Stabilization of the p53 transformation-related protein in mouse fibrosarcoma cell lines: effects of protein sequence and intracellular environment.

The transformation-related protein p53 is normally very labile. The stability of p53 is significantly increased in a number of fibrosarcoma cell lines derived from mouse tumors induced by treatment with physical or chemical agents. In many instances, p53 stabilization is correlated with the ability to form a stable complex with the heat shock protein cognate hsc70. We describe a line in which p53 is very stable yet has no detectable interaction with hsc70. The inability to form such a complex probably resides in the primary structure of the endogenous p53, since introduction of other p53 variants into those cells resulted in the appearance of a p53-hsc70 complex. The factors affecting p53 stability were investigated by stable transfection experiments. The results indicated that the primary structure of the p53 protein is a major determinant of its turnover rate; different p53 variants were degraded at distinct and characteristic rates in a number of transformed cell types. However, at least one p53 variant was degraded differently in nontransformed BALB/c-3T3 than in transformed fibrosarcoma cells, demonstrating that the specific cellular environment can also affect the stability of p53.     

Differences in epitope accessibility of p53 monoclonal antibodies suggest at least three conformations or states of protein binding of p53 protein in human tumor cell lines

The p53 tumor suppressor gene is deleted or mutated in over 50% of human tumors. Mutations frequently extend the half-life of the p53 protein; and a high level of nuclear p53 expression, detected by immunohistochemistry, has been used to predict the p53 status of tumors. We compared the sensitivity and reactivity of five frequently used, commercially available monoclonal antibodies (1801, DO1, DO7, BP53.12 and 421) in immunoblot and immunofluorescence assays, and found that results differed among the antibodies. Comparison of immunoblot analysis of denatured nuclear and cytoplasmic p53 protein were consistent with antibodies DO1, DO7 and BP53.12, each of which generated a strong specific signal in both cell fractions. However, in situ analysis demonstrated that although all antibodies recognized nuclear p53, only BP53.12 and 421 recognized p53 protein in the cytoplasm. In addition, 1801 produced a signal in p53-negative tumor cell lines. Differences in situ among the antibodies were probably due to the accessibility of their respective epitopes and suggested that nuclear and cytoplasmic p53 either have different three-dimensional conformations or are bound to different proteins. A third p53 protein conformation was also suggested by the observation that only two of the five antibodies (BP53.12 and DO7) detected induced levels of p53 in situ following exposure to ionizing radiation. In summary, except for the fact that DO7 does not recognize cytoplasmic p53 in situ, we found it to be the most specific, versatile, and reliable antibody. We conclude that the p53 antibody of choice depends upon the specific goal of a study and the method used to detect this protein.     

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.     

Sanguinarine causes DNA damage and p53-independent cell death in human colon cancer cell lines

The benzophenanthridine alkaloid sanguinarine has antimicrobial and possibly anticancer properties but it is not clear to what extent these activities involve DNA damage. Thus, we studied its ability to cause DNA single and double strand breaks, as well as increased levels of 8-oxodeoxyguanosine, in human colon cancer cells and found DNA damage consistent with oxidation. Since the tumor suppressor p53 is frequently involved in inducing apoptosis following DNA damage we investigated the effect of sanguinarine in wild type, p53-mutant and p53-null colon cancer cell lines. We found them to be equally sensitive to this plant compound, indicating that cell death is not mediated by p53 in this case. In addition, our observation that apoptosis induced by sanguinarine is initiated very rapidly raised the question whether there is enough time for cellular signaling in response to DNA damage. Moreover, the abundance of double strand breaks is not consistent with only oxidative damage to DNA. We conclude that the majority of DNA double strand breaks in sanguinarine-treated cells are likely the result, rather than the cause, of apoptotic cell death and that apoptosis induced by sanguinarine is independent of p53 and most likely independent of DNA damage.     

