Genetic alterations in the coding region of the bak gene in uterine cervical carcinoma.

A significant frequency of mutations (six missense and one silent) was found, for the first time, at the coding region of the bak gene (exons 3, 4 and 6) in 42 carcinomas of the uterine cervix, while no mutations were detected in 32 non-neoplastic cervix tissues. Bak mutations were observed more frequently in the advanced stage and mutated cancer tissues were more resistant to radiotherapy, although trends were not statistically significant because of small sample size.

Effects of N-methyl-N'-nitro-N-nitrosoguanidine on the human colorectal polyps consecutively maintained in SCID mice.

N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatments for a long period induced morphological and molecular alterations in the benign human colorectal polyps which were maintained in the severe combined immunodeficient C.B17/N-scid/scid mice. Thirty four xenografts of colorectal polyps from five solitary polyp and three familial polyposis patients were examined for K-ras and p53 mutations. Six K-ras mutations were induced in 16 grafts treated with MNNG more than five times, while no K-ras mutations were detected in 14 untreated grafts (P<0.05). Additional and new K-ras mutations were also induced in two polyps in which K-ras mutation had pre-existed. p53 mutations were not observed in both MNNG-treated and untreated groups. The mutations in K-ras gene were induced at codon 12 (GGT-->GAT) except one at codon 13 (GGC-->GGT). The results indicate that K-ras mutation plays an important role in human colorectal carcinogenesis as is the case in experimental animals.

Familial nasal NK/T-cell lymphoma and pesticide use.

Familial occurrence of nasal NK/T-cell lymphoma (NNKTCL) in pesticide users is presented. The proband (71 years old, male) and son (39 years old) were both diagnosed with NNKTCL within interval of 26 months. Laboratory data showed slight anemia, with no abnormal cells in peripheral blood. They and their wives were farmers and used large amounts of pesticides (fungicides and insecticides) in the hothouse. NNKTCL did not develop in the wives. Proband's father was diagnosed with malignant lymphoma of the neck and died of the disease. Genetic analyses of the peripheral blood leukocytes and tumor tissues did not show p53 and k-ras gene mutations and microsatellite instability. Metaphase cells from peripheral blood leukocytes bore specific marker chromosomes (father, 44XY,-14,-17,-18,-22,+2mar; son, 46XY,-17,+1mar). Environmental exposures to pesticides in conjunction with familial or genetic factors might increase the risk for NNKTCL.

Dedifferentiation of adenoid cystic carcinoma: report of a case implicating p53 gene mutation.

Adenoid cystic carcinoma is an indolent tumour with an unfavorable long-term prognosis. Dedifferentiation of adenoid cystic carcinoma, which is associated with an accelerated clinical course, has recently been described. We report a case with immunohistochemical and molecular workup to elucidate the likely mechanism of dedifferentiation. The patient, a 64-year-old woman, developed dedifferentiated adenoid cystic carcinoma of the submandibular gland ab initio, accompanied by cervical lymph node metastasis. Histologically, the low-grade adenoid cystic carcinoma merged gradually into an extensive dedifferentiated component that was composed of solid sheets and cords of anaplastic tumor cells with focal gland formation. Immunohistochemically, the dedifferentiated component, but not the adenoid cyst carcinoma component, showed strong overexpression of p53 protein and cyclin D1, as well as a higher Ki67 index. Molecular study confirmed the presence of p53 gene mutation selectively in the dedifferentiated component, suggesting a pivotal role of p53 gene alteration in the dedifferentiation process of adenoid cystic carcinoma.

Epstein-Barr virus associated B-cell lymphoma of brain developing in myelodysplastic syndrome with c-kit mutation (Try-557 -->stop).

