Replication-induced DNA damage after PARP inhibition causes G₂ delay, and cell line-dependent apoptosis, necrosis and multinucleation

<p>PARP inhibitors have been approved for treatment of tumors with mutations in or loss of <i>BRCA1/2</i>. The molecular mechanisms and particularly the cellular phenotypes resulting in synthetic lethality are not well understood and varying clinical responses have been observed. We have investigated the dose- and time-dependency of cell growth, cell death and cell cycle traverse of 4 malignant lymphocyte cell lines treated with the PARP inhibitor Olaparib. PARP inhibition induced a severe growth inhibition in this cell line panel and increased the levels of phosphorylated H2AX-associated DNA damage in S phase. Repair of the remaining replication related damage caused a G₂ phase delay before entry into mitosis. The G₂ delay, and the growth inhibition, was more pronounced in the absence of functional ATM. Further, Olaparib treated Reh and Granta-519 cells died by apoptosis, while U698 and JVM-2 cells proceeded through mitosis with aberrant chromosomes, skipped cytokinesis, and eventually died by necrosis. The TP53-deficient U698 cells went through several rounds of DNA replication and mitosis without cytokinesis, ending up as multinucleated cells with DNA contents of up to 16c before dying. In summary, we report here for the first time cell cycle-resolved DNA damage induction, and cell line-dependent differences in the mode of cell death caused by PARP inhibition.</p

CSPP-L Associates with the Desmosome of Polarized Epithelial Cells and Is Required for Normal Spheroid Formation

<div><p>Deleterious mutations of the Centrosome/Spindle Pole associated Protein 1 gene, <i>CSPP1</i>, are causative for Joubert-syndrome and Joubert-related developmental disorders. These disorders are defined by a characteristic mal-development of the brain, but frequently involve renal and hepatic cyst formation. CSPP-L, the large protein isoform of <i>CSPP1</i> localizes to microtubule ends of the mitotic mid-spindle and the ciliary axoneme, and is required for ciliogenesis. We here report the microtubule independent but Desmoplakin dependent localization of CSPP-L to Desmosomes in apical-basal polarized epithelial cells. Importantly, siRNA conferred depletion of CSPP-L or Desmoplakin promoted multi-lumen spheroid formation in 3D-cultures of non-ciliated human colon carcinoma Caco-2 cells. Multi-lumen spheroids of <i>CSPP1</i> siRNA transfectants showed disrupted apical cell junction localization of the cytoskeleton organizing RhoGEF ECT2. Our results hence identify a novel, non-ciliary role for CSPP-L in epithelial morphogenesis.</p></div

New distinct compartments in the G₂ phase of the cell cycle defined by the levels of γH2AX.

<p>Induction of DNA double strand breaks leads to phosphorylation and focus-formation of H2AX. However, foci of phosphorylated H2AX (γH2AX) appear during DNA replication also in the absence of exogenously applied injury. We measured the amount and the number of foci of γH2AX in different phases of the cell cycle by flow cytometry, sorting and microscopy in 4 malignant B-lymphocyte cell lines. There were no detectable γH2AX and no γH2AX-foci in G₁ cells in exponentially growing cells and cells treated with PARP inhibitor (PARPi) for 24 h to create damage and reduce DNA repair. The amount of γH2AX increased immediately upon S phase entry, and about 10 and 30 γH2AX foci were found in mid-S phase control and PARPi-treated cells, respectively. The γH2AX-labeled damage caused by DNA replication was not fully repaired before entry into G₂. Intriguingly, G₂ cells populated a continuous distribution of γH2AX levels, from cells with a high content of γH2AX and the same number of foci as S phase cells (termed “G₂H” compartment), to cells that there were almost negative and had about 2 foci (termed “G₂L” compartment). EdU-labeling of S phase cells revealed that G₂H was directly populated from S phase, while G₂L was populated from G₂H, but in control cells also directly from S phase. The length of G₂H in particular increased after PARPi treatment, compatible with longer DNA-repair times. Our results show that cells repair replication-induced damage in G₂H, and enter mitosis after a 2–3 h delay in G₂L.</p

Depletion of CSPP-L and Desmoplakin cause multi-lumen spheroid formation in Caco-2 spheroids.

<p>(A) Localization of CSPP-L (a-CSPP-L, green), filamentous actin (Phalloidin, white), E-cadherin (a-E-cadherin, red), and DNA (blue) during different stages of spheroid development of Caco-2 cells. Cells were grown in 3D-Matrigel culture and formalin fixed for IF. Images show projections of z-sections enclosing the entire lumen volume. CSPP-L shows prominent enrichment juxtapose to the apical filamentous actin throughout all stages of spheroid development (B) The apical CSPP-L staining pattern (a-CSPP-L, green) is CSPP1 siRNA sensitive and not altered by Desmoplakin depletion (a-α-tubulin, red; phalloidin, white). CSPP1 and Desmoplakin siRNA Caco-2 transfectants develop disorganized cell aggregates with multiple lumen.</p

CSPP-L localizes to the Desmosome.

