WAF1/p21 regulates proliferation, but does not mediate p53-dependent apoptosis in urothelial carcinoma cell lines

The WAF1/p21 gene product is an inhibitor of cyclin-dependent kinases which can be induced by the tumor suppressor p53 and mediate some of its effects, or function in p53-independent pathways of cell cycle regulation. Although a potential tumor suppressor gene, WAF1/p21 is expressed in bladder cancer. To elucidate the function of p21 in tumor cells we have investigated in urothelial carcinoma cell lines: i) WAF1/p21 mRNA and protein expression, ii) the biological effects of p21 overexpression or down-regulation and (iii) whether p21 can be induced by p53. WAF1/p21 mRNA levels examined in four cell lines were comparable to bladder mucosa. One cell line, HT1376, failed to express p21 protein due to a frame shift mutation. Overexpression of WAF1/p21 cDNA inhibited clone formation in three cell lines, whereas transfection with antisense WAF1 increased clone sizes and numbers. WAF1 sense clones showed diminished cell proliferation compared to the parental cell line. Apoptosis- induced wild-type p53 was not inhibited by overexpression of antisense WAF1/p21. In a cell clone derived from line VMCub1 by stable transfection with wild-type p53 under the control of a metallothionein promotor, p21 was induced along with p53 upon activation of the promoter with zinc chloride. This induction was accompanied by a decrease in cell proliferation but by little apoptosis. These data suggest that p21 inhibits proliferation in a p53-dependent or independent manner but does not mediate p53-induced apoptosis in urothelial carcinoma cells.     

TGF-beta 1 induces the cyclin-dependent kinase inhibitor p27Kip1 mRNA and protein in murine B cells

TGF-beta1 inhibits the cell cycle progression of many types of cells by arresting them in the G1 phase. This cell cycle arrest has been attributed to the regulatory effects of TGF-beta1 on both the levels and the activities of the G1 cyclins and their kinase partners. The activities of these kinases are negatively regulated by a number of proteins, such as p15INK4b, p21WAF1/Cip1, and p27Kip1, that physically associate with cyclins, cyclin-dependent kinases (Cdk), or cyclin-Cdk complexes. In epithelial cell lines, TGF-beta1 was previously shown to inhibit cell cycle progression through down-regulation of Cdk4 and/or up-regulation of p15INK4b and/or p21WAF1/Cip1. However, TGF-beta1 had little or no effect on the p27Kip1 mRNA and protein levels. In this report, we show that, in contrast to observations in epithelial cell lines, TGF-beta1 increased the p27Kip1 mRNA and protein levels in the murine B cell lines CH31 and WEHI231. This TGF-beta1-mediated induction of p27Kip1 also resulted in an increased association of p27Kip1 with Cdk2 and a decreased Cdk2 kinase activity. In contrast to epithelial cells, however, TGF-beta1 had little or no effect on the Cdk4 and p21WAF1/Cip1 protein levels in these B cells. Finally, although several studies suggested a direct role of p53 in TGF-beta1-mediated cell cycle arrest in epithelial cells, TGF-beta1 inhibited cell cycle progression in CH31 even in the absence of wild-type p53. Taken together, these results suggest that TGF-beta1 induces G1 arrest in B cells primarily through a p53-independent up-regulation of p27Kip1 protein.     

The geranylgeranyltransferase-I inhibitor GGTI-298 arrests human tumor cells in G0/G1 and induces p21(WAF1/CIP1/SDI1) in a p53-independent manner

