Download Gene knockouts reveal new hierarchy of cell cycle proteins: CNIO

Gene knockouts reveal new hierarchy of cell cycle proteins:
CNIO researchers report in Nature engineering of mouse strains
that shed new light on the role of Cdks in mammalian cell
proliferation
Madrid. 16 August 2007-. An article published in the August 16th issue of the journal
Nature mainly carried out in the Barbacid lab at the Spanish National Cancer Research
Centre (CNIO), demonstrates that the cyclin-dependent kinase Cdk1 is sufficient to
drive the mammalian cell cycle.
Basic Background
Cell division, a process by which a cell – the parent cell – divides into two - daughter
cells - is the most basic of biological processes. Without it, living organisms cannot
develop and would remain as one-celled zygotes. The diversity of organisms just as their
respective cell types is phenomenal, though this fundamental process has remained
constant throughout evolution.
In all eukaryotic organisms, the cell cycle requires the activation of cyclin-dependent
kinases (Cdks).While Cdks are usually present throughout the entirety of the cell cycle,
cyclins only synthesise at determined phases of each cell division or cycle. The discovery
of these basic processes earned Paul Nurse, Timothy Hunt and Lee Hartwell the Nobel
Prize for Medicine and Physiology just six years ago.
When a cell receives the signal to divide it abandons its rested stated, know as phase G0
and enters into the G1 phase of the cell cycle. In G1 the cell prepares to duplicate its
genome producing one identical copy of its DNA; phase S. The journey continues to
phase G2 to mitosis – the stage in which a cell duplicates its chromosomes to generate
two identical cells. This is then followed by cytokinesis which divides the cytoplasm and
cell membrane, resulting in two identical cells with an equal distribution of organelles
and other cellular components. Mitosis followed by cytokinesis complete the division of
the mother cell into two identical daughter cells, each with the genetic equivalent of the
parent cell.
The Tradition
The principle difference in cell division between unicellular and multicellular organisms is
determined by Cdks. In unicellular organisms just one sole Cdk (Cdk2, Cdk8, or Cdk1)
controls the entire process and can sequentially associate itself with the different cyclins
throughout the four phases of the cell cycle. Multicellular organisms however, such as
mammals, require multiple Cdks specific to each phase. In the past, it has been widely
accepted that mammalian cells use up to five different Cdks to complete the cell cycle.
In sequence these are Cdk4, Cdk6, Cdk3, Cdk2 and Cdk1.
According to this widely accepted model, Cdk4 and Cdk6, which interact with D cyclins,
are essential for the G1 phase. S phase is lead by Cdk2 that sequentially binds with
cyclins E and cyclins A to accomplish DNA duplication. Cdk1 interacts with cyclin A
during phase G2 and finally the complexes formed by Cdk1 and cyclins B have been
considered as responsible for proceeding through mitosis. Very little has been reported
about Cdk3 since laboratory mice lack functional Cdk3, and it has only therefore been
studied in human cells in vitro, suggesting a similar role to that of Cdk2.
The Turning Point
This previously affirmed series of events and processes however was first questioned
four years ago when the Experimental Oncology Group at the CNIO, directed by
Mariano Barbacid, published a study in Nature Genetics revealing that Cdk2, assumed to
be essential for genome replication in phase S, is not necessary since mice cell types
proliferate in the absence of this enzyme. Similar studies were also reported in Current
Biology by the Kaldis lab at NCI. A further study followed from the Barbacid group - this
time published in Cell. They demonstrated that Cdk4 and Cdk6 were not essential for
phase G1 of the cell cycle or for any other phase, but, they were necessary for the
division of just one cell type – haematopoietic (blood) cells.
