aphidicolin and Carcinoma--Renal-Cell

The FHIT gene spans approximately 1 Mb of DNA at chromosome band 3p14.2, which includes the familial renal cell carcinoma chromosome translocation breakpoint (between FHIT exons 3 and 4), the most frequently expressed human constitutive chromosomal fragile site (FRA3B, telomeric to the t(3;8) translocation), and numerous homozygous deletions in various human cancers, frequently involving FHIT exon 5. The FRA3B has previously been shown to represent more than one specific site, and some specific representatives of FRA3B breaks have been shown to fall in two regions, which we know to be in FHIT introns 4 and intron 5. Because breakage and integration of exogenous DNA in this chromosome region is frequent in aphidicolin-treated somatic cell hybrids, cancer cells, and, presumably, aphidicolin-treated normal lymphocytes that exhibit gaps or breaks, we determined by one- and two color fluorescence in situ hybridization, using cosmids covering specific regions of the FHIT gene, that most of the aphidicolin-induced gaps at FRA3B fall within the FHIT gene, with the highest frequency of gaps falling in intron 5 of the FHIT gene, less than 30 kb telomeric to FHIT exon 5. Gaps also occur in intron 4, where a human papillomavirus 16 integration site has been localized, and in intron 3, where the t(3;8) break point is located. These results suggest that the cancer-specific deletions, which frequently involve introns 4 and 5, originated through breaks in fragile sites.

Topics: Acid Anhydride Hydrolases; Aphidicolin; Carcinoma, Renal Cell; Chromosome Fragile Sites; Chromosome Fragility; Chromosome Mapping; Chromosome Walking; Chromosomes, Human, Pair 3; DNA Probes; Exons; Humans; In Situ Hybridization, Fluorescence; Kidney Neoplasms; Lymphocytes; Molecular Sequence Data; Neoplasm Proteins; Proteins

The constitutive fragile site at human chromosomal band 3p14.2, FRA3B, has been described as the most active common fragile site in the human genome. FRA3B is cytologically indistinguishable from the chromosome 3 breakpoint observed in the hereditary renal cell carcinoma (hRCC) translocation t(3;8) (p14.2;q24.13). Previous work demonstrated that a 1330-kb YAC clone, YC850A6, spans both the t(3;8) translocation and FRA3B and also encompasses FRA3B-associated breakpoints induced in hamster-human hybrids. This YAC was used to construct a multi-hit cosmid library. Screening of this library resulted in a 350-kb cosmid contig that extends distally from the t(3;8) translocation breakpoint. Seventeen aphidicolin-induced 3p14. 2 breakpoints derived from hamster-human hybrids were mapped within this cosmid contig. These breakpoints were found to localize as two distinct clusters, separated by 200 kb, which lie on either side of a region of frequent breakage within FRA3B as defined by FISH analysis using cosmids from the contigs. The most proximal of the breakpoint clusters lies approximately 100 kb distal to the hRCC t(3;8) breakpoint. The distribution of these breakpoints, together with the region of frequent chromosomal breakage mapped by FISH analysis, further confirms the position of FRA3B and helps to define the extent over which its fragility is exerted. These data indicate that FRA3B comprises several hundred kilobases of DNA sequence within 3p14.2. The 350-kb contig and the cosmid library constructed from YAC YC850A6 will be essential for further characterization of the region surrounding FRA3B and in experiments to determine the molecular basis of the fragility of FRA3B.

Topics: Animals; Aphidicolin; Base Sequence; Carcinoma, Renal Cell; Chromosome Fragile Sites; Chromosome Fragility; Chromosomes, Artificial, Yeast; Chromosomes, Human, Pair 3; Chromosomes, Human, Pair 8; Cosmids; Cricetinae; Enzyme Inhibitors; Gene Library; Humans; Hybrid Cells; Kidney Neoplasms; Molecular Sequence Data; Neoplastic Syndromes, Hereditary; Nucleic Acid Synthesis Inhibitors; Polymerase Chain Reaction; Translocation, Genetic; Trinucleotide Repeats

