Background Erythroid nuclear cells (ENC) of the bone marrow (BM) have not previously been considered as important producers of wide spectrum of haemo- and immunoregulatory cytokines. The aim of the current work was to confirm the production of the main hemo- and immunoregulatory cytokines in human ENC from BM.
Background
Erythroid nuclear cells (ENC) of the bone marrow (BM) have not previously been considered as important producers of wide spectrum of haemo- and immunoregulatory cytokines. The aim of the current work was to confirm the production of the main hemo- and immunoregulatory cytokines in human ENC from BM.Results We used native human BM ENC in our experiments. We for the first time have shown, that the unstimulated erythroblasts (Gl A +  or AG-EB + ) produced a wide spectrum of immunoregulatory cytokines. Human BM ENC produce cytokines such as interleukn (IL)-1β, IL-2, IL-4, IL-6, interferon (IFN)-γ, transforming growth factor (TGF)-β1, tumor necrosis factor (TNF)-α and IL-10. They can be sub-divided into glycophorin A positive (Gl A + ) and erythroblast antigen positive (AG-EB + ) cells. To study potential differences in cytokine expression between these subsets, ENC were isolated and purified using specific antibodies to Gl A and AG-EB and the separated cells were cultivated for 24 hours. The cytokine contents of the supernatant were measured by electrochemiluminescence immunoassay. Quantitative differences in TGF-β1 and TNF-α production were found between Gl A +  and AG-EB +  BM ENC. Furthermore, in vitro addition of erythropoietin (EPO) reduced IFN-γ and IL-2 production specifically by the AG-EB +  ENC. Thus, Gl A +  and AG-EB +  ENC produce IL-1β, IL-2, IL-4, IL-6, IFN-γ, TGF-β1 and TNF-α. Gl A +  ENC also produce IL-10.
Results
We used native human BM ENC in our experiments. We for the first time have shown, that the unstimulated erythroblasts (Gl A +  or AG-EB + ) produced a wide spectrum of immunoregulatory cytokines. Human BM ENC produce cytokines such as interleukn (IL)-1β, IL-2, IL-4, IL-6, interferon (IFN)-γ, transforming growth factor (TGF)-β1, tumor necrosis factor (TNF)-α and IL-10. They can be sub-divided into glycophorin A positive (Gl A + ) and erythroblast antigen positive (AG-EB + ) cells. To study potential differences in cytokine expression between these subsets, ENC were isolated and purified using specific antibodies to Gl A and AG-EB and the separated cells were cultivated for 24 hours. The cytokine contents of the supernatant were measured by electrochemiluminescence immunoassay. Quantitative differences in TGF-β1 and TNF-α production were found between Gl A +  and AG-EB +  BM ENC. Furthermore, in vitro addition of erythropoietin (EPO) reduced IFN-γ and IL-2 production specifically by the AG-EB +  ENC. Thus, Gl A +  and AG-EB +  ENC produce IL-1β, IL-2, IL-4, IL-6, IFN-γ, TGF-β1 and TNF-α. Gl A +  ENC also produce IL-10.Conclusion Cytokine production by erythroid nuclear cells suggests that these cells might be involved in regulating the proliferation and differentiation of hematopoietic and immunocompetent cells in human BM.
Conclusion
Cytokine production by erythroid nuclear cells suggests that these cells might be involved in regulating the proliferation and differentiation of hematopoietic and immunocompetent cells in human BM.

Background Haematopoesis is regulated by lymphoid and non-lymphoid cells through a complex network of paracrine and autocrine mechanisms involving cytokines, growth factors and their receptors. However, although stromal [ 1 - 6 ], endothelial [ 7 - 10 ], megakaryocytic [ 11 , 12 ] and osteogenic cells [ 13 - 16 ] and lymphocytes are known to express cytokines, erythroid nuclear cells (ENC), the major cell population of the BM, are not considered as important producers of hemo- and immunoregulatory cytokines. Experiments on ENC isolated from mouse spleen undergoing erythroid hyperplasia, and on cells separated from single erythroid colonies, have revealed that mRNAs for cytokines such as IL-1α, IL-1β, IL-4, IL-6, granulocyte-macrophage colony stimulating factor (GM-CSF), TGF-β1 and IFN-γ are present [ 17 , 18 ]. Production of GM-CSF and IFN-γ has also been reported [ 17 ]. Similarly, IL-2 and IL-3 mRNAs were found after erythroid cells were treated with erythropoietin (EPO) [ 17 - 20 ]. Human fetal liver erythroid cells have also been shown to produce such cytokines as IL-1, IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ and TGF-β1 [ 20 ]. Stopka and co-authors have demonstrated the ability of burst-forming unit erythroid cells (BFU-E) to express and produce EPO [ 21 ]. Also it has been shown that erythroid cells express and produce vascular-endothelial growth factor (VEGF)-A and placental growth factor (PlGF) both in vitro and in vivo. Production of these proteins varies during differentiation and is increased 100-fold by PlGF and 3-fold by VEGF-A [ 22 ]. Macrophage colony stimulating factor (M-CSF), fibroblast growth factor (FGF)-2, VEGF-A, hepatocyte (H)GF, insulin-like (I)GF-1, IL-1β, trombopoietin (TPO), TNF-α, IFN-γ, FAS-L, and macrophage inflammatory protein (MIP)-1α mRNAs are also expressed in BFU-E isolated from BM, and VEGF-A and TGF-β1 are produced [ 23 , 24 ]. All these data allow us to consider erythroid cells as cytokine producers involved in regulating hemo- and immunopoiesis. The aim of the current work was to confirm previous observations by studying the production of the main hemo- and immunoregulatory cytokines, IL-1β, IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ and TGF-β1, in human ENC from BM. Furthermore, we wished to determine whether different subsets of ENC (AG-EB+ and Gl A+ erythroid cells from human BM) might account for altered expression levels. Expression of both these markers is specific to erythroid cells; they are absent from the membranes of other blood-forming cells. AG-EB is expressed in a proportion of BFU-E cells (up to 4% of AG-EB +  BFU-E cells) and on colony-forming units – erythroid (CFU-E) (up to 36% of AG-EB +  CFU-E cells) [ 25 ]. Its expression on erythroblasts is further increased (up to 90% in AG-EB + erythroblasts). Later in development, AG-EB expression decreases, and only 4% of reticulocytes carry AG-EB [ 25 ]. In contrast, Gl A is expressed in a minor subpopulation of the CFU-E cells (less than 4%). On the erythroblasts this expression is increased and reach up to 100% in reticulocytes [ 26 , 27 ]. Sheme of AG-EB and Gl A expression during erythroid maturation present in Table  1 . Table 1 Expression of AG-EB and Gl A during erythroid maturation. The percentage of positive cells detected by flow cytometric analysis [25, 26, 27]. % of positive cells BFU-E CFU-E Proerythroblasts → erythroblasts reticulocytes AG-EB 0 – 4 36 93–95 4 Gl A 0 0 – 4 Increase with maturation 100 In this article we show which human BM ENC produce the cytokines IL-1β, IL-2, IL-4, IL-6, IFN-γ, TGF-β1, TNF-α and IL-10.
Background
Haematopoesis is regulated by lymphoid and non-lymphoid cells through a complex network of paracrine and autocrine mechanisms involving cytokines, growth factors and their receptors. However, although stromal [ 1 - 6 ], endothelial [ 7 - 10 ], megakaryocytic [ 11 , 12 ] and osteogenic cells [ 13 - 16 ] and lymphocytes are known to express cytokines, erythroid nuclear cells (ENC), the major cell population of the BM, are not considered as important producers of hemo- and immunoregulatory cytokines.Experiments on ENC isolated from mouse spleen undergoing erythroid hyperplasia, and on cells separated from single erythroid colonies, have revealed that mRNAs for cytokines such as IL-1α, IL-1β, IL-4, IL-6, granulocyte-macrophage colony stimulating factor (GM-CSF), TGF-β1 and IFN-γ are present [ 17 , 18 ]. Production of GM-CSF and IFN-γ has also been reported [ 17 ]. Similarly, IL-2 and IL-3 mRNAs were found after erythroid cells were treated with erythropoietin (EPO) [ 17 - 20 ]. Human fetal liver erythroid cells have also been shown to produce such cytokines as IL-1, IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ and TGF-β1 [ 20 ]. Stopka and co-authors have demonstrated the ability of burst-forming unit erythroid cells (BFU-E) to express and produce EPO [ 21 ]. Also it has been shown that erythroid cells express and produce vascular-endothelial growth factor (VEGF)-A and placental growth factor (PlGF) both in vitro and in vivo. Production of these proteins varies during differentiation and is increased 100-fold by PlGF and 3-fold by VEGF-A [ 22 ]. Macrophage colony stimulating factor (M-CSF), fibroblast growth factor (FGF)-2, VEGF-A, hepatocyte (H)GF, insulin-like (I)GF-1, IL-1β, trombopoietin (TPO), TNF-α, IFN-γ, FAS-L, and macrophage inflammatory protein (MIP)-1α mRNAs are also expressed in BFU-E isolated from BM, and VEGF-A and TGF-β1 are produced [ 23 , 24 ]. All these data allow us to consider erythroid cells as cytokine producers involved in regulating hemo- and immunopoiesis.The aim of the current work was to confirm previous observations by studying the production of the main hemo- and immunoregulatory cytokines, IL-1β, IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ and TGF-β1, in human ENC from BM. Furthermore, we wished to determine whether different subsets of ENC (AG-EB+ and Gl A+ erythroid cells from human BM) might account for altered expression levels. Expression of both these markers is specific to erythroid cells; they are absent from the membranes of other blood-forming cells. AG-EB is expressed in a proportion of BFU-E cells (up to 4% of AG-EB +  BFU-E cells) and on colony-forming units – erythroid (CFU-E) (up to 36% of AG-EB +  CFU-E cells) [ 25 ]. Its expression on erythroblasts is further increased (up to 90% in AG-EB + erythroblasts). Later in development, AG-EB expression decreases, and only 4% of reticulocytes carry AG-EB [ 25 ]. In contrast, Gl A is expressed in a minor subpopulation of the CFU-E cells (less than 4%). On the erythroblasts this expression is increased and reach up to 100% in reticulocytes [ 26 , 27 ]. Sheme of AG-EB and Gl A expression during erythroid maturation present in Table  1 .Expression of AG-EB and Gl A during erythroid maturation. The percentage of positive cells detected by flow cytometric analysis [25, 26, 27].In this article we show which human BM ENC produce the cytokines IL-1β, IL-2, IL-4, IL-6, IFN-γ, TGF-β1, TNF-α and IL-10.Results Erythroid cells were separated according to the presence of the surface markers AG-EB and Gl A. BM erythroid cells carrying AG-EB produce the cytokines IL-1β, IL-2, IL-4, TNF-α, IFN-γ and TGF-β1 (figure  1 ). Those carrying Gl A also produce these cytokines and in addition might be secrete IL-10 (figure  1 ). Interestingly, erythroid cells carrying AG-EB release more TGF-β1 (p < 0.05) than Gl A +  cells, whereas Gl A +  secrete more TNF-α (p < 0.05). Figure 1 Cytokine production by BM erythroid nuclear cells carrying Erythroblast Antigen and Glycophorin A.  □ – BM AG-EB +  erythroid nuclear cells; ■ – BM Gl A +  erythroid nuclear cells. All populations were cultivated for 24 h at concentrations of 10 6  cells per ml. Results present as (Mean ± SEM). n/d – not detected. * – P < 0.05. To the best of our knowledge, this difference between BM AG-EB +  and Gl A +  ENC has not previously been reported. To verify that the cytokines were truly expressed by these erythroid cells, we isolated erythoid colonies by cultivating single BM cells in MethoCult H4433 medium. As shown in Table  2 , the cytokines produced were comparable between ENC derived from erythroid colonies and ENC enriched from the BM. Table 2 Concentration of cytokines in conditioned medium derived from erythroid cells. Concentration of cytokines (pg/ml) in conditioned medium derived from enriched population of BM erythroid cells (1 × 10 6 /ml) and erythroid cells isolated from erythroid colonies (1 × 10 6 /ml). Results were obtained from at least three independent experiments (Mean ± SEM). Enriched erythroid nuclear cell population Erythroid nuclear cells derived from erythroid colonies IL-1β 78.64 ± 44.84 10.8 ± 0.39 IL-2 255.36 ± 88 237.73 ± 237.73 IL-4 62.66 ± 45.28 738.26 ± 640.15 IL-6 741.08 ± 395.95 324 ± 56.14 IL-10 121.26 ± 53.22 73.4 ± 73.4 TNF-α 2.59 ± 1.41 0.0 IFN-γ 766.35 ± 319.45 752.88 ± 492.65 TGF-β1 524.88 ± 372.59 479.25 ± 267.75 EPO regulates the proliferation and differentiation of erythroid cells [ 28 - 30 ]. Therefore, we assessed the effect of EPO on our different ENC cell populations and found that the production of some hemo- and immunoregulatory molecules was altered (figure  2 ). For instance, EPO treatment of AG-EB +  cells led to a 99% decrease in IFN-γ production and a 50% decrease in IL-2 production, and the production of IL-1β and TNF-α was enhanced. However, EPO treatment had no discernible effect on the cytokine profile of Gl A +  cells. Figure 2 Influence of EPO on cytokine production by BM AG-EB + erythroid nuclear cells.  □ – BM AG-EB +  erythroid nuclear cells; ■ – BM AG-EB +  erythroid nuclear cells after treatment with EPO. BM AG-EB +  erythroid nuclear cells (10 6  cells/ml) were treated with EPO (2 U/ml) for 24 h. Results present as (Mean ± SEM). n/d – not detected. * – P < 0.05.
