Clinical Cancer Research CTRC-AACR San Antonio Breast Cancer Symposium
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Opalinska, J. B.
Right arrow Articles by Gewirtz, A. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Opalinska, J. B.
Right arrow Articles by Gewirtz, A. M.
Clinical Cancer Research Vol. 11, 4948-4954, July 1, 2005
© 2005 American Association for Cancer Research

Cancer Therapy: Preclinical

Multigene Targeting with Antisense Oligodeoxynucleotides: An Exploratory Study Using Primary Human Leukemia Cells

Joanna B. Opalinska1, Boguslaw Machalinski2, Janina Ratajczak3, Mariusz Z. Ratajczak3 and Alan M. Gewirtz4

Authors' Affiliations: 1 Hematology and 2 Pathology, Pommeranian Medical University, Szczecin, Poland; 3 James Brown Cancer Center, University of Louisville, Louisville, Kentucky; and 4 Division of Hematology/Oncology, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania

Requests for reprints: Alan M. Gewirtz, Division of Hematology/Oncology, Department of Medicine, University of Pennsylvania School of Medicine, Room 713, BRB II/III, 421 Curie Boulevard, Philadelphia, PA 19104. Phone: 215-898-4499; Fax: 215-573-2078; E-mail: gewirtz{at}mail.med.upenn.edu.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: We previously reported that the c-myb and Vav proto-oncogenes are amenable to silencing with antisense oligodeoxynucleotides and that inhibition of either impairs leukemic cell growth. Because the expression of these genes is not known to be linked, we sought to determine the therapeutic value of silencing both genes simultaneously in K562 and primary patient (n = 9) chronic myelogenous leukemia cells.

Experimental Design: K562 and primary chronic myelogenous leukemia cells were exposed to antisense oligodeoxynucleotides (alone or in combination) for 24 or 72 hours and then cloned in methylcellulose cultures. Effects on K562 cluster, and blast-forming unit–erythroid colonies and granulocyte-macrophage colony-forming units were determined and correlated with the ability to down-regulate the targeted mRNA.

Results: After 24-hour exposure, K562 cell growth was inhibited in a sequence specific, dose-responsive manner with either c-myb or Vav antisense oligodeoxynucleotides. Exposure to both oligodeoxynucleotides simultaneously considerably enhanced growth inhibition and accelerated apoptosis. Primary cell results were more complex. After 24- and 72-hour exposures to either anti–vav or anti–myb antisense oligodeoxynucleotides, equivalent colony-forming unit inhibition was observed. Exposing cells to both antisense oligodeoxynucleotides simultaneously for 24 hours did not result in additional inhibition of colony formation. However, after 72-hour incubation with both oligodeoxynucleotides, colony formation was diminished significantly when compared with either oligodeoxynucleotides alone (from ~30% to ~78% for granulocyte-macrophage colony-forming unit; ~50% to ~80% for blast-forming unit–erythroid).

Conclusions: We hypothesize that exposing primary leukemic cells to antisense oligodeoxynucleotides targeted to two, or possibly more, genes might significantly augment the therapeutic utility of these molecules.

Myb and Vav are largely hematopoietic cell proteins with transcriptional activation and signal transduction roles, respectively, in normal cells. Both proteins have been intensively studied and their functions authoritatively reviewed (1–9).

c-Myb is one of three members of a family of transcription factors (A-myb, B-myb, and c-myb), which is defined by sequence homology within the DNA-binding domain and the functional ability to bind the consensus sequence [YG(A/G)C(A/C/G)GTT(G/A)]. Myb family of proteins play a vital role in cell cycle progression. They also regulate the expression of a growing list of cellular genes required for hematopoietic cell survival and function. It has been unequivocally established that Myb is required for both normal and malignant hematopoiesis (10–14). Accordingly, silencing c-myb expression with, for example, antisense oligodeoxynucleotides, may be of therapeutic use in patients with hematologic malignancies (15, 16).

