Studies on type II progesterone receptor in MCF-7 cells

These experiments demonstrate for the first time the existence of a Type II progesterone receptor (RpII) in MCF-7 human breast cancer cells. RpII was shown to have a lower affinity for tritiated progesterone ([3H]Pg) (Kd greater than or equal to 13 nM) than classical Rp (Kd less than or equal to 3 nM). RpII was detected by cytosolic, nuclear, and whole cell assays of MCF-7 cells. Scatchard analysis of [3H]Pg binding data revealed that classical Rp but not RpII could be recompartmentalized from the cytosolic to the nuclear pool by treating cells 1 h at 37 degrees C with 1 microM Pg. RpII levels were shown to be increased more than two-fold by growing MCF-7 cells for 4 days in 10 nM estradiol (E2) plus 100 nM Pg when compared to either untreated cells or to cells treated with only E2.     

Internalization of the E-Cadherin/Catenin Complex and Scattering of Human Mammary Carcinoma Cells MCF-7/AZ after Treatment with Conditioned Medium from Human Skin Squamous Carcinoma Cells COLO 16

Cytokines and other paracrine or autocrine factors functionally modulate the invasion-suppressor and signal-transducing E-cadherin/catenin complex. We have used conditioned medium from human squamous carcinoma COLO 16 cells (CM COLO 16) as a source of such factors to modulate the E-cadherin/catenin complex in human breast carcinoma MCF-7 cells.

CM COLO 16 induces scattering of MCF-7/AZ, but not of MCF-7/6 cells on tissue culture plastic substratum, and reduces aggregation of MCF-7/AZ cells in suspension. Insulin-like growth factor I counteracts this reduction of aggregation. Confocal laser scanning microscopy of immunocytochemical stainings shows loss of the honeycomb pattern of E-cadherin, α-catenin and β-catenin, and internalization of those elements. Cell surface biotinylation shows a decrease in membrane-bound E-cadherin. Immunoprecip-itation and cell fractionation show that the composition of the complex is maintained. Interleukin-1, interleukin-6, granulocyte-monocyte colony stimulating factor, stem cell factor, scatter factor/hepatocyte growth factor and transforming growth factor-β, added separately to MCF-7/AZ cells, could not mimic the effects of CM COLO 16. Neither could we find evidence that the 80 k Da extracellular fragment of E-cadherin is implicated in scattering of MCF-7/AZ cells. This fragment is present in CM COLO 16, but it is also produced by the MCF-7/AZ cells themselves, even at higher levels.

Our data point toward cytoplasmic internalization induced by paracrine factors as one of the downregulating mechanisms for the E-cadherin/catenin complex.     

Internalization of the E-cadherin/catenin complex and scattering of human mammary carcinoma cells MCF-7/AZ after treatment with conditioned medium from human skin squamous carcinoma cells COLO 16

Cytokines and other paracrine or autocrine factors functionally modulate the invasion-suppressor and signal-transducing E-cadherin/catenin complex. We have used conditioned medium from human squamous carcinoma COLO 16 cells (CM COLO 16) as a source of such factors to modulate the E-cadherin/catenin complex in human breast carcinoma MCF-7 cells. CM COLO 16 induces scattering of MCF-7/AZ, but not of MCF-7/6 cells on tissue culture plastic substratum, and reduces aggregation of MCF-7/AZ cells in suspension. Insulin-like growth factor I counteracts this reduction of aggregation. Confocal laser scanning microscopy of immunocytochemical stainings shows loss of the honeycomb pattern of E-cadherin, alpha-catenin and beta-catenin, and internalization of those elements. Cell surface biotinylation shows a decrease in membrane-bound E-cadherin. Immunoprecipitation and cell fractionation show that the composition of the complex is maintained. Interleukin-1, interleukin-6, granulocyte-monocyte colony stimulating factor, stem cell factor, scatter factor/hepatocyte growth factor and transforming growth factor-beta, added separately to MCF-7/AZ cells, could not mimic the effects of CM COLO 16. Neither could we find evidence that the 80 kDa extracellular fragment of E-cadherin is implicated in scattering of MCF-7/AZ cells. This fragment is present in CM COLO 16, but it is also produced by the MCF-7/AZ cells themselves, even at higher levels. Our data point toward cytoplasmic internalization induced by paracrine factors as one of the downregulating mechanisms for the E-cadherin/catenin complex.     

