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344
Derivation of a New Hematopoietic Cell Line with
Endothelial Features from a Patient with Transformed
Myeloproliferative Syndrome
A Case Report
Walter Fiedler, M.D.1
R. Peter Henke, M.D.2
Süleyman Ergün, M.D.3
Udo Schumacher, M.D.3
Ursula M. Gehling, M.D.1
Gabi Vohwinkel, M.Sc.1
Nerbil Kilic, M.D.3
Dieter K. Hossfeld, M.D.1
1
Department of Oncology/Hematology, University
Hospital Eppendorf, Hamburg, Germany.
2
Institute of Pathology, University Hospital Eppendorf, Hamburg, Germany.
3
Institute of Anatomy, University Hospital Eppendorf, Hamburg, Germany.
BACKGROUND. During embryonal development primitive hematopoiesis can be
observed first in the yolk sac, in which both hematopoietic and endothelial cells are
derived from a common precursor, the hemangioblast. Whether cells with this dual
differentiation potential persist during postnatal life is unknown.
METHODS. A cell line was derived from a patient with secondary acute leukemia.
Because of its ability to grow in soft agar and in SCID mice, this cell line was
analyzed for expression of differentiation antigens by fluorescence-activated cell
sorter analysis, immunocytochemistry, fluorescent in situ hybridization (FISH)
analysis with simultaneous cell surface staining, and polymerase chain reaction
(PCR).
RESULTS. A new cell line was established from a patient with essential thrombocytosis that transformed into acute leukemia. The patient’s initial clinical presentation included skin and lymph node infiltrations that were taken for an angiosarcoma due to positivity for CD34, CD31, and von Willebrand factor on
immunohistology. In addition to hematopoietic markers, leukemic cells expressed
endothelial antigens such as CD62E, CD105, and bound Ulex europäeus lectin-1.
Immunocytochemistry revealed positive staining for vascular endothelial growth
factor receptor type 2 (KDR), Tie-2/Tek, the angiopoietin receptor, and vascular
endothelial cadherin. These results were confirmed by PCR analysis. Simultaneous
staining for CD62E and FISH analysis showed that cells with endothelial characteristics belonged to the leukemia. FISH analysis of histologic sections of the lymph
node infiltration confirmed this manifestation as part of the leukemic process. The
derived cell line, UKE-1, forms colonies in soft agar and is tumorigenic in SCID
mice.
CONCLUSIONS. This new cell line, UKE-1, appears to combine hematopoietic and
endothelial features, indicating the close ontogenic relation of both lineages.
Cancer 2000;88:344 –51. © 2000 American Cancer Society.
KEYWORDS: essential thrombocytosis, leukemia, endothelial cell, cell line.
Supported in part by grants from Deutsche Forschungsgemeinschaft Fi 389/4-1 and Roggenbuck
Stiftung.
Address for reprints: Walter Fiedler, M.D., Department of Oncology/Hematology, University Hospital
Eppendorf, Martinistraße 52, 20246 Hamburg,
Germany.
Received April 26, 1999; revision received August
16, 1999.
© 2000 American Cancer Society
D
uring embryonal development mesenchymal cells give rise to the
formation of both endothelial and hematopoietic cells.1–3 This
process first can be observed in the mouse embryo at Day 7 of
gestation when blood islands are being formed in the yolk sac.4 The
close developmental relation between endothelial and hematopoietic
cells within this extraembryonic structure led to the hypothesis that
both cell systems are derived from a common stem cell, the hemangioblast.5,6 This concept has been supported by several mouse knockout models in which both endothelial and hematopoietic cells were
Hematopoietic Cell Line with Endothelial Features/Fiedler et al.
found to be deficient.7,8 In addition, hemangioblasts
have been derived from embryonic stem cells in vitro.9
Because of their common origin, endothelial and hematopoietic cells share several cell surface antigens,
such as CD34, CD31, and CD10510 and produce or
possess receptors for cytokines such as granulocytecolony stimulating factor (G-CSF), granulocyte-macrophage– colony stimulating factor, stem cell factor,
interleukin-6, tumor necrosis factor-␣, or vascular endothelial growth factor (VEGF).11–13
Whether this common precursor cell of hematopoiesis and angiogenesis persists during postnatal life
still is a matter of debate. Although endothelial cells
have been detected in peripheral blood and endothelial-like cells have been derived from progenitor cells
isolated from peripheral blood, to our knowledge definite proof of the existence of the putative hemangioblast still is lacking.14 –16 In the current study, we
present the medical history of a patient with leukemic
transformation of a myeloproliferative syndrome and
the derivation of a cell line that shows both endothelial and hematopoietic features. This observation adds
further support to the concept of a hemangioblast
being the precursor of both endothelial and hematopoietic cells.
