Long-term hematopoietic transfer of the anti-cancer and lifespan-extending capabilities of a genetically engineered blood system by transplantation of bone marrow mononuclear cells.

A causal relationship exists among the aging process, organ decay and disfunction, and the occurrence of various diseases including cancer. A genetically engineered mouse model, termed Klf1K74R/K74R or Klf1(K74R), carrying mutation on the well-conserved sumoylation site of the hematopoietic transcription factor KLF1/EKLF has been generated that possesses extended lifespan and healthy characteristics, including cancer resistance. We show that the healthy longevity characteristics of the Klf1(K74R) mice, as exemplified by their higher anti-cancer capability, are likely gender-, age-, and genetic background-independent. Significantly, the anti-cancer capability, in particular that against melanoma as well as hepatocellular carcinoma, and lifespan-extending property of Klf1(K74R) mice, could be transferred to wild-type mice via transplantation of their bone marrow mononuclear cells at a young age of the latter. Furthermore, NK(K74R) cells carry higher in vitro cancer cell-killing ability than wild-type NK cells. Targeted/global gene expression profiling analysis has identified changes in the expression of specific proteins, including the immune checkpoint factors PDCD and CD274, and cellular pathways in the leukocytes of the Klf1(K74R) that are in the directions of anti-cancer and/or anti-aging. This study demonstrates the feasibility of developing a transferable hematopoietic/blood system for long-term anti-cancer and, potentially, for anti-aging.

H3K9 histone methyltransferase, KMT1E/SETDB1, cooperates with the SMAD2/3 pathway to suppress lung cancer metastasis.

Aberrant histone methylation is a frequent event during tumor development and progression. KMT1E (also known as SETDB1) is a histone H3K9 methyltransferase that contributes to epigenetic silencing of both oncogenes and tumor suppressor genes in cancer cells. In this report, we demonstrate that KMT1E acts as a metastasis suppressor that is strongly downregulated in highly metastatic lung cancer cells. Restoring KMT1E expression in this setting suppressed filopodia formation, migration, and invasive behavior. Conversely, loss of KMT1E in lung cancer cells with limited metastatic potential promoted migration in vitro and restored metastatic prowess in vivo. Mechanistic investigations indicated that KMT1E cooperates with the TGFß-regulated complex SMAD2/3 to repress metastasis through ANXA2. Together, our findings defined an essential role for the KMT1E/SMAD2/3 repressor complex in TGFß-mediated lung cancer metastasis.

The self-interaction of native TDP-43 C terminus inhibits its degradation and contributes to early proteinopathies.

The degraded, misfolded C terminus of TAR DNA-binding protein-43 is associated with a wide spectrum of neurodegenerative diseases, particularly frontotemporal lobar degeneration with ubiquitin-positive inclusions and amyotrophic lateral sclerosis. However, the precise mechanism of pathological cleavage of the TAR DNA-binding protein-43 remains unknown. Here we show that the TAR DNA-binding protein-43 C-terminal protein physically interacts with itself or with the cellular-folded yeast prion domain of Sup35 forming dynamic aggregates. This prion-like nature governs known cellular functions of the TAR DNA-binding protein-43, including subcellular localisation and exon skipping of the cystic fibrosis transmembrane conductance regulator. Significantly, mutants with a failure to engage in prion-like interactions are processed into an ~24-kDa C-terminal fragment of the TAR DNA-binding protein-43. The estimated cleavage site of degraded TAR DNA-binding protein-43 fragments corresponds to the pathological cleavage site identified in patients with the TAR DNA-binding protein-43 proteinopathies. Consistently, epigallocatechin gallate constrains prion-like interactions, attenuating pathological-like degradation. Thus, the native folding of TAR DNA-binding protein-43 C terminus acts as a guardian of pathogenesis, which is directly associated with loss-of-function.

Regulation of autophagy by neuropathological protein TDP-43.

