Figure 3 - uploaded by Albert J Fornace
Content may be subject to copyright.
Western immunoblot analysis of p21 in untreated and adriamycin treated DLD1 cells. p21 was either kept repressed (lanes 1 and 2), transiently induced and then repressed at the time of adriamycin addition (lanes 3 and 4) or kept induced during the course of adriamycin treatment (lanes 5 and 6). Cell lysates from control or adriamycin (200 n M for 48 h) treated cells were
Source publication
Postulated roles for p21(Waf1/Cip1/Sdi1) (p21) in DNA repair and apoptosis remain controversial. Studies suggest both stimulatory and inhibitory effects of p21 in DNA repair. p21 has also been implicated in induction or protection from apoptosis. Using the tetracycline inducible expression system, we studied the role of p21 in DNA repair and apopto...
Contexts in source publication
Context 1
... to constantly elevated levels of exogenous p21. For example, (i) the majority of these cells generally grew slower and formed smaller colonies than those in which p21 was kept repressed. The average colony size after 5 and 8 days of p21 induction, was 15 cells and 48 cells respectively, while the average colony size in the absence of p21 was 23 cells and 90 cells on similar days. (ii) p21 also induced a complete growth arrest in a number of cells; those cells were clearly visible as well isolated one, two and three cells 5 days after p21 induction. (iii) Increasing number of well isolated, as well as individual cells growing within colonies, became giant cells; there was an approximately ®vefold increase in the number of single or multinucleated giant cells within 8 days of p21 induction (the quantitative data is illustrated in Figure 1a, while the photomicrographs in Figure 1b panels 2 and 3 show typical giant cells). The giant cells did not revert to usual morphology even when p21 expression was turned o; these giant cells ultimately succumbed to apoptotic death (Figure 1b). (iv) A number of well isolated as well as individual cells growing within colonies underwent apoptotic death (Figure 1b) within 8 days without prior giant cell formation. The dead cells exhibited characteristic morphological features such as condensed chromatin, nuclear fragmentation with intact membrane (see Figure 1b, compare panel 1 with panels 2 and 3). These cells ultimately became rounded and refractile prior to detaching from plates. All these features are a morphological hallmark of apoptotic death (Isaac, 1994). Clearly however, p21 overexpression did not induce massive cell killing. Since some cells underwent apoptosis in response to p21 induction, while others were apparently able to tolerate higher levels of exogenous p21, the quantitative data were obtained by counting (i) the number of well isolated apoptotic cells and (ii) the number of colonies exhibiting 50% or more apoptotic cells (Figure 1b panel 2 shows one representative colony with all cells exhibiting apoptotic features 8 days following p21 induction). The results presented in Table 1 show that the plates in which p21 expression was turned `on' had more apoptotic cells than the comparable plates in which it was turned `o'. The morphological features of cells in which p21 expression was kept induced or repressed are illustrated as representative photomicrographs in Figure 1b (compare the cells showing normal morphological features in panel 1 with apoptotic cells showing condensed chromatin and fragmented nuclei in panel 2 and 3). We next sought to investigate whether the presence or absence of high levels of p21 would aect adriamycin-induced cell death. Waldman et al . (1996) have recently reported that, in a short term non- clonogenic assay, the p21 knockout HCT 116 cells (p21 7 / 7 ) were more sensitive to acute killing by adriamycin than their p21 +/+ counterparts. After 30 h of adriamycin (200 n M ) treatment, the p21 +/+ cells were typically arrested in G 1 and G 2 while the p21 7 / 7 cells arrested only in G 2 ; a prolonged treatment with adriamycin, while not eecting p21 +/+ cells, induced polyploidy and apoptosis in p21 7 / 7 cells (Waldman et al ., 1996). Similar results were also reported in DLD1 colorectal carcinoma cells; DLD1 cells, carrying both mutated alleles of p53, constitutively express very low levels of endogenous p21 and were considered functionally equivalent to p21 7 / 7 cells (Waldman et al ., 1996). Adriamycin normally induces the expression of endogenous p21 in cells with wild-type p53 function but not in cells such as DLD1 that harbor mutant p53 (Waldman et al ., 1996). We investigated whether elevated levels of p21 would modulate the adriamycin eects in DLD1 colorectal carcinoma cells. Cells plated at low density were divided into three groups, in group one p21 was kept repressed, in group two p21 was transiently induced for 24 h and then repressed at the time of adriamycin addition, while group three cells had p21 `on' during the course of experiment. Cells were treated with 200 n M adriamycin for 48 h. The results presented in Table 2 show that there was no dierence in the number of apoptotic cells after adriamycin treatment irrespective of whether p21 was (i) kept repressed, (ii) transiently induced and then repressed or (iii) kept continuously induced through the course of adriamycin treatment. Representative photomicrographs in Figure 2 depict the apoptotic cells which constantly expressed p21 during the course of adriamycin treatment. Thus, overexpression of p21 during or prior to adriamycin treatment neither sensitized to nor protected from adriamycin-induced cell death. Figure 3 shows the expression pro®le of p21 in the three groups of cells described above. These cells constitutively express very low levels of endogenous p21, the expression of the p21 transgene can be controlled by addition or removal of tetracycline (Figure 3 compare lanes 1 ± 4 with lanes 5 ± 6) and adriamycin does not induce endogenous p21 in these (p53 mutant) cells. We then investigated the p21 eect on clonogenic survival following adriamycin treatment and u.v.