BACKGROUND

In our previous investigations of the role of the extracellular matrix (ECM) in promoting neurite growth we have observed that a permissive laminin (LN) substrate stimulates differential growth responses in subpopulations of mature dorsal root ganglion (DRG) neurons. DRG neurons expressing Trk and p75 receptors grow neurites on a LN substrate in the absence of neurotrophins, while isolectin B4-binding neurons (IB4+) do not display significant growth under the same conditions. We set out to determine whether there was an expression signature of the LN-induced neurite growth phenotype. Using a lectin binding protocol IB4+ neurons were isolated from dissociated DRG neurons, creating two groups - IB4+ and IB4-. A small-scale microarray approach was employed to screen the expression of a panel of ECM-associated genes following dissociation (t=0) and after 24 hr culture on LN (t=24LN). This was followed by qRT-PCR and immunocytochemistry of selected genes.

RESULTS

The microarray screen showed that 36 of the 144 genes on the arrays were consistently expressed by the neurons. The array analyses showed that six genes had lower expression in the IB4+ neurons compared to the IB4- cells at t=0 (CTSH, Icam1, Itgβ1, Lamb1, Plat, Spp1), and one gene was expressed at higher levels in the IB4+ cells (Plaur). qRT-PCR was carried out as an independent assessment of the array results. There were discrepancies between the two methods, with qRT-PCR confirming the differences in Lamb1, Plat and Plaur, and showing decreased expression of AdamTs1, FN, and Icam in the IB4+ cells at t=0. After 24 hr culture on LN, there were no significant differences detected by qRT-PCR between the IB4+ and IB4- cells. However, both groups showed upregulation of Itgβ1 and Plaur after 24 hr on LN, the IB4+ group also had increased Plat, and the IB4- cells showed decreased Lamb1, Icam1 and AdamTs1. Further, the array screen also detected a number of genes (not subjected to qRT-PCR) expressed similarly by both populations in relatively high levels but not detectably influenced by time in culture (Bsg, Cst3, Ctsb, Ctsd, Ctsl, Mmp14, Mmp19, Sparc. We carried out immunohistochemistry to confirm expression of proteins encoded by a number of these genes.

CONCLUSIONS

Our results show that 1B4+ and IB4- neurons differ in the expression of several genes that are associated with responsiveness to the ECM prior to culturing (AdamTs1, FN, Icam1, Lamb1, Plat, Plaur). The data suggest that the genes expressed at higher levels in the IB4- neurons could contribute to the initial growth response of these cells in a permissive environment and could also represent a common injury response that subsequently promotes axon regeneration. The differential expression of several extracellular matrix molecules (FN, Lamb1, Icam) may suggest that the IB4- neurons are capable of maintaining /secreting their local extracellular environment which could aid in the regenerative process. Overall, these data provide new information on potential targets that could be manipulated to enhance axonal regeneration in the mature nervous system.

Obese subjects with a similar body mass index (BMI) exhibit substantial heterogeneity in gluco- and cardiometabolic heath phenotypes. However, defining genes that underlie the heterogeneity of metabolic features among obese individuals and determining metabolically healthy and unhealthy phenotypes remain challenging. We conducted unsupervised hierarchical clustering analysis of subcutaneous adipose tissue transcripts from 30 obese men and women ⩾40 years old. Despite similar BMIs in all subjects, we found two distinct subgroups, one metabolically healthy (group 1) and one metabolically unhealthy (group 2). Subjects in group 2 showed significantly higher total cholesterol (P=0.005), low-density lipoprotein cholesterol (P=0.006), 2-h insulin during oral glucose tolerance test (P=0.015) and lower insulin sensitivity (SI, P=0.029) compared with group 1. We identified significant upregulation of 141 genes (for example, MMP9 and SPP1) and downregulation of 17 genes (for example, NDRG4 and GINS3) in group 2 subjects. Intriguingly, these differentially expressed transcripts were enriched for genes involved in cardiovascular disease-related processes (P=2.81 × 10(-11)-3.74 × 10(-02)) and pathways involved in immune and inflammatory response (P=8.32 × 10(-5)-0.04). Two downregulated genes, NDRG4 and GINS3, have been located in a genomic interval associated with cardiac repolarization in published GWASs and zebra fish knockout models. Our study provides evidence that perturbations in the adipose tissue gene expression network are important in defining metabolic health in obese subjects.

