Transglutaminase 2 as an independent prognostic marker for survival of patients with non-adenocarcinoma subtype of non-small cell lung cancer.

BACKGROUND: Expression of transglutaminase 2 (TGase 2) is related to invasion and resistance to chemotherapeutic agents in several cancer cells. However, there has been only limited clinical validation of TGase 2 as an independent prognostic marker in cancer. METHODS: The significance of TGase 2 expression as an invasive/migratory factor was addressed by in vitro assays employing down-regulation of TGase 2. TGase 2 expression as a prognostic indicator was assessed in 429 Korean patients with early-stage non-small cell lung cancer (NSCLC) by immunohistochemical staining. RESULTS: TGase 2 expression increased the invasive and migratory properties of NSCLC cells in vitro, which might be related to the induction of MMP-9. In the analysis of the immunohistochemical staining, TGase 2 expression in tumors was significantly correlated with recurrence in NSCLC (p = 0.005) or in the non-adenocarcinoma subtype (p = 0.031). Additionally, a multivariate analysis also showed a significant correlation between strong TGase 2 expression and shorter disease-free survival (DFS) in NSCLC (p = 0.029 and HR = 1.554) and in the non-adenocarcinoma subtype (p = 0.030 and HR = 2.184). However, the correlation in the adenocarcinoma subtype was not significant. CONCLUSIONS: TGase 2 expression was significantly correlated with recurrence and shorter DFS in NSCLC, especially in the non-adenocarcinoma subtype including squamous cell carcinoma.

Bioimaging for the monitoring of the in vivo distribution of infused mesenchymal stem cells in a mouse model of the graft-versus-host reaction.

Cell therapy using MSCs (mesenchymal stem cells) might be effective treatment for refractory GVHD (graft-versus-host disease). However, the fate and distribution of MSCs after transplantation remains unclear. In this study, an animal model was developed to monitor the dynamic distribution of MSCs in mice with GVHD. A GVHD mouse model was established by transplanting C57BL/6 donor bone marrow cells and C57BL/6 EGFP (enhanced green fluorescent protein) splenocytes into lethally irradiated BALB/c nude recipient mice. Donor MSCs were obtained from MHC-identical C57BL/6 RFP (red fluorescent protein) mice and infused into the recipient mice on the same transplantation day. In vivo movement of the donor splenocytes (EGFP) and MSCs (RFP) were evaluated by measuring the biofluorescence (IVIS-Xenogen system). Donor splenocytes and MSCs reached the lungs first, and then the gastrointestinal tract, lymph nodes and skin, in that order; the transit time and localization site of these cells were very similar. In the recipient mouse with GVHD, the number of detectable cells declined with time, as assessed by biofluorescence imaging and confirmed by RT (real-time)-PCR. This bioimaging system might be useful for preclinical testing and the design of therapeutic strategies for monitoring the dynamic distribution of MSCs with GVHD.

Mesenchymal stromal cells inhibit graft-versus-host disease of mice in a dose-dependent manner.

BACKGROUND AIMS: Graft-versus-host disease (GvHD) remains a major complication after allogeneic hematopoietic cell transplantation (HCT). Recent literature demonstrates a potential benefit of human mesenchymal stromal cells (MSC) for the treatment of refractory GvHD; however, the optimal dose remains uncertain. We set out to develop an animal model that can be used to study the effect of MSC on GvHD. METHODS: A GvHD mouse model was established by transplanting C3H/he donor bone marrow (BM) cells and spleen cells into lethally irradiated BALB/c recipient mice. MSC were obtained from C3H/he mice and the C3H/10T1/2 murine MSC line. RESULTS: The mRNA expression of Foxp3 in regional lymph nodes (LN) localized with T cells was markedly increased by the addition of C3H10T1/2 cells in a real-time polymerase chain reaction (PCR). Using a mixed lymphocyte reaction, we determined the optimal splenocyte proliferation inhibition dose (MSC:splenocyte ratios 1:2 and 1:1). Three different C3H10T1/2 cell doses (low, 0.5 x 10(6), intermediate, 1 x 10(6), and high, 2 x 10(6)) with a consistent splenocyte dose (1 x 10(6)) were evaluated for their therapeutic potential in an in vivo GvHD model. The clinical and histologic GvHD score and Kaplan-Meier survival rate were improved after MSC transplantation, and these results demonstrated a dose-dependent inhibition. CONCLUSIONS: We conclude that MSC inhibit GvHD in a dose-dependent manner in this mouse model and this model can be used to study the effects of MSC on GvHD.

