Induction of apoptosis and inhibition of cell growth by developmental regulator hTBX5

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Abstract

T box (Tbx) genes are a large family of transcription regulators that play critical roles in invertebrate and vertebrate development. Mutations in Tbx5 gene have been found to cause Holt–Oram syndrome (HOS) in humans. Partial dysfunction of TBX5 in mouse also causes HOS phenotype. Little is known about its molecular and cellular mechanism. Here, we report that ectopic expression of TBX5 inhibited colony formation, induced apoptosis, and decreased the growth rate of cells. The two point mutations in T domain and a truncated mutation in C-terminal found in human HOS patients produced TBX5 mutant proteins with a significantly reduction of colony suppression activity. Deletion of the DNA-binding domain, however, nearly completely abrogated its ability to suppress colony formation. These results reveal TBX5 as a new regulator of apoptosis and cell growth, suggesting a possible mechanism for Holt–Oram syndrome, and a potential reagent for controlling tumor growth.

Section snippets

Materials and methods

Plasmid constructs. To express hTBX5, hTBX5 mutatants, or the wild type SRP20 protein, cDNA tagged at the C terminus with the myc epitope followed by an internal ribosome entry site (IRES) was inserted into expression vector pCS2+. Human TBX5 cDNA was a gift from Dr. Brook [16]. The TBX5-G80R and -R237Q point mutations were generated by the two-step PCR strategy. The nucleotide sequences of all plasmid constructs were confirmed by automated sequencing on an ABI 377 sequencer.

Cell culture. U2OS

Effect of TBX5 on colony formation

To explore the potential effect of TBX5 on apoptosis and cell proliferation, we first examined the effect of TBX5 on colony formation. Colony formation assay has been extensively used to measure the growth suppression effect of a tested protein, such as tumor suppressor gene pRB and WT1 [31], [32]. Osteosarcoma U2OS cells derived from osteroblasts, which play important roles in limb development, have been used extensively for this assay [31], [32]. We constructed a TBX5-expression plasmid with

Discussion

TBX5 plays critical roles in vertebrate morphogenesis. Little is known about its specific functions in the cells. In this study, we showed that ectopic expression of TBX5 causes apoptosis and inhibits cell growth, and the intact transcription activity is essential for its functions.

The TBX5 activities on transcriptional regulation contribute to induction of apoptosis and/or inhibition of cell proliferation [30, and this study]. Mutations G80R, R237Q, and C-terminal truncation cause human

Acknowledgements

This work was supported by seed funds (to M.-L.H and H.-F. K) from the URC of the University of Hong Kong, RGC, and AoE grant from UGC of Hong Kong.

References (34)

  • V.E. Papaioannou et al.

    The T-box gene family

    Bioessays

    (1998)
  • M. Tada et al.

    T-targets: clues to understanding the functions of T-box proteins

    Dev. Growth Differ.

    (2001)
  • A. Kispert et al.

    The T protein encoded by Brachyury is a tissue-specific transcription factor

    EMBO J.

    (1995)
  • M. He et al.

    Transcription repression by Xenopus ET and its human ortholog TBX3, a gene involved in ulnar-mammary syndrome

    Proc. Natl. Acad. Sci. USA

    (1999)
  • M.E. Horb et al.

    Tbx5 is essential for heart development

    Development

    (1999)
  • C.J. Hatcher et al.

    Identification and localization of TBX5 transcription factor during human cardiac morphogenesis

    Dev. Dyn.

    (2000)
  • C.T. Basson et al.

    Mutations in human TBX5 [corrected] cause limb and cardiac malformation in Holt–Oram syndrome

    Nat. Genet.

    (1997)
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