HemoGenix, Inc, Colorado Springs, Colorado 80907
1 To whom correspondence should be addressed at HemoGenix, Inc, 4405 N. Chestnut Street, Suite D, Colorado Springs, CO 80907. Fax: (719) 264-6253. E-mail: ivannr{at}hemogenix.com.
Received May 24, 2005; accepted July 5, 2005
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: colony-forming assay; lympho-hematotoxicity; proliferation assay, Registry of Cytotoxicity.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although toxicity studies performed during late screening and lead optimization may shed light on potential problems to the blood-forming system, conventional hemotoxicity testing is performed only during preclinical animal testing. It is part of a large number of tests and usually consists of measuring peripheral blood cell parameters and pathological effects in the hematopoietic organs. These are certainly important parameters, but they have little, if any, predictive value. Serious toxic side effects observed during preclinical testing, or even worse, during clinical trials cost not only time and money, but potential danger to the patients.
Bradley and Metcalf (1966) and Pluznik and Saks (1966)
introduced the hematopoietic colony-forming assays (CFA). These in vitro assays detect clonal expansion of stem, progenitor, and precursor cells. This methodology has been part of the experimental and clinical hematology community for nearly four decades and has been validated worldwide. Without these assays, the biology and physiology of the blood-forming system and the lympho-hematopoietic hierarchy as we know it today, would not have been possible. Differentiation of various cell populations in vitro is made possible by the functional ability of these cells to proliferate, divide, and differentiate, in response to growth factors, into cells that are immobilized in a semisolid medium to form colonies that can be viewed and counted under the microscope. There is no other assay system that can detect and measure the different cell populations that comprise the stem cell and amplification compartments of the lympho-hematopoietic system. It is important to emphasize that the CFA is a differentiation assay; that is, it measures the capacity of cells to form colonies of differentiated cells in vitro. There are many reports using the colony-forming assays in drug development (Ghielmini et al., 1998
; Parchment, 1998
) and environmental studies (Parent-Massin et al., 1993
; Van Den Heuvel et al., 2001
), but with few exceptions, it has never been used routinely, despite the fact that it can provide greater predictive information regarding compound toxicity than conventional hemotoxicity (Parchment et al., 1998
). There are, however, a number of drawbacks to the CFA that have hindered its use in drug development. First, it is time consuming to perform. Manual enumeration of colonies is highly subjective and requires technical expertise. Furthermore, colony enumeration lacks standardization. Although side-by-side cell populations and species comparisons can be performed, this can only be done on a small scale, due to the low throughput capability of the system. This almost precludes the use of the assay during the drug development process, except where a drug is known to interfere with the differentiation process. These are usually relatively small studies, since the assay is limited by the manual enumeration process.
Realizing that no other assay system can be used to detect and measure different proliferating cell populations, the colony-forming assay methodology was redesigned into an ATP-based bioluminescence proliferation assay readout. The new assay is called HALO (Hemotoxicity Assays via Luminescence Output). HALO does not suffer from the drawbacks of the manual CFA. It is rapid, being completed in half the time of the manual CFA, highly sensitive, nonsubjective, and standardized. The 96-well plate format provides high-throughput capability. In addition, the system provides multifunctional capability so that up to 14 different lympho-hematopoietic populations from different hematopoietic sources (peripheral blood and bone marrow and cord blood from humans) from five different species (human, nonhuman primate, dog, rat, and mouse) can be detected and measured simultaneously. The HALO Platform has been validated against the manual CFA (Rich and Hall, submitted for publication).
As part of a further validation study, 11 reference compounds from the Registry of Cytotoxicity (RC) (NIH Publication, 2001) were tested on seven different lympho-hematopoietic cell populations from fresh human and mouse bone marrow. These results, together with those obtained from a number of different anticancer drugs tested on fresh human bone marrow, have provided evidence for a new prediction hemotoxicity paradigm that allows the assay system to be used during the early screening stages of drug development. This report describes the predictions and examples that make up this paradigm. In addition, validation of HALO against the Registry of Cytotoxicity Prediction Model allows the estimation of relevant starting doses for preclinical studies and even initial or clinically relevant dosing for clinical trials (NIH Publication, 2001
; Spielmann et al., 1999
).
