be13, a human t-leukemia cell line highly sensitive to ... · more than 90% of the be13 cells...

[CANCER RESEARCH 44, 4594-4601, October 1984]

Be13, a Human T-Leukemia Cell Line Highly Sensitive to Dexamethasone-induced Cytolysis1

Uri Calili,2 Aviva Peleg, Yoram Milner, and Naomi Calili

Department of Hematology, Hadassah University Hospital [U. G., A. P.], Department of Biological Chemistry, Hebrew University [Y. M.J, and Department of Virology,Hadassah Medical School [N. G.], Jerusalem, Israel

ABSTRACT

A unique human T-leukemia cell line highly sensitive to dexa-methasone-induced lysis is described. The cell line designatedBe13 is killed readily within 24 hr by 10~9 M dexamethasone. No

lysis is induced by nonglucocorticoid steroids. The lysis is mediated via specific cytoplasmic receptors and is efficiently blockedby the antagonist cortexolone. The inhibiting effect of actinomy-cin D and cycloheximide on the lytic process suggests theinvolvement of gene activation and destruction of the cells by an"autolytic protein." Kinetic studies imply that the lytic process is

induced during a distinct phase of the cell cycle. Dexamethasone,however, does not cause an arrest in a distinct phase of the cellcycle. The Be13 cell is a unique human cell line killed directly byglucocorticoids, and it may serve as a suitable in vitro model forstudying the lytic effect of glucocorticoids on the proliferatingcompartment of human leukemias.

INTRODUCTION

GC3 play a major role in the chemotherapeutic regimen of ALL.

Administration of these hormones results in a decrease in themalignant lymphoid cell population in a proportion of the ALLpatients (17). The GC effect in humans has been attributed tothe inhibition of cell proliferation rather than to a direct lytic effect(6). This concept emerged from the findings that GC do not killhuman thymocytes or peripheral blood lymphocytes but, rather,affect the mitogenic or allogeneic stimulation of peripheral bloodlymphocytes (3, 4). In contrast, murine and rat thymocytes andlymphocytes are killed readily by these hormones (5, 7, 28).Humans have thus been designated a "GC-resistant" species,whereas mice or rats are "sensitive" species (6). In a series of

studies, we have demonstrated recently several normal andmalignant human lymphoid subsets which are lysed by the upperphysiological and pharmacological concentrations of GC. Theseinclude human prothymocytes (13), activated T-cells (9, 15, 16),CLL cells (12, 14), and ALL cells from some of the patientstested (11,14). Myeloid leukemia cells were found to be resistantto the GC-induced lysis (11,14). In an attempt to understand themechanism of this specific GC-induced lysis, the nonproliferatingCLL cells and immunoactivated T-cells were utilized for a model

system (11,12). These studies indicated that, in the nonproliferating GC-sensitive cells, a short exposure to the hormone was

sufficient to induce an irreversible lytic process. In addition, the

1This work was supported also by the Samuel Wang award granted by the

Israel Cancer Research Fund.2Leukemia Society of America Special Fellow. To whom requests for reprints

should be addressed, at Hematology Research Laboratory, School of Medicine,Cancer Research Institute, University of California, San Francisco, CA 94143.

3The abbreviations used are: GC, glucocorticoids; ALL, acute lymphoblastic

leukemia; CLL, chronic lymphocytic leukemia.Received March 5, 1984; accepted July 6, 1984.

lysis involved specific binding of the GC molecule to a cytoplasmic receptor, with the subsequent activation of a specificgene(s), for the possible synthesis of an "autolytic protein" (11,

12).Since the major objective of chemotherapy regimens is the

erradication of the proliferating malignant clone, the best modelfor studies of GC-induced lysis is a proliferating human lymphoidcell line. Much search has been made for such a GC-sensitiveline to parallel the murine S49 cell line (30). Thus far, only theCEM clone 7 human T-leukemia cell line has been reported toexhibit proliferation inhibition when exposed to 10~6 M dexa

methasone (18, 25). These cells were arrested, in the presenceof GC, in the d phase of the cell cycle. The arrest resulted incell death commencing within 48 hr of incubation (18). We reporthere on a new human T-cell line, isolated from a T-ALL patient

and designated Be13, which is killed within 16 to 24 hr ofincubation with as little as 1CT9M dexamethasone. Analysis of

the lytic process in these cells revealed some difference in thepatterns of cell death from those found previously in the nonproliferating CLL malignant cells.

