Expression dynamics of periodic transcripts during cancer cell cycle progression and their correlation with anticancer drug sensitivity

Background:The cell cycle is at the center of cellular activities and is orchestrated by complex regulatory mechanisms,among which transcriptional regulation is one of the most important components.Alternative splicing dramatically expands the regulatory network by producing transcript isoforms of genes to exquisitely control the cell cycle.However,the patterns of transcript isoform expression in the cell cycle are unclear.Therapies targeting cell cycle checkpoints are commonly used as anticancer therapies,but none of them have been designed or evaluated at the alternative splicing transcript level.The utility of these transcripts as markers of cell cycle-related drug sensitivity is still unknown,and studies on the expression patterns of cell cycle-targeting drug-related transcripts are also rare.Methods:To explore alternative splicing patterns during cell cycle progression,we performed sequential transcriptomic assays following cell cycle synchronization in colon cancer HCT116 and breast cancer MDA-MB-231 cell lines,using flow cytometry and reference cell cycle transcripts to confirm the cell cycle phases of samples,and we developed a new algorithm to describe the periodic patterns of transcripts fluctuating during the cell cycle.Genomics of Drug Sensitivity in Cancer(GDSC)drug sensitivity datasets and Cancer Cell Line Encyclopedia(CCLE)transcript datasets were used to assess the correlation of genes and their transcript isoforms with drug sensitivity.We identified transcripts associated with typical drugs targeting cell cycle by determining correlation coefficients.Cytotoxicity assays were used to confirm the effect of ENST00000257904 against cyclin dependent kinase 4/6(CDK4/6)inhibitors.Finally,alternative splicing transcripts associated with mitotic(M)phase arrest were analyzed using an RNA synthesis inhibition assay and transcriptome analysis.Results:We established high-resolution transcriptome datasets of synchronized cell cycle samples from colon cancer HCT116 and breast cancer MDA-MB-231 cells.The results of the cell cycle assessment showed that 43,326,41,578 and 29,244 transcripts were found to be periodically expressed in HeLa,HCT116 and MDA-MB-231 cells,respectively,among which 1280 transcripts showed this expression pattern in all three cancer cell lines.Drug sensitivity assessments showed that a large number of these transcripts displayed a higher correlation with drug sensitivity than their corresponding genes.Cell cycle-related drug screening showed that the level of the CDK4 transcript ENST00000547281 was more significantly associated with the resistance of cells to CDK4/6 inhibitors than the level of the CDK4 reference transcript ENST00000257904.The transcriptional inhibition assay following M phase arrest further confirmed the M-phase-specific expression of the splicing transcripts.Combined with the cell cycle-related drug screening,the results also showed that a set of periodic transcripts,for example,ENST00000314392(a dolichylphosphate mannosyltransferase polypeptide 2 isoform transcript),was more associated with drug sensitivity than the levels of their corresponding gene transcripts.Conclusions:In summary,we identified a panel of cell cycle-related periodic transcripts and found that the levels of transcripts of drug target genes showed different values for predicting drug sensitivity,providing novel insights into alternative splicing-related drug development and evaluation.

Regulation of the G1 phase of the mammalian cell cycle

In any multi-cellular organism, the balance between cell division and cell death maintains a constant cell number. Both cell division cycle and cell death are highly regulated events. Whether the cell will proceed through the cycle or not, depends upon whether the conditions required at the checkpoints during the cycle are fulfilled. In higher eucaryotic cells, such as mammalian cells, signals that arrest the cycle usually act at a G1 checkpoint. Cells that pass this restriction point are committed to complete the cycle. Regulation of the GI phase of the cell cycle is extremely complex and involves many different families of proteins such as retinoblastoma family cyclin dependent kinases, cyclins, and cyclic kinase inhibitors.

