Download Human CD34+ Hematopoietic Progenitors Have

Human CD34+ Hematopoietic Progenitors Have Low, Cytokine-Unresponsive
06-Alkylguanine-DNA Alkyltransferase and Are Sensitive to
06-Benzylguanine Plus BCNU
By Stanton L. Gerson, Weldon Phillips, Michael Kastan, Luba L. Dumenco, and Cheryl Donovan
Human bone marrow (BM) cells contain low levels of the
DNA repair protein, 0'-alkylguanine-DNA alkyltransferase,
which may explain their susceptibility to nitrosourea-induced cytotoxicityand the development of secondary leukemia after nitrosourea treatment. IsolatedCD34+myeloid
progenitorswere also foundto have low levels of alkyltransferase activity. The level of alkyltransferase in CD34+ cells
or in mononuclearBM cells did not increase after incubation
with granulocyte-macrophage colony-stimulating factor, interleukin-3, stem cell factor,the combination, or 5637 conditioned medium. BCNU sensitivity remained unchanged as
well. In addition, 0'-benzylguanine depleted alkyltransfer-
ase activity in BM cells at concentrationsas low as 1.5 pmoll
L after a l-hour exposure. 0'-benzylguanine pretreatment
markedly sensitized hematopoietic progenitor colony-forming cells to BCNU, resulting in a reduction in the dose of
drug (termed the dose-modification factor) required to inhibit 50% of the colony formation (IC,) of threefold to fivefold. Since, unlike many other cell types, proliferating early
(CD34') hematopoieticprecursors do not induce alkyltransferase, myelosuppression may be the dose-limiting toxicity
of the combination of 0'-benzylguanine plus BCNUin clinical trials.
0 1996 by The American Societyof Hematology.
M
mans as a result of exposure to nitrosoureas and procarbaYELOSUPPRESSION IS a dose-limiting toxicity of
zine.I4Nonetheless, it is possible that growth factor stimulathe nitrosourea, tetrazine, and triazine classes of chetion would increase alkyltransferase and reduce the susceptimotherapeutic agents.'" This group of agents is unique in its
bility of hematopoietic cells to nitrosoureas and related
mechanismsof cytotoxicityand in thecellularcomponents
responsible for drug resistance. For instance, while these agents compounds. This susceptibility is particularly important because alkyltransferase inhibitors have been described which
induce a variety of DNA adducts, the most cytotoxic is that
are now entering clinical practice.
producedatthe O6 position of guanine?"Themethylating
agents, streptozotocin, procarbazine, temozolomide, and dacar- A few years ago, we observed that colony-forming units
of the myeloid and erythroid lineages were sensitized to
bazine, form 06-methylguanineDNA adducts."** This adduct,
when hydrogen-bonded to cytosine
or thymine (the preferential BCNU by pre-exposure to 06-methylguanine, an agent that
inactivated 80% to 90%of the alkyltran~ferase.'~
06-methylbase inserted by the DNA polymerases), is recognized by the
guanine pretreatment resulted in a dose-modification factor
DNA mismatch repair system. The ensuing attempt at repair
replaces the cytosine or thymine opposite the adducted guanine,
(the ratio of the concentration of the drug inhibiting 50%
but fails to repair the06-methylguanineDNA adduct, resulting colony formation [IC,,] of BCNU alone over the concentration required with the combination) of approximately twoin an abortive mismatch-repair process? If there are sufficient
fold to threefold but was less than that observed with acute
persistent 06-methylguanine DNA adducts, this cycle of remyelogenous leukemia samples in which the dose-modificapeated mismatch repair appears to lead to single- and doublestrand breaks, and ultimately in cell death. In contrast to the
tion factor was approximately 6.3-fold. 06-benzylguanine is
a much more potent inhibitor of alkyltransferase than 06large number of 06-methylguanineDNA adducts required for
cytotoxicityaftermethylatingagentexposure,avery
small
methylguanine, resulting in rapid and complete inactivation
