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Clinical Cancer Research Vol. 11, 7047s-7052s, October 1, 2005
© 2005 American Association for Cancer Research

Experimental Studies

Mechanisms of Cell Sensitization to {alpha} Radioimmunotherapy by Doxorubicin or Paclitaxel in Multiple Myeloma Cell Lines

Stephane Supiot1,2, Sebastien Gouard1, Josiane Charrier2, Christos Apostolidis3, Jean-Francois Chatal1,2, Jacques Barbet1, François Davodeau1 and Michel Cherel1,2

Authors' Affiliations: 1 Institut National de la Sante et de la Recherche Medicale U601, Moncousu, Nantes, France; 2 Centre René Gauducheau, Nantes-St. Herblain, France; and 3 Transuranium Institute, Karlsruhe, Germany

Requests for reprints: Michel Cherel, Institut National de la Sante et de la Recherche Medicale U601, 9 quai Moncousu, 44093 Nantes, France. Phone: 33-2-4008-4747; Fax: 33-3-4035-6697; E-mail: Michel.Cherel{at}univ-nantes.fr.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: The purpose of this study was to analyze different mechanisms (cell cycle synchronization, DNA damage, and apoptosis) that might underlie potential synergy between chemotherapy (paclitaxel or doxorubicin) and radioimmunotherapy with {alpha} radionuclides.

Experimental Design: Three multiple myeloma cell lines (LP1, RMI 8226, and U266) were treated with 213Bi-radiolabeled B-B4, a monoclonal antibody that recognizes syndecan-1 (CD138) 24 hours after paclitaxel (1 nmol/L) or doxorubicin (10 nmol/L) treatment. Cell survival was assessed using a clonogenic survival assay. Cell cycle modifications were assessed by propidium iodide staining and DNA strand breaks by the comet assay. Level of apoptosis was determined by Apo 2.7 staining.

Results: Radiation enhancement ratio showed that paclitaxel and doxorubicin were synergistic with {alpha} radioimmunotherapy. After a 24-hour incubation, paclitaxel and doxorubicin arrested all cell lines in the G2-M phase of the cell cycle. Doxorubicin combined with {alpha} radioimmunotherapy increased tail DNA in the RPMI 8226 cell line but not the LP1 or U266 cell lines compared with doxorubicin alone or {alpha} radioimmunotherapy alone. Neither doxorubicin nor paclitaxel combined with {alpha} radioimmunotherapy increased the level of apoptosis induced by either drug alone or {alpha} radioimmunotherapy alone.

Conclusion: Both cell cycle arrest in the G2-M phase and an increase in DNA double-strand breaks could lead to radiosensitization of cells by doxorubicin or paclitaxel, but apoptosis would not be involved in radiosensitization mechanisms.

{alpha}-Emitting radioisotopes might be more effective for metabolic radiotherapy than other radionuclides because of their high linear energy transfer and short pathways and because they can target radiation to individual cancer cells (1) and micrometastases (2). Early results with {alpha} radioimmunotherapy have been encouraging (1). Like other radionuclides, they could be combined with chemotherapeutic drugs as this strategy has a strong biological rationale (3). However, up to now, most studies have considered combining drugs with X-rays or {gamma}-rays, and radiosensitizing of cells by paclitaxel to {gamma}-rays or X-rays (4, 5) has been attributed to three mechanisms: interactions with the cell cycle, apoptosis, and tumor hypoxia (6), those of doxorubicin to induction of DNA damage (7, 8) and tumor hypoxia (9). Paclitaxel does not induce DNA damage but promotes microtubule polymerization (10). Our preliminary investigations on three myeloma cell lines (LP1, RPMI 8226, and U266) have shown that doxorubicin or paclitaxel combined with {alpha} radioimmunotherapy is more effective than drug alone or {alpha} radioimmunotherapy alone. In accordance with results obtained with other forms of radiation (6), the effect was stronger when {alpha} radioimmunotherapy was given 24 hours after doxorubicin or paclitaxel. Experiments are in progress to formally show synergism between {alpha} radioimmunotherapy and these drugs (11).

