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.     

Aberrant regulation and function of wild-type p53 in radioresistant melanoma cells.

Sporadic human tumors and the hereditary cancer predisposition syndrome Li-Fraumeni are frequently associated with mutations in the p53 tumor suppressor gene that compromise its ability to function as a DNA damage checkpoint. A subset of Li-Fraumeni patients with wild-type p53 alleles have mutations in chk2/hcds1, one of the genes signaling the presence of DNA damage to the p53 protein. This suggests that p53 may be kept inactive in human cancer by mutations targeting DNA damage signaling pathways. Melanoma cells are highly radioresistant, yet they express wild-type p53 protein, raising the possibility of defects in the pathways that activate p53 in response to DNA damage. We have described a chk2/hcds1-independent DNA damage signaling pathway that targets Ser-376 within the COOH terminus of p53 for dephosphorylation and leads to increased p53 functional activity. We now report that in several human melanoma cell lines that express wild-type p53, the phosphorylation state of Ser-376 was not regulated by DNA damage. In these cell lines, neither the endogenous wild-type p53 protein nor high levels of ectopic wild-type p53 led to cell cycle arrest or apoptosis. Thus, defective activation of p53 in response to DNA damage may underlie the radioresistance of human melanoma cells.     

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.     

A prognostic signature of defective p53-dependent G1 checkpoint function in melanoma cell lines

Melanoma cell lines and normal human melanocytes (NHM) were assayed for p53-dependent G1 checkpoint response to ionizing radiation (IR)-induced DNA damage. Sixty-six percent of melanoma cell lines displayed a defective G1 checkpoint. Checkpoint function was correlated with sensitivity to IR with checkpoint-defective lines being radio-resistant. Microarray analysis identified 316 probes whose expression was correlated with G1 checkpoint function in melanoma lines (P≤0.007) including p53 transactivation targets CDKN1A, DDB2, and RRM2B. The 316 probe list predicted G1 checkpoint function of the melanoma lines with 86% accuracy using a binary analysis and 91% accuracy using a continuous analysis. When applied to microarray data from primary melanomas, the 316 probe list was prognostic of 4-yr distant metastasis-free survival. Thus, p53 function, radio-sensitivity, and metastatic spread may be estimated in melanomas from a signature of gene expression.     

SW480, a p53 double-mutant cell line retains proficiency for some p53 functions

During certain types of cellular stress, the p53 tumor suppressor protein binds to DNA and transactivates a variety of genes that regulate critical responses including apoptosis, cell cycle checkpoints, differentiation, and angiogenesis. In addition, functional p53 is known to be required for efficient nucleotide excision repair (NER) of bulky DNA adducts generated through exposure to environmental mutagens such as UV light. Nonetheless, we previously showed that the model p53-mutated human adenocarcinoma strain SW480 is proficient in the removal of UV-induced cyclobutane pyrimidine dimers (CPD) via NER. We undertook the present study to begin probing the molecular basis for this unexpected repair phenotype. Cytogenetic analysis indicated that SW480 is stable at the chromosomal level, i.e. manifests a karyotypic profile very similar to that revealed for this line as far back as 14 years ago. After fluorescence in situ hybridization (FISH), using a probe complementary to the p53 gene, we found that 98% of the SW480 interphase nuclei contains three copies of the gene, later revealed to be localized on intact short arms of three chromosomes 17. DNA sequence analysis further showed that all three p53 copies in SW480 carry two point mutations (R273H and P309S), and levels of the corresponding mutated p53 protein are about 20-fold higher than in the closely related p53 wild-type strain LoVo. Using an electrophoretic mobility shift assay (EMSA), we demonstrated that R273H/P309S p53 is able to bind with wild-type affinity to its consensus DNA sequence in vitro. Analysis of p21(Cip1/WAF1) expression and in vivo footprinting by ligation-mediated PCR (LMPCR) showed that, in wild-type LoVo cells, an exposure to cellular stress (e.g. UV or ionizing radiation) is necessary for p53 activation of the p21(Cip1/WAF1) promoter. In contrast, the R273H/P309S-mutated p53 protein in SW480 constitutively activates p21(Cip1/WAF1) in the absence of stress through an unknown mechanism. A similar phenomenon whereby mutated p53 in SW480 is able to induce NER-related proteins might explain the normal DNA repair phenotype previously observed in this strain. For now we conclude that, in general, results obtained using SW480 as a p53-deficient cell line should be interpreted very cautiously.     

