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R Sager

Publications and source records attributed to R Sager.

At least 37 records · Page 2Linked to original sources
PubMed1998

Down-regulation of laminin-5 in breast carcinoma cells.

BACKGROUND: Laminin-5 (ln-5), a large heterotrimeric glycoprotein consisting of an alpha 3, beta 3, and gamma 2 chain, is a component of epithelial cell basement membranes that functions as a ligand of the alpha 3 beta 1 and alpha 6 beta 4 integrins to regulate cell adhesion, migration, and morphogenesis. The ln-5 chains show tissue-specific patterns of regulation in tumors derived from different tissues. For example, ln-5 is often up-regulated in gliomas, gastric carcinomas, and squamous carcinomas and down-regulated in prostate and basal cell carcinomas. Ln-5 expression patterns may represent useful tumor markers and help to elucidate the role of ln-5 in tumor progression in different tissue types. MATERIALS AND METHODS: We have studied ln-5 expression patterns in the breast. mRNA levels were examined in tumor and normal breast epithelial cell lines, tissue samples, and immunomagnetically sorted primary cultures using differential display, Northern blotting, and hybridization arrays. Protein levels were examined by immunoprecipitation. Gene integrity was assessed by Southern blotting of representative cell types. RESULTS: Ln-5 alpha 3, beta 3, and gamma 2 mRNA expression was found to be markedly down-regulated in a panel of breast tumor cell lines when compared with normal breast epithelial cells. Ln-5 mRNA was expressed at relatively high levels in MCF-10A immortal normal breast epithelial cells, long-term cultures of normal breast cells, and sorted primary cultures of normal breast luminal epithelial and myoepithelial cells. Reduced, but detectable, levels of ln-5 tended to be expressed in cell lines derived from early-stage breast tumors, whereas expression was generally not detected in cell lines derived from later-stage tumors. In breast tumor tissue specimens, expression of ln alpha 3 and beta 3 mRNAs tended to be reduced relative to levels observed in adjacent nontumor tissue, whereas in gamma 2 levels were elevated in specimens with increased amounts of myoepithelial cells. These ln-5 expression changes could not be attributed to large-scale mutations or gene rearrangements. Ln-5 protein levels were found to reflect mRNA levels in representative cell lines. At senescence, a growth state believed to suppress tumorigenesis, expression of all three ln-5 mRNAs was up-regulated. CONCLUSION: The down-regulation of ln-5 mRNA expression in breast tumors cells provides a new molecular marker and suggests that ln-5 functions to control tumor progression in the breast.

Breast↗
PubMed1997

Re-expression of elafin in 21MT2 breast carcinomas by phorbol 12-myristate 13-acetate is mediated by the Ap1 site in the elafin promoter.

elafin is a unique elastase inhibitor. It is differentially expressed at the transcriptional level in human normal mammary epithelial cells and carcinomas. The elafin gene is induced by PMA in 21MT2 breast tumor cells. By deletion analysis and mutagenesis, we have identified the Ap1 site in the promoter as the cis element mediating transcriptional activation of elafin in 70N normal breast cells and its induction by phorbol 12-myristate 13-acetate (PMA) in 21MT2 breast tumors. PMA treatment induces AP1 factor expression, which binds to the Ap1 site of the elafin promoter. Mutation of this Ap1 abolishes the capability of induction by PMA. Furthermore, our data provide a basis for therapeutic manipulation of proteinase inhibitors such as elafin.

Binding Sites↗
PubMed1997

Expression of maspin in prostate cells is regulated by a positive ets element and a negative hormonal responsive element site recognized by androgen receptor.

Prostate cancer is the most common cancer in men. The molecular mechanisms leading to its development are poorly understood. Maspin is a tumor-suppressing serpin expressed in normal breast and prostate epithelium. We have found that expression of maspin in normal and carcinoma-derived prostate epithelial cells is differentially regulated at the transcriptional level. We have identified two different kinds of cis elements, Ets and hormonal responsive element (HRE), in the maspin promoter. The Ets element is active in regulating maspin expression in normal prostate epithelial cells but inactive in tumor cells. The HRE site is a negative element that is active in both cell types. This negative DNA sequence can repress a heterologous promoter recognized by the androgen receptor. We conclude that expression of maspin is under the influence of both a positive Ets and a negative HRE element. Loss of maspin expression during tumor progression apparently results from both the absence of transactivation through the Ets element and the presence of transcription repression through the negative HRE element recognized by androgen receptor.

