OBJECTIVE: To investigate the value of using MDM2 amplification probe and DDIT3 dual-color, break-apart rearrangement probe fluorescence in situ hybridization (FISH) technique in the diagnosis of liposarcoma. METHODS: In the study, 62 cases of liposarcoma diagnosed in Peking University First Hospital from January 2015 to December 2019 were analysed for clinicopathological information. Of these 62 cases of liposarcoma, all were analysed for MDM2 amplification and 48 cases were analysed for DDIT3 rearrangement using a FISH technique. Our study aimed to evaluate the status of MDM2 and DDIT3 by FISH in liposarcoma and correlate it with diagnosis of different subtypes of liposarcoma. The subtypes of liposarcoma were classified according to the FISH results, combined with the relevant clinicopathological features. RESULTS: The patients aged 31-89 years (mean: 59 years) with a 1.75:1 male to female ratio. Histologically, there were 20 cases of atypical lipomatous tumour/well-differentiated liposarcoma (ALT/WDLPS), 26 cases of dedifferentiated liposarcoma (DDLPS), 13 myxoid liposarcoma (MLPS) and 3 pleomorphic liposarcoma (PLPS). Tumors with DDLPS (23/26) and WDLPS (8/20) were localized retroperitoneally, while both tumours of MLPS and PLPS were localized extra-retroperitoneally, and the difference of sites among the four subtypes of liposarcoma was statistically significant (P < 0.05). Histologically, varied mucoid matrix could be observed in the four subtypes of liposarcoma, and the difference was statistically significant (P < 0.05). MDM2 gene amplification was demonstrated in all cases of ALT/WDLPS and DDLPS (100%, 20/20 and 26/26 respectively); DDIT3 gene rearrangement was noted only in MLPS (100%, 13/13); most cases of DDLPS (96.2%, 25/26) and ALT/WDLPS (83.3%, 5/6, 6 cases selected for detection) demonstrated the picture of amplification of the DDIT3 telomeric tag. According to the instructions of DDIT3 break-apart rearrangement probe, the 5' telomere probe and 3' centromere probe spanned but did not cover the DDIT3 gene itself, on the contrary, the 5' telomere probe covered the CDK4 gene, while the DDIT3 and CDK4 gene were located adjacent to each other on chromosome, therefore, when the amplification signal appeared on the telomeric tag of the DDIT3 rearrangement probe, it indeed indicated the CDK4 gene amplification rather than the DDIT3 gene rearrangement. Then the 10 cases with DDIT3 telomeric tag amplification were selected for CDK4 and DDIT3 gene amplification probe FISH tests, and all the cases showed CDK4 gene amplification (100%, 10/10) and two of the 10 cases demonstrated co-amplification of CDK4 and DDIT3 (20%, 2/10); DDIT3 polysomy detected by DDIT3 gene rearrangement probe was found in 1 case of DDLPS and 2 cases of PLPS (66.7%, 2/3) with morphology of high-grade malignant tumour and poor prognosis. CONCLUSION Our results indicate that a diagnosis of different subtype liposarcoma could be confirmed based on the application of MDM2 and DDIT3 FISH, combined with clinicopathological findings. It is also noteworthy that atypical signals should be correctly interpreted to guide correct treatment of liposarcomas.
Male ; Female ; Humans ; In Situ Hybridization, Fluorescence/methods* ; Cyclin-Dependent Kinase 4/metabolism* ; Liposarcoma/pathology* ; Lipoma/pathology* ; Gene Amplification ; Transcription Factor CHOP/genetics* ; Proto-Oncogene Proteins c-mdm2/metabolism*

