Efficacy Endpoints in Clinical Trials in Actinic Keratosis

<p><b>Article full text</b></p><p><br></p><p>The full text of this article can be found here<b>. </b><a href="https://link.springer.com/article/10.1007/s13555-018-0247-0">https://link.springer.com/article/10.1007/s13555-018-0247-0</a></p><p></p><p><br></p><p><b>Provide enhanced content for this article</b></p><p><br></p><p>If you are an author of this publication and would like to provide additional enhanced content for your article then please contact <a href="http://www.medengine.com/Redeem/”mailto:[email protected]”"><b>[email protected]</b></a>.</p><p><br></p><p>The journal offers a range of additional features designed to increase visibility and readership. All features will be thoroughly peer reviewed to ensure the content is of the highest scientific standard and all features are marked as ‘peer reviewed’ to ensure readers are aware that the content has been reviewed to the same level as the articles they are being presented alongside. Moreover, all sponsorship and disclosure information is included to provide complete transparency and adherence to good publication practices. This ensures that however the content is reached the reader has a full understanding of its origin. No fees are charged for hosting additional open access content.</p><p><br></p><p>Other enhanced features include, but are not limited to:</p><p><br></p><p>• Slide decks</p><p>• Videos and animations</p><p>• Audio abstracts</p><p> </p><p>• Audio slides</p> <p> </p

Genome organization of RnPV2, MmuPV1 variant, and AsPV1.

<p>Boxes indicating PV genes are drawn to scale and genes are drawn in three lines representing three putative open reading frames relative to nt position zero at the beginning of the upstream regulatory region. The polyadenylation sites are indicated with triangles.</p

Maximum Likelihood (ML) tree of papillomaviruses (PV).

<p>ML tree comprising a representative set of 79 types including our two novel types (AsPV1, RnPV2) and one variant (MmuPV1 variant), as inferred from predicted E6, E7, E1, E2, L2, and L1 aa sequence analysis (3,720 aa positions, of which 69% were parsimony-informative). Generic PV clades <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047164#pone.0047164-Bernard1" target="_blank">[11]</a> are indicated by Greek lettering. Supertaxa are colored red (Alpha+Omikron-PV), green (Beta+Xi-PV), blue (Delta+Zeta-PV), and ocher (Lamda+Mu-PV), respectively. Branch lengths are drawn to scale, with the scale bar indicating the number of nt substitutions per site. Numbers on branches are bootstrap support values under the ML criterion; values under 50 are not shown. The novel PV types and variant of our present study are highlighted by arrows. According to the common PV-nomenclature our novel variant of <i>Mus musculus</i> is referred to MmuPV1 variant.</p

Co-immunoprecipitation analyses of Bcl-x_AK with Bcl-2 family members.

<p>(A) SK-Mel-13 melanoma cells were transiently transfected with each 5 µg of pcDNA3 plasmids encoding Bcl-x_L, Bcl-x_AK, Bax or empty vector (Mock). Cells lysates were immunoprecipitated with microbeads covered with anti-Myc antibody, and immunoprecipitates were analysed by Western blotting. Non-bound supernatants (S) were compared with the immunoprecipitated pellet fractions (P). Antibodies for immunodetection: anti- Myc, Bcl-2, Bax and Bad. The complete experiment was performed two times, which both gave the same result. (B) A model for apoptosis induction by Bcl-x_AK is suggested. It is based on mitochondrial translocation of Bcl-x_AK and activation of Bax/Bak. Bcl-2/Bcl-x_L prevent Bax/Bak activation but not Bcl-x_AK translocation. BH3-only proteins may mediate a BH3 domain-dependent pathway via inactivation of antiapoptotic Bcl-2 proteins and may also drive a BH3-independent pathway analogous to Bcl-x_AK (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034549#s4" target="_blank">discussion</a> part).</p

Bax and Bak activation after Bcl-x_AK overexpression.

<p>(A) For investigation of Bax and Bak clustering, DU145 cells stably transfected for expression of EGFP-Bax or EGFP-Bak were transduced with AdV-AK and cultured for 48 h under OFF or ON conditions. Doxorubicin-treated cells (2 µM, 24 h) were used as positive controls. Examples of fluorescence microscope images taken at 48 h after transduction and promoter induction are shown. (B) A quantitative evaluation of Bax and Bak clustering was performed (means and SDs of triplicate values of a representative experiment). A second experiment revealed comparable results. (C) Bax and Bak activation upon Bcl-x_AK expression was determined in Mel-2a at 48 after AdV-AK transduction (50 MOI), by flow cytometry after staining with conformation-specific antibodies against Bax and Bak N termini (Bax/Bak NT). The bars indicate the populations counted as positive for activated Bax and Bak, respectively. (D) A quantification of triplicate values (one experiment of two independent) is shown. Transduction with AdV-Nbk (50 MOI) is shown for comparison.</p

Bcl-x_AK-mediated apoptosis depends on Bax or Bak.

