Successful chemotherapy of experimental neuroendocrine lung tumors in hamsters with an antagonist of Ca2+/calmodulin

The chemotherapeutic effect of B859-35, the (-)-enantiomer of dihydropyrine 3-methyl-5-3-(4,4-diphenyl-1-piperidinyl)-propyl-1,4-dihydro-2,6-dimethy l-4- (3-nitrophenyl)-pyridine-3,5-dicarboxylate-hydrochloride (niguldipine), was tested on tumors induced in Syrian golden hamsters by N-nitrosodiethylamine (DEN). Peripheral pulmonary adenomas/adenocarcinomas were induced in hamsters maintained under ambient air conditions by multiple s.c. injections of DEN for 20 weeks. We have reproducibly shown that within this time interval lung adenomas develop in a significant number of the animals. The carcinogen treatment was discontinued at this point and one group of these hamsters was given B859-35 intragastrically 5 days/week for 20 weeks while the second group of such tumor-bearing hamsters were kept for an identical time interval without further treatment. Neuroendocrine lung tumors were induced in hamsters maintained in an atmosphere of 60% O2 by multiple s.c. injections of DEN for 8 weeks. We have reproducibly shown that within this short time interval neuroendocrine lung tumors develop in a significant number of the animals. The carcinogen treatment was discontinued at this point and the animals were returned to ambient air conditions. One group of these tumor-bearing hamsters was then given B859-35 intragastrically 5 days/week for 20 weeks while a second group of these hamsters was kept untreated for an identical time interval. A control group was given s.c. injections of saline for 20 weeks under ambient air conditions. A dramatic and selective anticarcinogenic effect of B859-35 was observed on the neuroendocrine lung tumors and nasal cavity tumors induced by DEN/hyperoxia while tumors of larynx/trachea were not affected. B859-35 had no effect on peripheral adenomas/adenocarcinomas, nasal cavity tumors, papillary polyps of larynx/trachea, or liver tumors induced by DEN under ambient air conditions.     

DNA ethylation in target and non-target organs of hamsters and rats treated with diethylnitrosamine

Kinetics of ethylation of target and non-target organ DNA in vivo by diethylnitrosamine (DEN) was compared in rats and Syrian golden hamsters, since published reports indicate a single dose of DEN induces both kidney and liver tumors in rats and almost exclusively respiratory tract tumors in hamsters. Following treatment with 200 mg DEN/kg, 7-ethylguanine (7-etG) was lost more rapidly from hamster than from rat liver DNA, while O6-ethylguanine (O6-etG) persisted longer in hamster than in rat liver DNA. DNA ethylation was not detected in rat lung (non-target organ), while both 7-etG and O6-etG were quantitated in hamster lung (target organ) following DEN treatment. DNA ethylation in rat kidney DNA was approximately 1/10 of that in liver by 200 mg DEN/kg, and the persistence of 7-etG and O6-etG differed only slightly in these tissues. Ethylation of hamster liver DNA by DEN at doses between 20 and 200 mg/kg, as measured by 7-etG and O6-etG was proportional to the dose of carcinogen up to 160 mg/kg; at larger doses DNA ethylation sharply increased. Differences in the persistence of O6-etG between DEN-treated rats and hamsters cannot solely account for species differences in the organotropism of DEN carcinogenesis.     

Promoting Effect of Sodium Chloride Solution Mist on the Induction of Papillomas on Syrian Hamster Tracheal Mucosa Following Administration of Diethylnitrosamine

The promoting effect of NaCI solution mist was studied in the induction of papillomas on Syrian hamster trachea following single subcutaneous injection of diethylnitrosamine (DEN). Sixty male hamsters were divided into three groups. Group A; Exposure to a 5% NaCl solution mist for 32 weeks following single subcutaneous injection of DEN at a dose of 3 mg/100 g body weight. Group B; Subcutaneous DEN injection alone. Group C; Exposure to the NaCI mist for 32 weeks alone. Total numbers of papillomas on the trachea in each group were 107 in group A, 64 in group B, and 0 in group C ( P 0.05 between group A and B, P <0.01 between either groups A or B and C). Comparing the incidence of large papillomas with a diameter of more than 2 mm, that in group A hamsters was significantly greater than that of group B (22 large papillomas vs. 8 large papillomas, P <0.01). No difference in the effects on the epithelial cells of the nasal cavity, the bronchial or the alveolar cells in the lung was recognized between groups A and B. The hamsters in group C were not observed to have any changes in any of the respiratory organs. Inhalation of NaCI solution mist is considered to have a promoting effect on the induction of papillomas in the hamster trachea following DEN injection.     

