Glycosylation of VSV glycoprotein is similar in cystic fibrosis, heterozygous carrier, and normal human fibroblasts.

The single envelope glycoprotein of vesicular stomatitis virus was used as a specific probe of glycosyltransferase activities in fibroblasts from two cystic fibrosis patients, an obligate heterozygous carrier and a normal individual. Gel filtration of pronase-digested glycopeptides from both purified virions and infected cell-associated VSV glycoprotein which had been labeled with[3H] glucosamine did not reveal any significant differences in the glycosylation patterns between the different cell cultures. All 4 cell lines were apparently able to synthesize the mannose- and glucosamine- containing core structure and branch chains terminating in sialic acid which are characteristic of asparagine-linked carbohydrate side chains in cellular glycoproteins. Analysis of tryptic glycopeptides by anion-exchange chromotography indicated that the same 2 major sites on the virus polypeptide were recognized and glycosylated in all 4 VSV-infected cell cultures. These studies suggest that the basic biochemical defect(s) in cystic fibrosis is not an absence or deficiency in enzymes responsible for the biosynthesis of complex carbohydrate side chains.     

Quantitative two-dimensional gel electrophoresis analysis of human fibroblasts transformed by ras oncogenes.

Quantitative two-dimensional gel electrophoresis was used to compare the cellular protein patterns of a normal foreskin-derived human fibroblasts cell line (LG1) and three immortal derivatives of LG1. One derivative, designated MSU-1.1 VO, was selected for its ability to grow in the absence of serum and is non-tumorigenic in athymic mice. The other two strains were selected for focus-formation following transfection with either Ha-ras or N-ras oncogenes and form high grade malignant tumors. Correspondence and cluster analysis provided a nonbiased estimate of the relative similarity of the different two-dimensional patterns. These techniques separated the gel patterns into three distinct classes: LG1, MSU-1.1 VO, and the ras transformed cell strains. The MSU-1.1 VO cells were more closely related to the parental LG1 than to the ras-transformed cells. The differences between the three classes were primarily quantitative in nature: 16% of the spots demonstrated statistically significant changes (P < 0.01, T test, mean ratio of intensity > 2) in the rate of incorporation of radioactive amino acids. The patterns from the two ras-transformed cell strains were similar, and variations in the expression of proteins that occurred between the separate experiments obscured consistent differences between the Ha-ras and N-ras transformed cells. However, while only 9 out of 758 spots were classified as different (1%), correspondence analysis could consistently separate the two ras transformants. One of these spots was five times more intense in the Ha-ras transformed cells than the N-ras.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pyrimidine biosynthesis in normal and transformed cells

We have developed procedures for sensitive measurement of specific radioactivities of pyrimidine nucleosides excreted from cells in culture. The changes in the observed values reflect dilution of the added isotope through de novo biosynthesis of nonradioactive pyrimidine nucleosides or by shifting and equilibration of other nucleotide pools into the free uridine pool. It is thus possible to monitor uridine biosynthesis occurring in intact cells without destroying or disrupting the cell population. On comparing a series of normal and and transformed lines, we have observed several growth-dependent patterns of change in specific activity and levels of uridine excretion and the temporal appearance of these changes. Hamster embryo fibroblasts slows pyrimidine biosynthesis at mid-growth while the hamster cell line V79 continues to dilute the pyrimidine pool at about 7% of the rate observed during exponential growth at confluence. Both cells exhibit Urd excretion beginning at one-half maximal growth. Passageable normal rat liver cells (IARC-20) also show a cessation of pyrimidine biosynthesis with a prior increase in uridine excretion. Two chemically transformed lines IARC-28 and IARC-19 derived from IARC-20 show different patterns. IARC-19 begins uridine excretion in early log growth and the specific activity continues to decrease at about 2% of the rate observed during exponential growth at confluence. The IARC-28 cells also begin excretion in early log growth but pyrimidine biosynthesis stops at about midlog. This method may prove to be an additional aid in recognizing and differentiating transformed cells in culture that do not exhibit the transformed phenotype.     