of the protein in tumor cell lines at concentrations of approxinumber of adducts formed after exposure to a chloroethylnimately 0.5 to 2 pm~"L.'~.''In addition, 06-benzylguanine
trosourea such as BCNU are sufficient for cytotoxicity. These
agents attack at the O6 positionof guanine, forming a chloropretreatment of xenograft colon, lung, prostate, and brain
ethyl DNA adduct that undergoes internal rearrangement first
tumors thatwere otherwise resistant toBCNU markedly
to N106-ethanoguanineand then to an interstrand crosslink?*Lo sensitized these tumors to BCNU, resulting in marked
growth delays and, in some instances, apparent
We have previously noted that human hematopoietic myeloid precursors in the BM have low levels of alkyltransferase activity-lower than most other human tissues, includFrom the Division of Hematology Oncology, the Department of
ing liver, lung, colon, kidney, and peripheral blood
Pharmacology and The Cancer Center, Case Western Reserve Unilymphocytes." We also observed that after nitrosourea inversity School of Medicine and University Hospitals of Cleveland,
duced DNA damage, alkyltransferase activity decreases in
Cleveland, OH: andthe Division of Pediatric Oncology andthe
hematopoietic cells and remains low even during a time of
Cancer Center, Johns Hopkins Medical Center, Baltimore, MD.
rapid DNA synthesis and cell proliferation, resulting in a
Submitted January 29, 1996: accepted April 25, 19%.
ratio of DNA repair capacity to DNA synthesis which is one
Supported inpart by Public Health Service GrantsNo. ROICA63193,
ROIES06288, UOlCA57725, POICA15183,P3OCA43703.
to two orders of magnitude lower in the marrow thanin
Address reprint requests to Stanton L. Gerson, MD, Division of
other tissues such as kidney and liver.' What was surprising
Hematology Oncology, Case Western Reserve University School of
wasthat hematopoietic cells do not appear to induce the
Medicine, I0900 Euclid Ave. Cleveland, OH 44106.
alkyltransferase during DNA synthesis, whereas induction is
The publication costs of this article were defrayed in part by page
observed in other cells either during cell proliferation after
charge payment. This article must therefore be hereby marked
growth factor exposure'2 or after DNA damage, at least in
"advertisement" in accordance with 18 U.S.C. section 1734 solely to
mammalian system^.'^ These data lead us to predict that the
indicate this fact.
low level of alkyltransferase may be a predisposing factor
0 1996 by The American Society of Hematology.
in the secondary leukemias that are known to occur in hu0006-4971/96/8805-0019$3.00/0
Blood, Vol 88, No 5 (September l ) , 1996: pp 1649-1655
1649
1650
GERSON ET AL
Based on these animal models of efficacy, clinical trials are
now in progress to evaluate the combination of 06-benzylguanine and BCNU.
However, during the course of the preclinical studies increased sensitivity to BCNU in the host animal has been
noted. In our own studies of xenograft bearing athymic mice
administered 90 mg/m2 06-benzylguanine, the maximum tolerated dose of BCNU was decreased from l10 to 50-70 mg/
m2.20In preclinical toxicology studies in the dog, myelosuppression was dose limiting and the maximum tolerated dose
of BCNU in dogs receiving 100 mg/m' 06-benzylguanine
was decreased from approximately 60 mg/m2 without pretreatment to 5 mg/m2.*' This suggests that significant bone
marrow (BM) suppression may be observed in clinical trials
as well. For this reason, we evaluated the alkyltransferase
activity in resting and growth-factor-stimulated BM cells
and determined the degree to which 06-benzylguanine sensitized cells to BCNU.
MATERIALS ANDMETHODS
Reagents. Complete medium for BM cells consisted of Iscove's
modified Dulbecco's Medium (IMDM; GIBCO/BRL, Gaithersburg,
MD) supplemented with 15% fetal bovine serum (FBS; Hyclone
Laboratories, Logan, UT) as previously described" and for the human breast cancer cell line, MCF-7, DMEMF12 medium (GIBCOI
BRL) supplemented with 10% bovine serum (Hyclone Laboratories).
