A Kinase Anchor Proteins ; genetics ; metabolism ; Animals ; Cell Cycle Proteins ; genetics ; metabolism ; Humans ; Neoplasms ; genetics ; metabolism

Animals ; Cell Cycle Proteins/metabolism* ; Insulin Resistance/genetics* ; Liver/physiopathology* ; Mice ; Sirtuin 1/metabolism*

Failure of mitotic checkpoint machinery leads to the chromosomal missegregation and nuclear endoreduplication, thereby driving the emergence of aneuploidy and tetraploidy population. Although abnormal nuclear ploidy and the resulting impairment of mitotic checkpoint function are typical physiological event leading to human hepatocellular carcinoma, any mutational change of mitotic checkpoint regulators has not yet been discovered. Therefore, we investigated the mutation of p31(comet), a recently identified mitotic checkpoint regulator, in human hepatocellular carcinoma. Of 51 human hepatocellular carcinoma tissue and 6 cell lines tested, five samples exhibited nucleotide sequence variations dispersed on four sites within the entire coding sequence. Among these sites with sequence substitutions, three were found to be missense mutation accompanied with amino acid change but one was a silent mutation. Of these sequence substitutions, two were present in both tumor and non-tumor liver tissues, suggesting the possibility of polymorphism. The present findings indicate that p31(comet) does not have an impact on the formation of aneuploidy and tetraploidy found in human hepatocellular carcinoma.
Adaptor Proteins, Signal Transducing ; Calcium-Binding Proteins/*metabolism ; Carcinoma, Hepatocellular/genetics/*metabolism ; Carrier Proteins/*genetics/metabolism ; Cell Cycle Proteins/*genetics/*metabolism ; Cell Line, Tumor ; Humans ; Liver Neoplasms/genetics/*metabolism ; *Mutation ; Nuclear Proteins ; Polyploidy ; Repressor Proteins/*metabolism

OBJECTIVETo investigate the effect of hepatitis B virus (HBV) X protein (HBx) on host cell cycle and HBV replication using cultured primary mouse hepatocytes to gain further insights into the mechanism of HBx-mediated modulation of cell cycle.

METHODSPrimary cultured mouse hepatocytes were transfected with HBx-expressing (pHBV) or HBx-selected (pHBV triangle X) plasmids, which were generated with sequences of the HBV ayw subtype 1.2 and included the green fluorescent protein (GFP) reporter gene. The levels of cell cycle proteins (p16, cyclin D1, p21, cyclin E and cyclin A) were measured with Western blotting, and HBV DNA was analyzed by Southern blotting and real-time PCR.

RESULTSThe freshly isolated hepatocytes showed no significant differences in levels of cell cycle proteins. However, at 48 hours post-transfection, the levels of cyclin D1, p21 and cyclin E were significantly higher and the level of p16 was significantly lower in the pHBV-transfected hepatocytes than in the pHBV triangle X-transfected hepatocytes (t = 15.713, 22.897, 14.680, and -19.584, respectively, P less than 0.05). The level of cyclin A was not different between the two groups (t = 0.142, P more than 0.05). At 72 hours post-transfection, the level of HBV DNA was higher in pHBV-transfected hepatocytes (rcDNA: 3288.336+/-448.011; dslDNA: 6458.318+/-182.163; ssDNA: 2760.613+/-393.561) than in pHBV triangle X-transfected hepatocytes (rcDNA: 515.721+/-62.530; dslDNA: 2122.228+/-28.347; ssDNA: 1632.013+/-207.021) and in the blank (untransfected) control group (P less than 0.05). Real-time PCR analysis of HBV DNA copy number per cell confirmed these results, (pHBV-transfected: 987.50+/-47.80 vs. pHBV triangle X-transfected: 303.67+/-33.94; t = 20.203, P less than 0.05).

CONCLUSIONSThe HBx protein can affect the levels of cell cycle proteins, which may induce quiescent hepatocytes to enter the G1 phase of the cell cycle and stay in this phase instead of entering the S phase, thereby promoting HBV intracellular replication.

Animals ; Cell Cycle ; Cell Cycle Proteins ; genetics ; metabolism ; Cell Line ; Hepatocytes ; cytology ; virology ; Male ; Mice ; Mice, Inbred C57BL ; Plasmids ; Trans-Activators ; genetics ; metabolism ; Transfection

