Search

Cell cycle-related molecules play crucial roles in maintaining genomic stability, and can also serve as biomarkers of cell cycle phase distribution at the same time. In this study, we used multiparameter analysis of various biomarkers to investigate their utility for the evaluation of tumor proliferation activities and the prognosis of patients with small-size lung adenocarcinoma. We performed immunohistochemical analysis using five cell cycle-related biomarkers (MCM7, Ki-67, Geminin, Aurora A and H3S10ph) for 102 surgically resected small-size lung adenocarcinomas. We classified them into three phenotypes based on the dominant cell cycle phase distribution of the tumor cell population, and evaluated whether these phenotypes were associated with clinicopathological factors and survival. Phenotype I (MCM7-negative tumors; n=56) was correlated with high or moderate differentiation and reduced local invasiveness (pleural and lymphovascular invasion) compared with phenotype II (MCM7-, Ki-67- and Geminin-positive tumors; n=23) and phenotype III (MCM7-, Aurora A- and H3S10ph-positive tumors; n=17). Five-year survival rates of phenotypes I, II and III were 89.8, 55.4 and 38.6%, respectively, with a significant difference between them (p<0.01). Multivariate analysis revealed that phenotypes II and III were independent prognostic factors in the 79 patients with stage I lung adenocarcinoma. Multiparameter analysis using cell cycle biomarkers for small-size lung adenocarcinoma provided novel insights into the cell cycle phase distribution of dynamic tumor cell populations in vivo; it may be possible to evaluate tumor proliferation activities and patient prognosis more precisely if this analytical procedure is used.

Bisphosphonates (BPs) inhibit bone resorption by preventing osteoclast maturation and apoptosis induction. Recently, BPs have also been shown to have antitumor effects against various types of carcinomas in vitro and in vivo. In this study, we investigated the antitumor effect of zoledronic acid (ZOL), a third generation bisphosphonate, on proliferation, cell cycle and apoptosis of oral cancer cells. Direct antitumor effects of ZOL against four oral carcinoma cell lines (squamous cell carcinoma, HSC3, HSC4, SCCKN; salivary adenocarcinoma, HSY) were measured by WST assay. Apoptosis-related molecules were analyzed by Western blot analysis and cell cycle was analyzed by flow cytometry. ZOL had a dose-dependent antitumor effect in the four oral cancer cell lines. ZOL activated caspase-3, -8 and -9 and induced cellular apoptosis. Western blot analysis showed that ZOL increased cleaved anti-human poly(ADP-ribose) polymerase expression and decreased Bcl-2 and Bid expression. Treatment with ZOL increased the number of cells in apoptosis, sub G1 phase and S phase, and reduced the number of cells in the G0/G1 and G2/M phase in a concentration-dependent manner. ZOL inhibits cell proliferation and induces apoptosis of oral cancer cells in vitro. These findings suggest that ZOL might be beneficial in the treatment of oral carcinoma patients.

Tyrosine phosphorylation of beta-catenin, an intracytoplasmic cadherin-binding protein, causes disruption of the cadherin-mediated cell adhesion system in cancer cells. We identified that the c-erbB-2 protein activated by TGFalpha was capable of binding to the 12th armadillo repeat (arm12) of beta-catenin with increased phosphorylation-state level. We produced a monoclonal antibody, 4G7, which recognizes a phosphorylated-tyrosine residue (Tyr-654) located at arm12 of beta-catenin. Tyrosine phosphorylation of the arm12 was detected after stimulation with TGFalpha in TMK-1. Immunoprecipitation analyses using 4G7, anti-beta-catenin, alpha-catenin, the c-erbB-2 gene product and phosphotyrosine antibodies indicated that tyrosine phosphorylation of the arm12 was closely correlated with dissociation between E-cadherin/beta-catenin and alpha-catenin or the c-erbB-2 gene product, but not with dissociation between beta-catenin and E-cadherin. Immunocytochemical staining of TMK-1 cells using the 4G7 antibody revealed that tyrosine phosphorylation of the arm12 was localized at cell-cell contact sites of cancer cells transiently and only after TGFalpha treatment. Immunohistochemical localization of the 4G7 antibody was observed in cancer cells at the invasive front where they detached from cancer nests. These findings indicated that the tyrosine phosphorylation of arm12 is a key marker of cadherin dysfunction and the 4G7 antibody may be useful in screening for a beta-catenin phosphorylation ligand or tyrosine kinases.

