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[Cancer Research 60, 728-732, February 1, 2000]
© 2000 American Association for Cancer Research

Tumor Biology

Selective Expression and Constitutive Phosphorylation of SHC Proteins in the CD34+ Fraction of Chronic Myelogenous Leukemias1

Antonio Bonati, Carmelo Carlo-Stella, Paolo Lunghi, Roberto Albertini, Silvana Pinelli, Enrica Migliaccio, Gabriella Sammarelli, Barbara Savoldo, Antonio Tabilio, Pier Paolo Dall’Aglio and Pier Giuseppe Pelicci2

The Institute of Medical Pathology [A. B., P. L., R. A., S. P., P. P. D., P. G. P.] and Chair of Haematology [C. C-S., G. S., B. S.], University of Parma, 43100 Parma; European Institute of Oncology, 20141 Milan [E. M., P. G. P.]; and Chair of Haematology, University of Perugia, 06100 Perugia [A. T.], Italy


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The BCR/ABL fusion protein is a constitutively active tyrosine kinase that is responsible for the pathogenesis of chronic myelogenous leukemia (CML). Clinically, CML is characterized by a chronic phase (CP) that eventually terminates into a blast crisis (BC). BC transformation is associated with accumulation of CD34+ blasts. We investigated the expression and phosphorylation of Src-homology-2 and collagen-homology domains (Shc) proteins in subpopulations of CML primary cells. Shc polypeptides are tyrosine kinase substrates that are constitutively tyrosine-phosphorylated in continuous cell lines of CML origin. High levels of Shc expression were found in the CD34+ cells from CML-BC, CML-CP and normal bone marrow. In contrast, CD34- fractions from CML-CP and normal bone marrow expressed low levels of p46Shc. Shc proteins were constitutively phosphorylated in the CD34+ fractions from CML cells (both CP and BC), but not in normal CD34+ cells. These data bear implications for the role of Shc in normal hemopoiesis and CML leukemogenesis: (a) dramatic changes of Shc expression during terminal differentiation of hemopoietic cells adds a further level of regulation to the signal transduction function of Shc; and (b) constitutive Shc tyrosine-phosphorylation in the rare CD34+ cells of CML-CP might contribute to the selection of this subpopulation during the blast crisis transformation of CMLs.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The mammalian Shc3 locus encodes for adaptor proteins, which are involved in the cytoplasmic transduction of mitogenic stimuli from RTKsto Ras (1) . The isolated Shc cDNA encoded two proteins of Mr 52,000 and 46,000 (p52/p46), which derive from differential translation initiation (2) . They share a COOH-terminal SH2 domain, a central collagen-homology domain (CH1), rich in proline/glycine residues, and an NH2-terminal phosphotyrosine-binding domain. Shc proteins associate with, and are phosphorylated by, a considerable number of RTKs (1) . In addition, Shc is rapidly phosphorylated after ligand stimulation of surface receptors that have no intrinsic TK activity and in cells transformed by v-src or v-fps (3 , 4) , suggesting that it is a common TK target in mitogenic signaling pathways. On phosphorylation, Shc interacts with the SH2 domain of Grb2 and functions as an alternative docking site for the Grb2/Sos complex (5, 6, 7) . Several lines of evidence suggest that the Shc/Grb2/Sos complex is involved in Ras activation. Shc overexpression induces transformation of fibroblasts (2) and, in PC12 cells, neurite extension (5) , a response that is blocked by expression of a dominant inhibitory Ras mutant.

Shc proteins have been shown to be constitutively tyrosine-phosphorylated in tumors carrying activation of oncogenes with TK activity (8 , 9) . CMLs are characterized by a chromosome translocation that involves the genes encoding bcr and the abl cytoplasmic TK (10) . As a consequence of the translocation, the bcr/abl fusion gene is formed on the recombinant Ph chromosome, which encodes for a bcr/abl fusion protein (11 , 12) . The bcr/abl fusion protein retains the catalytic domain of abl and is endowed with constitutive enzymatic activity (13) . Shc polypetides are good substrates of the bcr/abl TK, and constitutive tyrosine-phosphorylation of Shc is found in bcr/abl expressing cells (14, 15, 16) .

CML is clinically characterized by an indolent chronic phase (CML-CP), which finally undergoes transformation into an invariably fatal blast crisis (CML-BC) (17) Cytologically, the CML-CP is characterized by hyperproliferating cells at various stages of myeloid differentiation (17) . The fraction of immature cells, as defined by expression of the CD34 antigen, is <5% (18) . The CML-BC, instead, is indistinguishable from acute leukemias, with accumulation of morphologically homogenous hemopoietic precursors blocked at a very early stage of differentiation. Indeed, neoplastic cells from CML-BC homogeneously express the CD34 antigen (19) . The molecular mechanisms, however, that underlay the process of transformation of CML into an acute leukemia are currently unknown.

