Suppression of c-myc is a critical step in glucocorticoid-induced human leukemic cell lysis.

Glucocorticoids evoke cytolysis in clonal human leukemic CEM-C7 cells. Suppression of c-myc mRNA by dexamethasone closely correlates with cell lysis only in CEM clones with both glucocorticoid receptor and intact lysis functions. We tested the theory that c-myc repression is essential for glucocorticoid-induced lymphocytolysis by preventing down-regulation of c-myc gene in the presence of dexamethasone and by reducing c-myc mRNA levels with antisense oligonucleotides. We find that sustained expression of c-myc provides resistance to dexamethasone-induced lysis, and antisense c-myc oligomers induce cell lysis. The lethal effects of dexamethasone in these leukemic cells appear to involve reduction of c-myc below the levels required to maintain cellular growth and integrity.

It has been suggested that glucocorticoids induce the expression of so-called "lysis genes" critical for lysis andor suppress the expression of genes required for maintenance of cellular growth and integrity. Evidence for the repression of essential gene products by glucocorticoids has been obtained from studies in S49 mouse lymphoma, P1798 mouse lymphosarcoma, and CEM-C7 human leukemic cells. It has been reported that in S49 mouse lymphomas glucocorticoids selectively lower c-myc, c-myb, and c-Ki-rus mRNA (17). c-myc expression was rapidly suppressed both in P1798 lymphosarcomas (18) and in CEM-C7 human leukemic cells (191, but in the latter, constitutive expression of c-Ki-ras was unaffected by the steroid treatment. Thus of the protooncogenes tested, c-myc may be of particular interest.
The c-myc protooncogene plays an important role in the control of normal cell growth and differentiation; genetic alterations caused by chromosomal translocation, amplification, or retroviral insertion attest to its role in many neoplasms (20)(21)(22). Avariety of mutational studies on Myc protein has defined functional domains involved in cotransformation, nuclear localization, and inhibition of differentiation (23). The mapping of a C-terminal domain for helix-loop-helix and leucine zipper regions, important in oligomerization of Myc protein, has unraveled the interaction of Myc protein with another protein termed Max in human and Myn in mouse (24,251. Such heterodimeric interactions are thought to facilitate the binding of Myc protein via its basic region to certain regulatory sequences on target genes that might be subsequently altering cell growth and differentiation. Our laboratory studies glucocorticoid action in human leukemic CCRF-CEM cells, which were derived from a patient with childhood T-acute lymphoblastic leukemia (26). CEM-C7 is a glucocorticoid-sensitive clonal line obtained from parental CCRF-CEM cells. CEM-C7 cells have about 10,000 functional glucocorticoid receptor binding sitedcell (27). Upon addition of glucocorticoid, these cells begin to be growth-inhibited in the GI phase of the cell cycle after a delay of 24 h, following which they show a progressive loss of cell viability (28). Previous results have shown that among the eight detectable oncogendgrowthrelated genes studied so far in CEM-C7 cells, only c-myc mRNA levels were suppressed by glucocorticoids (19). Reduction in c-myc could be detected as early as 1 h after glucocorticoid addition and reached a maximum by 12-18 h. There was no change in c-myc mRNA in glucocorticoid-resistant CEM clones whether or not they contained functional glucocorticoid receptors. Hence, c-myc suppression is seen only in CEM cells with both functional glucocorticoid receptor and intact lysis functions.
The present study tests the hypothesis that glucocorticoid repression of c-myc is a critical step leading to the eventual lysis of CEM-C7 cells. If c-myc expression is a key step in the lysis process, then altering the cells so that its expression is sustained in the face of glucocorticoid addition should provide protection from lysis. Conversely, down-regulating c-myc by non-steroidal agents should prove lethal. We h a v e carried out both tests and report the results here.

EXPERIMENTAL PROCEDURES expression vectors, MMTV-myc (MMTV-Stumyc and MMTV-H3myc),
Mouse c-myc Gene Expression Vectors-The steroid-inducible c-myc were kindly provided by Dr. E. A. Thompson, University of Texas Medical Branch, Galveston, TX (29). The metal ion-inducible metallothionein c-myc expression vector (MTmyc) was a kind gift from Dr. B. Wold (CALTECH, Pasadena, CA). The constitutive c-myc expression vector pSVc-myc-1 was purchased from ATCC, Rockville, MD (30). These expression vectors are all capable of expressing functional Myc protein in transfected cells (29,31). For transient transfection assays, plasmid DNA was prepared by cesium chloride density gradient centrifugation (32). Synthetic DNA Oligonucleotides-The oligomers employed for the antisense experiments were synthesized by our departmental facility or by Genosys Biotechnologies Inc., Houston, TX.
