A cell regulatory sialoglycopeptide, CeReS-18, purified from intact bovine cerebral cortex cells, has exhibited the capability of reversibly inhibiting cellular DNA synthesis and the proliferation of a wide array of mammalian cells. In the present study, the effect of CeReS-18 on the proliferation of bacterial (Bacillus cereus and Escherichia coli) and yeast (Saccharomyces cerevisiae and Schizosaccharomyces pombe) cells was investigated. The results showed that replication and viability of the bacterial cells were not affected by CeReS-18 at any concentration tested, including 15-fold higher than that used for inhibiting mouse 3T3 cell proliferation. In contrast to bacterial cells, CeReS-18 was able to inhibit the replication of yeast cells, in a concentration-dependent, reversible manner, and the addition of calcium to the culture medium could abrogate the inhibitory effect of CeReS-18. A cytotoxic effect of CeReS-18 on both yeast cell species was observed when it was applied at higher concentrations.

We have purified a cell regulatory sialoglycopeptide, CeReS-18, from intact bovine cerebral cortex cells. This is an 18-kDa molecule that reversibly inhibits cellular DNA synthesis and the proliferation of a wide array of target cells. In the present study, the effect of CeReS-18 on mouse 3T6 host cell proliferation and polyomavirus replication was investigated. The results showed that CeReS-18 was able to inhibit 3T6 cell cycling in a concentration-dependent, calcium-sensitive, and reversible manner. Despite the inhibition of cell proliferation, CeReS-18 did not influence polyomavirus infection of 3T6 cells. Indirect immunofluorescent assays revealed that CeReS-18-treated, and cell cycle-arrested, 3T6 cells remained permissive to polyomavirus replication. Electron microscopy and immunogold labeling showed that new viral particles were assembled inside the nuclei of infected cells in the presence of CeReS-18 and during cell cycle arrest. The cellular requirements for the replication of polyomavirus DNA and the synthesis of viral proteins, as well as for the assembly of viral particles, therefore, remained available in CeReS-18-inhibited 3T6 cells. In addition, although polyomavirus infection can be mitogenic, infection of CeReS-18-treated 3T6 cells did not reverse the cell cycle arrest mediated by this cell cycle inhibitor.

A series of studies have tested the possibility that the microgravity environment may be superior to laboratories on earth for several biomedical applications. One such application is isoelectric focusing (IEF). The purpose of our research is to design, build, test, and employ an analytical IEF instrument for use in the laboratory on the International Space Station (ISS) and to demonstrate the advantages of space-based IEF. This paper describes IEF in general, discusses the design considerations that arise for IEF in low-gravity, and presents design solutions to some of the systems under development. Isoelectric focusing is a powerful technique that has applications for both analytical analysis the preparative purification of macromolecules. IEF resolves proteins by net charge separation, in either liquid or semi-solid substrates, where the molecules migrate to their isoelectric point (pI). In earth-based IEF, separation media are usually semi-solids such as polyacrylamide and agarose gels. The matrix structure of these media is used to offset the gravity-induced diffusion and convection that occurs in free solutions. With these effects being greatly reduced, a free solution could be used as a superior media. Because diffusion in liquids is reduced in microgravity (Snyder, 1986), a given electrical field should result in more tightly focused bands. This would allow for the separation of proteins that have very closely spaced pI’s. If superior results are achieved, there are numerous pharmaceutical and genetic engineering companies that would take advantage of this unique development. The design of the Commercial IsoElectric Focusing Apparatus (CIEFA) presents several significant engineering challenges specific to its operation in the microgravity environment. Three difficulties of particular importance are gases generated through electrolysis, temperature control and verification of protein separation. Gases generated through electrolysis must be isolated from electrodes to prevent current limiting. Special measures for temperature control must be made due to the absence of gravity-induced convective heat flow. In order for the experiment results to be examined, some mechanism must be in place to either document or preserve the protein bands. Preliminary testing aboard the space shuttle requires that the CIEFA be compatible with the shuttle’s middeck locker. This requirement poses limits in the physical parameters of size, mass, power consumption, and heat generation. In addition, the design must be NASA certifiable for shuttle flight. This diverse list of design obstacles requires integration of biological, electrical, and mechanical solutions.

