Functional studies of the canonical Bone Morphogenetic Protein (BMP) signalling pathway in human epidermal keratinocytes have been limited to the immortalized and p53-mutated HaCaT cells and are primarily dependent on BMP6 treatment in mouse epidermal keratinocytes. Despite these insightful analyses, the molecular mechanism underlying the role of BMP signalling in the precise balance between growth arrest and terminal differentiation of keratinocytes still remains not clearly defined. The current study first investigated the hitherto uncharacterized status and functions of BMP signalling in normal human keratinocytes by using three independent strains of primary interfollicular epidermal keratinocytes. Then we provided data demonstrating the role of BMP2 compared to BMP6 in the inhibition of growth and induction of subsequent terminal differentiation of these cells. A second relevant finding is based on the clonal analysis of colony types present in untreated and BMP2/6-treated cultures in absence of EGF. BMP treatment results in the clonal transition from proliferative to abortive colonies, suggesting that BMP signalling most likely inhibits stem cell proliferation and triggers cell cycle exit from transit amplifying cells. Third, we showed evidence that, of the three members of the Cip/Kip family of cyclin-dependent kinase inhibitors, only p57Kip2 and p21Cip1 have a BMP2/6-induced expression. One mechanism of inhibition of cell proliferation involves p57Kip2 as an immediate early response, in contradistinction with p21Cip1 which largely depends on de novo protein synthesis for its effect to proceed. All together, these results clarify the BMP signalling status in normal primary human keratinocytes and support a new mechanism of inhibition of the proliferation of interfollicular epidermal keratinocytes coupled with induction of their terminal differentiation following BMP2 or BMP6 addition.

As modern day society takes an increasing interest in outdoor activities, its exposure to sunlight has never been greater. As a consequence, countries throughout the world are experiencing a dramatic increase in the incidences of skin carcinomas and melanomas. From DNA photolesions to mutations, skin cancer and cell death provides an authoritative source of information for photobiologists interested in the series of genetic events that occur in the skin, and eventually lead to cancer. With contributions from eminent scientists in the field, this book includes the latest information on DNA photolesions and repair, as well as the key mechanisms of solar UV in skin cancer initiation and development. Significant information relating to UV-induced photolesions and mechanisms of skin tumour occurrence is also included. By providing the basic phenomena underlying the science and an overview of the biological events that take place when cells are exposed to solar UV radiation, From DNA photolesions to mutations, skin cancer and cell death is suitable to all researchers interested in the process of photocarcinogenesis.

The UVB component of the solar spectrum induces DNA lesions that, in the absence of error-free DNA repair, may give rise during DNA replication to mutations in caretaker and gatekeeper genes. The DNA repair genes are the best candidates for caretaker genes as exemplified by the human hereditary xeroderma pigmentosum (XP) syndrome. Cultured XP cells are hypermutable after UVB irradiation. This increased mutation frequency is also found in gatekeeper genes, which govern signalling pathways implicated in the control of cellular proliferation, differentiation and survival of human epidermal keratinocytes. We describe and discuss the role of mutated gatekeeper genes in five specific signalling pathways which have been implicated in skin carcinogenesis. The pathways we focus on in this review are: (i) P16(INK4A)-CDK4/6-RB; (ii) P14(ARF)-HDM2-P53; (iii) Sonic hedgehog (SHH)/GLI; (iv) WNT/beta-catenin; and (v) Bone Morphogenetic Protein (BMP)/SMAD. 70-80% of XP skin cancers exhibit one or several mutations in the P53, PTCH-1, SMO or CDKN2A genes, the type and frequency of mutated genes being different between squamous cell (SCCs) and basal cell carcinomas (BCCs). In XP cancers, the typically UVB-induced CC to TT tandem transitions represent approximately 60% of total mutations compared to 10-15% in skin tumours from DNA repair-proficient patients. Acquired activation of the pathways described herein can alter proliferation and differentiation of keratinocytes, allowing a damaged cell to replicate and give rise to mutated daughter cells, then eventually to the development of the carcinogenic process following clonal selection.

