ABSTRACT
BACKGROUND: CD26 is a multifunctional membrane-bound glycoprotein that regulates tumour growth in addition to its other activities. Because disease aggressiveness is correlated with CD26 expression in several T-cell malignancies, we decided to investigate the invasiveness of cells expressing different levels of CD26. METHODS: To assess CD26 involvement in cell invasion, we performed in vitro invasion assays with human T cell lines expressing different levels of CD26. These included the parental CD26-positive T-lymphoblast cell line HSB-2 and clones infected with a retrovirus expressing siRNA vectors that either targeted CD26 or encoded a missense siRNA, and the parental CD26-negative T-leukaemia cell line Jurkat and clones expressing CD26. CD26 expression in these cell lines was evaluated by flow cytometry and western immunoblotting. CXCR4 expression, phosphorylation of signalling kinases, and MMP-9 secretion were also evaluated by western immunoblotting, whereas MMP-9 activity and the effect of kinase and CD45 inhibitors on activity were measured by zymography of conditioned media. RESULTS: The presence of CD26 enhanced stromal-cell-derived factor-1-alpha (SDF-1-alpha)-mediated invasion of T cell lines. This process was regulated in part by the PI-3K and MEK1 pathways, as indicated by increased phosphorylation of p44/42 MAP kinase and Akt in the presence of SDF-1-alpha and the effect of their respective inhibitors on MMP-9 secretion and in vitro invasion. In addition, CD26-associated enhancement of SDF-1-alpha-induced invasion was decreased when CD45 was inhibited. CONCLUSIONS: Our results indicate that the expression of CD26 in T cell lines leads to increased SDF-1-alpha-mediated invasion in an in vitro system and that this is controlled in part by the PI-3K and MEK1 pathways. The data also suggest that CD26 enhancement of invasion may be mediated by CD45, however, more studies are required to confirm this involvement.
Subject(s)
Chemokine CXCL12/physiology , Dipeptidyl Peptidase 4/physiology , Chromones/pharmacology , Dipeptidyl Peptidase 4/analysis , Extracellular Signal-Regulated MAP Kinases/metabolism , Humans , Jurkat Cells , Leukocyte Common Antigens/antagonists & inhibitors , Matrix Metalloproteinase 9/biosynthesis , Morpholines/pharmacology , Neoplasm Invasiveness , Phosphorylation , Protein Kinase Inhibitors/pharmacology , Receptors, CXCR4/analysis , TransfectionABSTRACT
Human Rec2/Rad51L1 is a member of the Rad51 family of proteins. Although recombinase activity, typical of this family, could not be established, its overexpression in mammalian cells has been shown to cause a delay in G1. Moreover, since hsRec2/Rad51L1 has been found to be induced by both ionizing and UV irradiation, it is likely that hsRec2/Rad51L1 is elevated following any DNA damage and causes a G1 delay to allow time for DNA repair to occur. Limited homology with catalytic domains X and XI of protein kinase A suggested that kemptide, an artificial substrate containing one phosphorylatable residue, a serine, might serve as a substrate for hsRec2/Rad51L1. Here, we report that hsRec2/Rad51L1 can phosphorylate kemptide, as well as myelin basic protein, p53, cyclin E, and cdk2, but not a peptide substrate containing tyrosine only. The finding that hsRec2/Rad51L1 exhibits protein kinase activity is a first step toward identifying a mechanism whereby this protein affects the cell cycle.