Anti CD34+ antibody HPCA-1 (MY-10) was from Becton Dickinson
(San Jose, CA), and immunomagnetic beads were from Dynabeads
(Lake Success, NJ). Human recombinant (hr) granulocyte-macrophage colony-stimulating factor (GM-CSF), hr stem cell factor
(SCF), and hr erythropoietin were kindly provided by Amgen (Thousand Oaks, CA) and hr interleukin-3 (IL-3) was kindly provided by
Sandoz Research Institute (East Hanover, NJ). 06-benzylguanine
was kindly provided by Dr R. Moschel (Frederick Cancer Research
and Development Center, Frederick, MD) and stored at 2 mg/mL in
dimethyl sulfoxide at -20°C and diluted at the time of use into
serum-free culture medium. BCNU was obtained from the Drug
Synthesis and Chemistry Branch, Drug Therapeutics Program, National Cancer Institute. Tissue culture reagents were from Terry Fox
Laboratories (Vancouver, BC, Canada), ['Hlthymidine was from
New England Nuclear (Boston, MA), and general reagents were
from Sigma Chemicals (St Louis, MO).
BM cell cultures and drug treatment. BM was aspirated from
the posterior iliac crest of normal volunteers and patients with normal
BM undergoing BM harvest, after giving informed consent, under
protocols approved by the University Hospitals of Cleveland Institutional Review Board and were obtained through the Stem Cell Faciiity of the Case Western Reserve University Cancer Center. Lowdensity mononuclear cells were separated after Ficoll-Hypaque
(Sonofi-Winthrop Pharmaceuticals, New York, NY) centrifugation
and depleted of adherent cells by a 2-hour incubation on Primeria
plates (Falcon Plastics, San Jose, CA). To evaluate the dose-dependent inactivation of alkyltransferase by 06-benzylguanine,cells were
incubated for 1 hour at 37°C in serum-free culture medium at 1 X
loh cells/mL in 0 to 5 pmoVL O'-benzylguanine, recovered after
centrifugation, washed twice in phosphate-buffered saline (PBS)
containing S% calf serum at37°C as previously described'" and
twice in PBS-I mmoVL EDTA, before resuspension in 1 mL cell
extract buffer at 4°C" and storage at -80°C until assayed for alkyltransferase activity (see below). To determine the effect of growth
factor stimulation, cells were cultured in complete medium in the
absence or presence of 200 U/mL GM-CSF, 20 ng/mL IL-3, or 10
ng/mL SCF for up to 72 hours. Aliquots of cells were then immedi-
ately washed in PBS-ImmoVL EDTA and frozen at -80°C. To
determine the proportion of cells in cell cycle, some aliquots were
pulsed with 1 pCi ['Hjthymidine for 4 hours at 37°C before assay
for colony survival in methylcellulose (see below). To determine
the cytotoxicity of Ob-benzylguanineand/or BCNU, freshly obtained
or cultured human marrow cells were washed in serum-free RPMI
medium and resuspended in RPMI at 4 X IO6 cells/mL and exposed
to either 0.1% dimethyl sulfoxide (vehicle alone) or S pmoUL 0"benzylguanine for 1 hour before treatment with BCNU at concentrations of 0 to 40 pmol/L for 2 hours also in serum-free medium.
Serum-free medium was used because 0'"benzylguanine is heavily
protein bound and BCNU is rapidly decomposed in serum- or protein-containing solutions, increasing the variability of its DNA damaging effects. Cells were then washed in serum-free medium and
plated in 0.8% methylcellulose, 15% FBS, 1% deionized bovine
serum albumen, 100 pg/mL hemin, 200 U/mL GM-CSF, I O ng/mL
IL-3, and 2 U/mL erythropoietin at 37°C for 14 days and scored
for colony formation usingan inverted microscope as previously
described."
