Weeks Of Carcinogen Treatment Research Articles

Aberrant Crypt Foci - Endpoint in Colorectal Cancer

Abstract Aberrant crypt foci (ACF) can be used as the endpoint in colorectal cancer development because aberrant crypts are postulated to be the earliest identifiable potential precursors of colorectal cancer. ACF were first described by Bird and defined as crypt that: (1) have altered luminal openings; (2) exhibit thickened epithelia; (3) are larger than adjacent normal crypts. ACF may eventually evolve into polyps and, subsequently, colorectal cancer in the case of adenoma-carcinoma sequence; it provides a simple and economical tool for preliminary screening of potential chemopreventive agents, and it allows a quantitative assessment of the mechanisms of colon carcinogenesis. ACF are morphological lesions that represent an early stage in the stepwise progression of colon cancer. These surface abnormalities often appear in the distal colon within 2 weeks of carcinogen treatment. Sequential analyses suggest that these early lesions increase in size and multiplicity and often exhibit nuclear atypia and dysplasia. ACF also shows increased proliferative activity, growth factor signaling and K-Ras mutations, suggesting that at least a subset of ACF are putative precursors of colon cancer. Human ACF shares a similar morphology and is present in grossly normal-appearing colon tissue from patients with colorectal cancer (CRC). It has been suggested that the nature and order of acquired genetic changes can profoundly impact ACF morphology and the likelihood of tumor progression. Although limited in numbers, flat dysplastic ACF appeared to be more likely to become tumors than the elevated ACF. Clearly, the expanding diversity of ACF identified in rodent carcinogen models provides researchers with exciting new tools for studying the earliest stages of CRC. Lesions characterized in these models may ultimately become useful for predicting cancer risk in humans.

Abstract LB-358: Carcinogen-induced epigenetic silencing of miR-205 and miR-200 family promotes EMT and transformation of lung epithelial cells

Abstract An in vitro carcinogen-induced transformation model was developed using immortalized bronchial epithelial cells (HBECs) to identify key molecular changes in preneoplastic lung epithelial cells that occur during the development of lung cancer in smokers. HBECs were treated for twelve weeks (one exposure per week) with low-doses of tobacco carcinogens (MNU and BPDE) that resulted in transformation from genotoxic stress (DNA damage). The HBECs displayed a morphological change consistent with an epithelial-to-mesenchymal transition (EMT) after 4 weeks of carcinogen treatment that persisted in colonies that were recovered and expanded from soft agar (transformed cell line). Expression of mesenchymal markers vimentin, fibronectin, and N-cadherin increased significantly, while the epithelial marker E-cadherin was significantly decreased. The most notable changes associated with EMT included 4 - 55 fold reduced expression of miR-200b, -200c, and -205 that correlated with 2-1500 fold increased expression of E-box binding transcription factors (SNAI1, ZEB1, ZEB2) and basic helix-loop-helix transcription factor (TWIST1). Restoring the expression of miR-200b, -200c or -205 in carcinogen transformed cells prevented growth in soft agar; meanwhile the cells maintained a fibroblast-like mesenchymal appearance. Levels of DNA methyltransferase 1 (DNMT1) protein also increased significantly during carcinogen treatment and stable knockdown of DNMT1, but not DNMT3A and DNMT3B prior to carcinogen prevented EMT and transformation. ChIP for H3K4me2 (indicative of active/open chromatin) revealed a progressive decrease of this mark at the promoters of miR-200b, -200c, and -205 over 12 weeks of carcinogen treatment and during transformation. A modest enrichment for H3K9me2 (inactive/closed chromatin mark) was observed at all time points. In contrast, H3K27me3 (indicative of inactive/closed chromatin) was enriched at the miR promoters after 4 weeks of carcinogen treatment and enrichment declined over time. Bisulfite sequencing revealed CpG DNA methylation increased from 3% to 42% in the miR-200 family and miR-205 promoters during transformation. To examine the clinical relevance of miR-200b, -200c, and -205 their expression was analyzed in lung tumors relative to distant normal lung tissue from 24 patients. Reduced expression of at least one miR was observed in 50% of lung adenocarcinomas. These findings implicate a major role for epigenetic regulation of EMT that extends beyond that of cancer metastasis to causality for development of premalignant lung cancer. Supported by ES008801. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-358.

Presence of albumin-positive cells in the liver of analbuminemic rats and their increase on treatment with hepatocarcinogens.

The analbuminemic rat is a mutant in which albumin mRNA processing is blocked owing to a seven-base-pair deletion in the 9th intron of the albumin gene. A small amount of albumin is detected in the serum of this rat. Production of serum albumin in the liver of this mutant rat was studied by an immunohistochemical method. The results showed that a few albumin-positive cells were present in the liver of the mutant rat, whereas all the hepatocytes in normal rat liver were albumin-positive. In the mutant the number of albumin-positive cells increased with age: their frequency was 10(-5) at birth and 6 X 10(-3) at 45 weeks of age. On administration of 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) and 2-acetylaminofluorene, albumin-positive cells increased dramatically. Their numbers were 7 to 10 times those of untreated rats after 10 to 16 weeks of carcinogen treatment. The number of albumin-positive cells did not decrease after 3'-Me-DAB feeding was discontinued. In regenerating liver after partial hepatectomy, clusters of albumin-positive cells increased while the number of singlet albumin-positive cells decreased. These observations suggest that the appearance of albumin-positive cells in analbuminemic rat liver may be due to a somatic mutation of the hepatocytes.

