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

Mary G Egan,Sonia Sirlin,Brigitta G Rumberger,Timothy A Garrow,Barry Shane,Francis M Sirotnak

doi:10.1074/jbc.270.10.5462

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 Me2SO 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 Me2SO on rate of growth and the increase in appearance of nitro blue tetrazolium-positive cells, a marker of cellular maturation, and the decrease after 5 days of exposure to Me2SO was solely accounted for by a 7-fold decrease in value for Vmax. The same time and concentration dependence for Me2SO 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 Me2SO 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.

Highlights

FPGH activity was unchanged during maturation. This decline in FPGS activity, which was readily seen by 1 day of exposure to 210 mM MezSO, 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 [3H1MTX used as a model folate compound
While the extent of the decline in FPGS activity 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
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

Summary

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. 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. A preliminary report of these studies has been presented in abstract form [25]

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION