Granulocytic stem cells in Friend leukemia.

Friend virus induces a leukemia characterized by the proliferation of neoplastic hematopoietic cells believed to be erythroid precursors. In vitro studies were conducted with spleen cells from mice with terminal Firend leukemia in order to determine their capacity for leukocytic differentiation. Spleen cells were obtained from leukemic DBA/2 mice 1 to 2 days before anticipated death and cultured in the presence or absence of colony-stimulating activity (CSA). Growth in liquid culture in dissusion chambers was dependent on CSA and resulted in the generation of normally differentiated granulocytes and macrophages. Colony formation in agar was also dependent on CSA, and the cloning efficiency of leukemic spleen cells was found to be approximately 10 times normal. The colonies formed were composed of leukocytes, which appeared morphologically normal. Total in vitro colony-forming units per leukemic spleen exceeded normal by more than 300-fold, but cells elaborating CSA were decreased. Although it is uncertain whether the stem cells stimulated by CSA are "normal" or leukemic," it is clear that Friend leukemia has profound effects on the proliferation and differentiation of nonerythroid stem cells.     

Effects of hemopoietic growth factors on stem cells in vitro

The central feature of hemopoiesis is the lifelong, stable cell renewal. This process is supported by hemopoietic stem cells, which in the steady-state appear to be dormant in cell cycling. The entry into cell cycle of the dormant stem cells may be promoted by such factors as IL-1, IL-6, and G-CSF. Available evidence indicates that the effects of IL-1 on stem cells are indirectly mediated in part by IL-6 and G-CSF. Once the stem cells leave G0 and begin proliferation, the subsequent process is characterized by continued proliferation and differentiation. While several models of stem cell differentiation have been proposed, micromanipulation studies of individual progenitors suggest that the commitment of multipotential progenitors to single lineages is a stochastic (random) process. The proliferation of early hemopoietic progenitors appears to be supported by IL-3, IL-4, and/or GM-CSF. Once the progenitors are committed to individual lineages, the subsequent maturation process appears to be supported by late-acting, lineage-specific factors such as Ep (for erythropoiesis), G-CSF (for neutrophil production), and IL-5 (for eosinophilopoiesis). Thus, hemopoietic proliferation appears to be regulated by a cascade of factors directed at different developmental stages.     

Effects of cancer immunotherapy with indomethacin and interleukin-2 on murine hemopoietic stem cells.

We examined: (a) whether in vitro-generated lymphocyte-activated killer (LAK) cells from normal mice and splenic killer cells from tumor-bearing mice subjected to interleukin-2 (IL-2) therapy alone or in combination with chronic indomethacin therapy have any detrimental effects on the spleen colony-forming units (CFU-S) of the normal bone marrow (BM); and (b) the effects of these immunotherapy protocols on CFU-S numbers in host hemopoietic organs. Effects of in vitro-generated LAK cells (normal C3H/HeN mouse splenocytes cultured with 1000 units IL-2/10(6) cells for 72 h) on BM CFU-S were examined by incubating macrophage-depleted BM cells with LAK cells at 1:2.5 and 1:5 BM:LAK cell ratios or with LAK cell supernatant for 4 h. The cells were washed and subsequently injected into irradiated mice. Irradiated mice were also reconstituted with BM cells or LAK cells incubated alone. Spleen colonies were scored macroscopically and microscopically on day 7 after reconstitution of lethally irradiated mice with the various cell combinations. A comparison of colony numbers produced by LAK and BM cell mixture revealed that LAK cells at either dose had no suppressive effect on the colony-forming ability of BM at the macroscopic and microscopic levels of analysis. The supernatant of cultured LAK cells had a minor suppressive effect on colony formation at the macroscopic but not the microscopic level of analysis, indicating the presence of one or more suppressive factors capable of mediating a short-term inhibitory effect. In the immunotherapy experiment, C3H/HeN mice transplanted s.c. with 5 x 10(5) C3L5 mammary adenocarcinoma cells received either vehicle alone (controls), IL-2 (1.5 x 10(4) Cetus units i.p. every 8 h on days 10-14 and days 20-25), or chronic indomethacin therapy (10 micrograms/ml in drinking water from day 5 onwards) plus IL-2 as above. Animals were killed 24-25 days after tumor transplantation to examine: (a) the number of metastatic lung nodules; (b) the effects of co-incubating therapy-generated splenic effector cells with normal BM cells for 4 h on BM CFU-S, and (c) the CFU-S content of host BM and spleen. Results revealed a drop in spontaneous lung metastases from a mean of 50 in control mice to 18 with IL-2 therapy alone, and to 5 with chronic indomethacin therapy plus IL-2 therapy. Splenocytes from normal and tumor-bearing control or treated mice, when incubated with normal BM, had no effect on spleen colony formation at the macroscopic level.(ABSTRACT TRUNCATED AT 400 WORDS)     