Presently, four Vav protein family members have been described, three of which are expressed in mammalian cells (Vav, Vav2, and Vav3; ref. 17). Mammalian Vav proteins are encoded by genes located on different chromosomes but they all share a diverse array of structural motifs, including Dbl and Pleckstrin homology domains, as well as two SH3 domains flanking a single SH2 region (18–20). The presence of these domains suggested that Vav family members might play an important role in hematopoietic signal transduction cascades and this has proven to be correct. Activated Vav members interact with the Rho/Rac family of GTP hydrolases that rearrange cytoskeleton and ultimately up-regulate expression of genes that are important for cell proliferation and differentiation, as well as those that might enhance the metastatic potential of transformed cells (8, 9, 21–23). Interestingly, Vav (–/–) mice develop normally (24). The phenotype of the mice is remarkable for defective lymphocyte signaling in response to antigen stimulation, but perhaps surprisingly, they have otherwise normal myelopoiesis and lymphopoiesis. In agreement with these observations, we have reported that antisense oligodeoxynucleotide–mediated inhibition of Vav in normal adult human hematopoietic cells has no effect on myeloid or megakaryocytic colony-forming units, although erythropoiesis is somewhat compromised. In myeloproliferative disorders (chronic phase CML and P. Vera), the effects are more pronounced (25).

Because vav and c-myb are both important hematopoietic genes, and we have previously shown that expression of each may be diminished with antisense oligodeoxynucleotides (11, 12, 16, 26), we sought to determine if exposing malignant blood cells to antisense oligodeoxynucleotides targeting both mRNAs might have additive inhibitory effects on proliferation and viability. To examine this possibility, we exposed K562 cells and primary CML cells to one or both antisense oligodeoxynucleotides for 24 or 72 hours and determined effects on cell viability and proliferation.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Cells. K562 cells are derived from a patient with chronic myelogenous leukemia (CML; ref. 27) and were originally obtained from the American Type Culture Collection (Manassas, VA). Primary mononuclear cells were isolated from the bone marrow or peripheral blood from nine patients with CML as previously described (28–30). CD34+ cells were isolated using anti-CD34+ antibodies conjugated to microbeads and sorted on MACS columns (Miltenyi BioTech, Auburn, CA) according to the manufacturer's directions. Granulocyte-macrophage colony-forming unit (CFU-GM) assays were done on cells from all nine patients, and blast-forming unit–erythroid (BFU-E) assays on cells from four patients. Studies were done at least thrice using the bone marrow or blood from the same patients.

Oligodeoxynucleotides. Unmodified phosphodiester antisense oligonucleotides were synthesized and purified as previously described (11, 31). Oligodeoxynucleotides were lyophilized to dryness and redissolved in Iscove's modified Dulbecco's medium before use (1 mg/mL = 0.175 mmol/L). Oligodeoxynucleotides sequences used, corresponding to codons 2 to 7 of the human proto-Vav cDNA sequence 1, were as follows: 5'-AAG GCA CAG GAA CTG GGA-3' (antisense oligodeoxynucleotides) and 5'-AGC TCG AAA GAC AGG GGA-3' (scrambled-sequence oligodeoxynucleotides). The antisense sequence corresponding to human c-myb codons 2 to 9 was as follows: 5'-GCC CGA AGA CCC CGG CAC AGC ATA-3'. A scrambled antisense myb oligodeoxynucleotide sequence (5'-GCA CGC AGC TGA AGCACA AGC ACC-3') was utilized as a control.

Cell culture. K562 cells were cultured in liquid suspension or in methylcellulose. For suspension, cells (2.5 x 103/well) were deposited in 24-well plates in 0.4 mL Iscove medium containing heat-inactivated 10% artificial serum substitute (32). For low-dose oligodeoxynucleotide exposure, 40 µg/mL of the oligodeoxynucleotide were added at time 0, and 50% of the initial dose was added again 18 hours later. For high-dose exposure, oligodeoxynucleotides were added on the same time schedule, but the initial dose was at a concentration of 100 µg/mL. Cells were incubated (100% humidity, 37°C, 5% CO2) for 10 days and cells were counted at days 3, 7, and 10. Cell counts for each time point were derived from the content of four separate wells.

