Hiroko Fukami

Induction of Glandular Stomach Cancers in Helicobacter pylori-sensitive Mongolian Gerbils Treated with N-Methyl-N-nitrosourea and N-Methyl-N′-nitro-N-nitrosoguanidine in Drinking Water

An animal model of stomach carcinogenesis was established using Mongolian gerbils with N‐methyl‐N‐nitrosourea (MNU) and N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG) as the carcinogens. In addition, the sensitivity of these gerbils to Helicobacter pylori (H. pylori) was confirmed. One hundred and sixty specific pathogen‐free male MGS/Sea animals, 7 weeks old, were treated with MNU in the drinking water (30 ppm for alternate weeks to give 10 weeks exposure, or 10 ppm or 3 ppm for 20 weeks continuous exposure), or given MNNG in the drinking water at 400 ppm or 200 ppm for 20 weeks, or orally inoculated with ATCC43504 H. pylori (1.7×1088 CFUs/animal). Adenocarcinomas in the glandular stomach were found in 2 out of 12 effective animals (2/12) treated with 30 ppm MNU at week 20, although all were dead or moribund by week 30 due to MNU toxicity. At week 50, the incidences of gastric adenocarcinomas in groups treated with 10 ppm MNU, 3 ppm MNU, 400 ppm MNNG, and 200 ppm MNNG were 2/21 (9.5%), 1/23 (4.3%), 7/11 (63.6%), and 1/10 (10.0%). The lesions were generally well differentiated, although poorly differentiated adenocarcinoma was also found in a single gerbil in each of the 10 ppm MNU and 400 ppm MNNG groups. In control animals no tumors were found. In the infection study, the animals were killed at week 20, and H. pylori was detected in all cases, causing multiple erosions with marked inflammatory cell infiltration in the lamina propria and submucosa, and frequent formation of lymphoid follicles. Thus, MNU and MNNG in the drinking water induced neoplastic lesions in the glandular stomach epithelium of H. pylori‐sensitive gerbils.

Frequent somatic mutations of mitochondrial DNA in esophageal squamous cell carcinoma

Recent studies of various cancers, such as those of the breast, head and neck, bladder and lung, reported that 46–64% of somatic mutations in the D‐loop region of mitochondrial DNA (mtDNA) are observed. However, in esophageal cancer, only a low rate (5%) of somatic mutations has so far been reported in one article (Hibi, K. et al., Int J Cancer 2001;92:319–321). Thus, to confirm this we analyzed the somatic mutations for hypervariable regions (HVR‐I and HVR‐II) in the D‐loop of mtDNA to reevaluate the possibility of mitochondrial genetic instability in this cancer. We amplified both HVRs by PCR and DNA samples obtained from 38 esophageal tumors and matched normal tissues, and then sequenced them. Comparing the sequences of tumors to those of normal tissues, we found 14 somatic mutations in 13 patients (34.2%). Eleven mutations were at the C consecutive stretch from position 303 to 309 of MITOMAP in the mitochondria databank (http://www.mitomap.org/), 1 at position 215 in HVR‐II and 2 at positions 16,304 and 16,324 in HVR‐I. There were 41 types of germ line variations in HVR‐I including 2 not so far recorded in the mtDNA databank and 17 in HVR‐II including 1 not yet recorded. We also determined nuclear genome instability of these 38 specimens by analyzing 3 independent microsatellite sequences. While 4 specimens showed a single microsatellite change, which is tumor specific, we did not find any co‐relation between a somatic mtDNA mutation and microsatellite instability of nuclear genome DNA. These results suggest that mtDNA mutations might show a genetic instability in esophageal cancer independently from a nuclear genome instability.

Primary monoclonal and secondary polyclonal growth of colon neoplastic lesions in C3H/HeN→BALB/c chimeric mice treated with 1,2‐dimethylhydrazine: Immunohistochemical detection of C3H strain‐specific antigen and simple sequence length polymorphism analysis of DNA

To determine the clonality and cellular origin of colon pre‐neoplastic and neoplastic lesions, C3H/HeN→BALB/c chimeric mice treated with 1,2‐dimethylhydrazine (DMH) were investigated immunohistochemically using a specific antibody to C3H strain‐specific antigen (CSA) enabling immunohistochemical discrimination of C3H cells in histological sections of chimeric mouse tissues. To confirm the results of immunostaining, simple sequence length polymorphism (SSLP) analysis was performed on DNA samples extracted from histological sections of adenocarcinomas. C3H/HeN→BALB/c chimeric mice were produced by an aggregation procedure and together with BALB/c and C3H/HeN animals were given weekly s.c. injections of 20 mg/kg body weight DMH for up to 20 weeks. At weeks 20 and 35 animals were killed and autopsied. In normal colonic mucosa of the chimeras, each gland was composed entirely of either CSA‐positive or ‐negative cells and no mixed glands were found. Cells of all focal atypias in chimeric mice were, in each case, homogeneous for one or another of the parental types. Of 91 adenomas in chimeric mice, only one comprised both types of cell. Among 119 adenocarcinomas, 12 contained cells of both parental types. In these tumors, however, the 2 phenotypes were not mixed together at random but arranged in discrete areas, with intermingling limited to the junctions. SSLP analysis demonstrated DNAs extracted from CSA‐positive and ‐negative tumors to exhibit the polymorphic patterns of C3H and BALB/c, respectively, while mixed CSA‐positive and ‐negative tumors showed mixtures of both polymorphic DNA types.

