Clinical Cancer Research CTRC-AACR San Antonio Breast Cancer Symposium Infection and Cancer: Biology, Therapeutics, and Prevention
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Blumenthal, R. D.
Right arrow Articles by Goldenberg, D. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Blumenthal, R. D.
Right arrow Articles by Goldenberg, D. M.
Clinical Cancer Research Vol. 8, 1301-1309, May 2002
© 2002 American Association for Cancer Research

Experimental Therapeutics, Preclinical Pharmacology

Modulation of Marrow Proliferation and Chemosensitivity by Tumor-produced Cytokines from Syngeneic Pancreatic Tumor Lines1

Rosalyn D. Blumenthal2, Albert Reising, Evelyn Leon and David M. Goldenberg

Garden State Cancer Center, Belleville, New Jersey 07109


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: A dynamic process exists in which hematopoietic progenitor andstromal cells interact to maintain normal hematopoiesis or to adjust to hematopoietic needs under "stress" situations. The effect that tumor-produced growth factors have on hematopoiesis has not been addressed. We postulate that an excess of tumor-produced stimulatory or inhibitory cytokines could impact marrow proliferation and sensitivity to cytotoxic agents.

Methods: We used two tumor lines (TGP47 and TGP51) taken from a panel of syngeneic murine pancreatic carcinomas, in which each produces a unique array of cytokines, and evaluated their effect in vitro on marrow proliferation and chemosensitivity.

Results: TGP51- and TGP47-conditioned medium increased [3H]thymidine incorporation into cultured marrow cells by ~12-fold and 4.8-fold, respectively. The percent of cells in the S + G2-M phases of the cell cycle increased by 110% (TGP51) and 44% (TGP47), and the MCF for proliferating cell nuclear antigen expression increased by 104% (TGP51) and 45% (TGP47). Marrow proliferation of untreated cells could be reduced by interleukin 6 but not by granulocyte macrophage colony-stimulating factor neutralization. Conditioned medium-induced stimulation was unchanged by either interleukin 6{alpha} or granulocyte macrophage colony-stimulating factor {alpha}. FLT3-L{alpha} reduced marrow proliferation induced by TGP51 medium. Addition of FLT3-L to TGP47 medium additionally enhanced the marrow proliferation. Antitumor necrosis factor {alpha} additionally increased marrow proliferation induced by TGP47 and TGP51 conditioned medium, whereas addition of tumor necrosis factor {alpha} reduced marrow proliferation associated with TGP51 medium. The TGP51-induced increase in marrow proliferation resulted in increased marrow chemosensitivity to three myelosuppressive drugs: doxorubicin, cyclophosphamide, and CPT-11, decreasing the IC50 by 46%, 38%, and 95%, respectively.

Conclusion: Tumor-produced cytokines can affect marrow proliferative activity and, thus, chemosensitivity to three distinct classes of chemotherapeutics.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The hematopoietic system is a rapidly proliferating organ that maintains normal peripheral blood cell counts and must respond to stress situations such as infection, blood loss, and therapy-induced myelosuppression (1, 2, 3) . The steady state production of blood cells depends in part on the interaction between hematopoietic stem/progenitor cells and the different components of the marrow microenvironment. These components include stromal cells (e.g., fibroblasts, macrophages, endothelial cells, and adipocytes), accessory cells (T lymphocytes, monocytes), and their products (extracellular matrix proteins like collagen, laminin, and fibronectin, and cell-secreted cytokines). Positive and negative regulation of hematopoiesis occurs by direct interaction (cell-to-cell contact) or by indirect contact with secreted regulatory molecules (cytokines; Refs. 4 , 5 ). Cytokines trigger the quiescent CFU-S3 component of the stem cell compartment into active cell cycling until normal bone marrow cellularity is achieved (6 , 7) .

Under the permissive influence of these early and late-acting hematopoietic cytokines, differentiation and proliferation of multiple lineages from pluripotent stem cells is induced (8) , resulting in maintenance of the steady state between production and consumption of mature blood cells (9) . Under stressful conditions, (e.g., radiation, chemotherapy, blood loss, infection, or inflammation), they play a major role in cellular adaptation processes (3) . They trigger quiescent CFU-S of the stem cell compartment into active cell cycling until normal bone marrow cellularity is restored (6) . For example, in one study, serum G-CSF was inversely related to leukocyte blood counts in patients treated with chemotherapy. After chemotherapy, as blood counts declined, G-CSF peaked from undetectable levels (<20 pg/ml) to 1000–2000 pg/ml, and as blood counts were restored to normal, G-CSF fell back to undetectable levels (10) . The rise in serum cytokines correlates with the severity of myelosuppression (11) . In addition to published findings on the stimulatory cytokines, there is also important information emerging on the inhibitory cytokines. Negative regulation of myelopoiesis occurs through several inhibitory cytokines, most notably MIP-1{alpha} (12 , 13) and TGF-ß3 (14) acting on CD34+CD38- cells (15) . A 10-mg dose of MIP-1{alpha} results in a 98% decrease in S phase CFU-S in vivo. Combinations of two cytokines have a synergistic effect (16 , 17) .

Eighteen years ago, it was reported that hematopoietic abnormalities resulted from the presence of a solid tumor (18) . The hematopoietic system in tumor-bearing mice was more radiosensitive than in mice without tumors. In the presence of tumor, CFUs were suppressed after radiation therapy, and bone marrow repopulation was inhibited. The mechanism governing these observations could not be explained. Over the last few years, several reports have shown that many nonhematopoietic tumors have the ability to produce multiple cytokines in vivo. Approximately 80–100% of bladder, kidney, pancreas, prostate, cervical, endometrial, and ovarian cancers produce significant amounts of GM-CSF, IL-6, and M-CSF. Other cytokines produced in some but not all tumors include G-CSF, SCF, TNF-{alpha}, TGF-ß, IL-8, and IL-10 (19, 20, 21, 22) . These cytokines may act as autocrine growth factors, regulating proliferation and migration of endothelial, tumor, and immune cells. The type of cytokines produced varies as a function of the type of cancer and stage of disease (21, 22, 23) . We questioned whether tumor-produced cytokines could influence marrow physiology, i.e., cell proliferation and cell sensitivity to chemotherapy. Using a syngeneic mouse pancreatic tumor system, we address the biological effects of tumor-produced cytokines on bone marrow.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines.
Six pancreatic tumors derived from Tg(Ela-1-SV40E)Bri18 transgenic mice (24) were generously provided by Dr. Olive Pettengill, Dartmouth Medical Center, Hanover, NH). These six murine TPG lines were developed by introducing viral or human oncogenes under the control of a pancreas-specific elastase-1 promoter and SV-40 early tumor antigen into fertilized mouse ova. The carcinomas that formed within 3–8 months had an acinar cell type with a high incidence of insulinomas and were syngeneic for B6SJL/F1 mice. To produce conditioned medium, these cell lines were grown to confluence in RPMI 1640 with 10% FBS, 1% glutamine, 1% nonessential amino acids, and 1% penstrep.

