Clinico-pathological features and somatic gene alterations in refractory ceramic fibre-induced murine mesothelioma reveal mineral fibre-induced mesothelioma identities

Abstract

Although human malignant mesothelioma (HMM) is mainly caused by asbestos exposure, refractory ceramic fibres (RCFs) have been classified as possibly carcinogenic to humans on the basis of their biological effects in rodents' lung and pleura and in cultured cells. Hence, further investigations are needed to clarify the mechanism of fibre-induced carcinogenicity and to prevent use of harmful particles. In a previous study, mesotheliomas were found in hemizygous Nf2 (Nf2(+/-)) mice exposed to asbestos fibres, and showed similar alterations in genes at the Ink4 locus and in Trp53 as described in HMM. Here we found that Nf2(+/-) mice developed mesotheliomas after intra-peritoneal inoculation of a RCF sample (RCF1). Clinical features in exposed mice were similar to those observed in HMM, showing association between ascite and mesothelioma. Early passages of 12 mesothelioma cell cultures from ascites developed in RCF1-exposed Nf2(+/-) mice demonstrated frequent inactivation by deletion of genes at the Ink4 locus, and low rate of Trp53 point and insertion mutations. Nf2 gene was inactivated in all cultures. In most cases, co-inactivation of genes at the Ink4 locus and Nf2 was found and, at a lower rate, of Trp53 and Nf2. These results are the first to identify mutations in RCF-induced mesothelioma. They suggest that nf2 mutation is complementary of p15(Ink4b), p16(Ink4a) and p19(Arf) or p53 mutations and show similar profile of gene alterations resulting from exposure to ceramic or asbestos fibres in Nf2(+/-) mice, also consistent with the one found in HMM. These somatic genetic changes define different pathways of mesothelial cell transformation.

Fig. 1

Histology of peritoneal mesotheliomas found in ceram-Nf2^+/− mice. (a) Epithelial mesothelioma (M) exhibiting a typical papillary pattern. Well-differentiated mesothelial cells proliferate into the peritoneal cavity (PC). Arrowheads: adjacent mesothelium. Grid represents 100 μm. (b) Epithelial mesothelioma (M). Dense and nodular proliferation at the surface of stomachal peritoneum, invading the peritoneal cavity (PC). Mu: stomachal muscle and St: stomachal mucosa. Grid represents 100 μm. Insert: higher magnification of tumour. Bar represents 2 μm. (c) Fusiform mesothelioma (M) at the surface of intestine peritoneum. In, intestinal mucosa; Mu: intestinal muscle and PC: peritoneal cavity. Grid represents 100 μm. (d) Typical bone differentiation (B) of a mesothelioma (M). Tumour growth is seen at the vicinity of the diaphragm (Di). Li: liver. Grid represents 100 μm.

Fig. 2

Analysis of p15/Cdkn2b (Ink4b locus), p16/Cdkn2a and p19/Arf (Ink4a/Arf locus) in mesothelioma cell cultures obtained from ascites developed in ceram-Nf2^+/− mice. (a) Analysis of p15/Cdkn2b, p16/Cdkn2a and p19/Arf genes by PCR. Exons 1 and 2 (p15/Cdkn2b), 1α (p16/Cdkn2a), 1β (p19/Arf) and 2 (common to p16/Cdkn2a and p19/Arf) were amplified by PCR using appropriate primer sets (Table I). Loss of exons suggests that Ink4a and Ink4b inactivation occurred by loss of a part or whole chromosome. Only four cultures (145, 146, 186 and 255) demonstrated normal exon amplification. Positive control (+) was gDNA extracted from mice erythroleukemia cell line. TCRa amplification was used as PCR control. (b) Analysis of p15^Ink4b and p16^Ink4a protein expression by western blots. Each antibody used for immunoblot was specific for a non-conserved epitope of the corresponding protein. p15^Ink4b and p16^Ink4a proteins were detected in four cell cultures (145, 146, 186 and 255). Positive control (+) was proteins extracted from mice erythroleukemia cell line. α-Tubulin was used as control for equivalent protein loading. (c) Analysis of p19^Arf protein expression by western blots. p19^Arf protein expression was studied in cultures showing gene amplification and in a negative mesothelioma culture (302) obtained from an asb-Nf2^+/− mice. p19^Arf was accordingly expressed in cultures where p19/Arf exons were present. Positive control (+) was proteins extracted from mice erythroleukemia cell line. α-Tubulin was used as control for equivalent protein loading.

Fig. 3

Analysis of Trp53 in mesothelioma cell cultures obtained from ascites developed in ceram-Nf2^+/− mice. (a) Mutations in the Trp53 in murine mesothelioma cell cultures 145, 186 and 255. A missense mutation was found in culture 145, whereas in culture 255 the mutation introduced a stop codon. A duplication was detected in culture 186. Arrows indicate the position of the mutations. gDNA sequence was compared with data provided in GeneBank (top: normal gDNA and bottom: gDNA from mesothelioma cells). (b) Analysis of p53 protein expression by western blots. p53 was found to be expressed in cultures 145 and 186 in agreement with protein stabilization related to the occurrence of gene mutation. Negative control (−) was proteins extracted from mice erythroleukemia cell line. α-Tubulin expression served as a control for equivalent protein loading.

Fig. 4

Analysis of loss of heterozygosity of exon 3 in Nf2 by PCR, and of gene expression in murine mesothelioma cell cultures obtained from ascites developed in ceram-Nf2^+/− mice. (a) Analysis of loss of heterozygosity of exon 3 in Nf2 gene by PCR. Loss of heterozygosity was found in all but two cell cultures (178 and 201). Positive control (+) was gDNA extracted from mice erythroleukemia cell line. TCRa amplification was used as PCR control. (b) Reverse transcription–PCR analysis of Nf2 mRNA transcripts. mRNA was reverse transcribed and cDNA was amplified using appropriate primers (Table I). Seven cultures were analysed, demonstrating that culture 178 and 201 express faint or no Nf2⁺ transcript respectively. Controls were mRNAs extracted from kidney (K) from Nf2^+/+ (K^+/+) and Nf2^+/− (K^+/−) mice, respectively. β-Actin cDNA was used as reverse transcription–PCR control. (c) nf2 protein expression of schwannomin (or merlin) by western blots. Schwannomin expression was studied in cultures showing exon 3 amplification (178 and 201) and in five negative cultures used as negative controls. Schwannomin was not found to be expressed in these cultures. Positive control (+) was proteins extracted from mice erythroleukemia cell line. α-Tubulin expression served as a control for equivalent protein loading.