Reconstitution of p53 expression in a nonproducer Ab-MuLV-transformed cell line by transfection of a functional p53 gene

L12 are Ab-MuLV-transformed cells that express the abl p120 oncogene product but lack the cellularly encoded p53. The functional p53 gene in these cells has been inactivated by the insertion of Moloney virus-like sequences into the first p53 intron. Transfection of L12 cells with a functional p53 gene, contained in a 16 kb Eco RI genomic cloned fragment gave rise to L12-derived cell lines with novel p53 sequences of various sizes and copy number. A high percentage of L12-derived clones efficiently transcribed p53 mRNA and synthesized the p53 protein. Whereas injection of L12 parental cells into syngeneic mice caused the development of local tumors that later regressed, L12-derived clones that expressed p53 caused lethal tumors in syngeneic mice, thus behaving similarly to other Ab-MuLV-transformed p53-producer cell lines. These results suggest that the expression of p53 is essential for tumor cells to exhibit a fully transformed phenotype, manifested in lethal tumors in syngeneic mice.     

4-Hydroxynonenal induces p53-mediated apoptosis in retinal pigment epithelial cells

4-Hydroxynonenal (4-HNE) has been suggested to be involved in stress-induced signaling for apoptosis. In present studies, we have examined the effects of 4-HNE on the intrinsic apoptotic pathway associated with p53 in human retinal pigment epithelial (RPE and ARPE-19) cells. Our results show that 4-HNE causes induction, phosphorylation, and nuclear accumulation of p53 which is accompanied with down regulation of MDM2, activation of the pro-apoptotic p53 target genes viz. p21 and Bax, JNK, caspase3, and onset of apoptosis in treated RPE cells. Reduced expression of p53 by an efficient silencing of the p53 gene resulted in a significant resistance of these cells to 4-HNE-induced cell death. The effects of 4-HNE on the expression and functions of p53 are blocked in GSTA4-4 over expressing cells indicating that 4-HNE-induced, p53-mediated signaling for apoptosis is regulated by GSTs. Our results also show that the induction of p53 in tissues of mGsta4 (−/−) mice correlate with elevated levels of 4-HNE due to its impaired metabolism. Together, these studies suggest that 4-HNE is involved in p53-mediated signaling in in vitro cell cultures as well as in vivo that can be regulated by GSTs.     

p53 gene expression and activation of p53-dependent transcription in melanoma cell lines

Malignant melanoma has poor prognosis because of its high metastatic potential and resistance to chemotherapy. A possible approach to more effective therapy is induction of p53-dependent apoptosis. This approach is promising, since the wild-type p53 is expressed in most melanomas. An attempt was made to estimate the functional activity of p53 in several malignant melanoma cell lines. Most lines were characterized by a high protein level and nuclear localization of p53. All cell lines expressing the wild-type p53 showed stabilization of p53, its translocation into the nucleus, and activation of several target genes in response to DNA-damaging agents, suggesting that p53 was functionally active. A high-molecular-weight protein localized in the cytoplasm and mimicking a p53 epitope was found in several cell lines. It was shown that the DO-1 epitope of this protein does not derive from p53, ruling out cytoplasmic retention of p53 in melanoma cell lines. A mechanism of camptothecin-induced stabilization of p53 by decreasing the level of the HDM2 mRNA was described for melanoma cells but not for normal melanocytes, which suggested a differential effect of camptothecin on tumor-derived and primary cells.     

Establishment of a doxycycline-regulated cell line with inducible,doubly-stable expression of the wild-type p53 gene from p53-deleted hepatocellular carcinoma cells

p53 is important in the development of hepatocellular carcinoma (HCC) and in therapeutic approaches, but the mechanism whereby it inhibits HCC growth is still unclear. The aim of the present study was to establish a HCC cell system in which p53 levels can be regulated. Full-length wild-type p53 cDNA obtained by PCR was cloned into a retroviral response vector controlled by the tetracycline responsive element (RevTRE-p53). The regulatory vectors RevTet-Off and RevTRE-p53 were transfected into a packaging cell line, PT67. Hep3B cells in which the p53 gene was deleted were infected with RevTet-Off viral particles from the PT67. Three G418-resistant cell clones with high luciferase expression and low background were infected with RevTRE-p53. By screening dozens of RevTRE-p53-infected clones with hygromycin we identified the one with the highest expression of p53 and the lowest background after doxycycline treatment. The results showed that p53 expression in this cell clone could be simply turned on or off by removing or adding doxycycline. Furthermore, it was found that the level of p53 protein was negatively and sensitively related to the doxycycline concentration. In conclusion, we have established a HCC cell line in which p53 expression can be switched on or off and regulated in a dose- and time-dependent manner.     

