Escherichia coli are still unknown, including dinQ. In this work we characterize dinQ and two small RNAs, agrA and agrB, with antisense complementarity to dinQ. Northern analysis revealed five dinQ transcripts, but only one transcript (+44) is actively translated. The +44 dinQ transcript translates into a toxic single transmembrane peptide localized in the inner membrane. AgrB regulates dinQ RNA by RNA interference to counteract DinQ toxicity. Thus the dinQ-agr locus shows the classical features of a type I TA system and has many similarities to the tisB-istR locus. DinQ overexpression depolarizes the cell membrane and decreases the intracellular ATP concentration, demonstrating that DinQ can modulate membrane-dependent processes. Augmented DinQ strongly inhibits marker transfer by Hfr conjugation, indicating a role in recombination. Furthermore, DinQ affects transformation of nucleoid morphology in response to UV damage. We hypothesize that DinQ is a transmembrane peptide that modulates membrane-dependent activities such as nucleoid compaction and recombination.The functions of several SOS regulated genes in  Escherichia coli to DNA damaging agents induces the SOS response, which up-regulates gene functions involved in numerous cellular processes such as DNA repair, cell division, and replication. Most of the SOS regulated genes in E. coli have been characterized, but still there are several genes of unknown function. One of these uncharacterized genes is dinQ. In this work we characterize dinQ and two novel small RNAs, agrA and agrB, that regulate expression of dinQ. The DinQ peptide is localized in the inner membrane as a single transmembrane peptide of 27 amino acids. Small proteins of less than 50 amino acids are important in cellular processes such as regulation, signalling, and antibacterial action. Here we demonstrate that DinQ modulates recombination and transformation of nucleoid morphology in response to UV damage. Our results provide new insights into small hydrophobic peptides that could regulate important DNA metabolic processes dependent on the inner membrane of the cell.Exposure of the bacterium  E. coli to DNA damaging agents induces the SOS response, which is under control of the RecA and LexA regulatory proteins. The SOS response upregulates gene functions involved in numerous cellular processes such as nucleotide excision repair (NER), UV induced mutagenesis, recombination, inhibition of cell division and replication. The LexA repressor downregulates more than 50 SOS genes by binding to the operator sequence in their promoter regions Exposure of Both NER and recombination are required to maintain DNA integrity. NER repairs numerous lesions introducing helical distortions, in which UvrA, UvrB and UvrC work in sequential steps to recognize and remove the lesion. The RecBCD complex is the major component for initiation of recombinational repair (RR) of DNA double strand breaks (DSBs) by processing a blunt dsDNA end into a dsDNA molecule possessing a 3′-terminated ssDNA tail. As part of this process RecBCD mediates RecA filamentation required for presynaptic processing of dsDNA ends.E. coli, but there are still several genes of unknown function. One of these uncharacterized genes is dinQ, which is located in the 823 bp region between arsR and gor, 78.58 min on the E. coli chromosome. DinQ is predicted to encode an open reading frame (ORF) of 49 aa 139 nt downstream from a LexA operator sequence E. coliMost of the characterized LexA regulated genes play important roles in the physiology of arsR-gor intergenic region in which an endonucleolytic product of dinQ is translated into a small hydrophobic peptide of 27 aa. DinQ is under LexA control and antisense regulation by a novel small RNA, agrB. DinQ is localized in the inner membrane as a single transmembrane peptide that modulates nucleoid compaction and conjugal recombination.In this work we characterize the dinQ gene in E. coli was identified in a search for new LexA regulated genes dinQ gene was found in the 823 bp arsR-gor intergenic region , containing consensus like −10 and −35 sequences and rho independent terminator sequences , one double mutant (agrAB) and one triple mutant (dinQ agrAB) were generated  wild type and mutant strains  were hybridized with radiolabeled riboprobes against the respective genes , in which the main transcript (a) migrates according to the expected size of full-length dinQ, ∼330 nt. DinQ-b, -c, -d and -e migrates as transcripts of about 290 nt, 250 nt, 200 nt and 130 nt, respectively, according to the size marker. All signals are absent in the dinQ mutant demonstrating that all five transcripts are derived from dinQ. The full-length dinQ product is 3- and 4.6-fold upregulated in the agrA and agrB mutants respectively under normal growth (without UV exposure). Notably, the dinQ-b signal is 4.8- and 3-fold upregulated in the agrB mutant as compared to the wild type and agrA mutant respectively. The dinQ-c product is not detectable in the agrB mutant. Further, the dinQ transcripts are induced in response to UV in wild type and agrA but not in agrB. These data indicate a regulatory mechanism by RNA interference, in which the agrA and agrB interfere differently with the dinQ transcript. Further, primer extension and 3′ mapping of dinQ RNA revealed transcript starts at 0, +44 and +125 corresponding to the estimated size of dinQ-a, -b and –d, respectively (dinQ-b) is upregulated in the agrB mutant as compared to wild type and agrA mutant. The primer extension could not identify any products corresponding to dinQ-c or -e. The agrAB probes showed that the agrB transcript migrates slightly slower than the agrA transcript, and none of the transcripts were regulated in response to UV irradiation , indicating that the transcripts are processed/terminated differently at the 3′end. In summary, these data demonstrate that both agrA and agrB downregulate the level of dinQ full-length transcript whereas agrB is particularly important for down regulation of +44 dinQ (dinQ-b).To examine a potential role for the small non-coding RNAs enerated . To estive genes . The dinectively . In agrerB genes . 3′ mappagrB gene  showed the same viability during normal growth conditions (data not shown). In contrast, it was not possible to transform pKK232-8-dinQ into the agrB mutant  (agrA or agrB they did not increase sensitivity of the cells to UV (data not shown). Interestingly, wild type cells transformed with the plasmid carrying the entire arsR-gor locus, expressing all three genes, showed no UV sensitivity, demonstrating that agrAB can neutralize the UV sensitizing effect of dinQ. The agrB mutant  . When thB mutant  and the nt  were cloned into the expression vector pET28b(+) and expressed under control of IPTG. DinQ I displayed no increased sensitivity in absence or presence of UV, suggesting that the putative peptide translated from ORF I  constructs encoding the putative peptides predicted from DinQ I–IV. Expression of DinQ I could not be detected whereas DinQ II–IV were highly expressed  DinQ V construct produced a peptide corresponding to the predicted molecular weight of peptide V , in which the start site were determined for transcript a, b and d. To assess the translational activity of the in vivo dinQ transcripts a, b and d we synthesized the corresponding PCR products carrying the T7 RNA polymerase promoter and added E. coli T7 S30 extract. Only the dinQ-b/+44 transcript was translationally active, generating a peptide with a molecular weight similar to DinQ V, whereas the other transcripts were translationally inert  is translated from the post transcriptionally modified +44 dinQ RNA.Northern analysis identified five in vivo, a 3×FLAG tag was inserted chromosomally in frame with the C-terminal of the dinQ gene in the wild type and agrB mutant. Western analysis revealed a faint band for the FLAG tagged DinQ peptide in the agrB mutant while the peptide was barely detectable in wild type . Spot assays on LB agar containing IPTG showed that the FLAG tagged peptide induced the same toxicity as the native peptide, demonstrating that the N-terminal FLAG tag had no effect on DinQ toxicity (data not shown). Cells were harvested at several time points after IPTG induction to test the level of expression by western analysis of whole cell extracts. The FLAG-DinQ peptide could not be detected before induction, but showed strong signals 5 to 40 min after induction (data not shown). To examine subcellular fractionation, antibodies against Lep and TolC were used as positive markers for inner and outer membrane fractions, respectively. The western blot showed that the inner and outer membranes are completely separated whereas the cytoplasmic fraction contains some contamination from the inner membrane .To investigate a potential role of  E. coli . Next, wE. coli cells overexpressing DinQ to take up the dye DiBAC4(3) [bis--trimethine oxanol]. The quantity of dye entering cells is proportional to membrane polarization, the less polarised the membrane the greater the quantity entering the cells and so increased fluorescence intensity due to binding to the membrane and intracellular components 4(3) for 20 min and analyzed by flow cytometry (4(3) uptake  uptake , suggestE. coli showed that DinQ is localized to the inner membrane  and recombinational repair (RR) counteract the genotoxic effects of UV irradiation. NER is required for the repair of UV induced photoproducts such as thymine dimers and cyclopyrimidine dimers, while RR is required for the repair of strand gaps and double strand breaks. To examine if DinQ is involved in NER we analysed UV sensitivity of the uvrA agrB and uvrA dinQ double mutants as compared to the single mutants. The survival analysis showed an additive effect between agrB and uvrA  and (dinQ recB) were exposed to UV irradiation. The double mutant (recB agrB) was slightly more sensitive to UV than the agrB single mutant . Hfr conjugation of Tn10 was at least 400-fold more efficient in dinQ and uvrA mutants as compared to agrB . In sum, these results suggest that recombination is inhibited in the agrB mutant during Hfr conjugation, but not in the transfer and uptake of DNA or survival of the agrB recipient. It thus appears that elevated levels of DinQ affect the recombination process.To further examine the role of DinQ in recombination we performed Hfr conjugation assays with a donor strain containing  to agrB , suggestE. coli, UV lesions cause a delay in DNA synthesis for a period of time while stalled forks undergo repair. Fluorescence microscopy of Hoechst stained cells has demonstrated that the DNA often forms a compact structure during this phase, and suggests that the nucleoids undergo a major reorganization after UV exposure agrB mutant the degree of nucleoid compaction was similar to that of wild type cells at 15 min after UV exposure  is translationally active. Further, agrB appears to repress accumulation of dinQ-b by RNA interference. Unexpectedly, DinQ is not translated from any of the three ATG codons within the ORF but from an alternative GTG start codon, encoding a 27 aa peptide which is localized in the inner membrane. The agrB mutant, which expresses elevated levels of dinQ-b, displays increased sensitivity to UV induced DNA damage and an impaired frequency of conjugal recombination. It thus appears that DinQ could be involved in the modulation of homologous recombination. Small single transmembrane peptides such as DinQ may be key regulators of processes at the inner membrane, in which their expression are strictly regulated to avoid toxicity. In summary, the experiments presented in this paper provide insights into the complex regulation of dinQ and suggest a