Influence of G2 arrest on the cytotoxicity of DNA topoisomerase inhibitors toward human carcinoma cells with different p53 status

We here report the influence of the cell cycle abrogator UCN-01 on RKO human colon carcinoma cells differing in p53 status following exposure to two DNA damaging agents, the topoisomerase inhibitors etoposide and camptothecin. Cells were treated with the two drugs at the IC90 concentration for 24 h followed by post-incubation in drug-free medium. RKO cells expressing wild-type, functional p53 arrested the cell cycle progression in both the G1 and G2 phases of the cell cycle whereas the RKO/E6 cells, which lack functional p53, only arrested in the G2 phase. Growth-arrested cells did not resume proliferation even after prolonged incubation in drug-free medium (up to 96 h). To evaluate the importance of the cell cycle arrest on cellular survival, a non-toxic dose of UCN-01 (100 nM) was added to the growth-arrested cells. The addition of UCN-01 was accompanied by mitotic entry as revealed by the appearance of condensed chromatin and the MPM-2 phosphoepitope, which is characteristic for mitotic cells. G2 exit and mitotic transit was accompanied by a rapid activation of caspase-3 and apoptotic cell death. The influence of UCN-01 on the long-term cytotoxic effects of the two drugs was also determined. Unexpectedly, abrogation of the G2 arrest had no influence on the overall cytotoxicity of either drug. In contrast, addition of UCN-01 to cisplatin-treated RKO and RKO/E6 cells greatly increased the cytotoxic effects of the alkylating agent. These results strongly suggest that even prolonged cell cycle arrest in the G2 phase of the cell cycle is not necessarily coupled to efficient DNA repair and enhanced cellular survival as generally believed.     

Differential responses of proliferating versus quiescent cells to adriamycin.

The relative sensitivity of proliferating and quiescent cells to DNA-damaging agents is a key factor for cancer chemotherapy. Here we undertook a reevaluation of the way that proliferating and quiescent cells differ in their responses and fate to adriamycin-induced damage. Distinct types of assays that measure membrane integrity, metabolic activity, cell size, DNA content, and the ability to proliferate were used to compare growing and quiescent Swiss3T3 fibroblasts after adriamycin treatment. We found that immediately after adriamycin treatment of growing cells, p53 and p21(Cip1/Waf1) were induced but the cells remained viable. In contrast, less p53 and p21(Cip1/Waf1) were induced in quiescent cells after adriamycin treatment, but the cells were more prone to immediate cell death, possibly involving apoptosis. Adriamycin induced a G2/M cell cycle arrest in growing cells and a concomitant increase in cell size. In contrast, adriamycin induced an increase in sub-G1 DNA content in quiescent cells and a decrease in cell size. In contrast to the short-term responses, adriamycin-treated quiescent cells have a better long-term survival and proliferation potential than adriamycin-treated growing cells in colony formation assays. These data suggest that proliferating and resting cells are remarkably different in their short-term and long-term responses to adriamycin.     

Multiple response elements and differential p53 binding control Perp expression during apoptosis

The p53 tumor suppressor gene responds to cellular stress by activating either cell cycle arrest or apoptosis. A growing number of target genes involved in each of these pathways have been identified. However, the mechanism by which the apoptosis versus arrest decision is made remains to be elucidated. Perp is a proapoptotic target gene of p53 expressed to high levels in apoptotic cells compared with those undergoing cell cycle arrest. This pattern of expression is unusual among p53 target genes, many of which are induced to similar levels during arrest and apoptosis. Here, we describe the regulation of the Perp gene by p53 through at least three response elements in the Perp promoter and first intron. These sites are occupied in vivo in E1A-expressing mouse embryo fibroblasts undergoing apoptosis but not cell cycle arrest, in contrast to the p21 5' response element, which is occupied during both. The apoptosis-deficient p53 point mutant, p53V143A, displays a selective deficit in binding to the Perp elements, demonstrating that p53 can distinguish between Perp and p21 at the level of DNA binding. These results provide mechanistic insight into the selective expression of Perp during apoptosis and may provide a useful model for studying the p53-dependent cell cycle arrest versus apoptosis decision.     

