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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 4 (August 15), 1999:
pp. 1393-1400
Expression of Chromosome 21-Localized Genes in Acute Myeloid Leukemia:
Differences Between Down Syndrome and Non-Down Syndrome Blast Cells and
Relationship to In Vitro Sensitivity to Cytosine Arabinoside and
Daunorubicin
By
Jeffrey W. Taub,
Xi Huang,
Larry H. Matherly,
Mark L. Stout,
Steven A. Buck,
Gita V. Massey,
David L. Becton,
Myron N. Chang,
Howard
J. Weinstein, and
Yaddanapudi Ravindranath
From the Division of Pediatric Hematology/Oncology, Children's
Hospital of Michigan, Detroit, MI; the Experimental and Clinical
Therapeutics Program, Barbara Ann Karmanos Cancer Institute,
Departments of Pediatrics and Pharmacology, Wayne State University
School of Medicine, Detroit, MI; the Medical College of Virginia,
Richmond, VA; the University of Arkansas, Little Rock, AR; the
University of Florida, Gainesville, FL; the Department of Pediatrics,
Massachusetts General Hospital, Boston, MA; and the Pediatric Oncology
Group, Chicago, IL.
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ABSTRACT |
The high event-free survival rates of Down syndrome (DS) children
with acute myeloid leukemia (AML) are due, in part, to increased in
vitro sensitivity of DS myeloblasts to cytosine arabinoside (ara-C) and
daunorubicin and the greater generation of ara-C triphosphate (ara-CTP)
from ara-C compared with myeloblasts from non-DS patients (Taub et al,
Blood 87:3395, 1996). This study further explores the molecular
basis of chemotherapy sensitivity of DS AML patients by examining the
expression of chromosome 21-localized genes in myeloblasts from newly
diagnosed AML patients. Transcript levels of two chromosome
21-localized genes, cystathionine- -synthase (CBS) and superoxide
dismutase (SOD), measured by quantitative reverse
transcriptase-polymerase chain reaction (RT-PCR), were 12.0- and 3.8-fold higher in DS compared with non-DS myeloblasts (P < .0001 and P < .0001, respectively).
Conversely, there were no significant increases in transcripts for 2 other chromosome 21-localized genes, carbonyl reductase and the reduced
folate carrier. CBS transcript levels correlated with both in vitro
ara-C sensitivity measured by the
3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium-bromide (MTT)
assay (P = .003) and the generation of 3H-ara-C
triphosphate (ara-CTP) after in vitro incubations with 5 µmol/L
3H-ara-C (P = .0003). Transcripts of
deoxycytidine kinase were 2.6-fold higher in DS compared with non-DS
cells and may be a factor in the enhanced metabolism of ara-C in DS
cells. There was no significant correlation of SOD transcripts with in
vitro ara-C and daunorubicin sensitivities. Increased CBS transcripts could result in elevated CBS activity, which modulates ara-C metabolism by altering reduced folate pools, deoxycytidine triphosphate pools, S-adenosylmethionine levels, and/or methylation of the deoxycytidine kinase gene. The further identification of the molecular mechanisms of
chemotherapy sensitivity of DS AML patients may lead to significant improvements in the treatment and cure of AML.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
DESPITE SIGNIFICANT progress in the
treatment of many childhood cancers, only modest progress has been made
in the treatment of acute myeloid leukemia (AML), the second most
common form of childhood leukemia. Childhood AML has the worst
prognosis of all major childhood cancers, with 5-year relative survival rates of approximately 37%.1 This contrasts with the
approximate 80% survival rate for acute lymphoblastic leukemia (ALL),
the most common form of childhood cancer.
Although recent approaches have improved the overall outcome of
treatment and prognosis for AML, they have not significantly impacted
the outcome for the majority of patients.2,3 For instance,
allogeneic bone marrow transplantation for AML in first remission has
been more effective than chemotherapy but is limited in application due
to the lack of suitable transplant donors in the majority of children.
