ABSTRACT
An F-box protein, beta-TrCP recognizes substrate proteins and destabilizes them through ubiquitin-dependent proteolysis. It regulates the stability of diverse proteins and functions as either a tumor suppressor or an oncogene. Although the regulation by beta-TrCP has been widely studied, the regulation of beta-TrCP itself is not well understood yet. In this study, we found that the level of beta-TrCP1 is downregulated by various protein kinase inhibitors in triple-negative breast cancer (TNBC) cells. A PI3K/mTOR inhibitor PI-103 reduced the level of beta-TrCP1 in a wide range of TNBC cells in a proteasome-dependent manner. Concomitantly, the levels of c-Myc and cyclin E were also downregulated by PI-103. PI-103 reduced the phosphorylation of beta-TrCP1 prior to its degradation. In addition, knockdown of beta-TrCP1 inhibited the proliferation of TNBC cells. We further identified that pharmacological inhibition of mTORC2 was sufficient to reduce the beta-TrCP1 and c-Myc levels. These results suggest that mTORC2 regulates the stability of beta-TrCP1 in TNBC cells and targeting beta-TrCP1 is a potential approach to treat human TNBC.
Subject(s)
Female , Humans , Cell Line, Tumor , Cell Proliferation , Cell Survival/drug effects , Cyclin E/genetics , Dose-Response Relationship, Drug , Furans/pharmacology , Gene Knockdown Techniques , Models, Biological , Multiprotein Complexes/antagonists & inhibitors , Phosphatidylinositol 3-Kinases/antagonists & inhibitors , Phosphorylation/drug effects , Protein Kinase Inhibitors/pharmacology , Proteolysis/drug effects , Proto-Oncogene Proteins c-myc/genetics , Pyridines/pharmacology , Pyrimidines/pharmacology , TOR Serine-Threonine Kinases/antagonists & inhibitors , Triple Negative Breast Neoplasms/genetics , beta-Transducin Repeat-Containing Proteins/geneticsABSTRACT
Tumor associated microtubule associated protein (TMAP), also known as cytoskeleton associated protein 2 (CKAP2) is a mitotic spindle-associated protein whose expression is cell cycle-regulated and also frequently deregulated in cancer cells. Two monoclonal antibodies (mAbs) against TMAP/CKAP2 were produced: B-1-13 and D-12-3. Interestingly, the reactivity of mAb D-12-3 to TMAP/CKAP2 was markedly decreased specifically in mitotic cell lysate. The epitope mapping study showed that mAb D-12-3 recognizes the amino acid sequence between 569 and 625 and that phosphorylation at T596 completely abolishes the reactivity of the antibody, suggesting that the differential reactivity originates from the phosphorylation status at T596. Immunofluorescence staining showed that mAb D-12-3 fails to detect TMAP/CKAP2 in mitotic cells between prophase and metaphase, but the staining becomes evident again in anaphase, suggesting that phosphorylation at T596 occurs transiently during early phases of mitosis. These results suggest that the cellular functions of TMAP/CKAP2 might be regulated by timely phosphorylation and dephosphorylation during the course of mitosis.
