Generation time of leukaemic blast progenitor cells

Previous studies have indicated that the generation time of human leukaemic cells is longer than that of normal haematopoietic cells. We have employed a modification of the thymidine (TdR)-suicide technique to measure directly the generation time of leukaemic progenitor cells capable of colony formation. The results obtained with two human leukaemic cell lines (KG-1 and HL-60) and with blast progenitor cells from two patients with acute myelogenous leukaemia indicate generation times ranging from 9 X 0-22 X 0 hr and S-phase durations ranging from 5 X 5-8 X 0 hr. Using the same technique, the generation time of normal bone marrow CFU-c was determined to be 9-11 hr. These findings suggest that the proliferation rate of human leukaemic blast progenitor cells is similar to that of normal haematopoietic stem cells.     

STEM CELL KINETICS IN THE L 5222 RAT LEUKAEMIA

The behaviour of normal haemopoietic stem cells of the bone marrow during the development of the acute transferable rat leukaemia, L 5222, has been investigated. The granulopoietic committed stem cells, measured by the in vitro colony technique, showed a marked decrease to less than half normal levels. Pluripotent stem cells included in the small lymphocytes of the bone marrow, and labelled with ³H-thymidine by the complete labelling method, showed only a modest decrease in number and an unchanged labelling intensity. The results suggest that in this leukaemia the pluripotent stem cells may be affected in such a way that they are unable to react by proliferation to the depletion of the succeeding cell compartments. This might be due to inhibition by leukaemic cells or to a disturbed feedback regulation between the committed and pluripotent stem cell compartments.     

Cholesterol esters as growth regulators of lymphocytic leukaemia cells

Objective: Alterations in plasma lipid profile and in intracellular cholesterol homoeostasis have been described in various malignancies; however, significance of these alterations, if any, in cancer biology is not clear. The aim of the present study was to investigate a possible correlation between alterations in cholesterol metabolism and expansion of leukaemia cell numbers. Materials and methods: Lipid profiles in plasma and in primary leukaemia cells isolated from patients with acute or chronic lymphocytic leukaemia (ALL and CLL) were studied. Results and conclusions: Decreased levels of HDL‐C were observed in plasma of leukaemic patients, levels of total cholesterol, LDL‐C, triglycerides and phospholipids were unchanged or only slightly increased. As compared to normal lymphocytes, freshly isolated leukaemic cells showed increased levels of cholesterol esters and reduction in free cholesterol. Growth stimulation of ALL and CLL cells with phytohemagglutinin led to further increase in levels of cholesterol esters. Conversely, treatment with an inhibitor of cell proliferation such as the mTOR inhibitor, RAD, caused decline in population growth rate of leukaemia cells, which was preceded by sharp reduction in rate of cholesterol esterification. On the other hand, exposure of leukaemic cells to two inhibitors of cholesterol esterification, progesterone and SaH 58‐035, caused 60% reduction in their proliferation rate. In addition to demonstrating tight correlation between cell number expansion and cholesterol esterification in leukaemic cells, these results suggest that pathways that control cholesterol esterification might represent a promising targets for novel anticancer strategies.     

In vitro proliferation of normal and leukaemic human leukocytes controlled by an inhibitory endopeptide

GI-3, an endogenous inhibitory fraction isolated from leukocytes, selectively inhibits the proliferation of granuloid precursor cells in a non-toxic manner. Its active principle was determined as an acidic chlor-tolidine positive decapeptide [ 3 ]. The in vitro effect on normal and acute leukaemic human bone marrow and blood cells was examined. A dose dependent inhibition by GI-3 of 3H-TdR incorporation into myeloid cells of normal bone marrow was found, the sensitivity of human cells being higher than that of rat cells. The proliferation of the target leukaemic bone marrow and blood cells (AML, AMMoL) was also decreased by the endogenous inhibitor in a dose dependent manner in untreated subjects as well as in patients in remission or relapse. The rate of inhibition of leukaemic of well-known cytostatics (adriamycin hydrochloride, dianhydrogalactitol) applied for comparison. Beyond its direct cytostatic effect, GI-3 could be used in the differential diagnosis of blastic leukaemias, complementing the routine cytochemical methods.     