Hexosaminidase-altered aberrant crypts, carrying decreased hexosaminidase α and β subunit mrnas, in colon of 1,2-dimethylhydrazine-treated rats

Aberrant crypt foci (ACF), consisting of morphologically irregular crypts, are thought to be precancerous lesions for colon cancers. For their molecular analysis, it is necessary to avoid contamination with adjacent normal crypts and stromal cells. Decreased hexosaminidase activity in ACF, which has been histochemically demonstrated, was used in the present study to classify isolated crypts in combination with morphological changes. The length, rim diameter, and width (average±SD, μm) of hexosaminidase‐positive (Hex+) crypts were 238.6±40.4, 89.5±22.9, and 57.6±14.0, respectively. For hexosaminidase‐negative (Hex‐) crypts, the values were 314.4±77.8, 140.3±45.7, and 97.3±34.7, the width being 1.69 tunes greater (P<0.0001). Crypts wider than 115 μm (approximately 2 tunes the average size of Hex+ crypts) were all from ACF, judging from hexosaminidase staining. To analyze transcription levels of Hex α and β subunits (Hexa and Hexb, respectively), real‐tune relative quantitative reverse transcription‐polymerase chain reaction (RT‐PCR) analysis was performed using the LightCycler system. In aberrant crypts, both Hexa and Hexb were significantly down‐regulated to 0.266 (P<0.002) and 0.131 (P<0.001) units, respectively, compared with those in morphologically normal crypts, with β‐actin as the internal standard. This decrease could be a molecular marker for precancerous enzyme‐altered ACF.

Development and Distribution of 2‐Amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]‐pyridine (PhIP)‐induced Aberrant Crypt Foci in the Rat Large Intestine

Aberrant crypt foci (ACF) are generally considered to be preneoplastic lesions for colon cancer. To assess their induction by 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP), a colon carcinogen, we performed a sequential study of ACF morphology and localization. F344 male rats were given PhIP, and methylene blue‐stained colon epithelium and isolated crypts were analyzed at weeks 12, 25, 50, and 75. Each crypt was classified into 2 groups, “single” with round bottoms and “bifurcating” displaying V‐shaped clefts (indicating proliferation). In combination with the number of crypts in an ACF, this classification was a good indicator for the generation of ACF in line with the fission mechanism of growth. Increasing numbers of crypts in ACF through weeks 12 to 75 and decreased percentages of ACF with bifurcating crypts at the late time points indicated that proliferation of crypts occurs predominantly during the early stages. The distribution pattern showed a significant shift (P < 0.000005) from the distal to the proximal part of the large intestine between weeks 25 and 50. Adenocarcinomas were first found to develop at week 50 in the ascending colon and cecum where bifurcating crypts were generally lacking at weeks 12 and 25. These data suggest the existence of (1) proliferating ACF which contains bifurcating crypt(s) and (2) quiescent or senescent ACF which consists of only single crypts.

Mouse Strain Susceptibility to Diethylnitrosamine Induced Hepatocarcinogenesis Is Cell Autonomous Whereas Sex‐susceptibility Is Due to the Micro‐environment: Analysis with C3H ↔ BALB/c Sexually Chimeric Mice

