Different levels of p53 induced either apoptosis or cell cycle arrest in a doxycycline-regulated hepatocellular carcinoma cell line <Emphasis Type="Italic">in vitro</Emphasis>

Induction of p53 gene expression in cancer cells can lead to both cell cycle arrest and apoptosis. To clarify whether the level of p53 expression determines the apoptotic response of hepatocellullar carcinoma (HCC) cells, we assessed the effect of various levels of expression of p53 gene on a p53-deficient HCC cell line, Hep3B, utilizing a doxycycline (Dox)-regulated inducible p53 expression system. Our results showed that apoptosis was induced in HCC cells with high levels of p53 expression. However, lower level of p53 expression induced only cell cycle arrest but not apoptosis. Bax expression was up-regulated following high levels of p53 expression, while bcl-2 expression was not altered by the level of p53 expression. Moreover, p21 expression was observed in both high and low expression of p53. These results suggest the level of p53 expression could determine if the HCC cells would go into cell cycle arrest or apoptosis. Bax may participate, at least in part, in inducing p53-dependent apoptosis and the induction of p21 alone was able to cause cell cycle arrest but not apoptosis.     

Introduction of wild-type p53 in a human ovarian cancer cell line not expressing endogenous p53.

Utilizing a temperature sensitive p53 mutant (pLTRp53cGval135) which expresses mutant p53 at 37 degrees C and a wild-type like p53 at 32 degrees C, we transfected a human ovarian cancer cell line (SKOV3) which does not express endogenous p53. Among the different clones obtained, we selected three clones. Two were obtained from simultaneous transfection of p53 and neomycin resistance expression plasmids (SK23a and SK9), the other was obtained from transfection experiments utilizing the neomycin resistance gene only (SKN). Introduction of mutant p53 did not alter the morphology or growth characteristics of this ovarian cancer cell line. Upon shifting to the permissive temperature, a dramatic change in morphology and growth rate was observed in SK23a and SK9 cells that is associated with the presence of a wild-type like p53. SKN and SKOV3 cells maintained at 32 degrees C did not change morphology and only slightly reduced proliferation. Both SK23a and SK9 cells did not show evidence of apoptosis when measured up to 72 hours of maintenance at 32 degrees C. In contrast to what observed in other cell lines, SK23a and SK9 cells maintained at 32 degrees C were not blocked in G1, but they were accumulated in G2-M. This accumulation was transient and could be due either to a blockade or to a delay in the G2 progression. No down-regulation of c-myc was observed in p53 expressing clones when shifted to the permissive temperature. In these conditions gadd45 mRNA expression was highly stimulated in SK9 and SK23a cells but not in SKN cells. In both clones Gas1 mRNA was not detected either at 37 degrees C or 32 degrees C. This system represents a new and useful model for studying the effect of the absence of p53 (SKOV3 or SKN), presence of mutated p53 (SK23a and SK9 kept at 37 degrees C) or wild type p53 (SK23a and SK9 kept at 32 degrees C) on the mechanism of response of cancer cells to DNA damaging agents.     

Abelson murine leukemia virus-transformed cells that lack p53 protein synthesis express aberrant p53 mRNA species.

Cells of the Abelson murine leukemia virus-transformed line L12 that lack the p53 protein also lack polyadenylated mRNA capable of directing the synthesis of p53 in a cell-free system. Direct analysis of stable polyadenylated mRNA from a variety of cell lines shows that all p53 producers shared a common mRNA species (2.0 kilobases) which hybridized with a p53-specific cDNA probe. This species, which appears to be the mature, normal-sized p53 mRNA, was totally undetectable in L12 cells, which did not produce p53 in vivo. However, L12 cells contained two major p53-specific mRNA species of a substantially larger size (3.5 and 6.5 kilobases) than the p53-specific mRNA in the p53-producing cells. Genomic DNA analysis uncovered an apparent alteration in the 5' proximal part of only one p53 gene, which is unique to the L12 cell line. It is thus possible that the nonproducer phenotype of L12 cells is due at least in part to an alteration within a p53-specific DNA sequence. These findings define a system in which production of p53 appears to be efficiently regulated at the level of stable mRNA and which can be used to study the mechanisms controlling p53 expression in Abelson murine leukemia virus-transformed cells.     