Modulation of cell surface iron transferrin receptors by cellular density and state of activation.

This report describes investigations of plasma membrane transferring receptors on a variety of lymphoid cell lines and normal peripheral blood lymphocytes during activation and cell growth cycles. Transformed lymphoid cell lines have as many as 1,000 times the number of receptors found on normal resting lymphocytes. The number of iron transferrin receptors on continuous cell lines as well as normal human fibroblasts is down-regulated during the transition from log-phase growth to stationary plateau growth. When normal lymphocytes are transformed by mixed lymphocyte culture or mitogens, they rapidly express a 50-fold increase in the number of transferrin binding sites. This appearance of iron transferrin receptors anticipates nuclear changes during cell activation and subsequent mitosis of normal cells.     

Polyoma T (tumor) antigen species in abortively and stably transformed cells.

Stable neoplastic transformation of cells by polyoma virus requires the particpation of two viral genes, designated ts-a and hr-t. The effects of mutations in these two genes on the patterns of T-antigen synthesis during productive infection have been previously described: ts-a mutants are affected in the "large" (100K) nuclear T antigen, and hr-t mutants are affected in the "middle" (36K, 56K, 63K) and "small" (22K) T antigens. The latter are associated predominantly with the plasma membrane (56K) and cytosol fractions, respectively. Here we examine the expression of the various forms of polyoma T antigen in nonproductive infection (abortive transformation) as well as in stably transformed cell lines of different species. The results on abortive transformation are essentially the same as those described above for productive infection. In stably transformed cells, the middle and small T antigens are seen to various extents. The large T antigen, however, is often absent or present below the level of detection. Clones lacking the large T antigen are found most often among mouse transformants, but are also seen among rat transformants. Retention of the 100K species in transformed cells therefore appears to be, at least in part, an inverse function of the level of permissivity of the host toward productive viral infection. These findings indicate that the induction of the transformed phenotype in both abortively and stably transformed cells generally does not require the large T antigen, but rather the products of the hr-t gene.     

Neuron models.

The nervous system is made up of several kinds of cells which should ideally be examined separately in biochemical studies. Since we are not yet able to isolate the different kinds of cells, use is made of cell lines derived from a single cell which are isolated from tumors of the nervous system and which can be reproduced at will in the same way as bacteria. They include cell lines isolated from a mouse neuroblastoma which exhibit some of the properties of nerve cells. Hybrid cells obtained by fusion of mouse neuroblastoma cells with rat glioma cells display even more characteristics of nerve cells. This article considers properties which justify our regarding the hybrid cells as neuron models. Application of the cells to problems of the nervous system is then demonstrated: (1) processing of information arriving in the form of mutually opposing hormonal signals; (2) mode of action of morphine.     

Action of phorbol esters in cell culture: mimicry of transformation, altered differentiation, and effects on cell membranes.

The carcinogenic process is usually multifactor in its causation and multistep in its evolution. It is likely that entirely different molecular mechanisms underlie the many steps in this process. In contrast to initiating carcinogens, the action of the tumor-promoting phorbol esters does not appear to involve covalent binding to cellular DNA and they are not mutagenic. Recent studies in cell culture have revealed two interesting biologic effects of the phorbol esters and related macrocyclic plant diterpenes. The first is that at nanomolar concentrations they induce several changes that resemble those seen in cells transformed by chemical carcinogens or tumor viruses. These include altered morphology and increased saturation density, altered cell surface fucose-glycopeptides, decrease in the LETS protein, increased transport of deoxyglucose, and increased levels of plasminogen activator and ornithine decarboxylase. In transformed cells exposed to phorbol esters the expression of these features is further accentuated. Phorbol esters do not induce normal cells to grow in agar but they do enhance the growth in agar of certain transformed cells. The second effect of the phorbol esters is inhibition of terminal differentiation. This effect extends to a variety of programs of differentiation and is reversible when the agent is removed. With certain cell culture systems induction of differentiation, rather than inhibition, is observed. Both the transformation mimetic and the differentiation effects are exerted by plant diterpenes that have tumor-promoting activity but not by congeners that lack such activity. The primary target of phorbol esters appears to be the cell membrane. Early membrane-related effects include enhanced uptake of 2-deoxyglucose and other nutrients, altered cell adhesion, induction of arachidonic acid release and prostaglandin synthesis, inhibition of the binding of epidermal growth factor to cell surface receptors, altered lipid metabolism, and modifications in the activities of other cell surface receptors. A model of "two stage" carcinogenesis encompassing the known molecular and cellular effects of initiating carcinogens and tumor promoters is presented. According to this model, initiating carcinogens induce stable alterations in the cellular genome but these are not manifested until tumor promoters modulate programs of gene expression and induce the clonal outgrowth of the initiated cell.     

