Toshihiro Kimura

Regulation of E-cadherin Endocytosis by Nectin through Afadin, Rap1, and p120ctn

Adherens junctions (AJs) are a major cell-cell adhesion structure in epithelial cells that are formed by two major cell-cell adhesion molecules, E-cadherin and nectin. We have previously shown that nectin first forms cell-cell adhesion and then recruits non-trans-interacting E-cadherin to the nectin-based cell-cell adhesion sites, which gradually trans-interact there, eventually forming AJs. We have examined here the effect of trans-interacting nectin on non-trans-interacting E-cadherin endocytosis. Trans-interacting nectin capable of associating with afadin, but not trans-interacting nectin mutant incapable of associating with afadin, inhibited non-trans-interacting E-cadherin endocytosis in intact cells. Afadin is a nectin- and actin filament-binding protein that connects nectin to the actin cytoskeleton. Studies on the mode of action of the nectin-afadin system using cell-free assay revealed that afadin associated with nectin bound Rap1 activated by trans-interacting nectin, interacted with p120ctn, and strengthened the binding of p120ctn to E-cadherin, eventually reducing non-trans-interacting E-cadherin endocytosis. Afadin, which did not bind Rap1, was inactive in this capacity. These results indicate that trans-interacting nectin inhibits non-trans-interacting E-cadherin endocytosis through afadin, Rap1, and p120ctn and thereby further accumulates non-trans-interacting E-cadherin to the nectin-based cell-cell adhesion sites for the formation of AJs.

Involvement of the Ras-Ras-activated Rab5 guanine nucleotide exchange factor RIN2-Rab5 pathway in the hepatocyte growth factor-induced endocytosis of E-cadherin.

E-cadherin is a key cell-cell adhesion molecule at adherens junctions (AJs) and undergoes endocytosis when AJs are disrupted by the action of an extracellular signal, such as hepatocyte growth factor (HGF)/scatter factor. Rab5 small G protein has been implicated in the HGF-induced endocytosis of E-cadherin, but the molecular mechanism for the regulation of Rab5 activity remains unknown. We first studied this mechanism by using the cell-free assay system for the endocytosis of E-cadherin of the AJ-enriched fraction from rat livers. HGF induced activation of Ras small G protein, which then bound to RIN2, a Rab5 GDP/GTP exchange factor with the Vps9p-like guanine nucleotide exchange factor and Ras association domains, and activated it. Activated RIN2 then activated Rab5, eventually inducing the endocytosis of E-cadherin. We then studied whether RIN2 was involved in the HGF-induced endocytosis of E-cadherin in intact Madin-Darby canine kidney cells. RIN2 localized at the cell-cell adhesion sites, and its guanine nucleotide exchange factor activity was required for the HGF-induced endocytosis of E-cadherin in Madin-Darby canine kidney cells. These results indicate that RIN2 connects Ras to Rab5 in the HGF-induced endocytosis of E-cadherin.

Overexpression of LAMP3/TSC403/DC-LAMP Promotes Metastasis in Uterine Cervical Cancer

LAMP3 (DC-LAMP, TSC403, CD208) was originally isolated as a gene specifically expressed in lung tissues. LAMP3 is located on a chromosome 3q segment that is frequently amplified in some human cancers, including uterine cervical cancer. Because two other members of the LAMP family of lysosomal membrane glycoproteins, LAMP1 and LAMP2, were previously implicated in potentially modulating the interaction of vascular endothelial and cancer cells, we hypothesized that LAMP3 might also play an important part in metastasis. To clarify the metastatic potential of LAMP3 in cervical cancers, we transfected a LAMP3 expression vector into a human uterine cervical cancer cell line, TCS. In an in vitro invasion assay, the migration of LAMP3-overexpressing TCS cells was significantly higher than in control TCS cells. In an in vivo metastasis assay, distant metastasis was detected in 9 of 11 LAMP3-overexpressing TCS cell-injected mice and in only 1 of 11 control mice. Histologic study showed that LAMP3-overexpressing cells readily invaded into the lymph-vascular space. In clinical samples, quantitative real-time reverse transcription-PCR (RT-PCR) analyses showed that LAMP3 mRNA was significantly up-regulated in 47 of 47 (100%) cervical cancers and in 2 of 15 (13%) cervical intraepithelial neoplasias, compared with a low level of LAMP3 mRNA expressed in normal uterine cervixes. Interestingly, high LAMP3 expression was significantly correlated with the overall survival of patients with stage I/II cervical cancers. These findings indicate that LAMP3 overexpression is associated with an enhanced metastatic potential and may be a prognostic factor for cervical cancer.

