Procoagulant activity in cancer cells is dependent on tissue factor expression

Procoagulant activity of pairs of cell lines, which were derived from the same original cell type but which possess different growth characteristics and metastatic properties, was examined. The following characteristics were considered suggestive of a greater likelihood of metastatic potential: high histological grade; establishment of the line from a metastatic rather than a nonmetastatic cancer; increased tumorigenicity in nude mice; and/or estrogen receptor-negative mammary cancer. Procoagulant activity was evaluated by a two stage clotting assay. Procoagulant activity was highly variable, with up to a 1,300-fold difference, among the cancer cell lines examined. The rate of clot formation was factor VII dependent and was totally inhibited by an anti tissue factor monoclonal antibody, indicating that tissue factor was the only significant procoagulant present in these cancer cells. Tissue factor antigen expression, evaluated by ELISA, correlated with procoagulant activity. In all pairs of cancer cell lines, those with characteristics of increased proliferative potential had increased tissue factor levels compared to cell lines that originated from the same cell type, but which possess less aggressive characteristics. Tissue factor activity in these cancer cells was increased by cell lysis or by exposure of intact cells to a calcium ionophore, similar to results previously obtained in fibroblasts. Tissue factor mRNA was evaluated by northern blot analysis using a specific probe complementary to tissue factor mRNA. The previously described predominant tissue factor mRNA species of 2.2 kb was identified in the majority of cancer cell lines examined, but tissue factor mRNA species of 3.2 to 3.4 kb were also identified.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of glucosylceramide synthase, converting ceramide to glucosylceramide, confers adriamycin resistance in human breast cancer cells

Multidrug-resistant cancer cells display elevated levels of glucosylceramide (Lavie, Y., Cao, H. T., Volner, A., Lucci, A., Han, T. Y., Geffen, V., Giuliano, A. E., and Cabot, M. C. (1997) J. Biol. Chem. 272, 1682-1687). In this study, we have introduced glucosylceramide synthase (GCS) into wild type MCF-7 breast cancer cells using a retroviral tetracycline-on expression system, and we developed a cell line, MCF-7/GCS. MCF-7/GCS cells expressed an 11-fold higher level of GCS activity compared with the parental cell line. Interestingly, the transfected cells demonstrated strong resistance to adriamycin and to ceramide, whereas both agents were highly cytotoxic to MCF-7 cells. The EC50 values of adriamycin and ceramide were 11-fold (p < 0.0005) and 5-fold (p < 0.005) higher, respectively, in MCF-7/GCS cells compared with MCF-7 cells. Ceramide resistance displayed by MCF-7/GCS cells closely paralleled the activity of expressed GCS with a correlation coefficient of 0.99. In turn, cellular resistance and GCS activity were dependent upon the concentration of the expression mediator doxycycline. Adriamycin resistance in MCF-7/GCS cells was related to the hyperglycosylation of ceramide and was not related to shifts in the levels of either P-glycoprotein or Bcl-2. This work demonstrates that overexpression of GCS, which catalyzes ceramide glycosylation, induces resistance to adriamycin and ceramide in MCF-7 breast cancer cells.     

Transforming growth factor beta 1 can induce estrogen-independent tumorigenicity of human breast cancer cells in athymic mice

We have examined the effect of transforming growth factor beta 1 (TGF-beta 1) overexpression in human breast cancer cell tumorigenicity in athymic mice. Estrogen-dependent MCF-7 cells were stably transfected with pSVTGF beta 1. A clone was isolated which overexpressed TGF-beta 1 mRNA and secreted > 10-fold more TGF-beta activity into the tissue culture medium. Similar to the parent line, the MCF-7/TGF-beta 1 cells were relatively insensitive to exogenous TGF-beta 1 and exhibited low levels of TGF-beta receptors. Clonogenicity in soft agarose, doubling time, morphology, and sensitivity to 17 beta-estradiol and the antiestrogen tamoxifen were not altered in the transfected cells. Inoculation s.c. of MCF-7/TGF-beta 1 cells in ovariectomized nude mice resulted in 100% tumor formation which was totally abrogated by i.p. administration of the neutralizing anti-TGF-beta 2G7 IgG2B. The parent cells formed tumors only after estrogen supplementation. By immunohistochemistry, higher levels of TGF-beta 1 protein were detected in MCF-7/TGF-beta 1 tumors than in estrogen-induced parent MCF-7 tumors. Administration of 1 microgram TGF-beta 1 i.p. daily for 3 weeks after tumor cell inoculation transiently supported estrogen-independent growth of parent MCF-7 tumors in castrated nude mice. These data indicate that overexpression of TGF-beta 1 in human breast cancer cells can contribute to their escape from hormone dependence.     

