Human Mammary Adenocarcinoma Cell Line Research Articles

Proteomic Characterization of a 3D HER2+ Breast Cancer Model Reveals the Role of Mitochondrial Complex I in Acquired Resistance to Trastuzumab.

HER2-targeted therapies, such as Trastuzumab (Tz), have significantly improved the clinical outcomes for patients with HER2+ breast cancer (BC). However, treatment resistance remains a major obstacle. To elucidate functional and metabolic changes associated with acquired resistance, we characterized protein profiles of BC Tz-responder spheroids (RSs) and non-responder spheroids (nRSs) by a proteomic approach. Three-dimensional cultures were generated from the HER2+ human mammary adenocarcinoma cell line BT-474 and a derived resistant cell line. Before and after a 15-day Tz treatment, samples of each condition were collected and analyzed by liquid chromatography-mass spectrometry. The analysis of differentially expressed proteins exhibited the deregulation of energetic metabolism and mitochondrial pathways. A down-regulation of carbohydrate metabolism and up-regulation of mitochondria organization proteins, the tricarboxylic acid cycle, and oxidative phosphorylation, were observed in nRSs. Of note, Complex I-related proteins were increased in this condition and the inhibition by metformin highlighted that their activity is necessary for nRS survival. Furthermore, a correlation analysis showed that overexpression of Complex I proteins NDUFA10 and NDUFS2 was associated with high clinical risk and worse survival for HER2+ BC patients. In conclusion, the non-responder phenotype identified here provides a signature of proteins and related pathways that could lead to therapeutic biomarker investigation.

Schizophrenia plausible protective effect of microRNA-137 is potentially related to estrogen and prolactin in female patients.

Schizophrenia (SCZ) is a serious chronic mental disorder. Our previous case-control genetic association study has shown that microRNA-137 (miR-137) may only protect females against SCZ. Since estrogen, an important female sex hormone, exerts neuroprotective effects, the relationship between estrogen and miR-137 in the pathophysiology of SCZ was further studied in this study. Genotyping of single-nucleotide polymorphism rs1625579 of miR-137 gene in 1,004 SCZ patients and 896 healthy controls was conducted using the iMLDR assay. The effect of estradiol (E2) on the miR-137 expression was evaluated on the human mammary adenocarcinoma cell line (MCF-7) and the mouse hippocampal neuron cell line (HT22). The relationships between serum E2, prolactin (PRL), and peripheral blood miR-137 were investigated in 41 SCZ patients and 43 healthy controls. The miR-137 and other reference miRNAs were detected by real-time fluorescent quantitative reverse transcription-PCR. Based on the well-known SNP rs1625579, the distributions of protective genotypes and alleles of the miR-137 gene were not different between patients and healthy controls but were marginally significantly lower in female patients. E2 upregulated the expression of miR-137 to 2.83 and 1.81 times in MCF-7 and HT22 cells, respectively. Both serum E2 and blood miR-137 were significantly decreased or downregulated in SCZ patients, but they lacked expected positive correlations with each other in both patients and controls. When stratified by sex, blood miR-137 was negatively correlated with serum E2 in female patients. On the other hand, serum PRL was significantly increased in SCZ patients, and the female patients had the highest serum PRL level and a negative correlation between serum PRL and blood miR-137. The plausible SCZ-protective effect of miR-137 may be female specific, of which the underlying mechanism may be that E2 upregulates the expression of miR-137. This protective mechanism may also be abrogated by elevated PRL in female patients. These preliminary findings suggest a new genetic/environmental interaction mechanism for E2/miR-137 to protect normal females against SCZ and a novel E2/PRL/miR-137-related pathophysiology of female SCZ, implying some new antipsychotic ways for female patients in future.

