Isolation of tumor-derived endothelial cells.
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Animals↗
Biomedical subjects
S W McLeskey
Publications and source records attributed to S W McLeskey.
The Syk tyrosine kinase suppresses malignant growth of human breast cancer cells.
Syk is a protein tyrosine kinase that is widely expressed in haematopoietic cells. It is involved in coupling activated immunoreceptors to downstream signalling events that mediate diverse cellular responses including proliferation, differentiation and phagocytosis. Syk expression has been reported in cell lines of epithelial origin, but its function in these cells remains unknown. Here we show that Syk is commonly expressed in normal human breast tissue, benign breast lesions and low-tumorigenic breast cancer cell lines. Syk messenger RNA and protein, however, are low or undetectable in invasive breast carcinoma tissue and cell lines. Transfection of wild-type Syk into a Syk-negative breast cancer cell line markedly inhibited its tumour growth and metastasis formation in athymic mice. Conversely, overexpression of a kinase-deficient Syk in a Syk-positive breast cancer cell line significantly increased its tumour incidence and growth. Suppression of tumour growth by the reintroduction of Syk appeared to be the result of aberrant mitosis and cytokinesis. We propose that Syk is a potent modulator of epithelial cell growth and a potential tumour suppressor in human breast carcinomas.
Animals↗
Expression and function of angiopoietin-1 in breast cancer.
Angiopoietin-1 (Ang1) has been shown to act as an angiogenic promoter in embryonic angiogenesis by promoting vascular branching, pericyte recruitment and endothelial survival. We have investigated the role of Ang1 in tumour neovascularization under clinical conditions and in animal models. The expression of Ang1 in clinical breast cancer specimens was analysed by using laser-capture microdissection and reverse transcriptase-linked polymerase chain reaction (RT-PCR) on RNA isolated from the samples. Despite the expression of Ang1 in many human breast cancer cell lines, the gene was expressed in only three of 21 breast cancer clinical specimens, even though its receptor, Tie2, is abundant in the vasculature of all of these tumours. Ang1 was then overexpressed in a human breast cancer cell line (MCF-7) on its own and in conjunction with FGF1, an angiogenic factor shown to be able to increase the tumorigenicity of MCF-7 cells. High concentrations of Ang1 were produced in the conditioned media of the transfected cells (range 156-820 ng ml(-1)). However, in contrast to its physiological role as promoter of angiogenesis, overexpression of Ang1 did not enhance tumour growth, but instead caused up to a 3-fold retardation of tumour growth (P = 0.003).
Angiopoietin-1↗
Fibroblast growth factors, their receptors and signaling.
Fibroblast growth factors (FGFs) are small polypeptide growth factors, all of whom share in common certain structural characteristics, and most of whom bind heparin avidly. Many FGFs contain signal peptides for secretion and are secreted into the extracellular environment, where they can bind to the heparan-like glycosaminoglycans (HLGAGs) of the extracellular matrix (ECM). From this reservoir, FGFs may act directly on target cells, or they can be released through digestion of the ECM or the activity of a carrier protein, a secreted FGF binding protein. FGFs bind specific receptor tyrosine kinases in the context of HLGAGs and this binding induces receptor dimerization and activation, ultimately resulting in the activation of various signal transduction cascades. Some FGFs are potent angiogenic factors and most play important roles in embryonic development and wound healing. FGF signaling also appears to play a role in tumor growth and angiogenesis, and autocrine FGF signaling may be particularly important in the progression of steroid hormone-dependent cancers to a hormone-independent state.
Animals↗
Overexpression of fibroblast growth factor 1 in MCF-7 breast cancer cells facilitates tumor cell dissemination but does not support the development of macrometastases in the lungs or lymph nodes.
