Non-cancerous Breast Cell Line Research Articles

Protein kinase D1 regulates ERα-positive breast cancer cell growth response to 17β-estradiol and contributes to poor prognosis in patients.

About 70% of human breast cancers express and are dependent for growth on estrogen receptor α (ERα), and therefore are sensitive to antiestrogen therapies. However, progression to an advanced, more aggressive phenotype is associated with acquisition of resistance to antiestrogens and/or invasive potential. In this study, we highlight the role of the serine/threonine-protein kinase D1 (PKD1) in ERα-positive breast cancers. Growth of ERα-positive MCF-7 and MDA-MB-415 human breast cancer cells was assayed in adherent or anchorage-independent conditions in cells overexpressing or depleted for PKD1. PKD1 induces cell growth through both an ERα-dependent manner, by increasing ERα expression and cell sensitivity to 17β-estradiol, and an ERα-independent manner, by reducing cell dependence to estrogens and conferring partial resistance to antiestrogen ICI 182,780. PKD1 knockdown in MDA-MB-415 cells strongly reduced estrogen-dependent and independent invasion. Quantification of PKD1 mRNA levels in 38 cancerous and non-cancerous breast cell lines and in 152 ERα-positive breast tumours from patients treated with adjuvant tamoxifen showed an association between PKD1 and ERα expression in 76.3% (29/38) of the breast cell lines tested and a strong correlation between PKD1 expression and invasiveness (P < 0.0001). In tamoxifen-treated patients, tumours with high PKD1 mRNA levels (n = 77, 50.66%) were significantly associated with less metastasis-free survival than tumours with low PKD1 mRNA expression (n = 75, 49.34%; P = 0.031). Moreover, PKD1 mRNA levels are strongly positively associated with EGFR and vimentin levels (P < 0.0000001). Thus, our study defines PKD1 as a novel attractive prognostic factor and a potential therapeutic target in breast cancer.

Abstract 1472: The differential expression of miRNAs in breast cancer cell lines

Abstract Breast cancer is a heterogenous disease that is the second leading cause of death amongst women in the United States. Triple negative breast cancer (TNBC) is a very aggressive subtype of breast cancer characterized by its loss of estrogen, progesterone and HER2 receptor expression which limits its targets for effective drug therapies. Micro Ribonucleic Acids (miRNAs) are a class of small, endogenous non-coding ribonucleic acids that seem to play an essential role in gene regulation. Changes in the patterns of miRNA expression profiles are serving as potential biomarkers for tumor diagnosis, prognosis of disease-specific outcomes, and the prediction of therapeutic responses in cancer. We analyzed miRNA array (Agilent) expression profiles in triple negative breast cancer cell lines (HCC70, HCC1806, MDA-MB-157) and non-cancerous (AG11132) African American women breast cell lines. We revealed that the TNBC cells exhibited a different miRNA expression pattern when compared to the normal breast cancer cell line. We analyzed the hybridization data files using standard Microsoft Excel techniques and verified our findings using PARTEK Genomics Suite. We combined the data sets from the TNBC cell lines and compared the miRNA array expression profiles to the normal control cell line AG11132. We revealed that there were significant dysregulation of miRNAs including those of the let-7 family, miR-16, miR-31, and miR-25b-1, miR34a, and miR-95 in the triple negative cells as compared to the normal cells. These miRNAs have been described in previous scientific studies to be associated with tumorigenesis in breast cancer. These studies are initial observations that we will follow up with molecular techniques such as real-time PCR and siRNA to validate the results in order to establish more direct correlations to miRNAs and their roles in triple negative breast cancer. Citation Format: Checo J. Rorie, Brianna L. Arrington, Sherette S. Godfrey, Armeshia S. McCoy, Kashenya M. Gurley. The differential expression of miRNAs in breast cancer cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1472. doi:10.1158/1538-7445.AM2014-1472

Cytotoxic activity of the methanolic extract of leaves and rhizomes of Curcuma amada Roxb against breast cancer cell lines

