**Effects of Recombinant Human Tumor Necrosis Factor-α and Its Combination with Native Human Leukocyte Interferon-α on P3-X63- Ag8.653 Mouse Myeloma Cell Growth**

Andrej Plesničar1, Gaj Vidmar2, Borut Štabuc3 and Blanka Kores Plesničar4 *1University of Ljubljana, Faculty of Health Sciences, Ljubljana, 2Institute for Rehabilitation, Ljubljana, 3University of Ljubljana, Faculty of Medicine, Ljubljana, 4University of Maribor, Faculty of Medicine, Maribor, Slovenia* 

#### **1. Introduction**

60 Multiple Myeloma – An Overview

Youn (2), J.I., Nagaraj, S., Collazo, M. & Gabrilovich, D.I. (2008). Subsets of myeloid-derived

Zheng, S.G., Wang, J. & Horwitz, D.A. (2008). Cutting edge: Foxp3+CD4+CD25+ regulatory

(October 2008), pp. 5791-5802, ISSN 1550-6606

suppressor cells in tumor-bearing mice. *Journal of Immunology*, Vol.181, No.8,

T cells induced by IL-2 and TGF-beta are resistant to Th17 conversion by IL-6. *Journal of Immunology*, Vol.180, No.11, (June 2008), pp. 7112-7116, ISSN 0022-1767

> Multiple myeloma (MM) is a malignant B-cell disease, characterized by uncontrolled proliferation of differentiated plasma cells in bone marrow (BM), osteolytic bone lesions, monoclonal protein peaks in serum or urine and suppression of normal antibody production. Patients with MM usually present with a number of clinical signs and symptoms, including fatigue, infection, severe bone pain, bone fractures, hypercalcaemia, and renal disease (Bommert et al., 2006; Raman et al., 2007; Redzepovic et al., 2008). Despite clinical responses produced by conventional chemotherapy, radiotherapy, and an increasing number of new compounds and improvements in supportive therapy, MM remains largely incurable (Katzel et al., 2007; Ozdemir et al., 2004; Redzepovic et al., 2008).

> Tumor necrosis factor-α (TNF-α) is a known survival and proliferation factor for myeloma cell lines. It is produced by tumor and stromal cells in BM of patients with MM and induces tumor cell proliferation, migration, survival, drug resistance, and blood vessel proliferation (Harrison et al., 2006; Jourdan et al., 1999). Although TNF-α secreted by MM cells does not induce significant growth and drug resistance in tumor cells, it stimulates interleukin-6 (IL-6) secretion in bone marrow stromal cells more potently than vascular endothelial growth factor (VEGF) or transforming growth factor-β (TGF-β) (Yasui et al., 2005). Out of BM environment, circulating TNF-α levels are increased in MM patients with manifest bone disease, whose osteoblasts constitutively overexpress receptors for TNF-related apoptosisinducing ligand, intercellular adhesion molecule-1 (ICAM-1), and monocyte chemotactic protein-1 (MCP-1) (Silvestris et al., 2004).

> In our previous study, treatment with native human leukocyte interferon-α (nhIFN-α), recombinant human interferon-α2a (rhIFN-α2a) and recombinant human interferon-α2b (rhIFN-α2b) in doses of 500 IU/ml, 1000/ml and 2000 IU/ml resulted in differential effects on P3-X63-Ag8.653 mouse myeloma cells. A statistically significant dose-dependent decrease in

Effects of Recombinant Human Tumor Necrosis Factor-α and Its Combination

atmosphere of 5% CO₂ for 48 hours.

phase in approximately 72 hours (three days).

exclusion in 24 hour intervals (days 1-4).

**2.3 Statistical analysis** 

**3. Results** 

**culture study groups** 

with Native Human Leukocyte Interferon-α on P3-X63-Ag8.653 Mouse Myeloma Cell Growth 63

modified Eagle's medium (Sigma-Aldrich, St. Louis, MO, USA), supplemented with 10% fetal calf serum (FCS) (Sigma-Aldrich, St. Louis, MO, USA) and gentamycin (Krka, tovarna zdravil, d. d., Novo Mesto, Slovenia). The cells were incubated at 37 °C in a humidified

In preparation for this study, P3-X63-Ag8.653 mouse myeloma cell growth curves on logarithmic scale plots were established when the most convenient seeding density to be used was determined. Various time zero values ranged from 5 X 103 to 6 X 104 cells/ml and S-shaped growth curves were observed after cell concentrations measured in 24 hour intervals over the 96 hours were plotted on Keuffel & Esser 464970 Semi-Logarithmic Grids general purpose drawing paper. Time zero density of 104 P3-X63-Ag8.653 mouse myeloma cells/ml was found to be the most appropriate for the study. With the use of Keuffel & Esser 464970 graph paper it was also possible to observe that P3-X63-Ag8.653 mouse myeloma cells started to enter the log phase in approximately 24 hours (one day) and the plateau

**2.2 Recombinant human tumor necrosis factor-α, native human interferon-α and cell** 

Actively growing P3-X63.Ag8.653 mouse myeloma cells were seeded into 35 mm Petri dishes (Becton Dickinson, Franklin Lakes, NJ, USA) and incubated in each study group with three different concentrations of rTNF-α (Prospecbio, East Brunswick, NJ, USA). In the first study group the cells were incubated with 2, 10 and 20 IU/ml of rTNF-α, in the second with 30, 40 and 50 IU/ml of rTNF-α, in the third with 100, 200 and 300 IU/ml of rTNF-α, and in the fourth study group with 400, 800 and 1200 IU/ml of rTNF-α. After the experiments with rTNF-α, in one study group the cells were incubated with a combination of 10 IU/ml of rTNF-α and 2000 IU/ML of nhIFN-α (Institute of Immunology Inc., Zagreb, Croatia). The combination was compared to the corresponding doses of single cytokines. Matched negative controls that consisted of P3-X63-Ag8.653 mouse myeloma cells cultured in the absence of cytokines were established for each of the different cytokine study groups. All experiments were replicated five times and 20 Petri dishes were used for each cytokine cell culture study group and their negative controls. Cell viability was assessed by Trypan blue

In proliferating cell lines, it is difficult to distinguish between early cell loss and prolonged lag phase in which cells are still adapting to their new environment (Wilson, 1994). The effects of different concentrations of rTNF-α and its combination with nhIFN-α were thus estimated with the use of whole growth curves to reduce the possibility of misinterpretation. Statistical evaluation was performed using SPSS® software package, version 12.0 (SPSS Inc., Chicago, IL, USA) for Windows®. Analysis of variance (ANOVA) was used to assess the differences between and within different treatment groups and their negative control groups. *P*-values of < 0.05 were considered to be statistically significant.