Recently we have shown that in fibroblasts (NIH 3T3 and Rat-1 cells) inhibition of protein geranylgeranylation leads to a G0/G1 arrest, whereas inhibition of protein farnesylation does not affect cell cycle distribution. Here we demonstrate that in human tumor cells the geranylgeranyltransferase-I (GGTase-I) inhibitor GGTI-298 blocked cells in G0/G1, whereas the farnesyltransferase (FTase) inhibitor FTI-277 showed a differential effect depending on the cell line. FTI-277 accumulated Calu-1 and A-549 lung carcinoma and Colo 357 pancreatic carcinoma cells in G2/M, T-24 bladder carcinoma, and HT-1080 fibrosarcoma cells in G0/G1, but had no effect on cell cycle distribution of pancreatic (Panc-1), breast (SKBr 3 and MDAMB-231), and head and neck (A-253) carcinoma cells. Furthermore, treatment of Calu-1, Panc-1, Colo 357, T-24, A-253, SKBr 3, and MDAMB-231 cells with GGTI-298, but not FTI-277, induced the protein expression levels of the cyclin-dependent kinase inhibitor p21WAF. HT-1080 and A-549 cells had a high basal level of p21WAF, and GGTI-298 did not further increase these levels. Furthermore, GGTI-298 also induces the accumulation of large amounts of p21WAF mRNA in Calu-1 cells, a cell line that lacks the tumor suppressor gene p53. There was little effect of GGTI-298 on the cellular levels of another cyclin- dependent kinase inhibitor p27KIP as well as cyclin E and cyclin D1. These results demonstrate that GGTase-I inhibitors arrest cells in G0/G1 and induce accumulation of p21WAF in a p53-independent manner and that FTase inhibitors can interfere with cell cycle events by a mechanism that involves neither p21WAF nor p27KIP. The results also point to the potential of GGTase-I inhibitors as agents capable of restoring growth arrest in cells lacking functional p53.     

Radiation induced G1-block and p53 status in six human cell lines

Considerable attention has recently been focused on the fact that the tumor suppressor protein p53 is involved in the cellular response to radiation. In its wild-type form the protein appears to control a cell cycle checkpoint, preventing entry into S-phase following DNA damage. A number of authors observed a radiation induced G1-block in cells expressing wild-type p53, but not in p53 mutant cells. We obtained similar results with four human tumour cell lines as well as two strains of human fibroblasts, whose p53 status was ascertained at the protein as well as DNA levels. In addition to cell cycle delays in exponentially growing cell cultures, we have studied the possible role of the p53 in the transition from quiescence to active proliferation. Cells were irradiated after 6 days of serum-starvation and labelled with BrdU at different times after addition of fresh medium. Entry into S-phase was found to be delayed by several hours in the p53 wild-type cells, but no such effect was observed in the p53 mutants. Where a delay occurred, it was roughly proportional to the X-ray dose. Although it remains to be clarified, whether the cells were delayed only in G1 or also in G0, it is interesting to note that entry into S-phase can be delayed by irradiation in a quiescent state immediately before serum-stimulation, provided the cells are wild-type with respect to p53. Certain differences in the cell cycle response of transformed and untransformed cells were noted.     

PTHrP and cell division: expression and localization of PTHrP in a keratinocyte cell line (HaCaT) during the cell cycle

Parathyroid hormone-related protein (PTHrP) is highly expressed in normal skin keratinocytes, and its involvement in growth and differentiation processes in these cells has been implicated by several lines of evidence which include the use of antisense PTHrP (Kaiser et al., 1994, Mol. Endocrinol., 8:139-147). In this study, we have investigated whether PTHrP expression and its subcellular localization is linked to cell cycle progression in a human keratinocyte cell line (HaCat), which constitutively expresses and secretes PTHrP. PTHrP mRNA and immunoreactive PTHrP were assessed in asynchronous dividing cells and in cells blocked at G1 or G2 + M phases of the cell cycle using several different protocols. The response of PTHrP mRNA expression was examined following readdition of serum in the continued presence of cycle blockers, and after release from cell cycle block, or from cell synchronization by serum deprivation. PTHrP expression was greatest in actively dividing cells when cells were in S and G2 + M phases of the cell cycle and were lowest in quiescent G1 cells. Most notable were the high levels of PTHrP mRNA and protein in cells at G2 + M phase of the cell cycle at division. Furthermore, PTHrP was localized to the nucleolus in quiescent cells, but redistributed to the cytoplasm when cells were actively dividing. Taken together, these results support a role for PTHrP in cell division in keratinocytes. In asynchronously growing cells, PTHrP expression fell as cells became confluent at a time when cell growth is inhibited and cells begin to differentiate. Mitogen stimulation of HaCaT cells resulted in a rapid increase in PTHrP mRNA expression, but was dependent upon cells being in the G1 phase of the cell cycle. Cells blocked in G1 responded to mitogen both in the continued presence of aphidicolin or when released from block. Cells blocked at G2 + M with colcemid expressed high levels of PTHrP mRNA and protein, and PTHrP mRNA did not respond further to mitogen in the continued presence of blocker. However, in cells released from block at G2 + M by addition of serum, an increase in PTHrP expression was seen coincident with the progression of cells into G1. In contrast, in a squamous cancer cell line (COLO16), basal PTHrP expression was high and was not altered during the cell cycle or by cell cycle block, consistent with association of its dysregulated expression in malignant cells. The results of this study suggest that PTHrP may have two roles in the cell cycle; one in G1 in response to mitogen, and a second at cell division when its expression is high and it is relocated from the nucleolus to the cytoplasm.     

