Hsp 27 is better associated with the expression of inducible thermotolerance in human pancreatic tumor cell lines than hsp 70, p53 or p21/waf1/cip1

We evaluated the levels of p53, p21/waf1/cip1, hsp 27 and hsp 70 proteins in confluent cultures of human pancreatic tumor cell lines during the expression of inducible thermotolerance (IT). Two of these cell lines have wild type, whereas the other two have mutant, p53 status. Three cell lines expressed a significant amount of IT (p<0.05 and better, one-tailed Student's t-test); the fourth manifested little IT. Hsp 70 was upregulated in all cell lines. In contrast, hsp 27 was upregulated only in the cell lines that expressed IT. There was no correlation between either p53, or p21, protein levels and propensity to express IT. We conclude that hsp 27 is better associated with the expression of IT than hsp 70 in these cell lines.     

Complex formation between the lymphotropic papovavirus large tumor antigen and the tumor suppressor protein p53

The simian B-lymphotropic papovavirus (LPV) encodes a large tumor antigen (T antigen) which is 45% identical to both the simian virus 40 (SV40) and the polyomavirus (PyV) large T antigens. In transgenic mice, the transforming properties of the LPV T antigen are similar to those of the SV40 T antigen. However, little is known about its biochemical activities. Since SV40 T antigen forms a complex with and stabilizes the host cell tumor suppressor protein p53 while the PyV large T antigen does not, we characterized the LPV T antigen for its ability to complex p53. We demonstrate an association between LPV T antigen and p53 in both a tumor-derived cell line and BALB/c 3T3 cells transformed in culture. A third protein of approximately 68 kDa which was found associated with the LPV T antigen-p53 complex in tumor-derived cells appears to be heat shock protein 70 (hsp70). The half-life of p53 in all LPV T-antigen-transformed cells was extended significantly; i.e., it was 3 to 7 h compared with 19 minutes in BALB/c 3T3 cells. The half-life of the LPV T antigen itself was 5 to 9 h depending on the cell line origin. That p53 was stabilized because of association with LPV T antigen and not because of mutation was demonstrated with the p53 conformation-dependent monoclonal antibody PAb246. This antibody distinguishes between wild-type p53 (PAb246+) and mutant, oncogenic p53 (PAb246-). Sequential immunoprecipitation showed all detectable p53 to be of the PAb246+ class in each LPV-transformed cell line, suggesting that the stable p53 was indeed wild type.     

hsp70 binds specifically to a peptide derived from the highly conserved domain (I) region of p53.

Products of a number of mutant p53 genes bind with high affinity to members of the hsp70 family of chaperonin proteins, whereas wild type p53 lacks this type of association. Examination of the sequences of p53 genes from five different species enabled us to predict domains on p53 which may be involved in the association with hsp70 family members. A synthetic polypeptide (Pro-17-Gly) corresponding to the candidate hsp70 binding domain bound to in vitro translated hsp70 as determined by affinity chromatography and nondenaturing gel mobility shift assays. In addition, the Pro-17-Gly peptide competitively inhibited association between hsp70 and p53, an activity which was determined by immunoprecipitation with anti-p53 monoclonal antibody PAb240. The data indicate that p53 contains a hsp70 binding domain, which is located in a highly conserved region at the amino terminus of the protein, and may participate in the cellular function of wild-type p53 or in the transforming capacity of p53 mutants.     

Characterization of proteins associated with heat shock protein hsp27 in the squamous cell carcinoma cell line A431.

Heat shock protein hsp27 is a molecular chaperone and identification of hsp27-binding proteins might help to elucidate its functional role in keratinocyte biology. In the present investigation we used a human epidermal cell carcinoma cell line (A431) transfected with hsp27 (A431/16) to study interference between hsp27 protein and other proteins. Immunoprecipitation experiments with anti-hsp27 antibody revealed a multicomponent complex when analysed by silver staining. By immunoblotting analysis we could demonstrate that hsp27 associates with actin, the mutant form of p53, hsp70 and hsp90. Immunofluorescence analysis showed a co-localization between hsp27 and p53, hsp70 and hsp90. To control for the specificity of the observed interactions, immuno-precipitations with antibodies to actin, p53, hsp70 and hsp90 respectively, were performed. All of the tested proteins demonstrated a coimmunoprecipitation with hsp27. We conclude that hsp27, like the other heat shock proteins, is part of a complex system of molecular chaperones in epidermal keratinocytes.     

Characterization of mutant p53-hsp72/73 protein-protein complexes by transient expression in monkey COS cells.

