Fludarabine transcriptionally upregulates MMP-9 and induces its localization to the CLL cell membrane.

<p>(A,B) 10–15×10⁶ CLL in RPMI/0.1% FBS cells from two different patients were treated with 3 or 5 µM fludarabine (Fluda) for 48 h and MMP-9 mRNA expression was analyzed by RT-PCR (A) and qPCR (B). Normalized average values (fold change) are shown. (C) 1.5×10⁵ CLL cells from two different patients were incubated with or without 3 µM fludarabine for 48 h and MMP-9 surface expression was analyzed by flow cytometry. White areas, control/untreated cells; grey areas, fludarabine treated cells. Arrows indicate specific fluorescence (SF) values for each cell population. Normalized average values are also shown.</p

Upregulation and membrane localization of MMP-9 is an initial CLL cell response to the cytotoxic action of ATO.

<p>(A) Cell sorter biparametric diagrams of PI⁻ CLL cells (1.5×10⁵) treated or not with ATO for 24 h and analyzed for MMP-9 expression. Numbers indicate the percentage of cells expressing MMP-9 in the early apoptotic (Annexin V⁺, top) and live (Annexin V⁻, bottom) cell compartments. (B) Flow cytometric analysis of MMP-9 expression in control or ATO-treated CLL cells with or without previous incubation with 50 µM Z-VAD-FMK. Histograms from two representative cases are shown. White areas: control cells; grey areas: ATO-treated cells. Arrows indicate specific fluorescence (SF). Normalized average values for all five samples analyzed are shown. The average % of early apoptotic (Ann V⁺/PI⁻) cells in these samples is also shown. (C) 10–15×10⁶ CLL cells treated as in (B) were analyzed for MMP-9 mRNA expression by qPCR, using TBP as an internal control. Average normalized values (fold change) are shown. (D,E) 10–15×10⁶ CLL cells were treated with 1 µM ATO for 24 h and MMP-9 mRNA expression analyzed by RT-PCR (D) and qPCR (E). Normalized average values (fold change) are shown. (F) CLL cells treated as in (D, E) were analyzed for MMP-9 surface expression by flow cytometry with an anti-MMP-9 pAb or a control pAb. Histograms for the same samples used in (D, E) are shown. Arrows, white and grey areas are as in (B). Normalized average SF values of all four samples studied are shown. *P≤0.05; ***P≤0.001.</p

Clinical characteristics of CLL patients.

a<p>Clinical stage according to references <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099993#pone.0099993-Zenz1" target="_blank">[1]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099993#pone.0099993-Gaidano1" target="_blank">[2]</a>.</p>b<p>Percentage of cells expressing this marker.</p>c<p>The coexpression of CD38 and ZAP-70 has clinical prognostic value <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099993#pone.0099993-Zenz1" target="_blank">[1]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099993#pone.0099993-Gaidano1" target="_blank">[2]</a>.</p><p>ND, not determined.</p

MMP-9 expression in MEC-1 cells prevents downregulation of anti-apoptotic Bcl-2 family proteins in response to ATO.

<p>(A) 5×10⁶ Mock- or MMP-9-MEC-1 cells were treated or not with 5 µM ATO. After 24 h cells were lysed and the expression of the indicated proteins was analyzed by Western blotting, using vinculin as an internal control. A representative experiment is shown for each case and numbers indicate the average values from all experiments performed, after normalizing Mock control values to 1. (B) The indicated ratios of anti-apoptotic/pro-apoptotic proteins are shown. (C) 3×10⁷ Mock- or MMP-9-cells were treated or not with 5 µM ATO for 24 h. Cells were lysed and lysates immunoprecipitated with anti-Mcl-1 or control Abs and analyzed by Western blotting. Values indicate the amount of Bim found in the Mcl-1 immunoprecipitates in both types of MEC-1 transfectants. * or ^#P≤0.05; ** or ^##P≤0.01; *** or ^###P≤0.001. Symbols are: *, Mock- vs MMP-9-cells; ^#, Mock- or MMP-9-cells treated with ATO compared to their respective untreated counterparts.</p

MMP-9 localizes to the CLL cell surface in response to ATO and in correlation with induction of apoptosis.