We have investigated several mechanisms that might give rise to synergy between paclitaxel or doxorubicin and {alpha} radioimmunotherapy. We studied cell cycle arrest in the G2-M phase, DNA strand breaks, and apoptosis in the three multiple myeloma cell lines pretreated or not with doxorubicin or paclitaxel and exposed during 12 hours to {alpha} radioimmunotherapy using the anti-syndecan-1 (CD138) antibody B-B4 labeled with bismuth-213 (213Bi).


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Human myeloma cell lines. The human myeloma cell lines, RPMI 8226 and U266, were obtained from the American Type Culture Collection (Rockville, MD), and LP1 was obtained from DSMZ (Braunschweig, Germany). These cell lines were cultured in RPMI 1640 (Bio Whittaker Europe, Verviers, Belgium) supplemented with 10% heat-inactivated FCS (Biomedia, Boussens, France) and 2 mmol/L L-glutamine, at 37°C, 5% CO2 and 100% humidity. Cells were transferred into fresh culture medium, and cell concentration was adjusted to 5 x 105 cells/mL, 48 hours before each assay.

Monoclonal antibodies. B-B4 monoclonal antibody (mAb), a murine IgG that recognizes syndecan-1 (CD 138) antigen, was kindly provided by Dr. John Wijdenes (Diaclone Research, Besançon, France; ref. 12). A sample of this antibody (50 mg/g) was allowed to react with twice-crystallized pepsin (Sigma, St. Louis, MO) for 2 hours at 37°C. The mixture was fractionated by exclusion-diffusion on a Superdex G200 column (Pharmacia, Guyancourt, France) equilibrated with 100 nmol/L phosphate buffer to obtain F(ab')2 fragments for use in all experiments at a 2 nmol/L concentration.

Monoclonal antibody labeling. The 213Bi generator was kindly provided by the TransUranium Institute (Karlsruhe, Germany). 213Bi was selectively eluted by 2 mL of a solution containing 0.1 mol/L HCl and 0.1 mol/L NaI. B-B4 antibody was conjugated to the bifunctional chelating agent cyclohexyldiethylenetriaminepentaacetic acid, synthesized in our laboratory, as described by Brechbiel et al. (13). The antibody was incubated with 50 eq (mol/mol) cyclohexyldiethylenetriaminepentaacetic acid in HEPES buffer (0.1 mol/L, pH 8), and after overnight incubation at room temperature, purified by high-performance liquid chromatography on a Sephadex G200 gel-filtration column (Amersham Biosciences, Saclay, France). Mean chelate number per antibody, as assessed with 4 eq of buffered citrate-acetate (0.02-0.15 mol/L, pH 5.5) 111In solution, was 2. B-B4 immunoreactivity, determined as described by Lindmo et al. (14), was modified only slightly by addition of chelate (>90%). A 10- to 100-µg sample of B-B4-cyclohexyldiethylenetriaminepentaacetic acid was incubated in a solution of freshly eluted 213Bi, for 15 minutes at 37°C, as described by Kaspersen et al. (15). The specific activity of the radiolabeled antibody was 150 MBq/mg. Radiochemical purity, checked by instant TLC-SG using 10% TCA as solvent, was >90%.

Drugs. Doxorubicin was purchased from Pharmacia and prepared extemporaneously in 9% NaCl. Paclitaxel was purchased from Bristol-Myers Squibb (Puteaux, France). Both drugs were diluted in culture medium to the desired final concentration. Drug concentrations leading to ~50% cell survival were chosen (10 and 1 nmol/L for doxorubicin and paclitaxel, respectively).

Clonogenic survival assays. Cell survival was assessed using the limiting dilution method. After treatment with drug followed by {alpha} radioimmunotherapy 24 hours after, the cells were incubated at 37°C for 24 hours. They were then washed with fresh medium, diluted, and plated in 48 replicates in 96-well round-bottomed plates at concentrations of 1,000, 333, 111, 37, 12, 4, 1, and 0.5 cells per well. Each replicate was assayed for clonogenic growth by macroscopic and microscopic observation. A positive score was given only to replicates containing colonies of >64 cells. The best-fit survival curve was generated according to an exponential decreasing survival model and the mean inactivation dose (MID, area under survival curve) was calculated. The radiation enhancement ratio (RER) was defined as RER = MID {alpha} radioimmunotherapy / MID drug + {alpha} radioimmunotherapy. A RER value of >1 is indicative of radiosensitization. Survival curves for combined treatment were corrected for drug-induced cytoxicity.