Androgens increase osteoblast-stimulating activity of human breast cancer cells in vitro

Bone metastases of breast cancers produce not only osteolytic but also osteosclerotic lesions. The latter are often observed after androgenic treatment of the tumor. Potential production of osteoblast stimulating activity (ObSA) in breast cancer cell lines, and possible androgen control of this activity have been investigated. Conditioned media (CM) collected from 4 breast cancer cell lines (MCF-7, ZR75, MDA-MB 231, BT20) was tested in vitro on ROS 17/2,8 osteoblast-like cells and on osteoblasts derived from human bone biopsies. The parameters monitored in osteoblasts were [3H]thymidine incorporation, alkaline phosphatase activity, and osteocalcin secretion. Serum-free media conditioned during 24 h by MCF-7 cells presented the highest ObSA. CM decreased thymidine incorporation in DNA and increased alkaline phosphatase activity in a dose-dependent manner. Bone GLA protein (osteocalcin) secretion by human osteoblasts was not increased however in the presence of CM. MCF-7 cells were cultured in the presence of dihydrotestosterone (DHT) [1-100 nM] for 5 days. Serum-free, DHT-free CM collected after an additional 24 h, contained alkaline-phosphatase stimulating activity which was DHT dose-dependent. Estradiol and 1,25(OH)2D3 failed to elicit a comparable increase of the ObSA in the CM. In conclusion, MCF-7 cells product factor(s) that interfere with bone remodeling. The DHT modulation of ObSA parallels the estradiol control of MCF-7 cells osteolytic lesions in relation with Prostaglandin E secretion. Sex hormones at physiological and pharmacological levels might thus control both osteosclerotic and osteolytic lesions observed in bone deposits of hormone dependent cancers.     

Stellate cell apoptosis by a soluble mediator from immortalized human hepatocytes

Activated hepatic stellate cells (HSCs) are the major source of extracellular matrix in fibrosis and cirrhosis. In this study, we have investigated the role of hepatitis C virus (HCV) core protein induced immortalized human hepatocytes (IHH) on HSC growth. Preferential growth of IHH and apoptosis of activated human hepatic stellate cells (LX2) were observed upon coculture of these two cell types in a dual chamber or in the presence of conditioned medium (CM) from IHH. CM did not display a growth inhibitory role on other hepatic (Huh-7, HepG2, Hep3B and THLE) and non-hepatic (HeLa, MCF-7, and BHK) epithelial cells, indicating that the soluble mediator from IHH does not have a generalized effect on cell lines examined in our study. Further studies suggested that CM from IHH increased the expression of TRAIL receptors on LX2 cell surface, and induced apoptosis by a caspase dependent mechanism. Peptide mass fingerprinting of the purified soluble mediator from CM suggested that gelsolin fragments may play a role in apoptosis of LX2 cells. Taken together, our results suggested that a soluble mediator secreted from immortalized human hepatocytes plays an important role in hepatic stellate cell growth regulation.     