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Tissues used and their preparation.
Fresh primary whole bone marrow was obtained from normal donors and purchased from Cambrex BioSciences (Walkserville, MD) after they had signed a consent form approved by the Internal Review Board (IRB). Approval to use this tissue was also obtained from the Dorn Veteran Affairs Medical Center (Columbia, SC) and the BioMed IRB (San Diego, CA). The mononuclear cell (MNC) fraction was separated by density gradient centrifugation using NycoPrep 1.077 (Greiner BioOne, Longwood, FL). After washing once in phosphate-buffered saline (PBS), the cells were resuspended in Iscove's Modified Dulbecco's Medium (IMDM). Viability was detected by 7-aminoactinomycin D (7-AAD, Beckman Coulter, Miami, FL) using flow cytometry, and nucleated cell counts (Z2 Particle Analyzer, Beckman Coulter) were performed on the whole tissue and after resuspension of separated MNCs.
Use of mice for this study was approved after submission of an animal use protocol to Colorado State University, Ft. Collins, CO, from which the animals were obtained. Mouse bone marrow was obtained from C57Bl/6J female mice 1012 weeks old. After removing the femora, the bone marrow was flushed into IMDM according the procedure described previously (Rich and Kubanek, 1982).
Growth factors and cell populations.
Human growth factors, with the exception of erythropoietin (EPO), which was obtained from R&D Systems (Minneapolis, MN), were obtained from CellGenix, Inc (Gaithersburg, MD). Murine growth factors were obtained from R&D Systems. Stimulation of different cell populations used the following growth factor combinations. High-proliferative-potential stem and progenitor cells (HPP-SP; stem cell factor (SCF, 50 ng/ml), interleukin-3 (IL-3, 100 ng/ml), interleukin-6 (IL-6, 20 ng/ml), Flt3-ligand (50 ng/ml), colony-forming cell granulocyte, erythroid, macrophage, megakaryocyte (CFC-GEMM): Erythropoietin (EPO, 3U/ml), granulocyte-macrophage colony-stimulating factor (GM-CSF, 20 ng/ml), granulocyte colony-stimulating factor (G-CSF, 20 ng/ml), thrombopoietin (TPO, 30 ng/ml), IL-3 (10 ng/ml), IL-6, SCF, Flt3-L. Burst-forming unit-erythroid (BFU-E): EPO, IL-3 and SCF. Granulocyte-macrophage colony-forming cells (GM-CFC): GM-CSF, IL-3, SCF. Megakaryocyte colony-forming cell (Mk-CFC): TPO and SCF. T-lymphocyte colony-forming cells (T-CFC): interleukin-2 (IL-2, 50 ng/ml). B-lymphocyte colony-forming cell (B-CFC): interleukin-7 (IL-7, 20 ng/ml). Murine CFC-GEMM did contain G-CSF.
The seven-population prediction hemotoxicity paradigm (7-PPHP).
The HALO Platform (HemoGenix, Inc, Colorado Springs, CO) was used to detect and measure the seven populations described above, namely two stem cells (HPP-SP and CFC-GEMM), three hematopoietic progenitor cells (BFU-E, GM-CFC, Mk-CFC), and two lymphopoietic populations (T-CFC and B-CFC). The HALO 7-PPHP was performed by mixing three separate HALO reagent mixes consisting of a serum mix (four parts), a methyl cellulose mix (four parts), and a growth factor mix (one part) with the bone marrow target cells (one part) to form a master mix. The cell density used was 1.5 x 106/ml. One hundred µl of the master mix was dispensed into wells of a 96-well, white-walled, transparent bottom luminescence plate (Greiner BioOne, Longwood, FL) where the compound dose had been added to eight replicates. The 96-well plates were placed in a humidified incubator (Heraeus, Kendro Scientific Instruments, Ashville, NC) at 37°C containing an atmosphere of 5% CO2 and 5% O2 (Rich, 1986; Rich and Kubanek, 1982
). Mouse cells were incubated for 5 days, while human cells were incubated for 7 days.
Controls.
For each compound and for each cell population tested, four controls were included. These were: (a) spontaneous control which contained cells without growth factors or drug/compound addition, (b) vehicle-only control, (c) growth factor control, (d) growth-factor-plus-vehicle control.