Promoting effect of sodium chloride solution mist on the induction of papillomas on Syrian hamster tracheal mucosa following administration of diethylnitrosamine

The promoting effect of NaCl solution mist was studied in the induction of papillomas on Syrian hamster trachea following single subcutaneous injection of diethylnitrosamine (DEN). Sixty male hamsters were divided into three groups. Group A; Exposure to a 5% NaCl solution mist for 32 weeks following single subcutaneous injection of DEN at a dose of 3 mg/100 g body weight. Group B; Subcutaneous DEN injection alone. Group C; Exposure to the NaCl mist for 32 weeks alone. Total numbers of papillomas on the trachea in each group were 107 in group A, 64 in group B, and 0 in group C (P less than 0.05 between group A and B, P less than 0.01 between either groups A or B and C). Comparing the incidence of large papillomas with a diameter of more than 2 mm, that in group A hamsters was significantly greater than that of group B (22 large papillomas vs. 8 large papillomas, P less than 0.01). No difference in the effects on the epithelial cells of the nasal cavity, the bronchial or the alveolar cells in the lung was recognized between groups A and B. The hamsters in group C were not observed to have any changes in any of the respiratory organs. Inhalation of NaCl solution mist is considered to have a promoting effect on the induction of papillomas in the hamster trachea following DEN injection.     

Amplification and enhanced expression of the c-Ki-ras2 protooncogene in human embryonal carcinomas

Two cell lines of human embryonal carcinoma, Tera-1 and Tera-2, have been found to exhibit a 4- to 6-fold amplification of protooncogene c-Ki-ras2. The polyadenylic acid selected RNA also showed 8-fold or greater enhancement, showing marked elevation in the level of two major mRNAs, 5.7 and 4.0 kilobases, and two additional minor mRNAs, 2.3 and 1.2 kilobases, as compared with those of a normal human embryonic fibroblast cell line, MRC-5. More than one-half of the number of tumor samples obtained from metastatic human embryonal carcinomas also showed c-Ki-ras2 gene amplification and enhanced mRNA expression. However, the c-Ki-ras2 gene amplification did not always lead to enhanced mRNA expression, and some embryonal carcinomas showed mRNA overexpression without apparent c-Ki-ras2 gene amplification. These results suggest that human embryonal carcinomas may have c-Ki-ras2 amplification and/or overexpression before in vitro culture. Among various chromosomal changes observed in Tera-1 and Tera-2 cells, there were anomalies in chromosome 12 in which c-Ki-ras2 is located although these karyological changes alone could not account for the amplification observed. It is suggested that the genomic instability and active DNA replication during the early developmental period may give rise to changes involving c-Ki-ras2 which may contribute to oncogenic processes.     

Promoting effects of cigarette smoke on the respiratory tract carcinogenesis of Syrian golden hamsters treated with diethylnitrosamine.

The potential short-term promoting effects of cigarette smoke on the development of tumors in the respiratory system were investigated in male Syrian golden hamsters. Three groups (1, 2 and 3) of 30 animals each received a single s.c. injection of 100 mg/kg body wt of diethylnitrosamine (DEN) at the commencement of experiment 1. They were then exposed to non-filter cigarette (NC) smoke, filter-tip cigarette (FC) smoke and sham smoke respectively, in a Hamburg II type smoking machine from week 1 to week 12. In addition, groups 4, 5 and 6 (10 animals each) were exposed to the NC smoke, FC smoke or sham smoke respectively, for the same time period without prior DEN treatment. In the DEN-treated groups, epithelial hyperplasias and/or papillomas were induced, the incidences and numbers/animal of these lesions in groups 1 and 2 being significantly increased as compared to group 3 values. In experiment 2, two groups of 25 hamsters each were exposed to cigarette smoke or sham smoke for up to 12 weeks, five animals in each group being killed for immunohistochemical analysis using BrdU antibodies and measurement of lipid peroxides in the lung and serum at weeks 1, 2, 4, 8 and 12. Small aggregations of macrophages (smoke cells) in the lung alveoli was observed in the smoke-exposed group, but no significant increase in the numbers of BrdU positive cells in any compartment of the respiratory system was apparent. Animals of this group showed a tendency for increased lung malondialdehyde levels at weeks 2 and 12, but not weeks 4 and 8.     

