The human papillomavirus type 58 E7 oncoprotein modulates cell cycle regulatory proteins and abrogates cell cycle checkpoints

HPV type 58 (HPV-58) is the third most common HPV type in cervical cancer from Eastern Asia, yet little is known about how it promotes carcinogenesis. In this study, we demonstrate that HPV-58 E7 significantly promoted the proliferation and extended the lifespan of primary human keratinocytes (PHKs). HPV-58 E7 abrogated the G1 and the postmitotic checkpoints, although less efficiently than HPV-16 E7. Consistent with these observations, HPV-58 E7 down-regulated the cellular tumor suppressor pRb to a lesser extent than HPV-16 E7. Similar to HPV-16 E7 expressing PHKs, Cdk2 remained active in HPV-58 E7 expressing PHKs despite the presence of elevated levels of p53 and p21. Interestingly, HPV-58 E7 down-regulated p130 more efficiently than HPV-16 E7. Our study demonstrates a correlation between the ability of down-regulating pRb/p130 and abrogating cell cycle checkpoints by HPV-58 E7, which also correlates with the biological risks of cervical cancer progression associated with HPV-58 infection.     

Tissue-specific regulation of Chk1 expression by p53

The regulation of Chk1, a critical protein kinase involved in G(2) phase arrest, has been a subject of recent research. Chk1 phosphorylates tumor suppressor p53 at multiple sites, while p53 has been shown to downregulate Chk1 expression under stress conditions in vitro, suggesting negative feedback between the two checkpoint proteins. Using the p53 knockout mouse model, we demonstrate by Western blot and immunohistochemistry that mChk1 expression is induced in spleen, thymus, and dermal fibroblasts and is reduced in lung and testis in p53(-/-) mice compared to p53(+/+) controls. The mChk1 protein was undetectable in heart, kidney, and skin, whereas abundant expression was observed in brain and liver in both p53(+/+) and p53(-/-) mice. These data indicate that p53 regulates Chk1 expression in a tissue-specific manner.     

PPM1D silencing by RNA interference inhibits proliferation and induces apoptosis in breast cancer cell lines with wild-type p53

PPM1D is an oncogene that is amplified and overexpressed in many human tumors, including breast cancer. It functions as a negative regulator of the p38 MAP kinase-p53 signaling pathway and is also proposed to participate in other critical cell survival pathways. To define the functional significance of PPM1D specifically in breast cancer, we used RNA interference to inhibit PPM1D expression in BT-474, MCF7, and ZR-75-1 breast cancer cell lines harboring amplification and increased expression of PPM1D. Efficient downregulation of PPM1D resulted in significantly reduced cell proliferation in MCF7 and ZR-75-1 cells carrying wild-type p53 but not in BT-474 carrying mutant p53, which indicates that the antiproliferative effect of PPM1D silencing is dependent on the p53 status of the cells. This result is in excellent agreement with the notion that PPM1D activation is an alternative mechanism for p53 inactivation. Additionally, our data indicate that the reduced cell growth observed after PPM1D silencing is due at least in part to increased apoptotic cell death. Our findings demonstrate that PPM1D is involved in the regulation of cell proliferation in breast cancer in a p53-dependent manner and that overexpression of PPM1D contributes to malignant phenotype by promoting sustained cell growth and cell survival.     

Multiparameter immunohistochemical analysis of the cell cycle proteins cyclin D1, Ki-67, p21WAF1, p27KIP1, and p53 in mantle cell lymphoma

