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Blood, Vol. 93 No. 4 (February 15), 1999:
pp. 1197-1207
The Nuclear Topography of ABL, BCR, PML, and RAR Genes:
Evidence for Gene Proximity in Specific Phases of the Cell Cycle
and Stages of Hematopoietic Differentiation
By
Hélia Neves,
Carlos Ramos,
Maria Gomes da Silva,
António Parreira, and
Leonor Parreira
From the Institute of Histology and Embryology, Lisbon Medical
School, Lisbon, Portugal; and the Service of Hematology, Portuguese
Institute of Cancer, Lisbon, Portugal.
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ABSTRACT |
The mechanisms whereby chromosomal translocations are consistently
associated with specific tumor types are largely unknown. A generally
accepted hypothesis is that the physical proximity of the involved
chromosomal regions may be one important factor in the genesis of these
phenomena. Accordingly, a likely possibility is that such a proximity
may occur in a cell-lineage and cell-differentiation stage-specific
manner. In this work, we have addressed this issue using as models the
ABL and BCR genes of t(9;22) and the PML and RAR genes of t(15;17).
By using in situ hybridization and confocal microscopy, we have
measured the distances between these two pairs of genes in
three-dimensionally preserved hematopoietic cells belonging to
different cell lineages, at various stages of differentiation, and at
various stages of the cell cycle, with the following results. (1)
Intergenic distances vary periodically during the cell cycle and a
significant association of ABL with BCR and of PML with RAR is seen
at the transition between S and G2, which persists during G2 and
prophase (such a behavior is not observed for distances between ABL or
PML and the  -globin genes, used as a control). (2) The proportion of
cells in which PML and RAR or ABL and BCR are closely associated is
higher in hematopoietic precursors than in B-lymphoid cells (whereas
the distances between ABL or PML and the -globin genes are not
affected by cell type). (3) When intergenic distances in unstimulated
bone marrow CD34+ cells were compared with those in
CD34+ cells treated with interleukin-3 (IL-3), a trend
towards a higher proximity of the ABL and BCR genes in the former and
of the PML and RAR genes in the latter is observed. (4) Analysis of
B-lymphoid cells during mitosis shows that intergenic distances at
metaphase are strongly influenced by physical constraints imposed by
the chromosomal location of the gene, by the size of the respective chromosome, and by the geometry of the metaphase plate. These findings
suggest that intrinsic spatial dynamics, established early in
hematopoiesis and perpetuated differentially in distinct cell lineages,
may facilitate the collision of individual genes and thus reciprocal
recombination between them at subsequent stages of hematopoietic differentiation.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
SINCE THE DISCOVERY of the t (9;22) in
chronic myeloid leukemia (CML), several types of reciprocal chromosomal
translocations have been consistently associated with human
cancer.1,2 The molecular characterization of
chromosomal breakpoints has shown that the same genomic regions are
systematically involved in each specific type of translocation, but,
despite this knowledge, the mechanisms underlying interchromosomal
recombination phenomena in somatic cells are largely unknown. In those
circumstances in which loci of antigen-receptor genes are involved, it
has been postulated that the enzymatic systems responsible for the
normal somatic VDJ rearrangements of these genes in normal B- or T-cell ontogeny may be subverted to promote the illegitimate recombination with other genomic regions.1,3 Also, it has been suggested that the higher susceptibility of certain chromosomal regions to
breakage and rearrangement may be related to local structural features
of the chromatin fiber that make them more susceptible to damage than
other genomic regions.4 Recently, it has been further
proposed that reciprocal rearrangements such as those underlying the
formation of complex BCR-ABL genes in cells of CML patients might be
mediated by the mutual attraction of Alu sequences located in
heterologous chromosomes.5
Irrespective of the exact nature of the biological mechanisms
responsible for the occurrence of chromosomal translocations in somatic
cells, a widely accepted assumption is that the spatial proximity of
the involved chromosomal regions is likely to be an obligatory
requirement for the exchange event to occur.6 Recent
reports appear to support this view, because the ABL and BCR genes were
found to be in close proximity in two-dimensional human
lymphocytes7 and total bone marrow cells8 more
often than would have been expected by chance. However, a still open question is whether the spatial relationships between these or other
loci frequently engaged in reciprocal recombination phenomena in
leukemic cells relate to a particular cell lineage, stage of cell
differentiation, and/or to specific phases of the cell cycle.
In this work, we sought to investigate these issues using as models the
ABL and BCR genes from the t(9;22) of CML and the PML and RAR genes
from t(15;17) of acute promyelocytic leukemia. Apart from their
consistent involvement in specific types of human neoplasia, a further
reason making these genes specially suited for the addressing of these
questions is that the stage of hematopoietic differentiation in which
each translocation takes place can be predicted with reasonable
certainty: the t(9;22) is present in cells of all hematopoietic
lineages in patients with CML,9 whereas the t(15;17) has
been exclusively observed in malignant promyelocytes.10
Accordingly, the BCR-ABL rearrangement can be envisaged as representing
an oncogenic molecular lesion of a very-early hematopoietic progenitor,
whereas the PML-RAR rearrangement is likely to occur in a precursor
already committed to the myelo-erythroid11 or granulocytic12 differentiation.