CD34' cells were isolated from low-density human nonadherent
mononuclear cells by immunomagnetic adherence as previously described.?l-22Briefly, cells were incubated sequentially in 1 pgHPCA1 per 1 X IO6 cells followed by O S X 10' goat-antimouse IgGIcoated immunomagnetic beads per 1 X lo6 BM cells and separation
of bead-bound cells by exposure to a magnet. The purityof the
separated cells was determined by incubation of the cells with Tuk3 anti-CD34' antibody (IgG3) followed by incubation with 1 pg
phycoerythrin-labeled goat-antimouse IgG3 and analyzed by FACScan (Becton Dickinson). Alternatively, alkyltransferase activity was
estimated by FACScan after incubation for 30 minutes with 2 pg/mL
MT3.1 monoclonal antibody against human alkyltransferase (kindly
provided by D. Bigner, Duke University, Durham, NC, and T. Brent,
St Judes Childrens Hospital, Memphis, TN), followed by incubation
with goat-antimouse IgG-fluorescein isothiocyanate (FITC). The remaining separated cells were then either washed as described above
in PBS-I m m o K EDTA and stored at -80°C for alkyltransferase
assay or were cultured in medium supplemented with 30% FBS and
with 7% vol/vol conditioned medium from the 5637 bladder cancer
cell line (previously titered to produce maximal growth stimulation)
for 4 to 7 days and then processed for alkyltransferase activity.
MCF-7 cell culture and drug treatment. MCF-7 human breast
cancer cells were kindly provided by Dr Ken Cowan (National Cancer Institute). Cells were plated at 2 X IO5 cells per 100-mm' culture
dish in complete medium, for 48 hours until 40% to 60% confluent,
incubated for 1 hour at 37°C in serum-free culture medium in 0
to 5 pmol/L 06-benzylguanine,washed twice in complete medium
maintained at 37°C and twice in PBS-1 mmol/L EDTA as described,
before trypsinization and resuspension in cell extract buffer and
storage at -80°C until assayed for alkyltransferase activity.
Alkyltransferase assay. Tissue alkyltransferase was measured as
previously described."," Briefly, cells were frozen in 1 mL cell
extract buffer, thawed, and immediately sonicated. Enzyme activity
was measured in whole-cell extracts by the amount of ['HI-methyl
group removed from ['H]-06-methylguanine present in calf thymus
DNA alkylated with ['HI-methylnitrosourea (specific activity of
0.047 fmol Oh-methylguanine/pg DNA). The alkylated [%-O"methylguanine and [3H]N7-methylguaninebases were separated by
high-performance liquid chromatography and quantified by liquid
scintillation. ['H]N7-methylguanine was used as the internal standard. Alkyltransferase activity was expressed a femtomole of 0'methylguanine removed per microgram of DNA.
RESULTS
Alkyltransferase was measured in low-density, nonadherent mononuclear BM cells collected immediately after Fi-
IETIC
HUMAN CD34'
6
1651
B
A
6
.1 GM-CSF
C
. IL-BISCF
5-
5-
4-
4-
32-
2-
4
1v-
0 HR
I
I
48HR
48HR+
GM-CSF
v
I
I
l
OHR
48HR
48 HR+
OHR
48HR
48HR+
Fig 1. Change in alkyltransferase activity after growth factor stimulation. Isolated hematopoieticcells from 3 to 7 donors per growth factor
were obtained immediately after collection or after 48-hour incubation in culture medium with or without the growth factor indicated.