Subject(s)
CDC2-CDC28 Kinases , DNA-Binding Proteins/metabolism , Protein Kinases/metabolism , Proteins/metabolism , Cell Cycle , Cyclin E/metabolism , Cyclin-Dependent Kinase 2 , Cyclin-Dependent Kinases/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/isolation & purification , Gene Expression , Humans , Phosphorylation , Protein Kinases/genetics , Protein Kinases/isolation & purification , Protein Serine-Threonine Kinases/metabolism , Proteins/genetics , Proteins/isolation & purification , Rad51 Recombinase , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Tumor Suppressor Protein p53/metabolismABSTRACT
Specific recognition of a region of duplex DNA by triplex-forming oligonucleotides (TFOs) provides an attractive strategy for genetic manipulation. Based on this, we have investigated the ability of the triplex-directed approach to induce mutations at a chromosomal locus in living cells. A mouse fibroblast cell line was constructed containing multiple chromosomal copies of the lambdasupFG1 vector carrying the supFG1 mutation-reporter gene. Cells were treated with specific (psoAG30) or control (psoSCR30) psoralen-conjugated TFOs in the presence and absence of UVA irradiation. The results demonstrated a 6- to 10-fold induction of supFG1 mutations in the psoAG30-treated cells as compared with psoSCR30-treated or untreated control cells. Interestingly, UVA irradiation had no effect onthe mutation frequencies induced by the psoralen-conjugated TFOs, suggesting a triplex-mediated but photoproduct-independent process of mutagenesis. Sequencing data were consistent with this finding since the expected T.A-->A.T transversions at the predicted psoralen crosslinking site were not detected. However, insertions and deletions were detected within the triplex binding site, indicating a TFO-specific induction of mutagenesis. This result demonstrates the ability of triplex-forming oligonucleotides to influence mutation frequencies at a specific site in a mammalian chromosome.
Subject(s)
DNA , Mutagenesis , Oligodeoxyribonucleotides , Animals , Base Sequence , Cell Line , Chromosomes , Furocoumarins , Genetic Vectors , Mammals , Mice , Molecular Sequence Data , Nucleic Acid Conformation , Research DesignABSTRACT
HsRec2/Rad51B is a 350-amino acid protein with a molecular mass of 38,300 Da that appears to be involved in cell cycle regulation and UV-induced apoptosis. The mouse and human genes were isolated based on their homology to a recombinational repair gene from Ustilago maydis and contain functional domains to hRAD51 and hLIM 15 (M. C. Rice et al., Proc. Natl. Acad. Sci. USA, 94: 7417-7422, 1997). Here, we report the results of studies on the behavior of CHO cells containing a plasmid encoding a wild-type hsRec2/Rad51B, a full-length protein with a single mutation at residue 163, which lies in the putative src site, and a truncated version of hsRec2/Rad51B, containing only the first 100 amino acids at the NH2 terminus. Using fluorescence-activated cell sorting analysis to follow the progression of cells through the cell cycle, we find that stable transfectants constitutively overexpressing the wild-type human Rec2/Rad51B protein exhibit a G1 delay. In addition, when irradiated with UV at a dose of 15 J/m2, CHO cells transfected with the various hREC2/RAD51B vectors exhibited different responses. Cells expressing the wild-type human Rec2/Rad51B underwent apoptosis, with the greatest cell death occurring 24 h after irradiation. The control cells, which contained an empty vector, and the cells expressing truncated hsRec2/Rad51B or the full-length Rec2 with a mutation at residue 163 did not. In summary, these findings of cell cycle slowing and UV-induced apoptosis in CHO cells constitutively expressing the human Rec2/Rad51B protein suggest that hsRec2/Rad51B plays a role in a DNA damage surveillance pathway.
Subject(s)
DNA Damage , DNA-Binding Proteins/genetics , DNA/radiation effects , G1 Phase , Proteins/genetics , Animals , Apoptosis , CHO Cells , Cricetinae , Humans , Mice , Rad51 Recombinase , Structure-Activity Relationship , Transfection , Ultraviolet RaysABSTRACT
Chimeric oligonucleotides consisting of one DNA strand paired with an O-methylated RNA strand interrupted by six DNA residues have been used in gene targeting experiments. Here we demonstrate that these hairpins can form a heteroduplex (or joint molecule) with single-stranded DNA targets in a reaction mediated by the E. coli RecA protein. One end of the double hairpin may unwind to form a 14-base-RecA filament which initiates the reaction. Chimeric oligonucleotides containing only O-methylated RNA residues on one strand or truncated hairpins lacking this 14-base segment did not participate in RecA-driven heteroduplex formation under these reaction conditions. The results presented here represent a first step in studying one facet of a strategy which uses O-methylated RNA residues as participants in homologous pairing events.
