Clinical Cancer Research Vol. 6, 807-812, March 2000
© 2000 American Association for Cancer Research
Suppression of Telomerase Activity and Cytokine Messenger RNA Levels in Acute Myelogenous Leukemia Cells in Vivo in Patients by Amifostine and Interleukin 41
H. D. Preisler2,
B. Li,
J. Yang,
R. W. Huang,
E. Devemy,
P. Venugopal,
M. Tao,
H. Chopra,
S. A. Gregory,
S. Adler,
S. Sivaraman,
P. Toofanfard,
A. Jajeh,
A. Galvez and
E. Robin
Department of Medicine, Division of Bone Marrow Transplantation, Hematology, and Medical Oncology, Rush Cancer Institute, Chicago, Illinois [H. D. P., B. L., J. Y., R. W. H., E. D., P. V., M. T., H. C., S. A. G., S. A., S. S., P. T.]; Cook County Hospital, Chicago, Illinois [A. J.]; Illinois Masonic Medical Center, Chicago, Illinois [A. G.]; and Ingalls Memorial Hospital, Harvey, Illinois [E. R.]
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ABSTRACT
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High
levels of telomerase activity and high rates of cell proliferation are
associated with a poor prognosis in acute myelogenous leukemia.
Furthermore, cytokine production by leukemia cells is believed to play
an important role in determining the proliferative characteristics of
leukemia. The in vivo effects of two noncytotoxic agents
on these parameters were determined in 33 acute myelogenous leukemia
patients. Three daily doses of interleukin (IL) 4 or a single dose of
amifostine reduced telomerase activity in the leukemia marrow cells in
7 of 9 and 11 of 13 patients, respectively. The administration of a
single dose of amifostine resulted in a reduction in tumor necrosis
factor 
and IL-6 transcript levels in the marrow cells of 10 of 13
and 12 of 13 patients in which these transcripts were present. The
administration of only three doses of IL-4 or a single dose of
amifostine has a significant effect on leukemia cell parameters, which
are believed to have a significant impact on the in vivo
biology of the disease and on its response to remission induction
therapy.
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INTRODUCTION
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High proliferative potential and high proliferative rates of
leukemic cells contribute to the rate of progression of leukemia and to
one type of treatment resistance, regrowth resistance (1
, 2) . We have previously reported on studies demonstrating that
the administration of 13-cis-retinoic acid and IFN- can
slow the proliferation of myeloid leukemia cells in vivo in
patients (3
, 4)
. This study reports that both amifostine
and IL3
-4 can
reduce telomerase activity in AML cells in vivo in patients
and that amifostine can also reduce the TNF-, IL-6, and increase
flt3 transcript levels within AML cells. Given that telomerase
activity and cytokine production play a role in determining the
proliferative potential (5
, 6)
and proliferative rate of
AML cells (7)
, the administration of amifostine and/or
IL-4 between courses of chemotherapy has the potential to reduce or
abrogate regrowth resistance as a cause of treatment failure in AML.
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PATIENTS AND METHODS
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Patients.
A single dose of amifostine was administered to 22 patients. IL-4 was
administered to 11 patients daily for 3 days. A diagnosis of AML was
confirmed by standard methods including morphological assessment and
immunophenotyping. Informed consent was obtained from each patient.
Sample Collection.
BM aspirates were obtained from AML patients for study before the
administration of IL-4 or amifostine. Patients then received either one
dose of amifostine (kindly provided by Alza Corp., Palo Alto, CA) as an
i.v. infusion (100 mg/m2) or three doses of IL-4
(kindly provided by the Schering Plow Corp., Kenilworth, NJ; 2–4
mg/kg2 daily x 3). Seventy-two h
after the amifostine infusion or 24 h after the last dose of IL-4,
a second BM aspirate sample was obtained. Part of each BM aspirate was
mixed immediately with a 4 M guanidinium thiocyanate
solution and processed for assessment of cytokine mRNA levels as
described below. Light density mononuclear cells were also obtained for
study by Ficoll-Hypaque density gradient centrifugation (specific
gravity = 1.077).
Telomerase Activity Assay.