The first case of B-cell lymphoma of brain in a patient with myelodysplastic syndrome (MDS) was reported. A 68-year-old man was admitted because of anemia, fever, and thrombocytopenia and was diagnosed as having MDS (refractory anemia with excess of blasts) on the basis of the findings of bone marrow aspiration and chromosomal analysis. The patient was followed up without chemotherapy, but a brain tumor appeared after 3 years. Histologic and immunohistologic examinations revealed diffuse large B-cell lymphoma. Mutations of the c-kit proto-oncogene (stem cell factor receptor) and the p53 tumor-suppressor gene were examined in the MDS lesion and malignant lymphoma (ML) by the polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP) method followed by direct sequencing. The p53 mutation was not found in either MDS or ML, but a nonsense mutation (Try-557 --> stop) in exon 11 of the c-kit, which might lead to dysfunction of tyrosine kinase activity, was detected in MDS. This is the first report of c-kit mutation in MDS. Epstein-Barr virus (EBV) genome was demonstrated in the nucleus of brain ML cells by in situ hybridization with EBV-encoded RNA-1 probe. Immunohistochemistry showed that the tumor cells expressed latent infection gene products, including EBV nuclear antigen-2 and latent membrane protein-1. This pattern of latent gene expression was Lat III, which is usually found in malignant lymphomas developing in immunocompromised hosts. These findings suggest that a profound pancytopenia in MDS resulted in an immunodeficient condition, after which EBV-positive B-cell lymphoma of brain developed.

Specific c-kit mutations in sinonasal natural killer/T-cell lymphoma in China and Japan.

Sinonasal lymphoma is one of the constituents of lethal midline granuloma, which is a clinical term for progressive, destructive lesions affecting the midline of the face. The majority of sinonasal lymphomas, especially those showing polymorphous patterns of proliferation and thus termed polymorphic reticulosis, recently were categorized as sinonasal natural killer/T-cell lymphomas. They are more prevalent in Asia than Europe or North America and are associated with EBV infection. Twenty-three cases with sinonasal natural killer/T-cell lymphomas were collected from two high-incidence regions: Beijing, China (14 cases) and Osaka, Japan (9 cases). c-kit mutations were analyzed on paraffin-embedded specimens by PCR-single-strand conformation polymorphism followed by direct sequencing; the c-kit proto-oncogene encodes a receptor of tyrosine kinase, which plays an important role in the regulation of normal and neoplastic hematopoiesis by the interaction with its specific ligand, termed stem cell factor. Twelve single nucleotide substitution mutations were seen in 23 cases. Ten of 14 Chinese cases (71.4%) had mutations at exon 11 or exon 17, whereas only two of nine Japanese cases (22.2%) had mutations, showing a significant difference in frequency between Chinese and Japanese cases. Furthermore, seven of eight mutations (92%) in exon 17 occurred at codon 825 and three of four mutations (75%) in exon 11 occurred at codon 561. Such a specificity has not been reported before, and these results, taken together, suggest that location-specific differences in etiological factors cause specific mutations in c-kit gene.

Microsatellite instability and k-ras, p53 mutations in thyroid lymphoma.

Patho-epidemiological studies showed that thyroid lymphoma (TL) arises in inflammatory lesions of chronic lymphocytic thyroiditis (CLTH). Replication error (RER) is found in inflammatory lesions and associated cancer, suggesting that chronic inflammation could be a risk factor for neoplastic development through causing RER. To clarify whether RER is involved in the pathogenesis of TL, we examined the microsatellite instability (MSI) in 9 cases with CLTH and 19 with TL, including 10 diffuse large B-cell lymphoma (DLBL), 4 follicle center cell lymphoma, 3 marginal zone B-cell lymphoma of extranodal (MALT) type, and 2 lymphoplasmacytic type. Sixteen distinct microsatellite repeats were analyzed. Mutations of p53 and k-ras genes were also examined. When alterations at 2 or more microsatellite loci were judged as positive, only 5 DLBL cases exhibited MSI. The frequency of MSI in DLBL was significantly higher than that in other types of TL and CLTH (P < 0.05). Four of 19 cases (21.1%) showed point mutation of the k-ras gene. The k-ras mutations occurred in the cases with DLBL with RER, and four of five cases with RER had a k-ras mutation, indicating a close association between RER and k-ras mutation. p53 mutations were not found in the CLTH. Two of 19 TL cases showed mutations of p53 gene. There was no significant association between RER and p53 mutation. These findings indicate that genomic instability contributes to the progression of TL from low grade to high grade, but not to the development of low grade lymphoma in CLTH lesions.

Mutations of the p53 gene in nasal NK/T-cell lymphoma.

