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Experimental Therapeutics |
Department of Pediatrics and the Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, Maryland 21201 [L. F., A. E. E.]; Department of Pediatrics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642 [J. I., K. K. F.]; Pediatric Oncology Branch, National Cancer Institute, NIH, Gaithersburg, Maryland 20877 [S. Z.]; Molecular Carcinogenesis, Cancer Research Center of Hawaii and the Kapi’olani Health Research Institute, Honolulu, Hawaii 96813 [R. K. W.]; Department of Biochemistry and Molecular Biology, The George Washington University, Washington, DC 20037 [P. E. B.]; Division of Oncology, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania 19104 [N. I.]; Department of Biology, Wheaton College, Wheaton, Illinois 60187 [R. H. K.]; and Department of Medicine, Children’s Hospital and Department of Pediatric Oncology, Dana Farber Cancer Institute, Boston, Massachusetts 02115 [C. N. F.]
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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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Iron may play an unusually important role in the viability and proliferation of neuroblastoma. Iron is required by nearly all organisms, and it facilitates critical cellular biochemical processes including electron transfer and respiration reactions in mitochondria, conversion of ribonucleotides to deoxyribonucleotides by RR3 in DNA synthesis, O2 transfer by hemoglobin, and the activities of many other metalloenzymes. Inhibition of RR has been proposed as the cause of the growth arrest that occurs in cells deprived of iron (8, 9, 10, 11, 12) , because this enzyme requires iron to reduce ribonucleotides to form the substrates for DNA synthesis. The regulated uptake and availability of iron is closely tied to cellular proliferation. Three proteins, the iron-transport protein transferrin, the transferrin receptor on the cell surface, and ferritin, the intracellular iron storage protein, are essential for making iron available for cellular use and for detoxifying excess iron. Many neuroblastomas produce very large amounts of ferritin (13) . In addition to being incorporated into heme proteins and enzymes, iron may participate more directly in the regulation of cell growth and apoptosis of neuroblastoma. Neuroblastoma cells appear to be unusually sensitive to the cytotoxic and cytostatic effects of iron chelators (14) . The use of DFO, a compound that chelates iron, to treat neuroblastoma in children is being investigated in national clinical studies.
In light of the clinical importance of N-myc overexpression in neuroblastoma malignancy, we report here a correlation between DFO-induced apoptosis and decreased N-myc expression in neuroblastoma cell lines with an amplified N-myc oncogene. Different caspase activity in these cell lines does not seem to result directly from a similar decrease in N-myc cellular protein, suggesting that the decrease in N-myc expression is unlikely to directly mediate apoptosis. Remarkably, the DFO-induced decrease in N-myc protein is not mediated through inhibition of RR. We also show that iron chelation by DFO, although decreasing N-myc transcription, alters transcription of some other oncogenes, including c-fos and c-myb, but not housekeeping genes. The specific inhibitory effects of iron chelation by DFO were characterized further on N-myc transcription initiation and on the transient expression of N-myc promoter activity.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cells and Cell Culture.
The LA-N-1 and LA-N-5 neuroblastoma cell lines were originally obtained
from Dr. Robert C. Seeger, University of California at Los Angeles
School of Medicine, Los Angeles, CA. The IMR-32 neuroblastoma cell line
was purchased from American Type Culture Collection (Rockville, MD).
KAG and KANR were obtained from Dr. Patrick Reynolds (Children’s
Hospital of Los Angeles, Los Angeles, CA) and NGP from Dr. G. M.
Brodeur (Children’s Hospital of Philadelphia, Philadelphia, PA). Cells
were maintained in RPMI 1640 supplemented with 10% FBS (HyClone,
Logan, UT), 10 mM L-glutamine, and 50 units/ml
penicillin/50 µg/ml streptomycin. All cell lines were routinely
cultured at 37°C in a humidified atmosphere of 5%
CO2 in air. All experiments were performed on
LA-N-1, unless otherwise specified.
Plasmids.
An N-myc promoter deletion-CAT reporter
construct, N-myc 3.0 CAT containing the
N-myc sequence from -1887 to +1058, was used as described
(15)
.
TUNEL Assay.
