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R Sager

Publications and source records attributed to R Sager.

At least 55 records · Page 3Linked to original sources
PubMed1995

Differential expression of elafin in human normal mammary epithelial cells and carcinomas is regulated at the transcriptional level.

Elafin is an elastase inhibitor with a unique structure, not related to the serpin family, which includes the neutrophil elastase inhibitor. The gene was identified in this laboratory by subtractive hybridization between RNAs from human mammary tumor-derived cells and cDNAs from normal human mammary epithelial cells. Elafin is consistently expressed in normal mammary epithelial cells, but is down-regulated in most breast tumor cell lines. Restriction fragment analysis detected no gross deletions or rearrangement of the gene in any of the tumor cell lines examined. The elafin gene was cloned, and both the cDNA and the promoter region were sequenced. A major positive upstream promoter element was identified by chloramphenicol acetyltransferase assay and deletion analysis, active in normal cell extracts but not in extracts of tumor cells. These results demonstrate that differential expression of elafin in normal mammary epithelial cells and breast tumor cells is regulated at the transcriptional level. Cell synchronization experiments demonstrated that elafin mRNA is down-regulated in S phase in normal cells. These results suggest that elafin may act as an inhibitor of cell cycle progression.

Base Sequence↗
PubMed1995

Enhanced expression of an insulin growth factor-like binding protein (mac25) in senescent human mammary epithelial cells and induced expression with retinoic acid.

mac25, the subject of this report, was selected by the differential display of mRNA method in a search for genes overexpressed in senescent human mammary epithelial cells. mac25 had previously been cloned as a discrete gene, preferentially expressed in normal, leptomeningial cells compared with meningioma tumors. mac25 is another member of the insulin growth factor-binding protein (IGFBP) family. Insulin-like growth factors are potent mitogens for mammary epithelial cells, and the IGFBPs have been shown to modulate this mitogenic activity. We report here that mac25, unlike most IGFBPs, is down-regulated at the transcription level in mammary carcinoma cell lines, suggesting a tumor-suppressor role. The gene was mapped to chromosome 4q12. We found that mac25 accumulates in senescent cells and is up-regulated in normal, growing mammary epithelial cells by all-trans-retinoic acid or the synthetic retinoid fenretinide. These findings suggest that mac25 may be a downstream effector of retinoid chemoprevention in breast epithelial cells and that its tumor-suppressive role may involve a senescence pathway.

Amino Acid Sequence↗
PubMed1995

Transport of immunoglobulin G and its subclasses across the in vitro-perfused human placenta.

OBJECTIVE: The transport of immunoglobulin G and its subclasses 1 to 4 was investigated in the in vitro-perfused isolated cotyledon of the human placenta. STUDY DESIGN: An in vitro system with separate perfusion of the villous capillary system (fetal compartment) and the corresponding intervillous space (maternal compartment) was set up in an isolated cotyledon of human term placenta. After a 2-hour control phase with both compartments perfused in a closed circuit with NCTC-135 tissue culture medium together with Earl's balanced salt solution (2:1), media were exchanged in both circuits and for the experimental phase immunoglobulin G (Sandoglobulin) together with carbon 14-labeled bovine serum albumin (5-10 microCi) was added to the maternal compartment at a concentration of 6 gm/L. During the experimental phase, lasting between 2 and 5 hours, samples were taken from the maternal and fetal compartments every 30 minutes up to 2 hours and every 60 minutes thereafter. RESULTS: During the control phase immunoglobulin G appeared in the maternal perfusate and reached a plateau at 60 to 80 mg/L, whereas the concentration in the fetal perfusate did not exceed 20 mg/L. A similar pattern of release was observed for hemoglobin, suggesting a washout of remains of blood from the intervillous space and the villous vascular compartment. After addition of immunoglobulin G to the maternal circuit during the first 2 hours in three of four experiments, no change in immunoglobulin G concentration was seen in the fetal circuit, and only in the fourth and fifth hours did the fetal concentration increase to 0.6% of the maternal concentration. In contrast, carbon 14-labeled bovine serum albumin was already detectable in the fetal circuit after 1 hour, but the level remained constant at 0.1% of the maternal concentration. Total immunoglobulin G transfer was estimated at 0.5% of the amount added to the maternal circulation, which was five times higher than total transfer of bovine serum albumin. Transfer was shown for all four subclasses. At the end of the experiment the ratio of immunoglobulin G1 to immunoglobulin G2 in the fetal perfusate was significantly higher than in the maternal perfusate (3.8 vs 1.8), suggesting preferential transfer of immunoglobulin G1. CONCLUSION: Transfer of all four immunoglobulin G subclasses of a commercially available immunoglobulin G preparation across the human placenta from the maternal to the fetal side was demonstrated by the dual in vitro perfusion system. There is a preferential transfer for immunoglobulin G1.

