Differentiation-Associated Changes in CD44 Isoform - Blood Journal

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Differentiation-Associated Changes in CD44 Isoform Expression During
Normal Hematopoiesis and Their Alteration in Chronic Myeloid Leukemia
By S. Ghaffari, G.J. Dougherty, P.M. Lansdorp, A.C. Eaves, and C.J. Eaves
CD44 is a widely expressed, multifunctional, cell-surface glycoprotein thathas been implicated in the regulationof normal hematopoiesis. In addition, expression of particular isoforms of CD44 has been associated with malignant
transformation and/or the acquisition of metastatic potential. In this study, we used two recently developed monoclonal anti-CD44 antibodies, one reactive with an epitope
shared by many CD44 isoforms and the other with an epitope unique t o CD44 isoforms containing amino acids encoded by thealternatively spliced exon v10, t o compare the
expression of CD44 on primitive hematopoietic cells from
the marrowof normal individuals and theirneoplastic counterparts present in the peripheral blood of patients with
chronic myeloid leukemia (CML). Multiparameterfluorescence-activated cell sorter (FACS) analysis and cell sorting
studies showed thatCD44 is normally expressed at high t o
very high levels on both long-term culture-initiating cells
(LTC-IC) and granulopoietic colony-forming cells (granulocyte-macrophage colony-forming units [CFU-GM]). In contrast, primitive erythropoietic progenitors(burst-forming
units-erythroid [BFU-El) in normal marrow were more homogeneous in their expression of CD44, and very few (less
than 5%) showed thevery high levels of CD44 seen on 20%
to 2540 of LTC-IC and CFU-GM. Antibody staining showed
the expression of exon vl0-containing CD44 isoforms t o be
restricted t o a small subpopulation(490t o 896)of morphologically recognizable mature (CD34-) myeloid cells within the
light-density fractionof normal marrowcells. Reverse transcription-polymerase chain reaction (RT-PCR) analysis
showed the presence of two exon vl0-containing mRNA
species. In CML, a significantly greater proportion of the
circulating neoplasticCFU-GM expressed very high levels of
CD44. and these CFU-GM were accompanied by an increased
number oflight density v10' cells, including some that coexpressed CD34. Nonmalignanthematopoieticprogenitors
mobilized by prior chemotherapy and growth factor treatment of patients with Hodgkin's disease or acute myeloid
leukemia in remission showed no changes in CD44 expression relative t o normal marrow progenitors. These results
provide evidence of early differentiation-associated changes
in CD44 expression during normalhematopoiesis in vivo that
may be deregulatedin theneoplastic clone of patients with
CML.
0 1995 by The American Society of Hematology.
I
homing of primitive hematopoietic cells into the
Neoplastic transformation may also alter the adhesive characteristics of primitive hematopoietic cells with associated
changes in their turnover and tissue distribution. For example, in chronic myeloid leukemia (CML), a multilineage
clonal malignancy believed to arise as a result of the formation of a BCR-ABL fusion gene in a pluripotent hematopoietic stem cell,' the leukemic progenitors exhibit abnormal
adhesive properties, are found at abnormally elevated levels
in the blood, and are able to establish hematopoiesis in extramedullary sites.'."
CD44 is an adhesion molecule that is expressed on the
surface of many cells, including representatives of all hematopoietic cell lineages."-16 Hyaluronan is the mostwidely
recognized ligand of CD44, but evidence of binding to fibronectin, collagen, and serglycin has also been rep~rted.''.'~
The common form of CD44 expressed on hematopoietic
cells (CD44H) isan 85- to 90-kD glycoprotein. A large
number of higher-molecular-weight isoforms may also be
produced in specific cell types or under specific conditions
as a result of the alternative splicing of at least 10 contiguous
exons (v1 through v10) within the CD44 gene.'"'" CD44R1
is one of several vl0-containing CD44 cDNAs. It was cloned
from the KG- 1 a leukemic cell line and contains in the extracellular region of the molecule an insertion of 132 amino
acids encoded by exons v8, v9,and v10." This isoform
corresponds to domains IVand V of the original CD44
clones and to the epithelial form of CD44 (CD44E), from
which it differs by only three amino acids." CD44R2 (also
referred to as domain V) is avl0-containing isoform of
CD44 that shares only the last 69 amino acids present in the
unique region of CD44R1. Both the expression and binding
properties of CD44 on normal cells appear to be subject to
regulation.'6 In addition, changes in CD44 isoform expres-
N ADULT LIFE, THE proliferation and differentiation
of primitive hematopoietic progenitors is normally restricted to the bone marrow, where these cells interact with
various stromal cells of nonhematopoietic origin, aswell
as with a complex mixture of extracellular matrix (ECM)
components. Such interactions are believed to regulate the
accessibility of these cells to stimuli that control their viability, cell-cycle progression, and movement into and out of
the circulation. These concepts are based in part on the identification on the surface of primitive hematopoietic cells of
cytokine receptors and adhesion molecules with affinities for
various membrane or ECM-bound ligands.',' In addition,
in vivo experiments have shown that particular ligands, or
antibodies for specific cell adhesion molecules, may stimulate the rapid exodus of primitive hematopoietic cells from
themarrow into the blood or, conversely, may alter the
From the Terry Fox Laboratory, British Columbia Cancer Agency,
Vancouver; and the Departments of Pathology and Laboratory Medicine, Medicine,and Medical Genetics, University of British Columbia,Vancouver, Canada.