Measurement of cell cycle distribution. Cells (1 x 106) were pelleted, resuspended in 0.2 mL PBS, and fixed by addition of 2 mL of ice-cold 70% ethanol/30% PBS. Fixed cells were pelleted, vigorously suspended in PBS, and incubated for 30 minutes at 37°C with 100 µg/mL RNase A (Sigma-Aldrich, St. Louis, MO) and 40 µg propidium iodide (Sigma-Aldrich, Saint-Quentin Fallavier, France). The fluorescence of the stained cells was analyzed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA). Data were analyzed with ModFit LT2 (Becton Dickinson).

Detection of apoptosis by Apo 2.7 staining. The percentage of apoptotic cells was determined by flow cytometry using Apo 2.7 mAb as described by Koester et al. (16). Cells (2.5 x 105) were incubated at 4°C for 20 minutes with Apo 2.7 mAb (Immunotech, Marseilles, France) in PBS for simple staining and then washed and fixed in PBS 1% formaldehyde. Flow cytometry analysis was done using a FACSCan and the CELLQuest program (Becton Dickinson).

Single-cell gel electrophoresis. We used the protocol of Alapetite et al. (17) with minor modifications. After mild trypsination, a suspension of tumor cells was mixed with low melting point agarose held at 37°C to obtain a final concentration of 0.5% agarose and 5 x 104 cells/mL. The suspension (170 µL) was spread on a frosted microscope slide precoated with a layer of 200 µL of normal melting point agarose. DNA damage was determined 12 hours after irradiation (36 hours after drug addition). The slides coated with agarose-embedded tumor cells were immersed for 1 hour in a cold lysis solution [2.5 mol/L NaCl, 100 mmol/L EDTA, 10 mmol/L Tris, 1% N-lauryl sarcosine, 1% Triton X-100, 10% DMSO (pH 10, 4°C)] then removed and transferred for 40 minutes into fresh alkaline buffer [300 mmol/L NaOH, 1 mmol/L EDTA (pH 13)]. They were then transferred to a gel box containing the same buffer for horizontal electrophoresis (20 minutes at 25 V and 300 mA). After which, they were rinsed twice for 5 minutes with neutralization buffer (0.4 mol/L Tris, pH 7.5), stained with 65 µL ethidium bromide (8 ng/µL), covered with a glass, and left for 30 minutes in the dark at 4°C. During all these steps, they were screened from direct sunlight. The slides were examined with an epifluorescence microscope (Leica, Rueil Malmaison, France). Comets were randomly captured at a constant gel depth and away from the edge of the gel. Superimposed comets were avoided. The fluorescence image of each captured comet was acquired with a digital camera KP-M1 (Hitachi, Tokyo, Japan) and analyzed with image analysis software (Komet version 4.0 from Kinetic Imaging, BSI Optilas, Evry, France). The tail DNA content of the comets was assessed and compared with control. In each experiment, 25 comets per drug concentration were analyzed.

Statistical analysis. All values are reported as means ± SE. Data were analyzed using the Student's t test or the Mann-Whitney rank sum test (SIGMASTAT software, Jandel Scientific, Erbrath, Germany). The significance level was set at P < 0.05.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Combined treatment of myeloma cells with {alpha} radioimmunotherapy and paclitaxel or doxorubicin. Drugs were added to cells at time 0. Radiolabeled antibody was added 24 hours later. Cells were incubated with drugs for 34 hours. The 12-hour incubation time is sufficient for complete 213Bi decay (15 periods). As shown in Fig. 1, paclitaxel and {alpha} radioimmunotherapy (Fig. 1A) synergized strongly in all three cell lines. A 48-hour incubation with 1 nmol/L paclitaxel gives RER values of 1.66, 1.63, and 1.42, for LP1, RPMI 8226, and U266 cells, respectively. Doxorubicin and {alpha} radioimmunotherapy (Fig. 1B) synergized moderately in all three cell lines. With 10 nmol/L doxorubicin, survival curves gives a RER value of 1.17 for LP1, 1.20 for RPMI 8226, and 1.19 for U266 cells.