Trichothiodistrophy (TTD), xeroderma pigmentosum (XP), and Cockayne's syndrome (CS) are three distinct human diseases with sensitivity to ultraviolet (UV) radiation affected by mutations in genes involved in nucleotide excision repair (NER). Among the many responses of human cells to UV irradiation, both nuclear accumulation of p53, a tumor suppressor protein, and alterations in cell-cycle checkpoints play crucial roles. The purpose of this study was to define the signals transmitted after UV-C-induced DNA damage, which activates p53 accumulation in TTD/XP-D fibroblasts, and compare this with XP-D cell lines that carry different mutations in the same gene, XPD. Our results showed that p53 was rapidly induced in the nuclei of TTD/XP-D and XP-D fibroblasts in a dose-dependent manner after UV-C irradiation, as seen in XP-A and CS-A fibroblasts, much lower doses being required for the protein accumulation than in normal human fibroblasts, XP variant cells, and XP-C cells. The kinetics of accumulation of p53 and two effector proteins involved in cell-cycle arrest, WAF1 and GADD45, were also directly related to the repair potential of the cells, as in normal human fibroblasts their levels declined after 24 h, the time required for repair of UV-induced lesions, whereas NER-deficient TTD/XP-D cells showed p53, WAF1, and GADD45 accumulation for over 72 h after irradiation. Our results indicate that p53 accumulation followed by transcriptional activation of genes implicated in growth arrest is triggered in TTD/XP-D cells by the persistence of cyclobutane pyrimidine dimers, which are known to block transcription, on the transcribed strands of active genes.

In this study, rat embryo lung organ cultures were exposed to benzo[a]pyrene (B[a]P). After carcinogen-treatment the cells were dissociated and an epithelial cell line (BP) was developed from the primary cell culture derived from the carcinogen-treated explants. Investigations were performed on the sequential changes occurring in the course of neoplastic progression of BP cells and in the tumor cells that arose in vivo from implanted BP cells. During the neoplastic progression a mutation was shown to occur in p53 gene at codon 130 (AAG > AGG; Lys > Arg) in a single cell which expanded and gave rise to a predominant subpopulation. This mutational event was already detected at passage 14 but was probably not a direct consequence of a specific alteration caused by the carcinogen in the target cell. This mutation was retained through the subsequent progressional steps first as a heterozygous mutation, then converted to a homozygous state. From passage 18 on, it was possible in BP cell cultures to detect foci of larger morphologically distinct cells emerging on a background of cells maintaining the original morphology. These foci were shown to derive from a single cell carrying the p53 mutation in a homozygous state. During the neoplastic progression the mutant p53 allele frequency steadily increased and this mutant allele eventually came to predominate completely in the late stages of the neoplastic progression, including in the transplantation-induced tumors. The pattern of a directional selection for mutant p53 gene towards fixation is probably applicable to a wide range of human malignancies and may reflect the particular importance of this gene for tumorigenesis.

The nuclear phosphoprotein p53 is an important regulator of cell proliferation in normal cells. Interestingly, the gene encoding p53 has usually undergone mutations in a wide range of tumor types. Recent studies of the p53 gene in Burkitt's lymphomas have demonstrated that mutations are extremely common, and in fact it is rare that both alleles of the p53 gene in these tumors are not inactivated by mutation or deletion. We present here genetic data regarding the status of the p53 gene in the Burkitt lymphoma cell line, Raji. As is typical for this type of tumor, both alleles have undergone point mutations. Further, statistical analysis of available data from a large number of Burkitt's lymphomas indicates an apparent tumor-specific distribution of p53 mutations. The possibility that specific mutations of the p53 gene may be important for different tumor types is discussed.

The nuclear phosphoprotein p53 occurs at elevated levels in many transformed cells. Mutant forms of mouse p53 possess enhanced transforming activity compared with wild type p53. Mutant mouse p53 proteins form complexes with the 70 kDa family of heat shock proteins (HSPs). We previously demonstrated an association between p53 and the 70 kDa HSPs in the human osteosarcoma (HOS) derived cell line HOS-SL. We report here the molecular cloning and sequencing of the p53 gene from HOS-SL cells, and demonstrate that it is in fact mutant. Further, analysis of similar HOS-derived cell lines demonstrates that they also encode the same mutant form of p53, whereas the wild type form of p53 appears to be lost in these cells. Stability studies demonstrate an increased half life of the p53 protein in these cells, in keeping with its association with the HSP 70 proteins. A potential role for this p53 mutant in the transformation process is discussed.

