aphidicolin and Osteosarcoma

The rate of protein synthesis in higher eukaryotes is largely regulated at the level of eIF2alpha phosphorylation by its kinases. A cellular glycoprotein, p67, protects eIF2alpha from phosphorylation. An enzyme, p67-deglycosylase, when active, removes the carbohydrate moieties from p67 and inactivates it. Subsequently, protein synthesis is inhibited. During mitosis the overall rate of protein synthesis sharply declines. To understand the molecular mechanism underlying this inhibition of protein synthesis, we have examined the phosphorylation of eIF2alpha and the activity of p67. We find that the phosphorylation of eIF2alpha increases at the G2/M border of cycling U2-OS cells, and p67 is deglycosylated at the same period of the cell cycle. In addition, the level and the activity of p67-deglycosylase also increase at the G2/M boundary of cycling U2-OS cells. These results thus provide an important in vivo correlation between the increased phosphorylation of eIF2alpha and deglycosylation of p67 by p67-deglycosylase at the G2/M boundary of cycling U2-OS cells. This may explain in part the inhibition of protein synthesis in U2-OS cells approaching mitosis.

Topics: Acetylglucosaminidase; Aminopeptidases; Animals; Aphidicolin; Enzyme Inhibitors; Eukaryotic Initiation Factor-2; G2 Phase; Glycoproteins; Glycosylation; Humans; Methionyl Aminopeptidases; Mitosis; Osteosarcoma; Phosphorylation; Protein Biosynthesis; Rabbits; Tumor Cells, Cultured

Tissue homeostasis and the prevention of neoplasia require regulatory co-ordination between cellular proliferation and apoptosis. Several cellular proteins, including c-myc and E2F, as well as viral proteins such as E1A, have dual functions as positive regulators of apoptosis and proliferation. The product of the retinoblastoma tumor suppressor gene, pRb, binds these proteins and is known to function in growth suppression. To examine whether pRb may function as a negative regulator of both proliferation and apoptosis, we analyzed apoptosis induced in transfected derivatives of the human osteosarcoma cell line SAOS-2. Ionizing radiation induced apoptosis in a time- and dose-dependent manner in SAOS-2 cells, which lack pRb expression. In both a transient and stable transfection assay, SAOS-2 derivatives expressing wild-type (wt) pRb exhibited increased viability and decreased apoptosis following treatment at a variety of radiation doses. Expression in SAOS-2 of a mutant pRb that fails to complex with several known binding partners of pRb, including E1A and E2F, did not protect SAOS-2 cells from apoptosis. Radiation exposure induced a G2 arrest in SAOS-2 and in derivatives expressing pRb. Inhibition of DNA synthesis and cell cycle progression by aphidicolin treatment failed to protect SAOS-2 cells or pRb-expressing isolates from undergoing apoptosis. Our data document a novel function for pRb in suppressing apoptosis and suggest that several proteins shown to induce apoptosis, including E1A, E2F and c-myc, may do so by interfering with the protective function of pRb.

Topics: Aphidicolin; Apoptosis; Cell Cycle; Dose-Response Relationship, Radiation; Genes, Retinoblastoma; Humans; Osteosarcoma; Radiation Tolerance; Retinoblastoma Protein; Transfection; Tumor Cells, Cultured; X-Rays

The RB gene product is a nuclear phosphoprotein that undergoes cell cycle-dependent changes in its phosphorylation status. To test whether RB regulates cell cycle progression, purified RB proteins, either full-length or a truncated form containing the T antigen-binding region, were injected into cells. Injection of either protein early in G1 inhibits progression into S phase. Co-injection of anti-RB antibodies antagonizes this effect. Injection of RB into cells arrested at G1/S or late in G1 has no effect on BrdU incorporation, suggesting that RB does not inhibit DNA synthesis in S phase. These results indicate that RB regulates cell proliferation by restricting cell cycle progression at a specific point in G1 and establish a biological assay for RB activity. Neither co-injection of RB with a T antigen peptide nor injection into cells expressing T antigen prevents cells from progressing into S phase, which supports the hypothesis that T antigen binding has functional consequences for RB.

Topics: Animals; Antigens, Viral; Aphidicolin; Baculoviridae; Bromodeoxyuridine; Cell Line; Electrophoresis, Polyacrylamide Gel; Escherichia coli; G1 Phase; Humans; Hydroxyurea; Insecta; Microinjections; Microscopy, Fluorescence; Nocodazole; Osteosarcoma; Phosphorylation; Recombinant Proteins; Retinoblastoma Protein; Simian virus 40; Tumor Cells, Cultured