We report here our investigations aimed at characterizing levels of Shc expression and activation in CD34+ and CD34- cell populations from CML-CP and from CML-BC fresh samples. Our results bear implications with the function of Shc in normal and Ph+ hemopoiesis.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients and Samples.
CML patients in BC (n = 11) or CP (n = 4) and healthy donors (n = 4) were included in this study. The diagnosis of CML and the identification of the phases of the disease were performed according to standard criteria (17) . All CML patients were 100% Philadelphia positive by standard cytogenetic analysis. Bone marrow cells were analyzed in all but in two cases (5 and 10), where peripheral blood was used. Normal hematopoietic cells were obtained from healthy donors undergoing bone marrow harvest (n = 3) or peripheral blood progenitor cell mobilization (n = 1) for allogeneic transplantation. Marrow or blood mononuclear cells were separated by centrifugation (400 x g for 30 min at 4°C) on a Ficoll-Hypaque gradient (density =1.077 g/ml). Interface cells were washed in PBS. The samples were directly lysed for protein detection after the separation of CD34+ and CD34- fractions (see below). PMN granulocytes were obtained after Ficoll by resuspending the pellet in dextran. K562 and TOM1 Ph+ cell lines were also analyzed.

Enrichment of CD34+ Cells.
BC cells were >90% CD34+. CP CML and normal bone marrow cells were enriched according to CD34 expression by means of a magnetic cell-sorting methodology (Miltenyi Biotec, Bergisch Gladbach, Germany). Briefly, marrow or blood cells were labeled with a haptenized CD34 antibody (QBEND/10), which is then magnetically labeled in a second-step reaction with an antihapten antibody coupled to super-paramagnetic microbeads. Labeled cells are then separated using a high-gradient magnetic separation column placed in a strong magnetic field. The magnetically stained cells are retained in the column while unstained cells pass through. When the column is removed from the magnetic field, the magnetically retained cells are eluted. The mean (± SD) percentage of CD34+ cells within enriched fractions was 86 ± 14% and 85 ± 11% for CML and normal samples, respectively.

Cell Lysis.
The cells were lysed on ice in 50 mM Tris-HCl (pH 8), 1.5 mM MgCl2, 150 mM NaCl, 5 mM EGTA (pH 7.5), 5% (v/v) glycerol, 1% (v/v) and TritonX-100 containing freshly added protease inhibitors (2 µg/ml aprotinin, 2 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM phenylmethyl sulfonyl fluoride, 1 mM sodium orthovanadate, and 50 mM sodium fluoride). Insoluble materials were removed by centrifugation for 10 min at 12,000 x g at 4°C, and protein concentration determined by Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hercules, CA).

Immunoprecipitation.
For immunoprecipitation experiments, cell lysates were incubated with appropriate antibodies and immune complexes isolated with Protein A-Sepharose CL-4B (Pharmacia LKB, Uppsala, Sweden). Immune complexes were denatured by heating at 95°C in reducing Laemmli buffer and analyzed by SDS-PAGE.

Immunoblotting.
Either specific immunoprecipitates or total cell lysates were electrotransferred onto polyvinylidene difluoride filters (Millipore Intertech, Bedford, Ma) after SDS-PAGE. After blocking nonspecific reactivity, filters were probed 1 h at room temperature with specific antibodies diluted in TBS-T [25 mM Tris-HCl (pH 8), 150 mM NaCl, 0.05% Tween 20] containing 5% nonfat milk. For antiphosphotyrosine experiments, 0.02% Tween 20 and 1% BSA (fraction V; Boehringer Mannheim, Mannheim, Germany) concentrations were used. After extensive washing, immunocomplexes were detected with HRP-conjugated species-specific secondary antiserum followed by enhanced chemiluminescence reaction (Amersham International plc, Buckinghamshire, United Kingdom).

