title: Induced expression of human CCND1 alternative transcripts in mouse Cyl-1 knockout fibroblasts highlights functional differences.
abstract: Splicing of human cyclin D1 (CCND1) mRNA producing transcripts a and b is modulated by a common polymorphism (A --> G) located in a conserved splice donor region at nucleotide 870. CCND1 A/G(870) genotype is associated with tumour progression and clinical outcome in a variety of cancers. Although in vitro expression of cyclin D1 transcript a (CCND1(tra)) has been widely investigated, few studies have examined the expression of CCND1 transcript b (CCND1(trb)). We have studied the effects of inducible expression of human CCND1(trb) in comparison with human CCND1(tra) in a mouse fibroblast knock-out for cyclin D1 (MEF(Cyl-1-/-)). Inducible expression was in stable clones isolated from MEF(Cyl-1-/-) transfectants. Induction of CCND1(tra) produced a 36-kDa protein, which led to a significant increase in the proportion of cells in S-phase, as detected by BrdU incorporation after 32 hr, compared to non-induced cells (p = 0.012). Clones induced to express CCND1(tra) exhibited a significantly increased ability to grow in serum depleted (2% FCS) medium compared to non-induced clones (p = 0.0004). Induced expression of CCND1(trb) in MEF(Cyl-1-/-) transfectants produced a 31-kDa protein and resulted in no significant difference in DNA synthesis, neither did the cells acquire the ability to grow in serum-depleted conditions compared to non-induced cells. Induction of CCND1(trb) significantly enhanced the ability of MEF(Cyl-1-/-) transfectants to form colonies in soft agar, (average 30-fold increase) compared to non-induced clones or those induced to express CCND1(tra). Our data supports the emerging view that CCND1 alternate transcripts encode proteins with differing independent biological functions. We suggest that CCND1(tra) encodes a protein involved in regulating mitogen responsive, anchorage-dependent G(1) progression, whereas CCND1(trb) modulates the ability of the cell to grow in an anchorage-independent manner.
authors.affiliation: Human Genomics Research Group, Institute for Science and Technology in Medicine, Keele University School of Medicine, University Hospital of North Staffordshire, Stoke-on-Trent, United Kingdom.
authors.forename: Sarah L
authors.initials: SL
authors.lastname: Holley
authors.forename: Jim
authors.initials: J
authors.lastname: Heighway
authors.forename: Paul R
authors.initials: PR
authors.lastname: Hoban
mesh_terms: D000818:Animals; D002453:Cell Cycle / Q000235:genetics*; D019938:Cyclin D1 / Q000096:biosynthesis* / Q000235:genetics; D004247:DNA / Q000096:biosynthesis; D018450:Disease Progression; D005347:Fibroblasts / Q000502:physiology; D005838:Genotype; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D008934:Mitogens / Q000494:pharmacology; D009369:Neoplasms / Q000235:genetics / Q000503:physiopathology; D011110:Polymorphism, Genetic; D014162:Transfection
chemical_list: D008934:Mitogens; D019938:Cyclin D1; D004247:DNA