<p>(A, C) Expression of Bax and Bak is shown by Western blot analysis in subclones of HCT116 and DU145, respectively. Equal loading was confirmed by incubation with β-actin. Two independent series of protein extracts revealed largely comparable expression. (B) HCT116 parental cells (Bax⁺/Bak⁺) as well as subclones (Bax⁻/Bak⁺), (Bax⁺/Bak⁻) and (Bax⁻/Bak⁻) were transduced with AdV-AK (MOI=50) and cultured under OFF or ON conditions. Relative DNA fragmentation values (apoptosis ELISA) were normalized according to the values of parental cells under OFF conditions (set to 1). (D) DU145 parental cells (Bax⁻/EGFP-Bak⁻) as well as subclones (Bax⁻/EGFP-Bak⁺) and (EGFP-Bax⁺/EGFP-Bak⁻) were transduced with AdV-AK (MOI=50, 100) and cultured under OFF or ON conditions. The percentages of apoptotic cells (sub-G1 populations) are shown, as determined by flow cytometry at 48 h after transduction. (B, D) Means and SDs of triplicate values of a representative experiment are shown (each two independent experiments). Statistical significance as determined by Student's t-test is indicated by asterisks (*, p<0.05; **, p<0.005), when comparing parental cells and subclones under ON conditions.</p

Bcl-2 and Bcl-x_L block the proapoptotic effects of Bcl-x_AK.

<p>(A) Subclones of A-375 cells stably transfected with pIRES-Bcl-2 (A375-Bcl-2) or mock-transfected (A375-Mock) were transduced with AdV-AK under OFF or ON conditions. Non-transduced cells (−) were used as additional controls. Numbers of apoptotic cells (sub-G1 cell populations) were determined by flow cytometry after PI staining. (B) SK-Mel-13 melanoma cells were transiently transfected with either Bcl-x_L or Bcl-x_AK alone or with a combination of both (each 2.5 µg plasmid-DNA). Relative DNA fragmentation values, as determined at 24 h and 48 h after transfection, were calculated with respect to cells that had received only the transfection lipid (white bars). (A, B) Means and SDs of triplicate values of a representative experiment are shown (each two independent experiments). Overexpression of Bcl-2, Bcl-x_L and Bcl-x_AK, as determined by Western blot analyses, is shown in the insets. (C) The mitochondrial membrane potential (Δψ_m) was determined by flow cytometry after TMRM staining in A375-Mock and in A375-Bcl-2 at 24 h and 48 h. After transduction with AdV-AK, inducible and non inducible conditions were compared (On/Off). The experiment was performed three times, giving comparable results.</p

Bcl-2 blocks Bcl-x_AK-mediated cytochrome c release and Bax translocation.

<p>Mel-2a, A375-Mock and A375-Bcl-2 cells were transduced with AdV-AK (MOI=50) and were kept under OFF and ON conditions. At 24 h and 48 h, cytosolic fractions (Cyto) and mitochondrial fractions (Mito) were isolated and analysed by Western blotting. Non-transfected controls (−) are shown as controls. The whole experiment was performed two times, resulting in highly comparable results. (A) Cytosolic extracts were analyzed for showing expression of Bcl-x_AK and release of cytochrome c. Mitochondrial extracts serve as positive controls, the mitochondrial protein VDAC ruled out any contaminations of cytosolic extracts with mitochondria, and β-actin served as loading control. (B) Mitochondrial extracts were analyzed for showing mitochondrial translocation of Bcl-2 proteins. Here, cytosolic extracts served as controls, equal protein loading was confirmed by VDAC and the relative purity of mitochondrial extracts was examined by GAPDH. 5% of the total mitochondrial fractions and 2% of the total cytosolic fractions had been loaded on the gels.</p

Activation of caspases and mitochondria.

