Mutant conformation of p53 translated in vitro or in vivo requires functional HSP90

The p53 mutant, 143ala, was translated in vitro in either rabbit reticulocyte lysate (RRL) or wheat germ extract (WGE). In RRL, p53-143ala protein of both mutant and wild-type conformation, as detected immunologically with conformation-specific antibodies, was translated. The chaperone protein HSP90, present in RRL, was found to coprecipitate only with the mutated conformation of p53. Geldanamycin, shown previously to bind to HSP90 and destabilize its association with other proteins, decreased the amount of immunologically detectable mutated p53 and increased the amount of detectable wild-type protein, without affecting the total translation of p53. When translated in WGE, known to contain functionally deficient HSP90, p53-143ala produced p53 protein, which was not recognized by a mutated conformation-specific antibody. In contrast, the synthesis of conformationally detectable wild-type p53 in this system was not compromised. Reconstitution of HSP90 function in WGE permitted synthesis of conformationally detectable mutated p53, and this was abrogated by geldanamycin. Finally, when p53-143ala was stably tansfected into yeast engineered to be defective for HSP90 function, conformational recognition of mutated p53 was impaired. When stable transfectants of p53-143ala were prepared in yeast expressing wild-type HSP90, conformational recognition of mutated p53 was antagonized by macbecin I, a geldanamycin analog also known to bind HSP90. Taken together, these data demonstrate a role for HSP90 in the achievement and/or stabilization of the mutated conformation of p53-143ala. Furthermore, we show that the mutated conformation of p53 can be pharmacologically antagonized by drugs targeting HSP90.     

Toxicity of cytostatic drugs to normal bone marrow cells in vitro

Metastatic melanomas are often resistant to chemotherapy. To study whether the p53 mutational status affects chemosensitivity, we compared the responses to chemotherapy of four melanoma cell lines containing the wild-type p53 and four cell lines carrying the mutant p53. Cisplatin, at 10 microM, virtually killed all the cells in the wild-type p53 cell lines, while 57-95% of the cells in the mutant p53 cell lines survived (P = 0.005). After treatment with 100 nM of vincristine, on average 18% of the wild-type p53 melanoma cells survived compared with 55% of the mutant p53 cells (P = 0.04). After treatment with 40 nM, 200 nM or 1 microM of camptothecin the survival rates were, on average, 16%, 8% and 4% for the wild-type p53 melanoma cells, compared with 89%, 67% and 38% for the mutant p53 cells, respectively (P = 0.00004, P = 0.003 and P = 0.04, respectively). The anticancer agents were not toxic to normal melanocytes at doses inducing cytotoxicity in wild-type p53 melanoma cells. The main mechanism of cytotoxicity appears to be drug-induced apoptosis. Cisplatin, camptothecin and vincristine all induced apoptosis in wild-type p53 melanoma cells, but not in mutant p53 cells. Our results suggest that chemotherapy-induced apoptosis in melanoma cells is p53 dependent, and mutation of the p53 gene is an indicator of drug resistance in melanoma.     

Role of the p53 tumor suppressor gene in the tumorigenicity of Burkitt's lymphoma cells

Burkitt's lymphoma (BL) cell lines carry a translocated c-myc gene and, in 60-80% of cases, exhibit mutations in the p53 tumor suppressor gene. We examined the potential role of the p53 gene in BL tumorigenicity using an in vitro assay that measures p53-dependent cell cycle arrest in the G1 phase of the cell cycle and an in vivo athymic murine model that detects differences in the tumorigenicity of BL cell lines. A highly significant inverse correlation was found between the ability of BL cells to arrest in G1 after irradiation and their tumorigenicity in athymic mice, consistent with the notion that loss of p53 function is associated with increased tumorigenicity. Inactivation of wild-type (wt) p53 function by expression of the human papillomavirus E6 protein in the AG876V BL cell line, which carries both wt and mutant p53 proteins, rendered the cell line significantly more tumorigenic in athymic mice. Transfection of the wt p53 gene into the p53 mutant and highly tumorigenic BL-41 cell line caused it to acquire wt p53 function and rendered it less tumorigenic in mice. In addition to confirming a role for the loss of p53 function in tumor progression, the data demonstrate that wt p53 protein can reduce BL tumorigenicity in vivo.     

