Azo dyes prevent hydrocarbon-induced leukemia in the rat.
A set of intraveous injections of 7,8,12-trimethylbenz[a]anthracene consistently elicited leukemia in more than 75% of young adult Long-Evans female rats. There was a profound reduction in the incidence of leukemia in companion groups of rats fed small amounts (1--10 mg) of Sudan III or Sudan IV prior to each injection of the carcinogenic hydrocarbon. Repeated feedings of 1 mg of Sudan III induced cumulative increases in the concentration of menadione reductase (EC 18.104.22.168) in liver, whereas protein concentration was unchanged. A single feeding of 1 mg of Sudan III prevented fatal toxicity in all members of large groups of rats injected with massive doses of 7,12-dimethylbenz[a]anthracene, but 50% of the survivors developed leukemia; unprotected rats succumbed in 1--3 days. Sudan III was not carcinogenic under the experimental conditions. (+info
Development of viral vectors for gene therapy of beta-chain hemoglobinopathies: optimization of a gamma-globin gene expression cassette.
Progress toward gene therapy of beta-chain hemoglobinopathies has been limited in part by poor expression of globin genes in virus vectors. To derive an optimal expression cassette, we systematically analyzed the sequence requirements and relative strengths of the Agamma- and beta-globin promoters, the activities of various erythroid-specific enhancers, and the importance of flanking and intronic sequences. Expression was analyzed by RNase protection after stable plasmid transfection of the murine erythroleukemia cell line, MEL585. Promoter truncation studies showed that the Agamma-globin promoter could be deleted to -159 without affecting expression, while deleting the beta-globin promoter to -127 actually increased expression compared with longer fragments. Expression from the optimal beta-globin gene promoter was consistently higher than that from the optimal Agamma-globin promoter, regardless of the enhancer used. Enhancers tested included a 2.5-kb composite of the beta-globin locus control region (termed a muLCR), a combination of the HS2 and HS3 core elements of the LCR, and the HS-40 core element of the alpha-globin locus. All three enhancers increased expression from the beta-globin gene to roughly the same extent, while the HS-40 element was notably less effective with the Agamma-globin gene. However, the HS-40 element was able to efficiently enhance expression of a Agamma-globin gene linked to the beta-globin promoter. Inclusion of extended 3' sequences from either the beta-globin or the Agamma-globin genes had no significant effect on expression. A 714-bp internal deletion of Agamma-globin intron 2 unexpectedly increased expression more than twofold. With the combination of a -127 beta-globin promoter, an Agamma-globin gene with the internal deletion of intron 2, and a single copy of the HS-40 enhancer, gamma-globin expression averaged 166% of murine alpha-globin mRNA per copy in six pools and 105% in nine clones. When placed in a retrovirus vector, this cassette was also expressed at high levels in MEL585 cells (averaging 75% of murine alpha-globin mRNA per copy) without reducing virus titers. However, recombined provirus or aberrant splicing was observed in 5 of 12 clones, indicating a significant degree of genetic instability. Taken together, these data demonstrate the development of an optimal expression cassette for gamma-globin capable of efficient expression in a retrovirus vector and form the basis for further refinement of vectors containing this cassette. (+info
Evidence that a single replication fork proceeds from early to late replicating domains in the IgH locus in a non-B cell line.
In non-B cell lines, like the murine erythroleukemia cell line (MEL), the most distal IgH constant region gene, C alpha, replicates early in S; other heavy chain constant region genes, joining and diversity segments, and the most proximal Vh gene replicate successively later in S in a 3' to 5' direction proportional to their distance from C alpha. In MEL, replication forks detected in the IgH locus also proceed in the same 3' to 5' direction for approximately 400 kb, beginning downstream of the IgH 3' regulatory region and continuing to the D region, as well as within the Vh81X gene. Downstream of the initiation region is an early replicating domain, and upstream of Vh81X is a late replicating domain. Hence, the gradual transition between early and late replicated domains can be achieved by a single replication fork. (+info
An intronic enhancer containing an N-box motif is required for synapse- and tissue-specific expression of the acetylcholinesterase gene in skeletal muscle fibers.
