Comparison of preoperative chemotherapy using docetaxel, cisplatin and fluorouracil with cisplatin and fluorouracil in patients with advanced carcinoma of the thoracic esophagus.

We retrospectively compared preoperative docetaxel, cisplatin, and fluorouracil (DCF) with cisplatin and fluorouracil (CF) in patients with esophageal cancer. The study included patients with advanced thoracic esophageal carcinoma (excluding T4 tumors) receiving preoperative chemotherapy. In the DCF group, five patients received two courses of treatment every 4 weeks, and 33 patients received three courses every 3 weeks. In the CF group, 38 patients received two courses of treatment every 4 weeks. Patients underwent curative surgery 4-5 weeks after completing chemotherapy. Patient demographic characteristics did not differ between the two study groups. The incidence of a grade 3 or 4 hematologic toxicity was significantly higher in the DCF group (33 patients) than in the CF group (five patients; P < 0.001). Curative resection was accomplished in 79% of patients in the DCF group and 66% in the CF group (P = 0.305). There were no in-hospital deaths. The incidence of perioperative complications did not differ between the groups. A grade 2 or 3 histological response was attained in a significantly higher proportion of patients in the DCF group (63%) than in the CF group (5%; P < 0.001). Progression-free survival and overall survival were significantly higher in the DCF group (P = 0.013, hazard ratio 0.473; P = 0.001, hazard ratio 0.344). In conclusion, a grade 3 or 4 hematologic toxicity was common in the DCF group but was managed by supportive therapy. Histological response rate, progression-free survival, and overall survival were significantly higher in the DCF group compared with the CF group.

Quality of the interface of primary tooth dentin bonded with antibacterial fluoride-releasing adhesive.

OBJECTIVE: Limited information is available on the physical properties and ultrastructure of resin-dentin interfaces created in primary teeth. The interfacial quality of sound and caries-affected primary tooth dentin bonded with an antibacterial fluoride-releasing self-etch adhesive was examined in the present study. METHODS: Primary molars were bonded with Clearfil Protect Bond (Kuraray Medical). A nano-indentation tester was employed for creating indentations vertically across resin-dentin interfaces of the bonded sound and caries-affected primary dentin for determination of hardness (H) and Young's modulus (Y). Statistical analysis were performed using one-way and two-way ANOVA and Fisher's PLSD test at p<0.05. Similar resin-dentin interfaces were examined with SEM/EDX, and with TEM using ammoniacal silver nitrate tracer for nanoleakage. RESULTS: In both sound and caries-affected dentin, compared to the underlying dentin, significantly lower values were seen in the H and Y values of the interfacial dentin except for the H in caries-affected dentin. No significant difference between the interfacial dentin and the underlying dentin was observed in the H of caries-affected dentin and the Ca and P contents in both sound and caries-affected dentin. TEM revealed extensive interfacial nanoleakage in bonded sound dentin, while no silver deposit in bonded caries-affected dentin. However, silver deposits were observed in the porous caries-affected dentin beneath the interface. CONCLUSION: Within the limitation of this study, Clearfil Protect Bond cannot demonstrate evidence of remineralization, does not increase the hardness and elasticity of the interfacial dentin, and does not prevent nanoleakage along the resin-dentin interface.

Hardness, elasticity and ultrastructure of primary tooth dentin bonded with a self-reinforcing one-step self-etch adhesive.

OBJECTIVE: This study evaluated the interfacial quality of sound and caries-affected primary tooth dentin bonded with a self-reinforcing one-step self-etch adhesive. METHODS: Non-carious, sound dentin was prepared with water-cooled high-speed diamond burs. Caries-infected dentin was removed with water-cooled low-speed round steel burs and hand instrument. Dentin was bonded with Bond Force (Tokuyama Dental). A nano-indentation tester was employed for determination of hardness (H) and Young's modulus (Y) of resin-dentin interface. Similar resin-dentin interfaces were examined with a SEM/EDX, and with a TEM using ammoniacal silver nitrate tracer for nanoleakage. RESULTS: In the comparison of the H and Y values between the interfacial dentin and the underlying mineralized dentin, no significant difference was seen in caries-affected dentin, however, the values of the interfacial dentin were significantly lower in sound dentin. The H value of the interfacial dentin of sound dentin was significantly lower than that of caries-affected dentin with significantly higher Ca content. No significant difference was observed in the Y values of the interfacial dentin of the two substrates. For both sound and caries-affected dentin, TEM revealed silver deposits in the interfacial dentin and adhesive layer, and smear layer remained within the resin-dentin interface. However, Ca and P contents of the adhesive layer at 10mum above the dentin surface were the same as those present in the interfacial dentin. CONCLUSIONS: Both for sound teeth and caries teeth, Bond Force does not prevent the nanoleakage along the resin-dentin interface.

