Functional analysis of hamartin by use of Tscl knockout mice.

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To elucidate the function of tuberous sclerosis 1 (Tsc1) gene product (hamartin) in vivo, we generated a line of Tsci knockout mouse and compared its phenotypes with those of tuberous sclerosis 2 (Tsc2) gene knockout mice. Heterozvgous Tsc1 mutant mice developed renal tumors by 1.5 year of age. Most of these tumors were cyst. adenomas. Hepatic hemangiomas were also developed with high frequency. Some mice developed uterus leiomyosarcomas and tail hemangiomas. In these renal and extra-renalltumors, loss of wild type Tsc1 allele was detected, suggesting that 2-hit of Tsc1 was important step for tumor development. Homozygous Tsc1 mutants died at around embryonic day 10.5 and were frequently associated with neural tube unciosure. These phenotypes were similar to those of Tsc2 knockout mice, suggesting the functional interaction of these two gene products in vivo. However, the development of renal tumors in Tsc1 knockout mice was apparently slower than that of Tsc2 knockout mice. This suggests that mechanism of tumorigenesis associated with Tsc1 mutation may somewhat differ from that associated with Tsc2 mutation. Next, as an in vitro model system for functional analysis of hamartin, we established cell lines from a renal tumor from a Tsc1 knockout mouse. Established cell lines, CACL1s, exhibited epithelial phenotypes and showed the loss of wild-type Tsc1 allele. By western blot analysis using anti-mouse hamartin antibody, loss of hamartin expression was also confirmed. The Erc gene, which is highly expressed in Tsc2-deficient renal tumor cell lines from the Eker rat model, was also highly expressed in CACL1s. CACL1s were not tumorigenic when subcutaneously transplanted into nude mice. Tsc1 knockout mice and hamartin deficient cell lines established in this study will be usefull experimental models for analyses of hamartin function and mechanism of tumorigenesis associated with Tsc1 mutation.