PIWIL4 regulates cervical cancer cell line growth and is involved in down-regulating the expression of p14ARF and p53

Recently, PIWIL4 has been identified as a functional protein involved in tumorigenesis. Cervical cancer is the second most prevalent form of cancer worldwide. The relationship between PIWIL4 and cervical cancer is still unknown. Here, we found that PIWIL4 is up-regulated in human cervical cancer tissues in comparison to adjacent normal tissues, and it promotes cell growth and proliferation by inhibiting apoptosis through the p14ARF/p53 pathway. PIWIL4 can also promote the invasion of cervical cancer cells. These results suggest that PIWIL4 might play an oncogenic role in cervical cancer and be useful as a new therapeutic target in the future.     

Lack of expression of the protein p53 gene in a human T-cell leukemia line

Expression of the gene encoding the nuclear phosphoprotein p53 (a proto-oncogene classified in the same functional family as c-myc and E1a adenovirus gene) was examined in a human T-cell leukemia (KE-37R cell line). No p53 (or a modified product) could be detected by immunoprecipitation with monoclonal antibodies P Ab 421 and P Ab 122 in KE-37R cell extracts, and no p53-specific RNA was characterized by Northern blot analysis. Southern blot using a murine p53 cDNA clone as a probe, did not reveal any gross rearrangement in the structure of the gene. However, this molecular probe was not suited for investigating the 5' end of the gene which contains the promoter and the non coding exon 1. It is interesting to notice that in KE-37R cells, c-myc has been activated by a t(8; 14) (q24; q11) translocation, suggesting that the c-myc product might substitute to some functions normally requiring p53.     

Agonist selective modulation of tyrosine hydroxylase expression by cannabinoid ligands in a murine neuroblastoma cell line

Functional interactions between catecholamines and cannabinoid transmission systems could explain the influence of Delta(9)-tetrahydrocannabinol on several central activities. Hence, the presence of cannabinoid CB(1) receptors in tyrosine hydroxylase (TH) containing cells has been suggested, providing clue for a direct control of catecholamines synthesis. In the present study, we evidenced the constitutive expression of functional cannabinoid CB(1) receptors in N1E-115 neuroblastoma and reported on the use of this model to examine the influence of diverse cannabinoid ligands on TH expression. Exposure of the cells to the high-affinity agonist HU 210 (5 h) resulted in a significant decrease in TH content (pEC(50): 6.40). In contrast, no change was observed after a similar treatment with the structurally unrelated agonist CP 55,940. Besides, the use of a luciferase reporter assay revealed that these two agonists showed opposite influences on TH gene promoter activity. Thus, in cells expressing pTH-luc constructs, inhibition and induction of luciferase activity were respectively observed with HU 210 (pEC(50): 8.95) and CP 55,940 (pEC(50): 9.09). Pharmacological characterisation revealed that these reciprocal responses were both related to the specific activation of cannabinoid CB(1) receptor, suggesting an agonist-dependent modulation of distinct signalling pathways. While these data points out the possible pharmacological manipulation of TH expression by cannabinoid ligands, such approach should take into account the existence of agonist selective trafficking of cannabinoid CB(1) receptor signalling.     

Functional characterization of a new p53 mutant generated by homozygous deletion in a neuroblastoma cell line

p53 is a key modulator of a variety of cellular stresses. In human neuroblastomas, p53 is rarely mutated and aberrantly expressed in cytoplasm. In this study, we have identified a novel p53 mutant lacking its COOH-terminal region in neuroblastoma SK-N-AS cells. p53 accumulated in response to cisplatin (CDDP) and thereby promoting apoptosis in neuroblastoma SH-SY5Y cells bearing wild-type p53, whereas SK-N-AS cells did not undergo apoptosis. We found another p53 (p53DeltaC) lacking a part of oligomerization domain and nuclear localization signals in SK-N-AS cells. p53DeltaC was expressed largely in cytoplasm and lost the transactivation function. Furthermore, a 3'-part of the p53 locus was homozygously deleted in SK-N-AS cells. Thus, our present findings suggest that p53 plays an important role in the DNA-damage response in certain neuroblastoma cells and it seems to be important to search for p53 mutations outside DNA-binding domain.     