ObjectivesTo evaluate the methanol extract of both the leaves and the rhizomes of Curcuma amada (C. amada) for their cytotoxic activity against breast cancer cell lines MCF-7 and MDA MB 231. MethodsViability and cytotoxicity induced by the extracts were assessed using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, sulforhodamine B, and lactate dehydrogenase release assays. Various staining techniques such as acridine orange/ethidium bromide, Giemsa, ethidium bromide, propidium iodide, and Hoechst 33342 staining were employed to study the mechanism of cell death induced by the extract. ResultsThe results indicated that the methanol extract of both the leaves and the rhizomes of C. amada exhibited strong cytotoxicity towards breast cancer cell lines MCF-7 and MDA MB 231. The extract also showed less cytotoxicity towards non-cancerous breast cell line HBL-100. The results of staining revealed that the extracts induced cell death in cancer cells which are mediated through apoptotic pathway. ConclusionsThe results indicated that the methanol extract of leaves and rhizomes of C. amada possess anticancer and cytotoxic activity.

G-Protein Activated Inwardly Rectifying Potassium Channels Control Motility of Breast Cancer Cells

Mortality of breast cancer is very high when it metastases to distant organs. New early tumor markers and mechanistic insight into carcinogenesis and metastasisation are needed to treat the disease at curable stages. GIRK channels are activated by GPCRs and regulate resting potential and excitability of neurons, myocytes and other cells. Recently GIRK proteins have been associated with endocrine adenomas and also breast cancer (Stringer et al.2001, Brevet et a.l 2008 and Choi et al.2009). In this study we analyzed the vital parameters of cancerous and noncancerous breast cell lines when human GIRK1 and splice variants (hG1a, hG1c, hG1d) are overexpressed or silenced.GIRK1a, GIRK1c and GIRK1d were stably overexpressed and knocked out in breast cell lines MCF7, MDA-MB-231 and MCF 10A. Vital parameters including invasion, wound healing, adhesion, proliferation and motility were evaluated.Our findings revealed that overexpression of GIRK1a significantly increased the velocity 0.102±0.035 (μm/min) in overexpresssed vs. 0.05±0.02 in vector control cells (P˂0.001) and motility coefficient 0.2±0.3 (μm2/min) in overexpresssed vs. 0.05±0.06 in vector control cells (P˂0.001) of the MCF7 cell lines. Overexpression of hG1c in MCF7 significantly increased invasiveness of the cells (P˂0.001) as measured by matrigel assay. Overexpression of the GIRK1 and splice variants didn't have significant effect on proliferation, wound healing and adhesion of the MCF7 cells.Stably overexpression of GIRK1 and splice variants in MCF7 cause increased velocity, motility coefficient and invasion of the cells. Expression of GIRK might be a new diagnostic biomarker of breast cancer and possibly is causally involved in metastasisation.Acknowledgements: The research is funded by the Austrian Science Foundation (FWF; P22974-B19 to W. S and KLIF 182 to T. B).

Abstract P2-09-17: Allosteric inhibitors of the proteasome: A new concept for breast cancer treatment

Abstract Proteasome is an essential protease of the proteasome-ubiquitin pathway, responsible for maintaining intracellular proteostasis. It is a multifunctional, multisubunit enzyme built from interchangeable modules and intricately regulated on the molecular and cellular levels. Proteasome inhibitors display proapoptotic properties, preferentially aimed at cancerous cells. Consequently, targeted therapy with bortezomib, the competitive inhibitor of the proteasome, has been very successful with refractory/relapsed multiple myeloma and with lymphomas. Unfortunately, breast cancers performed disappointingly in clinical trials with bortezomib. We propose that a new generation of small-molecule proteasome inhibitors distinct from bortezomib may prove successful with breast cancers. In contrast to bortezomib which directly blocks proteasome active sites, the new inhibitors are allosteric ligands binding to noncatalytic sites and disrupting the enzyme's function by targeting long-distance transfer of structural signals within the enzyme's molecule. The advantages of allosteric ligands, now rapidly gaining popularity in targeted drug design, include versatility of molecular actions, defying drug resistance mechanisms, and usually high specificity with low toxicity. For example, one of the small-molecule allosteric ligands we created compromises interactions between the catalytic core proteasome and its regulatory sub-assembly 19S (“cap”, regulatory particle) responsible for recognizing and processing the majority of intracellular protein substrates tagged for degradation by polyubiquitin. We found that treatment with nanomolar concentration of the compound induced cell death in Mcf7 human breast cancer cultured cells. Interestingly, this cell line is known for its resistance to bortezomib treatment. Even more, treatment with the allosteric ligand together with bortezomib produced a synergistic effect and sensitized the Mcf7 cells to the actions of competitive inhibitor. Remarkable, the cytotoxic effects of allosteric ligand or its combination with bortezomib were not observed with the noncancerous human breast cell line, Mcf10A. Summarizing, allosteric regulation of the proteasome by small-molecule ligands, alone or in combination with established drugs competitively inhibiting proteasome, seem to be a promising strategy to utilize proteasome as a drug target in breast cancers. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-09-17.