Mutations of the p53 tumor suppressor gene are reported in various kinds of malignancies including lymphomas. However, p53 gene mutations in nasal NK/T-cell lymphoma have not been reported because most parts of tumors are necrotic and a small amount of living tumor tissues is available for the molecular study. Expression and mutations of the p53 gene were examined in the paraffin-embedded specimens of the nasal lesions from 42 Chinese (Beijing and Chengdu) and Japanese (Okinawa and Osaka) patients with nasal NK/T-cell lymphoma by the immunohistochemistry and single strand conformation polymorphism (SSCP) analysis of polymerase chain reaction (PCR) amplified products followed by direct sequencing. Thirty single-nucleotide substitution mutations were observed in 20 of 42 cases (47.6%). Among the 30 mutations, 18 were missense (mainly G:C to A:T transitions), 9 were silent, and 1 was a nonsense mutation. The remaining 2 mutations involved intron 5 and exon 5 terminal points. Abnormal expression of the p53 protein was also observed in 19 of 42 (45.2%) cases. The incidence was significantly (4-fold) higher in the cases of Osaka than those in other areas, although the incidence of p53 mutations in the cases of Osaka was one-half to one-third of those in the other three areas. The results may suggest some racial, environmental, or lifestyle differences in the cause of nasal tumorigenesis.

neu mutation in schwannomas induced transplacentally in Syrian golden hamsters by N-nitrosoethylurea: high incidence but low allelic representation.

Peripheral nerve tumors (PNT) and melanomas induced transplacentally on day 14 of gestation in Syrian golden hamsters by N-nitrosoethylurea were analyzed for activated oncogenes by the NIH 3T3 transfection assay, and for mutations in the neu oncogene by direct sequencing, allele-specific oligonucleotide hybridization, MnlI restriction-fragment-length polymorphism, single-strand conformation polymorphism, and mismatch amplification mutation assays. All (67/67) of the PNT, but none of the melanomas, contained a somatic missense T --> A transversion within the neu oncogene transmembrane domain at a site corresponding to that which also occurs in rat schwannomas transplacentally induced by N-nitrosoethylurea. In only 2 of the 67 individual hamster PNT did the majority of tumor cells appear to carry the mutant neu allele, in contrast to comparable rat schwannomas in which it overwhelmingly predominates. The low fraction of hamster tumor cells carrying the mutation was stable through multiple transplantation passages. In the hamster, as in the rat, specific point-mutational activation of the neu oncogene thus constitutes the major pathway for induction of PNT by transplacental exposure to an alkylating agent, but the low allelic representation of mutant neu in hamster PNT suggests a significant difference in mechanism by which the mutant oncogene acts in this species.

High incidence of esophageal cancer in esophageal achalasia by the oral administration of N-amyl-N-methylnitrosamine and its prevention by nicardipine hydrochloride in mice.

Esophageal achalasia (EA) is a rare disease in man and animals and there are many discussions on its higher risk of esophageal cancer. N-Amyl-N-methylnitrosamine (AMN) which specifically induces esophageal tumors in mice and rats was given to three mutant mouse strains, i.e. 101/N, STX/Le and BXH-8, which develop a high incidence of EA. The incidence of EA in 101/N, STX/Le, BXH-8 and normal C57BL/6J mice was 38.5% (110/286), 30.1% (43/143), 91.8% (190/207) and 0% (0/167), respectively. The average numbers of AMN-induced esophageal tumors in EA(+) were significantly higher than those of EA(-) in all of the 101/N, STX/Le and BXH-8 mice. Furthermore, significantly larger size tumors and invasive squamous cell carcinomas were found in EA(+) mice than in EA(-) mice. These results indicate the higher sensitivity of EA for both tumor induction and promotion, possibly due to the longer retention of AMN. In fact, relaxation of the lower esophagus by a smooth muscle relaxing calcium-channel blocker, nicardipine hydrochloride, significantly prevented the induction of esophageal tumors.

Frequent p53 mutations at dipyrimidine sites in patients with pyothorax-associated lymphoma.

