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Tuberous sclerosis complex (TSC) is a tumour suppressor gene syndrome whose manifestations include seizures, mental retardation, autism, and tumours of the brain, retina, kidney, heart, and skin. (1) Mutations in two tumour suppressor genes, TSC1 on chromosome 9q34 and TSC2 on chromosome 16p13, cause TSC. TSC2 encodes tuberin, a 190 kDa protein with homology to the catalytic domain of a GTPase activating protein (GAP) for Rapl. (2) TSC1 encodes hamartin, a 130 kDa protein. (3) Tuberin and hamartin have been shown to directly interact, both in mammalian cells (4 5) and in Drosophila. (6 7) This is consistent with the nearly identical spectrum of disease seen in humans with TSC1 and TSC2 germline mutations, and with the identical phenotypes of Drosophila TSC1 (6-8) and TSC2 (9) homologue mutants. The mouse models of TSC1 and TSC2 also have similar phenotypes; renal carcinoma and renal cysts develop in heterozygous animals of the Eker rat model of TSC2 (10 11) and in the knock out mouse models of both TSC1 (12) and TSC2, (13 14) all of which are embryonically lethal in the homozygous form.
The cellular pathways through which germline TSC1 or TSC2 mutations result in tumorigenesis are not completely understood. Mammalian tuberin and hamartin have been shown to suppress cell growth, accompanied by an increase in cells in the G1 phase of the cell cycle. (15-17) The importance of cell cycle regulation to human TSC is not known. In this study, we used a sensitive fluorescence activated cell sorting approach to investigate the cell cycle effects of wild type hamartin and tuberin, two patient derived mutant forms of tuberin, and a carboxy-terminus construct of tuberin containing the region of GTPase activating protein homology. Similar overexpression approaches using cell sorting have been used to elucidate the cell cycle effects of numerous proteins including the retinoblastoma protein and its family members, PTEN, cyclin dependent kinases, cyclin dependent kinase inhibitors, and substrates of the cyclin dependent kinases. (18-26)
Our goal was to determine the impact of transient expression of wild type tuberin and hamartin, and mutant and truncated forms of tuberin, on the cell cycle. Previously, transient expression of wild type full length tuberin was shown to increase the Gl fraction of cells (16 17) and stable expression of hamartin suppressed cell growth accompanied by an increase in G1. (15)
MATERIAL AND METHODS
We cotransfected cells with TSC1 and/or TSC2 in excess of a construct encoding the cell surface protein CD19, and sorted transfected cells by FACS using an FITC conjugated anti-CD19 antibody. Transfections were performed using calciumphosphate precipitation. The TSC1 and/or TSC2 constructs were transfected in five-fold molar excess of the expression vector encoding the cell surface protein CD19, as previously described. (27) After transfection, the cells were washed with phosphate buffered saline and cultured in fresh media until trypsinisation at 48 hours post-transfection. A construct encoding the cyclin dependent kinase inhibitor p21 (WAF1/CIP1) was used as a positive control for an increase in the G1 phase of the cell cycle. (27) A summary of the constructs used in these experiments is shown in fig lA. The C-terminus construct has been previously shown to reduce the in vitro proliferation and in vivo tumorigenicity of Eker rat renal carcinoma cells. (28) Site directed PCR based mutagenesis was performed u sing the QuickChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) to generate TSC2 mutants which were then confirmed by DNA sequencing. To generate the exon 16 (G1832A) TSC2 mutant construct, the primers were 5'-GAG CAG CAT CCA GCT GCA GGC C-3' and 5'-GGC CTG CAG CTG GAT GCT GCT C-3'. The G1832A missense change in exon 16 changes the amino acid at position 611 from arginine to glutamine (R61lQ). This is a frequent naturally occurring germline mutation in TSC patients (29) and has recently been found as a somatic mutation in tumour cells from women with the sporadic form of pulmonary lymphangiomyomatosis. (30) To generate the exon 38 …