Leukemic chromosome clone. I. Risk factor in the acute lymphatic leukemia of children]

As long as the aetiology of acute lymphatic leukaemia of children is not known its therapy is based on clinical experience. Among the values of experience those factors will play a part, the evidence of which during the ALL initial stage will be a risk for successful therapy and survival rate. This results in a choice of more aggressive variants of modern therapy schemes. In a cytogenetic study made in 35 children with ALL it was tested, whether even leukaemic chromosome clones will be a risk for the course of acute leukaemia. The duration of the first remission and survival rate were considered as criteria. The evidence of a leukaemic chromosome clone could be shown to be followed by a short survival rate, irrespective of the stage of the disease where the clone had been observed first. Thus, cytostatic therapy in those ALL patients who are affected with luekaemic chromosome aberration of stem line character should be aimed at the complete annihilation of the clone, irrespective of other remission criteria. The failure of blood and bone-marrow cultures as early as during the untreated initial stage indicated a primary cellular immuno-insufficiency. This combination of cell immuno-depression with high peripheral leukocytes connts and a primary mediastinal tumour or a generalizing lymphosarcoma respectively, was the highest risk up till now for the course of the disease. Judging from the duration of the first remission and the survival rates, the consecutive schemes of therapy did not differ in their effect on leukaemia with pathological stem lines. On the basis of the present study the impression could not be excluded that up till now long term survival rates could be attributed rather to individual manners of response than to the modern therapy scheme.     

The acid-base equilibrium during mouse lymphoblastic leukemia]

In transplantable and in spontaneous lymphoblastic mouse leukemia blood pH, PCO2, TCO2 and BB were examined. Spontaneous Gross-leukemia in AKR mice was found to develop in its final phase respiratory acidosis. Transplantable (TAL) leukemia of AKR mice presents from the onset a tendency toward respiratory acidosis. "L-1210"-leukemia, on the contrary, was shown to alcalise the recipients during the first 8 days after inoculation, later on it can bring about an acidosis. The pH-deviations in "L-1210"-leukemia display a respiratory character in syngeneic DBA/2J recipients, whereas in semiallogeneic recipients the observed changes are metabolic or mixt in nature. This finding strongly argues for the importance of histocompatibility. Intranodally inoculated animals distinctly differ in their parameters in comparison to intravenously and intraperitoneally recipients, hence, an important role of the tissular-milieu which is put first in contact with the leukemic factor must be concluded.     

Characteristics of beta-adrenoreceptors of L-1210 leukemia and its sarcolysine-resistant variant

Beta-adrenoceptors have been discovered on the surface of cells of leukaemia L1210 and its variant resistant to sarcolysine (D, L-melphalan). One type of functionally active receptors with dissociation constant Kd for L-[3H] dihydroalprenolol about 0.02 nM and 360 receptors per cell have been revealed in leukaemia L1210 cells. In the resistant cells two types of functionally inactive receptors with Kd1 approximately 0.02 nM (420 receptors per cell) and Kd2 approximately 2.5 nM (3000 receptors per cell) have been revealed. This property of beta-adrenoceptors may be one of the causes of tumour cell resistance to sarcolysine.     

外源H2O2对水稻幼苗抗寒性的调节作用

在常温下用不同浓度的外源H2O2(0～20 mmol·L-1)预处理水稻幼苗,再进行12 h 6℃低温胁迫,根据幼苗相对含水量和质膜相对透性筛选最佳外源H2O2处理浓度,并分析最佳外源H2O2浓度下幼苗的渗透调节物质和活性氧相关指标的变化.结果表明:(1)0～8 mmol·L-1 H2O2预处理可以增加水稻幼苗的相对含水量,降低其质膜相对透性,并以4 mmol·L-1 H2O2的效果最佳.(2)低温胁迫后,与对照组相比,4 mmol·L-1外源H2O2预处理降低了水稻幼苗萎蔫程度,并使其总呼吸速率、交替途径容量都有增加,同时还抑制了丙二醛的含量,增加了可溶性糖、可溶性蛋白质和脯氨酸的含量.(3)外源H2O2预处理对水稻幼苗的内源H2O2含量以及O(-)/(·)2产生速率没有显著影响.研究发现,外源H2O2可以通过提高呼吸速率、降低脂质过氧化程度、增加碳氮代谢来有效增强水稻幼苗的抗寒性,它可能以一种独立于内源活性氧系统之外的方式发挥作用.     