Cytokine Quantitation.
Medium from semiconfluent TGP cell lines was collected, cell debris pelleted, and cytokines in the conditioned medium measured by ELISA kits from Endogen (IL-1ß, IL-3, IL-6, GM-CSF, IFN-{gamma}, TNF-{alpha}) and R&D Systems (G-CSF and FLT3-L). All of the assay kits have high sensitivity (approximately 3–15 pg/ml), are specific, and show no cross-reactivity with any other murine or human cytokines. Measurements were made in triplicate and the study repeated twice from two different generations of TGP growing cells.

Intracellular Cytokine Expression.
Cultured cells were washed in cold fluorescence-activated cell sorter buffer (Dulbecco’s PBS, 1% FBS, and 0.01% sodium azide), and 106 were resuspended in Cytofix buffer (2014KZ; PharMingen, San Diego, CA) and incubated on ice for 30 min. Fixed cells were pelleted and then incubated in 250 µl Perm/Wash buffer (phycoerythrin 2091KZ; PharMingen) on ice for 20 min followed by a 45-min incubation with biotinylated antimouse SCF Ab. Cells were washed and incubated for 45 on ice in the dark with streptavidin-PE. Cells are washed in Perm/Wash buffer, resuspended in 1 ml fluorescence-activated cell sorter buffer, and the percentage of positive cells and MCF were determined by flow cytometric analysis.

Marrow Proliferation Assay.
Murine femoral bone marrow cells were flushed from young mice (6–12 weeks in age), counted, and seeded in triplicate into 96-well round-bottomed culture plates. One of four concentrations ranging from 50,000 to 400,000 cells/well was seeded in 100 µl of medium (RPMI 1640 + 15% FBS + 10 µg/ml insulin + 20 µg/ml transferrin + 0.1 mM 2-mercapto-ethanol + 2 mM glutamine) in the absence or presence of conditioned medium. The high number of cells was used because total marrow was being considered, rather than unique marrow subpopulations. Conditioned medium (100 µl) from the cell lines of interest (e.g., TGP 47, TGP49, TGP50, or TGP51) were added to each well and incubated for 72 h. During the last 4 h, cells were exposed to [3H]thymidine (1 µCi/well). Cells were collected on filter mats and washed using a Tomtec plate harvester, and counted using a Wallac Microbeta plate counter. Controls included background wells with no cells and cells incubated with fresh medium alone and no conditioned medium.

For some studies, the influence of marrow chronobiology was considered. Animals were placed on one of three 12:12 light:dark schedules as described previously (25, #3776; 26, #4558) for 3 weeks before the start of all studies to allow for standardization of chronobiological rhythms. Room temperature was kept at 22 ± 2° C. Marrow samples were collected from synchronized mice at three different HALOs: 3 HALO (9 a.m.), 12 HALO (6 p.m.), and 18 HALO (12 a.m.). The statistical difference between average values of treatment groups were compared using a two-tailed t test where Ho: µ1 = µ2 and H1: µ1 != µ2.

In some studies neutralizing antibodies to specific cytokines or addition of exogenous cytokines on marrow proliferation were evaluated. The concentration of neutralizing Ab used was based on manufacturer guidelines. The Ab to mouse IL-6 at 10 ng/ml will neutralize 70 pg/ml IL-6 (Biogenesis Clone 256/H9 Rat IgG1 antimouse IL-6). We used 3.6 µg/200 µl well containing TGP51 (~115 pg/ml IL-6). The Ab to GM-CSF at 10 µg/ml neutralizes 1 ng/ml of GM-CSF (Biogenesis 4740–0975 Clone 26–3/2 Rat IgG2a antimouse GM-CSF). We added 0.52 µg/200 µl well containing TGP51 conditioned medium (~261 pg/ml GM-CSF). Goat antimouse FLT3-L (R&D #AF427; 10 ng/well) at a dose of 0.4 µg/ml neutralizes 12.5 ng/ml. Rat antimouse TNF-{alpha} (10 ng/well) was purchased from Endogen #MM-350Dl. Exogenous mouse FLT3-L (90 pg/ml; R&D 427-FL) and mouse TNF-{alpha} (100 pg/ml; R&D 410-MT) were added to the conditioned medium in some wells. A comparison between treatment groups was done using a two-tailed t test.

PCNA Expression.
PCNA is essential for cellular DNA synthesis and has been used as a proliferation marker for both hematopoiesis and tumor growth. It is expressed in proliferating cells in the late G1 phase and early during the S phase of the cell cycle (25) . Femoral marrow was collected at 10 a.m.; the cells were washed and incubated with FITC-conjugated anti-PCNA (Clone PC10; PharMingen). The percentage of PCNA-positive cells was determined. Appropriate negative and positive controls were used. Samples were run in triplicate, and the study was repeated twice.

Cell Cycle Analysis.
Washed femoral marrow was fixed with drop-by-drop addition of 1 ml ice-cold 70% ethanol, covered with parafilm, and stored overnight. Cells are pelleted and washed with permeabilizing buffer (6.05 g Tris buffer, 495 ml dH2O, 4.5 g NaCl, 73.5 mg CaCl2, 23.8 mg MgCl2, and 5 ml NP40). One ml of a 0.5 µM SYTOX Green [1 µl 5 mM SYTOX Green (frozen, -) 20°C + 9 ml permeabilizing buffer + 1 ml 1000 Kunitz RNase A] is added and the sample in incubated at room temperature for 15 min with gentle rocking. Cells are pelleted (3000 rpm x 10 min) and resuspended in 1 ml of sheath fluid with 1% formalin.

