Borrelia and Chlamydia Can Form Mixed Biofilms in Infected Human Skin Tissues.

Our research group has recently shown that Borrelia burgdorferi, the Lyme disease bacterium, is capable of forming biofilms in Borrelia-infected human skin lesions called Borrelia lymphocytoma (BL). Biofilm structures often contain multiple organisms in a symbiotic relationship, with the goal of providing shelter from environmental stressors such as antimicrobial agents. Because multiple co-infections are common in Lyme disease, the main questions of this study were whether BL tissues contained other pathogenic species and/or whether there is any co-existence with Borrelia biofilms. Recent reports suggested Chlamydia-like organisms in ticks and Borrelia-infected human skin tissues; therefore, Chlamydia-specific polymerase chain reaction (PCR) analyses were performed in Borrelia-positive BL tissues. Analyses of the sequence of the positive PCR bands revealed that Chlamydia spp. DNAs are indeed present in these tissues, and their sequences have the best identity match to Chlamydophila pneumoniae and Chlamydia trachomatis. Fluorescent immunohistochemical and in situ hybridization methods demonstrated the presence of Chlamydia antigen and DNA in 84% of Borrelia biofilms. Confocal microscopy revealed that Chlamydia locates in the center of Borrelia biofilms, and together, they form a well-organized mixed pathogenic structure. In summary, our study is the first to show Borrelia-Chlamydia mixed biofilms in infected human skin tissues, which raises the questions of whether these human pathogens have developed a symbiotic relationship for their mutual survival.

Evidence of In Vivo Existence of Borrelia Biofilm in Borrelial Lymphocytomas.

Lyme borreliosis, caused by the spirochete Borrelia burgdorferi sensu lato, has grown into a major public health problem. We recently identified a novel morphological form of B. burgdorferi, called biofilm, a structure that is well known to be highly resistant to antibiotics. However, there is no evidence of the existence of Borrelia biofilm in vivo; therefore, the main goal of this study was to determine the presence of Borrelia biofilm in infected human skin tissues. Archived skin biopsy tissues from borrelial lymphocytomas (BL) were reexamined for the presence of B. burgdorferi sensu lato using Borrelia-specific immunohistochemical staining (IHC), fluorescent in situ hybridization, combined fluorescent in situ hybridization (FISH)-IHC, polymerase chain reaction (PCR), and fluorescent and atomic force microscopy methods. Our morphological and histological analyses showed that significant amounts of Borrelia-positive spirochetes and aggregates exist in the BL tissues. Analyzing structures positive for Borrelia showed that aggregates, but not spirochetes, expressed biofilm markers such as protective layers of different mucopolysaccharides, especially alginate. Atomic force microscopy revealed additional hallmark biofilm features of the Borrelia/alginate-positive aggregates such as inside channels and surface protrusions. In summary, this is the first study that demonstrates the presence of Borrelia biofilm in human infected skin tissues.

Effectiveness of Stevia Rebaudiana Whole Leaf Extract Against the Various Morphological Forms of Borrelia Burgdorferi in Vitro.

Lyme disease is a tick-borne multisystemic disease caused by Borrelia burgdorferi. Administering antibiotics is the primary treatment for this disease; however, relapse often occurs when antibiotic treatment is discontinued. The reason for relapse remains unknown, but recent studies suggested the possibilities of the presence of antibiotic resistant Borrelia persister cells and biofilms. In this study, we evaluated the effectiveness of whole leaf Stevia extract against B. burgdorferi spirochetes, persisters, and biofilm forms in vitro. The susceptibility of the different forms was evaluated by various quantitative techniques in addition to different microscopy methods. The effectiveness of Stevia was compared to doxycycline, cefoperazone, daptomycin, and their combinations. Our results demonstrated that Stevia had significant effect in eliminating B. burgdorferi spirochetes and persisters. Subculture experiments with Stevia and antibiotics treated cells were established for 7 and 14 days yielding, no and 10% viable cells, respectively compared to the above-mentioned antibiotics and antibiotic combination. When Stevia and the three antibiotics were tested against attached biofilms, Stevia significantly reduced B. burgdorferi forms. Results from this study suggest that a natural product such as Stevia leaf extract could be considered as an effective agent against B. burgdorferi.