Two-dimensional electrophoretic analysis of transformation-sensitive polypeptides during chemically, spontaneously, and oncogene-induced transformation of rat liver epithelial cells.

Recently, we described the establishment of a computerized database of rat liver epithelial (RLE) cellular polypeptides (Wirth et al., Electrophoresis, 1991, 12, 931-954). This database has now been expanded to include the analysis of cellular polypeptide alterations during chemically (aflatoxin B1; AFB), spontaneously, and oncogene (v-Ha-ras, v-raf, and v-myc/v-raf)-induced transformation of RLE cells. Two-dimensional mapping of [35S]methionine-labeled whole cell lysate, cell-free in vitro translation products and [32P]orthophosphate-labeled polypeptides revealed subsets of polypeptides specific for each transformation modality. A search of the RLE protein database indicated the specific subcellular location for the majority of these transformation-sensitive proteins. Significant alterations in the expression of the extracellular matrix protein, fibronectin, as well as tropomyosin- and intermediate filament-related polypeptides (vimentin, beta-tubulin, the cytokeratins, and actin) were observed among the various transformant cell lines. Immunoprecipitation and Western immunoblot analysis of tropomyosin expression in four individual AFB-, as well as four spontaneously induced, and each of the oncogene-transformed cell lines indicated that five major tropomyosin (Tm 1-5) isoforms were variably expressed in the various cell lines, including one polypeptide tentatively identified as Tm6. Whereas alterations in tropomyosin expression appeared to be transformation-specific, alterations in the individual intermediate filament polypeptides were related more to the differentiation state of the individual cell lines rather than to the transformation phenotype. These studies extend our earlier efforts toward the establishment of a comprehensive computerized database of RLE cellular proteins and demonstrates how such a database may serve as a useful source for studies concerning the regulation of growth and differentiation as well as transformation of RLE cells.     

Lunasin: a novel cancer preventive seed peptide that modifies chromatin

Lunasin is a novel cancer preventive peptide whose efficacy against chemical carcinogens and oncogenes has been demonstrated in mammalian cells and a skin cancer mouse model. In contrast, constitutive expression of the lunasin gene in mammalian cells leads to arrest of cell division and cell death. Isolated and characterized in soy, lunasin peptide is also documented in barley and wheat and is predicted to be present in many more seeds because of its possible role in seed development. Initial studies show that lunasin is bioavailable in mice when orally ingested. Lunasin internalizes into mammalian cells within minutes of exogenous application, and localizes in the nucleus after 18 h. It inhibits acetylation of core histones in mammalian cells but does not affect the growth rate of normal and established cancer cell lines. An epigenetic mechanism of action is proposed whereby lunasin selectively kills cells being transformed or newly transformed cells by binding to deacetylated core histones exposed by the transformation event, disrupting the dynamics of histone acetylation-deacetylation.     

Selection of malignant melanoma variant cell lines for ovary colonization

Murine melanoma line B16-F1, which shows some specificity for metastatic organ colonization of lung but rarely metastasizes to ovary, was used to select variant cell lines with increased preference for experimental ovary metastasis. Ovary-colonizing melanoma cell lines were sequentially selected in syngeneic C57BL/6 mice by repeated intravenous administration and surgical recovery of ovarian melanoma tumors for tissue culture. After ten selections for experimental ovary metastasis, line B16-010 was established which formed experimental metastatic ovary tumors in almost every test animal. In tissue culture B16-010 cells grew in circular colonies with rounded, smooth cell peripheries compared to B16-F1 cells which were flatter, grew in irregular patterns, and exhibited long cellular projections. Ovary-selected B16 lines contained less melanin pigment (B16-010 less than B16-05 less than B16-01 approximately equal to B16-F1) compared to the parental melanoma line. Together with previous cloning and selection data, these results are consistent with the preexistence of highly malignant cells in the parental tumor population that possess the ability to metastasize to specific organs.     