The requirement for long term storage of cell cultures previous to arrival on the International Space Station (ISS), as well as culture maintenance after the conduct of experiment in microgravity, necessitates inhibition of cell proliferation and metabolism pending return to earth-based laboratories. Transport of cells in a nonstabilized condition can lead to a loss of cell viability and/or a source of selection pressures for survival that can alter the overall cell population. We have isolated in our laboratory a reversible inhibitor of cell proliferation, a cell regulatory sialoglycopeptide (CeReS-18), that has the capability of stabilizing cells isolated from a wide phylogenetic range by arresting them in the G1 phase of the cell cycle. We show here that CeReS-18 is unusually stable and can be stored at ambient temperatures for weeks without a measurable loss in its biological activity. In addition we demonstrate that CeReS-18 is a superior cell-stabilizing agent as compared to other methods deployed for cell stabilization purposes, such as, decrease in the incubation temperature and serum down shifts. We also discovered that hybridoma cultures stabilized in their proliferative cycle by CeReS-18 produced 150%–300% more antibody per cell than that measured in the proliferating control cultures. The reversible inhibitory activity of CeReS-18, together with its unusual stability, as well as its wide target range lend themselves to use of this inhibitor as a cell stabilizing agent for cell transport to and storage on the ISS.

CeReS-18, a cell regulatory sialoglycopeptide, has been shown to inhibit proliferation of a wide array of target cells. In the present study, the effect of CeReS-18 on vascular smooth muscle cell (SMC) proliferation was characterized in cultured rat aorta SMCs (A7r5). More extensively, the effect of CeReS-18 on platelet-derived growth factor (PDGF)-induced SMC migration was examined using a modified Boyden's chamber assay. CeReS-18 inhibits both SMC proliferation and migration in a concentration-dependent, calcium-sensitive, and reversible manner. Furthermore, cells preincubated with the inhibitor had an increased sensitivity to CeReS-18-mediated inhibition of SMC migration. Immunoprecipitation and in vitro phosphorylation assays demonstrated that MAP kinase activity was inhibited in the CeReS-18-treated cells and pretreatment with CeReS-18 suppressed the activation of MAP kinase stimulated by PDGF. However, it is not likely that the suppression of the MAP kinase pathway was directly responsible for the ability of CeReS-18 to inhibit migration of the rat aorta smooth muscle cells since a MEK-specific inhibitor, PD98059, did not influence A7r5 cell migration.

BACKGROUND. Polypeptide growth factors are positive and negative regulators of prostatic growth and function, and many positive regulators of growth in the prostate have been extensively studied. However, very few inhibitors of prostate cell proliferation have been identified. We have isolated a unique 18-kDa sialoglycopeptide (CeReS-18) which inhibits cell proliferation of three separate lines of human prostate cancer cells, as well as inducing cellular cytotoxicity via an apoptotic pathway unrelated to the Bcl-2 family of proteins.METHODS. Cell cycle inhibition was analyzed by direct cell counts with a Coulter (Miami, FL) cell counter. Apoptotic cells were analyzed by electron microscopy, annexin V-fluorescein isothiocyanate (FITC) staining, fluorescence microscopy, and propidium iodide uptake measured with a fluorescence-activated cell sorter. Expression of the proteins of the Bcl-2 family was detected by Western blot analysis.RESULTS. We found that CeReS-18 inhibits cell proliferation of androgen-responsive, LNCaP.FGC human prostate cancer cells, as well as of androgen-nonresponsive DU-145 and PC3 human prostate cancer cells. Furthermore a, fivefold increase over the inhibitory concentration of CeReS-18 elicited a cytotoxic response by all three cell lines. We thus characterized the cytotoxic mechanism as apoptotic in nature, and we measured the expression of several members of the Bcl-2 family in PC3 cells upon treatment with CeReS-18.CONCLUSIONS. The data indicate that CeReS-18 is a potent inhibitor of cellular progression through the cell cycle by both androgen-responsive and androgen-nonresponsive human prostate cancer cells. In addition, treatment of both types of cells with increased concentrations of CeReS-18 induces cellular cytotoxicity, characterized as apoptosis. (C) 1999 Wiley-Liss, Inc.

CeReS-18 is a unique negative regulator of cell proliferation with a wide array of target cells. To elucidate the mechanism by which CeReS-18 mediates cell growth inhibition, the possibility that CeReS-18 alters the function of G1 cyclins and their respective cyclin-dependent kinases (cdks) has been examined in mouse fibroblasts (Swiss 3T3) synchronized by CeReS-18. We show here that cyclin D-associated cdk activity is significantly inhibited in the CeReS-18-treated cells. Corresponding to the inhibited cdk function, we demonstrate a low expression of cyclin D in mid G1 determined by Western blot analysis, and cyclin D was greatly reduced in the immunocomplex recovered with antibody to cdk4 and cdk6. Previously, we have shown that the retinoblastoma susceptibility gene product (pRb), a key substrate of cyclin D-cdk complex, was maintained in the hypophosphorylated state in the CeReS-18-inhibited cells. We conclude here that cyclin D/cdk4,6/pRb is the major pathway by which CeReS-18 mediates cell cycle arrest.

