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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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Representative photomicrographs of PC-3 cells labeled using the TUNEL procedure after 48 h of culture with either 100 (A) or 200 μg/ml (B) of rOvUS or 200 μg/ml of the control protein ovalbumin (C). Cells in panel D were treated with DNAse as a positive control.
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Figure 4: Representative photomicrographs of PC-3 cells labeled using the TUNEL procedure after 48 h of culture with either 100 (A) or 200 μg/ml (B) of rOvUS or 200 μg/ml of the control protein ovalbumin (C). Cells in panel D were treated with DNAse as a positive control.

Mentions: The TUNEL procedure was used to test whether rOvUS decreased cell proliferation by induction of DNA fragmentation characteristic of apoptosis and other forms of cell death. Representative images of TUNEL labeled cells are shown in Figure 4 and the average percent of cells that were TUNEL positive is shown in Figure 5. Treatment of PC-3 with either rOvUS or the control protein OVA did not increase the percent of cells that were TUNEL positive at either 24 or 48 h after treatment; the percentage of cells that were TUNEL positive was low for all groups (< 5.7%). The fact that rOvUS did not induce apoptosis makes the action of this serpin distinct from that of two other serpins that inhibit cell proliferation. Both maspin [6] and PEDF [7] are proapoptotic serpins.


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)

Representative photomicrographs of PC-3 cells labeled using the TUNEL procedure after 48 h of culture with either 100 (A) or 200 μg/ml (B) of rOvUS or 200 μg/ml of the control protein ovalbumin (C). Cells in panel D were treated with DNAse as a positive control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Representative photomicrographs of PC-3 cells labeled using the TUNEL procedure after 48 h of culture with either 100 (A) or 200 μg/ml (B) of rOvUS or 200 μg/ml of the control protein ovalbumin (C). Cells in panel D were treated with DNAse as a positive control.
Mentions: The TUNEL procedure was used to test whether rOvUS decreased cell proliferation by induction of DNA fragmentation characteristic of apoptosis and other forms of cell death. Representative images of TUNEL labeled cells are shown in Figure 4 and the average percent of cells that were TUNEL positive is shown in Figure 5. Treatment of PC-3 with either rOvUS or the control protein OVA did not increase the percent of cells that were TUNEL positive at either 24 or 48 h after treatment; the percentage of cells that were TUNEL positive was low for all groups (< 5.7%). The fact that rOvUS did not induce apoptosis makes the action of this serpin distinct from that of two other serpins that inhibit cell proliferation. Both maspin [6] and PEDF [7] are proapoptotic serpins.

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