Suppression by iron chelator phenanthroline of sodium chloride-enhanced gastric carcinogenesis induced by N-methyl- N′-nitro- N-nitrosoguanidine in Wistar rats

The effect of prolonged administration of iron chelator phenanthroline on sodium chloride-enhanced gastric carcinogenesis induced by N-methyl- N′-nitro- N-nitrosoguanidine, and the labeling and apoptotic indices in the gastric cancers was investigated in Wistar rats. After 25 weeks of carcinogen treatment, the rats were given chow pellets containing 10% sodium chloride and intraperitoneal injections of phenanthroline at doses of 15 or 30 mg/kg body weight every other day. At week 52, feeding of sodium chloride significantly increased the incidence of gastric cancers, as compared with the control group. Prolonged injections of phenanthroline at both doses significantly reduced the incidence of gastric cancers, which was enhanced by oral supplementation with sodium chloride. Phenanthroline at both doses significantly decreased the labeling index of gastric cancers, which was enhanced by sodium chloride, and significantly increased the apoptotic index of gastric cancers, which was lowered by sodium chloride. In vitro examination using electron spin resonance revealed that sodium chloride promotes the production of hydroxyl radical during Fe 2+ oxidation by Fenton's reaction. These findings suggest that enhancement by sodium chloride of gastric carcinogenesis may be mediated by hydroxyl radicals.

Attenuation by d-limonene of sodium chloride-enhanced gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine in Wistar rats.

The effects of prolonged administration of d-limonene, a monocyclic monoterpene, on sodium chloride-enhanced induction of gastric carcinogenesis by N-methyl-N'-nitro-N-nitrosoguanidine, the labeling and apoptotic indices, and ornithine decarboxylase (ODC) activity of gastric cancers were investigated in Wistar rats. After 25 weeks of carcinogen treatment, rats were given chow pellets containing 10% sodium chloride and 1% limonene ad libitum. In week 52, the incidence of gastric cancers, the labeling index and ODC activity were significantly higher and the apoptotic index was significantly lower in rats given sodium chlolide than in untreated control rats. However, in rats given both sodium chloride and d-limonene, the incidence of gastric cancers, the labeling index and ODC activity were significantly lower and the apoptotic index was significantly higher than in rats given sodium chloride alone. Our findings suggest that limonene attenuates the gastric carcinogenesis enhanced by sodium chloride via increased apoptosis and decreased ODC activity in gastric cancers.

Attenuation by all-trans-retinoic acid of sodium chloride-enhanced gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine in Wistar rats.

The effect of prolonged administration of all-trans-retinoic acid (RA) on sodium chloride-enhanced gastric carcinogenesis induced by N-methyl-N′-nitro-N-nitrosoguanidine, and the labelling and apoptotic indices and immunoreactivity of transforming growth factor (TGF) α in the gastric cancers was investigated in Wistar rats. After 25 weeks of carcinogen treatment, the rats were given chow pellets containing 10% sodium chloride and subcutaneous injections of RA at doses of 0.75 or 1.5 mg kg−1 body weight every other day. In week 52, oral supplementation with sodium chloride significantly increased the incidence of gastric cancers compared with the untreated controls. Long-term administration of RA at both doses significantly reduced the incidence of gastric cancers, which was enhanced by oral administration of sodium chloride. RA at both doses significantly decreased the labelling index and TGF-α immunoreactivity of gastric cancers, which were enhanced by administration of sodium chloride, and significantly increased the apoptotic index of cancers, which was lowered by administration of sodium chloride. These findings suggest that RA attenuates gastric carcinogenesis, enhanced by sodium chloride, by increasing apoptosis, decreasing DNA synthesis, and reducing TGF-α expression in gastric cancers. © 1999 Cancer Research Campaign

Effect of plyamine oxidase inhibition on the colonic malignant transformation process induced by 1.2-dimethylhydrazine