Measurement of luminescence.
The proliferative status in control and drug-treated cells is dependent on the intracellular ATP concentration. As the cells proliferate or when the cells are inhibited, the proportion of intracellular ATP varies accordingly. Using a multichannel pipette or liquid handler (Beckman Coulter, BioMek 2000), 50 µl of an ATP-releasing reagent (ATP-RR) was added and thoroughly mixed with the contents of each well. The plates were incubated at 23°C for 15 min, during which time the intracellular ATP was released from the cultured cells. Thereafter, 100 µl of luminescence monitoring reagent (ATP-MR) was added to each well and mixed. The plates were then transferred to a plate luminometer (Molecular Devices, Lmax) and the bioluminescence measured.
Prior to all sample measurements, a background and ATP standard curve was performed. The background consisted of 100 µl of medium (IMDM) added to four replicate wells followed by the addition of ATP-RR and ATP-MR. The ATP standard dose response was performed by serially diluting ATP to 1 µM, 0.5 µM, 0.1 µM, 0.05 µM, and 0.01 µM and dispensing each dilution into four replicate wells followed by the addition of ATP-RR and ATP-MR. Performing an ATP standard dose response ensured that the luminometer was working correctly and that the luminescence reagents were functional. In addition, it allows conversion of nonstandardized Relative Luminescence Units (RLU) into standardized ATP (µM) unit values, so that luminescence measured on different instruments can be compared. Another advantage of performing the ATP dose response is that results can be normalized so that experiments performed at different times and even in different laboratories can be compared.
Statistics.
Luminescence results were obtained using Softmax software (Molecular Devices). The software was programmed so that the background ATP value was subtracted from all raw data and converted directly to ATP (µM) values using the ATP standard dose response curve. The mean RLU and ATP values, standard deviation and percent variation coefficient were calculated. Results are expressed as the mean RLUbackground/well or mean normalized ATP (µM) production/well. Where possible, the inhibitory concentrations at 50% (IC50 values) were estimated from the dose response curves using a nonlinear, Hill or four-parameter logistic analysis regression. To calculate the IC75 or IC90 values, the RLU or ATP results were converted to percent from the growth-factor-plus-vehicle control. Nonlinear curve-fitting analysis was performed, and the IC values estimated using TableCurve 2D software (SPSS, Inc). Linear regression analysis and correlation was performed to compare results obtained from HALO with those of the registry of Cytotoxicity. Results were compared using analysis of variance (ANOVA).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Prediction 1
If no demonstrable affect occurs at the stem or on any of the proliferating progenitor or precursor cell populations, the compound will have no affect on lympho-hematopoiesis. This, however, does not exclude the possibility that the compound acts on nonproliferating or maturing cells or end cells, since HALO would not detect changes in this part of the system. In this latter case, the classical colony-forming assay would have to be employed. Both naladixic acid and antipyrine are examples of this first prediction.
Prediction 2
If a compound acts on one or more lineages of the amplification and differentiation compartment, which includes all progenitor and precursor cells, the corresponding mature functional cells would be affected. Figures 1A1C demonstrate this prediction using chloramphenicol. Figure 1A shows the effect on the human primitive and mature stem cell populations. Figure 1B shows the effect on the three hematopoietic progenitor populations, and Figure 1C shows the effect on the two lymphopoietic cell populations. It is first interesting to note that the primitive stem cell population, HPP-SP, exhibits a positive effect over the complete dose range to chloramphenicol. This positive effect is responsible for the partial stimulation in both the lymphopoietic populations. This specific effect, where the response of the HPP-SP population can lead to a concomitant response of the lymphopoietic cell populations is also described in Prediction 5 below. As far as the present prediction is concerned, there is no affect on the CFC-GEMM, GM-CFC, or Mk-CFC, but a specific inhibition is observed for the BFU-E population. The BFU-E, GM-CFC, and Mk-CFC are part of the hematopoietic amplification and differentiation compartment. Since only the BFU-E is inhibited, the prediction would be that depletion in erythrocytes would result, leading to an anemic situation. Indeed, the specific and reversible inhibition of chloramphenicol and thiamphenicol on in vitro erythropoiesis has been studied previously (Nijhof et al., 1977), but chloramphenicol toxicity and its role in the induction of aplastic anemia have been known for decades.