<p>(A) Processing of caspase-3, -8 and -9 is shown in Mel-2a cells at 24 h and at 48 h after transduction with AdV-AK (MOI=50). Expression of Bcl-x_AK was switched on in the absence of doxycycline (ON) or shut off with doxycycline (OFF). Equal protein loading (20 µg/lane) was confirmed by GAPDH. The whole experiment was performed twice. (B) Inhibition of apoptosis by preincubation with the pancaspase inhibitor zVAD-fmk (1 h, 10 µM) is shown. SK-Mel-13 cells had been transduced with AdV-AK (MOI=100, 48 h). Means and SDs of triplicate values of a representative experiment (one of two) are shown. (C) Decrease of the mitochondrial membrane potential (Δψ_m) is shown for Mel-2a cells at 48 h after transduction of AdV-AK, as determined by flow cytometry after JC-1 or TMRM⁺ staining. Cultures with doxycycline (OFF, grey) are compared to cultures grown in the absence of doxycycline (ON, open graphs). The experiment was performed three times, resulting in highly comparable results. (D) ROS levels were determined in Mel-2a cells at 24 h and 48 h after transduction with AdV-AK under ON and OFF conditions (flow cytometry after H₂DCFDA staining). Below, parallel cultures were pre-treated for 1 h with 200 µM NAC before transduction. (E) Relative DNA-fragmentation rates (apoptosis) at 48 h with or without NAC were determined in parallel. Non-transduced cells (−/+NAC) are shown as additional controls (open bars). Values had been normalized with regard to non-treated controls, set to 1. Means and SDs of triplicate values of a representative experiment are shown (two independent experiments). (F) Expression levels of Bcl-2 proteins, of p53 and Survivin were determined by Western blot analysis in Mel-2a cells at 24 h and 48 h after transduction with AdV-AK (ON and OFF conditions). Equal protein loading (20 µg/lane) was confirmed by GAPDH.</p

Efficient induction of cell death by Bcl-x_AK.

<p>(A) The structure of the adenoviral construct AdV-AK is shown. The adenoviral E1 region was replaced by the Bcl-x_AK cDNA driven by a tetracyclin-responsive promoter (P_TRE), and the E3 region was replaced by the tetracyclin-controlled transactivator (tTA) driven by a CMV promoter (P_CMV). The tTA mediates Tet-OFF regulation. Striped boxes indicate the poly(A)+ regions. (B) Bcl-x_AK expression as determined by Western blot analysis is shown in melanoma cell lines SK-Mel-13, A-375 and Mel-2a at 48 h after transduction with AdV-AK (MOI=50). Cells had received doxycycline (OFF condition) or were left without (ON condition). Equal protein loading was confirmed by β-actin. (C) Left, examples of cell cycle analysis after PI staining indicating sub-G1 apoptotic cell populations in Mel-2a at 48 h of transduction. Middle panel, detached and rounded cells indicating apoptosis are shown of Mel-2a at 48 h after transduction with AdV-AK under OFF and ON conditions. Right panel, chromatin condensation and nuclear fragmentation were visualized by bisbenzimide (DAPI) staining in Mel-2a at 48 h after AdV-AK transduction (MOI=50). D–F) Time course analyses of apoptosis (D, flow cytometry after PI staining), cytotoxicity (E, LDH release) and cell proliferation (F, WST-1 assay) are shown for SK-Mel-13, A-375 and Mel-2a cells at 24, 48 and 72 h after transduction with AdV-AK (50 MOI, +Dox=Off, −Dox=On). As positive controls for induced cytotoxicity, cell lines were completely lysed by triton X-100 (T=100%) or were treated with doxorubicin (D, 500 nM, 72 h). WST-1 values are expressed as percent of non-treated controls (=100%). (G) For comparison, apoptosis induction (sub-G1 cells) by AdV-Nbk is shown for Mel-2a cells at 24 h, 48 h and 72 h (MOI=50). AdV-Nbk shares the same backbone with AdV-AK. For induction, doxycycline was omitted (On). (H) A time course analysis of Bcl-x_AK expression (3–48 h) after AdV-AK transduction and promoter induction is shown for Mel-2a, as determined by Western blot analysis. (I) Cell survival was determined according to calcein staining in Mel-2a cells at 48 h of Bcl-x_AK induction. A shift to the left indicates calcein-negative (=non viable) cells. (J) Quantification of the calcein experiment. (D, E, F, G, J) Means and standard deviations of triplicate values of representative experiments are shown. A luciferase-encoding adenovirus (Ad5-CMV-Luc) applied at the same MOI served as mock control (M), for controlling adenovirus transduction. All experiments were performed at least twice, resulting in highly comparable results.</p