Autologous bone marrow transplantation in first remission does not
appear to be clearly superior to intensive chemotherapy.4
Salvage chemotherapy for relapsed/refractory AML has also not been very
effective.5
Cytogenetic abnormalities in AML have been identified as one of the
most important prognostic factors that predict treatment response in
both children and adults. Favorable cytogenetic abnormalities include
inv(16), t(8;21), and t(15;17), all of which are associated with higher
remission induction rates and event-free survival (EFS)
rates of greater than 40%.6,7
A recent unexpected finding is that Down syndrome (DS; trisomy 21)
children with AML have the highest EFS rates of any subgroup of AML
patients, with EFS rates of 68% to 100% when patients are treated
with current intensive protocols.8-13 These studies
reported that DS patients had leukemia relapse rates of less than 20%, indicating that significant leukemia cell kill occurs before the development of drug resistance. It was demonstrated by several groups
(Pediatric Oncology Group, Children's Cancer Group, Nordic Society of
Pediatric Hematology Oncology, and Berlin-Frankfurt-Münster) that
the improved response rate to therapy for DS children with AML
coincided with the use of high-dose cytosine arabinoside (ara-C) for
intensification,8,9,10,13 whereas Japanese and Canadian studies have used lower-dose ara-C successfully.11,12
In initial laboratory studies to investigate the basis for these
compelling clinical findings, we reported that myeloblasts from newly
diagnosed DS patients with AML were significantly more sensitive in
vitro to both ara-C and daunorubicin compared with myeloblasts from
non-DS patients.14 Furthermore, DS myeloblasts generated
significantly higher levels of the active intracellular ara-C
metabolite, ara-C triphosphate (ara-CTP), after in vitro incubations
with ara-C.14
The enhanced chemotherapy sensitivity of DS myeloblasts may reflect the
altered expression of chromosome 21-localized genes in DS cells. We
previously hypothesized that alterations in expression of at least two
such genes, cystathionine--synthase (CBS; 21q22.3)15 and
zinc/copper superoxide dismutase (SOD; 21q22.1),16 may
directly or indirectly contribute to the increased sensitivity of DS
cells to ara-C and daunorubicin.5,14 In this study, we
further explore the molecular basis of chemotherapy sensitivity of DS
AML patients by examining the expression of these key chromosome
21-localized genes in DS and non-DS myeloblasts.
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MATERIALS AND METHODS |
Patient specimens.
Myeloblasts were obtained from (1) newly diagnosed pediatric AML
patients registered for treatment on the Pediatric Oncology Group (POG)
9421 chemotherapy protocol (n = 53) before the initiation of therapy,
(2) AML patients treated at Children's Hospital of Michigan (CHM) from
1988 to 1998 (n = 7), and (3) newborn DS babies with the transient
myeloproliferative disorder (TMD; n = 3). The mononuclear cells were
isolated on a Ficoll-Hypaque gradient to obtain a highly purified
mononuclear cell fraction consisting of mostly leukemic blasts. The
majority of the specimens were obtained as bone marrow aspirates,
although peripheral blood was used when the peripheral white blood cell
(WBC) counts were sufficiently high (>50,000/µL). The
AML samples received from member POG institutions were collected in
sodium heparin diluted with equal amounts of RPMI, shipped overnight at
room temperature, and purified at CHM upon receipt; the samples were
then frozen in 20% fetal calf serum (FCS)/10% dimethyl sulfoxide
(DMSO) at  195°C until further analysis. The
leukemia samples were classified according to French-American-British (FAB) classification system as previously reported.8 The
diagnosis of megakaryocytic leukemia (M7) was based on the expression
of platelet-specific antigens recognized by the monoclonal antibodies CD61, CD41, and CD42. The protocol was approved by the hospital institutional review board.
Chemicals.
[5-3H]-Cytosine--D-arabinofuranoside (25 Ci/mmol) was
obtained from Moravek Biochemicals (Brea, CA). Unlabeled cytosine
arabinoside, daunorubicin, and
3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium-bromide (MTT)
were obtained from Sigma Chemical Co (St Louis, MO). Tissue culture
reagents and supplies were purchased from assorted vendors. Polymerase
chain reaction (PCR) primers were purchased from Genosys Biotechnologies, Inc (The Woodlands, TX).
Cell culture.
The CCRF-CEM and K562 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 U/mL
penicillin, and 100 µg/mL streptomycin in a humidified atmosphere at
37°C in the presence of 5% CO2/95% air.
