Regulation of cyclin-dependent kinase inhibitor p21<Superscript>WAF1/CIP1</Superscript> by protein kinase Cδ-mediated phosphorylation

Cyclin-dependent kinase (CDK) inhibitor p21^{WAF1/CIP1(-/-)}-null mice have an increased incidence of tumor formation. Here, we demonstrate that p21^WAF1/CIP1 is unstable in HeLa cells treated with siRNA duplexes that target PKCδ. PKCδ phosphorylates p21^WAF1/CIP1 at a serine residue (¹⁴⁶Ser) located in its C-terminal domain. In cells treated with 12-O-tetradecanoylphorbol 13-acetate, the levels of both p21^WAF1/CIP1 and its ¹⁴⁶Ser-phosphorylated form increased significantly. We also show that a substitution, resulting from a single nucleotide polymorphism (SNP) at ¹⁴⁹Asp found in certain cancer patients, strongly compromises PKCδ-mediated phosphorylation at ¹⁴⁶Ser and results in cells that are relatively resistant to TNFα-induced apoptosis. Thus, post-translational phosphorylation of p21^WAF1/CIP1 is important from an apoptotic cell death, and may also have patho-physiological relevance for the development of human cancer.     

p21WAF1 modulates drug-induced apoptosis and cell cycle arrest in B-cell precursor acute lymphoblastic leukemia

p21^WAF1 is a well-characterized mediator of cell cycle arrest and may also modulate chemotherapy-induced cell death. The role of p21^WAF1 in drug-induced cell cycle arrest and apoptosis of acute lymphoblastic leukemia (ALL) cells was investigated using p53-functional patient-derived xenografts (PDXs), in which p21^WAF1 was epigenetically silenced in T-cell ALL (T-ALL), but not in B-cell precursor (BCP)-ALL PDXs. Upon exposure to diverse cytotoxic drugs, T-ALL PDX cells exhibited markedly increased caspase-3/7 activity and phosphatidylserine (PS) externalization on the plasma membrane compared with BCP-ALL cells. Despite dramatic differences in apoptotic characteristics between T-ALL and BCP-ALL PDXs, both ALL subtypes exhibited similar cell death kinetics and were equally sensitive to p53-inducing drugs in vitro, although T-ALL PDXs were significantly more sensitive to the histone deacetylase inhibitor vorinostat. Transient siRNA suppression of p21^WAF1 in the BCP-ALL 697 cell line resulted in a moderate depletion of the cell fraction in G1 phase and marked increase in PS externalization following exposure to etoposide. Furthermore, stable lentiviral p21^WAF1 silencing in the BCP-ALL Nalm-6 cell line accelerated PS externalization and cell death following exposure to etoposide and vorinostat, supporting previous findings. Finally, the Sp1 inhibitor, terameprocol, inhibited p21^WAF1 expression in Nalm-6 cells exposed to vorinostat and also partially augmented vorinostat-induced cell death. Taken together, these findings demonstrate that p21^WAF1 regulates the early stages of drug-induced apoptosis in ALL cells and significantly modulates their sensitivity to vorinostat.     

Upregulation of p21(WAF1/Cip1) precedes tumor necrosis factor-induced necrosis-like cell death

The molecular mechanisms mediating death receptor-induced caspase-independent necrotic cell death are still largely unknown. We have previously reported that NIH3T3 cells are sensitized by caspase inhibition to death receptor-induced cytotoxicity leading to a necrosis-like cell death. In addition, we have identified an important role of cell cycle progression for this sensitization effect. Here, we report that tumor necrosis factor-induced necrotic death is preceded by an upregulation of the cyclin-dependent kinase inhibitor p21(WAF1/Cip1). Increased expression of p21(WAF1/Cip1) occurs prior to cell death in the nucleus, where it binds to a cyclin-dependent kinase indicating its functionality. The use of specific pharmacological inhibitors revealed a partial involvement of p38 mitogen-activated protein kinase in the upregulation of p21(WAF1/Cip1). Inhibition of p21(WAF1/Cip1) upregulation prevents a previously observed delay of the cells in the G2/M phase of the cell cycle thereby augmenting, not inhibiting cell death.     

