Cancer Research
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rybarczyk, B. J.
Right arrow Articles by Simpson-Haidaris, P. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rybarczyk, B. J.
Right arrow Articles by Simpson-Haidaris, P. J.
[Cancer Research 60, 2033-2039, April 1, 2000]
© 2000 American Association for Cancer Research

Tumor Biology

Fibrinogen Assembly, Secretion, and Deposition into Extracellular Matrix by MCF-7 Human Breast Carcinoma Cells1

Brian J. Rybarczyk and Patricia J. Simpson-Haidaris2

Departments of Pathology [B. J. R., P. J. S-H.], Medicine-Vascular Medicine Unit [B. J. R., P. J. S-H.], and Microbiology and Immunology [P. J. S-H.], University of Rochester School of Medicine and Dentistry, Rochester, New York 14642


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A hallmark of breast carcinoma is the deposition of fibrinogen (FBG) without subsequent conversion to fibrin in the tumor stroma. In this study, the ability of the MCF-7 human breast cancer epithelial cell line to synthesize, secrete, and deposit FBG into the extracellular matrix (ECM) was examined. Whereas MCF-7 cells produced low levels of intact FBG, abundant levels of FBG intermediate complexes or degraded A{alpha}, Bß, and {gamma} chain polypeptides were observed. Most of the Bß chain was degraded and missing an NH2-terminal peptide fragment. Reverse transcription-PCR analysis indicated that only {gamma} chain mRNA was present in detectable steady-state levels, although Southern hybridization revealed that the FBG A{alpha}, Bß, and {gamma} chain genes were intact in MCF-7 cells. Immunostaining showed that extracellular FBG was bound to the surface of MCF-7 cells in a punctate pattern, reminiscent of receptor binding, rather than a fibrillar pattern characteristic of mature ECM. A similar punctate pattern of staining was observed when MCF-7 FBG was added to fibroblasts that normally assemble exogenous FBG into an extensive, fibrillar ECM, suggesting that MCF-7 cells are defective in assembly of a fibrillar ECM. The loss of FBG Bß chain NH2-terminal peptides may contribute to the lack of intact FBG assembly in MCF-7 cells, which may further affect its ability to assemble FBG into a fibrillar ECM. Taken together, the data suggest that endogenous synthesis and secretion of FBG is, at least in part, the source of FBG deposition in the ECM of breast cell carcinomas.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The progression of a tumor from benign and localized to invasive and metastatic growth is the major cause of poor clinical outcome in cancer patients. This complex process requires many components for successful dissemination and growth of the tumor cell at secondary sites, including angiogenesis, enhanced ECM3 degradation via tumor and host-secreted proteases, tumor cell migration, and modulation of tumor cell adhesion (1) . Thus, the progression of solid tumors has been likened to "a wound that does not heal." The importance of angiogenesis to tumor growth is now well recognized; the establishment of a tumor beyond 1–2 mm3 requires the formation of new blood vessels from preexisting vessels (2, 3, 4, 5) . Much like in a healing wound, the deposition of FBG and/or fibrin, along with other adhesive glycoproteins, into a provisional matrix serves as a scaffold to support binding of growth factors and the cellular responses of adhesion, proliferation, and migration in angiogenesis and tumor cell growth (1 , 4 , 6, 7, 8) .

FBG is the major adhesive glycoprotein involved in the final stages of blood clotting in support of platelet aggregation and fibrin formation. FBG is synthesized constitutively by liver epithelium; however, upon induction of a systemic inflammatory response, FBG synthesis is up-regulated 2–10-fold. The increased levels of circulating FBG and resulting fibrin serve to restore homeostasis by augmenting the innate immune response to infection, tissue injury, or neoplasia (2 , 4 , 5 , 9, 10, 11) . Recently, we showed that the assembly of soluble FBG into the insoluble ECM of fibroblasts and lung alveolar epithelial cells occurs in the absence of proteolytic processing. This matrix-bound FBG is conformationally altered to expose a "fibrin-specific" epitope; however, the FBG protomer remains intact in that it is not converted to fibrin by thrombin cleavage. Furthermore, the matrix-bound FBG coaligns with mature matrix fibrils of both heparan sulfate proteoglycans and FN (12) .

This previously unrecognized feature of FBG as a component of the ECM becomes important in light of the many studies showing both FBG and fibrin localization to the tumor-host cell interface (3 , 13) . Fibrin is abundant in different types of tumors (2 , 4 , 5 , 9 , 10) , such as primary brain lesions (14) and prostate cancer (15) . However, FBG, not fibrin, deposition is a feature of mesothelioma (16) , colon cancer (17) , lymphoma (18) , and a predominant component in breast tumor stroma (13) . The primary source of this extracellular FBG is thought to be attributable to exudation of plasma from leaky blood vessels and deposition of fibrin(ogen) into the tumor stroma (2 , 4) . In contrast, it has been speculated that the presence of FBG in the connective tissue stroma of breast carcinoma may not be attributable to exudation from plasma because other plasma proteins of comparable or smaller size are not found (19) . Heretofore, endogenous synthesis and secretion of FBG by breast epithelial tumor cells has not been considered the source of FBG in the stroma of breast carcinomas. In this study, we show that MCF-7 human breast cancer cells synthesize and secrete FBG polypeptides, suggesting that the origin of FBG in the stroma of breast carcinoma may be, in part, attributable to endogenous synthesis and deposition in vivo.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Cultures.
All cell lines were obtained from the American Type Culture Collection (Rockville, MD). All culture media were supplemented with 10% fetal bovine serum (Intergen, Purchase, NY), 100 units/ml penicillin, 0.1 mg/ml streptomycin, and 2 mM L-glutamine (Life Technologies, Gaithersburg, MD). MCF-7 human breast adenocarcinoma cells (HTB 22) were grown in Eagle’s MEM (Sigma Chemical Co., St. Louis, MO), supplemented with 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, and 10 µg/ml bovine insulin; HepG2 (HB 8065) human hepatocarcinoma cells were cultured in Eagle’s MEM supplemented with 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, and 15 mM tricine (pH 7.4). HFFs were grown in McCoys 5A medium (Life Technologies; Ref. 12 ).

Metabolic Labeling of Cultured Cells and Immunoprecipitation of FBG.
MCF-7 and HepG2 cells were continuously labeled for 24 h in the presence of 40 µCi/ml [35S]methinonine + [35S]cysteine (Express Labeling; DuPont NEN, Boston, MA). The protease inhibitor aprotinin (Miles, Inc., Kankakee, IL; 100 units/ml) was added to all samples before immunoprecipitation. Intracellular and secreted FBG was immunoprecipitated using purified anti-FBG pAbs (DAKO Corp., Carpinteria, CA) coupled to protein A-Sepharose (Pharmacia Biotech, Piscataway, NJ; Refs. 12 and 20 ). The immunoprecipitated proteins were resolved by SDS-PAGE as described (21) .

