Internalizing disorders and leukocyte telomere erosion: a prospective study of depression, generalized anxiety disorder and post-traumatic stress disorder.

There is evidence that persistent psychiatric disorders lead to age-related disease and premature mortality. Telomere length has emerged as a promising biomarker in studies that test the hypothesis that internalizing psychiatric disorders are associated with accumulating cellular damage. We tested the association between the persistence of internalizing disorders (depression, generalized anxiety disorder and post-traumatic stress disorder) and leukocyte telomere length (LTL) in the prospective longitudinal Dunedin Study (n=1037). Analyses showed that the persistence of internalizing disorders across repeated assessments from ages 11 to 38 years predicted shorter LTL at age 38 years in a dose-response manner, specifically in men (ß=-0.137, 95% confidence interval (CI): -0.232, -0.042, P=0.005). This association was not accounted for by alternative explanatory factors, including childhood maltreatment, tobacco smoking, substance dependence, psychiatric medication use, poor physical health or low socioeconomic status. Additional analyses using DNA from blood collected at two time points (ages 26 and 38 years) showed that LTL erosion was accelerated among men who were diagnosed with internalizing disorder in the interim (ß=-0.111, 95% CI: -0.184, -0.037, P=0.003). No significant associations were found among women in any analysis, highlighting potential sex differences in internalizing-related telomere biology. These findings point to a potential mechanism linking internalizing disorders to accelerated biological aging in the first half of the life course, particularly in men. Because internalizing disorders are treatable, the findings suggest the hypothesis that treating psychiatric disorders in the first half of the life course may reduce the population burden of age-related disease and extend health expectancy.

p53-dependent cell death/apoptosis is required for a productive adenovirus infection.

The p53 tumor suppressor protein binds to both cellular and viral proteins, which influence its biological activity. One such protein is the large E1b tumor antigen (E1b58kDa) from adenoviruses (Ads), which abrogates the ability of p53 to transactivate various promoters. This inactivation of p53 function is believed to be the mechanism by which E1b58kDa contributes to the cell transformation process. Although the p53-E1b58kDa complex occurs during infection and is conserved among different serotypes, there are limited data demonstrating that it has a role in virus replication. However, loss of p53 expression occurs after adenovirus infection of human cells and an E1b58kDa deletion mutant (Onyx-015, also called dl 1520) selectively replicates in p53-defective cells. These (and other) data indicate a plausible hypothesis is that loss of p53 function may be conducive to efficient adenovirus replication. However, wild-type (wt) Ad5 grows more efficiently in cells expressing a wt p53 protein. These studies indicate that the hypothesis may be an oversimplification. Here, we show that cells expressing wt p53, as well as p53-defective cells, allow adenovirus replication, but only cells expressing wt p53 show evidence of virus-induced cytopathic effect. This correlates with the ability of adenovirus to induce cell death. Our data indicate that p53 plays a necessary part in mediating cellular destruction to allow a productive adenovirus infection. In contrast, p53-deficient cells are less sensitive to the cytolytic effects of adenovirus and as such raise questions about the use of E1b58kDa-deficient adenoviruses in tumor therapy.

p53 promotes adenoviral replication and increases late viral gene expression.

The tumor suppressor protein, p53, plays a critical role in viro-oncology. However, the role of p53 in adenoviral replication is still poorly understood. In this paper, we have explored further the effect of p53 on adenoviral replicative lysis. Using well-characterized cells expressing a functional p53 (A549, K1neo, RKO) and isogenic derivatives that do not (K1scx, RKOp53.13), we show that virus replication, late virus protein expression and both wtAd5 and ONYX-015 virus-induced cell death are impaired in cells deficient in functional p53. Conversely, by transfecting p53 into these and other cells (IIICF/c, HeLa), we increase late virus protein expression and virus yield. We also show, using reporter assays in IIICF/c, HeLa and K1scx cells, that p53 can cooperate with E1a to enhance transcription from the major late promoter of the virus. Late viral protein production is enhanced by exogenous p53. Taken together, our data suggest that functional p53 can promote the adenovirus (Ad) lytic cycle. These results have implications for the use of Ad mutants that are defective in p53 degradation, such as ONYX-015, as agents for the treatment of cancers.

Some p53-binding proteins that can function as arbiters of life and death.

