Methods
Cells Culture
BMPRA1.TUC3.BMRPA1 and B16 cells were cultured in complete medium (cRPMI=RPMI with 10%FBS and 1x Penicillin-Streptomycin). BMRPA1.TUC3 cells are BMRPA1 cells (normal rat pancreatic acinar cells) transformed by transfection with a human oncogenic k-rasval12 gene. Both cell lines are products of this laboratory. All other cell lines described were obtained from ATCC. B16 cells are mouse melanoma cells. MIAPaCa-2 are human pancreatic cancer cells. Ag13145 are primary human fibroblasts. A2058 cells are human melanoma cells. MIAPaCa-2, Ag13145 and A2058 cells were cultured in cDMEM (DMEM with 10%FBS and 1x Penicillin-Steptomycin).
Detection of Lactic acid dehydrogenase (LDH)
Cells grown in 24 well chambers were treated with Vectcytotratin and a control peptide at various concentrations. At predetermined time points, 600 of the supernatant were collected from each well and distributed into 96-well plates for the LDH Cytotoxicity Assay as the described by the manufacturer (Promega, WI, USA). Total cellular LDH was obtained by lysing the cells in each well, retrieving a 50ml sample from each lysed supernatant and processing the sample in the LDH Cytoxicity Assay. Substrate readings were done at nm. Results are presented in raw data (direct OD readings). LDH released as % of total LDH and % cytoxicity.
Cell Fractionation
Cells grown in 75cm2 flasks were washed extensively with ice cold PBS, followed by scraping and collection into PBS and centrifugation at 500Xg, 10 min, 60C. The supernatant was discarded and the pellet was resuspended protease inhibitors-containing (Pierce, USA) homogenization buffer equivalents to 10 times the volume of the cell pellet. The cells centrifuged at 140,000Xg, 6oC, 1h, using a SW55 rotor in a LB80M ultracentrifuge (Beckman Instr., CA, USA). The pellet was resuspended in 20 times its volume in PBS and ultra-centrifuged again for 30min at 30.000Xg. The supernatant was discarded and the pellet was dissolved in protease inhibitors and 1% Triton X-100-containing lysis buffer.
SDS-PAGE and Immunoblotting (IB)
Proteins in total cell ​​ lysates, cell plasma membrane fractions, and molecular weight standards were separated in 12% SDS-polyacrylamide using a Bio-Rad gel. Samples, each applied to near the same protein concentration as measured by the Bradford protein assay, were prepared with 2x Laemmli sample buffer under non-reducing and reducing conditions (5% 2-b-mercaptoethanol) and boiled for 3 minutes.
Gels were run at 60 mA, then briefly fixed and stained with Bio-Safe Coomassie Blue for 1 hour followed by staining in ddH2O until individual polypeptide bands were visible. For IB, gels were left unfixed and unstained, and separated polypeptide bands were electrophoretically transferred to a nitrocellulose membrane at 26 mA overnight and 150 mA for 2 hours. The NC membrane was detached from the gel and placed in blocking buffer (5% milk in TBS) for 30 min at room temperature. The blocked membrane was then washed with dH2O and incubated for 1 hour at room temperature with the primary antibody solution (5 mg/ml blocking buffer). The membrane was then washed with TBS-T and re-incubated with the secondary antibody solution (secondary HRPConjugated diluted 1:1000 in 0.1% milk in TBS-T) for 30 min at room temperature. Followed by extensive washing with TBS-T and dH2O. Substrate solution (Immun-Star HRP peroxide buffer + luminol/Immun-Star HRP enhancer 1:1) was then added to the membranes and the chemiluminescent reaction was exposed on X-ray film in complete darkness. Routinely, the nitrocellulose membrane was exposed for 30 seconds to 30 minutes to individual X-ray films.
