Anti-p53 autoantibodies in colon cancer patients.
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Autoantibodies↗
Biomedical subjects
N Goldfinger
Publications and source records attributed to N Goldfinger.
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.
Alternative Splicing↗
Differential expression of the regularly spliced wild-type p53 and its COOH-terminal alternatively spliced form during epidermal differentiation.
In the present study, we investigated the role of p53 in the differentiation of epidermal keratinocyte cells. The interrelationship between p53 expression and the various stages of epidermal differentiation and the role of the COOH terminus of the p53 molecule in this process were determined by comparing the expression of the regularly spliced p53 (RSp53) molecule and that of the COOH-terminal alternatively spliced (ASp53) form. p53 mRNA distribution was studied by in situ analysis of frozen skin sections and by reverse transcription-PCR analysis of the various wild-type p53 forms expressed in neonatal skin cell fractions separated by Percoll gradient. p53 protein levels were measured by fluorescence-activated cell sorting analysis and immunohistochemistry, using antibodies that recognize either the COOH terminus of RSp53 or ASp53. The results show that although less mature keratinocyte cells predominantly express the RSp53 form, the more mature cells preferentially express the ASp53 form. Therefore, it is possible that the two p53 forms are associated with different functions required at the various stages of keratinocyte differentiation. The results suggest that the COOH-terminal domain of the p53 molecule is important for its activity in the process of keratinocyte differentiation.
Alternative Splicing↗
Induction of apoptosis and p53 expression in immature thymocytes by direct interaction with thymic epithelial cells.
Apoptosis of normal thymocytes was shown to be triggered by several mechanisms (e.g. glucocorticoids, gamma-irradiation). In the present study the authors report on thymocyte apoptosis that is induced by thymic epithelial cells. The thymocytes undergo a massive apoptotic death within 24 h of cocultivation with thymic epithelial cell monolayers derived from primary cultures (PTEC) or from a thymic epithelial cell line (TEC). Non-thymic monolayers were inactive. Apoptosis induction in this experimental model requires direct contact between the thymocytes and the thymic epithelial monolayer and can be blocked by anti-CD2 and anti-LFA-1 antibodies. The immature CD3-/+dull CD4+CD8+ thymocytes were the cells which undergo apoptosis. The fact that the authors are dealing with a massive apoptotic process of immature cells in the absence of exogenous antigen suggests that it involves the nonselected thymocytes. The apoptotic pathway selected by thymocytes following their culturing on TEC involves p53 expression. Indeed it was found that TEC-induced apoptosis, led to the accumulation of p53 protein that preceded the step of DNA fragmentation in freshly isolated thymocytes as well as in a glucocorticoid resistant thymoma cell line. Since glucocorticoid-induced thymocyte apoptosis is p53-independent, glucocorticoids are conceivably not involved in TEC-induced thymocyte death. The in vitro experimental model presented here may reflect the physiological sequence of events leading to thymocyte death in the thymus.
Animals↗
Induction of HL-60 cells to undergo apoptosis is determined by high levels of wild-type p53 protein whereas differentiation of the cells is mediated by lower p53 levels.
The observation that wild-type p53 may induce cells to undergo either apoptosis or differentiation raises the question of whether these two events share similar p53-dependent pathways. To evaluate the interrelationship between these two p53-dependent processes, our study focused on the human HL-60, a promyelocytic p53 nonproducer cell line in which p53 expression was introduced and the induction of apoptosis and differentiation was followed under controlled conditions. p53 expression was induced in the HL-60 cell line by infection with the recombinant wild-type p53 (p53WT) vaccinia virus. Viral infection gave rise to cells expressing various levels of wild-type p53 protein. High levels of p53 protein induced cells to undergo rapid apoptosis, whereas lower levels of p53 protein induced cells to undergo cell differentiation at a more moderate rate of kinetics. These results suggest that p53 protein levels may determine whether a given cell should prefer one pathway over the other to exit the cell cycle. Accordingly, we propose that the p53 vaccinia virus may be used as a potential vector for cell therapy leading toward the exit of p53 null human primary hematopoetic tumors from the malignant state in vivo via the apoptotic or cell differentiation pathways.
Apoptosis↗
Spot-1, a novel NLS-binding protein that interacts with p53 through a domain encoded by p(CA)n repeats.