Mice bearing primary tumors produced by LacZ-tagged MCF-7 human breast carcinoma cells transfected with fibroblast growth factor (FGF) 1 have frequent micrometastases, but macrometastases are not observed. i.v. injection of FGF-1-transfected tumor cells produced no pulmonary macrometastases, and removal of primary tumors resulted in the disappearance of spontaneous micrometastases. Thus, failure of micrometastases to proliferate was not due to inhibitory factors released from the primary tumor, and the presence of the primary tumor is required for maintenance of the micrometastases. This indicates that the micrometastases result from continued seeding from the primary tumor balanced by clearance from the metastatic site. Tumor emboli trapped in the vessels of lungs and lymph nodes and single tumor cells observed in the pulmonary vein implied that FGF-1-overexpressing MCF-7 cells are deficient in their ability to extravasate. The frequency of tumor cells incorporating bromodeoxyuridine was consistently lower in lung tissues when compared with primary tumors, indicating that disseminated tumor cells were unable to maintain a high rate of proliferation. Increased angiogenesis resulting from FGF-1 production by the transfected cells with a concomitant increased rate of intravasation into developing blood vessels may be the underlying determinant of spontaneous micrometastasis produced by these cells when compared with parental MCF-7 cells.
Animals↗
Tumor growth of FGF or VEGF transfected MCF-7 breast carcinoma cells correlates with density of specific microvessels independent of the transfected angiogenic factor.
We have previously shown that fibroblast growth factor (FGF)-1-, FGF-4-, or vascular endothelial growth factor (VEGF/VPF)-transfected MCF-7 breast carcinoma cells growing as tumors in nude mice are tamoxifen resistant and/or estrogen independent. These transfectants provide opportunity for study of in situ tumor-induced angiogenesis promoted by the individual angiogenic factors under growth-promoting versus growth-inhibiting hormonal conditions. In the present study, vessels in tumors harvested at varying times after tumor cell injection were immunohistochemically highlighted and vessel morphology and topography were scored on a scale of 0 to 4 by blinded observers. In tumors produced by all cell lines under all growth-promoting hormonal conditions, there was significantly increased abundance (P < 0.05) of edge-associated and intratumor microvessels, but not of stromally located microvessels, when compared with tumor nodules harvested under growth-inhibiting conditions, regardless of the identity of the angiogenic factor or the hormonal treatment. Image analysis of bromodeoxyuridine (BrdU)-labeled nuclei of tumors produced by all cell lines under all hormonal conditions harvested at early time points showed that mean labeling indices were highest for hormonal conditions that produced the most robust growth in that particular cell line, implying that a high BrdU labeling index is a predictor of future tumor growth in individual tumors. These results confirm previous studies that established the importance of neovascularization for tumor growth and provide validation for use of these cell lines to study the process of angiogenesis in vivo. Study of gene expression in endothelial cells in edge-associated or intratumor vessels using this model might reveal mechanisms important in tumor-induced angiogenesis in human breast cancer.
Animals↗
Tamoxifen-resistant fibroblast growth factor-transfected MCF-7 cells are cross-resistant in vivo to the antiestrogen ICI 182,780 and two aromatase inhibitors.
Although the antiestrogen tamoxifen has been the mainstay of therapy for estrogen receptor (ER)-positive breast cancer, successful treatment of responsive tumors is often followed by the acquisition of tamoxifen resistance. Subsequently, only 30-40% of patients have a positive response to second hormonal therapies. This lack of response might be explained by mechanisms for tamoxifen resistance that sensitize ER pathways to small amounts of estrogenic activity present in tamoxifen or that bypass ER pathways completely. To elucidate one possible mechanism of tamoxifen resistance, we treated ovariectomized tumor-bearing mice injected with fibroblast growth factor (FGF)-transfected MCF-7 breast carcinoma cells with the steroidal antiestrogen ICI 182,780 or one of two aromatase inhibitors, 4-OHA or letrozole. These treatments did not slow estrogen-independent growth or prevent metastasis of tumors produced by FGF-transfected MCF-7 cells in ovariectomized nude mice. FGF-transfected cells had diminished responses to ICI 182,780 in vitro, suggesting that autocrine activity of the transfected FGF may be replacing estrogen as a mitogenic stimulus for tumor growth. ER levels in FGF transfectants were not down-regulated, and basal levels of transcripts for estrogen-induced genes or of ER-mediated transcription of estrogen response element (ERE) luciferase reporter constructs in the FGF expressing cells were not higher than parental cells, implying that altered hormonal responses are not due to down-regulation of ER or to FGF-mediated activation of ER. These studies indicate that estrogen independence may be achieved through FGF signaling pathways independent of ER pathways. If so, therapies directed at the operative mechanism might produce a therapeutic response or allow a response to a second course of antiestrogen treatment.
