Induction of immunogenic cell death in murine colon cancer cells by ferrocene-containing redox phospholipid polymers.

Immunogenic cell death (ICD), activated by damage-associated molecular patterns (DAMPs), is an apoptotic cell death process that elicits antitumor immunity. Although anticancer drugs that can induce ICD are promising for cancer treatment, the design strategy for ICD inducers remains unclear. In this study, we demonstrated the cell-penetrating redox phospholipid polymer poly(2-methacryloyloxyethyl phosphorylcholine-co-vinyl ferrocene) (pMFc) inducing ICD in murine colon cancer CT26 cells. pMFc produced oxidative stress by extracting electrons from CT26 cells and induced the release of DAMPs, such as calreticulin, adenosine triphosphate, and high-mobility group box 1. Moreover, the injection of pMFc-treated CT26 cells inhibited tumor formation in subsequently challenged CT26 cells, indicating that pMFc elicited antitumor immunity through ICD. Using in vivo therapy, intratumoral injections of pMFc induced complete tumor regression in 20% (1/5) of mice. These results suggested that the redox phospholipid polymer provides a new option for ICD-inducing anticancer polymers.

Clinical influence of anastomotic stricture caused by pancreatogastrointestinalstomy following pancreatoduodenectomy.

BACKGROUND AND PURPOSE: Pancreatic fistula after pancreatoduodenectomy (PD) is not uncommon, but few reports describe a stricture after pancreatogastrointestinalstomy. We investigated the clinical influence of anastomotic stricture caused by pancreatogastrointestinalstomy after PD. METHODS: The subjects of this prospective cohort study were 132 patients who underwent PD or pylorus-preserving PD. We reviewed the relationships between pancreatic duct dilatation of the remnant pancreas and several risk factors. We also compared pancreatic duct dilatation with pancreatic atrophy and analyzed nutrient parameters in the first postoperative year. RESULTS: Patients with a preoperative pancreatic duct diameter less than 3 mm had a significantly dilated postoperative pancreatic duct diameter (p = 0.0001). The average atrophy rate of the remnant pancreas was 26.3 %, with the lowest atrophy rate (7.3 %) seen in patients without pre- or postoperative pancreatic duct dilation. A normal pancreas in which pancreatic duct dilatation developed postoperatively had a high atrophy rate (34.9 %). Moreover, only patients without pre- or postoperative pancreatic dilatation gained body weight (3.9 %). CONCLUSION: This study shows a significant correlation between pancreatic atrophy rate and weight loss. Atrophy of the remnant pancreas caused by anastomotic stricture influences the exocrine function of patients after PD. The anastomotic method must be improved to prevent pancreatic duct dilatation and allow for early diagnosis and management of stenotic lesions.

Prokineticin 1 protein expression is a useful new prognostic factor for human sporadic colorectal cancer.

BACKGROUND: Hematogenous metastasis, regarded as closely related to angiogenic growth factors, is associated with colorectal cancer prognosis. The angiogenic growth factor prokineticin 1 (PROK1) has been cloned from endocrine cells. However, its protein expression in human malignant tumors has not been studied. The current study established the anti-PROK1 monoclonal antibody (mAb) and examined the relationship between the expression of PROK1 protein and human colorectal cancer. METHODS: The expression of PROK1 protein was assessed in 620 resected sporadic colorectal cancer tissue samples by immunohistochemical staining with in-house-developed human PROK1 mAb to investigate the relationship of PROK1 expression to clinicopathologic factors, recurrence, and survival rate and to evaluate its prognostic significance. RESULTS: The expression of PROK1 protein was detected in 36 % (223/620) of human primary colorectal cancer lesions but no in the healthy mucosa adjacent to the colorectal cancer lesions. According to the clinicopathologic examinations, the frequency of positive PROK1 expression was significantly higher in cases with serosal invasion, lymphatic invasion, venous invasion, lymph node metastasis, liver metastasis, hematogenous metastasis, and higher stage disease. The recurrence rate and prognosis for patients with PROK1 expression-positive lesions were significantly worse. In the Cox proportional hazard model, PROK1 expression was an independent prognostic factor. CONCLUSIONS: The expression of PROK1 protein was identified for the first time as a new prognostic factor in colorectal cancer.

