²¹²Pb-Radioimmunotherapy Induces G₂ Cell-Cycle Arrest and Delays DNA Damage Repair in Tumor Xenografts in a Model for Disseminated Intraperitoneal Disease

Targeted radiation therapy with monoclonal antibodies (mAb) using β⁻ emitting radionuclides has been shown to be an efficacious strategy for the treatment and management of cancer patients (1, 2). Due to the combination of shorter path length (50–80 μm) and higher linear energy transfer (100 KeV/μm), targeted α-particle radioimmunotherapy (RIT) offers the potential for more specific tumor cell killing, with less damage to surrounding normal tissue than β⁻ emitters. Although this strategy has been successfully applied toward the eradication of leukemia (3, 4), the same physical properties suggest that targeted α-therapy would be suitable for the elimination of minimal residual or micrometastatic disease in other types of human malignancies (5–7).

Targeted and pretargeted RIT using α-emitters such as ²¹²Bi (T_1/2 = 1.01 hours) and ²¹²Pb (T_1/2 = 10.6 hours) have shown significant therapeutic efficacy in both in vitro and in vivo model systems (8–10). ²¹²Pb is the longer lived parental radionuclide of ²¹²Bi and, as such, it serves as an in vivo generator of ²¹²Bi. The ²¹²Pb/²¹²Bi system, therefore, is a promising α-particle emitting source that provides an alternative option for the treatment and management of cancer (8, 11).

Trastuzumab (Herceptin) is a humanized mAb that targets HER2 and has been well documented to have antitumor activity for the management of breast cancer (12, 13). Previously, this laboratory showed the efficacy of α-particle RIT using the CHX-A” diethylenetriaminepentaacetic acid linker with ²¹³Bi in intraperitoneal models for pancreatic and ovarian cancer using trastuzumab as the targeting moiety (6). Complementary to those results, suitable chelation chemistry for the retention of ²¹²Pb with the protein was also designed and synthesized, that is, 2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraaza-1,4,7, 10-tetra-(2-carbamoylmethyl)-cyclododecane (4-NCS-Bz-TCMC; TCMC) to overcome the limitations associated with the direct use of the shorter half-life bismuth radioisotopes, ²¹³Bi or ²¹²Bi, and to obviate a source of toxicity originating from intracellular dissociation of ²¹²Pb from 1, 4, 7, 10-tetraazacyclododecane-N,N′N″,N′”-tetraacetic acid (8, 14). Studies using ²¹²Pb showed the feasibility of this isotope in RIT for the treatment of disseminated intraperitoneal disease; ²¹²Pb-TCMC-trastuzumab given as a single injection showed therapeutic efficacy, which increased with multiple injections given at approximately monthly intervals (8).

The mechanism by which α-particle RIT induces cell death is not completely understood (15–18). A recent report indicated that upon exposure to α-particles, cell survival was not impacted by traversals of the cytoplasm, but by traversal of nuclei (19). Seidl and colleagues reported that RIT with an α-emitting radionuclide induced nonapoptotic cell death at 72-hour posttreatment (20). It is noteworthy that there have been few studies related to the determination of the actual mechanisms involved in the α-particle RIT cytotoxicity. The literature to date has been effectively restricted to in vitro studies, which, by their very nature, are self-limiting and not reflective of RIT treatment of tumors in a complex environment (21, 22).

The studies reported herein were designed to gain an understanding of the underlying mechanism(s) of action of ²¹²Pb-TCMC-trastuzumab therapy in a systematic fashion using the murine model currently under investigation in our laboratory. The ultimate objective will be to incorporate the knowledge gained into the design of future therapy studies and to improve the therapeutic benefit of targeting HER2 with α-particle emitting radionuclides. The studies reported herein describe the apoptotic response, cell-cycle distribution, DNA repair, and changes in chromatin remodeling in LS-174T intraperitoneal xenograft tumors following RIT with ²¹²Pb. The studies suggested that ²¹²Pb-TCMC-trastuzumab therapy-induced cell killing in the LS-174T intraperitoneal xenograft model occurred principally by G₂-M arrest, accompanied by a delay in DNA damage repair.

The human colon carcinoma cell line (LS-174T) was used for all in vivo studies. LS-174T was grown in a supplemented Dulbecco's Modified Eagle's Medium (DMEM) as previously described (23). All media and supplements were obtained from Lonza. The cell line has been screened for Mycoplasma and other pathogens before in vivo use according to National Cancer Institute (NCI) Laboratory Animal Sciences Program policy. No authentication of the cell line was conducted by the authors.