The nonrestricting/nonmodifying strain Bacillus subtilis 222 (r-m-) can be induced to synthesize a DNA-modifying activity upon treatment with either mitomycin C (MC) or UV light. This is shown by the following facts. (i) Infection of MC-pretreated 222 cells with unmodified SPP1 phage yields about 3% modified phage that are resistant to restriction in B. subtilis R (r+m+). The induced modifying activity causes the production of a small fraction of fully modified phage in a minority class of MC-treated host cells. (ii) The MC-pretreated host cells contain a DNA cytosine methylating activity: both bacterial and phage DNAs have elevated levels of 5-methylcytosine. (iii) The MC-induced methylation of SPP1 DNA takes place at the recognition nucleotide sequences of restriction endonuclease R from B. subtilis R. (iv) Crude extracts of MC-pretreated 222 cells have enhanced DNA methyltransferase activities, with a substrate specificity similar to that found in modification enzymes present in (constitutively) modifying strains.

Current research focuses on developing alternative strategies to restore decreased liver mass prior to the onset of end-stage liver disease. Cell engraftment/repopulation requires regeneration in normal liver, but we have shown that severe liver injury stimulates repopulation without partial hepatectomy (PH). We have now investigated whether a less severe injury, secondary biliary fibrosis, would drive engraftment/repopulation of ectopically transplanted mature hepatocytes.

Ductular proliferation and progressive fibrosis in dipeptidyl-peptidase IV (DPPIV)(-) F344 rats was induced by common bile duct ligation (BDL). Purified DPPIV(+)/green fluorescent protein (GFP)(+) hepatocytes were infused without PH into the spleen of BDL rats and compared to rats without BDL.

Within one week, transplanted hepatocytes were detected in hepatic portal areas and at the periphery of expanding portal regions. DPPIV(+)/GFP(+) repopulating cell clusters of different sizes were observed in BDL rats but not untreated normal recipients. Surprisingly, some engrafted hepatocytes formed CK-19/claudin-7 expressing epithelial cells resembling cholangiocytes within repopulating clusters. In addition, substantial numbers of hepatocytes engrafted at the intrasplenic injection site assembled into multicellular groups. These also showed biliary "transdifferentiation" in the majority of intrasplenic injection sites of rats that received BDL but not in untreated recipients. PCR array analysis showed upregulation of osteopontin (SPP1). Cell culture studies demonstrated increased Itgβ4, HNF1β, HNF6, Sox-9, and CK-19 mRNA expression in hepatocytes incubated with osteopontin, suggesting that this secreted protein promotes dedifferentiation of hepatocytes.

Our studies show that biliary fibrosis stimulates liver repopulation by ectopically transplanted hepatocytes and also stimulates hepatocyte transition towards a biliary epithelial phenotype.

The small subunit of bacteriophages SPP1 and SF6 terminase, G1P, share 71% identity clustered in three conserved segments (I, II, and III). Within segment I the helix-turn-helix DNA-binding domain was mapped, whereas segment III was found to be nonessential. For terminase activity, chimeric G1Ps, obtained by domain swapping between gene 1 of SPP1 and the SF6 origin (Chi1 to Chi4), were purified. The chimeric proteins behave in all respects similarly to the G1P of SPP1 or SF6. The major determinant for G1P:G1P interactions was found to lie within segment II. We showed that a G1P derivative (G1P*) lacking the 62 N-terminal residues (segment I), and Chi1 lacking the 45 C-terminal residues (segment III) interact with G1P. The N-terminal domain of G1P is necessary for terminase subunit assembly, because the large subunit of the terminase (G2P) interacts only with G1P and Chi1, but fails to do so with G1P*. These results suggest that segment III and the extended C-terminal part of SPP1 G1P do not play a major role in DNA recognition and that G1P recognizes an extended nucleotide sequence and DNA structure.

All tailed bacteriophages follow the same general scheme of infection: they bind to their specific host receptor and then transfer their genome into the bacterium. DNA translocation is thought to be initiated by the strong pressure due to DNA packing inside the capsid. However, the exact mechanism by which each phage controls its DNA ejection remains unknown. Using light scattering, we analyzed the kinetics of in vitro DNA release from phages SPP1 and lambda (Siphoviridae family) and found a simple exponential decay. The ejection characteristic time was studied as a function of the temperature and found to follow an Arrhenius law, allowing us to determine the activation energy that governs DNA ejection. A value of 25-30 kcal/mol is obtained for SPP1 and lambda, comparable to the one measured in vitro for T5 (Siphoviridae) and in vivo for T7 (Podoviridae). This suggests similar mechanisms of DNA ejection control. In all tailed phages, the opening of the connector-tail channel is needed for DNA release and could constitute the limiting step. The common value of the activation energy likely reflects the existence for all phages of an optimum value, ensuring a compromise between efficient DNA delivery and high stability of the virus.

