Breast Adenocarcinoma Cell Line MCF-7 Research Articles

HLA‐G protein expression is not induced during malignant transformation

To evaluate the biological relevance of HLA-G expression during tumoral transformation, we analyzed its expression in different malignant cells and immune effector cells infiltrating solid tumors. Our analysis of 33 tumor cell lines and 53 tumoral biopsies demonstrated that: i) six tumor cell lines display HLA-G transcription with differential alternative splicing patterns and only Jeg3 choriocarcinoma and MCF-7 breast adenocarcinoma cell lines express HLA-G translated products; and ii) HLA-G antigens are not expressed in malignantly transformed cells derived from lung (n=18), liver (n=5), colon (n=5), breast (n=10), kidney (n=5), ovary (n=5), and larynx (n=5) tissues ex vivo. The healthy tissues surrounding these tumor tissues do not express HLA-G molecules either. On the other hand, surprisingly, HLA-G products were detected in activated macrophages and dendritic cells localized in tumoral biopsies of 5 out of 18 different lung carcinomas. No HLA-G labelling was observed in resident mononuclear phagocytes of surrounding healthy tissues. Our observations clearly demonstrate that HLA-G is not a marker of malignant cells but appears as a gene expressed in tumor-associated macrophages and dendritic cells, preferentially in those recruited in lung carcinomas. Our findings suggest that specific environmental factors around lung tumors could be involved in the induction of HLA-G protein expression.

Involvement of p21 in mitotic exit after paclitaxel treatment in MCF-7 breast adenocarcinoma cell line.

It has been shown recently that expression of p21 is enhanced by paclitaxel. This cytotoxic compound induces mitotic spindle damage resulting in blockade of the mitotic cell cycle associated or not with apoptotic cell death. In the present study, we showed that, in MCF-7 cells, paclitaxel induced accumulation of p21 in cells with a G2/M DNA content, corresponding to cells either in abnormal mitosis or in an interphase-like state (decondensed chromatin) with multiple nuclei. In MCF-7 cells, the increase in p21 was subsequent to the mitotic arrest and was associated with the exit from abnormal mitosis leading to formation of cells with micronuclei. In this cell line, we noted a relationship between the elevation of p21 expression and the inhibition of p34cdc2 activity. High levels of p21 protein were also found to be associated with inactive p34cdc2/cyclin B protein complex after treatment with paclitaxel. Treatment with p21 antisense oligonucleotide partially blocked induction of p21 expression by paclitaxel and significantly reduced survival of MCF-7 cells exposed to this agent. In NIH-OVCAR-3 cells, which are deficient in basal and paclitaxel-induced p21 expression, paclitaxel led to a prolonged activation of p34cdc2 and a delayed mitotic exit associated with apoptotic cell death. These observations suggest that p21 is not required for the mitotic arrest in response to paclitaxel, but argue in favor of a role for this inhibitor in facilitating the exit from abnormal mitosis. This effectively enhances cell survival after paclitaxel-induced spindle damage.

Enantioselective synthesis and antiproliferative properties of an ilmofosine analog, 2'-(trimethylammonio)ethyl 3-(hexadecyloxy)-2-(methoxymethyl)propyl phosphate, on epithelial cancer cell growth.

An asymmetric synthesis of the 1-alkyloxy analog of the thioether phosphocholine ilmofosine (BM 41.440, rac-1), 2'-(trimethylammonio)ethyl 3-(hexadecyloxy)-2-(methoxymethyl)propyl phosphate (2), is described. Stereoselectivity was obtained in an asymmetric hydroboration-oxidation sequence carried out on a 2,2-disubstituted 1-alkene, 3-(hexadecyloxy)-2-(methoxymethyl)-1-propene (9), which was prepared by starting with either ethyl acrylate or ethyl alpha-(hydroxymethyl)acrylate (3). (R)- and (S)-2 and rac-1 were highly effective in inhibiting the proliferation of the breast adenocarcinoma cell line MCF-7 (IC50, 2 microM), moderately effective against A549 (non-small-cell lung adenocarcinoma) (IC50, 8-10 icroM), and less effective against A427 (large cell lung carcinoma) (IC50, approximately 20 microM). The in vitro cytotoxicity against the three epithelial cancer cell lines was independent of the configuration about C-2 of the glycerol backbone of 2 and was also not altered by substitution of oxygen for sulfur in the sn-1 ether linkage of ilmofosine.