Comparison of transpulmonary thermodilution, transthoracic echocardiography and conventional hemodynamic monitoring in neonates and infants after open heart surgery: a preliminary study.

BACKGROUND: Transpulmonary thermodilution (TPTD) is an increasingly popular method used to monitor the complex hemodynamic changes in critically ill children. The purpose of our study was to examine the relationship between transthoracic echocardiographic (TTE) parameters and global hemodynamic variables derived from TPTD and those derived from conventional measurements in infants and neonates undergoing corrective cardiac surgery. METHODS: After approval from the Ethics Committee of Gottsegen György Hungarian Institute of Cardiology and individual parental consent were obtained, patients were prospectively enrolled. In parallel with continuous postoperative conventional monitoring, TPTD was measured four times daily, and TTE was performed once per day. Conventional hemodynamic, TPTD and TTE parameters were compared with weighted linear regression statistics and a Pearson correlation. RESULTS: One hundred forty-five TPTD measurements and 35 TTE examinations of thirteen enrolled patients were analyzed. Global end-diastolic volume index (GEDVI) was correlated with the fractional shortening (SF, r=0.67, P=0.001) measured by TTE. Among the preload parameters, the percentage change of GEDVI between two consecutive time points showed a pertinent correlation with changes of cardiac index (r=0.67, P=0.001) and changes of stroke volume index (r=0.57, P=0.008). Percentage changes in SF demonstrated a strong negative correlation with changes of left ventricular end-systolic diameter (r=-0.86, P<0.001). There was no significant relationship between alterations in arterial or central venous pressure values with TTE or TPTD parameters. CONCLUSION: Both TPTD and TTE may be used in the estimating volumetric preload parameters. The time course of TPTD-derived parameters may have clinical relevance in pediatric critical care practice.

The use of the L-plastin promoter for adenoviral-mediated, tumor-specific gene expression in ovarian and bladder cancer cell lines.

A 2.4-kb truncated L-plastin promoter was inserted either 5' to the LacZ gene (Ad-Lp-LacZ) or 5' to the cytosine deaminase (CD) gene (Ad-Lp-CD) in a replication-incompetent adenoviral vector backbone. Infectivity and cytotoxicity experiments with the LacZ and CD vectors suggested that the L-plastin promoter-driven transcriptional units were expressed at much higher levels in explants of ovarian cancer cells from patients and in established ovarian or bladder cancer cell lines than they were in normal peritoneal mesothelial cells from surgical specimens, in organ cultures of normal ovarian cells, or in the established CCD minimal deviation fibroblast cell line. Control experiments showed that this difference was not attributable to the lack of infectivity of the normal peritoneal cells, the normal ovarian cells, or the minimal deviation CCD fibroblast cell line, because these cells showed expression of the LacZ reporter gene when exposed to the replication-incompetent adenoviral vector carrying the cytomegalovirus (CMV)-driven LacZ gene (Ad-CMV-LacZ). The Ovcar-5 and Skov-3 ovarian cancer cell lines exposed to the Ad-Lp-CD adenoviral vector were much more sensitive to the prodrug 5-fluorocytosine (5FC), which is converted from the 5FC prodrug into the toxic chemical 5-fluorouracil, than was the CCD minimal deviation fibroblast cell line after exposure to the same vector. A mouse xenograft model was used to show that the Ad-Lp-CD vector/5FC system could prevent engraftment of ovarian cancer cells in nude mice. Finally, injection of the Ad-Lp-CD vector into s.c. tumor nodules generated a greater reduction of the size of the tumor nodules than did injection of the Ad-CMV-LacZ vectors into tumor nodules. The Ad-Lp-CD vectors were as suppressive to tumor growth as the Ad-CMV-CD vectors. These results suggest that an adenoviral vector carrying the CD gene controlled by the L-plastin promoter (Ad-Lp-CD) may be of potential value for the i.p. therapy of ovarian cancer.