BACKGROUND

It has been well documented that two factors, brain death (BD) and ischemia/reperfusion (I/R) injury, have distinct but overlapping adverse influences on the clinical outcome of renal transplantation.

METHODS

We previously established a rat model of renal isografting from brain dead donors. In the present study, we performed genomic expression profiling with a high-density oligonucleotide microarray to identify genes that were upregulated or downregulated by BD and/or I/R injury.

RESULTS

Among a total of 20,550 genes, most of those upregulated by BD were genes for adhesion molecules and cytokines or for chemokines such as Gro1 and IP-10. When overexpression of these genes was assessed by real-time reverse transcriptase-polymerase chain reaction, it was only observed one hr after the engraftment of kidneys from BD donors and returned to baseline thereafter, indicating the presence of an acute systemic inflammatory response to BD. Analysis of biologic networks demonstrated the activation of specific pathways that were clearly different for BD and I/R injury. The p53 and NFkappaB pathway was involved in the acute response to BD, whereas the Myc, Jun, and c-fos pathway was involved in I/R injury. Investigation of secretory protein genes identified LCN2 and SPP1 as candidate genes for biologic markers.

CONCLUSIONS

Because our experimental system is a good model of renal transplantation from brain dead or living human donors, our data may be useful for elucidating the pathologic processes involved and for identification of novel markers for graft dysfunction of renal transplantation.

Human mesenchymal stem cells (hMSCs) represent a promising source of cells for bone tissue engineering. However, their low frequencies and limited proliferation restrict their clinical utility. An alternative is the use of human embryonic stem cells (hESCs), but labor-intensive expansion with the need for coating support limits their clinical use. We have previously derived a cell line from hESCs denoted matrix-free growth (MFG)-hESC that are independent of coating support for expansion, and we here compare its osteogenic capacity to that of hMSCs. Microarray analysis of hMSCs and MFG-hESCs revealed differential expression of genes involved in ossification. MFG-hESCs have significantly higher expression of secreted phosphoprotein 1 (SPP1) during osteogenic differentiation, whereas the opposite was true for alkaline phosphatase (ALPL), transforming growth factor, beta 1 (TGFB2), runt-related transcription factor 2 (RUNX2), and forkhead box C1 (FOXC1), as well as the activity of the ALPL enzyme, demonstrating that these two cell types differentiate into the osteogenic lineage using different signaling pathways. von Kossa staining, time-of-flight secondary ion mass spectrometry, and measurement of calcium and phosphate in the extracellular matrix demonstrated a superior ability of the MFG-hESCs to produce a mineralized matrix compared to hMSCs. The superior ability of the MFG-hESCs to form mineralized matrix compared to hMSCs demonstrates that MFG-hESCs are a promising alternative to the use of adult stem cells in future bone regenerative applications.

The current study was undertaken with a view to identify differential biomarkers in chewing-tobacco-associated oral cancer tissues in patients of Indian ethnicity. The gene expression profile was analyzed in oral cancer tissues as compared to clinically normal oral buccal mucosa. We examined 30 oral cancer tissues and 27 normal oral tissues with 16 paired samples from contralateral site of the patient and 14 unpaired samples from different oral cancer patients, for whole genome expression using high-throughput IlluminaSentrix Human Ref-8 v2 Expression BeadChip array. The cDNA microarray analysis identified 425 differentially expressed genes with >1.5-fold expression in the oral cancer tissues as compared to normal tissues in the oral cancer patients. Overexpression of 255 genes and downregulation of 170 genes (p < 0.01) were observed. Further, a minimum twofold overexpression was observed in 32 genes and downregulation in 12 genes, in 30-83% of oral cancer patients. Biological pathway analysis using Kyoto Encyclopedia of Genes and Genome Pathway database revealed that the differentially regulated genes were associated with critical biological functions. The biological functions and representative deregulated genes include cell proliferation (AIM2, FAP, TNFSF13B, TMPRSS11A); signal transduction (FOLR2, MME, HTR3B); invasion and metastasis (SPP1, TNFAIP6, EPHB6); differentiation (CLEC4A, ELF5); angiogenesis (CXCL1); apoptosis (GLIPR1, WISP1, DAPL1); and immune responses (CD300A, IFIT2, TREM2); and metabolism (NNMT; ALDH3A1). Besides, several of the genes have been differentially expressed in human cancers including oral cancer. Our data indicated differentially expressed genes in oral cancer tissues and may identify prognostic and therapeutic biomarkers in oral cancers, postvalidation in larger numbers and varied population samples.

