Membrane-associated binding sites for estrogen contribute to growth regulation of human breast cancer cells.

Membrane-associated binding sites for estrogen may mediate rapid effects of estradiol-17beta that contribute to proliferation of human breast cancers. After controlled homogenization and fractionation of MCF-7 breast cancer cells, the bulk of specific estradiol binding is found in nuclear fractions. However, a significant portion of specific, high-affinity estradiol-17beta binding-sites are also enriched in plasma membranes. These estradiol binding-sites co-purify with 5'-nucleotidase, a plasma membrane-marker enzyme, and are free from major contamination by cytosol or nuclei. Electrophoresis of membrane fractions allowed detection of a primary 67-kDa protein and a secondary 46-kDa protein recognized by estradiol-17beta and by a monoclonal antibody directed to the ligand-binding domain of the nuclear form of estrogen receptor. Estrogen-induced growth of MCF-7 breast cancer cells in vitro was blocked by treatment with the antibody to estrogen receptor and correlated closely with acute hormonal activation of mitogen-activated protein kinase and Akt kinase signaling. Estrogen-promoted growth of human breast cancer xenografts in nude mice was also significantly reduced by treatment in vivo with the estrogen receptor antibody. Thus, membrane-associated forms of estrogen receptor may play a role in promoting intracellular signaling for hormone-mediated proliferation and survival of breast cancers and offer a new target for antitumor therapy.

Steroid hormone receptors in target cell membranes.

Numerous reports of rapid steroid hormone effects in diverse cell types cannot be explained by the generally prevailing theory that centers on the activity of hormone receptors located exclusively in the nucleus. Cell membrane forms of steroid hormone receptors coupled to intracellular signaling pathways may also play an important role in hormone action. Membrane-initiated signals appear to be the primary response of the target cell to steroid hormones and may be prerequisite to subsequent genomic activation. Recent dramatic advances in this area have intensified efforts to delineate the nature and biologic roles of all receptor molecules that function in steroid hormone-signaling pathways. This work has profound implications for our understanding of the physiology and pathophysiology of hormone actions in responsive cells and may lead to development of novel approaches for the treatment of many cell proliferative, metabolic, inflammatory, reproductive, cardiovascular, and neurologic defects.

Epidermal growth factor receptor and tyrosine phosphorylation of estrogen receptor.

Activation of estrogen receptor-alpha (ERalpha) by growth factors in the absence of estrogen is a well-documented phenomenon. To study further this process of ligand-independent receptor activation, COS-7 cells without ER were transfected with both ER and epidermal growth factor receptor (EGFR). In the absence of estrogen, epidermal growth factor (EGF) stimulated rapid tyrosine phosphorylation of ER in transfected COS-7 cells. Similarly, in MCF-7 breast cancer cells that have natural expression of ER and EGFR, EGF promoted acute phosphorylation of serine and tyrosine residues in ER, and a direct interaction between ER and EGFR after treatment with EGF was found. In confirmation of a direct interaction between ER and EGFR, activation of affinity-purified EGFR tyrosine kinase in vitro stimulated the phosphorylation of recombinant ER. The cross-communication between EGFR and ER appears to promote significant stimulation of cell proliferation and a reduction in the apoptotic loss of those cells that express both receptor signaling pathways. However, COS-7 cells transfected with both ER and EGFR show minimal stimulation of classical estrogen response element (ERE)-dependent transcriptional activity after stimulation by EGF ligand. This suggests that the proliferative and antiapoptotic activity of EGF-induced ER activation may be dissociated from ERE-dependent transcriptional activity of the ER.

Biologic effects of heregulin/neu differentiation factor on normal and malignant human breast and ovarian epithelial cells.