We have therefore analyzed the distances between these genes in
hematopoietic cells belonging to different cell lineages and at various
stages of differentiation and of the cell cycle. The analysis of gene
positioning in bidimensional cells may induce important artifacts that
make it difficult to interpret the results from in situ hybridization
techniques,13 thus requiring the use of mathematical
corrections for three-dimensional (3-D) reconstruction of the
data.7,8 Therefore, we have measured intergenic distances in 3-D preserved cells using nonisotopic in situ hybridization and
confocal microscopy. We show that, irrespective of their behavior in
earlier stages of the cell cycle, each of these pairs of genes tends to
become closely associated at the transition of late-S to G2 in all cell
types analyzed and, in hematopoietic precursors in interphase, the
interdistances PML-RAR and ABL-BCR are smaller than those observed
on cells of mature B phenotype. Also, when intergenic distances in
unstimulated CD34+ cells were compared with those in cells
treated with interleukin-3 (IL-3), a tendency towards a higher
proximity of the ABL and BCR genes in the former and of the PML and
RAR genes in the latter were observed.
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MATERIALS AND METHODS |
Cells
Hematopoietic precursors.
Hematopoietic progenitors were enriched from 20 to 30 mL heparinized
normal bone marrow obtained from donors of the bone marrow transplantation program, after informed consent had been obtained. Mononuclear cells were isolated by density gradient centrifugation (Ficoll-Paque; Pharmacia, Uppsala, Sweden) and
CD34+ cells subsequently obtained using the MiniMACS
separation column (Miltenyi Biotec Inc, Auburn, CA). Technical
procedures were performed according to the instructions included in the
CD34 Progenitor Cell Isolation Kit (QBEND/10, Auburn, CA). In
preliminary experiments, the phenotypic profile of the selected
CD34+ cells was assessed by flow cytometric analysis in
five independent bone marrow samples, with the following results (mean ± SEM): CD34+, 90.8% ± 2.1%; CD38+,
97.4 % ± 0.4%; HLA-DR+, 92% ± 1.6%;
CD33+, 59.7% ± 4.5%; CD7+, 6.2% ± 0.9%; CD3+, 1.8% ± 0.7%; CD19+, 24.5% ± 5.6%; CD56+, 1.0% ± 0.2 %; and
CD71+, 25.8% ± 7.1% (all antibodies from Becton
Dickinson, Mountain View, CA). Double-labeling experiments
performed in three of the five samples further showed that the
CD34+ cells simultaneously expressed HLA-DR in 97.1% ± 0.63%, CD38 in 98.9% ± 0.53%, and CD33 in 66.4% ± 4.85%. Accordingly, unstimulated cells with these characteristics were
used in this study as representative of a cell pool enriched in early
hematopoietic precursors.
To obtain a cell population preferentially committed to the myeloid
differentiation, CD34+ cells were cultured in 96-microwell
plaques at 105 cells/mL in Iscove's Modified
Dulbecco's Medium (IMDM) supplemented with 5% fetal calf serum (FCS;
GIBCO-BRL, Gaithersburg, MD), 1% penicillin/streptomycin (GIBCO-BRL),
and recombinant IL-3 (10 ng/mL; Sandoz-Pharma, Basel, Switzerland) at
37°C and 5% CO2. Morphological analysis of cytospin
preparations obtained from three independent samples of
CD34+ cells on 7 consecutive days and stained with
May-Grunwald-Giemsa showed the following: first 48 hours undifferentiated blasts, 70% to 82%; day 3undifferentiated
blasts (45% to 48%) and promyelocytes (52% to 55%); day 4undifferentiated blasts (30% to 33%), promyelocytes (55% to
65%), and monocytes (2% to 15%); day 5undifferentiated blasts
(12% to 15%), promyelocytes (40% to 45%), myelocytes (20% to
24%), and monocytes (17% to 19%); day 6undifferentiated blasts (7% to 8%), promyelocytes (18% to 20%), myelocytes (8% to 10%), and monocytes (40% to 55%); day 7undifferentiated blasts (5% to
7%), promyelocytes (18% to 23%), myelocytes (8% to 10%), and monocytes (46% to 50%). No erythroid differentiation was observed in
these conditions. On the basis of these results, CD34+
cells treated with IL-3 for 48 hours were subsequently used in the
study as a cell population enriched in progenitors already committed to
the myeloid differentiation.
Lymphoid cells.
A mature B-lymphoid cell line (IM-9; ECACC, Salisbury, UK) was used as
representative of a nonmyeloid cell population. This cell line has a
diploid modal chromosomal number and was cultured in suspension in
RPMI-1640 (GIBCO-BRL) supplemented with 10% FCS (GIBCO-BRL).
In Situ Hybridization in 3-D Preserved Cells
Cell preparation.
Unstimulated CD34+ cells (CD34+),
CD34+ stimulated with IL-3 for 48 hours (CD34+,
IL-3), and IM-9 cells were harvested by centrifugation at 50g for 5 minutes and applied onto poly-L-lysine-coated 10 mm/10 mm cover
slips. This was immediately followed by fixation and permeabilization under conditions that preserve the three-dimensionality of the cells.
Briefly, the cells were fixed and extracted in 3.7%
paraformaldehyde/0.5% Triton X-100/HPEM (65 mmol/L PIPES, 30 mmol/L
HEPES, 10 mmol/L EGTA, 2 mmol/L MgCl2, pH
6.9) for 15 minutes at room temperature,14 followed by permeabilization in 0.7% Triton X-100/0.1 N
HCl/phosphate-buffered saline (PBS) for 10 minutes, on ice, as
described.14 Subsequently, cells were incubated with 0.1 mg/mL RNase A in 2× SSC for 30 minutes at 37°C. Before the
hybridization, cells were denatured in 50% formamide in 2× SSC
for 20 minutes at 80°C.15
Molecular probes and in situ hybridization.