We investigated the effects of a water-soluble extract of Maitake (Grifola frondosa), a Japanese edible mushroom, on the proliferation and cell death of four human gastric cancer cell lines (TMK-1, MKN28, MKN45 and MKN74). The Maitake extract (ME) inhibited the proliferation of all four cell lines in a time-dependent manner. The inhibition was most pronounced in TMK-1 cells, which exhibited up to 90% inhibition after treatment with 10% ME for 3 days. Staining of ME-treated TMK-1 cells with Hoechst 33258 revealed increased numbers of nuclear condensations and apoptotic bodies. Induction of apoptosis was confirmed by fluorescence-activated cell sorting analyses. Western blot analyses of TMK-1 cells after ME treatment revealed increases in intracytoplasmic cytochrome c and cleavage of caspase-3 and poly(ADP-ribose) polymerase, but no expression of p21 or Bax. The caspase-3 protease activities in lysates of TMK-1 cells treated with 1% or 10% ME were about three times higher than those in control cells. The proliferation of TMK-1 cells was hardly affected by the caspase-3 inhibitor z-DEVD-fmk. Taken together, these results suggest that ME induces apoptosis of TMK-1 cells by caspase-3-dependent and -independent pathways, resulting in potential antitumor effects on gastric cancer.

DNA should be duplicated precisely once per cell cycle to maintain genome integrity. After DNA replication, geminin binding to Cdt1 inhibits uploading of the minichromosome maintenance (MCM) complex as DNA helicase onto chromatin and prevents DNA re-replication in the same cell cycle. Expression of geminin indicates poor prognosis in some malignancies, such as breast and renal cell carcinoma. We evaluated the expression of geminin to clarify its pathobiological and prognostic significance in colorectal cancer, compared with expression of MCM7 and Ki-67. We performed Western blot analyses of 5 human colorectal cancer cell lines and immunohistochemistry on 191 surgically removed specimens of Dukes' B and C stage colorectal cancer. Double-labeling immunofluorescence was also carried out to identify co-expression of geminin, MCM7 and Ki-67. Geminin proteins were detected in all the 5 cell lines examined. Geminin, MCM7 and Ki-67 were co-expressed and cells that stained only for geminin were not detected. Mean labeling indices (LIs) for geminin, MCM7 and Ki-67 were 26.3, 58.2 and 40.8%, respectively. Patients with high geminin LIs had significantly unfavorable prognosis in stage II and III colorectal cancer (P=0.04). Patients with a tumor with a higher proliferating nature (i.e. high LIs for three markers) showed significantly unfavorable prognosis in multivariate Cox analysis. Our results indicate that assessment of geminin, MCM7 and Ki-67 may be useful for predicting prognosis in patients with colorectal cancer.

Minichromosome maintenance (MCM) proteins are essential components for DNA replication, and also prognostic markers for various human tumors. MCM-positive but Ki67-negative cells (e.g. primary oocytes) are thought to be licensed non-proliferating populations, and are significantly correlated with the clinicopathological profiles of some human tumors. In the present study, we evaluated the expression levels of MCM7, MCM2 and Ki67 in colorectal cancer to clarify their pathobiological significance. We carried out Western blot analyses of 5 human colorectal cancer cell lines and performed immunohistochemistry on 202 surgically removed colorectal cancers of Dukes' B and C stages. Double-labeling immunofluorescence was also carried out on the cancer specimens to identify MCM-positive but Ki67-negative tumor cells. MCM proteins were detected in all the 5 cell lines examined. MCM7 and MCM2 were coexpressed in almost the same populations of tumor cells, whereas MCM7-negative but Ki67-positive tumor cells were absent in the double-labeled specimens, except for mitotic cells. The mean positive tumor labeling indexes (LIs) for MCM7, MCM2 and Ki67 were 58.1, 57.1 and 40.6%, respectively. The mean LI for MCM7-positive but Ki67-negative tumor cells was 17.6%, and significantly correlated with the N status (P=0.01), distant metastasis (P=0.01) and UICC stage (P=0.02). The high LI of >58.1% for MCM7 were independent prognostic factors in multivariate Cox regression analysis (relative risk = 2.12; P=0.02). Our results indicate that MCM7 expression is an independent prognostic factor for human colorectal cancer, and MCM7-positive but Ki67-negative tumor cells are correlated with tumor metastases.

