3-Hydrogenkwadaphnin induces monocytic differentiation and enhances retinoic acid-mediated granulocytic differentiation in NB4 cell line

Recently, we have reported that 3-hydrogenkwadaphnin (3-HK), a diterpene ester isolated from Dendrostellera lessertii (Thymealeaceae), is very effective against leukemia cell lines without any detectable effects on normal cells (Moosavi et al., 2005b). In this study, we report that 3-HK induces G1 cell-cycle arrest, differentiation and apoptosis in APL NB4 cell line. Indeed, the drug between 24 to 96 h induced 7-65% growth inhibition of NB4 cells. Cell viability was also decreased by 2-55% between 24 to 96 h treatments with the drug, respectively. These effects of the drug were also dose-dependent. According to flow cytomtry results, 3-HK (15 nM) induced a significant G1-arrest up to 24 h which was consequently followed with appearance of sub-G(1) peak at 72 to 96 h. Hoechst 33258 staining and DNA fragmentation assays confirmed the occurrence of apoptosis among the treated cells. On the other hand, NBT reducing assay, Wright-Giemsa staining, phagocytic activity and expression of cell surface markers (CD11b and CD14) confirmed that the inhibition of proliferation is associated with differentiation especially toward macrophage-like morphology. Interestingly, 3-HK at 5 and 10 nM enhanced the effects of all-trans retinoic acid (ATRA) in NB4 cells. Based on these results, 3-HK might become an ideal candidate for treatment of APL patients pending full exploration of its biological functions.     

Regulation of class II gene expression in a murine pre-B cell line

Class II major histocompatibility complex (MHC) gene expression has been studied in an Abelson virus-transformed pre-B cell line R8, and its Ia-negative variant R8205. These variant cells contained barely detectable levels of RNA specific for all class II genes, including the nonpolymorphic invariant chain gene (Ii), and did not express cell surface Ia. Fusion of this murine Ia-negative cell line to the human Ia-positive Raji cell produced an interspecies hybridoma that expressed the murine Ia. These data are further evidence for the existence of trans-acting factors that can regulate class II gene expression. Furthermore, the T cell-derived lymphokine B cell stimulatory factor 1 (BSF-1) induced expression of class II genes in the R8205 cells. Exposure of R8205 cells to an antibody that has been shown to mimic BSF-1 activity on normal B cells also resulted in expression of class II genes. These data demonstrate that three distinct signals--a lymphokine, an alloantibody binding to membrane structures, and an interspecies trans-acting factor--can induce expression of class II genes.     

Effects of neurotrophic factors and cell substrates on the differentiation of a sympathoadrenal progenitor cell line

The detailed spatial organization of cytoskeletal proteins in an immortalized sympathoadrenal precursor cell line, termed MAH, was studied when the cells were grown on cellular substrates and when treated with combinations of recombinant nerve growth factor, ciliary neurotrophic factor and basic fibroblast growth factor. In response to growth factors, MAH cells expressed appropriate distributions of phosphorylated and non-phosphorylated neurofilaments, and dendrite and axon specific microtubule associated proteins. Sequential stages of maturation and axon formation were identified as the MAH cells established neuronal polarity and developed into sympathetic-like neurons. Combinations of the growth factors initiated growth associated protein-43 expression in processes and promoted the MAH cells to acquire sympathetic-like neuron characteristics with long, thin processes that branched and often terminated in elaborate growth cones. When treated with the three trophic factors, 15% of the MAH cells differentiated into sympathetic-like neurons, in contrast to less than 10% when cultured with ciliary neurotrophic factor plus nerve growth factor. An enhanced cholinergic phenotype was evident in the MAH cells when grown with ciliary neurotrophic factor. MAH cells also expressed neuron-specific markers when co-cultured on enriched substrates of smooth muscle, fibroblasts or Schwann cells. The results indicate that this sympathoadrenal cell lineage, carrying the v-myc oncogene, can express appropriate cytoskeletal markers in the process of neuronal differentiation when induced by neurotrophic factors or by specific cellular conditions in vitro.     

Induction of the granulocyte-macrophage colony-stimulating factor (CSF) receptor by granulocyte CSF increases the differentiative options of a murine hematopoietic progenitor cell.