Biological Transport↗
PubMed1995

Differential expression of human tissue factor in normal mammary epithelial cells and in carcinomas.

BACKGROUND: Tissue factor (TF) is a glycoprotein which binds factor VIIa. The TF-VIIa complex serves as a potent initiator of the coagulation pathways. TF, an immediate early gene, may also play a role in cell growth. Expression of TF was correlated with some types of cancers. MATERIALS AND METHODS: Normal, immortalized, and tumor human mammary epithelial cells were used in the experiments. The differential display (DD) technique was used to identify genes differentially expressed in the cells. TF expression patterns were examined by Northern blot analysis, immunofluorescence staining of cultured cells, and immunohistochemical staining in human cryostat sections. RESULTS: In a 5-way display, an amplified polymerase chain reaction (PCR) product was found in normal and immortalized human mammary epithelial cells but not in the breast cancer cells. The PCR fragment was cloned and sequenced. The result showed that the fragment was identical to human tissue factor. Northern blot analysis showed that expression level of tissue factor mRNA remained high in growing, quiescent, and senescent normal mammary epithelial cells. Immunofluorescence staining also confirmed tissue factor expression pattern in the cell lines tested. Immunohistochemical staining showed that tissue factor was expressed in the normal luminal and myoepithelial cells of some ducts but not others. No staining was observed in invasive carcinoma cells. However, myoepithelial cell staining was seen in some residual ductal structures in invasive tumors. CONCLUSIONS: This study shows the use of DD to reveal the loss of TF expression pattern in human breast cancer cell lines. Immunohistochemical staining results showed breast carcinoma cells expressed little TF, if any, suggesting that TF is not required for breast tumor cell invasion. The results also indicated that TF expression was independent of the proliferation status of the expressing cells. The expression pattern of TF may be a meaningful marker in the development of breast cancer.

Base Sequence↗
PubMed1994

Production, purification, and characterization of recombinant maspin proteins.

Maspin, a novel mammary serine protease inhibitor, was shown to have tumor suppressing activity (Zou, Z., Anisowicz, A., Hendrix, M. J. C., Thor, A., Neveu, M., Sheng, S., Rafidi, K., Seftor, E., and Sager, R. (1994) Science 263, 526-529). In this paper, we report the production of recombinant glutathione S-transferase-maspin fusion protein, expressed in the bacterium Escherichia coli, and recombinant maspin, expressed in the insect Spodoptera frugiperda cells. The fusion protein was purified by glutathione affinity chromatography. Maspin expressed in insect cells was purified by a combination of Bio-Rad AG1-2X anion exchange chromatography and heparin affinity chromatography. The recombinant maspin from insect cells was cleaved at the putative reactive center, as confirmed by protein sequencing. Both recombinant proteins demonstrated strong inhibitory effects on the invasion by two breast tumor cell lines across reconstituted basement membranes and such inhibitory effect was abolished in the presence of the polyclonal antibody made against the reactive center region of maspin. The trypsin-cleaved recombinant maspin did not inhibit invasion, indicating that the inhibitory activity requires the intact putative reactive center. This paper provides evidence that recombinant maspin protein itself inhibits invasion, and supports the role of maspin as a tumor suppressor.

Amino Acid Sequence↗
PubMed1994

Maspin, a serpin with tumor-suppressing activity in human mammary epithelial cells.

A gene encoding a protein related to the serpin family of protease inhibitors was identified as a candidate tumor suppressor gene that may play a role in human breast cancer. The gene product, called maspin, is expressed in normal mammary epithelial cells but not in most mammary carcinoma cell lines. Transfection of MDA-MB-435 mammary carcinoma cells with the maspin gene did not alter the cells' growth properties in vitro, but reduced the cells' ability to induce tumors and metastasize in nude mice and to invade through a basement membrane matrix in vitro. Analysis of human breast cancer specimens revealed that loss of maspin expression occurred most frequently in advanced cancers. These results support the hypothesis that maspin functions as a tumor suppressor.

Amino Acid Sequence↗
PubMed1994

Lack of transport of erythropoietin across the human placenta as studied by an in vitro perfusion system.