Hepatic artery infusion therapy is effective for chemotherapy-resistant liver metastatic colorectal cancer.

BACKGROUND: Systemic FOLFOX (folinic acid (leucovorin (LV)), 5-fluorouracil (5-FU), and oxaliplatin), FOLFIRI (LV, 5-FU, and irinotecan), or FOLFOXIRI (5-FU, leucovorin, oxaliplatin, and irinotecan) chemotherapy regimens and additional molecular-target treatments, including anti-vascular endothelial growth factor, anti-epidermal growth factor receptor, and anti-multi-kinase antibodies, have been recommended for unresectable recurrent colorectal cancers. However, no effective treatments are currently available for cases refractory to these therapies. Therefore, the development of alternative therapies is desired. In the present study, we administered and observed the effectiveness of hepatic artery infusion therapy (HAIC) in patients with unresectable liver metastatic colorectal cancers refractory to systemic chemotherapy. In addition, we observed that in an experimental system with anticancer drug-resistant colorectal cancer lines, apoptosis and cell death could be induced by increasing anticancer drug concentrations. METHODS: The subjects had liver metastatic colorectal cancers that were unresponsive to systemic chemotherapy (FOLFOX/FOLFIRI) or to additional molecular-target therapies for progressive disease. Hepatic infusion tube placement was conducted according to the Seldinger method to insert a catheter with a side hole via the right femoral artery. A coiling procedure was performed to prevent drug influx into the gastroduodenal artery. Ten subjects were selected, and the results were evaluated after HAIC (5-FU and LV administered once weekly). Moreover, anticancer drug-resistant colorectal cancer lines were subsequently prepared to investigate whether increased anticancer drug concentrations could induce apoptosis or cell death. RESULTS: Of the 10 subjects, 3 (30 %) showed partial response and 4 (40 %) showed no change according to computed tomography imaging findings obtained after hepatic artery infusion. The disease control rate was 70 %. Eight subjects had improved quality of life. Survival time ranged from 2 to 16 months (median, 9 months). Meanwhile, we found that higher anticancer drug concentrations induced apoptosis and cell death in an anticancer drug-resistant colorectal cancer cell line. CONCLUSIONS: HAIC was effective in some systemic chemotherapy-resistant colorectal cancers with liver metastases and should be considered as an effective palliative therapy. This supports the finding that apoptosis and cell death could be induced in anticancer drug-resistant colorectal cancer cells in a drug concentration-dependent manner.

Anti-prokineticin1 (PROK1) monoclonal antibody suppresses angiogenesis and tumor growth in colorectal cancer.

BACKGROUND: The prokineticin1 (PROK1) gene has been cloned as an angiogenic growth factor from endocrine gland cells. However, we have not known about potentials of anti-PROK1 monoclonal antibody in human cancers. Here we investigated how the anti-PROK1 monoclonal antibody (mAb; established by our department) would affect the high-PROK1-expressing colorectal cancer (CRC) cells in vitro and vivo. METHODS: We confirmed PROK1 protein expression in the CRC cells by performing immunohistochemical staining and measured the amount of soluble PROK1 protein. Next, we mixed the CRC cell culture fluid with the anti-PROK1mAb to examine angiogenic activity in vitro and in vivo. Additionally, we investigated whether the anti-PROK1mAb would affect the tumor-forming capability of high PROK1-expressing CRC cells implanted into mice. RESULTS: PROK1 protein expression was confirmed in 3 CRC cell lines, and soluble PROK1 protein was also confirmed in the CRC cell culture fluid. The culture fluid increased angiogenesis in vitro and vivo, whereas the anti-PROK1mAb suppressed angiogenesis. Subcutaneous tumor formation and tumor angiogenesis in mice were suppressed by the anti-PROK1mAb treatment. The anti-PROK1mAb significantly suppressed the number of CD31 stained cells in mice. CONCLUSIONS: The in vitro and vivo experimental system indicated that the anti-PROK1mAb could suppress angiogenesis and tumor growth in the CRC strains.

