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Fresh, superficial transitional cell carcinomas (TCCs) of low-grade atypia (3 grade I, Ta; 6 grade II, Ta), as well as primary cultures derived from them were labeled with [35S]methionine for 16 h, between 2 and 6 days after inoculation. Whole protein extracts were subjected to IEF (isoelectric focusing) two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) followed by autoradiography. Proteins were identified by a combination of proteomic technologies that included microsequencing, mass spectrometry, 2-D PAGE immunoblotting and comparison with the bladder TCC protein database available on the internet (http://biobase.dk/cgi-bin/celis). Comparison of the IEF 2-D gel protein profiles of fresh tumors and their primary cultures showed that the overall expression profiles were strikingly similar, although differing significantly in the levels of several proteins whose rate of synthesis was differentially regulated in at least 85% of the tumor/culture pairs as a result of the short-term culturing. Most of the proteins affected by culturing were upregulated and among them we identified components of the cytoskeleton (keratin 18, gelsolin and tropomyosin 3), a molecular chaperone (hsp 28), aldose reductase, GST pi, metastasin, synuclein, the calreticulin precursor and three polypeptides of unknown identity. Only four major proteins were downregulated, and these included two fatty acid-binding proteins (FABP:FABP5 and A-FABP) which are thought to play a role in growth control, the differentiation-associated keratin 20, and the calcium-binding protein annexin V. Proteins that were differentially regulated in only some of the cultured tumors included alpha-enolase, triosphosphate isomerase, members of the 14-3-3 family, hnRNPs F and H, PGDH, hsp (heat-shock protein) 60, BIP, the interleukin-1 receptor antagonist, the nucleolar protein B23, as well as several proteins of yet unknown identity. The suitability of in vitro bladder tumor culture models to study complex biological phenomena such as malignancy and invasion is discussed.

In our laboratories we are exploring the possibility of using proteome expression profiles of fresh bladder tumors (transitional cell carcinomas, TCCs; squamous cell carcinomas, SCCs) and random biopsies as fingerprints to subclassify histopathological types and as a starting point to search for protein markers that may form the basis for diagnosis, prognosis, and treatment. Ultimately, the goal of these studies is to identify signaling pathways and components that are affected at various stages of bladder cancer progression and that may provide novel leads in drug discovery. Here we present our ongoing efforts to establish comprehensive two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) databases of TCCs and SCCs which are being constructed based on the proteomic and immunohistochemical analysis of hundreds of fresh tumors, random biopsies and cystectomies received shortly after operation (http://biobase.dk/cgi-bin/celis).

Psoriasin is a novel chemotactic inflammatory protein that possesses weak similarity to the S100 family members of Ca(2+)-binding proteins, and that is highly up-regulated in hyperproliferative psoriatic keratinocytes. Here we have used the psoriasin cDNA to express recombinant human (rh) psoriasin in Escherichia coli as a fusion protein containing a hexa His tag and a factor Xa cleavage site in the NH2-terminus. The protein was purified by affinity chromatography on Ni(2+)-nitrilotriacetic acid agarose, digested with factor Xa, further purified by ion-exchange chromatography and characterized by two-dimensional (2-D) gel electrophoresis and NH2-terminal sequencing. The ability of rh psoriasin to bind Ca2+, Zn2+, and Mg2+ was determined by dialysis experiments. We found that rh psoriasin may bind at least seven molecules of Ca2+ in KCl and several molecules in NaCl, with an affinity for the first bound molecule of 1.3-1.6 x 10(4) M-1. This indicates that psoriasin may cooperatively bind several molecules of Ca2+ when present in the extracellular space, or putatively, if localized in subcellular compartments where the concentration of Ca2+ is relatively high. At least eight molecules of Zn2+ were bound in KCl and four in NaCl, with an affinity just below 1 x 10(4) M-1 for the first molecule. Thus psoriasin does not bind significant amounts of Zn2+ at physiological concentrations. Mg2+ and Ca2+ are bound anti-cooperatively and binding of each of the ions (Ca2+, Zn2+, or Mg2+), is accompanied by conformational changes that move tyrosine residues to more hydrophobic areas.