Harvested cells ware assayed for alkyltransferaseactivity. Connected symbols indicate paired samplss from individual donors.
coll-Hypaque centrifugation and compared to cells grown in
the absence or presence of GM-CSF, IL-3, or SCF plus
IL-3 for 48 hours. Alkyltransferase measurements in paired
samples from the same donors showed no significantchange
after growth factor stimulation (Fig 1). Mean values of alkyltransferase before and after stimulation for each growth factor were: GM-CSF, 2.9 ? 1.2 v 3.3 ? 1.8 fmol/pg DNA (7
donors); IL-3, 2.0 ? 0.8 v 1.7 ? 0.65fmoVpgDNA
(6
donors); and IL-3/SCF, 3.8 5 0.3 v 3.6 ? 1.2 fmol/pg DNA
(3 donors). A similar lack of induction of alkyltransferase
was observed in cell cultures incubated in each growth factor
for 72 and 96 hours (data not shown). Finally, cells from
three donors were exposed to the combination of GM-CSF
and IL-3 and, again, no induction of alkyltransferase was
seen.
Isolated CD34+ marrow-derived cells were also analyzed
for alkyltransferase activity immediately after isolation or
after growth-factor stimulation for 7 days. The CD34+ cells
collected after immunomagnetic bead separation from three
donors were greater than 80% positive for CD34+ by FACS
analysis. The 7-day culture was chosen to enhance the proliferative response to 5637 conditioned medium. The mean
alkyltransferase activity in CD34+ cells was 3.1 -+ 1.6 fmol/
pg DNA, similar to that in the CD34- fraction recovered
after bead separation, 2.9 ? 1.1 fmol/pg DNA (Fig 2). This
was confirmed by FACScan analysis of alkyltransferase activity in unseparated and CD34+-enriched samples. Figure
3 shows identical histograms of the two preparations, indicating that CD34+ cells have low alkyltransferase activity and
that the distribution of activity in CD3+ cells is not skewed.
Mean alkyltransferase activity in the cultured CD34+ cells
decreased in all three donors to 1.6 t 0.7 fmol/pg DNA
after 7 days of growth factor stimulation. Similar results
were obtained with CD34+ cells cultured for 4 days (data
not shown).
Next, low-density mononuclear BM cells were grown in
medium containing 100 ng/mL IL-3 for 72 hours to determine whether growth factor stimulation increased resistance
to BCNU. In three separate experiments, the mean number
of colony-forming unit-granulocyte-macrophage (CFUGM) remained constant (72 t 21 v 75 -+ 23/105 cells) and
burst-forming units-erythroid increased (38 +. 11 v 86 ? 24/
lo5 cells) during growth in 100 ng/mL IL-3. The proportion
of progenitors in cell cycle also increased from a mean of
49% 2 12%to73% t 16% for BFU-Eand from 41% ?
17% to 80% ? 12% for CFU-GM. Despite these differences,
after 30 pmol/L BCNU exposure the percentage of progenitor cells surviving drug treatment was identical to that observed in unstimulated cultures (Fig 4). Pilot experiments
with two donors each were performed with either preexpo-
"1
5432-
1-
0
ODAYS
ODAYS
7DAYS
CD34CD34+
CD34+
Fig 2. Change in alkyltransferase activity in growth factor stimulated CD%+ cells.CD34+cellsor BM-derived cell suspensionsdepleted of CD%+ cells were isolated from 3 donors and were grown
in culture medium for 7 days in the presence of colony-stimulating
activity after which they were harvestedfor alkyltransferaseactivity.
Connected symbols indicate paired samples from individual donors.
GERSONET AL
1652
& MCF-7
0
0.001
FL2-Height
Fig 3.FACS
analysis of alkyltransferase expression in human
CD34 cells. Mononuclear hematopoietic cells (gray line) and isolated
CD34' cells (black line) were incubatedwith FITC-labeledmt3.1 antibody, recognizing humanalkyltransferase, or anisotype-specific antibody (dotted line)
t o indicate thelevel of background staining. In the
isolated CD34' cells, there isa higher proportion of nonstaining
cells.
sure to GM-CSF or the combination of GM-CSF and IL-3
for 48 or 72 hours before BCNU exposure. In each instance,
no protective effect was observed. This suggests that hematopoietic progenitor cells remain sensitive to BCNU during
growth factor exposure andthat potential mechanisms of
nitrosourea drug resistance, other than the alkyltransferase,
are unlikely to be induced by growth factor stimulation.