Subject(s)
DNA, Single-Stranded/chemistry , RNA/chemistry , Rec A Recombinases/chemistry , Base Sequence , Methylation , Molecular Sequence Data , Nucleic Acid Conformation , Oligonucleotides/chemical synthesis , Oligonucleotides/chemistry , Protein BindingABSTRACT
The insulin-like growth factor-I receptor (IGF-IR) plays a critical role in cell growth regulation and transformation. The radiosensitivity of NIH 3T3 fibroblasts overexpressing either wild-type or mutant IGF-IR was examined. High levels of wild-type IGF-IR conferred radioresistance, and mutational analysis revealed that this effect correlated with the transforming capacity but not the mitogenic activity of the receptor. The radioresistant phenotype was reversed when the cells were incubated with antisense oligonucleotides targeted to IGF-IR mRNA, demonstrating that IGF-IR directly influences radioresistance. The clinical significance of these findings was examined in an immunohistochemical analysis of primary breast tumors, revealing that high levels of IGF-IR in tumor samples were highly correlated with ipsilateral breast tumor recurrence (IBTR) following lumpectomy and radiation therapy (P = 0.001). Subgroup analysis revealed that, for early breast tumor relapses (within 4 years of initial breast tumor diagnosis), elevated levels of IGF-IR were strongly associated with IBTR (P = 0.004) but IGF-IR expression was not prognostic for IBTR from breast cancer patients with late relapses (P was not significant). These studies provide evidence for the influence of IGF-IR on cellular radioresistance and response to therapy and raise the possibility that the radiocurability of selected tumors may be improved by pharmaceutical strategies directed toward the IGF-IR.
Subject(s)
Breast Neoplasms/metabolism , Receptor, IGF Type 1/metabolism , 3T3 Cells , Animals , Breast Neoplasms/radiotherapy , Breast Neoplasms/therapy , Humans , Immunohistochemistry , Mastectomy, Segmental , Mice , Neoplasm Recurrence, Local/metabolism , Oligonucleotides, Antisense/pharmacology , Radiation Tolerance/drug effects , Radiotherapy, Adjuvant , Receptor, IGF Type 1/genetics , TransfectionABSTRACT
Light-activated psoralens can covalently modify DNA and are widely used to study nucleic acid secondary structure and mutagenesis. Sequence specificity can be added to the photoaddition reaction by attaching the psoralen to an oligonucleotide designed to recognize a double-stranded DNA binding site through formation of a triple helix. We have previously used this strategy to study targeted psoralen modification of a triplex binding site within the bacterial supF gene carried in viral genomes. In the present work we report the targeting of psoralen photoadducts in vitro to a specific site in the genome of a transgenic mouse. Both 10 base and 16 base oligonucleotide-psoralen conjugates were capable of sequence-specific modification of genomic mouse DNA, while a truncated 8 base conjugate was not. Light activation was necessary, and a dose dependence was demonstrated for target site modification and mutagenesis. The 10 base conjugate rapidly found its target, with sequence-specific binding occurring after just 10 min incubation in the presence of mouse DNA. The ability to target psoralen photoadducts within mammalian genomes may prove useful in the study of chromatin structure and DNA repair. Moreover, this work may lead to potential in vivo applications of targeted psoralen modification.
