Signal-dependent protection from apoptosis in mice expressing caspase-resistant Rb.

The retinoblastoma tumour suppressor protein RB is cleaved by caspases during apoptosis. Here we have mutated the caspase cleavage site in the carboxy terminus of the murine Rb protein in the mouse germ line to create the Rb-MI allele. After endotoxic shock, expression of Rb-MI inhibits apoptosis in the intestines, but not in the spleen, and promotes the survival of male mice. Fibroblasts expressing Rb-MI protein are protected from apoptosis induced by the tumour-necrosis factor-alpha type I receptor (TNFRI) but remain sensitive to cell death induced by DNA damage. Correspondingly, the release of cytochrome c and the activation of caspase-3 induced by TNFRI, but not by DNA damage, are defective in cells expressing Rb-MI. Our results highlight the importance of Rb cleavage in TNFRI-induced apoptosis.

Apoptotic function of human PMS2 compromised by the nonsynonymous single-nucleotide polymorphic variant R20Q.

Mismatch repair (MMR) corrects replication errors during DNA synthesis. The mammalian MMR proteins also activate cell cycle checkpoints and apoptosis in response to persistent DNA damage. MMR-deficient cells are resistant to cisplatin, a DNA cross-linking agent used in chemotherapy, because of impaired activation of apoptotic pathways. It is shown that postmeiotic segregation 2 (PMS2), an MMR protein, is required for cisplatin-induced activation of p73, a member of the p53 family of transcription factors with proapoptotic activity. The human PMS2 is highly polymorphic, with at least 12 known nonsynonymous codon changes identified. We show here that the PMS2(R20Q) variant is defective in activating p73-dependent apoptotic response to cisplatin. When expressed in Pms2-deficient mouse fibroblasts, human PMS2(R20Q) but not PMS2 interfered with the apoptotic response to cisplatin. Correspondingly, PMS2 but not PMS2(R20Q) enhanced the cytotoxic effect of cisplatin measured by clonogenic survival. Because PMS2(R20Q) lacks proapoptotic activity, this polymorphic allele may modulate tumor responses to cisplatin among cancer patients.

BCR-ABL-transformed GMP as myeloid leukemic stem cells.

During blast crisis of chronic myelogenous leukemia (CML), abnormal granulocyte macrophage progenitors (GMP) with nuclear beta-catenin acquire self-renewal potential and may function as leukemic stem cells (Jamieson et al. N Engl J Med, 2004). To develop a mouse model for CML-initiating GMP, we expressed p210(BCR-ABL) in an established line of E2A-knockout mouse BM cells that retain pluripotency in ex vivo culture. Expression of BCR-ABL in these cells reproducibly stimulated myeloid expansion in culture and generated leukemia-initiating cells specifically in the GMP compartment. The leukemogenic GMP displayed higher levels of beta-catenin activity than either the nontransformed GMP or the transformed nonGMP, both in culture and in transplanted mouse BM. Although E2A-deficiency may have contributed to the formation of leukemogenic GMP, restoration of E2A-function did not reverse BCR-ABL-induced transformation. These results provide further evidence that BCR-ABL-transformed GMP with abnormal beta-catenin activity can function as leukemic stem cells.

Radiation dose-dependent maintenance of G(2) arrest requires retinoblastoma protein.

In response to ionizing radiation (IR), cell cycle checkpoints are activated to provide time for DNA repair. Several different checkpoint mechanisms have been elucidated. However, mechanisms that regulate the duration of cell cycle arrest are not understood. Previous studies have shown that the retinoblastoma tumor suppressor protein (RB) is required for radiation-induced G1 arrest. Working with primary fibroblasts derived from Rb+/+ and Rb-/- mouse embryos, we show that RB also regulates the duration of G2 arrest. The initial G2 checkpoint response is enhanced in Rb-/- cells due to a defect in G1 arrest. However, the permanent arrest in G2 induced by higher doses of IR does not occur in Rb-/- cells. Rb-/- cells either resumed proliferation or underwent apoptosis at IR doses that caused the majority of Rb+/+ cells to arrest permanently in G2. The prolongation of G2 arrest in Rb+/+ cells correlated with a gradual accumulation of hypophosphorylated RB. Thus, regulation of the RB function may be an important aspect in the maintenance of cell cycle checkpoints in DNA damage response.

Blockade of tumor necrosis factor-induced Bid cleavage by caspase-resistant Rb.

Tumor necrosis factor-alpha (TNF) activates caspase-8 to cleave effector caspases or Bid, resulting in type-1 or type-2 apoptosis, respectively. We show here that TNF also induces caspase-8-dependent C-terminal cleavage of the retinoblastoma protein (Rb). Interestingly, fibroblasts from Rb(MI/MI) mice, in which the C-terminal caspase cleavage site is mutated, exhibit a defect in Bid cleavage despite caspase-8 activation. Recent results suggest that TNF receptor endocytosis is required for the activation of caspase-8. Consistent with this notion, inhibition of V-ATPase, which plays an essential role in acidification and degradation of endosomes, specifically restores Bid cleavage in Rb(MI/MI) cells. Inhibition of V-ATPase sensitizes Rb(MI/MI) but not wild-type fibroblasts to TNF-induced apoptosis and stimulates inflammation-associated colonic apoptosis in Rb(MI/MI) but not wild-type mice. These results suggest that Rb cleavage is required for Bid cleavage in TNF-induced type-2 apoptosis, and this requirement can be supplanted by the inhibition of V-ATPase.