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(Received for publication, September 13,
1994; and in revised form, December 22, 1994) From the
These studies in HL-60 cells examined the regulation of
folylpolyglutamate synthetase (FPGS) activity at the level of gene
expression during terminal maturation. Following addition of 210 mM Me
The process of folylpolyglutamylation in mammalian cells is
important to the conservation and efficient utility of folate coenzymes
required for macromolecular
biosynthesis(1, 2, 3, 4, 5) .
Cellular folates exist primarily as It has been suggested in the context of
earlier reports (reviewed in (17, 18, 19, 20) ) that one way in
which tumor cells may control their macromolecular synthesis is through
regulation of intracellular folate homeostasis. In addition to the
metabolic conversion of folate compounds, this could occur at the level
of mediated entry of exogenous folates (reviewed in (17) ) and
(or) through the biosynthesis of
folylpolyglutamates(1, 2, 3, 4, 5) .
As one approach to understanding the manner by which the expression of
these processes are regulated in tumor cells, we and others have
examined (19, 21, 22, 23) the
expression of a putative oncofetal property, the tumor-specific,
one-carbon, reduced folate transport system in the plasma membrane,
during induced maturation of murine erythroleukemia and HL-60
promyelocytic leukemia cells. These studies showed (19, 21, 22, 23) that the level of
inward transport of folates mediated by this system and synthesis of
the transporter rapidly declined during maturation of these cells. The
present work utilized a similar approach to studies of FPGS in the
context of this same system. Our data expand upon findings presented in
an earlier report (24) which showed a delayed onset in
reduction of FPGS activity during growth of HL-60 cells in the presence
of inducers of maturation. In contrast, our results appear to show that
FPGS activity in HL-60 cells is rapidly down-regulated during
Me
Figure 1:
Time course for aminopterin
polyglutamate formation at 37 °C by cell-free FPGS derived from
L1210 and S180 cells and from HL-60 cells before and after incubation
with Me
Initially, HL-60 cells were exposed to 210 mM Me In other experiments, we measured the level of FPGS activity in
cell-free extract and the relative number of NBT
Figure 2:
Time course for decrease of FPGS activity
and increase in NBT
A
summary of the kinetic properties for FPGS activity in cell-free
extract from HL-60 cells before and after exposure to 210 mM Me
Figure 3:
Northern blot analysis of FPGS mRNA in
HL-60 cells before and after growth in the presence of Me
Figure 4:
Northern blot analysis of FPGS mRNA in
HL-60 cells before and after growth in the presence of different
concentrations of Me
Figure 5:
Quantitative analysis of FPGS mRNA and
NBT
Figure 6:
Reversibility of effects on FPGS mRNA
level in HL-60 cells cultured with Me
Figure 7:
Onset of Me
The studies described here document an early and rapid
decline in FPGS activity in HL-60 cells in culture following induction
of maturation by Me Evidence was also provided
for coupling between the decrease in FPGS mRNA and commitment of HL-60
cells to terminal maturation. However, the initial effect on FPGS mRNA
observed during exposure to Me Our results
have further significance when viewed in the context of cellular folate
homeostasis. Folates play an important role in the biosynthesis of
macromolecules by mediating (reviewed in (17) -20, 36)
one-carbon transfer. Adequate access of tumor cells to exogenous
reduced folates is achieved by the one-carbon, reduced folate transport
system and conservation, and efficient utilization of these folates in
the cell (1, 2, 3, 4, 5) is
ensured through their
polyglutamylation(1, 2, 3, 4, 5) .
