Dalia Seger

Nucleosome core particle stability and conformational change: Effect of temperature, particle and NaCl concentrations, and crosslinking of histone H3 sulfhydryl groups☆

We have studied the reversible dissociation of core size DNA from chicken erythrocyte nucleosome core particles in solutions containing 0 X 1 M to 0 X 6 M-NaCl. Dissociation increases with increasing NaCl concentration, increasing temperature and decreasing particle concentration. At high particle concentrations, no free DNA is observed below 0 X 3 M-NaCl, whereas above 0 X 3 M-NaCl a lower limit of dissociation is reached. A theoretical analysis based on the migrating-octamer mechanism of Stein is in disagreement with his conclusions concerning dependence of core particle dissociation on particle concentration, but provides a good explanation for our observations, and those of others, using salt concentrations up to 1 M-NaCl. It appears that the core particle is not stabilized primarily by electrostatic interactions. DNA length is not critical for core particle stabilization. The conformation of remaining intact nucleosome core particles changes only moderately within the range of NaCl concentrations studied. Crosslinking by copper phenanthroline of the Cys110 histone H3 single sulfhydryl groups in the intact nucleosome core particle leads to a decrease in stability, yet essentially unchanged hydrodynamic properties are maintained at 0 X 6 M-NaCl, confirming conclusions derived from the behavior of the native core particles. Values for density increments of nucleosome core particles over a range of NaCl concentrations are also given. A method is described for studying binding of histones to nucleosome core particles in the ultracentrifuge by scanning at 230 and 260 nm.

Inhibition of Tumor Growth and Elimination of Multiple Metastases in Human Prostate and Breast Xenografts by Systemic Inoculation of a Host Defense–Like Lytic Peptide

We report on a short host defense-like peptide that targets and arrests the growth of aggressive and hormone-resistant primary human prostate and breast tumors and prevents their experimental and spontaneous metastases, respectively, when systemically inoculated to immunodeficient mice. These effects are correlated with increased necrosis of the tumor cells and a significant decrease in the overall tumor microvessel density, as well as newly formed capillary tubes and prostate-specific antigen secretion (in prostate tumors). Growth inhibition of orthotopic tumors derived from stably transfected highly fluorescent human breast cancer cells and prevention of their naturally occurring metastases were visualized in real time by using noninvasive whole-body optical imaging. The exclusive selectivity of the peptide towards cancer derives from its specific binding to surface phosphatidylserine and the killing of the cancer cells via cytoplasmic membrane depolarization. These data indicate that membrane disruption can provide a therapeutic means of inhibiting tumor growth and preventing metastases of various cancers.

Glycolysis and glucose transporter 1 as markers of response to hormonal therapy in breast cancer.

Estrogen plays a key role in the development and progression of breast cancer; hence, antiestrogens, such as tamoxifen, have a marked impact on the treatment and outcome of breast cancer patients. Estrogen‐induced growth requires continuous replenishment of energy, predominantly generated by glycolysis. Previous work from this laboratory demonstrated estrogen induction and tamoxifen inhibition of glycolysis in MCF7 human breast cancer cells in vitro (Furman et al., J Steroid Biochem Mol Biol 1992;43:189–95). We present here studies of estrogen vs. tamoxifen regulation of glycolysis in orthotopic MCF7 human breast cancer xenografts in vivo. In addition we investigated mediation of this metabolic regulation through glucose transporter 1, in the same cells, in vitro, as well as in 2 other hormone‐responsive human breast cancer cells. Tumor response and glycolysis were monitored noninvasively by means of magnetic resonance imaging and 13C spectroscopy, respectively. During estrogen‐stimulated tumor growth (from ≈0.5 to ≈1.3 cm3 in 10 days), the rate of glucose metabolism through glycolysis in vivo was high at 40 ± 4 μmole/g/min. However, treatment for 10 days with tamoxifen induced growth arrest and a concomitant decrease of 2‐fold in the rate of glycolysis. In congruence, glucose transporter 1 expression was stimulated by estrogen, reaching after 72 hr a 2‐ to 3‐fold higher level of expression relative to that in tamoxifen‐treated cells. Thus, estrogen‐induced changes in glycolysis appeared to be mediated via its regulation of glucose transporter 1 expression. The in vivo monitoring of glycolysis may serve as a tool to expose hormonal regulation of glucose transporter 1 expression in breast cancer tumors, as well as to assess response to hormonal therapy.