|
|
|
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The colony-forming assays have been used to study the effects of compounds in drug development and in the environmental toxicity arena on specific progenitor cells populations (Ghielmini et al., 1998; Parchment, 1998
; Parent-Massin et al., 1993
; Van Den Heuvel et al., 2001
). However, the European Center for Validation of Alternative Methods (ECVAM) has promoted and paved the way for routine and regulatory aspects using this assay technique in a standardized manner (Gribaldo, 2002
; Gribaldo et al., 1996
; NIH Publication, 2001
). Much of this work has focused on drug-induce neutropenia using the GM-CFC assay (Pessina et al., 2001
), and an international blind trial was carried out to validate a prediction model (Pessina et al., 2003
). This work demonstrated that an 87% prediction of the maximum tolerated dose (MTD) could be achieved when the IC90 value of the compound was determined. This was the reason for including the IC90 values in the studies reported above. Further studies have included other hematopoietic populations such as the Mk-CFC and the BFU-E (Parent-Massin, 2001
). A methyl cellulose microculture system for GM-CFC to assess drug-induced toxicity has also been reported (Pessina et al., 2004
). The latter report also tested whether the optical density, using an MTT test, could be used instead of colony counts, but results were not comparable. A MTT test for GM-CFC was reported in 2000 by Horowitz and King using agar as the semi-solid medium rather than methyl cellulose (Horowitz and King, 2000
). More recently, Greenwalt and colleagues (2001)
reported on a high-throughput assay for hematopoietic progenitor cells using a fluorescence-based signal detection system.
The manual colony-forming assay is useful if a compound acts specifically at the differentiation or maturation level. That is, the compound acts to block or inhibit differentiation and/or maturation, since the type (and possibly size) of the colonies produced can indicate the stage of differentiation and/or maturation at which this occurs. Furthermore, limiting the testing protocol to a single lineage (e.g., GM-CFC or MK-CFC or BFU-E) limits the information obtained, since although some compounds will act specifically on a particular lineage (e.g., chloramphenicol, phenylhydrazine), the majority will not. If two or more lineages are affected by the same compound, then according to the lympho-hematopoietic hierarchy, that compound must act on the cell(s) that is responsible for producing cells that enter these lineages. Thus, the compound must act at the stem cell level. Therefore, a compound tested on the CFC-GEMM population will provide information relevant to the whole of the hematopoietic system. Similarly, a compound tested on a primitive stem cell population (HPP-SP) capable of producing both lymphopoietic and hematopoietic stem cells will provide information relevant to the whole lympho-hematopoietic system. It therefore follows that, by screening compounds for their effects on the CFC-GEMM and/or HPP-SP population, a wealth of predictive information can be obtained (unpublished results).
The CFA procedure can certainly be used to asses the effects of compounds on both the CFC-GEMM and HPP-SP populations, but its use in compound/drug screening is limited. The assay is time-consuming and requires a high degree of technical skill to count and differentiate colonies manually. It is subjective and cannot be standardized. Although multiple populations and multiple species can be compared, the studies demonstrated in this report would be impossible to perform. These drawbacks of the manual clonogenic assay procedure led to the development of the HALO Platform and the use of a highly sensitive bioluminescence signal with high-throughput capability to measure the proliferative capacity of different cell populations from different sources and species simultaneously. Since HALO is based on the methyl cellulose colony-forming assay, it was first validated against the classical assay (Rich and Hall, submitted for publication). To be used for screening compounds, it was necessary to revalidate HALO against a different cytotoxicity test system that had been used to study a number of varied compounds.