Viral scaffolding proteins direct polymerization of major capsid protein subunits into icosahedral procapsid structures. The scaffolding protein of bacteriophage SPP1 was engineered with a C-terminal hexahistidine tag (gp11-His(6)) and purified. The protein is an alpha-helical-rich molecule with a very elongated shape as found for internal scaffolding proteins from other phages. It is a 3.3 S tetramer of 93.6 kDa at micromolar concentrations. Intersubunit cross-linking of these tetramers generated preferentially covalently bound dimers, revealing that gp11-His(6) is structurally a dimer of dimers. Incubation at temperatures above 37 degrees C correlated with a reduction of its alpha-helical content and a less effective intersubunit cross-linking. Complete loss of secondary structure was observed at temperatures above 60 degrees C. Refolding of gp11-His(6) thermally denatured at 65 degrees C led to reacquisition of the protein native ellipticity spectrum but the resulting population of molecules was heterogeneous. Its hydrodynamic behavior was compatible with a mix of 3.3 S elongated tetramers (approximately 90%) and a smaller fraction of 2.4 S dimers (approximately 10%). This population of gp11-His(6) was competent to direct polymerization of the SPP1 major capsid protein gp13 into procapsid-like structures in a newly developed assembly assay in vitro. Although native tetramers were active in assembly, refolded gp11-His(6) showed enhanced binding to gp13 revealing a more active species for interaction with the major capsid protein than native gp11-His(6).

We have shown previously that nontumorigenic NIH 3T3 cells can be made tumorigenic and metastatic by transfection and expression of activated ras, whereas in LTA cells, which are tumorigenic but nonmetastatic, the degree of malignancy is not altered by ras. To investigate possible mechanisms of natural ras resistance, we compared the expression patterns of several genes thought to be involved in ras-induced metastatic progression in LTA (ras-resistant) and NIH 3T3 (ras-sensitive) cells, before and after constitutive expression of transfected T24-H-ras. We examined the expression of the nuclear "early-response" genes jun and fos and the "tumor-suppressor" retinoblastoma (Rb) gene, as well as genes involved in invasion (major excreted protein [MEP], tissue inhibitor of metalloproteinases [TIMP]), and cell adhesion (secreted phosphoprotein 1 [SPP1; also known as osteopontin]). We found distinct differences in both the basal and ras-induced levels of expression of most of these genes in LTA versus NIH 3T3 cells. High levels of MEP and low levels of TIMP were induced in ras-transfected NIH 3T3 cells, whereas LTA cells showed intermediate levels of MEP and high levels of TIMP that were only marginally affected by the expression of transfected ras. Similarly, SPP1 expression was strongly induced by ras in NIH 3T3 cells but was repressed by ras in LTA cells. Enzymogram assays for functional gelatinase activity showed an increase in 67-kd and 62-kd bands in NIH 3T3 cells in the presence of ras. LTA cells showed no gelatinolytic activity in the presence or absence of ras. Data from an in vitro assay for chemoinvasiveness showed a pattern as predicted from the expression of invasion-related genes; chemoinvasiveness in ras-transfected NIH 3T3 was greater than in LTA and ras-transfected LTA cells, which was greater than in NIH 3T3 cells. Differences in expression of the genes examined are believed to contribute to the ras responsiveness of NIH 3T3 cells and the ras resistance of LTA cells.

Bacteriophage SPP1 infection of Bacillus subtilis cells bearing plasmids induces the synthesis of multigenome-length plasmid molecules. Two independent pathways can account for this synthesis. In one of those, homology to the phage genome is required, whereas in the other such homology is not a prerequisite. In wild type cells both modes overlap. In dnaB(Ts), at non permissive temperature, or in recE polA strains the main concatemeric plasmid replication mode is the homology-dependent plasmid (hdp) mode. The rate of recombination-dependent concatemeric plasmid DNA synthesis is a consequence of a phage-plasmid interaction which leads to chimeric phage::plasmid DNA. The second mode, which is an homology-independent plasmid (hip) mode seems to be triggered upon the synthesis of a phage encoded product(s) (e.g. inactivation of the exonuclease V enzyme).