Four sets of p53-binding proteins are discussed in this review. These are the E2F family, the ASPP family, Y-box-binding protein YB1, and the prolyl isomerase Pin1. Each appears to play a role in the decision by p53 to induce an arrest of cell proliferation or apoptosis and they may also be independent markers of cancer. Their activities appear to be linked with the cell cycle and they may also interact with each other. In this review, the properties of each protein class are discussed as well as how they affect p53 functions. A model is proposed as to how their activities might be coordinated.

Control functions of adenovirus transformation region E1A gene products in rat and human cells.

Altered control of the rat cell cycle induced by adenovirus requires expression of transformation region E1A, but not of E1B, E2A, E2B, or late genes. We show here that neither E3 nor E4 is required, so the effect results directly from an E1A product. Mutants with defects in the 289-amino-acid (aa) E1A product had little or no effect on the rat cell cycle even at 1,000 IU per cell. A mutant (pm975) lacking the 243-aa E1A product altered cell cycle progression, but less efficiently than did wild-type virus. The 289-aa E1A protein is therefore essential for cell cycle effects; the 243-aa protein is also necessary for the full effect but cannot act alone. Mutants with altered 289-aa E1A proteins showed different extents of leak expression of viral early region E2A as the multiplicity was increased; each leaked more in human than in rat cells. dl312, with no E1A products, failed to produce E2A mRNA or protein at 1,000 IU per cell in rat cells but did so in some experiments in human cells. There appears to be a very strict dependence of viral early gene expression on E1A in rat cells, whereas dependence on E1A is more relaxed in HeLa cells, perhaps due to a cellular E1A-like function. Altered cell cycle control is more dependent on E1A function than is early viral gene expression.

Efficient induction of cell death by adenoviruses requires binding of E1B55k and p53.

The use of an Elb55k-deficient adenovirus, ONYX-015, to selectively target tumor cells containing a mutated p53 gene has produced promising results. However, recent reports have questioned the selectivity of this virus, showing that ONYX-015 can replicate in cells containing a wild-type p53 and that p53 may actually be required for cell death. To address these apparent contradictions in the literature, we infected a number of mutant and wild-type p53-containing cell lines with ONYX-015 and wild-type adenovirus and observed their death profiles up to 10 days postinfection. We demonstrate that two distinct cell death phenotypes exist, one of which is rapid and dependent on the presence of p53 and one of which is p53 independent. Using adenoviruses expressing E1b55k proteins deficient in their ability to bind p53, we show that formation of a complex between p53 and the adenoviral Elb55k protein is necessary for the activation of the rapid cell death pathway. In the absence of p53 or the absence of complex formation between p53 and Elb55k, cell death is delayed considerably. These data suggest three things: that the selectivity of killing appears to be dependent on the presence of the E1b55k/p53 complex; that viruses lacking Elb55k (such as ONYX-015) kill cells in a delayed manner independent of p53; and that binding of E1b55k to p53 does not merely serve to inactivate p53, but rather is required for the induction of rapid cell death. The components of this complex that lead to rapid cell death remain to be determined.

The Δ133p53 isoform and its mouse analogue Δ122p53 promote invasion and metastasis involving pro-inflammatory molecules interleukin-6 and CCL2.

A number of naturally occurring isoforms of the tumour suppressor protein p53 have been discovered, which appear to have differing roles in tumour prevention or promotion. We are investigating the tumour-promoting activities of the Δ133p53 isoform using our mouse model of Δ133p53 (Δ122p53). Here, we report that tumours from Δ122p53 homozygous mice show evidence of invasion and metastasis and that Δ122p53 promotes migration though a 3-dimensional collagen matrix. We also show that Δ122p53 and Δ133p53 promote cell migration in scratch wound and Transwell assays, similar to the 'gain-of-function' phenotypes seen with mutant p53. Using the well-defined B16 mouse melanoma metastatic model, we show that Δ122p53 leads to faster generation of lung metastases. The increased migratory phenotypes are dependent on secreted factors, including the cytokine interleukin-6 and the chemokine CCL2. We propose that Δ122p53 (and Δ133p53) acts in a similar manner to 'gain-of-function' mutant p53 proteins to promote migration, invasion and metastasis, which may contribute to poor survival in patients with Δ133p53-expressing tumours.

