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Cancer Res

Genetic alterations in gastrinomas and nonfunctioning pancreatic neuroendocrine tumors: an analysis of p16/MTS1 tumor suppressor gene inactivation.

Muscarella P. Melvin WS. Fisher WE. Foor J. Ellison EC. Herman JG. Schirmer WJ. Hitchcock CL. DeYoung BR. Weghorst CM.
Division of General Surgery, College of Medicine and Public Health, The Ohio State University, Columbus 43210-1240, USA.
Neoplasms of the endocrine pancreas are extremely rare, and molecular mechanisms influencing their development are poorly understood. Nevertheless, gastrinomas have become a paradigm for the study of hormonally active tumors. In the present study, 12 gastrinoma and nonfunctioning pancreatic neuroendocrine tumor specimens were evaluated for genetic alterations of the p16/MTS1 tumor suppressor gene. DNA extracted from microdissected portions of paraffin-embedded tumor sections were examined for mutations and homozygous deletions using "Cold" single-strand conformation polymorphism and semiquantitative PCR-based analyses, respectively. Samples were also analyzed for the presence of 5' CpG island hypermethylation using methylation-specific PCR. The p16/MTS1 gene was found to be homozygously deleted in 41.7% of tumors and methylated in 58.3%, but no mutations were identified by single-strand conformation polymorphism analyses. Overall, 91.7% of the specimens demonstrated inactivating alterations in p16/MTS1. These data suggest that transcriptional silencing of p16/MTS1 is a frequent event in these rare and poorly understood tumors.

Localization and expression of p27KIP1 in multistage colorectal carcinogenesis.

Ciaparrone M. Yamamoto H. Yao Y. Sgambato A. Cattoretti G. Tomita N. Monden T. Rotterdam H. Weinstein IB.
Centro di Ricerche Oncologicalhe Giovanni XXXIII Universita Cattolica del Sacro Cuore, Rome, Italy.
The cyclin-dependent kinase inhibitor p27Kip1 can inhibit the G1 to S transition of the cell cycle and is a putative tumor suppressor. However, our laboratory found that a variety of human cancer cell lines express relatively high levels of this protein and that this is often associated with increased expression of cyclin D1 or cyclin E. Therefore, in the present study we analyzed by immunohistochemistry the expression of p27Kip1 in a series of human tissue samples representing various stages of colon carcinogenesis, using 20 samples of normal colon mucosa, 20 hyperplastic polyps, 19 samples of adenomatous polyps, and 40 samples of various types of colorectal carcinomas. Parallel immunostaining was done for cyclin D1 and also for Ki67 to evaluate cell proliferation. An additional 17 human colon carcinoma samples, together with paired adjacent normal mucosa samples, were analyzed for levels of expression of the p27Kip1 protein by Western blot analysis, and 7 of these pairs of samples were examined by Northern blot analysis for levels of p27Kip1 mRNA. We did not find a positive or negative correlation between p27Kip1 expression and cell proliferation in the normal mucosa and tumor samples. There was, however, an inverse correlation between p27Kip1 and Ki67 expression in the lymphoid follicles present in the colonic mucosa. There was no evidence for a consistent increase or decrease in p27Kip1 expression in the mucosal cells during colon carcinogenesis, because the mean values for percentage p27Kip1-positive cells were similar in the normal mucosa, adenomatous polyps, and carcinoma samples. This is in contrast to Ki67 and cyclin D1 expression, which did show significant increases in mean values with tumor development. A subset (35%) of the carcinomas displayed diffuse cytoplasmic staining, in addition to nuclear staining, for p27Kip1, and in these cases the percentage of cells that were positive for p27Kip1 was higher than in cases that had only nuclear staining. There was a significant correlation between p27Kip1 expression and tumor grade; ie., well and moderately differentiated carcinomas had high p27Kip1 expression, whereas poorly differentiated carcinomas had lower expression. The Western blot analysis data on p27Kip1 expression confirmed this correlation. Comparisons of Northern and Western blots did not show a correlation between the level of p27Kip1 mRNA and the corresponding protein, a finding consistent with evidence that the p27Kip1 protein is regulated mainly via a posttranscriptional mechanism. The immunostaining studies revealed a significant correlation between high p27Kip1 protein expression and high cyclin D1 expression in the adenomatous polyps and in the subset of carcinomas that had only nuclear p27Kip1 expression. This may reflect the existence of a homeostatic feedback mechanism that is lost in the high-grade carcinomas that express low levels of p27Kip1.

DNA adducts in human pancreatic tissues and their potential role in carcinogenesis.

Wang M. Abbruzzese JL. Friess H. Hittelman WN. Evans DB. Abbruzzese MC. Chiao P. Li D.
Department of Clinical Investigation, The University of Texas M.D. Anderson Cancer Center, Houston 77030, USA.
Pancreas cancer is the fourth and fifth leading cause of cancer death for men and women, respectively, in the United States. Although the etiology of this cancer is poorly understood, smoking and dietary fat have been implicated by epidemiological studies. To test the hypothesis that DNA damage derived from carcinogen exposure and diet is involved in pancreatic carcinogenesis, aromatic and lipid peroxidation-related DNA adducts in 13 normal tissues adjacent to tumor and 20 tumors from pancreatic cancer patients were analyzed by 32P-postlabeling. Normal pancreatic tissues from 5 nonpancreatic cancer patients and 19 healthy organ donors served as controls. To correlate the DNA adduct level with patients' characteristics, information on age, sex, body mass index, and smoking status of pancreatic cancer patients were collected from medical records. A significantly higher level of total DNA adducts was detected in pancreatic cancer patients as compared with controls. The mean level of adducts/10(8) nucleotides in adjacent normal pancreatic tissues from pancreatic cancer patients (A tissues) was 102 +/- 21 compared with 39 +/- 6 and 13 +/- 1 in pancreatic tumor tissues (T tissues) and normal pancreatic tissues from controls (C tissues), respectively. Among the adducts observed, one single aromatic adduct (spot 1) was present in 100, 90, and 0% of the A, T, and C tissues, respectively. Two novel clusters of adducts (spots 2 and 3) were observed in 11 of 13, 12 of 20, and 2 of 24 of A, T, and C tissues, respectively, and the presence of these adducts was positively correlated with smoking status. In addition, the previously defined smoking-related diagonal radioactive zone was detected in three A samples only, although 50% (10 of 20) of the patients with pancreatic cancers in this study were ever smokers. Putative lipid peroxidation-related adducts were detected in all samples examined and were significantly higher in A than in T and C samples. Multiple regression analyses showed that body mass index was positively correlated to the levels of spot 1 and the lipid peroxidation-related adducts in A tissues and the total aromatic adducts in tumors. Smoking was also positively correlated to the level of total adducts. These observations are consistent with previous epidemiological findings and support the hypothesis that DNA damage related to carcinogen exposure and lipid peroxidation is involved in human pancreatic carcinogenesis.

Novel mutations in the p16/CDKN2A binding region of the cyclin-dependent kinase-4 gene.

Tsao H. Benoit E. Sober AJ. Thiele C. Haluska FG.
Department of Dermatology, Massachusetts General Hospital and Dana-Farber/Partner Cancer Care, Boston 02114, USA.
Mutations in genes that lie in the retinoblastoma pathway have been implicated in the pathogenesis of many tumor types. Two critical components that determine progression from G1 to S include p16/CDKN2A and CDK4. Alterations in p16/CDKN2A have been well documented in multiple cancers, including melanoma. However, changes in CDK4 are apparently more rare. Only two alterations, both at codon 24, have been identified in CDK4: an activating arginine-to-cysteine transition and a germ-line arginine-to-histidine substitution in one French kindred. In a survey of 20 neuroblastomas, 17 uncultured metastatic melanomas, 33 uncultured primary uveal melanomas, 8 colon cancer cell lines, and 20 primary colon cancer samples, we found no evidence of mutations in exon 2 of CDK4. From our cell lines derived from metastatic melanomas, we detected two alterations in the functionally critical exon 2 of CDK4: a lysine-to-glutamine transition at codon 22 and the arginine-to-histidine mutation at codon 24. These findings document several novel changes in the p16-binding region of CDK4.

Cross-resistance to methotrexate and metals in human cisplatin-resistant cell lines results from a pleiotropic defect in accumulation of these compounds associated with reduced plasma membrane binding proteins.

Shen D. Pastan I. Gottesman MM.
Laboratory of Cell Biology, National Cancer Institute, NIH, Bethesda, Maryland 20892-4255, USA.
Cross-resistance to a wide array of toxic chemicals is a common phenomenon in cisplatin-resistant cell lines. In this study, two independently isolated cisplatin-resistant cell lines derived from a human hepatoma and a cervical adenocarcinoma were shown to be cross-resistant to methotrexate (MTX) and several metal salts, such as sodium arsenite, sodium arsenate, antimony potassium tartrate, and cadmium chloride. A pleiotropic defect resulting in reduced accumulation of cisplatin, 3[H]MTX, 73As3+, and 73As5+ was found in both cisplatin-resistant cell lines. Analysis by immunoblot, indirect immunofluorescence, and Northern hybridization showed dramatically reduced expression of the folate binding protein that mediates MTX uptake in both human cisplatin-resistant cell lines. By photoaffinity labeling with UV irradiation, specific binding proteins of Mr 230,000 and Mr 48,000 for 73As3+ and Mr 190,000 for 73As5+ were found in enriched plasma membrane of both human cisplatin-sensitive parental cell lines. Expression of these specific binding proteins was decreased in cells selected for cisplatin resistance. A protein band at Mr 36,000 that binds to 73As3+ was overexpressed in both human cisplatin-resistant cell lines. The finding of loss of distinct binding proteins for MTX, arsenate, and arsenite in association with decreased accumulation of these agents in cisplatin-resistant cells suggests a pleiotropic, possibly regulatory, alteration in these cells.

Modulation of apoptosis and Bcl-2 expression by prostaglandin E2 in human colon cancer cells.

Sheng H. Shao J. Morrow JD. Beauchamp RD. DuBois RN.
Department of Surgery, Vanderbilt University Medical Center, Nashville, Tennessee 37232-2279, USA.
Previously, we have shown that forced expression of prostaglandin endoperoxide synthase-2 [also called cyclooxygenase (COX) 2] leads to inhibition of programmed cell death in intestinal epithelial cells. More recently, we have demonstrated that growth of human colonic cancer xenografts is inhibited by treatment with a highly selective COX-2 inhibitor in tumors that express COX-2 (HCA-7) but not in those that lack COX-2 expression (HCT-116). To explore the biochemical mechanisms involved in these effects, we have evaluated the role of COX-2-derived eicosanoid products on programmed cell death in human colon cancer cells. Here we report that PGE2 treatment of human colon cancer cells leads to increased clonogenicity of HCA-7, but not HCT-116 cells. Treatment with a highly selective COX-2 inhibitor (SC-58125) decreases colony formation in monolayer culture and this growth inhibition was reversed by treatment with PGE2. Additionally, PGE2 inhibits programmed cell death caused by SC-58125 and induces Bcl-2 expression, but did not affect Bcl-x or Bax expression in human colon cancer (HCA-7) cells. Therefore, decreased cell death caused by PGE2 would enhance the tumorigenic potential of intestinal epithelial cells. Thus, these results may help to explain a component of the mechanism by which COX inhibitors prevent colorectal cancer in humans.

Consistent genetic alterations in xenografts of proximal stomach and gastro-esophageal junction adenocarcinomas.

El-Rifai W. Harper JC. Cummings OW. Hyytinen ER. Frierson HF Jr. Knuutila S. Powell SM.
Department of Medical Genetics, University of Helsinki, Finland.
The genetic alterations underlying the development of gastric and gastro-esophageal carcinoma remain largely undefined. DNA copy number changes were determined by comparative genomic hybridization in eight xenografts of proximal gastric and gastro-esophageal junction adenocarcinomas of the intestinal type. All tumors exhibited DNA copy number changes, with a total of 139 changes detected (range, 11-24 per tumor; mean = 17), indicating numerous and widespread alterations within these cancers. Gains (65%) in DNA copy number were more frequent than losses (35%). Our most striking finding was gain (all eight cases) or high-level amplification (four cases) in 20q, with a minimal common overlapping region at 20q13. Other frequent gains were observed at 6p, 7q, and 17q (six cases each) and at 1q, 2q, and 8q (five cases each). Frequent losses were observed at 4q and 5q (six cases each) and at 9p (five cases). No differences in DNA copy number changes were seen in tumors arising from the gastro-esophageal junction compared to those of the proximal stomach. The presence of common and consistent DNA copy number changes in these tumors implicate a number of chromosomal regions that may harbor important genes that are involved in tumorigenesis of the proximal stomach and gastro-esophageal junction.

Frequent nitric oxide synthase-2 expression in human colon adenomas: implication for tumor angiogenesis and colon cancer progression.

