Autoinduction of differentiation in myeloid leukemic cells: restoration of normal coupling between growth and differentiation in leukemic cells that constitutively produce their own growth-inducing protein

Growth and differentiation of normal myeloid haematopoietic cells are regulated by a family of macrophage- and granulocyte-inducing (MGI) proteins. Some of these proteins (MGI-1) induce cell growth and others (MGI-2) induce cell differentiation. Addition of MGI-1 to normal myeloid cells induces growth and also induces the endogenous production of MGI-2. This induction of differentiation-inducing protein by growth-inducing protein then ensures the coupling between growth and differentiation found in normal cells. There are myeloid leukemic cells that constitutively produce their own MGI-1, but the cells do not differentiate in culture medium containing horse or calf serum. By removing serum from the medium, or in medium with mouse or rat serum, these leukemic cells are induced to differentiate to mature cells, which like normal mature cells, then no longer multiply. Leukemic cells with constitutive production of MGI-1 continuously cultured in serum-free medium with transferrin were also induced to differentiate by removing transferrin. This induction of differentiation was in all these cases associated with the endogenous production of MGI-2 by the cells. The results indicate that changes in specific constituents of the culture medium can result in autoinduction of differentiation in these leukemic cells due to restoration of the induction of MGI-2 by MGI-1, which then restores the normal coupling of growth and differentiation.     

Pirarubicin nuclear uptake does not correlate with its induced cell death effect during reversal of multidrug resistance by quinine in human K562 and CEM leukemic cells

A number of small and lipophilic cations are able to reverse in vitro the resistance to anthracyclines and other natural products through their interaction with P-glycoprotein or P-gp. However, some modulators do not interact with P-gp. We have demonstrated in a previous a work, using confocal laser microspectrofluorometry, that quinine does not increase nuclear anthracycline uptake in multidrug-resistant Chinese hamster ovary LR73 cells. In this case the LR73 cells were transfected with the mdr1 gene. Moreover, quinine induced in these cells an increase of mdr1 gene expression. In the present study, we investigated verapamil and quinine for their ability to increase nuclear pirarubicin uptake in multidrug-resistant K562R and CEMR human leukemic cell lines. These two cell lines resist, respectively, to doxorubicin and vinblastine and both overexpress the P-gp. Verapamil was able to restore nuclear pirarubicin in both cell lines. On the other hand, quinine was unable to significantly increase nuclear pirarubicin uptake. Both modulators were able to restore pirarubicin sensitivity in both resistant cell lines. After treatment with quinine, mdr1 gene and P-gp expression was not significantly altered as observed previously in the LR73 cells. This suggest that the effect of quinine on mdr1 gene expression is dependent on the cell line studied. These data suggest that quinine could modify the molecular environment of anthracyclines and/or its binding to a possible cytoplasmic target, and that the mechanisms by which anthracyclines induce cell death, and ways by which chemotherapy fails in multidrug-resistant leukemic cells remain complex and are related to more than one target.     

Cyclosporin A has low potency as a calcineurin inhibitor in cells expressing high levels of P-glycoprotein