<p>IF of CSPP-L (a-CSPP-L, green) in the basal-like breast cancer cell line HCC1937 showed co-localization with the desmosomal protein Desmoplakin (A, a-DSP, red; upper panel) but not with the AJ associated protein β-catenin (B, a-CTNNB1, red; lower panel), as tested by co-localization analysis (linear dependence, Pearson’s coefficient). (C-E) 3D-super-resolution microscopy refined the localization of CSPP-L to single patches that frame Desmoplakin at the cell junction (C) and do not co-localize with β-catenin (D). Higher magnification image and line plots of signal intensities across individual desmosomes at cell junctions (E). Dashed line in overview image connects peaks in Desomplakin signals. Line plots show signal intensities along an 800nm line across desmosomes depicted below (dashed line in magnifications, scale bars magnifications = 200nm). Average distances (with standard deviation) of peak intensities of CSPP-L patches and Desmoplakin (26 cells with non-separated and eleven cells with separated Desmoplakin signals).</p

Microtubule independent localization of Desomoplakin and CSPP-L to the Desmosome.

<p>(A) 3D-super-resolution microscopy of CSPP-L (green) and MTs (α-tubulin, red). MTs are infrequently observed to localize with MT (+)-ends at junctional CSPP-L pairs (i), but predominantly align parallel to the cell cortex (ii). (B) Cell junction staining of CSPP-L (green) and Desmoplakin (red, right panel) in HCC1937 cells is resistant to nocodazole induced MT de-polymerization (α-tubulin, red, left panel). (C) MTs are not required for recruitment of Desmoplakin and CSPP-L to forming cell junctions in a calcium switch assay in HCC1937 cells.</p

Mis-localization of ECT2 in CSPP1 depleted multi-lumen Caco-2 spheroids.

<p>Spheroids of Caco-2 cells transfected with indicated siRNAs were stained for ECT2 (a-ECT2, green), the filamentous actin (Phalloidin, white) and α-tubulin (a-α-tubulin,red). ECT2 localized to apical cell-cell junctions in single-lumen spheroids of siGFP control transfectants and mal-organized spheroids of Desmoplakin depleted cells. The apical cell-cell junction ECT2 staining pattern is lost in multi-lumen spheroids of CSPP-L depleted cells. Bar diagram shows frequency of spheroids with lost or strongly reduced junctional ECT2 staining (100 spheroids per treatment scored in two experiments, error bars depict SEM, statistical significance was tested in paired t-test).</p

Desmoplakin is required for CSPP-L localization to the Desmosome.

<p>IF (A-C) and immunoblotting of total cell lysates (D) of HCC1937 cells treated with indicated siRNAs. Cells were transfected in low calcium medium. 72hrs post-transfection cells were allowed to form cell-cell junctions for 40 min by change to pre-warmed, normal calcium medium. Cell-cell junction staining of CSPP-L (green in overlay image) is siCSPP1 sensitive (A). Depletion of CSPP-L does not impair cell-cell junction localization of Desmoplakin (A, red) or β-catenin (B, red). Depletion of Desmoplakin (C, red) results in loss of CSPP-L (green) staining at cell-cell junctions. Knockdown efficacy was monitored by immunoblotting for Desmoplakin, CSPP-L and compared to the loading control γ-tubulin (D).</p

Apical-basal polarity is not disrupted in CSPP-L and Desmoplakin depleted multi-lumen Caco-2 spheroids.

<p>(A) Quantification of the multi-lumen phenotype in Caco-2 siRNA transfectants (siGFP, siCSPP1, siCSPP1 <i>s</i>mart <i>p</i>ool, siDSP). The bar diagram shows average of two experiments, error bars depict standard deviation. Statistical significance was tested by paired t-test. (B) CSPP-L and Desmoplakin depletion in Caco-2 spheroids was validated by immunoblotting (right panel). (C) Multi-lumen spheroids in CSPP1 and Desmoplakin depleted cells depict filamentous actin stabilization indicated by solid arrow heads (Phalloidin, white) and PKCζ enrichment (a-PKCζ, red) at the lumen facing apical membrane. Occasional weak PKCζ staining at the basal-side of outer-rim cells is seen in <i>siCSPP1</i> and <i>siDSP</i> transfectants (open arrowheads). (D) Centrosomes (a-Pericentrin, green) positioned in the lumen oriented cytoplasm (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134789#pone.0134789.s003" target="_blank">S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134789#pone.0134789.s004" target="_blank">S2</a>, and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134789#pone.0134789.s005" target="_blank">S3</a> Videos).</p

Internalization.

<p>Representative images of internalization of HH1 and Rituximab in Ramos cells after incubation with 10 μg/ml of HH1 or 20 μg/ml rituximab at 4°C or 37°C for 1 hour or 19 hours, respectively. HH1-DOTA bound to Alexa Fluor 488 is shown in green, Rituximab bound to Alexa Fluor 647 is shown in magenta and Hoechst 33342 bound to DNA in the cell nucleus is shown in blue. 20 to 40 cells were scanned for each treatment.</p