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Molecular and Cellular Biology Sep 1990By using a retrovirus-derived vector system, we generated derivatives of the human colon cancer cell line HT29 that stably overexpress a full-length cDNA encoding the...
By using a retrovirus-derived vector system, we generated derivatives of the human colon cancer cell line HT29 that stably overexpress a full-length cDNA encoding the beta 1 isoform of rat protein kinase C (PKC). Two of these cell lines, PKC6 and PKC7, displayed an 11- to 15-fold increase in PKC activity when compared with the C1 control cell line that carries the vector lacking the PKC cDNA insert. Both of the overexpresser cell lines exhibited striking alterations in morphology when exposed to the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Following exposure to TPA, PKC6 and PKC7 cells displayed increased doubling time, decreased saturation density, and loss of anchorage-independent growth in soft agar; but these effects were not seen with the C1 cells. Also, in contrast to the control cells, the PKC-overproducing cells failed to display evidence of differentiation, as measured by alkaline phosphatase activity, when exposed to sodium butyrate. In addition, the PKC-overexpresser cells displayed decreased tumorigenicity in nude mice, even in the absence of treatment with TPA. These results provide the first direct evidence that PKC can inhibit tumor cell growth. Thus, in some tumors, PKC might act as a growth-suppressor gene.
Topics: Animals; Cell Division; Cell Line; Colonic Neoplasms; Gene Expression; Genetic Vectors; Humans; Kinetics; Mice; Mice, Nude; Neoplasm Transplantation; Protein Kinase C; Rats; Tetradecanoylphorbol Acetate; Transfection; Transplantation, Heterologous; Tumor Cells, Cultured
PubMed: 2388620
DOI: 10.1128/mcb.10.9.4650-4657.1990 -
British Journal of Haematology Feb 1997P-glycoprotein (PGP) lung resistance protein (LRP) and multidrug resistance associated protein (MRP) expressions and function were evaluated by flow cytometry in 65...
P-glycoprotein (PGP) lung resistance protein (LRP) and multidrug resistance associated protein (MRP) expressions and function were evaluated by flow cytometry in 65 leukaemic patients (38 acute non-lymphocytic leukaemias, eight acute lymphocytic leukaemias, 19 Ph-positive chronic myeloid leukaemias in blastic phase). By using the MRK-16, the LRP-56 and the MRPm6 MoAbs, 34% of the cases did not over-express any proteins (-); 24.5% over-expressed (+) only PGP, 11% only LRP, 1.5% only MRP, 24.5% both PGP and LRP, and 4.5% both PGP and MRP. The mean intracellular daunorubicin accumulation (IDA) and rhodamine 123 (Rh123) retention in the presence or absence of the reversal agent SDZ PSC 833 (PSC) of the PGP-/LRP-/MRP- cases were comparable to the ones observed in normal leucocytes. With respect to the non-over-expressing cases, the PGP-/LRP+/MRP- cases showed only an impaired IDA (mean 204 +/- 29; P < 0.001). The PGP+/ LRP+/MRP- cases had a defect both in IDA (mean 166 +/- 47, P < 0.001) and Rh123 retention (mean 0.42 +/- 0.14: P < 0.001), which were both corrected by PSC. All the PGP+/LRP+/MRP- cases had a defect in IDA (mean daunorubicin (DNR) accumulation 192 +/- 44; P < 0.001). However, only in 8/16 of them an evident defect in Rh123 retention was found. In conclusion, both PGP and LRP over-expression were common in leukaemia. An impaired IDA was found in all cases over-expressing PGP, LRP or both. The study of Rh123 retention could give incorrect information about the blast cells' ability to accumulate cytotoxic drugs in patients over-expressing both PGP and LRP.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP-Binding Cassette Transporters; Daunorubicin; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Humans; Leukemia; Lymphocytes; Multidrug Resistance-Associated Proteins; Neoplasm Proteins; Rhodamine 123; Rhodamines; Vault Ribonucleoprotein Particles
PubMed: 9029025
DOI: 10.1046/j.1365-2141.1997.d01-2020.x -
Current Biology : CB Apr 2012Faithful chromosome segregation is required for cell and organism viability and relies on both the mitotic checkpoint and the machinery that corrects...