The ApopTag Direct In Situ Apoptosis Detection
Kit-Fluorescence (Oncor, Gaithersburg, MD) was used for detection of
DNA fragmentation in apoptotic cells by direct end-labeling of cellular
genomic DNA with a fluorescein-conjugated nucleotide using terminal
deoxynucleotidyltransferase enzyme. LA-N-1 cells, both floating
and in monolayer (detached by trypsinization), were pooled, washed in
PBS, and fixed in 4% formaldehyde in PBS for 15 min at room
temperature. Fixed cells were stored dry on aminoalkylsilane-coated
slides (Sigma, St. Louis, MO) at -20°C before processing according
to the manufacturer’s specifications. Propidium iodide dissolved in
antifade solution (2% n-propyl gallate in 70% glycerol; Oncor,
Gaithersburg, MD) was used for counterstaining. After mounting,
apoptotic cells that fluoresce bright green were visualized at an
excitation wavelength of 494 nm and an emission wavelength of 523 nm
under a fluorescence microscope. The apoptotic index was determined by
counting the number of bright green fluorescent cells among a total of
500 or more red or bright green fluorescent cells.
DNA Laddering.
LA-N-1 cells cultured with 50 µM DFO or water for 96 h in RPMI 1640 + 3% FBS were scraped and lysed in buffer
containing 10 mM Tris-Cl (pH 8.0), 0.1 M EDTA
(pH 8.0), 0.5% SDS, and 20 µg/ml RNase (DNase-free) at 37°C for
1 h. Cell lysates were treated with proteinase K at 100 µg/ml at
50°C overnight. Cellular genomic DNA was isolated with an equal
volume of neutral phenol and chloroform:isoamyl alcohol and
precipitated with 0.1 volume of 3 M sodium acetate (pH 5.2)
and 2 volumes of 100% ethanol at -20°C. DNA was dissolved in 10
µM Tris (pH 8.0) and 1 µM EDTA (pH 8.0)
buffer. DNA (
10 µg) was resolved by electrophoresis in 1% agarose
gel and stained in 5 µg/ml ethidium bromide.
Caspase Activity.
The CaspaTag Fluorescein Caspase Activity Assay (Intergen, Purchase,
NY) was used to detect DFO-induced activity of activated caspases in
neuroblastoma cells per the manufacturer’s instructions. The
FAM-VAD-FMK reagent provided in the kit is a carboxyfluorescein
(FAM) derivative of benzyloxycarbonyl valyl alanyl aspartic acid
fluoromethyl ketone (zVAD-FMK), which is a potent inhibitor of caspase
activity. It enters the cell and irreversibly binds to activated
caspases (caspases 1, 2, 3, 4, 5, 6, 7, 8, and 9) allowing for direct
detection of pan-caspase activity by green fluorescence in live cells.
Both floating cells and those in monolayer (detached by trypsinization)
were pooled and labeled in the dark with 1x FAM-VAD-FMK in the medium
for 1 h at 37°C. Cells containing the bound and labeled
inhibitor were washed and analyzed by single color flow cytometry at
488 nm for fluorescein on the FL1 channel after vortexing. Gating was
used for LA-N-5 to exclude clumped cells.
Western Blotting.
LA-N-1 cells cultured in complete RPMI 1640 with or without 50
µM DFO for 24 h were detached with EDTA + 0.05% trypsin and lysed in PBS containing NP40, phenylmethylsulfonyl
fluoride, aprotinin, and DNase. The Bio-Rad (Hercules, CA) Bradford
method was used to determine protein content in the cell lysates.
Western blotting was performed loading equal amounts of protein/lane as
described previously (14)
. The NCM II-100 monoclonal
antibody specific for N-myc was described previously (16)
.
Northern Blotting.
Cellular mRNA was prepared, electrophoresed, and blotted on Nytran
membrane as described by the manufacturer (Schleicher & Schuell,
Keene, NH). A panel of cDNA probes were used for detection of
N-myc exon 2 (EcoRI-BamHI fragment),
transferrin receptor (HindIII-HindIII fragment),
glyceraldehyde phosphate dehydrogenase exons 8 and 9
(EcoRI-HindIII fragment;
GAPDH), c-fos (0.6-kb fragment of exon 4
obtained by PCR), c-myb (EcoRI-EcoRI
cDNA fragment), or c-jun (1.9-kb cDNA fragment).
Nuclear Run-on.