Nuclear Localization Signals (NLS) have been found to mediate the import of proteins into the nucleus. Proteins interacting directly with NLS control the subcellular localization of nucleophilic proteins. The p53 protein is spatially regulated throughout the cell cycle and this regulation has been shown to be dependent on the presence of its NLS sequences. We identified three novel cDNA clones that were isolated from an expression library because they encode polypeptides that bind a synthetic peptide containing the major NLS of p53 (NLS I). These clones were found to share a common domain encoded by p(CA)n repeats; a simple sequence length polymorphism (SSLP). THis is the first report where p(CA)n repeats were found to encode protein. One cDNA clone encodes a full length, 16 kDa protein, designated spot-1, that is represented in cells predominantly as oligomers. spot-1 interacts with the NLS I of p53 through its p(CA)n repeat. Cell fractionation and immunofluorescence analysis demonstrated that spot-1 is a nuclear protein which, in fibroblasts, co-localizes with p53.
Amino Acid Sequence↗
Does wild-type p53 play a role in normal cell differentiation?
Inactivation of the p53 tumor suppressor gene plays a major role in malignant transformation. The central question in this issue is concerned with the understanding of the function of p53 in normal cells and its deregulation in cancer cells. Several in vitro and in vivo experimental models have indicated that induction of cells to undergo differentiation involve up-regulation in the expression of the p53. In the case of B cell differentiation, p53 was found to be involved in several steps of the differentiation pathway. The conclusion that p53 plays a role in normal development and differentiation in vivo is substantiated by the observation that p53 is expressed during embryonic development and is detected at low levels in a number of organs of adult mice. Accentuated levels of p53 in testes of adult mice, suggests that p53 plays a role in the meiotic process of spermatogenesis. B cell differentiation and spermatogenesis are biological pathways which normally involve DNA reshuffling and rearrangements. In accordance with the notion that p53 is associated with DNA repair it is tempting to speculate that at least in these physiological pathways p53 functions as a master gene that controls genome integrity.
Animals↗
Mice with reduced levels of p53 protein exhibit the testicular giant-cell degenerative syndrome.
Transgenic mice which carry hybrid p53 promoter-chloramphenicol acetyltransferase (CAT) transgenes were found to express CAT enzymatic activity predominantly in the testes. Endogenous levels of p53 mRNA and protein were lower than in the nontransgenic control mice. The various p53 promoter-CAT transgenic mice exhibited in their testes multinucleated giant cells, a degenerative syndrome resulting presumably from the inability of the tetraploid primary spermatocytes to complete meiotic division. The giant-cell degenerative syndrome was also observed in some genetic strains of homozygous p53 null mice. In view of the hypothesis that p53 plays a role in DNA repair mechanisms, it is tempting to speculate that the physiological function of p53 that is specifically expressed in the meiotic pachytene phase of spermatogenesis is to allow adequate time for the DNA reshuffling and repair events which occur at this phase to be properly completed. Primary spermatocytes which have reduced p53 levels are probably impaired with respect to DNA repair, thus leading to the development of genetically defective giant cells that do not mature.
Animals↗
Testicular tissue-specific expression of the p53 suppressor gene.
The hybrid transgene approach was adapted to study the physiological pathway(s) in which the p53 suppressor gene is involved. p53 promoter-CAT transgenic mice were found to express enzymatic CAT activity predominantly in the testes. In situ hybridization indicated that expression of the transgene as well as the endogenous p53 agreed with the typical wave and cycle patterns of spermatogenesis. p53 promoter-CAT transgenic mice expressed in the testes reduced levels of endogenous p53 mRNA that correlated with the copy number of the mouse or human transgene. The spatial and cyclical expression of the p53 gene which is confined to the primary spermatocytes in the seminiferous tubuli suggested that p53 may play a role in the meiotic process of spermatogenesis in vivo.
Animals↗
Wild type p53 functions as a control protein in the differentiation pathway of the B-cell lineage.