Faithful chromosome segregation is required for cell and organism viability and relies on both the mitotic checkpoint and the machinery that corrects kinetochore-microtubule (k-MT) attachment errors. Most solid tumors have aneuploid karyotypes and many missegregate chromosomes at high rates in a phenomenon called chromosomal instability (CIN). Mad2 is essential for mitotic checkpoint function and is frequently overexpressed in human tumors that are CIN. For unknown reasons, cells overexpressing Mad2 display high rates of lagging chromosomes. Here, we explore this phenomenon and show that k-MT attachments are hyperstabilized by Mad2 overexpression and that this undermines the efficiency of correction of k-MT attachment errors. Mad2 affects k-MT attachment stability independently of the mitotic checkpoint because k-MT attachments are unaltered upon Mad1 depletion and Mad2 overexpression hyperstabilizes k-MT attachments in Mad1-deficient cells. Mad2 mediates these effects with Cdc20 by altering the centromeric localization and activity of Aurora B kinase, a known regulator of k-MT attachment stability. These data reveal a new function for Mad2 to stabilize k-MT attachments independent of the checkpoint and explain why Mad2 overexpression increases chromosome missegregation to cause chromosomal instability in human tumors.
Topics: Aurora Kinase B; Aurora Kinases; Calcium-Binding Proteins; Cdc20 Proteins; Cell Cycle Proteins; Cell Line; Centromere; Chromosome Segregation; Green Fluorescent Proteins; Kinetochores; M Phase Cell Cycle Checkpoints; Mad2 Proteins; Microscopy, Fluorescence; Microtubules; Nuclear Proteins; Protein Serine-Threonine Kinases; Repressor Proteins; Spindle Apparatus; Transfection; Tubulin
PubMed: 22405866
DOI: 10.1016/j.cub.2012.02.030 -
Nan Fang Yi Ke Da Xue Xue Bao = Journal... Jan 2020To investigate the effect of overexpression of leukemia inhibitory factor (LIF) on cisplatin and paclitaxel resistance of endometrial cancer cells .
OBJECTIVE
To investigate the effect of overexpression of leukemia inhibitory factor (LIF) on cisplatin and paclitaxel resistance of endometrial cancer cells .
METHODS
Endometrial cancer cell lines HEC-1B and RL95-2 were infected with a recombinant lentivirus to overexpress LIF, and the changes in LIF expression was verified using RT-qPCR and ELISA. The viability of the LIF-overexpressing cells was assessed using CCK-8 assay, and the cell apoptosis and changes in mitochondrial membrane potential in response to cisplatin or paclitaxel treatment were analyzed with annexin V-FITC/PI staining and JC-1 assay, respectively. The effect of LIF overexpression on the expressions of Bcl-2 family proteins and STAT3 pathway was evaluated using Western blotting; dual-luciferase reporter gene assay was employed to detect the transcriptional activity of STAT3. The effect of STAT3 silencing on apoptosis of the LIF-overexpressing cells induced by cisplatin or paclitaxel was investigated.
RESULTS
The cell lines infected with the recombinant lentivirus showed significantly increased mRNA and protein levels of LIF ( < 0.05) without obvious changes in the cell viability (>0.05). LIF overexpression significantly attenuated cisplatin-or paclitaxel-induced apoptosis of the endometrial cancer cells ( < 0.05) and markedly increased mitochondrial membrane potential of the cells ( < 0.05). The expressions of Bcl-2, Bcl-xL and p-STAT3 proteins increased obviously while the expressions of Bax, Bad and STAT3 either decreased or showed no obvious changes in the LIF-overexpressing cells. Overexpressing LIF significantly enhanced the transcriptional activity of STAT3 ( < 0.05), and silencing STAT3 obviously enhanced apoptosis of the endometrial cancer cells overexpressing LIF ( < 0.05).
