Model for Ubp10 role on the modulation of PCNA ubiquitylation in <i>S. cerevisiae</i> cells.

<p>SUMOylated PCNA progress with the replisome at replication forks. Detection of bulky lesions on DNA impedes fork progression and induces Rad6/Rad18 ubiquitylation of PCNA; therefore, it enhances ubPCNA-TLS DNA polymerases interaction or further ubPCNA polyubiquitylation (by the Ubc13/Mms2/Rad5 ubiquitin ligase). After lesion bypass, Ubp10 deubiquitylates ubPCNA to allow remodelling of the replisome by switching back to replicative DNA polymerases, resuming rapid and processive DNA replication fork progression.</p

Cells lacking <i>UBP10</i> accumulate mono- and di-ubiquitylated PCNA in response to DNA damage and replicative stress.

<p>(A) A polyclonal rabbit antibody that specifically detects PCNA forms in yeast cell extracts. Immunoblot analysis with (affinity purified) rabbit α-PCNA antibody of TCA-protein extracts from wild-type, <i>rad18Δ</i> (unable to ubiquitylate PCNA), <i>pol30^K164R</i> (unable to ubiquitylate or SUMOylate PCNA), <i>mms2Δ</i> (unable to biubiquitylate PCNA) and <i>siz1Δ</i> (unable to SUMOylate PCNA) cells treated 90 minutes with 0.020% MMS and resolved in 10% or 12% polyacrylamide gels (as indicated), note that right lane of the 10% gel correspond to wild-type cells treated with 0.3% MMS (conditions where only SUMOylated PCNA forms are detected). (B) Di-ubiquitylated PCNA accumulation in MMS-treated single <i>ubp1</i> to <i>ubp17</i> deletions in <i>S.cerevisiae</i>. Graph of di-ubiquitylated PCNA accumulation in 0.020% MMS-treated single <i>UBP1-17</i> deletions in <i>S.cerevisiae</i>. Wild-type and single mutant cells exponentially grown at 30°C were treated 60 minutes with 0.020% MMS. TCA-cell extracts were analyzed for PCNA ubiquitylation by Western blot, quantitated and plotted. Average values from three independent assays are plotted. (C) Immunodetection of ubiquitylated forms of PCNA in wild-type, <i>ubp10Δ</i>, <i>pol30^K164R</i> and <i>ubp10Δ pol30^K164R</i> TCA-cell extracts to show that <i>UBP10</i> mutant cells accumulate K164 mono-ub and di-ubPCNA forms. Immunodetection of mono-ubiquitylated (D) and di-ubiquitylated PCNA (E) in wild-type and <i>ubp10Δ</i> cells treated with 0.020% MMS, 200 mM HU, 0.2 µg/ml 4-NQO and 100 J/m² UV-light (as indicated). Rad53 phosphorylation was used to test checkpoint activation upon treatments.</p

Increased Rev1–PCNA interaction in cells deleted for <i>UBP10</i>.

<p>(A) Co-immunoprecipitation assay showing physical interaction between Rev1-myc and PCNA. PCNA was immunoprecipitated from 0.020% MMS-treated cells, blots were incubated with α-myc (to detect Rev1) or α-PCNA. The immunoblots shown are those from MMS-treated cells (a comparable result was obtained with untreated cells). As indicated the strains used in this assays were <i>REV1-myc</i> and <i>REV1-myc ubp10Δ</i>. Immunoprecipitated Rev1-myc was quantitated, normalized (to immunoprecipitated PCNA) and plotted. In (A) as well as in (C), the average and standard deviation values obtained from three independent experiments are plotted. (B) Co-immunoprecipitation assay showing physical interaction between Rev1-myc and PCNA-FLAG. PCNA-FLAG was immunoprecipitated (from protein samples crosslinked with formaldehyde, see methods) from asynchronously growing or α-factor blocked cells (as indicated), blots were incubated with α-myc (to detect Rev1) or α-FLAG (to detect PCNA). As indicated the strains used in this assays were <i>REV1-myc POL30-FLAG</i> and <i>REV1-myc POL30-FLAG ubp10Δ</i>. (C) Plots of PCNA-FLAG-co-immunoprecipitated Rev1-myc from untreated and 0.02% MMS-treated cells. Rev1-myc samples were quantitated and normalized to immunoprecipitated PCNA-FLAG. Quantitation is shown in bar diagrams. (D) Increased number of chromatin-associated Rev1 foci in MMS-treated <i>UBP10</i> mutant yeast cells. Spread nuclei of wild-type and <i>ubp10Δ</i> strains carrying <i>REV1-myc</i> tagged were stained with DAPI (blue) and anti-myc antibodies (red). Cells were treated with 0.03% MMS for 1 h. The nuclei were classified in three categories according to the number of Rev1 foci. Representative <i>ubp10Δ</i> spread nuclei of each class and quantitation of wild-type and <i>ubp10Δ</i> nuclei are shown. 47 nuclei were scored for each strain.</p

Cells lacking Ubp10 accumulate ubiquitylated PCNA forms early during S-phase in response to HU-induced DNA replication blocks.

