©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Rapid Decline in Folylpolyglutamate Synthetase Activity and Gene Expression during Maturation of HL-60 Cells
NATURE OF THE EFFECT, IMPACT ON FOLATE COMPOUND POLYGLUTAMATE POOLS, AND EVIDENCE FOR PROGRAMMED DOWN-REGULATION DURING MATURATION (*)

(Received for publication, September 13, 1994; and in revised form, December 22, 1994)

Mary G. Egan (1) Sonia Sirlin (1) Brigitta G. Rumberger (1) Timothy A. Garrow (3) Barry Shane (3) Francis M. Sirotnak (1) (2)(§)

From the  (1)Program in Molecular Pharmacology and Therapeutics, Memorial Sloan-Kettering Cancer Center and the (2)Graduate School of Medical Sciences, Cornell University, New York, New York 10021 and the (3)Department of Nutritional Sciences, University of California, Berkeley, California 94720

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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(2)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 Me(2)SO 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 Me(2)SO was solely accounted for by a 7-fold decrease in value for V(max). The same time and concentration dependence for Me(2)SO 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 Me(2)SO 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 [^3H]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.


INTRODUCTION

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 -polyglutamate peptides of varying length. Their anabolism and that of folate analogues are mediated (1, 2, 3) by the enzyme folylpolyglutamate synthetase (FPGS) (^1)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.

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(2)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) .


EXPERIMENTAL PROCEDURES

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 times 10^3 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(2), 20 mM KCl, 100 mM MET, 4 mM [^3H]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 [^3H]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. [^3H]Methotrexate (specific activity = 22 Ci/mmol) was purchased from Moravek Biochemicals (City of Industry, CA). lL5-CHO-folateH(4) was purchased from Schirks Laboratory, Jonas, Switzerland. These compounds were purified to >97% by high pressure liquid chromatography(8) . Analyses of [^3H]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) .


RESULTS

FPGS and FPGH Activities in HL-60 Cells in the Presence and Absence of Me(2)SO

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, MgCl(2), and KCl in each case.


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(2)SO. HL-60 cells were incubated with 210 mM Me(2)SO 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%).



Initially, HL-60 cells were exposed to 210 mM Me(2)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 Me(2)SO reduced FPGS activity compared to control approximately 3- and 6-fold, respectively. The addition of 210 mM Me(2)SO 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 Me(2)SO 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 Me(2)SO to cultures of HL-60 cells (data not shown).

In other experiments, we measured the level of FPGS activity in cell-free extract and the relative number of NBT cells among HL-60 cells exposed to 210 mM Me(2)SO 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 Me(2)SO 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 Me(2)SO 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 Me(2)SO at a time when the number of NBT cells began to markedly increase and FPGS activity was already reduced 3-fold compared to control.


Figure 2: Time course for decrease of FPGS activity and increase in NBT cells during exposure of HL-60 cells to Me(2)SO in culture. Cells were incubated with 210 mM Me(2)SO in RPMI medium plus 10% fetal calf serum. A, cells were removed before and after various periods of time during incubation with Me(2)SO and assayed for FPGS activity and the percent of NBT cells. B, growth of HL-60 cells in the presence and absence of Me(2)SO 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%).



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(2)SO for 5 days is given in Table 1. The data show that a single saturable component and the same value for apparent K(m) was obtained in cell-free extract derived from HL-60 cells before and after exposure to Me(2)SO. However, the value for V(max) 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 Me(2)SO. Effects on either property were seen at a Me(2)SO 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 [^3H]MTX as a Model Folate Compound

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 [^3H]MTX before and after 3 days of growth in RPMI medium with 210 mM Me(2)SO. From the data, it can be seen (Table 3) that compared to control cells net intracellular accumulation of [^3H]MTX in cells grown with 210 mM Me(2)SO was reduced 7-fold and total [^3H]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 [^3H]MTX along with hypoxanthine and thymidine to avoid any effects of the drug on growth. Me(2)SO (210 mM) was added 24 h after initiation of growth and cells harvested 3 days later. In this case, higher levels of total [^3H]MTX polyglutamates were accumulated compared to Experiment 1. However, the results again show (Table 3) that net accumulation in Me(2)SO-treated cells of total [^3H]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 Me(2)SO 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 Me(2)SO. 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 Me(2)SO. The reduction in FPGS mRNA blotted from HL-60 cells exposed to 100 mM Me(2)SO 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 Me(2)SO, and showed the same relationship with time of exposure to Me(2)SO and fraction of NBT cells as that shown for FPGS activity.


Figure 3: Northern blot analysis of FPGS mRNA in HL-60 cells before and after growth in the presence of Me(2)SO for various intervals. Cells were cultured with 210 mM Me(2)SO for 1-5 days. Control and Me(2)SO exposed cells were removed for mRNA extraction. Poly(A) RNA was denatured in 1 M glyoxal, 50% Me(2)SO, 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 times 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.