- irradiation. Both adriamycin and u.v.-irradiation are genotoxic agents; adriamycin induces DNA strand breaks and other lesions repaired predominately via non-NER mechanisms, while u.v.-irradiation generates pyrimidine dimers and other photo-products (reviewed in Friedberg et al ., 1995). Cells growing at a very low density in the presence of tetracycline were washed with PBS and refed with identical medium with or without tetracycline. Approximately 6 h later cells were exposed to adriamycin or u.v.-irradiation and kept in medium with or without tetracycline for another 20 h. Time course analysis revealed that exogenous p21 can be readily detected within 6 h after tetracycline removal in these cells (Chen et al ., 1995). The rationale of this experimental strategy was to increase the p21 levels transiently at the time of DNA damage and a few hours thereafter. As shown previously, the transient induction of p21 for 24 h does not induce alterations in the growth pro®le of these cells (Chen et al ., 1995). We reasoned that if p21 enhances DNA repair, then elevated levels of p21 at the time of DNA damage should facilitate ecient DNA repair, which should result in enhanced clonogenic survival. Alter- natively, if p21 inhibits DNA repair, then that would be re ̄ected as a decrease in clonogenic survival. The results presented in Figure 4 demonstrate that the transient induction of p21, at the time of u.v.- irradiation and few hours thereafter, appreciably increased cell survival following 3.7 J/m 2 and 7 J/m 2 u.v. exposure; higher does of u.v-irradiation (10 J/m 2 and 20 J/m 2 ) were, however, highly toxic and the results were not informative (data not shown). Similar induction of p21, however, did not alter the clonogenic survival after adriamycin treatment (data not shown). Thus, p21 appears to protect cells from u.v.-type DNA damage, but not from other classes of DNA damage, at least as is shown for adriamycin in the present study. To further investigate the role of p21 in pathways that control the repair of u.v.-type DNA damage, the ability of these cells to repair u.v.-damaged plasmid DNA was assessed. In these `host cell reactivation' experiments (Smith et al ., 1995, 1996), u.v.-damaged or undamaged plasmids carrying the chloramphenicol acetyl transferase gene under the control of SV40 promoter enhancer were introduced into cells growing in the presence of tetracycline. Host cell reactivation is based on the principle that pyrimidine dimers in particular, strongly block the transcription of damaged reporter genes, thus a reporter can be expressed only to the extent that the damage is repaired when transfected into a given cell line (Klocker et al ., 1985; Protic et al ., 1988; Smith et al ., 1995, 1996). Twenty- four hours post-transfection, cells were harvested and divided into two groups. In one group the expression of exogenous p21 was turned on, while in the other group it was kept repressed. Approximately 48 h later cells were harvested and CAT activity was determined in the lysates prepared from both groups of cells. We reasoned that if p21 indeed enhances the repair of u.v.- type DNA damage, then those cells in which p21 was induced should display higher reporter activity than those in which it was kept repressed. Moreover, since the same pool of transfected cells were equally divided into two groups, this strategy ensured that the interpretation of results would not be confounded by the variations introduced due to multiple transfections. Figure 5 shows that indeed the group of cells, in which p21 was induced after transfection of damaged plasmid, exhibited higher CAT activity than the cells in which p21 was kept uninduced. Similar results were obtained when two other u.v.-damaged or undamaged plasmids, each expressing either an alkaline phosphatase gene from SV40 promoter enhancer or the green lantern gene under the control of CMV promoter, were used (Figure 5). Together these ®ndings demonstrate that the p21 induction resulted in the enhanced reactivation of three dierent u.v.-damaged reporter plasmids. In this study we have demonstrated that the overexpression of p21 neither sensitized to nor protected from adriamycin-mediated apoptosis. Similar results were obtained when other chemotherapeutic agents such as etoposide and taxol were tested (data not shown). Overexpression of p21 itself, however, induced apoptosis as well as giant cell formation. The p21-induction did not result into massive cell killing and there was an approximately ®vefold increase in the giant cell formation under p21 induced state; the giant cells ultimately underwent apoptosis. The p21-induced apoptosis and giant cell formation occurred late; the fact that not all the cells exhibited these features and ...