Cell Culture-The clonal glucocorticoid-sensitive cell line CEM-C7 used in this study (33) was derived from a parental line CCRF-CEM, obtained from a patient with acute lymphoblastic leukemia (26). The CEM-C7 cells were grown in RPMI 1640 medium, pH 7.4, with 5% heat-inactivated fetal bovine serum (FBS) at 37 "C in a humidified atmosphere of 95% air, 5% COz as stationary suspensions. Cells were maintained in the midlog phase of growth by appropriate subculturing at regular intervals.
Dansient Dansfection of CEM-C7 Cells by Electroporation -Transfections were carried out a s described in Harbour et al. (41). Briefly, logarithmically growing CEM-C7 cells were pelleted, washed three times with cold pD buffer (phosphate-buffered isotonic saline, pH 7.5, devoid of CaZ+ and Mg2+). The cells were then resuspended to 8 x lo6 in 0.8 ml of pD buffer. Twenty micrograms of plasmid DNA were mixed with the cells in electroporation cuvettes (Bio-Rad, Richmond, CA) and allowed to stand on ice for 15 min. The mixture of cells and DNAwas pulsed for 12-14 ms at room temperature with a setting of 200 V and 500 microfarads capacitance. The cells were then resuspended to 8 x 105/ml in RPMI 1640 with 5% FBS and allowed to recover for 24 h a t 37 "C in a humidified atmosphere as described earlier. All transfections were done in triplicate or quadruplicate and repeated at least three times.
To determine the extent of cell damage by electroporation itself, the following preliminary experiments were done. CEM-C7 cells were transfected with: ( a ) no DNA, (b) pBR322, or (c) pSVzneo, plasmid vectors for nonspecific DNA. In all these instances, the nonspecific lysis observed was less than 4%. Hence in various experiments one of the above DNAs was used as control for transfection of CEM-C7 cells. Under these conditions, the efficiency of transfection, determined by measuring autoradiographically the retention of 3ZP-labeled DNA vectors on a cell by cell basis was about 30%.2 Steroid Deatment and Cell Viability-Twenty-four h after transfection, cells were resuspended to about 2 x 105/ml in fresh RPMI 1640 containing 5% FBS. For MMTV-myc and pSVc-myc-1 transfectants, dexamethasone (Dex) (Sigma) in ethanol was added to a final concentration of 1 p~ or ethanol vehicle to a final concentration of 0.1%. For MTmyc transfectants, ZnSO., (Sigma) was added to a final concentration of 20 PM with or without Dex (1 PM) in ethanol or ethanol alone to a final concentration of 0.1%. We determined viable cell counts manually with a hemocytometer by the trypan blue dye exclusion method, immediately after transfection and 24, 48, 72, and 96 h later. For the trypan blue assay, 100 pl of dispersed cells were mixed with 100 pl of 0.1% trypan blue dye, and 15 pl of the mixture was added to the chambers of a hemocytometer. Those cells that excluded the dye were taken as viable cells while stained blue cells were counted as dead cells. It has been shown previously that viable cell numbers determined thus correlated with the soft agar cloning assay and that the trypan blue assay was a more accurate determinant of viability than cell counts obtained by Coulter counter (34).
Antibody Preparations-A hybridoma cell line CRL1729, secreting a monoclonal antibody MYC 1-9E10.2 (35), raised against a synthetic peptide C-terminal to Myc protein (purchased from ATCC) was grown in RPMI 1640 medium with 10% FBS a t cell concentrations between 5 x lo5 and 1 x lo6 cells/ml. The supernatant containing the secreted monoclonal antibody was aliquoted into 1-ml fractions and stored a t -20 "C. Antibody titer was determined on Myc protein in CEM-C7 cell extracts L. V. Nazareth and E. B. Thompson, unpublished observations. by serial dilutions of the antibody. This monoclonal antibody was used in the immunoblot analyses. Apolyclonal antibody R3586 raised against Myc protein, a generous gift by Dr. W. Bryce (Abbott Laboratories, Abbott Park, IL), gave confirmatory results in the immunoprecipitation experiments not included herein. The actin antibody (Ab-1) was purchased from Oncogene Science Inc., Mineola, N Y . The partially purified recombinant Myc protein was kindly provided by Dr. R. Koski, Amgen Inc., Thousand Oaks, CA.