Abstract 359: IWS1 is essential for mouse development

Abstract Iws1 (Interacts with Spt6) was originally identified in Saccharomyces Cerevisiae as a binding partner of Spt6, a histone H3/H4 chaperone.As a transcription factor, Iws1 plays a key role in defining the composition of the RNA polymerase II (RNAPII) elongation complex and in modulating the production of mature mRNA transcripts.Iws1 is phosphorylated, following stimulation with growth factors, by Akt3 and Akt1 at Ser720/Thr721 in human cells (Ser667/Thr668 in the mouse).Preliminary evidence supports a critical protumorigenic role of Iws1 and its phosphorylation in several types of malignancies such as lung cancer (NSCLC), mammary adenocarcinomas and melanomas.For example, it has been shown that, while IWS1 expression in normal human lung is very low, dramatically increases in many NSCLCs. Interestingly the level of IWS1 phosphorylation is variable between tumors and correlates with Akt3 expression.Moreover data from the TCGA database show that Iws1 is expressed at higher levels in tumors than in normal mammary gland specimens and also that basal and HER-2-enriched tumors express the highest levels. The genetic depletion of Iws1 in human mammary adenocarcinoma cell lines inhibits anchorage-independent growth in soft agar and mammosphere formation and growth. Similar experiment revealed that human melanoma cells were extraordinarily sensitive to the knockdown of Iws1. These experiments identify mammary adenocarcinomas and melanomas as tumors potentially addicted to Iws1. However, Iws1 biological functions are poorly characterized and there is no reported in vivo investigation about this RNA-processing factor.To begin to shed some light on the function of this protein at organismal level, we performed a thorough analysis of the expression of Iws1 and generated Iws1-deficient mice.The systematic analysis of expression in mouse tissues shows that Iws1 is ubiquitously expressed. We also report that ablation of Iws1 causes lethality at very early stages of mouse development. In fact, Iws1-deficient mouse embryos die at pre-implantation stage. Overall, these results show that Iws1 is an essential gene for mouse development and suggest a critical function in mammary adenocarcinomas as well as in melanoma. Citation Format: Arturo Orlacchio, Claudia Foray, Tyler Sheetz, Erika Reese, Foued Amari, Aaron E. Stark, Krista La Perle, Ioannis Sanidas, Philip N. Tsichlis, Dario Palmieri, Vincenzo Coppola. IWS1 is essential for mouse development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 359.

Breast cancer stem cells are involved in Trastuzumab resistance through the HER2 modulation in 3D culture.

Breast cancer human cells culture as spheroids develop autophagy and apoptosis, which promotes Trastuzumab resistance in HER2 overexpressing cells. Our aim was to study the association of the hostile environment developed in 3D with the breast cancer stem cells population and the HER2 modulation. Human mammary adenocarcinoma cell lines were cultured as spheroids using the hanging drop method. We generated hypoxia conditions by using a hypoxic chamber and CoCl2 treatment. Breast cancer stem cells were measured with mammosphere assays, the analysis of CD44 + CD24low population by flow cytometry and the pluripotent gene expression by RT-qPCR. HER2 expression was evaluated by flow cytometry and Western blot. MTS assays were conducted to study cell viability. Hostil environment developed in spheroids, defined by hypoxia and autophagy, modulated the response to Trastuzumab. In HER2+ cells with acquired resistance, we observed an increase in the breast cancer stem cell population. In BT474 spheroids, Trastuzumab induced the acquisition of resistance, along with an increase in breast cancer stem cells. Also, in 3D culture conditions we determined a modulation in the HER2 expression. Moreover, breast cancer stem cells showed enhanced HER2 expression. Finally, cells without HER2 gene amplification cultured as spheroids were sensitive to Trastuzumab, diminishing HER2 expression and cancer stem cells. Our findings show that 3D architecture is able to modulate breast cancer stem cell population and HER2 distribution, modifying the cell response to Trastuzumab.

Autophagy Protects from Trastuzumab-Induced Cytotoxicity in HER2 Overexpressing Breast Tumor Spheroids.

Multicellular tumor spheroids represent a 3D in vitro model that mimics solid tumor essential properties including assembly and development of extracellular matrix and nutrient, oxygen and proliferation gradients. In the present study, we analyze the impact of 3D spatial organization of HER2-overexpressing breast cancer cells on the response to Trastuzumab. We cultured human mammary adenocarcinoma cell lines as spheroids with the hanging drop method and we observed a gradient of proliferating, quiescent, hypoxic, apoptotic and autophagic cells towards the inner core. This 3D organization decreased Trastuzumab sensitivity of HER2 over-expressing cells compared to monolayer cell cultures. We did not observe apoptosis induced by Trastuzumab but found cell arrest in G0/G1 phase. Moreover, the treatment downregulated the basal apoptosis only found in tumor spheroids, by eliciting protective autophagy. We were able to increase sensitivity to Trastuzumab by autophagy inhibition, thus exposing the interaction between apoptosis and autophagy. We confirmed this result by developing a resistant cell line that was more sensitive to autophagy inhibition than the parental BT474 cells. In summary, the development of Trastuzumab resistance relies on the balance between death and survival mechanisms, characteristic of 3D cell organization. We propose the use of spheroids to further improve the understanding of Trastuzumab antitumor activity and overcome resistance.