Light density mononuclear BM cells obtained by density separation
(1.077) were frozen at -130°C. The frozen cells were thawed
rapidly in a 37°C water bath. After washing with PBS, protein was
extracted for 30 min with cold CHAPS lysis buffer
(106 cells/200 µl; 0.5% CHAPS, 10
mM Tris-HCl, 1 mM MgCl2,
1 mM EGTA, 0.1 mM benzamidine, and 10%
glycerol) and centrifuged at 12,000 x g for 30 min at
4°C. Each extract (2 µl; equivalent to 5,000 cells) was assayed in
a 50-µl reaction
mixture.4
To assay telomerase activity, a semiquantitative approach
established by our laboratory was used.4 Immortal
cells, which have high levels of telomerase activity (catalogue number
S7700; Oncor, Inc., Gaithersburg, MD), are mixed in different
proportions with cells that do not have telomerase activity (RBCs), and
a standard telomerase activity curve is generated (100% immortal
cells = 5000 cells; 50% dilution of immortal cells = 2500
immortal cells and 2500 RBCs). The telomerase activity in a patient
specimen is measured and matched with the corresponding telomerase
activity in the standard curve (expressed as the percentage of activity
of the pure population of immortal cells). Because we have found that
the slope of the curve that reflects telomerase activity is different
for cell suspensions containing 0–5% or >5% of immortal cells, the
former dilution was used as the standard for assaying low levels of
telomerase activity, whereas the latter curve was used to assay higher
activity levels (Fig. 1, A and B
).
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Fig. 1. Reliability of telomerase activity assessment
when low and high levels of activity are present. A,
modification of the standard TRAP assay to permit measurement of
low levels of telomerase activity. The TPG number is the arithmetic
mean of three tests. B, standard curve for the
conventional TRAP assay. The TPG number is the arithmetic mean
of three tests. Each unit of TPG corresponds to the number of TS
primers (in 1 x 103/mole or 600 molecules)
extended with at least four telomeric repeats by telomerase in the cell
extract. The formula TPG = (X -
X0/C(C -
C0)/Cr.
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The telomerase reaction products were amplified in a thermal cycler for
28 rounds at 94°C for 30 s, 58°C for 30 s, and 72°C for
30 s in the standard semiquantitative
assay,4 and to measure low levels of telomerase
activity, the telomerase reaction products were amplified in a thermal
cycler for 35 rounds at 94°C for 30 s, 62°C for 30 s, and
72°C for 30 s in our semimicroquantitative detection
assay.4 The reactions were conducted in the
presence of [32-P]ATP. The reaction product
was electrophoresed in 10% polyacrylamide nondenaturing gels and
analyzed in a PhosphorImager by ImageQuant software (Molecular
Dynamics, Sunnyvale, CA).
Assessment of Cytokine Transcript Profiles.
To assess the cytokine transcript profiles, the BM aspirate specimens
were mixed immediately after removal from the patient with an equal
volume of 4 M guanidinium thiocyanate solution to
lyse the cells and prevent RNA degradation. Total cellular RNA was
extracted according to the guanidinium thiocyanate-phenol chloroform
procedure of Chomczynski and Sacchi (8)
. Extracted RNAs
were stored in ethanol at -80°C.
Multiplex RT-PCR.
To detect multiple cytokine transcripts simultaneously, we designed a
multiplex RT-PCR to assess the transcripts of the following seven
cytokine or cytokine-related genes: (a) IL-1ß;
(b) TNF-; (c) IL-6; (d) GM-CSF;
(e) IL-1RA; (f) SCF; and (g)
flt3 (9)
. In the studies described here, ß-actin
transcripts were used as an internal control. Total cellular RNA was
first used as a template to synthesize total cDNA, as described
previously (9)
. The cDNA was then used for further PCR
amplification. To reduce possible differences in amplification
efficiencies due to the differences in primer lengths and composition,
primers were chosen carefully to ensure maximum homology in their
composition and annealing kinetics. The amplification condition for
each set of primers was established, and the multiplex reaction was
achieved by adding each set of primers one at a time. RNA
extracted from HL60 cells was used as positive cytokine transcript
control.
The sequences of the primers for each cytokine are listed in Table 1
. The final reaction conditions for PCR
amplification were 3.50 mM MgCl2,
0.32 mM deoxynucleotide triphosphate, and 5 units of Taq
polymerase cycled 26 times at 94°C for 50 s, 60°C for 45 s, and 72°C for 45 s, with a final extended elongation at
72°C for 10 min. The PCR-amplified products were then separated on a
1.5% agarose gel (FMC Bioproducts), stained, and photographed. The
band densities were measured using EagleSight software (Eagle Eye II;
Stratagene). The cytokine and cytokine-related transcript densities
were divided by the ß-actin transcript density of the same specimen.
