Deregulation of the p14ARF/Mdm2/p53 pathway and G1/S transition in two glioblastoma sets

Sixty-one glioblastomas have been studied, subdivided into the categories of classic glioblastomas (GBM) and glioblastomas with astrocytic (GBA) and oligodendroglial (GBO) differentiated areas. On surgical samples, TP53, Mdm2, CDKN2A/p16–p14 alterations were studied by molecular biology techniques and by immunohistochemistry. It has been found that Mdm2 amplification was more frequent in GBM than in GBA and GBO, that p14^ARF was inactivated in a high percentage of cases in the three tumor categories. Both these and other alterations did not reach a statistical significance, with the exception of CDKN2A/p16 homozygous deletion which showed the highest frequency in GBO. The latter finding could be in line with the observation that CDKN2A/p16 inactivation is a step in the molecular pathway to tumor progression in oligodendrogliomas. TP53 mutations and Mdm2 amplifications were mutually exclusive, whereas TP53 mutations and CDKN2A/p14 inactivation coexisted in 5 cases. The alterations of the p53/Mdm2/p14^ARF pathway occurred in 73% of cases and in 80% of cases if CDKN2A homozygous deletions were associated. All glioblastomas with gemistocytic areas showed p14^ARF inactivation. Immunohistochemistry showed higher percentages of positivity in comparison with molecular genetics, but with similar variations.     

Jaceosidin induces p53-dependent G2/M phase arrest in U87 glioblastoma cells

Flavonoid compounds have been shown to trigger cell cycle arrest at G0/G1, S and G2/M checkpoints, allowing cells to repair DNA damage before entry into mitosis. Jaceosidin, a flavonoid compound, has been reported to induce apoptosis in various cancer cell lines. In our previous study, we established that jaceosidin induces apoptosis in U87 glioblastoma cells through G2/M phase arrest. However the molecular mechanisms oremained unclear. In the present study, mRNA and protein expression levels of major cell cycle regulatory genes were analyzed by semi-quantitative RT-PCR and Western blot studies respectively. The results demonstrated that jaceosidin-induced G2/M phase arrest in U87 cells is associated with DNA fragmentation, up-regulation of p53 and p21 and subsequent down-regulation of cyclin B1 and CDK1 expression at mRNA as well as at protein level. These findings provide insights into jaceosidin-induced G2/M phase arrest in U87 glioblastoma cells.     

EGFR inhibition in glioblastoma cells induces G2/M arrest and is independent of p53

Mutations involving the TP53 gene are frequently identified in up to 50% of all human tumors, including glioblastomas. Analysis of expression patterns of TP53 in glioblastomas shows that it is mainly mutated in secondary glioblastomas and is less common in primary GBMs. However, the prognostic significance of TP53 loss of function in astrocytomas has always been controversial. In contrast, EGFR/erbB2 complexes have been implicated in the poor prognosis of several cancers, including glioblastomas. Our previous work showed that transforming phenotypes could be inhibited by interfering with active EGFR/erbB2 complex using mutant erbB2 proteins in wild-type p53 GBM cells. To assess the dependence of EGFR inhibited phenotype on p53, we used three mutant p53 glioblastoma cell lines in the present study and showed that mutant erbB2 can be exploited to inhibit EGFR-mediated oncogenic transformation irrespective of p53 status. Ectopic expression of a mutant erbB2 receptor (T691S) in mutant p53 GBM cells resulted in slower growth rate than empty vector controls. T691S-expressing clones exhibited a more flattened and nontransformed morphology. Consistently, T691S inhibited transformation in soft agar assays and tumor formation in nude mice independent of p53 status. Biochemical analysis showed reduced Akt and GSK-3 alpha/beta, but not p42/44MAPK phosphorylation, in T691S-expressing cells, when compared to parental controls, suggesting the P13-K pathway may be more relevant than MAPK for glial cell transformation. Cell cycle analysis showed reduced cyclin D1 and CDK6 and increased phospho-Cdc-2 (Tyr15) and p15INK4B in erbB2-inhibited cells, suggesting that nonfunctional EGFR/erbB2 complexes exert their inhibitory effects at various stages of the cell cycle to block the progression of cells through G2/M via Akt/GSK-3/Cdc2 pathway. Collectively, these observations provide a basis for receptor-based therapies that disable erbB receptors and inhibit proliferative signals in erbB-expressing human cancers including glioblastomas, regardless of their TP53 status.     

