No abstract

No abstract

The extracellular-regulated kinase (ERK) signaling pathway plays important roles in regulating the malignant potential of cancer cells in vitro. However, the effect of ERK signaling on the prognosis of human tumors is not clearly understood. The present study examined the expression of phosphorylated ERK1/2 (p-ERK1/2) as a hallmark of ERK activation, in relation to KRAS and BRAF mutations, in 63 endometrial cancer specimens with endometrioid-subtype, in order to clarify the prognostic value of p-ERK1/2 expression. Immmunohistochemical analysis revealed that 40 tumors (63%) expressed p-ERK1/2, with varying levels of expression. Total ERK1/2 expression was also evaluated in a subset of tumors; most cases expressed ERK1/2 constitutively but no correlation was observed with p-ERK expression, indicating that p-ERK1/2 staining was not due to ERK overexpression but to hyperactivation of ERK1/2. There was no statistically significant correlation between p-ERK1/2 expression and clinicopathological features, including patient age, International Federation of Gynecology and Obstetrics stage, pathological grade, myometrial invasion and lymph node metastasis. Sequencing analysis indicated that 23% of patients had a mutation in exon 1 of KRAS, whereas none of the patients had a mutation in exons 11 or 15 of BRAF, which are reportedly hot spots for mutation in many tumor types. There was no significant correlation between KRAS or BRAF status and p-ERK1/2 expression. Unexpectedly, patients with low p-ERK1/2 expression had significantly lower relapse-free survival (P = 0.041) and overall survival (P = 0.020). Multivariate Cox regression analysis indicated that p-ERK1/2 expression was an independent prognostic indicator for overall survival (P = 0.047). These findings suggest that ERK activation occurs in a KRAS-and BRAF-independent manner in endometrial cancer, and is associated with favorable prognosis. (Cancer Sci 2007; 98: 652-658)A denocarcinoma of the endometrium is the most common gynecological malignancy in the USA. In Japan, it is the second most common gynecological cancer, but its frequency has increased dramatically in the last decade. Although there are well-established surgical and chemotherapeutic treatments for endometrial cancer, the need for molecular-target therapy has increased, especially for recurrent disease that has acquired radioor chemoresistance; thus, there is a need for a better understanding of the molecular pathways of endometrial carcinogenesis. Several genetic alterations are reportedly associated with endometrial carcinogenesis, and the most frequent genetic abnormalities in cases of endometrial carcinogenesis are mutations in PTEN and KRAS. (1)(2)(3)(4)(5) A PTEN mutation activates the phosphatidylinositol-3-kinase (PI3K) pathway, and a KRAS mutation activates the extracellular-regulated kinase (ERK)-mitogen-activated protein kinase (MAPK) pathways; both mutations activate nuclear downstream targets and play major roles in most human carcinogenesis. (6) The ERK-MAPK pathway is activated by mitog...

Background:Epithelial cells of endometriotic tissues are difficult to propagate in vitro as experimental material is scarce owing to their limited life span. However, there is an increasing concern regarding their malignant transformation in ovaries. The present study sought to generate their stable culture system.Methods and results:Purified epithelial cells isolated from ovarian endometriomas using microscopic manipulation were successfully immortalised by combinatorial transfection of human cyclinD1, cdk4 and human telomerase reverse transcriptase (hTERT) genes, whereas the introduction of hTERT alone, or together with cdk4, was insufficient for immortalisation, leading to cellular senescence. We confirmed stable cytokeratin expression in the immortalised cells, proving their epithelial origin. These cells expressed progesterone receptor B and showed significant growth inhibition by various progestins. Oestrogen receptor (ER) expression was detected in these cells, albeit at low levels. Additional overexpression of ERα generated stable cells with oestrogen-dependent growth activation. Soft-agar colony formation assay and nude mice xenograft experiments demonstrated that these cells, even those with additional inactivation of p53, did not have transformed phenotypes.Conclusion:We for the first time generated immortalised epithelial cells from ovarian endometrioma that retained sex steroid responsiveness. These cells are invaluable tools not only for the consistent in vitro work but also for the study of molecular pathogenesis or carcinogenesis of endometriosis.

Despite tremendous development in chemotherapy for ovarian cancer over the past few decades, the prognosis of advanced cases with massive peritoneal dissemination is still unsatisfactory, and novel treatment modalities that can combine with chemotherapy are urgently needed. We recently developed virotherapy for solid tumors using telomerase-specific replication-selective adenoviruses (Telomelysin: OBP-301), in which the human telomerase reverse transcriptase (hTERT) gene promoter has been inserted to direct tumor-specific E1 gene expression. In this study, we investigated the anti-tumor effects of OBP-301, combined with cisplatin (CDDP), on ovarian cancer cells. In vitro treatment of SKOV3 cells with OBP-301 at a multiplicity of infection (MOI) of 0.01-100 induced significant cell death in a dose-dependent manner, with moderate cytotoxicity at an MOI of 1-10 and maximal cytotoxicity at an MOI of 100. In contrast, OBP-301 treatment of normal human cells showed no significant cell death at an MOI of 1-10 and exhibited modest cytotoxicity at an MOI of 100. The effects of low-dose CDDP at 0.5-1 mM, which induced only 20% cell death, were significantly augmented by combination with OBP-301 at an MOI of 1-10, finally achieving 40% cell death. Such enhancement of CDDP sensitivity was also observed in CDDP-resistant ovarian cancer cells. The combinatorial effects were further tested using a xenograft mouse model of SKOV3 with peritoneal dissemination. After intraperitoneal administration of OBP-301, we confirmed that injected OBP-301 fused with the green fluorescent protein (GFP) gene (OBP-401) was preferentially localized to peritoneal disseminations, as determined by fluorescence imaging. Treatment of mice with CDDP at low dose (0.5 mg kg -1 ) had modest effects, showing a 10% decrease in disseminations, whereas combination with intraperitoneal administration of OBP-301 at an MOI of 10 led to enhanced effects, achieving an approximately 80% decrease in disseminations. Kaplan-Meier analysis showed improved overall survival of mice treated with CDDP plus OBP-301 compared with CDDP alone. These findings support the therapeutic potential of intraperitoneal administration of OBP-301 to sensitize ovarian cancer cells to CDDP.

No abstract

Background:Recently developed detection system for circulating tumour cells (CTCs) using a telomerase-specific replicative adenovirus generated nonspecific green fluorescent protein (GFP) signals because of the co-presence of white blood cells (WBCs) nonspecifically infected by viruses. Here, we established a unique detection system for CTCs that completely excludes nonspecific signals.Methods:Blood obtained from the patients was subjected to haemolytic processes to eliminate red blood cells. The cell pellets were then infected with OBP-401, fixed, incubated with fluorescence-labelled anti-CD45 antibody to mark white blood WBCs, and examined on slides under a microscope.Results:Preparatory experiments with cancer cells artificially added to healthy donor samples confirmed that CD45 labelling could distinguish GFP-positive cancer cells from WBCs. In 53 patients with gynaecological cancers, CTCs were detected in 21 patients (39.6%) when CD45-positive cells were excluded as WBCs among GFP-positive cells. No CTCs were detected in samples from healthy volunteers. There was no significant correlation between CTC counts and known clinicopathological factors. The CTCs rapidly vanished after surgery or chemotherapy in most patients whose treatments were effective. In contrast, the persistence of CTCs even after treatments was tightly associated with poor response to the treatments (P<0.005).Conclusion:The presence of CTCs in our system may potentially be a novel therapeutic marker in gynaecological cancers.

No abstract