Stable formation of mutated p53 multimers in a Chinese hamster cell line causes defective p53 nuclear localization and abrogates its residual function

We have previously described a methotrexate-resistant cell line (MTX M) characterized by amplified dihydrofolate reductase (DHFR) genes, cytoplasmic p53 localization, and p53 stable tetramers. To investigate the p53 functionality in MTX M, the effect of chemical/physical agents was studied. In MTX M cells, DNA damage did not induce p53 or mdm-2 protein, while in the parental V79 cells, a residual p53 activity was found. cDNA sequencing showed that V79 and MTX M cells share the same mutations, indicating that the complete loss of p53 function in MTX M cells was due to cytoplasmic sequestration of a mutated p53 with residual activity. In Chinese hamster, both p53 and DHFR genes map on short arm of chromosome 2 suggesting that p53 itself might be amplified. However, fluorescence in situ hybridization with a hamster p53 probe showed only a single signal. Thus, the presence of p53 stable tetramers in MTX M cells, although correlated with DNA amplification, could not be the consequence of either p53 or DHFR gene amplification. Expression of a C-terminal human p53 peptide does not induce p53 nuclear accumulation, indicating that the cytoplasmic localization is due to a mechanism different from that already described in cancer cell lines. Treatments with Sodium Butyrate induced beta-tubulin polymerization, but did not apparently organize a normal microtubule network, which is shown to be important for the p53 localization. Our data indicated that in MTX M cells, p53 is sequestered in the cytoplasm by a novel mechanism that abrogates p53 residual function.     