OBJECTIVE: To study the effect of knocking down fascin on cervical cancer cell proliferation and tumorigenicity in nude mice. METHODS: Cervical cancer CaSki cells were infected with a lentiviral vector carrying fascin siRNA or with a negative control lentivirus, and fascin mRNA and protein expressions in the cells were detected using qRT-PCR and Western blotting. MTT assay was used to determine the proliferation of CaSki cells with fascin knockdown. CaSki cells transfected with fascin siRNA or the control lentiviral vector and non-transfected CaSki cells were inoculated subcutaneously in nude mice, and the volume and weight of the transplanted tumor were measured; Western blotting was used to detect the expressions of proliferating cell nuclear antigen (PCNA), survivin, cyclin dependent kinase 4 (CDK4) and p21 proteins in the tumor xenograft. RESULTS: Infection with the lentiviral vector carrying fascin siRNA, but not the negative control vector, caused significant reductions in the expression levels of fascin mRNA and protein in CaSki cells ( < 0.05). Fascin knockdown resulted in significantly reduced proliferation of CaSki cells ( < 0.05). The nude mice inoculated with CaSki cells with fascin knockdown showed reduced tumor volume and weight, lowered levels of PCNA, survivin and CDK4, and increased expression of p21 protein in the tumor xenograft compared with the control mice. The negative control lentivirus did not affect the proliferation or tumorigenicity of CaSki cells in nude mice or the expression levels of PCNA, survivin, CDK4 or p21 proteins in the xenografts. CONCLUSIONS Knocking down fascin can inhibit the growth and tumorigenicity of cervical cancer cells in nude mice.
Animals ; Apoptosis ; Carrier Proteins ; genetics ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Cyclin-Dependent Kinase 4 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Female ; Gene Knockdown Techniques ; Genetic Vectors ; Humans ; Mice ; Mice, Nude ; Microfilament Proteins ; genetics ; metabolism ; Proliferating Cell Nuclear Antigen ; metabolism ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; Survivin ; metabolism ; Transfection ; Tumor Burden ; Uterine Cervical Neoplasms ; etiology ; pathology

BACKGROUND: Angiogenesis is an important process in the development of tumor. PD 0332991, a cell cycle inhibitor, can specifically inhibit CD4/6 phosphorylation and cell cycle progression. In xeongraft mice models, PD 0332991 treated mice had significantly decreased angiogenesis and vascular density compared with the control group, but the mechanism remains unknown. The purpose of this study is to investigate the role and molecular mechanism of PD 0332991 on vascular endothelial cells. METHODS: EA.hy926 cells, a kind of vascular endothelial cell, were used as the research model. The effects of PD 0332991 on the activity and proliferation of EA.hy926 cells were detected by the MTT, EdU assays. Wound-healing assays and transwell assays were used to determine the effects of PD 0332991 on the mobility of EA.hy926. The influence of PD 0332991 on cell cycle and apoptosis of endothelial cells was tested by flow cytometry, and the Western blot was applied to observe the expression of cell cycle related proteins in EA.hy926 cells treated by PD 0332991. RESULTS: PD 0332991 significantly inhibited the proliferation and mobility of EA.hy926 cells, caused cell cycle arrest and apoptosis. At the same time, PD 0332991 inhibited the expression of CDK4/6 and phosphorylation of Rb, and thus inhibited the cell cycle progression of EA.hy926 cells. CONCLUSIONS PD 0332991 can inhibit the proliferation and activity of endothelial cells and induces apoptosis.
Angiogenesis Inhibitors ; pharmacology ; Animals ; Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Cyclin-Dependent Kinase 4 ; genetics ; metabolism ; Cyclin-Dependent Kinase 6 ; genetics ; metabolism ; Endothelial Cells ; cytology ; drug effects ; metabolism ; Humans ; Lung Neoplasms ; drug therapy ; genetics ; metabolism ; physiopathology ; Mice ; Piperazines ; pharmacology ; Pyridines ; pharmacology

