Correlations have been found between radiation exposure and thyroid carcinoma development, particularly in children. Recent studies on a large cohort of radiation-induced papillary thyroid carcinomas ...(PTC) after the Chernobyl reactor accident disclosed a common type of underlying genetic alteration. A high prevalence of rearrangements of the receptor tyrosine kinase (TK) c-RET was observed, besides some rearrangements involving NTRK1. Radiation-induced RET rearrangements in PTC consist most frequently of fusions to the H4 gene (RET/PTC1) or to the ELE1 (ARA70) gene (RET/PTC3). Both fusions are formed by balanced paracentric inversions on chromosome 10. An analysis of the fused genes in ELE1/RET rearrangements revealed DNA double-strand breaks spread over a distance of about 2.3 kb in two introns and the interposed exon of ELE1, exon 11 and intron 11 of RET, without significant clustering in these parts of the genes. Topoisomerase I sites were found exactly at or in close vicinity to all breakpoints, suggesting a role for this enzyme in formation of DNA strand breaks or inversions. The genes fuse at short regions of sequence homology and short direct or inverted repeats (microhomology-mediated DNA end joining). A minority of PTC cases contain novel types of RET rearrangement, with RIα, GOLGA2, HTIF, HTIF homolog, RFG8, ELKS, KTN1 and PCM-1 as the 5′-fused genes. These novel types of gene fusions are formed by interchromosomal translocation. The formation of these rare types of rearrangement seems to be highly related to radiation as they have rarely been found in sporadic PTC. All RET gene fusions seem to act similarly on RET function: The strict physiological control of RET TK activity is suspended through constitutive activation by 5′-fused parts of genes containing coiled-coil domains with dimerization potential. RET expression in thyrocytes, which under normal conditions, lack RET TK activity apparently triggers clonal expansion and early invasion of the affected cells. RET-fused genes, some of which are transcriptional coactivators, are important determinators of the peculiar phenotype of the tumour and for its clinical course. This is most significant in RET/PTC3 rearrangements with ELE1 as the RET-fused gene: this type of rearrangement leads more often to the phenotype of a solid variant of PTC, and to rapid tumour development and early lymph node metastasis. Up to now, no other genetic aberration has more frequently been observed in PTC than RET rearrangement, thus suggesting that RET rearrangement represents a genetic marker lesion of radiation history in the development of a PTC.
Papillary thyroid carcinomas (PTC) developed with a high incidence in children and young adults who had been exposed to radioactive fallout in contaminated regions of Belarus after the Chernobyl ...reactor accident. They are informative for a molecular genetic analysis of radiation-induced PTC. In contrast to spontaneous PTC, a high prevalence of gene aberrations was found with rearrangements of the receptor tyrosine kinase gene
RET in the majority of cases and a few
NTRK1 rearrangements. In the rearranged form of
RET, the transmembrane and extracellular parts are replaced by regulatory units of other genes. Among the fused genes,
ELE1 (
ARA70) is most prevalent in PTC at short latency periods after irradiation,
H4 gene fusions prevail in later-occurring PTC. Both types of rearrangement, PTC3 and PTC1, respectively, are formed by intrachromosomal inversions on chromosome 10. Analysing
ELE1/
RET chimeric genes, we found radiation-induced DNA breakpoints localized exactly at or in close vicinity to topoisomerase I binding sites indicating a role for this enzyme in the formation of DNA breaks after irradiation. Breakpoints are distributed in the affected introns of both genes without significant clustering. They do not contain larger deletions of insertions. Short regions of sequence homology and short direct or inverted repeats were observed at the breakpoints suggesting microhomology-mediated DNA end joining as a mechanism in the fusion process. In addition to PTC1 and PTC3, we and others described several rare novel types of
RET rearrangement, all formed by interchromosomal translocations, with parts of
RIα,
GOLGA5,
HTIF,
RFG7,
RFG8,
KTN1, and
ELKS fused at the 5' end of the
RET tyrosine kinase domain. These gene fusions appear to have in common an uncoupling of the
RET tyrosine kinase from its physiological control due to an inherent dimerization potential. This may lead to constitutive, ligand-independent activation of
RET tyrosine kinase in thyrocytes lacking this activity under normal conditions, and subsequent clonal expansion of the affected cells. It is evident from our comparative study on a large number of PTC with
ELE1/
RET and
H4/
RET rearrangements after irradiation that the type of the
RET-fused gene determines the tumor phenotype and may be decisive also for the clinical course of radiation-induced PTC.