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  		Fig. 1. Relative survival after treatment with {alpha} radioimmunotherapy ({alpha}-RIT) combined with 1 nmol/L paclitaxel (A) or 10 nmol/L doxorubicin (B). Specific activity of 213Bi-cyclohexyldiethylenetriaminepentaacetic acid (CHX DTPA)-B-B4: 96 MBq/mg for LP1, 150 MBq/mg for RPMI 8226, and 120 MBq/mg (paclitaxel, TXL) or 150 MBq/mg (doxorubicin, DOX) for U266. {square}, {alpha} radioimmunotherapy; {blacksquare}, drug + {alpha} radioimmunotherapy. Survival curves for combined treatment have been corrected for drug-induced cytoxicity. Means of at least two experiments; bars, SE.

 
Cell cycle arrest by paclitaxel and doxorubicin. As shown in Fig. 2, even low paclitaxel concentrations induced a major redistribution of the cell cycle. All three multiple myeloma cell lines were arrested in the G2-M phase when incubated with increasing concentrations of paclitaxel. The percentage of cells arrested increased with time but U266 cells remained unaffected until 6 hours of incubation. After 24 hours, >50% of cells were arrested in all three cell lines.



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  		Fig. 2. Cell synchronization by drug treatment. The proportion of cells arrested in the G2-M phase of the cell cycle after doxorubicin and paclitaxel treatment at different incubation times is shown as a function of concentration for the three multiple myeloma cell lines. Square, LP1; triangle, RPMI 8226; open circle, U266.

 
Cell arrest in the G2-M phase was slower with doxorubicin than with paclitaxel. Arrest of LP1 and RPMI 8226 cells did not occur before 6 hours of incubation. After 24 hours of incubation, all three cell lines were arrested. As for paclitaxel, the percentage of cells arrested at 24 hours was higher for RPMI 8226 and LP1 than for U266.

Doxorubicin-induced DNA damage. Cell response to potential strand-breaking agents was determined by the alkaline single-cell gel electrophoresis assay (or comet assay). Interrupted DNA strands of agarose-embedded cells may, after lysis and electrophoresis, migrate out of the nucleus toward the anode, forming a comet tail that can be visualized with a fluorescent DNA binding dye. The tail is related to the number of DNA breaks in the cells. Incubation of cells with 213Bi-cyclohexyldiethylenetriaminepentaacetic acid-B-B4 induced DNA strand breaks in RPMI 8226 cells (relative tail DNA: 1.95 ± 0.32, P = 0.01; Fig. 3). Incubation of cells with doxorubicin induced DNA strand breaks in RPMI 8226 cells (1.30 ± 0.25, P = 0,02). By contrast, no increase in DNA strand breaks was noted in LP1 or U266 cells, whether after incubation of {alpha} radioimmunotherapy (1.15 ± 0.35, 1.04 ± 0.48, respectively) or doxorubicin (1.17 ± 0.5, 0.78 ± 0.29, respectively).



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  		Fig. 3. Tail DNA content measured by the comet assay. Tail DNA content is given as a ratio compared with tail DNA content in untreated control cells. For all three cell lines, the bars correspond to C, untreated control; {alpha}-RIT, {alpha} radioimmunotherapy treatment with 2 nmol/L 213Bi-cyclohexyldiethylenetriaminepentaacetic acid (CHX-A-DTPA)-B-B4 at a specific activity of 150 MBq/mg for 12 hours; DOX, treatment with 10 nmol/L doxorubicin for 36 hours; {alpha} radioimmunotherapy + doxorubicin: treatment with doxorubicin for 36 hours and {alpha} radioimmunotherapy (213Bi-CHX-A-DTPA-B-B4) during the last 12 hours.

