Paula Sommer

Ultradian cortisol pulsatility encodes a distinct, biologically important signal.

Context Cortisol is released in ultradian pulses. The biological relevance of the resulting fluctuating cortisol concentration has not been explored. Objective Determination of the biological consequences of ultradian cortisol pulsatility. Design A novel flow through cell culture system was developed to deliver ultradian pulsed or continuous cortisol to cells. The effects of cortisol dynamics on cell proliferation and survival, and on gene expression were determined. In addition, effects on glucocorticoid receptor (GR) expression levels and phosphorylation, as a potential mediator, were measured. Results Pulsatile cortisol caused a significant reduction in cell survival compared to continuous exposure of the same cumulative dose, due to increased apoptosis. Comprehensive analysis of the transcriptome response by microarray identified genes with a differential response to pulsatile versus continuous glucocorticoid delivery. These were confirmed with qRT-PCR. Several transcription factor binding sites were enriched in these differentially regulated target genes, including CCAAT-displacement protein (CDP). A CDP regulated reporter gene (MMTV-luc) was, as predicted, also differentially regulated by pulsatile compared to continuous cortisol delivery. Importantly there was no effect of cortisol delivery kinetics on either GR expression, or activation (GR phosphoSer211). Conclusions Cortisol oscillations exert important effects on target cell gene expression, and phenotype. This is not due to differences in cumulative cortisol exposure, or either expression, or activation of the GR. This suggests a novel means to regulate GR function.

Glucocorticoid receptor overexpression exerts an antisurvival effect on human small cell lung cancer cells

Small cell lung cancer (SCLC) is an aggressive tumour with an abysmal prognosis. These cancers are characteristically resistant to glucocorticoid (Gc) action, owing to impaired expression of the glucocorticoid receptor (GR). We identified reduced GR expression in human SCLC cell lines, compared to a non-SCLC cell line. The SCLC cells also showed no Gc inhibition of proliferation, in contrast to non-SCLC cells. Retroviral overexpression of GR resulted in significantly increased cell death, which was partially blocked by the GR antagonist, RU486. Indeed, in cells sorted for GR expression, there was rapid, near complete loss of live cells by 72 h, in contrast to control cells that proliferated as expected. Flow cytometry using Annexin V revealed that cell death was by apoptosis. In addition, confocal analysis of fixed cells showed that cells overexpressing GR displayed a significant increase in fragmenting apoptotic nuclei. Microarray studies showed that transgenic GR expression upregulated the proapoptotic genes, BAD and BAX. We have, therefore, identified a profound apoptotic effect of GR in SCLC cells, which may explain the low levels of endogenous GR in SCLC cells. Understanding how GR overexpression leads to apoptotic cell death in SCLC cells may uncover new therapeutic strategies.

Loss of Glucocorticoid Receptor Expression by DNA Methylation Prevents Glucocorticoid Induced Apoptosis in Human Small Cell Lung Cancer Cells

Human small cell lung cancer (SCLC) is highly aggressive, and quickly develops resistance to therapy. SCLC cells are typically insensitive to glucocorticoids due to impaired glucocorticoid receptor (GR) expression. This is important as we have previously shown that expression of a GR transgene induces cell death in-vitro, and inhibits tumor growth in-vivo. However, the underlying mechanism for loss of GR expression is unknown. The SCLC cell line, DMS79, has low GR expression, compared to non-SCLC cell lines and normal bronchial epithelial cells. Retroviral GR expression in DMS79 cells caused activation of the apoptotic pathway as evidenced by marked induction of caspase-3 activity. Methylation analysis of the GR promoter revealed some methylation in the 1D, and 1E promoters of the GR gene, however the ubiquitous constitutively active 1C promoter was heavily methylated. In the 1C promoter there was a highly significant increase in DNA methylation in a panel of 14 human SCLC cell lines compared to a mixed panel of GR expressing, and non-expressing cell lines, and to peripheral blood mononuclear cells. Furthermore, within the panel of SCLC cell lines there was a significant negative correlation seen between methylation of the 1C promoter, and GR protein expression. Reversal of GR gene methylation with DNA methyltransferase inhibition caused increased GR mRNA and protein expression in SCLC but not non-SCLC cells. This resulted in increased Gc sensitivity, decreased Bcl-2 expression and increased caspase-3 activity in SCLC cells. These data suggest that DNA methylation decreases GR gene expression in human SCLC cells, in a similar manner to that for conventional tumor suppressor genes.

