Simultaneous Deletion of p21Cip1/Waf1 and Caspase-3 Accelerates Proliferation and Partially Rescues the Differentiation Defects of Caspase-3 Deficient Hematopoietic Stem Cells

Specialized blood cells are generated through the entire life of an organism by differentiation of a small number of hematopoietic stem cells (HSC). There are strictly regulated mechanisms assuring a constant and controlled production of mature blood cells. Although such mechanisms are not completely understood, some factors regulating cell cycle and differentiation have been identified. We have previously shown that Caspase-3 is an important regulator of HSC homeostasis and cytokine responsiveness. p21^cip1/waf1 is a known cell cycle regulator, however its role in stem cell homeostasis seems to be limited. Several reports indicate interactions between p21^cip1/waf1 and Caspase-3 in a cell type dependent manner. Here we studied the impact of simultaneous depletion of both factors on HSC homeostasis. Depletion of both Caspase-3 and p21^cip1/waf1 resulted in an even more pronounced increase in the frequency of hematopoietic stem and progenitor cells. In addition, simultaneous deletion of both genes revealed a further increase of cell proliferation compared to single knock-outs and WT control mice, while apoptosis or self-renewal ability were not affected in any of the genotypes. Upon transplantation, p21^cip1/waf1-/- bone marrow did not reveal significant alterations in engraftment of lethally irradiated mice, while Caspase-3 deficient HSPC displayed a significant reduction of blood cell production. However, when both p21^cip1/waf1 and Caspase-3 were eliminated this differentiation defect caused by Caspase-3 deficiency was abrogated.     

MDM2 promotes p21waf1/cip1 proteasomal turnover independently of ubiquitylation

The CDK inhibitor p21waf1/cip1 is degraded by a ubiquitin-independent proteolytic pathway. Here, we show that MDM2 mediates this degradation process. Overexpression of wild-type or ring finger-deleted, but not nuclear localization signal (NLS)-deleted, MDM2 decreased p21waf1/cip1 levels without ubiquitylating this protein and affecting its mRNA level in p53(-/-) cells. This decrease was reversed by the proteasome inhibitors MG132 and lactacystin, by p19(arf), and by small interfering RNA (siRNA) against MDM2. p21waf1/cip1 bound to MDM2 in vitro and in cells. The p21waf1/cip1-binding-defective mutant of MDM2 was unable to degrade p21waf1/cip1. MDM2 shortened the half-life of both exogenous and endogenous p21waf1/cip1 by 50% and led to the degradation of its lysine-free mutant. Consequently, MDM2 suppressed p21waf1/cip1-induced cell growth arrest of human p53(-/-) and p53(-/-)/Rb(-/-)cells. These results demonstrate that MDM2 directly inhibits p21waf1/cip1 function by reducing p21waf1/cip1 stability in a ubiquitin-independent fashion.     

Loss of Notch1-dependent p21Waf1/Cip1 expression influences the Notch1 outcome in tumorigenesis

Notch signaling plays a complex role in carcinogenesis, and its signaling pathway has both tumor-suppressor and oncogenic components. In this study we investigated the effects of reactive oxygen species (ROS) on Notch1 signaling outcome in keratinocyte biology. We demonstrate that Notch1 function contributes to the arsenic-induced keratinocyte transformation. We found that acute exposure to arsenite increases oxidative stress and inhibits proliferation of keratinocyte cells by upregulation of p21^waf1/Cip1. The necessity of p21^waf1/Cip1 for arsenite-induced cell death was demonstrated by targeted downregulation of p21^waf1/Cip1 by using RNA interference. We further demonstrated that on acute exposure to arsenite, p21^waf1/Cip1 is upregulated and Notch1 downmodulated, whereas on chronic exposure to arsenite, malignant progression of arsenite-treated keratinocytes cells was accompanied by regained expression and activity of Notch1. Notch1 activity in arsenite-transformed keratinocytes inhibits arsenite-induced upregulation of p21^waf1/Cip1 by sustaining c-myc expression. We further demonstrated that c-myc collaborates with Nrf2, a key regulator for the maintenance of redox homeostasis, to promote metabolic activities that support cell proliferation and cytoprotection. Therefore, Notch1-mediated repression of p21^waf1/Cip1 expression results in the inhibition of cell death and keratinocytes transformation. Our results not only demonstrate that sustained Notch1 expression is at least one key event implicated in the arsenite human skin carcinogenic effect, but also may provide mechanistic insights into the molecular aspects that determine whether Notch signaling will be either oncogenic or tumor suppressive.     