P.1.017 Histaminergic regulation of presumed serotonin neurons in the dorsal raphe nucleus

NOK (also known as STYK1) has been identified as an oncogene. However, its biochemical and biological activities as a molecular regulator are poorly defined. In the present study, we report that NOK overexpression led to enhanced phosphorylation of GSK-3β at its Ser9 residue via Akt phosphorylation at Thr308. NOK could make complexes with both Akt and GSK-3β. Moreover, the expression levels of NOK, p-Akt(Thr308) and p-GSK-3β(Ser9) were positively correlated in cancerous and non-cancerous breast cell lines. Thus, our data identified a novel functional molecular complex formed by NOK, Akt and GSK-3β that may relay a NOK-directed tumourigenic cascade.GSK3B physically interacts with NOK and Akt by anti tag coimmunoprecipitation (View interaction).GSK3B physically interacts with NOK by anti tag coimmunoprecipitation (View interaction).

NOK/STYK1 interacts with GSK-3β and mediates Ser9 phosphorylation through activated Akt

NOK (also known as STYK1) has been identified as an oncogene. However, its biochemical and biological activities as a molecular regulator are poorly defined. In the present study, we report that NOK overexpression led to enhanced phosphorylation of GSK-3β at its Ser9 residue via Akt phosphorylation at Thr308. NOK could make complexes with both Akt and GSK-3β. Moreover, the expression levels of NOK, p-Akt(Thr308) and p-GSK-3β(Ser9) were positively correlated in cancerous and non-cancerous breast cell lines. Thus, our data identified a novel functional molecular complex formed by NOK, Akt and GSK-3β that may relay a NOK-directed tumourigenic cascade. Structured summary of protein interactionsGSK3Bphysically interacts with NOK and Akt by anti tag coimmunoprecipitation (View interaction).GSK3Bphysically interacts with NOK by anti tag coimmunoprecipitation (View interaction).

Abstract 2029: Wild type BRCA2 mRNA splice variants in normal and breast tumor cells

Abstract BRCA2 is one of the two principal tumor suppressor genes responsible for Hereditary Breast and Ovarian Cancer (HBOC) Syndrome. Most deleterious mutations in BRCA2 are nonsense, frame-shifting insertions/deletions, or splicing mutations resulting in premature mRNA stop codons. However, numerous sequence variants of uncertain significance (VUS) are routinely seen during clinical testing. Some of these could potentially affect splicing fidelity by altering splice site choices or affecting the ratios of normally occurring splice variants, which have yet to be defined for BRCA2. To characterize the normally occurring splice variants and possible alternate protein products of BRCA2, we have performed a complete cDNA exon scan of BRCA2 cDNA in MCF7 (a breast cancer cell line), 184A1 (a non-cancerous breast cell line), and two lymphoblastoid cell lines from unaffected wild type. In all cell lines we identified three BRCA2 splice variants: delta exon 3, delta exon 12, and delta exons 6&amp;7+2bp. While the first two variants retain the BRCA2 translational reading frame and could theoretically result in alternate BRCA2 protein products, the third is an out-of-frame variant predicted to result in a premature stop codon. We have further developed a set of splice variant-specific detection assays that revealed all three splice variants were present in 14 breast tumor/adjacent normal tissue pairs. Surprisingly, all three BRCA2 splice variants were comparatively more abundant in the tumor cells than their normal adjacent tissues. To characterize the regulators of BRCA2 splicing in tumor cells, we repeated the exon scan in the presence of cyclohexamide or puromycin to inhibit nonsense-mediated mRNA decay (NMD). We saw no evidence of accumulated mis-spliced BRCA2 mRNA products in the presence of the inhibitors, indicating NMD does not play a major role in regulating the accumulation of normally occurring splice variants. To determine whether nonsense-associated alternate splicing (NAS) promotes skipping of exons containing frame shifting or nonsense mutations, we repeated the BRCA2 exon scan in four lymphoblastoid cell lines containing BRCA2 mutations 5578delAA, 852delC, 6174delT and S1955X. In all cases, the splicing patterns were the same as breast and wild type lymphoblastoid cell line patterns, indicating NAS does not generally promote alternate BRCA2 protein products in response to truncating mutations. We conclude that, in addition to full-length BRCA2 mRNA, there are three additional splice variants that accumulate to comparatively higher levels in tumors than in adjacent normal tissue. This suggests breast tumor tissues have altered splicing regulation, altered regulation of selective mRNA degradation, or possibly both, and could reflect a level of epigenetic misregulation of BRCA2 and possibly other genes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2029. doi:10.1158/1538-7445.AM2011-2029

