Scatter factor and the c-met receptor: a paradigm for mesenchymal/epithelial interaction.

Epithelia and mesenchyme interact during various physiologic and pathologic processes. Scatter factor is a mesenchyme-derived cytokine that stimulates motility, proliferation, and morphogenesis of epithelia. Recent studies suggest that scatter factor and its receptor (c-met) mediate mesenchyme/epithelia signalling and even interconversion. In this mini-review, we will discuss how scatter factor and c-met may mediate interactions between mesenchyme and epithelia during embryogenesis, organ repair, and neoplasia.

Regulation of scatter factor production via a soluble inducing factor.

Scatter factor (SF) (also known as hepatocyte growth factor [HGF]) is a fibroblast-derived cytokine that stimulates motility, proliferation, and morphogenesis of epithelia. SF may play major roles in development, repair, and carcinogenesis. However, the physiologic signals that regulate its production are not well delineated. We found that various human tumor cell lines that do not produce SF secrete factors that stimulate SF production by fibroblasts, suggesting a paracrine mechanism for regulation of SF production. Conditioned medium from these cell lines contained two distinct scatter factor-inducing factor SF-IF activities: a high molecular weight (> 30 kD), heat sensitive activity and a low molecular weight (< 30 kD) heat stable activity. Further studies revealed that SF-producing fibroblasts also secrete factors that stimulate their own SF production. We characterized the < 30-kD SF-IF activity from ras-3T3 (clone D4), a mouse cell line that overproduces both SF and SF-IF. The < 30-kD filtrate from ras-3T3 conditioned medium induced four- to sixfold increases in expression of SF biologic activity, immunoreactive protein, and mRNA by multiple SF-producing fibroblast lines. Ras-3T3 SF-IF activity was stable to boiling, extremes of pH, and reductive alkylation, but was destroyed by proteases. We purified ras-3T3 SF-IF about 10,000-fold from serum-free conditioned medium by a combination of ultrafiltration, cation exchange chromatography, and reverse phase chromatography. The purified protein exhibited electrophoretic mobility of about 12 kD (reduced) and 14 kD (nonreduced) by SDS-PAGE. The identity of the protein was verified by elution of biologic activity from gel slices. Purified SF-IF stimulated SF production in a physiologic concentration range (about 20-400 pM). Its properties and activities were distinct from those of IL-1 and TNF, two known inducers of SF production. We suggest that SF-IF is a physiologic regulator of SF production.

Vascular permeation of platelet factor 4 after endothelial injury.

Antibody to platelet factor 4 was used to demonstrate permeation of this factor into the blood vessel wall after endothelial injury in rabbits. The presence of platelet factor 4 antigen in the vessel wall after removal of the endothelium was shown by immunofluorescence 10 and 30 minutes after injury but not 240 minutes afterward. This study demonstrates that factors carried by platelets can enter the vessel wall and that the movement of these platelet products into the vasculature is a short-lived, self-limiting process.

Isolation and culture of a tetraploid subpopulation of smooth muscle cells from the normal rat aorta.

Smooth muscle cells with 4C (double diploid) DNA content have been found in major arteries. The proportion of 4C cells increases with normal aging and with hypertension. These cells may represent a state of arrest at the G2 phase of the cell cycle or may be examples of true tetraploidy. Flow cytometric cell sorting was used to isolate 4C smooth muscle cells from the rat aorta, and the cells were cultured. Flow cytometry, Feulgen microdensitometry, and karyotyping of the progeny of the 4C cells established the presence of true tetraploid cells. These findings demonstrate the presence of reproductively viable tetraploid cells in a normal mammalian tissue.

Vascular smooth muscle cell kinetics: a new assay for studying patterns of cellular proliferation in vivo.

