MyD genes in negative growth control

Two interrelated cellular processes are invoked simultaneously upon induction of differentiation, the regulated progression of cells through successive stages of cell differentiation and growth inhibition which ultimately leads to growth arrest. In tissues with rapid cell turnover terminally differentiated cells undergo programmed cell death. Terminal differentiation, thus, represents one form of negative growth control. It was surmised that the molecular engine which drives the differentiation process forward requires induction of positive regulators of terminal cell differentiation, to be found among differentiation primary response genes, as well as suppression of negative regulators, which correspond to genes which control cellular growth. This line of thought has prompted the isolation of myeloid differentiation primary response (MyD) genes activated in the absence of de novo protein synthesis, upon IL-6 induced terminal differentiation of murine M1 myeloblastic leukemia cells, where the cells growth arrest and ultimately undergo programmed cell death. As delineated in this review many of the genes identified as MyD genes, including both known genes [IRF-1, (AP-1)Fos/Jun.EGR-1] and novel ones (MyD88, MyD116, MyD118), turned out to play a role in negative growth control, including growth suppression and apoptosis, in many cell types, of both hematopoietic and non hematopoietic origins.     

Genomic organization of IFI16, an interferon-inducible gene whose expression is associated with human myeloid cell differentiation: correlation of predicted protein domains with exon organization

The human IFI16 gene is a member of an interferon-inducible family of mouse and human genes closely linked on syntenic regions of chromosome 1. Expression of these genes is largely restricted to hemopoietic cells, and is associated with the differentiation of cells of the myeloid lineages. As a prelude to defining the mechanisms governing IFI16 expression, we have deduced its genomic organization using a combination of genomic cloning and polymerase chain reaction amplification of genomic DNA. IFI16 consists of ten exons and nine intervening introns spanning at least 28 kilobases (kb) of DNA. The reiterated domain structure of IFI16 protein is closely reflected in its intron/exon boundaries, and may represent the evolutionary fusion of several independent functional domains. Thus, exon 1 consists of 5' untranslated (UT) sequences and contains sequence motifs that may confer interferon-inducibility, and exon 2 encodes the lysine-rich amino-terminal ("K") region, which possesses DNA-binding activity. Exon 3 codes for a domain which is poorly conserved between family members, except for a strongly retained basic motif likely to provide localization. The first of two 200 amino acid repeat domains that are the hallmark of this family (domain A) is represented jointly on exons 4 and 5, which are reiterated as exons 8 and 9, respectively, to encode the second 200 amino acid domain (B). Two intervening serine-threonine-rich domains (C and C'), unique to IFI16, are each encoded by single exons of identical length (exons 5 and 6). These domains are predicted to encode semi-rigid "spacer" domains between the 200 amino acid repeats. The reiterated nature of exons 4 to 6 and the insertion of introns into a single reading frame strongly suggest that IFI16 and related genes arose by a series of exon duplications, some of which antedated speciation into mouse and humans. Several alternative mRNA cap sites downstream of a TATA consensus sequence were defined, using primer extension analysis of mRNA. Sequencing of approximately 1.7 kb of DNA upstream of this region revealed no recognizable consensus elements for induction by interferon-alpha (interferon-alpha/beta-stimulated response elements), but two motifs resembling interferon-gamma activation sites were located. IFNs alpha and gamma both induce IFI16 mRNA expression in myeloid cells. Interferon-alpha inducibility of IFI16 may be regulated by an interferon-alpha/beta-stimulated response consensus element in the 5' UT exon, as a similar motif is conserved in the corresponding position in the related myeloid cell nuclear differentiation antigen gene.(ABSTRACT TRUNCATED AT 400 WORDS)     

Analysis of cell death in myeloid cells inducibly expressing the cell cycle protein p55Cdc

p55Cdc, a cell cycle protein is expressed in cycling mammalian cells and is required for normal cell division. Expression of this protein is regulated during the cell cycle, peaking in late G1 and S. We have previously shown that constitutive expression of p55Cdc results in inhibition of granulocyte differentiation. Degradation of p55Cdc is also required for apoptosis in growth factor and serum starved cells. In the present study we prepared stably transfected cells conditionally expressing p55Cdc in response to zinc stimulation to investigate the role of inducible p55Cdc expression in apoptosis of myeloid cells. We report that inducible expression of p55Cdc in the myeloid leukemic cell line 32Dc13 resulted in increased cell death. p55Cdc overexpression led to a statistically significant decrease in the viability of 32Dc13 cells compared with that of control cells. Furthermore, cell staining and flow cytometry analysis revealed that p55Cdc-overexpressing 32Dc13 cells progressed to apoptosis much earlier than uninduced cells. These results suggest that inducible expression of p55Cdc leads to earlier increases in cell death in the absence of growth factor and serum in myeloid leukemic cells.     