The role of p53 in growth of mouse fibroblast lines

To examine the hypothesis that p53 protein may play a central role in regulating reproduction of mammalian cells, we compared the absolute amounts and relative rates of synthesis of p53 protein in two pseudonormal cell lines, 3T3 and C3H 10T1/2, during quiescence, during log proliferation, and in quiescent cells stimulated with serum. The absolute amount of p53 protein per cell was found to be severalfold lower in quiescent cells than in log-phase cells. The ratio of the rate of synthesis of p53 protein to the rate of synthesis of total protein was slightly higher in quiescent cells than the same ratio in log-phase cells. Thus, entry into quiescence is not accompanied by a differential switch-off of synthesis of p53 protein. In quiescent cells stimulated with serum the amount of p53 protein per cell and its rate of synthesis increase, but only in proportion to the increase in total protein per cell and the increase in rate of total protein synthesis. Similarly, 12-14 h after serum stimulation, the time of the G1 to S transition, the accumulated increase in p53 protein per cell is about what would be expected for a short-lived protein whose rate of synthesis has increased in proportion to the increase in rate of synthesis of total protein. The results are not those expected for a protein that functions specifically in release from quiescence or in transition from G1 to S.     

Expression of the mouse p53 cellular tumor antigen in monkey cells.

DNA specific for the murine p53 cellular tumor antigen was linked to the early simian virus 40 promoter and introduced into monkey COS cells either by transfection with recombinant plasmids or by infection with virus. Recipient cells made substantial amounts of a protein apparently identical to mouse p53. Severalfold-larger quantities were detected when cells were transfected with an intron-containing p53-specific segment, as compared with transfection with intronless cDNA. The p53 encoded by the recombinant DNA was capable of complexing with the simian virus 40 T antigen. Transfected p53 was also probably associated with a cellular 68-kilodalton protein, which may be related to a protein coprecipitating with p53 in some transformed cells. These findings confirm the predicted reading frame and protein boundaries and demonstrate that apparently functional p53 can be produced in cells via experimentally introduced recombinant DNA.     

Anti-Fas antibody-induced apoptosis in human colorectal carcinoma cell lines: role of the p53 gene

The cell surface Fas antigen transducts an apoptotic signal by its crosslinking with Fas ligand or anti-Fas antibody in a variety of human cultured cells. In this study, we examined the expression of Fas antigen and its mediation of apoptosis in six human colorectal carcinoma cell lines. A flow cytometric analysis revealed that LoVo, DLD-1, WiDr and SW837 cell lines showed higher expression levels of Fas antigen, in contrast to lower expression in COLO201 and COLO320DM. Interferon- enhanced the expression of Fas antigen in all of the cell lines examined. Both Fas ligand and Fas-associated phosphatase-1 (FAP-1) were expressed only in COLO320DM. Anti-Fas antibody induced apoptosis in LoVo carrying wild-type p53 gene, but not in the other five cell lines carrying mutated p53 gene. The transfection of wild-type p53 gene using an adenovirous vector upregulated P53 protein in WiDr and SW837 cells, both of which showed, however, no increase in apoptotic cells by anti-Fas antibody treatment. These results indicated that (1) Fas antigen was variably expressed, regardless of the p53 gene status and (2) the susceptibility to anti-Fas antibody-mediated apoptosis did not correlate to Fas, Fas ligand or FAP-1 expression levels. Therefore, we conclude that wild-type P53 expression might not necessarily be essential for Fas-mediated apoptosis in human colorectal carcinoma cell lines.     

Immunological evidence for the association of p53 with a heat shock protein, hsc70, in p53-plus-ras-transformed cell lines.