Ambs S. Merriam WG. Bennett WP. Felley-Bosco E. Ogunfusika MO. Oser SM. Klein S. Shields PG. Billiar TR. Harris CC.
Laboratory of Human Carcinogenesis, National Cancer Institute, NIH, Bethesda, Maryland 20892-4255, USA.
An increased expression of nitric oxide synthase (NOS) has been observed in human colon carcinoma cell lines as well as in human gynecological, breast, and central nervous system tumors. This observation suggests a pathobiological role of tumor-associated NO production. Hence, we investigated NOS expression in human colon cancer in respect to tumor staging, NOS-expressing cell type(s), nitrotyrosine formation, inflammation, and vascular endothelial growth factor expression. Ca2+-dependent NOS activity was found in normal colon and in tumors but was significantly decreased in adenomas (P < 0.001) and carcinomas (Dukes' stages A-D: P < 0.002). Ca2+-independent NOS activity, indicating inducible NOS (NOS2), is markedly expressed in approximately 60% of human colon adenomas (P < 0.001 versus normal tissues) and in 20-25% of colon carcinomas (P < 0.01 versus normal tissues). Only low levels were found in the surrounding normal tissue. NOS2 activity decreased with increasing tumor stage (Dukes' A-D) and was lowest in colon metastases to liver and lung. NOS2 was detected in tissue mononuclear cells (TMCs), endothelium, and tumor epithelium. There was a statistically significant correlation between NOS2 enzymatic activity and the level of NOS2 protein detected by immunohistochemistry (P < 0.01). Western blot analysis of tumor extracts with Ca2+-independent NOS activity showed up to three distinct NOS2 protein bands at Mr 125,000-Mr 138,000. The same protein bands were heavily tyrosine-phosphorylated in some tumor tissues. TMCs, but not the tumor epithelium, were immunopositive using a polyclonal anti-nitrotyrosine antibody. However, only a subset of the NOS2-expressing TMCs stained positively for 3-nitrotyrosine, which is a marker for peroxynitrite formation. Furthermore, vascular endothelial growth factor expression was detected in adenomas expressing NOS2. These data are consistent with the hypothesis that excessive NO production by NOS2 may contribute to the pathogenesis of colon cancer progression at the transition of colon adenoma to carcinoma in situ.

Insulin-like growth factor II induced by hypoxia may contribute to angiogenesis of human hepatocellular carcinoma.

Kim KW. Bae SK. Lee OH. Bae MH. Lee MJ. Park BC.
Department of Molecular Biology, Pusan National University, Korea. kimkw@hyowon.cc.pusan.ac.kr
Insulin-like growth factor II (IGF-II) is highly expressed during hepatocarcinogenesis (P. Schirmacher et al., Cancer Res., 52: 2549-2556, 1992; B. C. Park et al., J. Hepatol., 22: 286-294, 1995). However, the mechanism of its enhanced expression is largely unknown. In this study, we show that IGF-II mRNA levels are increased within six h of exposing human hepatoma cell cultures to hypoxia, suggesting that hypoxia may be a strong stimulus for the induction of IGF-II expression in the process of hepatocarcinogenesis. This finding and the fact that hepatocellular carcinoma (HCC) is a typical hypervascular tumor (M. Mise et al., Hepatology, 23: 455-464, 1996) imply that IGF-II may play an important role in the development of neovascularization of HCC. Here we demonstrate that IGF-II substantially increases vascular endothelial growth factor (VEGF) mRNA and protein levels in a time-dependent manner in human hepatoma cells. The induction of VEGF by IGF-II was additively increased by hypoxia. Moreover, the direct angiogenic activity of IGF-II was observed in the quantitative chick chorioallantoic membrane assay (M. Nguyen et al., Microvasc. Res., 47: 31-40, 1994). These data suggest that IGF-II may be a hypoxia-inducible angiogenic factor in HCC.

Tumor cell-associated hyaluronan as an unfavorable prognostic factor in colorectal cancer.

Ropponen K. Tammi M. Parkkinen J. Eskelinen M. Tammi R. Lipponen P. Agren U. Alhava E. Kosma VM.
Department of Pathology, University of Kuopio, Finland.
Hyaluronan (HA) is a linear high molecular weight extracellular polysaccharide. It is thought to be involved in mitosis and the enhancement of wound healing, tumor invasion, and metastasis. Because its clinical relevance in cancer has not been explored, we scored HA in colorectal adenocarcinoma and studied its relationship with patient survival. A specific probe prepared from cartilage proteoglycan aggregates was used to stain paraffin-embedded tumor samples from 202 colorectal adenocarcinoma patients treated in Kuopio University Hospital and followed up for a mean of 14 years. The hypothesis that the percentage of HA-positive carcinoma cells (HA%) and HA intensity in cancer cells correlated with survival was tested with the log-rank test, hazard ratios, and their confidence intervals. Ninety-three % of tumors had at least a proportion of carcinoma cells positive for HA. HA intensity in tumor epithelium was stronger in Dukes' stages C and D tumors and in high-grade tumors. The cancer-related survival rate was lower among patients with strong HA intensity in tumor epithelium (P < 0.001) and high HA% (P < 0.001). Recurrence-free survival was also shorter in patients with an intense signal for HA (P = 0.001) and high HA% in tumor epithelium (P = 0.04). HA intensity in tumor epithelium independently predicted survival and recurrence-free survival (Cox's analysis). We conclude that a high proportion of HA-positive cancer cells and high intensity of the HA-signal predicts a poor survival rate. The abnormal expression of HA in the neoplastic colon epithelial cells is suggested to provide a distinct advantage for invasive growth and metastasis.

Mutation of the multiple endocrine neoplasia type 1 gene in nonfamilial, malignant tumors of the endocrine pancreas.

Hessman O. Lindberg D. Skogseid B. Carling T. Hellman P. Rastad J. Akerstrom G. Westin G.
Department of Surgery, University Hospital, Uppsala, Sweden. ola.hessman@kirurgi.uu.se
Endocrine pancreatic tumors are rare neoplasms that occur sporadically or as part of a multiple endocrine neoplasia type 1 (MEN1) syndrome. Germ-line mutations of the MEN1 gene, located at 11q13, have been demonstrated in MEN1 kindreds, and loss of heterozygosity (LOH) on 11q13 together with somatic MEN1 mutations have been detected in 20% of nonfamilial parathyroid tumors. Here, we examine 11 non-MEN1 malignant tumors of the endocrine pancreas, 9 nonfunctioning tumors, and 2 glucagonomas. LOH of at least one informative locus on 11q13 was found in 70% of the tumors. Three tumors displayed somatic mutations of the MEN1 gene together with LOH on 11q13, whereas the corresponding germ-line DNA was normal. These findings support the hypothesis that MEN1 gene mutations contribute to the tumorigenesis of nonfamilial, malignant endocrine pancreatic tumors.

Comparison of (111)In-labeled somatostatin analogues for tumor scintigraphy and radionuclide therapy.

de Jong M. Breeman WA. Bakker WH. Kooij PP. Bernard BF. Hofland LJ. Visser TJ. Srinivasan A. Schmidt MA. Erion JL. Bugaj JE. Macke HR. Krenning EP.
Department of Nuclear Medicine, University Hospital, Dijkigt, Rotterdam, The Netherlands. dejong@nuge.azr.nl
We evaluated the following (111)In-labeled somatostatin (SS) analogues (diethylenetriaminepentaacetic acid, DTPA; tetraazacyclododecanetetraacetic acid, DOTA): [DTPA0]octreotide, [DTPA0,Tyr3]octreotide, [DTPA0,D-Tyr1]octreotide, [DTPA0,Tyr3]octreotate [Thr(ol) in octreotide replaced with Thr], and [DOTA0,Tyr3]octreotide, in vitro and in vivo. In vitro, all compounds showed high and specific binding to SS receptors in mouse pituitary AtT20 tumor cell membranes, and IC50s were in the nanomolar range. Furthermore, all compounds showed specific internalization in rat pancreatic tumor cells; uptake of [(111)In-DTPA0,Tyr3]octreotate was the highest of the compounds tested, and that of [(111)In-DTPA0,D-Tyr1]octreotide was the lowest. Biodistribution experiments in rats showed that, 4, 24, and 48 h after injection of [(111)In-DTPA0,Tyr3]octreotide, [(111)In-DTPA0,Tyr3]octreotate, and [(111)In-DOTA0,Tyr3]octreotide, radioactivity in the octreotide-binding, receptor-expressing tissues and tumor-to-blood ratios were significantly higher than those after injection of [(111)In-DTPA0]octreotide. Uptake of [(111)In-DTPA0,Tyr3]octreotate in the target organs was also, in vivo, the highest of the radiolabeled peptides tested, whereas that of [(111)In-DTPA0,D-Tyr1]octreotide was the lowest. Uptake of [(111)In-DTPA0,Tyr3]octreotide, [(111)In-DTPA0,Tyr3]octreotate, and [(111)In-DOTA0,Tyr3]octreotide in target tissues was blocked by >90% by 0.5 mg of unlabeled octreotide, indicating specific binding to the octreotide receptors. Blockade of [(111)In-DTPA0,D-Tyr1]octreotide was >70%. In conclusion, radiolabeled [DTPA0,Tyr3]octreotide and, especially, [DTPA0,Tyr3]octreotate and their DOTA-coupled counterparts are most promising for scintigraphy and radionuclide therapy of SS receptor-positive tumors in humans.

Overexpression of the nonpancreatic secretory group II PLA2 messenger RNA and protein in colorectal adenomas from familial adenomatous polyposis patients.

Kennedy BP. Soravia C. Moffat J. Xia L. Hiruki T. Collins S. Gallinger S. Bapat B.
Department of Biochemistry and Molecular Biology, Merck Frosst Center for Therapeutic Research, Pointe Claire-Dorval, Quebec, Canada.
The synovial fluid or group II secretory phospholipase A2 (sPLA2) has been implicated in various inflammatory processes and has been shown to release arachidonic acid for prostaglandin biosynthesis. In human colorectal cancer, both arachidonic acid and eicosanoid levels are elevated. Recently, sPLA2 has been identified as a candidate gene that modifies the Apc gene in the Min mouse, a murine model for familial adenomatous polyposis (FAP). Loss of sPLA2 gene function results in susceptibility to the Min phenotype and the formation of multiple intestinal polyps, whereas mice expressing an active sPLA2 gene are resistant to polyp formation. Therefore, there are two potentially contrasting roles for sPLA2 in colon cancer; one is protection against polyp formation, and the other, the release of arachidonic acid for prostaglandin production and subsequent tumor promotion. To investigate these contrasting dual roles of sPLA2, we have examined the expression and sequence of the sPLA2 mRNA in normal mucosa and duodenal and colorectal polyps from FAP patients. In 11 of 14 patients, there was a significant increase in sPLA2 mRNA levels in the adenoma over the normal tissue. In some cases, there was over 100-fold increase in mRNA levels in the adenoma compared with normal tissue. Analysis of multiple adenomatous polyps from individual patients revealed that not all polyps contained elevated levels of sPLA2 mRNA. Immunoblot analysis also showed that sPLA2 protein expression was elevated in adenoma over normal tissue in five of six FAP patients analyzed. Furthermore, sequence analysis of sPLA2 mRNA present in these samples did not reveal mutations in the coding region. The implications of the up-regulation of sPLA2 in FAP is not clear, but unlike the Min mouse model, it does not seem to have a significant effect on polyp formation. In contrast, the high level of sPLA2 expression is more likely contributing to the elevated levels of arachidonic acid found in colorectal cancer and, in conjunction with the elevated expression of cyclooxygenase-2, could be another factor in tumor formation.

Analysis of PTEN/MMAC1 alterations in aerodigestive tract tumors.

Okami K. Wu L. Riggins G. Cairns P. Goggins M. Evron E. Halachmi N. Ahrendt SA. Reed AL. Hilgers W. Kern SE. Koch WM. Sidransky D. Jen J.
Department of Otolaryngology-Head and Neck Surgery, The Johns Hopkins University, Baltimore, Maryland 21205-2196, USA.
PTEN/MMAC1 is a candidate tumor suppressor gene recently identified at chromosomal band 10q23. It is mutated in sporadic brain, breast, and prostate cancer and in the germ line of patients with hereditary Cowden disease. We searched for genetic alterations of the PTEN/MMAC1 gene in 39 primary head and neck cancers (HNSCCs), 42 primary non-small cell lung cancers (NSCLCs), 80 pancreatic cancer xenografts, and 37 cell lines and xenografts from colon, lung, and gastric cancers. Microsatellite analysis revealed loss of heterozygosity at markers near the gene in 41% of primary HNSCCs, 50% of NSCLCs, and 39% of the pancreatic cancers. Three cases of HNSCCs displayed homozygous deletion involving the gene. We sequenced the entire coding region of the PTEN/MMAC1 gene in the remaining tumors displaying loss of heterozygosity and found one terminating mutation in a HNSCC sample. Thus, a second inactivation event was observed in 4 of 39 primary HNSCC cases. By use of a protein truncation assay, one terminating mutation was also identified in one of eight NSCLC cell lines. Our results suggest that PTEN/MMAC1 gene inactivation plays a role in the genesis of some tumor types.

Lewis and secretor gene dosages affect CA19-9 and DU-PAN-2 serum levels in normal individuals and colorectal cancer patients.