UV and melanoma: the TP53 link

Ultraviolet radiation (UVR) is a major risk factor for melanoma development, but it has been unclear exactly how UVR leads to melanomagenesis. In a recent publication in Nature, Viros et al. identify TP53/Trp53 as a UVR-target gene in melanoma and show that UVR-induced TP53/Trp53 mutations accelerate BRAF(V600E)-driven melanomagenesis.Melanoma is the deadliest skin cancer, and its incidence has relentlessly increased over recent decades. According to the American Cancer Society''s estimates for melanoma in the United States for 2014, about 76 100 new melanomas will be diagnosed and about 9 710 people are expected to die from melanoma. It is well known that UVR is the major environmental factor contributing to melanomagenesis¹. This suggests that there is at least a component of melanoma risk which may be preventable through UVR protective strategies — an issue of immense public health importance due to the availability of sunblocks and sun-safe behaviors. Importantly, while many studies have been conducted to elucidate the link between UVR and melanoma, the precise molecular mechanism(s) by which UVR triggers melanoma formation have remained incompletely understood.Recently, a powerful UVR-induced skin inflammatory response has been shown to provoke metastasis of melanoma² and the presence of UV signature mutations has also been reported throughout the melanoma exome, including recurrent melanoma genes such as RAC1, PPP6C, and STK19^3,4. Previous mouse models for UVR-induced melanoma revealed that UVR-induced inflammation promoted melanomagenesis in neonatal mice^5,6,7. These studies underlined UVR''s significant contribution to melanoma formation. In a recent study published in Nature, Viros et al.⁸ address the role of UVR in previously established BRAF(V600E)-expressing melanocytes in vivo, and demonstrate that significantly accelerated melanoma formation often associated with mutations in TP53/Trp53. To mimic both somatic mutation acquisition and mild sunburn in humans, BRAF(V600E) was expressed at physiological levels in adult mice which were subsequently exposed to repeated low doses of UVR. In addition, certain mice were partially covered with UVR-proof cloth or topically treated with SunSense Milk Sunscreen SPF50 (2.2 mg/cm²) 30 min before UVR exposure, to assess the impact of these protective strategies.UVR was seen to significantly accelerate melanoma formation in mice whose melanocytes express BRAF(V600E), but not in BRAF wild-type mice (which unlike BRAF(V600E)-expressing mice do not develop long latency melanomas independently of UVR). Application of UVR-proof cloth or sunscreen delayed the onset of UVR-driven melanoma and partially prevented acceleration of BRAF(V600E)-driven melanomagenesis by UVR, and sunscreen-protected UVR-exposed BRAF(V600E) mice developed a reduced number of melanomas compared with unprotected UVR-exposed BRAF(V600E) mice (Figure 1). More somatic single nucleotide variants and a significantly higher proportion of C-to-T transitions at the 3′ end of pyrimidine dimers were observed in UVR-exposed melanomas, providing direct evidence of UVR-induced DNA damage. In addition, Trp53 mutations (H39Y, S124F, R245C, R270C, C272G) were detected in UVR-exposed BRAF(V600E) mouse melanomas, indicating a direct role of UVR in the induction of Trp53 mutations in melanoma. The mutated corresponding residues (S127F/S124F, R248C/R245C, R273C/R270C, C275G/C272G) were also identified in TP53 mutations in human melanoma, suggesting that TP53 mutations are linked to evidence of UVR-induced DNA damage in human melanoma. These results are consistent with previous reports that p53 deletion accelerates BRAF(V600E)-driven melanomagenesis both in mice⁹ and in zebrafish¹⁰, but demonstrate the ability of UVR to inflict UV signature mutations within the gene as has been widely observed in non-melanoma skin cancers and also in human melanomas.Open in a separate windowFigure 1A diagram depicting feasible routes of BRAF(V600E)-driven melanomagenesis.This elegant study by Viros et al. clearly helps to establish key roles of UVR in melanomagenesis, and further validates the functional importance of TP53 as a UVR-targeted tumor suppressor gene in a fraction of melanomas. The study also raises several intriguing questions worthy of follow-up analysis. For example, through which mechanism(s) did sunscreen or sunshielding delay but not prevent UVR-induced melanoma? Induction of cutaneous inflammatory changes that are less anatomically restricted to UV irradiated fields, would seem to be an attractive mechanism. This may help to explain the known risk of melanoma in both sun-exposed and less-exposed skin of lightly pigmented people. It is also valuable to better understand the role of UVB vs UVA wavelengths in melanomagenesis. Mechanistically, these distinct regions of the UV spectrum inflict largely distinctive chemical alterations on the genome. Efforts to block UVA as well as UVB in commercial sunscreen products are currently being promoted by the US Food and Drug Administration, a welcome improvement to sun protection strategies. Still, the precise role(s) of UVA in melanomagenesis remain incompletely understood and may involve both cell-autonomous and non-cell-autonomous targets. In addition to the acceleration of BRAF(V600E)-driven melanoma formation by UVR, red pigment (pheomelanin) has also been observed to accelerate BRAF(V600E)-driven melanomagenesis even in the absence of UVR¹¹. Pheomelanin has been identified as an intrinsic risk factor for melanoma with the red pigment itself producing reactive oxygen species that cause DNA damage in the skin, and consequently promote melanomagenesis independently of UVR. UVR likely exacerbates red pigment-induced BRAF(V600E)-driven melanoma, and still remains as a major contributor to melanomagenesis. Therefore, along with UV shielding by sunscreens, further preventative strategies should be investigated to diminish UVR-independent melanoma risk mechanisms.Viros et al. provide intriguing answers to several controversial questions regarding melanomagenesis: Does UVR really trigger melanoma? And can sunscreen actually prevent melanoma? The studies by Viros et al. provide experimental evidence for acceleration of BRAF(V600E)-driven melanoma by UVR-induced TP53/Trp53 mutation and demonstrate that sunscreen delayed but did not completely block UVR-driven melanoma. The current study clearly shows that UVR boosts melanoma and sunscreens may provide partial UVR protection against melanoma — evidence which matches human epidemiologic data. Nevertheless, to protect the public from melanoma, Viros et al. advise that sunscreen should be utilized in combination with additional sun avoidance strategies. In addition, measures that may prevent UV-independent melanoma formation will require additional research and may also be needed in order to optimally battle the incidence of this life-threatening malignancy.     

转化的大鼠胚胎成纤维细胞系与p53val135功能关系的研究

A1-5是一株典型的温度敏感的p53^val135细胞系,许多实验室用它来研究p53的功能,我们首次报道了A1-5细胞表现出非常强的抗辐射性并伴随不同寻常强的G2延迟效应,B4是另一株温度敏感的p53^val135细胞系,A1-5与B4细胞都 是来源于同样的原代转化的大鼠胚胎成纤维细胞系,只是来自不同的转染实验,本阐明了A1-5细胞非常强的抗辐射性及不同寻常强的G2停滞效应与p53val135的功能无关,A1-5细胞系具有的特殊表型是研究放射敏感性新性质的独特的模型。     

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.     