Two standard tests for basal cytotoxicity measure the effect of neutral red uptake on the mouse fibroblast 3T3 A31 cell line and normal human keritinocytes (NHK). The Registry of Cytotoxicity (RC) (Halle, 1998; Spielmann et al., 1999
), a compendium of 347 chemicals for which the IC50 values for 3T3 and NHK cells using neutral red uptake have been measured and the oral rat or mouse LD50 values are known, was used to standardize the procedures for qualifying any cytotoxicity test and to determine whether such a cytotoxicity test can use the RC Prediction Model (Halle, 1998
; Spielmann et al., 1999
). Eleven reference chemicals from the RC are used for a cytotoxicity assay. These 11 chemicals were used in the second validation phase of the HALO Platform. However, rather than testing these chemicals on a single population from a single species, we utilized the multifunctionality and high-throughput capability of the procedure to study seven different lympho-hematopoietic populations from human and mouse bone marrow simultaneously. The results from this and a second study, which employed only human bone marrow to evaluate several different anticancer drugs, demonstrated a number of commonalities that could be brought together into a prediction paradigm for hemotoxicity testing. It should be emphasized that these predictions are based on our knowledge of the biology and physiology of the lympho-hematopoietic system. However, as far as we are aware, this is the first time that a systematic study of this kind has been performed and provides direct evidence that the effects of different compounds on stem and progenitor cells can be used to predict how the blood-forming system will respond and what the effects in the periphery will be.
The correlation produced by the IC50 values of nine of the tested compounds with the LD50 values provided by the Registry of Cytotoxicity validates the HALO Platform to be used as a cytotoxicity test. This, in turn, allows the RC Prediction Model to be applied to results produced by HALO. An important application of this is shown in Table 3, where the IC values for several anticancer drugs have been used to calculate and predict clinical doses. In the last column of this table, clinical doses that have been used in the past or are actually used today for the treatment of different cancers are shown. In most cases, the estimated doses are similar to those actually used in the clinic. Another important observation is that many of the anticancer drugs tested are not employed for treatment of blood malignancies. As such, HALO, even though it is used to measure responses to the lympho-hematopoietic system, can be used to extrapolate results applicable to other systems, most notably, systems that exhibit continuous proliferation. This is because these systems have a similar organizational structure to that of the lympho-hematopoietic system comprising a stem cell, amplification and differentiation, maturation, and functional mature end cell compartment.
Finally, the ability to incorporate primary human hematopoietic tissue into a validated, predictive platform that can be used at all stages of drug development, but especially during early drug screening, cannot be underestimated. By incorporating HALO into the drug screening process, a valuable decision-making tool can be utilized that can save money, time, animals, but above all, provide increase efficacy and safety for the patient.
![]() |
NOTES |
---|
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bleiberg, H. (1997). Colorectal canceris there an alternative to 5-FU? Eur. J Cancer 33, 536541.[CrossRef][Medline]
Bradley, T. R., and Metcalf, D. (1966). The growth of mouse bone marrow cells in vitro. Aust. J. Exp. Biol. Med. 44, 287299.[ISI][Medline]
Citron, M. L., Berry, D. A., Cirrincione, C., Hudis, C., Winer, E. P., Gradishar, W. J., Davidson, N. E., Martino, S., Livingston, R., Ingle, J. N., et al. (2003). Randomized trial of dose-dense versus conventionally scheduled and sequential versus concurrent combination chemotherapy as postoperative adjuvant treatment of node-positive primary breast cancer: First report of Intergroup Trial C9741/Cancer and Leukemia Group B Trial 9741. J. Clin. Oncol. 21, 14311439.
Demetri, G. D., von, M. M., Blanke, C. D., Van den Abbeele, A. D., Eisenberg, B., Roberts, P. J., Heinrich, M. C., Tuveson, D. A., Singer, S., Janicek, M., Fletcher, J. A., et al. (2002). Efficacy and safety of imatinib mesylate in advanced gastrointestinal stromal tumors. N. Engl. J. Med. 347, 472480.
FDA (2002). Guidance for Industry and Reviewers: Estimating the safe starting dose in clinical trials for therapeutics in adult healthy volunteers. Food and Drug Administration URL: http://www.fda.gov/cder/guidance/3814dft.pdf.
