Enhanced microtubule-dependent trafficking and p53 nuclear accumulation by suppression of microtubule dynamics

Abstract

The tumor suppressor protein p53 localizes to microtubules (MT) and, in response to DNA damage, is transported to the nucleus via the MT minus-end-directed motor protein dynein. Dynein is also responsible for MT-mediated nuclear targeting of adenovirus type 2 (Ad2). Here we show that treatment with low concentrations of MT-targeting compounds (MTCs) that do not disrupt the MT network but are known to suppress MT dynamics enhanced p53 nuclear accumulation, and the activation of the p53-downstream target genes. p53 nuclear accumulation required binding of MTCs to MTs and enhanced the induction of p53-up-regulated modulator of apoptosis (PUMA) mRNA and apoptosis on challenging cells with the DNA-damaging drug adriamycin. Low concentrations of MTCs enhanced the rate of movement of fluorescent Ad2 to the nucleus and increased the nuclear targeting efficiency of Ad2. We propose that suppression of MT dynamics by low concentrations of MTCs enhances MT-dependent trafficking toward the minus ends of MTs and facilitates nuclear targeting.

Fig 1.

(A) Low concentrations of PTX or VCR enhance p53 nuclear accumulation in A549 cells. A549 cells were treated with the indicated drugs for 18 hr and processed for double immunofluorescence labeling with antibodies against p53 (red, labeled with Rhodamine red X) or α-tubulin (green, labeled with FITC). Untreated A549 cells were included as control (CTRL). Staining was analyzed by CLSM. (Bar = 10 μm.) (B) Low concentrations of PTX or VCR induce mdm2 in A549 cells. Untreated A549 cells (0) or cells treated for 18 hr were processed for Western blotting. Cells treated with 400 ng/ml of ADR for 18 hr are included as a positive control for p53 and mdm2 increased levels. Thirty micrograms of total cellular protein from each sample were resolved in a 10% SDS/PAGE, transferred, and immunoblotted with antibodies against p53 and mdm2. Please notice that in the Westerns blots shown here, the control lane (CTRL) has been moved from the first position to the second to facilitate the direct comparison of the control lane with the ADR lane to the right and the PTX 3 lane to the left.

Fig 2.

Low concentrations of PTX or VCR enhance p53 nuclear accumulation, and induce mdm2 in A2780 (1A9) cells in a tubulin-dependent manner. 1A9 cells were plated in duplicate, subjected to the indicated drug treatments, and processed either for CLSM (A) or Western blotting (B). (A) Cells are stained with antibodies against p53 (red, labeled with Rhodamine red X) or α-tubulin (green, labeled with FITC). (Bar = 10 μm.) (B) Thirty micrograms of total cell protein from each sample were resolved in a 10% SDS/PAGE, transferred, and immunoblotted with antibodies against p53 and mdm2. Induction of mdm2 is consistent with enhanced p53 nuclear accumulation (A). (C) The β tubulin mutant A2780/Epo B cells were treated with the indicated drugs for 18 hr and processed for double immunofluorescence labeling with antibodies against p53 (red, labeled with Rhodamine red X) or α-tubulin (green, labeled with FITC). Untreated A2780/Epo B cells (CTRL) and cells treated with ADR are included as controls. Staining was analyzed by CLSM. (Bar = 10 μm.)

Fig 3.

Low concentrations of other MT-active agents enhance p53 nuclear accumulation and induce mdm2 in A549 cells. A549 cells were plated in duplicate, subjected to the indicated drug treatments, and processed either for CLSM (A) or Western blotting (B) (see Fig. 2). Arrows in A point to either bundled MTs (Epo A, Epo B) or depolymerized tubulin network (colchicine, Noc). (Bar = 10 μm.)

Fig 4.

Low concentrations of PTX or Noc enhance the nuclear targeting and the motility of Ad2 in A549 cells. (A) Untreated A549 cells (mock) or cells treated with 1 nM Noc or 1 nM PTX (Tax) for 18 hr were infected with Ad2-TR in RPMI-BSA at 37°C. In randomly selected untreated or drug-treated cells, the nuclear, perinuclear, cytoplasmic, and peripheral fluorescence of Ad2-TR was quantitated 15 min (Upper) or 40 min (Lower) p.i. The bars represent the average of fluorescence ± SEM of 20–40 independent measurements. (B) Untreated A549 cells (no drug) or cells treated with 1 nM of either PTX (Tax) or Noc for 18 hr were infected with Ad2-TR in RPMI-BSA at 37°C for 15 min followed by further incubation in RPMI-BSA in the presence of PTX or Noc but without the virus until imaging of Ad2-TR was completed. Ad2-TR was imaged in time-lapse mode, and ES were analyzed for each viral particle at 0–20 min (Upper) or 20–50 min (Lower) p.i. Population motilities (pop mot) represent the mean number of ES larger than 0.1 μm/s divided by the total of ES of each particle. Mot c and mot p were defined as the average fractions of the vectorial ES components larger than 0.1 μm/s directed toward the cell center (mot c) and toward the cell periphery (mot p), including the SEM and P values. For all measurements, (−) indicates motility toward the cell's center.

Fig 5.

Treatment with low concentrations of PTX or VCR followed by ADR enhances apoptosis in A549 cells. (A) A549 cells were treated with 3, 6, or 100 nM of PTX or VCR for 18 hr followed by treatment with 200 ng/ml of ADR for an additional 6 hr (PTX3→ADR, PTX6→ADR, PTX100→ADR, and VCR3→ADR, VCR6→ADR, VCR100→ADR). ADR was added to the medium containing PTX or VCR at the indicated concentrations. Untreated cells (CTRL) or cells treated with 200 ng/ml of ADR alone (ADR), PTX alone, or VCR alone for 24 hr (PTX3, PTX6, PTX100; VCR3, VCR6, VCR100) were included as controls. Western blots were probed with antibodies against the cleaved (p85) form of PARP, p53, mdm2, and actin as a loading control. (B) A549 cells were treated with 3, 6, or 100 nM PTX for 18 hr, followed by treatment with 200 ng/ml of ADR for an additional 6 hr (PTX3→ADR, PTX6→ADR, PTX100→ADR). Untreated cells (CTRL) or cells treated with PTX alone for 24 hr (PTX3, PTX6, PTX100) are included as controls. Ten micrograms of total RNA from each sample were analyzed by Northern blotting. Ethidium bromide staining of 28S RNA is shown as loading control.