Interaction between hormone-dependent and hormone-independent human breast cancer cells

We developed two different models based on in vitro co-culture of hormone-dependent and hormone-independent cell lines to simulate the cell population heterogeneity of human breast cancer tumours. Oestrogen-dependent (MCF-7, ZR 75.1) and oestrogen-independent cell lines (MDAMB-231 BT-20) were grown under serum-free conditions. Co-culture of hormone-dependent and hormone-independent cell lines resulted in an increased cell yield compared to single cell cultures carried out at the same seeding ratios. Such an increase was not affected by addition of oestradiol and single growth factors (EGF, bFGF and IGF-I). These results allow us to conclude that in a heterogeneous cell population like human breast tumours, interaction between hormone-dependent and hormone-independent cell lines may result in a complex regulation of cell growth.     

Interaction of hyperthermia with Taxol in human MCF-7 breast adenocarcinoma cells.

Hyperthermia treatments (43 degrees C, 1 h) were performed on exponentially growing MCF-7 breast adenocarcinoma cells at the beginning, middle, or end of 24 h incubations of the cells in vitro with Taxol (paclitaxel). When the cells were heated at the beginning or middle of the Taxol incubation, the hyperthermia treatment protected against the toxic effect of each of the Taxol concentrations examined (5, 10 and 100 nM). Consistent with earlier studies, Taxol treatment at 37 degrees C resulted in an accumulation of greater than 94% of the cells in G2/M at 24 h. Heating the cells at the middle or end of the Taxol treatment resulted in a similar accumulation. However, heat treatment during the first hour of Taxol exposure resulted in a significantly smaller percentage of cells (approximately 50%) in G2/M. HPLC analysis showed that at 37 degrees C, Taxol uptake into MCF-7 cells approached maximum within 0.25 h and increased only slightly more over the next 11.75 h. The parental Taxol level was markedly lower by 24 h. In contrast, 1 h hyperthermia treatments at the beginning or middle of the Taxol incubation resulted in higher Taxol concentrations at 12 and 24h, and higher intracellular concentrations overall than at 37 degrees C. These results indicate that hyperthermia inhibits Taxol related cell cycle effects and cytotoxicity, in spite of causing higher concentrations of Taxol to be present in heated cells.     

Heparin coating of human breast carcinoma cells in vivo

Mast cells, the only source of heparin in the body, are common in breast carcinoma. Due to metachromatic staining of heparin-proteoglycans released from the mast cells toluidine blue may stain connective tissue a reddish-purple. This study shows that tumour cells in the vicinity may also be coated with metachromatic substance. Such bl vivo coating does not appear to have been reported previously. In experimental studies in vitro coating has been described following suspension of mouse tumour cells in a heparin solution and preincubation of ascitic tumour cells in heparin has given reduction in survival time after transplantation. In human breast carcinoma stromal metachromasia may under certain circumstances be indicative of poor prognosis. The biological implications of in vivo tumour cell coating with heparin are under investigation.     

Differential expression of glycoproteins containing alpha-D-galactosyl groups on normal human breast epithelial cells and MCF-7 human breast carcinoma cells

Cell surface glycoproteins were isolated from the lysates of 125I-labeled normal human mammary epithelial cells (NHMEC) and from the human breast carcinoma cell line MCF-7, of blood-group O phenotype, by affinity chromatography on Griffonia simplicifolia I lectin-Sepharose. Specific elution of glycoproteins from the column with methyl alpha-D-galactoside suggests the presence of alpha-D-galactosyl groups on these moieties. SDS-PAGE analysis of isolated glycoproteins revealed both quantitative and qualitative differences between glycoproteins from normal and malignant cells. Three major glycoproteins of Mr 180 kDa, 85 kDa and the 44 kDa were obtained from MCF-7 cells. The 180-kDa glycoprotein was absent in NHMEC and the 44-kDa glycoprotein was very weakly expressed in these cells. The only glycoprotein which was found in almost equal amount in the lysate from both normal and malignant cells was the 85-kDa glycoprotein. These results indicate differences between normal human mammary epithelial cells and one kind of malignant human mammary epithelial cells, in the expression of glycoproteins containing alpha-D-galactosyl groups, irrespective of blood-group phenotype; they also demonstrate that alpha-D-galactosyl group are expressed in a very restrictive manner on the surface of this tumor cell line.     