An analysis of cell lines representing different stages of the B-cell differentiation pathway indicated that about 50% of the cell lines examined expressed exclusively wild type p53 protein. These lines therefore offer a convenient system to study the involvement of p53 in cell differentiation. When 70Z/3, a pre-B cell line which expresses wild type p53, was treated with the differentiation inducer lipopolysaccharide (LPS), it was seen that increased levels of p53 mRNA preceded specific changes in kappa (kappa) immunoglobulin expression. This increased expression of kappa specific mRNA, which was evaluated by specific PCR analysis, was blocked following transfection with mutant p53 coding plasmids. Treatment of 13A60, another cell line which endogenously expresses wild type p53, with LPS caused a secretion of IgA antibodies, also accompanied by increased p53 mRNA expression. The conclusion was that induction of B-cell differentiation involves the transcription of the p53 gene. This was further substantiated by experiments showing that differentiation of stable clones derived from the 70Z/3 cell line, harboring a p53-promoter-CAT plasmid, induced increased CAT activity. Furthermore, wild type p53 transactivated the promoter control sequences of the kappa light chain gene. Taken together, these results suggest that p53 is involved in B-cell differentiation, a pathway which involves DNA rearrangements that may be accompanied by generation of faulty DNA. The fact that wild type p53 was shown to function as a transcriptional factor, coupled with the notion that it is associated with DNA repair systems, may designate p53 as a control protein in the B-cell differentiation pathway.
Animals↗
Expression of p53 protein in spermatogenesis is confined to the tetraploid pachytene primary spermatocytes.
The various steps of differentiation and maturation in spermatogenesis are well characterized and offer a convenient system to explore the possibility that p53 plays a role in cell differentiation in vivo. In situ hybridization experiments indicate that the p53 gene is expressed in tetraploid primary spermatocytes at the meiotic pachytene stage of the first round of spermatogenesis in young mice. An analysis of spermatogenic cells treated with anti-p53 antibodies reveals that the p53 protein is expressed in a discrete tetraploid cell population, with size and cellular structure parameters characteristic of midpachytene spermatocytes. The specific kinetics of p53 expression in the first round of spermatogenesis and its localization in adult testicular sections, coupled with the fact that the protein is expressed in the largest cells with lower DNA density, suggest that p53 expression is confined to the tetraploid primary spermatocytes of the meiotic pachytene phase. These observations identify p53 protein as a potential member of the meiosis control protein family.
Animals↗
Expression of wild-type and mutant p53 proteins by recombinant vaccinia viruses.
To facilitate the purification of wild type p53 protein, we established a recombinant p53 vaccinia viral expression system. Using this efficient eukaryotic expression vector, we found that the expressed p53 proteins retained their specific structural characteristics. A comparison between wild type and mutant p53 proteins showed the conservation of the typical subcellular localization and the expression of specific antigenic determinants. Furthermore, wild type p53 exhibited a typical binding with large T antigen, whereas no binding was detected with mutant p53. Both wild type and mutant p53 proteins were highly stable and constituted 5-7% of total protein expressed in the infected cells. These expression recombinant viruses offer a simple, valuable system for the purification of wild type and mutant p53 proteins that are expressed abundantly in eukaryotic cells.
Antigens, Viral, Tumor↗
Involvement of wild-type p53 in pre-B-cell differentiation in vitro.
Wild-type p53 protein is a growth modulator whose inactivation has been found to be a key event in malignant transformation. Reconstitution of wild-type p53 in the p53-nonproducer, Abelson murine leukemia virus-transformed pre-B-cell line L12 gave rise to stably growing clones. Wild-type p53-producer derived cell lines exhibit an altered cell cycle, however. More cells with an extended G0/G1 phase were found than in the p53-nonproducer parental cell line. Furthermore, when injected into syngeneic mice, these cells induced a lower incidence of tumors and these tumors were less aggressive. Analysis of immunoglobulin expression revealed that wild-type p53 induced the expression of cytoplasmic immunoglobulin mu heavy chain. In addition, these derived cells lines exhibited increased levels of a B-cell-specific surface marker, B220. These results suggest that wild-type p53 may function as a cell differentiation factor that can induce development of pre-B cells into a more advanced stage in the pathway of B-cell maturation. In these pre-B cells, wild-type p53 may induce cell differentiation without terminal growth arrest of the cell population.
Abelson murine leukemia virus↗
Alterations in tumor development in vivo mediated by expression of wild type or mutant p53 proteins.
