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1.
Int J Oncol ; 30(6): 1369-80, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17487357

ABSTRACT

We previously isolated several clones that were closely-related genetically from a human colorectal tumor (HCT116) cell line. These clones displayed significantly different X-radiation response phenotypes. In this paper, we investigated how a single dose of X-radiation modulated the transcriptomic profiles of either the radiation-resistant (HCT116Clone2_XRR) or the radiation-sensitive (HCT116CloneK_XRS) clone when each was compared to a reference clone, HCT116Clone10_control. The latter represented a control clone that displayed a similar X-radiation response as the parental HCT116 cells. Pooled RNAs were obtained from HCT116Clone2_XRR, HCT116CloneK_XRS or HCT116Clone10_control cells either before or at 10 min, 6 or 24 h after treatment with 4-Gy X-radiation. Transcriptomic profiles were assessed by cDNA microarrays. At least three independent experiments were carried out for each time point and statistical analysis was performed by paired t-test (p<0.05). From 19,200 genes/ESTs examined, we identified only 120 genes/ESTs that were differentially expressed at any one of these four time points. Interestingly, different patterns of gene modulation were observed between the radiation-sensitive and radiation-resistant clones. However, the fold changes of gene modulation were generally small (2-3 fold). Surprisingly, only 12.7% of 79 genes involved in DNA damage sensor/repair and cell cycle and between 2.6 and 9.2% of 76 genes involved in apoptosis, were significantly modulated in these early time points following irradiation. By comparison, up to 10% of 40 known housekeeping genes were differentially expressed. Thus in our experimental model, we were able to detect the up-regulation or down-regulation of mostly novel genes and/or pathways in the acute period (up to 24 h) following a single dose of 4-Gy X-radiation.


Subject(s)
Colorectal Neoplasms/genetics , Gene Expression Profiling , Gene Expression Regulation/radiation effects , Radiation Tolerance/genetics , Blotting, Western , Cell Line, Tumor , Humans , Oligonucleotide Array Sequence Analysis , X-Rays
2.
Radiat Res ; 163(4): 357-63, 2005 Apr.
Article in English | MEDLINE | ID: mdl-15799689

ABSTRACT

This study was designed to examine the effects of treatment with N1, N13-diethylnorspermine (DENSPM), a spermine analog, and X radiation on survival and on the polyamine and spermidine/spermine N1-acetyltransferase (SSAT) levels in closely related human colorectal tumor (HCT116) clones exhibiting a wide range of X-radiation and drug responses. After treatment with DENSPM and X radiation, clonogenic cell survival was measured. SSAT protein levels were measured by Western blot analysis and SSAT enzymatic activities by the conversion of [1-14C]acetyl-CoA into [1-14C]acetylspermidine. Polyamine [i.e. putrescine (PUT), spermine (SPM) and spermidine (SPD)] levels were measured with high-performance liquid chromatography. DENSPM enhanced the efficacy of radiation treatment in HCT116, HCT116-Clone2 (a radiation-resistant clone) and HCT116-Clone10 (a clone with similar X-radiation response as the parental HCT116 cells) but not in HCT116-CloneK (an X-radiation-sensitive but relatively drug-resistant clone). Treatment with DENSPM without X radiation caused the most significant increase in SSAT activity (approximately 22-fold) and an almost complete depletion of SPD levels in HCT116-CloneK. Our results suggest that (a) the lack of sensitization of X-radiation treatment by DENSPM in HCT116-CloneK was likely due to the prior depletion of SPD levels by DENSPM alone, (b) natural polyamine contents and/or inducibility of SSAT may be important factors influencing cellular response to combined X-radiation and DENSPM treatments, and (c) more importantly, there may be a potentially novel role for combining polyamine analogs such as DENSPM with X rays.