Context 2
... Cells growing in the presence or absence of tetracycline were morphologically indistinguishable until day 3. However, on day 5, the p21 overexpressing cells visibly diered from those in which p21 was kept repressed and exhibited a heterogeneous response to constantly elevated levels of exogenous p21. For example, (i) the majority of these cells generally grew slower and formed smaller colonies than those in which p21 was kept repressed. The average colony size after 5 and 8 days of p21 induction, was 15 cells and 48 cells respectively, while the average colony size in the absence of p21 was 23 cells and 90 cells on similar days. (ii) p21 also induced a complete growth arrest in a number of cells; those cells were clearly visible as well isolated one, two and three cells 5 days after p21 induction. (iii) Increasing number of well isolated, as well as individual cells growing within colonies, became giant cells; there was an approximately ®vefold increase in the number of single or multinucleated giant cells within 8 days of p21 induction (the quantitative data is illustrated in Figure 1a, while the photomicrographs in Figure 1b panels 2 and 3 show typical giant cells). The giant cells did not revert to usual morphology even when p21 expression was turned o; these giant cells ultimately succumbed to apoptotic death (Figure 1b). (iv) A number of well isolated as well as individual cells growing within colonies underwent apoptotic death (Figure 1b) within 8 days without prior giant cell formation. The dead cells exhibited characteristic morphological features such as condensed chromatin, nuclear fragmentation with intact membrane (see Figure 1b, compare panel 1 with panels 2 and 3). These cells ultimately became rounded and refractile prior to detaching from plates. All these features are a morphological hallmark of apoptotic death (Isaac, 1994). Clearly however, p21 overexpression did not induce massive cell killing. Since some cells underwent apoptosis in response to p21 induction, while others were apparently able to tolerate higher levels of exogenous p21, the quantitative data were obtained by counting (i) the number of well isolated apoptotic cells and (ii) the number of colonies exhibiting 50% or more apoptotic cells (Figure 1b panel 2 shows one representative colony with all cells exhibiting apoptotic features 8 days following p21 induction). The results presented in Table 1 show that the plates in which p21 expression was turned `on' had more apoptotic cells than the comparable plates in which it was turned `o'. The morphological features of cells in which p21 expression was kept induced or repressed are illustrated as representative photomicrographs in Figure 1b (compare the cells showing normal morphological features in panel 1 with apoptotic cells showing condensed chromatin and fragmented nuclei in panel 2 and 3). We next sought to investigate whether the presence or absence of high levels of p21 would aect adriamycin-induced cell death. Waldman et al . (1996) have recently reported that, in a short term non- clonogenic assay, the p21 knockout HCT 116 cells (p21 7 / 7 ) were more sensitive to acute killing by adriamycin than their p21 +/+ counterparts. After 30 h of adriamycin (200 n M ) treatment, the p21 +/+ cells were typically arrested in G 1 and G 2 while the p21 7 / 7 cells arrested only in G 2 ; a prolonged treatment with adriamycin, while not eecting p21 +/+ cells, induced polyploidy and apoptosis in p21 7 / 7 cells (Waldman et al ., 1996). Similar results were also reported in DLD1 colorectal carcinoma cells; DLD1 cells, carrying both mutated alleles of p53, constitutively express very low levels of endogenous p21 and were considered functionally equivalent to p21 7 / 7 cells (Waldman et al ., 1996). Adriamycin normally induces the expression of endogenous p21 in cells with wild-type p53 function but not in cells such as DLD1 that harbor mutant p53 (Waldman et al ., 1996). We investigated whether elevated levels of p21 would modulate the adriamycin eects in DLD1 colorectal carcinoma cells. Cells plated at low density were divided into three groups, in group one p21 was kept repressed, in group two p21 was transiently induced for 24 h and then repressed at the time of adriamycin addition, while group three cells had p21 `on' during the course of