<p> (A) Experimental design, exponentially growing cultures of wild-type and <i>ubp10Δ</i> strains were synchronized with α-factor and then released in 0.2 M HU. Samples were taken at indicated intervals and processed for FACS and Western analysis. (B) FACS analysis showing the checkpoint-induced S phase arrest of asynchronous wild-type and <i>ubp10Δ</i> cells during the HU treatment. BI: budding index. (C) Western blot analysis of PCNA, Rad53, Sic1 and Clb5 protein levels in wild-type and <i>ubp10Δ</i> cells treated with 0.2 M HU (labeled as wt and 10, respectively). ubPCNA signals were quantitated and normalized to loading controls. Quantitation is shown in bar diagrams. Whereas <i>ubp10Δ</i> cells accumulate ubPCNA forms in response to HU, ubPCNA levels declined after the 40 minutes peak in wild-type cells.</p

<i>GAL1</i>-driven overproduction of <i>UBP10</i> reverts PCNA ubiquitylation in response to DNA damage.

<p>(A) Time-course analysis of active GST-Ubp10 induction. An asynchronously growing culture of <i>GAL1,10:GST-UBP10</i>, incubated in raffinose as unique carbon source, was incubated 30 minutes in the presence of 0.02% MMS. Expression of GST-Ubp10 was either repressed by adding glucose (GAL OFF) or induced with galactose (GAL ON) in the continuous presence of the alkylating chemical (as described). Samples were taken at indicated intervals and processed for immunodetection of modified PCNA forms, PCNA, GST-Ubp10 and Rad53. Ponceau staining of the blotted protein extracts is shown. Mono-ubiquitylated PCNA was quantitated, normalized and plotted. (B) Catalytically active Ubp10 reverts PCNA ubiquitylation <i>in vivo</i>. Immunodetection of ubiquitylated and di-ubiquitylated PCNA forms in wild-type cells and in cells reppressed (GAL OFF) or induced (GAL ON) for GST-Ubp10 or GST-Ubp10^CS expression, after a 90 minutes treatment with 0.020% MMS. TCA-obtained cells extracts were processed for immunoblotting with α-PCNA and α-GST antibodies. Ponceau staining of the blotted protein extracts is shown for loading control. (C) Ectopic expression of a catalytically active Ubp10 ubiquitin protease hypersensitizes cells to MMS-induced DNA damage. Ten-fold dilutions of equal numbers of cells of wild-type, <i>GAL1,10:GST-UBP10</i> and <i>GAL1,10:GST-ubp10^C371S</i> were incubated at 25°C in the absence or the presence of indicated percentages of MMS for 72 hours and photographed.</p

PCNA interacts <i>in vivo</i> with Ubp10.

<p>(A) The sliding clamp PCNA and Ubp10 specific-ubiquitin protease interact physically <i>in vivo</i>. Co-immunoprecipitation assay showing physical interaction between Ubp10-myc and FLAG tagged PCNA. PCNA-FLAG was immunoprecipitated from formaldehyde-crosslinked protein extracts (see methods) both from untreated or 0.020% MMS-treated cells, blots were incubated with α-myc (to detect Ubp10) or α-FLAG (to detect PCNA). The immunoblots shown are those from untreated cells (a similar result was obtained with MMS-treated cells). As indicated the strains used in this assays were <i>UBP10-myc POL30-FLAG</i> and <i>UBP10-myc POL30-FLAG rad18Δ</i>. Immunoprecipitated Ubp10-myc was quantitated, normalized and plotted. Each immunoprecipitation experiment was repeated three times to gain an estimate of error. (B) Co-immunoprecipitation assay showing physical interaction between Ubp10-myc and PCNA. PCNA was immunoprecipitated both from untreated or 0.020% MMS-treated cells, blots were incubated with α-myc (to detect Ubp10) or α-PCNA. The immunoblots shown are those from MMS-treated cells (a similar result was obtained with untreated cells). As indicated the strains used in this assays were <i>UBP10-myc</i> and <i>UBP10-myc rad18Δ</i>. Immunoprecipitated Ubp10-myc was quantitated, normalized and plotted. Note that in our experiments we detect Ubp10 interacting with unmodified PCNA (or unmodified PCNA-FLAG).</p

Analysis of Rev3-PCNA and Rev7-PCNA interactions in cells deleted for <i>UBP10</i>.

