Tropomyosin isoform switching in tumorigenic human fibroblasts.

We identified six tropomyosin (Tm) isoforms in diploid human fibroblasts. We used computerized microdensitometry of 2-dimensional protein profiles to measure the relative rates of synthesis and abundance of the individual Tm isoforms and actin, the two major structural constituents of microfilaments. In carcinogen-transformed human fibroblasts (HuT-14), the rates of synthesis of three Tm isoforms (Tm1, Tm2, and Tm6) were greatly decreased relative to normal diploid parental fibroblasts and to actin. In contrast, related nontumorigenic HuT fibroblasts which are "immortalized" and anchorage independent exhibited both slight down-regulation of Tm1 and Tm6 and 3.5-fold up-regulation of Tm3. Thus, Tm isoform switching from the predominance of the larger more avid Tm isoforms (Tm1, Tm2, Tm3, and Tm6) to the smaller, less avid Tm isoforms (Tm4 and Tm5) in microfilaments was a transformation-induced change correlated with tumorigenicity in human fibroblasts.

Expression of transfected mutant beta-actin genes: alterations of cell morphology and evidence for autoregulation in actin pools.

Two different mutant human beta-actin genes have been introduced into normal diploid human (KD) fibroblasts and their immortalized derivative cell line, HuT-12, to assess the impact of an abnormal cytoskeletal protein on cellular phenotypes such as morphology, growth characteristics, and properties relating to the neoplastic phenotype. A mutant beta-actin containing a single mutation (Gly-244----Asp-244) was stable and was incorporated into cytoskeletal stress fibers. Transfected KD cells which expressed the stable mutant beta-actin in excess of normal beta-actin were morphologically altered. In contrast, a second mutant beta-actin gene containing two additional mutations (Gly-36----Glu-36 and Glu-83----Asp-83, as well as Gly-244----Asp-244) did not alter cell morphology when expressed at high levels in transfected cells, but the protein was labile and did not accumulate in stress fibers. In both KD and HuT-12 cells, endogenous beta- and gamma-actin decreased in response to high-level expression of the stable mutant beta-actin, in a manner consistent with autoregulatory feedback of actin concentrations. Since the percent decreases in the endogenous beta- and gamma-actins were equal, the ratio of net beta-actin (mutant plus normal) to gamma-actin was significantly increased in the transfected cells. Antisera capable of distinguishing the mutant from the normal epitope revealed that the mutant beta-actin accumulated in stress fibers but did not participate in the formation of the actin filament-rich perinuclear network. These observations suggest that different intracellular locations differentially incorporate actin into cytoskeletal microfilaments. The dramatic impact on cell morphology and on beta-actin/gamma-actin ratios in the transfected diploid KD cells may be related to the acquisition of some of the characteristics of cells that underwent the neoplastic transformation event that originally led to the appearance of the beta-actin mutations.

Expression of transfected mutant beta-actin genes: transitions toward the stable tumorigenic state.

Mutant human beta-actin genes were introduced into normal human (KD) fibroblasts and the derivative cell line HuT-12, which is immortalized but nontumorigenic, to test their ability to promote conversion to the tumorigenic state. Transfected substrains of HuT-12 fibroblasts that expressed abundant levels of mutant beta-actin (Gly-244----Asp-244) produced subcutaneous tumors in athymic mice after long latent periods (1.5 to 3 months). However, transfected substrains of KD fibroblasts retained their normal finite life span in culture and consequently were incapable of producing tumors. Substrains of HuT-12 cells transfected with the wild-type beta-actin gene and some transfected strains that expressed low or undetectable levels of mutant beta-actin did not produce tumors. Cell lines derived from transfectant cell tumors always exhibited elevated synthesis of the mutant beta-actin, ranging from 145 to 476% of the level expressed by the transfected cells that were inoculated to form the tumor. In general, primary transfectant cells that expressed the highest levels of mutant beta-actin were more tumorigenic than strains that expressed lower levels. The tumor-derived strains were stable in tumorigenicity and produced tumors with shortened latent periods of only 2 to 4 weeks. These findings imply that the primary transfectant strains develop subpopulations of cells that are selected to form tumors because of their elevated rate of exogenous mutant beta-actin synthesis. Actin synthesis and accumulation of gamma-actin mRNA from the endogenous beta- and gamma-actin genes were diminished in tumor-derived strains, apparently to compensate for elevated mutant beta-actin synthesis and maintain the normal cellular concentration of actin. Synthesis of the transformation-sensitive tropomyosin isoforms was decreased along with mutant beta-actin expression. Such modulations in tropomyosin synthesis are characteristically seen in transformation of avian, rodent, and human fibroblasts. Our results suggest that this mutant beta-actin contributes to the neoplastic phenotype of immortalized human fibroblasts by imposing a cytoarchitectural defect and inducing abnormal expression of cytoskeletal tropomyosins.

