Cloning of self-major histocompatibility complex antigen-specific suppressor cells from adult bone marrow.

We examined if suppressor cell clones may be established from adult bone marrow that contains a population of cells capable of specifically downregulating the immune response directed toward self-major histocompatibility complex (MHC) antigens. Freshly prepared adult C3H (H-2k) marrow cells were cultured in medium containing interleukin 2 (IL-2), IL-3, or a mixture of IL-2 and IL-3. After 7-10 d, cells grown in IL-3-containing medium were screened for their capacity to suppress cytotoxic T lymphocyte (CTL) generation against self-MHC antigens in allogeneic mixed lymphocyte cultures. Cells capable of suppressing anti-C3H CTL generation were cloned by limiting dilution. Several suppressor clones were established that exhibited strong suppression of anti-H-2k, anti-H-2Kk/Ik, and anti-H-2Dk CTL generation, but failed to suppress anti-H-2d and anti-H-2b responses. When tested in a skin allograft model, intravenous injections of these bone marrow-derived anti-self suppressor cells (2.5 x 10(7) cells) together with IL-3 induced prolongation of C3H skin allografts in anti-mouse lymphocyte serum-treated B6AF1 mice. Injection of IL-3 alone had no effect on allograft survival. Moreover, these cells failed to prolong B10.AKM skin allografts on B6AF1 recipients. Northern blot analysis showed that these cells express full-length transcripts of the T cell receptor (TCR) gamma gene, but not those of TCR alpha, beta, or delta genes. However, no rearrangement of gamma gene was observed by Southern blot analysis. Flow cytometric analysis revealed that bone marrow-derived suppressor cells are strongly positive for Thy-1 antigen but negative for CD3, CD4 (L3T4), and CD8 (Lyt-2) surface markers, and express only class I MHC antigens. Suppressor cells derived from adult bone marrow may play an important role in extrathymic induction of self-tolerance.

A single institutional non-randomized retrospective comparison between definitive chemoradiotherapy and radical surgery in 82 Japanese patients with resectable esophageal squamous cell carcinoma.

This retrospective study was conducted to compare the treatment results between radical surgery and definitive chemoradiotherapy for resectable squamous cell carcinoma of the esophagus. Between June 2000 and May 2005, 82 consecutive patients were selected for this study in which 33 were treated with chemoradiotherapy and 49 with surgery. The patients in the chemoradiotherapy (CRT) group received 2-4 cycles of 5-fluorouracil (1000 mg/m(2)/day, day 1-4, continuous) combined with cisplatin (75 mg/m(2), day 1, bolus) plus 50.4 Gy of radiation, while those in the surgery group were treated by an esophagectomy with radical node dissection. Eighteen surgical patients received postoperative chemotherapy. The baseline clinical TNM stage was similar between the two groups. With a median follow-up period of 36 months (range: 23-84 months) with 47 survivors (57%), the 3-year overall survival rates (P = 0.22) and disease-free survival rates (P = 0.16) were 48% and 44% in the chemoradiotherapy group versus 65% and 59% in the surgery group, and lacked statistical significance. This non-randomized study on patients with resectable squamous cell carcinoma of the esophagus showed that chemoradiotherapy could result in survival comparable with conventional surgery in spite of selection bias of patients. There is a trend toward improved survival with surgery versus definitive CRT.

A novel variant of human Grb7 is associated with invasive esophageal carcinoma.

The cDNAs of a putative growth factor-bound (Grb) 7 signal transduction molecule and Grb7V novel splice variant were isolated from an invasive human esophageal carcinoma. Although both Grb7 isoforms share homology with the Mig-10 cell migration gene, the Grb7V isoform lacks 88 base pairs in the C terminus; the resultant frame shift leads to substitution of an SH2 domain with a short hydrophobic sequence. The wild-type Grb7 protein, but not the Grb7V isoform, is rapidly tyrosyl phosphorylated in response to EGF stimulation in esophageal carcinoma cells. Analysis of human esophageal tumor tissues and regional lymph nodes with metastases revealed that Grb7V was expressed in 40% of Grb7-positive esophageal carcinomas. More importantly, Grb7V expression was enhanced after metastatic spread to lymph nodes as compared to the original tumor tissues. Finally, transfection of an antisense Grb7 RNA expression construct lowered endogenous Grb7 protein levels and suppressed the invasive phenotype exhibited by esophageal carcinoma cells. These findings suggest that Grb7 isoforms are involved in cell invasion and metastatic progression of human esophageal carcinomas.