Submitted March 27, 1995; accepted June 14, 199.5.
Supported by the National Cancer Institute of Canada (NClCJ
and the Canadian CancerSociety. S.G. is a recipient of u Terry Fox
Physician-Scientist Fellowship of the NClC. G.J.D. is a Research
Scientist of the NCIC, and C.J.E. is a Terr). Fax Cancer Research
Scientist of the NCIC.
Address reprint requests to C.J. Eaves, PhD, Terty Fox Laboratory, 601 W 10th Ave, Vancouver, BC VSZ IL3, Canada.
The pubiicationcosts of this article were defrayed in partby 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.
0 1995 by The American Society of Hematology.
0006-4971/95/8608-0020$3.00/0
2976
Blood, Vol 86, No 8 (October 15). 1995: pp 2976-2985
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CD44EXPRESSIONONNORMAL
2977
AND CML CELLS
sion have been found to characterize various metastasizing
cells, such as those present in certain aggressive lymphomas23-25 and in a variety of transformed epithelial cell populat i o n ~ . *Evidence
~ - ~ ~ that CD44 may be involved in the regulation of early stages of normal hematopoiesis has been
suggested by the observations that primitive clonogenic cells
express CD44I5 and that the addition of anti-CD44 monoclonal antibodies to long-term marrow cultures (LTC) can
cause a marked and sustained decrease in the number of
mature cells subsequently found in the nonadherent fraction
of these ~ u l t u r e s . Whether
~ ~ ~ ~ ' this is due to an effect on the
primitive hematopoietic cells that normally become part of
the adherent cell layer in this culture system has not been
established. Indeed, the level of expression of CD44 on cells
that give rise to clonogenic progenitors under these conditions in vitro has not been previously characterized. Similarly, in spite of the well-known abnormal adhesive properties of CML cells, potential alterations in their expression
of CD44 have not been investigated.
In this study, we show that there are quantitative differences in the levels of CD44 expressed on primitive normal
erythroid (burst-forming unit-erythroid [BFU-E]) and granulopoietic(colony-forming
unit-granulocyte/macrophage
[CFU-GM]) progenitors and on their precursors identified
functionally as LTC-initiating cells (LTC-IC). We have also
identified a small subset of CD34- cells in normal marrow
that express CD44 isoform(s) containing the amino acids
encoded by the alternatively spliced exon v10. Interestingly,
quantitative changes in both of these aspects of CD44 expression were found when predominantly leukemic populations isolated from patients with CML were studied. These
changes include an increase in the proportion of CFU-GM
expressing very high levels of CD44 and the expression of
the exon vl0-containing CD44 isoform(s) on CD34' neoplastic cells.
MATERIALS AND METHODS
Cells. All samples of human peripheral blood (PB) and bone
marrow (BM) were obtained from informed and consenting individuals. Samples of normal BM were from harvests taken for allogeneic
transplantation or from normal cadaveric tissue. Some samples were
also obtained from leukapheresis harvests collected from patients
with hematologic diseases in remission (two with Hodgkin's disease
and one acute myeloid leukemia [AML]). The two patients with
Hodgkin's disease received chemotherapy (cyclophosphamide, 7 g/
m') followed by administration of interleukin-3 (IL-3; 2.5 to 5 pgi'
kg/d) and granulocyte-macrophage colony-stimulating factor (GMC S F 5 pgkgld) until the leukaphereses were completed. The patient
with AML received G-CSF (12 pg/kg/d) for 5 days followed by 2
days of leukapheresis. Heparinized PB samples were obtained from
patients with newly diagnosed Philadelphia chromosome-positive
(Ph') chronic phase CML undergoing routine hematologic assessment (Table 1). In all but one case, the CML cells had been cryopreserved in 90% fetal calf serum (FCS; StemCell Technologies, Vancouver, Canada) and 10% dimethyl sulfoxide and then stored at
-70В°C before use. To ensure that the results of analyses of CML
samples could be attributed to leukemic progenitor populations, only
PB samples that were found to contain clonogenic cells that were
present at frequencies at least 25-fold above themean for fresh
normal blood were used (Table 1). All analyses were performed on
Table 1. Initial WBC and Clonogenie Progenitor Concentrations in
Fresh or Cryopresewed PB Samples From Patients With CML
Sample
No.
WBC Count
(xlOe/mLi
la*
1 b'
180
180
2
250
3
450
350
300
4
5
Status When Used
F
T
T
T
T
T
BFU-EtmL
7,200
7,400
CFU-GMImL
2,700
2,200
400,000
150,OOO
150,000
970,000
780,000
380,000
190,000
120,000
Abbreviations: F. fresh; T, subsequently thawed.
* Progenitor values for samples la and l b are from the same patient.
cells in the light-density fraction (less than 1.077 g/mL) isolated
using Ficoll-hypaque (Pharmacia LKB, Uppsala, Sweden).