Narimatsu H. Iwasaki H. Nakayama F. Ikehara Y. Kudo T. Nishihara S. Sugano K. Okura H. Fujita S. Hirohashi S.
Division of Cell Biology, Institute of Life Science, Soka University, Hachioji, Toyko, Japan. hisashi@scc1.t.soka.ac.jp
The effect of doses of the secretor (Se) and Lewis (Le) genes on the serum levels of CA19-9 and DU-PAN-2 was investigated in 400 normal individuals. It was clearly demonstrated that the Se gene dosage negatively affected both the CA19-9 and DU-PAN-2 values, whereas the Le gene dosage positively affected the CA19-9 value and negatively affected the DU-PAN-2 value. The 400 normal individuals were separated into nine groups by their Le and Se genotypes, as follows: group 1, Le/Le and se/se; group 2, Le/le and se/se; group 3, Le/Le and Se/se; group 4, Le/le and Se/se; group 5, Le/Le and Se/Se; group 6, Le/le and Se/Se; group 7, le/le and se/se; group 8, le/le and Se/se; and group 9, le/le and Se/Se. The group 1 individuals, having homozygous inactive Se alleles (se/se) and homozygous active Le alleles (Le/Le), exhibited the highest mean CA19-9 value. The CA19-9 value clearly ranged from a high in group 1 to a low in group 9. All of the Le-negative individuals who had the le/le genotype (groups 7, 8, and 9) had completely negative CA19-9 values, i.e., under 1.0 unit/ml, irrespective of the Se genotype. Group 7 individuals (le/le and se/se) showed a higher mean DU-PAN-2 value than did individuals in other groups. The Le-negative individuals in groups 8 and 9 also showed a higher mean DU-PAN-2 value than did the Le-positive individuals in groups 1-6. We recommend that the revised Le and Se genotype-dependent positive/negative cutoff values for CA19-9 and DU-PAN-2, determined in this study, be applied for more accurate cancer diagnoses. The Le and Se genotypes of 168 patients with colorectal cancer were also examined, and the CA19-9 and DU-PAN-2 values were measured before surgical resection. All 15 Le-negative patients (le/le) with colorectal cancer again showed undetectable CA19-9 values, i.e., under 1.0 unit/ml, but many of them exhibited highly positive DU-PAN-2 values. In contrast, many of the Le-positive patients (Le/Le or Le/le) had positive CA19-9 values, whereas very few of them exhibited positive DU-PAN-2 values. CA19-9 measurement is more useful than is DU-PAN-2 measurement for Le-positive patients, but it is not useful for Le-negative ones. DU-PAN-2 measurement should be performed in Le-negative patients for cancer diagnosis.

An oligopeptide transporter is expressed at high levels in the pancreatic carcinoma cell lines AsPc-1 and Capan-2.

Gonzalez DE. Covitz KM. Sadee W. Mrsny RJ.
Department of Pharmaceutical Research and Development, Genentech, Inc., South San Francisco, California 94080, USA.
Carcinomas of the exocrine pancreas are poorly understood and have a poor prognosis because of their highly malignant nature. Using two human pancreatic cancer cell lines, AsPc-1 and Capan-2, we have investigated avenues that might be useful in targeting the delivery of antineoplastic agents to such cancers. Qualitative RNA PCRs established the presence of the oligopeptide transporter PEPT 1 in these pancreatic cell lines. Northern analysis confirmed the presence of a 3.3-kb transcript. The transporter is normally expressed primarily in small intestinal epithelial cells for nutrient absorption. It is also expressed in a human intestinal cell line, Caco-2. High levels of PEPT 1 protein expression in AsPc-1 and Capan-2, as multiple glycosylated forms (Mr approximately 90,000-120,000), were confirmed by Western immunoblotting, when compared with Caco-2 cell cultures. Absorption of the model dipeptide glycyl-L-sarcosine by AsPc-1 and Capan-2 cells was similar to glycyl-L-sarcosine absorption by Caco-2 cells and a Chinese hamster ovary cell line expressing human PEPT 1 (CHO-PEPT 1). Uptake was pH dependent and inhibited by several di/tripeptides and bestatin, but it remained unaffected by glycine and tetraglycine. Peptide solute transport by AsPc-1 and Capan-2 cells exhibited binding affinities (Kms) similar to those previously reported for PEPT 1, whereas the transport maximal velocity (Vmax) of the AsPc-1 cells was much greater than those of the Capan-2 and Caco-2 cells. Immunomicroscopy demonstrated PEPT 1 protein localized at the plasma membrane and in intracellular vesicular structures, similar to that observed for Caco-2 and CHO-PEPT 1 cells. These data suggest that the pancreatic cancer cells AsPc-1 and Capan-2 express surprisingly high levels of a solute transporter that was previously thought to be restricted in function to the absorption of nutrients from the small intestine.

Colon carcinoma cells use different mechanisms to escape CD95-mediated apoptosis.

von Reyher U. Strater J. Kittstein W. Gschwendt M. Krammer PH. Moller P.
Institute of Pathology, University of Ulm, Germany.
CD95(APO-1/Fas) is a cell surface receptor that, when oligomerized by natural ligand, CD95L, or antibody, confers an apoptotic signal to apoptosis-sensitive cells. Whereas CD95 is expressed in every colonocyte of normal colon mucosa, CD95 is down-regulated or lost in the majority of colon carcinomas. To investigate the sensitivity to CD95-mediated apoptosis of normal and neoplastic colonocytes, we applied cross-linking CD95(anti-APO-1) monoclonal antibody to freshly isolated colon crypts and colon carcinoma cell lines and monitored apoptosis by DNA fragmentation and morphology. Normal colonocytes were constitutively sensitive to CD95-mediated apoptosis. All carcinoma lines were constitutively resistant but were sensitized upon pretreatment with IFN-gamma. Transcription blocking, protein synthesis, and export in carcinoma cells indicated that even low surface levels of CD95 were sufficient to efficiently transmit the signal. Despite low CD95 surface levels of non-IFNgamma-treated cells, actinomycin D, cycloheximide, and brefeldin A each sensitized all cell lines, but at different rates and kinetics. In this context, it was observed that a greatly delayed apoptotic response of SW620 cells was associated with the absence of antibody-induced CD95 capping. Phorbol 12-myristate 13-acetate inhibited CD95-mediated apoptosis by counteracting the IFNgamma-, actinomycin D-, and cycloheximide-mediated but not the brefeldin A-mediated sensitization. This phorbol 12-myristate 13-acetate-induced protection against apoptosis was completely abolished by staurosporine and by a selective protein kinase C inhibitor, Goe 6983. We conclude that, during malignant transformation, colonocytes acquire different mechanisms to escape CD95-mediated apoptosis. These include abrogation of CD95, inhibition of CD95 capping, and activation of antiapoptotic programs, both governed by and independent of protein kinase C.

An inverse relation between cagA+ strains of Helicobacter pylori infection and risk of esophageal and gastric cardia adenocarcinoma.

Chow WH. Blaser MJ. Blot WJ. Gammon MD. Vaughan TL. Risch HA. Perez-Perez GI. Schoenberg JB. Stanford JL. Rotterdam H. West AB. Fraumeni JF Jr.
National Cancer Institute, Division of Cancer Epidemiology and Genetics, Bethesda, Maryland 20852, USA.
Gastric colonization with Helicobacter pylori, especially cagA+ strains, is a risk factor for noncardia gastric adenocarcinoma, but its relationship with gastric cardia adenocarcinoma is unclear. Although incidence rates for noncardia gastric adenocarcinoma have declined steadily, paralleling a decline in H. pylori prevalence, rates for adenocarcinomas of esophagus and gastric cardia have sharply increased in industrialized countries in recent decades. To clarify the role of H. pylori infection in these tumors with divergent incidence trends, we analyzed serum IgG antibodies to H. pylori and to a recombinant fragment of CagA by antigen-specific ELISA among 129 patients newly diagnosed with esophageal/gastric cardia adenocarcinoma, 67 patients with noncardia gastric adenocarcinoma, and 224 population controls. Cancer risks were estimated by odds ratios (OR) and 95% confidence intervals (CI) using logistic regression models. Infection with cagA+ strains was not significantly related to risk for noncardia gastric cancers (OR, 1.4; CI, 0.7-2.8) but was significantly associated with a reduced risk for esophageal/cardia cancers (OR, 0.4; CI, 0.2-0.8). However, there was little association with cagA- strains of H. pylori for either cancer site (OR, 1.0 and 1.1, respectively). These findings suggest that the effects of H. pylori strains on tumor development vary by anatomical site. Further studies are needed to confirm these results and to assess whether the decreasing prevalence of H. pylori, especially cagA+ strains, may be associated with the rising incidence of esophageal/gastric cardia adenocarcinomas in industrialized countries.

Hypermethylation can selectively silence individual p16ink4A alleles in neoplasia.

Myohanen SK. Baylin SB. Herman JG.
The Oncology Center, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21231, USA.
Inactivation of p16ink4A and other tumor suppressor genes has been associated with promoter region hypermethylation in neoplasia. However, direct proof for aberrant DNA methylation as an independent event for loss of gene function has been difficult to obtain. We addressed this question in the colon carcinoma cell line HCT116, which contains one allele of p16ink4A with a coding region frameshift mutation and one wild-type allele. Neither allele contains a mutation in the proximal promoter region. The promoter of the wild-type allele, but not the mutant allele, is hypermethylated, and only the mutant allele is expressed. Transcription from the methylated/wild-type allele was restored after cell treatment with the demethylating agent 5-aza-2'-deoxycytidine. Thus, in neoplastic cells, stable allele-specific loss of transcription may arise from aberrant methylation of a nonmutated promoter region, identifying hypermethylation as a direct mechanism for tumor suppressor gene inactivation.

Close correlation between mutations of E2F4 and hMSH3 genes in colorectal cancers with microsatellite instability.

Ikeda M. Orimo H. Moriyama H. Nakajima E. Matsubara N. Mibu R. Tanaka N. Shimada T. Kimura A. Shimizu K.
First Department of Surgery, Okayama University Medical School, Japan.
Defects in mismatch repair function can lead to the microsatellite instability (MI+; replication error) phenotype in certain human cancers. We previously reported that MI+ tumor-specific repeat number alteration at 13 consecutive trinucleotide (CAG) repeats within a coding exon of the E2F4 gene is a possible target of the defective repair pathway. Additional investigations revealed that E2F4 mutations are common (11 of 17 cases, 65%, mostly deletions) in a subset of human colorectal cancers with extensive MI+ phenotype, with respect to the proportion of loci affected and that most of these E2F4-mutated tumors (9 of 11, 82%) were accompanied by frameshift mutations in a polyadenine stretch within the seventh exon of the hMSH3 gene, a known mismatch repair gene that is responsible for repair of mismatch loops of two to four nucleotides. However, neither of these mutations was detected in 15 tumors with a lower incidence of MI+ loci. Similar repeat number alterations were less frequent in CAG repeats from other genes in all of the MI+ tumors we examined. These results indicate the presence of a novel cascade of mutational events that may be involved in acquisition of the malignant phenotype of human colorectal cancers with genetic instability.

Characterization of MAST9/Hevin, a SPARC-like protein, that is down-regulated in non-small cell lung cancer.

Bendik I. Schraml P. Ludwig CU.
Department of Research, University Hospital Basel, Switzerland.
In the search for genes down-regulated in non-small cell lung cancer (NSCLC), we have identified a cDNA fragment, termed MAST9. Cloning, sequencing, and characterization of the full-length MAST9 cDNA revealed that the entire 2808 nucleotide sequence had an open reading frame of 1992 nucleotides encoding a Mr 75,000 protein. Sequence analysis disclosed a striking homology to SPARC, known to be involved in tumorigenesis. The recently identified "Hevin" cDNA isolated from high endothelial venules is identical to MAST9. Using Northern and Western blot analysis, we showed that MAST9 was down-regulated in the tumor samples of nine non-small cell lung carcinoma patients. Furthermore, we demonstrated that both the bacterially expressed and the endogenous MAST9 proteins form homodimers. The lack of expression in non-small cell lung carcinomas and its homology to SPARC suggest a putative role of MAST9 in lung tumor formation.

Plasma tocopherol and prevalence of colorectal adenomas in a multiethnic population.

Ingles SA. Bird CL. Shikany JM. Frankl HD. Lee ER. Haile RW.
Department of Preventive Medicine, University of Southern California/Norris Cancer Center, Los Angeles 90033, USA. ingles@hsc.usc.edu
Although vitamin E can block mutagenesis and cell transformation in vitro and can reduce the number of chemically induced colonic adenomas in mice, previous clinical trials have found no protective effect of vitamin E supplements against colorectal adenomas, and epidemiological studies have found only weak protective effects of dietary or plasma alpha-tocopherol against colorectal cancer. We previously examined first diagnosis of colorectal adenomas in a sigmoidoscopy screening population and failed to find a protective effect of dietary vitamin E. Because measurements of dietary intake may not be a good proxy of vitamin E status, we assayed plasma alpha- and gamma-tocopherol concentration for 332 subjects with colorectal adenomas and 363 control subjects from this previous sigmoidoscopy-based study. Increasing alpha-tocopherol and decreasing gamma-tocopherol levels were associated with decreased occurrence of large (> or = 1 cm) but not of small (

Her-2/neu-derived peptides are tumor-associated antigens expressed by human renal cell and colon carcinoma lines and are recognized by in vitro induced specific cytotoxic T lymphocytes.