p53 gene expression of human hepatoma cell lines and their sensitivities to parvovirus H-1]

DNA structure and expression of p53 gene in human hepatoma cell lines SMMC-7721, YY-8103 and a spontaneously transformed liver cell line L-02 were analysed using the following method: analysis of allelic losses on chromosome 17p, PCR/SSCP, Northern blot and immunoprecipitation. There was no point mutation found in the exons 4-9 of the p53 gene, and a low level of expression of p53 gene was detected in the three cell lines. These observations were in agreement to the reported results of the relevant experiment using the human hepatoma cell line QGY-7703. Sensitivities of these cell lines and other eight human hepatoma cell lines (QGY-7703, PLC/PRF/5, Tong/HCC, Huh-7, FOCUS, Hep3B, SK-Hep-1, HepG2) with known p53 backgrounds to parvovirus H-1 was assayed using MTT method. Abnormality in the structure and/or function was observed in all of the cell lines examined except HepG2. The cell line HepG2 with normal structure and function of the p53 gene was found to be the least sensitive to H-1 in comparison to all the cell lines which have defeated structure and/or function of the p53 gene. The present study serves as a preliminary evidence that enhancement of the sensitivity of human hepatoma cell lines to H-1 is correlated to the abnormality of the structure and/or function of the p53 gene.     

人肝癌细胞p53基因的表达及其对细小病毒H-1的敏感性

本文利用单链构象多态性分析,17号染色体短臂等位基因杂合性分析,Northern印迹,免疫沉淀,p53基因第7外显子酶切等技术检测了两个中国人肝癌细胞系SMMC-7721,YY-8103和一个自发转化的人肝细胞系L-02的p53基因结构与表达。实验表明,这三个细胞系中没有出现17号染色体短臂等位基因杂合性缺失,第4—9外显子也没发生突变,但其mRNA和蛋白表达水平很低。利用MTT比色分析法研究了这三个细胞系和其他已知p53基因背景的八个人肝癌细胞系(QGY-7703、PLC/PRF/5、Huh-7、Hep3B、FOCUS、Tong/ HCC、SK-Hep-1、HepG2)对自主性细小病毒H-1的敏感性。除HepG2细胞外,其他十个细胞系p53基因的结构和/或表达都不正常。经H-1感染(moi=20)后,其敏感性均高于HepG2细胞。本研究初步表明了p53基因结构或表达的不正常可能导致人肝癌或转化细胞对H-1的敏感性的提高。     

Effects of exogenous wild-type p53 on a human lung carcinoma cell line with endogenous wild-type p53

Several studies have shown that expression of exogenous wild-type p53 is detrimental to the growth of cell lines with absent or mutant p53. In this study, wild-type p53 cDNA expression plasmids were transfected into A549 lung carcinoma cells which had previously been shown by sequencing to contain wild-type p53. When a constitutively expressed wild-type p53 plasmid containing the neomycin resistance gene was transfected into these cells, no G418-resistant colonies contained the exogenous p53 cDNA even though the neomycin resistance gene was integrated. When cells were transfected with a dexamethasone-inducible wild-type p53 cDNA expression plasmid, induction of p53 expression resulted in a decreased growth rate and a decreased proportion of S-phase cells. Continuous treatment with dexamethasone resulted in continued p53 expression for 16 days, but beyond that time expression ceased and could not be reinduced. These data indicated that although the A549 cell line could proliferate in the presence of endogenous wild-type p53 there was a strong selection pressure against continued expression of additional exogenous wild-type p53.     

