Stem Cells
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

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
Right arrow Citation Map
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 Feldman, E.
Right arrow Articles by Abraham, N.G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Feldman, E.
Right arrow Articles by Abraham, N.G.
Stem Cells, Vol. 15, No. 5, 386-395, September 1997
© 1997 AlphaMed Press

Adenovirus Mediated Alpha Interferon (IFN-{alpha}) Gene Transfer into CD34+ Cells and CML Mononuclear Cells

E. Feldmana, T. Ahmeda, J.D. Luttona, T. Farleya, K. Tanib, M. Freundc, S. Asanob, N.G. Abrahama

a Departments of Pharmacology and Medicine, New York Medical College, Valhalla, New York, USA;
b The Institute of Medical Science, The University of Tokyo, Tokyo, Japan;
c University of Rostock, Division of Hematology and Oncology, Rostock, Germany

Key Words. Gene transfer • {alpha} interferon • Leukemia • Stem cells

Correspondence: Dr. Nader G. Abraham, Department of Pharmacology, New York Medical College, Valhalla, NY 10595, USA.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Gene transfer or gene therapy has advantages in the treatment of a variety of disorders due to its selective expression within specific mammalian cells. Interferon-{alpha} (IFN-{alpha}) has been used in the management of leukemia but its diverse adverse activities with multiple potential side effects, possibly unrelated to therapeutic targets, may negatively influence the ability of IFN-{alpha} to treat this disorder. Therefore, we examined the ability of adenovirus (Ad)-IFN-{alpha} gene construct to transfect normal (CD34+ cells) and chronic myelogenous leukemia (CML) bone marrow mononuclear cells (BMMNC) and the transient overexpression of IFN-{alpha} in these cells. Ad-cytomegalovirus promoter driven IFN-{alpha} (AdCMV-IFN-{alpha}) at multiple doses was assessed to transfect highly purified CD34+ cells in liquid culture, and optimal transduction of CD34+ cells was achieved using 120 plaque forming units. Flow cytometric determinations revealed that there was no significant difference in cell viability for the 4 h or 24 h transfection periods. Immunoassay of IFN-{alpha} produced by CD34+ cells shows that IFN-{alpha} levels increased several fold in transfected cells. Transient expression of the IFN-{alpha} gene did not suppress proliferation of CD34+ progenitors as indicated by BFU-E or colony forming units-granulocyte-macrophage (CFU-GM) growth. Reverse transcriptase/polymerase chain reaction analysis of RNA from CD34+ harvested CFU-GM progenitor cells demonstrated transient IFN-{alpha} mRNA expression. Similarly, CML BMMNC were transfected with AdCMV-IFN-{alpha} under similar conditions as described for CD34+ cells. BMMNC cells exposed to adenovirus for 24 h and 48 h were found to express IFN-{alpha} at a substantial level. This in vitro data suggest that Ad-mediated gene transfer of IFN-{alpha} into hematopoietic stem cells can be achieved and that the IFN-{alpha} gene can be translated into its specific mRNA in CD34 progenitor cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Recently, this laboratory has utilized retrovirus vectors as vehicles for gene transfer into a variety of cells including hematopoietic stem cells in vitro and in vivo [1]. Furthermore, we have established novel conditions in which stromal adherent cells permit high efficiency of retrovirus-mediated gene transfer of adenosine deaminase into bone marrow (BM) hematopoietic progenitor cells [1]. Successful transplantation of the genes was further documented by their appearance in hematopoietic clones of cells from animals many months after reconstitution. Over the past several years, investigators have begun to apply retroviral gene transfer technology directed at hematopoietic stem cells in preliminary human clinical trials [2]. Recombinant adenoviruses are the second most commonly used viral vector for gene delivery [3-4], thus minimizing the risk of permanently altering the cellular genotype or of insertional mutagenesis. Adenoviruses are preferentially used when the transient expression of a transgene is sufficient to achieve the therapeutic goals [5-6]. Investigators found that adenovirus vectors can infect various BM hematopoietic cells including early progenitors, and that transduced cells are functionally normal in vitro. Several procedures have been reported using different exposure times with adenovirus vectors for in vitro gene transduction [7-8].

The current paper investigates the development of gene transfer vectors and assesses the effect of adenovirus-mediated gene transfer into CD34+ cells. We have previously utilized an adenovirus to mediate gene transfer of heme oxygenase into nonhematopoietic stem cells [9]. A similar recombinant adenoviral vector was used for interferon-{alpha} (IFN-{alpha}) gene transfer. The viral construct with the cytomegalovirus (CMV) promoter was also described by Ohwada et al. [10] and used for transfer of thrombopoietin into hematopoietic stem cells. A similar metabolic approach to construct adenovirus CMV-IFN-{alpha} gene was used to transfect both normal and chronic myelogenous leukemia (CML) hematopoietic stem cells. Successful adenovirus-CMV promoter driven IFN-{alpha} (Ad-CMV-IFN-{alpha}) gene transfer into CD34+ cells is monitored by Southern blot analysis and Northern blot analysis of genomic DNA and RNA, respectively, from the CD34+ cells and from hematopoietic clones, BFU-E and colony forming units-granulocyte-macrophage (CFU-GM) seeded by transfected CD34+ cells. The results allowed us to determine the feasibility of developing an adenovirus-mediated gene transfer into CD34+ cells. Since IFN-{alpha} has been used in the management of tumor growth and treatment of CML, we decided to use CML cells as a model tumor system for IFN-{alpha} gene transfer.

CML arises from a clonal expansion of a transformed stem cell capable of differentiation into mature hematopoietic cells [11-13]. The Philadelphia (Ph) chromosome is characteristic of the disease in which the reciprocal translocation t(9;22) results in the creation of a chimeric bcr/abl gene which plays a central role in leukemogenesis [14, 15]. Induction of clonal expansion associated with bcr/abl expression may be due in part to increased tyrosine kinase activity [16]. In addition, CD34+ marrow cells from patients with CML respond to colony-stimulating factors, but their adhesion to the stroma is impaired, resulting in a loss of sensitivity to stromal inhibitory signals [17-21].