Coexpression of Grb7 with epidermal growth factor receptor or Her2/erbB2 in human advanced esophageal carcinoma.

Growth factor receptors transmit intracellular signals that may be important in carcinogenesis. The Grb7 protein was recently identified as a substrate of the epidermal growth factor receptor and related Her2/ erbB2 receptor-linked tyrosine kinase activity. The Grb7 gene has been found to be coamplified with Her2/erbB2 in breast carcinomas. In this study, Grb7 expression was studied in 32 human esophageal cancers. A human Grb7 cDNA encoding for N-terminal amino acids was isolated and found to be 90% homologous to the murine counterpart. Although there was no amplification of the Grb7 gene in esophageal cancers, Grb7 mRNA was found to be overexpressed in 14 cancers (43.8%) but not in adjacent normal esophageal mucosa. It is noteworthy that coexpression of Grb7 with epidermal growth factor receptor or Her2/erbB2 was detected in 10 esophageal carcinomas (31.3%) and was significantly related to extramucosal tumor invasion (P = 0.02), whereas such a relationship was not shown by each sole expression. These findings suggest a possible relationship of Grb7 signaling in association with expression of tyrosine kinase receptors in aggressive human esophageal cancer.

Expression of a Mr 32,000 laminin-binding protein messenger RNA in human colon carcinoma correlates with disease progression.

Cell surface receptors for laminin may play an important role in tumor migration and metastasis. To evaluate laminin receptor/laminin-binding protein expression in human colon carcinoma, surgical specimens of primary colon cancers and liver metastases were examined by blot hybridization of total RNA with a complementary DNA clone which encodes a Mr 32,000 human laminin-binding protein. The mRNA level of the laminin-binding protein was higher in primary colon carcinoma than in adjacent normal colonic epithelium in 20 of 21 cases. In all 6 cases of colon cancer liver metastases, the laminin-binding protein mRNA level was more than 3-fold greater in tumor than in adjacent normal liver tissue. The tumor/normal ratio of this laminin-binding protein mRNA expression in primary colon cancer has significant correlation with Dukes' classification (P less than 0.001). Our results suggest that mRNA expression of the laminin-binding protein may be a marker of human colorectal cancer progression and biological aggressiveness.

Expression of human alpha-actinin in human hepatocellular carcinoma.

Little is known regarding gene expression during hepatocyte transformation. We have isolated an alpha-actinin complementary DNA from a human hepatocellular carcinoma library. This partial 2.4-kilobase complementary DNA has high homology with human placental and chicken nonmuscle alpha-actinins; our isolate contains the entire 3' noncoding region and it is within these sequences where the major differences between the vertebrate alpha-actinin complementary DNAs arise. Northern analysis revealed a 3.5-kilobase transcript in nonmuscle and a smaller 3.0-kilobase species in muscle tissue. Levels of alpha-actinin expression were low in normal liver and we investigated its expression during both hepatocyte proliferation and transformation. We found no increase during rat hepatocyte regeneration up to 24 h following two-thirds hepatectomy. However, high levels of alpha-actinin transcripts were observed in human hepatocellular carcinoma compared to noninvolved adjacent liver. We conclude that the alpha-actinin gene is highly expressed when hepatocytes have assumed the malignant phenotype.

Increased expression of human ribosomal phosphoprotein P0 messenger RNA in hepatocellular carcinoma and colon carcinoma.