Induction of PBP74/mortalin/Grp75, a member of the hsp70 family, by low doses of ionizing radiation: a possible role in induced radioresistance

The identification of genes whose expression is altered following exposure to a low dose of ionizing radiation (IR) is an important step in understanding the phenomenon of the adaptive response. Using the differential mRNA display method we have identified a gene whose expression is up-regulated following exposure to 0.25 Gy IR. Partial DNA sequence and restriction endonuclease analysis of this gene showed that it is identical to the gene encoding for the human peptide-binding protein 74 (PBP74/mortalin/Grp75), a member of the heat shock 70 protein family. Time-course measurement of the PBP74/mortalin/Grp75 mRNA showed that its level was elevated after a lag of at least 15 min. The maximum induction appears to be at 30 min following gamma -irradiation and there is then a steady decline to control levels within 5 h in the HT29 cell line. On the other hand, the level of the PBP74/mortalin/Grp75 mRNA in the human breast adenocarcinoma cell line MCF-7 is consistently elevated after gamma -irradiation for up to 6 h postirradiation. Furthermore, a cell line that does not demonstrate the induced radioresistance phenomenon (SW48) shows no induction of the PBP74/mortalin/Grp75 mRNA in contrast with HT29 or MCF-7. Treatment of the HT29 cells with antisense oligonucleotide directed towards the initiation codon of PBP74 sensitized cells to ionizing radiation.

Co-culture of human breast adenocarcinoma MCF-7 cells and human dermal fibroblasts enhances the production of matrix metalloproteinases 1, 2 and 3 in fibroblasts.

No measurable amounts of matrix metalloproteinases (MMPs) were produced by human breast adenocarcinoma cell lines MCF-7 and BT-20 in culture. When MCF-7 cells were co-cultured with human dermal fibroblasts enhanced production of precursors of MMP-1 (interstitial collagenase), MMP-2 (gelatinase A), MMP-3 (stromelysin 1) and tissue inhibitor of metalloproteinase type 1 (TIMP-1) was observed. Immunohistochemical studies indicated that these pro-MMPs originated primarily from the fibroblasts, suggesting that MCF-7 cells have a stimulatory effect on stromal cells to produce at least three pro-MMPs and TIMP-1. BT-20 cells also enhanced the production of pro-MMP-2 and TIMP-1 in the dermal fibroblasts, but not of pro-MMP-1 and pro-MMP-3. Normal mammary epithelial cells promoted only TIMP-1 production. To investigate further the stimulatory factors from MCF-7 cells, the conditioned medium and the cell membrane were prepared and examined. The cell membrane fraction enhanced the production of pro-MMP-1 and -3 and TIMP-1, but not of pro-MMP-2. The conditioned medium, on the other hand, augmented the production of all four proteins in the fibroblasts. These observations suggest that breast adenocarcinoma MCF-7 cells in culture produce both soluble and membrane-bound factor(s) which stimulate the production of pro-MMPs and TIMP-1 in neighbouring stromal cells, but the factor(s) released into the medium and that associated with cell membranes are probably different. Such communication between the normal and malignant cell types may, in part, assist the cancer cells to invade and metastasise.

P53 protein expression is correlated with benzo[a]pyrene-DNA adducts in carcinoma cell lines.

p53 inhibits cell cycle progression and DNA damaging cytostatics induce p53 protein expression, indicating that p53 responds to DNA damage. We have measured benzo[a]pyrene (BP)-induced DNA damage in association with p53 expression. The most relevant DNA adducts for carcinogenesis, benzo[a]pyrene-7,8-diol-9,10-epoxide-DNA adducts, were measured by synchronous fluorescence spectrophotometry and p53 immunohistochemistry using polyclonal antibody CM1, which detects both wild-type and mutated forms of p53. Activation of BP in A-549 lung carcinoma and MCF-7 breast adenocarcinoma cell lines containing wild-type p53 was followed by an increase in p53 protein expression. alpha-Naphthoflavone, an inhibitor of cytochrome P450 (CYP)1A1, decreased both the formation of diolepoxide metabolites and the p53 response. The cell lines not able to activate BP, A-427 and SK-LU-1 (both human lung carcinomas), SK-MES-1 (human lung squamous carcinoma) and human fibroblasts, did not show any increase in p53 immunohistochemistry. The OVCAR-3 ovarian adenocarcinoma cell line, containing a mutation in exon 7 of p53, and the SK-LU-1 cell line expressed very high levels of p53 protein before BP treatment and no increase in p53 immunohistochemistry was seen. These findings indicate that p53 protein is part of the response of the cells to BP-induced DNA damage.

Cloning and Expression of cDNA Encoding Human Betacellulin, a New Member of the EGF Family

Betacellulin (BTC) is a novel growth factor purified from the conditioned media of mouse pancreatic beta tumor cells and has been found to be a new member of the epidermal growth factor (EGF) family. The cDNA encoding human BTC has been cloned from a cDNA library prepared from human breast adenocarcinoma cell line MCF-7. The nucleotide sequence encodes a polypeptide which consists of 178 amino acid residues including a putative signal sequence, indicating that the structural organization of human BTC is similar to that of mouse BTC. The amino acid sequence of the human BTC precursor protein exhibits 79% similarity with that of the mouse precursor protein. The BTC gene was found to be expressed in several mouse tissues including kidney and liver as well as in a mouse beta tumor cell line and MCF-7 cells, suggesting that BTC might play a physiological role in normal tissues.

