Multiple myeloma uncovered by investigating a negative serum iron level.
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Aged↗
Biomedical subjects
L C Wu
Publications and source records attributed to L C Wu.
Treatment train for site remediation at a petrochemicals-contaminated site within a petroleum refinery.
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Extraction and Processing Industry↗
Inactivation of human SRBC, located within the 11p15.5-p15.4 tumor suppressor region, in breast and lung cancers.
A cDNA clone encoding human SRBC [serum deprivation response factor (sdr)-related gene product that binds to c-kinase] was isolated in a yeast two-hybrid screening, with amino acids 1-304 of BRCA1 as the probe. The human SRBC gene (hSRBC) was mapped to chromosome region 11p15.5-p15.4, close to marker D11S1323, at which frequent loss of heterozygosity (LOH) has been observed in sporadic breast, lung, ovarian, and other types of adult cancers as well as childhood tumors. hSRBC-coding region mutations including frame shift and truncation mutations were detected in a few ovarian and lung cancer cell lines. More significantly, the expression of hSRBC protein was down-regulated in a large fraction [30 (70%) of 43] of breast, lung, and ovarian cancer cell lines, whereas strong expression of hSRBC protein was detected in normal mammary and lung epithelial cells. The down-regulation of hSRBC expression in cancer cells was associated with hypermethylation of CpG dinucleotides in its promoter region, and 3 (60%) of 5 primary breast tumors and 11 (79%) of 14 primary lung tumors were also found to be hypermethylated. Treatment of breast cancer MCF7 cells with 5'azacytidine and Trichostatin A resulted in expression of hSRBC, confirming DNA methylation as the mode of inactivation. Our results suggest that epigenetic or mutational inactivation of hSRBC may contribute to the pathogenesis of several types of human cancers, marking hSRBC as a candidate tumor suppressor gene.
Amino Acid Sequence↗
Characterization of Sp1, AP-1, CBF and KRC binding sites and minisatellite DNA as functional elements of the metastasis-associated mts1/S100A4 gene intronic enhancer.
The mts1/S100A4 gene encodes a small acidic calcium-binding protein that is expressed in a cell-specific manner in development, tumorigenesis and certain tissues of adult mice. A composite enhancer that is active in murine mammary adenocarcinoma cells was previously identified in the first intron of the mts1/S100A4 gene. Here we present a detailed analysis of the structure and function of this enhancer in the Mts1/S100A4-expressing CSML100 and non-expressing CSML0 mouse adenocarcinoma cell lines. In CSML100 cells the enhancer activity is composed of at least six cis-elements interacting with Sp1 and AP-1 family members and CBF/AML/PEBP2 and KRC transcription factors. In addition, a minisatellite-like DNA sequence significantly contributes to the enhancer activity via interaction with abundant proteins, which likely have been described previously under the name minisatellite-binding proteins. Extensive mutational analysis of the mts1/S100A4 enhancer revealed a cooperative function of KRC and the factors binding minisatellite DNA. This is the first example of an enhancer where two nuclear factors earlier implicated in different recombination processes cooperate to activate transcription. In Mts1/S100A4-negative CSML0 cells the strength of the enhancer was 7- to 12.5-fold lower compared to that in CSML100 cells, when referred to the activities of three viral promoters. In CSML0 cells the enhancer could be activated by exogenous AP-1 and CBF transcription factors.
Allosteric Site↗
Regulation of clock and NPAS2 DNA binding by the redox state of NAD cofactors.
Clock:BMAL1 and NPAS2:BMAL1 are heterodimeric transcription factors that control gene expression as a function of the light-dark cycle. Although built to fluctuate at or near a 24-hour cycle, the clock can be entrained by light, activity, or food. Here we show that the DNA-binding activity of the Clock:BMAL1 and NPAS2:BMAL1 heterodimers is regulated by the redox state of nicotinamide adenine dinucleotide (NAD) cofactors in a purified system. The reduced forms of the redox cofactors, NAD(H) and NADP(H), strongly enhance DNA binding of the Clock:BMAL1 and NPAS2:BMAL1 heterodimers, whereas the oxidized forms inhibit. These observations raise the possibility that food, neuronal activity, or both may entrain the circadian clock by direct modulation of cellular redox state.