TDP-43 is a DNA/RNA-binding protein with multicellular functions. As a pathosignature protein of a range of neurodegenerative diseases, TDP-43 is also the major component of the polyubiquitinated inclusions in the pathological cellular samples of these diseases. In normal cells, TDP-43 is processed and degraded by both autophagy and the ubiquitin-proteasome systems. We have found, by microarray hybridization and RT-PCR analyses, that the level of the mRNA encoding the major autophagy component Atg7 is decreased upon depletion of TDP-43 by RNAi knockdown. This decrease of the Atg7 mRNA level could be rescued by overexpression of an siRNA-resistant form of TDP-43, and it appears to be the result of destabilization of the Atg7 mRNA, to which TDP-43 could bind through its RNA recognition motif 1 domain. Furthermore, depletion of TDP-43 with the consequent loss of the Atg7 mRNA/ATG7 protein causes impairment of the autophagy and facilitates the accumulation of polyubiquitinated proteins as well as the autophagy/ubiquitin-proteasome system substrate p62 in the cells. These data demonstrate the function of TDP-43 as a maintenance factor of the autophagy system, and they suggest the existence of a feedback regulatory loop between TDP-43 and autophagy. A scenario in which loss of function of TDP-43 contributes to the development of TDP-43 proteinopathies is presented.

Sumoylation of p45/NF-E2: nuclear positioning and transcriptional activation of the mammalian beta-like globin gene locus.

NF-E2 is a transcription activator for the regulation of a number of erythroid- and megakaryocytic lineage-specific genes. Here we present evidence that the large subunit of mammalian NF-E2, p45, is sumoylated in vivo in human erythroid K562 cells and in mouse fetal liver. By in vitro sumoylation reaction and DNA transfection experiments, we show that the sumoylation occurs at lysine 368 (K368) of human p45/NF-E2. Furthermore, p45 sumoylation enhances the transactivation capability of NF-E2, and this is accompanied by an increase of the NF-E2 DNA binding affinity. More interestingly, we have found that in K562 cells, the beta-globin gene loci in the euchromatin regions are predominantly colocalized with the nuclear bodies promyelocytic leukemia protein (PML) oncogenic domains that are enriched with the PML, SUMO-1, RNA polymerase II, and sumoylatable p45/NF-E2. Chromatin immunoprecipitation assays further showed that the intact sumoylation site of p45/NF-E2 is required for its binding to the DNase I-hypersensitive sites of the beta-globin locus control region. Finally, we demonstrated by stable transfection assay that only the wild-type p45, but not its mutant form p45 (K368R), could efficiently rescue beta-globin gene expression in the p45-null, erythroid cell line CB3. These data together point to a model of mammalian beta-like globin gene activation by sumoylated p45/NF-E2 in erythroid cells.

DNA methyltransferase Dnmt1 and mismatch repair.

We have examined the relationship between DNA mismatch repair and deficiency of DNA methylation in a mouse embryonic cell line, Dnmt1-/- ES, with homologous deletion of the gene coding for the maintenance DNA methyltransferase Dnmt1. With the use of a sensitive PCR for the assay of two mononucleotide- and three dinucleotide microsatellite markers that exhibited instabilities in mismatch repair-deficient cells, significantly higher frequencies of instability were detected at three of the five markers in Dnmt1-/- ES than the wild-type ES. The data suggest that Dnmt1 enzyme plays an integrating role in DNA replication and/or repair. The implication that Dnmt1 enzyme and/or cytosine methylation may participate in the strand discrimination of mismatch repair during eukaryotic DNA replication is discussed.

A candidate gene for Drosophila genome methylation.

Like vertebrates, the genome of Drosophila melanogaster also contains methylated cytosines. However, the enzyme(s) responsible for this methylation has been elusive. By DNA transfection and sodium bisulfite sequencing, we show here that overexpression of dDnmt2, which is the only expressed and cloned Drosophila protein consisting of motifs conserved among the DNA cytosine methyltransferases, results in genomic DNA methylation of Drosophila S2 cells. The data provide the first evidence for dDnmt2 being one candidate gene encoding the Drosophila DNA methyltransferase(s).