Induction of apoptosis by 5,7-dihydroxy-8-nitrochrysin in breast cancer cells: The role of reactive oxygen species and Akt

5,7-dihydroxy-8-nitrochrysin (NOC), a novel synthetic chrysin analogue, induces apoptosis in human cancer cells. We previously demonstrated that NOC possesses stronger cytotoxicity towards human colon carcinoma and human gastric carcinoma cells than chrysin. Herein, we demonstrate the mechanism by which NOC preferentially suppresses the viability of the MDA-MB-453 human breast cancer cell line (ER negative, Her2 overexpressing) and moderately suppresses the viability of the MCF-7 cell line (ER positive, Her2 low), but has little effect on the immortalized non-cancerous HBL-100 breast cell line (ER positive, Her2 low). Moreover, the results of our studies, for the first time, provide mechanistic evidence that NOC induces apoptosis by the generation of reactive oxygen species and Akt dephosphorylation. Our findings highlight a new mechanism responsible for NOC-induced apoptosis, and raise the possibility that NOC might be promising as a candidate for human breast cancer therapy.

Abnormal expression, localization and interaction of canonical transient receptor potential ion channels in human breast cancer cell lines and tissues: a potential target for breast cancer diagnosis and therapy

BackgroundCa2+ is known to be involved in a number of metastatic processes including motility and proliferation which can result in store-depletion of Ca2+. Up regulation of genes which contribute to store operated channel (SOC) activity may plausibly be necessary for these processes to take place efficiently. TRPC proteins constitute a family of conserved Ca2+-permeable channels that have been shown to contribute to SOC activity.ResultsIn breast cancer biopsy tissues, TRPC3 and TRPC6 were the predominant TRPC genes expressed with TRPC3 and TRPC6 being significantly up regulated compared to normal breast tissue. In the lowly metastatic breast cancer cell line MCF-7, TRPC6 was the chief TRPC gene expressed while in the highly metastatic breast cancer cell line MDA-MB-231 both TRPC3 and TRPC6 were the predominant TRPC genes expressed. Western blotting, immunoconfocal analysis and immunoprecipitation experiments confirmed that the MDA-MB-231 cell line expressed both TRPC3 and TRPC6 protein with the majority of protein being intracellular. TRPC3 and TRPC6 were found to be in an immunoprecipitatble complex and co-localize within the cell. To demonstrate the potential of targeting TRP channels in breast cancer, hyperforin reportably a specific activator of TRPC6 significantly reduced the growth and viability of the breast cancer cell lines but had no effect on the non-cancerous breast cell line. Silencing of TRPC6 in MDA-MB-231 cells resulted in a significant reduction in cell growth but not viability.ConclusionTRPC channels may be potential future targets for breast cancer diagnosis and therapy and deserve further investigation to evaluate their role in cancer cell physiology.