A new quantitative assay for studying the kinetics of vascular smooth muscle cells in vivo is reported. The assay was used to determine the specific activity of DNA from rabbit aortic smooth muscle cells stimulated to grow by removal of the endothelial layer. The specific activity of the DNA was correlated with the rate of tritiated thymidine incorporation as measured by autoradiography and with the rate of DNA synthesis as estimated by direct measurement of cellular proliferation. Smooth muscle cells exhibit a 24-hour latent period in vivo prior to DNA synthesis; the synthesis peaks at 48 hours and then rapidly declines. The decline in DNA synthesis is not related to endothelial regrowth, and may be of homeostatic significance in limiting luminal stenosis. The assay offers a rapid and reliable alternative to autoradiographic and morphometric techniques for evaluating growth kinetics and growth regulation in vivo.

BRCA1 inhibition of estrogen receptor signaling in transfected cells.

Mutations of the breast cancer susceptibility gene BRCA1 confer increased risk for breast, ovarian, and prostatic cancers, but it is not clear why the mutations are associated with these particular tumor types. In transient transfection assays, BRCA1 was found to inhibit signaling by the ligand-activated estrogen receptor (ER-alpha) through the estrogen-responsive enhancer element and to block the transcriptional activation function AF-2 of ER-alpha. These results raise the possibility that wild-type BRCA1 suppresses estrogen-dependent transcriptional pathways related to mammary epithelial cell proliferation and that loss of this ability contributes to tumorigenesis.

Effect of Akt inhibition on scatter factor-regulated gene expression in DU-145 human prostate cancer cells.

The cytokine scatter factor (SF) (hepatocyte growth factor) transduces various biologic actions, including cell motility, invasion, angiogenesis and apoptosis inhibition. The latter is relevant to understanding the role of SF in promoting tumor cell survival in different contexts, for example, detachment from basement membrane, growth in metastatic sites and responses to chemo- and radiotherapy. Previously, we showed that SF protects cells against apoptosis owing to DNA damage, by a mechanism involving phosphoinositol-3-kinase/c-Akt signaling. Here, we used DNA microarray assays to identify c-Akt-regulated genes that might contribute to cell protection. DU-145 human prostate cancer cells were transfected+/-a dominant-negative mutant Akt, treated+/-SF and analysed for gene expression using Affymetrix arrays. These studies identified SF-regulated genes for which induction was c-Akt-dependent vs -independent. Selected microarray findings were confirmed by semiquantitative and quantitative reverse transcription-polymerase chain reaction. We tested the contribution of four SF-inducible/c-Akt-dependent genes (AMPD3, EPHB2, MX1 and WNT4) to protection against adriamycin (a DNA topoisomerase IIalpha inhibitor) using RNA interference. Knockdown of each gene except EPHB2 caused a small but significant reduction in the SF cell protection. The lack of effect of EPHB2 knockdown may be due to the fact that DU-145 cells contain a single-mutant EPHB2 allele. A combination of three small interfering RNAs blocked most of the protection by SF in both DU-145 and T47D cells. These findings identify novel c-Akt-regulated genes, some of which contribute to SF-mediated cytoprotection.

Induction of c-Ha-ras gene expression by double-stranded RNA and interferon requirement.

The addition of double-stranded RNA (dsRNA) to NIH 3T3 cells led to an increase in the RNA levels of c-Ha-ras. The double-stranded configuration was required for the increase in c-Ha-ras mRNA levels, as heat-denatured dsRNA and single-stranded RNA did not have any effect. Nuclear run-on transcription experiments indicated that the increase in c-Ha-ras mRNA levels stimulated by dsRNA was due to transcriptional activation of the gene. The induction of c-Ha-ras gene expression by dsRNA was inhibited by anti-beta interferon antibodies, suggesting that interferon might mediate the induction.

The multisubstrate adapter Gab1 regulates hepatocyte growth factor (scatter factor)-c-Met signaling for cell survival and DNA repair.