It is noteworthy, therefore, that coordinate down-regulation of both of
these properties in addition to dihydrofolate reductase (22) occurs as early events during Me
Volume 270,
Number 10,
Issue of March 10, 1995 pp. 5462-5468
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
NATURE OF THE EFFECT, IMPACT ON FOLATE COMPOUND POLYGLUTAMATE
POOLS, AND EVIDENCE FOR PROGRAMMED DOWN-REGULATION DURING MATURATION (*)
SO to cultures of HL-60 cells at a concentration
that induces maturation of 85-90% of the cells, FPGS activity,
but not folylpolyglutamate hydrolase (FPGH) activity, was reduced
2-7-fold within 1-5 days. The initial decline in FPGS
activity preceded any effect of MeSO on rate of growth and
the increase in appearance of nitro blue tetrazoliumpositive cells, a
marker of cellular maturation, and the decrease after 5 days of
exposure to MeSO was solely accounted for by a 7-fold
decrease in value for V
. The same time and
concentration dependence for MeSO was shown for the decline
in FPGS activity, increase in nitro blue tetrazolium-positive cells,
and decline in the level of a 2.1-kilobase FPGS mRNA during exposure to
this inducer. This decline in FPGS mRNA was reversible when
MeSO was removed from the culture medium but only until
that time when an appreciable number of cells were committed to
terminal maturation. Following growth of HL-60 cells with
[
H]MTX, used as a model folate compound, a large
reduction in its intracellular polyglutamate pools was shown during
maturation which quantitatively reflected the decline in FPGS activity
as well as folate transport inward (Sirotnak, F. M., Jacobson, D. M.,
and Yang, C-H.(1986) J. Biol. Chem. 261, 11150-11156).
Other data showed that folate status or obviation of the folate
requirement during growth of these cells strongly influenced the
rapidity of the onset of maturation following exposure to inducer.
Overall, these results show that FPGS activity in HL-60 cells is a
marker for proliferative capacity and that the underlying basis for the
decline in FPGS activity during maturation is altered cognate gene
expression which is manifested as early reversible and late
irreversible phases. They also suggest that the coordinate reduction
observed in folate transport, FPGS activity, dihydrofolate reductase,
and probably other folate related enzymes by limiting macromolecular
biosynthesis may be early programmed events in the maturation process
that influence the switch from proliferation to senescence in these
cells.
-polyglutamate peptides of
varying length. Their anabolism and that of folate analogues are
mediated (1, 2, 3) by the enzyme
folylpolyglutamate synthetase (FPGS) (
)and metabolic
turnover of these anabolites appears to be modulated by
folylpolyglutamate hydrolase (FPGH) after their mediated entry
(reviewed in (6) ) into lysosomes. In tumors and normal
proliferative tissues of animals and man, the process of
polyglutamylation has pharmalogic
relevance(2, 3, 4, 5, 7, 8, 9, 10, 11) to the
therapeutic use of classical folate analogues. Intracellular levels and
preferences among folate analogues as substrate for FPGS and, probably,
levels of FPGH appear to partially determine (12, 13) the extent of cytotoxic action of these
analogues in these proliferative tissues. Moreover, decreased levels of
FPGS activity (14, 15) and increased levels of FPGH
activity (16) have been associated with acquired resistance of
tumors to these analogues.SO-induced maturation. In these studies, we examined this
effect and its time and concentration dependence during maturation at
the level of FPGS activity, the intracellular accumulation and
retention of model 4-amino-folylpolyglutamates and cognate gene
expression. We also show that the effects on FPGS mRNA levels after
removal of inducer are reversible until commitment of HL-60 cells to a
program of terminal maturation verifying that they are bona fide maturational events. Finally, we provide evidence to suggest that
folate homeostasis in general and programmed down-regulation of
dihydrofolate reductase, folate transport inward, and
polyglutamylation, in particular, may be involved in initiating the
switch from proliferative capacity to maturation in these cells. A
preliminary report of these studies has been presented in abstract form (25) .