The PML, RAR, ABL, and BCR (Mbcr) genes were visualized with
specific probes purchased from Oncor (Gaithersburg, MD) according to
the instructions of the manufacturer.
A c-DNA probe for the -globin gene (a 3.7-kb Cla
I/Kpn I fragment; kindly provided by Dr Mike Antoniou, Guy's
Hospital, London, UK) was nick-translated with dinitrophenyl-11-dUTP
(DNP; Molecular Probes, Leiden, The Netherlands), as described by
Johnson et al.16 The -globin genes (GLB) were used in
this study as a control, representing gene sequences that are not
usually involved in recombination phenomena in leukemic cells.
Chromosome painting probes for chromosomes 9 and 11 (digoxigenin-labeled; Oncor) and 15 (biotin-labeled; Cambio, Cambridge, UK) were hybridized and detected according to the instructions of the
manufacturer. For double-hybridization experiments, additional probes
for chromosomes 9, 17, and 22 were kindly provided by Dr J. Gray
(Biomedical Science Division, Lawrence Livermore National Laboratory,
San Francisco, CA)17 and nick-translated with biotin-16-UTP (Boehringer Mannheim, Mannheim, Germany; chromosome 9) or
digoxigenin-11-dUTP (Boehringer Mannheim; chromosomes 17 and 22). For
each sample, 30 ng/µL of each labeled probe (in the case of
noncommercial painting probes for chromosomes 15 and 17) or 100 ng/mL
(for chromosome 9 biotin-labeled probe) were combined with Human Cot 1 DNA (GIBCO-BRL; 0.560 mg/mL in the case of probe for chromosome 9 and 2 mg/mL for the remaining probes), ethanol-precipitated, air-dried, and dissolved in hybridization buffer (50% formamide, 2× SSC, 10% dextran sulphate, 50 mmol/L phosphate buffer, pH 7.0).
All the procedures for in situ hybridization, signal detection, and
simultaneous visualization of the nuclear envelope were performed as
previously described.13,18,19 The probe for the GLB genes
was denatured for 10 minutes at 75°C, combined with the other
gene-specific probes, and, after the hybridization procedure, detected
with a rabbit anti-DNP antibody (1:100; Molecular Probes) at 37°C
for 30 minutes and washed in 0.05% Tween 20/PBS (3× for 5 minutes), followed by incubation with a Texas-Red-conjugated goat
antirabbit Ig (1:50; Jackson Immunoresearch Laboratories, West Grove,
PA). For the simultaneous visualization of hybridization sites and the
nuclear envelope, the samples were incubated immediately after the
detection of the hybridization signals with a rabbit anti-lamin-B
antibody (kindly provided by Dr S. Georgatos, Heidelberg, Germany),
diluted at 1:100 at 37°C for 30 minutes and washed as described,
followed by incubation with goat Texas-Red-conjugated antirabbit Ig
(Jackson Immunoresearch Laboratories) at 37°C for 30 minutes.
Identification of the Cell Cycle Phases
Cells were cultivated under the presence of Bromodeoxyuridin (BrdU) as
previously described.13 Briefly, BrdU (Boehringer Mannheim)
was added at 10 µmol/L to the cell culture for 20 minutes, followed
by fixation and permeabilization procedures. The detection of
incorporated BrdU was performed after the in situ hybridization step
using a Cy5-conjugated anti-BrdU antibody (1:50; Jackson Immunoresearch
Laboratories). In these conditions, it is possible to identify cells in
the S-phase (those that incorporated BrdU) as well as to discriminate
different stages within the S-phase based on the morphological patterns
of BrdU incorporation.20
To identify cells in the G2 phase of the cell cycle, the following
pulse and chase strategy was used: the duration of the G2 phase was
first determined in the IM-9 cell line according to the methods
previously described21,22 and found to be approximately 2 hours and 30 minutes. On the basis of this information, the cells were
then cultured in the presence of BrdU at 10 µmol/L for 20 minutes,
followed by the removal of the culture medium by centrifugation at
50g for 5 minutes and subsequently cultured in fresh medium
without BrdU for a period of time inferior to the duration of G2 (2 hours and 15 minutes). At this time point, the cells were
harvested, fixed, permeabilized, and hybridized as described above. In
these conditions, those cells exhibiting a late-S pattern of BrdU
incorporation were considered to be in G2 and were selected for
analysis of intergenic distances (assuming that the duration of G2 does
not vary markedly in different cell types, the same value was accepted
for the duration of G2 in bone marrow cells).
In IM-9 cells, the intergenic distances were also analyzed along the
sequential phases of mitosis (prophase, metaphase, anaphase, and
telophase) that were directly identified under the microscope by DNA
staining with 4'6-diamidino-2-phenylindole (DAPI).
Determination of Intergenic Distances
Criterion for gene proximity.
