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Blood, Vol. 94 No. 10 (November 15), 1999:
pp. 3551-3558
Enforced CD19 Expression Leads to Growth Inhibition and Reduced
Tumorigenicity
By
Maged S. Mahmoud,
Ryuichi Fujii,
Hideaki Ishikawa, and
Michio M. Kawano
From the Department of Immunohematology, Yamaguchi University School
of Medicine, Ube, Japan.
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ABSTRACT |
In multiple myeloma (MM), the cell surface protein, CD19, is
specifically lost while it continues to be expressed on normal plasma
cells. To examine the biological significance of loss of CD19 in human
myeloma, we have generated CD19 transfectants of a tumorigenic human
myeloma cell line (KMS-5). The CD19 transfectants showed slower growth
rate in vitro than that of control transfectants. They also showed a
lower capability for colony formation as evaluated by
anchorage-independent growth in soft agar assay. The CD19 transfectants also had reduced tumorigenicity in vivo when subcutaneously implanted into severe combined immunodeficiency (SCID)-human interleukin-6 (hIL-6) transgenic mice. The growth-inhibitory effect was
CD19-specific and probably due to CD19 signaling because this effect
was not observed in cells transfected with a truncated form of CD19
that lacks the cytoplasmic signaling domain. The in vitro
growth-inhibitory effect was confirmed in a nontumorigenic human
myeloma cell line (U-266). However, introduction of the CD19 gene into
a human erythroleukemia cell line (K-562) also induced growth
inhibition, suggesting that this effect is CD19-specific, but not
restricted to myeloma cells. These data suggest that the specific and
generalized loss of CD19 in human myeloma cells could be an important
factor contributing to the proliferation of the malignant plasma cell
clones in this disease.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
HUMAN MULTIPLE MYELOMA (MM) is a
proliferative disorder of monoclonal plasma cells, which accumulate in
the bone marrow. The origin of the malignant clones in MM is still
controversial despite many efforts to identify the MM stem
cell.1 Recently, phenotypic analysis has been used as a
valuable method in the diagnosis and prognosis of MM. One of the
surface molecules that distinguish normal plasma cells from myeloma
cells is the B-cell marker, CD19. We have reported that normal plasma
cells express CD19, in contrast to primary myeloma cells and cell lines
that are CD19 .2 We have also
demonstrated that the Pax-5 gene, which positively controls CD19 gene
expression, was expressed in normal plasma cells, but not in myeloma
cells and cell lines.3 However, the significance of loss of
CD19 expression in human myeloma plasma cells is unclear.
The CD19 molecule is a 95-kD cell surface glycoprotein
expressed on the surface of B lymphocytes.4 Its
extracellular portion contains Ig-like domains and an Epstein-Barr
virus-related cytoplasmic tail,5,6 and the cytoplasmic
domain is highly conserved between human, mouse, and guinea
pig.7 CD19 is a key molecule in the B-lymphocyte signal
transduction complex, where it associates noncovalently with the
complement receptor type 2 (CR2) CD21,8 CD81 (TAPA-1), and
Leu-13.9
Targeted disruption of the CD19 gene in mice showed its importance in
T-cell-dependent antigen responses10 and B-cell
memory.11 CD19 can act as both a positive and negative
regulator of B-cell proliferation depending on the stimulating
signal.12 When coligated to the B-cell antigen receptor
(surface Ig [sIg]), CD19 lowers the threshold for antigen receptor
stimulation of B lymphocytes.13 CD19 has also been linked
to several related signal transduction pathways including the
activation of phospholipase C- -1 (PLC--1)14 and
phosphoinositol-3 kinase (PI3-K).15 Recently, it has been shown that 2 major signals emerging from PI3-K can target protein kinase B (PKB; also known as Akt) and mitogen-activated protein kinase
(MAPK).16 Tyrosine phosphorylated CD19
interacts with src-homolgy-2 (SH-2) domain-containing proteins linking
it to intracellular signaling pathways.17 CD19 molecules
phosphorylated at tyrosine-391 bind the proto-oncogene product Vav,
which contains an SH-2 domain, to mediate an increase in intracellular
Ca2+, activation of phosphatidylinositol 4-phosphate
5-kinase, and the c-Jun-N-terminal kinase (JNK), thus linking 2 distinct signaling pathways of lipid and protein kinases.18
Because CD19 can act as a positive or negative regulator in B-cell
signaling and because it is lost in MM plasma cell, we examined the
pathophysiologic effect and significance of loss of CD19 expression in
human myeloma by generating CD19 transfectants of 2 myeloma cell lines.
