Leucovorin antagonizes the effects of trimetrexate on mouse hemopoietic progenitors.

Trimetrexate (2, 4, diamino -5- methyl - 6 [3, 4, 5, trimethoxyanilino) methyl] quinazoline) (TMQ) is a non-classic folate antagonist that is used as an antineoplastic and antipneumocystis agent with promising results. TMQ and methotrexate (MTX) toxicities are comparable. Leucovorin (N-5-formyltetrahydrofolate) (LV) is used to prevent the toxic effects of MTX. In this study the effects of LV on TMQ induced hemopoietic progenitor damage are studied in a murine model. Changes of pluripotent stem cells (colony forming units spleen, CFU-S), granulocyte-macrophage committed progenitors (GM-CFC), erythroid committed progenitor (BFU-E) levels in the bone marrow were followed after administration to mice of a single dose of TMQ or of simultaneous injection of TMQ and LV. Results show that the latter significantly reduces the effects of the former on peripheral blood cells and on hemopoietic progenitors.     

Implications for improved high-dose methotrexate therapeutic effects in cultured human breast cancer and bone marrow cells

The cytotoxicity of high-dose methotrexate (MTX), 10 and 100 microM, and 5-fluorouracil (5-FU) combinations is independent of sequence in human MDA-MB-436 breast carcinoma cells. The growth inhibitory effects of 10 and 100 microM MTX are 22.54+/-1.56% and 16.20+/-0.74%, respectively, of the control rate. When the MTX and 5-FU concentrations are 10 microM, antiproliferative effects of MTX 2 hr before 5-FU (MTX/5-FU) and 5-FU 2 h before MTX (5-FU/MTX) are 25.17+/-1.23% and 25.60+/-1.28% of the control rate, respectively. The percentage of control rates for 5-FU alone is 94.89+/-1.35%. The growth rates of MDA-MB-436 cells in 100 microM MTX and 10 microM 5-FU are 15.19+/-0.62% (MTX/5-FU) and 16.53+/-0.85% (5-FU/MTX) of the control rate. The growth of cancer cells in the presence of 5-FU alone is 93.82+/-1.69% of the control rate. A comparison of the cell-killing effects of MTX and the nonpolyglutamable antifolate trimetrexate (TMQ) alone and in combination with 5-FU was performed to indirectly explore the role of polyglutamylation in breast cancer and bone marrow cells. The comparisons were made in equitoxic concentrations (10 microM) of MTX and TMQ and the time of exposure was the same. The inhibitory effects of TMQ, TMQ/5-FU, and 5-FU/TMQ in breast cancer cells were identical, but significantly less than MTX, MTX/5-FU, and 5-FU/MTX. The interaction between TMQ and MTX, TMQ/5-FU and MTX/5-FU, and 5-FU/TMQ and 5-FU/MTX was quantitatively similar in bone marrow. (Significant protection occurred in bone marrow cells exposed to 5-FU/TMQ and 5-FU/MTX.) Because the effects of 5-FU/MTX and 5-FU/TMQ on bone marrow were the same, it is unlikely that polyglutamylation plays a significant role in the protective effects of 5-FU. However, the greater inhibitory effect of MTX or MTX and 5-FU combinations, when compared with TMQ or TMQ and 5-FU, suggests that polyglutamylation of MTX may contribute to the cytotoxicity of this antifolate to breast cancer cells. Hence, these studies suggest that a priming and nontoxic dose of 5-FU before high-dose MTX sustains MTX cytotoxicity in breast cancer and protects against MTX toxicity to bone marrow progenitor cells.     

Effects of methotrexate and of the "nonclassical" folate antagonist trimetrexate on human leukemia cells