MTT Cytotoxicity Assay.
The assay was done according to the method of Mosmann (26) . Cells are plated at 104/well in a 96-well dish in 150 µl medium and incubated for 24 h in a humidified chamber. Three drugs were evaluated: doxorubicin (S phase-specific topoisomerase II inhibitor), cyclophosphamide (noncycle active alkylating agent), and CPT-11 (Camptosar; S phase-specific topoisomerase I inhibitor), which induce mild, moderate, and high myelosuppressive responses, respectively. Drugs were added at various concentrations in 150 µl and incubated for 3 days. The medium was removed, and 0.5 mg/ml MTT (100 µl) was added to each well and incubated for 2–4 h at 37°C. Acid-isopropanol (0.4 N HCl in isopropanol) was added to each well (100 µl) and mixed to dissolve purple crystals. The plate is read at 570 nm. Data were converted to percentage of control (no drug). The results of percentage of survival versus log of the drug concentration are plotted to obtain the IC50. Samples were run in triplicate, and all of the studies were repeated 4 to 7 times and the mean ± SD determined.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
We have evaluated the conditioned medium from six TGP murine pancreatic tumor lines by immunoassay for production of six stimulatory cytokines (GM-CSF, G-CSF, IL-1ß, IL-3, IL-6, and FLT3-L), two inhibitory cytokines (TNF-{alpha} and IFN-{gamma}), and one cytokine that can have both stimulatory and inhibitory effects (IL-10) The results of these studies are summarized in Table 1Citation . The expression of IL-3 and IFN-{gamma} was similar between the various lines. IL-1ß was not expressed by any of the lines (results not shown). The TGP47 line produced substantial GM-CSF (187 pg/ml) and G-CSF (37.97 pg/ml), and the TGP49 and TGP51 lines produced large amounts of IL-6 (250 and 115 pg/ml, respectively) and FLT3-L (87.5 and 46.8 pg/ml), but TGP-51 also produced some GM-CSF and had very low amounts of the constitutive inhibitory cytokine, TNF-{alpha} (17 pg/ml) compared with the TGP49 line (122.2 pg/ml). Expression of one additional stimulatory cytokine, SCF, was performed by flow cytometry, and the results are summarized in Table 2Citation . TGP48 and TGP54 demonstrated the highest percentage of positive cells (43.7% and 44.96%, respectively), and TGP48 had the highest MCF (496.61).


View this table:
[in this window]
[in a new window]
  		Table 1 Cytokine production of murine TGP tumor lines

 

View this table:
[in this window]
[in a new window]
  		Table 2 SCF expression by murine TGP tumor lines

 
To select the best model(s) to evaluate the effect of tumor cytokine production on hematopoiesis, we evaluated in vitro marrow proliferation ([3H]thymidine incorporation) in the presence of medium from cytokine-producing lines (Fig. 1)Citation . TGP51-conditioned medium increased marrow proliferation 12-fold, as demonstrated by a shift in marrow cell cpm from 172 ± 68 to 2068 ± 228 when 400,000 cells were plated (P < 0.001). TGP47 medium increased [3H]thymidine uptake by 4.8-fold (704 ± 55 cpm), whereas TGP49 and TGP50 medium stimulated uptake by 4.5- (601 ± 38 cpm) and 2.4-fold (404 ± 31 cpm), respectively. The results suggest that high levels of stimulatory cytokines coupled with low levels of TNF-{alpha} result in an increase in marrow proliferation (e.g., TGP51). These same stimulatory cytokines appear to be counterbalanced by high TNF-{alpha} (e.g., TGP49).



View larger version (20K):
[in this window]
[in a new window]
  		Fig. 1. Tumor-produced conditioned medium increases marrow proliferation in vitro. Marrow proliferation measured by [3H]thymidine incorporation (50,000 to 400,000 plated marrow cells) after a 72-h incubation in unconditioned medium or conditioned medium from various pancreatic tumor lines with different patterns of cytokine production. Results represent the mean of triplicates; bars, ±SD.

 
To additionally demonstrate that tumor-produced cytokines affect marrow proliferation, we evaluated cell cycle kinetics (Fig. 2Citation , top) and the expression (MCF) of PCNA (Fig. 2Citation , bottom) in femoral marrow cells grown for 48 h in control medium or in conditioned medium from the TGP47 or TGP51 pancreatic tumor lines. The percentage of marrow cells in the S or G2-M phases increased from 4.86 ± 0.6% in untreated cells to 6.98 ± 0.6 (P < 0.02) when grown in TGP47-conditioned medium to 10.25 ± 0.4% (P < 0.001) when grown in TGP51-conditioned medium. MCF increased from 92.7 ± 16.0 in untreated marrow cells to 134.2 ± 15.8 (P < 0.05) in TGP47-cultured cells, and to 188.8 ± 19.5 in TGP51-cultured cells (P < 0.01). These studies are in agreement with the thymidine incorporation studies presented in Fig. 1Citation .



View larger version (65K):
[in this window]
[in a new window]
  		Fig. 2. Tumor-produced conditioned medium increases marrow cell cycle kinetics and PCNA expression. Percentage (S + G2M) of marrow cells grown in unconditioned medium, or TGP47- or TGP51-conditioned medium (top panel). MCF of PCNA expression of cultured femoral marrow cells grown for 48 h in the absence or presence of tumor-produced cytokines using either TGP47 or TGP51-conditioned medium compared with control medium (bottom panel). Results represent the mean from two experiments each done in triplicate; bars, ±SD.

 
Because circadian fluctuations in bone marrow DNA synthesis, CFU-GM activity, cell number, and cytokine production exist, we assessed the effect of conditioned medium on marrow cells collected at three different times of the day: 3 HALO (9 a.m.), 12 HALO (6 p.m.), and 18 HALO (12 a.m.). For all four of the tumor lines studied, the greatest increase in marrow proliferation occurred at 18 HALO and the least at 3 HALO (Fig. 3)Citation . For example, for TGP-51 cultured marrow cells, [3H]thymidine incorporation increased 5.2 ± 0.5-fold at 3 HALO, 7.5 ± 0.5-fold at 12 HALO, and 9.6 ± 1.1-fold at 18 HALO (P < 0.01 between 3 and 18 HALO and between 12- and 3 HALO, and P < 0.05 between 12 HALO and 18 HALO). Similar results were obtained for marrow cells grown in TGP47-, TGP48-, and TGP50-conditioned medium.



View larger version (90K):
[in this window]
[in a new window]
  		Fig. 3. Time of marrow harvest influences baseline marrow proliferation but not conditioned-induced proliferation. Marrow proliferation ([3H]thymidine incorporation) into cultured marrow cells (300,000/well) collected at 3, 12, and 18 HALO (9 a.m., 6 p.m., and 12 a.m.) in unconditioned medium or conditioned medium from TGP47, TGP48, TGP50, and TGP51 tumor lines. Results represent the mean of triplicate values; bars, ±SD.

