Biochemical and Biophysical Research Communications
Induction of apoptosis and inhibition of cell growth by developmental regulator hTBX5
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.
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