Normal Human Myeloid Progenitor Cells Research Articles

Mechanism of thymidylate synthase inhibition by methotrexate in human neoplastic cell lines and normal human myeloid progenitor cells.

We have studied the roles of 5,10-methylenetetrahydrofolate (5,10-methylene-H4PteGlu) depletion and dihydrofolate (H2PteGlu) accumulation in the inhibition of de novo thymidylate synthesis by methotrexate (MTX) in human MCF-7 breast cancer cells. Using both a high pressure liquid chromatography system and a modification of the 5-fluoro-2'-deoxyuridine-5'-monophosphate radioenzymatic binding assay, we determined that the 5,10-methylene-H4PteGlu pool is 50-60% depleted in human MCF-7 breast cancer cells following exposure to 1 micron MTX for up to 21 h. Similar alterations in the 5,10-methylene-H4PteGlu pools were obtained when human promyelocytic HL-60 leukemia cells and normal human myeloid precursor cells were incubated with 1 micron MTX. The H2PteGlu pools within the MCF-7 cells increased significantly after 15 min of 1 micron MTX exposure, reaching maximal levels by 60 min. Thymidylate synthesis, as measured by labeled deoxyuridine incorporation into DNA, decreased to less than 20% of control activity within 30 min of 1 micron MTX exposure. The inhibition of thymidylate synthesis coincided temporally with the rapid intracellular accumulation of H2PteGlu, a known inhibitor of thymidylate synthase. Furthermore, inhibition of this pathway was associated in a log-linear fashion with the intracellular level of dihydrofolate. These studies provide further evidence that depletion of the thymidylate synthase substrate 5,10-methylene-H4PteGlu is inadequate to account completely for diminished thymidylate synthesis resulting from MTX treatment. Our findings suggest that acute inhibition of de novo thymidylate synthesis is a multifactorial process consisting of partial substrate depletion and direct enzymatic inhibition by H2PteGlu polyglutamates.

Interaction of deoxycytidine and deoxycytidine analogs in normal and leukemic human myeloid progenitor cells

The inhibitory effects of three deoxycytidine analogs, 1-B-D-arabinofuranosylcytosine (Ara-C), 5-aza-2′-deoxycytidine, (DAZ) and Ara-5-azacytosine (AAC) were compared with respect to the clonogenic behavior of human promyelocytic leukemic cells (HL-60), a deoxycytidine kinase deficient subvariant (HL-60/Ara-C), and normal human myeloid progenitor cells (CFU-GM). When cells were continuously exposed to each agent for 7 days, Ara-C was the most inhibitory, DAZ slightly less effective and AAC the least inhibitory on a molar basis. HL-60/Ara-C were also highly cross-resistant to both DAZ and AAC. In the absence of deoxycytidine, all three agents were either equally inhibitory or slightly more inhibitory to the growth of CFU-GM than to HL-60, whereas administration of deoxycytidine in ten to one-hundred fold excess protected CFU-GM to a greater extent than HL-60. In contrast, administration of high concentrations of drugs, e.g. 10 −5−10 −4 M, in conjunction with excess deoxycytidine exhibited greater toxicity toward CFU-GM than toward HL-60/Ara-C. Coadministration of deoxycytidine in ten-fold excess reduced the total intracellular accumulation and DNA incorporation of each analog in HL-60 cells by approx. 50%, whereas a hundred fold excess was associated with greater than a 90% reduction in these values. These studies demonstrate that deoxycytidine may antagonize the effects of Ara-C, DAZ and AAC in both normal and leukemic human myeloid cells, and that at low drug concentrations the degree of protection may be greater for normal elements. However, regimens employing high drug concentrations in conjunction with deoxycytidine do not appear to exert a selective inhibitory effect toward a highly resistant leukemic subvariant. These data suggest that alternative deoxycytidine/deoxycytidine analog dose relationships and schedules must be sought which are capable of selectively eradicating resistant cells.

In-vitro effects of antineoplastic prostaglandins on human leukemic cell growth and normal myelopoiesis

The effects of prostaglandin (PG)E 1, PGD 2 and 9-deoxy-Δ 9-PGD 2 (PGJ 2) on the clonogenic growth of six kinds of human leukemic cell lines (K562, KG1, HL60, U937, THP1 and Molt4) and normal human myeloid progenitor cells (CFU-GM) were studied using semisolid agar cultures. While the degree of suppression of leukemic growth by PGE 1 varied from cell line to cell line, PGD 2 and PGJ 2 equally suppressed the growth of all leukemic cell lines. The potency of growth inhibition was as follows: PGJ 2 > PGD 2 > PGE 1. The increase of cellular cAMP level induced by prostaglandin treatment did not parallel their cytotoxic potency. Normal myeloid colony formation was also suppressed by PGE 1, PGD 2 or PGJ 2. In contrast to the preferential inhibition of macrophage colony formation by PGE 1 such lineage-selective suppression was not observed for PGD 2 or PGJ 2. These findings suggest that PGD 2 and PGJ 2 potently inhibit the leukemic growth by a different mechanism from that of PGE 1 and by a cAMP-independent mechanism. These prostaglandins seem to be promising chemotherapeutic agents for acute leukemia.

Effect of 4-hydroperoxycyclophosphamide on leukemic and normal human myeloid progenitor cells

The sensitivity of myeloid progenitor cells from normal subjects (N-CFU-GM) and from leukemic patients in complete remission (LR-CFU-GM) to 4-hydroperoxycyclophosphamide (4-HC) were compared to the sensitivity of leukemic progenitor cells (L-CFU) to this drug. The results were expressed as the dose of 4-HC needed to kill 90% (TD 90) of the progenitor cells. The mean TD 90 were respectively for N-CFU-GM : 59(± 11 S.E.M.) nM ml −1 and for L-CFU 79(± 6 S.E.M.) nM ml −1. Thus, L-CFU were equally sensitive to 4-HC as N-CFU-GM. Moreover, the mean TD 90 for LR-CFU-GM was 87 (± 5 S.E.M.) nM ml −1. Thus, the sensitivity of N-CFU-GM and LR-CFU-GM did not differ significantly from that of L-CFU. These results are not encouraging for the use of 4-HC in vitro to eliminate the residual leukemic cells from autologous bone marrow of AML patients in complete remission. The sensitivity of L-CFU was modified neither by previous cytoreductive therapy (different from cyclophosphamide) nor by the time elapsed since diagnosis of AML.

Retinoic acid enhances colony-stimulating factor-induced clonal growth of normal human myeloid progenitor cells in vitro

We studied the effect of vitamin A and its analogues (retinoids) on the clonal growth in vitro of normal human myeloid progenitor cells. Normal human bone marrow cells were cultured in soft gel in the presence of a source of colony-stimulating factor (CSF), and various retinoids, and the number of granulocyte-macrophage colonies (CFU-GM) were scored. The addition of 3 × 10 −8 to 3 × 10 −6 M retinoic acid to culture plates containing CSF markedly increased the number of myeloid colonies as compared with culture plates containing CSF alone. Maximal stimulation occurred at a concentration of 3 × 10 −7 M retinoic acid which increased the mean number of colonies by 213 ± 8 % (±S.E.) over plates containing CSF alone. Retinal or retinyl acetate was less potent than retinoic acid, and retinol (vitamin A) had no effect on colony growth. Retinoic acid had no direct CSF activity nor did it stimulate CSF production by the cultured bone marrow cells. Our studies show for the first time that retinoids can stimulate granulopoiesis in vitro and we suggest that this stimulation may be mediated by increased responsiveness of the granulocyte-macrophage progenitors to the action of CSF.