Tumor necrosis factor alpha enhances secretion of transforming growth factor beta2 in MCF-7 breast cancer cells

We studied the effect of tumor necrosis factor alpha (TNF-alpha) on transforming growth factor beta (TGF-beta) secretion by human breast cell lines to further characterize the antitumor effects of TNF-alpha. We found that TNF-alpha increased the secretion of TGF-beta in two established breast cancer cell lines (MCF-7 and ZR-75-1) but not in two immortalized human mammary epithelial cell lines (184B5 and MCF-10A). In MCF-7 cells, TNF-alpha increased the secretion of total TGF-beta 6.1-fold within 72 h in a dose-dependent manner. The secretion of both latent and active forms of TGF-beta was increased, and their ratio altered from 25:1 to 12:1 in the medium. TNF-alpha converted the secretory pattern of TGF-beta by MCF-7 cells from the heterodimeric form TGF-beta1.2 to the homodimeric form TGF-beta2. Immunoblot analysis under nonreducing conditions identified four molecular mass species of TGF-beta secreted in the culture media of untreated MCF-7 cells (238, 210, 40-55, and 25 kDa). Under reducing conditions, three molecular mass species of TGF-beta were identified: 88, 44, and 12 kDa. Gel filtration analysis demonstrated that the secreted TGF-beta within the range of 12-88 kDa was biologically active. TNF-alpha treatment did not alter the size of molecular mass species secreted by MCF-7 cells and did not change steady-state levels of mRNA for TGF-beta1 or TGF-beta2. These findings indicate that TNF-alpha may regulate quantitatively and qualitatively TGF-beta secretion by human breast cancer cells in vitro. The diverse biological activities of TGF-beta may also allow TNF-alpha to regulate the growth and metabolism of human mammary epithelial cells and/or stromal cells in a paracrine manner.     

A macrophage-dependent factor that stimulates the proliferation of fibroblasts in vitro

Whole blood serum (HBS) stimulates the proliferation of fibroblasts in vitro, while platelet-poor plasma serum (PPPS) does not. Fibroblasts grown in the presence of PPPS are truly quiescent in that they are not deprived of nutrients in the culture medium and less than 3% of the cells synthesize DNA and divide. In vivo experiments have suggested that macrophages are necessary for stimulation of fibroplasia during wound repair. We have utilized the difference in growth-promoting activity between HBS and PPPS to study the ability of macrophages to produce growth-promoting activity in cell culture. Guinea pig peritoneal macrophages cultured in vitro in medium containing PPPS release into the medium, either directly or indirectly, a factor (or factors) that stimulates the proliferation of guinea pig wound fibroblasts. This macrophage-dependent, fibroblast-stimulating activity (MFSA) is nondialyzable, heat stable (56 C for 30 minutes), and requires culture in vitro for demonstration of activity. The relationship between MFSA and other growth factor(s) has not yet been determined. In contrast to the macrophage, lymphocytes prepared from mesenteric lymph nodes produced no figroblast-stimulating activity.     