The area-code hypothesis: the immune system provides clues to understanding the genetic and molecular basis of cell recognition during development

Numberous studies of embryogenesis have provided evidence for highly specific cell-surface recognition phenomena. These include both the interactions of neighboring cells and the specific cellular migrations which occur as the developmental program of the embryo progresses. The area-code hypothesis elaborate here is an attempt to provide a framework for understanding cell-recognition phenomena in development. This hypothesis is based on extensive genetic, molecular, and cellular studies of the immune system. These studies suggest that the following events occur during the differentiation of antibody-producing cells. 1) Somatic cell lines of antibody-producing cells undergo a modification of their DNA as they become committed to synthesize a particular type of antibody molecule. This chromosomal modification event is probably a DNA translocation which leads to a somatic rearrangement of certain antibody genes. 2) In each of the specific cell lineages the new arrangement of DNA is inherited by all subsequent generations of cells. 3) The developmental programs which control these genetic alterations may be employed in a programmed and reproducible fashion. This programming of antibody development is suggested because different embryos appear to become committed to the production of identical antibody molecules in the same developmental sequence. 4) Antibody molecules are initially displayed on the cell surface where they serve as highly specifici receptors to trigger the cell to proliferate and differentiate upon interacting with appropriate external molecular signals. 5) Antibody-producing cells display combinations of different molecules on their surfaces which cause each of a very large number of different cells to interact differently with their environment. 6) The genes which code for many of these cell-surface molecules are organized into multigene families. These observations as well as information from other developmental systems have led us to propose the area-code hypothesis. This hypothesis is concerned with the structure, function, and regulation of cell-surface molecules that mediate recognition phenomena during embryogenesis. Area-code molecules are cell-surface molecules which are involved in the specific recognition phenomena during growth and development. These molecules provide cells with distinct cell-surface addresses or phenotypes, and provide the basis for the specificity in cell-cell recognition during cell migrations and cell-cell interactions, as well as serving as receptors for diffusible differentiation signals. The area-code hypothesis has 3 main postulates. i) There is a progressive display of specific combinations of area-code molecules on the surfaces of cells during development. ii) The genetic programs which determine the specific expression of area-code molecules are in part controlled by DNA modifications. These chromosomal modifications are believed to channel cells into specific lineages uith progressively restricted developmental options...     

A near-infrared Fourier transform Raman spectroscopy of epidermal keratinocytes: changes in the protein-DNA structure following malignant transformation

We report here the use of near-infrared (NIR) Fourier transform (FT) Raman spectroscopy to analyze normal human epidermal keratinocytes prior to and following malignant transformation. Our analysis indicates specific Raman spectral differences between immortalized (HPK1A) and malignant ras transformed (HPK1A-ras) cells. In addition, striking spectral differences are seen in the DNA isolated from these cells and particularly in the 843/810 cm(-1) ratio with values of 1.6 +/- 0.13 in HPK1A cells and 0.68 +/- 0.09 in HPK1A-ras cells (mean +/- S.D., n = 12, P < 0.001) indicating specific alterations in the backbone conformation markers following malignant transformation. Subsequently, we analysed the effect of a strong inhibitor of keratinocyte growth, the Vitamin D analog EB1089, on the Raman spectra of intact cells and on the 843/810 cm(-1) ratio in the DNA isolated from both cell lines. Specific changes were observed in intact cells in the 1300-750 cm(-1) region. Furthermore, the 843/810 cm(-1) ratio of isolated DNA from HPK1A cells was not affected by EB1089 but significantly increased in DNA isolated from HPK1A-ras cells so much that it became closer to the value observed for HPK1A cells (1.07 +/- 0.10). Our data suggest that Raman analysis of DNA and in particular the 843/810 cm(-1) ratio can provide useful indices of malignant transformation and efficacy of anticancer agents.     