Mitochondrial ribosomal protein L41 mediates serum starvation-induced cell-cycle arrest through an increase of p21(WAF1/CIP1)

Ribosomal proteins not only act as components of the translation apparatus but also regulate cell proliferation and apoptosis. A previous study reported that MRPL41 plays an important role in p53-dependent apoptosis. It also showed that MRPL41 arrests the cell cycle by stabilizing p27(Kip1) in the absence of p53. This study found that MRPL41 mediates the p21(WAF1/CIP1)-mediated G1 arrest in response to serum starvation. The cells were released from serum starvation-induced G1 arrest via the siRNA-mediated blocking of MRPL41 expression. Overall, these results suggest that MRPL41 arrests the cell cycle by increasing the p21(WAF1/CIP1) and p27(Kip1) levels under the growth inhibitory conditions.     

c-Jun NH2-terminal kinase decreases ubiquitination and promotes stabilization of p21(WAF1/CIP1) in K562 cell

Proteasome-dependent degradation of regulatory proteins is a known mechanism of cell cycle control. p21(WAF1/CIP1) (p21), a negative regulator of the cell division cycle, exhibits proteasome-sensitive turnover and ubiquitination. In the present study, we analyzed the regulatory effects of JNK1 on p21 protein accumulation in p53 null K562 cells. We found that JNK1 (wild type, WT) mediated H(2)O(2)-induced p21 protein up-regulation. Over-expression of JNK1 (WT) could elevate endogenous p21 protein level but did not affect p21 mRNA level and also prolong the p21 half-life as well as inhibited the p21 ubiquitination. These findings indicated that JNK1 could regulate cellular p21 level via inhibiting ubiquitination of p21, which provided a new insight for analyzing the regulatory effect of JNK after stress.     

PKCdelta modulates p21WAF1/CIP1 ability to bind to Cdk2 during TNFalpha-induced apoptosis

Cyclin-dependent kinase 2 (Cdk2) activity is thought to be involved in cell death-associated chromatin condensation and other manifestations of apoptotic death. Here we show that during TNFalpha-induced apoptosis, PKCdelta is activated in a caspase-3-dependent manner and phosphorylates p21(WAF1/CIP1), a specific cyclin-dependent kinase inhibitor, on (146)Ser. This residue is located near a cyclin-binding motif (Cy2) that plays an important role in the interaction between p21(WAF1/CIP1) and Cdk2, and its phosphorylation modulates the ability of p21(WAF1/CIP1) to associate with Cdk2. The phosphorylation of p21(WAF1/CIP1) is temporally related to the activation kinetics of Cdk2 activity during the apoptosis. We propose that during TNFalpha-induced apoptosis, PKCdelta-mediated phosphorylation of p21(WAF1/CIP1) at (146)Ser attenuates the Cdk2 binding of p21(WAF1/CIP1) and thereby upregulates Cdk2 activity.     

ZNF313 is a novel cell cycle activator with an E3 ligase activity inhibiting cellular senescence by destabilizing p21WAF1

ZNF313 encoding a zinc-binding protein is located at chromosome 20q13.13, which exhibits a frequent genomic amplification in multiple human cancers. However, the biological function of ZNF313 remains largely undefined. Here we report that ZNF313 is an ubiquitin E3 ligase that has a critical role in the regulation of cell cycle progression, differentiation and senescence. In this study, ZNF313 is initially identified as a XIAP-associated factor 1 (XAF1)-interacting protein, which upregulates the stability and proapoptotic effect of XAF1. Intriguingly, we found that ZNF313 activates cell cycle progression and suppresses cellular senescence through the RING domain-mediated degradation of p21^WAF1. ZNF313 ubiquitinates p21^WAF1 and also destabilizes p27^KIP1 and p57^KIP2, three members of the CDK-interacting protein (CIP)/kinase inhibitor protein (KIP) family of cyclin-dependent kinase inhibitors, whereas it does not affect the stability of the inhibitor of CDK (INK4) family members, such as p16^INK4A and p15^INK4B. ZNF313 expression is tightly controlled during the cell cycle and its elevation at the late G1 phase is crucial for the G1-to-S phase transition. ZNF313 is induced by mitogenic growth factors and its blockade profoundly delays cell cycle progression and accelerates p21^WAF1-mediated senescence. Both replicative and stress-induced senescence are accompanied with ZNF313 reduction. ZNF313 is downregulated during cellular differentiation process in vitro and in vivo, while it is commonly upregulated in many types of cancer cells. ZNF313 shows both the nuclear and cytoplasmic localization in epithelial cells of normal tissues, but exhibits an intense cytoplasmic distribution in carcinoma cells of tumor tissues. Collectively, ZNF313 is a novel E3 ligase for p21^WAF1, whose alteration might be implicated in the pathogenesis of several human diseases, including cancers.     

