BRCA1, a familial breast and ovarian cancer susceptibility gene encodes nuclear phosphoproteins that function as tumor suppressors in human breast cancer cells. Previously, we have shown that overexpression of a BRCA1 splice variant BRCA1a accelerates apoptosis in human breast cancer cells. In an attempt to determine whether the subcellular localization of BRCA1 is cell cycle regulated, we have studied the subcellular distribution of BRCA1 in asynchronous and growth arrested normal, breast and ovarian cancer cells using dierent BRCA1 antibodies by immuno¯uorescence and immunohistochemical staining. Upon serum starvation of NIH3T3, some breast and ovarian cancer cells, most of the BRCA1 protein redistributed to the nucleus revealing a new type of regulation that may modulate the activity of BRCA1 gene. We have also characterized two new variant BRCA1 proteins (BRCA1a/p110 and BRCA1b/ p100) which are phosphoproteins containing phosphotyrosine. Immuno¯uorescence and Western blotting analysis indicate cytoplasmic and nuclear localization of BRCA1a and BRCA1b proteins. To elucidate the biological function of BRCA1, we created a bacterial fusion protein of glutathione-transferase (GST) and BRCA1 zinc ®nger domain and detected two cellular proteins with molecular weights of approximately 32 and 65 kD, one of which contains phosphotyrosine designated p32 and p65 BRCA1 interacting proteins (BIP) that speci®cally interact with BRCA1. Western blot analysis of BIP with cyclins/CDKs and E2F antisera indicated association with cdc2, cdk2, cdk4, cyclin B, cyclin D, cyclin A and E2F-4 but not with cdk3, cdk5, cdk6, E2F-1, E2F-2, E2F-3, E2F-5 and cyclin E. Furthermore, we have also demonstrated a direct interaction of in vitro translated BRCA1a and BRCA1b proteins with recombinant cyclin A, cyclin B1, cyclin D1, cdc2, cdk2 and E2F fusion proteins in vitro. Taken together these results seem to suggest that BRCA1 could be an important negative regulator of cell cycle that functions through interaction with E2F transcriptional factors and phosphorylation by cyclins/cdk complexes with the zinc ring ®nger functioning as a major protein-protein interaction domain. If the interactions we observe in vitro is also seen in vivo then it may be possible that lack or impaired binding of the disrupted BRCA1 proteins to E2F, cyclins/CDKs in patients with mutations in the zinc ®nger domain could deprive the cell of an important mechanism for braking cell proliferation leading to the development of breast and ovarian cancers.

SummaryThe cell cycle is regulated by the cyclin-dependent kinase (CDK), and CDK inhibitors can bind to CDKs and inhibit their activities. This review examines plant CDK inhibitors, with particular emphasis on their molecular and cellular functions, regulation and cellular localization. In plants, a family of ICK/KRP CDK inhibitors represented by ICK1 is known and another type of CDK inhibitor represented by the SIMESE (SIM) has recently been reported. Considerable understanding has been gained with the ICK/KRP CDK inhibitors. These plant CDK inhibitors share only limited sequence similarity in the Cterminal region with the KIP/CIP family of mammalian CDK inhibitors. The ICK/KRP CDK inhibitors thus provide good tools to understand the basic machinery as well as the unique aspects of the plant cell cycle. The ICK/KRP CDK inhibitors interact with D-type cyclins or A-type CDKs or both. Several functional regions and motifs have been identified in ICK1 for CDK inhibition, nuclear localization and protein instability. Clear evidence shows that ICK/KRP proteins are important for the cell cycle and endoreduplication. Preliminary evidence suggests that they may also be involved in cell differentiation and cell death. Results so far show that plant CDK inhibitors are exclusively localized in the nucleus. The molecular sequences regulating the localization and functional significance will be discussed.

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The organization of microtubules during interphase and prophase in embryogenic cultures of black spruce (Picea mariana) was investigated by indirect immunofluorescence. Somatic embryos of black spruce possessed an extensively branched and interconnecting network of fine interphase cortical microtubules. The development of pre-prophase bands (PPBs) in embryogenic black spruce cultures was compared with that in non-embryogenic cell cultures of jack pine (Pinus banksiana). PPBs in both species were initially arranged as a very broad array of microtubules, later (early to mid-prophase) becoming narrower and more intensely fluorescent. The occurrence of pre-prophase bands in relation to the number of phragmoplasts (i.e. PPB index) of black spruce somatic embryos was significantly higher (p<0.01) than that found for jack pine cells.