Regulation of cell survival by sphingosine-1-phosphate receptor S1P1 via reciprocal ERK-dependent suppression of Bim and PI-3-kinase/protein kinase C-mediated upregulation of Mcl-1.

Although the ability of bioactive lipid sphingosine-1-phosphate (S1P) to positively regulate anti-apoptotic/pro-survival responses by binding to S1P1 is well known, the molecular mechanisms remain unclear. Here we demonstrate that expression of S1P1 renders CCL39 lung fibroblasts resistant to apoptosis following growth factor withdrawal. Resistance to apoptosis was associated with attenuated accumulation of pro-apoptotic BH3-only protein Bim. However, although blockade of extracellular signal-regulated kinase (ERK) activation could reverse S1P1-mediated suppression of Bim accumulation, inhibition of caspase-3 cleavage was unaffected. Instead S1P1-mediated inhibition of caspase-3 cleavage was reversed by inhibition of phosphatidylinositol-3-kinase (PI3K) and protein kinase C (PKC), which had no effect on S1P1 regulation of Bim. However, S1P1 suppression of caspase-3 was associated with increased expression of anti-apoptotic protein Mcl-1, the expression of which was also reduced by inhibition of PI3K and PKC. A role for the induction of Mcl-1 in regulating endogenous S1P receptor-dependent pro-survival responses in human umbilical vein endothelial cells was confirmed using S1P receptor agonist FTY720-phosphate (FTY720P). FTY720P induced a transient accumulation of Mcl-1 that was associated with a delayed onset of caspase-3 cleavage following growth factor withdrawal, whereas Mcl-1 knockdown was sufficient to enhance caspase-3 cleavage even in the presence of FTY720P. Consistent with a pro-survival role of S1P1 in disease, analysis of tissue microarrays from ER(+) breast cancer patients revealed a significant correlation between S1P1 expression and tumour cell survival. In these tumours, S1P1 expression and cancer cell survival were correlated with increased activation of ERK, but not the PI3K/PKB pathway. In summary, pro-survival/anti-apoptotic signalling from S1P1 is intimately linked to its ability to promote the accumulation of pro-survival protein Mcl-1 and downregulation of pro-apoptotic BH3-only protein Bim via distinct signalling pathways. However, the functional importance of each pathway is dependent on the specific cellular context.

The in situ local immune response, tumour senescence and proliferation in colorectal cancer.

BACKGROUND: Immune cell infiltrates are important determinants of colorectal cancer (CRC) outcome. Their presence may be driven by tumour or host-specific factors. From previous studies in mice, senescence, a state of cell cycle arrest, may moderate tumour progression through upregulation of antitumour immune responses. The relationships between senescence and immune infiltrates have not previously been studied in humans. We explore whether a marker of senescence (p16(ink4a)) in combination with low level expression of a proliferation marker (ki-67) relate to T cell infiltrates in CRC, and whether p16(ink4a), Ki-67 and immune infiltrates have similar prognostic value. METHODS: Immunostaining of p16(inka) and Ki-67 was performed within a CRC tissue microarray. Nuclear p16(inka) and Ki-67 were categorised as high/low. T-cell markers, CD3, CD45RO, CD8 and FOXP3 were scored separately as high/low grade in three areas of the tumour: the invasive margin (IM), tumour stroma and cancer cell nests (CCNs). results: Two hundred and thirty stage I-III cancers were studied. High nuclear p16(ink4a) was expressed in 63% and high proliferation (Ki-67 >15%) in 61%. p16(ink4a) expression was associated with reduced CD45RO+ cells at the IM (P<0.05) and within the stroma (P<0.05) and reduced CD8+ cells at the IM (P<0.01). A low Ki-67 proliferative index was associated with reduced density of CD3+ cells in CCNs (P<0.01), reduced CD45RO+ cells at the IM (P<0.05) and within the CCNs (P<0.001), reduced FOXP3+ cells at the IM (P<0.001), within the stroma (P=0.001) and within CCNs (P<0.001) and reduced CD8+ cells at the IM (P<0.05) and within the CCNs (P<0.05). Tumours with both a low proliferative index and expression of p16(ink4a) demonstrated similar consistent relationships with reduced densities of T-cell infiltrates. On multivariate analysis, TNM stage (P<0.001), low CD3 cells at the IM (P=0.014), low CD8 cells at the IM (P=0.037), low proliferation (Ki-67; P=0.013) and low senescence (p16(ink4a); P=0.002) were independently associated with poorer cancer survival. CONCLUSION: Senescence, proliferation and immune cell infiltrates are independent prognostic factors in CRC. Although related to survival, p16(ink4a)-associated senescence is not associated with an upregulation of antitumour T-cell responses.

Is the biology of breast cancer changing? A study of hormone receptor status 1984-1986 and 1996-1997.