Clinical evaluation of polypropylene glycol-based caries detecting dyes for primary and permanent carious dentin.

OBJECTIVE: The aim of this study was to compare the clinical efficacy of new caries detecting dye Caries Check Blue (CCB) with Caries Check (CC) and Caries Detector (CD) using a laser fluorescence device (DIAGNOdent). METHOD: Primary and permanent teeth with dentin caries were stained with polypropylene glycol (MW=300) based new caries detecting dyes CCB, CC, or propylene glycol (MW=76) based CD. In the CCB and CC groups, stained dentin was completely removed. In the CD groups, pink-stained dentin was retained according to the manufacturers' instructions. Cavities before and after caries removal were measured with the DIAGNOdent. Data were analyzed using ANOVA and Fisher's PLSD multiple comparison test at alpha=0.05. Regression analyses were performed between DIAGNOdent readings and scores obtained from the clinical parameters. RESULTS: The DIAGNOdent readings after caries removal were: primary-CCB (13.2+/-10.4), primary-CC (14.3+/-16.7), primary-CD (9.0+/-5.2), permanent-CCB (22.7+/-13.4), permanent-CC (10.6+/-6.8) and permanent-CD (9.7+/-9.0). Significant differences were identified between the permanent-CCB and all other groups. Correlation coefficients between DIAGNOdent readings and clinical parameters were low. CONCLUSIONS: When dentin stained with Caries Check Blue or Caries Check was completely removed, the DIAGNOdent readings were higher than those recorded when palely-stained pink dentin was retained with the Caries Detector, with significant difference observed for the permanent-CCB group. Caries Check Blue may be used clinically to avoid excessive removal of caries-affected or sound dentin in permanent teeth but not in primary teeth.

A new operative technique for the resection of gastric tube cancer by means of lifting the anterior chest wall and videoscope-assisted surgery.

The prolonged survival of patients receiving surgery for esophageal cancer has led to an increased incidence of adenocarcinoma arising in the gastric tube used for reconstruction (gastric tube cancer). In patients with advanced gastric tube cancer, resection of the gastric tube should be considered, but currently available procedures are very invasive. In patients undergoing curative surgery for gastric tube cancer that has developed after reconstruction through the retrosternal route, the gastric tube is usually resected through a median sternotomy, followed by reconstruction with the colon. However, postoperative complications often occur and treatment outcomes remain poor. We developed a new surgical technique for gastric tube resection without performing a sternotomy in patients with gastric tube cancer who had previously undergone reconstruction through the retrosternal route. Our technique was used to treat two patients. Two Kirschner wires were passed subcutaneously through the anterior chest; the chest was lifted to extend the retrosternal space and secure an adequate surgical field. The stomach was separated from the surrounding tissue under videoscopic guidance. Total resection of the gastric tube was done. The retrosternal space was used to lift the jejunum. Roux-en-Y reconstruction was performed. Neither patient had suture line failure or surgical site infection. Their recovery was uneventful. Our surgical technique has several potential advantages including (i) reduced surgical stress; (ii) the ability to use the retrosternal space for reconstruction after gastric tube resection; and (iii) a reduced risk of serious infections such as osteomyelitis in patients with suture line failure. Our findings require confirmation by additional studies.

Minimum leakage rate (0.5%) of stapled esophagojejunostomy with sacrifice of a small part of the jejunum after total gastrectomy in 390 consecutive patients.