Adherent cell depleted marrow derived mononuclear cells (105 cells total) were incubated in 0.4 mL of serum-free Iscove medium containing 10 mmol/L HEPES buffer in polypropylene tubes (Fisher Scientific, Pittsburgh, PA). Oligodeoxynucleotides (100 µg/mL = 25 µmol/L) were added at time 0 and 50% of the initial dose was added again 18 hours later. After 24 or 72 hours, treated adherent cell depleted marrow derived mononuclear cells were prepared for plating in methylcellulose cultures as previously described. Cells (1 x 105) were not washed before plating. Control cultures contained cells manipulated in an identical manner but without exposure to oligodeoxynucleotides. Growth factors and concentrations used were as follows: 5.0 units/mL erythropoietin + 100 ng/mL stem cell factor for BFU-E colonies; 20 ng/mL interleukin-3 + 5 ng/mL granulocyte-macrophage colony-stimulating factor for CFU-GM. Cells were incubated (100% humidity, 37°C, 5% CO2) for 11 days in the case of CFU-GM, and 14 days for BFU-E. Colonies were counted with the aid of an inverted microscope.

Reverse transcription-PCR. Reverse transcription-PCR for detection of Vav and ß-actin mRNA was done as previously described (25). In brief, adherent cell depleted marrow derived mononuclear cells (2 x 106 to 5 x 106) were incubated (37°C; 5% CO2) in 0.4 mL serum-free Iscove medium. Oligodeoxynucleotides (100 µg/mL = 25 µmol/L) were added at time 0 and 50% of the initial dose was added again 18 hours later. After 36 hours, RNAzol (Teltest, Friendswood, TX) was used to isolate total cellular RNA, which was stored at –70°C until used. RNA was reverse transcribed with 500 units of Moloney murine leukemia virus reverse transcriptase and 50 pmol of a 21-nucleotide oligodeoxynucleotide 3' primer complementary to nucleotides 980 to 959 (GGA GGT GAT ATT TGA GAA CTC) of the proto-vav cDNA sequence. The resulting cDNA fragment was amplified for 30 cycles using 5 units of Thermus aquaticus (Taq) polymerase and a 21-nucleotide oligodeoxynucleotide 5' primer specific for nucleotides 610 to 631 (CCA TCC AGC ATT TCT TGA) and the 3' primer specific for nucleotides 980 to 959 (GGA GGT GAT ATT TGA GAA CTC). The ~370 bp product was then resolved on 1% agarose gel.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell viability assay. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was done according to the manufacturer's recommendations (Promega, Madison, WI). Briefly, cells were seeded in 96-well plates at 2 x 105 cells/well in 100 µL of RPMI medium containing 0.5% bovine serum albumin. Oligodeoxynucleotides (100 µg/mL) were added at time 0 and 50% of the initial dose was added again 18 hours later. After 48 hours, 20 mL of Cell Titer 96 Aqueous One Solution reagent was added to each well and the plates were incubated for 3 to 4 hours. Subsequently, plates were read at 490 nm using an automated plate reader.

Statistical analysis. Analysis of data was done using InStat 3 for Macintosh (GraphPad Software San Diego, CA).


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Effect of anti–myb and anti–vav oligodeoxynucleotides on K562 cell growth. We initially examined the effect of oligodeoxynucleotides targeted to Myb and Vav, alone and in combination, on K562 cell growth at low and high doses (40 + 20 and 100 + 50 µg/mL respectively). Assays were conducted on cells grown in liquid suspension or cloned in methylcellulose. Similar results were obtained in both assays. In the case of the colony-forming unit assay (Fig. 1), for example, low-dose anti–Vav antisense oligodeoxynucleotides inhibited colony formation by ~32% (P > 0.05), anti–Myb oligodeoxynucleotides inhibited colony-forming unit formation by ~55% (P < 0.01), whereas the combination of the antisense oligodeoxynucleotides inhibited colony-forming unit by ~75% (P < 0.001) when compared with the growth of colonies not exposed to oligodeoxynucleotides. At the high dose, inhibition of colony formation was even greater, being 58%, 76%, and 94%, respectively (P < 0.001 for all compared with control colony growth), suggesting that inhibition was dose related and that effects of the two oligodeoxynucleotides were additive. Sequence specificity was shown by the fact that the scrambled oligodeoxynucleotide sequences gave no inhibition when used alone and only minimal inhibition when used together. Accordingly, these results suggested that a combination of the two antisense oligodeoxynucleotides might be far superior for inhibition of cell growth than either oligodeoxynucleotides used alone. The mechanism for this additive effect seemed to be an enhanced induction of apoptosis as shown by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (Table 1). By 48 hours after exposure to both antisense oligodeoxynucleotides, 75 ± 7% of cells were dead or dying, versus 51 ± 14% of cells exposed to the anti–Myb oligodeoxynucleotides, and 27 ± 13% of cells exposed to the Vav antisense oligodeoxynucleotides alone.



