Characterization of mild whole-body hyperthermia protocols using human breast, ovarian, and colon tumors grown in severe combined immunodeficient mice.

OBJECTIVE: We have shown that one treatment of fever-like whole body hyperthermia (WBH) on mice bearing human breast tumors results in a tumor growth delay. Our goal was to repeat this study in mice bearing human ovarian or colon tumors. We further evaluated this WBH protocol by performing multiple and interrupted WBH treatments. METHODS: Human tumors were grown in severe combined immunodeficient (SCID) mice. For WBH, core body temperatures were maintained at 39.8+/-0.2 degrees C for 6-8 hours. Multiple treatments were given 6-7 days apart. Interrupted WBH consisted of three 2-hour heatings, 15 minutes apart. Tumor growth time (TGT) was the number of days to grow 1.5 or 2 times in volume. RESULTS: For WBH-treated ovarian tumors, TGT was 12+/-1.2d, compared with 5.0+/-0.1d for untreated mice (P < 0.05). For colon tumors with one WBH treatment TGT was 4.4+/-1.1d. Two and three treatments had TGTs of 9+/-2.3d and 8+/-1.6d. For the untreated tumors, TGT was 2+/-0.7d (P < 0.01 for one, two, and three treatments). Histological examination indicated that one and two treatments were associated with cellular damage within the tumors. With a slower growing colon tumor, the TGT was 24+/-3.3d with three WBH treatments, compared with 14+/-1.8d for controls (P < 0.01). The TGT of breast tumors treated with interrupted WBH was not significantly different than the noninterrupted, with TGT of 7.3+/-0.8d and 6.2+/-1.0d, respectively. CONCLUSIONS: These data illustrate that WBH causes a tumor growth delay in mice bearing human ovarian and colon tumors. This response is enhanced with a second treatment of WBH. Interrupted and noninterrupted WBH give comparable anti-tumor results. We will continue to evaluate WBH in various animal models to optimize its potential for clinical administration and maximize the anti-tumor response

Generation of Arbitrary Frequency Chirps with a Fiber-Based Phase Modulator and Self-Injection-Locked Diode Laser

We present a novel technique for producing pulses of laser light whose frequency is arbitrarily chirped. The output from a diode laser is sent through a fiber-optical delay line containing a fiber-based electro-optical phase modulator. Upon emerging from the fiber, the phase-modulated pulse is used to injection-lock the laser and the process is repeated. Large phase modulations are realized by multiple passes through the loop while the high optical power is maintained by self-injection-locking after each pass. Arbitrary chirps are produced by driving the modulator with an arbitrary waveform generator

Disruption of Spectrin-Like Cytoskeleton in Differentiating Keratinocytes by PKCδ Activation Is Associated with Phosphorylated Adducin

Spectrin is a central component of the cytoskeletal protein network in a variety of erythroid and non-erythroid cells. In keratinocytes, this protein has been shown to be pericytoplasmic and plasma membrane associated, but its characteristics and function have not been established in these cells. Here we demonstrate that spectrin increases dramatically in amount and is assembled into the cytoskeleton during differentiation in mouse and human keratinocytes. The spectrin-like cytoskeleton was predominantly organized in the granular and cornified layers of the epidermis and disrupted by actin filament inhibitors, but not by anti-mitotic drugs. When the cytoskeleton was disrupted PKCδ was activated by phosphorylation on Thr505. Specific inhibition of PKCδ(Thr505) activation with rottlerin prevented disruption of the spectrin-like cytoskeleton and the associated morphological changes that accompany differentiation. Rottlerin also inhibited specific phosphorylation of the PKCδ substrate adducin, a cytoskeletal protein. Furthermore, knock-down of endogenous adducin affected not only expression of adducin, but also spectrin and PKCδ, and severely disrupted organization of the spectrin-like cytoskeleton and cytoskeletal distribution of both adducin and PKCδ. These results demonstrate that organization of a spectrin-like cytoskeleton is associated with keratinocytes differentiation, and disruption of this cytoskeleton is mediated by either PKCδ(Thr505) phosphorylation associated with phosphorylated adducin or due to reduction of endogenous adducin, which normally connects and stabilizes the spectrin-actin complex

Combined Inactivation of MYC and K-Ras Oncogenes Reverses Tumorigenesis in Lung Adenocarcinomas and Lymphomas