Subject(s)
Animals , Humans , Mice , Amino Acid Sequence , Antibodies, Monoclonal/metabolism , Cell Cycle/physiology , Cells, Cultured , Cytoskeletal Proteins/chemistry , Epitope Mapping , HeLa Cells , Mitosis/physiology , Molecular Sequence Data , Phosphorylation , Protein Serine-Threonine Kinases/metabolism , Sequence Homology, Amino Acid , Threonine/metabolismABSTRACT
BACKGROUND AND OBJECTIVES: Elevated intracellular calcium level is known to play important roles in the apoptotic pathway. IP3 receptor (ligand-gated channels that release Ca2+ from intracellular stores) is emerging as a key site for regulation of apoptosis. 2-Aminoethoxydiphenyl borate (2-APB) is one of the reliable IP3 receptor antagonists. We examined the effect of 2-APB on gentamicin ototoxicity in vitro, using the HEI-OC1 cell line. MATERIALS AND METHOD: HEI-OC1 cells were trWWeated with 100micrometer gentamicin. Using a CaspACE assay, we measured the caspases-3 activity in the gentamicin treated hair cells with and without 2-APB pre-incubation. We also observed intra-cellular calcium concentrations in HEI-OC1 cells using a confocal microscopy (calcium green-1 stain). Live cell imaging was performed by using fluorescence video-time lapse system. RESULTS: Cytosolic calcium elevation by gentamicin was remarkably inhibited by 2-APB. Caspases-3 activities of gentamicin treated cells were higher than those of the control. After incubation with 2-APB, caspases-3 activities and cell death of gentamicin treated cells were shown to decrease. CONCLUSION: 2-APB reduces Caspases-3 activity in the gentamicin treated HEI-OC1 cells by inhibition of cytosolic calcium increase.
Subject(s)
Apoptosis , Calcium , Caspase 3 , Cell Death , Cell Line , Cytosol , Fluorescence , Gentamicins , Hair , Inositol 1,4,5-Trisphosphate Receptors , Microscopy, ConfocalABSTRACT
KM-HN-1 is a C-terminal coiled-coil domain containing protein previously referred to as image clone MGC33607. This protein has been previously identified as a cancer/testis antigen and reported as nuclear and chromatin localizing protein. We raised polyclonal antisera with the GST fusion protein and identified them as a 105 kDa protein. Motif analysis showed that this protein harbors the leucine zipper motif in internal 1/3 region and the coiled-coil domain in the C-terminal region. Using the full length and various deletion mutants, we determined the motif that governs the subcellular localization of KM-HN-1. Immunofluorescence staining of the endogenous KM-HN-1 and various kinds of GFP-tagged KM-HN-1 revealed that KM-HN-1 localizes to the centrosomes as well as nucleus. The centrosomal localization-determining region of this protein is C-terminal coiled-coil domain in which the leucine zipper motif and the nuclear export signal (NES) harbor.
Subject(s)
Humans , Amino Acid Motifs/physiology , Amino Acid Sequence , Antigens, Neoplasm/chemistry , Cells, Cultured , Centrosome/metabolism , Fluorescent Antibody Technique , Leucine Zippers/physiology , Molecular Sequence Data , Mutation , Nuclear Proteins/chemistry , Protein Conformation , Protein Structure, Tertiary , Sequence Analysis, ProteinABSTRACT
Allelic deletions involving the short arm of chromosome 3(3p13-21.1) have been observed frequently in cervical carcinomas. Recently the fragile histidine triad(FHIT) gene was cloned and mapped to this chromosomal region(3p14.2). From various studies involving tumor cell lines and primary cancers, the FHIT gene has been presumed to be a candidate for tumor suppressor gene involving various tumors. In FHIT gene, the most common aphidicolin-inducible fragile site, FRA3B exists and the FRA3B has been considered as a region of the spontaneous integration site of HPV 16. In order to elucidate the role of the FHIT in carcinogenesis of cervical cancer, this study was designed to investigate both the expression of FHIT protein in normal, preinvasive and invasive cancer samples employing immunohistochemical study and allelic loss of FHIT gene locus against several microsatellite markers employing the PCR analysis. Immunohistochemical studies of FHIT protein revealed following features. In normal ectocervical squamous epithelium, the expression of FHIT was relatively weak and confined to the basal layer, but in normal endocervical glandular epithelium it was very strong. The expression of FHIT was reduced as the tumor progressed from early lesion to invasive cancer. The koilocytosis was associated with diminished expression of FHIT protein. The study of allelic loss of FHIT gene locus was undertaken against two intragenic (D3S1300, D3S1234) and one extragenic (D3S1295) microsatellite markers. The 5th intron, D3S1300, showed allelic change in 6 of 15 assays and 7th intron, D3S1234 showed allelic change in 10 of 29 assays. There was no apparent LOH from 29 assays in D3S1295. In conclusion, the expression of FHIT protein was markedly reduced or absent in cervical squamous cell carcinoma and the chromosome breakage in FHIT region might be related to the diminished expression of FHIT. On the basis of the reduced expression of FHIT and its encompassment of FRA3B region, it is suggested that disruption of FHIT, a putative tumor suppressor gene, might be the mechanism by which HPV infection enhances cervical tumorigenesis and clonal outgrowth.