View larger version (50K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. Effect of oligodeoxynucleotides targeted to c-myb and Vav, alone and in combination, on K562 cell cloning efficiency in methylcellulose at low (A; 40 + 20 µg/mL) and high (B; 100 + 50 µg/mL) doses. Cells were cultured, and colonies scored as detailed in Materials and Methods. Cont, untreated control cell growth; V AS, proto-Vav–targeted antisense oligodeoxynucleotides; Myb AS, c-Myb–targeted antisense oligodeoxynucleotides; Scr, scrambled sequence; M + V, Myb + Vav oligodeoxynucleotides.

 

View this table:
[in this window]
[in a new window]

  		Table 1. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay on K-562 cells

 
Primary chronic myelogenous leukemia cell growth in vitro after oligodeoxynucleotide exposure. Whereas the cell line data was suggestive, it was clearly necessary to test the utility of the multigene targeting approach on primary cells. Accordingly, the effect of Vav and Myb antisense oligodeoxynucleotides and scrambled-sequence oligodeoxynucleotides on primary CML patient progenitor cell growth was assessed by exposing mononuclear cells to the oligodeoxynucleotides and then counting the resulting BFU-E and CFU-GM colonies as described in Materials and Methods.

Effect of 24-hour oligodeoxynucleotide exposure on progenitor cell growth. The effect of a 24-hour exposure to the c-myb, proto-vav, or oligodeoxynucleotide combinations on the growth of CFU-GM from nine patients is shown in Fig. 2. Although the cloning efficiency of the individual patient samples was quite variable, some trends were observable. When compared with colony growth from untreated (control) cells (arbitrary 100% growth), the number of colonies developing in the plates was reduced in all cases after exposure to one or both of the antisense molecules. Inhibition of colony formation was variable, averaging ~45% of control cell growth for both the Myb and the proto-vav targeted antisense molecules. Combining the two did not seem to augment inhibition any further. Nonetheless, it did seem to be sequence specific because, except for patient 3, exposure to the scrambled sequences, at the same total oligodeoxynucleotide concentrations, had no statistically significant effect on colony formation when compared with the untreated control.



View larger version (18K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. GM-CFU–derived colonies from nine CML patients after 24-hour exposure to Myb or proto-Vav targeted oligodeoxynucleotides. Individual patient results are shown along the abscissa; number of colonies per 105 CD34+ cells cloned is shown along the ordinate. Control, colony growth by untreated cells. Vav/c-myb, Myb + Vav oligodeoxynucleotides; AS, antisense. Colonies were counted on day 11 with an inverted microscope.

 
Similar results were obtained when BFU-E colony formation was assayed in four patients after a 24-hour oligodeoxynucleotides exposure (Fig. 3). The c-myb– and vav–targeted oligodeoxynucleotides variably inhibited colony formation in all four patients studied by an average of ~50% and 60%, respectively when compared with controls. As was noted with the CFU-GM colonies, combining the two oligodeoxynucleotides gave no further inhibition of colony formation in any of the patient samples. Nonspecific growth inhibition is unlikely to explain these results, because again except for patient 3, the scrambled oligodeoxynucleotides used at the same concentrations did not inhibit colony formation to any significant degree.



View larger version (15K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. BFU-E–derived colonies from four CML patients after 24-hour exposure to Myb or proto-Vav targeted oligodeoxynucleotides. Individual patient results are shown along the abscissa; number of colonies per 105 CD34+ cells cloned is shown along the ordinate. Colonies were counted on day 14 with an inverted microscope.

 
Effect of 72-hour oligodeoxynucleotide exposure on progenitor cell growth. As is true for many RNA-silencing strategies, we have previously reported that prolonged exposure to the targeting agent, in this case oligodeoxynucleotides, results in a more efficient knockdown of the mRNA target (16). For this reason, we investigated the effect of exposing the progenitor cells to the oligodeoxynucleotides for 72 hours. In these experiments, we saw similar degrees of inhibition of CFU-GM (Fig. 4) and BFU-E (Fig. 5) as noted with the 24-hour exposure. However, when cells were incubated for 72 hours with both molecules simultaneously, we observed a much more substantial reduction in colony numbers (an average of 78% for both CFU-GM and BFU-E compared with the ~30% and 50% average for CFU-GM and BFU-E, respectively, after 24-hour exposure). Note again that exposure to scrambled-sequence oligodeoxynucleotides at the same final DNA concentration had no significant effect on colony growth.