Recombination plays a significant role in bacteriophage biology. Functions promoting recombination are involved in key stages of phage multiplication and drive phage evolution. Their biological role is reflected by the great variety of phages existing in the environment. This work presents the role of recombination in the phage life cycle and highlights the discrete character of phage-encoded recombination functions (anti-RecBCD activities, 5' ->> 3' DNA exonucleases, single-stranded DNA binding proteins, single-stranded DNA annealing proteins, and recombinases). The focus of this review is on phage proteins that initiate genetic exchange. Importance of recombination is reviewed based on the accepted coli-phages T4 and lambda models, the recombination system of phage P22, and the recently characterized recombination functions of Bacillus subtilis phage SPP1 and mycobacteriophage Che9c. Key steps of the molecular mechanisms involving phage recombination functions and their application in molecular engineering are discussed.

To investigate the cell cycle checkpoint response to aberrant S phase-initiation, we analyzed mutations of the two DNA primase subunit genes of Schizosaccharomyces pombe, spp1(+) and spp2(+) (S. pombe primase 1 and 2). spp1(+) encodes the catalytic subunit that synthesizes the RNA primer, which is then utilized by Polalpha to synthesize the initiation DNA. Here, we reported the isolation of the fission yeast spp1(+) gene and cDNA and the characterization of Spp1 protein and its cellular localization during the cell cycle. Spp1 is essential for cell viability, and thermosensitive mutants of spp1(+) exhibit an allele-specific abnormal mitotic phenotype. Mutations of spp1(+) reduce the steady-state cellular levels of Spp1 protein and compromised the formation of Polalpha-primase complex. The spp1 mutant displaying an aberrant mitotic phenotype also fails to properly activate the Chk1 checkpoint kinase, but not the Cds1 checkpoint kinase. Mutational analysis of Polalpha has previously shown that activation of the replication checkpoint requires the initiation of DNA synthesis by Polalpha. Together, these have led us to propose that suboptimal cellular levels of polalpha-primase complex due to the allele-specific mutations of Spp1 might not allow Polalpha to synthesize initiation DNA efficiently, resulting in failure to activate a checkpoint response. Thus, a functional Spp1 is required for the Chk1-mediated, but not the Cds1-mediated, checkpoint response after an aberrant initiation of DNA synthesis.

SPO1 bacteriophage injects its DNA into minicells produced by Bacillus subtilis CU403 divIVB1. The injected DNA is partially degraded to small trichloracetic acid-precipitable material and trichloroacetic acid-soluble material. The injected DNA is not replicated; however, it serves as a template for RNA and protein synthesis. The RNA produced specifically hybridizes to SPO1 DNA, and the amount of RNA hybridized can be reduced by competition with RNA isolated at all stages of the phage cycle from infected nucleate cells of the B. subtilis CU403 divIVB1. An unrelated phage, SPP1, also induces phage-specific RNA in infected minicells. Translation occurs in SPO1-infected minicells resulting in at least eight proteins which have been separated by gel electrophoresis, and two of these proteins have mobilities similar to proteins found only in infected B. subtilis CU403 divIVB1 nucleate cells. A large proportion of the polypeptide material synthesized in infected minicells is very small and heterogeneous in size.

The temporal program of SPP1 transcription was examined by hybridizing RNA extracted from infected B. subtilis cells, pulse-labelled at various times after infection, to restriction fragments of SPP1 DNA. RNA made early after infection hybridises to contiguous fragments in the left part of the SPP1 molecule, whereas hybridization to fragments in the right part of the chromosome is found late in infection. Viral gene transcription proceeds from right to left on the H-strand throughout the lytic cycle. At late times transcription occurs also from left to right using the L-strand as template. These assignments follow from the established 5'--3' polarity of the complementary (H- and L-) strains of SPP1 DNA and the determination of strand specificity in SPP1 transcription. Early and late transcriptions are also defined physiologically: protein synthesis and phage DNA replication must precede late transcription.

Bacteriophages SPP1, 41c, 22a, rho 15 and SF6 of Bacillus subtilis share a common and specific host receptor site for adsorption. Experiments described here have established the relatedness between these phages. They were indistinguishable on the basis of host-range, plating efficiency, various growth parameters and serological properties. In addition, they shared the ability to carry out generalized transduction. They could be differentiated, however, by the restriction patterns of their DNAs, with the exception of 41c and 22a, which seemed to be identical. Recombination between 41c and SPP1 was demonstrated by transfection with mixed digests of their DNAs.