Conditional transgenic models have established that tumors require sustained oncogene activation for tumor maintenance, exhibiting the phenomenon known as "oncogene-addiction." However, most cancers are caused by multiple genetic events making it difficult to determine which oncogenes or combination of oncogenes will be the most effective targets for their treatment.To examine how the MYC and K-ras(G12D) oncogenes cooperate for the initiation and maintenance of tumorigenesis, we generated double conditional transgenic tumor models of lung adenocarcinoma and lymphoma. The ability of MYC and K-ras(G12D) to cooperate for tumorigenesis and the ability of the inactivation of these oncogenes to result in tumor regression depended upon the specific tissue context. MYC-, K-ras(G12D)- or MYC/K-ras(G12D)-induced lymphomas exhibited sustained regression upon the inactivation of either or both oncogenes. However, in marked contrast, MYC-induced lung tumors failed to regress completely upon oncogene inactivation; whereas K-ras(G12D)-induced lung tumors regressed completely. Importantly, the combined inactivation of both MYC and K-ras(G12D) resulted more frequently in complete lung tumor regression. To account for the different roles of MYC and K-ras(G12D) in maintenance of lung tumors, we found that the down-stream mediators of K-ras(G12D) signaling, Stat3 and Stat5, are dephosphorylated following conditional K-ras(G12D) but not MYC inactivation. In contrast, Stat3 becomes dephosphorylated in lymphoma cells upon inactivation of MYC and/or K-ras(G12D). Interestingly, MYC-induced lung tumors that failed to regress upon MYC inactivation were found to have persistent Stat3 and Stat5 phosphorylation.Taken together, our findings point to the importance of the K-Ras and associated down-stream Stat effector pathways in the initiation and maintenance of lymphomas and lung tumors. We suggest that combined targeting of oncogenic pathways is more likely to be effective in the treatment of lung cancers and lymphomas

Spectrin-based skeleton as an actor in cell signaling

This review focuses on the recent advances in functions of spectrins in non-erythroid cells. We discuss new data concerning the commonly known role of the spectrin-based skeleton in control of membrane organization, stability and shape, and tethering protein mosaics to the cellular motors and to all major filament systems. Particular effort has been undertaken to highlight recent advances linking spectrin to cell signaling phenomena and its participation in signal transduction pathways in many cell types

Knowledge-guided docking: accurate prospective prediction of bound configurations of novel ligands using Surflex-Dock

Prediction of the bound configuration of small-molecule ligands that differ substantially from the cognate ligand of a protein co-crystal structure is much more challenging than re-docking the cognate ligand. Success rates for cross-docking in the range of 20–30 % are common. We present an approach that uses structural information known prior to a particular cutoff-date to make predictions on ligands whose bounds structures were determined later. The knowledge-guided docking protocol was tested on a set of ten protein targets using a total of 949 ligands. The benchmark data set, called PINC (“PINC Is Not Cognate”), is publicly available. Protein pocket similarity was used to choose representative structures for ensemble-docking. The docking protocol made use of known ligand poses prior to the cutoff-date, both to help guide the configurational search and to adjust the rank of predicted poses. Overall, the top-scoring pose family was correct over 60 % of the time, with the top-two pose families approaching a 75 % success rate. Correct poses among all those predicted were identified nearly 90 % of the time. The largest improvements came from the use of molecular similarity to improve ligand pose rankings and the strategy for identifying representative protein structures. With the exception of a single outlier target, the knowledge-guided docking protocol produced results matching the quality of cognate-ligand re-docking, but it did so on a very challenging temporally-segregated cross-docking benchmark

A Remote Arene-Binding Site on Prostate Specific Membrane Antigen Revealed by Antibody-Recruiting Small Molecules

Prostate specific membrane antigen (PSMA) is a membrane-bound glutamate carboxypeptidase overexpressed in many forms of prostate cancer. Our laboratory has recently disclosed a class of small molecules, called ARM-Ps (antibody-recruiting molecule targeting prostate cancer) that are capable of enhancing antibody-mediated immune recognition of prostate cancer cells. Interestingly, during the course of these studies, we found ARM-Ps to exhibit extraordinarily high potencies toward PSMA, compared to previously reported inhibitors. Here, we report in-depth biochemical, crystallographic, and computational investigations which elucidate the origin of the observed affinity enhancement. These studies reveal a previously unreported arene-binding site on PSMA, which we believe participates in an aromatic stacking interaction with ARMs. Although this site is composed of only a few amino acid residues, it drastically enhances small molecule binding affinity. These results provide critical insights into the design of PSMA-targeted small molecules for prostate cancer diagnosis and treatment; more broadly, the presence of similar arene-binding sites throughout the proteome could prove widely enabling in the optimization of small-molecule–protein interactions