Dexamethasone enhances colonization of soft agar by tumorigenic mouse lung-derived cell lines

When cultured on plastic, tumorigenic mouse lung-derived cell lines exhibit different proliferative responses to glucocorticoids; some lines are inhibited while others are stimulated or unaffected. In contrast to the variable dexamethasone responses when cells are cultured on plastic, soft agar colonization by each of these cell lines is enhanced by dexamethasone. Enhanced soft agar growth is unlikely to result from expression of a mutant glucocorticoid receptor, since dexamethasone also enhanced colony formation in two cell lines that stably express a transfected normal glucocorticoid receptor gene. Thus, cell attachment influences the effect of glucocorticoids on cell cycle progression.     

Screening the p53 status of human cell lines using a yeast functional assay

We have screened the p53 status of 156 human cell lines, including 142 tumor cell lines from 27 different tumor types and 14 cell lines from normal tissues by using functional analysis of separated alleles in yeast. This assay enables us to score wild-type p53 expression on the basis of the ability of expressed p53 to transactivate the reporter gene HIS3 via the p53-responsive GAL1 promotor in Saccharomyces cerevisiae. Of 142 tumor cell lines, at least 104 lines (73.2%) were found to express the mutated p53 gene: 94 lines (66.2%) were mutated in both alleles, three lines (2.1%) were heterozygous, and no p53 cDNA was amplified from seven lines (4.9%). Of the 14 cell lines originating from normal tissues, all the transformed or immortalized cell lines expressed mutant p53 only. Yeast cells expressing mutant p53 derived from 94 cell lines were analyzed for temperature-sensitive growth. p53 cDNA from eight cell lines showed p53-dependent temperature-sensitive growth, growing at 30 degrees C but not at 37 degrees C. Four temperature-sensitive p53 mutations were isolated: CAT-->CGT at codon 214 (H214R), TAC-->TGC at codon 234 (Y234C), GTG-->ATG at codon 272 (V272M), and GAG-->AAG (E285K). Functionally wild-type p53 was detected in 38 tumor cell lines (26.8%) and all of the diploid fibroblasts at early and late population doubling levels. These results strongly support the previous findings that p53 inactivation is one of the most frequent genetic events that occurs during carcinogenesis and immortalization.     

p53 mutation and tamoxifen resistance in breast cancer

A substantial portion of patients with estrogen receptor-positive breast cancer fail to respond to estrogen depletion or to the antiestrogen tamoxifen. The molecular changes that lead to tamoxifen resistance and estrogen-independent growth are unknown. To test the hypothesis that a p53 mutation could result in tamoxifen resistance and estrogen-independent growth, the MCF-7 cell line was transfected with p53 cDNA which was mutated at codon 179 (histidine to glutamine). MCF-7 is an estrogen receptor-positive, estrogen-dependent, tamoxifen-sensitive cell line with only wild-type p53. The presence of transfected mutant p53 cDNA was verified by the PCR, and overexpression of p53 protein was assessed by Western blotting. Five separate mutant-transfected clones were selected and tested in subsequent growth experiments. In monolayer culture, there was no consistent evidence of estrogen-independent growth or tamoxifen resistance in the mutant transfectants compared with vector-only controls or the parental cell line. In soft agar growth experiments, four of five mutant transfectants remained sensitive to tamoxifen in a dose-dependent manner. In the presence of wild-type p53, mutant 179 p53 protein does not result in estrogen-independent growth or tamoxifen resistance. These results do not exclude the possibility that other p53 mutational types could result in tamoxifen resistance, or that loss of the remaining wild-type allele may be necessary to result in this phenotype.     

