Molecular cloning and characterization of plastin, a human leukocyte protein expressed in transformed human fibroblasts.

The phosphoprotein plastin was originally identified as an abundant transformation-induced polypeptide of chemically transformed neoplastic human fibroblasts. This abundant protein is normally expressed only in leukocytes, suggesting that it may play a role in hemopoietic cell differentiation. Protein microsequencing of plastin purified from leukemic T lymphocytes by high-resolution two-dimensional gel electrophoresis produced eight internal oligopeptide sequences. An oligodeoxynucleotide probe corresponding to one of the oligopeptides was used to clone cDNAs from transformed human fibroblasts that encoded the seven other oligopeptides predicted for human plastin. Sequencing and characterization of two cloned cDNAs revealed the existence of two distinct, but closely related, isoforms of plastin--l-plastin, which is expressed in leukocytes and transformed fibroblasts, and t-plastin, which is expressed in normal cells of solid tissues and transformed fibroblasts. The leukocyte isoform l-plastin is expressed in a diverse variety of human tumor cell lines, suggesting that it may be involved in the neoplastic process of some solid human tumors.     

Human smooth muscle myosin light chain-2 gene expression is repressed in ras transformed fibroblast cells.

We have previously characterized human smooth muscle myosin light chain (MLC)-2 isoform by complementary DNA cloning and have shown that this isoform is expressed in a number of nonmuscle cells such as fibroblast cells. In this report, we show that when human osteosarcoma derived clonal cells (TE 85 clone F-5) (HOS), which are immortalized and nontumorigenic, undergo transformation following infection by Kirsten murine sarcoma virus (K-HOS) or by a chemical carcinogen [N-methyl-N-nitro-N-nitrosoguanidine (MNNG-HOS)], the smooth muscle MLC-2 mRNA is repressed. Revertants of transformed K-HOS cells (K-HOS312H) show normal levels of smooth muscle MLC-2 mRNA. Transformation of HOS cells by Ha-ras oncogene sequences, either by retroviral infection or by transfection followed by selection for tumorigenic cells in nude mice, results in complete repression of smooth muscle MLC-2 mRNA level. Treatment of HOS cells with tumor promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, results in repression of smooth muscle MLC-2 mRNA. Smooth muscle MLC-2 mRNA level is repressed in many, but not all, transformed cell lines, suggesting that it is not an indirect consequence of transformation but is specific to the agent that brings about transformation. HOS cells synthesize three MLC-2 protein species resolved by the two-dimensional gel electrophoretic system. The identity of the smooth muscle MLC-2 isoform was established by coelectrophoresis of the in vitro synthesized MLC-2 protein corresponding to the cloned complementary DNA in the two-dimensional gel system along with total [35S]methionine labeled HOS cell proteins. Quantitative analysis of MLC-2 isoforms in different HOS cells indicates that the synthesis of smooth muscle MLC-2 isoform is specifically repressed to an undetectable level in ras transformed and MNNG transformed cells and also following treatment with 12-O-tetradecanoylphorbol-13-acetate.     

Proteins IEF (isoelectric focusing) 31 and IEF 46 are keratin-type components of the intermediate-sized filaments: keratins of various human cultured epithelial cells