In man, liver cancer is on the increase, especially in males. Sex differences also exist in rodent models. To elucidate the mechanisms, chimeric mice were produced by amalgamation of early embryos from high and low hepatocarcinogen‐susceptible strains, C3H and BALB/c. Tumor formation was initiated with 10 mg/kg of diethylnitrosamine at the ages of 7 and 14 days and mice were sacrificed at 30 and 45 weeks. The chimeras were classified into XY↔XY, XY↔XX, XX↔XY, and XX↔XX in terms of sex chromosomes by means of polymerase chain reaction‐simple sequence length polymorphism analysis (SSLP) using Y chromosome‐specific Sry primers in combination with the D3Mit21 marker. Liver lesions were analyzed histopathologically, by immunostaining using a C3H strain‐specific antibody and by DNA in situ hybridization with the Y chromosomespecific digoxigenin‐labeled Y353/B probe. Sex and strain genotyping by SSLP analysis matched histological observations, confirming the reliability of our system. The strain differences in liver tumor numbers of each strain type in XY↔XY and XX↔XX subtypes of C3H↔BALB/c chimeras were retained well (P< 0.0001 and P< 0.001, respectively), indicating a minimum influence of the C3H or BALB/c surrounding milieu on development of individual lesions. On the other hand, significant promotion of XX cell tumors was evident in phenotypically male sexually chimeric XY↔XX and XX↔XY chimeras for both C3H (P< 0.02) and BALB/c (P< 0.01) lesions compared to the XX↔XX case. The results suggest the presence of hormonal or micro‐environmental factors specific for males, which are not caused cell‐autonomously. Basic strain differences, however, are determined by intrinsic genetic factors rather than the strain‐dependent micro‐environment

Identification of growth-promoting factors from various glioma cell lines and partial purification of the factor from C6 solid tumor.

Growth-promoting factors in the extracts of various glioma cell lines (C6, LRM55 and 354A) were investigated. The cell extracts of astrocytoma (C6) and mixed glioma (LRM55) showed a high mitogenic activity to normal glioblasts. With its low content of intracellular growth-promoting factor, rat peripheral glioma (354A) exhibited a high proliferative response to C6 cell extracts. The factor which was partially purified from C6 solid tumor by ion exchange and gel filtration column chromatographies had two forms of different molecular weights (150,000 Mr and 35,000 Mr) and the low molecular weight form was further split into two acidic proteins (pl 5.0 and pl 6.0) by isoelectric focusing. The mitogenic activity of the factor was susceptible to heat and to proteases, and the factor showed no esteropeptidase activity. These physicochemical properties closely resemble those of glia maturation factor from porcine brains.

In vitro and in vivo growth characteristics of a new ascites-type neuroblastoma cell from mouse C1300 neuroblastoma.

A clonal ascited type cell, NAs-1, was obtained in culture from a mouse neuroblastoma C1300. The cells were adapted to anchorage-independently grow in the flask by the in vitro-in vivo alternate passage technique, and retained the ability of growing and producing ascites fluid when intraperitoneally injected into mice. Although the majority of growing cells in culture medium showed a small and round cell shape without any neuronal process, occasionally non-specific attachment onto the flask surface was observed, but devoid of the extrusion of processes. Karyotype analysis showed a homogeneous chromosome number, 40, with a marker chromosome [t(13:16)] and a minichromosome. Catecholamines, norepinephrine and dopamine, were found in the cell extracts and the contents of dopamine was particularly high as shown in another catecholaminergic neuroblastoma cell, N1E-115. Neuron specific enolase (?-subunit) was also detected. The treatment of the cells by dibutyryl cyclic AMP, prostaglandin E(1), or BL191 (phosphodiesterase inhibitor) induced the biochemical differentiation in terms of catecholamine and cyclic AMP contents, but failed to promote typical morphological differentiations including the extension of process or the significant promotion of adherence onto the flask surface.

Tumorigenicity, major histocompatibility antigens, and karyotypes of interspecific hybrids between mouse neuroblastoma and rat glioma or liver cells

Five interspecific hybrids of mouse neuroblastoma with rat glioma (NG108-15, 140-3, and 141-B) or with nontransformed rat liver cells (NBr-10A and NBr-20A) were examined for major histocompatibility (MHC) antigens and tumorigenicity in comparison with their karyotypes. Both mouse and rat MHC antigens were present in each hybrid population, as determined by a simple cytotoxicity test. All five hybrid cell lines produced tumors in athymic nude mice with varied take incidences. Four hybrid cells, NG108-15, 140-3, NBr-10A, and NBr-20A, were highly tumorigenic. Their karyotypes were characterized by a higher modal chromosome numbers than would be expected from the fusion of parent cells in which at least one parent contained an increased number of chromosomes. In contrast, 141-B cells, with massive loss of chromosomes from both malignant parents, were weakly tumorigenic. The results suggest that the retention of marker chromosomes as well as double minutes (DMs) or microchromosomes of neuroblastoma origin may be required for expression of malignancy in these hybrid cells. The survival time of tumor-bearing mice also varied within the five cell lines, but it was significantly short in NG108-15, which yielded lung metastases in the host animals.

Chromosome studies in mouse neuroblastoma cells.

Chromosome studies were carried out in cultured cells from a mouse neuroblastoma. C1300 tissue and in three clones established from this tumor. They possessed characteristic karyotypes with remarkable markers. Double minutes (DMs) were demonstrated in all cell lines, in addition to some other chromosomes aberrations, such as microchromosomes and chromosome pulverization.