 
RPMI 8226 cells exposed to doxorubicin and then to {alpha} radioimmunotherapy showed a higher number of DNA strand breaks (relative tail DNA: 2.86 ± 0.3) than those exposed to doxorubicin alone (P = 0.028) or to {alpha} radioimmunotherapy alone (P = 0.05). The number of strand breaks remained unchanged for LP1 cells (1.47 ± 1.1) and U266 cells (1.15 ± 0.6) exposed to doxorubicin and {alpha} radioimmunotherapy.

Apoptotic effects of paclitaxel or doxorubicin combined with {alpha} radioimmunotherapy. Apoptosis was monitored by fluorescence staining and flow cytometry with the antibody Apo2.7. The Apo 2.7 mAb recognizes 7A6, a 38-kDa antigen exposed on the outer mitochondrial membranes of cells undergoing apoptosis (18). 7A6 is detected on apoptotic cells, but not on normal-surface or digitonin-permeabilized cells. 7A6 antigen is revealed early during the apoptotic cascade before caspase-3 activation (19). Apo 2.7 mAb can discern apoptotic and incomplete apoptotic cells from necrotic cells (20) and has been used to quantify myeloma cell apoptosis in patients (21). {alpha} Radioimmunotherapy induced apoptosis of LP1 cells (18% versus 8%, P < 0.001) and RPMI 8226 cells (27% versus 15%, P < 0.001) compared with controls (Fig. 4). By contrast, the level of apoptosis in {alpha} radioimmunotherapy–treated U266 cells was not significantly different from that of controls (8% versus 6%, not significant). Paclitaxel induced significantly (P < 0.05) higher apoptosis levels (21%, 41%, and 15%) than doxorubicin (13%, 24%, and 8%) in LP1, RPMI 8226, and U266 cells, respectively. In U266 cells, paclitaxel doubled the number of apoptotic cells compared with controls (15% versus 6.2%, P = 0.015), whereas {alpha} radioimmunotherapy or doxorubicin had no significant effect.



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  		Fig. 4. Apoptosis after combined treatment. Apoptosis is given by the percentage of Apo 2.7–positive cells measured by fluorescence cytometry. For all three cell lines, the bars correspond to C, untreated control; {alpha}-RIT, {alpha} radioimmunotherapy treatment [2 nmol/L 213Bi-cyclohexyldiethylenetriaminepentaacetic acid (CHX-A-DTPA)-B-B4 at a specific activity of 150 Mbq/mg] for 12 hours; DOX, treatment with doxorubicin for 36 hours; {alpha} radioimmunotherapy + doxorubicin: treatment with doxorubicin for 36 hours and {alpha} radioimmunotherapy (213Bi-CHX-A-DTPA-B-B4) during the last 12 hours; TXL, treatment with 1 nmol/L paclitaxel for 36 hours; {alpha} radioimmunotherapy + paclitaxel, treatment with paclitaxel for 36 hours and {alpha} radioimmunotherapy (213Bi-CHX-A-DTPA-B-B4) during the last 12 hours.

 
Although the apoptosis level was slightly higher after combined treatment (paclitaxel or doxorubicin + {alpha} radioimmunotherapy) compared with treatment with paclitaxel (or doxorubicin) alone or {alpha} radioimmunotherapy alone, this increase was not statistically significant.


   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
RER measured for the combination of {alpha} radioimmunotherapy and paclitaxel or doxorubicin treatment clearly shows synergy. This synergy is especially strong with paclitaxel. This is similar to results obtained with external beam treatments or conventional radioimmunotherapy with ß-emitting radionuclides. This observation may seem puzzling as {alpha}-particles have been presented as capable of inducing unrepairable DNA damage. However, we observed more subtle effects, such as apoptosis or cell cycle arrest, in response to {alpha} particles. Thus, complex mechanisms are involved in {alpha}-particle irradiation that may be sensitive to radiosensitization. Experiments to formally show the synergy between paclitaxel or doxorubicin and {alpha}-immunotherapy are in progress and will be published elsewhere.

Here we have investigated three mechanisms that might underlie the synergy observed between paclitaxel or doxorubicin and {alpha} radioimmunotherapy. We shall consider each of these mechanisms in turn.

