Distribution of selenoproteins in mouse mammary epithelial cells in vitro and in vivo

Resolution of selenium-containing proteins synthesized by mouse mammary gland cells was achieved using the technique of two-dimensional gel electrophoresis. Radioactive selenium as H2[75Se]O3 was incorporated into relatively few proteins within mammary gland cells maintained in vitro and cells of mammary gland tissue in vivo. The pattern of selenoproteins obtained was identical qualitatively between a nontumorigenic differentiated cell line, COMMA-D, and a tumorigenic cell line, MOD. Eleven selenoproteins ranging in molecular weight from 12,000-78,000 were detected and a total of 25 spots were visible indicating charge heterogeneity of some of the proteins. A major selenoprotein (Mr 26,000) migrated identically with the subunit form of glutathione peroxidase, a well-characterized protein containing four selenocysteine residues. Other major selenoproteins had molecular weights of 58,000, 22,000, 18,000, and 14,000. Analysis of the total cellular protein extract and of each of the five major proteins indicated that selenium was incorporated as selenocysteine in the proteins. Incorporation of selenium as selenomethionine into cellular proteins was detected only when selenomethionine was provided in the culture medium. Cleavage of 75Se-labeled proteins with N-chlorosuccinimide produced polypeptides of different molecular weights indicating that the Mr 58,000, 26,000, and 22,000 selenoproteins were dissimilar in the amino acid sequences containing the selenoamino acid. The pattern of selenoproteins of mammary gland cells in vivo was similar to that obtained for cells in culture and most other tissues in vivo. These results provide evidence for the presence of multiple selenium-containing proteins in mammary epithelial cells. The possible significance of these proteins in selenium-mediated inhibition of cell growth awaits future clarification.     

Development of mammary preneoplasias in vivo from mouse mammary epithelial cell lines in vitro.

A series of mouse mammary epithelial cell lines has been established by a protocol that gives highly reproducible results. The mammary epithelial cell lines, designated as FSK lines, were judged to be epithelial based on positive immunostaining for keratin-intermediate filaments, negative immunostaining for vimentin-intermediate filaments, hormonal induction of casein, and the ability to exhibit ductal and alveolar mammary morphogenesis in vivo. The FSK cell lines are dependent on epidermal growth factor and insulin in a low serum (1%) medium. Conditioned medium from spindle cell cultures replaced the requirement for serum and increased the growth of FSK3 and FSK4 4-5 times in collagen gels and 12-14 times in monolayer culture, respectively. Following injection into the mammary fat pad at passages 2-11, the FSK cell lines generated stable transplantable hyperplastic alveolar outgrowth lines. The in vivo outgrowth lines were judged as preneoplastic based on their stable alveolar morphology in vivo and an increased susceptibility for tumorigenesis. The FSK cell lines and their derivative in vivo outgrowth lines provide a new and potentially productive system to examine critical molecular alterations involved in the development of mammary preneoplasias. Furthermore, the reproducibility of the in vitro culture system provides the assurance that stable cell lines of mouse mammary epithelial cells can be generated easily and at will.     

Heterogeneous expression of cytokeratins in metastatic mammary adenocarcinoma cells in vitro and in vivo

The expression of cytokeratins was investigated in rat 13762NF adenocarcinoma cell lines and clones growing in vitro and in vivo. The anti-cytokeratin monoclonal antibody PKK1 used in this study recognized one cytokeratin with a molecular weight of 54,000 in these cells. Immunofluorescence staining of cultured tumor cells with PKK1 detected cytokeratins in less than 1% of the tumor cells isolated from the locally growing parental tumors, but intense cytokeratin staining in the form of fibrils and networks was found in the cell cultures derived from spontaneous metastases. There were variable degrees of staining with the anti-cytokeratin antibody in cultured cells from metastases, indicating phenotypic diversity in the expression of cytokeratins. Staining with PKK1 of frozen sections from locally growing tumors obtained after injecting the cells into the mammary fat pads of syngeneic rats also demonstrated considerable heterogeneity in cytokeratin expression. The anti-cytokeratin reagent stained only a few, isolated cells in frozen sections of tumors established from parental tumor cells, while this reagent stained intensely cells in local tumors and their metastases established from tumor cells cultured from spontaneous lymph node or lung metastases.     

ENU-induced in vitro neoplastic transformation of rat mammary epithelial cells.