This value was then used to compare transcript levels in different
patient specimens. Normalization of cytokine transcript levels to the
ß-actin transcript levels permits a reproducible estimate to be made
of relative transcript levels independent of the efficiency of the
RT-PCR reactions.
Statistical Analyses.
Descriptive statistics such as counts, ranges, and percentages
were used to present the distribution of the measured parameters. The
median and the mean values were used to measure the variability of the
parameters. Confidence intervals of parameter means were also generated
based on 1 unit of SD. Considering the number of observations that were
available in the studies described here, nonparametric statistical
techniques were used as the primary analytical methods. Relationships
between parameters were determined by the Spearman rank correlation.
The significance of changes in parameters between pre- and posttherapy
specimens were determined by the Wilcoxon matched-pairs test, which
also tests for directional changes. A two-tailed level of 0.05 was
used as the criterion for statistical significance.
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RESULTS
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General Characteristics of the Patients and Leukemias Studied.
The median age of the patients studied was 61 years, with an age range
of 20–81 years. There were 15 female and 18 male patients. The FAB
classification of the AMLs that were studied are as follows:
(a) M1, 11 cases; (b) M2, 13 cases;
(c) no FAB M3 cases; (d) M4, 5 cases;
(e) no FAB M5 cases; and (f) M6, 1 case.
The FAB classification of three of the AMLs was unknown. The median WBC
count and platelet counts were 15,600/µl and 49,000/µl,
respectively.
Changes in telomerase activity levels were measured in 22 marrow
specimens, and changes in cytokine transcript levels were studied in 22
patients. Median and ranges for telomerase activity levels were 32%
(0–90%; Fig. 2A
). Fig. 2B
provides the ranges of cytokine transcript levels. As we
have described previously, AML cells in vivo in patients do
not contain either GM-CSF or SCF transcripts.
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Fig. 2. Telomerase activity and intracellular cytokine
transcript levels in AML patients. A, telomerase
activity as a percentage of 5000 immortal cells. B,
normalization of transcript levels achieved by dividing the density of
the PCR-amplified transcript by the density of ß-actin in the same
RT-PCR reaction.
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Effects of IL-4 on Telomerase Activity and Cytokine Transcript
Levels in Leukemia Cells.
Three daily doses of IL-4 were administered to 11 patients. IL-4
administration was associated with a fall in the level of telomerase
activity in seven of nine studies from 38.9 ± 11.6% (median,
30%) to 16.7 ± 7.4% (median, 10%; P = 0.02;
Fig. 3A
). The effects of IL-4
on cytokine transcript levels within leukemia cells were inconsistent.
Changes in telomerase activity were moderately correlated with changes
in both IL-1ß and TNF- transcript levels, but these were not
significant (r = 0.42, P = 0.30, and
n = 8 and r = 0.32, P =
0.44, and n = 8). Changes in IL-1ß and TNF-
transcript levels were highly correlated (r = 0.81,
P = 0.02, and n = 8; Fig. 3B
).
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Fig. 3. Effects of IL-4 on AML marrow aspirate cells
in vivo in patients. A, effects on
telomerase activity. B, relationship between
IL-4-induced changes in intracellular IL-1ß and TNF- transcript
levels.
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Effects of Amifostine on Telomerase Activity and on Cytokine
Transcript Levels.
A single dose of amifostine was administered to 22 patients. The effect
of this agent on telomerase activity was measured in 13 patients, with
a fall in activity occurring in 11 patients(Fig. 4)A
. The mean ± SE and
median activity values before amifostine administration were 40% ±
5% (42%), whereas the values after administration were 29.1% ±
3.8% (30%; P = 0.02).
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Fig. 4. Effects of amifostine on AML cells in
vivo in patients. A, telomerase activity;
B, IL-6 transcript levels; C, TNF-
transcript levels; D, flt3 transcript levels;
E, association of changes in IL-1ß and flt3 transcript
levels.