A high incidence of non-Hodgkin's lymphoma of the pleural cavity has developed in Japanese patients with long-standing pyothorax (38 years on average) resulting from artificial pneumothorax for the treatment of pulmonary tuberculosis or tuberculous pleuritis. Patients with pyothorax-associated lymphoma (PAL) have long been exposed to antituberculous drugs, antibiotics, bacterial or viral products, and frequent diagnostic radiation for the confirmation of pneumothorax and pyothorax. We analyzed p53 mutations on paraffin-embedded specimens from 21 patients with PAL by PCR-single-strand conformational polymorphism followed by direct sequencing. An unusually high frequency of p53 mutations (14 of 21 cases, 67%) was detected in the PAL specimens, and mutations consisted of 13 nucleotide substitutions and 1 deletion. Furthermore, 10 of 13 substitutions (77%) occurred at dipyrimidine sites (CC:GG to CT:GA substitution). Such specificity has not been reported, except for solar light-related skin cancer and AIDS-related lymphoma in some parts. An UV light mimetic agent may be produced in the long history of chronic inflammation in tuberculosis or immunodeficient patients.

Morphology and function of human benign tumors and normal thyroid tissues maintained in severe combined immunodeficient mice.

In the improved SCID (severe combined immunodeficient) mice, various human benign tumors of the head and neck region were well maintained morphologically and functionally for 3 years until the experiments were terminated, e.g. transplanted parathyroid adenoma secreted parathyroid hormone (PTH) in the SCID mice for more than 1 year. Normal human thyroid tissue was also well maintained in the SCID mice for 3 years. Rapid and high uptake of radioiodine into the transplanted human thyroid tissue was observed. Furthermore, transplanted human thyroid tissue secreted thyroid hormone (T3) and T3 secretion was stimulated by the injection of human thyroid stimulating hormone (TSH). These findings suggest that the improved SCID mice will provide an invaluable experimental system for investigating the function of normal human tissues and the influence of endogenous and exogenous factors on human tissues.

Molecular evidence that most but not all carcinosarcomas of the uterus are combination tumors.

The pathogenesis of carcinosarcoma is still a subject of controversy. In the present study, molecular techniques were applied to determine the pathogenesis of uterine carcinosarcomas. The patterns of chromosome X inactivation were analyzed, targeting a portion of exon 1 of the human androgen receptor (HUMARA) in malignant epithelial and mesenchymal components. The presence of p53 and K-ras mutations were also analyzed. H&E-stained sections of paraffin-embedded, formalin-fixed tissues were microdissected to obtain both epithelial and nonepithelial lesions from 25 carcinosarcomas, and DNAs were extracted by proteinase K digestion. Following treatment with methylation-sensitive restriction endonuclease (HhaI or HpaII), PCR amplification was performed using nested primers targeted to the HUMARA locus. Mutations in the p53 gene and K-ras gene were found in eight (32%) and six (24%) tumors, respectively. The patterns of chromosome X inactivation were different between the carcinomatous and sarcomatous components of three carcinosarcomas, indicating that these three tumors represent collision tumors. By contrast, the patterns of chromosome X inactivation, K-ras sequence, and p53 sequence were identical in both carcinomatous and sarcomatous components in 21 carcinosarcomas, indicating that these 21 tumors represent combination tumors. One case produced equivocal results that precluded determination of whether it represented a collision or combination tumor. These observations show that although most carcinosarcomas are combination tumors, some develop as collision tumors. The determination of histogenesis in individual cases of carcinosarcoma using molecular markers may be worthwhile, because the result could help predict the prognosis of individual cases and help guide clinical management.

Mutations of p53 tumor-suppressor gene in angiosarcoma.

Transgenic mice deficient for the p53 gene were reported to frequently develop angiosarcoma (AS), suggesting that alterations in the gene are associated with tumorigenesis of AS. However, little is known about genetic changes, including p53 gene alterations, in human AS because of its rarity. We analyzed p53 mutations on paraffin-embedded specimens from 33 patients with AS by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) followed by direct sequencing. Age of patients ranged from 18 to 91 (median 70) years, with a male to female ratio of 1.5:1. Sites of tumor were the head in 13 patients, the trunk in 4, the extremities in 4, the heart in 4, bones in 2 and others in 6. PCR-SSCP revealed aberrant mobility shifts of bands in 17 cases: 11 in exon 5, 5 in exon 7 and 4 in exon 8. Direct sequencing on these 17 cases revealed a total of 20 mutations. The frequency of p53 mutations was different by site of tumors: 7 of 13 in head, all 4 in extremities, 2 of 4 in heart and none of 4 in trunk. Our findings suggest that occurrence of p53 mutation is a major pathway for development of human AS.