Ghielmini, M., Colli, E., Bosshard, G., Pannella, G., Geroni, C., Torri, V., D'Incalci, M., Cavalli, F., and Sessa, C. (1998). Hematotoxicity on human bone marrow- and umbilical cord blood-derived progenitor cells and in vitro therapeutic index of methyoxymorpholinyldoxorubicin and its metabolites. Cancer Chemother. Pharmacol. 42, 235240.[CrossRef][ISI][Medline]
Greenwalt, D. E., Szabo, J., and Manchel, I. (2001). High throughput cell-based assay of hematopoietic progenitor differentiation. J. Biomol. Screen. 6, 383392.[CrossRef][ISI][Medline]
Gribaldo, L. (2002). Haematotoxicology: Scientific basis and regulatory aspects. ATLA 30(Suppl. 2), 111113.[ISI][Medline]
Gribaldo, L., Bueren, J., Deldar, A., Hokland, P., Meredith, C., Moneta, D., Mosesso, P., Parchment, R., Parent-Massin, D., Pessina, A., et al. (1996). The use of in vitro systems for evaluating haematotoxicity. ATLA 24, 211231.[ISI]
Halle, W. (1998). Toxizitaetspruefungen in Zellkulturen fuer eine Virhersage der akuten Toxizitaet (LD50) zur Einsparung von Tierversuchen. Life Sciences/ Lebenwissenschaften 1, 94.
Hehlmann, R., Heimpel, H., Hasford, J., Kolb, H. J., Pralle, H., Hossfeld, D. K., Queisser, W., Loffler, H., Hochhaus, A., and Heinze, B. (1994). Randomized comparison of interferon-alpha with busulfan and hydroxyurea in chronic myelogenous leukemia. The German CML Study Group. Blood 84, 40644077.
Horowitz, D., and King, A. G. (2000). Colorimetric determination of inhibition of hematopoietic progenitor cells in soft agar. J. Immunol. Methods 244, 4958.[CrossRef][ISI][Medline]
Khayat, D., Chollet, P., Antoine, E. C., Monfardini, S., Ambrosini, G., Benhammouda, A., Mazen, M. F., Sorio, R., Borg-Olivier, O., Riva, A., et al. (2001). Phase II study of sequential administration of docetaxel followed by doxorubicin and cyclophosphamide as first-line chemotherapy in metastatic breast cancer. J. Clin. Oncol. 19, 33673375.
Larson, R. A., Dodge, R. K., Linker, C. A., Stone, R. M., Powell, B. L., Lee, E. J., Schulman, P., Davey, F. R., Frankel, S. R., Bloomfield, C. D., et al. (1998). A randomized controlled trial of filgrastim during remission induction and consolidation chemotherapy for adults with acute lymphoblastic leukemia: CALGB study 9111. Blood 92, 15561564.
Lee, E. J., Petroni, G. R., Schiffer, C. A., Freter, C. E., Johnson, J. L., Barcos, M., Frizzera, G., Bloomfield, C. D., and Peterson, B. A. (2001). Brief-duration high-intensity chemotherapy for patients with small noncleaved-cell lymphoma or FAB L3 acute lymphocytic leukemia: Results of cancer and leukemia group B study 9251. J. Clin. Oncol. 19, 40144022.
Li, Q., Geiselhart, L., Mittler, J. N., Mudzinski, S. P., Lawrence, D. A., and Freed, B. M. (1996). Inhibition of human T lymphoblast proliferation by hydroquinone. Toxicol. Appl. Pharmacol. 139, 317323.[CrossRef][ISI][Medline]
Linker, C., Damon, L., Ries, C., and Navarro, W. (2002). Intensified and shortened cyclical chemotherapy for adult acute lymphoblastic leukemia. J. Clin. Oncol. 20, 24642471.
Meluch, A. A., Greco, F. A., Gray, J. R., Thomas, M., Sutton, V. M., Davis, J. L., Kalman, L. A., Shaffer, D. W., Yost, K., Rinaldi, D. A., et al. (2003). Preoperative therapy with concurrent paclitaxel/carboplatin/infusional 5-FU and radiation therapy in locoregional esophageal cancer: Final results of a Minnie Pearl Cancer Research Network phase II trial. Cancer J 9, 251260.[ISI][Medline]
Nabholtz, J. M., Falkson, C., Campos, D., Szanto, J., Martin, M., Chan, S., Pienkowski, T., Zaluski, J., Pinter, T., Krzakowski, M., et al. (2003). Docetaxel and doxorubicin compared with doxorubicin and cyclophosphamide as first-line chemotherapy for metastatic breast cancer: Results of a randomized, multicenter, phase III trial. J. Clin. Oncol. 21, 968975.