Cyclosporin A (CsA) is a widely-used immunosuppressant drug whose therapeutic and toxic actions are mediated through inhibition of calcineurin (CN), a calcium- and calmodulin-dependent phosphatase. Inhibition of CN by CsA requires drug binding to its protein cofactor in the inhibition, cyclophilin. Because cyclophilin is a high affinity target for CsA it is expected that this protein can act as a reservoir for the drug in the cell and may be able to inhibit cellular efflux of CsA. P-glycoprotein (P-gp) is known to increase the rate of CsA efflux from CsA loaded cells but it is not clear if the P-gp drug efflux pump can compete effectively with cyclophilin at therapeutically relevant concentrations of CsA. To test the hypothesis that increased expression of P-gp confers protection against CsA-dependent inhibition of CN phosphatase activity, KB-V cells expressing varying levels of P-gp were analyzed to determine the potency of CsA as a CN inhibitor. When intact cells were treated with CsA, a positive correlation was observed between P-gp expression and resistance to CsA-dependent inhibition of CN: the IC50 is approximately 20-fold higher in the multidrug resistant epidermal carcinoma cell line, KB-V, which expresses P-gp at a high level than in the parental, KB, cell line expressing very low levels of P-gp. The resistance displayed by KB-V cells is abrogated by co-administration of the P-gp inhibitor verapamil, whereas verapamil has no effect on CsA potency in control KB cells. In cell lysates from KB-V cells with different amounts of P-gp CsA exhibits equivalent potency, indicating that the difference in sensitivity to CsA among the cell types requires maintenance of cell integrity. These observations support the view that resistance to CN inhibition by CsA occurs in cells with moderately elevated P-gp activity. Therefore, P-gp activity appears to be an important determinant of CsA cellular specificity for both therapeutic and toxic effects.     

Efficient adenovirus-mediated gene transduction of normal and leukemic hematopoietic cells

We evaluated the efficiency of adenovirus-mediated gene transfer into normal and malignant human hematopoietic cells. An E-1 and E-3 deleted, replication-defective recombinant Ad.RSV beta gal vector was used and the transduction efficiency was studied at a multiplicity of infection of 13 p.f.u. per cell. Approximately 40-50% of normal monocytes were transduced, whereas purified normal resting T cells and B cells were resistant to infection. We showed that 50-80% of primary chronic myeloid leukemia cells (CML, n = 12) were efficiently transduced in contrast to CML, successful transduction of resting primary chronic B lymphocytic leukemia cells required appropriate preactivation of targeted cells. A novel protocol for the efficient transduction of adenovirus into B-CLL cells was presented. We showed that anti-CD40 mAb or CD40 ligand acts in synergy with rhIL-4 to enable the transduction of approximately 50-75% of B-CLL cells (B-CLL, n = 6). Expression of beta-galactosidase in transduced CML cells and B-CLL cells was detected for at least 15 days after transduction. The present studies underline the utility of adenovirus vectors for the construction of cytokine gene-modified tumor vaccines for the treatment of hematopoietic malignancies such as CML and B-CLL.     

Cyclosporin A and cyclosporin SDZ PSC 833 enhance anti-CD5 ricin A-chain immunotoxins in human leukemic T cells

Recent studies have shown that cyclosporin A (CsA) may affect ricin A-chain immunotoxin (RTA-IT) therapy. In this study, we evaluated the ability of CsA and its nonimmunosuppressive analog, SDZ PSC 833, to enhance anti-CD5 T101 RTA-ITs in vitro. Both 4 mumol/L CsA and 4 mumol/L SDZ PSC 833 significantly and specifically enhanced the cytotoxic activity of T101 RTA-IT on the human lymphoblastic T-cell line, CEM III (101-fold and 105-fold, respectively). Furthermore, these Cs also enhanced the cytotoxicity of the more potent T101 F(ab')2 RTA-IT (ninefold and eightfold, respectively). The effect of human plasma, originating from four patients enrolled in a phase I high-dose CsA regimen, was examined on T101 RTA-IT cytotoxicity on CEM III cells. In each case, with plasma CsA levels between 3,090 and 4,860 ng/mL (2.5 to 4 mumol/L), a significant increase in T101 RTA-IT-mediated cytotoxicity was observed ranging from 31% to 60%. Neither CsA nor SDZ PSC 833 affected the rate of RTA-IT binding, internalization, intracellular trafficking, or degradation. Analysis of internalized T101 RTA-IT molecules showed that these were essentially intact, which suggests that these enhancers may act only on a small population of RTA-ITs that escapes present investigational techniques. In conclusion, because the concentrations used are clinically achievable, Cs appear to be promising agents for in vivo enhancement of RTA-ITs.     