Twenty-five samples of fresh malignant cells of patients with acute leukemia (16 with acute myeloblastic leukemia, five with acute lymphoblastic leukemia, and four with chronic myelocytic leukemia in blast crisis) and of two patients with colon carcinoma were exposed for 1 hr to different concentrations of methotrexate (MTX) or trimetrexate (TMQ). In all samples, TMQ was at least one log more potent than MTX in inhibiting [3H]deoxyuridine incorporation into DNA. Cells relatively resistant in vitro to MTX also displayed decreased sensitivity to TMQ, although TMQ "resistance" was usually much less pronounced. In eight of the 25 leukemia samples, intracellular drug levels after exposure in vitro could also be measured. The intracellular levels of TMQ were 6 to 64 times higher than those of MTX, indicating that the difference in potency of these drugs may be related to the difference in intracellular accumulation. The intracellular levels of both agents varied widely and were not directly related to the degree of DNA synthesis inhibition. There was, however, a clear positive correlation between the intracellular ratio [TMQ]/[MTX] and the deoxyuridine incorporation after exposure to MTX. This observation suggests that 1) intracellular TMQ levels may be used as an internal standard to correct steady state intracellular MTX levels for variations such as cell size, and 2) in analogy to tissue-culture models, decreased intracellular accumulation of MTX may contribute to clinically encountered drug resistance.     

Cytotoxicity of methotrexate and trimetrexate and its reversal by folinic acid in human leukemic CCRF-CEM cells with carrier-mediated and receptor-mediated folate uptake

The cytotoxic effects of the antifolates methotrexate (MTX) and trimetrexate (TMQ) were investigated for two human leukemic CCRF-CEM cell lines, one expressing the "classical" reduced folate/MTX carrier (CEM-RF), another lacking this carrier but expressing a membrane associated folate binding protein (CEM-FBP). CEM-FBP cells were found to be highly resistant to MTX compared to CEM-RF cells, especially in short exposures. For example, after 4 h incubation, IC50 values for MTX were 251 microM and 0.98 microM for CEM-FBP and CEM-RF cells, respectively. On the other hand, CEM-FBP cells were much more sensitive to the lipophilic antifolate TMQ than CEM-RF cells as shown by IC50 values (after 4 h of exposure) of 0.059 microM and 7.5 microM, respectively. Finally, the reversal of TMQ cytotoxicity by folinic acid was significantly impaired for CEM-FBP cells, in contrast to CEM-RF cells. These results indicate that the nature of the membrane transport system for folates can be a critical determinant in tumor cell sensitivity or resistance to antifolates.     

突变的MTX耐药基因对骨髓造血干细胞的保护作用

目的研究转染耐药基因后对造血干细胞的保护作用方法以脂质体为载体，把二氢叶酸还原酶（ＤＨＦＲ）突变的对氨甲喋呤（ＭＴＸ）耐药的基因转染到小鼠造血干细胞，再输回至小鼠体内后，对小鼠进行为期９周的大剂量ＭＴＸ化疗。结果转染ＭＴＸ耐药基因组小鼠化疗前后的粒细胞总数及体重均较稳定，而未转染基因组小鼠的粒细胞总数及体重呈明显下降（Ｐ＜０．０５）。小鼠存活率在转染基因组较对照组有明显提高。从小鼠脾脏提取ＤＮＡ，经ＰＣＲ扩增后，证实转染基因组有耐药基因的高效表达。结论此实验为肿瘤患者在大剂量化疗时，进行保护造血功能，提高化疗疗效的实验研究和临床应用，提供有参考价值的资料。     

Enzyme studies of methotrexate-resistant human leukemic cell (K562) subclones.

Five methotrexate (MTX)-resistant K562 cell subclones (K562/MTX-1 approximately -5) were established and were examined for mechanisms of drug resistance. Impairment of MTX-polyglutamate formation, with membrane transport alteration, in the resistant cells was demonstrated in the previous studies (Koizumi, S. (1988) Jpn. J. Cancer Res. 79, 1230). Further analysis of sensitivity of the cells to trimetrexate (TMQ), which is not polyglutamated and does not require the reduced folate transporter, but is a potent inhibitor of human DHFR, revealed a modest decrease in sensitivity to TMQ (2.4- to 15-fold). Enzyme studies showed that the dihydrofolate reductase (DHFR) activities of these resistant subclones were very similar to that of the parent cells. The number of binding sites of these subclones for MTX calculated from Scatchard analysis was increased up to 7-fold in the K562/MTX-1 and -4 subclones and up to 3-fold in the other 3 subclones as compared to the parent cells. KD values of MTX for the DHFR in the K562/MTX-1 and -4 subclones also appeared to be altered relative to the parent cell line. Further, thymidylate synthase (TS) activity of the resistant subclones was reduced to 50% in K562/MTX-1 and -4 cells, and to 11-25% in the other subclones as compared to the parent cell line. These findings suggest that antifolate resistance in the newly established K562/MTX subclones in multifactorial with polyglutamation and transport defects accounting for the majority of resistance to MTX, and that alteration in the binding affinity of DHFR for MTX and diminished levels of TS may contribute to the 'residual' drug resistance to TMQ and have importance with respect to MTX.     