Proteinase-activated receptor 2 expression in breast cancer and its role in breast cancer cell migration

Proteinase-activated receptor 2 (PAR2) is a G protein-coupled receptor that is activated by trypsin-like proteinases. PAR2 is detected in breast tumor specimens; however, it is not clear how PAR2 level in breast cancer cell/tissues compares with normal cell/tissues. Here, we show the elevation of PAR2 protein level in 76 of 105 breast tumor specimens but only 5 of 24 normal breast tissues. PAR2 level is also higher in breast cancer cell lines than that in normal breast cells and non-cancerous breast cell lines. To determine the role of PAR2 in breast carcinogenesis, we examined the effect of PAR2 agonists on cell proliferation and migration. Our studies show that PAR2 agonists (PAR2-activating peptide and trypsin) are neither potent growth enhancers nor chemoattractants to breast cancer cells. Instead, PAR2 agonists induce significant chemokinesis. PAR2-mediated chemokinesis is G(alphai)-dependent, and inhibiting Src kinase activity or silencing c-Src expression blocks PAR2-mediated chemokinesis. These results suggest that c-Src works downstream of G(alphai) to mediate this PAR2 agonist-induced event. To characterize c-Src effector, we reveal that PAR2 agonists activate JNKs in a Src-dependent manner and that JNK activity is essential for PAR2-mediated chemokinesis. Moreover, PAR2 agonist stimulation leads to paxillin Ser(178) phosphorylation and paxillin(S178A) mutant inhibits PAR2-mediated chemokinesis. In conclusion, our studies show that PAR2 agonists facilitate breast cancer cell chemokinesis through the G(alphai)-c-Src-JNK-paxillin signaling pathway.

Identifying Peach and Plum Polyphenols with Chemopreventive Potential against Estrogen-Independent Breast Cancer Cells

Our objective was to evaluate the cancer suppression activity of extracts from a commercial variety of yellow-fleshed peach 'Rich Lady' (RL) and a red-fleshed plum 'Black Splendor' (BS) and identify the phenolic fractions that may possess potential as chemopreventive and/or chemotherapeutic natural compounds. The peach RL extract effectively inhibited the proliferation of the estrogen-independent MDA-MB-435 breast cancer cell line. The concentration to inhibit 50% of cell proliferation (IC(50)) was approximately 42 mg/L for this cell line compared to an IC(50) of approximately 130 and approximately 515 mg/L for the noncancerous breast cell line MCF-10A and the estrogen-dependent breast cancer cell line MCF-7, respectively. Similarly, BS extracts showed greater effects on MDA-MB-435 cells as compared to the other breast cancer or the normal breast cell lines. In general, BS extracts were less effective than RL extracts. Within all RL and BS fractions, fraction 3 (F3, flavonoids) and fraction 4 (F4, procyanidins) were more potent than fraction 1 (F1, phenolic acids) and fraction 2 (F2, anthocyanins) against the three cell lines. The order of potency of RL fractions against MDA-MB-435 was F(3) approximately F(4) > F(1) > F(2). The antiproliferative activity of pure compounds identified in F(3) and F(1) confirmed that quercetin 3beta-glucoside is the bioactive compound in F(3), with the same level of toxicity on the estrogen-independent MDA-MB-435 breast cancer and breast epithelial MCF-10A cells (IC(50) = 1.9 +/- 0.2 and 1.8 +/- 0.3, respectively). However, we confirmed that phenolic acids present in F(1): chlorogenic and neo-chlorogenic acids have potential as chemopreventive dietary compounds because of the relatively high growth inhibition exerted on the estrogen-independent MDA-MB-435 breast cancer cell line and low toxicity exerted in the normal MCF-10A cells.

Regulation of human tissue kallikrein-related peptidase expression by steroid hormones in 32 cell lines

Human tissue kallikrein-related peptidases(KLK), which are secreted serine proteases, are encoded by 15 genes located on chromosome 19q 13.4. Previous studies have shown that KLK expression is regulated by steroid hormones and many KLKs are dysregulated in hormone dependent malignancies. Some KLKs are proposed biomarkers for these cancers. We have characterized KLK hormonal regulation patterns using a large number of human cell lines. KLK levels were quantified in supernatants from 32 cell lines, each subjected to four hormonal stimulations (dexamethasone, norgestrel, dihydrotestosterone or estradiol), using ELISAs. Cell lines included breast, prostate, ovarian, lung, pancreatic, colon, and cervical cancer cells, T-lymphocytes, keratinocytes and non-cancerous epithelial breast cell line. KLKs were regulated in several cell lines not previously studied, such as keratinocytes (KLK 5, 6, and 7), ovarian cancer (KLK 5 and 9) and cervical cancer (KLK 3, 5, 6, 7,8, 10, 11, and 13) cells. Many KLKs were regulated by the synthetic glucocorticoid dexamethasone; specifically, KLK 5, 6, 8, 10, and 11 were upregulated in several breast cancer lines and downregulated in several cervical cancer lines. Knowledge of KLK hormonal regulation patterns will help to shed further light on their potential use as biomarkers and therapeutic targets for hormone-related malignancies.

