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Regulation of DNA synthesis and the cell cycle in human prostate cancer cells and lymphocytes by ovine uterine serpin.

Padua MB, Hansen PJ - BMC Cell Biol. (2008)

Bottom Line: Like some other serpins, these proteins do not appear to be functional proteinase inhibitors.The effect of OvUS in the production of the autocrine and angiogenic cytokine interleukin (IL)-8 by PC-3 cells was also determined.Treatment of PC-3 cells with OvUS did not cause cytotoxicity or apoptosis or alter interleukin-8 secretion into medium.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Animal Sciences, University of Florida, Gainesville, FL 32611-0910, USA. mpadua@ufl.edu

ABSTRACT

Background: Uterine serpins are members of the serine proteinase inhibitor superfamily. Like some other serpins, these proteins do not appear to be functional proteinase inhibitors. The most studied member of the group, ovine uterine serpin (OvUS), inhibits proliferation of several cell types including activated lymphocytes, bovine preimplantation embryos, and cell lines for lymphoma, canine primary osteosarcoma and human prostate cancer (PC-3) cells. The goal for the present study was to evaluate the mechanism by which OvUS inhibits cell proliferation. In particular, it was tested whether inhibition of DNA synthesis in PC-3 cells involves cytotoxic actions of OvUS or the induction of apoptosis. The effect of OvUS in the production of the autocrine and angiogenic cytokine interleukin (IL)-8 by PC-3 cells was also determined. Finally, it was tested whether OvUS blocks specific steps in the cell cycle using both PC-3 cells and lymphocytes.

Results: Recombinant OvUS blocked proliferation of PC-3 cells at concentrations as low as 8 mug/ml as determined by measurements of [3H]thymidine incorporation or ATP content per well. Treatment of PC-3 cells with OvUS did not cause cytotoxicity or apoptosis or alter interleukin-8 secretion into medium. Results from flow cytometry experiments showed that OvUS blocked the entry of PC-3 cells into S phase and the exit from G2/M phase. In addition, OvUS blocked entry of lymphocytes into S phase following activation of proliferation with phytohemagglutinin.

Conclusion: Results indicate that OvUS acts to block cell proliferation through disruption of the cell cycle dynamics rather than induction of cytotoxicity or apoptosis. The finding that OvUS can regulate cell proliferation makes this one of only a few serpins that function to inhibit cell growth.

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Related in: MedlinePlus

Inhibition of proliferation of PC-3 cells by recombinant ovine uterine serpin (rOvUS) as determined by ATP content/well. Ovalbumin (OVA) was used as a negative control. Data represent least-squares means ± SEM. Means that differ from untreated cells are indicated by symbols (†P < 0.1; **P < 0.01; ***P < 0.001).
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Figure 2: Inhibition of proliferation of PC-3 cells by recombinant ovine uterine serpin (rOvUS) as determined by ATP content/well. Ovalbumin (OVA) was used as a negative control. Data represent least-squares means ± SEM. Means that differ from untreated cells are indicated by symbols (†P < 0.1; **P < 0.01; ***P < 0.001).

Mentions: The antiproliferative effects of rOvUS on proliferation of PC-3 cells were evaluated by two different assays. In the first experiment, it was shown that rOvUS caused a concentration-dependent decrease in incorporation of [3H]thymidine into DNA (P < 0.001) with the minimum effective concentration being 8 μg/ml (Figure 1). The antiproliferative actions of OvUS using [3H]thymidine uptake as the measure of proliferation has been demonstrated previously for PC-3 cells and other cell types [18-22,24,25]. To confirm this effect of rOvUS reflected an inhibition in cell proliferation and not a disruption in [3H]thymidine uptake by the cells, antiproliferative effects were also evaluated by an assay in which the relative total number of cells per well was estimated by the ATP content per well. Treatment with rOvUS reduced ATP content per well at all concentrations tested (50, 100 and 200 μg/ml) (Figure 2). In contrast, the control serpin, ovalbumin, did not cause effect in the ATP content per well. The finding that rOvUS reduced ATP content per well confirms that the effects of OvUS to reduce [3H]thymidine incorporation reflect a reduction in cell proliferation rather than interference with [3H]thymidine transport into the cell.