Western Blotting.
Western blotting was performed as described (21) . Blots were incubated with the following mAbs: mAb 134B29, anti-FBG A{alpha}566–580 (a kind gift from Dr. Z. Ruggeri; Scripps Research Institute, La Jolla, CA); mAb 18C6, anti-FBG Bß1–21 (Accurate Chemicals, Westbury, NY); mAb D73H, anti-FBG Bß243–305;,zfoot;4>4and mAb J88B, anti-FBG {gamma}63–78 (22) . The blots were then incubated with horseradish peroxidase-conjugated rabbit antimouse IgG (Jackson, West Grove, PA), and signal development was performed using a chemiluminescence detection kit (DuPont NEN). After exposure to film, the blots were stripped according to kit instructions and reprobed with another mAb.

Tunicamycin and N-Glycosidase F Treatment.
To analyze the glycosylation of FBG, MCF-7 and HepG2 cells were pretreated for 4 h ± 10 µg/ml TUN (Calbiochem, San Diego, CA). Next, the cells were incubated in serum-free medium for 15 min, pulsed with 1 mCi/ml [35S]methionine + [35S]cysteine for 30 min, and then chased in complete media with or without 10 µg/ml TUN for 90 min. FBG was immunoprecipitated from culture media and cell lysates and subjected to SDS-PAGE, followed by fluorography. For N-Glycosidase F treatment, MCF-7 cells and HepG2 cells were metabolically labeled with 40 µCi/ml [35S]methionine + [35S]cysteine for 24 h. Media and cell lysates were collected, immunoprecipitated using anti-FBG pAb, heat denatured at 100°C in 1% SDS, and incubated with 0.5 unit/ml N-glycosidase F (Sigma) for 18 h at 37°C (23) .

Northern, RT-PCR, and Southern Analyses.
FBG mRNAs were analyzed by both Northern hybridization and RT-PCR (20) . Total RNA was isolated using TriReagent (Molecular Research Center, Cincinnati, OH), according to the manufacturer’s instructions. Northern blot hybridizations were performed using FBG-specific cDNAs as probes (20) . RT-PCR was performed using primer pairs described previously for human FBG Bß and {gamma} chains and the highly conserved primers for rat ß-actin, which amplify human ß-actin mRNA efficiently (20) . A{alpha} primer pairs were: sense, 5'-CCTACAACCGAGTGTCAGAGGATCTGAG; and antisense, 5'-CAGAGTTCCAGCTTCC AGTACTTCCAGG-3'. Each human FBG primer pair for amplification of mRNA spans an intron. First-strand cDNA synthesis was primed by oligo(dT) using 15 µg of total RNA from HepG2 or MCF-7 cells. Samples were denatured at 94°C for 2 min, annealed at 55°C for 3 min, and extended at 72°C for 3 min for 35 cycles (24) .

Genomic DNA was isolated from MCF-7 and HepG2 cells by SDS- Proteinase K digestion and multiple phenol:chloroform extractions (24) . Twenty µg of each genomic DNA were digested to completion with EcoRI and HindIII. After gel electrophoresis through 0.8% agarose, the DNA was denatured and neutralized and then transferred from the gel by capillary blotting onto Zeta-Probe nylon membrane (Bio-Rad, Richmond, CA). Probes were labeled with [{alpha}-32P]dCTP using a random primer kit (Life Technologies). After heat denaturation, the probe was added to the hybridization solution and incubated for 16 h at 65°C. The blots were then washed, air dried, and placed against film (24) .

Immunofluorescence Staining.
Immunofluorescence staining of HFF and MCF-7 cells cultured on glass coverslips was carried out as described (25) . Purified plasma FBG used in all experiments was depleted of FN by gelatin-Sepharose affinity chromatography. Cells were grown for 10–14 days, washed, overlaid with or without 50 µg/ml purified plasma FBG or Oregon Green (Molecular Probes, Eugene, OR)-labeled purified plasma FBG and incubated for an additional 3 days. The cells were washed, fixed in 3.7% formaldehyde, and stained with pAbs against FBG, followed by rhodamine-conjugated goat antirabbit IgG (Cappel, Durham, NC) or directly visualized by epifluorescence microscopy. Coverslips were mounted inverted onto glass slides with GelMount (Biomeda Corp., Foster City, CA). FBG was detected by indirect immunofluorescence staining. MCF-7 cells were grown on glass coverslips for 10–14 days. The cells were washed, fixed, and stained with pAb or mAb, followed by secondary rhodamine-conjugated goat antirabbit IgG or rhodamine-conjugated goat antimouse IgG (Cappel).

Competitive Inhibition of Nascent FBG Binding to the MCF-7 Cell Surface.
MCF-7 cells were incubated in serum-free medium for 15 min, pulsed with 1 mCi/ml [35S]methionine + [35S]cysteine for 5 min, washed once with PBS, and then chased with and without increasing concentrations of unlabeled purified plasma FBG for 45 min. The 45-min chase allowed ample time for nascent FBG to be synthesized and secreted from MCF-7 cells. After 45 min, the medium was removed, and the cells were washed with PBS and then lysed as described above. The cell lysate for each condition was precipitated in TCA (26) , and the protein concentration was measured by Bradford assay according to the kit instructions (Bio-Rad). The total TCA-precipitable cpm was divided by the µg of total protein. The results were graphed as a function of the amount of competing unlabeled FBG added.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
MCF-7 Cells Produce FBG Polypeptides.
FBG is composed of two sets of three nonidentical polypeptide chains that are products of three separate genes designated A{alpha}, Bß, and {gamma} (27) . Metabolic labeling and immunoprecipitation of FBG from control HepG2 cells showed intact FBG in both media and cell lysates (Fig. 1ACitation , Lanes 1). Under reducing conditions, all three FBG chains were seen in HepG2 cells at their expected molecular weights of Mr 66,000 (A{alpha}), Mr 52,000 (Bß), and Mr 46,000 ({gamma}; Fig. 1BCitation , Lanes 1). Metabolic labeling and immunoprecipitation of FBG from MCF-7 cells showed barely detectable levels of intact FBG either secreted in the medium or retained intracellularly (Fig. 1ACitation , Lanes 2, arrow). Under reducing conditions, only lower molecular weight immunoreactive FBG species were detected, particularly in the intracellular fraction (Fig. 1BCitation , Lanes 2). None of the detectable polypeptide chains immunoprecipitated from MCF-7 cells using the monospecific anti-FBG pAb corresponded in molecular size to the A{alpha}, Bß, and {gamma} chain polypeptides in HepG2 FBG. The numerous bands at and below the Mr 46,000–48,000 band most likely represent FBG degradation products.



