Bcl-2 Protein Targeting by the p53/p21 Complex—Letter
Liz J. Hernández BorreroRahmat K. SikderAmriti R. LullaPrashanth GokarePaulo Roberto Del ValleXiaobing TianShengliang ZhangPhilip H. AbboshWafik S. El‐Deiry
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Kim and colleagues reported a p53/p21 complex regulates cancer cell invasion and apoptosis by targeting the Bcl-2 family (1). Interaction of overexpressed p53 and p21 proteins in p53-null H1299 cells correlated with decreased cell invasion. No coimmunoprecipitation experiments documenting endogenous p53:p21 protein interaction were performed. There was no consideration of how the p53/p21/Bcl-w complex might relate to p53/MDM2 or p21/cyclin/CDK/PCNA complexes. The authors showed similar findings in IMR-32 cells that carry cytoplasmic wild-type (wt-p53). Although they demonstrated si-p53 or si-p21 affect Bcl-w:Bax interaction, they did not prove endogenous p53 interacts with p21.There was a modest effect of p53 on suppression of invasion (Fig. 1; ref. 1), and this phenotype was suppressed by si-p21. However, there was no effort to determine whether effects on invasion could be explained by altered cell-cycle phase or proliferation state. This is important as tumor cell growth and proliferation is required for invasiveness (2). On the other hand, some evidence suggests growth arrest may be required for invasion (3). Kim and colleagues state that cell invasion is regulated by p53/p21, but little insight is provided beyond cell-cycle and growth regulation. The authors suggest suppression of invasion is mediated by Bax. However, Bax is a direct target of p53, and its effects on apoptosis are well established (dead cells do not invade). The authors do not consider direct regulation of p53 toward Bax and whether this could explain their findings. Endogenous p53 decreases invasion by promoting MDM2:Slug interaction, resulting in Slug ubiquitin–mediated degradation (4). The authors did not experimentally consider these mechanisms.Kim and colleagues show results inconsistent with the paradigm in the p53 field. First, WT-p53 is stabilized following exposure of cells to DNA damage such as ionizing radiation (5). In Fig. 5B of ref. 1, lane 6 shows no increase in nuclear p53 expression following 20 Gy of radiation; rather, p53 remains cytoplasmic. Second, it is well known that overexpression of p53 suppresses cancer cell growth. Figure 6 of ref. 1 shows similar tumor growth of exogenously transfected wt-p53 versus vector control and greater than p53ΔC37 at 0 Gy. These unexpected results should be better documented, including WT-p53 localization by immunofluorescence and WT-p53 functionality in vivo, to validate whether these are cell type–specific phenotypes. Overexpression of p53ΔC37 was not characterized, and the mutant lacks one of two nuclear localization signal. Whether effects are due to failure of p21 interaction or inability of p53 nuclear import to activate transcription of p53 targets was not addressed.p21/CDKN1A is not infrequently mutated in bladder cancer (The Cancer Genome Atlas; ref. 6). We used p21-CRISPR in bladder cancer cells to directly test the hypothesis that p21 mutation might contribute to greater migration or invasiveness of tumor cells. CRISPR knockout of CDKN1A resulted in decreased cellular motility (Fig. 1A). This effect was muted when indels were targeted to the C-terminus of the protein, when a truncated peptide was still detectable and retaining the CDK-interacting domain (Fig. 1B). Importantly, SW780 cells carry WT-p53, and 50% of bladder cancers retain two intact copies of TP53. Thus, p21 ablation or mutation does not increase cell migration that is needed for invasion.p21 appears to protect from cell death through various mechanisms (6), including growth arrest and cytoplasmic effects of p21. The role of p21 in apoptosis following p53 overexpression in H1299 cells in Kim and colleagues should have been better substantiated as a cell type–specific phenotype.For multiple reasons, the evidence supporting the model put forth by Kim and colleagues is lacking in rigor and is not convincing due to alternative explanations. Kim and colleagues reported similar findings that have similar limitations (7, 8). It is important to document endogenous protein:protein interactions and to include critical controls to support novel paradigm-shifting results. It remains unclear whether a physiologic complex of p53/p21/Bcl-w regulates invasion or apoptosis.See the Response, p. 2772No potential conflicts of interest were disclosed.Keywords:
Retinoblastoma protein
Immunoprecipitation
CDK inhibitor
Cyclin B1
G1 phase
Retinoblastoma protein
Retinoblastoma
CDK inhibitor
Cyclin-dependent kinase 4
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Abstract Pre-mRNA splicing is an indispensable mechanism for eukaryotic gene expression. Splicing inhibition causes cell cycle arrest at G1 and G 2 /M phases, which is thought to be one of the reasons for the potent antitumor activity of splicing inhibitors. However, the molecular mechanisms underlying the cell cycle arrest have many unknown aspects. In particular, the mechanism of G 2 /M-phase arrest caused by splicing inhibition is completely unknown. Here, we found that lower and higher concentrations of pladienolide B caused M-phase and G2-phase arrest, respectively. We analyzed protein levels of cell cycle regulators and found that a truncated form of the p27 CDK inhibitor, named p27*, accumulates in G 2 -arrested cells. Overexpression of p27* caused partial G 2 -phase arrest. Conversely, knockdown of p27* accelerated exit from G2/M phase after washout of splicing inhibitor. These results suggest that p27* contributes to G2/M-phase arrest caused by splicing inhibition. We also found that p27* bound to and inhibited M-phase cyclins, although it is well known that p27 regulates G 1 /S transition. Intriguingly, p27*, but not full-length p27, was resistant to proteasomal degradation and remained in G 2 /M phase. These results suggest that p27*, which is a very stable truncated protein in G 2 /M phase, contributes to G 2 -phase arrest caused by splicing inhibition.