We studied the biological characteristics of CD19 transfected cells in
comparison to both neo-control transfectants and transfectants
expressing a truncated form of the molecule that lacks the cytoplasmic
domain. The possible biological role of loss of CD19 in the
proliferation of plasma cells in MM is discussed.
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MATERIALS AND METHODS |
Cell culture.
The tumorigenic KMS-519 and the nontumorigenic
U-26620 human myeloma cell lines and the human
erythroleukemia cell line K-562 were maintained in RPMI-1640 (Nissui,
Tokyo, Japan) supplemented with 10% fetal calf serum (FCS; M.A.
Bioproducts, Walkersville, MD). Growth curves of U-266 were performed
in the presence or absence of exogenous recombinant human interleukin-6
(rhIL-6; 2 ng/mL). For cell culture experiments in
serum-free medium, we used AlbuMAX (200 µg/mL), bovine insulin (2 µg/mL), and human transferrin (2 µg/mL; all purchased from GIBCO
BRL, Grand Island, NY).
Plasmid construction.
Full-length CD19 cDNA was cloned by reverse transcriptase-polymerase
chain reaction (RT-PCR) from the human Burkitt's lymphoma cell line
(Raji) using primers CD19 F1 and CD19 R1 described below. The cloned
PCR product was directly ligated into the TA vector pCR-TOPO
(Invitrogen, San Diego, CA) to give pCR-CD19. A truncated form of CD19,
which has only the extracellular and transmembrane domains, but lacks
the cytoplasmic domain, was amplified by PCR from pCR-CD19 using
primers CD19 F1 and CD19 R2 described below and directly cloned to the
TA-vector to give pCR- CD19 (Fig 1A). Both cDNA
inserts in pCR-CD19 and pCR-CD19 were sequenced by the dyedeoxy
termination method using (ABI PRISM) dye terminator cycle sequencing
ready reaction kit (Perkin Elmer, Applied Biosystems Division, Foster
City, CA) according to the manufacturer's instructions. The cloned
cDNA fragments were then eluted and subcloned into the
EcoRI site of the mammalian expression vector pCI-neo
(Promega, Madison, WI) driven by cytomegalovirus (CMV)
immediate early enhancer/promoter to give pCI-CD19 (full-length) and
pCI-CD19 (truncated form). The orientation of the inserts in the
expression vector was verified by Xho I restriction for the
full-length construct and Sma I for the truncated form
(pCI-CD19).
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| Fig 1.
Establishment of K-19+ myeloma cell clones.
(A) Schematic representation of the mammalian expression vectors
pCI-CD19 and pCI- 19. CMV, cytomegalovirus immediate early
enhancer/promoter; Int, chimeric intron; ED, TM, and CD, extracellular,
transmembrane, and cytoplasmic domains of CD19, respectively; Poly(A),
SV40 late polyadenylation signal; the arrows indicate the positions of
pCI-neo specific primers pCIF and pCIR. (B) Single color flow
cytometric analysis with PE-labeled mouse antihuman IgG as a negative
control (control IgG) and PE-labeled CD19 antibody showing the
expression level of the CD19 transgene on different clones. (C) RT-PCR amplification of mRNA from
K-19+ and K-19 clones using full-length CD19F1 and
CD19R1 primers (top) or truncated form CD19 F1 and CD19R2 primers
(middle). (D) PCR amplification of genomic DNA from the parental cell
line, KMS-5, and neo-control clones (M nos. 1, 2, and 3).