Granulocyte colony-stimulating factor (G-CSF) mediated mobilization of leukemic cells in Philadelphia chromosome positive acute lymphoblastic leukemia expressing myeloid antigens (my+Ph+ALL)

We report a male infant who has impaired penile development, hypospadias, and mild developmental delay with a 46,XY,t(1;18)(q32.1;q22.1) karyotype. Fluorescent in situ hybridization (FISH) was performed to more precisely map the translocation breakpoint. The translocation breakpoint maps to a region that has been implicated in genitourinary malformations in the 18q- syndrome. This case report suggests that a gene involved in genitourinary development maps at or near the chromosome 18 translocation breakpoint.     

Manganese uptake and release by cultured human hepato-carcinoma (Hep-G2) cells

The liver is the primary organ involved in manganese (Mn) homeostasis. The human hepato-carcinoma cell line, Hep-G2, shows many liver specific functions. Consequently, Hep-G2 cells were investigated as a possible model of hepatic metabolism of Mn. Initial experiments showed that the concentration of Mn in the diet, or culture medium, similarly affected the retention of Mn by isolated rat hepatocytes and Hep-G2 cells. Manganese uptake by Hep-G2 cells suggested that uptake was followed by release from the cell. Uptake was saturable and half-maximal at 2.0 micromol Mn/L, and was inhibited by iodoacetate, vanadate, cold, and bepridil. The cations Fe2+, Cu2+, Ni2+, Cd2+, and Zn2+ decreased Mn uptake. Uptake was dependent on Calcium (Ca) concentration in a manner that resembled saturation kinetics. Cells that were pulsed with 54Mn and then placed into nonradioactive medium quickly released a large portion of their internalized Mn. Release of internalized Mn could be inhibited by low temperature, nocodozole, quinacrine and sodium azide. These data show that Hep-G2 cells are a potentially good model of hepatic Mn metabolism. Mn is taken up by a facilitated process that may be related to Ca uptake. Release apparently is an active, controlled process, that may involve microtubules and lysosomes.     

In vitro induction of primary DNA double-strand breaks in leukemic and normal blood cells by ultraviolet irradiation

The yield of UV-induced DNA double-strand breaks was studied for white blood cells ("light" fraction) derived from peripheral blood, and from patients with lymphomas, chronic lymphoid leukemia (CLL), and chronic myeloid leukemia (CML). The method employed was constant-field electrophoresis of plug-embedded DNA in agarose gel. Characteristic dose-response curves were obtained for various cell populations. Lymphoid cells, both from healthy subjects and CLL patients, revealed less damage to DNA under UV-irradiation, whereas CML cells were much more affected. Possible interpretation of these results includes species-specific differences in UV-induced DNA damage, as well as sufficient DNA crosslinking, thus interfering with DNA dsbs detection in irradiated cells.     

Decreased feedback regulation of low density lipoprotein receptor activity by sterols in leukemic cells from patients with acute myelogenous leukemia

Leukemic cells from patients with acute myelogenous leukemia (AML) have higher low density lipoprotein (LDL) receptor activity than normal white blood and bone marrow cells. The underlying mechanism behind this is unclear. We studied the inhibitory effect of sterols on induction of LDL-receptor activity in leukemic cells from 27 patients with AML and in white blood cells from 13 healthy individuals. The high affinity degradation rate of 125I-labeled LDL was determined in mononuclear blood cells directly after isolation from blood and after incubation for 2 days in medium with 10% lipoprotein-deficient serum with or without various concentrations of 25-hydroxycholesterol + cholesterol. The median sterol concentration for 50% inhibition (IC50) of induction was more than five times higher for leukemic cells than for normal mononuclear cells. At the highest sterol concentration (0.400 microg/mL 25-hydroxycholesterol + 8 microg/mL cholesterol), the LDL-receptor activity was abolished in cells from all healthy individuals while the induction of LDL-receptor activity in cells from three AML patients was unaffected. The LDL-receptor activity of leukemic cells, directly after isolation from blood, correlated with IC50 values (r = 0.53, P = 0.007) and WBC counts (r = 0.72, P = 0.0001) but not with cellular cholesterol levels. The results demonstrate decreased feedback regulation of LDL-receptor activity by sterols in AML cells and support the conclusion that elevated LDL-receptor activity is associated with sterol resistance and cell proliferation. The findings are of potential interest for diagnosis and specific treatment of leukemia.     

