| Summary: | Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by mutation in either the TSC1 or TSC2 genes and characterised by the development of benign hamartomatous growths in multiple organ systems. We have inactivated Tsd in the mouse germ line by gene targeting in ES cells and confirmed that the mutant allele (Tsc1) has a recessive embryonic lethal phenotype. Tsc1+, mice developed macroscopically visible renal lesions as early as 3-6 months. Renal lesions progressed from cysts through cystadenomas to solid carcinomas. Eighty percent of Tsc1+/ mice on a Balb/c background exhibited solid renal cell carcinomas (RCC) by 15-18 months and in 41%, RCCs were _5mm, resulting in grossly deformed kidneys. Some RCCs had a sarcomatoid morphology of spindle cells in whorled patterns and metastasised to the lungs. This new murine model of hamartin deficiency exhibits a more severe phenotype than existing models. A Bloom's deficient mouse model (Blmm3/m3) has been shown to induce colorectal tumourigenesis when crossed with Apc+/M,n mice. Here, we investigate whether the Blmm3/m3 genotype could induce tumourigenesis in extra-colonic tissues in Tsc1+/ mice that are predisposed to renal cystadenomas and carcinomas. Tsc1+/ B mm3,m3 mice had significantly more macroscopic and microscopic renal lesions at 3-6 months compared to Tsc1+' Blm+/m3 mice. Tsc1+, Blrrf3*"3 mice tumours showed significantly increased levels of somatic LOH of the wild type Tsd (Tsrf *) allele, as compared to those from Tsc1+, Blm+/+ mice. This work demonstrates the utility of the Blmm3/m3 mice for inducing renal tumourigenesis and the high levels ( 87%) of LOH in the resultant tumours will help facilitate mapping of loci involved in tumour progression. TSC1 and TSC2 are generally considered to act as tumour suppressors that fulfil Knudson's '2-hit hypothesis'. Here, we identified somatic Tsd mutations (2nd hits) in -80% of CAs and RCCs, but only 31.6% of cysts from Tsc1+/- mice, raising the possibility that haploinsufficiency for Tsd plays a role in cyst formation. Consistent with this proposal, many cysts showed little or no staining for phosphorylated mTOR and phosphorylated S6 ribosomal protein, whereas >90% of CAs and RCCs showed strong staining for both markers. We also sought somatic mutations in renal lesions from Tsc1+/ Blm'A mice that have a high frequency of somatic LOH, thereby facilitating the detection of 2nd hits. We also found significantly less somatic mutations in cysts, as compared to CAs and RCCs from these mice. Our data indicate that although activation of the mTOR pathway is an important step in Tsc-associated renal tumourigenesis, it may not be the key initiating event in this process.
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