FITC immunostaining of apoptin expression in breast carcinoma cell lines.

<p>Immunostaining of apoptin protein showed that in can express in breast carcinoma cell lines after 12λ ZAP-CMV vector have not any apoptosis after 36 h as same as untreated cells.</p

Confirmation of apoptosis by flowcytometry.

<p>BT-474, SKBR-3 and ZR-75 cells were examined after 36 h of transfection by flowcytometry. All the cell lines were susceptible to NBPs apoptin-induced apoptosis. We have not apoptosis in vector treated group and untreated group.</p

Recovery of NBPs from nude mice tissues.

<p>There were no significant differences in recombinant NBPs titer that was recovered from the different tissues of the treated mice. The tumor site was the only part of the mice body to support the more NBPs accumulation.</p

Cytotoxicity evaluation of plasmid and vehicle.

<p>BT-474 breast carcinoma cell line transfected with λ ZAP-CMV-apoptin, λ ZAP-CMV vector and λ phage (vehicle) construct stained with FITC immunostaining and then visualized by fluorescence microscopy. There was no sign of cell necrosis after the treatments. There is only apoptotic morphology of cells after treatment with λ ZAP-CMV-apoptin.</p

Immunohistochemical results of NBPs treated animals.

<p>(<b>A</b>) Histochemistry analysis of tumor tissue sections showing apoptotic changes. The untreated tumor tissue contains many dividing cells. After 96 h treatment with NBPs there are a few cells maintained in the tumor tissue that could proliferate. Tumor growth was markedly suppressed in the apoptin treated group, (<b>B</b>) Histological examination of other organs (brain and heart) in tumor bearing mice that is not involved in the pathological changes of BT-474 cells. There are no changes in morphology of the brain and/or heart tissues and they are as same as control groups.</p

Determination of tumor size in nude mice.

<p>(<b>A</b>) Treatment of nude mice bearing human breast cancer xenografts with BT-474. Injection of BT-474 breast carcinoma cells to the nude mice resulted in the growth of xenografts. The Tumors were injected 3 times a week with 10⁹ PFU of recombinant NBPs, only vector and PBS. Hereafter, tumor growth was measured regularly, (<b>B</b>) Volumes of the BT-474 tumor tissue during treatment. Median tumor volume is determined over time in all treated groups. Tumor volumes were calculated using the equation: Length × width² ×0.52, (<b>C</b>) The effects of different treatments on change in body weight over the treatment period. Average body weight was calculated for each treatment group at the indicated time points.</p

Cell viability and apoptosis after NBPs treatment.

<p>(<b>A</b>) Apoptosis in BT-474 breast carcinoma cell line induced by λ NBPs. The right side indicates NBPs treated group and Left side image indicates control group, (<b>B</b>) Cell viability determined by MTT dye reduction assay.</p

Design and Construction of NBPs harboring λ ZAP-CMV-apoptin expression plasmid.

<p>(<b>A</b>) The expression vector was subsequently inserted into the λ phage, generating the recombinant NBPs, (<b>B</b>) Different cell types were infected with the indicated recombinant λ NBPs at an MOI of 10 PFU/cell and apoptin transcription was analyzed by RT-PCR, (<b>C</b>) RT-PCR result for λ ZAP-CMV vector treated cells that have no expression of apoptin, (<b>D</b>) Western blot analysis to detect apoptin protein from cells supernatants and lysates. To analyze apoptin expression, the cells were infected with the indicated recombinant NBPs. Purified apoptin was used as a positive control and CAV infected BT-474 cell was used as a negative control.</p