RU-486 can abolish glucocorticoid-induced increases in CSF-1 receptor expression in primary human breast carcinoma specimens.

OBJECTIVE: We present the results of the application of organ culture techniques previously described in this journal to the study of steroid hormone responsiveness of primary breast carcinoma specimens. METHODS AND RESULTS: Nearly all breast carcinomas that express macrophage colony-stimulating factor-1R (CSF-1R) at tissue harvest (15 of 18) had levels of CSF-1R expression lowered after incubation in steroid-free media. The decrease in CSF-1R expression was reversed by treatment with glucocorticoids; this glucocorticoid-induced increase in CSF-1R expression can be blocked by mifepristone (RU-486), a competitive inhibitor of glucocorticoid action. CONCLUSION: These results demonstrate that steroid hormone responsiveness of primary breast carcinomas can be assayed in vitro, a result which can not only be employed to better predict the responsiveness of breast carcinomas to therapies with steroid hormone agonists and antagonists, but also suggests that the therapeutic utility of mifepristone in breast cancer deserves further study.

Roles of Fas and Fas ligand during mammary gland remodeling.

Mammary involution is associated with degeneration of the alveolar structure and programmed cell death of mammary epithelial cells. In this study, we evaluated the expression of Fas and Fas ligand (FasL) in the mammary gland tissue and their possible role in the induction of apoptosis of mammary cells. FasL-positive cells were observed in normal mammary epithelium from pregnant and lactating mice, but not in nonpregnant/virgin mouse mammary tissue. Fas expression was observed in epithelial and stromal cells in nonpregnant mice but was absent during pregnancy. At day 1 after weaning, high levels of both Fas and FasL proteins and caspase 3 were observed and coincided with the appearance of apoptotic cells in ducts and glands. During the same period, no apoptotic cells were found in the Fas-deficient (MRL/lpr) and FasL-deficient (C3H/gld) mice. Increase in Fas and FasL protein was demonstrated in human (MCF10A) and mouse (HC-11) mammary epithelial cells after incubation in hormone-deprived media, before apoptosis was detected. These results suggest that the Fas-FasL interaction plays an important role in the normal remodeling of mammary tissue. Furthermore, this autocrine induction of apoptosis may prevent accumulation of cells with mutations and subsequent neoplastic development. Failure of the Fas/FasL signal could contribute to tumor development.

Absence of estrogen receptor-beta expression in metastatic ovarian cancer.

OBJECTIVE: To evaluate the expression of estrogen receptor (ER)alpha and ERbeta mRNA and protein in normal ovarian tissue and primary and metastatic tumors. METHODS: Estrogen receptor alpha and ERbeta expression was studied in normal ovarian biopsies (n = 9) and primary (n = 8) and metastatic ovarian epithelial cancers (n = 8). Ovarian tissue was collected from surgical samples. Estrogen receptor alpha and ERbeta mRNA expression was compared by coamplification of the mRNA of the ERs. Expression was confirmed at the protein level by Western blot analysis using antibodies specific for each receptor. RESULTS: Among eight primary ovarian cancer samples, three had only ERalpha, two had only ERbeta, and three had both. All eight metastatic ovarian cancer tissues expressed only ERalpha mRNA and protein. Biopsies from normal ovaries had ERalpha and ERbeta mRNA and protein. Two of the ovarian epithelial cancer samples were paired and showed the same results. CONCLUSION: We found varying amounts of ERalpha and ERbeta in normal ovaries, lower levels of ERbeta expression in ovarian epithelial cancer primary tumors, and only ERalpha in metastatic tumors. Our findings indicate that a fundamental difference might exist between primary and metastatic cells, which could be caused by intrinsic or extrinsic factors that regulate ER gene expression.

Effect of all-trans-retinoic acid on c-fms proto-oncogene [colony-stimulating factor 1 (CSF-1) receptor] expression and CSF-1-induced invasion and anchorage-independent growth of human breast carcinoma cells.