Monosialoganglioside (GM1) immunofluorescence in rat spinal roots studied with a monoclonal antibody

The rate of reaction of monochlorobimane with glutathione (GSH) was measured in native human mammary MCF-7 adenocarcinoma cells (MCF-7wt) and sublines displaying resistance to 4-hydroperoxycyclophosphamide (MCF-7hc) and adriamycin (MCF-7adr) prior to examination by epifluorescence and confocal microscopy. After a 60-min incubation period at 37 degrees C, essentially all GSH was conjugated in the MCF-7wt and MCF-7adr cell lines whereas only 80% of the GSH was conjugated in the MCF-7hc line. All three lines displayed significant export of the conjugate from the cell during this period, with the MCF-7adr line displaying the most rapid efflux with 85% of the conjugate exported within 60 min. Epifluorescence microscopy detected an approximately 20% increase in integrated fluorescence intensity in the nuclear region in all three lines. Confocal microscopy however, indicated that most of the cells examined showed a homogeneous fluorescence distribution. The cells grown in monolayers were found to be thicker in the nuclear region suggesting that the observed increase in fluorescence intensity in the nuclear region in the images from epifluorescence microscopy was probably derived from fluorescence from an out-of-focus plane. Cells depleted of GSH with buthionine sulfoximine followed by treatment with mBCl showed significant fluorescence intensity resulting from nonspecific binding of this probe. These studies illustrate the need for measuring the rate of GSH conjugate export and for determining probe specificity, and emphasizes the need for using confocal techniques for the quantitative evaluation of the distribution of intracellular fluorescence.     

Autocrine transforming growth factor alpha provides a growth advantage to malignant cells by facilitating re-entry into the cell cycle from suboptimal growth states

CBS human colon carcinoma cells are poorly tumorigenic in athymic nude mice, whereas FET colon carcinoma cells are non-tumorigenic. Both cell lines have well differentiated properties in tissue culture. Transforming growth factor alpha (TGF-alpha) was ectopically expressed by stable transfection of a TGF-alpha cDNA under repressible tetracycline control. The TGF-alpha-transfected cells showed enhanced clonal initiation and shortened lag phase growth in tissue culture without an alteration in doubling time in exponential phase relative to untransfected cells. Furthermore, the TGF-alpha transfectants showed increased independence from exogenous growth factors in clonal growth assays and induction of DNA synthesis after release from quiescence. Growth factor independence was associated with sustained epidermal growth factor receptor activation in quiescent TGF-alpha-transfected cells and the requirement of exogenous insulin for stimulation of quiescent cells to re-enter the cell cycle. Higher cloning, reduced lag time in tissue, and the acquisition of growth factor independence for DNA synthesis without a change in doubling time of TGF-alpha-transfected cells indicate that autocrine TGF-alpha functions by facilitating re-entry into the cell cycle from sub-optimal growth states rather than promoting or controlling the proliferation of actively cycling cells. The modulation of growth regulation by autocrine TGF-alpha was associated with increased malignant properties as TGF-alpha transfectants showed increased tumorigenicity in athymic nude mice. The administration of tetracycline reversed the effects of TGF-alpha expression in these cells both in vivo and in vitro, indicating that the alterations of the biological properties were due to the expression of TGF-alpha. Since these cells are continuously grown in a completely chemically defined medium without serum supplementation, it was possible to assign the mechanism underlying the generation of growth factor independence to the replacement of a requirement for exogenous insulin in parental cells by autocrine TGF-alpha.     

Expression of tissue factor pathway inhibitor in vascular smooth muscle cells and its regulation by growth factors

Tissue factor pathway inhibitor (TFPI) in vivo is thought to be synthesized mainly by endothelial cells. To date, no significant regulator of TFPI synthesis has been described. Vascular smooth muscle cells (VSMC) express tissue factor in vitro and in vivo, which may contribute to vascular thrombosis. We hypothesized that VSMC might also express TFPI. To determine this, we examined growth-arrested coronary VSMC in culture and found that VSMC secreted an amount of TFPI similar to that seen in endothelial cells. Immunohistochemistry of normal human coronary arteries showed TFPI staining throughout the media and intima of the vessel with localization to VSMC and endothelial cells. To determine regulation of TFPI expression in VSMC, we examined the effects of serum stimulation on TFPI secretion and found that FBS induced a 5-fold increase in TFPI antigen and activity levels in conditioned medium at 48 hours (P<0.001) when compared with serum-free conditions. A similar stimulatory effect was seen with 10% pooled human serum. Moreover, epidermal growth factor and platelet-derived growth factor-B increased TFPI secretion by 4- to 5-fold and 2- to 3-fold, respectively (P<0.05), and these growth factors accounted for approximately 50% of the TFPI secretion effects of human serum. The serum effect was associated with a 3-fold increase in TFPI mRNA 24 hours after release from growth arrest and a 50% decrease in TFPI secretion after treatment with actinomycin D. Taken together, this study suggests that there is significant TFPI expression in VSMC in culture and in VSMC within the intima and media of the normal coronary artery wall. We present the first evidence for TFPI regulation by serum in VSMC and more specifically by its constituent growth factors, epidermal growth factor and platelet-derived growth factor-B.     

