David W. Goodrich

S-phase entry upon ectopic expression of G1 cyclin-dependent kinases in the absence of retinoblastoma protein phosphorylation

In mammalian cells, the retinoblastoma protein (Rb) is thought to negatively regulate progression through the G1 phase of the cell cycle by its association with the transcription factor E2F [1-3]. Rb-E2F complexes suppress transcription of genes required for DNA synthesis ([4], reviewed in [3,5]), and the prevailing view is that phosphorylation of Rb by complexes of cyclin-dependent kinases (Cdks) and their regulatory cyclin subunits, and the subsequent release of active E2F, is required for S-phase entry [1-3]. This view is based, in part, on the fact that ectopic expression of cyclin-Cdks leads to Rb phosphorylation and that this modification correlates with S-phase entry [6-8]. In Drosophila, however, cyclin E expression can bypass a requirement for E2F, suggesting that cyclins may activate replication independently of the Rb/E2F pathway [9]. We sought to examine whether Rb phosphorylation is a prerequisite for S-phase entry in Rb-deficient SAOS-2 osteosarcoma cells, using a commonly used cotransfection assay [6-8,10]. We find that a G1 arrest in SAOS-2 cells mediated by an Rb mutant lacking all 14 consensus Cdk phosphorylation sites is bypassed by coexpressing G1-specific E-type or D-type cyclin-Cdk complexes, and that injection of purified cyclin-Cdks during G1 accelerates S-phase entry. Our results indicate that Rb phosphorylation is not essential for S-phase entry when G1 cyclin-Cdks are overexpressed, and that other substrates of these kinases can be rate-limiting for the G1 to S-phase transition. These data also reveal that the SAOS-2 cotransfection assay is complicated by Rb-independent effects of the coexpressed Cdks.

Apoptosis Induced by the Nuclear Death Domain Protein p84N5 Is Associated with Caspase-6 and NF-κB Activation

Although the mechanisms involved in responses to extracellular or mitochondrial apoptotic signals have received considerable attention, the mechanisms utilized within the nucleus to transduce apoptotic signals are not well understood. We have characterized apoptosis induced by the nuclear death domain-containing protein p84N5. Adenovirus-mediated N5 gene transfer or transfection of p84N5 expression vectors induces apoptosis in tumor cell lines with nearly 100% efficiency as indicated by cellular morphology, DNA fragmentation, and annexin V staining. Using peptide substrates and Western blotting, we have determined that N5-induced apoptosis is initially accompanied by activation of caspase-6. Activation of caspases-3 and -9 does not peak until 3 days after the peak of caspase-6 activity. Expression of p84N5 also leads to activation of NF-κB as indicated by nuclear translocation of p65RelA and transcriptional activation of a NF-κB-dependent reporter promoter. Changes in the relative expression level of Bcl-2family proteins, including Bak and Bcl-Xs, are also observed during p84N5-induced apoptosis. Finally, we demonstrate that p84N5-induced apoptosis does not require p53 and is not inhibited by p53 coexpression. We propose that p84N5 is involved in an apoptotic pathway distinct from those triggered by death domain-containing receptors or by p53.

Glutamic acid mutagenesis of retinoblastoma protein phosphorylation sites has diverse effects on function

The retinoblastoma tumor suppressor gene (Rb) has many functions within the cell including regulation of transcription, differentiation, apoptosis, and the cell cycle. Regulation of these functions is mediated by phosphorylation at as many as 16 cyclin-dependent kinase (CDK) phosphorylation sites in vivo. The contribution of these sites to the regulation of the various Rb functions is not well understood. To characterize the effect of phosphorylation at these sites, we systematically mutagenized the serines or threonines to glutamic acid. Thirty-five mutants with different combinations of modified phosphorylation sites were assayed for their ability to arrest the cell cycle and for their potential to induce differentiation. Only the most highly substituted mutants failed to arrest cell cycle progression. However, mutants with as few as four modified phosphorylation sites were unable to promote differentiation. Other mutants had increased activity in this assay. We conclude that modification of Rb phosphorylation sites can increase or decrease protein activity, that different Rb functions can be regulated independently by distinct combinations of sites, and that the effects of modification at any one site are context dependent.