ARNTL Transcription Factors↗
Structure of the human zinc finger protein HIVEP3: molecular cloning, expression, exon-intron structure, and comparison with paralogous genes HIVEP1 and HIVEP2.
Here we report the cloning and characterization of HIVEP3, the newest member in the human immunodeficiency virus type 1 enhancer-binding protein family that encodes large zinc finger proteins and regulates transcription via the kappaB enhancer motif. The largest open reading frame of HIVEP3 contains 2406 aa. and is approximately 80% identical to the mouse counterpart. The HIVEP3 gene is located in the chromosomal region 1p34 and is at least 300 kb with 10 exons. RNA studies show that multiple HIVEP3 transcripts are differentially expressed and regulated. Additionally, transcription termination occurs in the ultimate exon, exon 10, or in exon 6. Therefore, HIVEP3 may produce protein isoforms that contain or exclude the carboxyl DNA binding domain and the leucine zipper by alternative RNA splicing and differential polyadenylation. Sequence homologous to HIVEP3 exon 6 is not found in mouse nor are the paralogous genes HIVEP1 and HIVEP2. Zoo-blot analysis suggests that sequences homologous to the human exon 6 are present only in primates and cow. Therefore, a foreign DNA harboring a termination exon likely was inserted into the HIVEP3 locus relatively recently in evolution, resulting in the acquisition of novel gene regulatory mechanisms as well as the generation of structural and functional diversity.
Amino Acid Sequence↗
The DNA-binding ability of HIVEP3/KRC decreases upon activation of V(D)J recombination.
Somatic V(D)J recombination of the immune receptor genes is mediated by the recombination signal sequence (RSS) and the recombination-activating genes RAG1 and RAG2. Previously, proteins binding specifically to the RSS have been characterized in nuclear extracts of T and B lymphocytes. Further elucidation of the role of those RSS-binding proteins in V(D)J recombination, however, has been hampered by the fact that their identities have not been established. Here, we show that the major RSS-binding protein present in the nuclear extracts of B lymphocytes is an Mr 135,000 species. Notably, its affinity for the RSS decreased when RAG1 and RAG2 were induced. In immunoblot analyses and gel supershift assays, we showed that KRC antisera react with the Mr 135,000 RSS-binding protein. We previously cloned KRC from a thymocyte expression library using 32P-RSS as a ligand and showed that KRC fusion proteins bind specifically to the RSS and to the kappaB enhancer motif. The lymphoid expression and DNA-binding characteristics suggest that KRC may be involved in lymphocyte development.
Antibody Specificity↗
Optimized protocols in terms of equalizing the true and accidental coincidences of each bed position in static multiple bed position carbon-11 PET studies and a target single bed position study.
Quantitative carbon-11 (11C) positron emission tomography (PET) is gaining more attention in oncological applications. In addition, it was discovered that accidental coincidences (randoms) can play an important role in studies such as partial volume effects correction without needing to use anatomical information. However, because of the short half-life of 11C, a homogeneous multiple bed position 11C image is hard to obtain. This study aimed to determine whether innovative pseudogating, optimized pseudogating and optimized double-up methods could match the true counts and randoms to each bed position and to a target single bed position study. A long cylindrical phantom filled with [11C]acetate was scanned by a dedicated PET scanner using six protocols: whole body, frame-duration-compensated whole body, double-up, optimized double-up, pseudogating, and optimized pseudogating. The differences between the protocols are in the initial activity, frame duration and frame number for each bed position. The parameters for pseudogating were determined according to the theory that true counts and randoms are proportional to the radioactivity and its square, respectively. The results show that the stated goals could only be achieved by pseudogating and optimized pseudogating. We conclude that pseudogating or optimized pseudogating must be applied for a critical quantitative study. However, optimized double-up and double-up methods are good alternatives for semi-quantitative and qualitative studies, respectively.