Nuclei were harvested for nuclear run-on assay as described
(17)
with the modification that, to minimize loss of
weakly attached cells, monolayers were scraped into PBS without
washing. Labeled RNA was purified after incubation of the nuclei with
[32P]UTP. The targeted sequences, exon 1, exon
2, or exon 3 of N-myc as designated, were cloned into M13 mp
10 or mp 11 in each orientation to yield single-stranded targets for
detection of either sense or antisense transcription (a gift of
Geoffrey Krystal, Medical College of Virginia, Richmond, VA;
Ref. 17
). Double-stranded probes for human
ß-actin were used to normalize for extent of
hybridization. Single-stranded targets and double stranded targets were
dotted onto nitrocellulose using a Bio-Rad filter manifold and baked at
80°C for 2 h under vacuum. Prehybridization and hybridization
were carried out as described (18
, 19)
.
Transient Transfection.
Twenty million LA-N-1 cells were transfected by electroporation in 250
µl RPMI 1640 with 20% FBS and other medium supplements in 0.4-cm
cuvettes at 960 µF capacitance and 250 mV. After culturing for
48 h in RPMI 1640 + 10% FBS with or without 50 µM
DFO, cells were harvested in 1x Reporter Lysis Buffer (Promega,
Madison, WI). Lysate protein was assayed using bicinchoninic acid
(Pierce, Rockford, IL). CAT and ß-galactosidase activities were
assayed, respectively, by TLC using the CAT Enzyme Assay System and on
96-well plates using the ß-galactosidase Enzyme Assay System from
Promega (Madison, WI).
Half-life of mRNA and Protein of N-myc.
To determine N-myc protein half-life, cultures were incubated with and
without 50 µM DFO for 24 h. Cycloheximide
was added at 20 µM, duplicate cultures were
lysed at timed intervals, and N-myc was assayed by Western blotting as
described above. Cellular N-myc mRNA was assayed by Northern
blotting at timed intervals after the addition of actinomycin D at 5
µg/ml.
Statistical Analysis.
Differences between means of triplicates were evaluated by the
one-tailed Student’s t test and were considered significant
at P < 0.05.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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DFO Induces Apoptosis in Cultured Human Neuroblastoma Cells.
TUNEL assay was performed on LA-N-1 cells cultured with or without 50
µM DFO as described in "Materials and Methods." The
time course of appearance of apoptotic cells is shown in Fig. 1A
. Few apoptotic cells were observed 24 h after DFO
exposure. However, the number of apoptotic cells/culture started to
increase after 48 h of DFO exposure. It continued to rise
progressively until eventually >80% of cells were apoptotic at
96 h. In Fig. 1B
, laddering of cellular genomic DNA
from LA-N-1 cells treated with 50 µM DFO for
96 h confirmed that DFO induces apoptosis when compared with the
control. In addition, the total number of cells/culture decreased
between 24 and 48 h of DFO (not shown), confirming the cytotoxic
effects of DFO on neuroblastoma cells. In apoptosis, caspases are
responsible for proteolytic cleavages that lead to cell disassembly and
are involved in upstream regulatory events. Pan-caspase activity assay
was performed on IMR-32, LA-N-1, LA-N-5, and KAG cells cultured in the
presence of 50 µM DFO with or without 50
µM FeSO4 for 48 h as
described in "Materials and Methods." The percentage of cells with
pan-caspase activity was significantly higher in DFO-treated cultures
of LA-N-1, LA-N-5, or IMR-32, respectively, than in that of the
controls. Although no pan-caspase activity was detectable in either
treated or control cultures of KAG at 48 h (Fig. 1C)
, a
significant increase in the percentage of pan-caspase positive cells
(40%) was observed after DFO treatment for 96 h
(6.6% ± 0.3%) when compared with the control (4.7% ± 0.4%; P < 0.005). These consistent
and reproducible results suggest that, albeit with different degrees of
sensitivity, DFO induces apoptosis in multiple neuroblastoma cell
lines.