Phosphorylation of Vitronectin by Casein Kinase II IDENTIFICATION OF THE SITES AND THEIR PROMOTION OF CELL ADHESION AND SPREADING

The cell adhesion protein vitronectin (Vn) was previously shown to be the major target in human blood for an extracellular protein kinase A, which is released from platelets upon their physiological stimulation with thrombin and also prevails as an ectoenzyme in several other types of blood cells. Because plasma Vn was shown to have only one protein kinase A phosphorylation site (Ser378) but to contain ∼3 mol of covalently bound phosphate, and because human serum and blood cells were shown to contain also a casein kinase II (CKII) on their surface, we studied the phosphorylation of Vn by CKII attempting to find out whether such phosphorylation modulates Vn function, an acid test for its having a physiological relevance. Here we show (i) that the CKII phosphorylation of Vn has a K m of 0.5–2 μm (lower than the Vn concentration in blood, 3–6 μm), (ii) that it is targeted to Thr50 and Thr57, which are vicinal to the RGD site of Vn, and (iii) that the phosphorylation of Thr57 facilitates the phosphorylation of Thr50. The maximal stoichiometry of the CKII phosphorylation of plasma Vn was found to be low, which, in principle, could be due to its partial prephosphorylation in vivo. However, for the detection of a functional modulation, we needed a comparison between a fully phosphorylated Vn (at Thr57 and Thr50) and a nonphosphorylated Vn. Therefore, we expressed Vn in a baculovirus system and show (i) that the CKII phosphorylation of wt-Vn enhances the adhesion of bovine aorta endothelial cells; (ii) that the double mutant T50E/T57E (in which the neutral Thr residues are replaced by the negatively charged Glu residues considered analogs of Thr-P) has a significantly enhanced capacity to promote cell adhesion and to accelerate cell spreading when compared with either wild-type Vn or to the neutral T50A/T57A mutant; and (iii) that, at least in the case of bovine aorta endothelial cells, the T50E/T57E mutant exhibits an enhanced adhesion, which seems to be due to an increased affinity toward the αvβ3 Vn receptors.

Real-time Imaging of Lymphogenic Metastasis in Orthotopic Human Breast Cancer

Metastatic spread to regional lymph nodes is one of the earliest events of tumor cell dissemination and presents a most significant prognostic factor for predicting survival of cancer patients. Real-time in vivo imaging of the spread of tumor cells through the lymphatic system can enhance our understanding of the metastatic process. Herein, we describe the use of in vivo fluorescence microscopy imaging to monitor the progression of lymph node metastasis as well as the course of spontaneous metastasis through the lymphatic system of orthotopic MDA-MB-231 human breast cancer tumors in severe combined immunodeficient mice. High-resolution noninvasive visualization of metastasizing cancer cells in the inguinal lymph nodes was achieved using cells expressing high levels of red fluorescent protein. Sequential imaging of these lymph nodes revealed the initial invasion of the tumor cells through the lymphatic system into the subcapsular sinuses followed by intrusion into the parenchyma of the nodes. FITC-dextran injected i.d. in the tumor area enabled simultaneous tracking of lymphatic vessels, labeled in green, and disseminated red cancer cells within these vessels. Fast snapshots of spontaneously metastasizing cells in the lymphatic vessels monitored the movement of a few tumor cells and the development of clumps clustered at lymphatic vessel junctions. Quantification of high interstitial fluid pressure (IFP) in the tumors and fast drainage rates of the FITC-dextran into the peritumoral lymphatic vessels suggested an IFP-induced intravasation into the lymphatic system. This work presents unprecedented live fluorescence images that may help to clarify the steps occurring in the course of spontaneous lymphogenic metastasis.

Estrogen regulation of vascular endothelial growth factor in breast cancer in vitro and in vivo: the role of estrogen receptor α and c-Myc

The role of c-Myc in estrogen regulation of vascular endothelial growth factor (VEGF) and of the vasculature function has been investigated in breast cancer cells and tumors. The studies were performed on MCF7 wild-type cells and MCF7-35im clone, stably transfected with an inducible c-Myc gene. In vitro and ex vivo methods for investigating molecular events were integrated with in vivo magnetic resonance imaging of the vascular function. The results showed that the c-Myc upregulation by estrogen is necessary for the transient induction of VEGF transcription; however, overexpression of c-Myc alone is not sufficient for this induction. Furthermore, both c-Myc and the activated estrogen receptor alpha (ERalpha) were shown to co-bind the VEGF promoter in close proximity, indicating a novel mechanism for estrogen regulation of VEGF. Studies of long-term estrogen treatment and overexpression of c-Myc alone demonstrated regulation of stable VEGF expression levels in vitro and in vivo, maintaining steady vascular permeability in tumors. However, withdrawal of estrogen from the tumors resulted in increased VEGF and elevated vascular permeability, presumably due to hypoxic conditions that were found to dominate VEGF overexpression in cultured cells. This work revealed a cooperative role for ERalpha and c-Myc in estrogen regulation of VEGF and the ability of c-Myc to partially mimic estrogen regulation of angiogenesis. It also illuminated the differences in estrogen regulation of VEGF during transient and long-term sustained treatments and under different microenvironmental conditions, providing a complementary picture of the in vitro and in vivo results.