Partial response after transcatheter arterial infusion chemotherapy in a patient with systemic chemotherapy-resistant unresectable colon cancer and hepatic metastasis: (case report).

We report here a case of partial response to hepatic arterial infusion chemotherapy in a patient who developed serious hepatic failure due to unresectable colorectal cancer and hepatic metastasis and showed resistance to systemic chemotherapy with molecular targeted drugs, mFOLFOX6, and FOLFIRI. The patient was a 60-year-old woman who underwent sigmoidectomy for sigmoid colon cancer, lateral posterior hepatic segmentectomy for metastatic liver cancer, and postoperative radiation therapy for metastatic lung cancer. As first-line systemic chemotherapy, mFOLFOX6 (oxaliplatin, 5-fluorouracil, and leucovorin), bevacizumab + FOLFIRI (irinotecan, 5-fluorouracil, leucovorin), and anti-epidermal growth factor receptor antibody + irinotecan were administered, in that order. However, recurrent hepatic metastasis was exacerbated, which induced serious hepatic failure manifested by general malaise, jaundice, abnormal hepatic function, difficulty in walking due to bilateral lower extremity edema, and decreased appetite. The patient was admitted in a serious condition. After hospitalization, the patient received hepatic arterial infusion chemotherapy with 5-fluorouracil and l-leucovorin. After two complete courses, the symptoms improved. The patient's performance status also improved, and she was discharged from the hospital. Four months after discharge, the patient had continued outpatient chemotherapy and maintained excellent performance status. Although HAIC is not presently considered an alternative to systemic chemotherapy, it is sometimes effective in patients who show resistance to molecular targeted drug therapy, FOLFOX, and FOLFIRI, and in whom hepatic metastasis is a key factor in determining prognosis and serious hepatic failure. Further studies should be performed in the future to verify these findings.

[A case study of stable disease after hepatic arterial infusion chemotherapy in a patient with systemic chemotherapy-resistant unresectable hepatic metastases of colon cancer].

The patient was a 72-year-old man with cancer of the descending colon accompanied by multiple unresectable hepatic metastases. As first-line systemic chemotherapy, mFOLFOX6, anti-vascular endothelial growth factor (VEGF) antibody+FOLFIRI, and anti-epidermal growth factor receptor (EGFR) antibody+CPT-11 were administered in order mentioned. However, recurrent hepatic metastasis was exacerbated. As fourth-line chemotherapy, the patient received hepatic arterial infusion chemotherapy (HAIC) with 5-FU and LV. The chemotherapy regimen consisted of 5-FU 600 mg/m2 and LV 250 mg/m2 given once a week. After 6 weeks, CT revealed that the multiple liver metastases had not increased, and the level of the tumor marker CA19-9 significantly decreased from 1,980 (normal range, 0-37 U/mL)to 942.9 U/mL. HAIC was continued, and the patient maintained an excellent PS for 3.5 months. We report a case of stable disease in response to HAIC in a patient who exhibited resistance to systemic chemotherapy with an anti-EGFR antibody, an anti-EGFR antibody, mFOLFOX6, and FOLFIRI.

[A case of response to panitumumab as third-line chemotherapy for multiple liver metastases and portal venal tumor embolus of rectal cancer].

A 64-year-old man who underwent rectal amputation for rectal cancer was diagnosed with multiple liver metastases and tumor embolus in the portal vein 6 months after operation. Though the patient underwent chemotherapy, mFOLFOX6, and bevacizumab+FOLFIRI, liver metastases were diagnosed as progressive disease (PD). After panitumumab+FOLFIRI was administered for three months as third-line chemotherapy, the tumor embolus completely disappeared, and liver metastases became cytoreductive on CT. The patient was judged to have achieved a partial response (PR). This case indicated that panitumumab was effective as third-line chemotherapy for unresectable recurrent rectal cancer.

