Multiple response elements and differential p53 binding control Perp expression during apoptosis

The p53 tumor suppressor gene responds to cellular stress by activating either cell cycle arrest or apoptosis. A growing number of target genes involved in each of these pathways have been identified. However, the mechanism by which the apoptosis versus arrest decision is made remains to be elucidated. Perp is a proapoptotic target gene of p53 expressed to high levels in apoptotic cells compared with those undergoing cell cycle arrest. This pattern of expression is unusual among p53 target genes, many of which are induced to similar levels during arrest and apoptosis. Here, we describe the regulation of the Perp gene by p53 through at least three response elements in the Perp promoter and first intron. These sites are occupied in vivo in E1A-expressing mouse embryo fibroblasts undergoing apoptosis but not cell cycle arrest, in contrast to the p21 5' response element, which is occupied during both. The apoptosis-deficient p53 point mutant, p53V143A, displays a selective deficit in binding to the Perp elements, demonstrating that p53 can distinguish between Perp and p21 at the level of DNA binding. These results provide mechanistic insight into the selective expression of Perp during apoptosis and may provide a useful model for studying the p53-dependent cell cycle arrest versus apoptosis decision.     

Reactive oxygen species and mitochondrial sensitivity to oxidative stress determine induction of cancer cell death by p21

p21(Waf1/Cip1/Sdi1) is a cyclin-dependent kinase inhibitor that mediates cell cycle arrest. Prolonged p21 up-regulation induces a senescent phenotype in normal and cancer cells, accompanied by an increase in intracellular reactive oxygen species (ROS). However, it has been shown recently that p21 expression can also lead to cell death in certain models. The mechanisms involved in this process are not fully understood. Here, we describe an induction of apoptosis by p21 in sarcoma cell lines that is p53-independent and can be ameliorated with antioxidants. Similar levels of p21 and ROS caused senescence in the absence of significant death in other cancer cell lines, suggesting a cell-specific response. We also found that cells undergoing p21-dependent cell death had higher sensitivity to oxidants and a specific pattern of mitochondrial polarization changes. Consistent with this, apoptosis could be blocked with targeted expression of catalase in the mitochondria of these cells. We propose that the balance between cancer cell death and arrest after p21 up-regulation depends on the specific effects of p21-induced ROS on the mitochondria. This suggests that selective up-regulation of p21 in cancer cells could be a successful therapeutic intervention for sarcomas and tumors with lower resistance to mitochondrial oxidative damage, regardless of p53 status.     

Cleavage of p21/WAF1/CIP1 by proteinase 3 modulates differentiation of a monocytic cell line. Molecular analysis of the cleavage site

Proteinase 3 (PR3), also called myeloblastin, is involved in the control of myeloid cell growth, but the underlying molecular mechanisms have not been elucidated. In U937/PR3, stably transfected with PRCRSV/PR3 to overexpress PR3, PMA-induced p21 expression was significantly decreased as compared with control U937, and this phenomenon was reversed in the presence of the serine proteinase inhibitor, pefabloc. Conversely, when PR3 was inactivated by small interfering RNA, p21 protein was increased, and PMA-induced monocytic differentiation was potentiated. Mass spectrometry analysis identified Ala45 as the primary cleavage site on p21, and the recombinant mutated p21A45R, generated by site-directed mutagenesis and expressed in Escherichia coli, was resistant to in vitro PR3 cleavage. The U937 cells were then stably transfected with either PRCRSV/p21 or PRCRSV/p21A45R, to ectopically express wild type p21 or PR3-resistant p21, respectively. In U937/p21A45R treated with PS-341, a selective proteasome inhibitor, a significant decrease in the S phase and a blockade in the G0-G1 phase of cell cycle were observed when compared with U937/p21 or control U937. This suggested that both PR3 and the proteasome are efficiently involved in the proteolytic regulation of p21 expression in myeloid cells. Moreover, PMA-induced p21 expression was more pronounced in U937/p21A45R compared with U937/p21 and was concomitant with the morphological features of early differentiation. Our data demonstrated that p21 is one specific target of PR3 and that PR3-mediated p21 cleavage prevents monocytic differentiation.     