Transfection of wild-type but not mutant p53 induces early monocytic differentiation in HL60 cells and increases their sensitivity to stress

HL60 cells, which lack the p53 gene due to a deletion, were used as an in vitro model system to study the effect of wild-type p53 gene expression on hematopoietic differentiation. We transfected HL60 cells with wild-type p53 and two mutant p53 cDNAs encoding the Val to Ala mutation at codon 143 and the Arg to Trp mutation at codon 248. Flow cytometry, growth, and cytochemical analysis for alpha-napthyl butyrate esterase activity and nitroblue tetrazolium reduction indicated that wild-type p53 but not mutant p53 induced early monocytic differentiation in the transfected HL60 cells without terminal growth arrest. The wild-type p53 transfectants did not differentiate along the granulocytic pathway, even when induced with 1.25% DMSO for 6 days; rather, these cells resembled monocytic cells, confirming that wild-type p53 transfection caused these cells to become committed to differentiate along the monocytic pathway. HL60 cells transfected with wild-type p53 were more sensitive to stress, such as growth in serum-depleted medium and exposure to a chemotherapeutic agent, etoposide.     

Wild-type p53 and a p53 temperature-sensitive mutant suppress human soft tissue sarcoma by enhancing cell cycle control

Soft-tissue sarcomas are a heterogeneous group of tumors that are putatively mesenchymal in origin. Therapeutic advances in this disease have been limited over the past several decades. Approximately one-half of all patients will ultimately succumb, usually to uncontrollable pulmonary metastases. Although little is known about the underlying molecular determinants driving soft-tissue sarcoma inception, proliferation, and metastasis, mutation of the p53 gene is the most frequently detected molecular alteration in this disease. Accordingly, we were interested in determining whether transduction of wild-type (wt) p53 into soft-tissue sarcomas bearing mutated p53 genes might alter the malignant phenotype. SKLMS-1 is a human-derived leiomyosarcoma cell line with a codon 245 p53 point mutation. Cationic liposome was used to transfect wt p53 or 143Ala temperature-sensitive mutant p53 into this cell line. SKLMS-1 stable transfectants expressing wt p53 had decreased cell proliferation in vitro, decreased in vitro colony formation in soft agar, and decreased tumorigenicity in severe combined immunodeficient mice in vivo. Flow cytometric analysis of cell cycle components demonstrated markedly increased G1 cell cycle arrest and decreased entry into S phase, which corresponded to the induction of p21cip1 protein in the transfectants. Using SKLMS-1 stable transfectants expressing the 143Ala p53 temperature-sensitive mutant, we demonstrated the kinetics of and the causal relationship between wt p53 expression, the wt p53-dependent induction of cell cycle inhibitor p21cip1, and inhibition of cell cycle progression in p53-transfected SKLMS-1 cells. The ability to restore wt p53 growth-regulatory functions in soft-tissue sarcoma may ultimately be useful as a future therapy in patients with soft-tissue sarcomas.     

Inhibition of NNK-induced lung tumorigenesis by modulators of NNK activation

Xenografts originated from human tumours offer the most appropriate research material for in vivo experimental research. However, primary human breast carcinomas are difficult to grow when transplanted in athymic mice: tumour take is less than 15%. Recently, we have achieved 60% tumour take by injecting tumour cell suspensions mixed with Matrigel. Human breast xenografts originated from primary breast carcinoma also frequently show the potential to metastasize spontaneously. In the present study, we generated a human breast carcinoma xenograft line (UISO-BCA-NMT-18) that shows 100% tumorigenicity and 80-100% lung metastasis when transplanted s.c. in athymic mice. We have studied in detail the characteristics of the xenograft and the patient's tumour from which the xenograft line originated. Both the xenograft and the patient's tumour showed intense staining for mutant p53 nuclear protein, and high expression of U-PA, PAI and u-PAR. In vivo growth of the xenograft is stimulated by exogenous supplementation of oestrogen. This xenograft is continuously growing in mice and has shown 80-100% metastasis for the last three successive in vivo passages. This well-characterized, oestrogen-responsive, metastatic breast carcinoma xenograft line will provide excellent research material for metastasis-related research.     