Synchronized cells irradiated during the G2-M phase are more radiosensitive than cells irradiated in G1 or S (22). Maximal radiosensitivity occurs during mitosis. The powerful arrest of cells in the radiosensitive G2-M phase of the cell cycle induced by taxanes is thought to account for the synergy between taxanes and {gamma}-rays or X-rays (23–25). Paclitaxel or doxorubicin synchronized cells in the G2-M phase of the cell cycle in three multiple myeloma cell lines. G2-M arrest was faster with paclitaxel than with doxorubicin, but after 24 hours of incubation, the percentage of cells in G2-M was similar for 1 nmol/L paclitaxel and 10 nmol/L doxorubicin, except for U266 cells, which showed a lower percentage of cell-arrest than LP1 or RPMI 8226 cells. The observed cell cycle arrest in the G2-M phase may be an explanation for the increased cell killing efficacy of {alpha}-immunotherapy given 24 hours after the initiation of drug treatment.

An increase in DNA strand breaks and impaired DNA repair have been invoked to explain synergy between drugs and ionizing radiation (26). Doxorubicin had a additive effect with {alpha} radioimmunotherapy on induction of DNA damage in one of the three cell lines (RPMI 8226), as determined by the alkaline comet test, which measures both simple and double-strand breaks. This is in line with published observations on the synergy between doxorubicin and X-rays (8), which was attributed to residual DNA double-strand breaks (7). The comet assay thus probably measured mostly unrepaired DNA double-strand breaks. Doxorubicin or {alpha} radioimmunotherapy or their combination did not induce a measurable increase in DNA strand breaks in U266 and LP1 cell lines. Reasons for this could be that cells killed by {alpha} radioimmunotherapy disappear rapidly and those that survive are repaired before 24 hours. Unfortunately, measurement of DNA strand breaks at earlier time points are difficult to do because of technical constraints (delivery of the irradiation dose by the {alpha}-emitting radioisotopes takes several hours).

Paclitaxel does not induce DNA damage. In vivo studies have suggested that its antitumor effects are due to apoptosis rather than to cell cycle modifications (27). Paclitaxel act on apoptosis by inducing phosphorylation of BCL2 (28, 29), particularly in multiple myeloma cell lines (30, 31). Doxorubicin also acts through apoptotic mechanisms (32). Because {alpha} radioimmunotherapy increases the number of apoptotic multiple myeloma cells (33), apoptotic mechanisms could be related to the synergy between the drugs and {alpha} radioimmunotherapy. However, our results do not support this hypothesis. Increased apoptosis was observed for LP1 and RPMI 8226 cells treated with {alpha} radioimmunotherapy, doxorubicin, or paclitaxel. Apoptosis was also induced in U266 cells by paclitaxel but not by {alpha} radioimmunotherapy or doxorubicin even at high drug concentrations (5 nmol/L, data not shown) or after long observation times (up to 72 hours, data not shown). However, neither paclitaxel nor doxorubicin significantly increased {alpha} radioimmunotherapy–induced apoptosis in any cell line. Longer observation (up to 72 hours) did not reveal any significant difference between the three cell lines (data not shown).

In conclusion, synchronization in the G2-M phase seems the most prominent effect of drug treatment in the three myeloma cell lines. Such a synchronization has been described as the major course of synergy between chemotherapy and external beam irradiation, although radiosensitization may not be seen in some cell line (34). Thus, synchronization in the G2-M phase may also be the major cause of the observed synergy between paclitaxel and doxorubicin on {alpha} radioimmunotherapy in myeloma cell lines. By contrast, DNA breaks increase is significant only in RPMI 8226 cells after combined doxorubicin and {alpha} radioimmunotherapy treatment and apoptosis is not enhanced by combined treatment.


   Acknowledgments
 
We thank Guy Blain (Subatech) for technical support.


   Footnotes
 
Grant support: Association pour la Recherche sur le Cancer (grant 4752) and Institut National de la Sante et de la Recherche Medicale, "poste-accueil" (S. Supiot).

Presented at the Tenth Conference on Cancer Therapy with Antibodies and Immunoconjugates, October 21-23, 2004, Princeton, New Jersey.