To evaluate the regulating effect of Buyang Huanwu decoction and its simple prescription (Naojian tablet) on CDK4/Cyclin D1 expression in hippocampus tissues of rats with cerebral ischemia, SD rats were divided into the sham-operation group, the model group, the Buyang Huanwu decoction group (ig, 3.15 g · kg⁻¹) and the simple prescription group (ig, 2.41 g · kg⁻¹). Each group was further divided into five subgroups based on time points after the administration, i. e. 1 d, 3 d, 7 d, 14 d and 28 d, respectively. CDK4/Cyclin D1 expressions of the group at different time points were examined by using immunohistochemistry and real-time qPCR. According to the results, the cerebral ischemia model group showed higher CDK4/Cyclin D1 expression than the sham-operation groups (P < 0.05), suggesting that the cell cycle signal pathway would be activated by the cerebral ischemic injury. Both Buyang Huanwu decoction and simple prescription groups showed significantly lower cyclin expression than the model group at 3 d, 7 d, 14 d, 28 d (P < 0.05), indicating both Buyang Huanwu decoction and its simple prescription could play the neuroprotective effect through the cell cycle signal pathway.
Animals ; Brain Ischemia ; drug therapy ; genetics ; metabolism ; physiopathology ; Cell Cycle ; drug effects ; Cyclin D1 ; genetics ; metabolism ; Cyclin-Dependent Kinase 4 ; genetics ; metabolism ; Drugs, Chinese Herbal ; administration & dosage ; Humans ; Male ; Rats ; Rats, Sprague-Dawley

Neural stem cells (NSCs) proliferation can be influenced by repetitive transcranial magnetic stimulation (rTMS) in vivo via microRNA-106b-25 cluster, but the underlying mechanisms are poorly understood. This study investigated the involvement of microRNA-106b-25 cluster in the proliferation of NSCs after repetitive magnetic stimulation (rMS) in vitro. NSCs were stimulated by rMS (200/400/600/800/1000 pulses per day, with 10 Hz frequency and 50% maximum machine output) over a 3-day period. NSCs proliferation was detected by using ki-67 and EdU staining. Ki-67, p21, p57, cyclinD1, cyclinE, cyclinA, cdk2, cdk4 proteins and miR-106b, miR-93, miR-25 mRNAs were detected by Western blotting and qRT-PCR, respectively. The results showed that rMS could promote NSCs proliferation in a dose-dependent manner. The proportions of ki-67+ and Edu+ cells in 1000 pulses group were 20.65% and 4.00%, respectively, significantly higher than those in control group (9.25%, 2.05%). The expression levels of miR-106b and miR-93 were significantly upregulated in 600-1000 pulses groups compared with control group (P<0.05 or 0.01 for all). The expression levels of p21 protein were decreased significantly in 800/1000 pulses groups, and those of cyclinD1, cyclinA, cyclinE, cdk2 and cdk4 were obviously increased after rMS as compared with control group (P<0.05 or 0.01 for all). In conclusion, our findings suggested that rMS enhances the NSCs proliferation in vitro in a dose-dependent manner and miR-106b/p21/cdks/cyclins pathway was involved in the process.
Animals ; Animals, Newborn ; Biomarkers ; metabolism ; Cell Proliferation ; genetics ; Cyclin-Dependent Kinase 2 ; genetics ; metabolism ; Cyclin-Dependent Kinase 4 ; genetics ; metabolism ; Cyclin-Dependent Kinase Inhibitor p21 ; genetics ; metabolism ; Cyclin-Dependent Kinase Inhibitor p57 ; genetics ; metabolism ; Cyclins ; genetics ; metabolism ; Gene Expression Regulation ; Hippocampus ; cytology ; metabolism ; Ki-67 Antigen ; genetics ; metabolism ; Magnetic Fields ; MicroRNAs ; genetics ; metabolism ; Neural Stem Cells ; cytology ; metabolism ; Primary Cell Culture ; Rats ; Rats, Sprague-Dawley ; Signal Transduction

