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. 2007 Jun;98(6):909-15.
doi: 10.1111/j.1349-7006.2007.00467.x. Epub 2007 Apr 12.

von Willebrand factor type D domain mutant of SVS-1/SUSD2, vWD(m), induces apoptosis in HeLa cells

Tetsuo Sugahara  1 , Yzumi Yamashita, Masahito Shinomi, Yumiko Isobe, Banri Yamanoha, Hiroyoshi Iseki, Akihiko Takeda, Yasushi Okazaki, Kenji Kawai, Hiroshi Suemizu, Toshiwo Andoh
Affiliations

von Willebrand factor type D domain mutant of SVS-1/SUSD2, vWD(m), induces apoptosis in HeLa cells

Tetsuo Sugahara et al. Cancer Sci. 2007 Jun.

Abstract

SVS-1/SUSD2 is a novel gene, which inhibits growth and reverses tumorigenic phenotypes of cancer cells in vitro. Here we report identification of a mutant of SVS-1, designated SVS-1-vWD(m), in which conserved amino acids GLLG at positions 591-594 in von Willebrand factor type D (vWD) domain are replaced by AAAA. As observed by laser confocal microscope, intracellular localization of the mutant protein has changed such that both the N-terminus and the C-terminus of SVS-1-vWD(m) were localized in the inner surface of the plasma membrane, whereas the N-terminus of SVS-1 was localized in the outer surface of the plasma membrane. Additionally, SVS-1-vWD(m) was processed much less efficiently and in a slightly different manner. In in vitro studies, adenovirus-mediated transduction of the SVS-1-vWD(m)gene induced growth suppression of HeLa cells in a dose-dependent manner, as the wild-type gene and inhibition of anchorage-independent growth. Of great interest is the finding that the mutant protein, vWD(m), but not the wild-type one induced apoptosis, as observed by nuclear as well as DNA fragmentation. Activation of caspase-3 and -9, but not caspase-8 or -12, was also demonstrated in vWD(m)-expressing cells. An inhibition of Akt phosphorylation, a major survival signaling component, also occurred in vWD(m)-expressing HeLa cells. Together these data suggest that vWD(m) induces apoptosis by inactivation of survival signaling component Akt and activation of caspase cascade (mitochondrial pathway) in HeLa cells. We propose SVS-1-vWD(m)as an alternative gene for use in developing new therapeutic strategies for the treatment of cancer.

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Figures

Figure 1
Figure 1
Alignment of amino acid sequences of vWD of SVS‐1 and several vWD‐containing proteins. Amino acid sequences of the predicted vWD of SVS‐1 protein and several vWD‐containing proteins are shown using the NCBI ‘Conserved Domains’ database (http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtm). Only the latter half of the domains is aligned. The conserved consensus sequence GXXG motif is underlined and highlighted.
Figure 2
Figure 2
Subcellular localization of wild‐type and von Willebrand factor Type D domain mutant (vWDm) SVS‐1 protein. (a) HeLa cells were transfected with pcDNA3‐FSVS‐1 or pcDNA3‐vWDm. At 48 h after transfection cells were fixed and permeabilized with Triton X‐100 or not and stained with anti‐FLAG or anti‐V5 antibodies staining the N‐terminus and the C‐terminus, respectively, of SVS‐1. (b) Fixed cells were washed with PBS with or without 0.2% Triton X‐100, stained with anti‐FLAG antibody staining the N‐terminus of SVS‐1, and observed using a laser confocal microscope.
Figure 3
Figure 3
Induced expression of FSVS‐1 and von Willebrand factor Type D domain mutant (vWDm) by Ad/G4VP2 infection, as assayed by immunofluorescence. At 48 h after infection at 4 moi or mock infection, HeLa/G5‐FSVS‐1 and HeLa/G5‐vWDm cells were fixed, permeabilized and stained with anti‐FLAG antibody.
Figure 4
Figure 4
Apoptosis of HeLa/G5‐vWDm cells induced by expression of von Willebrand factor type D domain mutant (vWDm). HeLa/G5‐LUC, HeLa/G5‐SVS‐1 and HeLa/G5‐vWDm cells were infected with Ad/G4VP2 at 16 moi, harvested 1 day after infection, then monitored for nuclei fragmentation. (a) Open arrowheads indicate nuclei fragmentation. (b) The number of cells with fragmented nuclei per each microscopic field was counted.
Figure 5
Figure 5
Induced expression of FSVS‐1 and von Willebrand factor Type D domain mutant (vWDm) by Ad/G4VP2 infection, as assayed using western blotting. At 48 h after infection at 2 moi, HeLa/G5‐FSVS‐1 and HeLa/G5‐vWDm cells were harvested, lyzed and analyzed using western blotting with anti‐FLAG antibody. As a loading control the membrane was stained with Ponceau S.
Figure 6
Figure 6
Overexpression of FSVS‐1 and von Willebrand factor Type D domain mutant (vWDm) inhibits cell proliferation. Cells, HeLa/G5‐Luc, HeLa/G5‐FSVS‐1 and HeLa/G5‐vWDm, were infected with various moi of Ad/G4VP2 and the growth of cells was followed by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay at the indicated number of days after infection. Relative cell numbers were expressed relative to the number of cells on day 0.
Figure 7
Figure 7
Inhibition of anchorage‐independent growth by SVS‐1 and von Willebrand factor Type D domain mutant (vWDm). (a) At 48 h after Ad/G4VP2 infection of HeLa/G5‐Luc, HeLa/G5‐FSVS‐1 and HeLa/G5‐vWDm cells at 16 moi, 1 × 104 cells were seeded per dish in methylcellulose medium on polyHEMA coated plates. After 12 days of incubation the numbers of colonies were counted. (b) The average number of colonies from three dishes of mock and Ad/G4VP2‐infected cells was calculated.
Figure 8
Figure 8
DNA breakdown in HeLa/G5‐vWDm cells induced by expression of von Willebrand factor type D domain mutant (vWDm). HeLa/G5‐LUC, HeLa/G5‐SVS‐1 and HeLa/G5‐vWDm cells were infected with Ad/G4VP2 at 16 moi, harvested at the indicated number of hours after infection, and DNA was extracted from the cells and electrophoresed on agorose gels for DNA degradation.
Figure 9
Figure 9
Cell cycle distribution of cells induced by expression of FSVS‐1‐vWDm. HeLa/G5‐LUC, HeLa/G5‐SVS‐1 and HeLa/G5‐vWDm cells were infected with Ad/G4VP2 at indicated moi, harvested 2 days after infection and subjected to flow cytometric analysis.
Figure 10
Figure 10
Activation of caspases‐3 and ‐9, and inactivation of Akt in cells induced by expression of FSVS‐1‐vWDm. HeLa/G5‐LUC, HeLa/G5‐SVS‐1 and HeLa/G5‐vWDm cells were infected with Ad/G4VP2 at 8 moi, and harvested 2 days after infection. (a) Western blotting analysis was performed using antibodies for cleaved caspase‐3, ‐8, ‐9 and pro‐caspase‐12. (b) Cells were transfected with pcDNA‐HA‐Akt. At 24 h after transfection, cells were infected with Ad/G4VP2 at a moi of 8. At 48 h after infection, cell lysates were subjected to western blotting analysis with anti‐phospho‐Akt (Ser473) and anti‐Akt antibodies. (c) Band intensities of Akt and phospho‐Akt were quantified by NIH image. The values of phospho‐Akt are indicated relative to those of Akt.
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