345
tion of peroxidase only, and incubation of cells with
normal rabbit serum (Sigma, Deisenhofen, Germany)
in concentrations ranging from 0.1– 0.01% rather than
primary antiserum. Monoclonal or polyclonal antibodies were purchased as follows: KDR (Sigma), von
Willebrand factor (Dakopatts), vascular endothelial
(VE)-cadherin (Immunotech, Hamburg, Germany),
CD31 (Pharmingen, Hamburg, Germany), and Tie2/
Tek (Santa Cruz Biotechnology, Santa Cruz, CA).
Fluorescence-Activated Cell Sorter Analysis
Fluorescein isothiocyanate (FITC) or phycoerythrin
(PE) labeled monoclonal antibodies against CD antigens were purchased as follows: CD34, CD45, and
HLA-Ia from Becton Dickinson (Heidelberg, Germany); CD13, CD33, and CD36 from Coulter/Immunotech (Hamburg, Germany); CD15, myeloperoxidase,
and CD61 from Dakopatts; CD41 and CD42b from
Serotech (Kidlington, UK); and CD62E, CD105, and
CD106 from Cymbus Biotechnology (Hants, Canada).
Ulex europäeus agglutinin-1 coupled with FITC was
purchased from Sigma. Staining of the cells was performed as recommended by the supplier. Fluorescence-activated cell sorter (FACS) analyses were run
on a FACScan using Cell Quest software, both from
Beckton Dickinson.
MATERIALS AND METHODS
Histology and Immunohistology
Polymerase Chain Reaction Analysis
Immunohistology was performed using the alkaline
phosphatase-antialkaline phosphatase technique. The
immunostaining procedure was performed on a TechMate 500 immunostainer (Dakopatts, Copenhagen,
Denmark). Antibodies against leukocyte common
antigen (LCA), CD30, CD31, CD61, factor VIII, myeloperoxidase, desmin, S-100 (Dakopatts), CD34 (Novocastra, Newcastle-upon-Tyne, UK), actin (Enzo, Farmingdale, NY), vimentin (Linaris, Wertheim, Germany),
and cytokeratins (KL1; Dianova, Hamburg, Germany)
were used. Sections were counterstained with hematoxylin.
Total cellular RNA from fresh leukemic and cultured
cells was prepared using Qiagen minicolumns (Qiagen, Hilden, Germany) as described by the manufacturer. One microgram of RNA was used for c-DNA
synthesis employing avian myeloblastosis virus reverse transcriptase and oligo dT as primer. Different
aliquots of c-DNA were amplified with specific primers for KDR, Tie-2/Tek, von Willebrand factor, VEcadherin, and actin as controls for successful c-DNA
synthesis. For KDR, Tie-2/Tek, von Willebrand factor,
and cadherin 5, 2 rounds and for actin 1 round of 35
cycles of polymerase chain reaction (PCR) were performed in a programmable heat block at 94 °C for 1.5
minutes, at 60 °C for 3 minutes, and at 72 °C for 4
minutes. PCR products were separated on 1% agarose
gels, stained with ethidium bromide, and visualized
under ultraviolet light. Primer sequences are available
on request. To avoid cross-contamination, PCR reactions and gel electrophoresis were performed in different rooms using different sets of pipettes. Appropriate control reactions always remained negative.