In this study, one immortalized human normal prostatic epithelial cell line (BPH) and four human prostate cancer cell lines (LNCaP, 22Rv1, PC-3, and DU-145) were treated with Ganoderma Lucidum triterpenoids (GLT) at different doses and for different time periods. Cell viability, apoptosis, and cell cycle were analyzed using flow cytometry and chemical assays. Gene expression and binding to DNA were assessed using real-time PCR and Western blotting. It was found that GLT dose-dependently inhibited prostate cancer cell growth through induction of apoptosis and cell cycle arrest at G1 phase. GLT-induced apoptosis was due to activation of Caspases-9 and -3 and turning on the downstream apoptotic events. GLT-induced cell cycle arrest (mainly G1 arrest) was due to up-regulation of p21 expression at the early time and down-regulation of cyclin-dependent kinase 4 (CDK4) and E2F1 expression at the late time. These findings demonstrate that GLT suppresses prostate cancer cell growth by inducing growth arrest and apoptosis, which might suggest that GLT or Ganoderma Lucidum could be used as a potential therapeutic drug for prostate cancer.
Antineoplastic Agents, Phytogenic ; isolation & purification ; pharmacology ; Apoptosis ; drug effects ; Caspase 3 ; genetics ; metabolism ; Caspase 9 ; genetics ; metabolism ; Cell Line, Tumor ; Cell Survival ; drug effects ; Cyclin D1 ; genetics ; metabolism ; Cyclin-Dependent Kinase 4 ; genetics ; metabolism ; Cyclin-Dependent Kinase Inhibitor p21 ; genetics ; metabolism ; Dose-Response Relationship, Drug ; E2F1 Transcription Factor ; genetics ; metabolism ; G1 Phase Cell Cycle Checkpoints ; drug effects ; genetics ; Gene Expression Regulation, Neoplastic ; Humans ; Male ; Nucleosomes ; drug effects ; metabolism ; pathology ; Plant Extracts ; chemistry ; Prostate ; drug effects ; metabolism ; pathology ; Reishi ; chemistry ; Signal Transduction ; Triterpenes ; isolation & purification ; pharmacology

OBJECTIVETo assess the effect of small interfering RNA (siRNA)-mediated suppression of CDK6 expression on the proliferation and cell cycles of nasopharyngeal carcinoma (NPC) cells in vitro.

METHODSQRT-PCR was used to examine the differential expression of CDK6 in 30 NPC tissues and 18 normal nasopharyngeal tissues. A siRNA targeting CDK6 was transfected in NPC CNE2 cells, and MTT assay and flow cytometry were used to analyze the changes in cell proliferation and cell cycle distribution. Western blotting was used to examine the expressions of the cell cycle-related factors.

RESULTSCompared with normal nasopharyngeal tissues, NPC tissues showed an increased expression of CDK6 mRNA. Knocking down CDK6 expression obviously inhibited tumor cell growth and cell cycle transition from G1 to S phase and caused reduced expressions of CDK4, CCND1, and E2F1 and enhanced expression of the tumor suppressor p21.

CONCLUSIONNPC tissues overexpress CDK6. Knocking down CDK6 expression inhibits the growth and cell cycle transition of NPC cells in vitro by inhibiting the expressions of CDK4, CCND1, and E2F1 and upregulating tumor suppressor p21 expression.

Carcinoma ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Cyclin D1 ; metabolism ; Cyclin-Dependent Kinase 4 ; metabolism ; Cyclin-Dependent Kinase 6 ; genetics ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; E2F1 Transcription Factor ; metabolism ; Gene Expression Regulation, Neoplastic ; Gene Knockdown Techniques ; Humans ; Nasopharyngeal Neoplasms ; pathology ; RNA, Messenger ; RNA, Small Interfering ; Transfection ; Up-Regulation

Fucoidan is an active component of seaweed, which inhibits proliferation and induces apoptosis of several tumor cells while the detailed mechanisms underlying this process are still not clear. In this study, the effect of Fucoidan on the proliferation and apoptosis of human breast cancer MCF-7 cells and the molecular mechanism of Fucoidan action were investigated. Viable cell number of MCF-7 cells was decreased by Fucoidan treatment in a dose-dependent manner as measured by MTT assay. Fucoidan treatment resulted in G1 phase arrest of MCF-7 cells as revealed by flow cytometry, which was associated with the decrease in the gene expression of cyclin D1 and CDK-4. Annexin V/PI staining results showed that the number of apoptotic cells was associated with regulation of cytochrome C, caspase-8, Bax and Bcl-2 at transcriptional and translational levels. Both morphologic observation and Hoechst 33258 assay results confirmed the pro-apoptotic effect of Fucoidan. Meanwhile, the ROS production was also increased by Fucoidan treatment, which suggested that Fucoidan induced oxidative damage in MCF-7 cells. The results of present study demonstrated that Fucoidan could induce G1 phase arrest and apoptosis in MCF-7 cells through regulating the cell cycle and apoptosis-related genes or proteins expression, and ROS generation is also involved in these processes.
Antineoplastic Agents ; chemistry ; pharmacology ; Apoptosis ; drug effects ; genetics ; Blotting, Western ; Breast Neoplasms ; genetics ; metabolism ; pathology ; Caspase 8 ; genetics ; metabolism ; Caspases ; genetics ; metabolism ; Cell Proliferation ; drug effects ; Cell Size ; drug effects ; Cyclin D1 ; genetics ; metabolism ; Cyclin-Dependent Kinase 4 ; genetics ; metabolism ; Cytochromes c ; genetics ; metabolism ; Dose-Response Relationship, Drug ; Fucus ; chemistry ; G1 Phase Cell Cycle Checkpoints ; drug effects ; genetics ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; MCF-7 Cells ; Microscopy, Fluorescence ; Molecular Structure ; Polysaccharides ; chemistry ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; metabolism ; Reactive Oxygen Species ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; drug effects ; bcl-2-Associated X Protein ; genetics ; metabolism