Reverse transcriptase-PCR (RT-PCR) analysis of
transcripts of chromosome 21-localized genes.
Total RNA was isolated from 5 to 10 × 106
patient myeloblasts or cultured cells by the phenol/chloroform
extraction method of Chomczynski and Sacchi17 using Tri
Reagent (Molecular Research Center, Cincinnati, OH). CBS, SOD, carbonyl
reductase (CBR; localized to 21q22.1),18 reduced folate
carrier (RFC; localized to 21q22.3),19 and deoxycytidine
kinase (dCk; localized to chromosome 4)20 transcripts were
assayed from the same cDNA mixtures using the CBS-1/CBS-2, SOD-1/SOD-2,
CBR-1/CBR-2, RFC-1/RFC-2, and dCk-1/dCk-2 oligonucleotide primer
pairs (Table 1).
For the analysis of CBS expression, first-strand cDNAs were synthesized
from 2 µg of total RNA in 20 µL of a reaction mix containing 2.5 µmol/L of random hexadeoxynucleotide primers, 10 mmol/L Tris-HCl (pH
8.3), 50 mmol/L KCl, 5 mmol/L MgCl2, 0.5 mmol/L each dNTP,
and 50 U of Moloney murine leukemia virus (MuLV) reverse transcriptase
(Perkin Elmer, Foster City, CA). After 1 hour of incubation at
42°C, the reaction was stopped by heating at 99°C for 5 minutes. Aliquots of the cDNAs were amplified for PCR in a reaction
mixture consisting of 1 µL of reverse transcription cDNA product,
10× PCR buffer (Perkin Elmer), 1.5 mmol/L MgCl2, 0.2 mmol/L of each dNTP, 0.2 µmol/L of each CBS primer, and 1.25 U Taq
DNA polymerase (Promega, Madison, WI). PCR was performed for 28 cycles
of 1 minute at 94°C (denaturation), 1 minute at 70°C
(annealing), and 1 minute at 72°C (extension) in a Perkin Elmer
GeneAmp PCR System 9600 using the CBS-1/CBS-2 primers (Table 1).
Aliquots of the PCR reaction (10 µL) were analyzed on agarose gels
(1.0%) that were stained with SYBR green I (FMC Bioproducts, Rockland,
ME) for 1 hour. The fluorescent PCR products were quantitated with a
Molecular Dynamics Storm 860 fluorescence and radioactivity imaging
system and Image Quant software (Molecular Dynamics, Sunnyvale, CA). Levels of CBS transcripts were calculated from the
intensities of the fluorescent PCR products and were
normalized to the levels of 18S ribosomal RNA (localized to
chromosome 13; Ambion Inc, Austin, TX).
PCR amplifications for the SOD, CBR, and RFC genes were for 30 cycles
of 1 minute at 94°C, 1 minute at 60°C (SOD and RFC) or 66°C
(CBR), and 1 minute at 72°C, and the PCR products were analyzed as
for the CBS gene. PCR amplification of the dCk gene was for 28 cycles
for 1 minute at 94°C, 1 minute at 60°C, and 1 minute at
72°C. For all primer sets, the linear range of exponential amplification was determined by preliminary reactions using dilutions of a series of cDNAs and a range of PCR cycles. Controls for the PCR
reactions to detect CBS, SOD, and CBR transcripts included the K562
(positive) and CCRF-CEM (negative) leukemia cell lines.
PCR analysis of genomic DNA.
DNA was isolated from the AML samples using Tri Reagent according to
the manufacturer's guidelines. PCR amplification of the CBS gene was
performed with 40 ng of genomic DNA in a 50 µL reaction volume
containing 10 mmol/L Tris-HCl (pH 8.3), 1.5 mmol/L MgCl2, 50 mmol/L KCl, 5% DMSO, 200 µmol/L of each dNTP, 1.25 U Taq DNA polymerase, and 0.2 µmol/L of each CBS primer. -Actin (localized to chromosome 7)21 was used as an internal control and
amplified with gene-specific primers (0.2 µmol/L each; Table 1) in
the same reaction. PCR amplification was performed for 29 cycles of 1 minute at 95°C, 1 minute at 68°C, and 1 minute at 72°C,
followed by a final extension for 10 minutes using the CBS-3/CBS-4
primers and actin-1/actin-2 primers (Table 1). The PCR products were electrophoresed on a 2% agarose gel, stained with SYBR Green I, and
the gel was analyzed as in the RT-PCR assays with the relative CBS gene
copy number expressed as the ratio of CBS/-actin level.