Results
Erythroid cells were separated according to the presence of the surface markers AG-EB and Gl A. BM erythroid cells carrying AG-EB produce the cytokines IL-1β, IL-2, IL-4, TNF-α, IFN-γ and TGF-β1 (figure  1 ). Those carrying Gl A also produce these cytokines and in addition might be secrete IL-10 (figure  1 ). Interestingly, erythroid cells carrying AG-EB release more TGF-β1 (p < 0.05) than Gl A +  cells, whereas Gl A +  secrete more TNF-α (p < 0.05).To the best of our knowledge, this difference between BM AG-EB +  and Gl A +  ENC has not previously been reported. To verify that the cytokines were truly expressed by these erythroid cells, we isolated erythoid colonies by cultivating single BM cells in MethoCult H4433 medium. As shown in Table  2 , the cytokines produced were comparable between ENC derived from erythroid colonies and ENC enriched from the BM.Concentration of cytokines in conditioned medium derived from erythroid cells. Concentration of cytokines (pg/ml) in conditioned medium derived from enriched population of BM erythroid cells (1 × 10 6 /ml) and erythroid cells isolated from erythroid colonies (1 × 10 6 /ml). Results were obtained from at least three independent experiments (Mean ± SEM).EPO regulates the proliferation and differentiation of erythroid cells [ 28 - 30 ]. Therefore, we assessed the effect of EPO on our different ENC cell populations and found that the production of some hemo- and immunoregulatory molecules was altered (figure  2 ). For instance, EPO treatment of AG-EB +  cells led to a 99% decrease in IFN-γ production and a 50% decrease in IL-2 production, and the production of IL-1β and TNF-α was enhanced. However, EPO treatment had no discernible effect on the cytokine profile of Gl A +  cells.Discussion We have shown that the native BM ENC can produce a wide spectrum of cytokines, which are capable of both stimulating and inhibiting erythropoiesis. Cytokine production has been confirmed using ENC isolated from erythroid colonies cloned from a single BM cell. Earlier we demonstrated cytokine production by ENC derived from fetal tissues [ 19 , 20 ]. However, there was a great difference between the profiles of cytokines released by BM and fetal ENC. TGF-β1 is known to inhibit the proliferation of erythroid precursors and to provoke or accelerate the terminal differentiation of mature erythroid cells [ 31 - 33 ]. The fetal AG-EB +  and Gl A +  erythroid cells produced TGF-β1 in concentrations less than 5 pg/ml and we suggested that this could be related to the increased proliferative activity of erythroid cells in the fetal liver [ 20 ]. We propose that the high level of TGF-β1 production by BM erythroid cells is probably related to the need to restrain erythroid cell proliferation in healthy adult individuals. This hypothesis were declared also in review [ 24 ]. It is notable that BM erythroid cells produce not only inhibitors but also high levels of stimulators of erythropoiesis such as IL-4 and IL-6, especially in the presence of EPO [ 34 , 35 ]. The production of these hemoregulatory molecules suggests that erythroid cells have a capacity for self-regulation. Interestingly, IL-2, which inhibits erythropoiesis [ 36 ], is produced by these cells in smaller quantities or, like IL-10, is not produced at all. Besides the differences in production of some cytokines by BM erythroid cells, there are parallels with cytokine production from fetal erythroid cells. A similar situation can be observed when TNF-α production by BM erythroid cells is compared with those from fetal liver [ 20 ]. In both cases AG-EB +  cells produce significantly less TNF-α than Gl A +  (figure  1 ). TNF-α is a known inhibitor of precursor cell proliferation and an inducer of cell death in a proportion of these cells [ 37 , 38 ]. Therefore it is plausible that erythroid cells use this cytokine for autocrine self-regulation, by maintaining a negative feedback loop. If this is so, then relatively small increases in the number of more differentiated Gl A +  cells would lead to a decreased precursor proliferation rate, due to the elevated level of TNF-α. Negative feedback can also be maintained through IFN-γ, which is produced at the same level by both Gl A +  and AG-EB +  cells. However, IFN-γ initiates apoptosis only in BFU-E [ 39 , 40 ], while in CFU-E cells it increases the expression of Bcl-x and protects these cells from elimination. Thus, IFN-γ enhances the differentiation of erythroid precursors [ 41 , 42 ]. We found no statistical significant differences between AG-EB +  and Gl A +  cells in the production of IL-1β, IL-2, IL-4, IL-6 and IL-10. The capacity of erythroid cells to change their cytokine expression profiles quantitatively and qualitatively under different conditions is an important indicator of their active role in the regulation of hemo- and immunopoiesis. Similar data were previously obtained in mouse cells and are presented here for human BM erythroid cells. Treatment with EPO leads to significant changes in IL-2 and IFN-γ production. Notably, IFN-γ and IL-2 secretion by AG-EB +  cells is lowered in the presence of EPO, while Gl A +  cells show no such change. This difference could be related to EPO-R expression in AG-EB +  cells, which are mainly CFU-E and erythroblasts [ 43 - 45 ]. Expression of this receptor ceases in more differentiated early erythroblasts. Indeed, Gl A +  cells do not express EPO-R, since late erythroblasts and more differentiated forms of erythroid cells are prevalent in this population [ 28 , 43 , 46 ]. The EPO-induced changes in cytokine production mainly concern inhibitors of erythroid precursor proliferation, such as IL-2 and IFN-γ. Thus, one mechanism by which erythroid cell proliferation is stimulated could be a decrease in proliferation inhibitor production by these cells as a result of EPO treatment.
Discussion
We have shown that the native BM ENC can produce a wide spectrum of cytokines, which are capable of both stimulating and inhibiting erythropoiesis. Cytokine production has been confirmed using ENC isolated from erythroid colonies cloned from a single BM cell.Earlier we demonstrated cytokine production by ENC derived from fetal tissues [ 19 , 20 ]. However, there was a great difference between the profiles of cytokines released by BM and fetal ENC.TGF-β1 is known to inhibit the proliferation of erythroid precursors and to provoke or accelerate the terminal differentiation of mature erythroid cells [ 31 - 33 ]. The fetal AG-EB +  and Gl A +  erythroid cells produced TGF-β1 in concentrations less than 5 pg/ml and we suggested that this could be related to the increased proliferative activity of erythroid cells in the fetal liver [ 20 ]. We propose that the high level of TGF-β1 production by BM erythroid cells is probably related to the need to restrain erythroid cell proliferation in healthy adult individuals. This hypothesis were declared also in review [ 24 ].It is notable that BM erythroid cells produce not only inhibitors but also high levels of stimulators of erythropoiesis such as IL-4 and IL-6, especially in the presence of EPO [ 34 , 35 ]. The production of these hemoregulatory molecules suggests that erythroid cells have a capacity for self-regulation. Interestingly, IL-2, which inhibits erythropoiesis [ 36 ], is produced by these cells in smaller quantities or, like IL-10, is not produced at all.Besides the differences in production of some cytokines by BM erythroid cells, there are parallels with cytokine production from fetal erythroid cells. A similar situation can be observed when TNF-α production by BM erythroid cells is compared with those from fetal liver [ 20 ]. In both cases AG-EB +  cells produce significantly less TNF-α than Gl A +  (figure  1 ). TNF-α is a known inhibitor of precursor cell proliferation and an inducer of cell death in a proportion of these cells [ 37 , 38 ]. Therefore it is plausible that erythroid cells use this cytokine for autocrine self-regulation, by maintaining a negative feedback loop. If this is so, then relatively small increases in the number of more differentiated Gl A +  cells would lead to a decreased precursor proliferation rate, due to the elevated level of TNF-α. Negative feedback can also be maintained through IFN-γ, which is produced at the same level by both Gl A +  and AG-EB +  cells. However, IFN-γ initiates apoptosis only in BFU-E [ 39 , 40 ], while in CFU-E cells it increases the expression of Bcl-x and protects these cells from elimination. Thus, IFN-γ enhances the differentiation of erythroid precursors [ 41 , 42 ]. We found no statistical significant differences between AG-EB +  and Gl A +  cells in the production of IL-1β, IL-2, IL-4, IL-6 and IL-10.The capacity of erythroid cells to change their cytokine expression profiles quantitatively and qualitatively under different conditions is an important indicator of their active role in the regulation of hemo- and immunopoiesis. Similar data were previously obtained in mouse cells and are presented here for human BM erythroid cells. Treatment with EPO leads to significant changes in IL-2 and IFN-γ production. Notably, IFN-γ and IL-2 secretion by AG-EB +  cells is lowered in the presence of EPO, while Gl A +  cells show no such change. This difference could be related to EPO-R expression in AG-EB +  cells, which are mainly CFU-E and erythroblasts [ 43 - 45 ]. Expression of this receptor ceases in more differentiated early erythroblasts. Indeed, Gl A +  cells do not express EPO-R, since late erythroblasts and more differentiated forms of erythroid cells are prevalent in this population [ 28 , 43 , 46 ]. The EPO-induced changes in cytokine production mainly concern inhibitors of erythroid precursor proliferation, such as IL-2 and IFN-γ. Thus, one mechanism by which erythroid cell proliferation is stimulated could be a decrease in proliferation inhibitor production by these cells as a result of EPO treatment.Conclusion Erythroid nuclear cells appear to be active producers of hemo- and immunoregulatory cytokines, involved in regulating the proliferation and differentiation of hematopoietic and immunocompetent cells in human BM. Changes in the cytokines produced by erythroid cells in response to EPO suggests the ability of these cells to respond to microenvironmental changes by altering the cytokine production profile.