Sensitivity to camptothecin of human breast carcinoma and normal endothelial cells

Purpose: To assess parameters that might determine resistance to the topoisomerase I inhibitor, camptothecin (CPT), the sensitivities of three established human breast cancer cell lines (ER⁻) and of normal bovine endothelial cells to CPT in the free form and incorporated into liposomes (LCPT), were contrasted with topoisomerase I (topo I) content and activity, and with cell cycle response to CPT treatment. Methods: Drug sensitivities were determined using the tetrazolium dye assay and by ³H-thymidine incorporation. Topo I levels were determined by Western blot analysis, and catalytic activity was determined with a plasmid relaxation assay, using nuclear protein from each cell line. CPT stabilization of cleavable complexes in nuclear extracts was determined using a labeled oligonucleotide with a specific topo I cleavage site. Cell cycle response to CPT was determined by flow cytometric analysis of propidium iodide-stained nuclei. Results: CPT was extremely potent against MDA-MB-157 cells with an IC₅₀ value of 7 nM compared with IC₅₀ values of 150 nM for GI 101A and 250 nM for MDA-MB-231 cells. In contrast, CPT inhibited the incorporation of ³H-thymidine at very low doses in GI 101A and MDA-MB-231 cells with IC₅₀ values of 9 nM and 5 nM, respectively; while MDA-MB-157 cells did not stop incorporating ³H-thymidine until very high doses (500 nM ) of CPT were used. When incorporated into multilamellar liposomes (LCPT), CPT retained its potency, with IC₅₀ values similar to that of the free drug. No correlation was found between CPT-induced cytotoxicity and any of the topo I parameters determined. Cell cycle analysis, however, showed an accumulation of cells in G₂/M phase after 24 h treatment with low doses (5 nM) of CPT in only GI 101A and MDA-MB-231 cells with no arrest in normal endothelial or MDA-MB-157 cells. At higher doses (50 nM), however, a dramatic accumulation of cells in the S phase was observed in MDA-MB-157, MDA-MB-231 and GI 101A cells. In contrast, a G₂/M phase block was seen with the normal bovine endothelial cells using the higher doses of CPT. Conclusions: The results suggest that cell cycle regulation plays an important role in determining the effect of CPT on malignant and normal cells. The possible mechanisms explaining the sensitivities of the two cellular compartments to the action of CPT are discussed. Received: 21 October 1996 / Accepted: 5 March 1997     

Telomeric DNA induces apoptosis and senescence of human breast carcinoma cells

Introduction  

Cancer is a leading cause of death in Americans. We have identified an inducible cancer avoidance mechanism in cells that reduces mutation rate, reduces and delays carcinogenesis after carcinogen exposure, and induces apoptosis and/or senescence of already transformed cells by simultaneously activating multiple overlapping and redundant DNA damage response pathways.     

神经酰胺诱导人乳腺癌细胞凋亡的研究

目的 :观察神经酰胺对人乳腺癌细胞株MCF 7及耐多柔比星细胞株MCF 7/ADR的诱导凋亡作用及其对bcl 2基因表达的影响 ,探讨神经酰胺诱导凋亡的机制。方法 :采用MTT法检测细胞生存率 ,用流式细胞术检测凋亡率 ,运用流式细胞术、RT PCR方法检测bcl 2基因表达。结果 :神经酰胺对MCF 7、MCF 7/ADR细胞有明显的抑制生长作用。 2 5 μmol/LC6 神经酰胺作用48h后 ,MCF 7及MCF 7/ADR细胞凋亡率分别为( 2 0 70± 2 66) %、( 12 3 9± 1 76) % ,前者凋亡率高于后者 ,P <0 0 1。流式细胞术检测 ,MCF 7、MCF 7/ADR细胞bcl 2蛋白阳性率分别为 ( 2 7 5 9± 2 94) %、( 79 5 6±3 2 0 ) %。MCF 7/ADR细胞经神经酰胺作用后 ,bcl 2蛋白及mRNA水平均降低。结论 :神经酰胺对MCF 7、MCF 7/ADR细胞有抑制生长及诱导凋亡作用 ,神经酰胺诱导凋亡可能是通过下调bcl 2基因表达而实现的     

5-Fluorouracil incorporation in DNA of human breast carcinoma cells

We have demonstrated previously the presence of 5-fluorouracil (FUra) residues in L1210 DNA. These findings have been extended to the MCF-7 human breast carcinoma cell line. Cesium sulfate gradient centrifugation has been used to separate the MCF-7 RNA and DNA fractions. Alkali and RNase digests have also been used to remove any possible RNA contaminating the DNA fraction. The purified DNA has been analyzed by high-pressure liquid chromatography following digestion to nucleotides and nucleosides. The results demonstrate that FUra residues are detectable in the DNA of these human breast carcinoma cells following exposure to either FUra of 5-fluorodeoxyuridine. Further, the extend of FUra incorporation in both MCF-7 RNA and DNA is similar with either fluorinated pyrimidine. We also demonstrate that the FUra incorporation in DNA from this human cell line can be enhanced by concurrent incubation with thymidine.     

