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Carcinogenesis |
Division of Molecular Carcinogenesis, Department of Hematology/Oncology [B. H., A. v. W., A. R., R. d. J., J. G., N. H.], and Department of Pathology [V. K., P. K. P., I. G-G.], Childrens Hospital Los Angeles Research Institute and the Keck School of Medicine of the University of Southern California, Los Angeles, California 90027
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In 1992, Freed and Hunter (3) described a Mr 41,000 protein that is specifically tyrosine phosphorylated in the leukemic cells of CML patients. The protein was subsequently identified by us as Crkl, an adapter that consists only of an SH2 and two SH3 domains (4) . Crkl forms a constitutive complex with Bcr/Abl through the Crkl SH3 domain, which can bind to polyproline-rich regions within Abl (5) . Crkl is specifically and constitutively tyrosine phosphorylated in human patient material containing a Bcr/Abl fusion protein but not in normal bone marrow or peripheral blood (6, 7, 8) . We have established previously (9 , 10) a line of transgenic mice to model the leukemia caused by Bcr/Abl P190 in vivo. The lymphoblastic leukemia/lymphoma that invariably develops in these mice is characterized by an active P190 Bcr/Abl kinase that tyrosine phosphorylates Crkl (11) . In addition, Bcr/Abl phosphorylates tyrosine residues in Cas, Hef1, and Cbl, which subsequently can bind to Crkl via its SH2 domain in the leukemic cells of these mice (12 , 13) . Many studies (14, 15, 16) have shown that the normal cellular function of Crkl is closely associated with signal transduction through membrane-bound receptors for extracellular matrix, growth factors, and cytokines. One study (17) reported that overexpression of Crkl alone allows 293 epithelial cells to grow in soft agar, one characteristic of cellular transformation.
To examine the function of the Crkl-Bcr/Abl interaction, deletion mutants of Bcr/Abl have been made that lack the Crkl-binding sites. One study (17) reported that this mutant has an impaired ability to transform Rat2 fibroblasts. However, others have shown that it retains the ability to generate IL-3-independence in the murine myeloid 32D cell line. Because Crkl was still detected in complex with the Bcr/Abl mutant and became tyrosine phosphorylated, these results suggest that Crkl is an integral component of the transforming signal (18 , 19) . Antisense oligonucleotides against Crkl inhibited the growth of Ph-positive cell lines established from acute lymphoblastic leukemia and CML patients but not of a leukemia cell line from a Ph-negative patient (20) . Cell-penetrating peptides that bind with high selectivity to the first SH3 domain of Crkl strongly inhibited the proliferation of primary blasts from 11 of 16 CML patients (21) . Thus, the combined data suggest Crkl as a logical suspect for an important role in transducing the oncogenic signals of Bcr/Abl.
Because leukemogenesis caused by Bcr/Abl P190 in transgenic mice is an excellent in vivo model for the signal transduction perturbed by this oncogene, we wished to also address the role of Crkl in this process in vivo. To this end, we have generated CRKL transgenic mice that express the Crkl protein under control of the CRKL promoter. We report in this study that the overexpression of Crkl in vivo induces tumorigenesis and markedly enhances the development of lymphoblastic leukemia/lymphoma caused by Bcr/Abl P190. These data implicate Crkl in transduction of the leukemogenic signal of Bcr/Abl.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mice and Pathology.
All of the animals were maintained in accordance with the NIH Guide for
the Care and Use of Laboratory Animals. BCR/ABL P190
transgenic mice have been described previously (10
, 25
, 26)
. A line of P190 BCR/ABL mice established from
founder 623 has been bred to obtain animals that contained the
transgene on both chromosomes. These mice were mated to the
CRKL transgenic mice. Ensuing progeny was genotyped, which
yielded genotypically matched siblings that were either P190
BCR/ABL only transgenics or P190
BCR/ABL x CRKL double
transgenics. A defined cohort of animals of both genotypes was
followed. Also, 5- and 10-copy CRKL only transgenics and
their nontransgenic siblings were monitored.