Predisposition of genetic disease by modestly decreased expression of GCH1 mutant allele.

Recently it was shown that single nucleotide polymorphisms (SNPs) can explain individual variation because of the small changes of the gene expression level and that the 50% decreased expression of an allele might even lead to predisposition to cancer. In this study, we found that a decreased expression of an allele might cause predisposition to genetic disease. Dopa responsive dystonia (DRD) is a dominant disease caused by mutations in GCH1 gene. The sequence analysis of the GCH1 in a patient with typical DRD symptoms revealed two novel missense mutations instead of a single dominant mutation. Family members with either of the mutations did not have any symptoms of DRD. The expression level of a R198W mutant allele decreased to about 50%, suggesting that modestly decreased expression caused by an SNP should lead to predisposition of a genetic disease in susceptible individuals.

Transient phosphorylation of tumor associated microtubule associated protein (TMAP)/cytoskeleton associated protein 2 (CKAP2) at Thr-596 during early phases of mitosis.

Tumor associated microtubule associated protein (TMAP), also known as cytoskeleton associated protein 2 (CKAP2) is a mitotic spindle-associated protein whose expression is cell cycle-regulated and also frequently deregulated in cancer cells. Two monoclonal antibodies (mAbs) against TMAP/CKAP2 were produced: B-1-13 and D-12-3. Interestingly, the reactivity of mAb D-12-3 to TMAP/CKAP2 was markedly decreased specifically in mitotic cell lysate. The epitope mapping study showed that mAb D-12-3 recognizes the amino acid sequence between 569 and 625 and that phosphorylation at T596 completely abolishes the reactivity of the antibody, suggesting that the differential reactivity originates from the phosphorylation status at T596. Immunofluorescence staining showed that mAb D-12-3 fails to detect TMAP/CKAP2 in mitotic cells between prophase and metaphase, but the staining becomes evident again in anaphase, suggesting that phosphorylation at T596 occurs transiently during early phases of mitosis. These results suggest that the cellular functions of TMAP/CKAP2 might be regulated by timely phosphorylation and dephosphorylation during the course of mitosis.

Increased lysine N-methylation of a 23-kDa protein during hepatic regeneration.

The methylation of a 23-kDa nuclear protein increased after partial hepatectomy and methylation returned to basal levels after the initial stage of regeneration. The methylating enzyme was partially purified from rat liver by ammonium sulfate precipitation, DEAE-anion exchange chromatography and Butyl-Sepharose chromatography. The 23-kDa protein was purified from a nuclear fraction of liver tissue with SP-Sepharose. When the 23-kDa protein was methylated with the partially purified methyltransferase and analyzed on C(18) high performance liquid chromatography (HPLC), the methylated acceptor amino acid was monomethyl lysine (MML). Previously, only arginine N-methylation of specific substrate proteins has been reported during liver regeneration. However, in this report, we found that lysine N-methylation increased during early hepatic regeneration, suggesting that lysine N-methylation of the 23-kDa nuclear protein may play a functional role in hepatic regeneration. The methyltransferase did not methylate other proteins such as histones, hnRNPA1, or cytochrome C, suggesting the enzyme is a 23-kDa nuclear protein- specific lysine N-methyltransferase.

Immunohistochemical localization of LLC1 in human tissues and its limited expression in non-small cell lung cancer.