mode of action within the bacterial inner membrane  whereas both sRNA repressed the primary translationally inactive dinQ transcript. Further, only the agrB antisense RNA counteracts DinQ toxicity. As such the dinQ-agrAB system appears to conform to the definition of a classical type I toxin-antitoxin (TA) system. It thus appears that the dinQ/agrB complex inhibit the endonucleolytic cleavage producing the active mRNA (dinQ-b). Presumably, these antisense sRNAs have been tandemly duplicated in the genome, in which agrA has partly degenerated and appears to be non-functional due to less antisense sequence complementarity with the dinQ sequence as compared to agrB.Previous attempts to identify a translation product for dinQ in the arsR-gor region resembles regulation of another SOS induced TA system, tisABdinQ RNA, endonucleolytic processing of the primary tisAB transcript is required to generate an active mRNA producing the toxic TisB peptide. Further, the tisAB locus contains an antisense RNA, IstR-1 that inactivates the translationally active mRNA by RNaseIII dependent cleavage. It appears that agrB may have a similar role in RNase dependent cleavage of the translationally active dinQ mRNA (dinQ-b). In addition, Darfeuille et al revealed that the antisense RNA IstR-1 inhibits translation of the TisB toxin by competing with standby ribosomes binding upstream of the translation initiation region (TIR). It is proposed that binding to the “standby” site is required for initiation of protein synthesis at the highly structured tisB TIR by ribosome sliding to the transiently open TIR agrB antisense RNA regulates/inhibits translation from the active +44 transcript by binding a potential “standby” site upstream of the dinQ TIR.The genomic organization and mode of antisense regulation of agrB mutant. The DinQ concentration in the agrB mutant after UV treatment is elevated only two fold as compared to the wild type, indicating that the DinQ levels in the agrB mutant is physiologically relevant. Of particular interest was the 400-fold reduction in the recombination frequency in the agrB mutant as compared to wild type cells, suggesting that augmented DinQ inhibits recombination in the agrB mutant. However, genetic data suggests that DinQ may also play a role in UV protective mechanisms independent of recombination.In order to investigate DinQ biochemically, we have attempted to purify DinQ, including fusion peptides. However, all attempts to purify DinQ as well as chemical synthesis of the peptide failed because of the hydrophobic nature of the peptide. Further, a general feature of small hydrophobic peptides including DinQ is the lack of obvious phenotypes associated with their inactivation agrB mutant may contribute to the impairment of DNA repair processes. Although such a direct role for DinQ is speculative, several small hydrophobic peptides have been demonstrated to modulate membrane dependent processes. The B1500 protein (65 aa) interacts with the PhoQ sensor It appears that the large, ordered hyperstructures involved in homologous recombination are associated with the cell membrane agrB mutant compared to the wild type both before and after UV exposure. In wild type cells UV irradiation induces a two fold increase in ATP concentration during the first 20 to 30 min after exposure, and the increase is RecBC dependent agrB and thereby excess of DinQ limit the cellular energy supply and may also explain some of the observed phenotypes. Our FLAG tag experiments revealed that DinQ increases only two fold in an agrB mutant and this apparently modest increase is sufficient to mediate dramatic effects on conjugal recombination rates, membrane depolarization, ATP levels and nucleoid reorganization. In a wild type cell population the level of DinQ translation is kept strictly under control by the LexA repressor, antisense agrB RNA and dinQ RNA processing, so for the majority of cells DinQ may never reach a level high enough to mediate the effects observed in an agrB mutant. Heterogeneity in the expression of LexA repressed genes has been observed by studying SOS promoter fusions in combination with imaging techniques and a subpopulation of cells clearly have a stronger SOS induction agrB phenotype. Such an effect has been proposed for some toxin/antitoxin pairs in promoting formation of persister cells agrB mutant could be an excellent model for studying the effects of DinQ and similar hydrophobic peptides in bacterial subpopulations.The intracellular concentration of ATP was reduced in the agrA (BK4042), agrB (BK4043) and dinQ (BK4040) single mutants were made by deleting each of the genes and introducing a rkan cassette. Next, the agrAB double mutant (BK4041) was generated by deleting both genes and introducing a rkan cassette. To construct a triple mutant the entire arsR-gor intergenic region containing dinQ, agrA and agrB was deleted (BK4044) and replaced with the rkan cassette. E. coli) contains a promoter less cat gene allowing selection of DNA fragments containing promoter activity arsR-gor in MCS, resulting in a plasmid that expresses E. coli dinQ from its own SOS inducible promoter. Cloning primers are listed in NcoI-BamHI restriction sites of the pET28b(+) vector (Novagen). Chromosomal point mutations in dinQ to either introduce a premature translational stop codon in DinQ ORFV (K4stop) or introduce three point mutations in the agrB antisense region of dinQ  or to introduce a chromosomal DinQ C-terminal 3×FLAG tag were made by splicing PCR products with overlap extension (SOEing PCR) and recombine the final SOEing PCR product into a MG1655 background as described rkan cassette close to the arsR gene to facilitate selection of recombinants. To avoid unwanted recombination between the rkan cassette and the point mutations or the 3×FLAG tag during strain construction the SOEing products were transformed into strain BK5444-pKD46 which lacks the chromosomal dinQ-agrAB locus and where insertion/recombination of the SOEing products is possible only in the flanking homologous DNA sequences. The final PCR products were transformed into MG1655 containing pKD46. Cells were cured for pKD46 and insertions verified by PCR and sequencing. Details of strain construction and oligos used are listed in NcoI-BamHI restriction sites of the pET28b(+) vector (Novagen). Cells were grown in LB- or K-medium The experiments were carried out in an AB1157 background In vitro transcription/translation on circular pET28b(+) templates or linear PCR products were performed according to Promegas protocols E. coli T7 S30 Extract System for Circular DNA and E. coli S30 Extract System for Linear Templates, respectively, with [14C]-Leucine as radiolabeled amino acid. The translation products were analysed by SDS-PAGE and visualized on Typhoon 9410 (Amersham).Aliquots of exponentially growing ER2566/pET28b(+)-DinQ V were harvested by centrifugation 20 min after IPTG induction (1 mM). Cells were resuspended in 4 ml 50 mM phosphate buffer pH 7.2 and sonicated three times for 15 sec. Further fractionation was performed as described by 600≈0.4 in LB and induced with IPTG (1 mM). At 0, 5 and 20 min culture samples were diluted 1∶10 in filtered AB minimal medium 4(3) (Sigma-Aldrich). After 20 min incubation in the dark at room temperature, cells were analysed in a Flowcytometry LSRII (Becton Dickinson) equipped with an argon ion laser and a krypton laser (both Spectra Physics). DiBAC4(3) was detected using 488 nm laser. The distribution of DiBAC4(3) fluorescence was plotted on a logarithmic scale. The data obtained was analyzed by winMDI software.Cells were grown to OD600 for each sample.Cell aliquots were harvested before and 20 min after induction with IPTG (1 mM) and washed once in 50 mM Tris-acetate pH 7.75. ATP was extracted from washed cells by 1% trichloroacetic acid (TCA) in 50 mM Tris-acetate pH 7.75 for 10 min. Tris-acetate pH 7.75 was added 1∶10 to obtain optimal pH of 7.75 before mixing with rL/L reagent  at room temperature. The amount of ATP extracted  was measured with 20/20 Luminometer (Turner Designs) and related to the ODdinQ (BK4040), agrB (BK4043) and uvrA (BK4180) were mixed in equal volumes with donor strains BW7623  or ER2738 (carrying a tetracycline resistance conjugative plasmid) and incubated at 37°C for 30 minutes. BW7623 was used to examine chromosomal transfer to the recipient strains (recombination dependent) whereas ER2738 was used for plasmid conjugation. Cells were vortexed thoroughly and spread on selective LB plates. Hfr recombination rate and plasmid conjugation rate was calculated as number of recombinants/conjugated cells pr 106 cells.Aliquots of exponentially growing recipient strains dinQ (BK4040) and agrB (BK4043) cells were UV irradiated with 3 J/m2 while stirring. 1.5 ml samples were taken at 0, 15, 30, 60 and 90 min after irradiation. Washed once and resuspended in 100 µl cold, filtered TE buffer. Then 1 ml of cold, filtered 77% ethanol was added for fixation. Fixed cells were mounted on a poly-L-lysine coated microscope slide and the DNA was stained with Hoechst 33258  in mounting medium (40% glycerol in PBS pH 7.5). Visualization of stained cells was performed using a Leica DM6000B phase-contrast/fluorescence microscope equipped with a 63× objective and a BP340-380 excitation filter. Pictures were taken using a Leica DFC350 FX digital camera that was connected to a computerized image analysis system . The fluorescent image was merged with the phase-contrast image.Exponentially growing wt (AB1157), Figure S1−1–10−5cells ml−1) log phase cultures of wt (AB1157) or agrB (BK4043) with expression vector pKK232-8 or vector constructs with agrB (pKK232-8-agrB = pBK446) were spotted onto LB plates and exposed to 0 J/m2 UV (left panel) or 50 J/m2 UV (right panel). Pictures were taken 1 day after incubation at 37°C.Complementation. Serially diluted (10(TIF)Click here for additional data file.Figure S2agrB mutant cells. (A) Growth rates of wt  compared to dinQ , agrAB , agrA , agrB  and dinQ-agrAB .  