[A case of stomach metastasis of pleomorphic carcinoma of the lung with hypercalcemia].

A 69-year-old man was admitted with fatigue, anorexia, and slight fever. Gastroscopy showed a tumor in the stomach, and biopsy revealed a poorly differentiated adenocarcinoma. CT scan revealed a tumor of the stomach, a tumor in the lower lobe of the right lung, and multiple tumors in the liver. Moreover, he was drowsy, probably caused by severe hypocalcemia thought to be caused by parathyroid hormone related protein. We treated him with S-1, but the gastric tumors progressed rapidly and massive pleural effusion developed. He died on the 16th day after admission. At autopsy, the histology of the lung tumor was found to be pleomorphic carcinoma, and that had metastasized to the stomach, the liver, and other abdominal organs. We treated a rare case of pleomorphic carcinoma with hypercalcemia that was discovered due to gastric metastasis.

[Local resection of a gangliocytic paraganglioma near the papilla of Vater].

A 69-year-old man was admitted to our hospital with epigastric discomfort. Upper gastrointestinal endoscopy showed a submucosal tumor near the papilla of Vater. Abdominal CT and MRI showed a small, well-enhanced tumor. Endoscopic tumor biopsy was performed before the operation, but pathologic findings showed normal duodenal musosa. Nevertheless, since malignancy could not be ruled out, we resected the tumor with the sphincter of the papilla of Vater, followed by plasty of the orifice for the common bile duct and main pancreatic duct. We identified 3 parts with tumor cells; epithelioid cells, spindle cells, and ganglion-like cells. The tumor was diagnosed as gangliocytic paraganglioma of the duodenum. Treatment by resecting the tumor with the sphincter of the papilla of Vater, followed by the plasty of the orifice for the common bile duct and main pancreatic duct, was selected considering the patient's safety and to achieve radical cure.

RIN1-Ras-ERK pathway plays an important role in carcinogenesis in colon cancer cell line LoVo.

The RIN1 protein has SH2, three domains, and H-Ras binding domains; thus, it is presumed to be an important molecule in an intracellular signaling pathway. We examined the effect of the introduction of a membrane protein-encoding, mutated (S351A)RIN1 gene into a colon cancer. In the LoVo colon cancer cell line, endogenous RIN1 protein was strongly expressed in the cytoplasmic fraction, and the RIN1 protein in the cytoplasmic fraction was strongly bound to the 14-3-3 protein. In the mutated (S351A)RIN1-transfected LoVo cells, the mutated (S351A)RIN1 protein was identified in the cell membrane, and was bound to HRas protein. Also, in vitro the proliferative capacity of the mutated (S351A)RIN1-transfected LoVo cells was significantly inhibited, compared with that of their empty vector-transfected counterparts. In the mutated (S351A)RIN1-transfected LoVo cells, the phosphorylation of ERK1/2 proteins downstream of the H-Ras molecule was inhibited, compared with the counterparts. This study is the first to show that the localization of RIN1 protein plays an important role in the carcinogenesis in colon cancer cells LoVo (i.e., signal transduction in the Ras-ERK pathway).

Ruptured mucinous cystic neoplasm with an associated invasive carcinoma of pancreatic head in a pregnant woman: report of a case and review of literature.

Mucinous cystic neoplasm (MCN) of the pancreas is characterized by mucin-producing columnar epithelium and an ovarian-type stroma. It occurs almost exclusively in women and is almost always located in the pancreatic body or tail. Here, we report a case of large MCN located in the pancreatic head but not in the body nor tail in a 32-year-old pregnant woman, which was thought to have grown rapidly during pregnancy. It was ruptured at 34 weeks of gestation and the patient was admitted to the emergency department of the University of Fukui Hospital with an acute abdomen. Emergency cesarean section followed by pancreaticoduodenectomy was performed. The tumor consisted of many small cysts lined by a single-layer of mucinous epithelium with papillary growth and partial solid parts showing invasive growth and sarcomatoid changes, indicating mucinous cystic neoplasm with an associated invasive carcinoma (previously referred as mucinous cystadenocarcinoma). Thickened septa revealed ovarian-type stroma strongly positive for α-inhibin and partly positive for progesterone receptor immunohistochemically. We also review and discuss previous reports of MCNs including those with an associated invasive carcinoma in pregnant patients.