To search for differentially expressed gene products in selected cancers of endodermal origin, cDNA libraries derived from mRNA in human hepatocellular carcinoma and adjacent grossly normal tissue were generated. From these parent libraries, subtracted cDNA libraries of tumor minus normal and normal minus tumor tissues were constructed. After screening these subtracted libraries by +/- hybridization, a cDNA clone that is overexpressed in hepatocellular carcinoma and encodes the human acidic ribosomal phosphoprotein P0 (P0) was identified. We then evaluated the expression of this phosphoprotein P0 in human colon carcinoma samples. Surgical specimens of primary tumors and liver metastases were examined by Northern hybridization of total RNA with one of 2 32P-labeled P0 probes. The mRNA level of the P0 was greater in primary colon carcinoma than in paired adjacent normal colonic epithelium in 36 of 38 cases; the mean tumor/normal ratio was 2.7 (range, up to 13). The tumor/normal ratio, when plotted against the Dukes' stage of disease, gave evidence for increasing P0 expression with increasing stage of colon carcinoma (P = 0.02). In all 8 cases of paired colon carcinoma metastatic to liver and 2 cases of paired primary hepatocellular carcinoma, the P0 mRNA level was greater in tumor than in adjacent normal liver tissue. The mean tumor/normal ratio was 4.0 (range, up to 11) for the colon cancers metastatic to liver and 4.2 for the primary hepatocellular carcinoma samples. These findings support a common increased expression of selected gene products in different tumors of endodermal origin and suggest that increased P0 expression, in line with certain other ribosomal proteins, may be associated with human colorectal cancer progression and biological aggressiveness.

Ubiquitin-ribosomal protein S27a gene overexpressed in human colorectal carcinoma is an early growth response gene.

One of the extension proteins on the carboxy terminus of ubiquitin was reported as the ribosomal protein S27a. We have cloned a gene which encodes this ubiquitin hybrid protein from a complementary DNA library of a human colon carcinoma cell line. Northern blot analysis of surgical specimens from colon cancer patients showed that these messenger RNA levels were higher in tumor tissue than in adjacent normal mucosa. Furthermore, to investigate the role of this novel ubiquitin hybrid gene in cellular growth control, the responsiveness of this gene to serum growth factors was examined. Within 30 min after serum or 12-O-tetradecanoylphorbol-13-acetate stimulation, its messenger RNA expression in rat fibroblast cells (Rat 1) was increased. Nuclear runoff transcription studies showed that the kinetics of induction of this gene is almost identical to that of protooncogene c-jun or c-fos, the known early growth response genes. Thus, this ubiquitin hybrid gene appears to be a novel early growth response gene overexpressed in human colon cancer and warrants further studies in the pathogenesis of colorectal carcinoma.

Deletion of the carboxyl-terminal exons of K-sam/FGFR2 by short homology-mediated recombination, generating preferential expression of specific messenger RNAs.

The K-sam gene was first identified as an amplified gene from human gastric cancer cell line KATOIII, and its product is identical to fibroblast growth factor receptor 2. The K-sam gene is located on human chromosome 10q26 and is preferentially amplified in the poorly differentiated types, especially in the scirrhous type, of gastric cancers. During the course of studies on the structural characterization of the amplification units, we found that the carboxyl-terminal exons of K-sam were deleted in three of four of the scirrhous type of gastric cancer cell lines. These deletions generate preferential expression of mRNAs encoding K-sam proteins lacking the carboxyl-terminal region containing the tyrosine residues at positions 780, 784, and 813. The carboxyl-terminal region has been reported to have a sequence required for the inhibition of NIH3T3 transformation, indicating that cells with amplification of the truncated K-sam gene have a growth advantage during the carcinogenic process for the scirrhous type of gastric cancers. This is the first report showing the deletion of the carboxyl-terminal exons of the receptor-type of the protein tyrosine kinase gene. Sequence analysis of the DNA sequences surrounding the deletion junctions shows the presence of unique sequences and indicates the involvement of short homology-mediated recombination in the generation of these deletions.

