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Immunology |
Division of Pediatric Hematology/Oncology, Children’s Hospital of Michigan [J. W. T., M. L. S., Y. R.], Experimental and Clinical Therapeutics Program, Barbara Ann Karmanos Cancer Institute [J. W. T., X. H., Y. G. R. M. M., Y. R., L. H. M.], and Departments of Pediatrics [J. W. T., Y. R.], Pharmacology [L. H. M.], and Internal Medicine [J. A. D., R. M. M.], Wayne State University School of Medicine, Detroit, Michigan 48201
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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37% for
non-DS AML patients (15)
, which indicates that DS
myeloblasts have enhanced sensitivity to drugs used in AML therapy. We
have reported that DS myeloblasts are more sensitive in
vitro to ara-C and daunorubicin compared with non-DS myeloblasts
and generate higher intracellular levels of the active intracellular
ara-C metabolite ara-CTP, after in vitro incubation with
[3
H]ara-C compared with non-DS cells. This
suggests a biochemical basis for the high EFS rates of DS AML patients
(16, 17, 18)
. CBS (EC 4.2.1.22), a pyridoxal 5'-phosphate-dependent enzyme (gene localized to chromosome 21q22.3), is involved in the transsulfuration pathway which catalyzes the condensation of serine and homocysteine to form cystathionine (19) . Changes in CBS activity in DS cells has been proposed to alter folate metabolism by "trapping" 5-methyl tetrahydrofolate, leading to increased MTX toxicity (20) . ara-C metabolism and folate pools are linked by the known synergistic effect of sequential MTX and ara-C therapy, leading to lowering of dCTP pools and a greater generation of ara-CTP (21 , 22) . On this basis, we proposed that increased CBS activity may also enhance ara-C metabolism in DS cells (8 , 16 , 17) . In an analysis of myeloblasts obtained from newly diagnosed pediatric AML patients, we found that relative CBS transcripts detected by quantitative RT-PCR were a median 12-fold higher in DS myeloblasts compared with non-DS myeloblasts, and that relative CBS transcripts correlated with both in vitro ara-C sensitivity and the generation of ara-CTP in myeloblasts from both the DS and the non-DS patients (18) .
In this study, we extended these patient studies to a model system by transfecting CBS-null CCRF-CEM cells with the CBS cDNA. This permits the study of the relationships between CBS gene expression and ara-C metabolism/sensitivity as a model of DS blast cells, thus eliminating the confounding effect of other chromosome 21-localized genes.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Culture.
The CCRF-CEM, K562, KG-1, HL-60, and U937 leukemia cell lines were
obtained from the American Type Culture Collection (Rockville, MD).
Cells were maintained in RPMI 1640 containing 10% heat-inactivated
calf serum, 100 units/ml penicillin and 100 µg/ml streptomycin in a
humidified atmosphere at 37° in the presence of 5%
CO2/95% air.
Gene Transfection.
The hCBS cDNA was kindly provided by Dr. W. Kruger
(23)
. After digestion with the restriction enzyme
EcoRI, the 2.2-kb hCBS insert was directionally
ligated into the pcDNA3 expression vector (Invitrogen, San Diego, CA).
The resulting recombinant plasmid pcDNA3-CBS was linearized with
EcoRI and transfected into CCRF-CEM cells (20 µg of
DNA/5 x 106 cells) by
electroporation (300 V, 500 µf) using the Electroporator II
(Invitrogen). Forty-eight h after transfection, the cells were plated
at 20,000 cells/dish in soft agarose (0.35%) containing 1 mg/ml G418
(Sigma). Stably transfected cells were selected after 3–4 weeks,
expanded, and screened for CBS transcripts and activity by RT-PCR,
Northern analysis, and enzyme assays.
Gene Transcript Analysis by RT-PCR.
Total RNA was isolated from 1 x 107 cultured cells by the phenol/chloroform
method using TriReagent (Molecular Research Center, Cincinnati, OH;
Ref. 24
). The analysis of CBS transcripts by RT-PCR was
performed as described previously (18)
.