Conclusion
Erythroid nuclear cells appear to be active producers of hemo- and immunoregulatory cytokines, involved in regulating the proliferation and differentiation of hematopoietic and immunocompetent cells in human BM. Changes in the cytokines produced by erythroid cells in response to EPO suggests the ability of these cells to respond to microenvironmental changes by altering the cytokine production profile.Methods BM cells This work was approved by the local ethics committee. After obtaining informed consent, sternum BM samples were collected from 8 normal healthy volunteers. All samples used in this study exhibited normal myelograms. The enriched population of erythroid cells was isolated from BM mononuclear cells by depleting of adherent cells and granulocytes as described previously [ 19 ]. Isolation of erythroid cell population carrying a surface erythroid antigen and Glycophorin A We used indirect panning to obtain cells expressing either the erythroid antigen (AG-EB) or Glycophorin A (Gl A) [ 19 ]. Monoclonal mouse anti-red blood cell Glycophorin A (MAS518, Harlan-Sera Lab, England), and monoclonal HAE-9 antibodies against AG-EB (kindly provided by Prof. Mechetner, Russia) were used [ 25 ]. Affinity isolated polyclonal rabbit anti-mouse immunoglobulin (Biosan, Russia) was used as a secondary reagent to coat Petri dishes during the panning procedure. To avoid non-specific binding to FcRs, cells were blocked with aggregated normal human IgG and then incubated with anti-Gl A or HAE-9 antibodies for 50 min at 4°C. Cells were transferred to Petri dishes covered with rabbit anti-mouse antibodies for panning for 50 min at 4°C. Subsequently, immobilised cells were collected, cultivated for 24 h in RPMI-1640 with 10% horse serum (Sigma, USA) with and without EPO [ 19 , 47 ]. Samples were collected after cultivation. Cultivation of erythroid cells with EPO 1 × 10 6  cells were placed in culture medium containing 2 U/ml Epo (Boehringer Mannheim, Germany) for a 24-h incubation. CFU-E Cells from the enriched erythroid population were maintained as described above [ 19 , 20 ] at 2·10 5 /ml and then cultivated in MethoCult H4433 medium (Stemcell Tech. Inc. Vancouver, B.C.) for 14 days. Colonies were visually characterised as CFU-E using an inverted microscope. After cultivation, the colonies were harvested and washed twice to remove methyl cellulose. The resulting cellular suspension (1 × 10 6 /ml) was cultivated for 24 h in RPMI-1640 with 10% horse serum, then supernatants were collected. Samples were stored at -20°C. Cells purity Cell purity was assessed using Nocht-Maksimov staining of smears [ 48 ] and staining of haemoglobin with benzidin [ 49 ]. All cells (Gl A + , AG-EB +  and CFU-E cells) were characterised as erythroblasts and were haemoglobin positive. Electrochemiluminescence (ECL) method for quantitative determination of cytokines Quantitative determination of cytokines was performed by the electrochemiluminescent immunoassay [ 50 - 52 ] using an ORIGEN Analyzer (IGEN Inc., USA) according to the manufacturer's protocol. Calibration curves ranged from 10 to 10,000 pg/ml. Assay sensitivity was 2.8 pg/ml, 2 pg/ml, 2 pg/ml and 6 pg/ml for IL-1β, IL-2, IL-4 and IL-6, respectively. Assay sensitivities were 2 pg/ml, 1 pg/ml, 1 pg/ml and 2 pg/ml for IL-10, TNF-α, IFN-γ, and TGF-β1, respectively. The ruthenylated and biotinylated antibodies were diluted to working concentrations (μg/ml) 1:1, 1:1, 1:2 and 2:1 for IL-1β, IL-2, IL-4 and IL-6, respectively. The biotinylated and ruthenylated antibodies were diluted to working concentrations (μg/ml) 2:2, 1:1, 2:1 and 4:2 for IL-10, TNF-α, IFN-γ and TGF-β1, respectively. For TGF-β1 detection, samples were treated with HCl to obtain intact and easily detectable proteins as described [ 53 ]. Antibodies All polyclonal and monoclonal antibodies were purchased from R&D Systems (Abington, UK). The following antibodies were used: against recombinant IL-1β, IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ, TGF-β1 polyclonal and monoclonal: anti-hIL-1β #AB-201-NA, anti-hIL-1β #MAB201, anti-hIL-2 #AB-202-NA, anti-hIL-2 #MAB202, anti-hIL-4 #AB-204-NA, anti-hIL-4 #MAB204, anti-hIL-6 #AB-206-NA, anti-hIL-6 #MAB206, anti-hIL-10 #AB-217-NA, anti-hIL-10 #MAB217, anti-hTNF-α #AB-210-NA, anti-hTNF-α #MAB210, anti-hIFN-γ #AB-285-NA, anti-hIFN-γ #MAB285, anti-hTGF-β1 #AF-101-NA, anti-hTGF-β1 #MAB240. No antibody used displayed cross-reactivity with other cytokines. Cytokines Recombinant cytokines were purchased from R&D Systems (Abington, UK): rhIL-1β #201-IL, rhIL-2 #202-IL, rhIL-4 #204-IL, rhIL-6 #206-IL, rhTNF-α #210-TA, rhIFN-γ #285-IF, rhTGF-β1 #240-B-002; and PeproTech, Inc. (Rocky Hill, NJ): rhIL-10 #200-10 was used for calibration curves. All the recombinant cytokines were diluted to 2 μg/ml in PBS, pH 7.4, supplemented with 0.05% NaN 3  and 0.1% BSA (aliquots were kept at -70°C before use). Statistical analysis Results are presented as means ± SEM. Statistical significance was determined using the nonparametric Mann-Whitney U-test. P < 0.05 was considered to be significant.
Methods
BM cells This work was approved by the local ethics committee. After obtaining informed consent, sternum BM samples were collected from 8 normal healthy volunteers. All samples used in this study exhibited normal myelograms. The enriched population of erythroid cells was isolated from BM mononuclear cells by depleting of adherent cells and granulocytes as described previously [ 19 ].
BM cells
This work was approved by the local ethics committee. After obtaining informed consent, sternum BM samples were collected from 8 normal healthy volunteers. All samples used in this study exhibited normal myelograms. The enriched population of erythroid cells was isolated from BM mononuclear cells by depleting of adherent cells and granulocytes as described previously [ 19 ].Isolation of erythroid cell population carrying a surface erythroid antigen and Glycophorin A We used indirect panning to obtain cells expressing either the erythroid antigen (AG-EB) or Glycophorin A (Gl A) [ 19 ]. Monoclonal mouse anti-red blood cell Glycophorin A (MAS518, Harlan-Sera Lab, England), and monoclonal HAE-9 antibodies against AG-EB (kindly provided by Prof. Mechetner, Russia) were used [ 25 ]. Affinity isolated polyclonal rabbit anti-mouse immunoglobulin (Biosan, Russia) was used as a secondary reagent to coat Petri dishes during the panning procedure. To avoid non-specific binding to FcRs, cells were blocked with aggregated normal human IgG and then incubated with anti-Gl A or HAE-9 antibodies for 50 min at 4°C. Cells were transferred to Petri dishes covered with rabbit anti-mouse antibodies for panning for 50 min at 4°C. Subsequently, immobilised cells were collected, cultivated for 24 h in RPMI-1640 with 10% horse serum (Sigma, USA) with and without EPO [ 19 , 47 ]. Samples were collected after cultivation.
Isolation of erythroid cell population carrying a surface erythroid antigen and Glycophorin A
We used indirect panning to obtain cells expressing either the erythroid antigen (AG-EB) or Glycophorin A (Gl A) [ 19 ]. Monoclonal mouse anti-red blood cell Glycophorin A (MAS518, Harlan-Sera Lab, England), and monoclonal HAE-9 antibodies against AG-EB (kindly provided by Prof. Mechetner, Russia) were used [ 25 ]. Affinity isolated polyclonal rabbit anti-mouse immunoglobulin (Biosan, Russia) was used as a secondary reagent to coat Petri dishes during the panning procedure. To avoid non-specific binding to FcRs, cells were blocked with aggregated normal human IgG and then incubated with anti-Gl A or HAE-9 antibodies for 50 min at 4°C. Cells were transferred to Petri dishes covered with rabbit anti-mouse antibodies for panning for 50 min at 4°C. Subsequently, immobilised cells were collected, cultivated for 24 h in RPMI-1640 with 10% horse serum (Sigma, USA) with and without EPO [ 19 , 47 ]. Samples were collected after cultivation.Cultivation of erythroid cells with EPO 1 × 10 6  cells were placed in culture medium containing 2 U/ml Epo (Boehringer Mannheim, Germany) for a 24-h incubation.
Cultivation of erythroid cells with EPO
1 × 10 6  cells were placed in culture medium containing 2 U/ml Epo (Boehringer Mannheim, Germany) for a 24-h incubation.CFU-E Cells from the enriched erythroid population were maintained as described above [ 19 , 20 ] at 2·10 5 /ml and then cultivated in MethoCult H4433 medium (Stemcell Tech. Inc. Vancouver, B.C.) for 14 days. Colonies were visually characterised as CFU-E using an inverted microscope. After cultivation, the colonies were harvested and washed twice to remove methyl cellulose. The resulting cellular suspension (1 × 10 6 /ml) was cultivated for 24 h in RPMI-1640 with 10% horse serum, then supernatants were collected. Samples were stored at -20°C.
CFU-E
Cells from the enriched erythroid population were maintained as described above [ 19 , 20 ] at 2·10 5 /ml and then cultivated in MethoCult H4433 medium (Stemcell Tech. Inc. Vancouver, B.C.) for 14 days. Colonies were visually characterised as CFU-E using an inverted microscope. After cultivation, the colonies were harvested and washed twice to remove methyl cellulose. The resulting cellular suspension (1 × 10 6 /ml) was cultivated for 24 h in RPMI-1640 with 10% horse serum, then supernatants were collected. Samples were stored at -20°C.Cells purity Cell purity was assessed using Nocht-Maksimov staining of smears [ 48 ] and staining of haemoglobin with benzidin [ 49 ]. All cells (Gl A + , AG-EB +  and CFU-E cells) were characterised as erythroblasts and were haemoglobin positive.
Cells purity
Cell purity was assessed using Nocht-Maksimov staining of smears [ 48 ] and staining of haemoglobin with benzidin [ 49 ]. All cells (Gl A + , AG-EB +  and CFU-E cells) were characterised as erythroblasts and were haemoglobin positive.Electrochemiluminescence (ECL) method for quantitative determination of cytokines Quantitative determination of cytokines was performed by the electrochemiluminescent immunoassay [ 50 - 52 ] using an ORIGEN Analyzer (IGEN Inc., USA) according to the manufacturer's protocol. Calibration curves ranged from 10 to 10,000 pg/ml. Assay sensitivity was 2.8 pg/ml, 2 pg/ml, 2 pg/ml and 6 pg/ml for IL-1β, IL-2, IL-4 and IL-6, respectively. Assay sensitivities were 2 pg/ml, 1 pg/ml, 1 pg/ml and 2 pg/ml for IL-10, TNF-α, IFN-γ, and TGF-β1, respectively. The ruthenylated and biotinylated antibodies were diluted to working concentrations (μg/ml) 1:1, 1:1, 1:2 and 2:1 for IL-1β, IL-2, IL-4 and IL-6, respectively. The biotinylated and ruthenylated antibodies were diluted to working concentrations (μg/ml) 2:2, 1:1, 2:1 and 4:2 for IL-10, TNF-α, IFN-γ and TGF-β1, respectively. For TGF-β1 detection, samples were treated with HCl to obtain intact and easily detectable proteins as described [ 53 ].