Chromosome band 1p36 contains a putative tumor suppressor gene important in the evolution of chronic myelocytic leukemia

Chronic myelocytic leukemia (CML) is a common neoplasm of hematopoietic pluripotent stem cells. Although the evolution from chronic phase to blast crisis (BC) in CML patients is an inevitable clinical feature, little is understood about the mechanisms responsible for the transformation. We have previously performed allelotype analysis in CML BC and have detected frequent loss of heterozygosity (LOH) on the short arm of chromosome 1. To know the common region of LOH where a putative tumor suppressor gene may reside, deletional mapping was performed using 33 microsatellite markers spanning chromosome 1 in 30 patients with CML BC (21 myeloid and 9 lymphoid). DNA was extracted from slides of bone marrow smears or from bone marrow mononuclear cells. In each patient, DNA from chronic phase was analyzed alongside DNA from either their BC or accelerated phase. Allelic loss on 1p was observed in 14 of the 30 individuals (47%): 10 of the 21 myeloid and 4 of the 9 lymphoid BC cases. Serial cytogenetic information was available in 10 cases with LOH on 1p; interestingly, deletions in this region were not detected. Two samples showed LOH at all informative loci on 1p, whereas the other 12 samples showed LOH on at least one but not all loci on 1p. The common region of LOH resided proximal to D1S508 and distal to D1S507 (1p36). Our results suggest that a tumor suppressor gene that frequently plays an important role in the evolution to BC resides on 1p36 in CML.     

Pigmented ("black") extraadrenal paraganglioma

Murine Gas2 is a microfilament-associated protein whose expression is increased at growth arrest in mammalian cells. During apoptosis, Gas2 is specifically cleaved at its C-terminus by a still unknown ICE-like protease, and the processed protein induces dramatic rearrangements in the cytoskeleton when overexpressed in several cell types. Here we report the characterization of a cDNA encoding the human homologue of Gas2, showing high conservation with the murine counterpart at the protein level. Fluorescence in situ hybridization analysis and radiation hybrid mapping localized the GAS2 gene on human chromosome 11p14.3-p15.2, in a region homologous to the gas2 region on mouse chromosome 7.     

Cloning, expression and mapping of a novel RING-finger gene (RNF5), a human homologue of a putative zinc-finger gene from Caenorhabditis elegans

The RING-finger is a unique zinc-chelating domain involved in mediating protein-protein interactions. The extensive sequence homology within the RING-finger domain allowed us to clone a novel member of the RING-finger family of genes. This cDNA clone, designated RNF5 (Ring-finger protein 5), contained an open reading frame of 540 nucleotides. Its predicted amino acid sequence revealed significant homology to a hypothetical protein encoded by Caenorhabditis elegans cosmid C16C10.7. The expression of RNF5 was detected in a variety of human tissues. The RNF5 gene was mapped by fluorescence in situ hybridization to chromosome 6p21.31. Radiation hybrid mapping further assigned RNF5 to a region proximal to the major histocompatibility complex (MHC) on chromosome 6. RNF5 is the third RING-finger gene identified in the region proximal to MHC raising the possibility that the RING-finger family of genes may exist as a cluster in this region.     

Comparative receptor binding analyses of cannabinoid agonists and antagonists

Chromosomal segregation during mitosis as well as meiosis is considered to be regulated by multiple kinases, but the precise mechanism remains largely unknown. A mutation in Drosophila, designated aurora, was identified as a responsible gene for a chromosomal segregation defect and encodes a putative serine-threonine kinase. Here we have identified mammalian aurora homologues, designated aurora-related kinase (ARK) 1 and ARK2. Kinase domains of murine ARK1 and ARK2 showed 61 and 62% identity, respectively, to that of aurora at the amino acid levels, respectively. Cell cycle analysis revealed that the expression of ARK1 was correlated with G2/M phase, while ARK2 was expressed during S and G2/M phases. Immunofluorescence analysis demonstrated that ARK2 was mainly localized to the midbody, while ARK1 has been reported to be localized to the spindle pole during mitosis. Collectively, these results suggest that these two kinases may have distinct roles with different expression timing and subcellular localization during the cell cycle progression. Interspecific backcross mapping revealed that Ark1 is located in a distal region of mouse chromosome 2, while Ark2 is located in a central region of mouse chromosome 11.