Treatment of P3-X63-Ag8.653 mouse myeloma cells with rTNF-α showed a statistically significant reduction in cell viability in comparison with negative control cells. The

cell viability was observed in P3-X63-Ag8.653 mouse myeloma cells treated with nhIFN-α in comparison with matched negative controls. Conversely, a statistically significant increase in cell viability was observed in P3-X63-Ag8.653 mouse myeloma cells treated with rhIFN-α2a and rhIFN-α2b. This increase in cell viability occurred only in relation to their matched negative controls and was not dose-dependent (Plesničar et al., 2009). The differences in effects on P3-X63-Ag8.653 mouse myeloma cell viability between nhIFN-α and recombinant interferons probably occurred because nhIFN-α is composed of many subtypes of nhIFN-α and also contains trace amounts of IFN-γ, TNF-α, TNF-β, interleukin (IL)-1α, IL-1β, IL-2, IL-6, granulocyte-macrophage colony-stimulating factor and platelet-derived growth factor. Therefore, the decrease of cell viability in nhIFN-α treated P3-X63-Ag8.653 mouse myeloma cell cultures may have occurred in consequence of a synergistic effect of the various cytokines in nhIFN-α preparation. The quantities and the synergistic effect of the cytokines in nhIFN-α preparation are very small at lower concentrations and probably become active only at higher concentrations, thus accounting for the dose-dependent effects observed on cell growth (Plesničar et al., 2009; Šantak et al., 2007, Zidovec & Mažuran, 1999). In contrast to nhIFN-α, rhIFN-α2a and rhIFN-α2b are each preparations of only one subtype of IFN-α. The increase in cell viability in P3-X63-Ag8.653 mouse myeloma cell culture groups treated with rhIFN-α2a and rhIFN-α2b in our study was in accordance with a number of reports suggesting that IFN-α could induce uncontrolled cell proliferation in some patients with MM (Plesničar et al., 2009; Puthier et al., 2001; Sawamura et al., 1992). Interferon-α has been recognized as a survival factor in MM in some studies, the data supporting this claim are based on the results of studies using recombinant interferons-α (Cheriyath et al., 2007; Ferlin-Bezombes et al., 1998; Puthier et al., 2001).

The P3-X63-Ag8.653 mouse myeloma cell line is routinely cultured in several types of growth media. The cells in P3-X63-Ag8.653 mouse myeloma cell line propagate in suspension and do not secrete immunoglobulin. They can be used as fusion partners for producing hybridomas and show lymphocyte-like morphology (Kearney et al., 1979). Human myeloma blood cells were described as carrying surface membrane monoclonal or idiotypic immunoglobulin structures, and were morphologically classified as atypical small to medium-sized lymphocytes, lymphoblasts, lymphoplasmacytoid, plasmacytoid cells or myeloma cells (Mellstedt et al., 1984). With regard to morphology and despite the differences, it may be possible that P3-X63-Ag8.653 mouse myeloma cells, growing in suspension cell cultures, share at least some common properties with circulating clonogenic CD19 positive and CD138 negative cells, described as phenotypically resembling mature B cells (Cremer et al., 2001; Matsui et al., 2004).

The aim of the present study was to compare the effects of different doses of rTNF-α on the *in-vitro* growth of P3-X63-Ag8.653 mouse myeloma cells. Additionally, in one cell culture study group the aim was also to compare the effect of a combination of rTNF-α and nhIFNα with the effects of corresponding doses of single cytokines on the *in-vitro* growth of P3- X63-Ag8.653 mouse myeloma cells.

#### **2. Materials and methods**

#### **2.1 P3-X63-Ag8.653 mouse myeloma cell preparation**

The P3-X63-Ag8.653 mouse myeloma cells were retrieved from the frozen storage at -80 °C and cultured in 25 cm² cell culture flasks (Cole Parmer, Vernon Hills, IL, USA) in Dulbecco's modified Eagle's medium (Sigma-Aldrich, St. Louis, MO, USA), supplemented with 10% fetal calf serum (FCS) (Sigma-Aldrich, St. Louis, MO, USA) and gentamycin (Krka, tovarna zdravil, d. d., Novo Mesto, Slovenia). The cells were incubated at 37 °C in a humidified atmosphere of 5% CO₂ for 48 hours.

In preparation for this study, P3-X63-Ag8.653 mouse myeloma cell growth curves on logarithmic scale plots were established when the most convenient seeding density to be used was determined. Various time zero values ranged from 5 X 103 to 6 X 104 cells/ml and S-shaped growth curves were observed after cell concentrations measured in 24 hour intervals over the 96 hours were plotted on Keuffel & Esser 464970 Semi-Logarithmic Grids general purpose drawing paper. Time zero density of 104 P3-X63-Ag8.653 mouse myeloma cells/ml was found to be the most appropriate for the study. With the use of Keuffel & Esser 464970 graph paper it was also possible to observe that P3-X63-Ag8.653 mouse myeloma cells started to enter the log phase in approximately 24 hours (one day) and the plateau phase in approximately 72 hours (three days).

