Primary Breast Cell Research Articles

Immortalization protocols used in cell culture models of human breast morphogenesis

Defining the key players in normal breast differentiation is instrumental to understanding how morphogenesis becomes defective during breast cancer progression. During the past 2 decades much effort has been devoted to the development of technologies for purification and expansion of primary human breast cells in culture and optimizing a relevant microenvironment, which may help to define the niche that regulates breast differentiation and morphogenesis. In contrast to the general property of cancer, normal human cells have a finite lifespan. After a defined number of population doublings, normal cells enter an irreversible proliferation-arrested state referred to as replicative senescence. To overcome this obstacle for continuous long-term studies, replicative senescence can be bypassed by treatment of cells with chemical agents such as benzopyrene, by radiation or by transfection with viral oncogenes or the gene for human telomerase (human telomerase reverse transcriptase, hTERT). A drawback of some of these protocols is a concurrent introduction of chromosomal changes, which sometimes leads to a transformed phenotype and selection of a subpopulation, which may not be representative of the tissue of origin. In recent years, we have sought to establish immortalized primary breast cells, which retain crucial characteristics of their original in situ tissue pattern. This review discusses various approaches to immortalization of breast-derived epithelial and stromal cells and the application of such cell lines for studies on human breast morphogenesis.

EGFR and EGFRvIII expression in primary breast cancer and cell lines.

EGFRvIII is a constitutively activated truncated variant of the epidermal growth factor receptor (EGFR) which has been shown to increase tumorgenicity. There are conflicting reports on the extent of EGFRvIII expression in tissues which may in part stem from the use of different assay methodologies. We investigated the expression of both EGFRvIII and wild-type EGFR (EGFRwt) in cell lines and primary breast cancers. First, we used a RT-PCR assay that can simultaneously measure EGFRwt and EGFRvIII mRNA to screen 55 tumor cell lines. We show that except for EGFRvIII transfected cells, only EGFRwt was detected. We then validated a real-time PCR assay and used this to screen 170 formalin fixed paraffin-embedded primary breast cancers for evidence of EGFRwt and EGFRvIII expression. No samples were positive for EGFRvIII expression except for control transfectants and glioblastomas. In contrast, EGFRwt was expressed at varying levels in the majority of samples tested. We conclude that the expression of EGFRvIII is extremely rare in breast cancer and therefore it does not contribute to the malignant phenotype.

Recurrence in HER2 over-expressing breast cancer-a predictive role for PEA3 and SRC-1

Abstract Introduction: Regulation of HER2 through the transcription factor PEA3 is associated with tumour progression and resistance to endocrine treatment in human breast cancer. The mechanism of this resistance remains unclear. Activation of estrogen receptor co-activator proteins via the MAP-kinase pathway is thought to be central to tamoxifen insensitivity. We hypothesised that activation of HER2 by PEA3 may result in activation of co-activator proteins such as AIB1 (amplified-in-breast-cancer) and SRC-1 (steroid co-activator), resulting in endocrine resistance. Methods: In primary breast cultures, growth factor induction of PEA3-HER2 promoter interaction was assessed using mobility-shift analysis and protein expression by western-blot. Expression of PEA3 and the co-activators was localised and co-localised in a cohort of patients using immunohistochemistry and immunofluorescence. Expression was correlated with clinicopathological parameters. Results: Binding of PEA3 to the HER2 promoter was increased by growth factors, bFGF and EGF. Growth factors were found to up-regulate the protein expression of PEA3 and the co-activators in primary breast cell cultures (n = 12). PEA3 expression was localised to the breast epithelial cells and was found to be co-expressed with AIB1 and SRC-1. HER2 expression was found to be associated with PEA3, AIB1 and SRC-1 (n = 72, p=0.0041, 0.0038 and 0.001 respectively). Moreover, by the odds ratio, patients that were both HER2 positive and SRC-1 positive had a 97% probability of recurrence, compared with a 5% probability in HER2 patients that were SRC-1 negative. Conclusions: The mechanisms of recurrence in HER2 positive breast cancer may be explained by the activation of the co-regulatory protein SRC-1.

