We have established a spontaneously immortalised mammary epithelial cell line, BME65Cs. This cell line exhibits the majority of normal MECs features, whereas growth character, the ability to form colonies and expression of relevant breast tumor genes are significantly different from breast cancer cells (MCF-7). These data suggest that BME65Cs cells are not derived from malignant transformations. Whether or not in vitro spontaneous transformation is correlated with in vivo benign tumor transformation, the immortal BME65Cs cell line will be a useful tool for studying the molecular mechanism of tumorigenesis and cellular senescence. In contrast, TERT or SV40 gene mediated immortalization by the random integration of exogenous genes may bring an unforeseeable influence on natural gene expression and regulation.
There are three types of human mammary epithelial cell progenitors have been identified. The first is thought to be a luminal-restricted progenitor; the second type is a bipotent progenitor which is identified by its ability to produce "mixed"colonies in single cell assays; the third type of progenitor is thought to be myoepithelial restricted progenitor because it only produces cells with myoepithelial features . In this study, a part of normal cultured BMECs show a basal (myoepithelial) markers (CK14+) coinciding with the observation in human MECs . However, BME65Cs show a MUC+/ESA+ CK14- and no "mixed" population are found, which suggests BME65Cs is not derived from myoepithelial cell or bipotent progenitor. Although BME65Cs maintains many characters of luminal cells with stable proliferation behaviour over the long period of culture, it is unclear whether BME65Cs derived from a luminal epithelial lineage or from a progenitor cell with the capacity for luminal.
Transcriptional analysis of multiple genes sheds light on the number of genes involved in the immortalisation of BME65Cs cells. An interesting question raised by the data is the following: what are the main factors that contribute to initiation of the BME65Cs cells immortalisation? There are two signaling pathways, p16INK4a-pRb and p53-p21, that are mainly responsible for the control of cellular senescence [12, 31]. The tumor suppressor genes p16
and p53 are frequently inactivated in cancer cells; inhibition of them escapes cellular senescence leading to tumorgenesis [31, 32]. In this study, a significant change (but not loss) of p16INK4a and p53 were found in BME65Cs cells, suggesting that the inhibition of senescent-relevant pathways contributes to the BME65Cs cell line immortalisation. Additionally, the p16INK4a -pRb pathway is thought to control the first growth barrier (self-selection) of MECs , and downregulation of p16
is required for the TERT-mediated immortalisation of MECs . Therefore, downregulation of p16
is likely to be one of the key factors associating to BME65Cs cells immortalisation. However, there is no evidence suggesting that inactivation or downregulation of p16
alone can induce immortalisation of MECs. Thus, changes in expression of other genes may also be involved in the initiation of BME65Cs cells immortalisation. Although the absence of p53 is thought to be an important initial factor for numbers of cancers, and several genes in the Bcl-2 family are regulated by p53 [9, 24, 31], it is unclear what the cause for the shortage of p53 expression (compare to normal cell) in BME65Cs cells. It has been known that telomere shortening and DNA damage activate p53 expression [21, 24]. Thus, the maintenance of telomere length in BME65Cs cells may be the one reason for low expression level of p53. There remains a discrepancy between the relationship of p53 and p16INK4a. In most cancer cells, p16INK4a and p53 are two-independent signalling pathways . However, one report suggests that p16INK4a regulates p53 in human mammary epithelial cells . Despite this, the low expression of p53 might contribute to the coordination of BME65Cs cells immortalisation, because p53 plays a role in transcriptional repression of TERT. Together, we speculate that the low expression of both p16
and p53 is important for the maintenance of BME65Cs cells immortalisation.