Cell cycle analysis and flow cytometry of wt (AB1157) (B) and agrB (BK4043) (C). Cells were grown exponentially in AB minimal medium supplemented with 1 µg/ml thiamine, 0.2% glucose and 0.5% casamino acids at 37°C to OD450 = 0.15. Cells were either harvested or treated with 300 µg/ml rifampicin and 10 µg/ml cephalexin for 5 generations. The left and middle panels show DNA histograms of exponentially growing cells and cells treated with rifampicin (rif) and cephalexin (cpx), respectively, where the number of cells is plotted against the number of chromosome equivalents. The number of chromosome equivalents for rifampicin- and cephalexin-treated cells corresponds to the number of origins at the time of drug action. The right panel shows a schematic diagram of the cell cycle. The timeline at the bottom shows the generation time (τ) as well as the times for initiation (ai) and termination (at) of replication. Each horizontal bar represents one generation where the “current” generation is denoted k, the “mother” generation k-1 and the “grandmother” generation k-2. The C period (elongation) is colored red and the D period (segregation and cell division) is colored grey as one replication cycle is followed through three  and two  generations. Cells showing the replication pattern at the time of initiation and termination of replication are drawn on top of the diagram. A black dot and a grey circle represent the origin and the chromosome, respectively. The C+D period was determined from the initiation age, generation time and number of generations spanned by C+D. The initiation age and number of generations spanned were found from the rif/cpx histogram and the relative lengths of C and D were found from the exponential histogram. Relative DNA content per mass of the agrB mutant as compared to wt is calculated and presented in the table (D). For details on cell cycle analysis and flow cytometry, see agrB mutant (BK4043) cells (C) were found to initiate at two origins when newborn in the “mother” generation. Since the replication and segregation periods (C+D) spanned fewer generations, the C period was found to be shorter than in wt cells (41 minutes compared to 55 minutes). The D period was determined to last for one generation (41 minutes) as in wt cells. The values found for the cell cycle parameters are average values for the respective cell cultures. Especially the mutant cells have a large cell-to-cell variability as can be seen in the rif/cpx histogram which shows that some cells have initiated replication, but not divided, and some cells have divided but not initiated replication.Growth curves, DNA histograms, and cell cycle analysis of wild-type cells and (TIF)Click here for additional data file.Figure S3recA and lexA mRNA concentrations with regards to rrsB in various dinQ regulon mutant strains both before and after UV exposure. The graphs show the mRNA concentrations of the SOS response regulators recA mRNA (A) and lexA mRNA (B) relative to the rrsB transcript before and after UV exposure. The relative concentrations in various mutant strains: dinQ (BK4040), agrAB (BK4041), agrA (BK4042), agrB (BK4043), dinQ-agrAB (BK4044) and the isogenic wt reference strain (AB1157) are compared. No significant difference was seen between the different strains indicating that the disrupted regions do not affect the induction of the SOS response at the transcription level. cDNA was synthesized from DNaseI treated total RNA (1 µg) using the High Capacity cDNA Reverse Transcription Kit (ABI) according to the manufactures' instructions. Power SYBR Green PCR MasterMix together with a StepOnePlus Real-time PCR System (ABI), cDNA (5 ng) and rrsB, recA or lexA primers (tC) values. The primers were automatically selected using the Primer Express 3.0 software (ABI). Four independent samples were run in quadruplet to generate a mean tC value relative to the rrsB  transcript, ΔtC, which was used as the endogenous control. The ΔtC values fall after UV exposure due to the stable larger amount of rrsB transcript and a rising but reduced amount of recA or lexA transcript.RT–qPCR showing stable  primers  to gener(TIF)Click here for additional data file.Figure S42) strains as indicated followed by growth for 1 and 2.5 h. Cells were grown in K-medium and stained with acridine orange. Each image is quantified and median values in µm are given together with number of cells measured (in parentheses).Filamentation. Fluorescence microscopy of UV exposed (50 J/m(TIF)Click here for additional data file.Figure S5dinQ,recB) and  mutants. Serially diluted Serially diluted (10−1–10−5cells ml−1) log phase cultures of wt (AB1157), ΔdinQ (BK4140), uvrA::kan (BK4180), ΔdinQ,uvrA (BK4141), recB::kan (BK4110) or ΔdinQ,recB::kan (BK4142) were spotted onto LB plates and exposed to 4 J/m2 (right panel), unexposed panels to the left. Pictures were taken 1 day after incubation at 37°C.Survival of ((TIF)Click here for additional data file.Table S1Characteristics of bacterial strains and plasmids used in this study.(DOCX)Click here for additional data file.Table S2agrB (BK4043/BK4180) were transformed by electroporation with plasmids pKK232-8 and pBK444 (dinQ). Dilutions were spread on LB plates and transformed cells were counted.Transformation efficiency. Exponentially growing cells of wt (AB1157) and (DOCX)Click here for additional data file.Table S3Oligonucleotides used in this study.(DOCX)Click here for additional data file.


Moreover, sex hormone binding globulin (SHBG) was also simultaneously upregulated in these cells. When the SHBG gene was blocked by SHBG siRNA knock-down, the induction of Oct3/4, Nanog, CD44 and CD90 by DHT was also correspondingly blocked in these cells. Immunohistochemical evaluation of clinical samples disclosed weakly positive, and areas negative for SHBG expression in the benign prostate tissues, while most of the prostate carcinomas were strongly positive for SHBG. In addition, higher levels of SHBG expression were significantly associated with higher Gleason score, more seminal vesicle invasions and lymph node metastases. Collectively, our results show a role of SHBG in upregulating stemness of prostate cancer cells upon DHT exposure in vitro, and SHBG expression in prostate cancer samples is significantly associated with poor clinicopathological features, indicating a role of SHBG in prostate cancer progression.Androgen plays a vital role in prostate cancer development. However, it is not clear whether androgens influence stem-like properties of prostate cancer, a feature important for prostate cancer progression. In this study, we show that upon DHT treatment  Prostate cancer is a common malignancy in Western countries in situ hybridization for SHBG mRNA, suggesting that SHBG is locally regulated and produced Potent androgens such as testosterone and 5α-dihydrotestosterone (DHT) play an important role in the development of normal prostate and prostate cancer SHBG) on selected cell membranes. Thereafter, subsequent binding of an appropriate androgen or estrogen to the SHBG-RSHBG complex is activated which results in accumulation of cAMP in prostate cancer The initial step of androgen and estrogen signaling though SHBG requires binding to its specific receptor . All individuals involved in this project have given written informed consent for the original human work that produced the tissue samples and written informed consent to publish these case details.2 incubator at 37°C. After allowing cells to attach onto the flasks, the cells were transferred into phenol red-free RPMI 1640 supplemented with 10% charcoal-stripped FBS (androgen-free medium) for overnight. DHT  was dissolved in ethanol and added in androgen-free medium for cell culture and the corresponding concentration of ethanol was used as blank control Human prostate cancer cell lines PC-3 and LNCaP were obtained from the American Type Culture Collection (ATCC). All cells were cultivated in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 100 units/ml penicillin and 100 µg/ml streptomycin in a humidified 5% COPC-3 (1000/well) and LNCaP (2000/well) cells were planted in 96-well plates. After the cells attached to plate, 1 nM or 10 nM DHT was added into the androgen-free medium for variable times. At each time point as indicated, the cells were added with MTT (Sigma-Aldrich) and cultivated at 37°C for 4 hours. Then 200 µL of dimethyl sulfoxide  was added to each well and mixed thoroughly. The plates were shaken for 15 min and absorbance was determined using spectrophotometer at a wavelength of 570 nm.500/well single cells of PC-3 cells and 1000/well single cells of LNCaP cells were seeded in six-well plates with/without DHT (1 nM or 10 nM) in androgen-free medium as mentioned above for 14 days before the cells were gently washed with PBS and fixed by 4% buffered formalin for 15 min. Subsequently, 1% crystal violet was used to stain the colonies for 30 min. The plates were gently washed with PBS and dried before colony evaluation under microscope. Colony number which contained more than 30 cells was counted and colony formation efficiency was determined as colony formation efficiency = colonies/input cells × 100%.The sphere assay was performed as described in our previous study Based on our previous study Cells were washed with ice-cold PBS twice and homogenized in lyses buffer  added with protease inhibitors  immediately before use. The total protein content in samples was measured by the Bio-Rad protein assay  according to the manufacturer’s instruction. Equal amounts of protein were resolved by 10% SDS-PAGE and electro-transferred to polyvinylidene difluoride transfer membrane in a Trans-Blot apparatus . 5% non-fat milk was used to block the membranes and the primary antibodies with optimized concentrations including AR (1 µg/ml), Oct3/4 (2 µg/ml), Nanog (1 µg/ml), SHBG (1 µg/ml) and GAPDH (0.2 µg/m) from R&D system were added to incubate for overnight at 4 °C. The corresponding secondary antibodies conjugated with HRP were then used to incubate the membranes. Immuno-complexes were visualized by enhanced chemiluminescence detection system .The PC-3 and LNCaP cells were cultivated in the androgen-free medium added with 1 nM/10 nM DHT for variable periods. Cell culture media at different culture periods were then collected, centrifuged to remove the cellular debris and stored at −70°C for further PSA and SHBG measurement. Total PSA was measured by the time-resolved fluoroimmunometric dual-label assay performed with the AutoDELFIA instrument ,. The assay was calibrated against WHO standards, with an assay sensitivity of 0.05 ng/ml and interassay coefficient of variation below 5% over the standard range from 0 to 260 ng/ml. The human SHBG ELISA kit  was used for quantitative determination of SHBG in the medium according to the manufacturer’s instruction. A calibrator curve was established and serum positive control was also included for every run.5 cells per well were planted at six-well plates for 24 hours. SHBG siRNA (50 pmols) and siRNA control (50 pmols) were dissolved within OPTI-MEM Reduced Serum Medium and cultivated for 30 min at room temperature separately. Then the transfection mixtures were added onto the 60–80% confluent cells in six-well plates for 5 hours’ cultivation. The medium was aspirated and replaced with fresh normal growth medium for 24 hours. Then the medium was changed to androgen-free medium added with/without DHT (10 nM) for 48 hours. The cells were washed with PBS and harvested for further experiments.SHBG siRNA (sc-44847) and siRNA control (sc-33007) for transient transfection were obtained from Santa Cruz Biotechnology. 2×10Radical prostatectomy specimens from 117 patients with clinically localized prostate cancer were included in this study. All the patients underwent radical retropubic prostatectomy (RRP) in the period from 1985 until 2006 at Soerlandet County Hospital, Arendal, Norway. The clinical samples were routinely processed for histological diagnosis. Final histological classification in this study was done according to the International Society of Urological Pathology  consensus on Gleason grading of prostate cancer in this study. Gleason grade score, surgical margin (SM) status, seminal vesicle invasion (SVI) and extraprostatic extension (EPE) for the malignant tumors were recorded. In total, five of these patients were found to have lymph node metastasis upon histological re-examination. In addition, formalin-fixed and paraffin-embedded samples from 10 prostate hyperplasia samples were retrieved from the archives of The Norwegian Radium Hospital and included in this study as well.2O2) for 5 min to block the endogenous peroxidase. The antibodies for Oct3/4 , Nanog , AR  and two different antibodies for SHBG  were used. The sections were incubated with the primary antibody for 30 min at room temperature. The liver tissue was used as positive control according to the instructions and the non-immune corresponding IgG was used as negative control. After rinsed with DAKO wash buffer, corresponding EnVision FLEX+Linker reagent was added and incubated for 15 min at room temperature before the slides were incubated with the EnVision FLEX+HRP for 30 min at room temperature. The sections were rinsed, colour reaction developed with DAB reagent, counterstained with hematoxylin for 20 seconds, dehydrated and mounted with cover slips before microscopy evaluation. The results were