Kinase expression and chromosomal rearrangements in papillary thyroid cancer tissues: investigations at the molecular and microscopic levels

Abstract

Structural chromosome aberrations are known hallmarks of many solid tumors. In the papillary form of thyroid cancer (PTC), for example, activation of the receptor tyrosine kinase (RTK) genes, ret or the neurotrophic tyrosine kinase receptor type I (NTRK1) by intra- or interchromosomal rearrangements have been suggested as a cause of the disease. The 1986 accident at the nuclear power plant in Chernobyl, Ukraine, led to the uncontrolled release of high levels of radioisotopes. Ten years later, the incidence of childhood papillary thyroid cancer (chPTC) near Chernobyl had risen by two orders of magnitude. Tumors removed from some of these patients showed aberrant expression of the ret RTK gene due to a ret/PTC1 or ret/PTC3 rearrangement involving chromosome 10. However, many cultured chPTC cells show a normal G-banded karyotype and no ret rearrangement. We hypothesize that the "ret-negative" tumors inappropriately express a different oncogene or have lost function of a tumor suppressor as a result of chromosomal rearrangements, and decided to apply molecular and cytogenetic methods to search for potentially oncogenic chromosomal rearrangements in Chernobyl chPTC cases. Knowledge of the kind of genetic alterations may facilitate the early detection and staging of chPTC as well as provide guidance for therapeutic intervention.

Fig. 1

The three major forms of ret activation by chromosomal rearrangement. A) The translocation breakpoint in the ret gene is between the transmembrane domain (TM) and the tyrosine kinase domain (TK). The tk domain is then fused in frame to an expressed sequence (H4, R1∞ or RFG/ele1). B) The ret/PTC1 translocation is a paracentric inversion fusing the 5′-end of ret to H4(D10S170) which is located more distal on the long arm of chromosome 10 (12).

Fig. 2

FISH analysis of the PTC cell line S48TK. A) The SKY analysis demonstrated several rearranged chromosomes. The insert in the lower right corner shows the two complex marker chromosomes (m1, m2), which we analyzed. B) The results of CGH analysis suggest copy number changes along chromosomes 1 and 9. C) Hybridization of individual BAC-derived DNA probes suggests that markers m1 and m2 do not contain a significant portion of the short arm of chromosome 9, but gene duplication on m1. D) The in situ hybridization of a cDNA probe for NTRK-1 shows strong signals in the cytoplasm of S48TK cells. E-G) Our BAC-FISH approach to find occult translocations, rapidly characterize markers and delineate breakpoint regions. E) BAC clones were pooled such that all probes mapping to either the short or long arm of chromosome 9 were labeled with the same reporter molecule. F) Very clean hybridizations to S48TK metaphase cells support the notion that markers m1 and m2 carry only chromosome 9 material, which stems from the long arm. G) Hybridization of differently-labeled probes from the region at the interphase of pools 9-10 and 9-11 demonstrated signals of these BAC probes on m1 and well as m2. H) In silico analysis. The region apparently translocated to m2 is shown in the viewer of the UCSC Genome Browser window (25).