Toxic effect of polyriboinosinic-polyribocytidylic acid on mouse hemopoietic stem cells

Within a few hours following a single intraperitoneal injection of polyriboinosinic-polyribocytidylic acid (poly I:poly C) into C3H/He mice, numbers of bone-marrow and spleen-colony-forming cells are significantly decreased. The dose-response curve obtained shows an exponential decrease of the surviving CFU with increasing amounts of poly I:poly C, up to 200 μg; with higher doses of poly I:poly C, no further decrease of CFU is observed. This suggests that poly I:poly C kills colony-forming cells in replicating phase, and is devoid of activity on cells in resting phase. The lack of additive toxic effects observed when poly I:poly C and vinblastine sulfate are administred simultaneously is consistent with this hypothesis. The cytotoxic effect of the polynucleotide is related to its double-strandedness and is not mediated by interferon present in the serum.     

Effects of daunorubicin and doxorubicin, free and associated with DNA, on hemopoietic stem cells.

We have compared daunorubicin (DNR)-DNA with free DNR and doxorubicin (DOX)-DNA with free DOX for their effects in vivo in mice on pluripotent stem cells and granulocytic committed stem cells. Dose-survival, time-survival, and recovery curves were obtained after one i.v. injection of either drug. The dose-survival curves of colony-forming units-spleen (CFU-S) and colony-forming units-committed stem cells (CFU-C) were exponential in shape with both agents. DNR-DNA appeared more toxic to the hemopoietic precursor cells than did free DNR. In contrast, DOX-DNA was less toxic toward CFU-S and as toxic as DOX toward CFU-C. Time-survival curves indicated a minimum level of CFU-S and CFU-C at about 33 hr. After that, the recovery of CFU-S was rapid for DNR-treated mice but remained below 50% of the controls on Day 12 for the DNR-DNA-treated group. In mice previously given injections of DOX or DOX-DNA, the recovery of the CFU-S was more protracted in time with a better recovery in mice treated with DOX-DNA. Both DNR and DNR-DNA induced an initial CFU-C decrease followed by a rapid but transient rise with a maximum on Day 4 after chemotherapy. On Day 12, the CFU-C recovery was still incomplete in both DNR- and DNR-DNA-treated mice. In the groups treated with DOX, the CFU-C recovery was more important after DOX-DNA complex than after free DOX. The results are discussed in view of the "lysosomotropic chemotherapy" hypothesis.     

Cryopreservation of autologous marrow grafts in acute leukemia: survival of in vivo clonogenic leukemic cells and normal hemopoietic stem cells

The survival of pluripotent hemopoietic stem cells and in vivo clonogenic leukemic cells after cryopreservation was determined in a rat model for human acute myelocytic leukemia (BNML). These stem cell populations can be selectively quantified with modified spleen colony assays (day 8 and day 12 CFU-S; LCFU-S). It appeared that the most primitive rat hemopoietic stem cell (day 12 CFU-S) was significantly less vulnerable to the freezing and thawing procedure as compared with the clonogenic leukemic cell (30% and 1.4% survival, respectively; p = 0.0026). Survival of the day 8 CFU-S population fell between those percentages (8.6%). In view of autologous bone marrow transplantation (ABMT), an attempt was made to extrapolate these and previously reported BNML rat data to man. Taking into account that a) only 1% of the clonogenic leukemic cells survive cryopreservation; b) the fraction of clonogenic leukemic cells in man is approximately 0.001; c) leukemic cells reinfused with the autologous marrow graft may lodge at sites unfavourable for growth; and d) supralethal high-dose chemoradiotherapy significantly hampers the regrowth of leukemia, it becomes rather unlikely that leukemic cells in the autologous marrow graft significantly contribute to a leukemia relapse after ABMT. Therefore, residual leukemia in the host surviving high-dose chemoradiotherapy is the most crucial factor as regards the final outcome of ABMT in acute leukemia.     

The proliferative state of normal hemopoietic stem cells during progression of leukemia. Studies in the BN acute myelocytic leukemia (BNML)

The kinetics of proliferation of normal hemopoietic stem cells (HSC) were investigated during the course of leukemia in a rat model for human acute myelocytic leukemia (BNML). The fraction of stem cells in DNA synthesis phase was determined with the hydroxyurea suicide technique followed by modified spleen colony assays on bone marrow, blood and spleen cell suspensions. It was found that, besides a redistribution and an absolute numerical decrease in the total number of HSC (to 45% of the original number), the percentage of cycling HSC decreased significantly as the leukemia progressed. In the bone marrow the S-phase fraction of HSC decreased from 38% to almost zero. Proliferating stem cells are thought to emigrate from the blood and lodge at extramedullary sites. Proliferation of HSC is best preserved in the spleen (in the terminal stage: 32 to 53% in S-phase). However, normal hemopoiesis is still insufficient and the rats die from the consequences of a lack of mature end cells. A possible mechanism underlying the changes and differences in kinetic behaviour of HSC in the various organs studied is discussed.     