experiment. Cells were treated with 200 n M adriamycin for 48 h. The results presented in Table 2 show that there was no dierence in the number of apoptotic cells after adriamycin treatment irrespective of whether p21 was (i) kept repressed, (ii) transiently induced and then repressed or (iii) kept continuously induced through the course of adriamycin treatment. Representative photomicrographs in Figure 2 depict the apoptotic cells which constantly expressed p21 during the course of adriamycin treatment. Thus, overexpression of p21 during or prior to adriamycin treatment neither sensitized to nor protected from adriamycin-induced cell death. Figure 3 shows the expression pro®le of p21 in the three groups of cells described above. These cells constitutively express very low levels of endogenous p21, the expression of the p21 transgene can be controlled by addition or removal of tetracycline (Figure 3 compare lanes 1 ± 4 with lanes 5 ± 6) and adriamycin does not induce endogenous p21 in these (p53 mutant) cells. We then investigated the p21 eect on clonogenic survival following adriamycin treatment and u.v.- irradiation. Both adriamycin and u.v.-irradiation are genotoxic agents; adriamycin induces DNA strand breaks and other lesions repaired predominately via non-NER mechanisms, while u.v.-irradiation generates pyrimidine dimers and other photo-products (reviewed in Friedberg et al ., 1995). Cells growing at a very low density in the presence of tetracycline were washed with PBS and refed with identical medium with or without tetracycline. Approximately 6 h later cells were exposed to adriamycin or u.v.-irradiation and kept in medium with or without tetracycline for another 20 h. Time course analysis revealed that exogenous p21 can be readily detected within 6 h after tetracycline removal in these cells (Chen et al ., 1995). The rationale of this experimental strategy was to increase the p21 levels transiently at the time of DNA damage and a few hours thereafter. As shown previously, the transient induction of p21 for 24 h does not induce alterations in the growth pro®le of these cells (Chen et al ., 1995). We reasoned that if p21 enhances DNA repair, then elevated levels of p21 at the time of DNA damage should facilitate ecient DNA repair, which should result in enhanced clonogenic survival. Alter- natively, if p21 inhibits DNA repair, then that would be re ̄ected as a decrease in clonogenic survival. The results presented in Figure 4 demonstrate that the transient induction of p21, at the time of u.v.- irradiation and few hours thereafter, appreciably increased cell survival following 3.7 J/m 2 and 7 J/m 2 u.v. exposure; higher does of u.v-irradiation (10 J/m 2 and 20 J/m 2 ) were, however, highly toxic and the results were not informative (data not shown). Similar induction of p21, however, did not alter the clonogenic survival after adriamycin treatment (data not shown). Thus, p21 appears to protect cells from u.v.-type DNA damage, but not from other classes of DNA damage, at least as is shown for adriamycin in the present study. To further investigate the role of p21 in pathways that control the repair of u.v.-type DNA damage, the ability of these cells to repair u.v.-damaged plasmid DNA was assessed. In these `host cell reactivation' experiments (Smith et al ., 1995, 1996), u.v.-damaged or undamaged plasmids carrying the chloramphenicol acetyl transferase gene under the control of SV40 promoter enhancer were introduced into cells growing in the presence of tetracycline. Host cell reactivation is based on the principle that pyrimidine dimers in particular, strongly block the transcription of damaged reporter genes, thus a reporter can be expressed only to the extent that the damage is repaired when transfected into a given cell line (Klocker et al ., 1985; Protic et al ., 1988; Smith et al ., 1995, 1996). Twenty- four hours post-transfection, cells were harvested and divided into two groups. In one group the expression of exogenous p21 was turned on, while in the other group it was kept repressed. Approximately 48 h later cells were harvested and CAT activity was determined in the lysates prepared from both groups of cells. We reasoned that if p21 indeed enhances the repair of u.v.