Evaluation of DNA damage by the alkaline elution technique in liver, kidneys and lungs of rats and hamsters treated with N-nitrosodialkyl-amines

Induction of single-strand breaks in the DNA of three organsof BD-VI rats and Syrian golden hamsters was examined 4 h aftera single i.p. dose of N-nitrosodimethylamine (DMN) or N-nitrosodiethylamine(DEN). Damage was monitored in vivo by the alkaline elutionmethod, in which DNA is dosed fluorometrically. DNA damage wasinduced by DMN in the liver and kidneys of rats and in the liverand lungs of hamsters, and by DEN in rat and hamster liver.High doses of the hepatocarcinogen and hepatotoxic compoundcarbon tetrachloride, did not induce DNA damage in rat liver.A correspondence between DNA fragmentation (our study) and tumourinduction (reported in the literature) was found in the followingorgans: rat and hamster liver (DMN, DEN), rat kidney (DMN),rat lung (DMN, DEN), hamster kidney (DMN, DEN). In contrast,no such correlation was observed in rat kidney and hamster lungfollowing DEN-treatment and in hamster lung following DMN-treatment.Thus, the in vivo alkaline elution assay would appear to bemost useful for detecting DNA damage by chemicals that are activatedmetabolically in the liver and that bind to hepatic DNA.     

Pathobiology of lung tumors induced in hamsters by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and the modulating effect of hyperoxia

Neuroendocrine lung cancer is among the most common types of lung cancers in smokers. We have recently shown that exposure of hamsters to N-nitrosodiethylamine and hyperoxia causes a high incidence of this tumor type. In this study, we show that the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone also causes neuroendocrine lung tumors in hyperoxic hamsters. Animals maintained in ambient air while being treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone developed pulmonary adenomas composed of Clara cells and alveolar type II cells. Pathogenesis experiments provide evidence for the tumors caused by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in ambient air being derived from Clara cells. In the hyperoxic hamsters, the neuroendocrine carcinogenesis appears to involve two stages: (a) transformation of focal alveolar type II cells into neuroendocrine cells and (b) development of neuroendocrine lung tumors from such foci.     

c-Ki-ras gene amplification and malignant behavior in murine embryonal carcinoma cell lines

Embryonal carcinoma (EC) cells are malignant components of murine teratoma tumors. To extend our earlier findings concerning c-Ki-ras amplification in the embryonal carcinoma PCC4 cell line, we examined the c-Ki-ras protooncogene and its expression in other EC cell lines. We report here that c-Ki-ras amplification is not a general feature of EC cell lines since neither PCC3, PCC6, nor F9 cell lines have an amplified copy number of this protooncogene. Furthermore, molecular analysis of three independently passaged PCC4 cell lines showed marked heterogeneity for c-Ki-ras amplification. Two PCC4 cell lines showed amplified copy number and elevated expression of c-Ki-ras whereas the original one does not, suggesting that this gene amplification occurred through laboratory passage. Malignancy in syngeneic mice of PCC4 with or without an amplified c-Ki-ras gene was also examined. Our results indicate that c-Ki-ras amplification alone is not a determining factor in the malignant behavior of EC cells.     

DNA ethylation in hamster tissues during subchronic diethylnitrosamine administration and in the hamster trachea after acute diethylnitrosamine administration

