ERK regulates strain‐induced migration and proliferation from different subcellular locations

Repetitive deformation like that engendered by peristalsis or villous motility stimulates intestinal epithelial proliferation on collagenous substrates and motility across fibronectin, each requiring ERK. We hypothesized that ERK acts differently at different intracellular sites. We stably transfected Caco‐2 cells with ERK decoy expression vectors that permit ERK activation but interfere with its downstream signaling. Targeting sequences constrained the decoy inside or outside the nucleus. We assayed proliferation by cell counting and migration by circular wound closure with or without 10% repetitive deformation at 10 cycles/min. Confocal microscopy confirmed localization of the fusion proteins. Inhibition of phosphorylation of cytoplasmic RSK or nuclear Elk confirmed functionality. Both the nuclear‐localized and cytosolic‐localized ERK decoys prevented deformation‐induced proliferation on collagen. Deformation‐induced migration on fibronectin was prevented by constraining the decoy in the nucleus but not in the cytosol. Like the nuclear‐localized ERK decoy, a Sef‐overexpressing adenovirus that sequesters ERK in the cytoplasm also blocked the motogenic and mitogenic effects of strain. Inhibiting RSK or reducing Elk ablated both the mitogenic and motogenic effects of strain. RSK isoform reduction revealed isoform specificity. These results suggest that ERK must translocate to the nucleus to stimulate cell motility while ERK must act in both the cytosol and the nucleus to stimulate proliferation in response to strain. Selectively targeting ERK within different subcellular compartments may modulate or replace physical force effects on the intestinal mucosa to maintain the intestinal mucosal barrier in settings when peristalsis or villous motility are altered and fibronectin is deposited into injured tissue. J. Cell. Biochem. 109: 711–725, 2010. Published in 2010 Wiley‐Liss, Inc.     

E2F Inhibition Synergizes with Paclitaxel in Lung Cancer Cell Lines

The CDK/Rb/E2F pathway is commonly disrupted in lung cancer, and thus, it is predicted that blocking the E2F pathway would have therapeutic potential. To test this hypothesis, we have examined the activity of HLM006474 (a small molecule pan-E2F inhibitor) in lung cancer cell lines as a single agent and in combination with other compounds. HLM006474 reduces the viability of both SCLC and NSCLC lines with a biological IC₅₀ that varies between 15 and 75 µM, but with no significant difference between the groups. Combination of HLM006474 with cisplatin and gemcitabine demonstrate little synergy; however, HLM006474 synergizes with paclitaxel. Surprisingly, we discovered that brief treatment of cells with HLM006474 led to an increase of E2F3 protein levels (due to de-repression of these promoter sites). Since paclitaxel sensitivity has been shown to correlate with E2F3 levels, we hypothesized that HLM006474 synergy with paclitaxel may be mediated by transient induction of E2F3. To test this, H1299 cells were depleted of E2F3a and E2F3b with siRNA and treated with paclitaxel. Assays of proliferation showed that both siRNAs significantly reduced paclitaxel sensitivity, as expected. Taken together, these results suggest that HLM006474 may have efficacy in lung cancer and may be useful in combination with taxanes.     

Genetic analysis of somatic embryogenesis in carrot cell culture: Initial characterization of six classes of temperature-sensitive variants

Cell cultures of the carrot Daucus carota are a useful experimental system for studying the genetic regulation of plant embryogenesis. A modified filtration-enrichment procedure was used to isolate 21 temperature-sensitive variants in somatic embryogenesis; the variants display normal embryo development at the permissive temperature (24°C) and altered development at the restrictive temperature (33°C). Temperature-shift experiments were performed on these variants to determine the timing of gene action for the putative temperature-sensitive alleles. According to their phenotypes at the restrictive temperature, these variants can be divided into six classes: No Growth, Callus Proliferation, Globularstage Block, Oblong-stage Block, Lateral Growth, and Root Formation. Although many variants exhibit lengthy temperature-sensitive periods, the temperature sensitivity of some variants is restricted to one or two embryonic stages. These results plus those in the literature are incorporated into a preliminary model concerning the genetic regulation of carrot embryogenesis.     

The role of alpha 6 integrin in prostate cancer migration and bone pain in a novel xenograft model

Of the estimated 565,650 people in the U.S. who will die of cancer in 2008, almost all will have metastasis. Breast, prostate, kidney, thyroid and lung cancers metastasize to the bone. Tumor cells reside within the bone using integrin type cell adhesion receptors and elicit incapacitating bone pain and fractures. In particular, metastatic human prostate tumors express and cleave the integrin A6, a receptor for extracellular matrix components of the bone, i.e., laminin 332 and laminin 511. More than 50% of all prostate cancer patients develop severe bone pain during their remaining lifetime. One major goal is to prevent or delay cancer induced bone pain. We used a novel xenograft mouse model to directly determine if bone pain could be prevented by blocking the known cleavage of the A6 integrin adhesion receptor. Human tumor cells expressing either the wildtype or mutated A6 integrin were placed within the living bone matrix and 21 days later, integrin expression was confirmed by RT-PCR, radiographs were collected and behavioral measurements of spontaneous and evoked pain performed. All animals independent of integrin status had indistinguishable tumor burden and developed bone loss 21 days after surgery. A comparison of animals containing the wild type or mutated integrin revealed that tumor cells expressing the mutated integrin resulted in a dramatic decrease in bone loss, unicortical or bicortical fractures and a decrease in the ability of tumor cells to reach the epiphyseal plate of the bone. Further, tumor cells within the bone expressing the integrin mutation prevented cancer induced spontaneous flinching, tactile allodynia, and movement evoked pain. Preventing A6 integrin cleavage on the prostate tumor cell surface decreased the migration of tumor cells within the bone and the onset and degree of bone pain and fractures. These results suggest that strategies for blocking the cleavage of the adhesion receptors on the tumor cell surface can significantly prevent cancer induced bone pain and slow disease progression within the bone. Since integrin cleavage is mediated by Urokinase-type Plasminogen Activator (uPA), further work is warranted to test the efficacy of uPA inhibitors for prevention or delay of cancer induced bone pain.