p53 effects both the duration of G2/M arrest and the fate of temozolomide-treated human glioblastoma cells

Temozolomide (TMZ) is a DNA-methylating agent that has recently been introduced into Phase II and III trials for the treatment of gliomas. TMZ produces O6-methylguanine in DNA, which mispairs with thymine during the next cycle of DNA replication. Subsequent futile cycles of DNA mismatch repair can lead to a p53-associated apoptotic cell death, although this mechanism has been described mostly in hematopoietic neoplasms. We studied the action of TMZ in gliomas and the role p53 might play by using U87 glioma cells that were either p53-wild-type or p53-deficient (by virtue of expression of the viral oncoprotein E6). LN-Z308 cells, in which p53 gene is deleted, were also used. p53-proficient U87 MG cells underwent a prolonged, p53- and p21(Waf1/Cip1)-associated G2-M arrest beginning 2 days after TMZ treatment. Although very few of these cells underwent apoptosis, most underwent senescence over a 10-day period. p53-deficient (E6-transfected U87 and LN-Z308) cells similarly underwent G2-M arrest in response to TMZ, but this arrest was accompanied by only minor changes in p53 or p21(Waf1/Cip1) and was reversed within 7 days of TMZ treatment in association with the appearance of cells with either 8n or subG1 DNA content. These results suggest that glioma cells respond to TMZ by undergoing G2-M arrest. p53 is not necessary for this G2-M arrest to occur but is important in the duration of G2-M arrest and in the ultimate fate of TMZ-treated cells. Therefore, the integrity of the G2-M cell cycle checkpoint may be important in the cytotoxicity of TMZ in glioma cells.     

TPA-induced p21 expression augments G2/M arrest through a p53-independent mechanism in human breast cancer cells

The tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), has a differential role on the regulation of the cell cycle in a variety of tumor cells. The mechanism between TPA and the cell cycle in breast cancer is not fully understood. Therefore, we investigated the regulatory mechanism of TPA on control of the cell cycle of breast cancer cells. Our results showed that TPA increased the level of p21 expression in MCF-7 cells with wild-type p53 and MDA-MB-231 cells with mutant p53 in a dose-dependent manner. In contrast, TPA decreased the expression of p53 in MCF-7 cells, but did not affect MDA-MB-231 cells. We next examined the regulatory mechanism of TPA on p21 and p53 expression. Our results showed that the TPA-induced up-regulation of p21 and down-regulation of p53 was reversed by UO126 (a MEK1/2 inhibitor), but not by SP600125 (a JNK inhibitor) or SB203580 (a p38 inhibitor), although TPA increased the phosphorylation of ERK and JNK in MCF-7 cells. In addition, the TPA-induced arrest of the G2/M phase was also recovered by UO126 treatment. To confirm the expression of p21 through the MEK/ERK pathway, cells were transfected with constitutively active (CA)-MEK adenovirus. Our results showed that the expression of p21 was significantly increased by CA-MEK overexpression. Taken together, we suggest that TPA reciprocally regulates the level of p21 and p53 expression via a MEK/ERK-dependent pathway. The up-regulation of p21 in response to TPA is mediated through a p53-independent mechanism in breast cancer cells.     

