Discerning the functional role of the distinct pocket proteins has been greatly aided by the use of gene-targeted mouse models to specifically disrupt individual pocket proteins. Genetic disruption of the mouse Rb1 gene to produce Rb1-/- mice results in embryonic lethality between embryonic day (E) 13.5 and E15 (89). The Rb1 -/- embryos have increased proliferation and apoptosis in the central nervous system (CNS) and the peripheral nervous system (PNS) (89). These mice also have defects in hematopoiesis and altered development and proliferation of the lens (89-92). The inappropriate
proliferation observed in Rb1-/- mice can be partially rescued by combined disruption of E2F1 or E2F3, which suggests that E2F regulation is a critical function of pRB (93-95). The ectopic proliferation and apoptosis observed was thought to highlight a critical role for pRB in maintaining appropriate growth control in distinct developmental contexts.
22
However, many of the defects in Rb1-/- mice were later found to be secondary to proliferative defects that occur in the placenta of these mice (96).
Specifically, the trophoblast cells found within the labyrinth layer of the placenta were found to be hyperproliferative in Rb1-/- embryos (97). This over-proliferation results in a decrease in the space between the maternal and fetal blood supply (96). This in turn was found to result in decreased nutrient transport to the embryos (96). To investigate phenotypes of Rb1-/- mice that are independent of the placental defect Rb1-/- mice were produced with normal placenta using tetraploid aggregation (97). The provision of the normal placenta was found to rescue many of the phenotypes associated with loss of Rb1 (97). Most notably the mice were no longer embryonic lethal between E13.5-E15.5 but rather could survive until birth (96). The defects in hematopoiesis and apoptosis in the CNS were not observed in the rescued Rb1-/- animals (96). However, the excess
proliferation in the CNS and the lens was observed in the rescued animals suggesting that these defects occurred independently of the placental defects (96). The mice, however, died shortly after birth due to defects in skeletal muscle formation (96). This defect results in significant disruption of the diaphragm that prevents the lungs of newborn Rb1-
/- animals from inflating and resulted in an inability for the mice to respire. Experiments
using conditional deletion of pRB in myoblasts suggest that the defect in skeletal muscle results from an inability of the Rb1-/- cells to terminally differentiate into multinucleated myotubes (98). Fibroblasts generated form Rb1-/- embryos also display significant defects in proliferative control. Specifically the fibroblasts have a shorter G1 phase of the cell
23 serum starvation they are unable to respond to ectopic arrests induced by p16 (100) and TGF! (101).
Taken together mouse models of pRB have defined an essential role for pRB in mammalian development. Specifically pRB function is required for proper proliferative control, placental development and muscle differentiation. Many cell types are able to proliferate and respond normally in distinct developmental contexts, as Rb1-/-animals are viable until birth if supplied with a normal placenta. Given the essential role for pRB in tumorigenesis and the postulated role in the regulation of the G1-S transition the
development of these mice suggests that in some contexts other pathways can function in the absence of pRB to maintain cell cycle control.
1.4.2
Redundancy in the pocket protein family
pRB appears to have a clear role in regulating a distinct set of developmental processes as observed from the Rb1-/-mice. However, proliferative control is maintained in many tissues and death in animals with a normal placenta is the result of defective differentiation of muscle cells. The maintenance of proliferative control in Rb1-/- mice appears to be due in part to the activity of the other pocket proteins p107 and p130. Loss of pRB results in a deregulation of E2F target gene expression that induces the expression of p107 which is itself an E2F target gene (102). The increased levels of p107 can allow for compensation for loss of pRB in many contexts. Combined disruption of pRB and p107 or pRB and p130 results in more severe apoptotic and proliferative defects that result in earlier embryonic lethality between E11 and E13 (103, 104). Importantly loss of p107 or p130 alone does not alter the viability of mice in a mixed genetic background
24 (105). Combined disruption of both p107 and p130 results in neonatal lethality with severe defects in bone development that results in shorter bones (105, 106). This suggests that the pocket proteins control partially overlapping pathways and in some
circumstances function to compensate for the loss of other pocket proteins. To further test the compensation between pocket proteins, fibroblasts were generated that disrupted pRB, p107 and p130, called TKO cells (107, 108). These fibroblasts were generated from the differentiation of directly targeted ES into TKO fibroblasts (107, 108). The TKO cells are defective for proliferative control and do not arrest in the G1 under a variety of conditions (107, 108). However, more recently TKO embryos have been generated and survive until days 9-11 of gestation (109). Further the embryos and cultured TKO cells are capable of exiting the cell cycle in G1 and
differentiating into multiple epithelial and neural lineages (109). This suggests that in some contexts cell cycle exit can occur in the absence of pocket protein activity however, the mechanism by which this may occur is still unclear.
1.4.3
A unique role for pRB in cancer
Loss of pRB in the retina results in the generation of retinoblastoma early in life. Initial efforts using the mouse model of Rb1 disruption investigated whether a similar effect would be observed in pRB null mice. In contrast to humans the Rb1+/- mice do not develop retinoblastoma but rather develop pituitary tumors that arise from the
intermediate lobe of this gland. (89) Rb1+/- mice typically develop tumors by one year of age in either the intermediate lobe of the pituitary gland or less frequently in the thyroid gland (110). These tumors display loss of heterozygosity (LOH) of the remaining wild
25 type allele of Rb1 to produce a tumor that is nullizygous. Interestingly disruption of other cell cycle regulators such p27 or p18 also results in pituitary tumors in the intermediate lobe (111-114). This suggests that this region may be uniquely susceptible to loss of proliferative control that gives rise to the observed tumors. Furthermore, the intermediate lobe is rudimentary and likely non-functional in humans which may explain the fact that pRB loss in humans does not induce pituitary tumors (115).
In contrast, the induction of retinoblastoma in mice requires disruption of both pRB and p107 as Rb1-/- Rbl1-/- chimeras or mice with deletion of pRB and p107 in the retina develop spontaneous retinoblastomas (116, 117). This further supports the compensatory role of p107 in the absence of pRB. p107 and p130 themselves are rarely disrupted in human cancers and mice lacking p107 gene Rbl1 or the p130 gene Rbl2 are not prone to tumors (118). Further, Rbl1+/- Rbl2-/- and Rbl1-/- Rbl2+/- mice are not tumor prone
suggesting a unique role for the remaining pRB in tumor suppression (34). Taken together this suggests that pRB has a unique role in tumorigenesis but the other pocket proteins can function in the absence of pRB in certain contexts to maintain proliferative control. Given the ability of all pocket proteins to interact with E2F transcription factors the mechanistic basis for the unique role of pRB in tumorigenesis is not clearly defined.