Numerous clinical trails have documented the ability of IFN to induce hematologic and cytogenetic remissions in patients with CML [22]. A number of in vitro effects of IFN-{alpha} on CML cells and stroma have been well documented, including inhibition of CML marrow progenitor growth [23, 24], restoration of the adherence to stroma [25-27], and regulation of stromal cytokine production [28] and cellular immune surveillance [29-33] important in the control of growth of the leukemic clone in CML.

The systemic administration of IFN-{alpha} following cytotoxic therapy has been disappointing, due in part to the inability of patients to tolerate the exogenous doses required. It is possible that endogenous production of IFN-{alpha} by the host may be more effective at lower levels as compared to the levels required exogenously. One possible means of improving the efficacy of IFN-{alpha} is to deliver the agent as a gene directly into the hematopoietic progenitor cells that are reinfused (following ablative therapy) by means of a viral vector in which one could achieve efficient and constant expression of the gene or product over a specific period of time. In this way, overexpression of IFN-{alpha} may provide antileukemic effects that suppress CML growth beyond what is possible with systemic administration.

The present report is concerned with the development of efficient adenovirus-mediated gene transfer of the IFN-{alpha} gene into hematopoietic CD34+ cells and also CML bone marrow mononuclear cells (BMMNCs) in vitro. Such in vitro studies may then provide insight on how to effectively deliver the IFN-{alpha} gene into cells with possible therapeutic potential.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
The replication-deficient adenovirus vector encoding IFN-{alpha} (AdCMV-IFN-{alpha}) was constructed in our laboratory, as described previously [9]. The recombinant adenoviral vector was constructed by hemologous recombination between the E3 deleted adenovirus, dL700 1 and the plasmid containing the human IFN-{alpha}. The AdCMV-IFN-{alpha} plasmid was constructed as follows: Xho1-Xho1 fragment of the human IFN-{alpha} cDNA deleted from the BMGNeo IFN-{alpha} plasmid was inserted into the hind III site of the pRC/CMV (Invitrogen; San Diego, CA) to obtain CMV-IFN-{alpha}. A similar approach was used in our laboratory with expression of the vector [9]. The Nru I-Bam HI fragment containing CMV-IFN-{alpha} was inserted into the Stu 1-Bam H1 sites of the pBacPac 8 (Clontech; Palo Alto, CA) to obtain pBacPac 8-CMV-IFN-{alpha}. The Bam HI-Bgl III fragment containing CMV-IFN-{alpha} from pBacPac8-CMV-IFN-{alpha} was inserted into the Bgl II of the pAdv.Bgl II plasmid to obtain the AdCMV-IFN-{alpha}. A human embryonic cell line that expressed E1 (293 cells ATCC #1573 - CRL) was cotransfected with 10 mg of ECOR I-digested AdCMV-IFN-{alpha} and 1 mg of Cla I-digested dL7001 DNA using a mammalian transfection kit (Stratagene; La Jolla, CA). The virus was replicated and encapsidated into an infectious virus. After five days, plaque locations were marked on the plate, and the resultant cytopathic effect to the monolayer was observed microscopically until the plaque reached an adequate size (usually one week). The plaques were purified by ultracentrifugation through cesium chloride gradients and checked for the presence of human IFN-{alpha} by polymerase chain reaction (PCR) using IFN-{alpha} specific primers. The primers for IFN-{alpha} are 5'CAG TTC CAG AAG GCT CAA GC3' and 5'ACC TCC TGC ATC ATA CAG GC3'. These primers give a PCR product with a size of 222 bp. The virus was released from infected cells two days after infection by five freeze-thaw cycles, concentrated by centrifugation using Ultrafree-MC filters (Millipore; Bedford, MA), and dialyzed against phosphate-buffered saline (PBS). Titers of viral stocks were determined by plaque assay using 293 cells as previously described [9, 34]. Titers of the viral stocks used for transfection were 1 to 5 x 1010 infectious units/ml. Cells were incubated in triplicate dishes with AdCMV-IFN-{alpha} at 40, 80, 120, 160 and 200 plaque forming units (pfu)/cell. The viral stock was diluted 1:1 with glycerol and stored at –20°C. Concentration of the virus was determined by measuring its optical density (OD) at 260 nm. Viral particle content was then calculated from its OD at 260 nm under the assumption that 1 OD equals 1 x 1012 viral particles/ml.

BM Samples
Heparinized BM samples were aspirated from the posterior iliac crest of normal volunteers or CML patients after informed consent was obtained according to the guidelines established by the Human Investigation Committee of New York Medical College. Specimens of normal and CML BM cells were also obtained from the National Disease Research Interchange (The National Resources Center). Low-density BMMNC were isolated by density centrifugation of heparinized marrow layered over Ficoll-Paque (Pharmacia Fine Chemicals; Piscataway, NJ) at 500 g for 25 min.

Selection of Purified Progenitor Populations
BMMNC were prepared by sequential counterflow centrifugation elutriation [35], sheep erythrocyte rosetting [36] and immunomagnetic bead depletion [37]. Alternatively, CD34+ cells were selected from BMMNC using avidin-biotin immunoadsorbtion columns (CellPro Inc; Bothell, WA) [38]. The selected populations were labeled with anti-CD34-phycoerythrin and anti-HLA-DR-fluorescein isothiocyanate antibodies (Becton-Dickinson; San Jose, CA) and sorted on an EPICS Elite II laser flow cytometry system (Coulter; Hialeah, FL). Cells were selected for low vertical and horizontal light scatter properties and for expression of CD34 and HLA-DR antigens based on isotype control stains [37, 39]. In some cases, purified CD34+ cells were obtained from the Hudson Valley Blood Services (Valhalla, NY).