Base Sequence↗
PubMed1997

Expression genetics in cancer: shifting the focus from DNA to RNA.

Expression genetics is a conceptually different approach to the identification of cancer-related genes than the search for mutations at the genome level. While mutations lie at the heart of cancer, at least in its early stages, what is recognized here are phenotypic changes usually many steps removed from the initiating mutation. Classically cancer geneticists have concentrated on genomic changes and have ignored the productive potential of examining downstream events based on screening for differential gene expression between tumor cells and well matched normal counterparts. Genes involved in cancer affect the normal functions of many cellular processes: not only proliferation but cell-cell and cell-matrix interactions, DNA repair, invasion and motility, angiogenesis, senescence, apoptosis, and others. Yet very few cancer-related genes affecting these processes have been identified in human cancers by classical methods to find mutated genes despite enormous efforts. I report here our success in readily isolating more than 100 candidate tumor suppressor genes from human tissue, estimated to represent roughly 20% of the total genes recoverable by this approach. Half of the genes are unknown and the other half include representatives of most known cancer processes. Because their expression is lost during cancer progression, they may be useful tumor markers for diagnosis and prognosis. Because these genes are not mutated, they provide opportunities for pharmacological intervention by inducing their reexpression.

Breast Neoplasms↗
PubMed1997

Identification, cloning, and characterization of cystatin M, a novel cysteine proteinase inhibitor, down-regulated in breast cancer.

A novel human cystatin gene was identified in a differential display comparison aimed at the isolation of transcriptionally regulated genes involved in invasion and metastasis of breast cancer. Messenger RNAs from primary and metastatic tumor cells isolated from the same patient were compared. A partial cDNA was isolated that was expressed in the primary tumor cell line but not in the metastatic line. The full-length cDNA was cloned and sequenced, and the inferred amino acid sequence was found to encode a novel protein, which we named cystatin M, with 40% homology to human family 2 cystatins and similar overall structure. Cystatin M is expressed by normal mammary cells and a variety of human tissues. The mature cystatin M protein was produced in Escherichia coli as a glutathione S-transferase fusion protein using the pGEX-2T expression system and purified by affinity chromatography. The cystatin M fusion protein displayed inhibitory activity against papain. Native cystatin M protein of approximately 14.5 kDa is secreted and was immunoprecipitated from supernatants of mammary cell cultures using affinity-purified antisera raised against recombinant cystatin M. An N-glycosylated form of cystatin M of 20-22 kDa was co-immunoprecipitated and accounted for about 30-40% of total cystatin M protein. Both forms of native cystatin M also occurred intracellularly. Consistent with the mRNA differential expression, no cystatin M protein was detected in metastatic mammary epithelial tumor cells. Loss of expression of cystatin M is likely associated with the progression of a primary tumor to a metastatic phenotype.

Amino Acid Sequence↗
PubMed1997

Protein transport across the in vitro perfused human placenta.

