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First published online 30 October 2007
doi: 10.1242/jcs.013946

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Midkine and LDL-receptor-related protein 1 contribute to the anchorage-independent cell growth of cancer cells

¹ Department of Biochemistry, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan
² Department of Pediatrics, St Louis Children's Hospital, Washington University School of Medicine, St Louis, MO 63110, USA
³ Department of Cell Biology and Physiology, St Louis Children's Hospital, Washington University School of Medicine, St Louis, MO 63110, USA
⁴ Department of Bioresources Engineering, Okinawa National College of Technology, Okinawa 905-2192, Japan
⁵ Department of Health Science, Faculty of Psychological and Physical Sciences, Aichi Gakuin University, 12 Araike, Iwasaki-cho, Nisshin, Aichi 470-0195, Japan

View larger version (33K): [in this window] [in a new window]   Fig. 1. Ligand blot of MK. (A) Schematic representation of various sLRP fragments. (B) Ligand blot analysis of MK with various fragments. MK was electrophoresed on SDS gels and transferred to a nitrocellulose membrane. Strips of the membranes were then incubated with conditioned medium harvested from sLRP cDNA-transfected COS7 cells. The bound sLRPs were detected using anti-HA antibody. Upper panel, ligand blot result; bottom panel, expression levels of sLRPs. (C) Bar graph of the integrated intensity of each band in (B). The intensities of ligand blot bands were measured and then calculated as the relative intensity (measured intensity/CTN intensity ratio). Experiments were independently performed twice. The results showed the same tendency, and results from one experiment are presented here.

View larger version (49K): [in this window] [in a new window]   Fig. 2. MK specifically interacts with MK-TRAP in vitro. (A) MK-TRAP expression in COS7 cells transiently overexpressing MK-TRAP or in the G401-derived M1 cell line stably overexpressing MK-TRAP. The arrowhead indicates the secreted form of MK-TRAP; the arrow indicates the pre-secreted form of MK-TRAP; C, cell lysate; M, medium. (B) MK-TRAP-containing medium was mixed with MK-containing medium or control medium, and MK was pulled down with anti-MK antibody. MK-TRAP was detected with anti-HA antibody. (C) MK was pulled down from cellular extracts or medium of the M1 cell line. MK-TRAP was detected in the immunoprecipitates. (D) Expression of MK and MK tagged with a Myc epitope (designated as MK-myc) in COS7 cells transiently transfected with each construct. (E) Cellular extracts or medium from COS7 cells co-transfected with both MK-myc and MK-TRAP were immunoprecipitated with an anti-HA antibody and subsequently detected with anti-Myc antibody, and vice versa. The arrowhead indicates the secreted form of MK-TRAP; the arrow indicates the pre-secreted form of MK-TRAP. (F) MK-TRAP-containing medium was mixed with human recombinant PTN (left panel), bFGF (middle panel) or PDGF-BB (right panel). PTN, bFGF or PDGF-BB was immunoprecipitated with anti-PTN, anti-bFGF or anti-PDGF-BB, respectively. MK-TRAP in each precipitate was detected with anti-HA antibody. (G) Exogenous proteins (10 µg of MK, PTN, bFGF or PDGF-BB) were added to a medium containing MK-TRAP and MK-myc to detect their effects on the binding between MK-TRAP and MK-myc. MK-TRAP was pulled down with anti-HA antibody, and MK-myc in the precipitates was detected using anti-Myc antibody. (H) sLRP3-containing medium was mixed with MK. MK was then pulled down with anti-MK antibody, and sLRP3 in the precipitates was detected with anti-HA antibody.

View larger version (19K): [in this window] [in a new window]   Fig. 3. The effect of MK-TRAP on the proliferation of G401-derived cell lines in monolayer culture. (A) G401-derived cell lines M1 and M2 that stably overexpress MK-TRAP, and control cell lines C1 and C2 were seeded into 96-well dishes and then counted with a cell counting kit over 4 consecutive days. The growth rate was calculated as the ratio of the measured OD on each day to the OD on day one. n=8; error bars, ± s.e.m.; ****P<0.001 (MK-TRAP cells versus control cells). (B) Exogenous MK (100 ng/ml) was added to assess its effect on the growth of M1 cells versus control (C1) cells. n=8; error bars, ± s.e.m.; ***P<0.005; ****P<0.001.

View larger version (42K): [in this window] [in a new window]   Fig. 4. The effect of MK-TRAP on anchorage-independent growth of G401 and CMT-93 cells. (A) M1 and C1 cells (3000 cells per dish) were plated in 35-mm dishes in soft agar. Two weeks later, representative colonies were photographed. (B) The total numbers of colonies of M1 cells versus C1 cells were counted and calculated as the mean of three independent experiments (n=3); error bars indicate ± s.e.m.; ****P<0.001. (C) Exogenous MK (10 ng/ml or 100 ng/ml) was added to both upper and bottom gels to rescue decreased anchorage-independent growth of M1 cells. The results are presented as means (n=3); error bars, ± s.e.m.; **P<0.01. (D) G401 cells transiently transfected with MK-TRAP or empty vector (control) were subjected to soft agar colony-formation assay, and the numbers of colonies with a diameter () >50 µm or >200 µm in each group were counted and calculated as means. n=3; error bars, ± s.e.m.; *P<0.05; ****P<0.001. (E) MK-TRAP-containing medium was produced by transient overexpression in COS7 cells and then added to both upper and bottom gels, in order to evaluate effects on anchorage-independent growth of G401 cells and CMT-93 cells. Left, G401 cell line; right, CMT-93 cell line. n=3; error bars, ± s.e.m.; ***P<0.005; ****P<0.001. (F) Goat anti-MK antibody (15 µg/ml) was added to upper gels to evaluate its effects on anchorage-independent growth of G401 cells and CMT-93 cells. Left, G401 cell line; right, CMT-93 cell line. n=5; error bars, ± s.e.m.; ****P<0.001. (G) CMT-93 bulk stable transformants were generated as described in the Materials and Methods section. Cells were inoculated subcutaneously into nude mice. The volume of the tumors was measured at the indicated intervals (left panel). The data are shown as the means ± s.e.m. (n=8). Twenty-five days later, the tumors were isolated and weighed immediately (right panel). Data are given as the means ± s.e.m. (n=8); *P<0.05.

View larger version (34K): [in this window] [in a new window]   Fig. 5. MK-TRAP acts by blocking MK-LRP1 interaction. (A) CHO-derived cells were exposed to 125I-MK and treated with the chemical crosslinker BS3. The 125I-MK-crosslinked band appeared in CHO-mLRP4 but not CHO-LRP– cells. MK-TRAP- or sLRP3-containing medium was added to CHO-mLRP4 cells to assess their effects on crosslinking. CHO-LRP cells are deficient in LRP1 expression; CHO-mLRP4 is a stable transfectant expressing LRP1-binding domain IV in the background of CHO-LRP– cells. The arrowhead indicates the unprocessed form of the protein; the arrow indicates the mature form of the protein. (B) GST-RAP, an inhibitor of LRP1, and GST control were administered to M1 and control C1 cells, and the total numbers of colonies in each group were counted and compared by Student's t-test. n=3; error bars, ± s.e.m.; **, P<0.01.