The growth-inhibitory function of p53 is separable from transactivation, apoptosis and suppression of transformation by E1a and Ras.

p53 is known to suppress oncogenic cell transformation, inhibit cell growth, induce apoptosis and activate and repress gene transcription. To investigate the relationships between these functions, we have examined various mutant forms of p53 for their abilities to perform each activity. This study has shown that growth inhibition is not a prerequisite for apoptotic cell death as these two functions are separate and alternative activities of p53. Additionally, we have demonstrated that the ability of p53 to suppress transformation (by adenovirus E1a and activated Ras) correlates with its ability to induce apoptosis and not with its ability to inhibit cell growth. Although p53 is thought to inhibit growth through the transactivation of p21WAFI, our study has demonstrated that transcriptional activation and repression are neither sufficient nor necessary for growth inhibition. This indicates that p53 has more than one mechanism for inhibiting cell growth and that another type of biochemical function must be involved. Furthermore, we have shown that transcriptional activation and repression may each be necessary, and the combination of these activities may even be sufficient, for p53-dependent apoptosis. In summary, our results have provided new information about the cellular and biochemical mechanisms through which p53 acts as a tumor suppressor.

Wild-type mouse p53 down-regulates transcription from different virus enhancer/promoters.

The protein encoded by the tumour-suppressor gene p53 can complex with SV40 virus large T antigen, the adenovirus E1B 58-kDa protein and the E6 protein of human papillomavirus type 16. The functions of these complexes are unclear, but there is some evidence to suggest that binding of p53 to these viral proteins may inactivate p53 function. Recent reports have shown that p53 is involved in regulation of transcription. We have considered the possibility that p53 may regulate transcription of viral genes important for virus replication and/or transformation. Inactivation of p53 function by formation of such complexes might then permit correct expression of these viral genes. Since p53 can bind to the SV40 virus enhancer/promoter, we have investigated the effect of p53 on transcription from this promoter and report here that mouse p53 is a potent repressor of the SV40 enhancer/promoter. Mutations within p53 severely inhibited this activity and provided some evidence to show that the N-terminus of p53 contains residues essential for this function. We also show that mouse p53 represses transcription from the promoters of viruses that do not express proteins that complex with p53: the human cytomegalovirus early promoter and the Rous sarcoma virus long terminal repeat. By studying the effect of p53 on transcription in different cell lines, we show that the effects of p53 on promoters may be cell type specific.

p53 confers a selective advantage on transfected HeLa cells.

The p53 gene, which is frequently mutated in various tumors, encodes a phosphoprotein thought to have a key role in the regulation of cell proliferation. To explore their biological effects, the HeLa carcinoma line, which does not express p53, was co-transfected with plasmid constructs expressing wild-type or mutant p53 proteins, or unrelated proteins, along with a plasmid conferring resistance to a neomycin-kanamycin antibiotic analog (G418). Both wild-type and mutant forms of p53 stimulated the number of G418-resistant colonies between 5- and 36-fold. Further investigation of colony development revealed that p53 enhanced cell survival, leading to increased colony numbers, but did not stimulate cell growth. Nonetheless, we suggest that an initial slowing of cell growth caused by expression of the unintegrated p53 plasmids renders the transfectants resistant to selection with G418, thus causing a higher frequency of G418-resistant colonies. p53 constructs were found to be expressed transiently in HeLa cells as expected, but the G418-resistant colonies frequently failed to express p53. This loss of p53 expression may be due to negative regulatory effects of p53 on the cytomegalovirus promoter that drives the selection marker.

Adenovirus E1b-58 kD antigen binds to p53 during infection of rodent cells: evidence for an N-terminal binding site on p53.

We show using mild extraction procedures that the p53 proto-oncogene forms a complex with adenovirus 5 E1b-58 kD during infection. These complexes are detected as coimmunoprecipitates from radiolabeled extracts of adenovirus infected cells on SDS-PAGE. Furthermore, adenovirus mutants with defects in E1b-58 kD fail to form complexes, whereas mutants in other early region genes still show evidence of complex. Using a panel of monoclonal antibodies to mouse p53, we show that antibodies reacting with N-terminal epitopes on p53, displace E1b-58 kD. This result suggests that E1b-58 kD binds to an N-terminal region of mouse p53. In addition, in a transient transfection assay in monkey COS cells, we show that an N-terminal deletion mutant of mouse p53 does not bind to E1b-58 kD but wild-type mouse p53 does bind. This result again suggests that E1b-58 kD binds an N-terminal determinant on p53.