- type DNA damage, then those cells in which p21 was induced should display higher reporter activity than those in which it was kept repressed. Moreover, since the same pool of transfected cells were equally divided into two groups, this strategy ensured that the interpretation of results would not be confounded by the variations introduced due to multiple transfections. Figure 5 shows that indeed the group of cells, in which p21 was induced after transfection of damaged plasmid, exhibited higher CAT activity than the cells in which p21 was kept uninduced. Similar results were obtained when two other u.v.-damaged or undamaged plasmids, each expressing either an alkaline phosphatase gene from SV40 promoter enhancer or the green lantern gene under the control of CMV promoter, were used (Figure 5). Together these ®ndings demonstrate that the p21 induction resulted in the enhanced reactivation of three dierent u.v.-damaged reporter plasmids. In this study we have demonstrated that the overexpression of p21 neither sensitized to nor protected from adriamycin-mediated apoptosis. Similar results were obtained when other chemotherapeutic agents such as etoposide and taxol were tested (data not shown). Overexpression of p21 itself, however, induced apoptosis as well as giant cell formation. The p21-induction did not result into massive cell killing and there was an ...
Citations
... The effect of p21 on NER was also investigated in vivo using cell model systems in which p21 was ablated, such as the HCT116/p21 −/− [37,38] or the p53-deficient DLD1 cell line, in which p21 expression (virtually absent) was driven by a Tet-regulated expression plasmid [39]. In both model systems, the repair of a damaged plasmid reporter, i.e., the hostcell reactivation (HCR) assay, analyzed in the presence or in the absence of p21, showed that p21 facilitated the repair of UV-or cisplatin-damaged DNA [37][38][39]. ...
... The effect of p21 on NER was also investigated in vivo using cell model systems in which p21 was ablated, such as the HCT116/p21 −/− [37,38] or the p53-deficient DLD1 cell line, in which p21 expression (virtually absent) was driven by a Tet-regulated expression plasmid [39]. In both model systems, the repair of a damaged plasmid reporter, i.e., the hostcell reactivation (HCR) assay, analyzed in the presence or in the absence of p21, showed that p21 facilitated the repair of UV-or cisplatin-damaged DNA [37][38][39]. The dependence of these results on the interaction of p21-PCNA was demonstrated by the reversed effect when a truncated p21 peptide, lacking the C-terminus and thus unable to interact with PCNA, was reintroduced into HCT116/p21 −/− cells [37]. ...
... In particular, the interaction with PCNA might be necessary for regulatory purposes and not only instrumental to the substrate recognition by the CRL4 complex. This hypothesis, in addition to previous studies indicating deficiencies in DNA repair in the absence of p21 [37][38][39]44], is supported by the evidence that p21/PCNA interaction is required to prevent chromosomal aberrations after DNA damage, which was observed in cells expressing a p21 mutant unable to interact with PCNA [110]. ...
The p21CDKN1A protein is an important player in the maintenance of genome stability through its function as a cyclin-dependent kinase inhibitor, leading to cell-cycle arrest after genotoxic damage. In the DNA damage response, p21 interacts with specific proteins to integrate cell-cycle arrest with processes such as transcription, apoptosis, DNA repair, and cell motility. By associating with Proliferating Cell Nuclear Antigen (PCNA), the master of DNA replication, p21 is able to inhibit DNA synthesis. However, to avoid conflicts with this process, p21 protein levels are finely regulated by pathways of proteasomal degradation during the S phase, and in all the phases of the cell cycle, after DNA damage. Several lines of evidence have indicated that p21 is required for the efficient repair of different types of genotoxic lesions and, more recently, that p21 regulates DNA replication fork speed. Therefore, whether p21 is an inhibitor, or rather a regulator, of DNA replication and repair needs to be re-evaluated in light of these findings. In this review, we will discuss the lines of evidence describing how p21 is involved in DNA repair and will focus on the influence of protein interactions and p21 stability on the efficiency of DNA repair mechanisms.