Northern Blot Analysis.
Total RNA from each cell line (20 µg) was electrophoresed on 0.9%
agarose gels containing 2.2 M formaldehyde and 1x
4-morpholinepropanesulfonic acid buffer. The gel was
equilibrated in 10x SSC and capillary-transferred to GeneScreen Plus
membrane (NEN, Boston, MA) in 10x SSC; the membrane was baked
at 80°C under vacuum for 1.5 h. Membranes were prehybridized in
QuickHyb solution (Stratagene, La Jolla, CA) for 15 min, then
hybridized for 1 h with the addition of
[32P]dCTP-labeled dCK cDNA insert labeled by
random priming. Nonspecific hybridization was removed by washing
membranes in 2x SSC, 0.1% SDS at 42°C and, finally, in 0.1x SSC,
0.1% SDS at 60°C. Densitometry of the autoradiograms was performed
on a Molecular Dynamics Storm 860 fluorescence and radioactivity
imaging system and Image Quant software.
CBS Assay.
This radioisotopic assay measures the amount of cystathionine generated
by a reaction of homocysteine and serine catalyzed by CBS
(25)
. The cells suspended in a lysis buffer [50
mM Tris-HCl (pH 8.3), 1 mM mercaptoethanol, 1
mM pyridoxal phosphate, and proteolytic inhibitors] were
extracted by freeze-thaw lysis (three times). The CBS assay was
performed in a total volume of 100 µl containing 10 mM
[14C]serine, 15 mM homocysteine,
650 µM pyridoxal phosphate, 2.5 mM EDTA, 100
µg protein extract, 1 mM propargylglycine to inhibit
cystathionase, 0.5 mg/ml BSA, and 150 mM Tris-HCl (pH 8.3).
All of the constituents were equilibrated at 37°C for 5 min followed
by the addition of homocysteine to start the reaction. The reaction
mixture was incubated for 4 h at 37°C and then spotted onto
Whatman 3 M chromatography paper and the
[14C]cystathionine product separated from the
substrate [14C]serine by ascending
chromatography in isopropanol:formic acid:water (70:10:20). After
drying, 0.2% ninhydrin reagent was used to detect the marker serine
and L-cystathionine (Rf,
0.25). Radioactive compounds were detected by cutting the
chromatogram into 1-cm strips that were solubilized in water and
counted directly after the addition of scintillation fluid.
Radioactivity from an enzyme-free blank was subtracted to determine the
amount of enzymatically formed
[14C]cystathionine generated. Enzymatic
activity was expressed as pmol of cystathionine formed per mg protein
per h at 37°C. Proteins were assayed by a modification of the Lowry
assay.
In Vitro Drug Cytotoxicity Assay.
For the determination of cytotoxicity, cells were cultured in complete
medium with dialyzed FCS in 24-well culture dishes at a density of
50,000 cells/ml of media. Cells were cultured continuously with a range
of concentrations of ara-C, daunorubicin, gemcitabine, or MTX at 37°,
and the cell numbers were counted after 4 days with a Coulter counter
(Coulter Electronics, Hialeah, FL). The
IC50 values were calculated as the concentration
of drug necessary to inhibit 50% growth compared with control cells
grown in the absence of drug.
ara-C Incubations and Measurement of ara-CTP.
Incubation of leukemia cells with 5 µM
[3
H]ara-C and the measurement of intracellular
[3
H]ara-CTP levels by high-performance liquid
chromatography was performed as described previously
(16)
.
SCID Mice Xenografts and Drug Sensitivity.
Four-week-old immunodeficient Fox Chase ICR SCID mice
[Tac:Icr:Ha(ICR)-scidfDf] (male or female) were obtained
from Taconic (Germantown, NY) and maintained in specific pathogen-free
conditions in microisolator cages stored in laminar flow racks. The
mice were fed sterile water and autoclaved food pellets ad
libitum.