The eukaryotic DNMT2 genes encode a new class of cytosine-5 DNA methyltransferases.

DNMT2 is a subgroup of the eukaryotic cytosine-5 DNA methyltransferase gene family. Unlike the other family members, proteins encoded by DNMT2 genes were not known before to possess DNA methyltransferase activities. Most recently, we have shown that the genome of Drosophila S2 cells stably expressing an exogenous Drosophila dDNMT2 cDNA became anomalously methylated at the 5'-positions of cytosines (Reddy, M. N., Tang, L. Y., Lee, T. L., and Shen, C.-K. J. (2003) Oncogene, in press). We present evidence here that the genomes of transgenic flies overexpressing the dDnmt2 protein also became hypermethylated at specific regions. Furthermore, transient transfection studies in combination with sodium bisulfite sequencing demonstrated that dDnmt2 as well as its mouse ortholog, mDnmt2, are capable of methylating a cotransfected plasmid DNA. These data provide solid evidence that the fly and mouse DNMT2 gene products are genuine cytosine-5 DNA methyltransferases.

Erythroid gene suppression by NF-kappa B.

NF-kappa B/Rel transcription factors play essential roles to mediate the immune response and apoptosis, and they have also been implicated in cellular differentiation such as erythropoiesis. To elucidate the possible role(s) of NF-kappa B in erythroid gene regulation and erythropoiesis, we have carried out transient transfection studies of the human embryonic/fetal erythroid cell line K562 and mouse adult erythroid MEL cells. It is shown that tumor necrosis factor-alpha represses the transcription activity directed by either alpha or zeta globin promoter in a dose-dependent manner. Furthermore, different NF-kappa B family members could effectively repress the transfected alpha-like globin promoters in K562 as well as in MEL cells. The involvement of NF-kappa B pathway is supported by the ability of a NF-kappa B-specific, dominant negative mutant to block the tumor necrosis factor-alpha or p65-mediated suppression of the alpha-like globin promoter activities. The suppression appears to be mediated through cis-linked HS-40 enhancer. Finally, stably transfected K562 cells overexpressing p65 contain reduced amounts of the p45/NF-E2 RNA and functional NF-E2 proteins. Our studies have identified a new set of targets of NF-kappa B. We suggest that the relatively high activity of the NF-kappa B pathway in early erythroid progenitors is involved in the suppression of erythroid-specific genes. Later in differentiation, together with other changes, the decline of the amounts of the NF-kappa B family of factors leads to derepression and consequent increase of NF-E2, which in turn would activate a subset of erythroid-specific genes.

High-level erythroid-specific gene expression in primary human and murine hematopoietic cells with self-inactivating lentiviral vectors.

Use of oncoretroviral vectors in gene therapy for hemoglobinopathies has been impeded by low titer vectors, genetic instability, and poor expression. Fifteen self- inactivating (SIN) lentiviral vectors using 4 erythroid promoters in combination with 4 erythroid enhancers with or without the woodchuck hepatitis virus postregulatory element (WPRE) were generated using the enhanced green fluorescent protein as a reporter gene. Vectors with high erythroid-specific expression in cell lines were tested in primary human CD34(+) cells and in vivo in the murine bone marrow (BM) transplantation model. Vectors containing the ankyrin-1 promoter showed high-level expression and stable proviral transmission. Two vectors containing the ankyrin-1 promoter and 2 erythroid enhancers (HS-40 plus GATA-1 or HS-40 plus 5-aminolevulinate synthase intron 8 [I8] enhancers) and WPRE expressed at levels higher than the HS2/beta-promoter vector in bulk unilineage erythroid cultures and individual erythroid blast-forming units derived from human BM CD34(+) cells. Sca1(+)/lineage(-) Ly5.1 mouse hematopoietic cells, transduced with these 2 ankyrin-1 promoter vectors, were injected into lethally irradiated Ly5.2 recipients. Eleven weeks after transplantation, high-level expression was seen from both vectors in blood (63%-89% of red blood cells) and erythroid cells in BM (70%-86% engraftment), compared with negligible expression in myeloid and lymphoid lineages in blood, BM, spleen, and thymus (0%-4%). The I8/HS-40-containing vector encoding a hybrid human beta/gamma-globin gene led to 43% to 113% human gamma-globin expression/copy of the mouse alpha-globin gene. Thus, modular use of erythroid-specific enhancers/promoters and WPRE in SIN-lentiviral vectors led to identification of high-titer, stably transmitted vectors with high-level erythroid-specific expression for gene therapy of red cell diseases.