Recent studies of colon adenocarcinomas in humans and experimentally induced colonic tumors in rodents have demonstrated selective elevations in the level of N1-acetylspermidine in these malignant tissues. The exact relationship of these alterations in acetylated polyamine levels to the malignant transformation process, however, remains unclear. In order to clarify this issue, rats were given s.c. injections of 1,2-dimethylhydrazine (DMH; 20 mg/kg body wt/week) or diluent for up to 26 weeks. After 10 weeks of carcinogen treatment, one-half of the animals in each group were also concomitantly given i.p. injections of MDL 72527 (20 mg/kg body wt/week), a specific inhibitor of polyamine oxidase, until they were killed. Animals were killed after 15 weeks of DMH treatment and polyamine levels as well as the activities of polyamine oxidase, ornithine decarboxylase and spermidine-N1-acetyltransferase were measured and compared in rat proximal and distal colonic mucosa of each group. Polyamine levels were also assessed in each of these groups after 26 weeks of treatment with this carcinogen +/- MDL 72527. In addition, in view of recent studies that have indicated that polyamines may influence certain oncogenes in human colonic carcinoma cells, tumors from DMH +/- MDL 72527 were analyzed for K-ras mutations. The results of these experiments demonstrated for the first time that: (i) MDL 72527 was a specific inhibitor of polyamine oxidase in normal and malignant colonic tissue; (ii) concomitant administration of this agent with DMH enhanced the elevation of colonic N1-acetylspermidine and significantly reduced the mean colonic tumor burden, as assessed by total tumor area per rat, produced by this carcinogen alone; (iii) analysis of K-ras mutations revealed a similar incidence (62-69%) in adenocarcinomas for both groups (+/- MDL 72527); (iv) however, analysis of the K-ras-mutated and non-mutated tumors revealed that in both carcinogen-treated groups (+/- MDL 72527), tumors with such mutations were smaller than their counterparts without such genetic alterations. Moreover, MDL 72527 reduced the average size of tumors, with and without such mutations, to a similar extent.

The relationship between epithelial Ia expression and the inflammatory cell infiltrate during experimental oral carcinogenesis.

The development of oral epithelial expression of Ia antigens and its relationship to the presence of IL-2r+ (CD25+) cells was investigated in rats treated with the water soluble carcinogen 4-nitroquinoline-N-oxide (4NQO). Acetone fixed frozen sections of the palate and tongue were stained using an indirect immunoperoxidase technique and monoclonal antibodies to rat Ia (I-A & I-E) and IL-2 receptor. After 4 weeks 4NQO treatment all rats expressed oral epithelial Ia but thereafter (2-9 months) expression was present in only 20-40% of animals. Epithelial expression of Ia by histologically normal, dysplastic and neoplastic epithelium was always associated with the presence of an underlying inflammatory cell infiltrate containing CD25+ cells. Overall there were significantly more CD25+ cells in tissue specimens containing Ia+ epithelium compared with Ia- epithelium. Furthermore, during the first 4 weeks of carcinogen treatment, a significant positive correlation was found between the CD25+ cell density and occurrence of focal epithelial Ia expression. These results, together with analysis of the T cell, NK cell, macrophage and B cell content of the infiltrates induced by 4NQO, suggest that the CD25+ cells represent activated T cells. Thus, our results in this experimental model are consistent with the idea that epithelial expression of Ia is the result of production of IFN-gamma by locally activated T cells.

Immunological identity of a 60 KD oncofetal protein induced in rats by chemical carcinogens and released by transformed cells

A 60,000 dalton (60 kd) oncofetal protein was previously shown to be produced by tumors in tumor-bearing rats and by target tissues within 3 weeks of carcinogen treatment. The factor is released to and accumulates in the blood in vivo and in the conditioned medium of cultured transformed cells in vitro. A polyclonal antibody produced against the 60 kd factor purified from the plasma of a rat carrying the N-2-fluorenylphthalamic acid-induced transplantable Hepatoma 7777, was tested against the 60 kd factor from various sources. Based on the results of immunoprecipitation of biochemical activity associated with the 60 kd factor, it was determined that these anti-60 kd antibodies cross-reacted with the factor released by a dimethylbenzanthracene-induced rat mammary carcinoma, with the factor in rat tumor cytosol and with rat spontaneous lymphoma cells, but not with a 60 kd factor isolated from pooled cancer patient plasma. Furthermore, these antibodies cross-reacted with the 60 kd factor induced within 21 days of treatment of the rats with a range of carcinogens from 8 chemical structural groups. The anti-60 kd factor antibodies did not cross-react with a 35 kd factor having similar biochemical activity found in normal adult cells.

Changes in the fine structure of rat hepatocytes during the early phases of chronic 2-acetylaminofluorene intoxication

A diet containing 0.05 % of 2-acetylaminofluorene (2-AAF) was administered to male rats of the Leeds strain. Animals were killed after 7–10 days, 3–4 weeks and 8–12 weeks of carcinogen treatment and their hepatic tissue was examined by electron microscopy. 2-AAF appeared to differ, in its effect on the fine structure of the hepatocyte, from many other hepatocarcinogens in failing to produce visible nuclear changes. The changes produced by 2-AAF included glycogen depletion, proliferation of the agranular endoplasmic reticulum, reduction in the quantity and morphological alterations of the granular endoplasmic reticulum, progressive hypertrophy of the Golgi apparatus with an associated increase in lysosomal abundance and, during the later stages of treatment, a reduction in mitochondrial size. Changes affecting the plasma membrane were also observed, including dilatation of the bile canaliculi. Some of the observed changes appear to be related to those previously observed in preneoplastic liver cells and hepatoma cells of 2-AAF-treated rats and may, therefore, be related to the carcinogenic activity of 2-AAF.