NORMAL AND ABNORMAL LYMPHOCYTE GROWTH REGULATION 
Owen N. Witte, M.D., Investigator 
An understanding of the mechanisms used to 
regulate the normal production of lymphocytes in 
the bone marrow and thymus is necessary for the 
study of hypoproliferative states such as immuno- 
deficiencies, as well as hyperproliferative states 
such as leukemias and lymphomas. Dr. Witte's labo- 
ratory has concentrated on the establishment of in 
vitro culture models to evaluate B cell production 
from primitive stem and progenitor elements and 
the growth deregulation caused by the expression 
of oncogenes that play a central role in the devel- 
opment of specific human leukemias. 
L Expression of the BCR/ABL Gene Family in Phila- 
delphia Chromosome-Positive Leukemias. 
Previous work has detailed the molecular events 
by which the Philadelphia chromosome transloca- 
tion (t9:22), found in human chronic myelogenous 
leukemia and some cases of acute lymphocytic leu- 
kemia, creates related chimeric mRNAs encoding 
the P210 or P185 BCR/ABL oncogenes. The tran- 
scriptional regulatory apparatus and 5 '-untrans- 
lated region of the BCR gene controls the expres- 
sion of the gene, and the tyrosine kinase domain of 
the ABL segment is crucial for transforming activity. 
In collaboration with Dr. Christopher T. Denny 
(University of California at Los Angeles), the promo- 
tor segment of BCR has been isolated, sequenced, 
and functionally analyzed. It lacks a TATA box ele- 
ment and appears to be a GC-rich sequence with 
multiple SPl-binding sites that efficiently drives ex- 
pression of heterologous test genes in a wide vari- 
ety of cells. 
Early lymphoid and myeloid cells are directly 
transformed by cDNA copies of BCR/ABL mRNAs in- 
troduced into hematopoietic culture systems in 
vitro. A number of surprising observations have 
been made with this system. First, recent studies 
have documented that the 5 '-untranslated se- 
quences, which are exceptionally GC rich, are a ma- 
jor determinant of pathogenesis. Only constructs 
with long untranslated regions (>400 bases) effi- 
ciently transform murine hematopoietic cells in 
vitro or in vivo. Second, the potency of transforma- 
tion in all the in vitro and in vivo model systems 
evaluated correlates directly to the specific activity 
of the tyrosine kinase. The P185 gene product is 
more potent biologically than P210 in fibroblast 
and hematopoietic transformation models, and this 
is reflected in a 5- to 10-fold increase in specific ty- 
rosine kinase activity measured on enzyme purified 
from mammalian cells or hyperexpressed from a 
baculovirus vector in insect cells. 
The unique chimeric junctions of the BCR/ABL 
mRNAs were exploited to develop a sensitive poly- 
merase chain reaction (PCR) diagnostic test to 
detect small numbers of Philadelphia chromo- 
some-positive cells in clinical samples. This test has 
been used extensively in multiple centers to study 
the pathobiology of this group of leukemias. Recent 
observations on a group of chronic myelogenous 
leukemia patients treated by bone marrow trans- 
plantation have indicated that PCR analysis over the 
first six months post-transplant may be a useful 
indicator of cytogenetic, and eventually clinical, 
relapse. 
II. Development of Selective Culture Conditions for 
Growth of Clonal Populations of B Lymphoid Pro- 
genitor Cells. 
The natural history of chronic myelogenous leu- 
kemia and acute lymphocytic leukemia support the 
hypothesis that primitive stem cells are the primary 
target of the BCR/ABL oncogene. A major goal of Dr. 
Witte's laboratory has been to combine the action 
of specific oncogenes and selective in vitro grov^h 
conditions to isolate defined stages of hematopoi- 
etic development, including such stem cells. Previ- 
ous attempts to combine long-term culture tech- 
niques selective for early B lineage precursors and 
pre-B cells with infection by the BCR/ABL oncogene 
resulted in overgrowth by pre-B cell types with im- 
munoglobulin |JL heavy-chain segments already rear- 
ranged to the DJ or VDJ configuration. 
By modifying the specific culture conditions, in- 
cluding the use of clonally derived stromal cell 
feeder layers and oncogene expression constructs 
that minimize toxicity and maximize transformation 
activity. Dr. Witte and his colleagues have been able 
to isolate clonal lines of B lymphoid progenitor 
cells routinely. These continuous cell lines retain 
immunoglobulin |x heavy-chain genes in the germ- 
line configuration but can subsequently rearrange 
these genes and express immunoglobulin. Most im- 
portantly, although they contain and are growth- 
stimulated by a potent oncogene, these lines are 
nontumorigenic on transplantation into SCID (se- 
vere combined immunodeficient) mice and effec- 
Continued 
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