Human ITCH is a coregulator of the hematopoietic transcription factor NF-E2.

We have cloned a new protein that interacts with the hematopoietic DNA-binding transcription factor, p45/NF-E2, by screening a human erythroleukemia cell cDNA library with the yeast two-hybrid approach. Predicted peptide sequence and chromosomal mapping identified the cloned molecule to be the product of the human ortholog of the mouse Itch gene, which has been implicated previously in the regulation of growth and differentiation of erythroid and lymphoid cells. Transfection experiments indicate that this human ITCH protein can act as a transcriptional corepressor of p45/NF-E2. Our data provide novel insights into the functional roles of the mammalian ITCH proteins in the development of hematopoietic cell lineages.

Distinction between AP1 and NF-E2 factor-binding at specific chromatin regions in mammalian cells.

Specific nuclear factor-DNA complexes formed within the promoters and enhancers are essential for transcriptional regulation. For eukaryotic systems, however, some DNA motif(s) are capable of binding to a family of related factors, thus making it difficult to identify the factor actually binding on the chromatic DNA in vivo and modulating the local transcription processes. To resolve this matter, we have refined a chromatin immunoprecipitation assay. Using the assay, we could directly link the regulatory functions of two members of the AP1/NF-E2 transcription factor family and their stable binding in vivo within distinct chromatin regions. The study demonstrated the feasibility of a general scheme in the determination of the identity of specific factor(s), among a group of family members, bound at unique sequence(s) in living mammalian cells.

Blockage of apoptotic signaling of transforming growth factor-beta in human hepatoma cells by carboxyfullerene.

Transforming growth factor-beta (TGF-beta) has been shown to induce apoptosis in normal hepatocytes and hepatoma cells both in vivo and in vitro. However, the mechanism by which TGF-beta induces apoptosis is not clear. The antiapoptotic activity of antioxidants including N-acetyl-L-cysteine (Ac-Cys), ascorbic acid and a novel free radical scavenger, carboxyfullerene (C60) on TGF-beta-treated human hepatoma Hep3B cells was examined. Only the water-soluble hexacarboxylic acid derivative of C60 was found to prevent TGF-beta-induced apoptosis. Antiapoptotic activity of C60 correlated its ability to eliminate TGF-beta-generated reactive oxygen species (ROSs). However, C60 did not interfere with TGF-beta-activated PAI-1 promoter activity in the Hep3B cells. These results indicate that the signaling pathway of TGF-beta-induced apoptosis may be related to the generation of ROSs and may be uncoupled from the TGF-beta-activated gene promoter activity. Furthermore, the regioisomer of C60 with a C3 symmetry was more potent in protecting cells from apoptosis than that with a D3 symmetry, and the C3 isomer had stronger interactions with lipid bilayers than the D3 isomer. The spectroscopic analysis revealed that the C3 isomer had stronger interactions with artificial lipid bilayers than the D3 isomer. Therefore, our study indicates that C60 may interact with membrane to eliminate TGF-beta-induced ROSs and to prevent apoptosis occur in human hepatoma cells.

Genomic footprinting and sequencing of human beta-globin locus. Tissue specificity and cell line artifact.