Intergenic distances  2 µm were used as a criterion for gene
proximity. The rationale was the following. First, mathematical models
emerging from studies of clastogenesis induced by ionizing irradiation
indicate that "free ends from double strand breaks initially formed
as much as ~2 µm apart can apparently interact,"6 a
possibility that implies a certain degree of freedom as to the movement
of chromatin within the nucleoplasm. Second, the latter assumption is
supported by recent in vivo observations in which it is shown that
particular chromatin segments do indeed undergo free Brownian movements
within a constrained subregion, the radius of which is 0.5 µm in
yeast nuclei and 0.9 µm in Drosophila embryo nuclei.23
Accordingly, an absolute requirement for two loci to interact would be
the overlap of their confined regions,23 which could easily
happen if two chromatin segments are separated by less than 2 µm in a
human hematopoietic cell nucleus with a diameter of 7 to 8 µm. Third,
further evidence suggesting that a distance of 2 µm may be sufficient
for the establishment of functional (physical?) interactions between
two distinct chromatin regions is that provided by the work of LaSalle
and Lalande,24 in which it is shown that the imprinted
homologous loci of Prader-Willi and Angelman syndromes become
transiently associated, at distances 2 µm, in the nucleus of
T-lymphoid cells during a short period of the S phase. Taken together,
the above arguments indicate that what could have been considered, at
first sight, a substantial distance for an average nucleus with a
diameter of 8 µm may well be compatible with the physical interaction
of two distinct chromatin regions.
Confocal microscopy analysis.
The analysis of hybridization signals in 3-D preserved nuclei was
performed with the confocal microscope Zeiss LSM-410 (Carl Zeiss,
Oberkochen, Germany) as previously described.18,19 The nuclei were selected for analysis according to the following criteria: adequate morphological preservation of chromatin (as assessed by DAPI
staining); integrity of the nuclear envelope (visualized with an
anti-lamin B antibody); and identification of all alleles by the
presence of distinct hybridization signals.
Thirty-one optical sections were obtained for each nucleus. Because in
almost all cells the hybridization signal for each gene could be seen
in several sequential planes, the section containing the brightest
signal was selected and recorded for further analysis with a macro
specifically developed for the NIH Image 1.55 software (Wayne Rasbaud,
NIH, Springfield, VA). For each gene, the absolute coordinates of the
center of each hybridization signal were determined (15 pixels = 1 µm
in the x and y axes; the average increment between sections along the z
axis was 0.25 µm, varying from 0.2 to 0.3 µm). Each of the
distances between homologous genes (dx-x) and the minimum
distance between heterologous genes (dx-y) were then calculated. One example of optical series obtained in IM-9 cells with
closely associated ABL and BCR genes is shown in
Fig 1.
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| Fig 1.
Optical series obtained in IM-9 cells hybridized with
gene-specific probes showing a nucleus from a late-S cell with closely
associated ABL (green) and BCR (red) genes (arrows). The nuclear
envelope is labeled with an anti-lamin B antibody (red).
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Statistical Analysis
The  2 test was applied to investigate the hypothesis
that the proportion of cells with dx-y 2 µm was the
same throughout the subphases of the cell cycle for each pair of genes
studied. When the null hypothesis was rejected, multiple comparisons
were made between successive subphases using multiple 2
tests. A similar strategy was used to investigate whether the proportion of cells with dx-y 2 µm was the same between
each pair of genes and its control. The level of significance chosen for all the tests was = .05. A total of 2,864 nuclei were analyzed, 50 for each pair of genes and each subphase of the cell cycle, except
in G2 (30 to 35 nuclei) and distances between the ABL and PML genes and
the control gene GLB in interphase (30 nuclei for each subphase and
pair of genes). Because the procedures for BrdU pulse and chase
experiments (see above) were consistently accompanied by inadequate 3-D
preservation in freshly eluted CD34+ cells, the G2 phase of
the cell cycle was not investigated in unstimulated CD34+
cells. The data concerning the PML-RAR and ABL-BCR interdistances in
unstimulated CD34+ cells in G1 were obtained from two
independent bone marrow donors: cells with intergenic distances 2
µm for PML and RAR genes were 50% and 52%, respectively
(P = .932), and for ABL and BCR genes were 50% and
62%, respectively (P = .536).
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RESULTS |
Intergenic Distances PML-RAR and ABL-BCR in IM-9 Cells
Throughout the Cell Cycle
The analysis of intergenic distances in IM-9 cells grown in the
presence of BrdU, in conditions that allow the discrimination of G1,
early-S, middle-S, late-S, and G2, shows that
(Fig 2) (1) in all phases of the cell cycle
there are cells in which the intergenic distances are 2 µm and (2)
the proportion of cells with closely associated genes vary during the
cell cycle. As to the PML and RAR genes in interphase (Fig 2A), this
proportion decreases from G1 to early-S (proportion ± standard
error; 41% ± 0.074% and 17% ± 0.057%, respectively;
P = .013), remaining stable during early-S and middle-S (16% ± 0.056%). At the transition of late-S to G2, a gradual increase
in the proportion of cells with gene proximity was observed (21% ± 0.063% to 29% ± 0.071%, respectively) that persists in
G2 and prophase (30% ± 0.062% in prophase), reaches a maximum in
metaphase (64% ± 0.071%; prophase v metaphase, P < .001), decreases in anaphase (41% ± 0.068%; metaphase
v anaphase, P = .023), and returns to values similar to
those of G1 in telophase (31% ± 0.076%; Fig 2A).
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| Fig 2.
Distribution of IM-9 cells with intergenic distances 2
µm during the cell cycle. (A) PML-RAR interdistances. (B) ABL-BCR
interdistances. See text (Results) for statistically significant
differences. E-S, early-S; M-S, middle-S; L-S, late-S; Pro, prophase;
Met, metaphase; Ana, anaphase; Tel, telophase.
