Hepatoma-derived growth factor is associated with reduced sensitivity to irradiation in esophageal cancer.

Radiotherapy is a useful component of treatment for esophageal cancer. Identification of the genes that are differentially expressed between radiosensitive and radioresistant cancer cells is important for predicting clinical effectiveness of radiotherapy. We established human esophageal cancer cell lines resistant to X-ray. Using differential display, we obtained one gene that was expressed in radiosensitive cells but was rarely expressed in radioresistant cells, and that gene was identical with hepatoma-derived growth factor (HDGF), an acidic polypeptide with mitogenic activity for fibroblasts. The semiquantitative reverse transcription-PCR assay confirmed that HDGF mRNA expression was reduced in established radioresistant cells, and its reduction was associated with reduced sensitivity to irradiation. Radiotherapy was more effective in clinical cases with high HDGF mRNA expression compared with cases with low expression (P < 0.05). The findings demonstrate that HDGF may play an important role in radiosensitivity, and it could be a novel marker predicting effectiveness of radiotherapy in clinical cases.

Loss of chemokine SDF-1alpha-mediated CXCR4 signalling and receptor internalization in human hepatoma cell line HepG2.

Expression of the chemokine stromal cell-derived factor-1alpha (SDF-1alpha) is absent from many carcinomas, including hepatomas. We note an early signalling defect in the hepatocellular carcinoma (HCC) cell line HepG2 that expresses the CXCR4 receptor and binds biotin-labelled SDF, but fails to stimulate downstream signalling events after engagement with SDF. In HepG2, the SDF/CXCR4 interaction did not result in calcium influx, phosphorylation and internalization of CXCR4, nor in a rapid phosphorylation of p44/42 MAP kinase. There were no CXCR4 mutations in the second chemokine binding loop or C terminal phosphorylation and internalization domains. The downstream signalling machinery in HepG2 appears to be intact since transfection of wild-type CXCR4 restored functional responsiveness. We conclude that HepG2 is unresponsive to SDF stimulation because of a defect located after receptor binding but before the activation of the signalling cascade. A hypothetical blocking molecule could hinder receptor internalization or CXCR4 signalling.

Clinical significance of matrix metalloproteinase-7 expression in esophageal carcinoma.

Matrix metalloproteinase-7 (MMP-7) is a member of MMP family and has a wide variety of substrate spectra. It is reported to play an important role in carcinoma invasion and metastasis. There is, however, little information on the clinical significance of MMP-7 in human esophageal carcinoma. We thus studied 48 tumor/normal pair samples of human esophagus by Northern blot analysis. The results demonstrated that the tumor tissue (T) of esophageal carcinoma showed a higher expression of MMP-7 mRNA than the corresponding normal tissue (N) in 31 cases (65%). We also statistically evaluated tumor MMP-7 value (T value) corrected for MMP-7-positive control (KYSE150 transfected with the MMP-7 gene). Fourteen cases with T value > or = 0.3 showed a higher frequency of lymph node metastasis than 34 cases with T value < 0.3 (P < 0.05). The cases with T value > or = 0.3 showed a significantly poorer prognosis than those with T value < 0.3 (P < 0.01). Multivariate analysis demonstrated that the MMP-7 expression status was the independent factor relating to the prognosis (P = 0.0005). The findings indicated that MMP-7 might be a novel prognostic factor for patients with esophageal carcinoma.

Angiogenin expression in human colorectal cancer: the role of focal macrophage infiltration.

Tumor angiogenesis is essential for tumor growth and tumor metastasis, and it depends on angiogenic factors produced by tumor cells and/or infiltrating cells in tumor tissue. In this study, we evaluated the clinical significance of the expression of angiogenin, which is a potent angiogenic protein, and the relationship between its mRNA expression and focal macrophage infiltration in colorectal cancer. Furthermore, we investigated the induction of angiogenin mRNA expression by proinflammatory cytokines mainly produced by inflammatory cells in tumor tissues. When we examined the relationship between the mRNA expression of angiogenin, by semiquantitative reverse transcription-PCR, and clinicopathological features in 65 patients with colorectal cancer, there was a significant difference in the vascular involvement, lymph node metastasis, liver metastasis, and advanced stage in patients with high-expression of angiogenin compared with low expression (P < 0.05). With regard to prognosis, the survival time for subjects in the high angiogenin mRNA group (tumor:normal ratio >1.9) was significantly worse (P < 0.05). When we examined the localization of angiogenin in colorectal cancer, immunohistochemical analysis in 65 patients with colorectal cancer revealed that angiogenin was predominantly expressed in cancer cells compared with stromal cells or normal tissues. The intensity of staining of angiogenin was significantly correlated with microvessel counts and focal macrophage infiltration counts (P < 0.05). In an in vitro study, interleukin-1beta and tumor necrosis factor-alpha induced angiogenin mRNA expression in colon cancer cells in a dose- and time-dependent manner, and these cytokines significantly upregulated the expression of angiogenin mRNA, especially in colon cancer cells rather than in other cells in the stroma of tumor tissues (fibroblasts, tumor infiltrating lymphocytes, macrophages). These results suggest that tumor angiogenesis in colorectal cancer may be advanced, at least in part, by angiogenin induced by proinflammatory cytokines derived from infiltrating macrophages.