Abnormal expression of c-fms proto-oncogene, which encodes for the macrophage colony-stimulating factor-1 (CSF-1) receptor, has been observed in a variety of carcinomas of epithelial origin, including those of the breast. Here, we have investigated the effect of retinoic acid (RA), an important regulator of normal differentiation of mammary epithelial tissues, on the expression of the c-fms gene and CSF-1/CSF-1 receptor-induced invasion and anchorage-independent growth in breast carcinoma cells. We have demonstrated that all-trans-RA (atRA) significantly increases levels of c-fms transcripts in the estrogen receptor-negative but RA receptor alpha-positive breast carcinoma cell lines BT20 and SKBR3. The atRA-induced increase in fms transcript levels was completely abolished by RO41-5253, a synthetic RA receptor alpha antagonist. Our results indicate that atRA could enhance fms expression by up-regulating the activity of the first promoter of the fms gene. DNase I protection, mobility shift, and mutational analysis revealed that a potential activator protein 1 (AP-1) site in the first fms promoter sequence could mediate the observed atRA effect on fms transcription. Our results also showed that atRA, by itself and in the presence of CSF-1, can increase the ability of breast carcinoma cells to invade in vitro. Furthermore, we demonstrated that atRA is able to abolish the CSF-1-induced increase in anchorage-independent growth of breast carcinoma cells without affecting the anchorage-dependent growth. In summary, our findings suggest that retinoids may play conflicting roles throughout breast cancer progression, depending on the stage of cancer development. Although retinoids might suppress growth at the early stages of tumor formation, they might promote malignant transformation at later stages by stimulating the invasive capacity of certain cell variants in the breast tumor population.

Signal transduction pathways regulated by CSF-1 receptors modulate the in vitro radiosensitivity of mammary epithelial cells.

PURPOSE: CSF-1 and its receptor have both been previously implicated in the basic biology and clinical course of mammary and female reproductive tract neoplasms. A recent study (1) demonstrated that expression of this receptor correlated with local relapse in early-stage breast cancer patients. In this communication, we investigated the role that this receptor/ligand pair plays in modulating cellular responses to ionizing radiation in a mammary epithelial cell line HC11. METHODS AND MATERIALS: The radiosensitivity of HC11 clonal cells transfected to overexpress either the wild-type CSF-1 receptor or CSF-1 receptor mutated at one of the two major autophosphorylation sites (TYR-->PHE 807 or TYR-->PHE 721) was quantitated by standard in vitro clonogenic assays. RESULTS: We demonstrated that a signal transduction pathway regulated by the phosphorylation of TYR-807 of CSF-1 receptor appears to play a major role in controlling the radiosensitivity of murine mammary epithelial cells. CONCLUSIONS: Our observations offer insights into potential pharmacologic and gene-therapeutic approaches for the modification of radiation response of mammary neoplasms.

The role of CSF-1 in normal and neoplastic breast physiology.

Colony stimulating factor (CSF-1) and its receptor (CSF-1R, product of c-fms proto-oncogene) were initially implicated as essential for normal monocyte development as well as for trophoblastic implantation. However, recent findings have suggested that CSF-1 and CSF-1R might have additional roles in mammary gland development during pregnancy and lactation. Studies with osteopetrotic (op-/op-) mice, which bear a specific mutation that inactivates the CSF-1 gene, demonstrated that op-/op- mothers are incapable of normal milk production due to the incomplete development of their mammary glands during pregnancy. Also, significant increases in the levels of CSF-1 and CSF-1R proteins are observed in the epithelial cells of mammary gland during pregnancy and lactation. In vitro studies investigating the effect of the three major lactogenic hormones (prolactin, insulin, and glucocorticoids) on the expression of CSF-1 and CSF-1R have demonstrated that expression of CSF-1 can be regulated by prolactin and insulin whereas CSF-1R expression is regulated by glucocorticoids. This apparent role for CSF-1/CSF-1R in normal mammary gland development is very intriguing because this receptor/ligand pair has also been found to be important in the biology of breast cancer, where they regulate tumor cell invasion by a urokinase-dependent mechanism. This review aims to summarize recent findings on the role of CSF-1 and its receptor in normal and neoplastic mammary development which may elucidate potential relationships of growth factor-induced biological changes in the breast during pregnancy and tumor progression.