Normal mammary epithelial cells, originating from female Sprague-Dawley rats, were grown in Dulbecco's Modified Eagles Medium containing 25% horse serum and hormone supplements. Once established as an epithelial cell culture, the cells were treated with N-ethyl-N-nitrosourea (ENU) in various doses (25-500 ug/ml) to study the process of in vitro mammary epithelial cell neoplastic transformation. The ENU-treatment of primary mammary epithelial cell culture resulted in a sequence of phenotypic changes, termed stages I-V. The rat mammary epithelial cells, after a period of approximately 30 days post-ENU exposure, showed a marked proliferation of morphologically altered cells which formed multi-layered colonies. Subsequently, these cells acquired the capacity to form colonies in soft agar and eventually produced a high yield of palpable tumors when inoculated into newborn female isologous hosts or female athymic nude mice. The immediate effect of ENU on these cells was monitored by measurement of cellular DNA content, unscheduled DNA synthesis, cell proliferation and chromosomal aberrations. The ENU effect on cell proliferation and DNA synthesis was dose dependent; doses greater than 100 ug/ml reduced the cell number and DNA synthesis. Cytofluorometric histograms of non-ENU-treated rat mammary epithelial cells showed a near diploid population of cells. The ENU exposed cells subsequently became hyperdiploid (24-72 hours after ENU) and then regained their near diploid pattern at 120 hours after ENU exposure. The ENU-treated cells also showed a second peak of cells with DNA content in the tetraploid and octaploid range at 24-72 hours after ENU exposure. Single chromatid breaks, isochromatid breaks, chromosomal exchanges, multiple chromosomal breaks and double minutes were among the chromosomal aberrations seen in ENU-treated cells. Most of the chromosomal aberrations peaked at 6 hours post-ENU exposure. The ENU-induced model of in vitro meplastic transformation of rat mammary epithelium as described in this communication appears to provide a good model for the systematic study of the early critical cellular events prerequisite to this carcinogenic process.     

Influence of phytoestrogens on the proliferation and expression of adhesion receptors in human mammary epithelial cells in vitro.

Tumor metastasis is associated with integrin-mediated adhesion and hyaluronan receptor expression. Accumulating evidence suggests that phytoestrogens, which are naturally occurring, plant-derived phytochemicals, could inhibit tumorigenesis during the development of breast cancer. Less is known, however, about the regulation of adhesion receptors by phytoestrogens and, particularly, their potency to influence proliferation of primary human breast cells in comparison with the steroid hormone 17beta-estradiol. Throughout the proliferation experiments, we used primary human mammary epithelial cells from normal tissue that was derived from plastic surgery. For receptor expression (beta1, alpha2, alpha3, CD44), we used the cell line MCF-7. Both investigations were carried out by flow cytometry. The phenotype of primary human mammary epithelial cells was microscopically characterized by analyzing the distribution of ZO-1, cytokeratin and the estrogen receptors alpha and beta. The integrins and the hyaluronan receptor were significantly up-regulated with 17beta-estradiol in human MCF-7 cells. In contrast, genistein and daidzein did not affect the expression at a concentration of 100 micromol/l. In all proliferation experiments with a significant stimulation of the primary human mammary epithelial cell growth due to 17beta-estradiol, in general, genistein and daidzein did not influence S-phase and G2/M-phase cells. Additionally, the stimulative effect of 17beta-estradiol could be inhibited. As the phytoestrogens do not up-regulate adhesion receptors in human breast cells and, regarding proliferation, are able to abolish the stimulatory effect of 17beta-estradiol, we suggest that phytoestrogens could have beneficial effects for the prevention or inhibition of carcinogenesis in hormone-dependent malignancies.     

Brca1 regulates in vitro differentiation of mammary epithelial cells

Murine Brca1 is widely expressed during development in different tissues. Why alterations of BRCA1 lead specifically to breast and ovarian cancer is currently not clarified. Here we show that Brca1 protein expression is upregulated during mammary epithelial differentiation of HC11 cells, during differentiation of C2C12 myoblasts into myotubes and during neuronal differentiation of N1E-115 cells. Ectopic overexpression of BRCA1 and downregulation of endogenous Brca1 expression specifically affect the regulation of mammary epithelial cell differentiation. Accelerated mammary epithelial cell differentiation upon high ectopic BRCA1 expression is not a consequence of the anti-proliferative capacity of this tumor suppressor and independent of functional p53. Overexpression of the BRCA1 variant lacking the large central exon 11 has no effects on mammary epithelial cell differentiation. These data provide new insights into the cellular role of Brca1.     