Human chromosome band 3p14 contains two tightly linked cytogenetic markers of broad interest, FRA3B and the t(3;8) breakpoint associated with hereditary renal cell carcinoma (RCC). The common fragile site at 3p14.2 (FRA3B) is the most sensitive site on normal human chromosomes to breakage when DNA replication is perturbed by aphidicolin or folate stress. The t(3;8)(p14.2;q24.1) translocation segregates with RCC in a large family and could mark the location of a tumor suppressor gene involved in renal cancers. In studies aimed at positional cloning of FRA3B and the t(3;8) breakpoint, we have used multicolor fluorescence in situ hybridization analysis (FISH) on metaphase spreads and interphase nuclei to order 14 yeast artificial chromosomes (YACs) in 3p14. The YACs used in this study were identified by a group of unordered lambda clones that had been previously localized to the 3p14 region and mapped proximal or distal to the t(3;8) breakpoint. FISH analysis was used to order the YACs and to map them in relation both to the t(3;8) translocation breakpoint and to FRA3B induced on normal chromosomes by treatment with aphidicolin. YACs that closely flanked both the t(3;8) translocation breakpoint and the fragile site were identified. A YAC walk from the closest distal YAC allowed the identification of a 1.3-Mb YAC derived from the CEPH large insert YAC library that spans both the FRA3B and the t(3;8) breakpoint. The order of the YACs and cytogenetic landmarks in 3p14 is cen-(126E1/230B9)-181H6-B15-D20F4-258B7-++ +280D2-70E12-168A8- 403B2-143C5-413C6-468B10-[850A6/t(3;8)/ FRA3B]-74B2.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Aphidicolin; Carcinoma, Renal Cell; Cells, Cultured; Chromosome Fragile Sites; Chromosome Fragility; Chromosome Mapping; Chromosomes, Artificial, Yeast; Chromosomes, Human, Pair 3; Chromosomes, Human, Pair 8; Cloning, Molecular; Cricetinae; Cricetulus; Female; Fibroblasts; Humans; Hybrid Cells; In Situ Hybridization, Fluorescence; Interphase; Kidney Neoplasms; Male; Metaphase; Translocation, Genetic

The common fragile site at 3p14(FRA3B) is cytogenetically close to the positions of translocation and deletion breakpoints frequently observed in renal cell carcinoma (RCC) and small cell carcinoma of the lung. Possible involvement of this fragile site in the familial RCC t(3;8)(p14.2;q24.1) was investigated. Expression of FRA3B, induced by treatment of lymphocytes with aphidicolin, is altered by the translocation. These results suggest that the fragile site is very close to, if not coincident with, the translocation breakpoint.

Topics: Aphidicolin; Carcinoma, Renal Cell; Cells, Cultured; Chromosome Banding; Chromosome Fragile Sites; Chromosome Fragility; Chromosomes, Human, Pair 3; Chromosomes, Human, Pair 8; Diterpenes; Genetic Markers; Humans; Karyotyping; Kidney Neoplasms; Lymphocytes; Translocation, Genetic

The common fragile site in human chromosome band 3p14 is a constant cytogenetic marker present on every normal chromosome #3. Therefore, we selected a renal cell carcinoma with a deletion breakpoint in 3p14 for analysis of the 3p14 fragile site. Aphidicolin was used to induce the expression of the 3p14 fragile site. The fragile sites expressed in the renal carcinoma cells generally mirrored those expressed in lymphocytes. The normal chromosome #3 in the renal carcinoma cells expressed the common 3p14 fragile site. The partially deleted #3 did not. The deletion breakpoint, therefore, cannot be beyond the 3p14 fragile site. The common fragile site in 3p14 must be at or very near the deletion breakpoint in 3p14 in renal cell carcinoma. These results are consistent with this fragile site causing this cancer chromosome deletion.

Topics: Aphidicolin; Carcinoma, Renal Cell; Chromosome Banding; Chromosome Deletion; Chromosome Fragile Sites; Chromosome Fragility; Chromosomes, Human, Pair 3; Diterpenes; Genetic Markers; Humans; Karyotyping; Kidney Neoplasms; Tumor Cells, Cultured