Nielsen, O. S., Dombernowsky, P., Mouridsen, H., Crowther, D., Verweij, J., Buesa, J., Steward, W., Daugaard, S., van Glabbeke, M., Kirkpatrick, A., et al. (1998). High-dose epirubicin is not an alternative to standard-dose doxorubicin in the treatment of advanced soft tissue sarcomas. A study of the EORTC soft tissue and bone sarcoma group. Br. J. Cancer 78, 16341639.[ISI][Medline]
NIH Publication (2001). Guidance document on using in vitro data to estimate in vivo starting doses for acute toxicity. No: 014500. URL: http://iccvam.niehs.nih.gov/methods/invidocs/guidance/iv_guide.htm.
Nijhof, W., Wierenga, P. K., and Kardaun, S. (1977). The effect of thiamphenicol on the production of immature red blood cells under anaemic conditions. Br. J. Haematol. 36, 2940.[ISI][Medline]
Parchment, R. E. (1998). Alternative testing systems for evaluating noncarcinogenic, hematologic toxicity. Environ. Health Perspect. 106(Suppl. 2), 541557.[ISI][Medline]
Parchment, R. E., Gordon, M., Grieshaber, C. K., Sessa, C., Volpe, D., and Ghielmini, M. (1998). Predicting hematological toxicity (myelosuppression) of cytotoxic drug therapy from in vitro tests. Ann. Oncol. 9, 357364.[Abstract]
Parent-Massin, D. (2001). Relevance of clonogenic assays in hematotoxicology. Cell Biol. Toxicol. 17, 8794.[CrossRef][ISI][Medline]
Parent-Massin, D., and Thouvenot, D. (1993). In vitro study of pesticide hematotoxicity in human and rat progenitors. J. Pharmacol. Toxicol. Methods 30, 203207.[CrossRef][ISI][Medline]
Perez, E. A., Vogel, C. L., Irwin, D. H., Kirshner, J. J., and Patel, R. (2001). Multicenter phase II trial of weekly paclitaxel in women with metastatic breast cancer. J. Clin. Oncol. 19, 42164223.
Pessina, A., Albella, B., Bayo, M., Bueren, J., Brantom, P., Casati, S., Croera, C., Gagliardi, G., Foti, P., Parchment, R., et al. (2003). Application of the CFU-GM assay to predict acute drug-induced neutropenia: An international blind trial to validate a prediction model for the maximum tolerated dose (MTD) of myelosuppressive xenobiotics. Toxicol. Sci. 75, 355367.
Pessina, A., Albella, B., Bueren, J., Brantom, P., Casati, S., Gribaldo, L., Croer, C., Gagliardi, G., Foti, P., Parchment, R., et al. (2001). Prevalidation of a model for predicting acute neutropenia by colony forming unit granulocyte/macrophage (CFC-GM) assay. Toxicol. In Vitro 15, 729740.[CrossRef][ISI][Medline]
Pessina, A., Croera, C., Bayo, M., Malerba, I., Passardi, L., Cavicchini, L., Neri, M. G., and Gribaldo, L. (2004). A methylcellulose microculture assay for the in vitro assessment of drug toxicity on granulocyte/macrophage progenitors (CFU-GM). ATLA 32, 1723.[ISI][Medline]
Pluznik, D. H., and Sachs, L. (1966). The induction of clones of normal mast cells by a substance from conditioned medium. Exp. Cell Res. 43, 553563.[CrossRef][ISI][Medline]
Quitt, M., Cassel, A., Yoffe, A., Anatol, A. M., and Froom, P. (2004). Autonomous growth of committed hematopoietic progenitors from peripheral blood of workers exposed to low levels of benzene. J. Occup. Environ. Med. 46, 2729.[ISI][Medline]
Rich, I. N. (1986). A role for the macrophage in normal hemopoiesis II Effect of varying physiological oxygen tensions on the release of hemopoietic growth factors from bone-marrow-derived macrophages in vitro. Exp. Hematol. 14, 746751.[ISI][Medline]
Rich, I. N., and Hall, K. M. (2005). Multilineage proliferation and individual growth factor response to lympho-hematopoietic colony-forming cells detected by bioluminescence. Exp. Hematol. (submitted).