Subject(s)
DNA Adducts/metabolism , DNA/metabolism , DNA/radiation effects , Ficusin/metabolism , Ficusin/pharmacology , Nucleic Acid Conformation , Animals , Base Sequence , Binding Sites , DNA/drug effects , Genes, Suppressor/genetics , Genome , Mice , Mice, Transgenic , Molecular Sequence Data , RNA, Transfer/geneticsABSTRACT
Transgenic mice carrying multiple copies of a recoverable lambda phage shuttle vector carrying the supF mutation reporter gene (lambda supF) were constructed for the purpose of studying mutagenesis in a whole animal. Spontaneous mutations in rescued supF target genes from mouse liver and skin were analyzed. The mutation frequency was similar in both tissues (in the range of 2 x 10(-5)), but the spectrum of point mutations was distinct, with transitions common in the skin and transversions more prominent in the liver (P = 0.01). These results may help to elucidate pathways of endogenous mutagenesis in vivo, and they illustrate potentially important tissue-specific differences in genetic instability.
Subject(s)
Bacteriophage lambda/genetics , Mice, Transgenic/genetics , Organ Specificity , Point Mutation , RNA, Transfer/genetics , Animals , Base Sequence , Female , Genes, Suppressor , Liver/physiology , Mice , Molecular Sequence Data , Mutation , Sequence Analysis, DNA , Skin Physiological PhenomenaSubject(s)
DNA Primers/genetics , DNA, Recombinant/genetics , Mutagenesis, Site-Directed , Nucleic Acid Conformation , Animals , Base Sequence , Cell Line, Transformed , Chlorocebus aethiops , DNA Adducts , DNA Primers/chemistry , DNA, Recombinant/chemistry , Escherichia coli/genetics , Ficusin/pharmacology , Genetic Vectors/drug effects , Genetic Vectors/genetics , Genetic Vectors/radiation effects , Molecular Sequence Data , Mutagens/pharmacology , Transformation, Bacterial , Transformation, Genetic , Ultraviolet RaysABSTRACT
To study the pathways associated with genomic instability in cancer, we examined UV-induced and spontaneous mutagenesis in clonal cell lines expressing human papillomavirus (HPV) proteins, either high-risk (HPV16) E6 or E7 or low-risk (HPV11) E6, in comparison to the parental RKO cells, a colon carcinoma cell line expressing only normal p53. High-risk E6 and E7 bind and functionally inactivate tumor suppressor proteins p53 and Rb, respectively, and both disrupt the G1 arrest in response to DNA damage. Low-risk HPV E6 proteins bind p53 with much lower affinity than high-risk E6 and fail to mediate p53 degradation or to disrupt the G1 checkpoint. We found that cells expressing HPV16 E6 had reduced survival and increased mutagenesis at the hprt locus when treated with low doses of UV. However, this analysis was complicated by the unexpected observation of a very high background of spontaneous mutagenesis in the unirradiated cells expressing the HPV16 E6 gene. Fluctuation analysis revealed a 5-fold elevated mutation rate in the cells expressing HPV16 E6. HPV11 E6 conferred a 2-fold elevation in the mutation rate, but HPV16 E7 had no effect. The increased spontaneous mutagenesis, therefore, appeared to be mediated by p53 inactivation and to be independent of Rb (which acts downstream of p53 in the G1 arrest pathway following DNA damage). Taken together, these findings suggest that the effect of p53 inactivation on spontaneous mutagenesis is manifested at the level of DNA repair, recombination, or coupling of transcription with one of these processes instead of by an alteration in G1 arrest.
Subject(s)
Mutagenesis , Oncogene Proteins, Viral/genetics , Papillomaviridae/genetics , Repressor Proteins , Tumor Suppressor Protein p53/physiology , Cell Survival/radiation effects , DNA Damage , G1 Phase , Humans , Papillomavirus E7 Proteins , Tumor Cells, Cultured , Ultraviolet RaysABSTRACT
The tumor suppressor protein, p53, is proposed to have a critical role in maintaining the integrity of the genetic material. It has been established that p53 induces a cell cycle block in the G1 phase upon cellular DNA damage. Recent evidence also indicates the involvement of p53 directly and indirectly in nucleotide excision repair (NER). We have examined the role of p53 with respect to UV-induced mutagenesis. By gene transfer, we established a mouse fibroblast cell line overexpressing the val135 temperature-sensitive p53 allele. In this line, p53 activity can be modulated through temperature shift, as confirmed by Western blot and by cell cycle analysis. This cell line was also constructed to contain a recoverable lambda phage shuttle vector carrying the supF mutation reporter gene. Induction of p53 was found to enhance the clonogenic survival of the cells following UV-irradiation compared to the p53-deficient parental mouse cell line. The transfectant line also displayed a 4-fold reduction in the frequency of UV-induced mutations as measured in the chromosomally integrated supF reporter gene. Our results are consistent with a p53-induced cell cycle block at G1 allowing cells to repair chromosomal damage before DNA replication. However, our data may also reflect a more direct role of p53 in the repair of UV-induced lesions as suggested by studies showing that p53 can interact directly with repair factors.