Inhibition of SV40 large T antigen induced apoptosis by small T antigen.

It is well established that the expression of simian virus 40 (SV40) early gene products causes oncogenic transformation of rodent cells. An important aspect of this process is the inactivation of the p53 and retinoblastoma (pRb) tumour suppressor proteins through interaction with the SV40 large tumour antigen (LT). In addition, the SV40 small tumour antigen (ST) may enhance LT induced transformation. Here we show that LT induces apoptotic cell death in rat embryo fibroblast (REF) cells and that ST functions to inhibit this effect by a mechanism which is different from other known anti-apoptotic proteins. Mutational analysis of LT indicates that mutants defective in the pRb-binding domain are unable to induce apoptosis whereas LT mutants defective in the p53-binding domain are still competent to induce apoptosis. Thus, interaction between LT and one or more pRb family members must occur for induction of apoptosis and that binding of p53 by LT is insufficient to inhibit LT induced apoptosis in REFs. The data presented herein suggest that the anti-apoptotic function of ST may explain, at least in part, how ST contributes to SV40 early region induced transformation of REF cells.

Involvement of RB-1, p53, p16INK4 and telomerase in immortalisation of human cells.

Involvement of the retinoblastoma susceptibility (RB-1), p16INK4, p53 and telomerase genes in immortalisation was examined by determining their status in 15 human cell lines representing four immortalisation complementation groups. No abnormalities of RB-1, p53 and p16INK4 were detected in cell lines containing DNA tumour virus proteins known to bind to the protein products of the RB-1 and p53 genes. In contrast, in all other cell lines from each of the four groups either RB-1 was mutant or p16INK4 protein was undetectable and there were cell lines containing p53 mutations in three of the groups. Telomerase activity was detected in 12/15 lines, including some of the virally immortalised lines and in some lines from each group. Since none of these changes correlated with complementation group, other genetic changes must be required for immortalisation.

The immunomodulating agent gliotoxin causes genomic DNA fragmentation.

Gliotoxin, a member of the class of secondary fungal metabolites characterized by the presence of an epipolythiodioxopiperazine ring, caused fragmentation of spleen cell DNA as observed by flow cytometry and gel electrophoresis. Gliotoxin was found to cause substantial double-stranded DNA breakage in spleen cells which was dose- and time-dependent. The ability of gliotoxin to cause DNA breakage was also found to be specific to cell type. DNA breakage occurred in all cell types in which gliotoxin inhibited proliferation and so provides a general explanation as to how gliotoxin prevents cell proliferation. Other results showed that gliotoxin bound to a similar extent to both sensitive and resistant cells, indicating that differential uptake is not a likely mechanism to explain cell type selectivity. The results are discussed in terms of a mechanism for gliotoxin action involving genomic DNA as the central target.

Δ122p53, a mouse model of Δ133p53α, enhances the tumor-suppressor activities of an attenuated p53 mutant.

Growing evidence suggests the Δ133p53α isoform may function as an oncogene. It is overexpressed in many tumors, stimulates pathways involved in tumor progression, and inhibits some activities of wild-type p53, including transactivation and apoptosis. We hypothesized that Δ133p53α would have an even more profound effect on p53 variants with weaker tumor-suppressor capability. We tested this using a mouse model heterozygous for a Δ133p53α-like isoform (Δ122p53) and a p53 mutant with weak tumor-suppressor function (mΔpro). The Δ122p53/mΔpro mice showed a unique survival curve with a wide range of survival times (92-495 days) which was much greater than mΔpro/- mice (range 120-250 days) and mice heterozygous for the Δ122p53 and p53 null alleles (Δ122p53/-, range 78-150 days), suggesting Δ122p53 increased the tumor-suppressor activity of mΔpro. Moreover, some of the mice that survived longest only developed benign tumors. In vitro analyses to investigate why some Δ122p53/mΔpro mice were protected from aggressive tumors revealed that Δ122p53 stabilized mΔpro and prolonged the response to DNA damage. Similar effects of Δ122p53 and Δ133p53α were observed on wild-type of full-length p53, but these did not result in improved biological responses. The data suggest that Δ122p53 (and Δ133p53α) could offer some protection against tumors by enhancing the p53 response to stress.

Inhibition of mevalonate synthesis with compactin does not prevent DNA replication in cultured animal cells.