Inhibition of p53 by Lentiviral Mediated sh RNA Abrogates G1 Arrest and Apoptosis in Retinal Pigmented Epithelial Cell Line

We silenced p53 gene expression in ARPE-19, a human retinal pigmented epithelial cell line using RNA interference. The effect of silencing the p53 gene in proliferating ARPE-19 cells was studied. Four short hairpin RNAs (shRNAs) targeting different regions of human p53 mRNA were delivered individually into ARPE-19 cells using lentiviral vector to produce stable cell lines. p53 mRNA and protein levels were reduced to varying extents in the four shRNA-transduced ARPE-19 cell lines. The cell line that showed greatest reduction (85-90%) of p53 expression showed decreased p21 promoter activation after DNA damage with camptothecin, etoposide and MMS. Whereas treatment of wild type ARPE-19 cells with camptothecin resulted in apoptosis, silencing p53 expression increased their survival. Cell cycle analyses indicated that irradiation resulted in a G1 arrest in ARPE-19 cells, and that the arrest was significantly reduced in p53-silenced cells. Thus, p53 plays a central role in the response of ARPE-19 cells to DNA damaging agents that act via different mechanisms. Additionally, ARPE-19 cells with reduced p53 expression behave similar to tumor cell lines with mutated or non-functional p53. The present data demonstrate the utility of lentiviral vectors to create stable isogenic cell lines with reduced expression of a specific gene, thereby permitting the study of the function of a gene, the pathways controlled by it, and the effect of therapeutics on a cell with altered genetic makeup in a pair-wise fashion.     

Cell-cycle regulation of the p53-inducible gene B99

B99 is a p53-inducible gene whose accumulation upon p53 activation is restricted to late S/G2 cells. Here we have analyzed B99 regulation during the cell cycle in murine cells with or without functional p53. We report that B99 accumulates in late S/G2 phase, is phosphorylated in mitosis, and disappears in G1 phase, regardless of the status of p53. As a complement to this observation, we show that B99 is not induced by p53 in quiescent cells. Therefore, B99 expression is modulated both by cell-cycle regulatory mechanisms and by p53, and p53 can increase the cellular levels of B99 only during the window of the cell cycle when it is normally expressed. On the basis of these observations we rename B99 Gtse-1 (G-two- and S-phase-expressed).     

Changes in Cyclins and Cyclin-Dependent Kinases Induced by DNA Damaging Agents in a Human Ovarian Cancer Cell Line Expressing Mutated or Wild-Type P53

The expression of cyclins, cyclin-dependent kinases (cdk), and cdk inhibitors was evaluated in clones from a human ovarian cancer cell line transfected with a temperature-sensitive mutant of p53, after treatment with the anticancer agents doxorubicin (DX) and AMSA. The two drugs were selected on the basis of their activity in these clones, since AMSA is equally active in cells expressing mutated or wild-type (wt) p53, while DX was much less cytotoxic in cells expressing wt p53. In untreated cells, the expression of wt p53 induced an accumulation of cells in the G2 and perhaps also the G1 phase of the cell cycle. Concomitantly cyclin B1 and cdc2 increased. Cyclin E and particularly D1 levels were also raised by wt p53 expression. Treatment of mutated p53-expressing cells (SK23a cells kept at 37°C) with DX or, more so, with AMSA, resulted in a strong accumulation of cyclin B1 and cdc2, in accordance with their ability to block cells in G2 phase of the cell cycle. Wt p53-expressing cells (SK23a cells kept at 32°C) treated with the drugs showed an increase in p21 expression and consequently decreased kinase activity after immunoprecipitation with p21 antibodies. Cdc2-associated kinase activity was also reduced in these conditions. We could also observe a decrease in the percentage of cells in G1 and G2 phases and an accumulation of cells in S phase after both DX and AMSA. Cdk2, retinoblastoma, and p27 levels did not change significantly. Treatment with DX or AMSA caused similar effects, suggesting that p53-induced changes in cyclin, cdk, and cdk inhibitors after DNA damage are not responsible for the marked reduction in the cytotoxicity of DX we observed in wt p53-expressing cells.     

Diphenyleneiodonium induces ROS-independent p53 expression and apoptosis in human RPE cells

The diphenyleneiodonium (DPI) is widely used as an inhibitor of flavoenzymes, particularly NADPH oxidase. In this study, we investigated the effect of DPI on the apoptosis of human RPE cells. DPI treatment in ARPE-19 cells evoked a dose- and time-dependent growth inhibition, and also induced DNA fragmentation and protein content of the proapoptotic factor Bax. In addition, DPI significantly induced the expression and phosphorylation of p53, which induces proapoptotic genes in response to DNA damage or irreparable cell cycle arrest. ROS have been implicated as a key factor in the activation of p53 by many chemotherapeutic drugs. Recent data on the regulation of intracellular ROS by DPI are controversial. Therefore, we analyzed whether DPI could contribute to the generation of intracellular ROS. Although there was increase in ROS level from cells treated for 24h with DPI, it was not detectable at early time points, required to induce p53 expression. And DPI-induced p53 expression was not affected by the ROS scavenger NAC. We conclude that DPI induces the expression of p53 by ROS-independent mechanism in ARPE-19 cells, and renders cells sensitive to drug-induced apoptosis by induction of p53 expression.     

