Furthermore, we investigated the mechanism of TNIP1-induced keratinocyte proliferation

n situ hybridization karyotyping of UE6E7T-3 at three culture stages. Chromosome 13 is circled. Colony formation in soft agar at three culture stages. Graphical representation of the relative colony counts at four culture stages. Colonies !300 m in diameter were counted. Bars represent mean numbers of colonies of triplicates., p < 0.01. Hematoxylin and eosin staining of invasive sarcoma following i.m. injection of U3-DT. Scale bar, 500m.. Magnification of transformed U3-DT in mouse muscle tissue. Dividing cells, cells containing two nuclear bodies, and cells containing increased chromatin indicate tumorigenesis. Scale bar, 50 m. LGX-818 chemical information PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19784385 doi:10.1371/journal.pone.0126562.g002 8 / 23 Alteration in Gene Expression on Transformation and subsequently near-triploidy with 6472 chromosomes. At further passages; the population pattern remained stable with mostly near-triploid cells. We next analyzed the structural karyotypes of UE6E7T-3 by mFISH. At PDL 60, cells exhibited a structurally normal diploid karyotype. By contrast, at PDL 92147 there were several distinct populations. Most of the near-diploid population, with 45 chromosomes, had lost one copy of chromosome 13 and one p-arm of chromosome 16 without translocations or insertions, whereas the population with 44 chromosomes had lost an additional chromosome . The near-tetraploid population consistently maintained two deletions of two copies of chromosome 13 and two p-arms of chromosome 16, with random loss of other chromosomes, but exhibited few structural rearrangements. These results indicate that near-diploid aneuploidy arose through the loss of one or two chromosomes from a diploid cell; subsequently, near-diploid cells spontaneously became near-tetraploid via cleavage failure. After the formation of near-tetraploid, structural rearrangements such PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19785914 as translocations, insertions, and deletions occurred frequently, e.g., the fusion of chromosome 13 and chromosome 8, and the insertion of chromosome 13 into chromosome 14. This suggests that the deletion of chromosome 13 induces further chromosomal instability. Neoplastic transformation of UE6E7T-3 cell line Transformed cells lose contact inhibition, distinguishing them from normal cells, which cannot grow past confluence. To test the contribution of chromosomal instability to transformation in vitro and in vivo, we examined the ability of UE6E7T-3 cells to form colonies in soft agar and tumors in immunodeficient mice. When UE6E7T-3 at various stages were seeded in soft agar, no anchorage-independent growth could be detected at early PDL, even after 4 weeks in culture. By contrast, a considerable number of anchorage-independent colonies were detected when PDL 252 cells were seeded in soft agar. To determine whether this colony formation indicated tumorigenicity, cells at PDL 262 were injected subcutaneously or intramuscularly into immunodeficient nude mice. Four of the six mice injected with the PDL 262 cells formed sarcomas at the injection site after 4 weeks. Of the three mice injected intramuscularly, all formed sarcomas, although only one of the three mice injected subcutaneously did. Histological examination of the injected quadriceps femoris revealed these tumors to be invasive sarcomas, exhibiting spindle-shaped cells characteristic of fibroblasts. This observation indicates that UE6E7T-3 cells had undergone neoplastic transformation during long-term propagation in culture. Gene expression profiling characterized at four stages of transformati