The role of p53 in regulating antiviral T cell responses

It is now well established that viral infections can induce large expansions of Ag-specific CD8(+) T cells. These cells divide very rapidly with an estimated doubling time of approximately 6 h. When virus is cleared, the vast majority of these effector CD8 T cells undergo apoptosis. The remaining memory cells persist at constant levels and provide the basis for the accelerated recall response upon rechallenge. The molecular mechanisms that control the rapid proliferation and death of Ag-specific T cells are poorly understood. Because of its important role in controlling cell proliferation and death, we examined antiviral immune responses in p53(-/-) mice using lymphocytic choriomeningitis virus. We found that effector CD8 and CD4 responses were comparable but that memory levels were slightly higher in -/- mice compared with +/+ mice. The lack of a major difference in virus-specific T cell responses between +/+ and -/- mice suggests that p53 only plays a minor role in regulating the proliferation, apoptosis, and maintenance of Ag-specific T cells. Thus, it appears that the primary function of p53 is in controlling "illegitimate" proliferation and tumor development and not in regulating Ag-specific T cell responses.     

Morphologic conversion of a neuroblastoma-derived cell line by E6-mediated p53 degradation.

Neuroblastoma-derived tumor cells, unlike cells from other tumor types, characteristically express a wildtype but cytoplasmically sequestered p53 protein. To ascertain whether the p53 in these cells retained any physiological activity, we inactivated it in SK-N-SH cells, a neuroblastoma-derived cell line, by introducing the human papilloma virus type 16 E6 expression plasmid. Parent SK-N-SH cell cultures are composed of two cell types exhibiting characteristic morphologies designated neuroblastic (N-type) or substrate-adherent fibroblastic (S-type) cells, both of which have been shown to spontaneously transdifferentiate or interconvert. We report here that down-regulation of p53 resulted in conversion of SK-N-SH cells to the substrate-adherent fibroblast-like S-type cells. The morphologic conversion was accompanied by a loss of neurofilament expression, a marker for the neuronal N-type cells, an increase in the expression of vimentin, and a lack of responsiveness to retinoic acid-induced neuronal differentiation. Importantly, we did not observe N-type cells in the E6-transfected cell population, suggesting that they were incapable of transdifferentiating to the N-type morphology. We also tested the ability of these E6-transfected S-type cells to form colonies in soft agar and observed a markedly reduced capacity of these cells to do so when compared with the parent and mutant E6-transfected cells. These results suggest that p53 is required for the maintenance of the neuroblastic tumorigenic phenotype.     

Accumulation of p53 in a mutant cell line defective in the ubiquitin pathway.

The wild-type p53 gene product plays an important role in the control of cell proliferation, differentiation, and survival. Altered function is frequently associated with changes in p53 stability. We have studied the role of the ubiquitination pathway in the degradation of p53, utilizing a temperature-sensitive mutant, ts20, derived from the mouse cell line BALB/c 3T3. We found that wild-type p53 accumulates markedly because of decreased breakdown when cells are shifted to the restrictive temperature. Introduction of sequences encoding the human ubiquitin-activating enzyme E1 corrects the temperature sensitivity defect in ts20 and prevents accumulation of p53. The data therefore strongly indicate that wild-type p53 is degraded intracellularly by the ubiquitin-mediated proteolytic pathway.     

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.     

p53 prevents progression of nevi to melanoma predominantly through cell cycle regulation

p53 is the central member of a critical tumor suppressor pathway in virtually all tumor types, where it is silenced mainly by missense mutations. In melanoma, p53 predominantly remains wild type, thus its role has been neglected. To study the effect of p53 on melanocyte function and melanomagenesis, we crossed the ‘high-p53’Mdm4^+/− mouse to the well-established TP-ras^0/+ murine melanoma progression model. After treatment with the carcinogen dimethylbenzanthracene (DMBA), TP-ras^0/+ mice on the Mdm4^+/− background developed fewer tumors with a delay in the age of onset of melanomas compared to TP-ras^0/+ mice. Furthermore, we observed a dramatic decrease in tumor growth, lack of metastasis with increased survival of TP-ras^0/+: Mdm4^+/− mice. Thus, p53 effectively prevented the conversion of small benign tumors to malignant and metastatic melanoma. p53 activation in cultured primary melanocyte and melanoma cell lines using Nutlin-3, a specific Mdm2 antagonist, supported these findings. Moreover, global gene expression and network analysis of Nutlin-3-treated primary human melanocytes indicated that cell cycle regulation through the p21^WAF1/CIP1 signaling network may be the key anti-melanomagenic activity of p53.