evaluated by two pathologists without knowledge about the patient data. The immunohistochemical results for SHBG for the 117 patients with prostate cancers were assessed according to a previous study 4µm paraffin sections from paraffin blocks with either cell lines or clinical samples were prepared. After deparaffinization, the sections were treated with hydrogen peroxide  was used for data analysis and statistical significance was considered as Cell growth of prostate cancer cell lines LNCaP and PC-3 treated with two different concentrations (1 nM or 10 nM) of DHT for variable periods of time were examined by MTT assays. Both low and high concentrations of DHT could stimulate the proliferation of androgen-responsive LNCaP cells in both time-dependent and concentration-dependent manners with significantly statistical difference after 48 hours . The PC-CD44, a candidate stem cell marker for prostate cancer, was upregulated in both prostate cancer cell lines treated with 10 nM DHT for 48 hours, although relatively less prominent in PC-3 cells. As shown in In an attempt to further explore whether stem-like properties of prostate cancer cells were altered in response to DHT, the expressions of stemness factors Oct3/4 and Nanog were investigated in LNCaP and PC-3 cells cultivated with/without DHT for variable periods of time. Both Oct3/4 and Nanog expressions began to be upregulated after 24 hours’ DHT treatment in both cell lines in a concentration-dependent manner , althougIn consistent with a previous study Since the expressions of Oct3/4 and Nanog could be upregulated in both AR positive LNCaP cells and AR negative PC-3 cells, induction of Oct3/4 and Nanog by DHT through AR was ruled out, at least in the PC-3 cells. Considering the high SHBG affinity to DHT To further investigate whether SHBG was involved in the upregulation of Oct3/4, Nanog, CD44 and CD90 in response to DHT, RNA interference assay was used to suppress endogenous SHBG expression in LNCaP and PC-3 cells. The transient transfection effect was obtained using the siRNA SHBG compared to the siRNA control and the immunoblotting analyses demonstrated that the SHBG-specific siRNA successfully inhibited SHBG expression after transfection for 24 and 48 hours in both cell lines . Thus, tTaking the above results together, greater stem-like properties were displayed by DHT treatment in prostate cancer cell lines and SHBG might play an important role in the induction of stemness features in these cells upon DHT treatment, indicating a potential role of SHBG in prostate carcinomas. To address this question, SHBG expression was investigated in clinical samples including benign and malignant prostate tissues by immunohistochemistry. SHBG expression in liver tissue was always kept as positive control in each running . Weak anp = 0.013). It was also found that tumors with higher levels of SHBG expression were more likely to have seminal vesicle invasion compared to the patients with lower level SHBG expression (p = 0.017). Importantly, all the tumors with lymph node involvement were shown strong SHBG immunostaining (p = 0.009). All the results show that SHBG expression in prostate carcinomas is significantly associated with poor clinicopathological features.The clinical and pathological characteristics of the 117 patients with prostate cancer are summarized in Androgens are essential for prostate cancer development and prolonged administration of testosterone could induce prostate cancer in rodents in vitro. Indeed, by colony formation and sphere formation assays, both cell lines exhibited a significantly higher colony and sphere formation efficiency, in a dose-dependent manner within the range of 1 nM to 10 nM of DHT. These observations highly indicated an involvement of stemness related molecules.The LNCaP cell line is a human prostate adenocarcinoma cell line derived from lymph node metastasis. This cell line is AR positive. PC-3 is a classical prostatic cell line originally derived from advanced androgen independent bone metastatic prostate cancer. In consistent with the previous study bright cells of prostate cancer cell lines displayed higher clonogenicity with significantly higher expression levels of stemness-related factors than the corresponding CD44dim cells We further examined the DHT influence on the expressions of CD44, CD24 and CD90 by flow cytometry, since all these molecules have been linked to cells’ stemness. After 48 hours treatment of DHT, significantly higher level of CD44 expression could be repeatedly seen in the LNCaP cells, while the CD44 induction in the PC-3 cells was not the same prominent as in the LNCaP cells, although higher levels of CD44 expression were also observed. This difference may be explained with the fact that PC-3 cells already expressed higher level of CD44 , and theOct3/4 and Nanog play an important role in maintenance of self-renewal of embryonic stem cell and primordial germ cells. These stemness factors are frequently overexpressed in histologically poorly differentiated tumors than in those well-differentiated tumors in vitro. The fundamental biological role of DHT has been characterized via AR. Thus, we further determined to examine the AR status of these two cell lines, in case any AR positivity in the PC-3 cells due to any undiscovered reason or mechanism. However, we verified that the PC-3 cells were still AR negative and not inducible with DHT addition while the LNCaP cells were AR positive and DHT could upregulate the expression of AR in these cells as well, a finding well in line with the previous report . Therefore, we reasoned that it is unlikely the upregulation of the stemness factors Oct3/4, Nanog, CD44 and CD90 in these prostate cancer cell lines was through AR, since similar results were also observed in the AR-negative PC-3 cells. In addition to the genomic action like the classic androgen-AR signaling, a growing body of evidence has suggested that androgen could exert rapid non-genomic effects shbg transgene expression is androgen dependent and exogenous androgen increases human SHBG mRNA levels in the kidneys of female mice All the above results pinpoint to a possibility that DHT indeed upregulates cell stemness of prostate cancer cells Collectively, these results have linked the SHBG expression with cells’ stemness. To further verify this possibility, specific SHBG siRNA was used to suppress endogenous SHBG expression in both cell lines. We have repeatedly demonstrated that when SHBG was blocked down by specific siRNA in these cells, DHT could not upregulate the expressions of the stemness factors Oct3/4, Nanog, CD44 and CD90 in these cells longer, indicating a direct interaction between the induction of these factors by DHT and SHBG. Therefore our results suggest that DHT-SHBG pathway may play an important role in induction of stem-like properties in prostate cancer cells and SHBG expression in prostate cancer samples may be of clinical consequence.In clinical sample study, all the ten benign prostate tissues were weak and areas negative for SHBG expression. Generally higher levels of SHBG expression were identified in prostate cancer samples compared to the benign prostate tissues. In the prostate carcinomas, infiltrating tumor cells or tumor cells located in the poorly differentiated areas were always strongly positive, while the well-differentiated glandular structures of cancer were always weakly positive for SHBG. Further statistical analyses showed significant association of SHBG with poor pathological characteristics, including high Gleason grade score, involvement of seminal vesicle invasion and lymph node metastasis. In a serum SHBG study, Andrea et al. have also found a similar correlation and suggested that SHBG may be identified as a multivariate predictor of lymph node invasion in prostate cancer patients undergoing extended pelvic lymph node dissection in vitro via SHBG and SHBG expression in prostate carcinomas is significantly associated with higher Gleason grade score, seminal vesicle invasion and lymph node metastasis. All our in vitro and clinical sample results indicate an important role of SHBG in prostate cancer progression.In conclusion, we have demonstrated that DHT could upregulate prostate cancer cell stemness


Apoptosis is a major feature in neural development and important in traumatic diseases. The presence of active caspases is a widely accepted marker of apoptosis. We report here the development of a method to study neuronal apoptotic death in whole-mounted brain preparations using fluorochrome-labeled inhibitors of caspases (FLICA). As a model we used axotomy-induced retrograde neuronal death in the CNS of larval sea lampreys. Once inside the cell, the FLICA reagents bind covalently to active caspases causing apoptotic cells to fluoresce, whereas nonapoptotic cells remain unstained. The fluorescent probe, the poly caspase inhibitor FAM-VAD-FMK, was applied to whole-mounted brain preparations of larval sea lampreys 2 weeks after a complete spinal cord (SC) transection. Specific labeling occurred only in identifiable spinal-projecting neurons of the brainstem previously shown to undergo apoptotic neuronal death at later times after SC transection. These neurons also exhibited intense labeling 2 weeks after a complete SC transection when a specific caspase-8 inhibitor (FAM-LETD-FMK) served as the probe. In this study we show that FLICA reagents can be used to detect specific activated caspases in identified neurons of the whole-mounted lamprey brain. Our results suggest that axotomy may cause neuronal apoptosis by activation of the extrinsic apoptotic pathway. Studies in the basic neurosciences are heavily reliant upon rat and mouse models (for a review see ). SeventPetromyzon marinus L.) genome, and the adaptation and optimization of many established molecular biology and histochemistry techniques for use in this species have made it an emerging nonmammalian model organism of choice for investigations into the evolution of hallmark vertebrate characteristics. In addition, the sea lamprey has proven to be a valuable animal model in spinal cord (SC) injury and regeneration studies [Lampreys occupy a key position close to the root of the vertebrate phylogenetic tree  and are  studies , 3.Programmed cell death (apoptosis) of large numbers of immature neurons is a major feature in neural development. Neuronal cell death is also an important component of both acute and chronic neurodegenerative diseases and traumatic injuries. In apoptosis, caspases are responsible for proteolytic cleavages that lead to cell disassembly (effector caspases) and are involved in upstream regulatory events (initiator caspases) . In vertin situ in individual cells immunocytochemically with antibodies generated against the epitope that is characteristic of the cleaved, enzymatically active form of caspases [Activated caspases can be detected in histological preparations and caspases , 15. Howcaspases –18 when In this paper we propose to use the lamprey brain as a model system to develop a new method for detecting activated caspases in the whole-mounted CNS of nonmammalian species. Recent studies have shown that axotomy after a complete SC transection induces death of spinal-projecting neurons of the lamprey brainstem , 20. Theex vivo.We are reporting a new method for the detection of activated caspases based on the use of fluorochrome-labeled inhibitors of caspases (FLICA). When applied to live cells, cell-permeate FLICA reagents tagged with fluorescent molecules exclusively label cells that have active caspases and are undergoing apoptosis (see Petromyzon marinus L.), 10–14 cm in length (4–7 years old), were obtained from streams feeding Lake Michigan and maintained in freshwater tanks at 16°C with the appropriate aeration until the day of use. Experiments were approved by the Institutional Animal Care and Use Committee at Temple University. Before experiments, animals (n = 25) were deeply anesthetized by immersion in Ringer solution containing 0.1% tricaine methanesulfonate .Wild-type larval sea lampreys  and the Image-iT LIVE Green Caspase-8 Detection Kit  were used to detect activated caspases in identifiable reticulospinal neurons of larval sea lampreys after a complete SC transection. This kit contains 1 vial (component A of the kit) of the lyophilized FLICA reagent . The reagent associates a fluoromethyl ketone (FMK) moiety, which reacts covalently with a cysteine, with a caspase-specific aminoacid sequence  for the poly caspases reagent and leucine-glutamic acid-threonine-aspartic acid (LETD) for the caspase-8 reagent). A carboxyfluorescein group (FAM) is attached as a fluorescent reporter. The FLICA reagent interacts with the enzyme active center of an activated caspase via the recognition sequence, and then attaches covalently through the FMK moiety .