<p>(A) 5×10⁶ CLL cells in RPMI/0.1% FBS were treated or not with 3 µM ATO for 24 h. The conditioned media was collected, concentrated 20× and analyzed by gelatin zymography. The results from four representative samples and the average normalized values (arbitrary units, AU) from all six samples studied are shown. (B) 1.5×10⁵ CLL cells were incubated with or without 3 µM ATO for 24 h. MMP-9 surface expression was analyzed by flow cytometry using an anti-MMP-9 pAb or a control pAb. Histograms from six representative cases are shown, where white areas correspond to control/untreated cells and grey areas to ATO treated cells. Arrows indicate specific fluorescence (SF). Average normalized values from all ten samples analyzed are also shown. (C) 30×10⁶ CLL cells in RPMI/0.1%FBS were incubated with or without 3 µM ATO for 24 h. Membrane (Mb) and cytosolic (Cyt) fractions were separated and analyzed by gelatin zymography. RhoGDI and CD45 detected by Western blotting in the same lysates were used as controls for the procedure. The conditioned media (CM) of these cells was also analyzed by gelatin zymography and the normalized average values of the quantitated bands are shown (D) 1.5×10⁵ CLL cells in RPMI/0.1%FBS were incubated for 1 h with or without the indicated antibodies. 3 µM ATO was added and after 24 h, surface-bound MMP-9 was determined by flow cytometry using an anti-MMP-9 pAb or a control pAb. Histograms for two representative cases are shown. White areas correspond to untreated cells (−ATO) and grey areas (both light and dark) to ATO-treated cells (+ATO). Average normalized SF values are also shown. **P≤0.01; ***P≤0.001.</p

Effect of ATO and fludarabine on MEC-1 cells.

<p>(A, B) 7.5×10⁴ MEC-1 cells in IMDM/0.1% FBS were treated or not with the indicated concentrations of ATO (A) or fludarabine (Fluda) (B). After 24 h (ATO) or 48 h (Fluda), cell viability was determined by the MTT assay. Control cell viability was normalized to 100 and average values are shown. (C,D) 5×10⁶ MEC-1 cells were treated with 3 µM ATO for the indicated times and MMP-9 mRNA expression was analyzed by RT-PCR, using GAPDH as internal control (C) and qPCR, using TBP as internal control (D). Normalized average values (fold change) are shown. (E) 1.5×10⁵ MEC-1 cells were treated or not with 3 µM ATO for 24 h and MMP-9 surface expression was analyzed by flow cytometry, using an anti-MMP-9 pAb or a control pAb. Histograms from a representative experiment and normalized average values for the three experiments performed are shown. White areas, control/untreated cells; grey areas, ATO-treated cells. Arrows indicate the specific fluorescence. *P≤0.05; **P≤0.01; ***P≤0.001.</p

MMP-9, isolated or present in stroma, induces resistance of CLL cells to ATO and fludarabine.

<p>(A) 1.5×10⁵ CLL cells RPMI/0.1% FBS were cultured on 0.5% BSA or 150 nM MMP-9 for 1 h prior to adding the indicated concentrations of ATO or fludarabine (Fluda). After 24 h (ATO) or 48 h (Fluda) cell viability was determined by flow cytometry using FITC-Annexin V and PI. (B) 1.5×10⁵ CLL cells were treated or not with anti-MMP-9 pAbs or control pAbs for 1 h and added to wells coated with 0.5% BSA, HS-5 cells or primary stromal cells (BMSC). After 2 h at 37°C, 2 µM ATO was added and cells further incubated for 48 h. Cell viability was determined by flow cytometry using FITC-Annexin V and PI. The viability of CLL cells cultured over stroma in the absence of ATO was normalized to 100 and average values are shown. *P≤0.05; **P≤0.01; ***P≤0.001.</p

MMP-9 expression in MEC-1 cells prevents downregulation of anti-apoptotic Bcl-2 family proteins in response to fludarabine.

<p>(A,B) 5×10⁶ Mock- or MMP-9-cells were treated or not with 5 µM fludarabine (Fluda). After 48 h cells were lysed and the indicated proteins (A) and ratios (B) analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099993#pone-0099993-g009" target="_blank">Figure 9</a>. * or ^#P≤0.05; ** or ^##P≤0.01; *** or ^###P≤0.001. Symbols are: *, Mock- vs MMP-9 cells; ^#, Mock- or MMP-9-cells treated with Fluda compared to their respective untreated counterparts.</p

Clinical features, evolution and biological characteristics of the ten CLL patients studied.

<p>Summary of the prognosis factors considered in the clinic and genetic lesions that the ten patients harbor. ND = not done, del = deletion.</p

Validated somatic mutation.

<p>CDS: Coding sequence. NA: not annotated. Chr: chromosome.</p