Androstenedione↗
Understanding latex allergy.
Latex allergy is an increasingly important problem in both health-care workers and patients. Predisposing factors to development of latex allergies include a history of atopy or allergy and frequent exposure to latex products. Identified allergens include latex proteins from the rubber tree that remain in manufactured products, as well as smaller molecules that remain from the latex purification and manufacturing process. Latex proteins absorbed to powder in latex surgical and examination gloves may be aerosolized and inhaled. Powder-absorbed latex proteins are thought to be important in triggering of sensitization in susceptible individuals, as well as in elicitation of symptoms in previously sensitized patients. Allergic reactions to latex can include local dermal reactions or generalized immediate hypersensitivity (anaphylactic) reactions. Pathophysiology, signs and symptoms, and treatment of each type of reaction are discussed. Measures to address latex allergy, however, must include measures to decrease exposure to latex antigens both in latex-allergic subjects, to prevent symptoms, and in naive subjects, to prevent sensitization. These measures may include finding, acceptable substitutes for latex in many products.
Anti-Inflammatory Agents↗
Latex allergy and gloving standards.
In response to universal precautions, latex gloves are used more frequently in health care settings, and more health care workers are reporting latex sensitivity or allergic reactions to natural rubber latex (NRL). As a result, there continues to be controversy about what gloves should be used in clinical practice and what are the appropriate clinical recommendations about gloving practices. This article reviews the current issues about gloving practices, addresses national standards related to gloving, and makes recommendations about future gloving practices.
Gloves, Surgical↗
MCF-7 breast carcinoma cells overexpressing FGF-1 form vascularized, metastatic tumors in ovariectomized or tamoxifen-treated nude mice.
FGF-1 is expressed in a high proportion of breast tumors. While overexpression of FGF-4 in the MCF-7 breast carcinoma cell line confers the ability to form spontaneously metastasizing tumors in ovariectomized nude mice without estrogen supplementation and in mice that receive tamoxifen pellets, the response of a cell to individual FGFs can be controlled at multiple levels, and the significance of FGF-1 expression in human breast tumors is uncertain. To study the role of FGF-1, MCF-7 human breast cancer carcinoma cells, previously transfected with bacterial beta-galactosidase, were retransfected with FGF-1 expression vectors. FGF-1 transfectants formed large, vascularized tumors in ovariectomized nude mice without estrogen supplementation as well as in mice that received tamoxifen pellets. Lymphatic and pulmonary micrometastases were detected as deposits of X-gal-stained cells as early as 17 days after cell inoculation whereas no metastases were detected in estrogen-supplemented mice bearing similar-sized control tumors. When compared with controls, both clonal and polyclonal populations of FGF-1 overexpressing cells exhibited increased anchorage-independent growth and decreased population doubling times in estrogen-depleted or 4-hydroxytamoxifen containing medium. These results suggest that FGF signaling may be important in the transition of breast cancer cells from hormone-dependent to hormone-independent and from nonmetastatic to metastatic.
Animals↗
Fibroblast growth factor overexpressing breast carcinoma cells as models of angiogenesis and metastasis.