BACKGROUND: Mantle cell lymphoma (MCL) is characterized by overexpression of cyclin D1, a G1 cyclin that participates in the control of cell cycle progression at the G1 to S phase transition. In addition to cyclin D1, other cell cycle regulatory molecules may be involved in the proliferation and progression of MCL. Mutation of p53, deletion of p16(INK4a), and loss of p21(WAF1) expression have been reported in some cases of blastoid MCL. OBJECTIVE: We sought to examine levels of expression of these proteins in typical and blastoid MCL and to determine whether differences were present between these subtypes of lymphomas. DESIGN: A retrospective series of typical and blastoid MCLs was evaluated for expression of the cell cycle-related proteins cyclin D1, p21(WAF1), p27(KIP1), Ki-67, and p53, as well as mitotic index. Paraffin-embedded archival tissues from 24 MCL specimens (17 typical, 7 blastoid) were immunostained with antibodies to p21(WAF1), p27(KIP1), p53, Ki-67, and cyclin D1. The percentage of positive cells for each specimen was estimated by counting 1500 cells under oil immersion microscopy. Levels of antigen expression were compared for the typical and blastoid MCLs. The mitotic index was estimated using twenty 100x oil immersion fields (OIFs) for each specimen. RESULTS: Cyclin D1 expression was seen in 22/24 specimens (92%). Blastoid MCLs were characterized by a significantly higher mean mitotic index (>20 mitoses/20 OIFs) and Ki-67 index (>45%) when compared with typical MCLs (P <.001 and P <.008, respectively; Fisher's exact test). High expression of p27(KIP1) (>25% staining) was seen more frequently in typical MCLs than in the blastoid variants (P =.03; Fisher's exact test). No significant differences were found between typical and blastoid MCLs for the expression of p21(WAF1) or p53. CONCLUSIONS: A significantly higher mitotic index and Ki-67 index were found in blastoid MCLs as compared with typical MCLs. Low p27(KIP1) expression was associated with the blastoid MCL variant. These findings confirm the high proliferative nature of blastoid MCL and suggest a role for p27(KIP1) in the negative regulation of the cell cycle in MCL.     

The human homologs of checkpoint kinases Chk1 and Cds1 (Chk2) phosphorylate p53 at multiple DNA damage-inducible sites

Upon DNA damage, the amino terminus of p53 is phosphorylated at a number of serine residues including S20, a site that is particularly important in regulating stability and function of the protein. Because no known kinase has been identified that can modify this site, HeLa nuclear extracts were fractionated and S20 phosphorylation was followed. We discovered that a S20 kinase activity copurifies with the human homolog of the Schizosaccharomyces pombe checkpoint kinase, Chk1 (hCHK1). We confirmed that recombinant hCHK1, but not a kinase-defective version of hCHK1, can phosphorylate p53 in vitro at S20. Additional inducible amino- and carboxy-terminal sites in p53 are also phosphorylated by hCHK1, indicating that this is an unusually versatile protein kinase. It is interesting that hCHK1 strongly prefers tetrameric to monomeric p53 in vitro, consistent with our observation that phosphorylation of amino-terminal sites in vivo requires that p53 be oligomeric. Regulation of the levels and activity of hCHK1 in transfected cells is directly correlated with the levels of p53; expression of either a kinase-defective hCHK1 or antisense hCHK1 leads to reduced levels of cotransfected p53, whereas overexpression of wild-type hCHK1 or the kinase domain of hCHK1 results in increased levels of expressed p53 protein. The human homolog of the second S. pombe checkpoint kinase, Cds1 (CHK2/hCds1), phosphorylates tetrameric p53 but not monomeric p53 in vitro at sites similar to those phosphorylated by hCHK1 kinase, suggesting that both checkpoint kinases can play roles in regulating p53 after DNA damage.     

Cellular localization and cell cycle regulation by a temperature-sensitive p53 protein

Primary rat embryo fibroblasts were transformed by a p53 mutant (alanine to valine change at amino acid 135) plus ras. This p53val135 mutant is temperature sensitive for a conformational change detected by the binding of a monoclonal antibody, PAb246, which recognizes the wild-type protein or the great majority of p53val135 at 32.5 degrees C. At 37 degrees C, both mutant and wild-type p53 conformational forms co-exist in the cells, while at 39.5 degrees C, the majority of the p53val135 in the cell is in a mutant conformation not recognized by PAb246 antibody. At 39.5 degrees C, the mutant p53 is localized in the cytoplasm of the cell. At 32.5 degrees C, the p53 protein enters the nucleus and stops the growth of these cells. At 37 degrees C where there is a mixture of mutant and wild-type p53, the wild-type p53 protein is in a complex with hsc70 and mutant p53 protein in the cytoplasm of the cell during G1. This wild-type protein enters the nucleus as the cells enter the S-phase of the cell cycle. At 32.5 degrees C, the cells stop replication and arrest at the G1/S border. After 48 hr at 32.5 degrees C, 91% of the cells are in the G1 fraction of the cell cycle. The S-phase cells appear to be immune to the p53 negative regulation of growth until they enter the next G1 period.(ABSTRACT TRUNCATED AT 250 WORDS)     

