DNA binding specificity of proteins derived from alternatively spliced mouse p53 mRNAs.

The mouse p53 gene generates two alternative splice products encoding p53 protein and a naturally occurring protein (p53as) with changes at the C-terminus. In p53as the negative regulatory region for DNA binding and PAb421 antibody binding site are replaced, and p53as is constitutively active for sequence-specific DNA binding. Using the technique of randomized synthetic oligonucleotide in cyclic amplification and selection of targets, we have found that p53as and p53 proteins have the same DNA binding specificities but that these specificities frequently diverge from the consensus of two copies of PuPuPuCATGPyPyPy. The importance of tetranucleotide CATG was confirmed but there was a less rigorous requirement for patterns of flanking or intervening sequences. In particular, the three purines upstream and three pyrimidines downstream of CATG are not required for p53 or p53as binding, 29 or more intervening nucleotides are tolerated, and one CATG is sufficient where adjacent nucleotides contain a region of homology with certain previously reported non-consensus p53 binding sequences. These results suggested further definition of the non-consensus motifs, and database searches with these uncovered additional candidate genes for p53 protein binding. We conclude that p53as and perhaps other activated forms of p53 exert their effects on the same genes and that differential activities of p53 protein forms are not due to inherently different sequence selectivities of DNA binding.     

Wild-type alternatively spliced p53: binding to DNA and interaction with the major p53 protein in vitro and in cells.

A p53 variant protein (p53as) generated from alternatively spliced p53 RNA is expressed in normal and malignant mouse cells and tissues, and p53as antigen activity is preferentially associated with the G2 phase of the cell cycle, suggesting that p53as and p53 protein may have distinct properties. Using p53as and p53 proteins translated in vitro, we now provide evidence that p53as protein has efficient sequence-specific DNA-binding ability. DNA binding by p53 protein is inefficient in comparison and requires activation. Furthermore, p53as and p53 proteins formed hetero-oligomers when co-translated in vitro, resulting in inactivation of p53as DNA-binding activity. Gel filtration indicated that p53as translated in vitro, like p53, formed tetramers. In support of a functional role of p53as in cells, p53as/p53 hetero-oligomers were coimmunoprecipitated from mouse cells, and both protein forms were detectable in nuclear extracts by electrophoretic mobility shift assays. These results suggest that the biochemical functions of p53 are mediated by interaction between two endogenous protein products of the wild-type p53 gene.     

The murine C'-terminally alternatively spliced form of p53 induces attenuated apoptosis in myeloid cells.

The onset of p53-dependent apoptosis results from the accumulation of damaged DNA. Recently, it was shown that the C' terminus of the p53 protein plays a central role in sensing damaged DNA. In our present study, we examined the role of the C' terminus in the induction of apoptosis. A temperature-sensitive (ts) mutant of the alternatively spliced form of p53 (p53AS-ts) and the ts mutant of the regularly spliced form (p53RS-ts) were used to generate series of stable clones with increasing amounts of p53 protein. Apoptotic patterns induced by either the regularly spliced p53 product (p53RS) or a C'-terminally alternatively spliced p53 product (p53AS) were compared. We found that although both forms of p53 induced apoptosis following expression of the wild-type protein conformation, the kinetics were different. Apoptosis induced by the p53AS protein was attenuated compared to that induced by p53RS. The delay in the manifestation of the apoptotic features following p53AS expression was in agreement with a delay in the regulation of the expression of apoptosis-related genes. The observation that p53 with an altered C' terminus is still capable of inducing apoptosis suggests that the actual onset of the apoptotic process most probably involves structural domains other than the C' terminus of the p53 molecule. However, the fact that the apoptotic activity mediated by the p53AS product was slower than that mediated by the p53RS product suggests that the C' terminus indeed exerts a certain control on the apoptotic activity of the p53 molecule.     

Compensatory expression of p73 in PARP-deficient mouse fibroblasts as response to a reduced level of regularly spliced wild-type p53 protein

We have investigated the effect of PARP gene inactivation on the expression of wild-type (wt) p53 protein. Using immortalized fibroblasts from control and PARP knock-out mice we have found by immunoblotting with the PAb421 antibody a profound decrease of the p53 expression to a barely detectable level in PARP knock-out cells. Surprisingly, longer exposure of immunoblots revealed an immunoreactive band at about 75 kD which was stronger in PARP-deficient cells than in wt cells and was not affected upon doxorubicin treatment. The size of the PAb421 immunoreactive protein and the lack of its inducibility in response to DNA damage resembled those of p73, the first described p53 homologue. Therefore, we examined the reactivity of anti-p53 antibodies with in vitro translated p73 protein. Interestingly, p73 was efficiently immunoprecipitated with distinct antibodies recognizing the carboxy-terminus of p53. In Northern blots we observed p73 signals of comparable intensity in controls and PARP-deficient cells. We conclude that elevated expression of p73 may compensate the reduced level of p53 in PARP-deficient cells.     

Alternatively spliced p53 RNA in transformed and normal cells of different tissue types.