The heregulins are a family of ligands with ability to induce phosphorylation of the p185HER-2/neu receptor. Various investigators have reported a variety of responses of mouse and human breast and ovarian cells to this family of ligands including growth stimulation, growth inhibition, apoptosis and induction of differentiation in cells expressing the HER-2/neu receptor. Some of the disparity in the literature has been attributed to variations in the cell lines studied, ligand dose applied, methodologies utilized or model system evaluated (i.e. in vitro or in vivo). To evaluate the effects of heregulin on normal and malignant human breast and ovarian epithelial cells expressing known levels of the HER-2/neu receptor, this report presents the use of several different assays, performed both in vitro and in vivo, in vitro proliferation assays, direct cell counts, clonogenicity under anchorage-dependent and anchorage-independent conditions, as well as the in vivo effects of heregulin on human cells growing in nude mice to address heregulin activity. Using a total of five different biologic assays in nine different cell lines, across two different epithelia and over a one log heregulin dose range, we obtained results that clearly indicate a growth-stimulatory role for this ligand in human breast and ovarian epithelial cells. We find no evidence that heregulin has any growth-inhibitory effects in human epithelial cells. We also quantitated the amount of each member of the type I receptor tyrosine kinase family (RTK I, i.e. HER-1, HER-2, HER-3 and HER-4) in the cell lines employed and correlated this to their respective heregulin responses. These data demonstrate that HER-2/neu overexpression itself affects the expression of other RTK I members and that cells expressing the highest levels of HER-2/neu have the greatest response to HRG.

Monoclonal antibody to HER-2/neureceptor modulates repair of radiation-induced DNA damage and enhances radiosensitivity of human breast cancer cells overexpressing this oncogene.

The management of human breast cancer frequently includes radiation therapy as an important intervention, and improvement in the clinical efficacy of radiation is desirable. Overexpression of the HER-2 growth factor receptor occurs in 25-30% of human breast cancers and correlates with poor clinical outcome, including earlier local relapse following conservative surgery accompanied by radiation therapy. In breast cancer cells with overexpression of HER-2 receptor, recombinant humanized monoclonal antibodies (rhuMAbs) to HER-2 receptors (rhuMAb HER-2) decrease cell proliferation in vitro and reduce tumor formation in nude mice. Therapy with rhuMAb HER-2 enhances tumor sensitivity to radiation at doses of 1-5 Gy, exceeding remission rates obtained with radiation alone. This benefit is specific to cells with HER-2 overexpression and does not occur in cells without overexpression. Treatment of cells with radiation (2-4 Gy) alone provokes a marked increase in unscheduled DNA synthesis, a measure of DNA repair, but HER-2-overexpressing cells treated with a combination of rhuMAb HER-2 and radiation demonstrate a decrease of unscheduled DNA synthesis to 25-44% of controls. Using an alternate test of DNA repair, i.e., radiation-damaged or undamaged reporter DNA, we introduced a cytomegalovirus-driven beta3-galactosidase into HER-2-overexpressing breast cancer cells that had been treated with rhuMAb HER-2 or control. At 24 h posttransfection, the extent of repair assayed by measuring reporter DNA expression was high after exposure to radiation alone but significantly lower in cells treated with combined radiation and rhuMAb HER-2 therapy. To further characterize effects of rhuMAb HER-2 and the combination of antibody and radiation on cell growth, analyses of cell cycle phase distribution were performed. Antibody reduces the fraction of HER-2-overexpressing breast cancer cells in S phase at 24 and 48 h. Radiation treatment is also known to promote cell cycle arrest, predominantly at G1, with low S-phase fraction at 24 and 48 h. In the presence of rhuMAb HER-2, radiation elicits a similar reduction in S phase at 24 h, but a significant reversal of this arrest appears to begin 48 h postradiation exposure. The level of S-phase fraction at 48 h is significantly greater than that found at 24 h with the combined antibody-radiation therapy, suggesting that early escape from cell cycle arrest in the presence of antireceptor antibody may not allow sufficient time for completion of DNA repair in HER-2-overexpressing cells. Because it is well known that failure of adequate p21WAF1 induction after DNA damage is associated with failure of cell cycle arrest, we also assessed the activity of this critical mediator of the cellular response to DNA damage. The results show induction of p21WAF1 transcripts and protein product at 6, 12, and 24 h after radiation treatment; however, increased levels of p21WAF1 transcript and protein are not sustained in HER-2-overexpressing cells exposed to radiation in the presence of rhuMAb HER-2. Although transcript and protein levels increase at 6-12 h, they are both diminished by 24 h. Levels of p21WAF1 transcript and protein at 24 h are significantly lower than in cells treated by radiation without antibody. A reduction in the basal level of p21WAF1 transcript also occurred after 12-24 h exposure to antibody alone. The effect of HER-2 antibody may be related to tyrosine phosphorylation of p21WAF1 protein. Tyrosine phosphorylation of p21WAF1 is increased after treatment with radiation alone, but phosphorylation is blocked by combined treatment with antireceptor antibody and radiation. This dysregulation of p21WAF1 in HER-2-overexpressing breast cells after treatment with rhuMAb HER-2 and radiation appears to be independent of p53 expression levels but does correlate with reduced levels of mdm2 protein. (ABSTRACT TRUNCATED)