Transfection Protocol for CD34+ Cells and BMMNC in Liquid Culture
CD34+ cells were resuspended in Iscove's modified Dulbecco's medium (IMDM) containing 0.5% fetal bovine serum in T-25 flasks supplemented with cytokines (interleukin 6 [recombinant human interleukin 6, 50 U/ml]; stem cell factor [recombinant human stem cell factor, 100 ng/ml]; interleukin 3; 5 ng/ml, G-CSF [50 U/ml]; GM-CSF [50 U/ml] [Amgen; Thousand Oaks, CA]; and megakaryocyte growth factor [10 U/ml] [Immunex; Seattle, WA]). Various pfu/cell (20, 40, 80, 120, 160 and 240) of AdCMV-IFN-{alpha} were added directly to the cultures of CD34+. The cocultivation of adenovirus with CD34+ was continued for 4, 8, 12, 24 and 48 h. After each incubation time, samples were removed for measurement of cell viability by propidium iodide (PI) staining or for clonal efficiency in methylcellulose cultures. IFN-{alpha} mRNA levels were assessed on CD34+ samples by reverse transcription (RT)/PCR using human specific primers to yield a PCR product of 222 bp. A similar protocol was followed for transfection of progenitor cells obtained from CML donors. CML BMMNC from fresh leukapheresis samples were separated by counterflow centrifugation and sheep erythrocyte rosetting as described above. BMMNC were then plated in T-25 flasks containing IMDM and 0.5% fetal calf serum and adenovirus was added to the flasks at 120-300 pfu/cell for 24 h. After this time period, samples were removed and assessed for clonal efficiency and expression of IFN-{alpha} mRNA in CFU-GM and BFU-E after 12 and 14 days.

Human IFN-{alpha}
The presence of IFN-{alpha} in CD34+ cell media was determined using a commercially available quantitative enzyme-linked immunosorbent assay (ELISA) (Endogen Inc.; Cambridge, MA). After 24 h incubation, the culture medium was removed and centrifuged at 1,000 rpm (4°C) to remove floating cells. The resultant supernatant was passed through a 0.22 m filter and stored at –70°C. Human IFN-{alpha} levels in the supernatant were determined by radioimmunoassays using antihuman IFN-{alpha} monoclonal antibodies following the manufacturer's instructions. CD34+ cells were placed in fresh IMDM at a concentration of 5 x 106 cell/ml and assayed for IFN-{alpha} after 24 and 48 h.

Hematopoietic Colony Assays
Methylcellulose culture technique was used to assess the clonal efficiency of the transduced cells with the IFN-{alpha} gene. One thousand CD34+ cells were plated in methylcellulose (final concentration 1.12% [Fisher Scientific; Fairlawn, NY]) with IMDM supplemented with 15% fetal bovine serum and 100 U/ml GM-CSF for CFU-GM colony assay or in the presence of 2 U/ml erythropoietin ([EPO] Toyobo; Osaka, Japan) and antibiotics as described previously [1, 29]. Cells were incubated at 37°C in a humidified atmosphere containing 5% CO2 in air. Myeloid colonies consisting of 50 cells or more were counted as CFU-GM and more than two clusters of 20 or more hemoglobinized cells were counted as erythroid BFU-E using an inverted microscope. CFU-GM and BFU-E clones were picked from methylcellulose plates and used for measurement of IFN-{alpha} mRNA by RT/PCR.

Quantitative Measurement of IFN-{alpha} mRNA by RT/PCR
RNA is reverse-transcribed using the first strand cDNA synthesis kit from Clontech, Inc. (Palo Alto, CA). Briefly, RNA in 13.5 ml of DEPC-treated H2O containing 1 ml of oligo (dT)18 (final concentration 0.2 mM) is denatured at 70°C for two min. The denatured RNA is placed on ice, and 6.5 ml of the reverse transcription mixture containing 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 0.5 mM of each dNTP, 1 U/ml of RNase inhibitor, and 200 U Moloney murine leukemia virus reverse transcriptase (Bethesda Research Laboratories; Gaithersburg, MD) are added. The reaction tube is then incubated at 42°C for one h followed by heating to 95°C to stop the reaction and then placed on ice.

The PCR reaction is performed by adding the PCR mixture to a final volume of 100 ml to the RT reaction tube [40]. The PCR mixture contained 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 0.001% gelatin, 1.5 mM MgCl2, 250 mM of each dNTP, 1 mM of sense and antisense primers, 2.5 U of Taq DNA polymerase, and 1 mCi of 32P-dCTP [{alpha}-32P] dCTP (3,000 Ci/mmol and Ci-37 Gbq are from Amersham Corporation; Arlington Heights, IL; DNA molecular weight marker VI is from Boehringer Mannheim Biochemicals; Indianapolis, IN; dNTPs [100 mM in sterile deionized H2O] are from Promega Corporation; Madison, WI; Taq DNA polymerase and restriction enzymes PstI and PvuII are from Stratagene, and Nusieve GTG and SeaKem LE agarose are from FMC BioProducts; Rockland, ME; standard and primers are from Promega). The reaction mixture is overlaid with two drops of mineral oil and subjected to 40 cycles as follows: 95°C for 1 min, 55°C for 1 min and 72°C for 2 min. After the last cycle, a final extension will be performed at 72°C for 10 min.

Southern and Northern Blot Analysis of Transfected Cells
Genomic DNA was extracted from CD34+ cells, and CFU-GM and BFU-E clones. The cell preparations (2-5 x 106 cells) were digested with Bam HI, Hind III and Xho I. The digested DNA was electrophoresed on an agarose gel and then transferred to nitrocellulose filters as previously described [34]. Total RNA from 2-5 x 106 CD34+ cells was extracted according to the method of Chomczyski and Sacchi [41] and used for RT/PCR. IFN-{alpha} transcripts were amplified by RT/PCR using 2 ml of 100 ml cDNA mixture of total reaction volume of 25 ml. Aliquots of the reaction mixture (10 ml) were subjected to electrophoresis in a 0.18% agarose gel. Nucleic acids were transfected to nitrocellulose by blotting and probed with IFN-{alpha} cDNA probes. Filters were exposed to x-ray for 48 h at –80°C. The Bgl I fragment of IFN-{alpha} cDNA was labeled with [{alpha}-32P] dCTP using a mulitprimer DNA-labeling system. The filters were then hybridized with the 32P-labeled IFN-{alpha} cDNA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In Vitro Expression of IFN-{alpha} cDNA Assessment of IFN-{alpha} Protein and mRNA
In preliminary experiments, we examined the expression/overexpression of IFN-{alpha} in transfected CD34+ cells. Multiple doses of AdCMV-IFN-{alpha} were used to transfect human CD34+ cells. A schematic representation of the adenoviral vector containing the IFN-{alpha} gene is represented in Figure 1. We next evaluated the ability of the AdvCMV-IFN-{alpha} vector to express the IFN-{alpha} cDNA and direct production of IFN-{alpha} protein. After 24 h of induction, the cells were spun down, the medium harvested and IFN-{alpha} protein measured by radioimmunoassay. Results are presented in Table 1. As seen in Table 1, AdCMV-IFN-{alpha} caused elevation of IFN-{alpha} protein in the conditioned medium, suggesting that the IFN-{alpha} gene is expressed in CD34+ cells resulting in the release of IFN-{alpha} into the medium (4.1 U/ml). On the other hand, AdCMV-HO-1 (heme oxygenase gene) in control cells does not express significant levels of IFN-{alpha}. The value listed for these two cells represents only about 0.3 U/ml.



