Ariana Celis

Cyclin (PCNA, auxiliary protein of DNA polymerase δ) is a central component of the pathway(s) leading to DNA replication and cell division

Cyclin, also known as PCNA or the auxiliary protein of mammalian DNA polymerase δ, is a stable cell cycle regulated (synthesized mainly in S‐phase) nuclear protein of apparent M r 36 000 whose rate of synthesis correlates directly with the proliferative state of normal cultured cells and tissues. Cyclin (PCNA) is absent or present in very low amounts in normal non‐dividing cells and tissues, but it is synthesized in variable amounts by proliferating cells of both normal and transformed origin. All available information indicates that this ubiquitous and tightly regulated DNA replication protein is a central component of the pathway(s) leading to DNA replication and cell division.

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.

Nuclear patterns of cyclin (PCNA) antigen distribution subdivide S-phase in cultured cells--some applications of PCNA antibodies.

Immunofluorescence studies using PCNA autoantibodies specific for the proliferation-sensitive protein cyclin have revealed dramatic changes in the nuclear distribution of this protein during the S-phase of normal and transformed cells. Patterns of cyclin antigen distribution subdivide S-phase and have provided new cell cycle landmarks. Some of these (nucleolar exclusion or staining), mimic topographical patterns of DNA synthesis thus arguing for a role of this protein in some specific aspect of DNA replication. Cells outside S-phase (G0 included) stain only weakly with PCNA antibodies, stressing the usefulness of this reagent for identifying proliferating cells (S-phase cells) of both normal and malignant origins.

Proteomic Strategies to Reveal Tumor Heterogeneity among Urothelial Papillomas

Proteomics and immunohistochemistry were used to reveal tumor heterogeneity among urothelial papillomas (UPs) with the long term goal of predicting their biological potential in terms of outcome. First, we identified proteins that were deregulated in invasive fresh lesions as compared with normal urothelium, and thereafter we immunostained UPs with a panel of antibodies against some of the markers. Twenty-two major proteins showing variations of 2-fold or more in at least one-third of the invasive lesions were selected. Specific antibodies against several of the proteins were obtained, but only a few reacted positively in immunostaining. A panel consisting of antibodies against keratinocytes (CKs) 5, 13, 18, and 20 and markers of squamous metaplasia (CKs 7, 8, and 14) was used to probe normal urothelium and 30 UPs collected during a period of five years. Four UPs showed a normal phenotype, whereas the rest could be grouped in five major types that shared aberrant staining with the CK20 antibody. Type 1 heterogeneity (n = 4) showed preferred staining of the umbrella cells with the CK8 antibody. Type 2 (n = 11) was typified by the staining of the basal and intermediate layers with the CK20 antibody. Type 3 (n = 7) was characterized by the predominant staining of the basal cell layer with the CK5 antibody. Type 4 (n = 1) showed areas of CK7 negative cells, whereas type 5 (n = 3) showed loss of staining of the basal cells with the CK20. 29% of the patients experienced recurrences, but none progressed to invasive disease. Patients harboring phenotypic alterations in the basal cell compartment (types 3 and 5) showed the highest number of recurrences (4/7 and 2/3, respectively), and all type 3 lesions progressed to a higher degree of dedifferentiation. Even though a long term prospective study involving a larger sample size is required to assess the biological potential of these lesions, we believe that this approach will prove instrumental for revealing early phenotypic changes in different types of cancer.

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.

Distribution of HeLa cells polypeptides in cytoplasts and karyoplasts

The polypeptide composition of cytoplasts and karyoplasts prepared form HeLa cells prelabelled with [35S]-methionine and enucleated with Cytochalasin B has been analyzed using high resolution two dimensional gel electrophoresis (IEF and NEPHGE). Of the 259 major proteins followed in this study we have identified 73 polypeptides (30 acidic(IEF) and 43 basic (NEPHGE)) that are present mainly in karyoplasts. One of these polypeptides (IEF 49) has previously been shown to be a polypeptide marker for cycling cells. A total of 59 polypeptides (27 acidic and 32 basic) were found to be present mainly in cytoplasts. Many polypeptides (109 acidic and 18 basic) including Y and beta-actin (60% in cytoplasts), beta-tubulin (60% in cytoplasts), vimentin (75% in cytoplasts) and alpha-actinin (65% in cytoplasts) were found to be present in both cellular fragments. These results could be of value in assigning the cellular distribution of potential regulatory proteins.

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.

Microfilament bundles in transformed mouse CLID X transformed cho cell hybrids: Correlation with tumorigenicity in nude mice☆

Abstract Tumorigenic mouse CLID cells (TK−1, thymidine kinase) having a ‘crossed’ pattern of microfilament bundles were fused by means of polyethylene glycol (PEG) with low tumorigenic ∗∗ transformed Chinese hamster ovary cells (CHO) (HGPRT−) displaying a normal distribution of microfilament bundles. Eight independent hybrid clones were isolated by selection in HAT media and were studied for their karyotype, pattern of microfilament bundles and tumorigenicity in nude mice. Fluorescent staining of metaphase chromosomes with Hoechst 33258 confirmed with one exception the hybrid character of the clones and further showed that all these hybrids have segregated CHO chromosomes. Indirect immunofluorescence studies using human actin antibodies revealed the ‘crossed’ pattern of microfilament bundles in all hybrids apart from one (no. 12) which presented a pattern similar to that observed in the CHO parent. Subcutaneous injection of the hybrid clones into nude mice showed that only four were consistently tumorigenic when 106 cells were inoculated per mouse although the period between inoculation and appearance of a small palpable tumour (latent period) was considerably longer (ranging from 24 to 45 days) than that observed in animals inoculated with the parental CLID cells (10 days). Three other hybrids produced tumours in 16 and 33% of the animals whilst Hy 12 failed to produce any tumors at all after 4 months of inoculation. The results clearly showed that no direct relationship could be drawn between the presence of a ‘crossed’ pattern of microfilament bundles and tumorigenicity in the nude mice. As with our previous studies on non-virally transformed cells, these results indicate that an alteration or loss of microfilament bundles cannot be used as a general assay to assess tumorigenicity at the cellular level.

Human proteins IEF 58 and 57a are associated with the Golgi apparatus.

A mouse monoclonal antibody (mAB 22‐II‐D8B) raised against lysed transformed human amnion cells (AMA) has been characterized. The mAB decorated the Golgi apparatus in growing and quiescent cultured monolayer cells (fibroblasts and epithelial cells) of various species as determined by double immunofluorescence labeling and colocalization with galactosyltransferase antibodies. It reacted with the acidic human proteins IEF 58 (M r = 29 000) and 57a, respectively (M r = 30 000) (HeLa protein catalogue number; [(1982) Clin. Chem. 28, 766]), Golgi staining was also observed in BS‐C‐1 cells microinjected with mAB 22‐II‐DSB suggesting that the epitopes recognized by the antibody are most likely located on the cytoplasmic face of the membranes. The precise localization of the antigens to the various cisternae of the Golgi apparatus could not be demonstrated by immunogold cytochemistry on ultrathin cryosections due to either weak reactivity of the antibody or low concentration of the antigens. Immunofluorescence staining with mAB 22‐II‐D8B of lymphoid human Molt‐4 cells and some human tissues failed to reveal any significant staining even though these expressed high levels of both IEF 58 and 57a. These results are taken to imply that the epitopes recognized by mAB 22‐II‐D8B may be masked in some cell types.