μL of DMSO (component D of the kit) was added to the vial containing the lyophilized FLICA reagent and mixed until the reagent was completely dissolved. The unused portion of the 150x FLICA reagent can be stored in small aliquots protected from light at −20°C, and the reagent will be stable for several months.To prepare the 150x FLICA reagent stock solution, 50 μL of phosphate buffered saline (PBS) containing 1 μL of the 150x FLICA labeling solution at 37°C for 1 hour. Then, the brains were washed 9 × 15 min at room temperature (protected from light) on a nutator using 1x wash buffer. The 1x wash buffer was prepared from 10x apoptosis wash buffer (component F of the kit) by adding 9 parts of deionized water to 1 part of 10x apoptosis wash buffer. After washes, the posterior and cerebrotectal commissures of the brain were cut along the dorsal midline, and the alar plates were deflected laterally and pinned flat to a small strip of Sylgard . Brains were fixed in 4% paraformaldehyde in PBS overnight at 4°C. Next, the brains were washed 4 × 15 min with PBS, mounted on Superfrost Plus glass slides , and coverslipped using Prolong (Invitrogen) as an antifade reagent.Brains from control noninjured animals and animals that survived for two weeks posttransection were dissected out and stripped of choroid plexus. Fresh dissected brains were immediately incubated in 150 In all experiments, brains of noninjured animals were processed in parallel with brains of experimental animals. Photomicrographs were taken using a 10x objective and under the same conditions of exposure time, and camera/microscope settings for those animals processed in parallel.μM for 1 hour at 37°C. Following this treatment, brains were processed for poly caspase FLICA labeling (see above). As a control for the specificity of labeling, the brains of 2 SC transected animals were first incubated in PBS containing the pan caspase inhibitor Z-VAD-FMK  at a concentration of 40 Photomicrographs were taken using a Nikon Eclipse 80i microscope equipped with a CoolSNAP ES  camera. Brightness and contrast were minimally adjusted using Adobe Photoshop CS4 software and lettering was added. Schematic drawings were carried out using CS BioDraw Ultra software.Whole-mounted brain preparations preserve three-dimensional information, which allows the rapid and accurate identification of labeled neurons. The lamprey brainstem can be studied in whole-mounted preparations because of its flat shape and because the lack of myelin  makes thFAM-VAD-FMK FLICA reagent detects any active caspase. It is well known that alternative stimuli could activate different caspases. For example, the extrinsic or death receptor pathway activates the initiator caspase-8, whereas the intrinsic or mitochondrial pathway activates the initiator caspase-9. Therefore, in separate experiments we used a FLICA kit for the specific detection of caspase-8 to determine if we could detect specific active caspases in a wholemount brain preparation. The caspase-8 specific FLICA reagent, FAM-LETD-FMK, was the probe in these experiments. Neuronal apoptosis was again induced by axotomy due to a complete SC transection (see above). In animals studied two weeks after the SC transection, intense FAM-LETD-FMK labeling appeared in the same identifiable reticulospinal neurons as when we used the poly caspase FLICA reagent (the M1, M2, M3, I1, I2, Mth, B1, B3, and B4 neurons; The specificity of FLICA labeling was assessed in control experiments by preincubation of the lamprey whole-mounted brains with the pan-caspase competitive inhibitor Z-VAD-FMK. Prior exposure to this unlabeled inhibitor of caspases totally prevented subsequent labeling with the poly caspase FLICA reagent: specific poly caspase FLICA labeling did not appear in the brains of experimental animals two weeks after a complete SC transection , thus suex vivo in whole-mounted brain preparations of lampreys. We modified the standard manufacture's FLICA protocol by adding additional washes after incubation with the FLICA reagent and extending the time of each wash. We made these modifications because our experiments used whole-mounted brains rather than cells in culture. Importantly, we showed that, because FLICA labeling is not lost after paraformaldehyde fixation, it should also be possible to combine FLICA labeling with the subsequent detection of other apoptotic or molecular markers by immunohistochemistry or by in situ hybridization. This method for detecting activated caspases in the lamprey brain offers advantages over methods that use commercially available antibodies generated against caspase aminoacidic sequences of mammals, which may lack specificity and/or antibody penetration in whole-mounted lamprey brain preparations.Caspase activation is a hallmark of apoptosis and FLICA reagents have been previously used to detect activated caspases in cell culture . ExposurThe larval sea lamprey is an extremely useful model for studying retrograde neuronal death after axotomy. In lampreys, identifiable reticulospinal neurons with a low regenerative ability die after a complete SC transection . The appWe have developed a novel method for detecting activated caspases in a nonmammalian vertebrate brain that does not rely upon availability of specific antibodies. Application of this new methodology to whole-mounted brain preparations offers great opportunities for increasing our understanding of the molecular mechanisms responsible for apoptosis initiation in lampreys and, more generally, in the many additional nonmammalian vertebrates and invertebrates for which specific antiactivated caspase antibodies are not available.


Lymphedema is a chronic disorder that occurs commonly after lymph node removal for cancer treatment and is characterized by swelling, fibrosis, inflammation, and adipose deposition. Although previous histological studies have investigated inflammatory changes that occur in lymphedema, the precise cellular make up of the inflammatory infiltrate remains unknown. It is also unclear if this inflammatory response plays a causal role in the pathology of lymphedema. The purpose of this study was therefore to characterize the inflammatory response to lymphatic stasis and determine if these responses are necessary for the pathological changes that occur in lymphedema.We used mouse-tail lymphedema and axillary lymph node dissection (ANLD) models in order to study tissue inflammatory changes. Single cell suspensions were created and analyzed using multi-color flow cytometry to identify individual cell types. We utilized antibody depletion techniques to analyze the causal role of CD4+, CD8+, and CD25+ cells in the regulation of inflammation, fibrosis, adipose deposition, and lymphangiogenesis.Lymphedema in the mouse-tail resulted in a mixed inflammatory cell response with significant increases in T-helper, T-regulatory, neutrophils, macrophages, and dendritic cell populations. Interestingly, we found that ALND resulted in significant increases in T-helper cells suggesting that these adaptive immune responses precede changes in macrophage and dendritic cell infiltration. In support of this we found that depletion of CD4+, but not CD8 or CD25+ cells, significantly decreased tail lymphedema, inflammation, fibrosis, and adipose deposition. In addition, depletion of CD4+ cells significantly increased lymphangiogenesis both in our tail model and also in an inflammatory lymphangiogenesis model.Lymphedema and lymphatic stasis result in CD4+ cell inflammation and infiltration of mature T-helper cells. Loss of CD4+ but not CD8+ or CD25+ cell inflammation markedly decreases the pathological changes associated with lymphedema. In addition, CD4+ cells regulate lymphangiogenesis during wound repair and inflammatory lymphangiogenesis. Wuchereria bancrofti, these infections are rarely seen in developed countries where lymphedema occurs most commonly after cancer surgery. Lymphedema is a chronic disorder that is characterized by progressive tissue swelling and fat deposition secondary to congenital defects, infections, or injury to the lymphatic system. In its most advanced forms, lymphedema results in massive changes in the extremities referred commonly to as elephantiasis. Although the most common cause of lymphedema worldwide is parasitic infections with nematodes such as The lack of a clear understanding of the pathology of lymphedema has served as a significant barrier to the development of effective, targeted treatments or preventative options for this disabling complication. Instead, current treatments are palliative in nature with a goal of preventing disease progression and decreasing symptoms rather than curing the underlying pathology. The fact that lymphedema in most cases develops 8–12 months after surgery (rather than immediately following lymph node dissection) With these goals in mind, the purpose of this study was to determine how lymphatic stasis regulates tissue inflammatory changes. In addition, we sought to determine if these chronic inflammatory responses play a pathological role in lymphatic dysfunction, fibrosis, and adipose deposition in lymphedema. We show that specific T cell subpopulations are a critical component of the subacute and chronic inflammatory cell response to lymphatic fluid stasis. In addition, using antibody depletion studies, we show that T-helper cell inflammatory responses are necessary for fibrosis, lymphatic dysfunction, subcutaneous fat deposition, and chronic inflammation occurring in response to sustained lymphatic fluid stasis. Our findings are important because we show for the first time that lymphedema results in characteristic inflammatory responses. Further, we show that these responses play a role in the pathology of lymphedema rather than simply reflecting worsening pathology.Adult female (10–12 week old) wild-type C57B6 or transgenic C57B6 mice deficient in CD4+ cells  were used and all studies were performed according to IACUC standards and approved animal protocols at Memorial Sloan-Kettering Cancer Center. A total of 8–10 animals were used for each group/experiment.In order to study the effects of sustained, severe lymphatic stasis on inflammatory responses, we used a well-described mouse tail model of lymphedema in which the superficial and deep lymphatic system of the tail are disrupted by excising a 3 mm portion of the skin and microsurgically ligating the deep lymphatic channels that run along the lateral tail veins. We also used a mouse model of axillary lymph node dissection to study the consequences of lymphadenectomy and disruption of the