Histopathology of tumors induced by the <i>ras</i>/<i>myc</i> plasmid.

<p>Tumors induced by circular pMSV-T24-H-<i>ras</i>/MSV-c-<i>myc</i> (panels <b>A</b> and <b>B</b>), and tumors induced by linear pMSV-T24-H-<i>ras</i>/MSV-c-<i>myc</i> (panels <b>C</b>, <b>D</b>, <b>E</b>, and <b>F</b>). Tumors in panels <b>A</b> to <b>E</b> were undifferentiated sarcomas, while the tumor shown in panel F had the appearance of a differentiated fibrosarcoma.</p

Evaluating the oncogenicity of DNA from cell lines established from four human tumors.

<p>Newborn CD3 epsilon mice were inoculated with 100 µg of cellular DNA from HeLa cells, CEM cells, A549 cells, or HT-1080 cells in the absence or presence of 1 µg of linear pMSV-T24-H-<i>ras</i>/MSV-c-<i>myc</i> DNA. Tumors were found in 14/14 mice inoculated with HeLa DNA plus <i>ras</i>/<i>myc</i> DNA but in 0/55 inoculated with HeLa DNA alone; tumors were found in 19/19 mice inoculated with CEM DNA plus <i>ras</i>/<i>myc</i> DNA but in 0/55 inoculated with CEM DNA alone; tumors were found in 13/13 mice inoculated with A549 DNA plus <i>ras</i>/<i>myc</i> DNA but in 0/53 inoculated with A549 DNA alone; and tumors were found in 5/5 mice inoculated with HT-1080 DNA plus <i>ras</i>/<i>myc</i> DNA but in 0/19 inoculated with HT-1080 DNA alone. Tumors appeared within 8 weeks, and all were at the site of inoculation.</p

Multiple tumors induced in newborn CD3 epsilon mice.

<p><b>A</b>. Three tumors evident in a newborn CD3 epsilon mouse that was inoculated SC with 25 µg of circular pMSV-T24-H-<i>ras</i>/MSV-c-<i>myc</i> DNA. <b>B</b>. Seven tumors evident in a CD3 epsilon mouse inoculated SC with 1 µg of linear pMSV-T24-H-<i>ras</i>/MSV-c-<i>myc</i> DNA; some of the tumors were not apparent until necropsy.</p

Integration patterns of the <i>myc</i> and <i>ras</i> genes in cell lines derived from tumors induced by linear pMSV-T24-H-<i>ras</i>/MSV-c-<i>myc</i>.

<p>DNA was isolated from tumor cell lines, digested with <i>Bgl</i>II, an enzyme that cuts the plasmid once, transferred to a membrane, and probed first with a <i>myc</i> probe (<b>A</b>), and then (after stripping the membrane) with a <i>ras</i> probe (<b>B</b>). Lane a is mouse NIH 3T3 DNA; lanes b to n represent different tumor cell lines. The position of the endogenous mouse c-<i>myc</i> gene is indicated by the arrow.</p

Analysis of the integration sites of the <i>myc</i> and <i>ras</i> genes in cell lines derived from tumors induced by linear pMSV-T24-H-<i>ras</i>/MSV-c-<i>myc</i>.

<p>DNA was isolated from tumor cell lines, digested with <i>BsrGI</i>, an enzyme that does not cut the plasmid, transferred to a membrane, and probed with a <i>myc</i> probe (<b>A</b>), or with a <i>ras</i> probe (<b>B</b>). Lane a is mouse NIH/3T3 DNA; lanes b to n represent different tumor cell lines. The position of the endogenous mouse c-<i>myc</i> gene is indicated by the arrow. [The apparent band in lane a in Fig. 7B is background.]</p

Localization of H-Ras and c-Myc in tumor-cell lines.

<p>Cells in chamber slides were fixed with 3.7% paraformaldehyde and permeabilized by treating with ice-cold acetone/methanol (1∶1) on ice for 15 min. The slides were then washed with PBS containing 0.1% Tween 20 (0.1%PBST) three times and blocked for 1 h with 5% normal goat serum. The anti-H-Ras mouse monoclonal antibody F235 (sc-29) and the anti-c-Myc rabbit polyclonal antibody C-19 (sc-788) were used at 1∶100 dilution. The antibodies were mixed and allowed to react overnight at 4°C. The second antibodies (at 1∶2500 dilution) were goat anti-mouse conjugated with Alexa Fluor 594 (for H-Ras) and goat anti-rabbit conjugated with Alexa Fluor 488 (for c-Myc). The slides were visualized using an Olympus 1×51 microscope fitted with fluorescence. Pictures were taken using a 40x objective, and images were over-layered using Olympus CellSens software at a sensitivity of 800 ISO with a resolution of 680×512 for live images and 1360×1024 for snap images. Panels <b>A</b>, <b>B</b>, <b>C</b>: NIH/3T3 cells. Panels <b>D</b>, <b>E</b>, <b>F</b> and <b>G</b>, <b>H</b>, <b>I</b> corresponded to two lines derived from tumors induced by circular <i>ras/myc</i> plasmid, while panels <b>J</b>, <b>K</b>, <b>L</b> and <b>M</b>, <b>N</b>, <b>O</b> corresponded to two lines derived from tumors induced by linear <i>ras/myc</i> plasmid. Panels <b>A</b>, <b>D</b>, <b>G</b>, <b>J</b>, <b>M</b>: anti-H-Ras antibody. Panels corresponded to two lines derived from tumors induced by circular <i>ras/myc</i> plasmid, <b>E</b>, <b>H</b>, <b>K</b>, <b>N</b>: anti c-Myc antibody. Panels <b>C</b>, <b>F</b>, <b>I</b>, <b>L</b>, <b>O</b>: merged images.</p

Structure of pMSV-T24-H-ras/MSV-c-myc.

<p>The human T24-H-<i>ras</i> gene is shown in red, the murine c-<i>myc</i> gene is shown in yellow, the MSV LTR elements are shown in blue.</p

All tumor cell lines expressed both the H-Ras and the c-Myc proteins.

<p>Lysates of the tumor cell lines corresponding to 2.5 µg of protein for H-Ras detection and 30 µg of protein for c-Myc detection were resolved by SDS-PAGE on 4–20% gradient gels and the proteins revealed by western analysis. <b>A & B</b>: c-Myc. <b>C & D</b>: H-Ras. <b>A & C</b>: Lines from tumors induced by <i>Sca</i>I linear plasmid; <b>B & D</b>: Lines from tumors induced by <i>Eco</i>RI linear plasmid. Lanes a: NIH/3T3 mouse control cell line. Lanes b to n represent different tumor-cell lines; these lines correspond to the cell lines in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108926#pone-0108926-g006" target="_blank">Fig. 6</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108926#pone-0108926-g007" target="_blank">Fig. 7</a>. Membranes were subsequently reacted with an antibody to actin to assess protein loadings. The PVDF membranes were reacted with the anti-actin antibody sc-1616; a band migrating at approximately 43 kD is seen in all lanes.</p

Dose-response data in CD3 epsilon mice inoculated with circular or linear pMSV-T24-H-<i>ras</i>/MSV-c-<i>myc</i>.

<p>ND: Not done.</p><p>Dose-response data in CD3 epsilon mice inoculated with circular or linear pMSV-T24-H-<i>ras</i>/MSV-c-<i>myc</i>.</p

Linear DNA is more oncogenic than circular DNA.

<p>Data from the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108926#pone-0108926-t001" target="_blank">Table 1</a> were plotted using GraphPad Prizm 5.</p