Algorithms↗
[Molecular cloning and expression of the cDNA encoding angiogenesis inhibitor K4K5 with Pichia pastoris].
Kringles of human plasminogen except kringle 4 can inhibit the endothelial cell growth. To determine whether recombinant plasminogen kringle 4-5 (r-K4K5) can inhibit the growth of bovine capillary endothelial (BCE) cell and the angiogenesis of chick embryo chorioallantoic membrane (CAM), we cloned and constructed the modified cDNA region encoding kringles 4 and 5 (K4K5) of human plasmin (ogen) and expressed it with pichia multi-copy expression system. One clone, with the most productive expression was selected from hundreds of transformants. Our data showed that the expression product r-K4K5 (MW 21.5 kD) amounted to 150-250 mg/liter, over 80% of the total secreted protein. It could, dose-dependently, inhibit BCE cell proliferation and inhibit chick embryo CAM angiogenesis.
Angiogenesis Inhibitors↗
Downregulation of KRC induces proliferation, anchorage independence, and mitotic cell death in HeLa cells.
The large zinc finger protein KRC regulates transcription of target genes via the kappaB gene enhancer element. As an attempt to investigate the cellular function of KRC, we have established cell lines stably transfected with KRC expression vectors. Introduction of a vector directing expression of a transcript antisense to KRC mRNAs in several mammalian cell lines resulted in accelerated proliferation. Furthermore, in HeLa cells, downregulation of KRC conferred anchorage-independent growth and promoted cell cycle progression without an intervening cytokinesis, culminating in the formation of multinucleated giant cells. Ultimately these cells died.
Animals↗
The kappaB and V(D)J recombination signal sequence binding protein KRC regulates transcription of the mouse metastasis-associated gene S100A4/mts1.
A kappaB-like sequence, Sb, is integral to the composite enhancer located in the first intron of the metastasis-associated gene, S100A4/mts1. Oligonucleotides containing this sequence form three specific complexes with nuclear proteins prepared from S100A4/mts1-expressing CSML100 adenocarcinoma cells. Protein studies show the Sb-interacting complexes include NF-kappaB/Rel proteins, p50.p50 and p50.p65 dimers. Additionally, the Sb sequence was bound by an unrelated approximately 200-kDa protein, p200. Site-directed mutagenesis in conjunction with transient transfections indicate that p200, but not the NF-kappaB/Rel proteins, transactivates S100A4/mts1. To identify candidate genes for p200, double-stranded DNA probes containing multiple copies of Sb were used to screen a randomly primed lambdagt11 cDNA expression library made from CSML100 poly(A)(+) RNA. Two clones corresponding to the DNA-binding proteins KRC and Alf1 were identified. KRC encodes a large zinc finger protein that binds to the kappaB motif and to the signal sequences of V(D)J recombination. In vitro DNA binding assays using bacterially expressed KRC fusion proteins, demonstrate specific binding of KRC to the Sb sequence. In addition, introduction of KRC expression vectors into mammalian cells induces expression of S100A4/mts1 and reporter genes driven by S100A4/mts1 gene regulatory sequences. These data indicate that KRC positively regulates transcription of S100A4/mts1.
3T3 Cells↗
Autonomous protein folding units.
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Animals↗
In vitro and in vivo suppression of growth of rat liver epithelial tumor cells by antisense oligonucleotide against protein kinase C-alpha.