In May this year the Barbacid lab also reported in a new journal, Mol. Oncology, the
generation of mice in the absence of Cdk4 and Cdk2 – the two kinases that were
previously considered as being principally responsible for all phases of the cell cycle
(with the exception of mitosis) in the majority of cells. To the surprise of many, these
mice completed embryonic development and reached birth. These mice once again
manifested specific problems in certain cell types such as cardiomyocytes, but not in the
basic cell cycle process. CNIO researchers have also shown that eliminating both
kinases in the liver of adult mice, through sophisticated genetic recombination, did not
have any consequences in hepatocytes lacking CdK4 and CdK2, and proliferated as
efficiently as normal heptocytes. These findings suggest once more the participation of
Cdks in cellular proliferation processes in specialised cells, but not in the basic cell cycle.
These results are at variance with those of the Kaldis lab reported in Developmental Cell.
Similar mice generated in this lab did not develop to term and their cells did not
proliferate in vitro. The reasons for this severe phenotype remain to be determined.
Now, the Barbacid team led by David Santamaría in collaboration with other
investigators from the CNIO (M. Malumbres), the University of Bordeaux (P. Dubus)
and the Western General Hospital of Edinburgh (K. Newton and J. Cáceres) report in
the August 16th issue of Nature that mouse embryos lacking in all interphase Cdks
(Cdk2, Cdk3, Cdk4 and Cdk6) can complete the cell cycle. More importantly, embryos
with Cdk1 alone undergo organogenesis which shows that such embryos cannot only
complete embryonic development assuming maximum cell division activity (20 million
cells produced in two weeks), but can also produce all cell types, tissues and organs of
an adult organism. As suspected, embryos with only Cdk1 do not complete embryonic
development since they do not produce blood cells through the absence of Cdk4 and
Cdk6.
The work carried out by David Santamaria and colleagues at the CNIO goes further by
demonstrating that Cdk1, in the same way as yeasts and other unicellular organisms, is
essential to drive cell division. In collaboration with researchers in the UK, CNIO
scientists have generated mice without Cdk1 and showed that in the absence of this
enzyme embryos fail to develop even to the morula stage (embryos with 16-64 cells),
suggesting that Cdk1 is essential for the first embryonic divisions.
In conclusion, these findings, as with previous aforementioned work of the Barbacid lab,
demonstrate that mammalian cells are not mechanistically too different from unicellular
organisms and that one Cdk alone – Cdk1 – can bind to all cyclins and drive the cell
cycle. CNIO researchers have also shown that the role of Cdk1 in the cell cycle is far
more involved than its function in mitosis, just as that occurring in unicellular organisms.
Equally, it seems ever more evident that generating multiple Cdks in superior organisms
– such as mammals – derives from the need to provide specialized cells with the
sufficient Cdk kinase activity to enable cells to divide according to the specific needs of
organisms.
Toward Targets
These studies are critical to developing specific therapies to treat tumours in humans.
Cdks have been the object of much interest as therapeutic targets from within the
entire pharmaceutical industry since cancer as well as other diseases are caused by
aberrant cell proliferation. Unfortunately to date, no Cdk inhibitor has achieved the
results necessary to advance further than phase II in clinical trials.
A more precise understanding of how each Cdk contributes to driving cell division
should help us to determine what strategy to follow to ensure that the inhibition of one
or more Cdk can in turn have an inhibiting effect on the proliferation of tumour cells
without effecting normal cells of cancer patients. Forthcoming studies will allow us to
validate whether Cdks are targets of therapeutic interest in cancer - once and for all.
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EDITOR’S NOTES
1) The Spanish National Cancer Research Centre (CNIO) is one of the few
European Cancer Research Centres of international excellence that effectively
combines basic and applied research. For more detailed information surrounding
the CNIO, its rapidly expanding research programmes, list of publications,
international conferences and symposia, and the latest Scientific Report please
visit: www.cnio.es or contact us directly:
2) David Santamaría
Centro Nacional de Investigaciones Oncológicas
Melchor Fernández Almagro 3
E-28029 Madrid
Tel: +34 91 2246900, Fax: +34 91 2246980
Email: [email protected]