32DC13(G) is an interleukin-3-dependent murine hematopoietic precursor cell line which differentiates into neutrophilic granulocytes upon exposure to granulocyte colony-stimulating factor (G-CSF) but ceases to proliferate and dies when exposed to granulocyte-macrophage (GM)-CSF. Surface receptors for GM-CSF are undetectable on 32DC13(G) cells but can be induced by priming the cells with G-CSF. Exposure of the G-CSF-primed cells to GM-CSF then results in the generation of monocytes as well as granulocytes. The acquired competence to respond to GM-CSF remains irreversibly encoded in the primed cells, although the GM-CSF receptor can be down regulated by interleukin-3. This phenomenon suggests a mechanism by which hematopoietic precursors may obtain additional receptors, thereby increasing their differentiative potential.     

Neolacto-series gangliosides induce granulocytic differentiation of human promyelocytic leukemia cell line HL-60

Neolacto-series gangliosides having linear poly-N-acetyl-lactosaminyl oligosaccharide structure have been demonstrated to be increased characteristically during granulocytic differentiation of human promyelocytic leukemia cell line HL-60 cells induced by dimethyl sulfoxide or retinoic acid (Nojiri, H., Takaku, F., Tetsuka, T., Motoyoshi, K., Miura, Y., and Saito, M. (1984) Blood 64, 534-541). When HL-60 cells were cultured in the presence of neolacto-series gangliosides prepared from mature granulocytes, the cells were found to be differentiated into mature granulocytes on the basis of the changes of morphology, surface membrane antigens, nonspecific esterase activity, and the activity of phagocytosis and respiratory burst. The differentiation of cells was dependent on the concentration of gangliosides and accompanied with inhibition of cell growth. These findings suggest that the particular ganglioside molecules play an important role in regulation of cell differentiation and that the appearance of neolacto-series gangliosides on cell surface membrane not only triggers the differentiation but also determines the direction of differentiation in HL-60 cells.     

Effect of erythroid differentiation factor on megakaryocytic differentiation of L8057, a murine megakaryoblastic leukemia cell line.

To assess the potent effect of erythroid differentiation factor (EDF) on megakaryocytopoiesis, effect of EDF on megakaryocytic differentiation of L8057, a murine megakaryoblastic cell line, was examined. EDF potentiated AchE induction of L8057 in a dose dependent manner. The potency of EDF on megakaryocytic differentiation is comparable to that on erythroid differentiation reported previously. The present results suggest that EDF may play a regulatory role in megakaryocytopoiesis as well as in erythropoiesis.     

The in vitro differentiation of a bipotential glial progenitor cell.

We have studied the properties of a glial progenitor cell from 7-day-old rat optic nerve that differentiates in vitro into an oligodendrocyte if cultured in serum-free medium and into an astrocyte if cultured in foetal calf serum (FCS). Using galactocerebroside as a marker of oligodendrocyte differentiation and glial fibrillary acidic protein as a marker of astrocyte differentiation, we show that the acquisition of these marker molecules occurs rapidly in culture and requires both RNA and protein synthesis. We provide evidence that the effect of FCS on the development of the glial progenitor cell is not due to its influence on cell-substrate adherence or actin filament organization and is not mimicked by an increase in intracellular cyclic AMP, cyclic GMP or pH. The progenitor cell contains vimentin filaments and retains them on becoming an astrocyte but loses them on becoming an oligodendrocyte. Most importantly, we show that the choice of developmental pathway taken by the bipotential glial progenitor cells in culture is reversible for 1-2 days and then becomes fixed, at least under the conditions we studied.     

Regulation of protein synthesis and accumulation during murine erythroleukemia cell differentiation

We have examined the repertoire of cytoplasmic proteins present at different times during murine erythroleukemia (MEL) cell differentiation. Our laboratory has developed an improved differentiation system in which the use of rapidly inducing MEL subclones and culture conditions which stabilize terminally differentiated cells results in highly synchronous differentiation and the accumulation of large numbers of cells in the end stages of differentiation. Using two-dimensional gel electrophoresis, the proteins of MEL cell cytoplasm have been fractionated at different times of induction in the improved system. The protein composition of MEL cell cytoplasm changes dramatically during the differentiation program, in contrast to previously reported results. We observe patterns of changes that are consistent with alterations in the relative degradative rates as well as the relative synthetic rates of the different proteins. We find that the rate of incorporation of labeled amino acid into protein is reduced in induced cultures of MEL cells. We demonstrate that the contribution of uninduced cells to the protein patterns observed late in differentiation is minor in our system, and argue that the results previously obtained for differentiating MEL cells were influenced by the heterogeneity of the induced populations.     