MTT colorimetric drug sensitivity assay.
The MTT assay was based on our previously reported
method.14 Briefly, leukemia cells (2 × 104) were suspended in triplicate in 80 µL of RPMI
1640/20% dialyzed FCS in the presence of varying concentrations of
ara-C, daunorubicin, or no drug (controls). After 72 hours in a
humidified incubator at 37°C, MTT was added to a final
concentration of 1 mmol/L. After 6 hours, the colored formazan crystals
were dissolved with the addition of 100 µL acidified isopropranol and
the optical densities read by a microplate reader at 540 nm. The
absorbances were corrected for blank readings. Absorbance (A) at 540 nm
directly correlates with cell number; the percentage of cell survival
was calculated as follows: A (treated)/A (untreated control) × 100. The data were plotted as drug concentration versus percentage of
cell survival, and the IC50s were determined by the MINSQ
curve fitting program, (Dynacomp Inc, Livonia, NY),
corresponding to the drug concentration that decreased cell survival by
50%.
Ara-C incubations and measurement of ara-CTP.
Incubation of leukemia cells with ara-C and the measurement of
intracellular ara-CTP levels were performed as previously
described.14 Briefly, cells suspended in Hank's balanced
salt solution were incubated with 5 µmol/L 3H-ara-C (250 µCi/µmol) at 37°C for 3 hours. The incubations were stopped
with the addition of ice-cold phosphate-buffered saline (PBS; with 20 µmol/L dipyridamole to inhibit nucleoside transport). After
additional PBS washes, the cells were aliquotted and processed as
follows: (1) 1 sample was assayed for radioactivity and protein content
(representing total intracellular accumulation of ara-C and
metabolites) and (2) 1 sample was treated with 0.5 N ice-cold perchloric acid and the supernatant (acid-soluble fraction) was neutralized with 5 N KOH and 1 mol/L KH2PO4
before high-performance liquid chromatography (HPLC) analysis.
The neutralized acid soluble fractions were analyzed for ara-C
metabolites by an ion-pairing HPLC method on an Isco liquid chromatograph equipped with a 4-µm C-18 octadecylsilane column with a
C-18 precolumn. The mobile phase consisted of 90 mmol/L potassium
phosphate. The elution positions of the standard unlabeled ara-C
metabolites (ara-C, ara-CMP, and ara-CTP) were monitored at 280 nm and
correlated with those of the radioactive metabolites. The levels of
intracellular metabolites (expressed in picomoles per milligram of
protein) were calculated from the total intracellular radiolabel (in
picomoles per milligram), the fraction comprising the PCA soluble pool,
and the percentage of each compound from chromatographic analysis.
Statistical analysis.
Differences in age, WBC count, ara-C and daunorubicin IC50,
ara-CTP, and transcript levels were compared between the DS and non-DS
patient groups using the nonparametric Mann-Whitney 2-sample test. The
nonparametric Spearman rank correlation coefficient was used to analyze
transcript levels and their relationships to MTT and ara-CTP data.
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RESULTS |
Transcript levels of the chromosome 21-localized genes, CBS and SOD, in
myeloblasts from pediatric patients (DS and TMD [n = 3]; DS and AML
[n = 16]; and non-DS and AML [n = 44]) were assayed by
quantitative RT-PCR (Fig 1). The goal was
to determine whether differences in gene expression exist between DS
and non-DS samples that might contribute to the dramatically increased
EFS rates for DS over non-DS children with AML. Transcript levels were
correlated with (1) ara-CTP levels generated during in vitro
3H-ara-C incubations and (2) in vitro sensitivities to
ara-C and daunorubicin determined by MTT assays. Patient
characteristics and experimental results are summarized in
Tables 2, 3,
and 4.
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| Fig 1.