The intranuclear localization of SV40 T-antigen (T-Ag) and the cellular protein p53 was studied in SV40 abortively infected baby mouse kidney cells using two complementary methods of ultrastructural immunocytochemistry in combination with preferential staining of nuclear RNP components and electron microscope autoradiography. Both proteins were revealed in association with peri- and interchromatin RNP fibrils containing the newly synthesized hnRNA. In addition, T-Ag and p53 remained bound, at least in part, to the residual internal nuclear matrix following nuclease and salt extractions of infected cells. The localization of T-Ag was different in SV40 lytically infected monkey kidney cells since, in addition to hnRNP fibrils, the viral protein was also associated with cellular chromatin. However, when lytic infection was performed in conditions of blocked viral DNA replication, T-Ag was no longer associated with the cellular chromatin but remained bound to the hnRNP fibrils. We conclude that the transforming and lytic functions of T-Ag can be distinguished by different subnuclear distributions. The significance of the association of T-Ag and p53 with hnRNP fibrils and the internal nuclear matrix is discussed in relation to the role of these structures in the control of cellular mRNA biogenesis.

We report here immunological evidence for the specific association between p53 and hsp72/hsc73 heat shock proteins in a human cell line derived from an osteosarcoma. The same association between p53 and hsp72/hsc73 was observed in HOS-TE85 clone 5 from which the HOS-SL cell line was derived. This association was indicated by the co-immunoprecipitation from HOS-SL of both p53 and hsp72/hsc73 proteins observed with either an anti-p53 monoclonal antibody (PAb421) or an antiserum specifically reacting with hsp72/hsc73 heat shock proteins. Furthermore, Western blot analysis allowed us to show that hsp72/hsc73 proteins did not share an epitope with p53, confirming that the co-immunoprecipitation of p53 and hsp72/hsc73 was attributable to the physical association of the proteins. Data obtained from SDS-PAGE show that the HOS-SL cells expressed two forms of p53 with distinct molecular weights. Both forms contained several species with different isoelectric points ranging between pH 6.0 and 6.5. The data obtained from both 1D and 2D gel analyses consistently show that the p53 proteins involved in the association with hsp72/hsc73 were mainly the species that migrated with the slower mobility in the SDS dimension. The possibility is discussed that the HOS-SL p53 variant forming a complex with hsp72/hsc73 contains an activating mutation for transformation.

During abortive infection of Go/G1-arrested primary baby mouse kidney (BMK) cell cultures with simian virus 40 (SV40), expression of the viral large T antigen is followed by a mitotic host response including the stimulation of host macromolecular synthesis and induction into the cell cycle of Go/G1-arrested cells. We performed an extensive study of the sequential events taking place after SV40 infection of confluent BMK cell cultures. Thus study comprised a detailed kinetic analysis of transcription, synthesis, and accumulation of p53, in conjunction with the time course of large T antigen synthesis and SV40-induced cellular DNA replication. The monoclonal antibodies used for specifically recognizing mouse p53 were PAb 421, PAb 122, PAb 246, PAb 248, and RA3-2C2. Our results consistently show that under our experimental conditions, the stimulation of p53 synthesis and the accumulation of p53 occur well after the onset of T antigen-induced cellular DNA replication. This relatively late activation of p53 expression appears to be controlled at a level other than transcription. In conclusion, we suggest that, at least in certain cases, T antigen's mitogenic potential is not dependent on its interaction with p53.

Blood sinusoidal plasma membrane subfractions were isolated from normal mouse liver in the presence of the proteinase inhibitors PhMeSO2F and iodoacetamide. They were purified from smooth microsomal and Golgi vesicle contaminants. The phosphorylation reaction was studied at 33 degrees C, in the presence of 2 mM MnCl2. Addition of epidermal growth factor (EGF) to the preparations stimulated 32P incorporation from [gamma-32P]ATP or [gamma-32P]GTP essentially into one 170 000 Mr protein. Some incorporation was observed in a minor 120 000-Mr component which appears to be a degradation product of the 170 000-Mr component. No EGF-dependent phosphorylation of other membrane proteins or various exogenous proteins could be detected in vitro. The dephosphorylation of the 170 000-Mr component was observed after 4 min of incubation at 33 degrees C. This dephosphorylation reaction was inhibited by addition of 5 mM p-nitrophenyl phosphate but not by addition of micromolar Zn2+, Be2+ or orthovanadate. The 170 000-Mr protein specifically bound 125I-labeled EGF and thus appeared to be the hepatic EGF receptor. The EGF stimulatable kinase activity considerably enhances incorporation of 32P into tyrosine residues of the 170 000-Mr EGF receptor at 33 degrees C. Tryptic peptide maps of the 32P-labeled 170 000-Mr protein revealed a multiplicity of phosphorylated sites. Seven 32P-labeled phosphopeptides were observed after EGF stimulation, three of them being largely prominent. Tryptic peptide maps of the 170 000-Mr protein after it was covalently linked to 125I-labeled EGF showed only one 125I-labeled peptide, the migration of which appeared different from that of 32P-labeled phosphopeptides. These findings were confirmed by V8 protease unidimensional peptide mapping of the 170 000-Mr protein, labeled with 32P or 125I-EGF.