Regulation of transforming growth factor-beta type II receptor expression in human breast cancer MCF-7 cells by vitamin D3 and its analogues

In view of the tumor suppressor role of the transforming growth factor-beta (TGFbeta) type II receptor (RII), the identification and characterization of agents that can induce the expression of this receptor are of potential importance to the development of chemoprevention approaches as well as treatment of cancer. To date, the identification of exogenous agents that control RII expression has been rare. We demonstrated that proliferation of MCF-7 early passage cells (MCF-7 E), which express RII and are sensitive to TGFbeta growth inhibition activity, was significantly inhibited by vitamin D3 and its analogue EB1089. In contrast, proliferation of MCF-7 late passage cells (MCF-7 L), which have lost cell surface RII and are resistant to TGFbeta, was not affected by these two compounds. TGFbeta-neutralizing antibody was able to block the inhibitory effect on MCF-7 E cells by these compounds, indicating that treatment induced autocrine-negative TGFbeta activity. An RNase protection assay showed approximately a 3-fold induction of the RII mRNA, while a receptor cross-linking assay revealed a 3-4-fold induction of the RII protein. In contrast, there was no change in either RII mRNA or protein in the MCF-7 L cells.     

Quantitation of platelet-derived growth factor receptors in human arterial smooth muscle cells in vitro

Platelet-derived growth factor (PDGF) is suggested to play an important role in the development of atherosclerosis as a migratory and mitogenic stimulus to arterial smooth muscle cells (ASMCs). Stimulated and unstimulated ASMCs were studied with respect to PDGF receptor (PDGF-R) mRNA and protein expression. Quantitative RT-PCR was developed for simultaneous evaluation of both PDGF-R alpha and -R beta mRNA expression and a quantitative ELISA for estimation of corresponding PDGF-R subunits. On the mRNA level, the overall PDGF-R beta expression was approximately 100 times lower than that of PDGF-R alpha. Furthermore, although PDGF-R alpha mRNA levels were high irrespective of hASMC phenotype, PDGF-R beta mRNA was influenced by serum stimulation with lower copy numbers in proliferating and confluent cells compared with quiescent cells. On the protein level, quiescent hASMCs expressed 10 times more PDGF-R beta than PDGF-R alpha. Serum stimulation decreased cell surface PDGF-Rs, with most prominent loss of PDGF-R alpha (ELISA and immunohistochemistry). Our results suggest a differential regulatory pattern for PDGF-R alpha and -R beta and are compatible with the usage of alternative promoters for regulation of -R alpha expression. Further, it seems that the number of available receptor subunits is not the only determinant of variations in cell stimulation with different PDGF isoforms.     

In vitro secretion of cytokines by human bone marrow: effects of age and estrogen status