Antibodies. lgGl monoclonal antibodies (MoAbs) specific for
CD34 (8G12),32Thy-l (5E10),33and an epitope located on the common NH,-terminal region of different isoforms of CD44 (3C12))4
were purified from tissue culture supernatants using Protein G chromatography (Pharmacia LKB). MoAb 8G12 labeled with Cyanine5 (Cy5) has been described previously32and was used at 10 pg/mL.
MoAb 5E10 was labeled with phycoerythrin (PE; Pharmingen, San
Diego, CA) and used at 5 &mL, and 3C12 was used at 1 pg/mL.
MoAb 2G1 is an IgGl MoAb that reacts with an epitope encoded
by exon ~ 1 0 . (In
' ~ this study, cells expressing exon vl0-containing
CD44 isoforms are referred to as 2G1+ cells or vl0' cells.) MoAb
2G1 was used as a hybridoma tissue culture supernatant. An irrelevant IgGl MoAb (anti-de~tran)~~
was used as a control in all staining
and sorting experiments. A goat F(ab'), anti-mouse IgG (H + L)
preparation labeled with fluorescein isothiocyanate (GAM-FITC)
was purchased from Caltag Laboratories (South San Francisco, CA).
Staining andfrow cytometry. Cells were washed twice and resuspended in Hank's balanced salt solution with 2% FCS and 0.02%
sodium azide (NaN,) (HFN). All staining procedures were indirect
and were performed with the cells at a concentration of107/mL.
Cells were first incubated with HFN containing 5% human serum
(HS) at room temperature to block Fc receptors, then washed twice
with HFN, and incubated with an anti-CD44 MoAb (3C12, 2G1) or
the IgGl control MoAb for 30 minutes at 4"C, followed by two
washes with HFN. Samples were then resuspended in a 150 dilution
of GAM-FITC in HFN, incubated for 30 minutes at 4"C, washed
twice, and incubated for another 30 minutes at 4В°C with 200 pg/mL
of the irrelevant mouse IgG MoAb to block residual GAM-FITC.
(The use of a high concentration of this antibody was necessary to
block the residual GAM-FITC on the surface of cells expressing
high numbers of CD44 molecules.) The anti-CD34 MoAb (8G12Cy51 andanti-Thy-l MoAb (5ElO-PE) were then added to this solution without further washing in the presence of the irrelevant mouse
IgG for another 30 minutes at 4В°C. Cells were then washed twice
with HFN, with the second wash containing propidium iodide (PI;
Sigma Chemicals, St Louis, MO) at 2 wg/mL to stain dead cells.
Sorting of cells was performed on a FACStar Plus (Becron Dickinson, San Jose, CA) equipped with a 5-W argon and a 30-mW helium
neon laser (Spectra-physics, Mountain View, CA).In all experiments, gates were set to exclude dead cells (PI+) as well as most
erythrocytes and granulocytes as defined by their forward light scatter (FSC) and side scatter (SSC) characteristics. Sorted cells were
collected in Iscove's Dulbecco's modified Eagle's medium (DMEM)
(STI) containing 2% FCS and were kept on ice until plated.
Reverse transcription-polymerase chain reaction (RT-PCR) analysis. The following PCR primers corresponding to CD44 se-
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GHAFFARI ET AL
2978
A
B
..
-
.:.
I
n
FSC
FSC
D
C
d.
c')
n
0
ssc
quences located in the 5' common region and in the 3' alternatively
spliced exon v10 were used: 5'(5'-TGTACATCAGTCACAGACCT-3') and ~'(S-AGGAACGATTCIACATTAGAG-~').Total
RNA was isolated using TRIzol Reagent (Gibco BRL, Gaithersburg,
MD). The first cDNA strand was generated in a 50-pL reaction
containing 5 pg of total RNA, 0.5 mmol/L deoxynucleotide triphosphates (dNTPs), 200 pm01of random hexamers, 4 mmol/L dithiothreitol, 200 U of superscript reverse transcriptase (Gibco BRL), and
2.5 U of human placental RNase inhibitor (Gibco BRL). The reaction
was incubated at 42В°C for 1 hour followed by 5 minutes at 95В°C to
inactivate the enzyme. Then I O pL of this reaction was subjected
to PCR in a 100-pLvolume of 25 mmol/L KCI, 1.5 mmol/L MgC12,
0.2 mmol/L dNTPs, 1 0 0 pmolof each primer, and2.5 U ofTaq
polymerase, Thirty cycles were performed as follows: 30 seconds
of denaturation at 94В°C. 30 seconds of annealing at 48В°C.and 3
minutes of extension at 72В°C. This was followed by a final extension
at 72В°C for 5 minutes. The PCR products were separated on a 2%
agarose gel, transferred to a nylon membrane, and hybridized to a
196-bp vl0-specific probe obtained by PCR of the CD44R1 cDNA2'
using the primers 5'(5'-TAGGAATGATGTCACAGGTG-3')and
3'(5'-AGGAACGATTGACATTAGAG-3'). Hybridization was performed overnight at 60В°C in 6X saline sodium citrate (SSC; 1X SSC
is 0. I5 mol/L NaCI, 0.01 5 mol/L sodium citrate, pH 7.5). 1% sodium
dodecyl sulfate (SDS), 0.02% polyvinylpyrrolidone, 0.02%ficoll,
0.02% bovine serum albumin (BSA), 10 pg/mL of denatured salmon
sperm DNA, and 10' c p d m l of denatured probe. The filter was
then washed at a final concentration of 0.1 X SSC, 1% SDS at 65В°C.