The CCRF-CEM cell lines (wild-type and transfected) were maintained in RPMI 1640 with heat-inactivated 10% FCS, penicillin/streptomycin, and G418 (transfected lines) prior to the experiments. Initially, mice were implanted s.c. with 1 x 107 cells in serum-free RPMI into each flank. The incidence and growth rate of the tumors were calculated from serial measurement of the length and width of the tumors by Vernier calipers (26 , 27) .
After the development of palpable tumors (1500 mg), the mice were
killed by cervical neck dislocation, and small fragments of tumors
(30 mg), were implanted s.c. bilaterally into the flanks of a second
group of mice using a 12-gauge trocar. The mice resumed normal activity
after the procedure. Other tumor fragments were dissected and made into
single-cell suspensions; the leukemia phenotype was confirmed by
cytometry and the stability of expression of the transfected genes
assessed by RT-PCR analysis.
Antileukemic drug therapy was initiated 14 days after the implantation of the mice with tumors, with all of the drugs being administered in 0.2 ml of RPMI 1640 i.p.; ara-C was administered daily for 8 days (total dose, 328 mg/kg); and gemcitabine was administered daily for 10 days (total dose, 25 mg/kg). Treated and untreated (control) mice were randomly selected for therapy; each treatment group consisted of four to five mice. After drug therapy, the mice were observed daily to assess changes in tumor size, weight changes, and drug side effects.
Mice were killed in any given trial if the tumor size reached 1500 mg, and no tumor was allowed to exceed 10% of the mouse’s body weight. The assessment of antitumor activity of ara-C and gemcitabine was based on the determination of tumor weight calculated as follows: tumor weight (mg) = (A x B)2/2, where A and B represented the tumor width (mm) and length (mm), respectively.
This protocol was approved by the Wayne State University Animal Investigation committee.
Methylation-specific PCR.
Genomic DNA was isolated from leukemia cell lines using the Puregene
System (Gentra System, Minneapolis, MN), and Tri Reagent was used to
isolate genomic DNA from clinical AML specimens (18)
.
Bisulfite modification of DNA was performed to convert all unmethylated
cytosines to uracil, whereas methylated cytosines would remain
unmodified (28)
. Genomic DNA was initially linearized by
shearing through a fine needle followed by alkali denaturation. The
denatured DNA (4 µg) was incubated in a total volume of 1.2 ml with
3.1 M sodium bisulfite/0.5 mM hydroquinone (pH
5.0) for 20 h at 50°C and was purified with the QIAquick PCR
purification kit (Qiagen, Valencia, CA). CpG Ware primer design
software (Intergen, Purchase, NY) was used to modify the dCK DNA
sequence (GenBank accession no. L07485) and PCR primers were
designed using Oligo 5 software to amplify bisulfite-modified DNA,
which would discriminate between methylated and unmethylated DNA. The
sense primers were: for dCK1, 5'-TGTTTGGGTGTTTGGTTGTTTGGGGTAGAG-3' (nt
183–213) and for dCK2, 5'-CACAACACCCCAACCTTACATCCCACATT-3' (nt
654–683; modified). The antisense primers were: for dCK 3,
5'-TTTAGAGTTGGTTGAGAAAGATGGGTAGTT-3' (nt 102–132); and for dCK 4,
5'-CAACACCCTCAAACCTCTAAAATCC-3' (nt 556–581; modified). PCR
amplification was performed with 10 µl of bisulfite-modified DNA in a
total volume of 50 µl containing 10 mM Tris-HCl (pH 8.3),
1.5 mM MgCl2, 50 mM KCl,
5% DMSO, 200 µM each dNTP, 1.25 units of Taq DNA
polymerase, and 0.2 µM each dCK primer. PCR amplification
was denatured for 2 min at 94°C followed by 32 cycles of 1 min at
94°C, 1 min at 50°C, and 2 min at 72°C, followed by a final
extension for 10 min. To determine the methylation status of the dCK
promoter, the PCR products were subcloned into the pGEM T-easy vector
(Promega, Madison, WI) using the TA cloning kit (Invitrogen) and
were sequenced using the BigDye Terminator Cycle Sequencing Ready
Reaction kit (PE Applied Biosystems, Foster City, CA).