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. The cellular N-myc protein level started to decrease by
12 h of exposure to 50 µM DFO and
continued to decrease for 2 days until it was barely detectable. In
cells cultured without DFO, the level of N-myc expression remained
approximately constant, although a slight increase was seen after 3
days in some experiments. Increasing DFO concentration resulted in
proportionate decreases in cellular N-myc protein in cells cultured for
24 h (Fig. 2B)
. Other compounds that chelate iron well,
but not those that chelate iron weakly (EDTA and
diethylenetriaminepentaacetic acid), also decreased N-myc in a time-
and concentration-dependent
manner.4
The addition of ferrous sulfate to the medium at an equimolar
concentration prevented the decrease in N-myc induced by 50
µM DFO, but the addition of equimolar
Zn+2, Cu+2,
Ca+2, or Mg+2 did not (Fig. 2C)
, suggesting that chelation of Fe+2
is specifically involved in the decreased N-myc expression.
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but caused complete growth
arrest (data not shown). Therefore, the decrease in N-myc caused by
iron deprivation is not mediated by inhibition of RR. Another possible
mechanism of the regulation of N-myc by iron is by growth
arrest. Like hydroxyurea, aphidicolin induces growth arrest of cells,
in this case by inhibition of DNA polymerase 
(21)
. The
addition of aphidicolin to LA-N-1 cells completely inhibited cell
growth (data not shown) but had no effect on N-myc levels (Fig. 2D)
. Similarly, the arrest of neuroblastoma cell growth
caused by deprivation of an amino acid, isoleucine (22)
,
does not affect N-myc expression (data not shown). Thus, the mechanism
of N-myc regulation by iron chelation is not simply
attributable to the arrest of cell growth or the decrease in RR
activity.
DFO Decreases N-myc Cellular Protein in Multiple Neuroblastoma Cell
Lines.
We studied N-myc expression by Western blot in five other neuroblastoma
cell lines with amplified N-myc and found that there is a
common sensitivity to DFO among them. In all these neuroblastoma cell
lines, LA-N-5, IMR-32, KAG, KANR, and NGP, N-myc protein levels
decreased after 24 h exposure to 50 µM DFO
when compared with the control, although the degree of inhibition
varied among the cell lines (Fig. 3)
. The amount of residual cellular protein was similar in IMR-32 and
LA-N-5, although much higher pan-caspase activity was induced in IMR-32
than in LA-N-5 by the same concentration of DFO.
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without affecting the mRNA level of the housekeeping gene,
GAPDH. The decrease was 6-fold when measured by
densitometry after 24 h of incubation with DFO (not shown), and
the rate of decrease was similar to that seen with N-myc protein (Fig. 2A)
. Northern blot also showed that iron chelation resulted
in an increase in transferrin receptor mRNA (Fig. 4A)
,
demonstrating that DFO was effectively decreasing the intracellular
iron available to regulate biochemical processes in the cells
(23)
. We also examined cellular mRNA levels of
c-myb, c-fos, and c-jun. Although no
change was seen in c-myb and c-jun, DFO induced a
marked increase in c-fos (Fig. 4B)
, 7-fold as
measured by densitometry (not shown). These changes were prevented by
coincubation of cells with a concentration of ferrous sulfate equimolar
to that of DFO (Fig. 4B)
. The increase in c-fos
and the lack of change in c-jun contrasts with the
decrease in N-myc mRNA, demonstrating the specificity of the
changes. Similar changes in proto-oncogene mRNA levels were seen in
three other N-myc-amplified human neuroblastoma cell lines
incubated with DFO, except that c-myb mRNA decreased in one
cell line (data not shown).
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. The concentration of the N-myc mRNA was measured similarly
at timed intervals after the addition of actinomycin D at 5 µg/ml
(25)
. The half-life of the N-myc mRNA was found
to be 57 min with or 59 min without 50 µM DFO
(Table 1)
. The half-life for N-myc protein in the control was in the
same range, though four times longer for N-myc mRNA than
those reported in other neuroblastoma cell lines (24
, 25)
.
Thus, iron chelation has little effect on the stability of
N-myc protein or on mRNA in LA-N-1 cells.
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. Hybridization to each exon was reduced to a similar extent (Fig. 5)
.
Therefore, incubation with DFO decreased initiation of N-myc
transcription rather than elongation of initiated N-myc
transcripts. To determine whether iron chelation had generalized
effects on transcription, we examined the effect of DFO on actin
transcription and saw little or no effect (Fig. 5)
. Also, a great deal
of N-myc antisense transcription occurs, which has the
potential to affect N-myc mRNA stability or translation
(28)
. DFO also decreased antisense transcription (Fig. 5)
.