MATERIALS AND METHODS

Cell Lines. The Be13 cell line was derived from the bone marrow cellsof an 11-year-old patient with T-ALL in relapse (10). Other cell linesstudied were the T-cell line HD-Mar, derived from a patient with Hodgkin'slymphoma (2), the B-cell line Daudi, derived from a Burkitt's lymphoma

patient (20), and myeloid cell line K562, originally grown from a chronicmyeloid leukemia patient in blast crisis (22). All cell lines were grown at37Â° in suspension culture by using the nutrient medium RPMI 1640

(Grand Island Biological Co., Grand Island, NY) supplemented with 10%(v/v) heat-inactivated fetal calf serum.

Surface Markers. E-receptor, Fc-receptor, T-cell antigen, Smlg, and

la antigen detection were performed according to previously described,routine methods (13).

In Vitro GC-induced Cytolysis. The test was performed as describedpreviously (11-16). Briefly, the cells were incubated with various concentrations of steroids for 24 hr at 37Â°in a 5% COz humidified atmosphere.

All steroids used were purchased from Sigma Chemical Co. (St. Louis,MO). The percentage of cytolysis was calculated by means of the formula

(a-b)x 100

where a is the concentration of viable cells in control wells and Ã¶is theconcentration of viable cells in wells containing the steroid. Viability wasassessed by trypan blue exclusion test. The dye penetrates only intodead cells.

Measurement of Total Cellular Binding and Nuclear Translocationof Dexamethasone (24,32). Two ml of 50 x 106 cells/ml were incubatedwith 2 iiC\ of 7 x 10~8 M [3H]dexamethasone (New England Nuclear;

specific activity, 27 Ci/mmol), for 1 hr at 0Â°or 37Â°. High-affinity [3H]-

dexamethasone binding was measured by addition of 0.2-ml aliquots ofcells to 10 ml of medium (50-fold dilution) and incubation of the diluted

4594 CANCER RESEARCH VOL. 44

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A Human T-Cell Line Sensitive to Dexamethasone Lysis

cells for an additional 30 min. High-affinity dexamethasone binding remains unaffected in the diluted cells incubated at 0Â°,whereas complete

dissociation of the steroid-receptor complex occurs in cells incubated at37Â°.The cells were thereafter washed twice in chilled medium, and the

radioactivity within the cell pellet was measured in Tricarb scintillationcounter utilizing Instagel as scintillation fluid (Packard). Efficiency ofmeasuring radioactivity was 35%. The amount of [3H]dexamethasonebound in diluted cells at 37Â°was substracted from the amount of [3H]-dexamethasone at 0Â°to obtain the specific binding of the hormone.

The proportion of dexamethasone translocated into the nucleus wasassessed by measuring radioactivity within the nuclei. The procedurewas carried out as described above for cellular binding, except that 1.5mm MgCI2 was used instead of medium when cells were diluted. Underthese conditions, the cell surface membrane is selectively lysed, whilethe nucleus remains intact. Nuclear translocation of dexamethasone wasexpressed as the proportion of specific radioactivity within the nucleifraction.

For additional assessment of dexamethasone binding and comparisonwith cortisol binding to the Be13 cells, receptor analysis was performedby incubation of 1-ml aliquots of 3 x 106 cells/ml in RPM11640 mediumwith increasing concentrations (1 to 100 nw) of [3H]dexamethasone(Amersham International, England; specific activity, 87 Ci/mmol), or [3H]-

cortisol (Amersham International; specific activity, 85 Ci/mmol) as tracersalone or in the presence of 10~5 M of the corresponding unlabeled GC.

The cells were incubated for 120 min at 37Â° in a 5% CO2 humidified

atmosphere. At the end of the incubation period, the cells were washedtwice with chilled medium, and radioactivity was measured as describedabove. The equilibrium dissociation constant (Ka) of receptor sites andthe number of receptors per cell were obtained from Scatchard analysisof the GC interaction (26).

Electron Microscopy. Scanning and transmission electron microscopy studies were carried out according to techniques described in ourprevious studies (12).

Metabolic Inhibitors. Actinomycin D (Sigma), an inhibitor of RNAsynthesis, and cycloheximide (Sigma), an inhibitor of protein synthesis,were used to determine whether transcriptional and translational eventsoccur in the cells during the course of dexamethasone-induced cytolysis.

[3H]Thymidine Incorporation in Be13 cells. Aliquots of 1 ml containing 106 Be13 cells were incubated for 1 hr at 37Â°in presence of 1 nC\of [3H]thymidine (Nuclear Research Center, Negev, Israel). Thereafter,

the cells were harvested on glass fiber filters and precipitated with cold5% trichloroacetic acid. The filters were dried, and radioactivity wasmeasured in Tricarb scintillation counter (Packard).