The synthesis, characterization, and purification of the bifunctional ligand TCMC have been previously described (8, 14). Trastuzumab (Herceptin; Genentech) was conjugated with TCMC by established methods using a 10-fold molar excess of ligand to mAb as previously reported (8). The final concentration of trastuzumab was quantified by the method of Lowry (24). The number of TCMC molecules linked to the mAb was determined using a spectrophotometric based assay (25). A 10 mCi ²²⁴Ra/²¹²Pb generator was supplied by AlphaMed, Inc. The preparation of the generator and radiolabeling procedure has been previously described (8).

Studies were carried out with 19 to 21 g female athymic mice (NCI-Frederick) bearing 3-day intraperitoneal LS-174T xenografts as previously reported (8). The viability of the LS-174T cells (>95%) was determined using trypan blue. Mice were injected intraperitoneally with 1 × 10⁸ LS-174T cells in 1 mL of DMEM. The inoculum size for this cell line represented the minimum amount of cells required for tumor growth in 100% of the mice (6). ²¹²Pb-TCMC-trastuzumab (10 μCi in 0.5 mL PBS) was administered to the mice 3 days postimplantation of tumor (n = 10–15). This treatment group was compared with sets of mice that received ²¹²Pb-TCMC-HuIgG, unlabeled trastuzumab, or HuIgG, or no treatment. Mice receiving trastuzumab or HuIgG were injected 3 days after tumor implantation with 10 μg of the respective material. The mice used for the cell-cycle and proliferation studies were injected intraperitoneally with 5-bromo-2′-deoxyuridine (BrdUrd; 1.5 mg in 0.5 mL PBS; Sigma) 4 hours before euthanasia. Tumors were harvested from mice bearing intraperitoneal LS-174T xenografts at 6, 24, 48, 72, 96, and 120 hours. The amount of tumor collected was not measured; however, on the basis of previous studies, the tumor burden at 7 days is typically 128.5 ± 205.6 mg (26). The tumors at each time point were pooled together, macroscopically inspected, and adherent tissues were removed. The tumor tissues were then thoroughly rinsed in ice-cold PBS 3 times, divided, and processed accordingly for each assay. The tumor tissues were either stored at −80°C until use or paraffin embedded after fixing in 1% formalin. All animal protocols were approved by the National Cancer Institute Animal Care and Use Committee.

Cell-cycle distribution and DNA synthesis was determined by flow cytometry as previously described with some modifications (27). The tumors were fixed in cold 70% ethanol at 4°C, washed in PBS twice, minced finely, and incubated in 1 mL 0.04% pepsin (Sigma) w/v in 0.1 N HCl (Mallinckrodt, Inc.) for 1 hour at 37 °C with shaking. The digest was filtered through a 70-μm nylon mesh after passage through a 25-gauge needle. Following centrifugation at 10,000 × g for 10 minutes, the resulting pellet was resuspended in 1 mL of 2 N HCl (Mallinckrodt, Inc.) and incubated at 37°C for 20 minutes with shaking. The nuclear suspension was neutralized with 0.1 mol/L sodium tetraborate (Sigma), washed twice in PBS containing 0.5% bovine serum albumin (BSA) and 0.5% Tween-20 (PBTB), and resuspended in PBTB. The nuclei (100 μL) were incubated with 20 μL of fluorescein isothiocyanate (FITC)-labeled anti-BrdU mAb (BD Biosciences-Pharmingen) for 1 hour at 4°C, followed by 2 washes in cold PBS. The samples were then resuspended in 2 mL of propidium iodide (50 μg/mL in PBTB; Sigma) containing RNAse A (50 μg; Sigma) and incubated for 30 minutes at 4ºC. Flow cytometry was done using a FACSCalibur (BD Biosciences), collecting 15,000 events with cell debris excluded from data collection. DNA content (propidium iodide) and DNA synthesis (BrdUrd content) were analyzed using 2 parameter data collection with CellQuest (BD Biosciences) software, whereas single parameter DNA distribution was done and analyzed using Modfit LT ver. 3.0 (Verity Software House, Inc).

