Prasad S. Sunkara

Antiretroviral activity of castanospermine and deoxynojirimycin, specific inhibitors of glycoprotein processing

The objective of this study was to investigate the antiretroviral activity of specific inhibitors of glycosidases and mannosidases that are involved in N-linked oligosaccharide processing of glycoproteins. Castanospermine and 1-deoxynojirimycin, potent inhibitors of glucosidases I and II, showed significant activity against Moloney murine leukemia virus (IC50: 1.2 microgram/ml). Deoxymannojirimycin and swainsonine, inhibitors of mannosidase I and II, respectively, did not show any activity. These observations suggest that removal of the outermost glucose residue from high mannose asparagine-linked oligosaccharide may be essential for the replication of mouse leukemia virus. The relative nontoxic nature of these inhibitors and a novel mechanism of action suggest a potential for compounds of this type as chemopreventive and therapeutic agents in the treatment of acquired immune deficiency syndrome (AIDS).

Role of polyamines in cytokinesis of mammalian cells.

Abstract Inhibition of polyamine biosynthesis in mammalian cells with methylglyoxal bis -(guanylhydrazone) and α-methyl ornithine inhibits cytokinesis and induces the formation of binucleate cells. Further, these binucleate cells exhibited a diffused pattern of microfilaments compared with the control cells as evidenced by indirect immunofluorescence using anti-actin antibodies. These effects can be reversed by increasing the intracellular levels of the polyamines. The results of this study suggest that polyamines may have a role in the process of cytokinesis and cell division.

Ornithine decarboxylase induction and polyamine biosynthesis are required for the growth of interleukin-2- and interleukin-3-dependent cell lines

The objective of this study was to evaluate induction of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, and subsequent polyamine accumulation in interleukin-2 (IL-2)- and interleukin-3 (IL-3)-dependent growth. The CTLL-20 and FDC-P1 cell lines, which have been shown to be absolutely dependent on IL-2 and IL-3, respectively, were used in these studies. The CTLL-20 and FDC-P1 cells each had different temporal patterns of ODC induction following lymphokine stimulation. ODC levels increased rapidly in the FDC-P1 cells, peaking 4 hr after stimulation with IL-3. In contrast, peak ODC activity in the CTLL-20 cells occurred 18 hr following stimulation with IL-2 and reached eightfold higher levels than those observed in the FDC-P1 cells. Treatment with D,L-alpha-difluoromethylornithine X HCl X H2O (DFMO), a specific irreversible inhibitor of ODC activity, completely abrogated lymphokine-dependent ODC induction in both the CTLL-20 and FDC-P1 cell lines. Similarly, intracellular levels of the polyamines putrescine and spermidine were reduced in both cell lines following DFMO treatment. DFMO treatment reduced both IL-2- and IL-3-dependent proliferation in a dose-dependent manner. However, this inhibition could be reversed by the addition of exogenous putrescine. DFMO treatment had no effect on cell viability. Polyamine-depleted CTLL-20 and FDC-P1 cells showed decreased absorption of IL-2 and IL-3 activity, respectively. However, the addition of exogenous putrescine restored the ability of the cells to absorb the appropriate lymphokine. These data are the first to demonstrate that ODC induction and polyamine biosynthesis are required in lymphokine dependent growth.

Inhibition of polyamine biosynthesis by α-difluoromethyl ornithine potentiates the cytotoxic effects of arabinosyl cytosine in HeLa cells

Abstract The object of this study was to examine the effect of inhibition of polyamine biosynthesis on the cell cycle traverse of HeLa cells using α-difluoromethyl ornithine (DFMO), a catalytic irreversible inhibitor of ornithine decarboxylase. The results of this study indicate that DFMO inhibits HeLa cell growth by causing a decrease in the intracellular levels of putrescine and spermidine without any significant effect on concentration of spermine. The inhibition is readily reversible by exogenous supply of putrescine to the medium. The DFMO treatment also results in an accumulation of cells in S phase. Further, the use of an S phase-specific drug like Ara-C following DFMO treatment results in a synergistic killing of the tumor cells as revealed by the inhibition of cell growth. These observations suggest that exploitation of regulation of the cell cycle by the depletion of polyamines with the use of inhibitors like DFMO might help in designing better therapeutic regimes in combination with other cytotoxic drugs.

Putrescine biosynthesis in mammalial cells: essential for DNA synthesis but not for mitosis.

