Isolation of a SIR-like gene, SIR-T8, that is overexpressed in thyroid carcinoma cell lines and tissues - PubMed (original) (raw)

Isolation of a SIR-like gene, SIR-T8, that is overexpressed in thyroid carcinoma cell lines and tissues

F de Nigris et al. Br J Cancer. 2002.

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Abstract

We used subtractive library screening to identify the changes that occur in gene expression during thyroid cell neoplastic transformation. Complementary DNA from normal thyroid cells (HTC 2) was subtracted from a complementary DNA library constructed from a human thyroid papillary carcinoma cell line. The library was screened for genes upregulated in human thyroid papillary carcinoma cell line cells, and several cDNA clones were isolated. One of these clones has a sirtuin core and high homology with the human silent information regulator protein family. This clone, designated "SIR-T8", was overexpressed in human thyroid carcinoma cell lines and tissues, but not in adenomas. The human SIR-T8 protein has a molecular weight of 39 kDa and is primarily located in the cytoplasm under the nuclear membrane. The SIR-T8 gene is located on chromosome 17q25-1.

Copyright 2002 Cancer Research UK

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Figures

Figure 1

Figure 1

Northern blot analysis of Sir-T8 expression. (A) Total RNA (5 μg per lane) was hybridised with SIR-T8 probe. Lane 1: normal thyroid cell line HTC-2, lane 2: Normal tissue; and lane 3: NPA cell line. A GAPDH probe was used as internal control for uniform RNA loading. Lower part: densitometric analysis. Bars present the mean±s.d. of the results obtained in three experiments. *P<0.001 vs HTC-2 and normal thyroid tissue (NT). (B) cDNA and amino acid sequences of SIR-T8. Nucleotides are numbered from the beginning of the sequence, and the deduced amino acid sequence is indicated from the beginning of the open reading frame. An in-frame stop codon is indicated in bold. (C) Comparison of the amino acid sequence of SIR-T8 with other known human SIR genes; the conserved domains are boxed.

Figure 2

Figure 2

Expression of SIR-T8 in normal and tumoral thyroid cells. Total RNA (5 μg per lane) was size-fractionated on denaturing formaldehyde agarose gel, blotted onto nylon filters (hybond-N, Amersham) and probed with SIR-T8 cDNA. RNA was fractioned from the following sources: lane 1: normal thyroid primary culture cells; lane 2: NIM cell line; lane 3: TPC-1 cell line; lane 4: NPA cell line; lane 5: WRO cell line; lane 6: FRO cell line; lane 7: ARO cell line. A GAPDH probe was used as internal control for uniform RNA loading. Lower panel: densitometric analysis of SIR-T8 expression. Bars present the mean±s.d. of the results obtained in three experiments. *P<0.0001 vs HTC2, #P<0.005 vs NPA and **P<0.001 vs HTC2.

Figure 3

Figure 3

SIR-T8 expression in normal and neoplastic thyroid tissues. RT–PCR analysis of SIR-T8 in normal and neoplastic thyroid tissues. The SIR-T8 mRNA was coamplified with GAPDH as an internal control. RNA sources were divided by pathology as indicated.

Figure 4

Figure 4

Upper panel: Western blot analysis of protein SIR-T8. Total protein extracts from the NPA cell line transfected and untransfected with p/Flag-SIRT-8 construct were subjected to Western blot analysis with antiFlag monoclonal antibody. Lane 1: untransfected NPA cells; lane 2: NPA cells transfected with the p/Flag-SIRT-8 construct; lane 3: COS cells transfected with the p/Flag-SIRT-8 construct as control. Lower panel: Subcellular localisation of SIR-T8. NPA cells were grown on coverslips for 24 h, transfected with SIR-T8 expression vector and stained with Hoechst (A), or immunostained with anti-Flag antibodies (B) as described.

Figure 5

Figure 5

Northern blot analysis of SIR-T8 gene in different normal tissues. SIR-T8 gene expression was analysed in Poly(A)+ RNAs from various normal tissues as indicated.

Figure 6

Figure 6

FISH mapping of the SIR-T8 gene to chromosome 17q25.1. Left ideogram of chromosome 17. Right 4′,6-diamidino2-phenylindole (top) and corresponding propidium (bottom) FISH analysis of normal lymphocyte metaphase with a phage clone the SIR-T8 gene hybridisation. Note the signal on chromosome 17q25.1.

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