Abundant synthesis of the transformation-induced protein of neoplastic human fibroblasts, plastin, in normal lymphocytes.

The transformation-induced protein plastin (p219; Mr 68,000, pl 5.3) is a reliable cytosolic marker for neoplastic human fibroblasts. Fibroblasts transformed in vitro by chemical carcinogens or SV40 virus and tumor-derived cancer cells of fibroblastoid or epithelioid origin usually express plastin and p220, a minor phosphorylated form of plastin. We report here that plastin is expressed as one of the most abundant proteins of normal, untransformed lymphocytes. The phosphorylated form of plastin was detectable in adherent monocytes but not in purified T- or NK lymphocytes. We also demonstrate that an allelic variant or mutated form of plastin exhibiting altered charge is found at a reduced frequency in the human population. We discuss the possible significance of these observations in terms of evaluating the role of plastin induction in expression of the cancerous phenotype of fibroblasts.

From dose rate to websites: making measurements accessible, understandable and helpful to the lay public.

The key role of public information in emergency preparedness has more recently been corroborated by the experience of the Great Eastern Japan Earthquake and Tsunami and the subsequent nuclear accident at the Fukushima NPP. Information should meet quality criteria such as openness, accessibility and authenticity. Existing information portals of radiation monitoring networks were frequently used even in Europe, although there was no imminent radiation risk. BfS responded by increasing the polling frequency, publishing current data not validated, refurbishing the website of the BfS 'odlinfo.bfs.de' and adding explanatory text. Public feedback served as a valuable input for improving the site's design. Additional services were implemented for developers of smart phone apps. Websites similar to 'ODLInfo' are available both on European and international levels. NGOs and grass root projects established platforms for uploading and visualising private dose rate measurements in Japan after 11 March 2011. The BfS site is compared with other platforms. Government information has to compete with non-official sources. Options on information strategies are discussed.

Identification of polypeptides on two-dimensional electrophoresis gels by amino acid composition.

We present a method that can, in principle, provide tentative identification of a substantial proportion of the polypeptides resolvable on a given two-dimensional electrophoresis gel. Computerized microdensitometry of autoradiograms from 20 gels labeled in turn with each of the 20 common amino acids provides the data for simultaneously measuring the amino acid composition of all polypeptides of interest on the gel. These compositions are then compared with computer data bases of known protein compositions. Similarity between a known and an unknown polypeptide with comparable molecular mass indicates a potential identification, which can then be confirmed with conventional techniques. We illustrate this technique by applying it to the identification of proteins in a transformed human cell line (HuT-14).

Neoplastic human fibroblast proteins are related to epidermal growth factor precursor.

We report the amino acid composition of two polypeptides, p788 and p789. These polypeptides are reliable markers for neoplastic transformation in human fibroblasts. Their compositions are unusually rich in cysteine and serine. Because the recently reported amino acid sequence of mouse epidermal growth factor precursor (prepro-EGF) is also rich in those two amino acids and because the role of p788 and p789 as markers for neoplastic transformation is consistent with the fact that epidermal growth factor has been shown to play some role in transformation, we investigated the hypothesis that p788 and p789 are related to prepro-EGF. We compared the amino acid composition of p788 with that of all possible interior domains of prepro-EGF of appropriate length. We found that the composition of p788 is remarkably similar to that of residues 630-880 of prepro-EGF. The similarity is sufficiently strong to support the conclusion that it reflects amino acid sequence homology.

Normalization and reproducibility of mass profiles in the detection of individual differences from urine.

The mass profiles of urine samples and their reproducibility (coefficient of variation) obtained by field-ionization mass spectrometry (volcano source) depend upon the choice of normalization method. We compared six different normalization techniques in terms of improved reproducibility and sensitivity. Several simple procedures markedly improved reproducibility while preserving the high degree of sensitivity. Although these procedures gave similar and satisfactory results, others were clearly inferior and are not recommended for clinical profiling.

Quantitative protein changes in metastatic versus primary epithelial ovarian carcinoma.

Primary and metastatic ovarian carcinomas from six patients were obtained during primary exploratory laparotomy. Tumor cells were synthetically radiolabeled with [35S]methionine. Radiolabeled cellular proteins of the primary and metastatic cells were examined by two-dimensional polyacrylamide gel electrophoresis followed by autoradiography. Computer assisted analysis of the resultant autoradiograms revealed that the amounts of only two proteins, p35 and p36, were consistently and significantly decreased in the metastatic tumor cells. No other consistent differences in protein synthesis between primary and metastatic tumors were detected.