Development of cancer-induced spontaneous pain, tactile allodynia, and movement-evoked pain in animals 21 days after surgery.

<p>(A) Spontaneous pain as measured by flinching of the ipsilateral hindlimb was determined in sham injected animals (control) or those injected with tumor cells expressing the wild type integrin (PC3N-A6-WT) or those injected with tumor cells expressing the mutated integrin (PC3N-A6-RR). Elevation in flinching behavior is indicative of an increased pain response. (B) Tactile allodynia as measured by paw-withdrawal from von Frey filaments in sham injected animals (Control) or those injected with tumor cells expressing the wild type integrin (PC3N-A6-WT) or those injected with tumor cells expressing the mutated integrin (PC3N-A6-RR). A decrease in the withdrawal threshold is indicative of an increased pain response. (C) Movement evoked pain was observed in sham injected animals (control) or those injected with tumor cells expressing the wild type integrin (PC3N-A6-WT) or those injected with tumor cells expressing the mutated integrin (PC3N-A6-RR).</p

Histological examination of bone destruction, tumor cell distribution and verification of mutated integrin expression after injection of prostate tumor cells.

<p>(A) Hematoxylin-eosin staining of the normal bone (control) or bone injected with PC3N-A6-WT cells (WT, middle panel and inset) and PC3N-A6-RR cells (RR, bottom panel and inset). The growth plate of the bone (epiphyseal plate) is oriented at the top left of each panel for comparison purposes. (B) RT-PCR analysis to detect expression of the mutated 6 integrin in the bone marrow. Twenty one days following injection, bone marrow was harvested, RNA was extracted and analyzed. RNA from PC3N-A6-WT cells and PC3N-A6-RR cells growing in tissue culture was compared to the bone marrow isolated from mice injected with PC3N-A6-WT cells (Bone marrow-WT cells) or PC3NA6- RR cells (bone marrow-RR cells). GAPDH amplification was carried out as control and the kB markers are as shown.</p

Biochemical and migration phenotype of PC3N-A6-WT and PC3N-A6- RR cells expressing the wildtype(cleavable) and RR(uncleavable) integrin A6, respectively.

<p>(A) The expression of the full length 6 integrin ( 6) and uPA dependent production of the 6p variant ( 6p) was determined by western blot analysis. Integrin status within total cell lysates from doxycycline (Dox) or urokinase (uPA) untreated (−) and treated (+) PC3N-A6-WT and PC3N-A6-RR cell lines was determined. (B) Surface expression of wild type and mutated integrin 6 in doxycycline induced PC3N-A6-WT and PC3N-A6-RR cells was determined by flow cytometry. PC3N-A6-WT and PC3NA6- RR cells were incubated with primary Rat anti-integrin A6 antibody J1B5 followed by Alexa 488 anti-rat antibody and visualized using the BD FACScan. The grey peak indicates fluorescence signal from secondary antibody only. (C) Schematic to illustrate the cleavage of the full length form of the 6 integrin to yield the 6p variant. The definition of the PC3N-A6-WT and PC3N-A6-RR cells with regard to integrin status is shown. (D) Integrin mediated migration of PC3N-A6-WT cells (top panels) and PC3NA6- RR cells (bottom panels) on matrigel. The cells were placed on matrigel in the presence of a coverslip to create a cell free zone on the matrigel surface. After cell adhesion was complete, the coverslips were removed from the matrigel surface to allow migration into the cell free zone indicated by white or black curved line. Cells migration occurred under optimal growth conditions at 37 for approximately 18 hours. Cells were either allowed to migrate in the absence (left panels) or presence (right panels) of integrin blocking antibody AIIB2. Images were collected using a Zeiss Axiovert microscope equipped with a 2.5X objective.</p