The concentration-dependent inactivation of alkyltransfer-
I
I
0.01
0.1
1 -
1
3
06bG, pM
Fig 5. Concentration-dependent inhibition of alkyltransferase by
O'-benzylguanine in hematopoietic cells and in MCFJ breast cancer
cells. BM mononuclear cells and MCF-7 human breast cancer cells
were incubatedin 06-benzylguanine(O'bG) for 2 hours, washed, and
assayed for alkyltransferase activity. Values represent the mean of
t w o determinationsfrom a representative experiment. Baseline alkyltransferase activity in the MCF-7 cell line was 23.6 fmollpg DNA,
compared with 3.2 fmollpg DNA in the marrow cellpreparation.
ase by O'-benzylguanine was determined in BM mononuclear cells and in the MCF-7 cell line exposed to 0"-benzylguanine for 2 hours, obtained, and assayed for residual
alkyltransferase activity. In both the marrow cells and the
breast cancer cell line, the concentration inhibiting 50% of
the protein was approximately 0.14 pmol/L and the concentration inhibiting 99% of the protein was 1.5 pmoVL (Fig
5 ) . Thus, there appears to be no selectivity in the kinetics
of alkyltransferase depletion by 0"-benzylguanine between
tumor cells and normal hematopoietic cells. Using a dose of
5 pmol/L Oh-benzylguanine for 1 hour, we observed complete inactivation of the alkyltransferase in six different BM
samples, confirming that this dose would serve as an appropriate dose for further drug sensitization studies.
To determine the degree to which 0'"benzylguanine mediated depletion of alkyltransferase sensitized hematopoietic
progenitor colony-forming cells to BCNU, cells were exposed to 0'-benzylguanine for 1 hour followed byBCNU
for 2 hours in serum-free suspension culture, and then plated
for surviving CFUs in methylcellulose. Figure 6 shows that
complete depletion of alkyltransferase by Oh-benzylguanine
pretreatment markedly sensitized hematopoietic progenitors
to BCNU, with a decrease in the ICso of BCNU,or the dosemodification factor, of a mean of 3.3-fold for BFU-E, 4-fold
for CFU-GM, and 5-fold for CFU-granulocyte, erythroid,
monocyte, megakaryocyte (CFU-GEMM). The shoulder apparent in the curves of cells treated with BCNU alone is no
longer present in cells treated with the combination.
I
loo
A
0
80 -
U
5
60-
0
0
T
I L-3
-
+
BFU-E
T
T
+
CFU-GM
Fig 4. Effect of IL-3 stimulation on BCNU sensitivity. BM hematopoietic progenitors were grown in the presence or absence of 11-3
for 72 hours and then exposed t o 30 pmollL BCNU for 2 hours before
plating in methylcellulose. Colony survival is shownas a percent of
untreated control values. Mean ? SD of three experiments.
DISCUSSION
These studies show for the first time that purified CD34'
hematopoietic progenitors contain low levels of the DNA
repair protein, 0'"alkylguanine-DNA alkyltransferase, simi-
1653
HUMAN CD34' HEMATOPOIETICPROGENITORS
CFU-GM SURVIVAL
BFU-ESURVIVAL
CFU-GEMM SURVIVAL
l
l 10
m
~
+ 06BG
\:i, + 0 6 B G
1
0
0
)
BCNU pM
0
10
20
30 40
BCNU pM
1
0
10
20
30
40
BCNU pM
Fig 6. b-benzylguanine potentiation of BCNU cytotoxicity in hematopoietic progenitors. Mononuclear BM cells were incubated in culture
medium with or without 10 pmollL O'-benzylguanine for 1 hour and then to increasing concentrations of BCNU for 2 hours before being
washed in drug-free medium and cultured in methylcellulosewith growth factors to enumerate colonies. Data are the mean & SE from the
mean values of four separateexperiments. For some values, the error bars are smaller than the symbols.
lar to that observed in low-density mononuclear cells in the
Furthermore, these low levels are not increased
during growth factor stimulation. This is unlike the situation
that we and others have observed with cell lines and human
peripheral blood lymphocytes, in which growth factor stimulation and the transition from the resting to proliferating state
are associated with an increase in alkyltransferase activity."