Prognostic significance of colony-stimulating factor receptor expression in ipsilateral breast cancer recurrence.

The macrophage colony-stimulating factor receptor (CSF-1R), the product of the c-fms proto-oncogene, regulates normal proliferation and differentiation of macrophages and trophoblasts. Recent research found abnormal expression of CSF-1R in human carcinomas of the breast, endometrium, and ovary. Furthermore, activation of CSF-1R by its ligand has been shown to regulate invasiveness and anchorage-independent growth in breast carcinoma cells. To study the significance of CSF-1R expression in breast cancer, we designed a case-controlled immunohistochemical study. We chose 80 patients from a database of 1200 early stage I or II breast cancer patients treated with conservative surgery and radiation therapy. Expression of CSF-1R in the tumors of 40 patients who experienced an ipsilateral breast tumor recurrence (IBTR) as a primary site of relapse were compared with 40 patients who had not experienced an IBTR. The index and control patients were matched by age, clinical stage, nodal status, and follow-up. Paraffin-embedded sections were immunostained with antibodies directed toward CSF-1R. For the CSF-1R antibody, a total of 28 index cases (70%) demonstrated strong staining, whereas only 16 control cases (40%) demonstrated high immunoreactivity (P = 0.007). The CSF-1R antibody showed a positive correlation for local relapse, but no correlation was found between CSF-1R expression and distant metastasis. In summary, our findings provide evidence for the poor prognostic role of CSF-1R in IBTR.

Ets-2 transdominant mutant abolishes anchorage-independent growth and macrophage colony-stimulating factor-stimulated invasion by BT20 breast carcinoma cells.

Activation of the macrophage colony-stimulating factor receptor (CSF-1R) by its cognate ligand CSF-1 dramatically increases the tumorigenicity and invasive potential of both normal and neoplastic mammary epithelial cells. Recent studies have suggested that the Ets2 transcription factor plays a central role in mediating CSF-1R-induced mitogenesis in fibroblasts. To determine whether the Ets2 transcription factor can also mediate CSF-1- and CSF-1R-stimulated signaling pathways in mammary epithelial cells, we expressed a dominant negative mutant, Ets2, in the CSF-1R- and Ets2-positive BT20 breast carcinoma cell line and examined its effects on CSF-1-induced cellular invasion and on colony formation in soft agar. Our data show that stable expression of the mutant Ets2 in BT20 cells completely inhibits the formation of soft agar colonies and abolishes the CSF-1-stimulated invasion of these cells through a barrier of reconstituted basement membrane (Matrigel). We have also demonstrated that the expression of this Ets2 mutant is capable of interrupting the CSF-1R-regulated intracellular signaling pathways by inhibiting CSF-1-induced c-myc, c-fos, and c-jun expression in BT20 cells. Our results are the first demonstration of an important role for the Ets2 transcription factor in the regulation of the anchorage-independent growth and cellular invasiveness of neoplastic mammary epithelial cells.

Expression of CSF-I and CSF-I receptor by normal lactating mammary epithelial cells.