BACKGROUND/AIMS: It has been hypothesized that liver stem cells may be activated and proliferate upon liver injury and may participate in the development of liver cancer. GP7TB, a rat liver epithelial tumor cell line, possesses characteristics of liver stem-like cells and can develop into a tumor in syngeneic Fischer 344 rat. We found that protein kinase C-alpha (PKC-alpha) is overexpressed in GP7TB cells. The importance of PKC-alpha for this liver tumor cell was elucidated. METHODS: Antisense oligonucleotide (ODN) was applied to suppress the production of PKC-alpha in GP7TB cells in vitro and in vivo. Cell viability was measured by acid phosphatase assay. The cellular levels of PKC-alpha and Bcl-2 were monitored by Western-blot analysis. Activation of nuclear factor NF-kappaB was analyzed by electrophoretic mobility shift assay. Cell cycle phase distribution was monitored by FACScan. Cell apoptosis was detected by TUNEL assay and histochemical staining of tumor tissue sections. The in vivo experiment was conducted by implanting tumor mass of GP7TB in the liver of F-344 rat and continuous delivery of the ODN by a mini-osmotic pump. RESULTS: Antisense ODN effectively suppressed the level of PKC-alpha that resulted in the decrease of Bcl-2 and nuclear NF-kappaB. The cumulative viable cells also decreased dramatically for the antisense-treated group. FACScan showed that the cells were arrested at early S-phase. These cells in turn went into apoptosis without completing a cell cycle. It was found that growth of the tumor was suppressed efficiently by antisense ODN. Cell apoptosis was found in the orthotopic tumor. The normal liver cells were not affected. CONCLUSIONS: A lethal effect of depressing the level of PKC-alpha in GP7TB cells and success in suppressing orthotopic tumor growth in vivo suggests that PKC-alpha antisense ODN would be a promising therapeutic agent for some liver cancers.
Acid Phosphatase↗
Regulation by phosphorylation of the zinc finger protein KRC that binds the kappaB motif and V(D)J recombination signal sequences.
The DNA binding protein KRC (forkappaB binding andrecognitioncomponent of the V(D)J recombination signal sequence) belongs to a family of large zinc finger proteins that bind to the kappaB motif and contains two widely separated DNA binding structures. In addition to the kappaB motif, KRC fusion proteins bind to the signal sequences of V(D)J recombination to form highly ordered complexes. Here, we report that KRC may be regulated by post-translational modifications. Specific protein kinases present in the nucleus of pre-B cells phosphorylated a KRC fusion protein at tyrosine and serine residues. Such protein modifications increased DNA binding, thereby providing a mechanism by which KRC responds to signal transduction pathways. KRC is a substrate of epidermal growth factor receptor kinase and P34cdc2 kinase in vitro. Our results suggest that activation of the KRC family of transcription factors may provide a mechanism by which oncogenic tyrosine kinases regulate genes with kappaB-controlled gene regulatory elements.
Amino Acid Sequence↗
11C-acetate clearance in nasopharyngeal carcinoma.
This study assessed 11C-acetate turnover (clearance) in nasopharyngeal carcinoma (NPC). Data were acquired by dynamic PET after the intravenous injection of 4.625 MBq.kg-1 body weight of 11C-acetate for 30 min. Tomograms were reconstructed and evaluated visually. A time-activity curve of the nasopharynx and neck was generated and the clearance rate of 11C-acetate from the nasopharynx in the slow phase and from NPC was calculated using 0.693/T1/2. Ten patients with nasopharyngeal carcinoma and nine normal subjects were studied. The clearance of 11C-acetate from the normal nasopharynx was rapid and biexponential, in contrast to the rapid uptake followed by extremely slow clearance in patients with NPC. The clearance rate (mean +/- S.D.) was 0.0074 +/- 0.0042 in NPC and 0.0263 +/- 0.0152 in controls in the slow phase, being significantly different between the two groups with no overlap. All nasopharyngeal carcinomas were clearly visualized, in contrast to no obvious retention in the normal nasopharynx. Our initial results indicate that 11C-acetate clearance can be used to differentiate nasopharyngeal carcinoma from a normal nasopharynx. This finding may lead to new applications of 11C-acetate in oncology.
Acetates↗
Four ubiquitously expressed genes, RD (D6S45)-SKI2W (SKIV2L)-DOM3Z-RP1 (D6S60E), are present between complement component genes factor B and C4 in the class III region of the HLA.