View larger version (33K):
[in this window]
[in a new window]
  		Fig. 1. Metabolically labeled MCF-7 cell-derived FBG. HepG2 and MCF-7 cells were metabolically labeled continuously for 24 h. Nascent FBG was immunoprecipitated using purified anti-FBG pAb coupled to protein A-Sepharose, followed by SDS-PAGE under nonreducing conditions (A) and reducing conditions (B) and fluorography. Lanes 1, HepG2; Lanes 2, MCF-7. Arrow, intact FBG at Mr 340,000.

 
To further characterize the FBG immunoreactive species identified above, Western blotting with a panel of mAbs to FBG A{alpha}, Bß, and {gamma} chains was performed to identify the chain composition. Purified plasma FBG (Fig. 2ACitation , Lane 1) and FBG immunoprecipitated from HepG2 conditioned media and cell lysates (Fig. 2ACitation , Lanes 2 and 3) showed the expected FBG immunoreactivity at Mr 340,000 with all mAbs, as well as with FBG-specific bands between Mr ~140,000 and Mr 170,000 from both HepG2 and MCF-7 cells. These results suggest that {alpha}-{gamma} and ß-{gamma} complexes, as well as the half-molecule (A{alpha}-Bß-{gamma}) at Mr 170,000, were present (Fig. 2A)Citation . Only mAb J88B (anti-{gamma}63–78) identified FBG secreted from MCF-7 cells at the expected Mr 340,000 (Fig. 2ACitation , panel {gamma}63–78, Lane 4, arrow), albeit at very low levels compared with the amount identified in the HepG2 cells. The A{alpha}566–580 mAb, specific for the RGD cell-binding domain of the FBG A{alpha} chain (28) , did not recognize the FBG half-molecule species even in HepG2 cells, suggesting that this epitope is inaccessible. Taken together, these results suggest that MCF-7 cells synthesized and secreted only small amounts of intact FBG, while the majority of FBG remained in half-molecule, unassembled, or degraded form.



View larger version (58K):
[in this window]
[in a new window]
  		Fig. 2. Western blot analysis of MCF-7 FBG. HepG2 and MCF-7 cells were grown for 7 days with no change in medium. FBG was immunoprecipitated, resolved by SDS-PAGE under nonreducing (A) and reducing (B) conditions, and then analyzed by Western blot with FBG chain-specific mAb. The mAb and chain specificity (denoted above the panels) were from left to right: mAb 134B29 (A{alpha}566–580); mAb 18C6 (Bß1–21); mAb D73H (Bß243–305); and mAb J88B ({gamma}63–78). The lanes on each gel correspond to: Lanes 1, purified plasma FBG; Lanes 2, HepG2 medium; Lanes 3, HepG2 cell lysate; Lanes 4, MCF-7 medium; and Lanes 5, MCF-7 cell lysate. Arrow, intact FBG at Mr 340,000.

 
Under reducing conditions, purified plasma FBG (Fig. 2BCitation , Lanes 1), as well as FBG in the HepG2 media (Fig. 2BCitation , Lanes 2) and cell lysates (Fig. 2BCitation , Lanes 3) showed the expected immunoreactivity with the FBG-specific mAbs A{alpha}566–580 (134B29), Bß1–21 (18C6), Bß243–305 (D73H), and {gamma}63–78 (J88B) at their appropriate molecular weights. Low levels of intact A{alpha} chain were identified at Mr 66,000 only in the MCF-7 cell lysate (Fig. 2BCitation , panel A{alpha}566–580, Lane 5). Furthermore, in both HepG2 and MCF-7 medium and cell lysate and, to some extent, plasma FBG, lower molecular weight products between Mr 40,000 and Mr 48,000 were identified with mAb 134B29 indicating the presence of FBG RGDS-containing A{alpha} chain degradation products (Fig. 2BCitation , panel A{alpha}566–580, Lanes 1–5). Similarly, mAb J88B reacted with products between Mr 44,000 and Mr 46,000 in plasma FBG and both HepG2 and MCF-7 media and cell lysates, indicative of the FBG {gamma} chain and {gamma} chain degradation products (Fig. 2BCitation , panel {gamma}63–78, Lanes 1–5). Although {gamma}-{gamma} dimers were not detected by mAb J88B in MCF-7 medium and cell lysate, they were identified in plasma FBG and FBG in the secreted fraction of HepG2 cells (Fig. 2BCitation , panel {gamma}63–78, Lanes 1 and 2). FBG Bß chains of the expected Mr 52,000 were recognized by mAb 18C6 in plasma FBG and in FBG from HepG2 media and cell lysates (Fig. 2BCitation , panel Bß1–21, Lanes 1–3). However, only a lower molecular weight product was detected with mAb 18C6 in MCF-7 medium and cell lysate (Fig. 2BCitation , panel Bß1–21, Lanes 4 and 5). These data suggest that this band is a Bß chain degradation product with an intact NH2-terminal region but missing an undetermined portion of the COOH terminus. FBG Bß chain-specific mAb D73H showed significant amounts of immunoreactive products between Mr 46,000 and Mr 48,000 (Fig. 2BCitation , panel Bß243–305, Lanes 1–5) that were not reactive with mAb 18C6 in the MCF-7 cell lysate (Fig. 2BCitation , panel Bß1–21, Lanes 4 and 5), suggesting that at least Bß1–21 is missing from the majority of the Bß chain produced by MCF-7 cells.

MCF-7 FBG Is Not Glycosylated.
Plasma FBG is N-glycosylated at Asp364 on the Bß chain and Asp52 on the {gamma} chain (29) . Inhibition of N-linked glycosylation in HepG2 cells using TUN resulted in the expected shift to a lower molecular weight of the FBG Bß and {gamma} chains but not the A{alpha} chain (Fig. 3)Citation . Treatment of MCF-7 cells with TUN did not produce any shift in the molecular weight because the FBG polypeptides isolated from the TUN-treated samples were of the same molecular weight as the untreated MCF-7 cells (Fig. 3)Citation . The absence of carbohydrate side chain addition on MCF-7 FBG was confirmed by peptide:N-glycosidase F treatment. N-Linked carbohydrate side chains were cleaved from mature Bß and {gamma} chains of HepG2 and plasma FBG but not from MCF-7 FBG (not shown). These data indicate that the MCF-7 cells derived FBG Bß and {gamma} chain polypeptides were not glycosylated, suggesting that appropriate posttranslational processing of FBG through the Golgi apparatus does not occur in these breast carcinoma epithelial cells.



View larger version (64K):
[in this window]
[in a new window]
  		Fig. 3. N-linked glycosylation pattern of MCF-7 FBG. MCF-7 and HepG2 cells were preincubated with TUN, serum starved for 15 min, pulsed with 1 mCi/ml [35S]methionine + [35S]cysteine for 30 min, and chased in cold, complete media with or without 10 µg/ml TUN for 90 min. The resulting media and cell lysates were collected and immunoprecipitated with anti-FBG pAb and then subjected to SDS-PAGE and fluorography.