Induction of cancer, actinic keratosis, and specific p53 mutations by UVB light in human skin maintained in severe combined immunodeficient mice.

To study the mechanism and risk of human skin cancer from solar light, we exposed human skin transplanted to severe combined immunodeficient mice to daily doses of UVB for periods of approximately 2 years. We have succeeded for the first time in inducing cancer and solar (actinic) keratosis in human skin by UVB. Of 18 normal skins exposed to doses of 7.3 x 10(5) to 1.8 x 10(6) J/m2, 14 actinic keratoses (77.8%) and 3 squamous cell carcinomas (16.7%) developed, whereas neither actinic keratosis nor cancer was observed in 15 human skins not exposed to UVB. Each human skin showed a different susceptibility, and skins sensitive for actinic keratosis were also sensitive for cancer induction. Among p53 mutations at various sites, mutation at codon 242 (C TGC --> C CGC; Cys --> Arg) was specifically observed in both skin cancers and actinic keratoses. Furthermore, double or triple mutations were induced in all UVB-induced skin cancers and in three of eight actinic keratoses. Most of the mutations (17 of 20) occurred at dipyrimidine sites.

Consecutive maintenance of human solitary and hereditary colorectal polyps in SCID mice.

Recently, the sequential changes from adenoma to adenocarcinoma have been well studied in human colorectal carcinogenesis. To study the precise clonal changes from colorectal polyps to cancer, we have established an experimental system to maintain human colorectal polyps in severe combined immunodeficient (SCID) mice that have been improved by the selective inbreeding of C.B17-scid/scid homozygous male and female showing undetectable serum IgG and IgM (< 1 microgram/ml). Two of two solitary polyps from two nonhereditary colon polyp patients, four of five colon polyps from two Peutz-Jeghers' syndrome patients and one polypoid lesion from a familial polyposis coli (FAP) patient grew very slowly but steadily, at approximately one-tenth the rate of their malignant form, (i.e., adenocarcinoma), in the improved SCID mice and were maintained for a long period (more than 2 years), over several mouse generations. However, two polyps from FAP and Peutz-Jeghers' syndrome patients could not be transplanted further because of microinfection at the transplanted site due to incomplete sterilization of original human tumors prior to surgical operation (endoscopic polypectomy). Transplanted colon polyps had a semitransparent, soft and sticky appearance, with cells containing large amounts of mucin. Malignant transformation of human colon polyp to adenocarcinoma has not been observed during the maintenance period (about 2 years) in SCID mice. In the consecutively maintained human colon polyps, however, K-ras mutations were detected at codon 12, while these mutations were not found in their original polyps in the patients.

Cytogenotoxicities of sublimed urethane gas to the mouse embryo.

Urethane (ethyl carbamate) which has long been used for commonly used drugs and has proven to be useful in the formation of products in every-day use, is volatile, and small amounts sublime spontaneously. Pregnant ICR mice were maintained in the vinyl chamber (45 liter) which was ventilated 4 times per hour. To inhale urethane gas, air was passed first through a glass bottle containing 500 g of crystalline urethane and then into the vinyl chamber. Concentration of the sublimed urethane gas in the chamber was 1.28 +/- 0.08 mg/l, and sublimed urethane gas produced significantly high incidence of chromosomal aberrations in the cells of whole embryo, when mice inhaled it for 48 h from day 9 to day 11 of pregnancy. High and significant incidence of chromosomal aberrations (36.0%) was detected in the embryo 3 h after urethane gas inhalation, but decreased to 5.3% at 24 h after exposure and showed no significant differences from controls after 48 h, while the incidence in bone marrow cells from the adult (pregnant) mice was lower (21.5%) at 3 h after exposure but a significant increase remained until 72 h after exposure. A majority of chromosomal aberrations was chromatid types. As a consequence of cellular damages by urethane gas inhalation during pregnancy, significantly high incidence of fetal deaths and congenital malformations (cleft palate, polydactyly, tail anomaly etc.) was induced in the offspring. Thus, we must be aware of the risk of volatile chemicals, because it is difficult to perceive and avoid hazardous exposure via respiration.