Brossart P. Stuhler G. Flad T. Stevanovic S. Rammensee HG. Kanz L. Brugger W.
Department of Hematology, Oncology and Immunology, University of Tubingen, Germany.
The Her-2/neu oncogene encodes a Mr 185,000 transmembrane protein with homology to the epidermal growth factor receptor. It is overexpressed in 30-40% of breast and ovarian cancers, and this overexpression was shown to correlate with aggressiveness of malignancy and poor prognosis. Using tumor-associated lymphocytes isolated from patients with ovarian or breast cancer, several HLA-A2-restricted, Her-2/neu-derived peptides were identified. Further studies revealed that these tumor-associated CTLs can also lyse other tumors, including non-small cell lung and pancreatic cancer cells, suggesting that Her-2/neu epitopes are shared between several distinct types of epithelial tumors. To analyze whether Her-2/neu epitopes are tumor-associated antigens for renal cell carcinoma (RCC) and colon carcinoma, we induced Her-2/neu peptide-specific CTL responses by primary in vitro immunization and used these CTLs to determine the presentation of Her-2/neu epitopes on human tumor lines. Autologous dendritic cells (DCs) generated from peripheral blood monocytes were pulsed with Her-2/neu-derived peptides E75 and GP2 and used as antigen-presenting cells for CTL priming. High CTL activity toward peptide-pulsed targets was obtained after two weekly restimulations. CTLs induced with DCs generated in the presence of TNF-alpha elicited a higher cytotoxic activity when they were stimulated with the cognate peptide than did CTLs induced with DCs grown in granulocyte macrophage colony-stimulating factor and interleukin 4 alone. The cytotoxicity of induced CTLs was antigen specific and HLA-A2 restricted. Furthermore, these CTLs lysed, in a MHC- and antigen-restricted fashion, not only breast cancer cells but also colon carcinoma and RCC cell lines expressing Her-2/neu. The cytotoxic activity against tumor cells was blocked by cold HLA-A2-positive targets pulsed with the cognate peptide in cold target inhibition assay and by anti-HLA-A2 monoclonal Ab. These results suggest that epitopes derived from Her-2/neu protein might be attractive candidates for broadly applicable vaccines and may prove useful for adoptive immunotherapies designed for colon carcinoma or RCC.

KAI1 is unchanged in metastatic and nonmetastatic esophageal and gastric cancers.

Guo XZ. Friess H. Maurer C. Berberat P. Tang WH. Zimmermann A. Naef M. Graber HU. Korc M. Buchler MW.
Department of Visceral and Transplantation Surgery, University of Bern, Inselspital, Switzerland.
Down-regulation of KAI1 mRNA expression has been shown to be associated with the formation of metastases or disease progression in pancreatic cancer. Whether KAI1 possesses similar characteristics in other malignancies of the gastrointestinal tract is not known. Here, we compared the patterns of KAI1 mRNA expression in 41 esophageal cancers and 35 stomach cancers to assess whether KAI1 might also be of biological relevance in the metastatic ability of these tumors. By Northern blot analysis, KAI1 mRNA levels ranged widely in both normal and cancerous esophageal and gastric tissue samples, with no statistical differences. No association between KAI1 mRNA expression and tumor stage or tumor differentiation was seen in these tumors. In addition, KAI1 mRNA expression was similar in primary esophageal and gastric cancer samples with or without metastases. By in situ hybridization, KAI1 mRNA expression was evident in the cytoplasm of most squamous epithelial cells in the normal esophagus and in nonmucosal epithelial cells of the normal stomach. The staining intensity in the esophageal and gastric cancer cells was similar to that in the normal controls. This differential pattern of KAI1 mRNA expression in esophageal and gastric cancers in comparison to pancreatic cancer indicates that KAI1 seems to influence the potential of gastrointestinal cancer cells to metastasize differently. In esophageal and gastric cancers, the formation of metastases is not dependent on the reduction of KAI1 in the cancer cells.

Defective expression of the DNA mismatch repair protein, MLH1, alters G2-M cell cycle checkpoint arrest following ionizing radiation.

Davis TW. Wilson-Van Patten C. Meyers M. Kunugi KA. Cuthill S. Reznikoff C. Garces C. Boland CR. Kinsella TJ. Fishel R. Boothman DA.
Department of Human Oncology, University of Wisconsin-Madison, 53792, USA.
A role for the Mut L homologue-1 (MLH1) protein, a necessary component of DNA mismatch repair (MMR), in G2-M cell cycle checkpoint arrest after 6-thioguanine (6-TG) exposure was suggested previously. A potential role for MLH1 in G1 arrest and/or G1-S transition after damage was, however, not discounted. We report that MLH1-deficient human colon carcinoma (HCT116) cells showed decreased survival and a concomitant deficiency in G2-M cell cycle checkpoint arrest after ionizing radiation (IR) compared with genetically matched, MMR-corrected human colon carcinoma (HCT116 3-6) cells. Similar responses were noted between murine MLH1 knockout compared to wild-type primary embryonic fibroblasts. MMR-deficient HCT116 cells or embryonic fibroblasts from MLH1 knockout mice also demonstrated classic DNA damage tolerance responses after 6-TG exposure. Interestingly, an enhanced p53 protein induction response was observed in HCT116 3-6 (MLH1+) compared with HCT116 (MLH1-) cells after IR or 6-TG. Retroviral vector-mediated expression of the E6 protein did not, however, affect the enhanced G2-M cell cycle arrest observed in HCT116 3-6 compared with MLH1-deficient HCT116 cells. A role for MLH1 in G2-M cell cycle checkpoint control, without alteration in G1, after IR was also suggested by similar S-phase progression between irradiated MLH1-deficient and MLH1-proficient human or murine cells. Introduction of a nocodazole-induced G2-M block, which corrected the MLH1-mediated G2-M arrest deficiency in HCT116 cells, clearly demonstrated that HCT116 and HCT116 3-6 cells did not differ in G1 arrest or G1-S cell cycle transition after IR. Thus, our data indicate that MLH1 does not play a major role in G1 cell cycle transition or arrest. We also show that human MLH1 and MSH2 steady-state protein levels did not vary with damage or cell cycle changes caused by IR or 6-TG. MLH1-mediated G2-M cell cycle delay (caused by either MMR proofreading of DNA lesions or by a direct function of the MLH1 protein in cell cycle arrest) may be important for DNA damage detection and repair prior to chromosome segregation to eliminate carcinogenic lesions (possibly brought on by misrepair) in daughter cells.

Keratinocyte growth factor protects mice from chemotherapy and radiation-induced gastrointestinal injury and mortality.

Year 1998
Farrell CL. Bready JV. Rex KL. Chen JN. DiPalma CR. Whitcomb KL. Yin S. Hill DC. Wiemann B. Starnes CO. Havill AM. Lu ZN. Aukerman SL. Pierce GF. Thomason A. Potten CS. Ulich TR. Lacey DL.
Amgen Center, Thousand Oaks, California 91320-1789, USA.
Keratinocyte growth factor (KGF) stimulates the proliferation and differentiation of epithelial cells including those of the gastrointestinal tract. Although chemotherapeutics and radiation exposure kill rapidly proliferating tumor cells, rapidly dividing normal cells of the host's gastrointestinal tract are also frequently damaged, leading to the clinical condition broadly termed "mucositis." In this report, recombinant human KGF used as a pretreatment in several mouse models of chemotherapy and/or radiation-induced gastrointestinal injury significantly improved mouse survival. Using multiple-dose 5-fluorouracil, methotrexate, and radiation in combination and total body radiation alone models, KGF increased survival by 55% or greater. In the models that used chemotherapy with or without radiation, KGF significantly ameliorated weight loss after injury and accelerated weight gain during recovery. The basis of these systemic benefits appears to be due in part to the trophic effects of the growth factor on the intestinal epithelium because KGF pretreatment caused an increase in measures of mucosal thickness (villus height and crypt depth) that persisted during the course of 5-fluorouracil chemotherapy. Treatment with KGF also afforded a 3.5-fold improvement in crypt survival in the small intestine, suggesting that KGF also has a direct effect on the crypt stem cells. These data indicate that KGF may be therapeutically useful to lessen the intestinal side effects of current cancer therapy regimens.

Overexpression of cyclin A but not Skp 2 correlates with the tumor relapse of human hepatocellular carcinoma.

Year 1998
Chao Y. Shih YL. Chiu JH. Chau GY. Lui WY. Yang WK. Lee SD. Huang TS.
Department of Internal Medicine, Veterans General Hospital-Taipei, Taiwan, Republic of China.
Cyclin A is an S- and G2-M-phase regulatory protein, and its abnormal expression has been implicated in cellular transformation. This work was undertaken to investigate the frequency of cyclin A overexpression and the correlated clinical outcome in human hepatocellular carcinoma (HCC). Herein, 12 of 31 (39%) patients exhibited cyclin A overexpression in their tumorous tissues, resulting from gene amplification in 6 of 12 patients, (post)transcription in 4 of 12 patients, and (post)translation in 2 of 12 patients. Patients who overexpressed cyclin A had significantly more tumor cells in the S and G2-M phases compared with those expressing a normal cyclin A level (P = 0.007 and 0.039, respectively). Increased levels of Skp 2, a cyclin A-interacting protein, were also found in 17 of 31 (55%) of HCC patients who showed a trend to have more S-phase tumor cells (P = 0.07). By an unpaired Student's t test and a Fisher's exact or chi2 analysis, overexpression of cyclin A had a strong correlation with elevated Skp 2 expression and increased alpha-fetoprotein levels (P = 0.001 and 0.009, respectively), but it was not associated with patients' age, tumor size, cirrhosis, or the positive detection of hepatitis B virus surface antigen. In the disease-free survival analysis, patients whose tumors overexpressed cyclin A had a median disease-free survival of 6 months, whereas patients who lacked cyclin A overexpression exhibited a longer median period of 29 months (P = 0.046). The overall survival analysis revealed the same trend, i.e., cyclin A-overexpressing patients had shorter overall survival periods (median, 12 versus 50 months; P = 0.09). By multivariate analysis, the correlation of cyclin A overexpression with shorter disease-free periods remained significant after adjustment for Skp 2 overexpression and alpha-fetoprotein induction (P = 0.019). These data suggest that overexpression of cyclin A can be an independent prognostic factor for the tumor relapse of human HCC.

Somatic frameshift mutations in DNA mismatch repair and proapoptosis genes in hereditary nonpolyposis colorectal cancer.

Year 1998
Yamamoto H. Sawai H. Weber TK. Rodriguez-Bigas MA. Perucho M.
The Burnham Institute, La Jolla Cancer Research Center, California 92037, USA.
An exacerbated genomic instability at simple repeated sequences characterizes cancer of the microsatellite mutator phenotype (MMP). The majority of hereditary nonpolyposis colon cancers (HNPCCs) and about 15% of nonselected ("sporadic") gastrointestinal tumors belong to the MMP pathway of tumorigenesis. Colorectal MMP+ and MMP- tumors exhibit fundamental differences in genotype and phenotype. We have shown previously that "sporadic" MMP+ colon cancers exhibit a paradoxical low incidence of somatic mutations in the p53 tumor suppressor gene and the c-K-ras proto-oncogene. On the other hand, gastrointestinal MMP+ cancers frequently harbor frameshift mutations in genes containing mononucleotide repeats. These include the cell growth regulator gene TGFbetaRII and the proapoptotic gene BAX. We have also recently shown the frequent presence of frameshift mutations in (A)8 and (C)8 tracts within the hMSH3 and hMSH6 DNA mismatch repair genes in sporadic colon cancer of the MMP. Here, we describe the nearly identical incidence of somatic frameshift mutations in these genes in a panel of 27 HNPCC MMP+ cancers: 52% in hMSH3 and BAX and 33% in hMSH6. In contrast, no mutations in any of these genes were found in 10 MMP- cancers of HNPCC patients. These results show that the multistep model for the unfolding of the MMP also applies to HNPCC and further illustrate the importance of the escape from apoptosis in the MMP pathway for gastrointestinal cancer. They also underscore the differences in genotype between tumors with and without enhanced microsatellite instability and the similarities in genotype between tumors of the MMP regardless of their hereditary or sporadic nature.

Activation of the beta-catenin gene by interstitial deletions involving exon 3 in primary colorectal carcinomas without adenomatous polyposis coli mutations.

Year 1998
Iwao K. Nakamori S. Kameyama M. Imaoka S. Kinoshita M. Fukui T. Ishiguro S. Nakamura Y. Miyoshi Y.
Department of Medical Genetics, Biomedical Research Center, Osaka University Medical School, Suita City, Japan.
Among 222 primary colorectal cancers we examined, 58 showed no detectable APC mutations by the protein truncation test. We screened those 58 tumors for somatic mutations in the beta-catenin gene. Although amino acid substitutions in serine or threonine residues in exon 3 had been reported, we found no such mutations; however, in seven tumors, we detected somatic interstitial deletions of 234-760 bp, each of which included all or part of exon 3. Short nucleotide sequences at both ends of each deletion were either identical or complementary, indicating that repeated or inversely repeated sequences were involved in the somatic rearrangements. Reverse transcription-PCR experiments using RNAs isolated from three of these seven tumors detected transcripts that lacked exon 3, in addition to the normal transcript. In one of these cases, we confirmed accumulation of aberrant beta-catenin protein in cytoplasm and nuclei of cancer cells by Western and immunohistochemical analyses. This result suggested that, in the absence of a peptide encoded by exon 3, beta-catenin is stabilized and has a dominant oncogenic effect on colorectal tumorigenesis.

Eleutherobin, a novel cytotoxic agent that induces tubulin polymerization, is similar to paclitaxel (Taxol).

Year 1998
Long BH. Carboni JM. Wasserman AJ. Cornell LA. Casazza AM. Jensen PR. Lindel T. Fenical W. Fairchild CR.
Oncology Drug Discovery, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543, USA.
Eleutherobin is a novel natural product isolated from a marine soft coral that is extremely potent for inducing tubulin polymerization in vitro and is cytotoxic for cancer cells with an IC50 similar to that of paclitaxel. This compound is cross-resistant along with other multidrug-resistant agents against P-glycoprotein-expressing cells and is cross-resistant with paclitaxel against a cell line that has altered tubulin. In mechanistic studies, eleutherobin shares with paclitaxel the ability to induce tubulin polymerization in vitro and is most likely cytotoxic by virtue of this mechanism. Human colon carcinoma cells exposed to eleutherobin contain multiple micronuclei and microtubule bundles, and they arrest in mitosis, depending on concentration, cell line, and length of exposure. These morphological abnormalities appearing in cultured cells are indistinguishable from those induced by paclitaxel. Electron microscopy reveals that eleutherobin induces homogeneous populations of long, rigid microtubules similar to those formed by paclitaxel. Thus, eleutherobin is a new chemotype with a mechanism of action similar to that of paclitaxel and, as such, has promising potential as a new anticancer agent.

