L1]Cl gave log K = 3.55 (Æ0.03).
Fig. 2 Bright field (upper left and lower right) and fluorescence
microscopy images of HeLa cells undergoing mitosis, with an
illustrative schematic of the cell cycle (0.5 mM, [TbÁL2]Cl, 3 h, lexc
350 nm).
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Notes and references
1 D. Zink, N. Sadoni and E. Stelzer, Methods, 2003, 29, 42; F. Mora-
Bermudez and J. Ellenberg, Methods, 2007, 41, 158.
2 T. Haraguchi, T. Kaneda and Y. Hiraoka, Genes Cells, 1997, 2, 369.
3 T. Hirota, D. Gerlich, B. Koch, J. Ellenberg and J.-M. Peters,
J. Cell Sci., 2004, 117, 6435.
4 A. Khodjakov and C. L. Rieder, Methods, 2006, 38, 2.
5 (a) C. P. Montgomery, B. S. Murray, E. J. New, R. Pal and
D. Parker, Acc. Chem. Res., 2009, 42, 925; (b) E. J. New,
D. Parker, D. G. Smith and J. W. Walton, Curr. Opin. Chem. Biol.,
2010, DOI: 10.1016/j.cbpa.2009.10.003; (c) R. Pal and D. Parker,
Org. Biomol. Chem., 2008, 6, 1020; (d) B. S. Murray, E. J. New,
R. Pal and D. Parker, Org. Biomol. Chem., 2008, 6, 2085.
6 A. S. Chauvin, S. Comby, B. Song, C. D. Vandevyver, F. Thomas
and J.-C. G. Bunzli, Chem.–Eur. J, 2007, 13, 9515;
C. P. Montgomery, E. J. New, L. O. Palsson, D. Parker,
A. S. Batsanov and L. Lamarque, Helv. Chim. Acta, 2009, 92, 2186.
7 S. W. Botchway, M. Charnley, J. W. Haycock, A. W. Parker,
D. L. Rochester, J. A. Weinstein and J. A. G. Williams, Proc. Natl.
Acad. Sci. U. S. A., 2008, 105, 16071; P. Wu, E. L. M. Wong,
D. L. Ma, G. S. M. Tsang, K. M. Ng and C. M. Che, Chem.–Eur.
J., 2009, 15, 3652; C. A. Puckett and J. K. Barton, J. Am. Chem.
Soc., 2009, 131, 8738; V. Fernandez-Moreira, F. L. Thorp-
Greenwood and M. P. Coogan, Chem. Commun., 2010, 46, 186.
8 (a) N. A. O’Connor, N. Stevens, D. Samaroo, M. R. Soloman,
A. A. Marti, J. Dyer, H. Vishwasrao, D. L. Atkins, E. R. Kandel
and N. J. Turro, Chem. Commun., 2009, 2640; A. J. Amoroso,
R. J. Arthur, M. P. Coogan, J. B. Court, V. Fernandez-Moreira,
A. J. Hayes, D. Lloyd, C. Millet and S. J. A. Pope, New J. Chem.,
2008, 32, 1097; (b) For ruthenium-based nuclear DNA staining:
M. R. Gill, J. Garcia-Lara, S. J. Foster, C. Smythe, G. Battaglia
and J. A. Thomas, Nat. Chem., 2009, 1, 662.
9 P. Atkinson, K. S. Findlay, F. Kielar, R. Pal, D. Parker,
R. A. Poole, H. Puschmann, S. L. Richardson, P. A. Stenson,
A. L. Thompson and J. Yu, Org. Biomol. Chem., 2006, 4, 1707.
10 A. Thibon and V. C. Pierre, J. Am. Chem. Soc., 2009, 131, 434.
11 E. E. Kilbourn and M. C. Siedel, J. Org. Chem., 1972, 37, 1145.
12 G.-L. Law, D. Parker, S. L. Richardson and K.-L. Wong, Dalton
Trans., 2009, 8481.
13 A. Beeby, I. M. Clarkson, R. S. Dickins, S. Faulkner, D. Parker,
L. Royle, A. S. de Sousa, J. A. G. Williams and M. Woods,
J. Chem. Soc., Perkin Trans. 2, 1999, 493.
Fig. 3 Time course (5 min intervals) of fluorescence microscopy
images staining chromatin in a cell undergoing division, showing the
progression from interphase (top left for comparison) via prophase to
prometaphase and metaphase (50 nM [TbÁL2]Cl, scale bar 5 mM, HeLa
cell, lexc 300 nm).
illumination, leading to spindle checkpoint activation and a
pause in the observed cell cycle. In control experiments,
assessing the cellular toxicity of [TbÁL1]+ and [TbÁL2]+, IC50
values of >400 mM were estimated, based on the perturbation
of mitochondrial redox (using ‘MTT’ or ‘WST-1’ methods).
Therefore, future studies with such probes will focus on using
less intense irradiation or will use two photon microscopy
methods, with excitation wavelengths in the near-IR range of
710 to 780 nm that are less likely to create problems of
phototoxicity.
In summary, the low chemical toxicity and ability of the
terbium complexes to stain the nuclear DNA in mitotic cells
augur well for the development of these and related
metal-based optical probes8b that are capable of visualising
DNA events in living cells. Future work will also address the
mechanism of cell uptake at low added complex concentration
in various cell types. For the cells examined here, this is likely
to be different from the pathway of macropinocytosis5a
defined for many lanthanide complexes recently.
14 R. S. Dickins, J. I. Bruce, D. Parker and D. J. Tozer, Dalton
Trans., 2003, 1264; D. Parker, Chem. Soc. Rev., 2004, 33, 156.
15 G. Bobba, J.-C. Frias, M. J. Cann, D. Parker and C. J. Hutchison,
Org. Biomol. Chem., 2003, 1, 905; R. A. Poole, G. Bobba,
M. J. Cann, J.-C. Frias, D. Parker and R. D. Peacock, Org.
Biomol. Chem., 2005, 3, 1013.
We thank EPSRC and CISbio s.a. for support.
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This journal is The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 2391–2393 | 2393