Killing cancer cells with nanotechnology: the effect of CaS nanostructures on the cell replication rate and survival of human mammary adenocarcinoma cell lines (780.9)

Calcium sulfide (CaS) nanostructures have the potential to play an important role as cadmium‐free nanoscaled semiconductors with applications in bioimaging, in vivo labeling, and sensing, and for drug delivery systems. The objective of this work is to establish the effect of CaS nanostructures on cell replication rate and survival of human mammary adenocarcinoma cell lines. Human adenocarcinoma cell lines are grown in eagle’s minimum essential medium (EMEM). Single doses of dispersions containing traces of CaS nanostructures were added to human mammary adenocarcinoma cell lines with a 70 % confluence. After a 24 hour period, 200,000 cancer cells were subculture in T‐75 treated flasks. The number of dead cells in the cell culture media supernatant was determined in intervals of 24 hours using a Biorad T10 cell counter. The number of dead cells in the supernatant is found to increase from 2,000 in control cell cultures to over 70,000 in cancer cell cultures exposed to a single dose of the CaS dispersion in the first 24 hours: this represents 35 % of the initial cell seeding. The number of dead cells remains in the range of 60,000 to 30,000 in measurements performed between 48 and 72 hours following the single dose of the dispersion containing the CaS nanostructures. The number of replicating cells is found to decrease in the first 72 hours and to increase thereafter. We conclude that the CaS nanostructures affect the replication rate and survival of human adenocarcinoma cells and demonstrate the role CaS nanostructures may play to treat localized cancers.Grant Funding Source: NIH NIGMS‐R25GM088023 and NIGMS‐R25GM096955

Characterization of a Novel Activated Ran GTPase Mutant and Its Ability to Induce Cellular Transformation

Ran (Ras-related nuclear) protein, a member of the Ras superfamily of GTPases, is best known for its roles in nucleocytoplasmic transport, mitotic spindle fiber assembly, and nuclear envelope formation. Recently, we have shown that the overexpression of Ran in fibroblasts induces cellular transformation and tumor formation in mice (Ly, T. K., Wang, J., Pereira, R., Rojas, K. S., Peng, X., Feng, Q., Cerione, R. A., and Wilson, K. F. (2010) J. Biol. Chem. 285, 5815-5826). Here, we describe a novel activated Ran mutant, Ran(K152A), which is capable of an increased rate of GDP-GTP exchange and an accelerated GTP binding/GTP hydrolytic cycle compared with wild-type Ran. We show that its expression in NIH-3T3 fibroblasts induces anchorage-independent growth and stimulates cell invasion, as well as activates signaling pathways that lead to extracellular regulated kinase (ERK) activity. Furthermore, Ran(K152A) expression in the human mammary SKBR3 adenocarcinoma cell line gives rise to an enhanced transformed phenotype and causes a robust stimulation of both ERK and the N-terminal c-Jun kinase (JNK). Microarray analysis reveals that the expression of the gene encoding SMOC-2 (secreted modular calcium-binding protein-2), which has been shown to synergize with different growth factors, is increased by at least 50-fold in cells stably expressing Ran(K152A) compared with cells expressing control vector. Knocking down SMOC-2 expression greatly reduces the ability of Ran(K152A) to stimulate anchorage-independent growth in NIH-3T3 cells and in SKBR3 cells and also inhibits cell invasion in fibroblasts. Collectively, our findings highlight a novel connection between the hyper-activation of the small GTPase Ran and the matricellular protein SMOC-2 that has important consequences for oncogenic transformation.