Apoptosis and cell cycle: distinct checkpoints with overlapping upstream control

The question as to whether apoptosis (programmed cell death) is controlled by one or few checkpoints is still unresolved. A growing body of evidence suggests that (one of) the decisive event(s) of cell death consists in the permeabilization of mitochondrial membranes. Indeed, multiple pro-apopotic signal transduction pathways converge on the proteins of the Bcl-2/Bax family which, in concert with the so-called permeability transition pore complex (PTPC), regulate mitochondrial membrane barrier function. Mitochondrial permeabilization causes the release of soluble intermembrane proteins, some of which are involved in the activation of apoptotic proteases and nucleases. Thus, the putative checkpoint determining the death/life decision is clearly different from the known checkpoints of cell cycle progression. Prominent oncogenes (e.g., c-Myc, Ras, Raf, Bcl-2) and tumor suppressor genes (e.g., p53, Bax) have been shown to modulate apoptosis via a direct or indirect effect on mitochondrial membranes. All these oncoproteins and tumor suppressor proteins may simultaneously influence the cell cycle and the propensity to undergo apoptosis. Several cell cycle regulatory proteins (e.g., cyclins, cdk, etc.) can induce or inhibit apoptosis via yet unknown pathways.     

Chk2/hCds1 functions as a DNA damage checkpoint in G(1) by stabilizing p53

Chk2/hcds1, the human homolog of the Saccharomyces cerevisiae RAD53/SPK1 and Schizosaccharomyces pombe cds1 DNA damage checkpoint genes, encodes a protein kinase that is post-translationally modified after DNA damage. Like its yeast homologs, the Chk2/hCds1 protein phosphorylates Cdc25C in vitro, suggesting that it arrests cells in G(2) in response to DNA damage. We expressed Chk2/hCds1 in human cells and analyzed their cell cycle profile. Wild-type, but not catalytically inactive, Chk2/hCds1 led to G(1) arrest after DNA damage. The arrest was inhibited by cotransfection of a dominant-negative p53 mutant, indicating that Chk2/hCds1 acted upstream of p53. In vitro, Chk2/hCds1 phosphorylated p53 on Ser-20 and dissociated preformed complexes of p53 with Mdm2, a protein that targets p53 for degradation. In vivo, ectopic expression of wild-type Chk2/hCds1 led to increased p53 stabilization after DNA damage, whereas expression of a dominant-negative Chk2/hCds1 mutant abrogated both phosphorylation of p53 on Ser-20 and p53 stabilization. Thus, in response to DNA damage, Chk2/hCds1 stabilizes the p53 tumor suppressor protein leading to cell cycle arrest in G(1).     

Bromodeoxyuridine induces p53-dependent and -independent cell cycle arrests in human gastric carcinoma cell lines.