Protein Profiling of Human Breast Tumor Cells Identifies Novel Biomarkers Associated with Molecular Subtypes

Molecular subtypes of breast cancer with relevant biological and clinical features have been defined recently, notably ERBB2-overexpressing, basal-like, and luminal-like subtypes. To investigate the ability of mass spectrometry-based proteomics technologies to analyze the molecular complexity of human breast cancer, we performed a SELDI-TOF MS-based protein profiling of human breast cell lines (BCLs). Triton-soluble proteins from 27 BCLs were incubated with ProteinChip arrays and subjected to SELDI analysis. Unsupervised global hierarchical clustering spontaneously discriminated two groups of BCLs corresponding to "luminal-like" cell lines and to "basal-like" cell lines, respectively. These groups of BCLs were also different in terms of estrogen receptor status as well as expression of epidermal growth factor receptor and other basal markers. Supervised analysis revealed various protein biomarkers with differential expression in basal-like versus luminal-like cell lines. We identified two of them as a carboxyl terminus-truncated form of ubiquitin and S100A9. In a small series of frozen human breast tumors, we confirmed that carboxyl terminus-truncated ubiquitin is observed in primary breast samples, and our results suggest its higher expression in luminal-like tumors. S100A9 up-regulation was found as part of the transcriptionally defined basal-like cluster in DNA microarrays analysis of human tumors. S100A9 association with basal subtypes as well as its poor prognosis value was demonstrated on a series of 547 tumor samples from early breast cancer deposited in a tissue microarray. Our study shows the potential of integrated genomics and proteomics profiling to improve molecular knowledge of complex tumor phenotypes and identify biomarkers with valuable diagnostic or prognostic values.

Knockdown of TFIIS by RNA silencing inhibits cancer cell proliferation and induces apoptosis

BackgroundA common element among cancer cells is the presence of improperly controlled transcription. In these cells, the degree of specific activation of some genes is abnormal, and altering the aberrant transcription may therefore directly target cancer. TFIIS is a transcription elongation factor, which directly binds the transcription motor, RNA Polymerase II and allows it to read through various transcription arrest sites. We report on RNA interference of TFIIS, a transcription elongation factor, and its affect on proliferation of cancer cells in culture.MethodsRNA interference was performed by transfecting siRNA to specifically knock down TFIIS expression in MCF7, MCF10A, PL45 and A549 cells. Levels of TFIIS expression were determined by the Quantigene method, and relative protein levels of TFIIS, c-myc and p53 were determined by C-ELISA. Induction of apoptosis was determined by an enzymatic Caspase 3/7 assay, as well as a non-enzymatic assay detecting cytoplasmic mono- and oligonucleosomes. A gene array analysis was conducted for effects of TFIIS siRNA on MCF7 and MCF10A cell lines.ResultsKnockdown of TFIIS reduced cancer cell proliferation in breast, lung and pancreatic cancer cell lines. More specifically, TFIIS knockdown in the MCF7 breast cancer cell line induced cancer cell death and increased c-myc and p53 expression whereas TFIIS knockdown in the non-cancerous breast cell line MCF10A was less affected. Differential effects of TFIIS knockdown in MCF7 and MCF10A cells included the estrogenic, c-myc and p53 pathways, as observed by C-ELISA and gene array, and were likely involved in MCF7 cell-death.ConclusionAlthough transcription is a fundamental process, targeting select core transcription factors may provide for a new and potent avenue for cancer therapeutics. In the present study, knockdown of TFIIS inhibited cancer cell proliferation, suggesting that TFIIS could be studied as a potential cancer target within the transcription machinery.