BACKGROUND

Malignant pleural effusion (MPE) is a common complication of advanced lung cancer. Research has shown that secreted phosphoprotein-1 (SPP1) is essential in MPE associated with lung cancer. This retrospective study was performed to evaluate the prognostic significance of SPP1 in the MPE of patients with non-small cell lung cancer (NSCLC).

METHODS

MPE specimens were obtained from 85 NSCLC patients (study group), and pleural effusion specimens were obtained from 24 patients with benign lung disease (control group). Specimens were tested for SPP1 using enzyme-linked immunosorbent assay (ELISA). Based on the cutoff value of receiver operating characteristic (ROC) curve analysis, the study patients were divided into a high-SPP1-expression subgroup and a low-expression subgroup. The primary and secondary endpoints of this study were progression-free survival (PFS) and overall survival (OS).

RESULTS

The SPP1 levels of the study group were significantly higher compared to those of the controls (Mann-Whitney U test, P = 0.017). The number of extrapulmonary metastases was significantly higher in the high-SPP1-expressing patients than in the low-expressing patients (P = 0.03). Kaplan-Meier survival analysis showed that SPP1 levels were negatively associated with OS and PFS in both subgroups of study patients (P = 0.026; P = 0.039, respectively). Cox regression analysis showed that SPP1 was an independent prognostic factor in patients with NSCLC (HR = 1.832, 95% confidence interval: 1.003-3.345; P = 0.049).

CONCLUSIONS

SPP1 in pleural effusion can be used for the auxiliary diagnosis of MPE and used to determine the prognosis of patients with NSCLC.

Lung adenocarcinoma is caused by the combination of genetic and environmental effects, and smoking plays an important role in the disease development. Exploring the gene expression profile and identifying genes that are shared or vary between smokers and nonsmokers with lung adenocarcinoma will provide insights into the etiology of this complex cancer. We obtained RNA-seq data from paired normal and tumor tissues from 34 nonsmoking and 34 smoking patients with lung adenocarcinoma (GEO: GSE40419). R Bioconductor, edgeR, was adopted to conduct differential gene expression analysis between paired normal and tumor tissues. A generalized linear model was applied to identify genes that were differentially expressed in nonsmoker and smoker patients as well as genes that varied between these two groups. We identified 2273 genes that showed differential expression with FDR < 0.05 and |logFC| >1 in nonsmoker tumor versus normal tissues; 3030 genes in the smoking group; and 1967 genes were common to both groups. Sixty-eight and 70% of the identified genes were downregulated in nonsmoking and smoking groups, respectively. The 20 genes such as SPP1, SPINK1, and FAM83A with largest fold changes in smokers also showed similar large and highly significant fold changes in nonsmokers and vice versa, showing commonalities in expression changes for adenocarcinomas in both smokers and nonsmokers for these genes. We also identified 175 genes that were significantly differently expressed between tumor samples from nonsmoker and smoker patients. Gene expression profile varied substantially between smoker and nonsmoker patients with lung adenocarcinoma. Smoking patients overall showed far more complicated disease mechanism and have more dysregulation in their gene expression profiles. Our study reveals pathogenetic differences in smoking and nonsmoking patients with lung adenocarcinoma from transcriptome analysis. We provided a list of candidate genes for further study for disease detection and treatment in both smoking and nonsmoking patients with lung adenocarcinoma.

BACKGROUND

Lung cancer is the leading cause of cancer deaths in the world. The most common type of lung cancer is lung adenocarcinoma (AC). The genetic mechanisms of the early stages and lung AC progression steps are poorly understood. There is currently no clinically applicable gene test for the early diagnosis and AC aggressiveness. Among the major reasons for the lack of reliable diagnostic biomarkers are the extraordinary heterogeneity of the cancer cells, complex and poorly understudied interactions of the AC cells with adjacent tissue and immune system, gene variation across patient cohorts, measurement variability, small sample sizes and sub-optimal analytical methods. We suggest that gene expression profiling of the primary tumours and adjacent tissues (PT-AT) handled with a rational statistical and bioinformatics strategy of biomarker prediction and validation could provide significant progress in the identification of clinical biomarkers of AC. To minimise sample-to-sample variability, repeated multivariate measurements in the same object (organ or tissue, e.g. PT-AT in lung) across patients should be designed, but prediction and validation on the genome scale with small sample size is a great methodical challenge.