The addition of Epidermal growth factor (EGF) to mouse liver sinusoidal plasma membrane subfractions, isolated from the blood face of hepatocytes, resulted in a two to threefold increase of 32P incorporation from [γ-32P ]ATP into a 120 000 dalton protein component. The EGF-stimulation of the phosphorylation of this component was also evidenced in plasma membranes derived from the lateral and biliary faces of hepatocytes. A major phosphoamino acid residue after EGF stimulation was identified as tyrosine. An EGF-enhancement of the phosphorylation of threonine and serine residues also appears to be detectable.

Extensive purification of mouse liver gap junctions has been achieved with 1.2% deoxycholate, after clostripain pretreatment of plasma membranes. The presence of a major polypeptide of 26 000 daltons is confirmed. A component of molecular weight 34 000 which has been implicated as a possible constitutive junctional protein or suspected to represent a combination of the 26 000 dalton polypeptide with a proteolytic product, does not copurify with gap junctions. This 34 000 dalton component is not urate oxidase from detergent-insoluble peroxisomal cores which contaminate crude junctional preparations.

AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL References 1 M. Simionescu, N. Simionescu, G.E. Palade, J. Cell Biol., 67, (1975), 863– 885. 2 M.V.L. Bennett, Fed. Proc., 32, (1973), 65– 75. 3 L.A. Staehelin, Int. Rev. Cytol., 39, (1974), 191– 283. 4 R. Weinstein, F. Merk, J. Alroy, Adv. Cancer Res., 23, (1976), 23– 89. 5 E.L. Benedetti, P. Emmelot, J. Cell Biol., 38, (1968), 15– 24. 6 D.A. Goodenough, W. Stoeckenius, J. Cell Biol., 54, (1972), 646– 656. 7 D.A. Goodenough, J. Cell Biol., 61, (1974), 557– 563. 8 H. Bloemendal, A. Zweers, F. Vermorken, I. Dunia, E.L. Benedetti, Cell Diff., 1, (1972), 91– 106. 9 I. Dunia, Ghosh C. Sen, E.L. Benedetti, A. Zweers, H. Bloemendal, FEBS Lett., 45, (1974), 139– 144. 10 W.H. Evans, J.W. Gurd, Biochem. J., 128, (1972), 691– 699. 11 N.B. Gilula, J. Cell Biol., 63, (1974), 111a– 12 J.R. Duguid, J.P. Revel, Cold Spring Harbor Symp. Quant. Biol., 40, (1975), 45– 47. 13 D.A. Goodenough, J. Cell Biol., 68, (1970), 220– 231. 14 E.L. Benedetti, I. Dunia, C.J. Bentzel, A.J.M. Vermorken, M. Kibbelaar, H. Bloemendal, Biochim. Biophys Acta, 457, (1976), 353– 384. 15 W.H. Evans, Biochem. J., 166, (1970), 833– 842. 16 A. Monneron, G. Blobel, G.E. Palade, J. Cell Biol., 55, (1972), 104– 125. 17 F.W. Studier, J. Mol. Biol., 79, (1973), 237– 248. 18 U.K. Laemmli, J. Mol. Biol., 80, (1973), 575– 599. 19 N. Dwyer, G. Blobel, J. Cell Biol., 70, (1976), 581– 591. 20 R.M. Lopez, P. Siekevitz, Anal. Biochem., 53, (1973), 594– 602. 21 J.C. Ehrhart, J. Chauveau, Biochim. Biophys. Acta, 375, (1975), 434– 445. 22 J.C. Ehrhart, J. Chauveau, Biochimie, 57, (1975), 987– 989. Citing Literature Volume78, Issue2June 01, 1977Pages 295-299 ReferencesRelatedInformation