Hygrolidin induces p21 expression and abrogates cell cycle progression at G1 and S phases

Hygrolidin family antibiotics showed selective cytotoxicity against both cyclin E- and cyclin A-overexpressing cells. Among them, hygrolidin was the most potent and inhibited growth of solid tumor-derived cell lines such as DLD-1 human colon cancer cells efficiently more than that of hematopoietic tumor cells and normal fibroblasts. FACS analysis revealed that hygrolidin increased cells in G1 and S phases in DLD-1 cells. While hygrolidin decreased amounts of cyclin-dependent kinase (cdk) 4, cyclin D, and cyclin B, it increased cyclin E and p21 levels. Hygrolidin-induced p21 bound to and inhibit cyclin A-cdk2 complex more strongly than cyclin E-cdk2 complex. Furthermore, hygrolidin was found to increase p21 mRNA in DLD-1 cells, but not in normal fibroblasts. Thus, hygrolidin inhibited tumor cell growth through induction of p21. In respect to p21 induction, inhibition of vacuolar-type (H+)-ATPase by hygrolidin was suggested to be involved.     

雷氏大疣蛛毒液对人肝癌HepG2细胞p21基因表达的影响

本文研究了雷氏大疣蛛毒液对人肝癌细胞株HepG2增殖抑制作用及其分子机制。采用XTT法观察到雷氏大疣蛛毒液剂量依赖抑制HepG2细胞增殖;流式细胞仪检测发现,经过雷氏大疣蛛毒液作用的HepG2细胞周期发生明显的选择性改变;RT-PCR方法检测到p21基因表达增强;Western blot检测发现,p21蛋白表达增加。结果提示,雷氏大疣蛛毒液抑制人肝癌细胞HepG2增殖的可能机制之一是使p21基因和蛋白表达增加,G2IM细胞周期被阻滞,从而诱导细胞凋亡。     

Multifaceted p21 in carcinogenesis,stemness of tumor and tumor therapy

Cancer cells possess metabolic properties that are different from those of benign cells. p21, encoded by CDKN1A gene, also named p21^Cip1/WAF1, was first identified as a cyclin-dependent kinase regulator that suppresses cell cycle G1/S phase and retinoblastoma protein phosphorylation. CDKN1A (p21) acts as the downstream target gene of TP53 (p53), and its expression is induced by wild-type p53 and it is not associated with mutant p53. p21 has been characterized as a vital regulator that involves multiple cell functions, including G1/S cell cycle progression, cell growth, DNA damage, and cell stemness. In 1994, p21 was found as a tumor suppressor in brain, lung and colon cancer by targeting p53 and was associated with tumorigenesis and metastasis. Notably, p21 plays a significant role in tumor development through p53-dependent and p53-independent pathways. In addition, expression of p21 is closely related to the resting state or terminal differentiation of cells. p21 is also associated with cancer stem cells and acts as a biomarker for such cells. In cancer therapy, given the importance of p21 in regulating the G1/S and G2 check points, it is not surprising that p21 is implicated in response to many cancer treatments and p21 promotes the effect of oncolytic virotherapy.     

Confirmation of the human thymidine kinase locus, 17q21 → 17qter,by means of a man-mouse somatic cell hybrid,D98/AH-2 X LMTK−Cl-1D

Summary A large metacentric marker chromosome, m20, in a line of human D98/AH-2 cells was identified by Q bands as being a translocation (1;17)(p36;q21). This was confirmed by means of somatic cell hybridization between D98/AH-2 and thymidine kinase (TK) deficient mouse cells. The hybrid clones by HAT selective system retained m20, indicating the presence of TK locus on this chromosome. The results also provide evidence that TK gene is located on the distal region of the breakpoint in 17q21 but not on 17q21 17pter.     