PROBLEM: Placental transport of various proteins present in human serum, such as immunoglobulins (IgG, IgA), specific anti-tetanus IgG (anti-TT-IgG), and tetanus toxoid-antigen (TT-AG), was investigated. In addition, the transport of IgG modified with biotin (IgG-BT) and 14C-bovine serum albumin (14C-BSA, a permeability marker for macromolecules), was assessed. METHOD OF STUDY: During the perfusion of an isolated cotyledon from human term placenta the perfusate was recirculated on both maternal and fetal sides. After an initial stabilisation phase of 2 hr (control phase), media on both sides were exchanged and perfusion was continued comparing two different conditions (experimental phase). In the first group (control experiments [A, n = 3]), no test proteins were added during the experimental phase (4-6 hr). In the second group (B, n = 5), during the experimental phase (6 hr) the maternal perfusion medium contained IgG (Sandoglobuline, 6-10 g/L), anti-TT-IgG (21-25 mg/L), TT-AG (0.19-0.24 mg/L), and IgA (0.13-0.19 g/L). IgG-BT (2 g/L) and 14C-BSA (30-40 nCi/ml) were added to the medium on the maternal side. IgGs and TT-AG were determined by specific enzyme-linked immunosorbent assay. RESULTS: Both groups showed stable metabolic conditions with constant rates of glucose consumption, lactate production, and hormone (human chorionic gonadotropin, human placental lactogen) release observed throughout the experiment. Washout levels of endogenous IgG and IgA observed in the maternal circuit at the end of the control period were 5 and 1000 times higher than in the fetal circuit. In the experimental phase these levels remained constant at 50-80% of control levels with no change in the last 4 hr of perfusion (group A). In group B, with addition of extra proteins, trace amounts of IgG-BT, IgA, and 14C-BSA were detectable in the fetal circuit within 1 hr, with no significant further increase in circulating levels in the following 4 hr of the perfusion. In contrast, the detection of IgGs in the fetal circuit was delayed by 2 hr; thereafter, a continuous linear increase was observed for all IgGs. TT-AG in fetal perfusate was below the detection limit. TT-AG was found on the fetal side only after ultrafiltration of samples obtained at the end of the experiment. For permeability comparison, the ratio between concentrations on the fetal and maternal side multiplied by 100 ([F:M] x 100), as detected after 6 hr of perfusion, was assessed (n = 5, mean +/- SD). Labelling of IgG with biotin (IgG-BT) reduced its placental transfer by a factor 10 (0.04 +/- 0.01) when compared with the natural IgG (0.49 +/- 0.08) or the specific antibody (anti-TT-IgG). The relative fetal-to-maternal ratio found for TT-AG (0.48 +/- 0.12) was similar to anti-TT-IgG (0.46 +/- 0.11), and approximately 4 and 50 times that of 14C-BSA (0.12 +/- 0.03) and IgA (0.01 +/- 0.01), respectively. Considering that the molecular weights of TT-AG and anti-TT-IgG were at least twice that of BSA and similar to IgA, the difference in transfer suggests a specific mechanism of transport. CONCLUSIONS: Compared with other proteins there is a significantly increased transfer of IgGs across the in vitro perfused human placenta from the maternal to the fetal side, indicating a specific transport mechanism. The similarity in transfer of anti-TT-IgG and tetanus antigen may suggest the transport as antibody-antigen complex.

Antigens, Bacterial↗
PubMed1997

Transactivation through Ets and Ap1 transcription sites determines the expression of the tumor-suppressing gene maspin.

Tumor invasion and metastasis are processes poorly understood at the molecular level. Maspin is a serine protease inhibitor (serpin) with tumor-suppressing function in the mammary gland. Maspin gene expression is decreased with malignancy and is lost in metastatic cells. We show in this report that differential expression of maspin in normal and carcinoma-derived mammary epithelial cells is regulated at the transcriptional level. We have identified the Ets and Ap1 sites in the maspin promoter that are active in regulating maspin expression in normal mammary epithelial cells but inactive in tumor cells. The Ets site alone is sufficient to activate transcription in a heterologous promoter, whereas the Ap1 site cooperates with Ets in activation. The enhancing function by Ets and Ap1 elements is decreased in primary tumor cells (21NT) and is abolished in invasive tumor cells (MDA-231). Thus, loss of maspin expression during tumor progression results at least in part from the absence of transactivation through the Ets and Ap1 sites.

Adaptor Protein Complex 1↗
PubMed1997

mMaspin: the mouse homolog of a human tumor suppressor gene inhibits mammary tumor invasion and motility.