Autopsies were performed as described (9 , 10 , 24 , 25) on animals that appeared seriously ill. Involved tissues were dissected, and a portion was stored in formalin for routine histological analysis. Another aliquot was used to prepare protein lysates in Triton lysis buffer [25 mM sodium phosphate (pH 7.5), 5 mM EDTA, 150 mM NaCl, 1% Triton-X100, 50 mM NaF, 10 µg/ml aprotinin and leupeptin, 1 mM Na3VO4, and 1 mM PMSF] or SDS-sample buffer. For analysis of protein levels (CrkII, Crkl, C3G, Cas/Hef1, and Cbl) in the Crkl-only transgenics, mice were sacrificed at 19 days of age. For analysis of Crkl-C3G immunocomplexes, protein lysates were precleared with protein A/G agarose beads (Life Technologies, Inc., Gaithersburg, MD). The supernatant, recovered after centrifugation, was immunoprecipitated with anti-Crkl monoclonal antibodies. Immune complexes were analyzed by SDS-PAGE and immunoblotting using anti-C3G rabbit polyclonal antibodies. For analysis of activated small GTPases, tissue samples were dissected and stored on ice. Tissues were homogenized in a Brinkmann Polytron (10 s; speed setting at 3–4) in an approximate volume of 1 ml of modified RIPA buffer (see below)/18–20 µg of tissue and then briefly homogenized in a straight-wall tissue grinder (Radnoti Glass Technology, Monrovia, CA). After an incubation for 20 min on ice, samples were centrifuged (4°C; 12,500 rpm; SS34; Sorvall), and the supernatant was frozen in aliquots on dry ice and stored at -80°C.
Macrophage Motility Assays.
We isolated both bone marrow-derived and elicited peritoneal
macrophages. The latter were obtained by injection of 3 ml of a 4%
(w/v) solution of thioglycollate and by harvesting of the peritoneal
macrophages 6 days later.
Short-term bone marrow-derived macrophage cultures were established essentially as described (27) . In short, after isolation of bone marrow cells, 7 x 106 cells were incubated for 24 h in 5 ml of DMEM + 1 nM IL-3 + 0.44 nM CSF-1. Nonadherent cells were collected, Pronase-treated, washed in horse serum, and cultivated in 5 ml of DMEM +1 nM IL-3 + 0.44 nM CSF-1 for 2 days. After a second Pronase and horse serum treatment, cells were cultured for 2 days in DMEM + 4.44 nM CSF-1. Migration assays were performed for 8 h with 5 x 104 cells at 37°C and 8% CO2 in Transwell plates (8-µm pores; Corning Coster Corp., Cambridge, MA). The bottom of each well was coated with 50 µl of a solution of 10 µg/ml fibronectin (Sigma Chemical Co., St. Louis, MO). Isolation of macrophages and migration assays were performed three times independently with two (elicited macrophages) or one mouse (bone marrow-derived macrophages)/genotype/experiment. Migration assays in each experiment were performed in triplicate. The results with the elicited macrophages were comparable with those of the bone marrow-derived macrophages, except that the absolute number of migrating macrophages in the latter samples was larger.
Cell Lines and Transfections.
COS-1 cells were obtained from the American Type Culture Collection
(Rockville, MD). All of the tissue culture reagents were from Life
Technologies, Inc. To obtain positive controls for detection of
activated Ras or Rap1, COS-1 cells were transfected with DNA constructs
encoding v-H-Ras or EGFP-V12Rap1 using DEAE/dextran.
Mock-transfected COS-1 cells were used as negative controls. Cells were
harvested 40–42 h after transfection and lysed in modified RIPA buffer
[10 mM sodium phosphate (pH 7.5), 100 mM NaCl,
5 mM MgCl2, 1 mM EDTA,
1% Triton X-100, 0.5% deoxycholic acid, 0.1% SDS, 1 PMSF, 10 µg/ml
leupeptin, and 10 µg/ml aprotinin] for the detection of activated
small GTPases. Lysates were cleared by centrifugation, and the protein
concentration was measured using the BCA protein assay (Pierce
Chemical Co., Rockford, IL). All of the chemicals were from Sigma
Chemical Co. or Life Technologies, Inc. NIH 3T3 cells were kept in DMEM
supplemented with 10% FCS , grown until 80% confluency, and then
incubated for 40 min with or without 0.4 M sorbitol before
the preparation of protein lysates.