Induced p21Cip1 in premature baboons with CLD: implications for alveolar hypoplasia

Aberrant pulmonary epithelial and mesenchymal cell proliferation occurs when newborns are treated with oxygen and ventilation to mitigate chronic lung disease. Because the cyclin-dependent kinase inhibitor p21 inhibits proliferation of oxygen-exposed cells, its expression was investigated in premature baboons delivered at 125 days (67% of term) and treated with oxygen and ventilation pro re nata (PRN) for 2, 6, 14, and 21 days. Approximately 5% of all cells expressed p21 during normal lung development of which <1% of these cells were pro-surfactant protein (SP)-B-positive epithelial cells. The percentage of cells expressing p21 increased threefold in all PRN-treated animals, but different cell populations expressed it during disease progression. Between 2 and 6 days of treatment, p21 was detected in 30-40% of pro-SP-B cells. In contrast, only 12% of pro-SP-B cells expressed p21 by 14 and 21 days of treatment, by which time p21 was also detected in mesenchymal cells. Even though increased epithelial and mesenchymal cell proliferation occurs during disease progression, those cells expressing p21 did not also express the proliferative marker Ki67. Thus two populations of epithelial and mesenchymal cells can be identified that are either expressing Ki67 and proliferating or expressing p21 and not proliferating. These data suggest that p21 may play a role in disorganized proliferation and alveolar hypoplasia seen in newborn chronic lung disease.     

Inhibition of G1 to S Cell Cycle Progression by BCCIPb

The BCCIPa protein was identified as a BRCA2 and CDKN1A (p21, or p21Waf1/Cip1) Interacting Protein. It binds to a highly conserved domain proximate to the C-terminus of BRCA2 protein and the C-terminal domain of the CDK-inhibitor p21. Previous reports showed that BCCIPa enhances the inhibitory activity of p21 toward CDK2 and that BCCIPa inhibits the growth of certain tumor cells. Here we show that a second isoform, BCCIPb, also binds to p21 and inhibits cell growth. The growth inhibition by BCCIPb can be partially abrogated in p21 deficient cells. Overexpression of BCCIPb delays the G1 to S progression and results in an elevated p21 expression. These data suggest BCCIPb as a new regulator for the G1-S cell cycle progression and cell growth control.     

Inhibition of G1 to S cell cycle progression by BCCIP beta

The BCCIP alpha protein was identified as a BRCA2 and CDKN1A (p21, or p21(Waf1/Cip1)) Interacting Protein. It binds to a highly conserved domain proximate to the C-terminus of BRCA2 protein and the C-terminal domain of the CDK-inhibitor p21. Previous reports showed that BCCIP alpha enhances the inhibitory activity of p21 toward CDK2 and that BCCIP alpha inhibits the growth of certain tumor cells. Here we show that a second isoform, BCCIP beta, also binds to p21 and inhibits cell growth. The growth inhibition by BCCIP beta can be partially abrogated in p21 deficient cells. Overexpression of BCCIP beta delays the G1 to S progression and results in an elevated p21 expression. These data suggest BCCIP beta as a new regulator for the G1-S cell cycle progression and cell growth control.     

Alphavirus M1 induces apoptosis of malignant glioma cells via downregulation and nucleolar translocation of p21WAF1/CIP1 protein

