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Replication asynchronism and mathylation status at the retinoblastoma 1 locus in thyroid tumours
The temporal order of DNA replication in the mammalian genome is a highly programmed process associated with transcriptional activity. Transcriptionally active loci replicate early during S-phase, while the inactive ones replicate late. Normally homologous loci replicate synchronously, however monoallelically expressed genes, such as genes subjected to X inactivation and imprinted genes, display an asynchronous pattern of allelic replication, in which the active allele replicates earlier than the inactive counterpart. Recently replicative asynchrony has been observed in leukaemic cells or lymphomas, also for genes with biallelic expression, such as the cancer implicated genes TP53, C-MYC and RB1. It has been suggeted that, if asynchronous replication reflects monoallelical expression, as above mentioned, their occurrence at cancer genes could reflect some epigenetic alteration manifesting as functional gene hemizygosis. This alteration, actually different from the classic gene mutation, may constitute one of the processes by which a cell acquires the number of mutations necessary to be transformed into tumour cell.
Aim of this study is the evaluation of replication pattern of the gene conferring the susceptibility to retinoblastoma RB1, in thyroidal tumours, adenomas and papillary carcinomas.
RB1 maps on 13q14.2 and it encodes for a nucleoprotein controlling the cell cycle and apoptosis. Loss of function of this gene is described in retinoblastoma as well as in several other tumour types, and it is believed to be a relevant event also in the development of thyroidal tumours. Hypermethylation of the region of the gene promoter is one of the mechanisms of gene inactivation.
The replication timing has been analysed by fluorescence in situ hybridisation (FISH) using a locus-specific probe. Replication status is derived from the number of signals in cells. Before replication, the hybridised cells present a couple of single signals, one for each of the two homologous chromosomes, after replication a couple of double signals, two for each chromosome. The presence of one double and of one single signal within the same cell, indicates the existence of replication asynchronism.
FISH analysis has been performed on archive slides prepared from apposition or from smear of tumour and healthy tissues. Normal tissue for the evaluation of basal levels of asynchronism has been obtained, when possible, from healthy lobe of the thyroid gland, otherwise from
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thyroidal healthy tissue of the same patient. The particular conditions for the set up of slides required modification of the classic protocol for FISH technique. Analyses have been performed on coded slides of 12 subjects, simultaneously on healthy and tumour tissues. The results show a significantly increased frequency of replication asynchronism in tumour tissues in comparison with healthy tissues; papillary carcinomas show higher frequency than adenomas.
The relevance of an alteration of gene RB1 function in thyroid cancers may be suggested also by the presence in analysed cases of cells with anomalous number of signals. To evaluate if these variation of signals number was due to locus deletions or duplications or to aneuploidy of the whole chromosome 13, we performed a dual color FISH, using simultaneously a probe for RB1 and a telomeric-specific probe for chromosome 13. All the samples have the only presence of aneuploidies: in samples of tumour cells with only one signal for RB1, we found loss of chromosome 13, as also the signal for telomere was missing; in samples with more than two signals, extra copies of chromosome 13 were found.
In order to test the hypothesis that late replication of one allele of RB1 could be associated to monoallelic expression, we investigated the possible functional inactivation of RB1 in our samples, by analysing the methylation status of gene promoter and the presense of pRB1.
We evaluated the expression levels of the protein by immunoistochemical analysis, performed on samples of tumour tissues stored in paraffin; healthy tissue surrounding the tumour area was used as control. Results show that in normal tissue and in adenomas the protein is expressed to high levels, while in malignant tumours there is a complete loss of expression. This finding highlights a possible involvement of loss of function of RB1 in the development of thyroid tumours.
The methylation status of promoter has been investigated by a technique based on bisulphite modification of DNA, extracted from samples of normal and tumour tissue, that modifies unmethylated cytosines into uracils whilst methylated cytosines are protected from modification; this technique allows the evaluation of methylation status of small regions of DNA even when small amounts of methylated DNA are present. The technique was validated in our laboratory. The presence of methylation was verified by means of nested PCR using primers for the selected region of 233 bp in RB1 promoter, which contains 37 CpG that are binding sites for known transcription factors. These primers were appositely designed for the modified sequence and, not including CpG, they are able to amplify it, independently form its methylation status. Methylation level has been estimated after sequencing of amplified region, evaluating the ratio of cytosines and tymines. The results of this analysis show that methylation of RB1 gene promoter
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is absent in all normal and tumour tissues.
These results of this study show how high levels of asynchronism are potentially associable to loss of gene expression and that promoter methylation seems not to be the epigenetic alteration involved in these phenomena. Loss of expression, observed only in malignant tumours, seems to confer to this gene a possible role in the development of these tumours.