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Stable transfection and cloning.
The human CD19 KMS-5 myeloma cell line was
transfected by pCI-neo vector (neo-control), pCI-CD19
(K-19+), or pCI-CD19 (K-19) by lipofection. Cells
were plated at 3 × 105 in 6-well plates overnight,
and on the following day, cells were washed in serum-free medium and
transfected by a mixture of 5 µg DNA and 20 µg lipofectin (GIBCO
BRL) and incubated at 37°C with 5% CO2. After 10 hours
of incubation, complete medium with 10% FCS was added and incubation
was continued for a total of 72 hours. Transfected cells were then
diluted 1:10 in medium and selected in geniticin (GIBCO BRL) at 500 µg/mL. By 8 weeks, resistant clones started to appear, and subcloning
was performed at least twice to obtain single-cell clones by limiting
dilution in 96-well plates. The K-562 erythroleukemia cell line was
transfected by lipofection in an identical manner. The cells were
selected in 1 mg/mL geniticin. The U-266 human myeloma cell line was
transfected by electroporation of 3 × 107 cells with
300 µg of each recombinant plasmid in 600 µL of Hank's buffered
solution. Electroporation was performed by the Gene Pulser (Bio-Rad
Laboratories, Tokyo, Japan) at 210 V and 1,440 µF capacitance, followed by selection in medium containing 1 mg/mL geniticin. Transfected U-266 and K-562 expressing the surface CD19 were sorted after 2 weeks of selection, maintained in selection medium, and subsequently used for experiments.
Antibodies and flow cytometry.
Cells were harvested and stained with either phycoerythrin
(PE)-labeled anti-IgG isotypic control or PE-labeled
anti-CD19 (Coulter, Hialeah, FL) as described previously21
and were analyzed by the cell sorter (Epics Elite ESP; Coulter).
Extraction of mRNA from transfected cells.
mRNA was extracted by oligo-dT latex beads (Oligotex-dT30; Takara,
Kyoto, Japan) according to the manufacturer's instructions. Isolated
mRNA was reverse transcribed at 37°C for 60 minutes in a reaction
mixture containing 100 pmol/L random hexamer (Pharmacia Biotech, Tokyo,
Japan), 0.5 mmol/L each deoxynucleotide-5-triphosphate (dNTPs), RT
buffer (50 mmol/L Tris-HCl, pH 8.8, 7.5 mmol/L KCl, 3 mmol/L
MgCl2), 10 mmol/L dithiothreitol (DTT), 20 U of human placental ribonuclease inhibitor (Takara), and 20 U of reverse transcriptase Superscript II (GIBCO BRL, Gaithersburg, MD).
PCR.
Full-length CD19 cDNA was amplified by RT-PCR using primers designed
according to the published sequence5 as follows: CD19 F1:
5'-GGA GAG TCT GAC CAC CAT GCC ACC T-3' (nucleotides 1-25) and CD19 R1: 5'-AAG GGG ACT GGA AGT GTC ACT GGC AT-3'
(nucleotides 1878-1853). The truncated construct was amplified from
pCI-CD19 using primers CD19 F1 and CD19 R2: 5'-GGC TCT TTG AAG
ATG AAG AAT GCC CAC-3' (nucleotides 964-938). For detection of
integration of the pCI-neo into the genome of mock transfectants, the
following pCI-neo vector specific primers (see Fig 1A) were used to
amplify a 222-bp fragment from genomic DNA; pCIF: 5'-ATC CAC TTT
GCC TTT CTC TCC ACA-3' (nucleotides 998-1021), and pCIR:
5'-CTG CAT TCT AGT TGT GGT TTG TCC-3' (nucleotides
1219-1146). PCR cloning of the full-length CD19 was performed in a
2-step cycle of 94°C for 30 seconds denaturation and 72°C
annealing/extension for 4 minutes (7 cycles), followed by 32 cycles of
denaturation at 94°C for 30 seconds and annealing/extension at
67°C for 4 minutes with rTth (Perkin Elmer) and Vent (New England
Biolabs, Beverly, MA) mixture of DNA polymerases and a manual hot start
by AmpliWax (Perkin Elmer). Thermal cycling for the pCI-neo sequences
was performed by rTaq (Takara) at 94°C denaturation for 1 minute, annealing at 65°C for 1 minute, and extension at 72°C for 1 minute. The truncated fragment was amplified using the same conditions except for annealing at 57°C for 1 minute.
