Normal remodeling of the oxygen-injured lung requires the cyclin-dependent kinase inhibitor p21(Cip1/WAF1/Sdi1)

Alveolar cells of the lung are injured and killed when exposed to elevated levels of inspired oxygen. Damaged tissue architecture and pulmonary function is restored during recovery in room air as endothelial and type II epithelial cells proliferate. Although excessive fibroblast proliferation and inflammation occur when abnormal remodeling occurs, genes that regulate repair remain unknown. Our recent observation that hyperoxia inhibits proliferation through induction of the cyclin-dependent kinase inhibitor p21(Cip1/WAF1/Sdi1), which also facilitates DNA repair, suggested that p21 may participate in remodeling. This hypothesis was tested in p21-wild-type and -deficient mice exposed to 100% FiO(2) and recovered in room air. p21 increased during hyperoxia, remained elevated after 1 day of recovery before returning to unexposed levels. Increased proliferation occurred when p21 expression decreased. In contrast, higher and sustained levels of proliferation, resulting in myofibroblast hyperplasia and monocytic inflammation, occurred in recovered p21-deficient lungs. Cells with DNA strand breaks and expressing p53 were observed in hyperplastic regions suggesting that DNA integrity had not been restored. Normal recovery of endothelial and type II epithelial cells, as assessed by expression of cell-type-specific genes was also delayed in p21-deficient lungs. These results reveal that p21 is required for remodeling the oxygen-injured lung and suggest that failure to limit replication of damaged DNA may lead to cell death, inflammation, and abnormal remodeling. This observation has important implications for therapeutic strategies designed to attenuate long-term chronic lung disease after oxidant injury.     

Characterization of a panel of novel anti-p21Waf1/Cip1 monoclonal antibodies and immunochemical analysis of p21Waf1/Cip1 expression in normal human tissues.

As a universal inhibitor of cyclin-dependent kinases and one of the target genes of the tumor suppresser p53, p21Waf1/Cip1 can act as a tumor suppresser through its ability to control cell cycle progression. To study the function of p21Waf1/Cip1 protein and to investigate its tissue distribution, a panel of anti-p21Waf1/Cip1 monoclonal antibodies was generated. These anti-p21Waf1/Cip1 monoclonal antibodies were initially raised against a GST-p21Waf1/Cip1 fusion protein produced in bacteria. Detailed characterization of the antibodies showed that they can specifically detect p21Waf1/Cip1 by immunoblotting, immunoprecipitation, and immunostaining. The specific induction of p21Waf1/Cip1 expression in response to gamma-radiation in cells containing p53 was also detected by these antibodies. The ability to detect p21Waf1/Cip1 expression in conventionally fixed tissue sections allowed us to investigate the distribution of p21Waf1/Cip1 in 23 different types of normal human tissues, and p21Waf1/Cip1 expression was found in most tissues. A close inverse relationship between p21Waf1/Cip1 expression and proliferation was seen in some tissues, including gastrointestinal tract. However, such association is not universal. In tissues such as lung, kidney, thyroid, pancreatic ducts and acini, and liver, despite the fact that most of the cells are quiescent, expression of p21Waf1/Cip1 was detected only in occasional epithelial cells. All these suggest that the expression of p21Waf1/Cip1 varies among different human tissues. Finally, epitope mapping of the anti-p21Waf1/Cip1 antibodies using a peptide library covering the entire p21Waf1/Cip1 protein sequence indicates that two of the antibodies recognize a region of p21Waf1/Cip1 close to that bound by proliferating cell nuclear antigen. These two monoclonal antibodies will therefore be additionally useful in further understanding the functions of p21Waf1/Cip1 both in vitro and in vivo.     

Prognostic implications of p21 (Waf1/Cip1) immunolocalization in multiple myeloma

Protein p21 (Waf1/Cip1) plays a critical role in controlling the cell cycle especially as a check point of G1 and is intimately associated with important cellular activities including differentiation, senescence, tumorigenesis, and apoptosis. In the present study, we examined the expression of p21 in multiple myeloma (MM) cells for prognostic evaluation. The immunocytochemical localization of p21 could be categorized into nuclear and cytoplasmic types. The nuclear-type p21 localization was correlated with the severity of MM and the expression of proliferating cell nuclear antigen and p53. Patients with the nuclear-type p21 localization survived significantly shorter than those with the cytoplasmic-type localization. Thus, the present study suggests that p21-immunolocalization can be a useful prognostic marker of MM.     

p21(Waf1/Cip1) expression and the p53/MDM2 feedback loop in gastric carcinogenesis