RESULTS

Specific Dexamethasone-induced Lysis of Be13 Cells.More than 90% of the Be13 cells incubated with 10~5 to 10~7 M

dexamethasone were lysed in a 24-hr period (Chart 1). Loweringthe concentration of dexamethasone to 10~9 M resulted in a 60%lysis of the cells. Incubation of the cells for 48 hr with 10~9 M

dexamethasone induced the lysis of 99% of the Be13 cells (notshown). The other GC hormone tested, cortisol, was less effective, however, causing 65% lysis at 10~5 M and 40% lysis at 10~6

M. Sex hormones such as estradici, progesterone, or testosterone did not affect the viability of the Be13 cells at a concentrationof 10-5M.

The observed lytic effect is highly specific to the Be13 cell line,which is arrested at a differentiation state between the prothy-

mocyte and cortical thymocyte stages (Table 1) (10). Thus, whileit expresses the T-antigen, only a partial E-rosetting capacity isobserved. The other T-cell line, HD-Mar, bearing cortical thymocyte characteristics (18), including the T-antigen and E-rosetting

capacity (Table 1), proliferated at the same rate as did control

100

80

ioÂ¡7560

20

010" 10=io10 io9 io'8 io'7 io6

STEROID CONCENTRATION [M]

Chart 1. Cytolysis of Be13 cells by various steroids: O, dexamethasone; â€¢,cortisol; O, estradici; D, progesterone; A, testosterone; V, cortexolone. Cell deathwas determined after 24 hr of incubation with the steroids at 37Â°.

Table 1

Specific pH]oexamethasone binding to various human cell lines

% of nu-Specific [3H)dexa- clearmethasone binding transiÃ³-

CelllineBe13HD-MarDandiK562PhenotypeT-type,

Tag-100%,Eros.-20%,Smlg-neg.,

la-neg.T-type,Tag-100%,Eros.-98%,Smlg-neg..

la-neg.B-type,Tag-neg.,Eros.-neg.,Smlg-100%,

la-100%Myeloid,Tag-neg.,Eros.-neg.,Smlg-neg.,

la-100%Ref.1022022(dpm/1

0Â»cells)28,000

Â±1,500*31

,400Â±2,10030,1

00Â±1,80033,500

Â±2,600cation868758072

Calculated at proportion of specific nuclear labeling. No translocation of theradiolabeled hormone to nuclei was detected at 4Â°(<600 dpm).

" Mean Â±S.D. of triplicate cultures.

cultures, in the presence of 10~6 M dexamethasone (Chart 2).

Similarly, growth of the B-cell line Daudi and the myeloid cell lineK562 was not affected by 10"6 M dexamethasone.

Involvement of Specific GC Receptors in the Lytic Process.Steroid action in mammalian cells is mediated via the interactionof the rapidly penetrating steroid molecules with specific cyto-plasmic receptors. The formation of steroid-receptor complexesin the course of the GC-induced lysis of Be13 cells was studiedby the use of: (a) specific GC antagonists; and (b) 3H-labeled

dexamethasone.A 100-fold excess of cortexolone, a potent antagonist for

receptor binding which cannot elicit nuclear events (31), partiallyinhibited the lytic effect of 10~7 M dexamethasone (Chart 3).Complete inhibition was seen when 10"8 M or 10~9 M dexameth

asone was used. Similarly, complete inhibition of lysis inducedby 10~6 M cortisol was achieved by 10"5 M cortexolone. The sex

hormones estradici and testosterone, which are partial antagonists (1), exhibited effective inhibition at 10~5 M concentrationonly with the very low concentration of 10~9 M dexamethasone.

No inhibition effect was elicited by these sex hormones on thecytolytic activity of 10~8 M dexamethasone.

Additional evidence for the presence of specific cytoplasmicreceptors to GC was obtained by the binding of [3H]dexameth-

asone (Table 1). The amount of the bound steroids correspondsto about 10,000 receptor sites/cell. While GC-receptor interac-

OCTOBER 1984 4595



U. Calili et al.

1.4r

ZHIu

oo

LUU

DAYS OF INCUBATIONChart2. In vitro growth of various human cell lines in presence of IO"6 M

dexamethasone. O, Bel 3 cells; G, HD-Mar cells; A, Daudi cells; V, K562 cells.Number of viable cells/ml in all cell lines except Be13 did not differ in controlcultures from that observed in cultures containing the hormone. Growth curve ofBe13 control cultures is the same as that of Daudi cells.