Assay for Activated GTPases.
Plasmids encoding GST-RalGDS-RBD and GST-Raf1-RBD were obtained from
Dr. H. Bos (University Medical Center, Utrecht, The
Netherlands). GST-fusion proteins were expressed in
Escherichia coli. GST-RalGDS-RBD was purified on glutathione
agarose, then dialyzed to remove glutathione and stored at -80°C in
50 mM Tris-HCl (pH 8.0), 10% glycerol, and
0.01% NaN3. GST-RalGDS (50 µg) was precoupled
to 50 µl of a 50% slurry of glutathione agarose for 1 h at
4°C. Beads were washed three times in lysis buffer, after which the
sample to be assayed was added. Bacterial lysates of GST-Raf1-RBD were
obtained by sonication of the bacterial pellet on ice for 6 min in
PBS + 0.5 mM DTT, 2 µg/ml aprotinin,
1 µg/ml leupeptin, and 10 µg/ml trypsin inhibitor. Triton-X100 was
added to 1%, and the solution was mixed for 30 min at 4°C. After
centrifugation (10 min; 12,500 rpm; Sorvall SS34; 4°C), the
supernatant was collected and stored at -80°C after addition of 10%
(v/v) glycerol. Bacterial lysate (250 µl) was incubated with 500 µl
of a 33% slurry of glutathione agarose for 1 h at 4°C. Beads
were washed three times with 1 ml of lysis buffer [50
mM Tris-HCl (pH 7.5), 10% glycerol, 1% NP40,
200 mM NaCl, and 2 mM
MgCl2]. Beads were resuspended in 500 µl of
lysis buffer + inhibitors (1 mM PMSF, 2
µg/ml aprotinin, 1 µg/ml leupeptin, and 10 µg/ml trypsin
inhibitor), aliquoted into 50-µl amounts, frozen on dry ice, and
stored at -80°C.
Assays for GTP-bound Rap and Ras were essentially as described (28 , 29) . Briefly, lysates of COS-1 cells or of mouse tissues were thawed quickly and then placed on ice. Precoupled GST-Raf1-RBD or GST-RalGDS-RBD (50 µg) was incubated with 1 mg of tissue lysate or 5 µg (COS-1-transfected with v-H-Ras) or 100 µg (COS-1-transfected with EGFP-V12Rap1) of cell lysate in a final volume of 600 µl in modified RIPA buffer. After incubation, beads were washed three times in lysis buffer. The beads were boiled in SDS-sample buffer; protein samples were separated on a 15% SDS-PAA gel and then transferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore, Bedford, MA). In addition, 2–2.5% of each supernatant after pull-down was included to measure total (GTP- and GDP-bound) small GTPase levels.
Polyclonal rabbit pan-Ras antibodies (KAP-GP001E) were from StressGen Biotechnology Corp. Monoclonal Rap1 antibodies were from Transduction Laboratories (R22020). We obtained rabbit polyclonal antibodies to detect phosphorylated JNK, p38 MAPK, and p44/42 Erk1/2 from New England Biolabs. Rabbit polyclonal p38 MAPK (C-20) antibody, goat polyclonal ERK1 antibodies (K-23), and rabbit polyclonal C3G (C-19) antibody were from Santa Cruz Biotechnology. Peroxidase-conjugated goat antirabbit and goat antimouse antibodies were obtained from Bio-Rad (Hercules, CA). Immunocomplexes were detected by enhanced chemiluminescence and Hyperfilm-enhanced chemiluminescence (Amersham Corp., Arlington Heights, IL). When necessary, the filters were stripped using a Re-Blot kit (Chemicon International, Inc., Temecula, CA).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, the transgenic construct included around 7 kb of sequences
5' to CRKL exon 1 and around 8 kb of intron 1. We chose the
CRKL promoter to control transcription of the transgene,
because the crkl gene is abundantly expressed in
hematopoietic cells (11)
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We analyzed expression of the transgene in different tissues of progeny
of all of the four lines using Western blots. In all of the tissues
analyzed, including heart, brain, kidney, spleen, liver, lungs, thymus,
peripheral blood, and bone marrow, the expression level was correlated
with the transgene copy number (data not shown). When levels of
expression in different tissues within one line were compared with
those of nontransgenics, the ratios mimicked those of endogenous
crkl. As shown in Fig. 1B
, Crkl protein was
abundantly expressed in hematopoietic tissues including bone marrow,
peripheral blood, and thymus. Expression levels in the 5- and 10-copy
transgenics were approximately 4- and 6-fold that of the endogenous
crkl protein in thymus. The expression of CrkII, a
gene related to Crkl, was unaffected by Crkl overexpression,
nor were differences found in the levels of Cas/Hef1 or Cbl (data not
shown) in transgenics compared with nontransgenics.