Subject(s)
Arm , Carcinogenesis , Carcinoma, Squamous Cell , Cell Line, Tumor , Chromosome Breakage , Clone Cells , Epithelium , Genes, Tumor Suppressor , Histidine , Human papillomavirus 16 , Introns , Loss of Heterozygosity , Microsatellite Repeats , Polymerase Chain Reaction , Uterine Cervical NeoplasmsABSTRACT
Guanine aminohydrolase (GAH; Guanine deaminase, EC 3.5.4.3) is an enzyme that has a role in purine catabolism. This enzyme produces xanthine and ammonia by hydrolysis of guanine, and xanthine is further degraded to uric acid and hydrogen peroxide by another enzyme, xanthine oxidase. Most of the enzymes involved in purine catabolism have been studied for their biological functions, physiological roles and amino acid sequences, and biochemical activity of GAH is known to be detected in various organs such as liver, kidney, small intestine and brain. Its activity is also known to be changed during brain development. In this study, we hoped to reveal expression pattern of GAH in developing rat brain by western blotting and immunohistochemistry. In western blotting, GAH immunoreactivity was not detected on 14-, 16- and 18-days-old fetal rat brains. Its reactivity was first detected from 20-days-old fetal rat brain and highly increased after birth. And it was maintained at steady level from 2 weeks after birth. In immunohistochemistry, no positive cells were found on 14- and 16-days-old fetal rat brain sections. A few GAH-immunoreactive cells appeared from 18-days-old fetal rat brain and they were localized at olfactory bulb, cerebral cortex, midbrain, pons and medulla. The 20-days-old fetal rat brain also showed immunoreactive cells at hippocampus and the staining intensity was still weak. Postnatal 2-days-old rat brain also showed immunoreactive cells at basal ganglia and the number of positive cells and staining intensity were increased. Thereafter, immunoreactivity appeared on many neuronal cells around various areas in the brain and nerve fibers also showed reactivity on postnatal brains. The number of positive cells decreased from 1 week after birth and a few positive cells were observed on olfactory bulb and cerebellum from 2 weeks after birth. In mature brain most of GAH were localized on nerve fibers and few positive cells could be found on olfatory bulb only. From these, we can suspect that GAH may have some functional relationship with nerve fibers.
Subject(s)
Animals , Rats , Amino Acid Sequence , Ammonia , Basal Ganglia , Blotting, Western , Brain , Cerebellum , Cerebral Cortex , Guanine Deaminase , Guanine , Hippocampus , Hope , Hydrogen Peroxide , Hydrolysis , Immunohistochemistry , Intestine, Small , Kidney , Liver , Mesencephalon , Metabolism , Nerve Fibers , Neurons , Olfactory Bulb , Parturition , Pons , Uric Acid , Xanthine , Xanthine OxidaseABSTRACT
BACKGROUND: A sustained outbreak of multi-resistant Acinetobacter baumannii has been noticed in intensive care unit (ICU) of a newly opened hospital. METHODS: We retrospectively studied 72 patients in the ICU whose specimen grew A. baumannii from March to December 1995. To identify risk factors for infection, a case control study was conducted. Comparing clinical characteristics of 19 infected patients with those of 53 colonized cases. Environmental culture was performed in January 1996 to identify the source of infection. We analyzed antibiotic susceptibility of the isolates, and ribotyping was performed with 52 isolates. RESULTS: Nineteen out of 72 patients developed disease: primary sepsis 2, catheter related infection 2, catheter related infection and pneumonia 2, wound infection 5, wound infection and sepsis 2, pneumonia 6. On comparison of clinical characteristics between the infected and colonized groups, central venous catheterization was a significant risk factor for development of disease by A. baumannii (P<0.05) and duration of lCU stay was a factor independently associated with A. baumannii infection by logistic regression analysis. An epidemiologic investigation failed to identify the source of infection, but we found 2 of 3 sinks in lCU were heavily contaminated by the organism. Antibiogram of the isolates showed a multi-drug resistance including amikacin, which was found to increase gradually during the course of the outbreak. Ribotyping showed 3 major subtypes: 2A (18 isolates) 2B (16) 2B'(13) and other types (5). CONCLUSION: The findings from this study support the reports from many parts of the world that A. baumannii plays an increasingly important role as one of the major nosocomial pathogens.