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The analysis of the distances between the genes ABL and BCR shows that
the proportion of cells with intergenic distances 2 µm is stable
during G1 and all S phase (G1, 22% ± 0.061%; early-S, 23% ± 0.060%; middle-S, 27% ± 0.063%; and late-S, 18% ± 0.056%; Fig 2B). Again, after late-S, a progressive increase of cells with
close ABL and BCR genes, which persists in G2 and prophase (late-S
v G2, P = .016), can be observed (36% ± 0.074%
and 34% ± 0.072%, respectively). However, in contrast with the
PML and RAR genes, metaphase is the phase of the cell cycle in which the proportion of cells with close ABL-BCR genes reaches its minimum (11% ± 0.045%; prophase v metaphase, P = .007). A
return to values of G1 is again observed in telophase (14% ± 0.051% in anaphase and 27% ± 0.065% in telophase; Fig 2B).
In summary, the data show that the distances between PML and RAR and
between ABL and BCR genes vary along the sequential phases of the cell
cycle. Also observed is a common emerging pattern, which consists in an
increase of cells with gene proximity beyond the late stages of S phase
that persists during G2 and prophase and a return to a G1 pattern of
interdistances in telophase.
Topography of Chromosomes 15, 17, 9, and 22 During Mitosis in IM9
Cells
To investigate the reasons for the opposite behavior of PML-RAR and
ABL-BCR interdistances in metaphase, and assuming that they might be
caused by constraints inherent to the formation of the metaphase plate,
we subsequently proceeded to the analysis of the spatial relationships
between the chromosomes where these genes are located during the stages
of mitosis. Thus, genomic libraries specific to chromosomes 15, 17, 9, and 22 were used in double hybridization experiments in 3-D preserved
mitotic cells. The results obtained are shown in
Fig 3. A high percentage of cells with
close proximity of chromosomes 15 and 17 (Fig 3A) and chromosomes 9 and
22 (Fig 3B) was observed in all stages of mitosis, which differed
significantly from distances between chromosomes 15 and 11 and 9 and
11: proximities between chromosomes 15 and 17 versus chromosomes 11 and
17 were P < .05 in metaphase and anaphase; and
proximities between chromosomes 9 and 22 versus 9 and 11 were P < .05 in metaphase and telophase. It is noteworthy that, in
metaphase, chromosomes 9 and 22 were adjacent in more than 60% of
cells, contrasting with the ABL and BCR genes, which were found to be
in close proximity in only 11% of metaphase cells (see Figs 2B and
3B). The reason for this discrepancy became evident when observing the
arrangement of chromosomes in the metaphase plate. In fact, during
metaphase, the chromosomes always assume a spatial arrangement in
which the centromeres are oriented toward the center of the
rosette, whereas the long and short arms are extended towards the
periphery (see diagram in Fig 4). Because the ABL gene is a subtelomeric gene in the long arm of chromosome 9, which is substantially larger than chromosome 22, and the BCR gene is
located not very far from the centromere of chromosome 22, one of the
shortest human chromosomes, the two genes are forced to move apart
during metaphase, even if they have been in close proximity in earlier
stages of the cell cycle (see illustrating examples in Fig
4). Such a behavior is not observed for the PML and RAR genes,
because both are located approximately at the middle of two short
chromosomes with similar sizes.
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| Fig 3.
Distribution of IM-9 cells with adjacent chromosomes
during mitosis. (A) Proximities between chromosomes 15 and 17 versus
chromosomes 11 and 17: P < .05 in metaphase and anaphase. (B)
Proximities between chromosomes 9 and 22 versus chromosomes 9 and 11:
P < .05 in metaphase and telophase. The legends are
as in Fig 2.
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| Fig 4.
Optical sections from three IM-9 cells in metaphase
hybridized with painting probes for chromosomes 9 (red) and 22 (green).
Note that the signal corresponding to chromosome 22 is smaller and more
centrally located than that of chromosome 9. The diagram on the right
represents the arrangement of chromosomes in a metaphase rosette and
its potential effect on ABL-BCR and PML-RAR interdistances.
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It is thus possible to conclude that intergenic distances during
metaphase are strongly influenced by physical and geometrical constraints related to the location of the gene in the chromosome, the
size of the chromosome, and the geometry of the metaphase plate.
Comparison of PML-RAR and ABL-BCR Interdistances With
Those Between PML and -Globin and ABL and
-Globin Genes in IM-9 Cells
Having determined the dynamics of intergenic distances during the cell
cycle, we subsequently asked whether the observed proximity was
specific to these pairs of genes. Therefore, the PML-RAR and ABL-BCR
interdistances were compared with those between PML-GLB and ABL-GLB in
all stages of the cell cycle. As shown in
Fig 5A and B, there are no significant
differences during G1, early-S, and middle-S between the two cell
populations (all P > .05). However, a clear divergence was
observed after late-S, with a progressive reduction in the proportion
of cells with close PML-GLB (31% ± 0.076% in late-S and
19% ± 0.065% in G2; Fig 5A) and ABL-GLB (27% ± 0.075% in
late-S and 20% ± 0.080% in G2; Fig 5B) as opposed to the increase
of cells with close PML-RAR (21% ± 0.063% in late-S and 29% ± 0.071% in G2) and ABL-BCR (18% ± 0.056% in late-S and 36% ± 0.074% in G2). Therefore, it appears that the PML and RAR or
ABL and BCR are specifically associated during G2 and prophase in IM-9
cells.
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| Fig 5.