Two promoters within the human LMO4 gene contribute to its overexpression in breast cancer cells

LMO4, a member of the LIM-only family of zinc-finger proteins, is overexpressed in more than 50% of primary breast cancers and cell lines, implying a role in the pathogenesis of this cancer. Southern blot analysis of these cell lines did not reveal amplification or rearrangement of the LMO4 gene. To investigate further the mechanism underlying LMO4 overexpression and the generation of two patterns of transcripts, we isolated genomic clones spanning the human gene. Similar to the mouse Lmo4 gene, there are two 5′ noncoding exons, exon 1a and exon 1b, which we show are differentially expressed in breast epithelial cells. This reflects differential promoter usage in combination with alternative splicing. Two promoter regions were defined, one upstream of exon 1a and the other upstream of exon 1b. Both promoters exhibited strong activity in breast cancer cells, with up to 400-fold activity above basal levels. These promoters were significantly more active in T-47D and MCF-7 cells relative to SKBR3 cells, consistent with RNA levels. Thus, overexpression of the LMO4 gene in breast cancer cells reflects increased promoter activity and appears to involve aberrant activation of the second promoter in a subset of these cells.

Detection of breast cancer cell contamination in leukapheresis product by real-time quantitative polymerase chain reaction.

Identification of sensitive techniques for breast cancer cell detection might be relevant for high-dose chemotherapy programs with autologous stem cell transplantation. We investigated the feasibility of Maspin, Mammaglobin and c-ErbB-2 amplification by real-time quantitative polymerase chain reaction (RQ-PCR) for the detection of breast cancer cells in leukaphereses. Expression of the three markers was determined in primary breast cancers and cell lines. Peripheral blood (PB), bone marrow (BM), and leukapheresis samples from patients with malignancies other than breast cancer were used as controls. Sensitivity was evaluated by dilution of primary tumors and cell lines with mononuclear blood cells. We found expression of the three markers in all primary tumors and most cell lines. No blood specimen from control patients had the Maspin transcript, while only one was positive for Mammaglobin. Weak c-ErbB-2 expression was detectable in most PB, all BM and all leukapheresis samples from controls. We observed a low sensitivity of Maspin RQ-PCR and a sensitivity of Mammaglobin RQ-PCR up to one tumor cell in 10(6) mononuclear cells. One out of 18 leukaphereses from breast cancer patients screened for the presence of Mammaglobin mRNA was positive. We conclude that Mammaglobin RQ-PCR might be a useful tool for detection of leukapheresis contamination.

Cell proliferation markers in human solid tumors: Assessing their impact in clinical oncology

Publisher Summary Proliferation, a fundamental biological process, is involved in determining growth and maintenance of tissue homeostasis. This chapter describes several approaches used to evaluate the proliferative activity of human solid tumors. It examines the relevance of S-phase markers in breast, colorectal, and lung cancers. Cell proliferation of primary breast and non-small cell lung cancers is only weakly related to clinical and pathologic stage, and such a relation is generally observed in large case series in which minimal differences in mean or median thymidine labeling index (TLI) or flow cytometry within stage subsets reached statistical significance. For colorectal cancer, as for other solid tumors, surgical resection represents the best treatment option, but it does not prevent the risk of relapse, which depends on degree of local invasion, spread to regional lymph nodes, and tumor site and differentiation. Alterations in cell proliferating compartments have been shown to be the early event of colon mucosa transformation, and an increase in proliferating or S-phase cell fraction has been generally shown to be related to poor prognosis.

Mapping the amplification of EIF3S3 in breast and prostate cancer

Gain of chromosome arm 8q is a frequent genetic alteration in breast and prostate cancer. Two amplified subregions, 8q21 and 8q23–24, have been identified with comparative genomic hybridization (CGH). We have recently demonstrated that the EIF3S3 (eIF3-p40) gene, located at 8q23, is often amplified and overexpressed in both breast and prostate cancer. Here, we used fluorescence in situ hybridization (FISH) to map the amplified region around EIF3S3 in primary breast cancers and cell lines. The size of the common highly amplified region was about 2.5 Mb between the markers D8S1668 and WI-7959. Next, we analyzed the expression of all expressed sequence tags (ESTs) located within and near this region by RNA slot blot hybridization. In addition to EIF3S3, three anonymous ESTs and EXT1 were found to be highly expressed in cancer cell lines with the amplification at 8q23–q24. However, the anonymous ESTs were located outside the minimal highly amplified region and EXT1 was overexpressed only in one of the cancer cell lines with 8q amplification. Since EIF3S3 was the only consistently overexpressed gene located in the minimal highly amplified region, it is the strongest candidate target gene for 8q23–q24 amplification. Genes Chromosomes Cancer 28:203–210, 2000. © 2000 Wiley-Liss, Inc.