Cells and Culture Conditions
The origin and
properties of HL-60 cells have been described in detail
elsewhere(26) . The particular subline utilized here was
obtained as a gift from Dr. Janice Gabrilove of Memorial
Sloan-Kettering Cancer Center, New York, NY. For experiments in RPMI
medium with 10% fetal calf serum with and without dialysis (plus folate
compound), cells in early log phase of growth were employed as inoculum
(initial concentration was 50-60 
10 cells/ml). A dye exclusion assay (27) was employed for
estimating viability during these studies. Terminal maturation of HL-60
cells was quantitated morphologically and by determining the fraction
of cells which acquired the ability to reduce NBT, a measure of
phagocytic activity (21) in granulocytes.Assay for FPGS Activity
Processing of cells for
measurements of FPGS activity was described
earlier(2, 14) . Protein concentration of the
cell-free extract was determined by a modification (28) of the
method of Lowry et al.(29) . Each FPGS assay (2, 14) contained in a final volume of 250 µl, 100
mM Tris-HCl (pH 8.85), 10 mM ATP, 20 mM MgCl, 20 mM KCl, 100 mM MET, 4
mM [H]glutamate (20-30
µCi/mmol, New England Nuclear), 100 µM aminopterin,
and cell-free enzyme preparation (0.05-0.3 mg). Following
termination of the reaction with the addition of 1 ml of cold
(0-4° C) 0.5 mM glutamate solution (pH 7.5) with 25
mM MET, the unreacted [H]glutamate was
separated (2, 14) from the tritiated polyglutamates by
DEAE-cellulose chromatography on a minicolumn (1-1.5-ml bed
volume). Product formation (aminopterin + GI) was linear for at
least 2 h at 37 °C and these cell-free preparations under these
conditions exhibited (12) no detectable folylpolyglutamate
hydrolase activity.Northern Blot Analysis of FPGS mRNA
A method (30) utilizing rapid isolation of poly(A)
RNA
directly from the cell lysates by means of an oligo(dT) column was used
to isolate poly(A) RNA. The integrity of the RNA was
assessed by electrophoresis in 1.1% agarose containing 1 M glyoxal and staining with ethidium bromide. An aliquot of the same
RNA was analyzed (31) by Northern blotting and radioautography
using a human FPGS cDNA, PZT 18U936-10(32) , as a probe
and normalized to -actin mRNA content with a human -actin
probe, PCD-2-actin(33) . Labeling of each probe was by random
priming (Random Primers DNA Labeling Kit, Boehringer Manneheim) using
[-
P]dCTP (300 Ci/mmol and 10 µg of
insert). Direct measurement of total radioactivity in each blot minus
background was obtained with a Betagen 603 Blot Analyzer.Materials and Other Analytical Methods
Aminopterin
was provided by Dr. J. R. Piper of the Southern Research Institute.
[H]Methotrexate (specific activity = 22
Ci/mmol) was purchased from Moravek Biochemicals (City of Industry,
CA). lL5-CHO-folateH
was purchased from Schirks Laboratory,
Jonas, Switzerland. These compounds were purified to >97% by high
pressure liquid chromatography(8) . Analyses of
[H]MTX polyglutamates was also by high
performance liquid chromatography (8) using various
polyglutamates of MTX as standards. Folylpolyglutamate hydrolase
activity was measured as described
previously(12, 34) .
FPGS and FPGH Activities in HL-60 Cells in the Presence
and Absence of Me
Appreciable FPGS activity was
found in cell-free extract from HL-60 cells. The level of this activity
was only slightly (25-30%) lower (Fig. 1) than that found
in similar extracts derived from L1210 and S180 murine tumor cells with
100 µM AMT as substrate. In all cases, the major
polyglutamate formed was AMT + G1 (data not shown). Additionally,
product formation showed (data not given) the same requirements for the
concentration of substrate, ATP, MET, MgClSO, and KCl in each
case.
SO. HL-60 cells were incubated with 210 mM MeSO for 2 or 4 days in RPMI medium. Additional
experimental details are provided in the text. Average of three
experiments (S.E. of the mean = <
±13%).