A rabbit antiserum was prepared against the C-terminal peptide of 21 amino acids from the human heat shock protein hsp70. These antibodies were shown to be specific for this highly inducible heat shock protein (72 kilodaltons [kDa] in rat cells), and for a moderately inducible, constitutively expressed heat shock protein, hsc70 (74 kDa). In six independently derived rat cell lines transformed by a murine cDNA-genomic hybrid clone of p53 plus an activated Ha-ras gene, elevated levels of p53 were detected by immunoprecipitation by using murine-specific anti-p53 monoclonal antibodies. In all cases, the hsc70, but not the hsp70, protein was coimmunoprecipitated with the murine p53 protein. Similarly, antiserum to heat shock protein coimmunoprecipitated p53. Western blot (immunoblot) analysis demonstrated that the hsc70 and p53 proteins did not share detectable antigenic epitopes. The results provide clear immunological evidence for the specific association of a single heat shock protein, hsc70, with p53 in p53-plus-ras-transformed cell lines. A p53 cDNA clone, p11-4, failed to produce clonable cell lines from foci of primary rat cells transfected with p11-4 plus Ha-ras. A mutant p53 cDNA clone derived from p11-4, SVKH215, yielded a 2- to 35-fold increase in the number of foci produced after transfection of rat cells with SVKH215 plus Ha-ras. When cloned, 87.5% of these foci produced transformed cell lines. SVKH215 encodes a mutant p53 protein that binds preferentially to the heat shock proteins of 70 kDa compared with binding by the parental p11-4 p53 gene product. These data suggest that the p53-hsc70 protein complex could have functional significance in these transformed cells.     

Multiplex PCR screening detects small p53 deletions and insertions in human ovarian cancer cell lines

Mutations at the p53 tumor suppressor gene locus are a frequent genetic alteration associated with human ovarian carcinoma. Little information exists regarding whether mutational events occur other than point mutations and large deletions, causing loss of heterozygosity. Small intragenic deletions and insertions in the p53 gene have been observed in various human neoplasias. We developed a multiplex polymerase chain reaction (MPCR) screening assay to amplify the complete p53 coding region from genomic DNA in a single step. Deletions and/or insertions were found in six out of 11 newly established ovarian carcinoma cell lines. MPCR detected deletions as small as 2bp, as confirmed by nucleotide sequence analysis. Most of the observed alterations (6/7) were homozygous or hemizygous. Structural aberrations of the p53 gene possibly leading to loss of p53 cell cycle control may be a consequence of a slipped-mispairing mechanism in rapid DNA replication during repetitious ovulation and wound repair of ovarian epithelial cells. MPCR may be a valuable tool for screening for possible p53 deletion and insertion mutations not only in ovarian cancer but also in other malignancies.     

Reversal of human cellular senescence: roles of the p53 and p16 pathways

Telomere erosion and subsequent dysfunction limits the proliferation of normal human cells by a process termed replicative senescence. Replicative senescence is thought to suppress tumorigenesis by establishing an essentially irreversible growth arrest that requires activities of the p53 and pRB tumor suppressor proteins. We show that, depending on expression of the pRB regulator p16, replicative senescence is not necessarily irreversible. We used lentiviruses to express specific viral and cellular proteins in senescent human fibroblasts and mammary epithelial cells. Expression of telomerase did not reverse the senescence arrest. However, cells with low levels of p16 at senescence resumed robust growth upon p53 inactivation, and limited growth upon expression of oncogenic RAS. In contrast, cells with high levels of p16 at senescence failed to proliferate upon p53 inactivation or RAS expression, although they re-entered the cell cycle without growth after pRB inactivation. Our results indicate that the senescence response to telomere dysfunction is reversible and is maintained primarily by p53. However, p16 provides a dominant second barrier to the unlimited growth of human cells.     

Effects of electrogenetherapy with p53wt combined with cisplatin on curvival of human tumor cell lines with different p53 status

The aim of our study was to evaluate electrogenetherapy with p53wt alone or combined with cisplatin on two colorectal (HT-29 and LoVo) and two prostatic (PC-3 and Du145) carcinoma cell lines with different p53 status. In addition, the feasibility of electrogenetherapy with p53wt was tested also in vivo on PC-3 prostatic cancer xenografts. Electrogenetherapy with p53wt was dependent on the p53 status of the cell lines used. Electrogenetherapy was the most effective on the PC-3 (p53 null) and Du145 (p53mt) cells, and to the much lesser extent in LoVo cells (p53wt). The exception was the HT-29 cell line with overexpressed mutated p53, where electrogenetherapy with p53wt was the least effective. Sensitivity of the cell lines to cisplatin was independent of the p53 status. Furthermore, the presence of exogenous p53 due to electrogenetherapy did not enhance cisplatin cytotoxicity, since the combination of these therapies resulted in additive cytotoxic effect. The effectiveness of electrogenetherapy with p53wt was also demonstrated in vivo by successful treatment of subcutaneous PC-3 tumors in mice. In conclusion, our study shows that electrogenetherapy with p53wt is feasible, and resulted in comparable cytotoxic and antitumor effectiveness to viral-mediated p53wt gene therapy. This therapy was effective and dependent on the p53 status of the tumor cell lines. Combination of electrogenetherapy and cisplatin resulted in additional cell kill by cisplatin, and was not dependent on the p53 status.     