OBJECTIVES: This study was designed to clarify the effects of bromodeoxyuridine (BrdU) on cell cycle progression and induction of apoptosis, and to demonstrate the role of P53 in these processes. METHODS: We continuously exposed four human gastric carcinoma cell lines with different P53 status (P53 wild-type AGS and MKN-45, P53-mutated MKN-28 and P53-deleted KATO-III) to BrdU in asynchronous and synchronous culture conditions, and analyzed DNA histograms of apoptotic and nonapoptotic cells determined by static DNA cytofluorometry. RESULTS: Continuous exposure to 20 microM BrdU after synchronization with hydroxyurea resulted in S phase delay and G1 arrest in MKN-45 and an increase of apoptosis in the first S/G(2) phase in AGS and MKN-45. In the second S phase, a delay of 3-6 h was observed in all the four cell lines. In asynchronous cultures, continuous exposures to 20 and 200 microM BrdU for 72 h or more caused growth suppression with G(1) and G(2) arrests, respectively, in all the cell lines. CONCLUSIONS: These data suggested that the BrdU-induced growth suppression of the cell lines examined was mainly caused by cell cycle arrest rather than cell death, and that the cell cycle arrests in the first S and G(1) phases (elicited by BrdU in the single DNA strand) and those in the second S, G(2) and G(1) phases (elicited by BrdU in the double DNA strands) were mediated by p53-dependent and -independent pathways, respectively.     

重组腺病毒介导的p^16与p^53基因联合转移对人肺癌细胞系 …

目的 探讨ｐ＾１６和ｐ＾５３基因对肺癌细胞的协同抑制效应及凋亡诱导作用。方法 首先用同源重组技术构建重组ｐ＾１６和ｐ＾５３腺病毒载体,然后单独或联合感染人肺癌细胞系Ｈ３５８,用免疫组织法及Ｗｅｓｔｅｒｎ ｂｌｏｔ检测腺病毒介导的基因转移效率与表达水平,用克隆形成实验、原位末端标记及流式术观察它们对Ｈ３５８生长特性及凋亡的影响,结果 免疫组织化学染色结果表明重组腺病毒载体可高效地将外源基因ｐ＾５３转     

6]-Gingerol induces cell cycle arrest and cell death of mutant p53-expressing pancreatic cancer cells

[6]-Gingerol, a major phenolic compound derived from ginger, has anti-bacterial, anti-inflammatory and anti-tumor activities. While several molecular mechanisms have been described to underlie its effects on cells in vitro and in vivo, the underlying mechanisms by which [6]-gingerol exerts anti-tumorigenic effects are largely unknown. The purpose of this study was to investigate the action of [6]-gingerol on two human pancreatic cancer cell lines, HPAC expressing wild- type (wt) p53 and BxPC-3 expressing mutated p53. We found that [6]-gingerol inhibited the cell growth through cell cycle arrest at G1 phase in both cell lines. Western blot analyses indicated that [6]-gingerol decreased both Cyclin A and Cyclin-dependent kinase (Cdk) expression. These events led to reduction in Rb phosphorylation followed by blocking of S phase entry. p53 expression was decreased by [6]-gingerol treatment in both cell lines suggesting that the induction of Cyclin-dependent kinase inhibitor, p21cip1, was p53-independent. [6]-Gingerol induced mostly apoptotic death in the mutant p53-expressing cells, while no signs of early apoptosis were detected in wild type p53-expressing cells and this was related to the increased phosphorylation of AKT. These results suggest that [6]-gingerol can circumvent the resistance of mutant p53- expressing cells towards chemotherapy by inducing apoptotic cell death while it exerts cytostatic effect on wild type p53- expressing cells by inducing temporal growth arrest.     

Aberrant cell cycle checkpoint function and early embryonic death in Chk1(-/-) mice

The recent discovery of checkpoint kinases has suggested the conservation of checkpoint mechanisms between yeast and mammals. In yeast, the protein kinase Chk1 is thought to mediate signaling associated with the DNA damage checkpoint of the cell cycle. However, the function of Chk1 in mammals has remained unknown. Targeted disruption of Chk1 in mice showed that Chk1(-/-) embryos exhibit gross morphologic abnormalities in nuclei as early as the blastocyst stage. In culture, Chk1(-/-) blastocysts showed a severe defect in outgrowth of the inner cell mass and died of apoptosis. DNA replication block and DNA damage failed to arrest the cell cycle before initiation of mitosis in Chk1(-/-) embryos. These results may indicate that Chk1 is indispensable for cell proliferation and survival through maintaining the G(2) checkpoint in mammals.