Placental Kisspeptins Differentially Modulate Vital Parameters of Estrogen Receptor-Positive and -Negative Breast Cancer Cells

Kisspeptins (KPs) are major regulators of trophoblast and cancer invasion. Thus far, limited and conflicting data are available on KP-mediated modulation of breast cancer (BC) metastasis; mostly based on synthetic KP-10, the most active fragment of KP. Here, we report for the first time comprehensive functional effects of term placental KPs on proliferation, adhesion, Matrigel invasion, motility, MMP activity and pro-inflammatory cytokine production in MDA-MB-231 (estrogen receptor-negative) and MCF-7 (estrogen receptor-positive). KPs were expressed at high level by term placental syncytiotrophoblasts and released in soluble form. Placental explant conditioned medium containing KPs (CM) significantly reduced proliferation of both cell types compared to CM without (w/o) KP (CM-w/o KP) in a dose- and time-dependent manner. In MDA-MB-231 cells, placental KPs significantly reduced adhesive properties, while increased MMP9 and MMP2 activity and stimulated invasion. Increased invasiveness of MDA-MB-231 cells after CM treatment was inhibited by KP receptor antagonist, P-234. CM significantly reduced motility of MCF-7 cells at all time points (2–30 hr), while it stimulated motility of MDA-MB-231 cells. These effects were reversed by P-234. Co-treatment with selective ER modulators, Tamoxifen and Raloxifene, inhibited the effect of CM on motility of MCF-7 cells. The level of IL-6 in supernatant of MCF-7 cells treated with CM was higher compared to those treated with CM-w/o KP. Both cell types produced more IL-8 after treatment with CM compared to those treated with CM-w/o KP. Taken together, our observations suggest that placental KPs differentially modulate vital parameters of estrogen receptor-positive and -negative BC cells possibly through modulation of pro-inflammatory cytokine production.     

Distinct stress and cell destruction pathways are engaged by TNF and ceramide during apoptosis of MCF-7 cells

Ceramide has been proposed to be an important signaling intermediate in tumor necrosis factor (TNF)-induced apoptosis in human MCF-7 breast adenocarcinoma cells. We compared cell death and signal transduction pathways induced by TNF and ceramide in TNF-sensitive, parental MCF-7 cells to those in TNF-resistant, MCF-7 cells (3E9). TNF caused proteolysis of the caspase substrate, polyADP-ribose polymerase (PARP) in parental cells, but not in 3E9 cells. Both apoptosis and PARP cleavage were strongly prevented by co-incubation with caspase inhibitors. In contrast, ceramide-induced cell death was neither affected by TNF resistance nor was it associated with PARP cleavage, and death could not be prevented by co-incubation with caspase inhibitors in either cell line. TNF was able to activate JNK/SAPK approximately 30-fold and approximately 5-fold in parental MCF-7 and 3E9 cells, respectively; in contrast, cell-permeable ceramide only weakly stimulated JNK/SAPK activity in either cell type. Although JNK was activated by TNF, pharmacological blockade of the JNK pathway did not inhibit TNF- or ceramide-mediated cell death. Using mass spectroscopic analysis for ceramide, no increase, rather, a decrease in total ceramide content in TNF-treated parental cells was observed. These results suggest that the cell death signaling and execution pathways utilized by ceramide are distinct from those activated by TNF in MCF-7 cells.     

Osteoblast-derived oxysterol is a migration-inducing factor for human breast cancer cells

Bone metastasis is the major reason for death caused by breast cancer. We used human breast cancer (MCF-7) cells that are poorly metastatic but show highly inducible migration to determine bone-derived factors that induce migration of initially non-disseminating breast cancer cells. We have found that a lipid fraction from human osteoblast-like MG63 cell-conditioned medium (MG63CM) contains a migration-inducing factor for MCF-7 cells. In this fraction, we have identified oxysterol (OS) as a lipid mediator for tumor cell migration. In MCF-7 cells, insulin-like growth factor 1 elevates the expression of OS-binding protein-related protein 7. Binding of OS to OS-binding protein or OS-binding protein-related protein is known to trigger elevation of sphingomyelin, a sphingolipid that organizes lipid microdomains in the cell membrane. In MCF-7 cells, OS increases the intracellular concentration of sphingomyelin and other phospholipids and induces the translocation of the small GTPase p21Ras to GM1- and cholesterol-rich membrane areas. The induction of migration by MG63CM is prevented by incubation of MG63 cells with mevinolin, a statin-type cholesterol biosynthesis inhibitor that depletes the conditioned medium of OS. Osteoblast-derived OS may, thus, be a yet unrecognized lipid mediator for bone metastasis of breast cancer and a new target for anti-metastasis chemotherapy with statins.     