In order to gain further insights of the regulatory mechanisms of human beta-like globin gene switch during erythroid development, we have studied protein-DNA interaction in vivo at the human adult beta and fetal gamma globin promoters and their upstream enhancer, 5'HS-2, in purified human adult erythroblasts, in which the beta, but not gamma or epsilon, globin gene is actively transcribing. This genomic footprinting analysis of adult erythroblasts was carried out in conjunction with those of different non-erythroid human tissues, an embryonic/fetal erythroid cell line K562, and several non-erythroid human cell lines. Protein-DNA binding in the beta globin promoter, in particular at the two CACC promoter boxes and the CCAAT box, is detectable only in the adult erythroblasts. As expected, the gamma globin promoters were bound with specific nuclear factors in the expressing K562 cells, but not in non-erythroid tissues or cell lines. Relatively weak protein binding could also be detected in the vicinities of the two CCAAT boxes of the inactive gamma globin promoters in the adult erythroblasts. Although the patterns of nuclear factor-DNA interaction in vivo at the NF-E2/AP1, GATA-1, and GT-I motifs of 5'HS-2 enhancer in adult erythroblasts are similar to those in K562 cells, we have identified a previously undetected factor-binding motif of 5'HS-2 that is protected only in the adult erythroblasts. This motif is identical in sequence to the 3'-CACC box of the human beta globin promoter, and it is well conserved at the same location among all mammalian 5'HS-2 enhancers, suggesting an important regulatory role of this element in human beta globin gene transcription in adult erythroblasts. All of the above four motifs of 5'HS-2 are free of nuclear factor binding in non-erythroid tissues, but two of them, NF-E2/AP1 and GT-I, are bound with factors in some non-erythroid cell lines but not in others. The functional implications of these genomic footprinting data and the tissue-specific CpG methylation patterns of the beta-like globin promoters we obtained by genomic sequencing are discussed in terms of positive and negative regulation of the human beta-like globin switch during erythroid development.

Plasma nitric oxide levels in newborn infants with sepsis.

Nitric oxide is thought to play an important role in the mediation of the cardiovascular features of septic shock. We determined plasma levels of nitrite and nitrate (not differentiated in measurement) in neonates with sepsis and found these levels to be elevated at the time of entry compared with those of control subjects (p < 0.05); the levels were significantly higher in the patients with sepsis and shock than in those without shock (p < 0.05). Elevations of nitrite plus nitrate were correlated with tumor necrosis factor and severity of illness judged by pediatric risk of mortality (PRISM) scores at onset (p < 0.05). Of 8 newborn infants with a nitrite-plus-nitrate value > 200 mumol/L, 6 had septic shock; none of 12 not reaching that cutoff value had septic shock (p < 0.05). Levels of nitrite plus nitrate were elevated as much in gram-positive as in gram-negative sepsis. We conclude that the determination of circulating plasma levels of nitrite plus nitrate may be useful in forecasting the severity of illness and the occurrence of septic shock; therapeutic approaches associated with inhibition of nitric oxide synthesis may be worth trying in infants with septic shock.

Association between protective efficacy of antibodies to tumor necrosis factor and suppression of nitric oxide production in neonatal rats with fatal infection.

In a rat model of fatal infection caused by Pseudomonas aeruginosa, the circulating level of nitrite/nitrate (NO2-/NO3-), a good indicator for nitric oxide production, was remarkably increased after elevation of circulatory tumor necrosis factor (TNF). Anti-TNF MAb cotreatment was shown to blunt hypoglycemia and hyperlacticemia and was associated with decreased mortality of septic animals. Moreover, anti-TNF MAb significantly reduced not only plasma TNF but also plasma NO2-/NO3- levels. Dexamethasone had a similar effects, and when anti-TNF MAb was used in combination with dexamethasone, the suppression of nitric oxide production and the protective efficacy were more remarkable compared with therapy with either anti-TNF MAb or dexamethasone alone. Our present data suggested that the protective efficacy of anti-TNF MAb may correlate with the suppression of nitric oxide production and also with a modulation in metabolic abnormalities in the septic newborn rats.

Transcriptional activation of human zeta 2 globin promoter by the alpha globin regulatory element (HS-40): functional role of specific nuclear factor-DNA complexes.