Using archived tumours, those from 1984-1986 and 1996-1997 underwent immunohistochemistry for hormone receptors and grade analysis. A significant shift towards more ER-positive and low-grade disease was found; this appears to reflect screening practices, but could still influence survival.

Association of increased telomere lengths in limited scleroderma, with a lack of age-related telomere erosion.

OBJECTIVES: Telomere erosion, a feature of biological ageing, is implicated in a wide range of diseases. Its impact on autoimmune diseases remains unclear although autoantibodies against many telomere nucleoprotein components are prevalent in these diseases. We aimed to assess if telomere biology was abnormal in a cohort of patients with limited cutaneous systemic sclerosis (lcSSc). METHODS: Telomere lengths in peripheral blood leucocytes (PBL) were determined using Southern blotting methods in a cohort of lcSSc subjects (n=43; age range 37-80 years) and a control population (n=107; age range 21-65 years). RESULTS: Telomere lengths in lcSSc subjects were longer than controls (p<0.001), did not show age-related telomere erosion and differed significantly from age-matched controls only after 50 years of age (p<0.001). CONCLUSIONS: This is the first report of maintenance of telomere lengths in an autoimmune disease state. These data indicate aberrant telomere biology and irregular biological ageing from the fifth decade of life. These findings provide insight into compromised DNA damage repair in lcSSc. Whether these observations indicate a causal or consequential relationship requires further investigation. This in turn, may provide potential novel targets for therapeutic intervention.

Expression levels of the JAK/STAT pathway in the transition from hormone-sensitive to hormone-refractory prostate cancer.

The main cause of prostate cancer-related mortality is the development of hormone-refractory disease. Circulating serum levels of IL-6 are raised in hormone-refractory prostate cancer patients and evidence from cell line studies suggests that the IL-6R/JAK/STAT3 pathway may be involved in development of this disease. In the current study we investigate if expression levels of these family members are implicated in the development of hormone-refractory prostate cancer. Immunohistochemistry using IL-6R, JAK1, STAT3, pSTAT3(Tyr705) and pSTAT3(Ser727) antibodies was performed on 50 matched hormone-sensitive and hormone-refractory tumours pairs. An increase in expression of cytoplasmic IL-6 receptor, with the development of hormone-refractory prostate cancer was associated with reduced time to relapse (P=0.0074) while an increase in expression of cytoplasmic pSTAT3(Tyr705) was associated with reduced patient survival (P=0.0003). In addition, those patients with high expression of cytoplasmic pSTAT3(Tyr705) in their hormone-refractory tumours had significantly shorter time to death from biochemical relapse and overall survival in comparison to those patients with low expression of cytoplasmic pSTAT3(Tyr705) (P=0.002 and P=0.0027, respectively). Activation of STAT3, via phosphorylation is associated with reduced patient survival, suggesting that activation of the IL-6R/JAK/STAT3 pathway is involved with development of hormone-refractory prostate cancer.

Observer variation in immunohistochemical analysis of protein expression, time for a change?

AIM: Immunohistochemical analysis of protein expression is central to most clinical translational studies and defines patient treatment or selection criteria for novel drugs. Interobserver variation is rarely analysed despite recognition that this is a key area of potential inaccuracy. Therefore our aim was to examine observer variation and suggest the revision of current standards. METHODS AND RESULTS: We analysed inter- and intra-observer variation, by interclass correlation coefficient (ICCC) and kappa statistics, in 8661 samples. Intra-observer assessment of nuclear, cytoplasmic and membrane staining for seven proteins in 1323 samples resulted in an ICCC of 0.94 and a kappa-value of 0.787. Interobserver reproducibility, assessed on 28 proteins by seven observer pairs in 8661 carcinomas, gave an ICCC of 0.90 and a kappa-value of 0.70. No significant effect of either antibody or cellular compartmentalization was observed. CONCLUSION: We have demonstrated that ICCC is a consistent method to assess observer variation when a continuous scoring system is used, compared with kappa statistics, which depends on a categorical system. Given the importance of accurate assessment of protein expression in diagnostic and experimental medicine, we suggest raising thresholds for observer variation: ICCC of 0.7 should be regarded as the minimum acceptable standard, ICCC of 0.8 as good and ICCC of > or = 0.9 as excellent.

The canine factor VIII cDNA and 5' flanking sequence.