 
To establish which cytokines found in the conditioned medium were responsible for the observed changes in marrow proliferation, we performed several studies in which cytokine effects were neutralized by addition of specific Ab or exogenous cytokine was added directly to marrow cells grown in unconditioned medium. The incubation of marrow cells grown in unconditioned medium or TGP51-conditioned medium with neutralizing Ab to IL-6 decreased marrow proliferation by 40% (P < 0.02) and 14% (P = not significant), respectively. Anti-GM-CSF Ab did not affect marrow proliferation in either group (Fig. 4)Citation . In contrast to the effects observed with conditioned medium from mouse pancreatic tumor lines, normal mouse pancreas-conditioned medium was not able to induce marrow proliferation. Addition of FLT3-L{alpha} to TGP51 medium reduced marrow proliferation by 28% (P < 0.02), whereas addition of FLT3-L to TGP47 medium resulted in a 2.17-fold increase in proliferation above TGP47 (P < 0.01). Addition of TNF-{alpha} to TGP51 medium reduced marrow proliferation induced by the conditioned medium by 27% (P < 0.01), and anti-TNF-{alpha} increased marrow proliferation of TGP47 and TGP49 medium by 1.86-fold (P < 0.01) and 1.78-fold (P < 0.02), respectively (Fig. 5)Citation .



View larger version (57K):
[in this window]
[in a new window]
  		Fig. 4. Anti-IL-6 neutralizes part of baseline marrow proliferation but not conditioned-medium induced proliferation. The effect of neutralization of IL-6 (3.6 µg/well) or GM-CSF (0.52 µg/ml), or antibodies to both cytokines on untreated- and TGP51 tumor-produced cytokine effects on marrow proliferation is depicted. Isolated murine femoral marrow (250,000 cells/well) were cultured for 3 days with medium or TG51-conditioned medium with or without neutralizing antibodies. [3H]thymidine (1 µCi) was added to each well on day 3 and incubated for 24 h. Cells were harvested and counted. The results represent the mean of quadruplicate wells; bars, ±SD.

 


View larger version (42K):
[in this window]
[in a new window]
  		Fig. 5. Anti-FLT3-L and anti-TNF-{alpha} influence conditioned medium-induced marrow proliferation. The effect of neutralization of FLT3-L (5 ng/ml) or TNF-{alpha} (10 ng/ml) or addition of exogenous FLT3-L (90 pg/ml) or TNF-{alpha} (10 ng/ml) to TGP-conditioned medium. Isolated murine femoral marrow (250,000 cells/well) were cultured for 3 days with medium or conditioned medium from normal mouse pancreas, or TG51-conditioned medium with or without neutralizing antibodies. [3H]thymidine (1 µCi) was added to each well on day 3 and incubated for 24 h. Cells were harvested and counted. The results represent the mean of quadruplicate wells; bars, ±SD.

 
To ascertain whether increased marrow proliferation induced by tumor-conditioned medium influences chemosensitivity, we determined the IC50 of doxorubicin, cyclophosphamide, and CPT-11 on marrow cells when grown in the presence of medium without cytokines or in the presence of conditioned medium containing tumor-produced cytokines (TGP51) for 3 days. The results of these assays are shown in Fig. 6Citation . The IC50 for doxorubicin decreases from 0.24 ± 0.07 µg for untreated cells to 0.13 ± 0.05 µg for TGP51-treated cells (P < 0.02). For cyclophosphamide, the untreated IC50 was 5.38 ± 1.50 µg, and the TGP51-treated IC50 was 3.33 ± 0.88 µg (P < 0.01). The difference in the IC50 between untreated and TGP51-treated marrow cells exposed to the most myelosuppressive drug, CPT-11, was the most significant (P < 0.001), decreasing the IC50 from 425.3 ± 105.7 µg to 23.2 ± 10.3 µg. These results clearly demonstrate that the increase in marrow proliferation associated with tumor-produced cytokines translates into a significant increase in marrow chemosensitivity.



View larger version (28K):
[in this window]
[in a new window]
  		Fig. 6. Conditioned medium increases the chemosensitivity of marrow cells. Percentage survival of femoral marrow cells grown in unconditioned medium or TGP51- conditioned medium and given increasing doses of doxorubicin (top panel), cyclophosphamide (middle panel), or CPT-11 (Camptosar; bottom panel). Incubation with drug was for 3 days, and incubation with MTT was for 4 h. Crystals were pelleted and solubilized in DMSO and the A2570 nm determined. The results represent the mean of triplicates in one study for each drug that was typical of four to seven separate studies; bars, ±SD.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Chemotherapy-induced myelosuppression is the most common dose-limiting and potentially fatal complication of cancer treatment (1 , 2 , 29) . Drug-induced hematopoietic toxicity is a frequent reason for curtailing high-dose chemotherapy in cancer patients (30) . The grade of toxicity resulting from a fixed dose can vary substantially within the patient population. We have recently completed a thorough statistical analysis of the interacting factors potentially affecting hematological toxicity (31) . Both univariate and multivariate analyses have confirmed the significance of absorbed red marrow dose as the single most important factor affecting hematological toxicity after radioimmunotherapy. Baseline peripheral blood cell counts and presence of multiple bone marrow metastases were also significant factors affecting hematological toxicity. Aside from these three predictive factors, there were still many patients who experienced greater toxicity and many patients who experienced less toxicity than would have been predicted. Another critical factor regulating the magnitude of toxicity, which to date has not been addressed, is the effect that tumor-produced growth factors have on the marrow microenvironment. We postulated that differences in the expression of various tumor-produced cytokines might impact on the magnitude, duration, and slope of recovery after therapy-induced myelosuppression. Tumor production of excess stimulatory cytokines (e.g., G-CSF, GM-CSF, IL-3, IL-6, SCF, and FLT3L) would result in more active marrow proliferation, and the same dose of a therapeutic would induce more myelotoxicity. Similarly, a greater tumor production of inhibitory cytokines (e.g., MIP-1{alpha}, TGF-ß3, TNF-{alpha}, and IFN-{gamma}) would have the reverse effect.

Support for the idea that cytokines from distal sites to marrow or spleen affect hematopoiesis comes from the field of inflammation. Several older papers report that infection or inflammation induces changes in hematopoiesis (32 , 33) . Total number of peripheral nucleated cells, and marrow stem cells, stromal cells, and erythroblasts were increased in mice experiencing chronic inflammation (34) . These findings can now be explained by changes in the cytokine milieu associated with the expression of inflammatory mediators like IL-1ß, TNF-{alpha}, and IFN-{gamma} (35 , 36) . It is well established that cytokine expression in a particular tissue can be modified in response to exposure to other cytokines, e.g., TGF-ß or IL-4 can modulate tumor SCF production (37 , 38) .