Apoptotic bodies were scored using hematoxylin and eosin (H&E) staining as described previously (28). Five fields were analyzed per tumor section, and the number of apoptotic bodies per 100 nuclei scored expressed as a percentage. The following criteria were used to distinguish apoptotic bodies: (a) isolated distribution of apoptotic bodies; (b) shrunken cells usually with empty space between neighboring cells; (c) eosinophilic cytoplasm; (d) condensation of nuclei into dense particles; (e) fragmentation of the nuclei into several bodies; and (f) the absence of inflammatory reaction surrounding the apoptotic cells (nonnecrotic cells).

The presence of apoptotic bodies on tumor sections was also determined using the Dead End Fluorometric terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) System (Promega). The sections were incubated in 100 μL of a 20 μg/mL proteinase solution at room temperature for 10 minutes, washed twice in PBS, and fixed in 4% formaldehyde. The sections were labeled by incubation in 100 μL of 200 mmol/L potassium cacodylate, 25 mmol/L Tris-HCl, 0.2 mmol/L DTT, 2.5 mmol/L cobalt chloride, and 0.25 mg/mL BSA for 10 minutes, and then incubated with 50 μL of TdT reaction mixture for 60 minutes at 37°C in a humidified chamber; immersion in 2× SSC halted the reaction. Tissue sections were mounted using Vectashield with DAPI (4′,6-diamidino-2-phenylindole; Vector Laboratories) to counterstain DNA.

Immunohistochemistry (IHC) was done as described in the manufacturer's instructions (Cell Signaling) with some modifications. After formalin fixation, antigen unmasking was done by subjecting the sections to 80°C for 10 minutes in 10 mmol/L sodium citrate, pH 6.0. After washing in distilled water 3 times, the slides were treated with 3% hydrogen peroxide for 10 minutes followed by 1-hour incubation with 100 μL of PBS with Tween 20 (PBST) containing 5% normal goat serum at room temperature. Thereafter, 200 μL of γH₂AX antibody (Cell Signaling) in PBST with 5% normal goat serum was added to the sections and incubated overnight at 4°C. After 3 washes, the sections were developed with 100 μL 3,3′-diaminobenzidine substrate, washed twice, dehydrated in 95% and 100% ethanol, and dipped in xylene 2 times. Coverslips were mounted using Cytoseal XYL (Thermo Scientific).

Sample preparation was done according to the tissue preparation protocol in the manufacturer's instructions (Comet Assay; Trevigen). For the best test of whether cells are in a satisfactory condition for comet assay, control (untreated cells) should give comets with a background level of breaks (mostly class 0). The neutral comet assay was done as described in the manufacturer's instructions (Comet Assay; Trevisen). In brief, the incubation times (4°C) were as follows: 30 minutes lysis, 15 minutes in 1× TBE, and 40 minutes electrophoresis in 1× TBE at 1 V/cm. Sixty comets per individual preparation were scored, and all measurement parameters were calculated on Comet Score.

The chromatin immunoprecipitation assay (ChIP) was used to determine whether histone methylation was altered by ²¹²Pb-TCMC-trastuzumab therapy at the promoter region of the p21 gene. The ChIP assay was done with the ChIP Assay Kit (Upstate Biotechnology) according to the manufacturer's instructions with minor adjustments. Formaldehyde was added directly to the tissue immediately after harvesting, incubated for 15 minutes at 37°C, and washed 3 times in cold PBS. The lysates in 10 mmol/L Tris-HCl, pH 8.0, 1% SDS containing phosphatase and protease inhibitors were sonicated (Branson Sonifier 450; Branson) and the supernatant split into several aliquots. Ten microliters (1:100) of each antibody for H3 methyl K4 and H3 methyl K9 (Upstate Biotechnology) was added; the resulting DNA–protein complexes were isolated on protein G agarose beads and eluted with 1% SDS in 0.1 mol/L NaHCO₃. Cross-linking was reversed by incubation at 65°C for 5 hours. The samples were treated with proteinase K, the DNA extracted with phenol/chloroform, and dissolved in elution reagent. Immunoprecipitated DNA was analyzed by quantitative real-time PCR (qPCR) using p21 promoter–specific primers (Applied Biosystems).