The association of the HLA class III region with many diseases motivates the investigation of unidentified genes in the 30-kb segment between complement component genes Bf and C4. RD, which codes for a putative RNA binding protein, is 205 bp downstream of Bf. SKI2W (HGMW-approved symbol SKIV2L), a DEVH-box gene probably involved in RNA turnover, is 171 bp downstream of RD (HGMW-approved symbol D6S45). RP1 (HGMW-approved symbol D6S60E) is located 611 bp upstream of C4. The DNA sequence between human RD and RP1 was determined and the exon-intron structure of SKI2W elucidated. SKI2W consists of 28 exons. The putative RNA helicase domain of Ski2w is encoded by 9 exons. Further analysis of the 2.5-kb intergenic sequence between SKI2W and RP1 led to the discovery of DOM3Z. The full-length cDNA sequence of DOM3Z encodes 396 amino acids with a leucine zipper motif. Dom3z-related proteins are present in simple and complex eukaryotes. In Caenorhabditis elegans, Dom3z-related protein could be involved in the development of germ cells. Human RD-SKI2W and DOM3Z-RP1 are arranged as two head-to-head oriented gene pairs with unmethylated CpG sequences at the common 5' regulatory region of each gene pair. The ubiquitous expression pattern suggests that these four genes are probably housekeeping genes.
Amino Acid Sequence↗
The C-terminal (BRCT) domains of BRCA1 interact in vivo with CtIP, a protein implicated in the CtBP pathway of transcriptional repression.
The BRCA1 tumor suppressor encodes a polypeptide with two recognizable protein motifs: a RING domain near the N terminus and two tandem BRCT domains at the C terminus. Studies of tumor-associated mutations indicate that the RING and BRCT sequences are required for BRCA1-mediated tumor suppression. In addition, recent work has shown that BRCA1 is a potent regulator of RNA transcription and that the BRCT domains are also essential for this activity. Therefore, we used the Sos recruitment system to screen for proteins that bind this critical region of BRCA1. Our results show that the BRCT domains interact in vivo with CtIP, a protein originally identified on the basis of its association with the CtBP transcriptional co-repressor. This finding suggests that BRCA1 regulates gene expression, at least in part, by modulating CtBP-mediated transcriptional repression. Moreover, the in vivo interaction between BRCA1 and CtIP is completely ablated by each of three independent tumor-associated mutations affecting the BRCT motifs of BRCA1. These results indicate that the BRCA1-CtIP interaction may be required for tumor suppression by BRCA1.
Alcohol Oxidoreductases↗
The human DEVH-box protein Ski2w from the HLA is localized in nucleoli and ribosomes.
The human helicase gene SKI2W is located between RD and RP1 in the class III region of the major histocompatibility complex. Transcripts of SKI2W are detectable in RNA samples isolated from multiple tissues. The protein product Ski2w shares striking amino acid sequence similarities to the yeast antiviral protein Ski2p that controls the translation of mRNAs, probably based on the mRNA structural integrity. Whether this translational regulation mechanism for cellular and viral RNAs exists in mammals is under investigation. Antisera against human Ski2w were generated using fusion proteins produced in bacteria or insect cells. Western blot analysis showed that the endogenous Ski2w protein is approximately 140 kDa in size and is enriched in polysomal fractions of cytoplasmic extracts from HeLa cells. Ribosomal profile studies revealed that Ski2w distributed throughout the entire sucrose gradient in the presence of Mg2+, but co-sedimented with the 18S rRNA-containing 40S subunit and the small ribosomal subunit protein S27a in the presence of EDTA. The co-sedimentation of Ski2w with the 40S subunit is not affected by RNase A treatment of the cell extract, or the addition of KCl to 0.5 M, suggesting that Ski2w is associated with the 40S ribosomal subunit. Indirect immunofluorescence experiments showed that human Ski2w is localized in the nucleoli and in the cytoplasm. In essence, human Ski2w is present at the sites of ribosome biogenesis and protein synthesis.
Amino Acid Sequence↗