Triiodothyronine control of ATP-citrate lyase and malic enzyme during differentiation of a murine preadipocyte cell line.

In the Ob 17 preadipocyte cell line, during adipose differentiation, T3 amplified the progressive expression of two enzymes of the lipogenic pathway, ATP-citrate lyase (ATP-CL) and malic enzyme (ME) as previously described for fatty acid synthase (FAS) and fatty acid synthesis, and in the same time-period of development. However, the stimulation by T3 was sustained at late stages of differentiation whereas it declined in FAS studies. The stimulation was preceded by an increase in the relative abundance of the specific mRNAs. Two ME mRNA species were detected (21S and 27S) and found to be differently distributed. Their abundance was asynchronously increased by T3 with a predominant effect on the 21S species. Culture of the cells in a thyroid-hormone depleted medium prevented any significant increase of ME activity. Early inclusion of T3 largely restored ME development whereas late elimination of T3 only moderately impaired it. It is suggested that T3 plays a crucial role at an early step of adipose differentiation, this leading to an increased expression of a set of late adipose phenotypes such as several lipogenic enzymes.     

小鼠角膜上皮祖细胞系TKE2的表型研究

目的：观察小鼠角膜上皮祖细胞系TKE2在扩增以及分化状态下的角蛋白及干细胞标志物的表达情况。方法小鼠角膜上皮祖细胞系TKE2在无血清培养基Keratinocyte-SFM （KSFM）以及含10﹪胎牛血清（FBS）的DMEM培养基中培养,约70﹪融合时进行角蛋白10、12、14、15、16（K10、K12、K14、K15、K16）以及Connexin43、ABCG2的免疫荧光染色,以及Ki67、P63、PCNA的免疫细胞化学染色。结果无血清培养状态下的TKE2细胞呈克隆样生长,克隆内所有细胞呈ABCG2、K14、Ki67、PCNA以及P63阳性,K15阳性细胞散在分布,K16阳性细胞呈片状分布于克隆中央区,K10、K12以及Connexin43染色为阴性。在含有10﹪胎牛血清的DMEM中培养2 d后,细胞明显增大, ABCG2、K15、P63、Ki67以及PCNA转为阴性,克隆内只有少量细胞呈K16、K14阳性染色, K10、K12、Connexin43仍为阴性。结论 TKE2细胞具有角膜上皮干细胞特性,可以作为角膜缘上皮干细胞表型维持和分化诱导研究的良好工具。     

Immunity to leprosy. II. Genetic control of murine T cell proliferative responses to Mycobacterium leprae

T cell proliferative responses to Mycobacterium leprae were measured after immunization of mice at the base of the tail with antigen and challenging lymphocytes from draining lymph nodes in culture with M. leprae. This T cell response to M. leprae has been compared in 18 inbred strains of mice. C57BL/10J mice were identified as low responder mice. The congenic strains B10.M and B10.Q were found to be high responders, whereas B10.BR and B10.P were low responders. F1 (B10.M X C57BL/10J) and F1 (B10.Q X C57BL/10J) hybrid mice were found to be low responders, similar to the C57BL/10J parent, indicating that the low responsive trait is dominant. Whereas B10.BR mice were shown to be low responders to M. leprae, B10.AKM and B10.A(2R) were clearly high responders, indicating that the H-2D region influences the magnitude of the T cell proliferative response. Gene complementation within the H-2 region was evident. Genes outside the H-2 region were also shown to influence the response to M. leprae. C3H/HeN were shown to be high responder mice, whereas other H-2k strains, BALB.K, CBA/N, and B10.BR, were low responders. Gene loci that influence the T cell proliferation assay have been discussed and were compared to known background genes which may be important for the growth of intracellular parasites. Because mycobacteria are intracellular parasites for antigen-presenting cells, genes that affect bacterial growth in these cells will also influence subsequent immune responses of the host.     

Commitment in a murine embryonal carcinoma cell line during differentiation induced by retinoic acid

Murine embryonal carcinoma (EC) cells are induced to differentiate when cultured in the presence of retinoic acid (RA). Whereas the EC cells have a high plating efficiency, the differentiated cells have little or no colony-forming ability under the same conditions. We have assumed that the loss of colony-forming ability following exposure of EC cells to RA corresponds to the irreversible commitment of EC cells to differentiate. We found that uncommitted EC cells persist in RA-treated aggregates of EC cells and that the proportion of EC cells stabilizes at a level inversely related to the RA concentration. Both experimental evidence and mathematical modelling results are consistent with the interpretation that there is a dynamic equilibrium achieved by a balance between the processes of EC cell proliferation and differentiation. Since different cell types are induced by different RA concentrations, our results suggest that the commitment to differentiate is not related in any simple way to the developmental program which ensues.     