Regulated ectopic expression of cyclin D1 induces transcriptional activation of the cdk inhibitor p21 gene without altering cell cycle progression

Cyclin D1 plays a key regulatory role during the G1 phase of the cell cycle and its gene is amplified and overexpressed in many cancers. To address the relationship between cyclin D1 and other cell cycle regulatory proteins, we established human glioma and rodent fibroblast cell lines in which cyclin D1 expression could be regulated ectopically with tetracycline. In both of these cell lines, we found that ectopic expression of cyclin D1 in asynchronously growing cells was accompanied by increased levels of the p53 tumor suppressor protein and the cyclin/cdk inhibitor p21. Despite the induction of these cell cycle inhibitory proteins, cyclin D1-associated cdk kinase remained activated and the cells grew essentially like that of the parent cells. Although growth parameters were unchanged in these cells, morphological changes were clearly identifiable and anchorage independent growth was observed in NIH3T3 cells. In a first step toward elaborating the mechanism for cyclin D1-mediated induction of p21 gene expression we show that co-expression of E2F-1 and DP-1 can specifically transactivate the p21 promoter. In support of these findings and a direct effect of E2F on induction of p21 gene expression a putative E2F binding site was identified within the p21 promoter. In summary, our results demonstrate that ectopic expression of cyclin D1 can induce gene expression of the cdk inhibitor p21 through an E2F mechanism the consequences of which are not to growth arrest cells but possibly to stabilize cyclin D1/cdk function.     

Studies of X-irradiated bladder cancer cell lines showing differences in p53 status: absence of a p53-dependent cell cycle checkpoint pathway

Cell growth arrest is a common response to DNA damage by ionising irradiation and the p53 gene has been shown to play an important role in this mechanism, possibly in a tissue-specific manner. Mutations in the p53 gene are frequent in invasive bladder cancers, which are often treated by radiotherapy. In this paper we have investigated the growth response to X-irradiation of three bladder cancer cell lines with differing p53 status: UCRU-BL-17 overexpresses mutant p53, while UCRU-BL-13 and UCRU-BL-28 contain wt P53. We have also examined the expression of proteins reported to be part of the p53 control pathway in response to irradiation-induced DNA damage. No G1 arrest was detectable in any of the cell lines after ionising irradiation; furthermore, in a downstream event reported to be correlated with p53 function there was no increase in WAF-1 protein levels regardless of p53 status. Rather, ionising irradiation resulted in G2 arrest, but the extent of this was not related to p53 status. p16 levels were also not affected by irradiation. Our results suggest that the UCRU-BL-28 cell line may have a defect in the p53-cell control pathway upstream of p53, while UCRU-BL-13 cells may have a defect downstream between p53 and WAF-1.     

Expression of differentially phosphorylated Rb and mutant p53 proteins in myeloid leukemia cell lines

We studied the structure and expression of Rb and p53 genes in six myeloid leukemia cell lines (HL-60, KBM3, K562, KBM5, EM2, KBM7) in the light of the published reports that structural abnormalities of these genes are rarely seen in leukemic cells and also a recent finding that Rb gene expression can be regulated by the p53 protein. Except for HL-60 cells which have a truncated p53 gene, none of the other cell lines revealed any gross structural abnormalities in the Rb and p53 genes. KBM3, KBM5 and EM-2 expressed lower levels of Rb mRNA than HL-60, K562 and KBM7. The amount of Rb protein was lowest in KBM3 cells and in this and two other cell lines (KBM5, KBM7) Rb was markedly hypophosphorylated compared to the other three cell lines. HL-60 and K562 did not express p53 m-RNA, while the other four cell lines all expressed high levels of mutant p53 protein. Thus even in the absence of gross structural alterations, subtle abnormalities in the expression pattern of Rb and p53 genes occur in myeloid leukemia cells.     