deep lymphatic system. We have previously shown that similar to the clinical scenario, axillary lymph node dissection (ALND) in mice results in minor though significant increases in limb volume enabling us to study acute tissue changes that occur in response to lymphatic stasis. A 1 cm portion of tissue was harvested 1 cm distal to the site of axillary lymph node dissection or 1.5 cm distal to the site of tail skin excision. Flow cytometry and analysis was performed using a modification of our previously reported methods. Cell suspensions were analyzed using an LSR II flow cytometer  with BD FACSDiva software and subsequent data analysis performed using FlowJo software . Cell populations were analyzed and defined using the cell surface markers listed in Depletion experiments were performed using the tail model of lymphedema as described above. Following tail skin and lymphatic vessel excision, animals were allowed to recover for 2 weeks and then randomized to experimental or control groups (n = 8–10/group). Experimental animals were depleted of CD4, CD8, or CD25 cells using intraperitoneal administration of monoclonal neutralizing antibodies  at a concentration of 10 ug/g every 5 days for a total of 4 weeks (i.e. 6 weeks postoperatively) as previously described. Control animals were treated with non-specific isotype control antibodies delivered at the same dose and timing. Adequacy of depletion was confirmed by flow cytometry analysis of splenic single cell suspensions comparing experimental and control animals at the time of sacrifice (6 weeks postoperatively).99 m) sulfur colloid in the distal tail and analyzing decay adjusted uptake in the sacral lymph nodes using an X-SPECT camera  and region-of-interest analysis using ASIPro software . Tail lymphedema was monitored weekly using multiple digital caliper tail circumference measurements distal to the zone of lymphatic injury and calculation of tail volumes using the truncated cone formula as previously described. Lymph node lymphangiogenesis was used as previously described to determine how CD4 cells regulate inflammatory lymphangiogenesis independent of wound healing. Briefly, a mixture of complete Freund’s adjuvant (Sigma) and ovalbumin (1∶1) was emulsified and injected in the hind paws of adult female wild-type C57B6 mice and popliteal lymph nodes were harvested 7 days later. Experimental animals (n = 8) were depleted of CD4+ cells using monoclonal neutralizing antibodies beginning 3 days prior to CFA/OVA injection as outlined above while control animals (n = 8) were treated with an equivalent dose of non-specific isotype control antibodies. To confirm our findings with CD4 depletion, we performed identical experiments in wild-type C57B6 mice (n = 8) and compared them with transgenic C57B6 mice deficient in CD4 cells .Tail tissues were fixed in 4% paraformaldehyde and decalcified in Immunocal prior to paraffin embedding. Lymph nodes were embedded in OCT medium  and frozen at −80°C; 5 um sections were prepared from all tissues for analyses.Immunohistochemical staining using horseradish-peroxidase based staining techniques were performed according to our established techniques Immunofluorescent stains were performed as described above, excluding peroxidase quenching steps. Primary antibody to LYVE-1  and TRITC-conjugated secondary antibody were utilized for staining. For frozen lymph node sections, slides were fixed briefly in acetone followed by blocking and staining techniques as described above. Lymphatic vessel density was determined using Metamorph Scanner and Metamorph Offline software  as previously described. Subcutaneous tissue thickness analysis was performed in histological cross-sections located 1.5 cm distal to the surgical site by blinded reviewers (n = 6–8 per group). The distance from the basal layer of the epidermis to deep fascia was analyzed in 4 standardized regions per section. Lymphatic vessel area was measured as previously described. http://rsbweb.nih.gov/ij/). Normalized expression was determined as a ratio relative to controls. For ELISA, 20–30 mg of protein from pooled samples  was analyzed to quantify VEGF-A and VEGF-C according to the manufacturer’s directions . All experiments were performed in triplicate.Tail tissues or lymph nodes were lysed  and 20–30 mg of pooled protein  was used for western blot analysis using our previously published methods. Student’s T-test was used to compare differences between 2 groups; paired T-tests were performed for all matched ALND and sham control samples. Multi-group comparisons were performed using 2-way analysis of variance (ANOVA) with Tukey-Kramer post-hoc test. Data are presented as mean ± standard deviation unless otherwise noted with p<0.05 considered significant.+ cells consistent with tissue inflammation when compared with controls  was also dramatically increased in lymphedematous tissues . ALND in this model  results in mild but significant edema of the upper extremity at 3 weeks postop and resolves by 6 weeks; 2.) Tissue inflammatory responses to ALND are histologically evident beginning primarily 3 weeks after surgery. In order to determine the type of inflammatory responses that are initiated by lymphatic injury prior to the onset of chronic lymphedema, we analyzed tissue inflammation in the upper extremity of mice that had undergone axillary lymph node dissection (ALND) or axillary skin incision without lymphatic injury 3 or 6 weeks after surgery. This approach was based on our previous studies demonstrating that: ). Similarly, we found no significant differences in the percentage of neutrophils, monocytes, macrophages, or dendritic cells at this time point.At the 3-week time point, we noted a mild (1.3 fold) but insignificant increase in the percentage of CD45+ leukocytes in animals treated with ALND as compared to controls (Based on the finding that T cell inflammation is initiated by lymphatic injury (ALND) and persists in chronic lymphedema, we tested the hypothesis that these inflammatory reactions contribute to the pathology of lymphedema by performing antibody depletion studies. This hypothesis is supported by recent findings demonstrating that T cells can regulate lymphangiogenesis As expected, depletion of CD4 or CD8 cells with neutralizing antibodies was highly effective resulting in near complete absence of these cells in the spleen (+ leukocytes (2.5-fold), CD4+ cells (16.7-fold), and F4/80+ macrophages  and increased decay adjusted total uptake (23-fold) in the draining lymph nodes of CD4 depleted animals .To explore the mechanisms regulating improved lymphatic function in CD4 depleted animals, we determined the effect of CD4 depletion on lymphangiogenesis and lymphangiogenic cytokine expression. When we analyzed tail tissues located distal to the zone of lymphatic disruption 6 weeks after surgery, we noted a significant increase  in the number lymphatic capillaries (LYVE-1+ or podoplanin+) in CD4 depleted animals as compared with controls . We found similar results when we repeated these experiments in transgenic mice deficient in CD4+ cells (CD4KO). In addition, we found modest though significant increases in the expression of both VEGF-A (1.3 fold) and VEGF-C (1.6 fold) protein in the lymph nodes of CD4 depleted animals as compared with controls  in animals with chronic lymphedema. In order to determine how acute, low-grade lymphatic stasis resulting from lymph node resection regulates inflammatory responses, we analyzed single cell suspensions from the upper arm area of mice treated with axillary lymphadenectomy (ALND) versus axillary incision without lymph node removal 3 or 6 weeks after surgery. Interestingly, we found that ALND, similar to tail lymphedema, resulted in a persistent and significant increase in tissue inflammation even as long as 6 weeks after surgery. More importantly, we found that similar to tail lymphedema, inflammatory responses to ALND included a prominent increase in the percentage of a variety of T lymphocytes . However, in contrast to the inflammatory changes in the tail model, we found that ALND had only modest, and largely insignificant effects on macrophages, monocytes, and dendritic cells suggesting that T cell inflammatory responses precede and possibly contribute to the pathology of lymphedema.We next explored the hypothesis that inflammatory responses contribute to the pathology of lymphedema and regulate, either directly or indirectly, tissue edema, adipose deposition, fibrosis, and lymphatic dysfunction. We found that depletion of CD4+ but not CD8+ or CD25+ cells resulted in marked improvements in lymphedema and decreased tail swelling, adipose tissue deposition, and fibrosis. In addition, only depletion of CD4+ cells significantly decreased tissue inflammation (CD45+ cell infiltration) and expression of Th1 and Th2 markers including IFN-y, T-Bet, and GATA-3. The hypothesis that CD4+ cell inflammation contributes to the pathology of lymphedema is supported by our previous studies demonstrating that blockade of TGF-B1 activation in the mouse tail model significantly improves lymphedema and is associated with markedly decreased tissue inflammation, CD4+ cell infiltration, and expression of Th1/Th2 markers. In order to determine how CD4+ cell inflammation contributes to pathology of lymphedema, we analyzed tissue fibrosis and regulators of extracellular matrix deposition. Interestingly, and consistent with our previous observations, Recent studies by our lab and others have also shown that T cells are independent and important regulators of lymphangiogenesis. For example, we have previously shown that nude mice  have significantly improved lymphatic function and markedly decreased expression of anti-lymphangiogenic cytokines such as TGF-B1, IFN-y, and endostatin. An interesting finding of our study was the massive increase in the number of T-reg cells in response to lymphatic stasis both after ALND or in response to tail lymphedema. We did not observe significant changes in pathological findings of lymphedema after depletion of CD25+ cells  suggesting that T-regulatory cells may play additional or distinct roles in lymphatic stasis. Indeed, increased numbers of T-reg cells may simply represent a homeostatic response to the chronic inflammation induced by lymphatic stasis. This response may be responsible for immune disturbances in patients with lymphedema such as increased risk of infections (lymphangitis and cellulitis) and inability to mount immune responses or acquire immunity to vaccines when administered in the lymphedematous limb. Similarly, our findings provide a rationale for previous reports demonstrating improved allograft skin graft survival after ligation of afferent lymphatics and delayed rejection of allograft tumors in animal models. In conclusion, in the current study we have characterized the cellular immune response to lymphatic fluid stasis resulting from lymph node dissection and chronic lymphedema using mouse models. In addition, we have shown that depletion of CD4+ cells, but not CD8+ or CD25+ cells markedly decreases that pathological findings of lymphedema including swelling, fibrosis, adipose deposition, and lymphatic dysfunction. Although CD8+ T cells do not appear to centrally regulate the pathophysiology of lymphedema, they may play a role in other aspects of lymphatic fluid stasis and dysfunction. In addition, we have shown that CD4+ cells play an important role in the regulation of lymphangiogenesis in lymphedema and inflammatory lymphangiogenesis. Finally, we have shown that lymphatic stasis results in increased number of T-regulator cells, however, the precise role that these cells play in the regulation of the pathology of lymphedema remains unknown.