Up‐regulation of pro‐inflammatory genes as adaptation to hypoxia in MCF‐7 cells and in human mammary invasive carcinoma microenvironment

The role of tumor cells in synthesizing pro-inflammatory molecules is still controversial. Here we report that hypoxic treatment of the MCF-7 human mammary adenocarcinoma cell line induced activation of hypoxia-inducible factor 1alpha (HIF-1alpha) and nuclear factor-kappa B (NF-kappaB). Importantly, hypoxia regulated expression of alarmin receptors such as the receptor for advanced glycation end products (RAGE) and the purinoreceptor (P2X7R), and up-regulated inflammatory response (IR) genes such as the inducible enzymes nitric oxide synthase (NOS2), cycloxygenase (COX2), and the acute-phase protein pentraxin-3 (PTX3). Hypoxia also stimulated chemokine (C-X-C motif) receptor 4 (CXCR4) mRNA synthesis. In fact, the CXCR4 ligand stromal-derived factor-1alpha (SDF-1alpha) increased invasion and migration of hypoxic MCF-7 cells. Inhibition of HIF-1alpha by chetomin and NF-kappaB by parthenolide reduced mRNA and protein expression of the studied molecules and prevented invasion of hypoxic MCF-7 cells. Moreover, solid invasive mammary tumor microenvironment was analyzed after laser-capture microdissection (LCMD) comparing tumor versus host normal tissue. Nuclear translocation of HIF-1alpha and NF-kappaB and up-regulation of IR, CXCR4, estrogen receptor alpha (ERalpha), and epithelial growth factor receptor (EGFR) was observed in tumor but not in host normal tissue in the absence of a local inflammatory leukocyte infiltrate. We conclude that under hypoxic conditions MCF-7 cells acquire a pro-inflammatory phenotype, and that solid human mammary carcinoma evidenced a similar activation of HIF-1alpha, NF-kappaB, and IR genes in malignant tumor cells as compared to the normal host tissues. We suggest a role for IR activation in the malignant progression of transformed cells.

Metabolic activation promotes estrogenic activity of the phytoestrogen-rich plant

Objective There is evidence that metabolic activation can increase the estrogenic activity of the phytoestrogen-rich herb in tests with HepG2 cells. Variation in both plant genetics and harvest season may also influence estrogenic activity of the plant materials. We evaluated the influence of in vitro metabolic activation by S9 mixture on the estrogenic activity of tuberous samples of different cultivars of the phytoestrogen-rich herb, Pueraria mirifica, harvested in different seasons. Methods Plant extracts were derived from the tubers of five plant cultivars collected during summer, rainy season and winter and administered to MCF-7 cultures, an ERα-positive human mammary adenocarcinoma cell line for 3 days at dosages of 0.1, 1, 10, 100 and 1000 μg/ml. These data were compared with the major plant isoflavonoids puerarin, daidzin, genistin, daidzein and genistein and with 17β-estradiol, at concentrations of 10 −12 to 10 −6 M. The test system was done in the absence and presence of the S9 mixture. Results The major plant isoflavonoids and the plant extracts exhibited variable degrees of estrogenic activities as evaluated by altered proliferation of the MCF-7 cell line which were significantly enhanced in the presence of the S9 mixture. Conclusion Metabolic activation of plant isoflavonoids at least in vitro by S9 mixture plays a significant role in amplification of the estrogenic activity of the phytoestrogen-rich plant. In addition, the estrogenic activities of the plant samples were potentially influenced by both seasonal changes and plant genetics.

Proteasomes Mediate Prolactin-induced Receptor Down-regulation and Fragment Generation in Breast Cancer Cells

Prolactin regulates a variety of physiological processes, including mammary gland growth and differentiation, and recent findings support an important role in breast cancer development and progression. However, little is known about the trafficking of its receptor, a member of the cytokine receptor superfamily. In the present study, we examined the effect of ligand on the endogenous "long" isoform of the prolactin receptor in breast cancer cells. We found that prolactin caused rapid and prolonged down-regulation of this receptor. The prolactin-induced increase in degradation was blocked by inhibitors of both proteasomes and lysosomes. However, the ubiquitin-conjugating system was not required for internalization. Prolactin also resulted in the concomitant appearance of a cell-associated prolactin receptor fragment containing the extracellular domain. This latter process required proteasomal, but not metalloprotease, activity, distinguishing it from ectodomain "shedding" of other membrane receptors, which are secreted as binding proteins. The prolactin receptor fragment was labeled by surface biotinylation and independent of protein synthesis. Together, these data indicated that prolactin binding initiates limited proteasomal cleavage of its receptor, generating a cell-associated fragment containing the extracellular domain. Our findings described a new potential mediator of prolactin action and a novel mechanism whereby proteasomes modulate cellular processes.

