Cellular effects of the PAcP and its chimeric signal sequence constructs in LNCaP cells
(A) Line diagram of wtPAcP, IgG and Prl signal sequences. N (bold and underlined) and H (bold without underline) domains have been engineered in front of the C domain of the wt PAcP signal sequence. Numbers above are amino acids of the signal sequence preceding the cleavage site. Cleavage site is indicated with downward pointing arrow, followed by three residues of the mature protein sequence. (B) Growth rate of LNCaP clone C81 cells transfected with PAcP and chimeric signal sequence constructs. Two sets of cells were transfected with pcDNA3.1zeo+ vector harboring full-length human PAcP and chimeric signal sequence constructs, as described in ‘Methods’ section. Mock represents cells transfected with empty pcDNA3.1zeo+ vector. The first set of cells was harvested and then counted immediately after transfection (5 h) and this number was used as the baseline for growth rate determination. The second set was harvested and counted 72 h after transfection. Data shown are % of growth rate above baseline. Mean ± S.D. (n=7); *P<0.03, significantly different from control (C81). (C) Growth rate of LNCaP clone C81 cDNA transfected cells in present of androgen. Two sets of LNCaP C-81 cells were transfected as for (B). The first set was harvested and counted 72 h after transfection and cell number used as a baseline for growth rate. A total of 10 nM of DHT was added to the other set for the last 48 h of transfection and then cells harvested and counted. Data shown are % of growth rate above baseline at 72 h. Mean ± S.D. (n=5); *P<0.04, significantly different from control (C81). (D) Effect of the addition of androgen on the level of c-Erb-B2 (pp185) in different types of transfected cells. Cells were transfected and androgen was added to the medium as described in (C). Seventy-two hours after transfection cells were harvested, lysed, and equal amounts of total protein (50 µg) were electrophoresed on SDS/PAGE for WB analysis with the anti-pY clone 4G10. Leftward pointing arrowhead indicates position of c-Erb-B2 (pp185). (E) Cell growth arrest at G0/G1 phase when transfected with IgG(NH)PAcP. Cells transfected as described were harvested, lysed, and equal amount of total proteins analyzed by SDS/PAGE and WB with polyclonal antibody reactive to cyclin D (open bars) or Cdk4 (closed bars). Lysates were probed with anti tubulin as a loading control shown. Data are mean ± S.D. (n=4); *P<0.03, significantly different from control (C81). (F) Addition of Prl(NH)PAcP transfected cell medium overcomes androgen dependence of IgG(NH)PAcP transfected cells. Two sets of LNCaP clone C81 cells were transfected with Prl(NH)-PAcP or empty vector. Seventy-two hours after transfection medium from both sets was harvested and used as a replacement medium for another two sets of cells (transfected as indicated) upon completion of transfection (5 h). Seventy-two hours later, cells were harvested, counted. The baseline for calculation of percent growth was taken to be growth in the presence of C81 (mock transfection) medium. A control set of cells was transfected as indicated at the same time without replacement media. The number of cells in that control was not statistically significantly different from cells that received mock replacement medium. Data shown are % of growth rate above baseline. Mean ± S.D. (n=4); *P<0.01, significantly different from control (C-81). Abbreviations: DHT, 5-alpha dihydrotestosterone; SDS/PAGE, polyacrylamide gel electrophoresis in sodium dodecyl sulfate; WB, Western blotting.