Therefore, antisense transcripts are unlikely to play a role in the
changes in N-myc as described. Thus, the main effect of DFO
on N-myc is to block the initiation of transcription.
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. Thus, DFO decreases N-myc promoter activity.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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N-myc mRNA and protein have very short half-lives (24
, 25)
and constitutive transcription and translation of the
N-myc oncogene is required to maintain its over-expressed
level in neuroblastoma cells. Inhibition of N-myc promoter
activity by 60% (Fig. 6)
may lead to a dramatic decrease of
N-myc mRNA and protein levels over time (Figs. 2A
and 4A)
. Our results suggest that DFO is acting by chelating
iron because it results in increased cellular transferrin receptor
mRNA, which is consistent with a decreased availability of cellular
iron (23)
. Furthermore, the addition of iron, but not
other metals, to the culture prevents the effects of DFO on the
inhibition of N-myc expression (Fig. 2D)
as well
as on the inhibition of cell growth and the induction of apoptosis
(Fig. 1C)
.
Deregulation of N-myc expression, either up- or
down-regulated, has been associated with drug-induced apoptosis in
neuroblastoma (29
, 30)
, and three target genes of the
closely related transcription factor c-myc
(31, 32, 33)
have been demonstrated to directly mediate the
apoptotic effect of c-myc up-regulation. However, the
similar degrees of decrease in N-myc expression by the
9-cis or all-trans isomers of retinoic acid
(34)
do not appear to be directly linked to their
differential effects on apoptosis in neuroblastoma cells
(35)
. Our results showed that DFO-induced apoptosis is
preceded by a decrease in the endogenous expression of
N-myc. However, despite this close correlation, DFO-induced
apoptosis may not result directly from the decrease in N-myc
expression, because similar decreased levels of endogenous
N-myc expression in IMR-32 and LA-N-5 neuroblastoma cell
lines (Fig. 3)
do not seem to correspond with their differential
degrees of DFO-induced apoptosis measured by pan-caspase activity (Fig. 1C)
.
We demonstrated here that DFO decreases N-myc
expression specifically by inhibiting transcription initiation (Fig. 5)
and by inhibiting N-myc promoter activity (Fig. 6)
. Although
iron has been demonstrated to affect gene expression directly in
several ways, none of these direct mechanisms appears to mediate the
alterations in N-myc expression. First, DFO is known to
affect iron dependent regulation of transferrin receptor and ferritin
expression, which occur posttranscriptionally via iron regulatory
elements in the untranslated regions of their mRNAs
(36)
. Whereas the increase in transferrin receptor and the
decrease in N-myc messages occurred with similar time
courses (Fig. 4A)
and DFO concentration (data not shown),
the inhibitory effect of DFO on N-myc expression is not
mediated directly by inhibiting translation initiation or by
destabilizing the N-myc mRNA because DFO had no effect on
the half-lives of N-myc protein or message (Table 1)
, and
the conserved sequence of the iron regulatory elements
(36)
is not present in 5' or 3' untranslated region of the
N-myc mRNA (37
, 38)
. Second, the
iron-containing heme molecule or the heme-hemopexin complex enhances
transcription of heme oxygenase-1 (reviewed in Ref.
39
) or the MT-1 gene (40)
by
binding to the promoter regions at a heme-responsive element
(39)
or to the heme-hemopexin complex-related binding
sites. With these minimum elements (39)
absent in the
N-myc promoter or the known N-myc genomic
sequence (37
, 38)
, the inhibitory effect of DFO on
N-myc transcription is unlikely to be mediated directly by
the effects on iron in heme or heme-hemopexin interaction with the
N-myc promoter. Instead, iron chelation may affect
N-myc transcription initiation or promoter activity
indirectly via the effects on iron or heme-dependent gene expression or
through other effects of iron chelation.