These studies document that BM cells remain sensitive to
nitrosourea chemotherapy after growth factor stimulation
and that other potential mechanisms of resistance are not
induced in these cells. This is surprising because resting
stem cells have other protective mechanisms that prevent
cell death after exposure to other agents (see below). In
contrast, the exquisite sensitivity seen with the nitrosoureas
indicates that there is little intrinsic resistance inhuman
hematopoietic progenitors to this class of agents. Because
growth factors are commonly administered to patients receiving chemotherapy, these observations reinforce the need
to avoid coadministration of growth factors and nitrosoureas
and related agents such as triazines and tetrazines (procarbazine, dacarbazine, and temozolomide) because of potentially increased cytotoxic and mutagenic effects. In fact, a
recent report noted a similar ability of 06-benzylguanine to
sensitize hematopoietic progenitor cells to temozolomide, a
methylating agent attacking at the O6position of guanine and
repaired by the alkyltransferase,26as we noted with BCNU.
Furthermore, the use of alkyltransferase inhibitors with either the chloroethylating or methylating agents may increase
the level of DNA damage to early hematopoietic progenitors
and, we predict, increase the risk for secondary leukemia^.'^
Although it is possible that increased BM aplasia would
result from this approach, leading to less survival of damaged
progenitors, this is unlikely to decrease the risk of cell transformation since the proliferative stress on surviving cells
will be that much increased. In both clinical and experimental settings, repeated marrow damage followed by cell proliferation appears to increase the likelihood of secondary leukemia~.~~,~~
The sensitivity of hematopoietic progenitors to these
agents is unlike the protection from cyclophosphamide due
to endogenously high levels of aldehyde dehydrogenase in
CD34+cells" or toward naturally occumng compounds due
to the relatively high levels of multidrug resistance gene
expression in CD34+ cells.29 While these drug resistance
genes may be selectively expressed in hematopoietic precursors, alkyltransferase levels are not increased. Indeed, we
have made numerous attempts to induce expression of alkyltransferase in hematopoietic cells and have been unsuccessfu1.',30 Furthermore, the sensitivity to nitrosoureas suggests
that there are not other repair mechanisms present in human
hematopoietic progenitors which can provide a backup or
"redundant" mechanism of repair for lesions at the O6 position of guanine. This suggests that human CD34+cells are
exquisitely dependent on the alkyltransferase for repair of
these lesions and thus for protection from the nitrosoureas
and related compounds. Perhaps more so thanwith other
drug resistance genes, these data provide compelling rationale for a gene therapy approach to increase expression of
the alkyltransferase gene, MGMT, in early hematopoietic
progenitors, as we and others have d e ~ c r i b e d . ~ " ~ ~
We have previously proposed that the low level of alkyltransferase in hematopoietic progenitors may be an etiologic
mechanism in the induction of secondary leukemia after alkylating agent exposure, particularly the nitrosoureas.1~1'~30
What is most striking is the observation that the BM response
to nitrosourea exposure is to increase the rate of DNA synthesis when alkyltransferase is low compared with other tissues that increase alkyltransferase but maintain much lower
rates of DNA synthesis and cell proliferation.' We tested
this hypothesis in an animal model of secondary leukemia
and found that transgenic mice overexpressing the human
MGMT gene in the thymus were prevented from developing
methylnitrosourea-induced thymic le~kemia/lymphomas,~~
proving the importance of the alkyltransferase in the process
and establishing the 06-alkylguanine DNA adduct as the
major DNA lesion responsible for these leukemidymphomas.
