to examine the release of CSA, light-density marrowwereperformed ..... mEg/liter). mEq/liter). mEq/Iiter) ml)Unfractienated. 0 lie ±3b ios ± 4. 125 ± 5. ± 79.
[CANCER RESEARCH 39. 321 5-321 9, August1979] 0008-5472/79/O039-0000$02.OO
Possible Mechanisms of Action of Lithium on Augmentation of in Vitro Spontaneous Myeloid Colony Formation1 Gary Spitzer,2 Dharmvir S. Verma, Barthel Barlogie, Miloslav A. Beran, and Karel A. Dicke Deportment of Developmental Therapeutics, University of Texas System Cancer Center, M.D. Anderson Hospital and Tumor Institute, Houston, Texas 77030
ABSTRACT
to delineate these mechanisms in detail using human bone marrow cells.
To understand the possible mechanisms of lithium carbon ate-induced neutmophilia,the in vitro effect on human myeboid MATERIALS AND METHODS progenitor cells was examined. A significant increase in spon taneous colony formation (15 of 24 experiments) was observed These investigations were performed after approval by the with the addition of lithium. Increased colony formation seldom local Human Investigations Committee. All patients donating occurred when human placental conditioned media as a source their marrow were informed about the nature of the investiga of colony-stimulating activity (CSA) was simultaneously added tion. to the cultures. Further data suggest that lithium requires an Acquisition of Marrow Specimens. Human marrow speci adherent marrow cell population for this action and that in mens were routinely obtained from patients with nonhemato creases in CSA-containing cultures may be due to suboptimal logical cancers without bone marrow involvement on prior CSA concentrations. Lithium was shown to release CSA from chemotherapy at the time of diagnostic bone marrow aspina marrow cells and adherent cell population prepared from hu tions. Marrow was aspirated from the posterior iliac spine. man bone marrow. Lithium possibly increases spontaneous Approximately 1 ml of marrow was placed into a tissue culture human myeboid colony development indirectly through CSA tube containing preservative-free hepanin (300 units in 2 ml of release by adherent cells. phosphate-buffered saline). Marrow Cell Preparation Used in Various Experiments. INTRODUCTION Buffy coat cells were used for the experiments designed to elicit the enhancement of spontaneous colony formation by Various reports have described consistent elevation of gran lithium and the abrogation of this effect by removal of adherent ulocytes accompanying lithium administration in psychiatric cells. Buffy coat cells were also used in those experiments patients (12, 14, 18). In 1975, Gupta et a!. (8, 9) reported that showing the effect of lithium on HPCM-stimulated marrow cells. lithium increased the leukocyte count in patients with Felty's HPCM was prepared as described previously(3). syndrome. They also documented increases in CSA3 active Ficoll-Hypaque interface cells were used in the experiments against murine bone marrow cells in the urine of these patients. designed to show the release of CSA by lithium. Recently, several authors have reported that lithium when Preparation of Buffy Coat Cells. Marrow specimens were administered to patients with various cancers may ameliorate centrifuged at 1200 x g for 10 mm in plastic culture tubes chemotherapy-induced myebosuppression (4, 6, 19, 20, 22) (Falcon Plastics, Oxnard, Calif.; Model 3033). The buffy coat and reduce the duration of granulocytopenic phase induced by was aspirated gently with a Pasteur pipet and subsequently chemotherapy in acute myeboidleukemia (5). Lithium has also used for various experiments. been used to elevate leukocytes, platelets, and hemoglobin Preparation of Light-Density Cells. Marrow specimens were levels in aplastic anemia with some success (1). diluted in equal volumes of a-MEM with I 5% FCS and centni Recently, Rothstein et a!. (15) have shown that the increase fuged at 400 x g for 35 mm after layering over a cushion of in blood neutrophil count seen after lithium administration is Ficoll-Hypaque (density, 1.077 g/mb) contained in a conical not merely due to demangination but is a result of enlargement plastic tube (Falcon Plastics; Model 3033) (2). The interface of the total blood neutrophil mass and increased neutnophil cells were aspirated gently with a Pasteur pipet, washed with production. phosphate-buffered saline, resuspended in a-MEM, and used Studies in mice have revealed that lithium enhances the CSA for the experiments mentioned later. production by lung tissue (9, 10). Using human peripheral Culture Procedure. The culture procedure has been de leukocyte undenlayers, lithium has been shown to increase scnibed before except for using HPCM as a source of CSA colony formation in in vitro agar culture system (21 ). However, instead of peripheral blood beukocytes(19). Briefly, the marrow thus far, exact mechanism(s) involved have not been cleanly preparations were cultured in equal volumes of double-strength elucidated. Herein, we report in vitro experiments performed a-MEM with 30% FCS and 0.6% agar (Bacto-agam;Difco Lab oratories, Detroit, Mich.) giving a final concentration of 0.3% ,Supported inpart byGrants CA 11520,CA 14528, andCA 19856from agar with single-strength a-MEM and 15% FCS. For all cub NIH, Bethesda,Md. tunes, 0.1 ml HPCM was used as a source of colony-stimulating 2 To whom request for reprints should be addressed, at Developmental Ther factor in underlayers of 0.5% agan and a-MEM with 15% FCS. apeutics, University of Texas System Cancer Center, M. D. Anderson Hospital The same batch of HPCM was used throughout the study. All and Tumor Institute, 6723 Bertner Avenue, Houston, Texas 77030. 3 The abbreviations used are: CSA, colony-stimulating activity; HPCM, human cultures were plated in triplicate for 7 days in a fully humidified placental conditioned medium; a-MEM, modified Eagle's medium; FCS, fetal calf atmosphere of 7% CO2 and air at 37°. serum; CFU-C, human myeloid progenitor cell. Received November 21 , 1978; accepted May 15, 1979. Culture Scoring. Cultures were scored on Day 7 using an AUGUST 1979
