LONDON ¿ The centrosome, which organizes the dynamic skeleton of the cell, is regarded as one of the last mysteries of cell biology. Very little is known about its structure or how the cell controls what it does. It plays a crucial role in cell division, organizing the two poles of the spindle, and must therefore be duplicated once ¿ and only once ¿ in each cell cycle.
Knowing how the duplication process is regulated could lead to interesting insights into tumor biology, as recent reports have shown that, in many tumors, the cells have numerous centrosomes.
Researchers in Switzerland, Denmark and the U.K. have now published one of the first papers to examine the control of centrosome duplication. Patrick Meraldi, of the department of molecular biology at the University of Geneva, with colleagues from the Institute of Cancer Biology in Copenhagen, Denmark, and from the University of Leicester in Leicester, U.K., present their results in a paper in the June edition of Nature Cell Biology, titled ¿Centrosome duplication in mammalian somatic cells requires E2F and Cdk2-Cyclin A.¿
Erich Nigg, professor of molecular biology at the University of Geneva, Switzerland, and senior author of the paper, told BioWorld International, ¿We have found that the same pathway that controls DNA duplication also controls centrosome duplication. This makes a lot of sense ¿ if you want to coordinate these processes, you put them under control of the same pathway. In addition, this pathway, which involves the protein retinoblastoma, seems to be mutated in just about every human cancer there is.¿
The centrosome of the cell is a tiny organelle from which most microtubules grow. The microtubules are part of the cytoskeleton, helping the cell to maintain its shape, move, transport organelles, and carry out endocytosis and secretion. The shape of a neuron, for example, is determined by its microtubules, and these also transport neurotransmitters to the tip of the axon.
To study the control of centrosome duplication, Nigg and his colleagues developed an assay in which DNA duplication could be uncoupled from centrosome duplication. By adding hydroxyurea to Chinese hamster ovary cells, they found they could study the duplication of centrosomes using immunofluorescence microscopy. They then overexpressed genes in these cells to find out which proteins could block the process of centrosome duplication. The genes selected were those encoding known regulators of the cell cycle.
Their results show that centrosome duplication is regulated through the retinoblastoma (Rb) pathway. Rb is phosphorylated by a family of cyclin-dependent kinases, which in turn are inhibited by small proteins such as p16. Nigg and his colleagues were able to block centrosome duplication in their model by transfecting genes that overexpressed p16, or which expressed an Rb mutant that lacks sites at which it can be phosphorylated.
In addition, centrosome duplication was significantly inhibited when they transfected a gene encoding a protein inhibiting the function of E2F ¿ a transcription factor that plays an important role in the Rb pathway. They also found that the protein Cdk2 is required for centrosome duplication, in conjunction with cyclin A.
Writing in Nature Cell Biology, Nigg and his colleagues conclude, ¿Our results clearly show that centrosome duplication in somatic mammalian cells requires the phosphorylation of Rb. This implies that both DNA replication and centrosome duplication are controlled through the same pathway. In normal proliferating cells, this would seem to provide an efficient means of ensuring the coordinate execution of these two events. It also follows, however, that the loss of a functional Rb pathway, either through mutation or the action of viral oncogenes, might jeopardize the coordination between DNA replication and centrosome duplication and lead to genomic instability.¿
Nigg told BioWorld International that, in tumors, ¿something always seems to be wrong with this pathway. Depending on the tumor type, you may find a loss of p16, the small protein that inhibits the cyclin dependent kinases, or overexpression of the cyclin dependent kinases, or mutation or loss of the Rb protein. So, abolishing this pathway seems to be critical for tumor development. While this pathway undoubtedly controls DNA replication, our study suggests that it also controls centrosome duplication.
¿Thus, the time is right to revive the idea that aberrations in the pathways that control mitotic spindle assembly may constitute prime targets for cancer therapy. I am quite confident that our increasing knowledge in this field will eventually invite therapeutic applications.¿
The group now is working on using the same assay to identify additional components of the regulatory pathway. It also wants to devise alternative assays for centrosome duplication, Nigg said, because treating cells with a drug such as hydroxyurea is far from ideal.