Gretchen N. Schwartz

Thrombopoietin and Chemokine mRNA Expression in Patient Post-Chemotherapy and In Vitro Cytokine-Treated Marrow Stromal Cell Layers

CD34+ cells and megakaryocyte progenitors were lower in marrow from patients after hematological recovery from the first cycle of 5‐fluorouracil, leucovorin, adriamycin, cyclophosphamide (FLAC) chemotherapy plus PIXY321 (GM‐CSF/interleukin 3; IL‐3 hybrid) than in FLAC + GM‐CSF or pre‐FLAC marrows. Marrow stromal layers, an in vitro model of the marrow microenvironment, express a combination of stimulatory and inhibitory factors that modulate hematopoietic progenitor cell proliferation and differentiation. The TaqMan assay and quantitative reverse transcriptase‐polymerase chain reaction were used to measure monocyte chemoattractant protein‐1 (MCP‐1), melanoma stimulatory growth activity, and monokine inducible by interferon‐γ (Mig) (inhibitory chemokines for primitive or megakaryocyte progenitors) mRNA levels in in vitro PIXY and GM‐CSF‐treated and patient post‐FLAC marrow stromal layers. Chemokine mRNA was increased after in vitro GM‐CSF and to a lesser extent after PIXY treatment. MCP‐1 mRNA levels were fivefold higher in FLAC + PIXY than in FLAC + GM‐CSF layers, and Mig mRNA was elevated in FLAC + GM‐CSF layers. Thrombopoietin (TPO), insulin‐like growth factor I (IGF‐I), and IGF‐II (stimulatory factors for primitive and megakaryocyte progenitors) mRNA were also measured. TPO mRNA levels were 30% lower in GM‐CSF and PIXY‐pretreated than in control layers with no decrease in IGF mRNA. TPO mRNA in stromal layers of patients who developed grade 3 thrombocytopenia (platelets < 20 × 109/l) during the third cycle of FLAC was only 24% of levels in stromal layers of marrow from other post‐FLAC patients. Results demonstrate that patient and in vitro treatment had modulatory effects on TPO and chemokine mRNA expression in marrow stromal layers.

5-Fluorouracil-mediated Thymidylate Synthase Induction in Malignant and Nonmalignant Human Cells

Thymidylate synthase (TS, EC 2.1.1.45) is an important target enzyme for the fluoropyrimidines used in cancer chemotherapy. Studies have documented a 2- to 4-fold induction of TS protein following 5-fluorouracil (5-FU) treatment of malignant cells. We measured the effect that 5-FU exposure had on TS protein expression in nonmalignant human breast (MCF-10 and HBL-100), colorectal (ATCC Co18, Co112, and Co33), and bone marrow cells along with malignant breast (MCF-7) and colon (NCI-H630) cells. Twenty-four hours after plating, cells were treated with 0.01 to 10 microM of 5-FU for a period of 24 hr. TS was quantitated by Western immunoblot using monoclonal antibody TS106. Absolute levels of TS in nonmalignant cells were substantially lower than in the malignant lines, ranging from approximately 40% in HBL-100 cells to less than 10% in the colon lines. An approximately two-fold induction in the level of TS was found for all cell lines examined, and there was a strong dependence on 5-FU exposure concentration in free TS levels of MCF-WT, and total TS levels of H630-WT, normal bone marrow, and MCF-10 cells. The induction of TS following 5-FU exposure is a generally observed phenomenon in both malignant and nonmalignant cells, suggesting that a selective means for inhibiting this induction may be critical for the development of alternative therapeutic strategies using 5-FU and the antifolate TS inhibitors.