Formation of O⁶-ethylguanine (O⁶EG) in trachea DNA was measured12 h after acute i.p. administration of 50, 100 and 200 mg diethylnitrosamine(DEN)/kg body wt to Syrian golden hamsters. Purine bases werefractionated by h.p.l.c, and ethylated guanines were quantifiedoptically by fluorescence spectrophotometry. Significant levelsof this promuta-genic base were found after the various singledoses of DEN. O⁶EG in trachea DNA was also measured at varioustimes up to 48 h after an acute i.p. dose of 200 mg DEN/kg bodywt. The maximum level of C⁶EG was detected 12 h after DEN treatment.A rapid decrease in O⁶EG concentration was seen in trachea DNAbetween 12 and 24 h, but only a negligible decrease was detectedfrom 24 to 48 h post-treatment. The formation and accumulationof O⁶EG in trachea, liver, lung and kidney DNA were determinedin hamsters treated sub-chronically with DEN, 20 mg/kg bodywt, s.c. twice weekly up to 8 weeks. O6EG accumulated to significantlevels in trachea and liver DNA. 7-Ethylguanine did not accumulatein the DNA of any of the organs studied during subchronic DENexposure. The formation and persistence of O⁶EG in trachea DNAof DEN-treated hamsters correlated with the sensitivity of thistissue to the carcinogenic action of DEN.     

Pancreatic ductal adenocarcinomas induced in Syrian hamsters by N-nitrosobis(2-oxopropyl)amine contain a c-Ki-ras oncogene with a point-mutated codon 12

We have used the polymerase chain reaction and dideoxynucleotide sequencing to amplify and sequence exons 1 and 2 of the c-Ki-ras proto-oncogene from the normal Syrian golden hamster. Similar methods were employed to screen for the presence of point mutations in the c-Ki-ras oncogene in primary hamster pancreatic ductal adenocarcinomas (PDC) induced by N-nitrosobis(2-oxopropyl)amine (BOP). A GGT to GAT point mutation was detected in codon 12 of the c-Ki-ras gene in 10 primary hamster PDCs. This same point mutation was present in two nonclonal cell lines, PC-1 and PC-1-0, established from tumors that were produced in hamsters by subcutaneously implanting a preparation of minced BOP-induced PDC. Two clonal cell lines, Cl-3 and Cl-7, were cloned from the PC-1 cell line, and these cell lines also carried the GAT point mutation at codon 12. This point mutation was the same as that detected in greater than 75% of adenocarcinomas from the human exocrine pancreas. Thus, our findings provide further validation for the use of the BOP-induced hamster PDC model as a relevant experimental model for human pancreas cancer: not only did the hamster pancreatic ductal adenocarcinomas closely resemble their human counterpart in histopathological morphology and sequential development, but they also contained the same point mutation in codon 12 of the c-Ki-ras oncogene, as has been reported for human pancreatic adenocarcinomas.     

BCRP基因在非小细胞肺癌组织和非癌肺组织中的表达及意义

目的：F是一步研究从人类乳腺癌细胞株（MCF-7/AdrVp)中克隆出对蒽环类抗癌药耐药的BCRP基因在非癌肺组织及非小细胞肺癌组织中的表达。方法：从术后即刻获得的正常肺组织和有省略的非小细胞肺癌组织新鲜标本中及时提取细胞总RNA,通过RT-PCR及PCR法获得并扩增BCRP基因的cDNA产物,再经凝胶电泳分离BCRP基因cDNA带,然后再通过转膜技术把这些BCRP基因cDNA产物转移到杂交膜上,用Southern blot杂交技术检测BCRP基因的表达程度。结果：细胞总RNA分别从8个只有肺癌组织标本和12对同时获取的肺癌组织及非癌肺组织标本中成功提取,然后用变性凝胶电泳鉴定提取RNA的质量,其中4例标本因术中缺血时间太长,或有坏死炎症或术前放化疗等原因,所提RNA有降解而放弃进一步的检测,通过RT-PCR及PCR方法从16例可用标本中获得并扩增BCRP基因的cDNA产物,再通过转膜及Southern blot杂交技术,最终发现BCRP基因在正常肺组织中均有不同程度的表达（10/10）,而在非小细胞肺癌组织标本中只有一半病人的标本有不同程度的表达（8/16）。结论：BCRP基因可能是一个在非癌肺组织中常规表达的基因,而在部分病人的肺癌组织中可能此基因有缺失或被抑制。因此,在部分非小细胞肺癌患者中如用蒽环类抗癌药（如阿霉素,柔红霉素等）化疗时,可能会诱导BCRP基因的过度表达而产生对此类药物的耐药现象。     

Quantitation of thymidylate synthase, dihydrofolate reductase, and DT-diaphorase gene expression in human tumors using the polymerase chain reaction.