Colony assay.
Soft agar (1.6% wt/vol) was prepared by autoclaving Bacto-agar (DIFCO,
Detroit, MI) in distilled water just before use. The bottom agar layer
(2.1 mL/well) contained 1.6% agar:2× RPMI/20% FCS:1×
RPMI/10% FCS without cells in a volume ratio of 1:1:1, respectively,
as a final agar concentration of 0.53%. The top agar layer (0.9 mL/well) contained 1.6% agar:2× RPMI/20% FCS:1× RPMI/10%
FCS with cells in a ratio 1:1:2, respectively, with a final agar
concentration of 0.4%. The number of cells plated for each clone was 5 × 102/mL or 5 × 103/mL. Plates were incubated at 37°C with 5%
CO2 for 2 weeks and the colonies were counted and
photographed under a phase contrast microscope (Nikon,
Tokyo, Japan).
In vivo growth and tumorigenicity.
Severe combined immunodeficiency (SCID)-hIL-6 transgenic
mice22 provided by Chugai Pharmaceutical Co, Ltd (Shizuoka,
Japan) were used for experiments between 4 to 6 weeks after birth. The tumorigenic KMS-5 transfectants (K-19+ and
neo-control cells) were harvested (5 × 106),
suspended in 200 µL phosphate-buffered saline (PBS), and
subcutaneously inoculated into the flanks or the back of the mice on
the same day. Mice were observed for tumor formation every 4 days.
After 5 weeks, mice were killed, tumor was resected, and its growth was
evaluated by volume according to the formula: 1/2ab2 (in microliters), where "a" is the longest and "b" is the
shortest axes of the tumor. Statistical analysis was performed by the
Student's t-test (n = 5 for each clone). This experiment was
reviewed by the Committee of the Ethics on Animal Experiment in
Yamaguchi University School of Medicine and performed under the control of the Guideline for Animal Experiment in Yamaguchi University School
of Medicine and The Law (No. 105) and Notification (No. 6) of the Government.
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RESULTS |
Establishment of KMS-5 myeloma cell line clones stably expressing CD19
(K-19 cells).
To characterize the transfected cells and confirm the expression of the
CD19 transgene, we used flow cytometry to screen clones obtained by
limiting dilution. Twenty-seven single-cell clones expressing variable
levels of surface CD19 were identified, and of those, 3 representative
clones were further identified and examined. One clone that expressed
low-level CD19 (K-19+ clone #8) and 2 clones expressing
high levels (K-19++ clones #3 and #11) were used for the
following experiments. We also characterized another clone that
expressed high levels of truncated CD19 (K-19) on its surface
(Fig 1B). The mRNA expression of the
transgenes was examined by RT-PCR. Amplification of reverse transcribed
mRNA using the CD19 F1 and CD19 R1 primer pair produced a single band
of the expected length from K-19+ cells, but not from
K-19 or neo-control transfected cells (Fig 1C, top). On the other
hand, CD19 F1 and CD19 R2 primers produced amplification products from
all clones except neo-control transfected cells (Fig 1C, middle).
Neo-control transfected cells were also cloned by limiting dilution to
isolate single-cell clones. The integration of the expression vector
into the genome of 3 independent neo-control transfectants (M no.
