Mutations and reduced expression of the transforming growth factor-beta receptor II gene in rat lung adenocarcinomas induced by N-nitrosobis-(2-hydroxypropyl)amine.

Mutations and expression of the transforming growth factor-beta receptor type II (TGF-beta RII) gene were investigated in lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) in rats. Males of the Wistar strain, 6 weeks old, were given 2000 ppm of BHP in their drinking water for 12 weeks and then maintained without further treatment until killed at week 25. Total RNA was extracted from 12 adenocarcinomas and mutations in TGF-beta RII were investigated by RT-PCR-restriction-SSCP analysis followed by sequencing analysis. Two out of 12 adenocarcinomas showed band shifts, indicative of mutations (16.7%). One was a CTG-to-TTG (Leu to Leu) transition at codon 308 without amino acid alteration and the other a frameshift deletion of one of two guanines at nucleotides 1434 to 1435 (codon 477 to 478). Semi-quantitative RT-PCR analysis demonstrated significantly reduced TGF-beta RII expression in adenocarcinomas, as compared with normal lung tissue. These results suggest that TGF-beta RII alterations may play a role in the acquisition of growth advantage by lung adenocarcinomas induced by BHP in rats.     

Elevated Expression of Interleukins in Lung Adenocarcinomas Induced by N-Nitrosobis(2-hydroxypropyl)amine in Rats

The expression of interleukins (ILs) in lung adenocarcinomas induced by N -nitrosobis(2-hydroxypropyl)amine (BHP) in rats was investigated using a multiprobe RNase protection assay (RPA) followed by densitometric quantification. Male Wistar rats, 6 weeks old, were given 2000 ppm BHP in their drinking water for 12 weeks and maintained without further treatment until they were killed at week 25. Total RNAs were extracted from 14 individual adenocarcinomas and 2 specimens of normal lung tissue of untreated rats. In adenocarcinomas, elevated expression of IL-1α (6/14), IL-1β (14/14), IL-3 (7/14), IL-4 (11/14), IL-5 (9/14), IL-6 (11/14) and IL-10 (8/14) was observed, compared with normal lung tissues. In contrast, no expression of IL-2 was detected in any case. The results suggest that preferential expression of these ILs and their complex networks may contribute to the development and progression of lung adenocarcinomas induced by BHP in rats.     

Elevated expression of interleukins in lung adenocarcinomas induced by N-Nitrosobis(2-hydroxypropyl)amine in rats.

The expression of interleukins (ILs) in lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) in rats was investigated using a multiprobe RNase protection assay (RPA) followed by densitometric quantification. Male Wistar rats, 6 weeks old, were given 2000 ppm BHP in their drinking water for 12 weeks and maintained without further treatment until they were killed at week 25. Total RNAs were extracted from 14 individual adenocarcinomas and 2 specimens of normal lung tissue of untreated rats. In adenocarcinomas, elevated expression of IL-1alpha (6 / 14), IL-1beta (14 / 14), IL-3 (7 / 14), IL-4 (11 / 14), IL-5 (9 / 14), IL-6 (11 / 14) and IL-10 (8 / 14) was observed, compared with normal lung tissues. In contrast, no expression of IL-2 was detected in any case. The results suggest that preferential expression of these ILs and their complex networks may contribute to the development and progression of lung adenocarcinomas induced by BHP in rats.     

Frequent mutations of Ki-ras but no mutations of Ha-ras and p53 in lung lesions induced by N-nitrosobis(2-hydroxypropyl)amine in rats

Point mutations of the Ki-ras and p53 genes in rat lung lesions induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) were investigated by polymerase chain reaction-single-strand conformation polymorphism analysis followed by direct sequencing using paraffin-embedded tissues. Male Wistar rats 6 wk old were given 2000 ppm BHP in drinking water for 15 wk. Another group was given drinking water without BHP. The rats were killed 20–27 wk after the beginning of the experiment. Lung adenomatous and squamous lesions, including carcinomas, were induced. The frequencies of Ki-ras mutations were 40% (six of 15) in alveolar hyperplasias, 36% (five of 14) in adenomas, 72% (18 of 25) in adenocarcinomas, 20% (three of 15) in squamous metaplasias, 50% (three of six) in squamous cell carcinomas, and 50% (five of 10) in adenosquamous carcinomas. The mutations were all G → A transitions at the second position of codon 12; no other mutations were detected. However, Ha-ras mutations in exons 1 and 2 and p53 mutations in exons 5, 6, and 7 were not detected in adenocarcinomas and squamous cell carcinomas. These results indicate that Ki-ras mutation is an early genetic event in some adenomatous and squamous lung carcinogenesis and that Ki-ras mutations can cause benign lesions to convert to malignant lesions. The results also show that Ha-ras and p53 mutations are not involved in rat lung carcinogenesis induced by BHP. © 1996 Wiley-Liss, Inc.     