Figure 4: Northern blot analysis of FPGS mRNA in HL-60 cells before and after growth in the presence of different concentrations of Me(2)SO. Cells were cultured for 5 days in the presence of 100 mM Me(2)SO (5 days) or 210 mM Me(2)SO (2 and 5 days) and without Me(2)SO. 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.




Figure 5: Quantitative analysis of FPGS mRNA and NBT cells during growth of HL-60 cells for various periods of time in Me(2)SO in culture. Cells were incubated for various periods of time with 210 mM Me(2)SO 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 Me(2)SO 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 Me(2)SO 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 Me(2)SO but also the reversibility of these effects following removal of inducer. Up to 2 days of exposure to Me(2)SO, 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.


Figure 6: Reversibility of effects on FPGS mRNA level in HL-60 cells cultured with Me(2)SO for various periods of time. HL-60 cells were incubated with 210 mM Me(2)SO 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 Me(2)SO. 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(2)SO-induced Maturation of HL-60 Cells Is Influenced by Folate Status

In addition to the alteration of FPGS activity seen following exposure in culture of HL-60 cells to Me(2)SO, 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(4)), a physiological folate level, at five times that level and at five times that level plus 100 µM hypoxanthine and 10 µM thymidine. Me(2)SO 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(4)), 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 Me(2)SO. Despite these differences in onset, the level of NBT cells in each culture eventually reached 85-90% within 3-5 days after Me(2)SO addition. Also, the magnitude of the decrease in FPGS activity with time of exposure to Me(2)SO 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.


Figure 7: Onset of Me(2)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(4)) or 20 nM calcium leucovorin or 20 nM calcium leucovorin with 100 µM hypoxanthine and 10 µM thymidine. After the addition of 210 mM Me(2)SO 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.




DISCUSSION

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(2)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 Me(2)SO, subsequently reached 7-fold after 5 days of exposure, was characterized by a commensurate reduction in V(max) for FPGS activity and had a major impact on net accumulation of polyglutamylated [^3H]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 Me(2)SO, shown for changes in FPGS activity and increase in NBT cells.

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(2)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 Me(2)SO (3-5 days) when the majority of the cells exposed to Me(2)SO 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.

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(2)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.


FOOTNOTES

*
This work was supported in part by grants CA08748, CA22764, and CA56517 from the National Cancer Institute and the Elsa U. Pardee Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Memorial Sloan-Kettering Cancer Center, 1275 York Ave., New York, NY 10021. Tel.: 212-639-7952; Fax: 212-794-4342.

(^1)
The abbreviations used are: FPGS, folylpolyglutamate synthetase; NBT, nitro blue tetrazolium; AMT + G1, aminopterin with one additional glutamate; FPGH, folylpolyglutamate hydrolase; 5-CHO-folateH(4), lL-5-formyltetrahydrofolate, calcium leucovorin; MTX, methotrexate; MET, 2-mercaptoethanol.