Subject(s)
Mutagenesis/radiation effects , Tumor Suppressor Protein p53/biosynthesis , Ultraviolet Rays , Animals , Bacteriophage lambda , Base Sequence , Blotting, Western , Cell Cycle/radiation effects , Cell Line , DNA Damage , Gene Transfer Techniques , Genes, Suppressor , Genetic Vectors , Mice , Molecular Sequence Data , Point Mutation , RNA, Transfer/biosynthesis , RNA, Transfer/genetics , Recombinant Proteins/analysis , Recombinant Proteins/biosynthesis , Tumor Suppressor Protein p53/analysisABSTRACT
A polypurine tract in the supF gene of bacteriophage lambda (base pairs 167-176) was selected as the target for triple helix formation and targeted mutagenesis by an oligopurine (5'-AGGAAGGGGG-3') containing a chemically linked psoralen derivative (4'-hydroxymethyl-4,5',8-trimethylpsoralen) at its 5' terminus (psoAG10). The thymines at base pairs 166 and 167, a 5'ApT site, were targeted for photomodification. Exposure of the triple helical complex to long wavelength ultraviolet radiation led to the covalent binding of psoAG10 to the targeted region in the supF gene and to the induction of site-specific mutations. We report here experiments to characterize the photomodification of the targeted region of the supF gene in the context of triple helix formation. An electrophoretic mobility-shift assay showed that, at low radiation doses, monoadducts at base pair 166 were the major photoadducts. At higher doses the monoadducts were converted to crosslinks between base pairs 166 and 167. HPLC analysis of enzymatically hydrolyzed photoreaction mixtures was used to confirm the electrophoresis results. A strong strand preference for specific photoadduct formation was also detected.
Subject(s)
Bacteriophage lambda/genetics , DNA, Viral/chemistry , DNA, Viral/radiation effects , Furocoumarins , Genes, Viral , Ultraviolet Rays , Base Sequence , Binding Sites , DNA, Viral/metabolism , Dose-Response Relationship, Radiation , Light , Molecular Sequence Data , Mutagenesis, Site-Directed , Nucleic Acid Conformation , Oligodeoxyribonucleotides/chemistryABSTRACT
Triple-helical DNA can be formed by oligonucleotides that bind as third strands of DNA in a sequence-specific manner in the major groove in homopurine/homopyrimidine stretches in duplex DNA. Such triple helix-forming oligonucleotides have been used to inhibit gene expression by blocking transcription factor access to promoter sites in transient expression assays. In an alternative approach to genetic manipulation using triplex DNA, we show that triplex-forming oligonucleotides can be used to produce site-specific, targeted mutations in a viral genome in order to achieve a permanent, heritable effect on gene function and expression. We use a triplex-forming oligonucleotide linked to a psoralen derivative at its 5' end to achieve targeted mutagenesis in a simian virus 40 (SV40) vector genome. Site-specific triplex formation delivers the psoralen to the targeted site in the SV40 DNA. Photoactivation of the psoralen yields adducts and thereby mutations at that site. Mutations were produced in the target gene in over 6% of the viral genomes. DNA sequence analysis of the mutations in the target gene showed that all were in the targeted region, and 55% were found to be the same T:A-to-A:T transversion precisely at the targeted base pair. In control experiments, no mutagenesis above the background frequency in the assay was produced by a non-triplex-forming, psoralen-linked oligonucleotide unless a vast excess of this oligonucleotide was used, demonstrating the specificity of the targeted mutagenesis. This frequency of targeted mutagenesis of SV40 in monkey cells represents a 30-fold increase relative to similar experiments using lambda phage in bacteria, suggesting that fixation of the triplex-directed lesion into a mutation occurs more efficiently in mammalian cells. If the ability to reproducibly and predictably target mutations to sites in viral DNA in vitro by using modified oligonucleotides can be extended to DNA in vivo, this approach may prove useful as a technique for gene therapy, as a strategy for antiviral therapeutics, and as a tool for genetic engineering.
