Induced expression of human CCND1 alternative transcripts in mouse Cyl‐1 knockout fibroblasts highlights functional differences

• Published in: International journal of cancer 2005-04, Vol.114 (3), p.364-370
• Main Authors: Holley, Sarah L., Heighway, Jim, Hoban, Paul R.
• Format: Article
• Language: English
• Subjects: adherent growth; Animals; Biological and medical sciences; Carcinogenesis, carcinogens and anticarcinogens; Cell Cycle - genetics; cyclin D1; Cyclin D1 - biosynthesis; Cyclin D1 - genetics; Disease Progression; DNA - biosynthesis; Fibroblasts - physiology; General aspects; Genotype; Humans; Medical sciences; Mice; Mice, Knockout; Mitogens - pharmacology; Neoplasms - genetics; Neoplasms - physiopathology; Polymorphism, Genetic; proliferation; splicing; Transfection; Tumors
• ISSN: 0020-7136; 1097-0215
• DOI: 10.1002/ijc.20750

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/G870 genotype is associated with tumour progression and clinical outcome in a variety of cancers. Although in vitro expression of cyclin D1 transcript a (CCND1tra) has been widely investigated, few studies have examined the expression of CCND1 transcript b (CCND1trb). We have studied the effects of inducible expression of human CCND1trb in comparison with human CCND1tra in a mouse fibroblast knock‐out for cyclin D1 (MEFCyl‐1−/−). Inducible expression was in stable clones isolated from MEFCyl‐1−/− transfectants. Induction of CCND1tra 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 CCND1tra exhibited a significantly increased ability to grow in serum depleted (2% FCS) medium compared to non‐induced clones (p = 0.0004). Induced expression of CCND1trb in MEFCyl‐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 CCND1trb significantly enhanced the ability of MEFCyl‐1−/− transfectants to form colonies in soft agar, (average 30‐fold increase) compared to non‐induced clones or those induced to express CCND1tra. Our data supports the emerging view that CCND1 alternate transcripts encode proteins with differing independent biological functions. We suggest that CCND1tra encodes a protein involved in regulating mitogen responsive, anchorage‐dependent G1 progression, whereas CCND1trb modulates the ability of the cell to grow in an anchorage‐independent manner. © 2004 Wiley‐Liss, Inc.