Osteopontin induces increased invasiveness and plasminogen activator expression of human mammary epithelial cells.

Osteopontin (OPN) has been associated with enhanced malignancy in breast cancer, but its functional role in this disease is poorly understood. To study the effect of OPN on cellular invasiveness, basal OPN expression was first assessed in members of a progression series of human mammary epithelial cell lines (21PT: immortalized, non-tumorigenic; 21NT: weakly tumorigenic; 21MT-1: tumorigenic, weakly metastatic; MDA-MB-435 cells: tumorigenic, highly metastatic). The two lines which expressed lowest basal levels of OPN (21PT, 21NT) were then examined for up-regulation of invasive behavior in response to exogenous or transfected (endogenous) OPN. Both 21PT and 21NT showed increased invasiveness through Matrigel when human recombinant (hr)OPN was added to the lower chamber of transwells. Both also showed a cell migration response to hrOPN. Populations of 21PT and 21NT cells stably transfected with an OPN-expression vector showed higher levels of cell invasiness than control vector transfectants. Examination of transfectants for mRNA of a number of secreted proteases showed that only urokinase-type plasminogen activator (uPA) expression was closely associated with OPN expression and cellular invasiveness. Treatment of the parental 21PT and 21NT cells with exogenous hrOPN resulted in increased uPA mRNA expression and increased urokinase activity of the conditioned media. Both increased cell migration and induction of uPA expression are thus potential mechanisms of increased invasiness of breast epithelial cells in response to OPN.     

Inhibition of tenascin-C expression in mammary epithelial cells by thyroid hormone

Multiple data suggest a relationship between thyroid hormone (triiodothyronine (T3)) and carcinogenesis. Studies on breast cancer have been inconclusive, suggesting contradictory effects of thyroid status and diseases. Recently, we reported that expression of the extracellular matrix glycoprotein tenascin-C is modulated by T3 during rat brain development. Because tenascin-C has been reported to have growth-, motility-, and angiogenic-promoting activities and to become upregulated during tumorigenesis in breast carcinoma and stromal cells, we analyzed the effects of T3 on tenascin-C expression in mammary epithelial cells. In this study, we showed that tenascin-C RNA expression was inhibited by T3 in normal un-transformed EpH4 mouse mammary epithelial cells expressing appropriate receptors. T3's action appeared to be due to a decreased half-life of the tenascin-C mRNA, with a maximum effect (85% at 100 nM) 48 h after addition. T3 also downregulated tenascin-C in the human mammary tumor cell line SKBR-3, which expresses endogenous thyroid receptors. Immunoprecipitation experiments confirmed that tenascin-C protein content was also decreased by T3 in EpH4 cells (70% reduction at 100 nM). Dexamethasone had a similar inhibitory effect (70% at 100 nM), whereas estradiol, the antiestrogen ICI 164,384, progesterone, and all-trans retinoic acid did not alter tenascin-C expression. Our data demonstrate an inhibitory action of T3 on tenascin-C expression in mammary epithelial cells that may play a role in the physiological regulation of this gene and in neoplastic processes.     

乳腺癌细胞与乳腺上皮细胞MicroRNAs表达谱的差异性分析

背景与目的：已有研究表明,MicroRNAs（miRNAs）的异常表达参与了乳腺癌的发生和发展,常起到抑癌基因或癌基因作用。本研究旨在探讨乳腺癌细胞与乳腺上皮细胞miRNAs的差异性表达,并预测异常表达的miRNAs可能调控的靶基因。方法：培养乳腺癌细胞株MCF-7和正常乳腺上皮细胞株HBL-100．分别提取细胞总RNA,并进行质检;利用miRNAs芯片技术,检测乳腺癌细胞和正常乳腺上皮细胞miRNAs的表达,对其表达谱进行差异性分析：采用实时定量RT—PCR进行验证：运用MiRanda、TargetScan、PicTar、DIANA—microT软件预测miRNAs可能调控的靶基因。结果：从MCF-7细胞与HBL-100细胞提取到的总RNA纯度较高,其A260／A280为1．90,A260/A230为2．0;琼脂糖凝胶实验结果显示总RNA完整性好。通过miRNAs表达谱的差异性分析,获得173个与乳腺癌相关的miRNAs,其中113个表达上调,60个表达下调：实时定量PCR检测到mir-18a和mir-195在MCF-7细胞中高表达;对乳腺癌细胞显著异常表达的mir-200b进行信息学分析,共筛选出68个靶基因。结论：获得了乳腺癌细胞与乳腺上皮细胞miRNAs的差异表达谱,其中部分miRNAs可能通过调控其靶基因而参与乳腺癌的发病机制。     

