Regulation of Estradiol Synthesis by Aromatase Interacting Partner in Breast (AIPB).

Estradiol is essential for the development of female sex characteristics and fertility. Postmenopausal women and breast cancer patients have high levels of estradiol. Aromatase catalyzes estradiol synthesis; however, the factors regulating aromatase activity are unknown. We identified a new 22-kDa protein, aromatase interacting partner in breast (AIPB), from the endoplasmic reticulum of human breast tissue. AIPB expression is reduced in tumorigenic breast and further reduced in triple-negative tumors. Like that of aromatase, AIPB expression is induced by nonsteroidal estrogen. We found that AIPB and aromatase interact in nontumorigenic and tumorigenic breast tissues and cells. In tumorigenic cells, conditional AIPB overexpression decreased estradiol, and blocking AIPB availability with an AIPB-binding antibody increased estradiol. Estradiol synthesis is highly increased in AIPB knockdown cells, suggesting that the newly identified AIPB protein is important for aromatase activity and a key modulator of estradiol synthesis. Thus, a change in AIPB protein expression may represent an early event in tumorigenesis and be predictive of an increased risk of developing breast cancer.

Vital imaging of multicellular spheroids.

Cell behavior is significantly different in two-dimensional and three-dimensional culture conditions, and a number of methods have been developed to establish and study three-dimensional cellular arrays in vitro. When grown under nonadherent conditions, many types of cells form structures called multicellular spheroids (MCSs), which have been popular models to study cell behavior in a three-dimensional environment. The histoarchitecture of MCSs derived from malignant cells resembles that of tumors, and there is rapidly increasing interest in using these structures to more accurately understand the dynamics of cancer cells in situ, including their responses to chemotherapeutics. Confocal microscopy is an extremely useful method to investigate cell behavior in MCSs due to its ability to more clearly image fluorescent probes at some depth in three-dimensional structures. This chapter describes some basic approaches toward visualizing a variety of fluorescent probes in MCSs.

Downstream bioengineering of ACE chromosomes for incorporation of site-specific recombination cassettes.

Advances in mammalian artificial chromosome technology have made chromosome-based vector technology amenable to a variety of biotechnology applications including cellular protein production, genomics, and animal transgenesis. A pivotal aspect of this technology is the ability to generate artificial chromosomes de novo, transfer them to a variety of cells, and perform downstream engineering of artificial chromosomes in a tractable and rational manner. Previously, we have described an alternative artificial chromosome technology termed the ACE chromosome system, where the ACE platform chromosome contains a multitude of site-specific, recombination sites incorporated during the creation of the ACE platform chromosome. In this chapter we review a variant of the ACE chromosome technology whereby site-specific, recombination sites can be integrated into the ACE chromosome following its de novo synthesis. This variation allows insertion of user-defined, site-specific, recombination systems into an existing ACE platform chromosome. These bioengineered ACE platform chromosomes, containing user-defined recombination sites, represent an ideal circuit board to which an array of genetic factors can be plugged-in and expressed for various research and therapeutic applications.

Sanguinarine cytotoxicity on mouse melanoma K1735-M2 cells--nuclear vs. mitochondrial effects.

Sanguinarine (SANG) is an alkaloid recognized to have anti-proliferative activity against various human tumour cell lines. No data is available on the susceptibility of advanced malignant melanoma to SANG, although this disease has a very poor prognosis if not detected in time due to the resistance to conventional chemotherapy. The present work was designed to study the nuclear and mitochondrial involvement in the pro-apoptotic effect of SANG in an invasive mouse melanoma cell line. The results obtained show that SANG is primarily accumulated by the cell nuclei, causing inhibition of cell proliferation and inducing cell death, as confirmed by an increase in sub-G1 peaks. At low concentrations, SANG induces mitochondrial depolarization in a sub-population of melanoma cells, which also generally displayed strong nuclear labelling of phosphorylated histone H2AX. Western blotting revealed an increase in p53, but not Bax protein, in both whole-cell extracts and in mitochondrial fractions. Isolated hepatic mitochondrial fractions revealed that SANG affects the mitochondrial respiratory chain, and has dual effects on mitochondrial calcium loading capacity. We suggest that SANG is able to induce apoptosis in metastatic melanoma cells. The knowledge of mitochondrial vs. nuclear effects of SANG is important in the development of this promising compound for clinical use against aggressive melanoma.

Mitochondrially targeted effects of berberine [Natural Yellow 18, 5,6-dihydro-9,10-dimethoxybenzo(g)-1,3-benzodioxolo(5,6-a) quinolizinium] on K1735-M2 mouse melanoma cells: comparison with direct effects on isolated mitochondrial fractions.

Berberine [Natural Yellow 18, 5,6-dihydro-9,10-dimethoxybenzo(g)-1,3-benzodioxolo(5,6-a)quinolizinium] is an alkaloid present in plant extracts and has a history of use in traditional Chinese and Native American medicine. Because of its ability to arrest the cell cycle and cause apoptosis of several malignant cell lines, it has received attention as a potential anticancer therapeutic agent. Previous studies suggest that mitochondria may be an important target of berberine, but relatively little is known about the extent or molecular mechanisms of berberine-mitochondrial interactions. The objective of the present work was to investigate the interaction of berberine with mitochondria, both in situ and in isolated mitochondrial fractions. The data show that berberine is selectively accumulated by mitochondria, which is accompanied by arrest of cell proliferation, mitochondrial fragmentation and depolarization, oxidative stress, and a decrease in ATP levels. Electron microscopy of berberine-treated cells shows a reduction in mitochondria-like structures, accompanied by a decrease in mitochondrial DNA copy number. Isolated mitochondrial fractions treated with berberine had slower mitochondrial respiration, especially when complex I substrates were used, and increased complex I-dependent oxidative stress. It is also demonstrated for the first time that berberine stimulates the mitochondrial permeability transition. Direct effects on ATPase activity were not detected. The present work demonstrates a number of previously unknown alterations of mitochondrial physiology induced by berberine, a potential chemotherapeutic agent, although it also suggests that high doses of berberine should not be used without a proper toxicology assessment.