Antibodies.
For immunoprecipitation, the following antibodies were used: rabbit polyclonal antihuman Shc (Upstate Biotechnology, Inc., Lake Placid, NY) and mouse monoclonal antihuman Shc (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). For immunoblotting, the following antibodies were used: rabbit polyclonal antihuman Shc (Santa Cruz Biotechnology), mouse monoclonal anti-antiphosphotyrosine (Upstate Biotechnology), goat polyclonal antihuman actin (Santa Cruz Biotechnology), goat antirabbit IgG (H+L)-HRP conjugated (Bio-Rad), goat antimouse IgG (H+L)-HRP conjugated (Bio-Rad), and donkey antigoat IgG (H+L)-HRP conjugated (Santa Cruz Biotechnology).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Higher levels of Shc Protein Expression in CD34+ versus CD34- Ph+ Cells.
To evaluate levels of Shc expression in CML, we performed Western blotting analysis of cellular lysates from 4 cases of CML-CP and 11 cases of CML-BC, using anti-Shc antibodies that recognize p46, p52 and the recently identified p66 Shc isoform (20) . Shc expression was markedly higher in the 11 CML-BC cases (see representative Western blots in Fig. 1Citation ). In addition, whereas all three Shc isoforms were expressed in the CML-BC samples, only p46 was detected in the cases of CML-CP (Fig. 1)Citation . This was not due to the lower levels of Shc protein expression in the CML-CP cases because p46 was the less abundant Shc isoform in the CML-CB cases. Probing of the same Western blots with antiactin polyclonal antibodies revealed that similar amounts of cellular proteins were loaded on each lane (representative results are shown in Fig. 1Citation ).



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  		Fig. 1. Shc protein expression in cell lysates from CML-BC and CML-CP cases. High expression levels of the three Shc isoforms are detectable in the BC samples. Unseparated fractions of CML-CP, as well as PMN, show only low levels of p46Shc. CP, unseparated fraction. The numbers beneath the Lanes are the reference numbers of the cases examined. The same blots were used for anti-Shc and antiactin probing, as indicated.

 
CML-CP samples consist of a heterogenous population of hemopoietic cells at different stages of differentiation, whereas CML-BC cells mainly consist of immature, CD34+ myeloid precursors (17 , 19) . The fraction of CD34+ cells in CML-CP usually does not exceed 2% of bone marrow mononuclear cells (18) . To evaluate whether the different levels of Shc expression in CML-CP versus CML-BC samples reflected differences in cellular composition, we analyzed Shc expression in purified CD34+ and CD34- cells from four different CML-CP samples. Mononuclear cells from CML-CP bone marrow samples were fractionated using an anti-CD34 monoclonal antibody affinity column and the purity of the two fractions (respectively, >95% and <5%; data not shown) assessed by fluorescence-activated cell-sorting analysis. Western blotting revealed low levels of p46 Shc in the CD34- CML-CP fraction, whereas high levels of all three Shc isoforms were found in the CD34+ fraction from the same samples (Fig. 2A)Citation . Strikingly, levels of Shc expression in the CD34+ CML-CP cases were comparable with those found in the CML-BC samples (Fig. 2A)Citation . The fact that unfractionated CML-CP samples expressed very low levels of Shc expression is consistent with the low frequency of CD34+ cells in the same samples (<5%). Taken together, these results suggest that Ph+ CD34+ cells, either from BC or CP, express markedly higher levels of Shc expression than Ph+ CD34- cells.



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  		Fig. 2. Shc expression in CD34+ versus CD34- fractions of CML and normal-derived bone marrow. A, high levels of Shc expression in the CD34+ fraction of three CML-CP samples and in two CML-BC samples. p66Shc and p52Shc are not expressed in the CD34- fraction of CML-CP samples, where only low levels of p46Shc are detectable. The same blots were used for anti-Shc and antiactin probing, as indicated. B, side-to-side comparison of Shc expression in the CD34+ or CD34- fractions from CML-CP (case 4) and normal bone marrow (N.BM#16) samples, and in the unseparated fraction of one CML-BC sample (BC#14). Comparable high levels of Shc expression are visualized in the different CD34+ fractions.

 
Higher Levels of Shc Protein Expression in CD34+ versus CD34- Fractions from Normal Bone Marrow.
The finding of higher levels of Shc proteins in CD34+ Ph+ cells might reflect a physiological regulation of Shc during differentiation of myeloid cells. Therefore, we analyzed Shc expression in CD34+ and CD34- fractions from three different normal bone marrow samples. Western blotting analysis revealed high levels of Shc expression in the CD34+ fractions, whereas Shc polypeptides were almost undetectable in the CD34- fractions (representative results for two bone marrow samples are shown in Fig. 2BCitation ). Notably, low levels of Shc expression, comparable with those found in the CD34- fractions, were also found in cellular lysates from purified normal neutrophils (Fig. 2A)Citation . Side-to-side comparison of Shc expression levels in the CD34+ cell fractions from normal bone marrow and CML-CP samples and in cells from CML-BC revealed comparable high levels of Shc expression (Fig. 2B)Citation . Taken together, these results reveal that Shc is up-regulated in normal CD34+ cells, as compared with CD34- cells, suggesting that its expression declines in differentiated hemopoietic cells. Therefore, the heterogenous expression of Shc proteins in the CD34+ and CD34- fractions of CML-CP might reflect the expansion of cell populations with different levels of Shc expression. Accordingly, CML-BC cells, which are mainly CD34+, expressed high levels of Shc proteins.