3215
G. Spitzer et a!. achievedanalyzed lithium in the presence of HPCM oventhat Olympus dissecting microscope at X25 to 40. They wereulationwith HPCMalone. Because of this unexpected finding, we nextcolonyfor total number of colonies (pen plate). The finalwith the effect of lithium on spontaneous colony forma incidence was the mean of the colony incidence fromexamined each ofRemoval plate for that particular observation in the study.tion.Twelve experiments were performed at a cell number of Adherent Cells. Removalof adherent cell pop 5 X 105/plate, a dose which routinely induces spontaneousulation withml was achieved by incubating 2 x 106 buffy coat cells/icolonyformation; 10 incorporated lithium in culture plates of a-MEM and 15% FCS, total volume of 2 ml in 35-mmandwithout HPCM, and 2 examined spontaneous colony for Falcon Petmidishes (Falcon Plastics; Model 3001 ) for 3 hr.mationalone. theNonadhement In other experiments, we also examined cells for culture were obtained as describedeffect at other cell doses on usually both stimulated of below. experimentsPreparation andlithium unstimulated colony formation. A total of 2 experimentssigned wasperformed with a cell dose of 1 X 1O5/dish, 9 of Conditioned Media. In the experiments de andcellsto examine the release of CSA, light-density marrowwereperformed with a cell dose of 2.5 X 105/dish, were obtained by Ficoll-Hypaque gradient centnifugationbecauseof were(density, the limitation of cell numbers, 5 experiments 1.077 g/mb) as described above.performedwith cells/dish.To 7.5 X 1O@ prepare the conditioned media, 2 x 106 interface cellsChart1 , A and B, are representative of experiments exam from a Ficoll-Hypaque gradient were incubated per 1 ml of a-ining theeffect of lithium on both spontaneous colony formationMEM theseTo and 15% FCS (total volumeof 2 ml; total cells 4 x 106).and that induced with HPCM. As can be seen from spontaneousinterface induced variable enhancement of obtain adherent cell-conditioned media, 2 x 106/mI ofgraphs,lithium bydescribed For example, the experiment represented cells were subjected to adherence procedure ascolonyformation. formationwere 1Ashows enhancement of spontaneous colony above (4 x 106 cells/dish), and nonadhenent cellsChart 0.05.MEM. highertarget cell numbers (5 and 7.5 X 1O@ cells), p < nemoved by 2 vigorous washings of Petni dishes with a-at byincubated in spontaneous colony formation induced Subsequently, the Petni dishes with adherent cells wereThisdifference thefor was abrogated when HPCM was added to (each dish containing 2 ml of a-MEM with 15% FCS)lithium,however, dishes.at varying periods in a fully humidified atmosphere of 7% CO2culture experimentspared 37°.Conditioned media from nonadherent cells were pre Chart2A shows the cumulative data of the thising by centrifuging the aspirated nonadherent cells, remov performed with HPCM and lithium. As can be seen from variable.in theeffect of lithium on CSA-stimulated plates is the supemnatant,nesuspending the cells to a volume of 2 mlchart, theplastic 2Bis the cumulative data from experiments in which a-MEM and 15% FCS, and reseeding into a new 35-mmChart Petni dish.effect oflithium on spontaneous colony formation was exam Conditioned media were harvested at specific times andmed.Enhancement aprepared of spontaneous colony formation was 7through by centrifuging at 2000 rpm for 10 mm and passingcommonphenomenon. In 6 of 9 experiments at 2.5 X 1O@, a Millipone filter (0.45 sm). Conditioned media wereof lithiumstored 12 at 5 x 10@,and 2 of 5 at 7.5 x 1O@cells, at —20° until assayed on 0.5 x 1O@nonadherent, light moredensity increased spontaneous cloning efficiency by 50% on (“, .@o
15 Chart 5. The effect of lithium on CSA release by human marrow cells. A, nenadherent cells net re moved; and B, adherent cells alone. Lithium effect maximum release of CSA at 96 hr followed by decline in the activity
by 7 days
of incubation
0,
10
endotexin
(100 1fg/ml); t@,lithium(3 mEq/liter); 0, lithIum 1.5 mEg/liter; •,lithium (0.5 mEq/Iiter); A, cells alone. Bars, S.D. In this experiment, the CSA release from adherent cells (B) is much less than that released when nonadherent cells are not removed.
20
0@
E z 0
24
72
96
168
Periodof incubation (hours) clear cells. There are no reports that document lithium-induced CSA release by human marrow cell populations or the release of CSA active on human bone marrow. It has been suggested that the colony-stimulating cell population in marrow may be of more significance in vivo than are peripheral mononuclear cells. We, therefore, decided to examine the possibility that lithium may require a bone marrow-adherent cell population for its mechanism of action and that the mechanism of action may be through the release of CSA from that population.
When
bone marrow adherent cells are removed prior to culture with lithium, themewas a marked reduction of both spontaneous colony formation and lithium enhancement of spontaneous colony formation. When lithium was incubated with light-density (