OBJECTIVE: Previous studies suggested a potential role for macrophage colony stimulating factor (CSF-1) in lactogenic differentiation of the breast. The aim of this study was to define the regulation of CSF-1 and its receptor (CSF-1R, the product of c-fms proto-oncogene) by lactogenic hormones in the breast in vivo during pregnancy and lactation and in vitro in organ culture and mammary epithelial cell lines. METHODS: Immunohistochemical staining assays for the expression of CSF-1 and CSF-1R antigens were performed on sections of breast biopsies from nonpregnant (n = 10), prepartum (n = 4), and postpartum lactating patients (n = 7) and on sections of human mammary glands cultured in the presence of the lactogenic hormones insulin, prolactin, and glucocorticoids. Northern blot analyses were used to study the regulation of CSF-1 and CSF-1R by these same lactogenic hormones in normal and neoplastic mammary epithelial cell lines in cell culture. RESULTS: Normal, nonlactating mammary epithelium did not express CSF-1R and synthesized only low levels of CSF-1. During lactation, significant levels of both proteins could be observed in the epithelial cells that line actively lactating ducts and alveoli. Very similar increases in epithelial cell expression of CSF-1 and CSF-1R were observed in organ cultures of normal mammary gland biopsies exposed to prolactin, insulin, and glucocorticoids. Colony stimulating factor mRNA levels were increased by prolactin and/or insulin in a normal mammary epithelial cell line, while glucocorticoids had no apparent effect on CSF-1 mRNA levels. In contrast, we found that the levels of CSF-1R transcript are regulated primarily by glucocorticoids in breast carcinoma cells, while prolactin merely modulates the glucocorticoid effect. CONCLUSION: The observed increases in the expression of CSF-1 and its receptor during lactogenesis and the regulation of CSF-1/CSF-1R by lactogenic hormones suggests that this cytokine/receptor pair might play a regulatory role in the cellular events leading to the lactogenic differentiation of mammary epithelial cells.

Recognition of activated CSF-1 receptor in breast carcinomas by a tyrosine 723 phosphospecific antibody.

The macrophage colony stimulating factor receptor (CSF-1R), the product of the c-fms proto-oncogene, plays an important role in regulating the normal proliferation and differentiation of macrophages and trophoblasts. However, the abnormal expression of CSF-1R transcripts and protein by human breast carcinomas has been shown to correlate with advanced stage and poor prognosis. Ligand activated CSF-1R dimers transphosphorylate several tyrosines in their cytoplasmic domains which provide recognition sites for various effector proteins in multiple signal transduction pathways. In cells transformed by the c-fms oncogene, one of the major CSF-1R phosphotyrosines, pTyr723 is important for phenotypic expression of anchorage-independent growth and metastasis. In order to investigate the relationship between receptor activation/phosphorylation and cellular phenotypes in vitro and in vivo, we prepared a CSF-1R phosphorylation-state specific antibody raised against a specific phosphopeptide of CSF-1R, which included phosphorylated tyrosine 723. On immunoblots of lysates from cells expressing CSF-1R, this antibody recognizes phosphorylated CSF-IR in CSF-1 stimulated cells but not in unstimulated cells. As an immunohistochemical reagent, this antibody stained 52% of invasive human breast tumors (72% of CSF-1R positive cases) in a sample of 114 cases and 38% of carcinoma in situ. This data represents the first direct evidence of in vivo phosphorylation of CSF-1R in human breast carcinomas.

[Successful multi-step management of developmental heart defects after intrauterine diagnosis]. / Intrauterin diagnosztizált összetett szívfejlódési rendellenesség többlépcsós sikeres sebészi korrigálása.

At 28th week of gestation a conotruncal malformation with ventricular septal defect was diagnosed by fetal echocardiography. Postnatal echocardiographic and angiocardiographic examinations confirmed the diagnosis of conotruncal malformation (pulmonary atresia, ventricular septal defect, patent ductus arteriosus, aortopulmonary collateral arteries). The unifocalization (age: 11 months) and total correction with aortic homograft (age: 7 years) were performed. To our knowledge our case is the first whose intrauterine diagnosis of complex congenital heart disease was confirmed after delivery and had successful two-stage surgical management.

Evidence for a role for DNA polymerase beta in mammalian meiosis.

DNA polymerase beta (pol beta) is an enzyme possessing both polymerase and deoxyribose phophatase activities. Although pol beta is not believed to participate in the replication of genomic DNA, several studies have indicated a role for pol beta in DNA repair. The high level of expression of pol beta in mouse and rat testes raises the possibility that pol beta participates in mammalian meiosis. Using antibody localization, we detect foci that stain with pol beta antisera at discrete sites along homologous chromosomes as they synapse and progress through prophase of meiosis I. These data suggest that pol beta participates in meiotic events associated with synapsis and recombination.

[Total cavo-pulmonary anastomosis in heart surgery for the correction of univentricular heart]. / Teljes cavopulmonalis anasztomózis az univentricularis szív sebészi korrekciójában.