Mouse polyclonal antibodies have been raised against two human proteins (IEF [isoelectric focusing] 31, Mr = 50,000; IEF 46, Mr = 43,500) that have previously been shown to be present in HeLa cytoskeletons enriched in intermediate-sized filaments. Immunoprecipitation studies show that both proteins share common antigenic determinants with each other and with the putative human keratins IEF 36 and 44, also present in HeLa cytoskeletons. Indirect immunofluorescence studies showed that both antibodies revealed similar filamentous networks in various cultured epithelial cells of human origin. These included AMA (transformed amnion), HeLa (cervical carcinoma), normal amnion cells, Fl-amnion (transformed amnion), WISH-amnion (transformed amnion), Chang liver (liver), and Detroid-98 (sternal marrow). Human cells that did not react with both antibodies included skin fibroblasts, lung fibroblasts (WI-38), SV40-transformed lung fibroblasts, Molt 4 (leukemia), lymphocytes, and monocytes. These results were in complete agreement with the presence or absence of both proteins in two-dimensional gels of the different cell types. Exposure of AMA cells to demecolcine (24 h; 10 micrograms/ml) caused the total collapse of vimentin filaments but, as seen by indirect immunofluorescence, caused only a partial redistribution of the IEF 31 and 46 filaments. These results are taken to suggest that both proteins are components of the intermediate-sized filaments of the "keratin" type. The antibodies could be clearly differentiated by staining human bladder carcinoma EJ 19 cells, as only the IEF 46 antibody stained a filamentous network in these cells The occurrence of keratins IEF 31, 36, 44, and 46 in different cultured human epithelial cells has been studied using two-dimensional gel electrophoresis.     

Purified scatter factor stimulates epithelial and vascular endothelial cell migration

Fibroblasts and smooth muscle cells release a protein activity which causes epithelial sheets to "scatter" into isolated cells. Purification of scatter factor (SF) activity from ras-transformed 3T3 cells was reported recently. We purified ras-3T3 SF by a slightly different method with essentially similar findings. Purified factor showed a single band at 77 +/- 3 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. Scatter activity was eluted from gel slices at this molecular size. Reduction with mercaptoethanol caused the loss of activity and the appearance of two bands (58 and 31 kDa). We report the amino acid composition of ras-3T3 SF and sequences of several tryptic peptides. These sequences were not similar to the known proteins in the Protein Database. We have shown previously that partially purified ras-3T3 scatter activity stimulates migration of epithelial and vascular endothelial cells in a new migration assay utilizing microcarrier beads. We now demonstrate that the same purified ras-3T3 protein scatters epithelial cells and stimulates epithelial and endothelial migration in microcarrier bead and Boyden chamber assays. Partially purified human smooth muscle scatter activity shares these activities, but the protein(s) responsible has not been isolated. Migration-stimulating activity was maximal at ras-3T3 protein concentrations less than 10 ng/ml (0.13 nM). ras-3T3 SF had no collagenolytic activity and did not stimulate DNA synthesis in fibroblast growth factor-responsive human melanocytes. ras-3T3 SF appears to be a new protein which regulates endothelial and epithelial mobility; and, therefore, it may be involved in vascular repair and wound healing.     

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 (Mr = 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.     

A specific protein, p92, detected in flat revertants derived from NIH/3T3 transformed by human activated c-Ha-ras oncogene

Total proteins from a mouse embryo fibroblast cell line NIH/3T3, NIH/3T3 cells transformed by human activated c-Ha-ras (EJ-ras) oncogene (EJ-NIH/3T3), and the two flat revertant cell lines, R1 and R2, were analyzed by two-dimensional gel electrophoresis (IEF and NEPHGE). Several hundred polypeptides were resolved as seen by silver staining. Common alterations in four polypeptide spots were observed in the revertants when compared with NIH/3T3 and EJ-NIH/3T3 cells. In these alterations, a new polypeptide spot p92-5.7 (designated by molecular weight x 10(-3) and pI) was detected only in the revertants and not in NIH/3T3 and EJ-NIH/3T3 cells. Furthermore, the expression level of p92-5.7 seemed to be associated with the flat morphology and the reduced tumorigenicity of the revertants. Polypeptide p92-5.7 was also not detected in the total proteins extracted from BALB/3T3 cells, NIH Swiss mouse primary embryo fibroblasts, NRK (normal rat kidney) cells, and L6 (rat myoblast). Subcellular fractionation of total protein from R1 cells revealed that the p92-5.7 was present in the cytosol. Western blot analysis using an anti-gelsolin antibody demonstrated that the p92-5.7 might be a variant form of gelsolin which is thought to be an actin regulatory protein or a gelsolin-like polypeptide. These results may suggest that the expression of p92-5.7 detected only in the revertants is associated, at least in part, with the reversion. This may be the first demonstration of specific protein expression in the flat revertants.     