Distribution of IM-9 cells with intergenic distances 2
µm during the cell cycle. (A) Comparison of distances PML-RAR with
PML-GLB. (B) Comparison of distances ABL-BCR with ABL-GLB. (C)
Comparison of distances PML-RAR with PML-PML and RAR-RAR. (D)
Comparison of distances ABL-BCR with ABL-ABL and BCR-BCR. See text
(Results) for statistically significant differences. The legends are as
in Fig 2.
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The comparison of the distances PML-RAR and ABL-BCR with those
between homologous genes (Fig 5C and D) also shows that distances between heterologous genes are shorter than those between homologous genes. Statistically significant differences are as follows: for PML-RAR versus PML-PML, P < .05 in G1, G2, metaphase, and
anaphase; for PML-RAR versus RAR-RAR, P < .05 in G2,
prophase, metaphase, and anaphase; for ABL-BCR versus ABL-ABL,
P < .05 in G1, late-S, G2, prophase, and telophase and for
ABL-BCR versus BCR-BCR in G1 and middle-S.
PML-RAR and ABL-BCR Interdistances in Immature
Normal Hematopoietic Cells
Having studied the intergenic distances in B-lymphoid cells, the
analysis has subsequently progressed to normal hematopoietic progenitors in interphase.
The analysis of PML and RAR genes in unstimulated CD34+
in G1 and S phases (G2 not performed) is shown in
Fig 6A. As in IM-9 cells, variations along
the sequential stages of the cell cycle can be observed. The proportion
of cells with gene proximity were 52% ± 0.072% in G1, 30% ± 0.066% in early-S, 27% ± 0.061% in middle-S, and 39% ± 0.089% in late-S (P = .027 at the transitions of G1 to early-S
and of middle-S to late-S). However, the proportion of
CD34+ cells with close PML and RAR genes is always
higher than that observed in IM-9 cells in all subphases analyzed
(P = .007 in late-S; see Fig 8A). Furthermore, in contrast with
IM-9 cells, the proportion of cells with PML-RAR proximity is always
higher than that of cells with PML-GLB proximity (cells with close
PML-GLB genes were 38% ± 0.068% in G1, 23% ± 0.075% in
early-S, 16% ± 0.066% in middle-S, and 7% ± 0.089% in
late-S), a difference that becomes more noticeable after middle-S
(P < .0001 in late-S; Fig 6A).
View larger version (28K):
[in this window]
[in a new window]
| Fig 6.
Distribution of bone marrow cells with intergenic
distances 2 µm during interphase. (A) CD34+ cells.
Comparison of distances PML-RAR with PML-GLB. (B)
CD34+ cells. Comparison of ABL-BCR with ABL-GLB. (C)
CD34+ cells stimulated with IL-3. Comparison of distances
PML-RAR with PML-GLB. (D) CD34+ cells stimulated
with IL-3. Comparison of distances ABL-BCR with ABL-GLB. See text
(Results) for statistically significant differences. The legends are as
in Fig 2.
|
|
The analysis of ABL-BCR distances in unstimulated CD34+
cells is shown in Fig 6B. Cells with close ABL and BCR genes were 56% ± 0.068% in G1, 42% ± 0.069% in early-S, 37% ± 0.066%
in middle-S, and 41% ± 0.091% in late-S (P = .045 at the
transition of G1 to early-S). Similar to what was observed for the PML
and RAR genes, the proportion of cells with close ABL and BCR genes
is higher than those with ABL-GLB proximity (P < .05 in
early-S; cells with close ABL and GLB genes were 41% ± 0.091% in
G1, 14% ± 0.066% in early-S, 30% ± 0.083% in middle-S, and
20% ± 0.070% in late-S) and markedly higher than in IM-9 cells,
with statistically significant differences in G1 (P < .0001),
early-S (P = .044), and late-S (P = .026)
(see Fig 8B).
The results obtained in CD34+ stimulated with IL-3 for 48 hours, in interphase, are depicted in Fig 6C and D. There is again a
higher proportion of cells with close PML-RAR than with close PML-GLB (respectively: G1, 51% ± 0.070% and 30% ± 0.083%; early-S, 38% ± 0.067% and 19% ± 0.070%; middle-S,
35% ± 0.065% and 27% ± 0.080%; late-S, 42% ± 0.069%
and 23% ± 0.077%; G2, 55% ± 0.086% and 24% ± 0.079%;
P = .015 in G2; Fig 6C) and with close ABL-BCR genes than with
close ABL-GLB (respectively: G1, 57% ± 0.070% and 26% ± 0.078%; early-S, 30% ± 0.072% and 33% ± 0.086%;
middle-S, 33% ± 0.071% and 26% ± 0.078%; late-S, 28% ± 0.094% and 13% ± 0.062%; G2, 40% ± 0.086% and 20% ± 0.073%; P = .006 in G1 and P = .019 in late-S;
Fig 6D). The distances ABL-BCR differ significantly at the
transition G1-early S (P = .01). In IL-3-stimulated cells, the
proportions of cells with close PML and RAR genes were higher than
in IM-9 cells (P = .020 in early-S, P = .043 in
middle-S, and P = .036 in late-S; see Fig 8A), the same
happening for interdistances ABL-BCR (P = .001 in G1; see Fig
8B).
The proportion of cells with PML-RAR or ABL-BCR proximity is also
higher than that with proximity between homologous genes in
CD34+ cells with or without stimulation with IL-3, a
difference that is more conspicuous than that observed in the lymphoid
cell line (Fig 7; see also Fig 5). In
CD34+ cells, PML-RAR interdistances differed
significantly from PML-PML in all subphases (P < .005)
and from RAR-RAR in G1 and late-S (P < .005; Fig 7A);
the ABL-BCR distances differed significantly from ABL-ABL and from
BCR-BCR in all subphases (P < .0001 in G1 and P < .05 in the remaining phases for ABL-BCR v ABL-ABL; P < .005 in G1 and early-S and P < .05 in middle-S and late-S
for ABL-BCR v BCR-BCR; Fig 7B).