The alternatively spliced RNA species of tumor suppressor gene p53, containing an additional 96 bases derived from intron 10, is present at approximately 25 to 30% the level of regularly spliced p53 RNA in both normal epidermal and carcinoma cells. The presence of this alternatively spliced RNA in 10T1/2 fibroblast cells, mouse liver and testis suggests that this alternative splicing may be universal. The level of alternatively spliced p53 RNA was increased coordinately with that of regularly spliced p53 in 10T1/2 cells in response to epidermal growth factor. Immunoprecipitation analysis of epidermal cells using monoclonal antibodies which recognize different epitopes of p53 suggested that distinct p53 proteins may be translated from both RNA species. Considering previous observations on the potential importance of carboxyl terminal sequences in p53 function, knowledge of the ubiquitous presence of alternatively spliced p53 is important for future studies of p53 function in normal cells and in oncogenesis.     

AMOP, a protein module alternatively spliced in cancer cells

This article describes a new extracellular domain--AMOP, for adhesion-associated domain in MUC4 and other proteins. This domain occurs in putative cell adhesion molecules and in some splice variants of MUC4. MUC4 splice variants are overexpressed in several tumours; in particular, they are highly expressed in pancreatic carcinomas but not in normal pancreas. The presence of AMOP in cell adhesion molecules could be indicative of a role for this domain in adhesion.     

Regulation of protein synthesis by inducible wild-type p53 in human lung carcinoma cells

Activation of an over-expressed mutant form of the tumour suppressor protein p53 has been shown to inhibit protein synthesis. To determine whether this effect is due only to high level expression or the mutant nature of the protein, we have used a doxycycline-inducible lung carcinoma cell line capable of expressing wild-type p53. We now show that levels of wild-type p53 similar to those expressed endogenously also inhibit protein synthesis. The mechanism involves dephosphorylation and accumulation of the translational inhibitor 4E-BP1, and increased association of 4E-BP1 with initiation factor eIF4E. The inhibition of translation is not a consequence of p53-mediated apoptosis.     

Prolonged wild-type p53 protein accumulation and cisplatin resistance

The major limitation for the chemotherapeutic use of DNA-damaging agent cisplatin is the development of resistance in initially responsive tumors. One of the main pathways regulating cell survival following DNA damage is the p53 pathway. In this study we compared the cisplatin-induced response of p53 protein and its downstream targets p21WAF-1 and Mdm2 in the cisplatin-sensitive ovarian carcinoma cell line A2780 and its cisplatin-resistant derivative CP70. A higher dose of cisplatin and a longer exposure time was required to achieve the same level of p53, p21WAF-1, and Mdm2 protein accumulation in the cisplatin-resistant CP70 cells versus cisplatin-sensitive A2780 cells. A significant difference between the two cell lines was observed in cisplatin-induced stabilization of p53 protein. The p53 half-life increased 31-fold in CP70 cells compared to only 6-fold in A2780 cells. In contrast, there was no difference in p21WAF-1 half-life between the two cell lines. These results demonstrate that in A2780 and CP70 cells resistance to cisplatin correlates with prolonged p53 protein stabilization and accumulation.     

An alternatively spliced Interleukin 4 form in lymphoid cells

The nucleotide sequence data presented in this paper have been submitted to the EMBL nucleotide sequence database and have been assigned the accession number X81851     

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.     

Expression of wild-type p53 is not compatible with continued growth of p53-negative tumor cells.

Inactivation of the cellular p53 gene is a common feature of Friend virus-induced murine erythroleukemia cell lines and may represent a necessary step in the progression of this disease. As well, frequent loss or mutation of p53 alleles in diverse human tumors is consistent with the view of p53 as a tumor suppressor gene. To examine the significance of p53 gene inactivation in tumorigenesis, we have attempted to express transfected wild-type p53 in three p53-negative tumor cell lines: murine DP16-1 Friend erythroleukemia cells, human K562 cells, and SKOV-3 cells. We found that aberrant p53 proteins, which differ from wild-type p53 by a single amino acid substitution, were expressed stably in these cells, whereas wild-type p53 expression was not tolerated. The inability of p53-negative tumor cell lines to support long-term expression of wild-type p53 protein is consistent with the view that p53 is a tumor suppressor gene.     

New alternatively spliced variants of calmodulin-dependent protein kinase II from rabbit liver

Polymerase chain reaction analysis revealed four alternatively spliced variants of each of the γ and δ isoforms of calmodulin-dependent protein kinase II (CaM-kinase II) in rabbit liver. Among the four variants of the γ isoform, two were novel ones, designated as CaM-kinase II γ-H and γ-I. The γ-I variant possessed both of the two deletable exons, D2a and D2b, which had never been found together in any variant. Sequence analysis of the γ-I indicated that the D2a was upstream of the D2b and that they were contiguous with each other in the γ-I. Among the four variants of the δ isoform, two were also novel ones, designated as CaM-kinase II δ-11 and δ-12, and the other two were the already-reported ones, δ-2 and δ-6. The δ-11 and δ-12 were identical to the δ-2 and δ-6, respectively, except that three bases (CAG) located at a splicing junction was deleted in the δ-11 and δ-12, suggesting two splicing sites of a single intron. Thus, the diverse splicing patterns may produce many more variants than those so far considered.     

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.