Estrogen-induced apoptosis in a breast cancer model resistant to long-term estrogen withdrawal

Estrogen suppression through the use of an aromatase inhibitor is an effective endocrine treatment option for postmenopausal breast cancer patients with estrogen receptor (ER)-positive disease, however, there are concerns that long-term estrogen deprivation will inevitably lead to resistance. To address the issue of acquired resistance to long-term estrogen deprivation our laboratory has developed an ER+/PR- hormone-independent breast cancer cell line, MCF-7:5C which is a variant clone of wild-type MCF-7 cells. Originally, these cells were cultured in estrogen-free MEM containing 5% charcoal-stripped calf serum and were found to be resistant to both estradiol (E(2)) and antiestrogens. Interestingly, a completely different phenomenon was observed when MCF-7:5C cells were cultured in phenol red-free RPMI 1640 medium containing 10% charcoal-stripped fetal bovine serum (SFS). Using DNA quantitation assays, we examined the effect of E(2) on the growth of MCF-7:5C cells under different media conditions. Our results showed that 10(-9)M E(2) caused a dramatic 90% reduction in the growth of MCF-7:5C cells cultured in RPMI medium containing 10% SFS but did not have any significant inhibitory effects on cells cultured in MEM media. Additional experiments were performed to determine whether the medium or the serum facilitated the inhibitory effects of E(2) and the results indicated that it was the serum. Annexin V and DAPI staining confirmed that the E(2)-induced growth inhibition of MCF-7:5C cells was due to apoptosis. We also examined the tumorigenic potential of MCF-7:5C cells by injecting 1x10(7)cells/site into ovariectomized athymic mice and found that these cells, previously cultured in RPMI media, spontaneously grew into tumors in the absence of E(2). Overall, these results show that low concentrations (>10(-11)M) of E(2) are capable of inducing apoptosis in an aromatase resistant breast cancer cell model and that this effect is highly influenced by the medium in which the cells are grown.     

Role of estrogen receptors and antiestrogen binding sites in an early effect of antiestrogens, the inhibition of cholesterol biosynthesis

The effects of estradiol (E2), 4-hydroxy-tamoxifen (OH-Tam), and LY117018 on cholesterogenesis were investigated in two human breast cancer cell lines (MCF-7 and BT20), and in rat hepatoma (HTC) and fibroblastic (NRK-49F) cell lines. It was found that 10(-10) M E2 stimulated and 10(-8) M OH-Tam inhibited cholesterol synthesis in the estrogen-sensitive MCF-7 cell line. The OH-Tam effect occurred in less than 15 min whereas E2 only stimulated after 8 h. The inhibition of cholesterol synthesis was not reversed by E2. E2 was without effect in the HTC and estrogen-resistant BT20 cell lines whereas OH-Tam was as effective as in the MCF-7 cells. LY117018 had nearly as much effect on cholesterol synthesis as OH-Tam, in both MCF-7 and BT20 cells. Neither E2 nor OH-Tam had any effect on the NRK-49F cell line, even at micromolar concentrations. The three lines (MCF-7, BT20, HTC), whose cholesterol synthesis has been shown to be OH-Tam sensitive, appeared to contain high-affinity antiestrogen binding sites (AEBS); since the OH-Tam-resistant line (NRK) only contained low-affinity AEBS, there appears to be some relationship between OH-Tam sensitivity and high-affinity AEBS content. This suggests that the cholesterogenesis inhibition induced by antiestrogens is ER-independent and may involve AEBS. The cholesterogenesis stimulation induced by E2 occurred via a different pathway that appears to be related to the presence of ER in the cells.     