Autoradiography was performed at room temperature withKodak
XAR-5 film for 14 hours (Eastman-Kodak, Rochester, NY).
CD44
Fig 1. Gates used for analysis
of antibody reactivity with normalBM
cells. BM cells were
gated to exclude dead cells (A)
and cellswith high SSC and very
low FSC (B). The gating used to
select cells with high CD34expressionis shown in panel C.
The gating used to subdivide the
CD34'cellsaccording
to their
expression of CD44 and Thy-l is
shown in panel D. The four
quadrants shown in panel D
represent the fourCD34subpopulations analyzed functionally: CD44++Thy-l-, CD44"Thyl*,
CD44"*Thy-l-. and CD44"'Thy-l'. The same gates were
used for CML PB and mobilized
blood.
Hematopoietic progenitor assays. Cells from primary samples
or LTC harvests were assayed for clonogenic erythropoietic (BFUE), granulopoietic (CFU-GM), and multilineage (CFU-granulocyte,
erythroid, monocyte, megakaryocyte: CFU-GEMM) progenitors in
Iscove's DMEM-based methylcellulose cultures containing 3 UlmL
of human erythropoietin; 20 n g h L each of granulocyte colonystimulating factor (G-CSF; Amgen, Thousand Oaks, CA), granulocyte-macrophage CSF (GM-CSF; Sandoz, Basel, Switzerland), IL3 (Sandoz), and IL-6 (obtained from the supernatant of COS cells
transfected with a full copy human IL-6 cDNA; D. Hogge, Terry
Fox Laboratory, Vancouver, Canada); and 50 ng/mL of Steel factor
(SF; Amgen). The methodology and criteria for hematopoietic colony generation and recognition were the same as previously de~cribed.~'
The general procedure used for LTC-IC assays has also
been described in detail previously." Briefly, test cells were resuspended in long-term medium (an enriched Alpha medium containing
12.5% horse serum, 12.5% FCS,
mol/L 2-mercaptoethanol
[STI], to which freshly dissolved hydrocortisone sodium hemisuccinate [Sigma] was added just before use to give a final concentration
of
moVL) and then seeded onto semiconfluent feeder layers of
irradiated (80 Gy) mouse marrow-derived fibroblasts that had been
genetically engineered to produce human G-CSF, L-3, and SF.3q
These LTC-IC assay cultures were then maintained at 37В°C and fed
weekly by replacement of half of the medium containing half of the
nonadherent cells with the same volume of fresh long-term medium.
After a total of 6 weeks, the nonadherent cells were removed,
washed, and combined with the trypsinized and suspended adherent
layer cells. These pooled cells were then plated in methylcellulose
assays as described above. The total number of clonogenic cells
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CD44 EXPRESSIONON
2979
NORMAL AND CML CELLS
103
104
CD44
Fig 2. Expression of CD44 versus Thy-l on CML PBCD34* cells
located within t h e gates shown in Fig 1.
(BFU-E plus CFU-GM plus CFU-GEMM) present at 6 weeks provides a relative measure of the number of LTC-IC originally present
in the test suspension." Limiting dilution experiments have shown
that under the conditions used herefor LTC-IC detection, on average,
one LTC-IC will produce approximately eight clonogenic cells
(C.J.E., unpublished observation, August 1995). and this was, therefore, the value used to derive the absolute LTC-IC frequencies reported. In LTC-IC assays where no colonies were detected, the
minimum number of LTC-ICthat could have been detected was
calculated by assuming that one colony had been produced by the
entire aliquot of cells evaluated in the final methylcellulose assays.
This value (instead of zero) was then used to derive an estimate of
the upper limit of the mean 5 SEM number of LTC-IC in groups
where one or more values were below the limit of detection. In such
cases the mean is indicated as less than x 5 SEM.
RESULTS
Altered CD44 expression on phenorypically dejined populations of primitive CML cells. To compare the patterns of
expression of CD44, CD34, and Thy-l on primitive normal
and neoplastic (Ph') CML cells, we isolated various fractions of light-density bone marrow cells from normal individuals and compared their staining profiles with those obtained for cells in the light-density fraction of PB from a
series of patients with newly diagnosed CML and high white
blood cell (WBC) counts. The choice of this source of CML
cells for these comparisons was based on previous data
showing that the light-density cells in the PB of patients
with chronic phase CML and high WBC counts, on average,
will contain a greater than 10-fold increase in all types of
leukemic (Ph') progenitors (both clonogenic cells and LTCIC), such that they approach or even exceed the frequency
of the same types of primitive normal cells in the lightdensity fraction of normal BM cell^.^^"
Figure 1 shows representative scatter plots for a normal
BM sample, and Fig 2 shows analogous plots for a representative CML PB sample stained in the same way. These figures illustrate the general finding for all samples studied:
290%of the light-density cells in both normal BM and CML
PB were CD44'. In addition, among the CD34' populations
cells were detectable.