RT-PCR analysis of chromosome 21-localized transcript
levels in myeloblasts obtained from newly diagnosed patients with AML
or TMD. Lanes DS 1 through 10 and non-DS 1 through 20 correspond to the
patients listed in Tables 1 and 2, respectively. CBS,
cystathionine--synthase localized to 21q22.3 corresponding to a
655-bp product; SOD, superoxide dismutase localized to 21q22.1
corresponding to a 436-bp product. The transcript levels were
normalized to 18S RNA transcript levels.
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CBS mRNA transcripts were detectable in all 19 of the DS TMD/AML
samples and in 29 of 44 (66%) of the non-DS blast cell samples; however, transcript levels in the DS samples were significantly higher
compared with the non-DS samples (median, 0.660 v 0.055, respectively; P < .0001; the 15 non-DS samples in which CBS
transcripts were not detectable were assigned a value of 0.001, which
is the lower limit of sensitivity of our RT-PCR assay;
Fig 2A). In contrast, when CBS gene copy
numbers were measured for a subset of AML DNA samples (9 DS and 19 non-DS samples), gene copies differed by 1.3-fold (P = .0006),
approximating the expected value (1.5-fold) for chromosome 21-localized
genes between DS and non-DS cells.
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| Fig 2.
Transcript levels of chromosome 21-localized genes in
myeloblasts from newly diagnosed AML patients. Median CBS and SOD
transcript levels were 12.0- and 3.8-fold higher, respectively, in DS
myeloblasts compared with non-DS myeloblasts (A and B). Transcript
levels of the dCk gene were 2.6-fold higher in DS compared with non-DS
cells (C). Transcript levels were normalized to 18S RNA transcript
levels. Horizontal bars represent the median values in each group.
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SOD transcripts were detected in all 19 DS and 44 non-DS samples, and,
again, marked differences were observed between the DS and non-DS
samples (median, 2.390 v 0.634, respectively; P < .0001; Fig 2B). Conversely, there were no significant differences between the DS and non-DS samples in transcript levels for other chromosome 21-localized genes, including CBR (DS [n = 19] and non-DS
[n = 44]; median, 0.567 v 0.520, respectively; P = .72) and RFC (DS [n = 10] and non-DS [n = 20]; median, 0.185 v 0.139, respectively; P = .59). Deoxycytidine kinase
transcripts were a median 2.6-fold higher in DS cells (n = 19) compared
with non-DS cells (n = 44; median, 2.477 v 0.943, respectively;
P = .03; Fig 2C).
There was no relationship between CBS transcripts and presenting WBC
count for all of the patients (r = .178, P = .16); there was a significant correlation between SOD transcripts and
WBC count (r = .271, P = .03). There was a
significant correlation between age and transcript levels for the CBS
(r = .513; P < .0001) and SOD (r = .454; P = .0003) genes, but the results may be skewed,
because the DS patients were significantly younger in age compared with
the non-DS patients (1.1 v 9.0 years, respectively; P < .0001).
For a significant number of samples, there were sufficient blasts for
assays of drug sensitivities and ara-C metabolism. DS myeloblasts (n = 14) were appreciably more sensitive than non-DS myeloblasts (n = 44) in
vitro to both ara-C (median IC50, 100.5 v 420.8 nmol/L, respectively; P < .0001) and daunorubicin (median IC50, 6.75 v 107.4 nmol/L, respectively; P < .0001; Fig 3A and B). DS samples (n = 11) also generated significantly higher levels of
3H-ara-CTP than non-DS cells (n = 20) during
3-hour in vitro incubations with 5 µmol/L 3H-ara-C
(median, 735.0 v 141.1 pmol/mg protein, respectively; P < .0001; Fig 3C). This may result from enhanced dCk activity secondary to increased dCk transcripts, as previously
predicted.5,14
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| Fig 3.
In vitro sensitivity and metabolism of ara-C in
myeloblasts from AML patients. DS myeloblasts were significantly more
sensitive to both ara-C (A) and daunorubicin (B) compared with non-DS
myeloblasts (P < .0001 and P < .0001, respectively). 3H-Ara-CTP generation in DS myeloblasts was
significantly higher compared with non-DS myeloblasts after incubation
with 3H-ara-C (P < .0001; C).