Electrochemiluminescence (ECL) method for quantitative determination of cytokines
Quantitative determination of cytokines was performed by the electrochemiluminescent immunoassay [ 50 - 52 ] using an ORIGEN Analyzer (IGEN Inc., USA) according to the manufacturer's protocol. Calibration curves ranged from 10 to 10,000 pg/ml. Assay sensitivity was 2.8 pg/ml, 2 pg/ml, 2 pg/ml and 6 pg/ml for IL-1β, IL-2, IL-4 and IL-6, respectively. Assay sensitivities were 2 pg/ml, 1 pg/ml, 1 pg/ml and 2 pg/ml for IL-10, TNF-α, IFN-γ, and TGF-β1, respectively. The ruthenylated and biotinylated antibodies were diluted to working concentrations (μg/ml) 1:1, 1:1, 1:2 and 2:1 for IL-1β, IL-2, IL-4 and IL-6, respectively. The biotinylated and ruthenylated antibodies were diluted to working concentrations (μg/ml) 2:2, 1:1, 2:1 and 4:2 for IL-10, TNF-α, IFN-γ and TGF-β1, respectively.For TGF-β1 detection, samples were treated with HCl to obtain intact and easily detectable proteins as described [ 53 ].Antibodies All polyclonal and monoclonal antibodies were purchased from R&D Systems (Abington, UK). The following antibodies were used: against recombinant IL-1β, IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ, TGF-β1 polyclonal and monoclonal: anti-hIL-1β #AB-201-NA, anti-hIL-1β #MAB201, anti-hIL-2 #AB-202-NA, anti-hIL-2 #MAB202, anti-hIL-4 #AB-204-NA, anti-hIL-4 #MAB204, anti-hIL-6 #AB-206-NA, anti-hIL-6 #MAB206, anti-hIL-10 #AB-217-NA, anti-hIL-10 #MAB217, anti-hTNF-α #AB-210-NA, anti-hTNF-α #MAB210, anti-hIFN-γ #AB-285-NA, anti-hIFN-γ #MAB285, anti-hTGF-β1 #AF-101-NA, anti-hTGF-β1 #MAB240. No antibody used displayed cross-reactivity with other cytokines.
Antibodies
All polyclonal and monoclonal antibodies were purchased from R&D Systems (Abington, UK). The following antibodies were used: against recombinant IL-1β, IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ, TGF-β1 polyclonal and monoclonal: anti-hIL-1β #AB-201-NA, anti-hIL-1β #MAB201, anti-hIL-2 #AB-202-NA, anti-hIL-2 #MAB202, anti-hIL-4 #AB-204-NA, anti-hIL-4 #MAB204, anti-hIL-6 #AB-206-NA, anti-hIL-6 #MAB206, anti-hIL-10 #AB-217-NA, anti-hIL-10 #MAB217, anti-hTNF-α #AB-210-NA, anti-hTNF-α #MAB210, anti-hIFN-γ #AB-285-NA, anti-hIFN-γ #MAB285, anti-hTGF-β1 #AF-101-NA, anti-hTGF-β1 #MAB240. No antibody used displayed cross-reactivity with other cytokines.Cytokines Recombinant cytokines were purchased from R&D Systems (Abington, UK): rhIL-1β #201-IL, rhIL-2 #202-IL, rhIL-4 #204-IL, rhIL-6 #206-IL, rhTNF-α #210-TA, rhIFN-γ #285-IF, rhTGF-β1 #240-B-002; and PeproTech, Inc. (Rocky Hill, NJ): rhIL-10 #200-10 was used for calibration curves. All the recombinant cytokines were diluted to 2 μg/ml in PBS, pH 7.4, supplemented with 0.05% NaN 3  and 0.1% BSA (aliquots were kept at -70°C before use).
Cytokines
Recombinant cytokines were purchased from R&D Systems (Abington, UK): rhIL-1β #201-IL, rhIL-2 #202-IL, rhIL-4 #204-IL, rhIL-6 #206-IL, rhTNF-α #210-TA, rhIFN-γ #285-IF, rhTGF-β1 #240-B-002; and PeproTech, Inc. (Rocky Hill, NJ): rhIL-10 #200-10 was used for calibration curves. All the recombinant cytokines were diluted to 2 μg/ml in PBS, pH 7.4, supplemented with 0.05% NaN 3  and 0.1% BSA (aliquots were kept at -70°C before use).Statistical analysis Results are presented as means ± SEM. Statistical significance was determined using the nonparametric Mann-Whitney U-test. P < 0.05 was considered to be significant.
Statistical analysis
Results are presented as means ± SEM. Statistical significance was determined using the nonparametric Mann-Whitney U-test. P < 0.05 was considered to be significant.Authors' contributions IBK, NJD, ANZ and MIL carried out the selection of donors, sternum BM sampling and analysis of myelograms. TVI, ANS and SVK carried out all of the experiments and drafted the manuscript. SVS participated in design and coordination of the research and revision of the manuscript. ANS participated in the preparation and revision of the manuscript. VAK conceived of the study. All authors read and approved the final manuscript.
Authors' contributions
IBK, NJD, ANZ and MIL carried out the selection of donors, sternum BM sampling and analysis of myelograms. TVI, ANS and SVK carried out all of the experiments and drafted the manuscript. SVS participated in design and coordination of the research and revision of the manuscript. ANS participated in the preparation and revision of the manuscript. VAK conceived of the study. All authors read and approved the final manuscript.


In this study we have made novel observations with regards to potentiation of the tumoricidal activity of the oncolytic adenovirus, dl1520 (ONYX-015) in rat glioblastoma cell lines expressing heat shock protein 72 (HSP72) due to permissive virus replication. ONYX-015 is a conditionally replicating adenovirus that is deleted for the E1B 55 kDA gene product whose normal function is to interact with cell-cycle regulatory proteins to permit virus replication. However, many murine and rodent cell lines are not permissive for adenovirus replication. Previously, it has been reported that the heat shock response is necessary for adenovirus replication and that induction of heat shock proteins is mediated by E1 region gene products. Therefore, we hypothesized that HSP72 expression may allow for permissive replication of ONYX-015 in previously non-permissive cells. Rat glioma cell lines 9L and RT2 were transfected with a plasmids expressing HSP72 or GFP. After infection with ONYX-015, no tumoricidal activity is observed in GFP expressing cell lines despite adequate transduction. In contrast, HSP72 transfected cells show cytopathic effects by 72 hours and greater than 75% loss of viability by 96 hours. Burst assays show active virus replication in the HSP72 expressing cell lines. Therefore, 9L-HSP72 and RT2-HSP72 are ideal models to evaluate the efficacy of ONYX-015 in an immunocompetent rat model. Our study has implications for creating rodent tumor models for pre-clinical studies with E1 region deleted conditionally replicating adenovirus.

Background Adenovirus vectors are commonly utilized in cancer gene therapy experiments. They readily infect numerous tumor cell types and are easily manipulated allowing for transgene expression[ 1 ]. In an effort to improve selectivity of these vectors for malignant cells, replication selective or conditionally replicating adenoviruses were created[ 2 ]. With greater understanding in the molecular aspects of adenovirus replication, these viruses were designed such that replication was predicated on alterations in cell cycle regulation, thus rendering only malignant cells susceptible. These adenovirus systems rely on virus replication as a means of exerting tumoricidal effect. One of the first of these conditionally replicating adenoviruses was dl1520 or ONYX-015[ 3 ]. It has been used extensively in cancer gene therapy clinical trials[ 4 , 5 ]. This virus is deleted for the E1B-55 kDA gene[ 6 ]. The E1 region genes of the common serotypes of adenovirus optimize their own replication in target cells and interact with many cell cycle associated gene products. The E1a region genes promote transition of cells into S phase through their interaction with the retinoblastoma protein (pRB)[ 7 ]. pRB in conjunction with p53 can inhibit transition of cells into the S-phase of the cell cycle. Binding of E1a to pRB prevents association of pRB to the E2F transcription factor resulting in activation of E2F[ 8 ]. E2F then functions to transition cells into the S-phase of the cell cycle which is conducive for optimal adenovirus replication. In wild type adenoviruses, the role of the E1B-55 kDa gene product is to neutralize p53[ 9 , 10 ]. p53 induction is thought to promote cell cycle arrest resulting in termination of the virus replicative lifecycle in that cell[ 11 ]. Thus it was hypothesized that deletion of the E1B-55 kDa gene product would result in p53 induced termination of virus replication in normal cells while being replication permissive in malignant cells with abnormal p53 expression or regulation[ 9 ]. Early reports with ONYX-015 indicated replication selectivity for tumor cells with abnormal p53 functioning[ 12 ]. However, the exact mechanism of replication selectivity has been brought to question with subsequent reports of ONYX-015 replication independent of p53[ 13 ] and researchers have looked at other factors associated with adenovirus replication. Various heat shock proteins have been shown to be necessary for efficient adenovirus replication. The avian adenovirus CELO requires the induction of HSP70 and HSP40 for production of viral proteins[ 14 ]. CELO mutants lacking the E1 region genes were replication incompetent in A549 cells. However, heat shock protein induction or expression allowed for permissive replication of these mutants. Similarly in a number of human cell lines, both heat shock and HSP72 expression enhanced the oncolytic effect of E1 containing adenovirus but not for E1 deleted adenovirus[ 15 ]. Adenovirus E1 region genes from serotype 5 and 12 have previously been shown to induce HSP72 expression [ 16 ]. HSP72 is a molecular chaperone protein involved in the repairing denatured proteins. Through this role, it can inhibit apoptosis downstream of caspase activation but prior to loss of mitochondrial membrane potential[ 17 ]. Stably transfected or E1 transformed cell lines show constitutive expression of the inducible HSP72 [ 18 ]. HSP72 levels are correlated with cell-cycle with increases in HSP72 during S-phase with a maximum level in the post-S-phase period [ 19 ]. In 293 cells which are transformed with the adenovirus E1 region gene, E1A mRNA accumulated just prior to HSP72 expression suggesting that E1A is responsible for the cell cycle regulation of HSP72 expression [ 18 ]. Additionally, HSP72 co-localizes with E1A gene products in infected cells [ 20 ]. HSP72 is predominately cytoplasmic but translocates to the nucleus following adenovirus infection. Double labelling experiments with E1A and HSP72 in infected cells showed co-localization of these molecules within the nucleoli and exhibited similar reticular and punctuate nuclear staining patterns depending on cell cycle. HSP72 transport to the nucleus was E1A and virus infection dependent. In 293 (E1 region transformed) cells, HSP72 and E1A co-localization was not observed until after infection with virus [ 20 ]. These data suggest a physical complex is necessary between adenovirus E1A, HSP72 and other adenovirus gene products One of the difficulties with studying E1 adenovirus deletion mutants is the lack of appropriate animal tumor models which allow for permissive growth of these viruses. Common animal tumor models for glioblastoma are the cell lines 9L and RT2. Previously, it has been shown that replication competent E1 deletion mutants can transduce 9L cells and result in transgene expression but there is a block against virus replication[ 21 ]. In this study we addressed if HSP72 expression could allow for permissive replication of the ONYX-015 adenovirus in these rat glioblastoma cells.