Cytotoxicity of Trimetrexate against Antifolate-resistant Human T-Cell Leukemia Cell Lines Developed in Oxidized or Reduced Folate

Cytotoxicity of trimetrexate (TMQ), a lipophilic dihydrofolate reductase inhibitor, was examined in antifolate-resistant human T-cell leukemia cell lines developed in oxidized or reduced folate. An approximately 60-fold methotrexate (MTX)-resistant subline was developed in oxidized folate (pter-oylglutamic acid: PGA) (CCRF-CEM/MTX₆₀-PGA) from human T-cell leukemia cell line CCRF-CEM; this line exhibited impaired membrane transport of the drug. Further enhancement of MTX resistance resulted in selection of an approximately 5000-fold MTX-resistant subline (CCRF-CEM/ MTX₅₀₀₀-PGA), which showed increased dihydrofolate reductase activity due to gene amplification in addition to further impairment of MTX transport. An approximately 140-fold MTX-resistant subline, and then a 1500-fold MTX-resistant subline were developed in reduced folate (10 nM leucovorin) (CCRF-CEM/MTXm-LV and CCRF-CEM/MTX₁₄₀-LV); they exhibited increased dihydrofolate reductase due to gene amplification accompanied by increased intracellular drug accumulation of MTX. While CCRF-CEM/MTX₁₄₀-LV and CCRF-CEM/MTX₁₅₀₀-LV cells showed cross-resistance to TMQ, CCRF-CEM/MTX₆₀-PGA and CCRF-CEM/MTX₅₀₀₀-PGA cells were at least as sensitive to TMQ as the parent cells. TMQ was more potent against approximately 200-fold N¹⁰-propargyl-5,8-dideazafolic-acid (CB3717)-resistant human T-cell leukemia MOLT-3 sublines developed in PGA (MOLT-3/CB3717₂₀₀-PGA) or leucovorin (MOLT-3/CB3717₂₀₀-LV), as compared to the parent cells; MOLT-3/CB3717₂₀₀-PGA and MOLT-3/CB3717₂₀₀-LV cells were resistant to CB3717 by virtue of impaired transport, only the former possessing gene amplification of thymidylate synthase. The Cytotoxicity of TMQ in both MOLT-3/CB3717₂₀₀-PGA and MOLT-3/CB3717₂₀₀-LV cells was reduced by addition of leucovorin in a dose-dependent manner, suggesting intracellular folate deficiency as a cause of TMQ sensitivity. These results demonstrate that TMQ overcomes transport-impaired antifolate resistance, irrespective of gene amplification of dihydrofolate reductase or thymidylate synthase. Types of folate used during the development of antifolate resistance seem to be important in relation to the mechanism of TMQ responsiveness as well as that of antifolate resistance.     

Mechanism of the induction of the differentiation of HL-60 leukemia cells by antifolates

The classic inhibitor of dihydrofolate reductase (DHFR), methotrexate (MTX), has been shown to be an effective inducer of the differentiation of HL-60 promyelocytic leukemia cells (Bodner A.J. et al.; J. Natl. Cancer Inst. 67:1025-1030; 1981). We have obtained evidence that induction of the differentiation of these cells by MTX, as well as by other folic acid antagonists, is the result of the effects of these agents on purine and thymine nucleotide biosynthesis. Thymidine (10 microM) completely blocked both the cytotoxicity and induction of differentiation produced by the specific inhibitor of thymidylate synthase (TS), N10-propargyl-5,8-dideazafolic acid (CB-3717). Thymidine also blocked the acute cytotoxicity caused by MTX and trimetrexate (TMQ); the induction of differentiation and the loss of proliferative capacity, however, were only partially prevented by thymidine. Hypoxanthine (100 microM), which completely restored antifolate-depleted purine nucleotide levels, had no effect on either the cytotoxicity or the induction of maturation produced by these agents. The growth inhibitory effects and the induction of differentiation caused by dideazatetrahydrofolic acid (DDATHF), which acts on de novo purine nucleotide biosynthesis rather than on DHFR or TS, was completely prevented by hypoxanthine. Hypoxanthine also completely prevented the inhibition of cellular replication and induction of differentiation by MTX and TMQ when combined with thymidine. The findings suggest that the depletion of intracellular thymine nucleotide levels by the antifolates, MTX, TMQ, and CB-3717 is the primary event involved in the maturation of HL-60 leukemia cells produced by these agents and that maturation occurs concomitantly with a high level of cytotoxicity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Polyglutamylation, an important element in methotrexate cytotoxicity and selectivity in tumor versus murine granulocytic progenitor cells in vitro