present in these samples, no CD"
From these analyses, two additional similarities between normal BM and CML PB were consistently noted. First, the
relative proportion of CD34' cells that were also Thy-l'
was approximately the same. Second, the proportion of (total) CD34' cells in CML PB expressing very high (1,OOOX
as compared with the
background, designated CD44"'),
proportion expressing intermediate (IOOX background,
CD44++),levels of CD44 appeared indistinguishable from
those characteristic of the CD34' population in normal BM
(Table 2). However, in the CML samples, the proportion of
Thy-]' cells that were CD44"'
was significantly higher
than in normal BM (0.4% t 0.1% of CD34' cells in normal
BM compared with 1.2%? 0.2%of CD34' cells in CML
PB; P < .01, Student's t test).
In addition, we looked within the entire light-density fraction of normal BM and CML PB, as well as within their
respective CD34' subpopulations, for cells expressing exon
vl0-containing CD44 isoforms. For this, we used the 2G1
MoAb, which specifically recognizes an epitope present on
Table 2. Comparisons of the Distribution of Total CD34* Cells and the Total Light-Density Cells by Their Levels of Expression
of CD44 and Thy-l in Normal BM Versus CML PB
CD44"
Population Evaluated and
Origin of Cells
Total LDF
Normal BM
CML PB
CD44" *
no
Thy-l-
Thy-l*
6
351
321
80 e 1
77 e 3
82 e 1
80 2 1
0.4 2 0.1
1.2 t 0.2
17 2 1
18 t 3
0.2 z 0.1
0.4 ? 0.1
4t2
11 e 8
422
11 2 8
0.05 2 0.02
0.3 t 0.02
0.9 t 0.3
423
6
Total Thy-l-
Thy-l'
Total
18 t 1
19 2 1
CD34'
Normal BM
6
CML PB
6
1 2 0.3
4e3
Values shown are the mean 2 SEM of the individual values for the various populations evaluated, expressed in each case as a percent of
the total LDF.
Abbreviation: LDF, light-density fraction.
Number of samples, for six normalindividuals and five patients with CML. One of the samples from the first patient with CML was studied
twice (before and after thawing; see Table 1).
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2980
GHAFFARI
B
A
I
CD44 common region
Alternatively spliced
I
v2
Extracellular
-D
Domain
I v3
Cytoplasmic
Domain
I
I
v4
I
680 bp
I
v5
480 bp
sequences
4-
-
W
Fig 3. RT-PCR analysis of 2G1+ cells. (A) Primers (arrows) were designed t o amplify all vl0-containing CD44 isoforms and t o exclude all
isoforms lacking thisexon. The shaded area corresponds t o the transmembrane domain. Human v1 contains an in-frame stop
codon. (B) PCR
products were separated on a 2% agarose gel, transferred t o a nylon membrane, and hybridized t o a v10-specific probe (see Materials and
Methods). The arrows indicate bands corresponding t o expected reaction products forCD44R1 and CD44R2.
the extracellular portion of CD44 isoforms containing the
amino acids encoded by the v10 exon. In normal BM, 2G1+
cells were relatively rare, comprising from 4% to 8% of the
total light-density fraction and s l % of the CD34' cells. In
all of the six CML PB samples analyzed, 2GI' cells were
much more prevalent (up to 30% of the light-density cell
fraction) and included cells that were also expressing CD34
(up to 7%), although there was marked patient-to-patient
variability in the proportion of CD34' cells that were also
2G l +.Nevertheless, because of the large elevations in total
numbers of light-density myeloid cells in the blood of these
patients, these findings suggest that there is a marked in-
crease in the absolute number of 2GI' hematopoietic cells
in patients with CML. On the other hand, in both normal
BM and CML PB, allof the 2G1' cells appearedtobe
Thy- 1 -. Fluorescence-activated cell sorter (FACS) analysis
further showed that the light-density 2G 1 cells from either
normal BM or CML PB had lowSSC properties and intermediate to highFSC characteristics. After sorting andMayGrunwald Giemsa staining, all 2G1' cells, regardless of their
origin, appeared to be exclusively myeloid cells (and did not
include erythroblasts or other recognizable cell types). In
functional assays of the sorted CD34' 2G1+ CML cells, no
clonogenic progenitors of any kind were detected.
+
Table 3. Progenitor Distributions in Subpopulations of CD34' Cells in Normal BM by
Their Levels of Expression of CD44 and T
h
y
l
No. of Progenitors/105 Cells'
CD44"'
CD44"
Tvoe of
Progenitor Evaluated
Sample No.
BFU-E
2
3
4
5
6
Mean ? SEM
CFU-GM
2
3
4
5
6
Mean 2 SEM
LTC-IC*
2
3
4
5
6
Mean t SEM
Thy-l
+
5,000
ND
1,300
2,100
2,500
ND
2,700 2 800
3,800
ND
23,000
9,000
7,700
ND
11,000 2 4,200
ND
18,000
1,800
1,600
3,300
2,900
5,600 2 3,200
Thy-l-
7,500
8,000
5,000
5,700
13,000
9,700
8,100 2 1,200
15,000
13,000
19,000
6,700
6,200
9,500
11,000 2 2,000
ND
900
80
200
900
800
600 ? 200
Thy-l-
Thy-l'
ND
ND
< 1,000
ND
ND
ND
< 1,000
ND
ND
15,000
ND
ND
ND
ND
ND
500
400
1,800
13,000
<300
<3,300 -c 2,500
Abbreviation: ND, not done.