lmmunoblotAnalysis-CEM-C7 cells in RPMI 1640 medium with 5%
FBS (1 x lo7 celldml) were harvested at various times after the treatment of interest by centrifugation a t 200 x g and washed twice with isotonic phosphate-buffered saline (PBS), pH 7.4. Cell pellets were reconstituted in Tris-EDTA buffer, pH 8.0, sonicated to lyse the cells, and stored frozen a t -70 "C until preparation for immunoblot analysis. Protein samples were quantitated by the Bradford assay (Bio-Rad), and 20-pg samples were mixed with a 3 x volume of sodium dodecyl sulfate (SDS) sample buffer and incubated a t 95 "C for 3 min. The samples were electrophoresed on 12% SDS-polyacrylamide gel. The proteins were electroblotted onto Immobilon-N membranes (Millipore, Bedford, MA). Prestained Rainbow molecular weight markers or low molecular weight markers (Bio-Rad) were used as standards. The filters were "blocked" by incubation with 5% nonfat milk powder in PTG (PBS, pH 7.4, containing 0.25% Triton X-100, 0.02% gelatin) at 37 "C for 30 min. The filter was then washed with PTG for 10 min. Immunodetection was performed by incubating the blots in the primary antibody Myc 1-9E10.2 (1:lO dilution) at 37 "C for 1 h or at 4 "C overnight. The filters were then washed three times for 5 min each with PTG on a rocking platform and incubated with a 1:3,000 dilution of goat anti-mouse IgG linked to alkaline phosphatase (Bio-Rad) at room temperature for 30 min. The filters were then washed 3 times for 5 min with PTG, and the color was developed using BCIPNBT as substrate (Kirkegaard & Perry Labs Inc., Gaithersburg, MD). The densitometric scanning of the immunoblots was done on an image analyzer (Biological Visions Inc., San Mateo, CAI. Immunoprecipitation-Immunoprecipitation of Myc protein was done as described with some modifications (36). CEM-C7 cells a t various times after treatment were collected, washed with isotonic PBS, pH 7.4, resuspended in methionine-free IMENZO media (Hazelton Research Products, Kansan City, MO), and grown for 1 h. Then cells were washed in PBS and labeled in 1 ml of IMENZO medium containing 250 pCi of [35Slmethionine (Amersham Corp.) for 30 min a t 37 "C. Cells were washed three times in PBS and stored frozen at -70 "C until preparation for immunoprecipitation. Frozen cell pellets were solubilized in 100 pl of cold antibody buffer (0.01 M Tris, pH 7.5, 0.5 M NaC1, 0.5% Nonidet P-40, 0.5% deoxycholate, 0.5% SDS, and 10 mM iodoacetamide) and lysed by sonication. Protein concentrations were determined by Bradford assay (Bio-Rad). Eighty micrograms of cell extracts in antibody buffer were added to preswollen protein A-Sepharose and incubated on a rotating wheel for 1 h at 4 "C. The samples were centrifuged, and the supernatants were incubated with a polyclonal antibody raised against recombinant Myc protein R3586 overnight a t 4 "C. The antigen-antibody complex was precipitated with protein A-Sepharose. Then the immunoprecipitated complex was washed with RIPA buffer (50 mM Tris, pH 8.0, 150 mM NaCI, 1% Nonidet P-40, 0.5% deoxycholate, and 0.1% SDS at 4 "C), and the pellet was resuspended in SDS-sample loading buffer. A small portion of each sample was used to count for radioactivity in a scintillation counter. The samples were then subjected to SDS-polyacrylamide gel electrophoresis and enhanced for fluorography.