View larger version (23K):
[in this window]
[in a new window]
  		Figure 1. A schematic diagram of the adenoviral vector containing the IFN-{alpha} gene.

 

View this table:
[in this window]
[in a new window]
  		Table 1. Secretion of IFN-{alpha} by AdCMV-IFN-{alpha} transduced human CD34+ cells
 
Viability of the cells was determined using PI and flow cytometric analysis. Cells were stained with 20 ml of CD34-fluorescein isothiocyanate and CD45-PerCP at 4°C for 30 min. Usually 10,000 cells were analyzed for each condition. PI (5 mg) was added five min before completion of the antibody incubation. The cells were washed once, resuspended in PBS, and analyzed immediately by flow cytometry. The flow cytometric analysis was performed using an EPICS Profile II (Coulter). The viability of the CD34+ and CD34– cell populations was determined by restricting analysis based upon antibody reactivity (fluorescence). Histograms depict light scatter characteristics CD34+ analysis and PI uptake of the CD34+ cell population. Results are from the flow cytometric determination of CD34+ cell viability at 4, 8, 16 and 24 h transfection periods.

As shown in Figure 2, viabilities were determined for purified CD34+ cell populations after 4 h (Fig. 2A-C), 8 h (Fig. 2D-F), 16 h (Fig. 2G-I), and 24 h (Fig. 2J-L) incubation periods. The loss of membrane integrity (nonviable) was calculated by measurement of PI dye uptake for both CD34+ and CD34– cell populations. The size (forward scatter) and internal complexity (side scatter) for each event were plotted for the four incubation periods ( Fig. 2A , D, G, J). The percentage of CD34+ cells in the samples, which was corrected for debris, was 96.3% at 4 h ( Fig. 2B), 94.8% at 8 h ( Fig. 2E), 67.2% at 16 h ( Fig. 2H), and 57.0% at 24 h ( Fig. 2K). The percentage of cells that were CD34+ and maintained membrane integrity was 98.8% at 4 h ( Fig. 2C), 97.0% at 8 h ( Fig. 2F), 83.3% at 16 h ( Fig. 2I), and 73.5% at 24 h ( Fig. 2L). This was done by analysis of data in Figure 2 (B, E, H and K) in conjunction with PI uptake, represented in Figure 2 (C, F, I and L).



View larger version (32K):
[in this window]
[in a new window]
  		Figure 2. Results from the flow cytometric determination of CD34+ cell viability at 4 h (A, B, C), 8 h (D, E, F), 16 h (G, H, I), and 24 h (J, K, L) transfection periods. Cells were stained with 20 ml of CD34-fluorescein isothiocyanate and CD45-PerCP (Becton-Dickinson) at 4°C for 30 min. PI (5 mg) was added five min before completion of the antibody incubation. The cells were washed once, resuspended in PBS, and analyzed immediately by flow cytometry. Histograms depict light scatter characteristics ( Fig. 2A , D, G and J), CD34+ analysis ( Fig. 2B , E, H and K) and PI uptake of the CD34+ cell population ( Fig. 2C , F, I and L).

 
Establishment of the Dose Dependent Expression of AdCMV-IFN-{alpha} in CD34+ Cells
CD34+ cells were plated in IMDM at 2 x 106 cells per 25 cm2 flask and incubated with AdCMV-IFN-{alpha} at different levels of viral particles (40, 80, 120, 160, 240, pfu/cell) for 1-48 h (37°C, 5% CO2) and then washed and used for assessment of mRNA and clonal efficiency. IFN-{alpha} mRNA was measured by RT/PCR in CD34+ cells. As seen in Figure 3A, optimal expression of IFN-{alpha} in CD34+ cells could be achieved with 120 pfu/CD34+/cell, (81%), and there was little or no significant increase with 160-240 pfu (76%-91%). Cells exposed to 30-40 pfu yielded about 15%-27% increase in IFN-{alpha} (data not shown).




View larger version (197K):
[in this window]
[in a new window]
  		Figure 3. (A) Expression of IFN-{alpha} in CD34+ cells exposed to different plague forming units (pfu) of AdCMV-IFN-{alpha}.. (B) CFU-GM growth by CD34+ cells exposed to different pfu.

 
Toxicity of AdCMV-IFN-{alpha} on CD34+ Cells
We tested the effect of the dose response from 120-240 pfu/cell on CD34+ progenitor cell growth and differentiation. After 24 h incubation with AdCMV-IFN-{alpha}, aliquots of CD34+ cells (1,000 cells) were plated in methylcellulose cultures containing GM-CSF. In all experiments the number of CFU-GM clones of more than 50 cells were counted. Results show that cells transfected with 240 pfu grow less CFU-GM when plated in culture as contrasted with cells that were exposed to 120 pfu ( Fig. 3B). Furthermore, 120 pfu yielded colony growth similar or equal to control cells. These results show that an optimal expression of IFN-{alpha} can be achieved with CD34+ cells exposed to 120 pfu without any noticeable toxic effect on CFU-GM growth derived from the CD34+ cells.

Clonogenic Potential of IFN-{alpha} Transduced CD34+ Cells
To evaluate the effect of AdCMV-IFN-{alpha} on CFU-GM growth, we compared the amount of CFU-GM growth by infected and noninfected cells. Results in Figure 4 show that there was no significant difference in CFU-GM growth of infected versus control cells for any of the exposure times. Although colony numbers were slightly less for the 24 h cell groups as compared with four or eight h, maximal gene transfer occurred with the cells exposed for 24 h. However, the overall clonogenic potential of both transduced and nontransduced cells in 48 h groups was less than that of cells exposed to adenovirus for 24 h in the liquid culture. Experiments are being performed using adenovirus vectors carrying the bacterial lacZ gene to accurately monitor transduction efficiency. However, the AdCMV heme oxygenase gene is available in our laboratory, and we used it for comparison to the AdCMV-IFN-{alpha} gene. Preliminary results have shown, by in vitro hybridization, that the rate of transfection varied from 5% to 41% (data not shown).