Inhibition of tumor cell growth by RTP/rit42 and its responsiveness to p53 and DNA damage

Through a differential screening technique, we have identified a cDNA clone with differential expression in normal versus tumor cells. This clone, designated rit42 (reduced in tumor, 42 kDa), was previously isolated as a homocysteine-inducible gene in human endothelial cells (RTP), and the same or a highly related androgen-responsive gene in mouse has also been identified. Both Northern blot analysis and in situ hybridization demonstrated a significantly diminished expression in tumor cells, including those derived from breast and prostate when compared with normal cells. It was shown that RTP/rit42 mRNA cycles with cell division, peaking at G1 and G2-M, with lower expression in S phase. The biphasic expression of RTP/rit42 mRNA was absent in tumor cells. Introduction of rit42 cDNA into human cancer cells reduced cell growth both in vitro and in nude mice. Moreover, analysis of a tetracycline-regulated p53-inducible system in null-p53 cell lines showed that RTP/rit42 mRNA expression increased concomitantly with p53 expression and followed a similar time course. In addition, DNA-damaging agents induced RTP/rit42 expression in a p53-dependent manner but independent of a p53-mediated G1 arrest. Immunofluorescence analysis of a FLAG epitope-tagged RTP/rit42 protein revealed a cytoplasmic localization pattern with redistribution to the nucleus upon DNA damage. We have localized RTP/rit42 to human chromosome 8q24.3. Taken together, these results are consistent with a growth inhibitory role for RTP/rit42, and its down-regulation may contribute to the tumor malignant phenotype.     

Inhibition of radiation-induced G2 delay potentiates cell death by apoptosis and/or the induction of giant cells in colorectal tumor cells with disrupted p53 function

We have previously identified a p53-independent apoptotic response that is delayed until 48-72 h after irradiation of colorectal adenoma and carcinoma cells. Because the delay appears to be in part due to a transient G2 cell cycle arrest, the importance of this checkpoint in the mechanism of ionizing radiation (IR)-induced death of colorectal tumor cells was investigated. An adenoma cell line with (282Arg-->Trp) mutant p53 (S/RG/C2) and a carcinoma cell line (PC/JW/FI) lacking p53 protein treated with 5 Gy IR in the presence of 1.5 mm caffeine (CAF) reduced IR-induced G2 arrest and increased the level of apoptosis (1.5-1.6-fold) 24 h after treatment. Increased IR apoptotic cell death with CAF significantly reduced IR cell survival over a 7-day period in S/RG/C2 and PC/JW/FI. To investigate whether CAF radiosensitization correlated with lack of wild-type (wt) p53, we studied transfected derivatives of an adenoma-derived cell line (PC/AA/C1), in which the endogenous wt p53 activity was disrupted by the expression of a dominant negative (273Arg-->His) p53 mutant protein (designated AA/273p53/B). This p53-defective cell line was also radiosensitized by CAF, whereas the vector control (AA/PCMV/D), which retained wt p53 activity, was not. In addition, as with the S/RG/C2 and PC/JW/FI cell lines, the 7-day IR cell survival was reduced significantly in AA/273p53/B compared with the vector control cell line. This suggests that radiosensitization by CAF and increased cell death is dependent on loss of wt p53 function. Interestingly, radiosensitization of the AA/273p53/B cell line was not associated with accelerated apoptosis but correlated with increased polyploid giant cells, which have been associated with disruption of cell cycle checkpoints and genomic instability. These results demonstrate that G2 checkpoint inhibition with CAF leads to preferential IR cell killing in cell lines in which wt p53 is inactivated and that this increased cell killing is not necessarily dependent on increased IR-induced apoptosis.     

