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Humans and animals undergo ageing, and although their primary cells undergo cellular senescence in culture, the relationship between these two processes is unclear. Here we show that gamma-H2AX foci (gamma-foci), which reveal DNA double-strand breaks (DSBs), accumulate in senescing human cell cultures and in ageing mice. They colocalize with DSB repair factors, but not significantly with telomeres. These cryptogenic gamma-foci remain after repair of radiation-induced gamma-foci, suggesting that they may represent DNA lesions with unrepairable DSBs. Thus, we conclude that accumulation of unrepairable DSBs may have a causal role in mammalian ageing.

A related DNA fragment distinct from the epidermal growth factor receptor and ERBB2 genes was detected by reduced stringency hybridization of v-erbB to normal genomic human DNA. Characterization of the cloned DNA fragment mapped the region of v-erbB homology to three exons with closest identity of 64% and 67% to a contiguous region within the tyrosine kinase domains of the epidermal growth factor receptor and ERBB2 proteins, respectively. cDNA cloning revealed a predicted 148-kDa transmembrane polypeptide with structural features identifying it as a member of the ERBB gene family, prompting us to designate the gene as ERBB3. It was mapped to human chromosome 12q13 and was shown to be expressed as a 6.2-kilobase transcript in a variety of normal tissues of epithelial origin. Markedly elevated ERBB3 mRNA levels were demonstrated in certain human mammary tumor cell lines. These rmdings suggest that increased ERBB3 expression, as in the case of epidermal growth factor receptor and ERBB2, may play a role in some human malignancies.Protooncogenes encoding growth factor receptors constitute several distinct families with close overall structural homology. The highest degree of homology is observed in their catalytic domains, essential for the intrinsic tyrosine kinase activity of these proteins (1). Examples of such families include genes encoding epidermal growth factor receptor (EGF-R) and ERBB2, the colony-stimulating factor 1/ platelet-derived growth factor receptors, insulin/insulin-like growth factor 1 receptors, and EPH/ELK (2-12). Growth factor receptors in several of these families play critical roles in regulation of normal growth and development. Some of these molecules have been implicated in the neoplastic process as well. In particular, both the EGF-R gene and ERBB2 have been shown to be activated as oncogenes by mechanisms involving overexpression or mutations that constitutively activate the catalytic activity of their encoded proteins (13)(14)(15)(16) DNA and RNA Hybridization. High-stringency hybridization was conducted as described (20). Reduced-stringency hybridization of DNA was carried out in 30% (vol/vol) formamide followed by washes in 0.6x SSC, whereas intermediate stringency was achieved by hybridization in 40% (vol/vol) formamide and washing in 0.25x SSC.Molecular Cloning. An oligo(dT)-primed human placenta cDNA library was obtained from Clontech. The oligo(dT) primed MCF-7 cDNA library was constructed in ApCEV9 (21). After plaque purification, phage DNA inserts were subcloned into pUC-based plasmid vectors for further characterization.Nucleotide and Amino Acid Sequence Analysis. The nucleotide sequence was determined for both DNA strands by the dideoxy chain-termination method (22) using supercoiled plasmid DNA as template.t Amino acid sequence comparison was performed with the alignment program by Pearson and Lipman (23). Hydrophobic and hydrophilic regions in the predicted protein were identified according to Kyte and Doolittle (24).