 
MCF-7 Cell FBG Gene Expression.
Northern blot analysis did not show steady-state levels of any of the FBG mRNAs in MCF-7 cells (not shown); therefore, the more sensitive technique of RT-PCR was used. Products of the expected sizes for FBG A{alpha}, Bß, and {gamma} chain mRNAs were amplified from total RNA isolated from HepG2 cells (Fig. 4A)Citation . However, only FBG {gamma} chain mRNA could be reproducibly amplified by RT-PCR from MCF-7 cell total RNA. No products were seen for FBG A{alpha} and Bß chains, indicating that a steady-state level of these mRNAs does not accumulate in MCF-7 cells.



View larger version (63K):
[in this window]
[in a new window]
  		Fig. 4. RT-PCR analysis of FBG mRNAs and Southern analysis of genomic DNA from MCF-7 cells. Total RNA was reverse transcribed and amplified by PCR. The amplified products were resolved by electrophoresis in TAE (Tris-acetate/EDTA) buffer on 1.8% agarose gels. The amplified products and expected size are shown in A: Lanes 1, A{alpha}, 634 bp; Lanes 2, Bß, 300 bp; Lanes 3, {gamma}, 182 bp; and Lanes 4, ß-actin, 450 bp. Left, molecular size markers in 100-bp increments. B, genomic DNA from HepG2 and MCF-7 cells was digested with EcoRI (Lanes 1) and HindIII (Lanes 2) and analyzed by Southern hybridization using 32P-labeled cDNA probes for FBG A{alpha} and Bß chains. Left, molecular size markers in kb.

 
The inability to detect steady-state populations of A{alpha} and Bß chain mRNAs suggested that either the mRNAs are unstable or that the A{alpha} and Bß chain genes may be defective. To investigate whether there were gross rearrangements or deletions of the FBG A{alpha} and Bß chain genes in the MCF-7 cells, Southern blot analysis was performed. Comparison of restriction digests of HepG2 and MCF-7 genomic DNAs probed with 32P-labeled FBG A{alpha} and Bß cDNAs showed that identical digestion patterns for both EcoRI and HindIII were found for HepG2 and MCF-7 genomic DNA (Fig. 4B)Citation . These results suggest that the FBG genes are intact in MCF-7 cells. Together, these data indicated that rapid degradation of the FBG A{alpha} and Bß chain mRNAs and polypeptides likely contributes to the defective assembly of FBG in MCF-7 cells.

Extracellular Localization of MCF-7 FBG.
Indirect immunofluorescence staining using pAb anti-FBG and mAbs for FBG A{alpha}566–580 (134B29), Bß1–21 (18C6), and {gamma}63–78 (J88B) chains confirmed positive extracellular staining for all three FBG chains (Fig. 5)Citation . These results suggest that FBG synthesized and secreted by MCF-7 cells binds to the cell surface. To determine whether this cell surface binding was FBG specific and saturable, the binding of metabolically labeled nascent secreted FBG to the MCF-7 cells was performed in the presence of increasing concentrations of unlabeled, purified plasma FBG. The data are presented as the amount of TCA-precipitable cpm/µg of total protein as a function of increasing concentrations of cold competitor FBG in the chase medium. These results show that binding of nascent FBG to the MCF-7 cell surface was inhibited in a dose-dependent and saturable manner with exogenously added FBG, suggesting a receptor-mediated binding event (Fig. 6)Citation .



View larger version (172K):
[in this window]
[in a new window]
  		Fig. 5. Indirect immunofluorescent staining of endogenous FBG. MCF-7 cells were grown on glass coverslips for 14 days. The cells were washed extensively, fixed, and stained with anti-FBG pAb or FBG-specific mAb, followed by rhodamine-conjugated goat antirabbit or goat antimouse secondary antibodies, respectively. A, {alpha}-RGD; B, 18C6; C, J88B; D, anti-FBG pAb. Inset in B, staining with rabbit antimouse polyvalent immunoglobulin alone. Inset in D, staining with goat antirabbit IgG alone; bar, 25 µm.

 


View larger version (13K):
[in this window]
[in a new window]
  		Fig. 6. Cell surface FBG competition for endogenous protein from MCF-7 cells. MCF-7 cells were serum starved, pulsed for 5 min, and chased for 45 min in complete medium in the presence or absence of increasing concentrations of exogenous unlabeled FBG. The cells were washed and disrupted in lysing buffer. Cell lysates were precipitated in TCA. The total TCA-precipitable counts/µg of total cellular protein were graphed as a function of increasing cold, competitive FBG concentration. Bars, SE (n = 3).

 
Defective Assembly of MCF-7 FBG in the Extracellular Matrix of Fibroblasts.
To determine whether FBG produced by MCF-7 cells could be incorporated into a preformed, heterologous ECM, 7-day-old MCF-7 cell conditioned medium was overlaid onto HFF cells, followed by indirect immunofluorescent staining using anti-FBG pAb. In the absence of MCF-7 conditioned medium, there was no detectable immunostaining with anti-FBG pAb (Fig. 7A)Citation . In contrast, in the presence of MCF-7 conditioned medium, the results showed a punctate pattern of positive staining for FBG on the surface of the HFF (Fig. 7B)Citation . These results indicated that MCF-7 FBG bound to the cell surface but did not assemble into the ECM of HFFs in the fibrillar pattern indicative of mature matrix, whereas plasma FBG assembled into mature matrix fibrils in the ECM of HFFs, as shown previously (12) and in Fig. 8ACitation .



View larger version (93K):
[in this window]
[in a new window]
  		Fig. 7. Conditioned MCF-7 medium overlay onto HFF cells. HFF cells were grown on glass coverslips for 14 days to establish a mature ECM. The cells were washed, and MCF-7 conditioned medium was overlaid onto HFF cells and incubated for an additional 3 days. The cells were stained under nonpermeabilized conditions with pAb anti-FBG antibodies, followed by rhodamine conjugated-goat antirabbit secondary antibodies and visualized by fluorescence microscopy. A, HFF cells stained with anti-FBG pAb only. B, HFF cells with MCF-7 conditioned medium overlay and stained with anti-FBG pAb. Insets, secondary antibody staining only; bar, 15 µm.

 


View larger version (92K):
[in this window]
[in a new window]
  		Fig. 8. Assembly of plasma FBG into ECM of HFF and MCF-7 cells. HFF (A) and MCF-7 (B) cells were grown on glass coverslips for 10 days. Purified plasma FBG labeled with Oregon Green was added to cells and incubated for 24 h. The cells were washed extensively, fixed, mounted, and directly visualized for Oregon Green-FBG by epifluorescence microscopy. Bar in B, 15 µm.