#### **2.2 Recombinant human tumor necrosis factor-α, native human interferon-α and cell culture study groups**

Actively growing P3-X63.Ag8.653 mouse myeloma cells were seeded into 35 mm Petri dishes (Becton Dickinson, Franklin Lakes, NJ, USA) and incubated in each study group with three different concentrations of rTNF-α (Prospecbio, East Brunswick, NJ, USA). In the first study group the cells were incubated with 2, 10 and 20 IU/ml of rTNF-α, in the second with 30, 40 and 50 IU/ml of rTNF-α, in the third with 100, 200 and 300 IU/ml of rTNF-α, and in the fourth study group with 400, 800 and 1200 IU/ml of rTNF-α. After the experiments with rTNF-α, in one study group the cells were incubated with a combination of 10 IU/ml of rTNF-α and 2000 IU/ML of nhIFN-α (Institute of Immunology Inc., Zagreb, Croatia). The combination was compared to the corresponding doses of single cytokines. Matched negative controls that consisted of P3-X63-Ag8.653 mouse myeloma cells cultured in the absence of cytokines were established for each of the different cytokine study groups. All experiments were replicated five times and 20 Petri dishes were used for each cytokine cell culture study group and their negative controls. Cell viability was assessed by Trypan blue exclusion in 24 hour intervals (days 1-4).

#### **2.3 Statistical analysis**

In proliferating cell lines, it is difficult to distinguish between early cell loss and prolonged lag phase in which cells are still adapting to their new environment (Wilson, 1994). The effects of different concentrations of rTNF-α and its combination with nhIFN-α were thus estimated with the use of whole growth curves to reduce the possibility of misinterpretation. Statistical evaluation was performed using SPSS® software package, version 12.0 (SPSS Inc., Chicago, IL, USA) for Windows®. Analysis of variance (ANOVA) was used to assess the differences between and within different treatment groups and their negative control groups. *P*-values of < 0.05 were considered to be statistically significant.

#### **3. Results**

62 Multiple Myeloma – An Overview

cell viability was observed in P3-X63-Ag8.653 mouse myeloma cells treated with nhIFN-α in comparison with matched negative controls. Conversely, a statistically significant increase in cell viability was observed in P3-X63-Ag8.653 mouse myeloma cells treated with rhIFN-α2a and rhIFN-α2b. This increase in cell viability occurred only in relation to their matched negative controls and was not dose-dependent (Plesničar et al., 2009). The differences in effects on P3-X63-Ag8.653 mouse myeloma cell viability between nhIFN-α and recombinant interferons probably occurred because nhIFN-α is composed of many subtypes of nhIFN-α and also contains trace amounts of IFN-γ, TNF-α, TNF-β, interleukin (IL)-1α, IL-1β, IL-2, IL-6, granulocyte-macrophage colony-stimulating factor and platelet-derived growth factor. Therefore, the decrease of cell viability in nhIFN-α treated P3-X63-Ag8.653 mouse myeloma cell cultures may have occurred in consequence of a synergistic effect of the various cytokines in nhIFN-α preparation. The quantities and the synergistic effect of the cytokines in nhIFN-α preparation are very small at lower concentrations and probably become active only at higher concentrations, thus accounting for the dose-dependent effects observed on cell growth (Plesničar et al., 2009; Šantak et al., 2007, Zidovec & Mažuran, 1999). In contrast to nhIFN-α, rhIFN-α2a and rhIFN-α2b are each preparations of only one subtype of IFN-α. The increase in cell viability in P3-X63-Ag8.653 mouse myeloma cell culture groups treated with rhIFN-α2a and rhIFN-α2b in our study was in accordance with a number of reports suggesting that IFN-α could induce uncontrolled cell proliferation in some patients with MM (Plesničar et al., 2009; Puthier et al., 2001; Sawamura et al., 1992). Interferon-α has been recognized as a survival factor in MM in some studies, the data supporting this claim are based on the results of studies using recombinant interferons-α (Cheriyath et al., 2007; Ferlin-Bezombes et al., 1998; Puthier et

The P3-X63-Ag8.653 mouse myeloma cell line is routinely cultured in several types of growth media. The cells in P3-X63-Ag8.653 mouse myeloma cell line propagate in suspension and do not secrete immunoglobulin. They can be used as fusion partners for producing hybridomas and show lymphocyte-like morphology (Kearney et al., 1979). Human myeloma blood cells were described as carrying surface membrane monoclonal or idiotypic immunoglobulin structures, and were morphologically classified as atypical small to medium-sized lymphocytes, lymphoblasts, lymphoplasmacytoid, plasmacytoid cells or myeloma cells (Mellstedt et al., 1984). With regard to morphology and despite the differences, it may be possible that P3-X63-Ag8.653 mouse myeloma cells, growing in suspension cell cultures, share at least some common properties with circulating clonogenic CD19 positive and CD138 negative cells, described as phenotypically resembling mature B

The aim of the present study was to compare the effects of different doses of rTNF-α on the *in-vitro* growth of P3-X63-Ag8.653 mouse myeloma cells. Additionally, in one cell culture study group the aim was also to compare the effect of a combination of rTNF-α and nhIFNα with the effects of corresponding doses of single cytokines on the *in-vitro* growth of P3-

The P3-X63-Ag8.653 mouse myeloma cells were retrieved from the frozen storage at -80 °C and cultured in 25 cm² cell culture flasks (Cole Parmer, Vernon Hills, IL, USA) in Dulbecco's

al., 2001).

cells (Cremer et al., 2001; Matsui et al., 2004).

**2.1 P3-X63-Ag8.653 mouse myeloma cell preparation** 

X63-Ag8.653 mouse myeloma cells.