Comparison of the protein expression patterns of proliferating normal primary human keratinocytes plated in serum-free medium (SFKM), supplemented with epidermal growth factor (EGF) and bovine pituitary extract (BPE), and similar cultures induced to differentiate by the addition of Dulbecco's modified Eagle medium (DMEM), containing 10% fetal calf serum (FCS), revealed several known and unknown polypeptides that are abnormally regulated in the differentiated cells. Upregulated proteins included keratins (keratins 6, 10/11, 14 and 16), members of the S100 protein family psoriasin, MRP8, MRP14 and S100c), actin-binding proteins (gelsolin and tropomyosin 9220), annexins (annexins IV and VIII), hsp28, the fatty acid binding protein 5 (FABP5), the squamous cell carcinoma (SCC) antigen, members of the 14-3-3 family, involucrin, E-cadherin, cystatin A, desmoglein and integrins alpha 2 and beta 1, as well as several proteins of as yet unknown identity. The highest upregulated proteins corresponded to psoriasin (124.0 times), MRP8 (42.4 times), MRP14 (14.9 times), tropomyosin 9220 (11.5 times), involucrin (11.1 times), and FABP5 (9.1 times). FABP5, hsp28, and tropomyosin 9220 were also highly upregulated in quiescent keratinocytes indicating that their increased levels in the differentiated cells may be due to loss of proliferative activity. Highly downregulated proteins included PAI-2, tropomyosins 9213, 9121 and 9122, keratin 5, calnexin, 14-3-3 beta and eta, nucleoside diphosphate kinase A, Rho GDIs, hsp60, hnRNPs H and C2, alpha-enolase, eIF-4D, thioredoxin, annexins III and V, moesin, nucleolar protein B23, GST pi and PCNA/cyclin. Both the high expression of keratin 6 and 16--which are markers for an alternative pathway of keratinocyte differentiation--as well as the extremely high upregulation of some members of the S100 protein family indicate that the cells have differentiated via an abnormal pathway.

The master two-dimensional (2-D) gel database of human keratinocytes currently lists 3154 cellular proteins (2224 isoelectric focusing, IEF; and 930 nonequilibrium pH gradient electrophoresis, NEPHGE), many of which correspond to post-translational modifications. 1082 polypeptides have been identified (protein name, organelle components, etc.) using a procedure or a combination of procedures that include (i) comigration with known human proteins, (ii) 2-D gel immunoblotting using specific antibodies, (iii) microsequencing of Coomassie Brilliant Blue stained proteins, (iv) mass spectrometry, (v)vaccinia virus expression of full length cDNAs, and (vi) in vitro transcription/translation of full-length cDNAs. This year, special emphasis has been given to the identification of signal transduction components by using 2-D gel immunoblotting of crude keratinocyte lysates in combination with enhanced chemoluminescence (ECL) detection. Identified proteins are listed both in alphabetical order and with increasing SSP number, together with their M(r), pI, cellular localization and credit to the investigator(s) that aided in the identification. Ultimately, the aim of the comprehensive database is to gather--through a systematic study of ekeratinocytes--qualitative and quantitative information on proteins and their genes that may allow us to identify abnormal patterns of gene expression and to pinpoint signaling pathways and components affected in various skin diseases, cancer included.