View larger version (19K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 4. GM-CFU–derived colonies from five CML patients after 72-hour exposure to Myb or proto-Vav targeted oligodeoxynucleotides. Individual patient results are shown along the abscissa; number of colonies per 105 CD34+ cells cloned is shown along the ordinate. Colonies were counted on day 11 with an inverted microscope.

 


View larger version (13K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig 5. BFU-E–derived colonies from four CML patients after 72-hour exposure to Myb or proto-Vav oligodeoxynucleotides. Individual patient results are shown along the abscissa; number of colonies per 105 CD34+ cells cloned is shown along the ordinate. Colonies were counted on day 14 with an inverted microscope.

 
The effect of vav targeted antisense oligodeoxynucleotides on mRNA expression. We have previously shown in multiple studies that the anti–myb oligodeoxynucleotides used in the study specifically inhibit their respective targets in hematopoietic cells at the mRNA and protein levels (11, 16, 28, 30, 33, 34). We have also shown activity of the anti–Vav oligodeoxynucleotides in several studies (25, 35). Nonetheless, to confirm activity of the Vav-targeted oligodeoxynucleotides at the mRNA level, we examined oligodeoxynucleotide-treated cells for Vav mRNA expression. As shown in Fig. 6, Vav mRNA was detectable in untreated CML cells (lane C), as well as in cells exposed to Vav scrambled oligodeoxynucleotides (lane Scr). In contrast, exposure to Vav antisense oligodeoxynucleotides resulted in loss of amplifiable mRNA (lane AS). Simultaneous assay for ß-actin mRNA shows detectable message regardless of the Vav oligodeoxynucleotide sequence to which CML cells were exposed (Fig. 6, bottom).



View larger version (78K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 6. Cells incubated with antisense oligodeoxynucleotides against proto–vav were subjected to PCR reaction with vav-specific primers. The resulting ~370 bp product was resolved on 1% agarose gel. Lane C, untreated cells; lane S, cells treated with vav sense sequence; lane AS, cells treated with antisense oligodeoxynucleotides against vav; lane Scr, scrambled control. The negative control is shown in the last lane (H2O). Note that only cells treated with antisense oligodeoxynucleotides against vav are negative for the specific PCR product, which indicates that we were able to abolish vav expression in antisense oligodeoxynucleotide–treated cells.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
We have previously shown the ability of antisense oligodeoxynucleotides targeted to the c-myb and vav proto-oncogenes to suppress the growth of normal and malignant human hematopoietic cells (11, 25, 28, 30, 33, 35). With each mRNA target, the effects on malignant cell growth were much more profound than on normal cells, and in the case of c-myb this was shown in a bone marrow purging study done on patients with CML (16). In the present study, we addressed the simple question of whether exposing malignant cells to both antisense molecules would produce an additive effect on cell growth inhibition. The factors that regulate Myb expression in human lymphoid cells have become better known (36), but in myeloid hematopoietic cells they remain incompletely defined. Nonetheless, to the best of our knowledge, there are no studies linking Vav signaling directly with MYB expression. Accordingly, it seemed reasonable to hypothesize that if the activity of both genes was important for malignant hematopoietic cell growth, inhibiting both might create an additive block on cell growth and/or viability.

The results obtained in this small study are potentially informative because they were done on a well-established human hematopoietic cell line model, and with primary patient material as well. Although a 24-hour exposure to either antisense oligodeoxynucleotide inhibited progenitor cell growth, the inhibition observed was not increased when cells were exposed to both molecules simultaneously for 24 hours. Having previously observed that a more prolonged exposure to oligodeoxynucleotides enhanced their efficiency (16), we exposed cells to these molecules for 72 hours as well. Somewhat unexpectedly, a 72-hour exposure to either molecule alone gave inhibition similar to that observed at 24 hours. However, treating cells with both molecules simultaneously for 72 hours diminished colony growth considerably compared with either molecule alone, or to the various control combinations. The small numbers of patients studied and the wide variation in the cloning efficiency of some of these individuals' blood cells precluded our drawing formal statistical conclusions about the significance of these differences. Nonetheless, the highly significant results obtained in the cell line experiments, along with the trends observed in each of the patient studies (Figs. 2- 5), lend objective support to what is otherwise an appealing, and intuitively logical, hypothesis.