Western Blot Analysis.
Total protein was isolated from cell lines using the lysis buffer
[20 mmol/liter Tris-HCl, 0.9% NaCl (pH 7.6), 0.1% Triton X-100, 1
mmol/l phenylmethylsulphonyl fluoride and 0.01% leupeptin], and the
protein concentration of the lysates was determined by the Bio-Rad
protein assay (Bio-Rad, Richmond, CA). Forty-µg aliquots of each
lysate were fractionated on a 7.5% polyacrylamide gel with SDS and
electroblotted onto a nitrocellulose membrane. The blot was blocked
overnight at 4°C in TTBS [(Tween Tris-based saline with 0.3% Tween
20 (pH 7.5)] containing 8% fat-free dried milk powder and was then
incubated with an anti-sera dCK antibody (kindly provided by Dr. D.
Shewach), diluted 1:1000 in TTBS containing 1% fat-free dried milk
powder for 2 h at room temperature. The blot was washed with TTBS
and incubated with a second antibody (goat antirabbit IgG linked to
horseradish peroxidase conjugate, diluted 1:5000 in TTBS-1% milk
powder) for 1 h at room temperature, and detected by enhanced
chemiluminescence with the SuperSignal CL-HRP Substrate System kit.
(Pierce, Rockford, IL).
dCK Enzyme Activity.
The dCK assay measures the ara-C phosphorylation ability of a
cell-free extract (29)
. Cells (1 x 107) were extracted in 50 mM Tris by
freeze-thaw lysis (three times). The reaction mixture (120 µl)
consisted of 20 µl of cell free supernatant, 10 mM ATP,
10 mM MgCl2, 50 mM Tris
(pH 7.6), 15 mM NaF, 1 mM tetrahydrouridine (to
block cytidine deaminase activity) and an ATP regenerating system
consisting of 15 mM phosphoenol pyruvate, 3.6 units of
myokinase, and 1.4 units of pyruvate kinase. The reaction was started
with the addition of 5 µM
[3
H]ara-C and incubated at 37° for 30 min.
The reaction was terminated by immersion of the reaction mixture in
boiling water for 1 min followed by the spotting of 50 µl of the
mixture onto DE-81 filter paper. The paper was washed with ice-cold 1
mM ammonium formate followed by additional washes with 70%
ethanol. The ara-C nts were eluted off the filter paper in
scintillation vials containing 0.1 M HCl and 0.2
M KCl, and the radioactivity was measured and quantitated
as pmol/mg protein/min.
Deoxynucleotide Pool Assay.
Cultured cells (1 x 107) were
extracted with ice-cold perchloric acid and the endogenous dCTP pools
were measured by a DNA polymerase assay as described previously
(16)
.
Statistical Analysis.
Differences in drug sensitivity and parameters of drug metabolism were
analyzed by the paired t test. The comparison of survival of
the SCID mice groups was analyzed by the Kaplan-Meier method with the
Mantel-Cox (log-rank) test using StatView statistical software
(Berkeley, CA).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several G418-resistant cell clones were identified after transfection
of the CCRF-CEM cell line by electroporation; two of the cell line
clones, designated CEM-CBS 21 and CEM-CBS 28, were further
characterized for our studies. Both of these clones had CBS transcripts
identified by RT-PCR (Fig. 1)
. CBS enzyme studies revealed enzyme activities 6- and 10-fold higher
in the CBS 21 (5.99 pmol/mg protein/h) and CBS 28 (10.6 pmol/mg
protein/h) cell lines compared with wild-type (0.47 pmol/mg protein/h)
or mock-transfected (0.82 pmol/mg protein/h) CCRF-CEM cells (CEM cells
transfected with the pcDNA3 expression vector without the CBS cDNA
insert).