Relaxin influences the growth of MCF-7 breast cancer cells. Mitogenic and antimitogenic action depends on peptide concentration.

The effects of relaxin (RLX) on the human breast adenocarcinoma cell line MCF-7 have been evaluated. The cells were maintained in culture with Dulbecco's modified Eagle's medium with 1% and 10% fetal calf serum. Highly purified porcine RLX was added at concentrations ranging between 10(-11) and 10(-6) M, and, after 96-hour incubation in the presence of the peptide, cell proliferation, intracellular cyclic adenosine monophosphate (cAMP) content, percent cycling cells, and structural and ultrastructural pattern were studied. The results indicate that RLX is a direct modulator of MCF-7 cell proliferation, stimulating growth at low concentrations and inhibiting growth at high concentrations. Determinations of percent cycling cells and intracellular cAMP accumulation agree with the results of the growth studies. Addition of different concentrations of 8-Br-cAMP to the culture medium results in a dose-related stimulation of MCF-7 cell proliferation. Morphologic examination shows that, in the current experiments, RLX does not induce any clear-cut signs of differentiation of MCF-7 cells in terms of activation of secretion or intracellular lipid deposition. These findings indicate that RLX should be regarded as a novel agent involved in the control of growth of human breast cancer cells.

Amphiregulin-associated protein: Complete amino acid sequence of a protein produced by the 12-0-tetradecanoylphorbol-13-acetate-treated human breast adenocarcinoma cell line MCF-7

Amphiregulin-associated protein (ARAP) was purified from serum-free conditioned medium of MCF-7, human breast carcinoma cells, treated with 12-0- tetradecanoylphorbol-13-acetate (TPA). ARAP is a single-chain, extremely hydrophilic, heparin-binding protein. Its apparent molecular weight is approximately 21,500 as assessed by gel chromatography and approximately 15,500 as determined by polyacrylamide gel electrophoresis. The complete amino acid sequence of ARAP was determined. The larger form contains 123 amino acids, whereas a shorter form is missing two amino acids at the amino-terminal. ARAP contains 10 cysteines and 30 basic amino acids (23 lysines and 7 arginines). ARAP sequence has been found to be identical to protein encoded by human MK gene.

Association of mRNA and eIF-2α with the cytoskeleton in cells lacking vimentin

The human bladder carcinoma cell lines RT4 and T24 and the human breast adenocarcinoma cell line MCF-7 were found to be negative for vimentin when studied by means of immunofluorescence and immunoblotting. Northern blot analysis revealed that these cells lacked detectable levels of vimentin mRNA with the exception of T24, which contains trace amounts of vimentin mRNA compared to the RNA level in vimentin-containing HeLa cells. CAT assays performed on these cells showed that a hamster vimentin promoter is inactive in RT4 and MCF-7 cells. In the vimentin-lacking cells, the binding of polyribosomes, specific mRNAs, and translation factor eIF-2α to the cytoskeletal fraction was examined. Our results indicate that the presence of a vimentin network is not crucial for the association of the translation machinery with the cytoskeleton. Furthermore, in these vimentin-negative cell lines the immunofluorescence staining pattern of eIF-2α shows a fibro-granular structure that has no resemblance to the cytokeratin or actin cytoskeleton present in these cells.

Amphiregulin: a bifunctional growth-modulating glycoprotein produced by the phorbol 12-myristate 13-acetate-treated human breast adenocarcinoma cell line MCF-7.

A glycoprotein, termed amphiregulin (AR), inhibits growth of several human carcinoma cells in culture and stimulates proliferation of human fibroblasts and certain other tumor cells. It has been purified to apparent homogeneity from serum-free conditioned medium of MCF-7 human breast carcinoma cells that had been treated with phorbol 12-myristate 13-acetate. AR is a single-chain extremely hydrophilic glycoprotein containing cysteines in disulfide linkage(s) that are essential for biological activity; it is stable between pH 2 and pH 12 and after heating for 30 min at 56 degrees C but unstable at 100 degrees C. The apparent molecular weights of AR and N-Glycanase-treated AR are 14,000 and 15,000, respectively, as assessed by gel chromatography, and approximately 22,500 and approximately 14,000, respectively, as determined by polyacrylamide gel electrophoresis. Treatment of AR with N-Glycanase, O-Glycanase, or neuraminidase does not affect its activity. The pI of AR is approximately 7.8. The amino-terminal amino acid sequence of AR has been determined, and no significant sequence homology between AR and other proteins was found. The molecule thus appears to be a distinct growth regulatory protein.