View larger version (87K):
[in this window]
[in a new window]
  		Figure 4. CFU-GM growth by CD34+ cells exposed to AdCMV-IFN-{alpha} for different exposure time periods.

 
Presence of CMV cDNA in Transfected Cells
Restriction endonuclease analysis of DNA extracted from CFU-GM clones after infection of CD34+ cells with AdCMV-IFN-{alpha} gene was done. Results are depicted in Figure 5 where lane 1 represents CD34+ cells after 24 h transfection; lane 2 represents CFU-GM after 12 days; lane 3 is a positive control; lane 4 shows CD34+ not transfected and lane 5 is a DNA marker. It can be seen that the DNA patterns are similar in lanes 1, 2 and 3, indicating an adequate presence of CMV cDNA of adenovirus construct which transfected CD34+ cells as well as CD34+ progenitors (CFU-GM) derived from the CD34+ cells after 14 days of culture.



View larger version (21K):
[in this window]
[in a new window]
  		Figure 5. Restriction endonuclease analysis of DNA extracted from CFU-GM clones after infection of CD34+ cells with AdCMV-IFN-{alpha} gene. A) Lane 1, CD34+ cells after 24 h transfection; lane 2, CFU-GM after 12 days; lane 3, positive control; lane 4, CD34+ not transfected with AdCMV-IFN-{alpha}; and lane 5, a DNA marker. B) Represents the same preparations as A after hybridization and blotting with the probe.

 
Detection of IFN-{alpha} mRNA in Transduced Cells
Southern blot analysis of the PCR product obtained from total RNA isolated from cells (and colonies) transfected with AdCMV-IFN-{alpha} gene is presented in Figure 6. Total RNA was extracted from CD34+ cells, BFU-E and CFU-GM clones. Results in Figure 6 show that transfected CD34+ cells (lane 2) and clones of CFU-GM (lanes 3, 4) and BFU-E (lanes 5, 6) (grown for 12-14 days) all show the appropriate RNA bands. Lane 1 is a nontransfected control. The experiments could not determine how many copies of IFN-{alpha} mRNA are transcribed. However, quantitative RT/PCR methods are being developed to answer this question.



View larger version (27K):
[in this window]
[in a new window]
  		Figure 6. Southern blot analysis of PCR products obtained from total RNA isolated from CD34+ cells transfected with AdCMV-IFN-{alpha} gene. Lane 1, PCR products amplified from nontransfected (control) CD34+ cell RNA; lane 2, RNA obtained from CD34+ cells transfected with AdCMV-IFN-{alpha} after 24 h; lanes 3 and 4, RNA from CFU-GM clones after 12 and 14 days of culture, respectively; lanes 5 and 6, RNA from BFU-E clones after 12 and 14 days of culture. Total RNA was extracted from CD34+ cells, BFU-E or CFU-GM clones. IFN-{alpha} transcripts were amplified by PCR using 2 ml of 100 ml cDNA mixture of a total reaction volume of 25 ml. Aliquots of the reaction mixture (10 ml) were subjected to electrophoresis in a 0.8% agarose (Sigma; St. Louis, MO) gel. Nucleic acids were transferred to nitrocellulose by blotting and probed with IFN-{alpha} cDNA probes, (gift of Dr. Asano) using {alpha}-32d-CTP. Filters were exposed to x-ray for 48 h at –80°C.

 
CFU-GM Clonogenic Potential of Transduced CML-BMMNC
Previous results using adenovirus-mediated IFN-{alpha} transfer as a reporter gene without significant suppression of CD34+ growth encouraged us to test the ability of adenovirus on transfections of progenitor cells obtained from CML patients. For this purpose, we used similar doses of adenovirus (120-240 pfu) to transfect BMMNC in liquid culture for 24 h in the presence of cytokines. Aliquots of cells were removed and tested for the transfection ability and clonal efficiency of CML progenitor cells. The clonal efficiency of transfected cells was then assayed by plating BMMNC in methylcellulose. As seen in Table 2, BMMNC from CML transduced with IFN-{alpha} (120 pfu) generated 46.0 ± 5.9 CFU-GM as compared to 48.0 ± 5.0 in nontransduced cells. In contrast, adenovirus CMV-IFN-{alpha} transfection causes a decreased number of CFU-GM when the pfu/cell were 240 compared to the uninfected control cells. A substantial inhibition of CFU-GM was also seen when BMMNC were transfected with 300 pfu/cell. Expression of IFN-{alpha} mRNA in CFU-GM generated from CML was next determined. In an effort to assess the expression of IFN-{alpha} mRNA in progenitor cells, we performed RT/PCR on the RNA extracted from various clones similar to that performed on CD34+ progenitor cells. As seen in Figure 7, results of RT/PCR of total RNA of CFU-GM and BFU-E demonstrate IFN-{alpha} mRNA was expressed in CFU-GM clones after 12 and 14 days of culture (lanes 3 and 4) as compared to the control (lane 1). Similarly, BFU-E clones expressed a substantial level of IFN-{alpha} mRNA. These results demonstrate that recombinant adenovirus is an effective vehicle for transiently expressing genes in both hematopoietic BMMNC obtained from CML donors and in CD34+ cells obtained from normal donors. Further, the IFN-{alpha} gene is effectively carried to clones of hematopoietic elements (CFU-GM) derived from both primitive cells of normal and CML donors. Although the expression of the gene is transient, we have been able to identify conditions for adenovirus-mediated IFN-{alpha} gene transfer into hematopoietic cells, thus, enabling us to determine the effect of multiple copies of IFN-{alpha} and to evaluate its expression and toxicity on hemopoietic elements.