REFERENCES

  1. McBurney, M. W., and Whitmore, G. F. (1974) Cell 2, 173-182 [Medline] [Order article via Infotrieve]
  2. McGuire, J. A., and Bertino, J. R. (1981) Mol. Cell. Biochem. 38, 19-48 [Medline] [Order article via Infotrieve]
  3. McGuire, J. J., and Coward, K. (1984) in Folates and Pterins (Blakely, R. L., and Benkovic, S. J., eds) Vol. 1, pp. 135-190, Wiley Interscience, New York
  4. Kisliuk, L. R. (1981) Mol. Cell Biochem. 39, 331-346 [Medline] [Order article via Infotrieve]
  5. Moran, R. G., and Colman, P. D. (1984) Biochemistry 23, 4580-4589 [Medline] [Order article via Infotrieve]
  6. Barrueco, J. R., O'Leary, D. F., and Sirotnak, F. M. (1992) J. Biol. Chem. 267, 15356-15361 [Abstract/Free Full Text]
  7. Poser, R. G., Sirotnak, F. M., and Chello, P. L. (1981) Cancer Res. 41, 1488-1495 [Abstract]
  8. Samuels, L. L., Moccio, D. M., and Sirotnak, F. M. (1985) Cancer Res. 45, 1488-1495 [Abstract]
  9. Sirotnak, F. M., and Degraw, J. I. (1984) Folate Antagonists as Therapeutic Agents (Sirotnak, F. M., Burchall, J. J., Ensminger, W. D., and Montgomery, J. A., eds) Vol. 1, pp. 43-91, Academic Press, New York
  10. Fabre, I. Fabre G., and Goldman, I. D. (1984) Cancer Res. 44, 3190-3195 [Abstract]
  11. Moran, R. G., Colman, P. D., Rosowski, A., Forsch, R. A., and Chang, K. K. (1985) Mol. Pharmacol. 27, 156-166 [Abstract]
  12. Samuels, L. L., Goutas, L. J., Priest, D. G., Piper, J. R., and Sirotnak, F. M. (1986) Cancer Res. 46, 2230-2235 [Abstract]
  13. Rumberger, B. G., Barrueco, J. R., and Sirotnak, F. M. (1990) Cancer Res. 50, 4639-4643 [Abstract]
  14. Rumberger, B. G., Schmid, F. A., Otter, G. A., and Sirotnak, F. M. (1990) Cancer Commun. 2, 305-310 [Medline] [Order article via Infotrieve]
  15. McCloskey, D. E., McGuire, J. J., Russell, C. A., Rowan, B. G., Bertino, J. R., Pizzarno, G., and Mini, E. J. (1991) J. Biol. Chem. 266, 6181-6187 [Abstract/Free Full Text]
  16. Rhee, M. S., Wang, Y., Nair, M. G., and Galivan, J. (1993) Cancer Res. 53, 2227-2230 [Abstract]
  17. Kisliuk, R. L. (1984) Folate Antagonists as Therapeutic Agents (Sirotnak, F. M., Burchall, J. J., Ensminger, W. D., and Montgomery, J. A., eds) Vol. 1, pp. 2-55, Academic Press, New York
  18. Grindey, G. B., and Jacobson, R. C. (1984) Folate Antagonists as Therapeutic Agents (Sirotnak, F. M., Burchall, J. J., Ensminger, W. D., and Montgomery, J. A., eds) Vol. 1, pp. 290-311, Academic Press, New York
  19. Sirotnak, F. M. (1985) Cancer Res. 45, 3992-4000 [Medline] [Order article via Infotrieve]
  20. Shane, B. (1989) Vitamin. Horm. 45, 263-335
  21. Corin, R. E., Haspel, H. C., and Sonenberg, M. (1984) J. Biol. Chem. 259, 206-211 [Abstract/Free Full Text]
  22. Sirotnak, F. M., Jacobson, D. M., and Yang, C-H. (1986) J. Biol. Chem. 261, 11150-11155 [Abstract/Free Full Text]
  23. Yang, C-H., Pain, J., and Sirotnak, F. M. (1992) J. Biol. Chem. 267, 6628-6634 [Abstract/Free Full Text]
  24. Barredo, J., and Moran, R. G. (1992) Mol. Pharmacol. 42, 687-692 [Abstract]
  25. Egan, M. G., Sirlin, S., Rumberger, B. G., Shane, B., and Sirotnak, F. M. (1993) Proc. Am. Assoc. Cancer Res. 34, 275
  26. Gallagher, R., Collins, S., Trujillo, J., McCredie, K., Ahearn, M., Tsai, S., Metzger, R., Aulakh, G., Ting, R., Ruscetti, F., and Gallo, R. (1979) Blood 54, 713-733 [Abstract]
  27. Chello, P. L., Sirotnak, F. M., Wong, E., Kisliuk, R. L., Gaumont, Y., and Combepine, G. (1982) Biochem. Pharmacol. 31, 1527-1530
  28. Peterson, G. L. (1983) Methods Enzymol. 91, 95-119 [Medline] [Order article via Infotrieve]
  29. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275 [Free Full Text]
  30. Badley, J. E., Bishop, G. A., St. John, T., and Frelinger, J. A. (1988) BioTechniques 6, 114-116 [Medline] [Order article via Infotrieve]
  31. Thomas, P. S. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 5201-5205 [Abstract]
  32. Garrow, T. A., Adman, A., and Shane, B. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 9151-9155 [Abstract]
  33. Gunning, P., Ponte, P., Okayama, H., Engle, J., Blau, H., and Kedes, L. (1983) Mol. Cell. Biol. 3, 787-795 [Medline] [Order article via Infotrieve]
  34. Samuels, L. L., Goutas, L. J., Priest, D. G., Piper, J. R., and Sirotnak, F. M. (1987) Cancer Res. 46, 2230-2235 [Abstract]
  35. Sirotnak, F. M., Johnson, T. B., Samuels, L. L., and Galivan, J. (1988) Biochem. Pharmacol. 37, 4239-4241 [Medline] [Order article via Infotrieve]
  36. Goldman, I. D., and Matherly, L. H. (1986) International Encyclopedia of Pharmacology Therapeutics (Goldman, I. D., ed) pp. 283-302, Pergamon Press, New York
  37. Priest, D. G., Schmitz, J. C., Bunni, M. A., and Stuart, R. K. (1991) J. Natl. Cancer Inst. 83, 1806-1812 [Abstract]
  38. Bloch, A. (1993) Leukemia 7, 1219-1224 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.