Crystallization during bending of a Pd-based metallic glass detected by x-ray microscopy.

Without the availability of slip systems and dislocation glide as in crystalline materials, metallic glasses resist irreversible deformation to elastic strains of 2% or more before undergoing heterogeneous plastic flow via the formation of shear bands. Observation of crystallite formation under compressive load was previously obtained by transmission electron microscopy. In this Letter, we present results of nondestructive x-ray diffraction microprofiling of the section of a bent glassy Pd40Cu30Ni10P20 ribbon in transmission using a synchrotron microbeam. Crystallization was clearly detected but only on the compression side of the neutral fiber. The experimental results and crystal nucleation frequency analysis are consistent with massive nucleation in shear bands forming under compressive stress but mainly for metallic glasses that show a large supercooled liquid temperature range ΔT=T(x)-T(g) between glass transition at T(g) and crystallization at T(x). The phenomenon is sensitively dependent on the volume change that accompanies crystallization in the supercooled liquid temperature range where the much larger liquid-state thermal expansion coefficient significantly increases the specific volume difference between the liquid and crystalline states. The results are also consistent with the many reports of extensive strain to fracture of metallic glasses under compressive load but not under tension.

Immediate early gene X-1 (IEX-1), a hydroxytamoxifen regulated gene with increased stimulation in MCF-7 derived resistant breast cancer cells.

The efficacy of tamoxifen in breast cancer treatment only lasts a few years and the tumor eventually recurs. We performed selective subtractive hybridization to isolate mRNAs that were differentially expressed in MCF-7 derived cells, in which resistance had been induced through long-term culture in the presence of hydroxytamoxifen (OHT). Among the 15 mRNAs found to be overexpressed, we focused on Immediate early gene X-1 (IEX-1) mRNA because of the recognized contribution of its expression to apoptosis or cell cycle progression, depending on the cell type and culture conditions. We observed that IEX-1 expression was stimulated by OHT, that the degree of increase was greater in resistant cells (four-fold versus 1.5-fold) and that this OHT regulation was estrogen receptor dependent. A detailed study of the IEX-1 promoter indicated that it involved NF-kappaB. Our cells were not cross-resistant to faslodex, a pure antiestrogen, which moreover was inefficient in regulating IEX-1 expression. Altogether, our data suggest that the greater IEX-1 expression in OHT resistant cells is related to their ability to grow in the presence of OHT. Knowledge on the capacity of OHT to stimulate gene expression and its NF-kappaB dependence should contribute to a better understanding of tamoxifen pharmacology and allow new drug strategies to be designed that would delay antiestrogen resistance acquisition.

An estrogen-responsive element-targeted histone deacetylase enzyme has an antiestrogen activity that differs from that of hydroxytamoxifen.

We showed previously that prolonged treatment of a MCF-7-derived cell line with hydroxytamoxifen (OHT) induces the irreversible silencing of some estrogen-responsive genes, whereas OHT-resistant cell growth appears simultaneously (E. Badia et al., Cancer Res., 60: 4130-4138, 2000). Based on the hypothesis that particular gene silencings could be involved in triggering the resistance phenomenon, we focused our study on the mechanism of OHT-induced silencing. More precisely, we wished to determine to what extent the recruited histone deacetylase (HDAC) activity, which is known to be involved in the repressive effect induced by antagonist ligands of nuclear receptors, could participate in various aspects of OHT effects, particularly in gene silencing. A fusion protein (HDAC-EG) of human HDAC1 fused with the estrogen receptor DNA-binding domain and the glucocorticoid receptor ligand-binding domain allowed targeting of chimeric HDAC1 activity on estrogen-responsive elements (EREs) in the presence of glucocorticoid ligands. When HDAC-EG was transiently expressed in HeLa cells together with estrogen receptor, an antiestrogen-like effect was obtained on an ERE-controlled luciferase reporter gene in the presence of agonist or antagonist glucocorticoids. In MCF-7-derived cells stably expressing HDAC-EG and an estrogen-regulated luciferase, liganded HDAC-EG again produced an antiestrogenic effect on expression of natural estrogen-regulated genes such as pS2, progesterone receptor, and cathepsin D and cell growth together with chimeric luciferase gene expression. However, a prolonged HDAC-EG-mediated antiestrogen effect did not lead to irreversible luciferase gene silencing, as OHT does. It nevertheless accelerated the OHT-driven phenomenon. The antiestrogen effect of OHT thus differs from that of an ERE-targeted HDAC1 activity that might participate in irreversible silencing but is not sufficient to trigger it.

Effect of prolonged hydroxytamoxifen treatment of MCF-7 cells on mitogen activated kinase cascade.

Resistance to the antiestrogen tamoxifen is the main stumbling block for the success of breast cancer therapy. We focused our study on cellular alterations induced by a prolonged treatment with the active tamoxifen metabolite hydroxytamoxifen (OHT). We show that a prolonged OHT treatment (for up to 7 days) led to a progressive increase in the level of phosphorylated p44/42 mitogen activated kinase (MAP kinase) induced by 10(-7) M TPA stimulation, without any significant change in the protein level. This effect was also observed in MCF-7 cells grown first in medium containing dextran-coated charcoal-treated FCS (DCC medium) for 20 days prior to OHT treatment, indicating a specific effect of the antiestrogen and not an effect of estrogen deprivation. It was prevented by cotreatment with estradiol and not observed in the estrogen receptor negative HeLa cell line, suggesting that it was mediated by the estrogen receptor. TPA induced phosphorylation of MEK1/2 was also raised by OHT treatment, without any change in their protein level or Raf-1 and H-Ras levels. When the MCF-7R OHT resistant cell line was grown in antiestrogen containing medium, the level of phosphorylated p44/42 MAP kinase was also high but reversed when the antiestrogen was removed. The 2 other MAP kinase, JNK and P38 pathways were not affected in the same way by OHT treatment. In conclusion, our data reveal that a prolonged OHT treatment, by increasing p44/42 MAPK activity, affects a key step in the growth control of MCF-7 cells, although not sufficiently to overcome the growth inhibitory effect of the drug.