Representative karyotypes of neu-expressing cell lines. Spectral karyotyping (SKY) analysis was performed on metaphase preparations of each cell line. Full karyotypes of each tumor cell line were shown. Arrows indicate translocations summarized in Table 1.

Effect of RTKI or anti-neu mAb on neu⁺ tumor cell lines. (A) Effect of gefitinib or anti-neu mAb on neu expression. TUBO, Bam1a, Bam IR-5, and D2F2/neu cells in monolayer culture were incubated with 2.5 μg/ml of mAb 7.16.4 or 4 μM of gefitinib for 8 hours (open histogram, grey outline). Control groups were treated with DMSO or normal mouse serum (open histogram, black outline). After the incubation, cells were stained with Ab4 and PE-conjugated goat anti-mouse IgG Fc. Isotype control was shown as shaded histogram. (B) Effect of gefitinib or anti-neu mAb on Akt phosphorylation. Cells were incubated for 2 hours in the presence of gefitinib (μM) or anti-neu mAb 7.16.4 (μg/ml). Whole cell lysates were resolved in SDS-PAGE. Blots were probed with phospho-Akt or control actin Abs. The concentrations of gefitinib from lanes 1 to 4 were 0, 0.25, 1, and 4 μM, respectively. The concentrations of mAb 7.16.4 from lanes 2-4 were 0.025, 0.25, and 2.5 μg/ml, respectively. Lane 1 was IgG_2a isotype control at 2.5 μg/ml

Characterization of neu⁺ tumor cell lines. (A) Expression of neu and MHC class I K^d. TUBO, Bam1a, Bam IR-5, D2F2/neu, and D2F2 cells were stained with anti-neu mAb, 7.16.4 (top row, open histogram) or mAb SF1.1 for K^d (bottom row, open histogram). Isotype controls were represented as shaded histograms. Secondary antibody was PE-conjugated goat anti-mouse IgG Fc. (B) Inhibition of tumor cell proliferation by RTKI and anti-neu antibody. Cells were plated in monolayer and incubated with gefitinib (0.25-4 μM, top row) or anti-neu mAb (0.025-2.5 μg/ml, bottom row). Control groups received DMSO or isotype control Ab. Relative cell number, reflected by metabolic activity, was evaluated at indicated time with MTT assay and graphed as percent of control. Each symbol represents the mean±SD of 4 replicates.

Effect of pcytneu DNA vaccination on neu tumor growth. BALB/c mice were electrovaccinated twice, i.m., 2 weeks apart with pcytneu and pGM-CSF or control vector and pGM-CSF. (A) IFN-γ secreting T cell response in immunized mice was measured by ELISPOT assay. Splenocytes were collected 2 weeks after last vaccination. Splenocytes from individual mice were incubated with 3T3/KB or 3T3/NKB cells. Each symbol represents the mean ± SD for individual mouse in 4 replicate wells. Results were expressed as number of spots per 10⁶ splenocytes and analyzed by Student’s t test. (B) At 3 weeks after final immunization, the remaining mice were inoculated with TUBO, Bam1a, Bam IR-5, or D2F2/neu cells. Tumor growth was measured weekly by palpation and the results expressed as % tumor free mice. There were 6-7 mice in each group. Results were analyzed by Log rank test. * indicates p<0.05 when compared with the control group. ** indicates p<0.005 when compared with the control group.

Rejection of drug-sensitive and -resistant tumors by pneuTM DNA vaccination. BALB/c mice were electrovaccinated twice, i.m., 2 weeks apart, with either pneuTM and pGM-CSF or control vector. Sera were collected 2 weeks after the final immunization. Also at 2 weeks after the final immunization, some mice were treated with mAb GK1.5 and 2.43 twice in one week to deplete CD4 and CD8 T cells. mAb treatment continued weekly throughout the remainder of the experiment. (A) Anti-neu antibody was measured by flow cytometry and antibody concentration was calculated by regression analysis as described in materials and methods. (B) PBL were collected and cells from mice within each group were pooled and stimulated with 3T3/KB or 3T3/NKB. The ratio of PBL to APC was 10:1. IFN-γ producing cells were enumerated by ELISPOT assay. Results were expressed as number of spots per 10⁶ PBL and analyzed by Student’s t test; * indicates p<0.05 when compared with the pCMV vaccinated control group. (C) Mice were challenged with TUBO, Bam1a, Bam IR-5, or D2F2/neu, 3 weeks after the last vaccination. Tumor growth was measured weekly by palpation and the results expressed as % tumor free mice. There were 6-10 mice in each group. Results were analyzed by Log rank test. * indicates p<0.05, ** indicates p<0.005 and *** indicates p<0.0005 when compared with the pCMV vaccinated control group. (D) Expression of neu by Bam IR-5 and D2F2/neu tumor outgrowths. Tumors were removed from mice, single cell suspensions were prepared by enzymatic dissociation, and expression of neu on the cell surface was measured using flow cytometry (lower panel). Cells maintained in monolayer culture were used as controls (upper panel). Open histograms represent cells stained with mAb 7.16.4 and filled histograms are isotype controls.