Remission of human breast cancer xenografts on therapy with humanized monoclonal antibody to HER-2 receptor and DNA-reactive drugs.

HER-2 oncogene encodes a transmembrane growth factor receptor that is overexpressed in 25-30% of patients with primary breast and ovarian cancer. A murine monoclonal antibody, 4D5, to the extracellular domain of HER-2 receptor elicits cytostatic growth inhibition of tumor cells overexpressing HER-2 protein, but clinical use of this antibody is limited by genesis of human anti-mouse antibodies. To avoid this problem, a recombinant humanized 4D5 monoclonal antibody (rhuMAb HER-2) was developed and tested using a human tumor xenograft model. Human breast and ovarian cancer cells which overexpress HER-2 were inhibited in vivo by the rhuMAb HER-2 antibody. Tumor growth relative to control was reduced at all doses of antibody tested, and the magnitude of growth inhibition was directly related to dose of rhuMAb HER-2. Tumor growth resumed on termination of antibody therapy, indicating a cytostatic effect. To elicit a cytotoxic response, human breast tumor xenografts were treated with a combination of antibody and antitumor drugs, cisplatin or doxorubicin. The combination of antibody with either cisplatin or doxorubicin resulted in significantly greater growth inhibition, with the cisplatin combination demonstrating a greater response. In addition, therapy with cisplatin and antireceptor antibody elicited complete tumor remissions after 2-3 cycles of therapy. The schedule of administration of anti-receptor antibody and cisplatin was critical for occurrence of antibody-induced potentiation in cisplatin cytotoxicity. Enhanced killing of tumor cells was found only if antibody and drug were given in close temporal proximity. Since interference with DNA repair pathways may contribute to this receptor-enhanced chemosensitivity, repair of cisplatin-damaged reporter DNA (pCMV-beta) was determined in human breast cells. As in studies of antibody-enhanced cisplatin cytotoxicity in vivo, treatment with rhuMAb HER-2 blocked the repair of cisplatin-damaged DNA only if the antibody was administered in close temporal proximity to transfection of the drug-exposed reporter DNA. An alternative measure of DNA repair, unscheduled DNA synthesis, was also assessed. Treatment with either cisplatin or doxorubicin led to an increase in unscheduled DNA synthesis that was reduced by combined therapy with antireceptor antibody specific to HER-2-overexpressing breast cancer cells. Using a direct measure of DNA repair, therapy of HER-2-overexpressing cells with rhuMAb HER-2 also blocked the removal of cisplatin-induced DNA adducts. Expression of p21/WAF1, an important mediator of DNA repair, was disrupted in breast cancer cells with HER-2 overexpression, but not in control cells, after treatment with HER-2 antibody, thus suggesting cross-communication between the HER-2 signaling and DNA repair pathways. These data demonstrate an in vivo antiproliferative effect of rhuMAb HER-2 on tumors that overexpress HER-2 receptor and a therapeutic advantage in the administration of the antireceptor antibody in combination with chemotherapeutic agents.

The effect of HER-2/neu overexpression on chemotherapeutic drug sensitivity in human breast and ovarian cancer cells.