Rich, I. N., and Kubanek, B. (1982). The effect of reduced oxygen tension on colony formation of erythropoietic cells in vitro. Br. J. Haematol. 52, 579588.[ISI][Medline]
Rose, P. G., Bundy, B. N., Watkins, E. B., Thigpen, J. T., Deppe, G., Maiman, M. A., Clarke-Pearson, D. L., and Insalaco, S. (1999). Concurrent cisplatin-based radiotherapy and chemotherapy for locally advanced cervical cancer. N. Engl. J. Med. 340, 11441153.
Rothenberg, M. L., and Berlin, J. D. (2003). Therapeutic strategies for metastatic colorectal cancer: Simultaneous, sequential, or specific? J. Clin. Oncol. 21, 37163717.
Ruiz, M. A., Augusto, L. G., Vassallo, J., Vigorito, A. C., Lorand-Metze, I., and Souza, C. A. (1994). Bone marrow morphology in patients with neutropenia due to chronic exposure to organic solvents (benzene): Early lesions. Pathol. Res. Pract. 190, 151154.[ISI][Medline]
Sawyers, C. L., Hochhaus, A., Feldman, E., Goldman, J. M., Miller, C. B., Ottmann, O. G., Schiffer, C. A., Talpaz, M., Guilhot, F., Deininger, M. W., et al. (2002). Imatinib induces hematologic and cytogenetic responses in patients with chronic myelogenous leukemia in myeloid blast crisis: Results of a phase II study. Blood 99, 35303539.
Seidel, H. J., Barthel, E., Schafer, F., Schad, H., and Weber, L. (1991). Action of benzene metabolites on murine hematopoietic colony-forming cells in vitro. Toxicol. Appl. Pharmacol. 111, 128131.[CrossRef][ISI][Medline]
Selby, P., Patel, P., Milan, S., Meldrum, M., Mansi, J., Mbidde, E., Brada, M., Perren, T., Forgeson, G., and Gore, M. (1990). ChlVPP combination chemotherapy for Hodgkin's disease: Long-term results. Br. J. Cancer 62, 279285.[ISI][Medline]
Smith, R. E., Brown, A. M., Mamounas, E. P., Anderson, S. J., Lembersky, B. C., Atkins, J. H., Shibata, H. R., Baez, L., DeFusco, P. A., Davila, E., et al. (1999). Randomized trial of 3-hour versus 24-hour infusion of high-dose paclitaxel in patients with metastatic or locally advanced breast cancer: National Surgical Adjuvant Breast and Bowel Project Protocol B-26. J. Clin. Oncol. 17, 34033411.
Spielmann, H., Genschow, E., Liebsch, M., and Halle, W. (1999). Determination of the starting dose for acute oral toxicity (LD50) testing in the up and down procedure (UDP) from cytotoxicity data. ATLA 27, 957966.[ISI]
Sternberg, C. N., and Parmar, M. K. (2001). Neoadjuvant chemotherapy is not (yet) standard treatment for muscle-invasive bladder cancer. J. Clin. Oncol. 19, 21S26S.
Stewart, D. J., Evans, W. K., Shepherd, F. A., Wilson, K. S., Pritchard, K. I., Trudeau, M. E., Wilson, J. J., and Martz, K. (1997). Cyclophosphamide and fluorouracil combined with mitoxantrone versus doxorubicin for breast cancer: Superiority of doxorubicin. J. Clin. Oncol. 15, 18971905.
Urba, S. G., Orringer, M. B., Ianettonni, M., Hayman, J. A., and Satoru, H. (2003). Concurrent cisplatin, paclitaxel, and radiotherapy as preoperative treatment for patients with locoregional esophageal carcinoma. Cancer 98, 21772183.[CrossRef][ISI][Medline]
Van Den Heuvel, R. L., Leppens, H., and Schoeters, G. E. (2001). Use of in vitro assays to assess hematotoxic effects of environmental compounds. Cell Biol. Toxicol. 17, 107116.[CrossRef][ISI][Medline]
Velasco, L. R., Barrera, E. E., Munoz, T. A., Tapia, A. R., Gonzalez, R. C., Garcia, L. M., Ortiz, M. V., and Betancourt, R. M. (2001). A model for the induction of aplastic anemia by subcutaneous administration of benzene in mice. Toxicology 162, 179191.[CrossRef][ISI][Medline]
|