... Depletion of p21 was found to cause lung embryonic fibroblasts more susceptible to UV, together with a decrease in NER capacity (Stivala et al. 2001). Furthermore, p21 modulates the nucleotide excision repair progress to promote the repair of UV-induced DNA damage even in the deficiency of wild-type p53 (Sheikh et al. 1997). There are also several studies which claim that p21 recruits to DNA-damage sites and colocalizes with DNA repair proteins. ...
... Influence of p21 on DNA repair after irradiation recruited to DNA-damage sites and colocalized with PCNA and PCNA-interacting proteins involved in nucleotide excision repair (NER), such as DNA polymerase δ, XPG and CAF-1 modulate the nucleotide excision repair process to facilitate the repair of UV-induced DNA damage even in the absence of wild-type p53Sheikh et al. (1997) H1299 human lung epithelial carcinoma cells, HCT116 and RKO human colorectal cancer cell lines and WI38 human fibroblasts UV irradiation Downregulation of p21 after UV radiation was required for efficient PCNA ubiquitination, which was conducive to PCNA-dependent DNA repairSoria et al. (2006) Normal human lung embryonic fibroblasts UV irradiation Loss of p21 was found to induce an increase in UV sensitivity, together with a reduction in NER capacityStivala et al. (2001) Murine lung epithelial cell line, human cervical carcinoma cell line, human diploid lung fibroblast cell line, Chinese hamster ovary cell line Laser irradiation EGFP-p21 accumulated rapidly at irradiated sites, and colocalized with the DSB marker γ-H2AX and with the DSB sensor protein Ku80Normal human foreskin fibroblasts Heavy ion bombardment p21 bound to DNA damage sites and colocalized with hMre11 and ...
Background
Greater than half of cancer patients experience radiation therapy, for both radical and palliative objectives. It is well known that researches on radiation response mechanisms are conducive to improve the efficacy of cancer radiotherapy. p21 was initially identified as a widespread inhibitor of cyclin-dependent kinases, transcriptionally modulated by p53 and a marker of cellular senescence. It was once considered that p21 acts as a tumour suppressor mainly to restrain cell cycle progression, thereby resulting in growth suppression. With the deepening researches on p21, p21 has been found to regulate radiation responses via participating in multiple cellular processes, including cell cycle arrest, apoptosis, DNA repair, senescence and autophagy. Hence, a comprehensive summary of the p21’s functions in radiation response will provide a new perspective for radiotherapy against cancer.
Methods
We summarize the recent pertinent literature from various electronic databases, including PubMed and analyzed several datasets from Gene Expression Omnibus database. This review discusses how p21 influences the effect of cancer radiotherapy via involving in multiple signaling pathways and expounds the feasibility, barrier and risks of using p21 as a biomarker as well as a therapeutic target of radiotherapy.
Conclusion
p21’s complicated and important functions in cancer radiotherapy make it a promising therapeutic target. Besides, more thorough insights of p21 are needed to make it a safe therapeutic target.
... Previously, depending on the p21Waf1 status a significant difference in the cell fate following various DNA damages was observed. Thus, p21Waf1 appears to protect cells from UV-induced DNA damage, but not from adriamycin treatment (Sheikh et al., 1997). Some data suggest the possibility of p21Waf1 involvement in doublestrand DNA breaks (DSBs) repair due to an observed p21Waf1 recruitment to DSBs sites and co-localization with DSBs repair proteins (Jakob et al., 2002;Koike et al., 2011). ...