Radiation response and cell cycle regulation of p53 rescued malignant keratinocytes

Mutations in the tumor suppressor gene p53 were found in more than 90% of all human squamous cell carcinomas (SCC). To study the function of p53 in a keratinocyte background, a tetracycline-controlled p53 transgene was introduced into a human SCC cell line (SCC15), lacking endogenous p53. Conditional expression of wild-type p53 protein upon withdrawal of tetracycline was accompanied with increased expression of p21(WAF1/Cip1) resulting in reduced cell proliferation. Flow-cytometric analysis revealed that these cells were transiently arrested in the G1/S phase of the cell cycle. However, when SCC15 cells expressing p53 were exposed to ionizing radiation (IR), a clear shift from a G1/S to a G2/M cell cycle arrest was observed. This effect was greatly depending on the presence of wild-type p53, as it was not observed to the same extent in SCC15 cells lacking p53. Unexpectedly, the p53- and IR-dependent G2/M cell cycle arrest in the keratinocyte background was not depending on increased expression or stabilization of 14-3-3sigma, a p53-regulated effector of G2/M progression in colorectal cancer cells. In keratinocytes, 14-3-3sigma (stratifin) is involved in terminal differentiation and its cell cycle function in this cell type might diverge from the one it fulfills in other cellular backgrounds.     

p53 loss of function: implications for the processes of immortalization and tumorigenesis.

The complex process of cell immortalization and transformation is likely to involve the inactivation of growth regulatory genes. Mutations (deletions, missense mutations) in the p53 gene are the most frequently observed genetic alteration in human tumors, making p53 a candidate for a cellular protein involved in the control of cell growth. Two recent studies have examined the role of p53 in immortalization and tumorigenesis. In the first study, p53 expression was examined in both mortal and immortal chick embryo fibroblasts. All mortal clones expressed p53 but the loss of wild-type p53 expression was observed in every immortal cell line examined. In the second study, a line of mice carrying two null p53 alleles has been created and characterized. Although these mice develop normally, they show a predisposition to develop a variety of neoplasms at an early age (< 6 months). Although it is unclear whether p53 regulates the same, different, or overlapping pathways in the two experimental systems, these data demonstrate that p53 function is critical for the maintenance of normal growth control and support the current classification of p53 as a growth suppressive or tumor suppressor gene.     

Identification of genotoxic stress in human cells by fluorescent monitoring of p53 expression.

The tumor suppressor protein p53 is induced upon DNA damage essentially by post-translational regulatory mechanisms, which lead to a substantial increase of p53 levels. To exploit this essential property of p53, we developed a novel reporter system for monitoring accumulation and subcellular translocation of p53 protein, which is able to function as a simple test for detecting mutagenic and genotoxic stress in human cells. For this purpose, we constructed a plasmid with a specific translational TP53::EGFP gene fusion and selected stable transfected clones in the human cell line HEK293, in which p53 is functionally stabilized due to the expression of the transgenic adenoviral E1A oncoproteins. HEK293-TP53::EGFP clones may be used as a living cell system for monitoring not only of the induction of p53 protein in the cell, but also of its subcellular localization. Using this human reporter cell system, we examined levels of p53 by fluorescence microscopy and by FACS analysis following treatment with several classes of genotoxic and carcinogenic compounds. All tested DNA damaging agents caused a significant increase of intracellular p53-EGFP levels in a concentration-dependent manner. On the other hand, non-genotoxic carcinogens and stress conditions that cannot damage DNA were not able to induce p53-EGFP accumulation. The induction effect caused by genotoxic stress was found to be dependent on the endogenous p53 status, because it was not observed in p53-deficient cell lines. This corroborates the notion that p53 may be used as an universal sensor for genotoxic stress and demonstrates the usefulness of HEK293-p53-EGFP cells as a reporter system for identification of mutagens and genotoxic carcinogens in human cells by means of visualizing and monitoring intracellular p53 levels and localization.     

Involvement of wild-type p53 protein in the cell cycle requires nuclear localization.

Transfection of wild-type p53 into a pre-B, p53 nonproducer cell line yielded the generation of stable clones. Although constitutively expressing the growth-suppressor wild-type p53 protein, these cells proliferate continuously in vitro. However, expression of wild-type p53 in these cells altered their cell cycle pattern and reduced their growth in vivo. When the same parental cells were transfected with a plasmid coding for a wild-type p53 lacking nuclear localization signals, a wild-type cytoplasmic p53 protein was expressed. Expression of this cytoplasmic p53 product did not exert any changes in the growth of the parental cells, suggesting that wild-type p53 affects the cell cycle only when localized in the nuclear cell compartment.     

