Project description:In our study, we characterized the changes in poly-A gene expression atributed to the process of spontaneous immortalization using a mouse embryonic fibroblasts (MEF) model. Primary MEFs were established, serially passaged and spontaneously immortalized using a modified 3T3 NIH protocol. We then performed gene expression profiling analysis using RNA-seq data of 3 different clones at two time points (primary and immortalized).
Project description:Human papillomavirus (HPV) induced immortalization of human foreskin keratinocytes (HFK) is a two-step process, including 1) the bypass of replicative senescence and acquisition of an extended lifespan, and 2) the outgrowth of immortal cells. Our previous study showed that the immortalization capacity of HPV is type dependent, as reflected by the presence or absence of a crisis period before reaching immortality. In the present study we determined how the HPV-type specific immortalization capacity relates to DNA damage induction and overall genomic instability. Early passage HFKs transduced with HPV types 16, 18, 31, 33, 35, 45, 51, 59, 66 and 70 showed an increased number of double strand DNA breaks compared to controls, without significant differences between the various HPV-types. However, immortal descendants of HPV-transduced HFKs that underwent a crisis period (HPV45-, 51-, 59-, 66- and 70-transduced HFKs) showed significantly more chromosomal aberrations compared to those without a crisis period (HPV16-, 18-, 31-, and 35-transduced HFKs) (p<0.01). In particular, regions on chromosome 5p, 8, and 9q were significantly more frequently altered in cells with crisis. Interestingly, the hTERT locus at 5p was exclusively gained in cell lines with crisis. Chromothripsis was detected in one of the HPV16-immortalized cell lines in which multiple rearrangements within chromosome 8 resulted in a gain of c-MYC. In conclusion, the present study shows that upon HPV-induced immortalization, the number of chromosomal aberrations is inversely related to the immortalization capacity of the virus type. This suggests that hrHPV types with reduced immortalization capacity in vitro, as reflected by a crisis period, require more genetic host cell aberrations to trigger immortalization.
Project description:Human papillomavirus (HPV) induced immortalization of human foreskin keratinocytes (HFK) is a two-step process, including 1) the bypass of replicative senescence and acquisition of an extended lifespan, and 2) the outgrowth of immortal cells. Our previous study showed that the immortalization capacity of HPV is type dependent, as reflected by the presence or absence of a crisis period before reaching immortality. In the present study we determined how the HPV-type specific immortalization capacity relates to DNA damage induction and overall genomic instability. Early passage HFKs transduced with HPV types 16, 18, 31, 33, 35, 45, 51, 59, 66 and 70 showed an increased number of double strand DNA breaks compared to controls, without significant differences between the various HPV-types. However, immortal descendants of HPV-transduced HFKs that underwent a crisis period (HPV45-, 51-, 59-, 66- and 70-transduced HFKs) showed significantly more chromosomal aberrations compared to those without a crisis period (HPV16-, 18-, 31-, and 35-transduced HFKs) (p<0.01). In particular, regions on chromosome 5p, 8, and 9q were significantly more frequently altered in cells with crisis. Interestingly, the hTERT locus at 5p was exclusively gained in cell lines with crisis. Chromothripsis was detected in one of the HPV16-immortalized cell lines in which multiple rearrangements within chromosome 8 resulted in a gain of c-MYC. In conclusion, the present study shows that upon HPV-induced immortalization, the number of chromosomal aberrations is inversely related to the immortalization capacity of the virus type. This suggests that hrHPV types with reduced immortalization capacity in vitro, as reflected by a crisis period, require more genetic host cell aberrations to trigger immortalization. DNA copy number analysis of human keratinocytes transformed by high-risk HPV
Project description:Lentivirus containing simian virus 40 large T antigen (SV40T) is routinely used to induce cell immortalization. However, the roles of viral integration itself in this progress is still controversial. Here, we transformed primary mouse embryonic fibroblasts (MEFs) with SV40T lentivirus and studied the roles of viral integration in the immortalization using RNA-seq and whole genome sequencing (WGS). During the immortalization, differentially expressed genes (DGEs) are enriched in viral infection and several diverse activities. However, DEGs between immortalized and aging cells are significantly enriched in DNA/chromosome- and extracellular matrix (ECM)-associated activities. Gene regulatory network (GRN) analysis shows that although p53 is a key regulatory factor, many other transcription factors also play critical roles in the process, like STAT1. Of these DEGs, 32 genes have viral integration in their coding and/or regulatory regions. Our findings suggest that viral integration may promote SV40T-mediated immortalization by disturbing the expression of DNA/chromosome- and ECM-associated genes.
Project description:Progression to malignancy requires cells to overcome senescence and switch to an immortal phenotype. Thus, exploring the genetic and epigenetic changes that occur during senescence/immortalization may help elucidate crucial events that lead to cell transformation. In the present study, we have globally profiled DNA methylation in relation to gene expression in primary, senescent and immortalized mouse embryonic fibroblasts.
Project description:Gene expression profile of p53 knockdown MSCs or p53 knockdown+TERT MSCs was compared with that of control MSCs. Our data show p53 knockdown prolongs the lifespan of MSCs, and a combination of p53 knockdown and TERT overexpression is sufficient to immortalize MSCs. The results provide important information about the molecular basis underlying p53 knockdown in MSCs and immortalization-related genes of MSCs.
Project description:Progression to malignancy requires cells to overcome senescence and switch to an immortal phenotype. Thus, exploring the genetic and epigenetic changes that occur during senescence/immortalization may help elucidate crucial events that lead to cell transformation. In the present study, we have globally profiled DNA methylation in relation to gene expression in primary, senescent and immortalized mouse embryonic fibroblasts. Primary, senescent and immortalized mouse fibroblasts obtained from three independent embryos. Compare immortalized versus primary and senescent versus primary cells.
Project description:To characterize the sequence of events associated with RasV12 immortalization of Drosophila embryonic cells, we generated transcriptional time series during cell line establishment, from primary cultures until passage (P) 19. We generated two transcriptional time series from two cell lines (R3 and R7) by sampling the cultures at successive stages, early (P2-4), intermediate (P4-11), and late (P16-19), characterized by different passage times. Time points for the R3 time-series were: P2, P2 (replicate), P5, P6, P7, P8, P16, P17 and P19; for the R7 time-series: P2, P2 (replicate), P3, P4, P7, P8, P16, P17 and P19.