Inhibition of PCAF by Anacardic Acid Derivative Leads to Apoptosis and Breaks Resistance to DNA Damage in BCR-ABL-expressing Cells

Acetylation of histones and nonhistone proteins is a posttranslational modification which plays a major role in the regulation of intracellular processes involved in tumorigenesis. It was shown that different acetylation of proteins correlates with development of leukemia. It is proposed that histone acetyltransferases (HATs) are important novel drug targets for leukemia treatment, however data are still not consistent. Our previous data showed that a derivative of anacardic acid - small molecule MG153, which has been designed and synthesized to optimize the HAT inhibitory potency of anacardic acid, is a potent inhibitor of p300/CBP associated factor (PCAF) acetyltransferase. Here we ask whether inhibition of PCAF acetyltransferase with MG153 will show proapoptotic effects in cells expressing BCR-ABL, which show increased PCAF expression and are resistant to apoptosis. We found that inhibition of PCAF decreases proliferation and induces apoptosis, which correlates with loss of the mitochondrial membrane potential and DNA fragmentation. Importantly, cells expressing BCR-ABL are more sensitive to PCAF inhibition compared to parental cells without BCR-ABL. Moreover, inhibition of PCAF in BCR-ABL-expressing cells breaks their resistance to DNA damage-induced cell death. These findings provide direct evidence that targeting the PCAF alone or in combination with DNA-damaging drugs shows cytotoxic effects and should be considered as a prospective therapeutic strategy in chronic myeloid leukemia cells. Moreover, we propose that anacardic acid derivative MG153 is a valuable agent and further studies validating its therapeutic relevance should be performed

The role of nibrin in Doxorubicin-induced apoptosis and cell senescence in nijmegen breakage syndrome patients lymphocytes

Nibrin plays an important role in the DNA damage response (DDR) and DNA repair. DDR is a crucial signaling pathway in apoptosis and senescence. To verify whether truncated nibrin (p70), causing Nijmegen Breakage Syndrome (NBS), is involved in DDR and cell fate upon DNA damage, we used two (S4 and S3R) spontaneously immortalized T cell lines from NBS patients, with the founding mutation and a control cell line (L5). S4 and S3R cells have the same level of p70 nibrin, however p70 from S4 cells was able to form more complexes with ATM and BRCA1. Doxorubicin-induced DDR followed by cell senescence could only be observed in L5 and S4 cells, but not in the S3R ones. Furthermore the S3R cells only underwent cell death, but not senescence after doxorubicin treatment. In contrary to doxorubicin treatment, cells from all three cell lines were able to activate the DDR pathway after being exposed to γ-radiation. Downregulation of nibrin in normal human vascular smooth muscle cells (VSMCs) did not prevent the activation of DDR and induction of senescence. Our results indicate that a substantially reduced level of nibrin or its truncated p70 form is sufficient to induce DNA-damage dependent senescence in VSMCs and S4 cells, respectively. In doxorubicin-treated S3R cells DDR activation was severely impaired, thus preventing the induction of senescence

Structure and organisation of the human testis and seminiferous tubules.

<p><b>A.</b> Macroscopic appearance. The testis is divided into compartments (lobules) separated by connective tissue. There are an estimated 250 lobules per testis, which vary in size; for clarity only 7 lobules are illustrated here. <b>B.</b> (<b>i</b>) Diagrammatic representation of the highly convoluted organisation of 3 seminiferous tubules (blue, black, grey) within a lobule. (<b>ii</b>) A magnified cross section through the lobule in <b>i</b> reveals how the tubules would appear in thin microscopic sections. Dashed lines (above section) and solid lines (below section) join the contiguous tubules. <b>C.</b> Cross section through an individual seminiferous tubule. Spermatogonia, located at the basal lamina of the seminiferous tubules, comprise a heterogeneous population of diploid germ cells. These can be classified according to their morphologies and correspond to three main maturation stages: A_dark spermatogonia, which are considered to represent the reserve stem cell population; highly proliferating A_pale spermatogonia; and more mature B spermatogonia that give rise to primary spermatocytes. Primary spermatocytes undergo meiosis to form secondary spermatocytes that differentiate to form spermatids, which when fully formed are released into the central lumen as spermatozoa that progress to the rete testis. Spermatogenesis is supported by the presence of the somatic Sertoli cells. The vascular and connective tissue network lie external to the wall of the seminiferous tubule.</p

Proposed model linking the immunohistochemical observations to clonal expansion of PAE mutations in seminiferous tubules.

<p><b>A.</b> Tubule undergoing normal spermatogenesis: spermatogonia (grey) adjacent to basal lamina (blue line) proliferate and differentiate (arrows) to spermatozoa in the lumen. <b>B.</b> An activating PAE mutation arises in a single spermatogonial cell (red cell). <b>C.</b> Altered cellular signalling associated with the mutation confers a proliferative or survival advantage to the mutant cell, leading to clonal expansion of spermatogonia and relative enrichment of mutant sperm. The clonally expanded cells retain the immunohistochemical features of the originating cell (such as MAGEA4 and FGFR3) and may also have altered markers of signal activation (pAKT) - forming localised microclones (<b>i</b>) or expanding around the circumference and along the tubule (<b>ii</b>). Process (ii), compatible with the appearance of the immunopositive tubules identified in this study, would account for the distribution and number of mutant cells containing PAE mutations previously determined by DNA studies <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042382#pone.0042382-Qin1" target="_blank">[20]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042382#pone.0042382-Choi1" target="_blank">[21]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042382#pone.0042382-Choi2" target="_blank">[22]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042382#pone.0042382-DakouaneGiudicelli1" target="_blank">[24]</a>. <b>D.</b> In rare cases, additional mutational events may arise and lead to the formation of spermatocytic seminoma, possibly via an ISS intermediate state.</p

Immunopositive tubules in sample 2–1.