We studied the functional interaction between human embryonic zeta 2 globin promoter and the alpha globin regulatory element (HS-40) located 40 kb upstream of the zeta 2 globin gene. It was shown by transient expression assay that HS-40 behaved as an authentic enhancer for high-level zeta 2 globin promoter activity in K562 cells, an erythroid cell line of embryonic and/or fetal origin. Although sequences located between -559 and -88 of the zeta 2 globin gene were dispensable for its expression on enhancerless plasmids, they were required for the HS-40 enhancer-mediated activity of the zeta 2 globin promoter. Site-directed mutagenesis demonstrated that this HS-40 enhancer-zeta 2 globin promoter interaction is mediated by the two GATA-1 factor binding motifs located at -230 and -104, respectively. The functional domains of HS-40 were also mapped. Bal 31 deletion mapping data suggested that one GATA-1 motif, one GT motif, and two NF-E2/AP1 motifs together formed the functional core of HS-40 in the erythroid-specific activation of the zeta 2 globin promoter. Site-directed mutagenesis further demonstrated that the enhancer function of one of the two NF-E2/AP1 motifs of HS-40 is mediated through its binding to NF-E2 but not AP1 transcription factor. Finally, we did genomic footprinting of the HS-40 enhancer region in K562 cells, adult nucleated erythroblasts, and different nonerythroid cells. All sequence motifs within the functional core of HS-40, as mapped by transient expression analysis, appeared to bind a nuclear factor(s) in living K562 cells but not in nonerythroid cells. On the other hand, only one of the apparently nonfunctional sequence motifs was bound with factors in vivo. In comparison to K562, nucleated erythroblasts from adult human bone marrow exhibited a similar but nonidentical pattern of nuclear factor binding in vivo at the HS-40 region. These data suggest that transcriptional activation of human embryonic zeta 2 globin gene and the fetal/adult alpha globin genes is mediated by erythroid cell-specific and developmental stage-specific nuclear factor-DNA complexes which form at the enhancer (HS-40) and the globin promoters.

Erythroid differentiation of mouse erythroleukemia cells results in reorganization of protein-DNA complexes in the mouse beta maj globin promoter but not its distal enhancer.

Dimethyl sulfoxide (DMSO) induction of mouse erythroleukemia (MEL) cells represents a well-defined in vitro system of terminal erythroid differentiation. We have studied the molecular mechanisms of transcriptional activation of the mouse beta maj globin gene during MEL cell differentiation by analyzing nuclear factor-DNA interactions in vivo at the gene's upstream promoter and a distal enhancer, 5'HS-2. Genomic footprinting data indicate that three motifs, CAC, NF-E2/AP1, and GATA-1, of the 5'HS-2 enhancer are bound with nuclear factors in MEL cells both prior to and after DMSO induction. No obvious conformational change of these nuclear factor-DNA complexes could be detected upon terminal differentiation of MEL cells. On the other hand, DMSO induction of MEL cells leads to the formation of specific nuclear factor-DNA complexes at several transcriptional regulatory elements of the mouse beta maj globin upstream promoter. Our genomic footprinting data have interesting implications with respect to the molecular mechanisms of transcriptional regulation and chromatin change of the mouse beta maj globin gene during erythroid differentiation.

CACC box and enhancer response of the human embryonic epsilon globin promoter.

The functional interaction between the human epsilon globin promoter and an erythroid-specific transcription enhancer, 5' HS-2, has been analyzed by transient expression assay. While stepwise deletion of DNA sequences between -852 and -122 had only small effects, removal of the CACC box at position -111 greatly decreased epsilon-globin promoter activity, as well as its response to the enhancer function of 5' HS-2 in erythroid cells. Our data demonstrated that the three ubiquitous promoter elements, the CACC, CCAAT, and TATA boxes, of the epsilon-globin-encoding gene together form a minimal promoter that would interact efficiently with 5' HS-2, and that at least the CACC box is an essential functional component of this enhancer-promoter interaction.