Mutations of the DPC4/Smad4 gene in biliary tract carcinoma.

Year 1998
Hahn SA. Bartsch D. Schroers A. Galehdari H. Becker M. Ramaswamy A. Schwarte-Waldhoff I. Maschek H. Schmiegel W.
Department of Internal Medicine, University of Bochum, Germany.
A candidate tumor suppressor gene, DPC4, located at 18q21.1, has recently been shown to be inactivated in half of pancreatic adenocarcinomas. The close developmental relationship of the pancreas and biliary tract prompted us to determine the role of DPC4 in the multistep carcinogenesis of biliary tract carcinoma. A search for mutations in the genomic sequence of the highly conserved COOH-terminal domain of DPC4 (exons 8-11) was performed by single-strand conformational polymorphism analysis. Five of 32 (16%) primary biliary tract carcinomas had point mutations in the DPC4 sequence. Interestingly, inactivation of DPC4 was especially common in carcinomas originating from the common bile duct (four of eight specimens analyzed), suggesting an important role for DPC4 in the development of this subtype of biliary tract tumor.

Mutational analysis of the APC/beta-catenin/Tcf pathway in colorectal cancer.

Year 1998
Sparks AB. Morin PJ. Vogelstein B. Kinzler KW.
Johns Hopkins Oncology Center, Baltimore, Maryland 21231, USA.
Mutation of the adenomatous polyposis coli (APC) tumor suppressor gene initiates the majority of colorectal (CR) cancers. One consequence of this inactivation is constitutive activation of beta-catenin/Tcf-mediated transcription. To further explore the role of the APC/beta-catenin/Tcf pathway in CR tumorigenesis, we searched for mutations in genes implicated in this pathway in CR tumors lacking APC mutations. No mutations of the gamma-catenin (CTNNG1), GSK-3alpha (GSK3A), or GSK-3beta (GSK3B) genes were detected. In contrast, mutations in the NH2-terminal regulatory domain of beta-catenin (CTNNB1) were found in 13 of 27 (48%) CR tumors lacking APC mutations. Mutations in the beta-catenin regulatory domain and APC were observed to be mutually exclusive, consistent with their equivalent effects on beta-catenin stability and Tcf transactivation. In addition, we found that CTNNB1 mutations can occur in the early, adenomatous stage of CR neoplasia, as has been observed previously with APC mutations. These results suggest that CTNNB1 mutations can uniquely substitute for APC mutations in CR tumors and that beta-catenin signaling plays a critical role in CR tumorigenesis.

A distinct region of chromosome 19p13.3 associated with the sporadic form of adenoma malignum of the uterine cervix.

Year 1998
Lee JY. Dong SM. Kim HS. Kim SY. Na EY. Shin MS. Lee SH. Park WS. Kim KM. Lee YS. Jang JJ. Yoo NJ.
Department of Pathology and Cancer Research Institute, Catholic University Medical College, Seoul, Korea.
Adenoma malignum (AM) is known to be one of the malignant tumors that is commonly associated with Peutz-Jeghers syndrome. Recently, the genetic locus of Peutz-Jeghers syndrome was mapped to the telomeric region of chromosome 19p. We analyzed nine sporadic cases of AM with high-density loss of heterozygosity to study the region of chromosome 19p13.2-13.3 using eight microsatellite markers. Our deletion mapping data revealed a distinct region with 100% loss of heterozygosity frequency at marker D19S216. This result indicates that a putative tumor suppressor gene for AM is located at D19S216 on chromosomal band 19p13.3 and plays an important role in AM tumorigenesis.

Ki-ras mutation and p53 overexpression predict the clinical behavior of colorectal cancer: a Southwest Oncology Group study.

Year 1998
Ahnen DJ. Feigl P. Quan G. Fenoglio-Preiser C. Lovato LC. Bunn PA Jr. Stemmerman G. Wells JD. Macdonald JS. Meyskens FL Jr.
University of Colorado Health Sciences Center and Department of Veterans Affairs Medical Center, Denver 80220, USA.
We assessed Ki-ras mutations by single-strand conformation polymorphism followed by DNA sequencing, p53 expression by immunohistochemistry, ploidy status, and S-phase fraction in 66 stage II and 163 stage III colon cancer patients enrolled on a randomized trial of surgery followed by observation or adjuvant levamisole or 5-fluorouracil (5FU) plus levamisole (Intergroup Trial 0035) to see whether these factors were independently associated with survival or with differential effects of adjuvant therapy. A Cox proportional hazards survival model was used to describe marker effects and therapy by marker interactions, with adjustment for the clinical covariates affecting survival. A Bonferroni adjustment was used to account for multiple testing. Mutation of the Ki-ras gene was found in 41% of the cancers and was associated with a poor prognosis in stage II but not stage III. In stage II, 7-year survival was 86% versus 58% in those with wild type versus Ki-ras mutations. After adjustment for treatment and clinical variables, the hazard ratio (HR) for death was 4.5; 95% confidence interval (CI), 1.7-12.1 (P = 0.012). p53 overexpression was found in 63% of cancers and was associated with a favorable survival in stage III but not stage II. Seven-year survival in stage III was 56% with p53 overexpression versus 43% with no p53 expression (HR, 2.2; 95% CI, 1.3-3.6; P = 0.012). Aneuploidy was more common in stage III than in stage II (66 versus 47%; P = 0.009) but was not independently related to survival in either group. The proliferative rate was greater in aneuploid than in diploid cancers but was not related to survival. There was no benefit of adjuvant therapy in stage II nor in any of the stage II subgroups defined by mutational status. In stage III, adjuvant therapy with 5FU plus levamisole improved 7-year survival in patients with wild-type Ki-ras (76 versus 44%; HR, 0.4; 95% CI, 0.2-0.8) and in those without p53 overexpression (64 versus 26%; HR, 0.3; 95% CI, 0.1-0.7). Adjuvant therapy did not benefit those with Ki-ras mutations or p53 overexpression. The effects of adjuvant therapy did not differ according to ploidy status or proliferative rate. Ki-ras mutation is a significant risk factor for death in stage II, and the absence of p53 expression is a significant risk factor for death in stage III colon cancer after adjustment for treatment and clinical covariates. Exploratory analyses suggest that patients with stage III colon cancer with wild-type Ki-ras or no p53 expression benefit from adjuvant 5FU plus levamisole, whereas those with Ki-ras mutations or p53 overexpression do not. An independent study will be required to determine whether response to adjuvant therapy in colon cancer depends on mutational status.

Molecular mimicry of carcinoembryonic antigen by peptides derived from the structure of an anti-idiotype antibody.

Year 1998
Chatterjee SK. Tripathi PK. Chakraborty M. Yannelli J. Wang H. Foon KA. Maier CC. Blalock JE. Bhattacharya-Chatterjee M.
Department of Internal Medicine, and Lucille Parker Markey Cancer Center, University of Kentucky Medical Center, Lexington 40536-0096, USA.
Our goal was to use carcinoembryonic antigen (CEA) as a target for immunotherapy in CEA-positive cancer patients who are all immune tolerant to the native antigen. We isolated and characterized an anti-idiotype monoclonal antibody 3H1, which mimics a distinct and specific epitope of the Mr 180,000 CEA and can be used as a surrogate for CEA. In Phase Ib clinical trials in a group of 23 advanced colorectal cancer patients, 3H1 induced both humoral and cellular anti-3H1 responses, as well as anti-CEA immunity. To study the cellular immunity invoked by 3H1 at the molecular level, we have cloned and sequenced the cDNAs encoding the variable heavy and light chains of 3H1 and deduced the amino acid sequences of the encoded proteins. To identify any cross-reactive peptides of 3H1 and CEA, we compared the amino acid sequences of 3H1 with those of CEA and found several regions of homology in 3H1 heavy and light chain variable domains, as well as in the framework regions. To search for potential cross-reactive T-cell epitopes, a number of peptides were synthesized based on 3H1/CEA homology and were used as stimulants in cell proliferation assays, using peripheral blood mononuclear cells from a group of 3H1-immunized CEA-positive cancer patients in the adjuvant setting. Two partially homologous peptides, designated LCD-2 (from 3H1) and CEA-B (from CEA), were identified in 10 of 21 adjuvant patients by strong proliferation responses (stimulation index, 3-50-fold), which were extensively studied in five of these individuals over an extended period of time (12-24 months). We saw no correlation with the MHC class I haplotype of the patients. Analysis of the subtype of the responding T cells demonstrated that primarily CD4+ T cells were stimulated by both 3H1 and 3H1-derived peptides. Interleukin 2, interleukin 4, and IFN-gamma were assayed in the culture medium of peripheral blood mononuclear cells stimulated with 3H1, CEA, and LCD-2 to determine the T-cell helper subset induced by these stimulants. The in vitro responses were mainly associated with secretion of IFN-gamma, which suggested that the induced T cells were most likely CD4+ Th1 type. Future studies will include the design of second-generation LCD-2 and CEA peptides to further enhance antigenicity, to characterize the responding T-cell populations more fully, and to test refined peptides for immunogenicity.

Low frequency of somatic mutations in the LKB1/Peutz-Jeghers syndrome gene in sporadic breast cancer.

Year 1998
Bignell GR. Barfoot R. Seal S. Collins N. Warren W. Stratton MR.
Section of Cancer Genetics, Institute of Cancer Research, Sutton, Surrey, United Kingdom.
Germ-line mutations in the LKB1 gene on chromosome 19p are responsible for most cases of the Peutz-Jeghers syndrome, in which intestinal hamartomas are associated with elevated risks of several cancer types, including breast cancer. We have evaluated the role of somatic mutations in LKB1 in breast cancer. Of 40 informative primary breast cancers, 3 showed loss of heterozygosity on chromosome 19p in the vicinity of LKB1, and no somatic mutations of LKB1 were observed in 62 primary breast cancers and 17 established breast cancer cell lines. The results indicate that mutations in LKB1 do not play an important role in the development of sporadic breast cancer.

Molecular detection of genetic alterations in the serum of colorectal cancer patients.

Year 1998
Hibi K. Robinson CR. Booker S. Wu L. Hamilton SR. Sidransky D. Jen J.
Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2196, USA.
We have searched for the presence of genetic alterations in serum DNA obtained from 44 colorectal cancer patients. Microsatellite analysis using highly polymorphic markers revealed loss of heterozygosity and/or microsatellite instability in 35 of 44 (80%) primary tumors. No alterations were detected in the paired serum DNA. We next used an oligonucleotide-mediated mismatch ligation assay to detect tumor specific gene mutations in the serum. Among the 16 cases with a K-ras gene mutation in the tumor, the same mutation was detected in three paired serum samples. In the 10 cases with a p53 mutation in the tumor, the identical mutation was detected in seven corresponding serum samples. Comparison of the molecular analysis with clinical diagnosis of these patients revealed that none of the seven Dukes' stage B patients with a K-ras mutation in their tumors demonstrated a mutation in the serum. In contrast, five of seven stage B patients with a p53 mutation in the tumor demonstrated a mutation in the paired serum (P = 0.01, Fisher's exact test). Taken together, either a K-ras or p53 mutation was detected in the serum in 40% of the 25 patients (95% confidence interval, 21-61%), whose primary tumors contained a mutation and in 23% of the 44 patients (95% confidence interval, 12-38%) with colorectal cancer. The frequent detection of p53 mutation in the serum of patients with early stage tumors suggests a possible use of this approach for clinical prognosis and cancer monitoring of colorectal cancer patients.

Differential expression of methionine adenosyltransferase genes influences the rate of growth of human hepatocellular carcinoma cells.

Year 1998
Cai J. Mao Z. Hwang JJ. Lu SC.
Center for Liver Disease Research, Department of Medicine, University of Southern California School of Medicine, Los Angeles 90033, USA.
Methionine adenosyltransferase (MAT) catalyzes the formation of S-adenosylmethionine (SAM), the principal methyl donor, and is essential to normal cell function. The two forms of MAT, liver specific and non-liver specific, are products of two genes, MAT1A and MAT2A, respectively. We have reported a switch from MAT1A to MAT2A gene expression in human liver cancer cells. In the current work, we examined whether the type of MAT expressed by the cell influences cell growth. HuH-7 cells were stably transfected with MAT1A and were subsequently treated with antisense oligonucleotides directed against MAT2A. MAT2A antisense treatment reduced the amount of MAT2A mRNA by 99% but had no effect on MAT1A mRNA. Cell growth and DNA synthesis rates were reduced by approximately 20-25% after transfection with MAT1A and by an additional 30-40% after MAT2A antisense treatment. SAM level and SAM:S-adenosylhomocysteine (SAH) ratio increased by 50-75% after MAT1A transfection and by an additional 60-80% after MAT2A antisense treatment. DNA methylation changed in parallel to changes in SAM level and SAM:SAH ratio. Supplementing untransfected HuH-7 cells with SAM in the culture medium increased SAM level, SAM:SAH ratio, and DNA methylation and decreased cell growth and DNA synthesis. In conclusion, cell growth is influenced by the type of MAT expressed. The mechanism likely involves changes in SAM:SAH ratio and DNA methylation.

Clinicopathological significance of altered loci of replication error and microsatellite instability-associated mutations in gastric cancer.