Immunocytochemistry
Cells were cultured in chamber slides, washed twice
with phosphate-buffered saline (PBS) for 10 minutes,
fixed for 15 minutes with freshly prepared paraformaldehyde (4%) at room temperature, and further processed for immunocytochemistry. A detailed description of the methods used was reported elsewhere.17,18
In brief, an amplification combination of the peroxidase antiperoxidase and the avidin-biotin-peroxidase
complex techniques was used. The peroxidase activity
was visualized by means of the nickel-glucose oxidase
technique.19,20 Controls included replacement of primary and secondary antibodies with PBS, visualiza-
Fluorescent In Situ Hybridization Analysis
On histologic sections, enumeration of chromosomes
7 and 8 was performed using biotinylated alpha satellite probes (D7Z1 and D8Z1; Oncor Inc., Gaithersburg,
346
CANCER January 15, 2000 / Volume 88 / Number 2
MD) and a nonfluorescent technique as described earlier.21,22 Briefly, 6-␮m paraffin sections were adhered
to silanized glass slides and air-dried. Sections were
dewaxed, heated by exposure to microwaves in citric
acid monohydrate, and treated with sodium thiocyanate, followed by a pepsin digestion step. Each section
was covered with 0.1 ng/mL biotinylated probe in
freshly prepared hybridization solution. Sections were
covered with coverslips and denatured for 10 minutes
in a 78 °C waterbath. Hybridization was performed
overnight at 37 °C. Labeled DNA was detected with
subsequent incubations with a mouse monoclonal antibiotin antibody, a biotinylated goat antimouse antibody, peroxidase-conjugated streptavidin, and diaminobenzidine. The slides were counterstained with
hematoxylin, dehydrated, and then permanently
mounted. For evaluation of each probe, the signal
numbers in 200 nonoverlapping neoplastic cells were
counted. Nonneoplastic cells were used as controls.
Cell Culture
Primary leukemic cells were obtained from peripheral
blood after readmission. After separation on FicollHypaque they were cultured in Iscove modified Dulbecco medium (IMDM) supplemented with 10% fetal
calf serum (FCS), 10% horse serum, and 1 ␮M hydrocortisone.
Fluorescent In Situ Hybridization Analysis with
Simultaneous Cell Surface Staining
After trypsinization, 2 ⫻ 104 cells were incubated in
PBS with 2% FCS with anti CD62E (Cymbus Biotechnology). After a washing step, cells were stained with a
Cy 3 labeled anti-mouse antibody and after further
washing spun onto glass slides with a cytocentrifuge.
Glass slides were dried overnight in the dark and then
treated with Carnoy fixative supplemented with 1%
paraformaldehyde. Cells were hybridized with a
digoxigenin labeled alpha satellite probe (D7Z1; Oncor, Inc.) To visualize the chromosome 7 probe, cells
were treated with an FITC labeled antidigoxigenin antibody (Oncor, Inc.). 4⬘6-diamidine-2⬘ phenylindole
dihydrochloride (DAPI) was used as counterstain. Ultraviolet microscopic images were documented with
the Metasystem software package (Metasystems, Heidelberg, Germany).
SCID Mouse Experiments
One million tumor cells dissolved in 200 ␮L tissue
culture medium were injected subcutaneously between the scapula of each of 5 SCID mice. After 12
weeks, tumors developed and the mice were sacrificed. The tumors, lungs, livers, spleens, and bone
samples were excised and fixed in neutral-buffered
formalin and processed for routine histologic examination.
RESULTS
Case Report
In 1968 a 30-year-old woman developed carcinoma of
the uterine cervix that was treated surgically without
consecutive radiotherapy and had remained in remission since that time. In 1985, she was diagnosed with
essential thrombocythemia. Thrombocytosis was controlled with hydroxyurea without bleeding or thrombotic episodes. In May 1997, she experienced pain and
swelling of the right shoulder. Physical examination
revealed a 5-cm right supraclavicular mass and enlarged cervical and axillary lymph nodes of approximately 1 cm in greatest dimension. Intracutane nodules were noted in the region of the affected shoulder
and the ipsilateral lower thorax. The remainder of the
physical examination was unremarkable except for a
systolic murmur and no hepatomegaly or splenomegaly was found. Computed tomography scans of the
chest and abdomen revealed osteolysis of the distal
end of the right clavicle, a lymph node of 3 cm in
greatest dimension at the bifurcation of the trachea, a
2-cm lymph node in the right lung segment 2/3, and a
2-cm hyperdense structure in the spleen that was considered to be a hemangioma. The right supraclavicular
lymph node connected to the right external jugular
vein was removed. Histologic examination was consistent with an epitheloid angiosarcoma of UICC
Grade 2. Laboratory analysis revealed a leukocyte
count of 7.2/nL, hemoglobin of 10.9 g/dL, and thrombocytes of 527/nL. Six cycles of ifosfamide and adriblastin were administered between May and September 1997. Due to febrile neutropenia developing after
the first cycle, G-CSF was given after subsequent cycles. After two therapy cycles and at the end of chemotherapy, enlarged lymph nodes and dermal nodules had disappeared and the mediastinal lymph node
had regressed to 1 cm; however, the intrapulmonary
lymph node remained constant. Only slight pain over
the osteolysis at the tip of the clavicle persisted.