Progression of breast cancer from an estrogen-dependent, slowly growing tumor amenable to tamoxifen treatment to an aggressive, metastatic, estrogen-independent phenotype has been mimicked by the transfection of MCF-7 breast carcinoma cells with fibroblast growth factors 1 or 4. FGF-transfected cells are aggressively tumorigenic in ovariectomized or tamoxifen-treated nude mice, conditions under which the parental cells would not produce tumors. When detection of metastasis was enhanced by lacZ transfection, the FGF-transfected MCF-7 cells were reliably metastatic to lymph nodes and frequently metastatic to lungs, in further contrast to parental cells. An antiangiogenic drug, AGM-1470, given to mice bearing tumors produced by FGF-transfected MCF-7 cells, produced a decrease in tumor size. The decreased tumor size was not as marked as that produced by treatment with pentosan polysulfate, an agent which would abrogate all autocrine or paracrine effects of the transfected FGF. Thus, increased angiogenesis may be a component of the phenotypic change produced by the FGF transfection, but other autocrine or paracrine effects may also be important. Since a clonal FGF-4 and lacZ doubly-transfected cell line, MKL-4, progressively lost expression of the transfected lacZ gene in individual cells, we performed successive rounds of fluorescence-activated cell sorting to select high-expressing cells. High-expressing cell populations thus obtained rapidly lost expression of beta-gal activity in continued culture. High beta-gal expressing clonal cell lines of MKL-4 cells established by either one or two rounds of low-density cloning also lost lacZ expression with continued culture. Southern analysis of DNA from lacZ transfected cell lines showed the transfected sequences to be present and grossly intact in both high and low expressing populations. However, Northern analysis revealed that high-expressing populations of MKL-4 cells contained the most lacZ mRNA, implying that in the unstable MKL-4 cell line, individual cells are down-regulating mRNA levels of lacZ. Stable lacZ expression has been obtained in other FGF-transfected and parental MCF-7 cell lines using the same expression vector. Thus, the MKL-4 cell line is down-regulating mRNA encoding the transfected gene through a mechanism not dependent on the CMV promotor utilized in the expression vector. This evidence suggests that lacZ expression is not a benign modification in certain cells.
Animals↗
Effects of AGM-1470 and pentosan polysulphate on tumorigenicity and metastasis of FGF-transfected MCF-7 cells.
Previously, we described FGF-1- or FGF-4-transfected MCF-7 breast carcinoma cells which are tumorigenic and metastatic in untreated or tamoxifen-treated ovariectomised nude mice. In this study, we have assessed the effects of AGM-1470, an antiangiogenic agent, and pentosan polysulphate (PPS), an agent that abrogates the effects of FGFs, on tumour growth and metastasis produced by these FGF-transfected MCF-7 cells. Untreated or tamoxifen-treated ovariectomised mice were injected with FGF-transfected cells, treated with AGM-1470 or PPS, and tumour growth and metastasis analysed. The sensitivity of FGF-transfected and parental MCF-7 cells to AGM-1470 or PPS was also determined in vitro. Both AGM-1470 and PPS inhibited tumour growth in otherwise untreated or tamoxifen-treated mice injected with either FGF- or FGF-4-transfected MCF-7 cells. This effect was more reliably seen in tamoxifen-treated animals. AGM-1470 was about 10(5) times less potent in inhibiting the anchorage-dependent growth of parental MCF-7 or FGF-transfected MCF-7 cells than in inhibiting the growth of human umbilical vein endothelial cells. PPS did not affect the in vitro growth of the transfectants or parental cells. Thus, the growth-inhibitory effect on tumours was in excess of the effect of either drug on the same cells in tissue culture, implying that stromal elements are important determinants of the effects of these drugs. There was a positive correlation between tumour size and the extent of proximal lymph node metastasis. However, neither drug had a significant effect on the extent of metastasis to proximal or distal lymph nodes or lungs. AGM-1470 or PPS may be helpful in cases of breast carcinoma in which angiogenesis is due to expression of FGFs by the tumour cells and may be more effective when combined with tamoxifen.
Animals↗
Fibroblast growth factor-4 enhanced G2 arrest and cell survival following ionizing radiation.