Increased expression of ornithine decarboxylase messenger RNA in human esophageal carcinoma.

Ornithine decarboxylase (ODC) is a key enzyme in the biosynthesis of polyamines, which are essential for cell proliferation. The purpose of this study was to evaluate ODC expression in human esophageal cancer at the mRNA level. Sixty-four pairs of primary esophageal cancers and normal esophageal epithelia were examined by reverse transcription-PCR for ODC mRNA expression. The ODC mRNA levels were higher in primary esophageal carcinoma than in adjacent normal esophageal epithelium in 58 (90.6%) of 64 cases. The tumor:normal (T:N) ratio of ODC mRNA expression in esophageal specimens has a significant correlation with tumor-node-metastasis staging (P = 0.043), lymph node metastasis (P = 0.039), vascular vessel invasion (P = 0.035), and histology (P = 0.034) of the tumor. In well- and moderately differentiated squamous cell carcinoma, the patients with a higher T:N ratio showed a significantly poorer prognosis (P = 0.027), and multivariate analysis also confirmed that the T:N ratio has a significant correlation with poor prognosis (P = 0.043). The steady-state of ODC mRNA overexpression in esophageal carcinoma implies that the ODC gene may play an important role in tumorgenesis in squamous epithelium. Furthermore, ODC mRNA expression may be used as a prognostic marker, especially for well- and moderately differentiated squamous cell carcinoma.

Promoters of epithelialization induce expression of vascular endothelial growth factor in human gastric epithelial cells in primary culture.

Both epithelialization and angiogenesis are indispensable processes in gastric ulcer healing. Coordination between these processes has not been well studied. In the present study, we have established a new primary culture system of human gastric epithelial cells and investigated the effect of epithelialization stimulants on a specific angiogenic factor, vascular endothelial growth characterized as epithelial cells. Both epithelialization stimulants, hepatocyte growth factor (HGF) and epidermal growth factor (EGF), significantly stimulated vascular EGF expression in gastric epithelial cells. HGF and EGF receptors were expressed by the cells, suggesting that regulation may be mediated through specific receptors.

Correlation of telomere lengths in normal and cancers tissue in the large bowel.

The hypothesis that telomeres in colorectal cancer cells exhibit age-related shortening, as in normal cells of the colorectal epithelium, was tested with samples of non-cancerous mucosa and cancer tissue from 124 patients (aged 29-97 years). Shortening with aging could be demonstrated for both normal and cancer tissues; regression analysis showed rates for length reduction of 44 and 50 base pair/year, respectively. Straight, essentially parallel, lines were obtained for the two cases, normal tissue values being about 2 kilobase pairs (kbp) higher, with a significant correlation between data at the individual patient level.

Overexpression of MMP-9 correlates with growth of small hepatocellular carcinoma.

Matrix metalloproteinases (MMP) play an important role in tumor cell invasion and metastasis. We examined the expression of MMPs in hepatocellular carcinoma (HCC) to determine whether they may help indicate the progression of HCC and patient outcome. The expression of MMP-1, -2, -7, -9, and MT1-MMP were determined in 37 pairs of HCC and adjacent non-tumor tissue specimens by semiquantitative reverse transcription polymerase chain reaction (RT-PCR). The tumor to non-tumor (T/N) ratios for mRNA expression of each MMP were compared with the clinicopathological findings and two-year disease-free survival rates. Immunohistochemical analysis for MMP-1 and -9 was performed in 30 specimens. MMP-1 and -9 expression was significantly higher in tumor tissue than in non-tumor tissue (p=0.001 and 0.0078, respectively). By contrast, the expression of MMP-2 and -7 was significantly lower in tumor tissue than in non-tumor tissue (p=0. 003 and 0.03, respectively). A higher MMP-9 T/N ratio was significantly associated with small HCCs (/=20 ng/ml) (p=0.013 and 0.028, respectively). Immunohistochemical analysis suggested that well differentiated HCC showed stronger immunoreactivity for MMP-9 than moderately differentiated HCC. MMP T/N ratio was not significantly associated with the disease-free survival rates. Overexpression of MMP-9 mRNA (T/N ratio >/=2) may be associated with the progression of small HCC (

Significance of alpha-1-antitrypsin mRNA expression in esophageal carcinoma.

