NORMAL AND ABNORMAL LYMPHOCYTE GROWTH REGULATION 
Owen N. Witte, M.D., Investigator 
The proper functioning of the immune system de- 
pends on the careful regulation of lymphocyte pro- 
duction in the bone marrow. Pluripotential stem 
cells divide and develop into committed progeni- 
tors for the B and T lymphoid lineages. Abnormal 
regulation of this process can lead to hyperprolifer- 
ative states such as leukemia or hypoproliferative 
states such as immunodeficiencies. A major goal of 
Dr. Witte's laboratory has been to devise techniques 
for the isolation and characterization of such stem 
and progenitor cells. 
B Lymphocyte Development 
Previous work established culture conditions for 
the continuous cultivation of immature murine B 
lymphocytes and their progenitors. Recent improve- 
ments in these culture conditions have allowed a 
significant enrichment of progenitor cells for the B 
cell lineage. These cells express known B cell sur- 
face markers and retain immunoglobulin genes in 
the germ configuration. The progenitor cells are re- 
sponsive to a new growth factor produced by stro- 
mal cells in the culture system. Previously de- 
scribed growth factors known to stimulate pre-B 
lymphocytes, including the KIT ligand and interleu- 
kin-7, are incapable of stimulating the B lineage 
progenitor cells. 
Using such progenitor cells as a source of mRNA, 
Dr. Witte and his colleagues have isolated several B 
lineage-specific genes that are expressed at this 
early point in development. One in particular, a 
new member of the cytoplasmic tyrosine kinase fam- 
ily, has been evaluated in detail. This protein, B lin- 
eage progenitor kinase (BPK) , helps to define a new 
subclass of tyrosine-specific protein kinases. Al- 
though generally related to the src family of kinases 
by the presence of the tyrosine kinase catalytic do- 
main and the SH2 and SH3 regulatory domains, the 
BPK kinase has an unusually long amino-terminal 
segment and lacks the carboxyl-terminal regulatory 
domain found in the src family kinases. The precise 
role of this kinase in B cell development is under 
investigation. 
Further analysis of the relationship of B cell pro- 
genitors to pluri- and multipotential stem cells re- 
quires the development of stage-specific markers. A 
new project proposes to develop a large panel of 
hematopoietic-specific genes that can be assigned to 
different lineages and cell types. In collaboration 
with Dr. Leroy Hood, phage libraries were derived 
from human bone marrow RNA. Clones enriched to 
represent rare or moderately expressed mRNAs were 
isolated. Each cloned sequence defines a new gene 
that was monitored for pattern of expression by hy- 
bridization against a large panel of leukemic cell 
RNAs. With this approach one should be able to as- 
semble a panel of markers specific for different cell 
lineages and stages of maturation. Eventually, se- 
lected probes will be evaluated in greater depth on 
normal marrow specimens. 
Use of Oncogenes to Stimulate Primitive 
Hematopoietic Stem Cells 
Previous work from this laboratory had defined 
the BCR/ABL oncogene expressed in human 
chronic myelogenous leukemia as a valuable tool 
for the transformation of primitive lymphoid cells 
and myeloid cell types. Recent work has shown that 
this gene can also be introduced into murine pluri- 
or multipotential stem cells to create a syndrome 
with many of the hallmarks of chronic myelogenous 
leukemia. Further analysis has demonstrated that di- 
rect stimulation of the stem cell can be achieved in 
vitro, as monitored in agar colony assays. The 
growth stimulation imparted by this gene is rather 
subtle, and there is no block to differentiation of the 
cells. Thus this relatively weak oncogene can be 
used to expand populations of cells that retain full 
developmental potential. 
Analysis of the mechanisms of activation of the 
BCR/ABL gene has demonstrated that unique fea- 
tures of the BCR segment are required to interact 
with the ABL tyrosine kinase segment to achieve 
transformation activation. The BCR segment con- 
tains a serine-rich segment that can bind to the tyro- 
sine kinase SH2 regulatory domain. This putative cis 
interaction between the segments of the chimeric 
oncogene appears to be critical for the activation of 
the ABL tyrosine kinase. Site-directed mutagenesis 
has shown that the ABL tyrosine kinase and a major 
site of tyrosine phosphorylation within ABL are also 
essential for full transformation. (This work was 
supported by a grant from the National Cancer 
Institute.) 
To provide better test models for the analysis of 
human leukemias, tissue explants of peripheral 
blood and bone marrow have been cultured in the 
in vivo environment of the SCID (severe combined 
immune deficiency) mouse strain. The results are 
dramatic. All specimens of acute myelogenous leu- 
kemia and all blast crisis specimens of chronic my- 
elogenous leukemia grew well in the SCID mouse. 
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