SO during very early logarithmic phase of growth in
RPMI medium for a period of 2 or 4 days. This concentration was shown
earlier (22) to be effective in inducing maturation of these
cells in culture with no growth inhibitory effect. Also a decrease in
viability of only 3-6% was observed only after 5 days of exposure
to this agent compared to 2-4% for control cells. From data shown
in Fig. 1, it can be seen that these different exposures (2 and
4 days) to MeSO reduced FPGS activity compared to control
approximately 3- and 6-fold, respectively. The addition of 210 mM MeSO to HL-60 cells in culture just prior to
harvesting of the cells had no effect (data not shown) on the level of
FPGS activity measured in the cell free extract. Additionally, growth
of L1210 or S180 cells in the presence of 210 mM MeSO for as long as 1 week had no effect on FPGS
activity. In contrast to the results for FPGS activity, FPGH activity
was unaltered following the addition of 210 mM MeSO to cultures of HL-60 cells (data not shown). cells
among HL-60 cells exposed to 210 mM MeSO for
various periods of time over 1-5 days. A substantial effect
(approximately 2-fold reduction compared to control level) on FPGS
activity was observed (Fig. 2A) within 1 day of
exposure to inducer, continued to decrease upon further exposure and
after 5 days of exposure was reduced 7-fold. The data also show (Fig. 2A) that the percent of NBT cells did not begin to increase until after 1 day of exposure to
inducer. A rapid increase in NBT cells began only
after 2-3 days of exposure to MeSO at a time when
FPGS activity was already reduced 3-4-fold compared to control
and after 5 days of exposure the fraction of NBT cells
reached 87%. In Fig. 2B, it can be seen that the
increase in NBT cells and decrease in FPGS activity
during exposure to MeSO was unrelated to effects on growth
of HL-60 cells in culture. No appreciable effect on growth was observed
until after 2 days of exposure to MeSO at a time when the
number of NBT cells began to markedly increase and
FPGS activity was already reduced 3-fold compared to control.
cells during exposure of HL-60
cells to MeSO in culture. Cells were incubated with 210
mM MeSO in RPMI medium plus 10% fetal calf serum. A, cells were removed before and after various periods of time
during incubation with MeSO and assayed for FPGS activity
and the percent of NBT cells. B, growth of
HL-60 cells in the presence and absence of MeSO as measured
by means of a Coulter counter. Additional experimental details are
provided in the text and legend of Fig. 1. Average of three
experiments (S.E. of the mean = <
±16%).
SO for 5 days is given in Table 1. The data
show that a single saturable component and the same value for apparent K
was obtained in cell-free extract derived from
HL-60 cells before and after exposure to MeSO. However, the
value for V for FPGS activity was 7-fold lower
than that derived with extract from control cells. The same time course
for the appearance of NBT cells was obtained ((22) ) when HL-60 cells were cultured for 5 days with 1
µM retinoic acid. In other experiments, we showed (Table 2) a similar concentration-response for the induction of
NBT cells and the reduction in FPGS activity following
exposure of HL-60 cells to MeSO. Effects on either property
were seen at a MeSO concentration as low as 50 mM.
Further elevation in concentration increased the content of
NBT cells in the culture and decreased the level of
FPGS activity measured in cell-free extract in approximately the same
manner.
Induced Maturation and Intracellular Polyglutamates of
[
MTX
shares a transport route and is polyglutamylated in tumor cells in a
manner similar(2) - (5, 19) to natural folate
compounds. For this reason, we utilized this folate analogue as a model
permeant and substrate for FPGS to estimate the probable impact of
down-regulated transport and FPGS on intracellular pools of natural
folate compounds during maturation of HL-60 cells. First, we
pulse-exposed HL-60 cells for 3 h at 37 °C with
[H]MTX as a Model Folate CompoundH]MTX before and after 3 days of growth in RPMI
medium with 210 mM MeSO. From the data, it can be
seen (Table 3) that compared to control cells net intracellular
accumulation of [H]MTX in cells grown with 210
mM MeSO was reduced 7-fold and total
[H]MTX polyglutamates approximately 25-fold. In
addition, the proportion of higher polyglutamates (>+GI) among
the total was substantially lower than in control cells. Second, HL-60
cells were grown in RPMI medium in the presence of trace amounts of
[H]MTX along with hypoxanthine and thymidine to
avoid any effects of the drug on growth. MeSO (210
mM) was added 24 h after initiation of growth and cells
harvested 3 days later. In this case, higher levels of total
[H]MTX polyglutamates were accumulated compared
to Experiment 1. However, the results again show (Table 3) that
net accumulation in MeSO-treated cells of total
[H]MTX polyglutamates, and the higher
polyglutamylated forms were very substantially reduced compared to
control cells. Both results appear to be entirely consistent with the
extent of down-regulation of transport and polyglutamylation of these
folate compounds also seen.