<p><b>A.</b> Under low power magnification of section 05, several clusters of two or more tubules with strong MAGEA4 staining are visible. <b>B.</b> Higher power magnification of the boxed region in <b>A.</b> Five tubules with stronger MAGEA4 staining (*) are distinguishable from their neighbouring tubules with normal levels of MAGEA4 staining. In normal tubules, MAGEA4 stains the nucleus and cytoplasm of the spermatogonia on the basal lamina. In the immunopositive tubules additional MAGEA4-positive cells are present, forming a double row. <b>C.</b> Serial sections spanning 115 µm display consistently stronger staining for MAGEA4, FGFR3 and pAKT. Clusters of MAGEA4 positive cells are present in the lumen of this immunopositive tubule. No differences in staining for OCT2, Ki67, SAGE1 or SSX are observed. H&E staining (section 02) shows that the tubule in the centre contains few spermatocytes and no spermatids, whereas the tubule on the right hand side contains both spermatocytes and spermatids. A higher resolution image of the H&E staining is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042382#pone.0042382.s004" target="_blank">Figure S4A</a>. Scale bar: 100 µm.</p

3D reconstruction of immunopositive tubules in sample 1–1.

<p><b>A.</b> (<b>i</b>) Five immunopositive tubular cross-sections (black arrows) in MAGEA4-stained section 51. The immunopositive tubules were followed by staining further sections at intervals of 4 or 6 slides (20 and 30 µm, respectively), until section 111. (<b>ii</b>). Although other immunopositive tubular cross-sections are present in section 111 (white arrows) and the intermediate sections, only those which could be traced back to section 51 (black arrows) were included in the reconstruction. Scale bars: 100 µm. <b>B.</b> 3D reconstruction of the immunopositive tubule. (<b>i</b>) The five immunopositive tubular cross-sections in section 51 were colour coded (green, pink, purple, blue and yellow arrows). The resolved 3D structure (with section 51 at top and section 111 at bottom) reveals that 4 of the 5 immunopositive cross-sections in section 51 (blue, purple, yellow and green) are part of the same tubule (joining where the colour margins blur together)(<b>ii</b>, <b>iii</b>, <b>iv</b>). Although they are in close proximity, it could not be demonstrated that the pink and green tubules are contiguous (<b>iv</b>). As the structure is highly convoluted, for clarity the scale of the <i>z</i>-axis has been increased 3-fold. A movie displaying the rotating 3D structure (shown to scale) is available as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042382#pone.0042382.s008" target="_blank">Video S1</a> (for description see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042382#pone.0042382.s007" target="_blank">Text S1</a>).</p

Examples of microclones in samples 2–1 and 3–1.

<p><b>A.</b> Microclone 2–1_C40, located away from the basal lamina, is positive for MAGEA4 and OCT2 (single cell identified <i>post hoc</i>) but not for FGFR3. <b>B.</b> Microclone 3–1_C1, located within the lumen of the tubule is positive for MAGEA4, FGFR3 and OCT2 (2 cells identified <i>post hoc</i>). Panels above the set of images show the markers used to stain the sections and the analysis of the results, using the same scheme as in legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042382#pone-0042382-g002" target="_blank">Figure 2</a>. Scale bars: 100 µm.</p

Examples of putative microclones with different antigenic profiles in samples 1–1 and 1–2.

<p><b>A.</b> MAGEA4 positive only (microclone no: 1–2_C1): nuclear immunoreactivity to MAGEA4 was identified independently (tagged with flag) in sections no.168, 171 and 175. This cellular cluster is negative for five additional markers as shown. Weaker, cytoplasmic MAGEA4 staining of primary spermatocytes is also present. <b>B.</b> MAGEA4, SSX, SAGE1, FGFR3 and Ki67 positive (microclone 1–1_C36). Additional FGFR3 positivity was determined <i>post hoc</i>. <b>C.</b> MAGEA4 and FGFR3 (microclone 1–1_C2): a large cluster of cells occupying the periphery of the tubule expresses MAGEA4 and FGFR3 on adjacent serial sections, but is negative for SSX, Ki67, SAGE1 and OCT2. Further screening was not possible because section 01 was the first section of the tissue block. <b>D.</b> SSX and SAGE1 (microclone 1–1_C41): one of the few examples where a microclone was negative for MAGEA4 expression. In this case, the cluster of cells is positive for SSX and SAGE1 only. The specificity of all markers including MAGEA4 is confirmed by their expression in spermatogonia situated at the periphery of the tubule (internal positive control). Scale bars: 100 µm. Tables above each figure display the antigenic profile (positive  =  coloured box with 1 (independent identification), or 0* (<i>post hoc</i> identification); negative  =  white box with 0) and length (pink bar) of the microclone. n.s: not stained. Cell counts for each positive section are also detailed.</p