Dihydroartemisinin is cytotoxic to papillomavirus-expressing epithelial cells in vitro and in vivo

Nearly all cervical cancers are etiologically attributable to human papillomavirus (HPV) infection and pharmaceutical treatments targeting HPV-infected cells would be of great medical benefit. Because many neoplastic cells (including cervical cancer cells) overexpress the transferrin receptor to increase their iron uptake, we hypothesized that iron-dependent, antimalarial drugs such as artemisinin might prove useful in treating HPV-infected or transformed cells. We tested three different artemisinin compounds and found that dihydroartemisinin (DHA) and artesunate displayed strong cytotoxic effects on HPV-immortalized and transformed cervical cells in vitro with little effect on normal cervical epithelial cells. DHA-induced cell death involved activation of the mitochondrial caspase pathway with resultant apoptosis. Apoptosis was p53 independent and was not the consequence of drug-induced reductions in viral oncogene expression. Due to its selective cytotoxicity, hydrophobicity, and known ability to penetrate epithelial surfaces, we postulated that DHA might be useful for the topical treatment of mucosal papillomavirus lesions. To test this hypothesis, we applied DHA to the oral mucosa of dogs that had been challenged with the canine oral papillomavirus. Although applied only intermittently, DHA strongly inhibited viral-induced tumor formation. Interestingly, the DHA-treated, tumor-negative dogs developed antibodies against the viral L1 capsid protein, suggesting that DHA had inhibited tumor growth but not early rounds of papillomavirus replication. These findings indicate that DHA and other artemisinin derivatives may be useful for the topical treatment of epithelial papillomavirus lesions, including those that have progressed to the neoplastic state.     

Basal lamina formation by normal and transformed mouse mammary epithelial cells duplicated in vitro

Cells from low-passage (LP) cultures of a mouse mammary epithelial line (NMuMG cells) form a basal lamina when they are cultured on a type I collagen gel substratum. A high-passage (HP) strain of this line maintained the morphologic, serologic, and karyologic properties of the LP cells. For the determination of whether transformation of the NMuMG cells might lead to defects in the basal lamina, cells from LP cultures were compared in vivo and in vitro with cells of HP cultures for tumorigenicity, growth characteristics, and ability to form a lamina. The LP NMuMG cells had a typical epithelial morphology and showed no cytologic evidence of cancer. They formed an ultrastructurally normal continuous basal lamina in vivo when they were injected into athymic nude mice. In contrast, the HP cells were pleomorphic and highly invasive when injected into nude mice where they showed frequent and large basal lamina defects. These cells also accumulated only traces of lamina-like materials when cultured on a collagen gel, indicating that neoplastic transformation had markedly reduced the ability of NMuMG cells to form a basal lamina both in vivo and in vitro. Because the collagen gel culture system duplicated the in vivo situation with regard to basal lamina integrity, the basis for this lack of in vitro basal lamina formation may be physiologically relevant for the mechanism of malignant invasion.     

In vitro differentiation of epithelial cells from cervical neoplasias resembles in vivo lesions.

Cell lines derived from cervical neoplasias, as well as cells from normal cervix and neonatal foreskin were examined in an in vitro culture system (raft system) that allows for stratification and differentiation of epithelial cells at an air-liquid interface. Epithelial cells from human foreskin and ectocervix were observed to differentiate in a manner histologically similar to normal epithelium in vivo as indicated by a single layer of basal cells with a defined mid and upper zone. In contrast, cells expanded from cervical squamous carcinoma explants showed total absence of normal differentiation in the raft system with numerous cells in the upper portion of the stratum exhibiting mitotic figures. Cell cultures derived from low grade cervical intraepithelial neoplasia and condyloma acuminata exhibited partial differentiation in addition to perinuclear clearing, binucleate cells and individual cell keratinization. These studies show that in the raft system, cultured cells derived from tissue biopsies can duplicate many of the histological features observed in cervical neoplasias. In addition, epithelial cells derived from normal fetal ectocervix and electroporated with cloned human papillomavirus (HPV) type 16 DNA exhibited abnormal differentiation patterns similar to those of cervical intraepithelial neoplasia in vivo. This model system will thus be useful for examining the effects of HPV infection on epithelial maturation and for investigating the role of other factors in the progression of cervical malignancies.     