Factor VIII is a trace plasma glycoprotein involved as a cofactor in the activation of factor X by factor IXa. Inherited deficiency of factor VIII results in the X-linked bleeding disorder hemophilia A which has been documented in humans, horses, sheep and dogs. In this report, the putative proximal promoter, 5' untranslated region, complete coding sequence and 3' untranslated region of the canine factor VIII gene have been characterized. When compared to the human gene, the 5' flanking region shows conservation of transcription factor binding sites in the 5' untranslated region. Alignment of the amino acid sequence with that of the previously reported human, mouse and pig proteins demonstrates sequence identity of between 77 and 92% for the A1, A2, A3, C1 and C2 domains but an identity of only between 44 and 62% for the central B domain. The three thrombin cleavage sites are conserved in the canine sequence as are the protein C cleavage sites and the von Willebrand factor binding region. In addition, all six tyrosine residues that are known to undergo sulfation in the human protein are conserved in the dog. The 3' untranslated region of the canine gene extends 1.5 kilobases. The initial 700 basepairs of this sequence are highly GC rich and the sequence terminates with 2 alternative potential polyadenylation sites. The knowledge of this sequence, in combination with a well described canine model of hemophilia A, provides the necessary starting point for studies addressing the long-term evaluation of factor VIII gene therapy using a homologous transgene.

Role of the liver-enriched transcription factor hepatocyte nuclear factor 1 in transcriptional regulation of the factor V111 gene.

Coagulation factor VIII is an essential cofactor required for normal hemostatic function. A deficiency in factor VIII results in the bleeding disorder hemophilia A. Despite the fact that the factor VIII gene was cloned a decade ago, the mechanisms which control its transcription remain unresolved. In our studies, we have characterized 12 protein binding sites within the factor VIII promoter by DNase I protection assays performed with rat liver nuclear extracts. Three of these elements (sites 1 to 3) are situated within the 5' untranslated region of the gene, while three other sites (sites 4 to 6) lie within the first 100 bp upstream of the transcriptional start site. We have identified an additional site (site 7) approximately 300 bp upstream from site 6, as well as a cluster of five sites in a 250-bp region which terminates approximately 1 kb from the transcriptional start site. Seven of these binding sites (sites 2, 3, 4, 6, 7, 9, and 10) bind members of the C/EBP family of transcription factors. DBP also binds to five of these sites (sites 3, 4, 6, 7, and 9). Utilizing transient transfection studies in HepG2 cells, we have shown that deletion of the factor VIII promoter sequences distal to nucleotide -44 results in a significant but small increase in promoter activity. The activity of each of the various 5' deletion constructs is significantly enhanced by cotransfection of C/EBPalpha and D-site-binding protein expression plasmids, while cotransfection of both C/EBPalpha and C/EBPbeta plasmids resulted in a further enhancement of transactivation. These studies also provide evidence of a repressor element located between nucleotides -740 and -1002. Since the minimal promoter sequence (-44 to +148) maintains the transcriptional activity of the full-length promoter sequence, we proceeded to identify additional factors binding to sites 1 to 4. Competition studies revealed that a ubiquitous transcription factor, NF-Y, binds to site 4, while the liver-enriched transcription factor hepatocyte nuclear factor I (HNF-1) binds to site 1. Mutation analysis of the minimal promoter demonstrated that HNF-1 is critical for activating transcription of the factor VIII gene in vitro. Our results also suggest that the multiple upstream elements that we have identified may act as a backup regulatory region in the event of disruption of the HNF-1 element in the 5' untranslated region.

Impact of Pneumocystis carinii and cytomegalovirus on the course and outcome of atypical pneumonia in advanced human immunodeficiency virus disease.

Patients undergoing bronchoscopy for possible pneumocystis pneumonia were studied retrospectively to characterize the impact of common viral pathogens on the course of advanced human immunodeficiency virus (HIV) disease and atypical pneumonia. In 327 episodes, Pneumocystis carinii was found in 220 (67%), cytomegalovirus (CMV) in 145 (44%), and herpes simplex virus in 16 (5%). Early deterioration in oxygenation and use of intensive care was less common in CMV-positive patients. Neither CMV nor P. carinii was a predictor of mortality in multivariate analyses. CMV was not associated with an increased prevalence of later CMV disease. Isolation of CMV from the bronchoalveolar lavage fluid of these patients was not an indication for antiviral therapy. Pulmonary shedding of CMV may be associated with a decreased inflammatory response to P. carinii. The outcome of HIV-associated atypical pneumonia where no clear pulmonary pathogen is found on routine evaluation was no better than that of treated P. carinii pneumonia.

The impact of concomitant viral pathogens on the course of Pneumocystis carinii pneumonia.

A large retrospective study was conducted to evaluate the impact of culturing cytomegalovirus from the respiratory secretions of AIDS patients with Pneumocystis carinii pneumonia. Pneumocystis carinii was found in 220 (67%) of 327 episodes and cytomegalovirus was found in 106 (48%) of the P. carinii-positive patients. Cytomegalovirus-positive and -negative patients were similar at baseline and had a similar number of hospital days, but had a lower incidence of early deterioration in oxygenation, fewer intensive-care days, were less frequently intubated, and had a higher 30-day survival. The better short-term outcome of cytomegalovirus positive patients observed in this study may relate to the immunosuppressive effects of cytomegalovirus.