Deatment of CEM-C7 Cells with Antisense c-myc Oligomers-Four PM antisense c-myc oligomers complementary to the first five codons of human c-myc mRNA were added to CEM-C7 cells (2.5-5.5 x lo5 cells/ ml) grown in RPMI 1640 medium with 5% FBS. Preliminary experiments showed this to be the optimal condition for our study. As controls, the same concentration of sense c-myc oligomers or completely unrelated oligomers of the vesicular stomatitis virus (VSV) M protein mRNA was used. Viable cell counts were determined at intervals for 48 h by the trypan blue dye exclusion method.  The protection against Dex-induced cell kill by transfection with MMTV-myc correlates with sustained levels of Myc protein in these cells. Fig. 3 shows the result of an experiment that compares Myc protein expression in control DNA and MMTVmyc-transfected cells treated with ethanol or Dex. Myc protein was identified immunologically after SDS-polyacrylamide gel electrophoresis of CEM-C7 cell extracts. The Myc band from the immunoblot of control DNA (left) or MMTV-myc-transfected cells (right) shown in Fig. 3A was then quantitated by densitometry (Fig. 3B) were also assayed for actin protein levels using an actin antibody Ab-1. There was no significant change in actin protein levels upon transfection and Dex treatment. From these results we hypothesize that the resistance to the lytic effects of Dex conferred during the transient post-transfection period is due to the persistence of Myc protein levels adequate for cell viability and that a certain threshold level of Myc protein is required to maintain growth and viability of CEM-C7 cells.

Glucocorticoids
Transfection of CEM-C7 cells with Zn2+-inducible or constitutive mouse c-myc expression vectors also conferred resistance to the cytolysis induced by Dex. The mouse metallothionein I c-myc (MTmyc) expression vector shown in Fig. 4A was transfected into CEM-C7 cells. Varying concentrations of Zn2+ ranging between 0 and 50 PM were added to the cells. Viable cell numbers were determined and c-myc mRNA levels assayed by dot blot analysis using a 32P-labeled mouse-specific exon 3 oligomer probe. We have shown previously by Northern blotting that exon 3 probes hybridize exclusively with c-myc mRNA (19). Protection against Dex-induced cytolysis was also tested using the constitutive c-myc expression vector pSVc-myc-1 shown in Fig. 5 A . Twenty-four h after transfection, CEM-C7 cells were treated with Dex (1 PM) or ethanol vehicle (O.l%), and viable cell number was determined at intervals for 72 h. Fig. 5B shows that there were significantly more viable cells after Dex treatment in pSVc-myc-1 transfectants compared with control DNA vector transfectants ( p < 0.05).
Incubation of CEM-C7 Cells with Antisense Oligomers Induces Cell Lysis and Reduces Myc Protein Levels-The second test of the central role of c-myc in maintaining CEM-C7 cell viability was to down-regulate the c-myc gene by non-steroidal treatment. It is well known that when cells are incubated with antisense oligonucleotides complementary to specific mRNA, the oligomers form a duplex with the mRNA. This duplex formation can result in hybridization arrest of translation, depleting both mRNA and protein levels in the cells (37). We incubated CEM-C7 cells with each of the oligomers shown in Fig. 6 A. and assayed for cell viability at various time points. Treatment with the antisense oligomers resulted in dramatic cell kill. The data represented combine the results of two experiments, each done in duplicate. The results indicate that at 48 h after addition of the oligomers, only 10% of the cells treated with c-myc antisense oligomers remain viable, whereas greater than 50% of the cells after treatment with sense oligomers were viable and greater than 95% of the cells were viable afler treatment with VSV M nonspecific oligomers completely unrelated to the c-myc gene. The addition of the antisense oligomers reduced immunoprecipitable Myc protein levels to about 50% of control, while the addition of sense or nonspecific oligomer did not significantly alter Myc protein levels (Fig. 6). DISCUSSION Normal cell growth and proliferation are regulated by a balance between growth-promoting and growth-suppressing factors, which are the products of growth-regulating genes. Molecular alterations in growth-promoting cancer genes or oncogenes have been thought to be involved in a number of malignancies. The protooncogene c-myc is the cellular homologue of the avian cytomatosis retroviral (v-myc) transforming gene. The presence of rearranged c-myc gene in tumors such as leukemias, lymphomas, and small lung carcinomas suggested a role of altered c-myc gene in neoplastic transformation (20,22). The precise mechanism of action of the protein Myc, the product of c-myc gene, is not known. However, there is a close correlation between cellular proliferation and c-myc gene expression in a variety of cell types (20). Furthermore, c-myc mRNA levels are diminished in differentiating cells relative to proliferating cells. The c-myc mRNA levels are low in quiescent, serum-starved fibroblasts and peripheral lymphocytes but increase on stimulation with mitogens (38).