RESULTS

To analyse PT-AT relationships efficiently in the statistical modelling, we propose an Extreme Class Discrimination (ECD) feature selection method that identifies a sub-set of the most discriminative variables (e.g. expressed genes). Our method consists of a paired Cross-normalization (CN) step followed by a modified sign Wilcoxon test with multivariate adjustment carried out for each variable. Using an Affymetrix U133A microarray paired dataset of 27 AC patients, we reviewed the global reprogramming of the transcriptome in human lung AC tissue versus normal lung tissue, which is associated with about 2,300 genes discriminating the tissues with 100% accuracy. Cluster analysis applied to these genes resulted in four distinct gene groups which we classified as associated with (i) up-regulated genes in the mitotic cell cycle lung AC, (ii) silenced/suppressed gene specific for normal lung tissue, (iii) cell communication and cell motility and (iv) the immune system features. The genes related to mutagenesis, specific lung cancers, early stage of AC development, tumour aggressiveness and metabolic pathway alterations and adaptations of cancer cells are strongly enriched in the AC PT-AT discriminative gene set. Two AC diagnostic biomarkers SPP1 and CENPA were successfully validated on RT-RCR tissue array. ECD method was systematically compared to several alternative methods and proved to be of better performance and as well as it was validated by comparison of the predicted gene set with literature meta-signature.

CONCLUSIONS

We developed a method that identifies and selects highly discriminative variables from high dimensional data spaces of potential biomarkers based on a statistical analysis of paired samples when the number of samples is small. This method provides superior selection in comparison to conventional methods and can be widely used in different applications. Our method revealed at least 23 hundreds patho-biologically essential genes associated with the global transcriptional reprogramming of human lung epithelium cells and lung AC aggressiveness. This gene set includes many previously published AC biomarkers reflecting inherent disease complexity and specifies the mechanisms of carcinogenesis in the lung AC. SPP1, CENPA and many other PT-AT discriminative genes could be considered as the prospective diagnostic and prognostic biomarkers of lung AC.

An important objective in nowadays research is the discovery of new biomarkers that can detect colon tumours in early stages and indicate with accuracy the status of the disease. The aim of our study was to identify potential biomarkers for colon cancer onset and progression. We assessed gene expression profiles of a list of 10 candidate genes (MMP-1, MMP-3, MMP-7, DEFA 1, DEFA-5, DEFA-6, IL-8, CXCL-1, SPP-1, CTHRC-1) by quantitative real time PCR in triplets of colonic mucosa (normal, adenoma, tumoral tissue) collected from the same patient during surgery for a group of 20 patients. Additionally we performed immunohistochemistry for DEFA1-3 and SPP1. We remarked that DEFA5 and DEFA6 are key factors in adenoma formation (p<0.05). MMP7 is important in the transition from a benign to a malignant status (p <0.01) and further in metastasis being a prognostic indicator for tumor transformation and for the metastatic potential of cancer cells. IL8, irrespective of tumor stage, has a high mRNA level in adenocarcinoma (p< 0.05). The level of expression for SPP1 is correlated with tumor level. We suggest that high levels of DEFAS, DEFA6 (key elements in adenoma formation), MMP7 (marker of colon cancer onset and progression to metastasis), SPP1 (marker of progression) and IL8 could be used to diagnose an early stage colon cancer and to evaluate the prognostic of progression for colon tumors. Further, if DEFA5 and DEFA6 level of expression are low but MMP7, SPP1 and IL8 level are high we could point out that the transition from adenoma to adenocarcinoma had already occurred. Thus, DEFA5, DEFA6, MMP7, IL8 and SPP1 consist in a valuable panel of biomarkers, whose detection can be used in early detection and progressive disease and also in prognostic of colon cancer.

