Combinatorial roles for pRB, p107, and p130 in E2F-mediated cell cycle control

Abstract

Numerous studies have implicated the pRB family of nuclear proteins in the control of cell cycle progression. Although over-expression experiments have revealed that each of these proteins, pRB, p107, and p130, can induce a G(1) cell cycle arrest, mouse knockouts demonstrated distinct developmental requirements for these proteins, as well as partial functional redundancy between family members. To study the mechanism by which the closely related pRB family proteins contribute to cell cycle progression, we generated 3T3 fibroblasts derived from embryos that lack one or more of these proteins (pRB(-/-), p107(-/-), p130(-/-), pRB(-/-)/p107(-/-), pRB(-/-)/p130(-/-), and p107(-/-)/p130(-/-)). By comparing the growth and cell cycle characteristics of these cells, we have observed clear differences in the manner in which they transit through the G(1) and S phases as well as exit from the cell cycle. Deletion of Rb, or more than one of the family members, results in a shortening of G(1) and a lengthening of S phase, as well as a reduction in growth factor requirements. In addition, the individual cell lines showed differential regulation of a subset of E2F-dependent gene promoters, as well as differences in cell cycle-dependent kinase activity. Taken together, these observations suggest that the closely related pRB family proteins affect cell cycle progression through distinct biochemical mechanisms and that their coordinated action may contribute to their diverse functions in various physiological settings.

Figure 1

Establishment of cells, protein expression, and cell size profiles of 3T3 cells deficient in pRB, p107, and/or p130. (A) Two independent isolates of MEFs, prepared from 13.5 days postcoitus embryos of mixed background (SV129/C57BL6) were used for all genotypes, except for pRB^−/−/p107^−/− and pRB^−/−/p130^−/− cells where only one isolate from an 11.5 days postcoitus embryo was used. The figure shows growth curves from these cell lines during the immortalization process. The y axis represents -fold growth in 3 days when seeded at a density of 10⁶ cells per 10-cm dish, and the x axis is passage number (B). pRB, p107, p130 protein expression in wt 3T3 cells as well as lines deficient in p107, p130, pRB, p107/p130, and pRB/p107 at passage 45. (C) Comparison of the cell size of wt and p107-deficient 3T3 cells. Asynchronously growing wt (solid line) or p107^−/− cells (dashed lines) were harvested and analyzed by FACS. FSC-H histograms, a relative measure of cell size is shown.

Figure 2

Timing of the cell cycle in cells deficient in pRB, p107, and/or p130. Asynchronous cells were treated with nocodazole for 10 h, and mitotic cells were shaken off and replated. (Upper) Example of the direct FACS data (R3 = G₁, R5 = S phase, and R4 = G₂/M); these 3T3 cells enter endo-reduplication upon prolonged nocodazole treatment. (Lower) The timing of S phase entry measured by BrdUrd incorporation and two-dimensional FACS analysis. The y-axis represents % S phase cells and the x-axis, hours postrelease. A representative example of three experiments is shown.

Figure 3

Cell cycle arrest and derepression of E2F target genes in pRB, p107, and/or p130 deficient cells. (A) The % of cells in S phase, as measured by two-dimensional FACS in asynchronous (solid bars), serum-starved (striped bars), and confluent cells (gray bars) in the genotypically different cells, as indicated in the figure. (B) Relative luciferase activity from pools of cells harboring an integrated B-myb reporter under serum-starved (striped bars) and confluent conditions (gray bars) as compared with asynchronous populations (arbitrarily set to 100). A representative example from three different experiments is shown. (C) Relative luciferase activity from pools of cells harboring an integrated p107 promoter construct under serum-starved (striped bars) and confluent conditions (gray bars) as compared with asynchronous populations (arbitrarily set to 100). A representative example from three different experiments is shown. (D) (Upper) cdk2 kinase activity, using a His-tagged RB protein as a substrate in confluent (C) and asynchronous (A) wt cells and confluent knockout cell lines. (Lower) The corresponding levels of p27.