Inactivation of p53 in normal human cells increases G2/M arrest and sensitivity to DNA-damaging agents

p53 mutations are found in about 70% of human cancers. In order to evaluate the role of these mutations in response to chemotherapeutic agents, it is important to distinguish between p53 response to DNA-damaging agents in normal and in tumour cells. Here, using normal human fibroblasts (NHFs), we show that cisplatin and UV radiation induce G₂/M arrest which is temporally linked to p53-protein induction. To study the contribution of p53 to this G₂/M arrest, we inhibited p53 induction in NHFs using p53 anti-sense oligonucleotides. Following exposure of NHFs to UV radiation, the inhibition of p53-protein induction leads to a greater accumulation of cells in the G₂/M phase, but also to a decreased fraction of cells in the G₁ phase. We propose that p53 does not induce G₂/M arrest directly, and that the extent of this arrest may depend on the fraction of cells that do not stop at the G₁ phase following exposure to DNA-damaging agents. Furthermore, inhibition of p53-protein induction leads to increased sensitivity of NHFs to UV radiation. These results suggest that inhibition of p53 protein enhances sensitivity to DNA-damaging agents in normal human cells. Int. J. Cancer 75:432–438, 1998. © 1998 Wiley-Liss, Inc.     

Celecoxib leads to G2/M arrest by induction of p21 and down-regulation of cyclin B1 expression in a p53-independent manner

A unique in vitro system has been developed in our lab that consists of normal enterocytes derived from the rat ileum (IEC-18 cells) and their transformed derivatives with c-K-ras (R1 cells), anti-sense bak (B3 cells) and cyclin D1 (D1 cells). R1 and B3 cells express high level of COX-2 protein and PGE2. IEC 18 and D1 cells express negligible amount of COX-2, and produce very low level of PGE2. A relatively low dose of celecoxib (5-10 microM) induced G2/M arrest, followed by induction of apoptosis in the transformed but not in the normal cells. Down-regulation of cyclin B1 and up-regulation of p21 expressions independent of p53 might have cause this cell cycle block. Growth inhibition was related to COX-2 function with 90-95% reduction in PGE2 production. These findings may be of clinical importance, since low concentration of celecoxib can be achieved in human serum following standard anti-inflammatory (100-200 mg bid) regime.     

Failure of taxol-based combination chemotherapy for malignant glioma cannot be overcome by G2/M checkpoint abrogators or altering the p53 status

In an effort to develop more effective forms of adjuvant chemotherapy for malignant brain tumors, we sought to develop a taxol-based combination chemotherapy regimen for glioma cell lines in vitro. Here, we report that coexposure of LN-229 or T98G glioma cells to taxol and either doxorubicin, camptothecin, cytarabine, or VM26 resulted in antagonistic effects rather than additive or synergistic cytotoxicity. There were no interactions of taxol with the effects of carmustine (BCNU) or vincristine. Antagonism was more prominent in cytotoxic cell death assays than in clonogenic cell death assays and was not overcome by G₂/M checkpoint abrogators such as caffeine or pentoxyfilline. Ectopic expression of mutant and wild-type p53val¹³⁵ attenuated taxol cytotoxicity in both T98G cells, which are mutant for p53, and LN-229 cells, which exhibit functional wild-type p53 activity. Interestingly, wild-type p53val¹³⁵ abrogated the taxol-imposed G₂/M arrest in both cell lines. However, wild-type p53val¹³⁵ did not promote glioma cell killing by combinations of taxol and any of the other drugs. Further, an analysis of a panel of 12 human glioma cell lines revealed no relationship between genetic or functional p53 status and taxol sensitivity. In summary, combination either with other chemotherapy drugs, with abrogators of the G₂/M checkpoint, with wild-type p53 gene transfer was not a promising approach for a taxol-based combination chemotherapy regimen in malignant glioma. Received: 14 December 1998 / Accepted: 15 February 1999     