mRNAs encoding acetylcholinesterase (AChE; EC 22.214.171.124) are highly concentrated within the postsynaptic sarcoplasm of adult skeletal muscle fibers, where their expression is markedly influenced by nerve-evoked electrical activity and trophic factors. To determine whether transcriptional regulatory mechanisms account for the synaptic accumulation of AChE transcripts at the mammalian neuromuscular synapse, we cloned a 5.3-kb DNA fragment that contained the 5' regulatory region of the rat AChE gene and generated several constructs in which AChE promoter fragments were placed upstream of the reporter gene lacZ and a nuclear localization signal (nls). Using a recently described transient expression assay system in intact skeletal muscle, we show that this AChE promoter fragment directs the synapse-specific expression of the reporter gene. Deletion analysis revealed that a 499-bp fragment located in the first intron of the AChE gene is essential for expression in muscle fibers. Further analysis showed that sequences contained within this intronic fragment were (i) functionally independent of position and orientation and (ii) inactive in hematopoietic cells. Disruption of an N-box motif located within this DNA fragment reduced by more than 80% the expression of the reporter gene in muscle fibers. In contrast, mutation of an adjacent CArG element had no effect on nlsLacZ expression. Taken together, these results indicate that a muscle-specific enhancer is present within the first intron of the AChE gene and that an intronic N-box is essential for the regulation of AChE along skeletal muscle fibers. (+info
Bcl-XL induction during terminal differentiation of friend erythroleukaemia cells correlates with delay of apoptosis and loss of proliferative capacity but not with haemoglobinization.
Friend murine erythroleukaemia (F-MEL) cells are a useful model for studying the processes that regulate erythroid differentiation since exposure of these cells to chemical inducers (DMSO or HMBA) results in commitment to terminal cell division and synthesis of haemoglobin. This study examined the relationship between differentiation and apoptosis in DMSO sensitive and resistant F-MEL cells. Clear apoptosis was not observed in DMSO-treated sensitive F-MEL (strain 745A) cells during the induction of differentiation. In contrast, DMSO-induced 745A cells exhibited delayed apoptosis compared to uninduced cells. Since the Bcl-2 family members play a major role in the control of apoptosis and/or differentiation, we determined their expression before and after DMSO or HMBA treatment. Neither untreated nor chemically-induced 745A cells expressed the Bcl-2 protein. The levels of Bax and Bad proteins remained relatively constant during DMSO-induced differentiation. DMSO or HMBA treatment of 745A cells induced a marked increase of Bcl-XL expression during the late phase of differentiation which persisted even when the cells began to die. This upregulation of Bcl-XL was independent of cell density but was correlated with cell arrest in G0/G1. DMSO treatment induced a similar delay of apoptosis and enhancement of Bcl-XL expression in F-MEL (strain TFP10) cells which fail to synthesize haemoglobin in the presence of DMSO. Dexamethasone, which blocks DMSO-induced differentiation of F-MEL cells, prevented the induction of Bcl-XL. Inhibitors such as imidazole or succinylacetone, which inhibit haemoglobin synthesis but not commitment to terminal cell division, did not suppress Bcl-XL induction in DMSO-induced cells. Taken together, these results indicate that DMSO treatment of F-MEL cells induces a marked increase in Bcl-XL expression suggesting a role for this anti-apoptotic protein in the process of erythroid differentiation in F-MEL cells. Moreover, induction of Bcl-XL during this process seems to be associated with loss of proliferative capacity rather than with haemoglobin synthesis. (+info
Phosphatidylserine externalization during differentiation-triggered apoptosis of erythroleukemic cells.