The transfer of human recombinant erythropoietin (rhEPO) from the maternal to the fetal side was investigated using the technique of in vitro perfusion of an isolated cotyledon of human placenta, with recirculation of the perfusate (130 ml) in separate closed maternal and fetal circuits. rhEPO (221-512 U), together with [14C]BSA (bovine serum albumin, 44.8 kBq or 2,688,000 dpm), was added to the maternal circuit only. Despite a considerably lower molecular weight of EPO (mol. wt. = 30,400 Da) compared to BSA (mol. wt. = 69,000 Da), no difference was found in their transfer across the placenta from the maternal to the fetal side, which was very low for both macromolecules. The total transfer of rhEPO derived from the concentration measured in the samples taken from the fetal circuit at the end of 4-5 h of perfusion, was in the range of 0.04% of the amount initially added to the maternal compartment. A similar amount of transfer was determined for [14C]BSA (0.04-0.07%, n = 12). In conclusion, by direct determination in a dually in vitro perfused human placental cotyledon, no significant transfer of rhEPO from the maternal to the fetal side could be shown.

Biological Transport↗
PubMed1994

Maternal-fetal transport of immunoglobulin G and its subclasses during the third trimester of human pregnancy.

PROBLEM: We determined the maternal-fetal transport of immunoglobulin G (IgG) during the third trimester of human pregnancy. METHOD: The concentration of IgG and its subclasses (IgG1-4) was determined in sera of blood samples from 38 pregnancies collected at the time of delivery from a peripheral maternal vein (MV) and from the placental umbilical artery (UA) and vein (UV). Gestational age varied between 28 and 42 weeks (WG). RESULT: Whereas placental weight showed a significant correlation with gestational age, the maternal level of IgG and the ratio of its subclasses did not vary with gestational age. At 28-33 WG (n = 15) the mean values in the UA (5.91 +/- 1.53 g/l) and UV (6.41 +/- 1.57 g/l) for total IgG concentration were lower than in the MV (10.74 +/- 2.55 g/l). At the end of gestation (37-42 WG, n = 12), IgG in both UA (11.21 +/- 1.95 g/l) and UV (12.26 +/- 2.06 g/l) exceeded the maternal concentration (9.69 +/- 1.84 g/l). In addition to the significant positive correlation between IgG concentration in the fetal circulation (UV + UA) and gestational age (28-42 WG), an increase in the positive difference between UV and UA at the end of pregnancy indicates that there is a substantial rise in placental IgG transport capacity. CONCLUSION: All four subclasses IgG1-4 were detectable in the umbilical circulation (28-42 WG). Whereas IgG3 and IgG4 in UA and UV had a similar concentration as in MV and remained unchanged, IgG2 increased with gestation from 0.67/0.74 g/l (UA/UV, 28-33 WG) to 1.29/1.58 g/l (UA/UV, 37-42 WG), but nevertheless remained lower than the level found in the MV (2.65 +/- 1.12 g/l). The main increase in IgG concentration, however, was due to the substantial rise in the transport of IgG1, which increased from 4.37 +/- 1.24 (UA) and 4.94 +/- 1.52 g/l (UV) at 28-33 WG to 8.94 +/- 1.66 (UA) and 10.89 +/- 1.96 g/l (UV) at the end of gestation, which was even higher than the overall IgG concentration in MV.

Enzyme-Linked Immunosorbent Assay↗
PubMed1994

Down-regulation of retinoic acid receptor beta in mammary carcinoma cell lines and its up-regulation in senescing normal mammary epithelial cells.

Retinoids are important cellular, dietary factors that regulate differentiation and cellular growth. They serve as ligands for specific nuclear receptors, the retinoic acid receptors (RARs). Ligand-activated receptors regulate gene transcription through target retinoic acid-responsive elements (RAREs) found in promoter regions. We have investigated the expression of retinoic acid receptor genes (alpha, beta, gamma) and retinoid X receptor beta in normal, senescing, and tumorigenic human mammary epithelial cells. We find that most tumor cells show a loss of RAR-beta expression, but that RAR-alpha and -gamma as well as retinoid X receptor beta are variably expressed in both normal and tumor cells. RAR-beta gene expression is induced both by retinoic acid and by fenretinide in normal cells, but tumor cells fail to respond to either. In contrast, RAR-beta expression increases with serial passage in senescing cells. Paradoxically, both normal and tumor cells can trans-activate an exogenous beta-RARE, as demonstrated by reporter gene assays. Oligonucleotide mobility shift assays with the beta-RARE show a single discrete complex in normal cells, whereas tumor cells exhibit a heterogeneous set of larger complexes, which indicates that tumor cells utilize a different array of factors within the beta-RARE. Reporter gene assays with extended promoter regions indicate the presence of negative regulatory elements and/or factor binding sites that reside between -1500 and the RARE located at -59, and that the promoter is down-regulated in MCF-7 tumor cells. Our findings reveal a dichotomy: RAR-beta transcription is down-regulated in tumor cells compared with normal human mammary epithelial cells, and up-regulated in senescence.