Estrogenic activity of phenylpropanoids from Sarcomelicope megistophylla and structure determination of a new norneolignan.

Bioassay-guided fractionation of the neutral extract of the bark of Sarcomelicope megistophylla resulted in the isolation of the new nor-neolignan sarcomeginal ( 1), together with the known ailanthoidol ( 2) and (+/-)-seco-isolariciresinol ( 3). The structure of 1 was determined by spectroscopic means. Estrogenic activity of the isolated compounds was tested using estrogen receptor-positive MCF7 and estrogen receptor-negative MDA-MB-231 human mammary adenocarcinoma cell lines. Compound 3 displayed significant estrogenic activity.

Increased rate of glutathione synthesis from cystine in drug-resistant MCF-7 cells

The rate of glutathione synthesis was determined in drug-sensitive and -resistant MCF-7 cells by monitoring the rate of label uptake from [3,3′- 13 C 2 ]-cystine using NMR spectroscopy and mass spectrometry. Compared with the wild-type human mammary adenocarcinoma cell line (MCF-7wt), the isotope incorporation rate was increased 1.6-, 2.4-, and 5.3-fold in the etoposide-resistant MCF-7 cell line (MCF-7vp), doxorubicin-resistant MCF-7 cell line (MCF-7adr), and 4-hydroperoxycyclophosphamide-resistant MCF-7 cell line (MCF-7hc), respectively. The increase in glutathione metabolism in the MCF-7hc line correlated with steady-state levels as determined by biochemical assay. In contrast, both the MCF-7vp and MCF-7adr lines showed increased metabolic synthesis of glutathione but displayed lower steady-state levels compared with the MCF-7wt line. The increased synthetic rates of all resistant lines reflected, in part, contributions from the increased activities of both γ-glutamyltranspeptidase and γ-glutamylcysteine synthetase. These results emphasize the importance of monitoring glutathione metabolic rates, rather than steady-state levels of enzymes or substrates, for assessing drug resistance.

Ligand-independent activation of oestrogen receptor alpha by caveolin-1.

Expression of caveolin-1 in the human mammary adenocarcinoma cell line MCF-7 causes ligand-independent concentration of oestrogen receptor alpha (ERalpha) in the nucleus, and potentiates ligand-independent and ligand-dependent transcription from an oestrogen response element-driven reporter gene. Furthermore, caveolin-1 co-immunoprecipitates with ERalpha [Schlegel, Wang, Katzenellenbogen, Pestell and Lisanti (1999) J. Biol. Chem. 274, 33551-33556]. In the present study we show that caveolin-1 binds directly to ERalpha. This interaction is mediated by residues 82-101 of caveolin-1 (i.e. the caveolin scaffolding domain) and residues 1-282 of ERalpha. The caveolin-binding domain of ERalpha includes the ligand-independent transactivation domain, activation function (AF)-1, but lacks the hormone-binding domain and the ligand-gated transactivation domain, AF-2. In co-transfection studies, caveolin-1 potentiates the transcriptional activation of ERalpha(1-282), a truncation mutant that has intact AF-1 and DNA-binding domains. Since AF-1 activity is regulated largely by phosphorylation we determined that co-expression with caveolin-1 increased the basal phosphorylation of ERalpha(1-282), but blocked the epidermal growth factor-dependent increase in phosphorylation. Indeed, caveolin-1 interacted with and potentiated the transactivation of an ERalpha mutant that cannot be phosphorylated by extracellular signal-regulated kinase (ERK)1/2 [ERalpha(Ser(118)-->Ala)]. Thus caveolin-1 is a novel ERalpha regulator that drives ERK1/2-independent phosphorylation and activation of AF-1.