Infectivity of Friend murine leukemia virus for hamster cells

Hamster-adapted Friend murine leukemia virus (F-MuLV) infected hamster cells and produced an XC-positive progeny. However, the sensitivity of hamster-adapted F-MuLV's to hamster cells was about 100 times lower than that to mouse cells. Hamster cells chronically infected with F-MuLV in vitro produced progeny virus in titers comparable to those in similarly infected mouse cells. Xenotropic and amphotropic murine leukemia virus can infect hamster cells when phenotypically mixed with the F-MuLV. Murine sarcoma virus pseudotyped with the F-MuLV easily induced sarcomas in hamsters and transformed hamster cells in vitro.     

Cytogenetic modifications of Friend leukemia cells resistant to adriamycin

Chromosome analysis was performed on adriamycin-sensitive and resistant Friend leukemia cells. Resistance to ADM is associated with an increased number of metacentric chromosomes. With increasing level of drug resistance additional metacentric chromosomes and several chromosomal markers were observed. Furthermore, the C-banding patterns and the heterochromatin distribution differed in resistant, as compared to sensitive cells. When sensitive cells were exposed to a toxic dose of ADM, multiple chromosomal breaks were observed in 62% of cells. In contrast, when ADM resistant cells were exposed to cytotoxic concentrations, the pulverization phenomenon was not observed and 75-80% of cells were without breaks. This striking difference suggests a different mechanism for cytotoxicity in sensitive and resistant cells.     

Characterization of hemopoietic precursor cells in juvenile-type chronic myelocytic leukemia

In order to study the pathogenesis of juvenile-type chronic myelocytic leukemia (CML), we examined the colony-forming capacity and colony composition in the bone marrow (BM) and peripheral blood (PB) of three children with juvenile-type CML. Large numbers of granulocytes and macrophage colonies were formed by BM and PB cells. Whole agar culture staining revealed that especially macrophage colonies increased in comparison with normal controls.After removal of carbonyl iron-laden cells with a magnet or deprivation of cells adherent to glass from BM cells, the number of macrophage colonies markedly reduced in comparison with the number of colonies formed by untreated BM cells, suggesting that some of the macrophage colony-forming cells (M-CFC) may have phagocytic and/or adherent activity. Radiation sensitivity and thymidine suicide rate of these M-CFC were not different from those of granulocyte colony-forming cells (G-CFC).The predominance of M-CFC in juvenile-type CML may be one of the reflections of fetal-type myelopoiesis since M-CFC are predominant in cord blood and PB in the neonatal period. Moreover, considerable numbers of erythroid-colony-forming units (CFU-E) were present in PB of all patients. It may be concluded that juvenile-type CML is a panmyelopathy with the predominance of M-CFC.     

Reversal of resistance to rhodamine 123 in adriamycin-resistant Friend leukemia cells

Pleiotropic resistance to rhodamine 123 (Rho-123) in Adriamycin (ADM)-resistant Friend leukemia cells was circumvented by cotreatment with 10 microM verapamil. Increased cytotoxicity corresponded to higher intracellular Rho-123 levels. The verapamil-induced increase of drug accumulation in resistant cells is accounted for at least in part by the blockage or slowing of Rho-123 efflux from these cells. In contrast, accumulation and consequent cytotoxicity of Rho-123 in sensitive cells are not increased by verapamil. Similar results were obtained when ADM was used in this cell system. These results suggest that the efflux system for Rho-123 and ADM in sensitive cells is either reduced or absent. Although Rho-123 accumulates specifically in mitochondria and ADM mainly in the nucleus, the loss of these two different classes of compounds from resistant cells appears to occur via a similar or common mechanism. The similarities in drug transport between Rho-123 and ADM may have important implications when applied to an in vivo environment.     

A radiosensitive T-lymphocyte associated with resistance of DBA/2 mice harboring Friend leukemia virus-dormant infections to transplantable Friend leukemia virus-erythroleukemia cells

The immunobiology of Friend erythroleukemia virus (FLV) has been the focal point of much research into immunological control of leukemia. We have been studying a murine model in which a rapidly fatal FLV infection of DBA/2 mice is suppressed to a dormant state by treatment with statolon, a double stranded RNA extract of Mycoplasma stoloniferum. We report here that mice with FLV-dormant infections resist the accumulation of transplanted FLV-transformed erythroleukemia cells (FLC-745) and that FLC-745 cells persist in the spleen for a prolonged period. Winn assays revealed that the spleen of FLV-dormant mice contain radiosensitive T-lymphocytes with anti-FLC-745 cell activity. Whole body irradiation of FLV-dormant mice abrogated their resistance to transplanted FLC-745 cells and confirmed the radiosensitivity of the protective immune response.     

