Repression of SHP-1 expression by p53 leads to trkA tyrosine phosphorylation and suppression of breast cancer cell proliferation.

The nerve growth factor (NGF) receptor, trkA, the tumour suppressor p53 and the phosphatase SHP-1 are critical in cell proliferation and differentiation. SHP-1 is a trkA phosphatase that dephosphorylates trkA at tyrosines (Y) 674 and 675. p53 can induce trkA activation and tyrosine phosphorylation in the absence of NGF stimulation. In breast cancer tumours trkA expression is associated with increased patient survival. TrkA protein expression is higher in breast-cancer cell lines than in normal breast epithelia. In cell lines (but not in normal breast epithelia) trkA is functional and can be NGF-stimulated to promote cell proliferation. This study investigates the functional relationship between trkA, p53 and SHP-1 in breast-cancer, and reveals that in wild-type (wt) trkA expressing breast-cancer cells both endogenous wtp53, activated by therapeutic agents, and transfected wtp53 repress expression of SHP-1 through the proximal CCAAT sequence of the SHP-1-P1-promoter and the transcription factor NF-Y. In these cells trkA-Y674/Y675 phosphorylation is detected when SHP-1 protein levels decrease in a wtp53-dependent manner. Proliferation and cell-cycle assays, with cells expressing endogenous or transfected wt-trkA and a temperature-sensitive p53 grown at 32 degrees C (when p53 is in the wt configuration), show suppressed cell proliferation. Suppression is not detected when grown at 37 degrees C (when p53 is in the mutant configuration). A release from suppression is observed when these cells are transiently transfected with wt-SHP-1 and grown at 32 degrees C. Suppression is also detected when, as control, wt-trkA-expressing cells are transiently transfected with SHP-1-siRNA, but not when a dominant-negative (DN) mutant trkA is used to abolish wt-trkA activity. Importantly, suppression is not seen with control trkA-negative breast-cancer cells (expressing wtp53, wt-SHP-1 and undetectable trkA), transfected with Y674F/Y675F mutant-trkA. BrdU-incorporation experiments reveal lack of incorporation in cells expressing wt-trkA and wtp53, or wt-trkA and SHP-1-siRNA. However, BrdU is incorporated in the presence of Y674F/Y675F mutant trkA or DN mutant trkA. These results indicate that p53 repression of SHP-1 expression leads to trkA-Y674/Y675 phosphorylation and trkA-dependent suppression of breast-cancer cell proliferation. These data provide an explanation as to why high trkA levels are associated with favourable prognosis.

Analysis of trk A and p53 association.

trk A tyrosine kinase (the high affinity receptor for nerve growth factor) binds to the p53 tumour suppressor protein in vitro and in vivo. Our aim was to determine which regions of p53 are involved in trk A association. In vitro binding experiments using baculovirus expressed trk A and in vitro transcribed and translated C-terminus p53 deletion mutants show amino acids 327-338 critical for association. Also, analysis with mutants at the N-terminus, conserved regions II, III, IV and V or amino acid positions 173, 175, 181, 248 and 249 (which are amino acids frequently mutated in a variety of neoplasms and transformed cell lines), show that these sites are not involved in trk A binding. Importantly, similar results are obtained after immunoprecipitation of lysates from p53 negative fibroblasts expressing trk A and the above p53 mutant proteins. These data suggest that the amino-terminus of the oligomerisation domain of p53 is involved in p53/trk A association.

c-abl is involved in the association of p53 and trk A.

The p53 tumour suppressor phosphoprotein associates with proteins involved in DNA replication, transcription, cell cycle machinery and regulation of its own expression. Recently it has been shown that p53 can also bind to trk A tyrosine kinase which is the receptor for nerve growth factor (NGF). This study demonstrates that p53 appears to associate with trk A via c-abl. Endogenous c-abl was detected when the trk A and p53 complex was immunoprecipitated from lysates of NGF stimulated NIH3T3 cells expressing trk A or NIH3T3 cells expressing trk A and a temperature sensitive p53 (val 135). Endogenous c-abl and trk A association was observed in NGF stimulated p53 negative fibroblasts transfected with trk A alone; suggesting that c-abl can independently bind to trk A in the absence of p53. Interestingly, association between endogenous p53 and trk A was not detected in NGF stimulated abl negative fibroblasts transfected with trk A or when these cells were exposed to gamma radiation. This result suggests that p53 preferentially binds to trk A in the presence of c-abl and that p53 and trk A do not appear to associate directly even if p53 is activated and its levels increased by gamma radiation. Overall, these data suggest that c-abl is possibly acting as an adaptor or bridge between p53 and trk A. Oncogene (2000).

P53 associates with trk tyrosine kinase.