Ligand-independent activation of oestrogen receptor α by caveolin-1

Expression of caveolin-1 in the human mammary adenocarcinoma cell line MCF-7 causes ligand-independent concentration of oestrogen receptor α (ERα) in the nucleus, and potentiates ligand-independent and ligand-dependent transcription from an oestrogen response element-driven reporter gene. Furthermore, caveolin-1 co-immunoprecipitates with ERα [Schlegel, Wang, Katzenellenbogen, Pestell and Lisanti (1999) J. Biol. Chem. 274, 33551–33556]. In the present study we show that caveolin-1 binds directly to ERα. This interaction is mediated by residues 82–101 of caveolin-1 (i.e. the caveolin scaffolding domain) and residues 1–282 of ERα. The caveolin-binding domain of ERα includes the ligand-independent transactivation domain, activation function (AF)-1, but lacks the hormone-binding domain and the ligand-gated transactivation domain, AF-2. In co-transfection studies, caveolin-1 potentiates the transcriptional activation of ERα(1–282), a truncation mutant that has intact AF-1 and DNA-binding domains. Since AF-1 activity is regulated largely by phosphorylation we determined that co-expression with caveolin-1 increased the basal phosphorylation of ERα(1–282), but blocked the epidermal growth factor-dependent increase in phosphorylation. Indeed, caveolin-1 interacted with and potentiated the transactivation of an ERα mutant that cannot be phosphorylated by extracellular signal-regulated kinase (ERK)1/2 [ERα(Ser118 → Ala)]. Thus caveolin-1 is a novel ERα regulator that drives ERK1/2-independent phosphorylation and activation of AF-1.

Tumoricidal activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo.

To evaluate the utility of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) as a cancer therapeutic, we created leucine zipper (LZ) forms of human (hu) and murine (mu) TRAIL to promote and stabilize the formation of trimers. Both were biologically active, inducing apoptosis of both human and murine target cells in vitro with similar specific activities. In contrast to the fulminant hepatotoxicity of LZ-huCD95L in vivo, administration of either LZ-huTRAIL or LZ-muTRAIL did not seem toxic to normal tissues of mice. Finally, repeated treatments with LZ-huTRAIL actively suppressed growth of the TRAIL-sensitive human mammary adenocarcinoma cell line MDA-231 in CB.17 (SCID) mice, and histologic examination of tumors from SCID mice treated with LZ-huTRAIL demonstrated clear areas of apoptotic necrosis within 9-12 hours of injection.

EGF-induced programmed cell death of human mammary carcinoma MDA-MB-468 cells is preceded by activation AP-1.

MDA-MB-468 is a human mammary adenocarcinoma cell line that overexpresses the epidermal growth factor (EGF) receptor and undergoes programmed cell death (apoptosis) in response to EGF treatment. Programmed cell death was shown to be greatly enhanced when cells were growth-arrested prior to EGF treatment. Apoptosis was characterized by an initial rounding up and detachment of the cells from their substrate starting about 12 h after EGF treatment, followed by chromatin condensation, nuclear fragmentation and oligonucleosomal fragmentation of the DNA at about 24 to 48 h. Cell death was dependent on de novo protein synthesis. We found a rapid induction of c-fos, c-jun and junB at the mRNA level after about 30 min of EGF treatment and a more delayed upregulation of fosB and fra-1. The junD gene was expressed in the absence of EGF, and it was moderately induced within 30 min of growth factor addition. The increase of the different fos and jun mRNAs were paralleled by an increase of activator protein-1 (AP-1) DNA binding activity. A characterization of the AP-1 complex revealed similar levels of several Fos and Jun proteins. Based on the kinetics of AP-1 accumulation and cell death, it seems likely that AP-1 contributes to the apoptotic cell death of EGF receptor-overexpressing MDA-MB-486 cells.

MCF-7 human mammary adenocarcinoma cell death in vitro in response to hormone-withdrawal and DNA damage.

Anti-oestrogens exert a tumoristatic effect on estrogen-receptor-positive breast carcinomas in vivo. At a cellular level this may reflect inhibition of cell proliferation and/or cell death counterbalanced by continued proliferation of a cell subpopulation. We evaluated the MCF-7 human mammary adenocarcinoma cell line as an in vitro model to study the effects of the novel oestrogen antagonist ICI 182,720 on cell population dynamics (cell gain vs. cell loss). After oestrogen-withdrawal monolayer cell number declined over 10 days, accompanied by cell detachment. This decrease in viable cell number was elevated 2-fold by ICI 182,780. Detached cells exhibited DNA fragments of 50 and 300 kbp, typical of apoptotic cells. However, internucleosomal cleavage to 180 bp integer fragments was not seen, and these detached cells exhibited a morphology which was not consistent with apoptosis. The remaining attached monolayer cells were morphologically viable (> 99%) with regard to both nuclear morphology and plasma membrane integrity. There was no difference in cell cycle phase distribution between oestrogen-withdrawn and ICI 182,780-treated cells; both induced accumulation in G1 phase. MCF-7 cells were also exposed to a variety of DNA damaging agents known to induce apoptosis in other cell types. We could demonstrate only limited induction of morphologically recognisable apoptosis in MCF-7 cells treated with methyl methanesulphonate. Our results add to the controversy surrounding the ability of the MCF-7 cell line to undergo apoptosis in vitro in response to anti-oestrogen therapies.