For the population analyzed.
t Measured as the total clonogenic output in LTC after 6 weeks divided by 8 (see Material and Methods).
400
800
600
500
500
500
<600 ? 40
12,000
8,500
20,000
4,500
8,000
13,000
11,000 2 2,200
ND
700
100
300
800
200
400 2 100
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2981
CD44EXPRESSION ON NORMAL AND CML CELLS
1ll
T
CFU-GM
TOTAL
BFU-E
LTC-IC
CELLS
Fig 4. Comparison ofthe distributionof different types of progenitors between theCD34+ CD44++(01and CD34* CD44+++(.l fractions
of normal light-density marrow cells with thelight scatter characteristics shown in Fig 1. Values shown are the mean f SEM of measurements of the relative recovery ofprogenitorsfrom each of six experiments calculated in each caseby multiplying the percentages of cells
retrieved in each fraction by the corresponding progenitor enrichment observed in that fraction and then normalizingthe data to 100%
for the number of Progenitorsdetected in the totalCD34+ population.
To determine the nature of the vl0-containing CD44 isoform(s) expressed by 2G1+ cells, RT-PCR was performed.
Primers were designed to amplify all vl0-containing CD44
isoforms but to exclude all other sequences lacking this exon.
This was achieved by selecting a 5' primer from a region
common to all CD44 isoforms and a 3' primer in the last of
the alternatively spliced exons (v10). This analysis yielded
two major bands of approximately 680 and 480 bp (Fig 3).
These correspond to the expected sizes of the CD44 isoforms
RI and R2 (672 and 481 bp, respectively), which have been
previously demonstrated in various hematopoietic cells.21
Differential expression of CD44 on different normal progenitor populations. In view of the extensive variation in
the level of CD44 expression seen on the CD34+ cells present in normal BM, it was of interest to determine whether
any lineage-associated changes in CD44 expression might
be demonstrable. To examine this possibility, light-density
CD34+ cells from six normal BM samples were sorted according to their expression of very high(+ + +) or intermediate levels (+ +) of CD44 and detectable versus undetectable
levels of Thy-l. Cells from each of the four subpopulations
thus obtained (shown in Fig 1) were then assayed in both
clonogenic and LTC-IC assays. The resultant frequencies of
the various progenitor types measured are shown in Table
3. The greatest enrichment of BFU-E was obtained in the
CD44++Thy-I- fraction and, to a lesser extent, in the
CD44++Thy-l+fraction. Veryfew BFU-E were found in
the CD44+++fractions (either Thy-l+ or Thy-l -; Fig 4). In
contrast, comparing the absolute numbers of colonies recovered, we found that both CFU-GM and LTC-IC were more
heterogeneous in their levels of CD44 expression (Table 3
and Fig 4). Most CFU-GM were Thy-I-, and further separa-
tion of these cells according to their level of CD44 expression did not result in a selective enrichment of a subpopulation of Thy-l- CFU-GM. Some (8% ? 4%) CFU-GM were
also Thy- 1 +.These appeared to be confined primarily to the
CD44++ population of CD34+ cells. As found previously,
LTC-IC were usually more highly enriched in the Thy-l+
as opposed to the Thy-l- fractions of CD34+ normal BM
cells.33However, in terms of total LTC-IC yields, a substantial proportion (68% +- 13%) of all LTC-IC were Thy-l-.
Figure 4 shows a comparison of the relative distribution of
each of these normal progenitor types between the CD44"
and the CD44+++fractions. The difference in the ratio of
CD44++ to CD44+++progenitors between CFU-GM (or
LTC-IC) and BFU-E was statistically significant ( P < .02
in both cases, Student's t test).
Expression of CD44 on CML progenitors is altered. PB
samples from five patients with CML were analyzed to determine the level of expression of Thy-l and CD44 on various
primitive leukemic cell populations. The frequencies of
CFU-GM and BFU-E in each of the four CD34+ subpopulations defined by differences in Thy-l and CD44 expression
(gated as indicated in Fig 2) are shown in Table 4. The
relative recoveries of these progenitors in the CD44++versus
the CD44"' fractions by comparison with normal progenitors are shown in Fig 5. To control for the fact that CML cells
might exhibit features unique to circulating and/or activated
mobilized progenitors, a series of PB harvests collected after
treatment of patients with remission AML or Hodgkin's disease with chemotherapy and administration of G-CSF or
GM-CSF and IL-3 were also analyzed. Scatter plots of CD44
versus Thy-l expression by the CD34+ cells in these leukapheresis samples were not noticeably different (data not
shown) from those seen for normal BM (Fig 1) or CML PB
(Fig 2). Similar CD44 gates were, therefore, used to compare
the distribution of different progenitor types amongst the
two subpopulations of interest (defined by their levels of
CD44 expression) in the CML PB and mobilized normal PB
samples. The results of these studies are shown in Table 5
and in Fig 5.