The total cavopulmonary anastomosis is one of the alternative surgical procedures which can be performed in the management of the most complicated congenital heart diseases. It was the first time in Hungary that this surgical management was performed successfully in a girl with univentricular heart, pulmonary valve stenosis, malposition of the great arteries, who was operated on palliative procedure previously.

Independent regulation of invasion and anchorage-independent growth by different autophosphorylation sites of the macrophage colony-stimulating factor 1 receptor.

Invasion of tissue by macrophages and implantation into the uterine wall by placental trophoblasts are known to be regulated by the macrophage colony-stimulating factor (CSF-1) and its receptor (CSF-1R, the product of the c-fms proto-oncogene). Recently, the clinical importance of CSF-1 and CSF-1R in invasive breast carcinoma has been recognized, but the significance of coexpression of CSF-1 and CSF-1R in mammary epithelial cell invasion has not been explored. In the present study, we investigated the invasive potential of a noninvasive, CSF-1R-negative, mouse mammary epithelial cell line (HC11) expressing a high level of CSF-1, which was stably transfected with the mouse wild-type CSF-1R. Compared with parental cells, transfected cells expressing a wild-type CSF-1R invaded 100-fold more efficiently through a barrier of reconstituted basement membrane (Matrigel) and formed colonies in soft agar, whereas the cellular growth rate was only slightly increased. Analysis of cell-conditioned medium by zymography and quantitative enzyme activity assays showed that clones transfected with a wild-type CSF-1R expressed significantly higher levels of urokinase-type plasminogen activator than did untransfected clones. Furthermore, after injection into the tail veins of BALB/c mice, CSF-1R-expressing clones also produced a 10-fold higher incidence of lung tumors than the parental cell line. We also analyzed HC11 clones transfected with CSF-1R mutated at two major autophosphorylation sites (Tyr-->Phe807 and Tyr-->Phe721). Mutation at Tyr807 eradicated the stimulatory effect of Fms expression on the invasive ability of HC11 cells and substantially reduced the metastatic potential of the transfected clones but did not alter the Fms-induced anchorage-independent growth in soft agar. In contrast, mutation at Tyr721 of Fms had no effect on invasion as measured in the in vitro assay but markedly abolished Fms-induced colony formation in soft agar and eradicated the metastatic potential of the transfected clones. Our results suggest that expression of CSF-1R can facilitate cellular invasion and anchorage-independent growth in mammary epithelial cells, and these two processes are independently regulated by separate phosphotyrosine sites of CSF-1R.

Transcriptional regulation of the c-fms (CSF-1R) proto-oncogene in human breast carcinoma cells by glucocorticoids.

Expression of the macrophage colony stimulating factor CSF-1 and its receptor, the c-fms proto-oncogene, has been observed in macrophages, trophoblast and in a variety of neoplasms of epithelial origin including those of the breast. We have reported earlier (Oncogene, 1991, 6: 941-952) that c-fms transcript and protein expression were dramatically increased in several breast carcinoma cell lines by glucocorticoids which are essential humoral regulators of normal mammary epithelial cell differentiation. In this communication, we demonstrate that levels of c-fms transcript and protein increased significantly within the first few hours of glucocorticoid treatment, and that these increases were completely abolished by pretreatment of cells with mifepristone (RU486). We also demonstrate that such early increases in c-fms transcript levels could not be attributed to prolongation of transcript half-life. Both promoters of the c-fms gene were found to exhibit some basal activity in breast carcinoma cell lines and both were stimulated 2-3-fold by glucocorticoids. However the first promoter was shown to be responsible for more than 95% of the observed c-fms transcription. Sequence upstream of both promoters was found to contain potential 'glucocorticoid response elements' (GREs), and in each case, elimination of the GRE closest to the promoter abolished glucocorticoid stimulation. Our observations suggest that one mechanism by which glucocorticoids regulate the proliferation and differentiation of neoplastic mammary epithelial cells is through their regulation of transcription of the gene for the receptor of a ubiquitous cytokine, CSF-1.