BACKGROUND: The development of new surgical instruments and devices has facilitated the performance of esophagojejunostomy after total gastrectomy. However, total prevention of dehiscence of anastomoses remains difficult. We introduced a new procedure for esophagojejunostomy using a circular stapler, requiring sacrifice of only a small part of the jejunum. METHODS: The study group comprised 390 consecutive patients who underwent reconstruction by Roux-en-Y esophagojejunostomy, performed with a circular stapler, sacrificing a small part of the jejunum after total gastrectomy. We assessed anastomotic leakage and anastomotic stenosis after surgery. RESULTS: Only 2 patients (0.5%) had leakage and 4 (1.0%) had anastomotic stenosis after reconstruction. All the patients were cured by conservative therapy. CONCLUSIONS: Esophagojejunostomy performed with a circular stapler after total gastrectomy, with sacrifice of only a small part of the jejunum, is a useful and easy procedure, with a leakage rate of 0.5%.

Hardness, elasticity, and ultrastructure of bonded sound and caries-affected primary tooth dentin.

Biomechanical properties of bonded dentin are important factors for resin restoration. We evaluated the hardness and elastic modulus of bonded sound and caries-affected primary tooth dentin using a one-step adhesive system, and observed the microstructure of the bonded interface. Six sound and six carious primary teeth were used. For sound teeth, flat occlusal dentin surfaces were prepared with a water-cooled high-speed diamond bur. For carious teeth, infected dentin was stained with a caries detector and removed with a water-cooled low-speed round steel bur and hand instruments. The prepared dentin was bonded with One-Up Bond F Plus (Tokuyama Dental Co., Tokyo, Japan). The resin-dentin interface and dentin beneath the interface were measured with a nano-indentation tester and observed with SEM and TEM. For both the carious and sound teeth, there was no significant difference between the hardness of the interfacial dentin and dentin 10-80 microm beneath the interface. However, the Young's modulus of the interfacial dentin was significantly lower than the dentin 40-80 microm (carious teeth) or 50-80 microm (sound teeth) beneath the interface. Both the hardness and Young's modulus of the interfacial dentin were not significantly different between the carious and sound teeth. Compared to the sound dentin, the hybrid layer on the caries-affected dentin was thicker and exhibited more complicated morphologic features. The thickness of the hybrid layers was generally less than 1 microm.

Evaluation of a new caries detecting dye for primary and permanent carious dentin.

OBJECTIVE: This study evaluated the clinical efficacy of a new caries detecting dye using a laser fluorescence device (DIAGNOdent). METHOD: Primary and permanent teeth with dentin caries were stained with Caries Check (CC), containing 1% acid red in polypropylene glycol (MW=300) or Caries Detector (CD), containing 1% acid red in propylene glycol (MW=76). Primary-CC, primary-CD, permanent-CC and permanent-CD groups were prepared. In the CC groups, stained dentin was completely removed. In the CD groups, pink-stained dentin was retained according to the manufacturers' instructions. Cavities before and after caries removal were measured with the DIAGNOdent. Data were analyzed using ANOVA and Fisher's PLSD multiple comparison test at alpha=0.05. Regression analyses were performed between DIAGNOdent readings and scores obtained from the clinical parameters. RESULTS: For all groups, there were no significant differences in the DIAGNOdent readings before treatment. The DIAGNOdent readings after caries removal were: primary-CC (16.0+/-17.6), primary-CD (9.6+/-5.2), permanent-CC (11.0+/-7.0) and permanent-CD (7.1+/-3.8). Significant differences were identified between the permanent-CC and primary-CD, and permanent-CC and permanent-CD subgroups but not for the primary subgroups. Correlation coefficients between DIAGNOdent readings and clinical parameters were low. CONCLUSIONS: When dentin stained with Caries Check was completely removed, the DIAGNOdent readings were higher than those recorded when palely-stained pink dentin was retained with the Caries Detector, with significant difference observed for the permanent teeth. Caries Check may be used clinically to avoid excessive removal of caries-affected or sound dentin in permanent teeth but not in primary teeth.