Reduced expression of the CXCR4 receptor mRNA in hepatocellular carcinoma and lack of inducibility of its ligand alpha-chemokine hIRH/SDF1alpha/PBSF in vitro.

Differential cDNA displays between hepatocellular carcinoma and adjacent non-malignant tissues have previously detected a PCR product, hIRH (human intercrine reduced in hepatomas), equivalent to SDF1alpha/PBSF whose mRNA was lost from human hepatocellular carcinoma and other malignant and pre-malignant samples and malignant cell lines. There are no reports to date of the mRNA status of the receptor for hIRH/SDF1alpha/PBSF, CXCR4 in malignant tissues. We report here that there is a reduction in the mRNA expression of CXCR4 in hepatocellular carcinoma as estimated by Northern blot and RT-PCR and compared to the adjacent non-malignant tissue. The average (mean SD) tumor/normal ratio for CXCR4 mRNA expression, determined by RT-PCR, was 0.65 0.36 in 10 pairs of hepatocellular carcinomas. There was no consistent loss of CXCR4 mRNA expression in a range of malignant cell lines. The 3'-non-coding region of hIRH, had typical early response gene element sequences. Despite the presence of these 3'-elements there was no induction of hIRH gene expression in human lung carcinoma A549 cells by tumor necrosis factor alpha, interleukin-2, lipopolysaccharide or phorbol myristic acetate, nor in human melanoma cell line SB-2 by uv irradiation, under conditions which induced the homologue CXC intercrine IL-8 expression. Furthermore, there was no induction of hIRH gene expression, but rather a suppression, upon serum or cytokine addition to serum-deprived fibroblast cell lines, to an in vitro mouse bone marrow preparation, and to monocytic cell line THP-1.

Vascular endothelial growth factor/vascular permeability factor mRNA expression in patients with chronic hepatitis C and hepatocellular carcinoma.

Angiogenic factors such as vascular endothelial growth factor (VEGF) may be involved in neovascularization of malignant tumors. Our aim was to determine whether there is an increased VEGF mRNA expression in liver from patients with HCC and premalignant hepatitis C virus (HCV) with differing severity of inflammation. VEGF mRNA (VEGF165, VEGF189) was detected by reverse transcription and semi-quantitative polymerase chain reaction (RT-PCR) amplification in all liver samples. There was no difference in VEGF mRNA expression ratios (corrected for glyceraldehyde-3-phosphate dehydrogenase) among three groups: steatohepatitis, as a non-malignant non-viral control, 1. 05+/-0.35, n=8; chronic hepatitis C, 0.86+/-0.27, n=18; hepatocellular carcinoma, 1.06+/-0.43, n=10. VEGF mRNA expression was independent of the severity of HCV inflammation estimated by the histological activity index: low HAI (/=10, n=10), 0.93+/-0.31 vs. 0.81+/-0.24, p=ns. There was no significant difference in mean VEGF expression between HCC tumor (1.06+/- 0.43) and adjacent tissue (0. 85+/-0.42) although the tumors tended to have higher expression than adjacent non-malignant tissues. In conclusion, all liver samples of steatohepatitis, chronic HCV infection and HCC expressed VEGF mRNA, VEGF mRNA may be uniformly expressed in liver tissue, the level of expression is probably not related to virus infection or the severity of inflammation. Other angiogenic or angiostatic factors might be more involved in angiogenesis in HCC.

CXCR4 mRNA expression in colon, esophageal and gastric cancers and hepatitis C infected liver.