The retinoblastoma tumor suppressor protein is required for efficient processing and repair of trapped Topoisomerase II-DNA cleavable complexes

Type II topoisomerases (TOP2) introduce transient double-stranded DNA breaks through a covalent TOP2–DNA intermediate. Anticancer agents like etoposide kill cells by trapping covalent TOP2–DNA cleavable complexes. Pathways influencing the repair of cleavable complexes are expected to be major determinants of therapeutic response to etoposide. Rb1 is required to enforce cell cycle checkpoints in response to DNA damage, but evidence for a direct role in the processing and repair of DNA lesions is lacking. We observe that degradation of trapped TOP2-cleavable complexes, liberation of DNA strand breaks, and repair of those breaks occurs more efficiently in cells expressing Rb1 protein (pRb). Cells lacking pRb are more sensitive to etoposide-induced cytotoxicity. Rb1-mediated processing and repair of TOP2-cleavable complexes is genetically separable from its ability to bind E2F and enforce DNA damage-induced cell cycle checkpoints. Rb1 protein binds both TOP2 and BRCA1 in intact cells, and pRb is required for association between TOP2 and BRCA1. These results suggest that pRb facilitates processing and repair of TOP2-cleavable complexes by recruiting proteins like BRCA1 to the damaged site. The functional status of pRb, therefore, may influence sensitivity to etoposide by facilitating the repair of trapped TOP2–DNA complexes as well as by enforcing cell cycle checkpoints.

Adenovirus-mediated N5 gene transfer inhibits tumor cell proliferation by induction of apoptosis.

Gene therapy designed to initiate apoptotic cell death provides a potentially effective method to treat cancer. A prerequisite for this approach is the identification of genes that function in distinct apoptotic pathways. Although apoptotic pathways initiated by receptors such as tumor necrosis factor receptor-1 are well characterized, little is known about apoptotic pathways initiated within the nucleus in response to genotoxic stress. We have demonstrated previously that the nuclear, death domain-containing protein p84N5 can induce apoptosis upon transfection into cells, suggesting that it may play a role in an apoptotic pathway initiated within the nucleus. To test the possibility that N5 could be used in the gene therapy of cancer, we have generated a recombinant adenovirus engineered to express N5 and tested the effects of viral infection on the growth and tumorigenicity of tumor cells. N5 adenovirus infection significantly reduced the proliferation and tumorigenicity of breast, ovarian, and osteosarcoma tumor cell lines. Reduced proliferation and tumorigenicity were mediated by an induction of apoptosis as indicated by DNA fragmentation in infected cells. The results suggest that the N5 cDNA is a candidate for the gene therapy of cancer.

Nuclear localization is required for induction of apoptotic cell death by the Rb-associated p84N5 death domain protein.

The mechanisms utilized to transduce apoptotic signals that originate from within the nucleus, in response to DNA damage for example, are not well understood. Identifying these mechanisms is important for predicting how tumor cells will respond to genotoxic radiation or chemotherapy. The Rb tumor suppressor protein can inhibit apoptosis triggered by DNA damage, but how it does so is unclear. We have previously characterized a death domain protein, p84N5, that specifically associates with an amino-terminal domain of Rb protein. The p84N5 death domain is required for its ability to trigger apoptotic cell death. Association with Rb protein inhibits p84N5-induced apoptosis suggesting that it may be a mediator of Rbs effects on apoptosis. Unlike other death domain-containing apoptotic signaling proteins, however, p84N5 is localized predominantly within the nucleus of interphase cells. Here we test whether p84N5 requires nuclear localization in order to trigger apoptosis. We identify the p84N5 nuclear localization signal and demonstrate that nuclear localization is required for p84N5-induced apoptosis. To our knowledge, this identifies p84N5 as the first death-domain containing apoptotic signaling protein that functions within the nucleus. By analogy to other death domain containing proteins, p84N5 may play some role in apoptotic signaling within the nucleus. Further, p84N5 is a potential mediator of Rb proteins effects on DNA damage induced apoptosis.