OBJECTIVEUsing an adenoviral vector, the wild-type PTEN gene was transduced into activated hepatic stellate cell (HSC) cultured in vitro and cell cycle markers and were detect. Thereby, the potential mechanisms of inhibitory effect of the wild-type PTEN overexpression on the proliferation in activated HSC was investigated.

METHODSThe wild type PTEN gene was transduced into activated HSC (HSC-T6 ) cultured in vitro mediated by adenoviral vector. PTEN expression in HSC was measured by Western blot and Real-time fluorescent quantitation PCR. Flow cytometry (FCM) was then used to detect cell cycle phase of activated HSC. And the expressions of cyclinD1 and cyclin dependent kinase 4 (CDK4) in HSC were determined by Western blot.

RESULTSThe data showed that exogenous wild type PTEN gene was successfully transduced and expressed in activated HSC cultured in vitro. The over-expression of wild type PTEN resulted in the increased number of HSC at G0/G1 phase ( P less than 0.01), and the number of HSC at S phase and G2/M phase were decreased significantly, P less than 0.01. Furthermore, there were decreased cyclinD1 and CDK4 expression in HSC infected with Ad-PTEN, P less than 0.01.

CONCLUSIONThe over-expression of wild type PTEN inhibit transition of activated HSC in vitro from G1 to S phase and arrest cell cycle of them at G0/G1 phase via the down-regulated expressions of cyclinD1 and CDK4, and then inhibit HSC proliferation.

Adenoviridae ; genetics ; Animals ; Cell Cycle ; Cell Line ; Cell Proliferation ; drug effects ; Cyclin D1 ; metabolism ; Cyclin-Dependent Kinase 4 ; metabolism ; Genetic Vectors ; Hepatic Stellate Cells ; metabolism ; PTEN Phosphohydrolase ; pharmacology ; Rats ; Transfection

OBJECTIVETo investigate the effect of EBV immediate-early protein Zta on cell cycle of Daudi cells and the involved mechanisms.

METHODSThe expression vector encoding Zta was constructed and electroporated into Daudi cells. Flow cytometric analysis was used to detect the cell cycle, Western blot to the protein levels of p21, Rb and E2F-1.

RESULTSThe vector was constructed successfully, the expression of Zta protein inhibited the proliferation of Daudi cells and promoted cell cycle from G(0)/G(1) phase \[(30.0 ± 3.4)%\] to S phase \[(47.7 ± 1.1)%\]. Meanwhile, Rb expression was significantly downregulated, E2F-1 and p21 expression upregulated by Zta.

CONCLUSIONZta could promote G(0)/G(1) phase to S phase transition in Daudi cells, which might be associated with the reduced expression of Rb and increased expression of E2F-1 and p21 protein.

Cell Cycle ; genetics ; Cell Division ; Cell Line, Tumor ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; E2F1 Transcription Factor ; metabolism ; Genetic Vectors ; Herpesvirus 4, Human ; genetics ; Humans ; Immediate-Early Proteins ; genetics ; Retinoblastoma Protein ; metabolism ; Trans-Activators ; genetics ; Transcriptional Activation ; Viral Proteins ; genetics