Fibroblast growth factors (FGFs) bind to cell membrane receptors and activate signal transduction pathways related to cell growth, angiogenesis, and tumorigenesis. FGFs have been shown to be abundantly expressed in some of the human tumors, which are known to be poorly responsive to radiation therapy. Using adrenal cortical carcinoma cells genetically engineered to express FGF-4, we have tested cellular survival following exposure to ionizing radiation. We report here that FGF-4 enhances cellular capacity to survive ionizing radiation. Furthermore, cell cycle analysis shows a pronounced increase in the duration of G2 arrest, suggesting perturbation of a cell cycle checkpoint. These findings implicate fibroblast growth factor-mediated signal transduction in cellular resistance of human tumors to radiation therapy.
Cell Survival↗
MDA-MB-134 breast carcinoma cells overexpress fibroblast growth factor (FGF) receptors and are growth-inhibited by FGF ligands.
Overexpression of some transmembrane tyrosine kinase growth factor receptors in breast and other tumors has been found to correlate with poor prognosis. Following the cloning of the first two members of the fibroblast growth factor family of receptors (FGFRs), amplification of these receptors in breast carcinomas was found. We have examined 23 breast carcinoma cell lines to determine the extent of expression of mRNA for fgfrs 1 through 4. All breast carcinoma cell lines examined expressed mRNA for at least one fgfr and several expressed high mRNA levels for a particular receptor. MDA-MB-134, an estrogen receptor-positive cell line, expressed very high levels of mRNA for fgfr-1 and elevated levels of mRNA for fgfr-4. This cell line was found to have an amplified fgfr-1 gene, but the gene for fgfr-4 was not amplified. MDA-MB-453 cells were found to express high levels of mRNA for fgfr-4 without amplification of the gene. MDA-MB-134 cells were examined for their response to FGF ligands. Tyrosine phosphorylation of a M(r) 150,000 protein resulted when MDA-MB-134 cells were treated with FGF-1 or FGF-2, implying the presence of a functional FGFR-1. MDA-MB-134 cells were growth-inhibited by picomolar concentrations of FGF-1 or FGF-2 in a dose-dependent manner under both anchorage-independent and anchorage-dependent conditions. These results may provide insight into the consequences of FGFR overexpression in breast tumors and the development of treatment modalities which use manipulation of growth factor responses.
Antibodies↗
Transfected MCF-7 cells as a model for breast-cancer progression.
The MCF-7 human breast carcinoma cell line has been used as a recipient for eukaryotic plasmid expression vectors to determine the effects of growth factor and growth factor receptor overexpression on the estrogen-dependent, antiestrogen sensitive and poorly metastatic phenotypes exhibited by this line. Overexpression of some members of the erbB family of ligands and receptors were found to have some effects on these phenotypes. However, only when two members of the fibroblast growth factor family, FGF-1 and FGF-4, were overexpressed was progressive in vivo growth observed is either ovariectomized nude mice without estrogen supplementation or in mice that received tamoxifen treatment. FGF transfected cells also exhibited an increased ability to form micrometastases. The implications of these results with regard to the possible role of the paracrine and autocrine effects of angiogenic growth factor production in breast cancer progression are discussed.
Adenocarcinoma↗
Emergence of MCF-7 cells overexpressing a transfected epidermal growth factor receptor (EGFR) under estrogen-depleted conditions: evidence for a role of EGFR in breast cancer growth and progression.
