Absence of the c-Ha-ras and c-Ki-ras oncogene mutations in the hermaphroditic fish Rivulus marmoratus papillary thyroid carcinomas induced by N-methyl-N'-nitro-N-nitrosoguanidine

Abstract

Previously we reported that papillary thyroid carcinomas were predominantly induced at high frequency by a low dose of N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG) in the hermaphroditic fish Rivulus marmoratus. In the current study, polymerase chain reaction (PCR) amplification and direct sequencing were used to examine the point mutations of Ha‐ and Ki‐ras genes, which may be associated with papillary thyroid tumour development in rivulus. Thirty‐three tumour samples were tested, however, no mutations were detected in rivulus Ha‐ and Ki‐ras genes. In human and rodent models, it has been reported that ras gene mutations in papillary thyroid tumours occurred preferentially in the N‐ras gene in general, while follicular tumours contained activated Ha‐ or Ki‐ras gene mutations. This may explain why papillary thyroid carcinomas in rivulus were not mutated at codon 12, 13 or 61 of exon 1 or exon 2 of the rivulus Ha‐ or Ki‐ras gene. These results imply that another oncogene, such as the N‐ras gene and others, may be preferentially activated in rivulus papillary thyroid carcinomas, and also give valuable information for comparative studies of papillary thyroid carcinogenesis.

Nmethyl-N-nitrosourea (MNU) induces papillary thyroid tumours which lackras gene mutations in the hermaphroditic fishRivulus marmoratus

“…However, although we examined extensively Ha-and Ki-ras gene exon 1 and exon 2 regions, we were not able to detect ras mutations in these tumour DNAs. This result supports our earlier observation that Ha-and Ki-ras genes were not mutated in papillary thyroid tumours induced by another alkylating agent MNNG [4]. We have already suggested the possibility that Rivulus papillary thyroid tumours may be induced by pathways independent of Ha-and Ki-ras such as by ret/PTC gene-associated rearrangement [4].…”

“…This result supports our earlier observation that Ha-and Ki-ras genes were not mutated in papillary thyroid tumours induced by another alkylating agent MNNG [4]. We have already suggested the possibility that Rivulus papillary thyroid tumours may be induced by pathways independent of Ha-and Ki-ras such as by ret/PTC gene-associated rearrangement [4]. The involvement of several other oncogenes in papillary carcinogenesis is also possible.…”

“…marmoratus were bred and reared in our aquarium as described earlier [4]. Young fish (less than 1 month old) were fed brine shrimp nauplii (Artemia salina), and adult fish were given chicken liver and Drosophila.…”

“…Typical papillary carcinomas were selected after Hematoxylin and Eosin staining, and the genomic DNA was extracted for mutation analysis of ras genes. For direct sequencing of R. marmoratus Ha-and Ki-ras exon 1 and exon 2 regions, polymerase chain reactions (PCR) products were generated as described previously [4]. Primer sets used are as shown in Table 1.…”

“…Previously we reported that the alkylating agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) specifically induced papillary thyroid tumours in R. marmoratus [3,4]. The purpose of this experiment was firstly to check the reproducibility of papillary thyroid tumour induction by another alkylating agent MNU to establish if papillary thyroid tumours are a general feature of alkylating agent exposure.…”