Regulation of DNA synthesis and the cell cycle in human prostate cancer cells and lymphocytes by ovine uterine serpin.

Padua MB, Hansen PJ - BMC Cell Biol. (2008)

Inhibition of proliferation of PC-3 cells by recombinant ovine uterine serpin (rOvUS) as determined by ATP content/well. Ovalbumin (OVA) was used as a negative control. Data represent least-squares means ± SEM. Means that differ from untreated cells are indicated by symbols (†P < 0.1; **P < 0.01; ***P < 0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2254604&req=5

Figure 2: Inhibition of proliferation of PC-3 cells by recombinant ovine uterine serpin (rOvUS) as determined by ATP content/well. Ovalbumin (OVA) was used as a negative control. Data represent least-squares means ± SEM. Means that differ from untreated cells are indicated by symbols (†P < 0.1; **P < 0.01; ***P < 0.001).
Mentions: The antiproliferative effects of rOvUS on proliferation of PC-3 cells were evaluated by two different assays. In the first experiment, it was shown that rOvUS caused a concentration-dependent decrease in incorporation of [3H]thymidine into DNA (P < 0.001) with the minimum effective concentration being 8 μg/ml (Figure 1). The antiproliferative actions of OvUS using [3H]thymidine uptake as the measure of proliferation has been demonstrated previously for PC-3 cells and other cell types [18-22,24,25]. To confirm this effect of rOvUS reflected an inhibition in cell proliferation and not a disruption in [3H]thymidine uptake by the cells, antiproliferative effects were also evaluated by an assay in which the relative total number of cells per well was estimated by the ATP content per well. Treatment with rOvUS reduced ATP content per well at all concentrations tested (50, 100 and 200 μg/ml) (Figure 2). In contrast, the control serpin, ovalbumin, did not cause effect in the ATP content per well. The finding that rOvUS reduced ATP content per well confirms that the effects of OvUS to reduce [3H]thymidine incorporation reflect a reduction in cell proliferation rather than interference with [3H]thymidine transport into the cell.

Bottom Line: Like some other serpins, these proteins do not appear to be functional proteinase inhibitors.The effect of OvUS in the production of the autocrine and angiogenic cytokine interleukin (IL)-8 by PC-3 cells was also determined.Treatment of PC-3 cells with OvUS did not cause cytotoxicity or apoptosis or alter interleukin-8 secretion into medium.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Animal Sciences, University of Florida, Gainesville, FL 32611-0910, USA. mpadua@ufl.edu

ABSTRACT

Background: Uterine serpins are members of the serine proteinase inhibitor superfamily. Like some other serpins, these proteins do not appear to be functional proteinase inhibitors. The most studied member of the group, ovine uterine serpin (OvUS), inhibits proliferation of several cell types including activated lymphocytes, bovine preimplantation embryos, and cell lines for lymphoma, canine primary osteosarcoma and human prostate cancer (PC-3) cells. The goal for the present study was to evaluate the mechanism by which OvUS inhibits cell proliferation. In particular, it was tested whether inhibition of DNA synthesis in PC-3 cells involves cytotoxic actions of OvUS or the induction of apoptosis. The effect of OvUS in the production of the autocrine and angiogenic cytokine interleukin (IL)-8 by PC-3 cells was also determined. Finally, it was tested whether OvUS blocks specific steps in the cell cycle using both PC-3 cells and lymphocytes.

Results: Recombinant OvUS blocked proliferation of PC-3 cells at concentrations as low as 8 mug/ml as determined by measurements of [3H]thymidine incorporation or ATP content per well. Treatment of PC-3 cells with OvUS did not cause cytotoxicity or apoptosis or alter interleukin-8 secretion into medium. Results from flow cytometry experiments showed that OvUS blocked the entry of PC-3 cells into S phase and the exit from G2/M phase. In addition, OvUS blocked entry of lymphocytes into S phase following activation of proliferation with phytohemagglutinin.

Conclusion: Results indicate that OvUS acts to block cell proliferation through disruption of the cell cycle dynamics rather than induction of cytotoxicity or apoptosis. The finding that OvUS can regulate cell proliferation makes this one of only a few serpins that function to inhibit cell growth.

Show MeSH
Related in: MedlinePlus