View this table:
[in this window]
[in a new window]
  		Table 2. Clonogenic potential (CFU-GM) of CML BMMNC transfected with AdCMV-IFN-{alpha}*
 


View larger version (25K):
[in this window]
[in a new window]
  		Figure 7. Southern blot analysis of PCR products obtained from total RNA isolated from CML hematopoietic stem cells (BMMNC) transfected with AdCMV-IFN-{alpha} gene. Lane 1, PCR products amplified from nontransfected (control) cell RNA; lane 2, RNA obtained from cells transfected with AdCMV-IFN-{alpha} after 24 h; lane 3, RNA from CFU-GM clones after 12 days; lane 4, CFU-GM clones after 14 days of culture; lane 5, no RNA was added; and lane 6, positive control. Total RNA was extracted from stem cells or CFU-GM clones by one single extraction. cDNA was synthesized from total RNA. IFN-{alpha} transcripts were amplified by PCR using 2 ml of 100 ml cDNA mixture of a total reaction volume of 25 ml. Aliquots of the reaction mixture (10 ml) were subjected to electrophoresis in a 0.8% agarose (Sigma) gel. Nucleic acids were transferred to nitrocellulose by blotting and probed with IFN-{alpha} cDNA probes, using {alpha}-32P-CTP. Filters were exposed to x-ray for 48 h at –80°C.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this article, we have described successful transfection of the IFN-{alpha} gene into human CD34+ cells and CML BMMNCs by using adenovirus-mediated IFN-{alpha} gene transfer. We have shown successful transfection of hematopoietic progenitor cells with adenovirus with transgene expression lasting for at least two weeks. When CD34+ cells were exposed to adenovirus IFN-{alpha} gene, IFN-{alpha} protein was detected in the incubation medium within 24 h, indicating that IFN-{alpha} protein was released into the medium. Analysis of IFN-{alpha} gene in CD34+ cells demonstrated that adenovirus was able to deliver functional DNA since IFN-{alpha} mRNA was detected in both CD34+ cells and in colonies generated from the CD34+ cells. At the present time, we do not know the efficacy of adenoviral vectors for transduction of CML BMMNCs as compared with normal CD34+ cells. It is possible that virus infection may be taken up at a higher level in BMMNCs than in CD34+ cells. Experiments to determine the number of copies of IFN-{alpha} mRNA in the more phenotypically primitive CD34+/CD38– CML BMMNCs are to be done in the near future. However, it is possible that a different receptor is present on more mature cells, such as BMMNC, which could then facilitate the uptake of adenovirus. In fact, Huang et al. [42] have shown that upregulation of certain receptors such as integrins on monocytes, facilitates adenovirus mediated gene delivery. Identification and utilization of a specific receptor on CML cells may prove to be an excellent tool for induction of cell death by IFN-{alpha}. It is not know what the effectiveness of microenvironmental production (endogenous) of IFN-{alpha} will have on progenitor cell growth and differentiation. It remains possible that local production of IFN-{alpha} in the stromal microenvironment may be more effective than much higher levels of IFN-{alpha} supplied exogenously.

The antitumor effects of IFN-{alpha} have been extensively studied by many investigators, and the effectiveness of IFN as a therapeutic modality has been confirmed in the treatment of renal cell carcinoma, hairy cell leukemia, malignant melanoma and myeloma [4]. In addition to these neoplasms, recent studies have revealed that IFN-{alpha} is also effective in the treatment of Ph+ CML [22]. However, long-term continual parenteral administration of IFN-{alpha} is required to maintain therapeutic efficacy against these diseases. IFN-{alpha} therapy has been shown to be of great value to control the levels of bcr/abl mRNA, adhesion molecule expression and enhancement of cell differentiation. It would be beneficial for the patient if an appropriate amount of IFN-{alpha} could be expressed locally over a long period of time. Gene transfer of IFN-{alpha} into human CD34+ cells may prove to be of clinical value for continuous overexpression of IFN-{alpha} in CML patients.

In this report, we used adenovirus as a transient means of gene transfer. Adenovirus vectors provide an ideal vehicle for transient expression by which to deliver IFN-{alpha} for the purpose of abrogating Ph+ levels induced in CML cells. Results suggest that the transgene of IFN-{alpha} can be achieved using the AdCMV vector with a varied efficiency ranging from 5%-41%. Further, there is a direct relationship between increased adenovirus dose and toxicity, especially in MNCs of CML donors. The objective of these studies was to define conditions that enhance in vitro adenovirus-mediated gene transfer to hematopoietic cells and to examine the effects of various growth factors on transduction to improve transduction efficiency. We have applied the adenovirus IFN-{alpha}-mediated gene transfer into CD34+ cells with good success. These studies in our laboratory have shown that there is no suppressive effect on CD34+ cell growth and differentiation after transduction with IFN-{alpha} gene.

Data from different laboratories suggest that normal hematopoietic stem cells (HSCs), present in CML marrow, can be separated from leukemic HSCs by fluorescence-activated cell sorting [39, 43]. These findings suggest that CML progenitors may be targets for vectors which express antisense bcr/abl [44]. Also, retroviral vectors which confer drug resistance to autologous bcr/abl-negative HSCs may be of potential therapeutic benefit for CML. Retrovirus-mediated antisense of bcr/abl is currently being considered for treatment of CML [45]. Retroviral vectors are currently the only gene transfer system with the appropriate characteristics of chromosomal integration and the concurrent use of stable helper-free producer cell lines that can be used clinically in protocols targeted at HSCs. In rodent models, investigators have demonstrated efficient and reproducible gene transfer to a high percentage of long-term repopulating stem cells and achieved long-term expression of introduced genes in appropriate lineages [1, 46]. Efficient gene transfer to primitive human progenitor cells, such as CFU-granulocyte-erythroid-macrophage-megakaryocyte or long-term culture initiating cells, has been reported, with gene transfer efficiencies greatly increased by exposing target cells to hematopoietic growth factors during transduction with viral vectors [46].

Results in this study show that we can efficiently transfer the IFN-{alpha} gene into normal (CD34+) cells and CML primitive hematopoietic mononuclear stem cells, and that the gene transfer has little or no effect on normal CD34+ hematopoietic growth but does reduce growth by CML BMMNC. This in vitro data using transient gene transfer IFN-{alpha} suggest that selective expression of IFN-{alpha} may be of beneficial value for CML therapy.

In summary, this report demonstrates and confirms other investigations that adenovirus vector can be used to mediate gene transfer into CD34+ cells. We applied this approach to an important clinical setting in which IFN is of general use. We have shown that adenovirus-mediated IFN-{alpha} can be effective in gene transfer. However, the level of IFN-{alpha} to be used at therapeutic levels to enhance adhesion molecules or suppress bcr/abl gene expression is an important debate for upcoming investigations.


