Estrogen Receptor-positive Human Breast Cancer Research Articles

Effects of Hormones on Tumor Growth and Immunoreactive Insulin–like Growth Factor–1 of Estrogen Receptor–positive Human Breast Cancer (MCF‐7) Transplanted in Nude Mice

The effects of estrogens and tamoxifen were analyzed on estrogen receptor–positive human breast cancer (MCF–7) transplanted into athymic nude mice. It was found that (1) the tumor growth and the proportion of 3H–thymidine –labeled cells were significantly increased in the 17β– estradiol dipropionate (E2) group, but significantly decreased In the tamoxifen (TAM) group with respect to the control group, and (2) the tumor content of insulin–like growth factor–1 (IGF–1) and the rate of IGF–1–positive cells were significantly lower in the E2 group, but significantly higher in the TAM group than in the control group. It was concluded that the tumor content of IGF–1 and the proportion of IGF–I–positive cells were inversely correlated to the tumor growth and the 3H–thymidine labeling index in vivo,

Folate Transport and the Modulation of Antifolate Sensitivity in a Methotrexate-Resistant Human Breast Cancer Cell Line

The mechanism of acquired methotrexate-resistance in an estrogen-receptor positive human breast cancer cell line (MTX(R)ZR-75-1) was studied. MTX(R) ZR-75-1 cells are 250-fold resistant to methotrexate when grown in the presence of 1 microM folinic acid and 2,400-fold resistant in the presence of 1 microM folic acid. This drug resistant cell line also showed collateral sensitivity (10-fold) to trimetrexate (TMQ), when grown in the presence of folinic acid. Using fluoresceinated methotrexate (F-MTX), FACS analysis indicated that there is no intracellular accumulation of methotrexate into MTX(R) ZR-75-1 cells, as determined by competition of F-MTX and methotrexate binding to dihydrofolate reductase. These characteristics strongly indicate that the mechanism of resistance involved down regulation of the reduced-folate transporter. To investigate this further, the transport kinetics of parental and MTX(R) ZR-75-1 cells were examined. Although the V(max) for methotrexate transport in wild-type (WT) ZR-75-1 breast cancer cells was 1-2 orders of magnitude lower than that in the well characterized leukemia cell lines, such as L1210 and CCRF-CEM cells, kinetic analysis indicated that transport of methotrexate into WT ZR-75-1 cells involved a mechanism that was similar if not identical to the reduced folate transporter. In contrast, no specific uptake of methotrexate was detected in MTX(R) ZR-75-1cells. Furthermore, neither cell line expressed detectable levels of folate binding protein, a binding protein with high affinity for folic acid as well as for reduced folates and antifolates. These results indicate that the level of expression of the reduced-folate carrier may be an important factor in determining the sensitivity of breast cancer cells as well as leukemia cells to antifolate compounds.

DNA synthesis in estrogen receptor-positive human breast cancer takes place preferentially in estrogen receptor-negative cells

The primary breast tumors of 27 patients were analyzed for the expression of estrogen receptors (ER) and DNA synthesis. Seventeen tumors were ER-positive, and the simultaneous expression of ER and DNA synthesis could be analyzed in 14 ER-positive tumors. DNA synthesis was measured through the thymidine labeling index (TLI). ER expression was detected by immunohistochemistry with monoclonal antibodies. In these tumors, 38.6% +/- 13.1% of the cells were ER-positive (average TLI = 0.60% +/- 0.70%), as opposed to the presence of 61.4% +/- 13.1% of ER-negative cells (average TLI = 0.65% +/- 0.53%). In 12 of 14 tumors, both ER-positive and ER-negative cells were found to be engaged in DNA synthesis, whereas in two tumors only ER-negative cells were synthesizing DNA. On the basis of the TLI and the proportion of ER-negative and ER-positive cells in the total population, it is suggested that the ER-positive and ER-negative compartments are interrelated in most tumors. In five tumors, the ER-negative compartment would be a precursor of the ER-positive segment, whereas in six tumors the ER-positive segment appears to be a precursor of the ER-negative one. In three tumors, no evidence of an interrelationship between both segments could be found. In the 14 tumors analyzed, it also was found that 69.1% +/- 21.3% of the DNA-synthesizing cells were ER-negative; this probably accounts for the temporary remissions observed after hormonal treatment in breast cancer.