Subject(s)
DNA , Genes, Viral/genetics , Mutagenesis, Site-Directed , Oligodeoxyribonucleotides , Simian virus 40/genetics , Animals , Base Sequence , Cells, Cultured , Ficusin , Genetic Vectors/genetics , Molecular Sequence Data , Sequence Analysis, DNAABSTRACT
Oligonucleotides can bind as third strands of DNA in a sequence-specific manner in the major groove in homopurine/homopyrimidine stretches in duplex DNA. Here we use a 10-base triplex-forming oligonucleotide linked to a psoralen derivative at its 5' end to achieve site-specific, targeted mutagenesis in an intact, double-stranded lambda phage genome. Site-specific triplex formation delivers the psoralen to the targeted site in the lambda DNA, and photoactivation of the psoralen produces adducts and thereby mutations at that site. Mutations in the targeted gene were at least 100-fold more frequent than those in a nontargeted gene, and sequence analysis of mutations in the targeted gene showed that 96% were in the targeted region and 56% were found to be the same T.A to A.T transversion precisely at the targeted base pair. The ability to reproducibly and predictably target mutations to sites in intact duplex DNA by using modified oligonucleotides may prove useful as a technique for gene therapy, as an approach to antiviral therapeutics, and as a tool for genetic engineering.
Subject(s)
Bacteriophage lambda/genetics , DNA, Viral/genetics , Genes, Viral , Mutagenesis, Site-Directed , Trioxsalen/analogs & derivatives , Bacteriophage lambda/radiation effects , Base Sequence , Cross-Linking Reagents , DNA, Viral/radiation effects , Escherichia coli/genetics , Genes, Viral/radiation effects , Molecular Sequence Data , Oligodeoxyribonucleotides/chemical synthesis , Restriction Mapping , Ultraviolet RaysSubject(s)
DNA-Binding Proteins/metabolism , Genes, MHC Class II , HLA-DR Antigens/genetics , Pregnancy Proteins/metabolism , Promoter Regions, Genetic , Base Sequence , Binding Sites , DNA-Binding Proteins/isolation & purification , Female , Humans , Molecular Sequence Data , Oligonucleotide Probes , Organ Specificity , Placenta/physiology , Pregnancy , Pregnancy Proteins/isolation & purificationABSTRACT
We previously reported that an initial response to acute cardiac stress is temporary translational inhibition. Using an in vitro translational assay to follow activity, we purified a translation-inhibiting peptide (TIP) obtained from control canine hearts and hearts acutely stressed by ascending aortic banding and heat shock for 1 h. We isolated a 17-kDa peptide. The amino acid composition was determined and residues 3-20 were sequenced. Treatment of polysome preparations with TIP shifted the polysome distribution profile to that characteristic of the acutely stressed heart in which monosomes predominate. Translational inhibition acted by suppressing formation of the 80S initiation complex. Suppression curves indicate that translation is inhibited by approximately 60%, with the translation of protein over 30 kDa being highly suppressed. We postulate that translational inhibition is an essential initial reaction to acute stress that allows the cell to redirect energy into vita cell functions that permit long-term adaptation to the stress. We believe that translational inhibition is effected by intracellular activator of TIP.