 
Defective Assembly of Plasma FBG into MCF-7 ECM.
Because plasma FBG assembles into the ECM of heterologous cells such as lung epithelial cells and fibroblasts (12) , we wanted to determine whether exogenous plasma FBG incorporates into breast epithelial cell ECM. Medium containing Oregon Green labeled-purified plasma FBG was overlaid onto MCF-7 cells. As a control, HFFs were also overlaid with the FBG-Oregon Green. The results showed the expected fibrillar pattern of plasma FBG in the ECM of HFFs (Fig. 8A)Citation . In contrast, FBG-Oregon Green appeared in a punctate pattern of staining (Fig. 8B)Citation , indicative of FBG binding to the MCF-7 cell surface. Together, these results indicate a defect in the synthesis and processing of endogenous FBG by MCF-7 cells, as well as a defect in the ability of MCF-7 breast carcinoma cells to assemble exogenously added plasma FBG into mature matrix fibrils.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The origin of the fibrin(ogen) and corresponding degradation products associated with tumors is usually thought to be attributable to exudation of plasma because of the increased vascular permeability and subsequent procoagulant or fibrinolytic activity at the tumor site (6 , 13 , 30, 31, 32, 33, 34, 35, 36) . Because FBG deposition in the stroma, but not subsequent fibrin formation, is considered a hallmark of breast carcinoma, we investigated whether the MCF-7 human breast cancer cell line is capable of synthesizing and secreting FBG in vitro. In further support of this line of investigation, it has been shown that FBG is synthesized and secreted from established extrahepatic epithelial cell lines including human uterine cervical (37) , intestinal (38) , and alveolar (20) carcinoma cells. These extrahepatic epithelial cells synthesize FBG composed of A{alpha}, Bß, and {gamma} chain polypeptides with the characteristic Mr 340,000. Hepatocytes secrete mainly intact FBG; although, occasionally, very small amounts of intermediate chain complexes, unassembled fragments, or individual chains are observed (39) . However, bovine granulosa cells appear to synthesize and secrete only ß and {gamma} chains of FBG (40) . Furthermore, studies of COS cells transfected with FBG cDNAs show that FBG assembly is not required for its secretion and that intermediates such as {alpha}-{gamma} complexes are secreted along with fully assembled FBG (41) .

We show in this report that the intracellular assembly of intact, dimeric FBG is limited in MCF-7 cells. Analysis of the chain composition indicates that most of the FBG secreted by MCF-7 cells is intact {gamma} chain with partially degraded A{alpha} and Bß chains. The absence of intact Bß chain likely hinders the formation of fully assembled FBG, because these NH2-terminal regions are known to be important in disulfide bond formation between two FBG half-molecules (42, 43, 44) . Thus, abundant FBG fragments with limited amounts of intact FBG are secreted from MCF-7 breast carcinoma cells. FBG secreted from MCF-7 cells binds to the cell surface instead of assembling into mature ECM fibrils. Together, these data support the hypothesis that breast carcinoma cell FBG and FBG degradation products may contribute to the pathophysiology of breast cancer progression by providing additional adhesive cell binding domains or chemotactic peptides to modulate breast cancer cell responses to the ECM microenvironment.

We investigated the expression of the FBG transcripts in MCF-7 cells by both Northern hybridization and RT-PCR. The FBG A{alpha} and Bß chain mRNAs were undetectable by both procedures, suggesting that these mRNA species are either expressed at low levels or that they are very unstable and do not reach steady-state levels in MCF-7 breast epithelial cells. One possibility for limited Bß chain production may involve mRNA instability. Instability of mRNA is mediated primarily by the AU-rich region in the 3' untranslated region in many genes such as tumor necrosis factor-{alpha} (45) , granulocyte/macrophage-colony stimulating factor (46) , and interleukin 3 (47 , 48) . The Bß chain gene contains a degradation signal in the 3' untranslated region that is homologous to the nonamer described as being the key AU-rich sequence mediating mRNA degradation (49 , 50) . The presence of this conserved AU-rich motif may lead to rapid degradation of the Bß chain mRNA in the MCF-7 cells by RNases not present in HepG2 cells. Together, the data indicate that the low levels of the FBG A{alpha} and Bß chain mRNAs and the rapid degradation of the polypeptides likely contribute to the defective intracellular assembly of FBG into the intact Mr 340,000 molecule in MCF-7 cells.

We have shown that nascent FBG assembles into the ECM of the alveolar epithelial cells as fibrils indicative of mature matrix (12) . Therefore, we wanted to determine whether nascent secreted FBG assembled into the ECM of MCF-7 cells. Indirect immunofluorescence staining using mAbs for each of the chains confirmed that the A{alpha}, Bß, and {gamma} chains of nascent FBG, or their fragments, bound to the cell surface of MCF-7 cells but did not assemble into mature matrix fibrils. This cell surface binding is likely receptor mediated, because the binding of nascent FBG to the MCF-7 cells was competitively inhibited with unlabeled, purified plasma FBG. The majority of MCF-7 cell-derived FBG is missing the NH2-terminal domain containing Bß1–21. The loss of Bß1–21 also results in disruption of the heparin binding domain defined by Bß1–42 (51) , which may participate in FBG incorporation into a fibrillar ECM (52) . Thus, the loss of Bß1–21 sequences in MCF-7 FBG may contribute to its inability to assemble into mature ECM through heparan sulfate proteoglycan-dependent mechanisms.

Both alveolar epithelial cell FBG and plasma FBG assemble as mature matrix fibrils into the ECM of heterologous cell types such as HFFs (12) . However, the extracellular association of MCF-7 FBG by heterologous cells was illustrated by a punctate pattern of FBG staining; no fibril formation was observed. Because HFFs do not synthesize FBG endogenously, the positive staining was attributable to extracellular binding of FBG from the MCF-7 conditioned medium overlay. Although purified plasma FBG showed the characteristic fibrillar pattern of mature matrix in the ECM of HFFs, the punctate pattern of staining observed with plasma FBG on the MCF-7 cell surface indicates that MCF-7 cells are capable of binding intact plasma FBG but not assembling plasma FBG into a fibrillar matrix. These results suggest that although the MCF-7 cells are capable of binding FBG, they lack the additional cell surface and/or matrix constituents that support the assembly of FBG into mature matrix fibrils. Preliminary studies in our laboratory indicate that FBG assembly into mature matrix fibrils requires cell surface integrin receptors, matrix heparan sulfate proteoglycans, and active matrix assembly of FN.5