To study the mechanism of p53 involvement in malignant transformation, we compared the tumor development patterns induced by a parental p53 nonproducer pre-B cell line with those by cell lines generated from this parental cell line following transfection of either wild type or mutant p53. It was found that whereas mutant p53 facilitated tumor development, expression of wild type p53 restrained tumor development. Cell lines expressing the wild type p53 induced the development of faster regressing tumors than the parental cell line. The parental p53 nonproducer and the wild type p53 producer regressor tumors underwent in vivo cell differentiation, manifested as IgG production. Mutant p53, producer cell lines, on the other hand, failed to show any immunoglobulin synthesis and gave rise to highly proliferative lethal tumors. Our results support the conclusion that these pre-B cells develop regressor tumors because they have undergone differentiation. Whereas the wild type p53 facilitates this differentiation, mutant p53 cells block it. We suggest that, in addition to inactivating the growth-suppressive activity of wild type p53, the expression of mutant p53 facilitates malignant transformation.
Animals↗
Nuclear localization is essential for the activity of p53 protein.
p53 appears to be a growth regulator, the perturbation of which induces changes in normal cell proliferation. Wild-type p53 protein is thought to function as a growth arrest gene, whereas mutant p53, which accumulates in transformed cells, has been shown to enhance malignant transformation. Both wild-type and mutant p53 migrate into the cell nucleus by means of identical nuclear localization signals (NLS) inherent in their primary sequences. Results presented here show that the suppressive activity of wild-type p53 measured as the reduction of transformation of primary rat fibroblasts induced by co-transfection with ras and either E1A or mutant p53, as well as the transformation enhancement of mutant p53 estimated by cooperation with ras in transformation of primary rat fibroblasts, is dependent upon nuclear localization signals in p53 protein. While transfection of unmodified wild-type p53 significantly reduces the number of rat embryonic fibroblast-transformed foci induced by E1A and ras or mutant p53 and ras, the wild-type p53 protein without NLS has completely lost this suppressive activity. Partially defective NLS wild-type p53, with a reduced nuclear accumulation ability, still exhibits some suppressive activity. In addition, we found that plasmids coding for intact mutant p53 protein efficiently cooperate with the ras oncogene, whereas the corresponding plasmids without NLS are totally inert. On this basis we conclude that nuclear localization of both wild-type and mutant p53 is a fundamental feature for manifesting the activities of these proteins. Both the suppressor activity mediated by the wild-type p53 and enhancement of transformation mediated by the mutant p53 require nuclear localization of the proteins to function.
Amino Acid Sequence↗
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.
Animals↗
Nuclear accumulation of p53 protein is mediated by several nuclear localization signals and plays a role in tumorigenesis.
The basic carboxy terminus of p53 plays an important role in directing the protein into the nuclear compartment. The C terminus of the p53 molecule contains a cluster of several nuclear localization signals (NLSs) that mediate the migration of the protein into the cell nucleus. NLSI, the most active domain, is highly conserved in genetically diverged species and shares perfect homology with consensus NLS sequences found in other nuclear proteins. The other two NLSs, II and III, appear to be less effective and less conserved. Although nuclear localization is dictated primarily by the NLSs inherent in the primary amino acid sequence, the actual nuclear homing can be modified by interactions with other proteins expressed in the cell. Comparison between wild-type p53 and naturally occurring mutant p53 showed that both protein categories could migrate into the nucleus of rat primary embryonic fibroblasts by essentially similar mechanisms. Nuclear localization of both proteins was totally dependent on the existence of functional NLS domains. In COS cells, however, we found that NLS-deprived wild-type p53 molecules could migrate into the nucleus by complexing with another nuclear protein, simian virus 40 large-T antigen. Wild-type and mutant p53 proteins differentially complexed with viral or cellular proteins, which may significantly affect the ultimate compartmentalization of p53 in the cell; this finding suggests that the actual subcellular compartmentalization of proteins may differ in various cell type milieux and may largely be affected by the ability of these proteins to complex with other proteins expressed in the cell. Experiments designed to test the physiological significance of p53 subcellular localization indicated that nuclear localization of mutant p53 is essential for this protein to enhance the process of malignant transformation of partially transformed cells, suggesting that p53 functions within the cell nucleus.
Amino Acid Sequence↗
p53 increases experimental metastatic capacity of murine carcinoma cells.
Transfection of a cloned p53 gene into a murine bladder carcinoma cell with a low metastatic capacity led to elevated levels of p53 protein in clonal transfectants. After intravenous inoculation into syngeneic mice, p53-transfected clones showed significantly increased metastatic potential in comparison with control transfectants. The observed change did not seem to be due to a change in growth potential per se since the cell lines showed similar growth properties in vitro.
Animals↗