Interaction of liposome-associated all-trans-retinoic acid with squamous carcinoma cells

Summary Because of their antiproliferative and differentiation-inducing properties, retinoids have been used clinically as therapeutic and chemopreventive agents against squamous-cell carcinomas (SCC). As is the case for many therapeutic agents, however, the administration of retinoids is associated with toxic effects. Because encapsulation of certain drugs in lipid vesicles (liposomes) has been shown to result in reduced toxic effects, we studied the in vitro interaction of liposome-encapsulated all-trans-retinoic acid (L-ATRA) with a SCC line (MDA 886Ln) and its multicellular tumor spheroid (MTS) model. Various L-ATRA formulations were tested for incorporation of retinoic acid, toxic effects against human red blood cells, uptake and retention by tumor cells, and anti-proliferative effects against SCC. Of the different formulations tested, L-ATRA containing diphosphatidyl palmitoylcholine (DPPC) and stearylamine (SA; 9:1, w/w) showed optimal drug incorporation, high stability, and minimal toxicity toward red blood cells and was highly efficacious in delivering ATRA and, thus, in inhibiting the growth of MDA 886Ln and its MTS model. DPPC: SA L-ATRA inhibited the expression of the enzyme keratinocyte transglutaminase in epidermal cells as effectively as did the free drug. These results suggest that liposomes can serve as an effective carrier system for the delivery of retinoids to SCC.This work was supported in part by Public Health Service grants FDR000923 from the Orphan Products Division, Food and Drug Administration, and CA 57116 from the National Cancer Institute     

genistein多途径抑制乳腺癌细胞生长和侵袭

目的 研究ｇｅｎｉｓｔｅｉｎ在乳腺癌细胞生长和侵袭中的作用。方法 应用体外细胞培养和体内乳腺癌细胞移植裸鼠模型以及酶谱分析、Ｎｏｒｔｈｅｎ和Ｗｅｓｔｅｒｎ ｂｌｏｔ方法。结果 在体外实验中,ｇｅｎｉｓｔｅｉｎ不仅具有抑制乳腺癌细胞生长的作用,还具有抑制乳腺癌细胞侵袭的功能,这种抑制功能是伴ｇｅｎｉｓｔｅｉｎ能明显抑制蛋白金属酶ＭＭＰ－９的表达和增加组织金属蛋白酶抑制剂ＴＩＭＰ１的表达。ｇｅｎｉｓｔ     

Imatinib mesylate enhances the malignant behavior of human breast carcinoma cells

Purpose

Imatinib mesylate (Imatinib), clinically employed for chronic myeloid leukemia and gastrointestinal stromal tumors, is a selective inhibitor of the tyrosine kinases, c-abl, c-kit and PDGFRs. Due to the frequent expression of these genes in breast cancer cells, the clinical efficacy of Imatinib has recently been investigated in patients with advanced and metastatic breast cancer. Here, we have studied the effects of Imatinib on human MA-11 breast carcinoma cells, expressing both c-abl and PDGFRbeta, in vitro and in mouse xenografts.

Methods

The effects of Imatinib mesylate on the human MA-11 breast carcinoma cell line were studied in vitro and in xenografts.

Results

Daily intraperitoneal treatment with 60 mg/kg Imatinib for 9 days of athymic nude mice pre-implanted subcutaneously with MA-11 cells did not result in an anti-tumor effect, but rather increased the take rate of 3 × 10⁴ cells from 30.8 to 84.6% and caused the appearance of large abdominal masses in 30% of mice. To investigate the mechanism(s) of the observed effects of Imatinib on MA-11 tumors, we exposed the cells in vitro to Imatinib for 9 days. The surviving population, expanded in culture, showed increased motility and over-expressed a set of genes associated with aggressive behavior. Also, several genes belonging to the Wnt and the MAPK pathway were differentially expressed. In promoter activation assays, Imatinib increased the promoter activity driven by both Wnt and MAPK/ERK-1/2.

Conclusions

Our data suggest caution in the clinical use of Imatinib in breast cancer patients; the comparison of Imatinib-surviving breast cancer cells with parental cells may help define the regulatory pathways involved in the increased malignancy of residual tumor cells that survive therapy, ultimately providing important therapeutic targets.     

Cellular immune reaction to human malignant melanoma and breast carcinoma cells.

An in vitro microassay was used to study cytotoxic reactivity of lymphocytes of 19 patients with malignant melanoma, 8 patients with breast carcinoma, and 18 normal subjects on cell cultures of malignant melanoma and breast carcinoma. In each of the twelve experiments, peripheral blood lymphocytes from individuals with and without cancer were tested simultaneously on two or three different target cells. Cytotoxic reactivity, evaluated by a comparison of the number of target cells remaining after incubation with lymphocytes with those incubated with medium only, was found in 20 cancer patients (74%) and 13 individuals without cancer (72%). The strength of lymphocyte reactivity of the cancer and of the non-cancer group did not differ significantly. Of the 27 cancer patients, 8 were positive only on the homologous target cells, 7 only on the opposite cells, and 5 on both types; 7 were negative. Short-term melanoma cell cultures were more lysable than were established cell lines; however, no direct correlation between the growth rate during the test period and susceptibility to lysis was seen. The blood group of the lymphocyte donors had no influence on cytotoxic reactivity.     