Data are non-existent regarding coincidental alterations in the expression of p53 and its downstream target genes MDM2 and p21(Waf1/Cip1) in gastric carcinogenesis. An immunohistochemical study was therefore performed to examine the interrelationships of p53, MDM2, and p21(Waf1/Cip1) expression in a series of Caucasian early gastric carcinomas and precursor lesions. In normal gastric mucosa, chronic gastritis, and intestinal metaplasia, the surface cells expressed p21(Waf1/Cip1) in the absence of detectable nuclear p53 and MDM2 protein. Nuclear p53 protein accumulation was found in 60 per cent of the carcinomas, with significant differences in staining characteristics between the Lauren types in the absence of detectable MDM2 protein ( p< 0.005). Nearly 80 per cent of the carcinomas expressed p21(Waf1/Cip1), irrespective of Lauren type. Stratification of the carcinomas according to histological grade and growth pattern did not result in significant differences in p53 and p21(Waf1/Cip1) expression. Finally, no significant correlation was found between overall p53 and p21(Waf1/Cip1) expression in early gastric carcinomas. It is concluded that p21(Waf1/Cip1) expression in the non-neoplastic mucosa most likely relates to cell senescence and/or terminal differentiation, perhaps even in a p53-independent manner. In view of p53/MDM2 homeostasis, the differences in p53 staining characteristics between intestinal and diffuse-type carcinomas probably result, at least in part, from a difference in the prevalence of p53 gene mutations. Moreover, p53-independent induction of p21(Waf1/Cip1) expression apparently occurs in a considerable proportion of early carcinomas. Finally, in contrast to other carcinomas, p21(Waf1/Cip1) expression is not significantly correlated with histological grade in gastric carcinomas, suggesting possible defects downstream of p21(Waf1/Cip1) as an underlying cause for this apparent discrepancy.     

Proteasome-dependent downregulation of p21(Waf1/Cip1) induced by reactive oxygen species.

After hydrogen peroxide (H(2)O(2)) treatment, the p21 (p21(Waf1/Cip1)) protein level in GM00637 fibroblast cells was rapidly decreased, reaching its nadir around 3 h. However, it rebounded within 5 hours to a level higher than that before treatment. Fluorescence microscopic analyses revealed that nuclear p21 was downregulated during the initial oxidative stress. H(2)O(2)-induced downregulation of p21 protein was accompanied by a gradual increase in p21 mRNA levels. Other inducers of genotoxic stress, such as treatment with adriamycin, a DNA damage compound, did not cause a significant decrease in p21 protein levels. Pretreatment of GM00637 cells with the proteasome inhibitors, lactacystin or MG132, completely blocked H(2)O(2)-induced p21 downregulation, suggesting that H(2)O(2) treatment accelerated p21 degradation. Conversely, cotreatment of cells with a protein synthesis inhibitor, cycloheximide, and H(2)O(2) drastically shortened the half-life of p21. Moreover, p21 mRNA levels were not downregulated by treatment with proteasome or protein synthesis inhibitors. Taken together, our studies indicate that oxidative stress induces rapid, but reversible, downregulation of functional p21 by accelerating its protein turnover.     

Redox activation of p21Cip1/WAF1/Sdi1: a multifunctional regulator of cell survival and death

Cell division requires the coordinated assembly of cyclins and cyclin-dependent kinases that promote cell-cycle progression through S phase and mitosis. Two families of cyclin-dependent kinase inhibitors prevent abnormal or premature proliferation by blocking cyclin kinase activity. Expression of the cyclin-dependent kinase inhibitor p21, a member of the Cip/Kip family, increases when cells are damaged. In addition to controlling cell-cycle progression, p21 participates in DNA repair and apoptotic processes. The recent appreciation that p21 regulates cell survival and death implies that it is a master regulator of cell fate. This review discusses how p21 can affect the cellular response to oxidative stress.     