   Acknowledgments
 
This work was supported, in part, by National Institutes of Health Grants RO1-HL-5438. We thank Dr. R. Shadduck (University of Pittsburgh School of Medicine, Pittsburgh, PA) for his helpful suggestions and review of the manuscript.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Chertkov JL, Jiang S, Lutton JD et al. The hematopoietic stromal microenvironment promotes retrovirus-mediated gene transfer into hematopoietic stem cells. STEM CELLS 1993;11:218-227.[Abstract]

  2. Dunbar CE, Emmons RV. Gene transfer to hematopoietic progenitor and stem cells: progress and problems. STEM CELLS 1994;12:563-570.[Abstract]

  3. Mulligan RC. The basic science of gene therapy. Science 1994;260:926-932.

  4. Hanania EG, Kavanagh J, Hortogagyi G et al. Recent advance in the application of gene therapy to human disease. Am J Med 1995;99:537-552.[Medline]

  5. Christal RG, McElvaney NG, Rosenfeld MA et al. Administration of an adenovirus containing the human CFTR cDNA to the respiratory tract of individuals with cystic fibrosis. Nat Genet 1994;8:42-51.[Medline]

  6. Watanabe T, Kiszynski C, Ino K et al. Gene transfer into human bone marrow hematopoietic cells mediated by adenovirus vectors. Blood 1996;87:5032-5036.[Abstract/Free Full Text]

  7. Haddada H, Lopez M, Martinache C et al. Efficient adenovirus-mediated gene transfer into human blood monocyte-derived macrophages. Biochem Biophy Res Commun 1993;195:1174-1183.[Medline]

  8. Mitani K, Graham FL, Caskey CT. Transduction of human bone marrow by adenoviral vector. Hum Gene Ther 1994;5:941-948.[Medline]

  9. Abraham NG, da Silva J-L, Lavrovsky Y et al. Adenovirus-mediated heme oxygenase-1 gene transfer into rabbit ocular tissues. Invest Ophthalmol Vis Sci 1995;36:2202-2210.[Abstract/Free Full Text]

  10. Ohwada A, Rafii S, Moore MAS et al. In vivo adenovirus vector-mediated transfer of the human thrombopoietin cDNA maintains platelet levels during radiation- and chemotherapy-induced bone marrow suppression. Blood 1996;88:778-784.[Abstract/Free Full Text]

  11. Fialkow PJ, Jacobson RJ, Papayannopoulou T. Chronic myelocytic leukemia: clonal origin in a stem cell common to the granulocyte, erythrocyte, platelet and monocyte/macrophage. Am J Med 1977;63:125-130.[Medline]

  12. Martin PJ, Najfield V, Hansen JA et al. Involvement of the B-lymphoid system in chronic myelogenous leukemia. Nature 1980;287:49-50.[Medline]

  13. Nitta M, Kato Y, Strife A et al. Incidence of involvement of the B and T lymphocyte lineages in chronic myelogenous leukemia. Blood 1985;66:1053-1061.[Abstract/Free Full Text]

  14. McGlave PB, Bartsch G, Anasetti C et al. Unrelated donor bone marrow transplantation therapy for chronic myelogenous leukemia: initial experience of the National Marrow Donor Program (NMDP). Blood 1993;81:543-550.[Abstract/Free Full Text]

  15. Snyder DS, McGlave PB. Treatment of chronic myelogenous leukemia with bone marrow transplantation. Hematol Oncol Clin North Am 1990;4:53-57.

  16. McWhirter JR, Wang JYI. Activation of tyrosine kinase and microfilament-binding functions by c-able by bcr sequences in bcr/abl fusion protein. Mol Cell Biol 1991;11:1553-1565.[Abstract/Free Full Text]

  17. Gordon MY, Dowding CR, Riley GP et al. Altered adhesive interactions with marrow stroma of haematopoietic cells in chronic myeloid leukemia. Nature 1987;328:342-345.[Medline]

  18. Verfaillie CM, McCarthy JB, McGlave PB. Mechanisms underlying abnormal trafficking of malignant progenitors in chronic myelogenous leukemia. J Clin Invest 1992;90:1232-1241.

  19. Wetzler M, Kurzrock R, Lowe DG et al. Alteration in bone marrow adherent layer growth factor expression: a novel mechanism of chronic myelogenous leukemia progression. Blood 1991;78:2400-2406.[Abstract/Free Full Text]

  20. Bhatia R, McGlave PB, Dewald GW et al. Abnormal function of the bone marrow microenvironment in chronic myelogenous leukemia: role of malignant stromal macrophages. Blood 1995;85:3636-3645.[Abstract/Free Full Text]

  21. Bhatia R, McCarthy JB, Varfaillie CM. Interferon-{alpha} restores normal b1 integrin-mediated inhibition of hematopoietic progenitor proliferation by the marrow microenvironment in chronic myelogenous leukemia. Blood 1996;87:3883-3891.[Abstract/Free Full Text]

  22. Italian Cooperative Study Group on Chronic Myeloid Leukemia. Interferon alfa-2a as compared with conventional chemotherapy for the treatment of chronic myeloid leukemia. N Engl J Med 1994;330:820-825.[Abstract/Free Full Text]

  23. Broxmeyer HE, Lu E, Platzer E et al. Comparative analyses of the influences of human gamma, alpha and beta inteferons on human multipotential (CFU-GEMM), erythroid (BFU-E) and granulocyte macrophage (CFU-GM) progenitor cells. J Immunol 1983;131:1330-1335.

  24. Geissler D, Gastl G, Aulitzky W et al. Recombinant interferon-alpha-2C in chronic myelogenous leukemia: relationship of sensitivity of committed haematopoietic precursor cells in vitro (BFU-E, CFU-GM, CFU-Meg) and clinical response. Leuk Res 1990;14:629-636.[Medline]

  25. Upadhyaya G, Guba SC, Sih SA et al. Interferon-alpha restores the deficient expression of the cytoadhesion molecule lymphocyte function antigen-3 by chronic myelogenous leukemia progenitor cells. J Clin Invest 1991;88:2131-2136.