06-benzylguanine is a potent inhibitor of the alkyltransfer-
GERSON ET AL
1654
as well as intumorcells.
We
observed no selectivity in dose-dependent inactivation of the
alkyltransferase in hematopoietic cells compared with tumor
no selectivity
cells in culture, suggesting that there will be
The
in the increased cytotoxicity observed with this agent.
degree of sensitization, measured by dose-modification fac3.3- andS-foldweregreater
torsforBCNUofbetween
Ohaftercompleteinactivationofthealkyltransferaseby
benzylguanine than the 1.S- to 3-fold dose modification factor we previously observed with
O6-methy1guanine." The
a loweraffinityfortheproteinand
lattercompoundhas
85% to 90%. This
inactivated the alkyltransferase by only
suggests that small residual levels of alkyltransferase may
provide protection from nitrosourea-induced chemotherapy.
Of note, the dose-modification factors observed for hematopoietic progenitors were less than those observed with the
MCF-7 cell line.24 Because this tumor cell line has much
higher levels of alkyltransferase than hematopoietic progeniin vitro
tors and is markedly resistant to nitrosoureas both
and in vivo , our results suggest that the combination may
sensitize tumor cells expressing high levelsof alkyltransferase more than BM cells, which are sensitive to nitrosoureas
because of low levels of alkyltransferase. These differences
could provide a small degree of selectivity and a sufficient
therapeutic index to allow successful administration of Ohbenzylguanine in the clinic. However, these studies
d o predict that the dose-limiting toxicity for 06-benzylguanine followedby a nitrosourea, a triazine, or a tetrazinewillbe
myelosuppression and that growth factor administration will
not protect the marrow precursors. In the absence of other
techniques to protect the BM, it would appear that cautious
attention to marrow toxicity during dose-escalation will be
required. Based on our prior studies, further caution must
be raised that the myelosuppression will increase the rate
of DNA synthesis in a progenitor cell population prone to
development of secondary leukemias and that paralysis of
an important DNA repair mechanism will enhance the risk
of mutational events and perhaps increase the incidence of
leukemias in these patients.
ase inhematopoieticcells
ACKNOWLEDGMENT
Karin Johnson, Jane Schupp, and Kathy Vitantonio are thanked
for expert technical assistance. DrHillard Lazarus is thanked for
assistance in the collection of bone marrow samples.
REFERENCES
I . Gerson SL, Trey JE, Miller K, Benjamin E: Repair of Ohalkylguanine during DNA synthesis in murine bone marrow hematopoietic precursors. Cancer Res 47:89, 1987
2. Newlands E, Blackledge GRP, Slack JA, Rustin GJS, Smith
DB, Stuart NSA, Quarterman CP. Hoffman
R, Stevens MFG, Brampton MH, Gibson AC: Phase I trial of temozolomide (CCRG 81045:
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3. Phillips GL, Fay J W , Herzig GP, Herzig RH, Weiner RS,
Wolff SN, Lazarus HM, Karanes C, Ross W, Kramer B: Intensive
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cryopreserved autologous marrow transplantation for refractory cancer: A phase 1-11 study. Cancer S2:1792, 1983
4. Lodich J, Chawla PL, Frei E: 1,3-bis-(2 chloroethy1)-l-nitrosourea and streptozotocin chemotherapy. Clin Pharmacol Ther
17:374, 1975
S. Tong WP,KirkMC,
Ludlum DB: Formation ofthe crosslink I-[N3-deoxycyctidyl],2-[Nl-deoxyguanosinyl]-ethanein DNA
treated with 1,3-bis(2-chloroethyl)-l-nitrosourea. Cancer Res
42:3102, 1982
6. Erickson LC, Laurent G, Sharkey NA, Kohn KW: DNA crosslinking and monoadduct repair in nitrosourea-treated human tumour
cells. Nature 288:727, 1980
7. Frei J, Lawley PD: Methylation of DNA in various organs of
C57BL mice by a carcinogenic dose of N-methyl-N-nitrosourea and
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