Northern Blot Analysis of FPGS mRNA in HL-60 Cells during
Induced Maturation
Relative FPGS mRNA levels in HL-60 cells
before and after exposure to 210 mM MeSO in
culture for 1-5 days were analyzed (Fig. 3) in a Northern
blot of poly(A) RNA using as a probe a 2.1-kb cDNA
insert (32) in PTZ18U (PTZ18U936-10). It can be seen that
the intensity of the FPGS mRNA blot (2.1 kb) relative to the control
-actin mRNA blot decreased similarly to the decrease in FPGS
activity (Fig. 2) with time of exposure of HL-60 cells to
MeSO. Levels were significantly reduced within 2 days,
further reduced after 3 days, and by 5 days of exposure were markedly
reduced compared to control. We showed (Fig. 4) that the effect
on FPGS mRNA level like FPGS activity was also dependent on the
concentration of MeSO. The reduction in FPGS mRNA blotted
from HL-60 cells exposed to 100 mM MeSO for 5 days
compared to control was substantially less than that seen with 2 or 5
days exposure to 210 mM. Total radioactivity associated with
four separate FPGS mRNA blots was also quantitated (Fig. 5) with
a Betagen blot analyzer. Radioactivity associated with each FPGS mRNA
blot (average of four separate blots) that were normalized against
separate -actin blots run in parallel, decreased appreciably after
1 day of exposure to 210 mM MeSO, and showed the
same relationship with time of exposure to MeSO and
fraction of NBT cells as that shown for FPGS activity.
SO
for various intervals. Cells were cultured with 210 mM MeSO for 1-5 days. Control and MeSO
exposed cells were removed for mRNA extraction. Poly(A) RNA was denatured in 1 M glyoxal, 50% MeSO,
10 mM sodium phosphate (pH 6.8) at 50 °C for 1 h after
loading on a 1.1% agarose gel. Electrophoresis was carried out in 10
mM phosphate (pH 6.8) at 90 V for 6 h with constant
recirculation. RNA was transferred in 10 SSC on to a nylon
membrane (S+S, Nytran), cross-linked by exposure to short wave UV
and hybridized with FPGS cDNA (PTZ18U936-10) and rehybridized
with -actin cDNA (PCD-1-actin) under standard conditions (31) prior to radioautography. The relative positions of the
FPGS and -actin blots were arbitrarily adjusted for photography.
Additional experimental details are provided in the text. The figure
shows a typical result of several replicate
experiments.
SO. Cells were cultured for 5 days in
the presence of 100 mM MeSO (5 days) or 210 mM MeSO (2 and 5 days) and without MeSO.
Isolated poly(A) RNA was hybridized with FPGS cDNA
(PTZ18U936-10) and rehybridized with -actin cDNA
(PCD-2-actin) as described in the text and legend of Fig. 5. The
relative positions of the FPGS and -actin blots were arbitrarily
arranged for photography. The figure shows a typical result of separate
replicate experiments.
cells during growth of HL-60 cells for various
periods of time in MeSO in culture. Cells were incubated
for various periods of time with 210 mM MeSO and
blotted with FPGS cDNA as described in the text and legend of Fig. 3. The amount of radioactivity associated with each FPGS
mRNA blot was analyzed with a Betagen Blot Analyzer. The results
represent an average of the radioactivity found in blots for each of
the experiments shown in Fig. 3and of two additional
experiments corrected for background radioactivity and normalized with
respect to radioactivity associated with each -actin blot. S.E. of
the mean = < ±18%.
Further Evidence for the Effect on FPGS Activity and mRNA
Level as a Maturation-related Event
A true maturationrelated
event must at some point become nonreversible. For this reason, the
relationship between the reduction in FPGS mRNA level following
exposure to MeSO and the reversibility of the effect in
relation to commitment to terminal maturation was investigated in the
following manner. Levels of mRNA were examined by Northern blotting in
control cells and after various periods of exposure to 210 mM MeSO in culture and 1 day after removal of inducer
following each exposure. The initial results of the Northern blots
showed (Fig. 6) that not only the extent of the decrease in mRNA
differs with time of exposure to MeSO but also the
reversibility of these effects following removal of inducer. Up to 2
days of exposure to MeSO, there was substantial recovery in
mRNA level after removal of inducer, while after more than 4 days there
was little or no recovery when compared to control. The results of this
Northern blot were more accurately portrayed by an analysis of
radioactivity for this blot and an additional blot with a Betagen blot
analyzer (see Table 4). These data showed that removal of inducer
after 1 day of exposure of HL-60 cells (Experiment II) resulted in
complete restoration of FPGS mRNA level. In contrast, removal of
inducer after 2, 3, or 4 days of exposure (Experiments I and II)
resulted in recovery to 73, 27, and 23%, respectively, when compared to
control. Removal of inducer after 5 days of exposure (Experiment II)
did not result in any recovery of FPGS mRNA level.