Expressions of cell cycle regulators in human colorectal cancer cell lines

To study the altered mechanisms of cell cycle regulation in colorectal cancer, the expressions of cyclins, cyclin-dependent kinases (CDKs), CDK inhibitors, p53 and retinoblastoma (Rb) protein were analyzed by western blotting in a series of human colorectal cancer cell lines. The colorectal cancer cell lines exhibited various expression patterns of cell cycle regulators, which may reflect differences in the biological characteristics of cancer cells and in the genetic backgrounds of carcinogenesis. A correlation was found between p53 gene alteration and p21 expression, suggesting that p53 gene mutation usually suppresses p21 expression, though p21 expression could be induced via both a p53-dependent and a p53-independent pathway in colorectal cancer. None of the cell lines studied expressed p16 protein, suggesting that inactivation of p16 may be a common alteration in colorectal cancer. Moreover, all the D-type cyclins, especially D2 and D3, were expressed at a high level in most of the cell lines. Loss of p16 expression and increased expression of D-type cyclins promote CDK-mediated Rb phosphorylation. All of the colorectal cancer cell lines studied herein expressed Rb protein, but the growth-suppressive properties of Rb may be inactivated by the loss of p16 expression and increased expressions of D-type cyclins. In view of the pivotal role of Rb in cell cycle regulation, loss of p16 expression and overexpression of D-type cyclins may be critical alterations in colorectal cancer.     

Involvement of p53 and p21 in cellular defects and tumorigenesis in Atm-/- mice

Disruption of the mouse Atm gene, whose human counterpart is consistently mutated in ataxia-telangiectasia (A-T) patients, creates an A-T mouse model exhibiting most of the A-T-related systematic and cellular defects. While ATM plays a major role in signaling the p53 response to DNA strand break damage, Atm-/- p53(-/-) mice develop lymphomas earlier than Atm-/- or p53(-/-) mice, indicating that mutations in these two genes lead to synergy in tumorigenesis. The cell cycle G1/S checkpoint is abolished in Atm-/- p53(-/-) mouse embryonic fibroblasts (MEFs) following gamma-irradiation, suggesting that the partial G1 cell cycle arrest in Atm-/- cells following gamma-irradiation is due to the residual p53 response in these cells. In addition, the Atm-/- p21(-/-) MEFs are more severely defective in their cell cycle G1 arrest following gamma-irradiation than Atm-/- and p21(-/-) MEFs. The Atm-/- MEFs exhibit multiple cellular proliferative defects in culture, and an increased constitutive level of p21 in these cells might account for these cellular proliferation defects. Consistent with this notion, Atm-/- p21(-/-) MEFs proliferate similarly to wild-type MEFs and exhibit no premature senescence. These cellular proliferative defects are also rescued in Atm-/- p53(-/-) MEFs and little p21 can be detected in these cells, indicating that the abnormal p21 protein level in Atm-/- cells is also p53 dependent and leads to the cellular proliferative defects in these cells. However, the p21 mRNA level in Atm-/- MEFs is lower than that in Atm+/+ MEFs, suggesting that the higher level of constitutive p21 protein in Atm-/- MEFs is likely due to increased stability of the p21 protein.     

Retinoic acid-mediated down-regulation of Oct3/4 coincides with the loss of promoter occupancy in vivo

Oct3/4, a hallmark of the earliest stages of embryogenesis, is expressed in undifferentiated embryonal carcinoma (EC) and embryonic stem (ES) cells. Oct3/4 gene expression is dependent on the promoter region, the proximal enhancer and the newly identified distal enhancer. We have analysed in vivo occupancy of these elements. In undifferentiated EC and ES cells, strong footprints were detected at specific sites of all three regulatory elements. These were promptly lost upon RA treatment in ES cells and in P19 EC cells, in parallel with sharply reduced Oct3/4 mRNA levels. Thus, the occupancy of regulatory elements is coupled with Oct3/4 expression, and RA treatment causes coordinated factor displacement, leading to extinction of gene activity. In F9 EC cells, footprint was first abolished at the proximal enhancer. However, this loss of binding site occupancy did not result in a decrease in Oct3/4 mRNA levels. The partial factor displacement seen in F9 EC cells, combined with the observation that EC and ES cells utilize the proximal and distal enhancers in differential manner, indicate the complex pattern of Oct3/4 gene regulation, which could reflect a cell type- and lineage-specific expression of the gene in vivo.     