OBJECTIVE: To establish a stable A549 cell line transfected by RNA binding motif 5 (RBM5) expression vector, and to investigate the effect of RBM5 gene on proliferation of A549 cell line and the expression of DEAH box polypeptide 15 (DHX15). METHODS: The eukaryotic expression vector pcDNA3.1 (+)/RBM5 was constructed by a twostep PCR technique. Then, the recombinant plasmid pcDNA3.1 (+)/RBM5 was verified by DNA sequencing and transfected into the lung adenocarcinoma cell A549. The positive cells with overexpression of RBM5 gene were identified by Western blotting. Flow cytometry was used to analyze the cell cycles of the positive A549 cells [pcDNA3.1 (+)/RBM5-A549] and the negative controls [pcDNA3 .1 (+)- A549]. Finally, RT-PCR was used to detect the expression of DHX15, a splicing-related factor, in the positively transfected A549 cells and the negative controls. RESULTS: A pcDNA3.1 (+)/RBM5 eukaryotic expression vector has been constructed successfully, and the A549 cell line that stably transfected with RBM5 gene has been established. Compared with negative control cells, the percentage of G1 phase cells in the positive cells was increased, while the percentage of S phase was decreased (both P<0.01), and the expression of DHX15 is upregulated (P<0.01). CONCLUSION RBM5 gene can inhibit the cell cycle and upregulate the expression of DHX15 in A549 cells.
Cell Cycle ; Cell Cycle Proteins ; genetics ; Cell Line, Tumor ; Cell Proliferation ; DNA-Binding Proteins ; genetics ; Genetic Vectors ; Humans ; RNA Helicases ; metabolism ; RNA-Binding Proteins ; genetics ; Transfection ; Tumor Suppressor Proteins ; genetics

OBJECTIVETo investigate the effects of PLK1 gene silence by short hairpin RNA (shRNA) on PLK1 expression and apoptosis in K562 cells, and explore the role of PLK1 in the pathogenesis of leukemia.

METHODSThe shRNA fragment targeting at 1416-1436 bp of PLK1 mRNA was synthesized and cloned into pEGFP-H1 vector, named as pEGFP-H1/PLK1. The empty control, pEGFP-H1 and pEGFP-H1/PLK1 were transfected into K562 cells respectively via electroporation. 24 h or 48 h after transfection, gene and protein expression of PLK1 in the cells were assayed by RT-PCR and Western blot analysis respectively, cells viability by MTT assay, caspase-3 activity by colorimetry, cell cycle and apoptosis by FACS.

RESULTS24 and 48 h after transfection, PLK1 expression in K562 cells was 1.25 +/- 0.07 for control group, 0.52 +/- 0.04 and 0.25 +/- 0.02 for pEGFP-H1/PLK1 group, and 1.24 +/- 0.08 and 1.23 +/- 0.09 for pEGFP-H1 group respectively. The alteration status of PLK1 protein levels were similar to that of PLK mRNA levels. The apoptosis rate was (8.3 +/- 0.6)% in control group, (8.7 +/- 0.7)% in pEGFP-H1 group and (49.7 +/- 3.8)% and (82.3 +/- 6.9)% in pEGFP-H1/PKLK1 group at 24 and 48 h, respectively. In addition, cell fraction at G(2)/M phase was increased obviously compared with control and pEGFP-H1-transfected group.

CONCLUSIONThe constructed shRNA can remarkably inhibit PLK1 expression and transfected K562 cell proliferation, increase apoptosis and block cell-cycle, suggesting that PLK1 play important roles in apoptosis and cell-cycle control of leukemia cells.

Apoptosis ; genetics ; Cell Cycle ; Cell Cycle Proteins ; genetics ; metabolism ; Cell Proliferation ; Genetic Vectors ; Humans ; K562 Cells ; Protein-Serine-Threonine Kinases ; genetics ; metabolism ; Proto-Oncogene Proteins ; genetics ; metabolism ; RNA Interference ; RNA, Messenger ; genetics ; Transfection

Spindle assembly checkpoint (SAC) is a protective mechanism for cells to undergo accurate mitosis. SAC prevented chromosome segregation when kinetochores were not, or incorrectly attached to microtubules in the anaphase of mitosis, thus avoiding aneuploid chromosomes in daughter cells. Aneuploidy and altered expression of SAC component proteins are common in different cancers, including lung cancer. Therefore, SAC is a potential new target for lung cancer therapy. Five small molecule inhibitors of monopolar spindle 1 (MPS1), an upstream component protein of SAC, have entered clinical trials. This article introduces the biological functions of SAC, summarizes the abnormal expression of SAC component proteins in various cancers and the research progress of MPS1 inhibitors, and expects to provide a reference for the future development of lung cancer therapeutic strategies targeting SAC components.
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Humans ; Cell Cycle Proteins/metabolism* ; Spindle Apparatus/metabolism* ; Protein Serine-Threonine Kinases/metabolism* ; M Phase Cell Cycle Checkpoints/genetics* ; Lung Neoplasms/metabolism*

OBJECTIVETo investigate the expression of neural salient serine/arginine-rich protein 1 (NSSR1) in colorectal cancer.