Regulation of the secretion of proteins synthesized by the human breast cancer cell line, MCF-7

In two sublines of the estrogen receptor-positive human breast cancer cell line, MCF-7, propagated with 0.5% fetal calf serum (FCS) (subline 0.5) and without serum (subline 9), respectively, addition of 10% newborn calf serum (NCS) inhibits cell proliferation and stimulates the secretion of a 42 kDa protein. Both with and without NCS, estradiol (E 2) stimulates the secretion of a 61 kDa and a 52 kDa protein in subline 9, and an additional third protein (66 kDa) is stimulated in subline 0.5. E 2 inhibits the synthesis of the 42 kDa protein in both sublines. Different cell proliferation rates of MCF-7 cells can be established in cultures with 10% NCS and varying concentrations of E 2. In such experiments final cell number after 6 days is positively correlated to the relative amount of the 66 kDa, the 61 kDa and the 52 kDa proteins, whereas the relative amount of the 42 kDa protein is negatively correlated to the final cell number. These results indicate that the 52 kDa, the 61 kDa and the 66 kDa proteins may act as positive growth factors, whereas the 42 kDa protein may act as a negative growth factor. The 42 kDa, the 52 kDa and the 61 kDa proteins are apparently glycoproteins, and the 66 kDa protein is not glycosylated. The 61 kDa protein is immunoreactive with antitrypsin antibodies.

Transcriptional regulation of c-myc oncogene expression by estrogen in hormone-responsive human breast cancer cells.

Enhanced c-myc oncogene expression associated with peptide mitogen-stimulated cell growth is primarily a result of a post-transcriptional event involving increased mRNA stability. We have recently shown that estradiol stimulates c-myc expression in estrogen receptor-positive human breast cancer cells. In this report, we show that in estrogen-responsive MCF-7 cells, estradiol stimulated the c-myc gene exclusively at the transcriptional level, increasing c-myc mRNA transcription more than 10-fold within 20 min, while having no effect on the c-myc mRNA half-life of 18 min. In addition, pretreatment of the cells with cycloheximide did not prevent induction of the c-myc oncogene, indicating a primary effect of estrogen. Furthermore, the elevated level of c-myc mRNA in estrogen-independent MDA-MB-231 cells was due primarily to a more stable c-myc mRNA with a half-life of 49 min, in the absence of enhanced transcription. These results indicate that different mechanisms of regulation of c-myc oncogene expression exist in hormone-dependent and -independent human breast cancer cells.

A 46-kDa antigen associated with estrogen receptor in human breast cancer

A 65-kDa estrogen receptor (ER) protein has been demonstrated both by sucrose gradient analysis and by immunoblot, using anti-ER monoclonal antibodies (MAbs). Since the ER is denatured in many experimental situations, such as formaldehyde fixing of samples for histochemistry and electroimmunoblotting studies, in this work we used a denatured 60–70-kDa ER-rich protein preparation as antigen for mice immunization in order to raise anti-ER MAbs. That material was obtained by affinity purification on an allyl-estradiol matrix of the MCF-7 cytosolic ER, followed by further isolation and enrichment by PAGE. NS-1 myeloma cells and spleen lymphocytes from the immunized mice were fused, and resultant hybridoma colonies were screened by [ 125I]-estradiol-labelled nuclear ER immunoprecipitation. The isolated MAb, E476, shows a moderate ability to precipitate ER and reacts strongly with a 46-kDa antigen in Western blot assay. The 46-kDa antigen was not detectable in native cytosol but became reactive after 50% ammonium sulfate precipitation of cytosolic proteins. The 46-kDa antigen appeared concentrated in the NaSCN plus estradiol eluate of the affinity column used for cytosolic ER purification. Freshly prepared 60–70-kDa material from the preparative gel electrophoresis did not show any E476 reactivity. However, when the 60–70-kDa proteins were frozen, thawed and speed vacuum concentrated, the 46-kDa antigen became detectable. Storage increased the reactivity of the 60–70-kDa material with the E476 MAb. The 46-kDa antigen was present only in the ER positive cell lines, and was absent in all negative cell lines tested. The 46-kDa protein is also present in the ER positive human breast cancer specimens. We conclude that the 46-kDa protein identified with the E476 MAb in human breast cancer is probably a naturally occurring ER fragment.