Gluco-corticoid

dex 10." M

dex. 10rM

Antagonist

cortexolone 10 M

â€¢I,OF LYSIS

played by dexamethasone. The KÂ¿for cortisol was found to be2.73 nw using the first 5 points of the binding curve. The actualK,, is even lower, since the binding curve did not reach asaturation in the cortisol concentration used. Accordingly, at 10nw, GC concentration, dexamethasone occupied 6500 receptors, whereas cortisol bound to only 300 receptor molecules.

Kinetics of the Lytic Process. Be13 cells incubated with 10 7

M dexamethasone for 24 hr were almost entirely lysed (>90%).This lysis could not be observed in the light microscope before16 hr of incubation and, at 20 hr of incubation, only 60 to 80%of cells were killed (Chart 5). The presence of the hormone inthe medium for the entire incubation period was essential forlysis. When dexamethasone was removed after 8 hr, no lysiswas observed at the end of the 24- or 48-hr period. Exposure ofthe cells for 12 hr to 10~7 M dexamethasone, however, resulted

in 20% lysis at the end of an additional 12-hr incubation and

100% lysis following 20 hr of incubation in the absence of thehormone (Chart 5). Incubation for 16 hr with dexamethasoneresulted in complete lysis of the cells only after an additional 16hr of incubation without the hormone.

These findings suggest that the lytic process is induced duringspecific stages of the cell cycle. A substantial proportion of thecells must pass this specific stage before a measurable lyticeffect is seen. Dexamethasone does not, however, cause anarrest in the cell cycle of the nonlysed population of cells. This isdemonstrated clearly by the findings that DMA synthesis of the106 viable cells did not vary following 24 hr of incubation with10~7 M dexamethasone (Chart 6). Furthermore, while only 5% of

dex 10"8M

dex IO"8M

dex

dex

dex

dex

1Ã–9M

irj9M

irJ9M

irj9M

cortexolone 10

cortexolone 10" M

testosterone10"5M

estradici 10"5 M

)5M fj

cortisol 10~6M

cortisol 10" Mcortexolone 10

'.FCharts. Inhibition of GC-induced lysis of Bet3 cells by various antagonists.

dex., dexamethasone.

tion is a prerequisite for induction of lysis, it is not sufficient forultimate lysis. Thus, the binding of [3H]dexamethasone to the

resistant cell lines was of a level similar to that of the sensitiveBe13 cells. Furthermore, no significant difference in the proportion of nuclear translocation of the bound steroid was noted inthe different cell lines. In all cell lines tested, 70 to 80% of thereceptor-bound [3H]dexamethasone was translocated to the nucleus during incubation of cells in 37Â°but not at 4Â°(Table 1).

Further analysis of the dexamethasone binding curve in Be13cells demonstrates a high-affinity interaction between the hor

mone and the cytoplasmic receptors (Chart 4A). Scatchard analysis of the binding data yielded a straight line with a sloperepresenting a KO of 5.55 rriM. The X-intercept provided an

estimation of 6500 receptors/cell, which is lower than that foundin the above method, probably due to the difference in methodology. The binding curve of cortisol (Chart 4B) demonstrated amuch lower affinity to the cytoplasmic receptors than that dis-

10

8

E- 6l/Ã®Q

x ^LU OD CD

Z 6 IOBtpM)

0 20 Â¿0

MOLAR CONCENTRATION OF DEX.

60x10~9M

B5 2.0~

- 1.6

3 g08

ceOu

0.4

0" 0 20 40 60x109M

MOLAR CONCENTRATION OF CORTISOLChart 4. The specific binding of [3H]dexamethasone (DEX.) (A) and [3H]cortisol

(ÃŸ)to 10' Bei 3 cells as a function of tracer concentration. Inset, Scatchard analysis

of the obtained data. B, bound; B/F, bound/free.





100r

80

60

20

8

HR OF

12 16

INCUBATION

20 2i

Chart 5. Cytolysis of Be13 cells following 12-hr (â€¢)and 16-hr (D) preincubationwith 10~7 M dexamethasone. Time 0 represents removal of dexamethasone from

cultures. O, control cultures in which the hormone is present during the incubationperiod.

(/)LUKg0Ou.

o^3.02.41.81.20.6

n10080-

&60ÃŽ3-Ã«

4020n

- D 20

16

12"

12 16 20 24

HR 0F INCUBATIONChart 6. Bel 3 cell cycte parameters in the presence of 10"7 M dexamethasone.