Hardness and elasticity of bonded carious and sound primary tooth dentin.

OBJECTIVE: Although biomechanical properties of bonded dentin are important for resin restorations, no information for primary teeth has been reported. This study evaluated the hardness and elastic modulus of bonded carious and sound primary tooth dentin using two adhesive systems. METHODS: Twelve sound primary molars and 17 carious primary molars were bonded with Clearfil SE Bond (SE: Kuraray) or Single Bond (SB: 3M) and the resin-dentin interface was measured with a nano-indentator and observed with a SEM. Data was statistically analyzed using ANOVA subsequent application of Fisher's PLSD at p<0.01. RESULTS: Compared to the dentin beneath the interface, the hardness of the interface dentin was not significantly different (carious-SE, sound-SE and sound-SB groups) or was significantly higher (carious-SB group). Young's modulus of the interface dentin was not significantly different (carious-SE, carious-SB and sound-SB groups) or was significantly lower (sound-SE group) than that for the dentin beneath the interface. In the comparison of the measurement on the interface dentin, the hardness showed no significant differences among all the groups. The Young's modulus of the sound-SE group was significantly lower than that of the carious-SE and sounds-SB groups, but no significant difference was observed between the carious-SB and other groups. CONCLUSIONS: Physical properties of bonded primary tooth dentin might differ from that of permanent tooth dentin.

Influence of Carisolv on resin adhesion for two different adhesive systems to sound human primary dentin and young permanent dentin.

OBJECTIVE: This study evaluated the influence of Carisolv (Medi Team) for resin adhesion to sound human primary and young permanent dentin. METHODS: The buccal surfaces of 28 primary molars and 64 premolars were used. Two adhesive systems and resin composites were used; SE: Clearfil SE (Kuraray) and Clearfil APX (Kuraray), and SB: Single Bond (3M) and Z250 (3M). Six groups were prepared. Groups 1-2 were primary dentin and Groups 3-6 were permanent dentin. Groups 1 and 3: SE was used. Groups 2 and 4: treated with Carisolv and then primed, SE was used. Group 5: SB was used. Group 6: treated with Carisolv and then etched, SB was used. The microstructural effects of primer or etchant, and Carisolv plus primer or etchant applied to dentin were evaluated by SEM. In addition, the microstructure of the resin-dentin interfaces of each group was studied using SEM. Shear bond strengths (SBS) were tested, and the failed surfaces were observed using SEM. Data was statistically analyzed using ANOVA with subsequent application of Fisher's PLSD at p<0.05. RESULTS: The mean SBS (unit: MPa) of Groups 1-6 were: 27.8, 19.2, 21.3, 21.7, 6.7 and 7.6. The SBS of Group 2 was significantly lower than that of Group 1. There was no significant difference of the SBS among Groups 1 and 4, 2, 3 and 4, and 5 and 6. In SE groups, the hybrid layer for primary dentin was thicker than that for permanent dentin. CONCLUSIONS: Carisolv treatment before priming significantly decreased the SBS to primary dentin in SE groups, but did not influence the SBS to permanent dentin in both SE and SB groups.

The nano-hardness and elastic modulus of sound deciduous canine dentin and young premolar dentin--preliminary study.

The purpose of this study was to compare the nano-hardness and elastic modulus among deciduous and permanent dentin, buccal and lingual sides, incisal, center and cervical areas, and outer, middle and inner layers. Three premolars and three deciduous canines were bucco lingually (BL) sectioned, and three deciduous canines were mesio-distally (MD) sectioned parallel to the long axis at the center of the tooth. Hardness (H), plastic hardness (PH) and Young's modulus (Y) were measured using a nano-indentation tester. The H, PH and Y values from the deciduous canine dentin were significantly lower than those from the premolar dentin at most sites. For deciduous canine dentin, the H and PH values of the MD sectioned dentin were significantly higher than those of the BL sectioned dentin in many layers of many areas. Generally deciduous canine dentin had H, PH and Y values that decreased from outer toward the inner layers and significant differences were obtained among the layers in many areas. For MD sectioned deciduous canine and BD sectioned premolar dentin, the H, PH and Y values of the cervical area were significantly lower than those of the incisal and center areas in many layers. It is possible that optimum bonding may require different treatments for deciduous and permanent dentin and perhaps also for different intratooth locations.