Subject(s)
Myocardium/analysis , Peptides/isolation & purification , Protein Biosynthesis/drug effects , Amino Acid Sequence , Amino Acids/analysis , Animals , Centrifugation , Centrifugation, Density Gradient , Chromatography, Gel , Chromatography, High Pressure Liquid , Dogs , Electrophoresis, Polyacrylamide Gel , Molecular Sequence Data , Myocardium/ultrastructure , Peptides/pharmacology , Polyribosomes/ultrastructure , Stress, Physiological/metabolism , UltrafiltrationABSTRACT
Stress was induced in rat hearts by heat shock and aortic banding and in cardiac cell cultures by heat shock. Proteins were labeled with [35S]-methionine, resolved by electrophoresis, and isotope incorporation measured. Determinations were made during and following stress. The immediate response was stress protein (SP) synthesis, cessation of normally occurring protein synthesis and reduced isotope incorporation. Following recovery, the prestress protein pattern returned, SP synthesis remained high and label incorporation doubled. Response of hearts to the two stresses were similar. Initial suppression may be an important precursor to accelerated protein synthesis seen in hypertrophy.
Subject(s)
Cardiomegaly/metabolism , Heat-Shock Proteins/biosynthesis , Muscle Proteins/biosynthesis , Animals , Rats , Stress, Physiological/metabolism , Vascular ResistanceABSTRACT
The acute response to heat shock was examined in the intact canine heart. Ventricular samples were removed before the dogs were heated. After heat treatment, the hearts were removed. Total RNA was extracted from pre and post heat shocked samples, translated in vitro with [35S]methionine and visualized by autoradiography. Polysome profiles from pre and post heat shocked hearts were then analyzed. Heat shocked hearts synthesized mRNAs for 71 kD stress proteins. The polysome profile after heat treatment showed a steady decline of the polysomal material toward the heavier polysome region when compared to myocardium before heat treatment. Stress protein synthesis and polysomal disaggregation are initial responses to stress that, in the mammalian heart, may be precursors to hypertrophy.
Subject(s)
Myocardium/metabolism , Protein Biosynthesis , Stress, Physiological/metabolism , Transcription, Genetic , Animals , Dogs , Hot Temperature , Polyribosomes/metabolism , RNA, Messenger/biosynthesisABSTRACT
The nuclear pore complex-lamina (PCL), composed of nuclear pore structures attached to fibrous lamina, was isolated from bovine liver nuclei. We found that the highly aggregated PCL was disrupted and 75% of the constituent polypeptides could be solubilized by extraction for 1 h with 2% deoxycholate (DOC) and 3% 2-mercaptoethanol. While some differential solubilization was observed at lower detergent concentrations, all PCL proteins were solubilized equally at 2% DOC. The reducing agent was necessary to achieve maximum dispersal of the PCL and to prevent aggregation of the solubilized proteins. No tightly bound phospholipid or Triton X-100 could be detected in these preparations. Rapid removal of DOC, by dialysis or gel filtration, resulted in aggregation and precipitation of the PCL proteins, but the detergent could be removed by centrifugation through sucrose gradients. The sedimentation profiles indicated that the three major polypeptides, lamins A, B, and C, each sedimented as a single peak with a shoulder of more rapidly sedimenting material, possibly higher oligomeric forms. The sedimentation coefficient of lamins B and C, in the presence and absence of detergent, was 4.5 S. In the presence of DOC, lamin A had a sedimentation coefficient of 5.6 S, but this value was decreased to 4.1 S, when DOC was omitted from the gradient. These studies suggested that lamins B and C do not interact with or bind DOC, while lamin A may bind appreciable amounts of the detergent. The Stokes radii of lamins A, B, and C were found by gel filtration to be 75, 75, and 70 A, respectively. The molecular weights and frictional ratios estimated from the sedimentation and gel filtration data indicated that the lamins are dimeric, rod-shaped molecules.