Hepatocyte growth factor (scatter factor) (HGF/SF) is a pleiotrophic mediator of epithelial cell motility, morphogenesis, angiogenesis, and tumorigenesis. HGF/SF protects cells against DNA damage by a pathway from its receptor c-Met to phosphatidylinositol 3-kinase (PI3K) to c-Akt, resulting in enhanced DNA repair and decreased apoptosis. We now show that protection against the DNA-damaging agent adriamycin (ADR; topoisomerase IIalpha inhibitor) requires the Grb2-binding site of c-Met, and overexpression of the Grb2-associated binder Gab1 (a multisubstrate adapter required for epithelial morphogenesis) inhibits the ability of HGF/SF to protect MDCK epithelial cells against ADR. In contrast to Gab1 and its homolog Gab2, overexpression of c-Cb1, another multisubstrate adapter that associates with c-Met, did not affect protection. Gab1 blocked the ability of HGF/SF to cause the sustained activation of c-Akt and c-Akt signaling (FKHR phosphorylation). The Gab1 inhibition of sustained c-Akt activation and of cell protection did not require the Gab1 pleckstrin homology or SHP2 phosphatase-binding domain but did require the PI3K-binding domain. HGF/SF protection of parental MDCK cells was blocked by wortmannin, expression of PTEN, and dominant negative mutants of p85 (regulatory subunit of PI3K), Akt, and Pak1; the protection of cells overexpressing Gab1 was restored by wild-type or activated mutants of p85, Akt, and Pak1. These findings suggest that the adapter Gab1 may redirect c-Met signaling through PI3K away from a c-Akt/Pak1 cell survival pathway.

Expression of scatter factor in human bladder carcinoma.

BACKGROUND: Scatter factor (SF) is a protein secreted by stromal (supporting) cells that induces disruption of intercellular junctions and stimulates motility and invasiveness of carcinoma cells. SF is also a potent inducer of angiogenesis (new blood vessel formation), a process required for tumor growth and dissemination. Invasion and angiogenesis are characteristics of biologically aggressive tumors, suggesting that the accumulation of SF within tumors might promote progression to a more malignant phenotype. PURPOSE: This study was designed to determine if SF is overexpressed in carcinoma of the bladder and to evaluate the potential mechanisms that might account for such overproduction. METHODS: We measured the SF content in urine from 20 patients with carcinoma of the bladder and various control groups. We also measured expression of SF in bladder tumor extracts, histologic sections of tumors, and cell culture models, using a variety of techniques, including enzyme-linked immunosorbent assays, immunohistochemistry, and Western and Northern blot analyses. Statistical comparisons were performed using two-tailed t tests. RESULTS: Urinary SF content was found to be significantly elevated in patients with bladder carcinoma as compared with normal control subjects (P < .001), patients with benign prostatic hypertrophy (P = .0055), and patients with prostate carcinoma, another genitourinary malignancy (P = .002). Extracts of bladder cancers, especially those from high-grade, invasive tumors, contained very high levels of SF. Both SF and its proto-oncogene (c-met)-encoded receptor were detected in bladder carcinoma tissue sections by immunostaining. Three different bladder carcinoma cell lines produced no detectable SF but produced very high titers of a high-molecular-weight (> 30 kd), heat-sensitive protein that stimulates SF production by stromal cell types. High titers of a similar SF-inducing activity were detected in vivo, in bladder carcinoma extracts, and in the urine of patients with bladder carcinoma. CONCLUSIONS: Our results suggest that SF is overproduced in bladder carcinomas and accumulates within the tumor and in the urine. Overproduction of SF may result from an abnormal urothelial-stromal interaction in which dysplastic or carcinomatous urothelium secretes factors that stimulate SF expression by bladder wall stromal cells. IMPLICATION: Quantitation of SF in the urine and tumor deserves further study as a possible marker of urothelial malignancy.

Alcohol stimulates estrogen receptor signaling in human breast cancer cell lines.