Tracheoesophageal fistula secondary to chemotherapy for malignant B-cell lymphoma of the thyroid: successful surgical treatment with jejunal interposition and mesenteric patch.

We report a case of tracheoesophageal fistula (TEF) secondary to chemotherapy for primary thyroid lymphoma. A 65-year-old man with a short history of a rapidly enlarging neck mass was diagnosed as having thyroid lymphoma of diffuse, large B-cell type. The TEF occurred during the first course of chemotherapy including cyclophosphamide, doxorubicin, vincristine and prednisolone. After placing a feeding gastrostomy without oral intake, eight cycles of chemotherapy were completed and complete remission was achieved. Although the cervical mass disappeared, TEF and esophageal stenosis persisted. Total thyroidectomy and resection of the stenotic cervical esophagus were carried out followed by interposition of the revascularized jejunum and its mesenteric patch to cover the TEF. This seems to be the first report of a TEF caused by chemotherapy for primary thyroid B-cell lymphoma. A variety of treatments for TEF including simple closure, tracheal resection, colonic bypass and muscle flap have been reported with low success rates. Our procedure using a jejunal mesenteric patch seems to be unique and may be a new treatment strategy for TEF.

The nano-hardness and elastic modulus of carious and sound primary canine dentin.

This study measured the nanohardness and elastic modulus- of carious and sound primary canine dentin and compared the values obtained under the lesion and in sound regions of incisal, center and cervical areas, and outer, middle and inner layers. Six extracted or exfoliated primary canines (three with dentin caries on both proximal surfaces and three sound teeth) were mesiodistally sectioned parallel to the long axis of the tooth and polished. The hardness (H), plastic hardness (PH) and Young's modulus (Y) were measured by a nano-indentation tester. Ten indentations at intervals of 10 microm on all regions, areas and layers were made using a load of 1 gf for one second. All indentations were observed using a microscope attached to the tester. All data were statistically analyzed using ANOVA and Scheffe's test at p < 0.05. For sound teeth, the H, PH and Y values of the inner layer were significantly lower than the outer and middle layers in all areas. The H, PH and Y values of the cervical area were significantly lower than the incisal area in almost all of the outer, middle and inner layers. For carious teeth, the H, PH and Y values of the inner layer were significantly lower than the outer and middle layers in the center area. For the center area, the H, PH and Y values under the lesion were significantly lower than sound teeth in the outer and middle layers. Dentin under the lesion, near the pulp and cervical areas showed significantly lower nanohardness and elasticity.

Somatic mutation of the Caspase-5 gene in human lung cancer.

Using cDNA array-based gene expression profiling, we previously found reduced expression of the Caspase-5 gene in highly metastatic subpopulations of a lung cancer cell line. The Caspase-5 gene contained poly(A) repeats in its coding region, an area that has been reported to be mutated in both endometrial and gastrointestinal tumors displaying evidence of microsatellite instability. In order to determine the contribution of Caspase-5 gene inactivation to lung cancer development and progression, the mutational status of the Caspase-5 poly(A) tract in 30 primary lung cancers with distant metastasis and 30 lung cancer cell lines was determined by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis and direct sequencing. Three somatic mutations of the Caspase-5 gene were found in two out of 30 lung cancer tissues, although no mutations were found in other genes that also contain small nucleotide repeats, such as hMSH3, hMSH6 and BAX. The results of the present study, combined with our prior cDNA array-based gene expression profiling data, suggest that Caspase-5 might be a suppressor gene of highly metastatic potential in lung cancer.

Laparoscopic intragastric stapled resection of gastric submucosal tumors located near the esophagogastric junction.