Physiologic estrogen receptor alpha signaling in non-tumorigenic human mammary epithelial cells

Currently, a number of breast cancer cell lines exist that serve as models for both estrogen receptor alpha (ERalpha) positive and ERalpha negative disease. Models are also available for pre-neoplastic breast epithelial cells that do not express ERalpha; however, there are no ideal systems for studying pre-neoplastic cells that are ERalpha positive. This has been largely due to the inability to establish an estrogen growth stimulated, non-tumorigenic breast epithelial cell line, as most human breast epithelial cells engineered to overexpress ERalpha have been found to be growth inhibited by estrogens. We have developed independently derived clones from the non-cancerous MCF-10A human breast cell line that express ERalpha and are growth stimulated by 17-beta-estradiol (E2) in the absence of epidermal growth factor (EGF), a cytokine normally required for MCF-10A cell proliferation. This effect is blocked by the selective estrogen receptor modulator (SERM), Tamoxifen and the selective estrogen receptor downregulator, ICI 182,780 (Faslodex, Fulvestrant). Exposure of these cells to EGF and E2 results in a growth inhibitory phenotype similar to previous reports. These data present a reconciling explanation for the previously described paradoxical effects of ERalpha overexpression, and provide a model for examining the carcinogenic effects of estrogens in non-tumorigenic human breast epithelial cells.

The expression and functional characterization of sigma (σ) 1 receptors in breast cancer cell lines

Sigma (σ) receptors have been implicated in cancer. However, to date there is little molecular data demonstrating the role of σ1 receptors in cancer. Expression of σ1 receptors in various human cancer cell lines in comparison to non-cancerous cell lines was investigated, using real time RT-PCR and by western blotting with a σ1 receptor specific antibody. Our results indicate that cancer cells express higher levels of σ1 receptors than corresponding non-cancerous cells. Localization of the σ1 receptor was investigated in MDA-MB-231 cells by immunocytochemistry and confocal microscopy, expression was visualized predominantly at the cell periphery. We have tested the effect of σ1 and σ2 drugs and a σ1 receptor silencing construct on various aspects of the metastatic process on two breast cell lines of different metastatic potential and a normal breast cell line. Both σ1 and σ2 drugs and the σ1 receptor silencing construct had effects on proliferation and adhesion for breast cancer cell lines, compared to a non-cancerous breast cell line. This data suggests σ1 receptor plays a role in proliferation and adhesion of breast cancer cells. Therefore, it is likely to be a potential target for the diagnosis and therapy of breast cancer.

Monoclonal antibody 5C3 raised against formalin fixed paraffin-embedded invasive breast tumour tissue: Characterisation of its reactive antigen via immunoprecipitation and internal sequencing

Monoclonal antibodies (MAbs) provide a powerful tool for the identification of novel tumour associated antigens. In an attempt to identify such an antigen, MAbs were generated by immunization with paraffin wax-embedded formalin-fixed invasive ductal breast tumour tissue from a patient who relapsed following an initial response to adjuvant chemotherapy. Extensive immunocytochemical and Western blot analysis of a range of cell lines and tissues including a series of pre- and post-chemotherapy treated invasive ductal breast carcinomas, with one of these MAbs, antibody 5C3, indicated that the 5C3 reactive antigen displayed a wide spectrum of reactivity amongst various human tumours. A reduced level of 5C3 expression was observed in non-cancerous archival breast tissues and breast cell lines and normal murine tissues compared to the expression observed in infiltrating breast tumour cells. Immunoprecipitation studies using the human ductal breast carcinoma cell line, ZR-75-1 resulted in the isolation of a 175 kDa reactive band which was excised from an SDS-PAGE gel and subjected to internal sequencing. Sequencing analysis and database searching revealed that this 175 kDa band represented a cytokeratin heteropolymer, composed of type I cytokeratin 9 and type II cytokeratin 6. Further studies confirmed that antibody 5C3 recognised this heteropolymer of cytokeratin 9 and 6 but not the individual cytokeratins. This novel method of MAb generation may facilitate the isolation of further potentially interesting cellular antigens. Characterisation of these novel antigens may identify specific disease targets with possible prognostic or predictive significance.