A Limited role for p21Cip1/Waf1 in maintaining normal hematopoietic stem cell functioning

Several studies have suggested that the cyclin-dependent kinase (CDK) inhibitor p21 plays a crucial role in regulating hematopoietic stem and progenitor pool size. To allow assessment of long-term stem cell functioning in vivo, we have backcrossed a p21 null allele to C57BL/6 (B6) mice, the most commonly used mouse strain in hematopoietic stem cell research. In various in vitro assays, the homozygous deletion of the p21 allele did not affect the number of hematopoietic cells in B6 mice. Furthermore, the competitive repopulation ability was not different between p21-deficient and wild-type stem cells from both young and aged (20-month-old) mice. These results show that p21 is not essential for regulation of stem cell number in steady state. When proliferative stress was applied on p21-deficient stem cells by serial transplantation of 1,500 Lin(-)Sca-1(+)c-kit(+) (LSK) cells, again no detrimental effect was observed on cobblestone area-forming cell (CAFC) frequency and competitive repopulating ability. However, when bone marrow cells from mice that received 2 Gy of irradiation were transplanted, p21 deficiency resulted in a more than fourfold reduction in competitive repopulation index. Finally, we did not find major differences in cell cycle status and global gene expression patterns between LSK cells from p21-deficient and wild-type mice. Our findings indicate that the background of mice used for studying the function of a gene by genetic modification may determine the outcome. Cumulatively, our data fail to support the notion that p21 is essential for stem cell function during steady-state hematopoiesis, but may be relatively more important under conditions of cellular stress.     

Clinicopathologic and prognostic significance of p21 (Cip1/Waf1) expression in bladder cancer

Recent studies have shown that altered expression p21 is shown to associate with tumorigenesis and tumor progression. To investigate the clinicopathological significance and prognostic value of p21 in bladder cancer (BCa). A total of 48 patients with BCa were included in this study. The correlation between p21 expression and clinicopathologic features and survival was studied. Also, a meta-analysis was performed to investigate the relationship between the p21 and BCa survival. Low p21 expression was detected both in tumor tissues compared with adjacent normal tissues. The expression of p21 was closely associated with advanced pathologic TNM stage (P = 0.001) and tumor grade (P = 0.013). Moreover, patients with low p21 expression had shorter recurrence-free survival (P = 0.016) and overall survival rates (P = 0.039). Multivariate Cox regression analysis revealed that p21 low expression was an independent prognostic factor for recurrence free survival (P = 0.03). Additionally, our meta-analysis. The available outcome data from six articles were examined. A meta-analysis of the HR indicated a significantly poor overall survival (OS, HR: 1.75, 95% CI: 1.38-2.21), recurrence free survival (RFS, HR: 1.83, 95% CI: 1.57-2.15), progression free survival (PFS, HR: 2.02, 95% CI: 1.48-2.75), and cancer specific survival (CSS, HR: 1.89, 95% CI: 1.53-2.33) in patients with low expression levels of p21. Our present results indicated that low p21 expression predicated tumor recurrence and poor prognosis in bladder cancer.     

Expression of p21Cip1/Waf1 and p27Kip1 in small cell neuroendocrine carcinoma of the uterine cervix

Small-cell neuroendocrine carcinoma of the uterine cervix (SCCC), a rare but malignant cervical neoplasm, has a highly aggressive phenotype that requires more intensive treatment than other cervical tumors. Immunohistochemical methods were used to compare the expression of p21Cip1/Waf1 and p27Kip1 in SCCC and squamous cell carcinoma, the most common type of cervical cancer. In SCCC, p21 expression was significantly reduced compared with squamous cell carcinoma, whereas expression of p27 was similar in both carcinomas. Reduced expression of p21 could be a helpful diagnostic marker and may contribute to the invasive phenotype of SCCC.     

Analysis of p21Waf1/Cip1 expression in normal, premalignant, and malignant cells during the development of human lung adenocarcinoma.

Our studies suggested that adenocarcinoma of the peripheral lung mostly develops by several steps from atypical adenomatous hyperplasia through early adenocarcinoma to overt adenocarcinoma, and that some p53 abnormalities play an important role in this progression. In the present study, we examined by immunohistochemistry the expression of p53-inducible cyclin-dependent kinase inhibitor p21Waf1/Cip1 (p21) in the cells at various developmental stages of lung adenocarcinoma (32 lesions of adenomatous hyperplasia, 14 of early adenocarcinoma, 23 of well differentiated adenocarcinoma, and 17 of moderately or poorly differentiated adenocarcinoma) in comparison with 19 reactive proliferative lesions and analyzed the relationship between p53 and p21 expression. Bronchioalveolar cells in the normal lung expressed very little or no p21 and no p53 expression. In not only reactive but also neoplastic lesions regardless of their developmental stage, the cells expressed p21 at various frequencies. The average labeling indices ranged from 5.4 to 13.8%, and there was no significant difference between any of these categories. The expression of p21, however, tended to be relatively low in moderately and poorly differentiated adenocarcinomas (5.5%) compared to well differentiated adenocarcinomas (12.2%), and high-level p21 expressors (10% < or = positive cells) were more frequent in the latter group (1 of 17 (6%) versus 3 of 23 (35%), P < 0.05), suggesting that p21 expression is affected by the degree of differentiation of the neoplastic cells. Although the correlation was positive between the expression of p21 and p53 in reactive lesions (r = 0.88; P < 0.001), none was found in neoplastic lesions at any step or grade (-0.12 < or = r < or = 0.26). These results indicated that p21 expression depends upon p53 expression in reactive lung cells, whereas p21 expression is at least in part independent of that of p53 from the earliest to the most fully developed step of lung adenocarcinoma tumorigenesis. We concluded that disruption of the p53-dependent cell cycle regulation is a very early event in the tumorigenesis of lung adenocarcinoma.     