BACKGROUND: The human maspin gene encodes a protein in the serine proteinase inhibitor (serpin) family with tumor-suppressing functions in cell culture and in nude mice. In order to examine the role of maspin in an intact mammal, we cloned and sequenced the cDNA of mouse maspin. The recombinant protein was produced and its activity in cell culture was assessed. MATERIALS AND METHODS: Mouse maspin (mMaspin) was cloned by screening a mouse mammary gland cDNA library with the human maspin cDNA probe. Northern blot analysis was used to examine the expression patterns in mouse tissues, mammary epithelial cells, and carcinomas. Recombinant mMaspin protein was produced in E. coli. Invasion and motility assays were used to assess the biological function of mMaspin. RESULTS: mMaspin is 89% homologous with human maspin at the amino acid level. Like its human homolog, mMaspin is expressed in normal mouse mammary epithelial cells and down-regulated in mouse breast tumor cell lines. The expression is altered at different developmental stages in mammary gland. Addition of the recombinant mMaspin protein to mouse tumor cells was shown to inhibit invasion in a dose-dependent manner. As with the human protein, recombinant mMaspin protein also inhibited mouse mammary tumor motility. Deletion in the putative mMaspin reactive site loop (RSL) region resulted in the loss of its inhibitory functions. CONCLUSIONS: mMaspin is the mouse homolog of a human tumor suppressor gene. The expression of mMaspin is down-regulated in tumor cells and is altered at different developmental stages of mammary gland. mMaspin has inhibitory properties similar to those of human maspin in cell culture, suggesting that the homologous proteins play similar physiological roles in vivo.

Amino Acid Sequence↗
PubMed1997

Maspin. A tumor suppressing serpin.

Maspin, a serpin found in mammary epithelial cells, has been shown to have tumor suppressor activity. The gene is expressed in normal human mammary epithelial cells but down-regulated in invasive breast carcinomas. Similar patterns of expression at the RNA and protein levels are seen by Northern analysis with cells grown in culture and by immunostaining of tissues. Biological assays of invasion by tumor cells through Matrigel membranes and of motility have shown that recombinant maspin inhibits both processes, and that its inhibitory action is totally lost by a single cleavage at the reaction center. Tumor transfectants expressing maspin are inhibited in growth and metastasis in nude mice. Maspin is located in the cell membrane and extracellular matrix, and does not behave as a classical inhibitory serpin against any known target protease. Its mode of action is presently unknown.

Amino Acid Sequence↗
PubMed1996

Maspin acts at the cell membrane to inhibit invasion and motility of mammary and prostatic cancer cells.

Maspin, a novel serine protease inhibitor (serpin), inhibits tumor invasion and metastasis of mammary carcinoma. We show here that recombinant maspin protein blocks the motility of these carcinoma cells in culture over 12 h, as demonstrated by time-lapse video microscopy. Lamellopodia are withdrawn but ruffling continues. Both exogenous recombinant maspin and maspin expressed by tumor transfectants exhibit inhibitory effects on cell motility and cell invasion as shown in modified Boyden chamber assays. In addition, three prostatic cancer cell lines treated with recombinant maspin exhibited similar inhibition of both invasion and motility, suggesting a similar mode of maspin action in these two glandular epithelial cancers. When mammary carcinoma cells were treated with recombinant maspin, the protein was shown by immunostaining to bind specifically to the cell surface, suggesting that maspin activity is membrane associated. When pretreated with antimaspin antibody, maspin loses its inhibitory effects on both invasion and motility. However, when maspin is added to these cells preceding antibody treatment, the activity of maspin is no longer inhibited by subsequent addition of the antibody. It is concluded therefore that the inhibition of invasion and motility by maspin is initially localized to the cell surface.

Antineoplastic Agents↗
PubMed1996

Maspin: a tumor suppressing serpin.

Maspin, a serpin found in mammary epithelial cells, has been shown to have tumor suppressor activity. The gene is expressed in normal human mammary epithelial cells but down-regulated in invasive breast carcinomas. Similar patterns of expression at the RNA and protein levels are seen by Northern analysis with cells grown in culture and by immunostaining of tissues. Biological assays of invasion by tumor cells through matrigel membranes and of motility have shown that recombinant maspin inhibits both processes, and that its inhibitory action is totally lost by a single cleavage at the reaction center. Tumor transfectants expressing maspin are inhibited in growth and invasion in nude mice. Maspin is located in the cell membrane and extracellular matrix, and does not behave as a classical inhibitory serpin against any known target protease. Its mode of action is presently unknown.

Amino Acid Sequence↗
PubMed1996

Evolution of maternofetal transport of immunoglobulins during human pregnancy.