The master two-dimensional (2-D) gel database of human keratinocytes currently lists 3087 cellular proteins (2168 isoelectric focusing, IEF; and 919 none-quilibrium pH gradient electrophoresis, NEPHGE), many of which correspond to posttranslational modifications, 890 polypeptides have been identified (protein name, organelle components, etc.) using one or a combination of procedures that include (i) comigration with known human proteins, (ii) 2-D gel immunoblotting using specific antibodies (iii) microsequencing of Coomassie Brilliant Blue stained proteins, (iv) mass spectrometry and (v) vaccinia virus expression of full length cDNAs. These are listed both in alphabetical order and with increasing SSP number, together with their M(r), pI, cellular localization and credit to the investigator(s) that aided in the identification. Furthermore, we list 239 microsequenced proteins recorded in the database. We also report a database of proteins recovered from the medium of noncultured, unfractionated keratinocytes. This database lists 398 polypeptides (309 IEF; 89 NEPHGE) of which 76 have been identified. The aim of the comprehensive databases is to gather, through a systematic study of keratinocytes, qualitative and quantitative information on proteins and their genes that may allow us to identify abnormal patterns of gene expression and, ultimately, to pinpoint signaling pathways and components affected in various skin diseases, cancer included.

We have identified nine molecular chaperones in human keratinocytes by one or a combination of three methods: (i) reaction with antibodies raised against the purified proteins, (ii) microsequencing of two-dimensional (2-D) gel purified proteins, or (iii), by cloning of the cDNA and expression of its encoded protein in transformed human amnion cells using the vaccinia virus expression system. The expression levels of each of the molecular chaperones were analyzed in quiescent, normal proliferating, and simian virus SV40 transformed K14 keratinocytes by cutting the corresponding protein spots from dried 2-D gels and counting the radioactivity by liquid scintillation. The most striking observation was the strong up-regulation (936%) of the small heat shock protein HSP28 in the quiescent keratinocytes, a fact that is in line with recent data indicating that the murine homologue (HSP25) may act as a growth inhibitor. Several chaperones that localize to the endoplasmic reticulum and that are involved in the secretory pathway (GRP78, GRP78v, endoplasmin, and calnexin) were expressed at approximately similar levels in normal proliferating and K14 keratinocytes but were down-regulated by 50% or more in the quiescent cells, implying that these cells may possess an impaired ability to secrete certain proteins. Both GRP78 and endoplasmin genes have similar sequences in the promoter regions, suggesting that they may be partly co-regulated at the transcriptional level (McCauliffe et al., J. Biol. Chem. 1992, 267, 2557-2562).(ABSTRACT TRUNCATED AT 250 WORDS)

A comprehensive human keratinocyte two-dimensional (2-D) gel protein database has been established to study the expression levels and properties of the thousands of proteins that orchestrate various keratinocyte functions both in health and disease, cancer included. A major task in establishing such a database is to identify known proteins in the 2-D gel patterns as well as to reveal hitherto unknown proteins. To date, protein identification has been performed by one or a combination of the following methods: (i) comigration with known proteins, (ii) Western blotting using specific antibodies, (iii) microsequencing and (iv) vaccinia virus expression of full length cDNAs. Recently, the systematic identification of proteins has gained a new dimension with the advent of computer programs for searching peptide molecular mass databases with experimentally obtained peptide mass maps. Here we investigate this approach to identify proteins that are highly up- or down-regulated in simian virus SV40 transformed human keratinocytes (K14). Peptide mass maps of several proteins, including keratins 7, 8, 18 and 19 were obtained either by plasma desorption mass spectrometry (PDMS) analysis of high performance liquid chromatography (HPLC) purified peptides or by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of total digests. The results demonstrated that peptide mass maps can be used for a rapid and sensitive protein identification allowing fast screening of proteins recorded in 2-D gel databases. The mass spectrometric approach when combined with microsequencing strengthened identification, and added the possibility of full characterization of post-translational modifications and sequence variations.