Gap junctional intercellular communication and connexin43 expression in human ovarian surface epithelial cells and ovarian carcinomas in vivo and in vitro.

Gap junctional intercellular communication (GJIC) and the expression of gap junction proteins (connexins) are frequently decreased in neoplastic cells and have been increased by cAMP and retinoids. GJIC and connexin expression were investigated in early passage normal human ovarian surface epithelial (HOSE) cells, human ovarian adenocarcinoma cell lines (CaOV-3, NIH:OVCAR-3, SK-OV-3 and SW626) and surgical specimens of human serous cystadenocarcinomas. We hypothesized that GJIC and connexin expression would be decreased in neoplastic cells and would be increased by cAMP and retinoic acid. Cultured HOSE cells exhibited extensive fluorescent dye-coupling and connexin43 (Cx43) expression; other connexins were not detected. The ovarian adenocarcinoma cell lines had little dye-coupling or connexin expression. Deletions and rearrangements of the Cx43 gene were not detected by Southern blotting in the carcinoma lines. N6, 2'-O-dibutyryladenosine 3',5'-cyclic monophosphate and all-trans-retinoic acid inhibited cell proliferation, but did not enhance GJIC or Cx43 expression. Surface epithelial cells of benign ovaries expressed Cx43, but this expression was barely detectable in ovarian serous cystadenocarcinomas. Thus, normal HOSE cells had extensive GJIC and Cx43 expression whereas ovarian carcinoma cells had less and cAMP and retinoic acid did not change these, although both agents inhibited cell growth.     

Tropomyosins of human mammary epithelial cells: consistent defects of expression in mammary carcinoma cell lines

Suppression of synthesis of specific tropomyosin (TM) isoforms occurs commonly in human, murine, and avian fibroblasts transformed by retroviral oncogenes or other modalities. The resulting deficiency or altered distribution of TMs may predispose the cells to microfilament instability and contribute to expression of the transformed phenotype. In this study we have asked whether defects in TM expression had relevance to human neoplasia, which arises most often in cells of the epithelial lineage rather than in fibroblasts and often is unrelated to demonstrable expression of oncogenes. TMs were characterized in normal primary human mammary epithelial cells (HMEC) and in an immortalized nontumorigenic cell line derived from them. Seven TM isoforms were identified in primary HMEC, two of which may be unique to epithelial cells. Immortalized nontumorigenic HMEC expressed the same array of isoforms. Of six established human breast carcinoma cell lines studied, all failed to express the Mr 39,000 TM isoform and five of six also lacked expression of either the Mr 38,000 or 35,000 isoform. Northern blot analysis with probes specific for the 1.1-kilobase mRNA of fibroblast TM1 detected a mRNA of this size in normal HMEC. This mRNA, which probably encodes the Mr 39,000 TM missing from all the carcinoma lines, was absent from five of the six breast cancer cell lines. These results indicate that abnormalities in TM expression in neoplastic cells are not limited to fibroblasts. The high frequency and consistent nature of such abnormalities among cell lines derived from human breast cancer raises the possibility that such abnormalities in expression of a major cytoskeletal protein may play a role in human neoplasia.     

Difference in the thermotolerance of mouse mammary carcinoma cells in vivo and in vitro

The kinetics of thermotolerance development were compared for cells in the SCK mammary carcinoma of A/J mice, and the same cells cultured in vitro. Tumors in the legs of mice or cells in culture flasks were conditioned with heat in a water bath at 43 degrees C for 30 min and the cells were left in the tumors or the culture flasks for various lengths of time after heat conditioning. The magnitude of thermotolerance was determined by preparing single cells and subjecting them to a test heating in a controlled environment in vitro at 43 degrees C, and the survival of the cells was determined by measuring the colony-forming ability in vitro. Thermotolerance in tumors reached its maximum 12 h after heat conditioning, at which time the survival response was characterized by Do (the duration of heating required for exponential cell inactivation to 1/e) of 116 min. This changed to a Do of 170 min when the cells in tumors were incubated in culture medium of neutral pH during the development of thermotolerance. In contrast, the thermotolerance of cells cultured in vitro was fully developed within 8 h, at which time the Do was 306 min. The kinetics of thermotolerance development, therefore, varied significantly between the cells of the same origin but grown in vivo or in vitro. Killing of tumor cells in vivo caused by the heat-conditioning dose was about three times greater than that for tumor cells in vitro, but the greater damage did not result in a greater development of thermotolerance. The above results indicate that the development of thermotolerance in tumors is suppressed by the influence of intratumor environment.