O, % of Bel 3 cell lysis; A, % of mitotic figures among viable cells; D, [3H]thymidineincorporation to 10* alive Bel 3 cells. For assessment of [3H]thymidine incorporationat 16,20, and 24 hr, cell suspensions were brought to a concentration of 10* viable

cells/ml.

the cells remained viable after 24 hr with dexamethasone, theproportion of Be13 cells with mitotic figures was similar to thatfound in control culture. This can be seen by electron microscopyas well (Fig. 8). After 24 hr with the steroid, a proportion of theviable cells display mitotic figures, indicating an ongoing cellproliferation.

Ultrastructural Changes in the Course of Lysis. Analysis byscanning electron microscope showed that no changes occuredin the cells incubated with dexamethasone for 8 hr. At 16 hr, aloss of microvilli resulting in a smoothening of the cell surfacewas seen (Figs. 1 and 2). This was followed by the appearanceof pores in the cell membrane. Observations in a transmissionelectron microscope indicated disruption in polysomes and homogenous distribution of ribosomes, as one of the first morphological alterations in the affected cell cytoplasm. This occured at16 hr (Figs. 3 to 6). The loss of microvilli was a concurrent event.Pyknosis of the nucleus was noted only in advanced stages ofthe lytic process, together with the disruption of the cell membrane and destruction of cytoplasmic organization (Fig. 7).

Transcriptional and Translational Events in the Course ofLysis. The model of steroid action includes the synthesis ofspecific mRNA and protein molecules for the manifestation of

100r

80

Â¿7560

20

10'Â° io9 lÃ²6 lÃ²7 lo6 lÃ²'

DEX. CONCENTRATION [M]

Chart 7. Inhibitory effect of actinomycin D (0.1 Â»ig/ml)(D) and cycloneximide (1ng/ml) (A) on dexamethasone (DÂ£X.)-induced lysis of Be13 cells. O, culturescontaining dexamethasone without metabolic inhibitors.

the biological effect of the hormone (8). In Be13 cells, inhibitorsof RNA and protein synthesis reduced the lytic effect of dexamethasone. When the RNA inhibitor actinomycin D was addedat a concentration of 0.1 ^g/ml, complete inhibition of lysisinduced by 10~7 and 10~8 M dexamethasone was observed(Chart 7). A partial inhibition of the lysis occured with 10~5 and10~6 M of the hormone. The protein synthesis inhibitor cyclohex-

imide (1 nQlm\) exhibited a more potent inhibitory effect andabolished the lysis induced by dexamethasone, even at the highconcentration of 10~5 M.

DISCUSSION

The Be1 3 cell line, originating from a T-ALL patient, was foundto be highly sensitive to GC-induced lysis and, thus, it could be

used to study the lytic phenomenon in proliferating malignantlymphoid cells. In contrast to freshly isolated leukemic cells, inwhich cortisol was as effective as was dexamethasone in inducing lysis (12), a significant difference in the killing potency ofthese 2 hormones was observed when Be13 cells were thetarget cells. Dexamethasone at a concentration of 10~9 M was

sufficient to induce 60 to 70% lysis during 24 hr as compared to10~6 M cortisol. This difference seems to be due to a higher

affinity of the dexamethasone for the cytoplasmic receptors. Thedifferent affinities of these 2 GC hormones to the cytoplasmicreceptors of the Be13 cells was demonstrated clearly in thebinding curve experiments with the radiolabeled hormones andby the K<, values obtained. The competitive inhibition effectexerted by the antagonist cortexolone further indicated the formation of steroid-receptor complexes. A 1000-fold increase inthe antagonist concentration competed with 10~8 M dexameth

asone to abolish the lytic effect. A 10-fold increase was sufficient,however, to block the lytic effect of 10~6 M cortisol, further

suggesting that dexamethasone does, indeed, bind with a higheraffinity to the cytoplasmic receptor.

Both actinomycin D and cycloheximide inhibited the lysis ofBe13 cells by dexamethasone, suggesting that gene activationis involved in the lytic action of GC. In line with the general modelof steroid action, the killing of Be13 cells involves the translocation of the dexamethasone-receptor complex into the nucleus,

where it apparently activates a gene(s) to synthesize a specificmRNA and subsequent "autolytic" protein. The presence of this

OCTOBER 1984 4597



U. Galili et al.

protein is only hypothetical, since it has not as yet been isolatedand identified. The antagonistic effect of the metabolic inhibitorsraises the need, however, for a critical evaluation of the clinicalbenefit of the administration of drugs with such an activity,concurrently with GC in the course of chemotherapeutic regimens.