To address the question of the effect of tumor-produced cytokines on marrow activity and marrow chemosensitivity, we chose an in vitro model to establish a proof or principle. Each of the TGP cell lines (24) studied produces a unique panel of stimulatory and inhibitory cytokines. Conditioned medium for all of the TGP lines enhanced marrow proliferation, although TGP51 medium resulted in significantly greater stimulation of marrow cells. Interestingly, the magnitude of stimulation in marrow proliferation varies depending on the time of day that the marrow is harvested and placed into culture suggesting that the expression of receptor(s) needed to respond to the active component(s) in conditioned medium fluctuates. This is not surprising because circadian fluctuations in cell number, cell activity, cytokine production, immunomodulating hormone production, and cellular and humoral activities are known to occur (39, 40, 41, 42) . In humans, bone marrow DNA synthesis and myeloid progenitor cell (CFU-GM) activity is at a maximum at 0–8 HALO (~7:00 a.m. to 3:00 p.m.) and at a minimum at 12–20 HALO (7:00 p.m. to 3:00 a.m.). Peak lymphocyte counts occurs at 17–19 HALO (12:00–2:00 a.m.) in both mice and humans (43) . Recently, it was noted that the response of CFU-GM to CSFs (IL-3, GM-CSF, and G-CSF) is also governed by circadian rhythms, with peak stimulation occurring at 3 HALO for all of the factors (44) . We have also observed that marrow cytokine expression varies with the time of day, as does the expression of various cytokine receptors (45 , 46) . Thus, the net influence of tumor-produced cytokines on marrow activity has greater impact at night in mice than during the day. Because mice are nocturnal, the reverse may prove to be true in humans.

Because each tumor line produces an array of cytokines, we attempted to determine which of the many expressed cytokines was most critical for increasing. These studies were not intended to identify all of the tumor-produced cytokines responsible for hematopoietic changes. Rather, it was designed to demonstrate for the first time that cytokines synthesized by tumor cells could significantly impact the marrow environment and could be a source for differences in myelotoxicity in response to a given therapeutic. On the basis of the profiles of expression (Tables 1Citation and 2Citation ) and the induced proliferation (Fig. 1)Citation , we selected three stimulatory cytokines (GM-CSF, IL-6, and FLT3L) for neutralization studies. We found that GM-CSF, a later acting stimulatory factor, was not essential for either baseline proliferation or tumor-cytokine-induced stimulation of marrow. Blocking of IL-6 resulted in a small reduction in baseline proliferation but did not block proliferation in response to conditioned medium. FLT3-L, a relatively new stromal cell-produced positive stimulatory cytokine (47 , 48) , is thought to act at a very early stage before the separation of myeloid and megakaryocytic lineages. Addition of FLT3-L enhanced marrow proliferation induced by condition medium, whereas neutralization of FLT3-L reduced stimulation by conditioned medium. It is also possible that the balance of positive and negative regulators determines differences in induction of marrow activity. Because TGP51 medium induced greater proliferation than TGP47 medium, and TGP51 expresses less TNF-{alpha} than TGP47, it will be important to evaluate neutralization of TNF-{alpha} as well. We believe that production of TNF-{alpha} may have a key role in hematopoiesis regulation. Published work (49) has demonstrated that the stroma derived from patient marrow undergoing chemotherapy could not fully support growth of normal donor CD34+ cells that were placed in contact with it. The number of CFU-GM formed was significantly reduced in cocultures with stroma exposed to chemotherapy. The defect in the stroma was an overproduction of the inhibitory cytokine TNF-{alpha}, which could be neutralized by an Ab. Negative regulation of myelopoiesis is also controlled through other inhibitory cytokines, most notably MIP-1{alpha} (12 , 13) and TGF-ß3 (14 , 17) . Future work will require that expression of these cytokines in tumor-conditioned medium is also quantitated.

We have not yet attempted to identify lineage-specific effects or specific changes within the hematopoietic hierarchy. Nor is it known whether the effect on marrow-proliferative activity by tumor-produced cytokines is direct, indirect, or both. Tumor-produced cytokines may bind directly to receptors found on marrow cells. Alternatively, a sizeable body of literature demonstrates that exogenous cytokines can affect expression of adhesion molecules on marrow stromal fibroblasts (50, 51, 52) , on hematopoietic cells (53) , and on progenitor cells (54, 55, 56) . Exogenous cytokines can also influence secretion of cytokines by human marrow cells and expression of cytokine receptors by marrow cells (57, 58, 59, 60) . The specific mechanism(s) by which tumor-produced exogenous cytokines affect marrow proliferation remains to be determined. Clonal analysis of defined populations of primitive and committed progenitor cells is essential for future studies.

The increase in marrow proliferation exposed to conditioned medium translated to significant differences in chemosensitivity. A downshift in the IC50 was observed for all three of the drugs studied with the greatest difference occurring when the most cytotoxic drug (CPT-11) was used. An important consideration when performing these studies is the age of the animal when the marrow is harvested. Two isolated studies were done with marrow from older mice (9–12 months), and the conditioned medium was not able to reduce the IC50 values for either doxorubicin or cyclophosphamide (results not shown). Age-related changes in the proliferation of marrow, telomere length, and in cytokine secretion by marrow have been reported (61, 62, 63) . However, when marrow was collected from young animals the trend was constant; a lower dose of the drug in more active marrow produced the same toxicity as a high dose of the drug in more quiescent marrow.

Although the production of cytokines by tumor cells has long been established, this is the first time that a connection has been made between the tumor biological phenomenon and the clinical concern of variation in myelosuppressive responses to chemotherapy. These studies have been generated from an in vitro model, yet the striking results provide strong support for the next phase of studies, i.e., translation of the in vitro findings to in vivo preclinical models. We have grown TGP51 tumors in mice and measured the presence of greater levels of several cytokines in the serum from these mice with only moderate tumor load compared with nontumor-bearing mice.4 We are now ready to address the biological significance that these circulating tumor-produced cytokines have on marrow biology. In addition to studies on magnitude and duration of myelosuppression and myelorecovery from cytotoxic therapy, we will also explore the impact of tumor-produced cytokines on stem cell transplantation technology; i.e., mobilization, engraftment, and differentiation of transplanted stem cells. These studies should help explain the differences in the grade of toxicity in response to cytotoxic therapy observed in patients receiving similar doses of myelosuppressive therapy. It may also eventually permit the sorting of patients into subpopulations that are likely to experience different myelosuppressive responses and myelorecovery profiles in response to therapy.

