Autologous Bone Marrow Transplantation - Blog Science Connections

178 Pharmacological Manipulation and Purging
possibility that certain types of clinical resistance can be circumvented by
coadministration of such agents. Before these pharmacological manipulations
can be examined clinically in ABMT, selectivity must be established by
demonstrating that verapamil does not also sensitize normal human hematopoietic
stem cells to the anticancer drugs involved in multidrug resistance. If
not, coadministration with drugs frequently associated with multidrug resistance
(e.g., the vinca alkaloids) may lead to selective killing of drug-resistant
cells during the purging procedure.
A second approach involves the use of potent inhibitors of nucleoside
transport, such as nitrobenzylthioinosine (NBMPR), to selectively protect
hematopoietic stem cells from tubercidin (7-deazaadenosine), a nucleoside
analogue (5,6) with considerable activity against neoplastic cell lines (7,8). We
(9,10) and others (11,12) have observed that a number of cultured cell lines
differ in their sensitivity to NBMPR inhibition of nucleoside transport. Thus,
depending on the relative sensitivities of neoplastic and hematopoietic stem
cells to NBMPR or other potent inhibitors of nucleoside transport (dilazep,
dipyridamole), it should be possible to use such inhibitors, which by themselves
are nontoxic, to protect normal hematopoietic stem cells but not neoplastic
stem cells from the cytotoxic effects of tubercidin. Nucleoside transport
systems and their sensitivity to specific inhibitors have recently been reviewed
(13,14).
In this work we present evidence that pharmacological strategies to both
circumvent resistance of neoplastic cells and protect hematopoietic stem cells
may indeed find broad application in ABMT.
MATERIALS AND METHODS
Heparinized bone marrow aspirates were obtained from the sternum of
patients undergoing cardiac surgery. The light-density fraction was obtained by
centrifugation of mononuclear cells on 60% Percoll. For studies with vincristine
and verapamil, 6 x 10 5
cells were incubated for 4 hours (37°C) in Iscove's
modification of Dulbecco's medium (IMDM) containing graded concentrations
of vincristine (0.1-100 uIA), verapamil (2.5-80 JUM), or both. Cells were then
washed with drug-free media and plated in mixed-colony assay (15,16) in 1 ml
of IMDM supplemented with 0.8% methylcellulose, 30% human plasma, 1 unit
erythropoietin, and 5 x 10 5
M mercaptoethanol. Human pluripotent hematopoietic
and granulocyte-macrophage colony-forming units (CFGs-GEMM and
CFGs-GM) and erythroid burst-forming units (BFGs-E) were counted after
incubation for 14 days at 37°C in 5% C0 2
in air. For studies with nucleoside
transport inhibitors and tubercidin, 6 x 10 5
light-density fraction marrow cells
were preincubated for 30 minutes (37°C) with inhibitor (3 uPA NBMPR, dilazep,
or dipyridamole) in IMDM and then, still in the presence of transport inhibitor,
incubated for 1 hour (37°C) in graded concentrations of tubercidin (0.001 -100
JUM). Cells were then washed and plated in mixed-colony assay as above.