The analyses of the CML samples revealed a number of
interesting findings. First, as can be seen in Table 4, both
Thy-l+ and Thy-l BFU-E and CFU-GM were readily and
consistently detected in CML PB. Althoughgenotyping studies of the colonies produced by these sorted progenitors were
not performed, it is unlikely from the number originally
present in the samples used (Table 1) that any of these sorted
progenitors contained a significant proportion of normal
cells. Moreover, in a more extensive analysis of CD34+
subpopulations of CML progenitors reported elsewhere,"'
the presence of a significant population of Ph+ Thy- 1 CFUGM in CML PB has been directly demonstrated. Second,
the number of LTC-IC in the CML PB samples studied was
often below the level of detectability. More recent studies4'
have shown that cryopreservation selectively kills Ph+ LTCIC, which would explain the unexpectedly low yieldof LTCIC obtained in this study wheremost samples hadbeen
frozen before use. Finally, as shown in Fig 5 , the proportion
of CFU-GM found in the CD44+++fraction of CD34+ CML
~
+
From www.bloodjournal.org by guest on December 22, 2014. For personal use only.
2982
GHAFFARI ET AL
Table 4. Progenitor Distributionsin Subpopulations of CD34' CML PB Cells by Their Levels of Expression of CD44 and Thy-l
No. of Prooenitors/lOsCellst
CD44"
Type of
Progenitor Evaluated
BFU-E
Patient No.
Thy-l
la*
1b*
2
17,000 3
23,000 4
15,000 5
Mean t SEM
la*
1b*
2
3
4
5
Mean 2 SEM
5,000
CFU-GM
CD44'
Thy-l Thy-l'
Thy-l
+
ND
4,800
7,000
5,300
7,300
7,200
6,300 2 500
ND
9,000
13,000
9,900
21,000
12,000
13,000 5 2,100
13,000
3,500
11,000
2,500
14,000 2 2,500
900
2,700
6,700
3,800
6,000
4,100
4,000 t 900
ND
5,300
4,300
3,500
7,900
ND
5,300 2 1,000
ND
7,100
1,800
4 , 7 0 0 2 1,000
11,000
19,000
12,000
24,000
19,000
ND
19,000 2 2,500
18,000
16,000
13,000
20,000
23,000
17,000 2 1,800
700
<500
Patient numbers l a and l b refer to fresh and thawed cells from the same original sample (see Table
1).
t For the population analyzed.
PB cells was consistently and significantly ( P < .01) elevated by comparison to CFU-GM in normal BM. Interestingly, this was not true for the BFU-E present in CML PB.
Moreover, the alteration in CD44 expression by CML CFUGM was not found to be a general feature of normal CFUGM that had been mobilized into the circulation, because
the ratio of CD44++ to CD44+++
CFU-GM in leukapheresis
harvests was not different from that characteristic of CFUGM in normal BM.
DISCUSSION
The production of different CD44 isoforms in conjunction
with variable degrees of their glycosylation and chondroitin
v
CML NBM
MOB
CFU-GM
BFU-E
CYL MOB NBH
CML MOB NBH
TOTALCELLS
Fig 5. Comparison of the distribution ofBFU-E and CFU-GM in
the CD34+ CD44*+ ( 0 )and CD34+ CD44+++(m) fractions of CML PB
(CML;n = 6 five patients, one evaluated in duplicate), mobilized
blood (MOB; n = 3), and normal bone marrow [NBM; n = 6). Error
bars indicate 1 SEM above the mean.
sulfate attachment is believed to explain the diversity of
adhesion-dependent processes in which CD44 has been implicated."." In this study, we have obtained further evidence
that CD44 may be involved in the regulation of early stages
of hematopoiesis based on the demonstration of differentiation- and transformation-associated changes in the expression of this gene product on primitive normal andCML
cells. Analysis of the CD34+ population in normal BM and
CML PB indicated that CD44- cells are not present at a
detectable level within this fraction, in contrast with the total
light-density cell fraction, of which a small percent (up to
approximately 10%)may be identified as CD".
Functional
studies showed that the LTC-IC in normal marrow express
intermediate to very high levels of CD44, thus mirroring the
heterogeneous pattern of CD44 expression also exhibited by
normal CFU-GM. In contrast, BFU-E in normal BM represent a more homogenous population of cells in terms of
their pattern of CD44 expression. It will be interesting to
determine whether the subset of CFU-GM expressing very
high levels of CD44 are those that are able to bind immobilized hyaluronan4' or that cooperate with the cr4/3, integrin
in binding to the C-terminal heparin-binding domain of fibronectin.4' Kansas et all4 also have reported changes in
CD44 expression as hematopoietic cells mature, although in
their studies, CD44 was seen to be downregulated at later
stages of myeloid and erythroid cell maturation. The pattern
of CD44 expression on primitive CML cells was found to
differ in two respects from that exhibited by normal BM or
mobilized PB cells. First, a significantly larger proportion
of CML CFU-GM were found to express CD44 at a very
high level, and second, a subset of CD34+ cells expressing
an exon vl0-containing CD44 isoform(s)that was not detectable in normal BM could be readily detected in all CML PB
samples analyzed. Because all normal samples used in this
work were frozen, the changes in thepattern of CD44 expres-
From www.bloodjournal.org by guest on December 22, 2014. For personal use only.