Higher Levels of Shc Phosphorylation in CD34+ Ph+ Leukemic Cells versus CD34+ Normal Bone Marrow Cells.
We next analyzed levels of Shc phosphorylation in the same samples. Analysis of the phosphotyrosine content of Shc proteins requires larger amount of cells, to immunopurify Shc polypeptides from cellular lysates. Sufficient biological material was available from two CML-CP and five CML-BC cases. Antiphosphotyrosine blots of anti-Shc immunoprecipitates revealed phosphorylation of Shc polypeptides in the BC, but not in the CP, CML cases (Fig. 3ACitation , left and middle). Side-to-side comparison of phosphotyrosine blots of cellular lysates and anti-Shc immunoprecipitates from one CML-BC sample and two CML established cell lines (K562 and TOM-1) showed that tyrosine-phosphorylated Shc polypeptides can be directly visualized also in the cellular lysates (Fig. 3ACitation , left). Similarly, phosphorylated Shc proteins were visualized in the cellular lysates of the other four CML-BC samples (Fig. 3ACitation , right). These findings confirm that Shc proteins are abundant intracellular polypeptides that can be identified by antiphosphotyrosine blots of whole cellular lysates (21) . We, therefore, analyzed levels of Shc tyrosine-phosphorylation in the cellular lysates from the remaining five cases of CML-BC and two cases of CML-CP. High levels of tyrosine-phosphorylated Shc proteins were seen in all of the CML-BC lysates. In the CML-CP lysates, instead, only p46Shc was detectable, at low levels (Fig. 3B)Citation . These results paralleled those obtained with the anti-Shc antibodies in the same cell samples: levels of Shc phosphorylation, infact, correlated with the levels of Shc protein expression in the CML samples.



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  		Fig. 3. Tyrosine phosphorylation of Shc proteins in CML samples. A, left, antiphosphotyrosine blot of cellular lysates (Lysate) and anti-Shc immunoprecipitates ({alpha}Shc I.P.) from the K562 and TOM-1 CML cell lines and from one CML-BC sample (BC15). Middle, antiphosphotyrosine blot of anti-Shc immunoprecipitates ({alpha}Shc I.P.) from the indicated cases. UF, unfractionated; Ig, immunoglobulin cross-reactive polypeptides. Right, antiphosphotyrosine blot of cellular lysates (Lysate) from the indicated cases. B, antiphosphotyrosine blot of cellular lysates from the indicated CML-CP, CML-BC PMN and K562 samples. The same blot was used for antiactin probing (bottom).

 
We then performed antiphosphotyrosine blots of CD34+ and CD34- fractions from CML-CP (four cases) and side-to-side comparison with CML-BC samples. In line with Shc expression data, we detected consistently higher levels of tyrosine-phosphorylated Shc proteins in the CD34+ fractions from CML-CP cases, as compared with their corresponding CD34- fractions. In one case (CML-CP #3; Fig. 3ACitation ), data were confirmed by antiphosphotyrosine blots of anti-Shc immunoprecipitations. Levels of tyrosine-phosphorylated Shc proteins in the CML-CP CD34+ fractions were comparable with those found in the unfractionated CML-BC lysates (Fig. 4A)Citation . Shc phosphorylation was then measured in the CD34+ and CD34- fractions from normal bone marrow (three cases). Low or undetectable tyrosine-phosphorylated Shc polypeptides were observed in either of the two fractions (Fig. 4B)Citation . To ascertain that the low levels of phosphorylation of Shc proteins in normal CD34+ cells reflected a functional status, we analyzed CD34+ cells exposed, in vivo, to G-CSF. Lysates from CD34+ cells obtained from one patient treated with G-CSF were analyzed by Western blotting using anti-Shc and antiphosphotyrosine antibodies. As shown in Fig. 4CCitation , high levels of Shc polypeptides (left) and tyrosine-phosphorylated Shc proteins (right) were found in the G-CSF-stimulated CD34+ cells.