Background
Adenovirus vectors are commonly utilized in cancer gene therapy experiments. They readily infect numerous tumor cell types and are easily manipulated allowing for transgene expression[ 1 ]. In an effort to improve selectivity of these vectors for malignant cells, replication selective or conditionally replicating adenoviruses were created[ 2 ]. With greater understanding in the molecular aspects of adenovirus replication, these viruses were designed such that replication was predicated on alterations in cell cycle regulation, thus rendering only malignant cells susceptible. These adenovirus systems rely on virus replication as a means of exerting tumoricidal effect. One of the first of these conditionally replicating adenoviruses was dl1520 or ONYX-015[ 3 ]. It has been used extensively in cancer gene therapy clinical trials[ 4 , 5 ]. This virus is deleted for the E1B-55 kDA gene[ 6 ].The E1 region genes of the common serotypes of adenovirus optimize their own replication in target cells and interact with many cell cycle associated gene products. The E1a region genes promote transition of cells into S phase through their interaction with the retinoblastoma protein (pRB)[ 7 ]. pRB in conjunction with p53 can inhibit transition of cells into the S-phase of the cell cycle. Binding of E1a to pRB prevents association of pRB to the E2F transcription factor resulting in activation of E2F[ 8 ]. E2F then functions to transition cells into the S-phase of the cell cycle which is conducive for optimal adenovirus replication. In wild type adenoviruses, the role of the E1B-55 kDa gene product is to neutralize p53[ 9 , 10 ]. p53 induction is thought to promote cell cycle arrest resulting in termination of the virus replicative lifecycle in that cell[ 11 ]. Thus it was hypothesized that deletion of the E1B-55 kDa gene product would result in p53 induced termination of virus replication in normal cells while being replication permissive in malignant cells with abnormal p53 expression or regulation[ 9 ]. Early reports with ONYX-015 indicated replication selectivity for tumor cells with abnormal p53 functioning[ 12 ]. However, the exact mechanism of replication selectivity has been brought to question with subsequent reports of ONYX-015 replication independent of p53[ 13 ] and researchers have looked at other factors associated with adenovirus replication.Various heat shock proteins have been shown to be necessary for efficient adenovirus replication. The avian adenovirus CELO requires the induction of HSP70 and HSP40 for production of viral proteins[ 14 ]. CELO mutants lacking the E1 region genes were replication incompetent in A549 cells. However, heat shock protein induction or expression allowed for permissive replication of these mutants. Similarly in a number of human cell lines, both heat shock and HSP72 expression enhanced the oncolytic effect of E1 containing adenovirus but not for E1 deleted adenovirus[ 15 ].Adenovirus E1 region genes from serotype 5 and 12 have previously been shown to induce HSP72 expression [ 16 ]. HSP72 is a molecular chaperone protein involved in the repairing denatured proteins. Through this role, it can inhibit apoptosis downstream of caspase activation but prior to loss of mitochondrial membrane potential[ 17 ]. Stably transfected or E1 transformed cell lines show constitutive expression of the inducible HSP72 [ 18 ]. HSP72 levels are correlated with cell-cycle with increases in HSP72 during S-phase with a maximum level in the post-S-phase period [ 19 ]. In 293 cells which are transformed with the adenovirus E1 region gene, E1A mRNA accumulated just prior to HSP72 expression suggesting that E1A is responsible for the cell cycle regulation of HSP72 expression [ 18 ]. Additionally, HSP72 co-localizes with E1A gene products in infected cells [ 20 ]. HSP72 is predominately cytoplasmic but translocates to the nucleus following adenovirus infection. Double labelling experiments with E1A and HSP72 in infected cells showed co-localization of these molecules within the nucleoli and exhibited similar reticular and punctuate nuclear staining patterns depending on cell cycle. HSP72 transport to the nucleus was E1A and virus infection dependent. In 293 (E1 region transformed) cells, HSP72 and E1A co-localization was not observed until after infection with virus [ 20 ]. These data suggest a physical complex is necessary between adenovirus E1A, HSP72 and other adenovirus gene productsOne of the difficulties with studying E1 adenovirus deletion mutants is the lack of appropriate animal tumor models which allow for permissive growth of these viruses. Common animal tumor models for glioblastoma are the cell lines 9L and RT2. Previously, it has been shown that replication competent E1 deletion mutants can transduce 9L cells and result in transgene expression but there is a block against virus replication[ 21 ]. In this study we addressed if HSP72 expression could allow for permissive replication of the ONYX-015 adenovirus in these rat glioblastoma cells.Results Transduction Efficiency Prior to studying the efficacy of ONYX-015 to result in tumor cell lysis, we determined the optimal dose necessary for tissue transduction. Adenovirus infection efficiency is predicated on the expression of adenovirus surface receptors[ 22 ]. Using a recombinant adenovirus expressing green fluorescent protein (GFP), we transduced various glioma cell lines and analyzed them by fluorescence microscopy. Results are summarized in Table  1 . Both the rat cell lines, 9L and RT2 required a significantly high dose of adenovirus to result in effective transduction. In contrast, the human glioma cell lines, DBTRG and NB4, require much less virus for efficient transduction. Table 1 Transduction efficiency of various CNS tumor cell lines by AD-GFP Numbers represent percentage of cells transduced with a recombinant adenovirus vector expressing green fluorescent protein (AD-GFP) at the stated multiplicity of infection (MOI). Transduction was determined by fluorescence microscopy of cells on a hemacytometer and is represented as % of total cells. Multiplicity of Infection Cell Line 0 1 10 100 300 500 1000 9L 0 0 1 27 54 63 74 RT2 0 1 8 41 68 81 93 DBTRG 0 69 100 100 - - - NB4 0 74 100 100 - - - Cytopathic Effect The dramatic difference in tumoricidal effect between HSP72 transfected and control tumor cells is best observed by cytopathic effect. In Figure  1  we show the affects of virus infection on RT2-GFP and RT2-HSP72 cells 96 hours after infection with ONYX-015. Non-infected RT2-GFP and RT2-HSP72 cells showed normal glioma histological features and growth characteristics (Figure  1 , panel A and B). The cells were infected when 50% confluent. At 96 hours, RT2-GFP cells infected at MOI of 100 or 300 showed little cytopathic effect and very little difference was ascertained in comparison to non-infected cells (Figure  1 , panel C and E). In contrast, the majority of RT2-HSP72 cells were dead or dying (Figure  1 , panel D and F). The few remaining cells were larger in size with oval or round morphology. These cells showed morphological characteristics consistent with adenovirus replication as observed in 293 cells. Similar results were obtained when assessing 9L-HSP72 cells. In table  2  we show the time to full CPE in the rat glioblastoma cell lines and a variety of human controls. Given the differences in transduction efficiency, the human cell lines were infected at a lower multiplicity of infection. No CPE is detected in the 9L-GFP and RT2-GFP cell lines. In contrast, 9L-HSP72 and RT2-HSP72 show CPE in as little as 4 days comparable to human glioblastoma cells when infected at an MOI of 10 and show CPE more rapidly than the human cell lines despite a lesser transduction efficiency. Table 2 Time to Cytopathic Effect Cells grown in 96 well plates were infected with ONYX-015 at the stated multiplicity of infection per row of cells. Values represent days until 50% of wells in a row showed cytopathic effect. Multiplicity of Infection Cell Line 0 1 10 100 300 9L-GFP 0 0 0 0 0 9L-HSP72 0 0 8 5 4 RT2-GFP 0 0 0 0 0 RT2-HSP72 0 0 0 4 4 DBTRG 0 6 4 - - NB4 0 5 4 - - 293 0 2 1 - - Figure 1 Cytopathic effect of ONYX-015 on GFP and HSP72 transfected glioma cells.  Photographs (100× magnification) of RT2-GFP and RT2-HSP72 cells 96 hours after ONYX-015 infection at various dosages are represented. A – RT2-GFP cells mock infected with virus. B – RT2-HSP72 cells mock infected with virus. C – RT2-GFP cells infected at MOI of 100. D – RT2-HSP72 cells infected at MOI of 100. E – RT2-GFP cells infected at MOI of 300. F – RT2-HSP72 cells infected at MOI of 300. HSP72 Expression Augments ONYX-015 Toxicity When HSP72-transfected cells were infected with a low dose (MOI 100) of ONYX-015, there is no loss of cell viability. However, comparison of cell numbers to GFP-transfected cells or AD-GFP infected cells showed a dramatic cytostatic effect (Figure  2 ). 9L-GFP and 9L-HSP72 cells were transduced with either AD-GFP or ONYX-015. There is no effect on cell proliferation over time of the 9L-GFP cells infected with either AD-GFP or ONYX-015 (data not presented). In contrast, the 9L-HSP72 cells show normal proliferation when infected with AD-GFP infected cells and their cell numbers increase over time. However, ONYX-015 infected 9L-HSP72 cells showed loss of proliferative potential with very little increase in cell number over time. After 72 hours, the AD-GFP infected cells showed a decrease in proliferation but this is likely due to overcrowding of cells in the tissue culture vessel and/or the rapid utilization of consumable energy in the media. Figure 2 Cytostatic effect of ONYX-015 infection on HSP72 transfected 9L cells.  9L-HSP72 cells were transduced with either AD-GFP (□) or ONYX-015 (■) at a multiplicity of infection of 100. Cell numbers were determined over time (X-axis) by direct counting using a Coulter Counter. Values (Y-axis) represent log of cell number as a fraction of the initial cell number plated. When assessing for cell viability, 9L-GFP (figure  3 ) and RT2-GFP (figure  4 ) cells exhibited minimal toxicity when infected with ONYX-015. In contrast, both 9L-HSP72 and RT2-HSP72 cell lines were more susceptible to ONYX-015 cell lysis. In both HSP72 transfected cell lines, loss of cell viability began as early as 24 hours after infection. Both 9L-HSP72 and RT2-HSP72 show greater than 90% loss of viability by 96 hours. Figure 3 9L Cell viability after ONYX-015 infection.  Cell viability (as % viable cells; y-axis) of 9L-GFP (■) and 9L-HSP72 (●) cell lines after infection with ONYX-015 at a MOI of 300 over time (x-axis). There is statistical significance (p < 0.05) when comparing GFP and HSP72 transfected cell numbers by analysis of variance at the 72 and 96 hour time points. Figure 4 RT2 Cell viability after ONYX-015 infection.  Cell viability (as % viable cells; y-axis) of RT2-GFP (■) and RT2-HSP72 (●) cell lines after infection with ONYX-015 at a MOI of 300 over time (x-axis). There is statistical significance (p < 0.05) when comparing GFP and HSP72 transfected cell numbers by analysis of variance at the 72 and 96 hour time points. Burst Assays The mechanism of cell death associated with replication competent adenoviruses has been attributed to release of virus from cells following virus replication. Having observed cytopathic effects (figure  1 ) in association with ONYX-015 infection of 9L-HSP72 and RT2-HSP72 cells, we presumed the same mechanism applied. To confirm this hypothesis we evaluated adenovirus replication in GFP and HSP72 transfected tumor cells by burst assay. In a normal burst assay, an increase in virus titer after 72 hours is indicative of virus replication. Tumor cells were also infected with the replication incompetent virus (AD-GFP) as a negative control and no significant virus replication was measured (data not presented). Since HSP72 transfected cells were susceptible to ONYX-015 mediated cell death we expected an increase in virus titer if the cell death was a result of permissive adenovirus replication. After infection of cells with ONYX-015, the 9L-HSP72 and RT2-HSP72 cells showed an increase in virus titer confirming virus replication (figure  5 ) in contrast to 9L-GFP and RT2-HSP72 transfected cells. There was a direct correlation between virus titer and cell death. Virus titer in both GFP transfected and HSP72 transfected cells increased in proportion to cell death. Figure 5 Burst assays.  Burst ratio is given as the virus titer at 72 hours compared to 4 hours of various cell lines (x-axis) infected while 50% confluent with ONYX-015 at MOI of 100. Results are representative of a typical experiment of at least three performed. (*) represents statistical significance (p < 0.05) by Student's t-test between 9L-HSP72 and 9L-GFP. (**) represents statistical significance (p < 0.05) by Student's t-test between RT2-HSP72 and RT2-GFP.