Efficient repair of bulky anti-BPDE DNA adducts from non-transcribed DNA strand requires functional p53 but not p21(waf1/cip1) and pRb

Wild-type p53 protein is known to regulate the global genomic repair (GGR), removing bulky chemical DNA adducts as well as cyclobutane pyrimidine dimers from the genome overall and from non-transcribed strands (NTS) in DNA. To investigate the role of cellular factor(s) relevant to p53 regulated DNA repair processes, we examined the repair kinetics of chemical carcinogen, anti-benzo[a]pyrene-diol epoxide (anti-BPDE), induced bulky DNA adducts in normal human mammary epithelial cells (HMECs) and HMEC transformed by human papillomavirus (HPV)-16E6 or -16E7 oncoproteins, which, respectively targets p53 or pRb proteins for degradation. The results show that the removal of anti-BPDE DNA adducts from the genome overall and NTS by GGR was significantly reduced in HPV-16E6 protein expressing cells as compared to that in normal and HPV-16E7 protein expressing cells, indicating the role of p53 and not pRb in nucleotide excision repair (NER). We further determined the potential effects of the p53-regulated p21(waf1/cip1) gene product in NER in human colon carcinoma, HCT116 cells expressing wild-type p53 but different p21(waf1/cip1) genotypes (p21+/+, p21+/-, p21-/-). The results donot show a discernible difference in the removal of anti-BPDE DNA adducts from the genome overall and the transcribed strand (TS) and NTS irrespective of the presence or absence of p21(waf1/cip1) expression. Based on these results, we suggest that: (i) the wild-type p53 function but not p21(waf1/cip1) expression is necessary for GGR of chemical induced bulky DNA adducts; (ii) the Rb gene product does not play a significant role in NER; and (iii) the modulation of NER by p53 may be independent of its function in the regulation of cell cycle arrest upon chemically induced DNA damage.     

p21(cip1) mRNA is controlled by endogenous transforming growth factor-beta1 in quiescent human hematopoietic stem/progenitor cells

Transforming growth factor-beta1 (TGF-beta1) has been described as an efficient growth inhibitor that maintains the CD34(+) hematopoietic progenitor cells in quiescence. The concept of high proliferative potential-quiescent cells or HPP-Q cells has been introduced as a working model to study the effect of TGF-beta1 in maintaining the reversible quiescence of the more primitive hematopoietic stem cell compartment. HPP-Q cells are primitive quiescent stem/progenitor cells on which TGF-beta1 has downmodulated the cytokine receptors. These cells can be released from quiescence by neutralization of autocrine or endogenous TGF-beta1 with a TGF-beta1 blocking antibody or a TGF-beta1 antisense oligonucleotide. In nonhematopoietic systems, TGF-beta1 cooperates with the cyclin-dependent kinase inhibitor, p21(cip1), to induce cell cycle arrest. We therefore analyzed whether endogenous TGF-beta1 controls the expression of the p21(cip1) in the CD34(+) undifferentiated cells using a sensitive in situ hybridization method. We observed that addition of anti-TGF-beta1 is followed by a rapid decrease in the level of p21(cip1) mRNA whereas TGF-beta1 enhances p21(cip1) mRNA expression concurrently with an inhibitory effect on progenitor cell proliferation. These results suggest the involvement of p21(cip1) in the cell cycle control of early human hematopoietic quiescent stem/progenitors and not only in the differentiation of more mature myeloid cells as previously described. The modulation of p21(cip1) observed in response to TGF-beta1 allows us to further precise the working model of high proliferative potential-quiescent cells.     

Alpha(1) adrenergic agonist induction of p21(waf1/cip1) mRNA stability in transfected HepG2 cells correlates with the increased binding of an AU-rich element binding factor

Stimulation of transfected HepG2 cells (TFG2) with the alpha(1)-adrenergic agonist phenylephrine (PE) significantly activated p21(waf1/cip1) gene expression without affecting p53 gene expression. Northern blotting and reporter assay demonstrated that this induction was due to PE stimulation of p21(waf1/cip1) mRNA stability. To further define the underlying mechanism, we prepared a chloramphenicol acetyltransferase (CAT)-p21(waf1/cip1) 3'-untranslated region (3'-UTR) hybrid construct by inserting the 3'-UTR of p21(waf1/cip1) mRNA just downstream from the CAT coding sequence and transfected it into TFG2 cells. PE treatment enhanced the activity of this construct by 6-fold. Deletion analyses indicated that an AU-rich element (AURE) located between 553 to 625 within the p21(waf1/cip1) 3'-UTR was required for this induction. RNA gel shift assays demonstrated that this AURE bound an RNA-binding protein. This protein has been purified 5000-fold from PE-treated TFG2 cells by heparin-Sepharose and RNA affinity chromatography. SDS-polyacrylamide gel electrophoresis, UV cross-linking, and Northwestern analyses indicated the molecular mass of this protein as 24 and 52 kDa. Finally, PE treatment markedly enhanced this RNA-protein binding by a p42/44 mitogen-activated protein kinase-dependent mechanism. These data suggest that the AURE located between 553 and 625 within the p21(waf1/cip1) mRNA 3'-UTR, which binds an RNA-binding protein, is responsible for PE-induced p21(waf1/cip1) mRNA stability.     