When analyzed by electron microscopy, the morphologicalfeatures of the lytic process were found to resemble those ofthe nonproliferating CLL cells (11 ). Loss of membrane microvilliand the disappearance of polysomal organization preceded theappearance of membranal pores and nuclear destruction. Incontrast, the kinetics of cell lysis was found to differ greatly inthe 2 cell types. Whereas CLL death could be detected within 6hr of incubation with GC, only at 16 to 18 hr could lysis beobserved in the Be13 cell line. Exposure of CLL cells to thehormone for a short period of 30 min was sufficient to evoke anirreversible lytic process (12); in the Be13 cells, incubation of atleast 12 hr was required to induce cell lysis following an additional36 hr in culture.

These results suggest that the induction of lysis in a proliferating cell is dependent on a specific stage of the cell growthcycle. It is possible that the genes required for activation by thesteroid-receptor complex are "accessible" only at a specific

phase of the cell cycle. CLL cells, which are all at the samephase, require only a short exposure to the steroid for inductionof the lytic process. Studies on synchronized Be13 cells arepresently being conducted to clarify this point.

The sensitivity of a particular malignant cell to GC appears tobe dependent upon the state of differentiation in which the cellis arrested. The Be13 cell, arrested at the prothymocyte stageof T-cell differentiation (10), is highly sensitive to GC-inducedlysis. In contrast, the HD-Mar cell line, which reflects the more

mature cortical thymocyte differentiation state, is resistant to aconcentration as high as 10~6 M dexamethasone. The B- cell line

Daudi and the myeloid cell line K562 are similarly resistant. Thesefindings correlate well with our previous studies on normal humanlymphoid cells (12-16). Human prothymocytes and activated T-cells were found to be sensitive to GC-induced lysis, while thecortical thymocytes, peripheral T-cells, B-cells, and myeloid cells

were resistant. Thus, the transformation event seems not tochange the degree of sensitivity of the cell to steroid-induced

lysis. Establishment of additional human GC sensitive cell linesis needed for confirmation. It should be noted that dexamethasone was found to bind equally well to the resistant and sensitivecell lines. Furthermore, translocation to the nucleus was similarin all cell lines. This is in agreement with our previous findings(9) and those of other investigators (19, 33), demonstrating nosignificant difference in these 2 parameters in GC-resistant and-sensitive cells. It should be noted, however, that other studieshave shown that, in certain lymphoid cell lines and some freshlyisolated leukemic cells, the GC effect does relate to the numberof receptors per cell (21, 27). Various, not fully understood,cellular factors thus seem to be involved in rendering a cellsensitive to GC lytic action. Analysis of the variations in cyto-

plasmic receptors (23, 29) and identification of the relevantgene(s) in relation to specific differentiation states will be helpfulin understanding the cause of cell resistance to GC therapy.

Thus far, Be13 is the only known human cell line which isdirectly lysed by GC. Harmon ef a/. (18) have reported on ahuman T-cell line, CEM-7, which is arrested in the GÃ¬phasefollowing exposure to 10~6 M dexamethasone for 2 to 3 days. At

Day 4 to 5, cells become pyknotic and are lysed. The death ofthe CEM-7 cells seems to be secondary to the arrest in cell

cycle. Thus, the effect of dexamethasone on these cells wasmost prominent in reducing the plating efficiency of the cell line,analyzed after 10 to 14 days of incubation with the hormone. Noplating efficiency changes could be tested with the Be13 cells,since they were unable to form colonies in soft agar; however,the Be13 cells, unlike CEM-7 cells, are lysed already within 16

to 20 hr by a direct mechanism which seems to involve specificgene activation. Furthermore, the cell cycle does not seem to beaffected by the dexamethasone, as indicated by the unalteredproportion of Be13 cells in S- and M-phases within the small

viable cell population remaining after 24 hr of exposure to thehormone. Nevertheless, both studies indicate that proliferatingleukemic cells must be exposed for relatively prolonged periodsto GC in order to elicit a response. This suggests that effectivetherapeutic response is dependent on a continuous administration of the steroids. In view of the short half-life of steroids in

circulation, administration of GC in pulses may result in theselection of relatively resistant clones of proliferating leukemiccells. At present, we are attempting to isolate resistant clones ofBe13 cells in order to investigate the mechanisms enabling GC-sensitive human leukemias to escape total eradication by steroidtherapy.

ACKNOWLEDGMENTS

The skillful electron microscopy work of Rina Brandt is gratefully acknowledged.

REFERENCES

1. Baxter, J. D., and Tomkins, G. M. Specific cytoplasme glucocorticoid hormonereceptors in hepatoma tissue culture cells. Proc. Nati. Acad. Sci. USA, 68:932-937,1971.