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15-fold more sensitive by growth
inhibition assay in vitro to ara-C compared with the
wild-type and mock-transfected cell lines (ara-C
IC50, 6.2 nM and 6.8
nM versus 130.2
nM and 90.9 nM;
P = 0.02 and 0.01, respectively (Fig. 2A)
. The sensitivity of the cell lines to gemcitabine
(2',2'-difluorodeoxycytidine)—a representative nucleoside agent that
undergoes phosphorylation via dCK in a fashion similar to that of
ara-C—was also determined. The CBS 21 and CBS 28 lines
demonstrated 18-fold more sensitivity to gemcitabine, compared with
that of the wild-type and mock-transfected CEM lines (Fig. 2B)
. There were no significant differences in the percentage
of cells in S phase to account for the differences in drug
sensitivities (CEM, 49.1%; CBS-MOCK, 51.7%; CBS 21, 40.4%; CBS 28,
45.8%). Furthermore, this increased sensitivity was not observed with
agents not activated by dCK, because there was no increased sensitivity
of the CBS 21 and CBS 28 cell lines to either MTX or daunorubicin
compared with the wild-type or mock-transfected cell lines (MTX
IC50, 16.1 nM and 16.9
nM versus 19.8
nM and 20.3 nM,
respectively; daunorubicin IC50, 14.6
nM and 15.7 nM
versus 15.3 nM and 15.6
nM, respectively).
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). There were no differences in endogenous dCTP pools
between the wild-type CEM cells (0.08 pmol/µg protein) and the CBS 21
and CBS 28 sublines (0.072 and 0.067 pmol/µg protein, respectively).
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22-fold higher) in the
CBS-transfected cell lines compared with the CBS-null cells (Fig. 3B)
. To determine whether the increased dCK activity in
CBS-transfected cells was related to elevated levels of dCK expression,
possibly attributable to decreased CpG methylation in the dCK promoter,
dCK transcripts from total RNA were measured by Northern analysis.
There were no significant differences in the levels of dCK transcripts
between the CBS-transfected and CBS-null CEM cells to account for the
differences in dCK activity (Fig. 4
, upper panel). Likewise, there were no differences in
methylation of CpG islands in the dCK promoter, determined by PCR
amplification of bisulfite-modified DNA. Western blot analysis using a
dCK antibody did not demonstrate increased levels of immunoreactive dCK
protein in the CBS-transfected CEM cells compared with that in the
CBS-null CEM cells (Fig. 4
, lower panel).
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At the time of initiation of drug therapy (day 14), there were no
significant differences in the sizes of palpable tumors between mice
implanted with either the wild-type or the CBS 28 cells in both the
untreated and drug-treated groups. All of the mice implanted with
either wild-type or CBS-transfected cells and treated with RPMI
medium alone, readily developed palpable tumors and were killed
once the tumors reached 1500 mg in size. There was no significant
difference in survival in days, in mice implanted with wild-type CEM
cells and treated with either ara-C or gemcitabine (median, 32
versus 37 days (ara-C) and 35 days (gemcitabine);
P = 0.37; Fig. 5A
). In contrast, there was a significant difference in
survival in mice implanted with the CBS 28 cell line, with no evidence
of palpable tumors after treatment with either ara-C or gemcitabine
compared with the untreated control group (P < 0.0001); this group was considered cured (Fig. 5B)
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our previous studies indicated a basis of the high EFS rates of DS AML patients; DS myeloblasts are more sensitive in vitro to ara-C and generate higher intracellular ara-CTP levels (16 , 17) . We also found that relative transcripts, detected by RT-PCR, of the chromosome 21-localized gene, CBS, were significantly higher in DS myeloblasts compared with non-DS myeloblasts; and relative CBS transcripts correlated with both in vitro ara-C sensitivity and generation of ara-CTP in myeloblasts from both the DS and non-DS patients (18) .