**2. Materials and methods** 

Treatment of P3-X63-Ag8.653 mouse myeloma cells with rTNF-α showed a statistically significant reduction in cell viability in comparison with negative control cells. The

Effects of Recombinant Human Tumor Necrosis Factor-α and Its Combination

identical (Figures 1-5).

with Native Human Leukocyte Interferon-α on P3-X63-Ag8.653 Mouse Myeloma Cell Growth 65

whole 96 hours (four days) period over which cell viability was measured, the growth curves were S-shaped. P3-X63-Ag8.653 mouse myeloma cells started to enter the log phase at approximately 24 hours (one day) and reached the plateau phase at approximately 72 hours (three days) from incubation with the different concentrations of rTNF-α and with the combination of rTNF-α and nhIFN-α. The intermediate portions (log phase) of the S-shaped growth curves, approximately between 24 and 72 hours, were linear. The slopes of the growth curves in the treated cell culture study groups and their negative controls were not

Fig. 1. The effect of 2, 10 and 20 IU/ml of human recombinant TNF-α (rTNF-α) on *in-vitro* P3-X63-Ag8.653 mouse myeloma cell growth plotted on a logarithmic scale (logarithmic number of P3-X63-Ag8.653 cells/ml), showing a dose-dependent reduction in cell viability over four days of treatment. The reduction in cell growth observed with rTNF-α was

statistically significant in comparison with negative control (*P* = 0.001). Legend: black, day 0;

green, day 1; light blue, day 2; dark blue, day 3; violet, day 4.

reduction in cell viability occurred in dose-dependent manner, with higher doses having a greater effect (Table 1, Figures 1-4). Treatment of P3-X63-Ag8.653 mouse myeloma cells with 400, 800 and 1200 IU/ml of rTNF-α showed a complete cessation of cell growth (Table 1), with the cells being unable to enter the log phase of the S-shaped growth curve (Figure 4). Treatment of P3-X63-Ag8.653 mouse myeloma cells with a combination of rTNF-α and nhIFN-α showed a statistically significant reduction in cell viability in comparison with negative control cells and cells treated exclusively with either rTNF-α or nhIFN-α. The addition of a small dose of rTNF-α (10 IU/ml) to the treatment of P3-X63-Ag8.653 mouse myeloma cells with a relatively high dose of nhIFN-α (2000 IU/ml) resulted in a further, although small, reduction in cell viability (Table 1, Figure 5).


**<sup>1</sup>**Comparison between active treatment overall and the corresponding negative control in each cell study group.

Table 1. Effect of recombinant human tumor necrosis factor-α (rTNF-α) at different concentrations, native human interferon-α (nhIFN-α) and the combination of rTNF-α and nhIFN-α on *in-vitro* P3-X63-Ag8.653 mouse myeloma cell growth.

As expected, when the cell numbers for each cell study group of P3-X63-Ag8.653 mouse myeloma cells treated with the different concentrations of rTNF-α and with the combination of rTNF-α and nhIFN-α or their negative controls were plotted on a logarithmic scale for the

reduction in cell viability occurred in dose-dependent manner, with higher doses having a greater effect (Table 1, Figures 1-4). Treatment of P3-X63-Ag8.653 mouse myeloma cells with 400, 800 and 1200 IU/ml of rTNF-α showed a complete cessation of cell growth (Table 1), with the cells being unable to enter the log phase of the S-shaped growth curve (Figure 4). Treatment of P3-X63-Ag8.653 mouse myeloma cells with a combination of rTNF-α and nhIFN-α showed a statistically significant reduction in cell viability in comparison with negative control cells and cells treated exclusively with either rTNF-α or nhIFN-α. The addition of a small dose of rTNF-α (10 IU/ml) to the treatment of P3-X63-Ag8.653 mouse myeloma cells with a relatively high dose of nhIFN-α (2000 IU/ml) resulted in a further,

> **Cytokine concentration (IU/ml)**

**rTNF-α (1)** Negative control 20.400 +/- 0.83306 *P* = 0.001 **rTNF-α** 2 20.187 +/- 1.65707 **rTNF-α** 10 16.675 +/- 1.99607 **rTNF-α** 20 13.425 +/- 0.54608 **rTNF-α (2)** Negative control 23.3250 +/- 1.96338 *P* = 0.000

> **rTNF-α** 30 12.8750 +/- 0.55234 **rTNF-α** 40 10.4250 +/- 0.71709

**rTNF-α** 50 9.3750 +/- 0.20444 **rTNF-α (3)** Negative control 22.9000 +/- 0.79096 *P* = 0.000 **rTNF-α** 100 8.5125 +/- 0.32918 **rTNF-α** 200 5.9625 +/- 0.34403 **rTNF-α** 300 1.6750 +/- 0.07500 **rTNF-α (4)** Negative control 22.2000 +/- 1.16011 *P* = 0.000 **rTNF-α** 400 1.0375 +/- 0.05078 **rTNF-α** 800 0.9250 +/- 0.06673 **rTNF-α** 1200 0.6750 +/- 0.03644

**nhIFN-α** Negative control 25.9625 +/- 0.62581 *<sup>P</sup>* = 0.000

**nhIFN-α** 2000 5.8000 +/- 0.10346

**nhIFN-<sup>α</sup>** 10 and 2000 4.8500 +/- 0.35609

As expected, when the cell numbers for each cell study group of P3-X63-Ag8.653 mouse myeloma cells treated with the different concentrations of rTNF-α and with the combination of rTNF-α and nhIFN-α or their negative controls were plotted on a logarithmic scale for the

**<sup>1</sup>**Comparison between active treatment overall and the corresponding negative control in each cell

Table 1. Effect of recombinant human tumor necrosis factor-α (rTNF-α) at different concentrations, native human interferon-α (nhIFN-α) and the combination of rTNF-α and

nhIFN-α on *in-vitro* P3-X63-Ag8.653 mouse myeloma cell growth.

**rTNF-α** 10 19.6375 +/- 1.07591

**No. of cells (104/ml) Mean +/- SE over days 0-4** 

**Statistical significance1**

although small, reduction in cell viability (Table 1, Figure 5).

**rTNF-α** and

**Cell study** 

**rTNF-α** and

study group.

