Nicholas C. Popescu

Senescing human cells and ageing mice accumulate DNA lesions with unrepairable double-strand breaks

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 γ-H2AX foci (γ-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 γ-foci remain after repair of radiation-induced γ-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.

Telomerase contributes to tumorigenesis by a telomere length-independent mechanism

Once immortalized, human cells are susceptible to transformation by introduction of an oncogene such as ras. Several lines of evidence now suggest that the maintenance of telomere length is a major determinant of replicative lifespan in human cells and thus of the immortalized state. The majority of human tumor cells acquire immortality through expression of the catalytic subunit of telomerase (hTERT), whereas others activate an alternative mechanism of telomere maintenance (ALT) that does not depend on the actions of telomerase. We have examined whether ALT could substitute for telomerase in the processes of transformation in vitro and tumorigenesis in vivo. Expression of oncogenic H-Ras in the immortal ALT cell line GM847 did not result in their transformation. However, subsequent ectopic expression of hTERT in these cells imparted a tumorigenic phenotype. Indeed, this outcome was also observed after introduction of a mutant hTERT that retained catalytic activity but was incapable of maintaining telomere length. These studies indicate that hTERT confers an additional function that is required for tumorigenesis but does not depend on its ability to maintain telomeres.

TrnR2, a novel receptor that mediates neurturin and GDNF signaling through Ret

Glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) comprise a family of TGF-beta-related neurotrophic factors (TRNs), which have trophic influences on a variety of neuronal populations. A receptor complex comprised of TrnR1 (GDNFR alpha) and Ret was recently identified and found to be capable of mediating both GDNF and NTN signaling. We have identified a novel receptor based on homology to TrnR1, called TrnR2, that is 48% identical to TrnR1, and is located on the short arm of chromosome 8. TrnR2 is attached to the cell surface via a GPI-linkage, and can mediate both NTN and GDNF signaling through Ret in vitro. Fibroblasts expressing TrnR2 and Ret are approximately 30-fold more sensitive to NTN than to GDNF treatment, whereas those expressing TrnR1 and Ret respond equivalently to both factors, suggesting the TrnR2-Ret complex acts preferentially as a receptor for NTN. TrnR2 and Ret are expressed in neurons of the superior cervical and dorsal root ganglia, and in the adult brain. Comparative analysis of TrnR1, TrnR2, and Ret expression indicates that multiple receptor complexes, capable of mediating GDNF and NTN signaling, exist in vivo.

Antiproliferative activity of ecteinascidin 743 is dependent upon transcription-coupled nucleotide-excision repair

While investigating the novel anticancer drug ecteinascidin 743 (Et743), a natural marine product isolated from the Caribbean sea squirt, we discovered a new cell-killing mechanism mediated by DNA nucleotide excision repair (NER). A cancer cell line selected for resistance to Et743 had chromosome alterations in a region that included the gene implicated in the hereditary disease xeroderma pigmentosum (XPG, also known as Ercc5). Complementation with wild-type XPG restored the drug sensitivity. Xeroderma pigmentosum cells deficient in the NER genes XPG, XPA, XPD or XPF were resistant to Et743, and sensitivity was restored by complementation with wild-type genes. Moreover, studies of cells deficient in XPC or in the genes implicated in Cockayne syndrome (CSA and CSB) indicated that the drug sensitivity is specifically dependent on the transcription-coupled pathway of NER. We found that Et743 interacts with the transcription-coupled NER machinery to induce lethal DNA strand breaks.

Natural melatonin `knockdown' in C57BL/6J mice: rare mechanism truncates serotonin N-acetyltransferase

Pineal melatonin synthesis (serotonin --> N-acetylserotonin --> melatonin) is severely compromised in most inbred strains of mice, in many cases because serotonin is not acetylated by serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT). We have found that in the C57BL/6J strain, AANAT mRNA encodes a severely truncated AANAT protein, because a pseudo-exon containing a stop codon is spliced in. This is the first identification of a natural mutation which knocks down melatonin synthesis. The decrease in melatonin signaling may have been a selective factor in the development of laboratory strains of mice because melatonin can inhibit reproduction and modify circadian rhythmicity.

GTF2IRD1 in Craniofacial Development of Humans and Mice

Craniofacial abnormalities account for about one-third of all human congenital defects, but our understanding of the genetic mechanisms governing craniofacial development is incomplete. We show that GTF2IRD1 is a genetic determinant of mammalian craniofacial and cognitive development, and we implicate another member of the TFII-I transcription factor family, GTF2I, in both aspects. Gtf2ird1-null mice exhibit phenotypic abnormalities reminiscent of the human microdeletion disorder Williams-Beuren syndrome (WBS); craniofacial imaging reveals abnormalities in both skull and jaws that may arise through misregulation of goosecoid, a downstream target of Gtf2ird1. In humans, a rare WBS individual with an atypical deletion, including GTF2IRD1, shows facial dysmorphism and cognitive deficits that differ from those of classic WBS cases. We propose a mechanism of cumulative dosage effects of duplicated and diverged genes applicable to other human chromosomal disorders.