Down-regulation of messenger ribonucleic acid (mRNA) for the estrogen receptor (ER) by phorbol ester requires ongoing RNA synthesis but not protein synthesis. Is hormonal control of ER mRNA degradation mediated by an RNA molecule?

Treatment of MCF-7 cells, a human mammary adenocarcinoma cell line, with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) (10-7 M) was associated with a time-dependent reduction in the level of estrogen receptor (ER) mRNA (half-life approximately 3 h). In the presence of the RNA synthesis inhibitor actinomycin D [5.0 micrograms/ml (4.0 microM)], half-life of ER mRNA was much longer (approximately 12 h). Furthermore, the TPA-dependent down-regulation of ER mRNA was abolished by actinomycin D. Similar effects were observed with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (150 microM), an inhibitor of RNA polymerase. Inhibition of protein synthesis by cycloheximide (50 microM) or puromycin [50 micrograms/ml (92 microM)] did not alter the steady state level of ER mRNA during a period of 10-12 h. Furthermore, these inhibitors of protein synthesis did not prevent the down-regulation of ER mRNA in the presence of TPA. Our studies show that degradation of ER mRNA by TPA in MCF-7 cells is dependent on ongoing RNA synthesis but not on protein synthesis. This indicates that an RNA molecule with rapid turnover, which does not require translation, might be involved in the TPA-dependent ER mRNA decay.

Inhibition of epidermal growth factor/transforming growth factor‐α–stimulated cell growth by a synthetic peptide

Estrogen-stimulated growth of the human mammary adenocarcinoma cell line MCF-7 is significantly inhibited by monoclonal antibodies to the epidermal growth factor (EGF) receptor that act as antagonists of EGF's mitogenic events by competing for high-affinity EGF receptor binding sites. These antibodies likewise inhibit the EGF or transforming growth factor-alpha (TGF-alpha)-stimulated growth of these MCF-7 cells. An analogous pattern of specific EGF or TGF-alpha growth inhibitory activity was obtained using a synthetic peptide analog encompassing the third disulfide loop region of TGF-alpha, but containing additional modifications designed for increased membrane affinity [( Ac-D-hArg(Et)2(31),Gly32,33]HuTGF-alpha(31-43)NH2). The growth factor antagonism by this synthetic peptide was specific in that it inhibited EGF, TGF-alpha, or estrogen-stimulated growth of MCF-7 cells but did not inhibit insulin-like growth factor-1 (IGF-1)-stimulated cell growth. Altogether, these results suggest that a significant portion of the estrogen-stimulated growth of these MCF-7 cells is mediated in an autocrine/paracrine manner by release of EGF or TGF-alpha-like growth factors. The TGF-alpha peptide likewise inhibited EGF- but not fibroblast growth factor (FGF)- or platelet-derived growth factor (PDGF)-stimulated growth of NIH-3T3 cells in completely defined media; but had no effect on growth or DNA synthesis of G0-arrested cells, nor did it effect growth of NR-6 cells, which are nonresponsive to EGF. Although this synthetic peptide did not directly compete with EGF for cell surface receptor binding, it exhibited binding to a cell surface component (followed by internalization), which likewise was not competed by EGF. The peptide did not directly inhibit EGF-stimulated phosphorylation of the EGF receptor, nor did it inhibit phosphorylation of an exogenous substrate, angiotensin II, by activated EGF receptor. The TGF-alpha peptide did, however, affect the structure of laminin as manifested by laminin self-aggregation; this affect on laminin may, in turn, have a modulatory effect on EGF-mediated cell growth.