Enhanced Motility of CRKL Transgenic Macrophages.
Crkl has been implicated in integrin-mediated adhesion
(30, 31, 32)
. Uemura and Griffin (30)
demonstrated that in transfected hematopoietic BaF3 cells,
overexpression of Crkl leads to increased cell migration. To
investigate whether Crkl is involved in hematopoietic cell migration
in vivo, we isolated peritoneal macrophages as well as bone
marrow-derived monocyte/macrophage cells from 10-copy CRKL
transgenics and from control nontransgenic siblings. Migration assays
of these macrophages on fibronectin-coated Transwell plates showed that
the haptotactic migration of both elicited (data not shown) and bone
marrow-derived macrophages (Fig. 2A)
on fibronectin was significantly increased.
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lysates). However, there were significantly more Crkl-C3G
complexes in the 10-copy as compared with the 1-copy CRKL transgenic
sample (Fig. 2B
, Crkl I.P.). These results
demonstrate that, in vivo, the involvement of Crkl and C3G
in integrin-mediated motility in hematopoietic cells is biologically
relevant.
Phenotype of CRKL Transgenics.
Founders as well as the progeny of the 5- and 10-copy CRKL
lines, including both transgenic and nontransgenic siblings of both
sexes, were followed until the animals appeared seriously ill, at which
point in time autopsies were performed. The major features found only
in the CRKL transgenics are listed in Table 1
. This analysis showed that the overexpression of Crkl protein was
associated with an increased incidence of tumorigenesis in transgenic
mice.
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were seen. The animal was diagnosed with a high-grade
anaplastic lymphoma. In animal 5632 (10-copy transgenic), we found two
large s.c. tumors in close proximity to the spine at 10 months of age.
Histology revealed a fibrosarcoma/malignant fibrous histiosarcoma (Fig. 3B)
. In another 10-copy animal, 5619, we found metastases in
kidney, liver, spleen, and mesenteric lymph node (Fig. 3C)
of an adenocarcinoma with unknown primarius at the age of 26
months. At 17 months, transgenic 5729 (10-copy animal) died of a
mammary adenocarcinoma that was widely disseminated to other organs
(Fig. 3, D and E)
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. In contrast, both ERK1/2 and p38 MAPK were very markedly
phosphorylated in the primary tumor and also in the metastasis
(Fig. 4A)
. Then, we investigated the primary tumor for
activation of small GTPases. Samples that were prepared as described in
"Materials and Methods" were incubated with GST-Raf1-RBD to detect
Ras in its GTP-bound form. As a positive methodological control, we
transfected COS-1 cells with v-H-Ras, a mutated form of Ras that is
constitutively GTP-bound. As shown in Fig. 4B
, the
transfected COS-1 cells expressed high levels of GTP-bound Ras, whereas
no GTP-Ras was detectable in mock-transfected COS-1 cells. However, the
mammary carcinoma did contain a small amount of active Ras in
comparison with the transfectant. As described above, there is
increased complex formation between Crkl and C3G, an exchange factor
for the small GTPase Rap1 (33)
in the CRKL
transgenic mice. Therefore, we asked whether we could detect activated
Rap1 in the mammary carcinoma. As positive and negative controls for
Rap1 activation, we used lysates prepared from COS-1 cells transfected
with EGFP-tagged V12Rap1 or from mock-transfected COS-1 cells. We could
clearly detect GTP-Rap1 in the transfected COS-1 cells (Fig. 4C
, top panel). Remarkably, the mammary carcinoma
sample contained very high levels of GTP-bound Rap1.