CONCLUSIONS
s Overexpression of LIF can enhance cisplatin and paclitaxel resistance to endometrial cancer cells .
Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cisplatin; Drug Resistance, Neoplasm; Endometrial Neoplasms; Female; Humans; Leukemia Inhibitory Factor; Paclitaxel; Proto-Oncogene Proteins c-bcl-2; STAT3 Transcription Factor; Signal Transduction; bcl-X Protein
PubMed: 32376564
DOI: 10.12122/j.issn.1673-4254.2020.01.04 -
Molecular Medicine Reports May 2017Distal-less homeobox 2 (Dlx2) is a member of the homeodomain family of transcription factors and is important for the development of cranial neural crest cells...
Distal-less homeobox 2 (Dlx2) is a member of the homeodomain family of transcription factors and is important for the development of cranial neural crest cells (CNCCs)‑derived craniofacial tissues. Previous studies revealed that Dlx2 was expressed in the cementum and a targeted null mutation disrupted tooth development in mice. However, whether Dlx2 overexpression may impair in vivo tooth morphogenesis remains to be elucidated. The present study used a transgenic mouse model to specifically overexpress Dlx2 in neural crest cells in order to identify the dental phenotypes in mice by observation, micro‑computed tomography and histological examination. The Dlx2‑overexpressed mice exhibited tooth abnormalities including incisor cross‑bite, shortened tooth roots, increased cementum deposition, periodontal ligament disorganization and osteoporotic alveolar bone. Therefore, Dlx2 overexpression may alter the alveolar bone, cementum and periodontal ligament (PDL) phenotypes in mice.
Topics: Animals; Gene Expression Regulation, Developmental; Homeodomain Proteins; Mice; Mice, Transgenic; Organogenesis; Periodontium; Tooth; Transcription Factors
PubMed: 28447749
DOI: 10.3892/mmr.2017.6315 -
British Journal of Cancer Dec 1999The expression of cyclin D1 protein in tumour sections from 81 patients with epithelial ovarian cancer was analysed using immunohistochemistry. The tumours that... (Clinical Trial)
Clinical Trial
The expression of cyclin D1 protein in tumour sections from 81 patients with epithelial ovarian cancer was analysed using immunohistochemistry. The tumours that overexpressed cyclin D1 in more than 10% of neoplastic cells were considered positive. Thus overexpression of cyclin D1 was observed in 72/81 (89%) of the cases examined. Protein was detected in both the nucleus and the cytoplasm in 24/81 (30%) and localized exclusively in the cytoplasm in 48/81 (59%) of the tumours. Cyclin D1 was overexpressed in both borderline and invasive tumours. There was no association between protein overexpression and tumour stage and differentiation. Furthermore, no correlation between cyclin D1 expression and clinical outcome was observed. However, in tumours overexpressing cyclin D1 (n = 72), the proportion displaying exclusively cytoplasmic localization of protein was higher in those with serous compared with non-serous histology (P = 0.004, odds ratio 4.8, 95% confidence interval 1.4-19.1). Western analysis using a monoclonal antibody to cyclin D1 identified a 36 kDa protein in homogenates from seven tumours displaying cytoplasmic only and one tumour demonstrating both nuclear and cytoplasmic immunostaining. Using restriction fragment length polymorphism polymerase chain reaction and PCR-multiplex analysis, amplification of the cyclin D1 gene (CCND1 was detected in 1/29 of the tumours demonstrating overexpression of cyclin D1 protein. We conclude that deregulation of CCND1 expression leading to both cytoplasmic and nuclear protein localization is a frequent event in ovarian cancer and occurs mainly in the absence of gene amplification.