A polymerase chain reaction (PCR)-based method was used to quantitate the expression levels of low abundance genes relevant to cancer drug activity. RNA from tumor samples as small as 20 mg was isolated and converted to cDNA using random hexamers. The 5' primers for the PCR contained a T7 polymerase promoter sequence, allowing the PCR-amplified DNA to be transcribed to RNA fragments. In each sample, the linear ranges of amplification of each cDNA of interest were established. Relative gene expressions were calculated by extrapolating the amounts of PCR products generated within the linear amplification regions of each gene to equal volumes of the cDNA solution. The method was accurate to less than a 2-fold difference in expression levels. Using beta 2-microglobulin and beta-actin gene expressions as internal reference standards and cDNA from HT-29 cells as an external linearity standard, we measured the relative expressions of thymidylate synthase, dihydrofolate reductase, and DT-diaphorase in a number of clinical tumor samples. The expressions of these genes varied from 50- to 100-fold among different tumors, although most of the values were grouped within about a 10-fold range. The amount of thymidylate synthase gene expression in tumor tissues was directly proportional to the content of thymidylate synthase protein. Those tumors with the lowest thymidylate synthase expression had the best response to both the 5-fluorouracil-leucovorin and 5-fluorouracil-cisplatin combinations.     

Expression of BCRP gene in the normal lung tissue and lung cancer

The development of multidrug resistance during chemotherapy was found to be correlated with over-expression of two transmembrane xenobiotic transporter protein, P-glycoprotein (Pgp) and the multidrug resistance protein (MRP).[1(6] Because the discordance between drug efflux and P-glycoprotein expression in human leukemic cells and unclear drug resistance exhibited during chemotherapy in advanced breast cancer patients, a alternative multidrug resistance transporter might be existed and has b…     

肺癌组织中黑色素瘤抗原-A3基因mRNA的表达及其序列点突变分析

目的检测肺癌组织中黑色素瘤抗原-3基因(MAGE-A3)mRNA的表达。方法用逆转录-套式聚合酶链反应(RT-PCR)对31例肺癌患者癌组织和相应癌旁组织MAGE-A3mRNA表达情况进行测定;PE-377DNA测序仪对5例10个RT-PCR扩增产物中的目的基因片段进行DNA序列测定。结果31例肺癌患者癌组织中26例表达MAGE-A3mRNA,阳性率为83.9%;相应的癌旁组织均未表达。DNA序列测定证明PCR扩增产物中目的基因片段均为MAGE-A3cDNA序列,所测5例样本中4例样本有两个相同位置的碱基发生了点突变(C2773→T2773;G2807→A2807),导致一个氨基酸残基改变(E143→K)。结论MAGE-A3mRNA在肺癌中呈高比例表达,提示此抗原有可能作为肺癌患者免疫治疗的靶抗原。我国肺癌患者中存在MAGE-A3基因个别位点的变异。     

Metabolism of diethylnitrosamine by nasal mucosa and hepatic microsomes from hamster and rat: species specificity of nasal mucosa

The oxidative metabolism of diethylnitrosamine (DEN) was investigated by acetaldehyde determination using microsomes from nasal mucosa and liver of Sprague-Dawley rats and nasal mucosa and liver of Syrian Golden hamsters, to establish the role of metabolic activation in the organo-targets for the carcinogenicity of the nitrosamine. The hepatic microsomal de-ethylation of DEN followed simple and biphasic Michaelis-Menten kinetics for rat liver and hamster liver, respectively. Both de-ethylations were inducible by phenobarbital (PB) and the DEN-de-ethylase activities and the Michaelis constants were determined. Microsomes from hamster liver showed a higher metabolic rate (Vmax) and a better affinity (Km) towards DEN with respect to microsomes from rat liver. In hamster, microsomes from nasal tissue biotransformed DEN at a rate and affinity quite similar to those of liver. In contrast, nasal mucosa of rat metabolized DEN poorly. The effect of metyrapone, a classical inhibitor of P-450 monooxygenases, on DEN de-ethylation was studied. It inhibited both hepatic and nasal DEN-de-ethylase activity, with greater affinity towards the latter. In addition metyrapone had a greater inhibitory effect on the hepatic P-450 isozymes induced in PB-treated animals. These results correlate well with the organotrophy of DEN carcinogenesis in the nasal region of hamster, but not of rat. They suggest that for the nose the metabolic activation of DEN in situ is necessary to elicit its carcinogenic effect.