Induction of K-channel expression in a neuroblastoma cell line

Whole-cell currents were examined in mouse neuroblastoma cells of the N2AB-1 line. In standard culture medium, N2AB-1 cells exhibited large voltage-dependent Na currents but no discernible K currents. Treatment of N2AB-1 cells with either dimethylsulfoxide (DMSO) in low-serum medium or with retinoic acid (RA) caused the expression of delayed rectifier K currents. Currents from two types of K channel with single channel slope conductances of 15.0 pS and 6.4 pS were observed in outside-out patches from cells of both treatment groups. Thus, while N2AB-1 cells did not exhibit K currents under standard culture conditions, they did possess the gene(s) encoding K channels. The treatments caused other changes that were not directly linked to K-channel expression. RA treatment caused neurite extension in most, but not all, N2AB-1 cells; however, all RA-treated cells, including those without neurites, expressed K currents. RA treatment did not suppress cell division or cause hypertrophy. In contrast, treatment with DMSO/low serum suppressed cell division and caused cellular hypertrophy, but did not cause long neurites to form. Thus, the regulation of K channels was not coupled in a simple fashion to properties that have been associated with a differentiated neuronal phenotype: neurite elaboration, changes in cell size, and inhibition of cell division. These results suggest that N2AB-1 cells may be a good model system for investigating the processes regulating K-channel expression.     

Induction of apoptosis in p53-deficient human hepatoma cell line by wild-type p53 gene transduction: inhibition by antioxidant

We investigated the role of wild-type (wt)-p53 as an inducer of apoptotic cell death in human hepatoma cell lines. Following the retrovirus-mediated transduction of the wt-p53 gene, Hep3B cells lacking the endogenous p53 expression began to die through apoptosis in 4 h. They showed a maximal apoptotic death at 12 h, whereas HepG2 cells expressing endogenous p53 did not. However, the transduction of the wt-p53 gene elicited growth suppression of both Hep3B and HepG2 cells. P21(WAF1/CIP1), a p53-inducible cell cycle inhibitor, was induced, not only in Hep3B cells undergoing apoptosis, but also in HepG2 cells. The kinetics of the p21(WAF1/CIP1) induction, DNA fragmentation, and growth suppression of the Hep3B cells showed that DNA fragmentation and growth suppression progressed rapidly following p21(WAF1/CIP1) accumulation. N-acetyl-cysteine or glutathione, potent antioxidants, strongly inhibited the DNA fragmentation, but did not reduce the elevated level of p21(WAF1/CIP1). These findings suggested that p21(WAF1/CIP1) was not a critical mediator for the execution of p53-mediated apoptosis, although it contributed to the growth inhibition of cells undergoing apoptosis. Furthermore, p53-mediated apoptosis could be repressed by antioxidants.     

抑癌基因p53调控的微RNA-miR-34基因家族

微RNA（microRNA,miRNA）是一种内源性非编码小RNA,在转录后水平调控基因表达,在肿瘤的发生发展过程中起重要作用。p53是重要的抑癌基因,在DNA损伤和癌基因等刺激下活化,诱导下游基因表达,使细胞周期阻滞、DNA修复并促进细胞衰老或凋亡。本文主要介绍近期发现的直接受p53调控的miR-34基因家族,及其在生长阻滞和细胞凋亡方面的研究进展,揭示了蛋白质与非编码RNA在重要的p53抑癌网络中的相互关系,为肿瘤的研究提供了新的思路。     

Differential expression of the p65 gene family

The genome of the marine ray Discopyge ommata contains at least three p65-related genes. o-p65-A is 84% identical, o-p65-B is 78% identical, and o-p65-C is only 41% identical to a previously characterized rat p65. The cytoplasmic domain, particularly the two regions that are similar to the regulatory domain of protein kinase C, are most highly conserved. The three genes are expressed in different but overlapping patterns in the central nervous system. o-p65-A immunoreactivity is found predominantly in forebrain, cerebellum, and neuroendocrine cells, while o-p65-B immunoreactivity is predominantly localized to the spinal cord, brainstem, and midbrain. Many synaptic vesicle proteins are members of small gene families that are differentially expressed, resulting in several unique combinations of these molecules in specific brain regions.     