Mechanisms of gelsolin-dependent and -independent EGF-stimulated cell motility in a human lung epithelial cell line

Acquisition of motility is an important step in malignant progression of tumor cells and involves dynamic changes in actin filament architecture orchestrated by many actin binding proteins. A role for the actin-binding protein gelsolin has been demonstrated in fibroblast motility. In this report, we investigated the role of gelsolin in bronchial epithelial cell motility. The non-tumorigenic bronchial epithelial cell line, NL20 migrated towards EGF in a modified Boyden chamber cell motility assay. However, the tumorigenic NL20-TA cell line derived from the NL20 cells and lacking gelsolin, did not migrate towards EGF. Ectopic expression of gelsolin in NL20-TA cells restored the EGF response, while motility of NL20-TA derived cells towards serum, PDGF, and fibronectin was independent of gelsolin expression. PI3-kinase inhibition failed to block EGF-stimulated motility in gelsolin transfected NL20-TA cells. Furthermore, EGF stimulated a motility response in cells lacking gelsolin in the presence of fibronectin or fibrinogen that was blocked with PI3-kinase inhibition. Thus, EGF-stimulated motility in NL20 cells and its derivatives are gelsolin dependent and PI3-kinase independent, while fibronectin and fibrinogen enhances EGF-stimulated motility through a pathway independent of gelsolin and dependent upon PI3-kinase.     

Oncogenic Ras promotes butyrate-induced apoptosis through inhibition of gelsolin expression

Activation of Ras promotes oncogenesis by altering a multiple of cellular processes, such as cell cycle progression, differentiation, and apoptosis. Oncogenic Ras can either promote or inhibit apoptosis, depending on the cell type and the nature of the apoptotic stimuli. The response of normal and transformed colonic epithelial cells to the short chain fatty acid butyrate, a physiological regulator of epithelial cell maturation, is also divergent: normal epithelial cells proliferate, and transformed cells undergo apoptosis in response to butyrate. To investigate the role of k-ras mutations in butyrate-induced apoptosis, we utilized HCT116 cells, which harbor an oncogenic k-ras mutation and two isogenic clones with targeted inactivation of the mutant k-ras allele, Hkh2, and Hke-3. We demonstrated that the targeted deletion of the mutant k-ras allele is sufficient to protect epithelial cells from butyrate-induced apoptosis. Consistent with this, we showed that apigenin, a dietary flavonoid that has been shown to inhibit Ras signaling and to reverse transformation of cancer cell lines, prevented butyrate-induced apoptosis in HCT116 cells. To investigate the mechanism whereby activated k-ras sensitizes colonic cells to butyrate, we performed a genome-wide analysis of Ras target genes in the isogenic cell lines HCT116, Hkh2, and Hke-3. The gene exhibiting the greatest down-regulation by the activating k-ras mutation was gelsolin, an actin-binding protein whose expression is frequently reduced or absent in colorectal cancer cell lines and primary tumors. We demonstrated that silencing of gelsolin expression by small interfering RNA sensitized cells to butyrate-induced apoptosis through amplification of the activation of caspase-9 and caspase-7. These data therefore demonstrate that gelsolin protects cells from butyrate-induced apoptosis and suggest that Ras promotes apoptosis, at least in part, through its ability to down-regulate the expression of gelsolin.     