2. Ben-Bassat, H., Mitrani-Rosenbaum, S., Gamliel, H., Naparstek, E., Leizer-owitz, R., Korkesh, A., Sagi, M., Voss, R., Kohn, G., and Polliack, A. Establishment in continuous culture of a T-lymphoid cell line (HD-Mar) from a patientwith Hodgkin's lymphoma. Int. J. Cancer, 25: 583-589, 1980.

3. CarÃ³n, G. A. Prednisolone inhibition of DNA synthesis by human lymphocytesinduced in vitro by phytohemagglutinin. Int. Arch. Allergy Appi. Immunol., 32:191-200,1967.

4. Cheigh, J. S., Stanza. K. J., Riggto, R. R., Katz, E. D., and Rubin, A. L. Effectof intravenous methylprednisolone on mixed lymphocyte cultures in normalhumans. Transplant. Proc., 7: 31-35, 1975.

5. Claesson, M. H., and Ropke, C. Quantitative studies on cortisol-induced decayof lymphoid cells in the thymolymphatic system. Acta PathcH.Microbio!. Scand.,76:376-382,1969.

6. Claman, H. N. Corticosteroids and lymphoid cells. N. Engl. J. Med. 287: 388-397, 1972.

7. Cowan, W. L., and Sorenson, G. D. Electron microscopic observations ofacute thymic involution produced by hydrocortisone. Lab. Invest., 73: 353-370, 1964.

8. Edelman, I. S. Mechanism of action of steroid hormones. J. Steroid Biochem.,6:147-159, 1975.

9. Galili, N., Galili, U., Klein, E.. Rosenthal, L., and Nordenskjold, B. Human T-lymphocytes become glucocorticoid-sensitive upon immune activation. Cell.Immunol., 50: 440-444,1980.

10. Galili, N., Galili, U., Ravid, Z.. Schlesinger, M., and Goldblum, N. Induction ofdifferentiation with phorbol ester in a human T-cell line (Be13) expressing bothprothymocyte and thymocyte characteristics. Hum. Lymph. Differen., 1:123-130,1981.

11. Galili, U. Glucocorticoid-induced cytolysis of human normal and malignantlymphocytes. J. Steroid Biochem., 79: 483-490,1983.

12. Galili, U., Leizerowitz, R., Moreb, J., Gamliel, H., GÃ¼rtel.D., and Polliack, A.Metabolic and ultrastructural aspects of in vitro lysis of chronic lymphocyticleukemia cells by glucocorticoids. Cancer Res., 42:1433-1440,1982.

13. Galili, U., Polliack, A., Okon, E., Leizerowitz, R., Gamliel, H., Korkesh, A.,Shenker, J. C., and Izak, G. Human prothymocytes: membrane properties,differentiation patterns, glucocorticoid sensitivity, and ultrastructural features.J. Exp. Med., 752: 796-810, 1980.

14. Galili, U., Prokocimer. M., and Izak, G. The in vitro sensitivity of leukemic andnormal leukocytes to hydrocortisone-induced cytolysis. Blood, 56:1077-1081,




1980.15. Calili, U., Rosenthal, L., Calili, N., and Klein, E. Activated T-cells in the synovial

fluid of arthritic patients: characterization and comparison with in vitro activatedhuman and murine T-cells in cooperation with monocytes in cytotoxicity. J.Immunol., 122: 878-883, 1979.

16. Calili, U., Seely, J., Svedmyr, E., Klein, E., Klein, G., and Weiland, O. Bloodlymphocytes in infectious mononucleosis share the following characteristicswith activated T-cells: natural attachment, stable E-rosetting, and glucocorti-coid sensitivity. J. Clin. Lab. Immunol., 3:153-158,1980.

17. Goldin, A., Sandberg, J., Henderson, E., Newman, J., Frie, E., and Holland, J.The chemotherapy of human and animal acute leukemia. Cancer Chemother.Rep., 736:213-221,1962.

18. Harmon, J. M., Norman, M. R., Fowlkes, B. ., and Thompson, E. B. Dexa-methasone induces irreversible G, arrest and death of human lymphoid cellline. J. Cell. Phystol., 98: 267-278,1979.

19. lacobelli, S., Natoli, V., Longo, P., Ranelletti, F. 0., DeRossi, G., Pasqualetti,0., Mandelli, F., and Mastrangelo, R. Glucocorticoid receptor determination inleukemia patients using cytosol and whole cell assay. Cancer Res., 41:3979-

3984,1981.20. Klein, E., Klein, G., Nadkami, J. S., Nadkami, J. J., Wigzell, H., and Clifford, P.