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In contrast to IL-4, amifostine administration produced significant
effects on cellular cytokine transcript levels. A fall in IL-6
transcript levels occurred in 12 of 13 studies (P =
0.002; Fig. 4B
), a fall in TNF- transcript levels
occurred in 10 of 13 studies but lacked significance (P = 0.21; Fig. 4C
), and an increase in flt3 transcript levels
was produced in 9 of 13 studies (P = 0.03; Fig. 4D
). Amifostine administration did not produce consistent
effects on the other cytokine transcripts that were measured. Whereas
there were no apparent relationships between the effects of amifostine
and cytokine transcript levels and telomerase activity, there was a
correlation between the effects of amifostine on IL-1ß and flt3
transcript levels (r = 0.55, P = 0.06,
and n = 12; Fig. 4E
).
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DISCUSSION
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The studies described here demonstrate that two different
noncytotoxic agents have the capacity to reduce telomerase activity
within leukemia cells in vivo in patients. Amifostine has
the further capacity to reduce IL-6, TNF-, and increase flt3
transcript levels within leukemia cells in vivo. The level
of telomerase activity is highest in hematopoietic progenitor cells,
with the self-renewal capacity of these cells being related directly to
the level of telomerase activity (5
, 6)
. Furthermore, high
levels of telomerase activity are characteristic of many malignant cell
types (10)
and are necessary for the immortalization of
cells (10)
. These observations strongly suggest that high
levels of telomerase activity in leukemia cells are indicative of and
play a role in producing a high self-renewal capacity and proliferative
potential in these cells. Given the important role that regrowth
resistance plays in the poor responses obtained in a variety of
otherwise drug-sensitive malignancies, the administration of either
IL-4 or amifostine, by virtue of their effects on telomerase activity,
might be expected to reduce the proliferative potential of malignant
cells in vivo, thereby reducing regrowth resistance. A pilot
study of this proposition appears to confirm this
conjecture.5
Whereas the inhibitory effects of amifostine on cytokine
transcript levels do not appear to play a role in the effects of this
agent on telomerase activity, these effects are potentially useful.
There is evidence of a correlation between the level of TNF-
transcripts and the percentage of S-phase leukemia cells within a
leukemia cell population
(9)
.6
Furthermore, there have been many reports of the stimulatory effects of
IL-6 on the proliferation of AML cells (11
, 12)
. Therefore
the effects of amifostine on cytokine transcript levels and hence on
cytokine production might reduce the number of cells proliferating in
an AML marrow as well as their rate of proliferation.
Furthermore, the ability of amifostine to reduce TNF- transcript
levels might explain, at least in part, the reported benefit that this
agent produces when used to treat myelodysplasia (13)
.
Taken together, the above data strongly suggest that the administration
of amifostine and/or IL-4 between courses of cytotoxic therapy may play
a useful role in improving treatment outcome in AML. A study submitted
for publication is consistent with this
hypothesis.5, 6
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FOOTNOTES
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Supported by National Cancer Institute Grant
1-PO-1 75606-01, with partial support from the Alza Company. 
2 To whom requests for reprints should be
addressed, at Rush Cancer Institute, 1725 West Harrison Street, Suite
809 Professional Building I, Chicago, IL 60612. Phone: (312) 563-2190;
Fax: (312) 455-9635.
3 The abbreviations used are: IL, interleukin;
AML, acute myelogenous leukemia; CHAPS,
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; TPG, total
product generated; TNF-, tumor necrosis factor ; BM , bone
marrow; RT-PCR, reverse transcription-PCR; FAB,
French-American-British; GM-CSF, granulocyte macrophage
colony-stimulating factor.
4 B. Li, S. Bi, J. Yang, C. Andrews, E. Horvath,
P. Toofanfard, H. Chopra, X. Z. Gao, E. Devemy, X. K. Huang,
J. Cartlidge, A. Raza, and H. D. Preisler. Telomerase
activity in human secondary hematologic disorder, submitted for
publication.
5 H. D. Preisler, B. Li, P. Venugopal,
S. A. Gregory, W-T. Hsu, J. Loew, S. Adler, S. Gezer, R-W. Huang,
A. Galvez, D. Slyvnick, R. Larson, and A. Jaje. Poor prognosis
AML. I. Response to treatment with high-dose
cytarabine/mitoxantrone/amifostine, submitted for publication.
6 B. Li, J. Nayini, J. Yang, S. Song, S.
Perambakam, E. Horvath, M. Tao, Y. Chen, R. Huang, H. Chopra, P.
Venugopal, and H. D. Preisler. Poor prognosis AML. II. Biological
and molecular biological characteristics, submitted for publication.
Received 6/ 8/98;
revised 2/17/99;
accepted 2/17/99.
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ABSTRACT
INTRODUCTION