Results
Transduction Efficiency Prior to studying the efficacy of ONYX-015 to result in tumor cell lysis, we determined the optimal dose necessary for tissue transduction. Adenovirus infection efficiency is predicated on the expression of adenovirus surface receptors[ 22 ]. Using a recombinant adenovirus expressing green fluorescent protein (GFP), we transduced various glioma cell lines and analyzed them by fluorescence microscopy. Results are summarized in Table  1 . Both the rat cell lines, 9L and RT2 required a significantly high dose of adenovirus to result in effective transduction. In contrast, the human glioma cell lines, DBTRG and NB4, require much less virus for efficient transduction. Table 1 Transduction efficiency of various CNS tumor cell lines by AD-GFP Numbers represent percentage of cells transduced with a recombinant adenovirus vector expressing green fluorescent protein (AD-GFP) at the stated multiplicity of infection (MOI). Transduction was determined by fluorescence microscopy of cells on a hemacytometer and is represented as % of total cells. Multiplicity of Infection Cell Line 0 1 10 100 300 500 1000 9L 0 0 1 27 54 63 74 RT2 0 1 8 41 68 81 93 DBTRG 0 69 100 100 - - - NB4 0 74 100 100 - - -
Transduction Efficiency
Prior to studying the efficacy of ONYX-015 to result in tumor cell lysis, we determined the optimal dose necessary for tissue transduction. Adenovirus infection efficiency is predicated on the expression of adenovirus surface receptors[ 22 ]. Using a recombinant adenovirus expressing green fluorescent protein (GFP), we transduced various glioma cell lines and analyzed them by fluorescence microscopy. Results are summarized in Table  1 . Both the rat cell lines, 9L and RT2 required a significantly high dose of adenovirus to result in effective transduction. In contrast, the human glioma cell lines, DBTRG and NB4, require much less virus for efficient transduction.Transduction efficiency of various CNS tumor cell lines by AD-GFP Numbers represent percentage of cells transduced with a recombinant adenovirus vector expressing green fluorescent protein (AD-GFP) at the stated multiplicity of infection (MOI). Transduction was determined by fluorescence microscopy of cells on a hemacytometer and is represented as % of total cells.Multiplicity of InfectionCell Line01101003005001000Cytopathic Effect The dramatic difference in tumoricidal effect between HSP72 transfected and control tumor cells is best observed by cytopathic effect. In Figure  1  we show the affects of virus infection on RT2-GFP and RT2-HSP72 cells 96 hours after infection with ONYX-015. Non-infected RT2-GFP and RT2-HSP72 cells showed normal glioma histological features and growth characteristics (Figure  1 , panel A and B). The cells were infected when 50% confluent. At 96 hours, RT2-GFP cells infected at MOI of 100 or 300 showed little cytopathic effect and very little difference was ascertained in comparison to non-infected cells (Figure  1 , panel C and E). In contrast, the majority of RT2-HSP72 cells were dead or dying (Figure  1 , panel D and F). The few remaining cells were larger in size with oval or round morphology. These cells showed morphological characteristics consistent with adenovirus replication as observed in 293 cells. Similar results were obtained when assessing 9L-HSP72 cells. In table  2  we show the time to full CPE in the rat glioblastoma cell lines and a variety of human controls. Given the differences in transduction efficiency, the human cell lines were infected at a lower multiplicity of infection. No CPE is detected in the 9L-GFP and RT2-GFP cell lines. In contrast, 9L-HSP72 and RT2-HSP72 show CPE in as little as 4 days comparable to human glioblastoma cells when infected at an MOI of 10 and show CPE more rapidly than the human cell lines despite a lesser transduction efficiency. Table 2 Time to Cytopathic Effect Cells grown in 96 well plates were infected with ONYX-015 at the stated multiplicity of infection per row of cells. Values represent days until 50% of wells in a row showed cytopathic effect. Multiplicity of Infection Cell Line 0 1 10 100 300 9L-GFP 0 0 0 0 0 9L-HSP72 0 0 8 5 4 RT2-GFP 0 0 0 0 0 RT2-HSP72 0 0 0 4 4 DBTRG 0 6 4 - - NB4 0 5 4 - - 293 0 2 1 - - Figure 1 Cytopathic effect of ONYX-015 on GFP and HSP72 transfected glioma cells.  Photographs (100× magnification) of RT2-GFP and RT2-HSP72 cells 96 hours after ONYX-015 infection at various dosages are represented. A – RT2-GFP cells mock infected with virus. B – RT2-HSP72 cells mock infected with virus. C – RT2-GFP cells infected at MOI of 100. D – RT2-HSP72 cells infected at MOI of 100. E – RT2-GFP cells infected at MOI of 300. F – RT2-HSP72 cells infected at MOI of 300.
Cytopathic Effect
The dramatic difference in tumoricidal effect between HSP72 transfected and control tumor cells is best observed by cytopathic effect. In Figure  1  we show the affects of virus infection on RT2-GFP and RT2-HSP72 cells 96 hours after infection with ONYX-015. Non-infected RT2-GFP and RT2-HSP72 cells showed normal glioma histological features and growth characteristics (Figure  1 , panel A and B). The cells were infected when 50% confluent. At 96 hours, RT2-GFP cells infected at MOI of 100 or 300 showed little cytopathic effect and very little difference was ascertained in comparison to non-infected cells (Figure  1 , panel C and E). In contrast, the majority of RT2-HSP72 cells were dead or dying (Figure  1 , panel D and F). The few remaining cells were larger in size with oval or round morphology. These cells showed morphological characteristics consistent with adenovirus replication as observed in 293 cells. Similar results were obtained when assessing 9L-HSP72 cells. In table  2  we show the time to full CPE in the rat glioblastoma cell lines and a variety of human controls. Given the differences in transduction efficiency, the human cell lines were infected at a lower multiplicity of infection. No CPE is detected in the 9L-GFP and RT2-GFP cell lines. In contrast, 9L-HSP72 and RT2-HSP72 show CPE in as little as 4 days comparable to human glioblastoma cells when infected at an MOI of 10 and show CPE more rapidly than the human cell lines despite a lesser transduction efficiency.Time to Cytopathic Effect Cells grown in 96 well plates were infected with ONYX-015 at the stated multiplicity of infection per row of cells. Values represent days until 50% of wells in a row showed cytopathic effect.Multiplicity of InfectionCell Line0110100300HSP72 Expression Augments ONYX-015 Toxicity When HSP72-transfected cells were infected with a low dose (MOI 100) of ONYX-015, there is no loss of cell viability. However, comparison of cell numbers to GFP-transfected cells or AD-GFP infected cells showed a dramatic cytostatic effect (Figure  2 ). 9L-GFP and 9L-HSP72 cells were transduced with either AD-GFP or ONYX-015. There is no effect on cell proliferation over time of the 9L-GFP cells infected with either AD-GFP or ONYX-015 (data not presented). In contrast, the 9L-HSP72 cells show normal proliferation when infected with AD-GFP infected cells and their cell numbers increase over time. However, ONYX-015 infected 9L-HSP72 cells showed loss of proliferative potential with very little increase in cell number over time. After 72 hours, the AD-GFP infected cells showed a decrease in proliferation but this is likely due to overcrowding of cells in the tissue culture vessel and/or the rapid utilization of consumable energy in the media. Figure 2 Cytostatic effect of ONYX-015 infection on HSP72 transfected 9L cells.  9L-HSP72 cells were transduced with either AD-GFP (□) or ONYX-015 (■) at a multiplicity of infection of 100. Cell numbers were determined over time (X-axis) by direct counting using a Coulter Counter. Values (Y-axis) represent log of cell number as a fraction of the initial cell number plated. When assessing for cell viability, 9L-GFP (figure  3 ) and RT2-GFP (figure  4 ) cells exhibited minimal toxicity when infected with ONYX-015. In contrast, both 9L-HSP72 and RT2-HSP72 cell lines were more susceptible to ONYX-015 cell lysis. In both HSP72 transfected cell lines, loss of cell viability began as early as 24 hours after infection. Both 9L-HSP72 and RT2-HSP72 show greater than 90% loss of viability by 96 hours. Figure 3 9L Cell viability after ONYX-015 infection.  Cell viability (as % viable cells; y-axis) of 9L-GFP (■) and 9L-HSP72 (●) cell lines after infection with ONYX-015 at a MOI of 300 over time (x-axis). There is statistical significance (p < 0.05) when comparing GFP and HSP72 transfected cell numbers by analysis of variance at the 72 and 96 hour time points. Figure 4 RT2 Cell viability after ONYX-015 infection.  Cell viability (as % viable cells; y-axis) of RT2-GFP (■) and RT2-HSP72 (●) cell lines after infection with ONYX-015 at a MOI of 300 over time (x-axis). There is statistical significance (p < 0.05) when comparing GFP and HSP72 transfected cell numbers by analysis of variance at the 72 and 96 hour time points.
HSP72 Expression Augments ONYX-015 Toxicity
When HSP72-transfected cells were infected with a low dose (MOI 100) of ONYX-015, there is no loss of cell viability. However, comparison of cell numbers to GFP-transfected cells or AD-GFP infected cells showed a dramatic cytostatic effect (Figure  2 ). 9L-GFP and 9L-HSP72 cells were transduced with either AD-GFP or ONYX-015. There is no effect on cell proliferation over time of the 9L-GFP cells infected with either AD-GFP or ONYX-015 (data not presented). In contrast, the 9L-HSP72 cells show normal proliferation when infected with AD-GFP infected cells and their cell numbers increase over time. However, ONYX-015 infected 9L-HSP72 cells showed loss of proliferative potential with very little increase in cell number over time. After 72 hours, the AD-GFP infected cells showed a decrease in proliferation but this is likely due to overcrowding of cells in the tissue culture vessel and/or the rapid utilization of consumable energy in the media.When assessing for cell viability, 9L-GFP (figure  3 ) and RT2-GFP (figure  4 ) cells exhibited minimal toxicity when infected with ONYX-015. In contrast, both 9L-HSP72 and RT2-HSP72 cell lines were more susceptible to ONYX-015 cell lysis. In both HSP72 transfected cell lines, loss of cell viability began as early as 24 hours after infection. Both 9L-HSP72 and RT2-HSP72 show greater than 90% loss of viability by 96 hours.Burst Assays The mechanism of cell death associated with replication competent adenoviruses has been attributed to release of virus from cells following virus replication. Having observed cytopathic effects (figure  1 ) in association with ONYX-015 infection of 9L-HSP72 and RT2-HSP72 cells, we presumed the same mechanism applied. To confirm this hypothesis we evaluated adenovirus replication in GFP and HSP72 transfected tumor cells by burst assay. In a normal burst assay, an increase in virus titer after 72 hours is indicative of virus replication. Tumor cells were also infected with the replication incompetent virus (AD-GFP) as a negative control and no significant virus replication was measured (data not presented). Since HSP72 transfected cells were susceptible to ONYX-015 mediated cell death we expected an increase in virus titer if the cell death was a result of permissive adenovirus replication. After infection of cells with ONYX-015, the 9L-HSP72 and RT2-HSP72 cells showed an increase in virus titer confirming virus replication (figure  5 ) in contrast to 9L-GFP and RT2-HSP72 transfected cells. There was a direct correlation between virus titer and cell death. Virus titer in both GFP transfected and HSP72 transfected cells increased in proportion to cell death. Figure 5 Burst assays.  Burst ratio is given as the virus titer at 72 hours compared to 4 hours of various cell lines (x-axis) infected while 50% confluent with ONYX-015 at MOI of 100. Results are representative of a typical experiment of at least three performed. (*) represents statistical significance (p < 0.05) by Student's t-test between 9L-HSP72 and 9L-GFP. (**) represents statistical significance (p < 0.05) by Student's t-test between RT2-HSP72 and RT2-GFP.