CML
CD44 EXPRESSION
AND
ON NORMAL
CELLS
2983
Table 5. Progenitor Distributions in Circulating Subpopulationsof Chemotherapy and Growth Factor Mobilized CD34+ Cells
CD44++
Progenitor
Evaluated
2,000 BFU-E
4,300
13,000
CFU-GM
1,900
2,100
Sample No.
~
~ Cells
1
1
2
3
Mean ? SEM
1
2
3
Mean ? SEM
0
~ Enrichtl
CD44"'
Recoveryt
86
97
73
6,400 ?100
3,300
6,000
27
3,300 ? 1,300
935 66
5
38
31 2 13
? 4
73
84
97 62
73 ? 6
4
(%l
FWlO' Cells
14
Enricht"
Recovaryt (%l
1
5
3
610
290
680 2 260
4,300
16
800
4,100
3,100 5 1,100
9 72 56 4
17
38
39 27
5 29
5 6
Abbreviation: FQ, frequency.
* Calculated by dividing the FO/105 sorted cells by the FQ/105 unsorted light-density cells in each individual experiment.
t Calculated as described in the legend to Fig 4.
sion on CML cells could not be attributed to a general freezethaw artefact. Whether they may contribute to the abnormal
adhesive properties previously described for CML cells remains to be established.
The mechanism(s) underlying the increased expression of
CD44 on CML CFU-GM as well as the increased expression
of exon vl0-containing CD44 isoform(s) on more mature
CML cells, including some within the CD34+ population, is
also unknown. In particular, it is not clear whether these
increases are accompanied by concomitant changes in the
expression of other CD44 isoforms. Activation is one of
multiple mechanisms of CD44 isoform expression described
in different cell types.I6 The increase observed here in expression of CD44 on CML CFU-GM may, therefore, be
related to their constitutively activated
for example,
through BCR-ABL stimulation of the ras pathway as a result
of the association of p210BCR-ABL
with GRB-~/SOS?'~'The
product of the BCR-ABL gene has also been found to inhibit
p120 GAP
which would favor the formation of
GTP-bound ras and, hence, its accumulation in an activated
form. The results of BCR-ABL transfection experiments
have also implicated p210BCR-ABL
in the constitutive activation of ras,48 and several studies have indicated that ras activation may alter CD44 expression, by modulating CD44
promotor activity as well as via mechanisms that control
CD44 transcript s p l i ~ i n g .It~ is~ interesting
, ~ ~ ~ ~ ~to note that
modulations of CD44 expression were not observed in cells
obtained from patients treated with IL-3 in spite of the described ability of this cytokine to transiently activate the
ras pathway." Collectively, these findings suggest multiple
mechanisms by which the observed changes in CD44 expression seen at different levels of granulopoiesis in CML could
be related to the expression of the BCR-ABL gene in these
cells, although they do not explain why such changes should
be restricted to the granulopoietic lineage.
The identification of a small subset of maturing myeloid
cells in normal BM that appear to express different exon
vl0-containing isoforms of CD44 is concordant with previous reports demonstrating that a change in CD44 isoform
expression is not uniquely associated with malignant transf o r m a t i ~ n * but
~ .may
~~
be more closely correlated with
changes in properties that also occur during the development
or functional activation of normal
The observed
increase in expression of exon vl0-containing CD44 isoform(s) on CML cells could simply reflect a selective amplification in CML of a cell type that normally expresses these
isoforms. Alternatively, it could reflect a direct effect of
malignant transformation on cells that would not normally
express exon v10. Support for the latter possibility has been
recently suggested by the finding of alterations in CD44
expression on primary cells from patients with other types
of (acute) myeloid le~kemia.~'
In summary, we have documented the presence of CD44
on LTC-IC in normal human BM and have provided evidence of a heterogeneity in the level of the CD44 expressed
on these very primitive cells that is also shared by normal
CFU-GM. In contrast, most normal BFU-E express, on average, a lower level of CD44. We have also provided evidence
for the expression of different vl0-containing CD44 isoforms in normal human marrow cells. In CML, there is a
disproportionate increase in the type of CFU-GM that express very high levels of CD44. These findings, taken together with the abnormal production by CD34' CML cells
of exon vl0-containing isoform(s) of CD44, suggest that the
control of CD44 mRNA production and processing may be
influenced by both normal differentiation mechanisms and
those that mediate leukemic transformation.
ACKNOWLEDGMENT
We thank GayleThomburyandWieslawaDragowskafor
op
erating the FACS, Giovanna Cameron for the differential analysis,
and Irene Edelmann for assistance in manuscript preparation. We
and Amgen and Sandoz
also thank Dr J. Spinelli for statistical advice
for generous gifts of recombinant human growth factors.
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1995 86: 2976-2985
Differentiation-associated changes in CD44 isoform expression
during normal hematopoiesis and their alteration in chronic myeloid
leukemia
S Ghaffari, GJ Dougherty, PM Lansdorp, AC Eaves and CJ Eaves
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