Recent studies indicate that oncogenes may be involved in determining the sensitivity of human cancers to chemotherapeutic agents. To define the effect of HER-2/neu oncogene overexpression on sensitivity to chemotherapeutic drugs, a full-length, human HER-2/neu cDNA was introduced into human breast and ovarian cancer cells. In vitro dose-response curves following exposure to 7 different classes of chemotherapeutic agents were compared for HER-2- and control-transfected cells. Chemosensitivity was also tested in vivo for HER-2- and control-transfected human breast and ovarian cancer xenografts in athymic mice. These studies indicate that HER-2/neu overexpression was not sufficient to induce intrinsic, pleomorphic drug resistance. Furthermore, changes in chemosensitivity profiles resulting from HER-2/neu transfection observed in vitro were cell line specific. In vivo, HER-2/neu-overexpressing breast and ovarian cancer xenografts were responsive to different classes of chemotherapeutic drugs compared to control-treated xenografts with no statistically significant differences between HER-2/neu-overexpressing and nonoverexpressing xenografts. We found no instance in which HER-2/neu-overexpressing xenografts were rendered more sensitive to chemotherapeutic drugs in vivo. HER-2/neu-overexpressing xenografts consistently exhibited more rapid regrowth than control xenografts following initial response to chemotherapy suggesting that a high rate of tumor cell proliferation rather than intrinsic drug resistance may be responsible for the adverse prognosis associated with HER-2/neu overexpression in human cancers.

The relationship between perceived stress and self-reported illness-related absenteeism.

PURPOSE: To investigate the association between perceived stress and illness-related work absenteeism. DESIGN: A standardized health profile questionnaire developed by Johnson & Johnson Advanced Behavioral Technologies, Inc., was used to collect demographic and personal health data between June 1988 and January 1993. Chi-square, odds ratio, and stepwise regression tests were used to analyze perceived stress and self-reported absenteeism data. SETTING: Worksite health promotion programs in 250 U.S. companies. SUBJECTS: Subjects consisted of 79,070 employees. MEASURES: Stress data, grouped as low, moderate, and high, were correlated with absenteeism data grouped by annual days missed (None, 1 to 2, 3 to 4, and 5+). RESULTS: Significant relationships were found (p < or = .05) between high stress and absenteeism for both genders. Female workers reported higher stress levels and absenteeism than men. Those with high stress were 2.22 more likely to be absent 5+ days per year than those with low stress. Work, finances, and family were the highest stress sources. Greatest absenteeism predictors were health, legal, social, and financial stress. CONCLUSIONS: These data primarily represented self-selected white workers and may not apply to all employees. However, if high stress relates to absenteeism, these data may provide valuable information for program design in stress management.

HER-2 tyrosine kinase pathway targets estrogen receptor and promotes hormone-independent growth in human breast cancer cells.

Growth of human breast cells is closely regulated by steroid hormone as well as peptide hormone receptors. Members of both receptor classes are important prognostic factors in human breast cancer. Clinical data indicate that overexpression of the HER-2 gene is associated with an estrogen receptor-negative phenotype. In this study, we demonstrate that introduction of a HER-2 cDNA, converting non-overexpressing breast cancer cells to those which overexpress this receptor, results in development of estrogen-independent growth which is insensitive to both estrogen and the antiestrogen, tamoxifen. Moreover, activation of the HER-2 receptor in breast cancer cells by the peptide growth factor, heregulin, leads to direct and rapid phosphorylation of ER on tyrosine residues. This is followed by interaction between ER and the estrogen-response elements in the nucleus and production of an estrogen-induced protein, progesterone receptor. In addition, overexpression of HER-2 receptor in estrogen-dependent tumor cells promotes ligand-independent down-regulation of ER and a delayed autoregulatory suppression of ER transcripts. These data demonstrate a direct link between these two receptor pathways and suggest one mechanism for development of endocrine resistance in human breast cancers.

Antibody to HER-2/neu receptor blocks DNA repair after cisplatin in human breast and ovarian cancer cells.