2D difference gel electrophoresis analysis of different time points during the course of neoplastic transformation of human mammary epithelial cells

Cell culture models of oncogenesis that use cellular reprogramming to generate a neoplastic cell from a normal cell provide one of the few opportunities to study the early stages of breast cancer development. Human mammary epithelial cells (HMECs) were induced to undergo a neoplastic transformation using defined genetic elements to generate transformed HMECs (THMECs). To identify proteins that displayed significantly different levels of abundance at three consecutive time points in oncogenesis over an 80 day period, protein extracts were analyzed by two-dimensional difference gel electrophoresis (2D-DIGE). Nine proteins were found to be significantly different in abundance: keratin 1, keratin 7, heat shock protein 4A-like, t-complex protein 1, stathmin, gelsolin, FK506 binding protein 5, ribosomal protein P0, and maspin. Keratin 7 and maspin displayed a linear down-regulation over 80 days. All of these proteins have been shown to be involved in the maintenance of a metastatic state including cytoskeletal modifications and motility. We conclude that, following neoplastic induction, THMECs display an early and progressive increase in metastatic potential. Further investigations into the function and regulatory mechanisms of these proteins will provide an unparalleled understanding of the initial states through which a breast cancer cell transitions following acquisition of the genetic abnormalities required for oncogenesis.     

Epigenetic regulation of gelsolin expression in human breast cancer cells.

Gelsolin is a multifunctional, actin-binding protein that is greatly decreased in many transformed cell lines and tumor tissues, including breast cancers. Downregulation of gelsolin RNA occurs in most breast cancers of rats, mice, and humans, but gross mutations of the gelsolin gene have not been found. Here we demonstrate by PCR and RT-PCR analysis that there are no point mutations in putative regulatory regions or the entire coding region of the cytoplasmic isoform of the gelsolin gene in human breast cancer cells (BCC). To determine if epigenetic modification is involved in downregulating gelsolin expression in MDA-MB-231 (MDA231), MCF7, and T47D BCC, we have used Southern blot analysis, 5-azacytidine (5aza) treatment, and trichostatin A (TSA) treatment. Southern blot analysis performed on genomic DNA demonstrated altered CpG methylation within intron 1 in DNA from all BCC compared to normal, mortal human mammary epithelial cells (HMEC). Treatment of the BCC with 5aza converted the DNA restriction pattern to that seen in untreated HMEC genomic DNA and caused modest increases in gelsolin RNA and protein. Incubation with TSA, an inhibitor of histone deacetylase, induced a dramatic upregulation of gelsolin RNA and protein levels which preceded apoptotic death of all BCC within 48-60 h. Our data support a role for epigenetic changes in chromatin structure leading to downregulation of gelsolin expression in human breast cancer. To our knowledge, this is the first example of a tumor suppressor gene downregulated in human breast cancer by changes in histone acetylation.     

Smooth muscle gelsolin and a Ca2+-sensitive contractile cell model

Smooth muscle gelsolin, termed smooth muscle 90-kDa protein in our previous paper (Kanno et al. FEBS Lett. 1985; 184:202-206), was purified from bovine aorta. Antibody prepared against smooth muscle gelsolin was used to detect the presence of gelsolin in human lung fibroblast MRC-5 cells permeabilized with Triton X-100 (MRC-5 cell models). These cells contracted in the presence of MgATP and Ca2+ in doses over 1 microM. Immunofluorescence microscopy using phalloidin and antigelsolin antibody showed that gelsolin was distributed along the stress fibers, except for a marginal bundle of cells, when MRC-5 cells were growth-arrested in serum-depleted medium. Making use of immunoblotting and indirect immunofluorescence techniques, we demonstrated that gelsolin is not retained in the MRC-5 cell models. We used purified smooth muscle gelsolin as a specific agent to sever the actin filaments. Preincubation of MRC-5 cell models with gelsolin led to a destruction of stress fibers, in a dose- and Ca2+ -dependent manner. The contractility was also lost, in the same manner described above, thereby indicating that a continuous distribution of actin filaments within the stress fibers is required for cell contraction. Treatment of MRC-5 cells with the Ca2+ ionophore A23187 induced an extracellular Ca2+ -dependent contraction but not a massive destruction of stress fibers, thereby indicating that most of the endogenous gelsolin was inactive under these conditions. Our interpretation of these results is that increases in cytoplasmic Ca2+ concentrations are sufficient for the contraction but may be too transient to activate endogenous gelsolin and thereby disrupt the stress fibers. Indeed, the inhibition of contraction of the MRC-5 cell, as induced by smooth muscle gelsolin, required preincubation in the presence of Ca2+, before the addition of MgATP. These results suggest that destruction of the stress fibers by endogenous gelsolin, which leads to inhibition of cell contraction, may occur if the cytoplasmic Ca2+ is maintained at high concentrations for a few minutes.     

