Electrophoretogram of sequencing analysis. “D” and “A” designate sequencing results of DNA from diploid and aneuploid nuclei of tumor sample CHTN56, respectively. Arrow indicates position of somatic mutation.

RNA blot analysis of T47D with inducible DBC2. T-47D cells with inducible DBC2 were treated with 5 μM of Muristerone A. DBC2 expression was examined at various time points after induction. The DBC2 transcript was detected only after induction. The control is β actin.

Effects of gene induction on the growth of T47D. T-47D cells with various constructs were treated with 5 μM Muristerone A. Percent increase of the cell count from 12 h after induction is shown. Expression of wild-type DBC2 inhibits cell growth of T47D between 12 and 48 h after gene induction, whereas mutant DBC2 and the empty vector have no influence on T47D proliferation.

RT-PCR analysis of tumor specimens. (A) RT-PCR products from mRNA of T-47D with five primer sets. Lanes 1–5 contain RT-PCR products with primers of DBC1, DBC2, KIAA0273, TNFRSF10A, and p53, respectively. Transcripts of all genes but DBC2 were detected in T-47D. (B) RT-PCR products with the DBC2 primer pair. Lanes 6–11 contain RT-PCR products from NCI-H82, Saos-2, SK-BR-3, SW 982, T-47D, and T84, respectively. DBC2 expression was observed in NCI-H82, Saos-2, SK-BR-3, and SW 982 but not in T-47D or T84.

Deletion map of 8p21. The vertical lines indicate the localization of sequence tag sites and genes used for deletion mapping, all of which were tested against 200 tumor samples. Genes are represented by a horizontal line with an arrowhead. The STSs are listed above the line, and the lines with a square indicate probes derived from the genes. The arrow with double heads indicates approximately 300 kb. Horizontal lines in the lower half represent deletions found in the tumor samples (named, Left). The DBC2 probe and WI-16696 demonstrated the highest deletion frequency. Both DBC2 and TNFRSF10B genes are overlapping with different orientations and deleted in all tumor specimens.

Deletion analysis of tumor samples. (A) Real-time PCR analysis of a breast tumor sample CHTN41 is demonstrated. The abscissa is cycle number of PCR and the ordinate is quantity of PCR products. The control primer set in this figure is WB23 from chromosome 22. “D” and “A” represent DNA from diploid and aneuploid fractions, respectively. The amplification curve from aneuloid DNA with WD31 primer set has a several cycle difference from the others, indicating deletion of WD31 in this tumor. (B) Southern blot analysis of DpnII representation with WD31 probe is shown. Each lane contains 5 μg of DpnII restriction fragments. Lanes 1 and 2 are DNA from a tumor and a matched normal (CHTN40), respectively, showing no deletion. Lanes 3 and 4 contain DNA from a tumor and a matched normal pair (CHTN41), showing deletion. ER48 is a control probe derived from chromosome 3. Lane 3 has a very faint band for DBC2, whereas the control probe exhibits the same intensity for tumor and normal samples, representing homozygous deletion. Contamination of normal stromal cells is considered to contribute the faint band.

Structure of DBC2. (A) The intron–exon structure of DBC2 (GenBank accession no. AF315385) and predicted functional domains of DBC2 are shown. The rectangles with a number represent exons. The shaded area indicates coding sequences. Based on deduced amino acid sequence, molecular mass of the predicted DBC2 product is 83 kDa. The asterisks denote missense mutations we have discovered. There were three in exon 5: T to G (7762), causing Tyr-284→Asp in a lung cancer cell line (SK-Mes-1), G to A (7807), causing Asp-299→Asn in a primary breast tumor (CHTN56), and A to C (8015), causing Asp-368→Ala in a breast cancer cell line (BT483); and one in exon 9: C to A (16389), causing Phe-647→Thr in a primary breast tumor (P-1). (B) Phylogenic tree demonstrating overall homology between DBC2 and human RAS family members is shown. A table of the pairwise distances between the genes was prepared by GCG Wisconsin Package, and a phylogram was then created from the table by using the neighbor-joining method. The length of a horizontal line is proportional to estimated divergence along each branch. Accession numbers of the genes used for this analysis are: RRAS1 (P10301); RRAS2 (P17082); RIT (AAB42213); RAN (P17080); RAD (P55042); RIN (AAB42214); RAB3A (P20336); GEM (P55040); RHEB (Q15382); RRAS2 (O14807); RHO A (P06749); H-RAS (P01112); RAP A (P10113); RAC1 (P15154); G25K (P25763); and RAL A (P11233). (C) RAS-BTB proteins from different organisms are compared. Accession numbers of the genes are listed after the designation of the organism: Hs, Homo sapiens; Mm, M. musculus; Dm, D. melanogaster; and Dd, Dictyostelium discoideum.