Subject(s)
Humans , Acinetobacter baumannii , Amikacin , Case-Control Studies , Catheterization, Central Venous , Catheters , Central Venous Catheters , Colon , Drug Resistance, Multiple , Intensive Care Units , Critical Care , Logistic Models , Microbial Sensitivity Tests , Pneumonia , Retrospective Studies , Ribotyping , Risk Factors , Sepsis , Wound InfectionABSTRACT
BACKGROUND: A sustained outbreak of multi-resistant Acinetobacter baumannii has been noticed in intensive care unit (ICU) of a newly opened hospital. METHODS: We retrospectively studied 72 patients in the ICU whose specimen grew A. baumannii from March to December 1995. To identify risk factors for infection, a case control study was conducted. Comparing clinical characteristics of 19 infected patients with those of 53 colonized cases. Environmental culture was performed in January 1996 to identify the source of infection. We analyzed antibiotic susceptibility of the isolates, and ribotyping was performed with 52 isolates. RESULTS: Nineteen out of 72 patients developed disease: primary sepsis 2, catheter related infection 2, catheter related infection and pneumonia 2, wound infection 5, wound infection and sepsis 2, pneumonia 6. On comparison of clinical characteristics between the infected and colonized groups, central venous catheterization was a significant risk factor for development of disease by A. baumannii (P<0.05) and duration of lCU stay was a factor independently associated with A. baumannii infection by logistic regression analysis. An epidemiologic investigation failed to identify the source of infection, but we found 2 of 3 sinks in lCU were heavily contaminated by the organism. Antibiogram of the isolates showed a multi-drug resistance including amikacin, which was found to increase gradually during the course of the outbreak. Ribotyping showed 3 major subtypes: 2A (18 isolates) 2B (16) 2B'(13) and other types (5). CONCLUSION: The findings from this study support the reports from many parts of the world that A. baumannii plays an increasingly important role as one of the major nosocomial pathogens.
Subject(s)
Humans , Acinetobacter baumannii , Amikacin , Case-Control Studies , Catheterization, Central Venous , Catheters , Central Venous Catheters , Colon , Drug Resistance, Multiple , Intensive Care Units , Critical Care , Logistic Models , Microbial Sensitivity Tests , Pneumonia , Retrospective Studies , Ribotyping , Risk Factors , Sepsis , Wound InfectionABSTRACT
c-erbB-2 oncogene encodes a growth factor receptor whose amino acid sequence has extensive homology with human epidermal growth factor receptor. It is frequently overexpressed in human breast, ovary, lung, and stomach cancers, where its overexpression is related significantly to the prognosis. Tl investigate the possible role of c-erbB-2 oncogene in the oncogenesis of stomach cancer, we examined the genetic alterations of c-erbB-2 oncogene in 4 stomach cancer cell lines, SNU-1, SNU-5, SNU-16 and KATO III. There were no differences in c-erbB-2 mRNA level as well as c-erbB-2 gene copy number among them. But gp185-erbB-2, c-erbB-2 gene product, was increased from 2- to 4-fold in SNU-1 and SNU-5 cells, compared with that in SNU-16 or KATO III cells. Our results suggest that post-transcriptional regulation of gp185erbB-2 expression may underlie gp185erbB-2 overexpression in cancer cells.