HNF4α target genes was the most common pathway specific to AC, while SqCC demonstrated disruption of numerous histone modifying enzymes as well as the transcription factor E2F1. In silico screening of candidate therapeutic compounds using subtype-specific pathway components identified HDAC and PI3K inhibitors as potential treatments tailored to lung SqCC. Together, our findings suggest that AC and SqCC develop through distinct pathogenetic pathways that have significant implication in our approach to the clinical management of NSCLC.For therapeutic purposes, non-small cell lung cancer (NSCLC) has traditionally been regarded as a single disease. However, recent evidence suggest that the two major subtypes of NSCLC, adenocarcinoma (AC) and squamous cell carcinoma (SqCC) respond differently to both molecular targeted and new generation chemotherapies. Therefore, identifying the molecular differences between these tumor types may impact novel treatment strategy. We performed the first large-scale analysis of 261 primary NSCLC tumors (169 AC and 92 SqCC), integrating genome-wide DNA copy number, methylation and gene expression profiles to identify subtype-specific molecular alterations relevant to new agent design and choice of therapy. Comparison of AC and SqCC genomic and epigenomic landscapes revealed 778 altered genes with corresponding expression changes that are selected during tumor development in a subtype-specific manner. Analysis of >200 additional NSCLCs confirmed that these genes are responsible for driving the differential development and resulting phenotypes of AC and SqCC. Importantly, we identified key oncogenic pathways disrupted in each subtype that likely serve as the basis for their differential tumor biology and clinical outcomes. Downregulation of  Lung cancer is the leading cause of cancer-related deaths worldwide and despite current treatments, prognosis remains poor, with a five year survival of <18% EGFR mutations in this subtype NSCLC is a heterogeneous disease with squamous cell carcinoma (SqCC) and adenocarcinoma (AC) being the predominant histological subtypes. Traditionally, these subtypes have been treated as a single disease entity with treatment strategies determined solely by disease stage. However, recent evidence from clinical trials has demonstrated that histological subtypes of NSCLC respond differently to both targeted drugs and newly developed chemotherapies, possibly related to differences in cell derivation and pathogenetic origins SOX2 and TITF1/NKX2-1 have recently been identified as key events specific to the development of lung SqCC and AC, respectively Initial gene expression profiling studies were able to segregate AC and SqCC tumors into their respective histologic groupings based on multi-gene models; however, critical events in tumorgenesis may be masked by reactive changes when examining expression profiles alone in silico screening of candidate therapeutic compounds using subtype-specific pathway components, we show how these new findings may influence our approach to the clinical management of NSCLC.In this study, we performed the first large-scale analysis of primary NSCLC tumors , integrating high resolution DNA copy number, methylation and gene expression profiles to identify critical subtype-specific molecular features. The characterization of the genomic and epigenomic landscapes of AC and SqCC revealed an astounding number of differences at the DNA level with subsequent gene expression changes that are selected for during subtype-specific lung tumor development. Importantly, we identified key oncogenic pathways disrupted by these alterations that likely serve as the basis for differential behaviors in tumor biology and clinical outcomes. Lastly, through prognostic analysis and Carcinomas of all types are known to harbor many DNA-level alterations linked, in part, to carcinogen exposure p-values were corrected for multiple comparisons with a cut-off of ≤0.01 considered significant. In addition, we required regions to be altered in >20% of samples from a subtype group and a difference between groups of >10% to be considered “of interest”. Although the NSCLC subtypes exhibit similar levels of genomic instability, if specific genetic pathways are involved in their differential development, differences in the genomic alterations selected during tumorigenesis should be present. To determine if genetic alterations unique to each NSCLC subtype exist, we looked for recurrent non-random regions of aberration in each group. Samples were grouped by subtype and probes were aggregated into regions based on similar copy number status. The frequency of alteration across autosomes was determined and compared between subtypes using the Fisher's exact test and the resulting This analysis revealed 294 regions of copy number disparity between SqCC and AC, 205 of which were SqCC-specific, whereas 89 were AC specific . Althougin situ lesions, the assumed precursors to lung SqCC tumors, revealed that the vast majority  of SqCC-specific alterations are present at this stage of tumor development, suggesting that these events may commence early in SqCC tumorigenesis . For SqCC, 4669 unique genes mapped to the subtype-specific copy number alterations (∼23 genes per region), and 797 (17%) of these were differentially expressed between subtypes in the anticipated direction. In AC, 2050 unique genes were located in subtype-specific copy number alterations (∼23 per region) and 171 (8%) were differentially expressed between subtypes in the anticipated direction.Although some genes overlapped, their disruption patterns were specific to the individual cancer type, suggestive of opposing roles (oncogenic vs tumor suppressive) depending on cellular context. Thus, these genes were also considered to be subtype-specific targets. When combined, the SqCC and AC subtype-specific copy number regulated candidates represented 968 unique genes and showed a clear distinction in expression levels between the two subtypes.In addition to demonstrating a relationship between expression and copy number alteration, a candidate subtype-specific gene was also required to be deregulated in cancer tissues relative to normal tissue Cellular pathways and processes specifically disrupted in individual subtypes may reveal key oncogenic mechanisms driving the differential development of AC and SqCC. Thus, after identifying the genes responsible for the differences between the subtypes, we next wanted to investigate their biological functions. To discover subtype-related networks of biologically related genes we performed Ingenuity Pathway Analysis (IPA) of the 71 AC and 447 SqCC specific target genes . SqCCs ep<0.0001).Unlike the genome, which is identical for most normal cells in the body, the epigenome differs between tissue types To determine whether these trends were evident in tumor-specific epigenetic alterations, , we compared the differential methylation profiles of AC and SqCC tumors. These profiles were generated by subtracting the average normal DNA methylation profile for the references from each of the 30 AC and 13 SqCC tumors, respectively. Similar to the global assessment of copy number alterations (gain and loss), there were no significant differences between the AC differential profiles and the SqCC differential profiles  in CpG iAlthough no differences in global methylation changes were observed between subtypes, the two subtypes may possess differential alteration frequencies at individual loci. To determine whether AC and SqCC tumors possess locus-specific differences in DNA methylation, we examined the frequencies of methylation alteration at gene-associated loci in both subtypes. Tumor DNA methylation levels were compared to the average of available normal reference tissue profiles. The frequency of probe hypermethylation and hypomethylation  in AC and SqCC samples was compared using the Fisher's exact test. Following correction for multiple comparisons, 2708 probes corresponding to 2384 genes were found to be differentially methylated (p≤0.05). The SqCC group contained markedly more recurrently hyper- and hypomethylated loci than the AC group, similar to the disparity in the numbers of subtype-specific copy number-regulated genes observed in the analysis of genomic alterations. In fact, only 8% of the 2708 significant probes were more frequently altered in AC, the rest being more commonly hyper- or hypomethylated in SqCC.p<0.05. 32 AC candidate genes and 297 SqCC genes met these stringent criteria and were further analyzed as subtype-specific epigenetically regulated genes . As expected, we found that EZH2 was expressed at a significantly higher level in SqCC tumors than AC tumors, demonstrating the biological consequence of E2F1 disruption  analysis on a large, independent panel of >200 lung tumors. Protein levels for three subtype specific genes with available antibodies validated for IHC were analyzed: ERCC1 (inactivated in AC), KEAP1 (inactivated in AC) and SOX2 (activated in SqCC) . AverageDSG2, PLAC2, ATP9A, TPM4, CD9 and PSMD11) were significantly associated with survival in both subtypes; however, they displayed a completely opposite pattern in terms of survival with low expression associated with poor survival in one subtype and high expression with poor survival in the other  in general. To test this, we determined the survival associations using a Mantel-Cox log rank test for each of the 778 subtype specific genes in AC, SqCC and NSCLC in the dataset with overall survival information available  that were significantly correlated with both the AC and SqCC specific gene signatures identified in our study  is known to occur more frequently in SqCC than AC Lastly, after defining and validating our AC and SqCC specific cancer genes, we applied these findings to define potential treatment strategies tailored to each lung cancer subtype. To do this, we queried the Connectivity Map (CMAP) database using our subtype specific genes to identify compounds that could “reverse” the expression direction of each signature. The CMAP consists of thousands of gene expression profiles from different cancer cell lines treated with a vast collection of small molecules ur study . Remarkain silico analysis, SqCC cell lines (average IC50 = 69 nM) were, on average, five times more sensitive to Trichostatin A than AC cell lines (average IC50 = 346 nM), a statistically significant difference   with the HDAC inhibitor Trichostatin A, which was the most significantly negative correlated HDAC inhibitor from the SqCC analysis. Importantly, we selected the available cell lines that best represented their respective clinical tumor subtypes by performing principle component analysis with the subtype-specific genes using publically available gene expression profiles for a large panel of NSCLC cell lines . As predicted by the  U Test) .The emergence of tumor cells from normal precursors is thought to involve a complex interplay between genetics and cell lineage In this study, we provide the first comprehensive investigation of the key genetic and epigenetic alterations distinguishing AC and SqCC lung tumors. We achieved this by integrating whole-genome DNA copy number, DNA methylation, and gene expression data to identify genes altered in a subtype-specific manner. These genes are associated with distinct gene networks in each lung cancer subtype, flagging distinct signaling pathways as contributing to tumorigenesis. We also found subtype-type specific changes to be correlated with clinical outcomes and highlight putative treatment strategies based on the subtype specific molecular signatures.SOX2 and BRF2 were identified, validating our approach NOTCH3 and FOXM1 were overexpressed through increased gene dosage specifically in SqCC while the tumor suppressor KEAP1 was deleted and underexpressed specifically in AC The 294 subtype-specific copy number alterations detected in this study demonstrate that different genetic pathways are involved in the pathogenesis of AC and SqCC. Importantly, previously identified lineage specific oncogenes including ASF1B, PRMT1, SAE1, SET8, CHAF1A and UHRF1 may drive this phenomenon and play a key role during the development of lung SqCC. As histone modifications also play an essential role in DNA replication, there may be a synergistic effect between the histone modifying genes and replication/recombination associated genes that contribute to tumor development. Interestingly, histone modification alterations occur more frequently in lung SqCC than AC, consistent with our findings A broader gene network-based analysis of the copy number-regulated genes revealed additional insights into the differential oncogenic mechanisms driving the pathogenesis of AC and SqCC. The top SqCC gene network was mainly associated with DNA replication, recombination and repair. In addition to these functions, histone modification genes were represented as well. Histones are fundamental building blocks of eukaryotic chromatin and are involved in myriad cellular processes, including replication, repair, recombination and chromosome segregation HNF4α leads to increased cellular proliferation, progression and dedifferentiation HNF4α may act as a tumor suppressor in epithelial carcinogenesis HNF4α was not affected, we found that numerous