Knowing that the functions of FBG are important during wound healing, its role can be extrapolated and expanded to other pathological processes including tumorigenesis. The novel finding that breast tumor cells synthesize and secrete FBG that becomes extracellularly associated suggests that extrahepatic cell-derived FBG versus plasma FBG may have specialized and unique functions in the context of various disease processes including breast cancer. Endogenous production of ECM proteins such as FBG and deposition into the matrix or bound to the cell surface may promote immediate cellular responses affecting various autocrine and/or paracrine mechanisms. The binding of melanoma cells to FBG promotes cell spreading (53) , whereas proteolytic cleavage fragments of other ECM proteins, such as osteopontin (54) and laminin-5 (55) , promote migration of breast adenocarcinoma cells involving RGD-integrin receptor-mediated interactions. Fibrin(ogen) contains numerous functional cell binding domains including RGD sequences (56) and inflammatory cell-binding regions (57 , 58) , demonstrating its importance as a cell-binding molecule. The positive Western blot and immunofluorescent staining of MCF-7 cells using mAb 134B29 (anti-A{alpha}566–580) showed the extracellular presence of FBG fragments containing the RGD cell-binding domain that engages integrin cell surface receptors. Thus, FBG can be thought of as a scaffold on which cells attach and detach for tumor cell anchorage, growth, migration, angiogenesis, and metastasis (19 , 59 , 60) . Furthermore, FBG fragments function as chemotactic factors for inflammatory cell infiltration (61) ; the Bß1–42 fragment generated by plasmin cleavage serves as a neutrophil chemoattractant (62) . Immunostaining of MCF-7 cells with mAb 18C6 showed the presence of Bß1–21 containing fragments of FBG bound to the surface of the cells. Thus, NH2-terminal Bß chain peptides may act as chemoattractants in the processes of breast tumor cell-inflammatory cell interactions. In summary, the aberrant synthesis, secretion, and degradation of FBG, and its inability to assemble into a mature, fibrillar ECM by MCF-7 breast carcinoma cells, may signify the production of a specialized ECM molecule that, in turn, affects cellular processes of adhesion, proliferation, migration, and angiogenesis that modulate the progression of breast cancer.


   ACKNOWLEDGMENTS
 
We thank Dr. C. G. Haidaris for critical reading of the manuscript.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by USPHS Grants HL50615 and HL30616 from the National Heart, Lung, and Blood Institute of the NIH. Back

2 To whom requests for reprints should be addressed, at Department of Medicine-Vascular, P. O. Box 610, University of Rochester, 601 Elmwood Avenue, Rochester, NY 14642. Phone: (716) 275-8267; Fax: (716) 473-4314; E-mail: pj_simpsonhaidaris{at}urmc.rochester.edu Back

3 The abbreviations used are: ECM, extracellular matrix; FBG, fibrinogen; FN, fibronectin; HFF, human foreskin fibroblast; mAb, monoclonal antibody; pAb, polyclonal antibody; RT-PCR, reverse transcription-PCR; TCA, trichloroacetic acid; TUN, tunicamycin. Back