BACKGROUND: Laparoscopic resection cannot be applied easily to tumors located near the esophagogastric junction or the pyloric ring. We evaluated our laparoscopic intragastric surgical technique for gastric submucosal tumors located near the esophagogastric junction and the results of a clinical study. MATERIALS AND METHODS: We performed our technique in six patients: one man and five woman with a mean age of 61 years. Using the laparoscopic procedure, after inflation of the stomach, we inserted two or three balloon-type ports into the stomach through the abdominal wall. RESULTS: A stapled resection of gastric submucosal tumors using a laparoscopic linear stapler was performed successfully in all the patients. Without exception, stapled resections were successfully performed. The mean operation time was 168 min, and the blood loss was minimal There were no intra- or postoperative complications. The mean postoperative hospital stay was 9.8 days. The mean maximal diameter size of the resected specimens was 2.4 cm. Histopathologic diagnoses were gastrointestinal stromal tumors in five cases and enterogenous cyst in one. There were no recurrences during a mean follow-up period of 27 +/- 11.6 months. CONCLUSION: Although we need to evaluate the long-term outcomes, our procedure is considered technically feasible, safe, and useful for the resection of gastric submucosal tumors located near the esophagogastric junction.

Aberrations in the fragile histidine triad (FHIT) gene in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) seems to be closely associated with lung carcinogenesis. To identify the genetic characteristics of precancerous IPF lesions in the peripheral lung, we performed PCR-based microsatellite analysis with DNA extracted from microdissected tissues; fluorescent in situ hybridization (FISH) analysis of the fragile histidine triad (FHIT) gene and immunohistochemical analysis of Fhit protein expression in samples of metaplasias and bronchiolar epithelia obtained from patients with IPF. We used four microsatellite markers of the FHIT gene within or flanking the FHIT gene on chromosome 3p for loss of heterozygosity (LOH) analysis. LOH of the FHIT locus was frequently found among the lesions of metaplasias and bronchiolar epithelia in the patients with IPF [62 (52%) of 119 informative lesions]. Fifty-four (73%) of the 74 lesions of metaplasias and bronchiolar epithelia obtained from the IPF patients with lung carcinoma and 8 (17%) of the 46 samples obtained from the IPF patients without lung carcinoma showed LOH at the FHIT gene (P < 0.0001). We confirmed allelic loss in the metaplasias and bronchiolar epithelia of IPF by FISH analysis of the FHIT gene. Additionally, the level of Fhit protein expression in the metaplastic cells of IPF was frequently reduced. Our findings suggest that allelic loss of the FHIT gene may be involved in carcinogenesis in the peripheral lung of patients with IPF.

Ca(2+) elevation evoked by membrane depolarization regulates G protein cycle via RGS proteins in the heart.

Regulators of G protein signaling (RGS), which act as GTPase activators, are a family of cytosolic proteins emerging rapidly as an important means of controlling G protein-mediated cell signals. The importance of RGS action has been verified in vitro for various kinds of cell function. Their in situ modes of action in intact cells are, however, poorly understood. Here we show that an increase in intracellular Ca(2+) evoked by membrane depolarization controls the RGS action on G protein activation of muscarinic K(+) (K(G)) channel in the heart. Acetylcholine-induced K(G) current exhibits a slow time-dependent increase during hyperpolarizing voltage steps, referred to as "relaxation." This reflects the relief from the decrease in available K(G) channel number induced by cell depolarization. This phenomenon is abolished when an increase in intracellular Ca(2+) is prevented. It is also abolished when a calmodulin inhibitor or a mutant RGS4 is applied that can bind to calmodulin but that does not accelerate GTPase activity. Therefore, an increase in intracellular Ca(2+) and the resultant formation of Ca(2+)/calmodulin facilitate GTPase activity of RGS and thus decrease the available channel number on depolarization. These results indicate a novel and probably general pathway that Ca(2+)-dependent signaling regulates the G protein cycle via RGS proteins.

Successful en bloc resection of a large superficial gastric cancer by using sodium hyaluronate and electrocautery incision forceps.