Epidemiological studies suggest that moderate alcohol consumption increases the risk of breast cancer, and that alcohol combined with estrogen replacement therapy may synergistically enhance the risk. However, the mechanism(s) of alcohol-induced mammary cancer is unknown. In human breast cancer cell lines, we found that ethanol (EtOH) caused a dose-dependent increase of up to 10- to 15-fold in the transcriptional activity of the liganded estrogen receptor (ER-alpha), but did not activate the nonliganded receptor. Significant stimulation of ER-alpha activity was observed at EtOH concentrations comparable with or less than blood alcohol levels associated with intoxication and at doses below the threshold for in vitro cytotoxicity. These findings may be explained, in part, by an EtOH-induced down-regulation of the expression of BRCA1, a potent inhibitor of ER-alpha activity, and, in part, by a modest increase in the ER-alpha levels. Our findings suggest that inactivation of BRCA1 and increased estrogen-responsiveness might contribute to alcohol-induced breast cancer.

Altered gene expression pattern in cultured human breast cancer cells treated with hepatocyte growth factor/scatter factor in the setting of DNA damage.

The cytokine hepatocyte growth factor/scatter factor (HGF/SF) protects epithelial and cancer cells against DNA-damaging agents via a pathway involving signaling from c-Met --> phosphatidylinositol-3- kinase --> c-Akt. However, the downstream alterations in gene expression resulting from this pathway have not been established. On the basis of cDNA microarray and semiquantitative RT-PCR assays, we found that MDA-MB-453 human breast cancer cells preincubated with HGF/SF and then exposed to Adriamycin (ADR), a DNA topoisomerase II inhibitor, exhibit an altered pattern of gene expression, as compared with cells treated with ADR only. [HGF/SF+ADR]-treated cells showed altered expression of genes involved in the DNA damage response, cell cycle regulation, signal transduction, metabolism, and development. Some of these alterations suggest mechanisms by which HGF/SF may exert its protective activity, e.g., up-regulation of polycystic kidney disease-1 (a survival-promoting component of cadherin-catenin complexes), down-regulation of 51C (an inositol polyphosphate-5-phosphatase), and down-regulation of TOPBP1 (a topoisomerase IIB binding protein). We showed that enforced expression of the cdc42-interacting protein CIP4, a cytoskeleton-associated protein for which expression was decreased in [HGF/SF+ADR]-treated cells, inhibited HGF/SF-mediated protection against ADR. The cDNA microarray approach may open up new avenues for investigation of the DNA damage response and its regulation by HGF/SF.

Induction of mammary differentiation by mammary-derived growth inhibitor-related gene that interacts with an omega-3 fatty acid on growth inhibition of breast cancer cells.

We previously identified and characterized a novel tumor growth inhibitor and a fatty acid-binding protein in human mammary gland and named it the mammary-derived growth inhibitor-related gene (MRG). Here, the effects of MRG on mammary gland differentiation and its interaction with omega-3 polyunsaturated fatty acids (omega-3 PUFAs) on growth inhibition were investigated. MRG protein expression was associated with human mammary gland differentiation, with the highest expression observed in the differentiated alveolar mammary epithelial cells from the lactating gland. Overexpression of MRG in human breast cancer cells induced differentiation with changes in cellular morphology and a significant increase in the production of lipid droplets. Treatment of mouse mammary gland in organ culture with MRG protein resulted in a differentiated morphology and stimulation of beta-casein expression. Treatment of human breast cancer cells with the omega-3 PUFA docosahexaenoic acid resulted in a differential growth inhibition proportional to their MRG expression. MRG-transfected cells or MRG protein treated cells were much more sensitive to docosahexaenoic acid-induced growth inhibition than MRG-negative or untreated control cells. Our results suggest that MRG is a candidate mediator of the differentiating effect of pregnancy on breast epithelial cells and may play a major role in omega-3 PUFA-mediated tumor suppression.

Tumor necrosis factor stimulates epithelial tumor cell motility.