In this study we demonstrated that the transfection of wild-type CCRF-CEM leukemia cells (which have very low CBS enzyme activity and lack CBS transcripts on Northern and RT-PCR analysis) with the CBS cDNA resulted in a 15-fold increased sensitivity to ara-C and a 8.5-fold increased generation of [3 H]ara-CTP after in vitro incubation with [3 H]ara-C in CBS- transfected cell clones compared with wild-type cells. A similar increased sensitivity of CBS-transfected cells to the deoxycytidine analogue, gemcitabine, suggests a common mechanism, likely attributable to increased dCK activity. SCID mice that were implanted with CBS-transfected CEM cells had significantly greater lengths of survival after ara-C and gemcitabine therapy compared with the identical therapy in mice implanted with wild-type CEM cells (median, 150 versus 37 days; P < 0.0001), which suggests the applicability of our DS model in vivo.
Increased CBS activity in DS cells has been proposed as a mechanism that accounts for the significant MTX toxicity of DS patients (20) . Although our results suggest that the increased MTX sensitivity of DS patients is not directly linked to the CBS gene alone in our CBS-transfected cell line model, the combined effects of increased gene expression and protein activity of CBS and the reduced folate carrier (RFC; the intracellular transport protein of reduced folates including MTX whose gene is localized to 21q22; Ref. 31 ), may be linked to the MTX sensitivity in DS cells. Notably, the CBS-transfected cells did not demonstrate increased sensitivity to daunorubicin, which indicated that there are other mechanisms that account for the increased in vitro daunorubicin sensitivity of DS myeloblasts (17 , 18) .
Our hypothesis for the increased ara-C sensitivity of DS myeloblasts was based on a key role for the CBS enzyme interacting with both reduced folate pools and AdoMet/methylation pathways (16 , 30) . Increased CBS activity is associated with the low plasma homocysteine levels in DS individuals and the absence of atheroma in DS patients at autopsy (32 , 33) . By contrast, the genetic disorder, homocystinuria, characterized by mental retardation, lens dislocation, skeletal abnormalities, thromboembolism, and early onset atherosclerosis, is attributable to CBS deficiency, and is associated with elevated homocysteine and methionine levels (19) . Increased CBS activity associated with decreased homocysteine and AdoMet levels (34 , 35) , can result in altered allosteric regulation of the MTHF reductase enzyme and altered gene expression via methylation including the dCK gene. We have previously proposed that increased CBS activity results in enhanced ara-C metabolism and sensitivity by the following mechanism: (a) decreased inhibition of MTHF reductase secondary to lower AdoMet levels, leading to increased diversion of MTHF pools to 5-methyl tetrahydrofolate; (b) reduced synthesis of deoxythymidylate and dTTP resulting from lower MTHF pools; and (c) decreased dCTP pools attributable to reduced inhibition of deoxycytidylate deaminase via dTTP; lower dCTP pools would result in increased phosphorylation of ara-C and less competitive binding between dCTP and ara-CTP to DNA (8 , 16 , 17) .
Consistent with this hypothesis, dCK activity was significantly increased in CBS-transfected cells. The levels of dCK transcripts on Northern blots were nearly identical between CBS-transfected and wild-type cells; and DNA sequencing of PCR products, amplified from bisulfite-modified DNA, showed no differences in methylation status of the dCK promoter, as reported by others (36 , 37) . Thus, the increased dCK activity is not attributable to increased dCK expression via differences in methylation of the dCK promoter. There was, as well, no significant difference in levels of dCK protein identified by Western blot analysis between the wild-type and CBS-transfected CEM cells. Collectively, these results suggest a posttranscriptional regulation of dCK in CBS-overexpressing cells that contributes to increased ara-C phosphorylation and drug activity. Although the mechanism for this effect is uncertain and is under active investigation, this may, in part, be related to the intracellular localization of the dCK protein (38 , 39) or, possibly, effects of dCK phosphorylation (40) or catalytic activity. Although dCTP can allosterically regulate dCK, as noted above, we did not detect differences in dCTP pools between CBS-transfected and wild-type CEM cells; this may reflect the compartmentalization of intracellular dCTP pools (41) .