Mapping the amplification ofEIF3S3 in breast and prostate cancer

Gain of chromosome arm 8q is a frequent genetic alteration in breast and prostate cancer. Two amplified subregions, 8q21 and 8q23-24, have been identified with comparative genomic hybridization (CGH). We have recently demonstrated that the EIF3S3 (eIF3-p40) gene, located at 8q23, is often amplified and overexpressed in both breast and prostate cancer. Here, we used fluorescence in situ hybridization (FISH) to map the amplified region around EIF3S3 in primary breast cancers and cell lines. The size of the common highly amplified region was about 2.5 Mb between the markers D8S1668 and WI-7959. Next, we analyzed the expression of all expressed sequence tags (ESTs) located within and near this region by RNA slot blot hybridization. In addition to EIF3S3, three anonymous ESTs and EXT1 were found to be highly expressed in cancer cell lines with the amplification at 8q23-q24. However, the anonymous ESTs were located outside the minimal highly amplified region and EXT1 was overexpressed only in one of the cancer cell lines with 8q amplification. Since EIF3S3 was the only consistently overexpressed gene located in the minimal highly amplified region, it is the strongest candidate target gene for 8q23-q24 amplification.

Combined 4-hydroxy-ifosfamide and vinorelbine treatment in established and primary human breast cell cultures

BackgroundVinorelbine and ifosfamide are active drugs against breast cancer, but the best treatment schedule has yet to be defined by preclinical or clinical studies. The antitumor activity of 4-hydroxy-ifosfamide (4-OH-IF), the active form of ifosfamide, and vinorelbine (VNB) and their interaction were investigated in two established breast cancer cell lines (MCF-7 and BRC-230) and in 10 primary breast cancer cultures. Materials and methodsCytotoxic activity was evaluated by a highly efficient clonogenic assay (HECA). The median-effect principle was applied to evaluate synergistic and antagonistic interactions and the corresponding combination index values were calculated. Cell cycle perturbations were analysed by flow cytometry. ResultsIn MCF-7 and BRC-230 cell lines the sequence VNB for 4 hours followed by 4-OH-IF for 24 hours produced an antagonistic effect. Conversely, the inverse sequential scheme, 4-OH-IF→VNB provided synergistic effects on both cell lines. The synergism was associated with a strong block in the G2-M phase. Synergistic activity of 4-OH-IF→VNB sequence was confirmed in 7 of 10 primary breast cancer cultures. ConclusionsIn conclusion, the sequence 4-OH-IF→VNB appeared to be the most effective scheme both in established cell lines and in primary breast cancer cultures.

ARHI is the center of allelic deletion on chromosome 1p31 in ovarian and breast cancers.

In our previous work, we had characterized ARHI as an imprinted putative tumor-suppressor gene in ovarian and breast cancers. ARHI is expressed in primary breast and ovarian cell lines but largely absent from the corresponding malignant tumors. Moreover, the non-imprinted functional allele is typically deleted in malignant cells. Since ARHI had been mapped to 1p31, a common deletion site in breast and ovarian cancer and male germ-cell tumors, in this study, we set out to define precisely the physical location of ARHI at 1p31 and to determine if this location lies within the smallest common region of deletion in breast and ovarian cancers. To this end, we first carried out radiation hybrid mapping of ARHI and surrounding markers, followed by a high-resolution study of loss of heterozygosity at 1p31 in 49 ovarian and breast cancers. Combining a radiation hybrid map and a physical map of the region encompassing ARHI, 3 discrete regions of minimal deletion were found at 1p31 in breast and ovarian cancers. ARHI is the most common deletion region at 1p31. Two other less common regions of deletion were found centromeric to this gene. One of them centered on D1S207 and the other one included and was proximal to D1S488. We also confirmed the preferential loss of non-imprinted functional allele in 7 of 9 tumor specimens. These data support the possibility that ARHI is a tumor-suppressor gene and suggest that additional tumor-suppressor genes may lie proximal to ARHI at 1p31. The data obtained from our study should aid in the identification and characterization of genes in this novel imprinted region.