Cellular motility is a critical function in embryonic development, tissue repair, and tumor invasion. We used assays of scattering (epithelial colony dispersion), cell migration, and cell invasion to study cytokine-regulated motility in epithelial and carcinoma cell lines. Tumor necrosis factor (TNF) stimulated motility in 12 of 14 cell lines in one or more assay systems. The motility-stimulating activity of TNF did not correlate with its antiproliferative activity. In lines whose migration was stimulated by both TNF and scatter factor (SF), a fibroblast-derived cytokine which stimulates epithelial cell motility, saturating concentrations of TNF plus SF induced greater migration than either agent alone. Anti-TNF monoclonal antibody blocked TNF- but not SF-stimulated motility. While various other factors (basic fibroblast growth factor, interleukin 6, interleukin 2, colony-stimulating factor 1) had little or not motility-stimulating activity, phorbol-12-myristate-13-acetate (PMA), a tumor-promoting phorbol ester, scattered and/or stimulated migration in all cell lines studied. Combinations of saturating concentrations of TNF plus PMA or of SF plus PMA induced greater migration than did any agent alone. These findings suggest that (a) carcinoma cell motility may be mediated by multiple biochemical pathways and (b) TNF stimulates epithelial motility by a mechanism different from that of SF and PMA. In vivo, TNF might enhance invasiveness of some carcinomas or stimulate epithelial wound healing.

Stimulation of mammary tumorigenesis by systemic tissue inhibitor of matrix metalloproteinase 4 gene delivery.

Tissue inhibitors of matrix metalloproteinase (TIMPs) are multifunctional proteins with both matrix metalloproteinase (MMP) inhibitory effects and growth-regulatory activity. TIMPs inhibit MMP activity, suggesting a use for cancer gene therapy. However, here we report that systemic administration of human TIMP-4 by electroporation-mediated i.m. injection of naked TIMP-4 DNA stimulates tumorigenesis of human breast cancer cells in nude mice. Consistent with tumor stimulation, TIMP-4 up-regulates Bcl-2 and Bcl-X(L) protein. TIMP-4 also inhibits apoptosis in human breast cancer cells in vitro and mammary tumors in vivo. A synthetic MMP inhibitor BB-94 did not have such antiapoptotic effect. Analysis of TIMP-4 expression in human mammary specimens indicates that TIMP-4 protein is increased in mammary carcinoma cells compared with normal mammary epithelial cells. These data indicate an antiapoptotic activity in breast cancer cells and a tumor-stimulating effect of TIMP-4 when administrated systemically.

Mutant BRCA1 genes antagonize phenotype of wild-type BRCA1.

Unregulated expression of wild-type BRCA1 (wtBRCA1) confers an altered phenotype in cultured human prostate cancer cells, characterized by chemosensitivity, susceptibility to apoptosis, decreased DNA repair activity, and alterations of key cell regulatory proteins. We now report that the expression of truncated or mutant full-length BRCA1 genes can abrogate certain phenotypic characteristics and/or confer the opposite phenotype to the wild-type BRCA1 gene. In particular, several carboxyl-terminal truncated BRCA1 proteins conferred chemoresistance, decreased susceptibility to apoptosis, and decreased ability to suppress in vivo tumor growth. These truncated BRCA1 proteins also blocked the ability of ectopically expressed wtBRCA1 to induce chemosensitivity and to inhibit estrogen receptor transcriptional activity. Studies using epitope-tagged truncated proteins confirmed their expression, nuclear localization, and functionality. On the other hand, in cells with no endogenous wild-type BRCA1 (HCC1937 human breast cancer cells), the wtBRCA1 gene enhanced cellular DNA repair activity and rendered the cells resistant to DNA damage; while truncated BRCA1 proteins blocked the wtBRCA1-induced chemoresistance. Our findings suggest that truncated BRCA1 proteins can inhibit the function of wild-type BRCA1. They raise the possibility that some inherited BRCA1 mutations may actively promote oncogenesis by blocking the function of the remaining wild-type BRCA1 allele, although this hypothesis remains to be proved.