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. The presence of the CRKL transgene markedly enhanced the
rate with which leukemia/lymphoma developed in the P190
BCR/ABL transgenic mice. The average age at death of the
P190 BCR/ABL transgenics was 413 days, whereas this was 300
days for the P190 BCR/ABL x CRKL
transgenics.
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, there was no evidence that JNK was activated either in the
P190 or in the P190 + Crkl-expressing lymphomas, although a
positive control consisting of NIH 3T3 cells stimulated with sorbitol
clearly showed that phosphorylated JNK could be detected. In contrast,
the MAPKs p44 and p42 were phosphorylated in both types of lymphomas
(Fig. 6B)
. Similarly, we could detect phosphorylated p38
MAPK in both samples (Fig. 6D)
. There were no detectable
levels of GTP-bound Rap1 (data not shown).
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DISCUSSION |
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We have generated transgenic mice overexpressing Crkl to investigate the role of this adapter in cancer and found that several CRKL transgenics from distinct lines developed different types of tumors. However, there was a low incidence of tumorigenesis, and tumors that developed occurred later in life. Therefore, our findings do not support the concept that Crkl is oncogenic in vivo when overexpressed. Instead, it is more likely that other events lead to the onset of tumorigenesis and that overexpression of Crkl subsequently enhances this process.
How could Crkl contribute to tumor progression? Previous experiments (30, 31, 32 , 36) using cell lines have linked Crkl and the related Crk to cell motility and integrin-mediated adhesion. The assembly of a complex between Crk and the scaffolding protein Cas serves as a molecular switch for the induction of cell migration of the human pancreatic carcinoma cell line FG-M (36) . Uemura and Griffin (30) showed that overexpression of Crkl increased the spontaneous migration of murine pre-B Ba/F3 cells 2.8-fold. In the current study, we have found that, in vivo, CRKL transgenic macrophages show increased motility. In this context, it is of interest that all of the tumors that we have found thus far in the 5- and 10-copy CRKL transgenics are highly metastatic. Therefore, we suggest that the contribution of Crkl is to enhance the ability of the tumor cells to migrate from the site of the primary tumor to secondary locations.
There are several signaling pathways associated with Crkl that could transduce such signals. Crkl has been shown to form a constitutive complex with C3G in many cell types (37, 38, 39) . This exchange factor for Rap1 and R-Ras stimulates the conversion of these small GTPases into their active, GTP-bound form (33 , 40) . R-Ras is involved in the regulation of integrin activation (41) and cell adhesion (42) . Decreased adhesion is thought to promote cell invasion and tumor progression. C3G itself has also directly been implicated in cell motility, because the increased migration of BaF3 cells transfected with Crkl was enhanced even more when C3G was cotransfected into these cells (30) . Hepatocyte growth factor/scatter factor induces motility and scattering of c-Met-expressing epithelial cell lines (43) . Stimulation of a human embryonic kidney cell line with hepatocyte growth factor/scatter factor results in recruitment of Crkl to the c-Met receptor via a scaffolding complex and in the rapid activation of Rap1. Thus, the increased levels of constitutive complexes between C3G and Crkl that we observed in the CRKL transgenic tissue lysates could be a mechanism by which motility is enhanced. Similarly, the greatly increased levels of the GTP-bound form of Rap1 that we found in the highly metastatic mammary carcinoma could affect integrin-mediated adhesion and motility of these cells.
We examined the effect of Crkl in vivo on leukemogenesis caused by Bcr/Abl and found that it decreases the life span by an average of 3.8 months. Because mice have a maximal life span of around 36 months, this represents a very marked reduction in life expectancy. On a molecular level, we have examined possible activation of signaling pathways in vivo in the lymphomas but detected no differences between lymphomas of P190 only and P190 + Crkl-overexpressing transgenics. This is not entirely unexpected, because lymphomas represent end-stage metastatic disease, in which the Bcr/Abl-expressing cells have already accumulated all of the mutations they need to be fully oncogenic.