Several mutant, but not wild-type, p53 proteins form complexes with hsp72/73 heat shock-related proteins in simian virus 40-transformed monkey COS cells. We carried out a detailed biochemical and structural mapping analysis of p53 and report here that p53-hsp72/73 complex formation showed considerable structural specificity. Such complexes were remarkably stable, but unlike analogous complexes formed between p53 and simian virus 40 T antigen, they did not form in in vitro association assays. p53-hsp72/73 complex formation in vivo appears to be dependent on aspects of mutant p53 protein conformation. However, absence of the conformation-sensitive epitope recognized by monoclonal antibody PAb 246 was not reliably diagnostic of such complexes, nor was p53-hsp72173 binding reliably diagnostic of oncogenic activation.     

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.     

Resistance of senescent keratinocytes to UV-induced apoptosis.

Irradiation with ultraviolet (UV) triggers programmed cell death (apoptosis) in keratinocytes. This process is believed to protect against skin carcinogenesis since the cells with damaged DNA are selectively removed, limiting the likelihood of the development of a malignant keratinocyte clone. The p53 protein is able to detect mutation-bearing DNA fragments and is thus indispensable for the UV-induced apoptosis in the epidermis. Since age is a risk factor for the development of skin tumors we investigated whether ultraviolet induces apoptosis and p53 activation in senescent keratinocytes. Cultured senescent keratinocytes were irradiated with broad-band ultraviolet, apoptosis was assessed using TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling) technique and the p53 activation pattern was determined with Western blotting and immunofluorescent staining with a panel of anti-p53 antibodies recognising different conformational forms of the protein (PAb 122, PAb 240, DO-7). In senescent keratinocytes arrested in the G1 phase of cell cycle, ultraviolet irradiation (100-2000 J/m2) caused accumulation and nuclear translocation of p53. However, in contrast to young cells where UV induces apoptotic cell death in G1, apoptosis was not detected in senescent cells. There were subtle differences in the p53 activation pattern between senescent keratinocytes and known patterns in young keratinocytes and other cell types. In senescent keratinocytes a constitutional nuclear expression of p53 (conformational form recognized by PAb 240) was present and the p53 induction in response to ultraviolet radiation was rapid. Suppression of apoptosis in senescent keratinocytes may be an important mechanism responsible for enhanced skin carcinogenesis in old age.     

Repression of hsp90beta gene by p53 in UV irradiation-induced apoptosis of Jurkat cells

Tumor suppressor p53 has been implicated in cell stress response and determines cell fate of either growth arrest or apoptosis. Heat shock proteins (Hsps) expressed under stress usually confer survival protection to the cell or interruption in the apoptotic pathways. Although Hsp90 can physically interact with p53, whether or not the hsp90 gene is influenced downstream of p53 in UV irradiation-induced apoptosis remains unclear. We have found that the level of p53 is elevated with the decline of Hsp90 in UV-irradiated cells and that malfunction of Hsp90, as inhibited by geldanamycin, enhances the p53-involved UV irradiation-induced apoptosis. In addition, the expression of the hsp90beta gene was reduced in both UV-irradiated and wild type p53-transfected cells. These results suggest a negative correlation between the trans factor p53 and a chaperone gene hsp90beta in apoptotic cells. Mutation analysis demonstrated that the p53 binding site in the first exon was indispensable for p53 regulation on the hsp90beta gene. In addition, with p53 bound at the promoter of the hsp90beta gene, mSin3a and p300 were differentially recruited in UV irradiation-treated or untreated Jurkat cells in vivo. The evidence of p53-repressed hsp90beta gene expression in UV-irradiated cells shed light on a novel pathway of Hsp90 in the survival control of the stressed cells.     

Analysis of the most representative tumour-derived p53 mutants reveals that changes in protein conformation are not correlated with loss of transactivation or inhibition of cell proliferation.

In an effort to correlate the biological activity of the p53 protein with its conformation, we analysed 14 p53 mutants representative of the most frequently observed protein alterations in human cancers, at codons 175, 248 and 273 (22% of all mutations thus far reported), all three of which contained a CpG dinucleotide. Strikingly, most of the mutants at codons 248 and 273 did not display any change in their conformation, as probed by monoclonal antibodies PAb240 and PAb1620 or by binding to hsp70 protein. For all 14 mutants tested, we found a strict correlation between the transactivation properties of p53, tested either on RGC sequences or using the WAF-1 promoter, and inhibition of cell proliferation. All these mutants showed nuclear localization. Several mutants, present at a low incidence in human tumours, displayed wild-type activity in all our assays, suggesting that the presence of a mutation is not strictly correlated with p53 protein inactivation in tumours. Further analysis of nine thus far undescribed p53 mutants at codon 175 revealed a wild-type or mutant behaviour. All these results suggest that the occurrence of a mutation is dependent on two criteria: (i) the mutability of a given codon, such as those containing a CpG dinucleotide; (ii) the resulting amino acids, eventually leading to synthesis of a p53 conferring a growth advantage on the cell.     