Compactin is a competitive inhibitor of the enzyme that catalyses the synthesis of mevalonate. In this study we have investigated the effects of compactin on DNA replication and cell cycle progression in animal cell cultures. We have examined several different cell types for cell cycle inhibitory effects of compactin, and although we can demonstrate that compactin inhibits mevalonate synthesis in BHK cells, we have observed little or no effect on the cell cycle. Similar results were obtained using different synchronization procedures and by measuring cell cycle progression by [3H]dThd labelling of DNA and with flow cytometry. We conclude that compactin has no appreciable and general effect on DNA replication in animal cells. These results are discussed in terms of the implications for mevalonate being a universal regulator of cell cycle progression in cultured animal cells.

IL-1 genotype and adult periodontitis among young New Zealanders.

Several recent studies have investigated the association between interleukin-1 genotype and periodontitis in clinical samples, where generalizability is an issue. The aim of this study was to investigate the association between adult periodontitis and IL-1 genotype in a population-based sample of 26-year-olds. Based on probing depth (PD) measurements, participants were divided into three disease groups: "Severe" (1+ teeth with 5+mm PD; N = 25), "Moderate" (2+ teeth with 4+mm PD; N = 36), and "Controls" (the remainder; N = 800). The "periodontitis-associated genotype" (PAG; Kornman et al., 1997) was present in 20.0% of the "Severe" group and in 34.8% of "Controls", whereas the IL-1A(+4845) [1,1]/IL-1B(+3953) [2,2] genotype was present in 12.0% and 0.9%, respectively. After controlling for sex, smoking status, and plaque levels, we found that those with IL-1B(+3953) [1,1]/IL-1A(+4845) [2,2] had 12.3 times the odds of being in the "Severe" group. Analysis of these data suggests that the IL-1A(+4845) [1,1]/IL-1B(+3953) [2,2] genotype is associated with periodontal disease in this young population. Future periodontal data collections as this cohort ages are required to confirm the predictive value of that genotype.

Activation of p53 following ionizing radiation, but not other stressors, is dependent on the proline-rich domain (PRD).

The tumor suppressor protein, p53 is one of the most important cellular defences against malignant transformation. In response to cellular stressors p53 can induce apoptosis, cell cycle arrest or senescence as well as aid in DNA repair. Which p53 function is required for tumor suppression is unclear. The proline-rich domain (PRD) of p53 (residues 58-101) has been reported to be essential for the induction of apoptosis. To determine the importance of the PRD in tumor suppression in vivo we previously generated a mouse containing a 33-amino-acid deletion (residues 55-88) in p53 (mΔpro). We showed that mΔpro mice are protected from T-cell tumors but not late-onset B-cell tumors. Here, we characterize the functionality of the PRD and show that it is important for mediating the p53 response to DNA damage induced by γ-radiation, but not the p53-mediated responses to Ha-Ras expression or oxidative stress. We conclude that the PRD is important for receiving incoming activating signals. Failure of PRD mutants to respond to the activating signaling produced by DNA damage leads to impaired downstream signaling, accumulation of mutations, which potentially leads to late-onset tumors.

PAX8 promotes tumor cell growth by transcriptionally regulating E2F1 and stabilizing RB protein.

The retinoblastoma protein (RB)-E2F1 pathway has a central role in regulating the cell cycle. Several PAX proteins (tissue-specific developmental regulators), including PAX8, interact with the RB protein, and thus regulate the cell cycle directly or indirectly. Here, we report that PAX8 expression is frequent in renal cell carcinoma, bladder, ovarian and thyroid cancer cell lines, and that silencing of PAX8 in cancer cell lines leads to a striking reduction in the expression of E2F1 and its target genes, as well as a proteasome-dependent destabilization of RB protein, with the RB1 mRNA level remaining unaffected. Cancer cells expressing PAX8 undergo a G(1)/S arrest and eventually senesce following PAX8 silencing. We demonstrate that PAX8 transcriptionally regulates the E2F1 promoter directly, and E2F1 transcription is enhanced after RB depletion. RB is recruited to the PAX8-binding site, and is involved in PAX8-mediated E2F1 transcription in cancer cells. Therefore, our results suggest that, in cancer, frequent and persistent expression of PAX8 is required for cell growth control through transcriptional activation of E2F1 expression and upregulation of the RB-E2F1 pathway.