Base Sequence↗
PubMed1993

Estrogen inhibits the growth of estrogen receptor-negative, but not estrogen receptor-positive, human mammary epithelial cells expressing a recombinant estrogen receptor.

Estrogen is essential for the growth of the normal mammary gland and most estrogen receptor (ER)-positive mammary carcinomas. To better understand the differences between the estrogen response pathways in normal and tumor cells, we have stably transfected ER-negative immortal, nontumorigenic human mammary epithelial cells and ER-negative breast cancer cells with an ER-encoding expression vector. Unexpectedly, estrogen treatment (1.0 nM) inhibited the proliferation of ER-transfected nontumorigenic and tumor-derived cells. The control transfectants and parental cells exhibited no response to estrogen concentrations as high as 1.0 microM. This inhibitory effect was attributed to a decreased growth rate and a perturbation of the cell cycle distribution by estrogen treatment of the ER transfectants. The inhibitory response was blocked by cotreatment with the antiestrogen ICI 164,384 as predicted for a pure antagonist of estrogen action. However, treatment with the antiestrogen hydroxytamoxifen caused growth inhibition, implying that hydroxytamoxifen acts as an agonist of estrogen action in ER-transfected cells. Since estrogen is a mitogenic and not a growth-inhibitory stimulus for ER-positive breast cancers and cell lines, we tested the effect of constitutive, high level expression of the ER in ER-positive tumor cells. Stable transfection of ER-positive MCF-7 and T47D cells with the ER expression vector yielded cells with varying amounts of ER. At ER levels comparable to those found in the ER-negative transfected cells, the MCF-7 and T47D ER transfectants were not inhibited by estrogen. These data suggest that ER-positive breast cancer cells can tolerate higher constitutive levels of ER expression than ER-negative cells. The mechanism by which this is accomplished may be an essential step in the process which yields ER-positive tumors.

Animals↗
PubMed1993

Specificity of gap junction communication among human mammary cells and connexin transfectants in culture.

In a previous paper (Lee et al., 1992), it was shown that normal human mammary epithelial cells (NMEC) express two connexin genes, Cx26 and Cx43, whereas neither gene is transcribed in a series of mammary tumor cell lines (TMEC). In this paper it is shown that normal human mammary fibroblasts (NMF) communicate and express Cx43 mRNA and protein. Transfection of either Cx26 or Cx43 genes into a tumor line, 21MT-2, induced the expression of the corresponding mRNAs and proteins as well as communication via gap junctions (GJs), although immunofluorescence demonstrated that the majority of Cx26 and Cx43 proteins present in transfected TMEC was largely cytoplasmic. Immunoblotting demonstrated that NMEC, NMF, and transfected TMEC each displayed a unique pattern of posttranslationally modified forms of Cx43 protein. The role of different connexins in regulating gap junction intercellular communication (GJIC) was examined using a novel two-dye method to assess homologous and heterologous communication quantitatively. The recipient cell population was prestained with a permanent non-toxic lipophilic dye that binds to membranes irreversibly (PKH26, Zynaxis); and the donor population is treated with a GJ-permeable dye Calcein, a derivative of fluorescein diacetate (Molecular Probes). After mixing the two cell populations under conditions promoting GJ formation, cells were analyzed by flow cytometry to determine the percentage of cells containing both dyes. It is shown here that Cx26 and Cx43 transfectants display strong homologous communication, as do NMEC and NMF. Furthermore, NMEC mixed with NMF communicate efficiently, Cx26 transfectants communicate with NMEC but not with NMF, and Cx43 transfectants communicate with NMF. Communication between Cx26 TMEC transfectants and NMEC was asymetrical with preferential movement of calcein from TMEC to NMEC. Despite the presence of Cx43 as well as Cx26 encoded proteins in the GJs of NMEC, few Cx43 transfectants communicated with NMEC. No heterologous GJIC was observed between Cx26- and Cx43-transfected TMEC suggesting that heterotypic GJs do not form or that Cx26/Cx43 channels do not permit dye transfer.