This study aimed to explore a role of p21Waf1 in γH2AX foci formation and DNA repair as assessed by a Host-Cell Reactivation Assay in wild-type (p21Waf +/+ ) and p21Waf1-deficient E1A+Ras-transformed cells. p21Waf1 +/+ cells have low γH2AX background compared to p21Waf1 -/- cells. Correspondingly, NaBut treatment increases the γH2AX content in p21Waf +/+ cells with little effect in p21Waf -/- cells. Moreover, NaBut inhibits DNA repair in wt cells but not in p21Waf1 -/- cells. In consistence with this, the binding of GADD45 with PCNA that is a prerequisite for effective DNA repair is reduced in NaBut-treated p21Waf1- expressing cells but not in p21Waf1 -/- cells. We suggest that in wt-ERas cells NaBut activates both p21Waf1 gene expression and a release of p21Waf1 from the complexes with E1A that leads to suppression of DNA repair and promotes γH2AX persistency. The absence of p21Waf1 is by itself considered by the cell as stressful factor with formation of γH2AX. But the lack of p21Waf1 interferes with an inhibitory effect of NaBut to inhibit DNA repair and thereby to stop concomitant accumulation of harmful mutations. We conclude that p21Waf1 is directly involved in control of genome integrity and DNA repair acting through modulation of the components of the DNA repair machinery.
... When D-type cyclins are complexed with CDK4/6 phosphorylate serine and threonine residues on the retinoblastoma (Rb) protein, this tethers the Rb from E2F transcriptional factors, thereby enabling the E2F-mediated activation of a series of target genes essential for S phase entry. The overexpression of p21, however, causes the accumulation of hypophosphorylated Rb (pRb) and the sequestration of E2F, which causes the cell to be arrested in G21 phase [19]. If the genomic insult is extensive instead, p53 induces apoptosis in an effort to eliminate potentially trans-formed cells (Figure 1). ...
... The cyclin-dependent kinase inhibitor p21, a checkpoint regulator of cell cycle, was significantly overexpressed following neonatal hyperoxia. p21, in addition to being a regulator of cell cycle, is involved in cell differentiation and DNA repair (Sheikh et al. 1997;Stein et al. 1999). Previous studies have shown that hyperoxia induces p21 in the neonatal lung (McGrath 1998;O'Reilly et al. 1998O'Reilly et al. , 2001. ...
Premature infants with bronchopulmonary dysplasia (BPD), are at risk for frequent respiratory infections and reduced pulmonary function. We studied whether neonatal hyperoxia disrupts adaptive immune responses in adult mice, contributing to higher respiratory-related morbidities seen in these infants. Newborn mice litters were randomized at 3 days to 85% O2 or room air (RA) for 12 days. Whole lung mRNA was isolated in both the groups at 2 weeks and 3 months. Gene expression for T-cell and B-cell adaptive immune response was performed by real-time PCR and qRT-PCR; protein expression (p21, IL4, IL10, IL27, cd4) was performed by enzyme immunoassay along with p21 immunohistochemistry. Hyperoxia increased expression of p21 and decreased expression of 19 genes representing T/B-cell activation by ≥ fourfold; three of them significantly (Rag1, Cd1d1, Cd28) compared to the RA group at 2 weeks. Despite RA recovery, the expression of IFNγ, IL27, and CD40 was significantly reduced at 3 months in the hyperoxia group. Expression of p21 was significantly higher and IL27 protein lower at 2 weeks following hyperoxia. Adult mice exposed to neonatal hyperoxia had lower IL4 and IL10 in the lung at 3 months. Adaptive immune responses are developmentally regulated and neonatal hyperoxia suppresses expression of genes involved in T-/B-cell activation with continued alterations in gene expression at 3 months. Dysfunction of adaptive immune responses increases the risk for susceptibility to infection in premature infants.
... Studies devoted to the identification of components of the cellular response to DNA damage have been carried out for a number of years, and many of them used UVirradiation as a stressor. Some of these investigations identified several novel UV-induced genes using a UVtreated low-ratio hybridization subtraction-enriched cDNA hamster library, and they were followed by screenings of partial-length expressed sequence tag (EST) clones regulated by this type of stress [22][23][24] . This latter procedure rendered a UV-induced EST clone that hybridized with a 1.4 kb transcript, which was named PDRG1 [22] . ...