Characterization of sequence elements involved in p53 stability regulation reveals cell type dependence for p53 degradation

The growth suppressive properties of the tumor suppressor protein p53 are activated upon DNA damage. The activation of p53 is reflected in increased p53 levels which are, at least in part, the result of an extended half-life of the protein. Although this suggests that stabilization of p53 is an intrinsic feature of p53 activation, the mechanisms involved in p53 degradation and stabilization are poorly understood. Here we report on the identification of an internal deletion mutant of wild-type p53, termed delta62-96, which can be stably expressed in various cell lines. This deletion mutant has a turnover rate similar to wild-type p53 and its stability is upregulated by treatment with UV light. In cell lines that express endogenous mutant or no p53, however, delta62-96 appears to be stable, strongly indicating that these cell lines have lost the ability to degrade p53. Further characterization of delta62-96 by mutational analyses defines sequence and structural requirements for p53 degradation and indicates that none of the known p53 phosphorylation sites is essential with respect to p53 stability regulation upon UV-irradiation.     

Wild-type p53-mediated down-modulation of interleukin 15 and interleukin 15 receptors in human rhabdomyosarcoma cells

We recently reported that rhabdomyosarcoma cell lines express and secrete interleukin 15 (IL-15), a tightly regulated cytokine with IL-2-like activity. To test whether the p53-impaired function that is frequently found in this tumour type could play a role in the IL-15 production, wild-type p53 gene was transduced in the human rhabdomyosarcoma cell line RD (which harbours a mutated p53 gene), and its effect on proliferation and expression of IL-15 was studied. Arrest of proliferation was induced by wild-type p53; increased proportions of G1-arrested cells and of apoptotic cells were observed. A marked down-modulation of IL-15 expression, at both the mRNA and protein level, was found in p53-transduced cells. Because a direct effect of IL-15 on normal muscle cells has been reported, the presence of IL-15 membrane receptors was studied by cytofluorometric analysis. Rhabdomyosarcoma cells showed IL-15 membrane receptors, which are down-modulated by wild-type p53 transfected gene. In conclusion, wild-type p53 transduction in human rhabdomyosarcoma cells induces the down-modulation of both IL-15 production and IL-15 receptor expression.     

Gastric cancer with p53 overexpression has high potential for metastasising to lymph nodes

Overexpression of the tumour suppressor gene p53 was investigated immunohistochemically in 96 primary gastric carcinomas and 26 corresponding metastatic perigastric lymph nodes. Abnormalities in p53 expression were found in 52 (54%) of the 96 primary carcinomas. Tumours stained positively for p53 frequently metastasised to lymph nodes (the metastatic rate: 85%) compared to findings in those with negative p53 staining (64%, P < 0.05). Ninety-two percent (24/26) of the malignant cells in the lymph nodes stained positively for p53. When the DNA ploidy pattern of the tumour was determined by flow cytometry, the aneuploid tumours in p53 positive and negative groups accounted for 69% and 45%, respectively (P < 0.05). Proliferative activity of the tumour, as measured by Ki-67 labelling, was significantly higher (30.6 +/- 12.0%) in the p53 positive group than that (25.1 +/- 10.7%) in the p53 negative group (P < 0.05). Thus, gastric cancer with a mutant p53 has high proliferative activity and metastasis to lymph nodes will probably occur.     

Analysis of p53 in human cutaneous melanoma cell lines

Mutations in the p53 tumour suppressor gene have been detected in a variety of human malignancies. Mutations have been found predominantly in conserved regions two to five. Our aim was to analyse p53 at the protein and DNA level in seven melanoma cell lines of cutaneous origin (HMB-2, DX3, LT5.1, MJM, SK23, A375P and A375M), including two parental/metastatic derivatives (A375P and A375M; DX3 and LT5.1). By immunohistochemical staining with three mouse monoclonal antibodies and a rabbit polyclonal serum, it was possible to observe differential nuclear expression of p53. The quantitation of p53 protein levels by ELISA correlated with the nuclear staining pattern. Western blotting showed an intact p53 protein in all cell lines; p53 was polymorphic in three cell lines (MJM, A375P and A375M). DNA sequencing studies showed that all cell lines had wild type p53. These results suggest that p53 is unlikely to play a significant role in the genesis of cutaneous melanoma.