Identification and functional analysis of SKA2 interaction with the glucocorticoid receptor

Glucocorticoid (GC) receptors (GRs) have profound anti-survival effects on human small cell lung cancer (SCLC). To explore the basis of these effects, protein partners for GRs were sought using a yeast two-hybrid screen. We discovered a novel gene, FAM33A, subsequently identified as a SKA1 partner and involved in mitosis, and so renamed Ska2. We produced an anti-peptide antibody that specifically recognized full-length human SKA2 to measure expression in human cell lines and tissues. There was a wide variation in expression across multiple cell lines, but none was detected in the liver cell line HepG2. A xenograft model of human SCLC had intense staining and archival tissue revealed SKA2 in several human lung and breast tumours. SKA2 was found in the cytoplasm, where it co-localized with GR, but nuclear expression of SKA2 was seen in breast tumours. SKA2 overexpression increased GC transactivation in HepG2 cells while SKA2 knockdown in A549 human lung epithelial cells decreased transactivation and prevented dexamethasone inhibition of proliferation. GC treatment decreased SKA2 protein levels in A549 cells, as did Staurosporine, phorbol ester and trichostatin A; all agents that inhibit cell proliferation. Overexpression of SKA2 potentiated the proliferative response to IGF-I exposure, and knockdown with shRNA caused cells to arrest in mitosis. SKA2 has recently been identified in HeLa S3 cells as part of a complex, which is critical for spindle checkpoint silencing and exit from mitosis. Our new data show involvement in cell proliferation and GC signalling, with implications for understanding how GCs impact on cell fate.

Glucocorticoid receptor over-expression promotes human small cell lung cancer apoptosis in vivo and thereby slows tumor growth.

Small cell lung cancer (SCLC) is an aggressive tumor, associated with ectopic ACTH syndrome. We have shown that SCLC cells are glucocorticoid receptor (GR) deficient, and that restoration of GR expression confers glucocorticoid sensitivity and induces apoptosis in vitro. To determine the effects of GR expression in vivo, we characterized a mouse SCLC xenograft model that secretes ACTH precursor peptides, and so drives high circulating corticosterone concentrations (analogous to the ectopic ACTH syndrome). Infection of SCLC xenografts with GR-expressing adenovirus significantly slowed tumor growth compared with control virus infection. Time to fourfold initial tumor volume increased from a median of 9 days to 16 days (P=0.05; n=7 per group). Post-mortem analysis of GR-expressing tumors revealed a threefold increase in apoptotic (TUNEL positive) cells (P<0.01). Infection with the GR-expressing adenovirus caused a significant reduction in Bcl-2 and Bcl-xL transcripts. Furthermore, in both the GR-expressing adenovirus-infected cells and tumors, a significant number of uninfected cells underwent apoptosis, supporting a bystander cell killing effect. Therefore, GR expression is pro-apoptotic for human SCLCs in vivo, as well as in vitro, suggesting that loss of GR confers a survival advantage to SCLCs.

Signals from the lens and Foxc1 regulate the expression of key genes during the onset of corneal endothelial development

Correct formation of the corneal endothelium is essential for continued development of the anterior segment of the eye. Corneal endothelial development is initiated at E12 when precursor peri-ocular mesenchyme cells migrate into the space between the lens and the presumptive corneal epithelium and begin to respond to signals from the lens, undergoing a mesenchymal to epithelial transition (MET) that is complete by E15.5. To study the initiation of MET, peri-ocular mesenchyme cell lines were derived from E12.5 and E13.5 murine embryos. These cells expressed key transcription factors, Foxc1 and Pitx2, as well as Slug and Tsc22, genes involved in MET. We have shown that all these genes must be down-regulated by E13.5 for differentiation to proceed. Lens-derived signals play a role in this down-regulation with Tgfβ2 specifically down-regulating Foxc1 and Pitx2. Over-expression and knock-down of Foxc1 significantly and similarly affected the expression of Pitx2, Tsc22 and Slug while Foxc1 was shown to play a role in mediating the lens effects on Slug. Thus, for the progression of initial corneal endothelial development, the key transcription factors, Foxc1 and Pitx2, as well as genes involved in MET, Slug and Tsc-22, must be down-regulated, a process driven by the lens and Foxc1.

Improving HIV proteome annotation: new features of BioAfrica HIV Proteomics Resource