The master two-dimensional gel database of human keratinocytes currently lists 3038 cellular proteins (2127 isoelectric focusing, IEF; and 911 nonequilibrium pH gradient electrophoresis, NEPHGE) many of which correspond to post-translational modifications. 763 proteins have been identified (protein name, organelle components, etc.) and they are listed both in alphabetical order and with increasing SSP number, together with their M(r), pI, cellular localization and credit to the investigator(s) that aided in the identification. Furthermore we have listed 176 proteins that have been microsequenced so far and that are recorded in this database. We also include synthetic images depicting some interesting sets of proteins identified so far; these include components of hnRNP's, proteasomes or prosomes, ribosomes, as well as assorted organelle markers, GTP-binding proteins, calcium binding proteins, stress proteins, autoantigens, differentiation markers and psoriasis upregulated proteins. The aim of the comprehensive database is to gather, through a systematic study of keratinocytes, qualitative and quantitative information on proteins and their genes that may allow us to identify abnormal patterns of gene expression and ultimately to pinpoint signaling pathways and components affected in various skin diseases, cancer included.

Microsequencing of proteins recovered from two-dimensional (2-D) gels is being used systematically to identify proteins in the master human keratinocyte 2-D gel database. To date, about 250 protein spots recorded in human 2-D gel databases have been microsequenced and, of these, 145 are recorded in the keratinocyte database under the entry partial amino acid sequence. Coomassie Brilliant Blue-stained protein spots cut from several (up to 40) dry gels were concentrated by elution-concentration gel electrophoresis, electroblotted onto PVDF membranes and digested in situ with trypsin. Eluting peptides were separated by reversed-phase HPLC, collected individually and sequenced. Computer search using the FASTA and TFASTA programs from Genetics Computer Group indicated that 110 of the microsequenced polypeptides shared significant similarity with proteins contained in the PIR, Mipsx or GenEMBL databases. Only 35 polypeptides corresponded to hitherto unknown proteins. Peptide sequences of all 145 proteins are listed together with their coordinates (apparent molecular weight and pI) in the keratinocyte database.

The master two-dimensional gel database of human keratinocytes currently lists 2980 cellular proteins (2098 isoelectric focusing, IEF; and 882 nonequilibrium pH gradient electrophoresis, NEPHGE) many of which correspond to posttranslational modifications. About 20% of all recorded proteins have been identified (protein name, organelle components, etc.) and they are listed in alphabetical order together with their M(r), pI, cellular localization and credit to the investigator(s) that aided in the identification. Also, we have listed 145 microsequenced proteins that are recorded in this database. As an aid in localizing the polypeptides we have included blow-ups of the master images (IEF, NEPHGE) displaying all the protein numbers. In the long run, the master keratinocyte database is expected to link protein and DNA sequencing and mapping information (Human Genome Program) and to provide an integrated picture of the expression levels and properties of the thousands of proteins that orchestrate various keratinocyte functions both in health and disease.

The master two-dimensional gel database of human AMA cells currently lists 3801 cellular and secreted proteins, of which 371 cellular polypeptides (306 IEF; 65 NEPHGE) were added to the master images during the last 10 months. These include: (i) very basic and acidic proteins that do not focus under normal running conditions and (ii) low-abundant proteins that can only be detected after prolonged gel exposure. Annotation categories updated in this version include "protein name", "antibody against protein", "cellular localization", and "microsequenced proteins". New entries include "human autoantigens" and "cDNAs". For convenience we have included an alphabetical list of all known proteins recorded in this database. In the long run, the main goal of this database is to link protein and DNA sequencing and mapping information (Human Genome Program) and to provide an integrated picture of the expression levels and properties of the thousands of proteins that orchestrate various cellular functions both under physiological and abnormal conditions.

Sixty-six human proteins recorded in the master transformed human epithelial amnion cells (AMA) (55) and keratinocyte (11) two-dimensional gel protein databases have been microsequenced since the last publication of the AMA database (Electrophoresis 1990, 12, 989-1071). Coomassie Brilliant Blue stained protein spots cut from several (up to 40) dry gels were concentrated by elution-concentration gel electrophoresis, electroblotted onto polyvinylidene difluoride membranes and in situ digested with trypsin. The eluting peptides were separated by reversed-phase high performance liquid chromatography (HPLC), collected individually and sequenced. Computer searches using the FASTA and TFASTA programs from the Genetics Computer Group indicated that 29 of the analyzed polypeptides correspond to hitherto unknown proteins.