Year 1998
Wu MS. Lee CW. Shun CT. Wang HP. Lee WJ. Sheu JC. Lin JT.
Department of Internal Medicine, National Taiwan University Hospital, Taipei, Republic of China.
Replication errors (RERs) judged by microsatellite instability and its associated mutations have been recognized as an important mechanism in tumorigenesis of gastric cancers (GCs). To gain a deeper insight into its significance, we examined the frequency of RERs using nine microsatellite markers and screened mutations in the polydeoxyadenine tract of the transforming growth factor beta type II receptor gene (TGF-betaRII) and polydeoxyguanine tracts of insulin-like growth factor II receptor and BAX genes. Twenty-four (30%) of 80 patients with GC had RERs, of which 3, 8, and 13 had one, two, and three or more loci, respectively. In 13 tumors with RERs in three or more loci, frameshift mutations of TGF-betaRII, insulin-like growth factor II receptor, and BAX were identified in 12, 3, and 2, respectively. Compared with GC with none, one or two RER-positive loci as a group, GC with RERs in three or more loci showed a significantly higher frequency of antral location (12 of 13 versus 35 of 67; P = 0.01), intestinal subtype (11 of 13 versus 30 of 67; P = 0.01), and previous Helicobacter pylori infection (12 of 13 versus 41 of 67; P = 0.05) and a lower incidence of lymph node metastasis (5 of 13 versus 49 of 67; P = 0.02) and tended to be in an advanced stage (12 of 13 versus 54 of 67; P = 0.28). These data indicate that GC with multiple RERs manifest distinct clinicopathological characteristics, and that a high frequency of frameshift mutations involving the TGF-betaRII gene may be causatively linked with tumorigenesis and progression.

Role of nitric oxide and superoxide anion in elimination of low metastatic human colorectal carcinomas by unstimulated hepatic sinusoidal endothelial cells.

Year 1998
Edmiston KH. Shoji Y. Mizoi T. Ford R. Nachman A. Jessup JM.
Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02115, USA.
Human colorectal carcinoma (CRC) cell survival for the first 24 h after implantation in the hepatic sinusoid determines its potential to colonize the liver. Nearly 10-fold more highly metastatic CX-1 cells survive within the livers of nude mice 24 h after intrasplenic injection than weakly metastatic clone A cells. Because CRCs contact sinusoidal endothelial cells (SECs) during implantation, we sought to determine whether SECs were more toxic to clone A than to CX-1 cells. When 2 x 10(4) vital dye-labeled CRC cells were added to murine SEC monolayers, more than 30% of clone A cells lost calcein AM fluorescence compared to fewer than 5% of CX-1 cells after 24 h of coculture with SECs. Kupffer cells did not mediate this effect, because neither enriched Kupffer cells nor SECs treated with a Kupffer cell inhibitor altered the SEC-mediated toxic effect to clone A cells. Pretreatment with a nitric oxide synthase inhibitor, N(G)-monomethyl-L-arginine, superoxide dismutase, or dexamethasone, blocked SEC-mediated toxicity to clone A cells, whereas calcium chelation and catalase did not. In addition, clone A cells were more sensitive to a superoxide donor, 3-morpholinosydnonimine N-ethylcarbamide, than were CX-1 cells, and neither cell line was sensitive to sodium nitroprusside, a nitric oxide donor. Thus, unstimulated murine SECs produce reactive oxygen species that are selectively toxic to weakly metastatic clone A cells. This may be a mechanism by which host liver cells eliminate weakly metastatic neoplastic cells.

Frequent abnormalities of the putative tumor suppressor gene FHIT at 3p14.2 in pancreatic carcinoma cell lines.

Year 1998
Simon B. Bartsch D. Barth P. Prasnikar N. Munch K. Blum A. Arnold R. Goke B.
Department of Internal Medicine, Philipps-University, Marburg, Germany. simonb@post.med.uni-marburg.de
The FHIT gene is localized on chromosome 3p14, a region including a tumor cell-specific, commonly deleted region. To determine the role of the FHIT gene in pancreatic carcinogenesis, 14 pancreatic carcinoma cell lines were analyzed by reverse transcription-PCR and exon-specific PCR amplification of genomic DNA. The full-length FHIT transcript was lost in 70% of the pancreatic carcinoma cell lines analyzed, while 66% also revealed intragenic homozygous deletions of exons 3, 4, and 5. Truncated FHIT transcripts lacking a variable number of exons most likely represented alternative splicing products. Fhit protein expression was dependent on a full-length FHIT transcript. The results suggest that the FHIT gene may be a target tumor suppressor gene involved in pancreatic carcinogenesis.

Cathepsin L2, a novel human cysteine proteinase produced by breast and colorectal carcinomas.

Year 1998
Santamaria I. Velasco G. Cazorla M. Fueyo A. Campo E. Lopez-Otin C.
Departamento de Bioquimica y Biologia Molecular, Facultad de Medicina, Universidad de Oviedo, Spain.
We have identified and cloned a new member of the papain family of cysteine proteinases from a human brain cDNA library. The isolated cDNA codes for a polypeptide of 334 amino acids that exhibits all of the structural features characteristic of cysteine proteinases, including the active site cysteine residue essential for peptide hydrolysis. Pairwise comparisons of this amino acid sequence with the remaining human cysteine proteinases identified to date showed a high percentage of identity (78%) with cathepsin L; the percentage of identity with all other members of the family was much lower (

An apoptosis-inducing gene therapy for pancreatic cancer with a combination of 55-kDa tumor necrosis factor (TNF) receptor gene transfection and mutein TNF administration.

Year 1998
Sato T. Yamauchi N. Sasaki H. Takahashi M. Okamoto T. Sakamaki S. Watanabe N. Niitsu Y.
Fourth Department of Internal Medicine, Sapporo Medical University, School of Medicine, Japan.
Intratumoral injection of recombinant human tumor necrosis factor (TNF) for inoperable pancreatic cancer has shown some efficacy in suppressing tumor growth or decreasing tumor markers. However, complete regression has not yet been achieved, possibly due to a lack of TNF receptors on tumor cells or an abundance of intracellular resistance factors. Recently, two distinct types of TNF receptors, R55 and R75, were identified, which are responsible for signaling of cytotoxicity and of proinflammation, respectively. In this study, a novel type of suicide gene therapy is proposed that is based on transfection of the R55 gene into human pancreatic cancer cells (AsPC-1 and PANC-1) and subsequent administration of TNF. The transfectants from both cell lines showed higher TNF susceptibility than their parental cells. In vivo tumor formation of an AsPC-1 clone (clone 10) inoculated in nude mice was substantially suppressed by administration of TNF. For practical use of this strategy, however, the adverse effects of TNF may become an obstacle. We previously produced mutein TNF 471, which had a higher affinity for R55, superior antitumor activity, and fewer adverse effects. This mutein TNF 471 manifested greater antitumor activity against clone 10. Because the R55 receptor is known to be involved in augmentation of cellular immunity by TNF, mutein TNF 471 is also expected to be highly potent in this function. In fact, the mutein TNF 471 induced higher splenic natural killer cell activity in nude mice inoculated with clone 10 than did native TNF. This property of augumenting cellular responses may be advantageous in the eradication of viable tumor cells left untransfected in practical gene therapy regimens in which 100% transfection of the R55 gene into tumors is not feasible. Thus, gene therapy combining transfection of the TNF-R55 gene with administration of mutein TNF 471 may provide a new modality for the treatment of pancreatic cancer.

Microsatellite instability in colorectal cancer: different mutator phenotypes and the principal involvement of hMLH1.

Year 1998
Thibodeau SN. French AJ. Cunningham JM. Tester D. Burgart LJ. Roche PC. McDonnell SK. Schaid DJ. Vockley CW. Michels VV. Farr GH Jr. O'Connell MJ.
Department of Laboratory Medicine and Pathology, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905, USA.
Recent studies have demonstrated the presence of microsatellite instability (MSI) in tumors from patients with hereditary nonpolyposis colorectal cancer and in a large number of sporadic tumors. To further characterize the type of alterations at these loci and their frequency of involvement in colon cancer, we studied DNA extracted from paraffin-embedded tissue from 508 patients using 11 microsatellites localized to chromosomes 5, 8, 15, 17, and 18. Overall, MSI at each locus varied in character and frequency and was observed with at least one marker in 191 cases (37.6%). Based on the number of markers displaying instability per tumor, three groups of patients were defined: those with or = 30% (MSI-H, n = 82, 16.1%); and those showing no instability (MSS, n = 317, 62.4%). These groups were tested for correlations with a number of clinical and pathological parameters, including age, sex, stage, ploidy status, and site of tumor. Comparing across the three groups and verified by pair-wise comparisons, the MSI-H group was associated with tumor site (proximal colon, P = 0.001), sex (females, P = 0.005), stage (Dukes' B, P = 0.01), and ploidy status (diploid, P = 0.03). No significant differences were noted between the MSI-L and MSS group for any of the parameters tested. An additional 188 consecutive surgical colorectal cancer cases were examined for the presence of MSI and for the immunohistochemical expression of hMLH1 and hMSH2 proteins. Of this group, 129 (68.6%) were classified as MSS, 17 (9.0%) as MSI-L, and 42 (22.3%) as MSI-H. None of the MSS and none of the MSI-L tumors had altered expression of either hMLH1 or hMSH2. However, the majority of MSI-H (40 of 42, 95%) cases demonstrated absence of staining for these proteins. The most frequently altered protein was hMLH1, occurring in 95% of the tumors with altered expression. Cumulatively, these data suggest that the tumor phenotype MSI-H is distinct from tumor phenotypes MSI-L and MSS, with no apparent differences between MSI-L and MSS. Furthermore, altered hMLH1 protein expression appears to be responsible for the mutator phenotype in the vast majority of MSI-H tumors.

Loss or altered subcellular localization of p27 in Barretts associated adenocarcinoma.

Year 1998
Singh SP. Lipman J. Goldman H. Ellis FH Jr. Aizenman L. Cangi MG. Signoretti S. Chiaur DS. Pagano M. Loda M.
Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215, USA.
The cyclin-dependent kinase inhibitor p27 is a negative regulator of the cell division cycle. It is expressed at the highest levels during the quiescent (G0) and prereplicative (G1) phases, and its degradation is required for entry into the S phase. Because lack of p27 is associated with aggressive behavior in a variety of tumors of epithelial and lymphoid origin, we used immunohistochemistry and in situ hybridization to evaluate the expression of p27 in metaplastic and dysplastic Barrett's epithelium and to assess its prognostic significance in Barrett's associated adenocarcinoma (BAA) of the esophagus. In metaplastic Barrett's epithelium, p27 protein and mRNA were restricted to the superficial third of glands in all cases and extended to the lower third in 4 cases. In contrast, expression of p27 message and protein was both increased and full-thickness, in the 23 cases with high-grade dysplasia adjacent to BAA and in carcinoma in situ. Although all invasive carcinomas had elevated levels of p27 mRNA, 45 (83%) of 54 invasive carcinomas had low p27 protein levels (

Human pancreatic adenocarcinomas express Fas and Fas ligand yet are resistant to Fas-mediated apoptosis.

Year 1998
Ungefroren H. Voss M. Jansen M. Roeder C. Henne-Bruns D. Kremer B. Kalthoff H.
Research Unit Molecular Oncology, Clinic for General Surgery and Thoracic Surgery, Christian-Albrechts-University, Kiel, Germany.
The Fas system, comprising the Fas receptor (Fas, CD95, APO-1) and its ligand, Fas ligand (FasL), is a central mediator of programmed cell death in various physiological and pathological processes. Recent evidence indicated that tumor cells can exploit this system to their benefit in the dialogue with the host immune system. We have shown that all human pancreatic adenocarcinoma cell lines tested by fluorescence-activated cell sorting analysis (6 of 6) and immunocytochemistry (12 of 12) were positive for Fas expression, as were normal and malignant duct cells in pancreatic tissue sections. However, despite Fas expression, pancreatic tumor cells have become largely resistant toward recombinant FasL- or anti-APO-1 agonistic antibody-induced apoptosis. This resistance correlated with high levels in pancreatic tumor cells of mRNA for FAP-1, a Fas-associated phosphatase that can block the apoptotic function of Fas. Using a variety of methodological approaches, we also present evidence for the production of FasL by pancreatic tumor cells because 6 of 6 pancreatic tumor cell lines were found to contain FasL mRNA as well as the Mr 40,000 and Mr 26,000 forms of the FasL protein. Likewise, pancreatic tissue revealed FasL-specific immunostaining in pancreatic tumor cells but not in the surrounding stroma. In coculture experiments, pancreatic tumor cells displayed a cytotoxic effect toward the Fas-sensitive Jurkat T-cell line, which could be inhibited by a FasL-specific neutralizing antibody. Together, these results support the recently proposed "counterattack model" for local deletion of tumor-reactive T-cells by tumor cell-derived FasL.

Size-dependent increase in prostanoid levels in adenomas of patients with familial adenomatous polyposis.