Burst Assays
The mechanism of cell death associated with replication competent adenoviruses has been attributed to release of virus from cells following virus replication. Having observed cytopathic effects (figure  1 ) in association with ONYX-015 infection of 9L-HSP72 and RT2-HSP72 cells, we presumed the same mechanism applied. To confirm this hypothesis we evaluated adenovirus replication in GFP and HSP72 transfected tumor cells by burst assay. In a normal burst assay, an increase in virus titer after 72 hours is indicative of virus replication. Tumor cells were also infected with the replication incompetent virus (AD-GFP) as a negative control and no significant virus replication was measured (data not presented). Since HSP72 transfected cells were susceptible to ONYX-015 mediated cell death we expected an increase in virus titer if the cell death was a result of permissive adenovirus replication. After infection of cells with ONYX-015, the 9L-HSP72 and RT2-HSP72 cells showed an increase in virus titer confirming virus replication (figure  5 ) in contrast to 9L-GFP and RT2-HSP72 transfected cells. There was a direct correlation between virus titer and cell death. Virus titer in both GFP transfected and HSP72 transfected cells increased in proportion to cell death.Discussion ONYX-015 is a promising new cancer gene therapy vector which exerts its tumoricidal effect by selective replication in cancer cells[ 6 ]. Replication selectivity previously was attributed to interactions of E1 gene products with p53[ 3 ]. Subsequent reports have shown that ONYX-015 replicates in cells with wild-type p53 as efficiently as in cells with mutant-p53[ 13 ]. Although the mechanism of cancer cell selectivity is still under investigation, the various hypotheses consistently involve cell-cycle regulatory proteins and their interactions within apoptosis pathways. HSP72 is a potent regulator of apoptosis [ 23 ]. Also, there is evidence that activation of the heat shock response by adenovirus early region genes is necessary for virus replication[ 14 ]. Previously it has been shown that heat shock induced HSP72 expression resulted in increased production of adenovirus proteins[ 24 ]. Additionally, the E1A adenovirus gene products necessary for virus genome replication have previously been shown to co-localize with HSP72[ 20 ]. To study the role of HSP72 in augmenting ONYX-015 replication, we chose two glioma cell lines with different derivations. 9L is a chemically induced gliosarcoma while RT2 is a virally transformed glioblastoma. Compared to many human tumor cell lines, these cell lines show relative resistance to the cytotoxic affects of ONYX-015. Resistance in part was secondary to low adenovirus transduction efficiency. At the multiplicities of infection utilized in this study, we never achieved 100% cell transduction (table  1 ). In most cases, when less than 30% of permissive HSP72-transfected cells were transduced, tumor cell growth was rapid and outpaced ONYX-015 virus replication resulting in no discernable cytotoxic or cytostatic effect after 1 week in culture (data not presented). Therefore, replicative adenovirus cytotoxicity is likely a balance between virus transduction efficiency, replication efficiency and cell growth characteristics. HSP72 transfection had no bearing on cell doubling time of either 9L or RT2 cells (data not presented) RT2 cell-doubling time is approximately 8 hours while 9L cell-doubling time is approximately 14 hours. Both cell lines after HSP72 transfection showed equal susceptibility to ONYX-015. Further evidence for the balance between transduction efficiency, replication efficiency and cell doubling time is evident in that ONYX-015 infection resulted in a predominantly cytostatic affect resulting in decreased cell division before evidence of cytotoxicity. The most dramatic affect of the role HSP72 in promotion of virus replication is evident in figure  1 . At 96 hours, the GFP transduced cells showed very little cytotoxic effect of ONYX-015 infection. In contrast, there is a distinct cytopathic effect at the same doses of ONYX-015 infection in the HSP72 transduced cells. These cells were rendered susceptible to ONYX-015 toxicity. The mechanism of cell death is related to increased virus replication. Significantly more ONYX-015 was isolated from HSP72 transduced cells than the GFP-transfected controls. The E1a gene products of adenovirus are responsible for activation of the HSP72 promoter and may affect HSP72 levels during the cell cycle[ 18 ]. Conversely, there is no evidence that HSP72 interacts with adenovirus promoters to stimulate viral transcription. There is evidence of HSP72 interactions with adenovirus structural proteins such as hexon[ 25 ] and fiber[ 26 ]. Adenovirus assembly is known to be inefficient with only a small percentage of total structural proteins eventually utilized in the production of infectious virus [ 26 ]. In conclusion, our studies demonstrated that HSP72 expression in rodent glioma tumor cells potentiated ONYX-015 replication and oncolysis of tumor cells. Further studies on the role of HSP72 in tumor types with wild-type and mutant p53 tumor suppressor genes is warranted to understand the molecular interactions of this chaperone protein in promoting virus replication. Additionally, addressing the role of HSP72 as an inhibitor of apoptosis in virus replication would further help characterize the interaction of cell-cycle regulatory pathways with virus replication. Furthermore, we intend to evaluate the role of HSP72 associated permissive replication in other animal cell lines to establish animal models for the study of ONYX-015 and similar replication competent adenoviruses.
Discussion
ONYX-015 is a promising new cancer gene therapy vector which exerts its tumoricidal effect by selective replication in cancer cells[ 6 ]. Replication selectivity previously was attributed to interactions of E1 gene products with p53[ 3 ]. Subsequent reports have shown that ONYX-015 replicates in cells with wild-type p53 as efficiently as in cells with mutant-p53[ 13 ]. Although the mechanism of cancer cell selectivity is still under investigation, the various hypotheses consistently involve cell-cycle regulatory proteins and their interactions within apoptosis pathways. HSP72 is a potent regulator of apoptosis [ 23 ]. Also, there is evidence that activation of the heat shock response by adenovirus early region genes is necessary for virus replication[ 14 ]. Previously it has been shown that heat shock induced HSP72 expression resulted in increased production of adenovirus proteins[ 24 ]. Additionally, the E1A adenovirus gene products necessary for virus genome replication have previously been shown to co-localize with HSP72[ 20 ].To study the role of HSP72 in augmenting ONYX-015 replication, we chose two glioma cell lines with different derivations. 9L is a chemically induced gliosarcoma while RT2 is a virally transformed glioblastoma. Compared to many human tumor cell lines, these cell lines show relative resistance to the cytotoxic affects of ONYX-015. Resistance in part was secondary to low adenovirus transduction efficiency. At the multiplicities of infection utilized in this study, we never achieved 100% cell transduction (table  1 ). In most cases, when less than 30% of permissive HSP72-transfected cells were transduced, tumor cell growth was rapid and outpaced ONYX-015 virus replication resulting in no discernable cytotoxic or cytostatic effect after 1 week in culture (data not presented). Therefore, replicative adenovirus cytotoxicity is likely a balance between virus transduction efficiency, replication efficiency and cell growth characteristics. HSP72 transfection had no bearing on cell doubling time of either 9L or RT2 cells (data not presented) RT2 cell-doubling time is approximately 8 hours while 9L cell-doubling time is approximately 14 hours. Both cell lines after HSP72 transfection showed equal susceptibility to ONYX-015. Further evidence for the balance between transduction efficiency, replication efficiency and cell doubling time is evident in that ONYX-015 infection resulted in a predominantly cytostatic affect resulting in decreased cell division before evidence of cytotoxicity.The most dramatic affect of the role HSP72 in promotion of virus replication is evident in figure  1 . At 96 hours, the GFP transduced cells showed very little cytotoxic effect of ONYX-015 infection. In contrast, there is a distinct cytopathic effect at the same doses of ONYX-015 infection in the HSP72 transduced cells. These cells were rendered susceptible to ONYX-015 toxicity. The mechanism of cell death is related to increased virus replication. Significantly more ONYX-015 was isolated from HSP72 transduced cells than the GFP-transfected controls.The E1a gene products of adenovirus are responsible for activation of the HSP72 promoter and may affect HSP72 levels during the cell cycle[ 18 ]. Conversely, there is no evidence that HSP72 interacts with adenovirus promoters to stimulate viral transcription. There is evidence of HSP72 interactions with adenovirus structural proteins such as hexon[ 25 ] and fiber[ 26 ]. Adenovirus assembly is known to be inefficient with only a small percentage of total structural proteins eventually utilized in the production of infectious virus [ 26 ].In conclusion, our studies demonstrated that HSP72 expression in rodent glioma tumor cells potentiated ONYX-015 replication and oncolysis of tumor cells. Further studies on the role of HSP72 in tumor types with wild-type and mutant p53 tumor suppressor genes is warranted to understand the molecular interactions of this chaperone protein in promoting virus replication. Additionally, addressing the role of HSP72 as an inhibitor of apoptosis in virus replication would further help characterize the interaction of cell-cycle regulatory pathways with virus replication. Furthermore, we intend to evaluate the role of HSP72 associated permissive replication in other animal cell lines to establish animal models for the study of ONYX-015 and similar replication competent adenoviruses.Conclusion For clinical applicability, these findings have a number of implications. Currently, clinical trials are ongoing with ONYX-015. Due to the previous association of ONYX-015 replication with p53 status, the current tumor types chosen for clinical trials are those exhibiting abnormal p53 expression in a majority of cells. Our study indicates, that tumor types with high HSP72 may also be viable candidates to evaluate ONYX-015 efficacy, as they are likely to demonstrate enhanced susceptibility to this replication competent virus. As HSP72 expression in tumors is associated with a more aggressive tumor phenotype, ONYX-015 may be ideal as adjunctive therapy for advanced disease. Additionally, one of the greatest limitations to adenovirus gene therapy is patient safety at high doses of adenovirus administration. Since, HSP72 expression resulted in oncolysis at much lower MOIs, a lower dose of virus administered to a HSP72 positive tumor may have the same benefit as a higher dose, allowing one to use lower doses to achieve the same effect. In addition, for some tumor types such as breast cancer, hyperthermia has been shown to significantly improve response rates[ 27 ]. HSP72 is readily induced with hyperthermia. Therefore, HSP72 induction by hyperthermia may also serve as an effective strategy to augment ONYX-015 oncolytic activity. We are currently in the process of studying this hypothesis. Lastly, the role of HSP72 in cell cycle regulation suggests alternate pathways for the mechanism of E1b deleted adenovirus replication in tumorigenic cells. HSP72 overexpression in non-transformed fibroblasts did not result in ONYX-015 oncolysis (data not presented). Understanding the molecular interactions of adenovirus proteins with molecular chaperones would help determine cell selectivity to replication competent adenoviruses influencing the design of more potent viruses.