Methotrexate (MTX) cytotoxicity was assessed by clonogenic assay in agar with granulocytic progenitor cells from mouse bone marrow and in the Ehrlich ascites tumor, the K562 human chronic myelogenous leukemia, and the P388 murine leukemia. After a 2-hr exposure to MTX, the concentrations necessary to produce 50% inhibition of colony formation were 100, 25, 1.2, and 0.25 microM, respectively. This was inversely related to the ability of the tumor cells to accumulate MTX polyglutamyl derivatives and consistent with the observation that no polyglutamyl derivatives were observed in granulocytic progenitor cells after a 2-hr exposure to 5 micron MTX. Continuous exposure to glycine (200 microM)-adenosine (100 microM)-thymidine (10 microM) (GAT), along with MTX, protected cells from MTX cytotoxicity by circumventing the requirement for tetrahydrofolate cofactors. However, while the presence of GAT during a 2-hr exposure to 5 microM MTX is sufficient to protect granulocyte progenitor cells from MTX cytotoxicity, the presence of GAT, even after MTX is removed, is required to protect tumor cells. Indeed, if, after a 2-hr exposure of tumor cells to MTX and GAT, both MTX and GAT are removed before plating in agar, cytotoxicity to tumor cells was expressed. This sustained antitumor effect of MTX correlates with the rapid build-up of polyglutamyl derivatives that are retained in the cell even after extracellular and intracellular monoglutamate is eliminated. This is in contrast to granulocytic progenitor cells which appear to be susceptible to the drug only during the period of exposure to the monoglutamate under these conditions. The data strongly suggest that the marked differences in the accumulation of MTX polyglutamyl derivatives between the tumor cells studied and the murine bone marrow granulocytic progenitor cells are an important element in MTX selectivity.     

Hemopoietic features of splenectomized mice bearing IL-3 producing T cell leukemia

Hemopoietic responses were studied in splenectomized mice after transplantation of the hemopoietic colony stimulating activity producing leukemia, L8313. In non-splenectomized mice, the number of peripheral leukocytes was elevated to 4 × 10⁵/mm³ after transplantation. In contrast, it was below 1.7 × 10⁴/mm³ in splenectomized mice. Total cell, hemopoietic stem cell and progenitor cell numbers in the bone marrow decreased in both groups. Serum from splenectomized mice contained multi-colony stimulating activity although less effective than that from non-splenectomized mice. Extramedullary hemopoiesis was observed in splenectomized mice. These results suggest that invasion of leukemic cells to bone marrow results in a damage of hemopoietic microenvironment.     

Long-term effects of cytostatic agents on the hemopoietic stroma: a comparison of four different assays

We have compared four assays to detect hemopoietic stromal damage induced by various cytostatic agents in young (4-week old) and adult (12-week old) mice. These assays included: (a) quantitation of the hemopoietic stem cell content of subcutaneously implanted spleens and femurs, (b) quantitation of fibroblastic colony-forming units per femur and spleen, (c) quantitation of the growth of normal hemopoietic progenitor cells in irradiated cytostatic drug-treated mice, and (d) measurement of splenic hemopoietic stem cell accumulation in response to bacterial lipopolysaccharide-induced hemopoietic stress. Busulfan caused a short- and long-term hemopoietic stromal defect. However, the four assays used showed different kinetics and severity of the stromal damage. Cyclophosphamide treatment resulted in a short-term stromal damage which was repaired within one week to three months, depending on the assay used. Methotrexate and vincristine did not cause long-term stromal damage as measured by the four assays used, whereas a short-term splenic stromal damage was detected using the subcutaneous implantation technique. No significant differences in stromal sensitivity to drug treatment were observed between young and adult mice. The presented data suggest that the four assays used to study stromal integrity measure different components of the hemopoietic microenvironment, and indicate that the use of a single assay may well lead to erroneous interpretations.     