Because our studies were limited in scope, we can only speculate on the potential mechanisms for the enhanced cell killing observed at the 72-hour time point. The simplest explanation is that some time is required for inhibition to take place and for the effected cells to suffer the biological consequences. Classic pharmacokinetics (i.e., toxicity = concentration x time of exposure) would predict this. Nevertheless, this study was somewhat unusual in that oligodeoxynucleotides with natural phosphodiester backbones were used in an economical attempt to circumvent some of the nonspecific toxicity observed with phosphorothioate backbones (37, 38). For this reason, we must acknowledge that nonspecific toxicity may be playing a role in some of the growth inhibitory effects observed. Vaerman et al. (39) reported that stepwise degradation of oligodeoxynucleotides by 3' exonucleases leads to potentially toxic deoxynucleotide triphosphate concentrations in cell cultures. In Vaerman et al.'s study, antiproliferative effects were observed with deoxynucleotide triphosphate concentrations of 5 to 10 µmol/L with phosphorothioate oligodeoxynucleotides, and 25 µmol/L with phosphodiester backbone oligodeoxynucleotides. Of note, cytotoxicity was mitigated by the presence of cytosine residues in these ends. Examination of the 3' end of the proto–vav sequences used revealed the four penultimate bases were devoid of dCTPs, whereas the four penultimate bases of the 3' end of the c-myb sequences did contain cytosine residues (one for the myb antisense sequence and three for the myb scrambled sequence). If all effects on growth were due to nonspecific accumulation of deoxynucleotide triphosphates, one might plausibly explain, for example, why the antisense oligodeoxynucleotides were toxic, whereas the Myb scrambled sequence was not. However, one could not explain why exposure to the Vav antisense or scrambled oligodeoxynucleotide sequences did not yield equivalent cytotoxicity as the base content of their 3' ends are identical. Similarly, one might have expected the anti–myb and proto–vav mixture to be slightly less toxic than the Vav oligodeoxynucleotide because the former contains a toxicity ameliorating dCTP. This was clearly not the case. Accordingly, we cannot explain our results as a function of simple degradation of the oligodeoxynucleotides. Because we have documented sequence-specific inhibition of the targeted mRNA, we feel that it is likely that an antisense mechanism was at least partially responsible for the biological effects observed, and this effect seemed to be due to an increase in apoptosis among the treated cells.

It is becoming increasingly clear that regardless of the modality used, targeting single genes or their products will be of limited utility for anticancer therapeutics. With the very notable exception of monoclonal antibodies targeting surface receptors on lymphoma cells (which likely kill by complement-mediated effects; refs. 40, 41), and the abl/kit tyrosine kinase inhibitor Gleevec (42), most such therapies have failed to produce dramatic clinical responses. This is likely due to the complexity of the diseases called "cancer" and their pathogenesis. Targeting multiple pathways in tumor cells is intuitively appealing, and studies of the type reported here support the concept. Further experimentation using enhanced delivery of so-called "third-generation" antisense molecules would likely test this approach in a more rigorous manner. Demonstrating an acceptable therapeutic index will also be important in the evolution of this concept. We hope to embark on such studies in the near future.


   Footnotes
 
Grant support: NIH grant RO1CA 10859 (A.M. Gewirtz), Doris Duke Charitable Foundation (A.M. Gewirtz), and Polish Research Council grant 2PO5A 12326 (J.B. Opalinska).

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.

Note: J.B. Opalinska and B. Machalinski contributed equally to this work. A.M. Gewirtz is a Distinguished Clinical Scientist of the Doris Duke Charitable Foundation.