1, M no. 2, and M no. 3) was verified by amplifying genomic DNA
using vector specific primers from 3 mock clones, but not from the
KMS-5 parent cells (Fig 1D). These data show the expression of
varying levels of CD19 in stably transfected clones.
CD19 expression in myeloma cells confers a negative growth regulatory
effect in vitro.
To examine the biological effects of CD19 expression on myeloma cells,
we first evaluated the growth rate of transfected clones cultured in
RPMI 1640 supplemented with 10% FCS. Interestingly, K-19+
clones showed slower growth rate compared with either neo-control or
K-19 cells (Fig 2). Moreover, the
growth-inhibitory effect seemed to be related to the level of CD19
expression. High CD19 expression (clones #3 and #11) was associated
with markedly slower growth, and low expression (clone #8) was
associated with less marked growth inhibition. Three neo-control clones
(M no. 1, M no. 2, and M no. 3) that we used in our experiments had an
almost identical growth pattern. The growth rate of neo-control clones and K-19 cells was almost identical. These data show that CD19 appears to exert a growth-inhibitory effect on myeloma cells in an
expression level-dependent manner. These effects are likely related to
intracellular signaling through CD19, because they were not observed in
the truncated CD19 transfectants or in neo-control clones.
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| Fig 2.
CD19 expression in KMS-5 human myeloma cell line leads to
growth inhibition in vitro. K-19+, K-19, and
neo-control clones were adjusted to 1 × 103/mL and
plated in RPMI 1640 + 10% FCS. Cells were harvested and counted at
the indicated time points both automatically on the cell sorter and
manually on a hemocytometer. Data from 3 experiments are shown as
mean ± standard deviation (SD).
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CD19 expression in KMS-5 myeloma cell line is associated with reduced
colony formation.
Another biological feature of malignant cells that we have investigated
is anchorage-independent growth capability. As evaluated by soft agar
colony assay, K-19+ cells showed markedly lower capability
for colony formation. Plating 5 × 102 cells/mL in
triplicate experiments, both neo-control and K-19 cells formed at
least 3 large colonies in each well after 2 weeks, whereas
K-19+ cells formed lower numbers of smaller colonies
(Fig 3 and
Table 1). Low CD19 expression clone
(K-19+ #8) formed 2 small colonies in only 1 well, and high
CD19 expression clones (K-19++ #3 and #11) did not form any
colonies, even when the cultures were kept for a longer incubation
period. Increasing the number of plated cells to 5 × 103 cells/mL also showed a lower capability for colony
formation by K-19+ clones compared with neo-control clones
(Table 1). These data show that the expression of CD19 in myeloma cells
resulted in reduced capacity for anchorage-independent growth. The
level of this inhibitory effect appears to be well-correlated with the level of CD19 expression in myeloma cells.
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| Fig 3.
CD19-expressing KMS-5 cells have lower capacity for
colony formation. The indicated clones were plated in soft agar at 5 × 102/mL or 5 × 103/mL in
triplicates in 6-well plates and incubated at 37°C and 5%
CO2. Two weeks later, the colonies were counted and
photographed under a phase contrast microscope.
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CD19 exerts its growth-inhibitory effect on K-19
myeloma cells in vivo.
We next tried to verify if CD19 exerts a similar growth inhibitory
effect in vivo by subcutaneous inoculation of CD19 transfectants in
SCID-hIL-6 transgenic mice. Mice were observed for tumor formation every 4 days. The earliest observed tumors appeared after 17 days in
mice injected with neo-control clones. After 5 weeks, mice were killed,
and the tumor was resected and evaluated according to the equation
described in Materials and Methods. The time required for tumor
formation and the tumor volume correlated well with the in vitro growth
characteristics of the different K-19+ clones. The clones
expressing CD19 formed tumors at least 1 week (K-19+ #8) or
more (K-19++ #3 and #11) slower than neo-control clones.
