Cell lines were authenticated by 16-locus short tandem repeat profiling and were confirmed to be free of species by CellBank Australia (Childrens Medical Research Institute, Westmead, New South Wales, Australia). Transient overexpression of minigenes and plasmids pIRES-hTERT Intron 4 Major (G) and pIRES-hTERT Intron 4 Minor (A) were transiently transfected into HT1080, MCF7, A2780 and Fre-71s-1 cells using siPORT NeoFX Transfection Agent (Life Technologies) and harvested after 24 hours for RT-PCR analysis. For all other overexpression experiments, plasmid constructs were transfected into HEK293T, HT1080, MCF7 and GM847 cells using Fugene 6 Transfection Reagent (Promega) and harvested after 24C72 hours. GUID:?2FA25488-8471-4929-BFFB-5AD49D651E9E S3 Fig: The minor allele at rs10069690 confers defective telomere lengthening in MCF7 cells. (A) Schematics of hTERT intron 4 minigene constructs with each allele at rs10069690 and the potential proteins produced. (B) Growth curve analysis of MCF7 cells stably transfected with the minigene constructs. (C) Quantification of growth rates of each cell line calculated Gefitinib-based PROTAC 3 from time points every 2 to 3 3 days over 100 days (mean SEM; calculated by two-tailed Students test; *p0.05). (D) RT-PCR analysis of FL-hTERT and INS1b levels in the stably-transfected lines over a range of population doublings. (E) Terminal restriction fragment (TRF) analysis of stable MCF7 cell cultures at increasing population doublings and the parental cell line.(TIF) pgen.1005286.s003.tif (1.1M) GUID:?2CD92631-25B6-4A38-A446-1A2D67FBAAB0 S4 Fig: Full-length hTERT and INS1b do not affect the Wnt signaling pathway. PCR array analysis of 84 human Wnt pathway genes in MCF7 cells transfected with hTERT intron 4 minigene and INS1b overexpression constructs for 48 hours. Results are plotted as a scatter plot where each point represents a gene; the x-axis is the empty vector control transcript levels and the y-axis is (A) the hTERT Intron 4 Major G allele, (B) the hTERT Intron 4 Minor A allele, and (C) the INS1b transfected sample transcript levels. Both axes are in logarithmic scale (n = 3).(TIF) pgen.1005286.s004.tif (267K) GUID:?0D614230-2E81-42ED-B112-EF9006E68B9D S1 Table: Genotypes of cell lines at rs10069690 and rs2242652. (PDF) pgen.1005286.s005.pdf (40K) GUID:?C48AC5D0-5343-4A42-8A7A-9FD9217DCC0A Data Availability StatementAll relevant data are within the paper Gefitinib-based PROTAC 3 and its Supporting Information files. Abstract The region of chromosome 5p15.33 is a multi-cancer susceptibility locus that encodes the reverse transcriptase subunit, hTERT, of the telomerase enzyme. Numerous cancer-associated single-nucleotide polymorphisms (SNPs), including rs10069690, have been identified within the hTERT gene. The minor allele (A) at rs10069690 creates an additional splice donor site in intron 4 of hTERT, and is associated with an elevated risk of multiple cancers including Rabbit Polyclonal to VEGFR1 (phospho-Tyr1048) breast and ovarian carcinomas. We previously demonstrated that the presence of this allele resulted in co-production of full length (FL)-hTERT and an alternatively spliced, INS1b, transcript. INS1b does not encode the reverse transcriptase domain required for telomerase enzyme activity, but we show here that INS1b protein retains its ability to bind to the telomerase RNA subunit, hTR. We also show that INS1b expression results in decreased telomerase activity, telomere shortening, and an increased telomere-specific DNA damage response (DDR). We employed antisense oligonucleotides to manipulate endogenous transcript expression in favor of INS1b, which resulted in a decrease in telomerase activity. These data provide the first detailed mechanistic insights into a cancer risk-associated SNP in the locus, which causes cell type-specific expression of INS1b transcript from the presence Gefitinib-based PROTAC 3 of an additional alternative splice site created in intron 4 by the risk allele. We predict that INS1b expression levels cause subtle inadequacies in telomerase-mediated telomere maintenance, resulting in an increased risk of genetic instability and therefore of tumorigenesis. Author Summary Multiple cancer-associated single nucleotide polymorphisms (SNPs) associated with risk of a wide variety of cancers have been identified in the region of 5p15.33, identifying this as a multi-cancer susceptibility locus. encodes the catalytic subunit of the enzyme telomerase, which is responsible for telomere length maintenance in the germline and in most immortalised cancer cells. To date, very little is known regarding the mechanisms by which specific SNPs predispose to cancer. In this study, we carried out detailed functional analyses on the intron 4 SNP rs10069690, which is associated with a small, but highly significant risk for many types of cancer. We show that the risk-associated minor allele of this SNP results in an hTERT mRNA splice variant, encoding a catalytically inactive protein which acts as a dominant negative inhibitor of telomerase activity and therefore decreases total telomerase activity. We propose that individuals who carry the rs10069690 minor allele have less telomerase activity in some cell types due to cell type-specific alternative splicing, which may result in slightly shorter telomeres, and hence an increased risk of genetic instability and tumorigenesis. Introduction Telomeres are nucleoprotein structures, which protect the ends of linear chromosomes from being recognized as DNA double-strand breaks . Telomeres shorten with each round of cell division due to the end-replication problem. Normal human somatic cells replicate until their telomeres diminish to a critical threshold, at which point they enter permanent cell cycle Gefitinib-based PROTAC 3 arrest and are constrained to a senescent state . Bypass of senescence due to loss of function of the p53 and pRB tumor suppressor pathways results in further telomere shortening which eventually becomes catastrophic, causing.