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.

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