K562 erythroleukemia cells undergo apoptosis when induced to differentiate along the erythroid lineage with hemin. This event, characterized by DNA fragmentation, correlated with downregulation of the survival protein, BCL-xL, and decrease in mitochondrial transmembrane potential (deltapsi[m]) that ultimately resulted in cell death. Reorientation of phosphatidylserine (PS) from the cells inner-to-outer plasma membrane leaflet and inhibition of the aminophospholipid translocase was observed upon hemin-treatment. Constitutive expression of BCL-2 did not inhibit hemin-induced alterations in lipid asymmetry or decrease in deltapsi[m], and only moderately prevented DNA fragmentation. BCL-2, on the other hand, effectively inhibited actinomycin D-induced DNA fragmentation, the appearance of PS at the cells outer leaflet and the decrease in deltapsi[m]. The caspase inhibitor, z.VAD.fmk, blocked DNA fragmentation by both hemin and actinomycin D, but inhibited PS externalization only in the actinomycin D-treated cells. These results suggest that, unlike pharmacologically-induced apoptosis, PS externalization triggered by differentiation-induced apoptosis occurs by a mechanism that is associated with a decrease in deltapsi[m], but independent of BCL-2 and caspases. (+info
Extracellular-regulated kinase 1/2, Jun N-terminal kinase, and c-Jun are involved in NF-kappa B-dependent IL-6 expression in human monocytes.
In the present study we investigated the possible involvement of the mitogen-activated protein kinase family members extracellular-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) in mediating IL-6 gene expression in human monocytes, in particular their role in enhancing NF-kappa B activity. Freshly isolated monocytes treated with the protein phosphatase inhibitor okadaic acid secreted high levels of IL-6 protein, which coincided with enhanced binding activity of NF-kappa B as well as with phosphorylation and activation of the ERK1/2 and JNK proteins. The ERK pathway-specific inhibitor PD98059 inhibited IL-6 secretion from monocytes. Transient overexpression of inactive mutants of either Raf-1 or JNK1 showed that both pathways were involved in kappa B-dependent IL-6 promoter activity. By using PD98059, we demonstrated that the Raf1/MEK1/ERK1/2 pathway did not affect the DNA binding of NF-kappa B but, rather, acted at the level of transcriptional activity of NF-kappa B. Interestingly, it was shown that NF-kappa B-mediated gene transcription, both in the context of the IL-6 promoter as well as on its own, was dependent on both serine kinase activity and interaction with c-Jun protein. We conclude that okadaic acid-induced IL-6 gene expression is at least partly mediated through the ERK1/2 and JNK pathway-dependent activation of NF-kappa B transcriptional capacity. Our results suggest that the JNK pathway may regulate NF-kappa B-mediated gene transcription through its phosphorylation and activation of c-Jun. (+info
Induction of erythroid differentiation by altered Galpha16 activity as detected by a reporter gene assay in MB-02 cells.
Heterotrimeric G proteins may assume modulatory roles in cellular proliferation and differentiation. The G protein alpha-subunit Galpha16, which is specifically expressed in hematopoietic cells, is highly regulated during differentiation of normal and leukemic cells. In human erythroleukemia cells, suppression of Galpha16 inhibited cellular growth rates. A reporter gene system was established to assess the role of Galpha16 on erythroid differentiation of MB-02 erythroleukemia cells. It is based on transient transfection with a plasmid that expresses green fluorescent protein under the control of the beta-globin promoter. Expression of Galpha16 led to a significant increase in green fluorescent protein-positive cells, as did transfection with a Galpha16 antisense plasmid (154 and 156% of controls, respectively). The GTPase-deficient, constitutively active mutant of Galpha16, Galpha16R186C, further stimulated differentiation to 195% of control values. Because the effect of Galpha16 is triggered most efficiently by the GTP-bound protein, an indirect action through interference of overexpressed Galpha16 with G protein betagamma-subunits can be excluded. The corresponding mutant of Galphaq (GalphaqR182C), the phylogenetically closest family member of Galpha16, had no effect. The data define a specific role for Galpha16-dependent signal transduction in cellular differentiation: deviations from optimal levels of Galpha16 functional activity lead to reduced growth rates and promote differentiation in hematopoietic cells. (+info