Roscovitine up-regulates p53 protein and induces apoptosis in human HeLaS(3) cervix carcinoma cells

Exposure of human HeLaS(3) cervix carcinoma cells to high doses of conventional cytostatic drugs, e.g. cisplatin (CP) strongly inhibits their proliferation. However, most cytostatic agents are genotoxic and may generate a secondary malignancy. Therefore, therapeutic strategy using alternative, not cytotoxic drugs would be beneficial. Inhibition of cyclin-dependent kinases (CDKs) by pharmacological inhibitors became recently a promising therapeutic option. Roscovitine (ROSC), a selective CDK inhibitor, efficiently targets human malignant cells. ROSC induces cell cycle arrest and apoptosis in human MCF-7 breast cancer cells. ROSC also activates p53 protein. Activation of p53 tumor suppressor protein is essential for induction of apoptosis in MCF-7 cells. Considering the fact that in HeLaS(3) cells wt p53 is inactivated by the action of HPV-encoded E6 oncoprotein, we addressed the question whether ROSC would be able to reactivate p53 protein in them. Their exposure to ROSC for 24 h induced cell cycle arrest at G(2)/M and reduced the number of viable cells. Unlike CP, ROSC in the used doses did not induce DNA damage and was not directly cytotoxic. Despite lack of detectable DNA lesions, ROSC activated wt p53 protein. The increase of p53 levels was attributable to the ROSC-mediated protein stabilization. Further analyses revealed that ROSC induced site-specific phosphorylation of p53 protein at Ser46. After longer exposure, ROSC induced apoptosis in HeLaS(3) cells. These results indicate that therapy of HeLaS(3) cells by ROSC could offer an advantage over that by CP due to its increased selectivity and markedly reduced risk of generation of a secondary cancer.     

Retained platinum uptake and indifference to p53 status make novel transplatinum agents active in platinum-resistant cells compared to cisplatin and oxaliplatin

Despite the clinical success of platinum-containing drugs in the treatment of solid tumors, acquired resistance remains a major obstacle. We previously identified a group of novel transplanaramine or transplatinum compounds based on distinct activity profiles in the NCI-60 panel. In the present study, parental KB-3.1 cells with wild-type p53 and its cisplatin- and oxaliplatin-resistant sublines harboring mutant p53 proteins were used to contrast several transplatinum compounds with cisplatin and oxaliplatin. The transplatinum compounds retained cytotoxic activity in the resistant cell lines. While intracellular accumulation and DNA platination of cisplatin and oxaliplatin was decreased in the resistant cells, the transplatinum compounds both accumulated intracellularly and platinated DNA at comparable levels in all cell lines. Cytoflow analysis confirmed that cisplatin and oxaliplatin alter the cell cycle distribution and result in apoptosis; however, at comparably toxic concentrations, the transplatinum compounds did not alter the cell cycle distribution. Analysis of the cytoplasmic fraction treated with acetone showed that cisplatin and oxaliplatin readily bound to macromolecules in the pellet, whereas a larger percentage of the transplatinum compounds remained in the supernatant. We concluded that, distinct from platinum compounds currently in use, transplatinum compounds accumulate intracellularly in resistant cells at levels comparable to those in drug-sensitive cells, do not affect the cell cycle and thus retain cytotoxicity independent of p53 status and likely have cytoplasmic targets that are important in their activity.     

A flow cytometry-based screen of nuclear envelope transmembrane proteins identifies NET4/Tmem53 as involved in stress-dependent cell cycle withdrawal

Disruption of cell cycle regulation is one mechanism proposed for how nuclear envelope protein mutation can cause disease. Thus far only a few nuclear envelope proteins have been tested/found to affect cell cycle progression: to identify others, 39 novel nuclear envelope transmembrane proteins were screened for their ability to alter flow cytometry cell cycle/DNA content profiles when exogenously expressed. Eight had notable effects with seven increasing and one decreasing the 4N∶2N ratio. We subsequently focused on NET4/Tmem53 that lost its effects in p53^−/− cells and retinoblastoma protein-deficient cells. NET4/TMEM53 knockdown by siRNA altered flow cytometry cell cycle/DNA content profiles in a similar way as overexpression. NET4/TMEM53 knockdown did not affect total retinoblastoma protein levels, unlike nuclear envelope-associated proteins Lamin A and LAP2α. However, a decrease in phosphorylated retinoblastoma protein was observed along with a doubling of p53 levels and a 7-fold increase in p21. Consequently cells withdrew from the cell cycle, which was confirmed in MRC5 cells by a drop in the percentage of cells expressing Ki-67 antigen and an increase in the number of cells stained for ß-galactosidase. The ß-galactosidase upregulation suggests that cells become prematurely senescent. Finally, the changes in retinoblastoma protein, p53, and p21 resulting from loss of NET4/Tmem53 were dependent upon active p38 MAP kinase. The finding that roughly a fifth of nuclear envelope transmembrane proteins screened yielded alterations in flow cytometry cell cycle/DNA content profiles suggests a much greater influence of the nuclear envelope on the cell cycle than is widely held.