Axonal protein synthesis and degradation are necessary for efficient growth cone regeneration.

Axonal regeneration can occur within hours of injury, the first step being the formation of a new growth cone. For sensory and retinal axons, regenerative ability in vivo correlates with the potential to form a new growth cone after axotomy in vitro. We show that this ability to regenerate a new growth cone depends on local protein synthesis and degradation within the axon. Axotomy in vitro leads to a fourfold to sixfold increase in 3H-leucine incorporation in both neurones and axons, starting within 10 min and peaking 1 h after axotomy. Application of protein synthesis inhibitors (cycloheximide and anisomycin) to cut axons, including axons whose cell bodies were removed, or proteasome inhibitors (lactacystin and N-acetyl-Nor-Leu-Leu-Al) all result in a reduction in the proportion of transected axons able to reform growth cones. Similar inhibition of growth cone formation was observed on addition of target of rapamycin (TOR), p38 MAPK (mitogen-activated protein kinase), and caspase-3 inhibitors. Comparing retinal and sensory axons of different developmental stages, levels of ribosomal protein P0 and phosphorylated translation initiation factor are high in sensory axons, lower in embryonic axons, and absent in adult retinal axons. Conditioning lesions, which increase the regenerative ability of sensory axons, lead to increases in intra-axonal protein synthetic and degradative machinery both in vitro and in vivo. Collectively, these findings suggest that local protein synthesis and degradation, controlled by various TOR-, p38 MAPK-, and caspase-dependent pathways, underlie growth cone initiation after axotomy.

Mismatch repair gene mutations in renal cell carcinoma.

We investigated the spectrum and genetic basis for mismatch repair (MMR) deficiency in renal cell carcinoma (RCC) by examining expression of four MMR genes important for hereditary and sporadic carcinogenesis. MMR deficiency was assessed using microsatellite instability (MSI) and genetic analyses of 25 cell lines derived from renal tumors. MMR gene alterations were detected using reverse transcription of RNA coupled with polymerase chain reaction (RT-PCR) and DNA sequencing. Three RCC lines with undetectable MLH1 were identified and investigated for MSI and inactivating mutations in the hMLH1 MMR gene. Genetic instability and hMLH1 mutations were identified in two RCC lines and their corresponding tumors. Genetic alterations affecting expression were limited to MLH1 since other MMR proteins (MSH2, MSH6 and PMS2) were detectable in our RCC lines. Complete inactivation of MMR is apparently uncommon in RCC and occurs predominantly through inactivating mutations in the hMLH1 gene.