The precise mechanism whereby granulocytes proliferate when haematopoietic colony stimulating factors (CSFs) are used in neutropenic cancer patients is poorly understood. The purpose of this study was to investigate whether these cytokines bring about leucocyte proliferation by increasing the levels of multiple forms of dihydrofolate reductase (DHFR). Blood samples were collected from 36 cancer patients (25 males and 11 females) with chemotherapy-induced neutropenia. One sample of blood from each patient was obtained before therapy either with CSF, such as granulocyte colony stimulating factor (G-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF) or with placebo, and another one at the time of resolution of neutropenia. Peripheral blood leucocytes in these blood samples were counted, separated and lysed. From lysates, cytoplasmic samples were prepared and analyzed for active DHFR by a methotrexate-binding assay and for total immunoreactive DHFR by an enzyme linked immunosorbent assay. The increase in total leucocyte count (TLC) was most prominent (P < 0.005) in the CSF group and less so (P < 0.05) in the placebo group. The mean +/- SD concentration values of active DHFR before and after stimulation with GM-CSF found were to be 0.34 +/- 0.4 ng/mg protein and 0.99 +/- 0.82 ng/mg protein, respectively, and in the group treated with G-CSF, 0.24 +/- 0.32 ng/mg protein and 1.18 +/- 2.4 ng/mg protein, respectively. This increase in active DHFR after stimulation with CSF was statistically significant (P <0.05). Similarly, concentration values of immunoreactive but nonfunctional form of DHFR (IRE) were 110 +/- 97 ng/mg protein and 605 +/- 475 ng/mg protein before and after stimulation with GM-CSF, and 115 +/- 165 ng/mg protein and 1,054 +/- 1,095 ng/ mg protein before and after stimulation with G-CSF. This increase in concentration of IRE after stimulation with GM-CSF or G-CSF was statistically significant (P < 0.005). In the control group, there was an increase in the concentration of both active DHFR and IRE after treatment with placebo. However, this was not statistically significant. Resolution of neutropenia was quicker in the groups treated with CSF compared to the control group. Results of this study indicate that colony stimulating factors (G-CSF and GM-CSF) induce white cell proliferation by increasing the levels of multiple forms of DHFR.
Adolescence ; Adult ; Cell Division/drug effects ; Child ; Female ; Granulocyte Colony-Stimulating Factor/therapeutic use ; Granulocyte Colony-Stimulating Factor/pharmacology* ; Granulocyte Colony-Stimulating Factor/adverse effects ; Granulocyte-Macrophage Colony-Stimulating Factor/therapeutic use ; Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology* ; Granulocyte-Macrophage Colony-Stimulating Factor/adverse effects ; Human ; Isoenzymes/metabolism ; Isoenzymes/biosynthesis ; Leukocyte Count ; Leukocytes/pathology ; Leukocytes/enzymology ; Leukocytes/drug effects ; Male ; Middle Age ; Neoplasms/enzymology ; Neoplasms/drug therapy ; Neoplasms/blood* ; Neutropenia/metabolism* ; Neutropenia/chemically induce ; Neutropenia/blood ; Tetrahydrofolate Dehydrogenase/metabolism* ; Tetrahydrofolate Dehydrogenase/biosynthesis

OBJECTIVETo study the growth inhibition and apoptosis induction effects of bufalin on human osteosarcoma cell lines in vitro.

METHODSU-2OS and U-2OS/methotrexate (MTX) 300-resistant cell lines were treated with bufalin. Cell viability was assessed by MTT assay. Cell-cycle status, apoptosis-inducing effects, and the expression of apoptosis-related proteins were evaluated by flow cytometry, fluorescent staining, DNA fragmentation assay, and Western blotting.

RESULTSBufalin inhibited cell growth in both U-2OS and U-2OS/MTX300 cells. The IC(50) values of bufalin for U-2OS and U-2OS/MTX300 cells were (8.49 ± 2.1) ng/ml and (10.19 ± 1.7) ng/ml, respectively. The induction of G(2)/M cell-cycle arrest was also seen in the bufalin-treated cells. The bufalin-induced apoptosis was confirmed by increased expression of tumor suppressor protein p53, bax and decreased expression of bcl-2.

CONCLUSIONBufalin inhibits the growth of and induces apoptosis in both MTX-sensitive and MTX-resistant human osteosarcoma U-2OS cell lines. The apoptosis-inducing effect of bufalin is not influenced by the presence of high levels of DHFR.

Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Bone Neoplasms ; metabolism ; pathology ; Bufanolides ; pharmacology ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Drug Resistance, Neoplasm ; Humans ; Methotrexate ; pharmacology ; Osteosarcoma ; metabolism ; pathology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Tetrahydrofolate Dehydrogenase ; metabolism ; Tumor Suppressor Protein p53 ; metabolism ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo explore the feasibility of transfecting DHFR (human double-mutant dihydrofolate reductase) gene into mouse bone marrow cells and the effect of resistance to high dose MTX chemotherapy.

METHODSAfter DHFR gene was transfected into mouse bone marrow cells with retroviral vector, the cells were treated with methotrexate (MTX) and then CFU-GM (granulocyte-macrophage colony-forming unit) assay was performed. Peripheral blood leucocytes and platelets, body weight and survival rate were observed. After treatment with high dose MTX, the expression of drug resistance gene was checked by RT-PCR in the transfected bone marrow cells.

RESULTSSFG-F/S-NeoR gene-transfected mice bone marrow cells yielded drug-resistance colonies to MTX (donor mice: 15.8%, recipient mice: 18.0%, control: 0) The peripheral blood leucocytes and platelets, body weight recovered gradually and the survival rate was 83.3% at the 40th day, while 0 in controls in gene transfected mice after large dose MTX treatment. RT-PCR of transgenic mouse marrow cells showed the band of F/S gene (400 bp).

CONCLUSIONDHFR gene can not only be integrated and expressed in bone marrow cells but also improve their drug-resistence to MTX.

Animals ; Antimetabolites, Antineoplastic ; pharmacology ; Bone Marrow Cells ; cytology ; drug effects ; metabolism ; Bone Marrow Transplantation ; Cells, Cultured ; Drug Resistance, Neoplasm ; genetics ; Erythrocyte Count ; Genetic Vectors ; Leukocyte Count ; Male ; Methotrexate ; pharmacology ; Mice ; Mice, Inbred BALB C ; Mutation ; Retroviridae ; genetics ; Survival Analysis ; Tetrahydrofolate Dehydrogenase ; genetics ; metabolism ; Transfection