Cross resistance of pleiotropically drug resistant P338 leukemia cells to the lipophilic antifolates trimetrexate and BW 301U

Several antifolate compounds were examined for their cytotoxic activity in a pleiotropically resistant P388 cell line (P388R). The sensitivity of P388R cells to methotrexate (MTX) and the lipophilic antifols, metoprine and methotrexate gamma-mono t-butyl ester (MTX-gamma-t-butyl ester) were comparable with that activity observed in the parental cell line (P388S). P388R cells were, however, resistant to 2 other lipophilic antifols, trimetrexate (TMQ) and BW 301U. The degree of resistance to TMQ and BW 301U was 22-fold and 15-fold, respectively and could be partially overcome by the calcium channel blocker, verapamil (VER) or the detergent Tween 80. Transport studies showed that net accumulation of trimetrexate was markedly reduced in P388R cells resulting in a steady-state level which was 25% of the sensitive line. This impaired uptake was reversed by 5 micrograms/ml VER which increased the steady-state to a level comparable to P388S. P388R also exhibited a 50% reduction in the unindirectional influx rate, however, this defect could not be reversed by VER. The resistance of P388R cells to TMQ and BW 301U and their potentiation by VER extends pleiotropic resistance to yet another class of drugs which have important clinical implications.     

Postirradiation treatment with granulocyte colony-stimulating factor and preirradiation WR-2721 administration synergize to enhance hemopoietic reconstitution and increase survival.

These studies tested whether WR-2721 could be used to protect hemopoietic stem cells, which after irradiation could be stimulated by granulocyte colony-stimulating factor (G-CSF) to proliferate and reconstitute the hemopoietic system. Female C3H/HeN mice were administered WR-2721 (4 mg/mouse, i.p.) 30 min before 60Co irradiation and G-CSF (2.5 micrograms/mouse/day, s.c.) from days 1-16 after irradiation. In survival studies, saline, G-CSF, WR-2721, and WR-2721 + G-CSF treatments resulted in LD50/30 values of 7.85 Gy, 8.30 Gy, 11.30 Gy, and 12.85 Gy, respectively. At these LD50/30 values, the dose reduction factor (DRF) of 1.64 obtained in combination-treated mice was more than additive between the DRF's of G-CSF-treated mice (1.06) and WR-2721-treated mice (1.44). Bone marrow and splenic multipotent hemopoietic stem cell (CFU-s) and granulocyte-macrophage progenitor cell (GM-CFC) recoveries were also accelerated most in mice treated with WR-2721 + G-CSF. In addition, mice treated with WR-2721 + G-CSF exhibited the most accelerated peripheral blood white cell, platelet, and red cell recoveries. These studies (a) demonstrate that therapeutically administered G-CSF accelerates hemopoietic reconstitution from WR-2721-protected stem and progenitor cells, increasing the survival-enhancing effects of WR-2721 and (b) suggest that classic radioprotectants and recombinant hemopoietic growth factors can be used in combination to reduce risks associated with myelosuppression induced by radiation or radiomimetic drugs.     

High proliferation of granulocyte-macrophage progenitors in tumor-bearing mice

Cancer may affect hemopoiesis by altering the proliferative status of hemopoietic progenitor cells. In Lewis lung carcinoma (LLC), the proliferative rate of the granulocyte-macrophage colony-forming unit (culture) (GM-CFUc) was studied using in vivo hydroxyurea techniques. The disposal of mature elements to the periphery was also monitored during tumor growth. Neutrophilia, anemia, and splenic hypertrophy developed during the course of the disease. By Day 6 post-tumor implant, myeloid hyperplasia of the marrow was evident, but the content of GM-CFUc in LLC mice was similar to that of control. However, by Day 11, the marrow of LLC mice displayed an increased concentration of GM-CFUc, which tripled by Day 19. There was an increased percentage of proliferating GM-CFUc in LLC mice by Day 6 which was highest by Day 11 and thereafter declined. The level of colony-stimulating activity was higher in the serum of tumor bearers than in that of controls. The early increase in proliferative rate of these early hemopoietic precursors can account for the later accumulation of GM-CFUc and myeloid elements in the marrow. Increased cycling of hemopoietic stem cells raises questions concerning the potential for early exhaustion of hemopoietic progenitor cells in these animals.     

