The Endoreduplication Cell Cycle: Regulation and Function 79
Analysis of endoreduplicating cells by pulse labeling with [^3 H]thymidine
(Lilly and Duronio 2005) or flow cytometry (Galbraith et al. 1983) shows that
endoreduplication consists, similarly to mitotic cells, of DNA synthesis phases
alternating with gap phases. This observation strongly suggests that the con-
trols that normally regulate entry into S-phase and prevent rereplication in
mitotic cells are largely operating during endoreduplication. Additional ge-
netic and molecular data also support the current view that endoreduplication
can be considered a derivation of the mitotic cell cycle, that endoreduplica-
tion requires more than just relaxation of the restrictions on origin licensing,
and that many of the pathways involving CDK, SCF, and APC activities need
reprogramming for endoreduplication to occur (DePamphilis et al. 2006).
2.2
The Retinoblastoma/E2F Pathway
The retinoblastoma (RB)/E2F pathway controls the expression of a plethora
of S-phase genes and plays a pivotal role in regulating endoreduplication. RB
plays a negative cell cycle role by repressing E2F/DP transcription factors (see
chapter by Chen W-H, this volume). In animals, the negative role of RB in
rereplication and the endoreduplication cell cycle may involve its interaction
with replicated origins, the downregulation of factors required for origin li-
censing, or enforcing the normal expression of mitotic checkpoint proteins
(Niculescu et al. 1998; Royzman et al. 1999; Bosco et al. 2001; Cobrinik 2005).
Phosphorylation and consequent inactivation of RB and related (RBR) pro-
teins, which relieves its inhibitory effect on transcription of genes involved
in DNA synthesis and S-phase progression, is associated with endoredupli-
cation in mammals and plants (Grafi et al. 1996; Harbour and Dean 2000).
The direct downregulation ofRBR1expression either using a virus-based, si-
lencing approach in tobacco or inhibition of RBR1 protein by wheat dwarf
virus (WDV) RepA expression inArabidopsisresults in increased expression
of E2F targets and the specific stimulation of endoreduplication in differenti-
ating organs/tissues (Park et al. 2005; Desvoyes et al. 2006). However, a rather
different situation has been found inDrosophila, where RB/E2F complexes
regulate replication origin licensing by cyclically downregulating CycE in the
gap phase during endoreduplication (Weng et al. 2003). Thus, integrity of the
RB/E2F pathway is required for normal endoreduplication inDrosophila.
S-phase gene expression and endoreduplication are also stimulated by
the upregulation of RBR-repressed E2F/DP transcription factors, such as
E2Fa and DPa inArabidopsis(De Veylder et al. 2002) and tobacco (Kosugi
and Ohashi 2003). In addition, RNAi-mediated downregulation of another
RBR1-inhibited member of the E2F family,E2Fc, leads to decreased endo-
reduplication in mature leaves, possibly by controlling certain aspects of the
G2/M-phase transition (del Pozo et al. 2006). Alteration in the balance within
the E2F family of genes affects endoreduplication as shown by the loss of