c-myc Suppression in Dexamethasone-induced Cell
Glucocorticoid-induced lymphocytolysis has been studied in cultures of rat thymocytes and a variety of other cell lines, including S49 mouse lymphoma, P1798 mouse lymphosarcoma, WEHI 7 mouse T-lymphoma, and CEM-C7 human T-cell leukemia (39). The lethal effects of glucocorticoids are dramatic in certain rodent cells; appropriate cells from rats or mice were killed by corticosteroids within hours (40). Although the exact mechanism(s) by which glucocorticoids induce cytolysis is still under investigation, the glucocorticoid receptor plays an important role in the cytolytic process. We have shown that restoration of glucocorticoid holoreceptor (41) or minimal receptor fragments (42) to glucocorticoid-resistant CEM cells containing mutant receptors results in restoration of the lytic response.
Furthermore, in P1798 mouse lymphosarcoma (18), S49 mouse lymphoma (171, and CEM-C7 human leukemia cells (19) steroids rapidly decrease c-myc mRNA. In CEM-C7 cells, c-myc mRNA levels are rapidly down-regulated, with measurable diminution within an hour and a minimum reached by about 18 h (19). Since both c-myc mRNA and protein have a very short half-life (about 30 min) in all cells studied (43), it is often presumed that Myc protein is expressed in a parallel fashion with its mRNA. On the contrary, it has been reported that in mouse erythroleukemic cells post-translational regulation of Myc protein results in a dramatic decrease in the accumulation of Myc protein during the postcommitment phase of differentiation despite high levels of Myc protein synthesis (36).
We show in this report that Myc protein follows a time course appropriate with the mRNA in CEM-C7 cells. Although other bands are obtained with the monoclonal antibody employed, the effect of Dex is observed only on the Myc holoprotein band. The nonspecific bands were also observed in immunoblots from other laboratories upon using anti-Myc antibodies and are presumed to be either specific breakdown products of c-myc protein (36) or nonspecific cross-reactions. Since there was no other band showing parallel or reciprocal change with Myc holoprotein, we assume in our case that the other bands are predominantly cross-reactions. Furthermore, there was no significant change in the constitutively expressed protein, actin. It is striking that the onset of cell death in CEM-C7 cells occurs shortly after c-myc mRNA and protein have reached a minimum. The lag period between the rapid decrease in c-myc mRNA and loss of cell viability suggests that one of the earlier steps in the cascade of events taking place during the process of Dex-induced lysis of CEM-C7 cells is c-myc down-regulation.
In the present studies, we have tested the theory that suppression of c-myc gene expression is a key step in glucocorticoid evocation of cell growth inhibition and lysis of a clonal human leukemic cell line. We find that transient transfection of these cells with three different inducible or constitutive mouse c-myc expression vectors confers significant resistance to the lytic effects of Dex. At present, we do not have the data to see if there is a precise correlation between Myc levels from each of these vectors and degree of resistance. But the qualitative conclusions seem inescapable. In considering these data, there are four points that need to be considered. 1) The resistance conferred was transient, and the best results were seen at 48 h after Dex addition, i.e. 72 h after transfection. We interpret this to be the result of variable uptake, retention, and expression of the transfected DNA depending on the efficiency of transfection in subsets of cells. The transfected DNA remains episomal for 72-96 h, during which period it is either lost from the cells or undergoes gradual degradation by endogenous nucleases. Thus in transient transfection assays, the expression of the c-myc gene was studied during the transient post-transfection period of 48-72 h after Dex treatment or 72-96 h after transfection. 2) Upon averaging the results obtained with the three c-myc expression vectors used in this study, the Dex-induced cell lysis of CEM-C7 cells was inhibited by about 50% in c-myc-transfected cells compared with control DNA vector-transfected cells. As observed from these transfection results the resistance conferred to Dex-induced cytolysis was not 100%. Since in these cells the efficiency of transfection by electroporation was 30%,2 we suggest that there are two populations of cells at any given time: one set that retained sufficient c-myc expression vectors to be resistant to Dex-induced cytolysis and the other set sensitive to Dex. 3) The extent of resistance conferred by the two steroid-inducible MMTV-myc expression vectors Stumyc and H3myc was similar within experimental errors. This suggests that irrespective of the presence (Stumyc) or absence (H3myc) of mouse c-myc constitutive promoters P1 and P2, the expression of c-myc gene driven by MMTV-LTR is responsible for the increase in viable cell number in MMTV-myc-transfected CEM-C7 cells. Such c-myc gene-transfected cells also maintained relatively higher levels of Myc protein. 4) There was no significant change in the rate of proliferation of cells transfected with any of the c-myc expression vectors as depicted by the growth curves in Figs. 2B, 4B, and 5B. We hypothesize that a certain threshold level of c-myc gene expression is required in these CEM-C7 cells for sustained cell growth and viability.