Overexpression of epidermal growth factor receptor (EGFR) is correlated with loss of estrogen receptor and poor prognosis in breast cancer. To investigate this phenomenon, we transfected a cytomegalovirus expression vector directing the expression of EGFR into estrogen receptor-positive MCF-7 breast cancer cells and into a clone of MCF-7 cells previously transfected with transforming growth factor alpha. Cells arising from single clones or pooled polyclonal populations maintained in charcoal-stripped calf serum, a medium devoid of estrogen, overexpressed EGFR. Switching these cells to a medium containing fetal calf serum or charcoal-stripped calf serum plus 17 beta-estradiol resulted in the emergence of a population expressing low EGFR levels. Loss of expression was not a consequence of nonspecific repression of the cytomegalovirus promoter, because expression of the fibroblast growth factor (FGF)-4 complementary DNA in a similar vector was not lost in fetal calf serum. While loss of EGFR overexpression in fetal calf serum was seen at both the protein and mRNA levels, Southern blotting shows that this was not due to loss of the transfected gene. Subclones of a cell population with low EGFR expression were capable of increasing expression upon estrogen withdrawal, demonstrating that the changes in EGFR expression were reversible and suggesting a growth advantage conferred by EGFR overexpression under these restrictive growth conditions. Overexpression of EGFR did not result in loss of ER expression. These results suggest a role for overexpression of EGFR in the growth of estrogen receptor-positive breast cancer cells in the absence of estrogen.
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Fibroblast growth factor 4 transfection of MCF-7 cells produces cell lines that are tumorigenic and metastatic in ovariectomized or tamoxifen-treated athymic nude mice.
Successful antiestrogen treatment in patients with tamoxifen-responsive breast tumors is often followed by an outgrowth of tumors cells that are antiestrogen resistant, implying that estrogen-dependent tumors can become estrogen-independent. In an effect to mimic this progression, we have transfected fibroblast growth factor 4 into MCF-7 cells, a human breast carcinoma cell line that is estrogen-dependent for growth in nude mice. This transfection results in cell lines that form progressively growing, metastatic tumors when injected s.c. into untreated or tamoxifen-treated ovariectomized nude mice. In contrast to the parental cell line, growth of transfected cells in ovariectomized nude mice is stimulated by tamoxifen treatment and inhibited by estrogen treatment of the mice. Parental MCF-7 cells were transfected with an expression vector for beta-galactosidase, conferring the ability to convert the chromogenic substrate, 5-bromo-4-chloro-3-indoyl-beta-galactoside, to a blue color and allowing the detection of their presence within tumors developing after coinoculation with fibroblast growth factor 4-transfected cells. The fibroblast growth factor 4-transfected cells could support growth and metastasis of the beta-galactosidase-expressing parental cell line when both lines were coinjected into the same site in untreated or tamoxifen-treated, ovariectomized mice. These data suggest a possible role for fibroblast growth factors in the progression of breast tumors to an estrogen-independent, antiestrogen-resistant, metastatic phenotype. They also support a role for paracrine factors in mixed populations of tumor cells of differing states of malignant progression.
Animals↗
Quantitative demonstration of spontaneous metastasis by MCF-7 human breast cancer cells cotransfected with fibroblast growth factor 4 and LacZ.
We recently established transfectants of MCF-7 human breast cancer cells with fibroblast growth factor 4 (fgf-4) that showed rapid growth and spontaneous metastasis in ovariectomized and tamoxifen-treated nude mice. To establish a spontaneous metastatic model of human breast cancer cells in nude mice with a sensitive marker for detection of micrometastasis, the transfection of fgf-4 was combined with transfection of the bacterial lacZ gene encoding beta-galactosidase. MKL-4 cells, a lacZ transfectant of an fgf-4-transfected cell line, showed the same level of fgf-4 expression as parental cells and expressed a high level of beta-galactosidase activity. When MKL-4 cells were injected s.c. into female nude mice, rapidly growing tumors developed. Whole organ staining for beta-galactosidase activity was able to detect even small numbers of metastatic tumor cells. Micrometastases in lymph nodes, lung, and brain were detected 3 weeks after the tumor cell injections, the first time point tested. Within 12 weeks, metastases were observed in lymph nodes, lung, brain, kidney, perirenal fatty tissues, liver, spleen, retroperitoneum, heart, and gallbladder. The frequency of metastasis and number of foci were correlated with the volume of the primary tumors. The distribution of metastatic sites was similar to that in breast cancer patients. MKL-4 cells may be a useful model for studying the malignant progression of hormone-dependent breast cancer, antimetastatic drugs, or early events in metastasis.
Animals↗