Determination of turnover of androstenedione to estrone via aromatase in estrogen receptor positive and negative human breast cancer cell lines [Poster 48

In one estrogen receptor (ER) negative (MDA-MB-231) and two ER positive human breast cancer cell lines (T-47-D,SK-BR-3) we measured aromatase activity by [ 3H]water assay and estrone (E1) production by thin-layer chromatography. Compared with ether extraction and charcoal method, lyophilization proved to be the most sensitive technique to measure the quantity of [ 3H]water. The extremely low contamination of the water soluble phase by [1ß- 3H]androstenedione (0.02%), as well as the lack of errors due to conjugated steroids, offers the possibility to measure changes of cellular aromatase activity even at very low levels. In contrast to SK-BR-3 and MDA-MB-231 cells, we found no aromatase activity in T-47-D cells. There was no coincidence between ER status and aromatase activity. Proliferation of tumor cells was parallel with a continuous increase of aromatase activity and E1 production during mitogenic growth phase reaching highest levels at the transition from log to plateau-phase.

Intrinsic and extrinsic factors in estrogen action in human breast cancer: Role of polamines and pituitary factors

Although polyamines are important in regulating proliferation of mammalian cells, their role in hormone induction of cell growth has not been delineated. In the estradiol-responsive human breast cancer cell line, T-47D clone 11, estradiol (10 −10 M) was able to stimulate cell proliferation and the activity of ornithine decarboxylase (ODC), the first and rate-limiting enzyme in the biosynthesis of polyamines. α-Difluoromethylornithine (DFMO), a specific inhibitor of ODC, blocked the estradiol-induced cell proliferation and ODC activity. Exogenous addition of putrescine, the natural product of ODC, rescued the inhibitory effect of DFMO. In addition, DFMO abolished the estradiol-induced growth of several other estrogen-responsive human breast cancer cell lines but did not affect the growth of hormoneindependent cell lines. Further, a serum factor was found to be required for estradiol to exert its effect. To gain insight into the nature of this and possibly other extrinsic factors involved, the effect of estradiol on the proliferation of T-47D cells transplanted into athymic nude mouse was evaluated. In this in vivo system, estradiol alone produced only moderate growth of the human breast tumor. The simultaneous transplantation of a prolactin (PRL)- and growth hormone (GH)-secreting rat pituitary tumor or normal rat pituitary glands at a different site dramatically potentiated the effect of estradiol on the growth of the breast tumor xenograft. Purified PRL or GH were without effect, indicating that the active pituitary factor is neither PRL nor GH. Further, conditioned medium from rat pituitary tumor cells potentiated the mitogenic effect of estradiol on T-47D and several other estrogen receptor-positive human breast cancer cell lines in vitro under serum-free condition. In conclusion, we have identified both intrinsic (polyamines) and extrinsic (pituitary/serum) factors that are importance for estrogen to exert its mitogenic action. The next goal will be to elucidate the mechanisms of action of these molecules in the modulation of estrogen action.

Stimulation by 17 beta-estradiol of a secreted glycoprotein in MCF-7 cells and human breast cancer.

At present there is no single test which can accurately predict the effectiveness of hormonal therapy in patients with breast cancer. Even patients whose tumors contain both estrogen and progesterone receptors show variations in sensitivity to therapy [5]. Therefore other indicators of hormone action are needed to further rationalize treatment. Westley and Rochefort [7, 8] have recently reported that estrogens induce the synthesis of a 46,000 daltons secreted protein (46 K) in three estrogen receptor-positive human breast cancer cell lines. In this paper we present evidence that a protein similar to the 46 K is present in freshly obtained human breast cancer tissue. This finding may prove to be extremely useful for both diagnostic and therapeutic purposes.

Combination hormonal, chemotherapy and radiotherapy in vitro: Implications for therapy in vivo

Hormonal therapy in breast cancer is widely employed, but its mechanism of action is poorly understood. We therefore studied the effect of 17β-estradiol (E2) and tamoxifen on an estrogen receptor-positive human breast cancer cell line (MCF-7). Neither E2 nor tamoxifen was significantly cytotoxic, even at pharmacologic doses. Physiologic doses of E2 enhanced proliferation, shortened overall cell cycle time and increased the proportion of cells in DNA synthesis. Pharmacologic concentrations of E2 decreased proliferation as well as the proportion of S-phase cells. When S-phase agents were used (ARA-C and MTX), physiologic concentrations of E2 enhanced killing whereas pharmacologic doses did not. Radiation survival curve parameters were unaffected by either concentration of the hormone. Sublethal (two-dose) damage repair was unaffected as well. The combination of S-phase agents with pharmacologic doses of hormones may lower the therapeutic ratio, whereas more physiologic concentrations may enhance the therapeutic efficacy of these agents.