Overexpression of TERT protects telomere repeats from erosion and, consequently, from induction of replicative senescence or apoptosis . Thus, expression of bTERT may be another important factor for immortalisation of BME65Cs cells. TERT is positively regulated by c-Myc , but negatively regulated by p53 . Therefore, the low expression of p53 and upregulation of c-Myc in BME65Cs cells might contribute to the activation of bTERT, and expression of bTERT may subsequently lead to stable telomere length in BME65Cs cells, aiding cells in overcoming senescence and leading to immortalisation. As a transcriptional factor, c-Myc is believed to regulate the expression of 15% of all genes, including genes involved in cell division, cell growth, and apoptosis . In addition to the high expression in a number of cancer cells, c-Myc is also expressed in stem cells, and is thought to be a requirement for the maintenance of stemness . Hence, we presume that the function pattern of cell linage-specific regulation of c-Myc might be dependent on its expression level. Curiously, the detection of TRF length shows discontinuity in BME65Cs. We suppose that it might be due to some non-immortal cells existing in BME65Cs, since some BME65Cs still died in each passage and showed SA-β-gal positive. It is interest to point out that the telomere length in BME65Cs is similar to that in early passage BMECs, this suggests immortalisation event for BME65Cs might occur in early passage cell.
In addition to c-Myc, two other genes, TRPS-1 and Bag-1, are also highly expressed in MCF-7 cells but undetectable in BME65C cells. TRPS-1 is called tricho-rhino-phalangeal syndromes type 1 gene, which mutations are previously shown to be associated with three rare autosomal dominant genetic disorders known as the tricho-rhino-phalangeal syndromes (TRPS). Recently, TRPS-1 protein has been found to be dramatically overexpressed in >90% of early and late-stage breast cancers . Bag-1 is a multifunctional protein that interacts with a wide range of cellular targets and regulates cell survival, signaling, metastasis, proliferation and transcription [26, 39]. In breast cancer, overexpression of Bag-1 has been detected in a majority of cases . Taken together, the expression of TRPS-1 and Bag-1 may be used to evaluate malignant or non-malignant transformation of immortalised MECs. However, low expression of TRPS-1 was detected in senescent BMECs cells, implying that expression of TRPS-1 could be regulated by the cellular growth state, and TRPS-1 protein probably has other function that differs from its function in cancer cells.
Bcl-2 and Bax are important oncoproteins involved in regulation of cellular apoptosis via the mitochondrial apoptosis pathway. Bcl-2 functions in suppressing cell apoptosis, whereas Bax promotes cell apoptosis [23, 41]. Paradoxically, expression of Bcl-2 has been consistently associated with a better prognosis of breast cancer patients [41–43]. It has been suggested that the Bax/Bcl-2 ratio determines whether apoptosis will occur . In this study, Bax is deficient in BME65Cs cells, whereas Bcl-2 is highly expressed. Hence, immortalisation of BME65Cs cells is also accompanied by the inhibition of the mitochondrial apoptosis pathway. Interestingly, expression of another member of the Bcl-2 family, Bag-1, is undetectable in three different bovine MECs including BME65Cs cells and excluding MCF-7 cells.
Although we have analysed changes in expression of several genes from different cell strains, it is unclear what the initial mechanism is responsible for the change of these genes. Based on the fact that both p16
and p53-p21 are low expressed but not silenced in BME65Cs cells, we propose that these two genes are not mutated during immortalisation. In addition, epigenetic control is another important mechanism in regulation of genes expression. Abnormal DNA methylation patterns have been shown in a number of cancer cells . Overexpression of DNMT1 has been detected in several human cancers [45–47]. Carcinogenesis is often accompanied by increases of DNMT1 mRNA, DNMT1 protein, and DNA methyltransferase activity [48–50]. Moreover, epigenetic change has been shown the important role in immortalisation of human MECs [51, 52]. In this study, DNMT1 is significantly increased in BME65Cs cells and highly expressed in MCF-7 cells, suggesting that DNA methylation status is likely to be linked to immortalisation. Whether the epigenetic changes conclusively lead to the altered expression of "immortalisation-initial genes" in BME65Cs cells requires further investigation.