芹菜素诱导乳腺癌T47D细胞系p53依赖性凋亡及G2/M期阻滞

  目的  探讨芹菜素（apigenin）对乳腺癌T47D细胞系凋亡及细胞周期的影响。  方法  常规培养T47D细胞, 用四甲基偶氮唑盐法（MTT法）检测芹菜素对乳腺癌T47D细胞系细胞增殖的影响。荧光染色法观察凋亡细胞的形态变化。流式细胞仪检测细胞凋亡率及周期分布。Western blot检测不同浓度（0、10、20、40、80 μM）芹菜素对凋亡及周期相关蛋白表达的影响。  结果  随着芹菜素浓度的增加, 芹菜素对T47D细胞的增殖抑制作用明显增强（P < 0.05）; 凋亡荧光染色及流式细胞仪显示, 芹菜素能够诱导乳腺癌T47D细胞凋亡, 随着其浓度的增高, 各组凋亡率分别为（2.41±0.072）%、（10.87±0.028）%、（18.02±0.056）%、（37.05± 0.092）%和（78.38±0.082）%, 与对照组相比, 差异有统计学意义（P < 0.05）; 流式周期分析显示, 芹菜素能够使T47D细胞系G₂/M期阻滞, 随着芹菜素浓度的增高, 不同浓度组细胞G₂/M期所占的比例逐渐增加, 差异有统计学意义（P < 0.05）; Western blot显示p53、p21、Bax和p-Cdc2的含量及Caspase-3, PARP剪切明显增加, 而Bcl-2、CyclinB1及Cdc2的含量却显著减少。  结论  芹菜素能够诱导乳腺癌T47D细胞系p53依赖性凋亡及G₂/M期阻滞, 从而明显抑制其增殖。      

Cdc25C interacts with PCNA at G2/M transition

Cdc25 activates maturation promoting factor (MPF) and promotes mitosis by removing the inhibitory phosphate from the Tyr-15 of Cdc2 in human cells. In this study, we searched the interacting protein(s) of human Cdc25C using the yeast two-hybrid screen and identified proliferating cell nuclear antigen (PCNA) as an interacting partner of Cdc25C. The interaction between Cdc25C and PCNA was confirmed in vitro and in vivo. Co-immunoprecipitation analyses using human T cell line, Jurkat, further revealed that Cdc25C interacted with PCNA transiently when cells began to enter mitosis. Immunofluorescence analysis also showed that Cdc25C and PCNA were transiently co-localized in the nucleus at the beginning of M phase. Together with the previous observations of the interaction between various cdc/cyclin and PCNA, our findings strongly suggested a potential role of PCNA at the G2 to M phase transition of cell cycle.     

Regulation of cancer stem cells by p53

The hypothesis that cancer stem cells are responsible for the chemoresistant and metastatic phenotypes of many breast cancers has gained support using cell-sorting strategies to enrich the tumor-initiating population of cells. The mechanisms regulating the cancer stem cell pool, however, are less clear. Two recent publications suggest that loss of p53 permits expansion of presumptive cancer stem cells in mouse mammary tumors and in human breast cell lines. These results add restriction of cancer stem cells as a new tumor suppressor activity attributed to p53.     

LATS1 tumor suppressor regulates G2/M transition and apoptosis

The LATS1 gene is a mammalian member of the novel lats tumor suppressor family. Both lats mosaic flies and LATS1 deficient mice spontaneously develop tumors. Our previous studies have shown that inactivation of Drosophila lats leads to up-regulation of cyclin A in the fly, and the human LATS1 protein associates with CDC2 in early mitosis in HeLa cells, suggesting that the lats gene family may negatively regulate cell proliferation by modulating CDC2/Cyclin A activity. We demonstrate here that transduction of the human breast cancer cell MCF-7 with recombinant LATS1 adenovirus (Ad-LATS1), but not with EGFP adenovirus (Ad-EGFP), inhibits in vitro cell proliferation. Ectopic expression of LATS1 in MCF-7 cells specifically down-regulates Cyclin A and Cyclin B protein levels and dramatically reduces CDC2 kinase activity, leading to a G2/M blockade. Furthermore, Ad-LATS1 suppresses anchorage-independent growth of MCF-7 cells in soft agar and tumor formation in athymic nude mice. We also demonstrate that ectopic expression of LATS1 in MCF-7 cells and human lung cancer cell H460 up-regulates the level of BAX proteins and induces apoptosis. Finally, we show that LATS1 kinase activity is required for its ability to inhibit cell growth and induce apoptosis. The results indicate that the LATS1 tumor suppressor may play an important role in the control of human tumor development and that LATS1 suppresses tumorigenesis by negatively regulating cell proliferation and modulating cell survival.     