We have shown both LLC1 expression in the lung epithelium by in situ hybridization and its inactivation in lung cancer by epigenetic modification. However, LLC1 protein's cellular localization or its role in normal lung or cancer tissues has not yet been evaluated. In the present study, a monoclonal antibody against recombinant LLC1 was produced, and immunohistochemical staining was performed on arrays including various human tissues, normal lung and non-small cell lung cancer (NSCLC) tissues for LLC1 localization. The immunohistochemical results showed LLC1 expression in the cilia of normal-airway epithelial cells and in the cytoplasm of type II pneumocytes in bronchiectatic patients, but no expression in most of the NSCLC tissues, which is consistent with our previous report positing LLC1 as a tumor suppressor. However, LLC1 over-expression in NSCLC cell lines NCI-H1299 and NCI-H23 did not show any change in proliferation or migration, which does not indicate any LLC1 tumor-suppressor role. As for the other human tissues, LLC1 was localized in renal tubular cells, pancreatic acinar cells, and epithelial cells of the stomach, duodenum, and gallbladder. In summary, our findings suggest that LLC1 is not a tumor suppressor, and that it is localized in the cilia of the normal lung epithelium but is absent in most NSCLC cases, probably due to the loss of cilia during lung carcinogenesis.

Transglutaminase 2 inhibitor abrogates renal cell carcinoma in xenograft models.

PURPOSE: To test whether transglutaminase 2 (TGase 2) inhibitor GK921 alone reverses renal cell carcinoma (RCC) tumor growth. RCC is resistant to both radiation and chemotherapy, and the prognosis remains poor. Despite the recent therapeutic success of vascular endothelial growth factor inhibition in RCC, approximately one-third of RCC patients develop metastatic disease. The expression of TGase 2 is markedly increased in most RCC cell lines, as well as in clinical samples. METHODS: Previously, we introduced the quinoxaline derivative GK13 as a lead compound for TGase 2 inhibitor. The inhibitory effect of GK13 on TGase 2 was improved in GK921 (3-(phenylethynyl)-2-(2-(pyridin-2-yl)ethoxy)pyrido[3,2-b]pyrazine). GK921 efficacy was tested using sulforhodamine in vitro as well as a xenograft tumor models using ACHN and CAKI-1 RCC cells. RESULTS: GK921 showed cytotoxicity to RCC (average GI50 in eight RCC cell lines: 0.905 µM). A single treatment with GK921 almost completely reduced tumor growth by stabilizing p53 in the ACHN and CAKI-1 preclinical xenograft tumor models. CONCLUSION: TGase 2 inhibitor GK921 abrogates RCC growth in xenograft tumor models, suggesting the possibility of a new therapeutic approach to RCC.

Chromatin CKAP2, a new proliferation marker, as independent prognostic indicator in breast cancer.

BACKGROUND: The level of proliferation activity is a strong prognostic or predictive indicator in breast cancer, but its optimal measurement is still in debate, necessitating new proliferation markers. In the present study, the prognostic significance of the CKAP2-positive cell count (CPCC), a new proliferation marker, was evaluated, and the results were compared with those for the mitotic activity index (MAI). METHODS: This study included 375 early-stage breast cancer samples collected from two institutions between 2000 and 2006. Immunohistochemical staining was performed using a CKAP2 monoclonal antibody. Cox proportional hazard regression models were fitted to determine the association between the CPCC and relapse-free survival (RFS) amongst three groups formed on the basis of the CPCC or MAI value: groups 2 and 3 showing the middle and highest values, respectively, and group 1 the lowest. RESULTS: After adjustment for age, T stage, N stage, HER2 status, estrogen receptor status, progesterone receptor status, institution, and year of surgical resection, the CPCC was associated with a significantly worse RFS {hazard ratio [HR]  = 4.10 (95% CI: 1.64-10.29) for group 2; HR  = 4.35 (95% CI: 2.04-10.35) for group 3}. Moreover, its prognostic significance was similar to or higher than that based on the MAI {HR  = 2.05 (95% CI: 0.94-4.65) for group 2; HR  = 2.35 (95% CI: 1.09-5.10) for group 3}. In subgroup analyses, the CPCC showed a prognostic significance in the luminal A and triple-negative subgroups, but not in the HER2-positive subgroup. CONCLUSIONS: Chromatin CKAP2 is an independent prognostic marker for RFS in early-stage breast cancer, and could potentially replace the MAI in clinical evaluation of proliferation activity. Additionally, our study results suggest that the prognostic significance of proliferation activity differs among the various subgroups of breast cancer.

