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.

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.

Computation of radiation dose distributions for shielded cervical applicators.

While cervical applicators with shielded ovoids are used widely in brachytherapy, we know of no system for calculating dose distributions for them. For shielded sources, because of a lack of symmetry and because of a rapid variation of dose as a function of position relative to the source, extensive measured data in three dimensions are required. In the method we have developed, the dose at a given point from a source in a shielded ovoid is calculated by multiplying the dose from an unshielded source by the "effective attenuation factor" of the shields. The latter quantity is obtained by linear-interpolation in a three-dimensional table generated from measurements described in an earlier paper. The unshielded-source dose is calculated as the product of source strength, time of implant, distance-dependent geometry factor and a tabulated quantity called the "relative dose rate factor". Relative dose rate factor is obtained by dividing measured dose rate by the product of geometry factor and source strength. Division by the geometry factor reduces the amount of data required with respect to accuracy in linear-interpolation. Input localization data must include not only the position of the end points defining the source but also a third reference point to define the orientation of the shields.

Measurements of radiation dose distributions for shielded cervical applicators.

Cervical applicators with shielded ovoids are employed to reduce dose to the rectum and bladder. Because of asymmetries introduced by the shields, dose distribution calculations for individual patients will require extensive computer reference data for the ovoid sources. Requisite 3-D dose distributions were measured for an unshielded and a shielded ovoid containing a Cs-137 source, using a computerized system employing a diode in a water phantom. The probe stops at each measurement point and accumulates dose for several seconds. The system automates horizontal positioning of the detector and angular motion of the ovoid to obtain dose in one plane. The detector is moved manually to other planes for a complete three dimensional set of measurements. In order to suppress the energy and directional dependence of the diode, final dose distributions are calculated from ratios of shielded to unshielded data in conjunction with independently measured TLD data for unshielded sources.

Arbitrary oblique image sections for 3-D radiation treatment planning.

Methods for selecting and computing arbitrary image sections for displaying anatomic and isodose information for three-dimensional treatment planning are investigated. Selection of the desired plane may be made by defining a plane that is perpendicular to an existing image section (called the base image) and passing through a line on the base image. Alternatively, the anatomic structures displayed perspectively in three dimensions as a series of contours that can be rotated and translated may be used to define an arbitrary plane for image reconstruction. The viewing screen is considered to be the plane of interest. As a typical three-dimensional image of 30 to 60 sections requires considerable computer storage (on the order of 25 megabytes), a reconstruction algorithm may need extensive memory space or CPU and disk I/O time. Of the schemes examined, we believe the following is the most efficient. One pair of images is read from the disk at a time in sequence and intersections of the rows of the cutting plane with the box formed by the consecutive images are computed. Pixel values of all points between the given images are computed by interpolation. Special cases, such as the cutting plane being parallel to or coincident with an existing image, must be considered separately.

MCF-7 breast cancer cells overexpressing transfected c-erbB-2 have an in vitro growth advantage in estrogen-depleted conditions and reduced estrogen-dependence and tamoxifen-sensitivity in vivo.

A c-erbB-2 expression vector was transfected into the estrogen receptor positive (ER+) MCF-7 human breast cancer cell line to determine if overexpression of this transmembrane tyrosine kinase could increase the malignant phenotype of this cell line. Loss of transfected c-erbB-2 expression was observed when cells were carried in medium containing estrogen. Homogeneous populations stably overexpressing levels of the 185 kDa c-erbB-2 observed in the SKBR-3 a breast cancer cell line which overexpresses c-erbB-2 as a result of gene amplification could be obtained by continually maintaining the transfected cell lines in estrogen-free conditions. Levels of constitutively activated c-erbB-2 varied among clonal isolates. Whereas some overexpressing lines did acquire the ability to form transient tumor nodules in ovariectomized nude mice without estrogen supplementation, as well as in mice that received the antiestrogen tamoxifen, one cell line that exhibited the highest levels of constitutively activated c-erbB-2 was able to form static tumors of a larger size under both conditions. This same cell line formed progressively growing tumors in estrogen-supplemented mice that were much larger than observed in mice injected with control cell lines, and also showed reduced sensitivity to antiestrogens in vitro, but it continued to have a low metastatic phenotype. These results suggest that signal transduction mediated by the c-erbB-2 tyrosine kinase can partially overcome the estrogen dependence of ER+breast cancer cells for growth and that c-erbB-2 overexpression confers a selective advantage to such cells in the absence of estrogen.

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.