**group Cytokine type** 

whole 96 hours (four days) period over which cell viability was measured, the growth curves were S-shaped. P3-X63-Ag8.653 mouse myeloma cells started to enter the log phase at approximately 24 hours (one day) and reached the plateau phase at approximately 72 hours (three days) from incubation with the different concentrations of rTNF-α and with the combination of rTNF-α and nhIFN-α. The intermediate portions (log phase) of the S-shaped growth curves, approximately between 24 and 72 hours, were linear. The slopes of the growth curves in the treated cell culture study groups and their negative controls were not identical (Figures 1-5).

Fig. 1. The effect of 2, 10 and 20 IU/ml of human recombinant TNF-α (rTNF-α) on *in-vitro* P3-X63-Ag8.653 mouse myeloma cell growth plotted on a logarithmic scale (logarithmic number of P3-X63-Ag8.653 cells/ml), showing a dose-dependent reduction in cell viability over four days of treatment. The reduction in cell growth observed with rTNF-α was statistically significant in comparison with negative control (*P* = 0.001). Legend: black, day 0; green, day 1; light blue, day 2; dark blue, day 3; violet, day 4.

Effects of Recombinant Human Tumor Necrosis Factor-α and Its Combination

with Native Human Leukocyte Interferon-α on P3-X63-Ag8.653 Mouse Myeloma Cell Growth 67

Fig. 3. The effect of 100, 200 and 300 IU/ml of human recombinant TNF-α (rTNF-α) on *in-*

(logarithmic number of P3-X63-Ag8.653 cells/ml), showing a dose-dependent reduction in cell viability over four days of treatment. The reduction in cell growth observed with rTNFα was statistically significant in comparison with negative control *(P* = 0.000). Legend: black,

*vitro* P3-X63-Ag8.653 mouse myeloma cell growth plotted on a logarithmic scale

day 0; green, day 1; blue, day 2; light violet, day 3; dark violet, day 4.

Fig. 2. The effect of 30, 40 and 50 IU/ml of human recombinant tumor necrosis factor-α (rTNF-α) on *in-vitro* P3-X63-Ag8.653 mouse myeloma cell growth plotted on a logarithmic scale (logarithmic number of P3-X63-Ag8.653 cells/ml), showing a dose-dependent reduction in cell viability over four days of treatment. The reduction in cell growth observed with rTNF-α was statistically significant in comparison with negative control *(P* = 0.000). Legend: black, day 0; green, day 1; light blue, day 2; dark blue, day 3; violet, day 4.

Fig. 2. The effect of 30, 40 and 50 IU/ml of human recombinant tumor necrosis factor-α (rTNF-α) on *in-vitro* P3-X63-Ag8.653 mouse myeloma cell growth plotted on a logarithmic scale (logarithmic number of P3-X63-Ag8.653 cells/ml), showing a dose-dependent

reduction in cell viability over four days of treatment. The reduction in cell growth observed with rTNF-α was statistically significant in comparison with negative control *(P* = 0.000). Legend: black, day 0; green, day 1; light blue, day 2; dark blue, day 3; violet, day 4.

Fig. 3. The effect of 100, 200 and 300 IU/ml of human recombinant TNF-α (rTNF-α) on *invitro* P3-X63-Ag8.653 mouse myeloma cell growth plotted on a logarithmic scale (logarithmic number of P3-X63-Ag8.653 cells/ml), showing a dose-dependent reduction in cell viability over four days of treatment. The reduction in cell growth observed with rTNFα was statistically significant in comparison with negative control *(P* = 0.000). Legend: black, day 0; green, day 1; blue, day 2; light violet, day 3; dark violet, day 4.

Effects of Recombinant Human Tumor Necrosis Factor-α and Its Combination

with Native Human Leukocyte Interferon-α on P3-X63-Ag8.653 Mouse Myeloma Cell Growth 69

Fig. 5. The effect of 10 IU/ml of human recombinant TNF-α (rTNF-α), 2000 IU/ML of native human leukocyte interferon-α (nhIFN-α), and of a combination of 10 IU/ml of rTNF-α and 2000 IU/ML of nhIFN-α on *in-vitro* P3-X63-Ag8.653 mouse myeloma cell growth plotted on a logarithmic scale (logarithmic number of P3-X63-Ag8.653 cells/ml), showing a reduction in cell viability over four days of treatment. The reduction in cell growth observed after treatment with rTNF-α, nhIFN-α, and with the combination of rTNF-α and nhIFN-α, was statistically significant in comparison with negative control (*P* = 0.000). Legend: black, day 0;

Treatment with rTNF-α at different doses had a negative effect on *in vitro* P3-X63-Ag8.653 mouse myeloma cell growth. A statistically significant dose-dependent reduction in cell viability was observed in P3-X63-Ag8.653 mouse myeloma cells treated with rTNF-α in comparison with negative controls. Additionally, a slightly enhanced reduction in P3-X63- Ag8.653 mouse myeloma cell viability was observed in cells treated with the combination of rTNF-α and nhIFN-α, in comparison with negative controls and cells treated exclusively

The results of this study are surprising, as the treatment of P3-X63-Ag8.653 mouse myeloma with rTNF-α showed statistically significant reduction in cell viability compared with untreated control cells, with higher doses having greater effect. These results are in contradiction with numerous reports describing TNF-α as a survival and proliferation factor in MM (Harrison et al., 2006; Hideshima et al., 2004; Jourdan et al., 1999; Yasui et al., 2006;

green, day 1; light blue, day 2; dark blue, day 3; violet, day 4.

**4. Discussion** 

with either rTNF-α or nhIFN-α.

Fig. 4. The effect of 400, 800 and 1200 IU/ml of human recombinant TNF-α (rTNF-α) on *invitro* P3-X63-Ag8.653 mouse myeloma cell growth plotted on a logarithmic scale (logarithmic number of P3-X63-Ag8.653 cells/ml), showing a dose-dependent reduction in cell viability over four days of treatment. The reduction in cell growth observed with rTNF-α was statistically significant in comparison with negative control (*P* = 0.000). Legend: black, day 0; green, day 1; light blue, day 2; dark blue, day 3; violet, day 4.