Mutations of the K-ras and p53 genes in gastric adenocarcinomas from a high-incidence region around Florence, Italy.

K-ras and p53 gene mutations in intestinal-type gastric carcinomas from a high-incidence area around Florence, Italy, were studied by single strand conformation polymorphism and DNA sequencing analysis. Single-strand conformation polymorphism analysis of K-ras indicated aberrant bands in 13 of 34 cases. Sequencing revealed point mutations in 7 (including two at a previously unreported site in codon 11), a significantly higher frequency than reported in countries other than Japan. No K-ras mutations were identified in stage III tumors. Single-strand conformation polymorphisms in p53 exons 5-8 occurred in 30 of 34 cases, with mutations identifiable by direct sequencing in 65% of the cases. Of these, 91% were base substitutions, a value similar to that usually reported, but the percentage of G:C to A:T transitions (90% in this study, 89% in all published European cases combined) differed significantly from that in Oriental cases (48%). The percentage of A:T to G:C transitions was greater in Oriental (22%) than European cases (2%), as was also true for transversions (30% in Oriental tumors, 9% in European tumors). The frequency of mutations at CpG sites (14%) varied significantly from the 67% in cases from a neighboring region in Italy. Helicobacter pylori infection was established in 19 cases and was somewhat more common in cases lacking a p53 mutation.

[Effect of mesalazine microgranules on experimental colitis].

Mesalazine microgranules are an ethylcellulose-coated formulation from which mesalazine is released throughout the intestinal tract and are expected to be effective for idiopathic inflammatory bowel disease, ulcerative colitis and Crohn's disease. Mesalazine microgranules were administered orally to investigate the distribution of mesalazine throughout the intestinal tract in rats. Mesalazine microgranules distributed sufficient amounts of mesalazine and its metabolite, N-acetyl-mesalazine, to the intestinal tissues, while pure mesalazine delivered lower amounts of both. In acetic acid-induced colitis in rats, mesalazine microgranules administered orally reduced the damage score significantly (P < 0.05) at a dose of 50 mg/kg as assessed by macroscopic observation and at 100 mg/kg as assessed by histological evaluation. The number of ulcers in carrageenan-induced colitis in guinea pigs was inhibited at doses of 50, 100, 200 mg/kg, p.o. The colonic wet weight of rats in 2,4,6-trinitrobenzenesulfonic acid (TNB)-induced colitis was reduced significantly (P < 0.05) at a dose of 50 mg/kg, p.o. Mesalazine microgranules showed the ability to distribute mesalazine efficiently throughout the intestinal tract and showed effectiveness against acetic acid-, carrageenan- and TNB-induced colitis. These studies strongly suggest that mesalazine microgranules are effective for idiopathic inflammatory bowel disease.

'Cold SSCP': a simple, rapid and non-radioactive method for optimized single-strand conformation polymorphism analyses.

A rapid (< 2.5 hrs) method for single-strand conformation polymorphism (SSCP) analysis of PCR products that allows the use of ethidium bromide staining is described. PCR products ranging in size from 117 to 256 bp were evaluated for point mutations and polymorphisms by 'cold SSCP' in commercially available pre-cast polyacrylamide mini-gels. Several electrophoretic parameters (running temperature, buffers, denaturants, DNA concentration, and gel polyacrylamide concentration) were found to influence the degree of strand separation and appeared to be PCR fragment specific. Use of the 'cold' SSCP technique and the mini-gel format allowed us to readily optimize the electrophoretic conditions for each PCR fragment. This greatly increased our ability to detect polymorphisms compared to conventional, radioisotope-labeled 'hot' SSCP, typically run under two standard temperature conditions. Excellent results have been obtained in resolving mutant PCR fragments from human p53 exons 5 through 8, human HLA-DQA, human K-ras exons 1 and 2, and rat K-ras exon 3. Polymorphisms could be detected when mutant DNA comprised as little as 3% of the total gene copies in a PCR mixture. Compared to standard 'hot' SSCP, this novel non-isotopic method has additional advantages of dramatically increased speed, precise temperature control, reproducibility, and easily and inexpensively obtainable reagents and equipment. This new method also lacks the safety and hazardous waste management concerns associated with radioactive methods.