Immunoblot analysis following standard procedures was done with total protein isolates using T-PER tissue protein extraction reagent (Thermo Scientific) with protease inhibitors (Roche). Fifty micrograms of total protein per lane was separated on a 4% to 20% tris-glycine gel and transferred to a nitrocellulose membrane. Antibodies against cleaved caspase-3 (Cell Signaling), Rad51, and DNA-PKcs (Abcam) were used at a dilution of 1:1,000 in PBS containing 5% BSA and 0.05% Tween-20. Horseradish peroxidase–conjugated rabbit secondary antibodies were used at 1:5,000 in 3% nonfat dry milk. The blots were developed using the ECL Plus Chemoluminescent Detection Kit (GE Healthcare) and the images acquired by Fuji LAS 4000.

At least 3 independent experiments were done for each point described. All values were expressed as mean ± SD. Student t test was used for paired data, and multiple comparisons were done with the ANOVA. A P value less than 0.05 was considered statistically significant.

Anticancer drugs and radiation have been shown to activate apoptosis pathways in solid tumors (29, 30). To determine whether ²¹²Pb-TCMC-trastuzumab induces apoptosis in the human colon carcinoma LS-174T xenografts, the TUNEL assay was done using paraffin-embedded tumor sections. The TUNEL assay results obtained at 24 hours after exposure to ²¹²Pb-TCMC-trastuzumab indicated a clear induction of apoptosis (Fig. 1A) that was greater than the other treatments. Next, the apoptotic bodies were quantitated using IHC and H&E staining. Apoptosis was noted on the basis of morphologic criteria described in the Methods. Quantitation of the apoptotic bodies showed that ²¹²Pb-TCMC-trastuzumab treatment significantly increased in apoptotic rates (Fig. 1B) compared with the ²¹²Pb-TCMC-HuIgG nonspecific control treatment at 24 to 72 hours (P < 0.05), suggesting greater effective targeted cell killing by ²¹²Pb-TCMC-trastuzumab in the tumor tissues.

Radiation causes DNA damage, the most harmful in terms of cellular cytotoxicity being double-strand breaks (DSB). An increase in DNA DSBs and impaired DNA damage repair has been invoked to explain the synergy between drugs and ionizing radiation (31). To investigate DNA damage by ²¹²Pb-TCMC-trastuzumab, IHC was done using paraffin-embedded tumor sections. Induction of DNA DSB damage was evident as measured by phosphorylated H₂AX, a marker for DNA double-strand damage (Fig. 2A). The neutral comet assay was also used to show physical DNA strand damage. Enhancement of the fluorescence intensity of tail was observed in both ²¹²Pb-TCMC-trastuzumab- and ²¹²Pb-TCMC-HuIgG–treated tumors. Next, to determine the effect of ²¹²Pb-TCMC-trastuzumab on DNA damage repair, DNA content in the tails were compared at different time points up to 96 hours after the various treatments. After 24 hours, ²¹²Pb-TCMC-trastuzumab showed significantly higher percent DNA in the tail than any other treatment, including ²¹²Pb-TCMC-HuIgG. At 24 hours, 18% of the tumor sample presented with DNA strand breaks. There was a steady decrease in the percentage of DNA damage and by 96 hours the percentage was 10. Thus, by 96 hours, inhibition of DNA damage repair elicited by ²¹²Pb-TCMC-trastuzumab was not yet been completely reversed. In comparison, the ²¹²Pb-TCMC-HuIgG resulted in 13% DNA strand breaks at 24 hours. By the 96-hour time point, DNA in the tail for ²¹²Pb-TCMC-HuIgG was similar to that of unlabeled HuIgG, indicating that DNA damage repair had returned to normal levels following treatment. This showed that DNA damage repair was compromised to a greater extent by the treatment with ²¹²Pb-TCMC-trastuzumab (Fig. 2B). These results suggested that there was a general effect attributable to α-radiation; however, there was a greater specific effect attributable to the ²¹²Pb-TCMC-trastuzumab (P < 0.001).

DSBs can be repaired by different pathways, the most important of which are homologous repair (HR) and nonhomologous end joining (NHEJ; ref. 32). To identify the DNA repair pathways involved in the study presented herein, Rad51 and DNA-PKcs, which play important roles in HR and NHEJ, respectively, were investigated. The densitometric analysis of the Western blots illustrated in Fig. 2C shows that at the protein expression level, Rad51 was significantly downregulated by ²¹²Pb-TCMC-trastuzumab (P < 0.05) treatment. However, Rad51 was not affected in the presence of the unlabeled trastuzumab, HuIgG, or ²¹²Pb-TCMC-HuIgG at 24 hours. DNA-PKcs was not affected following any of the treatments, suggesting that differences in induction of the HR pathway protein Rad51 may, at least in part, be responsible for the delayed DNA damage repair following the ²¹²Pb-TCMC-trastuzumab treatment.