BACKGROUND: The advisability of endoscopic mucosal resection (EMR) for treatment of large superficial gastric cancers has been challenged. For more reliable en bloc resection, a new method of EMR was developed that uses a viscous substance, sodium hyaluronate, and two newly designed devices. METHODS: A large superficial gastric cancer was treated with this new EMR technique. Sodium hyaluronate was injected into the submucosa and mucosal incisions were made with a needle-knife. The newly developed incision forceps and flat-ended transparent hood were used for submucosal incisions. RESULTS: The large cancer was successfully resected endoscopically as a single piece of mucosa 6 cm in diameter without complication. Histopathologic evaluation of the specimen confirmed that the resection was curative. CONCLUSIONS: EMR with sodium hyaluronate along with two new devices may be a reliable method for en bloc resection of large superficial gastric lesions.

Altered expression of several genes in highly metastatic subpopulations of a human pulmonary adenocarcinoma cell line.

Non-small cell lung cancer is associated with approximately 85% mortality due to its high metastatic potential. Therapeutic efforts have failed to produce a significant improvement in prognosis. In this situation, a better understanding of the key factors of metastasis may be useful for designing new molecular targets of therapy. In order to identify these factors, we compared the expression profiles of two subpopulations of an adenocarcinoma cell line with a high metastatic potential, PC9/f9 and PC9/f14, with the parent cell line, PC9, using a cDNA array. The expression of 15 genes was found to be significantly enhanced or reduced in the highly metastatic subpopulations. The expression of matrix metalloproteinase-2 (MMP-2), plasminogen activator inhibitor-1 (PAI-1) and interleukin-1 (IL-1 alpha) were upregulated in the highly metastatic subpopulations, while the expression of carcinoembryonic antigen (CEA), caspase-5, Fas ligand, Prk/FNK, cyclin E, cyclin B1, Ki-67, proliferating cell nuclear antigen (PCNA), Smad4, macrophage proinflammatory human chemokine-3 alpha (MIP-3 alpha)/LARC, Met and CD44 were downregulated. Data from the literature suggest that the altered expression of MMP-2, PAI-1, IL-1 alpha, CEA, caspase-5, Fas ligand, Prk/FNK and Smad4 promotes the highly metastatic phenotype. The differential expression of these genes was confirmed by Northern blot analysis, standard reverse transcription-polymerase chain reaction (RT-PCR) and real-time quantitative RT-PCR. This analysis in subpopulations of a lung cancer cell line indicated that the highly metastatic potential of lung cancer may be induced not by an alteration in the expression of a single gene, but by the accumulation of alterations in the expression of several genes involved in extracellular matrix (ECM) adhesion disruption, ECM degradation, escape from apoptosis, and resistance to transforming growth factor-beta(1) (TGF-beta(1)). Strategies for inhibiting metastasis of pulmonary adenocarcinoma should be designed accordingly.

Genomic structure of the human MAD2 gene and mutation analysis in human lung and breast cancers.

Some of the many human cancers that exhibit chromosomal instability also carry mutations in mitotic checkpoint genes and/or reveal reduced expression of some of those genes, such as hMAD2. To facilitate investigation of alterations of hMAD2, we determined its genomic structure and intronic primers designed to amplify the entire coding region. Since general impairment of the mitotic checkpoint is frequently reported in lung cancers, and reduced expression of hMAD2 has been reported in breast cancers as well, we searched for mutations throughout the coding sequence of this gene in the genomic DNA of 30 primary lung tumors, 30 lung-cancer cell lines and 48 primary breast cancers. Our approach, which involved polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis and direct sequencing, revealed nucleotide variants in only two of the 108 specimens. One was a cytosine-to-adenine substitution 3 bp upstream of exon 4 that occurred in one lung cancer cell line and one primary breast tumor, a change that did not alter transcriptional sequence. The other was an adenine-to-guanine substitution within exon 4, of the same lung cell line; this change already had been reported as a polymorphism. The results suggested that the hMAD2 gene is not commonly mutated in either lung nor breast cancers. Further studies should focus on other mechanisms that might account for reduced expression of the hMAD2 gene, and/or pursue analyses of other mitotic checkpoint genes for mutations in human cancer. Nevertheless, the genomic structure, the intronic primer sequences, and polymorphisms of the hMAD2 gene presented here will facilitate future studies to determine the full spectrum and frequency of the genetic events that can affect expression of the hMAD2 gene in human tumors.