Fig. 5. The effect of 10 IU/ml of human recombinant TNF-α (rTNF-α), 2000 IU/ML of native human leukocyte interferon-α (nhIFN-α), and of a combination of 10 IU/ml of rTNF-α and 2000 IU/ML of nhIFN-α on *in-vitro* P3-X63-Ag8.653 mouse myeloma cell growth plotted on a logarithmic scale (logarithmic number of P3-X63-Ag8.653 cells/ml), showing a reduction in cell viability over four days of treatment. The reduction in cell growth observed after treatment with rTNF-α, nhIFN-α, and with the combination of rTNF-α and nhIFN-α, was statistically significant in comparison with negative control (*P* = 0.000). Legend: black, day 0; green, day 1; light blue, day 2; dark blue, day 3; violet, day 4.

#### **4. Discussion**

68 Multiple Myeloma – An Overview

Fig. 4. The effect of 400, 800 and 1200 IU/ml of human recombinant TNF-α (rTNF-α) on *in-*

(logarithmic number of P3-X63-Ag8.653 cells/ml), showing a dose-dependent reduction in cell viability over four days of treatment. The reduction in cell growth observed with rTNF-α was statistically significant in comparison with negative control (*P* = 0.000). Legend: black, day 0; green, day 1; light blue, day 2; dark blue, day 3; violet, day 4.

*vitro* P3-X63-Ag8.653 mouse myeloma cell growth plotted on a logarithmic scale

Treatment with rTNF-α at different doses had a negative effect on *in vitro* P3-X63-Ag8.653 mouse myeloma cell growth. A statistically significant dose-dependent reduction in cell viability was observed in P3-X63-Ag8.653 mouse myeloma cells treated with rTNF-α in comparison with negative controls. Additionally, a slightly enhanced reduction in P3-X63- Ag8.653 mouse myeloma cell viability was observed in cells treated with the combination of rTNF-α and nhIFN-α, in comparison with negative controls and cells treated exclusively with either rTNF-α or nhIFN-α.

The results of this study are surprising, as the treatment of P3-X63-Ag8.653 mouse myeloma with rTNF-α showed statistically significant reduction in cell viability compared with untreated control cells, with higher doses having greater effect. These results are in contradiction with numerous reports describing TNF-α as a survival and proliferation factor in MM (Harrison et al., 2006; Hideshima et al., 2004; Jourdan et al., 1999; Yasui et al., 2006;

Effects of Recombinant Human Tumor Necrosis Factor-α and Its Combination

consequently in important cost savings (Drexler & Matsuo, 2000).

patients' own cytokine-producing cells become available.

No.2, (February 2010), pp. 116-120, ISSN 1875-5992

cells in such patients.

**5. Conclusion** 

**6. References** 

9232

0268-960X

to obtain further information.

with Native Human Leukocyte Interferon-α on P3-X63-Ag8.653 Mouse Myeloma Cell Growth 71

associated with poor prognosis (Alexandrakis et al., 2004; Fillela et al., 1996; Jourdan et al., 1999). Hypothetically, it may be possible to speculate that the increased serum levels of TNF-α in patients with active MM represent a part of a complex negative control loop mechanism that regulates and negatively affects the quantity of circulating clonogenic B-

A heterologous system was used to evaluate the effects of rTNF-α and its combination with nHIFN-α on MM cells *in-vitro*. Recombinant human tumor necrosis factor-α and nHIFN-α used in this study were active in P3-X63-Ag8.653 mouse myeloma cells, again confirming the observations that cytokines synthesized in cells of one species may have a considerable effect in cells of another closely related species (Desmyter et al., 1968; Greenberg & Mosny, 1977; Ozdemir et al., 2004). Moreover, MM cells are difficult to grow in vitro (Barker et al., 1993). An important advantage of the P3-X63-Ag8.653 mouse myeloma cell line may also lie in its easy reproducibility, unlimited supply, infinite storability and recoverability, and

The results of this study point to the importance of the study of differential effects TNF-α may exert on malignant cells in MM during specific phases of their development and differentiation. It is possible that TNF-α may have a role in future carefully planned personalized therapy approaches based on genetic features, age, and other risk factors in patients with MM (Durie, 2008; Ludwig et al., 2008). Such therapy could perhaps include patients' own TNF-α, IFN-α, other substances and their combinations, provided that effective procedures for the establishment and maintenance of *ex vivo* cell cultures of

The results of this study point to the importance of assessing the role of TNF-α in study and therapy of MM. Additional studies with other cytokines and human MM cells are required

Agarwal, J.A. & Matsui, W. (2010). Multiple Myeloma: A Paradigm for Translation of the

Alexandrakis, M.G., Passam, F.J., Ganotakis, E., Dafnis, E., Dambaki, C., Konsolas, J.,

Barker, H.F., Ball, J., Drew, M. & Franklin, I.M. (1993). Multiple Myeloma: The Biology of

*Medicine*, Vol.42, No.10, (October 2004), pp. 1122-1126, ISSN 1434-6621 Baker, S.J. & Reddy, E.P. (1996). Transducers of life and death: TNF Receptor Superfamily

Cancer Stem Cell Hypothesis. *Anti-Cancer Agents in Medicinal Chemistry*, Vol.10,

Kyriakou, D.S. & Stathopoulos, E. (2004). Bone Marrow Microvascular Density and Angiogenic Growth Factors in Multiple Myeloma. *Clinical Chemistry and Laboratory* 

and Associated Proteins. *Oncogene*, Vol.12, No.1, (January 1996), pp. 1-9, ISSN 0950-

Malignant Plasma Cells. *Blood Reviews*, Vol.7, No.1 (March 1993), pp.19-23. ISSN

Westendorf et al., 1996). However, TNF-α has previously also been described as an apoptotic factor in MM. The TNF-dependent trimerization of TNF receptors may lead to the recruitment of TRADD (TNF-R1 associated death domain protein), FADD (Fas-associated death domain protein) or RIP (receptor interacting protein) adapter proteins, resulting in activation and acceleration of caspase cascade (Baker & Reddy, 1996; Dai et al., 2003; Jourdan et al., 1999). This mechanism may further lead to apoptosis in MM cells (Jourdan et al., 1999).

