[Frontiers in Bioscience 3, d944-960, September 1, 1998]
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BIOLOGICAL AND MOLECULAR BASIS OF HUMAN BREAST CANCER

Jose Russo, Xiaoqi Yang, Yun-Fu Hu, Betsy A. Bove, Yajue Huang, Ismael D.C.G. Silva, Quivo Tahin, Yuli Wu, Nadia Higgy, Abdel Zekri, and Irma H. Russo

Breast Cancer Research Laboratory, Fox Chase Cancer Center, Philadelphia, Pennsylvania, USA

Received 12/17/97 Accepted 7/21/98

5. MOLECULAR MECHANISMS OF CELL IMMORTALIZATION

5.1. Activation of telomerase

There is evidence that the repetitive TTAGGG sequences located at the ends of human chromosomes (i.e., telomeres) may act as a molecular mitotic clock (39). It is generally believed that each successive genome replication is accompanied by gradual shortening of 50-200 bp due to incomplete replication of the 3 ends and cellular senescence occurs when telomeres reach a critically-short length that replication of the genome can not be maintained (22). Stabilization of the telomeric sequences at the ends of chromosomes, which is required for the continuous proliferation of immortal cells, involves the activation of the enzyme telomerase, which adds TTAGGG repeats to the 3 ends of chromosomes (40, 41). The genetic nature of cellular senescence implicates repression of telomerase as a key element of cell immortalization (41). Elevated levels of telomerase activities have been detected in a number of immortal cell lines and human tumor tissues (42, 43). Our observation indicates that cell telomerase is expressed in immortal MCF-10F cells but not in the mortal #130 cells, and telomere lengths that have become shorter in the #130 cells, have been maintained with no further shortening due to telomerase activation in the immortal MCF-10F cells (44), suggesting that telomerase reactivation may be one of the mechanisms leading to the spontaneous immortalization of MCF-10F cells. In addition, we have evidence that telomerase activity may be regulated by the Ca++ concentration in the medium (44).

5.2. Abrogation of cell cycle control

Result from cell fusion studies indicates that the phenotype of cellular senescence is dominant and immortality results from recessive changes in normal regulatory genes (45). Conceivably, inactivation of the genes that restrict cell cycle progression is essential to cell immortalization. Cyclin-dependent kinase (CDK) complexes and their inhibitors are essential components of cell cycle machinery, controlling cell cycle arrest in the G1 phase of the cell cycle. Since p53 acts to regulate cell cycle progression through transcriptional activation of p21WAF-1, an inhibitor of all G1 CDKs (46), abrogation of p53 function has been implicated in the immortalization of HBEC. Insertional mutation at codon 247 of the p53 gene has been implicated in spontaneous immortalization of MCF-10F cells (47). Spontaneous immortalization in vitro has been observed in HBEC from a Li-Fraumeni patient with a point mutation in the p53 gene (41) and introduction of a single-amino acid deletion mutant (del239) of p53 gene abrogates wild-type p53-mediated cellular responses and induces immortalization of HBEC (48). A recent study indicates that alterations in p53 appear to be important in overcoming the M1 blockade (49). However, introduction of seven missense mutants of p53 genes failed to induce immortalization in the same cell line (48), even though all of these p53 mutants have been shown to abrogate p53-mediated transactivation in other cell types (50). In addition, the immortalized MCF-10F cells are still able to produce the wild-type p53 protein (7) and maintain wild-type p53-mediated functional responses, such as expression of p21WAF-1 (51) and mdm2 (7). Therefore, the role of p53 in immortalization of HBEC needs further evaluation.

The CDK-4 inhibitor (CDKN2), commonly referred to as p16, is also an inhibitor of the cell cycle and has been localized to 9p21-22 (52). Homozygous deletion of this chromosomal subregion has been observed in the immortalized MCF-10F cells (53), which contain a balanced reciprocal translocation, t(3;9) (3p13;9p22) (ref 12). Similarly, loss of the 9p21 subregion has been correlated with the acquisition of an immortal phenotype of neoplastic human head and neck keratinocyte cell lines (54). Clearly, these results suggest a potential role of CDK2 in the control of immortalization of human breast epithelial cells.

Another inhibitor of the cell cycle, prohibitin, has been implicated in the process of cell immortalization (55, 56). Prohibitin gene is localized to chromosome 17q21 (57) where mutations have been reported in certain forms of breast cancer (58), suggesting that it may be a tumor suppressor gene. Lack of heterozygosity has been documented in immortalized cell lines (55). Expression of prohibitin gene produces a 30kD-protein that inhibits cell cycle transition and DNA synthesis in normal cells (59). The 3 untranslated region of prohibitin gene has been shown to function as a trans-acting regulatory RNA (i.e. riboregulator) crucial to its antiproliferative activity (56).

5.3. Genes preferentially expressed during cell immortalization

As further efforts to identify genes underlying the process of immortalization, we have performed subtractive hybridization and differential display analysis between immortal MCF-10F and its parental mortal #130 cells. Using a 10F(+)/130(-) subtractive cDNA library, we isolated more than 15 clones. Analysis of these clones showed that one of these clones contains sequences identical to H-ferritin (Figure 5) (60). Up-regulation of H-ferritin may be a source of iron necessary for growth and clonal expansion. 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. Amplification of H-ferritin gene (61) and overexpression of H-ferritin protein (62) have been associated with the progression of human breast cancer. The role of H-ferritin in the immortalization of human breast epithelial cells is unclear and will be fully evaluated by gene transfection studies.

Figure 5. Differential expression of mRNA of H-ferritin in the transformed HBEC (Reproduced with permission from Ref. 60).

Recently, we have observed an increase in the expression of a cDNA, namely, calcium binding protein (CaBP), in the immortalized cells by differential display analysis (Figure 6) (63). Sequence analysis revealed that the CaBP cDNA is S100P Ca++-binding protein. Since Ca++ plays an important role in the spontaneous immortalization of MCF-10F human breast epithelial cells (13), it is conceivable that an increase in the expression of the S100P Ca++-binding protein may facilitate the process of cell immortalization. Further characterization of other cDNA clones identified by differential analysis is still in progress.

Figure 6. Northern blot hybridization using total RNA from immortal (MCF-10F), mortal (#130) and primary (#244) human breast epithelial cells showing a 0.6 kb transcript from the isolated calcium-binding protein gene (10F-D or S100P). The gene is expressed in the immortalized cells but not in the mortal or primary cells.