SO for various periods
of time. HL-60 cells were incubated with 210 mM MeSO for 2 or 4 days in RPMI medium. Cells were then
processed for mRNA extraction along with control cells before and after
a further 1-day incubation in the absence of MeSO. The
inducing agent was removed by centrifuging and resuspending cells in
fresh medium. Further experimental details are given in the text and
legend of Fig. 5. The results shown are typical for one of a
number of experiments.
The Onset of Me
In addition to the
alteration of FPGS activity seen following exposure in culture of HL-60
cells to MeSO-induced Maturation of HL-60
Cells Is Influenced by Folate StatusSO, there are large, rapid declines (22, 23) in both folate transport inward and
dihydrofolate reductase activity. The concerted effect of these three
early alterations alone should ultimately have a profound impact on
overall folate homeostases and proliferative capacity in these cells as
maturation proceeds. Moreover, it is entirely possible that other
aspects of folate anabolism may be similarly altered. Would such
changes occur rapidly enough to influence the onset of maturation as
early programmed events in this process? This question was addressed in
the following experiment. HL-60 cells were cultured for several days
(two to three passages) in 4 nM calcium leucovorin (as the
natural diastereoisomer, lL5-CHO-folateH), a physiological
folate level, at five times that level and at five times that level
plus 100 µM hypoxanthine and 10 µM thymidine.
MeSO at 210 mM was then added to each culture and
the onset of maturation monitored by means of the increase in
NBT cells with time. From the results shown in Fig. 7, it can be seen that initially the rate of growth of
HL-60 cells was the same (Fig. 7B) under these various
conditions. However, the onset in the increase in NBT cells and subsequent cessation of growth (Fig. 7B) under these different conditions varied
markedly (Fig. 7A). At a physiological folate level (4
nM of lL5-CHO-folateH), the number of
NBT cells increased rapidly, almost immediately. By
comparison, at a higher level of this folate, there was an appreciable
delay in the increase of NBT cell, and with this level
of folate in the presence of hypoxanthine and thymidine or with
hypoxanthine or thymidine alone (data not shown) there was an even
longer delay. In this latter case, no increase in NBT cells was observed until 2 days after the addition of
MeSO. Despite these differences in onset, the level of
NBT cells in each culture eventually reached
85-90% within 3-5 days after MeSO addition.
Also, the magnitude of the decrease in FPGS activity with time of
exposure to MeSO under conditions which obviate the folate
requirement (plus hypoxanthine and thymidine) was the same (data not
given) as that shown in Fig. 2A. This was consistent
with the decrease in folate compound polyglutamate pools documented in Table 3for cells grown in RPMI medium (Experiment I) or with
added hypoxanthine and thymidine (Experiment II) although the method
used for measurement of polyglutamate pools was somewhat different in
each case.
SO-induced
maturation of HL-60 cells grown at different folate levels or in the
presence of hypoxanthine and thymidine. Cells were grown in RPMI medium
with dialyzed fetal calf serum containing either 4 nM calcium
leucovorin (lL5-CHO-folateH) or 20 nM calcium
leucovorin or 20 nM calcium leucovorin with 100 µM hypoxanthine and 10 µM thymidine. After the addition
of 210 mM MeSO to each culture, measurements (A) of NBT HL-60 cells were made
periodically. The growth of HL-60 under these different conditions is
shown in B. Additional details are provided in the text. The
data are an average with a variance of ± 18% of two experiments
done on separate days.
