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Cell line Ad-312/SV40, which was derived from a primary pleomorphic salivary gland adenoma with t(1;12)(p22;q15), was used in fluorescence in situ hybridization (FISH) analysis to characterize its translocation breakpoint region on chromosome 12. Results of previous studies have indicated that the chromosome 12 breakpoint in Ad-312/SV40 is located proximally to locus D12S8 and distally to the CHOP gene. We here describe two partially overlapping yeast artificial chromosome (YAC) clones, Y4854 (500 kbp) and Y9091 (460 kbp), which we isolated in the context of a chromosome walking project with D12S8 and CHOP as starting points. We present a composite long-range restriction map encompassing the inserts of these two YAC clones and show by FISH analysis that both YACs span the chromosome 12 breakpoint as present in Ad-312/SV40 cells. Subsequently, we have isolated cosmid clones corresponding to various sequence-tagged sites (STSs) mapping within the inserts of these YAC clones. These included cRM51, cRM69, cRM85, cRM90, cRM91, cRM110, and cRM111. In FISH studies, cosmid clones cRM85, cRM90, and cRM111 appeared to map distally to the chromosome 12 breakpoint, whereas cosmid clones cRM51, cRM69, cRM91, and cRM110 were found to map proximally to it. These results assign the chromosome 12 breakpoint in Ad-312/SV40 to a DNA region of less than 165 kbp. FISH evaluation of the chromosome 12 breakpoints in five other pleomorphic salivary gland adenoma cell lines indicated that these are located proximally to the one in Ad-312/SV40, at a distance of more than 0.9 Mbp from STS RM91. These results, while pinpointing a potentially critical region on chromosome 12, also provide evidence for the possible involvement of 12q13-q15 sequences located elsewhere.

Specific DNA repeats serve as a molecular protection shield at the telomeric ends of mammalian chromosomes. The absence of telomerase activity leads to a gradual decrease of telomeric repeat length in normal somatic cells. In contrast, immortalized cells from malignant tumors are usually thought to re-express telomerase to overcome a self-limited growth. Following this hypothesis, re-expression of telomerase is due to a rare mutational event. Herein we describe our results of telomeric length determination in two newly established breast cancer cell lines. During in vitro establishment from pleural effusions, both cell lines showed a marked decrease of the upper border range of telomeric repeat length distribution. The lower border remained within a constant range characteristic for each cell line. In no case was decrease of repeat length accompanied by an increased incidence of telomeric associations or fusions. The results show that a constant telomeric repeat length does not constitute a characteristic feature of immortalized cells. Furthermore, the kinetics of repeat length decrease and the constant range of the lower border reveal that the onset of telomerase activity is not necessarily due to a rare, i.e., mutational, event.

We report here the physical mapping of recurrent chromosome 12q13-q15 breakpoints in cell lines derived from primary myxoid liposarcoma, lipoma, uterine leiomyoma, and pleomorphic adenoma of the salivary glands. In fluorescence in situ hybridization (FISH) experiments, we first mapped the position of the chromosome 12 translocation breakpoint in uterine leiomyoma cell line LM-30.1/SV40 relative to loci COL2A1, D12S4, D12S17, D12S6, D12S6, D12S19, D12S8, and D12S7. It mapped between linkage probes CRI-C86 (D12S19) and p7G11 (D12S8). We then isolated YAC clones using CRI-C86- and p7G11-derived sequence-tagged sites, constructed corresponding YAC contigs of 310 and 800 kb, respectively, and established long-range physical maps of these. Cosmid clones LLNL12NCO1-98C10 and LLNL12NCO1-113D12 were isolated using STSs within the CRI-C86- and the p7G11-derived YAC contigs, respectively, and a mixture of them was used to routinely study the various tumor cell lines by FISH analysis. The chromosome 12 breakpoints of all tumor cell lines tested mapped between cosmids LLNL12NCO1-98C10 and LLNL12NCO1-113D12. None of the breakpoints appeared to map within any of the isolated YAC clones. Furthermore, FISH analysis using cosmid LLNL12-NCO1-144G3, which maps at the CHOP locus, revealed that the chromosome 12 breakpoints in all cell lines of the three benign solid tumors that were tested were located distal to the chromosome 12 translocation breakpoint with the CHOP gene in myxoid liposarcoma cells with t(12;16). In conclusion, our studies seem to indicate that the chromosome 12 breakpoints of myxoid liposarcoma, lipoma, uterine leiomyoma, and pleomorphic adenoma of the salivary glands are all clustered within the 7-cM interval between D12S19 and D12S8, with those of the benign solid tumors distal to CHOP. Finally, the MYF5 gene mapped telomeric to LLNL12NCO1-113D12, and the MIP gene mapped centromeric to the chromosome 12 translocation breakpoint in myxoid liposarcoma cells.