Delayed kinetics of DNA double-strand break processing in normal and pathological aging.

Accumulation of DNA damage may play an essential role in both cellular senescence and organismal aging. The ability of cells to sense and repair DNA damage declines with age. However, the underlying molecular mechanism for this age‐dependent decline is still elusive. To understand quantitative and qualitative changes in the DNA damage response during human aging, DNA damage‐induced foci of phosphorylated histone H2AX (γ‐H2AX), which occurs specifically at sites of DNA double‐strand breaks (DSBs) and eroded telomeres, were examined in human young and senescing fibroblasts, and in lymphocytes of peripheral blood. Here, we show that the incidence of endogenous γ‐H2AX foci increases with age. Fibroblasts taken from patients with Werner syndrome, a disorder associated with premature aging, genomic instability and increased incidence of cancer, exhibited considerably higher incidence of γ‐H2AX foci than those taken from normal donors of comparable age. Further increases in γ‐H2AX focal incidence occurred in culture as both normal and Werner syndrome fibroblasts progressed toward senescence. The rates of recruitment of DSB repair proteins to γ‐H2AX foci correlated inversely with age for both normal and Werner syndrome donors, perhaps due in part to the slower growth of γ‐H2AX foci in older donors. Because genomic stability may depend on the efficient processing of DSBs, and hence the rapid formation of γ‐H2AX foci and the rapid accumulation of DSB repair proteins on these foci at sites of nascent DSBs, our findings suggest that decreasing efficiency in these processes may contribute to genome instability associated with normal and pathological aging.

Human mitochondrial topoisomerase I

Tension generated in the circular mitochondrial genome during replication and transcription points to the need for mtDNA topoisomerase activity. Here we report a 601-aa polypeptide highly homologous to nuclear topoisomerase I. The N-terminal domain of this novel topoisomerase contains a mitochondrial localization sequence and lacks a nuclear localization signal. Therefore, we refer to this polypeptide as top1mt. The pattern of top1mt expression matches the requirement for high mitochondrial activity in specific tissues. top1mt is a type IB topoisomerase that requires divalent metal (Ca2+ or Mg2+) and alkaline pH for optimum activity. The TOP1mt gene is highly homologous to the nuclear TOP1 gene and consists of 14 exons. It is localized on human chromosome 8q24.3.

Localization of the human erbB-2 gene on normal and rearranged chromosomes 17 to bands q12–21.32

Through the use of a cDNA probe, the human erbB-2 gene was localized by in situ hybridization of normal human chromosomes at 17q11-q21. In situ hybridization of chromosomes derived from fibroblasts carrying a constitutional 15;17t(q22.3;q11.21) translocation showed that the erbB-2 gene was relocated on the rearranged chromosome 15. These results as well as grain localization on prophase chromosomes locate the erbB-2 gene at 17q12-q21.32. This localization may facilitate the search for human malignancies with chromosome changes involving the erbB-2 gene.

DLC-1 gene inhibits human breast cancer cell growth and in vivo tumorigenicity

The human DLC-1 (deleted in liver cancer 1) gene was cloned from a primary human hepatocellular carcinoma (HCC) and mapped to the chromosome 8p21–22 region frequently deleted in common human cancers and suspected to harbor tumor suppressor genes. DLC-1 was found to be deleted or downregulated in a significant number of HCCs. We expanded our investigations to other cancers with recurrent deletions of 8p22, and in this study examined alterations of DLC-1 in primary human breast tumors, human breast, colon, and prostate tumor cell lines. Genomic deletion of DLC-1 was observed in 40% of primary breast tumors, whereas reduced or undetectable levels of DLC-1 mRNA were seen in 70% of breast, 70% of colon, and 50% of prostate tumor cell lines To see whether DLC-1 expression affects cell growth and tumorigenicity, two breast carcinoma cell lines lacking the expression of endogenous gene were transfected with the DLC-1 cDNA. In both cell lines, DLC-1 transfection caused significant growth inhibition and reduction of colony formation. Furthermore, introduction of the DLC-1 cDNA abolished the in vivo tumorigenicity in nude mice, suggesting that the DLC-1 gene plays a role in breast cancer by acting as a bona fide tumor suppressor gene.