To examine the involvement of caspase-3, which plays an important role in the condensation and degradation of chromatin of apoptotic cells in ²¹²Pb-TCMC-trastuzumab–induced apoptosis, cleaved caspase-3 was analyzed using immunoblot techniques. Cleaved caspase-3 was observed in the tumor tissues 24 hours after treatment with ²¹²Pb-TCMC-HuIgG (Fig. 2D). In contrast to this result, it seemed that ²¹²Pb-TCMC-trastuzumab treatment significantly reduced the level of cleaved caspase-3 (P < 0.001), suggesting that ²¹²Pb-TCMC-trastuzumab–induced apoptosis occurred via a caspase-3–independent mechanism.

In response to radiation, cells typically require cell-cycle arrest at G₁ and a slow S-phase to allow DNA repair. As a consequence of reduced intracellular signaling, trastuzumab as a sole agent induces early escape from this cycle arrest and thereby promotes an accumulation of DNA damage (33). The effect of ²¹²Pb-TCMC-trastuzumab treatment on cell-cycle distribution was examined. In the same study, mice were injected with BrdUrd 4 hours prior to tumor collection to pulse label the tumor xenografts to evaluate DNA synthesis. DNA content and BrdUrd incorporation was then determined using a 2-parameter analysis by flow cytometry. As shown in Table 1, tumor cells harvested from untreated mice were found to have the expected uptake of BrdUrd (23.8 ± 1.3%); the resulting cell-cycle distribution was also in the expected range (Table 2). On the other hand, 6 hours after ²¹²Pb-TCMC-trastuzumab treatment, there was a noticeable decrease in BrdUrd incorporation (13.7 ± 1.2%). BrdUrd incorporation decreased further to 2.6 ± 0.7% at 48 hours and remained at this low level throughout the remainder of the 120-hour study period. In contrast to ²¹²Pb-TCMC-trastuzumab treatment, however, DNA synthesis after treatment with ²¹²Pb-TCMC-HuIgG did reinitiate by 120 hours after decreasing at the early time points. The continuation of DNA synthesis by the tumors that were treated with unlabeled trastuzumab, or HuIgG alone, provided evidence that the cessation of DNA synthesis is specific to the α-radiation and that continued depression of DNA synthesis after 120 hours was specific to the targeted ²¹²Pb-TCMC-trastuzumab treatment. In addition, there was a decrease in the S-phase fraction with a corresponding increase in the G₂-M phase fraction beginning at 24 hours compared with the unlabeled analogs. The lower S-phase and the elevated G₂-M phase fractions were maintained throughout the 120-hour study period for the tumors upon ²¹²Pb-TCMC-trastuzumab treatment. In contrast, the cell-cycle distribution rebounded in those tumors that were collected from mice treated with ²¹²Pb-TCMC-HuIgG. The initial depression of the S-phase fraction and elevation of G₂-M phase fraction seemed to be α-radiation related because tumors collected from mice treated with either antibody alone (not labeled with ²¹²Pb) did not seem to experience any alteration in cell-cycle distribution.

Chromatin remodeling is significantly altered in tumors, suggesting a direct role for methylation in cellular transformation. γH₂AX facilitates the recruitment of damage-responsive proteins and chromatin remodeling complexes to the sites of DNA damage and influences both the efficiency and fidelity of DNA repair (34). To determine whether changes occur in chromatin remodeling following ²¹²Pb-TCMC-trastuzumab treatment, the ChIP assay was employed using p21 promoter–specific primers, one of the known radiation response genes, and immunoprecipitated DNA was analyzed by a qPCR. The ratio between H3K4 methylation and H3K9 methylation was used as a measurement of change (open/close) in chromatin structure (35–37). The abundance of histone modifications identified with transcriptionally activated chromatin states, such as H3 methylated at lysine 4, was observed at 48 hours after treatment with ²¹²Pb-TCMC-HuIgG and at 72 hours after ²¹²Pb-TCMC-trastuzumab treatment (Fig. 3). It seems that at earlier time points, histone modifications associated with transcriptionally repressed chromatin states, such as H3 methylated at lysine 9, prevailed on treatment with ²¹²Pb-TCMC-trastuzumab, indicating that ²¹²Pb-TCMC-trastuzumab induced the delayed open chromatin structure until 72 hours.

