Early RB94-produced cytotoxicity in cancer cells is independent of caspase activation or 50 kb DNA fragmentation

Abstract

RB94, which lacks the N-terminal 112 amino-acid residues of the full-length retinoblastoma protein (RB110) is a more potent inhibitor of cancer cell growth than RB110, being cytotoxic to all cancer cell lines studied, independent of their genetic abnormalities. Although we initially thought RB94-induced cell death was caspase-dependent, such caspase activation now appears to be a late event. Cells that remained attached 48 h after transduction with Ad-RB94 showed, among other changes, nuclear enlargement, peripheral nuclear chromatin condensation and often micronucleation. In addition, the cells were TdT-mediated dUTP nick end labeling (TUNEL) positive but showed no cleavage of caspase 3 or 9. Only after the cells detached was cleavage of both caspase 3 and 9 observed. These TUNEL-positive cells showed neither cytochrome c mitochondrial translocation usually found in typical apoptotic cells nor DNA laddering indicative of oligonucleosomal DNA fragmentation. In addition, although 50 kb DNA fragmentation was produced in these TUNEL-positive cells, which was dependent on apoptosis-inducing factor (AIF), inhibiting this fragmentation by siAIF did not inhibit TUNEL formation or cytotoxicity. As RB94 will soon be used for gene therapy further understanding the molecular basis of these early changes in killing cancer cells is one of our particularly important present goals.

Figures

Figure 1

Morphological microscopic changes in RB negative UC-14 cells following Ad-RB94 transfection. A. Normal morphology in untreated cells B. Typical morphological changes seen 48 hours after Ad-RB94 treatment of UC-14 cells. These included micronucleation (red arrows) seen in approximately 10 per cent of the cells and approximately 30% of the cells with enlarged nuclei showing a peripheral nuclear condensed ring-like appearance (black arrows). Note the round floating cells also seen, which represent RB94 induced cells detaching from the dish (green arrows). Magnification 400X. The insert in the lower right shows DAPI staining of a cell with the peripheral nuclear condensation noted in phase contrast by black arrows.

Figure 2

RB94 and TUNEL staining after Ad-RB94 treatment. A. RB94 staining (brown nuclear staining) was seen in approximately 40–50% of the initially RB negative UC14 cells 48 hrs after Ad-RB94 treatment. B. TUNEL staining (brown nuclear staining) was similarly observed in approximately the percentage of cells 48hr afterAd-RB94 treatment that were RB94 positive as shown in 2A. Magnification 400X. C. Confocal examination showing both RB94 and TUNEL staining in the same cells. Plate 1-DAPI staining (blue), Plate 2- TUNEL staining (green), Plate 3-RB94 staining (red), Plate 4-TUNEL and RB94 staining (merged).

Figure 3

Lack of caspase 3, and caspase 9 cleavage or cytochrome C translocation in attached cells 48 h after Ad-RB94 treatment. A and B. No caspase 3 or caspase 9 cleavage, respectively was seen in the cells which were still attached 48 hr after treatment, although many of these cells were TUNEL positive (see Fig. 2B). Cleavage of caspases 3, and 9 was observed in the detached, floating cells, however C. No cytochrome C translocation from the mitochonrdria to the cytoplasm was observed after Ad-RB94 treatment unlike that observe after treatment with staurosporine (STS) shown as a postitve control which produced which is known to produce typical caspase –dependent apoptosis. In addition cytochrome C is only found in the mitochondria of control cells as expected illustrating the success of the purity of the mitochondrial and cytoplasmic fractions.

Figure 4

Ad-RB94 produced 50 kilbase DNA fragmentation involves AIF but is not a mechanism of RB94 produced cytotoxicity. A. AIF siRNA blocks AIF protein production in control and Ad-RB94 treated cells (arrow). B. Ad-RB94 produces 50 kb DNA fragmentation in UC9 and UC14 cells which was inhibited by AIF si RNA as shown by FIGE C. Morphological changes seen in Ad-RB94 treated cells, including enlarged nuclei with a peripheral nuclear condensed ring-like appearance (black arrows) and cellular detachment (see Fig. 1B), were not blocked by AIF siRNA. Magnification 400X. All cells were evaluated 48 hrs after Ad-RB94 treatment in each experiment.

Figure 5

Lack of cytotoxicity produced by Ad-RB94 in normal human urothelial cells (TERT-NHUCs). Left Plate. No decrease in cell growth is seen in the TERT-NHUCs after Ad-RB-94 treatment. Ad-βgal was also used as a control at the same MOI as Ad-RB94 and in which approximately 50% of the cells show βgal staining 48 hr after treatment. Right Plates, A and B. No morphological changes are seen 48 hrs after Ad-RB94 treatment compared to untreated control TERT-NHUCs. C and D. Only rare TUNEL positive cells were seen in either control or Ad-RB94 treated cells (arrows) indicating that Ad-RB94 did not produce apoptosis as measured by TUNEL in TERT-NHUCs compared to cancer cells (see Fig. 2B). Magnification 400X.