PROBLEM: We determined the evolution of the maternal-fetal transport of immunoglobulins during human pregnancy. METHOD: Paired blood samples were collected between 17-41 weeks of gestation (WG) by puncture of a peripheral maternal vein and by cordocentesis (17-36 WG, n = 91) or directly at delivery (37-41 WG n = 16) from the umbilical vein. Additional maternal samples were collected from the same individual (n = 16) at 10, 20, 30 WG, and at term. The concentration of IgG and its four subclasses and of IgA were determined in the sera using ELISA method. RESULTS: The mean level of IgG and IgA in maternal sera at 9-16 WG was 13.72 +/- 2.53 g/L and 3.95 +/- 1.23 g/L, respectively. Both, IgG and IgA throughout pregnancy decreased to a level of 60-70% (37-41 WG) of the initial concentration in early pregnancy. The ratio of IgG1:IgG2 in the maternal circulation was 2-3 and remained constant throughout pregnancy (17-41 WG). IgG3 and IgG4 levels remained constant and together were less than 10% of total IgG. In the fetal circulation a continuous rise in the level of both IgG and IgA was observed between 17 and 41 WG. Fetal level of IgG at 17-22 WG was only 5-10% of the maternal level and at term exceeded the maternal level reaching a value of 11.98 +/- 2.18 g/L. IgG1 at 17-22 WG was 0.93 +/- 0.42 g/L, which is approximately three times higher than IgG2. IgG1 showed an exponential rise and at 37-41 WG its concentration was seven times higher than IgG2. IgG3 and IgG4 also showed an exponential rise and at term reached a similar level as in the maternal circulation. Striking was the difference in results for IgG2 with a slow linear rise throughout gestation. The fetal IgG2 level at term remained significantly below the maternal concentration. The IgG subclasses when characterized according to the differences in transport capacity gave the following sequence: IgG1 > IgG4 > IgG3 > IgG2. Fetal IgA showed a slow linear rise with fetal levels at term remaining approximately 1,000 times lower than the concentration in the maternal circulation. CONCLUSIONS: Comparison of fetal and maternal levels of immunglobulines indicate that the human placenta during pregnancy develops a specific transport mechanism for IgG. There are differences for the four subclasses with preferential transfer of IgG1 while the slowest transfer is seen for IgG2.

Female↗
PubMed1996

Gap junction genes Cx26 and Cx43 individually suppress the cancer phenotype of human mammary carcinoma cells and restore differentiation potential.

Normal human mammary epithelial cells express hcx43 and hCx26 proteins, which form functional gap junction channels. Both Cx genes are transcriptionally down-regulated in mammary carcinoma cell lines; consequently, no protein is made and gap junctions are absent. This result suggests that the loss of gap junctional communication may play an important role in carcinogenesis. To address this question, two sets of stable transfectants were produced in a recloned line of human mammary carcinoma cells (MDA-MB-435). One set expressed hCx26, and the other expressed hCx43. Studies of transfectants that contain functional gap junctions showed that they grew more slowly in culture than controls, and that their tumor-forming ability was strongly suppressed. In studies designed to examine their differentiation capacity, these transfectants were found to have regained the capacity to form three-dimensional structures in a matrigel matrix. This property is characteristic of normal mammary epithelial cells, but it is lost in the parental tumor cells and neo-transfectant controls. Thus, junctional communication is shown here to play a decisive role in the morphogenesis of mammary gland structures. The hCx26 and hCx43 genes behave as classical tumor suppressor genes both in culture and in animal tests in restoring growth regulatory properties to metastatic mammary carcinoma cells. Expression of these genes further induces the ability to differentiate as shown by the formation of three-dimensional structures when transfected cells are embedded in a matrigel matrix. These findings suggest that the reexpression of gap junctions may play a vital role in normalizing tumor cell behavior.

Breast Neoplasms↗
PubMed1996

A novel protease homolog differentially expressed in breast and ovarian cancer.