Immunofluorescence (IF) and Confocal Microscopy (CFM)
Cells were released with trypsin from their TCFs and grown on glass cover slips in 24-well dishes until they reached 50-60% density. After removal of the spent medium and PBS washing buffer they were treated for up to 15min at 37 degrees C in a humidified 5% CO2 – 95% Air incubator environmental chamber with Vectcytotratin and control at 50 ug/ml incubation medium. At the end of incubation the cells were washed and fixed in 3% paraformaldehyde in PBS (pH 7.2) supplemented with 0.01% glutaraldehyde for 1.5h followed by extensive washing and transfer into PBS for storage until mounting on glass slides for microscopy. In order to quench free aldehyde groups, cells were in glycine and sodium borohydride (NaBH4), followed by washing in PBS. Cells were then stained (direct staining) for 2h, 4 degrees C, with flourescein-labeled mouse monocional antibody against p53 [FITC-mAba-p53 (DO-1)](51g/ml) and modamin-labeled (TRITC-) mAba-against h/t/mdm2 (51g/ml). After removal of non-reactive Ab and extensive washing, the cover slips were mounted over antifade (Molecular Probes – Invitrogen, CA) on glass slides and examined with a laser-equipped Olympus Confocal microscope 1X76. Results were digitally recorded. The colocalization of the two Abs was confirmed by overlapping green (anti-p53) and red (anti-hrmdm2) fluorescent labels which produced a yellow color. Real-time Live Cell Imaging with a Spinning Disk Confocal Laser Microscopy (Zeiss Perkin-Emer, CT) was performed at The Rockefeller University using an Inverted Zeiss Axiovert 200 Microscope linked to Hamatsu Orca ER cooled CCd Camera and 491nm and 561nm Lasers. Image acquisition and analysis were done with the UIC’s MetaMorph software. For these experiments, cells were released as described above, seeded into and grown overnight in 35mm TC dishes with glass overslip (#1.5) bottoms (Mattek, MA, USA) to reach a density of 60%. For each timed observation a dish with cells was placed in the correct medium or buffer (PBS) into the specimen carrier of the Zeiss microscope, the cells brought into focus a DIC picture taken to mark t=0 (untreated), the desired marker dye (Propidum Icode or PI) added and the recording begun at 10-30sec intervals. After a predetermined recorded time interval the peptide (Vectcytotratin) was added at the desired concentration and observation and recording (at 10-30sec intervals) of PI distribution and changes in cell morphology was continued for another predetermined time.
Transmission Electronmicroscopy (TEM)
Cells (1X 106) were grown in 6-well dishes overnight when spent medium was removed, the cells washed with PBS and treated at 37 degrees C with Vectcytotratin at 50 ug/mi in PBS. After 15min the cells were washed and fixed in 3% buffered paraformaldehyde supplemented with 0.01% glutaraldehyde for 1.5h. After extensive washing, the cells were treated with glycine and NaBH4 as described above followed by 3 times washing in PBS. Immunogold staining was performed with primary antibodies against p53 (Rab lgG a-p53) and M mAb ainfrimom2 (each at 5lg/ml) (Santa Cruz, CA, USA) at 4 degrees C, overnight. After removal of non-bound Ab and extensive washing with PBS the cells were incubated for 8h with gold-conjugated secondary antibodies (6nm goldconjugated Goat a-M lgG and 12nm gold-conjugated Donkey a-Rab lgG). After extensive washing cells were post-fixed in 1% glutaraldehyde in cacodylate buffer (o.113M, pH7.2) overnight, at which time the cells were scraped into PBS and centrifuged into a pellet. The cells were washed in cacodylate buffer and postfixed in 1% Osmium tetraoxide for 1h., RT. The fixed cells were dehydrated through sequential passagings in increasing concentrations of ethanol and embedded in agar which was then exchange for Epon. After hardening the Epon for 72h, thin sections (60nm) were cute on an ultramicrotome, stained with uranyl acetate and examined in a Zeiss EM1D transmission electron microscope.
Induction times of total cytotoxicity by CPVC in malignant cell lines
course of time
Dose Response