Received 1/17/04; revised 3/25/05; accepted 9/ 5/05.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Graf T. Myb: a transcriptional activator linking proliferation and differentiation in hematopoietic cells. Curr Opin Genet Dev 1992;2:249–55.[CrossRef][Medline]
  2. Wolff L. Myb-induced transformation. Crit Rev Oncog 1996;7:245–60.[Medline]
  3. Lipsick JS, Wang DM. Transformation by v-Myb. Oncogene 1999;18:3047–55.[CrossRef][Medline]
  4. Friedman AD. Runx1, c-Myb, and C/EBP{alpha} couple differentiation to proliferation or growth arrest during hematopoiesis. J Cell Biochem 2002;86:624–9.[CrossRef][Medline]
  5. Ramsay RG, Barton AL, Gonda TJ. Targeting c-Myb expression in human disease. Expert Opin Ther Targets 2003;7:235–48.[CrossRef][Medline]
  6. Bustelo XR. The VAV family of signal transduction molecules. Crit Rev Oncog 1996;7:65–88.[Medline]
  7. Romero F, Fischer S. Structure and function of vav. Cell Signal 1996;8:545–53.[CrossRef][Medline]
  8. Bustelo XR. Vav proteins, adaptors and cell signaling. Oncogene 2001;20:6372–81.[CrossRef][Medline]
  9. Bustelo XR. Regulation of Vav proteins by intramolecular events. Front Biosci 2002;7:d24–30.[Medline]
  10. Mucenski ML, McLain K, Kier AB, et al. A functional c-myb gene is required for normal murine fetal hepatic hematopoiesis. Cell 1991;65:677–89.[CrossRef][Medline]
  11. Gewirtz AM, Calabretta B. A c-myb antisense oligodeoxynucleotide inhibits normal human hematopoiesis in vitro. Science 1988;242:1303–6.[Abstract/Free Full Text]
  12. Ratajczak MZ, Kant JA, Luger SM, et al. In vivo treatment of human leukemia in a scid mouse model with c-myb antisense oligodeoxynucleotides. Proc Natl Acad Sci U S A 1992;89:11823–7.[Abstract/Free Full Text]
  13. Goto NK, Zor T, Martinez-Yamout M, Dyson HJ, Wright PE. Cooperativity in transcription factor binding to the coactivator CREB-binding protein (CBP). The mixed lineage leukemia protein (MLL) activation domain binds to an allosteric site on the KIX domain. J Biol Chem 2002;277:43168–74.[Abstract/Free Full Text]
  14. Ohmine K, Nagai T, Tarumoto T, et al. Analysis of gene expression profiles in an imatinib-resistant cell line, KCL22/SR. Stem Cells 2003;21:315–21.[Abstract/Free Full Text]
  15. Gewirtz AM. Myb targeted therapeutics for the treatment of human malignancies. Oncogene 1999;18:3056–62.[CrossRef][Medline]
  16. Luger SM, O'Brien SG, Ratajczak J, et al. Oligodeoxynucleotide-mediated inhibition of c-myb gene expression in autografted bone marrow: a pilot study. Blood 2002;99:1150–8.[Abstract/Free Full Text]
  17. Moores SL, Selfors LM, Fredericks J, et al. Vav family proteins couple to diverse cell surface receptors. Mol Cell Biol 2000;20:6364–73.[Abstract/Free Full Text]
  18. Katzav S, Martin Zanca D, Barbacid M. vav, a novel human oncogene derived from a locus ubiquitously expressed in hematopoietic cells. EMBO J 1989;8:2283–90.[Medline]
  19. Katzav S. vav: a molecule for all haemopoiesis? Br J Haematol 1992;81:141–4.[Medline]
  20. Zugaza JL, Lopez Lago MA, Caloca MJ, Dosil M, Movilla N, Bustelo XR. Structural determinants for the biological activity of Vav proteins. J Biol Chem 2002;277:45377–92.[Abstract/Free Full Text]
  21. DeFranco AL. Vav and the B cell signalosome. Nat Immunol 2001;2:482–4.[CrossRef][Medline]
  22. Turner M, Billadeau DD. VAV proteins as signal integrators for multi-subunit immune-recognition receptors. Nat Rev Immunol 2002;2:476–86.[CrossRef][Medline]
  23. Ardouin L, Bracke M, Mathiot A, et al. Vav1 transduces TCR signals required for LFA 1 function and cell polarization at the immunological synapse. Eur J Immunol 2003;33:790–7.[CrossRef][Medline]
  24. Zhang R, Tsai FY, Orkin SH. Hematopoietic development of vav–/– mouse embryonic stem cells. Proc Natl Acad Sci U S A 1994;91:12755–9.[Abstract/Free Full Text]
  25. Luger SM, Ratajczak J, Ratajczak MZ, et al. A functional analysis of protooncogene Vav's role in adult human hematopoiesis. Blood 1996;87:1326–34.[Abstract/Free Full Text]
