Heideh K. Fattaey

Modulation of growth-related gene expression and cell cycle synchronization by a sialoglycopeptide inhibitor

When an 18-kDa cell surface sialoglycopeptide (SGP), isolated from intact bovine cerebral cortex cells, was incubated with exponentially growing Swiss 3T3 cells, cell proliferation was efficiently arrested. The inhibition was totally reversible since after removal of the SGP the arrested cells resumed their progress in the cell cycle in a synchronized manner for at least two divisions. Readdition of the GSP 4 h after reversal of the inhibition did not, however, affect the commitment of the cells to advance through metaphase, although progress through the cell cycle was once again inhibited after the cells reentered the G1 phase. The efficient nature of the SGP-mediated cell cycle arrest in G1 provided us with a basis to examine potential changes in the expression of several competence genes, and genes associated with mid and late G1, that have been implicated in cell cycle progression. Upon serum stimulation of quiescent Swiss 3T3 cells, the induction of c-myc and c-fos expression was not influenced by the SGP at concentrations highly inhibitory to cell cycling. Expression of JE was induced by serum, and the presence of the SGP had little effect on the expression of this growth-related gene. KC expression was not appreciably stimulated by serum although, surprisingly, the addition of the SGP resulted in a significant increase in expression. In addition, we learned that the SGP did not alter expression of ornithine decarboxylase, c-ras, or thymidine kinase, which are induced later than the genes associated with the initial stages of competence.

Identification of a cell surface component of Swiss 3T3 cells associated with an inhibition of cell division

Polyclonal IgG, prepared to a purified bovine cell surface sialoglycopeptide (SGP) inhibitor of cell division, was used to identify an antigenically related molecule on the surface of Swiss 3T3 cells. SDS-PAGE and Western analyses showed that the anti-SGP antibody was monospecific and primarily recognized a 66-kDA protein of 3T3 cell membranes. Treatment of intact 3T3 cells or 3T3 cell membranes with either broad and phosphatidylinositol-specific phospholipase C enzymes suggested that the antigenic material most likely existed as an integral membrane molecule, or associated as a multimeric complex, and was not anchored at the cell surface by a phospholipid. The addition of anti-SGP IgG to 3T3 cell monolayer cultures was shown to promote cell division, suggesting a regulatory function for the membrane-associated molecule.

Cell cycle arrest and cellular differentiation mediated by a cell surface sialoglycopeptide

Cell cycling by a relatively wide variety of cell lines was shown to be reversibly inhibited by a cell surface sialoglycopeptide (SGP) isolated and purified from intact bovine cerebral cortex cells. Cell cycle arrest, mediated by the bovine SGP inhibitor, was shown to be completely reversible with mouse Swiss 3T3, mouse 1316 fibrosarcoma, mouse N2a neuroblastoma, bovine MDBK and monkey BSC-1 cells. These cell lines represented both fibroblast and epithelial-like cells, transformed and nontransformed cells, as well as their being derived from a broad array of species. In contrast to the others tested, human HL-60 leukemic cells were sensitive to the inhibitory effects of the SGP but did not reenter the mitotic cycle after the removal of the inhibitor. Instead, the mitotic arrest of HL-60 cells appeared to enhance entry into a terminal and irreversible state of cellular differentiation.

Murine polyomavirus infection of 3T6 mouse cells shows evidence of predominant necrosis as well as limited apoptosis

The current study was developed to determine if polyomavirus infected 3T6 mouse cells evoked an apoptotic or a necrotic mechanism during infection. Infected cells were analyzed by flow cytometry, transmission electron microscopy (TEM), DNA electrophoresis and by measuring caspase-3 enzymatic activity. Infected cells that were analyzed at 72 h post-infection showed the following: flow cytometry analysis revealed a 5% increase in apoptotic cells and a 46% increase in necrotic cells when compared to uninfected cells; electron microscopy showed 10% cells with characteristic apoptotic morphology and 40% with necrotic appearance; caspase-3 activity was found to increase two fold when compared to uninfected cells and DNA fragmentation (laddering) was clearly evident late in infection. It was concluded that infected cells predominantly showed necrosis, although some cells showed apoptosis in late infection. Recombinant capsid-like particles composed of the polyomavirus structural proteins were not able to induce cell death.

The role of a cell surface inhibitor in early signal transduction associated with the regulation of cell division and differentiation

Serum stimulation of quiescent human fibroblast cultures resulted in a hyperphosphorylation of the nuclear retinoblastoma gene susceptibility product (RB). However, serum stimulation in the presence of 9 x 10(-8) M of a purified bovine sialoglycopeptide (SGP) cell surface inhibitor abrogated the hyperphosphorylation of the RB protein and the subsequent progression of cells through the mitotic cycle. The experimental results suggest that the SGP mediated its cell cycle arrest at a site in the cell cycle that was at the time of RB phosphorylation or somewhat upstream of the modification of this regulatory protein of cell division. Both cells serum-deprived and serum stimulated in the presence of the SGP displayed only a hypophosphorylated RB protein, consistent with the SGP-mediated cell cycle arrest point being near the G1/S interface.

CeReS-18 inhibits growth and induces apoptosis in human prostatic cancer cells

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.

CeReS-18, a novel cell surface sialoglycopeptide, induces cell cycle arrest and apoptosis in a calcium-sensitive manner

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.

The stabilization of cell proliferation and its reversal in the microgravity environment

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.

Inhibition of hormone and growth factor responsive and resistant human breast cancer cells by CeReS-18, a cell regulatory sialoglycopeptide.

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.

Model system for studies on bone matrix formation by osteogenic cells in microgravity

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 bone 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 ...