Chemotherapy cytotoxicity of human MCF-7 and MDA-MB 231 breast cancer cells is altered by osteoblast-derived growth factors

One-third of women with breast cancer will develop bone metastases and eventually die from disease progression at these sites. Therefore, we analyzed the ability of human MG-63 osteoblast-like cells (MG-63 cells), MG-63 conditioned media (MG-63 CM), insulin-like growth factor I (IGF-I), and transforming growth factor beta 1 (TGF-beta1) to alter the effects of adriamycin on cell cycle and apoptosis of estrogen receptor negative (ER-) MDA-MB-231 and positive (ER+) MCF-7 breast cancer cells, using cell count, trypan blue exclusion, flow cytometry, detection of DNA fragmentation by simple agarose gel, and the terminal deoxynucleotidyl transferase (TdT)-mediated nick end-labeling method for apoptosis (TUNEL assay). Adriamycin arrested MCF-7 and MDA-MB-231 cells at G2/M phase in the cell cycle and inhibited cell growth. In addition, adriamycin arrested the MCF-7 cells at G1/G0 phase and induced apoptosis of MDA-MB-231 cells. Exogenous IGF-I partially neutralized the adriamycin cytotoxicity/cytostasis of cancer cells. MG-63 CM and TGF-beta1 partially neutralized the adriamycin cytotoxicity of MDA-MB-231 cells but enhanced adriamycin blockade of MCF-7 cells at G1/G0 phase. MG-63 osteoblast-like cells inhibited growth of MCF-7 cells while promoting growth and rescued MDA-MB-231 cells from adriamycin apoptosis in a collagen co-culture system. These data suggest that osteoblast-derived growth factors can alter the chemotherapy response of breast cancer cells. Conceivably, host tissue (bone)-tumor cell interactions can modify the clinical response to chemotherapy in patients with advanced breast cancer.     

Purification and characterization of an alpha 1-antichymotrypsin-like 66 kDa protein from the human breast cancer cell line, MCF-7.

The synthesis of a 66 kDa protein immunoreactive with antibodies to human alpha 1-antichymotrypsin (alpha 1-ACT) is induced by estradiol (E2) in the human breast cancer cell line MCF-7. We have purified this alpha 1-ACT-like 66 kDa protein from medium conditioned by MCF-7 cells, performed a comparative physico-chemical characterization with serum alpha 1-ACT, and analysed its presumed positive regulatory effect on growth of MCF-7 cells. The 66 kDa protein is a functional antiproteinase which is antigenically identical to serum alpha 1-ACT. The 66 kDa protein does however deviate from serum alpha 1-ACT with respect to mol. wt. and pattern of microheterogeneity, the molecular mechanism for this is probably an incomplete glycoprotein processing in the MCF-7 cells. The results of our growth experiments suggest that the 66 kDa protein is a minor positive growth regulatory factor, which may contribute to breast carcinoma cell proliferation in a cooperative manner.     

MDR1 P-glycoprotein reduces influx of substrates without affecting membrane potential

MDR1 (multidrug resistance) P-glycoprotein (Pgp; ABCB1) decreases intracellular concentrations of structurally diverse drugs. Although Pgp is generally thought to be an efflux transporter, the mechanism of action remains elusive. To determine whether Pgp confers drug resistance through changes in transmembrane potential (E(m)) or ion conductance, we studied electrical currents and drug transport in Pgp-negative MCF-7 cells and MCF-7/MDR1 stable transfectants that were established and maintained without chemotherapeutic drugs. Although E(m) and total membrane conductance did not differ between MCF-7 and MCF-7/MDR1 cells, Pgp reduced unidirectional influx and steady-state cellular content of Tc-Sestamibi, a substrate for MDR1 Pgp, without affecting unidirectional efflux of substrate from cells. Depolarization of membrane potentials with various concentrations of extracellular K(+) in the presence of valinomycin did not inhibit the ability of Pgp to reduce intracellular concentration of Tc-Sestamibi, strongly suggesting that the drug transport activity of MDR1 Pgp is independent of changes in E(m) or total ion conductance. Tetraphenyl borate, a lipophilic anion, enhanced unidirectional influx of Tc-Sestamibi to a greater extent in MCF-7/MDR1 cells than in control cells, suggesting that Pgp may, directly or indirectly, increase the positive dipole potential within the plasma membrane bilayer. Overall, these data demonstrate that changes in E(m) or macroscopic conductance are not coupled with function of Pgp in multidrug resistance. The dominant effect of MDR1 Pgp in this system is reduction of drug influx, possibly through an increase in intramembranous dipole potential.     