downstream targets of this gene are downregulated specifically in AC. Thus, this may have the same net affect as inactivation of HNF4α itself and lead to increased cellular proliferation during AC tumorigenesis.The gene network detected as perturbed in AC subtype tumors contained genes mainly involved in regulating tissue development and cell-to-cell signaling and known to be targeted by the transcription factor HNF4α. HNF4α regulates a large set of genes in a cell-specific manner and is necessary for cell differentiation and maintenance of a differentiated epithelial phenotype E2F1, was found to exhibit SqCC-specific hypomethylation and overexpression. E2F1 is upregulated in SCLC tumors EZH2, an oncogenic polycomb histone-methyltransferase EZH2 expression is significantly higher in SqCC than AC  and SqCC (activated) tumors may be an important consideration in determining the efficacy of this treatment against lung cancer subtypes Importantly, numerous genomic regions showed opposite patterns of alteration in each lung cancer subtype. For example, a discrete alteration spanning 2.4 Mbp on chromosome bands 8p12-p11.23 was commonly gained in SqCC and lost in AC, implying that genes in these regions may play opposite roles during the development of the individual NSCLC subtypes, acting as TSGs in AC and as oncogenes in SqCC. Such diametric alteration is seen when including epigenomic alterations as well. This is the case for ber loss . This inCD9 was gain/overexpressed in SqCC and high expression of this gene correlated with favorable survival in this subtype as well  is known to occur more frequently in SqCC than AC the finding of multiple PI3K/mTOR inhibitors as potential therapeutics for SqCC is also logical Furthermore, in order to demonstrate how these findings can be used to define treatment strategies tailored to the individual lung cancer subtypes, we performed CMAP analysis using our AC and SqCC specific gene signatures to identify compounds that can potentially reverse the expression of these genes. Although the results for AC were uninformative, the SqCC CMAP analysis identified numerous HDAC and PI3K/mTOR inhibitors as compounds that could potentially induce a gene expression signature negatively correlated with that associated with SqCC . The HDALastly, it is important to note that although they display broadly unifying characteristics, AC and SqCC themselves are very heterogeneous tumor types, with many molecular, pathologic and clinical subtypes Fundamental discrepancies in tumor biology may be a primary factor determining the differential outcomes of lung cancer patients. Biological differences that segregate with cell lineages may also lead to differences in response to therapies All patient samples were collected under informed, written patient consent and anonymized as approved by the University of British Columbia–British Columbia Cancer Agency Research Ethics Board (REB number H04-60060).Formalin-fixed, paraffin embedded (FFPE) and fresh-frozen tissues were collected from St. Paul's Hospital, Vancouver General Hospital and Princess Margaret Hospital following approval by the Research Ethics Boards. Hematoxylin and eosin stained sections for each sample were graded by a lung pathologist for use in selecting regions for microdissection. DNA was isolated using standard procedure with proteinase K digestion followed by phenol-chloroform extraction as previously described Array hybridization was performed as previously described SoftWoRx Tracker Spot Analysis software . Systematic biases were removed from all array data files using a stepwise normalization procedure as previously described SeeGH software was used to combine replicates and visualize all data as log2 ratio plots aCGH-SmoothHybridized arrays were imaged using a charge-coupled device (CCD) camera system and analyzed using p-value of ≤0.05 were used. When comparing tumor samples (AC/SqCC) and normal non-malignant samples , probes were deemed aberrantly methylated if the absolute difference between tumor and the average of the appropriate normal samples was ≥0.15.For 30 AC samples, 30 patient-matched non-malignant lung samples, 13 SqCC samples and 18 non-patient matched bronchial epithelia samples , DNA methylation profiling was performed using the Illumina HumanMethylation27 chip. Five hundred nanograms of DNA from each sample were analyzed by this technology. Normalized β-values were obtained and only those with a detection R statistical computing environment on a 3×2 contingency table as previously described, generating a p-value for each probe Regions of differential copy number alteration between AC and SqCC genomes were identified as follows. Each array element was scored as 1 (gain/amplification), 0 , or −1 (loss/deleted) for each individual sample. Values for elements filtered based on quality control criteria were inferred by using neighbouring probes within 10 Mb. Probes were then aggregated into genomic regions if the similarity in copy number status between adjacent probes was at least 90% across all samples from the same subtype. The occurrence of copy number gain/amplification, loss/deletion, and retention at each locus was then compared between AC and SqCC data sets using the Fisher's exact test. Testing was performed using the p-value cut-off of p<0.05 was used to deem a probe differentially methylated between the two groups.A similar approach was used for determining subtype-specific DNA methylation alterations. Frequencies of hypermethylation and hypomethylation for each probe were compared using a Fisher's exact test, followed by a Benjamini-Hochberg multiple testing correction. A corrected Gene Expression Omnibus were used: Affymetrix U133 Plus 2 expression data was downloaded for accession numbers GSE3141 Fresh-frozen lung tumors were obtained from Vancouver General Hospital as described above. Microdissection of tumor cells was performed and total RNA was isolated using RNeasy Mini Kits . Samples were labeled and hybridized to a custom Affymetrix microarray according to the manufacture's protocols . In addition, RNA was obtained from exfoliated bronchial cells of lung cancer free individuals obtained during fluorescence bronchoscopy ranksum function in Matlab. As the direction of gene expression difference was predicted to match the direction of copy number difference, one tailed p-values were calculated. A Benjamini-Hochberg multiple hypothesis testing correction was applied based on the total number of gene expression probes analyzed for each region. Probes with a corrected p-value≤0.001 were considered significant. If multiple probes mapped to the same gene, the one with the lowest p-value was used. Resulting genes were then mapped to the corresponding probes on the Affymetrix U133 Plus 2 array in order to compare their expression in a second set of NSCLC tumors (GSE3141 above) against normal bronchial epithelial cells. If multiple probes were present for a gene, the one with the strongest p-value was used. All comparisons were performed using a one-tailed t-test with unequal variances in Excel and genes with a p<0.001 were considered significant. The fold-change for tumors versus normal tissues was then determined in order to determine genes expressed in the direction predicted by copy number.Gene expression probes were mapped to March 2006 (hg 18) genomic coordinates and those within the regions of copy number difference between the subtypes were determined. Comparisons between expression levels for AC and SqCC tumors were performed using the Mann-Whitney U test and computed with the GraphPad Prism software.Principle component analysis was performed using expression data for the three independent tumor data sets (described above) in MATLAB. All genes of interest with probes on the corresponding arrays were used. Briefly, the first and second principal components were generated from the original dataset. In the subsequent validation in secondary datasets, these principal components are then used to weight the expression data for a gene based on the original distribution. The Receiver Operating Characteristic (ROC) area under the curve (AUC) analysis was performed to determine the ability of principle component 1 to separate the AC and SqCC samples into their appropriate histological groups. Calculations were performed using the http://www.broad.mit.edu/cmap/) analysis was performed using the up and downregulated genes specific to each subtype as previously described Connectivity Map . AC and SqCC specific gene lists were imported as individual experiments using the Core Analysis tool. The analysis was performed using Ingenuity Knowledge Database with the Affymetrix U133 Plus 2 platform as the reference set and was limited to direct and indirect relationships.Functional identification of gene networks and canonical signalling pathways was performed using Ingenuity Pathway Analysis program  from surgically resected lung cancer specimens from the Lung Cancer Specialized Program of Research Excellence TissueBank at The University of Texas M.D. Anderson Cancer Center. We used archived, formalin-fixed, paraffin-embedded (FFPE) tumor tissue samples placed in tissue micro-array (TMA). The tumor tissue samples were collected between 1997 and 2003, and were histologically analyzed and classified using the 2004 WHO classification system The immunohistochemical analysis was done using commercially available antibodies against KEAP1 , ERCC1  and SOX2 . Immunohistochemical staining was done using an automated stainer  with 5-µm-thick TMA sections from FFPE tissues. Tissue sections were deparaffinized and hydrated. Antigen retrieval was done in pH 6.0 citrate buffer in a decloaking chamber  and washed on Tris buffer. Peroxide blocking was done at ambient temperature with 3% H2O2 in methanol. The slides were incubated with primary antibody  at ambient temperature and washed with Tris buffer, followed by incubation with biotin-labeled secondary antibody for 30 minutes . The immunostaining was developed with 0.5% 3,3′- diaminobenzidine, freshly prepared with imidazole-HCl buffer (pH 7.5) containing hydrogen peroxide and an antimicrobial agent (Dako) for 5 minutes, and then the slides were counterstained with hematoxylin, dehydrated, and mounted.Nuclear ERCC1, cytoplasmic KEAP1, and nuclear SOX2 expressions were quantified using a four-value intensity score  and the percentage (0–100%) of the extent of reactivity. An immunohistochemical expression score was obtained by multiplying the intensity and reactivity extension values , and these expression scores were used to determine expression levels.50 values were generated in Graph Pad v5 using the proportionate response of all 20 drug concentrations. Experiments were repeated in quadruplicate and differences in IC50 values were determined using a student's t-test with a p-value<0.05 considered significant.The effect of HDAC inhibitor Trichostatin A,  on six NSCLC cell lines; three adenocarcinoma  and three SqCC lines  was assessed by cell viability assays. Cells were plated in triplicate in 96 well plates at optimal densities for growth . Cells were subjected to a series of 2-fold dilutions of Trichostatin A prepared in cell growth media and DMSO. The experimental inhibitor concentrations ranged from 100 uM to 109 pM and the final DMSO concentration for treated and untreated (control) cells was 1%. Blank wells contained equal volumes of growth media with 1% DMSO. Cells were incubated for 72 hours at 37°C and then treated with 10 µl of Alamar Blue cell viability reagent  according to manufacturer's instructions. The reaction product was quantified by measuring absorbance at 570 nm with reference to 600 nm using an EMax plate reader . The response of treated cells was measured as a proportion of the viability of untreated cells, with the mean background subtracted treatment absorbance divided by the mean background subtracted untreated absorbance for each inhibitor concentration. Dose response curves and ICFigure S1Principle component analysis of NSCLC cell lines using subtype-specific genes.(TIF)Click here for additional data file.Table S1Clinical Samples used in Analyses.(DOCX)Click here for additional data file.Table S2List of copy number alterations specific to each subtype.(XLSX)Click here for additional data file.Table S3SqCC-Specific Copy Number Alteration Present in CIS Cases.(XLSX)Click here for additional data file.Table S4Genes deregulated by copy number specifically in each subtype.(XLSX)Click here for additional data file.Table S5IPA network analysis of subtype specific copy number alteration target genes.(XLSX)Click here for additional data file.Table S6Genes deregulated by DNA methylation in each subtype.(XLSX)Click here for additional data file.Table S7IPA network analysis of subtype specific methylation target genes.(XLSX)Click here for additional data file.Table S8Genes disrupted by gene-dosage in one subtype and DNA methylation in the other.(XLSX)Click here for additional data file.Table S9Genes demonstrating concerted genetic, epigenetic and transcriptional disruption in SqCC.(XLSX)Click here for additional data file.Table S10Survival analysis of subtype-specific genes.(XLSX)Click here for additional data file.Table S11CMAP analysis of subtype-specific genes.(XLS)Click here for additional data file.