Alpha-1-antitrypsin (alpha 1AT) is present not only in the normal gastrointestine, but in malignant gastrointestinal tissue as well. We studied the expression of alpha 1AT mRNA in 30 cases of esophageal carcinoma using a Northern blot analysis. In addition, we also examined the expression of matrix metalloproteinase 7 (MMP7) mRNA in the latest 15 cases by reverse transcriptase-polymerase chain reaction (RT-PCR) method to clarify the relationship between the alpha 1AT and MMP7. In 25 of the 30, the expression of alpha 1AT mRNA in esophageal carcinomatous tissue (T) was lower than that in the corresponding normal tissue (N) of the esophagus. The T/N ratio of alpha 1AT mRNA expression showed a significant inverse correlation with the depth of tumor invasion, lymph node metastasis and stage of disease (p=0.042, p=0.0001 and p=0.002, respectively). The T/N ratio of alpha 1AT bad a converse correlation with that of MMP7 (p=0.034). The current study thus suggested that i) the lower expression of T/N expression may be a marker for the biological aggressiveness of esophageal carcinoma, and ii) the reduction of alpha 1AT may be partially correlated with the overexpression of MMP7 in tumor tissue.

High frequency of the expression of the MAGE gene family in human esophageal carcinoma.

The human gene MAGE encodes tumor specific peptide antigens and consists of at least 12 families. Some antigens coded by the MAGE genes may be potentially useful for cancer specific immunotherapy. There is, however, so far little information on the expression of these gene families in human esophageal carcinomas. We investigated the expression of MAGE-1, -2, -3, -4, -6, -8, -9, -10, -11, and -12 genes in 24 human esophageal carcinoma cell lines, and in 50 pairs of tumor and corresponding normal tissue specimens from the human esophagus by means of reverse transcription polymerase chain reaction. The expression rate varied from 13% of MAGE-6 and 8 to 79% of MAGE-4 in the esophageal carcinoma cell lines, and from 6% of MAGE-6 to 62% of MAGE-4 in clinical tumor samples. The most frequently and the least expressed gene were the MAGE-4 and MAGE-6 genes, respectively, in both the cell lines and the clinical samples. Forty-seven of the 50 clinical tumors expressed at least one MAGE gene. No significant clinicopathologic difference between the tumor cases was observed, regardless of the presence or absence of MAGE gene expression. The findings of this study thus demonstrated that the MAGE gene family is frequently expressed in clinical samples as well as in the cell lines of human esophageal carcinomas. Therefore, to identify the MAGE gene family may be useful, not only for esophageal tumor specific immunotherapy but for molecular diagnostic usage as well.

alpha6 integrin expression in esophageal carcinoma.

One of the genes that the authors have isolated by a differential display of hepatocellular carcinoma compared to adjacent liver is the alpha6 integrin. alpha6 integrin is the major adhesion receptor for laminin and is suggested to be involved in tumor cell invasion and metastasis. To our knowledge, however, there are no reports on alpha6 integrin expression in esophageal carcinoma. We thus conducted a study to determine its clinicopathologic significance in human esophageal carcinoma. The tumor/normal (T/N) ratio of alpha6 integrin expression was calculated by Northern hybridization in 45 cases of esophageal carcinoma. In selected cases the expression of the alpha6 integrin variants A and B was also investigated by reverse transcriptase-polymerase chain reaction, and immunostaining for alpha6 integrin was performed. The expression levels of alpha6 integrin mRNA in the tumor tissue were greater than those of the corresponding normal tissue in 39 of 45 cases (87%). The overexpression of alpha6 integrin was also recognized by immunostaining. Fifteen cases with a high T/N ratio demonstrated a deeper invasion into the esophageal wall than the 30 cases with a low T/N ratio. Although there was no significant difference, the 15 cases with a high T/N ratio had a tendency for a worse prognosis. The ratio of the two variants (alpha6A/alpha6B) did not show any relationship to survival. The findings imply that alpha6 integrin is overexpressed in human esophageal carcinomas and that alpha6 integrin may play an important role in esophageal tumor invasion.

