Mapping of the der(X)t(X;17) breakpoint.

<p>(<b>A</b>) A scheme of the bacterial artificial chromosome (BAC) probes used for the FISH analysis in order to localize the translocation. Filled squares represent probes detecting 17q material on both 17q and Xq, empty squares represent probes detecting 17q material only on 17q and not on Xq. A magnification of the breakpoint region is illustrated on the left-hand side of the panel. (<b>B</b>) The breakpoint region is localized in the 17q24.3 region. Fluorescence <i>in situ</i> hybridization analysis along the 17q arm of WI-38T^HP-1 reveals the breakpoint region in the interval between two probes: RP11-387O17 (left-hand side – red) which is detected on chrX in addition to its normal position on chr17. In contrast, probe RP11-304I14 (right-hand side – red) is detected only on the two normal copies of chr17 and not on chrX. ChrX material is marked in green. (<b>C</b>) No aberrations in chromosome Xq material are detected. Fluorescence <i>in situ</i> hybridization along Xq arm. The two probes detecting the most distant area of the Xq arm (RP11-304H and RP11-26A - red) are visible only on the two copies of chrX and not on chr17. The chr17 centromeric region is marked in green.</p

Highly proliferating WI-38T (WI-38T^HP-1/2) exhibit pre-malignant phenotypes.

<p>(<b>A</b>) The growth curve of WI-38T and WI-38T^HP-1/2 cells over 10 days is depicted. (<b>B</b>) The colony formation capability of WI-38T and WI-38T^HP-1/2 is depicted.</p

Gain of the BPTF genomic locus and mRNA levels in human tumors.

<p>(<b>A</b>) Representative pictures of the various chromosomal rearrangements in the BPTF locus in six human tumor types. FISH analysis using probe RP11-387O17 (detecting 17q24.3 region in the BPTF locus) and a probe detecting chr17 centromeric material to distinguish between polysomy and gain of the BPTF locus. The nuclei presented are of lung adenocarcinoma cells and are representative of the different probe patterns detected in the other tumors (colon, neuroblastomas and leukimias). a) normal ploidy. b) chr17 polysomy. c) partial trisomy of 17q24.3. d) gain of 17q24.3. (<b>B</b>) A graph depicts the percentage of samples containing gain of 17q24.3 (a sample in which over 30% of cells exhibit gain was considered a positive sample) as assessed by FISH of a paraffin tissue array containing 143 tumor samples. (<b>C</b>) Expression of BPTF in several human tumors. Depicted is the relative expression of BPTF in several human cancers compared to normal tissues as obtained from the ONCOMINE database.</p

The der(X)t(X;17) translocation is selected in tumors-derived from WI-38T^HP-1 cells harboring the GSE56 and H-Ras^V12 constructs.

<p>(<b>A</b>) The WI-38T^HP-1 cells that express the GSE56 and oncogenic H-Ras^V12 exhibit genomic instability. Spectral karyotyping (SKY) analysis of WI-38T^HP-1 cells which were introduced with GSE56 and oncogenic H-Ras^V12 reveals genomic instability manifested by polysomy and several genomic aberrations. (<b>B</b>) A SKY analysis of WI-38T^HP-1 tumor-derived cells is shown, the der(X)t(X;17) translocation is circled. (<b>C</b>) A FISH analysis on WI-38T^HP-1 tumor-derived cells that harbor a shRNA against p53 (shp53) and oncogenic H-Ras^V12. The probe detecting chr17q24.3 (RP11-387O17 - red) is visible on two copies of chr17 and on one copy of chrX. A probe detecting the most telomeric X chromosomal region (RP11-26A4) is marked in green.</p

Localization of the breakpoint region within the fourth intron of BPTF gene.

<p>(<b>A</b>) DNA samples from primary WI-38 and WI-38T^HP-1 were labeled and hybridized to Agilent custom arrays covering the 0.5-Mb region (the distance between probe RP11-387O17 and RP11-304I14) on chromosome 17 with an average probe spacing of 0.5 kb. (<b>B</b>) Quantitative Real-Time PCR analysis on genomic DNA from WI-38T^HP-1/2 in the BPTF gene revealed a 1.5 fold increase in BPTF genomic dosage compared to WI-38T cells. Two sets of primers were used to detect the gene dosage of BPTF. One set annealing to the 5-prime end of the BPTF gene (BPTF-exon 2) and the second set annealing to the 3-prime end of it (BPTF-exon 30).</p