Decreased basal, noninsulin-stimulated glucose uptake and metabolism by skeletal soleus muscle isolated from obese-hyperglycemic (ob/ob) mice

Insulin resistance of diaphragms of ob/ob mice has been repeatedly demonstrated previously both in vitro and in vivo. In the present study, transport and metabolism of glucose with and without insulin stimulation were compared in a skeletal muscle more likely than diaphragm or heart to be representative of the overall striated muscle mass, i.e. isolated soleus muscle. Compared with soleus muscle from lean controls, unstimulated lactate release in the presence of exogenous glucose was depressed from 16.2 to 12.3 nmol/60 min per mg wet wt in soleus from ob/ob mutants; glycolysis was decreased from 6.6 to 3.7 and [14C]glucose oxidation to 14CO2 from 0.90 to 0.33 nmol glucose/60 min per mg wet wt. Uptake of 2-deoxyglucose (2-DOG), both with and without insulin, was very much less for soleus from ob/ob than from lean mice, at 2-DOG concentrations ranging from 0.1 to 10 mM, and in mice of 6-15 wk. When 2-DOG concentration was 1 mM, its basal uptake was 0.53 nmol/30 min per mg wet wt for soleus of ob/ob as against 0.96 for soleus of lean mice. The absolute increment due to 1 mU/ml insulin was 0.49 in muscle of ob/ob as against 1.21 in that of lean mice. When the resistance to insulin action was decreased by pretreatment in vivo by either streptozotocin injection or fasting, the decreased basal 2-DOG uptake of subsequently isolated soleus muscle was not improved. Inhibition of endogenous oxidation of fatty acids by 2-bromostearate, while greatly increasing 14CO2 production from [14C]glucose, did not affect basal [5-3H]glucose metabolism or 2-DOG uptake. It is suggested that transport and/or phosphorylation of glucose under basal, unstimulated conditions are depressed in soleus muscle of ob/ob mice, whether or not resistance to insulin and hyperinsulinemia are also present. Although the origin of the decreased basal glucose uptake remains unknown it might be related to a similar decrease in basal glucose uptake by ventromedial hypothalamic cells, an event presumably resulting in a tendency to hyperphagia. Decreased basal glucose uptake by soleus muscle of ob/ob mice might explain the hyperglycemia, and hence partly the hyperinsulinemia and excessive fat deposition of those animals.     

A comparative study on the uptake of alpha-aminoisobutyric acid by normal and immortalized human embryonic kidney cells from proximal tubule

To understand the pathogenesis of AIDS, we must first understand the unique features of HIV that make it such a formidable pathogen. These features and their different qualities are discussed in this article.     

Growth inhibition of leukemic cells by (-)-epigallocatechin gallate, the main constituent of green tea

In this report, we presented the results that EGCG, the main constituent of the polyphenols present in Japanese green tea inhibited growth of leukemic cell lines of both human and mice. The proliferation of human leukemic cell lines and mouse NFS60 cell line was inhibited by EGCG. Sensitivity of each line to EGCG was different, and more than 50% of DNA synthesis was reduced in all the cell lines in the presence of 50 microM EGCG. On the other hand, normal hematopoietic progenitor cells retained their natural function of supplying mature cells of various lineages in the presence of less than 10 microM EGCG in vitro. Even in the presence of 100 microM EGCG, half the colonies containing all the lineages of cells were developed. All the dead cells of each line showed characteristics of apoptosis, which might be due to inhibition by EGCG of growth factors' signaling. Besides anticarcinogenic activity, EGCG is expected to have a new function for leukemia therapy without side effects.     

