2. Introduction. 3. Human Breast Epithelial Cells (HBEC) in Culture 4. Factors Influencing Susceptibility of HBEC to Cell Transformation 4.1. Lobular differentiation 4.2. Genetic predisposition 4.3. Cell immortalization 5. Molecular Mechanisms of Cell Immortalization 5.1. Activation of telomerase 5.2. Abrogation of cell cycle control 5.3. Genes preferentially expressed during cell immortalization 6. Molecular Mechanisms of Cell Transformation 6.1. Epigenetic mechanisms 6.2. Genetic mechanisms 7. Genomic changes in Immortalization and Transformation of HBEC 7.1. Genomic changes in cell immortalization 7.2. Genomic changes in cell transformation 8. Genomic Changes in Human Breast Lesions 9. Functional Roles of Chromosomes 11 and 17 in Transformed Phenotype Expression of HBEC 10. Summary and Perspectives 11. Acknowledgments 12. References

Subtractive hybridization was used to isolate genes expressed uniquely in the immortalized human breast epithelial cell (HBEC) line MCF-10F and not in the mortal HBEC line S-130, from which MCF-10F cells were derived. We identified a 233-bp cDNA that was expressed in MCF-10F cells and not in their mortal counterpart S-130 cells. Sequence comparison with the GenBank database revealed that the cDNA was identical to the gene encoding human ferritin heavy H chain. Northern blot analysis using the isolated cDNA as a probe showed a differentially expressed 1.1-kb transcript of ferritin H in total RNA from the immortal MCF-10F cells, MCF-10F cells treated with the chemical carcinogens 7,12-dimethylbenz[a]anthracene and benzo[a]pyrene, and the breast cancer cell lines MCF-7, HBL-100, T-47D, and BT-20. No ferritin H transcript was detected in the mortal line S-130 or in other primary HBEC cultures. Increased levels of mRNA transcript signals were also detected in total RNA from breast cancer tissue samples. Tissue with ductal hyperplasia had higher expression levels than normal adjacent mammary tissue. In situ hybridization showed high levels of ferritin H transcript in mammary tissue areas with ductal hyperplasia, carcinoma in situ, and infiltrating ductal carcinoma. This is the first report of the differential expression and upregulation of human ferritin H chain gene in immortal HBECs. It may be an important factor in the process of immortalization, possibly an early stage of malignant transformation of HBECs, providing cells with iron necessary for growth and clonal expansion. Also, ferritin iron, once released, may increase the level of reactive iron, leading to an increase in oxygen free-radical generation, oxidative DNA damage, and mutation.

The RT7 gene recently cloned by us is expressed as an abundant RNA in round spermatids. In vitro transcription-translation showed that the RT7 gene encodes a protein of 26-27 kDa on SDS-polyacrylamide gels. Here we report the development of monoclonal antibodies (mAbs) raised against a peptide from the predicted N-terminal amphipathic alpha-helix of the rat RT7 protein. All mAbs recognize RT7 protein or N-terminal parts of it. To investigate RT7 in vivo, mAbs were used in immunofluorescence microscopy and confocal laser immunofluorescence microscopy. Several mAbs recognize RT7 protein in elongating spermatids: the observed staining pattern suggests a nonrandom localization in these cells. Two mAbs recognize the protein only in sperm tails. Using co-immunoprecipitation assays, we found that RT7 can form stable complexes with itself that are associated through a region located in the N-terminal half of RT7. Our results identify the RT7 protein as a major sperm tail component and suggest that it may be a structural component of sperm tail outer dense fibers (ODF).

Possible functions of the c-mos proto-oncogene during spermatogenesis were investigated through perturbations of its expression in transgenic mice. Two promoters, one from the pre-meiotic male germ cell-specific mouse phosphoglycerate kinase 2 gene, and the other from the post-meiotic male germ cell-specific rat RT7 gene were used to direct expression of c-mos. Northern blot analysis of testis RNA from transgenic PGK-c-mos mice indicated elevated levels of c-mos RNA in spermatocytes and spermatids compared to controls. No transgene expression was detected in any other tissue examined, suggesting that the mouse PGK2 promoter, like the previously used human PGK2 promoter, confers correct cell-specific expression onto c-mos. The promoter from a newly characterized rat gene, RT7, was shown to direct expression specific to post-meiotic spermatids. Transgenic mice carrying an RT7-lacZ construct displayed immunoreactive bacterial beta-galactosidase as well as enzyme activity in round spermatids. The cellular specificity for beta-galactosidase expression observed in RT7-lacZ transgenic animals was in agreement with endogenous RT7 transcript expression. Northern blot analysis of testis RNA of RT7-c-mos transgenic mice showed elevated levels of c-mos in spermatids, but not in other cells or tissues examined. Western blot analysis demonstrated elevated levels of p43c-mos in spermatids of both PGK-c-mos and RT7-c-mos transgenic animals, but only PGK-c-mos transgenics had increased p43c-mos levels in spermatocytes. Both RT7-c-mos and PGK-c-mos transgenic mice are fertile and show no tendency toward transformation. RT7-c-mos mice have no discernible phenotype associated with the c-mos overexpression in spermatids. However, PGK-c-mos transgenic males exhibited a significant increase in germ cell number, as determined by cell counts using total germ cells and germ cells fractionated by centrifugal elutriation. Because mitotic divisions of germ cells occur prior to PGK-c-mos transgene expression, our observations suggest that c-mos overexpression in spermatocytes causes an alteration in cell-cell interactions.