持续吸入高浓度氧对新生大鼠肺的影响

目的探讨持续吸入高浓度氧对新生大鼠发育中肺的影响.方法新生鼠生后分别于85%以上氧和空气中持续暴露,于1、3、7、14、21d,动态观察肺组织病理学改变和胶原染色面积的变化;肺组织SOD活性和MDA含量.结果与空气对照组比较,高氧组肺泡发育过程受阻,肺损伤逐渐加重,最终纤维化;胶原染色阳性面积7d时高于对照组,随时间延长而逐渐增加;MDA含量在吸氧3d时升高,7d时最高,以后下降;SOD活性的改变较迟,7d时增高,持续增加,21d仍高于对照.结论高氧可引起肺的氧化损伤,使新生鼠肺发育受阻,最终出现胶原增加的纤维化改变.     

p21(Cip1/WAF1/Sdi1) does not affect expression of base excision DNA repair enzymes during chronic oxidative stress

Exposure to chronic oxidative stress during elevated oxygen (hyperoxia) damages DNA and inhibits cell proliferation in G(1) through induction of the cyclin-dependent kinase inhibitor p21. Cells that fail to express p21 growth-arrest in S phase. The observation that growth arrest in G(1) is associated with reduced DNA damage and enhanced survival suggests that p21 may affect expression of base excision repair (BER) enzymes used to repair oxidized DNA. This hypothesis was tested in p21 wild-type and p21-deficient mice and human lung adenocarcinoma H1299 cells with tetracycline-on regulated expression of p21. The mRNA levels of Ogg1, Tdg, Udg, Mpg, Nth1, and Mgmt remained constant during 3 days of hyperoxia. The expression of Ogg1, Nth1, and APE protein also remained unchanged. Although hyperoxia increased p21, its absence did not significantly affect expression of these repair enzymes. These findings reveal that hyperoxia induces p21 without significantly altering BER enzyme expression. This suggests that p21 may protect oxidized cells by affecting the activity of BER enzymes and/or through other mechanisms, such as apoptosis.     

MicroRNAs regulate p21(Waf1/Cip1) protein expression and the DNA damage response in human embryonic stem cells

Studies of human embryonic stem cells (hESCs) commonly describe the nonfunctional p53-p21 axis of the G1/S checkpoint pathway with subsequent relevance for cell cycle regulation and the DNA damage response (DDR). Importantly, p21 mRNA is clearly present and upregulated after the DDR in hESCs, but p21 protein is not detectable. In this article, we provide evidence that expression of p21 protein is directly regulated by the microRNA (miRNA) pathway under standard culture conditions and after DNA damage. The DDR in hESCs leads to upregulation of tens of miRNAs, including hESC-specific miRNAs such as those of the miR-302 family, miR-371-372 family, or C19MC miRNA cluster. Most importantly, we show that the hESC-enriched miRNA family miR-302 (miR-302a, miR-302b, miR-302c, and miR-302d) directly contributes to regulation of p21 expression in hESCs and, thus, demonstrate a novel function for miR-302s in hESCS. The described mechanism elucidates the role of miRNAs in regulation of important molecular pathway governing the G1/S transition checkpoint before as well as after DNA damage.     