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.     

The OGF-OGFr axis utilizes the p16INK4a and p21WAF1/CIP1 pathways to restrict normal cell proliferation

Opioid growth factor (OGF) is an endogenous opioid peptide ([Met⁵]enkephalin) that interacts with the OGF receptor (OGFr) and serves as a tonically active negative growth factor in cell proliferation of normal cells. To clarify the mechanism by which OGF inhibits cell replication in normal cells, we investigated the effect of the OGF–OGFr axis on cell cycle activity in human umbilical vein endothelial cells (HUVECs) and human epidermal keratinocytes (NHEKs). OGF markedly depressed cell proliferation of both cell lines by up to 40% of sterile water controls. Peptide treatment induced cyclin-dependent kinase inhibitor (CKI) p16^INK4a protein expression and p21^WAF1/CIP1 protein expression in HUVECs and NHEKs, but had no effect on p15, p18, p19, or p27 protein expression in either cell type. Inhibition of either p16^INK4a or p21^WAF1/CIP1 activation by specific siRNAs blocked OGF inhibitory action. Human dermal fibroblasts and mesenchymal stem cells also showed a similar dependence of OGF action on p16^INK4a and p21^WAF1/CIP1. Collectively, these results indicate that both p16^INK4a and p21^WAF1/CIP1 are required for the OGF–OGFr axis to inhibit cell proliferation in normal cells.     

Chloroquine reverses chemoresistance via upregulation of p21WAF1/CIP1 and autophagy inhibition in ovarian cancer

Overcoming drug-resistance is a big challenge to improve the survival of patients with epithelial ovarian cancer (EOC). In this study, we investigated the effect of chloroquine (CQ) and its combination with cisplatin (CDDP) in drug-resistant EOC cells. We used the three EOC cell lines CDDP-resistant A2780-CP20, RMG-1 cells, and CDDP-sensitive A2780 cells. The CQ-CDDP combination significantly decreased cell proliferation and increased apoptosis in all cell lines. The combination induced expression of γH2AX, a DNA damage marker protein, and induced G2/M cell cycle arrest. Although the CQ-CDDP combination decreased protein expression of ATM and ATR, phosphorylation of ATM was increased and expression of p21^WAF1/CIP1 was also increased in CQ-CDDP-treated cells. Knockdown of p21^WAF1/CIP1 by shRNA reduced the expression of γH2AX and phosphorylated ATM and inhibited caspase-3 activity but induced ATM protein expression. Knockdown of p21^WAF1/CIP1 partly inhibited CQ-CDDP-induced G2/M arrest, demonstrating that knockdown of p21^WAF1/CIP1 overcame the cytotoxic effect of the CQ-CDDP combination. Ectopic expression of p21^WAF1/CIP1 in CDDP-treated ATG5-shRNA/A2780-CP20 cells increased expression of γH2AX and caspase-3 activity, demonstrating increased DNA damage and cell death. The inhibition of autophagy by ATG5-shRNA demonstrated similar results upon CDDP treatment, except p21^WAF1/CIP1 expression. In an in vivo efficacy study, the CQ-CDDP combination significantly decreased tumor weight and increased expression of γH2AX and p21^WAF1/CIP1 in A2780-CP20 orthotopic xenografts and a drug-resistant patient-derived xenograft model of EOC compared with controls. These results demonstrated that CQ increases cytotoxicity in combination with CDDP by inducing lethal DNA damage by induction of p21^WAF1/CIP1 expression and autophagy inhibition in CDDP-resistant EOC.Subject terms: Cancer therapeutic resistance, Ovarian cancer, Translational research