Recently a number of papers have been published demonstrating the involvement of the c-myc gene in the induction of programmed cell death. Constitutive expression of c-myc suppressed cell cycle arrest and accelerated apoptotic events upon IL-3 withdrawal in IL-3-dependent myeloid cells (44). In IL-3 dependent pre-B-lymphocytes, IL-3 deprivation suppressed cmyc expression and induced growth arrest and cell death (45). In rat-1 fibroblasts, constitutive expression of c-myc induced apoptosis upon serum deprivation, and the rate of cell death was determined by the level of Myc protein expressed (46). These results have led to the proposal that there may be at least two signal systems in cells, both controlled by c-myc, one for proliferation and the other for programmed cell death. When the necessary growth factors or cytokines are available, the cells proliferate, which may be via appropriately increased c-myc expression. Under conditions of serum starvation or cytokine removal or absence of exogenous growth factors and when the cells are not able to down-regulate c-myc expression, the cells undergo programmed cell death. It has been suggested that growth factors signal the cell to inhibit cell death and allow c-myc to promote continued movement throughout the cell cycle.
In contrast, differentiation inducers such as glucocorticoids suppress c-myc expression and hence induce cell death. Our results suggest that such glucocorticoid-induced cell death is dependent on dropping below a certain threshold level of c-myc expression. The resolution of the contrasts between these two systems should reveal important principles about the control of vital cellular processes.
The second test of the central role of c-myc in maintaining CEM-C7 cell viability was to down-regulate the c-myc gene by non-steroidal agents. Synthetic antisense oligonucleotides complementary to specific mRNAs have been employed by several investigators to study the importance of the respective gene expression. Antisense oligomers complementary to c-myc mRNA have been shown to inhibit mitogen-induced human T-lymphocyte entry into S phase but not progress from Go to GI phase of the cell cycle (47). Antisense c-myc oligomers also inhibited proliferation of HL-60 promyelocytic cells and induced differentiation (37). Constitutive expression of c-myc antisense transcripts accelerated dimethyl sulfoxide-induced differentiation in MEL cells (49). Our results indicate that expression of the growth-related gene c-myc and resistance to glucocorticoid-induced cell lysis are coupled. Induction of significant lysis of CEM-C7 cells and reduction in Myc protein levels by the addition of c-myc antisense oligomers further supports this hypothesis. CEM-C7 cells are primitive or poorly differentiated T-lymphoblasts, while other cell lines such as HL-60 promyelocytic leukemia, F9 teratocarcinoma, and MEL mouse erythroleukemia are more differentiated or chronic forms of leukemias. Hence the response to down-regulation of c-myc expression is different for CEM-C7 cells and other cell lines such as HL-60, F9, and MEL. In the latter, suppression of c-myc gene inhibits cellular proliferation and induces differentiation while in the former, down-regulation of c-myc gene also stops the cell cycle but further induces cell lysis. In the CEM-C7 cells, the partial cell kill we observed with sense oligomer could be a result of inhibition of the product of antisense transcription of the c-myc gene or could be a nonspecific effect. Antisense transcription of c-myc has been demonstrated by nuclear run-on experiments both in human and mouse (48,50).
In summary, our results show that suppression of both c-myc mRNA and protein by Dex in a line of human leukemic T-cells is an early event beginning within an hour of the addition of Dex. Restoration of c-myc levels by transfection with three different mouse c-myc expression vectors protects these CEM-C7 cells from the Dex-induced cell kill. We also demonstrate that such transfected cells also maintained relatively high levels of Myc protein. Furthermore, suppression of c-myc expression by non-steroidal agents induced cell kill. These data confirm our hypothesis that a critical level of c-myc expression is required for not only the growth but also the viability of these cells. We hypothesize that either the protein product of the early gene c-myc, regulated by glucocorticoids, is a trans-acting factor that inhibits expression of the genes critical for cell lysis or that Myc protein may modify the functions of other trans-acting factors regulating the process of lysis induced by glucocorticoids. These data provide new insights in the search for the mechanism(s) of the Dex-induced cell lysis of human leukemic cells.