The Hematological Effects of Folate Analogs: Implications for Using the Dihydrofolate Reductase Gene for In Vivo Selection

Previous studies have shown that dihydrofolate reductase (DHFR) gene transfer protects marrow from methotrexate (MTX)-mediated toxicity; however, MTX treatment in vivo has not convincingly been shown to enrich DHFR-transduced progenitors or stem cells. Experiments were performed to better characterize the hematological effects of MTX, and maneuvers were tested with the aim of improving the utility of MTX as an agent for in vivo selection. Progenitors were assayed as colony forming unit cells in culture (CFU-C) and in the spleens of irradiated mice (day 11 CFU-S). A single injection of MTX at doses up to 250 mg/kg (more than three times the LD₁₀) failed to reduce CFU-C numbers significantly in the femur or spleen assayed 1–3 days later. However, consistent declines in the number of mononuclear cells per femur reflected a significant depletion of nonclonogenic precursor cells. Preceding administration of pegylated stem cell factor (SCF), 100 μg/kg per day, increased CFU-C killing by a single dose of 5-fluorouracil (5-FU) 15- to 65-fold in the femur, and 5- to 15-fold in the spleen, consistent with previous reports. In contrast, despite preceding SCF administration there was no significant progenitor killing by MTX. Similar results were obtained using a second folate analog, trimetrexate. These results suggest that the mechanism by which folate analogs exert their hematological toxicity is through the depletion of relatively mature, nonclonogenic precursor cells, and not by killing progenitors. This information is relevant to the use of DHFR in gene therapy protocols, and suggests that folate analogs are poorly suited agents for selection at the level of clonogenic progenitor cells in vivo.

Overview summary

Dihydrofolate reductase (DHFR) gene transfer has potential clinical applications for the prevention of methotrexate (MTX)-mediated myelosuppression and for use as a dominant selectable marker to permit the enrichment of genetically modified, drug-resistant hematopoietic cell populations in vivo. Although DHFR gene transfer clearly protects marrow from MTX toxicity, evidence for the in vivo enrichment of DHFR-containing clonogenic progenitors or stem cells is lacking. Studies presented here provide a likely basis for this phenomenon. We show that clonogenic progenitor cells are resistant to extraordinarily high doses of MTX. In contrast, the total number of mononuclear cells per femur falls significantly in response to MTX, indicating a significant depletion of relatively mature, nonclonogenic precursor cells. Although preceding administration of stem cell factor (SCF) significantly augments 5-fluorouracil (5-FU)-mediated progenitor depletion, this strategy fails to significantly augment MTX's effect on progenitors. Similar results were obtained using a second folate analogue. Because of the resistance of clonogenic progenitor cells to killing by folate analogues, these results suggest that this class of drug is poorly suited for the selection of transduced progenitors in vivo.