Antiproliferative effect of phorbol esters on MCF-7 human breast adenocarcinoma cells: relationship with enhanced expression of transforming growth-factor-beta 1.

In human breast carcinoma MCF-7 cells, phorbol diesters inhibit proliferation and induce cell maturation. We have recently reported that exogenous TGF-beta 1 reverses the resistance of a breast adenocarcinoma MCF-7 subline (MCF-7:RPh-4) to these phorbol ester effects. Here, we investigated the involvement of TGF-beta 1 in the PKC-mediated inhibition of breast-cancer cell proliferation. Parental MCF-7-conditioned medium contained a 20-fold higher transforming activity on NRK-49F fibroblasts than the TPA-resistant subline. TPA increased TGF-beta activity in MCF-7 conditioned medium. MCF-7 cells also expressed more TGF-beta 1 mRNA than the resistant subline. TPA induced a dose-dependent increase in TGF-beta 1 mRNA levels that paralleled the inhibitory effect on MCF-7 proliferation. The lower level of TGF-beta mRNA expression in TPA resistant subline was not modified after addition of TPA, but was significantly increased in the presence of exogenous TGF-beta 1. These data argue in favor of a role of endogenous TGF-beta 1 in the maturation process induced by protein kinase C activation.     

Synergistic proliferative action of insulin-like growth factor I and 17 beta-estradiol in MCF-7S breast tumor cells.

We have analyzed the mechanism by which the combination of insulin-like growth factor I (IGF-I) and 17 beta-estradiol (E2) induces cell cycle progression in MCF-7S cells. This cell line differs from many other breast cancer-derived cell lines in that E2 (1 nM) does not induce cell cycle progression, whereas the combination of submitogenic concentrations of IGF-I (2 ng/ml) and E2 does. We find that addition of IGF-I to MCF-7S cells leads to a dose-dependent activation of the IGF type I receptor and of the MAP kinase and PI3-kinase signaling pathways. No synergy of IGF-I and E2 was detected in the activation of these signaling cascades. In terms of cell cycle-related molecules, we find that IGF-I dose-dependently raises cyclin D1 levels in serum-starved cells. Subsequent activation of cyclin E/CDK2, hyperphosphorylation of pRb, and DNA synthesis are only induced by mitogenic concentrations of IGF-I (> or =20 ng/ml). Treatment of the cells with E2 also results in the induction of cyclin D1, but in the absence of IGF-I the cells remain arrested in G1 phase. We conclude that in MCF-7S cells, the synergistic action of E2 and IGF-I derives from the ability of both hormones to induce cyclin D1 expression. The action of IGF-I is required in these cells to induce activity of the cyclin D1/CDK4 complex, which triggers progression through the cell cycle.     

Recombinant Analogs of a Novel Milk Pro-Apoptotic Peptide,Lactaptin, and Their Effect on Cultured Human Cells

We recently isolated and characterized a human milk peptide, lactaptin, which induced apoptosis of cultured human MCF-7 cells. Lactaptin was identified as a proteolytic fragment of human kappa-casein. Here, we generated two recombinant analogs of the peptide, RL1 and RL2, containing truncated and complete amino acid sequences of lactaptin, respectively. Analogs were produced in E.coli, purified and assayed for biological activity on cultured human MCF-7 cells. RL1 was shown to induce only a small decrease in cell viability, whereas RL2 lowered the viability of MCF-7 cells by 60%. This reduction in MCF-7 cell viability was associated with apoptosis, which was indicated by phosphatidilserine externalization and caspase-7 activation. The viability of A549 and Hep-2 cells was also reduced by RL2, albeit to a lesser degree than seen with MCF-7 cells; this reduced viability was not accompanied by apoptosis. Non-malignant human mesenchymal stem cells (MSC) were completely resistant to RL2 action.     