Atypical metastatic carcinoid of the uterine cervix and review of the literature.

Atypical carcinoid tumors of the uterine cervix represent rare neuroendocrine tumors and are highly aggressive, showing early lymphatic invasion and hematogenous distant metastases. Because of the small number of cases, there are currently no recommendations regarding treatment, and little is known about the response to chemotherapeutic agents. A 39-year-old woman was diagnosed with a primary atypical carcinoid of the uterine cervix with numerous metastases to the liver. After radical hysterectomy, she underwent hepatic arterial chemoembolization with streptozotocin and 5-fluorouracil. Complete response was achieved in numerous liver metastases. At the 2-year follow up of chemotherapy, the patient remains alive. Treatment for atypical carcinoid tumors remains elusive, however hepatic arterial chemoembolization with streptozotocin and 5-fluorouracil was effective in the present primary atypical carcinoid with liver metastases. A review of the previous reports is also presented.

Prokineticin-1 induces normal lymphangiogenic activity and is involved in lymphangiogenesis and lymph node metastasis in colorectal cancer.

BACKGROUND: Prokineticin family correlates with important roles in several biological processes, including homeostasis. We discovered novel functions of prokineticin1 (PROK1) in lymphangiogenesis and lymphnode metastasis in colorectal cancer. MATERIALS AND METHODS: We examined changes in the number of lymphatic endothelial cells after PROK1 stimulation. PROK1 protein was stimulated with subcutaneously implanted in mice. Also a high-PROK1-expressing colorectal cancer cell line and anti-PROK1 antibody(Ab) were subcutaneously implanted in mice, and then examine lymphangiogenesis. PROK1 expression and the number of lymph vessels were examined in the primary lesion of 391 patients whose colorectal tumors had been resected. RESULTS: When PROK1 was used as a stimulus, the number of lymphatic cells increased compared to unstimulated cells. And the number of lymph vessels in the skin of mice increased compared to mice implanted without PROK1. The number of lymph vessels in the primary tumor tissue increased when PROK1 was highly expressed compared to cases with non-detectable PROK1 expression. When PROK1 was expressed in human colorectal tumors, the rate of lymphnode metastasis was significantly higher than that in cases with non-detectable PROK1 expression. CONCLUSIONS: PROK1 is a lymphangiogenic factor involved in the formation of new lymph vessels and lymphnode metastasis in human colorectal cancer.

The activation of Proteinase-Activated Receptor-1 (PAR1) mediates gastric cancer cell proliferation and invasion.