4 P. J. Simpson-Haidaris, unpublished data. Back

5 P. J. Simpson-Haidaris, unpublished data. Back

Received 10/ 5/99. Accepted 1/31/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Zetter B. R. Angiogenesis and tumor metastasis. Annu. Rev. Med., 49: 407-424, 1998.[Medline]
  2. Dvorak H. F., Senger D. R., Dvorak A. M. Fibrin as a component of the tumor stroma: origins and biological significance. Cancer Metastasis Rev., 2: 41-73, 1983.[Medline]
  3. Brown L. F., Van de Water L., Harvey V. S., Dvorak H. F. Fibrinogen influx and accumulation of cross-linked fibrin in healing wounds and in tumor stroma. Am. J. Pathol., 130: 455-465, 1988.[Abstract]
  4. Nagy J. A., Brown L. F., Senger D. R., Lanir N., Van de Water L., Dvorak A. M., Dvorak H. F. Pathogenesis of tumor stroma generation: a critical role for leaky blood vessels and fibrin deposition. Biochim. Biophys. Acta, 948: 305-326, 1988.
  5. Dvorak H. F., Nagy J. A., Berse B., Brown L. F., Yeo K. T., Yeo T. K., Dvorak A. M., van de Water L., Sioussat T. M., Senger D. R. Vascular permeability factor, fibrin, and the pathogenesis of tumor stroma formation. Ann. NY Acad. Sci., 667: 101-111, 1992.[Medline]
  6. Price J. T., Bonovich M. T., Kohn E. C. The biochemistry of cancer dissemination. Crit. Rev. Biochem. Mol. Biol., 32: 175-253, 1997.[Medline]
  7. Zetter B. R. Cell motility in angiogenesis and tumor metastasis. Cancer Investig., 8: 669-671, 1990.[Medline]
  8. Bach T. L., Barsigian C., Chalupowicz D. G., Busler D., Yaen C. H., Grant D. S., Martinez J. VE-Cadherin mediates endothelial cell capillary tube formation in fibrin and collagen gels. Exp. Cell Res., 238: 324-334, 1998.[Medline]
  9. Dvorak H. F., Harvey V. S., Estrella P., Brown L. F., McDonagh J., Dvorak A. M. Fibrin containing gels induce angiogenesis. Implications for tumor stroma generation and wound healing. Lab. Investig., 57: 673-686, 1987.[Medline]
  10. Clark R. A., Lanigan J. M., DellaPelle P., Manseau E., Dvorak H. F., Colvin R. B. Fibronectin and fibrin provide a provisional matrix for epidermal cell migration during wound reepithelialization. J. Investig. Dermatol., 79: 264-269, 1982.[Medline]
  11. Clark, R. A. F. Wound repair. In: R. A. F. Clark (ed.), The Molecular and Cellular Biology of Wound Repair, Ed. 2 , p. 617. New York: Plenum Publishing Corp., 1996.
  12. Guadiz G., Sporn L. A., Simpson-Haidaris P. J. Thrombin cleavage-independent deposition of fibrinogen in extracellular matrices. Blood, 90: 2644-2653, 1997.[Abstract/Free Full Text]
  13. Costantini V., Zacharski L. R., Memoli V. A., Kisiel W., Kudryk B. J., Rousseau S. M. Fibrinogen deposition without thrombin generation in primary human breast cancer tissue. Cancer Res., 51: 349-353, 1991.[Abstract/Free Full Text]
  14. Bardos H., Molnar P., Csecsei G., Adany R. Fibrin deposition in primary and metastatic human brain tumours. Blood Coagul. Fibrinolysis, 7: 536-548, 1996.[Medline]
  15. Wojtukiewicz M. Z., Zacharski L. R., Memoli V. A., Kisiel W., Kudryk B. J., Moritz T. E., Rousseau S. M., Stump D. C. Fibrin formation on vessel walls in hyperplastic and malignant prostate tissue. Cancer (Phila.), 67: 1377-1383, 1991.[Medline]
  16. Wojtukiewicz M. Z., Zacharski L. R., Memoli V. A., Kisiel W., Kudryk B. J., Rousseau S. M., Stump D. C. Absence of components of coagulation and fibrinolysis pathways in situ in mesothelioma. Thromb. Res., 55: 279-284, 1989.[Medline]
  17. Wojtukiewicz M. Z., Zacharski L. R., Memoli V. A., Kisiel W., Kudryk B. J., Rousseau S. M., Stump D. C. Indirect activation of blood coagulation in colon cancer. Thromb. Haemostasis, 62: 1062-1066, 1989.[Medline]
  18. Costantini V., Zacharski L. R., Memoli V. A., Kisiel W., Kudryk B. J., Rousseau S. M., Stump D. C. Fibrinogen deposition and macrophage-associated fibrin formation in malignant and nonmalignant lymphoid tissue. J. Lab. Clin. Med., 119: 124-131, 1992.[Medline]
  19. Costantini V., Zacharski L. R. The role of fibrin in tumor metastasis. Cancer Metastasis Rev., 11: 283-290, 1992.[Medline]
  20. Simpson-Haidaris P. J. Induction of fibrinogen biosynthesis and secretion from cultured pulmonary epithelial cells. Blood, 89: 873-882, 1997.[Abstract/Free Full Text]
  21. Lawrence S. O., Wright T. W., Francis C. W., Fay P. J., Haidaris P. J. Purification and functional characterization of homodimeric {gamma}B-{gamma}B fibrinogen from rat plasma. Blood, 82: 2406-2413, 1993.[Abstract/Free Full Text]
  22. Odrljin T. M., Rybarczyk B. J., Francis C. W., Lawrence S. O., Hamaguchi M., Simpson-Haidaris P. J. Calcium modulates plasmin cleavage of the fibrinogen D fragment {gamma} chain N-terminus: mapping of monoclonal antibody J88B to a plasmin sensitive domain of the {gamma} chain. Biochim. Biophys. Acta, 1298: 69-77, 1996.[Medline]
  23. Tarentino A. L., Gomez C. M., Plummer T. H., Jr. Deglycosylation of asparagine-linked glycans by peptide: N-glycosidase F. Biochemistry, 24: 4665-4671, 1985.[Medline]
  24. Haidaris P. J., Wright T. W., Gigliotti F., Haidaris C. G. Expression and characterization of a cDNA clone encoding an immunodominant surface glycoprotein of Pneumocystis carinii. J. Infect. Dis., 166: 1113-1123, 1992.[Medline]
  25. Haidaris P. J., Francis C. W., Sporn L. A., Arvan D. S., Collichio F. A., Marder V. J. Megakaryocyte and hepatocyte origins of human fibrinogen biosynthesis exhibit hepatocyte-specific expression of {gamma} chain-variant polypeptides. Blood, 74: 743-750, 1989.[Abstract/Free Full Text]
  26. Guadiz G., Sporn L. A., Goss R. A., Lawrence S. O., Marder V. J., Simpson-Haidaris P. J. Polarized secretion of fibrinogen by lung epithelial cells. Am. J. Respir. Cell Mol. Biol., 17: 60-69, 1997.[Abstract/Free Full Text]
  27. Doolittle R. F. Fibrinogen and fibrin. Annu. Rev. Biochem., 53: 195-229, 1984.[Medline]
  28. Cheresh D. A., Berliner S. A., Vicente V., Ruggeri Z. M. Recognition of distinct adhesive sites on fibrinogen by related integrins on platelets and endothelial cells. Cell, 58: 945-953, 1989.[Medline]
  29. Hantgan, R. R., Simpson-Haidaris, P. J., Francis, C. W., and Marder, V. J. Fibrinogen structure and physiology. In: R. W. Colman, J. Hirsh, V. J. Marder, and E. W. Salzman (eds.), Haemostasis and Thrombosis: Basic Principles and Clinical Practice, Ed. 4. Philadelphia: J. B. Lippincott Co., in press, 2000.
  30. Dvorak H. F., Detmar M., Claffey K. P., Nagy J. A., van de Water L., Senger D. R. Vascular permeability factor/vascular endothelial growth factor: an important mediator of angiogenesis in malignancy and inflammation. Int. Arch. Allergy Immunol., 107: 233-235, 1995.[Medline]
  31. Brown L. F., Berse B., Jackman R. W., Tognazzi K., Guidi A. J., Dvorak H. F., Senger D. R., Connolly J. L., Schnitt S. J. Expression of vascular permeability factor (vascular endothelial growth factor) and its receptors in breast cancer. Hum. Pathol., 26: 86-91, 1995.[Medline]
  32. Costantini V., Sidoni A., Deveglia R., Cazzato O. A., Bellezza G., Ferri I., Bucciarelli E., Nenci G. G. Combined overexpression of urokinase, urokinase receptor, and plasminogen activator inhibitor-1 is associated with breast cancer progression: an immunohistochemical comparison of normal, benign, and malignant breast tissues. Cancer (Phila.), 77: 1079-1088, 1996.[Medline]
  33. Costantini V., Zacharski L. R. Fibrin and cancer. Thromb. Haemostasis, 69: 406-414, 1993.[Medline]
  34. Zacharski L. R., Wojtukiewicz M. Z., Costantini V., Ornstein D. L., Memoli V. A. Pathways of coagulation/fibrinolysis activation in malignancy. Semin. Thromb. Hemost., 18: 104-116, 1992.[Medline]