Subject(s)
Cell Survival/drug effects , Cell Survival/radiation effects , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/radiotherapy , Spermine/analogs & derivatives , Spermine/administration & dosage , Antineoplastic Agents/administration & dosage , Cell Line, Tumor , Cloning, Molecular/methods , Colorectal Neoplasms/classification , Colorectal Neoplasms/genetics , Combined Modality Therapy , Dose-Response Relationship, Drug , Dose-Response Relationship, Radiation , Drug Resistance, Neoplasm/radiation effects , Humans , Radiation Tolerance/drug effects , Treatment Outcome , X-Ray Therapy/methods
3.
BMC Microbiol ; 4: 13, 2004 Mar 30.
Article in English | MEDLINE | ID: mdl-15050034

ABSTRACT

BACKGROUND: The Bacillus subtilis glucokinase operon was predicted to be comprised of the genes, yqgP (now named gluP), yqgQ, and glcK. We have previously established a role for glcK in glucose metabolism. In the absence of enzymes that phosphorylate glucose, such as GlcK and/or enzyme IIGlc, accumulated cytoplasmic glucose can be transported out of the cell. Genes within the glucokinase operon were not previously known to play a role in glucose transport. Here we describe the expression of gluP and its function in glucose transport. RESULTS: We found that transcription of the glucokinase operon was regulated, putatively, by two promoters: sigmaA and sigmaH. Putative sigmaA and sigmaH-recognition sites were located upstream of and within gluP, respectively. Transcriptional glucokinase operon--lacZ fusions and Northern blotting were used to analyze the expression of gluP. GluP was predicted to be an integral membrane protein. Moreover, the prediction of GluP structure revealed interesting signatures: a rhomboid domain and two tetracopeptide repeat (TPR) motifs. Microscopic analysis showed that GluP minus cells were unable to divide completely, resulting in a filamentous phenotype. The cells were grown in either rich or minimal medium. We found GluP may be involved in glucose transport. [14C]-glucose uptake by the GluP minus strain was slightly less than in the wild type. On the other hand, trehalose-derived glucose in the growth medium of the GluP minus strain was detected in very low amounts. Experimental controls comprised of single or multiple genes mutations within the glucose transporting phosphotransferase system. CONCLUSIONS: gluP seems to be regulated only by a putative sigmaA-dependent promoter. The glucose uptake and export assays suggest that GluP is important for glucose export and may act as an exporter. This also supports the role of the glucokinase operon in glucose utilization.


Subject(s)
Bacillus subtilis/metabolism , Bacterial Proteins/physiology , Glucose/metabolism , Monosaccharide Transport Proteins/physiology , Amino Acid Motifs , Amino Acid Sequence , Bacillus subtilis/cytology , Bacillus subtilis/genetics , Bacillus subtilis/physiology , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Biological Transport , Cell Division , Gene Expression Regulation, Bacterial , Genes, Bacterial , Glucokinase/genetics , Molecular Sequence Data , Monosaccharide Transport Proteins/chemistry , Monosaccharide Transport Proteins/genetics , Operon , Peroxisome-Targeting Signal 1 Receptor , Promoter Regions, Genetic , Protein Structure, Tertiary , Receptors, Cytoplasmic and Nuclear/chemistry , Repetitive Sequences, Amino Acid , Spores, Bacterial/physiology
4.
BMC Microbiol ; 4: 6, 2004 Feb 03.
Article in English | MEDLINE | ID: mdl-15018644

ABSTRACT

BACKGROUND: Bacillus subtilis glucokinase (GlcK) (GenBank NP_390365) is an ATP-dependent kinase that phosphorylates glucose to glucose 6-phosphate. The GlcK protein has very low sequence identity (13.7%) to the Escherichia coli glucokinase (Glk) (GenBank P46880) and some other glucokinases (EC 2.7.1.2), yet glucose is merely its substrate. Our lab has previously isolated and characterized the glcK gene. RESULTS: Microbial glucokinases can be grouped into two different lineages. One of the lineages contains three conserved cysteine (C) residues in a CXCGX(2)GCXE motif. This motif is also present in the B. subtilis GlcK. The GlcK protein occurs in both monomer and homodimer. Each GlcK monomer has six cysteines. All cysteine residues have been mutated, one-by-one, into alanine (A). The in vivo GlcK enzymatic activity was assayed by functional complementation in E. coli UE26 (ptsG ptsM glk). Mutation of the three motif-specific residues led to an inactive enzyme. The other mutated forms retained, or in one case (GlcKC321A) even gained, activity. The fluorescence spectra of the GlcKC321A showed a red shift and enhanced fluorescence intensity compare to the wild type's. CONCLUSIONS: Our results emphasize the necessity of cysteines within the CXCGX(2)GCXE motif for GlcK activity. On the other hand, the C321A mutation led to higher GlcKC321A enzymatic activity with respect to the wild type's, suggesting more adequate glucose phosphorylation.