BACKGROUND: In addition to regulating platelet function, the G protein-coupled sub-family member Proteinase-activated receptor-1 (PAR1) has a proposed role in the development of various cancers, but its exact role and mechanism of action in the invasion, metastasis, and proliferation process in gastric cancer have yet to be completely elucidated. Here, we analyzed the relationship between PAR1 activation, proliferation, invasion, and the signaling pathways downstream of PAR1 activation in gastric cancer. METHODS: We established a PAR1 stably transfected MKN45 human gastric cancer cell line (MKN45/PAR1) and performed cell proliferation and invasion assays employing this cell line and MKN28 cell line exposed to PAR1 agonists (alpha-thrombin and TFLLR-NH2). We also quantified NF-kappaB activation by electrophoretic mobility shift assay (EMSA) and the level of Tenascin-C (TN-C) expression in conditioned medium by ELISA of MKN45/PAR1 following administration of alpha-thrombin. A high molecular weight concentrate was derived from the resultant conditioned medium and subsequent cultures of MKN45/PAR1 and MKN28 were exposed to the resultant concentrate either in the presence or absence of TN-C-neutralizing antibody. Lysates of these subsequent cells were probed to quantify levels of phospholyrated Epidermal Growth Factor Receptor (EGFR). RESULT: PAR1 in both PAR1/MKN45 and MKN28 was activated by PAR1 agonists, resulting in cell proliferation and matrigel invasion. We have shown that activation of NF-kappaB and EGFR phosphorylation initially were triggered by the activation of PAR1 with alpha-thrombin. Quantitative PCR and Western blot assay revealed up-regulation of mRNA and protein expression of NF-kappaB target genes, especially TN-C, a potential EGFR activator. The suppressed level of phosphorylated EGFR, observed in cells exposed to concentrate of conditioned medium in the presence of TN-C-neutralizing antibody, identifies TN-C as a putative autocrine stimulatory factor of EGFR possibly involved in the sustained PAR1 activation responses observed. CONCLUSION: Our data indicate that in gastric carcinoma cells, PAR1 activation can trigger an array of responses that would promote tumor cell growth and invasion. Over expression of NF-kappaB, EGFR, and TN-C, are among the effects of PAR1 activation and TN-C induces EGFR activation in an autocrine manner. Thus, PAR1 is a potentially important therapeutic target for the treatment of gastric cancer.

Survivin-3B gene decreases the invasion-inhibitory effect of colon cancer cells with 5-fluorouracil.

The expression of survivin molecules has been confirmed in many types of cancer cells, including colon cancer cells, and they are considered important antiapoptotic molecules. Recent studies have revealed the existence of different splicing forms of survivin molecules; however, no studies have examined their expression in gastrointestinal cancers. In 2004, we reported the existence of the survivin-3B gene, a novel splice variant of survivin. In this study, we investigated the relationship between human colon cancer and our recently cloned survivin-3B gene with a coding region of 594 bp. In the first examination, survivin-3B expression was analyzed by RT-PCR in human colon cancer and adjacent normal mucosal tissues. The associations of its expression status with clinicopathological parameters and the prognosis were also examined. Survivin-3B mRNA expression was observed in 37 (46.3%) of 80 primary colon cancers, but not in the adjacent normal colonic mucosal tissue. The rate of survivin-3B gene expression was significantly higher in colon cancer with serosal invasion. The 5-year survival rate of patients with survivin-3B gene-positive primary colon cancer was significantly poorer, at 48.7%, than that (75.4%) of survivin-3B gene-negative patients. In the second examination, after the introduction of the survivin-3B gene into cells of the colon cancer cell line DLD-1, 5-fluorouracil-induced changes in their invasive capacity was examined. The invasion-inhibitory effect of 5-fluorouracil on survivin-3B gene-transfected DLD-1 cells was significantly lower than their empty vector gene-transfected counterparts. We speculate that survivin-3B expression in colon cancer is an important factor involved in the invasive capacity of cancer cells in the presence of anticancer drug.

The effectiveness of transverse coloplasty in patients with ultra-lower rectal cancer.

Rectal cancer accounts for 40% of colon cancer, and postoperative defecatory function is considered to markedly affect the patients' quality of life. We performed transverse coloplasty in 33 patients with rectal cancer who had undergone an anal function preservation operation in which the anastomotic site was within 1 cm of the dentate line (ultra-low anterior resection) and evaluated its effectiveness in controlling the patients' defecatory function. The average daily defecation frequency 1, 6, and 12 months postoperatively was 7.8, 5, and 3.6 times daily following straight colorectal reconstruction (the anastomotic site was more than 5 cm from the dentate line) and 7.5, 3.5, and 2.4 times daily following transverse coloplasty, respectively. Concerning postoperative complications, anastomotic leakage, soiling, and constipation were observed in 1, 1, and 1 cases, respectively. Transverse coloplasty can be performed in a short time, and it is considered a safe and useful method to manage defecatory

Enhanced desalination performance in compacted carbon-based reverse osmosis membranes.

