Generation of a Novel HLA Class I Transgenic Mouse Model Carrying a Knock-in Mutation at the ß2-Microglobulin Locus.

We generated a series of monochain HLA class I knock-in (KI) mouse strains, in which a chimeric HLA class I molecule (α1/α2 domain of HLA-A*0201, HLA-A*0301, HLA-A*2402, or HLA-A*3101 and α3 domain of H-2Db) was covalently linked with 15 aa to human ß2-microglobulin (ß2m) and introduced into the endogenous mouse ß2m locus. In homozygous KI mice, mouse ß2m gene disruption resulted in loss of the endogenous H-2 class I molecules and reduction in the peripheral CD8+ T cell population that was partially restored by monochain HLA class I expression. A gene dosage-dependent expression of HLA, similar to that in human PBMCs, was detected in heterozygous and homozygous HLA KI mice. Upon vaccination with various virus epitopes, HLA-restricted, epitope-specific CTLs were induced in HLA KI mice, similar to the response in the commonly used HLA transgenic mice. Importantly, the CTL responses induced in heterozygous KI mice were similar to those in homozygous KI mice. These results suggest that coexpression of H-2 class I does not affect HLA-restricted CTL responses in HLA KI mice, which differs from the situation reported for monochain HLA Tg × ß2m-/- mice. Furthermore, we generated double KI mice harboring two different HLA (HLA-A*2402 and HLA-A*0301) KI alleles, which showed a CTL response against both HLA-A24 and HLA-A3 epitopes when immunized with a mixture of both peptides. These results indicated that this HLA class I KI mouse model provides powerful research tools not only for the study of HLA class I-restricted CTL responses, but also for preclinical vaccine evaluation.

High Potency VEGFRs/MET/FMS Triple Blockade by TAS-115 Concomitantly Suppresses Tumor Progression and Bone Destruction in Tumor-Induced Bone Disease Model with Lung Carcinoma Cells.

Approximately 25-40% of patients with lung cancer show bone metastasis. Bone modifying agents reduce skeletal-related events (SREs), but they do not significantly improve overall survival. Therefore, novel therapeutic approaches are urgently required. In this study, we investigated the anti-tumor effect of TAS-115, a VEGFRs and HGF receptor (MET)-targeted kinase inhibitor, in a tumor-induced bone disease model. A549-Luc-BM1 cells, an osteo-tropic clone of luciferase-transfected A549 human lung adenocarcinoma cells (A549-Luc), produced aggressive bone destruction associated with tumor progression after intra-tibial (IT) implantation into mice. TAS-115 significantly reduced IT tumor growth and bone destruction. Histopathological analysis showed a decrease in tumor vessels after TAS-115 treatment, which might be mediated through VEGFRs inhibition. Furthermore, the number of osteoclasts surrounding the tumor was decreased after TAS-115 treatment. In vitro studies demonstrated that TAS-115 inhibited HGF-, VEGF-, and macrophage-colony stimulating factor (M-CSF)-induced signaling pathways in osteoclasts. Moreover, TAS-115 inhibited Feline McDonough Sarcoma oncogene (FMS) kinase, as well as M-CSF and receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation. Thus, VEGFRs/MET/FMS-triple inhibition in osteoclasts might contribute to the potent efficacy of TAS-115. The fact that concomitant dosing of sunitinib (VEGFRs/FMS inhibition) with crizotinib (MET inhibition) exerted comparable inhibitory efficacy for bone destruction to TAS-115 also supports this notion. In conclusion, TAS-115 inhibited tumor growth via VEGFR-kinase blockade, and also suppressed bone destruction possibly through VEGFRs/MET/FMS-kinase inhibition, which resulted in potent efficacy of TAS-115 in an A549-Luc-BM1 bone disease model. Thus, TAS-115 shows promise as a novel therapy for lung cancer patients with bone metastasis.

Down-regulation of the large-conductance Ca(2+)-activated K+ channel, K(Ca)1.1 in the prostatic stromal cells of benign prostate hyperplasia.

Large-conductance Ca(2+)-activated K(+) (BK(Ca)) channel encoded by K(Ca)1.1 plays an important role in the control of smooth muscle tone by modulating membrane potential and intracellular Ca(2+) mobilization. BK(Ca) channel is functionally expressed in prostatic smooth muscle cells, and is activated by α(1)-adrenoceptor agonists. The main objective of this study was to elucidate the pathophysiological significance of changes in prostatic K(Ca)1.1 expressions in benign prostatic hyperplasia (BPH). Our previous study has shown that K(Ca)3.1 encoding intermediate-conductance K(Ca) (IK(Ca)) channel is up-regulated in stromal cells of implanted urogenital sinuses (UGSs) of stromal hyperplasia BPH model rats and in those of prostatic tissues from BPH patients. In the present study, the results from real-time polymerase chain reaction (PCR), Western blot, and immunohistochemical analyses showed significant down-regulation of K(Ca)1.1 transcripts and proteins and negative correlation between K(Ca)1.1 and K(Ca)3.1 transcript expressions in prostatic stromal cells of both BPH model rats and BPH patients. Corresponding to down-regulation of K(Ca)1.1 expression in stromal cells of implanted UGSs, membrane depolarization by application of the BK(Ca) channel blocker was disappeared. Down-regulation of K(Ca)1.1 may be involved in the phenotype switch from contractile profile to proliferative one in prostatic stromal cells of BPH patients.