A myelosclerotic syndrome in mice engrafted with cells producing high levels of leukemia inhibitory factor (LIF)

DBA/2 mice engrafted with FDC-P1 cells producing high levels of the leukemia-inhibitory factor (LIF) developed high circulating levels of LIF and a fatal syndrome including the accumulation of excess osteoblasts in the marrow and new bone formation. The mice developed a neutrophil leucocytosis, an enlarged spleen, and excess numbers of hemopoietic cells in the spleen and liver. Marrow cellularity was reduced with selective survival of granulocytic cells, but the frequency of hemopoietic progenitor cells in both the marrow and spleen was higher than in control mice. Megakaryocyte numbers were reduced in marrows with pronounced sclerosis. The disease state may represent a useful model of myelosclerosis, but it remains to be established whether the hemopoietic abnormalities in these mice are direct effects of LIF or secondary changes following occlusion of the marrow by osteosclerotic tissue.     

Comparison of leucovorin protection from variety of antifolates in human lymphoid cell lines

Summary Leucovorin requirements for protection of the T cell line CCRF-CEM and the B cell line LAZ-007 against the cytotoxic effects of a variety of antifolates were studied. Differential leucovorin protection for DDMP-induced growth suppression occurred in the opposite direction to that for MTX, with CCRF-CEM requiring less leucovorin than LAZ-007 for equivalent protection. A pattern of differential protection from DDMP different from that of protection from MTX was also seen for the cell lines RAJI and MOLT-4. Differential leucovorin protection was observed for the chain-extended MTX analogue PT441. The degree of differential protection was similar to that seen for MTX, and transport studies showed that PT441 was a weak inhibitor of tritiated MTX uptake into CCRF-CEM cells. Differential leucovorin protection was observed for the lipophilic antifolate trimetrexate glucoronate (TMQ) but the degree of differential protection was smaller than that seen for PT441 or for MTX. Since TMQ is not transported into cells by the reduced folate system, while PT441 is a weak competitive inhibitor of [³H]MTX transport, and since neither is polyglutamylated, these results support the conclusion reached in previous experiments that differential leucovorin protection of MTX is unlikely to be a transport-related phenomenon and is not due to an effect on polyglutamylation. In addition, the different patterns of relative leucovorin requirements for DDMP and MTX protection suggest that differential metabolism or catabolism of leucovorin does not account for differential protection.Abbreviations MTX methotrexate - HDMTX high dose MTX - HPLC high pressure liquid chromatography - [³H]dUrd tritiated deoxyuridine - DDMP 2,4-diamino-5-(3, 4 dichlorophenyl)-6-methylpyrimidine - PT44 N-(4-amino-4-deoxy-N¹⁰-methylpteroyl)-L--aminoadipate - TMO 2,4-diamino-5-methyl-6-(3,4,5-trimethoxy-anilino)methyl) quinazoline Supported by the National Cancer Institute of Canada and by Grant CA25394 from the National Cancer Institute     

Biological Activity and Intracellular Metabolism of ZD1694 in Human Leukemia Cell Lines with Different Resistance Mechanisms to Antifolate Drugs

