In breast cancer cells, growth factor stimulation of membrane protrusion was a better predictor of 3D migration than 2D motility, cognate receptor expression, or receptor activation.

Replication Protein A (RPA) is the primary eukaryotic ssDNA binding protein utilized in diverse DNA transactions in the cell. RPA is a heterotrimeric protein with seven globular domains connected by flexible linkers, which enable substantial inter-domain motion that is essential to its function. Small angle X-ray scattering (SAXS) experiments on two multi-domain constructs from the Nterminus of the large subunit (RPA70) were used to examine the structural dynamics of these domains and their response to the binding of ssDNA. The SAXS data combined with molecular dynamics simulations reveal substantial interdomain flexibility for both RPA70AB (the tandem high affinity ssDNA binding domains A and B connected by a 10-residue linker) and RPA70NAB (RPA70AB extended by a 70-residue linker to the RPA70N protein interaction domain). Binding of ssDNA to RPA70NAB reduces the interdomain flexibility between the A and B domains, but has no effect on RPA70N. These studies provide the first direct measurements of changes in orientation of these three RPA domains upon binding ssDNA. The results support a model in which RPA70N remains structurally independent of RPA70AB in the DNA bound state and therefore freely available to serve as a protein recruitment module.RPA is the primary eukaryotic ssDNA binding protein utilized for diverse DNA transactions in the replication and maintenance of the genome (reviewed by Fanning and coworkers [1]). RPA functions by binding and protecting ssDNA from degradation by endonucleases, inhibiting formation of ssDNA secondary structure, and providing a scaffold for DNA processing machinery by interacting with numerous DNA processing proteins. RPA biochemical functions and biological activities have been intensively investigated and the structures of its domains determined [2][3][4][5][6][7][8][9]. Despite this detailed information, the mechanisms for RPA function remain poorly understood, largely due to the inherent difficulties of *To whom correspondence should be addressed: Center for Structural Biology, Vanderbilt University, 465 21 st Avenue, Suite 5140, Nashville, Telephone: (615) 936-2210; Fax: (615) 936-2211; walter.chazin@vanderbilt.edu.• These authors contributed equally to this work.Experimental and theoretical scattering profiles, P(r) functions, SAXS envelopes and atomic models will be deposited in the BIOISIS database (www.bioisis.net) under accession code 61. SUPPORTING INFORMATION AVAILABLE. SEC profiles and Guinier analysis for RPA70AB, RPA70NAB and their ssDNA complexes. This material is available free of charge via the internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2011 April 6. Published in final edited form as:Biochemistry. 2010 April 6; 49(13): 2880-2889. doi:10.1021/bi9019934. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript characterizing proteins with modular organization and the fact that RPA function is integrated within complex multi-protein machinery.RPA is a mod...

Magnetic fluid hyperthermia as a cancer treatment method is an attractive alternative to other forms of hyperthermia. It is based on the heat released by magnetic nanoparticles subjected to an alternating magnetic field. Recent studies have shown that magnetic fluid hyperthermia-treated cells respond significantly better to chemotherapeutic treatment compared with cells treated with hot water hyperthermia under the same temperature conditions. We hypothesized that this synergistic effect is due to an additional stress on the cellular membrane, independent of the thermal heat dose effect that is induced by nanoparticles exposed to an alternating magnetic field. This would result in an increase in Cis-diammine-dichloroplatinum (II) (cDDP, cisplatin) uptake via passive transport. To test this hypothesis, we exposed cDDPtreated cells to extracellular copper in order to hinder the human cell copper transporter (hCTR1)-mediated active transport of cDDP. This, in turn, can increase the passive transport of the drug through the cell membrane. Our results did not show statistically significant differences in surviving fractions for cells treated concomitantly with magnetic fluid hyperthermia and cDDP, in the presence or absence of copper. Nonetheless, significant copper-dependent variations in cell survival were observed for samples treated with combined cDDP and hot water hyperthermia. These results correlated with platinum uptake studies, which showed that cells treated with magnetic fluid hyperthermia had higher platinum uptake than cells treated with hot water hyperthermia. Changes in membrane fluidity were tested through fluorescence anisotropy measurements using trimethylamine-diphenylhexatriene. Additional uptake studies were conducted with acridine orange and measured by flow cytometry. These studies indicated that magnetic fluid hyperthermia significantly increases cell membrane fluidity relative to hot water hyperthermia and untreated cells, and hence this could be a factor contributing to the increase of cDDP uptake in magnetic fluid hyperthermia-treated cells. Overall, our data provide convincing evidence that cell membrane permeability induced by magnetic fluid hyperthermia is significantly greater than that induced by hot water hyperthermia under similar temperature conditions, and is at least one of the mechanisms responsible for potentiation of cDDP by magnetic fluid hyperthermia in Caco-2 cells.

The induction of hyperthermia using nanoparticles, known as magnetic fluid hyperthermia (MFH) in combination with anti-cancer drugs is an attractive method because of the potential for enhanced anti-cancer effects. Recent studies have shown that cells treated with MFH are more sensitive to the proteasome inhibitor bortezomib (BZ) than cells treated by hot water hyperthermia (HWH) under the same temperature conditions. We hypothesized that enhanced proteotoxic stress, caused by a combination of microtubule damage and an increase in the amount of aggregated proteins, may be partially responsible for this observation. To test this hypothesis MCF-7 cells were exposed to hyperthermic treatment (MFH or HWH) at 43 °C or 45 °C for 30 minutes. Then, aggresome formation and microtubule disruption studies at 30 minutes or 2.5 hours of recovery time were performed to evaluate the progressive effects induced by the two treatments. Cell viability at short and long times was evaluated. Aggresome formation and microtubule disruption results suggested that one of the mechanisms by which MFH enhances BZ cytotoxicity is the formation and subsequent accumulation of aggregated proteins in the cytosol due to the interruption of their transport to the perinuclear area through microtubules. Our data show evidence that MFH induces a more toxic and unmitigated proteotoxic stress than HWH under similar temperature conditions.