The p53 tumor suppressor gene encodes a phosphoprotein which when overexpressed can induce growth arrest at the G1 and G2/M phases of the cell cycle, promote differentiation and apoptosis. This paper demonstrates that p53 can associate with trk tyrosine kinase. Expression of a murine temperature-sensitive (ts) p53 mutant in PC12 cells overexpressing trk (a model system to analyse cellular differentiation and signal transduction induced by NGF) induces morphological changes in the absence of NGF stimulation at 32 degrees C but not at 37 degrees C. In cells differentiated by p53, trk, but not EGFr, was hyperphosphorylated on tyrosine. Furthermore trk was not phosphorylated when expressed in Saos-2 cells (human osteosarcoma cells that lack expression of both endogenous trk and p53) at either temperature. However, transfection of ts p53 into these cells induces trk phosphorylation at 32 degrees C in the absence of NGF stimulation. Association of trk and p53 can be detected in NIH3T3 and PC12 cells co-expressing trk and the ts p53 mutant, in NIH3T3 and PC12 cells transfected with trk alone, and in untransfected PC12 cells, showing that overexpressed and/or endogenous trk associates with endogenous, low levels of p53. These data suggest a novel function for p53 which involves the stimulation of signal transduction pathways (mediating morphological properties of cells), possibly through association with and hyperphosphorylation of trk.

Analysis of intron 4 of the p53 gene in human cutaneous melanoma.

DNA sequencing of intron 4 of the p53 gene from seven cutaneous melanoma cell lines showed an absence of mutations. However, both control and melanoma cell lines sequences were different from the reference source obtained from GenBank databank (accession No. X54156). Base pairs 101 and 689 were determined to be T (instead of A) and C (instead of G). Also, an additional C was not detected at position 371. Comparative analysis with p53 DNA-binding sequences, a sequence recognized by a p53 intron 4-binding protein and consensus sequences recognized by transcription factors demonstrated that intron 4 contains putative sequences for NF-kappa B, SP1, AP1 and TFIID binding. Binding of transcription factors could be one of the mechanisms by which intron 4 modulates human p53 expression.

Activation of c-Jun-NH2-kinase by UV irradiation is dependent on p21ras.

We have demonstrated previously that Jun-NH2-kinase (JNK) activation in vitro is potentiated by association with the p21(ras) protein. To determine if in vivo activation of JNK also depends on p21(ras), we have used M1311 cells that carry the cDNA for the neutralizing antibody to p21(ras), Y13-259, under a dexamethasone-inducible promoter. The ability of UV to activate JNK gradually decreased over a 4-day period of cell growth in dexamethasone. This decrease coincides with weaker transcriptional activation measured via gel shift and chloramphenicol acetyltransferase assays. Peptides corresponding to amino acids 96-110 on p21(ras), which were shown to block Ras-JNK association, inhibited UV-mediated JNK activation in mouse fibroblast 3T3-4A cells as well as in M1311 cells, further supporting the role of p21(ras) in UV-mediated JNK activation. Overall, the present studies provide in vivo confirmation of the role p21(ras) plays in JNK activation by UV irradiation.

Intracellular expression of the monoclonal anti-ras antibody Y13-259 blocks the transforming activity of ras oncogenes.

Microinjection of the anti-ras antibody Mab Y13-259 modifies ras function and can induce temporary reversion of the transformed phenotype in mutant ras-transformed cells. Intracellular production of neutralizing antibodies represents an approach to investigate the regulation of gene function. The genes coding for the heavy and light chains of Mab Y13-259 were isolated from a cDNA library. NIH3T3 cells transfected with heavy and light chain expression vectors produced functional anti-ras antibody. The production of functional antibody did not require glycosylation. To ensure that the antibody entered the cytoplasm and not the secretory pathway, the hydrophobic leader sequences of both chains were removed and replaced with synthetic initiator sequences. The modified heavy chain gene was cloned under the control of the murine sarcoma virus long terminal repeat, and the light chain gene under the control of the mouse mammary tumor virus long terminal repeat, which allows the induction of light chain expression in the presence of dexamethasone. When both heavy and light chain genes were expressed in cells with activated ras (morphologically transformed) in the presence of dexamethasone, we observed phenotypic reversion to characteristics of nontransformed cells. These experiments show that intracellular expression of antibodies can also be used as an alternative to analyze biological functions of a given protein.

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.

Simian virus 40 small tumor antigen and an amino-terminal domain of large tumor antigen share a common transforming function.

The 82-residue amino-terminal sequences of simian virus 40 large tumor antigen (TAg) and small tumor antigen (tAg) are identical. Genetic analysis of TAg lacking amino acids 1-82 revealed that it was transformation-defective, as revealed by the agar growth assay, except when introduced in the presence of tAg. Since the latter, alone, lacks overt transforming activity, it would appear that the function of the sequence common to TAg and tAg is necessary, but not sufficient, for TAg transforming activity and that tAg can provide that function or its equivalent in trans. Thus, tAg may, in part, be viewed as a "portable" copy of a TAg functional domain.