Blotting, Western↗
PubMed1993

Identification by differential display of alpha 6 integrin as a candidate tumor suppressor gene.

A new method of differential expression cloning called differential display (DD) has been used to screen for novel tumor suppressor genes involved in breast cancer. The screen is based on positive selection at the mRNA level for genes expressed in normal mammary epithelial cells but decreased or lost in corresponding tumor cells. A candidate tumor suppressor gene recovered by DD is integrin alpha-6 (alpha 6), a component of the heterodimeric integrin receptors alpha 6 beta 1 and alpha 6 beta 4. Loss of alpha 6 expression was confirmed in total RNAs by Northern blot analysis and by immunostaining with alpha 6 antibodies. Consistent with these cell culture findings, previous immunostaining of mammary tissue sections has identified decreased alpha 6 protein expression during breast tumor progression. Southern blot analysis demonstrated that alpha 6 gene is present in tumor cell lines, suggesting that reexpression may be inducible by pharmacological intervention. The likelihood that alpha 6 may have tumor suppressing activity is supported by growing evidence of a central role for integrins in transducing growth control and differentiation signals from growth factors and the extracellular matrix (ECM).

Base Sequence↗
PubMed1992

Differential display and cloning of messenger RNAs from human breast cancer versus mammary epithelial cells.

Identification of the genes that are specifically expressed in tumor cells but not in normal cells (oncogenes), or vice versa (tumor suppressor genes), is important for understanding the molecular basis of cancer. The differential display technique was applied to compare mRNAs from normal and tumor-derived human mammary epithelial cells, cultured under the same conditions. Complementary DNA fragments corresponding to several apparently differentially expressed mRNAs were recovered and sequenced. They exhibit characteristics of the 3' end of eukaryotic mRNA, as predicted by the method. A complementary DNA fragment seen only in the normal cell was used as a probe to isolate its corresponding complementary DNA clone from a library. Northern analysis confirmed its differential expression. Thus, this method can be used for detecting, cloning, and sequencing of genes that are unique to a host of biological and disease processes.

Base Sequence↗
PubMed1992

Down-regulation of a member of the S100 gene family in mammary carcinoma cells and reexpression by azadeoxycytidine treatment.

A cDNA clone, designated CaN19 (originally called clone 19), isolated by subtractive hybridization, contains sequences that are preferentially expressed in normal mammary epithelial cells but not in breast tumor cells. Comparison of its deduced amino acid sequence with sequences in the GenBank data base revealed similarity with the S100 protein family, a group of small Ca(2+)-binding modulator proteins involved in cell cycle progression and cell differentiation. CaN19 expression is down-regulated in normal cells by A23187, a calcium ionophore, suggesting that its regulation is calcium-dependent. We have assigned CaN19 to human chromosome 1q21-q24, a region containing four other S100-related genes. In contrast to CaN19 mRNA expression, most members of the S100 protein family are activated or overexpressed in tumor cells. Synchronization experiments by growth-factor deprivation demonstrated a biphasic induction of CaN19 expression in normal cells, approximately 2-fold in early G1 phase and another 2- to 3-fold at the G1/S boundary. Exposure of mammary tumor cells to 5-aza-2'-deoxycytidine, an inhibitor of DNA methylation, reactivated the expression of CaN19 mRNA.

Amino Acid Sequence↗
PubMed1992

Preferential chromosome loss in human papilloma virus DNA-immortalized mammary epithelial cells.

Human papilloma virus (HPV) DNA-immortalized human mammary epithelial cells may provide a model system for studying the molecular basis of immortalization and its role in breast neoplasia. Cytogenetic analyses were performed on clones derived from HPV 16- and HPV 18-immortalized human mammary epithelial cells. The majority of the clones contained near-diploid karyotypes. The single most frequent whole-chromosome loss was that of chromosome 19. Regions that showed preference for deletion and/or translocation included 2pter, 11qter, and 15pter. Evidence of chromosome 19 loss was confirmed by polymerase chain reaction (PCR)-generated, chromosome 19-specific, dinucleotide microsatellite repeat polymorphism analysis.

Breast↗
PubMed1992

Tumor suppressor genes in the cell cycle.

In the past year, two tumor suppressor genes, retinoblastoma and p53, have been established as important players in cell-cycle control, mediated by phosphorylation and by stage-specific transcription complexes. Evidence that they also participate in other transcription complexes is accumulating and searches are underway for the downstream genes under their regulation. Genes down-regulated in tumor cells are being screened by subtractive hybridization to bridge the gap between transcription factors and their targets.

Animals↗