In November 1997, the patient presented with
multiple skin infiltrations, enlarged cervical and axillary lymph nodes, and bilateral pleural effusions. Laboratory investigation showed a leukocyte count of 5.6/
nL, a hemoglobin of 6.4 g/dL, and thrombocytes of
14/nL. Bone marrow biopsy showed infiltration with
54% blast cells. Pleural effusions contained numerous
blast cells. A few days after readmission, bilateral
pneumonia developed causing respiratory insufficiency. The patient was transferred to an intensive
care unit and became dependent on mechanical ventilation. Chemotherapy with cytosine arabinoside and
Hematopoietic Cell Line with Endothelial Features/Fiedler et al.
347
FIGURE 1. (a) Blood film of the patient
showing a circulating leukemic cell
(Pappenheim, stain, ⫻ 1000). (b) Cell
line obtained from this patient showing
large, adherent vacuolized blast cells
and some smaller cells, one resembling
a granulocyte (Pappenheim, stain, ⫻
1000). (c) Histologic section of the patient’s skin infiltration showing a highly
vascularized sarcomatoid tumor with
marked nuclear pleomorphism. A capillary can be seen at the center (H & E, ⫻
630). (d) Histologic section of bone marrow demonstrating infiltration with atypical, polymorphic cells with similar nuclear features as seen in panel c. (H & E,
⫻ 630).
mitoxanthrone did not result in regression of the skin
infiltration or normalization of the blood findings. The
patient refused further therapy and died in December
1997.
Laboratory Investigations
Biopsy of a right supraclavicular lymph node revealed
a highly vascularized and pleomorphic tumor displaying areas with large atypical cells with hyperchromatic, often vesicular nuclei (Fig. 1). Immunohistology revealed that tumor cells were positive for
vimentin, CD31, and factor VIII. Cytokeratins, S-100,
CD30, actin, and desmin were not expressed. Based on
these histologic findings, an epitheloid angiosarcoma
was diagnosed.
At the patient’s second presentation 6 months
later, the peripheral blood contained 54% blast cells.
These cells had a wide cytoplasm that was filled with
multiple vacuoles. The nuclei contained one to three
nucleoli (Fig. 1A). Bone marrow biopsy revealed infiltration by atypical immature cells with a massive concomitant fibrosis (Fig. 1D). Slides of the supraclavicular lymph node were reviewed after determining that
the patient had from a myeloproliferative syndrome
with leukemic transformation. Additional immunohistochemical staining revealed that tumor cells expressed CD34 and myeloperoxidase. Rare large cells
were positive for CD61. Taking all data into consideration, the diagnosis of a myelosarcoma was found to
be more appropriate. In the interim, the patient had
developed a pleural effusion and blasts cells were obtained from this effusion.
FACS analysis of leukemic cells obtained by pleural puncture revealed coexpression of myeloid and
endothelial antigens. In addition to the presence of
CD34 (81%) and CD31 (72%), as already mentioned
earlier, the cells were positive for the myeloid markers
CD33 (91%), CD13 (74%), CD15 (84%), CD36 (53%),
CD45 (97%), HLA-DR (76%), and myeloperoxidase
(9%). Endothelial antigens included CD62E (56%),
CD105 (16%), and Ulex europäeus agglutinin-1 (33%)
(Fig. 2). Megakaryocytic markers such as CD41,
CD42b, and CD61 were expressed by ⬍3% of the cells,
excluding the possibility that essential thrombocytosis
had transformed into acute myeloid leukemia (AML)
of French–American–British type M7.
To further prove the endothelial nature of these
leukemic blasts, cultured cells were investigated for
expression of additional antigens by PCR and immunocytochemistry. By PCR analysis, m-RNA was detected for von Willebrand factor, VE-cadherin, VEGF
receptor 2 (KDR), and the angiopoietin-1 receptor
(Tie-2/Tek) (Fig. 3). The PCR results were confirmed
by immunocytochemistry (Fig. 4).