A classic proinflammatory T helper cell type 1 (TH1) response directed against intracellular pathogens includes the cytokine osteopontin, which acts predominantly on macrophages, where it induces the secretion of interleukin (IL)-12 and suppresses the secretion of IL-10. As cell-mediated immune responses play an important role in the resistance to Lyme arthritis, a manifestation of infection by the extracellular pathogen Borrelia burgdorferi, we tested the hypothesis that osteopontin may be required to induce T(H)1 responses and inflammation. The role of osteopontin was tested in vivo and using ex vivo macrophages in B6129F3 mice susceptible to experimental Lyme arthritis. Mice of this genetic background and those fully backcrossed to C57BL/6, which lacked osteopontin expression (spp1-/-), were as susceptible to B. burgdorferi-induced arthritis as littermate controls. Furthermore, equal numbers of spirochetes, as measured by quantitative polymerase chain reaction of the B. burgdorferi gene recA in spp1-/- and B6129F3 wild-type littermates, suggested that susceptibility to infection was not dependent on this cytokine. Neither of the B6129F3 parental mouse strains lacked the ability to secrete osteopontin. spp1-/- mice and controls had immunoglobulin G2 titers, suggestive of a TH1 response. B. burgdorferi was able to directly stimulate the secretion of the proinflammatory cytokines IL-12 and tumor necrosis factor alpha from wild-type and spp1-/- macrophages alike. These results indicate that the usually critical role of osteopontin in the induction of cellular immune responses to intracellular pathogens was circumvented by the ability of the extracellular pathogen B. burgdorferi to induce macrophages directly to produce proinflammatory cytokines.

Early full-term pregnancy affords lifetime protection against the development of breast cancer. Parity-induced protection can be reproduced in a carcinogen-induced rat mammary carcinoma model, but the molecular mechanisms of this protection against carcinogenic stimuli in rat mammary glands have not been fully characterized. To gain a better understanding of these molecular mechanisms, we used an oligonucleotide microarray to examine gene expression in parous and age-matched virgin (AMV) mammary glands of Lewis rats before and after carcinogen (N-methyl-N-nitrosourea; MNU) treatment. Parous mammary glands before MNU treatment showed up-regulation of multiple differentiation-related genes, such as whey acidic protein (Wap), casein beta (Csn2), casein gamma (Csng), lipopolysaccharide binding protein (Lbp), secreted phosphoprotein 1 (Spp1) and glycosylation-dependent cell adhesion molecule 1 (Glycam1). Also, parous mammary glands before MNU treatment exhibited down-regulation of growth-related genes such as regenerating islet-derived 3 alpha (Reg3a), mesothelin (Msln), insulin-like growth factor 2 (Igf2) and insulin-like growth factor binding protein 4 (Igfbp4). After MNU treatment, AMV mammary glands exhibited up-regulation of growth-related genes, such as Msln, cell division cycle 2 homolog A (Cdc2a), Igf2, Igfbp4, stathmin 1 (Stmn1) and homeobox, msh-like 1 (Msx1), whereas expression of these genes remained low in parous mammary glands. AMV mammary glands also exhibited marked up-regulation of Cdc2a and Stmn1 in response to MNU. After MNU treatment, the PCNA labeling index increased significantly in AMV mammary epithelial cells (13.7+/-1.1%), but remained low in parous mammary glands (3.6+/-0.4%). The response of AMV mammary glands to carcinogenic stimuli includes up-regulation of growth-related genes and increased cell proliferation. The lack of a similar response in parous mammary glands may explain parity-induced protection against mammary tumor development.

Twenty-four individuals were investigated that spanned six generations in a Chinese family affected with an apparently autosomal dominant form of dentinogenesis imperfecta type II (DGI-II, OMIM #125490). All affected individuals presented with typical, clinical and radiographic features of DGI-II, but without bilateral progressive high-frequency sensorineural hearing loss. To investigate the mutated molecule, a positional candidate approach was used to determine the mutated gene in this family. Genomic DNA was obtained from 24 affected individuals, 18 unaffected relatives of the family and 50 controls. Haplotype analysis was performed using leukocyte DNA for 6 short tandem repeat (STR) markers present in chromosome 4 (D4S1534, GATA62A11, DSPP, DMP1, SPP1 and D4S1563). In the critical region between D4S1534 and DMP1, the dentin sialophosphoprotein (DSPP) gene (OMIM *125485) was considered as the strongest candidate gene. The first four exons and exon/intron boundaries of the gene were analyzed using DNA from 24 affected individuals and 18 unaffected relatives of the same family. DNA sequencing revealed a heterozygous deletion mutation in intron 2 (at positions -3 to -25), which resulted in a frameshift mutation, that changed the acceptor site sequence from CAG to AAG (IVS2-3C->>A) and may also have disrupted the branch point consensus sequence in intron 2. The mutation was found in the 24 affected individuals, but not in the 18 unaffected relatives and 50 controls. The deletion was identified by allele-specific sequencing and denaturing high-performance liquid chromatography (DHPLC) analysis. We conclude that the heterozygous deletion mutation contributed to the pathogenesis of DGI-II.