Cytokine profile of conditioned medium from human tumor cell lines after acute and fractionated doses of gamma radiation and its effect on survival of bystander tumor cells

Cytokines are known to play pivotal roles in cancer initiation, progression and pathogenesis. Accumulating evidences suggest differences in basal and stress-induced cytokine profiles of cancers with diverse origin. However, a comprehensive investigation characterising the cytokine profile of various tumor types after acute and fractionated doses of gamma-irradiation, and its effect on survival of bystander cells is not well known in literature. In the present study, we have evaluated the cytokine secretion profile of human tumor cell lines (HT1080, U373MG, HT29, A549 and MCF-7) either before (basal) or after acute (2, 6 Gy) and fractionated doses (3 × 2 Gy) of gamma-irradiation in culture medium obtained from these cells by multiplex bead array/ELISA. Moreover, clonogenic assays were performed to evaluate the effect of conditioned medium (CM) on the survival and growth of respective cells. Based on the screening of 28 analytes, our results showed that the basal profiles of these cell lines varied considerably in terms of the number and magnitude of secreted factors, which was minimum in MCF-7. Interestingly, TNF-α, IL-1β, PDGF-AA, TGF-β1, fractalkine, IL-8, VEGF and GCSF were found in CM of all the cell lines. However, secretion of certain cytokines was cell line-specific. Moreover, CM caused increase in clonogenic survival of respective tumor cells (in the order HT1080 > U373MG > HT29 > A549 > MCF-7), which was correlated with the levels of IL-1β, IL-6, IL-8, GMCSF and VEGF in their CM. After irradiation, the levels of most of the cytokines increased markedly in a dose dependent manner. The fold change in cytokine levels was lower in irradiated conditioned medium (ICM) of tumor cells collected after fractionated than respective acute dose, except in MCF-7. Interestingly, amongst these cell lines, the radiation-induced fold increase in cytokine levels was maximum in ICM of A549 cells. Moreover, bystander A549 cells treated with respective ICM showed dose dependent decrease in clonogenic survival. In conclusion, present study revealed the similarities and subtle differences in basal and radiation-induced cytokine profile of different tumor cell lines, and its influence on growth and survival of respective bystander cells. These findings may add a new dimension to our current understanding about role of cytokines in cancer biology.     

Identification of estrogen-induced genes downregulated by AhR agonists in MCF-7 breast cancer cells using suppression subtractive hybridization

Aryl hydrocarbon receptor (AhR) agonists inhibit 17beta-estradiol (E2) induced growth of MCF-7 human breast cancer cells in vitro and rodent mammary tumor growth in vivo. Genes associated with inhibitory AhR-estrogen receptor (ER) crosstalk were investigated in MCF-7 human breast cancer cells using poly(A)(+)RNA from cells treated with either 1 nM E2 (target) or E2 plus 1 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (reference) or 25 microM diindolylmethane (DIM) as AhR agonists in MCF-7 cells. Suppression subtractive hybridization (SSH) was subsequently used to identify 33 genes with sequence homology to known human genes that are induced by E2 and inhibited by AhR agonists in MCF-7 cells; two unknown genes were also identified. Many of these genes are involved in cell proliferation and these include cell cycle regulators (cdc28/cdc2-associated protein), nucleotide synthases (thymidylate synthase), early intermediate genes (early growth response alpha, EGRalpha) and other proteins involved in signaling pathways (calmodulin, ATP synthase alpha subunit). Thus SSH has identified a diverse spectrum of new genes that are affected by inhibitory AhR-ER crosstalk and among this group are a subset of genes that may be critical for the in vivo antitumorigenic effects of AhR agonists.