Intermediate-conductance Ca2+-activated K+ channel, KCa3.1, as a novel therapeutic target for benign prostatic hyperplasia.

Recently, a new experimental stromal hyperplasia animal model corresponding to clinical benign prostatic hyperplasia (BPH) was established. The main objective of this study was to elucidate the roles of the intermediate-conductance Ca(2+)-activated K(+) channel (K(Ca)3.1) in the implanted urogenital sinus (UGS) of stromal hyperplasia BPH model rats. Using DNA microarray, real-time polymerase chain reaction, Western blot, and/or immunohistochemical analyses, we identified the expression of K(Ca)3.1 and its transcriptional regulators in implanted UGS of BPH model rats and prostate needle-biopsy samples and surgical prostate specimens of BPH patients. We also examined the in vivo effects of a K(Ca)3.1 blocker, 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34), on the proliferation index of implanted UGS by measurement of UGS weights and proliferating cell nuclear antigen immunostaining. K(Ca)3.1 genes and proteins were highly expressed in implanted UGS rather than in the normal host prostate. In the implanted UGS, the gene expressions of two transcriptional regulators of K(Ca)3.1, repressor element 1-silencing transcription factor and c-Jun, were significantly down- and up-regulated, and the regulations were correlated negatively or positively with K(Ca)3.1 expression, respectively. Positive signals of K(Ca)3.1 proteins were detected exclusively in stromal cells, whereas they were scarcely immunolocalized to basal cells of the epithelium in implanted UGS. In vivo treatment with TRAM-34 significantly suppressed the increase in implanted UGS weights compared with the decrease in stromal cell components. Moreover, significant levels of K(Ca)3.1 expression were observed in human BPH samples. K(Ca)3.1 blockers may be a novel treatment option for patients suffering from BPH.

New histopathological experimental model for benign prostatic hyperplasia: stromal hyperplasia in rats.

PURPOSE: Histological observations of clinical benign prostatic hyperplasia specimens show that benign prostatic hyperplasia tissue is mainly composed of stromal components, smooth muscle and fibrous tissue, so-called stromal hyperplasia. However, little is understood regarding the pathogenesis of this stromal hyperplasia due to no suitable stromal hyperplasia model to elucidate the pathology of benign prostatic hyperplasia. We created a novel model of benign prostatic hyperplasia accompanied by clinically relevant stromal hyperplasia. MATERIALS AND METHODS: The urogenital sinus isolated from male rat 20-day embryos was implanted into pubertal male rat ventral prostates. Two to 8 weeks after the operation the implanted urogenital sinus was isolated, weighed and subjected to histochemical analysis. To distinguish between and characterize the epithelial and stromal components we stained for collagen, smooth muscle components, growth factors and proliferating cell nuclear antigen. In addition, to determine whether the implanted urogenital sinus had differentiated into functional prostate we stained for androgen receptor and dorsolateral prostatic secretory protein. RESULTS: Urogenital sinuses removed from male rat 20-day embryos initially weighed approximately 1 mg. After implantation into host rat ventral prostates they grew in time dependent fashion with no apparent change in the original ventral prostate weight in the host rat. Implanted urogenital sinus weight was more than 100 mg 3 weeks after implantation. Histological observation demonstrated that the ratio of stromal to total area was approximately 70%, which was much higher than that in age matched rat ventral prostates and in a testosterone induced epithelial hyperplasia model (approximately 20% and 15%, respectively). This predominantly stromal tissue composition was maintained up to 8 weeks after implantation. Proliferating cell nuclear antigen staining revealed that the ratio of proliferating cells in stroma was equal to or greater than that in epithelium. In this model the antiandrogen agent chlormadinone acetate (Wako Pure Chemicals Industries, Osaka, Japan) at a dose of 10 mg/kg prevented the increase in implanted urogenital sinus weight (19.1%) but its potency was less than that seen in the testosterone induced epithelial hyperplasia model, that is 93.4% at the 10 mg/kg dose. CONCLUSIONS: We have established a new experimental stromal hyperplasia model corresponding to clinical benign prostatic hyperplasia in terms of the composition of stromal components and functional differentiation of the prostate. Furthermore, the localization and time course of growth factor expression were also similar to those in men with benign prostatic hyperplasia.