Topics: Blotting, Western; Carcinoma; Cyclin D1; Female; Humans; Immunohistochemistry; Neoplasm Proteins; Ovarian Neoplasms; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; Subcellular Fractions; Treatment Outcome
PubMed: 10584879
DOI: 10.1038/sj.bjc.6690826 -
Nucleic Acids Research Jun 2018Overexpression of the flap endonuclease FEN1 has been observed in a variety of cancer types and is a marker for poor prognosis. To better understand the cellular...
Overexpression of the flap endonuclease FEN1 has been observed in a variety of cancer types and is a marker for poor prognosis. To better understand the cellular consequences of FEN1 overexpression we utilized a model of its Saccharomyces cerevisiae homolog, RAD27. In this system, we discovered that flap endonuclease overexpression impedes replication fork progression and leads to an accumulation of cells in mid-S phase. This was accompanied by increased phosphorylation of the checkpoint kinase Rad53 and histone H2A-S129. RAD27 overexpressing cells were hypersensitive to treatment with DNA damaging agents, and defective in ubiquitinating the replication clamp proliferating cell nuclear antigen (PCNA) at lysine 164. These effects were reversed when the interaction between overexpressed Rad27 and PCNA was ablated, suggesting that the observed phenotypes were linked to problems in DNA replication. RAD27 overexpressing cells also exhibited an unexpected dependence on the SUMO ligases SIZ1 and MMS21 for viability. Importantly, we found that overexpression of FEN1 in human cells also led to phosphorylation of CHK1, CHK2, RPA32 and histone H2AX, all markers of genome instability. Our data indicate that flap endonuclease overexpression is a driver of genome instability in yeast and human cells that impairs DNA replication in a manner dependent on its interaction with PCNA.
Topics: Cell Line, Tumor; DNA Damage; Flap Endonucleases; Genomic Instability; HEK293 Cells; Humans; Lung Neoplasms; Proliferating Cell Nuclear Antigen; Saccharomyces cerevisiae Proteins; Small Cell Lung Carcinoma; Sumoylation; Ubiquitination
PubMed: 29741650
DOI: 10.1093/nar/gky313 -
The Journal of Cell Biology Jun 2011High expression of the protein kinase Bub1 has been observed in a variety of human tumors and often correlates with poor clinical prognosis, but its molecular and...
High expression of the protein kinase Bub1 has been observed in a variety of human tumors and often correlates with poor clinical prognosis, but its molecular and cellular consequences and role in tumorigenesis are unknown. Here, we demonstrate that overexpression of Bub1 in mice leads to near-diploid aneuploidies and tumor formation. We found that chromosome misalignment and lagging are the primary mitotic errors responsible for the observed aneuploidization. High Bub1 levels resulted in aberrant Bub1 kinase activity and hyperactivation of Aurora B kinase. When Aurora B activity is suppressed, pharmacologically or via BubR1 overexpression, chromosome segregation errors caused by Bub1 overexpression are largely corrected. Importantly, Bub1 transgenic mice overexpressing Bub1 developed various kinds of spontaneous tumors and showed accelerated Myc-induced lymphomagenesis. Our results establish that Bub1 has oncogenic properties and suggest that Aurora B is a critical target through which overexpressed Bub1 drives aneuploidization and tumorigenesis.
Topics: Aneuploidy; Animals; Aurora Kinase B; Aurora Kinases; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Chromosome Segregation; Fibroblasts; HeLa Cells; Humans; Mice; Mice, Transgenic; Neoplasms; Protein Serine-Threonine Kinases; Survival Rate; Tissue Distribution
PubMed: 21646403
DOI: 10.1083/jcb.201012035 -
The Journal of Biological Chemistry May 1997Cell adhesion to substratum has been shown to regulate cyclin A expression as well as cyclin D- and E-dependent kinases, the latter via the up-regulation of cyclin D1...