Human telomerase reverse transcriptase (hTERT) and epidermal growth factor receptor (EGFR) play an important role in many cancers including gynecological cancers. We previously reported the usefulness of a quantitative highly sensitive detection method for hTERT mRNA in the serum of cancer patients. By this method, we attempted to elucidate the diagnostic evaluation of serum hTERT mRNA for gynecologic malignancies. In 174 female patients with gynecological lesions (47 with ovarian lesions, 63 with uterine lesions, 2 with malignancies in other gynecological lesions, and 62 benign lesions) and 20 healthy individuals, we measured serum hTERT mRNA and EGFR mRNA by using the newly developed real-time quantitative RT-PCR. We examined their sensitivity and specificity in cancer diagnosis, clinical significance in comparison with conventional tumor markers, and their correlations with the clinical parameters by using multivariate analyses. Serum hTERT mRNA showed higher values in patients with gynecologic cancers than in those with benign diseases and healthy individuals. The hTERT mRNA level independently correlated with the presence of cancers (P=0.004 for both ovarian and uterine cancer) and clinical stage (P<0.001). The sensitivity and specificity of hTERT mRNA in cancer diagnosis was 74.4% and 74.1%, respectively. The hTERT mRNA level showed a significant correlation with CA125 by Pearson's relative test (P=0.035) and with histological findings in ovarian cancer by the Friedman test (P<0.004). EGFR mRNA did not display any differences between the diseases. hTERT mRNA is useful for diagnosing gynecologic cancer and is superior to conventional tumor markers. Therefore, serum hTERT mRNA is a novel and available biomarker for gynecologic malignancies.

This study examined the clinicopathological significance of minichromosome maintenance-2 (MCM2) expression in 38 human malignant fibrous histiocytomas (MFHs) and 36 benign fibrohistiocytic tumors (BFHTs) immunohistochemically, and in 9 human sarcoma or carcinoma cell lines, as well as 7 surgical specimens by Western blotting. MCM2 was detected in all the cell lines and surgical specimens as a single band at 120 kDa, while P53 expression was variable. Nuclear expression of MCM2 was noted in tumor but not mitotic cells of all the MFHs and 26 (72.2%) of the BFHTs, the labeling indices (LIs) being 62.0% in the 28 ordinary types, 38.5% in the 10 myxoid types, and 11.2% in the BFHTs with significant difference. Moreover, the LI was significantly higher for MCM2 than that for Ki-67 in the MFHs of both types (p<0.05). No correlation was noted between the MCM2-LI and P53 expression or apoptotic indices, which were significantly higher in the MFHs than BFHTs (p<0.01). These results indicate that MCM2 would correlate with cell proliferation rather than apoptosis in MFHs, and the expression is ubiquitous in proliferating cells, regardless of the expression of P53. Thus, MCM2 might be a reliable marker of proliferating cells in human MFH.

We examined the role of the p53 gene in hyperthermia-induced apoptosis using three human gastric carcinoma cell lines, MKN-28 (carrying mutated type p53 gene), MKN-74 (wild-type), and KATO-III (complete deletion). The results indicate that i) long-term hyperthermia causes necrosis, and short-term treatment induces apoptosis in a gradual time dependent fashion, ii) hyperthermia-triggered apoptosis can occur both in a p53 gene-dependent and -independent manner, and iii) up-regulation of Hsp70 might enhance the function of wild-type p53 protein in hyperthermia-induced apoptosis.