Identification of a type 6 protein ser/thr phosphatase regulated by interleukin-2 stimulation

We have identified a 36 kD phosphoprotein that forms a complex with spliceosomal small nuclear ribonucleoproteins in lymphocyte extracts. This 36 kD protein is differentially phosphorylated in transformed human lymphoid cell lines and is regulated by IL-2 in peripheral blood T cells. We purified the 36 kD protein from human lymphocytes by employing a combination of immuno-affinity chromatography and preparative two-dimensional gel electrophoresis. Internal amino acid sequence analysis of the purified protein yielded two peptides that had perfect matches with sequences in the human protein serine/threonine phosphatase 6 (PP6). Using degenerate primers corresponding to the peptides, we obtained from a human T lymphocyte cDNA library a DNA fragment whose sequence is homologous to an EST cDNA clone (R05547). The predicted amino acid sequence of this clone showed over 98% sequence identity to human PP6. The identification of an IL-2 regulated type 6 protein serine/threonine phosphatase in lymphocytes was further substantiated by immunoblotting with anti-peptide antibodies. These findings suggest that PP6 is a component of a signaling pathway regulating cell cycle progression in response to IL-2 receptor stimulation.     

Identification of gelsolin, a Ca2+-dependent regulatory protein of actin gel-sol transformation, and its intracellular distribution in a variety of cells and tissues

Antiserum prepared against gelsolin, a major Ca2+-dependent regulatory protein of actin gel-sol transformation in rabbit lung macrophages, was used to detect the presence of proteins immunologically related to gelsolin in a variety of cells and tissues. Cell extracts were electrophoresed on polyacrylamide gels, and replicas of the gels on cellulose nitrate paper were stained by an indirect immunohistochemical technique. A single band of crossreactive material which comigrates with macrophage gelsolin is found in at least nine different kinds of cells and tissues derived from rabbits and humans and in four lines of cultured cells from humans and rats. Gelsolin was also identified in human serum and plasma, raising the possibility that it may contribute to the clearance of actin from the circulatory system. Using this antiserum, we demonstrated, by indirect immunofluorescent staining of acetone-fixed macrophages and polymorphonuclear leukocytes, that gelsolin resides in the cortical cytoplasm and that during phagocytosis it is concentrated in pseudopodia engulfing particles to be ingested, an area of the cytoplasm actively engaged in movement. In longitudinal cryostat sections of contracted rabbit skeletal muscle, antigelsolin staining was associated with the I-band of the myofibril, suggesting that it may be involved, by an as yet undefined mechanism, in skeletal muscle function. In rabbit intestinal epithelial cells, gelsolin was associated with the cytoplasm and the terminal web region of the brush border, a localization distinct from that previously reported for villin, a structurally and functionally similar protein isolated from the brush borders of chicken intestinal epithelial cells. In conclusion, our findings support the idea that gelsolin is involved in the regulation of movement and suggest that gelsolin-mediated Ca2+- regulation of actin cytoskeletal structure, first characterized in macrophages, may be of general importance.     

Proteomic profiling of bone marrow mesenchymal stem cells upon transforming growth factor beta1 stimulation