The biological activity and cellular metabolism of ZD1694, a novel folate-based thymidylate synthase (TS) inhibitor, were analyzed in a human leukemia cell line, MOLT-3, and its antifolate-resistant sublines with different mechanisms of resistance to methotrexate (MTX), trimetrexate (TMQ) and N¹⁰-propargyl-5,8-dideazafolic acid (CB3717). MOLT-3/CB3717₄₀, which was selected for CB3717 resistance, demonstrated impaired membrane drug transport via reduced folate carrier (RFC) and lower accumulation of [³H]ZD1694-polyglutamates in the cells with a shift in the polyglutamate distribution profile to shorter chain length polyglutamates, indicating an alteration in polyglutamation capacity in this subline. Impaired RFC and reduced rate of polyglutamation could explain the cross-resistance (12-fold) of this subline to ZD1694. On the other hand, there was little or no cross-resistance to this drug in a subline (MOLT-3/TMQ₈₀₀) reportedly resistant to TMQ through impaired membrane transport for TMQ and an increase in dihydrofolate reductase (DHFR) activity. Total amount of ZD1694 polyglutamated to a level higher than diglutamate was approximately 1.7-fold higher in the TMQ-resistant cells than that in the parent cells, but a low degree of increase in TS activity in the cells counteracted the supposed increase in sensitivity to ZD1694. MOLT-3/TMQ₈₀₀-MTX₁₀₀₀₀ cells, which were established by sequential exposure of the TMQ-resistant cells to MTX and were previously shown to amplify mutated DHFR with low affinity for MTX, showed a decreased accumulation of polyglutamated ZD1694 as compared with the parent line and this was consistent with cross-resistance to ZD1694 in this subline. Overproduction of variant DHFR scarcely influenced the sensitivity to this drug. These results indicate that ZD1694 could overcome antifolate resistance through a mechanism such as amplified DHFR activity, and the biological activity of this drug against the cells paralleled the amount of polyglutamated drug inside the cells. Determination of polyglutamation capacity in tumor cells may allow prediction of sensitivity to this drug.     

Effects of prenatal irradiation on fetal, neonate, and young adult murine hemopoiesis

B6D2F1 mice received cobalt-60 radiation on day 10.5 of gestation at doses of 50 to 300 rad at a dose rate of 40 rad per min. These animals were studied at four selected age periods: (a) day 14.5 of gestation, (b) neonate, (c) juvenile, and (d) 13 week-old adult. Fetal liver cellularity, morphology, and hemopoietic progenitor cell concentration reflected injury after 200 rad. The 15 day-old mouse spleen cellularity was affected more than bone marrow cellularity, but greater radiation injury was reflected by bone marrow hemopoietic progenitor cells. Fluctuations from normal hematopoietic values were greater in the 15 day-old juvenile than in the 9 day-old neonate, commencing with 50 rad. These included peripheral blood parameters and marrow- and spleen-derived erythroid-, granulocytic- and megakaryocytic-progenitor cells. The consequences of prenatal irradiation (150 rad) were evident in the 13 week-old mouse. This was manifested by a reduced spleen cellularity and perturbations in concentrations of hemopoietic progenitor cells in the bone marrow.     

Amifostine promotes hemopoietic progenitor cell mobilization in patients with myelodysplastic syndrome

The multilineage hematological effects of the chemoprotective agent amifostine suggested administration of the drug to patients with myelodysplastic syndromes. Partial but encouraging responses were seen. The aim of this research was to further evaluate amifostine activity on the hemopoietic progenitor cells of myelodysplastic patients. We determined amifostine effects on in vitro growth of pre-treatment peripheral blood and bone marrow hemopoietic progenitor cells from myelodysplastic patients, and colony forming unit granulocyte-macrophage and burst forming unit erythroid levels in the peripheral blood of 7 myelodysplastic patients during and following a 3-week treatment with amifostine (200 mg/m2 i.v., three times a week). No variations were observed for hemoglobin, platelets, white blood cells and reticulocyte values. The peripheral blood levels of both colony forming unit granulocyte-macrophage and burst forming unit erythroid, however, showed a significant and sustained increase. In vitro a significant effect exerted by the drug was observed in single cases, but the in vitro effect did not predict the in vivo increase of peripheral blood levels of hemopoietic progenitor cells. These results appear to provide evidence that amifostine has a significant impact on early events of hemopoiesis in myelodysplastic patients.     

二氢叶酸还原酶耐药基因对人外周血CD34+细胞的保护作用

目的 探讨突变的二氢叶酸还原酶（ｍＤＨＦＲ）耐药基因在人外周血ＣＤ３４＾＋细胞中抗甲氨喋呤（ＭＴＸ）的效应。方法 应用免疫磁珠分选系统（ＭＡＣＳ）分离纯化外周血ＣＤ３４＾＋细胞后,用含ｍＤ－ＨＦＲ基因的逆转录病毒上清转染人外周血ＣＤ３４＾＋细胞,采用造血祖细胞集落形成实验进行转导后细胞抗ＭＴＸ分析。结果 在ＭＴＸ（２０ｎｍｏｌ／Ｌ）存在的条件下,转导ｍＤＨＦＲ耐药基因的外周血ＣＤ３４＾＋细胞集落形