A two-dimensional (2-D) gel database of cellular proteins from noncultured, unfractionated normal human epidermal keratinocytes has been established. A total of 2651 [35S]methionine-labeled cellular proteins (1868 isoelectric focusing, 783 nonequilibrium pH gradient electrophoresis) were resolved and recorded using computer-aided 2-D gel electrophoresis. The protein numbers in this database differ from those reported in an earlier version due to changes in the scanning hardware (Celis et al., Electrophoresis 1990, 11, 242-254). Annotation categories reported include: "protein name" (listing 207 known proteins in alphabetical order), "basal cell markers", "differentiation markers", "proteins highly up-regulated in psoriatic skin", "microsequenced proteins" and "human autoantigens". For reference, we have also included 2-D gel (isoelectric focusing) patterns of cultured normal and psoriatic keratinocytes, melanocytes, fibroblasts, dermal microvascular endothelial cells, peripheral blood mononuclear cells and sweat duct cells. The keratinocyte 2-D gel protein database will be updated yearly in the November issue of Electrophoresis.

A new version of the MRC-5 two-dimensional gel cellular protein database (Celis et al., Electrophoresis 1989, 10, 76-115) is presented. Gels were scanned with a Molecular Dynamics laser scanner and processed by the PDQUEST II software. A total of 1895 [35S]methionine-labeled cellular polypeptides (1323 with isoelectric focusing and 572 with nonequilibrium pH gradient electrophoresis) are recorded in this database, containing quantitative and qualitative data on the relative abundance of cellular proteins synthesized by quiescent, proliferating and SV40 transformed MRC-5 fibroblasts. Of the 592 proteins quantitated so far, the levels of 138 were up- or down-regulated (51 and 87, respectively) by two times or more in the transformed cells as compared to their normal proliferating counterparts, while only 14 behaved similarly in quiescent cells. Seven MRC-5 SV40 proteins, including plastin and two interferon-induced proteins, were not detected in the master MRC-5 images. The identity of 36 of the transformation-sensitive proteins whose levels are up or down regulated by two times or more was determined and additional information can be transferred from the master transformed human epithelial amnion cells (AMA) database (Celis et al., Electrophoresis 1990, 11, 989-1071) for those polypeptides of known and unknown identity that have been matched to AMA polypeptides. As more information is gathered in this and other laboratories, including data on oncogene proteins and transcription factors, this comprehensive database will outline an integrated picture of the expression levels and properties of the thousands of protein components of organelles, pathways and cytoskeletal systems that may be directly or indirectly involved in properties associated with the transformed state.

A total of 3430 polypeptides (2592 cellular; 838 secreted) from transformed human amnion cells (AMA) labeled with [35S]methionine were separated and recorded using computer-aided two-dimensional (2-D) gel electrophoresis. A master 2-D gel database of cellular protein information that includes both qualitative and quantitative annotations has been established. The protein numbers in this database differ from those reported in an earlier version (Celis et al. Leukemia 1988, 2,561-602) as a result of changes in the scanning hardware. The reported information includes: percentage of total radioactivity recovered from the gels (based on quantitations of polypeptides labeled with a mixture of 16 14C-amino acids), protein name (including credit to investigators that aided identification), antibody against protein, cellular localization, (nuclear, 40S hnRNP, 20S snRNP U5, proteasomes, endoplasmic reticulum, mitochondria, Golgi, ribosomes, intermediate filaments, microfilaments and microtubules), levels in fetal human tissues, partial protein sequences (containing information on 48 human proteins microsequenced so far), cell cycle-regulated proteins, proteins sensitive to interferons alpha, beta, and gamma, heat shock proteins, annexins and phosphorylated proteins. The results presented should be considered as the initial phase of a joint effort between our laboratories to undertake a general and systematic analysis of human proteins. Using this integrated approach it will be possible to identify phenotype-specific proteins, to microsequence them and store the information in the database, to identify the corresponding genes, to search for homology with previously characterized proteins and to study the function of groups of proteins (pathways, organelles, etc.) that exhibit interesting regulatory properties. In particular, the 2-D gel protein database may become increasingly important in view of the concerted effort to map and sequence the entire human genome.