We would like to emphasize that this research fits well with decades of work demonstrating changes in hematopoiesis (e.g., abnormal total and differential blood cell counts and splenomegaly) as tumor burden increases. The contribution is this paper on the influence of tumor-elaborated cytokines on the marrow environment need to be evaluated in the context of other hematopoietic perturbations such as cytokine cascades initiated by the endogenous hematopoietic cells of the host reacting to the presence of a tumor, or metabolites produced by live or dying tumor cells.

In summary, we have used an in vitro system to demonstrate that tumor-produced cytokines can affect marrow proliferative activity and, thus, chemosensitivity to three distinct classes of chemotherapeutics. Certain cytokines are more important at affecting marrow activity and, indeed, the balance of positive and negative regulators are an important consideration.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported in part by United States Public Health Service grants RO1 CA39995 and RO1 CA54425 from the NIH. Back

2 To whom requests for reprints should be addressed, at Garden State Cancer Center, 520 Belleville Avenue, Belleville, NJ 07109. Phone: (973) 844-7014; Fax: (973) 844-7020; E-mail: rblumenthal.gscancer{at}att.net Back

3 The abbreviations used are: CFU-S, spleen colony-forming unit; CFU, colony-forming unit; G-CSF, granulocyte colony-stimulating factor; MIP, macrophage inflammatory protein; TGF, transforming growth factor; IL, interleukin; GM-CSF, granulocyte macrophage colony-stimulating factor; FBS, fetal bovine serum; MCF, mean channel fluorescence; HALO, hours after light onset; Ab, antibody; PCNA, proliferating cell nuclear antigen; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Back

4 R. D. Blumenthal, L. Osorio, A. Reisins, and D. M. Goldenberg. Differences in levels of serum cytokine of tumor-bearing mice versus mice without tumors, manuscript in preparation. Back