Conclusion
For clinical applicability, these findings have a number of implications. Currently, clinical trials are ongoing with ONYX-015. Due to the previous association of ONYX-015 replication with p53 status, the current tumor types chosen for clinical trials are those exhibiting abnormal p53 expression in a majority of cells. Our study indicates, that tumor types with high HSP72 may also be viable candidates to evaluate ONYX-015 efficacy, as they are likely to demonstrate enhanced susceptibility to this replication competent virus. As HSP72 expression in tumors is associated with a more aggressive tumor phenotype, ONYX-015 may be ideal as adjunctive therapy for advanced disease. Additionally, one of the greatest limitations to adenovirus gene therapy is patient safety at high doses of adenovirus administration. Since, HSP72 expression resulted in oncolysis at much lower MOIs, a lower dose of virus administered to a HSP72 positive tumor may have the same benefit as a higher dose, allowing one to use lower doses to achieve the same effect. In addition, for some tumor types such as breast cancer, hyperthermia has been shown to significantly improve response rates[ 27 ]. HSP72 is readily induced with hyperthermia. Therefore, HSP72 induction by hyperthermia may also serve as an effective strategy to augment ONYX-015 oncolytic activity. We are currently in the process of studying this hypothesis. Lastly, the role of HSP72 in cell cycle regulation suggests alternate pathways for the mechanism of E1b deleted adenovirus replication in tumorigenic cells. HSP72 overexpression in non-transformed fibroblasts did not result in ONYX-015 oncolysis (data not presented). Understanding the molecular interactions of adenovirus proteins with molecular chaperones would help determine cell selectivity to replication competent adenoviruses influencing the design of more potent viruses.Methods Cell Lines 9L and RT2 cells are rat glioma cell lines with different derivations. 9L cells are chemically induced gliosarcoma cells[ 28 ] while RT2 are virally transformed and have glioblastoma histology[ 29 ]. These cell lines were obtained from Dr. Martin Graf (Medical College of Virginia, Richmond, VA). Cells were kept at 37°C, 5% CO 2  and 95% humidity in Dulbecco's modified eagle medium (Cellgro, Herndon, VA) supplemented with 10% (v/v) heat inactivated fetal bovine serum (BioWhittaker, Walkersville, MD), 2 mM L-glutamine and 100 units/ml Penicillin and 1000 ug/ml Streptomycin (Invitrogen). 9L and RT2 cells were transfected with the plasmids pEGFP-N2 (Clontech) or pHSP72 using Lipofectamine (Invitrogen) according to the manufacturers protocol followed by selection in 0.2 mg/ml G418 (Invitrogen). 293 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA; CRL-1573) were used for virus propagation and purification. Plasmid Creation pHSP72 was created by ligation of the DNA Polymerase blunt ended 2.3 KB Bam H1-Hind III fragment from plasmid pH2.3 (ATCC) into the blunt ended Xho1-XbaIA vector pEGFP-N2. Adenovirus Creation of ONYX-015 has previously been described[ 6 ]. The virus was propagated on 293 cells and purified by cesium chloride gradient followed by dialysis. Virus was stored at -80°C until use. Titer was determined by tissue culture infectious dose-50 method (TCID-50). Briefly, 293 cells were plated at 10 4  cells/well in 96 well flat-bottomed tissue culture plates. Virus titer from combined supernatant and freeze-thawed cell lysate was determined by serial 10-fold dilution into different rows of the titer plate. After 10 days, wells were scored for cytopathic effect. The titer was calculated using the formula T = 10 1+d(S-0.5)  where d is the Log 10 of the dilution and S is the sum of the ratios of positive wells in each row. A more lengthy description of this method is provided in the adenovirus application manual at  . Adenovirus Infection Cells were infected when 50% confluent with the dose of adenovirus given in the appropriate table. The infection was performed in serum free DMEM media for 90 minutes at 37°C and 5% CO 2 . Cells were subsequently washed with phosphate buffered saline and then cultured in complete medium. Cell Viability Determination Cell viability was determined by the standard Trypan-blue exclusion test. Cells were washed with PBS and then lifted from tissue culture plates with Trypsin-EDTA solution (Invitrogen). Cells were stained with 0.2% Trypan blue solution for 5 minutes and then counted on a hemacytometer.
Methods
Cell Lines 9L and RT2 cells are rat glioma cell lines with different derivations. 9L cells are chemically induced gliosarcoma cells[ 28 ] while RT2 are virally transformed and have glioblastoma histology[ 29 ]. These cell lines were obtained from Dr. Martin Graf (Medical College of Virginia, Richmond, VA). Cells were kept at 37°C, 5% CO 2  and 95% humidity in Dulbecco's modified eagle medium (Cellgro, Herndon, VA) supplemented with 10% (v/v) heat inactivated fetal bovine serum (BioWhittaker, Walkersville, MD), 2 mM L-glutamine and 100 units/ml Penicillin and 1000 ug/ml Streptomycin (Invitrogen). 9L and RT2 cells were transfected with the plasmids pEGFP-N2 (Clontech) or pHSP72 using Lipofectamine (Invitrogen) according to the manufacturers protocol followed by selection in 0.2 mg/ml G418 (Invitrogen). 293 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA; CRL-1573) were used for virus propagation and purification.
Cell Lines
9L and RT2 cells are rat glioma cell lines with different derivations. 9L cells are chemically induced gliosarcoma cells[ 28 ] while RT2 are virally transformed and have glioblastoma histology[ 29 ]. These cell lines were obtained from Dr. Martin Graf (Medical College of Virginia, Richmond, VA). Cells were kept at 37°C, 5% CO 2  and 95% humidity in Dulbecco's modified eagle medium (Cellgro, Herndon, VA) supplemented with 10% (v/v) heat inactivated fetal bovine serum (BioWhittaker, Walkersville, MD), 2 mM L-glutamine and 100 units/ml Penicillin and 1000 ug/ml Streptomycin (Invitrogen). 9L and RT2 cells were transfected with the plasmids pEGFP-N2 (Clontech) or pHSP72 using Lipofectamine (Invitrogen) according to the manufacturers protocol followed by selection in 0.2 mg/ml G418 (Invitrogen). 293 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA; CRL-1573) were used for virus propagation and purification.Plasmid Creation pHSP72 was created by ligation of the DNA Polymerase blunt ended 2.3 KB Bam H1-Hind III fragment from plasmid pH2.3 (ATCC) into the blunt ended Xho1-XbaIA vector pEGFP-N2.
Plasmid Creation
pHSP72 was created by ligation of the DNA Polymerase blunt ended 2.3 KB Bam H1-Hind III fragment from plasmid pH2.3 (ATCC) into the blunt ended Xho1-XbaIA vector pEGFP-N2.Adenovirus Creation of ONYX-015 has previously been described[ 6 ]. The virus was propagated on 293 cells and purified by cesium chloride gradient followed by dialysis. Virus was stored at -80°C until use. Titer was determined by tissue culture infectious dose-50 method (TCID-50). Briefly, 293 cells were plated at 10 4  cells/well in 96 well flat-bottomed tissue culture plates. Virus titer from combined supernatant and freeze-thawed cell lysate was determined by serial 10-fold dilution into different rows of the titer plate. After 10 days, wells were scored for cytopathic effect. The titer was calculated using the formula T = 10 1+d(S-0.5)  where d is the Log 10 of the dilution and S is the sum of the ratios of positive wells in each row. A more lengthy description of this method is provided in the adenovirus application manual at  .
Adenovirus
Creation of ONYX-015 has previously been described[ 6 ]. The virus was propagated on 293 cells and purified by cesium chloride gradient followed by dialysis. Virus was stored at -80°C until use. Titer was determined by tissue culture infectious dose-50 method (TCID-50). Briefly, 293 cells were plated at 10 4  cells/well in 96 well flat-bottomed tissue culture plates. Virus titer from combined supernatant and freeze-thawed cell lysate was determined by serial 10-fold dilution into different rows of the titer plate. After 10 days, wells were scored for cytopathic effect. The titer was calculated using the formula T = 10 1+d(S-0.5)  where d is the Log 10 of the dilution and S is the sum of the ratios of positive wells in each row. A more lengthy description of this method is provided in the adenovirus application manual at  .Adenovirus Infection Cells were infected when 50% confluent with the dose of adenovirus given in the appropriate table. The infection was performed in serum free DMEM media for 90 minutes at 37°C and 5% CO 2 . Cells were subsequently washed with phosphate buffered saline and then cultured in complete medium.
Adenovirus Infection
Cells were infected when 50% confluent with the dose of adenovirus given in the appropriate table. The infection was performed in serum free DMEM media for 90 minutes at 37°C and 5% CO 2 . Cells were subsequently washed with phosphate buffered saline and then cultured in complete medium.Cell Viability Determination Cell viability was determined by the standard Trypan-blue exclusion test. Cells were washed with PBS and then lifted from tissue culture plates with Trypsin-EDTA solution (Invitrogen). Cells were stained with 0.2% Trypan blue solution for 5 minutes and then counted on a hemacytometer.
Cell Viability Determination
Cell viability was determined by the standard Trypan-blue exclusion test. Cells were washed with PBS and then lifted from tissue culture plates with Trypsin-EDTA solution (Invitrogen). Cells were stained with 0.2% Trypan blue solution for 5 minutes and then counted on a hemacytometer.Authors' contributions JM performed all of the experiments outlined in this study. JAK independently replicated results for validity. JAK and MRS contributed equally to the molecular cloning of the plasmids and adenovirus propagation and purification. All authors participated in maintenance of cells in culture. The hypothesis, study design and statistical analysis were all performed by MRS. MRS drafted the entire manuscript with editing and approval by all of the authors.
Authors' contributions
JM performed all of the experiments outlined in this study. JAK independently replicated results for validity. JAK and MRS contributed equally to the molecular cloning of the plasmids and adenovirus propagation and purification. All authors participated in maintenance of cells in culture. The hypothesis, study design and statistical analysis were all performed by MRS. MRS drafted the entire manuscript with editing and approval by all of the authors.


The essential difference between cancer cells and normal cells is that cancer cells evolve. Most cancers arise from a single cell through a sequential evolutionary process of mutation and selection. Cancer cells harbor mutations in a number of critical genes that, at various stages during the evolution of the tumor, provide those cells with a selective advantage. Many of the phenotypes, or physical outcomes, conferred by these mutant genes are subverted from a normal cell's repertoire, including proliferation, invasion, migration, loss of differentiation, and loss of apoptosis (programmed cell death); other phenotypes, such as immortalization, are novel. Tumor evolution is thought to adhere to Darwinian principles, with mutations arising randomly within an individual cell, followed by selection for mutant clones with favorable traits.Support for this idea stems from the observation that end-stage tumors have mutations in a number of genes. But linking mutations in particular genes with defined stages is difficult for most human cancers and especially so for the last and deadliest stage: when cancer disseminates throughout the body during metastasis. It's unclear, for example, whether there are mutations in particular genes or sets of genes that enhance metastasis. That is, do metastatic lesions develop through a continuation of Darwinian evolution, or is metastasis an intrinsic property of the primary tumor, meaning that further genetic evolution is not required? It is also unclear whether there is a “preferred” sequence of mutations, such that selective pressure for particular mutations depends on preexisting mutations.Since the earliest days of research on oncogenes—genes that can cause a cell to become cancerous—it has been known that certain oncogenic mutations cooperate to transform normal cells into cancer cells. For example, an “activating” mutation in the oncogene  Ras  and the loss of the tumor suppressor  p53  cooperate to transform cells.Rasp53The paper by Christopher Kemp and his colleagues at the Fred Hutchinson Cancer Research Center sheds light on some of these questions, and many of the issues center around the most notorious oncogene:  Ras . Using a well characterized mouse model of squamous cell carcinogenesis, which generates a form of skin cancer, the authors examine both the functional and evolutionary relationships between three cancer genes that play major roles in most human cancers:  Ras  and the tumor suppressors  Arf  and  p53 .RasRasArfp53Two seminal early observations set the stage: mutational activation of  Ras  is the initiating genetic event in this cancer model, while mutation of  p53  occurs later, during the benign to malignant transition; and expression of mutant  Ras  in cells activates  p53  via signaling through the protein encoded by  Arf .Rasp53Rasp53ArfKemp et al. confirm that this pathway is active in “autochthonous” tumors—which grow and develop where they are initiated—by showing that  p53  expression in tumors with  Ras  mutations is dependent on the presence of  Arf . Thus, during the early benign stages of tumor growth,  Ras  activates  Arf , which in turn activates  p53 , thereby inhibiting tumor progression. This provides strong selective pressure in favor of cells with mutations in either  Arf  or  p53 , and these mutations are indeed observed as the tumors progress to malignancy. That  Arf  and  p53  function as tumor suppressors was confirmed by demonstrated accelerated tumor progression in mice lacking either  Arf  or  p53 . This answers a longstanding question concerning the nature of the signal that activates  p53  during autochthonous tumor development: Mutation of  Ras  not only initiates tumor development but, through its intracellular signaling through  Arf  and  p53 , directly influences the subsequent evolutionary trajectory of the tumors. In this view, secondary evolutionary events are determined by the preexisting genetic lesion, as a result of direct signaling interactions.p53RasArfRasArfp53Arfp53Arfp53Arfp53p53RasArfp53The authors go on to show that tumors lacking  Arf  or  p53  show accelerated metastatic dissemination, a phenomenon rarely seen in mouse squamous cell cancer models. Thus both benign and malignant tumors lacking these tumor suppressors are at high risk for metastasis. As  Ras  is well known to confer many phenotypes required for the metastatic process, it appears that  Ras , together with loss of its inhibitors,  Arf  and  p53 , may be sufficient to drive this process. More direct evidence that metastasis does or does not require further genetic evolution awaits.Arfp53RasRasArfp53