Expressions of cyclin E, cyclin dependent kinase 2 and p57^(KIP2) in human gastric cancer

Objective To investigate the expressions of cyclin E, cyclin dependent kinase 2(CDK-2)and cyclin-dependent kinase inhibitor p57 KIP2 in human gastric cancer, and to evaluate the relationships between protein levels and clinicopathological parameters. Methods Western blot was used to measure the expressions of cyclin E, CDK-2 and p57 KIP2 proteins in the surgically resected gastric carcinoma, adjacent normal mucosa and metastatic lymph nodes from 36 patients. Results Cyclin E and CDK-2 protein levels were higher in gastric cancer tissues in comparison with normal tissues (P<0.05). Overexpression of cyclin E was correlated with lymph node involvement, poor histological grade and serosa invasion (P<0.05). Overexpression of CDK-2 was correlated with lymph nodes involvement (P<0.05). No statistically significant difference between cyclin E and CDK-2 expression was found when samples were stratified according to tumor size (P>0.05). Expression of cyclin E and CDK-2 showed a positive linear correlation (r=0.451, P=0.01). Protein levels of p57 KIP2 were lower in gastric cancer tissues than in the normal mucosa (P<0.05). Decreased expression of p57 KIP2 was correlated with lymph node involvement (P<0.05). No statistically significant difference in p57 KIP2 expression was found when sample were stratified according to tumor size, histological grade or serosa invasion (P>0.05). In metastatic lymph nodes, expression of cyclin E was increased and the expression of p57 KIP2 decreased. Conclusion Overexpressions of cyclin E, CDK-2 and downregulated expression of p57 KIP2 may play important roles in tumorigenesis and metastatic potential of gastric cancer.

Failure to Inactivate CDK Activity Is Responsible for the Enhanced Apoptotic Response in U937 Cells Mediated by Silencing ATM Gene

Silencing ATM gene gave rise to enhanced apoptotic response to irradiation and i rradiation like chemotherapy agents, this paper explored the crucial identities of the molecular elements responsible for the enhanced apoptotic response in U937 cells mediated by silencing ATM gene. Two U937 cell mutants named U937 ASPI3K (ATM, negative) and U937 pZeosv2(+) (ATM, wild type) were used as a cell model system to identify the critical molecule(s) responsible for the varied apoptotic response in the absence or presence of ATM gene. Apoptosis was examined by measuring concentrations of free nucleosome in U937 cells. Western blot was employed to measure nuclear protein abundance of CDC25A, CDC25B, CDC25C, total p34cdc2, p34cdc2 (Thr 161) or p34cdc2 (Thr 14,Tyr 15). RT PCR was used to estimate CDC25 transcript levels. U937 ASPI3K exhibited an enhanced apoptotic response to lower dosage of irradiation, which could not be blocked by protein synthesis inhibitor. Protein serine threonine phosphatase inhibitor or cyclin dependent kinase (CDK) inhibitors, on the other hand, abolished the enhancement indicated that protein phosphorylation/dephosphorylation modification and CDK activity are required for the enhanced apoptotic response in the absence of ATM gene. Upon irradiation, p34cdc2 in U937 pZeosv2 (+) was maintained in an inactive state by phosphorylation on threonine 14 (Thr 14) and tyrosine 15 (Tyr 15), which was associated with a dramatic decrease of nuclear CDC25A, CDC25B and CDC25C proteins. In contrast, p34cdc2 in U937 ASPI3K maintained in an active state by dephosphorylation on threonine 14 (Thr 14) and tyrosine 15 (Tyr 15), which was associated with constant nuclear CDC25A, CDC25B and CDC25C protein abundance before and after irradiation. The responsive decrease of nuclear CDC25 proteins occurred at the post transcription level. Silencing ATM gene blocks the responsive decrease of nuclear CDC25 proteins, which is responsible for failure to inactivate p34cdc2 after irradiation. Active p34cdc2 and CDK2, in turn, acts as the death executors to trigger apoptosis. In abstract, aberrantly activated CDK activity is the critical molecular mechanism central to enhanced apoptotic responses in the absence of ATM gene.