Treatment of P3-X63-Ag8.653 mouse myeloma cells with the combination of rTNF-α and nHIFN-α resulted in an enhancement of the reduction in cell viability in comparison with negative control cells and cells treated exclusively with either rTNF-α or nhIFN-α. The nHIFN-α used in this study contains traces of a number of other cytokines produced by human peripheral blood leukocytes infected by Sendai virus (Šantak et al., 2007, Zidovec & Mažuran, 1999). The differences between the slopes of the S-shaped growth curves in the rTNF-α treated P3-X63-Ag8.653 mouse myeloma cell cultures and their controls, and equally prominent differences between the slopes of the growth curves of cells treated with the combination of rTNF-α and nHIFN-α and corresponding doses of single cytokines and their controls, may indicate that the active mechanisms associated with rTNF-α and nHIFN-α, and involved in reduction of cell viability, share some similarities and may possibly benefit from the synergy between rTNF-α, various subtypes of IFN-α and the small amounts of a number of other cytokines in the nHIFN-α preparation (Desmyter et al., 1968; Plesničar et al., 2009). In this context, it would be interesting to identify whether TNF-α and IFN-α share any signaling pathways leading to the reduction in MM cell viability and MM cell death.

Contrary to expectations, in this study treatment of P3-X63-Ag8.653 mouse myeloma cells with rTNF-α showed no increase, but a significant dose-dependent reduction in their cell viability. The P3-X63-Ag8.653 mouse myeloma cells propagate in suspension and show lymphocyte-like morphology (Kearney et al., 1979), and with this in mind, these cells may perhaps be useful in assessment of the effects rTNF-α may have on the growth of clonogenic B-cells in blood of patients with MM. Clonogenic B-cells represent the proliferating compartment in MM and possibly also a biologically distinct, drug-resistant MM progenitor population responsible for cell growth in tumor relapse after the treatment (Matsui et al., 2004; Matsui et al., 2008). In comparison to terminally differentiated plasma cells in MM, clonogenic B-cells appear to be relatively resistant to a number of anti-cancer agents, including dexamethasone, bortezomib, lenalidomide, and 4-hydroxycyclophosphamide (Agarwal & Matsui, 2010; Matsui et al., 2008). Possible similarities between P3-X63-Ag8.653 mouse myeloma cells and clonogenic B-cells in patients with MM, and because clonogenic B-cells are insensitive to standard cytotoxic chemotherapy and dexamethasone (Matsui et al., 2008), render the results observed in this study quite intriguing.

It is known that the activity of TNF-α as a survival and proliferation factor for MM is a part of a complex network of interactions between MM plasma cells, stromal cells and other cells in BM (Jourdan et al., 1999; Matsui et al., 2008). In this *in vitro* study, P3-X63-Ag8.653 mouse myeloma cells were grown in suspension culture, probably resembling the circumstances in which clonogenic B-cells in patients with MM grow without influences of BM microenvironment (Matsui et al., 2008). In a number of studies, serum levels of TNF-α were shown to be increased in patients with active MM and manifest bone disease, and to be associated with poor prognosis (Alexandrakis et al., 2004; Fillela et al., 1996; Jourdan et al., 1999). Hypothetically, it may be possible to speculate that the increased serum levels of TNF-α in patients with active MM represent a part of a complex negative control loop mechanism that regulates and negatively affects the quantity of circulating clonogenic Bcells in such patients.

A heterologous system was used to evaluate the effects of rTNF-α and its combination with nHIFN-α on MM cells *in-vitro*. Recombinant human tumor necrosis factor-α and nHIFN-α used in this study were active in P3-X63-Ag8.653 mouse myeloma cells, again confirming the observations that cytokines synthesized in cells of one species may have a considerable effect in cells of another closely related species (Desmyter et al., 1968; Greenberg & Mosny, 1977; Ozdemir et al., 2004). Moreover, MM cells are difficult to grow in vitro (Barker et al., 1993). An important advantage of the P3-X63-Ag8.653 mouse myeloma cell line may also lie in its easy reproducibility, unlimited supply, infinite storability and recoverability, and consequently in important cost savings (Drexler & Matsuo, 2000).

The results of this study point to the importance of the study of differential effects TNF-α may exert on malignant cells in MM during specific phases of their development and differentiation. It is possible that TNF-α may have a role in future carefully planned personalized therapy approaches based on genetic features, age, and other risk factors in patients with MM (Durie, 2008; Ludwig et al., 2008). Such therapy could perhaps include patients' own TNF-α, IFN-α, other substances and their combinations, provided that effective procedures for the establishment and maintenance of *ex vivo* cell cultures of patients' own cytokine-producing cells become available.