Mutations of adenomatous polyposis coli and beta-catenin genes during progression of lung tumors induced by N-nitrosobis(2-hydroxypropyl)amine in rats

In the present study, we investigated mutations of the adenomatous polyposis coli (APC) and beta-catenin genes to clarify possible molecular mechanisms underlying development of lung tumors induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) in rats. Male Wistar rats, 6 weeks of age, were given 2000 ppm BHP in drinking water for 12 weeks and then maintained without further treatment until sacrifice at week 25 DNA was extracted from paraffin-embedded tissues, and PCR-single-strand conformation polymorphism analysis, followed by nucleotide sequencing, was performed. No APC mutations were detected in 17 hyperplasias, but 2 of 15 adenomas (13.3%) and 8 of 20 adenocarcinomas (40.0%) showed changes within exon 1 to the mutation cluster region in exon 15. For beta-catenin, no mutations were detected in 17 hyperplasias, but 3 of 15 adenomas (20.0%) and 5 of 20 adenocarcinomas (25.0%) had alterations within or flanking codons corresponding to important phosphorylation sites. Immunohistochemical staining showed beta-catenin protein localized in the cell membranes in the surrounding normal-appearing lung and 216 hyperplasias and localized mainly in the cytoplasm and/or nucleus in 10 of 37 adenomas (27.0%) and 21 of 40 adenocarcinomas (52.5%). These results suggest that the APC-beta-catenin-T-cell factor signaling pathway is involved in the acquisition of growth advantage from adenomas to adenocarcinomas in BHP-induced rat lung carcinogenesis.     

Expression of vascular endothelial growth factor and its receptors during lung carcinogenesis by N-nitrosobis(2-hydroxypropyl)amine in rats.

The expression of vascular endothelial growth factor (VEGF) and its receptors (VEGFRs), VEGFR-1/Flt-1 and VEGFR-2/Flk-1, was investigated by immunohistochemical and northern blot analysis during lung carcinogenesis by N-nitrosobis(2-hydroxypropyl)amine (BHP) in male Wistar rats. After BHP was given in the drinking water for 12 wk, the rats were maintained without further treatment until they were killed at 20-28 wk. Immunohistochemical studies revealed VEGF expression in almost all malignancies, the reaction being strongly positive in most adenocarcinomas (15 of 18; 83.3%) and squamous cell carcinomas (four of five; 80.0%), but less so in a total of 120 adenomas and 136 alveolar hyperplasias. In addition, VEGF mRNA and VEGFR mRNAs were found to be overexpressed in most adenocarcinomas and squamous cell carcinomas as well as in one to three of the five adenomas tested. The results indicated that VEGF and VEGFRs play important roles in the acquisition of malignant potential by preneoplastic lung lesions induced by BHP in rats. Moreover, overexpression of VEGF was related to upregulation of VEGFR-1/Flt-1 and VEGFR-2/Flk-1 expression in malignant and premalignant lung lesions.     

Frequent Microsatellite Instabilities and Analyses of the Related Genes in Familial Gastric Cancers

Microsatellite instability or replication error seems to be related to defective DNA mismatch repair genes, such as hMSH2 , hMLH1 , hPMS1 and hPMS2 , which have been identified as causative genes of hereditary nonpolyposis colorectal cancers (HNPCC). Recently, it was reported that mutations at the simple repeated sequences in the transforming growth factor-β type II receptor ( TGF-β RII ) gene occurred in replication error-positive colorectal cancers. To determine genetic alterations in familial gastric cancers (FGC), we examined replication error using eight microsatellite DNA markers, and screened mutations in the hMSH2 , hMLH1 and TGF-β RII genes in six cases from four FGC kindreds. Moreover, hMTH1 , a human homolog of the bacterial mutT gene, was also screened. Four of six (67%) cancers showed the replication error-positive phenotype, indicating that microsatellite instability is highly associated with not only HNPCC, but also FGC. No germline mutation was found in the whole coding sequences of hMSH2 and hMTH1 , or in the conservative regions of hMLH1 in any patient, while one cancer DNA showed a somatic mutation at codon 682 (threonine to alanine) in hMSH2 . No alteration was found at the small repeated sequences in TGF-β RII in FGC tumor DNA. These results indicate that the carcinogenetic process of FGC may be different from that of HNPCC.