It has been proposed that cytokines mediate the acceleration of bone loss following menopause. Because of the intimate relationship between bone marrow stromal cells and bone tissue, it is possible that marrow cells and their products contribute to the bone microenvironment and influence the regulation of bone cell differentiation and activity. We examined the production of cytokines by bone marrow stromal cells from a total of 37 women and 15 men undergoing total hip replacement for noninflammatory joint disease. Low-density mononuclear cells were isolated from bone marrow and were cultured in phenol red-free alpha MEM medium supplemented with 10% FBS and antibiotics. Constitutive secretion of interleukin-6 (IL-6) was positively correlated with age in a series of 8 women and 5 men measured by bioassay (r = 0.98; P < 0.01) and in a series of 18 women and 10 men measured by immunoassay (r = 0.56; P < 0.01). The pattern of cytokine production by bone marrow stromal cells was examined in detail in 23 postmenopausal women, aged 49-88 yr. Basal secretion of immunoreactive IL-6 and IL-11, but not granulocyte-macrophage colony-stimulating factor, increased with time in culture. Exogenous IL-1 beta stimulated secretion of IL-6 and IL-11 in a saturable, dose-dependent manner. Secretion of soluble IL-6 receptor was not correlated with secretion of IL-6, either constitutively or in the presence of IL-1 beta. In 4 of 14 samples, IL-1 beta also stimulated secretion of granulocyte-macrophage colony-stimulating factor. IL-1 beta was undetectable in 7 of 9 cultures during the 2-week culture period. IL-6 did not stimulate secretion of IL-1 beta in the 7 cultures tested. Cells were dependent upon serum for viability and growth and were not sustained by a serum substitute (1% insulin-transferrin-selenium-BSA). Cells grown in medium with 10% FBS and supplemented with 1% insulin-transferrin-selenium-BSA secreted 10-fold more IL-6 than cells grown in serum alone. Marrow from 7 women receiving estrogen replacement therapy showed lower constitutive secretion of IL-6 (75%; P < 0.006) and IL-11 (43%; P < 0.05) than marrow from age-matched controls and had blunted stimulation of IL-6 and IL-11 secretion by exogenous IL-1 beta. These data indicate distinct patterns of cytokine production by human marrow stromal cultures dependent upon age and estrogen status.     

Generation of a monoclonal antibody that inhibits the procoagulant activity of various cancer cell lines

BACKGROUND: Tumor procoagulant is one of the factors responsible for disseminated intravascular coagulation and metastasis. The authors found procoagulant activity in LK52 human squamous cell carcinoma cells, which they designated cancer cell-derived blood coagulating activity 1 (CCA-1). A monoclonal antibody (MoAb) was generated to characterize this CCA-1 procoagulant activity. To date, antibodies that show an inhibitory effect on procoagulant activity as well as high reactivity in cancer cells are well known for their tissue factor specificity. METHODS: Characterization of the procoagulant activity of CCA-1 was performed and an anti-CCA-1 MoAb, FS01, was generated. CCA-1 expression on the cancer cell surface was examined by flow cytometry. Procoagulant activity of various cancer cell lines and the inhibitory effect of the FS01 MoAb on this procoagulant activity was monitored by a clot timer. RESULTS: The enzymologic character differed from that of cancer procoagulant (CP). The FS01 MoAb inhibited the procoagulant activity of CCA-1, but did not inhibit that of tissue factor. A positive correlation was observed between the expression intensity of CCA-1 and the inhibitory effect of the FS01 MoAb on the procoagulant activity of cancer cell lines. Expression of CCA-1 was observed more frequently than that of tissue factor in human cancer cell lines. CONCLUSIONS: The FS01 MoAb generated in the current study is a new antibody that reacts with various cancer cell lines, but not with normal cells. FS01 inhibits cancer cell-derived procoagulant activity and does not react with tissue factor and CP. CCA-1, which is recognized by the FS01 MoAb, appears to play a major role in cancer cell-derived procoagulant activity.     

A multidrug resistance transporter from human MCF-7 breast cancer cells

MCF-7/AdrVp is a multidrug-resistant human breast cancer subline that displays an ATP-dependent reduction in the intracellular accumulation of anthracycline anticancer drugs in the absence of overexpression of known multidrug resistance transporters such as P glycoprotein or the multidrug resistance protein. RNA fingerprinting led to the identification of a 2.4-kb mRNA that is overexpressed in MCF-7/AdrVp cells relative to parental MCF-7 cells. The mRNA encodes a 655-aa [corrected] member of the ATP-binding cassette superfamily of transporters that we term breast cancer resistance protein (BCRP). Enforced expression of the full-length BCRP cDNA in MCF-7 breast cancer cells confers resistance to mitoxantrone, doxorubicin, and daunorubicin, reduces daunorubicin accumulation and retention, and causes an ATP-dependent enhancement of the efflux of rhodamine 123 in the cloned transfected cells. BCRP is a xenobiotic transporter that appears to play a major role in the multidrug resistance phenotype of MCF-7/AdrVp human breast cancer cells.     