We have recently demonstrated by Northern blot and RT-PCR that the mRNA expression of the alpha-chemokine hIRH/SDF-1alpha is reduced in hepatocellular carcinoma (HCC), several digestive tract cancers and premalignant colon adenomas, and that its receptor CXCR4 mRNA expression is reduced in HCC. Here we investigate the expression of CXCR4 mRNA expression in several digestive tract cancers and hepatitis C viral (HCV) infected liver, a premalignant condition. There was no difference in the CXCR4 mRNA expression in colon, esophageal or gastric cancers compared to non-cancerous tissues. This is significantly different from the reduced expression we have seen with hepatocellular carcinoma (p<0.05). To better refine regional tumor or hepatic cytokine mRNA analysis within a biopsy sample we describe a micro-isolation technique for RNA extraction from portal and triad areas of liver biopsies or other small malignant or non-malignant biopsy samples suitable for use in RT-PCR and differential display reactions. In HCV liver biopsies, the expression of hIRH and its receptor CXCR4 mRNA, corrected for G3PDH, was not significantly different from that of control non-HCV (steatosis) biopsies. CXCR4 is expressed on leukocytes and its expression was predicted to correlate with hepatic inflammation. CXCR4 receptor mRNA expression did correlate significantly with that of its ligand hIRH/SDF-1alpha (p=0.001), and with the severity of fibrosis (p<0.05), but not with portal inflammation (p<0.10), piecemeal necrosis (p<0.10), lobular inflammation (p>0.10), the presence of lymphoid aggregates (p>0.10), or the total histological activity index (p=0.07). There was no difference in expression of hIRH or CXCR4 between responders and non-responders to interferon (IFN) treatment, while as a control, the responder group of patients did show a higher expression of IFNalpha receptor than the non-responder group (p=0.05).

Glyceraldehyde-3-phosphate dehydrogenase mRNA expression in hepatocellular carcinoma.

Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) is often used as a control gene for mRNA expression, however it has been proposed to be overexpressed in all hepatocellular carcinomas (HCC). Equal amounts of tumor and paired normal (T/N) RNA, based on OD260/280 nm, were compared using ethidium bromide staining, poly-T probing, gene-specific dot blot and Northern blots using control probes G3PDH, actin and histone H4. Using mRNA blots 13/20 surgical HCC pairs did not overexpress G3PDH. Those 7/20 intact samples which did appear to overexpress G3PDH on Northern blot could not be detected by poly-T probing of dot blots. The apparent overexpression was not specific for the control gene G3PDH nor for the malignancy HCC. It may represent partial mRNA degradation, or the presence of as yet unknown substances which interfere with absorption at 260/280 nm. We advise caution in selecting human T/N pairs for differential gene expression studies. For HCC, no clinicopathological variables, including cirrhosis, predicted whether a T/N sample pair was likely to be balanced or not.

Expression of prothymosin-alpha and c-myc mRNA in human gastric cancer.

Prothymosin-alpha (PT-alpha) is a nuclear protein involved in cell proliferation. c-myc is implicated in the carcinogenesis of many human cancers. PT-alpha gene transcription is reported to be regulated by the c-myc gene in vitro. However, little has been reported on the PT-alpha and c-myc mRNA expressions in gastric cancer. We semi-quantitatively determined the PT-alpha and c-myc mRNA expressions in 60 pairs of gastric cancer tissue (T) and corresponding normal tissue (N) using the reverse transcriptase-polymerase chain reaction method. The average of T/N ratio was 1.20 for PT-alpha and 1.30 for c-myc. Cases demonstrating a T/N ratio of more than 1.0 were seen in 33 (55%) and 30 (50%) cases for PT-alpha and c-myc, respectively. No significant correlation was observed between either of these two mRNA expressions and any of the examined clinicopathologic factors for gastric cancer. However, a significant correlation was seen between the expressions of both genes (p<0.0001). The findings support the hypothesis that, regarding human gastric cancer, the transcription of PT-alpha is considered to be under the control of c-myc gene, however, the value of these gene expressions do not reflect biological behavior.

Multiple tumor-suppressor-1 gene and esophageal-carcinoma.

The multiple tumor suppressor 1 (MTS1) gene is homozygously deleted frequently in cell lines derived from a wide variety of tumors. We investigated the deletion of the MTS1 gene in esophageal cancer cell lines and primary esophageal squamous carcinomas using the polymerase chain reaction. Sixteen and 15 of 23 esophageal cancer cell lines showed homozygous deletion of MTS1 exon 1 and exon 2, respectively, while none of 21 primary esophageal carcinomas showed the deletion. An analysis of MTS1 gene mutations was carried out by direct DNA sequencing in 8 cell lines and 21 primary carcinomas showing no homozygous deletion. In contrast to previous reports of esophageal carcinoma, there were no mutations recognized in the region sequenced. Our study suggests that the inactivation of the MTS 1 gene may play an important role in esophageal carcinoma cell lines but may be less important in primary carcinomas of the human esophagus.