The cellular effects of iron chelation with DFO have been attributed extensively to the inhibition of RR (8, 9, 10, 11, 12) . However, the decrease in N-myc expression by DFO is not mediated through the inhibition of RR because hydroxyurea, a specific inhibitor of this enzyme (20) , does not decrease N-myc expression in our study. Mammalian RR catalyzes the reduction of ribonucleotides to their corresponding deoxyribonucleotides. Its smaller (R2) subunit contains a binuclear ferric iron center and a tyrosyl-free radical. The latter is stabilized by the iron center and is essential for enzymatic activity (8) . DFO chelates the intracellular iron pool (8) , whereas hydroxyurea reduces the RR iron center, inducing iron loss, and scavenges the tyrosyl radical of active R2 protein (41) . As a result, both DFO and hydroxyurea prevent formation of the iron-radical center in newly synthesized apo-R2 protein, and hydroxyurea removes iron from and inactivates active R2 protein (8 , 41) . In addition, it is not known whether the effects of DFO on the inhibition of DNA synthesis and the induction of apoptosis are mediated by the inhibition of RR. Both DFO and hydroxyurea lead to the inhibition of DNA synthesis and cell growth as well as to the induction of apoptosis in mammalian cells (42 , 43) . They both inhibit cell growth of cultured neuroblastoma cell lines.4 DFO arrests cultured neuroblastoma cell lines, in a cell line-dependent fashion, predominantly in either G1 or S phase, whereas hydroxyurea consistently arrests them in S phase,4 suggesting that DFO may have effects on cell growth in addition to its inhibition of RR. Because chelation of the intracellular iron pool by DFO may disrupt iron-dependent processes other than RR, iron chelation may lead to apoptosis by routes independent of the inhibition of RR in neuroblastoma cells.
We demonstrated that DFO treatment markedly and specifically increased
the mRNA level of c-fos, but not c-jun, in LA-N-1
neuroblastoma cells (Fig. 4B)
. DFO treatment was also shown
by others to enhance c-fos expression in another
neuroblastoma cell line, IMR-32 (44)
. In some cell types,
Fos/Jun (AP-1) transcription factor complex may function to modulate
stress-induced apoptosis (45)
. The effects of DFO on AP-1
functional activity as well as the significance of c-fos
expression on N-myc expression and apoptosis are unknown.
Also, iron chelation has been reported by others to lead to an increase
in p53 expression in a human leukemic cell line (46)
, and
the p53 tumor suppressor protein may induce apoptosis via a complex
network of interacting pathways. In human primary neuroblastomas, p53
is usually wild-type (47)
, and its signal transduction
pathway is intact (48)
; but in some tumors it may be
aberrantly translocated to the cytoplasm, thus compromising its
suppressor function (49)
. Currently, the status of p53 in
DFO-treated LA-N-1 neuroblastoma cells and its relationship to
DFO-induced apoptosis is not known.
N-myc amplification results in overexpression of N-myc in human neuroblastoma. Whereas the extent of expression of the amplified N-myc genes is quite variable, DFO decreases N-myc expression in all cell lines that we examined. N-myc amplification may be responsible for treatment failure in those neuroblastoma patients receiving cisplatin or VP-16 (50) . Given the clinical importance of N-myc expression, combined use of these drugs and an iron chelator to decrease N-myc expression might be clinically useful.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 We acknowledge the support of the American
Institute for Cancer Research (to L. F.), the Children’s Cancer
Foundation (to C. N. F.), and NIH Grant NS34432 (to R. K. W.).
Shaoxian Zhu was a fellow of the World Health Organization. 
2 To whom requests for reprints should be
addressed, at Department of Pediatrics, University of Maryland School
of Medicine, Room 10-044A, BRB, 655 W. Baltimore Street, Baltimore, MD
21201.
3 The abbreviations used are: RR, ribonucleotide
reductase; DFO, deferoxamine mesylate; FBS, fetal bovine serum; CAT,
chloramphenicol acetyltransferase; TUNEL, terminal deoxynucleotidyl
transferase-mediated nick end labeling; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase.
4 C. N. Frantz, unpublished data.
Received 5/24/99. Accepted 11/20/00.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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.. Nature (Lond.), 275: 458-460, 1978.[Medline]
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, control with water;
, with 50 µM DFO. Bar, SE.
B, DNA fragmentation by DNA laddering. Cells were
cultured with 50 µM DFO or water in RPMI 1640 + 3% FBS for 96 h. DNA was isolated separately from three cultures
and dissolved in 10 µM Tris (pH 8.0) and 1
µM EDTA (pH 8.0) buffer. DNA (