Year 1998
Yang VW. Shields JM. Hamilton SR. Spannhake EW. Hubbard WC. Hylind LM. Robinson CR. Giardiello FM.
Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA. vyang@welchlink.welch.jhu.edu
Recent studies indicate that nonsteroidal anti-inflammatory drugs have a chemopreventive effect against colorectal neoplasia. Nonsteroidal anti-inflammatory drugs inhibit cyclooxygenases, principal enzymes that mediate the formation of prostanoids. To determine whether prostanoids are involved in the pathogenesis of colorectal adenomas, we compared the levels of five major stable metabolic products of the cyclooxygenase pathway in the normal-appearing mucosa and in adenomas of patients with familial adenomatosis polyposis. Of 12 patients tested, 6 had elevated levels of at least one prostanoid in the adenomas. More importantly, the relative levels of three prostanoids [prostaglandin (PG)D2, PGE2, and 6-keto-PGF1alpha] were elevated in adenomas compared to normal-appearing mucosa from the same patients, and the resulting ratios were correlated with the size of the adenoma. These results suggest a role for prostanoids in progression of colorectal polyposis in familial adenomatosis polyposis patients.

Up-regulation of Fas (APO-1/CD95) ligand and down-regulation of Fas expression in human esophageal cancer.

Year 1998
Gratas C. Tohma Y. Barnas C. Taniere P. Hainaut P. Ohgaki H.
Unit of Molecular Pathology, International Agency for Research on Cancer, Lyon, France.
Fas (APO-1/CD95) is a cell surface receptor that mediates apoptosis when it reacts with Fas ligand (FasL) or Fas antibody. In this study, we analyzed Fas and FasL expression in normal esophageal mucosa and esophageal squamous cell carcinomas. Reverse transcriptase-PCR revealed that Fas, soluble Fas, and FasL were expressed in all eight esophageal squamous carcinoma cell lines analyzed. Furthermore, it was demonstrated that FasL expressed in esophageal carcinoma cells is functional because coculture experiments using FasL-expressing TE-15 esophageal carcinoma cells resulted in apoptosis of Jurkat T leukemia cells, which are sensitive to Fas-mediated apoptosis. Immunohistochemistry of Fas and FasL showed that they are constitutively expressed in normal esophageal mucosa, FasL being predominantly in the basal and suprabasal layers, whereas Fas is in more differentiated layers, i.e., rows of polyhedral cells of the intermediate layers and squamous cells forming the outer layers. In 18 of 19 invasive esophageal squamous cell carcinomas, FasL expression was found in >50% of tumor cells. In contrast, most tumors (15 of 19, 79%) either showed no Fas expression or showed expression in

Somatic mutations in LKB1 are rare in sporadic colorectal and testicular tumors.

Year 1998
Avizienyte E. Roth S. Loukola A. Hemminki A. Lothe RA. Stenwig AE. Fossa SD. Salovaara R. Aaltonen LA.
Department of Medical Genetics, Haartman Institute, University of Helsinki, Finland.
Germ-line mutations in a serine/threonine kinase gene, LKB1, were recently shown to underlie Peutz-Jeghers syndrome (PJS), a hereditary disorder that predisposes to benign and malignant tumors of multiple organ systems. Most mutations that have been described thus far dramatically change the predicted protein and are likely to be of an inactivating nature. This observation and a previous observation that the LKB1 locus is often deleted in PJS polyps suggest that the gene may function as a tumor suppressor. We examined whether somatic mutations in this gene are present in sporadic carcinomas of the colon and testis, tumors that are characteristic of PJS. First, 20 randomly selected colorectal and 28 testicular tumors were analyzed by single-strand conformation polymorphism analysis. No mutations in LKB1 were found in colorectal tumors. One testicular tumor displayed a heterozygous missense type variant, in which glycine 163 was changed to aspartic acid. This change was absent in the DNA of normal tissue. To better focus our efforts, we tested 75 additional colon carcinomas for loss of heterozygosity at 19p, where LKB1 is localized. Of 75 samples analyzed, 50 were informative with a closely linked marker, D19S886, and 13 (26%) of these displayed loss of heterozygosity. The 13 tumors were scrutinized for LKB1 mutations by genomic sequencing. This analysis revealed no changes. Together, these findings suggest that somatic mutations of LKB1 are not frequent in colorectal and testicular cancer.

Cloning, characterization, and chromosomal localization of a gene frequently deleted in human liver cancer (DLC-1) homologous to rat RhoGAP.

Year 1998
Yuan BZ. Miller MJ. Keck CL. Zimonjic DB. Thorgeirsson SS. Popescu NC.
Laboratory of Experimental Carcinogenesis, Division of Basic Sciences, National Cancer Institute, NIH, Bethesda, Maryland 20892-4255, USA.
The isolation of genes involved in cancer development is critical for uncovering the molecular basis of cancer. We report here the isolation of the full-length cDNA and chromosomal localization of a new gene frequently deleted in liver cancer (DLC-1) that was identified by representational difference analysis. Loss of heterozygosity was detected for DLC-1 in 7 of 16 primary hepatocellular carcinomas (HCCs) and in 10 of 11 HCC cell lines. Although mRNA for DLC-1 was expressed in all normal human tissues, it was not expressed in 4 of 14 HCC cell lines. Full-length cDNA for DLC-1 of 3800 bp encodes a protein of 1091 amino acids, has 86% homology with rat p122 RhoGAP gene, and was localized by fluorescence in situ hybridization on chromosome 8 at bands p21.3-22. Deletions on the short arm of chromosome 8 are recurrent in liver, breast, lung, and prostate cancers, suggesting the presence of tumor suppressor genes. DLC-1 may be a tumor suppressor gene in liver cancer as well as in other cancers.

Expression of urokinase-type plasminogen activator (u-PA), u-PA receptor, and tissue-type PA messenger RNAs in human hepatocellular carcinoma.

Year 1998
De Petro G. Tavian D. Copeta A. Portolani N. Giulini SM. Barlati S.
Department of Biomedical Sciences and Biotechnology, University of Brescia, Italy.
Expression of plasminogen activators (PAs) and urokinase-type PA receptor (u-PAR) is associated with tumor growth and invasion. For in vivo human tumor tissues, there is no information on gene expression of PAs in hepatocellular carcinoma (HCC) or other hepatic pathophysiological conditions. In this study we examined the relative levels of u-PA, tissue-type PA (t-PA), and u-PAR mRNA expression in human HCC by reverse transcription-PCR compared with those expressed in peritumoral hepatic tissues. Twenty-five of 25 HCCs expressed u-PA mRNA, as well as 16 of 25 hepatic peritumoral tissues. However, none of the 14 cases of nontumorous liver samples (i.e., normal parenchyma, steatosis, and nonspecific reactive and chronic hepatitis) showed detectable levels of u-PA mRNA. The same samples analyzed for uPAR and t-PA mRNAs exhibited higher levels of these mRNAs in the malignant tissues compared with nontumorous ones. A strong correlation was found between the relative levels of u-PA and t-PA mRNAs detected in the tumor and in the corresponding peritumoral tissues (P < 0.001 for u-PA; P < 0.02 for t-PA). However, there was no correlation between the expression of u-PA and t-PA in HCC (P = 0.565). Furthermore, a significant inverse correlation was found between survival months of male patients and the relative level of u-PA mRNA (P < 0.05) detected at the time of biopsy, whereas no correlation was found in the case of t-PA mRNA. These results are in line with the possible differential biological role of u-PA and t-PA in the tumor etiopathogenesis and suggest that the detection of relative levels of u-PA mRNA may be a useful prognostic factor for male HCC patients.

Adenovirus-mediated wild-type p53 gene transfer down-regulates vascular endothelial growth factor expression and inhibits angiogenesis in human colon cancer.

Year 1998
Bouvet M. Ellis LM. Nishizaki M. Fujiwara T. Liu W. Bucana CD. Fang B. Lee JJ. Roth JA.
Department of Surgical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston 77030, USA.
Recent studies have indicated that angiogenesis may be regulated, in part, by p53 tumor suppressor gene function. We hypothesized that wild-type p53 replacement would down-regulate vascular endothelial growth factor (VEGF) expression and inhibit angiogenesis. KM12L4 and SW620, human colon cancer cell lines with p53 mutations, were transduced with a replication-defective adenoviral vector containing the wild-type p53 gene (Ad5/CMV/p53). Reverse transcription-PCR confirmed the presence of p53 in Ad5/CMV/p53-transduced cells. Transduction of colon cancer cells with wild-type p53 decreased VEGF RNA expression compared with that of controls. The decrease in VEGF expression in SW620 cells was dose dependent, with a 49% decrease observed at a multiplicity of infection of 50, and a 71% decrease observed at a multiplicity of infection of 100. Similar effects were seen in KM12L4 cells. VEGF supernatant protein levels were significantly reduced compared with those in nontransduced controls 48 h after the introduction of wild-type p53. Ad5/CMV/p53 inhibited tumor cell-induced angiogenesis in vivo. Restoration of wild-type p53 expression may decrease tumor growth by inhibiting the angiogenic response. These findings may explain, in part, the bystander effect seen with p53 tumor suppressor gene therapy.

Antioxidants reduce cyclooxygenase-2 expression, prostaglandin production, and proliferation in colorectal cancer cells.

Year 1998
Chinery R. Beauchamp RD. Shyr Y. Kirkland SC. Coffey RJ. Morrow JD.
Department of Medicine, The Vanderbilt Cancer Center, Vanderbilt University Medical Center, Nashville, Tennessee 37232, USA.
Increased expression of cyclooxygenase (COX) and overproduction of prostaglandins (PGs) have been implicated in the development and progression of colorectal cancer (CRC). Recent observations suggest that reactive oxygen intermediates play a role in tumor cell growth regulation and expression of the inducible COX, COX-2. We therefore evaluated the effects of various antioxidants on COX expression and cellular growth in the human CRC cell line HCA-7. The antioxidants pyrrolidinedithiocarbamate (PDTC), N-acetylcysteine, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), and U74006 decreased PG production, intracellular redox status, and cellular growth in a concentration-dependent manner. The decrease in cellular growth was associated with the induction of apoptosis. Unlike the selective COX inhibitors 1-[(4-methylsulfonyl)phenyl]-3-trifluoromethyl-5-[(4-fluoro)phenyl]pyraz ole (SC 58125) and (2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide (NS 398) that inhibit COX-2 catalytic activity, these antioxidants decreased COX-2 expression at the transcriptional level. Combined treatment of HCA-7 cells with PDTC and SC 58125 resulted in an additive decrease in PG levels and anchorage-dependent and -independent growth. Furthermore, whereas antioxidants or SC 58125 reduced tumor growth in vivo, coadministration of PDTC and SC 58125 resulted in actual tumor regression. These results suggest that combined therapy with NSAIDs and antioxidants might be useful in the prevention and/or treatment of CRC.

Alterations in pancreatic, biliary, and breast carcinomas support MKK4 as a genetically targeted tumor suppressor gene.

Year 1998
Su GH. Hilgers W. Shekher MC. Tang DJ. Yeo CJ. Hruban RH. Kern SE.
Department of Pathology, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21205-2196, USA.
Mitogen-activated protein kinase (MAPK) kinase 4 (MKK4) is a component of a stress and cytokine-induced signal transduction pathway involving MAPK proteins. The MKK4 protein has been implicated in activation of JNK1 and p38 MAPK on phosphorylation by conserved kinase pathways. A recent report on the deletion and mutation of the MKK4 gene in human pancreatic, lung, breast, testicle, and colorectal cancer cell lines suggests an additional role for MKK4 in tumor suppression. Both the gene function and the infrequency of mutations might be considered atypical for many human tumor suppressor genes, and constitutional DNA was not previously available to determine whether the reported sequence variants had preceded tumor development. Here, we report that homozygous deletions are detected in 2 of 92 pancreatic adenocarcinomas (2%), 1 of 16 biliary adenocarcinomas (6%), and 1 of 22 breast carcinomas (when combined with reported sequence alterations, 3 of 22 or 14%). In addition, in a panel of 45 pancreatic carcinomas prescreened for loss of heterozygosity, one somatic missense mutation of MKK4 is observed and confirmed in the primary tumor (2%). Mapping of the homozygous deletions further indicated MKK4 to lie at the target of deletion. The finding of a somatic missense mutation in the absence of any other nucleotide polymorphisms or silent nucleotide changes continues to favor MKK4 as a mutationally targeted tumor suppressor gene. Coexistent mutations of other tumor suppressor genes in MKK4-deficient tumors suggest that MKK4 may participate in a tumor suppressive signaling pathway distinct from DPC4, p16, p53, and BRCA2.

Purification and characterization of a tumor lipid-mobilizing factor.

Year 1998
Todorov PT. McDevitt TM. Meyer DJ. Ueyama H. Ohkubo I. Tisdale MJ.
Pharmaceutical Sciences Institute, Aston University, Birmingham, United Kingdom.
Cancer patients with weight loss showed urinary excretion of a lipid-mobilizing factor (LMF), determined by the ability to stimulate lipolysis in isolated murine epididymal adipocytes. Such bioactivity was not detectable in the urine of cancer patients without weight loss or in normal subjects. The LMF was purified using a combination of ion exchange, exclusion, and hydrophobic interaction chromatographies to give a single component of apparent Mr 43,000, which showed homology in amino acid sequence with human plasma Zn-alpha2-glycoprotein. Both substances showed the same mobility on denaturing and nondenaturing gels and the same chymotrypsin digestion pattern, both stained heavily for carbohydrate, and they showed similar immunoreactivity. Polyclonal antisera to human plasma Zn-alpha2-glycoprotein was also capable of neutralization of the bioactivity of human LMF in vitro. Using competitive PCR to quantify expression of Zn-alpha2-glycoprotein, we found that only those tumors that were capable of producing a decrease in carcass lipid expressed mRNA for Zn-alpha2-glycoprotein. These results provide strong evidence to suggest that tumor production of Zn-alpha2-glycoprotein is responsible for the lipid catabolism seen in cancer patients.

Biological evaluation of a lipid-mobilizing factor isolated from the urine of cancer patients.