Inhibition of MDM2 by hsp90 contributes to mutant p53 stabilization

Stabilization and overexpression are hallmarks of mutant p53 found in nearly 50% of human tumors. Mutations in the conformation-sensitive core domain of p53 often lead to association with molecular chaperones such as hsp70 and hsp90. Inhibition of hsp90 function accelerates mutant p53 degradation. We recently found that expression of p53 core domain mutants inhibits MDM2 degradation, suggesting that mutant p53 can modulate MDM2 functions. In this report, we show that mutant p53 mediates formation of MDM2-p53-hsp90 complexes. Release of MDM2 from the p53-hsp90 complex after DNA damage restores MDM2 but not p53 turnover, whereas dissociation of hsp90 by geldanamycin increases the degradation of both MDM2 and mutant p53. Mutant p53 degradation after hsp90 inhibition requires MDM2 expression. The interaction between MDM2 and hsp90 is disrupted by the 2A10 antibody, which recognizes a site on MDM2 important for binding to alternative reading frame (ARF). Expression of mutant p53 prevents MDM2 from binding ARF and accumulating in the nucleolus in an hsp90-dependent fashion. These results suggest that hsp90 recruited by mutant p53 conceals the ARF-binding site on MDM2 and inhibits its ubiquitin-protein isopeptide ligase function, resulting in the stabilization of both mutant p53 and MDM2.     

Biochemical characterization of different conformational states of the Sf9 cell-purified p53His175 mutant protein

In this study, we expressed and purified the p53 mutant encoded by the His175 allele (p53His175) in a baculovirus expression system in order to study the folding and the DNA binding activity of the protein. A two-site ELISA revealed that purified p53His175 protein preferentially displayed a PAb1620 conformation, which appeared to be not sufficient to interact specifically with DNA. The cryptic DNA binding activity of this mutant was then investigated by electrophoretic mobility shift assay in the presence of anti-p53 antibodies, and shown to be refractory to significant activation by PAb421 (a potent allosteric activator of wild-type p53's DNA binding activity). Nevertheless, p53His175 DNA binding was regulated by antibodies targeting the N-terminal region of the protein. Furthermore, while the protein preferentially displayed a PAb1620 conformation, our data suggested the existence of an equilibrium between at least two folding states of the protein (PAb1620 and PAb240 conformations). A model rationalizing the conformation, antibody-interacting ability and DNA binding regulation potential of p53His175 is presented.     

Activating mutations for transformation by p53 produce a gene product that forms an hsc70-p53 complex with an altered half-life.

The 11-4 p53 cDNA clone failed to transform primary rat fibroblasts when cotransfected with the ras oncogene. Two linker insertion mutations at amino acid 158 or 215 (of 390 amino acids) activated this p53 cDNA for transformation with ras. These mutant cDNAs produced a p53 protein that lacked an epitope, recognized by monoclonal antibody PAb246 (localized at amino acids 88 to 110 in the protein) and preferentially bound to a heat shock protein, hsc70. In rat cells transformed by a genomic p53 clone plus ras, two populations of p53 proteins were detected, PAb246+ and PAb246-, which did or did not bind to this monoclonal antibody, respectively. The PAb246- p53 preferentially associated with hsc70, and this protein had a half-life 4- to 20-fold longer than free p53 (PAb246+). These data suggest a possible functional role for hsc70 in the transformation process. cDNAs for p53 derived from methylcholanthrene-transformed cells transform rat cells in cooperation with the ras oncogene and produce a protein that bound with the heat shock proteins. Recombinant clones produced between a Meth A cDNA and 11-4 were tested for the ability to transform rat cells. A single amino acid substitution at residue 132 was sufficient to activate the 11-4 p53 cDNA for transformation. These studies have identified a region between amino acids 132 and 215 in the p53 protein which, when mutated, can activate the p53 cDNA. These results also call into question what the correct p53 wild-type sequence is and whether a wild-type p53 gene can transform cells in culture.     