Microsatellite instability in Japanese colorectal carcinoma.

Recent studies have shown that microsatellite instability (MSI) play an important role in the development of various types of cancer. To clarify the clinicopathologic significance of MSI in colorectal carcinoma (CRC), the presence of MSI was examined in 54 Japanese cases of CRC using the polymerase chain reaction-based method. The incidence of MSI in CRC cases was 13 out of 54 cases (24%). CRC with MSI also showed a significant tendency not to have lymph node metastasis (P<0.05), although neither the survival nor the prognosis of the cases examined in this study were available due to the short period of follow-up. The present study showed that the incidence of MSI in Japanese CRC was 24% and suggests that CRC with MSI may behave in a less malignant manner.

Surgical treatments for oesophageal cancer concomitant with gastric adenoma with severe epithelial atypia.

The various surgical treatments for oesophageal cancer concomitant with gastric adenoma with severe epithelial atypia (SEA) are discussed by presenting three such cases. As gastric adenoma with SEA had been considered to be a precancerous condition but not normally an indication for gastrectomy, we devised an operative strategy of laparotomy and thoracotomy. For Case 1, with early oesophageal cancer and gastric adenoma with SEA in the body of stomach, a laparotomy and local resection of the gastric lesion were carried out prior to thoracotomy in order to determine the safest reconstructive method after measuring the length of the well-nourished gastric tube. In Case 2, with advanced oesophageal cancer, a thoracotomy was first performed to assess the curability of surgery for the oesophageal cancer and a gastric adenoma was removed. In contrast, for Case 3 with advanced oesophageal cancer, and in whom post-operative survival was deemed to be short, a gastric adenoma was not resected. The most appropriate operative methods should thus be decided after careful consideration of the stage of the oesophageal cancer and characteristics of the gastric adenoma.

A bag carrier for continuous intravenous hyperalimentation.

We designed a new bag-carrier device system for continuous intravenous hyperalimentation. The patient carries it on his shoulder and can both walk up and down stairs and go out. The use of this device is simple and easy, and was found to increase the patient's opportunity to engage in physical activity.

Pancreaticoduodenectomy under epidural anesthesia without endotracheal intubation for the elderly.

To evaluate the efficacy of a single application of epidural anesthesia without endotracheal intubation for pancreaticoduodenectomy (PD), the data on 30 patients who underwent PD were analyzed for their operative morbidity and mortality. These patients were classified into two groups according to the type of anesthesia performed: 15 received epidural anesthesia alone (Group I) and 15, general anesthesia under endotracheal intubation (Group II). The clinical characteristics of the patients in both groups were comparable at the time of operation, except that Group I included a significantly larger proportion of elderly patients than Group II (p < 0.05). Postoperative pulmonary complications (PPCs) occurred in 5 (33.3%) of the Group II patients, especially in elderly patients who underwent lengthy operations, whereas no such complications occurred in the Group I patients (p < 0.05), even in elderly patients with a long operating time. The curability of the malignant tumors and the incidences of other complications were not significantly different between the two groups. These findings suggest that a single application of epidural anesthesia is effective in preventing PPCs when performing a time-consuming PD, especially in elderly patients.

Surgical management of patients with radiation enteritis.

To improve the surgical outcome in patients with radiation enteritis, 18 female cases were analyzed. Out of the 18 cases, 3 (16.7%) were treated conservatively while 15 (83.3%) underwent surgical procedures. Thirteen out of 14 patients (92.8%) with ileus underwent an operation. The overall mortality was 22.2% (4 out of 18 cases). However, no significant difference in the mortality between the operated and non-operated cases was observed. Although an analysis of the 15 operated cases did not reveal any significant factors that might have affected the prognosis, all four patients who underwent a bypass operation showed a good postoperative course, with only one excepting being a patient suffering from malnutrition. Although only a small number of patients were included in this study, these results suggest that 1) surgeons should not hesitate to operate on patients with radiation enteritis demonstrating ileus, and 2) a bypass operation may be one surgical alternative in the presence of massive adhesion or for patients at high risk for a standard operation.

[Psychophysical and vergence responses of normal and stereoanomalous observers to pulse-disparity stimulus].