The Human Immunodeficiency Virus (HIV) is one of the pathogens that cause the greatest global concern, with approximately 35 million people currently infected with HIV. Extensive HIV research has been performed, generating a large amount of HIV and host genomic data. However, no effective vaccine that protects the host from HIV infection is available and HIV is still spreading at an alarming rate, despite effective antiretroviral (ARV) treatment. In order to develop effective therapies, we need to expand our knowledge of the interaction between HIV and host proteins. In contrast to virus proteins, which often rapidly evolve drug resistance mutations, the host proteins are essentially invariant within all humans. Thus, if we can identify the host proteins needed for virus replication, such as those involved in transporting viral proteins to the cell surface, we have a chance of interrupting viral replication. There is no proteome resource that summarizes this interaction, making research on this subject a difficult enterprise. In order to fill this gap in knowledge, we curated a resource presents detailed annotation on the interaction between the HIV proteome and host proteins. Our resource was produced in collaboration with ViralZone and used manual curation techniques developed by UniProtKB/Swiss-Prot. Our new website also used previous annotations of the BioAfrica HIV-1 Proteome Resource, which has been accessed by approximately 10 000 unique users a year since its inception in 2005. The novel features include a dedicated new page for each HIV protein, a graphic display of its function and a section on its interaction with host proteins. Our new webpages also add information on the genomic location of each HIV protein and the position of ARV drug resistance mutations. Our improved BioAfrica HIV-1 Proteome Resource fills a gap in the current knowledge of biocuration. Database URL: http://www.bioafrica.net/proteomics/HIVproteome.html

The N-terminal transactivation domain of the glucocorticoid receptor mediates apoptosis of human small cell lung cancer cells.

Small cell lung cancer (SCLC) is an aggressive disease with a poor prognosis. These cancers are deficient in glucocorticoid receptor (GR) expression, and therefore, resistant to glucocorticoids. Overexpression of the GR both in vivo and in vitro leads to apoptotic cell death suggesting that loss of GR is favorable for cancer growth. Indeed, the GR promoter is silenced in SCLC cells by methylation. We now show that treatment of the SCLC cell line (DMS79) cells with the demethylating agent, 5‐aza‐2′‐deoxycytidine (5‐aza), results in significant endogenous re‐expression of both GRα and the ligand‐independent GR‐P. The GR gene has a complex promoter region comprising nine alternative promoters, the proximal seven of which lie within a CpG island. The endogenous re‐expression seen is attributed to the constitutive promoters 1B and 1C and 1J but predominantly 1F, which we show to be heavily methylated in SCLC cells. Flow cytometric analysis using the apoptotic marker, Annexin V, shows that this endogenous re‐expression is sufficient to drive the SCLC cells to apoptosis. Apoptotic induction is specific to GR re‐expression as cotreatment with 5‐aza and the GR antagonist, RU486 prevented apoptosis. Of the three functional GR domains (the DNA binding domain, ligand binding domain, and transactivation domain), we identified that the transactivation domain is essential for apoptosis in SCLC. The discovery that endogenous re‐expression of the GR in SCLC cells is sufficient to induce apoptotic cell death by reversing a cancer‐driven DNA methylation effect may lead to the development of novel adjunct therapies.

Can stem cells really regenerate the human heart? Use your noggin, dickkopf! Lessons from developmental biology : review article

Abstract The human heart is the first organ to develop and its development is fairly well characterised. In theory, the heart has the capacity to regenerate, as its cardiomyocytes may be capable of cell division and the adult heart contains a cardiac stem cell niche, presumably capable of differentiating into cardiomyocytes and other cardiac-associated cell types. However, as with most other organs, these mechanisms are not activated upon serious injury. Several experimental options to induce regeneration of the damaged heart tissue are available: activate the endogenous cardiomyocytes to divide, coax the endogenous population of stem cells to divide and differentiate, or add exogenous cell-based therapy to replace the lost cardiac tissue. This review is a summary of the recent research into all these avenues, discussing the reasons for the limited successes of clinical trials using stem cells after cardiac injury and explaining new advances in basic science. It concludes with a reiteration that chances of successful regeneration would be improved by understanding and implementing the basics of heart development and stem cell biology.

The tobacco carcinogen NNK drives accumulation of DNMT1 at the GR promoter thereby reducing GR expression in untransformed lung fibroblasts

Small cell lung cancer (SCLC) is a highly aggressive, predominantly cigarette smoke-induced tumour with poor prognosis. The glucocorticoid receptor (GR), a SCLC tumour suppressor gene, is typically reduced in SCLC. We now show that SCLC cells express high levels of DNA methyltransferase 1 (DNMT1) which accumulates at the GR promoter. DNMT1 expression is further increased by exposure to the tobacco carcinogen NNK. In the untransformed human lung fibroblast cell line, MRC-5, short term NNK treatment decreases GRα mRNA and protein expression due to accumulation of DNMT1 at the GR promoter. Long term NNK treatment results in persistently augmented DNMT1 levels with lowered GR levels. Long term exposure to NNK slows cell proliferation and induces DNA damage, while the GR antagonist RU486 stimulates proliferation and protects against DNA damage. Although both NNK and RU486 treatment increases methylation at the GR promoter, neither are sufficient to prevent senescence in this context. NNK exposure results in accumulation of DNMT1 at the GR promoter in untransformed lung cells mimicking SCLC cells, directly linking tobacco smoke exposure to silencing of the GR, an important step in SCLC carcinogenesis.