Towards a fully synthetic MUC1-based anticancer vaccine: efficient conjugation of glycopeptides with mono-, di-, and tetravalent lipopeptides using click chemistry

The membrane‐bound tumor‐associated glycoprotein MUC1 is aberrantly glycosylated in cancer cells compared with normal cells, and is therefore considered an attractive target for cancer immunotherapy. However, tumor‐associated glycopeptides from MUC1 do not elicit a sufficiently robust immune response. Therefore, antitumor vaccines were developed, which consist of MUC1 glycopeptides as the B epitopes and immune‐stimulating toll‐like receptor 2 (TLR 2) lipopeptide ligands. These fully synthetic vaccine candidates were prepared by solid‐phase synthesis of the MUC1 glycopeptides. The Pam₃Cys lipopeptide, also synthesized on solid‐phase, was C‐terminally coupled to oligovalent lysine cores, which N‐terminally incorporate O‐propargyl oligoethylene glycol acyl side chains. The MUC1 glycopeptides and lipopeptide lysine constructs were then conjugated by click chemistry to give oligovalent synthetic vaccines. Oligovalent glycopeptide–lipopeptide conjugates are considered more immunogenic than their monovalent analogues.     

Metabonomic analysis of serum reveals antifatigue effects of Yi Guan Jian on fatigue mice using gas chromatography coupled to mass spectrometry

Yi Guan Jian (YGJ), one of the most commonly used traditional Chinese medicines, has been reported to possess significant antifatigue effects. However, the mechanisms underlying its antifatigue effects remain largely unresolved. In this study, a metabonomics approach, involving gas chromatography coupled to mass spectrometry and a multivariate statistical technique, was developed to estimate the extent to which YGJ alleviated the exhausting swimming‐induced fatigue of mice. High‐dose treatment with YGJ significantly extended the swimming time of fatigued mice. Significant alterations of metabolites involving amino acids, organic acids and carbohydrates were observed in the serum of fatigued mice, which were reversed by YGJ treatment while biochemical indexes returned to normal. These metabolic changes suggest that the antifatigue effect of YGJ is associated with the impairement of amino acid, organic acids and carbohydrates. It also appears that YGJ can induce significant metabolic alterations independent of the exhausting swimming‐induced metabolic changes. The significantly altered metabolites induced by YGJ intervention include l‐ 2‐amino‐acetoacetate, taurine, fumaric acid, malic acid, oxoadipic acid and l‐ aspartate, all of which are associated with antifatigue properties. This suggests that YGJ exerts chemopreventive effects via antifatigue mechanisms.     

TRANSFORMATION OF BALB 3T3 CELLS EXPOSED TO A GERMICIDAL UV LAMP AND A SUNLAMP

Abstract— The effect of germicidal UV and sunlamp exposure on direct and simian virus-40 (SV-40) transformation of Balb 3T3 cells was studied. Transformation was determined by the ability of transformed cells to grow as clones in agar. Radiation from these lamps enhanced direct transformation, and enhanced viral transformation to approximately the same degree. Enhanced transformation was seen with exposures of light that caused no measurable cell killing, which suggests that the induction of new transformants is involved rather than the selection of pre-existing transformants. Induction is also suggested by post-irradiation growth kinetics experiments.     

