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Blood, Vol. 94 No. 1 (July 1), 1999:
pp. 373-374
CORRESPONDENCE
Tyrosine Phosphorylation of Shc Proteins in Normal
CD34+ Progenitor Cells and Leukemic Cells
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LETTER |
To the Editor:
After the Shc gene was identified, biological function studies have
focused on the oncogenic potentials of Shc proteins, especially their
role in the Ras-dependent mitogen-activated protein kinase activation.1,2 Jucker et al3 reported in
Blood that Shc proteins are constitutively
tyrosine-phosphorylated in primary acute myelogenous leukemia (AML)
cells but not in primary cell cultures or normal
tissues.3,4 The presence of constitutively phosphorylated
Shc proteins found in the AML peripheral blood (PB) cells, but not in
normal PB cells, led to the suggestion that the Ras pathway may be
constitutively activated in AML.3 However, it should be
noted that in these studies Shc phosphorylation was compared in PB
cells of AML and PB cells of healthy donors. One potential problem with
their conclusion is that AML cells are immature myeloid cells while
normal PB cells are end-stage mature cells. Hence, the Shc
phosphorylation difference between these two groups of cells might
reflect difference in the level of cell maturation rather than a
difference between leukemic and normal myeloid cells. To distinguish
between Shc phosphorylation in these two groups of cells, one must
compare Shc phosphorylation in AML cells and in normal bone marrow (BM)
cells that are at a comparable level of maturation.
We evaluated Shc protein expression and phosphorylation in normal
hematopoietic progenitor cells and leukemia hematopoietic cells. Five
normal individuals and 10 AML patients participated in this study.
Three leukemic cell lines (HL-60, K562, and KG-1) were also studied. BM
and PB specimens were collected before any treatment from the 10 patients with newly diagnosed AML. BM aspirates were obtained from the
five normal donors. Standard Ficoll density centrifugation
was performed to collect mononuclear cells (MNCs). MNCs from BM
specimens of two patients with AML and five normal donors were
subjected to CD34 separation by MACS separation columns (Miltenyi
Biotec Inc, Auburn, CA). Cell pellets of PB and BM were lysed in cell
lysis buffer. The cell lysates were diluted to 1 mg/mL total cell
protein measured by the Bio-Rad Protein Assay. Polyclonal
anti-human Shc antibody (5 µg) (Upstate Biotechnology Inc, Lake
Placid, NY) was added. The immunocomplex was captured by Protein
A/G Plus agarose beads. Total cell lysates or
immunoprecipitates were subjected to Western blotting. Blots were
probed with antiphosphotyrosine (4G10) (0.5 µg/mL) (Upstate
Biotechnology Inc). Antiphosphotyrosine blots were stripped and
reprobed with anti-Shc anti-serum (3 µg/mL).
Our data show that among the 10 AML patients reported here,
tyrosine-phosphorylated Shc proteins were expressed constitutively in
PB cells of all 10 AML patients. Phosphorylated p66Shc,
p52Shc, and p46Shc was found in 8, 10, and 7 of
10 AML PB samples, respectively. Shc tyrosine phosphorylation was
examined in CD34+ and CD34 cells separated
from BM aspirates of two AML patients. The expression of Shc proteins
was found in both subsets. However, tyrosine-phosphorylated Shc
proteins were expressed only in CD34+ cells and not in the
CD34 leukemia cells. Among five normal BM specimens,
tyrosine-phosphorylated Shc proteins were expressed exclusively in the
CD34+ cell compartment, but not in CD34
cells or in the unseparated cells. Phosphorylated p66Shc,
p52Shc, and p46Shc was found in 2, 5, and 5 of
5 normal CD34+ samples, respectively. With respect to
tyrosine-phosphorylated Shc proteins, no significant difference was
observed between CD34+ leukemia cells and CD34+
normal cells (Fig 1). Phosphorylation of
p66Shc, p52Shc, and p46Shc on
tyrosine was detected in K562 and KG-1 cells, but not in HL-60 cells.
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| Fig 1.
Tyrosine phosphorylation of Shc proteins in normal
CD34+ progenitor cells and leukemic CD34+
cells. Total cell lysates (20 µg protein) were subjected to Western
blot analysis. Phosphotyrosine was identified by antiphosphotyrosine
antibody (4G10). Representative blots are shown. The positions of
p66Shc, p52Shc, and p46Shc are
indicated on the right. Molecular-weight marker bands are indicated on
the left. Lanes 1 and 4, enhanced chemiluminescence protein
molecular-weight markers; lane 2, Nonstimulated A431 cell
lysate; lane 3, epidermal growth factor-stimulated A431 cell lysate;
lane 5, CD34+ cells from normal BM; lane 6, CD34 cells from normal BM; lane 7, CD34+
cells from AML patient BM; lane 8, CD34 cells from AML
patient BM.
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Our study shows that the presence of constitutively
tyrosine-phosphorylated Shc proteins does not distinguish between
normal progenitor cells and AML cells, suggesting that phosphorylation of Shc proteins might be associated more with cell maturity than with
malignant transformation.
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ACKNOWLEDGMENT |
Supported by National Cancer Institute Grant No. 1-PO-1
CA75606-01.
Jie Yang
Biao-Ru Li
Jaya Nayini
Parameswaran Venugopal
Min Tao
Colleen B. Andrews
Harvey D. Preisler
Rush Cancer Institute
Rush Presbyterian-St Luke's
Medical Center
Chicago, IL
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REFERENCES |
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Association of the Shc and Grb2/Sem5 SH2-containing proteins is implicated in activation of the Ras pathway by tyrosine kinases.
Nature
360:689, 1992[Medline]
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Egan SE, Giddings BW, Brooks MW, Buday L, Sizeland AM, Weinberg RA:
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Nature
363:45, 1993[Medline]
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Jucker M, Schiffer CA, Feldman RA:
A tyrosine-phosphorylated protein of 140 kD is constitutively associated with the phosphotyrosine binding domain of Shc and the SH3 domains of Grb2 in acute myeloid leukemia cells.
Blood
89:2024, 1997[Abstract/Free Full Text]
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11:899, 1995[Medline]
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