  26. Dowding C, Guo AP, Osterholz J et al. Interferon-alpha overrides the deficient adhesion of chronic myelogenous leukemia primitive progenitor cells to bone marrow stromal cells. Blood 1991;78:499-505.[Abstract/Free Full Text]

  27. Bhatia R, McGlave P, Verfaillie CM. Treatment of marrow stroma with interferon-{alpha} restores normal b1 integrin-dependent adhesion of chronic myelogenous leukemia hematopoietic progenitors. Role of MIP-1{alpha}. J Clin Invest 1995;96:931.

  28. Aman MJ, Keller U, Derigs G et al. Regulation of cytokine expression by interferon alpha in human bone marrow stroma cells: inhibition of hematopoietic growth factors and induction of IL-1 receptor antagonist. Blood 1994;84:4142-4150.[Abstract/Free Full Text]

  29. Bhatia R, McGlave PB, Verfaillie CM. Interferon-{alpha} treatment of marrow stroma results in enhanced adhesion of chronic myelogenous leukemia progenitors via mechanism involving MIP-1{alpha} and TGF-ß. Exp Hematol 1994;22:797a.

  30. Zoumbos N, Gascon P, Young NS. The induction of lymphocytes in normal and suppressed hematopoiesis. Blut 1984;48:1-9.[Medline]

  31. Emerson SG, Antin JH. Bone marrow progenitor cells induce a regulatory autologous proliferative T lymphocyte response. J Immunol 1989;142:766-772.[Abstract]

  32. Eaves CJ, Cashman JD, Eaves AC et al. Mechanisms that regulate the cell cycle status of very primitive hematopoietic cells in long-term human marrow cultures. II. Analysis of positive and negative regulators produced by stromal cells within the adherent layer. Blood 1991;78:110-117.[Abstract/Free Full Text]

  33. Hanabuchi S, Koyanagi M, Kawasaki A et al. Fas and its ligand is a general mechanism of T-cell-mediated cytotoxicity. Proc Natl Acad Sci USA 1994;91:4930-4934.[Abstract/Free Full Text]

  34. Ausubel FM, Brent R, Kingston RE et al. Current Protocols in Molecular Biology. New York: John Wiley & Sons, 1987:121-132.

  35. Brandt J, Srour EF, van Besien K et al. Cytokine dependent long term culture of highly enriched precursors of hematopoietic progenitor cells from human bone marrow. J Clin Invest 1990;86:932-941.

  36. Verfaillie C, McGlave PB. Leukemia inhibitory factor/human interleukin for DA cells: a growth factor that stimulates the in vitro development of multipotential human hemopoietic progenitors. Blood 1991;77:263-270.[Abstract/Free Full Text]

  37. Verfaillie C, Blakholmer K, McGlave P. Purified primitive human hematopoietic progenitors with long term in vitro repopulating capacity adhere selectively to irradiated bone marrow stroma. J Exp Med 1990;179:509-520.

  38. Berenson RJ, Andrews RG, Bensinger WI et al. Antigen CD34+ marrow cells engraft lethally irradiated baboons. J Clin Invest 1988;81:951-956.

  39. Verfaillie CM, Miller WJ, Boylan K et al. Selection of benign primitive hematopoietic progenitors in chronic myelogenous leukemia on the basis of DR antigen expression. Blood 1992;79:1003-1008.[Abstract/Free Full Text]

  40. Goodman AI, Choudhury M, da Silva J-L et al. Quantitative measurement of heme oxygenase-1 in the human adenocarcinoma. J Cell Biochem 1996;63:342-348.[Medline]

  41. Chomczyski R, Sacchi N. Single-step method of RNA isolation by acid guanidium isothiocyanate-phenol-chloroform extraction. Anal Biochem 1987;162:156-159.[Medline]

  42. Huang S, Endo RI, Nemerow GR. Upregulation of integrins alpha v beta 3 and alpha v beta 5 on human monocytes and T lymphocytes facilitates adenovirus-mediated gene delivery. J Virol 1995;69:2257-2263.[Abstract]

  43. Talpaz M, Kantarjiian H, Kurzrock R et al. Interferon-alpha produced sustained cytogenetic responses in chronic myelogenous leukemia. Ann Intern Med 1991;114:532-538.

  44. Leemhuis T, Liebowitz D, Cox G et al. Identification of BCR/ABL-negative primitive hematopoietic progenitor cells within chronic myeloid leukemia marrow. Blood 1993;81:801-807.[Abstract/Free Full Text]

  45. Martiat P, Lewalle P, Taj AS et al. Retrovirally transduced antisense sequences stably suppress P210BCR-ABL expression and inhibit the proliferation of BCR/ABL-containing cell lines. Blood 1993;81:502-509.[Abstract/Free Full Text]

  46. Conneally E, Bardy P, Eaves CJ et al. Rapid and efficient selection of human hematopoietic cells expressing murine heat state antigen as an indicator of retroviral-mediated gene transfer. Blood 1996;87:456-464.[Abstract/Free Full Text]

accepted for publication on June 9, 1997.




This article has been cited by other articles:


Home page
 Cancer Res.Home page
T. Fukazawa, Y. Maeda, F. M. Sladek, and L. B. Owen-Schaub
Development of a Cancer-Targeted Tissue-Specific Promoter System
Cancer Res., January 1, 2004; 64(1): 363 - 369.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
D. M. Shayakhmetov, T. Papayannopoulou, G. Stamatoyannopoulos, and A. Lieber
Efficient Gene Transfer into Human CD34+ Cells by a Retargeted Adenovirus Vector
J. Virol., March 15, 2000; 74(6): 2567 - 2583.
[Abstract] [Full Text]

Home page
 Clin. Cancer Res.Home page
A. S. Pearson, P. E. Koch, N. Atkinson, M. Xiong, R. W. Finberg, J. A. Roth, and B. Fang
Factors Limiting Adenovirus-mediated Gene Transfer into Human Lung and Pancreatic Cancer Cell Lines
Clin. Cancer Res., December 1, 1999; 5(12): 4208 - 4213.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
L. Yang, S. Quan, and N. G. Abraham
Retrovirus-mediated HO gene transfer into endothelial cells protects against oxidant-induced injury
Am J Physiol Lung Cell Mol Physiol, July 1, 1999; 277(1): L127 - L133.
[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
Right arrow Citation Map
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 Feldman, E.
Right arrow Articles by Abraham, N.G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Feldman, E.
Right arrow Articles by Abraham, N.G.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
STEM CELLS THE ONCOLOGIST CME ALPHAMED PRESS JOURNALS