The chaperone function of hsp70 is required for protection against stress-induced apoptosis

Cellular stress can trigger a process of self-destruction known as apoptosis. Cells can also respond to stress by adaptive changes that increase their ability to tolerate normally lethal conditions. Expression of the major heat-inducible protein hsp70 protects cells from heat-induced apoptosis. hsp70 has been reported to act in some situations upstream or downstream of caspase activation, and its protective effects have been said to be either dependent on or independent of its ability to inhibit JNK activation. Purified hsp70 has been shown to block procaspase processing in vitro but is unable to inhibit the activity of active caspase 3. Since some aspects of hsp70 function can occur in the absence of its chaperone activity, we examined whether hsp70 lacking its ATPase domain or the C-terminal EEVD sequence that is essential for peptide binding was required for the prevention of apoptosis. We generated stable cell lines with tetracycline-regulated expression of hsp70, hsc70, and chaperone-defective hsp70 mutants lacking the ATPase domain or the C-terminal EEVD sequence or containing AAAA in place of EEVD. Overexpression of hsp70 or hsc70 protected cells from heat shock-induced cell death by preventing the processing of procaspases 9 and 3. This required the chaperone function of hsp70 since hsp70 mutant proteins did not prevent procaspase processing or provide protection from apoptosis. JNK activation was inhibited by both hsp70 and hsc70 and by each of the hsp70 domain mutant proteins. The chaperoning activity of hsp70 is therefore not required for inhibition of JNK activation, and JNK inhibition was not sufficient for the prevention of apoptosis. Release of cytochrome c from mitochondria was inhibited in cells expressing full-length hsp70 but not in cells expressing the protein with ATPase deleted. Together with the recently identified ability of hsp70 to inhibit cytochrome c-mediated procaspase 9 processing in vitro, these data demonstrate that hsp70 can affect the apoptotic pathway at the levels of both cytochrome c release and initiator caspase activation and that the chaperone function of hsp70 is required for these effects.     

Monoclonal antibody analysis of p53 expression in normal and transformed cells.

The cellular phosphoprotein p53 binds tightly and specifically to simian virus 40 T antigen and the 58,000-molecular-weight adenovirus E1b protein. Many human and murine tumor cell lines contain elevated levels of the p53 protein even in the absence of these associated viral proteins. Recently the cloned p53 gene, linked to strong viral promoters, has been shown to complement activated ras genes in transformation of primary rodent cell cultures. Overexpression of the p53 gene alone rescues some primary rodent cell cultures from senescence. We isolated three new monoclonal antibodies to the p53 protein, designated PAb242, PAb246, and PAb248, and mapped the epitopes they recognized on p53 in comparison with other previously isolated antibodies. At least five sterically separate epitopes were defined on murine p53. One of the antibodies, PAb246, recognizes an epitope on p53 that is unstable in the absence of bound simian virus 40 T antigen. This effect is demonstrable in vivo and in newly developed in vitro assays of T-p53 complex formation. Using the panel of anti-p53 antibodies and sensitive immunocytochemical methods, we found that p53 has a predominantly nuclear location in established but not transformed cells as well as in the vast majority of transformed cell lines. Several monoclonal antibodies to p53 showed cross-reactions with non-p53 components in immunocytochemical staining.     

RTN4-C基因在肝癌组织中的表达及其对SMMC7721细胞生长和凋亡的影响

RTN4-C属于编码内质网蛋白的基因家族(RTNs)。为研究RTN4-C在肝癌及其癌旁组织中的表达情况及其对SMMC7721肝癌细胞株的影响,利用RT-PCR检测RTN4-C在肝癌及其癌旁组织中的表达。构建RTN4-C-pcDNA3.1真核表达重组质粒,用脂质体介导转染SMMC7721肝癌细胞,通过G418稳定筛选,建立转染RTN4-C/SMMC7721稳定细胞株。MTT法测定转染前后细胞生长改变、吖啶橙染色检测细胞凋亡改变、免疫细胞化学检测肿瘤相关蛋白p53、Hsp70和c-Fos表达改变。结果表明,RTN4-C/SMMC7721细胞生长减慢,与对照组相比,第2、3、4d细胞生长抑制率分别为33·8%±0·026、56·2%±0·094、34·8%±0·077;凋亡明显增多。突变型p53蛋白从核内转移到细胞质且表达减弱,c-Fos、Hsp70蛋白表达量明显减弱。提示RTN4-C基因在肝癌组织中存在表达差异,促进突变型p53的核转移并减少其表达量、抑制癌基因c-Fos和Hsp70的表达,从而抑制SMMC7721细胞的生长,促进其凋亡。     

Activating mutations in p53 produce a common conformational effect. A monoclonal antibody specific for the mutant form.