Different kinases desensitize the human delta-opioid receptor (hDOP-R) in the neuroblastoma cell line SK-N-BE upon peptidic and alkaloid agonists

In a previous work, we described a differential desensitization of the human δ-opioid receptor (hDOP-R) by etorphine (a non-selective and alkaloid agonist) and δ-selective and peptidic agonists (DPDPE ([d-Pen^2,5]enkephalin) and deltorphin I (Tyr-d-Ala-Phe-Asp-Val-Val-Gly-NH₂)) in the neuroblastoma cell line SK-N-BE (Allouche et al., Eur. J. Pharmacol., 371, 235, 1999). In the present study, we explored the putative role of different kinases in this differential regulation.

First, selective chemical inhibitors of PKA, PKC and tyrosine kinases were used and we showed a significant reduction of etorphine-induced opioid receptor desensitization by the bisindolylmaleimide I (PKC inhibitor) while genistein (tyrosine kinase inhibitor) was potent to impair desensitization induced by the different agonists. When the PKA was inhibited by H89 pretreatment, no modification of opioid receptor desensitization was observed whatever the agonist used.

Second, we further studied the role of G protein-coupled receptor kinases (GRKs) and by using western-blot experiments we observed that only the GRK2 isoform was expressed in the SK-N-BE cells. Next, the neuroblastoma cells were transfected with the wild type GRK2 or its dominant negative mutant GRK2-K220R and the inhibition on cAMP level was determined in naïve and agonist-pretreated cells. We showed that over-expression of GRK2-K220R totally abolished etorphine-induced receptor desensitization while no effect was observed with peptidic agonists and over-expression of GRK2 selectively impaired cAMP inhibition promoted by etorphine suggesting that this kinase was involved in the regulation of hDOP-R activated only by etorphine.

Third, correlation between functional experiments and phosphorylation of the hDOP-R after agonist activation was assessed by western-blot using the specific anti-phospho-DOP-R Ser³⁶³ antibody. While all agonists were potent to increase phosphorylation of opioid receptor, we showed no impairment of receptor phosphorylation level after PKC inhibitor pretreatment. Upon agonist activation, no enhancement of receptor phosphorylation was observed when the GRK2 was over-expressed while the GRK2-K220R partially reduced the hDOP-R Ser³⁶³ phosphorylation only after peptidic agonists pretreatment.

In conclusion, hDOP-R desensitization upon etorphine exposure relies on the GRK2, PKC and tyrosine kinases while DPDPE and deltorphin I mediate desensitization at least via tyrosine kinases. Although the Ser³⁶³ was described as the primary phosphorylation site of the mouse DOP-R, we observed no correlation between desensitization and phosphorylation of this amino acid.     

Contributions of differential p53 expression in the spontaneous immortalization of a chicken embryo fibroblast cell line

Background

The present study was carried out to determine whether the p53 pathway played a role in the spontaneous immortalization of the SC-2 chicken embryo fibroblast (CEF) cell line that has been in continuous culture for over three years.

Results

The SC-2 cell line emerged from an extended crisis period with a considerably slower growth rate than primary CEF cells. The phenotype of the SC-2 cells changed dramatically at about passage 80, appearing smaller than at earlier passages (e.g., passage 43) and possessing a small, compact morphology. This morphological change coincided with an increase in growth rate. Passage 43 SC-2 cells expressed undetectable levels of p53 mRNA, but by passage 95, the levels were elevated compared to primary passage 6 CEF cells and similar to levels in senescent CEF cells. However, the high level of p53 mRNA detected in passage 95 SC-2 cells did not correlate to functional protein activity. The expression levels of the p53-regulated p21^WAF1 gene were significantly decreased in all SC-2 passages that were analyzed. Examination of the Rb pathway revealed that E2F-1 and p15^INK4b expression fluctuated with increasing passages, with levels higher in passage 95 SC-2 cells compared to primary passage 6 CEF cells.

Conclusion

The present study suggests that altered expression of genes involved in the p53 and Rb pathways, specifically, p53 and p21^WAF1, may have contributed to the immortalization of the SC-2 CEF cell line.