Autocrine erythropoietin signaling inhibits hypoxia-induced apoptosis in human breast carcinoma cells

Disordered perfusion and the resulting hypoxia are important features conferring tumor heterogeneity, which may contribute to relapse. Hypoxic tumor cells have been associated with resistance both to radiation and to cytotoxic drugs. Hypoxia may also serve as a selection pressure in tumors by promoting apoptosis of some cells and expanding variants with decreased apoptotic potential, and thus play a role in the development of a more aggressive phenotype. Erythropoietin (Epo), induced by hypoxia, controls erythropoiesis and plays a role in protection of neurons from hypoxic damage. We have recently demonstrated hypoxia-stimulated expression of Epo and Epo receptor (EpoR) in human breast and cervix cancers, suggesting a role for autocrine Epo signaling in the hypoxic adaptations of carcinomas. In the current study we provide evidence that increased autocrine Epo signaling induced by moderate levels of hypoxia inhibits hypoxia-induced apoptosis and promotes survival in MCF-7 human breast cancer cells. The anti-apoptotic effect of Epo correlates with upregulation of bcl-2 and bcl-XL, suggesting a mechanism similar to those described in hematopoietic cells. The resulting decreased apoptotic potential of hypoxic tumor cells may contribute to increased aggressiveness and therapy resistance of breast cancers.     

An antitumor antibody in normal human serum: reaction of anti-T with breast carcinoma cells

Carcinoma of the breast is now the most common malignant disease of females in Europe and North America. The most critical single factor in determining the success or failure of treatment is the extent of the disease at diagnosis. A sensitive immunoperoxidase technique was utilized to (1) detect the presence in normal human serum of antibody directed against breast carcinoma cells (anti-T antibody), and (2) determine whether this antibody could be used to discriminate between benign and malignant breast epithelium in sections of formalin-fixed, paraffin-embedded tissue. A total of 11 benign and 11 malignant breast lesions were examined for evidence of binding of human immunoglobulin, before and after application of purified anti-T antibody or normal human AB serum (containing anti-T antibody). Malignant cells showed significant binding of human immunoglobulin (anti-T); benign or normal cells did not. Clear immunohistological separation of benign and malignant breast lesions appears to be potentially feasible by this method.     

Systematic optimization of the clonal growth of human primary breast carcinoma cells

Human breast carcinomas have been one of the most difficult tumor types to culture in agar-based clonogenic assays. This fact has limited their clinical applicability. We have used statistically motivated experimental designs to systematically improve the clonal culture of enzymatically monodispersed primary human carcinoma cells in an anchorage-independent agar system. Based upon an initial comparison of two basal media, we selected one which gave the best colony growth and then sought to optimize the individual additives in the medium. Hydrocortisone, fetal bovine serum, and red blood cells all improved both plating efficiency and median size of colonies derived from breast carcinoma cells. Next, the concentrations of these three components were simultaneously idealized using response surface methodology. By these methods, it was found that the optimal concentration of hydrocortisone was 0.35 microgram/ml, fetal bovine serum was 6.5%, and red blood cells was 2.1 X 10(7) cells/ml. Using these culture conditions, we have achieved plating efficiencies of 0.39% and 0.19% for colonies with diameters greater than 50 (50 cells) or 70 (130 cells) micron, respectively.     

Down-regulation of galectin-3 suppresses tumorigenicity of human breast carcinoma cells.

Galectin-3 is an endogenous beta-galactoside-binding protein with specificity for type I and II ABH blood group epitopes and poly-N-acetyllactosamine glycan-containing cell surface glycoproteins and is the major nonintegrin cellular laminin-binding protein. Galectin-3 is expressed at an elevated level in a wide range of neoplasms, and expression was shown to be associated in some tumor cell systems with metastases. Here we determined the functional consequence of blocking galectin-3 expression in highly malignant human breast carcinoma MDA-MB-435 cells. Inhibition of galectin-3 expression led to reversion of the transformed phenotype as determined by altered morphology, loss of serum-independent growth, acquisition of growth inhibition properties by cell contact, and abrogation of anchorage-independent growth. The blockage of galectin-3 expression led to a significant suppression of tumor growth in nude mice. These results provide direct evidence that galectin-3 expression is necessary for the maintenance of the transformed and tumorigenic phenotype of MDA-MB-435 breast carcinoma cells.