Year 1998
Hirai K. Hussey HJ. Barber MD. Price SA. Tisdale MJ.
Department of Obstetrics and Gynecology, Osaka City University Medical School, Japan.
We have previously shown human lipid-mobilizing factor (LMF) to be homologous with the plasma protein Zn-alpha2-glycoprotein in amino acid sequence, electrophoretic mobility, and immunoreactivity. In this study, both LMF and Zn-alpha2-glycoprotein have been shown to stimulate glycerol release from isolated murine epididymal adipocytes with a comparable dose-response profile. Both LMF and Zn-alpha2-glycoprotein caused a stimulation of adenylate cyclase in murine adipocyte plasma membranes in a GTP-dependent process, with maximum stimulation at 0.1 microM GTP and with saturation at protein concentrations of >5 microg/assay. Administration of LMF to exbreeder male mice over a 89-h period produced a decrease in body weight without a change in food and water intake. Body composition analysis showed a 42% reduction in carcass lipid when compared with controls. Treatment of ob/ob mice with human LMF over a 160-h period also produced a decrease in body weight, with a 19% reduction in carcass fat, without a change in body water or nonfat mass. Serum levels of glycerol and 3-hydroxybutyrate were significantly increased, as was oxygen uptake by interscapular brown adipose tissue, providing evidence of increased lipid mobilization and utilization. Human white adipocytes responded to both LMF and isoprenaline to the same extent, although the maximal response was lower than that for murine white adipocytes. These results suggest that LMF not only has the capacity to induce lipid mobilization and catabolism in mice, but it also has the potential to exert similar effects in cachectic cancer patients.

Determination of somatostatin receptor subtype 2 in carcinoid tumors by immunohistochemical investigation with somatostatin receptor subtype 2 antibodies.

Year 1998
Janson ET. Stridsberg M. Gobl A. Westlin JE. Oberg K.
Department of Internal Medicine, University Hospital, Uppsala, Sweden. Eva.TiensuuvJanson@medicin.uu.se
We have shown previously that expression of mRNA for somatostatin receptor subtype 2 (sst2) detected by in situ hybridization correlates to therapeutic outcome in patients with carcinoid tumors treated with somatostatin analogues. However, in situ hybridization is laborious and not practical in clinical routine work. We have, therefore, developed polyclonal antibodies directed against sst2 that may be used for immunohistochemistry on tissue specimens. The staining is specific and is highly correlated to expression of mRNA for sst2 (P < 0.01) as well as to tracer uptake at somatostatin receptor scintigraphy (P < 0.01). There is also a good correlation to the therapeutic response in carcinoid patients treated with somatostatin analogues (P < 0.05). Of 35 patients with carcinoid tumors included in this investigation, 25 stained positive with the antibodies. Twenty-two of these were investigated by somatostatin receptor scintigraphy and showed tracer uptake in metastases. An additional two patients that did not stain with the antibodies showed pathological uptake of the tracer in metastases, which might indicate binding to somatostatin receptor subtype 5. None of the 10 patients without positive immunostaining responded to somatostatin analogue treatment, whereas patients with a positive stain had a biochemical response or remained stable during treatment. Thus, these antibodies may be used to determine the presence of sst2 in carcinoid tumors and to select patients suitable for somatostatin analogue treatment. The method is easily applicable in clinical practice.

Identification of a novel gene marker specific for epithelial cells by utilizing a 3-directed cDNA library.

Year 1998
Ohnishi T. Tomita N. Miyoshi Y. Shinoki N. Tanaka T. Kawabata Y. Sekimoto M. Monden T. Okubo K. Matsubara K. Monden M.
Department of Surgery II, Biomedical Research Center, Osaka University Medical School, Suita, Japan.
To achieve reliability of molecular diagnosis using reverse transcription-PCR (RT-PCR), we established a unique method to search for a novel gene marker specific for colonic epithelial cells. Of eight candidate genes selected from a 3'-directed cDNA library in colonic mucosa, two genes were expressed in normal mucosa and cancer of the colon but not in either normal lymph node or normal liver tissue. Known sequences of these genes were reported to be located in the 3' noncoding region, and an additional sequence just upstream to gs04094 (one of the candidate genes) was determined. According to the newly identified sequence, we designed a new set of primers so that we could distinguish the DNA fragment amplified in RT-PCR from that in genomic PCR. RT-PCR using these primers demonstrated that gs04094 was expressed in all of 10 primary colon cancers and 4 liver metastases from colon cancer but in none of 5 normal lymph nodes, 10 peripheral blood samples, and 2 normal liver tissues. Sensitivity of this method was so high as to detect gs04094 mRNA in 10(-6) microg of colon cancer RNA per 1 microg of normal lymph node RNA. Thus, our strategy to search for a novel gene marker using 3'-directed cDNA library proved to be highly efficient.

Identification of two common regions of allelic loss in chromosome arm 12q in human pancreatic cancer.

Year 1998
Kimura M. Furukawa T. Abe T. Yatsuoka T. Youssef EM. Yokoyama T. Ouyang H. Ohnishi Y. Sunamura M. Kobari M. Matsuno S. Horii A.
Department of Molecular Pathology, Tohoku University School of Medicine, Sendai, Japan.
Using the method of microsatellite analysis, we studied 40 tissues with pancreatic ductal adenocarcinoma and identified two commonly deleted regions on the long arm of chromosome 12. One (region A) was found between D12S81 and D12S1719 at 12q21 at a frequency of 67.5%, and the other (region B) was located between D12S360 and D12S78 at 12q22-q23.1 at a frequency of 60%; the latter was reported previously (M. Kimura, et al. Genes Chromosomes Cancer, 17: 88-93, 1996). The results of microsatellite analyses were verified by fluorescence in situ hybridization. We further analyzed 19 pancreatic cancer cell lines by fluorescence in situ hybridization and found that 10 of them showed allelic loss at D12S81 and 6 showed allelic loss at D12S360. Yeast artificial chromosome contigs were constructed to cover the deleted regions. Region B was completely covered by a 650-kb yeast artificial chromosome clone. The frequently deleted regions in chromosome 12q in pancreatic cancer that were identified here may provide new avenues for isolating novel tumor suppressor genes.

Activation of the beta-catenin gene in primary hepatocellular carcinomas by somatic alterations involving exon 3.

Year 1998
Miyoshi Y. Iwao K. Nagasawa Y. Aihara T. Sasaki Y. Imaoka S. Murata M. Shimano T. Nakamura Y.
Department of Medical Genetics, Biomedical Research Center, Osaka University Medical School, Japan.
We screened 75 primary hepatocellular carcinomas for somatic mutations in the entire coding region of the beta-catenin gene. We detected somatic mutations in 14 tumors; 12 were considered to cause amino acid substitutions and 2 were interstitial deletions of 51 or 195 nucleotides of genomic DNA, corresponding to exon 3. Among the 12 point mutations, 6 occurred at potential serine/threonine phosphorylation residues of codons 33, 41, or 45. The remaining six tumors contained a mutation at codon 32 (aspartic acid) or 34 (glycine), flanking to the serine residue at codon 33. By Western blot analysis, we confirmed accumulation of beta-catenin in five tumors for which frozen tissues were available; the five included tumors in which amino acid alterations had occurred at codons 32, 34, or 45, and one with a 17-amino acid deletion. Our results suggested that accumulation of beta-catenin due to amino acid substitutions at potential serine/threonine phosphorylation residues or at their neighboring codons or interstitial deletions involving exon 3 could contribute to hepatocellular carcinogenesis.

Epitope mapping of a series of human thymidylate synthase monoclonal antibodies.

Year 1998
Behan KA. Johnston PG. Allegra CJ.
National Cancer Institute, Medicine Branch at the National Naval Medical Center, Bethesda, Maryland 20889-5105, USA.
We have reported previously the development and application of several monoclonal antibodies to thymidylate synthase (TS). In this study, we used a series of overlapping 17-mer peptides that spanned the entire TS protein to map the epitope recognized by three TS monoclonal antibodies (TS 106, TS 109, and TS 110). Using an ELISA, we identified two peptides (R126-F142 and L131-R147) that bound all three antibodies, which suggests that each antibody recognized a similar epitope on TS. A second set of peptides, representing sequential single-residue truncations from either the amino terminus or the carboxyl terminus starting with a G129-E145 17-mer, was synthesized. A 10-amino acid sequence P133-F142 (PVYGFQWRHF) was identified as the binding epitope for all three antibodies. Further investigation via substitution mutational analysis of each residue within this epitope revealed that residues F137, W139, R140, H141, and F142 were critical for maximal binding of TS 106 and TS 110. TS 109 showed a similar pattern except in regard to R140, with which there was no apparent loss of binding. In addition to the utility of the three antibodies in detecting and measuring TS levels, identification of the binding locus permits the potential application of these antibodies in the investigation of TS enzymatic and regulatory function.

Expression of a novel antiapoptosis gene, survivin, correlated with tumor cell apoptosis and p53 accumulation in gastric carcinomas.

Year 1998
Lu CD. Altieri DC. Tanigawa N.
Department of General and Gastroenterological Surgery, Osaka Medical College, Takatsuki City, Japan.
A novel inhibitor of apoptosis designated survivin has recently been found in many common human cancers but not in normal tissues. A potential distribution of survivin in gastric cancer and its implication for apoptosis inhibition have been investigated. Recombinant survivin expressed in Escherichia coli as a glutathione S-transferase fusion protein was used to raise a novel panel of mouse monoclonal antibodies. In an immunohistochemical analysis of 174 cases of gastric carcinomas (stages I-III), anti-survivin monoclonal antibody 8E2 (IgG1) reacted with 34.5% of cases (60 of 174 cases) with a variable number of tumor cells stained (20-100%). In contrast, no expression of survivin in neighboring normal tissues was observed. When stratified for p53 and bcl-2 expression and apoptotic index, the expression of survivin significantly segregated with p53- and bcl-2-positive cases [56.1 versus 15.2% (P = 0.001) and 69.2 versus 31.6% (P = 0.006), respectively] and with a decreased apoptotic index as compared with that of survivin-negative tumors (0.97 +/- 0.64 versus 0.62 +/- 0.39%, P < 0.001). These data identify a role for survivin in promoting aberrantly increased cell viability in gastric cancer and suggest a potential correlation between accumulated p53 and survivin expression in neoplasia.

Transactivation of transforming growth factor alpha gene by hepatitis B virus preS1.

Year 1998
Ono M. Morisawa K. Nie J. Ota K. Taniguchi T. Saibara T. Onishi S.
The First Department of Internal Medicine, Kochi Medical School, Nankoku Kochi, Japan.
Hepatitis B virus (HBV) causes acute and chronic liver injury, and integration of HBV DNA is considered to be an important pathogenic determinant for hepatocarcinogenesis and tumor development. Transforming growth factor alpha (TGF-alpha) drastically accelerates hepatocarcinogenesis when it is overexpressed in TGF-alpha transgenic mice (C. Jhappan et al., Cell, 61: 1137-1146, 1990). In HBV-infected patients, hepatocellular carcinoma (HCC) cells show elevated expression of TGF-alpha (C. C. Hsia et al., J. Med. Virol., 43: 216-221, 1994), the mechanism for which, however, has not been clarified yet. We here show that preS1, a part of the HBV large surface protein, carries a transcriptional transactivation domain and activates the transcription of the TGF-alpha gene by 2-fold in human HCC HuH6 cells. The responsive elements are restricted to the 315-bp segment of the proximal TGF-alpha promoter (-373 to -59). Furthermore, the expression of TGF-alpha was markedly increased in permanently preS1-producing HuH6 transformants. The crucial role for HBV preS1 in hepatocarcinogenesis and tumor development through transactivation of the TGF-alpha gene may give us new insight into the understanding of pathogenesis and therapy of viral hepatocarcinogenesis.

Absence of a radiation-induced first-cycle G1-S arrest in p53+ human tumor cells synchronized by mitotic selection.

Year 1998
Nagasawa H. Keng P. Maki C. Yu Y. Little JB.
Department of Cancer Cell Biology, Harvard School of Public Health, Boston, Massachusetts 02115, USA.
It is well known that normal human diploid fibroblasts undergo a significant, p53-dependent arrest in the G1 phase of the cell cycle after exposure to ionizing radiation. The presence and magnitude of a G1 arrest in human tumor cell lines, however, has been controversial, particularly in cells derived from solid tumors and irradiated during exponential growth. To examine this question more precisely, we synchronized cells by mitotic selection and irradiated them in very early G1 prior to any of the described G1 checkpoints. Progression of cells from G1 into the S phase was monitored by autoradiographic measurement of cumulative labeling indices and by flow cytometric analysis. Three different human tumor cell lines confirmed as expressing normal p53 function were examined, i.e., lines derived from an adenocarcinoma of the colon (RKO), a breast cancer (MCF-7), and a squamous cell carcinoma (SCC61). Following irradiation with 4-8 Gy, there was a transient delay in progression from G1 into S phase, lasting approximately 2 h, and in two of the three cell lines (RKO and MCF-7), a small fraction of cells (5-8%) never entered the first S phase. Although there was no evidence for a prolonged G1 arrest, the expected G2 delay was observed in all three cell lines. When irradiated RKO cells were resynchronized at the next mitosis, approximately 30% of the cells did not enter the second S phase. This latter finding is consistent with earlier reports on the kinetics of radiation-induced reproductive failure in mammalian cells. These results indicate that cells derived from human solid tumors that express normal p53 may respond to irradiation quite differently than do normal cells in terms of G1 checkpoint control.

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