Received 1/10/02; accepted 2/14/02.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Maxwell M., Maher K. Chemotherapy-induced myelosuppression. Semin. Oncol. Nurs., 8: 113-123, 1992.[CrossRef][Medline]
  2. Blijham G. Prevention and treatment of organ toxicity during high dose chemotherapy: an overview. Anti-Cancer Drugs, 4: 527-533, 1993.[Medline]
  3. Cannistra S., Griffin J. Regulation of the production and function of granulocytes and monocytes. Semin. Hematol., 25: 173-188, 1988.[Medline]
  4. Mayani H., Guilbert L., Janowska-Wiesczorek A. Biology of the hematopoietic microenvironment. Eu. J. Haematol., 49: 225-233, 1992.
  5. Morrison S., Uchida N., Weissman I. The biology of hematopoietic stem cells. Annu. Rev. Cell Dev. Biol., 11: 35-71, 1995.[CrossRef][Medline]
  6. Becker A., McCulloch E., Siminovich L., Till J. The effect of differing demands for blood cell production on DNA synthesis by hemopoietic colony-forming cells of mice. Blood, 26: 296-304, 1965.[Abstract/Free Full Text]
  7. Wagemaker G., Neelis K., Wognum A. Surface markers and growth factor receptors of immature hematopoietic stem cell subsets. Stem Cells, 13: 165-171, 1995.
  8. Lowry P. Hematopoietic stem cell cytokine response. J. Cell Biochem., 58: 410-415, 1995.[CrossRef][Medline]
  9. Whetton A., Dexter T. Myeloid hematopoietic growth factors. Biochem. Biophys. Acta, 989: 111-132, 1994.
  10. Sallerfors B., Olofsson T. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) in serum during induction treatment of acute leukemia. Br. J. Hematol., 78: 343-351, 1991.[Medline]
  11. Chen Y., Whang-Peng J., Ming-Lu J., Ing-Tiau Kuo B., Wang S-Y., Tsai C-M., Perng R. Serum cytokine level fluctuations in chemotherapy-induced myelosuppression. Jpn. J. Clin. Oncol., 26: 18-23, 1996.[Abstract/Free Full Text]
  12. Cooper S., Mantel C., Broxmeyer H. Myelosuppressive effects in vivo with very low dosages of monomeric recombinant murine macrophage inflammatory protein-1a. Exp. Hematol., 22: 186-193, 1994.[Medline]
  13. Dunlop D., Wright E., Lorimore S., Graham G., Holyoake T., Kerr D., Wolpe S., Pragnell I. Demonstration of stem cell inhibition and myeloprotective effects of SI/rhMIP1a in vivo. Blood, 79: 2221-2225, 1992.[Abstract/Free Full Text]
  14. Jacobsen S., Keller J., Ruscetti F., Kondaiah P., Roberts A., Falk L. Bidirectional effects of transforming growth factor b (TGFb) on colony-stimulating factor-induced myelopoiesis in vitro: differential effects of distinct TGFb isoforms. Blood, 78: 2239-2247, 1991.[Abstract/Free Full Text]
  15. van Ranst P., Snoeck H., Lardon F., Lenjou F., Nijs G., Weekx S., Rodrigus I., Berneman Z., van Bockstaele D. TGF-ß and MIP-1{alpha} exert their main inhibitory activity on very primitive CD34+CD38- cells but show opposite effects on more mature CD34-CD38+ human hematopoietic progenitors. Exp. Hematol., 24: 1509-1515, 1996.[Medline]
  16. Broxmeyer H. E., Sherry B., Cooper S., Lu L., Maze R., Beckmann M., Cerami A., Ralph P. Comparative analysis of the human macrophage inflammatory protein family of cytokines (chemokines) on proliferation of human myeloid progenitor cells. J. Immunol., 150: 3448-3459, 1993.[Abstract]
  17. Maze R., Sherry B., Kwon B., Cerami A., Broxmeyer H. Myelosuppressive effects in vivo of purified recombinant murine macrophage inflammatory protein 1a. J. Immunol., 149: 1004-1009, 1992.[Abstract]
  18. DeGowin R., Gibson D., Knapp S. Tumor bearing impairs hemopoietic recovery and survival after irradiation. Exp. Hematol., 11: 305-314, 1983.[Medline]
  19. Steube K., Meyer C., Drexler H. Secretion of functional hematopoietic growth factors by human carcinoma cell lines. Int. J. Cancer, 78: 120-124, 1998.[CrossRef][Medline]
  20. Bronte V., Chappell D., Apolloni E., Cabrelle A., Wang M., Hwu P., Restifo N. Unopposed production of granulocyte-macrophage colony stimulating factor by tumor inhibits CD8+ T cell responses by dysregulating antigen-presenting cell maturation. J. Immunol., 162: 5728-5737, 1999.[Abstract/Free Full Text]
  21. Chopra V., Dinh T., Hannigan E. V. Angiogenin, interleukins, and growth factor levels in serum of patients with ovarian cancer. Correlation with angiogenesis. Cancer J. Sci. Am., 2: 279-285, 1996.[Medline]
  22. Chopra V., Dinh T., Hannigan E. Circulating serum levels of cytokines and angiogenic factors in patients with cervical cancer. Cancer Investig., 16: 152-159, 1998.[Medline]
  23. Pekarek L., Weichselbaum R., Beckett M., Nachman J., Schreiber H. Footprinting of individual tumors and their variants by constitutive cytokine expression patterns. Cancer Res., 53: 1978-1981, 1993.[Abstract/Free Full Text]
  24. Pettengill O. S., Memoli V. A., Brinck-Johnson T., Longnecker D. S. Cell lines derived from pancreatic tumors of Tg(Ela-1-SV40E)Bri18 transgenic mice express somatostatin and T antigen. Carcinogenesis (Lond.), 15: 61-65, 1994.[Abstract/Free Full Text]
  25. Blumenthal R., Reising A., Lew W., Dunn R., Ying Z., Goldenberg D. Chronotolerance of experimental radioimmunotherapy: clearance, toxicity and maximal tolerated dose of l-131-anti-CEA IgG as a function of time of day of dosing in a murine model. Eur J. Cancer, 35: 815-824, 1999.
  26. Blumenthal R., Ochakovskaya R., Osorio L., Ying Z., Goldenberg D. Regulation of tumour drug delivery by blood flow chronobiology. Eur. J. Cancer, 36: 1876-1884, 2001.[CrossRef]
  27. Mathews M., Bernstein R., Franza R., Garrels J. Identity of the proliferating cell nuclear antigen and cyclin. Nature (Lond.), 309: 374-376, 1984.[CrossRef][Medline]
  28. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity. J. Immunol. Methods, 65: 55-63, 1983.[CrossRef][Medline]
  29. Vriesendorp H. M., Quadri S. M., Andersson B. S., Dicke K. A. Hematologic side effects of radiolabeled immunoglobulin therapy. Exp Hematol., 24: 1183-1190, 1996.[Medline]
  30. Boesen J., Nooter K., Valerio D. Circumvention of chemotherapy-induced myelosuppression by transfer of the mdr-1 gene. Biotherapy, 6: 291-302, 1993.[CrossRef][Medline]
  31. Juweid M., Zhang C., Blumenthal R., Sharkey R. M., Dunn R., Dunlop D., Goldenberg D. M. Factors influencing hematologic toxicity of radioimmunotherapy with 131I-labeled anti-carcinoembryonic antigen antibodies. Cancer (Phila.), 80: 2749-2753, 1997.[CrossRef][Medline]
  32. Lucia H., Booss J. Immune stimulation, inflammation, and changes in hematopoiesis. Host responses of the murine spleen to infection with cytomegalovirus. Am. J. Pathol., 104: 90-97, 1981.[Abstract]
  33. Werts E., Gibson D., Degowin R. Chronic inflammation suppresses bone marrow stromal cells and medullary erythropoiesis. J. Lab. Clin. Med., 93: 995-1003, 1979.[Medline]
  34. DeGowin R., Lass S. Chronic inflammation impairs hematopoiesis and survival after irradiation. J. Lab. Clin. Med., 105: 299-304, 1985.[Medline]
  35. Crippen T., Klasing K., Hyde D. Regulation of cytokine-induced neutrophil chemoattractant in rat bone marrow-derived macrophages by inflammatory mediators. Pathobiology, 66: 293-301, 1998.[CrossRef][Medline]
  36. Shao L., Frigon N., Yu A., Paiyash J., Yu J. Contrasting effects of inflammatory cytokines and glucocorticoids on the production of activin A in human marrow stromal cells and their implications. Cytokine, 10: 227-235, 1998.[CrossRef][Medline]