The cytokine hepatocyte growth factor/scatter factor inhibits apoptosis and enhances DNA repair by a common mechanism involving signaling through phosphatidyl inositol 3' kinase.

Scatter factor (SF) [aka. hepatocyte growth factor (HGF)] (designated HGF/SF) is a multifunctional cytokine that stimulates tumor cell invasion and angiogenesis. We recently reported that HGF/SF protects epithelial and carcinoma cells against cytotoxicity from DNA-damaging agents and that HGF/SF-mediated cytoprotection was associated with up-regulation of the anti-apoptotic protein Bcl-XL in cells exposed to adriamycin. We now report that in addition to blocking apoptosis, HGF/SF markedly enhances the repair of DNA strand breaks caused by adriamycin or gamma radiation. Constitutive expression of Bcl-XL in MDA-MB-453 breast cancer cells not only simulated the HGF/SF-mediated chemoradioresistance, but also enhanced the repair of DNA strand breaks. The ability of HGF/SF to induce both chemoresistance and DNA repair was inhibited by wortmannin, suggesting that these activities of HGF/SF are due, in part, to a phosphatidylinositol-3'-kinase (PI3K) dependent signaling pathway. Consistent with this finding, HGF/SF induced the phosphorylation of c-Akt (protein kinase-B), a PI3K substrate implicated in apoptosis inhibition; and an expression vector encoding a dominant negative kinase inactive Akt partially but significantly inhibited HGF/SF-mediated cell protection and DNA repair. These findings suggest that HGF/SF activates a cell survival and DNA repair pathway that involves signaling through PI3K and c-Akt and stabilization of the expression of Bcl-XL; and they implicate Bcl-XL in the DNA repair process.

BRCA1 as a potential human prostate tumor suppressor: modulation of proliferation, damage responses and expression of cell regulatory proteins.

In addition to breast and ovarian cancer in women, recent evidence suggests that germ-line mutations of the breast cancer susceptibility gene-1 (BRCA1) also confer an increased life-time risk for prostate cancer in male probands. However, it is not known if and how BRCA1 functions in prostate cancer. We stably expressed wild-type (wt) and tumor-associated mutant BRCA1 transgenes in DU-145, a human prostate cancer cell line with low endogenous expression of BRCA1. As compared with parental cells and vector transfected clones, wtBRCA1 clones exhibited: (1) a slightly decreased proliferation rate (doubling time = 25 h as compared with 22 h for control cells); (2) a (3-6)-fold increase in sensitivity to chemotherapy drugs (adriamycin, camptothecin, and taxol); (3) increased susceptibility to drug-induced apoptosis; (4) reduced repair of single-strand DNA strand breaks; and (5) alterations in expression of key cellular regulatory proteins (including BRCA2, p300, Mdm-2, p21(WAF1/CIP1), Bcl-2 and Bax). Clones transfected with the 5677insA breast cancer-associated mutant BRCA1 (insBRCA1) displayed a similar phenotype to wtBRCA1 clones, except that insBRCA1 clones had a significantly decreased proliferation rate (doubling time = 42 h). On the other hand, cells transfected with with 185delAG mutant BRCA1 showed no obvious phenotype as compared with parental or vector transfected cells. These findings suggest that BRCA1 may function as a human prostate tumor suppressor by virtue of its ability to modulate proliferation and various components of the cellular damage response. They also suggest several potential target gene products for a BRCA1 prostate tumor suppressor function.

Regulation of BRCA1 and BRCA2 expression in human breast cancer cells by DNA-damaging agents.