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  		Fig. 4. Tyrosine phosphorylation of Shc proteins in CD34+ and CD34- fractions from CML and normal bone marrow or peripheral blood samples. A, antiphosphotyrosine blot of cellular lysates from CD34+ and CD34- CML-CP fractions compared with CML-BC, PMN, and K562 samples. The same blot was used for antiactin probing (bottom). B, antiphosphotyrosine blots of cellular lysates from CD34+ and CD34- fractions derived from normal human bone marrow (N.BM) and compared with CD34+ and CD34- CML-CP and CML-BC. C, anti-Shc (left) and anti-phosphotyrosine (right) blots of cellular lysates from G-CSF mobilized CD34+ cells derived from normal human peripheral blood (P.B.), compared with PMN and K562 lysates. To mobilize CD34+ cells, the donors received rhG-CSF (Neupogen, Roche, Milan, Italy) at a dose of 8 µg/Kg body weight s.c. twice daily for at least 5 days.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The main findings of our investigation are: (a) markedly higher levels of Shc protein expression in CD34+ than CD34- cell populations from both normal and Ph+ hemopoiesis; and (b) constitutive tyrosine-phosphorylation of Shc proteins in the CD34+ cell populations from CML cells, but not in the CD34- Ph+ cells or in the normal bone marrow CD34+ cells. These data bear implications for the role of Shc proteins in both normal hemopoiesis and Ph+ leukemogenesis.

The finding of marked variations of Shc expression into different cell populations of the bone marrow, regardless whether normal or transformed, was unexpected, because p52/p46 Shc was initially reported to be ubiquitously expressed (2 , 22) . The fact that higher levels of Shc were found in CD34+ cells, as compared with CD34- cells and neutrophils, suggest that Shc expression correlates with the status of differentiation. A similar correlation has been recently found on developing brain tissue samples, where the proliferative and postmitotic areas can easily be identified. Surprisingly, these analyses revealed marked variations in Shc levels at the transition from proliferation to differentiation, with almost undetectable levels of Shc expression in the postmitotic neurones (23) . As Shc possesses no catalytic domains, these data suggest that variations in the availability of adaptor proteins might influence the response of different cells to the external stimuli. Accordingly, changes in Shc levels during maturation of hematopoietic precursors may affect the ability of a given factor to activate downstream components of the signaling cascade. This hypothesis would imply that adaptor molecules such as Shc may be regarded not only as passive components of cytoplasmic signaling cascades but, rather, as active partners in the specification of cellular responses.

We found in the Ph+ cells a close correlation among expression of the CD34+ antigen, high levels of Shc expression, and Shc tyrosine-phosphorylation. High levels of tyrosine-phosphorylated Shc proteins were, in fact, found in the CD34+ fractions of CML-CP samples, as well as in the CML-BC samples, which homogenously express the CD34 antigen (17, 18, 19) . Tyrosine-phosphorylation of Shc expression in the Ph+ cells reflects the expression of the constitutively active bcr-abl tyrosine kinase (14, 15, 16) . The lack of easily detectable tyrosine-phosphorylated Shc proteins in the CD34- Ph+ cells, instead, may simply reflect the fact that Shc is expressed at very low levels in these samples. It seems, therefore, that the CD34+ fraction of Ph+ cells is the only that expresses detectable levels of Shc proteins and that Shc is functionally active in these cells (a Shc-Grb2 complex was consistently detected in our CD34+ Ph+ samples; data not shown).

The progression from CML-CP to CML-BC involves the selection of a Ph+ CD34+ cell population. Constitutive tyrosine-phosphorylation of Shc proteins may contribute to the more aggressive phenotype of CD34+ Ph+ cells. However, it remains to be established whether tyrosine-phosphorylated Shc proteins contribute to the expansion of the small CD34+ fraction of CML-CP during the BC transformation.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by grants from Associazione Italiana per la Ricerca sul Cancro (AIRC), "Consiglio Nazionale delle Ricerche" (Progetto Finalizzato ACRO), and "Ministero dell’Università e della Ricerca Scientifica e Tecnologica" (MURST-40% & 60%). P. L. is supported by a grant provided by "Chiara Tassoni Association against Leukemia." Back

2 To whom requests for reprints should be addressed, at European Institute of Oncology, Via Ripamonti 435, 20141 Milan, Italy. Back

3 The abbreviations used are: Shc, Src-homology-2 and collagen-homology domains; TK, tyrosine kinase; RTK, TK receptor; CML, chronic myeloid leukemia; CP, chronic phase; BC, blast crisis; HRP, horseradish peroxidase; PMN, polymorphonuclear; G-CSF, granulocyte colony-stimulating factor. Back

Received 9/22/99. Accepted 11/29/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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P. Lunghi, A. Costanzo, M. Levrero, and A. Bonati
Treatment with arsenic trioxide (ATO) and MEK1 inhibitor activates the p73-p53AIP1 apoptotic pathway in leukemia cells
Blood, July 15, 2004; 104(2): 519 - 525.
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