Effect of myleran on murine hemopoiesis. I. Granulocytic cell line specificity of action on progenitor cells.

Time- and dose-dependent patterns of depletion and regeneration of hemopoietic progenitor cells in mouse femora and spleens following treatment with the antileukemic agent Myleran (Busulphan, MY) were studied using the murine spleen colony system and the agar gel in vitro colony system. MY was found to depress granulopoiesis selectively, as manifested by the development of marked prolonged neutropenia, hypoplasia of the bone marrow and (to a lesser degree) of the spleen, reduction of the incidence of multipotential hemopoietic progenitor cells (CFU-S) and of granulocytic progenitor cells (CFU-C) in both femora and spleens, and impairment of the capacity of CFU-S from either tissue to generate granulocytic colonies in the spleens of irradiated hosts. The severity and duration was greatest at high dose levels of MY (800 microgram). The action of MY on CFU-S was more pronounced than that on CFU-C, suggesting that MY is a cycle-independent agent. Repopulation of the CFU-C pool preceded that of the CFU-S pool. Development of neutropenia and maximal marrow hypoplasia followed the onset of depression of CFU-S and CFU-C incidence, while recovery of normal nucleated cellularity in the blood, femur and spleen preceded repopulation of the CFU-S and CFU-C pools. MY treatment resulted in transitory stimulation of colony stimulating factor (CSF) generation by the femur but had no effect on serum CSF levels. The peak of femoral CSF generation coincided with the nadir of CFU-C depression. These findings indicated that the prolonged neutropenia following MY treatment was secondary to depletion of the progenitor cell pools, that during recovery granulopoietic repopulation took precedence over self-maintenance of the hemopoietic progenitor cell pools, and that increased generation of CSF may play a role in the early phase of granulopoietic recovery.     

Effect of AML serum on normal human myeloid differentiation and hexamethylene bisacetamide-induced granulocytic differentiation of the human HL-60 promyelocytic leukaemic cell line.

The effect of serum from 32 AML patients on the normal human myeloid differentiation and the hexamethylene-bisacetamide induced granulocytic differentiation of HL-60 promyelocytic leukaemic cell line was studied. Nonadherent normal mononuclear marrow cells were cultured in vitro at a concentration of 5 x 10(5) cells/ml for 6 days with each of the 32 AML sera. Ten normal human AB sera were used as control. The results showed an inhibitory activity on both morphological and functional differentiation of normal human myeloid immature marrow cells by 29 out of the 32 AML sera tested. These 29 AML sera were added to cultures of HL-60 (2.5 x 10(5)/ml) leukaemia cell line which incorporated 2 mM hexamethylene-bisacetamide for 6 days. The results showed no significant inhibition of hexamethylene-bisacetamide induced granulocytic differentiation by any of the 29 AML sera. The efficacy of hexamethylene-bisacetamide in inducing differentiation in the presence of inhibitory factors suggests a possible role in the treatment of AML patients.     

Oxidative stress enhances granulocytic differentiation in HL 60 cells,an acute promyelocytic leukemia cell line

Abstract

This paper studied the effects of physiologically available oxidants on HL 60 differentiation induced by all-trans retinoic acid (ATRA) or dimethyl sulfoxide (DMSO). Hydrogen peroxide (15 μM) and taurine chloramine (200 μM) induced HL 60 differentiation, which was detected by CD11b expression and superoxide production. Cd11b and p67^phox mRNA expression was also augmented by these oxidants. In contrast, reducing chemicals, such as dithiothreitol, 2,3-dimercapto-1-propanol and N-acetylcysteine inhibited CD11b expression. Notably, DMSO inhibited methionine sulfoxide reductase activity, induced heme oxygenase-1 (ho-1) mRNA and enhanced oxidant-induced cell death, which indicated that DMSO intensified oxidative stress. After the addition of oxidants, ho-1 expression preceded the cd11b expression. Vicinal dithiol-reactive phenylarsine oxide (50 nM) also increased CD11b expression induced by DMSO or ATRA. These observations suggested that oxidative stress enhanced granulocytic differentiation of HL 60 cells and that leukaemic cell differentiation was affected by cellular redox status.