Quantification of glycopeptides by multiple reaction monitoring liquid chromatography/tandem mass spectrometry

Protein glycosylation has a major influence on functions of proteins. Studies have shown that aberrations in glycosylation are indicative of disease conditions. This has prompted major research activities for comparative studies of glycoproteins in biological samples. Multiple reaction monitoring (MRM) is a highly sensitive technique which has been recently explored for quantitative proteomics. In this work, MRM was adopted for quantification of glycopeptides derived from both model glycoproteins and depleted human blood serum using glycan oxonium ions as transitions. The utilization of oxonium ions aids in identifying the different types of glycans bound to peptide backbones. MRM experiments were optimized by evaluating different parameters that have a major influence on quantification of glycopeptides, which include MRM time segments, number of transitions, and normalized collision energies. The results indicate that oxonium ions could be adopted for the characterization and quantification of glycopeptides in general, eliminating the need to select specific transitions for individual precursor ions. Also, the specificity increased with the number of transitions and a more sensitive analysis can be obtained by providing specific time segments. This approach can be applied to comparative and quantitative studies of glycopeptides in biological samples as illustrated for the case of depleted blood serum sample. Copyright © 2012 John Wiley & Sons, Ltd.     

Molecules Present in Plant Essential Oils for Prevention and Treatment of Colorectal Cancer (CRC)

Essential oils (EOs) are a complex mixture of hydrophobic and volatile compounds synthesized from aromatic plants, commonly present in the human diet. In recent years, many in vitro studies have suggested possible anticancer properties of single EO compounds, on colorectal cancer (CRC) cells. However, the majority of these studies did not compare the effects of these compounds on normal and cancer colon cells. By using NCM-460, a normal human mucosal epithelial cell line, Caco-2, a human colon epithelial adenocarcinoma cell line, and SW-620, colon cancer cells derived from lymph node metastatic site, we identified cinnamaldehyde, derived from cinnamon EO and eugenol, derived from bud clove EO, as compounds with a specific anticancer action selectively targeting the transformed colonic cells. Both cinnamaldehyde (75 µM) and eugenol (800 µM), after 72 h of treatment, were capable to induce apoptosis, necrosis and a cell cycle slowdown in Caco-2 and in SW-620, but not in NCM-460 cells. If associated with a targeted delivery to the colon, these two compounds could prove effective in the prevention or treatment of CRC.     

IDENTIFICATION OF A TRANSFORMING ras ONCOGENE IN AN ULTRAVIOLET RADIATION-INDUCED CORNEAL TUMOR OF Monodelphis domestica

Chronic exposure of the gray, short-tailed oppossum, Monodelphis domestica to ultraviolet radiation (UVR) induces mesenchymal tumors of the cornea. High molecular weight DNA samples from 6 UVR-induced corneal tumors were assayed for their ability to transform NIH 3T3 cells to tumorigenicity. NIH 3T3 cells transfected with DNA from 5 of the corneal tumors produced 14 tumors in nude mice. Cell lines were established from these tumors. DNA from 13 of 14 tumor cell lines contained repetitive opossum DNA sequences. Southern blot analysis revealed that DNA from 3 of 4 cell lines derived from tumorigenic NIH 3T3 cells transfected with DNA from a single oppossum tumor contained opossum Ki-ras oncogene sequences in addition to the murine Ki-ras gene. Northern blot analysis of mRNA from a mouse tumor cell line containing opossum Ki-ras gene sequences showed mRNA species identical in size to opossum Ki-ras mRNA, as well as murine Ki-ras mRNA species. These results suggest that an activated Ki-ras oncogene was present in one of the original opossum corneal tumors tested. Thus, activation of Ki-ras may play a role in the development of UVR-induced corneal tumors in Monodelphis domestica. Further characterization of ras oncogenes in these opossum tumors may provide information on the molecular mechanisms by which UVR induces corneal tumors in this species.     

In vivo INDUCTION OF MALIGNANT TRANSFORMATION OF NUDE MOUSE B LYMPHOCYTES BY A HUMAN MULTIPLE MYELOMA

The hone marrow puncture fluid of human multiple myeloma was inoculated subcutaneously in BALB/C/nu nude mice. After passage of the xenograft from nude mice in vitro, a murine malignant lymphosarcoma cell line was unexpectedly established, which was named NLB-SK. The murine NLBSK cell line has been cultivated in vitro over 72 passages for 10 months. Repeated cryopreservation showed that the murine NLB-SK cell line revived satisfactorily. Based on cell biological characteristics the malignant transformation migh the attributed to horizontal oncogenesis between human malignant tumor cells and nude mouse normal somatic cells.