Also, the tumor volume of K-19+ clones was significantly
smaller than that of mock clones. The statistical data are drawn from 5 animals injected with each clone, and the P value is shown
above the horizontal bars (Fig 4). When K-19+ clones (K-19+ #8, K-19++ #3,
and #11) were compared with each other, there was significant difference between them, specifically between clone #8, expressing low-level CD19, and the high expression clones #3 and #11. These in
vivo data support and confirm the in vitro observations of the
growth-inhibitory effect of CD19 in myeloma cells. It also shows that
the level of expression of CD19 correlates with the growth inhibitory
effect.
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| Fig 4.
CD19 exerts its growth-inhibitory on KMS-5 in vivo. A
total of 5 × 106 cells of each clone was inoculated
subcutaneously in SCID-hIL-6 transgenic mice. After 5 weeks mice, were
killed and tumors resected and measured as described in Materials and
Methods. The statistical data are drawn from 5 animals injected with
each clone, and the P value is shown above the horizontal bars.
The mean ± SD values for each group were as follows: neo-control
(5,007 ± 1,212); K-19+ #8 (2,756 ± 1,877);
K-19++ #3 (854 ± 375); and K-19++ #11
(269 ± 336). Each point represents 1 animal.
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The in vitro growth-inhibitory effect of CD19 is observed in another
myeloma (U-266) and nonmyeloma (K-562) cell lines.
To verify whether the growth-inhibitory effect of CD19 is specific to
myeloma cells, we have used 2 cell lines, U-266 nontumorigenic myeloma
cell line and K-562 human erythroleukemia cell line, to express the
intact and truncated forms of CD19. Because the U-266 cells proliferate
in response to exogenous IL-6, we have tested these cells both in the
presence and absence of exogenous rhIL-6 (2 ng/mL). Intact CD19 induced
growth inhibition in U-266 cell line both in the presence or absence of
exogenous IL-6 (Fig 5A). However, this
growth-inhibitory effect was also observed in K-562 (Fig 5B). Taken
together, these data further support the observation of the
growth-inhibitory effect exerted by CD19 and suggest that this effect
is not restricted to myeloma or B-lineage cells.
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| Fig 5.
The growth-inhibitory effect is specific to
CD19, but not restricted to myeloma cells. (A) The expression vectors
pCI-CD19 and pCI-19 were electroporated into the human myeloma cell
line, U-266, to generate U-19+ and U-19 cells,
respectively, as detected by flow cytometry after staining with
PE-labeled anti-CD19 (top). Cells expressing surface CD19 were sorted
after 2 weeks of selection in G-418 (1 mg/mL) and used for evaluation
of growth pattern (bottom). Data are from 3 independent experiments.
The mean ± SD of cell number at each time is shown. (B) The pCI-CD19
and pCI-19 vectors were transfected by lipofection into the human
erythroleukemia cell line, K-562, to generate K562-19+
and K562-19, respectively. After 2 weeks of selection in G-418 (1 mg/mL), surface CD19 expression was confirmed by flow cytometry after
staining with PE-labeled anti-CD19 (top), and the in vitro growth curve
of these cells is shown as mean ± SD values of cell numbers at the
indicated time points (bottom).
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DISCUSSION |
An important feature of human MM plasma cells is the
specific loss of the B-cell surface protein, CD19, in contrast to
normal plasma cells, which do express this molecule.2 The
loss of CD19 in human myeloma cells was correlated with the absence of its positive regulatory factor, B-cell-specific activator protein (BSAP), encoded by Pax-5 gene.3 However, the biological
significance and the consequences of loss of CD19 in human myeloma has
been unclear. In this study, we have generated clones of myeloma cell lines that stably express CD19 and studied the effects of transgene expression on cell growth and tumorigenicity. We observed a striking growth inhibition in cells expressing intact CD19, with the degree of
inhibition related to the level of expression of the transgene (Fig 2);
high expression clones had slower growth rates. This in vitro growth
inhibition was further confirmed by in vivo studies performed by
subcutaneous inoculation in SCID-hIL-6 transgenic mice (Fig 4). We
observed a good correlation between in vitro and in vivo growth
characteristics of the CD19-transfected cells. The parental cell line,
KMS-5, is IL-6 independent and rapidly proliferates with a doubling
time of less than 2 days.23 This might explain the fact
that cell growth was not completely arrested, thus allowing isolation
of stable transfectants.