Monoclonal antibody analysis of simian virus 40 small t-antigen expression in infected and transformed cells.

The monoclonal antibody PAb280 binds to small t antigen but not to large T antigen. Its binding site within the unique region of small t antigen was localized by studying its reaction with simian virus 40 mutants, other papovaviruses, and bacterial expression vectors coding for fragments of small t antigen. The antibody was used to define the cellular location of small t antigen by immunocytochemistry and by immunoprecipitation of subcellular extracts of infected cells. PAb280 reacts strongly with a cytoplasmic form of small t antigen that appears to be associated with the cytoskeleton and is not detected by antibodies directed to the common N terminus of small t and large T antigens. Immunoperoxidase staining of cells infected by the simian virus 40 defective strain SV402 with PAb280 and other anti-T antibodies demonstrated that this virus produced an N-terminal fragment of large T antigen as well as small t antigen. In cells infected by the virus, this fragment was located in the cell nucleus but was very unstable. These results suggest that the activity of the SV402 virus in transformation assays may not be entirely due to the action of small t antigen alone.

The adenovirus Ela gene induces differentiation of F9 teratocarcinoma cells.

Undifferentiated F9 cells transfected with plasmids encoding adenovirus E1a gene products underwent radical morphological changes. They ceased to express the SSEA-1 stem cell marker antigen and started to express a number of the characteristics of the differentiated state that is induced in F9 cells by treatment with retinoic acid. In particular, they expressed keratin intermediate filaments and acquired the ability to synthesise simian virus 40 tumor antigens after virus infection. The transfected cells expressed the E1a proteins, and this expression was necessary to induce the phenotypic changes, since a coisogenic plasmid encoding only a truncated 70-amino-acid E1a polypeptide and the transfection procedure itself did not detectably after the morphology or marker expression of the F9 stem cells. The phenotypic change was induced by both 13S and 12S cDNA plasmids. We discuss these results in the context of known E1a functions and with reference to the other oncogenes and external factors that can cause F9 cell differentiation.

SV40 small t antigen enhances the transformation activity of limiting concentrations of SV40 large T antigen.

A murine recombinant Neo(r) retrovirus encoding the SV40 small t antigen was used to infect Balb/c 3T3 CIA31 cells. From analyses of G418-resistant clones containing at least as much intact t as Cos-1 cells, we found that t, alone, had no detectable A31 transforming activity. In contrast, we noted that SV40 large T promoted A31 agar colony formation when present over a 5- to 7.5-fold concentration range. However, at the low end of the spectrum, its transforming effect was manifest inefficiently except in the presence of t. Thus a major role for t in the SV40 transforming mechanism is to enhance directly or indirectly the transforming function of T.

Monoclonal antibody to simian virus 40 small t.

A monoclonal antibody, PAb280, was produced that recognizes simian virus 40 (SV40) small t but does not react with SV40 large T. The specificity of the antibody was analyzed by immunoprecipitation of labeled cell extracts, Western blotting, and immunocytochemistry. Small t was found to accumulate late in the SV40 lytic cycle and was localized in both the cytoplasm and the nucleus of cells infected with wild-type SV40. Importantly, antibodies against determinants common to SV40 large T and small t did not appear to be able to recognize the cytoplasmic form of SV40 small t at the immunocytochemical level. The localization of small t within the nucleus appeared to be distinct from that of large T.

Localizacion en los trofozoitos de Entamoeba histolytica de una lipopeptidofosfoglicana extraida por fenol-agua de la cepa HK-9. / Identification in the trophozoites of Entamoeba histolytica of a lipopeptidophosphoglycan extracted by phenol-water from the HK-9 strain

Una lipopeptidofosfoglicana (LPFG) de trofozoitos de la cepa HK-9 de Entamoeba histolytica fue extraida por el metodo de fenol-agua Los anticuerpos contra esta LPFG fueron producidos inmunizando a conejos con la molecula y el titulo de los mismos fueron medidos por hemolisis pasiva. Posteriormente, los anticuerpos fueron purificados precipitando el suero con sulfato de amonio y pasandolos a traves de una columna de DEAE-celulosa. Los anticuerpos IgG anti-LPFG se utilizaron como primer anticuerpo para que se fijaran a las moleculas de lipopeptidofosfoglicana presentes en los trofozoitos de E. histolytica; posteriormente se agrego un suero de cabra anti-IgG de conejo marcando con fluoresceina para hacer visible el sitio de reaccion del primer anticuerpo. Los resultados de inmunofluorescencia indirecta demostraron que la LPFG esta presente en la superficie de los trofozoitos