1alpha,25-dihydroxyvitamin D3 actions in LNCaP human prostate cancer cells are androgen-dependent

We and others have recently shown that 1alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] significantly inhibits cell proliferation and increases secretion of prostate-specific antigen (PSA) in LNCaP cells, an androgen-responsive human prostate cancer cell line. The present study was designed to investigate the possible interactions between 1,25-(OH)2D3 and androgens in the regulation of LNCaP cellular function. LNCaP cell growth was dose-dependently inhibited by 1,25-(OH)2D3 (60% inhibition at 10 nM) when cells were cultured in medium supplemented with FBS (FBS medium). 1,25-(OH)2D3-treated cells showed a 5-fold increase in PSA secretion, similar to the increase seen in dihydrotestosterone (DHT)-treated cells. In combination, 1,25-(OH)2D3 and DHT synergistically enhanced PSA secretion 22-fold. This synergistic effect was even greater when cells were cultured in medium supplemented with charcoal-stripped serum (CSS medium), where endogenous steroids are substantially depleted. Under these conditions, 1,25-(OH)2D3 and DHT together stimulated PSA secretion up to 50-fold over the untreated control. Radioligand binding assays and Western blot analyses showed that the androgen receptor (AR) content was increased significantly by 1,25-(OH)2D3 at 48 h. Furthermore, the steady-state mRNA level of AR was up-regulated approximately 2-fold by 1,25-(OH)2D3 at 24 h. When cells were grown in CSS medium, 1,25-(OH)2D3 alone no longer inhibited cell growth or induced PSA secretion. Titration experiments revealed that the addition of DHT at 1 nM to the medium restored the antiproliferative activity of 1,25-(OH)2D3. Conversely, an antiandrogen, Casodex, completely blocked 1,25-(OH)2D3 antiproliferative and PSA stimulation activities when cells were cultured in FBS medium. In conclusion, these results demonstrate that the antiproliferative and PSA induction activities of 1,25-(OH)2D3 in LNCaP cells are dependent upon androgen action and that AR up-regulation by 1,25-(OH)2D3 likely contributes to the synergistic actions of 1,25-(OH)2D3 and DHT in these cells.     

Doxorubicin cellular pharmacokinetics plays no role in chemosensitizing effect of verapamil on Swiss-3T3 cells

AIM: To find whether or not the doxorubicin (Dox) cellular pharmacokinetics plays a role in chemosensitizing effect of verapamil (Ver) on drug sensitive cells. METHODS: Cytotoxicity and cellular Dox contents (during accumulation and retention periods) were measured in the absence and presence of verapamil in Swiss-3T3 cells and compared with those in multidrug resistant (MDR) MCF-7Adr cells and drug sensitive MCF-7WT cells. mdr-1 mRNA expression in Swiss-3T3 cells was analyzed. RESULT: Dox cytotoxicity was enhanced 2.0-fold in Swiss-3T3 cells by Ver (3 mumol.L-1) and 3.6-fold in MCF-7Adr cells by Ver (6 mumol.L-1), but not in MCF-7WT cells (Ver 6 mumol.L-1). Cellular accumulation of equi-effective concentrations of Dox increased at 6-h incubation in the presence of Ver in Swiss-3T3 (1.5-fold)i and MCF-7WT cells (2.1-fold) but decreased rapidly in MCF-7Adr cells by 20% to 50% compared to that in the absence of Ver. Cellular retention of Dox decreased after 10-min increase in the presence of Ver in Swiss-3T3 cells compared to that in the absence of Ver, that was similar to that in MCF-7WT cells, while the retention was augmented by Ver in MCF-7Adr cells. Slot blot analysis of RNA revealed no mdr-1 gene expression in Swiss-3T3 cells. CONCLUSION: Changes in cellular accumulation and retention of Dox did not account for the chemosensitizing effect of Ver on Swiss-3T3 cells.