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SR protein
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The retinoblastoma (RB) tumor suppressor protein is a negative regulator of cell proliferation that is functionally inactivated in the majority of human tumors. Elevated Cdk activity via RB pathway mutations is observed in virtually every human cancer. Thus, Cdk inhibitors have tremendous promise as anticancer agents although detailed mechanistic knowledge of their effects on RB function is needed to harness their full potential. Here, we illustrate a novel function for Cdks in regulating the subcellular localization of RB. We present evidence of significant cytoplasmic mislocalization of ordinarily nuclear RB in cells harboring Cdk4 mutations. Our findings uncover a novel mechanism to circumvent RB-mediated growth suppression by altered nucleocytoplasmic trafficking via the Exportin1 pathway. Cytoplasmically mislocalized RB could be efficiently confined to the nucleus by inhibiting the Exportin1 pathway, reducing Cdk activity, or mutating the Cdk-dependent phosphorylation sites in RB that result in loss of RB-Exportin1 association. Thus RB-mediated tumor suppression can be subverted by phosphorylation-dependent enhancement of nuclear export. These results support the notion that tumor cells can modulate the protein transport machinery thereby making the protein transport process a viable therapeutic target.
Retinoblastoma protein
Retinoblastoma
CDK inhibitor
Nuclear export signal
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Artepillin C, 3, 5-diprenyl-4-hydroxycinnamic acid, is one of the bioactive constituents in Brazilian propolis. In the present study, the anticarcinogenic activity of this compound was investigated in human hepatoma HepG2 cells. Artepillin C inhibited the cell proliferation in a dose- and time-dependent manner accompanied by G0/G1 phase arrest in the cell cycle. This compound caused a decrease in the phosphorylation levels of the retinoblastoma protein at Ser780 and Ser807/811 and a decrease in the kinase activity of the cyclinD and CDK4 complex without any change in these protein levels. Artepillin C increased the protein level of p27Kip1, known as a CDK inhibitor. This up-regulation was regulated by both the transcriptional and post-transcriptional levels, i.e., the treatment increased the mRNA of p27Kip1 and decreased the proteosome activity. Thus, artepillin C induces cell cycle arrest at G0/G1 phase accompanied by up-regulation of p27Kip1, resulting in the inhibition of cell proliferation in HepG2 cells. This study suggested that artepillin C will be a promising anti-cancer agent against hepatoma cancer.
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G1 phase
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Retinoblastoma protein
CDK inhibitor
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Retinoblastoma
Retinoblastoma protein
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Cells switch between quiescence and proliferation states for maintaining tissue homeostasis and regeneration. At the restriction point (R-point), cells become irreversibly committed to the completion of the cell cycle independent of mitogen. The mechanism involving hyper-phosphorylation of retinoblastoma (Rb) and activation of transcription factor E2F is linked to the R-point passage. However, stress stimuli trigger exit from the cell cycle back to the mitogen-sensitive quiescent state after Rb hyper-phosphorylation but only until APC/CCdh1 inactivation. In this study, we developed a mathematical model to investigate the reversible transition between quiescence and proliferation in mammalian cells with respect to mitogen and stress signals. The model integrates the current mechanistic knowledge and accounts for the recent experimental observations with cells exiting quiescence and proliferating cells. We show that Cyclin E:Cdk2 couples Rb-E2F and APC/CCdh1 bistable switches and temporally segregates the R-point and the G1/S transition. A redox-dependent mutual antagonism between APC/CCdh1 and its inhibitor Emi1 makes the inactivation of APC/CCdh1 bistable. We show that the levels of Cdk inhibitor (CKI) and mitogen control the reversible transition between quiescence and proliferation. Further, we propose that shifting of the mitogen-induced transcriptional program to G2-phase in proliferating cells might result in an intermediate Cdk2 activity at the mitotic exit and in the immediate inactivation of APC/CCdh1. Our study builds a coherent framework and generates hypotheses that can be further explored by experiments.
Restriction point
Retinoblastoma protein
E2F
CDK inhibitor
Cyclin A
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The G1-S phase transition is critical to maintaining proliferative control and preventing carcinogenesis. The retinoblastoma tumor suppressor is a key regulator of this step in the cell cycle.Here we use a structure-function approach to evaluate the contributions of multiple protein interaction surfaces on pRB towards cell cycle regulation. SAOS2 cell cycle arrest assays showed that disruption of three separate binding surfaces were necessary to inhibit pRB-mediated cell cycle control. Surprisingly, mutation of some interaction surfaces had no effect on their own. Rather, they only contributed to cell cycle arrest in the absence of other pRB dependent arrest functions. Specifically, our data shows that pRB-E2F interactions are competitive with pRB-CDH1 interactions, implying that interchangeable growth arrest functions underlie pRB's ability to block proliferation. Additionally, disruption of similar cell cycle control mechanisms in genetically modified mutant mice results in ectopic DNA synthesis in the liver.Our work demonstrates that pRB utilizes a network of mechanisms to prevent cell cycle entry. This has important implications for the use of new CDK4/6 inhibitors that aim to activate this proliferative control network.
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E2F
G1 phase
Ectopic expression
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