Alphavirus, a genus of arthropod-borne togavirus, is well-known for its pro-apoptotic capability. However, the underlying mechanism remains to be further clarified. Here, we have identified that M1, an alphavirus isolated in 1960s, targeted C6 malignant glioma cells for apoptosis. Flow cytometry analysis showed that more cells enter S-phase post M1 infection, and subsequently undergo a classic apoptosis. To elucidate the mechanism of S-phase arrest and its relationship to apoptosis, we tested the expression of several critical cell cycle regulatory proteins and found elevated phosphorylation of cyclin-dependent kinase 2 (CDK2), decreased expression of cyclin A and proliferating cell nuclear antigen (PCNA). Notably, the protein level of p21^WAF1/CIP1 was downregulated earliest and most effectively among all tested changes of cell cycle regulators, though its mRNA level was strongly upregulated. To evaluate the role of p21^WAF1/CIP1 in S-phase accumulation and subsequent apoptosis, we confirmed that exogenous p21^WAF1/CIP1 overexpression or treatment with roscovitine (a selective chemical inhibitor of CDK2) efficiently protected against apoptosis with a reduced S-phase accumulation Thus, it is indicated that the downregulation of p21^WAF1/CIP1 mediated C6 apoptosis via overactivation of CDK2. In addition, confocal microscopy showed that p21^WAF1/CIP1 totally translocated to nucleolus during M1-induced C6 apoptosis. Altogether, downregulation and nucleolar translocation of the p21^WAF1/CIP1 protein played an active role in M1-induced C6 apoptosis.     

Chalcone HTMC causes in vitro selective cytotoxicity, cell-cycle G1 phase arrest through p53-dependent pathway in human lung adenocarcinoma A549 cells, and in vivo tumor growth suppression

The present Letter identified 2'-hydroxy-2,3,4',6'-tetramethoxychalcone (HTMC) as a potent in vitro cytotoxic agent with selective activity against cell lines derived from human lung cancer. In A549 lung adenocarcinoma cells, HTMC caused G1 phase cell-cycle arrest. HTMC treatment also led to an inhibition of cell-cycle regulatory proteins phosphorylation of cdc2 (Tyr(15) and Tyr(161)) and Rb (Ser(795) and Ser(807/811)), which was accompanied by the accumulation of tumor suppressor genes p53 and p21. In addition, in vivo data demonstrated that HTMC act as a tumor growth suppressing agent.     

IL-1-induced ERK1/2 activation up-regulates p21 protein by inhibition of degradation via ubiquitin-independent pathway in human melanoma cells A375

IL-1 inhibits the proliferation of human melanoma cells A375 by arresting the cell cycle at G0/G1 phase, which accompanies the increase of p21^Waf1/Cip1 (p21) protein. Here, we demonstrate that IL-1 induces the stabilization of p21 protein via ERK1/2 pathway. The degradation of p21 was inhibited by IL-1, however the ubiquitination level of p21 was not affected. In addition, the degradation of non-ubiquitinated form of lysine less mutant p21-K6R was also inhibited by IL-1, suggesting that IL-1 stabilized p21 protein via ubiquitin-independent pathway. Furthermore, the inhibition of p21 protein degradation was prevented by a selective inhibitor of ERK1/2 pathway, PD98059. These results suggest that IL-1-induced ERK1/2 activation leads to the up-regulation of p21 by inhibiting degradation via ubiquitin-independent pathway in human melanoma cells A375.     

Cell Cycle Regulation Targets of MYCN Identified by Gene Expression Microarrays

Background: We have previously shown that MYCN knockdown causes a G1 arrest in MYCN amplified (MNA), p53 wild type (wt) and p53 mutant MNA neuroblastoma cell lines, with increases in p21WAF1 and hypo RB in p53 wt cell lines. 1 Hypothesis: MYCN acts by inhibiting p21WAF1, and also by p21 independent mechanisms to override the G1 checkpoint in exponentially growing cells. Methods: Genes potentially regulated by MYCN were identified using gene expression microarrays in p53 wt MNA IMR-32 and p53 mutant MNA SKNBE(2c) neuroblastoma cell lines treated with MYCN or scrambled siRNA. Results were validated using qRT-PCR and confirmed using the regulatable MYCN expression system (SHEP Tet21N). Results: MYCN knockdown altered the expression of several cell cycle related genes. SKP2 was down regulated in both cell lines, and up regulated in MYCN+ Tet21N cells. Expression of the WNT antagonist DKK3 increased in both cell lines and decreased in MYCN+ Tet21N cells. Expression of CDKN1C (p57cip2) and TP53INP1 also increased after MYCN knockdown. Conclusions: MYCN may override the G1 checkpoint through down-regulation of SKP2 and TP53INP1 resulting in reduced p21WAF1 expression in p53 wt cell lines, and in addition may act through the WNT signalling pathway in a p53 independent manner.     