We have hypothesized that metacentric and submetacentric chromosomes frequently observed in malignant canine tumors are a result of telomeric fusions. Therefore cells from a canine mammary pleomorphic adenoma were transformed with a plasmid containing the SV40 'early region', known to cause telomeric associations. Compared with non-transformed adenoma cells, the cells had a higher proliferative capacity and expressed the large SV40-T-antigen. Karyotype studies showed the conversion from a normal to an aberrant karyotype with an increase of bi-armed chromosomes resulting from fusions of acrocentric chromosomes. In addition, the length of the telomeric repeats (TTAGGG) was determined for an early and a late passage of the transformed cells by Southern hybridization. The length of the telomeric repeats was apparently longer in the 5th than in the 38th passage. In situ hybridization with a telomere-specific DNA revealed interstitial telomeric repeats in the bi-armed chromosomes. We have concluded that these findings reflect the clonal expansion of head-to-head-telomeric fusions of canine acrocentric chromosomes leading to dicentric chromosomes with a very short distance between the two centromeres. Our results support the idea that the apparent centric fusions that have been described in some canine tumors may in fact be the cytogenetic products of head-to-head-telomeric fusions.

We developed cell line LIS-3/SV40 from a primary myxoid liposarcoma with the t(12;16)(q13;p11.2) and trisomy 8. Following fusion of LIS-3/SV40 to mouse A9 cells, we obtained hybrid LIS-3/SV40/A9-B4 which contained the der(16) but neither the der(12) nor some of the normal human chromosomes, including 12 and 16. Furthermore, microclone library ML12q1315 was constructed from microdissected fragments of chromosome bands 12q13-15 and its microclones were found to span the breakpoint in LIS-3/SV40.

Chromosomal aberrations involving 8q12 or 12q13-15 characterize two cytogenetic subgroups of salivary gland pleomorphic adenomas. As the tumors of the two groups differ in their clinical and histologic characteristics, we decided to determine their susceptibility to SV40 transformation. We transfected cell cultures from 13 adenomas with aberrations involving 8q12 and from seven adenomas with involvement of 12q13-15 using an SV40 plasmid coding for the early region of the viral genome. Whereas all cultures with aberrations of 12q13-15 showed transformed foci, only 4 of the 13 cultures with 8q12 abnormalities showed foci of transformed cells. We also observed a much higher immortalization rate in the first group (3/7 vs. 1/13). All successfully transformed tumor cell cultures showed a relatively stable karyotype in the pre-crisis stage and a high mitotic index, were T-antigen positive, and had an extended life span in vitro.

12q13-15 changes are the most frequent cytogenetic abnormalities in human tumor cells. To test their biological significance we used an assay based on lipoma cells with a limited in vitro lifetime and this type of chromosomal aberration. Lipoma cells with a reciprocal translocation t(3;12)(q28;q14) were transformed by transfection with a plasmid containing the SV40 "early region". The transformed cells showed an altered morphology with loss of contact inhibition, formation of foci, and T-antigen expression. They were immortalized after a growth crisis. The karyotypic patterns before and after the crisis show that the translocation together with expression of SV40 T-antigen is not sufficient for direct immortalization.

Tumor cells from three uterine leiomyomas showing translocations involving 12q14-15 were transformed by transfection using the "early regions" of the SV40 genome. The cells had a higher proliferative capacity, were able to form colonies in soft agar, and showed an increased growth potential. Karyotype analyses of these transformed leiomyoma cells showed that the cells had retained the initial t(12;14) and t(12;15).

Cells from a salivary gland pleomorphic adenoma with a reciprocal translocation, t(8;12)(q22;q14), were transformed by transfection with a plasmid containing the SV40 "early region." Compared with nontransformed adenoma cells, the cells expressed large-T antigen, had a higher proliferative capacity, showed a drastically increased or even unlimited growth potential in vitro, and formed colonies in soft agar. The amount of cellular material produced in this way should make possible many experiments at the molecular level that could not be performed before.