We have used two-dimensional gel electrophoresis as a general "preparative" method to purify proteins for microsequencing analysis. In the first experiments, proteins derived from a total extract of Nicotiana tabacum leaf tissue were directly blotted from the gel onto poly(4-vinyl-N-methylpyridinium iodide)-coated glass fiber sheets. The major spots were excised and subjected to NH2-terminal sequence analysis, which made it possible to identify five of the eight selected proteins, while two more were recognized by generated internal sequences. In a second set of experiments, proteins of human origin were separated on multiple two-dimensional gels and the Coomassie Brilliant Blue-stained spots were excised from the gels. The combined spots were re-eluted and concentrated in a new gel and blotted on Immobilon. They were fragmented by in situ proteolysis and the generated peptides were separated by reverse phase-high performance liquid chromatography and sequenced. At the average, the internal sequences that were obtained covered 35 residues per protein and allowed unambiguous identification of 13 of the 23 proteins analyzed so far. The sequence information obtained of the unidentified proteins is sufficient for further cloning. These results demonstrate that systematic sequence analysis of the major proteins seen in two-dimensional gels is within the reach of current technologies. This offers a unique opportunity to link information contained in protein databases with known or forthcoming DNA sequence data.

A two-dimensional (2-D) gel database of proteins from noncultured total normal human epidermal keratinocytes has been established. A total of 1449 [35S]methionine labelled proteins (1112 isoelectric focusing, 337 nonequilibrium pH gradient electrophoresis) were resolved and recorded using computer assisted (PDQ-SCAN and PDQUEST software) 2-D gel electrophoresis. By matching the protein patterns of total keratinocytes and transformed human amnion cells (master database; Celis et al., Leukemia 1988, 2, 561-602) as well as by 2-D immunoblotting and microsequencing of keratinocyte proteins, it was possible to identify 72 polypeptides in the keratinocyte database. The database also includes data on polypeptides that are synthesized at a higher level by keratinocytes enriched in basal cells, and on six secreted proteins which are produced, albeit at a reduced rate, by normal keratinocytes and that are strongly up-regulated in psoriatic epidermis (Celis et al., FEBS Letters, in press).

Databases of protein information from human embryonal lung fibroblasts (MRC-5) have been established using computer analyzed two-dimensional gel electrophoresis. One thousand four hundred and eighty-two cellular proteins (1060 with isoelectric focusing and 422 with nonequilibrium pH gradient electrophoresis, in the first dimension) ranging in molecular mass between 8 and 234 kDa were separated and numbered. Information entered in the database (in most cases for major proteins) includes: protein name, HeLa protein catalog number, mouse protein catalog number, proteins matched in transformed human epithelial amnion cells (AMA) and peripheral blood mononuclear cells (PBMC), transformation and/or proliferation sensitive proteins, synthesis in quiescent cells, cell cycle regulated proteins, mitochondrial and heat shock proteins, cytoskeletal proteins and proteins whose synthesis is affected by interferons. Additional information entered for a few transformation-sensitive proteins that have been selected for future studies includes levels of synthesis and amounts in fetal human tissues. A total of four hundred and seventy-six [35S]methionine labeled polypeptides (258 isoelectric focusing; 218, nonequilibrium pH gradient electrophoresis) secreted by MRC-5 fibroblasts were separated and recorded (J. E. Celis et al., Leukemia 1987, 1, 707-717). Information entered in this database includes molecular weight and transformation sensitive proteins. These databases, as well as those of epithelial and lymphoid cell proteins (J. E. Celis et al., Leukemia 1988, 9, 561-601), represent the initial stages of a systematic effort to establish comprehensive databases of human protein information. In the long run, these databases are expected to offer a useful framework in which to focus the human genome sequencing effort.