Insights into the mechanism of cell death induced by saporin delivered into cancer cells by an antibody fusion protein targeting the transferrin receptor 1

Abstract

We previously developed an antibody-avidin fusion protein (ch128.1Av) that targets the human transferrin receptor 1 (TfR1) and exhibits direct cytotoxicity against malignant B cells in an iron-dependent manner. ch128.1Av is also a delivery system and its conjugation with biotinylated saporin (b-SO6), a plant ribosome-inactivating toxin, results in a dramatic iron-independent cytotoxicity, both in malignant cells that are sensitive or resistant to ch128.1Av alone, in which the toxin effectively inhibits protein synthesis and triggers caspase activation. We have now found that the ch128.1Av/b-SO6 complex induces a transcriptional response consistent with oxidative stress and DNA damage, a response that is not observed with ch128.1Av alone. Furthermore, we show that the antioxidant N-acetylcysteine partially blocks saporin-induced apoptosis suggesting that oxidative stress contributes to DNA damage and ultimately saporin-induced cell death. Interestingly, the toxin was detected in nuclear extracts by immunoblotting, suggesting the possibility that saporin might induce direct DNA damage. However, confocal microscopy did not show a clear and consistent pattern of intranuclear localization. Finally, using the long-term culture-initiating cell assay we found that ch128.1Av/b-SO6 is not toxic to normal human hematopoietic stem cells suggesting that this critical cell population would be preserved in therapeutic interventions using this immunotoxin.

Figure 1. Time course protocol for mRNA isolation and verification of the cytotoxic effects of the fusion protein alone and conjugated to b-SO6

Cells were treated with 10 nM ch128.1 alone or conjugated to b-SO6 for the indicated times in duplicate samples in a 48-well plate at 200,000 cells/well (A). At each time point RNA was collected for microarray analysis. Control wells were treated with buffer alone. Additional wells for each treatment were incubated in parallel for 48 hours to measure the anti-proliferative (B) and pro-apoptotic (C) effect of ch128.1Av and ch128.1Av/b-SO6 on IM-9 and U266 cells. The anti-proliferative effect was measured by [³H]-thymidine incorporation and values expressed as the % of buffer control. Error bars represent standard deviation. Apoptosis was determined by flow cytometry in cells labeled with Annexin-V/Propidium Iodide. Percentage of total cells is shown in the corner of each quadrant.

Figure 2. Gene expression analysis in cells treated with ch128.1Av complexed to b-SO6

Changes in gene expression with a greater than 1.0 Log base 2-fold change (LFC) after 1, 3, 9, or 24 hours of ch128.1Av/b-SO6 treatment with respect to their time matched control (buffer alone) are shown for IM-9 (A) and U266 (B) cells clustered by time point and magnitude of change. Changes in IM-9 greater than 1.0 LFC at 9 hours (C) post treatment are also shown for comparison with changes in U266 at 24 hours. Clustering was conducted using the Cluster program and visualized using Java TreeView.

Figure 3. Comparison of gene expression changes in IM-9 and U266 at the various time points

Direct comparison in gene expression changes between IM-9 and U266 cells treated with ch128.1Av/b-SO6 in which genes with a variance greater than 0.4 are clustered over the various time points and the magnitude of difference between the RNA levels in each cell line. In this case genes whose transcripts are present at a higher level in U266 than in IM-9 are shown in yellow. Clustering was conducted using the Cluster program and visualized using Java TreeView.

Figure 4. Gene clustering according to ontologies

The same list of genes shown in Figure 3 are now shown clustered by their corresponding ontologies, with statistically enriched ontologies listed at right alongside their corresponding p-values. Clustering was conducted using the Cluster program and visualized using Java TreeView.

Figure 5. Validation of gene expression changes by real time PCR

Freshly isolated RNA from U266 (A) and IM-9 (B) cells treated with either 10 nM ch128.1Av, b-SO6, or ch128.1Av/b-SO6 for 24 hours was converted to cDNA and gene expression determined using relative quantification analysis on a Roche LightCycler 480 System. Data is shown as the fold change in target gene expression compared to the housekeeping gene GAPDH. All samples were tested in triplicate and the standard deviations are shown. Data are representative of two independent experiments carried out with different RNA preparations.

Figure 6. Protection of ch128.1Av/b-SO6-induced cell death by the antioxidant NAC

IM-9 (left panels) and U266 (right panels) cells were treated with 10 nM (top panels) or 1 nM (bottom panels) ch128.1Av, b-SO6, or ch128.1Av/b-SO6 for 48 hours with or without the addition of 2 mM NAC. CHX was used as a common protein synthesis inhibitor. The percentage of Annexin V positive cells was determined using flow cytometry. Data are the average of three independent experiments. Error bars indicate the standard deviation. * p < 0.01, Student’s t-test.

Figure 7. Detection of b-SO6 in nuclear extracts of cells treated with ch128.Av/b-SO6 conjugate

U266 (left panels) and IM-9 (right panels) were treated with 10 nM ch128.1Av/b-SO6 for either 1 hour or 16 hours followed by the preparation of nuclear and cytoplasmic extracts. Saporin within these extracts was detected by Western Blot analysis. Control cells (“C”) were incubated with an equal amount of buffer for 16 hours. GAPDH was used a control for cytoplasmic protein, while TBP was used as a control for nuclear protein.