A new, easy method for the immunodetection of specific antigens in two-dimensional electrophoresis (2-DE) is described. Areas of 2-DE gels containing antigens of interest are electrophoretically ...transferred to polyvinylidene difluoride membranes, immunostained with specific antibodies using Fast Red or 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium as detection systems and counterstained with Coomassie Brilliant Blue. In contrast to conventional methods, it is possible to use this procedure to exactly assign immunoreacting proteins on a single blot to their corresponding and surrounding blue-stained protein spots.
Heterotrimeric G proteins participate in the signal transduction cascade. Adult thyroid tumors have been shown to harbor specific point mutations in codons 201 and 227 of the G(s)alpha subunit of the ...adenylate cyclase stimulator. This protein affects the GDP/GTP turnover and finally results in an enhanced activation of G(s) and thus adenylate cyclase. We attempted to find out if G(s)alpha gene mutations were present in thyroid tumors of children from Belarus after the Chernobyl nuclear accident. Paraffin sections of 20 thyroid tumors were used for PCR amplification by oligonucleotide intron primers flanking exons 8 and 9, encompassing codon 201 and 227, respectively. By direct sequencing of the 274-bp amplification product, we did not detect any mutations of the G(s)alpha gene in codon 201 or 227. In contrast to thyroid neoplasia of adults, G(s)alpha gene mutations do not play a role in the development of childhood thyroid tumors after the Chernobyl reactor accident.
c-myc expression, amplification, and rearrangement were studied in neoplastic hepatic nodules of rats after diethylnitrosamine treatment, in hepatocellular carcinomas induced by ...N-methyl-N-nitrosourea, and in tumorigenic liver cell lines derived from rat hepatocytes transformed by diethylnitrosamine in vivo. Steady-state levels of c-myc transcripts, as measured by Northern blot hybridization, were slightly increased in neoplastic hepatic nodules and showed high levels in some hepatocellular carcinomas and some tumorigenic liver cell lines. DNA of the samples was analyzed after restriction nuclease treatment by Southern blot hybridization, using different probes specific for the three exons of the c-myc gene. The results suggest rearrangements of the complete gene and/or amplification in some cases. Rearrangements include the 5'-part of the first exon and a stretch upstream c-myc supposed to contain control elements of c-myc expression. Aberrations of this kind are most pronounced in advanced metastasizing hepatocellular carcinomas and highly tumorigenic, metastasizing liver cell lines. Heterogenicity of genomic alterations and c-myc transcript levels were found among different samples of the same hepatocellular carcinoma indicating subclonal diversification.
A chromosomal analysis was performed on two cell lines which were derived from the liver of two rats exposed to diethylnitrosamine in vivo. The cells were obtained by collagenase perfusion of the ...liver at an early stage of development of ATPase-deficient putative preneoplastic populations, and propagated from foci of epithelial cells which started growth in vitro. Cell line CL 38 proved to be tumorigenic after transplantation into nude mice, giving rise to hepatocellular carcinomas and metastases. Cell line CL 44 was nontumorigenic after transplantation into nude mice and was therefore considered preneoplastic. The diploid nontumorigenic line CL 44, with a modal number of 42 chromosomes, showed a deletion of chromosome 1 and a translocation of chromosomes 3 and 14 t(3q12;14q21). The hyperdiploid neoplastic cell line CL 38 has a modal chromosome number of 52 and showed tri- or tetrasomy of chromosomes 3, 7, 9, 11, and 12 and a marker chromosome that might have originated from aberrant chromosome 1. One or two homologues of chromosome 3 showed terminal deletions (q42, q41, or q35). In both cell lines rearrangements of chromosome 11 were observed rob(11q;?) or +11 or -11 or del(11)(q12). Some of these karyotype abnormalities are located on the same chromosome as described for transplantable hepatomas and for other chemically induced tumors of the rat.