Low-expression of E-cadherin in leukaemia cells causes loss of homophilic adhesion and promotes cell growth

E-cadherin (epithelial cadherin) belongs to the calcium-dependent adhesion molecule superfamily and is implicated in the interactions of haematopoietic progenitors and bone marrow stromal cells. Adhesion capacity to bone marrow stroma was impaired for leukaemia cells, suggesting that a breakdown of adhesive mechanisms governed by an adhesion molecule may exist in leukaemic microenvironment. We previously found that E-cadherin was low expressed in primary acute leukaemia cells compared with normal bone marrow mononuclear cells. In this study, we investigate the functional importance of low E-cadherin expression in leukaemia cell behaviours and investigate its effects in the abnormal interaction of leukaemic cells with stromal cells. After expression of E-cadherin was restored by a demethylating agent in leukaemia cells, E-cadherin-specific adhesion was enhanced. Additionally, siRNA (small interfering RNA)-mediated silencing of E-cadherin in Raji cells resulted in a reduction of cell homophilic adhesion and enhancement of cell proliferation and colony formation. These results suggest that low expression of E-cadherin contributes to the vigorous growth and transforming ability of leukaemic cells.     

Bortezomib suppresses self-renewal and leukemogenesis of leukemia stem cell by NF-ĸB-dependent inhibition of CDK6 in MLL-rearranged myeloid leukemia

Acute myeloid leukaemia (AML) with chromosomal rearrangements involving the H3K4 methyltransferase mixed-lineage leukaemia (MLL) is an aggressive subtype with low overall survival. Bortezomib (Bort) is first applied in multiple myeloma. However, whether bort possesses anti-self-renewal and leukemogenesis of leukaemia stem cell (LSC) in AML with MLL rearrangements is still unclear. Here, we found that bort suppressed cell proliferation and decreased colony formation in human and murine leukaemic blasts. Besides, bort reduced the frequency and function of LSC, inhibited the progression, and extended the overall survival in MLL-AF9 (MF9) -transformed leukaemic mice. Furthermore, bort decreased the percentage of human LSC (CD34⁺CD38^-) cells and extended the overall survival in AML blasts-xenografted NOD/SCID-IL2Rγ (NSG) mice. Mechanistically, cyclin dependent kinase 6 (CDK6) was identified as a bort target by RNA sequencing. Bort reduced the expressions of CDK6 by inhibiting NF ĸB recruitment to the promoter of CDK6, leading to the abolishment of NF ĸB DNA-binding activity for CDK6 promoter. Overexpression of CDK6 partially rescued bort-induced anti-leukemogenesis. Most importantly, bort had little side-effect against the normal haematological stem and progenitor cell (HSPC) and did not affect CDK6 expression in normal HSPC. In conclusion, our results suggest that bort selectively targets LSC in MLL rearrangements. Bort might be a prospective drug for AML patients bearing MLL rearrangements.     

Regulation of megakaryocytes in W/Wv mice

W/Wv mice were injected with antiplatelet serum to produce thrombocytopenia or with platelet transfusions to induce thrombocytosis. The responses of their platelets and megakaryocytes were followed to determine if proliferative abnormalities of the megakaryocytic system would be detected. W/Wv mice responded normally to the stimulation from thrombocytopenia with rebound thrombocytosis, macromegakaryocytosis, and macrothrombocytosis. The megakaryocytes of these mice became smaller than normal in response to post-thrombocytopenic rebound thrombocytosis but not to transfusion-induced thrombocytosis. Thus, endogenous thrombocytosis appeared to be a more potent suppressor of megakaryocyte growth than exogenous. These results failed to reveal an effective abnormality of the thrombocytopoietic regulatory system of W/Wv mice in spite of their intrinsically reduced numbers of megakaryocytes and the well known defect of stem cell proliferation. Thrombocytopoietic regulation appeared, therefore, to occur mainly at the committed, rather then pluripotential, stem cell level, and normal responses of the platelet system were observed in spite of severe abnormalities at the pluripotential stem cell level.     