Cytogenetic analysis of cells from the pleural effusion showed biclonality with the following karyotype: 1) 48, XX, ⫹8, ⫹19 [14] and 2) 45, XX, ⫺7,
del(11)(p14)[8].nish 7 cen (D7Z1x1)(data not shown).
To demonstrate that cells expressing endothelial antigens belonged to the same clone, simultaneous fluo-
348
CANCER January 15, 2000 / Volume 88 / Number 2
FIGURE 2. Fluorescence-activated
cell sorter analysis of cultured cells. Left
upper panel shows staining with phycoerythrin (PE) and fluorescein isothiocyanote (FITC) labeled control antibodies.
Detection of subgroups of positive cells
for (upper right panel) CD13 and CD62E,
(lower left panel) CD31 and CD15, and
(lower right panel) CD34 and Ulex europäeus lectin-1 are shown. Ig: immunoglobulin.
FIGURE 3. Polymerase chain reaction
analysis of cultured cells indicating expression for all tested genes (actin, von
Willebrand factor [VWF], vascular endothelial-cadherin [VE-CAD], Tie-2/Tek,
and vascular endothelial growth factor
receptor type 2 (KDR)] by patient cells.
Lane indications: M: size marker 100
basepair; C: negative control lanes without c-DNA; H: positive control lanes in
which c-DNA from human umbilical vein
endothelial cells was used; P: lanes in
which c-DNA from patient cells was
used.
rescent in situ hybridization (FISH) analysis with cell
surface staining of cultured cells was performed. As
shown in Figure 5A, cells positive for CD 62E displayed
monosomy 7.
To determine whether the initial skin and lymph
node infiltration represented the same disease as the
later developing leukemia, interphase cytogenetic
analysis on histologic sections was performed using
probes for chromosomes 7 and 8. Three signals for
chromosome 8 were found in 35.8% of cells in the
tumor, whereas only 3.7% of cells showed ⱖ4 signals
for this chromosome. The probe for chromosome 7
displayed 1 signal in 70.7% of cells and 2 signals in
9.3% of cells in the tumor tissue. Thus, the signal
distributions were consistent with a monosomy 7 and
a trisomy 8 (Fig. 5B).
Leukemic cells from peripheral blood were cultured in vitro in IMDM supplemented with 10% FCS,
10% horse serum, and 1 ␮M hydrocortisone. After
approximately 6 weeks cells started to proliferate at
a higher rate so that they could be split once a week.
At last follow-up the cells had been in culture for
approximately 9 months and were continuing to
grow. Morphologically, these cells can be subdi-
Hematopoietic Cell Line with Endothelial Features/Fiedler et al.
349
FIGURE 4.
Immunocytochemistry of
cultured cells using the immunoperoxidase technique showing positive staining for (a) von Willebrand factor, (b) the
vascular endothelial growth factor receptor type 2 (KDR), (c) the angiopoietin
receptor Tie-2/Tek, and (d) negative
control staining without specific primary
antibody (⫻ 1000).
FIGURE 5. (a) Analysis of cultured cells with simultaneous cells surface staining with a monoclonal antibody against CD62E and interphase fluorescent in situ
hybridization analysis with a chromosome 7 specific probe. Both cells show monosomy 7, but only one cell was positive for CD62E (⫻ 1000). (b) Interphase
cytogenetic analysis of the lymph node with a chromosome 8 specific probe shows a trisomic signal distribution. Several cells with three signals are indicated by
arrows representing trisomy 8 (⫻ 1000).
vided into large, adherent cells with prominent
vacuoles and into smaller nonadherent cells. Some
cells of the nonadherent fraction appear to be capable of differentiating spontaneously along the
myelomonocytic lineage because a few cells resemble mature granulocytes (Fig. 1B). Adherent and
nonadherent cells continuously show monosomy 7
by interphase FISH analysis. This cell line has been
called UKE-1. Cultured cells form colonies in soft
agar. Subcutaneous transplantation of these cells
into SCID mice led to tumor formation after approximately 2–3 months. On histologic examination, tumors in SCID mice closely resembled the original
skin infiltration (data not shown). No spread to the
bone marrow, liver, or spleen was observed in SCID
mice. However, in one of five SCID mice a solitary
lung metastasis was detected.