Germline mutations in the breast cancer susceptibility genes BRCA1 and BRCA2 have been linked to the development of breast cancer, ovarian cancer, and other malignancies. Recent studies suggest that the BRCA1 and BRCA2 gene products may function in the sensing and/or repair of DNA damage. To investigate this possibility, we determined the effects of various DNA-damaging agents and other cytotoxic agents on the mRNA levels of BRCA1 and BRCA2 in the MCF-7 and other human breast cancer cell lines. We found that several agents, including adriamycin (a DNA intercalator and inhibitor of topoisomerase II), camptothecin (a topoisomerase I inhibitor), and ultraviolet radiation induced significant decreases in BRCA1 and BRCA2 mRNA levels. Decreased levels of BRCA1 and BRCA2 mRNAs were observed within 6-12 h after treatment with adriamycin and persisted for at least 72 h. Adriamycin also induced decreases in BRCA1 protein levels; but these decreases required several days. U.V. radiation induced dose-dependent down-regulation of BRCA1 and BRCA2 mRNAs, with significant decreases in both mRNAs at doses as low as 2.5 J/m2, a dose that yielded very little cytotoxicity. Adriamycin-induced down-regulation of BRCA1 and BRCA2 mRNAs was first observed at doses that yielded relatively little cytotoxicity and little or no apoptotic DNA fragmentation. Adriamycin and U.V. radiation induced distinct dose- and time-dependent alterations in the cell cycle distribution; but these alterations did not correlate well with corresponding changes in BRCA1 and BRCA2 mRNA levels. However, the adriamycin-induced reduction in BRCA1 and BRCA2 mRNA levels was correlated with p53 functional status. MCF-7 cells transfected with a dominant negative mutant p53 (143 val-->ala) required at least tenfold higher doses of adriamycin to down-regulate BRCA1 and BRCA2 mRNAs than did parental MCF-7 cells or control-transfected MCF-7 clones. These results suggest that BRCA1 and BRCA2 may play roles in the cellular response to DNA-damaging agents and that there may be a p53-sensitive component to the regulation of BRCA1 and BRCA2 mRNA expression.

Scatter factor protects epithelial and carcinoma cells against apoptosis induced by DNA-damaging agents.

Scatter factor (SF) (hepatocyte growth factor) is a cytokine that may play a role in human breast cancer invasiveness and angiogenesis. We now report that SF can block the induction of apoptosis by various DNA damaging-agents, including cytotoxic agents used in breast cancer therapy. SF protected MDA-MB-453 human breast cancer cells, EMT6 mouse mammary tumor cells and MDCK renal epithelial cells against apoptosis induced by adriamycin (ADR), X-rays, ultraviolet radiation, and other agents. Protection was observed in assays of DNA fragmentation, cell viability (MTT), and clonogenic survival. Protection of MDA-MB-453 cells against ADR was dose- and time-dependent; maximal protection required pre-incubation with 75-100 ng/ml of SF for 48 h or more. Protection required functional SF receptor (c-Met), but was not dependent on p53. Western blotting analysis revealed that pre-treatment of MDA-MB-453 cells with SF inhibited the ADR-induced decreases in the levels of Bcl-XL, an anti-apoptotic protein related to Bcl-2; and the dose-response and time course characteristics for SF-mediated increases in the Bcl-XL protein levels of ADR-treated cells were consistent with the degrees of protection against apoptosis observed under the same conditions. Furthermore, Bcl-XL levels were not down-regulated by ADR in MDA-MB-231 breast cancer cells, consistent with the finding that SF failed to protect these cells against ADR, despite the fact that they contain functional c-Met receptor. In contrast to Bcl-XL, SF blocked ADR-induced increases in c-Myc and inhibited the expression of p21WAF1/CIP1 and of the BRCA1 protein in MDA-MB-453 cells. However, SF did not cause significant changes in the cell cycle distribution of ADR-treated cells. These findings suggest that SF-mediated protection of human breast cancer cells may involve inhibition of one or more pathways required for the activation of apoptosis and may particularly target the anti-apoptotic mitochondrial membrane pore-forming protein Bcl-XL as a component of the protective mechanism. By implication, the accumulation of SF within human breast cancers may contribute to the development of a radio- or chemoresistant phenotype.