3H-Estradiol uptake and retention by target tissues of light-sterilized female rats

Tissue distribution of radioactivity was studied at 2, 4 and 6 h after intravenous injection of 3H-estradiol-17beta (41.7 ng/100 g b.w.) in 12 light-sterilized and 11 control female rats ovariectomized 72 h prior to injection. Female rats were light-sterilized by exposure to continuous illumination for 82 days and, based on the duration of continuous vaginal cornification and the absence of corpora lutea at post-mortem histological examination of the ovaries, were anovulatory for at least 30 days prior to injection. Control rats were housed under conditions similar to the experimentals except that they were exposed to alternating lighting (14 h light:10 h dark). They remained ovulatory throughout the experiment. Uterine 3H-estradiol uptake and retention were significantly depressed in the light-sterilized group. There were no significant differences in 3H-estradiol uptake or retention as a result of light-sterilization in any of the other tissues studied, including anterior, middle, and posterior hypothalamus, hippocampus, amygdala, cerebrum, anterior pituitary and plasma. The failure to detecta reduction in neural 3H-estradiol uptake demonstrates that the anovulatory state can exist without a concomitant reduction in the hypothalamic estrogen binding capacity. The possibility is discussed that the decreased target tissue binding noted in various types of anovulatory animals is the result, not the cause, of altered ovarian function.     

Daunorubicin (DNR) accumulation in fresh leukemic cells: correlation with clinical and biological features

The DNR accumulation (DNR Acc) and the verapamil (VRP) index (percent increase of VRP on DNR accumulation) was studied by using flow cytometry. Fresh leukemic mononuclear bone marrow blasts from 80 unselected ANLL patients' samples were incubated with DNR in the presence or absence of VRP. The DNR accumulation was determined by flow cytometry. The median DNR Acc was 28 (range: 4-101) and the median VRP index was 4% (range 0-53). VRP significantly enhanced DNR Acc in 42 of the ANLL samples (52.5%). DNR Acc or VRP index were not influenced by age, sex, or WBC counts. Only the FAB subclassification and the blast immunophenotyping were found to influence the parameters studied here. The lowest DNR Acc was found in M0 and M6 blast cells (15 range 0-46 and 10.5 range 8-13 respectively). M4 and M5 ANLL samples accumulated significantly more DNR than M0 and M6 blast cells. The VRP index was significantly higher in M0 compared with M1 and M2 samples, as well as in M4 compared with M1 samples. A slightly positive correlation was found between the percentage of CD34-positive cells in the CD34-positive samples and DNR Acc. In this study, DNR Acc and the VRP index were not significantly correlated with the response to chemotherapy or survival. In conclusion, this study shows that ANLL leukemic cells differ in anthracyclin accumulation and response to VRP in vitro.     

Interleukin 10 (IL-10) upregulates functional high affinity IL-2 receptors on normal and leukemic B lymphocytes

Interleukin 10 (IL-10) has recently been shown to induce normal human B lymphocytes to proliferate and differentiate into immunoglobulin (Ig)-secreting cells. Herein, we show that IL-10 also promotes DNA synthesis and IgM production by anti-CD40 activated B cell chronic lymphocytic leukemia (B-CLL). Most strikingly, IL-2 and IL-10 were found to synergize to induce the proliferation and differentiation of B-CLL cells. This synergy between IL-2 and IL-10 was also observed with normal B cells which proliferated strongly and secreted large amounts of IgM, IgG, and IgA. The observed synergy is likely to be due to the IL-10-induced increase of high affinity IL-2 receptors on both normal and leukemic B cells. This increase of high affinity receptor is associated to an increase of Tac/CD25 expression that can be detected by flow cytometric analysis. Taken together, these results indicate that IL-10 permits anti-CD40 activated B cells to respond to IL-2 through an induction of high affinity IL-2 receptors. This effect of IL-10 may partly explain how T cells, which activate B cells in a CD40-dependent fashion, induce B cell proliferation and differentiation mostly through IL-2.