Reverse osmosis membranes typically suffer compaction during the initial stabilization stage due to the applied hydraulic pressure, altering the desalination performance. The elucidation of the underlying transformations during compaction is key for further development of new membranes and its deployment in real-world scenarios. Hydraulic compaction of amorphous carbon (a-C) based membranes under cross-flow operation for water purification and desalination has been observed experimentally, and analysed employing molecular dynamics simulations. The previous outstanding separation performance for carbon membranes, especially for the nitrogen-containing (a-C:N) type, has been studied during compaction using lab-scale cross-flow desalination membrane systems. Our results indicate that the high-water pressure induces an overall reduction in the interstitial spaces within the a-C structure. Remarkably, the compacted a-C:N membrane exhibits improved performance in salt rejection and water permeability, compared to the a-C based membrane. Our analysis shows that performance improvement can be related to the higher mechanical stability of the carbon structure due to the presence of nitrogen sites, which also promote water diffusion and permeability. These results show that a-C:N based membranes are a feasible alternative to conventional polymeric membranes.

Cloning of a novel splicing variant of RIN1 and its expression in gastric and colon cancer.

The regular RIN1 gene is a molecule located on chromosome 1lq13.2, and contains a coding region of 2352 bp with a 3' domain that binds to H-Ras protein, suggesting that it is an important molecule in the intracellular signaling pathway. In this study, we confirmed the existence of a novel form of the RIN1 gene with a different splicing pattern, successfully cloned it, and examined its expression in gastric and colon cancer cell lines. A 612-bp band (the RIN1 variant mRNA) was identified in the RT-PCR product from the colon cancer cell line Colo320D. (A 2352-bp band representing the regular RIN1 gene in HT29 cell line.) The 612-bp band was sequenced and compared with that of the regular RIN1 gene. As a result, the 612-bp product was found to contain a tyrosine phosphorylation site on the 5' side and Ras and 14-3-3 binding domains on the 3' side, indicating that it is a product with a different splicing pattern. The expression of the RIN1 variant mRNA was observed in two of six gastric cancer cell lines and four of five colon cancer cell lines. We identified a novel RIN1 gene with a splicing pattern different from that of the regular RIN1 gene. Comparison of both genes revealed that the novel RIN1 products had a structure conserving the Ras and 14-3-3 binding domains, but lacking two tyrosine phosphorylation sites. Novel RIN1 variant protein was expressed primarily in the cytoplasm and no expression in the cell membrane, and RIN1 variant protein was bound to 14-3-3 protein. In addition, the novel RIN1 mRNA was found to be expressed in gastric and colon cancer cell lines, suggesting that it is an important gene for the function of cancer cells.

Rat islet culture in serum-free medium containing silk protein sericin.

BACKGROUND: The development of islet cultures is desirable for successful clinical islet transplantation. Fetal bovine serum (FBS) has been used as a supplement in islet culture medium, but it may be an unsuitable supplement due recent animal health problems. We have evaluated the use of the silk protein, sericin, derived from Bombyx mori as a replacement for FBS in islet culture medium. METHODS: Twenty rat islets were cultured in medium containing either sericin or FBS, or no supplement, for 14 days, during which time viable islets were counted in order to evaluate islet survival. Insulin secretion was measured in vitro by static incubation on days 3 and 7. In vivo function of cultured islets was tested by syngeneic transplantation. The islets were evaluated histologically and immunohistochemically after culture and transplantation. RESULTS: Ninety-five percent of islets were viable after culture for 14 days in culture medium supplemented with either FBS or sericin, while no islets survived beyond 7 days in culture without supplement. No significant differences in stimulated insulin secretion were noted between two groups of islets grown on supplemented media. Following transplantation, islets cultured in FBS or sericin rapidly reversed hyperglycemia and maintained normal glycemic control. Histologically, islets cultured with sericin displayed a well-preserved structure and strong insulin staining before and after transplantation. CONCLUSION: Serum-free medium containing sericin appears to be useful for islet culture.