Can CBS expression be altered in leukemia cells in patients to mimic our DS cell line model to improve the effectiveness of ara-C therapy for AML? Translating our in vitro/ex vivo model to the clinic is clearly hampered in vivo by difficulties in selectively targeting leukemia cells for gene transfer because of leukemia being a systemic disease (42) . Selective targeting of leukemia cells in vivo has been accomplished using monoclonal antibodies that recognize leukemia-specific surface antigens (e.g., anti-CD 33 monoclonal antibody therapy for AML; Refs. 43 and 44 ). On this basis, the conjugation of a CBS cDNA/expression vector system complex (45) to an anti-CD 33 monoclonal antibody could potentially result in systemic CBS gene transfer into leukemia cells, thus "sensitizing" myeloblasts prior to ara-C therapy.
Our results demonstrating dramatic increases in both ara-C sensitivity and the metabolism of ara-C to ara-CTP in leukemia cell lines transfected with the CBS cDNA, along with increased ara-C sensitivity of transfected cell lines implanted in SCID mice, provide compelling evidence as to the relationship of CBS gene expression and ara-C metabolism in our model. This mechanism is likely a major factor that accounts for the increased chemotherapy sensitivity of pediatric DS AML patients and provides the first molecular basis for the high cure-rate of DS children with AML. Although our model was developed in a T-cell ALL cell line, the development of a CBS-transfected AML cell line would more closely be a model of DS and AML. Studies in progress are currently developing these cell line models. The further identification of the molecular mechanisms of chemotherapy sensitivity of DS AML patients and the development of new therapeutic approaches based on these mechanisms may lead to significant improvements in the treatment and cure of AML.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by The Leukemia and Lymphoma
Society Research Translational Grant (6203-98), Children’s Leukemia
Foundation of Michigan, Children’s Research Center of Michigan, Art
Gagnon Memorial Fund, BenePro Corporation, Litvak Foundation, and
Leukemia Research, Life, Inc. (Detroit, MI). 
2 To whom requests for reprints should be
addressed, at Children’s Hospital of Michigan, 3901 Beaubien
Boulevard, Detroit, MI 48201. Phone: (313) 745-5515; Fax: (313)
745-5237.
3 The abbreviations used are: DS, Down
syndrome; SCID, severe combined immunodeficient; EFS, event-free
survival; AML, acute myeloid leukemia; ara-C,
1-ß-D-arabinofuranosylcytosine; ara-CTP, ara-C
triphosphate; AdoMet, S-adenosylmethionine; ALL, acute
lymphoblastic leukemia; MTX, methotrexate; CBS;
cystathionine-ß-synthase; RT, reverse transcription; dCK,
deoxycytidine kinase; nt, nucleotide; MTHF,
5,10-methylenetetrahydrofolate.
Received 12/28/99. Accepted 9/18/00.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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C. M. Zwaan, G. J. L. Kaspers, R. Pieters, K. Hahlen, G. E. Janka-Schaub, C. H. van Zantwijk, D. R. Huismans, E. de Vries, M. G. Rots, G. J. Peters, et al. Different drug sensitivity profiles of acute myeloid and lymphoblastic leukemia and normal peripheral blood mononuclear cells in children with and without Down syndrome Blood, January 1, 2002; 99(1): 245 - 251. [Abstract] [Full Text] [PDF]
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 Y. Ge, L. H. Matherly, and J. W. Taub Transcriptional Regulation of Cell-specific Expression of the Human Cystathionine beta -Synthase Gene by Differential Binding of Sp1/Sp3 to the -1b Promoter J. Biol. Chem., November 16, 2001; 276(47): 43570 - 43579. [Abstract] [Full Text] [PDF]
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