Loss of nuclear p21(Cip1/WAF1) during neoplastic progression to metastasis in gamma-irradiated p21 hemizygous mice

p21(Cip1/WAF1) localizes to the nucleus in response to gamma-irradiation induced DNA damage and mediates a G(1) checkpoint arrest. Although gamma-irradiated p21(+/-) mice develop a broad spectrum of tumors, gamma-irradiated p21(-/-) mice develop significantly more metastatic cancers. To evaluate the expression of p21 in tissues prone or resistant to tumorigenesis as a function of gamma-irradiation, and to determine whether phenotypic loss of p21 heterozygosity occurs during tumor progression in p21(+/-) mice, tissues and tumors from gamma-irradiated mice were evaluated immunohistochemically. The percentage of tumors in p21(+/-) mice that were nuclear p21-positive declined with progression to metastasis (p<0.0001). Benign tumors were more often p21-positive and comprised of larger subsets of nuclear p21-positive cells than were malignant tumors of the same histopathological type, while metastatic cancers were nuclear p21-negative (p=0.0003). Even when a primary cancer was comprised of a subset of nuclear p21-positive cells, the metastatic foci of that same cancer were nuclear p21-negative. Mesenchymal tumors, though rare, were more likely metastatic than were epithelial tumors (p=0.0004), and these were invariably nuclear p21-negative. Prepubescent epithelial tissues from which most tumors later originated in mice with reduced p21 gene dosage (i.e., harderian gland, ovary, small intestine, and lung) were p21 expressive within 4 h of gamma-irradiation (p=0.0625), so that p21/Ki67 ratios increased post-gamma-irradiation (p=0.03). In contrast, p21 did not localize to nuclei of cortical thymocytes, a tissue where tumorigenesis was not augmented by reduced p21 gene dosage. Cellular subclones of malignant tumors, especially those of mesenchymal cell origin, which lack nuclear p21 may more readily acquire the genetic alterations of the metastatic phenotype.     

p21Cip1 protection against hyperoxia requires Bcl-XL and is uncoupled from its ability to suppress growth

The cyclin-dependent kinase inhibitor p21Cip1/Waf1/Sdi1 protects the lung against hyperoxia, but the mechanism of protection remains unclear because loss of p21 does not lead to aberrant cell proliferation. Because some members of the Bcl-2 gene family have been implicated in hyperoxia-induced cell death, the current study investigated their expression as well as p21-dependent growth suppression and cytoprotection. Conditional overexpression of full-length p21, its amino-terminal cyclin-binding (p211-82NLS) domain or its carboxy-terminal PCNA-binding (p2176-164) domain inhibited growth of human lung adenocarcinoma H1299 cells, but only the full-length protein was cytoprotective. Low levels of p21 inhibited cell proliferation, whereas higher levels were required for protection. Expression of the anti-apoptotic protein Bcl-XL declined during hyperoxia but was maintained in cells expressing p21. RNA interference (RNAi) knockdown of Bcl-XL enhanced hyperoxic death of cells expressing p21, whereas overexpression of Bcl-XL increased cell survival. Consistent with growth suppression and cytoprotection requiring different levels of p21, hyperoxia inhibited PCNA expression in p21+/+ and p21+/- mice but not in p21-/- mice. In contrast, p21 was haplo-insufficient for maintaining expression of Bcl-XL and protection against hyperoxia. Taken together, these data show that p21-mediated cytoprotection against hyperoxia involves regulation of Bcl-XL and is uncoupled from its ability to inhibit proliferation.     

Combined examination of p27(Kip1), p21(Waf1/Cip1) and p53 expression allows precise estimation of prognosis in patients with gastric carcinoma

AIMS: In order to estimate the prognostic values of p27(Kip1), p21(Waf1/Cip1), and p53, alone and in combination, we investigated immunohistochemically the expression of p27(Kip1), p21(Waf1/Cip1), and p53 proteins in gastric carcinomas. METHODS AND RESULTS: The expression of p27(Kip1), p21(Waf1/Cip1), and p53 was immunohistochemically examined in 140 gastric carcinomas. Positive expression of p27(Kip1) and p21(Waf1/Cip1) correlated significantly with a favourable prognosis (P < 0.05), whereas, positive expression of p53 tended to correlate with poor prognosis. Multivariate survival analysis revealed that TNM stage of tumour (P < 0.001), lymph node state (P=0.005), and p27(Kip1) expression (P=0.006) were independent prognostic factors. A striking stratification of mortality rate was found when patients were divided into four groups according to the expression of p21(Waf1/Cip1) and p27(Kip1). The mortality rate was higher in patients with both p21(Waf1/Cip1)- and p27(Kip1)-negative gastric carcinoma than in patients with one or both positive carcinomas (P < 0.01). In addition, if the four p21(Waf1/Cip1)/p27(Kip1) groups were compared based on p53 status, p53+ cases tended to have a higher mortality rate than p53- cases. CONCLUSION: Our results suggest that low expression of both p27(Kip1) and p21(Waf1/Cip1), could be useful as markers of poorer prognosis, and the combined examination of p27(Kip1), p21(Waf1/Cip1) and p53 expression allows reliable estimation of prognosis for patients with gastric carcinoma.