Effect of human papillomavirus on cell cycle-related proteins p16INK4A, p21waf1/cip1, p53 and cyclin D1 in sinonasal inverted papilloma and laryngeal carcinoma. An in situ hybridization study

Human papillomavirus (HPV) infection is implicated as an important risk factor in the development of head and neck cancers. Many studies focusing on the relationships between HPV infection and cell cycle proteins immunoexpression in laryngeal lesions have provided contradictory results. The aim of this study was to evaluate the relationships between HPV DNA presence and p16INK4a, p21waf1/cip1, p53 and cyclin D1 immunoexpression in heterogenous HPV-positive and HPV-negative groups of laryngeal cancers and inverted papillomas. The HPV DNA expression was detected using an in situ hybridization method and immunoexpression of p16INK4a, p21waf1/cip1, p53 and cyclin D1 using immunohistochemistry. The immunoexpression of p21waf1/ /cip1 and p53 proteins was lower in the HPV-positive group compared to the HPV-negative group, although only the difference of p53 staining was statistically significant. The immunoexpression of p16INK4a and cyclin D1 was significantly increased in the HPV-positive group compared to the HPV-negative group. The increased immunoexpression of p16INK4a and cyclin D1, and the lower immunoexpression of p21waf1/cip1 and p53 in the HPV-positive group compared to the HPV-negative group, supports the hypothesis that HPV may play an important role in cell cycle dysregulation.     

Chloramphenicol-induced mitochondrial stress increases p21 expression and prevents cell apoptosis through a p21-dependent pathway

Pretreatment of HepG2 and H1299 cells with chloramphenicol rendered the cells resistant to mitomycin-induced apoptosis. Both mitomycin-induced caspase 3 activity and PARP activation were also inhibited. The mitochondrial DNA-encoded Cox I protein, but not nuclear-encoded proteins, was down-regulated in chloramphenicol-treated cells. Cellular levels of the p21(waf1/cip1) protein and p21(waf1/cip1) mRNA were increased through a p53-independent pathway, possibly because of the stabilization of p21(waf1/cip1) mRNA in chloramphenicol-treated cells. The p21(waf1/cip1) was redistributed from the perinuclear region to the cytoplasm and co-localized with mitochondrial marker protein. Several morphological changes and activation of the senescence-associated biomarker, SA beta-galactosidase, were observed in these cells. Both p21(waf1/cip1) antisense and small interfering RNA could restore apoptotic-associated caspase 3 activity, PARP activation, and sensitivity to mitomycin-induced apoptosis. Similar effects were seen with other antibiotics that inhibit mitochondrial translation, including minocycline, doxycycline, and clindamycin. These findings suggested that mitochondrial stress causes resistance to apoptosis through a p21-dependent pathway.     

Roles for p21waf1/cip1 and p27kip1 during the adaptation response to massive intestinal resection

The magnitude of gut adaptation is a decisive factor in determining whether patients are able to live independent of parenteral nutrition after massive small bowel loss. We previously established that the cyclin-dependent kinase inhibitor (CDKI) p21(waf1/cip1) is necessary for enterocyte proliferation and a normal adaptation response. In the present study, we have further elucidated the role of this CDKI in the context of p27(kip1), another member of the Cip/Kip CDKI family. Small bowel resections (SBRs) or sham operations were performed in control (C57/BL6), p21(waf1/cip1)-null, p27(kip1)-null, and p21(waf1/cip1)/p27(kip1) double-null mice. Morphological (villus height/crypt depth) alterations in the mucosa, the kinetics of enterocyte turnover (rates of enterocyte proliferation and apoptosis), and the protein expression of various cell cycle-regulatory proteins were recorded at various postoperative times. Enterocyte compartment-specific mRNA expression was investigated using laser capture microdissection. Resection-induced adaptation in control mice coincided with increased protein expression of p21(waf1/cip1) and decreased p27(kip1) within 3 days postoperatively. Identical changes in mRNA expression were detected in crypt but not in villus enterocytes. Adaptation occurred normally in control and p27(kip1)-null mice; however, mice deficient in both p21(waf1/cip1) and p27(kip1) failed to increase baseline rates of enterocyte proliferation and adaptation. The expression of p21(waf1/cip1) protein and mRNA in the proliferative crypt compartment is necessary for resection-induced enterocyte proliferation and adaptation. The finding that deficient expression of p27(kip1) does not affect adaptation suggests that these similar CDKI family members display distinctive cellular functions during the complex process of intestinal adaptation.