Sensitive detection of viable circulating tumor cells using a novel conditionally telomerase-selective replicating adenovirus in non-small cell lung cancer patients.

Circulating tumor cells (CTCs) have a crucial role in the clinical outcome of cancer patients. Detection of non-small cell lung cancer (NSCLC) using an antibody against epithelial cell adhesion molecule (EpCAM) in captured CTCs has low sensitivity; the loss of epithelial markers leads to underestimation of CTCs with mesenchymal phenotype. We propose a new approach for detection of viable CTCs, including those with epithelial-mesenchymal transition status (EMT-CTCs), using the new telomerase-specific replication-selective adenovirus (OBP-1101), TelomeScan F35. Peripheral venous blood samples and clinicopathological data were collected from 123 NSCLC patients. The sensitivity of CTC detection was 69.1%, and for patients with stage I, II, III and IV, it was 59.6%, 40.0%, 85.7%, and 75.0%, respectively. Among the EMT-CTC samples, 46% were vimentin positive and 39.0% of non-EMT-CTC samples were EpCAM positive. Patients testing positive for EMT-CTCs at baseline had poor response to chemotherapy (P = 0.025) and decreased progression-free survival (EMT-CTC positive vs. negative: 193 ± 47 days vs. 388 ± 47. days, P = 0.040) in comparison to those testing negative. TelomeScan F35 is a highly sensitive CTC detection system and will be a useful screening tool for early diagnosis of NSCLC patients. Mesenchymal-phenotype CTCs are crucial indicators of chemotherapeutic efficacy in NSCLC patients.

Efficient detection of human circulating tumor cells without significant production of false-positive cells by a novel conditionally replicating adenovirus.

Circulating tumor cells (CTCs) are promising biomarkers in several cancers, and thus methods and apparatuses for their detection and quantification in the blood have been actively pursued. A novel CTC detection system using a green fluorescence protein (GFP)-expressing conditionally replicating adenovirus (Ad) (rAd-GFP) was recently developed; however, there is concern about the production of false-positive cells (GFP-positive normal blood cells) when using rAd-GFP, particularly at high titers. In addition, CTCs lacking or expressing low levels of coxsackievirus-adenovirus receptor (CAR) cannot be detected by rAd-GFP, because rAd-GFP is constructed based on Ad serotype 5, which recognizes CAR. In order to suppress the production of false-positive cells, sequences perfectly complementary to blood cell-specific microRNA, miR-142-3p, were incorporated into the 3'-untranslated region of the E1B and GFP genes. In addition, the fiber protein was replaced with that of Ad serotype 35, which recognizes human CD46, creating rAdF35-142T-GFP. rAdF35-142T-GFP efficiently labeled not only CAR-positive tumor cells but also CAR-negative tumor cells with GFP. The numbers of false-positive cells were dramatically lower for rAdF35-142T-GFP than for rAd-GFP. CTCs in the blood of cancer patients were detected by rAdF35-142T-GFP with a large reduction in false-positive cells.

Overexpression of gamma-glutamyltransferase in transgenic mice accelerates bone resorption and causes osteoporosis.

We previously identified gamma-glutamyltransferase (GGT) by expression cloning as a factor inducing osteoclast formation in vitro. To examine its pathogenic role in vivo, we generated transgenic mice that overexpressed GGT in a tissue-specific manner utilizing the Cre-loxP recombination system. Systemic as well as local production of GGT accelerated osteoclast development and bone resorption in vivo by increasing the sensitivity of bone marrow macrophages to receptor activator of nuclear factor-kappaB ligand, an essential cytokine for osteoclastogenesis. Mutated GGT devoid of enzyme activity was as potent as the wild-type molecule in inducing osteoclast formation, suggesting that GGT acts not as an enzyme but as a cytokine. Recombinant GGT protein increased receptor activator of nuclear factor-kappaB ligand expression in marrow stromal cells and also stimulated osteoclastogenesis from bone marrow macrophages at lower concentrations. Thus, GGT is implicated as being involved in diseases characterized by accelerated osteoclast development and bone destruction and provides a new target for therapeutic intervention.

Urinary gamma-glutamyltransferase (GGT) as a potential marker of bone resorption.

We recently identified gamma-glutamyltransferase (GGT) as a novel bone-resorbing factor. The present study was undertaken to determine whether GGT is a marker of bone resorption in two genetic models of hyper- and hypo-function of osteoclasts, as well as in postmenopausal women with accelerated bone resorption, using type I collagen N-telopeptide (NTX) and deoxypyridinoline (DPD) as established biochemical markers. Urinary excretion of GGT, corrected for creatinine, was found to be increased in osteoprotegerin (OPG)-deficient osteoporotic mice as well as in patients with postmenopausal osteoporosis (67-83 years of age); in both cases the urinary level decreased after treatment of patients or mice with alendronate, a selective inhibitor of bone resorption, concomitantly with a reduction in DPD and NTX. Conversely, in osteopetrotic op/op mice, urinary GGT increased in parallel with DPD after induction of osteoclasts with M-CSF injection. Constant infusion of parathyroid hormone (PTH) also increased urinary GGT along with DPD. In a survey of 551 postmenopausal women (50-89 years of age) at their regular health checkup, urinary GGT excretion exhibited a high correlation with DPD (rho = 0.49, p < 0.0001). The calculated sensitivity and specificity for diagnosing elevated bone resorption, as determined by a DPD value higher than 7.6 nM/mM Cr, were 61% and 92%, respectively, when a cut-off value of 40 IU/g Cr was assigned for urinary GGT. Since GGT activity can be measured inexpensively in large numbers in a very short time, the measurement of urinary level may provide a convenient and useful method for mass screening to identify those with increased bone turnover and hence at increased risk for bone fracture.

c-Fos protein as a target of anti-osteoclastogenic action of vitamin D, and synthesis of new analogs.

Although active vitamin D drugs have been used for the treatment of osteoporosis, how the vitamin D receptor (VDR) regulates bone cell function remains largely unknown. Using osteoprotegerin-deficient mice, which exhibit severe osteoporosis due to excessive receptor activator of NF-kappaB ligand/receptor activator of NF-kappaB (RANKL/RANK) stimulation, we show herein that oral treatment of these mice with 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] inhibited bone resorption and prevented bone loss, suggesting that VDR counters RANKL/RANK signaling. In M-CSF-dependent osteoclast precursor cells isolated from mouse bone marrow, 1alpha,25(OH)2D3 potently and dose-dependently inhibited their differentiation into multinucleate osteoclasts induced by RANKL. Among signaling molecules downstream of RANK, 1alpha,25(OH)2D3 inhibited the induction of c-Fos protein after RANKL stimulation, and retroviral expression of c-Fos protein abrogated the suppressive effect of 1alpha,25(OH)2D3 on osteoclast development. By screening vitamin D analogs based on their c-Fos-suppressing activity, we identified a new analog, named DD281, that inhibited bone resorption and prevented bone loss in ovariectomized mice, more potently than 1alpha,25(OH)2D3, with similar levels of calcium absorption. Thus, c-Fos protein is an important target of the skeletal action of VDR-based drugs, and DD281 is a bone-selective analog that may be useful for the treatment of bone diseases with excessive osteoclastic activity.