Abstract The object of this study was to examine the role of putrescine in the regulation of DNA synthesis and mitosis in synchronized Chinese hamster ovary cells using 1,3-diaminopropane (DAP) which is a potent inhibitor of ornithine decarboxylase (EC 4.1.17). Inhibition of putrescine biosynthesis significantly reduced the incorporation of [ 3 H ]-TdR into DNA but had no effect on the progression of cells from G1 to S phase. However, inhibition of putrescine synthesis in synchronized S phase cells did not affect their progression to mitosis. In these experiments, the DAP treatment had little or no effect on the levels of spermidine and spermine. These results indicate that putrescine biosynthesis is essential for the completion of DNA synthesis but not required for mitosis and cell division.

Selective killing of transformed cells in combination with inhibitors of polyamine biosynthesis and S-phase specific drugs

The objective of this study was to examine the effect of inhibition of polyamine biosynthesis on the cell cycle traverse of normal and transformed cells using α-difluoromethyl ornithine (DFMO), a catalytic irreversible inhibitor of ornithine decarboxylase. The results of this study indicate that DFMO brings about a significant decrease in the intracellular levels of putrescine and spermidine followed by an inhibition of cell growth in both the normal and transformed cells. The DFMO treatment results in a differential effect on the cell cycle traverse of normal and transformed cells. A majority of the normal cells were arrested in G1 phase whereas their transformed counterparts were found to be accumulated in S phase of the cell cycle. Further, treatment of both types of cells with an S phase specific drug like Ara-C after polyamine depletion with DFMO resulted in a preferential synergistic killing of the transformed cells while the normal cells were protected from the cytotoxicity of Ara-C. These observations suggest the use of inhibitors like DFMO might help in designing better therapeutic regimens in combination with other cytotoxic drugs.

Swainsonine, an inhibitor of glycoprotein processing, enhances mitogen induced interleukin 2 production and receptor expression in human lymphocytes

Swainsonine, an inhibitor of mannosidase II, enhanced Con A induced lymphocyte IL-2 receptor expression, IL-2 production, and proliferation. Mitogen activated lymphocytes treated with swainsonine and subsequently restimulated with IL-2 showed a three-fold increase in proliferation. Castanospermine, 1-deoxynojirimycin, bromoconduritol and 1-deoxymannojirimycin, inhibitors of glucosidase 1, glucosidases 1 and II, glucosidase II, and mannosidase 1, respectively, did not exhibit any immunoenhancing activity. These results indicate that specific inhibition of mannosidase II during glycoprotein processing can enhance IL-2 mediated lymphocyte mitogenesis.

A potent inhibitor of β-N-acetylglucosaminidases: 6-acetamido-6-deoxycastanospermine

Abstract A facile synthesis of 6-acetamido-6-deoxycastanospermine ( 4 ) is described. The target compound inhibits mammalian β- N -acetylglucosaminidases at or below 1 μM.

The relationship between levels and rates of synthesis of polyamines during mammalian cell cycle

Abstract The objective of this study was to examine the rate of synthesis and the intracellular levels of polyamines as a function of the HeLa cell cycle. The intracellular levels of ornithine, which were high during mitosis and early G1 phase, decreased rapidly during late G1 phase when the ornithine decarboxylase activity was at its peak. The activities of ornithine decarboxylase and S-adenosyl methionine decarboxylase reached a peak during G1 and decreased rapidly during the S phase. The levels of polyamines were maximum in mitosis and S phase. In constrast, the rate of polyamine synthesis during S phase was 5–10 fold lower than that in mitosis or G1 phase. We have also observed fluctuations in diamine-oxidase activity during the cell cycle. The enzyme activity was high during mitosis and late G1 and low during S phase. Thus, the results of this study suggest an important role for the catabolic enzymes in the regulation of polyamine levels during the mammalian cell cycle.

An essential role for putrescine biosynthesis during meiotic maturation of amphibian oocytes

Abstract The objective of this study was to investigate the role of polyamines during meiotic maturation of Xenopus oocytes. The results indicate a rapid and significant increase in the activity of ornithine decarboxylase (ODC), the rate-limiting enzyme in the polyamine biosynthetic pathway, during the meiotic maturation induced by either progesterone or human chorionic gonadotropin (HCG). This increase in the enzyme activity was followed by an accumulation of putrescine without any effect on the levels of spermidine or spermine. The inhibition of ODC activity and the accumulation of putrescine levels by α-difluoromethyl ornithine (DFMO), a catalytic irreversible inhibitor of ODC, also resulted in the inhibition of maturation mediated by progesterone in Xenopus oocytes. DFMO caused an inhibition of both maturation and ovulation induced by HCG in ovarian fragments. This inhibition was readily reversible by exogenous supply of putrescine to the medium. These observations suggest that putrescine plays an important role during the meiotic maturation of amphibian oocytes.