PDRG1 is a small oncogenic protein of 133 residues. In normal human tissues, the PDRG1 gene exhibits maximal expression in the testis and minimal levels in the liver. Increased expression has been detected in several tumor cells and in response to genotoxic stress. High-throughput studies identified the PDRG1 protein in a variety of macromolecular complexes involved in processes that are altered in cancer cells. For example, this oncogene has been found as part of the RNA polymerase II complex, the splicing machinery and nutrient sensing machinery, although its role in these complexes remains unclear. More recently, the PDRG1 protein was found as an interaction target for the catalytic subunits of methionine adenosyltransferases. These enzymes synthesize S-adenosylmethionine, the methyl donor for, among others, epigenetic methylations that occur on the DNA and histones. In fact, downregulation of S-adenosylmethionine synthesis is the first functional effect directly ascribed to PDRG1. The existence of global DNA hypomethylation, together with increased PDRG1 expression, in many tumor cells highlights the importance of this interaction as one of the putative underlying causes for cell transformation. Here, we will review the accumulated knowledge on this oncogene, emphasizing the numerous aspects that remain to be explored.
... Initial studies using p21 +/+ or p21 À/À HCT116 human colon cancer cells exposed to ultraviolet (UV) or cis-platinum revealed a positive effect of p21 on DNA processing and repair of DNA lesions handled primarily by nucleotide excision repair (NER) [24]. Similarly, using a tetracycline-inducible expression system, another report demonstrated that p21 might positively affect NER in the repair of UV-induced DNA lesions in the absence of wildtype p53 in DLD1 colorectal carcinoma cells [25]. It is now accepted that the capability of p21 to inhibit the function of PCNA in DNA replication, but not in (PCNAdependent) NER, might help explain in vivo data showing that genetic damage can lead to the inactivation of replication, while at the same time permitting DDR and repair [26]. ...
Upon DNA damage or other stressors, the tumor suppressor p53 is activated, leading to transient expression of the cyclin-dependent kinase inhibitor (CKI) p21. This either triggers momentary G1 cell cycle arrest or leads to a chronic state of senescence or apoptosis, a form of genome guardianship. In the clinic, the presence of p21 has been considered an indicator of wildtype p53 activity. However, recent evidence suggests that p21 also acts as an oncogenic factor in a p53-deficient environment. Here, we discuss the controversial aspects of the two-faced involvement of p21 in cancer and speculate on how this new information may increase our understanding of its role in cancer pathogenesis. Prevailing notions indicate that p21 might also act as antiapoptotic agent, which may have relevant implications for future therapeutic strategies.
... In addition, early studies using ectopic expression of the protein showed that p21 did not inhibit NER [135,136]. In particular, cells expressing a p21 mutant form unable to bind PCNA were deficient in NER, but when the wild type protein was expressed, cells became proficient for repair [135]. ...
... It has been found that AKBA-induced apoptosis does not involve the generation of reactive oxygen species; however, AKBA activated p21 expression suggesting a role for p21 as an effector of AKBA-induced apoptosis. Interestingly, p21 has been found to accumulate in gliomas in vivo, and p21 has crucial role in inhibiting drug-induced apoptosis in glial and non-glial tumor cells (Jung et al., 1995;Sheikh et al., 1997;Ruan et al., 1998) and it mediates the cytoprotective effects of steroids in glioma cells (Naumann et al., 1998). In addition, boswellic acids synergize with the cytotoxic cytokine, CD95 ligand (which is a novel target for malignant glioma therapy), in inducing glioma cell apoptosis (Glaser et al., 1999). ...
Epidemiological studies have showed that regular consumption of phytochemicals is strongly associated with a reduced risk of cancer. The gum resin of the Boswellia species has been used traditionally in Ayurvedic medicine to treat inflammatory conditions. Recent experimental data from cell culture, preclinical and clinical studies strongly suggests the potential use of Boswellia essential oil (frankincense) for the prevention and/or treatment of wide variety of cancers including pancreatic, breast, prostate, blood, colorectal, brain, skin, bladder and hepatic cancers. Analysis of the ingredients of these extracts revealed that the pentacyclic triterpenes boswellic acids (BAs) possess biological activities and appear to be responsible for the respective anticancer activity. One such agent is acetyl-11-keto-beta-boswellic acid (AKBA), which shows promise for potential use as an effective anticancer agent.
... In addition, early studies using ectopic expression of the protein showed that p21 did not 10 inhibit NER [135,136]. In particular, cells expressing a p21 mutant form unable to bind ...