The relations between psychophysical and oculo-motor responses to retinal disparity were examined in two experiments. Experiment I examined the subject's discriminability of the depth produced by 2 degrees crossed, 2 degrees uncrossed, and zero disparities with the durations of 100 and 1000 ms. As a result, six stereonormal, one crossed stereoanomalous, and one stereoblind observers were identified. In Experiment II, eye movement responses of three subjects with the different types of stereoscopic vision to the disparity pulses were monitored by the photo-electric method. The results of Experiment II indicated that the normal observer showed normal vergence, the crossed stereoanomalous observer showed anomalous convergence, and the stereoblind observer did not show any vergences but saccadic movements. Several theoretical implications of the results are discussed.

PKCgamma in Vc and C1/C2 is involved in trigeminal neuropathic pain.

The aim of the present study was to clarify the involvement of protein kinase Cγ (PKCγ) in the facial neuropathic pain following infraorbital nerve injury. We analyzed the change in PKCγ expression in the trigeminal spinal subnucleus caudalis (Vc) and upper cervical spinal cord (C1/C2) following chronic constriction injury of the infraorbital nerve (ION-CCI). We also studied ION-CCI-mediated mechanical nocifensive behavior in rats. The mechanical head-withdrawal threshold significantly decreased 1 to 14 days after ION-CCI compared with that before ION-CCI and in sham rats. The expression of PKCγ was significantly larger in the ipsilateral Vc compared with the contralateral side in ION-CCI rats 3, 7, and 14 days after ION-CCI. Intrathecal (i.t.) administration of the PKCγ inhibitor chelerythrine prevented an increase in the PKCγ expression in the ipsilateral Vc. Moreover, i.t. administration of chelerythrine annulled ION-CCI-mediated reduction in the head-withdrawal threshold. Taken together, these findings suggest that PKCγ expression in the Vc played an important role in the mechanism of orofacial static mechanical allodynia following trigeminal nerve injury.

A new detection method for the K variant of butyrylcholinesterase based on PCR primer introduced restriction analysis (PCR-PIRA).

The K variant of human butyrylcholinesterase is caused by a G/A transition in the butyrylcholinesterase gene, which neither creates nor destroys any restriction site. In an attempt to detect the K variant both simply and rapidly, we developed a two step method of "PCR primer introduced restriction analysis" (PCR-PIRA). The first step was used to introduce a new Fun4HI site into the normal allele for a screening test, while the second step was performed to create a new MaeIII site on the variant allele for a specific test. This method thus enabled us to distinguish clearly the K variant from the normal allele, and also showed that the frequency of the K variant allele is 0.164 in the Japanese population.

Occlusion as a depth cue in the Wheatstone-Panum limiting case.

We examined the hypothesis (Ono & Wade, 1985) that occlusion of far stimuli by a near one on the same visual line can operate as a depth cue in stereograms containing different numbers of targets in the two eyes. By controlling eye positions, we created conditions in which the visual system could interpret the retinal images as originating from stimuli on the visual axis of one eye and also created other conditions in which the origin of the retinal images was ambiguous. In Experiment 1, we presented two lines to one eye and a single line to the other eye. When the image of the line on the temporal side of the line pair on one retina fused with the image of the single line on the other retina, the nonfused line appeared farther away more often than it did when the image on the nasal side fused. In Experiment 2, we used two differently shaped stimuli. In the condition in which the nonfused stimulus represented an object being occluded, it appeared farther away more often than in the four conditions in which it did not. In Experiment 3, we extended the idea to three different objects. When the middle of the three images fused with the single image, the nonfused stimulus appeared farther when it could be interpreted as being occluded than when it could not. In the condition in which the most temporal image fused with the single image, the nonfused stimuli appeared farther than in the condition in which the most nasal one fused. The results supported the hypothesis that occlusion plays a role in depth perception in the Wheatstone-Panum limiting case.

The interaction of perceived distance with the perceived direction of visual motion during movements of the eyes and the head.

A horizontally moving target was followed by rotation of the eyes alone or by a lateral movement of the head. These movements resulted in the retinal displacement of a vertically moving target from its perceived path, the amplitude of which was determined by the phase and amplitude of the object motion and of the eye or head movements. In two experiments, we tested the prediction from our model of spatial motion (Swanston, Wade, & Day, 1987) that perceived distance interacts with compensation for head movements, but not with compensation for eye movements with respect to a stationary head. In both experiments, when the vertically moving target was seen at a distance different from its physical distance, its perceived path was displaced relative to that seen when there was no error in perceived distance, or when it was pursued by eye movements alone. In a third experiment, simultaneous measurements of eye and head position during lateral head movements showed that errors in fixation were not sufficient to require modification of the retinal paths determined by the geometry of the observation conditions in Experiments 1 and 2.