<p>(A) Rev3 (DNA polymerase ζ catalytic subunit) interacts with PCNA similarly in wild-type and <i>ubp10Δ</i> cells. Co-immunoprecipitation assay showing physical interaction between Rev3-myc and PCNA-FLAG. PCNA-FLAG was immunoprecipitated from asynchronously growing or 0.02% MMS-treated cells (as indicated), blots were incubated with α-myc (to detect Rev3) or α-FLAG (to detect PCNA). As indicated the strains used in this assays were <i>REV3-myc POL30-FLAG</i>, <i>REV3-myc POL30-FLAG ubp10Δ</i> and single tagged <i>POL30-FLAG</i> or <i>REV3-myc</i> controls. Whole cell extracts (WCE) and mock Ip controls are also shown as indicated. (B) The interaction of PCNA with Rev7 (an accessory subunit of DNA polymerase ζ) is reduced in cells deleted for <i>UBP10</i>. Co-immunoprecipitation assay of Rev7-myc and PCNA-FLAG. PCNA-FLAG was immunoprecipitated from asynchronously growing or 0.02% MMS-treated cells (as indicated), blots were incubated with α-myc (to detect Rev7) or α-FLAG (to detect PCNA). As indicated, the key strains used in this assays were <i>REV7-myc POL30-FLAG</i> and <i>REV7-myc POL30-FLAG ubp10Δ.</i> Appropriate single tagged, input (WCE) and mock Ip controls are shown. (C) Deletion of <i>UBP10</i> alters the interaction of PCNA with Rev7. To assure that deletion of <i>UBP10</i> reduced significantly Rev7-PCNA interaction, <i>UBP10</i> was deleted in the <i>REV7-myc POL30-FLAG</i> strain used in B. Five different <i>REV7-myc POL30-FLAG ubp10Δ</i> deletion strains and a <i>REV7-myc POL30-FLAG</i> control were used in the co-immunoprecipitation analysis. PCNA-FLAG was immunoprecipitated from asynchronously growing cells, blots were incubated with α-myc (to detect Rev7) or α-FLAG (to detect PCNA). The strains used in this assays were either <i>REV7-myc POL30-FLAG (1)</i> or <i>REV7-myc POL30-FLAG ubp10Δ</i> (2a, 2b, 2c, 2d and 2e). Input whole cell extracts (WCE) and mock Ip controls are shown. Note that similar amounts of Rev7 are present in whole cell extracts of <i>REV7-myc POL30-FLAG</i> (1) and <i>REV7-myc POL30-FLAG ubp10Δ</i> (2c) cells.</p

Simultaneous Inhibition of EGFR/VEGFR and Cyclooxygenase-2 Targets Stemness-Related Pathways in Colorectal Cancer Cells

<div><p>Despite the demonstrated benefits of anti-EGFR/VEGF targeted therapies in metastatic colorectal cancer (mCRC), many patients initially respond, but then show evidence of disease progression. New therapeutic strategies are needed to make the action of available drugs more efficient. Our study aimed to explore whether simultaneous targeting of EGFR/VEGF and cyclooxygenase-2 (COX-2) may aid the treatment and management of mCRC patients. The dual tyrosine kinase inhibitor AEE788 and celecoxib were used to inhibit EGFR/VEGFR and COX-2, respectively, in colorectal cancer cells. COX-2 inhibition with celecoxib augmented the antitumoral and antiangiogenic efficacy of AEE788, as indicated by the inhibition of cell proliferation, induction of apoptosis and G1 cell cycle arrest, down-regulation of VEGF production by cancer cells and reduction of cell migration. These effects were related with a blockade in the EGFR/VEGFR signaling axis. Notably, the combined AEE788/celecoxib treatment prevented β-catenin nuclear accumulation in tumor cells. This effect was associated with a significant downregulation of FOXM1 protein levels and an impairment in the interaction of this transcription factor with β-catenin, which is required for its nuclear localization. Furthermore, the combined treatment also reduced the expression of the stem cell markers Oct 3/4, Nanog, Sox-2 and Snail in cancer cells, and contributed to the diminution of the CSC subpopulation, as indicated by colonosphere formation assays. In conclusion, the combined treatment of AEE788 and celecoxib not only demonstrated enhanced anti-tumoral efficacy in colorectal cancer cells, but also reduced colon CSCs subpopulation by targeting stemness-related pathways. Therefore, the simultaneous targeting of EGFR/VEGF and COX-2 may aid in blocking mCRC progression and improve the efficacy of existing therapies in colorectal cancer.</p></div

Combined AEE788/celecoxib treatment downregulates stemness-related pathways in colorectal cancer cells.

<p>The expression of stem cell markers Oct 3/4, Nanog and Sox-2 was analyzed by western blot in total cell extracts of colon cancer cells after 6h of indicated treatments. The expression of -actin is included as loading control. The corresponding densitometric analysis is also shown. Data are means ± SEM of three independent experiments (*p <0.05, compared with the control; # p<0.05, compared with AEE788-treated cells).</p

AEE788 inhibits cell proliferation, induces apoptosis and alters cell cycle in colorectal cancer cells.

<p>A) Cell proliferation was evaluated after 72h of treatment with different doses of AEE788. B) Inhibition of cell proliferation by AEE788 was tested in cells growing in the presence of EGF (100 ng/ml). C) The fraction of apoptotic cells was estimated after 48 h of treatment with different doses of AEE788 of cells growing in the presence of EGF (100 ng/mL). D) Analysis of cell cycle was performed by flow cytometry after 48 h of treatment with different doses of AEE788 of cells growing in the presence of EGF (100 ng/mL). Data are means ± SEM of three independent experiments (*p <0.05, compared with the control).</p