  35. Costantini V., Zacharski L. R., Memoli V. A., Kudryk B. J., Rousseau S. M., Stump D. C. Occurrence of components of fibrinolysis pathways in situ in neoplastic and nonneoplastic human breast tissue. Cancer Res., 51: 354-358, 1991.[Abstract/Free Full Text]
  36. Brown L. F., Guidi A. J., Schnitt S. J., Van De Water L., Iruela-Arispe M. L., Yeo T. K., Tognazzi K., Dvorak H. F. Vascular stroma formation in carcinoma in situ, invasive carcinoma, and metastatic carcinoma of the breast. Clin. Cancer Res., 5: 1041-1056, 1999.[Abstract/Free Full Text]
  37. Lee S. Y., Lee K. P., Lim J. W. Identification and biosynthesis of fibrinogen in human uterine cervix carcinoma cells. Thromb. Haemostasis, 75: 466-470, 1996.[Medline]
  38. Molmenti E. P., Ziambaras T., Perlmutter D. H. Evidence for an acute phase response in human intestinal epithelial cells. J. Biol. Chem., 268: 14116-14124, 1993.[Abstract/Free Full Text]
  39. Amrani D. L., Plant P. W., Pindyck J., Mosesson M. W., Grieninger G. Structural analysis of fibrinogen synthesized by cultured chicken hepatocytes in the presence or absence of dexamethasone. Biochim. Biophys. Acta, 743: 394-400, 1983.[Medline]
  40. Parrott J. A., Whaley P. D., Skinner M. K. Extrahepatic expression of fibrinogen by granulosa cells: potential role in ovulation. Endocrinology, 133: 1645-1649, 1993.[Abstract]
  41. Hartwig R., Danishefsky K. J. Studies on the assembly and secretion of fibrinogen. J. Biol. Chem., 266: 6578-6585, 1991.[Abstract/Free Full Text]
  42. Zhang J. Z., Redman C. M. Role of interchain disulfide bonds on the assembly and secretion of human fibrinogen. J. Biol. Chem., 269: 652-658, 1994.[Abstract/Free Full Text]
  43. Xu W., Chung D. W., Davie E. W. The assembly of human fibrinogen. The role of the amino-terminal and coiled-coil regions of the three chains in the formation of the {alpha}{gamma} and ß{gamma} heterodimers and {alpha}ß{gamma} half-molecules. J. Biol. Chem., 271: 27948-27953, 1996.[Abstract/Free Full Text]
  44. Zhang J. Z., Redman C. M. Assembly and secretion of fibrinogen. Involvement of amino-terminal domains in dimer formation. J. Biol. Chem., 271: 12674-12680, 1996.[Abstract/Free Full Text]
  45. Kim Y., Rus H. G., Fisher S. N., Pitha P. M., Shin M. L. Binding of a protein to an AU-rich domain of tumour necrosis factor {alpha} mRNA as a 35 kDa complex and its regulation in primary rat astrocytes. Biochem. J., 316: 455-460, 1996.
  46. Aharon T., Schneider R. J. Selective destabilization of short-lived mRNAs with the granulocyte-macrophage colony-stimulating factor AU-rich 3' noncoding region is mediated by a cotranslational mechanism. Mol. Cell. Biol., 13: 1971-1980, 1993.[Abstract/Free Full Text]
  47. Chen C. Y., Shyu A. B. AU-rich elements: characterization and importance in mRNA degradation. Trends Biochem. Sci., 20: 465-470, 1995.[Medline]
  48. Chen C. Y., Shyu A. B. Selective degradation of early-response-gene mRNAs: functional analyses of sequence features of the AU-rich elements. Mol. Cell. Biol., 14: 8471-8482, 1994.[Abstract/Free Full Text]
  49. Ross J. mRNA stability in mammalian cells. Microbiol. Rev., 59: 423-450, 1995.[Abstract/Free Full Text]
  50. Zubiaga A. M., Belasco J. G., Greenberg M. E. The nonamer UUAUUUAUU is the key AU-rich sequence motif that mediates mRNA degradation. Mol. Cell. Biol., 15: 2219-2230, 1995.[Abstract]
  51. Odrljin T. M., Shainoff J. R., Lawrence S. O., Simpson-Haidaris P. J. Thrombin cleavage enhances exposure of a heparin binding domain in the N-terminus of the fibrin ß chain. Blood, 88: 2050-2061, 1996.[Abstract/Free Full Text]
  52. Simpson-Haidaris P. J., Guadiz G., Odrljin T. M. Cell surface heparan sulfate proteoglycan-dependent fibrinogen assembly in extracellular matrix. Blood, 90: 464 1997.[Abstract/Free Full Text]
  53. Felding-Habermann B., Ruggeri Z. M., Cheresh D. A. Distinct biological consequences of integrin {alpha}vß3-mediated melanoma cell adhesion to fibrinogen and its plasmic fragments. J. Biol. Chem., 267: 5070-5077, 1992.[Abstract/Free Full Text]
  54. Senger D. R., Perruzzi C. A. Cell migration promoted by a potent GRGDS-containing thrombin-cleavage fragment of osteopontin. Biochim. Biophys. Acta, 1314: 13-24, 1996.[Medline]
  55. Giannelli G., Falk-Marzillier J., Schiraldi O., Stetler-Stevenson W. G., Quaranta V. Induction of cell migration by matrix metalloprotease-2 cleavage of laminin-5. Science (Washington DC), 277: 225-228, 1997.[Abstract/Free Full Text]
  56. Chang M. C., Wang B. R., Huang T. F. Characterization of endothelial cell differential attachment to fibrin and fibrinogen and its inhibition by Arg-Gly-Asp-containing peptides. Thromb. Haemostasis, 74: 764-769, 1995.[Medline]
  57. Altieri D. C., Duperray A., Plescia J., Thornton G. B., Languino L. R. Structural recognition of a novel fibrinogen {gamma} chain sequence (117–133) by intercellular adhesion molecule-1 mediates leukocyte-endothelium interaction. J. Biol. Chem., 270: 696-699, 1995.[Abstract/Free Full Text]
  58. Altieri D. C., Plescia J., Plow E. F. The structural motif glycine 190-valine 202 of the fibrinogen {gamma} chain interacts with CD11b/CD18 integrin ({alpha}Mß2, Mac-1) and promotes leukocyte adhesion. J. Biol. Chem., 268: 1847-1853, 1993.[Abstract/Free Full Text]
  59. Hynes R. O. Integrins: versatility, modulation, and signaling in cell adhesion. Cell, 69: 11-25, 1992.[Medline]
  60. McCarthy J. B., Skubitz A. P., Iida J., Mooradian D. L., Wilke M. S., Furcht L. T. Tumor cell adhesive mechanisms and their relationship to metastasis. Semin. Cancer Biol., 2: 155-167, 1991.[Medline]
  61. Sueishi K., Nanno S., Tanaka K. Permeability enhancing and chemotactic activities of lower molecular weight degradation products of human fibrinogen. Thromb. Haemostasis, 45: 90-94, 1981.[Medline]
  62. Skogen W. F., Senior R. M., Griffin G. L., Wilner G. D. Fibrinogen-derived peptide B ß1–42 is a multidomained neutrophil chemoattractant. Blood, 71: 1475-1479, 1988.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
H. O. Duan and P. J. Simpson-Haidaris
Cell Type-specific Differential Induction of the Human {gamma}-Fibrinogen Promoter by Interleukin-6
J. Biol. Chem., May 5, 2006; 281(18): 12451 - 12457.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Chem.Home page
A.-J. Cheng, L.-C. Chen, K.-Y. Chien, Y.-J. Chen, J. T.-C. Chang, H.-M. Wang, C.-T. Liao, and I-H. Chen
Oral Cancer Plasma Tumor Marker Identified with Bead-Based Affinity-Fractionated Proteomic Technology
Clin. Chem., December 1, 2005; 51(12): 2236 - 2244.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
Y.-J. Tan, P.-Y. Tham, D. Z. L. Chan, C.-F. Chou, S. Shen, B. C. Fielding, T. H. P. Tan, S. G. Lim, and W. Hong
The Severe Acute Respiratory Syndrome Coronavirus 3a Protein Up-Regulates Expression of Fibrinogen in Lung Epithelial Cells
J. Virol., August 1, 2005; 79(15): 10083 - 10087.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
M. L. Criscuoli, M. Nguyen, and B. P. Eliceiri
Tumor metastasis but not tumor growth is dependent on Src-mediated vascular permeability
Blood, February 15, 2005; 105(4): 1508 - 1514.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
B. J. Rybarczyk, S. O. Lawrence, and P. J. Simpson-Haidaris
Matrix-fibrinogen enhances wound closure by increasing both cell proliferation and migration
Blood, December 1, 2003; 102(12): 4035 - 4043.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rybarczyk, B. J.
Right arrow Articles by Simpson-Haidaris, P. J.
Right arrow