   References
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 Abstract
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 Results
 Discussion
 References
 

  1. Jurcic JG, Larson SM, Sgouros G, et al. Targeted {alpha} particle immunotherapy for myeloid leukemia. Blood 2002;100:1233–9.[Abstract/Free Full Text]
  2. Kennel SJ, Boll R, Stabin M, Schuller HM, Mirzadeh S. Radioimmunotherapy of micrometastases in lung with vascular targeted 213Bi. Br J Cancer 1999;80:175–84.[CrossRef][Medline]
  3. McDevitt MR, Ma D, Lai LT, et al. Tumor therapy with targeted atomic nanogenerators. Science 2001;294:1537–40.[Abstract/Free Full Text]
  4. Milas L, Hunter NR, Mason KA, Kurdoglu B, Peters LJ. Enhancement of tumor radioresponse of a murine mammary carcinoma by paclitaxel. Cancer Res 1994;54:3506–10.[Abstract/Free Full Text]
  5. Milas L, Saito Y, Hunter N, Milross CG, Mason KA. Therapeutic potential of paclitaxel-radiation treatment of a murine ovarian carcinoma. Radiother Oncol 1996;40:163–70.[CrossRef][Medline]
  6. Milas L, Milas MM, Mason KA. Combination of taxanes with radiation: preclinical studies. Semin Radiat Oncol 1999;9:12–26.[Medline]
  7. Bonner JA, Lawrence TS. Doxorubicin decreases the repair of radiation-induced DNA damage. Int J Radiat Biol 1990;57:55–64.[Medline]
  8. Byfield JE, Lee YC, Tu L. Molecular interactions between Adriamycin and X-ray damage in mammalian tumor cells. Int J Cancer 1977;19:186–93.[Medline]
  9. Durand RE, LePard NE. Tumour blood flow influences combined radiation and doxorubicin treatments. Radiother Oncol 1997;42:171–9.[CrossRef][Medline]
  10. Schiff PB, Horwitz SB. Taxol stabilizes microtubules in mouse fibroblast cells. Proc Natl Acad Sci U S A 1980;77:1561–5.[Abstract/Free Full Text]
  11. Supiot S, Rio E, Gouard S, et al. {alpha}-Radioimmunotherapy of multiple myeloma could be improved by concomitant chemotherapy, #4814. In: San Francisco, California: 93rd annual meeting of the American Association for Cancer Research; 2002 April 6–10. p. 972.
  12. Wijdenes J, Vooijs WC, Clement C, et al. A plasmocyte selective monoclonal antibody (B-B4) recognizes syndecan-1. Br J Haematol 1996;94:318–23.[CrossRef][Medline]
  13. Brechbiel MW, Gansow OA. Synthesis of C-functionalized trans- cyclohexyldiethylnenetriaminepenta-acetic acid for labelling of monoclonal antibodies with the bismuth-212 {alpha}-particle emitter. J Chem Soc Perkin Trans 1992;1:1173–8.
  14. Lindmo T, Boven E, Cuttitta F, Fedorko J, Bunn PA, Jr. Determination of the immunoreactive fraction of radiolabeled monoclonal antibodies by linear extrapolation to binding at infinite antigen excess. J Immunol Methods 1984;72:77–89.[CrossRef][Medline]
  15. Kaspersen FM, Bos E, Doornmalen AV, Geerlings MW, Apostolidis C, Molinet R. Cytotoxicity of 213Bi- and 225Ac-immunoconjugates. Nucl Med Commun 1995;16:468–76.[Medline]
  16. Koester SK, Roth P, Mikulka WR, Schlossman SF, Zhang C, Bolton WE. Monitoring early cellular responses in apoptosis is aided by the mitochondrial membrane protein-specific monoclonal antibody APO2.7. Cytometry 1997;29:306–12.[CrossRef][Medline]
  17. Alapetite C, Wachter T, Sage E, Moustacchi E. Use of the alkaline comet assay to detect DNA repair deficiencies in human fibroblasts exposed to UVC, UVB, UVA and {gamma}-rays. Int J Radiat Biol 1996;69:359–69.[CrossRef][Medline]
