Senescence and genomic integrity are thought to be important barriers in the development of malignant lesions. Human fibroblasts undergo a limited number of cell divisions before entering an irreversible arrest, called senescence. Here we show that human mammary epithelial cells (HMECs) do not conform to this paradigm of senescence. In contrast to fibroblasts, HMECs exhibit an initial growth phase that is followed by a transient growth plateau (termed selection or M0; refs 3-5), from which proliferative cells emerge to undergo further population doublings (approximately 20-70), before entering a second growth plateau (previously termed senescence or M1; refs 4-6). We find that the first growth plateau exhibits characteristics of senescence but is not an insurmountable barrier to further growth. HMECs emerge from senescence, exhibit eroding telomeric sequences and ultimately enter telomere-based crisis to generate the types of chromosomal abnormalities seen in the earliest lesions of breast cancer. Growth past senescent barriers may be a pivotal event in the earliest steps of carcinogenesis, providing many genetic changes that predicate oncogenic evolution. The differences between epithelial cells and fibroblasts provide new insights into the mechanistic basis of neoplastic transformation.

Expression of 14-3-3 () is induced in response to DNA damage, and causes cells to arrest in G2. By SAGE (serial analysis of gene expression) analysis, we identified as a gene whose expression is 7-fold lower in breast carcinoma cells than in normal breast epithelium. We verified this finding by Northern blot analysis. Remarkably, mRNA was undetectable in 45 of 48 primary breast carcinomas. Genetic alterations at such as loss of heterozygosity were rare (1͞20 informative cases), and no mutations were detected (0͞34). On the other hand, hypermethylation of CpG islands in the gene was detected in 91% (75͞82) of breast tumors and was associated with lack of gene expression. Hypermethylation of is functionally important, because treatment of -non-expressing breast cancer cell lines with the drug 5-aza-2-deoxycytidine resulted in demethylation of the gene and synthesis of mRNA. Breast cancer cells lacking expression showed increased number of chromosomal breaks and gaps when exposed to ␥-irradiation. Therefore, it is possible that loss of expression contributes to malignant transformation by impairing the G 2 cell cycle checkpoint function, thus allowing an accumulation of genetic defects. Hypermethylation and loss of expression are the most consistent molecular alterations in breast cancer identified so far.A lthough many studies have identified critical genetic and epigenetic changes that mark the transformation of cells in tissues such as colon, pancreas, and lung, similar studies in breast cancer have met with limited success. In this paper we report the identification of a gene, 14-3-3 (), whose expression is undetectable in 94% (45͞48) of breast tumors.was originally identified as an epithelial-specific marker, HME1, which was down-regulated in a few breast cancer cell lines but not in cancer cell lines derived from other tissue types (1). Later studies showed that protein (also called stratifin) was abundant in differentiated squamous epithelial cells, but decreased by 95% in simian virus 40-transformed epithelial cells and in primary bladder tumors (2-4).We investigated the molecular mechanism underlying the low expression of in breast cancers. We find that genetic alterations such as loss of heterozygosity (LOH) and intragenic mutations are not major contributing mechanisms for lack of expression. Instead, we show that hypermethylation of the CpG-rich region in the gene is associated with its transcriptional silencing in the majority of breast tumors. Treatment of breast cancer cell lines that do not express with the DNA methyltransferase inhibitor, 5-aza-2Ј-deoxycytidine (5-aza-dC), leads to partial demethylation of this CpG island and synthesis of mRNA. Thus, hypermethylation appears to be responsible for silencing of gene expression.Recent studies have shed light on the function of . It was originally identified as a p53-inducible gene that is responsive to DNA damaging agents (5). apparently sequesters the mitotic initiation complex, cdc2-cyclin B1, in the cytoplasm after DNA damage (6). This prevents cdc...

Although rodent cells have been immortalized following transfection with a mutant p53 gene, the role of p53 in the immortalization of human cells is unknown. Therefore, human epithelial cell lines were examined for p53 mutations in exons 4-9 which include the evolutionarily conserved regions. A spontaneously immortalized skin keratinocyte cell line, HaCat, and three ras-transfected clones, have a p53 mutational spectrum that is typical of ultraviolet light induced mutations. A normal finite lifespan cell strain (184) and two benzo[a]pyrene immortalized mammary epithelial cell lines derived from 184 (184A1 and 184B5) contain wild type p53 sequences in exons 4-9, although elevated levels of nuclear p53 indicate an alteration in the stability of the normally transient protein. Wild type p53 was found in human bronchial, esophageal and hepatic epithelial cells immortalized by SV40 T antigen gene and human renal epithelial cells immortalized by adenovirus 5. BEAS-2B, an SV40 T antigen immortalized bronchial epithelial cell line and two subclones, have a germline polymorphism at codon 47. Inactivation of p53 by mechanisms such as mutation or complexing with proteins of DNA tumor viruses appears to be important in the immortalization of human epithelial cells.