Surface IgM-kappa specificity on Burkitt's lymphoma cells in vivo and in derived

culture lines. Cancer Res., 28:1300-1310,1968.21. uppman, M. E., Yarbro, G. K., and Leventhal, B. G. Clinical implications of

glucocorticoid receptors in human leukemia and lymphoma cells. Cancer Res.,38:4251-4257,1978.

22. Lozzio, C. B., and Lozzio, B. B. Human chronic myelogenous leukemia cellline with positive Philadelphia chromosome. Blood, 45: 321-328, 1975.

23. McCaffrey, R., ullquist, A., and Bell, R. Abnormal glucocorticoid receptors in


acute leukemia cells. Blood, 59. 393-400,1982.24. McPartland, R. P., Milholland, R. J., and Rosen, F. Nuclear binding of steroid

receptor complexes to lymphosarcoma P1798-resistant and -sensitive cellsand effect of concanavalin A on receptor levels. Cancer Res., 37:4256-4260,

1977.25. Norman, M. R., and Thompson, E. B. Characterization of a glucocorticoid-

sensitive human lymphoid cell line. Cancer Res., 37: 3785-3791,1977.26. Scatchard, G. The attractions of proteins for small molecules and ions. Ann.

NY Acad. Sci., 51: 660-666,1949.27. Schmidt, T. J., Harmon, J. M., and Thompson, E. B. Activation "labile"

glucocorticoid-receptor complexes of a steroid-resistant variant of CEM-C7human lymphoid cells. Nature (Lond.), 286: 507-510,1980.

28. Schrek, R. Cytotoxicity of adrenal cortex hormones on normal and malignantlymphocytes in man and rat. Proc. Soc. Exp. Bio). Med., 108:328-332,1961.

29. Sherman, M. R., Pickering, L. A., Rollwagen, R. M., and Miller, L. K. Merore-ceptors: proteotytic fragments of receptors containing steroid binding site.Fed. Proc., 37. 167-173, 1978.

30. Sibtey, C. H., and Yamamoto, K. R. Mouse lymphoma cells: mechanisms ofresistance to glucocorticoids. Monogr. Endocrinol., 12: 357-376,1979.

31. Tumell, R. W., Kaiser, N., Milholland, R. J., and Rosen, F. Glucocorticoidreceptors in rat thymocytes. Interaction with the antiglucocorticoid cortexotoneand mechanism of its action. J. Bid. Chem., 249:1133-1138,1974.

32. Wira, C. R., and Munck, A. Glucocorticoid-receptor complexes in rat thymuscells, "cytoplasmic'-nuclear transformations. J. Biol. Chem., 249: 5328-5336,

1974.33. Zawydiwski, R., Harmon, J. M., and Thompson, E. B. Glucocorticoid-resistant

human acute lymphoblastic leukemic cell line with functional receptor. CancerRes., 43: 3865-3873,1983.

OCTOBER 1984 4599



U. Galili et al.

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Vif '. .:*>â€¢* vÂ«; .

mFig. 1. Bel 3 cells Â¡ncontrol cultures; note the elongated microvHIi.x 6000. Figs. 1 and 2, scanning electron microscope of Be13 cells in the course of 10 ' M

dexamethasone-inducedlysis.

Fig. 2. Be13 cells incubated for 16 hr with dexamethasone.One of the cells displays normal features and the second has tost microvilli,whereas the third cell is dead,as indicated by the porous cell membrane, x 5500.

Fig.3. Be13 cells in control cultures; note the convoluted nucleus characteristic to thymic lymphocytes and T-leukemia cells, x 12,000. Figs. 3 to 8, transmissionelectron microscope of Be13 cells in the course of 10 ' Mdexamethasone-inducedlysis.

Fig.4. Portion of a Be13 cell; note the distinct polysomes comprised of 4 to 6 ribosomes. x 45,000.Fig. 5. Cells incubated for 16 hr with dexamethasone; note the toss of microvilli. x 9500.Fig.6. A portion of a Be13 cell following 16 hr of incubation with dexamethasone;note the absence of polysomes and the homogenousdispersion of free ribosomes.

x 45,000.Fig.7. Be13 cells following 20 hr of incubation with dexamethasone; note the disintegration of membranal structure and of the intranuclear heterochromatin

organization, x 7500.Fig. 8. Dividing cells containing distinct chromosomes within the culture incubated for 20 hr with the hormone, x 6500.





8

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OCTOBER 1984 4601



1984;44:4594-4601. Cancer Res Uri Galili, Aviva Peleg, Yoram Milner, et al. Dexamethasone-induced CytolysisBe13, a Human T-Leukemia Cell Line Highly Sensitive to

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