  18. Zhang C, Ao Z, Seth A, Schlossman SF. A mitochondrial membrane protein defined by a novel monoclonal antibody is preferentially detected in apoptotic cells. J Immunol 1996;157:3980–7.[Abstract]
  19. Bussing A, Vervecken W, Wagner M, Wagner B, Pfuller U, Schietzel M. Expression of mitochondrial Apo2.7 molecules and caspase-3 activation in human lymphocytes treated with the ribosome-inhibiting mistletoe lectins and the cell membrane permeabilizing viscotoxins. Cytometry 1999;37:133–9.[CrossRef][Medline]
  20. Koester SK, Schlossman SF, Zhang C, Decker SJ, Bolton WE. APO2.7 defines a shared apoptotic-necrotic pathway in a breast tumor hypoxia model. Cytometry 1998;33:324–32.[CrossRef][Medline]
  21. Le Gouill S, Pellat-Deceunynck C, Harousseau JL, et al. Farnesyl transferase inhibitor R115777 induces apoptosis of human myeloma cells. Leukemia 2002;16:1664–7.[CrossRef][Medline]
  22. Sinclair WK, Morton RA. X-ray sensitivity during the cell generation cycle of cultured Chinese hamster cells. Radiat Res 1966;29:450–74.[Medline]
  23. Tishler RB, Schiff PB, Geard CR, Hall EJ. Taxol: a novel radiation sensitizer. Int J Radiat Oncol Biol Phys 1992;22:613–7.[Medline]
  24. Asosingh K, De Raeve H, Van Riet I, Van Camp B, Vanderkerken K. Multiple myeloma tumor progression in the 5T2MM murine model is a multistage and dynamic process of differentiation, proliferation, invasion, and apoptosis. Blood 2003;101:3136–41.[Abstract/Free Full Text]
  25. Leonard CE, Chan DC, Chou TC, Kumar R, Bunn PA. Paclitaxel enhances in vitro radiosensitivity of squamous carcinoma cell lines of the head and neck. Cancer Res 1996;56:5198–204.[Abstract/Free Full Text]
  26. Lawrence TS, Blackstock AW, McGinn C. The mechanism of action of radiosensitization of conventional chemotherapeutic agents. Semin Radiat Oncol 2003;13:13–21.[CrossRef][Medline]
  27. Milross CG, Mason KA, Hunter NR, Chung WK, Peters LJ, Milas L. Relationship of mitotic arrest and apoptosis to antitumor effect of paclitaxel. J Natl Cancer Inst 1996;88:1308–14.[Abstract/Free Full Text]
  28. Haldar S, Chintapalli J, Croce CM. Taxol induces bcl-2 phosphorylation and death of prostate cancer cells. Cancer Res 1996;56:1253–5.[Abstract/Free Full Text]
  29. Haldar S, Jena N, Croce CM. Inactivation of Bcl-2 by phosphorylation. Proc Natl Acad Sci U S A 1995;92:4507–11.[Abstract/Free Full Text]
  30. Kroning R, Lichtenstein A. Taxol can induce phosphorylation of BCL-2 in multiple myeloma cells and potentiate dexamethasone-induced apoptosis. Leuk Res 1998;22:275–86.[CrossRef][Medline]
  31. Gazitt Y, Rothenberg ML, Hilsenbeck SG, Fey V, Thomas C, Montegomrey W. Bcl-2 overexpression is associated with resistance to paclitaxel, but not gemcitabine, in multiple myeloma cells. Int J Oncol 1998;13:839–48.[Medline]
  32. Panaretakis T, Pokrovskaja K, Shoshan MC, Grander D. Activation of Bak, Bax, and BH3-only proteins in the apoptotic response to doxorubicin. J Biol Chem 2002;277:44317–26.[Abstract/Free Full Text]
  33. Couturier O, Faivre_Chauvet A, Filippovich IV, et al. Validation of 213Bi-{alpha} radioimmunotherapy for multiple myeloma. Clin Cancer Res 1999;5:3165–70s.
  34. Liebmann J, Cook JA, Fisher J, Teague D, Mitchell JB. In vitro studies of Taxol as a radiation sensitizer in human tumor cells. J Natl Cancer Inst 1994;86:441–6.[Abstract/Free Full Text]




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