Higher tyrosine protein kinase activity in resting lymphocytes than in proliferating normal or leukaemic blood cells

We have studied tyrosine phosphorylation in particulate fractions from 11 leukaemic cell lines by using as substrate either a synthetic tyrosine containing peptide or the endogenous proteins. The results were compared with those obtained using similar fractions from normal lymphocytes and bone marrow cells. Particulate fractions from all the leukaemic cell lines and normal bone marrow cells exhibited lower levels of tyrosine protein kinase activity compared to normal lymphocytes. When the phosphorylation of endogenous substrates was assayed, proteins were phosphorylated on tyrosine residues (rather than serine or threonine residues) to a larger extent in normal lymphocytes than in leukaemic cell lines. Separation of labelled endogenous substrates on sodium dodecyl sulfate-polyacrylamide gels showed a number of phosphorylated alkali-resistant bands in the range 14-175kd in the lymphoid cell lines; normal lymphocytes exhibited a smaller number of strongly phosphorylated bands. Normal lymphocytes from different individuals showed reproducible patterns of phosphorylated substrates. Normal bone marrow cells and myeloid leukaemia lines showed weak, if any, phosphorylation. Among the leukaemic cell lines no particular pattern of phosphorylated substrates common to cells of similar phenotype could be detected. We suggest that the level of overall tyrosine protein kinase activity in these fractions reflects their position in the cell cycle rather than their normal or malignant status.     

Stem cell and kinase activity-independent pathway in resistance of leukaemia to BCR-ABL kinase inhibitors

BCR-ABL tyrosine kinase inhibitors, such as imatinib (Gleevec) are highly effective in treating human Philadelphia chromosome-positive (Ph+) chronic myeloid leukaemia (CML) in chronic phase but not in terminal acute phase; acquired drug resistance caused mainly by the development of BCR-ABL kinase domain mutations prevents cure of the leukaemia. In addition, imatinib is ineffective in treating Ph+ B-cell acute lymphoblastic leukaemia (B-ALL) and CML blast crisis, even in the absence of the kinase domain mutations. This type of drug resistance that is unrelated to BCR-ABL kinase domain mutations is caused by the insensitivity of leukaemic stem cells to kinase inhibitors such as imatinib and dasatinib, and by activation of a newly-identified signalling pathway involving SRC kinases that are independent of BCR-ABL kinase activity for activation. This SRC pathway is essential for leukaemic cells to survive imatinib treatment and for CML transition to lymphoid blast crisis. Apart from BCR-ABL and SRC kinases, stem cell pathways must also be targeted for curative therapy of Ph+ leukaemia.     

Hypoxia‐induced proliferation in mesenchymal stem cells and angiotensin II‐mediated PI3K/AKT pathway

Hypoxia could stimulate proliferation of mesenchymal stem cells (MSCs) under certain conditions. This study determined angiotensin II mechanisms and PI3K/AKT pathway in hypoxia‐induced proliferation of MSCs. Hypoxia (3% oxygen) induced cellular proliferation in mouse MSCs and upregulated endogenous angiotensin II and angiotensin‐converting enzyme in the cell culture and expression of AT1 receptors. The expressions of Sox2, not Oct4 and Rex1, were significantly increased by the hypoxia. The blockade of AT1 receptors, not AT2 receptors, depressed hypoxia induced the proliferative effects. Both hypoxia and exogenous angiotensin II activated p‐AKT. Moreover, AT1 receptor inhibitor blocked the effects of hypoxia‐mediated p‐AKT upregulation. The data demonstrated that the hypoxia at 3% oxygen level could induce mouse MSC proliferation, probably as a result of the activation of PI3K signalling pathways via AT1 receptors. Copyright © 2015 John Wiley & Sons, Ltd.     

Cancer stem cells: lessons from leukaemia

Increasing evidence suggests that leukaemias are sustained by leukaemic stem cells. Leukaemia can indeed be viewed as aberrant haematopoietic processes initiated by rare leukaemic stem cells that have maintained or re-acquired the capacity for indefinite proliferation through accumulated mutations and/or epigenetic changes. Yet, despite their critical importance, much remains to be learned about the developmental origin of leukaemic stem cells and the molecular pathways underlying the transformation of normal cells into leukaemic stem cells. This report will review our current knowledge on leukaemic stem cells development and finally demonstrate how these discoveries provide a paradigm for identification of cancer stem cells from solid tumours.     