View larger version (30K):
[in this window]
[in a new window]
| Fig 7.
Distribution of bone marrow cells with intergenic
distances 2 µm during interphase. (A) CD34+ cells.
Distances PML-RAR versus PML-PML and PML-RAR versus
RAR-RAR. (B) CD34+ cells. Distances ABL-BCR versus
ABL-ABL and ABL-BCR versus BCR-BCR. (C) CD34+ cells
stimulated with IL-3. Distances PML-RAR versus PML-PML and
PML-RAR versus RAR-RAR. (D) CD34+ cells
stimulated with IL-3. Distances ABL-BCR versus ABL-ABL and ABL-BCR
versus BCR-BCR. See text (Results) for statistically significant
differences. The legends are as in Fig 2.
|
|
In CD34+ cells stimulated with IL-3, interdistances
PML-RAR differed significantly from PML-PML and RAR-RAR in all
subphases (P < .05 in G1 and P < .0005 in the
remaining phases for PML-RAR v PML-PML; P < .0001 in G1, early-S, late-S, and G2 and P < .05 in middle-S for
PML-RAR v RAR-RAR; Fig 7C). The distances ABL-BCR differed from ABL-ABL in all subphases except middle-S (P < .0001 in G1, early-S, late-S, and G2) and from BCR-BCR in G1 (P < .0001), early-S (P < .05), and middle-S (P < .0005) (Fig 7D).
When the distances PML-RAR and ABL-BCR in unstimulated
CD34+ cells were compared with those observed in
IL-3-stimulated CD34+ cells, no statistically significant
differences were found. However, a higher proximity for the ABL and BCR
genes was observed in unstimulated CD34+ cells
(Fig 8B), whereas the distances between PML
and RAR were shorter in cells stimulated with IL-3 (Fig 8A).
View larger version (15K):
[in this window]
[in a new window]
| Fig 8.
Comparison of bone marrow and IM-9 cells with intergenic
distances 2 µm during interphase. (A) Distances PML-RAR in IM-9
cells, CD34+ cells and CD34+ cells
stimulated with IL-3. (B) Distances ABL-BCR in IM-9 cells,
CD34+ cells and in CD34+ cells stimulated
with IL-3. See text (Results) for statistically significant
differences. The legends are as in Fig 2.
|
|
In summary, the analysis of intergenic distances in early hematopoietic
precursors shows that, similar to what happens in IM-9 cells, there are
variations in intergenic distances during interphase. It was also
observed that the proportion of bone marrow cells in interphase with
close proximity between PML and RAR or ABL and BCR genes is higher
than that observed in IM-9 cells. Furthermore, a higher ABL-BCR
proximity in unstimulated CD34+ cells and a higher
PML-RAR proximity was observed in cells that were stimulated with
IL-3 for 48 hours.
| |
DISCUSSION |
In this work, we have measured the distances between PML and RAR and
between ABL and BCR genes in 3-D preserved hematopoietic cells
belonging to different cell lineages at various stages of differentiation and of the cell cycle. We observed that each pair of
genes tends to become associated at the transition of late-S to G2 in
all cell types analyzed, a behavior not observed for distances between
ABL or PML and the -globin genes. Furthermore, the proportion of
cells in which PML and RAR or ABL and BCR are closely associated is
higher in hematopoietic precursors than in B-lymphoid cells, in
contrast with distances between ABL or PML and the -globin genes,
which are not affected by cell type.
Because the cell cycle phase is known to influence the topography of
chromatin within the nucleus,25-27 we first asked whether intergenic distances change during the sequential phases of the cell
cycle. The results in IM-9 cells show that distances between heterologous genes vary periodically during the cell cycle as a return
to a G1-pattern of gene interdistances was consistently observed at
telophase. Furthermore, the observed fluctuations in the percentage of
cells with gene proximity indicate that in each phase of the cell cycle
there is a proportion of cells in which all the different pairs of loci
under analysis are in close proximity. However, when PML-RAR or
ABL-BCR interdistances were compared with those between PML, or ABL,
and the control gene, the emerging pattern was that, starting at the
transition of late-S to G2, there is a trend towards a greater
proximity between the PML and RAR, or ABL and BCR genes, as opposed
to distances between PML or ABL and the -globin gene. These findings
contrast with those reported by Kozubek et al7 in which
distances between the ABL and BCR genes were found to increase during
S/G2 in human lymphocytes. This discrepancy might be explained, at
least partly, by cells in S and G2 phases having been merged into a
single cell population and analyzed as such.7 This could
result in a dilution effect of G2 cells, due to the short duration of
G2 as compared with S phase28 that, according to our
results, would bias the measured distances towards the greater values
we consistently observed in S phase cells. Consequently, the trend
towards greater gene proximity observed at the transition of late-S to
G2 would have been missed had not these subphases been specifically discriminated.