Synaptojanin 2 is a druggable mediator of metastasis and the gene is overexpressed and amplified in breast cancer.

Small-molecule inhibitors of the lipid phosphatase synaptojanin 2 may prevent breast cancer metastasis. Blocking Receptor Recycling to Prevent Metastasis Blocking cancer cell metastasis can prolong patient survival. Ben-Chetrit et al. found that many patients with aggressive breast cancer have tumors with increased expression of SYNJ2, which encodes the lipid phosphatase synaptojanin 2. In cultured breast cancer cells, epidermal growth factor (EGF) triggered the localization of SYNJ2 to lamellipodia and invadopodia, which are cellular protrusions associated with invasive behavior. Knocking down SYNJ2 inhibited recycling of the EGF receptor to the cell surface and decreased the invasive behavior of cultured breast cancer cells. Expressing a phosphatase-deficient mutant of SYNJ2 in xenografted breast cancer cells suppressed tumor growth and lung metastasis in mice. A chemical screen identified SYNJ2 inhibitors that reduced cell invasion through a 3D matrix, suggesting that targeting SYNJ2 may prevent metastasis in breast cancer patients. Amplified HER2, which encodes a member of the epidermal growth factor receptor (EGFR) family, is a target of effective therapies against breast cancer. In search for similarly targetable genomic aberrations, we identified copy number gains in SYNJ2, which encodes the 5′-inositol lipid phosphatase synaptojanin 2, as well as overexpression in a small fraction of human breast tumors. Copy gain and overexpression correlated with shorter patient survival and a low abundance of the tumor suppressor microRNA miR-31. SYNJ2 promoted cell migration and invasion in culture and lung metastasis of breast tumor xenografts in mice. Knocking down SYNJ2 impaired the endocytic recycling of EGFR and the formation of cellular lamellipodia and invadopodia. Screening compound libraries identified SYNJ2-specific inhibitors that prevented cell migration but did not affect the related neural protein SYNJ1, suggesting that SYNJ2 is a potentially druggable target to block cancer cell migration.

Evidence showing that the two-chain form of vitronectin is produced in the liver by a selective furin cleavage.

The adhesive protein vitronectin (75 kDa) occurs in human blood fluid in a one‐chain (Vn75) or a two‐chain form (Vn65+10), and is produced by a specific cleavage (at Arg379–Ala380), by a proteinase not identified hitherto. These two forms were shown to be functionally different and therefore, this cleavage may have a regulatory significance in vivo. Here, we report the use of a tailored one‐chain recombinant Vn, a specific protein kinase A phosphorylation at Ser378, and sequence analysis to show: (1) that none of the proteinases originating from blood, previously thought to be the endogenous proteinase (plasmin, thrombin, tPA, and uPA), is indeed the in vivo convertase; and (2) that furin, a serine endoproteinase residing in the secretory pathway of hepatocytes, where Vn is synthesized, specifically cleaves Vn at the endogenous cleavage site. Consequently, we propose that the Vn75 to Vn65+10 conversion takes place in the liver (not in blood) and is carried out by furin.

DNA CONFORMATION AND FOLDING: FROM SOLUTION TO THE HIGHER ORDER STRUCTURE OF CHROMATIN

We describe the folding of DNA in solution, in the nucleosome and in chromatin. From measurements of plasmid ColE1-III linear LiDNA in LiCl solutions we find a limiting value for the persistence length a of 29 nm at high concentrations of salt. No condensation of DNA occurs up to 5 M LiCl. DNA in chromatin, at low concentrations of salt, is compacted because of the folding of the DNA in the nucleosome. To DNA of chromatin comprising about 45 nucleosomes, would correspond a radius of gyration Rg of 253 nm, if free in solution. The measured value for the “10 nm” fiber is 74 nm. Considering the folding due to the linker DNA we calculate about 83 nm for Rg. Upon addition of up to 75 mM NaCl or 0.3 mM MgCl2 chromatin folds into the “30 nm” higher order fiber and Rg for this sample is reduced to about 28 nm. We calculate 5.9 nucleosomes in the helical repeat, if a regular solenoidal structure is assumed.