Cell adhesion to substratum has been shown to regulate cyclin A expression as well as cyclin D- and E-dependent kinases, the latter via the up-regulation of cyclin D1 and the down-regulation of cyclin-Cdk inhibitors p21 and p27, respectively. This adhesion-dependent regulation of cell cycle is thought to be mediated by integrins. Here we demonstrate that stable transfection and overexpression of the integrin-linked kinase (ILK), which interacts with the beta1 and beta3 integrin cytoplasmic domains, induces anchorage-independent cell cycle progression but not serum-independent growth of rat intestinal epithelial cells (IEC18). ILK overexpression results in increased expression of cyclin D1, activation of Cdk4 and cyclin E-associated kinases, and hyperphosphorylation of the retinoblastoma protein. In addition, ILK overexpression results in the expression of p21 and p27 Cdk inhibitors with altered electrophoretic mobilities, with the p27 from ILK-overexpressing cells having reduced inhibitory activity. The transfer of serum-exposed IEC18 cells from adherent cultures to suspension cultures results in a rapid down-regulation of expression of cyclin D1 and cyclin A proteins as well as in retinoblastoma protein dephosphorylation. In marked contrast, transfer of ILK-overexpressing cells from adherent to suspension cultures results in continued high levels of expression of cyclin D1 and cyclin A proteins, and a substantial proportion of the retinoblastoma protein remains in a hyperphosphorylated state. These results indicate that, when overexpressed, ILK induces signaling pathways resulting in the stimulation of G1/S cyclin-Cdk activities, which are normally regulated by cell adhesion and integrin engagement.
Topics: Animals; CDC2-CDC28 Kinases; Cell Adhesion; Cell Cycle; Cell Cycle Proteins; Cell Line; Cyclin D1; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Cyclins; Enzyme Inhibitors; G1 Phase; Microtubule-Associated Proteins; Oncogene Proteins; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Proteins; Proto-Oncogene Proteins; Rats; S Phase; Tumor Suppressor Proteins; Up-Regulation
PubMed: 9153256
DOI: 10.1074/jbc.272.21.13937 -
The Journal of Biological Chemistry Jul 2006The mitochondrial genome of trypanosomes, termed kinetoplast DNA (kDNA), contains thousands of minicircles and dozens of maxicircles topologically interlocked in a...
The mitochondrial genome of trypanosomes, termed kinetoplast DNA (kDNA), contains thousands of minicircles and dozens of maxicircles topologically interlocked in a network. To identify proteins involved in network replication, we screened an inducible RNA interference-based genomic library for cells that lose kinetoplast DNA. In one cloned cell line with inducible kinetoplast DNA loss, we found that the RNA interference vector had aberrantly integrated into the genome resulting in overexpression of genes down-stream of the integration site (Motyka, S. A., Zhao, Z., Gull, K., and Englund, P. T. (2004) Mol. Biochem. Parasitol. 134, 163-167). We now report that the relevant overexpressed gene encodes a mitochondrial cytochrome b(5) reductase-like protein. This overexpression caused kDNA loss by oxidation/inactivation of the universal minicircle sequence-binding protein, which normally binds the minicircle replication origin and triggers replication. The rapid loss of maxicircles suggests that the universal minicircle sequence-binding protein might also control maxicircle replication. Several lines of evidence indicate that the cytochrome b(5) reductase-like protein controls the oxidization status of the universal minicircle sequence-binding protein via tryparedoxin, a mitochondrial redox protein. For example, overexpression of mitochondrial tryparedoxin peroxidase, which utilizes tryparedoxin, also caused oxidation of the universal minicircle sequence-binding protein and kDNA loss. Furthermore, the growth defect caused by overexpression of cytochrome b(5) reductase-like protein could be partially rescued by simultaneously overexpressing tryparedoxin.
Topics: Animals; Cytochrome-B(5) Reductase; DNA Replication; DNA, Kinetoplast; Gene Expression Regulation; Oxidation-Reduction; Protozoan Proteins; Thioredoxins; Trypanosoma brucei brucei
PubMed: 16690608
DOI: 10.1074/jbc.M602880200