Proteomic analysis of factors released from p21-overexpressing tumour cells

The p21Waf1/Cip1/Sdi1 cyclin-dependent kinase inhibitor is a key regulator of cell cycle progression and has also been observed to influence the expression of genes associated with several age-related disorders. Previous work has shown that expression of p21 in tumour cells mediates an antiapoptotic and mitogenic paracrine effect, which is in contrast to the arrested state of p21-expressing cells. Here, we have employed SELDI-MS technology to characterise, at a proteomic level, factors released from HT-1080 human fibrosarcoma cells displaying inducible p21 expression. Conditioned media from induced and noninduced cells were profiled on a range of diverse ProteinChip arrays and subjected to SELDI-MS analysis. Evaluation of proteins binding onto IMAC, Q10 or CM10 surfaces led to the discovery of a number of putative p21-regulated factors. We further validated three p21-regulated proteins observed at 10.2, 11.7 and 13.4 kDa. Using Q Ceramic HyperD fractionation columns, we were able to selectively enrich for each of these three proteins. Subsequent SDS-PAGE and MS analysis of tryptic digests identified the 13.4 kDa protein as cystatin C and the 10.2 kDa protein as pro-platelet basic protein (PPBP). Judging by the apparent MW and the pI of the 11.7 kDa protein, we reasoned that it may be beta-2-microglobulin, which was confirmed by subsequent identification. Increased levels of cystatin C and beta-2-microglobulin in conditioned media from p21-expressing cells was confirmed by antibody capture experiments using anticystatin C and anti-beta-2-microglobulin antibodies on preactivated PS-20 arrays. Western blot analysis demonstrated increased expression of intracellular and extracellular cystatin C and beta-2-microglobulin in p21-expressing cells, compared to noninduced controls. Increased levels of PPBP were validated in cell lysates from p21-expressing cells. The three secreted factors that we have identified in this study, have all been shown previously to have growth modulating effects and, as such, may contribute to the observed mitogenic and anti-apoptotic paracrine activity of p21-expressing [corrected] cells.     

Immunodominant T cell determinants of aquaporin-4, the autoantigen associated with neuromyelitis optica

Autoantibodies that target the water channel aquaporin-4 (AQP4) in neuromyelitis optica (NMO) are IgG1, a T cell-dependent Ig subclass. However, a role for AQP4-specific T cells in this CNS inflammatory disease is not known. To evaluate their potential role in CNS autoimmunity, we have identified and characterized T cells that respond to AQP4 in C57BL/6 and SJL/J mice, two strains that are commonly studied in models of CNS inflammatory diseases. Mice were immunized with either overlapping peptides or intact hAQP4 protein encompassing the entire 323 amino acid sequence. T cell determinants identified from examination of the AQP4 peptide (p) library were located within AQP4 p21-40, p91-110, p101-120, p166-180, p231-250 and p261-280 in C57BL/6 mice, and within p11-30, p21-40, p101-120, p126-140 and p261-280 in SJL/J mice. AQP4-specific T cells were CD4+ and MHC II-restricted. In recall responses to immunization with intact AQP4, T cells responded primarily to p21-40, indicating this region contains the immunodominant T cell epitope(s) for both strains. AQP4 p21-40-primed T cells secreted both IFN-γ and IL-17. The core immunodominant AQP4 21-40 T cell determinant was mapped to residues 24-35 in C57BL/6 mice and 23-35 in SJL/J mice. Our identification of the AQP4 T cell determinants and characterization of its immunodominant determinant should permit investigators to evaluate the role of AQP4-specific T cells in vivo and to develop AQP4-targeted murine NMO models.