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Based on our hypothesis,5,14 increased CBS transcripts may
lead to greater generation of ara-CTP and correlate with ara-C sensitivity; increased SOD transcripts may be associated with increased
in vitro ara-C and daunorubicin sensitivities.
When the correlation between CBS transcript levels and ara-C
sensitivity was evaluated for the DS and non-DS patients separately, the expected relationships were not seen. This may be due to sample size of the group and the fact that no CBS transcripts could be detected in up to one third of non-DS samples. As seen in Fig 2, there
was a striking separation of the CBS transcript levels between DS and
non-DS cases; only 8 of 44 non-DS cases exceeded the lowest transcript
levels seen in the DS group and only 1 exceeded the median value for
the DS cases. Interestingly, when the DS and non-DS groups were
analyzed as a whole, CBS expression closely correlated with both in
vitro ara-C sensitivity (14 DS and 44 non-DS samples; Spearman
correlation coefficient r = .397, P = .003) and
in vitro 3H-ara-CTP generation (11 DS and 20 non-DS
samples; r = .666, P = .0003;
Fig 4A and B), suggesting a causal
relationship. There was no significant correlation between SOD
transcripts and sensitivities to ara-C and daunorubicin for the group
analyzed together (14 DS and 44 non-DS samples; ara-C: r = .236, P = .07; daunorubicin: r = .217,
P = .10; Fig 4C and D). Five DS samples were not included in
the correlation analysis due to lack of MTT data secondary to
insufficient cell numbers.
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| Fig 4.
Correlation between CBS and SOD transcripts measured by
RT-PCR in myeloblasts and in vitro drug activity and sensitivity. CBS
transcripts correlated with both 3H-ara-CTP generation
(Spearman correlation coefficient r = .666, P = .0003) and ara-C sensitivity (r = .397, P = .003) (A and B, respectively). There were no significant correlations
between SOD transcripts and ara-C (r = .236, P = .07) or daunorubicin (r = .217, P = .10) sensitivities (C and D, respectively). Transcript levels were
normalized to 18S RNA transcript levels. ( ) DS; ( ) non-DS.
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DISCUSSION |
The significantly higher EFS rates of DS AML patients compared with
non-DS patients are likely multifactorial and related to specific
genetic alterations present in DS cells. Based on this premise, we
previously speculated that key enzymes encoded by genes localized to
chromosome 21 likely contribute to the sensitivity of myeloblasts to
the chemotherapy drugs currently used in AML therapy, notably ara-C and
daunorubicin.5,14 We previously reported that DS
myeloblasts are more sensitive in vitro to both ara-C and daunorubicin
compared with non-DS myeloblasts and hypothesized that at least 2 chromosome 21-localized genes, CBS and SOD, may be
responsible.5,14
In this study, we found that the median CBS and SOD transcript levels
in DS myeloblasts were 12.0- and 3.8-fold higher, respectively, than
the levels in non-DS myeloblasts. For ara-C, increased drug sensitivity
was associated with dramatically elevated synthesis of ara-CTP as well.
Increased CBS expression and enzyme activity could enhance the
metabolism of ara-C to ara-CTP by 2 mechanisms5,14: (1) via
effects on folate metabolism, decreased generation of deoxythymidine triphosphate, and, ultimately, decreased deoxycytidine triphosphate (dCTP), a feedback inhibitor of dCk required for activation of ara-C;
and (2) via decreased generation of S-adenosylmethionine (AdoMet) and
hypomethylation of the dCk gene.22,23 We previously reported that endogenous dCTP levels were lower in DS
cells.14 In this study, we found that dCk transcript levels
were also higher in DS cells than non-DS cells and likely contribute to
differences in ara-CTP generation. It has been suggested that the in
vitro cytostatic effects of ara-C were reversed by the administration of AdoMet, providing evidence that ara-C activity and AdoMet levels are
linked,24 perhaps at the level of dCk. Most recently, we found that CCRF-CEM leukemia cells transfected with the CBS cDNA are
more sensitive to ara-C due to higher levels of ara-CTP accumulation over wild-type cells.25
Although we did not detect a significant relationship between SOD
expression and sensitivities to either ara-C or daunorubicin, a
relationship may be more apparent with the analysis of a greater number
of samples. Preliminary experiments from our laboratory have shown that
transfection of CCRF-CEM cells with the SOD cDNA results in increased
ara-C sensitivity, although there was no change in daunorubicin
sensitivity compared with wild-type cells (unpublished
data). Our results may not be entirely unexpected either
in that SOD is not directly involved in the metabolism of either ara-C
or daunorubicin. Rather, increased free radical generation may have an
additive effect through lipid peroxidation and damage to cellular
membranes and organelles.26,27 Imbalances of oxygen
radicals secondary to increased SOD activity are known to be involved
in the pathophysiology of DS.28 Increased SOD activity has
been reported to be associated with increased apoptosis in thymic
tissue of transgenic mice with elevated levels of SOD in vivo and DS
fetal neurons in vitro that can be prevented by treatment with
free-radical scavengers.29,30 This suggests that DS cells
may have increased susceptibility to undergo apoptosis after exposure
to apoptosis-inducing agents,31 thereby contributing to the
enhanced sensitivity of DS cells to chemotherapy agents.