Approximately 30% of human breast and ovarian cancers have amplification and/or overexpression of HER-2/neu gene which encodes a cell surface growth-factor receptor. Overexpression of this receptor, p185HER-2/neu, is associated with poor outcome and may predict clinical response to chemotherapy. Antibodies to HER-2/neu receptor have a cytostatic effect in suppressing growth of cells with overexpression of p185HER-2/neu. To elicit a cytocidal effect, therapy with antireceptor antibody was used in combination with the DNA-damaging drug, cisplatin, and this combined treatment produced a synergistic decrease in cell growth. In addition, antibody mediated an increased sensitivity to cisplatin in drug-resistant ovarian carcinoma cells containing multiple copies of HER-2/neu gene. To evaluate the mechanism for this synergy, unscheduled DNA synthesis was measured in cancer cells using incorporation of [3H]thymidine and autoradiography, and formation and repair of cisplatin-induced DNA adducts was also measured. Treatment with cisplatin led to a marked, dose-dependent increase in unscheduled DNA synthesis which was significantly reduced by combined treatment with antireceptor antibody in HER-2/neu-overexpressing cells. Therapy with antibody to HER-2/neu receptor also led to a 35-40% reduction in repair of cisplatin-DNA adducts after cisplatin exposure and, as a result, promoted drug-induced killing in target cells. This phenomenon which we term receptor-enhanced chemosensitivity may provide a rationale for more selective targeting and exploitation of overexpressed growth factor receptors in cancer cells, thus leading to new strategies for clinical intervention.

Validation of ultrafast computed tomographic left ventricular volume measurement.

Left ventricular volume has been measured with ultrafast computed tomography. However, the accuracy with which this can be done is unknown. We therefore imaged with ultrafast computed tomography 11 rectangular phantoms, 20 to 225 ml, and 17 left ventricular casts, 15 to 112 ml. Two observers planimetered serial tomographic images and computed volume from sequential tomograms. There was no significant inter- or intraobserver difference in measurement of phantoms. Deviation of ultrafast computed tomographic volume from true phantom volume was -0.1 +/- 3.5% SD, range 9.0 to -7.6%. Correlation of true phantom volume with ultrafast computed tomographic volume was 0.99, SEE = 1.9 ml. No significant difference was observed between merged and single ultrafast computed tomographic scanning sequences. Left ventricular cast volume determined by ultrafast computed tomography deviated from true volume by 6% +/- 20%, range 54% to -45%. Correlation of true volume with ultrafast computed tomographic volume was 0.99, SEE = 5.1 ml. There was no interobserver significant difference in measurement of left ventricular cast volume. Correlation between ultrafast computed tomographic volume and cineradiographic volume of the same left ventricular casts was 0.99, SEE = 4.4 ml. Thus, phantom volumes can be measured accurately without significant intra- or interobserver variation. Merged scanning sequences did not influence volume determination. Left ventricular cast volume determination was comparable to that obtained with cineradiography.

Quantitative validation of cineangiographic axial oblique biplane left ventricular volume measurement.

Axial oblique left ventriculography allows unique visualization of acquired and congenital cardiac lesions. However, validation of the accuracy of left ventricular (LV) volume with axial oblique projections is limited and clouded by orthogonal violations between biplane projections. Biplane cineradiographic volume measurement of 17 LV casts employing the axial projection 35 degrees right anterior oblique/55 degrees left anterior oblique/30 degrees cranial (35 degrees RAO/55 degrees LAO/30 degrees Cr) was performed and compared to the conventional postero-anterior/lateral (PA/Lat) and 30 degrees right anterior oblique/60 degrees left anterior oblique (30 degrees RAO/60 degrees LAO) views. LV volume was calculated from biplane cineradiograms by area length and Simpson's rule method. True LV volume by water displacement was 33 +/- 28 (mean +/- S.D.), range 15 to 112 ml. LV cast volume calculated by the area length method from cineradiograms was overestimated (p less than 0.002) but no different by Simpson's rule method (pNS). The ideal correlation was best approximated by the 35 degrees RAO/55 degrees LAO/30 degrees Cr biplane view calculated by Simpson's rule, r = 0.99, y = 3.5 + 0.9x, and standard error of estimate (SEE) = 4.3 ml. Biplane LV angiography with the axial projection permitted accurate LV volume measurement, and Simpson's rule provided the best representation of true volume.

Quantitative validation of cineangiographic biplane axial oblique right ventricular volume measurement.