Clonal analysis of the actions of the murine leukemia inhibitory factor on leukemic and normal murine hemopoietic cells

The in vitro actions of leukemia inhibitory factor (LIF) purified from Krebs tumor conditioned medium, were analyzed on murine leukemic M1 and WEHI-3B D+ cells and on normal hemopoietic progenitor cells. LIF has no observable effects on WEHI-3B D+ cells but rapidly induced macrophage differentiation and loss of clonogenicity in M1 cells, resulting in the formation of abortive clones or differentiating colonies of reduced size and number. These effects were observable within one to two cell divisions in the presence of LIF and were irreversible. Addition of macrophage-colony-stimulating factor (CSF) but not granulocyte/macrophage-CSF, granulocyte-CSF, or multi-CSF reduced the LIF-induced suppression of colony numbers and size. G-CSF had a slower differentiation-inducing action on M1 cells than LIF but potentiated the differentiation-inducing effects of low concentrations of LIF. LIF had no colony-stimulating activity for normal granulocyte-macrophage progenitor cells and did not alter their quantitative responsiveness to CSF. However, culture of normal progenitor cells in the presence of LIF, but initial absence of CSF, reduced the survival of these cells. The differing actions of LIF and G-CSF on M1 leukemic cells suggest the existence of distinct mechanisms for inducing macrophage differentiation in these leukemic cells.     

Specificities of acylglycerols and phospholipids for interaction with phorbol ester receptor on Friend leukemia cells

For the purpose of characterizing the nature of the phorbol ester receptor on intact cells, the effects of acylglycerols and phospholipids, which are known as activators of protein kinase C, on the binding of 3H-phorbol dibutyrate to Friend erythroid leukemia cells (FLC) were examined. Even when intact cells were used, diolein inhibited the binding of phorbol ester, and the degrees of inhibition by several kinds of acylglycerols paralleled their abilities to activate protein kinase C. On the other hand, phosphatidylserine enhanced the binding. Among phospholipids tested, the order of activities for increasing the binding was almost the same as that for activating protein kinase C. These results provide support for the hypothesis that the phorbol ester receptor in intact cells is protein kinase C, and that the ability of intact cells to bind phorbol ester is affected by endogenous lipids.     

DS/Cu对急性髓系白血病干细胞增殖与凋亡影响及其机制的探讨

目的:探讨双硫仑(disulfiram,DS)单药及DS联合Cu(DS/Cu)在体外对白血病干细胞的影响及其分子机制。方法:应用流式细胞仪分选KG1a细胞中具有LSCs特性的CD34+CD38-亚群;MTT检测DS和DS/Cu对CD34+CD38-KG1a细胞的抑制增殖作用;Annexin-Ⅴ/PI法检测DS及DS/Cu对CD34+CD38-KG1a细胞的诱导凋亡作用;蛋白质印迹法检测药物处理后NF-кb通路和JNK通路蛋白表达的变化。结果:CD34+CD38-亚群占KG1a细胞的(59.4±6.2)%,流式细胞分选后的CD34+CD38-细胞可达(93.16±2.7)%,其中(65.3±6.4)%处于G0/G1期。DS和DS/Cu能抑制CD34+CD38-KG1a细胞增殖能力,24h的IC50分别为(0.54±0.18)μmol/L和(0.21±0.03)μmol/L,DS/Cu的作用显著强于DS,P=0.036。DS和DS/Cu对CD34+CD38-KG1a细胞均有诱导凋亡的作用,0.05、0.5和5μmol/L的DS和DS/Cu作用细胞24h后凋亡率分别为〔(7.69±3.44)%、(15.06±4.58)%、(32.67±6.06)%〕和〔(28.83±6.34)%、(42.33±3.21)%、(58.93±1.53)%〕,DS/Cu诱导CD34+CD38-KG1a细胞凋亡的比例显著高于DS单药,P<0.01;DS及DS/Cu均能抑制p65蛋白及下游蛋白c-myc、Survivin的表达、并上调磷酸化JNK及下游转录因子p-c-jun的表达,DS/Cu的作用更加显著。结论:DS在体外能抑制LSCs增殖及诱导其凋亡,Cu可以显著增强这种作用,其作用机制与抑制NF-κb通路及激活JNK通路相关。