Two Distinct Pathways to Development of Squamous Cell Carcinoma of the Vulva

Squamous cell carcinoma (SCC) accounts for approximately 95% of the malignant tumors of the vaginal vulva and is mostly found in elderly women. The future numbers of patients with vulvar SCC is expected to rise, mainly because of the proportional increase in the average age of the general population. Two different pathways for vulvar SCC have been put forth. The first pathway is triggered by infection with a high-risk-type Human Papillomavirus (HPV). Integration of the HPV DNA into the host genome leads to the development of a typical vulvar intraepithelial neoplasia (VIN), accompanied with overexpression of p14ARF and p16INK4A. This lesion subsequently forms a warty- or basaloid-type SCC. The HPV vaccine is a promising new tool for prevention of this HPV related SCC of the vulva. The second pathway is HPV-independent. Keratinizing SCC develops within a background of lichen sclerosus (LS) through a differentiated VIN. It has a different set of genetic alterations than those in the first pathway, including p53 mutations, allelic imbalances (AI), and microsatellite instability (MSI). Further clinical and basic research is still required to understand and prevent vulvar SCC. Capsule. Two pathway for pathogenesis of squamous cell carcinoma of the value are reviewed.

Analysis of Clonality and HPV Infection in Benign, Hyperplastic, Premalignant, and Malignant Lesions of the Vulvar Mucosa

To elucidate the pathogenesis of vulvar carcinomas, we studied clonality and human papillomavirus (HPV) infection in vulvar epithelial diseases. Monoclonal composition was demonstrated in all 9 invasive tumors (squamous cell carcinoma [SCC], 6; basal cell carcinoma, 1; malignant melanoma, 2), 15 of 20 cases of vulvar intraepithelial neoplasia (VIN), 7 of 9 cases of Paget disease, 2 of 6 cases of lichen sclerosus (LS), and 2 of 3 cases of squamous cell hyperplasia (SCH); high-risk type HPV was revealed in 5 of 6 SCCs and 17 of 20 VINs. These observations might imply that a subset of cases of LS and SCH result from a neoplastic proliferation, similar to VINs but not related to infection with high-risk type HPV. In 1 case of SCC with concurrent VIN 3 in an adjacent lesion, both lesions showed the same pattern of X chromosome inactivation and the presence of HPV-16 in episomal and integrated forms, suggesting that monoclonal expansion triggered by high-risk type HPV integration is an early event for carcinogenesis of HPV-associated SCC.

Is laparoscopic colorectal surgery less invasive than classical open surgery? Quantitation of physical activity using an accelerometer to assess postoperative convalescence

Background: With the technical advances of recent years, the number of operative manipulations in the abdominal cavity by laparoscopic surgery is now considered to be the same as that using classical open surgery. The question has been raised whether laparoscopic colorectal surgery with lymphadenectomy improves the recovery compared to open surgery. Methods: We compared patients’ physical activity for 7 days postoperatively as measured with an accelerometer between laparoscopic-assisted colorectal resection (LAC, n = 32) and classical open colorectal surgery (OC, n = 30). Results: Physical activity expressed as cumulative acceleration was significantly higher in the LAC than in the OC group on each postoperative day. The recovery time, defined as the day on which the cumulative acceleration recovered to 90% of the preoperative level, was significantly shorter (p < 0.05) in the LAC (3.4 ± 1.2 days) than in the OC group (6.8 ± 1.7 days). Conclusion: Our results showed that the duration of convalescence with LAC was significantly shorter than that with the OC procedure. Laparoscopic colorectal surgery appears to allow an earlier recovery after the operation than the classical open procedure, and it is less invasive as assessed by convalescence.