Bone marrow mesenchymal stem cells (MSCs) can differentiate into different types of cells and have tremendous potential for cell therapy and tissue engineering. Transforming growth factor beta1 (TGF-beta) plays an important role in cell differentiation and vascular remodeling. We showed that TGF-beta induced cell morphology change and an increase in actin fibers in MSCs. To determine the global effects of TGF-beta on MSCs, we employed a proteomic strategy to analyze the effect of TGF-beta on the human MSC proteome. By using two-dimensional gel electrophoresis and electrospray ionization coupled to quadrupole/time-of-flight tandem mass spectrometers, we have generated a proteome reference map of MSCs, and we identified approximately 30 proteins with an increase or decrease in expression or phosphorylation in response to TGF-beta. The proteins regulated by TGF-beta included cytoskeletal proteins, matrix synthesis proteins, membrane proteins, metabolic enzymes, etc. TGF-beta increased the expression of smooth muscle alpha-actin and decreased the expression of gelsolin. Overexpression of gelsolin inhibited TGF-beta-induced assembly of smooth muscle alpha-actin; on the other hand, knocking down gelsolin expression enhanced the assembly of alpha-actin and actin filaments without significantly affecting alpha-actin expression. These results suggest that TGF-beta coordinates the increase of alpha-actin and the decrease of gelsolin to promote MSC differentiation. This study demonstrates that proteomic tools are valuable in studying stem cell differentiation and elucidating the underlying molecular mechanisms.     

Localization of gelsolin proximal to ABL on chromosome 9.

Gelsolin is a plasma and cytoskeletal protein that severs actin filaments and is regulated by both Ca+2 and polyphosphoinositides. The two forms of gelsolin are encoded by a single gene and derived through alternative message splicing. By Southern blot analysis of somatic cell hybrids and in situ chromosomal localization, we demonstrate that the gelsolin gene is present on human chromosome 9 in bands q32-q34. In situ hybridization of gelsolin to cells containing a Philadelphia chromosome [(9;22)(q34;q11)], as well as Southern blot analysis of K562 cell DNA, indicates that gelsolin is centromeric to the ABL locus in 9q34. Southern blot analysis of NotI-digested, pulsed-field gel electrophoresis-separated DNA indicates the gelsolin gene is greater than or equal to 40 kb centromeric to ABL. These studies and standard Southern blot analysis of digested DNA also indicate that the NotI restriction site contained in the gelsolin gene is uncleavable in DNA from white blood cells and hematopoietic cell lines.     

A mutation in actin associated with neoplastic transformation

A new protein was recognized in a chemically transformed human fibroblast cell line when its proteins labeled with [35S]methionine were compared with those from normal human fibroblasts by two-dimensional gel electrophoresis. The new protein was found in the Triton-insoluble cytoskeletal fraction as well as in the Triton-soluble fraction, and it migrated very closely to beta- and gamma-actins on the gels. This new protein was identified as a variant form of actin by its reaction with antiactin antibody and its tryptic peptide pattern, which was identical to actin. mRNA coding for the variant actin was detected only in this particular transformed line. The size and cross- hybridizability with Dictyostelium actin cDNA of mRNA coding for the variant actin and complete amino acid sequence of the variant actin indicate that the new variant actin is the product of a mutated beta-actin gene. Only a single amino acid (glycine) at position 244 was replaced by aspartic acid. This substitution corresponds to a GC----AT transition, a point mutation. On the other hand, a highly malignant cell variant was isolated from the transformed line. The mutated beta-actin was further altered in this highly malignant subclone: it showed a more negative charge, rapid synthetic rate, and a short half-life in the cells. Incorporation into the cytoskeleton was significantly reduced in the mutated beta-actin. A hypothesis on the relationship between a mutation in the actin gene and oncogenic transformation was proposed.     

Differential response of stress fibers and myofibrils to gelsolin

The actin-severing activity of human platelet gelsolin was analyzed on embryonic skeletal and cardiac myofibrils, and on stress fibers in non-muscle cells. These subcellular structures, although in all three cell types composed of contractile proteins arranged in sarcomeric units, were found to respond differently to gelsolin. The myofibrils in permeabilized myotubes or cardiac cells, as well as in living, microinjected muscle cells proved resistant to a wide concentration range of gelsolin. The same was found for the "mini-sarcomeres" which are seen in developing muscle cells. In contrast, stress fibers in microinjected fibroblasts or epithelial cells, as well as in permeabilized cells, were broken down rapidly by the platelet gelsolin. We conclude from these results that the mini-sarcomeres in embryonic myotubes and cardiac myocytes are not identical with stress fibers.     