The data obtained in IM-9 cells show that the proximity between the ABL
and BCR and between PML and RAR genes persists during G2 and
prophase. At metaphase, the pattern of intergenic distances changes
abruptly, with cells having PML and RAR genes in close proximity
reaching its maximum (64% of metaphase cells had PML-RAR interdistances 2 µm), whereas cells with close ABL and BCR genes reached its minimum (10%). The analysis of the spatial relationships between the chromosomes where those genes are located showed that at
least one chromosome 9 (containing the ABL gene) and one chromosome 22 (containing the BCR gene) were in adjacent positions in more than 60%
of the metaphases analyzed. It was thus clear that the increase in
distance between the ABL and BCR genes in metaphase was not due to a
separation of the respective chromosomes during this stage of the cell
cycle, but rather to physical constraints imposed by the size of the
chromosome, the chromosomal location of the gene, and the geometrical
arrangement of the metaphase rosette.
Having studied the intergenic distances in B-lymphoid cells, the
analysis has subsequently progressed to normal hematopoietic progenitors in distinct stages of differentiation. We observed that,
similar to IM-9 cells, intergenic distances change during interphase. A
high percentage of cells with closely associated genes was always
present in G1 (for each pair of genes), which decreased at early-S to
start augmenting again in middle-S or late-S. These findings thus show
that a tendency towards a reassociation of these genes in the late
stages of interphase is present in hematopoietic cells of different
cell lineages and in distinct stages of cell differentiation. However,
the most striking aspects of this analysis are the higher gene
proximity in bone marrow cells than in IM-9 cells and the trend toward
a closer association of ABL and BCR in unstimulated CD34+
when compared with IL-3-treated cells, the opposite pattern being observed for the PML and RAR genes. It seems then that, in
hematopoietic cells of different lineages and in distinct stages of
differentiation, there is always a proportion of cells, varying in
different cell types and stages of the cell cycle, in which the ABL and
BCR, or PML and RAR genes, are separated by a distance that is
theoretically compatible with their physical contact (see criterion for
gene proximity in Materials and Methods) and, presumably, with their reciprocal recombination. The higher proximity of these genes in
hematopoietic precursors would favor their recombination in those
cells. Likewise, the higher proximity of the ABL and BCR genes in
CD34+ cells (which correspond to an immature cell
population that contains the pool of hematopoietic stem cells), and of
the PML and RAR genes in cells treated with IL-3 (mostly committed
to myeloid differentiation), shows an interesting correlation with the
type of cells in which these gene rearrangements are thought to occur in vivo. However, the fact that in some cells the control gene is also
in close proximity to the ABL or PML genes equally reinforces the
notion that the proximity between loci, necessary as it may be, cannot
account solely for recombination phenomena in somatic cells. In this
context, the pattern of variation of intergenic distances during the
cell cycle is of special interest. In fact, if the nature of
chromosomal arrangements in prometaphase and metaphase explain the
variations observed for the PML-RAR and ABL-BCR interdistances at
the prophase-metaphase transition, the same cannot be said about their
behavior in interphase, where little is known as to the spatial
distribution of chromosomes and the rules that underlie that
arrangement (see previous reviews29-32). Especially intriguing was the association of these genes at the S-G2
transition and its persistence during G2 and prophase. The specificity
of the phenomenon raises the possibility that such an association might
obey some, as yet unknown, functional imperative. The already mentioned
transient association between homologous imprinted genes, presumably
for trans-regulation effects,24 is an example that
phenomena of this type may occur and be part of the cell strategies for
epigenetic regulation of gene transcription. If this is so, it is then
likely that the recombinogenic potential of gene proximity may differ
in distinct phases of the cell cycle and/or in different cell
types. In other words, the specific association of these loci in G2 and
prophase in hematopoietic precursors might reflect an intrinsic
dynamical behavior that is established early in hematopoietic
development and perpetuated in different cell lineages, but whose
putative functional meaning may be restricted to a short window of
hematopoietic differentiation. If, for the sake of functionality, the
spatial association of these genes is accompanied by local chromatin
decondensation, then the conditions that might facilitate their
reciprocal rearrangement may well be created. There are, of course,
other alternatives. One is that the recombination may occur with
similar frequencies in different hematopoietic cell types, but only
cause cell immortalization in specific moments of differentiation. This
possibility would explain the rare finding of BCR-ABL transcripts in
the peripheral blood of healthy individuals.33 Another
possibility is that these translocations may occur rarely, being
frequently observed due to the selective advantage that the fusion
protein may confer in a specific cell type.
The uncovering of the biological role of the proteins coded by these
genes in normal hematopoiesis will hopefully help to clarify these issues.
| |
ACKNOWLEDGMENT |
The authors are grateful to Dr S. Georgatos for the anti-lamin B
antibody, to Dr Michael Antoniou for the -globin probe, to Dr Joe
Gray for the chromosome-specific libraries, to Amgen (Thousand Oaks,
CA) for the MiniMACS isolation system, and to Sandoz-Pharma for the gift of recombinant IL-3.
| |
FOOTNOTES |
Submitted August 5, 1998; accepted October 8, 1998.
Supported by Program PRAXIS XXI. H.N. is supported by a fellowship from
Junta Nacional de Investigação Científica.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Leonor Parreira, MD, PhD, Instituto de
Histologia e Embriologia, Faculdade de Medicina de Lisboa, Av. Prof.
Egas Moniz 1699 Lisboa Codex, Portugal; e-mail: hleonor{at}fml.fm.ul.pt.
| |
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J. Anastasi, R. Moinuddin, and C. Daugherty
The Juxtaposition of ABL With BCR and Risk for Fusion May Come at the Time of BCR Replication in Late S-Phase
Blood,
August 1, 1999;
94(3):
1137 - 1138.
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Abstract
Introduction