Previous experiments (44) have shown that Ras becomes activated in Bcr/Abl-transfected fibroblasts and hematopoietic cell lines, and a number of studies (45, 46, 47, 48) have investigated the multiple pathways used by Bcr/Abl to activate Ras. In concordance with this, we have detected Ras activation in lymphomas of P190 BCR/ABL transgenic mice5 and, in the current study, we find that downstream signaling mediators of Ras, ERK1/2, become phosphorylated both in tumors expressing P190 alone and in tumors expressing P190 and Crkl. Because this was also found in the mammary carcinoma of the CRKL transgenic mouse, phosphorylation of ERK1/2 may occur more commonly in tumorigenesis and may be a general indicator of increased mitogenesis. However, in contrast to what was reported for Bcr/Abl-transfected murine myeloid DAGM cells (48) , we could not detect prominent signs of JNK activation in the P190 lymphomas. This may reflect differences in the cell type involved (myeloid versus lymphoid) or other differences between a cell line and an animal model.
In CML, patients invariably progress from chronic phase to blast crisis. A relatively large percentage of patients have a duplicated Ph-chromosome and, because of this, an extra copy of the BCR/ABL gene. Because CRKL is located on chromosome 22q11 proximal to the t(9;22) breakpoint, it remains on the Ph-chromosome and will also be duplicated in patients with an extra Ph-chromosome (4) . Our current results suggest that in such patients, CRKL gene dosage may be a contributing factor in disease progression.
Hematopoiesis is a steady state process occurring in the bone marrow microenvironment, in which hematopoietic stem cells selectively adhere to the stroma and to the extracellular matrix. The proliferation, differentiation, and maturation of the progenitor cells in this environment are tightly regulated by growth factors, cytokines, and adhesion molecules expressed on stroma cells and the extracellular matrix (49) . In CML patients, the hematopoietic stem cells are released prematurely from the bone marrow because of an apparent defect in the interaction with the microenvironment. Also, in contrast to their normal counterparts, CML progenitor cells continue to proliferate when in contact with bone marrow stroma (50) . Compared with normal hematopoietic stem cells, stem cells from CML patients show decreased colony formation after adhesion to bone marrow stroma and/or fibronectin (51 , 52) . It is evident that motile cells will show diminished stable adhesion. Therefore, based on the current in vivo results, we speculate that the role of Crkl in disease progression of Bcr/Abl-positive leukemia is to decrease stable adhesion of the Bcr/Abl-positive cells to stroma and to increase motility.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by Public Health Service Grant
CA 50248 (to N. H.), a Grant from the T. J. Martell Foundation (to
N. H.) and by Grant 6KB-0015 from the California Breast Cancer
Research Program (to V. K.). 
2 Present address: Idun Pharmaceuticals, La Jolla,
CA.
3 To whom requests for reprints should be
addressed, at the Division of Hematology/Oncology MS#54, Childrens
Hospital Los Angeles, 4650 Sunset Boulevard, Los Angeles, CA 90027.
Phone: (323) 669-4595; Fax: (323) 671-3613; E-mail: heisterk{at}usc.hsc.edu
4 The abbreviations used are: Ph, Philadelphia;
CML, chronic myeloid leukemia; RIPA, radioimmunoprecipitation assay;
GST, glutathione S-transferase; PMSF,
phenylmethylsulfonyl fluoride; JNK, c-Jun-NH2-terminal
kinase; MAPK, mitogen-activated protein kinase; ERK, extracellular
signal-regulated kinase.
5 N. Heisterkamp, J. W. Voncken, D. Senadheera, B.
Hemmeryckx, I. Gonzalez-Gomez, A. Reichert, P. K. Pattengale, and J.
Groffen. The Bcr N-terminal oligomerization domain
contributes to the full oncogenicity of Pi90 Bcr/Abl in transgenic
mice, manuscript in preparation.
Received 10/ 9/00. Accepted 12/15/00.
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L. Lamorte, I. Royal, M. Naujokas, and M. Park Crk Adapter Proteins Promote an Epithelial-Mesenchymal-like Transition and Are Required for HGF-mediated Cell Spreading and Breakdown of Epithelial Adherens Junctions Mol. Biol. Cell, May 1, 2002; 13(5): 1449 - 1461. [Abstract] [Full Text] [PDF]
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