#### **5. Conclusion**

70 Multiple Myeloma – An Overview

Westendorf et al., 1996). However, TNF-α has previously also been described as an apoptotic factor in MM. The TNF-dependent trimerization of TNF receptors may lead to the recruitment of TRADD (TNF-R1 associated death domain protein), FADD (Fas-associated death domain protein) or RIP (receptor interacting protein) adapter proteins, resulting in activation and acceleration of caspase cascade (Baker & Reddy, 1996; Dai et al., 2003; Jourdan et al., 1999). This mechanism may further lead to apoptosis in MM cells (Jourdan et

Treatment of P3-X63-Ag8.653 mouse myeloma cells with the combination of rTNF-α and nHIFN-α resulted in an enhancement of the reduction in cell viability in comparison with negative control cells and cells treated exclusively with either rTNF-α or nhIFN-α. The nHIFN-α used in this study contains traces of a number of other cytokines produced by human peripheral blood leukocytes infected by Sendai virus (Šantak et al., 2007, Zidovec & Mažuran, 1999). The differences between the slopes of the S-shaped growth curves in the rTNF-α treated P3-X63-Ag8.653 mouse myeloma cell cultures and their controls, and equally prominent differences between the slopes of the growth curves of cells treated with the combination of rTNF-α and nHIFN-α and corresponding doses of single cytokines and their controls, may indicate that the active mechanisms associated with rTNF-α and nHIFN-α, and involved in reduction of cell viability, share some similarities and may possibly benefit from the synergy between rTNF-α, various subtypes of IFN-α and the small amounts of a number of other cytokines in the nHIFN-α preparation (Desmyter et al., 1968; Plesničar et al., 2009). In this context, it would be interesting to identify whether TNF-α and IFN-α share any signaling pathways leading to the reduction in MM cell viability and MM cell death.

Contrary to expectations, in this study treatment of P3-X63-Ag8.653 mouse myeloma cells with rTNF-α showed no increase, but a significant dose-dependent reduction in their cell viability. The P3-X63-Ag8.653 mouse myeloma cells propagate in suspension and show lymphocyte-like morphology (Kearney et al., 1979), and with this in mind, these cells may perhaps be useful in assessment of the effects rTNF-α may have on the growth of clonogenic B-cells in blood of patients with MM. Clonogenic B-cells represent the proliferating compartment in MM and possibly also a biologically distinct, drug-resistant MM progenitor population responsible for cell growth in tumor relapse after the treatment (Matsui et al., 2004; Matsui et al., 2008). In comparison to terminally differentiated plasma cells in MM, clonogenic B-cells appear to be relatively resistant to a number of anti-cancer agents, including dexamethasone, bortezomib, lenalidomide, and 4-hydroxycyclophosphamide (Agarwal & Matsui, 2010; Matsui et al., 2008). Possible similarities between P3-X63-Ag8.653 mouse myeloma cells and clonogenic B-cells in patients with MM, and because clonogenic B-cells are insensitive to standard cytotoxic chemotherapy and dexamethasone (Matsui et

It is known that the activity of TNF-α as a survival and proliferation factor for MM is a part of a complex network of interactions between MM plasma cells, stromal cells and other cells in BM (Jourdan et al., 1999; Matsui et al., 2008). In this *in vitro* study, P3-X63-Ag8.653 mouse myeloma cells were grown in suspension culture, probably resembling the circumstances in which clonogenic B-cells in patients with MM grow without influences of BM microenvironment (Matsui et al., 2008). In a number of studies, serum levels of TNF-α were shown to be increased in patients with active MM and manifest bone disease, and to be

al., 2008), render the results observed in this study quite intriguing.

al., 1999).

The results of this study point to the importance of assessing the role of TNF-α in study and therapy of MM. Additional studies with other cytokines and human MM cells are required to obtain further information.

#### **6. References**


Effects of Recombinant Human Tumor Necrosis Factor-α and Its Combination

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**4** 

*Republic of Korea* 

**Cellular Immunotherapy Using Dendritic** 

*Chonnam National University Hwasun Hospital, Hwasun, Jeollanamdo,* 

Je-Jung Lee1,2,3, Youn-Kyung Lee1,3 and Thanh-Nhan Nguyen-Pham1,2

Multiple myeloma (MM) is a clonal B cell malignant disease that is characterized by the proliferation of plasma cells in the bone marrow (BM) in association with monoclonal protein in the serum and/or urine, immune paresis, skeletal destruction, renal dysfunction, anemia, hypercalcemia and lytic bone diseases (Kyle & Rajkumar, 2004; Sirohi & Powles, 2004). Although the introduction of conventional chemotherapy, high-dose therapy with hematopoietic stem cell transplantation (HSCT), and the development of novel molecular target agents has resulted in a marked improvement in overall survival, the disease still remains incurable (Attal & Harousseau, 2009; Lonial & Cavenagh, 2009). Alternative approaches are clearly needed to prolong the disease-free survival as well as the overall survival of patients with MM. To prolong the survival of patients with MM who are undergoing allogeneic HSCT, donor lymphocyte infusion can be used successfully as a salvage therapy, which is based on the graft-versus myeloma effect in some cases of MM that relapse after allogeneic HSCT (Harrison & Cook, 2005; Perez-Simon et al., 2003). This role of immune effector cells provides the framework for the development of immune-based therapeutic options that use antigen-presenting cells (APCs) with increased potency, such as

DCs are the most potent APCs for initiating cellular immune responses through the stimulation of naive T cells. Immature DCs are good at antigen uptake and processing, but for a stimulatory T cell response they must mature to become fully activated DCs, which express high levels of cell surface-related major histocompatibility complex (MHC)-antigen and costimulatory molecules. Because of their ability to stimulate T cells, DCs act as a link in antitumor immune responses between innate immunity and adaptive immunity (Banchereau & Steinman, 1998). These DCs play a central role in various immunotherapy protocols by generation of cytotoxic T lymphocytes (CTLs) (Reid, 2001). DC-based vaccines have become the most attractive tool for cancer immunotherapy and have been used in the treatment of more than 20 malignancies, most commonly melanoma, renal cell carcinoma, prostate cancer and colorectal carcinoma (Palucka et al., 2011; Ridgway, 2003). In MM, cellular immunotherapy using DCs is emerging as a useful immunotherapeutic modality to

**1. Introduction** 

dendritic cells (DCs), in MM (Harrison & Cook, 2005).

**Cells Against Multiple Myeloma** 

*1Research Center for Cancer Immunotherapy 2Department of Hematology-Oncology,* 

*3Vaxcell-Bio Therapeutics, Hwasun, Jeollanamdo,* 

Myeloma. *British Journal of Haematology*, Vol.82, No.3, (November 1992), pp. 631, ISSN 1365-2141