p53-independent downregulation of histone gene expression in human cell lines by high- and low-let radiation

Using microarrays to analyze differential gene expression as a function of p53 status and radiation quality, we observed downregulation of a large set of histone genes in p53 wild-type TK6 cells 24 h after exposure to equitoxic doses of high-LET (1.67 Gy 1 GeV/amu (56)Fe ions) or low-LET (2.5 Gy γ rays) radiation. Quantitative real-time PCR of specific subtypes of core (H2A, H2B, H3 and H4) and linker (H1) histones confirmed this result. DNA synthesis and histone gene expression are tightly coordinated during the S phase of the cell cycle, and both processes are regulated by cell cycle checkpoints in response to DNA damage caused by ionizing radiation. However, we observed similar repression of histone gene expression in both TK6 cells and their p53-null derivative NH32 after radiation exposure, although the histone gene expression was not decreased to the same extent in NH32 cells as it was in TK6 cells. We also found decreased histone gene expression that was dose- and time-dependent in the colon cancer cell line HCT116 and its p53-null derivative. These results show that both high- and low-LET radiation exposure negatively regulate histone gene expression in human lymphoblastoid and colon cancer cell lines independent of p53 status.     

Selective COX-2 inhibitor (celecoxib) decreases cellular growth in prostate cancer cell lines independent of p53

Abstract

Celecoxib is a clinically available COX-2 inhibitor that has been reported to have antineoplastic activity. It has been proposed as a preventative agent for several types of early neoplastic lesions. Earlier studies have shown that sensitivity of prostatic carcinoma (PCa) to celecoxib is associated with apoptosis; however, these studies have not demonstrated adequately whether this effect is dependent on p53 status. We studied the relation between sensitivity to celecoxib and the phenotypic p53 status of PCa cells lines, LNCaP (wild type p53), PC3 (null p53) and DU145 (mutated p53). Cellular growth was assessed at 24, 48, 72 and 96 h after celecoxib treatment at concentrations of 0, 10, 30, 50, 70 and 100 μM using an MTT assay. Cellular proliferation (Ki-67 expression) was determined by immunocytochemistry. Phenotypic expression of p53 was analyzed by western blotting. The effects of celecoxib on cellular growth and its association with p53 were assessed after down-regulation of p53 using synthetic interfering RNAs (siRNA) in LNCaP cells. Expression of p53 and COX-2 at mRNA levels was assessed by quantitative real time polymerase reaction (qRT-PCR). We found that celecoxib inhibited cellular growth and proliferation in a dose-dependent manner in all three cell lines; LNCaP cells with a native p53 were the most sensitive to celecoxib. We observed a down- regulation effect on p53 in LNCaP cells exposed to ≥ 30 μM celecoxib for 72 h, but found no significant changes in the p53 levels of DU145 cells, which have a mutated p53. Reduced COX-2 expression was found with decreased p53 in LNCaP and PC-3 cells that were exposed to ≥ 20 μM of celecoxib for 72 h, but COX-2 expression was increased in DU145 cells. All three cell lines demonstrated pan-cytotoxicity when exposed to 100 μM celecoxib. When p53 expression was inhibited using siRNA in LNCaP cells, the inhibitory effects on cellular growth usually exerted by celecoxib were not changed significantly. Celecoxib reduces the growth of prostate cancer cell lines in part by decreasing proliferation, which suggests that the inhibition of growth of LNCaP cells by celecoxib is independent of normal levels of native p53.