To assess the induction of p21 in response to DNA damage, Western blot analysis of p21 was done. Enhanced protein expression of p21 was observed at 24 to 48 hours after ²¹²Pb-TCMC-HuIgG treatment, whereas ²¹²Pb-TCMC-trastuzumab treatment resulted in enhanced p21 expression at 72 hours (Fig. 4A). These results suggested there were correlations between induction of p21 protein and modification in chromatin structure in response to ²¹²Pb-TCMC-trastuzumab and ²¹²Pb-TCMC-HuIgG treatment. Selecting the 24-hour time point, further analysis was done to compare the effect of all the treatments on the LS-174T xenograft. There was also a significant reduction of p21 protein at 24 hours (P < 0.05) compared with a nonspecific control, indicating that reduction of p21 protein expression was specific to ²¹²Pb-TCMC-trastuzumab treatment (Fig. 4B).

Unlike β⁻ particle or photon irradiation, α-particle radiation is cytotoxic at dose rates as low as 1 Gy/h. The shorter path of α particles may also have the advantage of limiting toxicity to normal tissue adjacent to tumor. Studies from this laboratory have shown the exquisite effectiveness of both ²¹³Bi- and ²¹²Pb-labeled trastuzumab for the treatment of low HER2-expressing intraperitoneal disease (6–8). A unique advantage of RIT versus monotherapy with trastuzumab is that neither high expression nor homogenous expression of HER2 throughout the tumor is required to affect therapy. One strategy to overcome the limitations of the shorter half-lives of the bismuth radioisotopes is to treat with ²¹²Pb (t_1/2 = 10.6 hours), which essentially serves as an in vivo generator. The application of ²¹²Pb-labeled trastuzumab in the appropriate setting has shown advantages over ²¹³Bi-labeled trastuzumab (6–8). These studies provided sufficient impetus for the in vivo assessment of the mechanisms of therapeutic efficacy of ²¹²Pb-TCMC-trastuzumab therapy.

The effect of ²¹²Pb-TCMC-trastuzumab treatment on apoptosis seemed to be more pronounced in the targeted cells at 24 hours (P < 0.05) compared with the nonspecific control, ²¹²Pb-TCMC-HuIgG. In prior observations, trastuzumab was reported to enhance radiation-induced apoptosis of cells in an HER2 level–dependent manner (38). Although the results obtained in this study with ²¹²Pb-TCMC-trastuzumab treatment seems to be similar to the prior report with respect to the enhancement of apoptosis, the 2 situations are inherently different. In the experiments presented here, trastuzumab was used as a vector to specifically direct α-particle radiation payload to the tumor. In this instance, trastuzumab was not expected to exert any pharmacologic effects. Irradiated cells release signals and induce responses in cells whose nuclei were not hit by radiation, resulting in genetic damage, genomic instability, or cell death. A high apoptotic rate was also observed for the nonspecific control ²¹²Pb-TCMC-HuIgG–treated group. Consideration must be given to this indirect effect of radiation because transmissible biologic effects resulting from the radiation insult are pronounced following high LET radiation, such as α-particle irradiation.

Direct injury to DNA is generally attributed to radiation therapy. Increased DNA repair has been shown in tumor cells resistant to radiation and anticancer drugs, in comparison with tumor cells sensitive to these modalities. Synergy between drugs and ionizing radiation is attributed to impaired repair of residual DNA DSBs (39). As shown from the percent DNA in the comet tail comparison, DNA damage following ²¹²Pb-HuIgG radiation showed gradual recovery beginning at 24 hours posttreatment. On the other hand, DNA repair inhibition was more pronounced in the mice treated with ²¹²Pb-TCMC-trastuzumab (P < 0.001), as indicated by the persistently higher % DNA in the comet tail at all time points studied. There seemed to be recovery of DNA repair for the ²¹²Pb-TCMC-HuIgG–treated tumors, although recovery was not evident after the ²¹²Pb-TCMC-trastuzumab treatment. These results were further investigated to understand which repair systems were involved. NHEJ and HR are predominant mechanisms in DNA DSB repair. High-level Rad51 expression has been reported in chemoresistant or radioresistant carcinomas (40). Upregulation of Rad51 after irradiation has been previously observed in eukaryotic cells (41). In this study, Rad51 was not affected in the presence of ²¹²Pb-TCMC-HuIgG at 24 hours. In contrast to this result, densitometric analysis of the Western blots clearly indicated that the ²¹²Pb-TCMC-trastuzumab treatment significantly reduced Rad51 at the protein level (P < 0.05). Therefore, inhibition of DNA damage repair induced by ²¹²Pb-TCMC-trastuzumab treatment, evidenced by the reduction of Rad51 protein expression and by the persisted inhibition of DNA damage repair, may be an explanation for the increased cell killing efficacy of ²¹²Pb-TCMC-trastuzumab treatment.