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.

Primary undifferentiated small cell carcinoma of the esophagus.

We histologically examined undifferentiated small cell carcinoma of the esophagus from 21 patients and used immunohistochemical methods for detection of chromogranin A and p53, bcl-2, and Rb oncoproteins. Nine (43%) of the 21 carcinomas consisted solely of undifferentiated cells, but heterogeneous components of in situ or invasive squamous cell carcinoma or mucoepidermoid carcinoma were observed in the other 12 (57%) tumors. Squamous cell carcinoma in situ was observed in the mucosa adjacent to the main tumor in 7 (50%) of the 14 resected esophageal specimens. An admixture of invasive squamous cell carcinoma and undifferentiated carcinoma was observed in 4 (19%) of the 21 tumors, and mucoepidermoid carcinoma was noted in one case. Chromogranin A staining yielded a positive reaction in two (10%) undifferentiated components but was negative in all heterogeneous components. Multiple sites of p53 immunopositivity were seen in the undifferentiated component of 17 (81%) of the 21 tumors, as well as in the in situ or invasive squamous cell carcinoma or mucoepidermoid carcinoma components of 9 (75%) of 12 tumors. Seven (33%) of the 21 tumors showed positive bcl-2 immunoreactivity in the small cell component, but all of the heterogeneous components were negative. Rb protein immunoreactivity was observed in the small cell component of one (5%) case and in 9 (75%) of the 12 heterogeneous components. Six (86%) of the seven in situ squamous cell carcinoma components were positive for Rb protein. Eighteen (86%) of the 21 patients died within 24 months of diagnosis. Two patients (10%) who survived for more than 24 months had received chemotherapy.

Telomere shortening with aging in human liver.

Progressive telomere shortening with aging was studied in the normal liver tissue of 94 human subjects aged between 0 and 101 years old to determine the rate of telomere loss in 1 year. Telomere length demonstrated accelerated shortening with reduction of 55 base pairs (bp) per year. The mean telomere length in five neonates was 12.9 +/- 2.6 kilobase pairs (kbp), and that in one centenarian was 8.3 kbp. Mean telomere lengths by age group were 13.2 +/- 2.0 kbp (< or = 8 years; 10 subjects), 7.8 +/- 1.9 kbp (40-79 years; 29 subjects), and 7.5 +/- 2.0 kbp (> or = 80 years; 53 subjects), with reduction thus appearing to show slowing on the attainment of middle age. The difference of mean telomere lengths for two groups with or without advanced malignancies of other than liver origin was not significant in the older two groups. Despite the slow turnover of liver tissue, the overall reduction rate of telomere length decrease in 1 year was almost the same as that of digestive tract mucosa, with its very rapid renewal.

Ubiquitin hybrid protein gene expression during human colon cancer progression.

Ubiquitin is involved in cell-cycle control and DNA replication through a specific proteolytic pathway. Our previous studies demonstrated selected higher expression of a gene encoding ubiquitin-ribosomal protein S27a in poorly differentiated colon carcinoma cell lines. In this study, we evaluated this ubiquitin hybrid protein gene expression in surgical specimens of colon cancers. Northern blot analysis showed that ubiquitin hybrid protein messenger RNA was overexpressed in primary colon cancers compared with adjacent normal colon mucosae in 17 of 20 patients. Dot blot analysis of RNA of 27 tumor samples revealed significantly greater expression in higher Dukes' stage primary colon tumors and liver metastases. These data imply that protein translation machinery is highly activated during progression and metastasis of colon tumors, and that ubiquitin hybrid protein may be useful as a marker of biological aggressiveness.