BACKGROUND: Using differential display (DD), we discovered a new member of the serine protease family of protein-cleaving enzymes, named protease M. The gene is most closely related by sequence to the kallikreins, to prostate-specific antigen (PSA), and to trypsin. The diagnostic use of PSA in prostate cancer suggested that a related molecule might be a predictor for breast or ovarian cancer. This, in turn, led to studies designed to characterize the protein and to screen for its expression in cancer. MATERIALS AND METHODS: The isolation of protease M by DD, the cloning and sequencing of the cDNA, and the comparison of the predicted protein structure with related proteins are described, as are methods to produce recombinant proteins and polyclonal antibody preparations. Protease M expression was examined in mammary, prostate, and ovarian cancer, as well as normal, cells and tissues. Stable transfectants expressing the protease M gene were produced in mammary carcinoma cells. RESULTS: Protease M was localized by fluorescent in situ hybridization analysis to chromosome 19q13.3, in a region to which other kallikreins and PSA also map. The gene is expressed in the primary mammary carcinoma lines tested but not in the corresponding cell lines of metastatic origin. It is strongly expressed in ovarian cancer tissues and cell lines. The enzyme activity could not be established, because of difficulties in producing sufficient recombinant protein, a common problem with proteases. Transfectants were selected that overexpress the mRNA, but the protein levels remained very low. CONCLUSIONS: Protease M expression (mRNA) may be a useful marker in the detection of primary mammary carcinomas, as well as primary ovarian cancers. Other medical applications are also likely, based on sequence relatedness to trypsin and PSA.

Amino Acid Sequence↗
PubMed1995

The human papilloma virus 16E6 gene sensitizes human mammary epithelial cells to apoptosis induced by DNA damage.

Programmed cell death (apoptosis) is a normal physiological process, which could in principle be manipulated to play an important role in cancer therapy. The key importance of p53 expression in the apoptotic response to DNA-damaging agents has been stressed because mutant or deleted p53 is so common in most kinds of cancer. An important strategy, therefore, is to find ways to induce apoptosis in the absence of wild-type p53. In this paper, we compare apoptosis in normal human mammary epithelial cells, in cells immortalized with human papilloma virus (HPV), and in mammary carcinoma cell lines expressing wild-type p53, mutant p53, or no p53 protein. Apoptosis was induced with mitomycin C (MMC), a DNA cross-linking and damaging agent, or with staurosporine (SSP), a protein kinase inhibitor. The normal and HPV-transfected cells responded more strongly to SSP than did the tumor cells. After exposure to MMC, cells expressing wild-type p53 underwent extensive apoptosis, whereas cells carrying mutated p53 responded weakly. Primary breast cancer cell lines null for p53 protein were resistant to MMC. In contrast, two HPV immortalized cell lines in which p53 protein was destroyed by E6-modulated ubiquitinylation were highly sensitive to apoptosis induced by MMC. Neither p53 mRNA nor protein was induced in the HPV immortalized cells after MMC treatment, although p53 protein was elevated by MMC in cells with wild-type p53. Importantly, MMC induced p21 mRNA but not p21 protein expression in the HPV immortalized cells. Thus, HPV 16E6 can sensitize mammary epithelial cells to MMC-induced apoptosis via a p53- and p21-independent pathway. We propose that the HPV 16E6 protein modulates ubiquitin-mediated degradation not only of p53 but also of p21 and perhaps other proteins involved in apoptosis.

Alkaloids↗
PubMed1995

The tumor suppressor maspin does not undergo the stressed to relaxed transition or inhibit trypsin-like serine proteases. Evidence that maspin is not a protease inhibitory serpin.

The role of tumor suppressor proteins in the development of malignancy has made the understanding of their molecular mechanisms of action of great importance. Maspin is a tumor suppressor produced by a number of cell types of epithelial origin. Exogenous recombinant maspin has been shown to block the growth, motility, and invasiveness of breast tumor cell lines in vitro and in vivo. Although belonging to the the serine proteinase inhibitor (serpin) superfamily of proteins, the molecular mechanism of maspin is currently unknown. Here we show that the reactive site loop of maspin exists in an exposed conformation that does not require activation by cofactors. The reactive site loop of maspin, however, does not act as an inhibitor of proteinases such as chymotrypsin, elastase, plasmin, thrombin, and trypsin but rather as a substrate. Maspin is also unable to inhibit tissue and urokinase type plasminogen activators. Stability studies show that maspin cannot undergo the stressed-relaxed transition typical of proteinase-inhibitory serpins, and the protein is capable of spontaneous polymerization induced by changes in pH. It is likely, therefore, that maspin is structurally more closely related to ovalbumin and angiotensinogen, and its tumor suppressor activity is independent of a latent or intrinsic trypsin-like serine proteinase-inhibitory activity.

Amino Acid Sequence↗