Subject(s)
Bacillus subtilis/enzymology , Bacterial Proteins/metabolism , Cysteine/metabolism , Glucokinase/metabolism , Amino Acid Sequence , Bacillus subtilis/genetics , Bacterial Proteins/classification , Bacterial Proteins/genetics , Cysteine/genetics , Glucokinase/classification , Glucokinase/genetics , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , Phylogeny , Sequence Homology, Amino Acid
5.
BMC Genomics ; 4(1): 32, 2003 Aug 09.
Article in English | MEDLINE | ID: mdl-12908878

ABSTRACT

BACKGROUND: As part of our investigation into the genetic basis of tumor cell radioresponse, we have isolated several clones with a wide range of responses to X-radiation (XR) from an unirradiated human colorectal tumor cell line, HCT116. Using human cDNA microarrays, we recently identified a novel gene that was down-regulated by two-fold in an XR-resistant cell clone, HCT116Clone2_XRR. We have named this gene as X-ray radiation resistance associated 1 (XRRA1) (GenBank BK000541). Here, we present the first report on the molecular cloning, genomic characterization and over-expression of the XRRA1 gene. RESULTS: We found that XRRA1 was expressed predominantly in testis of both human and macaque. cDNA microarray analysis showed three-fold higher expression of XRRA1 in macaque testis relative to other tissues. We further cloned the macaque XRRA1 cDNA (GenBank AB072776) and a human XRRA1 splice variant from HCT116Clone2_XRR (GenBank AY163836). In silico analysis revealed the full-length human XRRA1, mouse, rat and bovine Xrra1 cDNAs. The XRRA1 gene comprises 11 exons and spans 64 kb on chromosome 11q13.3. Human and macaque cDNAs share 96% homology. Human XRRA1 cDNA is 1987 nt long and encodes a protein of 559 aa. XRRA1 protein is highly conserved in human, macaque, mouse, rat, pig, and bovine. GFP-XRRA1 fusion protein was detected in both the nucleus and cytoplasm of HCT116 clones and COS-7 cells. Interestingly, we found evidence that COS-7 cells which over-expressed XRRA1 lacked Ku86 (Ku80, XRCC5), a non-homologous end joining (NHEJ) DNA repair molecule, in the nucleus. RT-PCR analysis showed differential expression of XRRA1 after XR in HCT116 clones manifesting significantly different XR responses. Further, we found that XRRA1 was expressed in most tumor cell types. Surprisingly, mouse Xrra1 was detected in mouse embryonic stem cells R1. CONCLUSIONS: Both XRRA1 cDNA and protein are highly conserved among mammals, suggesting that XRRA1 may have similar functions. Our results also suggest that the genetic modulation of XRRA1 may affect the XR responses of HCT116 clones and that XRRA1 may have a role in the response of human tumor and normal cells to XR. XRRA1 might be correlated with cancer development and might also be an early expressed gene.


Subject(s)
Colorectal Neoplasms/metabolism , Proteins/genetics , Testis/metabolism , Amino Acid Sequence , Animals , Antigens, Nuclear/metabolism , Base Sequence , COS Cells , Cattle , Cell Line , Cell Line, Tumor , Cell Nucleus/metabolism , Chlorocebus aethiops , Clone Cells , Cloning, Molecular , Colorectal Neoplasms/genetics , Cytoplasm/metabolism , DNA-Binding Proteins/metabolism , Gene Components , Gene Expression , Genomics , Humans , Ku Autoantigen , Macaca fascicularis , Male , Mice , Molecular Sequence Data , Promoter Regions, Genetic , Proteins/metabolism , Rats , Sequence Alignment , Swine , X-Rays
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