Expression of a truncated first exon BCR sequence in chronic myelogenous leukemia cells blocks cell growth and induces cell death.
We have shown that a deletion mutant form of Bcr [Bcr(64-413)] is a strong inhibitor of the tyrosine kinase of Bcr-Abl in vitro and also inhibits its oncogenic growth effects (Liu et al., Cancer Res., 56: 5120-5124, 1996). To determine the effects of this Bcr-Abl kinase inhibitor on chronic myelogenous leukemia (CML) cells, we cloned BCR(64-413) into a recombinant, replication-defective adenovirus to express useful quantities of Bcr(64-413) in a wide variety of cells in culture. Infection of Cos1 cells with plaque-purified virus at a multiplicity of infection of 20-40 induced high expression of Bcr(64-413) as detected by Western blotting. Infection of hematopoietic cells at modest multiplicities of infection (20-40) required special conditions involving shifting cycling cells to a nongrowing condition involving serum starvation and cell crowding. Under these conditions, both Bcr-Abl-positive and -negative hematopoietic cells can be efficiently infected by adenovirus, as demonstrated by 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside staining of cells infected by beta-galactosidase (beta-GAL) adenovirus. We found that expression of Bcr(64-413) in Bcr-Abl-positive K562 and BV-173 cells, but not Bcr-Abl-negative SMS-SB cells, increased cell-cell clumping and inhibited cell growth. In contrast to the effects of the Bcr(64-413) adenovirus, the beta-GAL adenovirus, despite infecting both types of cells, did not block growth or increase cell-cell clumping of Bcr-Abl-positive and -negative hematopoietic cells. Expression of Bcr(64-413) protein in primary cultures of cells from CML patients with active disease interfered with cell growth, induced apoptosis (as measured by annexin staining), and increased cell-cell clumping, whereas the beta-GAL adenovirus and mock-infected cells lacked these effects. In contrast, normal marrow cells did not exhibit these effects on infection with Bcr(64-413) adenovirus. We conclude from these findings that Bcr(64-413) interferes with the oncogenic effects of Bcr-Abl and therefore has the potential for use in therapy of CML.
Wang
Y
Y
Department of Molecular Pathology, The University of Texas M. D. Anderson Cancer Center, Houston 77030, USA.
Liu
J
J
Wu
Y
Y
Luo
W
W
Lin
S H
SH
Lin
H
H
Hawk
N
N
Sun
T
T
Guo
J Q
JQ
Estrov
Z
Z
Talpaz
M
M
Champlin
R
R
Arlinghaus
R B
RB
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D017209:Apoptosis / Q000235:genetics; D019556:COS Cells; D002455:Cell Division / Q000235:genetics; D002470:Cell Survival / Q000235:genetics; D005091:Exons; D016044:Fusion Proteins, bcr-abl / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000821:virology; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000235:genetics* / Q000473:pathology*; D015513:Oncogene Proteins / Q000096:biosynthesis / Q000235:genetics*; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases; D011518:Proto-Oncogene Proteins; D051562:Proto-Oncogene Proteins c-bcr; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism
D015513:Oncogene Proteins; D011518:Proto-Oncogene Proteins; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D016044:Fusion Proteins, bcr-abl; C496613:BCR protein, human; D051562:Proto-Oncogene Proteins c-bcr