Axial cranial oblique biplane views of the right ventricle (RV) permit unique assessment of the RV outflow tract, relationship of the great vessels, and anatomy of the proximal pulmonary arteries. This view may also be useful in assessment of RV free wall and RV septal wall motion. However, the accuracy of volumes derived from biplane angiocardiography with the use of the axial cranial oblique projection has not been adequately validated or compared to conventional views. Nineteen RV animal casts whose volume was determined by water displacement were filmed in conventional posteroanterior/lateral (PA/Lat), 30-degree right anterior oblique/60-degree left anterior oblique (30 degrees RAO/60 degrees LAO) views and in angulated 35-degree right anterior oblique/55-degree left anterior oblique/30-degree cranial (35 degrees RAO/55 degrees LAO/30 degrees Cr) view. Tracings of biplane cast images were analyzed for RV volume by Simpson's rule. RV cast volume was significantly overestimated by 12.2 +/- 6.8, 6.0 +/- 5.3, and 9.3 +/- 9.5 ml in PA/Lat, 30 degrees RAO/60 degrees LAO, and 35 degrees RAO/55 degrees LAO/30 degrees Cr views, respectively. The correlation coefficient (r) and the standard error of the estimate (SEE) for true vs calculated volume was 0.96, 0.98, and 0.97, and 6.8, 5.2, and 7.3 ml, respectively, for PA/Lat, 30 degrees RAO/60 degrees LAO, and 35 degrees RAO/55 degrees LAO/30 degrees Cr views. Although there was a high correlation of angiographic volumes with true volume in all three views, the 30 degrees RAO/60 degrees LAO projection had the most ideal regression characteristics with highest r value and the lowest SEE.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative angiographic right and left ventricular volumes.

Comparative angiographic right and left ventricular volumes and right and left ventricular ejection fractions have been reported in the same normal infants and children. This relationship was assessed in adult patients to determine if these pediatric observations persist in later life. Seventeen adults, who had both right and left ventricular angiograms and who had no demonstrable organic heart disease, were studied. Right ventricular end-diastolic volume ranged from 54 to 98 (76 +/- 14, mean +/- SD) cc/m2 and left ventricular end-diastolic volume ranged from 48 to 90 (70 +/- 12) cc/m2; p less than 0.03. Right ventricular end-systolic volume ranged from 22 to 47 (33 +/- 8.0) cc/m2 and left ventricular end-systolic volume ranged from 13 to 34 (22 +/- 5.3) cc/m2; p less than 0.00005. Calculated right ventricular stroke volume ranged from 31 to 60 (43 +/- 8.3) cc/m2 and left ventricular stroke volume ranged from 29 to 70 (48 +/- 11) cc/m2; p = NS. Calculated right ventricular ejection fraction ranged from 0.48 to 0.62 (0.57 +/- 0.04) and the left ventricular ejection fraction ranged from 0.57 to 0.84 (0.68 +/- 0.07; p less than 0.00005. Both right ventricular end-systolic and end-diastolic volumes were greater than left ventricular end-systolic and end-diastolic volumes. This resulted in decreased right ventricular ejection fraction compared to left ventricular ejection fraction. The difference between the two ventricles may be due to compliance, muscle mass, and anatomic configuration with a net result of one chamber more completely emptying than the other. Thus it appears that the relationships between right and left ventricular volumes noted in infancy and childhood persist in adult life.

Specific internalization of estrogen and binding to nuclear matrix in isolated uterine cells.

Fractionation of rat uterine cells incubated at 22 degrees C with 0.2 nM [3H]-estradiol-17 beta (E2 beta) was performed to analyze the subcellular distribution of internalized hormone. The postnuclear supernatant of homogenates was resolved in Percoll density gradients into six major fractions defined by enzyme markers. Within 10 s, E2 beta concentrates at the density of plasma membranes and also at a more buoyant density (p = 1.052 +/- 0.001) with peak accumulation of hormone by 2 min. Thereafter, binding in the latter fraction declines concomitantly with appearance of a portion of hormone at higher densities corresponding to Golgi and lysosomes. E2 beta exhibits preferential accumulation in nuclear matrix from 5 to 60 min. Microfiltration and scanning electron microscopy of the buoyant 2-min peak fractions reveal organelles, 50-200 nm. These may represent endocytotic vesicles that serve as vehicles for nuclear transfer of hormone.