Inhibition of tumor growth in a mouse xenograft model by the humanized anti-HGF monoclonal antibody YYB-101 produced in a large-scale CHO cell culture.

The humanized anti-hepatocyte growth factor (HGF) monoclonal antibody (mAb) YYB-101 is a promising therapeutic candidate for treating various cancers. In this study, we developed a bioprocess for large-scale production of YYB-101 and evaluated its therapeutic potential for tumor treatment using a xenograft mouse model. By screening diverse chemically defined basal media formulations and by assessing the effects of various feed supplements and feeding schedules on cell growth and antibody production, we established an optimal medium and feeding method to produce 757 mg/l of YYB-101 in flask cultures, representing a 7.5-fold increase in titer compared with that obtained under non-optimized conditions. The optimal dissolved oxygen concentration for antibody production was 70% pO2. A pH shift from 7.2 to 7.0, rather than controlled pH of either 7.0 or 7.2, resulted in productivity improvement in 5 L and 200 L bioreactors, yielding 737 and 830 mg/ml of YYB-101, respectively. The YYB-101 mAb highly purified by affinity chromatography using a Protein A column and two-step ion exchange chromatography effectively neutralized HGF in a cell-based assay and showed potent tumor suppression activity in a mouse xenograft model established with human glioblastoma cells.

Condensed chromatin staining of CKAP2 as surrogate marker for mitotic figures.

PURPOSE: Proliferation activity has long been known to be one of the strongest prognostic factors in many different cancers. Nevertheless, microscopic evaluation of mitotic figures remains time-consuming and, furthermore, is relatively subjective. As the expression of cytoskeleton-associated protein 2 (CKAP2) is closely related to the mitotic phase, CKAP2 was evaluated as a surrogate mitotic figure (MF) marker. METHODS: A monoclonal antibody specific to human CKAP2 was produced, and immunohistochemistry was performed on normal tissue array sections and 30 breast cancer tissues. RESULTS: The expression of CKAP2 in the normal human tissues was limited to well-known cell proliferation zones. Strong, readily visible, condensed chromatin staining of CKAP2 was observed specifically in mitotic cells, and the number of these cells was tightly correlated with the MF count in breast cancer tissues (P < 0.001, ρ = 0.743), suggesting its usefulness as a surrogate marker for MF counting. CONCLUSION: Immunohistochemical staining with CKAP2 monoclonal antibody can be considered to be a new, effective approach to the assessment of proliferation activity in cancer tissues.

Transglutaminase 2 as a cisplatin resistance marker in non-small cell lung cancer.

PURPOSE: Recently, it was reported that expression of transglutaminase 2 plays an important role in doxorubicin/cisplatin resistance in breast and ovarian cancer. The aims of this study were to verify the role of transglutaminase 2 in cisplatin response in non-small cell lung cancer (NSCLC) and to study if transglutaminase 2 gene (TGM2) methylation can be a molecular marker for good response to cisplatin. METHODS: TGM2 promoter methylation was analyzed by sodium bisulfite sequencing. Cisplatin sensitivity was analyzed by treatment of cisplatin in NSCLC cell lines with/without TGM2 or TGM2 siRNA transfection. RESULTS: In one-third of NSCLC cell lines, TGase 2 gene (TGM2) was silenced by promoter methylation. The TGM2 promoter-methylated cell lines (HCC-95 and HCC-1588) showed relatively higher sensitivity to cisplatin than the TGM2-expressing cell lines (NCI-H1299 and HCC-1195). Down-regulation and over-expression of TGM2 in those NSCLC cells also suggested a positive correlation of cisplatin sensitivity and TGM2 inhibition. With doxorubicin, the relationship was quite similar. CONCLUSIONS: We showed that good responders of cisplatin in NSCLC could be identified by the promoter methylation of TGM2 and that TGase 2 inhibition appears to be an effective cisplatin-sensitizing modality in NSCLC.