Polyoma virus-human cell interactions: persistence of T-antigen in two cell lines with and without transformation.

The interaction of polyoma virus and human cells was investigated. Abortive infection as evidenced by the synthesis of T-antigen was observed in normal fibroblast and abnormal (transformed) cells but not in normal epithelial cells. A high percentage of simian virus 40-transformed WI-18 Va2 and spontaneously transformed BE skin cells produced T-antigen after high-multiplicity infection, but most of the cells rapidly lost antigen-producing capacity upon cell passage, and the cultures became negative by passage 3. All fibroblast cells displayed varying degrees of susceptibility to infection, but most of the cell lines became negative for T-antigen except for two. In one, T-antigen persisted in a small percentage of the cells throughout the lifetime of the culture, without cellular transformation occurring. In the other, the entire culture became morphologically transformed and eventually consisted of 100% T-antigen-positive cells. This is the first time that normal diploid human fibroblast cells have been transformed by polyoma virus.     

Protein targets of 1,4-benzoquinone and 1,4-naphthoquinone in human bronchial epithelial cells

Many aspects of the toxicity of xenobiotic compounds have been attributed to the consequences of covalent modification of specific proteins, but the nature and specificity of protein targets for classes of electrophilic toxins remain largely uncharacterized. For inhaled toxicants, the point of exposure or absorption lies with epithelial cells lining the pulmonary tree. In this study, abundant proteins in human bronchial epithelial cells that are arylated in vitro by two quinonoid compounds, 1,4-benzoquinone (BQ) and 1,4-naphthoquinone (NQ) have been detected using (14)C-labeled quinones and two-dimensional gel electrophoresis. These proteins were identified using matrix assisted laser desorption/ionization mass spectrometry for tryptic mass mapping followed by sequence database searching. Corroborative identification of protein targets was obtained from the apparent isoelectric points, molecular weights, and the use of antibody probes. There were subtle differences in the protein targets of BQ and NQ, but both associated with the following abundant proteins, nucleophosmin, galectin-1, probable protein disulfide isomerase, protein disulfide isomerase, 60 kDa heat shock protein, mitochondrial stress-70 protein, epithelial cell marker protein, and S100-type calcium binding protein A14. We further delineate the properties of these proteins that make them preferred targets and the evidence these adducts present for delivery of these quinones to subcellular compartments.     

Two-dimensional protein profiles of cultured stromal and epithelial cells from hyperplastic human prostate

Studies were undertaken to compare and contrast the two-dimensional protein profiles of epithelial and stromal cells from hyperplastic human prostate to establish the protein composition of the two major cellular components of the prostate. Epithelial and stromal cells were isolated from human prostate obtained from patients undergoing open prostatectomy for benign prostatic hyperplasia (BPH). Proteins, isolated from the two cell populations and separated by two-dimensional (2D) electrophoresis, were analyzed by silver staining, fluorography of [35S]-methionine-labeled proteins, and immunoprotein blotting. Isolated prostatic epithelial cells, but not stromal cells, contained cytokeratin polypeptides 5, 6, 7, 8, 13, 14, 15, 16, 17, 18, and 19. Although vimentin could not be identified in silver stained 2D gels and fluorographs of cultured prostatic epithelial cells, a low level of immunoreactivity was noted following immunoblot analysis of epithelial cells proteins by the use of an anti-vimentin polyclonal. Vimentin was prominently expressed in cultured prostatic stromal cells and could be identified on silver stained 2D gels, fluorographs, and immunoblots of stroma-derived proteins. In addition, stromal marker proteins SM1, SM2, and SM3 were identified in 2D gels of stromal cells to distinguish them from epithelial cells. These studies demonstrate (1) the two-dimensional protein profile and cytokeratin polypeptide composition of cultured epithelial cells from hyperplastic human prostate and (2) the 2D protein profile of cultured prostatic stromal cells and identification of specific stromal marker proteins.