METHODSRT-PCR, Western blot and immunohistochemical staining were used to detect the expression of NSSR1 mRNA and protein in different mouse tissues and human colorectal cancer, respectively.

RESULTSNSSR1 mRNA was expressed in mouse cerebrum, cerebellum, heart, liver, intestine, kidney and lung tissue, but NSSR1 protein was only expressed in neural tissues. In normal human intestinal mucosa, NSSR1 was expressed in the colorectal epithelial cells. In colorectal cancer, NSSR1 was highly expressed in the nucleus of tumor cells.

CONCLUSIONThe extensive expression of NSSR1 in colorectal cancer cells may hint it's roles in the biological function of colorectal cancer.

Animals ; Cell Cycle Proteins ; genetics ; metabolism ; Colon ; metabolism ; Colorectal Neoplasms ; metabolism ; Humans ; Mice ; Neoplasm Proteins ; genetics ; metabolism ; RNA, Messenger ; genetics ; RNA-Binding Proteins ; genetics ; metabolism ; Rectum ; metabolism ; Repressor Proteins ; genetics ; metabolism ; Serine-Arginine Splicing Factors

OBJECTIVETo construct the recombinant plasmid pcDNA3.0-RGC32 and evaluate the effect of the response gene to complement-32 (RGC32) on cell cytoskeleton in vitro.

METHODSThe full-length cDNA of RGC32 was obtained by RT-PCR and inserted into the eukaryotic expression vector pcDNA3.0 to generate the recombinant plasmid pcDNA3.0-RGC32. After transfection of the recombinant plasmid into SW480 cells, the expression of RGC32 in the cells was detected by Western blotting. The cytoskeleton of SW480 cells was visualized before and after the transfection, and the changes in the cell migration ability was assessed by wound-healing assay.

RESULTSThe recombinant plasmid pcDNA3.0-RGC32 was successfully constructed. The expression of RGC32 was significantly increased in SW480 cells after transfection with pcDNA3.0-RGC32. Before the transfection, the microfilaments of SW480 cells were few and short without obvious polarity, but after the transfection, the microfilaments were increased and elongated with also an obvious polarity, and the invasive structures of lamellae and lamellipodia occurred. The migration ability of the cells was enhanced after transfection with pcDNA3.0-RGC32.

CONCLUSIONOverexpression of RGC32 can cause the reorganization of cytoskeleton and promotes the cell migration, which can be an important mechanism of RGC32 in promoting cancer metastasis.

Cell Cycle Proteins ; biosynthesis ; genetics ; Cell Line, Tumor ; Cell Movement ; Colorectal Neoplasms ; genetics ; metabolism ; pathology ; Cytoskeleton ; chemistry ; metabolism ; Genetic Vectors ; Humans ; Muscle Proteins ; biosynthesis ; genetics ; Neoplasm Metastasis ; genetics ; Nerve Tissue Proteins ; biosynthesis ; genetics ; Plasmids ; genetics ; Recombinant Proteins ; biosynthesis ; genetics

OBJECTIVETo elucidate the effect of hSav1 expression on Mst1-mediated apoptosis in HeLa cells.

METHODSPlasmids pCMV-HA-hSav1 and pcDNA/4TO-Flag-Mst1 were constructed and cotransfected into HeLa cells. Triple immunofluorescent labeling of hSav1, Mst1 and nucleus was performed to determine their subcellular localization. Plasmids pCMV-HA-hSav1 and/or pcDNA/4TO-Flag-Mst1 were transfected into HeLa cells, and 36 hours later cisplatin (50 micromol/L) as a pro-apoptotic agent was added for 14 hours. Cell apoptosis was analyzed by annexin V/PI assay.

RESULTSPlasmids pCMV-HA-hSav1 and pcDNA/4TO-Flag-Mst1 were constructed and the authenticity of constructs was verified by sequencing. The binding in vitro showed that hSav1 could be detect from the anti-Mst1 immunoprecipitation complex. The immunofluorescent labeling showed that hSav1 and Mst1 had the same localization in cells. Overexpressed protein hSav1 did not induce a significant cell apoptosis. However, co-expression of hSav1 with Mst1 resulted in a significant increase of apoptosis above the level seen with Mst1 alone (24.5% +/- 2.4% vs. 39.3% +/- 4.0%, P < 0.05).

CONCLUSIONOur findings indicate that hSav1 is a newly identified protein that interacts with Mst1 and augments Mst1-mediated apoptosis.

Apoptosis ; Cell Cycle Proteins ; genetics ; metabolism ; Cytoplasm ; metabolism ; HeLa Cells ; Hepatocyte Growth Factor ; genetics ; metabolism ; Humans ; Plasmids ; Proto-Oncogene Proteins ; genetics ; metabolism ; Transfection