  37. Sugimoto Y., Koji T., Miyoshi S. Modification of expression of stem cell factor by various cytokines. J. Cell. Physiol., 181: 285-294, 1999.[CrossRef][Medline]
  38. Lahm H., Amstad P., Vilmaz A., Borbenyi Z., Wyniger J., Fischer J., Suardet L., Givel J., Odartchenko N. Interleukin 4 down regulates expression of c-kit and autocrine stem cell factor in human colorectal carcinoma cells. Cell Growth Differ., 6: 1111-1118, 1995.[Abstract]
  39. Haus E., Nicolau G. Y. Rhythms in bone marrow cell proliferation: how to apply to the chronotherapy of cancer?. Chronobiol. Int., 9: 393-398, 1992.[Medline]
  40. Young M., Matthews J., Kanabrocki E., Sothern R., Roitman-Johnson B., Scheving L. Circadian rhythmometry of serum interleukin-2, interleukin-10, tumor necrosis factor {alpha}, and granulocyte-macrophage colony stimulating factor. Chronobiol. Int., 12: 19-27, 1995.[Medline]
  41. Hayashi O., Kikuchi M. The effects of light-dark cycle on humoral and cell-mediated immune responses of mice. Chronobiologia, 9: 291-300, 1982.[Medline]
  42. Gudewill S., Pollmacher T., Vedder H., Schreiber W., Fassbender K., Holsboer F. Nocturnal plasma levels of cytokines in healthy men. Eur. Arch. Psychiatry Clin. Neurosci., 242: 53-56, 1992.[CrossRef][Medline]
  43. Swoyer J., Haue E., Sackett-Lundeen L. Circadian reference values for hematologic parameters in several strains of mice. Prog. Clin. Biol. Res., 227A: 281-296, 1987.
  44. Pierpoint B., Le Bousse-Kerdiles C., Clay D., Smadja-Joffe F., Depres-Brumme P., Laporte-simitsidis S., Jasmin C., Levi F. In vitro chronopharmacology of recombinant mouse IL-3, mouse GM-CSF, and human G-CSF on murine myeloid progenitor cells. Exp. Hematol., 23: 362-368, 1995.[Medline]
  45. Blumenthal R., Reising A., Goldenberg D. Effect of tumor-producing cytokines on bone marrow proliferation. Exp. Hematol., 28: 50 2000.
  46. Alisauskas R., Flynn M., Goldenberg D., Blumenthal R. Chronobiological principles associated with preclinical IL-12-based therapy. Proc. Amer. Assoc. Cancer Res., 40: 78 1999.
  47. Brasel K., McKenna H., Morrissey P., Charrier K., Morris A., Lee C., Williams D., Lyman S. Hematologic effects of flt-3 ligand in vivo in mice. Blood, 88: 2004-2012, 1996.[Abstract/Free Full Text]
  48. Lisovsky M., Estrov Z., Zhang X., Consoli U., Sanchez-Williams G., Snell V., Munker R., Goodacre A., Savchenko V., Andreeff M. Flt3 ligand stimulates proliferation and inhibits apoptosis of acute myeloid leukemia cells: regulation of Bcl-2 and Bax. Blood, 88: 3987-3997, 1996.[Abstract/Free Full Text]
  49. Schwartz G., Warren M., Rothwell S., Zujewski J., Halverson D., Cowan K., Tolcher A., O’Shaughnessy J., Gress R. Post-chemotherapy and cytokine pretrated marrow stromal cell layers suppress hematopoiesis from normal stromal cell layers suppress hematopoiesis from normal donor CD34+ cells. Bone Marrow Transplant., 22: 457-468, 1998.[CrossRef][Medline]
  50. Stolze B., Ebell W., Welte K. Different binding mechanisms of myeloid leukemic cells to adhesion molecules on bone marrow stromal fibroblasts induced by TNF-{alpha} and IL-4. Ann. Hematol., 77: 265-274, 1998.[CrossRef][Medline]
  51. Bendall L., Kortlepel K., Gottlieb D. Bone marrow fibroblast exposure to the inflammatory cytokines tumor necrosis factor-{alpha} and interferon-{gamma} increases adhesion of acute myeloid leukemia cells and alters the adhesive mechanism. Exp. Hematol., 25: 132-139, 1997.[Medline]
  52. Schmitz B., Park I., Kaufmann R., Thiele J., Fischer R. Influence of cytokine stimulation (granulocyte macrophage-colony stimulating factor, interleukin-3 and transforming growth factor-ß-1) on adhesion molecule expression in normal human bone marrow fibroblasts. Acta Haematol., 94: 173-181, 1995.[Medline]
  53. Kovach N., Lin N., Yednock T., Harlan J., Broudy V. Stem cell factor modulates aidity of {alpha} 4 ß 1 and {alpha} 5 ß 1 integrins expressed on hematopoietic cell lines. Blood, 85: 159-167, 1995.[Abstract/Free Full Text]
  54. Mohle R., Murea S., Kirsch M., Haas R. Differential expression of L-selectin, VLA-4 and LFA-1 on CD34+ progenitor cells from bone marrow and peripheral blood during G-CSF-enhanced recovery. Exp. Hematol., 23: 1535-1542, 1995.[Medline]
  55. Watanabe T., Dave B., Heimann D., Lethaby E., Kessinger A., Talmadge J. GM-CSF-mobilized peripheral blood CD34+ cells differ from steady-state bone marrow CD34+ cells in adhesion molecule expression. Bone Marrow Transplant., 19: 1175-1181, 1997.[CrossRef][Medline]
  56. Levesque J., Haylock D., Simmons P. Cytokine regulation of proliferation and cell adhesion are correlated events in human CD34+ hemopoietic progenitors. Blood, 88: 1168-1176, 1996.[Abstract/Free Full Text]
  57. Besse A., Trimoreau F., Praloran V., Denizot Y. Effect of cytokines and growth factors on the macrophage colony-stimulating factor secretion by human bone marrow stromal cells. Cytokine, 12: 522-525, 2000.[CrossRef][Medline]
  58. Denizot Y., Besse A., Raher S., Nachat R., Trimoreau F., Praloran V., Godard A. Interleukin-4 (IL-4), but not IL-10, regulates the synthesis of IL-6, IL-8 and leukemia inhibitory factor by human bone marrow stromal cells. Biochim. Biophys. Acta, 1449: 83-92, 1999.[Medline]
  59. Caldwell J., Emerson S. Interleukin-1 {alpha} upregulates tumor necrosis factor receptors expressed by human bone marrow stromal cell strain: implications for cytokine redundancy and synergy. Blood, 86: 64-72, 1995.
  60. Shieh J., Peterson R., Moore M. Granulocuye colony-stimulating factor modulation of cytokine receptors on murine bone marrow cells. In vivo and in vitro studies. J. Immunol., 147: 2984-2990, 1991.[Abstract]
  61. Ogawa T., Kitagawa M., Hirokawa K. Age-related changes of human bone marrow: a histometric estimation of proliferative cells, apoptotic cells. T cells, B cells and macrophages. Mech. Ageing Dev., 117: 57-68, 2000.[CrossRef][Medline]
  62. Pedrazzini E., Mamev N., Slavutsky I. Age related decrease in NOR activity in bone marrow metaphase chromosomes from healthy individuals. Mol. Pathol., 51: 39-42, 1998.[Abstract]
  63. Cheleuitte D., Mizuno S., Glowacki J. In vitro secretion of cytokines by human bone marrow: effects of age and estrogen status. J. Clin. Endocrinol. Metab., 83: 2043-2051, 1998.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Cancer Res.Home page
U. Emmenegger, S. Man, Y. Shaked, G. Francia, J. W. Wong, D. J. Hicklin, and R. S. Kerbel
A Comparative Analysis of Low-Dose Metronomic Cyclophosphamide Reveals Absent or Low-Grade Toxicity on Tissues Highly Sensitive to the Toxic Effects of Maximum Tolerated Dose Regimens
Cancer Res., June 1, 2004; 64(11): 3994 - 4000.
[Abstract] [Full Text] [PDF]

Home page
 JNMHome page
J. A. Siegel, D. Yeldell, D. M. Goldenberg, M. G. Stabin, R. B. Sparks, R. M. Sharkey, A. Brenner, and R. D. Blumenthal
Red Marrow Radiation Dose Adjustment Using Plasma FLT3-L Cytokine Levels: Improved Correlations Between Hematologic Toxicity and Bone Marrow Dose for Radioimmunotherapy Patients
J. Nucl. Med., January 1, 2003; 44(1): 67 - 76.
[Abstract] [Full Text] [PDF]

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

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