A New Parameter for Assessing Postoperative Recovery of Physical Activity Using an Accelerometer

AbstractPurpose. While many retrospective and prospective observational studies have shown laparoscopic surgery to be less invasive than conventional open surgery, this issue has not been evaluated by objective parameters. Currently available clinical parameters, such as the day of first ambulation, the day food intake is commenced, and the length of postoperative hospital stay, are subjective. The purpose of this study was to investigate whether measuring postoperative physical activity with an accelerometer is a useful parameter for evaluating postoperative recovery after surgical stress. Methods. The subjects included 20 patients who underwent laparoscopic partial gastrectomy (LPG group), 35 patients who underwent open distal gastrectomy (ODG group), and 20 patients who underwent open total gastrectomy (OTG group). The cumulative acceleration of voluntary movement, measured by an Active tracer AC-301 (ACT) accelerometer for 7 days postoperatively, was compared among these three groups. Results. The cumulative acceleration of physical activity for 24 h was significantly better in the LPG group than in the ODG and OTG groups on each postoperative day. The recovery time, defined as the day that cumulative acceleration had recovered to more than 90% of the preoperative level, was significantly shorter in the LPG group (2.8 ± 0.9 days) than in the ODG (6.6 ± 2.1 days) and OTG (7.8 ± 1.2 days) groups. Conclusion. These results showed that convalescence differed with the degree of surgical stress, and that measurement of the cumulative acceleration of voluntary movement by using an accelerometer could be a useful objective and quantitative parameter for evaluating postoperative recovery.

Completely Laparoscopic Total Colectomy for Chronic Constipation: Report of a Case

Laparoscopic surgery has had a remarkable impact on the practice of colorectal surgery. However, most operations are performed using a technique of laparoscopic assistance, whereby extracorporeal bowel division and anastomosis are made following laparoscopic mobilization of the bowel. To our knowledge, this is the first report to describe a case of chronic constipation managed by total colectomy with ileorectal anastomosis, performed completely laparoscopically. The diagnosis of slow transit constipation was made by a transit time study. After dissection of the entire colon, the colon to be resected was delivered through the open rectal stump and brought out transanally. The anvil of an intraluminal circular stapler was passed through the rectum into the peritoneal cavity and the end of the open distal rectum was closed with a linear cutting stapler. The anvil of the circular stapler was inserted into the end of the open terminal ileum and fixed with an Endo-Loop, following which an intracorporeal double-stapling anastomosis was performed. By 3 months following surgery, the patient was passing 3–4 stools a day. Thus, we highly recommend this technique as it eliminates the need for a small incision to deliver the resected colon, thereby minimizing the operative time and risk of wound infection.

Serum Biomarkers for Early Detection of Gynecologic Cancers

Ovarian, endometrial, and cervical cancers are three of the most common malignancies of the female reproductive organs. CA 125, historically the most reliable serum marker for ovarian cancer, is elevated in 50% of early-stage ovarian tumors. For endometrial cancers, there are no established serum markers. SCC, which is the best studied serum marker for squamous cell carcinomas, has been unreliable; SCC is elevated in cervical squamous cell carcinomas ranging from 28–85% of the time. Recent proteomics-based analyses show great promise for the discovery of new and more useful biomarkers. In this review, we will discuss the currently utilized serum tumor markers for gynecologic cancers and the novel biomarkers that are now under investigation.

Frequent inactivation of RUNX3 in endometrial carcinoma

OBJECTIVE Our objective was to determine whether RUNX3 tumor suppressor is inactivated in endometrial carcinoma. METHODS We have investigated 24 endometrial carcinomas, 3 endometrial carcinoma cell lines, and 9 normal endometria for genetic and epigenetic alterations of RUNX3. Reverse-transcription PCR (RT-PCR), methylation-specific PCR (MS-PCR) analysis, and loss of heterozygosity (LOH) analysis were performed. We also tested RUNX3 protein expression by immunohistochemistry. RESULTS Using RT-PCR technique, we observed a significant loss of RUNX3 mRNA expression in nine of 24 endometrial carcinomas (38%) and in all 3-cell lines (100%). In contrast, all nine of the normal endometria showed an abundant expression of RUNX3 mRNA. Methylation-specific PCR (MS-PCR) analysis of the CpG islands of RUNX3 showed the promoter region to be hypermethylated in 18 of 21 analyzed carcinomas (86%), whereas only two of nine normal endometria (22%) were methylated (p<0.01). By using two polymorphic microsatellite markers, D1S199 and D1S1676, we detected 1p36 LOH in 7 of 21 carcinomas (33%). We observed a significant relationship between the loss of RUNX3 mRNA expression and this regional LOH (p<0.01). Immunohistochemical staining showed that RUNX3 protein expression was lost in 12 of 21 endometrial carcinomas (57%). We observed a significantly more frequent loss of RUNX3 protein expression in the histologically higher-grade tumors (Grade 3) than in Grade 1 or 2 tumors (p<0.01). CONCLUSION These findings indicate that RUNX3 inactivation may play an important role in carcinogenesis of the endometrium, especially in high-grade endometrial carcinoma.