Role of p53 and p38 MAP kinase in nitric oxide-induced G2/M arrest and apoptosis in the human lung carcinoma cells

The authors, Jui-I.Chao and Pao-Chen Kuo, found critical problemswith some of the data they reported in the above paper and wishto withdraw it from publication. The paper has been publishedonline     

Regulation of p53 stability

Leading the way in imposing a policy of zero tolerance of cellular abnormalities that might lead to tumor development is the p53 protein. The efficiency of p53 in preventing cell growth is a strong deterrent to malignant progression, but this activity must be kept tightly restrained to allow normal cell growth and development. Essential components of this regulation are the mechanisms by which the p53 protein is degraded, and efficient turnover of p53 in normal cells prevents the accumulation of the protein. Modulation of these degradation pathways in response to stress leads to the rapid stabilization and accumulation of p53, and activation of the p53 response. It is now becoming clear that the Mdm2 protein is central to the regulation of p53 stability and multiple pathways exist through which the activity of Mdm2 can be inhibited. Defects in the ability to stabilize p53 are likely to contribute to malignant development, and restoration of this activity represents an extremely attractive possibility for tumor therapy.     

Deregulation of the p14ARF/MDM2/p53 pathway is a prerequisite for human astrocytic gliomas with G1-S transition control gene abnormalities

Deregulation of G1-S transition control in cell cycle is one of the important mechanisms in the development of human tumors including astrocytic gliomas. We have previously reported that approximately two-thirds of glioblastomas (GBs) had abnormalities of G1-S transition control either by mutation/homozygous deletion of RB1 or CDKN2A p16INK4A), or amplification of CDK4 (K. Ichimura et al., Oncogene, 13: 1065-1072, 1996). However, abnormalities of G1-S transition control genes may induce p53-dependent apoptosis in cells. Recent investigations suggest that p14ARF is induced in response to abnormal cell cycle entry and results in p53 accumulation by inhibiting MDM2-mediated transactivational silencing and degradation of p53. To investigate the roles of the G1-S transition control system and the p14ARF/MDM2/p53 pathway in the development of astrocytic gliomas, we examined abnormalities of genes involved in these regulatory pathways in a total of 190 primary human astrocytic gliomas of different malignancy grades [136 GBs, 39 anaplastic astrocytomas (AAs) and 15 astrocytomas (As)]. Sixty-seven percent of GBs (91/136) and 21% of AAs (8/39) had abnormalities of the G1-S control system either by mutation/homozygous deletion of RB1, CDKN2A or CDKN2B, or amplification of CDK4. Seventy-six percent of GBs (103 of 136), 72% of AAs (28 of 39), and 67% of As (10 of 15) had deregulated p53 pathway either by mutation of TP53, amplification of MDM2, or homozygous deletion/mutation of p14ARF. When all of the data were combined and compared, 96% of GBs (87 of 91) and 88% of AAs (7 of 8) with abnormal G1-S transition control also had deregulated p53 pathway. Thus, we demonstrate that deregulation of the G1-S transition control system was almost always accompanied by inactivation of the p53 pathway, clearly illustrating the cooperative roles of these two systems in the development/progression of primary human astrocytic gliomas.