Tumor-associated protein variants were detected by two-dimensional gel electrophoresis (2-DE) in soluble proteins from chemically
induced rat hepatomas and transformed rat liver cell lines. Among ...them, a series of 8 protein variants was found localized
at similar sites in 2-DE gels (33-35 kDa, with different pI values). We characterized four of them. In situ peptide mapping
with limited proteolysis disclosed a significant relationship between these four individual protein spots. Their different
position in 2-DE gels might be due to posttranslational modifications: one of the variants was phosphorylated, three others
were modified by glycosylation. The most prominent tumor-associated protein variant of this series (spot 17) was further studied
by amino acid analysis and internal amino acid microsequencing. It became evident that this variant is identical to aldose
reductase (EC 1.1.1.21). This enzyme of the sorbitol pathway is expressed in the liver during embryogenesis, but is absent
in adult rat liver. Our results suggest that it is reexpressed and functionally active during liver carcinogenesis.
Purpose and Methods: To investigate the potential role of an aberrant cellular DNA repair in target cells during malignant transformation we studied cell type-specific mRNA expression of the DNA ...repair protein O super(6)-alkylguanine-DNA alkyltransferase (O super(6)-AGT) in normal and regenerating rat liver, chronic hepatitis and preneoplastic liver lesions by in situ hybridization and semiautomatic image analysis. Results: O super(6)-AGT mRNA expression was found to be four to five times higher in hepatocytes than in nonparenchymal cells. A 1.9-fold increase in O super(6)-AGT mRNA was observed after partial hepatectomy. Intraperitoneal injection of diethylnitrosamine led to a 1.3-fold and 2.6-fold rise in periportal and perivenous hepatocytes, respectively. Ethylnitrosourea produced an enhancement of mRNA levels up to 1.6-fold in hepatocytes without regional differences. In megalocytic hepatocytes of Long-Evans Cinnamon rats with chronic hepatitis, a 4.4-fold mRNA induction was found. In small preneoplastic lesions induced after chronic diethylnitrosamine-exposure, O super(6)-AGT mRNA expression was identical to that of adjacent normal tissue. Intermediate and large lesions revealed 1.5- to 1.6-fold higher mRNA levels. Conclusions: These results suggest an induction of O super(6)-AGT mRNA expression in hepatocellular target tissue under conditions of increased carcinogen sensitivity . The O super(6)-AGT expression in early preneoplastic lesions did not differ from normal surrounding liver tissue, thus excluding the possibility that progression of preneoplasia in rat liver is associated with a deficient mRNA expression of this DNA repair protein. On the contrary, enhanced O super(6)-AGT mRNA expression in more advanced foci and early neoplastic nodules may confer a selective advantage upon early malignant hepatocytes with regard to further tumor progression.
Purpose and Methods: To investigate the potential role of an aberrant cellular DNA repair in target cells during malignant transformation we studied cell type-specific mRNA expression of the DNA ...repair protein O^sup 6^-alkylguanine-DNA alkyltransferase (O^sup 6^-AGT) in normal and regenerating rat liver, chronic hepatitis and preneoplastic liver lesions by in situ hybridization and semiautomatic image analysis. Results: O^sup 6^-AGT mRNA expression was found to be four to five times higher in hepatocytes than in nonparenchymal cells. A 1.9-fold increase in O^sup 6^-AGT mRNA was observed after partial hepatectomy. Intraperitoneal injection of diethylnitrosamine led to a 1.3-fold and 2.6-fold rise in periportal and perivenous hepatocytes, respectively. Ethylnitrosourea produced an enhancement of mRNA levels up to 1.6-fold in hepatocytes without regional differences. In megalocytic hepatocytes of Long-Evans Cinnamon rats with chronic hepatitis, a 4.4-fold mRNA induction was found. In small preneoplastic lesions induced after chronic diethylnitrosamine-exposure, O^sup 6^-AGT mRNA expression was identical to that of adjacent normal tissue. Intermediate and large lesions revealed 1.5- to 1.6-fold higher mRNA levels. Conclusions: These results suggest an induction of O^sup 6^-AGT mRNA expression in hepatocellular target tissue under conditions of increased carcinogen sensitivity . The O^sup 6^-AGT expression in early preneoplastic lesions did not differ from normal surrounding liver tissue, thus excluding the possibility that progression of preneoplasia in rat liver is associated with a deficient mRNA expression of this DNA repair protein. On the contrary, enhanced O^sup 6^-AGT mRNA expression in more advanced foci and early neoplastic nodules may confer a selective advantage upon early malignant hepatocytes with regard to further tumor progression.PUBLICATION ABSTRACT
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