Epigenetic inactivation of a RAS association domain family protein from the lung tumour suppressor locus 3p21.3

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Allelic loss at the short arm of chromosome 3 is one of the most common and earliest events in the pathogenesis of lung cancer, and is observed in more than 90% of small-cell lung cancers (SCLCs) and in 50–80% of non-small-cell lung cancers1,2 (NSCLCs). Frequent and early loss of heterozygosity and the presence of homozygous deletions suggested a critical role of the region 3p21.3 in tumorigenesis2,3,4 and a region of common homozygous deletion in 3p21.3 was narrowed to 120 kb (ref. 5). Several putative tumour-suppressor genes located at 3p21 have been characterized, but none of these genes appear to be altered in lung cancer. Here we describe the cloning and characterization of a human RAS effector homologue (RASSF1) located in the 120-kb region of minimal homozygous deletion. We identified three transcripts, A, B and C, derived from alternative splicing and promoter usage. The major transcripts A and C were expressed in all normal tissues. Transcript A was missing in all SCLC cell lines analysed and in several other cancer cell lines. Loss of expression was correlated with methylation of the CpG-island promoter sequence of RASSF1A. The promoter was highly methylated in 24 of 60 (40%) primary lung tumours, and 4 of 41 tumours analysed carried missense mutations. Re-expression of transcript A in lung carcinoma cells reduced colony formation, suppressed anchorage-independent growth and inhibited tumour formation in nude mice. These characteristics indicate a potential role for RASSF1A as a lung tumour suppressor gene.

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Figure 1: Location of genes and deletions in 3p21.3 and amino acid comparison.
Figure 2: Northern-blot analysis of RASSF1A, RASSF1B and RASSF1C transcripts in human tissues and cancer cell lines.
Figure 3: Expression of RASSF1 in normal and tumour cell lines.
Figure 4: Methylation analysis of RASSF1A.
Figure 5: Re-expression of transcript A by treatment with 5-aza-2′-deoxycytidine (5-Aza-CdR).
Figure 6: Effect of RASSF1A expression on cell growth characteristics.

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We thank C.A. Tessler for technical support. This work was supported by a grant from the University of California Tobacco Related Disease Research Program (6RT-0361 to G.P.P.).

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Correspondence to Gerd P. Pfeifer.

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