Relationship of p53 overexpression to other cell cycle regulatory proteins in oral squamous cell carcinoma

Aberrations of the p53 gene and the overexpression of its protein are described in a variety of neoplasms, including oral and other head and neck cancers. Here we report the association of p53 (over)expression with a downstream cell cycle inhibitor p21/waf 1 in oral squamous cell carcinoma (SCC). The loss of expression of p16 and p27, two other cyclin-dependent kinase (cdk) inhibitors, was also examined. In this panel of tumours, 10/24 carcinomas were p53-immunopositive. Heterogeneous expression of p21 and p27 was seen in 10/24 SCC and 9/16 SCC, respectively, and this was not correlated to p53 status. The expression of p21 and p27 in these SCCs suggests the existence of mechanisms by which some growing tumour cells may tolerate these cell cycle inhibitors; eight SCCs lacked expression of both inhibitors but only two of these cancers overexpressed p53, suggesting that accumulation of p21/p27 can be independent of the functional status of the p53 gene. Data do not support a clear example of a phenotype that shows an overexpression of p53 with downregulation of p21 or p27 leading to cell cycle alterations. Furthermore, only three SCCs were p16-negative and p53-positive. This suggests that these two tumour suppressors may act in separate pathways.     

Role of the p53 tumor suppressor gene in the tumorigenicity of Burkitt's lymphoma cells

Burkitt's lymphoma (BL) cell lines carry a translocated c-myc gene and, in 60-80% of cases, exhibit mutations in the p53 tumor suppressor gene. We examined the potential role of the p53 gene in BL tumorigenicity using an in vitro assay that measures p53-dependent cell cycle arrest in the G1 phase of the cell cycle and an in vivo athymic murine model that detects differences in the tumorigenicity of BL cell lines. A highly significant inverse correlation was found between the ability of BL cells to arrest in G1 after irradiation and their tumorigenicity in athymic mice, consistent with the notion that loss of p53 function is associated with increased tumorigenicity. Inactivation of wild-type (wt) p53 function by expression of the human papillomavirus E6 protein in the AG876V BL cell line, which carries both wt and mutant p53 proteins, rendered the cell line significantly more tumorigenic in athymic mice. Transfection of the wt p53 gene into the p53 mutant and highly tumorigenic BL-41 cell line caused it to acquire wt p53 function and rendered it less tumorigenic in mice. In addition to confirming a role for the loss of p53 function in tumor progression, the data demonstrate that wt p53 protein can reduce BL tumorigenicity in vivo.     

Cell cycle re-entry following chemically-induced cell cycle synchronization leads to elevated p53 and p21 protein levels

Mimosine (MIM) and aphidicolin (APH) are two agents frequently used in tissue culture-based experiments to achieve cell synchronization at late G1 and S phases. Following MIM or APH treatment of human cancer cell lines, a reversible growth arrest in late G1 and S phases of the cell cycle was correlated with moderate increases in p53 and p21 protein levels. Both p53-dependent and -independent increases in p21 were observed following treatment with either agent. However, a striking increase in p21 protein levels and a continuous elevation in both p53 and p21 protein levels were observed over 48 h after cells re-entered the cell cycle following the chemically-induced synchronization. In addition, the increase in p21 protein levels typically seen following treatment of cells with DNA damaging agents, was enhanced when cells were treated with genotoxic agents following MIM or APH synchronization. These findings suggest that caution should be exercised when interpreting results from experiments using cell synchronization agents, in particular, studies designed to investigate p53- and p21-regulatory pathways.     

Loss of a p53-associated G1 checkpoint does not decrease cell survival following DNA damage

Cell cycle checkpoints regulate progression through the cell cycle. In yeast, loss of the G2 checkpoint by mutation of the rad9 gene results in increased genetic instability as well as increased sensitivity to ionizing radiation. In contrast, comparing clonogenic survival of cells which are isogeneic except for p53 functional status, we find that loss of a G1 checkpoint in mammalian cells is not associated with increased sensitivity to the lethal effects of ionizing radiation or a topoisomerase I inhibitor, camptothecin. These results indicate that increased sensitivity to DNA-damaging agents is not necessarily a defining feature of a mammalian cell cycle checkpoint. Furthermore, in light of a recent link of p53 function to radiation-induced apoptosis in hematopoietic cells, these observations suggest that p53-dependent apoptosis is a cell type-specific phenomenon and thus predict that the biological consequences of loss of p53 function will be cell type specific.