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 effect of light and 2,4-D on anthocyanin production in Oxalis reclinata callus

In vitro cultures of O. reclinata accumulate red anthocyanin pigments. Two callus lines were established from O. reclinata, one red and the other non-pigmented. The red callus accumulated cyanidin-3-glucoside as a major pigment. Light irradiation induced anthocyanin synthesis in white callus, resulting in a heterogenous red callus line being formed. The incubation of red and white callus cultures in the dark or at low-light resulted in the repression of red pigment accumulation. The application of 2,4-D (1.0 mg l^-1) inhibited pigment production in the white callus and decreased anthocyanin accumulation in the red callus. The polypeptide composition of the red and white callus lines from O. reclinata were compared using two-dimensional electrophoresis. The red callus had a larger subset of neutral and acidic polypeptides.     

Alteration of cellular adhesion by heat shock

Chinese hamster ovary (CHO) cells were analyzed for their ability to reassemble microfilament bundles, to remain attached to a tissue culture surface, or to initiate and complete attachment onto a substrate after heat shock (45 degrees C/10 min). The cells remained attached to the tissue culture surface during and after the heat shock while the actin microfilament bundles were reversibly disrupted. Heat shock inhibited the ability of the cells to initiate and complete attachment onto a new tissue culture surface or onto a plastic surface coated with vitronectin. An inspection of the proteins present in substrate-attached material (SAM) revealed 11 major proteins containing glucosamine whose apparent Mr values were 250,000, 200,000, 150,000, 140,000, 90,000, 86,000, 82,000, 68,000, 54,000, 47,000, and 46,000. Three of the proteins (p200, p150, and p46) bound to wheat germ agglutinin while p150 and p140 bound to concanavalin A. The composition of the 11 proteins of the SAM fraction synthesized previous to the heat shock was not altered during heat shock. However, the appearance of the newly synthesized proteins in the SAM fraction was delayed by heat shock (0.5 h for p150 and 6 h for p82). The ability of heat-shocked cells to reattach onto a vitronectin-coated surface correlated with the appearance of newly synthesized p150 and p82 in the SAM fraction. Our results suggest that in addition to the microfilament bundles, heat shock may reversibly disrupt the cellular adhesion site. Further, p150 and p82, proteins whose appearance in the SAM fraction is delayed by heat shock, may be involved in the cellular attachment onto substrates, including vitronectin.