Responses of CDKs and p53 in Delayed Ischemic Neuronal Death

Stroke is a debilitating disease that affects millions each year. While in many cases cerebral ischemic injury can be limited by effective resuscitation or thrombolytic treatment, the injured neurons wither in a process known as delayed neuronal death (DND). Mounting evidence indicates that DND is not simply necrosis played out in slow motion but apoptosis is triggered. Of particular interest are two groups of signal proteins that participate in apoptosis cyclin dependent kinases (CDKs) and p53 among a myriad of signaling events after an ischemic insult. Recent investigations have shown that CDKs, a family of enzymes initially known for their role in cell cycle regulation, are activated in injured neurons in DND. As for p53, new reports suggest that its up regulation may represent a failed attempt to rescue injured neurons, although its up regulation was previously considered an indication of apoptosis. These observations thus rekindle an old quest to identify new neuroprotective targets to minimize the stroke damage. In this review, the author will examine the evidence that indicates the participation of CDKs and p53 in DND and then introduce pre clinical data to explore CDK inhibition as a potential neuroprotective target. Finally, using CDK inhibition as an example, this paper will discuss the pertinent criteria for a viable neuroprotective strategy for ischemic injury.

Effect of Cell Cycle Inhibitor Olomoucine on Astroglial Proliferation and Scar Formation after Focal Cerebral Infarction in Rats

Background:Astrocytes become reactive following many types of CNS injuries.Excessive astrogliosis is detrimental and contributes to neuronal damage.We sought to determine whether inhibition of cell cycle could decrease the proliferation of astroglial cells and therefore reduce excessive gliosis and glial scar formation after focal ischemia.Methods:Cerebral infarction model was induced by photothrombosis method.Rats were examined using MRI,and lesion volumes were estimated on day 3 post-infarction.The expression of glial fibrillary acidic protein(GFAP) and proliferating cell nuclear antigen(PCNA) was observed by immunofluorescence staining.Protein levels for GFAP,PCNA,Cyclin A and Cyclin B1 were determined by Western blot analysis from the ischemic and sham animals sacrificed at 3,7,30 days after operation.Results:Cell cycle inhibitor olomoucine significantly suppressed GFAP and PCNA expression and reduced lesion volume after cerebral ischemia.In parallel studies,we found dense astroglial scar in boundary zone of vehicle-treated rats at 7 and 30 days.Olomoucine can markedly attenuate astroglial scar formation.Western blot analysis showed increased protein levels of GFAP,PCNA,Cyclin A and Cyclin B1 after ischemia,which was reduced by olomoucine treatment.Conclusion: Our results suggested that astroglial activation,proliferation and subsequently astroglial scar formation could be partially inhibited by regulation of cell cycle.Cell cycle modulation thereby provides a potential promising strategy to treat cerebral ischemia.

Neuronal cdc-2 like kinase in developing kindling rat hippocampus

Objective To test the hypothesis that neuronal cdc2 like kinase (Cdk5/p35 nck5a ) plays an important role in neuronal maturation and sprouting Methods Changes kinase activity, expression levels and subcellular localizations of Cdk5 and p35 nck5a in the rat hippocampus were studied during kindling progression by Western blot analysis, immunohistochemitry, immunoprecipitation and kinase assay Results Kinase activity in kindling rats was significantly higher than that in normal adult rats The kinase activity at stage 3 was most prominent among all stages of kindling progression The changes in kinase activity coincided with those of p35 nck5a expression in kindling rats In contrast, the expression of Cdk5 was constant throughout the progression of kindling stages However, subcellular localization of Cdk5 dramatically changed in the hippocampal neurons of kindling rats Cdk5 was translocated from axon to soma when kinase activity was high p35 nck5a was always localized in the soma throughout kindling progression Conclusions Neuronal cdc2 like kinase plays an important role in synaptic reorganization, and the translocation of Cdk5 to the soma from the axon may be a novel regulatory mechanism to control kinase activity

Effects of transforming growth factor beta 1 on the growth of rhabdomyosarcoma cell line RD