p53DINP1, a p53-inducible gene, regulates p53-dependent apoptosis

Using the differential display method combined with a cell line that carries a well-controlled expression system for wild-type p53, we isolated a p53-inducible gene, termed p53DINP1 (p53-dependent damage-inducible nuclear protein 1). Cell death induced by DNA double-strand breaks (DSBs), as well as Ser46 phosphorylation of p53 and induction of p53AIP1, were blocked when we inhibited expression of p53DINP1 by means of an antisense oligonucleotide. Overexpression of p53DINP1 and DNA damage by DSBs synergistically enhanced Ser46 phosphorylation of p53, induction of p53AIP1 expression, and apoptotic cell death. Furthermore, the protein complex interacting with p53DINP1 was shown to phosphorylate Ser46 of p53. Our results suggest that p53DINP1 may regulate p53-dependent apoptosis through phosphorylation of p53 at Ser46, serving as a cofactor for the putative p53-Ser46 kinase.     

Isolation of a full-length mouse cDNA clone coding for an immunologically distinct p53 molecule.

Transfection of a cloned p53 gene into a p53 nonproducer Abelson murine leukemia virus-transformed cell line, L12, reconstituted p53 expression. The protein expressed in these cells was indistinguishable from that naturally expressed in p53 producer tumor cells. Conversely, p53 protein expressed in L12-derived clones that were established by transfection with a full-length p53 cDNA clone (pM8) exhibited a discrete immunological form. Immunoprecipitation of p53 with a panel of monoclonal anti-p53 antibodies showed that L12-derived clones that were transfected with the genomic p53 clone contained the same antigenic determinants as those found in the p53 protein expressed in tumor cells. These p53 proteins bound all monoclonal antibody types as well as the polyclonal anti-p53 tested. However, L12-derived clones established by transfection of the p53 cDNA clone (pM8) expressed a p53 protein that bound the RA3-2C2 and PAb200.47 anti-p53 monoclonal antibodies as well as polyclonal anti-p53 serum but totally lacked the antigenic receptor for the PAb122 and PAb421 monoclonal antibodies. The p53 proteins expressed by either genomic or cDNA p53 clones exhibited the same apparent molecular sizes and identical partial peptide maps. We suggest that transfection of the p53 gene induced expression of the entire group of the possible mRNA species, whereas cloned p53 cDNA (pM8) represented a single mRNA molecule that codes for a discrete species of p53 protein.     

ARF triggers cell G1 arrest by a P53 independent ERK pathway

In this study, in order to investigate the p53-independent function of p14ARF, we established p14ARF-inducible clones in the p53-deficient HCT cell line using the doxycycline-inducible expression system. A strong cell growth inhibition and G1/S arrest were observed after doxycycline induction in p53-/-HCT cells, and the cells also exhibited an obvious decrease of DNA synthesis. We further examined if the MEK/ERK pathway is involved in the G1 arrest induced by p14ARF in p53-/-HCT cells. The results indicate that ERK1/2 and p21 were activated upon p14ARF induction. Totally, the functional roles of ERK and p21 for ARF in p53-independent tumor suppression were demonstrated.     

p53 Protein expression and proliferative activity in ductal breast carcinomas

The immunocytochemical expression of p53 protein and Ki-67 labelling index in tumour cells of 100 ductal breast carcinomas of different histological grade and stage was evaluated in cytological material. In order to investigate p53 expression and Ki-67 expression an avidin-extravidin immunocytochemical technique was applied to imprints. Monoclonal antibody (MoAb) DO-p53 and proliferating cell monoclonal antibody were used as primary antibodies. A statistically significant difference was observed between p53 protein expression and grade of malignancy and clinical stage (P = 0.001, P < 0.001, respectively). A statistically significant difference was also observed between Ki-67 LI and histological grade and stage of the tumours (P < 0.001, P < 0.001 correspondingly). A correlation was observed between p53 protein expression and Ki-67 LI (P < 0.001). The immunocytochemical study of p53 protein and Ki-67 expression in cytological material represents a simple method which can be applied in routine cytological laboratories for the investigation of potential malignancy of ductal breast cancer.