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Abstract
The substructure within the eukaryotic nucleus is dynamic. Motion within the nuclear environment must be considered in the context of the variety of organelles and organizing matrixes facilitating and impeding translocation within a confined space. The Harlow lab has previously reported the perinucleolar co-localization of the retinoblastoma susceptibility protein (pRb) and lamin A in human diploid primary fibroblasts. These nuclear foci represent the convergence of replication and transcription regulation. The observations presented in this thesis result from an initial effort to examine nuclear interactions in a defined physical location that coordinate organization of the nuclear substructure and cell cycle progression. Here I submit data supporting the proposal that colocalization of lamin A and pRb has functional significance for cell proliferation and for pRb regulatory activities.
We examined pRB function in cells lacking lamin A/C, finding that pRB levels are dramatically decreased and that the remaining pRB is mis-localized. We also demonstrate that pRb levels increase markedly when lamin A/C null cells are treated with proteasome inhibitors. Induced overexpression of endogenous pRB in a lamin A null background by proteasome inhibitors fails to form perinucleolar foci in G1. Both pRB levels and localization are restored upon re-introduction of lamin A. Moreover, Lmna−/− cells phenotypically resemble Rb−/− cells, exhibiting altered cell cycle properties and reduced capacity to undergo cell cycle arrest in response to DNA damage. These findings establish a functional link between a core nuclear structural component and an important cell cycle regulator.
Lamin A-dependent tethering of pRb to specific G1-S perinucleolar foci may thus be related to pRb function and, by extension, to tumor suppression. This raises the possibility that naturally-occurring lamin A mutations may lead to inactivation of pRb in tumors. We asked the question: are there LMNA mutations in human tumor-derived cell lines? We found multiple lamin A mutations in the colon cancer cell line, HCT15. pRb is less frequently localized to the perinucleolar foci in this cell line than in the other similar colorectal lines examined, suggesting that the localization of human pRb in human tumors is affected by changes in lamin A.