Acquired extra copies of chromosome 21 in pediatric B-precursor ALL are
frequently associated with a favorable prognosis.32,33 Although the basis for this effect in ALL is not well established, it
partly reflects increased chromosome numbers, because expression of RFC
(localized to 21q22.3) is elevated in hyperdiploid blasts containing
acquired extra copies of chromosome 21.34 Acquired trisomy
21 was associated with an intermediate prognosis in a study of 1,612 pediatric and adult AML patients; however, in another study, this did
not confer an improved treatment outcome due to the presence of
additional cytogenetic abnormalities.7,35 In our study, 2 non-DS samples had acquired trisomy 21 (NDS 13 and 39, Table 3) and
there was no pattern of increased transcript levels in these samples;
CBS transcripts were undetectable in NDS 13 and approximately 4-fold
higher in NDS 39 compared with the median non-DS value, whereas SOD
transcripts in NDS 39 were below the median SOD value and only 1.1-fold
higher for NDS 13. This suggests that the mere presence of extra
chromosome 21 copies does not account for differences in CBS and SOD
gene expression. Likewise, relative transcripts for CBS and SOD in DS
myeloblasts far exceeded the minimum level in non-DS cells and the
expected 1.5-fold increase from the 3 copies of chromosome 21 in DS
cells. The lack of detectable CBS transcripts by RT-PCR in many of the non-DS AML samples was due to low levels of gene expression, rather than to gene deletions not apparent by karyotype analysis. By contrast,
the median values for CBR and RFC more closely approximated the
expected differences. Selective and disproportionate expression of
chromosome 21-localized genes may reflect the presence of variable deletions in the 21q region that are not detectable by karyotype analysis36 and their effects on gene transcription. Similar findings of a disproportionate expression of chromosome 21-localized genes have been described in DS fetal tissue compared with non-DS tissue,37,38 and a 3.3-fold higher expression of the TIAM
gene was reported in DS leukemia cells compared with a control
group.39
In conclusion, this is the first study to demonstrate increased gene
expression of chromosome 21-localized genes relevant to chemotherapy
response in DS myeloblasts compared with non-DS myeloblasts from newly
diagnosed AML patients. Our results imply that increased gene
expression of chromosome 21-localized genes in DS myeloblasts
contributes to the increased chemotherapy sensitivities of pediatric DS
AML patients. The elevated CBS transcript levels are accompanied by
increased sensitivities of DS myeloblasts to ara-C. The further
identification of the molecular mechanisms of chemotherapy sensitivity
of DS AML patients may lead to new approaches in the treatment and cure
of AML.
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ACKNOWLEDGMENT |
The authors thank Rita Russo and Christine Menig for the patient data
collection and Dr Long Zhang for providing the RFC primers.
| |
FOOTNOTES |
Submitted December 8, 1998; accepted April 13, 1999.
Supported in part by the Leukemia Society of America Research
Translational Grant (6203-98), Children's Cancer Research Fund, Children's Research Center of Michigan, The Litvak Foundation, BenePro
Corp, and Leukemia Research, Life, Inc (Detroit, MI).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Jeffrey W. Taub, MD, Children's Hospital
of Michigan, 3901 Beaubien Blvd, Detroit, MI 48201.
| |
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ABSTRACT
INTRODUCTION