  26. Calabretta B, Gewirtz AM. Functional requirements of c-myb during normal and leukemic hematopoiesis. Crit Rev Oncog 1991;2:187–94.[Medline]
  27. Lozzio BB, Lozzio CB. Properties of the K562 cell line derived from a patient with chronic myeloid leukemia. Int J Cancer 1977;19:136.[Medline]
  28. Calabretta B, Sims RB, Valtieri M, et al. Normal and leukemic hematopoietic cells manifest differential sensitivity to inhibitory effects of c-myb antisense oligodeoxynucleotides: an in vitro study relevant to bone marrow purging. Proc Natl Acad Sci U S A 1991;88:2351–5.[Abstract/Free Full Text]
  29. Ratajczak MZ, Hijiya N, Catani L, et al. Acute- and chronic-phase chronic myelogenous leukemia colony-forming units are highly sensitive to the growth inhibitory effects of c-myb antisense oligodeoxynucleotides. Blood 1992;79:1956–61.[Abstract/Free Full Text]
  30. Ratajczak MZ, Perrotti D, Melotti P, et al. Myb and ets proteins are candidate regulators of c-kit expression in human hematopoietic cells. Blood 1998;91:1934–46.[Abstract/Free Full Text]
  31. Gewirtz AM, Boghosian-Sell L, Catani L, Ratajczak MZ, Shen YM, Schreiber AD. Expression of Fc{gamma}RII and CD4 receptors by normal human megakaryocytes. Exp Hematol 1992;20:512–6.[Medline]
  32. Ratajczak MZ, Ratajczak J, Machalinski B, Mick R, Gewirtz AM. In vitro and in vivo evidence that ex vivo cytokine priming of donor marrow cells may ameliorate posttransplant thrombocytopenia. Blood 1998;91:353–9.[Abstract/Free Full Text]
  33. Gewirtz AM, Anfossi G, Venturelli D, Valpreda S, Sims R, Calabretta B. G1/S transition in normal human T-lymphocytes requires the nuclear protein encoded by c-myb. Science 1989;245:180–3.[Abstract/Free Full Text]
  34. Opalinska JB, Kalota A, Gifford LK, et al. Oxetane modified, conformationally constrained, antisense oligodeoxyribonucleotides function efficiently as gene silencing molecules. Nucleic Acids Res 2004;32:5791–9.[Abstract/Free Full Text]
  35. Clevenger CV, Ngo W, Sokol DL, Luger SM, Gewirtz AM. Vav is necessary for prolactin-stimulated proliferation and is translocated into the nucleus of a T-cell line. J Biol Chem 1995;270:13246–53.[Abstract/Free Full Text]
  36. Lauder A, Castellanos A, Weston K. c-Myb transcription is activated by protein kinase B (PKB) following interleukin 2 stimulation of T cells and is required for PKB-mediated protection from apoptosis. Mol Cell Biol 2001;21:5797–805.[Abstract/Free Full Text]
  37. Srinivasan SK, Iversen P. Review of in vivo pharmacokinetics and toxicology of phosphorothioate oligonucleotides. J Clin Lab Anal 1995;9:129–37.[Medline]
  38. Crooke RM. In vitro toxicology and pharmacokinetics of antisense oligonucleotides. Anticancer Drug Des 1991;6:609–46.[Medline]
  39. Vaerman JL, Moureau P, Deldime F, et al. Antisense oligodeoxyribonucleotides suppress hematologic cell growth through stepwise release of deoxyribonucleotides. Blood 1997;90:331–9.[Abstract/Free Full Text]
  40. Maloney DG, Smith B, Rose A. Rituximab: mechanism of action and resistance. Semin Oncol 2002;29:2–9.
  41. Cheson BD. Hematologic malignancies: new developments and future treatments. Semin Oncol 2002;29:33–45.[Medline]
  42. Druker BJ. STI571 (Gleevec) as a paradigm for cancer therapy. Trends Mol Med 2002;8:S14–8.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Nucleic Acids ResHome page
X. Tang, J. Swaminathan, A. M. Gewirtz, and I. J. Dmochowski
Regulating gene expression in human leukemia cells using light-activated oligodeoxynucleotides
Nucleic Acids Res., February 2, 2008; 36(2): 559 - 569.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Opalinska, J. B.
Right arrow Articles by Gewirtz, A. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Opalinska, J. B.
Right arrow Articles by Gewirtz, A. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online