SO. In contrast, FPGH activity was
unchanged during maturation. This decline in FPGS activity, which was
readily seen by 1 day of exposure to 210 mM MeSO,
subsequently reached 7-fold after 5 days of exposure, was characterized
by a commensurate reduction in V for FPGS
activity and had a major impact on net accumulation of polyglutamylated
[H]MTX used as a model folate compound. Since
both MTX and folate coenzymes share the same transport route and are
substrates for FPGS and the alterations observed (here and (23) ) on each process relate specifically to folate
transporter and FPGS protein expression it is reasonable to assume that
similar alterations would occur for other folate polyglutamate pools.
While the extent of the decline in FPGS activity ultimately correlated
with the appearance and accumulation of NBT cells, its
onset in this HL-60 cell population occurred prior to any detectable
effect on growth of the cells in culture and prior to any increase in
NBT cells. It should again be mentioned here that FPGS
activity was examined in an earlier study (24) during HL-60
cell maturation. This study documented a very delayed effect and more
complex time course for a gradual decline in FPGS activity with time of
exposure to retinoic acid and dimethyl formamide which occurred only
after the number of NBT cells approached maximum. To
what degree these disparate results represent a difference in each case
in the inducers of maturation used, in the source of HL-60 cells, their
folate status, culture conditions, or other factors is unclear. In
other experiments, we provide data which suggest that the decline in
FPGS activity in HL-60 cells during maturation reflects an alteration
in cognate gene expression. Analysis by Northern blot and with a blot
analyzer of FPGS mRNA revealed rapid changes in the level of this mRNA
with the same concentration and time dependence, during exposure of
cells to MeSO, shown for changes in FPGS activity and
increase in NBT cells.SO occurred before any
appreciable increase in NBT cells and was reversible.
Thus, the onset of this change appeared to precede commitment as an
early event in the maturation process. Ultimately, the decrease in FPGS
mRNA level became essentially nonreversible during continued exposure
to MeSO (3-5 days) when the majority of the cells
exposed to MeSO were NBT. These results
along with others showing that the reduction in FPGS activity and FPGS
mRNA level occurs well before there is any effect on HL-60 cell growth
rules out growth arrest as a basis for these effects and supports the
notion that these effects on this enzyme activity are a property of
maturating cells. They assume additional interest in light of our
earlier studies (35) which revealed similar reductions in FPGS
activity but no change in FPGH activity, in maturating luminal
epithelial cells from mouse small intestine. Thus, the same selective
effect of maturation on folate compound accumulation and anabolism, but
not folate polyglutamate catabolism, was observed in each case. In
addition, recent studies by others (24) documented remarkable
differences in the level of FPGS activity in various tissues at
different developmental stages and proliferative capacities. Generally,
the highest levels of FPGS activity were found in fetal tissues and
rapidly proliferating normal and neoplastic tissues. As was suggested
in this earlier report by Barredo and Moran(24) , it would
appear from these studies (24) and our own presented here and
in a prior report (35) that the expression of FPGS activity is
under stringent regulation among these various tissues.SO-induced
maturation of HL-60 cells. It is very likely, as well, that other
folate-related anabolic properties are similarly down-regulated. In
contrast, we have shown that ATP-dependent efflux (22) and FPGH
are not down-regulated during maturation. The extent to which early
down-regulation of all of the former properties through their effect on
folate homeostasis, might, themselves, influence the onset of terminal
maturation was also addressed in the context of the current studies.
Our results showed that onset of maturation was delayed in the presence
of folate concentrations beyond 1-10 nM that is
considered physiological(37) , and obviation of the folate
requirement by the addition of hypoxanthine and thymidine markedly
delayed this onset further. However, the time course for the decrease
in FPGS activity in the latter case was the same as that shown in Fig. 2A. These results suggest that early
down-regulation of these and, probably, other folate anabolic processes
is programmed to limit macromolecular biosynthesis and, thus,
facilitate the switch (38) from a program of proliferation to
terminal maturation. That is the reason why obviating the folate
requirement will delay onset of maturation. In any event, this cellular
system appears to offer an opportunity to further pursue this question.
), lL-5-formyltetrahydrofolate, calcium leucovorin; MTX,
methotrexate; MET, 2-mercaptoethanol.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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