DISCUSSION
Since the advent of bone marrow transplantation it
has been clear that human hematopoietic stem cells
can differentiate into four lineages: lymphoid, erythroid, myelomonocytic, and megakaryocytic. Recently it has been suggested that during embryonal life
both hematopoietic and endothelial cells are derived
from a common precursor cell, the hemagioblast.5– 8
Experimental studies provide evidence that such cells
350
CANCER January 15, 2000 / Volume 88 / Number 2
persist during postnatal life. It has been shown that
endothelial cells can be derived from immature cells
that have been purified from peripheral blood.15 In a
canine model, bone marrow transplantation was performed simultaneously with the implantation of a Dacron graft into the aorta. After 12 weeks the endothelial cells lining the Dacron graft were of donor origin.16
These experimental observations now have been extended by the description in the current study of a
case of immature leukemia. Blast cells from this patient showed features of both hematopoietic and endothelial cells. This dual differentiation potential was
observed in the clinical presentation, in which formation of a solid tumor resembling angiosarcoma and
bone marrow infiltration with circulating malignant
cells occurred. Accordingly simultaneous expression
of hematopoietic and endothelial markers was detected by cell surface marker analysis. Although few
cell surface antigens are completely specific for endothelial cells, the detection of multiple markers such as
CD31, CD34, CD62E, CD105, von Willebrand factor,
VE-cadherin, and Ulex europäeus agglutinin-1 on a
subgroup of the cells clearly establishes the endothelial nature of the circulating cells. The receptors for
VEGF and the angiopoietins (KDR and Tie-2/Tek, respectively) also were identified on cultured leukemic
cells from our patient by means of PCR analysis and
immunocytochemistry. They normally are expressed
on endothelial cells such as those obtained from the
human umbilical cord. Rarely, these receptors can be
found on leukemic blasts from patients with acute
myeloid leukemia.13,23 Furthermore, one of the endothelial markers, CD62E, could be located unambiguously on the leukemic cells by simultaneous FISH and
immunofluorescent cell surface staining, indicating
that cells with endothelial features belonged to the
leukemic clone.
Although it is tempting to speculate that this patient’s tumor represented transformation of the preexisting myeloproliferative syndrome, definitive proof
is lacking. The leukemic stem cell of myeloproliferative syndromes including chronic myelogenous leukemia has been shown to be the malignant counterpart
of very immature hematopoietic cells. Because the
patient’s tumor showed features of hematopoietic and
endothelial lineages, its clonogenic cell presumably
represented an early common precursor. Conversely,
the chromosomal status of the patient’s essential
thrombocythemia was not known. The majority of
patients with essential thrombocythemia have a normal karyotype although patients with trisomy 8 have
been described.24 The complex chromosomal status of
the disease in the patient in the current study also is
compatible with a secondary neoplasm possibly in-
duced by long term cytostatic therapy. The frequency
of transformation of essential thrombocythemia
caused by hydroxyurea monotherapy has been estimated to be between the spontaneous transformation
rate and 3.5%.25,26 To our knowledge, the most recurrent chromosomal abnormality detected in hydroxyurea-induced transformation of essential thrombocythemia is rearrangement of chromosome 17 with
possible alteration of the p53 gene.25 Because we did
not find a chromosome 17 abnormality in the leukemic cells of our patient, the influence of hydroxyurea
on the transformation process appears unlikely. To
our knowledge, alterations of chromosome 8 in transformed essential thrombocythemia, as in the case presented in the current study, have been described in
two patients,27,28 but the relation of these changes to
previous therapy could not be established.
To our knowledge, few chromosomal analyses
have been reported in classic angiosarcoma. In a recent publication summarizing a series of patients with
Stewart-Treves syndrome, monosomy 22 or X and trisomy 2 have been described as recurrent numeric
deviations.29 These are different from those deviations
found in the patient described in the current study,
underlining the more immature developmental stage
of this patient’s tumor. The exact relation of the neoplasm described in the current study to more mature
angiosarcoma and potential differentiation pathways
toward endothelial cells remain to be elucidated.
Stimulated by this report, we believe more cases
of immature leukemia with dual potential for endothelial and hematopoietic differentiation possibly will
be identified, thereby shedding more light onto the
biology of stem cells.
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