Introduction of intact and truncated CD19 transgenes into another
myeloma cell line, U-266, and a myeloid cell line, K-562, confirmed
that the growth-inhibitory effect is specific to CD19 (Fig 5). The in
vivo effect of CD19 could not be confirmed in U-266 because this line
is nontumorigenic. However, these experiments showed that this effect
was not specifically restricted to myeloma cells because it could be
observed in the erythroleukemia cell line.
Another biological feature clearly modified by the introduction of CD19
into myeloma cells was anchorage-independent growth in soft agar. The
number and size of colonies formed by CD19 expressing clones was
significantly lower than that of neo-control and truncated CD19 clones
(Fig 3 and Table 1). The failure of high expression clones to form
colonies in soft agar was not merely due to their slower growth rates,
because they did not form colonies even when kept in culture for longer
periods. A 10-fold increase in the number of plated cells did not
result in marked increase of the number of neo-control colonies, but
intact CD19 transfectants could form few small colonies (Table 1).
Also, this effect seems to be related to intact CD19 cytoplasmic domain
and hence to CD19 signaling, because the truncated mutant form failed
to exert similar effects.
The mechanism(s) by which CD19 induces these effects remains to be
elucidated. Systematic study of the major candidate signaling pathways
(mainly PI-3 kinase and MAP kinase) downstream of CD19 is ongoing in
our laboratory. Preliminary experiments on K-19+ cells
stimulated with serum showed that phosphorylated ERK-1 and ERK-2 MAP
kinases were downregulated in K-19++ clones, but were
constitutively phosphorylated in neo-control and K-19 clones (data
not shown). This suggests that CD19 might be working as a
molecular switch, which turns the growth signals off in appropriate
time, and its absence may leave constitutively activated
proliferation-promoting signals on, at least regarding the MAPK
pathway. Nevertheless, it is surprising that expression of a surface
protein such as CD19, unlike other known tumor suppressor gene
products, could inhibit the growth of these cell lines both in vitro
and in vivo. Taken together, CD19 may act in human myeloma as a new
type of tumor suppressor gene product that is a transmembrane-signal transducing molecule.
We also observed decreased sensitivity of CD19 transfected cells to
apoptotic stimuli such as dexamethasone, insulin, and serum starvation
in comparison to neo-control and truncated CD19 clones. The difference
was less striking than that of growth inhibition and tumorigenicity.
Because the parental cell line, KMS-5, is already resistant to
apoptotic stimuli, the observed effect could be due to CD19 expression,
CD19-mediated amplification of antiapoptotic signals elicited by
another pathway, or alternatively, due to the proliferation-inhibitory
effect of CD19 expression (data not shown).
Our data suggest that loss of CD19 expression in human MM could be an
important factor leading to the expansion of the malignant plasma
cells. The availability of these clones should help in the dissection
of signal transduction pathways in plasma cells, which no longer
express the B-cell antigen receptor, and hence their signaling pathways
via CD19 could be different from those of mature B cells. Finally,
although the growth-inhibitory effect of CD19 is not specifically
exerted on myeloma cells, the probability of specific transfer of CD19
gene into myeloma cells could contribute to the development of new
strategies for the treatment of human MM.
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FOOTNOTES |
Submitted December 9, 1998; accepted July 15, 1999.
Supported in part by grants from the Japanese Ministry of Education,
Science, and Culture, and the Japanese Ministry of Health and Welfare.
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 Michio M. Kawano, MD, Department of
Immunohematology, Yamaguchi University School of Medicine, 1-1-1 Minami-kogushi, Ube, Yamaguchi 755-8505, Japan; e-mail: mkawano{at}po.cc.yamaguchi-u.ac.jp.
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