A considerable amount of attention has been focused on the physiological factors that are responsible for the reduction of bone mineralization and mass during prolonged periods in the microgravity environment. Although bane mineralization can be reduced by one percent per month as shown to result from shuttle flights and Mir habitation, the reasons for this phenomenon remain unclear. Changes in specific markers of bone cells upon differentiation indicate that the induction of bone matrix formation is dependent upon these cells reaching confluency. In our laboratory, we have isolated a reversible inhibitor of cellular growth (CeReS-18) that could be important in cell contact inhibition and thus may mimic the signals involved in growth confluency. Preliminary experiments with osteogenic cells have revealed the potential capability of CeReS-18 to inhibit these cells in a reversible manner. We are developing a series of studies, designed at the cellular level, to quantitatively measure the production of bone matrix by osteogenic cells propagated in culture. The use of CeReS-18 would facilitate the study of several factors being assessed regarding matrix formation including the rate of cell population density, hormone induction events, calcium availability, and cell cycle arest. The studies are being conducted in a manner that will allow comparable measurements in the microgravity environment with flight hardware designed and deployed by BioServe Space Technologies.

The development of a linear pH gradient is necessary for the precise and consistent separation of biomolecules via isoelectric focusing techniques. Existing applications have used various voltage potentials in performing the separations, but the exact relationship between the voltage and the required time duration on the development of a linear pH gradient has not been determined. Certain applications, such as separations within the microgravity environment, can benefit from the use of low voltages, whereas other applications are not constrained by voltage limitations. In general, as the voltage increases, the time duration required for high-definition separation appears to be reduced, but the effect of these changes on the linearity of the pH gradient was not previously known. A mixture of ampholytes with a pH range of 3 to 10 was used in the investigation of this relationship, and voltage levels from 125 V to 1000 V were employed. A 6% polyacrylamide gel in a cylindrical configuration was used as the medium in all the experiments. The pH profiles of the gels were determined immediately after the conclusion of each time-duration experiment, without disturbing the integrity of the gel. This was accomplished by using a specialized iridium-oxide pH sensor that can be placed directly on the gel surface. The authors present their findings on the ability to produce linear pH gradients as a function of the voltage level applied and the time duration of the isoelectric focusing experiment.

Very few growth inhibitors have been identified whichcan inhibit the proliferation of a broad spectrumof human breast cancer cell lines. CeReS-18, anovel cell surface sialoglycopeptide growth inhibitor, can reversiblyinhibit the proliferation of both estrogen receptor positive(MCF-7) and negative (BT-20) human breast cancer celllines. In addition, at concentrations above those requiredfor the reversible inhibition of cell proliferation, CeReS-18can also induce cell death in MCF-7 cells.Changes in nuclear and cytoplasmic morphology, characteristic ofapoptosis, were detected in MCF-7 cells treated witha cytotoxic concentration of CeReS-18, and internucleosomal DNAcleavage was also observed. The sensitivity of MCF-7and BT-20 cells to the biological properties ofCeReS-18 could be influenced by altering the calciumconcentration in the extracellular growth medium, such thatwhen the calcium concentration in the environment wasdecreased, an increased sensitivity to CeReS-18-induced growth inhibitionand cytotoxicity were observed. The addition of thecalcium chelating agent EGTA to MCF-7 cells, culturedin a normal calcium environment, could mimic theincreased sensitivity to the biological effects of CeReS-18observed under reduced calcium conditions.