Mechanisms of haemopoietic stem cell proliferation control

The control of stem cell (CFU-S) proliferation is mediated by short-range acting factors which can be detected by the proliferation modifying activities present in media conditioned by haemopoietic cells. A specific inhibitor of stem cell proliferation is obtained from haemopoietic tissue containing minimally proliferating CFU-S, whilst stimulatory material is obtained from cell suspensions containing rapidly proliferating CFU-S. Used competitively, these factors, which are detected in different molecular weight range fractions, manipulate the rate of CFU-S proliferation in a manner compatible with a physiological control mechanism. In addition, a long-term bone marrow culture system has been shown to provide an in vitro model of stem cell control. Fractionation of cell populations from haemopoietic tissues reveals marked concentration differences of the CFU-S proliferation modifying activities depending on the proliferative state of the CFU-S. However, irrespective of whether the tissue contains stem cells that are actively or minimally proliferating, both stimulatory and inhibitory activities are detected. From dose-response studies it is concluded that stem cell proliferation is controlled by an appropriate balance of stimulatory and inhibitory factors which, however, are not produced by the stem cells themselves.     

Granulocytopoiesis in children with acute lymphoblastic leukaemia.

Numbers, proliferative potential, and differentiative capacity of bone marrow granulocyte-macrophage precursor cells were studied in 130 children with acute lymphoblastic leukaemia (ALL), including 77 children in an acute phase of the disease and 53 in remission. Bone marrow samples from 65 children without haematopoietic abnormalities were used as controls. The numbers of clonogenic precursors were found to be below normal in all phases of ALL, particularly during the acute period when the bone marrow was heavily infiltrated with leukaemic cells. It is shown that the decreases in the numbers and proliferative potential of the precursor cells during the acute phases was associated with the effects of leukaemic blast cells, but that in remission the observed reduction in the precursor cell pool was due to the cytostatic effect of therapy. The differentiative capacity of clonogenic granulocyte and macrophage precursors was not altered in children with ALL.     

Differences in electrophoretic mobility and cytochemistry of leukemic T-cells with identical immunologic phenotype (CD1 positive)

Leukaemic cells of acute T-lymphoblastic leukaemia and T-lymphoblastic lymphoma with secondary leukaemic course of disease which showed the same immunological phenotype (cortical thymocytes, thymocyte stage II), could be further differentiated according to their cytochemical pattern and electrophoretic mobility (EPM). Leukaemic cells of T-ALL usually reveal a high EPM, whereas EPM of leukaemic cells of T-lymphoblastic lymphoma was significantly lowered. In cytochemical respect acid phosphatase was positive in both cases of leukaemia. An activity of acid esterase, however, could only be demonstrated in leukaemic cells of T-lymphoblastic lymphoma. The findings are interpreted as manifestation of a different stage of maturity of both T-cell clones with the same immunological phenotype.     

Uptake and metabolism of myo-inositol by L1210 leukaemia cells.

The initial rate of uptake of [3H]myo-inositol by L1210 murine leukaemia cells is directly proportional to the extracellular concentration and unaffected by several analogues of myo-inositol even at millimolar concentrations. Scyllitol, a geometric isomer of myo-inositol, partially inhibited the uptake of myo-inositol (40% at 0.1 mM). A portion of the uptake of myo-inositol was not inhibited even at 5 mM-scyllitol. At steady-state the intracellular concentration of [3H]myo-inositol is directly proportional to the extracellular concentration. Addition of myo-inositol to medium does not enhance the growth of L1210 cells; these cells can maintain an extracellular concentration of 20 microM-myo-inositol even when grown in myo-inositol-free medium. Synthesis of myo-inositol from glucose by L1210 cells was demonstrated by use of [13C]glucose and m.s. L1210 cells maintain myo-inositol pools by a combination of synthesis de novo and uptake of exogenous myo-inositol by either passive diffusion or a low affinity carrier.