After irradiation with ²¹²Pb, cleaved caspase-3 was observed, albeit at a lower level, after ²¹²Pb-TCMC-trastuzumab treatment (P < 0.001) compared with ²¹²Pb-TCMC-HuIgG treatment, suggesting that apoptosis induced by ²¹²Pb-TCMC-trastuzumab treatment was not dependent on caspase-3. Various studies also reported that high LET α-particle immunoconjugates induced apoptosis and that the mode of cell death triggered by α-particle emitters seemed to be dependent on the type of cells irradiated (42–46).

The lower levels of DNA synthesis that were observed following ²¹²Pb-TCMC-trastuzumab treatment at the early time points persisted beyond 120 hours, but seemed to rebound for the ²¹²Pb-TCMC-HuIgG–treated tumors by the same time point. A similar temporal progression was observed for the phase distribution of cells in which cells were arrested in the G₂-phase, with a severely depressed S-phase at the early time points on ²¹²Pb treatment that seemed to rebound for ²¹²Pb-TCMC-HuIgG–treated mice by 120 hours, but not for the group treated with ²¹²Pb-TCMC-trastuzumab. Notably, tumors from mice given ²¹²Pb-TCMC-trastuzumab remained arrested at the G₂-M phase with depressed S-phase beyond 120 hours. Synchronization in the G₂ phase after 120-hour treatment, together with persistent reduced DNA synthesis, seemed the most prominent difference between the targeted ²¹²Pb-TCMC-trastuzumab and the control ²¹²Pb-TCMC-HuIgG treatment. Such synchronization has been described as the major course of synergy between chemotherapy and external beam irradiation, although radiosensitization may not be achieved in all cell lines or tumors (47). The arrest of cells in the radiosensitive G₂-M phase of the cell cycle induced by ²¹²Pb-TCMC-trastuzumab treatment may be another explanation for the increased cell killing efficacy of α-particle RIT. These results are bolstered by the persistently delayed DNA repair on ²¹²Pb-TCMC-trastuzumab treatment indicated by the comet assay compared with the recovery of DNA repair at later time points on ²¹²Pb-TCMC-HuIgG treatment.

Epigenetic markers for open and closed chromatin status using H3K4/H3K9 ratio revealed correlations between the induction of p21 protein and modification in chromatin structure of p21 in response to ²¹²Pb-TCMC-trastuzumab and ²¹²Pb-TCMC-HuIgG treatment. In contrast to the results of ²¹²Pb-TCMC-HuIgG control, induction of p21 protein and open chromatin structure was delayed until 72 hours by ²¹²Pb-TCMC-trastuzumab, indicating that inhibition of p21 protein at early time points was associated with histone modifications that correlated with repressed transcription. In fact, there was a significant reduction of p21 at the protein level at 24 hours by ²¹²Pb-TCMC-trastuzumab (P < 0.05). These results indicate that the increased killing efficacy of ²¹²Pb-TCMC-trastuzumab treatment was, in part, associated with the delay in open chromatin structure of p21 that correlated to less active transcription at earlier time points. Wendt and colleagues reported that radiation-induced p21 expression and G₂ arrest results in resistance to apoptosis and inhibition of p21 displayed enhanced radiation-induced apoptosis (48).

Understanding the mechanisms of cell death and DNA repair is critical to the design of novel strategies that combine chemotherapy with targeted α-particle radiation. Studies of this nature help refine and optimize all of the components to improve efficacy and minimize toxicity. Carefully planned preclinical investigation and improved targeting strategies will facilitate translation into clinical evaluation to move the field forward. Studies are currently underway to evaluate the potential mechanism(s) of chemotherapeutics in combination with α-particle RIT.

No potential conflicts of interest were disclosed.

This research was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.