We have previously documented that CeReS-18, a cellregulatory sialoglycopeptide, inhibits the cellular proliferation of normaland transformed cell types from a diverse rangeof species. Most cell types studies exhibit asimilar sensitivity to the reversible but growth inhibitoryeffects of CeReS-18 at 7 × 10 -8 Mconcentration, while at higher concentrations CeReS-18 can elicitcytotoxicity. The present study was conducted to examinethe effect of CeReS-18 on the proliferation ofhuman mammary epithelial carcinoma cells. MCF-7 cells, whichare estrogen receptor positive (ER + ), and BT-20 cells,which are estrogen receptor negative (ER - ), were utilized.Both cell lines show equal sensitivity to growthinhibition elicited by CeReS-18. Complete cessation of cellcycling was achieved with 7 × 10 -8 MCeReS-18, and the arrest was shown to becompletely reversible. Flow cytometric analysis, performed on CeReS-18treated cells from both cell types, revealed thatthe majority of these cells were arrested inthe G1 phase of the cell cycle. Whencells were treated simultaneously with inhibitor and stimulatoryconcentrations of mitogens such as epidermal growth factor(EGF), basic fibroblast growth factor (b-FGF), estrogen, insulin-likegrowth factors I and II (IGFI and IGFII),no alteration of the inhibitory activity of CeReS-18was observed. CeReS-18 clearly abrogated the mitogenic activitythat these growth factors elicited with human mammarycarcinoma cells.

Studies conducted on two recent shuttle missions (STS-69 and STS-73) have shown that cell cycle arrest of human myeloma IM-9 cells, mediated by a purified cell regulatory sialoglycopeptide (CeReS-18), can maintain cells in a stabilized condition for several days prior to launch. In addition, when the orbiter reached the microgravity environment, the inhibition was readily reversed by a simple dilution of the concentration of the inhibitory CeReS-18. This was accomplished by the introduction of fresh culture medium, without inhibitor. The previously stabilized cell population then rapidly initiated a renewal of cell cycling. Since it has been previously shown that the ability of CeReS-18 to stabilize cell populations in a nonproliferating state was calcium ion dependent (Betz et al. 1994, Betz et al. 1995) the current studies included the use of culture medium containing either high (0.42 mM) or low (0.042 mM) calcium concentrations.

Cell growth is controlled by the complex interactions of both positive and negative growth modulators. Studies were performed to directly compare the growth inhibitory properties of TGF-β1 and CeReS-18, a novel cell surface sialoglycopeptide growth inhibitor. Growth inhibition by CeReS-18 and that by TGF-β1 shared some similarities although significant differences were apparent. Similarities included a dose-responsive inhibition of BALB/c mouse keratinocyte (MK) cell proliferation that could be nontoxic and reversible. Both CeReS-18 and TGF-β1 could equally inhibit the stimulation of DNA synthesis induced by serum or keratinocyte growth factor in MK cells, and in both cases the inhibition was not due to decreased KGF binding to the target cell surface receptor. Inhibition of cell proliferation with CeReS-18, followed by an immediate reincubation with either CeReS-18 or TGF-β1, suggested that the sites of arrest mediated by both inhibitors were similar but not necessarily identical. However, recovery from CeReS-18-induced growth inhibition could be achieved by either removing the CeReS-18 or adding calcium directly to CeReS-18-inhibited MK cells, while recovery from TGF-β1-induced growth arrest could be reversed only by TGF-β1 removal. In addition, the sensitivity of MK cells to CeReS-18-induced cell cycle arrest could be altered by changing the extracellular calcium concentration, but sensitivity to TGF-β1 was unaffected by the calcium environment of the MK cells. While recovery from cell cycle arrest was rapid and complete with MK cell cultures inhibited with CeReS-18, removal of TGF-β1 led to a slower and incomplete recovery of cell cycling.

A number of homeodomain proteins have been shown to regulate cellular development by stimulating the transcription of specific target genes. In contrast to their distinct activities in vivo, however, most homeodomain proteins bind indiscriminately to potential target sites in vitro, suggesting the involvement of cofactors which specify target site selection. One such cofactor, termed extradenticle, has been shown to influence segmental morphogenesis in Drosophila melanogaster by binding cooperatively with certain homeodomain proteins to target regulatory elements. Here we demonstrate that STF-1, an orphan homeodomain protein required for pancreatic development in mammals, binds cooperatively to DNA with Pbx, the mammalian homolog of extradenticle. Cooperative binding with Pbx requires a pentapeptide motif (FPWMK) which is well conserved among a large subset of homeodomain proteins, The FPMWK motif is not sufficient to confer Pbx cooperativity on other homeodomain proteins, however; the N-terminal arm of the STF-1 homeodomain is also essential, As cooperative binding with Pbx occurs on only a subset of potential STF-1 target sites, our results suggest that Pbx may specify target gene selection in the developing pancreas by forming heterodimeric complexes with STF-1.

