Gitte P. Ratz

Short‐term culturing of low‐grade superficial bladder transitional cell carcinomas leads to changes in the expression levels of several proteins involved in key cellular activities

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 π, 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.

High-Resolution Two-Dimensional Gel Electrophoresis of Proteins: Isoelectric Focusing and Nonequilibrium pH Gradient Electrophoresis (NEPHGE)

Publisher Summary This chapter discusses high-resolution two-dimensional (2D) gel electrophoresis method. High-resolution two-dimensional (2D) gel electrophoresis is at present considered the method with the highest resolution for the separation of complex protein mixtures such as those present in eukaryotic cells. The technique, which separates proteins in terms of their isoelectric points and molecular weights, is commonly used to identify new cellular components for example cytoskeletal proteins, organelle components, etc., and to detect alterations in their expression using qualitative and quantitative comparisons. The method allows resolving proteins having apparent molecular weights between 8.5 and 230 kDa and pI values from 4 to 12.

Identification of a group of proteins that are strongly up‐regulated in total epidermal keratinocytes from psoriatic skin

Analysis using two‐dimensional (2D) gel electrophoresis of the [35S]‐methionine‐labelled proteins synthesized by non‐cultured total epidermal keratinocytes obtained from normal and psoriatic skin revealed 6 proteins that are strongly up‐regulated (5 times or more) in psoriatic skin. These proteins are synthesized at albeit lower levels by keratinocytes from normal and normal‐appearing (uninvolved) skin of psoriatic patients, and correspond to isoelectric focusing sample spot numbers 4311 (40.3 kDa), 4003 (12.4 kDa), 5008 (11.9 kDa), 3012 (11.6 kDa), 6016 (11.6 kDa) and 1015 (10;1 kDa) in the normal keratinocyte 2D gel protein database [Celis et al, (1990) Electrophoresis, in press]. These proteins are also detected in the labelling medium indicating that they are at least in part secreted. Given their striking regulatory behavior, these proteins may play a role in the pathogenesis of psoriasis.

Comprehensive, human cellular protein databases and their implication for the study of genome organization and function

Comprehensive, computerized databases of cellular protein information derived from the analysis of two‐dimensional gels, together with recently developed techniques to microsequence proteins offer a new dimension to the study of genome organization and function. In particular, human protein databases provide an ideal framework in which to focus the human genome sequencing effort.

Progressin: a novel proliferation-sensitive and cell cycle-regulated human protein whose rate of synthesis increases at or near the G1/S transition border of the cell cycle

A novel proliferation‐sensitive and cell cycle‐specific basic protein, termed progressin (M r,=33 000), has been identified in proliferating human cells of epithelial, fibroblast and lymphoid origin. Progressin is synthesized almost exclusively during the S‐phase of transformed human amnion cells (AMA). Increased synthesis of this protein is first detected late in G1, at or near the G1/S transition border, reaches a maximum in mid to late S‐phase, and declines thereafter. Contrary to histones, progressin synthesis is not coupled to DNA replication. As expected for an S‐phase‐specific protein, no detectable synthesis of progressin was observed in non‐proliferating human MRC‐5 fibroblasts and epidermal basal keratinocytes. Elevated, but variable levels of this protein were observed in proliferating normal fibroblasts and transformed cells of fibroblast, epithelial and lymphoid origin. Taken together the above observations suggest that progressin may be a component of the common pathway leading to DNA replication and cell division.

Electroelution of Proteins from Two-Dimensional Gels

Publisher Summary There are many situations in which it may be required to recover proteins from acrylamide gels for further analysis. For example, polypeptides extracted from gels can be readily used to immunize rabbits or mice to prepare antibodies. This chapter discusses a simple and effective method for electroelution of proteins from fixed, unstained, and Coomassie brilliant blue-stained dry two-dimensional (2D) gels. The method uses a silver-stained gel of electroeluted glutathione S-transferase π cut from a dry Coomassie brilliant blue-stained 2D gel of SV40-transformed keratinocytes. The sample was co-run with a small amount of 35 S-methionine-labeled proteins from the same cell type. However, the solution in the buffer chamber should be stirred to prevent bubbles from sticking to the bottom of the dialysis membrane. Sometimes, electroeluted proteins give rise to artifactual variants.