Backgroud Transforming growth factor-β (TGF-β) can inhibit the growth of most epithelial and endothelial cells. The growth regulative role of TGF-β on soft tissue sarcoma was seldom reported. Here we examined TGF-β1 effects on the growth of human rhabdomyosarcoma cell RD and searched the relative molecular mechanism. Methods The viability of RD was examined by [ 3H]-thymidine incorporation and [3-(4,5-dimethylthiazol-z-yl)-2,5-diphenyltetraz olium bromide] (MTT) assay. RD cell cycle was analysed by flow cytometry. The protein and mRNA of cell cycle regulative factors in RD were detected by Western blot and reverse transcription-polymerase chain reation (RT-PCR), respectively. The kinase activity of cdk2 or cdk4 was examined by immunoprecipitation and kinase assay. Immunofluorescent staining was used to detect the location of cell cycle regulative factors in RD by laser scanning confocal microscope.Results TGF-β1 inhibits RD proliferation by G1-arrest in cell cycle progression. TGF-β1 can prominently up-regulate P27 of RD, then augment P27 to bind cyclinE-cdk2 complexes, which effectively suppress cdk2 kinase activity. P21 increased and c-myc decreased in RD due to TGF-β1. Both P15 and cdk4 have not been involved in the growth inhibitory event. TGF-β1 treatment induced P27 to congregate around nucleus. P21 pervaded from nucleus to both nucleus and cytoplasm by TGF-β1 treatment.Conclusion TGF-β1 inhibits the proliferation of human rhabdomyosarcoma cell line RD and induces RD G1-arrest. This course is accomplished by TGF-β1 up-regulating P27 to suppress cdk2 kinase activity. The induction of P21 and down-regulation of C-myc might participate in the growth-arrest event.

Human stem cell model to study signal transduction and molecular regulation mechanisms in CML

Abstract:Objective To develop a primary human hematopoietic stem/progenitor cell model for chronic myeloid leukemia (CML) and study signal transduction and molecular regulation mechanisms in CML. Methods We developed a human model of p210BCR/ABL positive CML by transducing normal human umbilical cord blood CD34+ cells with a retroviral vector containing the b3a2 bcr/abl cDNA. We also examined whether this model recreated the cellular phenotype of CML by assessing cell adhesion, cell migration, cell proliferation and cell survival. Results We found that significantly more myeloid colony forming units grew from p210BCR/ABL expressing cells, adhesion of p210BCR/ABL expressing CD34+ cells to fibronectin was decreased but migration over fibronectin was enhanced compared with mock transduced CD34+ cells. In this model, we showed that the presence of p210BCR/ABL leads to elevated levels of p27kip in p210BCR/ABL expressing CD34+ cells. We also showed that multidrug resistance-1 (MDR-1) Pgp was upregulated in the p210BCR/ABL expressing cells which correlates with the expression of p210BCR/ABL. Conclusion This primary human CML model recreates most of the features of CML and provides a useful tool to study signal transduction and downstream molecular regulation drived by the p210BCR/ABL oncogene in normal CD34+ cells.

Expression of CDKI p27^(Kip1) in trophoblastic neoplasm

To evaluate the role of p27 Kip1 in tumorigenesis and the development of trophoblastic cell disease MethodsUsing immunohistochemistry, the expression of p27 protein was investgated in 10 normal chorionic villi in the first trimester of pregnancy, 15 complete hydatidiform moles (HM), 7 invasive moles (IM) and 7 choriocarcinomas (CC) Results In all cases, immunohistochemical staining localized p27 protein in the plasma Decreased expression of p27 Kip1 was observed in malignant trophoblastic neoplasms with a positive rate of 21 43%, which is significantly less than that in normal chorionic villi (80%) and in complete HM (73 33%) ( P <0 05) The positive rate of p27 Kip1 in those complete HM with large uterine size for gestational age was lower than that in those with normal or small uterus (42 86% vs 100%, P <0 05) Conclusion p27 Kip1 may be involved in the tumorigenesis of gestational trophoblastic neoplasm as a negative regulator of the cell cycle The expression level of p27 Kip1 in trophoblastic cells may be a prognostic factor for complete HM