A series of studies have shown that a purified cell regulatory sialoglycopeptide (CeReS) that arrests cell division and induces cellular differentiation is fully capable of functionally interacting with target insect and mammalian cells in the microgravity environment. Data from several shuttle missions suggest that the signal transduction events that are known to be associated with CeReS action function as well in microgravity as in ground‐based experiments. The molecular events known to be associated with CeReS include an ability to interfere with Ca2+ metabolism, the subsequent alkalinization of cell cytosol, and the inhibition of the phosphorylation of the nuclear protein product encoded by the retinoblastoma (RB) gene. The ability of CeReS to function in microgravity opens a wide variety of applications in space life sciences.

Previous attempts to physically separate the cell cycle inhibitory and protease in activities in preparations of a purified cell regulatory sialoglycopeptide (CeReS) inhibitor were largely unsuccessful. Gradient elution of the inhibitor preparation from a DEAE HPLC column separated the cell growth inhibitor from the protease, and the two activities have been shown to be distinct and non‐overlapping. The additional purification increased the specific biological activity of the CeReS preparation by approximately two‐fold. The major inhibitory fraction that eluted from the DEAE column was further analyzed by tricine‐SDS‐PAGE and microbore reverse phase HPLC and shown to be homogeneous in nature. Two other fractions separated by DEAE HPLC, also devoid of protease activity, were shown to be inhibitory to cell proliferation and most likely represented modified relatives of the CeReS inhibitor. The highly purified CeReS was chemically characterized for amino acid and carbohydrate composition and the role of the carbohydrate in cell proliferation inhibition, stability, and protease resistance was assessed. © 1995 Wiley‐Liss, Inc.

While studies concerning mitogenic factors have been an important area of research for many years, much less is understood about the mechanisms of action of cell surface growth inhibitors. We have purified an 18 kDa cell surface sialoglycopeptide growth inhibitor (CeReS‐18) which can reversibly inhibit the proliferation of diverse cell types. The studies discussed in this article show that three mouse keratinocyte cell lines exhibit sixtyfold greater sensitivity than other fibroblasts and epithelial‐like cells to CeReS‐18‐induced growth inhibition. Growth inhibition induced by CeReS‐18 treatment is a reversible process, and the three mouse keratinocyte cell lines exhibited either single or multiple cell cycle arrest points, although a predominantly G0/G1 cell cycle arrest point was exhibited in Swiss 3T3 fibroblasts. The sensitivity of the mouse keratinocyte cell lines to CeReS‐18‐induced growth inhibition was not affected by the degree of tumorigenic progression in the cell lines and was not due to differences in CeReS‐18 binding affinity or number of cell surface receptors per cell. However, the sensitivity of both murine fibroblasts and keratinocytes could be altered by changing the extracellular calcium concentration, such that increased extracellular calcium concentrations resulted in decreased sensitivity to CeReS‐18‐induced proliferation inhibition. Thus the increased sensitivity of the murine keratinocyte cell lines to CeReS‐18 could be ascribed to the low calcium concentration used in their propagation. Studies are currently under way investigating the role of calcium in CeReS‐18‐induced growth arrest. The CeReS‐18 may serve as a very useful tool to study negative growth control and the signal transduction events associated with cell cycling. © 1994 Wiley‐Liss, Inc.

A novel cell regulatory sialoglycopeptide (CeReS‐18), purified from the cell surface of bovine cerebral cortex cells has been shown to be a potent and reversible inhibitor of proliferation of a wide array of fibroblasts as well as epithelial‐like cells and nontransformed and transformed cells. To investigate the possible mechanisms by which CeReS‐18 exerts its inhibitory action, the effect of the inhibitor on the posttranslational regulation of the retinoblastoma susceptibility gene product (RB), a tumor suppressor gene, has been examined. It is shown that CeReS‐18 mediated cell cycle arrest of both human diploid fibroblasts (HSBP) and mouse fibroblasts (Swiss 3T3) results in the maintenance of the RB protein in the hypophosphorylated state, consistent with a late G1 arrest site. Although their normal nontransformed counterparts are sensitive to cell cycle arrest mediated by CeReS‐18, cell lines lacking a functional RB protein, through either genetic mutation or DNA tumor virus oncoprotein interaction, are less sensitive. The refractory nature of these cells is shown to be independent of specific surface receptors for the inhibitor, and another tumor suppressor gene (p53) does not appear to be involved in the CeReS‐18 inhibition of cell proliferation. The requirement for a functional RB protein product, in order for CeReS‐18 to mediate cell cycle arrest, is discussed in light of regulatory events associated with density‐dependent growth inhibition. © 1994 Wiley‐Liss, Inc.