Point mutations in the p53 gene are the most frequently identified genetic change in human cancer. They convert murine p53 from a tumour suppressor gene into a dominant transforming oncogene able to immortalize primary cells and bring about full transformation in combination with an activated ras gene. In both the human and murine systems the mutations lie in regions of p53 conserved from man to Xenopus. We have developed a monoclonal antibody to p53 designated PAb240 which does not immunoprecipitate wild type p53. A series of different p53 mutants all react more strongly with PAb240 than with PAb246. The PAb240 reactive form of p53 cannot bind to SV40 large T antigen but does bind to HSP70. In contrast, the PAb246 form binds to T antigen but not to HSP70. PAb240 recognizes all forms of p53 when they are denatured. It reacts with all mammalian p53 and chicken p53 in immunoblots. We propose that immunoprecipitation of p53 by PAb240 is diagnostic of mutation in both murine and human systems and suggest that the different point mutations which convert p53 from a recessive to a dominant oncogene exert a common conformational effect on the protein. This conformational change abolishes T antigen binding and promotes self-oligomerization. These results are consistent with a dominant negative model where mutant p53 protein binds to and neutralizes the activity of p53 in the wild type conformation.     

Interaction of heat-shock protein 70 with p53 translated in vitro: evidence for interaction with dimeric p53 and for a role in the regulation of p53 conformation.

In intact cells, hsp70 proteins selectively complex with mutant p53. We report here that rabbit reticulocyte lysate contains hsp70 which selectively complexes with the mutant p53 translated in vitro. Hsp70 complexes with dimers and possibly monomers of p53 in a manner that requires the terminal 28 amino acids of p53. Using murine p53Val135, which is temperature-sensitive for phenotype, we demonstrate that p53-hsp70 complexes can occur after post-translational switching from wild-type to mutant p53 phenotype. Moreover, the temperature-induced switch of full-length p53Val135 from wild-type to mutant phenotype is ATP-independent, whereas the switch from mutant to wild-type form requires ATP hydrolysis and involves hsp70. These results imply that hsp70 is involved in the regulation of p53 conformation.     

Conformational and molecular basis for induction of apoptosis by a p53 C-terminal peptide in human cancer cells

A p53-derived C-terminal peptide induced rapid apoptosis in breast cancer cell lines carrying endogenous p53 mutations or overexpressed wild-type (wt) p53 but was not toxic to nonmalignant human cell lines containing wt p53. Apoptosis occurred through a Fas/APO-1 signaling pathway involving increased extracellular levels of Fas/FasL in the absence of protein synthesis, as well as activation of a Fas/APO-1-specific protease, FLICE. The peptide activity was p53-dependent, and it had no effect in three tumor cell lines with null p53. Furthermore, the C-terminal peptide bound to p53 protein in cell extracts. Thus, p53-dependent, Fas/APO-1 mediated apoptosis can be induced in breast cancer cells with mutant p53 similar to the recently described Fas/APO-1 induced apoptosis by wt p53. However, mutant p53 without p53 peptide does not induce a Fas/APO-1 activation or apoptosis. Docking of the computed low energy conformations for the C-terminal peptide with those for a recently defined proline-rich regulatory region from the N-terminal domain of p53 suggests a unique low energy complex between the two peptide domains. The selective and rapid induction of apoptosis in cancer cells carrying p53 abnormalities may lead to a novel therapeutic modality.     

DNA damage in human B cells can induce apoptosis, proceeding from G1/S when p53 is transactivation competent and G2/M when it is transactivation defective.

Cisplatin treatment of Epstein-Barr virus-immortalized human B lymphoblastoid cell lines (LCLs) results in p53-mediated apoptosis which occurs largely in a population of cells at the G1/S boundary of the cell cycle. Cell cycle progression appears to be required for this apoptosis because arresting cells earlier in G1 inhibited apoptosis despite the accumulation of p53. Overexpression of wild-type p53 also induces apoptosis in an LCL. Therefore six mutant genes derived from Burkitt's lymphoma (BL) cells were assayed for their ability to induce apoptosis when similarly overexpressed. The same genes were analysed in transient transfection assays for their ability to transactivate appropriate reporter plasmids. A correlation between the ability of p53 to transactivate and induce apoptosis was revealed. The only mutant capable of transactivation also induced apoptosis. Further analysis of the BL lines in which p53 had been characterized showed that whereas some lines were essentially resistant to cisplatin, three were rapidly induced to undergo apoptosis. All three have a single p53 allele encoding a mutant which is incapable of transactivation or (for two tested) mediating apoptosis when expressed in an LCL. Cell cycle analysis revealed that this apparently p53-independent apoptosis did not follow G1 arrest but in fact occurred largely in cells distributed in the G2/M phase of the cell cycle. These data suggest the existence of a second checkpoint in the G2 or M phase which, in the absence of a functional p53, is the primary point of entry into the apoptosis programme following DNA damage.