Numerous epidemiological observations point to sex differences in lung cancer etiology and progression. The present study was aimed at understanding the bases of these sex differences. To test the effect of estradiol on tumor progression, we used a mouse model based on conditional Kras expression and concurrent deletion of Tp53 following inhalation of an adenoviral vector expressing Cre recombinase (AdeCre). Ovariectomized females and males were treated with estradiol via a continuous-release capsule. Tumor multiplicity, tumor volume, and histological grade were determined at 10 weeks after AdeCre administration. Cell proliferation was monitored by Ki67 immunohistochemistry at 4 and 10 weeks after AdeCre administration. At 10 weeks, female mice had more than twice the number of tumors evident on the surface of the lungs than male mice; ovariectomy eliminated this sex difference. The estrogen treatment significantly increased tumor number and volume in ovariectomized females and in males. Histological character of the tumors ranged from adenoma to adenocarcinoma. Ovary-intact females exhibited higher grade tumors than ovariectomized females or males. Progression to higher histological grade was stimulated by estrogen in male mice but not in ovariectomized females. At 10 weeks after AdeCre administration, tumor cell Ki67-labeling varied widely, precluding assessment of an estrogen effect; however, at 4 weeks, Ki67 labeling of lung parenchymal cells was increased 3.5-fold by estrogen. In conclusion, estrogen acts as a promoter for lung adenocarcinoma in a genetically defined lung cancer model; estrogen-induced cell proliferation in the oncogene-initiated cells is likely to play a role in this tumor promoter activity.

In vitro studies suggest that activation of class I A phosphatidylinositol 3 (PI-3) kinase is necessary for normal erythroid cell development. However, when class I A PI-3 kinase-deficient mice were generated by a targeted deletion of the p85␣ regulatory subunit, fetal erythropoiesis was reportedly unaffected. Given the discrepancies between these studies, we performed a more detailed in vivo analy- IntroductionErythropoiesis is a coordinated process regulated by specific signaling pathways. In vitro studies suggest that activation of class I A phosphatidylinositol 3 (PI-3) kinase following binding of erythropoietin (Epo) and kit-ligand (KitL) to their receptors is required for the proliferation, survival, and differentiation of erythroid progenitors. 1-10 However, the physiologic role of class I A PI-3 kinase in regulating fetal and adult erythropoiesis is not known. A class I A PI-3 kinase knock-out mouse was generated by a targeted deletion of the p85␣ regulatory subunit of this kinase. 11,12 Although p85␣ Ϫ/Ϫ mice die shortly after birth secondary to hepatic necrosis and chylous ascites, 12 we used p85␣ Ϫ/Ϫ embryos to investigate the effect of p85␣ deficiency on fetal liver erythropoiesis in vivo. Study designMice and fetal hematopoietic cell isolation p85␣ ϩ/Ϫ mice (129/SV ϫ C57BL/6) were obtained from Dr Lewis Cantley at Harvard University (Boston, MA). Studies were conducted with a protocol approved by the Indiana University Animal Care and Use Committee. The p85␣ allele was genotyped by polymerase chain reaction (PCR) as previously described. 11,12 p85␣ ϩ/Ϫ mice were mated to produce day-14.5 p85␣ Ϫ/Ϫ and p85␣ ϩ/ϩ embryos. Fetal liver cells were isolated as previously described. 13 Single cell suspensions were prepared by pushing the hepatic tissues through a 23-gauge needle. Peripheral blood analysis and fetal liver erythropoiesisEmbryonic blood was obtained from day-14.5 fetal hearts for peripheral smears, and fetal liver touch preps were performed as previously described 14 and stained with Wright-Giemsa (Dade Behring, Newark, DE). Photomicrographs of peripheral smears and touch preps were taken with an Olympus DP11 microscope (Melville, NY). Colony assaysRecombinant KitL and Epo were obtained from Peprotech (Rocky Hill, NJ) and Amgen (Thousand Oaks, CA), respectively. Erythroid burst-forming unit (BFU-E) and erythroid colony-forming unit (CFU-E) assays were performed exactly as previously described. 15 c-kit ؉ cell isolationFetal liver cells were incubated with 1 g phycoerythrin (PE)-conjugated c-kit monoclonal antibody (Pharmingen, San Diego, CA) per 10 6 cells, placed on ice for 20 minutes, pelleted, washed, and resuspended in phosphate-buffered saline (PBS). C-kit ϩ cells were purified by immunomagnetic bead enrichment as previously described. 15Apoptosis and proliferation assays c-kit ϩ fetal liver cells were stained with fluorescein isothiocyanate (FITC)-annexin V (Pharmingen) and propidium iodide (Sigma, St Louis, MO) exactly per manufacturer's protocol followed by flow cytometric analysis as ...

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Autotaxin is a secreted enzyme that catalyzes the conversion of lysophosphatidyl choline into the bioactive lipid mediator lysophosphatidic acid (LPA). It is the primary enzyme responsible for LPA production in plasma. It is upregulated in inflammatory conditions and inhibition of autotaxin may have antiinflammatory activity in a variety of inflammatory diseases. To determine the role of autotaxin and LPA in the pathophysiology of inflammatory disease states, we used a potent and orally bioavailable inhibitor of autotaxin that we have recently identified, and characterized it in mouse models of inflammation, inflammatory bowel disease (IBD), multiple sclerosis (MS), and visceral pain. Compound-1, a potent inhibitor of autotaxin with an IC 50 of ∼2 nM, has good oral pharmacokinetic properties in mice and results in a substantial inhibition of plasma LPA that correlates with drug exposure levels. Treatment with the inhibitor resulted in significant anti-inflammatory and analgesic effects in the carrageenaninduced paw inflammation and acetic acid-induced visceral pain tests, respectively. Compound-1 also significantly inhibited disease activity score in the dextran sodium sulfate-induced model of IBD, and in the experimental autoimmune encephalomyelitis model of MS. In conclusion, the present study demonstrates the anti-inflammatory and analgesic properties of a novel inhibitor of autotaxin that may serve as a therapeutic option for IBD, MS, and pain associated with inflammatory states.

A splicing variant of rat striatin-3 (rSTRN3g) was found to associate with estrogen receptor-a (ERa) in a ligand-dependent manner. In two-hybrid and pull-down analyses, estradiol induced an interaction between rSTRN3g and ERa. STRN3g protein was found in nuclear extracts from rat uterus and human cell lines. Overexpression of rSTRN3g induced a decrease in ERa transcriptional activity but had no effect on ERb activity. Immunoprecipitation analyses showed that rSTRN3g interacts with both the ERa and the catalytic subunit of protein phosphatase 2A (PP2A(C)). The transrepressor action of rSTRN3g was overcome by okadaic acid, an inhibitor of PP2A(C), and by cotransfection of PP2A(C) siRNA. rSTRN3g caused dephosphorylation of ERa at serine 118 and this was abrogated by okadaic acid. ERa lacking phosphorylation sites at either serine 118 or 167 was insensitive to the corepressor action of rSTRN3g. These observations suggest that an rSTRN3g-PP2A(C) complex is recruited to agonist-activated ERa, thereby leading to its dephosphorylation and inhibiting transcription.